# Patent

## Title
Proteins, polynucleotides encoding them and methods of using the same

## Abstract

Disclosed herein are nucleic acid sequences that encode novel polypeptides. Also disclosed are polypeptides encoded by these nucleic acid sequences, and antibodies, which immunospecifically-bind to the polypeptide, as well as derivatives, variants, mutants, or fragments of the aforementioned polypeptide, polynucleotide, or antibody. The invention further discloses therapeutic, diagnostic and research methods for diagnosis, treatment, and prevention of disorders involving any one of these novel human nucleic acids and proteins.

## Background
RELATED APPLICATIONS
This application claims priority to U.S. Ser. No. 60/270,523, filed Feb. 21 2001; U.S. Ser. No. 60/322,712, filed Sep. 17, 2001; U.S. Ser. No. 60/311,980, filed Aug. 13, 2001; U.S. Ser. No. 60/330,307, filed Oct. 18, 2001; U.S. Ser. No. 60/278,796, filed Mar. 26, 2001; U.S. Ser. No. 60/281,521, filed Apr. 4, 2001; U.S. Ser. No. 60/276,677, filed Mar. 16, 2001; U.S. Ser. No. 60/311,595, filed Aug. 10, 2001, U.S. Ser. No. 60/270,220, filed Feb. 21, 2001; U.S. Ser. No. 60/274,295, filed Mar. 8, 2001; U.S. Ser. No. 60/318,526, filed Sep. 10, 2001; U.S. Ser. No. 60/286,548, filed Apr. 25, 2001; U.S. Ser. No. 60/291,765, filed May 17, 2001; U.S. Ser. No. 60/270,797, filed Feb. 23, 2001; U.S. Ser. No. 60/276,400, filed Mar. 16, 2001; and U.S. Ser. No. 60/270,810, filed Feb. 23, 2001 each of which is incorporated by reference in its entirety.


FIELD OF THE INVENTION
The invention relates to polynucleotides and the polypeptides encoded by such polynucleotides, as well as vectors, host cells, antibodies and recombinant methods for producing the polypeptides and polynucleotides, as well as methods for using the same.
BACKGROUND OF THE INVENTION
The present invention is based in part on nucleic acids encoding proteins that are new members of the following protein families: Androgen-Regulated Short-Chain Dehydrogenase/Reductase-like, Aryl-Acylamidase-like, Insulysin-like, Integrin Beta-7 Precursor-like, Membrane protein-like, BCSC-1-like, Amino Acid Transporter-like, Lymphocyte Antigen Precursor-like, Lymphocyte Antigen LY-6F-like, Early B-Cell Factor-like, High-Affinity Camp-Specific and IBMX-Insensitive-like, KIAA0216-like, TWIK 3-like, TASK 4-like, Copper Transporter-like, Cytokeratin-like, Protocadherin-like, Protocadherin Beta-like, Cadherin 23-like, Transforming Growth Factor Beta 2-like, Ebnerin-like, Fatty Acid Binding-like, Platelet glycoprotein V-like, GARPIN-like, Centaurin Beta 2-like, Sorting Nexin 9-like, Katanin-like, Calpain-like, Keratin 18-like, Polycystic Kidney Disease Associated, Cholinephosphate Cytidylyltransferase-like and mac25/IGFBP7-like. More particularly, the invention relates to nucleic acids encoding novel polypeptides, as well as vectors, host cells, antibodies, and recombinant methods for producing these nucleic acids and polypeptides.
SUMMARY OF THE INVENTION
The invention is based in part upon the discovery of nucleic acid sequences encoding novel polypeptides. The novel nucleic acids and polypeptides are referred to herein as NOVX, or NOV1, NOV2, NOV3, NOV4, NOV5, NOV6, NOV7, NOV8, NOV9, NOV10, NOV11, NOV12, NOV13, NOV14, NOV15, NOV16, NOV17, NOV18, NOV19, NOV20, NOV21, NOV22, NOV23, NOV24, NOV25, NOV26, NOV27, NOV28, NOV29 and NOV30 nucleic acids and polypeptides. These nucleic acids and polypeptides, as well as derivatives, homologs, analogs and fragments thereof, will hereinafter be collectively designated as “NOVX” nucleic acid or polypeptide sequences.
In one aspect, the invention provides an isolated NOVX nucleic acid molecule encoding a NOVX polypeptide that includes a nucleic acid sequence that has identity to the nucleic acids disclosed in SEQ ID NOS:1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91 and 93. In some embodiments, the NOVX nucleic acid molecule will hybridize under stringent conditions to a nucleic acid sequence complementary to a nucleic acid molecule that includes a protein-coding sequence of a NOVX nucleic acid sequence. The invention also includes an isolated nucleic acid that encodes a NOVX polypeptide, or a fragment, homolog, analog or derivative thereof. For example, the nucleic acid can encode a polypeptide at least 80% identical to a polypeptide comprising the amino acid sequences of SEQ ID NOS:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92 and 94. The nucleic acid can be, for example, a genomic DNA fragment or a cDNA molecule that includes the nucleic acid sequence of any of SEQ ID NOS:1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91 and 93.
Also included in the invention is an oligonucleotide, e.g., an oligonucleotide which includes at least 6 contiguous nucleotides of a NOVX nucleic acid (e.g. SEQ ID NOS:1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41,43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91 and 93) or a complement of said oligonucleotide. Also included in the invention are substantially purified NOVX polypeptides (SEQ ID NOS:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92 and 94). In certain embodiments, the NOVX polypeptides include an amino acid sequence that is substantially identical to the amino acid sequence of a human NOVX polypeptide.
The invention also features antibodies that immunoselectively bind to NOVX polypeptides, or fragments, homologs, analogs or derivatives thereof.
In another aspect, the invention includes pharmaceutical compositions that include therapeutically- or prophylactically-effective amounts of a therapeutic and a pharmaceutically-acceptable carrier. The therapeutic can be, e.g., a NOVX nucleic acid, a NOVX polypeptide, or an antibody specific for a NOVX polypeptide. In a further aspect, the invention includes, in one or more containers, a therapeutically- or prophylactically-effective amount of this pharmaceutical composition.
In a further aspect, the invention includes a method of producing a polypeptide by culturing a cell that includes a NOVX nucleic acid, under conditions allowing for expression of the NOVX polypeptide encoded by the DNA. If desired, the NOVX polypeptide can then be recovered.
In another aspect, the invention includes a method of detecting the presence of a NOVX polypeptide in a sample. In the method, a sample is contacted with a compound that selectively binds to the polypeptide under conditions allowing for formation of a complex between the polypeptide and the compound. The complex is detected, if present, thereby identifying the NOVX polypeptide within the sample.
The invention also includes methods to identify specific cell or tissue types based on their expression of a NOVX.
Also included in the invention is a method of detecting the presence of a NOVX nucleic acid molecule in a sample by contacting the sample with a NOVX nucleic acid probe or primer, and detecting whether the nucleic acid probe or primer bound to a NOVX nucleic acid molecule in the sample.
In a further aspect, the invention provides a method for modulating the activity of a NOVX polypeptide by contacting a cell sample that includes the NOVX polypeptide with a compound that binds to the NOVX polypeptide in an amount sufficient to modulate the activity of said polypeptide. The compound can be, e.g., a small molecule, such as a nucleic acid, peptide, polypeptide, peptidomimetic, carbohydrate, lipid or other organic (carbon containing) or inorganic molecule, as further described herein.
Also within the scope of the invention is the use of a therapeutic in the manufacture of a medicament for treating or preventing disorders or syndromes including, e.g., trauma, regeneration (in vitro and in vivo), viral/bacterial/parasitic infections, Von Hippel-Lindau (VHL) syndrome, Alzheimer's disease, stroke, Tuberous sclerosis, hypercalceimia, Parkinson's disease, Huntington's disease, Cerebral palsy, Epilepsy, Lesch-Nyhan syndrome, Multiple sclerosis, Ataxia-telangiectasia, Leukodystrophies, behavioral disorders, addiction, anxiety, pain, actinic keratosis, acne, hair growth diseases, allopecia, pigmentation disorders, endocrine disorders, connective tissue disorders, such as severe neonatal Marfan syndrome, dominant ectopia lentis, familial ascending aortic aneurysm, isolated skeletal features of Marfan syndrome, Shprintzen-Goldberg syndrome, genodermatoses, contractural arachnodactyly, inflammatory disorders such as osteo- and rheumatoid-arthritis, inflammatory bowel disease, Crohn's disease; immunological disorders, AIDS; cancers including but not limited to lung cancer, colon cancer, Neoplasm; adenocarcinoma; lymphoma; prostate cancer; uterus cancer, leukemia or pancreatic cancer; blood disorders; asthma; psoriasis; vascular disorders, hypertension, skin disorders, renal disorders including Alport syndrome, immunological disorders, tissue injury, fibrosis disorders, bone diseases, Ehlers-Danlos syndrome type VI, VII, type IV, S-linked cutis laxa and Ehlers-Danlos syndrome type V, osteogenesis imperfecta, Neurologic diseases, Brain and/or autoimmune disorders like encephalomyelitis, neurodegenerative disorders, immune disorders, hematopoietic disorders, muscle disorders, inflammation and wound repair, bacterial, fungal, protozoal and viral infections (particularly infections caused by HIV-1 or HIV-2), pain, acute heart failure, hypotension, hypertension, urinary retention, osteoporosis, Treatment of Albright Hereditary Ostoeodystrophy, angina pectoris, myocardial infarction, ulcers, benign prostatic hypertrophy, arthrogryposis multiplex congenita, osteogenesis imperfecta, keratoconus, scoliosis, duodenal atresia, esophageal atresia, intestinal malrotation, Pancreatitis, Obesity Systemic lupus erythematosus, Autoimmune disease, Emphysema, Scleroderma, allergy, ARDS, Neuroprotection, Fertility Myasthenia gravis, Diabetes, obesity, Growth and reproductive disorders Hemophilia, Hypercoagulation, Idiopathic thrombocytopenic purpura, Immunodeficiencies, Graft vesus host, Adrenoleukodystrophy, Congenital Adrenal Hyperplasia, Endometriosis, Xerostomia, Ulcers, Cirrhosis, Transplantation, Diverticular disease, Hirschsprung's disease, Appendicitis, Arthritis, Ankylosing spondylitis, Tendinitis, Renal artery stenosis, Interstitial nephritis, Glomerulonephritis, Polycystic kidney disease, erythematosus, Renal tubular acidosis, IgA nephropathy, anorexia, bulimia, psychotic disorders, including anxiety, schizophrenia, manic depression, delirium, dementia, severe mental retardation and dyskinesias, such as Huntington's disease and/or other pathologies and disorders of the like.
The therapeutic can be, e.g., a NOVX nucleic acid, a NOVX polypeptide, or a NOVX-specific antibody, or biologically-active derivatives or fragments thereof.
For example, the compositions of the present invention will have efficacy for treatment of patients suffering from the diseases and disorders disclosed above and/or other pathologies and disorders of the like. The polypeptides can be used as immunogens to produce antibodies specific for the invention, and as vaccines. They can also be used to screen for potential agonist and antagonist compounds. For example, a cDNA encoding NOVX may be useful in gene therapy, and NOVX may be useful when administered to a subject in need thereof. By way of non-limiting example, the compositions of the present invention will have efficacy for treatment of patients suffering from the diseases and disorders disclosed above and/or other pathologies and disorders of the like.
The invention further includes a method for screening for a modulator of disorders or syndromes including, e.g., the diseases and disorders disclosed herein and/or other pathologies and disorders of the like. The method includes contacting a test compound with a NOVX polypeptide and determining if the test compound binds to said NOVX polypeptide. Binding of the test compound to the NOVX polypeptide indicates the test compound is a modulator of activity, or of latency or predisposition to the aforementioned disorders or syndromes.
Also within the scope of the invention is a method for screening for a modulator of activity, or of latency or predisposition to disorders or syndromes including, e.g., the diseases and disorders disclosed above and/or other pathologies and disorders of the like by administering a test compound to a test animal at increased risk for the aforementioned disorders or syndromes. The test animal expresses a recombinant polypeptide encoded by a NOVX nucleic acid. Expression or activity of NOVX polypeptide is then measured in the test animal, as is expression or activity of the protein in a control animal which recombinantly-expresses NOVX polypeptide and is not at increased risk for the disorder or syndrome. Next, the expression of NOVX polypeptide in both the test animal and the control animal is compared. A change in the activity of NOVX polypeptide in the test animal relative to the control animal indicates the test compound is a modulator of latency of the disorder or syndrome.
In yet another aspect, the invention includes a method for determining the presence of or predisposition to a disease associated with altered levels of a NOVX polypeptide, a NOVX nucleic acid, or both, in a subject (e.g., a human subject). The method includes measuring the amount of the NOVX polypeptide in a test sample from the subject and comparing the amount of the polypeptide in the test sample to the amount of the NOVX polypeptide present in a control sample. An alteration in the level of the NOVX polypeptide in the test sample as compared to the control sample indicates the presence of or predisposition to a disease in the subject. Preferably, the predisposition includes, e.g., the diseases and disorders disclosed above and/or other pathologies and disorders of the like. Also, the expression levels of the new polypeptides of the invention can be used in a method to screen for various cancers as well as to determine the stage of cancers.
In a further aspect, the invention includes a method of treating or preventing a pathological condition associated with a disorder in a mammal by administering to the subject a NOVX polypeptide, a NOVX nucleic acid, or a NOVX-specific antibody to a subject (e.g., a human subject), in an amount sufficient to alleviate or prevent the pathological condition. In preferred embodiments, the disorder, includes, e.g., the diseases and disorders disclosed above and/or other pathologies and disorders of the like.
In yet another aspect, the invention can be used in a method to identity the cellular receptors and downstream effectors of the invention by any one of a number of techniques commonly employed in the art. These include but are not limited to the two-hybrid system, affinity purification, co-precipitation with antibodies or other specific-interacting molecules.
NOVX nucleic acids and polypeptides are further useful in the generation of antibodies that bind immuno-specifically to the novel NOVX substances for use in therapeutic or diagnostic methods. These NOVX antibodies may be generated according to methods known in the art, using prediction from hydrophobicity charts, as described in the “Anti-NOVX Antibodies” section below. The disclosed NOVX proteins have multiple hydrophilic regions, each of which can be used as an immunogen. These NOVX proteins can be used in assay systems for functional analysis of various human disorders, which will help in understanding of pathology of the disease and development of new drug targets for various disorders.
The NOVX nucleic acids and proteins identified here may be useful in potential therapeutic applications implicated in (but not limited to) various pathologies and disorders as indicated below. The potential therapeutic applications for this invention include, but are not limited to: protein therapeutic, small molecule drug target, antibody target (therapeutic, diagnostic, drug targeting/cytotoxic antibody), diagnostic and/or prognostic marker, gene therapy (gene delivery/gene ablation), research tools, tissue regeneration in vivo and in vitro of all tissues and cell types composing (but not limited to) those defined here.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In the case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.
Other features and advantages of the invention will be apparent from the following detailed description and claims.


DETAILED DESCRIPTION OF THE INVENTION
The present invention provides novel nucleotides and polypeptides encoded thereby. Included in the invention are the novel nucleic acid sequences and their encoded polypeptides. The sequences are collectively referred to herein as “NOVX nucleic acids” or “NOVX polynucleotides” and the corresponding encoded polypeptides are referred to as “NOVX polypeptides” or “NOVX proteins.” Unless indicated otherwise, “NOVX” is meant to refer to any of the novel sequences disclosed herein. Table A provides a summary of the NOVX nucleic acids and their encoded polypeptides.







TABLE A







Sequences and Corresponding SEQ ID Numbers













SEQ ID






NO


NOVX

(nucleic
SEQ ID NO


Assignment
Internal Identification
acid)
(polypeptide)
Homology














 1
CG57413-01
1
2
Androgen-Regulated Short-Chain






Dehydrogenase/Reductase-like


 2a
CG57391-01
3
4
Aryl-Acylamidase-like


 2b
CG57391-02
5
6
Aryl-Acylamidase-like


 3a
CG57433-01
7
8
Insulysin-like


 3b
CG57433-02
9
10
Insulysin-like


 4a
CG57185-01
11
12
Integrin Beta-7 Precursor-like


 4b
CG57185-02
13
14
Integrin Beta-7 Precursor-like


 5
CG57360-01
15
16
Membrane protein-like


 6
CG57362-01
17
18
BCSC-1 like


 7a
CG57387-01
19
20
Amino Acid Transporter-like


 7b
CG57387-02
21
22
Amino Acid Transporter-like


 7c
CG57387-03
23
24
Amino Acid Transporter-like


 8a
CG56417-01
25
26
Lymphocyte Antigen Precursor-like


 8b
CG56417-02
27
28
Lymphocyte Antigen Precursor-like


 8c
CG56417-03
29
30
Lymphocyte Antigen Precursor-like


 8d
CG56417-04
31
32
Lymphocyte Antigen LY-6F-like


 8e
172885384
33
34
Lymphocyte Antigen LY-6F-like


 9a
CG57480-01
35
36
Early B-Cell Factor-like


 9b
CG57480-02
37
38
Early B-Cell Factor-like


10
CG57389-01
39
40
High-Affinity Camp-Specific and






IBMX-Insensitive-like


11
CG57337-01
41
42
KIAA0216-like


12a
CG57220-01
43
44
TWIK 3-like


12b
CG57220-02
45
46
TWIK 3-like


12c
CG57220-03
47
48
TWIK 3-like


13
CG57220-04
49
50
TASK 4-like


14
CG57458-01
51
52
Copper Transporter-like


15
CG57454-01
53
54
Cytokeratin-like


16a
CG57448-01
55
56
Protocadherin-like


16b
CG57446-01
57
58
Protocadherin-like


16c
CG57444-01
59
60
Protocadherin-like


16d
CG57442-01
61
62
Protocadherin Beta-like


17a
CG57429-01
63
64
Cadherin 23-like


17b
CG57429-02
65
66
Cadherin 23-like


18
CG55887-02
67
68
Transforming Growth Factor Beta






2-like


19
CG57333-01
69
70
Ebnerin-like


20a
CG57556-01
71
72
Fatty Acid Binding-like


20b
CG57556-02
73
74
Fatty Acid Binding-like


21
CG57436-01
75
76
Platelet glycoprotein V-like


22
CG57529-01
77
78
GARPIN-like


23
CG57351-01
79
80
Centaurin Beta 2-like


24
CG57515-01
81
82
Sorting Nexin 9-like


25
CG57568-01
83
84
Katanin-like


26
CG57509-01
85
86
Calpain-like


27
CG57484-01
87
88
Keratin 18-like


28
CG57236-01
89
90
Polycystic Kidney Disease






Associated-like


29
CG57589-01
91
92
Cholinephosphate






Cytidylyltransferase-like


30
CG57558-01
93
94
mac25/IGFBP7-like









NOVX nucleic acids and their encoded polypeptides are useful in a variety of applications and contexts. The various NOVX nucleic acids and polypeptides according to the invention are useful as novel members of the protein families according to the presence of domains and sequence relatedness to previously described proteins. Additionally, NOVX nucleic acids and polypeptides can also be used to identify proteins that are members of the family to which the NOVX polypeptides belong.
The NOVX nucleic acids and polypeptides can also be used to screen for molecules, which inhibit or enhance NOVX activity or function. Specifically, the nucleic acids and polypeptides according to the invention may be used as targets for the identification of small molecules that modulate or inhibit, e.g., neurogenesis, cell differentiation, cell proliferation, hematopoiesis, wound healing and angiogenesis.
Additional utilities for the NOVX nucleic acids and polypeptides according to the invention are disclosed herein.
NOV1
A disclosed NOV1 nucleic acid of 872 nucleotides (also referred to as CG57413-01) encoding a novel Androgen-Regulated Short-Chain Dehydrogenase/Reductase-like protein is shown in Table 1A. An open reading frame was identified beginning with an ATG initiation codon at nucleotides 11-13 and ending with a TAA codon at nucleotides 845-847. Putative untranslated regions, if any, upstream from the initiation codon and downstream from the termination codon are underlined in Table 1A, and the start and stop codons are in bold letters.







TABLE 1A





NOV1 Nucleotide Sequence.















(SEQ ID NO:1)







CTCGTGAAGGATGGTACGTGATGCTCTTGTTTCCCTTGCCGATAGGAAGTTCTTTGCTGGTGGAGTGTGTAG


AACAAATGTGCAGCTTCCTGGCAAGGTAGTGGTGATCACTGGCGCCAACACGGGCATTGGCAAGGAGACGGC


CAGAGAGCTCGCTAGCCGAGGAGCCCGAGTCTATATTGCCTGCAGAGATGTACTGAAGGGGGAGTCTGCTGC


CAGTGAAATCCGAGTGGATACAAAGAACTCCCAGGTGCTGGTGCGGAAATTGGACCTATCCGACACCAAATC


TATCCGAGCCTTTGCTGAGGGCTTTCTGGCAGAGGAAAAGCAGCTCCATATTCTGATCAACAATGCGGGAGT


AATGATGTGTCCATATTCCAAGACAGCTGATGGCTTTGAAACCCACCTGGGAGTCAACCACCTGGGCCACTT


CCTCCTCACCTACCTGCTCCTGGAGCGGCTAAAGGTGTCTGCCCCTGCACGGGTGGTTAATGTGTCCTCGGT


GGCTCACCACATTGGCAAGATTCCCTTCCACGACCTCCAGAGCGAGAAGCGCTACAGCAGGGGTTTTGCCTA


TTGCCACAGCAAGCTGGCCAATGTGCTTTTTACTCGTGAGCTGGCCAAGAGGCTCCAAGGCACCGGGGTCAC


CACCTACGCAGTGCACCCAGGCGTCGTCCGCTCTGAGCTGGTCCGGCACTCCTCCCTGCTCTGCCTGCTCTG


GCGGCTCTTCTCCCCCTTTGTCAAGACGGCACGGGAGGGGGCGCAGACCAGCCTGCACTGCGCCCTGGCTGA


GGGCCTGGAGCCCCTGAGTGGCAAGTACTTCGGTGTCTCCAAGGGCCCGAAATAACAAAACAGCTGAGCGCC


TATGGAAT









The NOV1 nucleic acid sequence maps to chromosome 14 and has 584 of 797 bases (73%) identical to a gb:GENBANK-ID:AX041971|acc:AX041971.1 mRNA from Homo sapiens (Sequence 1 from Patent WO0065067) (E=2.1 e−84). Similiarity information was assessed using public nucleotide databases including all GenBank databases and the GeneSeq patent database. Chromosome information was assigned using OMIM and the electronic northern tool from Curatools to derive the the chromosomal mapping of the SeqCalling assemblies, Genomic clones, and/or EST sequences that were included in the invention.
In all BLAST alignments herein, the “E-value” or “Expect” value is a numeric indication of the probability that the aligned sequences could have achieved their similarity to the BLAST query sequence by chance alone, within the database that was searched. For example, the probability that the subject (“Sbjct”) retrieved from the NOV1 BLAST analysis, e.g., mRNA from Homo sapiens (Sequence 1 from Patent WO0065067), matched the Query NOV1 sequence purely by chance is 2.1e−84. The Expect value (E) is a parameter that describes the number of hits one can “expect” to see just by chance when searching a database of a particular size. It decreases exponentially with the Score (S) that is assigned to a match between two sequences. Essentially, the E value describes the random background noise that exists for matches between sequences.
The Expect value is used as a convenient way to create a significance threshold for reporting results. The default value used for blasting is typically set to 0.0001. In BLAST 2.0, the Expect value is also used instead of the P value (probability) to report the significance of matches. For example, an E value of one assigned to a hit can be interpreted as meaning that in a database of the current size one might expect to see one match with a similar score simply by chance. An E value of zero means that one would not expect to see any matches with a similar score simply by chance. See, e.g., http://www.ncbi.nlm.nih.gov/Education/BLASTinfo/. Occasionally, a string of X's or N's will result from a BLAST search. This is a result of automatic filtering of the query for low-complexity sequence that is performed to prevent artifactual hits. The filter substitutes any low-complexity sequence that it finds with the letter “N” in nucleotide sequence (e.g., “NNNNNNNN”) or the letter “X” in protein sequences (e.g., “XXX”). Low-complexity regions can result in high scores that reflect compositional bias rather than significant position-by-position alignment. Wootton and Federhen, Methods Enzymol 266:554-571, 1996.
The disclosed NOV1 polypeptide (SEQ ID NO:2) encoded by SEQ ID NO:1 has 278 amino acid residues and is presented in Table 1B using the one-letter amino acid code. Signal P, Psort and/or Hydropathy results predict that NOV1 does not contain a signal peptide and is likely to be localized to the mitochondrial matrix space with a certainty of 0.4843 and to the microbody (peroxisome) with a certainty of 0.3713.







TABLE 1B





Encoded NOV1 protein sequence.















(SEQ ID NO:2)







MVRDALVSLADRKFFAGGVCRTNVQLPGKVVVITGANTGIGKETARELASRGARVYIACRDVLKGESAASEI


RVDTKNSQVLVRKLDLSDTKSIRAFAEGFLAEEKQLHILINNAGVMMCPYSKTADGFETHLGVNHLGHFLLT


YLLLERLKVSAPARVVNVSSVAHHIGKIPFHDLQSEKRYSRGFAYCHSKLANVLFTRELAKRLQGTGVTTYA


VHPGVVRSELVRHSSLLCLLWRLFSPFVKTAREGAQTSLHCALAEGLEPLSGKYFGVSKGPK









The NOV1 amino acid sequence has 197 of 260 amino acid residues (75%) identical to, and 228 of 260 amino acid residues (87%) similar to, the 318 amino acid residue ptnr:SPTREMBL-ACC:Q9NRW0 protein from Homo sapiens (Androgen-Regulated Short-Chain Dehydrogenase/Reductase 1) (E=4.6e−105).
NOV1 is expressed in at least the following tissues: kidney, pancreas, retina and vulva. This information was derived by determining the tissue sources of the sequences that were included in the invention including but not limited to SeqCalling sources, public EST sources, and/or RACE sources.
Possible small nucleotide polymorphisms (SNPs) found for NOV1 are listed in Table 1C. Depth, when shown, represents the number of clones covering the region of the SNP. The putative allele frequence (PAF), when shown, is the fraction of these clones containing the SNP. A dash, when shown, means that a base is not present. The sign “>” means “is changed to.” Silent, when indicated, means that the SNP did not result in an amino acid change.







TABLE 1C







SNPs
















Amino





Nucleotide
Base
Acid
Base



Variant
Position
Change
Position
Change







13375950
327
A > G
106
Lys > Arg










NOV1 has homology to the amino acid sequences shown in the BLASTP data listed in Table 1D.







TABLE 1D







BLAST results for NOV1












Gene Index/

Length
Identity
Positives



Identifier
Protein/Organism
(aa)
(%)
(%)
Expect





gi|12861668|dbj|BAB32258.1|
data source: SPTR,
316
217/264
227/264
e−117


(AK020927)
source key: Q9NRW0,

(82%)
(85%)



evidence: ISS ̃homolog



to ANDROGEN-



REGULATED SHORT-



CHAIN



DEHYDROGENASE/REDUCTASE



1 ̃putative



[Mus musculus]


gi|16740649|gb|AAH16204.1|
Similar to RIKEN
304
207/264
216/264
e−109


AAH16204
cDNA A930033N07

(78%)
(81%)


(BC016204)
gene [Mus musculus]


gi|9622124|gb|AAF89632.1|
androgen-regulated
318
187/260
217/260
e−105


AF167438_1
short-chain

(71%)
(82%)


(AF167438)
dehydrogenase/reductase



1 [Homo sapiens]


gi|7705791|ref|NP_057110.1|
CGI-82 protein;
318
187/260
217/260
e−105


(NM_016026)
likely ortholog of

(71%)
(82%)



mouse cell line



MC/9.IL4 derived



transcript 1 [Homo



sapiens]


gi|12835589|dbj|BAB23296.1|
cell line MC/9.IL4
316
184/266
218/266
e−103


(AK004413)
derived transcript

(69%)
(81%)



1 ̃data source: MGD,



source key: MGI: 



102581, evidence: 



ISS ̃putative [Mus



musculus]









The homology between these and other sequences is shown graphically in the ClustalW analysis shown in Table 1E. In the ClustalW alignment of the NOV1 protein, as well as all other ClustalW analyses herein, the black outlined amino acid residues indicate regions of conserved sequence (i.e., regions that may be required to preserve structural or functional properties), whereas non-highlighted amino acid residues are less conserved and can potentially be altered to a much broader extent without altering protein structure or function.







TABLE 1E





Clustal Analysis of NOV1















1) NOV1 (SEQ ID NO:2)


2) gi 12861668|dbj|BAB32258.1| (AK020927) data source:SPTR, source key:Q9NRW0,


evidence:ISS ̃homolog to ANDROGEN-REGULATED SHORT-CHAIN DEHYDROGENASE/REDUCTASE


1 ̃putative [Mus musculus] (SEQ ID NO:95)


3) gi′16740649|gb|AAH16204.1|AAH16204 (BC016204) Similar to RIKEN cDNA A930033N07


gene [Mus musculus] (SEQ ID NO:96)


4) gi|9622124|gb|AAF89632.1|AF167438_1 (AF167438) androgen-regulated short-chain


dehydrogenase/reductase 1 [Homo sapiens]] (SEQ ID NO:97)


5) gi.7705791|refNP_057110.1| (NM_016026) CGI-82 protein; likely ortholog of mouse


cell line MC/9.1L4 derived transcript 1 [Homo sapiens] (SEQ ID NO:98)


6) gi.12835589|dbj|BAB23296.1| (AK004413) cell line MC/9.IL4 derived transcript


1 ̃data source:MGD, source key: MGI: 102581,evidence: ISS ̃putative [Mus musculus] (SEQ ID NO:99)












































The presence of identifiable domains in NOV1, as well as all other NOVX proteins, was determined by searches using software algorithms such as PROSITE, DOMAIN, Blocks, Pfam, ProDomain, and Prints, and then determining the Interpro number by crossing the domain match (or numbers) using the Interpro website (http:www.ebi.ac.uk/interpro). DOMAIN results for NOV1, as disclosed in Table 1F, were collected from the Conserved Domain Database (CDD) with Reverse Position Specific BLAST analyses. This BLAST analysis software samples domains found in the Smart and Pfam collections. For Table 1F and all successive DOMAIN sequence alignments, fully conserved single residues are indicated by black shading or by the sign (|) and “strong” semi-conserved residues are indicated by grey shading or by the sign (+). The “strong” group of conserved amino acid residues may be any one of the following groups of amino acids: STA, NEQK, NHQK, NDEQ, QHRK, MILV, MILF, HY, FYW.
Table 1F lists the domain description from DOMAIN analysis results against NOV1. This indicates that the NOV1 sequence has properties similar to those of other proteins known to contain these domains.







TABLE 1F





Domain Analysis of NOV1















gnl|Pfam|pfam00106, adh_short, short chain dehydrogenase. This family


contains a wide variety of dehydrogenases. (SEQ ID NO:100)


CD-Length = 249 residues, 99.6% aligned


Score = 124 bits (310), Expect = 9e−30










Query:
27
PGKVVVITGANTGIGKETARELASRGARVYIACRDVLKGESAASEIRVDTKNSQVLVRKL
 86




 ||| ++|||++|||   |+ ||  ||+| +  |   | |+||          + |  +|


Sbjct:
1
TGKVALVTGASSGIGLAIAKRLAEEGAKVVVVDRREEKAEAAAELKA--ELGDRALFIQL
 58


Query:
87
DLSDTKSIRAFAEGFLAEEKQLHILINNAGVM--MCPYSKTADGFETHLGVNHLGHFLLT
144




|++| +||+|     + |  +| +|+||||++    |+  + | +|  + ||  | ||||


Sbjct:
59
DVTDEESIKAAVAQAVEELGRLDVLVNNAGILGPGEPFELSEDDWERVIDVNLTGVFLLT
118


Query:
145
YLLLERLKVSAPARVVNVSSVAHHIGKIPFHDLQSEKRYSRGFAYCHSKLANVLFTRELA
204




  +|  +   +  |+||+||||  +                       | | | ||| ||


Sbjct:
119
QAVLPHMLKRSGGRIVNISSVAGLVP------------SPGLSAYSASKAAVVGFTRSLA
166


Query:
205
KRLQGTGVTTYAVHPGVVRSELV-RHSSLLCLLWRLFSPFVK---------TAREGAQTS
254




  |   |+   |+ || | +++     |+      |               |  | |


Sbjct:
167
LELAPHGIRVANAIAPGGVDTDMTKALRSIAPADPELVERITSALVPLGRYGTPEEVANAV
226


Query:
255
LHCALAEGLEPLSGKYFGVSKG
276




|  |       ++|+   |  |


Sbjct:
227
LFLASDGASYSVTGQTLNVDGG
248









Most mammalian hydroxysteroid dehydrogenases known thus far belong to the protein superfamilies of short-chain dehydrogenases/reductases (SDR) and aldo-keto reductases (AKR). Whereas members of the AKR family are soluble, cytoplasmic enzymes, SDR-type hydroxysteroid dehydrogenases are also located to other subcellular compartments, i.e. endoplasmic reticulum, mitochondria or peroxisomes. Differential localization might play an important role in influencing the reaction direction of hydroxy dehydrogenase/oxo reductase pathways by determining the available nucleotide cofactor pool. Targeting signals for different subcellular organelles in human hydroxysteroid dehydrogenases have been identified, however, in several enzymes localization signals remain to be determined. The short-chain dehydrogenases/reductases family (SDR) is a very large family of enzymes, most of which are known to be NAD- or NADP-dependent oxidoreductases. As the first member of this family to be characterized was Drosophila alcohol dehydrogenase, this family used to be called ‘insect-type’, or ‘short-chain’ alcohol dehydrogenases. Most members of this family are proteins of about 250 to 300 amino acid residues. Most dehydrogenases possess at least 2 domains, the first binding the coenzyme, often NAD, and the second binding the substrate. This latter domain determines the substrate specificity and contains amino acids involved in catalysis. Little sequence similarity has been found in the coenzyme binding domain although there is a large degree of structural similarity, and it has therefore been suggested that the structure of dehydrogenases has arisen through gene fusion of a common ancestral coenzyme nucleotide sequence with various substrate specific domains.
The NOV1 nucleic acid of the invention encoding a Androgen-Regulated Short-Chain Dehydrogenase/Reductase-like protein includes the nucleic acid whose sequence is provided in Table 1A, or a fragment thereof. The invention also includes a mutant or variant nucleic acid any of whose bases may be changed from the corresponding base shown in Table 1A while still encoding a protein that maintains its Androgen-Regulated Short-Chain Dehydrogenase/Reductase-like activities and physiological functions, or a fragment of such a nucleic acid. The invention further includes nucleic acids whose sequences are complementary to those just described, including nucleic acid fragments that are complementary to any of the nucleic acids just described. The invention additionally includes nucleic acids or nucleic acid fragments, or complements thereto, whose structures include chemical modifications. Such modifications include, by way of non-limiting example, modified bases, and nucleic acids whose sugar phosphate backbones are modified or derivatized. These modifications are carried out at least in part to enhance the chemical stability of the modified nucleic acid, such that they may be used, for example, as antisense binding nucleic acids in therapeutic applications in a subject. In the mutant or variant nucleic acids, and their complements, up to about 27% of the residues may be so changed.
The NOV1 protein of the invention includes the Androgen-Regulated Short-Chain Dehydrogenase/Reductase-like protein whose sequence is provided in Table 1B. The invention also includes a mutant or variant protein any of whose residues may be changed from the corresponding residue shown in Table 1B while still encoding a protein that maintains its Androgen-Regulated Short-Chain Dehydrogenase/Reductase-like activities and physiological functions, or a functional fragment thereof. In the mutant or variant protein, up to about 25% of the bases may be so changed.
The NOV1 nucleic acids and proteins of the invention are useful in potential diagnostic and therapeutic applications implicated in various diseases and disorders described below and/or other pathologies. For example, the compositions of the present invention will have efficacy for treatment of patients suffering from: diabetes, autoimmune disease, renal artery stenosis, interstitial nephritis, glomerulonephritis, polycystic kidney disease, systemic lupus erythematosus, renal tubular acidosis, IgA nephropathy, hypercalceimia, Lesch-Nyhan syndrome, Von Hippel-Lindau (VHL) syndrome, pancreatitis, obesity, tuberous sclerosis, fertility and other diseases, disorders and conditions of the like.
NOV1 nucleic acids and polypeptides are further useful in the generation of antibodies that bind immunospecifically to the novel substances of the invention for use in therapeutic or diagnostic methods. These antibodies may be generated according to methods known in the art, using prediction from hydrophobicity charts, as described in the “Anti-NOVX Antibodies” section below. For example the disclosed NOV1 protein have multiple hydrophilic regions, each of which can be used as an immunogen. This novel protein also has value in development of powerful assay system for functional analysis of various human disorders, which will help in understanding of pathology of the disease and development of new drug targets for various disorders.
NOV2
NOV2 includes two novel Aryl-acylamidase-like proteins disclosed below. The disclosed proteins have been named NOV2a and NOV2b.
NOV2a
A disclosed NOV2a nucleic acid of 1527 nucleotides (also referred to as CG57391-01) encoding a novel Aryl-acylamidase-like protein is shown in Table 2A. An open reading frame was identified beginning with an ATG initiation codon at nucleotides 37-39 and ending with a TAA codon at nucleotides 1240-1242. Putative untranslated regions, if any, upstream from the initiation codon and downstream from the termination codon are underlined in Table 2A, and the start and stop codons are in bold letters.







TABLE 2A





NOV2a nucleotide sequence.















(SEQ ID NO:3)







TCTCAGTACTGTGAAGAAGCTGGAAAAAGGGATATTATGGGGCTAAAAGCTCTCTGTTTGGGGCTGCTTTGT


GTTCTTTTTGTCTCTCATTTTTACACACCCATGCCAGACAACATTGAAGAAAGCTGGAAAATAATGGCCTTG


GATGCCATCGCTAAAACTTTGTTCTTACAGGCTATGTGTTTTGAAAATATGCGTATTATGAGATATGAAGAG


TTTATATCCATGATATTCAGGCTGGATTATACCCAACCACTTTCAGATGAATACATCACAGTGACTGATACA


ACATTTGTTGACATTCCAGTACGATTGTACTTGCCAAAAAGAAAGTCAGAAACCCGAAGGCGAGCTGTGATA


TATTTTCATGGTGGTGGTTTTTGTTTTGGAAGTTCCAAACAGAGGGCTTTTGACTTCCTGAATAGATGGACG


GCAAACACGCTTGATGCTGTTGTTGTAGGCGTGGACTATAGGCTGGCTCCTCAACACCACTTTCCTGCTCAG


TTTGAAGATGGCCTTGCTGCAGTCAAATTTTTTCTTTTGGAAAAAATTCTTACAAAATATGGAGTGGATCCC


ACCCGAATCTGCATTGCGGGAGACAGTTCTGGGGGCAATTTAGCAACAGCGGTCACTCAACAGGTGCAGAAT


GATGCTGAAATAAAACATAAAATCAAGATGCAAGTCTTACTTTACCCTGGCTTACAGATAACAGATTCTTAT


TTGCCATCTCACCGAGAAAATGAGCATGGTATAGTTTTGACCAGGGATGTAGCCATAAAACTCGTGAGCTTA


TATTTCACCAAGGATGAAGCACTTCCCTGGGCAATGAGAAGAAACCAACACATGCCTCTGGAGTCAAGACAT


CTGTTTAAGTTTGTTAACTGGAGTATTCTTCTTCCTGAGAAGTATAGAAAAGACTATGTATATACTGAACCA


ATTCTTGGAGGACTTAGTTATTCATTGCCAGGACTTACAGACAGCAGAGCATTACCCTTGTTGGCCAATGAT


TCTCAGTTACAGAATTTGCCACTAACCTATATTCTTACTTGTCAACATGATCTCTTAAGAGATGATGGACTT


ATGTATGTTACAAGACTTCGAAATGTTGGAGTCCAAGTTGTTCATGAACATATTGAGGATGGAATTCATGGA


GCTTTATCATTCATGACTTCACCATTTTATTTACGTCTAGGTCTTAGGATAAGAGATATGTATGTAAGTTGG


CTGGATAAGAATTTATAAATATGTGATGTGTATGTATAGCCCTTACATAGTGGATTGTAATTTGTGATATTT


TGTGGTTTTGGAGCAAAGAACAATGTCATTTGAGTTATCTAAATCTACATTTGCAACATTTGTAGCAGTTAA


TGTGTGTCCTTGAAGAGTTATTAAATTTTCTGACTTGCAGACCCTGAATATGTAAAATGTATGTAATCCTGC


CTATTTTCTCCTTACTTATAATTTATTATAATTATGTTGGTTCTAATAAGAACCAATGCTTATTAAAGTTGA


GAAATAAGAGTGGTT









The disclosed NOV2a nucleic acid sequence, localized to chromsome 3, has 770 of 1205 bases (63%) identical to a gb:GENBANK-ID:HUMARDE|acc:L32179.1 mRNA from Homo sapiens (Human arylacetamide deacetylase mRNA, complete cds) (E=1.3e−75).
A NOV2a polypeptide (SEQ ID NO:4) encoded by SEQ ID NO:3 has 401 amino acid residues and is presented using the one-letter code in Table 2B. Signal P, Psort and/or Hydropathy results predict that NOV2a contains a signal peptide and is likely to be localized to the cytoplasm with a certainty of 0.8524 or extracellularly with a certainty of 0.7714. The most likely cleavage site for a NOV2a peptide is between amino acids 18 and 19: VSH-FY.







TABLE 2B





Encoded NOV2a protein sequence.















(SEQ ID NO:4)







MGLKALCLGLLCVLFVSHFYTPMPDNIEESWKIMALDAIAKTLFLQAMCFENMRIMRYEEFISMIFRLDYTQ


PLSDEYITVTDTFFVDIPVRLYLPKRKSETRRRAVIYFHGGGFCFGSSKQRAFDFLNRWTANTLDAVVVGVD


YRLAPQHHFPAQFEDGLAAVKFFLLEKILTKYGVDPTRICIAGDSSGGNLATAVTQQVQNDAEIKHKIKMQV


LLYPGLQITDSYLPSHRENEHGIVLTRDVAIKLVSLYFTKDEALPWAMRRNQHMPLESRHLFKFVNWSILLP


EKYRKDYVYTEPILGGLSYSLPGLTDSRALPLLANDSQLQNLPLTYILTCQHDLLRDDGLMYVTRLRNVGVQ


VVHEHIEDGIHGALSFMTSPFYLRLGLRIRDMYVSWLDKNL









The NOV2a amino acid sequence has 205 of 401 amino acid residues (51%) identical to, and 282 of 401 amino acid residues (70%) similar to, the 399 amino acid residue ptnr:pir-id:A53856 protein from human (aryl-acylamidase (EC 3.5.1.13)) (E=2.7e−107).
The disclosed NOV2a is expressed in at least the following tissues: heart, uterus and colon. This information was derived by determining the tissue sources of the sequences that were included in the invention including but not limited to SeqCalling sources, Public EST sources, Literature sources, and/or RACE sources.
NOV2b
A disclosed NOV2b nucleic acid of 1324 nucleotides (also referred to as CG57391-02) encoding a novel Aryl-acylamidase-like protein is shown in Table 2C. An open reading frame was identified beginning with an ATG initiation codon at nucleotides 34-36 and ending with a TAA codon at nucleotides 1237-1239. Putative untranslated regions, if any, upstream from the initiation codon and downstream from the termination codon are underlined in Table 2C, and the start and stop codons are in bold letters.







TABLE 2C





NOV2b nucleotide sequence.















(SEQ ID NO:5)







CAGTACTGTGAAGAAGCTGGAAAAAGGGATATTATGGGGCTAAAAGCTCTCTGTTTGGGGCTGCTTTGTGTT


CTTTTTGTCTCTCATTTTTACACACCCATGCCAGACAACATTGAAGAAAGCTGGAAAATAATGGCCTTGGAT


GCCATCGCTAAAACTTGTACATTTACGGCTATGTGTTTTGAAAATATGCGTATTATGAGATATGAAGAGTTT


ATATCCATGATATTCAGGCTGGATTATACCCAACCACTTTCAGATGAATACATCACAGTGACTGATACAACA


TTTGTTGACATTCCAGTACGATTGTACTTGCCAAAAAGAAAGTCAGAAACCCGAAGGCGAGCTGTGATATAT


TTTCATGGTGGTGGTTTTTGTTTTGGAAGTTCCAAACAGAGGGCTTTTGACTTCCTGAATAGATGGACGGCA


AACACGCTTGATGCTGTTGTTGTAGGCGTGGACTATAGGCTGGCTCCTCAACACCACTTTCCTGCTCAGTTT


GAAGATGGCCTTGCTGCAGTCAAATTTTTTCTTTTGGAAAAAATTCTTACAAAATATGGAGTGGATCCCACC


CGAATCTGCATTTCGGGAGACAGTTCTGGGGGCAATTTAGCAACAGCGGTCACTCAACAGGTGCAGAATGAT


GCTGAAATAAAACATAAAATCAAGATGCAAGTCTTACTTTACCCTGGCTTACAGATAACAGATTCTTATTTG


CCATCTCACCGAGAAAATGAGCATGGTATAGTTTTGACCAGGGATGTAGCCATAAAACTCGTGAGCTTATAT


TTCACCAAGGATGAAGCACTTCCCTGGGCAATGAGAAGAAACCAACACATGCCTCTGGAGTCAAGACATCTG


TTTAAGTTTGTTAACTGGAGTATTCTTCTTCCTGAGAAGCATAGAAAAGACTATGTATATACTGAACCAATT


CTTGGAGGACTTAGTTATTCATTGCCAGGACTTACAGACAGCAGAGCATTACCCTTGTTGGCCAATGATTCT


CAGTTACAGAATTTGCCACTAACCTATATTCTTACTTGTCAACATGATCTCTTAAGAGATGATGGACTTATG


TATGTTACAAGACTTCGAAATGTTGGAGTCCAAGTTGTTCATGAACATATTGAGGATGGAATTCATGGAGCT


TTATCATTCATGACTTCACCATTTTATTTACGTCTAGGTCTTAGGATAAGAGATATGTATGTAAGTTGGCTG


GATAAGAATTTATAAATATGTGATGTGTATGTATAGCCTTTACATAGTGGATTGTAATTTGTGATATTTTGT


GGTTTTGGAGCAAAGAACAATGTCATTT









The disclosed NOV2b nucleic acid sequence, localized to chromsome 3, 805 of 1261 bases (63%) identical to a gb:GENBANK-ID:HUMARDE|acc:L32179.1 mRNA from Homo sapiens (Human arylacetamide deacetylase mRNA, complete cds) (E=1.4e−76).
A NOV2b polypeptide (SEQ ID NO:6) encoded by SEQ ID NO:5 has 401 amino acid residues and is presented using the one-letter code in Table 2D. Signal P, Psort and/or Hydropathy results predict that NOV2b contains a signal peptide and is likely to be localized to the cytoplasm with a certainty of 0.8524 or extracellularly with a certainty of 0.7714. The most likely cleavage site for a NOV2b peptide is between amino acids 18 and 19: VSH-FY.







TABLE 2D





Encoded NOV2b protein sequence.















(SEQ ID NO:6)







MGLKALCLGLLCVLFVSHFYTPMPDNIEESWKIMALDAIAKTCTFTAMCFENMRIMRYEEFISMIFRLDYTQ


PLSDEYITVTDTTFBDIPVRLYLPKRKSETRRRAVIYFHGGGFCFGSSKQRAFDFLNRWTANTLDAVVVGVD


YRLAPQHHFPAQFEDGLAAVKFFLLEKILTKYGVDPTRICISGDSSGGNLATAVTQQVQNDAEIKHKIKMQV


LLYPGLQITDSYLPSHRENEHGIVLTRDVAIKLVSLYFTKDEALPWAMRRNQHMPLESRHLFKFVNWSILLP


EKHRKDYVYTEPILGGLSYSLPGLTDSRALPLLANDSQLQNLPLTYILTCQHDLLRDDGLMYVTRLRNVGVQ


VVHEHIEDGIHGALSFMTSPFYLRLGLRIRDMYVSWLDKNL









The NOV2b amino acid sequence has 206 of 401 amino acid residues (51%) identical to, and 280 of 401 amino acid residues (69%) similar to, the 399 amino acid residue ptnr:pir-id:A53856 protein from human (aryl-acylamidase (EC 3.5.1.13)) (E=3.9e−107).
The disclosed NOV2b is expressed in at least the following tissues: heart, uterus and colon. This information was derived by determining the tissue sources of the sequences that were included in the invention including but not limited to SeqCalling sources, Public EST sources, Literature sources, and/or RACE sources.
Possible small nucleotide polymorphisms (SNPs) found for NOV2a are listed in Table 2E.







TABLE 2E







SNPs
















Amino





Nucleotide
Base
Acid
Base



Variant
Position
Change
Position
Change

















13377048
179
T > A
48
Met > Lys



13377047
839
A > T
268
Gln > Leu



13377046
853
G > A
273
Glu > Lys



13377045
907
T > C
291
Tyr > His



13377044
1044
T > C
Silent
N/A



13377043
1140
T > C
Silent
N/A



13377042
1210
A > G
392
Met > Val



13377041
1266
C > T
Silent
N/A










NOV2a and NOV2b are very closely homologous as is shown in the amino acid alignment in Table 2F.







TABLE 2F





Amino Acid Alignment of NOV2a and NOV2b
































































Homologies to any of the above NOV2 proteins will be shared by the other NOV2 proteins insofar as they are homologous to each other as shown above. Any reference to NOV2 is assumed to refer to both of the NOV2 proteins in general, unless otherwise noted.
NOV2a has homology to the amino acid sequences shown in the BLASTP data listed in Table 2G.







TABLE 2G







BLAST results for NOV2a












Gene Index/
Protein/
Length
Identity
Positives



Identifier
Organism
(aa)
(%)
(%)
Expect





gi|11433103|ref|XP_
arylacetamide
399
201/386
272/386
e−113


003002.1|
deacetylase

(52%)
(70%)


(XM_003002)
[Homo



sapiens]


gi|4557227|ref|NP_001077.1|
arylacetamide
399
200/386
270/386
e−112


(NM_001086)
deacetylase

(51%)
(69%)



[Homo



sapiens]


gi|75135571|pir||A58922
esterase/N-
398
199/386
264/386
e−111



deacetylase

(51%)
(67%)



(EC 3.5.1.-),



50K hepatic



[Oryctolagus



cuniculus]


gi|18043412|gb|AAH19999.1|
arylacetamide
398
200/386
262/386
e−105


AAH19999
deacetylase

(51%)
(67%)


(BC019999)
(esterase)



[Mus musculus]


gi|13184050|ref|NP_
arylacetamide
398
199/386
261/386
e−105


075872.1|
deacetylase

(51%)
(67%)


(NM_023383)
(esterase)



[Mus musculus]









The homology of these sequences is shown graphically in the ClustalW analysis shown in Table 2H.







TABLE 2H





ClustalW Analysis of NOV2a















1) NOV2a (SEQ ID NO:4)


2) gi 11433103|ref|XP_003002.1| (XM_003002) arylacetamide deacetylase [Homo sapiens]


(SEQ ID NO:101)


2) gi|4557227|refNP_001077.1| (NM_001086) arylacetamide deacetylase [Homo sapiens]


(SEQ ID NO:102)


3) gi|7513557|pir..A58922 esterase/N-deacetylase (EC 3.5.1.-), 50K hepatic


[Orycotagus cuniculus] (SEQ ID NO:103)


4) gi 18043412|gb AAH19999.1 AAH19999 (BC019999) arylacetamide deacetylase (esterase)


[Mus musculus] (SEQ ID NO:104)


5) gi 13184050|ref|NP_075872.1| (NM_023383) arylacetamide deacctylase (esterase)


[Mus musculus] (SEQ ID NO:105)



















































The NOV2 nucleic acid of the invention encoding a Aryl-acylamidase-like protein includes the nucleic acid whose sequence is provided in Tables 2A and 2C, or a fragment thereof. The invention also includes a mutant or variant nucleic acid any of whose bases may be changed from the corresponding base shown in Tables 2A and 2C while still encoding a protein that maintains its Aryl-acylamidase-like activities and physiological functions, or a fragment of such a nucleic acid. The invention further includes nucleic acids whose sequences are complementary to those just described, including nucleic acid fragments that are complementary to any of the nucleic acids just described. The invention additionally includes nucleic acids or nucleic acid fragments, or complements thereto, whose structures include chemical modifications. Such modifications include, by way of non-limiting example, modified bases, and nucleic acids whose sugar phosphate backbones are modified or derivatized. These modifications are carried out at least in part to enhance the chemical stability of the modified nucleic acid, such that they may be used, for example, as antisense binding nucleic acids in therapeutic applications in a subject. In the mutant or variant nucleic acids, and their complements, up to about 37% of the residues may be so changed.
The NOV2 protein of the invention includes the Aryl-acylamidase-like protein whose sequence is provided in Tables 2B and 2D. The invention also includes a mutant or variant protein any of whose residues may be changed from the corresponding residue shown in Tables 2B and 2D while still encoding a protein that maintains its Aryl-acylamidase-like activities and physiological functions, or a functional fragment thereof. In the mutant or variant protein, up to about 49% of the bases may be so changed.
The NOV2 nucleic acids and proteins of the invention are useful in potential diagnostic and therapeutic applications implicated in various diseases and disorders described below and/or other pathologies. For example, the compositions of the present invention will have efficacy for treatment of patients suffering from: Cardiomyopathy, Atherosclerosis, Hypertension, Congenital heart defects, Aortic stenosis, Atrial septal defect (ASD), Atrioventricular (A-V) canal defect, Ductus arteriosus, Pulmonary stenosis, Subaortic stenosis, Ventricular septal defect (VSD), valve diseases, Tuberous sclerosis, Scleroderma, Obesity, Transplantation, Endometriosis, Fertility and other diseases, disorders and conditions of the like.
NOV2 nucleic acids and polypeptides are further useful in the generation of antibodies that bind immunospecifically to the novel substances of the invention for use in therapeutic or diagnostic methods. These antibodies may be generated according to methods known in the art, using prediction from hydrophobicity charts, as described in the “Anti-NOVX Antibodies” section below. For example the disclosed NOV2 protein have multiple hydrophilic regions, each of which can be used as an immunogen. This novel protein also has value in development of powerful assay system for functional analysis of various human disorders, which will help in understanding of pathology of the disease and development of new drug targets for various disorders.
NOV3
NOV3 includes two novel Insulysin-like proteins disclosed below. The disclosed proteins have been named NOV3a and NOV3b.
NOV3a
A disclosed NOV3a nucleic acid of 4321 nucleotides (also referred to as CG57433-01) encoding a novel Insulysin-like protein is shown in Table 3A. An open reading frame was identified beginning with an ATG initiation codon at nucleotides 58-60 and ending with a TGA codon at nucleotides 3115-3117. Putative untranslated regions, if any, upstream from the initiation codon and downstream from the termination codon are underlined in Table 3A, and the start and stop codons are in bold letters.







TABLE 3A





NOV3a nucleotide sequence.















(SEQ ID NO:7)







CCGGCTCGAAGCGCAACGAGGAAGCGTTTGCGGTGATCCCGGCGACTGCGCTGGCTAATGCGGTACCGGCTA


GCGTGGCTTCTGCACCCCGCACTGCCCAGCACCTTCCGCTCAGTCCTCGGCGCCCGCCTGCCGCCTCCGGAG


CGCCTGTGTGGTTTCCAAAAAAAGACTTACAGCAAAATGAATAATCCAGCCATCAAGAGAATAGGAAATCAC


ATTACCAAGTCTCCTGAAGACAAGCGAGAATATCGAGGGCTAGAGCTGGCCAATGGTATCAAAGTACTTCTT


ATCAGTGATCCCACCACGGATAAGTCATCAGCAGCACTTGATGTGCACATAGGTTCATTGTCGGATCCTCCA


AATATTGCTGGCTTAAGTCATTTTTGTGAACATATGCTTTTTTTGGGAACAAAGAAATACCCTAAAGAAAAT


GAATACAGCCAGTTTCTCAGTGAGCATGCAGGAAGTTCAAATGCCTTTACTAGTGGAGAGCATACCAATTAC


TATTTTGATGTTTCTCATGAACACCTAGAAGGTGCCCTAGACAGGTTTGCACAGTTTTTTCTGTGCCCCTTG


TTCGATGAAAGTTGCAAAGACAGAGAGGTGAATGCAGTTGATTCAGAACATGAGAAGAATGTGATGAATGAT


GCCTGGAGACTCTTTCAATTGGAAAAAGCTACAGGGAATCCTAAACACCCCTTCAGTAAATTTGGGACAGGT


AACAAATATACTCTGGAGACTAGACCAAACCAAGAAGGCATTGATGTAAGACAAGAGCTACTGAAATTCCAT


TCTGCTTACTATTCATCCAACTTAATGGCTGTTTGTGTTTTAGGTCGAGAATCTTTAGATGACTTGACTAAT


CTGGTGGTAAAGTTATTTTCTGAAGTAGAGAACAAAAATGTTCCATTGCCAGAATTTCCTGAACACCCTTTC


CAAGAAGAACATCTTAAACAACTTTACAAAATAGTACCCATTAAAGATATTAGGAATCTCTATGTGACATTT


CCCATACCTGACCTTCAGAAATACTACAAATCAAATCCTGGTCATTATCTTGGTCATCTCATTGGGCATGAA


GGTCCTGGAAGTCTGTTATCAGAACTTAAGTCAAAGGGCTGGGTTAATACTCTTGTTGGTGGGCAGAAGGAA


GGAGCCCGAGGTTTTATGTTTTTTATCATTAATGTGGACTTGACCGAGGAAGGATTATTACATGTTGAAGAT


ATAATTTTGCACATGTTTCAATACATTCAGAAGTTACGTGCAGAAGGACCTCAAGAATGGGTTTTCCAAGAG


TGCAAGGACTTGAATGCTGTTGCTTTTAGGTTTAAAGACAAAGAGAGGCCACGGGGCTATACATCTAAGATT


GCAGGAATATTGCATTATTATCCCCTAGAAGAGGTGCTCACAGCGGAATATTTACTGGAAGAATTTAGACCT


GACTTAATAGAGATGGTTCTCGATAAACTCAGACCAGAAAATGTCCGGGTTGCCATAGTTTCTAAATCTTTT


GAAGGAAAAACTGATCGCACAGAAGAGTGGTATGGAACCCAGTACAAACAAGAAGCTATACCGGATGAAGTC


ATCAAGAAATGGCAAAATGCTGACCTGAATGGGAAATTTAAACTTCCTACAAAGAATGAATTTATTCCTACG


AATTTTGAGATTTTACCGTTAGAAAAAGAGGCGACACCATACCCTGCTCTTATTAAGGATACAGCTATGAGC


AAACTTTGGTTCAAACAAGATGATAAGTTTTTTTTGCCGAAGGCTTGTCTCAACTTTGAATTTTTCAGTCGC


TACATTTATGCTGATCCTCTCCATTGCAACATGACATACCTGTTTATCAGGTTATTGAAGGATGATTTAAAA


GAGTATACATATGCAGCACGCCTCTCAGGTTTGAGCTATGGCATTGCATCAGGAATGAATGCAATACTTCTT


TCAGTGAAAGGTTACAATGACAAGCACCCAATTTTACTAAAGAAGATTATTGAGAAAATGGCTACCTTTGAG


ATTGATGAAAAAAGATTTGAAATTATCAAAGAAGCATATATGCGATCTCTTAACAATTTCCGGGCTGAACAG


CCTCACCAGCATGCCATGTACTACCTCCGCTTGCTGATGACTGAAGTGGCCTGGACTAAAGATGAGTTAAAA


GAAGCTCTGGATGATGTAACCCTTCCTCGCCTTAAGGCCTTCATACCTCAGCTCCTGTCACGGCTGCACATT


GAAGCCCTTCTCCATGGAAACATAACAAAGCAGGCTGCATTAGGAATTATGCAGATGGTTGAAGACACCCTC


ATTGAACATGCTCATACCAAACCTCTCCTTCCAAGTCAGCTGGTTCGGTATAGAGAAGTTCAGCTCCCTGAC


AGAGGATCGTTTGTTTATCAGCAGAGAGATGAAGTTCACAATAACTGTGGCATCGAGATATACTACCAAACA


GACATGCAAAGCACCTCAGAGAATATGTTTCTGGAGCTCTTCTGTCAGATTATCTCGGAACCTTGCTTCAAC


ACCCTGCGCACCAAGGAGCAGTTGGGCTATATCGTCTTCAGCGGGCCACGTCGAGCTAATGGCATACAGGGC


TTGAGATTCATCATCCAGTCAGAAAAGCCACCTCACTACCTAGAAAGCAGAGTGGAAGCTTTCTTAATTACC


ATGGAAAAGTCCATAGAGGACATGACAGAAGAGGCCTTCCAAAAACACATTCAGGCATTAGCAATTCGTCGA


CTAGACAAACCAAAGAAGCTATCTGCTGAGTGTGCTAAATACTGGGGAGAAATCATCTCCCAGCAATATAAT


TTTGACAGAGATAACACTGAGGTTGCATATTTAAAGACACTTACCAAGGAAGATATCATCAAATTCTACAAG


GAAATGTTGGCAGTAGATGCTCCAAGGAGACATAAGGTATCCGTCCATGTTCTTGCCAGGGAAATGGATTCT


TGTCCTGTTGTTGGAGAGTTCCCATGTCAAAATGACATAAATTTGTCACAAGCACCAGCCTTGCCACAACCT


GAAGTGATTCAGAACATGACCGAATTCAAGCGTGGTCTGCCACTGTTTCCCCTTGTGAAACCACATATTAAC


TTCATGGCTGCAAAACTCTGAAGATTCCCCATGCATGGGAAAGTGCAAGTGGATGCATTCCTGAGTCTTCCA


GAGCCTAAGAAAATCATCTTGGCCACTTTAATAGTTTCTGATTCACTATTAGAGAAACAAACAAAAAATTGT


CAAATGTCATTATGTAGAAATATTATAAATCCAAAGTAAATTACAAAATCTTATAGATGTAGAATATTTTTT


AAATACATGCCTCTTAAATATTTTAAAATTTTTCTTTTGATTACTGAGAGAAATTTCCCCAATATAACAATG


CTTAAAATGAATGATATTCCTATAGAATCTTCCTTCCCTATTCTGTAAAATAGTCACTTGTCCGAAGAAAGT


TAAAAGTTAGCTCTTTTCTAAAAGCCTCCTAGCTTGACATAGAAGGCTTCACAACATTTAGAAAGGTAATAA


CTTTTTAAAAATTGATCCTCAAATTTGCTTTCTACTTGATGGTTTCATGTAAATCAGTGGAAAACATTACAT


TTGGCAGATGATAAAGCAATGTCATCTTTTATTAGTGAAATGCTGGTTATATAAGGCATGGTTTTAATCTTT


TTATAAAATTTGAACATGTTTTTTATGCCAACTCGTAAAATGCTAGAAAACCCTACTTATTTACAATGCTAG


AAATACAGACTTACCTTACATCAATTTTGTCCTAAACCGAATTTCTCAGGATTACTGTGGTTTCTTTCATTC


TGATTGAATTATATTGACCTACTTCTTCATAGTTGGTTTGCAGTGTTCCATGAGTTTTACTTTTCCTCATCA


ACATATTGCTTTAACACAACATATTTATTTAACACGTACAAATAGGGTCAACTTCAGATCCTACTGAGTGTG


TGACATGCTTTTCCAACATCAGCTTTTTGTAACCACCTGTATAACTTTTTATTACAGTGAAATTGCAGTCAG


TATGTGAACCAAAATATCTTGCCCCTTTATGAATTTAAAAGGCAGCCAATACAAAGCCACCTTTTTGGAAAT


ATAAAAAGTAAAGCCTTGCATTCTTATATAGCAGGTCTTCATAAAACTCTAAAATCCCTTGTTGCTACCAGT


CTAATCTTGCCTTAAATGTTAAGTTATTTTTTGAATATATAAATATAAACATATAAACACAGATGATGACTG


GAGTAGACTTTTAAAAAAATATTTTTTTCATGAGATACTATTTTAGGTGAAATTGTACTGTAGATTTACAGC


T









The disclosed NOV3a nucleic acid sequence, localized to chromsome 10, has 3265 of 3336 bases (97%) identical to a gb:GENBANK-ID:HUMIDE|acc:M21188.1 mRNA from Homo sapiens (Human insulin-degrading enzyme (IDE) mRNA, complete cds) (E=0.0).
A NOV3a polypeptide (SEQ ID NO:8) encoded by SEQ ID NO:7 has 1019 amino acid residues and is presented using the one-letter code in Table 3B. Signal P, Psort and/or Hydropathy results predict that NOV3a contains a signal peptide and is likely to be localized to the peroxisome (microbody) with a certainty of 0.8347 or the mitochondrial matrix space with a certainty of 0.6517. The most likely cleavage site for a NOV3a peptide is between amino acids 22 and 23: VLG-AR.







TABLE 3B





Encoded NOV3a protein sequence.















(SEQ ID NO:8)







MRYRLAWLLHPALPSTFRSVLGARLPPPERLCGFQKKTYSKMNNPAIKRIGNHITKSPEDKREYRGLELANG


IKVLLISDPTTDKSSAALDVHIGSLSDPPNIAGLSHFCEHMLFLGTKKYPKENEYSQFLSEHAGSSNAFTSG


EHTNYYFDVSHEHLEGALDRFAQFFLCPLFDESCKDREVNAVDSEHEKNVMNDAWRLFQLEKATGNPKHPFS


KFGTGNKYTLETRPNQEGIDVRQELLKFHSAYYSSNLMAVCVLGRESLDDLTNLVVKLFSEVENKNVPLPEF


PEHPFQEEHLKQLYKIVPIKDIRNLYVTFPIPDLQKYYKSNPGHYLGHLIGHEGPGSLLSELKSKGWVNTLV


GGQKEGARGFMFFIINVDLTEEGLLHVEDIILHMFQYIQKLRAEGPQEWVFQECKDLNAVAFRFKDKERPRG


YTSKIAGILHYYPLEEVLTAEYLLEEFRPDLIEMVLDKLRPENVRVAIVSKSFEGKTDRTEEWYGTQYKQEA


IPDEVIKKWQNADLNGKFKLPTKNEFIPTNFEILPLEKEATPYPALIKDTAMSKLWFKQDDKFFLPKACLNF


EFFSRYIYADPLHCNMTYLFIRLLKDDLKEYTYAARLSGLSYGIASGMNAILLSVKGYNDKQPILLKKIIEK


MATFEIDEKRFEIIKEAYMRSLNNFRAEQPHQHAMYYLRLLMTEVAWTKDELKEALDDVTLPRLKAFIPQLL


SRLHIEALLHGNITKQAALGIMQMVEDTLIEHAHTKPLLPSQLVRYREVQLPDRGWFVYQQRDEVHNNCGIE


IYYQTDMQSTSENMFLELFCQIISEPCFNTLRTKEQLGYIVFSGPRRANGIQGLRFIIQSEKPPHYLESRVE


AFLITMEKSIEDMTEEAFQKHIQALAIRRLDKPKKLSAECAKYWGEIISQQYNFDRDNTEVAYLKTLTKEDI


IKFYKEMLAVDAPRRHKVSVHVLAREMDSCPVVGEFPCQNDINLSQAPALPQPEVIQNMTEFKRGLPLFPLV


KPHINFMAAKL









The NOV3a amino acid sequence has 989 of 1019 amino acid residues (97%) identical to, and 999 of 1019 amino acid residues (98%) similar to, the 1019 amino acid residue ptnr:pir-id:SNHUIN protein from human (insulysin (EC 3.4.24.56)) (E=0.0).
The disclosed NOV3a is expressed in at least the following tissues: Cervix, Liver, Spleen, Testis, Melanocyte, Heart and Uterus. This information was derived by determining the tissue sources of the sequences that were included in the invention including but not limited to SeqCalling sources, Public EST sources, Literature sources, and/or RACE sources.
NOV3b
A disclosed NOV3b nucleic acid of 3153 nucleotides (also referred to as CG57433-02) encoding a novel Insulysin-like protein is shown in Table 3C. An open reading frame was identified beginning with an ATG initiation codon at nucleotides 37-39 and ending with a TGA codon at nucleotides 3094-3096. Putative untranslated regions, if any, upstream from the initiation codon and downstream from the termination codon are underlined in Table 3C, and the start and stop codons are in bold letters.







TABLE 3C





NOV3b nucleotide sequence.















(SEQ ID NO:9)







AAGCGTTTGCGGTGATCCCGGCGACTGCGCTGGCTAATGCGGTACCGGCTAGCGTGGCTTCTGCACCCCGCA


CTGCCCAGCACCTTCCGCTCAGTCCTCGGCGCCCGCCTGCCGCCTCCGGAGCGCCTGTGTGGTTTCCAAAAA


AAGACTTACAGCAAAATGAATAATCCAGCCATCAAGAGAATAGGAAATCACATTACCAAGCCTCCTGAAGAC


AAGCGAGAATATCGAGGGCTAGAGCTGGCCAATGGTATCAAAGTACTTCTTATCAGTGATCCCACCACGGAT


AAGTCATCAGCAGCACTTGATGTGCACATAGGTTCATTGTCGGATCCTCCAAATATTGCTGGCTTAAGTCAT


TTTTGTGAACATATGCTTTTTTTGGGAACAAAGAAATACCCTAAAGAAAATGAATACAGCCAGTTTCTCAGT


GAGCATGCAGGAAGTTCAAATGCCTTTACTAGTGGAGAGCATACCAATTACTATTTTGATGTTTCTCATGAA


CACCTACAAGGTGCCCTACACAGGTTTGCACAGTTTTTTCTGTGCCCCTTGTTCGATGAAAGTTGCAAAGAC


AGAGAGGTGAATGCAGTTGATTCAGAACATGAGAAGAATGTGATGAATGATGCCTCGAGACTCCTTCAATTG


GAAAAAGCTACAGGGAATCCTAAACACCCCTTCAGTAAATTTGGGACAGGTAACAAATATACTCTGGAGACT


AGACCAAACCAAGAAGGCATTGATGTAAGACAAGAGCTACTGAAATTCCATTCTGCTTACTATTCATCCAAC


TTAATGGCTGTTTGTGTTTTAGGTCGAGAATCTTTAGATGACTTGACTAATCTGGTGGTAAAGTTATTTTCT


GAAGTAGAGAACAAAAATGTTCCATTGCCAGAATTTCCTGAACACCCTTTCCAAGAAGAACATCTTAAACAA


CTTTACAAAATAGTACCCATTAAAGATATTAGGAATCTCTATGTGACATTTCCCATACCTGACCTTCAGAAA


TACTACAAATCAAATCCTGGTCATTATCTTGGTCATCTCATTGGGCATGAAGGTCCTGGAAGTCTGTTATCA


GAACTTAAGTCAAAGGGCTGGGTTAATACTCTTGTTGGTGGGCAGAAGGAAGGAGCCCGAGGTTTTATGTTT


TTTATCATTAATGTGGACTTGACCGAGGAAGGATTATTACATGTTGAAGATATAATTTTGCACATGTTTCAA


TACATTCAGAGGTTACGTGCAGAAGGACCTCAAGAATGGGTTTTCCAAGAGTGCAAGGACTTGAATGCTGTT


ACTTTTAGGTTTAAAGACAAAGAGAGGCCACGGGGCTATACATCTAAGATTGCAGGAATATTGCATTATTAT


CCCCTAGAAGAGGTGCTCACAGCGGAATATTTACTGGAAGAATTTAGACCTGACTTAATAGAGATGGTTCTC


GATAAACTCAGACCAGAAAATGTCCGGGTTGCCATAGTTTCTAAATCTTTTGAAGGAAAAACTGATCGCACA


GAAGAGTGGTATGGAACCCAGTACAAACAAGAAGCTATACCGGATGAAGTCATCAAGAAATGGCAAAATGCT


GACCTGAATGGGAAATTTAAACTTCCTACAAAGAATGAATTTATTCCTACGAATTTTGAGATTTTACCGTTA


GAAAAAGAGGCGACACCATACCCTGCTCTTATTAAGGATACAGCTATGAGCAAACTTTGGTTCAAACAAGAT


GATAAGTTTTTTTTGCCGAAGGCTTGTCTCAACTTTGAATTTTTCAGTCGCTACATTTATGCTGATCCTCTC


CATTGCAACATGACATACCTGTTTATCAGGTTATTGAAGGATGATTTAAAAGAGTATACATATGCAGCACGC


CTCTCAGGTTTGAGCTATGGCATTGCATCAGGAATGAATGCAATACTTCTTTCAGTGAAAGGTTACAATGAC


AAGCAGCCAATTTTACTAAAGAAGATTATTGAGAAAATGGCTACCTTTGAGATTGATGAAAAAAGATTTGAA


ATTATCAAAGAAGCATATATGCGATCTCTTAACAATTTCCGGGCTGAACAGCCTCACCAGCATGCCATGTAC


TACCTCCGCTTGCTGATGACTGAAGTGGCCTGGACTAAAGATGAGTTAAAAGAGGCTCTGGATGATGTAACC


CTTCCTCGCCTTAAGGCCTTCATACCTCAGCTCCTGTCACGGCTGCACATTGAAGCCCTTCTCCATGGAAAC


ATAACAAAGCAGGCTGCATTAGGAATTATGCAGATGGTTGAAGACACCCTCATTGAACATGCTCATACCAAA


CCTCTCCTTCCAAGTCAGCTGGTTCGGTATAGAGAAGTTCAGCTCCCTGACAGAGGATGGTTTGTTTATCAG


CAGAGAAATGAAGTTCACAATAACTGTGGCATCGAGATATACTACCAAACAGACATGCAAAGCACCTCAGAG


AATATGTTTCTGGAGCTCTTCTGTCAGATTATCTCGGAACCTTGCTTCAACACCCTGCGCACCAAGGAGCAG


TTGGGCTATATCGTCTTCAGCGGGCCACGTCGAGCTAATGGCATACAGGGCTTGAGATTCATCATCCAGTCA


GAAAAGCCACCTCACTACCTAGAAAGCAGAGTGGAAGCTTTCTTAATTACCATGGAAAAGTCCATAGAGGAC


ATGACAGAAGAGGCCTTCCAAAAACACATTCAGGCATTAGCAATTCGTCGACTAGACAAACCAAAGAAGCTA


TCTGCTGAGTGTGCTAAATACTGGGGAGAAATCATCTCCCAGCAATATAATTTTGACAGAGATAACACTGAG


GTAGCATATTTAAAGACACTTACCAAGGAAGATATCATCAAATTCTACAAGGAAATGTTGGCAGTAGATGCT


CCAAGGAGACATAAGGTATCCGTCCATGTTCTTGCCAGGGAAATGGATTCTTGTCCTGTTGTTGGAGAGTTC


CCATGTCAAAATGACATAAATTTGTCACAAGCACCAGCCTTGCCACAACCTGAAGTGATTCAGAACATGACC


GAATTCAAGCGTGGTCTGCCACTGTTTCCCCTTGTGAAACCACATATTAACTTCATCGCTGCAAAACTCTGA


AGATTCCCCATGCATGGGAAAGTGCAAGTGGATGCATTCCTGAGTCTTCCAGAGCCT









The disclosed NOV3b nucleic acid sequence, localized to chromsome 10q23-q25, 3078 of 3153 bases (97%) identical to a gb:GENBANK-ID:HUMIDE|acc:M21188.1 mRNA from Homo sapiens (Human insulin-degrading enzyme (IDE) mRNA, complete cds) (E=0.0).
A NOV3b polypeptide (SEQ ID NO:10) encoded by SEQ ID NO:9 has 1019 amino acid residues and is presented using the one-letter code in Table 3D. Signal P, Psort and/or Hydropathy results predict that NOV3b contains a signal peptide and is likely to be localized to the peroxisome (microbody) with a certainty of 0.8347 or the mitochondrial matrix space with a certainty of 0.6517. The most likely cleavage site for a NOV3b peptide is between amino acids 22 and 23: VLG-AR.







TABLE 3D





Encoded NOV3b protein sequence.















(SEQ ID NO:10)







MRYRLAWLLHPALPSTFRSVLGARLPPPERLCGFQKKTYSKMNNPAIKRIGNHITKPPEDKREYRGLELANG


IKVLLISDPTTDKSSAALDVHIGSLSDPPNIAGLSHFCEHMLFLGTKKYPKENEYSQFLSEHAGSSNAFTSG


EHTNYYFDVSHEHLEGALDRFAQFFLCPLFDESCKDREVNAVDSEHEKNVMNDAWRLLQLEKATGNPKHPFS


KFGTGNKYTLETRPNQEGIDVRQELLKFHSAYYSSNLMAVCVLGRESLDDLTNLVVKLFSEVENKNVPLPEF


PEHPFQEEHLKQLYKIVPIKDIRNLYVTFPIPDLQKYYKSNPGHYLGHLIGHEGPGSLLSELKSKGWVNTLV


GGQKEGARGFMFFIINVDLTEEGLLHVEDIILHMFQYIQRLRAEGPQEWVFQECKDLNAVTFRFKDKERPRG


YTSKIAGILHYYPLEEVLTAEYLLEEFRPDLIEMVLDKLRPENVRVAIVSKSFEGKTDRTEEWYGTQYKQEA


IPDEVIKKWQNADLNGKFKLPTKNEFIPTNFEILPLEKEATPYPALIKDTAMSKLWFKQDDKFFLPKACLNF


EFFSRYIYADPLHCNMTYLFIRLLKDDLKEYTYAARLSGLSYGIASGMNAILLSVKGYNDKQPILLKKIIEK


MATFEIDEKRFEIIKEAYMRSLNNFRAEQPHQHAMYYLRLLMTEVAWTKDELKEALDDVTLPRLKAFIPQLL


SRLHIEALLHGNITKQAALGIMQMVEDTLIEHAHTKPLLPSQLVRYREVQLPDRGWFVYQQRNEVHNNCGIE


IYYQTDMQSTSENMFLELFCQIISEPCFNTLRTKEQLGYIVFSGPRRANGIQGLRFIIQSEKPPHYLESRVE


AFLITMEKSIEDMTEEAFQKHIQALAIRRLDKPKKLSAECAKYWGEIISQQYNFDRDNTEVAYLKTLTKEDI


IKFYKEMLAVDAPRRHKVSVHVLAREMDSCPVVGEFPCQNDINLSQAPALPQPEVIQNMTEFKRGLPLFPLV


KPHINFMAAKL









The NOV3b amino acid sequence has 986 of 1019 amino acid residues (96%) identical to, and 996 of 1019 amino acid residues (97%) similar to, the 1019 amino acid residue ptnr:pir-id:SNHUIN protein from human (insulysin (EC 3.4.24.56)) (E=0.0).
The disclosed NOV3b is expressed in at least the following tissues: Lung, skin, uterus, colon, breast, liver, spleen, testis, and pediatric pre-B cell acute lymphoblastic leukemia. This information was derived by determining the tissue sources of the sequences that were included in the invention including but not limited to SeqCalling sources, Public EST sources, Literature sources, and/or RACE sources.
Possible SNPs found for NOV3a are listed in Table 3E and possible SNPs found for NOV3b are listed in Table 3F.







TABLE 3E







SNPs
















Amino





Nucleotide
Base
Acid
Base



Variant
Position
Change
Position
Change

















13375689
177
C > T
Silent
N/A



13375688
184
A > G
43
Asn > Asp



13375672
223
A > T
56
Lys > End



13375687
226
T > C
57
Ser > Pro



13375686
270
T > C
Silent
N/A



13375685
322
G > T
89
Ala > Ser



13375684
330
T > C
Silent
N/A



13375683
355
C > T
100
Pro > Ser



13375682
435
A > G
Silent
N/A



13375681
450
C > T
Silent
N/A



13375326
661
T > C
202
Phe > Leu



13375680
685
A > G
210
Asn > Asp



13375679
905
T > C
283
Val > Ala



13375327
1256
A > G
400
Lys > Arg



13375328
1318
G > A
421
Ala > Thr



13375329
1412
C > T
452
Ala > Val



13375678
2686
A > G
877
Met > Val



13375677
2751
G > A
Silent
N/A



13375676
2815
A > G
920
Arg > Gly



13375675
2895
A > G
Silent
N/A



13375674
2910
A > G
Silent
N/A



13375673
2984
A > G
976
Asn > Ser



13377049
3077
T > A
1007
Leu > His

















TABLE 3F







SNPs












Consensus

Base




Position
Depth
Change
PAF







656
7
C > T
0.429










NOV3a and NOV3b are very closely homologous as is shown in the amino acid alignment in Table 3G.







TABLE 3G





Amino Acid Alignment of NOV3a and NOV3b































































































































Homologies to any of the above NOV3 proteins will be shared by the other NOV3 proteins insofar as they are homologous to each other as shown above. Any reference to NOV3 is assumed to refer to both of the NOV3 proteins in general, unless otherwise noted.
NOV3a has homology to the amino acid sequences shown in the BLASTP data listed in Table 3H.







TABLE 3H







BLAST results for NOV3a












Gene Index/
Protein/
Length
Identity
Positives



Identifier
Organism
(aa)
(%)
(%)
Expect















gi|4826770|ref|NP_004960.1|
insulysin;
1019
974/1019
984/1019
0.0


(NM_004969)
insulinase

(95%)
(95%)



[Homo



sapiens]


gi|6981076|ref|NP_037291.1|
insulin
1019
934/1019
965/1019
0.0


(NM_013159)
degrading

(91%)
(94%)



enzyme



[Rattus



norvegicus]


gi|13621162|ref|NP_
insulin
1019
929/1019
963/1019
0.0


112419.1|
degrading

(91%)
(94%)


(NM_031156)
enzyme [Mus



musculus]


gi|18576366|ref|XP_
insulysin
554
530/554
539/554
0.0


051153.2|
[Homo

(95%)
(96%)


(XM_051153)
sapiens]


gi|7296294|gb|AAF51584.1|
Ide gene
990
439/966
634/966
0.0


(AE003591)
product

(45%)
(65%)



[Drosophila



melanogaster]









The homology of these sequences is shown graphically in the ClustalW analysis shown in Table 3I.







TABLE 3I





ClustalW Analysis of NOV3a















1) NOV3a (SEQ ID NO:8)


2) gi|4826770|ref|NP_004960.1| (NM_004969) insulysin; insulinase [Homo sapiens]


(SEQ ID NO:106)


2) gi|6981076|ref|NP_013159.1| (NM_013159) insulin degrading enzyme


[Rattus norvegicus] (SEQ ID NO:107)


3) gi|13621162|ref|NP_112419.1| (NM_031156) insulin degrading enzyme [Mus musculus]


(SEQ ID NO:108)


4) gi|18576366|ref|XP_051153.2| (XM_051153) insulysin [Homo sapiens] (SEQ ID NO:109)


5) gi|7296294|gb AAF51584.1| (AE003591) Ide gene product [Drosophila melanogaster]


(SEQ ID NO:110)


















































































































Table 3J lists the domain description from DOMAIN analysis results against NOV3a. This indicates that the NOV3a sequence has properties similar to those of other proteins known to contain these domains.







TABLE 3J





Domain Analysis of NOV3a















gnl|Pfam|pfam00675, Peptidase_M16, Insulinase (Peptidase family M16).


(SEQ ID NO:111)


CD-Length = 149 residues, 99.3% aligned


Score = 157 bits (396), Expect = 4e−39










Query:
74
KVLLISDPTTDKSSAALDVHIGSLSDPPNIAGLSHFCEHMLFLGTKKYPKENEYSQFLSE
133




+| |  ||  | |+  | |  ||  +| +  ||+|| ||| | ||||||  ||  + | +


Sbjct:
1
RVALEHDPPADTSAVGLWVDAGSRYEPDDNNGLAHFLEHMAFKGTKKYP-SNELEEELEK
 59


Query:
134
HAGSSNAFTSGEHTNYYFDVSHEHLEGALDRFAQFFLCPLFDESCKDR-------EVNAV
186




  || ||+|| ||| || +| ++ |  |+|| | ||| |||  |  +|       || ||


Sbjct:
60
LGGSLNAYTSREHTAYYVEVLNDDLPKAVDRLADFFLNPLFSPSEVERERLVVLYEVEAV
119


Query:
187
DSEHEKNVMN----DAWRLFQLEKATGNP
211




|+| +  +++     |+|   | ++   |


Sbjct:
120
DAEPQAVLLDNLHAAAYRGTPLGRSLLGP
148









After binding to its receptor on the cell surface, insulin is internalized by receptor-mediated endocytosis and degraded within components of the endosomal apparatus. Degradation of insulin is important in the termination of signaling and clearance of the circulating hormone. It has been proposed that insulin-degrading enzyme (IDE), an evolutionarily conserved, neutral thiol-metalloendopeptidase, plays a crucial role in the degradation of internalized insulin in many types of cells. Despite the substantial evidence supporting the importance of IDE in cellular insulin degradation, there is controversy over its mode and site of action, mainly because of its cytosolic location. Its physiological location in cells has recently been elucidated through subcellular fractionation of liver parenchyma and through immunofluorescence microscopy of stably transfected Chinese hamster ovary cells that overexpress IDE. These experiments have excluded the presence of the enzyme in endosomes and have defined a peroxisomal location, consistent with the presence of a peroxisomal targeting sequence at the carboxyl terminus of the protein. Recently, researchers have demonstrated the functional significance of peroxisome-associated IDE (type I peroxisomal enzyme) in degrading cleaved leader peptides of peroxisomal proteins targeted by the type II motif. IDE is the first cloned and characterized proteinase to be localized to peroxisomes. Moreover, IDE appears to be a member of a newly identified superfamily of metalloendopeptidases that has an HXXEH active-site motif. Although fundamental questions concerning the biological role of IDE remain, its high degree of evolutionary conservation suggests that it must have important functions and multifaceted biological significance (Authier et al., Clin Invest Med 19(3):149-60, 1996)
Insulin degradation is a regulated process that plays a role in controlling insulin action by removing and inactivating the hormone. Abnormalities in insulin clearance and degradation are present in various pathological conditions including type 2 diabetes and obesity and may be important in producing clinical problems. The uptake, processing, and degradation of insulin by cells is a complex process with multiple intracellular pathways. Most evidence supports IDE as the primary degradative mechanism, but other systems (PDI, lysosomes, and other enzymes) undoubtedly contribute to insulin metabolism. Recent studies support a multifunctional role for IDE, as an intracellular binding, regulatory, and degradative protein. IDE increases proteasome and steroid hormone receptor activity, and this activation is reversed by insulin. This raises the possibility of a direct intracellular interaction of insulin with IDE that could modulate protein and fat metabolism. The recent findings would place intracellular insulin-IDE interaction into the insulin signal transduction pathway for mediating the intermediate effects of insulin on fat and protein turnover (Duckworth et al., Endocr Rev 19(5):608-24, 1998).
A number of proteases dependent on divalent cations for their activity have been shown to belong to one family, on the basis of sequence similarity. These enzymes include those listed below.
Insulinase (EC 3.4.24.56) (also known as insulysin or insulin-degrading enzyme or IDE), a cytoplasmic enzyme which seems to be involved in the cellular processing of insulin, glucagon and other small polypeptides.
Escherichia coli protease III (EC 3.4.24.55) (pitrilysin) (gene ptr), a periplasmic enzyme that degrades small peptides.
Mitochondrial processing peptidase (EC 3.4.24.64) (MPP). This enzyme removes the transit peptide from the precursor form of proteins imported from the cytoplasm across the mitochondrial inner membrane. It is composed of two nonidentical homologous subunits termed alpha and beta. The beta subunit seems to be catalytically active while the alpha subunit has probably lost its activity.
Nardilysin (EC 3.4.24.61) (N-arginine dibasic convertase or NRD convertase) this mammalian enzyme cleaves peptide substrates on the N-terminus of Arg residues in dibasic stretches.
Klebsiella pneumoniae protein pqqF. This protein is required for the biosynthesis of the coenzyme pyrrolo-quinoline-quinone (PQQ). It is thought to be protease that cleaves peptide bonds in a small peptide (gene pqqA) thus providing the glutamate and tyrosine residues necessary for the synthesis of PQQ.
Yeast protein AXL1, which is involved in axial budding.
Eimeria bovis sporozoite developmental protein.
Escherichia coli hypothetical protein yddC and HI1368, the corresponding Haemophilus influenzae protein.
Bacillus subtilis hypothetical protein ymxG.—Caenorhabditis elegans hypothetical proteins C28F5.4 and F56D2.1.
It should be noted that in addition to the above enzymes, this family also includes the core proteins I and II of the mitochondrial bcl complex (also called cytochrome c reductase or complex III), but the situation as to the activity or lack of activity of these subunits is quite complex. In mammals and yeast, core proteins I and II lack enzymatic activity. In Neurospora crassa and in potato core protein I is equivalent to the beta subunit of MPP. In Euglena gracilis, core protein I seems to be active, while subunit II is inactive.
These proteins do not share many regions of sequence similarity; the most noticeable is in the N-terminal section. This region includes a conserved histidine followed, two residues later by a glutamate and another histidine. In pitrilysin, it has been shown that this H-x-x-E-H motif is involved in enzyme activity; the two histidines bind zinc and the glutamate is necessary for catalytic activity. Non active members of this family have lost from one to three of these active site residues. A signature pattern has been developed that detect active members of this family as well as some inactive members.
The NOV3 nucleic acid of the invention encoding a Insulysin-like protein includes the nucleic acid whose sequence is provided in Tables 3A and 3C, or a fragment thereof. The invention also includes a mutant or variant nucleic acid any of whose bases may be changed from the corresponding base shown in Tables 3A and 3C while still encoding a protein that maintains its Insulysin-like activities and physiological functions, or a fragment of such a nucleic acid. The invention further includes nucleic acids whose sequences are complementary to those just described, including nucleic acid fragments that are complementary to any of the nucleic acids just described. The invention additionally includes nucleic acids or nucleic acid fragments, or complements thereto, whose structures include chemical modifications. Such modifications include, by way of non-limiting example, modified bases, and nucleic acids whose sugar phosphate backbones are modified or derivatized. These modifications are carried out at least in part to enhance the chemical stability of the modified nucleic acid, such that they may be used, for example, as antisense binding nucleic acids in therapeutic applications in a subject. In the mutant or variant nucleic acids, and their complements, up to about 3% of the residues may be so changed.
The NOV3 protein of the invention includes the Insulysin-like protein whose sequence is provided in Tables 3B and 3D. The invention also includes a mutant or variant protein any of whose residues may be changed from the corresponding residue shown in Tables 3B and 3D while still encoding a protein that maintains its Insulysin-like activities and physiological functions, or a functional fragment thereof. In the mutant or variant protein, up to about 4% of the bases may be so changed.
The NOV3 nucleic acids and proteins of the invention are useful in potential diagnostic and therapeutic applications implicated in various diseases and disorders described below and/or other pathologies. For example, the compositions of the present invention will have efficacy for treatment of patients suffering from: Cardiomyopathy, Atherosclerosis, Hypertension, Congenital heart defects, Aortic stenosis, Atrial septal defect (ASD), Atrioventricular (A-V) canal defect, Ductus arteriosus, Pulmonary stenosis, Subaortic stenosis, Ventricular septal defect (VSD), valve diseases, Tuberous sclerosis, Scleroderma, Obesity, Transplantation, Endometriosis, Fertility, Von Hippel-Lindau (VHL) syndrome, Cirrhosis, Transplantation, Hemophilia, Hypercoagulation, Idiopathic thrombocytopenic purpura, Immunodeficiencies, Retinitis pigmentosa, autosomal dominant; Retinitis pigmentosa, autosomal recessive; SEMD, Pakistani type; Urofacial syndrome; Alzheimer disease 6; Cholesteryl ester storage disease; Corneal dystrophy, Thiel-Behnke type; Dubin-Johnson syndrome; Leukemia, T-cell acute lymphocytic; Leukemia, T-cell acute lymphocytic; Spinocerebellar ataxia, infantile-onset, with sensory neuropathy; Split hand/foot malformation, type 3; Tolbutamide poor metabolizer; Warfarin sensitivity; Wolman disease; Anterior segment mesenchymal dysgenesis and cataract; Cataract, congenital; Neurofibrosarcoma; Diabetes mellitus, insulin-dependent, 17; Diabetes mellitus, insulin-dependent, 17; obesity, insulin resistance, Graft vesus host and other diseases, disorders and conditions of the like.
NOV3 nucleic acids and polypeptides are further useful in the generation of antibodies that bind immunospecifically to the novel substances of the invention for use in therapeutic or diagnostic methods. These antibodies may be generated according to methods known in the art, using prediction from hydrophobicity charts, as described in the “Anti-NOVX Antibodies” section below. For example the disclosed NOV3 protein have multiple hydrophilic regions, each of which can be used as an immunogen. This novel protein also has value in development of powerful assay system for functional analysis of various human disorders, which will help in understanding of pathology of the disease and development of new drug targets for various disorders.
NOV4
NOV4 includes two novel Integrin Beta-7 Precursor-like proteins disclosed below. The disclosed proteins have been named NOV4a and NOV4b.
NOV4a
A disclosed NOV4a nucleic acid of 2798 nucleotides (designated CuraGen Acc. No. CG57185-01) encoding a novel Integrin Beta-7 Precursor-like protein is shown in Table 4A. An open reading frame was identified beginning with an ATG initiation codon at nucleotides 152-154 and ending with a TAA codon at nucleotides 2545-2547. Putative untranslated regions upstream from the initiation codon and downstream from the termination codon are underlined in Table 4A, and the start and stop codons are in bold letters.







TABLE 4A





NOV4a Nucleotide Sequence















(SEQ ID NO:11)







CGTTGCTGTCGCTCTGCACGCACCTATGTGGAAACTAAAGCCCAGAGAGAAAGTCTGACTTGCCCCACAG


CCAGTGAGTGACTGCAGCAGCACCAGAATCTGGTCTGTTTCCTGTTTGGCTCTTCTACCACTACGGCTTG


GGATCTCGGGCATGGTGGCTTTGCCAATGGTCCTTGTTTTGCTGCTGGTCCTGAGCAGAGGTGAGAGTGA


ATTGGACGCCAAGATCCCATCCACAGGGGATGCCACAGAATGGCGGAATCCTCACCTGTCCATGCTGGGG


TCCTGCCAGCCAGCCCCCTCCTGCCAGAAGTGCATCCTCTCACACCCCAGCTGTGCATGGTGCAAGCAAC


TGAACTTCACCGCGTCGGGAGAGGCGGAGGCGCGGCGCTGCGCCCGACGAGAGGAGCTGCTGGCTCGAGG


CTGCCCGCTGGAGGAGCTGGAGGAGCCCCGCGGCCAGCAGGAGGTGCTGCAGGACCAGCCGCTCAGCCAG


GGCGCCCGCGGAGAGGGTGCCACCCAGCTGGCGCCGCAGCGGGTCCGGGTCACGCTGCGGCCTGGGGAGC


CCCAGCAGCTCCAGGTCCGCTTCCTTCGTGCTGAGGGATACCCGGTGGACCTGTACTACCTTATGGACCT


GAGCTACTCCATGAAGGACGACCTGGAACGCGTGCGCCAGCTCGGGCACGCTCTGCTGGTCCGGCTGCAG


GAAGTCACCCATTCTGTGCGCATTGGTTTTGGTTCCTTTGTGGACAAAACGGTGCTGCCCTTTGTGAGCA


CAGTACCCTCCAAACTGCGCCACCCCTGCCCCACCCGGCTGGAGCGCTGCCAGTCACCATTCAGCTTTCA


CCATGTGCTGTCCCTGACGGGGGACGCACAAGCCTTCGAGCGGGAGGTGGGGCGCCAGAGTGTGTCCGGC


AATCTGGACTCGCCTGAAGGTGGCTTCGATGCCATTCTGCAGGCTGCACTCTGCCAGGAGCAGATTGGCT


GGAGAAATGTGTCCCGGCTGCTGGTGTTCACTTCAGACGACACATTCCATACAGCTGGGGACGGGAAGTT


GGGCGGCATTTTCATGCCCAGTGATGGGCACTGCCACTTGGACAGCAATGGCCTCTACAGTCGCACCACA


GAGTTTGACTACCCTTCTGTGGGTCAGGTAGCCCAGGCCCTCTCTGCAGCAAATATCCAGCCCATCTTTG


CTGTCACCAGTGCCGCACTGCCTGTCTACCAGGAGCTGAGTAAACTGATTCCTAAGTCTGCAGTTGGGGA


GCTGAGTGAGGACTCCAGCAACGTGGTACAGCTCATCATGGATGCTTATAATAGCCTGTCTTCCACCGTG


ACCCTTGAACACTCTTCACTCCCTCCTGGGGTCCACATTTCTTACGAATCCCAGTGTGAGGGTCCTGAGA


AGAGGGAGGGTAAGGCTGAGGATCGAGGACAGTGCAACCACGTCCGAATCAACCAGACGGTGACTTTCTG


GGTTTCTCTCCAAGCCACCCACTGCCTCCCAGAGCCCCATCTCCTGAGGCTCCGGGCCCTTGGCTTCTCA


GAGGAGCTGATTGTGGAGTTGCACACGCTGTGTGACTGTAATTGCAGTGACACCCAGCCCCAGGCTCCCC


ACTGCAGTGATGGCCAGGGACACCTACAATGTGGTGTATGCAGCTGTGCCCCTGGCCGCCTAGGTCGGCT


CTGTGAGTGCTCTGTGGCAGAGCTGTCCTCCCCAGACCTGGAATCTGGGTGCCGGGCTCCCAATGGCACA


GGGCCCCTGTGCAGTGGAAAGGGTCACTGTCAATGTGGACGCTGCAGCTGCAGTGGACAGAGCTCTGGGC


ATCTGTGCGAGTGTGACGATGCCAGCTGTGAGCGACATGAGGGCATCCTCTGCGGAGGCTTTGGTCGCTG


CCAATGTGGAGTATGTCACTGTCATGCCAACCGCACGGGCAGAGCATGCGAATGCAGTGGGGACATGGAC


AGTTGCATCAGTCCCGAGGGAGGGCTCTGCAGTGGGCATGGACGCTGCAAATGCAACCGCTGCCAGTGCT


TGGACGGCTACTATGGTGCTCTATGCGACCAATGCCCAGGCTGCAAGACACCATGCGAGAGACACCGGGA


CTGTGCAGAGTGTGGGGCCTTCAGGACTGGCCCACTGGCCACCAACTGCAGTACAGCTTGTGCCCATACC


AATGTGACCCTGGCCTTGGCCCCTATCTTGGATGATGGCTGGTGCAAAGAGCGGACCCTGGACAACCAGC


TGTTCTTCTTCTTGGTGGAGGATGACGCCAGAGGCACGGTCGTGCTCAGAGTGAGACCCCAAGAAAAGGG


AGCAGACCACACGCAGGCCATTGTGCTGGGCTGCGTAGGGGGCATCGTGGCAGTGGGGCTGGGGCTGGTC


CTGGCTTACCGGCTCTCGGTGGAAATCTATGACCGCCGGGAATACAGTCGCTTTGAGAAGGAGCAGCAAC


AACTCAACTGGAAGCAGGACAGTAATCCTCTCTACAAAAGTGCCATCACGACCACCATCAATCCTCGCTT


TCAAGAGGCAGACAGTCCCACTCTCTGAAGGAGGGAGGGACACTTACCCAAGGCTCTTCTCCTTGGAGGA


CAGTGGGAACTGGAGGGTGAGAGGAAGGGTGGGTCTGTAAGACCTTGGTAGGGGACTAATTCACTGGCGA


GGTGCGGCCACCACCCTACTTCATTTTCAGAGTGACACCCAAGAGGGCTGCTTCCCATGCCTGCAACCTT


GCATCCATCTGGGCTACCCCACCCAAGTATACAATAAAGTCTTACCTCAGAAAAAAAAAAAAAAAAAA









A NOV4a polypeptide (SEQ ID NO:12) encoded by SEQ ID NO:11 is 798 amino acid residues and is presented using the one letter code in Table 4B.







TABLE 4B





NOV4a protein sequence















(SEQ ID NO:12)







MVALPMVLVLLLVLSRGESELDAKIPSTGDATEWRNPHLSMLGSCQPAPSCQKCILSHPSCAWCKQLNFTASGE


AEARRCARREELLARGCPLEELEEPRGQQEVLQDQPLSQGARGEGATQLAPQRVRVTLRPGEPQQLQVRFLRAE


GYPVDLYYLMDLSYSMKDDLERVRQLGHALLVRLQEVTHSVRIGFGSFVDKTVLPFVSTVPSKLRHPCPTRLER


CQSPFSFHHVLSLTGDAQAFEREVGRQSVSGNLDSPEGGFDAILQAALCQEQIGWRNVSRLLVFTSDDTFHTAG


DGKLGGIFMPSDGHCHLDSNGLYSRSTEFDYPSVGQVAQALSAANIQPIFAVTSAALPVYQELSKLIPKSAVGE


LSEDSSNVVQLIMDAYNSLSSTVTLEHSSLPPGVHISYESQCEGPEKREGKAEDRGQCNHVRINQTVTFWVSLQ


ATHCLPEPHLLRLRALGFSEELIVELHTLCDCNCSDTQPQAPHCSDGQGHLQCGVCSCAPGRLGRLCECSVAEL


SSPDLESGCRAPNGTGPLCSGKGHCQCGRCSCSGQSSGHLCECDDASCERHEGILCGGFGRCQCGVCHCHANRT


GRACECSGDMDSCISPEGGLCSGHGRCKCNRCQCLDGYYGALCDQCPGCKTPCERHRDCAECGAFRTGPLATNC


STACAHTNVTLALAPILDDGWCKERTLDNQLFFFLVEDDARGTVVLRVRPQEKGADHTQAIVLGCVGGIVAVGL


GLVLAYRLSVEIYDRREYSRFEKEQQQLNWKQDSNPLYKSAITTTINPRFQEADSPTL









NOV4b

A disclosed NOV4b nucleic acid of 2211 nucleotides (designated CuraGen Acc. No. CG57185-02) encoding a novel Integrin Beta-7 Precursor-like protein is shown in Table 4C. An open reading frame was identified beginning with an ATG initiation codon at nucleotides 13-15 and ending with a TAA codon at nucleotides 1801-1803. Putative untranslated regions upstream from the initiation codon and downstream from the termination codon are underlined in Table 4C, and the start and stop codons are in bold letters.







TABLE 4C





NOV4b Nucleotide Sequence















(SEQ ID NO:13)







GTGGCTTTGCCAATGGTCCTTGTTTTGCTGCTGGTCCTGAGCAGAGGTGAGAGTGAATTGGACGCCAAGA


TCCCATCCACAGGGGATGCCACAGAATGGCGGAATCCTCACCTGTCCATGCTGGGGTCCTGCCAGCCAGC


CCCCTCCTGCCAGAAGTGCATCCTCTCACACCCCAGCTCTGCATGGTGCAAGCAACTGAACTTCACCGCG


TCGGGAGAGGCGGAGGCGCGGCGCTGCGCCCGACGAGAGGAGCTGCTGGCTCGAGGCTGCCCGCTGGAGG


AGCTGGAGGAGCCCCGCGGCCAGCAGGAGGTGCTGCAGGACCAGCCGCTCAGCCAGGGCGCCCGCGGAGA


GGGTGCCACCCAGCTGGCGCCGCAGCGGGTCCGGGTCACGCTGCGGCCTGGGGAGCCCCAGCAGCTCCAG


GTCCGCTTCCTTCGTGCTGAGGGATACCCGGTGGACCTGTACTACCTTATGGACCTGAGCTACTCCATGA


AGGACGACCTGGAACGCGTGCGCCAGCTCGGGCACGCTCTGCTGGTCCGGCTGCAGGAAGTCACCCATTC


TGTGCGCATTGGTTTTGGTTCCTTTGTGGACAAAACGGTGCTGCCCTTTGTGAGCACAGTACCCTCCAAA


CTGCGCCACCCCTGCCCCACCCGGCTGGAGCGCTGCCAGTCACCATTCAGCTTTCACCATGTGCTGTCCC


TGACGGGGGACGCACAAGCCTTCGAGCGGGAGGTGGGGCGCCAGAGTGTGTCCGGCAATCTGGACTCGCC


TGAAGGTGGCTTCGATGCCATTCTGCAGGCTGCACTCTGCCAGGAGCAGATTGGCTGGAGAAATGTGTCC


CGGCTGCTGGTGTTCACTTCAGACGACACATTCCATACAGCTGGGGACGGGAAGTTGGGCGGCATTTTCA


TGCCCAGTGATGGGCACTGCCACTTGGACAGCAATGGCCTCTACAGTCGCAGCACAGAGTTTGACTACCC


TTCTGTGGGTCAGGTAGCCCAGGCCCTCTCTGCAGCAAATATCCAGCCCATCTTTGCTGTCACCAGTGCC


GCACTGCCTGTCTACCAGGAGCTGAGTAAACTGATTCCTAAGTCTGCAGTTGGGGAGCTGAGTGAGGACT


CCAGCAACGTGGTACAGCTCATCATGGATGCTTATAATAGCCTGTCTTCCACCGTGACCCTTGAACACTC


TTCACTCCCTCCTGGGGTCCACATTTCTTACGAATCCCAGTGTGAGGGTCCTGAGAAGAGGGAGGGTAAG


GCTGAGGATCGAGGACAGTGCAACCACGTCCGAATCAACCAGACGGTGACTTTCTGGGTTTCTCTCCAAG


CCACCCACTGCCTCCCAGAGCCCCATCTCCTGAGGCTCCGGGCCCTTGGCTTCTCAGAGGAGCTGATTGT


GGAGTTGCACACGCTGTGTGACTGTAATTGCAGTGACACCCAGCCCCAGGCTCCCCACTGCAGTGATGGC


CAGGGACACCTACAATGTGGTGTATGCAGCTGTGCCCCTGGCCGCCTAGGTCGGCTCTGTGAGTGCTCTG


TGGCAGAGCTGTCCTCCCCAGACCTGGAATCTGGGTGCCGGGCTCCCAATGGCACAGGGCCCCTGTGCAG


TGGAAAGGGTCACTGTCAATGTGGACGCTGCAGCTGCAGTGGACAGAGCTCTGGGCATCTGTGCGAGTGT


GACGATGCCAGCTGTGAGCGACATGAGGGCATCCTCTGCGGAGGGACTGTGCAGAGTGTGGGGCCTTCAG


GACTGGCCCACTGGCCACCAACTGCAGTACAGCTTGTGCCCATACCAATGTGACCCTGGCCTTGGCCCCT


ATCTTGGATGATGGCTGGTGCAAAGAGCGGACCCTGGACAACCAGCTGTTCTTCTTCTTGGTGGAGGATG


ACGCCAGAGGCACGGTCGTGCTCAGAGTGAGACCCCAAGAAAAGGGAGCAGACCACACGCAGGCCATTGT


GCTGGGCTGCGTAGGGGGCATCGTGGCAGTGGGGCTGGGGCTGGTCCTGGCTTACCGGCTCTCGGTGGAA


ATCTATGACCGCCGGGAATACAGTCGCTTTGAGAAGGAGCAGCAACAACTCAACTGGAAGCAGGTGAGGA


GACTTCCTGGTTAGGCCCCTTTTTAGCTGTTCCCCCACCACAAGACCAGCCCTGATTCCTCCCACTGGGT


TCCCCCAGCCCCTAGCACATGTAACCAACCCCTCTGCTAAC









The nucleic acid sequence of NOV4b maps to chromosome 12 and has 1813 of 1881 bases (96%) identical to a gb:GENBANK-ID:HUMINTB7|acc:M68892.1 mRNA from Homo sapiens (Human integrin beta-7 subunit mRNA, complete cds) (E=0.0).
A NOV4b polypeptide (SEQ ID NO:14) encoded by SEQ ID NO:13 is 596 amino acid residues and is presented using the one letter code in Table 4D. Signal P, Psort and/or Hydropathy results predict that NOV4b contains a signal peptide and is likely to be localized to the mitochondrial matrix space with a certainty of 0.4542 and the mitochondrial intermembrane space with a certainty of 0.4030. The most likely cleavage site for a NOV4b polypeptide is between amino acids 14 and 15: GES-EL.







TABLE 4D





NOV4b protein sequence















(SEQ ID NO:14)







MVLVLLLVLSRGESELDAKIPSTGDATEWRNPHLSMLGSCQPAPSCQKCILSHPSCAWCKQLNFTASGEAEARR


CARREELLARGCPLEELEEPRGQQEVLQDQPLSQGARGEGATQLAPQRVRVTLRPGEPQQLQVRFLRAEGYPVD


LYYLMDLSYSMKDDLERVRQLGHALLVRLQEVTHSVRIGFGSFVDKTVLPFVSTVPSKLRHPCPTRLERCQSPF


SFHHVLSLTGDAQAFEREVGRQSVSGNLDSPEGGFDAILQAALCQEQIGWRNVSRLLVFTSDDTFHTAGDGKLG


GIFMPSDGHCHLDSNGLYSRSTEFDYPSVGQVAQALSAANIQPIFAVTSAALPVYQELSKLIPKSAVGELSEDS


SNVVQLIMDAYNSLSSTVTLEHSSLPPGVHISYESQCEGPEKREGKAEDRGQCNHVRINQTVTFWVSLQATHCL


PEPHLLRLRALGFSEELIVELHTLCDCNCSDTQPQAPHCSDGQGHLQCGVCSCAPGRLGRLCECSVAELSSPDL


ESGCRAPNGTGPLCSGKGHCQCGRCSCSGQSSGHLCECDDASCERHEGILCGGTVQSVGPSGLAHWPPTAVQLV


PIPM









The NOV4b amino acid sequence has 571 of 571 amino acid residues (100%) identical to, and 571 of 571 amino acid residues (100%) similar to, the 798 amino acid residue ptnr:SWISSNEW-ACC:P26010 protein from Homo sapiens (Human) (Integrin Beta-7 Precursor) (E=5.9e−318).
NOV4b is expressed in at least the following tissues: Colon, Lung, Lymph node, Lymphoid tissue, Peripheral Blood, Placenta, Salivary Glands, Spleen, Thyroid and Tonsils. Expression information was derived from the tissue sources of the sequences that were included in the derivation of NOV4b.
Possible small nucleotide polymorphisms (SNPs) found for NOV4a are listed in Table 4E.







TABLE 4E







SNPs
















Amino





Nucleotide
Base
Acid
Base



Variant
Position
Change
Position
Change

















13377057
423
G > A
 91
Cys > Tyr



13377058
1133
C > T
328
Pro > Ser



13377059
1261
G > A
Silent
N/A



13377056
2126
A > G
659
Thr > Ala



13377055
2241
T > C
697
Leu > Pro



13377054
2285
C > T
712
Leu > Phe



13377053
2303
G > A
718
Glu > Lys



13377052
2414
C > T
755
Arg > Cys



13377051
2443
G > A
Silent
N/A



13377050
2447
C > T
766
Gln > End










NOV4a and NOV4b are very closely homologous as is shown in the amino acid alignment in Table 4F.







TABLE 4F





Amino Acid Alignment of NOV4a and NOV 4b










































































































Homologies to any of the above NOV4 proteins will be shared by the other NOV4 proteins insofar as they are homologous to each other as shown above. Any reference to NOV4 is assumed to refer to both of the NOV4 proteins in general, unless otherwise noted.
NOV4a also has homology to the amino acid sequences shown in the BLASTP data listed in Table 4G.







TABLE 4G







BLAST results for NOV4a












Gene Index/

Length
Identity
Positives



Identifier
Protein/Organism
(aa)
(%)
(%)
Expect





gi|4504777|ref|NP_000880.1|
integrin, beta 7
798
737/798
737/798
0.0


(NM_000889)
[Homo sapiens]

(92%)
(92%)


gi|400075 sp|P26011|
INTEGRIN BETA-7
806
632/784
673/784
0.0


ITB7|MOUSE
PRECURSOR

(80%)
(85%)



(INTEGRIN BETA-P)



(M290 IEL ANTIGEN)



[Mus musculus]


gi|255139|gb|AAB23193.1|
beta 7 integrin
806
631/784
672/784
0.0


(S44607)
[Mus musculus]

(80%)
(85%)


gi|7305193|ref|NP_038594.1|
integrin beta 7
805
631/784
672/784
0.0


(NM_013566)
[Mus musculus]

(80%)
(85%)


gi|2196784|gb|AAB61241.1|
integrin beta-7
640
513/632
551/632
0.0


(AF003598)
subunit [Rattus

(81%)
(87%)



norvegicus]









The homology of these sequences is shown graphically in the ClustalW analysis shown in Table 4H.







TABLE 4H





ClustalW Analysis of NOV4















1) NOV4a (SEQ ID NO:12)


2) gi|4504777|refNP_000880.1| (NM_000889) integrin, beta 7 [Homo sapiens]


(SEQ ID NO:112)


3) gi|400075|sp|P26011|ITB7_MOUSE INTEGRIN BETA-7 PRECURSOR (INTEGRIN BETA-P)


(M290 IEL ANTIGEN) [Mus musculus] (SEQ ID NO:113)


4) gi 255139|gb|AAB23193.1| (S44607) beta 7 integrin [Mus musculus] (SEQ ID NO:114)


5) gi 7305193|ref|NP_038594.1| (NM_013566) integrin beta 7 [Mus musculus]


(SEQ ID NO:115)


6) gi 2196784|gb|AAB61241.1| (AF003598) integrin beta-7 subunit [Rattus norvegicus]


(SEQ ID NO:116)





























































































Tables 4I and 4J lists the domain description from DOMAIN analysis results against NOV4a. This indicates that the NOV4a sequence has properties similar to those of other proteins known to contain these domains.







TABLE 4I





Domain Analysis of NOV4a















gnl|Pfam|pfam00362, integrin_B, Integrins, beta chain.


Sequences cut off at repeats due to overlap with EGF. (SEQ ID NO: 117)


CD-Length = 428 residues. 100.0% aligned


Score = 633 bits (1632), Expect = 0.0










Query:
50
SCQKCILSHPSCAWCKQLNFTASGEAEARRCARREELLARGCPLEELEEPRGQQEVLQDQ
109




||++|+|| | |||| + + +||+ || ++ ||  |  ||++||||+ +| ||   |+|+||


Sbjct:
1
SCEECLLSGPGCAWCTKEDFTSPGEPDSERCDTRANLLSKGCPLDSIENPRSSAEILEDQ
 60


Query:
110
PLSQGARGEGATQLAPQRVRVTLRPGEPQQLQVRFLRAEGYPVDLYYLMDLSYSMKDDLE
169




|||     + +|||+|| ||+ ||||| |  |+   ||| ||||||||||||||||||||


Sbjct:
61
PLSNKGSRDSSTQLSPQEVRLRLRPGEEQTFQLTVRRAEDYPVDLYYLMDLSYSMKDDLE
120


Query:
170
RVRQLGHALLVRLQEVTHSVRIGFGSFVDKTVLPFVSTVPSKLRHPCPTRLERCQSPFSF
229




 |+ ||  |   + ++|   |||||||||||| ||||| | |||+||| + + || || |


Sbjct:
121
NVKSLGTDLAREMSKLTSDFRIGFGSFVDKTVSPFVSTHPEKLRNPCPNKEKNCQPPFGF
180


Query:
230
HHVLSLTGDAQAFEREVGRQSVSGNLDSPEGGFDAILQAALCQEQIGWRN-VSRLLVFTS
288




 |||||| |   |  |||+| +|||||+||||||||+|||+|+|+||||| |+|||||++


Sbjct:
181
KHVLSLTDDVDEFNEEVGKQRISGNLDAPEGGFDAIMQAAVCKEKIGWRNDVTRLLVFST
240


Query:
289
DDTFHTAGDGKLGGIFMPSDGHCHLDSNGLYSRSTEFDYPSVGQVAQALSAANIQPIFAV
348




|  || |||||||||  |+|| |||||| +|+ ||  |||||||+ + ||  ||||||||


Sbjct:
241
DAGFHFAGDGKLGGIVQPNDGRCHLDSNNMYTMSTTMDYPSVGQLVEKLSENNIQPIFAV
300


Query:
349
TSAALPVYQELSKLIPKSAVGELSEDSSNVVQLIMDAYNSLSSTVTLEHSSLPPGVHISY
408




|   + +|++|++||| |||||||||||||||||+|||| | | |  || | || |+++|


Sbjct:
301
TEKQVHLYEKLTELIPGSAVGELSEDSSNVVQLIIDAYNKLRSEVELEVSDLPEGLNLSY
360


Query:
409
ESQCEGPEKREGKAEDRGQCNHVRINQTVTFWVSLQATHCLPEP--HLLRLRALGFSEEL
466




 | |+      |  + ||+|+ |+|  ||+| ||+ |  | ||   | + ++ ||||+ |


Sbjct:
361
TSFCKNGVSSPG--QRRGKCSGVQIGDTVSFEVSVTARECPPEGQKHSIIIKPLGFSDTL
418


Query:
467
IVELHTLCDC
476




 | +|  |||


Sbjct:
419
EVHVHPNCDC
428
















TABLE 4J





Domain Analysis of NOV4a















gnl|Smart|smart00187, INB, Integrin beta subunits (N-terminal portion


of extracellular region); Portion of beta integrins that lies N-terminal


to their EGF-like repeats. Integrins are cell adhesion


molecules that mediate cell-extracellular matrix and cell-cell


interactions. They contain both alpha and beta subunits. Beta


integrins are proposed to have a von Willebrand factor type-A “insert”


or “I”-like domain (SEQ ID NO:118)


CD-Length = 423 residues, 100.0% aligned


Score = 614 bits (1584), Expect = 5e−177










Query:
50
SCQKCILSHPSCAWCKQLNFTASGEAEARRCARREELLARGCPLEELEEPRGQQEVLQDQ
109




|| +|| |||+||||   |||+ | +              ||  | +|+|+ + |||+||


Sbjct:
1
SCGECIQSHPNCAWCTDENFTSGGSSARCDSRANLLAK--GCSPESIEDPKSEIEVLEDQ
 58


Query:
110
PLSQGARGEGATQLAPQRVRVTLRPGEPQQLQVRFLRAEGYPVDLYYLMDLSYSMKDDLE
169




|||       | |++|||||+ |||||||   +   +|| |||||||||||||||||||+


Sbjct:
59
PLSDSESSGQAVQVSPQRVRLKLRPGEPQNFTLTVRQAEDYPVDLYYLMDLSYSMKDDLD
118


Query:
170
RVRQLGHALLVRLQEVTHSVRIGFGSFVDKTVLPFVSTVPSKLRHPCPTRLERCQSPFSF
229




 ++ ||  |   ++ +| + |+|||||||||| ||||| | || +|||     |+ |+ |


Sbjct:
119
NLKSLGDDLAREMKGLTSNFRLGFGSFVDKTVSPFVSTRPEKLENPCPNYNLTCEPPYGF
178


Query:
230
HHVLSLTGDAQAFEREVGRQSVSGNLDSPEGGFDAILQAALCQEQIGWRN-VSRLLVFTS
288




 |||||| |   |  || +| +|||||+||||||||+|||+| ||||||    |||||++


Sbjct:
179
KHVLSLTDDTDEFNEEVKKQRISGNLDAPEGGFDAIMQAAVCTEQIGWREDARRLLVFST
238


Query:
289
DDTFHTAGDGKLGGIFMPSDGHCHLDSNGLYSRSTEFDYPSVGQVAQALSAANIQPIFAV
348




|  || |||||| ||  |+|| ||||+|| |+ ||  ||||+||+ | |+  || |||||


Sbjct:
239
DAGFHFAGDGKLAGIVQPNDGQCHLDNNGEYTMSTTQDYPSIGQLNQKLAENNINPIFAV
298


Query:
349
TSAALPVYQELSKLIPKSAVGELSEDSSNVVQLIMDAYNSLSSTVTLEHSSLPPGVHISY
408




|   + +|+||| ||| |+|| |||||||||+|| |||| +|| | || +||| || ++|


Sbjct:
299
TKKQVSLYKELSALIPGSSVGVLSEDSSNVVELIKDAYNKISSRVELEDNSLPEGVSVTY
358


Query:
409
ESQCEGPEKREGKAEDRGQCNHVRINQTVTFWVSLQATHCLPE-PHLLRLRALGFSEELI
467




 | | |     |      +|  |+|  ||+| |++ || | ||  | +|+| +|||| |


Sbjct:
359
TSSCPGGVVGPGTR----KCEGVKIGDTVSFEVTVTATKCPPEKEHSIRIRPVGFSETLE
414


Query:
468
VELHTLCDC
476




|||  ||||


Sbjct:
415
VELTFLCDC
423









The integrins are a large family of heterodimeric cell-surface glycoproteins that play key roles in the adherence of cells to other cells and to extracellular matrix proteins. Leukocytes express a variety of integrins that are crucial participants in the inflammatory and immune responses. All integrins are composed of 1 alpha subunit and 1 beta subunit that are translated separately and are noncovalently associated. The cDNA sequences of 6 mammalian beta subunits, numbered beta-1 through beta-6, had previously been reported. Erle et al. (J. Biol. Chem. 266: 11009-11016, 1991) determined the complete sequence of a novel integrin beta subunit from leukocytes, designated beta-7. The cDNA contained a single large open reading frame predicted to encode a 798-amino acid protein precursor (signal peptide plus mature protein). Like other beta-subunit proteins, it was predicted to contain a large extracellular domain, a transmembrane domain, and a cytoplasmic tail. The deduced beta-7 amino acid sequence was 32 to 46% identical to the 6 previously sequenced subunits. It was most similar to the leukocyte integrin common beta subunit (ITGB2; CD18; 116920). Erle et al. (1991) predicted that integrin beta-7 plays a role in adhesive interactions of leukocytes. Krissansen et al. (Immunogenetics 35: 58-61, 1992) assigned the ITGB7 gene to human chromosome 12 by analysis of somatic cell hybrids. Krissansen et al. (1992) commented on the fact that the gene encoding the alpha subunit of the fibronectin receptor (FNRA; 135620) resides on chromosome 12 also. They stated that the ‘extent and evolutionary significance of linkages between integrin genes . . . remains to be determined.’ Baker et al. (Mammalian Genome 2:272-273, 1992) assigned the ITGB7 gene to 12q13.13 by nonisotopic in situ hybridization. Yuan et al. (Immunogenetics 35: 403-407, 1992) mapped the mouse homolog Itgb-7 to chromosome 15 by study of mouse-hamster somatic cell hybrids and by interspecific backcrosses.
The NOV4 nucleic acid of the invention encoding a Integrin Beta-7 Precursor-like protein includes the nucleic acid whose sequence is provided in Tables 4A and 4C, or a fragment thereof. The invention also includes a mutant or variant nucleic acid any of whose bases may be changed from the corresponding base shown in Tables 4A and 4C while still encoding a protein that maintains its Integrin Beta-7 Precursor-like activities and physiological functions, or a fragment of such a nucleic acid. The invention further includes nucleic acids whose sequences are complementary to those just described, including nucleic acid fragments that are complementary to any of the nucleic acids just described. The invention additionally includes nucleic acids or nucleic acid fragments, or complements thereto, whose structures include chemical modifications. Such modifications include, by way of non-limiting example, modified bases, and nucleic acids whose sugar phosphate backbones are modified or derivatized. These modifications are carried out at least in part to enhance the chemical stability of the modified nucleic acid, such that they may be used, for example, as antisense binding nucleic acids in therapeutic applications in a subject. In the mutant or variant nucleic acids, and their complements, up to about 4% of the residues may be so changed.
The NOV4 protein of the invention includes the Integrin Beta-7 Precursor-like protein whose sequence is provided in Tables 4B and 4D. The invention also includes a mutant or variant protein any of whose residues may be changed from the corresponding residue shown in Tables 4B and 4D while still encoding a protein that maintains its Integrin Beta-7 Precursor-like activities and physiological functions, or a functional fragment thereof.
The NOV4 nucleic acids and proteins of the invention are useful in potential diagnostic and therapeutic applications implicated in various diseases and disorders described below and/or other pathologies. For example, the compositions of the present invention will have efficacy for treatment of patients suffering from: systemic lupus erythematosus, autoimmune disease, asthma, emphysema, scleroderma, allergy, ARDS; atherosclerosis, thrombosis, cancer, asthma, lymphedema; fertility, xerostomia, hyperthyroidism, hypothyroidism and other diseases, disorders and conditions of the like.
NOV4 nucleic acids and polypeptides are further useful in the generation of antibodies that bind immunospecifically to the novel substances of the invention for use in therapeutic or diagnostic methods. These antibodies may be generated according to methods known in the art, using prediction from hydrophobicity charts, as described in the “Anti-NOVX Antibodies” section below. For example the disclosed NOV4 protein have multiple hydrophilic regions, each of which can be used as an immunogen. This novel protein also has value in development of powerful assay system for functional analysis of various human disorders, which will help in understanding of pathology of the disease and development of new drug targets for various disorders.
NOV5
A disclosed NOV5 nucleic acid of 1360 nucleotides (also referred to as CG57360-01) encoding a novel membrane protein-like protein is shown in Table 5A. An open reading frame was identified beginning with an ATG initiation codon at nucleotides 244-246 and ending with a TGA codon at nucleotides 1282-1284. Putative untranslated regions upstream from the intitation codon and downstream from the termination codon are underlined in Table 5A, and the start and stop codons are in bold letters.







TABLE 5A





NOV5 Nucleotide Sequence















(SEQ ID NO:15)







GTCGAATATCCATGCAGAGTACCTGGAATACATCAGGGATCCTTGCTCTCATGAGGTCAGAGATGAGGAATTC


CATAGGATGAGTCTTCTGTTCTTAAACCGAGTGTAAGGATAAGGCTGTCGGAGAGGAGGGAGTAGGATTTGGG


CATCAGAGCCCCCCAGCTGCTGCTCGTGGGGGTGAGGACACCAGGCCCCTGACTTTGCTCTGGTCTCTCTCCC


AGCTCCCTGCCCAGGCCCACAGCCATGGCCATGGCCCAGAAACTCAGCCACCTCCTGCCGAGTCTGCGGCAGG


TCATCCAGGAGCCTCAGCTATCTCTGCAGCCAGAGCCTGTCTTCACGGTGGATCGAGCTGAGGTGCCGCCGCT


CTTCTGGAAGCCGTACATCTATGCGGGCTACCGGCCGCTGCATCAGACCTGGCGCTTCTATTTCCGCACGCTG


TTCCAGCAGCACAACGAGGCCGTGAATGTCTGGACCCACCTGCTGGCGGCCCTGGCACTGCTGCTGCGGCTGG


CCCTCTTTGTGGAGACCGTGGACTTCTGGGGAGACCCACACGCCCTGCCCCTCTTCATCATTGTCCTTGCCTC


TTTCACCTACCTCTCCTTCAGTGCCTTGGCTCACCTCCTGCAGGCCAAGTCTGAGTTCTGGCATTACAGCTTC


TTCTTCCTGGACTATGTGGGGGTGGCCGTGTACCAGTTTGGCAGTGCCTTGGCACACTTCTACTATGCTATCG


AGCCCGCCTGGCATGCCCAGGTGCAGGCTGTTTTTCTGCCCATGGCTGCCTTTCTCGCCTGGCTTTCCTGCAT


TGGCTCCTGCTATAACAAGTACATCCAGAAACCAGGCCTGCTGGGCCGCACATGCCAGGAGGTGCCCTCCGTC


CTGGCCTACGCACTGGACATTAGTCCTGTGGTGCATCGTATCTTCGTGTCCTCCGACCCCACCACGGATGATC


CAGCTCTTCTCTACCACAAGTGCCAGGTGGTCTTCTTTCTGCTGGCTGCTGCCTTCTTCTCTACCTTCATGCC


CGAGCGCTGGTTCCCTGGCAGCTGCCATGTCTTCGGGCAGCGCCACCAACTTTTCCACATCTTCTTGGTGCTG


TGCACGCTGGCTCAGCTGGAGGCTGTGGCACTGGACTATGAGGCCCGACGGCCCATCTATGAGCCTCTGCACA


CGCACTGGCCTCACAACTTTTCTGGCCTCTTCCTGCTCACGGTGGGCAGCAGCATCCTCACTGCATTCCTCCT


GAGCCAGCTGGTACAGCGCAAACTTGATCAGAAGACCAAGTGAAGGGGGATGGCATCTGGTAGGGAGGGAGGT


ATAGTTGGGGGACAGGGGTCTGGGTTTGGCTCCAGGTGGGAACAAG









The NOV5 nucleic acid was identified on chromosome 1 and has 308 of 508 bases (60%) identical to a gb:GENBANK-ID:RNU87960|acc:U87960.1 mRNA from Rattus norvegicus (Rattus norvegicus leukocyte common antigen receptor (LAR) gene, trans-spliced alternative untranslated exon) (E=1.5e−06).
A disclosed NOV5 polypeptide (SEQ ID NO:16) encoded by SEQ ID NO:15 is 346 amino acid residues and is presented using the one-letter code in Table 5B. Signal P, Psort and/or Hydropathy results predict that NOV5 does not contain a signal peptide and is likely to be localized at the plasma membrane with a certainty of 0.6000 and the Golgi body with a certainty of 0.4000.







TABLE 5B





Encoded NOV5 protein sequence















(SEQ ID NO:16)







MAMAQKLSHLLPSLRQVIQEPQLSLQPEPVFTVDRAEVPPLFWKPYIYAGYRPLHQTWRFYFRTLFQQHNEA


VNVWTHLLAALALLLRLALFVETVDFWGDPHALPLFIIVLASFTYLSFSALAHLLQAKSEFWHYSFFFLDYV


GVAVYQFGSALAHFYYAIEPAWHAQVQAVFLPMAAFLAWLSCIGSCYNKYIQKPGLLGRTCQEVPSVLAYAL


DISPVVHRIFVSSDPTTDDPALLYHKCQVVFFLLAAAFFSTFMPERWFPGSCHVFGQGHQLFHIFLVLCTLA


QLEAVALDYEARRPIYEPLHTHWPHNFSGLFLLTVGSSILTAFLLSQLVQRKLDQKTK









The NOV5 amino acid sequence has 171 of 349 amino acid residues (48%) identical to, and 230 of 349 amino acid residues (65%) similar to, the 359 amino acid residue ptnr:TREMBLNEW-ACC:CAC05478 protein from Homo sapiens (Human) (DJ304B14.1 (NOVEL PROTEIN) (E=3.4e−84).
NOV5 is expressed in at least the following tissues: Adrenal Gland/Suprarenal gland, Bone Marrow, Kidney, Liver, Prostate and Testis. This information was derived by determining the tissue sources of the sequences that were included in the invention including but not limited to SeqCalling sources, Public EST sources, genomic clone sources, literature sources, and/or RACE sources. The NOV5 sequence is also predicted to be expressed in the following tissues because of the expression pattern of (GENBANK-ID: gb:GENBANK-ID:RNU87960|acc:U87960.1) a closely related Rattus norvegicus leukocyte common antigen receptor (LAR) gene, trans-spliced alternative untranslated exon homolog in species Rattus norvegicus: leukocyte.
Possible small nucleotide polymorphisms (SNPs) found for NOV5 are listed in Table 5C.







TABLE 5C







SNPs
















Amino





Nucleotide
Base
Acid
Base



Variant
Position
Change
Position
Change

















13377060
967
A > G
242
Lys > Glu



13377061
1233
A > G
Silent
N/A










NOV5 has homology to the amino acid sequences shown in the BLASTP data listed in Table 5D.







TABLE 5D







BLAST results for NOV5












Gene Index/

Length
Identity
Positives



Identifier
Protein/Organism
(aa) 
(%)
(%)
Expect





gi|18549110|ref|XP_
hypothetical
346
324/346
324/346
e−168


089356.1|
protein XP_089356

(93%)
(93%)


(XM_089356)
[Homo sapiens]


gi|12844255|dbj|BAB26296.1|
data source: SPTR,
345
266/346
283/346
e−130


(AK009450)
source key: 

(76%)
(80%)



Q9HD02, evidence: 



ISS ̃homolog to



DJ304B14.1 (NOVEL



PROTEIN)



(FRAGMENT)  ̃putative



[Mus musculus]


gi|12839033|dbj|BAB24412.1|
data source: SPTR,
354
158/351
215/351
4e−69


(AK006107)
source

(45%)
(61%)



key: Q9HD02,



evidence: ISS ̃homolog



to DJ304B14.1



(NOVEL PROTEIN)



(FRAGMENT)  ̃putative



[Mus musculus]


gi|9955433|emb|CAC05478.1|
dJ304B14.1 (novel
359
146/316
199/316
2e−66


(AL136125)
protein) [Homo

(46%)
(62%)



sapiens]


gi|18564176|ref|XP_
hypothetical
354
146/316
199/316
2e−66


094815.1|
protein XP_094815

(46%)
(62%)


(XM_094815)
[Homo sapiens]









The homology of these sequences is shown graphically in the ClustalW analysis shown in Table 5E.







TABLE 5E





Clustal W Sequence Alignment















1) NOV5 (SEQ ID NO:16)


2) gi|18549110|ref|XP_089356.1| (XM_089356) hypothetical protein XP_089356


[Homo sapiens] (SEQ ID NO:119)


3) gi|12844255|dbj|BAB26296.1| (AK009450) data source:SPTR, source key:Q9HD02,


evidence:ISS ̃to DJ304B14.1 (NOVEL PROTEIN) (FRAGMENT) ̃putative [Mus musculus]


(SEQ ID NO:120)


4) gi|12839033|dbj|BAB24412.1| (AK006107) data source:SPTR, source key:Q9HD02,


evidence:ISS ̃to DJ304B14.1 (NOVEL PROTEIN) (FRAGMENT) ̃putative [Mus musculus]


(SEQ ID NO:121)


5) gi|9955433|emb|CAC05478.1| (AL136125) dJ304B14.1 (novel protein) [Homo sapiens]


(SEQ ID NO:122)


6) gi|18564176|ref|XP_094815.1| (XM_094815) hypothetical protein XP_094815


[Homo sapiens] (SEQ ID NO:123)



















































Table 5F list the domain description from DOMAIN analysis results against NOV5. This indicates that the NOV5 sequence has properties similar to those of other proteins known to contain these domains.







TABLE 5F





Domain Analysis of NOV5















gnl Pfam pfam03006, UPF0073, Uncharacterised protein family (Hly-III/


UPF0073). Members of this family are integral membrane proteins. This


family includes a protein with hemolytic activity from Bacillus


cereus. It is not clear if all the members of this family are


hemolysins. (SEQ ID NO:124)


CD-Length = 238 residues, 99.2% aligned


Score = 81.6 bits (200), Expect = 7e−17










Query:
57
TWRFYFRTLFQQHNEAVNVWTHLLAALALLLRLALFVETVDFWGDPHALPLFIIVL---A
113




+++  |+++|   |||  |+|||||  +     | ||   +            +


Sbjct:
2
SFKKCFKSIFSWHNETSNIWTHLLGFILFFFLLILFFLFLLPPILATWQDRVVFGFFLAG
 61


Query:
114
SFTYLSFSALAHLLQAKSEFWHYSFFFLDYVGVAVYQFGSALAHFYYAIEPAWHAQVQAV
173




+|  |  |++ |     ||     |  |||+ |+++    | +   |||     |   + +


Sbjct:
62
AFLCLLLSSIYHTFSCHSEKHSEFFLKLDYLGISLLIVASFIPIIYYAFYC--HPFFRTL
119


Query:
174
FLPMAAFLAWLSCIGSCYNKYIQKPGLLGRTCQEVPSVLAYALDISPVVHRIFVSSD-PT
232




++ +   |  ++    |  +|++       |  +     ||     + |++| + +


Sbjct:
120
YISIILVLGLIAIYVSLSDKFVSPKF---RPLRAGFFVLLGCSGVIPLLHALILFGGHEN
176


Query:
233
TDDPALLYHKCQVVFFLLAAAFFSTFMPERWFPGSCHVFGQGHQLFHIFLVLCTLAQLEA
292




    || +     | +++ |  |+ | +|||+|||   ++|  |||||+ +||       |


Sbjct:
177
LVRIALPWVLLMAVLYIVGAVFYGTRIPERFFPGKFDIWGHSHQLFHLLVVLAAFYHYRA
236


Query:
293
V
293


Sbjct:
237
G
237









The NOV5 nucleic acid of the invention encoding a membrane protein-like protein includes the nucleic acid whose sequence is provided in Table 5A, or a fragment thereof. The 10 invention also includes a mutant or variant nucleic acid any of whose bases may be changed from the corresponding base shown in Table 5A while still encoding a protein that maintains its membrane protein-like activities and physiological functions, or a fragment of such a nucleic acid. The invention further includes nucleic acids whose sequences are complementary to those just described, including nucleic acid fragments that are complementary to any of the nucleic acids just described. The invention additionally includes nucleic acids or nucleic acid fragments, or complements thereto, whose structures include chemical modifications. Such modifications include, by way of non-limiting example, modified bases, and nucleic acids whose sugar phosphate backbones are modified or derivatized. These modifications are carried out at least in part to enhance the chemical stability of the modified nucleic acid, such that they may be used, for example, as antisense binding nucleic acids in therapeutic applications in a subject. In the mutant or variant nucleic acids, and their complements, up to about 40% of the residues may be so changed.
The NOV5 protein of the invention includes the membrane protein-like protein whose sequence is provided in Table 5B. The invention also includes a mutant or variant protein any of whose residues may be changed from the corresponding residue shown in Table 5B while still encoding a protein that maintains its membrane protein-like activities and physiological functions, or a functional fragment thereof. In the mutant or variant protein, up to about 52% of the bases may be so changed.
The NOV5 nucleic acids and proteins of the invention are useful in potential diagnostic and therapeutic applications implicated in various diseases and disorders described below and/or other pathologies. For example, the compositions of the present invention will have efficacy for treatment of patients suffering from: cancer, trauma, regeneration (in vitro and in vivo), viral/bacterial/parasitic infections, adrenoleukodystrophy, congenital adrenal hyperplasia, Von Hippel-Lindau (VHL) syndrome, cirrhosis, transplantation, hemophilia, hypercoagulation, idiopathic thrombocytopenic purpura, autoimmune disease, allergies, immunodeficiencies, fertility, hypogonadism, diabetes, autoimmune disease, renal artery stenosis, interstitial nephritis, glomerulonephritis, polycystic kidney disease, systemic lupus erythematosus, renal tubular acidosis, IgA nephropathy, hypercalceimia, Lesch-Nyhan syndrome and other diseases, disorders and conditions of the like.
NOV5 nucleic acids and polypeptides are further useful in the generation of antibodies that bind immunospecifically to the novel substances of the invention for use in therapeutic or diagnostic methods. These antibodies may be generated according to methods known in the art, using prediction from hydrophobicity charts, as described in the “Anti-NOVX Antibodies” section below. For example the disclosed NOV5 protein have multiple hydrophilic regions, each of which can be used as an immunogen. This novel protein also has value in development of powerful assay system for functional analysis of various human disorders, which will help in understanding of pathology of the disease and development of new drug targets for various disorders.
NOV6
A disclosed NOV6 nucleic acid of 3509 nucleotides (also referred to as CG57362-01) encoding a novel BCSC-1-like protein is shown in Table 6A. An open reading frame was identified beginning with an ATG initiation codon at nucleotides 155-157 and ending with a TGA codon at nucleotides 3206-3208. Putative untranslated regions upstream from the intitation codon and downstream from the termination codon are underlined in Table 6A, and the start and stop codons are in bold letters.







TABLE 6A





NOV6 Nucleotide Sequence















(SEQ ID NO:17)







GGTCCGAGCGGGGCTCTGTGACGACAGCCCCACCAGCTGCTTCGGGGTGGGCAGCCTTCAGGAGGAAGGGCTG


GCCTGGGAGGAGCTGGCTGCCCCTCGGGACGTGTTCTCAGGCCCTGCCCGCTGCCCTGCCCCATATACCTTCT


CCTTCGAGATGCTGGTGACTGGGCCATGCCTGCTTGCAGGCCTGGAGAGCCCCTCTCATGCTCTGCGGGCAGA


TGCCCCCCCTCATGCCAGCTCTGCAGCCACCATCTGTGTCACACTGGCAGAGGGCCACCACTGTGACCGGGCC


TTGGAGATCCTGCTGCACCCCAGTGAGCCCCATCAGCCACACCTGATGCTGGAGGGCGGCAGCCTGAGCTCAG


CAGAATATGAGGCCCGGGTGAGGGCCCGCCGAGATTTTCAGAGGCTACAGCGAGGGGACAGTGATGGGGACCG


GCAGGTGTGGTTCCTGCAGCGACGCTTCCACAAGGACATCCTGCTGAACCCCGTGCTGGCGCTGAGCTTCTGC


CCAGACCTGAGCTCCAAGCCCGGACACCTGGGGACAGCTACTCGGGAGCTACTCTTCCTTTTGGATAGCAGCA


GCGTGGCACACAAGGATGCCATTGTTTTGGCTGTGAAGTCCCTCCCACCCCAGACGCTTATCAACCTGGCCGT


GTTTGGGACGTTGGTGCAGCCACTCTTCCCAGAGAGCCGGCCTTGCAGTGATGATGCTGTGCAGCTGATCTGC


GAGAGCATTGAGACCCTGCAGGTTCCGAGTGGGCCCCCAGACGTGCTGGCTGCTCTGGACTGGGCCGTGGGGC


AGCCCCAGCACAGGGCCTACCCTCGGCAGCTGTTCCTGCTCACTGCTGCCTCACCCATGGCCGCCACTACCCA


CCGAACCCTGGAGCTCATGAGGTGGCACAGGGGGACAGCCAGGTGCTTCTCCTTTGGGCTGGGGCCCACCTGC


CACCAGCTGCTCCAGGGTTTATCTGCCCTCAGCAGCCGAGGTCGCGCCACTGCACTCCAGCCTGGCCGACAGA


GCAAGCCACAGCTGGTACAGGCTCTGCGGAAGGCACTGGAGCCTGCTTTGAGTGACATCTCTGTGGACTGGTT


TGTGCCCGACACTGTGGAGGCACTGCTGACCCCTCGGGAGATCCCAGCACTCTACCCTGGGGACCAGCTGCTC


GGTTACTGCTCACTCTTCAGGGTGGATGGCTTCCGGTCCCGCCCACCAGGGGGCCAAGAGCCTGGCTGGCAGA


GCTCGGGTGGGTCCGTGTTTCCATCCCCAGAAGAGGCCCCGTCTGCTGCCAGCCCTGGCACTGAGCCCACTGG


CACCTCAGAGCCACTGGGAACAGGCACTGTCTCAGCAGAACTGTCCAGCCCATGGGCTGCCAGGGACTCGGAG


CAGAGTGGTACTGATGCTCTGACAGACCCAGTCACGGATCCTGGACCCAACCCCTCTGACACAGCCATATGGC


GCCGCATCTTTCAGTCCTCGTACATTCGGGAGCAGTATGTGCTCACCCACTGCTCTGCCAGCCCCGAGCCAGG


CCCAGGCTCCACAGGCAGCAGTGAGTCCCCAGGCTCACAGGGCCCTGGCTCCCCCGAAGGTAGTGCTCCCTTG


CAGCCCCCTTCTCAGCAGGGCTGCCGCAGTCTGGCCTGGGGAGAACCTGCAGGCTCCCGCTCCTGTCCCCTGC


CTGCACCCACACCAGCTCCATTCAAGGTGGGGGCCTTGAGTACTGAGGTGCTGGGCCGTCAGCACAGAGCGGC


TCTGGCTGGCCGAAGCCTCTCATCCCCTCCAGGCCGGGCAAACCAAGTCCCCGGCCGACCCCGGAAACCCTCT


TTGGGTGCAATACTAGATGGCCCAAGTCCTGAGCCAGGCCAACAGTTGGGACAAGGCCTGGATGACTCAGGTA


ACCTGCTCTCCCCAGCCCCTATGGACTGGGACATGCTGATGGAACCACCCTTCTTATTCACGGCTGTGCCTCC


TAGTGGGGAGTTGGCCCCTCCAGCAGTGCCTCCCCAGGCTCCACGCTGCCATGTGGTGATCCGGGGCCTGTGT


GGGGAGCAGCCCATGTGCTGGGAGGTGGGTGTTGGGCTGGAGACACTGTGGGGACCTGGAGATGGCTCACAGC


CTCCCTCACCTCCTGTAAGAGAAGCTGCTTGGGACCAAGCACTCCATCGGCTGACAGCAGCCTCTGTGGTCCG


GGACAATGAGCAGCTGGCCCTCCGAGGAGGGGCAGAGACCACAGCTGACCGGGGCCATGCCCGGAGGTGCTGG


CTTCGAGCCCTTCAAACAAGTAAGGTCAGCTCTGCCCCCTCCTGCTTCACTTGCCCTGTAGCTGTGGATGCTA


CTACTAGGGAGGTCCTGCCTGGGGCCCTGCAGGTGTGCAGCTCAGAGCCCGCTGAGCCCCCAGGAACCCCTCC


TGCCTCTCACAGCCATCTAGATGCAGCTCCTCTGCCCACTGTTGTCTACTCTAAAGGTGCCTGGGACTCGGAC


CAAAATGGCAACTCCAAGCGTGCTTTGGGGGACCCTGCCACTCCCACGGAAGGTCCTCGCCGCCCACCTCCCC


GTCCTCCCTGTCGGCTCAGCATGGGCCGCCGTCACAAACTCTGTAGCCCTGACCCGGGCCAGGCCAACAACAG


TGAAGGCAGCGACCATGACTACCTGCCCTTGGTGCGGCTGCAGGAGGCACCAGGCTCCTTCCGCCTGGACGCG


CCCTTCTGCGCCGCTGTGCGCATCTCGCAGGAGCGCCTCTGCCGTGCCTCGCCCTTTGCCGTGCACCGCGCCA


GCCTCAGCCCCACCTCGGCCTCATTGCCCTGGGCACTTCTGGGCCCTGGTGTTGGCCAGGGTGACAGTGCCAC


GGCCTCCTGCAGCCCGTCCCCCAGCTCGGGCTCTGAGGGGCCAGGCCAGGTGGACAGTGGGCGGGGCTCAGAC


ACCGAGGCCTCCGAGGGGGCGGAAGGGCTGGGCGGCACCGACCTGCGGGGCCGGACCTGGGCCACTGCCGTAG


CACTCGCCTGGCTGGAGCACCGATGCGCCGCTGCCTTCGACGAGTGGGAACTGACAGCGGCCAAGGCTGATTG


CTGGCTGCGGGCCCAGCACTTGCCTGACGGCCTTGACCTGGCCGCCCTCAAGGCCGCAGCCCGAGGGCTCTTC


CTGCTACTGCGCCACTGGGACCAAAACCTGCAGCTACACCTGCTGTGCTACAGCCCAGCGAACGTGTGAAGGC


TGCCCCCTGCTGCTTGGGCTGGCGCCCCACCCAACACACTCAAGTCACTGCCGCCCAGGGCTGGCCTCTTGGT


GCTGGGAAAGTGTAGGCTGGTTCCAGCCTGTCCCCCACTGCTTCTTACTCCCTCCCTAGAGCCCTCTTGCCCC


CACAAAAAGTGCCTGCCTGTGCTCTCTCCCTCTCCTCCCACCCCACTCACACTCCCCTCCATCCTTTGAGCTC


CCTGCAACACAGTGGAAGGGTAGAGAGCCACAGTCCCCAAATCCTATGCAATAAAGTGCCTCTTAGGGAAAAA


AAAAA









The NOV6 nucleic acid was identified on chromosome 3 and has 2669 of 2799 bases (95%) identical to a gb:GENBANK-ID:AB047829|acc:AB047829.1 mRNA from Macaca fascicularis (Macaca fascicularis brain cDNA, clone:QccE-10361) (E=0.0).
A disclosed NOV6 polypeptide (SEQ ID NO:18) encoded by SEQ ID NO:17 is 1017 amino acid residues and is presented using the one-letter code in Table 6B. Signal P, Psort and/or Hydropathy results predict that NOV6 contains a signal peptide and is likely to be localized to the nucleus with a certainty of 0.6000. The most likely cleavage site is between amino acids 18 and 19: SHA-LR.







TABLE 6B





Encoded NOV6 protein sequence















(SEQ ID NO:18)







MLVTGPCLLAGLESPSHALRADAPPHASSAATICVTLAEGHHCDRALEILLHPSEPHQPHLMLEGGSLSSAE


YEARVRARRDFQRLQRGDSDGDRQVWFLQRRFHKDILLNPVLALSFCPDLSSKPGHLGTATRELLFLLDSSS


VAHKDAIVLAVKSLPPQTLINLAVFGTLVQPLFPESRPCSDDAVQLICESIETLQVPSGPPDVLAALDWAVG


QPQHRAYPRQLFLLTAASPMAATTHRTLELMRWHRGTARCFSFGLGPTCHQLLQGLSALSSRGRATALQPGR


QSKPQLVQALRKALEPALSDISVDWFVPDTVEALLTPREIPALYPGDQLLGYCSLFRVDGFRSRPPGGQEPG


WQSSGGSVFPSPEEAPSAASPGTEPTGTSEPLGTGTVSAELSSPWAARDSEQSGTDALTDPVTDPGPNPSDT


AIWRRIFQSSYIREQYVLTHCSASPEPGPGSTGSSESPGSQGPGSPEGSAPLEPPSQQGCRSLAWGEPAGSR


SCPLPAPTPAPFKVGALSTEVLGRQHRAALAGRSLSSPPGRANQVPGRPRKPSLGAILDGPSPEPGQQLGQG


LDDSGNLLSPAPMDWDMLMEPPFLFTAVPPSGELAPPAVPPQAPRCHVVIRGLCGEQPMCWEVGVGLETLWG


PGDGSQPPSPPVREAAWDQALHRLTAASVVRDNEQLALRGGAETTADRGHARRCWLRALQTSKVSSAPSCFT


CPVAVDATTREVLPGALQVCSSEPAEPPGTPPASHSHLDAAPLPTVVYSKGAWDSDQNGNSKRALGDPATPT


EGPRRPPPRPPCRLSMGRRHKLCSPDPGQANNSEGSDHDYLPLVRLQEAPGSFRLDAPFCAAVRISQERLCR


ASPFAVHRASLSPTSASLPWALLGPGVGQGDSATASCSPSPSSGSEGPGQVDSGRGSDTEASEGAEGLGGTD


LRGRTWATAVALAWLEHRCAAAFDEWELTAAKADCWLRAQHLPDGLDLAALKAAARGLFLLLRHWDQNLQLH


LLCYSPANV









The NOV6 amino acid sequence has 767 of 803 amino acid residues (95%) identical to, and 779 of 803 amino acid residues (97%) similar to, the 803 amino acid residue ptnr:TREMBLNEW-ACC:BAB12255 protein from Macaca fascicularis (Crab eating macaque) (Cynomolgus monkey) (Hypothetical 84.7 Kda Protein) (E=0.0).
NOV6 is expressed in at least the following tissues: lung. This information was derived by determining the tissue sources of the sequences that were included in the invention including but not limited to SeqCalling sources, Public EST sources, genomic clone sources, literature sources, and/or RACE sources. The NOV6 sequence is also predicted to be expressed in the following tissues because of the expression pattern of (GENBANK-ID: gb:GENBANK-ID:AB047829|acc:AB047829.1) a closely related Macaca fascicularis brain cDNA, clone:QccE-10361 homolog in species Macaca fascicularis: brain.
Possible small nucleotide polymorphisms (SNPs) found for NOV6 are listed in Table 6C.







TABLE 6C







SNPs
















Amino





Nucleotide
Base
Acid
Base



Variant
Position
Change
Position
Change







13377063
3196
A > G
Silent
N/A










NOV6 has homology to the amino acid sequences shown in the BLASTP data listed in Table 6D.







TABLE 6D







BLAST results for NOV6












Gene Index/

Length
Identity
Positives



Identifier
Protein/Organism
(aa)
(%)
(%)
Expect















gi|12654717|gb|AAH01200.1|
Unknown (protein for
1001
826/972
831/972
0.0


AAH01200
IMAGE: 3356192)

(84%)
(84%)


(BC001200)
[Homo sapiens]


gi|9967105|dbj|BAB12255.1|
hypothetical
803
646/804
657/804
0.0


(AB047829)
protein [Macaca

(80%)
(81%)



fascicularis]


gi|15295425|ref|XP_
similar to
620
494/611
494/611
0.0


029078.2|
Unknown (protein for

(80%)
(80%)


(XM_029078)
IMAGE: 3356192)



[Homo sapiens]


gi|11231113|dbj|BAB18151.1|
hypothetical
260
192/247
194/247
5e−97


(AB051125)
protein [Macaca

(77%)
(77%)



fascicularis]


gi|12855146|dbj|BAB30227.1|
data source: SPTR,
1209
142/346
229/346
2e−72


(AK016425)
source key: O75668,

(41%)
(66%)



evidence: ISS ̃homolog



to DJ745E8.1



(BREAST CANCER



SUPPRESSOR



CANDIDATE 1



(BCSC-1) LIKE)



(FRAGMENT)  ̃putative



[Mus musculus









The homology of these sequences is shown graphically in the ClustalW analysis shown in Table 6E.







TABLE 6E





Clustal W Sequence Sequence Alignment















1) NOV6 (SEQ ID NO:18)


2) gi|12654717|gb|AAH01200.1|AAH01200 (BC001200) Unknown (protein for IMAGE:3356192)


[Homo sapiens] (SEQ ID NO:125)


3) gi|9967105|dbj|BAB12255.1| (AB047829) hypothetical protein [Macaca fascicularis]


(SEQ ID NO:126)


4) gi|15295425|refXP_029078.2| (XM_029078) similar to Unknown (protein for


IMAGE:3356192) [Homo sapiens](SEQ ID NO:127)


5) gi|1231113|dbj|BAB18151.1| (AB051125) hypothetical protein [Macaca fascicularis]


(SEQ ID NO:128)


6) gi|12855146|dbj|BAB30227.1| (AK016425) data source:SPTR, source key:O75668,


evidence:ISS ̃homolog to DJ745E8.1 (BREAST CANCER SUPRESSOR CANDIDATE 1 (BCSC-1) LIKE)


(FRAGMENT) ̃putative [Mus musculus] (SEQ ID NO:129)














































































































































Table 6F list the domain description from DOMAIN analysis results against NOV6. This indicates that the NOV6 sequence has properties similar to those of other proteins known to contain these domains.







TABLE 6F





Domain Analysis of NOV6















gnl|Smart|smart00327, VWA, von Willebrand factor (vWF) type A domain;


VWA domains in extracellular eukaryotic proteins mediate adhesion via


metal ion-dependent adhesion sites (MIDAS). Intracellular VWA domains


and homologues in prokaryotes have recently been identified. The


proposed VWA domains in integrin beta subunits have recently been


substantiated using sequence-based methods (Ponting et al. Adv Prot


Chem (2000) in press). (SEQ ID NO:130)


CD-Length = 180 residues, 97.2% aligned


Score = 42.0 bits (97), Expect = 2e−04










Query:
134
RELLFLLDSS-SVAH------KDAIVLAVKSL---PPQTLINLAVFGTLVQPLFPESRPC
183




 +++|||| |  |+        |+ ++  |+ |    |    + |  | +  + ||| +


Sbjct:
2
LDVVFLLDGSGSMGGNRFELAKEFVLKLVEQLDIGPDGDRVGLVTFSSDARVLFPLNDSQ
 61


Query:
184
SDDAVQLICESIETLQV-PSGPPDVLAALDWAVGQPQH------RAYPRQLFLLTAASPM
236




| ||+    |++ +|      |  ++ |||++|+            |  |+ | |+|


Sbjct:
62
SKDALL---EALASLSYSLGGGTNLGAALEYALENLFSESAGSRRGAPKVLILITDGESN
118


Query:
237
AATTHRTLELMRWHRGTARCFSFGLGPTCHQLLQGLSALSSRGRATALQPGRQSKPQLVQ
296




              |   + |  |+|       + |  |+|       +     |   |+


Sbjct:
119
DGGEDILKAAKELKRSGVKVFVVGVGN--DVDEEELKKLASAPGGVFVVEDLPSLLDLLI
176









NOV6 described herein bears resemblance to BCSC-1 a molecule that is a candidate for suppression of breast cancer and this molecule may play a similar role in the lung and brain.
The NOV6 nucleic acid of the invention encoding a BCSC-1-like protein includes the nucleic acid whose sequence is provided in Table 6A, or a fragment thereof. The invention also includes a mutant or variant nucleic acid any of whose bases may be changed from the corresponding base shown in Table 6A while still encoding a protein that maintains its BCSC-1-like activities and physiological functions, or a fragment of such a nucleic acid. The invention further includes nucleic acids whose sequences are complementary to those just described, including nucleic acid fragments that are complementary to any of the nucleic acids just described. The invention additionally includes nucleic acids or nucleic acid fragments, or complements thereto, whose structures include chemical modifications. Such modifications include, by way of non-limiting example, modified bases, and nucleic acids whose sugar phosphate backbones are modified or derivatized. These modifications are carried out at least in part to enhance the chemical stability of the modified nucleic acid, such that they may be used, for example, as antisense binding nucleic acids in therapeutic applications in a subject. In the mutant or variant nucleic acids, and their complements, up to about 5% of the residues may be so changed.
The NOV6 protein of the invention includes the BCSC-1-like protein whose sequence is provided in Table 6B. The invention also includes a mutant or variant protein any of whose residues may be changed from the corresponding residue shown in Table 6B while still encoding a protein that maintains its BCSC-1-like activities and physiological functions, or a functional fragment thereof. In the mutant or variant protein, up to about 5% of the bases may be so changed.
The NOV6 nucleic acids and proteins of the invention are useful in potential diagnostic and therapeutic applications implicated in various diseases and disorders described below and/or other pathologies. For example, the compositions of the present invention will have efficacy for treatment of patients suffering from: cancer, trauma, regeneration (in vitro and in vivo), viral/bacterial/parasitic infections, systemic lupus erythematosus, autoimmune disease, asthma, emphysema, scleroderma, allergy, ARDS and other diseases, disorders and conditions of the like.
NOV6 nucleic acids and polypeptides are further useful in the generation of antibodies that bind immunospecifically to the novel substances of the invention for use in therapeutic or diagnostic methods. These antibodies may be generated according to methods known in the art, using prediction from hydrophobicity charts, as described in the “Anti-NOVX Antibodies” section below. For example the disclosed NOV6 protein have multiple hydrophilic regions, each of which can be used as an immunogen. This novel protein also has value in development of powerful assay system for functional analysis of various human disorders, which will help in understanding of pathology of the disease and development of new drug targets for various disorders.
NOV7
NOV7 includes three novel Amino Acid Transporter-like proteins disclosed below. The disclosed proteins have been named NOV7a, NOV7b and NOV7c.
NOV7a
A disclosed NOV7a nucleic acid of 1513 nucleotides (also referred to as CG57387-01) encoding a novel Amino Acid Transporter-like protein is shown in Table 7A. An open reading frame was identified beginning with an ATG initiation codon at nucleotides 2-4 and ending with a TAG codon at nucleotides 1502-1504. Putative untranslated regions, if any, upstream from the initiation codon and downstream from the termination codon are underlined in Table 7A, and the start and stop codons are in bold letters.







TABLE 7A





NOV7a nucleotide sequence.















(SEQ ID NO:19)







TATGGAGCTAAAGGCTCCAGCTGCAGGAGGTCTTAATGCTGGCCCTGTCCCCCCAGCTGCCATGTCCACGCA


GAGACTTCGGAATGAAGACTACCACGACTACAGCTCCACGGACGTGAGCCCTGAGGAGAGCCCGTCGGAAGG


CCTCAACAACCTCTCCTCCCCGGGCTCCTACCAGCGCTTTGGTCAAAGCAATAGCACAACGTGGTTCCAGAC


CTTGATCCACCTGTTAAAAGGCAACATTGGCACAGGACTCCTGGGACTCCCTCTGGCGGTGAAAAATGCAGG


CATCGTGATGGGTCCCATCAGCCTGCTGATCATAGGCATCGTGGCCGTGCACTGCATGGGTATCCTGGTGAA


ATGTGCTCACCACTTCTGCCGCAGGCTGAATAAATCCTTTGTGGATTATGGTGATACTGTGATGTATGGACT


AGAATCCAGCCCCTGCTCCTGGCTCCGGAACCACGCACACTGGGGAAGACGTGTTGTGGACTTCTTCCTGAT


TGTCACCCAGCTGGGATTCTGCTGTGTCTATTTTGTGTTTCTGGCTGACAACTTTAAACAGGTGATAGAAGC


GGCCAATGGGACCACCAATAACTGCCACAACAATGAGACGGTGATTCTGACGCCTACCATGGACTCGCGACT


CTACATGCTCTCCTTCCTGCCCTTCCTGGTGCTGCTGGTTTTCATCAGGAACCTCCGAGCCCTGTCCATCTT


CTCCCTGTTGGCCAACATCACCATGCTGGTCAGCTTGGTCATGATCTACCAGTTCATTGTTTTCAGGTACAT


GCTTTCTGTCTTTCAGAGGATCCCAGACCCCAGCCACCTCCCCTTGGTGGCCCCTTGGAAGACCTACCCTCT


CTTCTTTGGCACAGCGATTTTTTCATTTGAAGGCATTGGAATGGTACTGCCCCTGGAAAACAAAATGAAGGA


TCCTCGGAAGTTCCCACTCATCCTGTACCTGGGCATGGTCATCGTCACCATCCTCTACATCAGCCTGGGGTG


TCTGGGGTACCTGCAATTTGGAGCTAATATCCAAGGCAGCATAACCCTCAACCTGCCCTTGTACCAGTCAGT


TAAGCTGCTGTACTCCATCGGGATCTTTTTCACCTACGCACTCCAGTTCTACGTCCCGGCTGAGATCATCAT


CCCCTTCTTTGTGTCCCGAGCGCCCGAGCACTGTGAGTTAGTGGTGGACCTGTTTGTGCGCACAGTGCTGGT


CTGCCTGACAATCTTGGCCATCCTCATCCCCCGCCTGGACCTGGTCATCTCCCTGGTGGGCTCCGTGAGCAG


CAGCGCCCTGGCCCTCATCATCCCACCGCTCCTGGAGGTCACCACCTTCTACTCAGAGGGCATGAGCCCCCT


CACCATCTTTAAGGACGCCCTGATCAGCATCCTGGGCTTCGTGGGCTTTGTGGTGGGGACCTATGAGGCTCT


CTATGAGCTGATCCAGCCAAGCAATGCTCCCATCTTCATCAATTCCACCTGTGCCTTCATATAGGGATCTGG


G









The disclosed NOV7a nucleic acid sequence, localized to chromsome 5, has 94 of 104 bases (90%) identical to a gb:GENBANK-ID:HSU42412|acc:U42412.1 mRNA from Homo sapiens (Human 5′-AMP-activated protein kinase, gamma-1 subunit mRNA, complete cds) (E=2.4e−08).
A NOV7a polypeptide (SEQ ID NO:20) encoded by SEQ ID NO:19 has 500 amino acid residues and is presented using the one-letter code in Table 7B. Signal P, Psort and/or Hydropathy results predict that NOV7a does not contain a signal peptide and is likely to be localized to the plasma membrane with a certainty of 0.6000.







TABLE 7B 





Encoded NOV7a protein sequence.















(SEQ ID NO:20)







MELKAPAAGGLNAGPVPPAAMSTQRLRNEDYHDYSSTDVSPEESPSEGLNNLSSPGSYQRFGQSNSTTWFQT


LIHLLKGNIGTGLLGLPLAVKNAGIVMGPISLLIIGIVAVHCMGILVKCAHHFCRRLNKSFVDYGDTVMYGL


ESSPCSWLRNHAHWGRRVVDFFLIVTQLGFCCVYFVFLADNFKQVIEAANGTTNNCHNNETVILTPTMDSRL


YMLSFLPFLVLLVFIRNLRALSIFSLLANITMLVSLVMIYQFIVFRYMLSVFQRIPDPSHLPLVAPWKTYPL


FFGTAIFSFEGIGMVLPLENKMKDPRKFPLILYLGMVIVTILYISLGCLGYLQFGANIQGSITLNLPLYQSV


KLLYSIGIFFTYALQFYVPAEIIIPFFVSRAPEHCELVVDLFVRTVLVCLTILAILIPRLDLVISLVGSVSS


SALALIIPPLLEVTTFYSEGMSPLTIFKDALISILGFVGFVVGTYEALYELIQPSNAPIFINSTCAFI









The NOV7a amino acid sequence has 127 of 276 amino acid residues (46%) identical to, and 175 of 276 amino acid residues (63%) similar to, the 486 amino acid residue ptnr:SPTREMBL-ACC:Q9VLM4 protein from Drosophila melanogaster (Fruit fly) (CG13384 PROTEIN)) (E=9.3e−84).
The disclosed NOV7a is expressed in at least the following tissues: Adrenal Gland/Suprarenal gland, Cerebral Medulla/Cerebral white matter, Ovary and Synovium. This information was derived by determining the tissue sources of the sequences that were included in the invention including but not limited to SeqCalling sources, Public EST sources, Literature sources, and/or RACE sources.
NOV7b
A disclosed NOV7b nucleic acid of 1501 nucleotides (also referred to as CG57387-02) encoding a novel Amino Acid Transporter-like protein is shown in Table 7C. An open reading frame was identified beginning with an ATG initiation codon at nucleotides 2-4 and ending with a TAG codon at nucleotides 1490-1492. Putative untranslated regions, if any, upstream from the initiation codon and downstream from the termination codon are underlined in Table 7C, and the start and stop codons are in bold letters.







TABLE 7C





NOV7b nucleotide sequence.















(SEQ ID NO:21)







TATGGAGCTAAAGGCTCCAGCTGCAGGAGGTCTTAATGCTGGCCCTGTCCCCCCAGCTGCCATGTCCACGCA


GAGACTTCGGAATGAAGACTACCACGACTACAGCTCCACGGACGTGAGCCCTGAGGAGAGCCCGTCGGAAGG


CCTCAACAACCTCTCCTCCCCGGGCTCCTACCAGCGCTTTGGTCAAAGCAATAGCACAACATGGTTCCAGAC


CTTGATCCACCTGTTAAAAGGCAACATTGGCACAGGACTGCTGGGACTCCCTCTGGCGGTGAAAAATGCAGG


CATCGTGATGGGTCCCATCAGCCTGCTGATCATAGGCATCGTGGCCGTGCACTGCATGGGTATCCTGGTGAA


ATGTGCTCACCACTTCTGCCGCAGGCTGAATAAATCCTTTGTGGATTATGGTGATACTGTGATGTATGGACT


AGAATCCAGCCCCTGCTCCTGGCTCCGGAACCACGCACACTGGGGAAGACGTGTTGTGGACTTCTTCCTGAT


TGTCACCCAGCTGGTATTCTGCTGTGTCTATTTTGTGTTTCTGGCTGACAACTTTAAACAGGTGATAGAAGC


GGCCAATGGGACCACCAATAACTGCCACAACAATGAGACGGTGATTCTGACGCCTACCATGGACTCGCGACT


CTACATGCTCTCCTTCCTGCCCTTCCTGGTGCTGCTGGTTTTCATCAGGAACCTCCGAGCCCTGTCCATCTT


CTCCCTGTTGGCCAACATCACCATGCTGGTCAGCTTGGTCATGATCTACCAGTTCATTGTTCAGAGGATCCC


AGACCCCAGCCACCTCCCCTTGGTGGCCCCTTGGAAGACCTACCCTCTCTTCTTTGGCACAGCGATTTTTTC


ATTTGAAGGCATTGGAATGGTTCTGCCCCTGGAAAACAAAATGAAGGATCCTCGGAAGTTCCCACTCATCCT


GTACCTGGGCATGGTCATCGTCACCATCCTCTACATCAGCCTGGGGTGTCTGGGGTACCTGCAATTTGGAGC


TAATATCCAAGGCAGCATAACCCTCAACCTGCCCAACTGCTGGTTGTACCAGTCAGTTAAGCTGCTGTACTC


CATCGGGATCTTTTTCACCTACGCACTCCAGTTCTACGTCCCCGCTGAGATCATCATCCCCTTCTTTGTGTC


CCGAGCGCCCGAGCACTGTGAGTTAGTGGTGGACCTGTTTGTGCGCACAGTGCTGGTCTGCCTGACATGCAT


CTTGGCCATCCTCATCCCCCGCCTGGACCTGGTCATCTCCCTGGTGGGCTCCGTGAGCAGCAGCGCCCTGGC


CCTCATCATCCCACCGCTCCTGGAGGTCACCACCTTCTACTCAGAGGGCATGAGCCCCCTCACCATCTTTAA


GGACGCCCTGATCAGCATCCTGGGCTTCGTGGGCTTTGTGGTGGGGACCTATGAGGCTCTCTATGAGCTGAT


CCAGCCAAGCAATGCTCCCATCTTCATCAATTCCACCTGTGCCTTCATATAGGGATCTGGG









The disclosed NOV7b nucleic acid sequence, localized to chromsome 5, has 1364 of 1428 bases (95%) identical to a gb:GENBANK-ID:AX049362|acc:AX049362.1 mRNA from Homo sapiens (Sequence 32 from Patent WO0071709) (E=1.4e−290).
A NOV7b polypeptide (SEQ ID NO:22) encoded by SEQ ID NO:21 has 496 amino acid residues and is presented using the one-letter code in Table 7D. Signal P, Psort and/or Hydropathy results predict that NOV7b does not contain a signal peptide and is likely to be localized to the plasma membrane with a certainty of 0.6000.







TABLE 7D





Encoded NOV7b protein sequence.















(SEQ ID NO:22)







MELKAPAAGGLNAGPVPPAAMSTQRLRNEDYHDYSSTDVSPEESPSEGLNNLSSPGSYQRFGQSNSTTWFQT


LIHLLKGNIGTGLLGLPLAVKNAGIVMGPISLLIIGIVAVHCMGILVKCAHHFCRRLNKSFVDYGDTVMYGL


ESSPCSWLRNHAHWGRRVVDFFLIVTQLVFCCVYFVFLADNFKQVIEAANGTTNNCHNNETVILTPTMDSRL


YMLSFLPFLVLLVFIRNLRALSIFSLLANITMLVSLVMIYQFIVQRIPDPSHLPLVAPWKTYPLFFGTAIFS


FEGIGMVLPLENKMKDPRKFPLILYLGMVIVTILYISLGCLGYLQFGANIQGSITLNLPNCWLYQSVKLLYS


IGIFFTYALQFYVPAEIIIPFFVSRAPEHCELVVDLFVRTVLVCLTCILAILIPRLDLVISLVGSVSSSALA


LIIPPLLEVTTFYSEGMSPLTIFKDALISILGFVGFVVGTYEALYELIQPSNAPIFINSTCAFI









The NOV7b amino acid sequence has 407 of 476 amino acid residues (85%) identical to, and 443 of 476 amino acid residues (93%) similar to, the 475 amino acid residue ptnr:TREMBLNEW-ACC:AAK67316 protein from Rattus norvegicus (Rat) (Lysosomal Amino Acid Transporter 1) (E=3.1e−221).
The disclosed NOV7b is expressed in at least the following tissues: Adrenal Gland/Suprarenal gland, Cerebral Medulla/Cerebral white matter, Ovary and Synovium. This information was derived by determining the tissue sources of the sequences that were included in the invention including but not limited to SeqCalling sources, Public EST sources, Literature sources, and/or RACE sources.
NOV7c
A disclosed NOV7c nucleic acid of 953 nucleotides (also referred to as CG57387-03) encoding a novel Amino Acid Transporter-like protein is shown in Table 7E. An open reading frame was identified beginning with an ATG initiation codon at nucleotides 2-4 and ending with a TGA codon at nucleotides 791-793. Putative untranslated regions, if any, upstream from the initiation codon and downstream from the termination codon are underlined in Table 7E, and the start and stop codons are in bold letters.







TABLE 7E





NOV7c nucleotide sequence.















(SEQ ID NO:23)







TATGGAGCTAAAGGCTCCAGCTGCAGGAGGTCTTAATGCTGGCCCTGTCCCCCCAGCTGCCATGTCCACGCA


GAGACTTCGGAATGAAGACTACCACGACTACAGCTCCACGGACGTGAGCCCTGAGGAGAGCCCGTCGGAAGG


CCTCAACAACCTCTCCTCCCCGGGCTCCTACCAGCGCTTTGGTCAAAGCAATAGCACAACATGGTTCCAGAC


CTTGATCCACCTGTTAAAAGGCAACATTGGCCACAGGACTGCTGGGACTCCCTCTGGCGGTGAAAATGCAGG


CATCGTGATGGGTCCCATCAGCCTGCTGATCATAGGCATCGTGGCCGTGCACTGCATGGGTATCCTGGTGAA


ATGTGCTCACCACTTCTGCCGCAGGCTGAATAAATCCTTTGTGGATTATGGTGATACTGTGATGTATGGACT


AGAATCCAGCCCCTGCTCCTGGCTCCGGAACCACGCACACTGGGGAAGACGTGTTGTGGACTTCTTCCTGAT


TGTCACCCAGCTGGGATTCTGCTGTGTCTATTTTGTGTTTCTGGCTGACAACTTTAAACAGGTGATAGAAGC


GGCCAATGGGACCACCAATAACTGCCACAACAATGAGACGGTGATTCTGACGCCTACCATGGACTCGCGACT


CTACATGCTCTCCTTCCTGCCCTTCCTGGTGCTGCTGGTTTTCATCAGGAACCTCCGAGCCCTGTCCATCTT


CTCCCTGTTGGCCAACATCACCATGCTCGTCAGCTTGGTCATGATCTACCAGTTCATTGTTCAGATCCTGTG


AATGGATTTACAGCCCATGTAGCAGACCAAGGTCTTCCACAGAGAGCAGGTTCCTCTCTGTCTTCAGCATGT


GGAGTCTCAAATGGAACAGTTCTGGGCAGAGTGCTTTGCACAGAGGGTGCTCCCAATAAATGTTTTATCACT


GCAAAAAAAAAAAAAAA









The disclosed NOV7c nucleic acid sequence, localized to chromsome 5, has 723 of 724 bases (99%) identical to a gb:GENBANK-ID:AX049362|acc:AX049362.1 mRNA from Homo sapiens (Sequence 32 from Patent WO0071709) (E=3.5e−157).
A NOV7c polypeptide (SEQ ID NO:24) encoded by SEQ ID NO:23 has 263 amino acid residues and is presented using the one-letter code in Table 7F. Signal P, Psort and/or Hydropathy results predict that NOV7c does not contain a signal peptide and is likely to be localized to the plasma membrane with a certainty of 0.6000.







TABLE 7F





Encoded NOV7c protein sequence.















(SEQ ID NO:24)







MELKAPAAGGLNAGPVPPAAMSTQRLRNEDYHDYSSTDVSPEESPSEGLNNLSSPGSYQRFGQSNSTTWFQT


LIHLLKGNIGTGLLGLPLAVKNAGIVMGPISLLIIGIVAVHCMGILVKCAHHFCRRLNKSFVDYGDTVMYGL


ESSPCSWLRNHAHWGRRVVDFFLIVTQLGFCCVYFVFLADNFKQVIEAANGTTNNCHNNETVILTPTMDSRL


YMLSFLPFLVLLVFIRNLRALSIFSLLANITMLVSLVMIYQFIVQIL









The NOV7c amino acid sequence has 209 of 241 amino acid residues (86%) identical to, and 228 of 241 amino acid residues (94%) similar to, the 475 amino acid residue ptnr:TREMBLNEW-ACC:AAK67316 protein from Rattus norvegicus (Rat) (Lysosomal Amino Acid Transporter 1) (E=6.8e−114).
The disclosed NOV7c is expressed in at least the following tissues: Adrenal Gland/Suprarenal gland, Cerebral Medulla/Cerebral white matter, Ovary and Synovium. This information was derived by determining the tissue sources of the sequences that were included in the invention including but not limited to SeqCalling sources, Public EST sources, Literature sources, and/or RACE sources.
Possible SNPs found for NOV7a are listed in Table 7G.







TABLE 7G







SNPs
















Amino





Nucleotide
Base
Acid
Base



Variant
Position
Change
Position
Change

















13377064
256
C > G
Silent
N/A



13377065
519
G > T
173
Gly > Val



13377066
1153
C > T
Silent
N/A










NOV7a, NOV7b and NOV7c are very closely homologous as is shown in the amino acid alignment in Table 7H.







TABLE 7H





Amino Acid Alignment of NOV7a, NOV7b and NOV7c














































































Homologies to any of the above NOV7 proteins will be shared by the other NOV7 proteins insofar as they are homologous to each other as shown above. Any reference to NOV7 is assumed to refer to the NOV7 proteins in general, unless otherwise noted.
NOV7a has homology to the amino acid sequences shown in the BLASTP data listed in Table 7I.







TABLE 7I







BLAST results for NOV7a












Gene Index/
Protein/
Length
Identity
Positives



Identifier
Organism
(aa)
(%)
(%)
Expect















gi|4826770|ref|NP_004960.1|
Amino Acid
1019
974/1019
984/1019
0.0


(NM_004969)
Transporter;

(95%)
(95%)



insulinase



[Homo



sapiens]


gi|6981076|ref|NP_037291.1|
insulin
1019
934/1019
965/1019
0.0


(NM_013159)
degrading

(91%)
(94%)



enzyme



[Rattus



norvegicus]


gi|13621162|ref|NP_
insulin
1019
929/1019
963/1019
0.0


112419.1|
degrading

(91%)
(94%)


(NM_031156)
enzyme [Mus



musculus]


gi|18576366|ref|XP_
Amino Acid
554
530/554
539/554
0.0


051153.2|
Transporter

(95%)
(96%)


(XM_051153)
[Homo



sapiens]


gi|7296294|gb|AAF51584.1|
Ide gene
990
439/966
634/966
0.0


(AE003591)
product

(45%)
(65%)



[Drosophila



melanogaster]









The homology of these sequences is shown graphically in the ClustalW analysis shown in Table 7J.







TABLE 7J





ClustalW Analysis of NOV7a















1) NOV7a (SEQ ID NO:20)


2) gi|4826770|ref|NP_004960.1| (NM_004969) Amino Acid Transporter; insulinase


[Homo sapiens] (SEQ ID NO:131)


2) gi|6981076|ref|NP_037291.1| (NM_013159) insulin degrading enzyme


[Rattus norvegicus] (SEQ ID NO:132)


3) gi|13621162|ref|NP_112419.1| (NM_031156) insulin degrading enzyme [Mus musculus]


(SEQ ID NO:133)


4) gi|18576366|ref|XP_051153.2| (XM_051153) Amino Acid Transporter [Homo sapiens]


(SEQ ID NO:134)


5) gi|7296294|gb|AAF51584.1| (AE003591) Ide gene product [Drosophila melanogaster]


(SEQ ID NO:135)

































































Table 7K lists the domain description from DOMAIN analysis results against NOV7a. This indicates that the NOV7a sequence has properties similar to those of other proteins known to contain these domains.







TABLE 7K





Domain Analysis of NOV7a















gnl|Pfam|pfam01490, Aa_trans, Transmembrane amino acid transporter


protein. This transmembrane region is found in many amino acid


transporters including UNC-47 and MTR. UNC-47 encodes a vesicular


amino butyric acid (GABA) transporter, (VGAT). UNC-47 is predicted to


have 10 transmembrane domains. MTR is a N system amino acid


transporter system protein involved in methyltryptophan resistance.


Other members of this family include proline transporters and amino


acid permeases. (SEQ ID NO:136)


CD-Length = 370 residues, 85.1% aligned


Score = 119 bits (299), Expect = 3e−28










Query:
91
AVKNAGIVMGPISLLIIGIVAVHCMGILVKCAHHFCRRLNKSFVDYGDTVMYGLESSPCS
150




| |  | + | + || + | + ++   +| +|  +    + | |++| | +   |      |


Sbjct:
1
AFKQLGWIPGLVLLLLAGFITLYTGLLLSECYEYVPGKRNDSYLDLGRSAYGGKGLLLTS
 60


Query:
151
WLRNHAHWGRRVVDFFLIVTQLGFCCVYFVFLADNFKQVIEAANGTTNNCHNNETVILTP
210




++                |   |    | +   |   ++|       ++ | +|


Sbjct:
61
FVG-------------QYVNLFGVNIGYLILAGDLLPKII------SSFCGDNCD-----
 96


Query:
211
TMDSRLYMLSFLPFLVLLVFIRNLRALSI--FSLLANITMLVSLVMIYQFIVFRYMLSVF
268




 +|   +++ |    ++ | || |   |||   |  +++  |   +  +    |   +  +


Sbjct:
97
HLDGNSWIIIFAAIIITLSFIPNFNLLSISSLSAFSSLAYLSIISFLIIVAVIAGIFVLL
156


Query:
269
QRIPDPSHLPLVAPWKTYPLFFGTAIFSFEGIGMVLPLENKNKDPR--KFPLILYLGMVI
326




  +      |         |  |  +|+|||   ++||++| || |    ||  +| + ++|


Sbjct:
157
GAVYGILWSPSFTKLTGLFLAIGIIVFAFEGHAVLLPIQNTMKSPSAKKFKKVLNVAIII
216


Query:
327
VTILYISLGCLGYLQFGANIQGSITLNLP---LYQSVKLLYSIGIFFTYALQFYVPAEII
383




||+||| +|   ||| || |++|+| ||||    +  | |   + |  |+  || +    ||


Sbjct:
217
VTVLYILVGFFGYLTFGNNVKGNILLNLPNNPFWLIVNLNLVVAILLTFPLQAFPIVRII
276


Query:
384
IPFFV---SRAPEHCELVVDLFVRTVLVCLTILAILIPR
419




        + ||   +| +  +    ++|    ++|||+|


Sbjct:
277
ENLLTKKNNFAPNKSKLLRVVIRSGLVVFTLLIAILVPF
315









NOV7 has significant homology in the transmembrane region found in many amino acid transporters including UNC-47 and MTR. UNC-47 encodes a vesicular amino butyric acid(GABA) transporter, (VGAT). UNC-47 is predicted to have 10 transmembrane domains. MTR is a N system amino acid transporter system protein involved in methyltryptophan resistance. Other members of this family include proline transporters and amino acid permeases. Amino acid transporters may play an important role in a number of diseases as exemplified below.
Because most cancer deaths result from disseminated disease, understanding the regulation of tumor invasion and metastasis is a central theme in tumor cell biology. Interactions between extracellular matrices (ECM) and cellular microenvironment play a crucial role in this process. Selected amino acids and polyamines have been tested for their ability to regulate RL95-2 cell invasion through both intact human amniotic basement membrane and a novel human ECM (Amgel). Three major systems for neutral amino acid transport, systems L, A, and ASC, are operational in these neoplastic cells. Amino acids entering the cell via transport system A or N, i.e., (methyl amino)-isobutyrate (MeAIB) or Asn, markedly enhanced invasiveness of these human adenocarcinoma cells as measured by a standard 72-hr amnion or Amgel invasion assay. Addition of 2-amino-2-norborane carboxylic acid (BCH; 1 mM), a model substrate of the L transport system, caused a significant decrease in invasive activity when tested in the Amgel assay. Interestingly, Val lowers steady-state levels of MeAIB uptake and blocks the increase in cell invasion elicited by MeAIB. At the same time, these amino acids do not influence cell proliferation activity. Neither the charged amino acid Lys or Asp (not transported by A/N/L systems) nor the polyamines putrescine, spermidine, or spermine modulate invasiveness under similar experimental conditions. Moreover, the observed time-dependent stimulation of system A activity (cellular influx of MeAIB) by substrate depletion is prevented by the addition of actinomycin D (5 microM) or cycloheximide (100 microM), suggesting the involvement of de novo RNA and protein synthesis events in these processes. MeAIB treatment of tumor cells selectively increased the activities of key invasion-associated type IV collagenases/gelatinases. These results indicate that in the absence of defined regulators (growth factors or hormones), certain amino acids may contribute to the epigenetic control of human tumor cell invasion and, by extension, metastasis. This study suggests that amino acids, acting via specific signaling pathways, modulate phenotypic cell behavior by modulating the levels of key regulatory enzymatic proteins (Singh et al., Cancer Invest 14(1):6-18, 1996).
To investigate the function of a basic and neutral amino acid transporter-like protein (rBAT) which is a candidate gene for cystinuria, the rBAT gene was analysed in cystinuric patients. Patient 1 is a compound heterozygote with mutations in the rBAT gene causing a glutamine-to-lysine transition at amino acid 268, and a threonine-to-alanine transition at amino acid 341, who inherited these alleles from his mother (E268K) and father (T341A), respectively. Injection of T341A and E268K mutant cRNAs into oocytes decreased transport activity to 53.9% and 62.5% of control (L-cystine transport activity in oocytes injected with wild-type rBAT cRNA), respectively. Co-injection of E268K and T341A into oocytes strongly decreased amino acid transport activity to 28% of control. On the other hand, co-injection of wild-type and mutant rBAT did not decrease transport activity. Furthermore, immunological studies have demonstrated that the reduction of amino acid transport is not due to a decrease in the amount of rBAT protein expressed in oocyte membranes. These results indicate that mutations in the rBAT gene are crucial disease-causing lesions in cystinuria. In addition, co-injection experiments suggest that rBAT may function as a transport activator orregulatory subunit by homo- or hetero-multimer complex formation (Miyamoto et al., Biochem J 310 (Pt 3):951-5, 1995).
The spinal cord of 20 patients with amyotrophic lateral sclerosis (ALS) and 5 patients with lower motor neuron disease (LMND) were investigated immunohistochemically using anti-human excitatory amino acid transporter 1 (EAAT1) and EAAT2 antibodies which are the astrocytic transporters. The purpose of the study was to examine relationships between EAAT1 and EAAT2 immunoreactivity and degeneration of anterior horn neurons. Specimens from 20 patients without any neurological disease served as controls. In controls, spinal cord gray matter was densely immunostained by antibodies, whereas the white matter was generally not immunostained. In motor neuron disease (MND) patients, EAAT1 immunoreactivity was relatively well preserved in the gray matter despite neuronal loss of anterior horn cells. On the other hand, EAAT2 immunoreactivity in anterior horns correlated with the degree of neuronal loss of anterior horn cells: in the patients with mild neuronal depletion, anterior horns were densely immunostained by the antibody, whereas in the patients with severe neuronal loss, EAAT2 expression was markedly reduced. Degenerated anterior horn cells frequently showed a much denser EAAT1 and EAAT2 immunoreactivity around the surface of the neurons and their neuronal processes than that observed in normal-appearing neurons. There was no difference in the expression of EAAT1 and EAAT2 immunoreactivity between LMND and ALS patients. These findings suggest that in the early stage of degeneration of anterior horn cells, EAAT1 and EAAT2 immunoreactivity is preserved in the astrocytic foot directly attached to normal-appearing neurons, whereas levels of EAAT1 and EAAT2 protein rather increase in the astrocytic foot directly attached to degenerated anterior horn neurons; the latter effect most probably reduces the elevated glutamate level, compensates for the reduced function of astroglial glutamate transporters, or represents a condensation of EAAT1 and EAAT2 immunoreactivity secondary to loss of neurites and greater condensation of astrocytic processes. Thus, the study demonstrates a difference in EAAT1 and EAAT2 immunoreactivity in different stages of progression in ALS, as a feature of the pathomechanism of this disease (Sasaki et al., Acta Neuropathol (Berl) 100(2):138-44, 2000).
The NOV7 nucleic acid of the invention encoding a Amino Acid Transporter-like protein includes the nucleic acid whose sequence is provided in Tables 7A, 7C and 7E, or a fragment thereof. The invention also includes a mutant or variant nucleic acid any of whose bases may be changed from the corresponding base shown in Tables 7A, 7C and 7E while still encoding a protein that maintains its Amino Acid Transporter-like activities and physiological functions, or a fragment of such a nucleic acid. The invention further includes nucleic acids whose sequences are complementary to those just described, including nucleic acid fragments that are complementary to any of the nucleic acids just described. The invention additionally includes nucleic acids or nucleic acid fragments, or complements thereto, whose structures include chemical modifications. Such modifications include, by way of non-limiting example, modified bases, and nucleic acids whose sugar phosphate backbones are modified or derivatized. These modifications are carried out at least in part to enhance the chemical stability of the modified nucleic acid, such that they may be used, for example, as antisense binding nucleic acids in therapeutic applications in a subject. In the mutant or variant nucleic acids, and their complements, up to about 10% of the NOV7a residues, about 5% of the NOV7b residues and about 1% of the NOV7c residues may be so changed.
The NOV7 protein of the invention includes the Amino Acid Transporter-like protein whose sequence is provided in Tables 7B, 7D and 7F. The invention also includes a mutant or variant protein any of whose residues may be changed from the corresponding residue shown in Tables 7B, 7D and 7F while still encoding a protein that maintains its Amino Acid Transporter-like activities and physiological functions, or a functional fragment thereof. In the mutant or variant protein, up to about 54% of the NOV7a bases, about 15% of the NOV7b bases and about 14% of the NOV7c bases may be so changed.
The NOV7 nucleic acids and proteins of the invention are useful in potential diagnostic and therapeutic applications implicated in various diseases and disorders described below and/or other pathologies. For example, the compositions of the present invention will have efficacy for treatment of patients suffering from: cancer, trauma, regeneration (in vitro and in vivo), viral/bacterial/parasitic infections, Von Hippel-Lindau (VHL) syndrome, Alzheimer's disease, stroke, tuberous sclerosis, hypercalceimia, Parkinson's disease, Huntington's disease, cerebral palsy, epilepsy, Lesch-Nyhan syndrome, multiple sclerosis, ataxia-telangiectasia, leukodystrophies, behavioral disorders, addiction, anxiety, pain, neurodegeneration, fertility, adrenoleukodystrophy, congenital adrenal hyperplasia and other diseases, disorders and conditions of the like.
NOV7 nucleic acids and polypeptides are further useful in the generation of antibodies that bind immunospecifically to the novel substances of the invention for use in therapeutic or diagnostic methods. These antibodies may be generated according to methods known in the art, using prediction from hydrophobicity charts, as described in the “Anti-NOVX Antibodies” section below. For example the disclosed NOV7 protein have multiple hydrophilic regions, each of which can be used as an immunogen. This novel protein also has value in development of powerful assay system for functional analysis of various human disorders, which will help in understanding of pathology of the disease and development of new drug targets for various disorders.
NOV8
NOV8 includes five novel Lymphocyte antigen precursor-like or Lymphocyte antigen LY-6F-like proteins disclosed below. The disclosed proteins have been named NOV8a-NOV8e.
NOV8a
A disclosed NOV8a nucleic acid of 468 nucleotides (also referred to as CG56417-01) encoding a novel Lymphocyte antigen precursor-like protein is shown in Table 8A. An open reading frame was identified beginning with an ATG initiation codon at nucleotides 31-33 and ending with a TGA codon at nucleotides 439-441. Putative untranslated regions, if any, upstream from the initiation codon and downstream from the termination codon are underlined in Table 8A, and the start and stop codons are in bold letters.







TABLE 8A





NOV8a nucleotide sequence.















(SEQ ID NO:25)







TGAAGTTTGTCTGTGCACTAGCACCCTGGAATGAGCAGTCTCCAGGCCATGAAGACCTTGTCCCTGGTCCTG


CTGGTGGCCCTGCTGAGCATGGAGAGAGCTCAGGGTCTGCGCTGCTACAGATGCTTGGCGGTCTTGGAAGGG


GCCTCCTGCAGCGTGGTCTCGTGCCCCTTCCTGGATGGGGTCTGTGTCTCCCAGAAAGTGAGCTTAAGTCTG


AGCAAGAAAAGAAGAAAAGAAAAAAACAAGCTCTCCCTCCTCTCCTGCCAGAAGGACGTCGGATTCCCCCTG


CTGAAACTTACAAGTGCCGTTGTGGACTCCCAGATCTCTTGCTGCAAGGGAGACCTCTGCAATGCGGTGGTC


CTGGCAGCCAGCAGCCCCTGGGCCCTGTGCGTACAGCTCCTGCTCAGCCTGGGGTCAGTCTTCCTCTGGGCC


CTGCTGTGAGGGCCCTTTCCCGCCCTCTCCCCCGCA









A NOV8a polypeptide (SEQ ID NO:26) encoded by SEQ ID NO:25 has 136 amino acid residues and is presented using the one-letter code in Table 8B.







TABLE 8B





Encoded NOV8a protein sequence.















(SEQ ID NO:26)







MSSLQAMKTLSLVLLVALLSMERAQGLRCYRCLAVLEGASCSVVSCPFLDGVCVSQKVSLSLSKKRRKEKNK


LSLLSCQKDVGFPLLKLTSAVVDSQISCCKGDLCNAVVLAASSPWALCVQLLLSLGSVFLWALL









NOV8b

A disclosed NOV8b nucleic acid of 610 nucleotides (also referred to as CG56417-02) encoding a novel Lymphocyte antigen precursor-like protein is shown in Table 8C. An open reading frame was identified beginning with an ATG initiation codon at nucleotides 47-49 and ending with a TAG codon at nucleotides 515-517. Putative untranslated regions, if any, upstream from the initiation codon and downstream from the termination codon are underlined in Table 8C, and the start and stop codons are in bold letters.







TABLE 8C





NOV8b nucleotide sequence.















(SEQ ID NO:27)







CTTGTAAGGGCGAGACAGCAGAGACTGGCACCAGGGAGGCTCCTCCATGGGAGTCTTCCATGATTACTCACA


GCGGGTGGGCAGAGGTGTTGCTAGGAAGCATGTTCTGGGGGGGTCCTCTGGGTGCACACGTGCAGTAGCTGC


ACCTGCTTGCTCATACGTCGCATGTGTCATTAGCATCTTAAATCTCCACCAGGGGTGTGTTTTCTTGCCCTC


TCTCCCAGCTCAGGGTCTGCGCTGCTACAGATGCTTGGCGGTCTTGGAAGGGGCCTCCTGCAGCGTGGTCTC


GTGCCCCTTCCTGGATGGGGTCTGTGTCTCCCAGAAAGTGAGCGTTTTGGCAGTGAGTCCCTGGGGTGCCAG


GGCAGAGGGCAGGTTAAGTGCCGTTGTGGACTCCCAGATCTCTTGCTGCAAGGGAGACCTCTGCAATGCGGT


GGTCCTGGCAGCCGGCAGCCCCTGGGCCCTGTGCGTACAGCTCCTGCTCAGCCTGGGGTCAGTCTTCCTCTG


GGCCCTGCTGTGAGGGCCTTTCCCGCCCTCTCCCCCGCAGGCCTACCCTCTGTCCCTGTGCGTCACCAGCTG


CTTGGTTTTGAAGAGCTGCCTCACTGAATTGCAG









The disclosed NOV8b nucleic acid sequence, localized to chromsome 8, has 192 of 249 bases (77%) identical to a gb:GENBANK-ID:HSU85047|acc:U85047.1 mRNA from Homo sapiens (Human FRA3B region corresponding to the breakpoint junction in lung tumor cell line NCIH211) (E=1.1e−22).
A NOV8b polypeptide (SEQ ID NO:28) encoded by SEQ ID NO:27 has 156 amino acid residues and is presented using the one-letter code in Table 8D. Signal P, Psort and/or Hydropathy results predict that NOV8b does not contain a signal peptide and is likely to be localized to the plasma membrane with a certainty of 0.6000.







TABLE 8D





Encoded NOV8b protein sequence.















(SEQ ID NO:28)







MGVFHDYSQRVGRGVARKHVLGGSSGCTRAVAAPACSYVACVISILNLHQGCVFLPSLPAQGLRCYRCLAVL


EGASCSVVSCPFLDGVCVSQKVSVLAVSPWGARAEGRLSAVVDSQISCCKGDLCNAVVLAAGSPWALCVQLL


LSLGSVFLWALL









The NOV8b amino acid sequence has 53 of 126 amino acid residues (42%) identical to, and 69 of 126 amino acid residues (54%) similar to, the 134 amino acid residue ptnr:SWISSNEW-ACC:P35460 protein from Mus musculus (Mouse) (Lymphocyte Antigen LY-6F.1 Precursor) (E=2.3e−16).
The disclosed NOV8b is expressed in at least the following tissues: Adrenal Gland/Suprarenal gland, Brain, Kidney, Lung, Pituitary Gland, Placenta, Trachea and Whole Organism. This information was derived by determining the tissue sources of the sequences that were included in the invention including but not limited to SeqCalling sources, Public EST sources, Literature sources, and/or RACE sources.
NOV8c
A disclosed NOV8c nucleic acid of 406 nucleotides (also referred to as CG56417-03) encoding a novel Lymphocyte antigen precursor-like protein is shown in Table 8E. An open reading frame was identified beginning with an ATG initiation codon at nucleotides 2-4 and ending with a TGA codon at nucleotides 404-406. Putative untranslated regions, if any, upstream from the initiation codon and downstream from the termination codon are underlined in Table 8E, and the start and stop codons are in bold letters.







TABLE 8E





NOV8c nucleotide sequence.















(SEQ ID NO:29)







AATGAGCAGTCTCCAGGCCATGAAGACCTTGTCCCTGGTCCTGCTGGTGGCCCTGCTGAGCATGGAGAGAGC


TCAGGGTCTGCGCTGCTACAGATGCTTGGCGGTCTTGGAAGGGGCCTCCTGCAGCGTGGTCTCGTGCCCCTT


CCTGGATGGGGTCTGTCTCTCCCAGAAAGTGAGCGTCTTTGGCAGTAAAGTGAGAGGGGAGAACAAGCTCTC


CCTCCTCTCCTGCCAGAAGGACGTCGGATTCCCCCTGCTGAAACTTACAAGTGCCGTTGTGGACTCCCAGAT


CTCTTGCTGCAAGGGAGACCTCTGCAATGCGGTGGTCCTGGCAGCCGGCAGCCCCCGGGCCCTGTGCGTACA


GCTCCTGCTCAGCCTGGGGTCAGTCTTCCTCTGGGCCCTGCTGTGA









The disclosed NOV8c nucleic acid sequence, localized to chromsome 8, has 260 of 403 bases (64%) identical to a gb:GENBANK-ID:RATLY6CA|acc:M30691.1 mRNA from Rattus norvegicus (Rat Ly6-C antigen mRNA, exon 2) (E=1.2e−16).
A NOV8c polypeptide (SEQ ID NO:30) encoded by SEQ ID NO:29 has 134 amino acid residues and is presented using the one-letter code in Table 8F. Signal P, Psort and/or Hydropathy results predict that NOV8c contains a signal peptide and is likely to be localized to the plasma membrane with a certainty of 0.9190. The most likely cleavage site for a NOV8c polypeptide is between amino acids 26 and 27: AQG-LR







TABLE 8F





Encoded NOV8c protein sequence.















(SEQ ID NO:30)







MSSLQAMKTLSLVLLVALLSMERAQGLRCYRCLAVLEGASCSVVSCPFLDGVCVSQKVSVFGSKVRGENKLS


LLSCQKDVGFPLLKLTSAVVDSQISCCKGDLCNAVVLAAGSPRALCVQLLLSLGSVFLWALL









The NOV8c amino acid sequence has 61 of 134 amino acid residues (45%) identical to, and 79 of 134 amino acid residues (58%) similar to, the 134 amino acid residue ptnr:SPTREMBL-ACC:Q63318 protein from Rattus norvegicus (Rat) (RAT LY6-C ANTIGEN) (E=4.4e−22).
NOV8d
A disclosed NOV8d nucleic acid of 611 nucleotides (also referred to as CG56417-04) encoding a novel Lymphocyte antigen LY-6F-like protein is shown in Table 8G. An open reading frame was identified beginning with an ATG initiation codon at nucleotides 47-49 and ending with a TGA codon at nucleotides 602-604. Putative untranslated regions, if any, upstream from the initiation codon and downstream from the termination codon are underlined in Table 8G, and the start and stop codons are in bold letters.







TABLE 8G





NOV8d nucleotide sequence.















(SEQ ID NO:31)







CTTGTAAGGGCGAGACAGCAGAGACTGGCACCAGGGAGGCTCCTCCATGGGAGTCTTCCATGATTACTCACA


GCGGGTGGGCAGAGGTGTTGCTAGGAAGCATGTTCTGGGGGGGTCCTCTGGGTGCACACGTGCAGTAGCTGC


ACCTGCTTGCTCATACGTCGCATGTGTCATTAGCATCTTAAATCTCCACCAGGGGTGTGTTTTCTTGCCCTC


TCTCCCAGCTCAGGGTCTGCGCTGCTACAGATGCTTGGCGGTCTTGGAAGGGGCCTCCTGCAGCGGGGTCTC


GTGCCCCTTCCTGGATGGGGTCTGTGTCTCCCAGAAAGTGAGCGTCTTTGGCAGTGAGTCCCTGGGGTGCCA


GGGCAGAGGGCAGGTTAAGTGCCGTTGTGGACTCCCAGATCTCTTGCTGCAAGGGAGACCTCTGCAATGCGG


TGGTCCTGGCAGCCGGCAGCCCCTGGGCCCTGTGCGTACAGCTCCTGCTCAGCCTGGGGTCAGTCTTCCTCT


GGGCCCTGCTGTGAGGGCCTTTCCCGCCCTCTCCCCCGCAGGCCTACCCTCTGTCCCTGTGCGTCACCAGCT


GCTTGGTTTTGAAGAGCTGCCTCACTGAATTGCAG









The disclosed NOV8d nucleic acid sequence, localized to chromsome 8, has 198 of 256 bases (77%) identical to a gb:GENBANK-ID:AP000509|acc:AP000509.1 mRNA from Homo sapiens (Homo sapiens genomic DNA, chromosome 6p21.3, HLA Class I region, section 8/20) (E=5.4e−23).
A NOV8d polypeptide (SEQ ID NO:32) encoded by SEQ ID NO:31 has 185 amino acid residues and is presented using the one-letter code in Table 8H. Signal P, Psort and/or Hydropathy results predict that NOV8d does not contain a signal peptide and is likely to be localized to the plasma membrane with a certainty of 0.7900.







TABLE 8H





Encoded NOV8d protein sequence.















(SEQ ID NO:32)







MGVFHDYSQRVGRGVARKHVLGGSSGCTRAVAAPACSYVACVISILNLHQGCVFLPSLPAQGLRCYRCLAVL


EGASCSVVSCPFLDGVCVSQKVSVFGSESLGCQGRGQVKCRCGLPDLLLQGRPLQCGGPGSRQPLGPVRTAP


AQPGVSLPLGPAVRAFPALSPAGLPSVPVRHQLLGFEELPH









The NOV8d amino acid sequence has 24 of 57 amino acid residues (42%) identical to, and 35 of 57 amino acid residues (61%) similar to, the 134 amino acid residue ptnr:SWISSNEW-ACC:P35460 protein from Mus musculus (Mouse) (Lymphocyte Antigen LY-6F. 1 Precursor) (E=2.4e−07).
The disclosed NOV8d is expressed in at least the following tissues: Brain, Pituitary Gland, Placenta, Lung, Trachea, Kidney, Colon and Whole Organism. This information was derived by determining the tissue sources of the sequences that were included in the invention including but not limited to SeqCalling sources, Public EST sources, Literature sources, and/or RACE sources.
NOV8e
A disclosed NOV8e nucleic acid assembly (also referred to as 172885384) of NOV8d (CG56417-04) encoding a novel Lymphocyte antigen LY-6F-like protein is shown in Table 8I. The cDNA coding for the mature form of the full length NOV8d from residue 49 to 183 was targeted for “in-frame” cloning by PCR. The insert 172885384 was found to encode an open reading frame between residues 49 and 183 of the target sequence of NOV8d.







TABLE 8I





NOV8e nucleotide sequence.















(SEQ ID NO:33)







GGATCCCACCAGGGGTGTGTTTTCTTGCCCTCTCTCCCAGCTCAGGGTCTGCGCTGCTACAGATGCTTGGCG


GTCTTGGAAGGGGCCTCCTGCAGCGTGGTCTCGTGCCCCTTCCTGGATGGGGTCTGTGTCTCCCAGAAAGTG


AGCGTCTTTGGCAGTGAGTCCCTGGGGTGCCAGGGCAGAGGGCAGGTTAAGTGCCGTTGTGGACTCCCAGAT


CTCTTGCTGCAAGGGAGACCTCTGCAATGCGGTCGTCCTGGCACCCGGCAGCCCCTGGGCCCTGTGCGTACA


GCTCCTGCTCAGCCTGGGGTCAGTCTTCCTCTGGGCCCTGCTGTGAGGGCCTTTCCCGCCCTCTCCCCCGCA


GGCCTACCCTCTGTCCCTGTGCGTCACCAGCTGCTTGGTTTTGAAGAGCTGCCTCACCTCGAG









A NOV8e polypeptide (SEQ ID NO:34) encoded by SEQ ID NO:33 has 136 amino acid residues and is presented using the one-letter code in Table 8J.







TABLE 8J





Encoded NOV8e protein sequence.















(SEQ ID NO:34)







GSHQGCVFLPSLPAQGLRCYRCLAVLEGASCSVVSCPFLDGVCVSQKVSVFGSESLGCQGRGQVKCRCGLPD


LLLQGRPLQCGGPGSRQPLGPVRTAPAQPGVSLPLGPAVRAFPALSPAGLPSVPVRHQLLGFEELPHLE









Possible SNPs found for NOV8a are listed in Table 8K and possible SNPs for NOV8d are listed in Table 8L.







TABLE 8K







SNPs
















Amino





Nucleotide
Base
Acid
Base



Variant
Position
Change
Position
Change

















13377070
53
A > G
8
Lys > Arg



13377074
161
T > C
44
Val > Ala



13377075
166
T > C
46
Cys > Arg



13377076
194
C > A
55
Ser > Tyr



13377077
196
C > T
56
Gln > End



13377078
300
A > G
Silent
N/A



13377068
362
T > C
111
Leu > Pro



13377079
370
A > G
114
Ser > Gly



13377067
371
G > A
114
Ser > Asn



13377080
431
C > T
134
Ala > Val



13377081
468
A > G
Silent
N/A

















TABLE 8L







SNPs










Consensus

Base



Position
Depth
Change
PAF













61
31
G > —
0.065


79
32
A > G
0.062


89
33
A > G
0.061


105
48
G > A
0.042


166
51
C > T
0.157


174
51
A > G
0.039


192
51
A > C
0.039


207
51
C > T
0.039


236
51
T > C
0.059


237
51
A > G
0.039


265
52
T > A
0.038


294
49
G > T
0.327


322
49
A > G
0.041


327
49
A > G
0.061


360
48
A > C
0.042


373
48
A > G
0.042


419
47
G > A
0.043


448
47
G > A
0.064









NOV8a-NOV8e are very closely homologous as is shown in the amino acid alignment in Table 8M.







TABLE 8M





Amino Acid Alignment of NOV8a-NOV8e











































Homologies to any of the above NOV8 proteins will be shared by the other NOV8 proteins insofar as they are homologous to each other as shown above. Any reference to NOV8 is assumed to refer to the NOV8 proteins in general, unless otherwise noted.
NOV8a has homology to the amino acid sequences shown in the BLASTP data listed in Table 8N.







TABLE 8N







BLAST results for NOV8a












Gene Index/
Protein/
Length
Identity
Positives



Identifier
Organism
(aa)
(%)
(%)
Expect





gi|17505223|ref|NP_
lysosomal
434
405/483
409/483
0.0


510968.1|
amino acid

(83%)
(83%)


(NM_078483)
transporter 1



[Homo sapiens]


gi|18426842|ref|NP_
lysosomal
475
371/484
406/484
0.0


569099.1|
amino acid

(76%)
(83%)


(NM_130415)
transporter 1



[Rattus norvegicus]


gi|17473038|ref|XP_
similar to
504
231/514
307/514
e−101


058449.1|
lysosomal

(44%)
(58%)


(XM_058449)
amino acid



transporter 1



[Homo sapiens]


gi|17449820|ref|XP_
similar to
165
147/162
147/162
6e−76


059717.1|
lysosomal

(90%)
(90%)


(XM_059717)
amino acid



transporter 1



(H. sapiens)



[Homo sapiens]


gi|18467570|ref|XP_
CG13384 gene
504
169/433
234/433
9e−69


079250.1|
product

(39%)
(54%)


(XM_079250)
[Drosophila melanogaster]









The homology of these sequences is shown graphically in the ClustalW analysis shown in Table 8O.







TABLE 8O





ClustalW Analysis of NOV8a















1) NOV8a (SEQ ID NO:26)


2) gi|17505223|refNP_510968.1| (NM_078483) lysosomal amino acid transporter 1


[Homo sapiens] (SEQ ID NO:137)


2) gi|18426842|refNP_569099.1| (NM_130415) lysosomal amino acid transporter 1


[Rattus norvegicus] (SEQ ID NO:138)


3) gi|17473038|refXP_058449.1| (XM_058449) similar to lysosomal amino acid


transporter 1 [Homo sapiens] (SEQ ID NO:139)


4) gi|17449820|refXP_059717.1| (XM_059717) similar to lysosomal amino acid


transporter 1 (H. sapiens) [Homo sapiens] (SEQ ID NO:140)


5) gi|18467570|refXP_079250.1| (XM_079250) CG13384 gene product


[Drosophila melanogaster] (SEQ ID NO:141)

































































Table 8P lists the domain description from DOMAIN analysis results against NOV8a. This indicates that the NOV8a sequence has properties similar to those of other proteins known to contain these domains.







TABLE 8P





Domain Analysis of NOV8a















gnl|Pfam|pfam01490, Aa_trans, Transmembrane Lymphocyte antigen


precursor protein. This transmembrane region is found in many


Lymphocyte antigen precursors including UNC-47 and MTR. UNC-47 encodes


a vesicular amino butyric acid (GABA) transporter, (VGAT). UNC-47 is


predicted to have 10 transmembrane domains. MTR is a N system


Lymphocyte antigen precursor system protein involved in


methyltryptophan resistance. Other members of this family include


proline transporters and amino acid permeases. (SEQ ID NO:142)


CD-Length = 370 residues, 85.1% aligned


Score = 119 bits (299), Expect = 3e−28










Query:
91
AVKNAGIVMGPISLLIIGIVAVHCMGILVKCAHHFCRRLNKSFVDYGDTVMYGLESSPCS
150




| |  | + | + || + | + ++   +| +|   +   + | |++| | +   |      |


Sbjct:
1
AFKQLGWIPGLVLLLLAGFITLYTGLLLSECYEYVPGKRNDSYLDLGRSAYGGKGLLLTS
 60


Query:
151
WLRNHAHWGRRVVDFFLIVTQLGFCCVYFVFLADNFKQVIEAANGTTNNCHNNETVILTP
210




++                |   |    | +   |    ++|      ++ | +|


Sbjct:
61
FVG-------------QYVNLFGVNIGYLILAGDLLPKII------SSFCGDNCD-----
 96


Query:
211
TMDSRLYMLSFLPFLVLLVFIRNLRALSI--FSLLANITMLVSLVMIYQFIVFRYMLSVF
268




 +|   +++ |    ++ | || |   |||   |  +++   |  +  +    |   +  +


Sbjct:
97
HLDGNSWIIIFAAIIITLSFIPNFNLLSISSLSAFSSLAYLSIISFLIIVAVIAGIFVLL
156


Query:
269
QRIPDPSHLPLVAPWKTYPLFFGTAIFSFEGIGMVLPLENKMKDPR--KFPLILYLGMVI
326




  +      |         |  |  +|+|||   ++||++| || |   ||  +|  + ++|


Sbjct:
157
GAVYGILWSPSFTKLTGLFLAIGIIVFAFEGHAVLLPIQNTMKSPSAKKFKKVLNVAIII
216


Query:
327
VTILYISLGCLGYLQFGANIQGSITLNLP---LYQSVKLLYSIGIFFTYALQFYVPAEII
383




||+||| +|   ||| || |++|+| ||||    +  | |   + |  |+ || +    ||


Sbjct:
217
VTVLYILVGFFGYLTFGNNVKGNILLNLPNNPFWLIVNLNLVVAILLTFPLQAFPIVRII
276


Query:
384
IPFFV---SRAPEHCELVVDLFVRTVLVCLTILAILIPR
419




        + ||   +|+  +     ++|    ++|||+|


Sbjct:
277
ENLLTKKNNFAPNKSKLLRVVIRSGLVVFTLLIAILVPF
315









Murine Ly-6 molecules are a family of cell surface glycoproteins which have interesting patterns of tissue expression during haematopoiesis from multipotential stem cells to lineage committed precursor cells, and on specific leucocyte subpopulations in the peripheral lymphoid tissues. These interesting patterns of tissue expression suggest an intimate association between the regulation of Ly-6 expression and the development and homeostasis of the immune system. Ly-6 molecules are low molecular weight phosphatidyl inositol anchored glycoproteins with remarkable amino acid homology throughout a distinctive cysteine rich protein domain that is associated predominantly with O-linked carbohydrate. The in vivo functions of Ly-6 molecules are not known although in vitro studies suggest a role in cellular activation (Gumley et al., Immunol Cell Biol.;73(4):277-96, 1995).
The Ly-6 alloantigens have been shown to play a critical role in T lymphocyte activation. LeClair et al. (LeClair et al., EMBO J. 5(12):3227-34, 1986) isolated a Ly-6 cDNA, synthetic oligonucleotides, based on the partial amino acid sequence of purified Ly-6E.1 protein, were used to probe a cDNA library. The synthetic oligonucleotides or the isolated cDNA detected a 1.1-kb RNA species. Sequence analysis of the cDNA clone revealed that the Ly-6E.1 protein consists of a 26-amino acid leader followed by a 108-residue, cysteine-rich, core protein with no N-linked glycosylation sites. Southern blot analysis of genomic DNAs revealed multiple bands indicating a family of related genes. Using recombinant inbred and Ly-6 congenic strains of mice, restriction fragment length polymorphisms were demonstrable, and correlated with the Ly-6 allotype of the DNA donors. This probe will enable further molecular genetic analysis of the role of Ly-6-linked proteins in the process of T lymphocyte activation. Isolation of Ly-6 genomic clones may promote a further understanding of the complex tissue-specific expression patterns characteristic of Ly-6-linked genes (LeClair et al., 1986).
NOV8 will have similar properties as other lymphocyte antigen precursor and Ly-6 proteins and may be involved in the regulation of T lymphocyte activation.
The NOV8 nucleic acid of the invention encoding a Lymphocyte antigen precursor-like protein includes the nucleic acid whose sequence is provided in Tables 8A, 8C, 8E, 8G and 8I or a fragment thereof. The invention also includes a mutant or variant nucleic acid any of whose bases may be changed from the corresponding base shown in Tables 8A, 8C, 8E, 8G and 8I while still encoding a protein that maintains its Lymphocyte antigen precursor-like activities and physiological functions, or a fragment of such a nucleic acid. The invention further includes nucleic acids whose sequences are complementary to those just described, including nucleic acid fragments that are complementary to any of the nucleic acids just described. The invention additionally includes nucleic acids or nucleic acid fragments, or complements thereto, whose structures include chemical modifications. Such modifications include, by way of non-limiting example, modified bases, and nucleic acids whose sugar phosphate backbones are modified or derivatized. These modifications are carried out at least in part to enhance the chemical stability of the modified nucleic acid, such that they may be used, for example, as antisense binding nucleic acids in therapeutic applications in a subject. In the mutant or variant nucleic acids, and their complements, up to about 23% of the NOV8b residues, about 36% of the NOV8c residues and about 23% of the NOV8d residues may be so changed.
The NOV8 protein of the invention includes the Lymphocyte antigen precursor-like protein whose sequence is provided in Tables 8B, 8D, 8F, 8H and 8J. The invention also includes a mutant or variant protein any of whose residues may be changed from the corresponding residue shown in Tables 8B, 8D, 8F, 8H and 8J while still encoding a protein that maintains its Lymphocyte antigen precursor-like activities and physiological functions, or a functional fragment thereof. In the mutant or variant protein, up to about 58% of the NOV8b bases, about 55% of the NOV8c bases and about 58% of the NOV8d bases may be so changed.
The NOV8 nucleic acids and proteins of the invention are useful in potential diagnostic and therapeutic applications implicated in various diseases and disorders described below and/or other pathologies. For example, the compositions of the present invention will have efficacy for treatment of patients suffering from: adrenoleukodystrophy, congenital adrenal hyperplasia, Von Hippel-Lindau (VHL) syndrome, Alzheimer's disease, stroke, tuberous sclerosis, hypercalceimia, Parkinson's disease, Huntington's disease, cerebral palsy, epilepsy, Lesch-Nyhan syndrome, multiple sclerosis, ataxia-telangiectasia, leukodystrophies, behavioral disorders, addiction, anxiety, pain, neurodegeneration, diabetes, autoimmune disease, renal artery stenosis, interstitial nephritis, glomerulonephritis, polycystic kidney disease, systemic lupus erythematosus, renal tubular acidosis, IgA nephropathy, hypercalceimia, Lesch-Nyhan syndrome, growth and reproductive disorders, systemic lupus erythematosus, autoimmune disease, asthma, emphysema, scleroderma, allergy, ARDS and other diseases, disorders and conditions of the like.
NOV8 nucleic acids and polypeptides are further useful in the generation of antibodies that bind immunospecifically to the novel substances of the invention for use in therapeutic or diagnostic methods. These antibodies may be generated according to methods known in the art, using prediction from hydrophobicity charts, as described in the “Anti-NOVX Antibodies” section below. For example the disclosed NOV8 protein have multiple hydrophilic regions, each of which can be used as an immunogen. This novel protein also has value in development of powerful assay system for functional analysis of various human disorders, which will help in understanding of pathology of the disease and development of new drug targets for various disorders.
NOV9
NOV9 includes two novel Early B-Cell Factor-like proteins disclosed below. The disclosed proteins have been named NOV9a and NOV9b.
NOV9a
A disclosed NOV9a nucleic acid of 1873 nucleotides (also referred to as CG57480-01) encoding a novel Early B-Cell Factor-like protein is shown in Table 9A. An open reading frame was identified beginning with an ATG initiation codon at nucleotides 25-27 and ending with a TGA codon at nucleotides 1813-1815. Putative untranslated regions, if any, upstream from the initiation codon and downstream from the termination codon are underlined in Table 9A, and the start and stop codons are in bold letters.







TABLE 9A





NOV9a nucleotide sequence.















(SEQ ID NO:35)







CTGCGGCCGCCGCCAGCAGTTTTCATGTTTGGGATTCAGGAGAATATTCCGCGCGGGGGGACGACCATGAAG


GAGGAGCCGCTGGGCAGCCGCATGAACCCGGTGCGCTCGTGGATGCACACGGCGGGCGTGGTGGACGCCAAC


ACGGCCGCCCAGAGCGGCGTGGGGCTGGCGCGGGCGCACTTCGAGAAGCAGCCGCCTTCCAACCTCCGGAAA


TCCAATTTCTTCCACTTCGTGCTGGCGCTCTACGATAGGCAGGGGCAGCCGGTGGAGATTGAAAGGACCGCT


TTTGTGGACTTTGTGGAGAAAGAGAAAGAGCCAAACAACGAGAAAACCAACAACGGCATCCACTATAAACTC


CAGTTATTGTACAGCAACGGTGTCAGAACAGAGCAAGATCTGTATGTTCGCCTCATAGATTCAATGACCAAA


CAGGCCATCGTCTACGAGGGCCAGGACAAGAACCCGGAGATGTGCCGTGTGCTGCTGACCCACGAGATCATG


TGCAGCCGGTGCTGTGACAAGAAAAGTTGTGGCAATAGAAACGAAACGCCCTCAGACCCTGTAATCATTGAC


AGGTTCTTTCTAAAGTTTTTCCTCAAGTGCAATCAGAACTGTTTGAAGAATGCAGGCAACCCTCGAGATATG


CGGAGATTCCAGGTAGTTGTATCGACAACAGTCAACGTGGACGGCCACGTGCTGGCCGTGTCAGACAACATG


TTTGTGCACAACAATTCCAAACACGGGAGGCGGGCCCGCCGCCTAGACCCGTCAGAAGGTACGGCCCCTTCT


TATCTGGAAAATGCCACTCCGTGCATCAAGGCCATCAGTCCCAGTGAAGGCTGGACCACGGGGGGTGCCACC


GTCATCATAATTGGCGACAACTTCTTTGACGGGCTGCAAGTTGTATTCGGAACTATGTTGGTGTGGAGCGAG


CTGATAACTCCCCATGCCATCCGAGTCCAGACCCCGCCGAGGCACATTCCTGGCGTCGTCCAAGTGACCCTC


TCCTACAAATCCAAGCAGTTCTGCAAAGGTGCTCCTGGGCGCTTTGTCTACACCGCCCTTAATGAACCAACC


ATAGATTACGGCTTTCAGAGGTTGCAGAAAGTGATCCCAAGACATCCGGGTGATCCCGAAAGGTTACCCAAG


GAGGTGTTACTGAAGCGGGCGGCGGACCTGGTGGAAGCCTTATACGGAATGCCTCACAACAACCAGGAGATC


ATCTTGAAGCGAGCGGCGGACATCGCCCAGGCGCTGTACAGCGTTCCCCGCAATCACAACCAGATCCCCACC


CTGGGCAACAACCCTGCACACACGGGCATGATGGGCGTCAACTCCTTCAGCAGCCAGCTAGCCGTCAACGTG


TCAGAGACGTCACAAGCCAACGACCAAGTCGGCTACAGTCGCAATACAAGCAGCGTGTCCCCGCGAGGCTAC


GTCCCCAGCAGTACTCCCCAGCAGTCCAATTACAACACAGTCAGCACTAGCATGAATGGATATGGAAGTGGC


GCCATGGCCAGTCTAGGGGTCCCTGGCTCGCCTGGATTTCTTAATGGCTCCTCCGCTAACTCTCCCTACGGC


GTAGTGCCGTCCAGCCCCACCATGGCAGCCTCTTCGGTCACCCTCCCTTCAAACTGTAGCAGCACACACGGC


ATTTTCTCATTCTCACCTGCCAATGTCATCTCCGCAGTGAAACAGAAGAGCCCCTTCGCGCCCGTGGTCCGG


CCCCAAGCCTCTCCTCCTCCTTCCTGCACCAGCGCCAACGGGAATGGACTGCAAGCTATGTCTGGGCTGGTA


GTCCCGCCAATGTGAGGGACTTCTGTTTACCTTCCGCAGCACCCAGCATCAAAGGACGGACTTCAGGGGACA


C









The disclosed NOV9a nucleic acid sequence, localized to chromsome 10, has 1769 of 1871 bases (94%) identical to a gb:GENBANK-ID:MMU92702|acc:U92702.1 mRNA from Mus musculus (Mus musculus Olf-1/EBF-like-2(9L) transcription factor (O/E-2(9L)) mRNA, complete cds) (E=0.0).
A NOV9a polypeptide (SEQ ID NO:36) encoded by SEQ ID NO:35 has 596 amino acid residues and is presented using the one-letter code in Table 9B. Signal P, Psort and/or Hydropathy results predict that NOV9a does not contain a signal peptide and is likely to be localized to the cytoplasm with a certainty of 0.6500.







TABLE 9B





Encoded NOV9a protein sequence.















(SEQ ID NO:36)







MFGIQENIPRGGTTMKEEPLGSGMNPVRSWMHTAGVVDANTAAQSGVGLARAHFEKQPPSNLRKSNFFHFVL


ALYDRQGQPVEIERTAFVDFVEKEKEPNNEKTNNGIHYKLQLLYSNGVRTEQDLYVRLIDSMTKQAIVYEGQ


DKNPEMCRVLLTHEIMCSRCCDKKSCGNRNETPSDPVIIDRFFLKFFLKCNQNCLKNAGNPRDMRRFQVVVS


TTVNVDGHVLAVSDNMFVHNNSKHGRRARRLDPSEGTAPSYLENATPCIKAISPSEGWTTGGATVIIIGDNF


FDGLQVVFGTMLVWSELITPHAIRVQTPPRHIPGVVEVTLSYKSKQFCKGAPGRFVYTALNEPTIDYGFQRL


QKVIPRHPGDPERLPKEVLLKRAADLVEALYGMPHNNQEIILKRAADIAEALYSVPRNHNQIPTLGNNPAHT


GMMGVNSFSSQLAVNVSETSQANDQVGYSRNTSSVSPRGYVPSSTPQQSNYNTVSTSMNGYGSGAMASLGVP


GSPGFLNGSSANSPYGVVPSSPTMAASSVTLPSNCSSTHGIFSFSPANVISAVKQKSAFAPVVRPQASPPPS


CTSANGNGLQAMSGLVVPPM









The NOV9a amino acid sequence has 593 of 596 amino acid residues (99%) identical to, and 595 of 596 amino acid residues (99%) similar to, the 596 amino acid residue ptnr:SWISSPROT-ACC:008791 protein from Mus musculus (Mouse) (Early B-Cell Factor-3 (Early B-Cell Factor 3) (EBF-3) (OLF-1/EBF- Like 2) (OE-2) (O/E-2)) (E=0.0).
The disclosed NOV9a is expressed in at least the following tissue: because of the expression pattern of (GENBANK-ID: gb:GENBANK-ID:MMU92702|acc:U92702.1) a closely related Mus musculus Olf-1/EBF-like-2(9L) transcription factor (O/E-2(9L)) mRNA, complete cds homolog in species Mus musculus: adult olfactory epithelium; in embryo, expressed in epithalamus, hypothalamus, throughout the brainstem and near the ventricular zones of mesencephalon, in rostral rhombencephalon, in vomeronasal organ, at high level in developing retina, developing spinal cord, at low level in dorsal root ganglia, trigeminal ganglia, glossopharyngeal nerve ganglia.
NOV9b
A disclosed NOV9b nucleic acid of 1667 nucleotides (also referred to as CG57480-02) encoding a novel Early B-Cell Factor-like protein is shown in Table 9C. An open reading frame was identified beginning with an ATG initiation codon at nucleotides 5-7 and ending with a TAA codon at nucleotides 1625-1627. Putative untranslated regions, if any, upstream from the initiation codon and downstream from the termination codon are underlined in Table 9C, and the start and stop codons are in bold letters.







TABLE 9C





NOV9b nucleotide sequence.















(SEQ ID NO:37)







TTTCATGTTTCCGATTCAGGAGAATATTCCGCGCGGGGGGACGACCATGAAGGAGGAGCCGCTGGGCAGCGG


CATGAACCCGGTGCGCTCGTGGATGCACACGGCGGGCGTGGTGGATGCCAACACGGCCGCCCAGAGCGGCGT


GGGGCTGGCGCGGGCGCACTTCGAGAAGCAGCCGCCTTCCAACCTCCGGAAATCCAATTTCTTCCACTTCGT


GCTGGCGCTCTACGATAGGCAGGGGCAGCCGGTGGAGATTGAAAGGACCGCTTTTGTGGACTTTGTGGAGAA


AGAGAAAGAGCCAAACAACGAGAAAACCAACAACGGCATCCACTATAAACTCCAGTTATTGTACAGCAACGG


AGTCAGAACAGAGCAAGATCTGTATGTTCGCCTCATAGATTCAATGACCAAACAGGCCATCGTCTACGAGGG


CCAGGACAAGAACCCGGAGATGTGCCGTGTGCTGCTGACCCACGAGATCATGTGCAGCCGGTGCTGTGACAA


GAAAAGTTGTGGCAATAGAAACGAAACGCCCTCAGACCCTGTAATCATTGACAGATTCTTTCTAAAGTTTTT


CCTCAAGTGCAATCAGAACTGTTTGAAGAATGCAGGCAACCCTCGAGATATGCGGAGATTCCAGGTTGTTGT


ATCGACAACAGTCAACGTGGACGGCCACGTGCTGGCCGTGTCAGACAACATGTTTGTGCACAACAATTCCAA


ACACGGGAGGCGGGCCCGCCGCCTAGACCCGTCAGAAGCCACTCCGTGCATCAAGGCCATCAGTCCCAGTGA


AGGCTGGACCACGGGGGGTGCCACCGTCATCATAATTGGCGACAACTTCTTTGACGGGCTGCAAGTTGTATT


CGGAACTATGTTGGTGTGGAGCGAGCTGATAACTCCCCATGCCATCCGAGTCCAGACCCCGCCGAGGCACAT


TCCTGGCGTCGTCGAAGTGACCCTCTCCTACAAATCCAAGCAGTTCTGCAAAGGTGCTCCTGGGCGCTTTGT


CTACACCGCCCTTAATGAACCAACCATAGATTACGGCTTTCAGAGGTTGCAGAAAGTGATCCCAAGACATCC


GGGTGATCCCGAAAGGTTACCCAAGGAGGTGTTACTGAAGCGGGCGGCGGACCTGGTGGAAGCCTTATACGG


AATGCCTCACAACAACCAGGAGATCATCTTGAAGCGAGCGGCGGACATCGCCGAGGCGCTGTACAGCGTTCC


CCGCAATCACAACCAGATCCCCACCCTGGGCAACAACCCTGCACACACGGGCATGATGGGCGTCAACTCCTT


CAGCAGCCAGCTAGCCGTCAACGTGTCAGAGACGTCACAAGCCAACGACCAAGTCCGCTACAGTCGCAATAC


AAGCAGCGTGTCCCCGCGAGGCTACGTCCCCAGCAGTACTCCCCAGCAGTCCAATTACAACACAGTCAGCAC


TAGCATGAATGGATATGGAAGTGGCGCCATGGCCAGTCTAGGGGTCCCTGGCTCGCCTGGATTTCTTAATGG


CTCCTCCGCTAACTCTCCCTACGGCATGAAACAGAAGAGCGCCTTCGCGCCCGTGGTCCGGCCCCAAGCCTC


TCCTCCTCCTTCCTGCACCAGCGCCAACGGGAATGGACTGTAAGCTATGTCTGGGCTGGTAGTCCCGCCAAT


GTGAGGGACTT









The disclosed NOV9b nucleic acid sequence, localized to chromsome 10, has 1568 of 1665 bases (94%) identical to a gb:GENBANK-ID:MMU92704|acc:U92704.1 mRNA from Mus musculus (Mus musculus Olf-1/EBF-like-2(0S) transcription factor (O/E-2(0S)) mRNA, alternative splice variant, complete cds) (E=0.0).
A NOV9b polypeptide (SEQ ID NO:38) encoded by SEQ ID NO:37 has 540 amino acid residues and is presented using the one-letter code in Table 9D. Signal P, Psort and/or Hydropathy results predict that NOV9b does not contain a signal peptide and is likely to be localized to the plasma membrane with a certainty of 0.6500.







TABLE 9D





Encoded NOV9b protein sequence.















(SEQ ID NO:38)







MFGIQENIPRGGTTMKEEPLGSGMNPVRSWMHTAGVVDANTAAQSGVGLARAHFEKQPPSNLRKSNFFHFVL


ALYDRQGQPVEIERTAFVDFVEKEKEPNNEKTNNGIHYKLQLLYSNGVRTEQDLYVRLIDSMTKQAIVYEGQ


DKNPEMCRVLLTHEIMCSRCCDKKSCGNRNETPSDPVIIDRFFLKFFLKCNQNCLKNAGNPRDMRRFQVVVS


TTVNVDGHVLAVSDNMFVHNNSKHGRRARRLDPSEATPCIKAISPSEGWTTGGATVIIIGDNFFDGLQVVFG


TMLVWSELITPHAIRVQTPPRHIPGVVEVTLSYKSKQFCKGAPGRFVYTALNEPTIDYGFQRLQKVIPRHPG


DPERLPKEVLLKRAADLVEALYGMPHNNQEIILKRAADIAEALYSVPRNHNQIPTLGNNPAHTGMMGVNSFS


SQLAVNVSETSQANDQVGYSRNTSSVSPRGYVPSSTPQQSNYNTVSTSMNGYGSGAMASLGVPGSPGFLNGS


SANSPYGMKQKSAFAPVVRPQASPPPSCTSANGNGL









The NOV9b amino acid sequence has 526 of 526 amino acid residues (100%) identical to, and 526 and 526 amino acid residues (100%) similar to, the 537 amino acid residue ptnr:TREMBLNEW-ACC:CAC16113 protein from Homo sapiens (Human) (BA234G16.1.1 (Novel Protein Similar Early B-Cell Factor (EBF)) (E=6.5e−289).
The disclosed NOV9b is expressed in at least the following tissues: olfactory tissue. This information was derived by determining the tissue sources of the sequences that were included in the invention including but not limited to SeqCalling sources, Public EST sources, Literature sources, and/or Race sources.
Possible SNPs found for NOV9a are listed in Table 9E and possible SNPs for NOV9b are listed in Table 9F.







TABLE 9E







SNPs














Amino




Nucleotide
Base
Acid
Base


Variant
Position
Change
Position
Change














13377086
138
C > T
Silent
N/A


13377087
172
G > A
 50
Ala > Thr


13377088
506
G > A
161
Cys > Tyr


13377089
1088
A > C
355
Tyr > Ser


13377084
1394
A > T
457
Gln > Leu


13377083
1671
A > G
Silent
N/A


13377085
1780
C > T
586
Gln > End
















TABLE 9F







SNPs










Consensus

Base



Position
Depth
Change
PAF













133
41
A > G
0.049


268
41
A > G
0.049


324
41
A > G
0.049


372
41
A > G
0.049


376
41
A > —
0.049


456
40
T > C
0.050


488
32
A > G
0.344









NOV9a and NOV9b are very closely homologous as is shown in the amino acid alignment in Table 9G.







TABLE 9G





Amino Acid Alignment of NOV9a and NOV9b





















































































Homologies to any of the above NOV9 proteins will be shared by the other NOV9 proteins insofar as they are homologous to each other as shown above. Any reference to NOV9 is assumed to refer to both of the NOV9 proteins in general, unless otherwise noted.
NOV9a has homology to the amino acid sequences shown in the BLASTP data listed in Table 9H.







TABLE 9H







BLAST results for NOV9a












Gene Index/
Protein/
Length
Identity
Positives



Identifier
Organism
(aa)
(%)
(%)
Expect





gi|13959320|sp|Q9H4
TRANSCRIPTION
596
582/596
583/596
0.0


W6|COE3_HUMAN
FACTOR COE3

(97%)
(97%)



(EARLY B-CELL



FACTOR 3)



(EBF-3) (OLF-



1/EBF-LIKE 2)



(OE-2) (O/E-2)


gi|6226802|sp|O08791|
TRANSCRIPTION
596
580/596
582/596
0.0


COE3_MOUSE
FACTOR COE3

(97%)
(97%)



(EARLY B-CELL



FACTOR 3)



(EBF-3) (OLF-



1/EBF-LIKE 2)



(OE-2) (O/E-2)


gi|12314255|emb|CAC16112.1|
bA234G16.1.2
582
568/582
569/582
0.0


(AL354950)
(novel protein

(97%)
(97%)



similar to



early B-cell



factor (EBF))



[Homo sapiens]


gi|13959679|sp|O73742|
Transcription
598
558/589
566/589
0.0


COE3_XENLA
factor COE3

(94%)
(95%)



(XCOE3) (OLF-



1/EBF-like 2)



(OE-2) (XOE-



2) (XEBF-3)


gi|6753706|ref|NP_034226.1|
early B-cell
551
535/596
537/596
0.0


(NM_010096)
factor 3;

(89%)
(89%)



Olf-1/EBF-



like 2 [Mus



musculus]









The homology of these sequences is shown graphically in the ClustalW analysis shown in Table 9I.







TABLE 9I





ClustalW Analysis of NOV9a















1) NOV9a (SEQ ID NO:36)


2) gi|13959320|sp|Q9H4W6 COE3_HUMAN TRANSCRIPTION FACTOR COE3 (EARLY B-CELL


FACTOR 3) (EBF-3) (OLF-1/EBF-LIKE 2) (OE-2) (O/E-2) [Homo sapiens] (SEQ ID NO:143)


2) gi|6226802|sp.O08791 COE3_MOUSE TRANSCRIPTION FACTOR COE3 (EARLY B-CELL FACTOR 3)


(EBF-3) (OLF-1/EBF-LIKE 2) (OE-2) (O/E-2) [Mus musculus] (SEQ ID NO:144)


3) gi 12314255|emb|CAC16112.1 (AL354950) bA234G16.1.2 (novel protein similar to early


B-cell factor (EBF)) [Homo sapiens] (SEQ ID NO:145)


4) gi|13959679|sp|O73742 COE3_XENLA Transcription factor COE3 (XCOE3) (OLF-1/EBF-like


2) (OE-2) (XOE-2) (XEBF-3) [Xenopus laevis] (SEQ ID NO:146)


5) gi 6753706|refNP_034226.1| (NM_010096) early B-cell factor 3; Olf-1/EBF-like 2


[Mus musculus] (SEQ ID NO:147)








































































Tables 9J, 9K and 9L lists the domain description from DOMAIN analysis results against NOV9a. This indicates that the NOV9a sequence has properties similar to those of other proteins known to contain these domains.







TABLE 9J





Domain Analysis of NOV9a















gnl|Pfam|pfam01833, TIG, IPT/TIG domain. This family consists of a


domain that has an immunoglobulin like fold. These domains are found


in cell surface receptors such as Met and Ron as well as in


intracellular transcription factors where it is involved in DNA


binding. CAUTION: This family does not currently recognise a


significant number of members. (SEQ ID NO:148)


CD-Length = 85 residues, 100.0% aligned


Score = 51.2 bits (121), Expect = 2e−07










Query:
263
PCIKAISPSEGWTTGGATVIIIGDNFF--DGLQVVFGTMLVWSELITPHAIRVQTPPRHI
320




| | +|||| |  +||  + | | |     + ++| ||             |  +|||


Sbjct:
1
PVITSISPSSGPLSGGTEITITGSNLGSGEDIKVTFGGTECDVVSQEASQIVCKTPPYAN
 60


Query:
321
PGVVEVTLSYKSKQFCKGAPGRFVYT
346




 |   ||+|         +|  | |


Sbjct:
61
GGPQPVTVSLDGGGLS-SSPVTFTYV
 85
















TABLE 9K





Domain Analysis of NOV9a















gnl|Smart|smart00429, IPT, ig-like, plexins, transcription factors


(SEQ ID NO:149)


CD-Length = 93 residues, 98.9% aligned


Score = 39.3 bits (90), Expect = 6e−04










Query:
263
PCIKAISPSEGWTTGGATVIIIGDNFFDGLQVVFGTMLVWS-------ELITPHAIRVQT
315




| |  |||+ |   +|| | | +     | + |||  + |            ++  ||  +|


Sbjct:
2
PVITRISPNSGPLSGG-TRITLCGKNLDSISVVFVEVGVGEVPCTFLPSDVSQTAIVCKT
 60


Query:
316
PPRH---IPGVVEVTLSYKSKQFCKGAPGRFVYT
346




|| |       | | +  ++     | |  | |


Sbjct:
61
PPYHNIPGSVPVRVEVGLRNGGV-PGEPSPFTYV
 93
















TABLE 9L





Domain Analysis of NOV9a















gnl|Smart smart00353, HLH, helix loop helix domain. (SEQ ID NO:150)


CD-Length = 53 residues, 75.5% aligned


Score = 37.0 bits (84), Expect = 0.003










Query:
353
IDYGFQRLQKVIPRHPGDPERLPKEVLLKRAADLVEALYGM
393




|  +  |  |+ ++|  | + ++| |  +|+ | | +++|


Sbjct:
11
INEAFDELRSLLPPLPNN-KKLSKASILRLAIDYIKSLQEQ
 50









Murine B lymphocytes, adipocytes, and olfactory neurons contain a DNA-binding protein that participates in the regulation of genes encoding tissue-specific components of signal transduction. Purification and cloning of this protein, termed early B-cell factor (EBF), from murine B lymphocytes and independent cloning of a protein, termed Olf-1, from olfactory neuronal cells revealed virtual complete amino acid sequence identity between these proteins.
Early B-cell factor (EBF) is a tissue-specific and differentiation stage-specific DNA-binding protein that participates in the regulation of the pre-B and B lymphocyte-specific MB1 gene. Travis et al. (Molec. Cell. Biol. 13: 3392-3400, 1993) purified the mouse Ebf protein from pre-B cells and found that it is composed of two 62- to 65-kD subunits. Hagman et al. (Genes Dev. 7: 760-773, 1993) determined partial amino acid sequences of Ebf and used them to isolate mouse pre-B-cell cDNAs encoding Ebf. The predicted 591-amino acid protein has 2 functional domains: an N-terminal cysteine-rich region essential for DNA binding, and a C-terminal dimerization region containing two 15-amino acid repeats with similarity to the dimerization domains of basic helix-loop-helix (bHLH) proteins. The calculated molecular mass of the encoded Ebf protein is 64.4 kD. The authors found that recombinant Ebf binds to DNA as a homodimer, forms complexes with the Mb1 promoter, and is a strong activator of transcription. Northern blot analysis detected multiple Ebf transcripts in pre-B- and early B-cell lines but not in other hematopoietic cells. S1 nuclease protection analysis of adult mouse RNAs showed high levels of Ebf expression in lymph node, spleen, and adipose tissues and low levels in several nonlymphoid tissues. By Southern blot analysis of somatic cell hybrid DNAs using a murine Ebf cDNA as a probe, and by fluorescence in situ hybridization using human genomic cosmids, Milatovich et al. (Genome 5: 211-215, 1994) mapped the human EBF gene to 5q34. The study mapped the mouse Ebf gene to proximal chromosome 11 by Southern blot analysis of somatic cell hybrid DNAs and by analysis of recombinant inbred strains.
The mammalian olfactory system has the remarkable ability to detect odorants with high sensitivity and specificity. The initial events in the olfactory signal transduction pathway occur in the specialized cilia of the sensory neurons. Unlike other neurons, the olfactory sensory cells are continually replaced throughout adult life. Cells within the olfactory epithelium follow an orderly developmental program resulting in the high level of expression of gene products essential for odorant signal transduction. The mature neurons express several olfactory-specific genes, some of which appear to mediate the odorant signal transduction cascade. Evidence supporting the involvement of a G protein-coupled receptor pathway in odorant signal transduction was provided by the isolation of olfactory-specific components that correspond to each step in the pathway, e.g., G-alpha-olf. Additional olfactory neuron-specific genes have been identified. The establishment of the mature olfactory neuronal phenotype probably results from the coordinated expression of olfactory-specific genes. In the investigation of these genes, Wang and Reed (Nature 364: 121-126, 1993) found that each contains at least 1 binding site for the DNA-binding protein Olf1. The binding of an olfactory-specific factor, Olf1, was first described in the olfactory marker protein gene (OMP). Olf1 activity was detectable in nuclear extracts from nasal epithelium and absent from nuclear extracts of a variety of other tissues. Using a novel genetic selection in yeast, Wang and Reed (1993) isolated a cDNA for the rat transcriptional activator Olf1, which binds to the regulatory sequences of several olfactory-specific genes. Expressed exclusively in the olfactory receptor neurons and their precursors, the Olf1 protein contains a new helix-loop-helix motif and functions as an apparent homodimer. They suggested that Olf1 may be the first member of a family of related proteins that may direct cellular differentiation in a variety of neuronal tissues.
Using homology screening methods, two additional Olf-1/EBF-like cDNAs were identified from a mouse embryonic cDNA library. The Olf-1/EBF-like (O/E) proteins O/E-1, O/E-2, and O/E-3 define a family of transcription factors that share structural similarities and biochemical activities. Although these O/E genes are expressed within olfactory epithelium in an identical pattern, they exhibit different patterns of expression in the developing nervous system. Although O/E-1 mRNA is present in several tissues in addition to olfactory neurons and developing B-cells, O/E-2 and O/E-3 are expressed at high levels only in olfactory tissue. In O/E-1 knock-out animals, the presence of two additional O/E family members in olfactory neurons may provide redundancy and allow normal olfactory neurodevelopment. Further, the identification of the O/E family of HLH transcription factors and their embryonic expression patterns suggest that the O/E proteins may have a more general function in neuronal development.
The independent cloning of rodent EBF/Olf-1 and Drosophila Collier has defined a family of transcription factors, the Collier or COE family. COE proteins have various functions in different organisms. Proteins currently known to belong to this family include: mammalian COE1 (OLF-1; EBF-1; OE-1), COE2 (EBF-2; OE-3) and COE3 (EBF-3; OE-2); Xenopus XCOE2 and XCOE3; zebrafish ZCOE2; Drosophila Collier; and Caenorhabditis elegans UNC-3. In mouse, COE1 has a role in B-cell differentiation, and could also perform a role in neuronal differentiation. All three COE are expressed in immature olfactory neuronal precursors and mature olfactory neurons as well as in developping nervous system during embryogenesis. In Drosophila, Collier is involved in the formation of the embryonic somatic muscle DA3, in the patterning of the wing by mediating Hedgehog activity. It could also act as a second-level regulator in the patterning of embryonic head. In Xenopus, XCOE2 is expressed in precursors of primary neurons, and may play a pivotal role in the transcriptional cascade specifying primary neurons in embryo. It promotes neuronal differentiation by activating XNeuroD expression. In Zebrafish, ZCOE2 is expressed in a subset of primary neuroblasts in the spinal cord and at later stages is a marker of the olfactory placodes. Caenorhabditis elegans mutants for UNC-3 move abnormally, suggesting that UNC-3 might regulate expression of genes involved in growth cone pioneering long the ventral cord or in fasciculation.
The NOV9 nucleic acid of the invention encoding a Early B-Cell Factor-like protein includes the nucleic acid whose sequence is provided in Tables 9A and 9C, or a fragment thereof. The invention also includes a mutant or variant nucleic acid any of whose bases may be changed from the corresponding base shown in Tables 9A and 9C while still encoding a protein that maintains its Early B-Cell Factor-like activities and physiological functions, or a fragment of such a nucleic acid. The invention further includes nucleic acids whose sequences are complementary to those just described, including nucleic acid fragments that are complementary to any of the nucleic acids just described. The invention additionally includes nucleic acids or nucleic acid fragments, or complements thereto, whose structures include chemical modifications. Such modifications include, by way of non-limiting example, modified bases, and nucleic acids whose sugar phosphate backbones are modified or derivatized. These modifications are carried out at least in part to enhance the chemical stability of the modified nucleic acid, such that they may be used, for example, as antisense binding nucleic acids in therapeutic applications in a subject. In the mutant or variant nucleic acids, and their complements, up to about 6% of the NOV9 residues may be so changed.
The NOV9 protein of the invention includes the Early B-Cell Factor-like protein whose sequence is provided in Tables 9B and 9D. The invention also includes a mutant or variant protein any of whose residues may be changed from the corresponding residue shown in Tables 9B and 9D while still encoding a protein that maintains its Early B-Cell Factor-like activities and physiological functions, or a functional fragment thereof. In the mutant or variant protein, up to about 1% of the NOV9 bases may be so changed.
The NOV9 nucleic acids and proteins of the invention are useful in potential diagnostic and therapeutic applications implicated in various diseases and disorders described below and/or other pathologies. For example, the compositions of the present invention will have efficacy for treatment of patients suffering from: Von Hippel-Lindau (VHL) syndrome, Alzheimer's disease, stroke, tuberous sclerosis, hypercalceimia, Parkinson's disease, Huntington's disease, cerebral palsy, epilepsy, Lesch-Nyhan syndrome, multiple sclerosis, ataxia-telangiectasia, leukodystrophies, behavioral disorders, addiction, anxiety, pain, neuroprotection, multiple sclerosis, myasthenia gravis, diabetes, obesity, neuronal development and other diseases, disorders and conditions of the like.
NOV9 nucleic acids and polypeptides are further useful in the generation of antibodies that bind immunospecifically to the novel substances of the invention for use in therapeutic or diagnostic methods. These antibodies may be generated according to methods known in the art, using prediction from hydrophobicity charts, as described in the “Anti-NOVX Antibodies” section below. For example the disclosed NOV9 protein have multiple hydrophilic regions, each of which can be used as an immunogen. This novel protein also has value in development of powerful assay system for functional analysis of various human disorders, which will help in understanding of pathology of the disease and development of new drug targets for various disorders.
NOV10
A disclosed NOV10 nucleic acid of 3892 nucleotides (also referred to as CG57389-01) encoding a novel High-Affinity CAMP Specific and IBMX-Insensitive-like protein is shown in Table 10A. An open reading frame was identified beginning with an ATG initiation codon at nucleotides 137-139 and ending with a TAG codon at nucleotides 2624-2626. Putative untranslated regions upstream from the intitation codon and downstream from the termination codon are underlined in Table 10A, and the start and stop codons are in bold letters.







TABLE 10A





NOV10 Nucleotide Sequence















(SEQ ID NO:39)







ACGCGAGATCCGCGCTCGCCGCCGCCCGCCCAGGCGGCGATGACACGGCGCCCGCGGCGGCCCGGAGGCGCCG


GGTGGGCCGTTTGCTGACCGGATCGCGGCTACCCGCCAGCGTGTCCGCGGCGCCGCCGCCAGCATGGGCTGTG


CCCCGAGCATCCACATTTCCGAGCGCCTGGTGGCCGAGGACGCGCCTAGCCCCGCGGCACCGCCGCTGTCGTC


CGGCGGGCCGCGCCTCCCGCAGGGCCAGAAGACGGCCGCCTTGCCCCGGACCCGCGGCGCCGGCCTCTTGGAG


TCGGAGGTTCGCGACGGCAGCGGCAAGAAGGTAGCAGTAGCTGATGTGCAGTTTGGCCCCATGAGATTTCATC


AAGATCAACTTCAGGTACTTTTAGTGTTTACCAAAGAAGATAACCAATGTAATGGATTCTGCAGGGCATGTGA


AAAAGCAGGGTTTAAGTGTACAGTTACCAAGGAGGCTCAGGCTGTCCTTGCCTGTTTCCTGGACAAACATCAT


GACATTATCATCATAGACCACAGAAATCCTCGACAGCTGGATGCAGAGGCACTGTGCAGGTCTATCAGATCAT


CAAAACTCTCAGAAAACACAGTTATTGTTGGTGTAGTACGCAGGGTGGATAGAGAAGAGTTGTCCGTAATGCC


TTTCATTTCTGCTGGATTTACAAGGAGGTATGTAGAAAACCCCAACATCATGGCCTGCTACAATGAACTGCTC


CAGCTGGAGTTTGGAGAGGTGCGATCACAACTGAAACTCAGGGCTTGTAACTCAGTATTCACTGCATTAGAAA


ACAGTGAAGATGCAATTGAAATTACAAGCGAAGACCGTTTTATACAGTATGCAAATCCTGCATTTGAAACAAC


AATGGGCTATCAGTCAGGTGGAATTAATAGGGAAGGAGTTAGGAGAAGTGCCTATAAATGAAAAAAGGCTGAC


TTGCTCGATACTATAAATTCATGCATCAGGATAGGCAAGGAGTGGCAAGGAATTTACTATGCCAAAAAGAAAA


ACGGAGATAATATACAACAAAATGTGAAGATAATACCTGTCATTGGACAGGGAGGAAAAATTAGACACTATGT


GTCCATTATCAGAGTGTGCAATGGCAACAATAAGGCTGAGAAAATATCCGAATGTGTTCAGTCTGACACTCGT


ACAGATAATCAGACAGGCAAACATAAAGACAGGAGAAAAGGCTCACTAGACGTCAAAGCTGTTGCCTCCCGTG


CAACTGAAGTTTCCAGCCAGAGACGACACTCTTCCATGGCCCGGATACATTCCATGACAATTGAGGCGCCCAT


CACCAAGGTAATCAATATTATCAATGCTGCCCAGGAAAGTAGTCCCATGCCTGTGACAGAAGCCCTAGACCGT


GTGCTGGAAATTCTAAGAACCACTGAGTTATATTCACCACAGTTTGGTGCTAAAGATGATGATCCCCATGCCA


ATGACCTTGTTGGGGGCTTAATGTCTGATGGTTTGCGAAGACTATCAGGGAATGAATATGTTCTTTCAACAAA


AAACACTCAAATGGTTTCAAGCAATATAATCACTCCCATCTCCCTTGATGATGTCCCACCACGGATAGCTCGG


GCCATGGAAAATGAGGAATACTGGGACTTTGATATTTTTGAACTGGAGGCTGCCACCCACAATAGGCCTTTGA


TTTATCTTGGTCTCAAAATGTTTGCTCGCTTTGGAATCTGTGAATTCTTACACTGCTCCGAGTCAACGCTAAG


ATCATGGTTACAAATTATCGAAGCCAATTATCATTCCTCCAATCCCTACCACAATTCTACACATTCTGCTGAT


GTGCTTCATGCCACTGCCTATTTTCTCTCCAAGGAGAGGATAAAGGAAACTTTAGATCCAATTGATGAGGTCG


CTGCACTCATCGCAGCCACCATTCATGATGTGGATCACCCTGGGAGAACCAACTCCTTCCTGTGTAATGCTGG


AAGTGAGCTGGCCATTTTGTACAATGACACTGCTGTGCTGGAGAGCCACCATGCGGCCTTGGCCTTCCAGCTG


ACCACTGGAGATGATAAATGCAATATATTTAAAAACATGGAGAGGAATGATTATCGGACACTGCGCCAGGGGA


TTATCGACATGGTCTTAGCCACAGAAATGACAAAGCACTTTGAGCATGTCAACAAATTTGTCAACAGCATCAA


CAAACCCTTGGCAACACTAGAAGAAAATGGGGAAACTGATAAAAACCAGGAAGTGATAAACACTATGCTTAGG


ACTCCAGAGAACCGGACCCTAATCAAACGAATGCTGATTAAATGTGCTGATGTGTCCAATCCCTGCCGACCCC


TGCAGTACTGCATCGAGTGGGCTGCACGCATTTCGGAAGAATATTTTTCTCAGACTGATGAAGAGAAGCAGCA


GGGCTTACCTGTGGTGATGCCAGTGTTTGACAGAAATACCTGCAGCATCCCCAAATCCCAAATCTCTTTCATT


GATTACTTCATCACAGACATGTTTGATGCTTGGGATGCCTTTGTAGACCTGCCTGATTTAATGCAGCATCTTG


ACAACAACTTTAAATACTGGAAAGGACTGGACGAAATGAAGCTGCGGAACCTCCGACCACCTCCTGAATAGTG


GGAGACACCACCCAGAGCCCTGAAGCTTTGTTCCTTCGGTCATTTGGAATTCCTGAGGGCAGCCAGAGCTCCT


TGGTCCTTTCAGTACTAGGCAGAACAGCCCCCGATCTGCATAGCCTGTGAAAGCCCACGGGGACATCAGTAAC


CTTCTGCAGCCACCATCCAATGCCATTACTGTCAAGTGAGACTTGGCCACTGTAGCCTGGGCCTCCTGCAGGA


GCTCTTCAGAAAGGCACATGAGGACCACGGTTTGCCTCAGTTTCTGGTAAAACACAAGGTCTGGAGTGCCCCT


GCAAAGGGTATTGATGGACTTCCTGCCAGTGACAGAGCATGTCTATTGCAAACAATTCTCTCAGTTACGTTCA


GCACTTAAGAACGGCTAATGGCAATAGGATCTTTAGCAACTTTTTCACATCATAGAAGGTGCAATCGCTCACT


TGGGAACACTACTGAGAGTGACTTCTCTTTTAAAATTGAGTAGCAGATGAAAAATTAAAATTTGAACTTGATT


ATTAATATCAATTAAAATGTTTTATTTATTTTATTAAAAGCTCAATATTTTCTATGAATTCAAAAATACTTCA


GAGCCAAAGCCAACTTCAAATACCGTGACCAAATTTACATGATTCATATTCATTATGCATTACTTGGTATACA


GACTTATTTTCATAATGCAAATTAATAAAATGACACTTTTACTGCACTATAGAAATATTCATGTATGTTAAAC


TTTTCTGATTGAGGCTAACTGGAAAAAGCTGGGGTCGTATTCTAAGTGCTAAAGAAGGCTGCTTCTACTGTAT


AGAACCCAGGGCTCTGAAACAGCTCTAGCCGCCTAATGCACTTCACAGGTAACTCCCCAAGGTAAAACTAGAC


TCTCTTGTTGGTTCGCAAAGAAAAGTTAGGACTTAACACTTTTTTCTAAAATTTTATAATTCAATTTCCAAAA


GTCTACTCTATTTTATACTGTTTCTACAAAATATTCCTTATAAAAACAAAGAACAAAAATTGAATATTTAATG


AATTGACATTTTATAACCAACCTGTTTTTATCTACGGTGGGAATCTTTGATGCCAGAAATTTATAAAGAGGTT


CTGTATCTTCACACCTTGAATAAGCATAATACCATAAAAAATGACACTTGACATGTCAATGTATTTGTCATTT


CATTTTAAACTCGTATTTGTGGTTTTTTTCCCAGATAAAAATGAAATTAAACCATTTCTTTTTAAGAAATCAA


AAAAAAAAAAAAAAAAAAAAAAA









The NOV10 nucleic acid was identified on chromosome 15 and has 3393 of 3396 bases (99%) identical to a gb:GENBANK-ID:AF056490|acc:AF056490.1 mRNA from Homo sapiens (Homo sapiens cAMP-specific phosphodiesterase 8A (PDE8A) mRNA, partial cds) (E=0.0).
A disclosed NOV10 polypeptide (SEQ ID NO:40) encoded by SEQ ID NO:39 is 829 amino acid residues and is presented using the one-letter code in Table 10B. Signal P, Psort and/or Hydropathy results predict that NOV10 contains a signal peptide and is likely to be localized to the cytoplasm with a certainty of 0.4500.







TABLE 10B





Encoded NOV10 protein sequence















(SEQ ID NO:40)







MGCAPSIHISERLVAEDAPSPAAPPLSSGGPRLPQGQKTAALPRTRGAGLLESEVRDGSGKKVAVADVQFGP


MRFHQDQLQVLLVFTKEDNQCNGFCRACEKAGFKCTVTKEAQAVLACFLDKHHDIIIIDHRNPRQLDAEALC


RSIRSSKLSENTVIVGVVRRVDREELSVMPFISAGFTRRYVENPNIMACYNELLQLEFGEVRSQLKLRACNS


VFTALENSEDAIEITSEDRFIQYANPAFETTMGYQSGELIGKELGEVPINEKKADLLDTINSCIRIGKEWQG


IYYAKKKNGDNIQQNVKIIPVIGQGGKIRHYVSIIRVCNGNNKAEKISECVQSDTRTDNQTQKHKDRRKGSL


DVKAVASRATEVSSQRRHSSMARIHSMTIEAPITKVINIINAAQESSPMPVTEALDRVLEILRTTELYSPQF


GAKDDDPHANDLVGGLMSDGLRRLSGNEYVLSTKNTQMVSSNIITPISLDDVPPRIARAMENEEYWDFDIFE


LEAATHNRPLIYLGLKMFARFGICEFLHCSESTLRSWLQIIEANYHSSNPYHNSTHSADVLHATAYFLSKER


IKETLDPIDEVAALIAATIHDVDHPGRTNSFLCNAGSELAILYNDTAVLESHHAALAFQLTTGDDKCNIFKN


MERNDYRTLRQGIIDMVLATEMTKHFEHVNKFVNSINKPLATLEENGETDKNQEVINTMLRTPENRTLIKRM


LIKCADVSNPCRPLQYCIEWAARISEEYFSQTDEEKQQGLPVVMPVFDRNTCSIPKSQISFIDYFITDMFDA


WDAFVDLPDLMQHLDNNFKYWKGLDEMKLRNLRPPPE









The NOV10 amino acid sequence has 712 of 713 amino acid residues (99%) identical to, and 713 of 713 amino acid residues (100%) similar to, the 713 amino acid residue ptnr:SWISSNEW-ACC:060658 protein from Homo sapiens (Human) (High-Affinity CAMP-Specific And IBMX-Insensitive 3′,5′-Cyclic Phosphodiesterase 8A (EC 3.1.4.17) (E=0.0).
NOV10 is expressed in at least the following tissues: kidney, lung, uterus, ovary and heart. This information was derived by determining the tissue sources of the sequences that were included in the invention including but not limited to SeqCalling sources, Public EST sources, genomic clone sources, literature sources, and/or RACE sources.
NOV10 has homology to the amino acid sequences shown in the BLASTP data listed in Table 10C.







TABLE 10C







BLAST results for NOV10












Gene Index/

Length
Identity
Positives



Identifier
Protein/Organism
(aa)
(%)
(%)
Expect





gi|14248761|gb|AAK57641.1|
cAMP-specific
829
803/829
804/829
0.0


AF332653_1
cyclic nucleotide

(96%)
(96%)


(AF332653)
phosphodiesterase



PDE8A1 [Homo



sapiens]


gi|16417190|gb|AAL18610.1|
cAMP
829
802/829
803/829
0.0


AF388183_1
phosphodiesterase

(96%)
(96%)


(AF388183)
PDE8A1 [Homo



sapiens]


gi|17477753|ref|XP_
phosphodiesterase
757
745/757
745/757
0.0


031443.2|
8A [Homo sapiens]

(98%)
(98%)


(XM_031443)


gi|16417192|gb|AAL18611.1|
cAMP
783
756/829
757/829
5e−97


AF388184_1
phosphodiesterase

(91%)
(91%)


(AF388184)
PDE8A2 [Homo



sapiens]


gi|5921805|sp|O60658|
High-affinity
713
701/713
702/713
2e−72


CN8A_HUMAN
cAMP-specific and

(98%)
(98%)



IBMX-insensitive



3′,5′-cyclic



phosphodiesterase



8A [Homo sapiens]









The homology of these sequences is shown graphically in the ClustalW analysis shown in Table 10D.







TABLE 10D





Clustal W Sequence Alignment















1) NOV10 (SEQ ID NO:40)


2) gi 14248761|gb AAK57641.1 AF332653_1 (AF332653) cAMP-specific cyclic nucleotide


phosphodiesterase PDE8A1 [Homo sapiens] (SEQ ID NO:151)


3) gi 16417190|gb|AAL18610.1|AF388183_1 (AF388183) cAMP phosphodiesterase PDE8A1


[Homo sapiens] (SEQ ID NO:152)


4) gi|17477753|refXP_031443.2| (XM_031443) phosphodiesterase 8A [Homo sapiens]


(SEQ ID NO:153)


5) gi|16417192|gb|AAL18611.1|AF388184_1 (AF388184) cAMP phosphodiesterase PDE8A2


[Homo sapiens] (SEQ ID NO:154)


6) gi|5921805|sp|O60658|CN8A_HUMAN High-affinity cAMP-specific and IBMX-insensitive


3′,5′-cyclic phosphodiesterase 8A [Homo sapiens] (SEQ ID NO:155)





























































































Tables 10E, 10F and 10G list the domain description from DOMAIN analysis results against NOV10. This indicates that the NOV10 sequence has properties similar to those of other proteins known to contain these domains.







TABLE 10E





Domain Analysis of NOV10















gnl|Pfam|pfam00233, PDEase, 3′5′-cyclic nucleotide phosphodiesterase.


(SEQ ID NO:156)


CD-Length = 240 residues, 99.6% aligned


Score = 202 bits (514), Expect = 6e−53










Query:
555
YHNSTHSADVLHATAYFLSKERIKETLDPIDEVAALIAATIHDVDHPGRTNSFLCNAGSE
614




|||  |+ ||   |   |    +   |  ++ +| + ||  ||||| |  |||   + ||


Sbjct:
1
YHNWRHAFDVTQTTHLLLLTLALDRYLTDLEILALVFAAACHDVDHRGTNNSFQIQSKSE
 60


Query:
615
LAILYNDTAVLESHHAALAFQLTTGDDKCNIFKNMERNDYRTLRQGIIDMVLATEMTKHF
674




||||||| +|||+|| |   +|   |++|||| |+ + |++|||  +|+|+|||+|+ ||


Sbjct:
61
LAILYNDESVLENHHLAQGIKLLQ-DEECNIFINLSKKDFQTLRDLVIEMILATDMSLHF
119


Query:
675
EHVNKFVNSINKPLATLEENGETDKNQEVINTMLRTPENRTLIKRMLIKCADVSNPCRPL
734




+   +                     ++    ||    ++ |+  +++  ||+||| +  


Sbjct:
120
QKEKRLKTM----------------VEQKKTYMLDNQTHKILLMSLIMTAADLSNPTKSW
163


Query:
735
QYCIEWAARISEEYFSQTDEEKQQGLPVVMPVFDRNTCSIPKSQISFIDYFITDMFDAWD
794




     ||  | ||+| | | |++ |||   |+ || +  +||||| |||+ +  +|    


Sbjct:
164
SVHRRWAELIMEEFFDQGDLERELGLP-PSPMCDRTSAYVPKSQIGFIDRIVEPIFKLLA
222


Query:
795
AFV--DLPDLMQHLDNN
809




     |+  |+  +++|


Sbjct:
223
DVTEKDIIPLLDRIEDN
239
















TABLE 10F





Domain Analysis of NOV10















gnl|Pfam|pfam00989, PAS, PAS domain. CAUTION. This family does not


currently match all known examples of PAS domains. PAS motifs appear


in archaea, eubacteria and eukarya. Probably the most surprising


identification of a PAS domain was that in EAG-like K+-channels. (SEQ ID NO:157)


CD-Length = 65 residues, 87.7% aligned


Score = 40.8 bits (94), Expect = 3e−04










Query:
221
LENSEDAIEITSEDRFIQYANPAFETTMGYQSGELIGKELGEVPINEKKADLLDTIN
277




||+  | | +  ||  | | | | |   |    |+||| | ++   |  | + + + 


Sbjct:
7
LESLPDPIFVVDEDGRILYWNAAAEELTGLSREEVIGKSLLDLVHEEDLARVREILQ
 63
















TABLE 10G





Domain Analysis of NOV10















gnl Smart|smart00091, PAS, PAS domain; PAS motifs appear in archaea,


eubacteria and eukarya. Probably the most surprising identification of


a PAS domain was that in EAG-like K+-channels. (SEQ ID NO:158)


CD-Length = 67 residues, 92.5% aligned


Score = 37.4 bits (85), Expect = 0.003










Query:
220
ALENSEDAIEITSEDRFIQYANPAFETTMGYQSGELIGKELGEVPINEKKADLLDTINSC
279




 ||+  | + +   |  | ||||| |  +||   ||||| | |+   | + +| + +   


Sbjct:
6
ILESLPDGVFVLDLDGRILYANPAAEELLGYSPEELIGKSLLELIHPEDREELQERLQRL
 65


Query:
280
IR
281




+


Sbjct:
66
LS
67









The NOV10 nucleic acid of the invention encoding a High-Affinity CAMP Specific and IBMX-Insensitive-like protein includes the nucleic acid whose sequence is provided in Table 10A, or a fragment thereof. The invention also includes a mutant or variant nucleic acid any of whose bases may be changed from the corresponding base shown in Table 10A while still encoding a protein that maintains its High-Affinity CAMP Specific and IBMX-Insensitive-like activities and physiological functions, or a fragment of such a nucleic acid. The invention further includes nucleic acids whose sequences are complementary to those just described, including nucleic acid fragments that are complementary to any of the nucleic acids just described. The invention additionally includes nucleic acids or nucleic acid fragments, or complements thereto, whose structures include chemical modifications. Such modifications include, by way of non-limiting example, modified bases, and nucleic acids whose sugar phosphate backbones are modified or derivatized. These modifications are carried out at least in part to enhance the chemical stability of the modified nucleic acid, such that they may be used, for example, as antisense binding nucleic acids in therapeutic applications in a subject. In the mutant or variant nucleic acids, and their complements, up to about 1% of the residues may be so changed.
The NOV10 protein of the invention includes the High-Affinity CAMP Specific and IBMX-Insensitive-like protein whose sequence is provided in Table 10B. The invention also includes a mutant or variant protein any of whose residues may be changed from the corresponding residue shown in Table 10B while still encoding a protein that maintains its High-Affinity CAMP Specific and IBMX-Insensitive-like activities and physiological functions, or a functional fragment thereof. In the mutant or variant protein, up to about 1% of the bases may be so changed.
The NOV10 nucleic acids and proteins of the invention are useful in potential diagnostic and therapeutic applications implicated in various diseases and disorders described below and/or other pathologies. For example, the compositions of the present invention will have efficacy for treatment of patients suffering from: Cardiomyopathy, Atherosclerosis, Hypertension, Congenital heart defects, Aortic stenosis, Atrial septal defect (ASD), Atrioventricular (A-V) canal defect, Ductus arteriosus, Pulmonary stenosis, Subaortic stenosis, Ventricular septal defect (VSD), valve diseases, Tuberous sclerosis, Scleroderma, Obesity, Transplantation, Endometriosis, Fertility, Systemic lupus erythematosus, Autoimmune disease, Asthma, Emphysema, Scleroderma, allergy, Diabetes, Autoimmune disease, Renal artery stenosis, Interstitial nephritis, Glomerulonephritis, Polycystic kidney disease, Systemic lupus erythematosus, Renal tubular acidosis, IgA nephropathy, Hypercalceimia, Lesch-Nyhan syndrome and other diseases, disorders and conditions of the like.
NOV10 nucleic acids and polypeptides are further useful in the generation of antibodies that bind immunospecifically to the novel substances of the invention for use in therapeutic or diagnostic methods. These antibodies may be generated according to methods known in the art, using prediction from hydrophobicity charts, as described in the “Anti-NOVX Antibodies” section below. For example the disclosed NOV10 protein have multiple hydrophilic regions, each of which can be used as an immunogen. This novel protein also has value in development of powerful assay system for functional analysis of various human disorders, which will help in understanding of pathology of the disease and development of new drug targets for various disorders.
NOV11
A disclosed NOV11 nucleic acid of 6524 nucleotides (also referred to as CG57337-01) encoding a novel KIAA0216-like protein is shown in Table 11A. An open reading frame was identified beginning with an ATG initiation codon at nucleotides 485-487 and ending with a TAG codon at nucleotides 5273-5275. Putative untranslated regions upstream from the intitation codon and downstream from the termination codon are underlined in Table 11A, and the start and stop codons are in bold letters.







TABLE 11A





NOV11 Nucleotide Sequence















(SEQ ID NO:41)







AGTCTCCTTGCTCTGGGCTGCTCCAGGCCAGGCCATGCGGAAAGTGTGTTGTTATGAGGATTCAATGAGGAGA


CCTGTATGAAGTGCCTTCCCCTGTGCCTGGCTCATAGCGAAAACAGAAGAACAGATTGCAGCAGAAGAGGCCT


GGAATGAGACGGAGAAGGTGTGGCTGGTCCATAGGGACGGCTTCTCACTGGCCAGTCAACTCAAATCTGAGGA


GCTCAACTTGCCTGAGGGGAAGGTGCGTGTGAAGCTGGACCACGATGGGGCCATCCTGGATGTGGATGAGGAT


GACGTTGAGAAGGCTAATGCTCCCTCCTGCGACCGTCTGGAGGATCTGGCCTCACTGGTGTACCTCAATGAGT


CCAGCGTCCTGCACACCTTGCGCCAGCGCTATGGCGCTAGCCTGCTGCACACGTATGCTGGCCCCAGCCTGCT


GGTTCTTGGCCCCCGTGGGGCCCCTGCTGTGTACTCTGAGAAGGTGATGCACATGTTCAAGGGTTGTCGGCGG


GAGGACATGGCACCCCACATCTATGCAGTGGCCCAGACCGCATACAGGGCGATGCTGATGAGCCGTCAGGATC


AGTCAATCATCCTCCTGGGCAGTAGTGGCAGTGGCAAGACCACCAGCTGCCAGCATCTGGTGCAGTACCTGGC


CACCATCGCGGGCATCAGCGGGAACAAGGTGTTTTCTGTGGAGAAGTGGCAGGCTCTGTACACCCTCCTGGAA


GCCTTTGGGAACAGCCCCACCATCATTAATGGCAATGCCACCCGCTTCTCCCAGATCCTCTCCCTGGACTTTG


ACCAAGCTGGCCAGGTGGCCTCAGCCTCCATTCAGACAATGCTTCTGGAGAAGCTGCGTGTGGCTCGGCGCCC


AGCCAGTGAAGCCACATTCAACGTCTTCTACTACCTGCTGGCCTGTGGGGATGGCACCCTCAGGACAGAGCTC


CACCTCAACCACTTGGCAGAGAACAATGTGTTTGGGATTGTGCCACTGGCCAAGCCTGAGGAAAAGCAGAAGG


CAGCTCAGCAGTTTAGTAAGCTGCAGGCGGCCATGAAGGTGCTGGGCATCTCCCCCGATGAACAGAAGGCCTG


CTGGTTCATTCTGGCTGCCATCTACCACCTGGGGGCTGCGGGAGCCACCAAAGAAGCTGCTGAAGCTGGGCGC


AAGCAGTTTGCCCGCCATGAGTGGGCCCAGAAGGCTGCGTACCTACTGGGCTGCAGCCTGGAGGAGCTGTCCT


CAGCCATCTTCAAGCACCAGCACAAGGGTGGCACCCTGCAGCGCTCCACCTCCTTCCGCCAGGGCCCCGAGGA


GAGTGGCCTGGGAGATGGGACAGGCCCGAAACTGAGTGCACTGGAGTGCCTTGAGGGCATGGCGGCCGGCCTC


TACAGCGAGCTCTTCACCCTTCTCGTCTCCCTGGTGAATAGGGCTCTCAAGTCCAGCCAGCACTCACTCTGCT


CCATGATGATTGTCGACACCCCGGGCTTCCAGAACCCTGAGCAGGGTGGGTCAGCCCGCGGAGCCTCCTTTGA


GGAGCTGTGCCACAACTACACCCAAGACCGGCTGCAGAGGCTCTTCCACGAGCGCACCTTCGTGCAGGAGTTG


GAAAGATACAAGGAGGAGAACATCGAGCTGGCGTTTGACGACTTGGAACCCCCCACGGATGACTCTGTGGCTG


CTGTGGACCAGGCCTCCCATCAGTCCCTGGTCCGCTCGCTGGCCCGCACAGACGAGGCGAGGGGCCTGCTCTG


GCTATTGGAAGAGGAGGCTCTGGTGCCAGGGGCCAGTGAGGACACCCTCCTGGAGCGCCTTTTCTCCTATTAT


GGCCCCCAGGAAGGTGACAAAAAAGGCCAAAGCCCCCTTCTGCACAGCAGCAAACCACACCACTTTCTCCTGG


GCCACAGCCATGGCACCAACTGGGTAGAGTACAATGTGACTGGCTGGCTGAACTACACCAAGCAGAACCCAGC


CACCCAGAATGTCCCCCGGCTCCTGCAGGACTCCCAGAAAAAAATCATCAGCAACCTGTTTCTGGGCCGCGCA


GGCAGTGCCACGGTGCTCTCTGGCTCCATCGCGGGCCTGGAGGGCGGCTCGCAGCTGGCACTGCGCCGGGCCA


CCAGCATGCGGAAAACCTTTACCACAGGCATGGTGGCTGTCAAAAAGAAGTCACTGTGCATCCAGATGAAGCT


ACAGGTGGACGCCCTCATCGACACCATCAAGAAGTCAAAGCTGCATTTTGTGCACTGCTTCCTGCCTGTAGCT


GAGGGCTGGGCTGGGGAGCCCCGTTCCGCCTCCTCCCGCCGAGTCAGCAGCAGCAGTGAGCTGGACCTGCCCT


CGGGAGACCACTGCGAGGCTGGGCTCCTGCAGCTCGACGTGCCCCTGCTCCGCACCCAGCTCCGCGGCTCCCG


CCTGCTCGATGCCATGCGCATGTACCGCCAAGGTTACCCTGACCACATGGTGTTTTCCGAGTTCCGCCGCCGC


TTTGATGTCCTGGCCCCGCACCTGACCAAGAAACACGGGCGTAACTACATCGTGGTGGATGAAAGGCGGGCAG


TGGAGGAGCTGCTGGAGTGCTTGGATCTGGAGAAGAGCAGCTGCTGCATGGGCCTGAGCCGGGTGTTCTTCCG


GGCGGGCACCTTGGCACGGCTGGAGGAGCAGCGGGATGAACAAACCAGCAGGAACCTAACCCTGTTCCAAGCA


GCCTGCAGGGGCTACCTGGCCCGCCAGCACTTCAAGAAGAGAAAGATCCAGGACCTGGCCATTCGCTGTGTAC


AGAAGAACATCAAGAAGAACAAAGGGGTGAAGGACTGGCCCTGGTGGAAGCTTTTTACCACAGTGAGGCCCCT


CATCGAAGTACAGCTGTCAGAGGAGCAGATCCGGAACAAAGACGAGGAGATCCAGCAGCTGCGGAGCAAGCTC


GAGAAGGCGGAGAAGGAGAGGAACGAGCTGCGGCTCAACAGTGACCGGCTGGAGAGCCGGATCTCAGAGCTGA


CATCGGAGCTGACAGATGAGCGTAACACAGGAGAGTCCGCCTCCCAGCTGCTGGACGCGGAGACAGCAGAGAG


GCTCCGGGCTGAGAAGGAGATGAAGGAACTGCAGACCCAGTACGATGCACTGAAGAAGCAGATGGAGGTTATG


GAAATGGAGGTGATGGAGGCCCGTCTCATCCGGGCAGCGGAGATCAACGGGGAAGTGGATGATGATGATGCAG


GTGGCGAGTGGCGGCTGAAGTATGAGCGGGCTGTGCGGGAGGTGGACTTCACCAAGAAACGGCTCCAGCAGGA


GTTTGAGGACAAGCTGGAGGTGGAGCAGCAGAACAAGAGGCAGCTGGAACGGCGGCTCGGGGACCTGCAGGCA


GATAGTGAGGAGAGTCAGCGGGCTCTGCAGCAGCTCAAGAAGAAGTGCCAGCGACTGACGGCTGAGCTGCAAG


ACACCAAGCTGCACCTGGAGGGCCAGCAGGTCCGCAACCACGAACTGGAGAAGAAGCAGAGGAGGTTTGACAG


TGAGCTCTCGCAGGCACATGAGGAGGCCCAGCGGGAGAAGCTGCAGCGGGAGAAGCTGCAGCGGGAGAAGGAC


ATGCTCCTCGCTGAGGCTTTCAGCCTGAAGCAGCAACTAGAGGAAAAAGACATGGACATTGCAGGGTTCACCC


AGAAGGTTGTGTCTCTAGAGGCAGAGCTCCAGGACATTTCTTCCCAAGAGTCCAAGGATGAGGCTTCTCTGGC


CAAGGTCAAGAAACAGCTCCGGGACCTGGAGGCCAAAGTCAAGGATCAGGAAGAAGAGCTGGATGAGCAGGCA


GGGACCATCCAGATGCTGGAACAGGCCAAGCTGCGTCTGGAGATGGAGATGGAGCGGATGAGACAGACCCATT


CTAAGGAGATGGAGAGTCGGGATGAGGAGGTGGAGGAGGCCCGGCAGTCGTGTCAGAAGAAGTTAAAACAGAT


GGAGGTGCAGCTAGAGGAAGAGTATGAGGACAAGCAGAAGGTTCTGCGAGAGAAGCGGGAGCTGGAGGGCAAG


CTCGCCACCCTCAGCGACCAGGTGAACCGGCGGGACTTTGAGTCAGAGAAGCGGCTGCGGAAGGACCTGAAGC


GCACCAAGGCCCTGCTGGCAGATGCCCAGCTCATGCTGGACCACCTGAAGAACAGTGCTCCCAGCAAGCGAGA


GATTGCCCAGCTCAAGAACCAGCTGGAGGAGTCAGAGTTCACCTGTGCGGCAGCCGTGAAAGCACGGAAAGCA


ATGGAGGTGGAGATCGAAGACCTGCACCTGCAGATTGATGACATCGCCAAAGCCAAGACAGCGCTGGAGGAGC


AGCTGAGCCGCCTTCAGCGTGAGAAGAATGAGATCCAGAACCGGCTGGAGGAAGATCAGGAAGACATGAACGA


ATTGATGAAGAAGCACAAGGCTGCCGTGGCTCAGGCTTCCCGGGACCTGGCTCAGATAAATGATCTCCAAGCT


CAGCTAGAAGAAGCCAACAAAGAGAAGCAGGAGCTGCAGGAGAAGCTACAAGCCCTCCAGAGCCAGGTGGAGT


TCCTGGAGCAGTCCATGGTGGACAAGTCCCTGGTGAGCAGGCAGGAAGCTAGGATACGGGAGATGGAGACACG


CCTGGAGTTTGAAAGGACGCAAGTGAAACGGCTGGAGAGCCTGGCTAGCCGTCTCAAGGAAAACATGGAGAAG


CTGACTGAGGAGCGGGATCAGCGCATTGCAGCCGAGAACCGGGAGAAGGAACAGAACAAGCGGCTACAGAGGC


AGCTCCGGGACACCAAGGAGGAGATGGGCGAGCTTGCCAGGAAGGAGGCCGAGGCGAGCCGCAAGAAGCACGA


ACTGGAGATGGATCTAGAAAGCCTGGAGGCTGCTAACCAGAGCCTGCAGGCTGACCTAAAGTTGGCATTCAAG


CGCATCGGGGACCTGCAGGCTGCCATTGAGGATGAGATGGAGAGTGATGAGAATGAGGACCTCATCAACAGTT


TGCAGGACATGGTGACAAAGTATCAGAAAAGAAAGAATAAACTTGAGGGAGACTCTGATGTGGACTCGGAGCT


GGAGGACCGTGTTGACGGGGTCAAGTCCTGGTTGTCAAAAAACAAGGGACCTTCCAAGGCAGCTTCTGATGAT


GGCAGCTTAAAGAGTTCCAGCCCCACCAGCTACTGGAAGTCCCTTGCCCCTGATCGGTCAGATGATGAGCACG


ACCCTCTCGACAACACCTCCAGACCGCGATACTCCCACAGTTATCTGAGTGACAGCGACACAGAGGCCAAGCT


GACGGAGACTAACGCATAGCCCAGGGGAGTGGTTGGCAGCCCTCTCACCCCAGGGCCTGTGGCTGCCTGGGCA


CCTCTCCCAGGAAGTGGTGGGGCACCGGTCTCCCCCACCCGACTGCTGATCTGCATGGGAAACACCCTGACCT


TCTTCTGTCAGGGGCACTTTCCAGGCTATGGGTGTCTGATGTCTCCACGTGGAAGAGGTGGGGGAAAGAGGAG


TTTCTGAAGAGAACTTTTTGCTCCTCTGTCTCAAAATGCCAGACTCTTGGCTTCTACCCTGTGTCACCGTGGG


CAGTGGCAGGTGGCCTGGCACTGCATGGAGCCAGCACGTTGACCTCCCTCTCAGCTCCCTGCTCAGGGACGGT


GGACAGGTTGCCTACTGGGACACTCTAGGTTGCTGGGTCCATGGGGAGGATTGGGGGAGGAGAAGCAGTGCCT


TCCCTCTCGTGTGGGGTGGGGGCTCTCTCTTCTTGGTGCCTGCTGTCTTTCTACTTTTTAATTTAAATACCCA


ACCTCTCCATCACAGCTGCATCCCTGAGAGTGGGAGGGGGCTGTAGTGGTAGCTGGGGCTCCCAAGAACGACT


CGGGAATGTCATCTCCATCTTCACCCTTCAGAGAGCAGTCCTTTCTCTGTGCAGCTGGAGACGCTGGTGAGGA


GAGCCGGGTCCAGGTTCTTAAGAATGAGGTGCGGAGGGGCTCTCCGGTGCTGCTGGGCTGGGTTGAGCAAGCC


TACGCAGACAAGTGTGTGTGTGGACCATCCGCACCTCCAGCCCCCACCCCACCCTCTTTGTCTCAGCGTGTTA


TGTGCAATGACCTATTTAAGGTAAACCCATTCCAACTACAGCAGTTCAGGGCTGATCCAAGCACTGCCTCCCT


CCTGCTCTGTCCAGGTGGTCTGGACCATAAACTCAACTTGAGAGGGAAGGCTTGGGGTTGAGGACTTGTGATC


AGAAAAACTGAAGATGGAAGTTTTGGCCGGTGCTCATTAGACATGAGTCCTCACTCTGTGTCCTGAGCCCGTG


TCATTCTTCCAACCTCCCTGCCCCCACACACTTATCCCAGACACAACACCATGTGGTCTGGAGGTCCCAGCCC


CCACCCTAAAAAGGTTATCCCTGAGAACTCCACCAGACTTGGGAGCCCAAGTGCAGTGCCTGGTGCTGCTCCC


ATCTGCCGCCCCCCTTCTCTCCTGCAATTGGTTTGTACTCACTGGGCTGTGCTCTCCCCTGTTTACCCGATGT


ATGGAAATAAAGGCCCTTTTCCTCCTG









The NOV11 nucleic acid was identified on chromosome 17 and has 4969 of 4979 bases (99%) identical to a gb:GENBANK-ID:D86970|acc:D86970.1 mRNA from Homo sapiens (Human mRNA for KIAA0216 gene, complete cds) (E=0.0).
A disclosed NOV11 polypeptide (SEQ ID NO:42) encoded by SEQ ID NO:41 is 1596 amino acid residues and is presented using the one-letter code in Table 11B. Signal P, Psort and/or Hydropathy results predict that NOV11 contains a signal peptide and is likely to be localized to the nucleus with a certainty of 0.9800. Although SignalP, Psort and/or hydropathy suggest that the NOV11 protein may be localized in the nucleus, the NOV11 protein is similar to the myosin family, some members of which are expected to have intracellular sub-cellular localization (Trends Cell Biol April 1998; 8(4):138-41). Therefore it is likely that NOV11 is available at the same sub-cellular localization and hence accessible to a diagnostic probe and for various therapeutic applications.







TABLE 11B





Encoded NOV11 protein sequence















(SEQ ID NO:42)







MHMFKGCRREDMAPHIYAVAQTAYRAMLMSRQDQSIILLGSSGSGKTTSCQHLVQYLATIAGISGNKVFSVE


KWQALYTLLEAFGNSPTIINGNATRFSQILSLDFDQAGQVASASIQTMLLEKLRVARRPASEATFNVFYYLL


ACGDGTLRTELHLNHLAENNVFGIVPLAKPEEKQKAAQQFSKLQAAMKVLGISPDEQKACWFILAAIYHLGA


AGATKEAAEAGRKQFARHEWAQKAAYLLGCSLEELSSAIFKMQHKGGTLQRSTSFRQGPEESGLGDGTGPKL


SALECLEGMAAGLYSELFTLLVSLVNRALKSSQHSLCSMMIVDTPGFQNPEQGGSARGASFEELCHNYTQDR


LQRLFHERTFVQELERYKEENIELAFDDLEPPTDDSVAAVDQASHQSLVRSLARTDEARGLLWLLEEEALVP


GASEDTLLERLFSYYGPQEGDKKGQSPLLHSSKPHHFLLGHSHGTNWVEYNVTGWLNYTKQNPATQNVPRLL


QDSQKKIISNLFLGRAGSATVLSGSIAGLEGGSQLALRRATSMRKTFTTGMVAVKKKSLCIQMKLQVDALID


TIKKSKLHFVHCFLPVAEGWAGEPRSASSRRVSSSSELDLPSGDHCEAGLLQLDVPLLRTQLRGSRLLDAMR


MYRQGYPDHMVFSEFRRRFDVLAPHLTKKHGRNYIVVDERRAVEELLECLDLEKSSCCMGLSRVFFRAGTLA


RLEEQRDEQTSRNLTLFQAACRGYLARQHFKKRKIQDLAIRCVQKNIKKNKGVKDWPWWKLFTTVRPLIEVQ


LSEEQIRNKDEEIQQLRSKLEKAEKERNELRLNSDRLESRISELTSELTDERNTGESASQLLDAETAERLRA


EKEMKELQTQYDALKKQMEVMEMEVMEARLIRAAEINGEVDDDDAGGEWRLKYERAVREVDFTKKRLQQEFE


DKLEVEQQNKRQLERRLGDLQADSEESQRALQQLKKKCQRLTAELQDTKLHLEGQQVRNHELEKKQRRFDSE


LSQAHEEAQREKLQREKLQREKDMLLAEAFSLKQQLEEKDMDIAGFTQKVVSLEAELQDISSQESKDEASLA


KVKKQLRDLEAKVKDQEEELDEQAGTIQMLEQAKLRLEMEMERMRQTHSKEMESRDEEVEEARQSCQKKLKQ


MEVQLEEEYEDKQKVLREKRELEGKLATLSDQVNRRDFESEKRLRKDLKRTKALLADAQLMLDHLKNSAPSK


REIAQLKNQLEESEFTCAAAVKARKAMEVEIEDLHLQIDDIAKAKTALEEQLSRLQREKNEIQNRLEEDQED


MNELMKKHKAAVAQASRDLAQINDLQAQLEEANKEKQELQEKLQALQSQVEFLEQSMVDKSLVSRQEARIRE


METRLEFERTQVKRLESLASRLKENMEKLTEERDQRIAAENREKEQNKRLQRQLRDTKEEMGELARKEAEAS


RKKHELEMDLESLEAANQSLQADLKLAFKRIGDLQAAIEDEMESDENEDLINSLQDMVTKYQKRKNKLEGDS


DVDSELEDRVDGVKSWLSKNKGPSKAASDDGSLKSSSPTSYWKSLAPDRSDDEHDPLDNTSRPRYSHSYLSD


SDTEAKLTETNA









The NOV11 amino acid sequence has 1579 of 1596 amino acid residues (98%) identical to, and 1581 of 1596 amino acid residues (99%) similar to, the 1581 amino acid residue ptnr:SPTREMBL-ACC:Q92614 protein from Homo sapiens (Human) (MYELOBLAST KIAA0216) (E=0.0). The NOV11 amino acid sequence has additional 15 internal amino acids, when compared to ptnr:SPTREMBL-ACC:Q92614 protein from Homo sapiens (Human) (MYELOBLAST KIAA0216).
NOV11 is expressed in at least the following tissues: adrenal gland, bone marrow, brain—amygdala, brain—cerebellum, brain—hippocampus, brain—substantia nigra, brain—thalamus, brain—whole, fetal brain, fetal kidney, fetal liver, fetal lung, heart, kidney, lymphoma—Raji, mammary gland, pancreas, pituitary gland, placenta, prostate, salivary gland, skeletal muscle, small intestine, spinal cord, spleen, stomach, testis, thyroid, trachea and uterus, aorta, ascending colon, bronchus, cervix, colon, coronary artery, epidermis, gall bladder, hypothalamus, lung, lymph node, lymphoid tissue, muscle, ovary, peripheral blood, pharynx, pineal gland, retina, right cerebellum, tonsils and whole organism. This information was derived by determining the tissue sources of the sequences that were included in the invention including but not limited to SeqCalling sources, Public EST sources, genomic clone sources, literature sources, and/or RACE sources.
Possible SNPs found for NOV11 are listed in Tables 11C and 11D.







TABLE 11C







SNPs














Amino




Nucleotide
Base
Acid
Base


Variant
Position
Change
Position
Change














13377093
491
A > G
  3
Met > Val


13377092
570
T > C
 29
Met > Thr


13377091
1783
G > A
Silent
N/A


13377090
3657
T > C
1058
Val > Ala


13374495
4848
C > T
1455
Ala > Val


13374496
4939
T > C
Silent
N/A


13374497
4998
A > T
1505
Lys > Met


13374498
5114
A > G
1544
Ser > Gly
















TABLE 11D







SNPs










Consensus

Base



Position
Depth
Change
PAF













455
24
C > T
0.083


546
27
T > C
0.074


605
27
A > T
0.074


721
27
A > G
0.074


753
26
C > T
0.077


891
13
C > T
0.154









NOV11 has homology to the amino acid sequences shown in the BLASTP data listed in Table 11E.







TABLE 11E







BLAST results for NOV11












Gene Index/

Length
Identity
Positives



Identifier
Protein/Organism
(aa)
(%)
(%)
Expect





gi|17978507|ref|NP_
TGFB1-induced
1581
1511/1596
1513/1596
0.0


510880.1|
anti-apoptotic

(94%)
(94%)


(NM_078471)
factor 1, isoform



1; TGF-beta-1-



induced



antiapoptotic



factor 1;



molecule



associated with



Jak-3 N-terminal



[Homo sapiens]


gi|7416032|dbj|BAA93660.1|
myosin containing
2035
1425/1596
1469/1596
0.0


(AB026497)
PDZ domain [Mus

(89%)
(91%)



musculus]


gi|18587640|ref|XP_
similar to TGFB1-
1355
1257/1318
1259/1318
0.0


031413.3|
induced anti-

(95%)
(95%)


(XM_031413)
apoptotic factor



1, isoform 1;



TGF-beta-1-



induced



antiapoptotic



factor 1;



molecule



associated with



Jak-3 N-terminal



[Homo sapiens]


gi|15718364|emb|CAC
myosin heavy
2566
 651/1530
 996/1530
5e−97


70712.1| (AJ310931)
chain [Homo

 (42%)
(64%)



sapiens]


gi|18250662|emb|CAC
myosin heavy
2566
 651/1530
 996/1530
2e−72


70714.2| (AJ310932)
chain [Homo

 (42%)
(64%)



sapiens]









The homology of these sequences is shown graphically in the ClustalW analysis shown in Table 11F.







TABLE 11F





Clustal W Sequence Alignment















1) NOV11 (SEQ ID NO:42)


2) gi|7978507|refNP_510880.1| (NM_078471) TGFB1-induced anti-apoptotic 1, isoform 1;


TGF-beta-1-induced antiapoptotic factor 1; molecule associated with Jak-3


N-terminal [Homo sapiens] (SEQ ID NO:159)


3) gi 7416032|dbj|BAA93660.1| (AB026497) myosin containing PDZ domain [Mus musculus]


(SEQ ID NO:160)


4) gi|18587640|refXP_031413.3| (XM_031413) similar to TGFB1-induced anti-apoptotic


factor 1, isoform 1; TGF-beta-1-induced antipoptotic factor 1; molecular associated


with Jak-3 N-terminal [Homo sapiens] (SEQ ID NO:161)


5) gi 15718364|emb CAC70712.1| (AJ310931) myosin heavy chain [Homo sapiens]


(SEQ ID NO:162)


6) gi|18250662|emb|CAC70714.2| (AJ310932) myosin heavy chain [Homo sapiens]


(SEQ ID NO:163)



















































































































































































































































































Tables 11G, 11H, 11I and 11J list the domain description from DOMAIN analysis results against NOV11. This indicates that the NOV11 sequence has properties similar to those of other proteins known to contain these domains.







TABLE 11G





Domain Analysis of NOV11















gnl|Smart|smart00242, MYSc, Myosin. Large ATPases.; ATPase; molecular


motor. Muscle contraction consists of a cyclical interaction between


myosin and actin. The core of the myosin structure is similar in fold


to that of kinesin. (SEQ ID NO:164)


CD-Length = 688 residues, 91.4% aligned


Score = 326 bits (836), Expect = 5e−90










Query:
1
MHMFKGCRREDMAPHIYAVAQTAYRAMLMSRQDQSIILLGSSGSGKTTSCQHLVQYLATI
 60




+  ++|  | ++ ||++|+|  ||| ||  +++||||+ | ||+||| + + ++|||| +


Sbjct:
60
IKKYRGKSRGELPPHVFAIADNAYRNMLNDKENQSIIISGESGAGKTENTKKIMQYLAAV
119


Query:
61
AGISGNKVFSVEKWQALYT--LLEAFGNSPTIINGNATRFSQILSLDFDQAGQVASASIQ
118




+| ||+   |||  | | +  +||||||+ |+ | |++|| + + + ||  |++  | |+


Sbjct:
120
SGSSGSVG-SVED-QILESNPILEAFGNAKTLRNNNSSRFGKFIEIHFDAKGKIVGAKIE
177


Query:
119
TMLLEKLRVARRPASEATFNVFYYLLACGDGTLRTELHLNHLAENNVFGIVPLAKPEEKQ
178




| |||| ||  +   |  +++|| |||     |+ +| |    |+  +         +


Sbjct:
178
TYLLEKSRVVSQAKGERNYHIFYQLLAGASEELKKKLGLKKSPEDYRYLNQGGCLTVDGI
237


Query:
179
KAAQQFSKLQAAMKVLGISPDEQKACWFILAAIYHLGAAGATKEAAEAGRKQFARHEWAQ
238




  |++| +   ||+||| | +||++ + ||||| |||     +   +   +     |


Sbjct:
238
DDAEEFKETLNAMRVLGFSEEEQESIFKILAAILHLGNIEFEEGRNDNAAETVKDKEELD
297


Query:
239
KAAYLLGCSLEELSSAIFKHQHKGGTLQRSTSFRQGPEESGLGDGTGPKLSALECLEGMA
298




 || |||   |||  |+          +|            |         ||+  + +|


Sbjct:
298
NAAELLGVDPEELEKAL---------TKRKIKTGGEVITVPLT-----VEQALDARDALA
343


Query:
299
AGLYSELFTLLVSLVNRALK-SSQHSLCSMMIVDTPGFQNPEQGGSARGASFEELCHNYT
357




  +|| ||  ||  +|++|      |   + ++|  ||+  |+       |||+|| ||


Sbjct:
344
KAIYSRLFDWLVKRINQSLSFKRDGSTNFIGVLDIYGFEIFEKN------SFEQLCINYA
397


Query:
358
QDRLQRLFHERTFVQELERYKEENIELAFDDLEPPTDDSVAAVDQASHQSLVRSLARTDE
417




 ++||+ |++  |  | | |+ | ||                +|   +|  +  +    +


Sbjct:
398
NEKLQQFFNQHVFKLEQEEYEREGIE-------------WTFIDFFDNQDCIDLI--EKK
442


Query:
418
ARGLLWLLEEEALVPGASEDTLLERLFSYYGPQEGDKKGQSPLLHSSK---PHHFLLGHS
474




  |+| ||+||   |  ++ | ||+|             + |     |      |++ |


Sbjct:
443
PLGILSLLDEECRFPKGTDQTFLEKLNKQ-------HLKKHPHFSKPKKNGRTEFIIKHY
495


Query:
475
HGTNWVEYNVTGWLNYTKQNPATQNVPRLLQDSQKKIISNLFLGRAGSATVLGSIAGLE
534




 |   | |+|||+|   | +  + ++  ||| |+  +|+ ||   ||


Sbjct:
496
AGD--VTYDVTGFLEKNK-DTLSDDLIELLQSSKNPLIALLFPEEAGQT----------
541


Query:
535
GGSQLALRRATSMRKTFTTGMVAVKKKSLCIQMKLQVDALIDTIKKSKLHFVHCFLPVAE
594




  |     ++   +             ++  | |  ++ |+||+  +  ||+ |  |  |


Sbjct:
542
--SSAPKEKSAKKKFQ-----------TVGSQFKESLNELMDTLNSTNPHFIRCIKPNEE
588


Query:
595
GWAGEPRSASSRRVSSSSELDLPSGDHCEAGLLQLDVPLLRTQLRGSRLLDAMRMYRQGY
654




   |                               |  |+  |||   +|+ +|+ | |+


Sbjct:
589
KKPG-----------------------------DFDSSLVLHQLRYLGVLETIRIRRAGF
619


Query:
655
PDHMVFSEFRRRFDVLAPHLTKKHGRNYIVVDERRAVEELLECLDLEKSSCCMGLSRVFF
714




|  + | || +|+ || |      |      | + | | ||+ | |++    +| ++||


Sbjct:
620
PYRLPFDEFLQRYRVLLPDTWPPWG-----GDAKEACELLLQSLGLDEDEYQIGKTKVFL
674


Query:
715
RAGTLARLEEQRDE
728




| | || ||| |+|


Sbjct:
675
RPGQLAELEELREE
688
















TABLE 11H





Domain Analysis of NOV11















gnl|Pfam|pfam00063, myosin_head, Myosin head (motor domain). (SEQ ID NO:165)


CD-Length = 670 residues, 92.2% aligned


Score = 283 bits (723), Expect = 7e−77










Query:
1
MHMFKGCRREDMAPHIYAVAQTAYRAMLMSRQDQSIILLGSSGSGKTTSCQHLVQYLATI
 60




+  ++| || ++ |||+|+|  |||+||  +++|||++ | ||+||| + + ++|||| +


Sbjct:
53
IKKYRGKRRYELPPHIFAIADEAYRSMLSDKENQSILISGESGAGKTENTKKVMQYLAAV
112


Query:
61
AGISGNKVFSVEKWQALYT--LLEAFGNSPTIINGNATRFSQILSLDFDQAGQVASASIQ
118




+| +| ||  ||  | | +  +||||||+ |  | |++|| + + + ||+ |++  | |+


Sbjct:
113
SGGNGGKVGRVED-QILQSNPILEAFGNAKTTRNNNSSRFGKYIEIQFDKTGKIVGAKIE
171


Query:
119
TMLLEKLRVARRPASEATFNVFYYLLACGDGTLRTELHLNHLAENNVFGIVPLAKPEEKQ
178




  |||| ||  +   |  |++|| |||     |+ ||+|    ++  +         +


Sbjct:
172
NYLLEKSRVVYQTPGERNFHIFYQLLAGASQQLKKELNLTD-PDDYHYLNQGGCYTVDGI
230


Query:
179
KAAQQFSKLQAAMKVLGISPDEQKACWFILAAIYHLGAAGATKEAAEAGRKQFARHEWAQ
238




  +++| +   || +|| | +|| + + |+||| ||      |+  +    +    +  |


Sbjct:
231
DDSEEFKETDKAMDILGFSDEEQLSIFRIVAAILHL-GNIKFKQRRKEEAAEPDDTKALQ
289


Query:
239
KAAYLLGCSLEELSSAIFKHQHKGGTLQRSTSFRQGPEESGLGDGTGPKLSALECLEGMA
298




 || |||   +||  |+   + | |    +    |  |+            |    + +|


Sbjct:
290
IAAELLGVDAKELEKALLSRRIKTGGEGVTVP--QNVEQ------------ANYARDALA
335


Query:
299
AGLYSELFTLLVSLVNRALKSSQHSLCSMMIV-DTPGFQNPEQGGSARGASFEELCHNYT
357




  ||| ||  +|+ +|++|        + + | |  ||+  |+       |||+|| |||


Sbjct:
336
KALYSRLFDWIVNRINKSLDFKAKEGANFIGVLDIYGFEIFEKN------SFEQLCINYT
389


Query:
358
QDRLQRLFNERTFVQELERYKEENIELAFDDLEPPTDDSVAAVDQASHQSLVRSLARTDE
417




 ++||+ |+   |  | | || | ||  | |      |+   +|    +


Sbjct:
390
NEKLQQFFNHHMFKLEQEEYKREGIEWTFIDF----GDNQPCIDLIEKKPP---------
436


Query:
418
ARGLLWLLEEEALVPGASEDTLLERLPSYYGPQEGDKKCQSPLLHSSKPHHFLLGHSHGT
477




  |+| ||+||   | |++ | |++|+| +         + |     +   |++ |  |


Sbjct:
437
--GILSLLDEECRFPKATDQTFLDKLYSEF---SNHPHFKKP--RFRQKKSFIIKHYAGD
489


Query:
478
NWVEYNVTGWLNYTKQNPATQNVPRLLQDSQKKIISNLFLGRAGSATVLSGSIAGLEGGS
537




  ||||| |+|   | +|   ++  ||+ |   +++ ||                    |


Sbjct:
490
--VEYNVEGFLEKNK-DPLFDDLIELLKSSSNPLLAELF------------PDYEEADPS
534


Query:
538
QLALRRATSMRKTFTTGMVAVKKKSLCIQMKLQVDALIDTIKKSKLHFVHCFLPVAEGWA
597




 |+ +|  + +  | |         +  | |  ++ |+ |+  +  ||| |  |  |


Sbjct:
535
SLSKKRKITKKSNFIT---------VGAQFKESLNTLMKTLSSTNPHFVRCIKPNEEKKP
585


Query:
598
GEPRSASSRRVSSSSELDLPSGDHCEAGLLQLDVPLLRTQLRGSRLLDAMRMYRQGYPDH
657




|                               |  |+  |||   +|+ +|+ | |+|


Sbjct:
586
G-----------------------------VFDASLVLHQLRCLQVLEGIRIRRAGFPSR
616


Query:
658
MVFSEFRRRFDVLAPHLTKKHGRNYIVVDERRAVEELLECLDLEKSSCCMGLSRVFFR
715




+ | || +|+ +|||    |    +    ++ | | ||+ |+|+|     | +++|||


Sbjct:
617
ITFDEFLQRYRILAPKTWPK----WSGDAKKGACELLLQALNLDKEEYQFGKTKIFFR
670
















TABLE 11I





Domain Analysis of NOV11















gnl|Pfam|pfam01576, Myosin tail, Myosin tail. The myosin molecule is a


multi-subunit complex made up of two heavy chains and four light


chains it is a fundamental contractile protein found in all eukaryote


cell types. This family consists of the coiled-coil myosin heavy chain


tail region. The coiled-coil is composed of the tail from two


molecules of myosin. These can then assemble into the macromolecular


thick filament. The coiled-coil region provides the structural


backbone the thick filament. (SEQ ID NO:166)


CD-Length = 860 residues, 77.1% aligned


Score = 87.4 bits (215), Expect = 6e−18










Query:
791
VQLSEEQIRNKDEEIQQLRSKLEKAEKERNELRLNSDRLESRISELTSELTDERNTGESA
 850




|   +++|+  +  |++|  +||     | +       |   + ||+  | +      +


Sbjct:
34
VAQLQKKIKELEARIRELEEELEAERAARAKAEKARADLSRELEELSERLEEAGGATAAQ
 93


Query:
851
SQLLDAETAERLRAEKEMKELQTQYD----ALKKQMEVMEMEVMEARLIRAAEINGEVDD
 906




 |+     ||  +  |+++|   |++     |+|+ +               |++ +++


Sbjct:
94
IELNKKREAELAKLRKDLEEANLQHEEALATLRKKHQD-----------AINELSEQIEQ
 142


Query:
907
-DDAGGEWRLKYERAVREVDFTKKRLQQEFEDKLEVEQQNKRQLERRLGDLQADSEESQR
 965




      +   +  +   |||    +|    + ||  |++ | ||| +| +||   +| ||


Sbjct:
143
LQKQKAKAEKEKSQLQAEVDDLLAQLDSITKAKLNAEKKAK-QLESQLSELQVKLDELQR
 201


Query:
966
ALQQLKKKCQRLTAELQDTKLHLEGQQVRNHELEKKQRRFDSELSQAHEEAQREKLQREK
1025




 |  |  +  || +|  |    ||  + +   | | + + +|+| +|    + |  +|


Sbjct:
202
QLNDLTSQKSRLQSENSDLTRQLEEAEAQVSNLSKLKSQLESQLEEAKRSLEEESRERAN
 261


Query:
1026
LQREKDMLLAEAFSLKQQLEEKDMDIAGFTQKVVSLEAELQDISSQ-ESKDEASLAKVKK
1084




|| +   |  +  ||+|||||+    |   +++    ||+|   |+ ||+      ++++


Sbjct:
262
LQAQLRQLEHDLDSLREQLEEESEAKAELERQLSKANAEIQQWRSKFESEGALRAEELEE
 321


Query:
1085
QLRDLEAKVKDQEEELDEQAGTIQMLEQAKLRLEMEMERMR------QTHSKEMESRDEE
1138




  + |  |+ + ||  +        ||+ | ||+ |+| ++         + |+| + +


Sbjct:
322
LKKKLNQKISELEEAAEAANAKCDSLEKTKSRLQSELEDLQIELERANAAASELEKKQKN
 381


Query:
1139
VEEARQSCQKKLKQMEVQLEEEYEDKQKVLREKRELEGKLATLSDQVNRRDFESEKRLRK
1198




 ++     ++|+ +++ +|+    + + +  |   |+ +|  | |||     |  | |+


Sbjct:
382
FDKILAEWKRKVDELQAELDTAQREARNLSTELFRLKNELEELKDQVEALRRE-NKNKQD
 440


Query:
1199
DLKRTKALLADAQLMLDHL-KNSAPSKREIAQLKNQLEESEFTCAAAVKARKAMEVEIED
1257




++      | +    +  | |     + |  +|+  |||+|             +||+


Sbjct:
441
EIHDLTDQLGEGGRNVHELEKARRRLEAEKDELQAALEEAEAALELEESKVLFAQVELSQ
 500


Query:
1258
LHLQIDDIAKAKTALEEQLSR-LQREKNEIQNRLEEDQEDMNELMKKHKAAVAQASRDLA
1316




+  +|+     |    |   +  ||    +|  || + +   |  +  |       +


Sbjct:
501
IRSEIERRLAEKEEEFENTRKNHQRAIESLQATLEAETKGKAEASRLK--------KLEG
 553


Query:
1317
QINDLQAQLEEANKEKQELQEKLQALQSQVEFLEQSMVDKS-LVSRQEARIREMETRLEF
1375




 ||+|                     +  ++   ++  +    | + + +++|++|++|


Sbjct:
554
DINEL---------------------EIALDHANKANAEAQKNVKKYQQQVKELQTQVEE
 592


Query:
1376
ERTQVKRLESLASRLKENMEKLTEERDQRIAAENREKEQNKRLQRQLRDTKEEMGELARK
1435




|+   +      +  +     |  | ++  +|  + +   |+ + +| +  | + ||  +


Sbjct:
593
EQRAREDAREQLAVAERRATALEAELEELRSALEQAERARKQAETELAEASERVNELTAQ
 652


Query:
1436
EAEASRKKHELEMDLESLEAANQSLQADLKLAFKRIGDLQAAIE
1479




 |     +| +|| +| +|++      +|| | +|    ||


Sbjct:
653
NSSLIAQKRKLEGELAALQSDLDEAVNELKAAEERAEKAQADAA
 696
















TABLE 11J





Domain Analysis of NOV11















gnl Pfam pfam00038, filament, Intermediate filament protein. (SEQ ID NO:167)


CD-Length = 312 residues, 89.7% aligned


Score = 43.9 bits (102), Expect = 7e−05










Query:
1041
KQQLEEKDMDIAGFTQKVVSLEAELQDISSQESKDEASLAKVKKQLRDLEAKVKDQEEEL
1100




|+|++  +  +| +  ||  || + +++   | | |    |    +  | +     | |+


Sbjct:
3
KEQMQNLNDRLASYIDKVEFLEQQNKEL---EVKIEELRQKQAPSVSRLYSLY---ETEI
 56


Query:
1101
DEQAGTIQMLEQAKLRLEMEMERMRQTHSKEMESRDEEVEEARQSCQKKLKQMEVQLEEE
1160




+|    |  |   + ||++|++ +|+      +  ++|+   ||  +  |  +   |+|


Sbjct:
57
EELRRQIDQLTNERARLQLEIDNLREAAEDFRKKYEDEINL-RQEAENDLVGLRKDLDEA
 115


Query:
1161
YEDKQKVLREKRELEGKLATLSDQVN--RRDFESE-KRLRKDLKRTKALLADAQLMLD--
1215




   +        +|| |+ +| +++   +++ | | | |+  ++ |  +  ||   ||


Sbjct:
116
TLARV-------DLENKVESLQEELEFLKKNHEEEVKELQAQIQDTVNVEMDAARKLDLT
 168


Query:
1216
---------HLKNSAPSKREI-AQLKNQLEESEFTCAAAVKARKAMEVEIEDLHLQIDDI
1265




         + + +  +++|     |++||| +   |   +| ++ + || +|  ||  +


Sbjct:
169
KALREIRAQYEEIAKKNRQEAEEWYKSKLEELQTAAARNGEALRSAKEEITELRRQIQSL
 228


Query:
1266
AKAKTALEEQLSRLQREKNEIQNRLEEDQEDMNELMKKHKAAVAQASRDLAQIN
1319




     +|+ | + |+|+  |++ | | +      |+ + +  + |   ++|+


Sbjct:
229
EIELQSLKAQNASLERQLAELEERYELELRQYQALISQLEEELQQLREEMARQL
 282









In an effort to sequence human cDNA clones which correspond to relatively long and nearly full-length transcripts, the sequence of KIAA0216 has been determined. It has been obtained from human immature myeloid cell line KG-1. KIAA0216 contains a myosin head (motor domain). Myosins are molecular motors that upon interaction with actin filaments convert energy from ATP hydrolysis into mechanical force. Evidence has emerged for the existence of a large, widely expressed and evolutionarily ancient superfamily of myosin genes.
In addition to the well-catheterized conventional, filament-forming, two-headed myosin-II of muscle and nonmuscle cells, at least ten additional classes of myosins have been identified. In vertebrates, at least seven classes are expressed, and many myosins can be expressed in a single cell type. Distance matrix and maximum parsimony methods have been used to study the evolutionary relationships between members of the myosin superfamily of molecular motors. Amino acid sequences of the conserved core of the motor region have been used in the analysis.
Myosins can be divided into at least three main classes, with two types of unconventional myosin being no more related to each other than they are to conventional myosin. Myosins have traditionally been classified as conventional or unconventional, with many of the unconventional myosin proteins thought to be distributed in a narrow range of organisms. It has been found that members of all three of these main classes are likely to be present in most (or all) eukaryotes. The structure of the trees suggests that these ungrouped proteins and some of the subclasses of the main classes are also likely to be widely distributed, implying that most eukaryotic cells contain many different myosin proteins. The groupings derived from phylogenetic analysis of myosin head sequences agree strongly with those based on tail structure, developmental expression, and (where available) enzymology, suggesting that specific head sequences have been tightly coupled to specific tail sequences throughout evolution.
Analysis of the relationships within each class has interesting implications. For example, smooth muscle myosin and striated muscle myosin seem to have independently evolved from nonmuscle myosin. Furthermore, brush border myosin I, a type of protein initially thought to be specific to specialized metazoan tissues, probably has relatives that are much more broadly distributed. Myosin II, the conventional two-headed myosin that forms bipolar filaments, is directly involved in regulating cytokinesis, cell motility and cell morphology in nomnuscle cells. To understand the mechanisms by which nonmuscle myosin-II regulates these processes, investigators are looking at the regulation of this molecule in vertebrate nomnuscle cells. The identification of multiple isoforms of nonmuscle myosin-II, whose activities and regulation differ from that of smooth muscle myosin-II, suggests that, in addition to regulatory light chain phosphorylation, other regulatory mechanisms control vertebrate nonmuscle myosin-II activity. It has been shown that nonmuscle myosin II, along with other myosins and cytoskeletal proteins, assembles on Golgi membranes. Nonmuscle myosin II associates transiently with membranes of the trans-Golgi network during the budding of a subpopulation of transport vesicles. The exact role of myosin II in vesicular trafficking is not yet understood, but its participation heralds a novel role for actin-based motors in vesicle budding.
In the aortic wall of mammalian species, the maturation phase of smooth muscle cell (SMC) lineage is characterized by two temporally correlated but opposite regulatory processes of gene expression: upregulation of SM type SM2 myosin isoform and downregulation of brain (myosin heavy chain B)- and platelet (myosin heavy chain A(pla))-type nonmuscle myosins. There is propensity of the immature type SMC population to be activated in experimental models and human vascular diseases that are characterized by proliferation and migration of medial SMCs into the subendothelial space. Neointimal proliferation leading to restenosis frequently develops after coronary angioplasty. This process is associated with a change in vascular smooth-muscle cells from a contractile (quiescent) phenotype to a synthetic or proliferating (activated) one. The expression of the B isoform of nonmuscle myosin heavy chain is increased in some coronary atherosclerotic plaques and that this increase in expression identifies a group of lesions at high risk for restenosis after atherectomy. The human homologue of the mouse dilute gene combines elements from both nonmuscle myosin type I and nonmuscle myosin type II. Mutations in the mouse dilute gene result not only in the lightening of coat color, but also in the onset of severe neurological defects shortly after birth, indicating that this gene is important in maintaining the normal neuronal function.
The NOV11 nucleic acid of the invention encoding a KIAA0216-like protein includes the nucleic acid whose sequence is provided in Table 11A, or a fragment thereof. The invention also includes a mutant or variant nucleic acid any of whose bases may be changed from the corresponding base shown in Table 11A while still encoding a protein that maintains its KIAA0216-like activities and physiological functions, or a fragment of such a nucleic acid. The invention further includes nucleic acids whose sequences are complementary to those just described, including nucleic acid fragments that are complementary to any of the nucleic acids just described. The invention additionally includes nucleic acids or nucleic acid fragments, or complements thereto, whose structures include chemical modifications. Such modifications include, by way of non-limiting example, modified bases, and nucleic acids whose sugar phosphate backbones are modified or derivatized. These modifications are carried out at least in part to enhance the chemical stability of the modified nucleic acid, such that they may be used, for example, as antisense binding nucleic acids in therapeutic applications in a subject. In the mutant or variant nucleic acids, and their complements, up to about 1% of the residues may be so changed.
The NOV11 protein of the invention includes the KIAA0216-like protein whose sequence is provided in Table 11B. The invention also includes a mutant or variant protein any of whose residues may be changed from the corresponding residue shown in Table 11B while still encoding a protein that maintains its KIAA0216-like activities and physiological functions, or a functional fragment thereof. In the mutant or variant protein, up to about 2% of the bases may be so changed.
The NOV11 nucleic acids and proteins of the invention are useful in potential diagnostic and therapeutic applications implicated in various diseases and disorders described below and/or other pathologies. For example, the compositions of the present invention will have efficacy for treatment of patients suffering from: restenosis, neurological, glomerular diseases and other diseases, disorders and conditions of the like. Enhanced embryonic nonmuscle myosin heavy chain isoform and matrix metalloproteinase expression in aortic abdominal aneurysm with rapid progression has been found (Cardiovasc Pathol 1999 September-October; 8(5):291-5). In addition, the expression of a nonmuscle myosin heavy chain in glomerular cells differentiates various types of glomerular disease in rats. The nonmuscle type myosin heavy chain, Smemb, is especially useful to detect both mesangial and glomerular epithelial cell activation in these glomerular disease models. Understanding the functional difference and regulatory mechanisms of these cytoskeletal proteins will provide insight into the pathogenesis and progression of glomerular diseases (Kidney Int 1996 May;49(5): 1231-41). The expression of the B isoform of nomnuscle myosin heavy chain is increased in some coronary atherosclerotic plaques and this increase in expression identifies a group of lesions at high risk for restenosis after atherectomy (N Engl J Med 1993 March 4;328(9):608-13). The human homologue of the mouse dilute gene, which may be important in maintaining the normal neuronal function in the mouse, combines elements from both nonmuscle myosin type I and nonmuscle myosin type II (Genomics 1994 February;19(3):407-16).
NOV11 nucleic acids and polypeptides are further useful in the generation of antibodies that bind immunospecifically to the novel substances of the invention for use in therapeutic or diagnostic methods. These antibodies may be generated according to methods known in the art, using prediction from hydrophobicity charts, as described in the “Anti-NOVX Antibodies” section below. For example the disclosed NOV11 protein have multiple hydrophilic regions, each of which can be used as an immunogen. This novel protein also has value in development of powerful assay system for functional analysis of various human disorders, which will help in understanding of pathology of the disease and development of new drug targets for various disorders.
NOV12
NOV12 includes three novel TWIK 3-like proteins disclosed below. The disclosed proteins have been named NOV12a, NOV12b and NOV12c.
NOV12a
A disclosed NOV12a nucleic acid of 1011 nucleotides (also referred to as CG57220-01) encoding a novel TWIK 3-like protein is shown in Table 12A. An open reading frame was identified beginning with an ATG initiation codon at nucleotides 17-19 and ending with a TAG codon at nucleotides 953-955. Putative untranslated regions, if any, upstream from the initiation codon and downstream from the termination codon are underlined in Table 12A, and the start and stop codons are in bold letters.








TABLE 12A





NOV12a nucleotide sequence.
















(SEQ ID NO:43)









CCCCGCCTCTCCCGCTATGTACCGACCGCGAGCCCGGGCGGCTCCCGAGGGCAGGGTCCGGGGCTGCGCGGT



GCCCGGCACCGTGCTCCTGCTGCTCGCCTACCTGGCTTACCTGGCGCTGGGCACCGGCGTGTTCTGGACGCT


GGAGGGCCGCGCGGCGCAGGACTCCAGCCGCAGCTTCCAGCGCGACAAGTGGGAGCTGTTGCAGAACTTCAC


GTGTCTGGACCGCCCGGCGCTGGACTCGCTGATCCGGGATGTCGTCCAAGCATACAAAAACGGAGCCAGCCT


CCTCAGCAACACCACCAGCATGGGGCGCTGGGAGCTCGTGGGCTCCTTCTTCTTTTCTGTGTCCACCATCTT


CTTTGCCCTTGTGGGGATCCCACTCAACCTCGTGGTGCTCAACCGACTGGGGCATCTCATGCAGCAGGGAGT


AAACCACTGGGCCAGCAGGCTGGGGGGCACCTGGCAGGATCCTGACAAGGCGCGGTGGCTGGCGGGCTCTGG


CGCCCTCCTCTCGGGCCTCCTGCTCTTCCTGCTGCTGCCACCGCTGCTCTTCTCCCACATGGAGGGCTGGAG


CTACACAGAGGGCTTCTACTTCGCCTTCATCACCCTCAGCACCGTGGGCTTCGGCGACTACGTGATTGGAAT


GAACCCCTCCCAGAGGTACCCACTGTGGTACAAGAACATGGTGTCCCTGTGGATCCTCTTTGGGATGGCATG


GCTGGCCTTGATCATCAAACTCATCCTCTCCCAGCTGGAGACGCCAGGGAGGGTATGTTCCTGCTGCCACCA


CAGCTCTAAGGAAGACTTCAAGTCCCAAAGCTGGAGACAGGGACCTGACCGGGAGCCAGAGTCCCACTCCCC


ACAGCAAGGATGCTATCCAGAGGGACCCATGGGAATCATACAGCATCTGGAACCTTCTGCTCACGCTGCAGG


CTGTGGCAAGGACAGCTAGTTATACTCCATTCTTTGGTCGTCGTCCTCGGTAGCAAGACCCCTGATTTTAAG


CTT









The disclosed NOV12a nucleic acid sequence, localized to chromsome 6p21.1-21.2, has 384 of 641 bases (59%) identical to a gb:GENBANK-ID:AF006823|acc:AF006823.1 mRNA from Homo sapiens (Homo sapiens TWIK-related acid-sensitive K+ channel (TASK) mRNA, complete cds) (E=3.1e−15).
A NOV12a polypeptide (SEQ ID NO:44) encoded by SEQ ID NO:43 has 312 amino acid residues and is presented using the one-letter code in Table 12B. Signal P, Psort and/or Hydropathy results predict that NOV12a contains a signal peptide and is likely to be localized to the plasma membrane with a certainty of 0.6400. The most likely cleavage site for a NOV12a polypeptide is between amino acids 48 and 49: RAA-QD.








TABLE 12B





Encoded NOV12a protein sequence.
















(SEQ ID NO:44)









MYRPRARAAPEGRVRGCAVPGTVLLLLAYLAYLALGTGVFWTLEGRAAQDSSRSFQRDKWELLQNFTCLDRP



ALDSLIRDVVQAYKNGASLLSNTTSMGRWELVGSFFFSVSTIFFALVGIPLNLVVLNRLGHLMQQGVNHWAS


RLGGTWQDPDKARWLAGSGALLSGLLLFLLLPPLLFSHMEGWSYTEGFYFAFITLSTVGFGDYVIGMNPSQR


YPLWYKNMVSLWILFGMAWLALIIKLILSQLETPGRVCSCCHHSSKEDFKSQSWRQGPDREPESHSPQQGCY


PEGPMGIIQHLEPSAHAAGCGKDS









The NOV12a amino acid sequence has 113 of 114 amino acid residues (99%) identical to, and 113 of 114 amino acid residues (99%) similar to, the 229 amino acid residue ptnr:TREMBLNEW-ACC:CAC07335 protein from Homo sapiens (Human) (DJ137F1.1 (Novel Member Of The Potassium Channel Subfamily K)) (E=2.4e−108).
The disclosed NOV12a is expressed in at least the following tissues: amygdala, brain—cerebellum, brain—hippocampus, brain—substantia nigra, brain—thalamus, brain—whole, fetal brain, fetal kidney, fetal liver, fetal lung, heart, kidney, lymphoma—Raji, mammary gland, pancreas, pituitary gland, placenta, prostate, salivary gland, skeletal muscle, small intestine, spinal cord, spleen, stomach, testis, thyroid, trachea and uterus. This information was derived by determining the tissue sources of the sequences that were included in the invention including but not limited to SeqCalling sources, Public EST sources, Literature sources, and/or RACE sources.
NOV12b
A disclosed NOV12b nucleic acid of 1083 nucleotides (also referred to as CG57220-02) encoding a novel TWIK 3-like protein is shown in Table 12C. An open reading frame was identified beginning with an ATG initiation codon at nucleotides 17-19 and ending with a TAG codon at nucleotides 1025-1027. Putative untranslated regions, if any, upstream from the initiation codon and downstream from the termination codon are underlined in Table 12C, and the start and stop codons are in bold letters.








TABLE 12C





NOV12b nucleotide sequence.
















(SEQ ID NO:45)









CCCCGCCTCTCCCGCTATGTACCGACCGCGAGCCCGGGCGGCTCCCGAGGGCAGGGTCCGGGGCTGCGCGGT



GCCCGGCACCGTGCTCCTGCTGCTCGCCTACCTGGCTTACCTGGCGCTGGGCACCGGCGTGTTCTGGACGCT


GGAGGGCCGCGCGGCGCAGGACTCCAGCCGCAGCTTCCAGCGCGACAAGTGGGAGCTGTTGCAGAACTTCAC


GTGTCTGGACCGCCCGGCGCTGGACTCGCTGATCCGGCCTCTTCCCCAGGATGTCGTCCAAGCATACAAAAA


CGGAGCCAGCCTCCTCAGCAACACCACCAGCATGGGGCGCTGGGAGCTCGTGGGCTCCTTCTTCTTTTCTGT


GTCCACCATCACCACCATTGGCTATGGCAACCTGAGCCCCAACACGATGGCTGCCCGCCTCTTCTGCATCTT


CTTTGCCCTTGTGGGGATCCCACTCAACCTCGTGGTGCTCAACCGACTGGGGCATCTCATGCAGCAGGGAGT


AAACCACTGGGCCAGCAGGCTGGGGGGCACCTGGCAGGATCCTGACAAGGCGCGGTGGCTGGCGGGCTCTGG


CGCCCTCCTCTCGGGCCTCCTGCTCTTCCTGCTGCTGCCACCGCTGCTCTTCTCCCACATGGAGGGCTGGAG


CTACACAGAGGGCTTCTACTTCGCCTTCATCACCCTCAGCACCGTGGGCTTCGGCGACTACGTGATTGGAAT


GAACCCCTCCCAGAGGTACCCACTGTGGTACAAGAACATGGTGTCCCTGTGGATCCTCTTTGGGATGGCATG


GCTGGCCTTGATCATCAAACTCATCCTCTCCCAGCTGGAGACGCCAGGGAGGGTATGTTCCTGCTGCCACCA


CAGCTCTAAGGAAGACTTCAAGTCCCAAAGCTGGAGACAGGGACCTGACCGGGAGCCAGAGTCCCACTCCCC


ACAGCAAGGATGCTATCCAGAGGGACCCATGGGAATCATACAGCATCTGGAACCTTCTGCTCACGCTGCAGG


CTGTGGCAAGGACAGCTAGTTATACTCCATTCTTTGGTCGTCGTCCTCGGTAGCAAGACCCCTGATTTTAAG


CTT









The disclosed NOV12b nucleic acid sequence, localized to chromsome 6p21.1-21.2, has 496 of 795 bases (62%) identical to a gb:GENBANK-ID:AF006823|acc:AF006823.1 mRNA from Homo sapiens (Homo sapiens TWIK-related acid-sensitive K+ channel (TASK) mRNA, complete cds) (E=1.2e−27).
A NOV12b polypeptide (SEQ ID NO:46) encoded by SEQ ID NO:45 has 336 amino acid residues and is presented using the one-letter code in Table 12D. Signal P, Psort and/or Hydropathy results predict that NOV12b contains a signal peptide and is likely to be localized to the plasma membrane with a certainty of 0.6400. The most likely cleavage site for a NOV12a polypeptide is between amino acids 48 and 49: RAA-QD.








TABLE 12D





Encoded NOV12b protein sequence.
















(SEQ ID NO:46)









MYRPRARAAPEGRVRGCAVPGTVLLLLAYLAYLALGTGVFWTLEGRAAQDSSRSFQRDKWELLQNFTCLDRP



ALDSLIRPLPQDVVQAYKNGASLLSNTTSMGRWELVGSFFFSVSTITTIGYGNLSPNTMAARLFCIFFALVG


IPLNLVVLNRLGHLMQQGVNHWASRLGGTWQDPDKARWLAGSGALLSGLLLFLLLPPLLFSHMEGWSYTEGF


YFAFITLSTVGFGDYVIGMNPSQRYPLWYKNMVSLWILFGMAWLALIIKLILSQLETPGRVCSCCHHSSKED


FKSQSWRQGPDREPESHSPQQGCYPEGPMGIIQHLEPSAHAAGCGKDS









The NOV12b amino acid sequence has 228 of 233 amino acid residues (97%) identical to, and 228 of 233 amino acid residues (97%) similar to, the 229 amino acid residue ptnr:TREMBLNEW-ACC:CAC07335 protein from Homo sapiens (Human) (DJ137F1.1 (Novel Member Of The Potassium Channel Subfamily K)) (E=1.1e−119).
The disclosed NOV12b is expressed in at least the following tissues: adrenal gland, bone marrow, brain—amygdala, brain—cerebellum, brain—hippocampus, brain—substantia nigra, brain—thalamus, brain—whole, fetal brain, fetal kidney, fetal liver, fetal lung, heart, kidney, lymphoma—Raji, mammary gland, pancreas, pituitary gland, placenta, prostate, salivary gland, skeletal muscle, small intestine, spinal cord, spleen, stomach, testis, thyroid, trachea and uterus. This information was derived by determining the tissue sources of the sequences that were included in the invention including but not limited to SeqCalling sources, Public EST sources, Literature sources, and/or RACE sources.
NOV12c
A disclosed NOV12c nucleic acid of 1256 nucleotides (also referred to as CG57220-03) encoding a novel TWIK 3-like protein is shown in Table 12E. An open reading frame was identified beginning with an ATG initiation codon at nucleotides 17-19 and ending with a TAG codon at nucleotides 953-955. Putative untranslated regions, if any, upstream from the initiation codon and downstream from the termination codon are underlined in Table 12E, and the start and stop codons are in bold letters.








TABLE 12E





NOV12c nucleotide sequence.
















(SEQ ID NO:47)









CCCCGCCTCTCCCGCTATGTACCGACCGCGAGCCCGGGCGGCTCCCGAGGGCAGGGTCCGGGGCTGCGCGGT



GCCCGGCACCGTGCTCCTGCTGCTCGCCTACCTGGCTTACCTGGCGCTGGGCACCGGCGTGTTCTGGACGCT


GGAGGGCCGCGCGGCGCAGGACTCCAGCCGCAGCTTCCAGCGCGACAAGTGGGAGCTGTTGCAGAACTTCAC


GTGTCTGGACCGCCCGGCGCTGGACTCGCTGATCCGGGATGTCGTCCAAGCATACAAAAACGGAGCCAGCCT


CCTCAGCAACACCACCAGCATGGGGCGCTGGGAGCTCGTGGGCTCCTTCTTCTTTTCTGTGTCCACCATCTT


CTTTGCCCTTGTGGGGATCCCACTCAACCTCGTGGTGCTCAACCGACTGGGGCATCTCATGCAGCAGGGAGT


AAACCACTGGGCCAGCAGGCTGGGGGGCACCTGGCAGGATCCTGACAAGGCGCGGTGGCTGGCGGGCTCTGG


CGCCCTCCTCTCGGGCCTCCTGCTCTTCCTGCTGCTGCCACCGCTGCTCTTCTCCCACATGGAGGGCTGGAG


CTACACAGAGGGCTTCTACTTCGCCTTCATCACCCTCAGCACCGTGGGCTTCGGCGACTACGTGATTGGAAT


GAACCCCTCCCAGAGGTACCCACTGTGGTACAAGAACATGGTGTCCCTGTGGATCCTCTTTGGGATGGCATG


GCTGGCCTTGATCATCAAACTCATCCTCTCCCAGCTGGAGACGCCAGGGAGGGTATGTTCCTGCTGCCACCA


CAGCTCTAAGGAAGACTTCAAGTCCCAAAGCTGGAGACAGGGACCTGACCGGGAGCCAGAGTCCCACTCCCC


ACAGCAAGGATGCTATCCAGAGGGACCCATGGGAATCATACAGCATCTGGAACCTTCTGCTCACGCTGCAGG


CTGTGGCAAGGACAGCTAGTTATACTCCATTCTTTGGTCGTCGTCCTCGGTAGCAAGACCCCTGATTTTAAG


CTTTGCACATGTCCACCCAAACTAAAGACTACATTTTCCATCCACCCTAGAGGCTGGGTGCAGCTATATGAT


TAATTCTGCCCAATAGGGTATACAGAGACATGTCCTGGGTGACATGGGATGTGACTTTCGGGTGTCGGGGCA


GCATGCCCTTCTCCCCCACTTCCTTACTTTAGCGGGCTGCAATGCCGCCGATATGATGGCTGGGAGCTCTGG


CAGCCATACGGCACCATGAAGTAGCGGCAATG









The disclosed NOV12c nucleic acid sequence, localized to chromsome 6p21.1-21.2, has 384 of 641 bases (59%) identical to a gb:GENBANK-ID:AF006823|acc:AF006823.1 mRNA from Homo sapiens (Homo sapiens TWIK-related acid-sensitive K+ channel (TASK) mRNA, complete cds) (E=1.3e−14).
A NOV12c polypeptide (SEQ ID NO:48) encoded by SEQ ID NO:47 has 312 amino acid residues and is presented using the one-letter code in Table 12F. Signal P, Psort and/or Hydropathy results predict that NOV12c contains a signal peptide and is likely to be localized to the plasma membrane with a certainty of 0.6400. The most likely cleavage site for a NOV12a polypeptide is between amino acids 48 and 49: RAA-QD.








TABLE 12F





Encoded NOV12c protein sequence.
















(SEQ ID NO:48)









MYRPRARAAPEGRVRGCAVPGTVLLLLAYLAYLALGTGVFWTLEGRAAQDSSRSFQRDKWELLQNFTCLDRP



ALDSLIRDVVQAYKNGASLLSNTTSMGRWELVGSFFFSVSTIFFALVGIPLNLVVLNRLGHLMQQGVNHWAS


RLGGTWQDPDKARWLAGSGALLSGLLLFLLLPPLLFSHMEGWSYTEGFYFAFITLSTVGFGDYVIGMNPSQR


YPLWYKNMVSLWILFGMAWLALIIKLILSQLETPGRVCSCCHHSSKEDFKSQSWRQGPDREPESHSPQQGCY


PEGPMGIIQHLEPSAHAAGCGKDS









The NOV12c amino acid sequence has 67 of 132 amino acid residues (50%) identical to, and 97 of 132 amino acid residues (73%) similar to, the 294 amino acid residue ptnr:SPTREMBL-ACC:Q9H591 protein from Homo sapiens (Human) (DJ137F1.2 (Novel Member Of The Potassium Channel Subfamily K)) (E=2.1e−47).
The disclosed NOV12c is expressed in at least the following tissues: amygdala, brain—cerebellum, brain—hippocampus, brain—substantia nigra, brain—thalamus, brain—whole, fetal brain, fetal kidney, fetal liver, fetal lung, heart, kidney, lymphoma—Raji, mammary gland, pancreas, pituitary gland, placenta, prostate, salivary gland, skeletal muscle, small intestine, spinal cord, spleen, stomach, testis, thyroid, trachea and uterus. This information was derived by determining the tissue sources of the sequences that were included in the invention including but not limited to SeqCalling sources, Public EST sources, Literature sources, and/or RACE sources.
Possible SNPs found for NOV12a are listed in Table 12G.







TABLE 12G







SNPs














Amino




Nucleotide
Base
Acid
Base


Variant
Position
Change
Position
Change














13377098
77
G > A
 21
Gly > Ser


13377097
593
T > C
193
Tyr > His


13377096
718
A > G
Silent
N/A


13377095
856
C > T
Silent
N/A









NOV12a, NOV12b and NOV12c are very closely homologous as is shown in the amino acid alignment in Table 12H.







TABLE 12H





Amino Acid Alignment of NOV12a, NOV12b and NOV12c

























































Homologies to any of the above NOV12 proteins will be shared by the other NOV12 proteins insofar as they are homologous to each other as shown above. Any reference to NOV12 is assumed to refer to the NOV12 proteins in general, unless otherwise noted.
NOV12a has homology to the amino acid sequences shown in the BLASTP data listed in Table 12I.







TABLE 12I







BLAST results for NOV12a












Gene Index/
Protein/
Length
Identity
Positives



Identifier
Organism
(aa)
(%)
(%)
Expect





gi|17025230|ref|NP_
potassium
332
276/332
276/332
 e−155


113648.2|
channel,

(83%)
(83%)


(NM_031460)
subfamily K,



member 17; 2P



domain



potassium



channel Talk-



2; potassium



channel TASK-



4; potassium



channel TALK-



2 [Homo



sapiens]


gi|13507377|gb|AAK28551.1|
potassium
343
204/259
204/259
 e−108


AF339912_1
channel TASK-

(78%)
(78%)


(AF339912)
4 [Homo



sapiens]


gi|9988111|emb|CAC07335.1|
dJ137F1.1
229
173/229
173/229
5e−90


(AL136087)
(novel member

(75%)
(75%)



of the



potassium



channel



subfamily K)



[Homo sapiens]


gi|9988112|emb|CAC07336.1|
dJ137F1.2
294
 75/226
118/226
3e−30


(AL136087)
(novel member

(33%)
(52%)



of the



potassium



channel



subfamily K)



[Homo sapiens]


gi|14149764|ref|NP_
pancreatic 2P
309
 75/226
118/226
4e−30


115491.1|
domain

(33%)
(52%)


(NM_032115)
potassium



channel TALK-



1; potassium



family,



subfamily K,



member 16



[Homo sapiens]









The homology of these sequences is shown graphically in the ClustalW analysis shown in Table 12J.







TABLE 12J





ClustalW Analysis of NOV12a















1) NOV12a (SEQ ID NO:44)


2) gi|17025230|refNP_113648.2| (NM_031460) potassium channel, subfamily K, member 17;


2P domain potassium channel Talk-2; potassium channel TASK-4; potassium channel


TALK-2 [Homo sapiens] (SEQ ID NO:168)


2) gi 13507377|gb AAK28551.1 AF339912_1 (AF339912) potassium channel TASK-4


[Homo sapiens] (SEQ ID NO:169)


3) gi|9988111|emb CAC07335.1| (AL136087) dJ137F1.1 (novel member of the potassium


channel subfamily K) [Homo sapiens] (SEQ ID NO:170)


4) gi|9988112|emb CAC07336.1| (AL136087) dJ137F1.2 (novel member of the potassium


channel subfamily K) [Homo sapiens] (SEQ ID NO:171)


5) gi 14149764|refNP_115491.1| (NM_032115) pancreatic 2P domain potassium channel


TALK-1; potassium family, subfamily K, member 16 [Homo sapiens]


(SEQ ID NO:172)












































Potassium channels are amongst the most heterogeneous class of ion channels known and are responsible for mediating a diverse range of biological functions. The most recently described family of K(+) channels, the ‘two pore-domain family’, contain four membrane spanning domains and two pore-forming domains, suggesting that two channel subunits associate to form a functional K(+) pore. Several sub-families of the two pore domain potassium channel family have been described, including the weakly inward rectifying K(+) channel (TWIK), the acid-sensitive K(+) channel (TASK), the TWIK-related K(+) channel (TREK) and the TWIK-related arachidonic acid stimulated K(+) channel (TRAAK). TWIK-1 and the TWIK-1-like channel KCNK7 were predominantly expressed in the CNS, in contrast to TWIK-2 which was preferentially expressed in peripheral tissues such as pancreas, stomach, spleen and uterus. TASK-1 was expressed in the CNS and some peripheral tissues, whereas TASK-2 was exclusively expressed in the periphery except for mRNA expression observed in dorsal root ganglion and spinal cord. In addition, mRNA expression of the recently identified TASK-3, was almost completely exclusive to cerebellum with little or no mRNA detected in any other tissues. TREK-1 and TRAAK mRNA expression was predominantly CNS specific in contrast to the closely related TREK-2, which was expressed in both CNS and peripheral tissues. (Medhurst et al., Brain Res Mol Brain Res 86:101-114).
The two-pore-domain K(+) channels, or K(2P) channels produce currents with unusual characteristics. They are quasi-instantaneous and noninactivating, and they are active at all membrane potentials and insensitive to the classic K(+) channel blockers. These properties designate them as background K(+) channels. They are expected to play a major role in setting the resting membrane potential in many cell types. Another salient feature of K(2P) channels is the diversity of their regulatory mechanisms. The weak inward rectifiers TWIK-1 and TWIK-2 are stimulated by activators of protein kinase C and decreased by internal acidification, the baseline TWIK-related acid-sensitive K(+) (TASK)-1 and TASK-2 channels are sensitive to external pH changes in a narrow range near physiological pH, and the TWIK-related (TREK)-1 and TWIK-related arachidonic acid-stimulated K(+) (TRAAK) channels are the first cloned polyunsaturated fatty acids-activated and mechanogated K(+) channels. The recent demonstration that TASK-1 and TREK-1 channels are activated by inhalational general anesthetics, and that TRAAK is activated by the neuroprotective agent riluzole, indicates that this novel class of K(+) channels is an interesting target for new therapeutic developments (Lesage and Lazdunski, Am J Physiol Renal Physiol 2000, 279(5):F793-801).
TWIK-1, the founding member of the 2P domain mammalian family, is widely expressed in human tissues and is particularly abundant in brain and heart. hTWIK-1 currents expressed in Xenopus oocytes are K+-selective, are time-independent, and present a nearly linear I-V relationship that rectifies for depolarizations positive to 0 mV. TWIK-1 is blocked by Ba2+, quinine, and quinidine (Lesage et al., 1996 EMBO J. 15: 1004-1011). Recently, hTWIK-2 (also called hTOSS), a TWIK-1-related gene, was cloned by two independent groups. Although both hTWIK-2 and hTOSS sequences are identical, conflicting results were published concerning functional expression in heterologous systems (Chavez, et al., 1999 J. Biol. Chem. 274: 7887-7892; Pountney, et al., 1999 FEBS Lett. 450: 191-196). hTWIK-2 expressed in Xenopus oocytes was shown to be a noninactivating, time-independent, weak inward rectifier with biophysical properties identical to TWIK-1. Pharmacologically, hTWIK-2 was reported to be different from TWIK-1 with a lack of sensitivity to quinine, quinidine, and Ba2+. On the contrary, no significant current was observed in hTOSS cRNA-injected Xenopus laevis oocytes or in hTOSS cDNA-transfected HEK293T cells. Co-injection of equimolar concentrations of hTWIK-1 and hTOSS cRNA also failed to generate currents in Xenopus oocytes. These negative findings have led these authors and others to propose that hTOSS may be targeted to locations other than the plasma membrane or that it may possess a regulatory function, modulating the properties of other principal channel-forming subunits with tissue-specific implications (Chavez, et al., 1999, J. Biol. Chem. 274: 7887-7892; Pountney, et al., 1999 FEBS Lett. 450: 191-196; Rajan, et al., 2000 J. Biol. Chem. 275: 16650-16657).
The NOV12 nucleic acid of the invention encoding a TWIK 3-like protein includes the nucleic acid whose sequence is provided in Tables 12A, 12C and 12E, or a fragment thereof. The invention also includes a mutant or variant nucleic acid any of whose bases may be changed from the corresponding base shown in Tables 12A, 12C and 12E while still encoding a protein that maintains its TWIK 3-like activities and physiological functions, or a fragment of such a nucleic acid. The invention further includes nucleic acids whose sequences are complementary to those just described, including nucleic acid fragments that are complementary to any of the nucleic acids just described. The invention additionally includes nucleic acids or nucleic acid fragments, or complements thereto, whose structures include chemical modifications. Such modifications include, by way of non-limiting example, modified bases, and nucleic acids whose sugar phosphate backbones are modified or derivatized. These modifications are carried out at least in part to enhance the chemical stability of the modified nucleic acid, such that they may be used, for example, as antisense binding nucleic acids in therapeutic applications in a subject. In the mutant or variant nucleic acids, and their complements, up to about 41% of the NOV12a residues, about 38% of the NOV12b residues and about 41% of the NOV12c residues may be so changed.
The NOV12 protein of the invention includes the TWIK 3-like protein whose sequence is provided in Tables 12B, 12D and 12F. The invention also includes a mutant or variant protein any of whose residues may be changed from the corresponding residue shown in Tables 12B, 12D and 12F while still encoding a protein that maintains its TWIK 3-like activities and physiological functions, or a functional fragment thereof. In the mutant or variant protein, up to about 1% of the NOV12a, about 3% of the NOV12b and about 50% of the NOV12c bases may be so changed.
The NOV12 nucleic acids and proteins of the invention are useful in potential diagnostic and therapeutic applications implicated in various diseases and disorders described below and/or other pathologies. For example, the compositions of the present invention will have efficacy for treatment of patients suffering from: central nervous system disorder; dementia; epilepsy; Alzheimer's disease; Parkinson's disease; multiple sclerosis; depression; amyotrophic lateral sclerosis; progressive supranuclear palsy; mania; Creutzfeldt-Jacob disease; psychiatric disorder; schizophrenic disorder; Korsakoff's psychosis; anxiety disorder; phobic disorder; amnesia; learning disorder; memory disorder; age-related memory loss; obesity; neurological disorder; cardiac disorder and other diseases, disorders and conditions of the like.
NOV12 nucleic acids and polypeptides are further useful in the generation of antibodies that bind immunospecifically to the novel substances of the invention for use in therapeutic or diagnostic methods. These antibodies may be generated according to methods known in the art, using prediction from hydrophobicity charts, as described in the “Anti-NOVX Antibodies” section below. For example the disclosed NOV12 protein have multiple hydrophilic regions, each of which can be used as an immunogen. This novel protein also has value in development of powerful assay system for functional analysis of various human disorders, which will help in understanding of pathology of the disease and development of new drug targets for various disorders.
NOV13
A disclosed NOV13 nucleic acid of 1260 nucleotides (also referred to as CG57220-04) encoding a novel TASK 4-like protein is shown in Table 13A. An open reading frame was identified beginning with an ATG initiation codon at nucleotides 107-109 and ending with a TAG codon at nucleotides 1163-1165. Putative untranslated regions upstream from the intitation codon and downstream from the termination codon are underlined in Table 13A, and the start and stop codons are in bold letters.








TABLE 13A





NOV13 Nucleotide Sequence
















(SEQ ID NO:49)









CTCAAGCAGGCGTTTGCGAGAGGAGATACGAGCTGGACGCCTGGCCCTTCCCTCCCACCGGGTCCTAGTCCAC



CGCTCCCGGCGCCGGCTCCCCGCCTCTCCCGCTATGTACCGACCGCGAGCCCGGGCGGCTCCCGAGGGCAGGG


TCCGGGGCTGCGCGGTGCCCGGCACCGTGCTCCTGCTGCTCGCCTACCTGGCTTACCTGGCGCTGGGCACCGG


CGTGTTCTGGACGCTGGAGGGCCGCGCGGCGCAGGACTCCAGCCGCAGCTTCCAGCGCGACAAGTGGGAGCTG


TTGCAGAACTTCACGTGTCTGGACCGCCCGGCGCTGGACTCGCTGATCCGGGATGTCGTCCAAGCATACAAAA


ACGGAGCCAGCCTCCTCAGCAACACCACCAGCATGGGGCGCTGGGAGCTCGTGGGCTCCTTCTTCTTTTCTGT


GTCCACCATCACCACCATTGGCTATGGCAACCTGAGCCCCAACACGATGGCTGCCCGCCTCTTCTGCATCTTC


TTTGCCCTTGTGGGGATCCCACTCAACCTCGTGGTGCTCAACCGACTGGGGCATCTCATGCAGCAGGGAGTAA


ACCACTGGGCCAGCAGGCTGGGGGGCACCTGGCAGGATCCTGACAAGGCGCGGTGGCTGGCGGGCTCTGGCGC


CCTCCTCTCGGGCCTCCTGCTCTTCCTGCTGCTGCCACCGCTGCTCTTCTCCCACATGGAGGGCTGGAGCTAC


ACAGAGGGCTTCTACTTCGCCTTCATCACCCTCAGCACCGTGGGCTTCGGCGACTACGTGATTGGAATGAACC


CCTCCCAGAGGTACCCACTGTGGTACAAGAACATGGTGTCCCTGTGGATCCTCTTTGGGATGGCATGGCTGGC


CTTGATCATCAAACTCATCCTCTCCCAGCTGGAGACAAACCCATCCTCTCCCAGCTGGAGACAACAGGGAGGG


TATGTTCCTGCTGCCACCACAGCTCTAAGGAAGACTTCAAGTCCCAAAGCTGGAGACAGGGACCTGACCGGGA


GCCAGAGTCCCACTCCCCACAGCAAGGATGCTATCCAGAGGGACCCATGGGAATCATACAGCATCTGGAACCT


TCTGCTCACGCTGCAGGCTGTGGCAAGGACAGCTAGTTATACTCCATTCTTTGGTCGTCGTCCTCGGTAGCAA


GACCCCTGATTTTAAGCTTTGCACATGTCCACCCAAACTAAAGACTACATTTTCCATCCACCCTAGAGGCTGG


GTGCAGCTATATGATTAAT









The NOV13 nucleic acid was identified on chromosome 6 and has 508 of 827 bases (61%) identical to a gb:GENBANK-ID:AF006823|acc:AF006823.1 mRNA from Homo sapiens (Homo sapiens TWIK-related acid-sensitive K+ channel (TASK) mRNA, complete cds) (E=1.2e−29).
A disclosed NOV13 polypeptide (SEQ ID NO:50) encoded by SEQ ID NO:49 is 352 amino acid residues and is presented using the one-letter code in Table 13B. Signal P, Psort and/or Hydropathy results predict that NOV13 contains a signal peptide and is likely to be localized to the extracellularly with a certainty of 0.6400. The most likely cleavage site for a NOV13 polypeptide is between amino acids 48 and 49: RAA-QD.








TABLE 13B





Encoded NOV13 protein sequence
















(SEQ ID NO:50)









MYRPRARAAPEGRVRGCAVPGTVLLLLAYLAYLALGTGVFWTLEGRAAQDSSRSFQRDKWELLQNFTCLDRP



ALDSLIRDVVQAYKNGASLLSNTTSMGRWELVGSFFFSVSTITTIGYGNLSPNTMAARLFCIFFALVGIPLN


LVVLNRLGHLMQQGVNHWASRLGGTWQDPDKARWLAGSGALLSGLLLFLLLPPLLFSHMEGWSYTEGFYFAF


ITLSTVGRGDYVIGMNPSQRYPLWYKNMVSLWILFGMAWLALIIKLILSQLETNPSSPSWRQQGGYVPAATT


ALRKTSSPKAGDRDLTGSQSPTPHSKDAIQRDPWESYSIWNLLLTLQAVARTASYTPFFGRRPR









The NOV13 amino acid sequence has 340 of 352 amino acid residues (96%) identical to, and 342 of 352 amino acid residues (97%) similar to, the 343 amino acid residue ptnr: TREMBLNEW-ACC:AAK28551 protein from Homo sapiens (Human) (Potassium Channel TASK-4) (E=5.0e−183).
NOV13 is expressed in at least the following tissues: liver, lung, pancreas, placenta, aorta and heart. This information was derived by determining the tissue sources of the sequences that were included in the invention including but not limited to SeqCalling sources, Public EST sources, genomic clone sources, literature sources, and/or RACE sources. The sequence is predicted to be expressed in the following tissues because of the expression pattern of (GENBANK-ID: gb:GENBANK-ID:AF006823|acc:AF006823.1) a closely related Homo sapiens TWIK-related acid-sensitive K+ channel (TASK) mRNA, complete cds homolog in species Homo sapiens: pancreas, placenta, brain, lung, prostate, heart, kidney, uterus, small intestine and colon.
NOV13 has homology to the amino acid sequences shown in the BLASTP data listed in Table 13C.







TABLE 13C







BLAST results for NOV13












Gene Index/

Length
Identity
Positives



Identifier
Protein/Organism
(aa)
(%)
(%)
Expect





gi|13507377|gb|AAK28551.1|
potassium channel
343
305/352
307/352
 e−164


AF339912_1
TASK-4 [Homo

(86%)
(86%)


(AF339912)
sapiens]


gi|17025230|ref|NP_
potassium
332
233/269
233/269
 e−128


113648.2|
channel,

(86%)
(86%)


(NM_031460)
subfamily K,



member 17; 2P



domain potassium



channel Talk-2;



potassium channel



TASK-4; potassium



channel TALK-2



[Homo sapiens]


gi|9988111|emb|CAC07335.1|
dJ137F1.1 (novel
229
193/229
193/229
 e−103


(AL136087)
member of the

(84%)
(84%)



potassium channel



subfamily K)



[Homo sapiens]


gi|9988112|emb|CAC07336.1|
dJ137F1.2 (novel
294
 87/226
136/226
1e−41


(AL136087)
member of the

(38%)
(59%)



potassium channel



subfamily K)



[Homo sapiens]


gi|14149764|ref|NP_
pancreatic 2P
309
 87/226
136/226
1e−41


115491.1|
domain potassium

(38%)
(59%)


(NM_032115)
channel TALK-1;



potassium family,



subfamily K,



member 16 [Homo



sapiens]









The homology of these sequences is shown graphically in the ClustalW analysis shown in Table 13D.







TABLE 13D





Clustal W Sequence Alignment















1) NOV13 (SEQ ID NO:50)


2) gi|13507377|gb|AAK28551.1 AF339912_1 (AF339912) potassium channel TASK-4


[Homo sapiens] (SEQ ID NO:173)


3) gi|17025230|ref|NP_113648.2|(NM_031460) potassium channel, subfamily K, member 17;


2P domain potassium channel Talk-2; potassium channel TASK-4; potassium channel


TALK-2 [Homo sapiens] (SEQ ID NO:174)


4) gi|9988111|cmb|CAC07335.1|(AL136087) dJl37F1.1 (novel member of the potassium


channel subfamily K) [Homo sapiens] (SEQ ID NO: 175)


5) gi|9988112|emb|CAC07336.1|(AL136087) dJ137F1.2 (novel member of the potassium


channel subfamily K) [Homo sapiens] (SEQ ID NO: 176)


6) gi|14149764|ref|NP_115491.1|(NM_032115) pancreatic 2P domain potassium channel


TALK-1; potassium family, subfamily K, member 16 [Homo sapiens] (SEQ ID NO: 177)



















































The NOV13 nucleic acid of the invention encoding a TASK 4-like protein includes the nucleic acid whose sequence is provided in Table 13A, or a fragment thereof. The invention also includes a mutant or variant nucleic acid any of whose bases may be changed from the corresponding base shown in Table 13A while still encoding a protein that maintains its TASK 4-like activities and physiological functions, or a fragment of such a nucleic acid. The invention further includes nucleic acids whose sequences are complementary to those just described, including nucleic acid fragments that are complementary to any of the nucleic acids just described. The invention additionally includes nucleic acids or nucleic acid fragments, or complements thereto, whose structures include chemical modifications. Such modifications include, by way of non-limiting example, modified bases, and nucleic acids whose sugar phosphate backbones are modified or derivatized. These modifications are carried out at least in part to enhance the chemical stability of the modified nucleic acid, such that they may be used, for example, as antisense binding nucleic acids in therapeutic applications in a subject. In the mutant or variant nucleic acids, and their complements, up to about 39% of the residues may be so changed.
The NOV13 protein of the invention includes the TASK 4-like protein whose sequence is provided in Table 13B. The invention also includes a mutant or variant protein any of whose residues may be changed from the corresponding residue shown in Table 13B while still encoding a protein that maintains its TASK 4-like activities and physiological functions, or a functional fragment thereof. In the mutant or variant protein, up to about 4% of the bases may be so changed.
The NOV13 nucleic acids and proteins of the invention are useful in potential diagnostic and therapeutic applications implicated in various diseases and disorders described below and/or other pathologies. For example, the compositions of the present invention will have efficacy for treatment of patients suffering from: cardiomyopathy, atherosclerosis, hypertension, congenital heart defects, aortic stenosis, atrial septal defect (ASD), atrioventricular (A-V) canal defect, ductus arteriosus, pulmonary stenosis, subaortic stenosis, ventricular septal defect (VSD), valve diseases, tuberous sclerosis, sclerodermia, obesity, transplantation, aneurysm, fibromuscular dysplasia, stroke, systemic lupus erythematosus, autoimmune disease, asthma, emphysema, allergy, ARDS, Von Hippel-Lindau (VHL) syndrome, cirrhosis, diabetes, pancreatitis, fertility, cancer, tissue degeneration, bacterial/viral/parasitic infections and other diseases, disorders and conditions of the like.
NOV13 nucleic acids and polypeptides are further useful in the generation of antibodies that bind immunospecifically to the novel substances of the invention for use in therapeutic or diagnostic methods. These antibodies may be generated according to methods known in the art, using prediction from hydrophobicity charts, as described in the “Anti-NOVX Antibodies” section below. For example the disclosed NOV13 protein have multiple hydrophilic regions, each of which can be used as an immunogen. This novel protein also has value in development of powerful assay system for functional analysis of various human disorders, which will help in understanding of pathology of the disease and development of new drug targets for various disorders.
NOV14
A disclosed NOV14 nucleic acid of 627 nucleotides (also referred to as CG57458-01) encoding a novel Copper transporter-like protein is shown in Table 14A. An open reading frame was identified beginning with an ATG initiation codon at nucleotides 31-33 and ending with a TGA codon at nucleotides 598-600. Putative untranslated regions upstream from the intitation codon and downstream from the termination codon are underlined in Table 14A, and the start and stop codons are in bold letters.








TABLE 14A





NOV14 Nucleotide Sequence
















(SEQ ID NO:51)









TCTGCTGACTCTCAACTTTTCTTGGAAAAAATGGATCATTCCCATCATACGGGGATGAGCTGTATGGACTCCA



ACAGTACCATGCAACGTCCTCACCATCACCCAACCACTTCAGCCTCACACTCCCGTGGTGGAGGAGACAGCAA


CATGATGATGATGATGCCTATGACCTTCTACTTTGGCTTTAAGAATGTGGAACTACTGTTTTCCAGTTTGGTG


ATCAATACAGCTGGAGAAATGGCTGGAGCTTTTGTGGCAGTGCTTTTGCTACAATTCCATGCCTGTCCCATAG


CCCAAGAGAGCCTGCTGTGTAAGTCACAAGTCAGCTTTTGCTACAATTCCATGCCTGTCCCAGGACCAAATGG


AACCATCCTTATGGAGACACACAAAACTGTTGGGCAGCAGATGCTGAGCTTTCCTCACCTCCTGCAAACAGTG


CTGCACATCATCCAGGTGGTCATAAGCTACCTCCTCATGCTCATCTTCATGACCTACAATGGGTACCTCTGCA


TTGCAGTAACAGCAGGGGCCGGTACAAGATACTTCCTCTTCAGCTGGAAGAAGGCAGTGGTAGTGGACATCAC


AGAGTATTGCCATTGACGTCAAACTCTATGGCATGGCCTTATC









The NOV14 nucleic acid was identified on chromosome 3.
A disclosed NOV14 polypeptide (SEQ ID NO:52) encoded by SEQ ID NO:51 is 189 amino acid residues and is presented using the one-letter code in Table 14B. Signal P, Psort and/or Hydropathy results predict that NOV14 contains a signal peptide and is likely to be localized to the plasma membrane with a certainty of 0.6000. The most likely cleavage site for a NOV14 polypeptide is between amino acids 68 and 69: TAG-EM.








TABLE 14B





Encoded NOV14 protein sequence
















(SEQ ID NO:52)









MDHSHHTGMSCMDSNSTMQRPHHHPTTSASHSRGGGDSNMMMMMPMTFYFGFKNVELLFSSLVINTAGEMAG



AFVAVLLLQFHACPIAQESLLCKSQVSFCYNSMPVPGPNGTILMETHKTVGQQMLSFPHLLQTVLHIIQVVI


SYLLMLIFMTYNGYLCIAVTAGAGTRYFLFSWKKAVVVDITEYCH









The NOV14 amino acid sequence has 166 of 190 amino acid residues (87%) identical to, and 170 of 190 amino acid residues (89%) similar to, the 190 amino acid residue ptnr:SWISSNEW-ACC:015431 protein from Homo sapiens (Human) (High-Affinity Copper Uptake Protein 1 (HCTR1) (E=1.9e−83).
NOV14 is expressed in at least the following tissues: brain. This information was derived by determining the tissue sources of the sequences that were included in the invention including but not limited to SeqCalling sources, Public EST sources, genomic clone sources, literature sources, and/or RACE sources.
NOV14 has homology to the amino acid sequences shown in the BLASTP data listed in Table 14C.







TABLE 14C







BLAST results for NOV14












Gene Index/

Length
Identity
Positives



Identifier
Protein/Organism
(aa)
(%)
(%)
Expect





gi|4507015|ref|NP_001850.1|
solute carrier
190
149/191
153/191
2e−67


(NM_001859)
family 31 (copper

(78%)
(80%)



transporters),



member 1; hCTR1;



copper



transporter 1



[Homo sapiens]


gi|17940111|gb|AAL49494.1|
high-affinity
189
135/191
141/191
6e−59


AF320815_1
copper uptake

(70%)
(73%)


(AF320815)
protein [Sus



scrofa]


gi|12229733|sp|Q9JK41|
High-affinity
187
133/187
139/187
1e−58


COP1_RAT
copper uptake

(71%)
(74%)



protein 1 (rCTR1)



(Copper



transporter 1)


gi|18652812|gb|AAL76092.1|
high affinity
188
113/189
130/189
3e−50


(AY077715)
copper uptake

(59%)
(67%)



protein [Danio



rerio]


gi|17462341|ref|XP_
similar to solute
175
118/189
126/189
6e−50


067707.1|
carrier family 31

(62%)
(66%)


(XM_067707)
(copper



transporters),



member 1; hCTR1;



copper



transporter 1



[Homo sapiens]









The homology of these sequences is shown graphically in the ClustalW analysis shown in Table 14D.







TABLE 14D





Clustal W Sequence Alignment















1) NOV14 (SEQ ID NO:52)


2) gi|4507015|ref|NP_001850.1|(NM_001859) solute carrier family 31 (copper


transporters), member 1; hCTR1; copper transporter 1 [Homo sapiens] (SEQ ID NO:178)


3) gi|17940111|gb|AAL49494.1|AF320815_1 (AF320815) high-affinity copper uptake protein


[Sus scrofa] (SEQ ID NO:179)


4) gi|12229733|sp|Q9JK41|COP1_RAT High-affinity copper uptake protein 1 (rCTR1)


(Copper transporter 1) (SEQ ID NO:180)


5) gi|18652812|gb AAL76092.1|(AY077715) high affinity copper uptake protein


[Danio rerio] (SEQ ID NO:181)


6) gi|17462341|ref|XP_067707.1|(XM_067707) similar to solute carrier family 31


(copper transporters), member 1; hCTR; copper transporter 1 [Homo sapiens]


(SEQ ID NO:182)






























Copper is an element essential for life, but excessive copper can be toxic or even lethal to the cell. Therefore, cells have developed sophisticated ways to maintain a critical copper balance, with the intake, export, and intracellular compartmentalization or buffering of copper strictly regulated. The two related genes ATP7A and ATP7B, responsible for the human diseases Menkes syndrome and Wilson disease (WND), respectively, are involved in copper export. In S. cerevisiae, three copper uptake genes CTR1, CTR2, and CTR3 have been identified.
In mammals, however, the molecular basis for copper uptake is unknown. Zhou and Gitschier (Proc Natl Acad Sci U S A. 94(14):7481-6, 1997) isolated a human cDNA encoding COPT1, which they called CTR1, by functional complementation of the yeast high-affinity copper uptake mutant ctr1. The deduced 190-amino acid human CTR1 protein is similar to yeast CTR1 and Arabidopsis COPT1, a copper transporter also isolated by functional complementation of yeast ctr1. All 3 predicted proteins have 3 transmembrane domains and an N terminus that is rich in methionine and serine residues; the N terminus of human CTR1 is also abundant in histidines. The authors proposed that human CTR1 is a high-affinity copper uptake gene because it can complement the yeast ctr1 mutation, it can rescue multiple defects in ctr1 yeast, its expression in ctr1 yeast increases the concentration of cellular copper, and its overexpression in yeast leads to a vulnerability to the toxicity of copper overload. Northern blot analysis detected 2 major CTR1 transcripts of approximately 2 kb and 5.5 kb and a less abundant transcript of about 8.5 kb in all human organs and tissues examined. Zhou and Gitschier (1997) found that the 3-prime untranslated region of the human CTR1 gene contains a CA repeat marker (D9S262) that had been previously mapped to 9q31-q32. By analysis of YAC clones, they showed that CTR1 and CTR2 (COPT2) which is also located in 9q31-q32, are not adjacent genes. The molecular mechanisms responsible for the cellular uptake of copper in mammalian cells are unknown. The isolation of a human gene involved in this process by complementation of the yeast high-affinity copper uptake mutant, ctr1 is described. Besides complementing ctr1 growth defect on nonfermentable media, the human gene also rescues iron transport and SOD1 defects in ctr1 yeast. Overexpression of the gene in yeast leads to vulnerability to the toxicity of copper overload. In addition, its expression in ctr1 yeast significantly increases the level of cellular copper, as demonstrated by atomic absorption. It is proposed that this gene is a candidate for high-affinity copper uptake in humans and by analogy have named it hCTR1. The hCTR1 and yeast CTR1 predicted transmembrane proteins are 29% identical, but the human protein is substantially smaller in both the extracellular metal-binding and intracellular domains. An additional human gene similar to hCTR1, here named hCTR2, was identified in a database search. Both hCTR1 and hCTR2 are expressed in all human tissues examined, and both genes are located in 9q31/32. These studies, together with the previously recognized functional and sequence similarity between the Menkes/Wilson copper export proteins and CCC2 in yeast, demonstrate that similar copper homeostatic mechanisms are used in these evolutionarily divergent organisms.
The NOV14 nucleic acid of the invention encoding a Copper transporter-like protein includes the nucleic acid whose sequence is provided in Table 14A, or a fragment thereof. The invention also includes a mutant or variant nucleic acid any of whose bases may be changed from the corresponding base shown in Table 14A while still encoding a protein that maintains its Copper transporter-like activities and physiological functions, or a fragment of such a nucleic acid. The invention further includes nucleic acids whose sequences are complementary to those just described, including nucleic acid fragments that are complementary to any of the nucleic acids just described. The invention additionally includes nucleic acids or nucleic acid fragments, or complements thereto, whose structures include chemical modifications. Such modifications include, by way of non-limiting example, modified bases, and nucleic acids whose sugar phosphate backbones are modified or derivatized. These modifications are carried out at least in part to enhance the chemical stability of the modified nucleic acid, such that they may be used, for example, as antisense binding nucleic acids in therapeutic applications in a subject. In the mutant or variant nucleic acids, and their complements, up to about 13% of the residues may be so changed.
The NOV14 protein of the invention includes the Copper transporter-like protein whose sequence is provided in Table 14B. The invention also includes a mutant or variant protein any of whose residues may be changed from the corresponding residue shown in Table 14B while still encoding a protein that maintains its Copper transporter-like activities and physiological functions, or a functional fragment thereof. In the mutant or variant protein, up to about 13% of the bases may be so changed.
The NOV14 nucleic acids and proteins of the invention are useful in potential diagnostic and therapeutic applications implicated in various diseases and disorders described below and/or other pathologies. For example, the compositions of the present invention will have efficacy for treatment of patients suffering from: Von Hippel-Lindau (VHL) syndrome, Alzheimer's disease, Stroke, Tuberous sclerosis, hypercalceimia, Parkinson's disease, Huntington's disease, Cerebral palsy, Epilepsy, Lesch-Nyhan syndrome, Multiple sclerosis, Ataxia-telangiectasia, Leukodystrophies, Behavioral disorders, Addiction, Anxiety, Pain, Neuroprotection and other diseases, disorders and conditions of the like.
NOV14 nucleic acids and polypeptides are further useful in the generation of antibodies that bind immunospecifically to the novel substances of the invention for use in therapeutic or diagnostic methods. These antibodies may be generated according to methods known in the art, using prediction from hydrophobicity charts, as described in the “Anti-NOVX Antibodies” section below. For example the disclosed NOV14 protein have multiple hydrophilic regions, each of which can be used as an immunogen. This novel protein also has value in development of powerful assay system for functional analysis of various human disorders, which will help in understanding of pathology of the disease and development of new drug targets for various disorders.
NOV15
A disclosed NOV15 nucleic acid of 1478 nucleotides (also referred to as CG57454-01) encoding a novel Cytokeratin-like protein is shown in Table 15A. An open reading frame was identified beginning with an ATG initiation codon at nucleotides 21-23 and ending with a TGA codon at nucleotides 1464-1466. Putative untranslated regions upstream from the intitation codon and downstream from the termination codon are underlined in Table 15A, and the start and stop codons are in bold letters.







TABLE 15A





NOV15 Nucleotide Sequence
















(SEQ ID NO:53)









ACTCCACTCCTGCCTCCACCATGTCCATCAGGGTGACCCAGAAGTCCTACAAGGTGTCCACCTCTGGCCCCCA



GGCCTTTAGCAGCCGCTTTTACACGAATGGGCCTGGTGCCCACATCAGCTCTTCAAGCCTCTCCCGAGTGAGC


AGCAGCAGCTTCCGGGGTGGCCTGGGCAGAGGCTATGGTGGGGCCAGCGGCATAGGAGGCATCACCACTGTCA


CGTTCAACCAGAGCCTGCTGAGCCCTGTTAACCTGGAGGTAGATCCCAATATCCAGGCCATGCACACCCAGGA


GAAGGAGCAGATCAAGACCCTCAACAACAAGTTTGCCTCCTTCATCGACAAGGTACGGTTCCTGCAGCAGAAG


AACAAGATGCTGGAGACCAAGTGGAGCCTCCTGCAGCAGCAGAAGATGGCTCGGATCAACGTATTTGAGAGCT


ACATGAACAACCTTAGGCGGCAGCTGGAGGCTCTGGGCCAGGAGAAGCTGAAGCTGGAGGCGGAGCTTGGCAA


TATGCAGGGGCTGGTGGAGGACTTCAAGAACAAGTATGAGGATGAGATCAATAAGCGTACAGAAACGGAGAAT


GAATTTGTCCTCATCAAGAAGGACATGGATGAAGCTTACATGAACAAGGCAGAGCTGGAGTCTCGCCTGGAAG


GGCTGACTGACGAGATCAACTTCCTCAGGCAACTGCATGAAGAGGAGATCCAGGAGCTGCAGTCCCAGATCTC


GGGCACGTCTGCGGTGCTGTCCATGGACAACAGCCTCTCCCTGGACATGGACAGCATCATCGCTGAGGTCAAG


GCACAGGAGGAGGAGATCGCCAACCGCAGCTGGGCTGAGGCTGAGAGCATGTACCAGATCAAGTATGCAGAGC


TGCAGACGCTGGCTGGCAAGCACGGGGATGACCTGCGGTGTACAAAGACTAAGATCTCCGAGATGAACCGGAA


CATCAGCCGGCTCCAGGCTGAGATTGAGGGCCTCAAAGGCCAGAGGGCTTCCCTGGAGGCCCCCATCGCAGAT


ACCGAGCAGCGTGGGGAGCTGGCCGTTAAGGATGCCAGCGCCAAGCGGTCGGAGCTGGAGGCCGCCCTGCAGC


GGGCCAAGCAGGACATGGCGCAGCAGCTGCGTGAGTACCAGGAGCTGATGAACGTCAAACTGGCCCTGGACAT


GGAGATCGCCACCTACAGGAAGCTGCTGGAGGGCGAGGAGAGCGCCCGGCTGGAGTCTGGGATGCAGAACACG


AGTATCCATAGGAAGACCACCAGCGGCTATGCAGGTGGTCTGAGTTCCGCCTATGGGGGCCTCACAAGCCCTG


GCCTCAGCTATGGCCTAAGCTCCAGCTTTGGCTCTGTCGCGGGCTCCAGCTCATTCAGCCGCACCGGCTCCGC


CAGGGCCATGGTTGTGCAGAAGATTGAGACCCGCGATGGGAAGCTGGTATCCGAGTCCTCTGACGTCCTGCCC


AAGTGAAGAGCTGCGGCA









The NOV15 nucleic acid was identified on chromosome 3 and has 1187 of 1344 bases (88%) identical to a gb:GENBANK-ID:AF213884S2|acc:AF224669.1 mRNA from Homo sapiens (Homo sapiens mannosidase, beta A, lysosomal (MANBA) gene, and ubiquitin-conjugating enzyme E2D 3 (UBE2D3) genes, complete cds) (E=7.3e−228).
A disclosed NOV15 polypeptide (SEQ ID NO:54) encoded by SEQ ID NO:53 is 481 amino acid residues and is presented using the one-letter code in Table 15B. Signal P, Psort and/or Hydropathy results predict that NOV15 contains a signal peptide and is likely to be localized to the plasma membrane with a certainty of 0.3600.







TABLE 15B





Encoded NOV15 protein sequence
















(SEQ ID NO:54)









MSIRVTQKSYKVSTSGPQAFSSRFYTNGPGAHISSSSLSRVSSSSFRGGLGRGYGGASGIGGITTVTFNQSL



LSPVNLEVDPNIQAMHTQEKEQIKTLNNKFASFIDKVRFLQQKNKMLETKWSLLQQQKMARINVFESYMNNL


RRQLEALGQEKLKLEAELGNMQGLVEDFKNKYEDEINKRTETENEFVLIKKDMDEAYMNKAELESRLEGLTD


EINFLRQLHEEEIQELQSQISGTSAVLSMDNSLSLDMDSIIAEVKAQEEEIANRSWAEAESMYQIKYAELQT


LAGKHGDDLRCTKTKISEMNRNISRLQAEIEGLKGQRASLEAPIADTEQRGELAVKDASAKRSELEAALQRA


KQDMAQQLREYQELMNVKLALDMEIATYRKLLEGEESARLESGMQNTSIHRKTTSGYAGGLSSAYGGLTSPG


LSYGLSSSFGSVAGSSSFSRTGSARAMVVQKIETRDGKLVSESSDVLPK









The NOV15 amino acid sequence has 424 of 482 amino acid residues (87%) identical to, and 448 of 482 amino acid residues (92%) similar to, the 482 amino acid residue ptnr:SWISSPROT-ACC:P05787 protein from Homo sapiens (Human) (Keratin, Type II Cytoskeletal 8 (Cytokeratin 8) (K8) (CK 8)) (E=4.9e−213).
NOV15 is expressed in at least the following tissues: Heart, Kidney, Liver, Lung, Muscle, Nose, Ovary, Pancreas, Parathyroid, Placenta, Pooled, Prostate, Stomach, Synovial membrane, Testis, Thyroid, Tonsil and Uterus. This information was derived by determining the tissue sources of the sequences that were included in the invention including but not limited to SeqCalling sources, Public EST sources, genomic clone sources, literature sources, and/or RACE sources.
NOV15 has homology to the amino acid sequences shown in the BLASTP data listed in Table 15C.







TABLE 15C







BLAST results for NOV15












Gene Index/

Length
Identity
Positives



Identifier
Protein/Organism
(aa)
(%)
(%)
Expect





gi|105815|pir||A34720
keratin 8, type
483
335/484
355/484
 e−145



II cytoskeletal

(69%)
(73%)



[Homo sapiens]


gi|2506774|sp|P05787|
KERATIN, TYPE II
483
335/484
354/484
 e−145


K2C8_HUMAN
CYTOSKELETAL 8

(69%)
(72%)



(CYTOKERATIN 8)



(K8) (CK 8) [Homo



sapiens]


gi|4504919|ref|NP_002264.1|
keratin 8;
483
335/484
354/484
 e−145


(NM_002273)
Keratin-8 [Homo

(69%)
(72%)



sapiens]


gi|181400|gb|AAA35748.1|
cytokeratin 8
483
333/484
354/484
1e−144


(M34225)
[Homo sapiens]

(68%)
(72%)


gi|87303|pir||JS0487
cytokeratin 8
482
335/484
354/484
1e−143



(version 1) [Homo

(69%)
(72%)



sapiens]









The homology of these sequences is shown graphically in the ClustalW analysis shown in Table 15D.







TABLE 15D





Clustal W Sequence Alignment















1) NOV15 (SEQ ID NO:54)


2) gi|105815|pir||A34720 keratin 8, type II cytoskeletal [Homo sapiens]


(SEQ ID NO:183)


3) gi|2506774|sp|P05787|K2C8_HUMAN KERATIN, TYPE II CYTOSKELETAL 8 (CYTOKERATIN 8)


(K8) (CK 8) [Homo sapiens] (SEQ ID NO:184)


4) gi|4504919|refNP_002264.1|(NM_002273) keratin 8; Keratin-8 [Homo sapiens]


(SEQ ID NO:185)


5) gi|181400|gb AAA35748.1|(M34225) cytokeratin 8 [Homo sapiens] (SEQ ID NO:186)


6) gi|87303 pir||JS0487 cytokeratin 8 (version 1) [Homo sapiens] (SEQ ID NO:187)


























































Table 15E lists the domain description from DOMAIN analysis results against NOV15. This indicates that the NOV15 sequence has properties similar to those of other proteins known to contain these domains.







TABLE 15E





Domain analysis of NOV15
















gnl|Pfam|pfam00038, filament, Intermediate filament protein. (SEQ ID NO:188)



CD-Length = 312 residues, 99.7% aligned


Score = 261 bits (668), Expect = 5e-71











Query:
90
QEKEQIKTLNNKFASFIDKVRFLQQKNKMLETKWSLLQQQKMARI----NVFESYMNNLR
145





 ||||++ ||++ ||+|||||||+|+|| || |   |+|++   +    +++|+ +  ||


Sbjct:
1
NEKEQMQNLNDRLASYIDKVRFLEQQNKELEVKIEELRQKQAPSVSRLYSLYETEIEELR
 60


Query:
146
RQLEALGQEKLKLEAELGNMQGLVEDFKNKYEDEINKRTETENEFVLIKKDMDEAYMNKA
205




||++ |  |+ +|+ |+ |++   |||+ ||||||| | | ||+ | ++||+||| + + 


Sbjct:
61
RQIDQLTNERARLQLEIDNLREAAEDFRKKYEDEINLRQEAENDLVGLRKDLDEATLARV
120


Query:
206
ELESRLEGLTDEINFLRQLHEEEIQELQSQISGTSAVLSMDNSLSLDMDSIIAEVKAQEE
265




+||+++| | +|+ ||++ ||||++|||+||  |  |              + |++|| |


Sbjct:
121
DLENKVESLQEELEFLKKNHEEEVKELQAQIQDTVNVEMDAARKLDLTK-ALREIRAQYE
179


Query:
266
EIANRSWAEAESMYQIKYAELQTLAGKHGDDLRCTKTKISEMNRNISRLQAEIEGLKGQR
325




||| ++  |||  |+ |  |||| | ++|+ ||  | +|+|+ | |  |+ |++ || | 


Sbjct:
180
EIAKKNRQEAEEWYKSKLEELQTAAARNGEALRSAKEEITELRRQIQSLEIELQSLKAQN
239


Query:
326
ASLEAPIADTEQRGELAVKDASAKRSELEAALQRAKQDMAQQLREYQELMNVKLALDMEI
385




||||  +|+ |+| || ++   |  |+||  ||+ +++||+||||||||++||||||+||


Sbjct:
240
ASLERQLAELEERYELELRQYQALISQLEEELQQLREEMARQLREYQELLDVKLALDIEI
299


Query:
386
ATYRKLLEGEES
397




||||||||||||


Sbjct:
300
ATYRKLLEGEES
311









Keratin 8 is a type II keratin (Moll et al., 1982). Endo A is the mouse equivalent. Endo B, which is the equivalent of human keratin 18, a type I keratin, is coexpressed with Endo A; the 2 appear to be the first intermediate filament (IF) proteins expressed during murine development (Jackson et al., Differentiation. 17(3):161-79, 1980). Yamamoto et al. (Mol Endocrinol. 4(3):370-4, 1990) studied a full-length cDNA for cytokeratin 8 from placenta. They determined the distribution of cytokeratin 8 mRNA in various fetal tissues and in placentae of different gestational ages. Keratins 8 and 18 of simple epithelia differ from the keratins of stratified epithelium in tissue expression and regulation.
Using PCR to study DNAs from somatic cell hybrids, Waseem et al. (Genomics. 7(2):188-94, 1990) located a single active gene for keratin 8 on chromosome 12. This chromosome contains several genes for type II keratins and also the gene for keratin 18, the type I keratin that is coexpressed with keratin 8. This location of both members of a keratin pair on a single chromosome is unique among keratin genes; it is consistent with the hypothesis that keratins 8 and 18 may be closer to an ancestral gene than the keratins of more highly differentiated epithelia. Casanova et al. (J Clin Invest. 103(11):1587-95, 1999) generated mice expressing the human KRT8 gene, leading to a moderate increase in the content of keratin in simple epithelia. These mice displayed progressive exocrine pancreas alterations, including dysplasia and loss of acinar architecture, redifferentiation of acinar to ductal cells, inflammation, fibrosis, and substitution of exocrine by adipose tissue, as well as increased cell proliferation and apoptosis. The phenotype was very similar to that reported for transgenic mice expressing a dominant-negative mutant TGF-beta type II receptor. Casanova et al. (1999) showed that these Tgfbr2 mutant mice also had elevated KRT8/KRT18 levels. The results indicated that simple epithelial keratins play a relevant role in the regulation of exocrine pancreas homeostasis and supported the idea that disruption of mechanisms that normally regulate keratin expression in vivo could be related to inflammatory and neoplastic pancreatic disorders.
The NOV15 nucleic acid of the invention encoding a Cytokeratin-like protein includes the nucleic acid whose sequence is provided in Table 15A, or a fragment thereof. The invention also includes a mutant or variant nucleic acid any of whose bases may be changed from the corresponding base shown in Table 15A while still encoding a protein that maintains its Cytokeratin-like activities and physiological functions, or a fragment of such a nucleic acid. The invention further includes nucleic acids whose sequences are complementary to those just described, including nucleic acid fragments that are complementary to any of the nucleic acids just described. The invention additionally includes nucleic acids or nucleic acid fragments, or complements thereto, whose structures include chemical modifications. Such modifications include, by way of non-limiting example, modified bases, and nucleic acids whose sugar phosphate backbones are modified or derivatized. These modifications are carried out at least in part to enhance the chemical stability of the modified nucleic acid, such that they may be used, for example, as antisense binding nucleic acids in therapeutic applications in a subject. In the mutant or variant nucleic acids, and their complements, up to about 12% of the residues may be so changed.
The NOV15 protein of the invention includes the Cytokeratin-like protein whose sequence is provided in Table 15B. The invention also includes a mutant or variant protein any of whose residues may be changed from the corresponding residue shown in Table 15B while still encoding a protein that maintains its Cytokeratin-like activities and physiological functions, or a functional fragment thereof. In the mutant or variant protein, up to about 13% of the bases may be so changed.
The NOV15 nucleic acids and proteins of the invention are useful in potential diagnostic and therapeutic applications implicated in various diseases and disorders described below and/or other pathologies. For example, the compositions of the present invention will have efficacy for treatment of patients suffering from: Cardiomyopathy, Atherosclerosis, Hypertension, Congenital heart defects, Aortic stenosis, Atrial septal defect (ASD), Atrioventricular (A-V) canal defect, Ductus arteriosus, Pulmonary stenosis, Subaortic stenosis, Ventricular septal defect (VSD), valve diseases, Tuberous sclerosis, Scleroderma, Obesity, Transplantation, Von Hippel-Lindau (VHL) syndrome, Alzheimer's disease, Stroke, Tuberous sclerosis, hypercalceimia, Parkinson's disease, Huntington's disease, Cerebral palsy, Epilepsy, Lesch-Nyhan syndrome, Multiple sclerosis, Ataxia-telangiectasia, Leukodystrophies, Behavioral disorders, Addiction, Anxiety, Pain, Neuroprotection and other diseases, disorders and conditions of the like.
NOV15 nucleic acids and polypeptides are further useful in the generation of antibodies that bind immunospecifically to the novel substances of the invention for use in therapeutic or diagnostic methods. These antibodies may be generated according to methods known in the art, using prediction from hydrophobicity charts, as described in the “Anti-NOVX Antibodies” section below. For example the disclosed NOV15 protein have multiple hydrophilic regions, each of which can be used as an immunogen. This novel protein also has value in development of powerful assay system for functional analysis of various human disorders, which will help in understanding of pathology of the disease and development of new drug targets for various disorders.
NOV16
NOV16 includes four novel Protocadherin-like proteins disclosed below. The disclosed proteins have been named NOV16a, NOV16b, NOV16c and NOV16d.
NOV16a
A disclosed NOV16a nucleic acid of 2907 nucleotides (also referred to as CG57448-01) encoding a novel Protocadherin-like protein is shown in Table 16A. An open reading frame was identified beginning with an ATG initiation codon at nucleotides 10-12 and ending with a TGA codon at nucleotides 2896-2898. Putative untranslated regions, if any, upstream from the initiation codon and downstream from the termination codon are underlined in Table 16A, and the start and stop codons are in bold letters.







TABLE 16A





NOV16a nucleotide sequence.
















(SEQ ID NO:55)









GGCCGCTGAATGCTAGCTAGAATTCAGCGGCCGCTGAATTCTAGGCGCTGCCGAGGGAATGCGCGCAGCTCA



CAGGCCCTGGGAGTGAGCTGGTGCCCGGCGACCTGGCACCCGCGCCTGGATATGGGGCGTCTACATCGTCCC


AGGAGCAGCACCAGCTACAGGAACCTGCCGCATCTGTTTCTGTTTTTCCTCTTCGTGGGACCCTTCAGCTGC


CTCGGGAGTTACAGCCGGGCCACCGAGCTTCTGTACAGCCTAAACGAGGGACTACCCGCGGGGGTGCTCATC


GGCAGCCTGGCCGAGGACCTGCGGCTGCTGCCCAGGTCTGCAGGGAGGCCGGACCCGCAGTCGCAGCTGCCA


GAGCGCACCGGTGCTGAGTGGAACCCCCCTCTCTCCTTCAGCCTGGCCTCCCGGGGACTGAGTGGCCAGTAC


GTGACCCTAGACAACCGCTCTGGGGAGCTGCACACTTCAGCTCAGGAGATCGACAGGGAGGCCCTGTGTGTT


GAAGGGGGTGGAGGGACTGCGTGGAGCGGCAGCGTTTCCATCTCCTCCTCTCCTTCTGACTCTTGTCTTTTG


CTGCTGGATGTGCTTGTCCTGCCTCAGGAATACTTCAGGTTTGTGAAGGTGAAGATCGCCATCAGAGACATC


AATGACAACGCCCCGCAGTTCCCTGTTTCCCAGATCTCGGTGTGGGTCCCGGAAAATGCACCTGTAAACACC


CGACTGGCCATAGAGCATCCTGCTGTGGACCCAGATGTAGGCATTAATGGGGTACAGACCTATCGCTTACTG


GACTACCATGGTATGTTCACCCTGGACGTGGAGGAGAATGAGAATGGGGAGCGCACCCCCTACCTAATTGTC


ATGGGTGCTTTGGACAGGGAAACCCAGGACCAGTATGTGAGCATCATCATAGCTGAGGATGGTGGGTCTCCA


CCACTTTTGGGCAGTGCCACTCTCACCATTGGCATCAGTGACATTAATGACAATTGCCCTCTCTTCACAGAC


TCACAAATCAATGTCACTGTGTATGGGAATGCTACAGTGGGCACCCCAATTGCAGCTGTCCAGGCTGTGGAT


AAAGACTTGGGGACCAATGCTCAAATTACTTATTCTTACAGTCAGAAAGTTCCACAAGCATCTAAGGATTTA


TTTCACCTGGATGAAAACACTGGAGTCATTAAACTTTTCAGTAAGATTGGAGGAAGTGTTCTGGAGTCCCAC


AAGCTCACCATCCTTGCTAATGGACCAGGCTGCATCCCTGCTGTAATCACTGCTCTTGTGTCCATTATTAAA


GTTATTTTCAGACCCCCTGAAATTGTCCCTCGTTACATAGCAAACGAGATAGATGGTGTTGTTTATCTGAAA


GAACTGGAACCCGTTAACACTCCCATTGCGTTTTTCACCATAAGAGATCCAGAAGGTAAATACAAGGTTAAC


TGCTACCTGGATGGTGAAGGGCCGTTTAGGTTATCACCTTACAAACCATACAATAATGAATATTTACTAGAG


ACCACAAAACCTATGGACTATGAGCTACAGCAGTTCTATGAAGTAGCTGTGGTGGCTTGGAACTCTGAGGGA


TTTCATGTCAAAAGGGTCATTAAAGTGCAACTTTTAGATGACAATGATAATGCTCCAATTTTCCTTCAACCC


TTAATAGAACTAACCATCGAAGAGAACAACTCACCCAATGCCTTTTTGACTAAGCTGTATGCTACAGATGCC


GACAGCGAGGAGAGAGGCCAAGTTTCATATTTTCTGGGACCTGATGCTCCATCATATTTTTCCTTAGACAGT


GTCACAGGAATTCTGACAGTTTCTACTCAGCTGGACCGAGAAGAGAAAGAAAAGTACAGATACACTGTCAGA


GCTGTTGACTGTGGGAAGCCACCCAGAGAATCAGTAGCCACTGTGGCCCTCACAGTGTTGGATAAAAATGAC


AACAGTCCTCGGTTTATCAACAAGGACTTCAGCTTTTTTGTGCCTGAAAACTTTCCAGGCTATGGTGAGATT


GGAGTAATTAGTGTAACAGATGCTGACGCTGGACGAAATGGATGGGTCGCCCTCTCTGTGGTGAACCAGAGT


GATATTTTTGTCATAGATACAGGAAAGGGTATGCTGAGGGCTAAAGTCTCTTTGGACAGAGAGCAGCAAAGC


TCCTATACTTTGTGGGTTGAAGCTGTTGATGGGGGTGAGCCTGCCCTCTCCTCTACAGCAAAAATCACAATT


CTCCTTCTAGATATCAATGACAACCCTCCTCTTGTTTTGTTTCCTCAGTCTAATATGTCTTATCTGTTAGTA


CTGCCTTCTACTCTGCCAGGCTCCCCGGTTACAGAAGTCTATGCTGTCGACAAAGACACAGGCATGAATGCT


GTCATAGCTTACAGCATCATAGGGAGAAGAGGTCCTAGGCCTGAGTCCTTCAGGATTGACCCTAAAACTGGC


AACATTACTTTGGAAGAGGCATTGCTGCAGACAGATTATGGGCTCCATCGCTTACTGGTGAAAGTGAGTGAT


CATGGTTATCCCGAGCCTCTCCACTCCACAGTCATGGTGAACCTATTTGTCAATGACACTGTCAGTAATGAG


AGTTACATTGAGAGTCTTTTAAGAAAAGAACCAGAGATTAATATAGAGGAGAAAGAACCACAAATCTCAATA


GAACCGACTCATAGGAAGGTAGAATCTGTGTCTTGTATGCCCACCTTAGTAGCTCTGTCTGTAATAAGCTTG


GGTTCCATCACACTGGTCACAGGGATGGGCATATACATCTGTTTAAGGAAAGGGGAAAAGCATCCCAGGGAA


GATGAAAATTTGGAAGTACAGATTCCACTGAAAGGAAAAATTGACTTGCATATGCGAGAGAGAAAGCCAATG


GATATTTCTAATATTTGATATTTCATG









The disclosed NOV16a nucleic acid sequence was localized to chromsome 5.
A NOV16a polypeptide (SEQ ID NO:56) encoded by SEQ ID NO:55 has 962 amino acid residues and is presented using the one-letter code in Table 16B. Signal P, Psort and/or Hydropathy results predict that NOV16a contains a signal peptide and is likely to be localized to the mitochondrial inner membrane with a certainty of 0.6916, to the mitochondrial intermembrane space with a certainty of 0.6185, to the plasma membrane with a certainty of 0.6000 and to the mitochondrial matrix space with a certainty of 0.5077. The most likely cleavage site for a NOV16a polypeptide is between amino acids 13 and 14: SRR-CR







TABLE 16B





Encoded NOV16a protein sequence.
















(SEQ ID NO:56)









MLARIQRPLNSRRCRGNARSSQALGVSWCPATWHPRLDMGRLHRPRSSTSYRNLPHLFLFFLFVGPFSCLGS



YSRATELLYSLNEGLPAGVLIGSLAEDLRLLPRSAGRPDPQSQLPERTGAEWNPPLSFSLASRGLSGQYVTL


DNRSGELHTSAQEIDREALCVEGGGGTAWSGSVSISSSPSDSCLLLLDVLVLPQEYFRFVKVKIAIRDINDN


APQFPVSQISVWVPENAPVNTRLAIEHPAVDPDVGINGVQTYRLLDYHGMFTLDVEENENGERTPYLIVMGA


LDRETQDQYVSIIIAEDGGSPPLLGSATLTIGISDINDNCPLFTDSQINVTVYGNATVGTPIAAVQAVDKDL


GTNAQITYSYSQKVPQASKDLFHLDENTGVIKLFSKIGGSVLESHKLTILANGPGCIPAVITALVSIIKVIF


RPPEIVPRYIANEIDGVVYLKELEPVNTPIAFFTIRDPEGKYKVNCYLDGEGPFRLSPYKPYNNEYLLETTK


PMDYELQQFYEVAVVAWNSEGFHVKRVIKVQLLDDNDNAPIFLQPLIELTIEENNSPNAFLTKLYATDADSE


ERGQVSYFLGPDAPSYFSLDSVTGILTVSTQLDREEKEKYRYTVRAVDCGKPPRESVATVALTVLDKNDNSP


RFINKDFSFFVPENFPGYGEIGVISVTDADAGRNGWVALSVVNQSDIFVIDTGKGMLRAKVSLDREQQSSYT


LWVEAVDGGEPALSSTAKITILLLDINDNPPLVLFPQSNMNYLLVLPSTLPGSPVTEVYAVDKDTGMNAVIA


YSIIGRRGPRPESFRIDPKTGNITLEEALLQTDYGLHRLLVKVSDHGYPEPLHSTVMVNLFVNDTVSNESYI


ESLLRKEPEINIEEKEPQISIEPTHRKVESVSCMPTLVALSVISLGSITLVTGMGIYICLRKGEKHPREDEN


LEVQIPLKGKIDLHMRERKPMDISNI









The NOV16a amino acid sequence has 946 of 947 amino acid residues (99%) identical to, and 946 of 947 amino acid residues (99%) similar to, the 947 amino acid residue ptnr:SPTREMBL-ACC:Q9NRT9 protein from Homo sapiens (Human) (Protocadherin 10) (E=0.0).
The disclosed NOV16a is expressed in at least the following tissues: Uterus, colon, eye and retina. This information was derived by determining the tissue sources of the sequences that were included in the invention including but not limited to SeqCalling sources, Public EST sources, Literature sources, and/or RACE sources.
NOV16b
A disclosed NOV16b nucleic acid of 2801 nucleotides (also referred to as CG57446-01) encoding a novel Protocadherin-like protein is shown in Table 16C. An open reading frame was identified beginning with an ATG initiation codon at nucleotides 6-8 and ending with a TAA codon at nucleotides 2724-2726. Putative untranslated regions, if any, upstream from the initiation codon and downstream from the termination codon are underlined in Table 16C, and the start and stop codons are in bold letters.







TABLE 16C





NOV16b nucleotide sequence.
















(SEQ ID NO:57)









ACCGGATGCACCCGGAGATCTCCTACTCAATTCCTGAGGAAAGAGAGAAAGGCTCTTTCGTGGGCAACATCT



CCAAGGACTTGGGTCTGGCGCCCCGGGAGCTGGCGGAGCGCGGAGTCCGCATAGTCTCCAGAGGTAGGACGC


AGCTTTTCTCTCTGAACCCGCGCAGCGGCAGCTTGGTCACCGCGGGCAGGATAGACCGGGAGGAGCTCTGCG


CTCAGAGCGCGCGGTGCGTGGTGAGTTTTAATATCCTTGTGGAAGACAGGGTGAAACTTTTTGGGATAGAAA


TAGAAGTAACTGATATCAATGACAATGCTCCAAAATTCCAAGCAGAAAATCTAGACGTAAAAATTAATGAAA


ATGTCGCTGCGGGAATGCGTTTTCCTCTCCCGGAAGCTATTGATCCGGATATAGGTGTAAACTCCCTGCAGG


GTTACCAGCTCAACTCAAACGGTTACTTTTCCCTGGACGTGCAAAGTGGGGCCGATGGGATTAAGTACCCAG


AGCTGGTGCTGGAACGCGCTCTAGATCGCGAGGAAGAGGCGGTTCACCACCTGGTCCTTACTGCCATGGATG


GCGGCGACCCTCTCCGCTCAAGCGTCGCCCAAATTCTGGTAACAGTTCTAGATGTGAATGACAACACTCCAA


TGTTTACTCAGCCTGTCTACCGTGTAAGTGTTCCTGAAAACCTGCCAGCTCCCGGAACTCGGGTGCTGATGG


TTAATGCAACGGATCCAGATGAAGGAGTCAATGCGGAAGTAATGTATTCATTTCGGAAAGTCAGAGACGAAA


GAGCACAGCTATTACAGTTGTTTTATCTGAGTGCGGAGATAACGATAATGAGGGGTCTGGAGGATGTGGACT


ATGGATACTATGACATAGATGACATAGACGATGAAGGCCATGGTGTCCGTGCTAGAAGAGCGGTACGCAAGG


TAGTGGTGGAAGTTTTGGATGAAAATGACAACGCCCCAGAAATCACAGTCACCTCCGTCACCACCGCAGTCC


CCGAAGCTGCTTCTGGAACTGCCATTATTTTCCTCAATGATAGTGACCGAGAGGACGGGGGGAACAGTCCAT


TTATCAGTTCTGTCAATCCGGGTCTTTCATTCAAAAAATTAGATAAAAAAGATGATTATTTCATTTTTAAAA


CGACTCAAGACATAGACCGAAAAACTGTGTCCGAATACAACATCACCGCAATAGCCCCAGAAACCCGAGCTC


CTTCCCCTTCAACTCATATTACACTCCTTGTGCTAGTGATCGACATCAATGAAAACCCTCCCCCTTATTCTC


AATCCTCCTACTACGTTTACGTAAACGAAAACAACGGCGCCGGCACTTCAATTATGACCGTTAATGACTCTG


ACCCCGATGACAATTCTAGTGTTATTTACTCCTTGGCAGAGGCTACCCAAGGAGCTCCTCCCTCCTCCACCT


ATGCCTCTATCACATCAAACACTGGTGTGCTTGATGTGTTGTCCTTCTTCTACTATGAGTATTTTGATTTTC


TGCAAATGCAGATGACGGCTAATGACAGTGGCAGCCCACCACTTAGCAGTAATGCGTCAATGAGATTGTTTG


TGTTGGACAAGGATGACAATGCCCAAGACCTCCAGTACCCTGCCCCCCCCACTGGTGGTACTGCTGTTGAGG


TGCTGCCCCGCTCTGCAGCGCCTGGCTACCTGGTGACCAAGGTGGTGGCAGTGGACGGAGACTCAGGCCAGA


ATGCTTGGCTCTCCTACCGCCTATTCGAGGCCAGTGAGCCGGGGCTCTTCTCGGTGGGGCTGCACACAGGTG


GAAAAGTGCGCACCGCTCGGGCCCTGCTAGATAGAGATGCGCTCAAACAGAGCCTTGTGGTGGCTGTACAGG


ACCATGGCCAGCCCCCTCTCTCCGCCGCCACCCTCACTGTGGCGGTTGCCGACGACTCCCTCCAAGACCTCG


CGGATTTCTTCGGCAGCCTCACGCCTTCAGAACACCAAGACGACTCCGGCCTCACACTCCTTCTTGTGGTAG


TAGTGGCTGCAGTCTGCTTCGTCTTCCCGGTCTTCGTCGTCGTGCTGCTAGTACTCAAGCTGAGGCGCTGGC


ACAAGTCCCGCCTGCTTCACGCTGAAGGCAGCAGGTTGGCAGGGGTGGCTGCCTCCCACTTTGGGGGCGTGG


AGGGGGTTGGGGTTTTCCTGCCGAACTATTCCCACGAGGTCTCCCTCACCGCGGACTCGCGGAAGAGCCCCC


TGATCCCCACCCAACCCCGCGCTGAAATTCCCCTCAGCAACCGGGAGAGTGGAGAGAGAAGCCGCCGTCTGG


TGATACTTAAGGATGTGCTTGAAACAGAGGGCGACCCTAGTGGACAGCAAGCCCCGCCCAACACGGACTGGC


GTTTCTCTCAGGCCCAGAGACCCGGCACCAGCGGCTCCCAAAATGGCGATGACACCGGCACCTGGCCCAACA


ACCAGTTTGACACAGAGATGCTGCAAGCCATGATCTTGGCGTCCGCCAGTGAAGCTGCTGATGGGAGCTCCA


CCCTGGGAGGGGGTGCCGGCACCATGGGATTGAGCGCCCGCTACGGACCCCAGTTCACCCTGCAGCACGTGC


CCGACTACCGCCAGAATGTCTACATCCCAGGCAGCAATGCCACACTGACCAACGCAGCTGGCAAGCGGGATG


GCAAGGCCCCAGCAGGTGGCAATGGCAACAAGAAGAAGTCGGGCAAGAAGGAGAAGAAGTAACATGGAGGCC


AGGCCAAGAGCCACAGGGCGGCCTCTCCCCAACCAGCCCAGCTTCTCCTTACCTGCACCCAGGCC









The disclosed NOV16b nucleic acid sequence, localized to chromsome 5, has 2214 of 2786 bases (79%) identical to a gb:GENBANK-ID:AF152324|acc:AF152324.1 mRNA from Homo sapiens (protocadherin gamma A4 (PCDH-gamma-A4) mRNA, complete cds) (E=0.0).
A NOV16b polypeptide (SEQ ID NO:58) encoded by SEQ ID NO:57 has 906 amino acid residues and is presented using the one-letter code in Table 16D. Signal P, Psort and/or Hydropathy results predict that NOV16b does not contain a signal peptide and is likely to be localized to the plasma membrane with a certainty of 0.7000 and to the nucleus with a certainty of 0.6000.







TABLE 16D





Encoded NOV16b protein sequence.
















(SEQ ID NO:58)









MHPEISYSIPEEREKGSFVGNISKDLGLAPRELAERGVRIVSRGRTQLFSLNPRSGSLVTAGRIDREELCAQ



SARCVVSFNILVEDRVKLFGIEIEVTDINDNAPKFQAENLDVKINENVAAGMRFPLPEAIDPDIGVNSLQGY


QLNSNGYFSLDVQSGADGIKYPELVLERALDREEEAVHHLVLTAMDGGDPLRSSVAQILVTVLDVNDNTPMF


TQPVYRVSVPENLPAPGTRVLMVNATDPDEGVNAEVMYSFRKVRDERAQLLQLFYLSAEITIMRGLEDVDYG


YYDIDDIDDEGHGVRARRAVRKVVVEVLDENDNAPEITVTSVTTAVPEAASGTAIIFLNDSDREDGGNSPFI


SSVNPGLSFKKLDKKDDYFIFKTTQDIDRKTVSEYNITAIAPETRAPSPSTHITLLVLVIDINENPPPYSQS


SYYVYVNENNGAGTSIMTVNDSDPDDNSSVIYSLAEATQGAPPSSTYASITSNTGVLDVLSFFYYEYFDFLQ


MQMTANDSGSPPLSSNASMRLFVLDKDDNAQDLQYPAPPTGGTAVEVLPRSAAPGYLVTKVVAVDGDSGQNA


WLSYRLFEASEPGLFSVGLHTGGKVRTARALLDRDALKQSLVVAVQDHGQPPLSAATLTVAVADDSLQDLAD


FFGSLTPSEHQDDSGLTLLLVVVVAAVCFVFPVFVVVLLVLKLRRWHKSRLLHAEGSRLAGVAASHFGGVEG


VGVFLPNYSHEVSLTADSRKSPLIPTQPRAEIPLSNRESGERSRRLVILKDVLETEGDPSGQQAPPNTDWRF


SQAQRPGTSGSQNGDDTGTWPNNQFDTEMLQAMILASASEAADGSSTLGGGAGTMGLSARYGPQFTLQHVPD


YRQNVYIPGSNATLTNAAGKRDGKAPAGGNGNKKKSGKKEKK









The NOV16b amino acid sequence has 635 of 903 amino acid residues (70%) identical to, and 721 of 903 amino acid residues (79%) similar to, the 931 amino acid residue ptnr:SPTREMBL-ACC:Q9Y5G9 protein from Homo sapiens (Human) (Protocadherin Gamma A4) (E=0.0).
The disclosed NOV16b is expressed in at least the following tissues: Uterus, colon, eye and retina. This information was derived by determining the tissue sources of the sequences that were included in the invention including but not limited to SeqCalling sources, Public EST sources, Literature sources, and/or RACE sources.
NOV16c
A disclosed NOV16c nucleic acid of 2836 nucleotides (also referred to as CG57444-01) encoding a novel Protocadherin-like protein is shown in Table 16E. An open reading frame was identified beginning with an ATG initiation codon at nucleotides 15-17 and ending with a TAA codon at nucleotides 2802-2804. Putative untranslated regions, if any, upstream from the initiation codon and downstream from the termination codon are underlined in Table 16E, and the start and stop codons are in bold letters.







TABLE 16E





NOV16c nucleotide sequence.
















(SEQ ID NO:59)









AACCCGAGCGAACGATGGGAGGGAGCTGCGCGCAGAGGCGCCGGGCCGGCCCGCGGCAGGTACTATTTCCTT



TGCTGCTGCCTTTGTTCTACCCCACGCTGTGTGAGCCGATCCGCTACTCGATTCCGGAGGAGCTGGCCAAGG


GCTCGGTGGTGGGGAACCTCGCTAAGGATCTAGGGCTTAGTGTCCTGGATGTGTCGGCTCGCGAGCTGCGAG


TGAGCGCGGAGAAGCTGCACTTCAGCGTAGACGCGCAGAGCGGGGACTTACTTGTGAAGGACCGAATAGACC


GTGAGCAAATATGCAAAGAGAGAAGAAGATGTGAGTTGCAATTGGAAGCTGTGGTGGAAAATCCTTTAAATA


TTTTTCATGTCATTGTGGTGATTGAGGATGTTAATGACCACGCCCCTCAATTCCGGAAAGATGAAATAAACT


TAGAAATCAGTGAATCCGTCAGCCTGGGGATGGGAACAATTCTTGAGTCTGCAGAAGATCCTGATATTAGTA


TGAATTCGCTGAGCAAATACCAACTAAGTCCTAACGAGTATTTCTCATTGGTGGAGAAAGACAATCCTGATG


GTGGCAAATATCCAGAATTAGTATTGCAGAAGACTCTGGACCGAGAAACGCAGAGCGCTCACCACTTGATAT


TGACCGCCTTGGACGGAGGGGACCCACCAAGAAGTGCCACCGCTCACATAGAAATTTCTGTCAAGGATACCA


ATGATAACCCCCCGGTTTTCAGCAGAGACGAATATAGAATTAGTCTTAGTGAAAATCTGCCCCCTGGGTCCC


CTGTGTTGCAAGTGACAGCCACTGACCAGGATGAGGGGGTCAATGCTGAGATAAACTACTACTTCCGAAGCA


CTGCCCAGAGCACAAAACATATGTTCTCATTGGATGAGAAAACAGGTATGATTAAGAATAACCAGTCATTTG


ATTTTGAAGATGTAGAAAGGTACACCATGGAAGTGGAAGCGAAGGACGGAGGTGGTCTCTCTACCCAGTGTA


AAGTAATCATAGAAATCCTTGATGAAAACGACAACAGCCCAGAAATAATCATCACTTCTCTCTCTGATCAGA


TTTTGGAGAATTCACCTCCAGGAATGGTTGTTGCCCTCTTCAAAACACGGGATCTGGATTTCGGAGGAAATG


GAGAAGTCAGGTGTAATATAGAAACAGACATTCCATTCAAGATTTATTCTTCTTCCAATAACTACTACAAAC


TGGTGACAGATGGAGCCCTGGACCGAGAGCAGACACCAGAATACAATGTCACCATCGTAGCCACTGACAGGG


GCAAGCCGCCTCTTTCTTCCAGTAGAAGCATCACCTTGTATGTCGCTGACATCAACGACAACGCCCCAGTTT


TCGACCAGACGTCCTACGTGGTCCACGTGGCCGAGAACAACCCGCCAGGAGCCTCCATTGCGCAAGTGAGCG


CCTCTGACCCGGATTTGGGGCTCAATGGCCACATCTCCTACTCTCTCATTGCCAGCGACCTGGAGTCACGAA


CGCTGTCGTCCTACGTGTCCGTGAGCGCGCAGAGCGGGGTGGTGTTCGCGCAGCGCGCCTTCGACCACGAGC


AGCTGCGCGCCTTCGCGCTCACGCTGCAGGCCCGCGACCAGGGCTCGCCCGCGCTCAGCGCGAACGTGAGCC


TGCGCGTGTTAGTGGACGACCGCAACGACAATGCGCCACGGGTGCTGTACCCAGCTCTGGGTCCTGACGGCT


CCGCGTTCTTCGATATGGTACCTCGCTCTGCAGAGCCCGGCTACCTAGTGACTAAGGTGGTAGCGGTGGACG


CCGACTCGGGACACAACGCCTGGCTGTCCTACCACGTGCTGCAGGCCAGTGAGCCCGGGCTCTTCAGCCTGG


GGCTGCGAACAGGCGAGGTGCGCATGGTGCGTGCTTTGGGTGACAAGGACTCGGTCCGCCAGCGCCTGCTAG


TCGCTATAAGAGATGGAGGACAGCCACCCCTTTCAGCCACTGCCACGCTGCACCTGGTGTTCGCAGATAGCT


TGCAAGAGGTACTGCCGGATTTCAGCGACCATCCCACACCCTCTGACTCCCAGGCTGAGATGCAGTTTTACC


TGGTGGTGGCCTTGGCCTTGATTTCTGTGCTCTTTCTCCTCGCGGTGATTCTAGCTATTGCTCTACGCCTGC


GACAGTCTTTCAGCCCTACTGCAGGAGACTGCTTTGAGTCAGTTCTCTGCTCCAAGTCCGGACCTGTGGGTC


CCCCCAACTACAGTGAGGGAACGTTGCCCTATGCCTATAATTTTTGTGTGCCTGGGGATCAAATGAATCCAG


AATTTAATTTTTTCACATCTGTTGATCATTGTCCAGCCACACAAGATAACCTCAACAAAGATAGCATGCTAC


TGGCTAGCATTTTAACTCCCAGCGTTGAAGCAGATAAGAAGATTCTTAAACAGCAAGCCCCGCCCAACACGG


ACTGGCGTTTCTCTCAGGCCCAGAGACCCGGCACCAGCGGCTCCCAAAATGGCGATGACACCGGCACCTGGC


CCAACAACCAGTTTGACACAGAGATGCTGCAAGCCATGATCTTGGCGTCCGCCAGTGAAGCTGCTGATGGGA


GCTCCACCCTGGGAGGGGGTGCCGGCACCATGGGATTGAGCGCCCGCTACGGACCCCAGTTCACCCTGCAGC


ACGTGCCCGACTACCGCCAGAATGTCTACATCCCAGGCAGCAATGCCACACTGACCAACGCAGCTGGCAAGC


GGGATGGCAAGGCCCCAGCAGGTGGCAATGGCAACAAGAAGAAGTCGGGCAAGAAGGAGAAGAAGTAACATG


GAGGCCAGGCCAAGAGCCACAGGGCGGC









The disclosed NOV16c nucleic acid sequence, localized to chromsome 5, has 2584 of 2822 bases (91%) identical to a gb:GENBANK-ID:AF152336|acc:AF152336.1 mRNA from Homo sapiens (protocadherin gamma B7 (PCDH-gamma-B7) mRNA, complete cds) (E=0.0).
A NOV16c polypeptide (SEQ ID NO:60) encoded by SEQ ID NO:59 has 929 amino acid residues and is presented using the one-letter code in Table 16F. Signal P, Psort and/or Hydropathy results predict that NOV16c does not contain a signal peptide and is likely to be localized to the plasma membrane with a certainty of 0.5140. The most likely cleavage site for a NOV16c polypeptide is between amino acids 30 and 31: TLC-EP







TABLE 16F





Encoded NOV16c protein sequence.
















(SEQ ID NO:60)









MGGSCAQRRRAGPRQVLFPLLLPLFYPTLCEPIRYSIPEELAKGSVVGNLAKDLGLSVLDVSARELRVSAEK



LHFSVDAQSGDLLVKDRIDREQICKERRRCELQLEAVVENPLNIFHVIVVIEDVNDHAPQFRKDEINLEISE


SVSLGMGTILESAEDPDISMNSLSKYQLSPNEYFSLVEKDNPDGGKYPELVLQKTLDRETQSAHHLILTALD


GGDPPRSATAHIEISVKDTNDNPPVFSRDEYRISLSENLPPGSPVLQVTATDQDEGVNAEINYYFRSTAQST


KHMFSLDEKTGMIKNNQSFDFEDVERYTMEVEAKDGGGLSTQCKVIIEILDENDNSPEIIITSLSDQILENS


PPGMVVALFKTRDLDFGGNGEVRCNIETDIPFKIYSSSNNYYKLVTDGALDREQTPEYNVTIVATDRGKPPL


SSSRSITLYVADINDNAPVFDQTSYVVHVAENNPPGASIAQVSASDPDLGLNGHISYSLIASDLESRTLSSY


VSVSAQSGVVFAQRAFDHEQLRAFALTLQARDQGSPALSANVSLRVLVDDRNDNAPRVLYPALGPDGSAFFD


MVPRSAEPGYLVTKVVAVDADSGHNAWLSYHVLQASEPGLFSLGLRTGEVRMVRALGDKDSVRQRLLVAIRD


GGQPPLSATATLHLVFADSLQEVLPDFSDHPTPSDSQAEMQFYLVVALALISVLFLLAVILAIALRLRQSFS


PTAGDCFESVLCSKSGPVGPPNYSEGTLPYAYNFCVPGDQMNPEFNFFTSVDHCPATQDNLNKDSMLLASIL


TPSVEADKKILKQQAPPNTDWRFSQAQRPGTSGSQNGDDTGTWPNNQRDTEMLQAMILASASEAADGSSTLG


GGAGTMGLSARYGPQFTLQHVPDYRQNVYIPGSNATLTNAAGKRDGKAPAGGNGNKKKSGKKEKK









The NOV16c amino acid sequence has 842 of 929 amino acid residues (90%) identical to, and 881 of 929 amino acid residues (94%) similar to, the 929 amino acid residue ptnr:SPTREMBL-ACC:Q9Y5F8 protein from Homo sapiens (Human) (Protocadherin Gamma B7) (E=0.0).
The disclosed NOV16c is expressed in at least the following tissues: Uterus, colon, eye and retina. This information was derived by determining the tissue sources of the sequences that were included in the invention including but not limited to SeqCalling sources, Public EST sources, Literature sources, and/or RACE sources.
NOV16d
A disclosed NOV16d nucleic acid of 2436 nucleotides (also referred to as CG57442-01) encoding a novel Protocadherin Beta-like protein is shown in Table 16G. An open reading frame was identified beginning with an ATG initiation codon at nucleotides 1-3 and ending with a TGA codon at nucleotides 2398-2400. Putative untranslated regions, if any, upstream from the initiation codon and downstream from the termination codon are underlined in Table 16G, and the start and stop codons are in bold letters.







TABLE 16G





NOV16d nucleotide sequence.
















(SEQ ID NO:61)









ATGGCTGTCAGAGAGTTGTGCTTCCCAAGACAAAGGCAAGTCCTGTTTCTTTTTCTTTTTTGGGGAGTGTCC



TTGGCAGGTTCTGGGTTTGGACGTTATTCGGTGACTGAGGAAACAGAGAAAGGATCCTTTGTGGTCAATCTG


GCAAAGGATCTGGGACTAGCAGAGGGGGAGCTGGCTGCAAGGGGAACCAGGGTGGTTTCCGATGATAACAAA


CAATACCTGCTCCTGGATTCACATACCGGGAATTTGCTCACAAATGAGAAACTGGACCGAGAGAAGCTGTGT


GGCCCTAAAGAGCCCTGTATGCTGTATTTCCAAATTTTAATGGATGATCCCTTTCAGATTTACCGGGCTGAG


CTGAGAGTCAGGGATATAAATGATCACGCGCCAGTATTTCAGGACAAAGAAACAGTCTTAAAAATATCAGAA


AATACAGCTGAAGGGACAGCATTTAGACTAGAAAGAGCACAGGATCCAGATGGAGGACTTAACGGTATCCAA


AACTACACGATCAGCCCCAACTCTTTTTTCCATATTAACATTAGTGGCGGTGATGAAGGCATGATATATCCA


GAGCTAGTGTTGGACAAAGCACTGGATCGGGAGGAGCAGGGAGAGCTCAGCTTAACCCTCACAGCGCTGGAT


GGTGGGTCTCCATCCAGGTCTGGGACCTCTACTGTACGCATCGTTGTCTTGGACGTCAATGACAATGCCCCA


CAGTTTGCCCAGGCTCTGTATGAGACCCAGGCTCCAGAAAACAGCCCCATTGGGTTCCTTATTGTTAAGGTA


TGGGCAGAAGATGTAGACTCTGGAGTCAACGCGGAAGTATCCTATTCATTTTTTGATGCCTCAGAAAATATT


CGAACAACCTTTCAAATCAATCCTTTTTCTGGGGAAATCTTTCTCAGAGAATTGCTTGATTATGAGTTAGTA


AATTCTTACAAAATAAATATACAGGCAATGGACGGTGGAGGCCTTTCTGCAAGATGTAGGGTTTTAGTGGAA


GTATTGGACACCAATGACAATCCCCCTGAACTGATCGTATCATCATTTTCCAACTCTGTTGCTGAGAATTCT


CCTGAGACGCCGCTGGCTGTTTTTAAGATTAATGACAGAGACTCTGGAGAAAATGGAAGGATGGTGTGCTAC


ATTCAAGATGATCTGCCATTCCTACTAAAACCTTCTGTTGAGAATTTTTACATCCTAATGACTGAAGGCGCG


CTGGACAGAGAAGCAAGAGCTGAATATAATATCACCCTCACCGTCACAGATATGGGGACTCCAAGGCTGAAA


ACGGAGCACAACATAACAGTGCAGATATCAGATGTCAATGATAACGCCCCCACTTTCACCCAAACCTCCTAC


GCCCTGTTCGTCCGCGAGAACAACAGCCCCGCCCTGCACATCGGCAGCGTCAGCGCCACAGACAGAGACTCA


GGCACCAACGCCCAGGTCACCTACTCGCTGCTGCCGCCCCAGGACCCGCACCTGCCCCTCGCCTCCCTGGTC


TCCATCAACGCAGACAACGGCCACCTGTTCGCCCTCAGGTCGCTGGACTACGAGGCCCTGCAGGCTTTCGAG


TTCCGCGTGGGCGCCACAGACCGCGGCTCCCCCGCGCTGAGCAGAGAGGCGCTGGTGCGCGTGCTGGTGCTG


GACGCCAACGACAACTCGCCCTTCGTGCTGTACCCGCTGCAGAACGGCTCCGCGCCCTGCACTGAGCTGGTG


CCCCGGGCGGCCGAGCCGGGCTACCTGGTGACCAAGGTGGTGGCGGTGGACGGCGACTCGGGCCAGAACGCC


TGGCTGTCGTACCAGCTGCTCAAGGCCACGGAGCCCGGGCTGTTCGGTGTGTGGGCGCACAATGGGGAGGTG


CGCACCGCCAGGCTGCTGAGCGAGCGCGACGCAGCCAAGCACAGGCTCGTGGTGCTTGTCAAGGACAATGGC


GAGCCTCCTCGCTCGGCCACCGCCACGCTGCACTTGCTCCTGGTGGACGGCTTCTCCCAGCCCTACCTGCCT


CTCCCGGAGGCGGCCCCGGCCCAGGCCCAGGCCGAGGCCGACTTGCTCACCGTCTACCTGGTGGTGGCGTTG


GCCTCGGTGTCTTCGCTCTTCCTCCTCTCGGTGCTCCTGTTCGTGGCGGTGCGGCTGTGCAGGAGGAGCAGG


GCGGCCTCGGTGGGTCGCTGCTCGGTGCCCGAGGGTCCTTTTCCAGGGCATCTGGTGGACGTGAGCGGCACC


GGGACCCTGTTCCAGAGCTACCAGTACGAGGTGTGTCTGACTGGAGGTTCAGAGACCGGCGAGTTCAAGTTC


TTGAAGCCGATTACCCCCCACCTCCCGCCCCATAGGGGTGGGAAAGAAATAGAGGAAAATTCTACTCTCCCC


AATAGCTTTGGATTTAATTATTGAAAGGAACCCACTTAATAAAGACATTTACTTCTTTAA









The disclosed NOV16d nucleic acid sequence, localized to chromsome 5, has 2319 of 2400 bases (96%) identical to a gb:GENBANK-ID:AF152489|acc:AF152489.1 mRNA from Homo sapiens (Homo sapiens protocadherin beta 10 (PCDH-beta10) mRNA, complete cds) (E=0.0).
A NOV16d polypeptide (SEQ ID NO:62) encoded by SEQ ID NO:61 has 799 amino acid residues and is presented using the one-letter code in Table 16H. Signal P, Psort and/or Hydropathy results predict that NOV16d does not contain a signal peptide and is likely to be localized to the plasma membrane with a certainty of 0.4600. The most likely cleavage site for a NOV16d polypeptide is between amino acids 26 and 27: SLA-GS.







TABLE 16H





Encoded NOV16d protein sequence.
















(SEQ ID NO:62)









MAVRELCFPRQRQVLFLFLFWGVSLAGSGFGRYSVTEETEKGSFVVNLAKDLGLAEGELAARGTRVVSDDNK



QYLLLDSHTGNLLTNEKLDREKLCGPKEPCMLYFQILMDDPFQIYRAELRVRDINDHAPVFQDKETVLKISE


NTAEGTAFRLERAQDPDGGLNGIQNYTISPNSFFHINISGGDEGMIYPELVLDKALDREEQGELSLTLTALD


GGSPSRSGTSTVRIVVLDVNDNAPQFAQALYETQAPENSPIGFLIVKVWAEDVDSGVNAEVSYSFFDASENI


RTTFQINPFSGEIFLRELLDYELVNSYKINIQAMDGGGLSARCRVLVEVLDTNDNPPELIVSSFSNSVAENS


PETPLAVFKINDRDSGENGRMVCYIQDDLPFLLKPSVENFYILMTEGALDREARAEYNITLTVTDMGTPRLK


TEHNITVQISDVNDNAPTFTQTSYALFVRENNSPALHIGSVSATDRDSGTNAQVTYSLLPPQDPHLPLASLV


SINADNGHLFALRSLDYEALQAFEFRVGATDRGSPALSREALVRVLVLDANDNSPFVLYPLQNGSAPCTELV


PRAAEPGYLVTKVVAVDGDSGQNAWLSYQLLKATEPGLFGVWAHNGEVRTARLLSERDAAKHRLVVLVKDNG


EPPRSATATLHLLLVDGFSQPYLPLPEAAPAQAQAEADLLTVYLVVALASVSSLFLLSVLLFVAVRLCRRSR


AASVGRCSVPEGPFPGHLVDVSGTGTLFQSYQYEVCLTGGSETGEFKFLKPITPHLPPHRGGKEIEENSTLP


NSFGFNY









The NOV16d amino acid sequence has 765 of 798 amino acid residues (95%) identical to, and 776 of 798 amino acid residues (97%) similar to, the 800 amino acid residue ptnr:SPTREMBL-ACC:Q9UN67 protein from Homo sapiens (Human) (Protocadherin Beta 10) (E=0.0).
The disclosed NOV16d is expressed in at least the following tissues: Uterus, colon, eye and retina. This information was derived by determining the tissue sources of the sequences that were included in the invention including but not limited to SeqCalling sources, Public EST sources, Literature sources, and/or RACE sources.
Possible SNPs found for NOV16c are listed in Table 16I.







TABLE 16I







SNPs














Amino




Nucleotide
Base
Acid
Base


Variant
Position
Change
Position
Change





13377099
218
G > T
Silent
N/A









NOV16a-NOV16d are very closely homologous as is shown in the amino acid alignment in Table 16J.







TABLE 16J





Amino Acid Alignment of NOV16a-NOV16d































































































































Homologies to any of the above NOV16 proteins will be shared by the other NOV16 proteins insofar as they are homologous to each other as shown above. Any reference to NOV16 is assumed to refer to the NOV16 proteins in general, unless otherwise noted.
NOV16a has homology to the amino acid sequences shown in the BLASTP data listed in Table 16K.







TABLE 16K







BLAST results for NOV16a












Gene Index/
Protein/
Length
Identity
Positives



Identifier
Organism
(aa)
(%)
(%)
Expect





gi|9622242|gb|AAF89690.1|
protocadherin
 947
911/947
911/947
0.0


AF169693_1
13 [Homo

(96%)
(96%)


(AF169693)
sapiens]


gi|15302224|ref|XP—
protocadherin
 924
889/924
889/924
0.0


054521.1|
20 [Homo

(96%)
(96%)


(XM_054521)
sapiens]


gi|14589939|ref|NP—
protocadherin
 924
888/924
888/924
0.0


073754.1|
20 precursor;

(96%)
(96%)


(NM_022843)
protocadherin



13 [Homo



sapiens]


gi|9966883|ref|NP_065136.1|
protocadherin
1203
359/815
495/815
e−178


(NM_020403)
9 precursor;

(44%)
(60%)



cadherin



superfamily



protein VR4-



11 [Homo



sapiens]


gi|14388339|dbj|BAB60731.1|
hypothetical
1032
358/815
495/815
e−178


(AB062939)
protein

(43%)
(59%)



[Macaca



fascicularis]









The homology of these sequences is shown graphically in the ClustalW analysis shown in Table 16L.







TABLE 16L





ClustalW Analysis of NOV16a















1) NOV16a (SEQ ID NO:56)


2) gi 9622242|gb|AAF89690.1|AF169693_1 (AF169693) protocadherin 13 [Homo sapiens]


(SEQ ID NO:189)


2) gi 15302224|refXP_054521.1|(XM_054521) protocadherin 20 [Homo sapiens]


(SEQ ID NO:190)


3) gi 14589939|refNP_073754.1|(NM_022843) protocadherin 20 precursor; protocadherin 13


[Homo sapiens] (SEQ ID NO:191)


4) gi|9966883|ref|NP_065136.1|(NM_020403) protocadherin 9 precursor; cadherin


superfamily protein VR4-11 [Homo sapiens] (SEQ ID NO:192)


5) gi|14388339|dbj|BAB60731.1|(AB062939) hypothetical protein [Macaca fascicularis]


(SEQ ID NO:193)














































































































































Tables 16M and 16N list the domain description from DOMAIN analysis results against NOV16a. This indicates that the NOV16a sequence has properties similar to those of other proteins known to contain these domains.







TABLE 16M





Domain Analysis of NOV16a
















gnl|Smart smart00112, CA, Cadherin repeats.; Cadherins are



glycoproteins involved in Ca2+-mediated cell-cell adhesion. Cadherin


domains occur as repeats in the extracellular regions which are


thought to mediate cell-cell contact when bound to calcium. (SEQ ID NO:194)


CD-Length = 82 residues, 100.0% aligned


Score = 89.4 bits (220), Expect = 9e-19











Query:
672
ISVTDADAGRNGWVALSVV--NQSDIFVIDTGKGMLRAKVSLDREQQSSYTLWVEAVDGG
729





+| ||||+| || |  |++  |    +| ||    |++      ||||+|| ||| ||| |||


Sbjct:
1
VSATDADSGENGKVTYSILSGNDGGLFSIDPETGIITTTKPLDREEQSEYTLTVEATDGG
 60


Query:
730
EPALSSTAKITILLLDINDNPP
751




 | ||||| +|+ +||+||| |


Sbjct:
61
GPPLSSTATVTVTVLDVNDNAP
 82
















TABLE 16N





Domain Analysis of NOV16a
















gnl|Pfam|pfam00028, cadherin, Cadherin domain. (SEQ ID NO:195)



CD-Length = 92 residues, 100.0%  aligned


Score = 75.5 bits (184), Expect = 1e-14











Query:
655
FSFFVPENFPGYGEIGVISVTDADAGRNGWVALSVVNQ--SDIFVIDTGKGMLRAKVSLD
712





+|  |||| |   |+  ++ |||| | || +  |++        | ||   | |     ||


Sbjct:
1
YSASVPENAPVGTEVLTVTATDADLGPNGRIFYSILGGGPGGWFRIDPDTGDLSTTKPLD
 60


Query:
713
REQQSSYTLWVEAVDGGEPALSSTAKITILLL
744




||    | | | | | | | || |  +|| +|


Sbjct:
61
RESIGEYELTVLATDSGGPPLSGTTTVTITVL
 92









Cadherins, a family of calcium-dependent cell-cell adhesion molecules, mediate neural cell-cell interactions. Sperry (1963) proposed that neurons recognize their synaptic partners through lock-and-key interactions mediated by millions of specifier molecules. Cadherins were likely candidates for components of the lock-and-key mechanism based on their neural location, their adhesive diversity, and the structural biology of their adhesive interactions maintaining a synapse gap size of approximately 200 angstroms (Shapiro and Colman, Neuron. 23(3):427-30, 1999). Neural cadherins (CDH2, 114020), as well as epithelial (CDH1, 192090), placental (CDH3, 114021), and retinal (CDH4, 603006) cadherins, have homophilic binding specificities in that they preferentially adhere to cells expressing the same cadherin type. Cadherins of the ‘classic’ type have a highly conserved extracellular sequence motif of approximately 110 amino acids that is repeated 5 times as well as a highly conserved cytoplasmic domain of approximately 200 amino acids that associates with intracellular actin microfilaments via catenins (see CTNNA1). ‘Nonclassic’ cadherins differ in that they may have 6 or 7 repeated extracellular domains or have cytoplasmic domains that connect to intermediate filaments instead of actin.
Protocadherins constitute a subfamily of the nonclassic cadherins. Kohmura et al. (Neuron. 20(6):1137-51, 1998) described cadherin-related neuronal receptors (CNR) in the mouse. By EST database searching for cadherin-like sequences, Wu and Maniatis (Cell 97(6):779-90, 1999) identified 52 novel genes organized into 3 closely linked tandem clusters on human chromosome 5q31. A distinct large exon of approximately 2,400 nucleotides encodes the 6 N-terminal extracellular domains and the transmembrane domain of each cadherin. In contrast, the C terminus of each of these proteins is identical within each cluster and is encoded by 3 small exons located downstream from the array of N-terminal exons. Wu and Maniatis (1999) designated the clusters alpha, beta and gamma. Each large exon is independently spliced to the first exon encoding the intracellular domain. The authors also denoted the extracellular portion as the variable region and the cytoplasmic portion as the constant region. The alpha cluster contains at least 15 proteins encoded by large, uninterrupted exons whose sequences most closely resemble those of the mouse CNR proteins. Wu and Maniatis (1999) proposed 4 models to explain protocadherin gene regulation and noted that several neurologic disorders map to chromosome 5q31.
The NOV16 nucleic acid of the invention encoding a Protocadherin-like protein includes the nucleic acid whose sequence is provided in Tables 16A, 16C, 16E and 16G, or a fragment thereof. The invention also includes a mutant or variant nucleic acid any of whose bases may be changed from the corresponding base shown in Tables 16A, 16C, 16E and 16G while still encoding a protein that maintains its Protocadherin-like activities and physiological functions, or a fragment of such a nucleic acid. The invention further includes nucleic acids whose sequences are complementary to those just described, including nucleic acid fragments that are complementary to any of the nucleic acids just described. The invention additionally includes nucleic acids or nucleic acid fragments, or complements thereto, whose structures include chemical modifications. Such modifications include, by way of non-limiting example, modified bases, and nucleic acids whose sugar phosphate backbones are modified or derivatized. These modifications are carried out at least in part to enhance the chemical stability of the modified nucleic acid, such that they may be used, for example, as antisense binding nucleic acids in therapeutic applications in a subject. In the mutant or variant nucleic acids, and their complements, up to about 21% of the NOV16b residues, about 9% of the NOV16c residues and about 4% of the NOV16d residues may be so changed.
The NOV16 protein of the invention includes the Protocadherin-like protein whose sequence is provided in Tables 16B, 16D, 16F and 16H. The invention also includes a mutant or variant protein any of whose residues may be changed from the corresponding residue shown in Tables 16B, 16D, 16F and 16H while still encoding a protein that maintains its Protocadherin-like activities and physiological functions, or a functional fragment thereof. In the mutant or variant protein, up to about 1% of the NOV16a bases, about 30% of the NOV16b bases, about 10% of the NOV16c bases and about 5% of the NOV16d bases may be so changed.
The NOV16 nucleic acids and proteins of the invention are useful in potential diagnostic and therapeutic applications implicated in various diseases and disorders described below and/or other pathologies. For example, the compositions of the present invention will have efficacy for treatment of patients suffering from: Hirschsprung's disease, Crohn's Disease, Appendicitis, Von Hippel-Lindau (VHL) syndrome, Diabetes, Tuberous sclerosis, Endometriosis, Fertility and other diseases, disorders and conditions of the like.
NOV16 nucleic acids and polypeptides are further useful in the generation of antibodies that bind immunospecifically to the novel substances of the invention for use in therapeutic or diagnostic methods. These antibodies may be generated according to methods known in the art, using prediction from hydrophobicity charts, as described in the “Anti-NOVX Antibodies” section below. For example the disclosed NOV16 protein have multiple hydrophilic regions, each of which can be used as an immunogen. This novel protein also has value in development of powerful assay system for functional analysis of various human disorders, which will help in understanding of pathology of the disease and development of new drug targets for various disorders.
NOV17
NOV17 includes two novel Cadherin 23-like proteins disclosed below. The disclosed proteins have been named NOV17a and NOV17b.
NOV17a
A disclosed NOV17a nucleic acid of 1303 nucleotides (also referred to as CG57429-01) encoding a novel Cadherin 23-like protein is shown in Table 17A. An open reading frame was identified beginning with an ATG initiation codon at nucleotides 31-33 and ending with a TAG codon at nucleotides 1291-1293. Putative untranslated regions, if any, upstream from the initiation codon and downstream from the termination codon are underlined in Table 17A, and the start and stop codons are in bold letters.







TABLE 17A





NOV17a nucleotide sequence.
















(SEQ ID NO:63)









ATATCCAATGGGCTGATTTATCTGACGGTCATGGCCATGGATGCTGGCAACCCCCCTCTCAACAGCACCGTC



CCTGTCACCATCGAGGTGTTTGATGAGAATGACAACCCTCCCACCTTCAGCAAGCCCGCCTACTTCGTCTCC


GTGGTGGAGAACATCATGGCAGGAGCCACGGTGCTGTTCCTGAATGCCACAGACCTGGACCGCTCCCGGGAG


TACGGCCAGGAGTCCATCATCTACTCCTTGGAAGGCTCCACCCAGTTTCGGATCAATGCCCGCTCAGGGGAA


ATCACCACCACGTCTCTGCTTGACCGAGAGACCAAGTCTGAATACATCCTCATCGTTCGCGCAGTGGACGGG


GGTGTGGGCCACAACCAGAAAACTGGCATCGCCACCGTAAACATCACCCTCCTGGACATCAACGACAACCAC


CCCACGTGGAAGGACGCACCCTACTACATCAACCTGGTGGAGATGACCCCTCCAGACTCTGACGTGACCACG


GTGGTGGCTGTTGACCCAGACCTGGGGGAGAATGGCACCCTGGTGTACAGCATCCAGCCACCCAACAAGTTC


TACAGCCTCAACAGCACCACGGGCAAGATCCGCACCACCCACGCCATGCTGGACCGGGAGAACCCCGACCCC


CATGAGGCCGAGCTGATGCGCAAAATCGTCGTCTCTGTTACTGACTGTGGCAGGCCCCCTCTGAAAGCCACC


AGCAGTGCCACAGTGTTTGTGAACCTCTTGGATCTCAATGACAATGACCCCACCTTTCAGAACCTGCCTTTT


GTGGCCGAGGTGCTTGAAGGCATCCCGGCGGGGGTCTCCATCTACCAAGTGGTGGCCATCGACCTCGATGAG


GGCCTGAACGGCCTGGTGTCCTACCGCATGCCGGTGGGCATGCCCCGCATGGACTTCCTCATCAACAGCAGC


AGCGGCGTGGTGGTCACCACCACCGAGCTGGACCGCGAGCGCATCGCGGAGTACCAGCTGCGGGTGGTGGCC


AGTGATGCAGGCACGCCCACCAAGAGCTCCACCAGCACGCTCACCATCCATGTGCTGGATGTGAACGACGAG


ACGCCCACCTTCTTCCCGGCCGTGTACAATGTGTCTGTGTCCGAGGACGTGCCACGCGAGTTCCGGGTGGTC


TGGCTGAACTGCACGGACAACGACGTGGGCCTCAATGCAGAGCTCAGCTACTTCATCACAGGTGCTGCCCCG


GCCTCCGCCCACCTGTGCAGGCCTCCTGGGGCCCTGCCTCCACCCCTCCCAGATGGACAGCCAGACTAGGTG


GGGGCAG









The disclosed NOV17a nucleic acid sequence, localized to chromsome 10, has 702 of 703 bases (99%) identical to a gb:GENBANK-ID:AY010111|acc:AY010111.1 mRNA from Homo sapiens (Homo sapiens cadherin-23 (CDH23) mRNA, partial cds) (E=6.9e−152).
A NOV17a polypeptide (SEQ ID NO:64) encoded by SEQ ID NO:63 has 420 amino acid residues and is presented using the one-letter code in Table 17B. Signal P, Psort and/or Hydropathy results predict that NOV17a does not contain a signal peptide and is likely to be localized to the plasma membrane with a certainty of 0.7900.







TABLE 17B





Encoded NOV17a protein sequence.
















(SEQ ID NO:64)









MAMDAGNPPLNSTVPVTIEVFDENDNPPTFSKPAYFVSVVENIMAGATVLFLNATDLDRSREYGQESIIYSL



EGSTQFRINARSGEITTTSLLDRETKSEYILIVRAVDGGVGHNQKTGIATVNITLLDINDNHPTWKDAPYYI


NLVEMTPPDSDVTTVVAVDPDLGENGTLVYSIQPPNKFYSLNSTTGKIRTTHAMLDRENPDPHEAELMRKIV


VSVTDCGRPPLKATSSATVFVNLLDLNDNDPTFQNLPFVAEVLEGIPAGVSIYQVVAIDLDEGLNGLVSYRM


PVGMPRMDFLINSSSGVVVTTTELDRERIAEYQLRVVASDAGTPTKSSTSTLTIHVLDVNDETPTFFPAVYN


VSVSEDVPREFRVVWLNCTDNDVGLNAELSYFITGAAPASAHLCRPPGALPPPLPDGQPD









The NOV17a amino acid sequence has 233 of 234 amino acid residues (99%) identical to, and 234 of 234 amino acid residues (100%) similar to, the 2552 amino acid residue ptnr:TREMBLNEW-ACC:AAG27034 protein from Homo sapiens (Human) (CADHERIN-23) (E=1.8e−120).
The disclosed NOV17a is expressed in at least the following tissues: lung, pancreas, spinal chord and testis. This information was derived by determining the tissue sources of the sequences that were included in the invention including but not limited to SeqCalling sources, Public EST sources, Literature sources, and/or RACE sources.
NOV17b
A disclosed NOV17b nucleic acid of 1278 nucleotides (also referred to as CG57429-02) encoding a novel Cadherin 23-like protein is shown in Table 17C. An open reading frame was identified beginning with an ATG initiation codon at nucleotides 6-8 and ending with a TAG codon at nucleotides 1266-1268. Putative untranslated regions, if any, upstream from the initiation codon and downstream from the termination codon are underlined in Table 17C, and the start and stop codons are in bold letters.







TABLE 17C





NOV17b nucleotide sequence.
















(SEQ ID NO:65)









TGGTCATGGCCATGGATGCTGGCAACCCCCCTCTCAACAGCACCGTCCCTGTCACCATCGAGGTGTTTGATG



AGAATGACAACCCTCCCACCTTCAGCAAGCCCGCCTACTTCGTCTCCGTGGTGGAGAACATCATGGCAGGAG


CCACGGTGCTGTTCCTGAATGCCACAGACCTGGACCGCTCCCGGGAGTACGGCCAGGAGTCCATCATCTACT


CCTTGGAAGGCTCCACCCAGTTTCGGATCAATGCCCGCTCAGGGGAAATCACCACCACGTCTCTGCTTGACC


GAGAGACCAAGTCTGAATACATCCTCATCGTTCGCGCAGTGGACGGGGGTGTGGGCCACAACCAGAAAACTG


GCATCGCCACCGTAAACATCACCCTCCTGGACATCAATGACAACCACCCCACGTGGAAGGACGCACCCTACT


ACATCAACCTGGTGGAGATGACCCCTCCAGACTCTGATGTGACCACGGTGGTGGCTGTTGACCCAGACCTGG


GGGAGAATGGCACCCTGGTGTACAGCATCCAGCCACCCAACAAGTTCTACAGCCTCAACAGCACCACGGGCA


AGATCCGCACCACCCACGCCATGCTGGACCGGGAGAACCCCGACCCCCATGAGGCCGAGCTGATGCGCAAAA


TCGTCGTCTCTGTTACTGACTGTGGCAGGCCCCCTCTGAAAGCCACCAGCAGTGCCACAGTGTTTGTGAACC


TCTTGGATCTCAATGACAATGACCCCACCTTTCAGAACCTGCCTTTTGTGGCCGAGGTGCTTGAAGGCATCC


CGACGGGGGTCTCCATCTACCAAGTGGTGGCCATCGACCTCGATGAGGGCCTGAACGGCCTGGTGTCCTACC


GCATGCCGGTGGGCATGCCCCGCATGGACTTCCTCATCAACAGCAGCAGCGGCGTGGTGGTCACCACCACCG


AGCTGGACCGCGAGCGCATCGCGGAGTACCAGCTGCGGGTGGTGGCCAGTGATGCAGGCACGCCCACCAAGA


GCTCCACCAGCACGCTCACCATCCATGTGCTGGATGTGAACGACGAGACGCCCACCTTCTTCCCGGCCGTGT


ACAATGTGTCTGTGTCCGAGGACGTGCCACGCGAGTTCCGGGTGGTCTGGCTGAACTGCACGGACAACGACG


TGGGCCTCAATGCAGAGCTCAGCTACTTCATCACAGGTGCTGCCCCGGCCTCCGCCCACCTGTGCAGGCCTC


CTGGGGCCCTGCCTCCACCCCTCCCAGATGGACAGCCAGACTAGGTGGGGGCAG









The disclosed NOV17b nucleic acid sequence, localized to chromsome 10, has 1188 of 1190 bases (99%) identical to a gb:GENBANK-ID:AF312024|acc:AF312024.1 mRNA from Homo sapiens (cadherin related 23 (CDH23) mRNA, complete cds) (E=6.6e−263).
A NOV17b polypeptide (SEQ ID NO:66) encoded by SEQ ID NO:65 has 420 amino acid residues and is presented using the one-letter code in Table 17D. Signal P, Psort and/or Hydropathy results predict that NOV17b does not contain a signal peptide and is likely to be localized to the plasma membrane with a certainty of 0.7900.







TABLE 17D





Encoded NOV17b protein sequence.
















(SEQ ID NO:66)









MAMDAGNPPLNSTVPVTIEVFDENDNPPTFSKPAYFVSVVENIMAGATVLFLNATDLDRSREYGQESIIYSL



EGSTQFRINARSGEITTTSLLDRETKSEYILIVRAVDGGVGHNQKTGIATVNITLLDINDNHPTWKDAPYYI


NLVEMTPPDSDVTTVVAVDPDLGENGTLVYSIQPPNKFYSLNSTTGKIRTTHAMLDRENPDPHEAELMRKIV


VSVTDCGRPPLKATSSATVFVNLLDLNDNDPTFQNLPFVAEVLEGIPTGVSIYQVVAIDLDEGLNGLVSYRM


PVGMPRMDFLINSSSGVVVTTTELDRERIAEYQLRVVASDAGTPTKSSTSTLTIHVLDVNDETPTFFPAVYN


VSVSEDVPREFRVVWLNCTDNDVGLNAELSYFITGAAPASAHLCRPPGALPPPLPDGQPD









The NOV17b amino acid sequence has 394 of 395 amino acid residues (99%) identical to, and 394 of 395 amino acid residues (99%) similar to, the 3354 amino acid residue ptnr:SPTREMBL-ACC:Q9H251 protein from Homo sapiens (Human) (Cadherin Related 23) (E=3.9e−208).
The disclosed NOV17b is expressed in at least the following tissues: lung, pancreas, spinal chord and testis. This information was derived by determining the tissue sources of the sequences that were included in the invention including but not limited to SeqCalling sources, Public EST sources, Literature sources, and/or RACE sources.
Possible SNPs found for NOV17a are listed in Table 17E.







TABLE 17E







SNPs














Amino




Nucleotide
Base
Acid
Base


Variant
Position
Change
Position
Change





13377100
495
C > T
Silent
N/A


13377100
820
G > Z
264
Ala > Thr









NOV17a and NOV17b are very closely homologous as is shown in the nucleic acid alignment in Table 17F.







TABLE 17F





Nucleic Acid Alignment of NOV17a and NOV17b



























































































































































Homologies to any of the above NOV17 nucleic acids and encoded proteins will be shared by the other NOV17 nucleic acids and encoded proteins insofar as they are homologous to each other as shown above. Any reference to NOV17 is assumed to refer to both of the NOV17 nucleic acids and encoded proteins in general, unless otherwise noted.
NOV17a has homology to the amino acid sequences shown in the BLASTP data listed in Table 17G.







TABLE 17G







BLAST results for NOV17a












Gene Index/
Protein/
Length
Identity
Positives



Identifier
Organism
(aa)
(%)
(%)
Expect





gi|18576726|ref|XP_
similar to
3399
395/395
395/395
0.0


057519.3|
cadherin

(100%)
(100%)


(XM_057519)
related 23



(H. sapiens)



[Homo



sapiens]


gi|16507962|ref|NP_
cadherin
3354
395/395
395/395
0.0


071407.2|
related 23,

(100%)
(100%)


(NM_022124)
isoform 1



precursor;



cadherin-23;



otocadherin



[Homo



sapiens]


gi|17366834|sp|Q9H251|
Cadherin 23
3354
395/395
395/395
0.0


CADN_HUMAN
precursor

(100%)
(100%)



(Otocadherin)



[Homo



sapiens]


gi|14017841|dbj|BAB47441.1|
Cadherin 23
 803
395/395
395/395
0.0


(AB058715)
[Homo

(100%)
(100%)



sapiens]


gi|17865345|ref|NP_
cadherin
3317
377/394
385/394
0.0


446096.1|
related 23

(95%)
(97%)


(NM_053644)
[Rattus



norvegicus]









The homology of these sequences is shown graphically in the ClustalW analysis shown in Table 17H.







TABLE 17H





ClustalW Analysis of NOV17a















1) NOV17a (SEQ ID NO:64)


2) gi 18576726|refXP_057519.3|(XM_057519) similar to cadherin related 23 (H. sapiens)


[Homo sapiens] (SEQ ID NO:196)


2) gi 16507962|refNP_071407.2|(NM_022124) cadherin related 23, isoform 1 precurso;


cadherin-23; otocadherin [Homo sapiens] (SEQ ID NO:197)


3) gi 17366834|sp|Q9H251.CADN_HUMAN Cadherin 23 precursor (Otocadherin)[Homo sapiens]


(SEQ ID NO:198)


4) gi 14017841|dbj|BAB47441.1|(AB058715) KIAA1812 protein [Homo sapiens] (SEQ ID NO:199)


5) gi 17865345|refNP_446096.1|(NM_053644) cadherin related 23 [Rattus norvegicus]


(SEQ ID NO:200)
































































































































































































































































































































































Tables 17I and 17J list the domain description from DOMAIN analysis results against NOV17a. This indicates that the NOV17a sequence has properties similar to those of other proteins known to contain these domains.







TABLE 17I





Domain Analysis of NOV17a
















gnl|Smart|smart00112, CA, Cadherin repeats.; Cadherins are



glycoproteins involved in Ca2+-mediated cell-cell adhesion. Cadherin


domains occur as repeats in the extracellular regions which are


thought to mediate cell-cell contact when bound to calcium. (SEQ ID NO:201)


CD-Length =  82 residues, 100.0%  aligned


Score =  85.1 bits (209), Expect =  7e-18











Query:
271
VVAIDLDEGLNGLVSYRMPVGMPRMDFLINSSSGVVVTTTELDRERIAEYQLRVVASDAG
330





| | | | | || |+| +  |     | |+  +|++ ||  ||||  +|| | | |+| |


Sbjct:
1
VSATDADSGENGKVTYSILSGNDGGLFSIDPETGIITTTKPLDREEQSEYTLTVEATDGG
 60


Query:
331
TPTKSSTSTLTIHVLDVNDETP
352




 |  |||+|+|+ ||||||  |


Sbjct:
61
GPPLSSTATVTVTVLDVNDNAP
 82
















TABLE 17J





Domain Analysis of NOV17a
















gnl|Pfam|pfam00028, cadherin, Cadherin domain. (SEQ ID NO:202)



CD-Length = 92 residues, 100.0%  aligned


Score = 80.9 bits (198), Expect = 1e-16











Query:
254
FVAEVLEGIPAGVSIYQVVAIDLDEGLNGLVSYRMPVGMPRMDFLINSSSGVVVTTTELD
313





+ | | |  | |  +  | | | | | || + | +  | |   | |+  +| + ||  ||


Sbjct:
1
YSASVPENAPVGTEVLTVTATDADLGPNGRIFYSILGGGPGGWFRIDPDTGDLSTTKPLD
 60


Query:
314
RERIAEYQLRVVASDAGTPTKSSTSTLTIHVL
345




|| | ||+| |+|+|+| |  | |+|+|| ||


Sbjct:
61
RESIGEYELTVLATDSGGPPLSGTTTVTITVL
 92









Cadherins are a family of animal glycoproteins responsible for calcium-dependent cell-cell adhesion. Cadherins preferentially interact with themselves in a homophilic manner in connecting cells; thus acting as both receptor and ligand. A wide number of tissue-specific forms of cadherins are known, including epithelial (E-cadherin, also known as uvomorulin or L-CAM; CDH1), neural (N-cadherin; CDH2), placental (P-cadherin; CDH3), retinal (R-cadherin; CDH4), vascular endothelial (VE-cadherin; CDH5), kidney (K-cadherin; CDH6), cadherin-8 (CDH8), osteoblast (OB-cadherin; CDH11), brain (BR-cadherin; CDH12), T-cadherin (truncated cadherin; CDH13), muscle (M-cadherin; CDH14), liver-intestine (LI-cadherin), and EP-cadherin.
Structurally, cadherins are built of the following domains: a signal sequence, followed by a propeptide of about 130 residues, then an extracellular domain of around 600 residues, then a transmembrane region, and finally a C-terminal cytoplasmic domain of about 150 residues. The extracellular domain can be sub-divided into five parts: there are four repeats of about 110 residues followed by a region that contains four conserved cysteines. It is suggested that the calcium-binding region of cadherins is located in the extracellular repeats. Cadherins are evolutionary related to the desmogleins which are component of intercellular desmosome junctions involved in the interaction of plaque proteins.
A form of nonsyndromic autosomal recessive deafness is caused by mutation in the cadherin-23 gene (CDH23), which is also the site of mutation in a form of Usher syndrome, USH1D. Chaib et al. (Hum Mol Genet. 5(7):1061-4, 1996) reviewed the frequency and inheritance of congenital isolated deafness and causes for difficulties associated with mapping of deafness genes. They noted that in the U.S., deafness affects 1 in 1000 children at birth or during infancy. About 75% of the inherited forms of congenital isolated deafness have an autosomal recessive mode of transmission. Chaib et al. (1996) noted that difficulties in localization of deafness genes are due to several factors, including extreme genetic heterogeneity of the defect (there are an estimated 40 deafness genes segregating in the population); the absence of clinical criteria to allow differentiation between inner ear defects; and the high proportion of marriages between deaf persons in Western countries leading to coexistence of several defective genes responsible for clinically indistinguishable phenotypes in one family. In an effort to simplify mapping of deafness genes, Chaib et al. (1996) carried out studies in consanguineous families living in isolated regions. Marker data from these families was initially analyzed using a lod-score approach. Subsequently they performed homozygosity mapping. They localized a ‘new’ gene for nonsyndromal recessive deafness (symbolized DFNB12) to chromosome 10q21-q22 in a consanguineous Sunni family who lived in an isolated village in Syria. Affected members in this family have profound prelingual sensorineural hearing impairment. A significant lod score (6.40 at theta=0.00) was obtained with the marker D10S535. Analysis of adjacent markers placed the gene distal to D10S529 and proximal to D10S532 in a 11- to 15-cM region. All affected individuals were homozygous for polymorphic markers located in this region. Chaib et al. (1996) stated that the gene encoding the mitochondrial transcription factor 6-like 2 is a possible candidate gene for DFNB12. They also noted that the homologous murine region for DFNB12 contains 3 deaf mouse mutants, including Jackson circler (jc), Waltzer (v), and Ames Waltzer (av). Bork et al. (Am J Hum Genet. 68(1):26-37, 2001) demonstrated that DFNB12 and a form of Usher syndrome characterized by deafness associated with retinitis pigmentosa and vestibular dysfunction (USH1D) are allelic disorders due to different mutations in the cadherin-23 gene.
Usher syndrome type I is an autosomal recessive disorder characterized by profound congenital hearing impairment with unintelligible speech, early retinitis pigmentosa, and constant vestibular dysfunction. Three different loci had been found by linkage analysis: USH1A on 14q34, USH1B on 11q13, and USH1C on 11p15. Gerber et al. (J Med Genet. 33(1):77-9, 1996) suggested the existence of yet a fourth form of Usher syndrome type I from the fact that the 3 previously reported loci were excluded by linkage studies in 2 families of Moroccan and Pakistani ancestry. Wayne et al. (Hum Mol Genet. 5(10): 1689-92, 1996) reported that a first-cousin union in a family of Pakistani origin produced 4 children with clinical signs of Usher syndrome, including profound prelingual auditory impairment of sensorineural type, congenital vestibular dysfunction, and progressive pigmentary retinopathy. Wayne et al. (1996) prepared 2 genomic DNA pools, one from the affected children and the other from the parents, and screened 161 polymorphic markers evenly spaced across the autosomal genome. The only region showing homozygosity by descent in the affected sibs was a 15-cM interval on chromosome 10 bounded by D10S529 and D10S573. Wayne et al. (1996) concluded that this was the location of the gene responsible for Usher syndrome in this family. They symbolized the locus USH1D. Bolz et al. (Nat Genet. 27(1): 108-12, 2001) identified mutations in the CDH23 gene in a Cuban family and a German patient with USH1D. Di Palma et al. (Nat Genet. 27(1):103-7, 2001; Gene. 281(1-2):31-41, 2001) demonstrated that mutations in the mouse Cdh23 gene are responsible for the ‘waltzer’ mutation, thus establishing it as a model for USH1D. Bork et al. (2001) showed that USH1D and DFNB12 are allelic disorders due to different mutations in the cadherin-23 gene.
Truncated or alternatively-spliced cadherins have been identified, and some may play roles in disease. Berx et al. (Hum Mutat. 12(4):226-37, 1998) found reports of 69 somatic mutations of the CDH1 gene. These comprised, in addition to a few missense mutations, mainly splice site mutations and truncation mutations caused by insertions, deletions, and nonsense mutations. There was a major difference in mutation type between diffuse gastric and infiltrative lobular breast cancers. In diffuse gastric tumors, the predominant defects were exon skippings, which caused in-frame deletions. By contrast, most mutations found in infiltrating lobular breast cancers were out-of-frame mutations, which were predicted to yield secreted truncated E-cadherin fragments. Two different forms of human OB-cadherin cDNA were cloned; one was a counterpart of the mouse gene and the other encoded a protein with a truncated cytoplasmic domain. Sequence analysis demonstrated that Ksp-cadherin, like LI-cadherin (CDH17), lacks the prosequence and tripeptide HAV adhesion recognition sequence typical of most classical cadherins, and possesses a truncated cytoplasmic domain.
The NOV17 nucleic acid of the invention encoding a Cadherin 23-like protein includes the nucleic acid whose sequence is provided in Tables 17A and 17C, or a fragment thereof. The invention also includes a mutant or variant nucleic acid any of whose bases may be changed from the corresponding base shown in Tables 17A and 17C while still encoding a protein that maintains its Cadherin 23-like activities and physiological functions, or a fragment of such a nucleic acid. The invention further includes nucleic acids whose sequences are complementary to those just described, including nucleic acid fragments that are complementary to any of the nucleic acids just described. The invention additionally includes nucleic acids or nucleic acid fragments, or complements thereto, whose structures include chemical modifications. Such modifications include, by way of non-limiting example, modified bases, and nucleic acids whose sugar phosphate backbones are modified or derivatized. These modifications are carried out at least in part to enhance the chemical stability of the modified nucleic acid, such that they may be used, for example, as antisense binding nucleic acids in therapeutic applications in a subject. In the mutant or variant nucleic acids, and their complements, up to about 1% of the NOV17 residues may be so changed.
The NOV17 protein of the invention includes the Cadherin 23-like protein whose sequence is provided in Tables 17B and 17D. The invention also includes a mutant or variant protein any of whose residues may be changed from the corresponding residue shown in Tables 17B and 17D while still encoding a protein that maintains its Cadherin 23-like activities and physiological functions, or a functional fragment thereof. In the mutant or variant protein, up to about 1% of the NOV17 bases may be so changed.
The NOV17 nucleic acids and proteins of the invention are useful in potential diagnostic and therapeutic applications implicated in various diseases and disorders described below and/or other pathologies. For example, the compositions of the present invention will have efficacy for treatment of patients suffering from: nonsyndromic autosomal recessive deafness, Usher syndrome type I, systemic lupus erythematosus, autoimmune disease, asthma, emphysema, scleroderma, allergy, ARDS, diabetes, Von Hippel-Lindau (VHL) syndrome, pancreatitis, obesity, multiple sclerosis, leukodystrophies, pain, neuroprotection, fertility and other diseases, disorders and conditions of the like.
NOV17 nucleic acids and polypeptides are further useful in the generation of antibodies that bind immunospecifically to the novel substances of the invention for use in therapeutic or diagnostic methods. These antibodies may be generated according to methods known in the art, using prediction from hydrophobicity charts, as described in the “Anti-NOVX Antibodies” section below. For example the disclosed NOV17 protein have multiple hydrophilic regions, each of which can be used as an immunogen. This novel protein also has value in development of powerful assay system for functional analysis of various human disorders, which will help in understanding of pathology of the disease and development of new drug targets for various disorders.
NOV18
A disclosed NOV18 nucleic acid of 1614 nucleotides (also referred to as CG55887-02) encoding a novel Transforming Growth Factor Beta 2 (TGF Beta 2)-like protein is shown in Table 18A. An open reading frame was identified beginning with an ATG initiation codon at nucleotides 182-184 and ending with a TAA codon at nucleotides 1343-1345. Putative untranslated regions upstream from the intitation codon and downstream from the termination codon are underlined in Table 18A, and the start and stop codons are in bold letters.







TABLE 18A





NOV18 Nucleotide Sequence
















(SEQ ID NO:67)









CAAGCAGGATACGTTTTTCTGTTGGGCATTGACTAGATTGTTTGCAAAAGTTTCGCATCAAAAACAAACAACA



ACAACAAAAAACCAAACAACTCTCCTTGATCTATACTTTGAGAATTGTTGATTTCTTTTTTTTTATTCTGACT


TTTAAAAACAACTTTTTTTTCCACTTTTTAAAAAATGCACTACTGTGTGCTGAGCGCTTTTCTGATCCTGCA


TCTGGTCACGGTCGCGCTCAGCCTGTCTACCTGCAGCACACTCGATATGGACCAGTTCATGCGCAAGAGGATC


GAGGCGATCCGCGGGCAGATCCTGAGCAAGCTGAAGCTCACCAGTCCCCCAGAAGACTATCCTGAGCCCGAGG


AAGTCCCCCCGGAGGTGATTTCCATCTACAACAGCACCAGGGACTTGCTCCAGGAGAAGGCGAGCCGGAGGGC


GGCCGCCTGCGAGCGCGAGAGGAGCGACGAAGAGTACTACTTCAGAATTGTTCGATTTGACGTCTCAGCAATG


GAGAAGAATGCTTCCAATTTGGTGAAAGCAGAGTTCAGAGTCTTTCGTTTGCAGAACCCAAAAGCCAGAGTGC


CTGAACAACGGATTGAGCTATATCAGATTCTCAAGTCCAAAGATTTAACATCTCCAACCCAGCGCTACATCGA


CAGCAAAGTTGTGAAAACAAGAGCAGAAGGCGAATGGCTCTCCTTCGATGTAACTGATGCTGTTCATGAATGG


CTTCACCATAAAGACAGGAACCTGGGATTTAAAATAAGCTTACACTGTCCCTGCTGCACTTTTGTACCATCTA


ATAATTACATCATCCCAAATAAAAGTGAAGAACTAGAAGCAAGATTTGCAGGTATTGATGGCACCTCCACATA


TACCAGTGGTGATCAGAAAACTATAAAGTCCACTAGGAAAAAAAACAGTGGGAAGACCCCACATCTCCTGCTA


ATGTTATTGCCCTCCTACAGACTTGAGTCACAACAGACCAACCGGCGGAAGAAGCGTGCTTTGGATGCGGCCT


ATTGCTTTAGAAATGTGCAGGATAATTGCTGCCTACGTCCACTTTACATTGATTTCAAGAGGGATCTAGGGTG


GAAATGGATACACGAACCCAAAGGGTACAATGCCAACTTCTGTGCTGGAGCATGCCCGTATTTATGGAGTTCA


GACACTCAGCACAGCAGGGTCCTGAGCTTATATAATACCATAAATCCAGAAGCATCTGCTTCTCCTTGCTGCG


TGTCCCAAGATTTAGAACCTCTAACCATTCTCTACTACATTGGCAAAACACCCAAGATTGAACAGCTTTCTAA


TATGATTGTAAAGTCTTGCAAATGCAGCTAAAATTCTTGGAAAAGTGGCAAGACCAAAATGACAATGATGATG


ATAATGATGATGACGACGACAACGATGATGCTTGTAACAAGAAAACATAAGAGAGCCTTGGTTCATCAGTGTT


AAAAAATTTTTGAAAAGGCGGTACTAGTTCAGACACTTTGGAAGTTTGTGTTCTGTTTGTTAAAACTGGCATC


TGACACAAAAAAAGTTGAAGGCCTTATTCTACATTTCACCTACTTTGTAAGTGAGAGAGACAAGAAGCAAATT


TTTTTAAA









The NOV18 nucleic acid was identified on chromosome 1 and has 1140 of 1140 bases (100%) identical to a gb:GENBANK-ID:HSGTSF|acc:Y00083.1 mRNA from Homo sapiens (Human mRNA for glioblastoma-derived T-cell suppressor factor G-TsF (transforming growth factor-beta2, TGF-beta2)) (E=0.0).
A disclosed NOV18 polypeptide (SEQ ID NO:68) encoded by SEQ ID NO:67 is 387 amino acid residues and is presented using the one-letter code in Table 18B. Signal P, Psort and/or Hydropathy results predict that NOV18 contains a signal peptide and is likely to be localized to the extracellularly with a certainty of 0.8200. The most likely cleavage site for a NOV18 polypeptide is between amino acids 20 and 21: ALS-LS.







TABLE 18B





Encoded NOV18 protein sequence
















(SEQ ID NO:68)









MHYCVLSAFLILHLVTVALSLSTCSTLDMDQFMRKRIEAIRGQILSKLKLTSPPEDYPEPEEVPPEVISIYN



STRDLLQEKASRRAAACERERSDEEYYFRIVRFDVSAMEKNASNLVKAEFRVFRLQNPKARVPEQRIELYQI


LKSKDLTSPTQRYIDSKVVKTRAEGEWLSFDVTDAVHEWLHHKDRNLGFKISLHCPCCTFVPSNNYIIPNKS


EELEARFAGIDGTSTYTSGDQKTIKSTRKKNSGKTPHLLLMLLPSYRLESQQTNRRKKRALDAAYCFRNVQD


NCCLRPLYIDFKRDLGWKWIHEPKGYNANFCAGACPYLWSSDTQHSRVLSLYNTINPEASASPCCVSQDLEP


LTILYYIGKTPKIEQLSNMIVKSCKCS









The NOV18 amino acid sequence 289 of 289 amino acid residues (100%) identical to, and 289 of 289 amino acid residues (100%) similar to, the 414 amino acid residue ptnr:SWISSPROT-ACC:P08112 protein from Homo sapiens (Human), and (Transforming Growth Factor Beta 2 Precursor (TGF-BETA 2) (Glioblastoma-Derived T-Cell Suppressor Factor) (G-TSF) (BSC-1 Cell Growth Inhibitor) (Polyergin) (Cetermin)) (E=1.2e−210).
NOV18 is expressed in at least the following tissues: Lung, Mammary gland/Breast, Placenta, Thymus, Uterus, Whole OrganismAorta, Brain, Cervix, Foreskin, Heart, Kidney, Prostate, Retina, Right Cerebellum and Spinal Chord. This information was derived by determining the tissue sources of the sequences that were included in the invention including but not limited to SeqCalling sources, Public EST sources, genomic clone sources, literature sources, and/or RACE sources.
Possible SNPs found for NOV18 are listed in Table 18C.







TABLE 18C







SNPs














Amino




Nucleotide
Base
Acid
Base


Variant
Position
Change
Position
Change





13377104
785
T > C
202
Cys > Arg









NOV18 has homology to the amino acid sequences shown in the BLASTP data listed in Table 18D.







TABLE 18D







BLAST results for NOV18












Gene Index/

Length
Identity
Positives



Identifier
Protein/Organism
(aa)
(%)
(%)
Expect





gi|557563|gb|AAA50405.1|
transforming
413
357/413
357/413
0.0


(M19154)
growth factor

(86%)
(86%)



beta 2 [Homo



sapiens]


gi|4507463|ref|NP_003229.1|
transforming
414
357/414
357/414
0.0


(NM_003238)
growth factor,

(86%)
(86%)



beta 2 [Homo



sapiens]


gi|1729919|sp|P09858|
Transforming
435
353/414
355/414
0.0


TGF2_PIG
growth factor

(85%)
(85%)



beta 2 precursor



(TGF-beta 2) [Sus



scrofa]


gi|164689|gb|AAB03850.1|
transforming
434
352/413
354/413
0.0


(L08375)
growth factor

(85%)
(85%)



beta 2 [Sus



scrofa]


gi|15029892|gb|AAH11170.1|
Similar to
414
344/414
349/414
0.0


AAH11170
transforming

(83%)
(84%)


(BC011170)
growth factor,



beta 2 [Mus



musculus]









The homology of these sequences is shown graphically in the ClustalW analysis shown in Table 18E.







TABLE 18E





Clustal W Sequence Alignment















1) NOV 18 (SEQ ID NO:68)


2) gi 557563|gbAAA50405.1|(M19154) transforming growwth factor beta 2 [Homo sapiens]


(SEQ ID NO:203)


3) gi 4507463|refNP_003229.1|(NM_003238) transforming growth factor, beta 2


[Homo sapiens] (SEQ ID NO:204)


4) gi 1729919|sp|P09858|TGF2_PIG Transforming growth factor beta 2 precursor


(TGF-beta 2) [Sus scrofa] (SEQ ID NO:205)


5) gi|15029892|gb|AAH11170.1|AAH11170 (BC011170) Similar to transforming growth


factor, beta 2 [Mus musculus] (SEQ ID NO:206)


6) gi|18250662|emb|CAC70714.2| (AJ310932) myosin heavy chain [Homo sapiens]


(SEQ ID NO:207)


























































Tables 18F, 18G and 18H list the domain description from DOMAIN analysis results against NOV18. This indicates that the NOV18 sequence has properties similar to those of other proteins known to contain these domains.








TABLE 18F





Domain Analysis of NOV18
















gnl Pfam pfam00688, TGFb_propeptide, TGF-beta propeptide. This



propeptide is known as latency associated peptide (LAP) in TGF-beta.


LAP is a homodimer which is disulfide linked to TGF-beta binding


protein. (SEQ ID NO:208)


CD-Length = 227 residues, 100.0% aligned


Score = 152 bits (383), Expect = 4e−38











Query:
24
CSTLDMDQFMR-KRIEAIRGQILSKLKLTSPPEDYPEPEEVPPEVISIYNSTRDLLQEKA
82





|  ||+ +  +  |+||| ||||||| |   |    ||  ||  ++ +||+  +| + | 


Sbjct:
1
CRPLDLRRSQKQDRLEAIEGQILSKLGLRRRPRPSKEPMVVPEYMLDLYNALSELEEGKV
 60


Query:
83
SRRAAACERERSDEE----------------------YYFRIVRFDVSAMEKNASNLVKA
120




 |     + +  +                         + +  ||++|++ +  +    |


Sbjct:
61
GRVPEISDYDGREAGRANTIRSFSHLESDDFEESTPESHRKRFRFNLSSIPEGETLTA-A
119


Query:
121
EFRVFRLQNPKARVPEQRIELYQILKSKDLTSPTQRYIDSKVVKTRAEGEWLSFDVTDAV
180




| |++|           |+|+||+||     ||  | +||++|  |  | ||||||| ||


Sbjct:
120
ELRLYRDPLALRSRATVRVEIYQLLKPGSDGSPDTRLLDSRLVDARDSGGWLSFDVTSAV
179


Query:
181
HEWLHHKDRNLGFKISLHCPCCTFVPSNNYIIPNKSEELEARFAGIDG
228




+ || + + ||| ++ + | |    ||   +|     |          


Sbjct:
180
NRWLSNPESNLGLQLEVECLCGHVRPSRAGLIGEPGPEQLQPLLVTFF
227

















TABLE 18G





Domain Analysis of NOV18
















gnl|Pfam|pfam00019, TGF-beta, Transforming growth factor beta like



domain. (SEQ ID NO:209)


CD-Length = 105 residues, 100.0% aligned


Score = 124 bits (310), Expect = 1e−29











Query:
287
QDNCCLRPLYIDFKRDLGWK-WIHEPKGYNANFCAGACPYLWSSDTQ---HSRVLSLYNT
342





   | || ||+|| |||||  ||  |+|| ||+|+|+||+    |     |+ + +|   


Sbjct:
1
ARPCRLRSLYVDG-RDLGWGDWIIAPEGYIANYCSGSCPFPLRDDLNLSNHAILQTLVRL
 59


Query:
343
INPEASASPCCVSQDLEPLTILYYI-GKTPKIEQLSNMIVKSCKCS
387




 || |   ||||   | ||++||        +    || || | | 


Sbjct:
60
RNPRAVPQPCCVPTKLSPLSMLYLDDNSNVVLRLYPNMSVKECGCR
105

















TABLE 18H





Domain Analysis of NOV18
















gnl|Smart|smart00204, TGFB, Transforming growth factor-beta (TGF-beta)



family; Family members are active as disulphide-linked homo- or


heterodimers. TGFB is a multifunctional peptide that controls


proliferation, differentiation, and other functions in many cell


types. (SEQ ID NO:210)


CD-Length = 102 residues, 100.0% aligned


Score = 109 bits (273), Expect = 3e−25











Query:
290
CCLRPLYIDFKRDLGWK-WIHEPKGYNANFCAGACPYLWSSD---TQHSRVLSLYNTINP
345





|    ||+||| ||||  ||  |||||| +| | ||+  |     | |+ | || + ++|


Sbjct:
1
CRRHDLYVDFK-DLGWDDWIIAPKGYNAYYCEGECPFPLSERLNATNHAIVQSLVHALDP
 59


Query:
346
EASASPCCVSQDLEPLTILYYI-GKTPKIEQLSNMIVKSCKCS
387




 |   ||||   | ||++|||       +    ||+|+ | | 


Sbjct:
60
GAVPKPCCVPTKLSPLSMLYYDDDGNVVLRNYPNMVVEECGCR
102









The human (de Martin et al., EMBO J. 6(6):1633-6, 1987; de Martin et al., EMBO J. 6(12):3673-7, 1987; Madisen et al., DNA. 7(1):1-8, 1988) and simian (Hanks et al., Proc Natl Acad Sci U S A. 85(1):79-82, 1988) forms of TGF-beta-2 have identical C-terminal 112 amino acid residues. The beta-2 form was derived from human glioblastoma cells. It has suppressive effects on interleukin-2-dependent T-cell growth. For these reasons it is also called glioblastoma-derived T-cell suppressor factor (G-TSF). It may have an autocrine function in enhancing tumor growth and/or reducing immunosurveillance of tumor development. By Southern blot analysis of somatic cell hybrid lines and, for the human locus, also by in situ chromosomal hybridization, Barton et al. (Oncogene Res. 3(4):323-31, 1988) mapped TGFB2 to 1q41 in the human and to chromosome 1 in the mouse, most likely in the known conserved syntenic region. Dickinson et al. (Genomics. 6(3):505-20, 1990) also assigned the mouse Tgfb-2 gene to chromosome 1. Nishimura et al. (Genomics. 15(2):357-64, 1993) identified 4 RFLPs and SSCPs (single strand conformation polymorphisms) for TGFB2 in humans and gorillas. Using these, they localized the gene within a framework map of distal 1q and showed close linkage to homeo box gene HLX1 (142995); maximum lod score of 14.49 at theta=0.031.
The NOV18 nucleic acid of the invention encoding a Transforming Growth Factor Beta 2-like protein includes the nucleic acid whose sequence is provided in Table 18A, or a fragment thereof. The invention also includes a mutant or variant nucleic acid any of whose bases may be changed from the corresponding base shown in Table 18A while still encoding a protein that maintains its Transforming Growth Factor Beta 2-like activities and physiological functions, or a fragment of such a nucleic acid. The invention further includes nucleic acids whose sequences are complementary to those just described, including nucleic acid fragments that are complementary to any of the nucleic acids just described. The invention additionally includes nucleic acids or nucleic acid fragments, or complements thereto, whose structures include chemical modifications. Such modifications include, by way of non-limiting example, modified bases, and nucleic acids whose sugar phosphate backbones are modified or derivatized. These modifications are carried out at least in part to enhance the chemical stability of the modified nucleic acid, such that they may be used, for example, as antisense binding nucleic acids in therapeutic applications in a subject.
The NOV18 protein of the invention includes the Transforming Growth Factor Beta 2-like protein whose sequence is provided in Table 1 8B. The invention also includes a mutant or variant protein any of whose residues may be changed from the corresponding residue shown in Table 18B while still encoding a protein that maintains its Transforming Growth Factor Beta 2-like activities and physiological functions, or a functional fragment thereof.
The NOV18 nucleic acids and proteins of the invention are useful in potential diagnostic and therapeutic applications implicated in various diseases and disorders described below and/or other pathologies. For example, the compositions of the present invention will have efficacy for treatment of patients suffering from: cancer and other diseases, disorders and conditions of the like.
NOV18 nucleic acids and polypeptides are further useful in the generation of antibodies that bind immunospecifically to the novel substances of the invention for use in therapeutic or diagnostic methods. These antibodies may be generated according to methods known in the art, using prediction from hydrophobicity charts, as described in the “Anti-NOVX Antibodies” section below. For example the disclosed NOV18 protein have multiple hydrophilic regions, each of which can be used as an immunogen. This novel protein also has value in development of powerful assay system for functional analysis of various human disorders, which will help in understanding of pathology of the disease and development of new drug targets for various disorders.
NOV19
A disclosed NOV19 nucleic acid of 2939 nucleotides (also referred to as CG57333-01) encoding a novel Ebnerin-like protein is shown in Table 19A. An open reading frame was identified beginning with an ATG initiation codon at nucleotides 20-22 and ending with a TAG codon at nucleotides 2912-2914. Putative untranslated regions upstream from the intitation codon and downstream from the termination codon are underlined in Table 19A, and the start and stop codons are in bold letters.







TABLE 19A





NOV19 Nucleotide Sequence
















(SEQ ID NO:69)









GAATGGAGGGAAGGCCTAGATGGATGAGTGGAAAGGAAGATGGGGGGAGGGAAAGGTGGATGAAGAGTCAGGA



GGCTGGGCGGCTTCCCCGACTCTAATTCTTTGGGTCTCTTCTACCCCAGGGTCCCCCCAGCTGCGCCTGGTGG


CTGGGCCCAGCAAGTGCTCAGGTCGACTGGAGGTGTGGCATGACCAGCGCTGGGGGACCGTGTGTGACGATAG


CTGGGACATGCGGGATTCAGCTGTGGTCTGCCGGGAGCTGGGCTGTGGTGGACCTCAGCAGCCAGACCCTGCT


GCCGGCCGCCTGGAGGTCTGGCACGGGGGTCGCTGGGGGTCGGTGTGTGACGACGCCTGGGACCTGCGAGACG


CCGCTGTGGCCTGCCGAGAGCTGGGCTGCGGAGGGGCGCTGGCCGCCCCCGGGGGCGCCTTCTTTGGGGAGGG


GTCTGGACCCATCATCCTGGACGACCTTCGGTGTCGGGGAAACGAGACGGCCTTACGATTCTGCCCAGCTCGG


CCCTGGGGCCAGCATGACTGTCACCACCGCGAGGACGCCGGGGCCGTGTGTGACGGCATGCCCCTGGGCTACG


TCCCTCCCACGGCCCCCACGGACAGCAACAACTCCACGCCCAGGGAGGCTGCCTCCAGGCCCCCGTCCACCAT


GACGAGCCAGGCTCCAGGGACGGCAGGCGTTTCACCTCCTCCAGCCTCCCCTACTGTCCTTTGGGAGCCTGGA


CCGGAAGCCGGGTCCCCCCAGCTGCGCCTGGTGGCTGGGCCCAGCAAGTGCTCAGGTCGACTGGAGGTGTGGC


ATGACCAGCGCTGGGGGACCGTGTGTGACGATAGCTGGGACATGCGGGATTCAGCTGTGGTCTGCCGGGAGCT


GGGCTGTGGTGGACCTCAGCAGCCAGACCCTGCTGCTGGCCGCTTTGGCTGGGGTGCGGGCCCCATCTGGCTA


GATGATGTGAGCTGTGTGGGGACCGAGGCTTCACTGTCCGACTGCCCTGCTGCTCCCTGGGGAAAGCACAACT


GCGCTCACAATGAGGATGTTGGGGTCACCTGCACTGGGCCCCCAGGCCTGGACTCCATCTCAGACCCCTTCAG


CTGGAGCTGGATTCCTGGACTGGGGAGAGATCGGGATGCCTGGCTCCCGGGAGAGCTGGCCACCAAGCCCTCT


GCAAGTGTGACTGCCAGTGTTCTGGAGAAAACAACCACGAAGGCCCCAGGGAAAATGCCTAAGAGTACTAAGA


AGTGGGTGACAAAAAATGCAAAGAGACCAACCACTCAACCCCCAGTGATGCCAACCACGAAACACTCCAGGGC


CCAAAGCCCCCCAGACCTAACTTCACAGACCACTGCAGCACTGACCACTGAGGCCTCCCGAAGACCTACCTCT


GAGTTTACCAGAAGGCCGACCACGGAGGCCCCCCAGAGATGGACCTCTCACACCACTGCCACGCTGACCCCTC


AGGCCCCCCGAGAACGGACCACTAAGACCATGGCAATGCTGACCACTCAAGGCCCCCAAGAAATGACCTCTGA


GTCCACTATCAAGAGTATCCCTCAGGCCTCCCTGGAGCCATCTGCTGAGATCCCAGAAGGGTCTCCAGAGTCA


CCCAAAGACCCGGCCCCCTCTCCCAGTGTTAGCACCACTGGGGAATCAGGCCTGTTCCGGGTTCGTCTGGCCG


ATGGGCCCAACCGCTGTGCTGGCCGGCTGGAAGTGTGGCATGCCGGACGCTGGGGAACAGTGTGTGATGACAA


CTGGGACCTGCGGGACGCCACTGTGGCCTGCTGGGAACTGGGCTGTGGAAAGGTCCGGCCTCGAGTAGGCAAA


ACCCATTACGGTCCTGGGACTGGGCCCATCTGGCTGGATGACATGGGCTGTAAGGGAAGCGAGGCCTCACTGA


GCGACTGCCCCTCGGGGGCTTGGGGGAAGCACAACTGTGACCACGAGGAAGACGTGGGGCTCACCTGCACTGG


CTACACAGACTATGACGATTATCCCCCCTGGACCTGGGACCCCACCTCAAGAGAGGACCTGGCCAAGGGGACT


ACCACAGCGGGGGTACCTGGACACACTCTCCCCTGGAGGACCACCCGGCGCCCGGGTAGCTCCTCCCCAGCAA


TAAGGCGCCTGCCGGACACAGGCAGCAAAGATGGTTACAAGCTTCCCTGGACGTGGGACACACCATCAGGAAG


GGGCCTGGCTGAGGGGACCCCTACCGCAGGCAAACTAGGACCAACTCTTGGGGCTGGCACCACCAGGAGCCCA


GGCAGTCCTCCAACTCCGAGAGTCCATGGAGACACAGGTTCCCCGAGGAAACCGTGGCCCGAGCGCCGGCCAC


CGCGGCCCGCTGCGACCAGGACAGCGCCCCCAACCCCGTCCCCAGGTCCCTCCGCCTCTCCGGGACCCCCAGG


CCCAGCGCTGACCTCTGACTCCAGTCGAGAGCTCACTCCCCACTCAGCCTTGACGTCCGAGGCGACCTCTGAC


GCTCCGGACACTTCACCACCCACCCCAGACCCGGCCTCCCGGACGAACCCCGACCTCATCTTGACAAGCCCTG


ACTTTGCTTTGTCCACCCCTGACTCCAGTGTGGTTCCCGCGTTGACCCCGGAGCCCTCACCCACGCCCTTACC


CACCTTGCCCAAAGAGCTGACCTCTGACCCTTCTACACCGTCGGAGGTGACCAGCCTTTCCCCTACCTCAGAG


CAGGTCCCAGAATCTGACACAACCCCAGATTTGGACACAACTCCATACTCCAGTACANGACTCCTGACCCCAC


CACGACCCCTTACCCCACCACTACTCCTGATCCCACCACGACCCCTCACCCCACAACTCCTGACCCTTCCTCA


ACCCCTGTCATCACTACTGTGTCCCTTCCAACCTCCTTGGGGACAGAACTCTCCTCTCCCACTCTAGCACCAA


CAGTCAAGCCCAGTCTGCA









The NOV19 nucleic acid and has 313 of 490 bases (63%) identical to a gb:GENBANK-ID:HSA243224|acc:AJ243224.1 mRNA from Homo sapiens (Homo sapiens mRNA for DMBT1 protein 8 kb transcript variant 1 (DMBT1/8kb.1)) (E=7.0e−46).
A disclosed NOV19 polypeptide (SEQ ID NO:70) encoded by SEQ ID NO:69 is 964 amino acid residues and is presented using the one-letter code in Table 19B. Signal P, Psort and/or Hydropathy results predict that NOV19 does not contain a signal peptide and is likely to be localized to the cytoplasm with a certainty of 0.4500.







TABLE 19B





Encoded NOV19 protein sequence
















(SEQ ID NO:70)









MDEWKGRWGEGKVDEESGGWAASPTLILWVSSTPGSPQLRLVAGPSKCSGRLEVWHDQRWGTVCDDSWDMRD



SAVVCRELGCGGPQQPDPAAGRLEVWHGGRWGSVCDDAWDLRDAAVACRELGCGGALAAPGGAFFGEGSGPI


ILDDLRCRGNETALRFCPARPWGQHDCHHREDAGAVCDGMPLGYVPPTAPTDSNNSTPREAASRPPSTMTSQ


APGTAGVSPPPASPTVLWEPGPEAGSPQLRLVAGPSKCSGRLEVWHDQRWGTVCDDSWDMRDSAVVCRELGC


GGPQQPDPAAGRFGWGAGPIWLDDVSCVGTEASLSDCPAAPWGKHNCAHNEDVGVTCTGPPGLDSISDPFSW


SWIPGLGRDRDAWLPGELATKPSASVTASVLEKTTTKAPGKMPKSTKKWVTKNAKRPTTQPPVMPTTKHSRA


QSPPDLTSQTTAALTTEASRRPTSEFTRRPTTEAPQRWTSHTTATLTPQAPRERTTKTMAMLTTQGPQEMTS


ESTIKSIPQASLEPSAEIPEGSPESPKDPAPSPSVSTTGESGLFRVRLADGPNRCAGRLEVWHAGRWGTVCD


DNWDLRDATVACWELGCGKVRPRVGKTHYGPGTGPIWLDDMGCKGSEASLSDCPSGAWGKHNCDHEEDVGLT


CTGYTDYDDYPPWTWDPTSREDLAKGTTTAGVPGHTLPWRTTRRPGSSSPAIRRLPDTGSKDGYKLPWTWDT


PSGRGLAEGTPTAGKLGPTLGAGTTRSPGSPPTPRVHGDTGSPRKPWPERRPPRPAATRTAPPTPSPGPSAS


PGPPGPALTSDSSRELTPHSALTSEATSDAPDTSPPTPDPASRTNPDLILTSPDFALSTPDSSVVPALTPEP


SPTPLPTLPKELTSDPSTPSEVTSLSPTSEQVPESDTTPDLDTTPYSSTXLLTPPRPLTPPLLLIPPRPLTP


QLLTLPQPLSSLLCPFQPPWGQNSPLPL









The NOV19 amino acid sequence has 57 of 109 amino acid residues (52%) identical to, and 75 of 109 amino acid residues (68%) similar to, the 1594 amino acid residue ptnr:SPTREMBL-ACC:Q95218 protein from Oryctolagus cuniculus (Rabbit) (HENSIN) (E=2.2e−40).
NOV19 is expressed in at least the following tissues: adrenal gland, bone marrow, brain—amygdala, brain—cerebellum, brain—hippocampus, brain—substantia nigra, brain—thalamus, brain—whole, fetal brain, fetal kidney, fetal liver, fetal lung, heart, kidney, lymphoma—Raji, mammary gland, pancreas, pituitary gland, placenta, prostate, salivary gland, skeletal muscle, small intestine, spinal cord, spleen, stomach, testis, thyroid, trachea and uterus. This information was derived by determining the tissue sources of the sequences that were included in the invention including but not limited to SeqCalling sources, Public EST sources, genomic clone sources, literature sources, and/or RACE sources.
NOV19 has homology to the amino acid sequences shown in the BLASTP data listed in Table 19C.







TABLE 19C







BLAST results for NOV19












Gene Index/

Length
Identity
Positives



Identifier
Protein/Organism
(aa)
(%)
(%)
Expect





gi|13434994|dbj|BAB39761.1|
PIT 54 [Gallus
 470
115/331
160/331
2e−49


(AB051832)
gallus]

(34%)
(47%)


gi|7513558|pir||T30549
hensin - rabbit
1594
119/328
153/328
4e−47





(36%)
(46%)


gi|18152779|ref|NP_
scavenger
 575
114/262
138/262
4e−47


542782.1|
receptor

(43%)
(52%)


(NM_080744)
cysteine-rich



protein SRCRB-S4D



[Homo sapiens]


gi|14765505|ref|XP_
similar to
1051
123/349
157/349
6e−47


051448.1|
deleted in

(35%)
(44%)


(XM_051448)
malignant brain



tumors 1



(H. sapiens) 



[Homo sapiens]


gi|2135587|pir||I38005
M130 antigen
1156
124/361
168/361
8e−47



precursor, splice

(34%)
(46%)



form 4 [Homo



sapiens]









The homology of these sequences is shown graphically in the ClustalW analysis shown in Table 19D.







TABLE 19D





Clustal W Sequence Alignment















1) NOV19 (SEQ ID NO:70)


2) gi 13434994|dbj|BAB39761.1|(AB051832) PIT 54 [Gallus gallus] (SEQ ID NO:211)


3) gi 7513558|pir T30549 hensin - rabbit (SEQ ID NO:212)


4) gi 18152779|refNP_542782.1|(NM_080744) scavenger receptor cysteine-rich protein


SRCRB-S4D [Homo sapiens] (SEQ ID NO:213)


5) gi 14765505|refXP_051448.1|(XM_051448) similar to deleted in malignant brain tumors 


1 (H. sapiens) [Homo sapiens] (SEQ ID NO:214)


6) gi|2135587|pirp51 I38005 M130 antigen precursor, splice form 4 [Homo sapiens]


(SEQ ID NO:215)




































































































Tables 19E and 19F list the domain description from DOMAIN analysis results against NOV19. This indicates that the NOV19 sequence has properties similar to those of other proteins known to contain these domains.








TABLE 19E





Domain Analysis of NOV19
















gnl|Smart|smart00202, SR, Scavenger receptor Cys-rich; The sea ucrhin



egg peptide speract contains 4 repeats of SR domains that contain 6


conserved cysteines. May bind bacterial antigens in the protein MARCO.


(SEQ ID NO:216)


CD-Length = 101 residues, 100.0% aligned


Score = 142 bits (359), Expect = 7e−35











Query:
550
VRLADGPNRCAGRLEVWHAGRWGTVCDDNWDLRDATVACWELGCGKVRPRVGKTHYGPGT
609





|||  | + | ||+||+| |+||||||| |||||| | | +|| |      |  ++|||+


Sbjct:
1
VRLVGGGSPCEGRVEVYHNGQWGTVCDDGWDLRDANVVCRQLGFGGAVSASGSAYFGPGS
 60


Query:
610
GPIWLDDMGCKGSEASLSDCPSGAWGKHNCDHEEDVGLTCT
650




||||||++ | |+||||||||   || ||| | || |+ |+


Sbjct:
61
GPIWLDNVRCTGTEASLSDCPHSGWGSHNCSHSEDAGVVCS
101

















TABLE 19F





Domain Analysis of NOV19
















gnl Pfam|pfam00530, SRCR, Scavenger receptor cysteine-rich domain.



These domains are disulphide rich extracellular domains. These domains


are found in several extracellular receptors and may be involved in


protein-protein interactions. (SEQ ID NO:217)


CD-Length = 95 residues, 96.8% aligned


Score = 95.9 bits (237), Expect = 9e−21











Query:
250
GPSKCSGRLEVWHDQRWGTVCDDSWDMRDSAVVCRELGCGGPQQPDPAAGRFGWGAGPIW
309





| |+| ||+||    +|||||| || +||+ ||||+|||||          |  | ||| 


Sbjct:
3
GSSRCEGRVEVRDGSKWGTVCDSSWTLRDANVVCRQLGCGGALSS-LGGPYFSEGGGPIP
 61


Query:
310
LDDVSCVGTEASLSDCPAAPWGHKNCAHNEDVGVTC
345




|| |+| | |+||| |   |     |+| || || |


Sbjct:
62
LDGVNCSGNESSLSQC---PHRSRQCSHGEDAGVVC
 94









Saliva is the first digestive fluid secreted by the gastrointestinal pathway and performs a variety of functions. It is essential in the formation of small boluses of food, provides lubrication for swallowing and speech, dissolves a number of chemicals in food substances, and provides digestive enzymes such as amylase and lipase (Hamosh & Scow, 1973, J. Clin. Invest.52:88-95; Field & Hand, 1987, Am. J. Physiol.253:G217-G225). About 90% of saliva is produced by three major glands, the parotid, the submaxillary, and the sublingual glands, whose secretions drain into the oral cavity.
von Ebner's glands are unique salivary glands contained within the tongue that drain directly into the clefts of the circumnvallate and foliate papillae, which contain the major taste buds. Secretions of von Ebner's gland directly modulate taste perception (Gurkan & Bradley, 1988, Chem. Senses 13: 655-661). Xiao-Jiang Li and Solomon H. Snyder (1995, J Biol Chem 270:17674-9) have identified a novel von Ebner's gland (VEG) protein, designated Ebnerin, which is formed in the ducts of von Ebner's gland and released into fluid bathing the taste buds contained in the taste papillae. Ebnerin possesses a putative single transmembrane domain at the C terminus with 17 amino acids in the cytoplasmic area. The extracellular region of Ebnerin contains a number of repeated domains with homology to the scavenger receptor cysteine-rich domain and to a repeated domain of bone morphogenetic protein-I and other related proteins. Western blot analysis reveals that Ebnerin exists in particulate and soluble forms in VEG and is present in secretions from VEG. The unique structure and localization of Ebnerin suggest that it may function as a binding protein in saliva for the regulation of taste sensation.
Ebnerin is a modular protein containing a signal peptide, SRCR (“scavenger receptor, cysteine rich”) domains. Other proteins known to have this domain include CRP-ductin, a cDNA expressed at high levels in mouse intestine (8 SRCR), hensin, the polarity reversal protein cloned from a rabbit kidney (8SRCR), and DMBT1, a sequence in human chromosome 10q25-26 frequently deleted in malignant gliomas (9 SRCR) (Cheng, et al., 1996, Anat. Rec. 244: 327-343; Mollenhauer, 1997, Nat. Genet. 17: 32-39; Takito et al., 1999, Am J Physiol 277:F277-89). Hensin, DMBT1, CRP-ductin, and ebnerin are alternately spliced products from a single gene located on human chromosome 10q25-26, a region often deleted in several cancers, especially malignant gliomas. Hensin is expressed in many epithelial cell types, and it plays a critical role in terminal differentiation of the intercalated cell and perhaps other epithelia (Al-Awqati et al., 2000, Exp Nephrol 8:66-71).
Vomeroglandin, a subform of mouse CRP-ductin, is a protein strongly expressed in the glands of mouse vomeronasal system. Both the proteins contain several of scavenger receptor cysteine-rich and CUB domains and one ZP domain. This domain arrangement is similar to those of rat Ebnerin, human DMBT1, and rabbit hensin. In situ hybridization analysis shows strong expression of vomeroglandin mRNA in the glands of vomeronasal system. Immunological analyses detect both membrane-bound and secreted forms of vomeroglandin. The secreted protein seems to be localized in the lumen of the vomeronasal organ, playing a certain role in the pheromone perception (Matsushita et al., 2000, Biochem Biophys Res Commun 268:275-81).
The NOV19 nucleic acid of the invention encoding a Ebnerin-like protein includes the nucleic acid whose sequence is provided in Table 19A, or a fragment thereof. The invention also includes a mutant or variant nucleic acid any of whose bases may be changed from the corresponding base shown in Table 19A while still encoding a protein that maintains its Ebnerin-like activities and physiological functions, or a fragment of such a nucleic acid. The invention further includes nucleic acids whose sequences are complementary to those just described, including nucleic acid fragments that are complementary to any of the nucleic acids just described. The invention additionally includes nucleic acids or nucleic acid fragments, or complements thereto, whose structures include chemical modifications. Such modifications include, by way of non-limiting example, modified bases, and nucleic acids whose sugar phosphate backbones are modified or derivatized. These modifications are carried out at least in part to enhance the chemical stability of the modified nucleic acid, such that they may be used, for example, as antisense binding nucleic acids in therapeutic applications in a subject. In the mutant or variant nucleic acids, and their complements, up to about 37% of the residues may be so changed.
The NOV19 protein of the invention includes the Ebnerin-like protein whose sequence is provided in Table 19B. The invention also includes a mutant or variant protein any of whose residues may be changed from the corresponding residue shown in Table 19B while still encoding a protein that maintains its Ebnerin-like activities and physiological functions, or a functional fragment thereof. In the mutant or variant protein, up to about 48% of the bases may be so changed.
The NOV19 nucleic acids and proteins of the invention are useful in potential diagnostic and therapeutic applications implicated in various diseases and disorders described below and/or other pathologies. For example, the compositions of the present invention will have efficacy for treatment of patients suffering from: several cancers, especially malignant gliomas, glioblastoma multiforme, medulloblastoma and other diseases, disorders and conditions of the like.
NOV19 nucleic acids and polypeptides are further useful in the generation of antibodies that bind immunospecifically to the novel substances of the invention for use in therapeutic or diagnostic methods. These antibodies may be generated according to methods known in the art, using prediction from hydrophobicity charts, as described in the “Anti-NOVX Antibodies” section below. For example the disclosed NOV19 protein have multiple hydrophilic regions, each of which can be used as an immunogen. This novel protein also has value in development of powerful assay system for functional analysis of various human disorders, which will help in understanding of pathology of the disease and development of new drug targets for various disorders.
NOV20
NOV20 includes two novel Fatty Acid Binding-like proteins disclosed below. The disclosed proteins have been named NOV20a and NOV20b.
NOV20a
A disclosed NOV20a nucleic acid of 400 nucleotides (also referred to as CG57556-01) encoding a novel Fatty Acid Binding-like protein is shown in Table 20A. An open reading frame was identified beginning with an ATG initiation codon at nucleotides 2-4 and ending with a TAG codon at nucleotides 398-400. Putative untranslated regions, if any, upstream from the initiation codon and downstream from the termination codon are underlined in Table 20A, and the start and stop codons are in bold letters.







TABLE 20A





NOV20a nucleotide sequence.
















(SEQ ID NO:71)









TATGGTAAGGGTGGAGGAGGCTTTCTGTGCTACCTGGAAGCTGACCAACAGTCAGAACTTTGATGAGTACAT



GAAGGCTCTAGGCGTGGGCTTTGCCACTAGGCAGGTGGGAAATGTGACCAAACCAACGGTAATTATCAGTCA


AGAAGGAGACAAAGTGGTCATCAGGACTCTCAGCACATTCAAGAACACGGAGATTAGTTTCCAGCTGGGAGA


AGAGTTTGATGAAACCACTGCAGATGATAGAAACTGTAAGTCTGTTGTTAGCCTGGATGGAGACAAACTTGT


TCACATACAGAAATGGGATGGCAAAGAAACAAATTTTGTAAGAGAAATTAAGGATGGCAAAATGGTTATGAC


CCTTACTTTTGGTGATGATGTGGTTGCCGTTCACCACTAG









The disclosed NOV20a nucleic acid sequence, localized to chromsome 6q22-23, has 389 of 398 bases (97%) identical to a gb:GENBANK-ID:HSAJ2962|acc:AJ002962.1 mRNA from Homo sapiens (Homo sapiens mRNA for hB-FABP) (E=2.5e−79).
A NOV20a polypeptide (SEQ ID NO:72) encoded by SEQ ID NO:71 has 132 amino acid residues and is presented using the one-letter code in Table 20B. Signal P, Psort and/or Hydropathy results predict that NOV20a does not contain a signal peptide and is likely to be localized to the microbody (peroxisome) with a certainty of 0.4849 and to the mitochondrial matrix space with a certainty of 0.4750. Although PSORT suggests that the NOV20a protein may be localized in the intracellularly, the NOV20a protein is similar to the Fatty Acid Binding Protein family, some members of which are localized at the plasma membrane. Therefore it is likely that this novel Fatty Acid Binding Protein-like protein is localized to the same sub-cellular compartment.







TABLE 20B





Encoded NOV20a protein sequence.
















(SEQ ID NO:72)









MVRVEEAFCATWKLTNSQNFDEYMKALGVGFATRQVGNVTKPTVIISQEGDKVVIRTLSTFKNTEISFQLGE



EFDETTADDRNCKSVVSLDGDKLVHIQKWDGKETNFVREIKDGKMVMTLTFGDDVVAVHH









The NOV20a amino acid sequence has 125 of 127 amino acid residues (98%) identical to, and 125 of 127 amino acid residues (98%) similar to, the 131 amino acid residue ptnr:SWISSNEW-ACC:O15540 protein from Homo sapiens (Human) (Fatty Acid-Binding Protein, Brain (B-FABP) (Brain Lipid-Binding Protein) (BLBP) (Mammary Derived Growth Inhibitor Related)) (E=1.9e−62).
The disclosed NOV20a is expressed in at least the following tissues: Bone, Brain, Heart, Kidney, Lung, Retina, Temporal Lobe and Uterus. This information was derived by determining the tissue sources of the sequences that were included in the invention including but not limited to SeqCalling sources, Public EST sources, Literature sources, and/or RACE sources. The disclosed NOV20a is also predicted to be expressed in the following tissues because of the expression pattern of (gb:GENBANK-ID:HSAJ2962|acc:AJ002962.1) a closely related Homo sapiens mRNA for hB-FABP homolog in species Homo sapiens: brain.
NOV20b
A disclosed NOV20b nucleic acid of 417 nucleotides (also referred to as CG57556-02) encoding a novel Fatty Acid Binding-like protein is shown in Table 20C. An open reading frame was identified beginning with an ATG initiation codon at nucleotides 5-7 and ending with a TAA codon at nucleotides 410-412. Putative untranslated regions, if any, upstream from the initiation codon and downstream from the termination codon are underlined in Table 20C, and the start and stop codons are in bold letters.







TABLE 20C





NOV20b nucleotide sequence.
















(SEQ ID NO:73)









TATAATGGTAAGGGTGGAGGAGGCTTTCTGTGCTACCTGGAGGCTGACCAACAGTCAGAACTTTGATGAGTA



CATGAAGGCTCTAGGCGTGGGCTTTGCCACTAGGCAGGTGGGAAATGTGACCAAACCAACGGTAATTATCAG


TCAAGAAGGAGACAAAGTGGTCATCAGGACTCTCAGCACATTCAAGAACACGGAGATTAGTTTCCAGCTGGG


AGAAGAGTTTGATGAAACCACTGCAGATGATAGAAACTGTAAGTCTGTTGTTAGCCTGGATGGAGACAAACT


TGTTCACATACAGAAATGGGATGGCAAAGAAACAAATTTTGTAAGAGAAATTAAGGATGGCAAAATGGTTAT


GACCCTTACTTTTGGTGATGTGGTTGCCGTTCACCACTATAAGAAGGCATAAAAATA









The disclosed NOV20b nucleic acid sequence, localized to chromsome 6q22-23, has 405 of 413 bases (98%) identical to a gb:GENBANK-ID:HSAJ2962|acc:AJ002962.1 mRNA from Homo sapiens (Homo sapiens mRNA for hB-FABP) (E=6.6e−84).
A NOV20b polypeptide (SEQ ID NO:74) encoded by SEQ ID NO:73 has 135 amino acid residues and is presented using the one-letter code in Table 20D. Signal P, Psort and/or Hydropathy results predict that NOV20b does not contain a signal peptide and is likely to be localized to the mitochondrial matrix space with a certainty of 0.5834 and to the peroxisome (microbody) with a certainty of 0.4915.







TABLE 20D





Encoded NOV20b protein sequence.
















(SEQ ID NO:74)









MVRVEEAFCATWRLTNSQNFDEYMKALGVGFATRQVGNVTKPTVIISQEG



DKVVIRTLSTFKNTEISFQLGEEFDETTADDRNCKSVVSLDGDKLVHIQK


WDGKETNFVREIKDGKMVMTLTFGDVVAVHHYKKA









The NOV20b amino acid sequence has 127 of 130 amino acid residues (97%) identical to, and 129 of 130 amino acid residues (99%) similar to, the 131 amino acid residue ptnr:SWISSNEW-ACC:O15540 protein from Homo sapiens (Human) (Fatty Acid-Binding Protein, Brain (B-FABP) (Brain Lipid-Binding Protein) (BLBP) (Mammary Derived Growth Inhibitor Related)) (E=6.8e−65).
The disclosed NOV20b is expressed in at least the following tissues: Bone, Brain, Heart, Kidney, Lung, Retina, Temporal Lobe and Uterus. This information was derived by determining the tissue sources of the sequences that were included in the invention including but not limited to SeqCalling sources, Public EST sources, Literature sources, and/or RACE sources. The disclosed NOV20b is also predicted to be expressed in the following tissues because of the expression pattern of (gb:GENBANK-ID:HSAJ2962|acc:AJ002962.1) a closely related Homo sapiens mRNA for hB-FABP homolog in species Homo sapiens: brain.
Possible SNPs found for NOV20a are listed in Table 20E.







TABLE 20E







SNPs










Consensus

Base



Position
Depth
Change
PAF













42
6
A > G
0.333


170
6
A > G
0.333


388
6
C > T
0.333









NOV20a and NOV20b are very closely homologous as is shown in the amino acid alignment in Table 20F.







TABLE 20F





Amino Acid Alignment of NOV20a and NOV20b




































Homologies to any of the above NOV20 proteins will be shared by the other NOV20 proteins insofar as they are homologous to each other as shown above. Any reference to NOV20 is assumed to refer to the NOV20 proteins in general, unless otherwise noted.
NOV20a has homology to the amino acid sequences shown in the BLASTP data listed in Table 20G.







TABLE 20G







BLAST results for NOV20a












Gene Index/
Protein/
Length
Identity
Positives



Identifier
Organism
(aa)
(%)
(%)
Expect





gi|4557585|ref|NP_001437.1|
fatty acid
132
125/127
125/127
2e−61


(NM_001446)
binding

(98%)
(98%)



protein 7,



brain; B-FABP



[Homo



sapiens]


gi|15826067|pdb|1FDQ|A
Chain A,
131
125/127
125/127
3e−61



Crystal

(98%)
(98%)



Structure Of



Human Brain



Fatty Acid



Binding



Protein [Homo



sapiens]


gi|2605596|dbj|BAA23324.1|
fatty acid
132
124/127
125/127
1e−60


(D50373)
binding

(97%)
(97%)



protein [Homo



sapiens]


gi|12224842|emb|CAC21646.1|
hypothetical
166
114/114
114/114
2e−58


(AL512688)
protein [Homo

(100%)
(100%)



sapiens]


gi|462065|sp|Q05423|
FATTY ACID-
132
114/127
119/127
9e−57


FABB_CHICK
BINDING

(89%)
(92%)



PROTEIN,



RETINA (R-



FABP) [Gallus



gallus]









The homology of these sequences is shown graphically in the ClustalW analysis shown in Table 20H.







TABLE 20H





ClustalW Analysis of NOV2Oa















1) NOV2Oa (SEQ ID NO:72)


2) gi 4557585|refNP_0014371.1|(NM_001446) fatty acid binding protein 7, brain; B-FABP


[Homo sapiens] (SEQ ID NO:218)


2) gi 15826062|pdb|1FDQ|A Chain A, Crystal Structure Of Human Brain Fatty Acid


Binding Protein [Homo sapiens] (SEQ ID NO:219)


3) gi 2605596|dbj|BAA23324.1| (D50373) fatty acid binding protein [Homo sapiens]


(SEQ ID NO:220)


4) gi 12224842|emb|CAC21646.1 (AL512688) hypothetical protein [Homo sapiens]


(SEQ ID NO:221)


5) gi 462065|sp Q05423|FABB_CHICK FATTY ACID-BINDING PROTEIN, RETINA (R-FABP)


[Gallus gallus] (SEQ ID NO:222)






























Table 20I lists the domain description from DOMAIN analysis results against NOV20a. This indicates that the NOV20a sequence has properties similar to those of other proteins known to contain these domains.







TABLE 20I





Domain Analysis of NOV20a
















gnl|Pfam|pfam00061, lipocalin, Lipocalin/cytosolic fatty-acid



binding protein family. Lipocalins are transporters for small


hydrophobic molecules, such as lipids, steroid hormones, bilins, and


retinoids. Alignment subsumes both the lipocalin and fatty acid


binding protein signatures from PROSITE. This is supported on


structural and functional grounds. Structure is an eight-stranded beta


barrel. (SEQ ID NO:223)


CD-Length = 145 residues, 86.2% aligned


Score = 63.2 bits (152), Expect = 9e-12











Query:
7
AFCATWKLTNSQNFDEYMK-ALGVGFATRQVGNVTK-PTVIISQEGDKVVIRTLSTFKNT
 64





 |   | |  | |||  +|  |||  |||+     |   + |  +|||  |   +  |  


Sbjct:
1
KFAGKWYLVASANFDPELKEELGVLEATRKEITPLKEGNLEIVFDGDKNGICEETFGKLE
 60


Query:
65
EISFQLGEEFDETTADDRNCKSVVSLDGDKLVHIQKWDGKETNFVREIKDGKMVMTLTFG
124




+   +|| |||  | |+|        |   || +|| || ||+   |+      ++    


Sbjct:
61
KTK-KLGVEFDYYTGDNRFVVLDTDYDNYLLVCVQKGDGNETSRTAELYGRTPELSPEAL
119


Query:
125
DDVVAV
130




+


Sbjct:
120
ELFETA
125









The murine brain fatty acid binding protein (B-FABP) is encoded by a developmentally regulated gene that is expressed in radial glial cells and immature astrocytes. The human B-FABP gene has been cloned and mapped to chromosome 6q22-23. B-FABP mRNA is expressed in human malignant glioma tumor biopsies and in a subset of malignant glioma cell lines, as well as in human fetal retina and brain. Malignant glioma tumors are characterized by cytoplasmic bundles of glial fibrillary acidic protein (GFAP), a protein normally expressed in mature astrocytes. Establishment of malignant glioma cell lines often results in loss of GFAP. The subset of malignant glioma cell lines that express GFAP mRNA also express B-FABP mRNA. Co-localization experiments in cell lines indicate that the same cells produce both GFAP and B-FABP. These data suggest that some malignant gliomas may be derived from astrocytic precursor cells which can express proteins that are normally produced at different developmental stages in the astrocytic differentiation pathway (Godbout et al., Oncogene. 16(15):1955-62, 1998).
The NOV20 nucleic acid of the invention encoding a Fatty Acid Binding-like protein includes the nucleic acid whose sequence is provided in Tables 20A and 20C, or a fragment thereof. The invention also includes a mutant or variant nucleic acid any of whose bases may be changed from the corresponding base shown in Tables 20A and 20C while still encoding a protein that maintains its Fatty Acid Binding-like activities and physiological functions, or a fragment of such a nucleic acid. The invention further includes nucleic acids whose sequences are complementary to those just described, including nucleic acid fragments that are complementary to any of the nucleic acids just described. The invention additionally includes nucleic acids or nucleic acid fragments, or complements thereto, whose structures include chemical modifications. Such modifications include, by way of non-limiting example, modified bases, and nucleic acids whose sugar phosphate backbones are modified or derivatized. These modifications are carried out at least in part to enhance the chemical stability of the modified nucleic acid, such that they may be used, for example, as antisense binding nucleic acids in therapeutic applications in a subject. In the mutant or variant nucleic acids, and their complements, up to about 3% of the NOV20a residues and up to about 2% of the NOV20b residues may be so changed.
The NOV20 protein of the invention includes the Fatty Acid Binding-like protein whose sequence is provided in Tables 20B and 20D. The invention also includes a mutant or variant protein any of whose residues may be changed from the corresponding residue shown in Tables 20B and 20D while still encoding a protein that maintains its Fatty Acid Binding-like activities and physiological functions, or a functional fragment thereof. In the mutant or variant protein, up to about 2% of the NOV20a bases and up to about 3% of the NOV20b bases may be so changed.
The NOV20 nucleic acids and proteins of the invention are useful in potential diagnostic and therapeutic applications implicated in various diseases and disorders described below and/or other pathologies. For example, the compositions of the present invention will have efficacy for treatment of patients suffering from: malignant glioma tumors, Arthropathy, progressive pseudorheumatoid, of childhood; Muscular dystrophy, congenital merosin-deficient; Nephropathy, IgA type; Oculodentodigital dysplasia; Ossification of posterior longitudinal ligament of spine; Syndactyly, type III; Hepatic fibrosis susceptibility due to Schistosoma mansoni infection; Hepatic fibrosis susceptibility due to Schistosoma mansoni infection; endometriosis, fertility; Von Hippel-Lindau (VHL) syndrome, Alzheimer's disease, stroke, tuberous sclerosis, hypercalceimia, Parkinson's disease, Huntington's disease, cerebral palsy, epilepsy, Lesch-Nyhan syndrome, multiple sclerosis, ataxia-telangiectasia, leukodystrophies, behavioral disorders, addiction, anxiety, pain, neurodegeneration; cardiomyopathy, atherosclerosis, hypertension, congenital heart defects, aortic stenosis, atrial septal defect (ASD), atrioventricular (A-V) canal defect, ductus arteriosus, pulmonary stenosis, subaortic stenosis, ventricular septal defect (VSD), valve diseases, tuberous sclerosis, scleroderma, obesity, transplantation; diabetes, autoimmune disease, renal artery stenosis, interstitial nephritis, glomerulonephritis, polycystic kidney disease, systemic lupus erythematosus, renal tubular acidosis, IgA nephropathy, hypercalceimia, systemic lupus erythematosus, autoimmune disease, asthma, emphysema, scleroderma, allergy, ARDS and other diseases, disorders and conditions of the like.
NOV20 nucleic acids and polypeptides are further useful in the generation of antibodies that bind immunospecifically to the novel substances of the invention for use in therapeutic or diagnostic methods. These antibodies may be generated according to methods known in the art, using prediction from hydrophobicity charts, as described in the “Anti-NOVX Antibodies” section below. For example the disclosed NOV20 protein have multiple hydrophilic regions, each of which can be used as an immunogen. This novel protein also has value in development of powerful assay system for functional analysis of various human disorders, which will help in understanding of pathology of the disease and development of new drug targets for various disorders.
NOV21
A disclosed NOV21 nucleic acid of 1931 nucleotides (also referred to as CG57436-01) encoding a novel Platelet Glycoprotein V-like protein is shown in Table 21 A. An open reading frame was identified beginning with an ATG initiation codon at nucleotides 135-137 and ending with a TAA codon at nucleotides 1878-1880. Putative untranslated regions upstream from the intitation codon and downstream from the termination codon are underlined in Table 21A, and the start and stop codons are in bold letters.







TABLE 21A





NOV21 Nucleotide Sequence
















(SEQ ID NO:75)









AGCATGCGTTCAGTATGTGATGATGGGGTTCAAAGCCAAGCCAGCATTGGCACTGGAGTTGATGTTGGCTTAA



AACTTGCCAGTGTCAGGTAGAGGACCCTGACTAACGTGTGCTCTCTCCCCTTGCACAGGCTATGCCACTGAAG


CATTATCTCCTTTTGCTGGTGGGCTGCCAAGCCTGGGGTGCAGGGTTGGCCTACCATGGCTGCCCTAGCGAGT


GTACCTGCTCCAGGGCCTCCCAGGTGGAGTGCACCGGGGCACGCATTGTGGCGGTGCCCACCCCTCTGCCCTG


GAACGCCATGAGCCTGCAGATCCTCAACACGCACATCACTGAACTCAATGAGTCCCCGTTCCTCAATATTTCA


GCCCTCATCGCCCTGAGGATTGAGAAGAATGAGCTGTCGCGCATCACGCCTGGGGCCTTCCGAAACCTGGGCT


CGCTGCGCTATCTCAGCCTCGCCAACAACAAGCTGCAGGTTCTGCCCATCGGCCTCTTCCAGGGCCTGGACAG


CCTTGAGTCTCTCCTTCTGTCCAGTAACCAGCTGTTGCAGATCCAGCCGGCCCACTTCTCCCAGTGCAGCAAC


CTCAAGGAGCTGCAGTTGCACGGCAACCACCTGGAATACATCCCTGACGGAGCCTTCGACCACCTGGTAGGAC


TCACGAAGCTCAATCTGGGCAAGAATAGCCTCACCCACATCTCACCCAGGGTCTTCCAGCACCTGGGCAATCT


CCAGGTCCTCCGGCTGTATGAGAACAGGCTCACGGATATCCCCATGGGCACTTTTGATGGGCTTGTTAACCTG


CAGGAACTGGCTCTACAGCAGAACCAGATTGGACTGCTCTCCCCTGGTCTCTTCCACAACAACCACAACCTCC


AGAGACTCTACCTGTCCAACAACCACATCTCCCAGCTGCCACCCAGCATCTTCATGCAGCTGCCCCAGCTCAA


CCGTCTTACTCTCTTTGGGAATTCCCTGAAGGAGCTCTCTCTGGGGATCTTCGGGCCCATGCCCAACCTGCGG


GAGCTTTGGCTCTATGACAACCACATCTCTTCTCTACCCGACAATGTCTTCAGCAACCTCCGCCAGTTGCAGG


TCCTGATTCTTAGCCGCAATCAGATCAGCTTCATCTCCCCGGGTGCCTTCAACGGGCTAACGGAGCTTCGGGA


GCTGTCCCTCCACACCAACGCACTGCAGGACCTGGACGGGAATGTCTTCCGCATGTTGGCCAACCTGCAGAAC


ATCTCCCTGCAGAACAATCGCCTCAGACAGCTCCCAGGGAATATCTTCGCCAACGTCAATGGCCTCATGGCCA


TCCAGCTGCAGAACAACCAGCTGGAGAACTTGCCCCTCGGCATCTTCGATCACCTGGGGAAACTGTGTGAGCT


GCGGCTGTATGACAATCCCTGGAGGTGTGACTCAGACATCCTTCCGCTCCGCAACTGGCTCCTGCTCAACCAG


CCTAGGTTAGGGACGGACACTGTACCTGTGTGTTTCAGCCCAGCCAATGTCCGAGGCCAGTCCCTCATTATCA


TCAATGTCAACGTTGCTGTTCCAAGCGTCCATGTACCTGAGGTGCCTAGTTACCCAGAAACACCATGGTACCC


AGACACACCCAGTTACCCTGACACCACATCCGTCTCTTCTACCACTGAGCTAACCAGCCCTGTGGAAGACTAC


ACTGATCTGACTACCATTCAGGTCACTGATGACCGCAGCGTTTGGGGCATGACCCATGCCCATAGCGGGCTGG


CCATTGCCGCCATTGTAATTGGCATTGTCGCCCTGGCCTGCTCCCTGGCTGCCTGCGTCGGCTGTTGCTGCTG


CAAGAAGAGGAGCCAAGCTGTCCTGATGCAGATGAAGGCACCCAATGAGTGTTAAAGAGGCAGGCTGGAGCAG


GGCTGGGGAATGATGGGACTGGAGGACCTGGGA









The NOV21 nucleic acid was identified on chromosome 1 and has 779 of 1317 bases (59%) identical to gb:GENBANK-ID:AF163101|acc:AF163101.1 mRNA from Mus musculus (Mus musculus platelet glycoprotein V gene, partial cds) (E=4.4e−43).
A disclosed NOV21 polypeptide (SEQ ID NO:76) encoded by SEQ ID NO:75 is 581 amino acid residues and is presented using the one-letter code in Table 21B. Signal P, Psort and/or Hydropathy results predict that NOV21 contains a signal peptide and is likely to be localized to the plasma membrane with a certainty of 0.4600. The most likely cleavage site for a NOV21 polypeptide is between amino acids 21 and 22: GLA-YH.







TABLE 21B





Encoded NOV21 protein sequence
















(SEQ ID NO:76)









MPLKHYLLLLVGCQAWGAGLAYHGCPSECTCSRASQVECTGARIVAVPTPLPWNAMSLQILNTHITELNESP



FLNISALIALRIEKNELSRITPGAFRNLGSLRYLSLANNKLQVLPIGLFQGLDSLESLLLSSNQLLQIQPAH


FSQCSNLKELQLHGNHLEYIPDGAFDHLVGLTKLNLGKNSLTHISPRVFQHLGNLQVLRLYENRLTDIPMGT


FDGLVNLQELALQQNQIGLLSPGLFHNNHNLQRLYLSNNHISQLPPSIFMQLPQLNRLTLFGNSLKELSLGI


FGPMPNLRELWLYDNHISSLPDNVFSNLRQLQVLILSRNQISFISPGAFNGLTELRELSLHTNALQDLDGNV


FRMLANLQNISLQNNRLRQLPGNIFANVNGLMAIQLQNNQLENLPLGIFDHLGKLCELRLYDNPWRCDSDIL


PLRNWLLLNQPRLGTDTVPVCFSPANVRGQSLIIINVNVAVPSVHVPEVPSYPETPWYPDTPSYPDTTSVSS


TTELTSPVEDYTDLTTIQVTDDRSVWGMTHAHSGLAIAAIVIGIVALACSLAACVGCCCCKKRSQAVLMQMK


APNEC









The NOV21 amino acid sequence 165 of 434 amino acid residues (38%) identical to, and 235 and 434 amino acid residues (54%) similar to, the 567 amino acid residue ptnr:SPTREMBL-ACC:Q9QZU3 protein from Mus musculus (Mouse) (Platelet Glycoprotein V) (E=7.5e−73).
NOV21 is expressed in at least the following tissues: Mammary Gland/Breast. This information was derived by determining the tissue sources of the sequences that were included in the invention including but not limited to SeqCalling sources, Public EST sources, genomic clone sources, literature sources, and/or RACE sources.
Possible SNPs found for NOV21 are listed in Table 21C.







TABLE 21C







SNPs
















Amino





Nucleotide
Base
Acid
Base



Variant
Position
Change
Position
Change

















13377106
166
T > C
 11
Val > Ala



13377107
287
G > A
Silent
N/A



13377108
399
C > T
Silent
N/A



13377109
576
T > C
148
Cys > Arg



13377110
1423
A > G
430
Asp > Gly



13377111
1517
C > T
Silent
N/A



13377112
1595
A > G
Silent
N/A



13377113
1618
G > A
495
Ser > Asn



13377114
1749
C > T
Silent
N/A










NOV21 has homology to the amino acid sequences shown in the BLASTP data listed in Table 21D.







TABLE 21D







BLAST results for NOV21
















Pos-



Gene Index/
Protein/
Length
Identity
itives



Identifier
Organism
(aa)
(%)
(%)
Expect





gi|18554959|ref|XP_
similar to
787
510/581
510/
0.0


067453.2|
putative (H.

(87%)
581


(XM_067453)
sapiens)


(87%)



[Homo 



sapiens]


gi|18677767|ref|NP_
leucine-rich
581
508/581
508/
0.0


570843.1|
repeat

(87%)
581


(NM_130830)
protein


(87%)



induced by



beta



amyloid



[Homo



sapiens]


gi|18565266|dbj|
Lib [Rattus
578
426/581
462/
0.0


BAB 84586.1|
norvegicus]

(73%)
581


(AB071036)



(79%)


gi|12856544|dbj|
Leucine Rich
391
294/367
317/
 e−156


BAB 30702.1|
Repeat

(80%)
367


(AK017350)
containing


(86%)



protein-data



source:



Pfam,



source



key:



PF00560,



evidence:



ISS-puta



tive [Mus



musculus]


gi|6980974|ref|NP_0
platelete
567
157/452
224/
6e−61 


36927.1|
glyco-

(34%)
452


(NM_012795)
protein 5


(48%)



[Rattus



norvegicus]









The homology of these sequences is shown graphically in the ClustalW analysis shown in Table 21E.







TABLE 21E





Clustal W Sequence Alignment















1) NOV21 (SEQ ID NO:76)


2) gi 18554959|refXP_067453.2|(XM_067453) similar to putative (H. sapiens)


[Homo sapiens] (SEQ ID NO:224)


3) gi 18677767|refNP_570843.1|(NM_130830) leucine-rich repeat protein induced by beta


amyloid [Homo sapiens] (SEQ ID NO:225)


4) gi 18565266|dbj|BAB84586.1|(AM071036) Lib [Rattus norvegicus](SEQ ID NO:226)


5) gi 12856544|dbj|BAB30702.1|(AK017350) Leucine Rich Repeat containing protein ̃data


source: Pfam, source key:PF00560, evidence:ISS ̃putative [Mus musculus]


(SEQ ID NO:227)


6) gi 6980974|refNP_036927.1|(NM_012795) platelete glycoprotein 5 [Rattus norvegicus]


(SEQ ID NO:228)






















































































Table 21F lists the domain description from DOMAIN analysis results against NOV21. This indicates that the NOV21 sequence has properties similar to those of other proteins known to contain these domains.







TABLE 21F





Domain Analysis of NOV21
















gnl Smart|smart00082, LRRCT, Leucine rich repeat C-terminal



domain (SEQ ID NO:229)


CD-Length = 51 residues, 76.5% aligned


Score = 36.6 bits (83), Expect = 0.004


Query:   423  NPWRCDSDILPLRNWLLLNQPRLGTDTVPVCFSPANVRGQ  462


              ||+ || ++  |  ||  |+  |       | || ++|| 


Sbjct:     1  NPFICDCELRWLLRWLQANR-HLQDPVDLRCASPESLRGP   39









Following injury to a blood vessel, platelets rapidly adhere to the site of exposed subendothelial matrix in the vessel wall, become activated, and secrete granule contents that will recruit and activate other platelets, which then aggregate at the site (thrombus) and seal the defect in the vessel wall. The clot that forms prevents further blood loss (Lopez and Dong, Curr Opin Hematol 4(5):323-9, 1997). Cell-cell and cell-extracellular matrix adhesion are critical aspects of platelet function, regulating interactions between circulating platelets in the bloodstream with the blood vessel wall. The platelet glycoprotein Ib-IX-V complex plays crucial roles in both of these processes by mediating platelet adhesion to sites of blood vessel injury and by participating in the thrombin induced aggregation of platelets in both haemostasis and thrombosis under conditions of high shear blood flow (Lopez and Dong, 1997; Andrews and Berndt, Histol Histopathol 13(3):837-44, 1998). Emerging evidence suggests GP Ib-IX-V-dependent signalling may involve receptor cross-linking and the cytoplasmic signalling protein, 14-3-3 zeta. (Andrews and Berndt, 1998; Williamson et al., Aust N Z J Med 29(3):452-61, 1999).
In addition to this normal physiological response, platelet adhesion is critical in the pathological process of thrombosis, where circulating platelets adhere to sclerotic lesions or undergo shear-induced aggregation within vessels occluded by atherosclerotic plaque (Andrews and Berndt, 1998). Under these circumstances, the resulting thrombus may result in acute myocardial infarction or stroke. The critical role of platelets in the development of the acute coronary syndromes is now well recognised, and a great deal of effort has therefore focused on elucidating the key adhesion receptors mediating platelet-vessel wall and platelet-platelet interactions. The vascular adhesion protein von Willebrand factor (vWf) plays a key role in supporting platelet adhesion to the damaged vessel wall and binds to two adhesion receptors on the platelet surface, the glycoprotein (GP) Ib-V-IX complex and glycoprotein IIb-IIa (Lopez and Dong, 1997; Andrews et al., Thromb Haemost 82(2):357-64, 1999). The GP Ib-V-IX complex is a unique adhesion receptor which enables platelets to roll on a vWf matrix under conditions of rapid blood flow as well as transducing signals leading to the activation of GP IIb-IIIa (Andrews and Berndt, 1998).
Genetic defects of the blood platelet membrane glycoproteins, GPIIb-IIIa (alpha IIb/beta 3; CD41/CD61) and GPIb-V-IX (CD42) are the origin of several rare bleeding disorders, the best known of which are Glanzmann's thrombasthenia, Bernard-Soulier syndrome, and platelet-type von Willebrand's disease (Hayashi and Suzuki, Semin Thromb Hemost 26(1):53-9, 2000; Clemetson and Clemetson, Curr Opin Hematol 1(5):388-93, 1994). In Glanzmann's thrombasthenia, GPIIb-IIa are missing or defective and platelet aggregation is lacking or reduced. Either gene can be affected and mutations leading to lack of expression or to expression of poorly functional form have been described. In Bernard-Soulier syndrome, GPIb-V-IX are missing or defective, leading to poor platelet adhesion at high-shear stress to damaged vessel wall and reduced platelet response to thrombin. Mutations in both GPIb alpha (CD42b) and GPIX (CD42a) have been described. Mutations in GPIb alpha can also lead to platelet-type von Willebrand's disease in which GPIb-V-IX are expressed normally but bind von Willebrand's factor spontaneously, which leads to platelet aggregation and thrombocytopenia. (Clemetson and Clemetson, 1994). Given the homology of the novel protein in this invention to platelet glycoprotein V, which has a critical role in thrombosis, the novel protein in this invention is an excellent antibody target in thrombosis, arterial thrombotic disorders, bleeding disorders, stroke and atherosclerosis.
The NOV21 nucleic acid of the invention encoding a Platelet Glycoprotein V-like protein includes the nucleic acid whose sequence is provided in Table 21A, or a fragment thereof. The invention also includes a mutant or variant nucleic acid any of whose bases may be changed from the corresponding base shown in Table 21A while still encoding a protein that maintains its Platelet Glycoprotein V-like activities and physiological functions, or a fragment of such a nucleic acid. The invention further includes nucleic acids whose sequences are complementary to those just described, including nucleic acid fragments that are complementary to any of the nucleic acids just described. The invention additionally includes nucleic acids or nucleic acid fragments, or complements thereto, whose structures include chemical modifications. Such modifications include, by way of non-limiting example, modified bases, and nucleic acids whose sugar phosphate backbones are modified or derivatized. These modifications are carried out at least in part to enhance the chemical stability of the modified nucleic acid, such that they may be used, for example, as antisense binding nucleic acids in therapeutic applications in a subject. In the mutant or variant nucleic acids, and their complements, up to about 41% of the NOV21 residues may be so changed.
The NOV21 protein of the invention includes the Platelet Glycoprotein V-like protein whose sequence is provided in Table 21B. The invention also includes a mutant or variant protein any of whose residues may be changed from the corresponding residue shown in Table 21B while still encoding a protein that maintains its Platelet Glycoprotein V-like activities and physiological functions, or a functional fragment thereof. In the mutant or variant protein, up to about 62% of the NOV21 bases may be so changed.
The NOV21 nucleic acids and proteins of the invention are useful in potential diagnostic and therapeutic applications implicated in various diseases and disorders described below and/or other pathologies. For example, the compositions of the present invention will have efficacy for treatment of patients suffering from: cancer, fertility and other diseases, disorders and conditions of the like.
NOV21 nucleic acids and polypeptides are further useful in the generation of antibodies that bind immunospecifically to the novel substances of the invention for use in therapeutic or diagnostic methods. These antibodies may be generated according to methods known in the art, using prediction from hydrophobicity charts, as described in the “Anti-NOVX Antibodies” section below. For example the disclosed NOV21 protein have multiple hydrophilic regions, each of which can be used as an immunogen. This novel protein also has value in development of powerful assay system for functional analysis of various human disorders, which will help in understanding of pathology of the disease and development of new drug targets for various disorders.
NOV22
A disclosed NOV22 nucleic acid of 2189 nucleotides (also referred to as CG57529-01) encoding a novel GARPIN-like protein is shown in Table 22A. An open reading frame was identified beginning with an ATG initiation codon at nucleotides 10-12 and ending with a TGA codon at nucleotides 2056-2058. Putative untranslated regions upstream from the intitation codon and downstream from the termination codon are underlined in Table 22A, and the start and stop codons are in bold letters.







TABLE 22A





NOV22 Nucleotide Sequence
















(SEQ ID NO:77)









CCGGAGACGATGCGCCCCCCTTCAAGCCTTCTCATTATTGCTGTTCTCTGTGCACTGGCCAGGCAAGCGGGCT



GGCAACCACGTTTCGTTAACCACCTTTATTCTTGCCGAGGGCAGAGCCTCGCTTCGGTGCCCAGCAGCCTCCC


GCCCCACGCCCGGATGCTCACCCTGGATGCCAACCCTCTCAAGACCCTGTGGAATCACTCCCTCCAGCCTTAC


CCTCTCCTGGAGAGCCTCAGCCTGCACAGCTGCCACCTGGAGCGCATCAGCCGCGGCGCCTTCCAGGAGCAAG


GTCACCTGCGCAGCCTGGTCCTGGGGGACAACTGCCTCTCAGAGAACTACGAAGAGACGGCAGCCGCCCTCCA


CGCCCTGCCGGGCCTGCGGAGGCTGGACTTGTCAGGAAACGCCCTGACGGAGGACATGGCAGCCCTCATGCTC


CAGAACCTCTCCTCGCTGCGGTCCGTGTCCCTGGCGGGGAACACCATCATGCGGCTGGACGACTCCGTCTTCG


AGGGCCTGGAGCGTCTCCGGGAGCTGGATCTGCAGAGGAACTACATCTTCGAGATCGAGGGCGGCGCTTTCGA


CGGCCTGGCTGAGCTGAGGCACCTCAACCTGGCCTTCAACAACCTCCCCTGCATCGTGGACTTCGGGCTCACG


CGGCTGCGGGTCCTCAACGTCAGCTACAACGTCCTGGAGTGGTTCCTCGCGACCGGGGGAGAGGCTGCCTTCG


AGCTGGAGACGCTGGACCTGTCTCACAACCAGCTGCTGTTCTTCCCGCTGCTGCCCCAGTACAGCAAGTTGCG


GACCCTCCTGCTGCGCGACAACAACATGGGCTTCTACCGGGACCTGTACAACACCTCGTCGCCGAGGGAGATG


GTGGCCCAGTTCCTCCTCGTGGACGGCAACGTGACCAACATCACCACCGTCAGCCTCTGGGAAGAATTCTCCT


CCAGCGACCTCGCAGATCTCCGCTTCCTGGACATGAGCCAGAACCAGTTCCAGTACCTGCCAGACGGCTTCCT


GAGGAAAATGCCTTCCCTCTCCCACCTGAACCTCCACCAGAATTGCCTGATGACGCTTCACATTCGGGAGCAC


GAGCCCCCCGGAGCGCTCACCGAGCTGGACCTGAGCCACAACCAGCTGTCGGAGCTGCACCTGGCTCCGGGGC


TGGCCAGCTGCCTGGGCAGCCTGCGCTTGTTCAACCTGAGCTCCAACCAGCTCCTGGGCGTCCCCCCTGGCCT


CTTCGCCAATGCTAGGAACATCACTACACTTGACATGAGCCACAATCAGATCTCACTTTGTCCCCTGCCAGCT


GCCTCGGACCGGGTGGGCCCCCCTAGCTGTGTGGATTTCAGGAATATGGCATCTTTAAGGAGCCTGTCTCTGG


AGGGCTGTGGCCTGGGGGCATTGCCAGACTGCCCATTCCAAGGGACCTCCCTGACCTACTTAGACCTCTCAAG


CAACTGGGGGGTTCTGAATGGGAGCCTCGCCCCACTCCAGGATGTTGCCCCCATGTTACAGGTCCTGTCTCTC


AGGAACATGGGCCTCCACTCCAGCTTTATGGCGTTGGACTTCTCTGGGTTTGGGAATCTCAGGGACTTAGATC


TGTCGGGGAATTGCTTGACCACCTTCCCAAGGTTTGGGGGCAGCCTGGCCCTGGAGACCCTGGATCTCCGTAG


AAACTCGCTCACAGCCCTTCCCCAGAAGGCTGTGTCTGAGCAGCTCTCGAGAGGTCTGCGGACCATCTACCTC


AGTCAGAATCCATATGACTGCTGTGGGGTGGATGGCTGGGGGGCCCTGCAGCATGGGCAGACGGTGGCCGACT


GGGCCATGGTCACCTGCAACCTCTCCTCCAAGATCATCCGCGTGACGGAGCTGCCCGGAGGTGTGCCTCGGGA


CTGCAAGTGGGAGCGGCTGGACCTGGGCCTGCTCTACCTCGTGCTCATCCTCCCCAGCTGCCTCACCCTGCTG


GTGGCCTGCACTGTCATCGTCCTCACTTTTAAGAAGCCTCTGCTTCAGGTCATCAAGAGCCGCTGCCACTGGT


CCTCCGTTTACTGACCTGGCTGTGTGCCAAGACTCGAAATTCGGTCCGCACACAACAGGACACTTTCTCTGCC


AGCTTTCAAGATGTGATGCAGAGGCCAAGTCTGACGAATTGAAGTTTCAATTAAAATTTAATATGTTTCCAT









The NOV22 nucleic acid was identified on chromosome 3 and has 671 of 1132 bases (59%) identical to a gb:GENBANK-ID:HUMIGFACID|acc:M86826.1 mRNA from Homo sapiens (Human IGF binding protein complex acid-labile subunit a mRNA, complete cds) (E=2.5e−08).
A disclosed NOV22 polypeptide (SEQ ID NO:78) encoded by SEQ ID NO:77 is 682 amino acid residues and is presented using the one-letter code in Table 22B. Signal P, Psort and/or Hydropathy results predict that NOV22 contains a signal peptide and is likely to be localized to the plasma membrane with a certainty of 0.4600. The most likely cleavage site for a NOV22 polypeptide is between amino acids 22 and 23: AGW-QP.







TABLE 22B





Encoded NOV22 protein sequence
















(SEQ ID NO:78)









MRPPSSLLIIAVLCALARQAGWQPRFVNHLYSCRGQSLASVPSSLPPHARMLTLDANPLKTLWNHSLQPYPL



LESLSLHSCHLERISRGAFQEQGHLRSLVLGDNCLSENYEETAAALHALPGLRRLDLSGNALTEDMAALMLQ


NLSSLRSVSLAGNTIMRLDDSVFEGLERLRELDLQRNYIFEIEGGAFDGLAELRHLNLAFNNLPCIVDFGLT


RLRVLNVSYNVLEWFLATGGEAAFELETLDLSHNQLLFFPLLPQYSKLRTLLLRDNNMGFYRDLYNTSSPRE


MVAQFLLVDGNVTNITTVSLWEEFSSSDLADLRFLDMSQNQFQYLPDGFLRKMPSLSHLNLHQNCLMTLHIR


EHEPPGALTELDLSHNQLSELHLAPGLASCLGSLRLFNLSSNQLLGVPPGLFANARNITTLDMSHNQISLCP


LPAASDRVGPPSCVDFRNMASLRSLSLEGCGLGALPDCPFQGTSLTYLDLSSNWGVLNGSLAPLQDVAPMLQ


VLSLRNMGLHSSFMALDFSGFGNLRDLDLSGNCLTTFPRFGGSLALETLDLRRNSLTALPQKAVSEQLSRGL


RTIYLSQNPYDCCGVDGWGALQHGQTVADWAMVTCNLSSKIIRVTELPGGVPRDCKWERLDLGLLYLVLILP


SCLTLLVACTVIVLTFKKPLLQVIKSRCHWSSVY









The NOV22 amino acid sequence 253 of 660 amino acid residues (38%) identical to, and 341 of 660 amino acid residues (51%) similar to, the 662 amino acid residue ptnr:SWISSNEW-ACC:Q14392 protein from Homo sapiens (Human) (GARP Protein Precursor (GARPIN) (Glycoprotein A Repetitions Predominant) (E=1.7e−82).
NOV22 is expressed in at least the following tissues: ovary, colon, breast and testis. This information was derived by determining the tissue sources of the sequences that were included in the invention including but not limited to SeqCalling sources, Public EST sources, genomic clone sources, literature sources, and/or RACE sources and because of the expression pattern of (gb:GENBANK-ID:HUMIGFACID|acc:M86826.1) a closely related Human IGF binding protein complex acid-labile subunit a mRNA, complete cds homolog in species Homo sapiens. 
Possible SNPs found for NOV22 are listed in Table 22C.







TABLE 22C







SNPs
















Amino





Nucleotide
Base
Acid
Base



Variant
Position
Change
Position
Change







13377115
1038
C > T
Silent
N/A










NOV22 has homology to the amino acid sequences shown in the BLASTP data listed in Table 22D.







TABLE 22D







BLAST results for NOV22
















Pos-



Gene Index/
Protein/
Length
Identity
itives



Identifier
Organism
(aa)
(%)
(%)
Expect





gi/18599627|ref|XP_
hypothetical
1558
617/658
621/
 0.0


093473.1|
protein

(93%)
658


(XM_093473)
XP_093473


(93%)



[Homo



sapiens]


gi|5031707|ref|NP_0
glycoprotein
 662
229/616
305/
2e−70


05503.1|
A repetitions

(37%)
616


(NM_005512)
predominant


(49%)



precursor;



garpin



[Homo



sapiens]


gi|7287886|gb|
hypothetical
1471
146/505
217/
3e−22


AAF44 924.1|
protein

(28%)
505


AE003406_129
[Drosophila


(42%)


(AE003416)
melano-



gaster]


gi|10728795|gb|
BG:
1216
146/505
217/
7e−22


AAF5 3442.2|
DS03192.2

(28%)
505


(AE003646)
gene product


(42%)



[Drosophila



melano-



gaster]


gi|7301872|gb|
CG7896
1348
145/555
230/
3e−20


AAF56 980.1|
gene product

(26%)
555


(AE003772)
[Drosophila


(41%)



melano-



gaster]









The homology of these sequences is shown graphically in the ClustalW analysis shown in Table 22E.







TABLE 22E





Clustal W Sequence Alignment















1) NOV22 (SEQ ID NO:78)


2) gi 18599627|refXP_093473.1|(XM_093473) hypothetical protein XP_093473


[Homo sapiens] (SEQ ID NO:230)


3) gi 5031707|refNP_005503.1|(NM_005512) glycoprotein A repetitions predominant


precursor; garpin [Homo sapiens] (SEQ ID NO:231)


4) gi 7287886|gb AAF44924.1 AE003406_129 (AE003416) hypothetical protein


[Drosophila melanogaster] (SEQ ID NO:232)


5) gi 10728795|gb AAF53442.2|(AE003646) BG:DS03192.2 gene product


[Drosophila melanogaster] (SEQ ID NO:233)


6) gi 7301872|gb AAF56980.1|(AE003772) CG7896 gene product [Drosophila melanogaster]


(SEQ ID NO:234)










































































































































































The NOV22 protein described herein belongs to the leucine-rich repeat protein family. It is homologous to GARPIN, insulin-like growth factor-binding protein, and platelet glycoprotein V. The leucine-rich repeat (INTERPRO IPR001611) is a recently characterized structural motif used in molecular recognition processes as diverse as signal transduction, cell adhesion, cell development, DNA repair and RNA processing. All proteins containing these repeats are thought to be involved in protein-protein interactions (Deisenhofer and Kobe, Trends Biochem. Sci. 19: 415-421, 1994).
Ollendorff et al. reported a new human gene, named GARP, encodes a putative transmembrane protein of 662 amino acids, the extracellular portion of which is almost entirely made of leucine-rich repeats. The molecular weight of the protein immunoprecipitated from transfected cells is 80,000. The GARP protein has structural similarities with the human GP Ib alpha and GP V platelet proteins, and with the Chaoptin, Toll, and Connectin adhesion molecules of Drosophila (Ollendorff et al., Cell Growth Differ 5(2):213-9, 1994).
Human platelet glycoprotein (GP) V, is a part of the Ib-V-IX system of surface glycoproteins (GPs Ib alpha, Ib beta, V, IX) that constitute the receptor for von Willebrand factor (vWf) and mediate the adhesion of platelets to injured vascular surfaces in the arterial circulation, a critical initiating event in hemostasis. System members share physical associations, leucine-rich glycoprotein (LRG) structures, and a congenital deficiency state, Bernard-Soulier syndrome (Hickey et al., Proc Natl Acad Sci U S A 90(18):8327-31, 1993). A 16-amino acid signal peptide is present. Mature GP V is a 544-amino acid transmembrane protein with a 504-amino acid extracellular domain that encompasses a set of 15 tandem LRG repeats in a “flank-LRG center-flank” array (Roth, Blood 77, 5-19, 1991) along with eight putative N-linked glycosylation sites and cleavage sites for thrombin and calpain. GP V is a transmembrane, adhesive LRG protein that plays an undefined, but potentially critical, role in the expression and/or function of the Ib-V-IX receptor for vWf/shear-dependent platelet adhesion in arteries (Hickey et al., Proc Natl Acad Sci U S A 90(18):8327-31, 1993).
Nearly all of the insulin-like growth factor (IGF) in the circulation is bound in a heterotrimeric complex composed of IGF, IGF-binding protein-3, and the acid-labile subunit (ALS). Full-length clones encoding ALS have been isolated from human liver cDNA libraries by using probes based on amino acid sequence data from the purified protein. These clones encode a mature protein of 578 amino acids preceded by a 27-amino acid hydrophobic sequence indicative of a secretion signal. Expression of the cDNA clones in mammalian tissue culture cells results in the secretion into the culture medium of ALS activity that can form the expected complex with IGF-I and IGF-binding protein-3. The amino acid sequence of ALS is largely composed of 18-20 leucine-rich repeats of 24 amino acids. These repeats are found in a number of diverse proteins that, like ALS, participate in protein-protein interactions (Leong et al., Mol Endocrinol 6(6):870-6, 1992).
Because of the presence of the Leucine rich repeat domains and the homology to the GARPIN, platelet glycoprotein (GP) V, and IGFBP, it is anticipated that NOV22 described here will have useful properties and functions similar to these genes.
The NOV22 nucleic acid of the invention encoding a GARPIN-like protein includes the nucleic acid whose sequence is provided in Table 22A, or a fragment thereof. The invention also includes a mutant or variant nucleic acid any of whose bases may be changed from the corresponding base shown in Table 22A while still encoding a protein that maintains its GARPIN-like activities and physiological functions, or a fragment of such a nucleic acid. The invention further includes nucleic acids whose sequences are complementary to those just described, including nucleic acid fragments that are complementary to any of the nucleic acids just described. The invention additionally includes nucleic acids or nucleic acid fragments, or complements thereto, whose structures include chemical modifications. Such modifications include, by way of non-limiting example, modified bases, and nucleic acids whose sugar phosphate backbones are modified or derivatized. These modifications are carried out at least in part to enhance the chemical stability of the modified nucleic acid, such that they may be used, for example, as antisense binding nucleic acids in therapeutic applications in a subject. In the mutant or variant nucleic acids, and their complements, up to about 41% of the NOV22 residues may be so changed.
The NOV22 protein of the invention includes the GARPIN-like protein whose sequence is provided in Table 22B. The invention also includes a mutant or variant protein any of whose residues may be changed from the corresponding residue shown in Table 22B while still encoding a protein that maintains its GARPIN-like activities and physiological functions, or a functional fragment thereof. In the mutant or variant protein, up to about 62% of the NOV22 bases may be so changed.
The NOV22 nucleic acids and proteins of the invention are useful in potential diagnostic and therapeutic applications implicated in various diseases and disorders described below and/or other pathologies. For example, the compositions of the present invention will have efficacy for treatment of patients suffering from: fertility, hypogonadism, endometriosis and other diseases, disorders and conditions of the like.
NOV22 nucleic acids and polypeptides are further useful in the generation of antibodies that bind immunospecifically to the novel substances of the invention for use in therapeutic or diagnostic methods. These antibodies may be generated according to methods known in the art, using prediction from hydrophobicity charts, as described in the “Anti-NOVX Antibodies” section below. For example the disclosed NOV22 protein have multiple hydrophilic regions, each of which can be used as an immunogen. This novel protein also has value in development of powerful assay system for functional analysis of various human disorders, which will help in understanding of pathology of the disease and development of new drug targets for various disorders.
NOV23
A disclosed NOV23 nucleic acid of 2484 nucleotides (also referred to as CG57351-01) encoding a novel Centaurin Beta 2-like protein is shown in Table 23A. An open reading frame was identified beginning with an ATG initiation codon at nucleotides 15-17 and ending with a TAG codon at nucleotides 2307-2309. Putative untranslated regions upstream from the intitation codon and downstream from the termination codon are underlined in Table 23A, and the start and stop codons are in bold letters.







TABLE 23A





NOV23 Nucleotide Sequence
















(SEQ ID NO:79)









CGGCCGCCGCAGCCATGACCGTGGAGTTCGAGAAGTGCGTCAAGAACTCCCCGCGCTTCAGGGCGACCATTGA



CGAGGTGGAGACGGACGTGGTGGAGATTGAGGCCAAACTGGACAAGCTGGTGAAGCTGTGCAGTGGCATGGTG


GAAGCCGGTAAGGCCTACGTCAGCACCAGCAGGCTTTTCGTGAGCGGCGTCCGCGACCTGTCCCAGCAGTGCC


AGGGCGACACCGTCATCTCGGAATGTCTGCAGAGGTTCGCTGACAGCCTACAGGAGGTGGTGAACTACCACAT


GATCCTGTTTGACCAGGCCCAGAGGTCCGTGCGGCAGCAGCTCCAGAGCTTTGTCAAAGAGGATGTGCGGAAG


TTCAAGGAGACAAAGAAGCAGTTTGACAAGGTGCGGGAGGACCTGGAGCTGTCCCTGGTGAGGAACGCCCAGG


CCCCGAGGCACCGGCCCCACGAGGTGGAGGAAGCCACCGGGGCCCTCACCCTCACCAGGAAGTGCTTCCGCCA


CCTGGCACTGGACTATGTGCTCCAGATCAATGTTCTGCAGGCCAAGAAGAAGTTTGAGATCCTGGACTCTATG


CTGTCCTTCATGCACGCCCAGTCCAGCTTCTTCCAGCAGGGCTACAGCCTCCTGCACCAGCTGGACCCCTACA


TGAAGAAGCTGGCAGCCGAGCTGGACCAGCTGGTGATCGACTCTGCGGTGGAAAAGCGTGAGATGGAGCGAAA


GCACGCCGCCATCCAGCAGCGGACCCTTAGGGACTTCTCCTACGATGAGTCCAAAGTGGAGTTTGACGTGGAC


GCGCCCAGTGGGGTGGTGATGGAGGGCTACCTCTTCAAGAGGGCCAGCAACGCTTTCAAGACATGGAACCGGC


GCTGGTTCTCCATTCAGAACAGCCAGCTGGTCTACCAGAAGAAGCTCAAGGATGCCCTCACCGTGGTGGTGGA


TGACCTCCGCCTGTGCTCTGTGAAGCCGTGTGAGGACATCGAGCGGAGGTTCTGCTTCGAGGTGCTGTCACCC


ACCAAGAGCTGCATGCTGCAGGCTGACTCCGAGAAGCTGCGGCAAGCCTGGGTCCAGGCTGTGCAGGCCAGCA


TCGCCTCCGCCTACCGCGAGAGCCCTGACAGTTGCTATAGCGAGAGGCTGGACCGCACAGCATCCCCGTCCAC


GAGCAGCATCGACTCCGCCACCGACACTCGGGAGCGTGGCGTGAAGGGCGAGAGTGTGCTGCAGCGTGTGCAG


AGTGTGGCCGGCAACAGCCAGTGCGGCGACTGCGGCCAGCCGGACCCCCGCTGGGCCAGCATCAACCTGGGCG


TGCTGCTCTGCATTGAGTGCTCCGGCATCCACAGGAGCCTGGGTGTCCACTGCTCCAAGGTGCGGTCCCTGAC


GCTGGACTCGTGGGAGCCTGAGCTGCTAAAGCTGATGTGTGAGCTTGGAAACAGCGCTGTGAATCAGATCTAT


GAGGCCCAGTGTGAGGGTGCAGGCAGCAGGAAACCCACAGCCAGCAGCTCCCGGCAGGACAAGGAGGCCTGGA


TCAAGGACAAATACGTGGAAAAGAAGTTTCTGCGGAAGGCGCCCATGGCACCAGCCCTGGAGGCCCCAAGACG


CTGGAGGGTGCAGAAGTGCCTGCGGCCCCACAGCTCTCCCCGCGCTCCCACTGCCCGCCGCAAGGTCCGGCTT


GAGCCCGTTCTGCCCTGTGTGGCCGCTCTGTCCTCAGAGGGTGCAGAGTCGGAGGAGTCCAGCGGTGAGGCAG


ACGGGGACACTGAGGCCGAGGCCTGGGGCCTGGCGGACGTGCGCGAGCTGCACCCGGGGCTCTTGGCGCACCG


CGCAGCGCGTGCCCGCGACCTTCCTGCGCTGGCGGCGGCGCTGGCCCACGGGGCCGAGGTCAACTGGGCGGAC


GCGGAGGATGAGGGCAAGACGCCGCTGGTGCAGGCCGTGCTAGGGGGCTCCTTGATCGTCTGTGAGTTCCTGC


TGCAAAACGGAGCGGACGTGAACCAAAGAGACAGCCGGGGCCGGGCGCCCCTGCACCACGCCACGCTGCTGGG


CCGCACCGGCCAGGTTTGCCTGTTCCTGAAGCGGGGCGCGGACCAGCACGCCCTGGACCAAGAGCAGCGGGAC


CCGTTGGCCATCGCAGTGCAGGCGGCCAACGCTGACATCGTGACACTGCTCCGTCTGGCGCGCATGGCGGAGG


AAATGCGCGAGGCCGAGGCTGCCCCTGGTCCCCCGGGCGCCCTGGCGGGCAGCCCCACGGAGCTCCAGTTCCG


CAGGTGTATCCAGGAGTTCATCAGCCTCCACCTGGAAGAGAGCTAGGGCCGGGCAGGCCGGGCAGCTGCCACC


CCGCCCGGCCCGACGCCCCGCATGCCCCGAAGTCCCTGGCGCCCACCCGGCCGCGGCCCTGCGTGTGACCCGC


GGGTCGATACCTGGCAGCCCCAGTGCTGGGGCGCCGCGGCCCTGCTCGCCCAGGAGGAGAGCGAGGGCCCCAC


AC









The NOV23 nucleic acid was identified on chromosome 1p36.33 and has 608 of 891 bases (68%) identical to a gb:GENBANK-ID:HSA238248|acc:AJ238248.1 mRNA from Homo sapiens (Homo sapiens mRNA for centaurin beta2) (E=5.9e−149).
A disclosed NOV23 polypeptide (SEQ ID NO:80) encoded by SEQ ID NO:79 is 764 amino acid residues and is presented using the one-letter code in Table 23B. Signal P, Psort and/or Hydropathy results predict that NOV23 contains a signal peptide and is likely to be localized to the plasma membrane with a certainty of 0.7000 and to the nucleus with a certainty of 0.6000.







TABLE 23B





Encoded NOV23 protein sequence
















(SEQ ID NO:80)









MTVEFEKCVKNSPRFRATIDEVETDVVEIEAKLDKLVKLCSGMVEAGKAYVSTSRLFVSGVRDLSQQCQGDT



VISECLQRFADSLQEVVNYHMILFDQAQRSVRQQLQSFVKEDVRKFKETKKQFDKVREDLELSLVRNAQAPR


HRPHEVEEATGALTLTRKCFRHLALDYVLQINVLQAKKKFEILDSMLSFMHAQSSFFQQGYSLLHQLDPYMK


KLAAELDQLVIDSAVEKREMERKHAAIQQRTLRDFSYDESKVEFDVDAPSGVVMEGYLFKRASNAFKTWNRR


WFSIQNSQLVYQKKLKDALTVVVDDLRLCSVKPCEDIERRFCFEVLSPTKSCMLQADSEKLRQAWVQAVQAS


IASAYRESPDSCYSERLDRTASPSTSSIDSATDTRERGVKGESVLQRVQSVAGNSQCGDCGQPDPRWASINL


GVLLCIECSGIHRSLGVHCSKVRSLTLDSWEPELLKLMCELGNSAVNQIYEAQCEGAGSRKPTASSSRQDKE


AWIKDKYVEKKFLRKAPMAPALEAPRRWRVQKCLRPHSSPRAPTARRKVRLEPVLPCVAALSSEGAESEESS


GEADGDTEAEAWGLADVRELHPGLLAHRAARARDLPALAAALAHGAEVNWADAEDEGKTPLVQAVLGGSLIV


CEFLLQNGADVNQRDSRGRAPLHHATLLGRTGQVCLFLKRGADQHALDQEQRDPLAIAVQAANADIVTLLRL


ARMAEEMREAEAAPGPPGALAGSPTELQFRRCIQEFISLHLEES









The NOV23 amino acid sequence 456 of 744 amino acid residues (61%) identical to, and 576 of 744 amino acid residues (77%) similar to, the 778 amino acid residue ptnr:SPTREMBL-ACC:Q9UQR3 protein from Homo sapiens (Human) (CENTAURIN BETA2) (E=3.4e−237).
NOV23 is expressed in at least the following tissues: Brain, mammary gland/breast, pituitary gland, germ cell, ovary, testis and muscle. This information was derived by determining the tissue sources of the sequences that were included in the invention including but not limited to SeqCalling sources, Public EST sources, genomic clone sources, literature sources, and/or RACE sources.
Possible SNPs found for NOV23 are listed in Table 23C.







TABLE 23C







SNPs
















Amino





Nucleotide
Base
Acid
Base



Variant
Position
Change
Position
Change

















13377116
1390
T > C
459
Leu > Pro



13376624
1464
G > A
484
Ala > Thr



13376623
1480
C > T
489
Ala > Val



13376622
1576
C > T
521
Pro > Leu










NOV23 has homology to the amino acid sequences shown in the BLASTP data listed in Table 23D.







TABLE 23D







BLAST results for NOV23
















Pos-



Gene Index/
Protein/
Length
Identity
itives



Identifier
Organism
(aa)
(%)
(%)
Expect





gi|12697977|dbj|
KIAA1716
804
665/770
666/
0.0


BAB 21807.1|
protein

(86%)
770


(AB051503)
[Homo


(86%)



sapiens]


gi|16945966|ref|NP_
centaurin,
759
654/759
655/
0.0


085152.1|
beta 5 

(86%)
759


(NM_030649)
[Homo


(86%)



sapiens]


gi|174348171|ref|XP_
KIAA1716
834
547/566
550/
0.0


027698.2|
protein

(96%)
566


(XM_027698)
[Homo


(96%)



sapiens]


gi|17977656|ref|NP_
centaurin,
778
328/519
410/
0.0


036419.1|
beta 2;

(63%)
519


(NM_012287)
centaurin


(78%)



beta2; Arf



GAP with



coiled coil,



ANK repeat



and PH



domains 2 



[Homo



sapiens]


gi|7661880|ref|NP_0
centaurin
740
350/728
454/
e−168


55531.1|
betal;

(48%)
728


(NM_014716)
KIAA0050


(62%)



gene



product;



Arf GAP



with coiled



coil, ANK



repeat and



PH domains



1 [Homo



sapiens]









The homology of these sequences is shown graphically in the ClustalW analysis shown in Table 23E.







TABLE 23E





Clustal W Sequence Alignment















1) NOV23 (SEQ ID NO:80)


2) gi 12697977|dbj|BAB21807.1|(AB051503) KIAA1716 protein [Homo sapiens]


(SEQ ID NO:235)


3) gi 16945966|ref|NP_085152.1|(NM_030649) centaurin, beta 5 [Homo sapiens]


(SEQ ID NO:236)


4) gi 17434817|ref|XP_027698.2|(XM_027698) KIAA1716 protein [Homo sapiens]


(SEQ ID NO:237)


5) gi 17977656|ref|NP_036419.1|(NM_012287) centaurin, beta 2; centaurin beta2; Arf


GAP with coiled coil, ANK repeat and PH domains 2 [Homo sapiens]


(SEQ ID NO:238)


6) gi 7661880|refNP_055531.1|(NM_014716) centaurin beta1; KIAA0050 gene product; Arf


GAP with coiled coil, ANK repeat and PH domains 1 [Homo sapiens]


(SEQ ID NO:239)




































































































Tables 23F, 23G and 23H list the domain description from DOMAIN analysis results against NOV23. This indicates that the NOV23 sequence has properties similar to those of other proteins known to contain these domains.







TABLE 23F





Domain Analysis of NOV23















gnl Pfam:pfam01412, ArfGap, Putative GTP-ase activating protein for


Arf. Putative zinc fingers with GTPase activating proteins (GAPs)


towards the small GTPase, Arf. The GAP of ARD1 stimulates GTPase


hydrolysis for ARD1 but not ARFs. (SEQ ID NO:240)


CD-Length = 122 residues, 97.5% aligned


Score = 153 bits (387), Expect = 3e−38










Query:
402
ESVLQRVQSVAGNSQCGDCGQPDPRWASINLGVLLCIECSGIHRSLGVHCSKRVSLTLDS
461




  ||+ ++|+ || +| ||| |+| |||+|||| +|||||||||||||| |||||||||+


Sbjct:
1
RRVLKLLRSLDGNKKCFDCGAPNPTWASVNLGVFICIECSGIHRSLGVHISKVRSLTLDT
 60


Query:
462
WEPELLKLMCELGNSAVNQIYEAQCEGAGSRKPTASSSRQDKEAWIKLKYVEKKFLRKAP
521




| || |+ | + ||   |  +|+      | ||  |  ||  |++|  || || |+ |


Sbjct:
61
WTPEELRKMEKGGNENANSFWESN-LDDFSLKPKDSDDRQKYESFIAAKYEEKLFVLKEG
119
















TABLE 23G





Domain Analysis of NOV23















gnl Smart|smart00233, PH, Pleckstrin homology domain.; Domain commonly


found in eukaryotic signalling proteins. The domain family possesses


multiple functions including the abilities to bind inositol


phosphates, and various proteins. PH domains have been found to


possess inserted domains (such as in PLC gamma, syntrophins) and to be


inserted within other domains. Mutations in Brutons tyrosine kinase


(Btk) within its PH domain cause X-linked agammaglobulinaemia (XLA) in


patients. Point mutations cluster into the positively charged end of


the molecule around the predicted binding site for


phosphatidylinositol lipids. (SEQ ID NO:241)


CD-Length = 104 residues, 100.0% aligned


Score = 66.6 bits (161), Expect = 5e−12










Query:
268
VVMEGYLFKRASNAFKTWNRRWFSIQNSQLVYQKKLKDAL------TVVVDDLRLCSVKP
321




|+ ||+| |++|   |+| +|+| + |  |+| |  |         ++ +    +


Sbjct:
1
VIKEGWLLKKSSGGKKSWKKRYFVLFNGVLLYYKSKKKKSSSKPKGSIPLSGCTVREAPD
 60


Query:
322
CEDIERRFCFEVLSPTKSCM-LQADSEKLRQAWVQAVQASIASA
364




 +  +++ |||+++| +  + |||+||+ |+ ||+|++ +||


Sbjct:
61
SDSDKKKNCFEIVTPDRKTLLLQAESEEERKEWVEALRKAIAKL
104
















TABLE 23H





Domain Analysis of NOV23







gnl|Pfam pfam00023, ank, Ank repeat. Ankyrin


repeats generally consist of a beta, alpha, alpha,


beta order of secondary structures. The repeats


associate to form a higher order structure.


(SEQ ID NO:242)


CD-Length = 33 residues, 97.0% aligned


Score = 45.1 bits (105), Expect = 2e−05










Query:
632
EGKTPLVQAVLGGSLIVCEFLLQNGADVNQRD
663




+| |||  |   | | | + ||+ ||||| ||


Sbjct:
1
DGNTPLHLAARNGHLEVVKILLEAGADVNARD
 32









Phosphoinositide (PI) 3-kinases play a role in the regulation of cell growth and survival, metabolism, transcription, vesicular trafficking and cytoskeletal organization. Receptor-regulated (class I) PI 3-kinases phosphorylate phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2] to phosphatidylinositol 3,4,5-trisphosphate [PtdIns(3,4,5)P3]. Proteins in the centaurin family are regulated by phosphoinositide and are regulatory proteins for Arfs (ADP ribosylation factors) (Jackson et al., Trends Biochem. Sci. 25: 489-95, 2000). Arfs are small GTP-binding proteins that function in vesicular trafficking and cytoskeletal regulation. Centaurin family members are GTPase-activating proteins (GAPs) for Arf or have homology to Arf GAPs and Arf effectors. ARFs are essential and ubiquitous in eukaryotes, being involved in vesicular transport and functioning as activators of phospholipase D. The functions of ARF proteins in membrane traffic and organelle integrity are intimately tied to their reversible association with membranes and specific interactions with membrane phospholipids.
The NOV23 protein described herein is predicted to share the attributes of the other centaurin family members and is thus implicated in signal transduction pathways that regulate cell growth and survival, metabolism and transcription. Its potential role in the regulation of ARF activity suggests that the centaurin beta 2-like protein may play an important role in vesicular trafficking and cytoskeletal regulation. Therefore, the NOV23 protein is an attractive target for drug intervention in the treatment of human metabolic diseases, central nervous system disorders, and cancer, among others.
The NOV23 nucleic acid of the invention encoding a Centaurin Beta 2-like protein includes the nucleic acid whose sequence is provided in Table 23A, or a fragment thereof. The invention also includes a mutant or variant nucleic acid any of whose bases may be changed from the corresponding base shown in Table 23A while still encoding a protein that maintains its Centaurin Beta 2-like activities and physiological functions, or a fragment of such a nucleic acid. The invention further includes nucleic acids whose sequences are complementary to those just described, including nucleic acid fragments that are complementary to any of the nucleic acids just described. The invention additionally includes nucleic acids or nucleic acid fragments, or complements thereto, whose structures include chemical modifications. Such modifications include, by way of non-limiting example, modified bases, and nucleic acids whose sugar phosphate backbones are modified or derivatized. These modifications are carried out at least in part to enhance the chemical stability of the modified nucleic acid, such that they may be used, for example, as antisense binding nucleic acids in therapeutic applications in a subject. In the mutant or variant nucleic acids, and their complements, up to about 32% of the NOV23 residues may be so changed.
The NOV23 protein of the invention includes the Centaurin Beta 2-like protein whose sequence is provided in Table 23B. The invention also includes a mutant or variant protein any of whose residues may be changed from the corresponding residue shown in Table 23B while still encoding a protein that maintains its Centaurin Beta 2-like activities and physiological functions, or a functional fragment thereof. In the mutant or variant protein, up to about 39% of the NOV23 bases may be so changed.
The NOV23 nucleic acids and proteins of the invention are useful in potential diagnostic and therapeutic applications implicated in various diseases and disorders described below and/or other pathologies. For example, the compositions of the present invention will have efficacy for treatment of patients suffering from: cancer, trauma, bacterial and viral infections, in vitro and in vivo regeneration, Von Hippel-Lindau (VHL) syndrome, Alzheimer's disease, stroke, tuberous sclerosis, hypercalceimia, Parkinson's disease, Huntington's disease, cerebral palsy, epilepsy, Lesch-Nyhan syndrome, multiple sclerosis, ataxia-telangiectasia, leukodystrophies, behavioral disorders, addiction, anxiety, pain, neurodegeneration endocrine dysfunctions, diabetes, obesity, growth and reproductive disorders, endometriosis, fertility, hypogonadism, muscular dystrophy, Lesch-Nyhan syndrome, and myasthenia gravis and other diseases, disorders and conditions of the like.
NOV23 nucleic acids and polypeptides are further useful in the generation of antibodies that bind immunospecifically to the novel substances of the invention for use in therapeutic or diagnostic methods. These antibodies may be generated according to methods known in the art, using prediction from hydrophobicity charts, as described in the “Anti-NOVX Antibodies” section below. For example the disclosed NOV23 protein have multiple hydrophilic regions, each of which can be used as an immunogen. This novel protein also has value in development of powerful assay system for functional analysis of various human disorders, which will help in understanding of pathology of the disease and development of new drug targets for various disorders.
NOV24
A disclosed NOV24 nucleic acid of 1930 nucleotides (also referred to as CG57515-01) encoding a novel Sorting Nexin 9-like protein is shown in Table 24A. An open reading frame was identified beginning with an ATG initiation codon at nucleotides 32-34 and ending with a TAA codon at nucleotides 1904-1906. Putative untranslated regions upstream from the intitation codon and downstream from the termination codon are underlined in Table 24A, and the start and stop codons are in bold letters.







TABLE 24A





NOV24 Nucleotide Sequence















(SEQ ID NO:81)







AGTCGGGACCGCCAGTCGGGGCGCCGGGACCATGGCGCTGCGCGCCCGGGCGCTGTACGACTTCAGGTCGGAG


AACCCAGGAGAGATCTCGCTGCGAGAGCACGAGGTGCTGAGCCTGTGCAGCGAGCAGGACATCGAGGGCTGGC


TCGAGGGGGTCAACAGCCGCGGCGACCGCGGCCTCTTCCCGGCCTCCTATGTGCAGGTGATCCGCGCCCCCGA


GCCTGGCCCGGCGGGAGACGGCGGCCCGGGCGCCCCGGCCCGCTACGCCAATGTGCCCCCCGGGGGCTTCGAG


CCCCTGCCCGTCGCGCCCCCCGCCTCCTTCAAGCCGCCGCCTGACGCCTTCCAGGCGCTGCTGCAGCCACAGC


AGGCGCCGCCTCCGAGCACCTTCCAGCCGCCCGGCGCGGGCTTCCCGTACGGCGGGGGCGCCCTGCAGCCGTC


GCCTCAGCAGCTCTACGGCGGCTACCAGGCCAGCCAAGGCAGCGATGATGACTGGGACGACGAGTGGGACGAC


AGCTCCACGGTGGCGGACGAGCCGGGCGCTCTGGGCAGCGGAGCATACCCGGACCTCGACGGCTCGTCTTCGG


CGGGTGTGGGCGCAGCCGGCCGCTACCGCCTGTCCACGCGCTCCGACCTGTCCCTGGGCTCCCGCGGCGGCTC


GGTCCCCCCGCAGCACCACCCGTCGGGGCCCAAGAGCTCGGCCACCGTGAGCCGCAACCTCAATCGCTTCTCC


ACCTTCGTCAAGTCCGGCGGGGAGGCCTTCGTGCTGGGGGAGGCGTCAGGCTTCGTGAAGGACGGGGACAAGC


TGTGCGTGGTGCTGGGGCCCTATGGCCCCGAGTGGCAGGAGAACCCCTACCCGTTCCAGTGCACCATCGACGA


CCCCACCAAGCAGACCAAGTTCAAGGGCATGAAGAGCTACATCTCCTACAAGCTGGTGCCCACGCACACGCAG


GTGCCGGTGCATCGGCGCTACAAGCACTTCGACTGGCTGTACGCGCGCCTGGCGGAGAAGTTCCCGGTCATCT


CCGTGCCCCACCTGCCCGAGAAGCAGGCCACCGGCCGCTTCGAGGAGGACTTCATCTCTAAGCGCAGGAAGGG


CCTGATCTGGTGGATGAACCACATGGCCAGCCACCCAGTGCTGGCGCAGTGCGACGTCTTCCAGCACTTCCTG


ACGTGCCCCAGCAGCACCGACGAGAAAGCCTGGAAGCAGGGCAAGAGGAAGGCCGAGAAGGACGAGATGGTGG


GCGCCAACTTCTTCCTGACCCTTAGCACGCCCCCCGCCGCTGCCCTTGACCTGCAGGAGGTGGAGAGCAAGAT


AGACGGCTTCAAGTGCTTCACCAAGAAGATGGACGACAGCGCGCTGCAGCTCAACCACACGGCCAACGAGTTC


GCGCGCAAGCAGGTGACCGGCTTCAAAAAGGAGTATCAGAAGGTGGGCCAGTCCTTCCGCGGCCTCAGCCAGG


CCTTTGAGCTGGACCAGCAGGCCTTCTCGGTGGGCCTGAACCAGGCTATCGCCTTCACCGGAGATGCCTATGA


CGCCATTGGCGAGCTCTTCGCGGAGCAGCCCAGGCAGGACCTGGATCCCGTCATGGACCTATTAGCGCTGTAT


CAGGGGCATCTGGCTAACTTCCCGGACATCATCCACGTTCAGAAAGGAGCTCTTACCAAAGTCAAGGAGAGTA


GGCGACACGTGGAGGAAGGGAAGATGGAGGTGCAGAAGGCTGACGGCATTCAGGATCGCTGTAACACTATTTC


TTTTGCCACTTTGGCTGAAATTCACCACTTCCATCAAATTCGAGTGAGAGACTTTAAATCACAGATGCAGCAT


TTCTTACAACAACAAATAATATTTTTCCAAAAAGTTACCCAGAAGTTGGAAGAAGCTCTTCACAAATATGATA


GTGTTTAATGACTGGACGTTGGATTATGGACT









The NOV24 nucleic acid was identified on chromosome 5 and has 275 of 435 bases (63%) identical to a gb:GENBANK-ID:AF121859|acc:AF121859.1 mRNA from Homo sapiens (Homo sapiens sorting nexin 9 (SNX9) mRNA, complete cds) (E=1.7e−14).
A disclosed NOV24 polypeptide (SEQ ID NO:82) encoded by SEQ ID NO:81 is 624 amino acid residues and is presented using the one-letter code in Table 24B. Signal P, Psort and/or Hydropathy results predict that NOV24 contains a signal peptide and is likely to be localized to the cytoplasm with a certainty of 0.7284.







TABLE 24B





Encoded NOV24 protein sequence















(SEQ ID NO:82)







MALRARALYDFRSENPGEISLREHEVLSLCSEQDIEGWLEGVNSRGDRGLFPASYVQVIRAPEPGPAGDGGP


GAPARYANVPPGGFEPLPVAPPASFKPPPDAFQALLQPQQAPPPSTFQPPGAGFPYGGGALQPSPQQLYGGY


QASQGSDDDWDDEWDDSSTVADEPGALGSGAYPDLDGSSSAGVGAAGRYRLSTRSDLSLGSRGGSVPPQHHP


SGPKSSATVSRNLNRFSTFVKSGGEAFVLGEASGFVKDGDKLCVVLGPYGPEWQENPYPFQCTIDDPTKQTK


FKGMKSYISYKLVPTHTQVPVHRRYKHFDWLYARLAEKFPVISVPHLPEKQATGRFEEDFISKRRKGLIWWM


NHMASHPVLAQCDVFQHFLTCPSSTDEKAWKQGKRKAEKDEMVGANFFLTLSTPPAAALDLQEVESKIDGFK


CFTKKMDDSALQLNHTANEFARKQVTGFKKEYQKVGQSFRGLSQAFELDQQAFSVGLNQAIAFTGDAYDAIG


ELFAEQPRQDLDPVMDLLALYQGHLANFPDIIHVQKGALTKVKESRRHVEEGKMEVQKADGIQDRCNTISFA


TLAEIHHFHQIRVRDFKSQMQHFLQQQIIFFQKVTQKLEEALHKYDSV









The NOV24 amino acid sequence 171 of 401 amino acid residues (42%) identical to, and 239 of 401 amino acid residues (59%) similar to, the 595 amino acid residue ptnr:SWISSNEW-ACC:Q9Y5X1 protein from Homo sapiens (Human) (Sorting Nexin 9 (SH3 and PX Domain-Containing Protein 1) (SDP1 Protein)) (E=1.0e−99).
NOV24 is expressed in at least the following tissues: lung, colon, mammary gland/breast, peripheral blood, pituitary gland, thyroid gland, and vulva. This information was derived by determining the tissue sources of the sequences that were included in the invention including but not limited to SeqCalling sources, Public EST sources, genomic clone sources, literature sources, and/or RACE sources.
NOV24 has homology to the amino acid sequences shown in the BLASTP data listed in Table 24C.







TABLE 24C







BLAST results for NOV24
















Pos-



Gene Index/
Protein/
Length
Identity
itives



Identifier
Organism
(aa)
(%)
(%)
Expect





gi|16418371|ref|NP_
sorting nexin
628
441/541
441/
0.0


443102.1|
18 [Homo

(81%)
541


(NM_052870)
sapiens]


(81%)


gi|18644890|ref|NP_
sorting nexin
614
462/633
468/
0.0


570614.1|
associated

(72%)
633


(NM_130796)
golgi protein


(72%)



1 [Mus



nusculus]


gi|17511850|gb|
Unknown
574
243/464
310/
 e−134


AAH1 8775.1|
(protein for

(52%)
464


AAH18775
MGC:32065)


(66%)


(BC018775)
[Homo



sapiens]


gi|13385132|ref|NP_
sorting nexin
395
166/394
221/
3e−80 


079940.1|
9 [Mus

(42%)
394


(NM_025664)
musculus]


(55%)


gi|15928697|gb|
Similar to
595
166/394
221/
6e−80 


AAH1 4814.1|
sorting nexin

(42%)
394


AAH14814
9 [Mus


(55%)


(BC014814)
musculus]









The homology of these sequences is shown graphically in the ClustalW analysis shown in Table 24D.







TABLE 24E





Clustal W Sequence Alignment















1) NOV24 (SEQ ID NO:82)


2) gi 16418371|ref|NP_443102.1|(NM_052870) sorting nexin 18 [Homo sapiens]


(SEQ ID NO:243)


3) gi 18644890|ref|NP_570614.1|(NM_130796) sorting nexin associated golgi protein 1


[Mus musculus] (SEQ ID NO:244)


4) gi 17511850|gb|AAH18775.1 AAH18775 (BC018775) Unknown (protein for MGC:32065)


[Homo sapiens] (SEQ ID NO:245)


5) gi 13385132|refNP_079940.1|(NM_025664) sorting nexin 9 [Mus musculus]


(SEQ ID NO:246)


6) gi 15928697|gb|AAH14814.1 AAH14814 (BC014814) Similar to sorting nexin 9


[Mus musculus] (SEQ ID NO:247)






















































































TABLE 24F





Domain Analysis of NOV24















gnl|Pfam pfam00018, SH3, SH3 domain. SH3 (Src homology 3) domains are


often indicative of a protein involved in signal transduction related


to cytoskeletal organization. First described in the Src cytoplasmic


tyrosine kinase. The structure is a partly opened beta barrel. (SEQ ID


NO :249)


CD-Length = 57 residues, 98.2% aligned


Score = 60.1 bits (144), Expect = 4e−10










Query:
4
RARALYDFRSENPGEISLREHEVLSLCSEQDIEGWLEGVNSRGDRGLFPASYVQVI
59




+  ||||+++    |+| ++ +++ +  + |  || +|       || |++||+ +


Sbjct:
2
KVVALYDYQARESDELSFKKGDIIIVLEKSDDGGWWKGRLKGTKEGLIPSNYVEPV
57









Sorting nexins are a family of phox homology (PX) domain containing proteins that are homologous to yeast proteins involved in protein trafficking. Sorting nexin-1 (SNX1) was identified based on its ability to bind to a region of the epidermal growth factor receptor (EGFR) containing the lysosomal targeting code (Kurten et al., Science 272:1008-10, 1996). The SNX1 protein contains a region of homology to a yeast vacuolar sorting protein, and overexpression of SNX1 decreased the amount of EGFR on the cell surface as a result of enhanced rates of constitutive and ligand-induced degradation. Human SNX1, SNX1A, SNX2, SNX3, and SNX4 are part of a larger family of hydrophilic molecules including proteins identified in Caenorhabditis elegans and Saccharomyces cerevisiae (Haft et al., Mol. Cell. Biol. 18:7278-87, 1998). Despite their hydrophilic nature, these sorting nexins are found partially associated with cellular membranes. They are widely expressed, although the tissue distribution of each sorting nexin mRNA varies. The sorting nexins have been demonstrated to interact with a variety of receptor types (including EGFR, platelet derived growth factor receptor, and leptin) suggesting that these proteins may be involved in several stages of intracellular trafficking in mammalian cells.
The NOV24 protein described in this invention is predicted to share the attributes of the other sorting nexin family members and is thus implicated in protein trafficking. Its potential role in the regulation of growth factor receptor localization and levels suggests that the NOV24 protein may play an important role in cell growth and differentiation. Therefore, the NOV24 protein is an attractive target for drug intervention in the treatment of human metabolic diseases, immune disorders, and cancer, among others.
The NOV24 nucleic acid of the invention encoding a Sorting Nexin 9-like protein includes the nucleic acid whose sequence is provided in Table 24A, or a fragment thereof. The invention also includes a mutant or variant nucleic acid any of whose bases may be changed from the corresponding base shown in Table 24A while still encoding a protein that maintains its Sorting Nexin 9-like activities and physiological functions, or a fragment of such a nucleic acid. The invention further includes nucleic acids whose sequences are complementary to those just described, including nucleic acid fragments that are complementary to any of the nucleic acids just described. The invention additionally includes nucleic acids or nucleic acid fragments, or complements thereto, whose structures include chemical modifications. Such modifications include, by way of non-limiting example, modified bases, and nucleic acids whose sugar phosphate backbones are modified or derivatized. These modifications are carried out at least in part to enhance the chemical stability of the modified nucleic acid, such that they may be used, for example, as antisense binding nucleic acids in therapeutic applications in a subject. In the mutant or variant nucleic acids, and their complements, up to about 37% of the NOV24 residues may be so changed.
The NOV24 protein of the invention includes the Sorting Nexin 9-like protein whose sequence is provided in Table 24B. The invention also includes a mutant or variant protein any of whose residues may be changed from the corresponding residue shown in Table 24B while still encoding a protein that maintains its Sorting Nexin 9-like activities and physiological functions, or a functional fragment thereof. In the mutant or variant protein, up to about 58% of the NOV24 bases may be so changed.
The NOV24 nucleic acids and proteins of the invention are useful in potential diagnostic and therapeutic applications implicated in various diseases and disorders described below and/or other pathologies. For example, the compositions of the present invention will have efficacy for treatment of patients suffering from: cancer, trauma, bacterial and viral infections, in vitro and in vivo regeneration, systemic lupus erythematosus, autoimmune disease, asthma, emphysema, scleroderma, allergy, ARDS, Hirschsprung's disease, Crohn's Disease, appendicitis, hyperthyroidism, hypothyroidism, fertility, anemia, ataxia-telangiectasia, immunodeficiencies, endocrine dysfunctions, diabetes, obesity, growth and reproductive disorders and other diseases, disorders and conditions of the like.
NOV24 nucleic acids and polypeptides are further useful in the generation of antibodies that bind immunospecifically to the novel substances of the invention for use in therapeutic or diagnostic methods. These antibodies may be generated according to methods known in the art, using prediction from hydrophobicity charts, as described in the “Anti-NOVX Antibodies” section below. For example the disclosed NOV24 protein have multiple hydrophilic regions, each of which can be used as an immunogen. This novel protein also has value in development of powerful assay system for functional analysis of various human disorders, which will help in understanding of pathology of the disease and development of new drug targets for various disorders.
NOV25
A disclosed NOV25 nucleic acid of 1514 nucleotides (also referred to as CG57568-01) encoding a novel Katanin-like protein is shown in Table 25A. An open reading frame was identified beginning with an ATG initiation codon at nucleotides 102-104 and ending with a TGA codon at nucleotides 1191-1193. Putative untranslated regions upstream from the intitation codon and downstream from the termination codon are underlined in Table 25A, and the start and stop codons are in bold letters.







TABLE 25A





NOV25 Nucleotide Sequence















(SEQ ID NO:83)







AGTAGTTTTAAACATATTTTAAAGAATAATAAATGTTTTAAAGTCAGTGCTGAAAATTTTTAAAAAGTCATTA


TATTTTATGTTTAGGTCTCTGAAAGAAGATGAATTTGGCTGAGATTTGTGATAATGCAAAGAAAGGAAGAGAA


TATGCCCTTCTTGGAAATTACGACTCATCAATGGTATATTACCAGGGGGTGATGCAGCAGATTCAGAGACATT


GCCAGTCAGTCAGAGATCCAGCTATCAAAGGCAAATGGCAACAGGTAAGATGGGGTTATGATAAGGATCTGGT


GGAAGCCCTTGAAAGAGACATTGTATCCAGGAATCCTAGCATTCATTGGGATGACATAGCAGATCTGGAAGAA


GCTAAGAAGTTGCTAAGGGAAGCTGTTGTTCTTCCAATGTGGATGCCTGACTTTTTCAAAGGGATTAGAAGGC


CATGGAAGGGTGTACTGATGGTTGGACCCCCAGGCACTGGTAAAACTATGCTAGCTAAAGCTGTTGCCACTGA


ATGTGGTACAACATTCTTCAACGTTTCGTCTTCTACACTGACATCTAAATACAGAGGTGAATCTGAGAAGTTA


GTTCGTCTGTTGTTTGAGATGGCTAGATTTTATGCCCCTACCACGATCTTCATTGATGAGATAGATTCTATCT


GCAGTCGAAGAGGAACCTCTGATGAACATGAGGCAAGTCGCAGGGTCAAGTCTGAACTGCTCATTCAGATGGA


TGGTGTTGGAGGAGCTTTAGAAAATGATGATCCTTCCAAAATGGTTATGGTATTGGCTGCTACTAATTTCCCG


TGGGACATTGATGAAGCTTTGCGAAGAAGGTTAGAAAAAAGGATATATATACCTCTCCCAACAGCAAAAGGAA


GAGCTGAGCTTCTGAAGATCAACCTTCGTGAGGTCGAATTAGATCCTGATATTCAACTGGAAGATATAGCCGA


GAAGATTGAGGGCTATTCTGGTGCTGACATCACTAATGTTTGCAGGGATGCCTCTTTAATGGCAATGAGACGG


CGTATCAATGGCTTAAGTCCAGAAGAAATCCGTGCACTTTCTAAAGAGGAACTTCAGATGCCTGTTACCAAAG


GAGACTTTGAATTGGCCCTAAAAAAAATTGCTAAGTCTGTCTCTGCTGCAGACTTGGAGAAGTATGAAAAATG


GATGGTTGAATTTGGATCTGCTTGAATTTCTGTCAGCTCTTTAATTTCTGGTATTTTTGTTGATAAAATACGA


AGAAATTCCTGCAATTTTTAAAAAACAAGTTTGGAACTTTTTTCAGTGGAGTGGTTTTCGCAAAAAAAAAAAA


AAAATCTAAAACTGCGAAGAATACTAAATGTAGTTGAGAAATAATTGATGGCGAGAGTTTGCTAGTCTCCCTC


CCCGGCTTTGTGCTGGTATTCCACGTATTCCTGCATTAATATTGCACACCCAAACCAGTCTATCAGGGAGGCT


GAAGCAGGGCGCAGTGTGATATTTAGGAATCAGAAGATTAGAAATCCCCTATTC









The NOV25 nucleic acid was identified on chromosome 13 and has 720 of 953 bases (75%) identical to a gb:GENBANK-ID:AF056022|acc:AF056022.1 mRNA from Homo sapiens (Homo sapiens p60 katanin mRNA, complete cds) (E=1.6e−123).
A disclosed NOV25 polypeptide (SEQ ID NO:84) encoded by SEQ ID NO:83 is 363 amino acid residues and is presented using the one-letter code in Table 25B. Signal P, Psort and/or Hydropathy results predict that NOV25 does not contain a signal peptide and is likely to be localized to cytoplasm with a certainty of 0.6500.







TABLE 25B





Encoded NOV25 protein sequence















(SEQ ID NO:84)







MNLAEICDNAKKGREYALLGNYDSSMVYYQGVMQQIQRHCQSVRDPAIKGKWQQVRWGYDKDLVEALERDIV


SRNPSIHWDDIADLEEAKKLLREAVVLPMWMPDFFKGIRRPWKGVLMVGPPGTGKTMLAKAVATECGTTFFN


VSSSTLTSKYRGESEKLVRLLFEMARFYAPTTIFIDEIDSICSRRGTSDEHEASRRVKSELLIQMDGVGGAL


ENDDPSKMVMVLAATNFPWDIDEALRRRLEKRIYIPLPTAKGRAELLKINLREVELDPDIQLEDIAEKIEGY


SGADITNVCRDASLMAMRRRINGLSPEEIRALSKEELQMPVTKGDFELALKKIAKSVSAADLEKYEKWMVEF


GSA









The NOV25 amino acid sequence 260 of 305 amino acid residues (85%) identical to, and 285 of 305 amino acid residues (93%) similar to, the 491 amino acid residue ptnr:SPTREMBL-ACC:O75449 protein from Homo sapiens (Human) (P60 Katanin) (E=1.2e−155).
NOV25 is expressed in at least the following tissues: lung, testis and b-cell. This information was derived by determining the tissue sources of the sequences that were included in the invention including but not limited to SeqCalling sources, Public EST sources, genomic clone sources, literature sources, and/or RACE sources.
Possible SNPs found for NOV25 are listed in Table 25C.







TABLE 25C







SNPs
















Amino





Nucleotide
Base
Acid
Base



Variant
Position
Change
Position
Change

















13377117
1050
A > G
317
Ile > Val



13377118
1061
T > C
Silent
N/A



13377119
1073
A > G
Silent
N/A



13377120
1074
C > T
325
Leu > Phe



13377121
1085
T > C
Silent
N/A



13377122
1111
C > T
337
Ala > Val



13377123
1127
T > C
Silent
N/A



13377124
1178
A > G
Silent
N/A



13377125
1190
T > C
Silent
N/A



13377126
1297
T > C
Silent
N/A










NOV25 has homology to the amino acid sequences shown in the BLASTP data listed in Table 25D.







TABLE 25D







BLAST results for NOV25
















Pos-



Gene Index/
Protein/
Length
Identity
itives



Identifier
Organism
(aa)
(%)
(%)
Expect





gi|14149767|ref|NP_
hypothetical
490
306/306
306/
 e−170


115492.1|
protein

(100%)
306


(NM_032116)
MGC2599


(100%)



similar to



katanin p60



subunit A 1



[Homo



sapiens]


gi|5825592|gb|
katanin p60
486
257/304
288/
e−149


AAD53 310.1|
[Xenopus

(84%)
304


AF177942 1
laevis]


(94%)


(AF177942)


gi|5901990|ref|NP_0
katanin p60
491
260/305
285/
e−148


08975.1|
subunit A 1

(85%)
305


(NM_007044)
[Homo


(93%)



sapiens]


gi|7594619|emb|
dJ12G14.4
384
260/305
285/
e−148


CAB88114.1|
(katanin p60

(85%)
305


(AL078581)
(ATPase-


(93%)



containing)



subunit A 1)



[Homo



sapiens]


gi|6754410|ref|NP_0
katanin p60
491
259/305
285/
e−147


35965.1|
(ATPase-

(84%)
305


(NM_011835)
containing)


(92%)



subunit A1



[Mus



musculus]









The homology of these sequences is shown graphically in the ClustalW analysis shown in Table 25E.







TABLE 25E





Clustal W Sequence Alignment















1) NOV25 (SEQ ID NO:84)


2) gi|14149767|refNP_115492.1|(NM_032116) hypothetical protein MGC2599 similar to


katanin p60 subunit A 1 [Homo sapiens] (SEQ ID NO:250)


3) gi 5825592|gb AAD53310.1|AF177942_1 (AF177942) katanin p60 [Xenopus laevis]


(SEQ ID NO:251)


4) gi 5901990|refNP_008975.1|(NM_007044) katanin p60 subunit A 1 [Homo sapiens]


(SEQ ID NO:252)


5) gi 7594619|emb|CAB88114.1|(AL078581) dJ12G14.4 (katanin p60 (ATPase-containing)


subunit A 1) [Homo sapiens] (SEQ ID NO:253)


6) gi 6754410|refNP_035965.1|(NM_011835) katanin p60 (ATPase-containing) subunit A1


[Mus musculus] (SEQ ID NO:254)

































































Table 25F lists the domain description from DOMAIN analysis results against NOV25. This indicates that the NOV25 sequence has properties similar to those of other proteins known to contain these domains.







TABLE 25F





Domain Analysis of NOV25















gnl|Pfam pfam00004, AAA, ATPase family associated with various


cellular activities (AAA). AAA family proteins often perform


chaperone-like functions that assist in the assembly, operation, or


disassembly of protein complexes. (SEQ ID NO:255)


CD-Length = 186 residues, 100.0% aligned


Score = 199 bits (505), Expect = 3e−52










Query:
116
GVLMVGPPGTGKTMLAKAVATECGTTFFNVSSSTLTSKYRGESEKLVRLLFEMARFYAPT
175




|+|+ ||||||||+|||||| | |  |  +| | | ||| |||||||| || +||  ||


Sbjct:
1
GILLYGPPGTGKTLLAKAVAKELGVPFIEISGSELLSKYVGESEKLVRALFSLARKSAPC
 60


Query:
176
TIFIDEIDSICSRRGTSDEHEASRRVKSELLIQMDGVGGALENDDPSKMVMVLAATNFPW
235




 |||||||++  +||     + | || ++|| +|||        +    |+|+ ||| |


Sbjct:
61
IIFIDEIDALAPKRGDVGTGDVSSRVVNQLLTEMDGF-------EKLSNVIVIGATNRPD
113


Query:
236
DIDEALRR--RLEKRIYIPLPTAKGRAELLKINLREVELDPDIQLEDIAEKIEGYSGADI
293




 +| || |  | ++|| +|||  + | |+|||+|++  |+ |+ |++|| +  |+||||+


Sbjct:
114
LLDPALLRPGRFDRRIEVPLPDEEERLEILKIHLKKKPLEKDVDLDEIARRTPGFSGADL
173


Query:
294
TNVCRDASLMAMR
306




  +||+|+| |+|


Sbjct:
174
AALCREAALRAIR
186









Microtubule disassembly at centrosomes is involved in mitotic spindle function. The microtubule-severing protein katanin, a heterodimer of 60- and 80-kD subunits, was purified and shown to localize to centrosomes in vivo (McNally and Vale, Cell 75: 419-429, 1993; McNally et al., J. Cell Sci. 109: 561-567, 1996). By homology to the sequence of S. purpuratus, Hartman et al. (Cell 93: 277-287, 1998) cloned the cDNA for the human p80 subunit. This protein contains WD40 repeats, which are frequently involved in protein-protein interactions. The p80 WD40 domain does not participate in p60 dimerization, but localizes to centrosomes in transfected mammalian cells. The results of the studies of Hartman et al. (1998) in yeast indicated that the activities of katanin are segregated into a subunit (p60) that possesses enzymatic activity and a subunit (p80) that targets the enzyme to the centrosome.
Microtubule disassembly at centrosomes is involved in mitotic spindle function. The microtubule-severing protein katanin, a heterodimer of 60 and 80 kDa subunits, was previously purified and shown to localize to centrosomes in vivo. The sequences and activities of the katanin subunits were reported. p60 is a new member of the AAA family of ATPases, and expressed p60 has microtubule-stimulated ATPase and microtubule-severing activities in the absence of p80. p80 is a novel protein containing WD40 repeats, which are frequently involved in protein-protein interactions. The p80 WD40 domain does not participate in p60 dimerization, but localizes to centrosomes in transfected mammalian cells. These results indicate katanin's activities are segregated into a subunit p60) that possesses enzymatic activity and a subunit (p80) that targets the enzyme to the centrosome (Hartman et al., Cell 93: 277-287, 1998).
The assembly and function of the mitotic spindle involve specific changes in the dynamic properties of microtubules. One such change results in the poleward flux of tubulin in which spindle microtubules polymerize at their kinetochore-attached plus ends while they shorten at their centrosome-attached minus ends. Since free microtubule minus ends do not depolymerize in vivo, the poleward flux of tubulin suggests that spindle microtubules are actively disassembled at or near their centrosomal attachment points. The microtubule-severing ATPase, katanin, has the ability actively to sever and disassemble microtubules and is thus a candidate for the role of a protein mediating the poleward flux of tubulin. The subcellular localization of katanin by immunofluorescence was identified as a preliminary step in determining whether katanin mediates the poleward flux of tubulin. Katanin is highly concentrated at centrosomes throughout the cell cycle. Katanin's localization is different from that of gamma-tubulin in that microtubules are required to maintain the centrosomal localization of katanin. Direct comparison of the localization of katanin and gamma-tubulin reveals that katanin is localized in a region surrounding the gamma-tubulin-containing pericentriolar region in detergent-extracted mitotic spindles. The centrosomal localization of katanin is consistent with the hypothesis that katanin mediates the disassembly of microtubule minus ends during poleward flux (McNally and Vale, Cell 75: 419-429, 1993).
The NOV25 nucleic acid of the invention encoding a Katanin-like protein includes the nucleic acid whose sequence is provided in Table 25A, or a fragment thereof. The invention also includes a mutant or variant nucleic acid any of whose bases may be changed from the corresponding base shown in Table 25A while still encoding a protein that maintains its Katanin-like activities and physiological functions, or a fragment of such a nucleic acid. The invention further includes nucleic acids whose sequences are complementary to those just described, including nucleic acid fragments that are complementary to any of the nucleic acids just described. The invention additionally includes nucleic acids or nucleic acid fragments, or complements thereto, whose structures include chemical modifications. Such modifications include, by way of non-limiting example, modified bases, and nucleic acids whose sugar phosphate backbones are modified or derivatized. These modifications are carried out at least in part to enhance the chemical stability of the modified nucleic acid, such that they may be used, for example, as antisense binding nucleic acids in therapeutic applications in a subject. In the mutant or variant nucleic acids, and their complements, up to about 25% of the NOV25 residues may be so changed.
The NOV25 protein of the invention includes the Katanin-like protein whose sequence is provided in Table 25B. The invention also includes a mutant or variant protein any of whose residues may be changed from the corresponding residue shown in Table 25B while still encoding a protein that maintains its Katanin-like activities and physiological functions, or a functional fragment thereof. In the mutant or variant protein, up to about 15% of the NOV25 bases may be so changed.
The NOV25 nucleic acids and proteins of the invention are useful in potential diagnostic and therapeutic applications implicated in various diseases and disorders described below and/or other pathologies. For example, the compositions of the present invention will have efficacy for treatment of patients suffering from: developmental disorders, fertility, hypogonadism, systemic lupus erythematosus, autoimmune disease, asthma, emphysema, scleroderma, allergy, ARDS, immunological diseases and other diseases, disorders and conditions of the like.
NOV25 nucleic acids and polypeptides are further useful in the generation of antibodies that bind immunospecifically to the novel substances of the invention for use in therapeutic or diagnostic methods. These antibodies may be generated according to methods known in the art, using prediction from hydrophobicity charts, as described in the “Anti-NOVX Antibodies” section below. For example the disclosed NOV25 protein have multiple hydrophilic regions, each of which can be used as an immunogen. This novel protein also has value in development of powerful assay system for functional analysis of various human disorders, which will help in understanding of pathology of the disease and development of new drug targets for various disorders.
NOV26
A disclosed NOV26 nucleic acid of 2442 nucleotides (also referred to as CG57509-01) encoding a novel Calpain-like protein is shown in Table 26A. An open reading frame was identified beginning with an ATG initiation codon at nucleotides 77-79 and ending with a TGA codon at nucleotides 2396-2398. Putative untranslated regions upstream from the intitation codon and downstream from the termination codon are underlined in Table 26A, and the start and stop codons are in bold letters.







TABLE 26A





NOV26 Nucleotide Sequence















(SEQ ID NO:85)







TGCAGTTGCTTCCTTTCCTTGAAGGTAGCTGTATCTTATTTTCTTTAAAAAGCTTTTTCTTCCAAAGCCACTT


GCCATGCCGACCGTCATTAGCGCATCTGTGGCTCCAAGGACAGCGGCTGAGCCCCGGTCCCCAGGGCCAGTTC


CTCACCCGGCCCAGAGCAAGGCCACTGAGGCTGGGGGTGGAAACCCAAGTGGCATCTATTCAGCCATCATCAG


CCGCAATTTTCCTATTATCGGAGTGAAAGAGAAGACATTCGAGCAACTTCACAAGAAATGTCTAGAAAAGAAA


GTTCTTTATGTGGACCCTGAGTTCCCACCGGATGAGACCTCTCTCTTTTATAGCCAGAAGTTCCCCATCCAGT


TCGTCTGGAAGAGACCTCCGGAAATTTGCGAGAATCCCCGATTTATCATTGATGGAGCCAACAGAACTGACAT


CTGTCAAGGAGAGCTAGGGGACTGCTGGTTTCTCGCAGCCATTGCCTGCCTGACCCTGAACCAGCACCTTCTT


TTCCGAGTCATACCCCATGATCAAAGTTTCATCGAAAACTACGCAGGGATCTTCCACTTCCAGTTCTGGCGCT


ATGGAGAGTGGGTGGACGTGGTTATAGATGACTGCCTGCCAACGTACAACAATCAACTGGTTTTCACCAAGTC


CAACCACCGCAATGAGTTCTGGAGTGCTCTGCTGGAGAAGGCTTATGCTAAGCTCCATGGTTCCTACGAAGCT


CTGAAAGGTGGGAACACCACAGAGGCCATGGAGGACTTCACAGGAGGGGTGGCAGAGTTTTTTGAGATCAGGG


ATGCTCCTAGTGACATGTACAAGATCATGAAGAAAGCCATCGAGAGAGGCTCCCTCATGGGCTGCTCCATTGA


TACAATCATTCCGGTTCAGTATGAGACAAGAATGGCCTGCGGGCTGGTCAGAGGTCACGCCTACTCTGTCACG


GGGCTGGATGAGGTCCCGTTCAAAGGTGAGAAAGTGAAGCTGGTGCGGCTGCGGAATCCGTGGGGCCAGGTGG


AGTGGAACGGTTCTTGGAGTGATAGATGGAAGGACTGGAGCTTTGTGGACAAAGATGAGAAGGCCCGTCTGCA


GCACCAGGTCACTGAGGATGGAGAGTTCTGGATGTCCTATGAGGATTTCATCTACCATTTCACAAAGTTGGAG


ATCTGCAACCTCACGGCCGATGCTCTGCAGTCTGACAAGCTTCAGACCTGGACAGTGTCTGTGAACGAGGGCC


GCTGGGTACGGGGTTGCTCTGCCGGAGGCTGCCGCAACTTCCCAGATACTTTCTGGACCAACCCTCAGTACCG


TCTGAAGCTCCTGGAGGAGGACGATGACCCTGATGACTCGGAGGTGATTTGCAGCTTCCTGGTGGCCCTGATG


CAGAAGAACCGGCGGAAGGACCGGAAGCTAGGGGCCAGTCTCTTCACCATTGGCTTCGCCATCTACGAGGTTC


CCAAAGAGATGCACGGGAACAAGCAGCACCTGCAGAAGGACTTCTTCCTGTACAACGCCTCCAAGGCCAGGAG


CAAAACCTACATCAACATGCGGGAGGTGTCCCAGCGCTTCCGCCTGCCTCCCAGCGAGTACGTCATCGTGCCC


TCCACCTACGAGCCCCACCAGGAGGGGGAATTCATCCTCCGGGTCTTCTCTGAAAAGAGGAACCTCTCTGAGG


AAGTTGAAAATACCATCTCCGTGGATCGGCCAGTGAAAAAGAAAAAAACCAAGCCCATCATCTTCGTTTCGGA


CAGAGCAAACAGCAACAAGGAGCTGGGTGTGGACCAGGAGTCAGAGGAGGGCAAAGGCAAAACAAGCCCTGAT


AAGCAAAAGCAGTCCCCACAGCCACAGCCTGGCAGCTCTGATCAGGAAAGTGAGGAACAGCAACAATTCCGGA


ACATTTTCAAGCAGATAGCAGGAGATGACATGGAGATCTGTGCAGATGAGCTCAAGAAGGTCCTTAACACAGT


CGTGAACAAACACAAGGACCTGAAGACACACGGGTTCACACTGGAGTCCTGCCGTAGCATGATTGCGCTCATG


GATACAGATGGCTCTGGAAAGCTCAACCTGCAGGAGTTCCACCACCTCTGGAACAAGATTAAGGCCTGGCAGA


AAATTTTCAAACACTATGACACAGACCAGTCCGGCACCATCAACAGCTACGAGATGCGAAATGCAGTCAACGA


CGCAGGATTCCACCTCAACAACCAGCTCTATGACATCATTACCATGCGGTACGCAGACAAACACATGAACATC


GACTTTGACAGTTTCATCTGCTGCTTCGTTAGGCTGGAGGGCATGTTCAGAGCTTTTCATGCATTTGACAAGG


ATGGAGATGGTATCATCAAGCTCAACGTTCTGGAGTGGCTGCAGCTCACCATGTATGCCTGAACCAGGCTGGC


CTCATCCAAAGCCATGCAGGATCACTCAGGATT









The NOV26 nucleic acid was identified on chromosome 15 and has 1717 of 1720 bases (99%) identical to a gb:GENBANK-ID:AF127765|acc:AF127765.3 mRNA from Homo sapiens (Homo sapiens calpain 3 (CAPN3) mRNA, complete cds, alternatively spliced) (E=0.0).
A disclosed NOV26 polypeptide (SEQ ID NO:86) encoded by SEQ ID NO:85 is 773 amino acid residues and is presented using the one-letter code in Table 26B. Signal P, Psort and/or Hydropathy results predict that NOV26 does not contain a signal peptide and is likely to be localized to the cytoplasm with a certainty of 0.6526. The most likely cleavage site for a NOV26 polypeptide is between amino acids 15 and 16: TAA-EP







TABLE 26B





Encoded NOV26 protein sequence















(SEQ ID NO:86)







MPTVISASVAPRTAAEPRSPGPVPHPAQSKATEAGGGNPSGIYSAIISRNFPIIGVKEKTFEQLHKKCLEKK


VLYVDPEFPPDETSLFYSQKFPIQFVWKRPPEICENPRFIIDGANRTDICQGELGDCWFLAAIACLTLNQHL


LFRVIPHDQSFIENYAGIFHFQFWRYGEWVDVVIDDCLPTYNNQLVFTKSNHRNEFWSALLEKAYAKLHGSY


EALKGGNTTEAMEDFTGGVAEFFEIRDAPSDMYKIMKKAIERGSLMGCSIDTIIPVQYETRMACGLVRGHAY


SVTGLDEVPFKGEKVKLVRLRNPWGQVEWNGSWSDRWKDWSFVDKDEKARLQHQVTEDGEFWMSYEDFIYHF


TKLEICNLTADALQSDKLQTWTVSVNEGRWVRGCSAGGCRNFPDTFWTNPQYRLKLLEEDDDPDDSEVICSF


LVALMQKNRRKDRKLGASLFTIGFAIYEVPKEMHGNKQHLQKDFFLYNASKARSKTYINMREVSQRFRLPPS


EYVIVPSTYEPHQEGEFILRVFSEKRNLSEEVENTISVDRPVKKKKTKPIIFVSDRANSNKELGVDQESEEG


KGKTSPDKQKQSPQPQPGSSDQESEEQQQFRNIFKQIAGDDMEICADELKKVLNTVVNKHKDLKTHGFTLES


CRSMIALMDTDGSGKLNLQEFHHLWNKIKAWQKIFKHYDTDQSGTINSYEMRNAVNDAGFHLNNQLYDIITM


RYADKHMNIDFDSFICCFVRLEGMFRAFHAFDKDGDGIIKLNVLEWLQLTMYA









The NOV26 amino acid sequence 506 of 506 amino acid residues (100%) identical to, and 506 of 506 amino acid residues (100%) similar to, the 821 amino acid residue ptnr:SWISSNEW-ACC:P20807 protein from Homo sapiens (Human) (Calpain 3 Large Subunit (EC 3.4.22.17) (Calpain L3) (Calpain P94, Large [Catalytic] Subunit) (Calcium-Activated Neutral Proteinase 3) (CANP 3) (Muscle-Specific Calcium-Activated Neutral Protease 3 Large Subunit)) (E=0.0). The NOV26 amino acid lacks 48 internal amino acids when compared to ptnr:SWISSNEW-ACC:P20807 protein from Homo sapiens (Human) (Calpain 3 Large Subunit (EC 3.4.22.17) (Calpain L3) (Calpain P94, Large [Catalytic] Subunit) (Calcium-Activated Neutral Proteinase 3) (CANP 3) (Muscle-Specific Calcium-Activated Neutral Protease 3 Large Subunit)).
NOV26 is expressed in at least the following tissues: adrenal gland, bone marrow, brain—amygdala, brain—cerebellum, brain—hippocampus, brain—substantia nigra, brain—thalamus, brain—whole, fetal brain, fetal kidney, fetal liver, fetal lung, heart, kidney, lymphoma—Raji, mammary gland, pancreas, pituitary gland, placenta, prostate, salivary gland, skeletal muscle, small intestine, spinal cord, spleen, stomach, testis, thyroid, trachea, uterus, aorta, epidermis, foreskin, heart, hypothalamus, liver, lung, lymph node, lymphoid tissue, muscle, peripheral blood, skin and whole organism. This information was derived by determining the tissue sources of the sequences that were included in the invention including but not limited to SeqCalling sources, Public EST sources, genomic clone sources, literature sources, and/or RACE sources. NOV26 is predicted to be expressed in leukocytes because of the expression pattern of (gb:GENBANK-ID:AF127765|acc:AF127765.3) a closely related Homo sapiens calpain 3 (CAPN3) mRNA, complete cds, alternatively spliced homolog in species Homo sapiens. 
Possible SNPs found for NOV26 are listed in Table 26C and Table 26D.







TABLE 26C







SNPs
















Amino





Nucleotide
Base
Acid
Base



Variant
Position
Change
Position
Change

















13377127
72
T > C
Silent
N/A



13377128
78
T > C
 1
Met > Thr



13377129
201
T > C
 42
Ile > Thr



13377130
412
C > T
Silent
N/A



13377131
2253
A > G
726
His > Arg

















TABLE 26D







SNPs












Consensus

Base




Position
Depth
Change
PAF







2258
29
A > G
0.379










NOV26 has homology to the amino acid sequences shown in the BLASTP data listed in Table 26E.







TABLE 26E







BLAST results for NOV26
















Pos-



Gene Index/
Protein/
Length
Identity
itives



Identifier
Organism
(aa)
(%)
(%)
Expect





gi/4557405|ref|NP_0
calpain,
821
738/821
738/
0.0


00061.1|
large poly-

(89%)
821


(NM_000070)
peptide L3;


(89%)



p94 [Homo



sapiens]


gi|4704752|gb|
calpain 3;
815
732/821
732/
0.0


AAD28 253.1|
calcium

(89%)
821


AF127764_1
activated


(89%)


(AF127764)
neutral



protease;



CAPN3;



CL1 [Homo



sapiens]


gi|11037484|gb|
calpain 3
815
720/821
727/
0.0


AAG2 7599.1|
[Macaca

(87%)
821


AF277376_1 
fascicularis]


(87%)


(AF277376)


gi|7684607|gb|
calpain 3;
729
710/773
713/
0.0


AAD28 254.3|
calcium

(91%)
773


AF127765_1 
activated


(91%)


(AF127765)
neutral



protease;



CAPN3;



CL1 [Homo



sapiens]


gi|8393041|ref|NP_0
calpain 3
821
699/821
718/
0.0


58813.1|
[Rattus

(85%)
821


(NM_017117)
norvegicus]


(87%)









The homology of these sequences is shown graphically in the ClustalW analysis shown in Table 26F.







TABLE 26E





Clustal W Sequence Alignment















1) NOV26 (SEQ ID NO:86)


2) gi 4557405|refNP_000061.1|(NM_000070) calpain, large polypeptide L3; p94


[Homo sapiens] (SEQ ID NO:256)


3) gi 4704752|gb AAD28253.1|AF127764_1 (AF127764) calpain 3; calcium activated


neutral protease; CAPN3; CL1 [Homo sapiens] (SEQ ID NO:257)


4) gi 11037484|gb AAG27599.1 AF277376_1 (AF277376) calpain 3 [Macaca fascicularis]


(SEQ ID NO:258)


5) gi 7684607|gb AAD28254.3|AF127765_1 (AF127765) calpain 3; calcium activated


neutral protease; CAPN3; CL1 [Homo sapiens] (SEQ ID NO:259)


6) gi 8393041|refNP_058813.1|(NM_017117) calpain 3 [Rattus norvegicus]


(SEQ ID NO:260)





























































































Tables 26G, 26H, 26I and 26J list the domain description from DOMAIN analysis results against NOV26. This indicates that the NOV26 sequence has properties similar to those of other proteins known to contain these domains.







TABLE 26G





Domain Analysis of NOV26















gnl|Pfam pfam00648, Peptidase_C2, Calpain family cysteine protease.


(SEQ ID NO:261)


CD-Length = 298 residues, 100.0% aligned


Score = 518 bits (1335), Expect = 4e−148










Query:
74
LYVDPEFPPDETSLFYSQKFPIQFVWKRPPEICENPRFIIDGANRTDICQGELGDCWFLA
133




|+||| ||    || |    |    |||| || |||+||+ || ||||||| ||||| ||


Sbjct:
1
LFVDPSFPAAPKSLGYKPLGPRGIEWKRPHEINENPQFIVGGATRTDICQGALGDCWLLA
 60


Query:
134
AIACLTLNQHLLFRVIPHDQSFIENYAGIFHFQFWRYGEWVDVVIDDCLPTYNNQLVFTK
193




|+| ||||+ || ||+|||||| |||||||||+||++|||||||+|| ||| + +|+|


Sbjct:
61
ALASLTLNEPLLLRVVPHDQSFQENYAGIFHFRFWQFGEWVDVVVDDLLPTKDGKLLFVH
120


Query:
194
SNHRNEFWSALLEKAYAKLHGSYEALKGGNTTEAMEDFTGGVAEFFEIRDAPS---DMYK
250




|  ||||||||||||||||+| |||| ||+||||+|| |||| | +|++ |||   ++


Sbjct:
121
SAERNEFWSALLEKAYAKLNGCYEALSGGSTTEALEDLTGGVCESYELKLAPSSMLNLGN
180


Query:
251
IMKKAIERGSLMGCSIDTIIPVQYETRMACGLVRGHAYSVTGLDEVPFKGEKVKLVRLRN
310




|+|| +|||||+|||||   ||  | ||| |||+||||||||+ || ++|| |||+||||


Sbjct:
181
IIKKMLERGSLLGCSIDITSPVDMEARMAKGLVKGHAYSVTGVKEVNYRGEGVKLIRLRN
240


Query:
311
PWGQVEWNGSWSDRWKDWSFVDKDEKARLQHQVTEDGEFWMSYEDFIYHFTKLEICNLT
369




||||||| | |||   ||+ || ||||||| +  ||||||||+|||+ ||++|||||||


Sbjct:
241
PWGQVEWTGDWSDSSPDWNIVDPDEKARLQLKF-EDGEFWMSFEDFLRHFSRLEICNLT
298
















TABLE 26H





Domain Analysis of NOV26















gnl|Smart|smart00230, Cyspc, Calpain-like thiol protease family.;


Calpain-like thiol protease family (peptidase family C2). Calcium


activated neutral protease (large subunit). (SEQ ID NO:262)


CD-Length = 323 residues, 100.0% aligned


Score = 491 bits (1263), Expect = 8e−140










Query:
56
VKEKTFEQLHKKCLEKKVLYVDPEFPPDETSLFYSQKFPIQFVWKRPPEICENPRFIIDG
115




 + + +|+| ++|||+  |+||| ||   +|||+||      ||||| || |+|  |+ |


Sbjct:
1
FENQDYEELRQECLEEGGLFVDPLFPAKPSSLFFSQLQRKFVVWKRPHEIFEDPPLIVGG
 60


Query:
116
ANRTDICQGELGDCWFLAAIACLTLNQHLLFRVIPHDQSFIENYAGIFHFQFWRYGEWVD
175




|+||||||| ||||| |||+| ||| + || |||| || | ||||||+||+|||||+|||


Sbjct:
61
ASRTDICQGVLGDCWLLAALAALTLREELLARVIPKDQEFSENYAGIYHFRFWRYGKWVD
120


Query:
176
VVIDDCLPTYNNQLVFTKSNHRNEFWSALLEKAYAKLHGSYEALKGGNTTEAMEDFTGGV
235




||||| |||||  |+|  || |||||||||||||||| | |||||||+||||+|| ||||


Sbjct:
121
VVIDDRLPTYNGDLLFMHSNSRNEFWSALLEKAYAKLRGCYEALKGGSTTEALEDLTGGV
180


Query:
236
AEFFEIRDA---PSDMYKIMKKAIERGSLMGCSIDTIIPVQYETRMACGLVRGHAYSVTG
292




||  |++     | +++| +||| ||||||||||     |+ | +   |||+|||||||


Sbjct:
181
AESIELKKISKDPDELFKDLKKAFERGSLNGCSIGAGTAVEEEEQKRNGLVKGHAYSVTD
240


Query:
293
LDEVPFKGEKVKLVRLRNPWGQVEWNGSWSDRWKDWSFVDKDEKARLQHQVTEDGEFWMS
352




+ ||  +    ||+|||||||+ |||| |||   +|  |  +||  |   + +|||||||


Sbjct:
241
VREVDGRRR-QKLLRLRNPWGESEWNGPWSDDSPEWRSVSAEEKKNLGLTMDDDGEFWMS
299


Query:
353
YEDFIYHFTKLEICNLTADALQSD
376




+|||+ ||||+|||||  |  +


Sbjct:
300
FEDFLRHFTKVEICNLRPDWFEYR
323
















TABLE 26I





Domain Analysis of NOV26















gnl Pfam pfam01067, Calpain_III, Calpain large subunit, domain III.


The function of the domain III and I are currently unknown. Domain II


is a cysteine protease and domain IV is a calcium binding domain.


Calpains are believed to participate in intracellular signaling


pathways mediated by calcium ions. (SEQ ID NO:263)


CD-Length = 148 residues, 100.0% aligned


Score = 223 bits (567), Expect = 4e−59










Query:
380
TWTVSVNEGRWVRGCSAGGCRNFPDTFWTNPQYRLKLLEEDDDPDDSEVICSFLVALMQK
439




 |  ++ || | || +||||||+|||||||||| + | | ||| +||   || |||||||


Sbjct:
1
KWEEAIVEGEWTRGSTAGGCRNYPDTFWTNPQYIITLPEPDDDDEDS---CSVLVALMQK
 57


Query:
440
NRRKDRKLGASLFTIGFAIYEVPKEMHGNKQHLQKDFFLYNASKARSKTYINMREVSQRF
499




+||++|++|| + |||||+|+|| |++    || +|||||| |+||| ||||+|||| ||


Sbjct:
58
DRRRERRMGADMLTIGFAVYKVPDELN----HLSRDFFLYNQSRARSSTYINLREVSLRF
113


Query:
500
RLPPSEYVIVPSTYEPHQEGEFILRVFSEKRNLSE
534




|||| |||+||||+||++||||||||||||   +


Sbjct:
114
RLPPGEYVLVPSTFEPNEEGEFILRVFSEKPINTR
148
















TABLE 26J





Domain Analysis of NOV26















gnl|Pfam pfam00036, efhand, EF hand. The EF-hands


can be divided into two classes: signaling


proteins and buffering/transport proteins. The


first group is the largest and includes the most


well-known members of the family such as


calmodulin, troponin C and S100B. These proteins


typically undergo a calcium-dependent conforma-


tional change which opens a target binding site.


The latter group is represented by calbindin D9k


and do not undergo calcium dependent conforma-


tional changes. (SEQ ID NO:264)


CD-Length = 29 residues, 96.6% aligned


Score = 36.2 bits (82), Expect = 0.007










Query:
649
CRSMIALMDTDGSGKLNLQEFHHLWNKI
676




 + +    | || ||++ +||  |  |+


Sbjct:
2
LKEIFKEFDKDGDGKISFEEFKELLKKL
 29









Calpains are intracellular cysteine proteases that are regulated by calcium. Cysteine protease activity is dependent on an active dyad of cysteine and histidine, the order and spacing of these residues varying in the 20 or so known families. Families C1, C2 and C10 are loosely termed papain-like, and nearly half of all cysteine proteases are found exclusively in viruses. Calpain is an intracellular protease involved in many important cellular functions that are regulated by calcium.
The protein is a complex of 2 polypeptide chains (light and heavy), with three known forms in mammals: a highly calcium-sensitive (i.e., micro-molar range) form known as mu-calpain, mu-CANP or calpain I; a form sensitive to calcium in the milli-molar range, known as m-calpain, m-CANP or calpain II; and a third form, known as p94, which is found in skeletal muscle only. All three forms have identical light but different heavy chains. The heavy chain comprises four domains: domain 2 contains the catalytic region; domain 4 binds calcium and regulates activity. Domain 2 shows low levels of sequence similarity to papain; although the catalytic His has not been located by biochemical means, it is likely that calpain and papain are related. Domain 4 has four EF hand calcium-binding regions and is simmilar to sorcin and the Ca2+-binding region of calpain light chain. Calpain shows preferential cleavage for Tyr- with leucine or valine as the P2 residue.
The product of the Drosophila gene sol has also been shown to be similar to calpain. Many calcium-binding proteins belong to the same evolutionary family and share a type of calcium-binding domain known as the EF-hand. This type of domain consists of a twelve residue loop flanked on both side by a twelve residue alpha-helical domain. In an EF-hand loop the calcium ion is coordinated in a pentagonal bipyramidal configuration. The six residues involved in the binding are in positions 1, 3, 5, 7, 9 and 12; these residues are denoted by X, Y, Z, −Y, −X and −Z. The invariant Glu or Asp at position 12 provides two oxygens for liganding Ca (bidentate ligand).
Calpains are known to be involved in a number of cellular processes, such as apoptosis, protein processing, cell differentiation, metabolism etc. As such, their role in pathophysiologies extends to—but is not restricted to—tissue remodeling and regeneration (in response to a variety of injury models in the eye, brain, spinal cord, kidney etc.), fertility, tumorigenesis and myopathies. One of the genes identified in susceptibility to type II diabetes is a calpain (calpain-10). Polymorphisms within this gene are correlated with insulin resistance. Therapies targeting calpain are relevant to disease areas such as cataract, spinal cord injury, Alzheimer's disease, muscular dystrophy, acoustic trauma, diabetes, cancer, learning and memory defects and infertility. Knockout and transgenic models of various calpains also point to a potential role for this family of proteases in a number of cellular and disease processes.
The NOV26 nucleic acid of the invention encoding a Calpain-like protein includes the nucleic acid whose sequence is provided in Table 26A, or a fragment thereof. The invention also includes a mutant or variant nucleic acid any of whose bases may be changed from the corresponding base shown in Table 26A while still encoding a protein that maintains its Calpain-like activities and physiological functions, or a fragment of such a nucleic acid. The invention further includes nucleic acids whose sequences are complementary to those just described, including nucleic acid fragments that are complementary to any of the nucleic acids just described. The invention additionally includes nucleic acids or nucleic acid fragments, or complements thereto, whose structures include chemical modifications. Such modifications include, by way of non-limiting example, modified bases, and nucleic acids whose sugar phosphate backbones are modified or derivatized. These modifications are carried out at least in part to enhance the chemical stability of the modified nucleic acid, such that they may be used, for example, as antisense binding nucleic acids in therapeutic applications in a subject. In the mutant or variant nucleic acids, and their complements, up to about 1% of the NOV26 residues may be so changed.
The NOV26 protein of the invention includes the Calpain-like protein whose sequence is provided in Table 26B. The invention also includes a mutant or variant protein any of whose residues may be changed from the corresponding residue shown in Table 26B while still encoding a protein that maintains its Calpain-like activities and physiological functions, or a functional fragment thereof. In the mutant or variant protein, up to about 1% of the NOV26 bases may be so changed.
The NOV26 nucleic acids and proteins of the invention are useful in potential diagnostic and therapeutic applications implicated in various diseases and disorders described below and/or other pathologies. For example, the compositions of the present invention will have efficacy for treatment of patients suffering from: obesity, diabetes, autoimmune disease, Von Hippel-Lindau (VHL) syndrome, cirrhosis, transplantation disorders, pancreatitis, renal artery stenosis, interstitial nephritis, glomerulonephritis, polycystic kidney disease, systemic lupus erythematosus, renal tubular acidosis, IgA nephropathy, hypercalcemia, Lesch-Nyhan syndrome, developmental defects, cataract, spinal cord injury, Alzheimer's disease, muscular dystrophy, acoustic trauma, cancer, learning and memory defects, infertility and other diseases, disorders and conditions of the like.
NOV26 nucleic acids and polypeptides are further useful in the generation of antibodies that bind immunospecifically to the novel substances of the invention for use in therapeutic or diagnostic methods. These antibodies may be generated according to methods known in the art, using prediction from hydrophobicity charts, as described in the “Anti-NOVX Antibodies” section below. For example the disclosed NOV26 protein have multiple hydrophilic regions, each of which can be used as an immunogen. This novel protein also has value in development of powerful assay system for functional analysis of various human disorders, which will help in understanding of pathology of the disease and development of new drug targets for various disorders.
NOV27
A disclosed NOV27 nucleic acid of 1327 nucleotides (also referred to as CG57484-01) encoding a novel Keratin 18-like protein is shown in Table 27A. An open reading frame was identified beginning with an ATG initiation codon at nucleotides 67-69 and ending with a TAA codon at nucleotides 1273-1275. Putative untranslated regions upstream from the intitation codon and downstream from the termination codon are underlined in Table 27A, and the start and stop codons are in bold letters.







TABLE 27A





NOV27 Nucleotide Sequence















(SEQ ID NO:87)







GCTTCACCACTCCCTCCACCTTCTCCACCAACTACCAGTCCCTGGGCTCTGTCCAGCCGCCCAGCTATGGCAC


CTGGCCGGTCAGCAGCGCAGCCAGCATCTATGCAGGCACTGGGGGGCTTGGGATCCCAGATCTCCATGTCCTG


TTCTACCAGTTTCTGGGGCGGCTTGGGGTCTGGGGGCCTGGCCACAGAGATGGCTGGGGGTCTGGCAGAAATG


GGGGGCATCCAGAATGAGAAGGAGACCATGCAAAGCCTGAACGACCACCTGGACTACCTGGACAGAGTGAGGA


ACCTGGAGACCGAGAACTGGAGGCTGGAGAGCAAAATCCAGGAGTATCTGGAGAAGAGACCCCATGTCAGAGA


CTGGGGCCATTACTTCAAGACCATCAAGGAACTGAGGGCTCAGATCTTCGCAAATACTGTGGACAATGTCCAC


ATCATTCTGCAGATCGACAATGCCCGTCTTGCTGCTGATGACTTCAGAGTCAAGTATGAGACAAGAGCTGGCC


ATGCGCCAGTCTGTGGAGAGAACATCCATGGGCTCTCCAAGGTCATTGATGACACCAATGTCACTCTGCTGCA


GCTGGAGACAGAGATGGGCGCTCTCAAGGAGGAGCTGCTCCTCATGAAGAAGAACCATGAAGAGGAAGTAAAA


GGCTTGCAAGTCCAGATTGCCAACTCTGGGTTGGCCGTGGAGGTAGATGCCCCCAAATCTCAAGTCCTCGCCA


AGGTCATGGCAGACATCAGGGCCCAAGATGAGCTGTCTCAGAAGAACTCAGAGAAGCTAGGCAAGTACTGGTC


TCAGCAGACTGAGGAGAGCACCACAGTGGTCACCACACACTCTGCCAAGGTCAGAGCTGCTGAGATGACAACG


GAGCTGAGACGTACAGTCCAGTGCTTGGAGATTGACCTGGACTCAATGAGAAATCTGAAGACCAGCTTGAACA


GCCTGAGGGAGGTGGAGGCCCGCTACGCCCTGCAGATGGAGCAGCTCAACAGAATCCTGCTGTACTTGGAGTC


AAAGCTGGCACAGAACTGGGCAGAGGGCCAGCGCAAGGTCCAGGAGTACAAGGACTTGCTGAACATCAGGGTC


AAGCTGGAGGCTGAGATCGCCACCTACCGCCGCCTGCTGGAAGACAGCGAGGGCCTCAATCTTGGTGATGCCC


TGGACAGCAGCAACTCCATGCAAACCATCCAAAAGACCACCACCCGCCAGATAGTGGATAGCAAAGTGGTGTC


TGAGATCAGTGACACCAAAGTTCTGAGACATTAAGCCAGCAGAAGCAGGGTACCCTGTGGGGAGTAAGAGGCC


AATAAAAAGTTCA









The NOV27 nucleic acid was identified on chromosome 12 and has 367 of 415 bases (88%) identical to a gb:GENBANK-ID:AF179904|acc:AF179904.1 mRNA from Homo sapiens (Homo sapiens keratin 18 (KRT18) gene, complete cds) (E=5.1e−180).
A disclosed NOV27 polypeptide (SEQ ID NO:88) encoded by SEQ ID NO:87 is 402 amino acid residues and is presented using the one-letter code in Table 27B. Signal P, Psort and/or Hydropathy results predict that NOV27 contains a signal peptide and is likely to be localized to the cytoplasm with a certainty of 0.4500.







TABLE 27B





Encoded NOV27 protein sequence















(SEQ ID NO:88)







MAPGRSAAQPASMQALGGLGSQISMSCSTSFWGGLGSGGLATEMAGGLAEMGGIQNEKETMQSLNDHLDYLD


RVRNLETENWRLESKIQEYLEKPRHVRDWGHYFKTIKELRAQIFANTVDNVHIILQIDNARLAADDFRVKYE


TRAGHAPVCGENIHGLCKVIDDTNVTLLQLETEMGALKEELLLMKKNHEEEVKGLQVQIANSGLAVEVDAPK


SQVLAKVMADIRAQDELSQKNSEKLGKYWSQQTEESTTVVTTHSAKVRAAEMTTELRRTVQCLEIDLDSMRN


LKTSLNSLREVEARYALQMEQLNRILLYLESKLAQNWAEGQRKVQEYKDLLNIRVKLEAEIATYRRLLEDSE


GLNLGDALDSSNSMQTIQKTTTRQIVDSKVVSEISDTKVLRH









The NOV27 amino acid sequence 315 of 404 amino acid residues (77%) identical to, and 344 of 404 amino acid residues (85%) similar to, the 429 amino acid residue ptnr:SWISSNEW-ACC:P05783 protein from Homo sapiens (Human) (Keratin, Type I Cytoskeletal 18 (Cytokeratin 18) (K18) (CK 18)) (E=2.9e−151).
NOV27 is expressed in at least the following tissues: adrenal gland, bone marrow, brain—amygdala, brain—cerebellum, brain—hippocampus, brain—substantia nigra, brain—thalamus, brain—whole, fetal brain, fetal kidney, fetal liver, fetal lung, heart, kidney, lymphoma—Raji, mammary gland, pancreas, pituitary gland, placenta, prostate, salivary gland, skeletal muscle, small intestine, spinal cord, spleen, stomach, testis, thyroid, trachea and uterus. This information was derived by determining the tissue sources of the sequences that were included in the invention including but not limited to SeqCalling sources, Public EST sources, genomic clone sources, literature sources, and/or RACE sources.
NOV27 has homology to the amino acid sequences shown in the BLASTP data listed in Table 27C.







TABLE 27C







BLAST results for NOV27
















Pos-



Gene Index/
Protein/
Length
Identity
itives



Identifier
Organism
(aa)
(%)
(%)
Expect





gi|4557888|ref|NP_0
keratin 18
430
303/404
332/
e−142


00215.1|
[Homo

(75%)
404


(NM_000224)
sapiens]


(82%)


gi|106851|pir||S068
keratin 18,
424
303/404
332/
e−142


89
cytoskeletal

(75%)
404



[Homo


(82%)



sapiens]


gi|12653819|gb|
keratin 18
430
302/404
332/
e−141


AAH00698.1|
[Homo

(74%)
404


AAH00698
sapiens]


(81%)


(BC000698)


gi|14602495|gb|
Similar to
375
286/372
310/
e−132


AAH09754.1|
keratin 18

(76%)
372


AAH09754
[Homo


(82%)


(BC009754)
sapiens]


gi|532610|gb|
cytokeratin
423
268/405
313/
e−121


AAA375 52.1|
[Mus

(66%)
405


(M22832)
musculus]


(77%)









The homology of these sequences is shown graphically in the ClustalW analysis shown in Table 27D.







TABLE 27E





Clustal W Sequence Alignment















1) NOV27 (SEQ ID NO:88)


2) gi 4557888|refNP_000215.1|(NM_000224) keratin 18 [Homo sapiens] (SEQ ID NO:265)


3) gi 106851|pir||S06889 keratin 18, cytoskeletal [Homo sapiens] (SEQ ID NO:266)


4) gi 12653819|gb AAH00698.1 AAH00698 (BC000698) keratin 18 [Homo sapiens]


(SEQ ID NO:267)


5) gi 14602495|gb|AAH09754.1|AAH09754 (BC009754) Similar to keratin 18 [Homo sapiens]


(SEQ ID NO:268)


6) gi 532610|gb AAA37552.1|(M22832) cytokeratin [Mus musculus] (SEQ ID NO:269)


























































Table 27E lists the domain description from DOMAIN analysis results against NOV27. This indicates that the NOV27 sequence has properties similar to those of other proteins known to contain these domains.







TABLE 27E





Domain Analysis of NOV27















gnl Pfam.pfam00038, filament, Intermediate filament protein. (SEQ ID


NO:270)


CD-Length = 312 residues, 99.4% aligned


Score = 204 bits (520), Expect = 6e−54










Query:
56
NEKETMQSLNDEL-DYLDRVRNLETENWRLESKIQEYLEKRPHV--RDWGHYFKTIKELR
112




|||| ||+||| |  |+|+|| || +|  || ||+|  +|+     | +  |   |+|||


Sbjct:
1
NEKEQMQNLNDRLASYIDKVRFLEQQNKELEVKIEELRQKQAPSVSRLYSLYETEIEELR
 60


Query:
113
AQIFANTVDNVHIILQIDNARLAADDFRVKYETRAGHAPVCGENIHGLCKVIDDTNVTLL
172




 ||   | +   + |+||| | ||+||| |||           ++ || | +|+  +  +


Sbjct:
61
RQIDQLTNERARLQLEIDNLREAAEDFRKKYEDEINLRQEAENDLVGLRKDLDEATLARV
120


Query:
173
QLETEMGALKEELLLMKKNHEEEVKGLQVQIANSGLAVEVDAPKSQVLAKVMADIRAQ-D
231




 || ++ +|+|||  +||||||||| || || ++   ||+|| +   | | + +|||| +


Sbjct:
121
DLENKVESLQEELEFLKKNHEEEVKELQAQIQDTVN-VEMDAARKLDLTKALREIRAQYE
179


Query:
232
ELSQKNSEKLGKYWSQQTEESTTVVTTHSAKVRAA-EMTTELRRTVQCLEIDLDSMRNLK
290




|+++|| ++  +++  + ||  |    +   +|+| |  ||||| +| |||+| |++


Sbjct:
180
EIAKKNRQEAEEWYKSKLEELQTAAARNGEALRSAKEEITELRRQIQSLEIELQSLKAQN
239


Query:
291
TSL-NSLREVEARYALQMEQLNRILLYLESKLAQNWAEGQRKVQEYKDLLNIRVKLEAEI
349




 ||   | |+| || |++ |   ++  || +| |   |  |+++||++||++++ |+ ||


Sbjct:
240
ASLERQLAELEERYELELRQYQALISQLEEELQQLREEMARQLREYQELLDVKLALDIEI
299


Query:
350
ATYRRLLEDSE
360




||||+|||  |


Sbjct:
300
ATYRKLLEGEE
310









Keratin 8 (K8) and keratin 18 (K18) are the most common and characteristic members of the large intermediate filament gene family expressed in ‘simple’ or single layer epithelial tissues of the body. Their persistent expression in tumor cells derived from these epithelia has led to the wide spread use of keratin monoclonal antibodies as aids in the detection and identification of carcinomas. Oncogenes which activate ras signal transduction pathways stimulate expression of the K18 gene through transcription factors including members of the AP-1 (jun and fos) and ETS families. The persistent expression of K8 and K18 may reflect the integrated transcriptional activation of such transcription factors and, in the cases of ectopic expression, an escape from the suppressive epigenetic mechanisms of DNA methylation and chromatin condensation. Comparison of the mechanisms of transcriptional control of K18 expression with expression patterns documented in both normal and pathological conditions leads to the proposal that persistent K8 and K18 expression is a reflection of the action of multiple different oncogenes converging on the nucleus through a limited number of transcription factors to then influence the expression of a large number of genes including these keratins. Furthermore, correlation of various tumor cell characteristics including invasive behavior and drug sensitivity with K8 and K18 expression has stimulated consideration of the possible functions of these proteins in both normal development and in tumorigenesis. Recent developments in the analysis of the functions of these intermediate filament proteins provide new insights into diverse functions influenced by K8 and K18 (Oshima et al., Cancer Metastasis Rev 15:445-71, 1996).
Keratin 8 (K8) and keratin 18 (K18) form intermediate filaments characteristic of liver and other single cell layered, internal epithelia and their derivative cancers. K8-deficient (K8(−)) mice, which escape embryonic lethality, develop inflammatory colorectal hyperplasia, mild liver abnormalities, and tolerate hepatectomy poorly. Normal and malignant epithelial cells deficient in K8 and K18 have been shown to be approximately 100 times more sensitive to TNF-induced death. K8 and K18 both bind the cytoplasmic domain of TNFR2 and moderate TNF-induced, Jun NH(2)-terminal kinase (JNK) intracellular signaling and NFkappaB activation. Furthermore, K8(−) and K18(−) mice are much more sensitive to TNF dependent, apoptotic liver damage induced by the injection of concanavalin A. This moderation of the effects of TNF may be the fundamental function of K8 and K18 common to liver regeneration, inflammatory bowel disease, hepatotoxin sensitivity, and the diagnostic, persistent expression of these keratins in many carcinomas (Caulin et al., J Cell Biol 149:17-22, 2000).
The NOV27 nucleic acid of the invention encoding a Keratin 18-like protein includes the nucleic acid whose sequence is provided in Table 27A, or a fragment thereof. The invention also includes a mutant or variant nucleic acid any of whose bases may be changed from the corresponding base shown in Table 27A while still encoding a protein that maintains its Keratin 18-like activities and physiological functions, or a fragment of such a nucleic acid. The invention further includes nucleic acids whose sequences are complementary to those just described, including nucleic acid fragments that are complementary to any of the nucleic acids just described. The invention additionally includes nucleic acids or nucleic acid fragments, or complements thereto, whose structures include chemical modifications. Such modifications include, by way of non-limiting example, modified bases, and nucleic acids whose sugar phosphate backbones are modified or derivatized. These modifications are carried out at least in part to enhance the chemical stability of the modified nucleic acid, such that they may be used, for example, as antisense binding nucleic acids in therapeutic applications in a subject. In the mutant or variant nucleic acids, and their complements, up to about 12% of the NOV27 residues may be so changed.
The NOV27 protein of the invention includes the Keratin 18-like protein whose sequence is provided in Table 27B. The invention also includes a mutant or variant protein any of whose residues may be changed from the corresponding residue shown in Table 27B while still encoding a protein that maintains its Keratin 18-like activities and physiological functions, or a functional fragment thereof. In the mutant or variant protein, up to about 23% of the NOV27 bases may be so changed.
The NOV27 nucleic acids and proteins of the invention are useful in potential diagnostic and therapeutic applications implicated in various diseases and disorders described below and/or other pathologies. For example, the compositions of the present invention will have efficacy for treatment of patients suffering from: inflammatory colorectal hyperplasia, mild liver abnormalities, skin abnormalities, inflammatory bowel disease, hepatotoxin sensitivity, chronic hepatitis, breast cancer, pancreatic cancer, mammary gland tumours, benign prostatic hyperplasia, prostate cancer, cryptogenic cirrhosis and other diseases, disorders and conditions of the like.
NOV27 nucleic acids and polypeptides are further useful in the generation of antibodies that bind immunospecifically to the novel substances of the invention for use in therapeutic or diagnostic methods. These antibodies may be generated according to methods known in the art, using prediction from hydrophobicity charts, as described in the “Anti-NOVX Antibodies” section below. For example the disclosed NOV27 protein have multiple hydrophilic regions, each of which can be used as an immunogen. This novel protein also has value in development of powerful assay system for functional analysis of various human disorders, which will help in understanding of pathology of the disease and development of new drug targets for various disorders.
NOV28
A disclosed NOV28 nucleic acid of 12992 nucleotides (also referred to as CG57236-01) encoding a novel Polycystic Kidney Disease Associated-like protein is shown in Table 28A. An open reading frame was identified beginning with an ATG initiation codon at nucleotides 53-55 and ending with a TAG codon at nucleotides 12950-12952. Putative untranslated regions upstream from the intitation codon and downstream from the termination codon are underlined in Table 28A, and the start and stop codons are in bold letters.







TABLE 28A





NOV28 Nucleotide Sequence















(SEQ ID NO:89)







CGCGCGCGGGCGGGCCTGGGGACGGCGGGGCCATGCGCGCGCTGCCCTAACGATGCCGCCCGCCGCGCCCGCC


CGCCTGGCGCTGGCCCTGGGCCTGGGCCTGTGGCTCGGGGCGCTGGCGGGGGGCCCCGGGCGCGGCTGCGGGC


CCTGCGAGCCCCCCTGCCTCTGCGGCCCAGCGCCCGGCGCCGCCTGCCGCGTCAACTGCTCGGGCCGCGGGCT


GCGGACGCTCGGTCCCGCGCTGCGCATCCCCGCGGACGCCACAGCGCTAGACGTCTCCCACAACCTGCTCCGG


GCGCTGGACGTTGGGCTCCTGGCGAACCTCTCGGCGCTGGCAGAGCTGGATATAAGCAACAACAAGATTTCTA


CGTTAGAAGAAGGAATATTTGCTAATTTATTTAATTTAAGTGAAATAAACCTGAGTGGGAACCCGTTTGAGTG


TGACTGTGGCCTGGCGTGGCTGCCGCGATGGGCGGAGGAGCAGCAGGTGCGGGTGGTGCAGCCCGAGGCAGCC


ACGTGTGCTGGGCCTGGCTCCCTGGCTGGCCAGCCTCTGCTTGGCATCCCCTTGCTGGACAGTGGCTGTGGTG


AGGAGTATGTCGCCTGCCTCCCTGACAACAGCTCAGGCACCGTGGCAGCAGTGTCCTTTTCAGCTGCCCACGA


AGGCCTGCTTCAGCCAGAGGCCTGCAGCGCCTTCTGCTTCTCCACCGGCCAGGGCCTCGCAGCCCTCTCGGAG


CAGGGCTGGTGCCTGTGTGGGGCGGCCCAGCCCTCCAGTGCCTCCTTTGCCTGCCTGTCCCTCTGCTCCGGCC


CCCCGCCACCTCCTGCCCCCACCTGTAGGGGCCCCACCCTCCTCCAGCACGTCTTCCCTGCCTCCCCAGGGGC


CACCCTGGTGGGGCCCCACGGACCTCTGGCCTCTGGCCAGCTAGCAGCCTTCCACATCGCTGCCCCGCTCCCT


GTCACTGCCACACGCTGGGACTTCGGAGACGGCTCCGCCGAGGTGGATGCCGCTGGGCCGGCTGCCTCGCATC


GCTATGTGCTGCCTGGGCGCTATCACGTGACGGCCGTGCTGGCCCTGGGGGCCGGCTCAGCCCTGCTGGGGAC


AGACGTGCAGGTGGAAGCGGCACCTGCCGCCCTGGAGCTCGTGTGCCCGTCCTCGGTGCAGAGTGACGAGAGC


CTTGACCTCAGCATCCAGAACCGCGGTGGTTCAGGCCTGGAGGCCGCCTACAGCATCGTGGCCCTGGGCGAGG


AGCCGGCCCGAGCGGTGCACCCGCTCTGCCCCTCGGACACGGAGATCTTCCCTGGCAACGGGCACTGCTACCG


CCTGGTGGTGGAGAAGGCGGCCTGGCTGCAGGCGCAGGAGCAGTGTCAGGCCTGGGCCGGGGCCGCCCTGGCA


ATGGTGGACAGTCCCGCCGTGCAGCGCTTCCTGGTCTCCCGGGTCACCAGGAGCCTAGACGTGTGGATCGGCT


TCTCGACTGTGCAGGGGGTGGAGGTGGGCCCAGCGCCGCAGGGCGAGGCCTTCAGCCTGGAGAGCTGCCAGAA


CTGGCTGCCCGGGGAGCCACACCCAGCCACAGCCGAGCACTGCGTCCGGCTCGGGCCCACCGGGTGGTGTAAC


ACCGACCTGTGCTCAGCGCCGCACAGCTACGTCTGCGAGCTGCAGCCCGGAGGCCCAGTGCAGGATGCCGAGA


ACCTCCTCGTGGGAGCGCCCAGTGGGGACCTGCAGGGACCCCTGACGCCTCTGGCACAGCAGGACGGCCTCTC


AGCCCCGCACGAGCCCGTGGAGGTCATGGTATTCCCGGGCCTGCGTCTGAGCCGTGAAGCCTTCCTCACCACG


GCCGAATTTGGGACCCAGGAGCTCCGGCGGCCCGCCCAGCTGCGGCTGCAGGTGTACCGGCTCCTCAGCACAG


CAGGGACCCCGGAGAACGGCGAGCGAGCCTGAGAGCAGGTCCCCGGACAACAGGACCCGCTGGCCCCCGCGTG


CATGCCAGGGGGACGCTGGTGCCCTGGAGCCAACATCTGCTTGCCGCTGGACGCCTCTTGCCACCCCCAGGCC


TGCGCCAATGGCTGCACGTCAGGGCCAGGGCTACCCGGGGCCCCCTATGCGCTATGGAGAGAGTTCCTCTTCT


CCGTTGCCGCGGGGCCCCCCGCGCAGTACTCGGTCACCCTCCACGGCCAGGATGTCCTCATGCTCCCTGGTGA


CCTCGTTGGCTTGCAGCACGACGCTGGCCCTGGCGCCCTCCTGCACTGCTCGCCGGCTCCCGGCCACCCTGGT


CCCCAGGCCCCGTACCTCTCCGCCAACGCCTCGTCATGGCTGCCCCACTTGCCAGCCCAGCTGGAGGGCACTT


GGGCCTGCCCTGCCTGTGCCCTGCGGCTGCTTGCAGCCACGGAACAGCTCACCGTGCTGCTGGGCTTGAGGCC


CAACCCTGGACTGCGGATGCCTGGGCGCTATGAGGTCCGGGCAGAGGTGGGCAATGGCGTGTCCAGGCACAAC


CTCTCCTGCAGCTTTGACGTGGTCTCCCCAGTGGCTGGGCTGCGGGTCATCTACCCTGCCCCCCGCGACGGCC


GCCTCTACGTGCCCACCAACGGCTCAGCCTTGGTGCTCCAGGTGGACTCTGGTGCCAACGCCACGGCCACGGC


TCGCTGGCCTGGGGGCAGTGTCAGCGCCCGCTTTGAGAATGTCTGCCCTGCCCTGGTGGCCACCTTCGTGCCC


GGCTGCCCCTGGGAGACCAACGATACCCTGTTCTCAGTGGTAGCACTGCCGTGGCTCAGTGAGGGGGAGCACG


TGGTGGACGTGGTGGTGGAAAACAGCGCCAGCCGGGCCAACCTCAGCCTGCGGGTGACGGCGGAGGAGCCCAT


CTGTGGCCTCCGCGCCACGCCCAGCCCCGAGGCCCGTGTACTGCAGGGAGTCCTAGTGAGGTACAGCCCCGTG


GTGGAGGCCGGCTCGGACATGGTCTTCCGGTGGACCATCAACGACAAGCAGTCCCTGACCTTCCAGAACGTGG


TCTTCAATGTCATTTATCAGAGCGCGGCGGTCTTCAAGCTCTCACTGACGGCCTCCAACCACGTGAGCAACGT


CACCGTGAACTACAACGTAACCGTGGAGCGGATGAACAGGATGCAGGGTCTGCAGGTCTCCACAGTGCCGGCC


GTGCTGTCCCCCAATGCCACGCTAGCACTGACGGCGGGCGTGCTGGTGGACTCGGCCGTGGAGGTGGCCTTCC


TGTGGAACTTTGGGGATGGGGAGCAGGCCCTCCACCAGTTCCAGCCTCCGTACAACGAGTCCTTCCCGGTTCC


AGACCCCTCGGTGGCCCAGGTGCTGGTGGAGCACAATGTCATGCACACCTACGCTGCCCCAGGTGAGTACCTC


CTGACCGTGCTGGCATCTAATGCCTTCGAGAACCTGACGCAGCAGGTGCCTGTGAGCGTGCGCGCCTCCCTGC


CCTCCGTGGCTGTGGGTGTGAGTGACGGCGTCCTGGTGGCCGGCCGGCCCGTCACCTTCTACCCGCACCCGCT


GCCCTCGCCTGGGGGTGTTCTTTACACGTGGGACTTCGGGGACGGCTCCCCTGTCCTGACCCAGAGCCAGCCG


GCTGCCAACCACACCTATGCCTCGAGGGGCACCTACCACGTGCGCCTGGAGGTCAACAACACGGTGAGCGGTG


CGGCGGCCCAGGCGGATGTGCGCGTCTTTGAGGAGCTCCGCGGACTCAGCGTGGACATGAGCCTGGCCGTGGA


GCAGGGCGCCCCCGTGGTGGTCAGCGCCGCGGTGCAGACGGGCGACAACATCACGTGGACCTTCGACATGGGG


GACGGCACCGTGCTGTCGGGCCCGGAGGCAACAGTGGAGCATGTGTACCTGCGGGCACAGAACTGCACAGTGA


CCGTGGGTGCGGCCAGCCCCGCCGGCCACCTGGCCCGGAGCCTGCACGTGCTGGTCTTCGTCCTGGAGGTGCT


GCGCGTTGAACCCGCCGCCTGCATCCCCACGCAGCCTGACGCGCGGCTCACGGCCTACGTCACCGGGAACCCG


GCCCACTACCTCTTCGACTGGACCTTCGGGGATGGCTCCTCCAACACGACCGTGCGGGGGTGCCCGACGGTGA


CACACAACTTCACGCGGAGCGGCACGTTCCCCCTGGCGCTGGTGCTGTCCAGCCGCGTGAACAGGGCGCATTA


CTTCACCAGCATCTGCGTGGAGCCAGAGGTGGGCAACGTCACCCTGCAGCCAGAGAGGCAGTTTGTGCAGCTC


GGGGACGAGGCCTGGCTGGTGGCATGTGCCTGGCCCCCGTTCCCCTACCGCTACACCTGGGACTTTGGCACCG


AGGAAGCCGCCCCCACCCGTGCCAGGGGCCCTGAGGTGACGTTCATCTACCGAGACCCAGGCTCCTATCTTGT


GACAGTCACCGCGTCCAACAACATCTCTGCTGCCAATGACTCAGCCCTGGTGGAGGTGCAGGAGCCCGTGCTG


GTCACCAGCATCAAGGTCAATGGCTCCCTTGGGCTGGAGCTGCAGCAGCCGTACCTGTTCTCTGCTGTGGGCC


GTGGGCGCCCCGCCAGCTACCTGTGGGATCTGGGGGACGGTGGGTGGCTCGAGGGTCCGGAGGTCACCCACGC


TTACAACAGCACAGGTGACTTCACCGTTAGGGTGGCCGGCTGGAATGAGGTGAGCCGCAGCGAGGCCTGGCTC


AATGTGACGGTGAAGCGGCGCGTGCGGGGGCTCGTCGTCAATGCAAGCCGCACGGTGGTGCCCCTGAATGGGA


GCGTGAGCTTCAGCACGTCGCTGGAGGCCGGCAGTGATGTGCGCTATTCCTGGGTGCTCTGTGACCGCTGCAC


GCCCATCCCTGGGGGTCCTACCATCTCTTACACCTTCCGCTCCGTGGGCACCTTCAATATCATCGTCACGGCT


GAGAACGAGGTGGGCTCCGCCCAGGACAGCATCTTCGTCTATGTCCTGCAGCTCATAGAGGGGCTGCAGGTGG


TGGGCGGTGGCCGCTACTTCCCCACCAACCACACGGTACAGCTGCAGGCCGTGGTTAGGGATGGCACCAACGT


CTCCTACAGCTGGACTGCCTGGAGGGACAGGGGCCCGGCCCTGGCCGGCAGCGGCAAAGGCTTCTCGCTCACC


GTGCTCGAGGCCGGCACCTACCATGTGCAGCTGCGGGCCACCAACATGCTGGGCAGCGCCTGGGCCGACTGCA


CCATGGACTTCGTGGAGCCTGTGGGGTGGCTGATGGTGACCGCCTCCCCGAACCCAGCTGCCGTCAACACAAG


CGTCACCCTCAGTGCCGAGCTGGCTGGTGGCAGTGGTGTCGTATACACTTGGTCCTTGGAGGAGGGGCTGAGC


TGGGAGACCTCCGAGCCATTTACCACCCATAGCTTCCCCACACCCGGCCTGCACTTGGTCACCATGACGGCAG


GGAACCCGCTGGGCTCAGCCAACGCCACCGTGGAAGTGGATGTGCAGGTGCCTGTGAGTGGCCTCAGCATCAG


GGCCAGCGAGCCCGGAGGCAGCTTCGTGGCGGCCGGGTCCTCTGTGCCCTTTTGGGGGCAGCTGGCCACGGGC


ACCAATGTGAGCTGGTGCTGGGCTGTGCCCGGCGGCAGCAGCAAGCGTGGCCCTCATGTCACCATGGTCTTCC


CGGATGCTGGCACCTTCTCCATCCGGCTCAATGCCTCCAACGCAGTCAGCTGGGTCTCAGCCACGTACAACCT


CACGGCGGAGGAGCCCATCGTGGGCCTGGTGCTGTGGGCCAGCAGCAAGGTGGTGGCGCCCGGGCAGCTGGTC


CATTTTCAGATCCTGCTGGCTGCCGGCTCAGCTGTCACCTTCCGCCTGCAGGTCGGCGGGGCCAACCCCGAGG


TGCTCCCCGGGCCCCGTTTCTCCCACAGCTTCCCCCGCGTCGGAGACCACGTGGTGAGCGTGCGGGGCAAAAA


CCACGTGAGCTGGGCCCAGGCGCAGGTGCGCATCGTGGTGCTGGAGGCCGTGAGTGGGCTGCAGATGCCCAAC


TGCTGCGAGCCTGGCATCGCCACGGGCACTGAGAGGAACTTCACAGCCCGCGTGCAGCGCGGCTCTCGGGTCG


CCTACGCCTGGTACTTCTCGCTGCAGAAGGTCCAGGGCGACTCGCTGGTCATCCTGTCGGGCCGCGACGTCAC


CTACACGCCCGTGGCCGCGGGGCTGTTGGAGATCCAGGTGCGCGCCTTCAACGCCCTGGGCAGTGAGAACCGC


ACGCTGGTGCTGGAGGTTCAGGACGCCGTCCAGTATGTGGCCCTGCAGAGCGGCCCCTGCTTCACCAACCGCT


CGGCGCAGTTTGAGGCCGCCACCAGCCCCAGCCCCCGGCGTGTGGCCTACCACTGGGACTTTGGGGATGGGTC


GCCAGGGCAGGACACAGATGAGCCCAGGGCCGAGCACTCCTACCTGAGGCCTGGGGACTACCGCGTGCAGGTG


AACGCCTCCAACCTGGTGAGCTTCTTCGTGGCGCAGGCCACGGTGACCGTCCAGGTGCTGGCCTGCCGGGAGC


CGGAGGTGGACGTGGTCCTGCCCCTGCAGGTGCTGATGCGGCGATCACAGCGCAACTACTTGGAGGCCCACGT


TGACCTGCGCGACTGCGTCACCTACCAGACTGAGTACCGCTGGGAGGTGTATCGCACCGCCAGCTGCCAGCGG


CCGGGGCGCCCAGCGCGTGTGGCCCTGCCCGGCGTGGACGTGAGCCGGCCTCGGCTGGTGCTGCCGCGGCTGG


CGCTGCCTGTGGGGCACTACTGCTTTGTGTTTGTCGTGTCATTTGGGGACACGCCACTGACACAGAGCATCCA


GGCCAATGTGACGGTGGCCCCCGAGCGCCTGGTGCCCATCATTGAGGGTGGCTCATACCGCGTGTGGTCAGAC


ACACGGGACCTGGTGCTGGATGGGAGCGAGTCCTACGACCCCAACCTGGAGGACGGCGACCAGACGCCGCTCA


GTTTCCACTGGGCCTGTGTGGCTTCGACACAGAGGGAGGCTGGCGGGTGTGCGCTGAACTTTGGGGCCCGCGG


GAGCAGCACGGTCACCATTCCACGGGAGCGGCTGGCGGCTGGCGTGGAGTACACCTTCAGCCTGACCGTGTGG


AAGGCCGGCCGCAAGGAGGAGGCCACCAACCAGACGGTGCTGATCCGGAGTGGCCGGGTGCCCATTGTGTCCT


TGGAGTGTGTGTCCTGCAAGGCACAGGCCGTGTACGAAGTGAGCCGCAGCTCCTACGTGTACTTGGAGGGCCG


CTGCCTCAATTGCAGCAGCGGCTCCAAGCGAGGGCGGTGGGCTGCACGTACGTTCAGCAACAAGACGCTGGTG


CTGGATGAGACCACCACATCCACGGGCAGTGCAGGCATGCGACTGGTGCTGCGGCGGGGCGTGCTGCGGGACG


GCGAGGGATACACCTTCACGCTCACGGTGCTGGGCCGCTCTGGCGAGGAGGAGGGCTGCGCCTCCATCCGCCT


GTCCCCCAACCGCCCGCCGCTGGGGGGCTCTTGCCGCCTCTTCCCACTGGGCGCTGTGCACGCCCTCACCACC


AAGGTGCACTTCGAATGCACGGGCTGGCATGACGCGGAGGATGCTGGCGCCCCGCTGGTGTACGCCCTGCTGC


TGCGGCGCTGTCGCCAGGGCCACTGCGAGGAGTTCTGTGTCTACAAGGGCAGCCTCTCCAGCTACGGAGCCGT


GCTGCCCCCGGGTTTCAGGCCACACTTCGAGGTGGGCCTGGCCGTGGTGGTGCAGGACCAGCTGGGAGCCGCT


GTGGTCGCCCTCAACAGGTCTTTGGCCATCACCCTCCCAGAGCCCAACGGCAGCGCAACGGGGCTCACAGTCT


GGCTGCACGGGCTCACCGCTAGTGTGCTCCCAGGGCTGCTGCGGCAGGCCGATCCCCAGCACGTCATCGAGTA


CTCGTTGGCCCTGGTCACCGTGCTGAACGAGTACGAGCGGGCCCTGGACGTGGCGGCAGAGCCCAAGCACGAG


CGGCAGCACCGAGCCCAGATACGCAAGAACATCACGGAGACTCTGGTGTCCCTGAGGGTCCACACTGTGGATG


ACATCCAGCAGATCGCTGCTGCGCTGGCCCAGTGCATGGGGCCCAGCAGGGAGCTCGTATGCCGCTCGTGCCT


GAAGCAGACGCTGCACAAGCTGGAGGCCATGATGCTCATCCTGCAGGCAGAGACCACCGCGGGCACCGTGACG


CCCACCGCCATCGGAGACAGCATCCTCAACATCACAGGAGACCTCATCCACCTGGCCAGCTCGGACGTGCGGG


CACCACAGCCCTCAGAGCTGGGAGCCGAGTCACCATCTCGGATGGTGGCGTCCCAGGCCTACAACCTGACCTC


TGCCCTCATGCGCATCCTCATGCGCTCCCGCGTGCTCAACGAGGAGCCCCTGACGCTGGCGGGCGAGGAGATC


GTGGCCCAGGGCAAGCGCTCGGACCCGCGGAGCCTGCTGTGCTATGGCGGCGCCCCAGGGCCTGGCTGCCACT


TCTCCATCCCCGAGGCTTTCAGCGGGGCCCTGGCCAACCTCAGTGACGTGGTGCAGCTCATCTTTCTGGTGGA


CTCCAATCCCTTTCCCTTTGGCTATATCAGCAACTACACCGTCTCCACCAAGGTGGCCTCGATGGCATTCCAG


ACACAGGCCGGCGCCCAGATCCCCATCGAGCGGCTGGCCTCAGAGCGCGCCATCACCGTGAAGGTGCCCAACA


ACTCGGACTGGGCTGCCCGGGGCCACCGCAGCTCCGCCAACTCCGTTGTGGTCCAGCCCCAGGCCTCCGTCGG


TGCTGTGGTCACCCTGGACAGCAGCAACCCTGCGGCCGTGCTGCATCTGCAGCTCAACTATACGCTGCTGGAC


GGCCACTACCTGTCTGAGGAACCTGAGCCCTACCTGGCAGTCTACCTACACTCGGAGCCCCGGCCCAATGAGC


ACAACTGCTCGGCTAGCAGGAGGATCCGCCCAGAGTCACTCCAGGGTGCTGACCACCGGCCCTACACCTTCTT


CATTTCCCCGGGGAGCAGAGACCCAGCGGGGAGTTACCATCTGAACCTCTCCAGCCACTTCCGCTGGTCGGCG


CTGCAGGTGTCCGTGGGCCTGTACACGTCCCTGTGCCAGTACTTCAGCGAGGAGGACATGGTGTGGCGGACAG


AGGGGCTGCTGCCCCTGGAGGAGACCTCGCCCCGCCAGGCCGTCTGCCTCACCCGCCACCTCACCGCCTTCGG


CGCCAGCCTCTTCGTGCCCCCAAGCCATGTCCGCTTTGTGTTTCCTGAGCCGACAGCGGATGTAAACTACATC


GTCATGCTGACATGTGCTGTGTGCCTGGTGACCTACATGGTCATGGCCGCCATCCTGCACAAGCTGGACCAGT


TGGATGCCAGCCGGGGCCGCGCCATCCCTTTCTGTGGGCAGCGGGGCCGCTTCAAGTACGAGATCCTCGTCAA


GACAGGCTGGGGCCGGGGCTCAGGTACCACGGCCCACGTGGGCATCATGCTGTATGGGGTGGACAGCCGGAGC


GGCCACCGGCACCTGGACGGCGACAGAGCCTTCCACCGCAACAGCCTGGACATCTTCCGGATCGCCACCCCGC


ACAGCCTGGGTAGCGTGTGGAAGATCCGAGTGTGGCACGACAACAAAGGGCTCAGCCCTGCCTGGTTCCTGCA


GCACGTCATCGTCAGGGACCTGCAGACGGCACGCAGCGCCTTCTTCCTGGTCAATGACTGGCTTTCGGTGGAG


ACGGAGGCCAACGGGGGCCTGGTGGAGAAGGAGGTGCTGGCCGCGAGCGACGCAGCCCTTTTGCGCTTCCGGC


GCCTGCTGGTGGCTGAGCTGCAGCGTGGCTTCTTTGACAAGCACATCTGGCTCTCCATATGGGACCGGCCGCC


TCGTAGCCGTTTCACTCGCATCCAGAGGGCCACCTGCTGCGTTCTCCTCATCTGCCTCTTCCTGGGCGCCAAC


GCCGTGTGGTACGGGGCTGTTGGCGACTCTGCCTACAGCACGGGGCATGTGTCCAGGCTGAGCCCGCTGAGCG


TCGACACAGTCGCTGTTGGCCTGGTGTCCAGCGTGGTTGTCTATCCCGTCTACCTGGCCATCCTTTTTCTCTT


CCGGATGTCCCGGAGCAAGGTGGCTGGGAGCCCGAGCCCCACACCTGCCGGGCAGCAGGTGCTGGACATCGAC


AGCTGCCTGGACTCGTCCGTGCTGGACAGCTCCTTCCTCACGTTCTCAGGCCTCCACGCTGAGGCCTTTGTTG


GACACATGAAGAGTGACTTGTTTCTGGATGATTCTAAGAGTCTGGTGTGCTGGCCCTCCGGCGAGGGAACGCT


CAGTTGGCCGGACCTGCTCAGTGACCCGTCCATTGTGGGTAGCAATCTGCGGCAGCTGGCACGGGGCCAGGCG


GGCCATGGGCTGGGCCCAGAGGAGGACGGCTTCTCCCTGGCCAGCCCCTACTCGCCTGCCAAATCCTTCTCAG


CATCAGATGAAGACCTGATCCAGCAGGTCCTTGCCGAGGGGGTCAGCAGCCCAGCCCCTACCCAAGACACCCA


CATGGAAACGGACCTGCTCAGCAGCCTGTCCAGCACTCCTGGGGAGAAGACAGAGACGCTGGCGCTGCAGAGG


CTGGGGGAGCTGGGGCCACCCAGCCCAGGCCTGAACTGGGAACAGCCCCAGGCAGCGAGGCTGTCCAGGACAG


GACTGGTGGAGGGTCTGCGGAAGCGCCTGCTGCCGGCCTGGTGTGCCTCCCTGGCCCACGGGCTCAGCCTGCT


CCTGGTGGCTGTGGCTGTGGCTGTCTCAGGGTGGGTGGGTGCGAGCTTCCCCCCGGGCGTGAGTGTTGCGTGG


CTCCTGTCCAGCAGCGCCAGCTTCCTGGCCTCATTCCTCGGCTGGGAGCCACTGAAGGTCTTGCTGGAAGCCC


TGTACTTCTCACTGGTGGCCAAGCGGCTGCACCCGGATGAAGATGACACCCTGGTAGAGAGCCCGGCTGTGAC


GCCTGTGAGCGCACGTGTGCCCCGCGTACGGCCACCCCACGGCTTTGCACTCTTCCTGGCCAAGGAAGAAGCC


CGCAAGGTCAAGAGGCTACATGGCATGCTGCGGAGCCTCCTGGTGTACATGCTTTTTCTGCTGGTGACCCTGC


TGGCCAGCTATGGGGATGCCTCATGCCATGGGCACGCCTACCGTCTGCAAAGCGCCATCAAGCAGGAGCTGCA


CAGCCGGGCCTTCCTGGCCATCACGCGGTCTGAGGAGCTCTGGCCATGGATGGCCCACGTGCTGCTGCCCTAC


GTCCACGGGAACCAGTCCAGCCCAGAGCTGGGGCCCCCACGGCTGCGGCAGGTGCGGCTGCAGGAAGCACTCT


ACCCAGACCCTCCCGGCCCCAGGGTCCACACGTGCTCGGCCGCAGGAGGCTTCAGCACCAGCGATTACGACGT


TGGCTGGGAGAGTCCTCACAATGGCTCGGGGACGTGGGCCTATTCAGCGCCGGATCTGCTGGGGGCATGGTCC


TGGGGCTCCTGTGCCGTGTATGACAGCGGGGGCTACGTGCAGGAGCTGGGCCTGAGCCTGGAGGAGAGCCGCG


ACCGGCTGCGCTTCCTGCAGCTGCACAACTGGCTGGACAACAGGAGCCGCGCTGTGTTCCTGGAGCTCACGCG


CTACAGCCCGCCCGTGGGGCTGCACGCCGCCGTCACGCTGCGCCTCGAGTTCCCGGCGGCCGGCCGCGCCCTG


GCCGCCCTCAGCGTCCGCCCCTTTGCGCTGCGCCGCCTCAGCGCGGGCCTCTCGCTGCCTCTGCTCACCTCGG


TGTGCCTGCTGCTGTTCGCCGTGCACTTCGCCGTGGCCGAGGCCCGTACTTGGCACAGGGAAGGGCGCTGGCG


CGTGCTGCGGCTCGGAGCCTGGGCGCGGTGGCTGCTGGTGGCGCTGACGGCGGCCACGGCACTGGTACGCCTC


GCCCAGCTGGGTGCCGCTGACCGCCAGTGGACCCGTTTCGTGCGCGGCCGCCCGCGCCGCTTCACTAGCTTCG


ACCAGGTGGCGCAGCTGAGCTCCGCAGCCCGTGGCCTGGCGGCCTCGCTGCTCTTCCTGCTTTTGGTCAAGGC


TGCCCAGCAGCTACGCTTCGTGCGCCAGTGGTCCGTCTTTGGCAAGACATTATGCCGAGCTCTGCCAGAGCTC


CTGGGGGTCACCTTGGGCCTGGTGGTGCTCGGGGTAGCCTACGCCCAGCTGGCCATCCTGCTCGTGTCTTCCT


GTGTGGACTCCCTCTGGAGCGTGGCCCAGGCCCTGTTGGTGCTGTGCCCTGGGACTGGGCTCTCTACCCTGTG


TCCTGCCGAGTCCTGGCACCTGTCACCCCTGCTGTGTGTGGGGCTCTGGGCACTGCGGCTGTGGGGCGCCCTA


CGGCTGGGGGCTGTTATTCTCCGCTGGCGCTACCACGCCTTGCGTGGAGAGCTGTACCGGCCGGCCTGGGAGC


CCCAGGACTACGAGATGGTGGAGTTGTTCCTGCGCAGGCTGCGCCTCTGGATGGGCCTCAGCAAGGTCAAGGA


GTTCCGCCACAAAGTCCGCTTTGAAGGGATGGAGCCGCTGCCCTCTCGCTCCTCCAGGGGCTCCAAGGTATCC


CCGGATGTGCCCCCACCCAGCGCTGGCTCCGATGCCTCGCACCCCTCCACCTCCTCCAGCCAGCTGGATGGGC


TGAGCGTGAGCCTGGGCCGGCTGGGGACAAGGTGTGAGCCTGAGCCCTCCCGCCTCCAAGCCGTGTTCGAGGC


CCTGCTCACCCAGTTTGACCGACTCAACCAGGCCACAGAGGACGTCTACCAGCTGGAGCAGCAGCTGCACAGC


CTGCAAGGCCGCAGGAGCAGCCGGGCGCCCGCCGGATCTTCCCGTGGCCCATCCCCGGGCCTGCGGCCAGCAC


TGCCCAGCCGCCTTGCCCGGGCCAGTCGGGGTGTGGACCTGGCCACTGGCCCCAGCAGGACACCCCTTCGGGC


CAAGAACAAGGTCCACCCCAGCAGCACTTAGTCCTCCTTCCTGGCGGGGGTGGGCCGTGGAGTCGGAGTGG









The NOV28 nucleic acid was identified on chromosome 16p13.3 and has 8732 of 8732 bases (100%) identical to a gb:GENBANK-ID:HUMPKD1A|acc:L33243.1 mRNA from Homo sapiens (Homo sapiens polycystic kidney disease 1 protein (PKD1) mRNA, complete cds) (E=0.0).
A disclosed NOV28 polypeptide (SEQ ID NO:90) encoded by SEQ ID NO:89 is 4299 amino acid residues and is presented using the one-letter code in Table 28B. Signal P, Psort and/or Hydropathy results predict that NOV28 contains a signal peptide and is likely to be localized to the plasma membrane with a certainty of 0.8200. The most likely cleaveage site for a NOV28 polypeptide is between amino acids 23 and 24: ALA-GG.







TABLE 28B





Encoded NOV28 protein sequence















(SEQ ID NO:90)







MPPAAPARLALALGLGLWLGALAGGPGRGCGPCEPPCLCGPAPGAACRVNCSGRGLRTLGPALRIPADATAL


DVSHNLLRALDVGLLANLSALAELDISNNKISTLEEGIFNALFNLSEINLSGNPFECDCGLAWLPRWEEQQ


VRVVQPEAATCAGPGSLAGQPLLGIPLLDSGCGEEYVACLPDNSSGTVAAVSFSAAHEGLLQPEACSAFCFS


TGQGLAALSEQGWCLCGAAQPSSASFACLSLCSGPPPPPAPTCRGPTLLQHVFPASPGATLVGPHGPLASGQ


LAAFHIAAPLPVTATRWDFGDGSAEVDAAGPAASHRYVLPGRYHVTAVLAIGAGSALLGTDVQVEAAPAALE


LVCPSSVQSDESLDLSIQNRGGSGLEAAYSIVALGEEPARAVHPLCPSDTEIFPGNGHCYRLVVEKAAWLQA


QEQCQAWAGAALAMVDSPAVQRFLVSRVTRSLDVWIGFSTVQGVEVGPAPQGEAFSLESCQNWLPGEPHPAT


AEHCVRLGPTGWCNTDLCSAPHSYVCELQPGGPVQDAENLLVGAPSGDLQGPLTPLAQQDGLSAPHEPVEVM


VFPGLRLSREAFLTTAEFGTQELRRPAQLRLQVRLLSTAGTPENGSEPESRSPDDNRTQLAPACMPGGRWCP


GANICLPLDASCHPQACANGCTSGPGLPGAPYALWREFLFSVAAGPPAQYSVTLHGQDVLMLPGDLVGLQHD


AGPGALLHCSPAPGHPGPQAPYLSANASSWLPHLPAQLEGTWACPACALRLLAATEQLTVLLGLRPNPGLRM


PGRYEVRAEVGNGVSRHNLSCSFDVVSPVAGLRVIYPAPRDGRLYVPTNGSALVLQVDSGANATATARWPGG


SVSARFENVCPALVATFVPGCPWETNDTLFSVVALPWLSEGEHVVDVVVENSASRANLSLRVTAEEPICGLR


ATPSPEARVLQGVLVRYSPVVEAGSDMVFRWTINDKQSLTFQNVVFNVIYQSAAVFKLSLTASNHVSNVTVN


YNVTVERNNRMQGLQVSTVPAVLSPNATLALTAGVLVDSAVEVAFLWNFGDGEQALHQFQPPYNESFPVPDP


SVAQVLVEHNVMHTYAAPGEYLLTVLASNAFENLTQQVPVSVRASLPSVAVGVSDGVLVAGRPVTFYPHPLP


SPGGVLYTWDFGDGSPVLTQSQPAANHTYASRGTYHVRLEVNNTVSGAAAQADVRVFEELRGLSVDMSLAVE


QGAPVVVSAAVQTGDNITWTFDMGDGTVLSGPEATVEHVYLRAQNCTVTVGAASPAGHLARSLHVLVFVLEV


LRVEPAACIPTQPDARLTAYVTGNPAHYLFDWTFGDSSNTTVRGCPTVTHNFTRSGTFPLALVLSSRVNRA


HYFTSICVEPEVNVTLQPERQFVQLGDEAWLVACAWPPFPYRYTWDFGTEEAAPTRARGPEVTFIYRDPGS


YLVTVTASNNISAANDSALVEVQEPVLVTSIKVNGSLGLELQQPYLFSAVGRGRPASYLWDLGDGGWLEGPE


VTHAYNSTGDFTVRVAGWNEVSRSEAWLNVTVKRRVRGLVVNASRTVVPLNGSVSFSTSLEAGSDVRYSWVL


CDRCTPIPGGPTISYTFRSVGTFNIIVTAENEVGSAQDSIFVYVLQLIEGLQVVGGGRYFPTNHTVQLQAVV


RDGTNVSYSWTAWRDRGPALAGSGKGFSLTVLEAGTYHVQLRATNMLGSAWADCTMDFVEPVGWLMVTASPN


PAAVNTSVTLSAELAGGSGVVYTWSLEEGLSWETSEPFTTHSFPTPGLHLVTMTAGNPLGSANATVEVDVQV


PVSGLSIRASEPGGSFVAACSSVPFWGQLATGTNVSWCWAVPGGSSKRGPHVTMVFPDAGTFSIRLNASNAV


SWVSATYNLTAEEPIVGLVLWASSKVVAPGQLVHFQILLAAGSAVTFRLQVGGANPEVLPGPRFSHSFPRVG


DHVVSVRGKNHVSWAQAQVRIVVLEAVSGLQMPNCCEPGIATGTERNFTARVQRGSRVAYAWYFSLQKVQGD


SLVILSGRDVTYTPVAAGLLEIQVRAFNALGSENRTLVLEVQDAVQYVALQSGPCFTNRSAQFEAATSPSPR


RVAYHWDFGDGSPGQDTDEPRAEHSYLRPGDYRVQVNASNLVSFFVAQATVTVQVLACREPEVDVVLPLQVL


MRRSQRNYLEAHVDLRDCVTYQTEYRWEVYRTASCQRPGRPARVALPGVDVSRPRLVLPRLALPVGHYCFVF


VVSFGDTPLTQSIQANVTVAPERLVPIIEGGSYRVWSDTRDLVLDGSESYDPNLEDGDQTPLSFHWACVAST


QREAGGCALNFGPRGSSTVTIPRERLAAGVEYTFSLTVWKAGRKEEATNQTVLIRSGRVPIVSLECVSCKAQ


AVYEVSRSSYVYLEGRCLNCSSGSKRGRWAARTFSNKTLVLDETTTSTGSAGMRLVLRRGVLRDGEGYTFTL


TVLGRSGEEEGCASIRLSPNRPPLGGSCRLFPLGAVHALTTKVHFECTGWHDAEDAGAPLVYALLLRRCRQG


HCEEFCVYKGSLSSYGAVLPPGFRPHFEVGLAVVVQDQLGAAVVALNRSLAITLPEPNGSATGLTVWLHGLT


ASVLPGLLRQADPQHVIEYSLALVTVLNEYERALDVAAEPKHERQHRAQIRKNITETLVSLRVHTVDDIQQI


AAALAQCMGPSRELVCRSCLKQTLHKLEAMMLILQAETTAGTVTPTAIGDSILNITGDLIHLASSDVRAPQP


SELGAESPSRMVASQAYNLTSALMRILMRSRVLNEEPLTLAGEEIVAQGKRSDPRSLLCYGGAPGPGCHFSI


PEAFSGALJANLSDVVQLIFLVDSNPFPFGYISNYTVSTKASMAFQTQACAQIPIERLASEPAITVKVPNNS


DWAARGHRSSANSVVVQPQASVGAVVTLDSSNPAAVLHLQLNYTLLDGHYLSEEPEPYLAVYLHSEPRPNEH


NCSASRRIRPESLQGADHRPYTFFISPGSRDPAGSYHLNLSSHFRWSALQVSVGLYTSLCQYFSEEDMVWRT


EGLLPLEETSPRQAVCLTRHLTAFGASLFVPPSHVRFVFPEPTADVNYIVMLTCAVCLVTYMVMAAILHKLD


QLDASRGRAIPFCGQRGRFKYEILVKTGWGRGSGTTAHVGIMLYGVDSRSGHRHLDGDRAFHRNSLDIFRIA


TPHSLGSVWKIRVWHDNKGLSPAWFLQHVIVRDLQTARSAFFLVNDWLSVETEANGGLVEKEVLAASDAALL


RFRRLLVAELQRGFFDKHIWLSIWDRPPRSRFTRIQRATCCVLLICLFLGANANWYGAVGDSAYSTGHVSRL


SPLSVDTVAVGLVSSVVVYPVYLAILFLFRMSRSKVAGSFSPTPAGQQVLDIDSCLDSSVLDSSFLTFSGLH


AFAFVGQMKSDLFLDDSKSLVCWPSGEGTLSWPDLLSDPSIVGSNLRQLARGQAGHGLGPEBDGFSLASPYS


PAKSFSASDEDLIQQVLAEGVSSPAPTQDTHMETDLLSSLSSTPGEKTETLALQRLCELGPPSPGLNWEQPQ


AARLSRTGLVEGLRKRLLPAWCASLAHGLSLLLVAVAVAVSGWVGASFPPGVSVAWLLSSSASFLASFLGWE


PLKVLLEALYFSLVAKRLHPDEDDTLVESPAVTPVSARVPRVRPPHGFALFLAKEEARKVKRLHGMLRSLLV


YMLFLLVTLLASYGDASCHGHAYRLQSAIKQELHSRAFLAITRSEELWPWMAHVLLPYVHGNQSSPELGPPR


LRQVRLQEALYPDPPGPRVHTCSAAGGFSTSDYDVGWESPHNGSGTWAYSAPDLLGAWSWGSCAVYDSGGYV


QELGLSLEESRDRLRFLQLHNWLDNRSRAVFLELTRYSPAVGLHAAVTLRLEFPAAGRALAALSVRPFALRR


LSAGLSLPLLTSVCLLLFAVHFAVAEARTWHREGRWRVLRLGAWARWLLVALTAATALVRLAQLGAADRQWT


RFVRGRPRRFTSFDQVAQLSSAARGLAASLLFLLLVKAAQQLRFLRQWSVFGKTLCRALPELLGVTLGLVVL


GVAYAQLAILLVSSCVDSLWSVAQALLVLCPGTGLSTLCPAESWHLSPLLCVGLWALRLWGALRLGAVILRW


RYHALRGELYRPAWEPQDYEMVELFLRRLRLWMGLSKVKEFRHKVRFEGMEPLPSRSSRGSKVSPDVPPPSA


GSDASHPSTSSSQLDGLSVSLGRLGTRCEPEPSRLQAVFEALLTQFDRLNQATEDVYQLEQQLHSLQGRRSS


RAPAQSSRGPSPGLRPALPSRLAPASRGVDLATGPSRTPLRAKNKVHPSST









The NOV28 amino acid sequence 4298 of 4302 amino acid residues (99%) identical to, and 4298 of 4302 amino acid residues (99%) similar to, the 4302 amino acid residue ptnr:SPTREMBL-ACC:Q15140 protein from Homo sapiens (Human) (Polycystic Kidney Disease 1 Protein) (E=0.0).
NOV28 is expressed in at least the following tissues: adrenal gland, bone marrow, brain—amygdala, brain—cerebellum, brain—hippocampus, brain—substantia nigra, brain—thalamus, brain—whole, fetal brain, fetal kidney, fetal liver, fetal lung, heart, kidney, lymphoma—Raji, mammary gland, pancreas, pituitary gland, placenta, prostate, salivary gland, skeletal muscle, small intestine, spinal cord, spleen, stomach, testis, thyroid, trachea and uterus. This information was derived by determining the tissue sources of the sequences that were included in the invention including but not limited to SeqCalling sources, Public EST sources, genomic clone sources, literature sources, and/or RACE sources. NOV28 is also predicted to be expressed in the following tissues because of the expression pattern of (gb:GENBANK-ID:HUMPKD1A|acc:L33243.1) a closely related Homo sapiens polycystic kidney disease 1 protein (PKD 1) mRNA, complete cds homolog in species Homo sapiens: kidney.
NOV28 has homology to the amino acid sequences shown in the BLASTP data listed in Table 28C.







TABLE 28C







BLAST results for NOV28
















Pos-



Gene Index/
Protein/
Length
Identity
itives



Identifier
Organism
(aa)
(%)
(%)
Expect





gi|4505833|ref|NP_0
polycystic
4302
3847/
3847/
0.0


00287.1|
kidney

4254
4254


(NM_000296)
disease 1

(90%)
(90%)



(autosomal



dominant);



Polycystin-1



[Homo



sapiens]


gi|1586345|prf||220
polycystin
4302
3844/
3844/
0.0


3412A
[Homo

4254
4254



sapiens]

(90%)
(90%)


gi|903758|gb|
polycystic
4292
3836/
3836/
0.0


AAC417 65.1|
kidney

4254
4254


(L43619)
disease

(90%)
(90%)



1 protein



[Homo



sapiens]


gi|1730587|sp|P9816
Polycystin
4303
3833/
3838/
0.0


1|PKD1_HUMAN
precursor

4255
4255



(Autosomal

(90%)
(90%)



dominant



polycystic



kidney



disease



protein



1) [Homo



sapiens]


gi|11320978|gb|
Pkd1 [Rattus
4283
3009/
3292/
0.0


AAG33986.1|
norvegicus]

4254
4254
4254


(AF277452)


(70%)









The homology of these sequences is shown graphically in the ClustalW analysis shown in Table 28D.







TABLE 28E





Clustal W Sequence Alignment















1) NOV28 (SEQ ID NO:90)


2) gi 4505833|refNP_000287.1|(NM_000296) polycystic kidney disease 1 (autosomal


dominant); Polycystin-1 [Homo sapiens] (SEQ ID NO:271)


3) gi 1586345|prf|2203412A polycystin [Homo sapiens] (SEQ ID NO:272)


4) gi 903758|gb AAC41765.1|(L43619) polycystic kidney disease 1 protein [Homo sapiens]


(SEQ ID NO:273)


5) gi 1730587|sp P98161 PKD1_HUMAN Polycystin precursor (Autosomal dominant polycystic


kidney disease protein 1) [Homo sapiens] (SEQ ID NO:274)


6) gi 11320978|gb AAG33986.1 (AF277452) Pkd1 [Rattus norvegicus] (SEQ ID NO:275)



























































































































































































































































































































































































































































Tables 28E, 28F, 28G and 28H list the domain description from DOMAIN analysis results against NOV28. This indicates that the NOV28 sequence has properties similar to those of other proteins known to contain these domains.







TABLE 28E





Domain Analysis of NOV28















gnl|Pfam.pfamC2010, REJ, REJ domain. The REJ (Receptor for Egg Jelly)


domain is found in PKDl, and the sperm receptor for egg jelly. The


function of this domain is unknown. The domain is 600 amino acids long


so is probably composed of multiple structural domains. There are six


completely conserved cysteine residues that may form disulphide


bridges. (SEQ ID NO:276)


CD-Length = 547 residues, 100.0% aligned


Score = 557 bits (1435), Expect = 5e−159










Query:
2168
YLEAHVDLRDCVTYQTEYRWEVYRTASCQR-PGRPARVALPGVDVSRPRLVLPRLALPV-
2225




 ||| |||+ |  |  +|+|+++   ||   |||   + || ||||| | +


Sbjct:
1
TLEASVDLKCCDAYTIDYKWQIFSVPSCDDDPGRTTPIPLPAVDVSRTRQLHIPRGALPY
  60


Query:
2226
GEYCFVPVVSFG----DTP-LTQSIQANVTVAPERLVPIIEGGSYRVWSDTRDLVLDGSE
2280




| | | | +|        | | +|| + + |    || +||||+||||| |+||+||||


Sbjct:
61
GVYVFNFTLSIRLKTSGEPTLKKSIISYLWVQAPPLVAVIEGGAyRVWSFTQDLILDGSN
 120


Query:
2281
SYDPNLEDGDQTPLSFHWACVAS------------TQREAGGCALNFGPRGSST--VTIP
2326




||||+ + | |+ | ||| ||              +|     | |+||  |||   +|||


Sbjct:
121
SYDPDADPGSQSNLQFHWYCVTVPRDYMGSSLDEASQNVCHPCQLSFGWPGSSGSVLTIP
 180


Query:
2327
RERLAAGVEYTFSLTVWKAGRKEEATNQTVLIRSGRVPIVSLECVSCKAQAVYEVSRSSY
2386




 | | | ||||| | + | ||  | |+||| + || +||| + |+|  || +||||| |


Sbjct:
181
PETLEANVEYTFRLVISKEGRITEFTDQTVHVLSGLLPIVHISCISNCAQYLYEVSRFSL
 240


Query:
2387
VYLEGRCLNCSSGSKRGRWAARTFSNKTLVLDET-TTSTGSAGMRLVLRRGVLRDGEGYT
2445




|||   | ||  |   |||+| + || |+||| +  |+||| |  |||+ |  | || |+


Sbjct:
241
VYL---CTNC-DGDDSGRWSALSLSNTTVVLDWSGQTTTGSNGPYLVLKPGAFRSGEEYS
 296


Query:
2446
FTLTVLGRSGEEEGCASIRLSP--NRPPLCGSCRLFPLGAVHALTTKVHFECTGWHDAED
2503




|||+| | | + || ||| | |  | || ||||+| |     || ||   ||+ +    |


Sbjct:
297
FTLSVTGSSWDSEGYASISLHPPINRPPAGGSCKLNPAE-GIALQTKFTVECSNF---RD
 352


Query:
2504
AGAPLVYALLLRRCR-----QGHCEEFCVYKGSLSSY-GAVLPPGF-RPHFEVGLAVVVQ
2556




   || | +++ |||       +|| | +|+||     || || ||     +| | | |


Sbjct:
353
VDEPLTYKIIVSRCRSVGMISSYCENFLLYEGSAPIKPGAFLPVGFGTDQHDVSLYVQVY
 412


Query:
2557
DQLGAAVVALNRSLAITLPEPNGSATGLTVWLRGLT--ASVLPGLLRQADPQHVIEYSLA
2614




| ||||   || +      +|  |   |   |  |    |+|  ||+| |||   | |||


Sbjct:
413
DSLGAASQVLNSATVHVPTDPASSKNVLQQLLSMLLLPESLLSTLLQQGDPQQAGELSLA
 472


Query:
2615
LVTVLNEYERALDVAAEPKHERQHRAQIRKNITETLVSLRVHTVDDIQQIAAALAQCMGP
2674




|++|||| |+  |     + ||  ||++|||+ + | +| |+||||||| +||||||


Sbjct:
473
LISVLNEIEQEDDS----EVERDDRARLRKNLVDQLAALPVNTVDDIQQSSAALAQCTQK
 528


Query:
2675
SRELVCRSCLKQTLHKLEA
2693




|||  |   +  ||  |||


Sbjct:
529
SREFSCHVQMIATLRLLEA
 547
















TABLE 28F





Domain Analysis of NOV28















gnl|Pfam′fam01477, PLAT, PLAT/LH2 domain. This domain is found in a


variety of membrane or lipid associated proteins. It is called the


PLAT (Polycystin-1, Lipoxygenase, Alpha-Toxin) domain or LH2


(Lipoxygenase homology) domain. The known structure of pancreatic


lipase shows this domain binds to procolipase pfam01114, which


mediates membrane association. So it appears possible that this domain


mediates membrane attachment via other protein binding partners. The


structure of this domain is known for many members of the family and


is composed of a beta sandwich. (SEQ ID NO:277)


CD-Length = 113 residues, 92.0% aligned


Score = 100 bits (248), Expect = 2e−2l










Query:
3115
FKYEILVKTGWGRGSGTTAHVGINLYGVDSRSGHRHLDGDRAFHRNSLDIFRIATPHSLG
3174




 +|+++| ||   |+|||  | | ||| +  ||   |         |   |       ||


Sbjct:
1
VRYQLVVATGGDEGAGTTGKVSISLYGEEGESGKIPLLKGEGAGPGSTFSFTFDVDEDLG
  60


Query:
3175
SVWKIRVWHDNKGL-SPAWFLQHVIVRDLQTARSAFFLVNDWLS
3217




 +  +|+ +++ || || |||+ + | |  |     |  | |+


Sbjct:
61
ELGAVRIPMEHSGLFSPEWFLKSITVEDGGTQGKVHFPCNSWVY
 104
















TABLE 28G





Domain Analysis of NOV28















gnl|Smart|smartc00308, LH2, Lipoxygenase homology 2 (beta barrel)


domain (SEQ ID NO:278)


CD-Length = 120 residues, 87.5% aligned


Score = 87.4 bits (215), Expect = 1e-17










Query:
3115
FKYEILVKTGWGRGSGTTAHVGIMLYGVDSRSGHRHLDG---DRAFHRNSLDIFRIATPH
3171




 ||++ | ||    +|||| | + | | + |     ||       | | |   |


Sbjct:
1
AKYKVTVTTGVLDFAGTTASVSLSLIGAEGRGKESKLDYLERPLLFARGSTYSFTFDVDV
  60


Query:
3172
SLGSVWKIRVIDNKGLSPAWFLQHVIVRDLQTARSAFFLVNDWL
3216




  | +  +++ +++ || | |||+ + |+|  |     |  | |+


Sbjct:
61
DFGELGAVKIKNEHAGLHPEWFLKSITVKDGPTGGKVHFPCNSWV
 105
















TABLE 28H





Domain Analysis of NOV28















gnl|Pfam pfam00801, PKD, PKD domain. This domain was first identified


in the Polycystic kidney disease protein PKDl. This domain has been


predicted to Contain an Ig-like fold. (SEQ ID NO:279)


CD-Length = 84 residues, 100.0% aligned


Score = 79.0 bits (193), Expect = 5e−15










Query:
1716
EPVGWLMVTASPNPAAVNTSVTLSAELAGGSGVVYTWSLEEGLSWETSEPFTTHSFPTPG
1775




|||  | |+|||+  |+  +|| +|    || | | |   +     ++ |  ||++  ||


Sbjct:
1
EPVPGLSVSASPSVVALGLTVTFTATSPDGSIVSYLWDFGDSPGLTSTGPNVTHTYLKPG
  60


Query:
1776
LHLVTMTAGNPLGSANATVEVDVQ
1799




 + ||+|| | +|||+||+ | |


Sbjct:
61
TYTVTLTATNDVGSASATLTVTVV
  84









Polycystic Kidney Disease Associated 8 (K8) and keratin 18 (K18) are the most common and characteristic members of the large intermediate filament gene family expressed in ‘simple’ or single layer epithelial tissues of the body. Their persistent expression in tumor cells derived from these epithelia has led to the wide spread use of keratin monoclonal antibodies as aids in the detection and identification of carcinomas. Oncogenes which activate ras signal transduction pathways stimulate expression of the K18 gene through transcription factors including members of the AP-1 (jun and fos) and ETS families. The persistent expression of K8 and K18 may reflect the integrated transcriptional activation of such transcription factors and, in the cases of ectopic expression, an escape from the suppressive epigenetic mechanisms of DNA methylation and chromatin condensation. Comparison of the mechanisms of transcriptional control of K18 expression with expression patterns documented in both normal and pathological conditions leads to the proposal that persistent K8 and K18 expression is a reflection of the action of multiple different oncogenes converging on the nucleus through a limited number of transcription factors to then influence the expression of a large number of genes including these keratins. Furthermore, correlation of various tumor cell characteristics including invasive behavior and drug sensitivity with K8 and K18 expression has stimulated consideration of the possible functions of these proteins in both normal development and in tumorigenesis. Recent developments in the analysis of the functions of these intermediate filament proteins provide new insights into diverse functions influenced by K8 and K18 (Oshima et al., Cancer Metastasis Rev 15:445-71, 1996).
Polycystic Kidney Disease Associated 8 (K8) and keratin 18 (K18) form intermediate filaments characteristic of liver and other single cell layered, internal epithelia and their derivative cancers. K8-deficient (K8(−)) mice, which escape embryonic lethality, develop inflammatory colorectal hyperplasia, mild liver abnormalities, and tolerate hepatectomy poorly. Normal and malignant epithelial cells deficient in K8 and K18 have been shown to be approximately 100 times more sensitive to TNF-induced death. K8 and K18 both bind the cytoplasmic domain of TNFR2 and moderate TNF-induced, Jun NH(2)-terminal kinase (JNK) intracellular signaling and NFkappaB activation. Furthermore, K8(−) and K18(−) mice are much more sensitive to TNF dependent, apoptotic liver damage induced by the injection of concanavalin A. This moderation of the effects of TNF may be the fundamental function of K8 and K18 common to liver regeneration, inflammatory bowel disease, hepatotoxin sensitivity, and the diagnostic, persistent expression of these keratins in many carcinomas (Caulin et al., J Cell Biol 149:17-22, 2000).
The NOV28 nucleic acid of the invention encoding a Polycystic Kidney Disease Associated-like protein includes the nucleic acid whose sequence is provided in Table 28A, or a fragment thereof. The invention also includes a mutant or variant nucleic acid any of whose bases may be changed from the corresponding base shown in Table 28A while still encoding a protein that maintains its Polycystic Kidney Disease Associated-like activities and physiological functions, or a fragment of such a nucleic acid. The invention further includes nucleic acids whose sequences are complementary to those just described, including nucleic acid fragments that are complementary to any of the nucleic acids just described. The invention additionally includes nucleic acids or nucleic acid fragments, or complements thereto, whose structures include chemical modifications. Such modifications include, by way of non-limiting example, modified bases, and nucleic acids whose sugar phosphate backbones are modified or derivatized. These modifications are carried out at least in part to enhance the chemical stability of the modified nucleic acid, such that they may be used, for example, as antisense binding nucleic acids in therapeutic applications in a subject. In the mutant or variant nucleic acids, and their complements, up to about 1% of the NOV28 residues may be so changed.
The NOV28 protein of the invention includes the Polycystic Kidney Disease Associated-like protein whose sequence is provided in Table 28B. The invention also includes a mutant or variant protein any of whose residues may be changed from the corresponding residue shown in Table 28B while still encoding a protein that maintains its Polycystic Kidney Disease Associated-like activities and physiological functions, or a functional fragment thereof. In the mutant or variant protein, up to about 1% of the NOV28 bases may be so changed.
The NOV28 nucleic acids and proteins of the invention are useful in potential diagnostic and therapeutic applications implicated in various diseases and disorders described below and/or other pathologies. For example, the compositions of the present invention will have efficacy for treatment of patients suffering from: polycystic kidney disease and renal carcinoma and other diseases, disorders and conditions of the like.
NOV28 nucleic acids and polypeptides are further useful in the generation of antibodies that bind immunospecifically to the novel substances of the invention for use in therapeutic or diagnostic methods. These antibodies may be generated according to methods known in the art, using prediction from hydrophobicity charts, as described in the “Anti-NOVX Antibodies” section below. For example the disclosed NOV28 protein have multiple hydrophilic regions, each of which can be used as an immunogen. This novel protein also has value in development of powerful assay system for functional analysis of various human disorders, which will help in understanding of pathology of the disease and development of new drug targets for various disorders.
NOV29
A disclosed NOV29 nucleic acid of 1222 nucleotides (also referred to as CG57589-01) encoding a novel Cholinephosphate Cytidylyltransferase-like protein is shown in Table 29A. An open reading frame was identified beginning with an ATG initiation codon at nucleotides 67-69 and ending with a TAA codon at nucleotides 1156-1158. Putative untranslated regions upstream from the intitation codon and downstream from the termination codon are underlined in Table 29A, and the start and stop codons are in bold letters.







TABLE 29A





NOV29 Nucleotide Sequence















(SEQ ID NO:91)







ATTGCGGGCGGCGGCGTTCCGAGTCGCCGGGAGCTGCCAGGCTGTCCGCGCCGCCGCTGCGCGGCCATGATCC


CGAACGGGCGCGGGGCTGCAGGCGGCGCAGAGCAGCCGGGCCCGGGGGGCAGGCGCGCCGTGAGGGTGTGGTG


CGATGGCTGCTATGACATGGTGCATTACGGCCACTCCAACCAGCTGCGCCAGGCACGGGCCATGGGTGACTAC


CTCATCGTAGGCGTGCACACCGATGAGAGATCGCCAAGCACAAGGGGCCCCCGGTGTTCACTCAGGAGGGAGA


GATACAAGATGGTGCAGGCCATCAAATGGGTGGACGAGGTGGTGCCAGCGGCTCCCTACGTCACTCACTAGA


GACCCTGGACAAATACAACTGTGACTTCTGTGTTCACGGCAATGACATCACCCTGACTGTAGATGGCCGGGAC


ACCTATGAGGAAGTAAAGCAGGCTGGGAGGTACAGAGAATGCAAGCGCACGCAAGGGGTGTCCACCACAGACC


TCGTGGGCCGCATGCTGCTGGTAACCAAAGCCCATCACAGCAGCCAGGAGATGTCCTCTGAGTACCGGGAGTA


TGCAGACAGTTTTGGCAAGTGCCCTGGTGGGCGCACCCCTGGACCGGGGTATCCCAGTTCCTGCAGACATCT


CAGAAGATCATCCAGTTTGCTTCTGGGAAGGAGCCCCAGCCAGGGGAGACAGTCATCTATGTGGCTGGTGCCT


TCGACCTGTTCCACATCGGGCATGTGGACTTCCTGGAGAAGGTGCACAGGCTGGCAGAGAGGCCCTACATCAT


CGCGGGCTTACACTTTGACCAGTACGTGTCAGAAGTGGTGATTGGAGCCCCGTACGCGGTCACAGCAGAGCTC


CTAAGTCACTTCAAGGTGGACCTGGTGTGTCACGGCAAGACAGGAATTATCCCTGACAGGGATGGCTCCGACC


CATACCAGGAGCCCAAGAGAAGGGGCATCTTCCGTCAGATTGACAGTGGCAGCAACCTCACCACAGACCTCAT


CGTCCAGCGGATCATCACCACAGGTTGGAGTATGAGGCGCGAAACCAGAAGAAGGAAGCCAAGGACCTGGCC


TTCCTGGACGGTGCCAGGCAGCAGGCGGCACAGCCCCTGGGGGAGCGCGATGGTGACTTCTAACCTGGCAGAG


GCCCTGGCCGGCCCTCCCCCTGCTCTGCTTCTGCGCCTTCTGCGTTTGGACATA









The NOV29 nucleic acid was identified on chromosome 17 and has 828 of 830 bases (99%) identical to a gb:GENBANK-ID:D84307|acc:D84307.1 mRNA from Homo sapiens (mRNA for phosphoethanolamine cytidylyltransferase, complete cds) (E=6.4e−181).
A disclosed NOV29 polypeptide (SEQ ID NO:92) encoded by SEQ ID NO:91 is 363 amino acid residues and is presented using the one-letter code in Table 29B. Signal P, Psort and/or Hydropathy results predict that NOV29 contains a signal peptide and is likely to be localized to the nucleus with a certainty of 0.3000.







TABLE 29B





Encoded NOV29 protein sequence















(SEQ ID NO:92)







MIRNGRGAAGGAEQPGPGGRRAVRVWCDGCYDMVHYGHSNQLRQARAMGDYLIVGVHTDEEIAKHKGPPVFT


QEERYKMVQAIKWVDEVVPAAPYVTTLETLDKYNCDFCVHGNDITLTVDGRDTYEEVKQAGRYRECKRTQGV


STTDLVGMLLVTKAHHSSQEMSSEYREYADSFGKCPGGRNPWTGVSQFLQTSQKIIQFASGKEPQPGETVI


YVAGAFDLFHIGHVDFLEKVHRLAERPYIIAGLHFDQYVSEVVIGAPYAVTAELLSHFKVDLVCHGKTGIIP


DRDGSDPYQEPKRRGIFRQIDSGSNLTTDLIVQRIITNRLEYEARNQKKEAKRLAFLEAARQQAAQPLGERD


GDF









The NOV29 amino acid sequence 254 of 256 amino acid residues (99%) identical to, and 255 of 256 amino acid residues (99%) similar to, the 389 amino acid residue ptnr:SWISSNEW-ACC:Q99447 protein from Homo sapiens (Human) (Ethanolamine-Phosphate Cytidylyltransferase (EC 2.7.7.14) (Phosphorylethanolamine Transferase) (CTP:Phosphoethanolamine Cytidylyltransferase)) (E=2.3e−196).
NOV29 is expressed in at least the following tissues: adrenal gland/suprarenal gland, bone marrow, brain, bronchus, coronary artery, dermis, epidermis, heart, hypothalamus, kidney, left cerebellum, liver, lung pleura, lymphoid tissue, mammary gland/breast, ovary, pancreas, peripheral blood, pituitary gland, placenta, prostate, skeletal muscle, spinal chord, spleen, stomach, substantia nigra, testis, thalamus, thymus, thyroid, uterus and whole organism. This information was derived by determining the tissue sources of the sequences that were included in the invention including but not limited to SeqCalling sources, Public EST sources, genomic clone sources, literature sources, and/or RACE sources.
NOV29 has homology to the amino acid sequences shown in the BLASTP data listed in Table 29C.







TABLE 29C







BLAST results for NOV29
















Pos-



Gene Index/
Protein/
Length
Identity
itives



Identifier
Organism
(aa)
(%)
(%)
Expect





gi|4505651|ref|NP_0
phosphate
389
331/389
331/
0.0


02852.1|
cytidylyl-

(85%)
389


(NM_002861)
transferase


(85%)



2,



ethanolamine



[Homo



sapiens]


gi|14603223|gb|
phosphate
389
330/389
331/
0.0


AAH10075.1|
cytidylyl-

(84%)
389


AAH10075
transferase


(84%)


(BC010075)
2,



ethanolamine



[Homo



sapiens]


gi|6758340|ref|NP_
phosphate
404
310/404
323/
e−174


446020.1|
cytidylyl-

(76%)
404


(NM_053568)
transferase


(79%)



2,



ethanolamine



[Rattus



norvegicus]


gi|14198445|gb|
RIKEN
404
309/404
322/
e−174


AAH08276.1|
cDNA

(76%)
404


AAH08276
1110033E03


(79%)


(BC008276)
gene [Mus



musculus]


gi|13195654|ref|NP_
RIKEN
404
308/404
321/
e−172


077191.1|
cDNA

(76%)
404


(NM_024229)
1110033E03


(79%)



[Mus



musculus]









The homology of these sequences is shown graphically in the ClustalW analysis shown in Table 29D.







TABLE 29D





Clustal W Sequence Alignment















1) NOV29 (SEQ ID NO:92)


2) gi 4505651|refNP_002852.1|(NM_002861) phosphate cytidylyltransferase 2,


ethanolamine [Homo sapiens] (SEQ ID NO:280)


3) gi 14603223|gb AAH10075.1 AAH10075 (BC010075) phosphate cytidylyltransferase 2,


ethanolamine [Homo sapiens] (SEQ ID NO:281)


4) gi 16758340|refNP_446020.1|(NM_053568) phosphate cytidylyltransferase 2,


ethanolamine [Rattus norvegicus] (SEQ ID NO:282)


5) gi 14198445|gbAAH08276.1 AAH08276 (BC008276) RIKEN cDNA 1110033E03 gene


[Mus musculus] (SEQ ID NO:283)


6) gi 13195654|refNP_077191.1|(NM_024229) RIKEN cDNA 1110033E03 [Mus musculus]


(SEQ ID NO:284)



















































Table 29E lists the domain description from DOMAIN analysis results against NOV29. This indicates that the NOV29 sequence has properties similar to those of other proteins known to contain these domains.







TABLE 29E





Domain Analysis of NOV29















gnl.Pfam:pfam01467, Cytidylyltransf, Cytidylyltransferase. This family


includes: Cholinephosphate cytidylyltransferase and Glycerol-3-


phosphate cytidylyltransferase. (SEQ ID NO:285)


CD-Length = 129 residues, 99.2% aligned


Score = 128 bits (321), Expect = 6e−31










Query:
23
VRVWCDGCYDMVHYGHSNQLRQARAMGDYLIVGVHTDEEIAKHKGPPVFTQEERYKMVQA
 82




 ||   | +|  | || + | +|+ +|| ||||| +||   | |  |   +| |  +++|


Sbjct:
2
KRVIYGGTFDPFHLGHLDLLERAKELGDELIVGVASDESKKKLK--PFTAEERRKMLLEA
 59


Query:
83
IKWVDEVVPAAPYVTTLETLDKYNCDFCVRGNDITLTVDGRDTYEEVKQAG-RYRECKRT
141




+| ||||   ||   |+| + +   |  | | |+  + +    |  ||+||       ||


Sbjct:
60
LKDVDEVYVFAPDDLTVEFIKEIKPDVIVRGLDVV-SFEYELLYALVKRAGLEVVFLPRT
118


Query:
142
QGVSTTDLVGR
152




+| ||+|+| |


Sbjct:
119
EGFSTSDIVKR
129









Ross et al. (Neuroscience 2001; 102:899-904) reported that the activities of phospholipase A(2), phosphocholine cytidylyltransferase and phosphoethanolamine cytidylyltransferase, key phospholipid metabolic enzymes, are low in substantia nigra of normal human brain and that this might reduce the ability of nigral neurons to repair damage to cell membranes. To determine whether adaptive changes in nigral phospholipid metabolism can occur in idiopathic Parkinson's disease they compared activities of 11 catabolic and anabolic enzymes in autopsied brain of 10 patients with Parkinson's disease to those in control subjects. Nigral activity of the catabolic enzyme phospholipase A(2) was normal in the Parkinson's disease group, whereas that of the biosynthetic enzymes phosphoethanolamine cytidylyltransferase, phosphocholine cytidylyltransferase, and phosphatidylserine synthase were elevated 193, 48 and 38%, respectively, possibly representing a compensatory response to repair membrane phospholipids. Enzyme activities were normal in all other brain areas with the exception of increased (+26%) activity of calcium-stimulated phospholipase A(2) in putamen, a change which could be consequent to either decreased dopaminergic striatal input or to a dopamine nerve terminal degenerative process. Their data indicate that the normally low rate of membrane phospholipid synthesis in the substantia nigra, the primary area of neurodegeneration in Parkinson's disease, is increased during the course of the disorder. It was suggested that pharmacotherapies which augment this compensatory response might have utility as a treatment for Parkinson's disease.
The NOV29 nucleic acid of the invention encoding a Cholinephosphate Cytidylyltransferase-like protein includes the nucleic acid whose sequence is provided in Table 29A, or a fragment thereof. The invention also includes a mutant or variant nucleic acid any of whose bases may be changed from the corresponding base shown in Table 29A while still encoding a protein that maintains its Cholinephosphate Cytidylyltransferase-like activities and physiological functions, or a fragment of such a nucleic acid. The invention further includes nucleic acids whose sequences are complementary to those just described, including nucleic acid fragments that are complementary to any of the nucleic acids just described. The invention additionally includes nucleic acids or nucleic acid fragments, or complements thereto, whose structures include chemical modifications. Such modifications include, by way of non-limiting example, modified bases, and nucleic acids whose sugar phosphate backbones are modified or derivatized. These modifications are carried out at least in part to enhance the chemical stability of the modified nucleic acid, such that they may be used, for example, as antisense binding nucleic acids in therapeutic applications in a subject. In the mutant or variant nucleic acids, and their complements, up to about 1% of the NOV29 residues may be so changed.
The NOV29 protein of the invention includes the Cholinephosphate Cytidylyltransferase-like protein whose sequence is provided in Table 29B. The invention also includes a mutant or variant protein any of whose residues may be changed from the corresponding residue shown in Table 29B while still encoding a protein that maintains its Cholinephosphate Cytidylyltransferase-like activities and physiological functions, or a functional fragment thereof. In the mutant or variant protein, up to about 1% of the NOV29 bases may be so changed.
The NOV29 nucleic acids and proteins of the invention are useful in potential diagnostic and therapeutic applications implicated in various diseases and disorders described below and/or other pathologies. For example, the compositions of the present invention will have efficacy for treatment of patients suffering from: Parkinson's disease and other diseases, disorders and conditions of the like.
NOV29 nucleic acids and polypeptides are further useful in the generation of antibodies that bind immunospecifically to the novel substances of the invention for use in therapeutic or diagnostic methods. These antibodies may be generated according to methods known in the art, using prediction from hydrophobicity charts, as described in the “Anti-NOVX Antibodies” section below. For example the disclosed NOV29 protein have multiple hydrophilic regions, each of which can be used as an immunogen. This novel protein also has value in development of powerful assay system for functional analysis of various human disorders, which will help in understanding of pathology of the disease and development of new drug targets for various disorders.
NOV30
A disclosed NOV30 nucleic acid of 893 nucleotides (also referred to as CG57558-01) encoding a novel mac25/IGFBP7-like protein is shown in Table 30A. An open reading frame was identified beginning with an ATG initiation codon at nucleotides 49-51 and ending with a TAA codon at nucleotides 871-873. Putative untranslated regions upstream from the intitation codon and downstream from the termination codon are underlined in Table 30A, and the start and stop codons are in bold letters.







TABLE 30A





NOV30 Nucleotide Sequence















(SEQ ID NO:93)







GTACCTTAAAGACAACAAACAAGCAAACACAACTTATAATTAAAAAACATGCAAAGGGCTCACCTTCCACTTC


CTTCTGGTCCTGCTCCTCTTCTTCCTCCTCTCCTGCCTCCTCTTCTCCCTGTTCCATCAGACCTTCTGGGGCC


CCTTTCAATAAGCAGCTGCTGGCCGGCCAGCCCTTGGGGGCAGGGCTGGAACCGGGGCAGGGGAGGCTGCGGG


GCCACTCGCTGGAGAGGCAAACAAGGAAGGACTGCCCCCTGAGCGCCAGGCTTCGGGCCCGGGAATCGCCGCCG


CCGCCGCCGCAGAGCTGCAGCTCGGGGCCGAGGGTAAGGAGGCGAGCCGGGAGCGGGAGGCCCGGGAGAGCTC


CGCGGGTCCCCGCGCCCAGTCCCCAGCCGCGCCCCGACCCCGCCGCCCCGGGCCTAACGCGGCGGCGAGGCC


TACGCGGCGGCCGCCGTCACCGTGCTGGAGCCGCCGGCCTCCGACCCCGAGCTGCAGCCCGCCGAGCGCCCGC


TGCCATCGCCGCCGGGGTCCGGGAGGGCGCCCCGGTCTTCCTCACGGGGCCTCGATCCCAGTGGGTGCTGCGGGG


GGCGGAGGTGGTGCTGACGTGCCGGGCGGGGGGCCTCCCCGAGCCCACACTGTACTGGGAGAAGGACGGGATG


GCCCTGGACGAAGTGTGGGACAGCAGCCACTTCGCGCTCCAGCCGGGCCGCGCCGAGGACGGCCCCGGCGCGA


GCCTGGCACTGCGCATCCTGGCGGCTCGGCTGCCGGATTCCGGCGTCTACGTGTGCCACGCCCGCAACGCGCA


CGGCCACGCGCAGGCGGGGGCGCTGCTCCAGGTGCTGACCCCCACCTTCCTGCCGCCAAGACAGCCCTAACCA


AGGCCCAGAAAGGGTAG









The NOV30 nucleic acid was identified on chromosome 2 and has 564 of 779 bases (72%) identical to a gb:GENBANK-ID:S56581|acc:S56581.1 mRNA from Rattus sp. (alpha inhibin gene {5′ region} [rats, Genomic, 2141 nt]) (E=1.3e−68).
A disclosed NOV30 polypeptide (SEQ ID NO:94) encoded by SEQ ID NO:93 is 274 amino acid residues and is presented using the one-letter code in Table 30B. Signal P, Psort and/or Hydropathy results predict that NOV30 contains a signal peptide and is likely to be localized to the extracellularly with a certainty of 0.3700. The most likely cleavage site for a NOV30 polypeptide is between amino acids 32-33: LLG-PL.







TABLE 30B





Encoded NOV30 protein sequence















(SEQ ID NO:94)







MQRAHLFLPSGPAPLLPPLLFPLLPVPSDLLGPLSISSCWPASPWGQGWNRGRGGCGATRWRGKQEGLPPER


QASGPGIAAAAAAELQLGAEGKEASREREARESSAGPRAQSPAAPRPRRPGPNAAGEAYAAAAVTVLEPPAS


DPELQPAERPLPSPGSGEGAPVFLTGPRSQWVLRGAEVVLTCRAGGLPEPTLYWEKDGMALDEVWDSSHFAL


QPGRAEDGPGASLALRILAARLPDSGVYVCHAHNAHGHAQAGALLQVLTPTFLPPRQP









The NOV30 amino acid sequence 80 of 266 amino acid residues (30%) identical to, and 112 of 266 amino acid residues (42%) similar to, the 277 amino acid residue ptnr:SPTREMBL-ACC:Q07822 protein from Homo sapiens (Human) (mac25 protein) (E=2.0e−17).
NOV30 is expressed in at least the following tissues: brain, ovary, breast and testis. This information was derived by determining the tissue sources of the sequences that were included in the invention including but not limited to SeqCalling sources, Public EST sources, genomic clone sources, literature sources, and/or RACE sources and the expression pattern of (gb:GENBANK-ID:S56581|acc:S56581.1) a closely related alpha inhibin gene {5′ region} [rats, Genomic, 2141 nt] homolog in species Rattus sp.
NOV30 has homology to the amino acid sequences shown in the BLASTP data listed in Table 30C.







TABLE 30C







BLAST results for NOV30
















Pos-



Gene Index/
Protein/
Length
Identity
itives


Identifier
Organism
(aa)
(%)
(%)
Expect





gi|14734071|ref|XP_
K1AA0657
1025
120/139
120/
1e−60


051017.1|
protein

(86%)
139


(XM_051017)
[Homo


(86%)



sapiens]


gi|13938170|gb|
Unknown
1044
120/139
120/
3e−60


AAH07201.1|
(protein for

(86%)
139


AAH07201
IMAGE: 


(86%)


(BC007201)
2961284)



[Homo



sapiens]


gi|18552587|ref|XP_
similar to
 180
38/58
39/
1e−11


087161.1|
Unknown

(65%)
58


(XM_087161)
(protein for


(66%)



IMAGE:



2961284)



[Homo



sapiens]


gi|14043550|gb|
hypothetical
 304
 37/126
 49/
9e−06


AAH07758.1|
protein

(29%)
126


AAH07758
FKSG28


(38%)


(BC007758)
[Homo



sapiens]


gi|15026974|emb|
obscurin
6620
 40/101
 48/
1e−05


CAC44768.1|
[Homo

(39%)
101


(AJ002535)
sapiens]


(46%)









The homology of these sequences is shown graphically in the ClustalW analysis shown in Table 30D.







TABLE 30D





Clustal W Sequence Alignment















1) NOV30 (SEQ ID NO:94)


2) gi 14734071|refXP_051017.1|(XM_051017) KIAA0657 protein [Homo sapiens]


(SEQ ID NO:286)


3) gi 13938170|gb AAH07201.1 AAH07201 (BC007201) Unknown (protein for IMAGE:2961284)


[Homo sapiens] (SEQ ID NO:287)


4) gi 18552587|refXP_087161.1|(XM_087161) similar to Unknown (protein for


IMAGE:2961284) [Homo sapiens] (SEQ ID NO:288)


5) gi 14043550|gbAAH07758.1 AAH07758 (BC007758) hypothetical protein FKSG28


[Homo sapiens] (SEQ ID NO:289)


6) gi 15026974|emb CAC44768.1 (AJ002535) obscurin [Homo sapiens] (SEQ ID NO:290)









































































































































































































































































































































































































































































































































































































































































































Tables 30E and 30F list the domain description from DOMAIN analysis results against NOV30. This indicates that the NOV30 sequence has properties similar to those of other proteins known to contain these domains.







TABLE 30E





Domain Analysis of NOV30















gnl Smart|smart00409, IG, Immunoglobulin (SEQ ID NO:291)


CD-Length = 86 residues, 100.0% aligned


Score = 57.4 bits (137), Expect = 1e−09










Query:
171
PRSQWVLRGAEVVLTCRAGGLPEPTLYWEKLGMALDEVWDSSHFALQPGRAE-DGPGASL
229




| |  |  |  | |+| | | | ||+ |                  + ||      | +


Sbjct:
1
PPSVTVKEGESVTLSCEASGNPPPTVTW---------YKQGGKLLAESGRFSVSRSGGNS
 51


Query:
230
ALRILAARLPDSGVYVCHARNAHGHAQAGALLQVL
264




 | |      ||| | | | |+ | | +|  | ||


Sbjct:
52
TLTISNVTPEDSGTYTCAATNSSGSASSGTTLTVL
 86
















TABLE 30F





Domain Analysis of NOV30















gnl Smart|smart00408, IGc2, Immunoglobulin C-2 Type (SEQ ID NO:292)


CD-Length = 63 residues, 96.8% aligned


Score = 48.1 bits (113), Expect = 6e−07










Query:
179
GAEVVLTCRAGGLPEPTLYWEKDGMALDE-VWDSSEFALQPGRAEDGPGASLALRILAAR
237




|  | ||| | | | | + | |||  | |    +|   |


Sbjct:
3
GESVTLTCPASGDPVPNITWLKDGKPLPESRVVASGSTLTIKNVSLE-------------
 49


Query:
238
LPDSGVYVCHARNAHG
253




  |||+| | |||+ |


Sbjct:
50
--DSGLYTCVAPNSVG
 63









NOV30 described here has homology to mac25, a potent tumor inhibitor. Mac25 is a follistatin (FS)-like protein that has a growth-suppressing effect on a p53-deficient osteosarcoma cell line (Saos-2). The protein exhibits a strong homology to FS, an activin-binding protein, and part of its sequence includes the consensus sequence of the member of the Kazal serine protease inhibitor family. The mac25 protein was localized in the cytoplasm and secreted into culture medium (Kato, Mol Med 6(2):126-35, 2000). Addition of recombinant mac25 protein (10-7 M) into the culture medium induced significant suppression of the growth of human cervical carcinoma cells (HeLa) and murine embryonic carcinoma cells (P19), as well as osteosarcoma cells (Saos-2). The mac25 protein was co-immunoprecipitated with activin A, a result that suggests that mac25 may be a secreted tumor-suppressor that binds activin A. The mac25 exhibits homology to insulin-like growth factor-binding proteins (IGF-BPs) and to fibroblast growth factor receptor. The multi-functional nature of mac25 protein may be important for growth-suppression and/or cellular senescence (Kato, 2000).
The mac25 is strongly induced in senescent epithelial cells, whereas CTGF stimulates angiogenesis and wound healing. Using in situ hybridization and immunohistochemistry, Wandji et al. examined the possibilities that mac25 is inhibited, whereas CTGF is induced during active periods of follicular development and luteogenesis (Wandji et al., Endocrinology 141(7):2648-57, 2000). Ovaries were collected during the follicular and early luteal phases from prostaglandin F2alpha-treated mature pigs and from slaughterhouse sows. CTGF transcripts were induced during the late preantral stage in granulosa and theca cells concomitantly with the appearance of endothelial cells in the theca. CTGF mRNA expression increased in granulosa cells to a maximum (P<0.01) in mid-antral follicles but was down regulated (P<0.01) in preovulatory follicles. In contrast, granulosa cell mac25 mRNA expression was undetectable between the preantral and mid-antral stage but was strongly induced in terminally differentiated granulosa cells of preovulatory follicles. CTGF mRNA and peptide were also detected in the theca externa/interstitium and in vascular endothelial cells of ovarian blood vessels, whereas mac25 transcripts, which were also abundant in ovarian blood vessels increased in the theca interna with follicular development. Transcripts of cyclin D 1, a marker of cell proliferation, appeared during the early antral stage and were moderate in granulosa cells but abundant in capillary endothelial cells in the theca interna, underneath the basement membrane. Following ovulation, CTGF and cyclin D1 mRNAs were associated with the migration of endothelial cells into the CL. Subsequently, there was a marked up-regulation of CTGF mRNA expression in granulosa luteins concomitantly with an increase in endothelial cell proliferation within the CL. Those data suggested that CTGF may promote ovarian cell growth and blood vessel formation during follicular and luteal development whereas mac25, a tumor inhibitor, may promote terminal differentiation of granulosa cells in preovulatory follicles (Wandji et al., 2000).
It is important to know the regulation of the expression of the mac25 gene because of its reduced expression in several cancer cells and of its induction by some hormonal factors. Kanemitsu et al. cloned the promoter region of the murine mac25 gene and found five repeats of CCAAT sequences, four Sp1 sites, a TATA-like sequence, and an initiator (INR) sequence. Analysis using luciferase reporter plasmids indicated that CCAAT repeats have a strong enhancer activity and the second to fourth Sp1 sites are essential for basal activity of the expression of the mac25 gene. The 1 kb region that contains the promoter and exon 1 of the mac25 gene was in a typical CpG island. As hypermethylation and reduced expression of the mac25 gene were reported in murine liver tumors, methylation of this CpG island may be directly associated with the expression of the mac25 gene and tumorigenesis (Kanemitsu et al., Biochem Biophys Res Commun 279(1):251-7, 2000).
Komatsu et al., used restriction landmark genomic scanning for methylation (RLGS-M) to detect alterations in DNA methylation associated with murine SV40 T/t antigen-induced hepatocarcinogenesis (Komatsu et al., Biochem Biophys Res Commun 267(1):109-17, 2000). An altered locus/spot (S130) was cloned and found to correspond to sequences in the 5′ flanking region and 5′ portion of the cDNA for the murine mac25/insulin-like growth factor binding protein-7 (Igfbp-7) gene. IGFBPs are believed to be capable of binding insulin, Igf1, and Igf2 and modulating mitogenic effects. Previous studies have shown that Igf2 has an important role in promoting liver tumorigenesis. Quantitative PCR was used to access the methylation status of the NotI site just 5′ to the coding region and the expression level of the mac25/igfbp-7 gene. The results indicated that the degree of methylation was inversely related to the expression level and is consistent with a role for DNA methylation in silencing mac25/Igfbp-7 gene expression and function for mac25/Igfbp-7 as a tumor suppressor gene (Komatsu et al., 2000).
Because of the homology to mac25/IGFBP7, the NOV30 described here will have useful properties and functions similar to these genes.
The NOV30 nucleic acid of the invention encoding a mac25/IGFBP7-like protein includes the nucleic acid whose sequence is provided in Table 30A, or a fragment thereof. The invention also includes a mutant or variant nucleic acid any of whose bases may be changed from the corresponding base shown in Table 30A while still encoding a protein that maintains its mac25/IGFBP7-like activities and physiological functions, or a fragment of such a nucleic acid. The invention further includes nucleic acids whose sequences are complementary to those just described, including nucleic acid fragments that are complementary to any of the nucleic acids just described. The invention additionally includes nucleic acids or nucleic acid fragments, or complements thereto, whose structures include chemical modifications. Such modifications include, by way of non-limiting example, modified bases, and nucleic acids whose sugar phosphate backbones are modified or derivatized. These modifications are carried out at least in part to enhance the chemical stability of the modified nucleic acid, such that they may be used, for example, as antisense binding nucleic acids in therapeutic applications in a subject. In the mutant or variant nucleic acids, and their complements, up to about 28% of the NOV30 residues may be so changed.
The NOV30 protein of the invention includes the mac25/IGFBP7-like protein whose sequence is provided in Table 30B. The invention also includes a mutant or variant protein any of whose residues may be changed from the corresponding residue shown in Table 30B while still encoding a protein that maintains its mac25/IGFBP7-like activities and physiological functions, or a functional fragment thereof. In the mutant or variant protein, up to about 70% of the NOV30 bases may be so changed.
The NOV30 nucleic acids and proteins of the invention are useful in potential diagnostic and therapeutic applications implicated in various diseases and disorders described below and/or other pathologies. For example, the compositions of the present invention will have efficacy for treatment of patients suffering from: Von Hippel-Lindau (VHL) syndrome, Alzheimer's disease, stroke, tuberous sclerosis, hypercalceimia, Parkinson's disease, Huntington's disease, cerebral palsy, epilepsy, Lesch-Nyhan syndrome, multiple sclerosis, ataxia-telangiectasia, leukodystrophies, behavioral disorders, addiction, anxiety, pain, neurodegeneration, fertility, hypogonadism, endometriosis, hemophilia, hypercoagulation, idiopathic thrombocytopenic purpura, immunodeficiencies, graft versus host disease and other diseases, disorders and conditions of the like.
NOV30 nucleic acids and polypeptides are further useful in the generation of antibodies that bind immunospecifically to the novel substances of the invention for use in therapeutic or diagnostic methods. These antibodies may be generated according to methods known in the art, using prediction from hydrophobicity charts, as described in the “Anti-NOVX Antibodies” section below. For example the disclosed NOV30 protein have multiple hydrophilic regions, each of which can be used as an immunogen. This novel protein also has value in development of powerful assay system for functional analysis of various human disorders, which will help in understanding of pathology of the disease and development of new drug targets for various disorders.
NOVX Nucleic Acids and Polypeptides
One aspect of the invention pertains to isolated nucleic acid molecules that encode NOVX polypeptides or biologically active portions thereof. Also included in the invention are nucleic acid fragments sufficient for use as hybridization probes to identify NOVX-encoding nucleic acids (e.g., NOVX mRNAs) and fragments for use as PCR primers for the amplification and/or mutation of NOVX nucleic acid molecules. As used herein, the term “nucleic acid molecule” is intended to include DNA molecules (e.g., cDNA or genomic DNA), RNA molecules (e.g., mRNA), analogs of the DNA or RNA generated using nucleotide analogs, and derivatives, fragments and homologs thereof. The nucleic acid molecule may be single-stranded or double-stranded, but preferably is comprised double-stranded DNA.
An NOVX nucleic acid can encode a mature NOVX polypeptide. As used herein, a “mature” form of a polypeptide or protein disclosed in the present invention is the product of a naturally occurring polypeptide or precursor form or proprotein. The naturally occurring polypeptide, precursor or proprotein includes, by way of nonlimiting example, the full-length gene product, encoded by the corresponding gene. Alternatively, it may be defined as the polypeptide, precursor or proprotein encoded by an ORF described herein. The product “mature” form arises, again by way of nonlimiting example, as a result of one or more naturally occurring processing steps as they may take place within the cell, or host cell, in which the gene product arises. Examples of such processing steps leading to a “mature” form of a polypeptide or protein include the cleavage of the N-terminal methionine residue encoded by the initiation codon of an ORF, or the proteolytic cleavage of a signal peptide or leader sequence. Thus a mature form arising from a precursor polypeptide or protein that has residues 1 to N, where residue 1 is the N-terminal methionine, would have residues 2 through N remaining after removal of the N-terminal methionine. Alternatively, a mature form arising from a precursor polypeptide or protein having residues 1 to N, in which an N-terminal signal sequence from residue 1 to residue M is cleaved, would have the residues from residue M+1 to residue N remaining. Further as used herein, a “mature” form of a polypeptide or protein may arise from a step of post-translational modification other than a proteolytic cleavage event. Such additional processes include, by way of non-limiting example, glycosylation, myristoylation or phosphorylation. In general, a mature polypeptide or protein may result from the operation of only one of these processes, or a combination of any of them.
The term “probes”, as utilized herein, refers to nucleic acid sequences of variable length, preferably between at least about 10 nucleotides (nt), 100 nt, or as many as approximately, e.g., 6,000 nt, depending upon the specific use. Probes are used in the detection of identical, similar, or complementary nucleic acid sequences. Longer length probes are generally obtained from a natural or recombinant source, are highly specific, and much slower to hybridize than shorter-length oligomer probes. Probes may be single- or double-stranded and designed to have specificity in PCR, membrane-based hybridization technologies, or ELISA-like technologies.
The term “isolated” nucleic acid molecule, as utilized herein, is one, which is separated from other nucleic acid molecules which are present in the natural source of the nucleic acid. Preferably, an “isolated” nucleic acid is free of sequences which naturally flank the nucleic acid (i.e., sequences located at the 5′- and 3′-termini of the nucleic acid) in the genomic DNA of the organism from which the nucleic acid is derived. For example, in various embodiments, the isolated NOVX nucleic acid molecules can contain less than about 5 kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5 kb or 0.1 kb of nucleotide sequences which naturally flank the nucleic acid molecule in genomic DNA of the cell/tissue from which the nucleic acid is derived (e.g., brain, heart, liver, spleen, etc.). Moreover, an “isolated” nucleic acid molecule, such as a cDNA molecule, can be substantially free of other cellular material or culture medium when produced by recombinant techniques, or of chemical precursors or other chemicals when chemically synthesized.
A nucleic acid molecule of the invention, e.g., a nucleic acid molecule having the nucleotide sequence SEQ ID NOS:1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91 and 93, or a complement of this aforementioned nucleotide sequence, can be isolated using standard molecular biology techniques and the sequence information provided herein. Using all or a portion of the nucleic acid sequence of SEQ ID NOS:1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37,3 9, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91 and 93 as a hybridization probe, NOVX molecules can be isolated using standard hybridization and cloning techniques (e.g. as described in Sambrook, et al., (eds.), MOLECULAR CLONING: A LABORATORY MANUAL 2nd Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989; and Ausubel, et al., (eds.), CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, New York, N.Y., 1993.)
A nucleic acid of the invention can be amplified using cDNA, mRNA or alternatively, genomic DNA, as a template and appropriate oligonucleotide primers according to standard PCR amplification techniques. The nucleic acid so amplified can be cloned into an appropriate vector and characterized by DNA sequence analysis. Furthermore, oligonucleotides corresponding to NOVX nucleotide sequences can be prepared by standard synthetic techniques, e.g., using an automated DNA synthesizer.
As used herein, the term “oligonucleotide” refers to a series of linked nucleotide residues, which oligonucleotide has a sufficient number of nucleotide bases to be used in a PCR reaction. A short oligonucleotide sequence may be based on, or designed from, a genomic or cDNA sequence and is used to amplify, confirm, or reveal the presence of an identical, similar or complementary DNA or RNA in a particular cell or tissue. Oligonucleotides comprise portions of a nucleic acid sequence having about 10 nt, 50 nt, or 100 nt in length, preferably about 15 nt to 30 nt in length. In one embodiment of the invention, an oligonucleotide comprising a nucleic acid molecule less than 100 nt in length would further comprise at least 6 contiguous nucleotides SEQ ID NOS:1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91 and 93, or a complement thereof. Oligonucleotides may be chemically synthesized and may also be used as probes.
In another embodiment, an isolated nucleic acid molecule of the invention comprises a nucleic acid molecule that is a complement of the nucleotide sequence shown in SEQ ID NOS:1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91 and 93, or a portion of this nucleotide sequence (e.g., a fragment that can be used as a probe or primer or a fragment encoding a biologically-active portion of an NOVX polypeptide). A nucleic acid molecule that is complementary to the nucleotide sequence shown NOS:1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91 and 93 is one that is sufficiently complementary to the nucleotide sequence shown NOS:1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91 and 93 that it can hydrogen bond with little or no mismatches to the nucleotide sequence shown SEQ ID NOS:1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91 and 93, thereby forming a stable duplex.
As used herein, the term “complementary” refers to Watson-Crick or Hoogsteen base pairing between nucleotides units of a nucleic acid molecule, and the term “binding” means the physical or chemical interaction between two polypeptides or compounds or associated polypeptides or compounds or combinations thereof. Binding includes ionic, non-ionic, van der Waals, hydrophobic interactions, and the like. A physical interaction can be either direct or indirect. Indirect interactions may be through or due to the effects of another polypeptide or compound. Direct binding refers to interactions that do not take place through, or due to, the effect of another polypeptide or compound, but instead are without other substantial chemical intermediates.
Fragments provided herein are defined as sequences of at least 6 (contiguous) nucleic acids or at least 4 (contiguous) amino acids, a length sufficient to allow for specific hybridization in the case of nucleic acids or for specific recognition of an epitope in the case of amino acids, respectively, and are at most some portion less than a full length sequence. Fragments may be derived from any contiguous portion of a nucleic acid or amino acid sequence of choice. Derivatives are nucleic acid sequences or amino acid sequences formed from the native compounds either directly or by modification or partial substitution. Analogs are nucleic acid sequences or amino acid sequences that have a structure similar to, but not identical to, the native compound but differs from it in respect to certain components or side chains. Analogs may be synthetic or from a different evolutionary origin and may have a similar or opposite metabolic activity compared to wild type. Homologs are nucleic acid sequences or amino acid sequences of a particular gene that are derived from different species.
Derivatives and analogs may be full length or other than full length, if the derivative or analog contains a modified nucleic acid or amino acid, as described below. Derivatives or analogs of the nucleic acids or proteins of the invention include, but are not limited to, molecules comprising regions that are substantially homologous to the nucleic acids or proteins of the invention, in various embodiments, by at least about 70%, 80%, or 95% identity (with a preferred identity of 80-95%) over a nucleic acid or amino acid sequence of identical size or when compared to an aligned sequence in which the alignment is done by a computer homology program known in the art, or whose encoding nucleic acid is capable of hybridizing to the complement of a sequence encoding the aforementioned proteins under stringent, moderately stringent, or low stringent conditions. See e.g. Ausubel, et al., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, New York, N.Y., 1993, and below.
A “homologous nucleic acid sequence” or “homologous amino acid sequence,” or variations thereof, refer to sequences characterized by a homology at the nucleotide level or amino acid level as discussed above. Homologous nucleotide sequences encode those sequences coding for isoforms of NOVX polypeptides. Isoforms can be expressed in different tissues of the same organism as a result of, for example, alternative splicing of RNA. Alternatively, isoforms can be encoded by different genes. In the invention, homologous nucleotide sequences include nucleotide sequences encoding for an NOVX polypeptide of species other than humans, including, but not limited to: vertebrates, and thus can include, e.g., frog, mouse, rat, rabbit, dog, cat cow, horse, and other organisms. Homologous nucleotide sequences also include, but are not limited to, naturally occurring allelic variations and mutations of the nucleotide sequences set forth herein. A homologous nucleotide sequence does not, however, include the exact nucleotide sequence encoding human NOVX protein. Homologous nucleic acid sequences include those nucleic acid sequences that encode conservative amino acid substitutions (see below) in SEQ ID NOS:1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91 and 93, as well as a polypeptide possessing NOVX biological activity. Various biological activities of the NOVX proteins are described below.
An NOVX polypeptide is encoded by the open reading frame (“ORF”) of an NOVX nucleic acid. An ORF corresponds to a nucleotide sequence that could potentially be translated into a polypeptide. A stretch of nucleic acids comprising an ORF is uninterrupted by a stop codon. An ORF that represents the coding sequence for a full protein begins with an ATG “start” codon and terminates with one of the three “stop” codons, namely, TAA, TAG, or TGA. For the purposes of this invention, an ORF may be any part of a coding sequence, with or without a start codon, a stop codon, or both. For an ORF to be considered as a good candidate for coding for a bona fide cellular protein, a minimum size requirement is often set, e.g., a stretch of DNA that would encode a protein of 50 amino acids or more.
The nucleotide sequences determined from the cloning of the human NOVX genes allows for the generation of probes and primers designed for use in identifying and/or cloning NOVX homologues in other cell types, e.g. from other tissues, as well as NOVX homologues from other vertebrates. The probe/primer typically comprises substantially purified oligonucleotide. The oligonucleotide typically comprises a region of nucleotide sequence that hybridizes under stringent conditions to at least about 12, 25, 50, 100, 150, 200, 250, 300, 350 or 400 consecutive sense strand nucleotide sequence SEQ ID NOS:1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91 and 93; or an anti-sense strand nucleotide sequence of SEQ ID NOS:1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91 and 93; or of a naturally occurring mutant of SEQ ID NOS:1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91 and 93.
Probes based on the human NOVX nucleotide sequences can be used to detect transcripts or genomic sequences encoding the same or homologous proteins. In various embodiments, the probe further comprises a label group attached thereto, e.g. the label group can be a radioisotope, a fluorescent compound, an enzyme, or an enzyme co-factor. Such probes can be used as a part of a diagnostic test kit for identifying cells or tissues which mis-express an NOVX protein, such as by measuring a level of an NOVX-encoding nucleic acid in a sample of cells from a subject e.g., detecting NOVX mRNA levels or determining whether a genomic NOVX gene has been mutated or deleted.
“A polypeptide having a biologically-active portion of an NOVX polypeptide” refers to polypeptides exhibiting activity similar, but not necessarily identical to, an activity of a polypeptide of the invention, including mature forms, as measured in a particular biological assay, with or without dose dependency. A nucleic acid fragment encoding a “biologically-active portion of NOVX” can be prepared by isolating a portion SEQ ID NOS:1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91 and 93, that encodes a polypeptide having an NOVX biological activity (the biological activities of the NOVX proteins are described below), expressing the encoded portion of NOVX protein (e.g., by recombinant expression in vitro) and assessing the activity of the encoded portion of NOVX.
NOVX Nucleic Acid and Polypeptide Variants
The invention further encompasses nucleic acid molecules that differ from the nucleotide sequences shown in SEQ ID NOS:1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91 and 93 due to degeneracy of the genetic code and thus encode the same NOVX proteins as that encoded by the nucleotide sequences shown in SEQ ID NOS:1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91 and 93. In another embodiment, an isolated nucleic acid molecule of the invention has a nucleotide sequence encoding a protein having an amino acid sequence shown in SEQ ID NOS:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92 and 94.
In addition to the human NOVX nucleotide sequences shown in SEQ ID NOS:1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91 and 93, it will be appreciated by those skilled in the art that DNA sequence polymorphisms that lead to changes in the amino acid sequences of the NOVX polypeptides may exist within a population (e.g., the human population). Such genetic polymorphism in the NOVX genes may exist among individuals within a population due to natural allelic variation. As used herein, the terms “gene” and “recombinant gene” refer to nucleic acid molecules comprising an open reading frame (ORF) encoding an NOVX protein, preferably a vertebrate NOVX protein. Such natural allelic variations can typically result in 1-5% variance in the nucleotide sequence of the NOVX genes. Any and all such nucleotide variations and resulting amino acid polymorphisms in the NOVX polypeptides, which are the result of natural allelic variation and that do not alter the functional activity of the NOVX polypeptides, are intended to be within the scope of the invention.
Moreover, nucleic acid molecules encoding NOVX proteins from other species, and thus that have a nucleotide sequence that differs from the human SEQ ID NOS:1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91 and 93 are intended to be within the scope of the invention. Nucleic acid molecules corresponding to natural allelic variants and homologues of the NOVX cDNAs of the invention can be isolated based on their homology to the human NOVX nucleic acids disclosed herein using the human cDNAs, or a portion thereof, as a hybridization probe according to standard hybridization techniques under stringent hybridization conditions.
Accordingly, in another embodiment, an isolated nucleic acid molecule of the invention is at least 6 nucleotides in length and hybridizes under stringent conditions to the nucleic acid molecule comprising the nucleotide sequence of SEQ ID NOS:1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91 and 93. In another embodiment, the nucleic acid is at least 10, 25, 50, 100, 250, 500, 750, 1000, 1500, or 2000 or more nucleotides in length. In yet another embodiment, an isolated nucleic acid molecule of the invention hybridizes to the coding region. As used herein, the term “hybridizes under stringent conditions” is intended to describe conditions for hybridization and washing under which nucleotide sequences at least 60% homologous to each other typically remain hybridized to each other.
Homologs (i.e., nucleic acids encoding NOVX proteins derived from species other than human) or other related sequences (e.g., paralogs) can be obtained by low, moderate or high stringency hybridization with all or a portion of the particular human sequence as a probe using methods well known in the art for nucleic acid hybridization and cloning.
As used herein, the phrase “stringent hybridization conditions” refers to conditions under which a probe, primer or oligonucleotide will hybridize to its target sequence, but to no other sequences. Stringent conditions are sequence-dependent and will be different in different circumstances. Longer sequences hybridize specifically at higher temperatures than shorter sequences. Generally, stringent conditions are selected to be about 5° C. lower than the thermal melting point (Tm) for the specific sequence at a defined ionic strength and pH. The Tm is the temperature (under defined ionic strength, pH and nucleic acid concentration) at which 50% of the probes complementary to the target sequence hybridize to the target sequence at equilibrium. Since the target sequences are generally present at excess, at Tm, 50% of the probes are occupied at equilibrium. Typically, stringent conditions will be those in which the salt concentration is less than about 1.0 M sodium ion, typically about 0.01 to 1.0 M sodium ion (or other salts) at pH 7.0 to 8.3 and the temperature is at least about 30° C. for short probes, primers or oligonucleotides (e.g., 10 nt to 50 nt) and at least about 60° C. for longer probes, primers and oligonucleotides. Stringent conditions may also be achieved with the addition of destabilizing agents, such as formamide.
Stringent conditions are known to those skilled in the art and can be found in Ausubel, et al., (eds.), CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, N.Y. (1989), 6.3.1-6.3.6. Preferably, the conditions are such that sequences at least about 65%, 70%, 75%, 85%, 90%, 95%, 98%, or 99% homologous to each other typically remain hybridized to each other. A non-limiting example of stringent hybridization conditions are hybridization in a high salt buffer comprising 6×SSC, 50 mM Tris-HCl (pH 7.5), 1 mM EDTA, 0.02% PVP, 0.02% Ficoll, 0.02% BSA, and 500 mg/ml denatured salmon sperm DNA at 65° C., followed by one or more washes in 0.2×SSC, 0.01% BSA at 50° C. An isolated nucleic acid molecule of the invention that hybridizes under stringent conditions to the sequences SEQ ID NOS:1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91 and 93, corresponds to a naturally-occurring nucleic acid molecule. As used herein, a “naturally-occurring” nucleic acid molecule refers to an RNA or DNA molecule having a nucleotide sequence that occurs in nature (e.g., encodes a natural protein).
In a second embodiment, a nucleic acid sequence that is hybridizable to the nucleic acid molecule comprising the nucleotide sequence of SEQ ID NOS:1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91 and 93, or fragments, analogs or derivatives thereof, under conditions of moderate stringency is provided. A non-limiting example of moderate stringency hybridization conditions are hybridization in 6×SSC, 5×Denhardt's solution, 0.5% SDS and 100 mg/ml denatured salmon sperm DNA at 55° C., followed by one or more washes in 1×SSC, 0.1% SDS at 37° C. Other conditions of moderate stringency that may be used are well-known within the art. See, e.g., Ausubel, et al. (eds.), 1993, CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, NY, and Kriegler, 1990; GENE TRANSFER AND EXPRESSION, A LABORATORY MANUAL, Stockton Press, NY.
In a third embodiment, a nucleic acid that is hybridizable to the nucleic acid molecule comprising the nucleotide sequences SEQ ID NOS:1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91 and 93, or fragments, analogs or derivatives thereof, under conditions of low stringency, is provided. A non-limiting example of low stringency hybridization conditions are hybridization in 35% formamide, 5×SSC, 50 mM Tris-HCl (pH 7.5), 5 mM EDTA, 0.02% PVP, 0.02% Ficoll, 0.2% BSA, 100 mg/ml denatured salmon sperm DNA, 10% (wt/vol) dextran sulfate at 40° C., followed by one or more washes in 2×SSC, 25 mM Tris-HCl (pH 7.4), 5 mM EDTA, and 0.1% SDS at 50° C. Other conditions of low stringency that may be used are well known in the art (e.g., as employed for cross-species hybridizations). See, e.g., Ausubel, et al. (eds.), 1993, CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, NY, and Kriegler, 1990, GENE TRANSFER AND EXPRESSION, A LABORATORY MANUAL, Stockton Press, NY; Shilo and Weinberg, 1981. Proc Natl Acad Sci USA 78: 6789-6792.
Conservative Mutations
In addition to naturally-occurring allelic variants of NOVX sequences that may exist in the population, the skilled artisan will further appreciate that changes can be introduced by mutation into the nucleotide sequences SEQ ID NOS:1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91 and 93, thereby leading to changes in the amino acid sequences of the encoded NOVX proteins, without altering the functional ability of said NOVX proteins. For example, nucleotide substitutions leading to amino acid substitutions at “non-essential” amino acid residues can be made in the sequence SEQ ID NOS:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92 and 94. A “non-essential” amino acid residue is a residue that can be altered from the wild-type sequences of the NOVX proteins without altering their biological activity, whereas an “essential” amino acid residue is required for such biological activity. For example, amino acid residues that are conserved among the NOVX proteins of the invention are predicted to be particularly non-amenable to alteration. Amino acids for which conservative substitutions can be made are well-known within the art.
Another aspect of the invention pertains to nucleic acid molecules encoding NOVX proteins that contain changes in amino acid residues that are not essential for activity. Such NOVX proteins differ in amino acid sequence from SEQ ID NOS:1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91 and 93 yet retain biological activity. In one embodiment, the isolated nucleic acid molecule comprises a nucleotide sequence encoding a protein, wherein the protein comprises an amino acid sequence at least about 45% homologous to the amino acid sequences SEQ ID NOS:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92 and 94. Preferably, the protein encoded by the nucleic acid molecule is at least about 60% homologous to SEQ ID NOS:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92 and 94; more preferably at least about 70% homologous SEQ ID NOS:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92 and 94; still more preferably at least about 80% homologous to SEQ ID NOS:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92 and 94; even more preferably at least about 90% homologous to SEQ ID NOS:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92 and 94; and most preferably at least about 95% homologous to SEQ ID NOS:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92 and 94.
An isolated nucleic acid molecule encoding an NOVX protein homologous to the protein of SEQ ID NOS:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92 and 94 can be created by introducing one or more nucleotide substitutions, additions or deletions into the nucleotide sequence of SEQ ID NOS:1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91 and 93, such that one or more amino acid substitutions, additions or deletions are introduced into the encoded protein.
Mutations can be introduced into SEQ ID NOS:1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91 and 93 by standard techniques, such as site-directed mutagenesis and PCR-mediated mutagenesis. Preferably, conservative amino acid substitutions are made at one or more predicted, non-essential amino acid residues. A “conservative amino acid substitution” is one in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined within the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g. threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, a predicted non-essential amino acid residue in the NOVX protein is replaced with another amino acid residue from the same side chain family. Alternatively, in another embodiment, mutations can be introduced randomly along all or part of an NOVX coding sequence, such as by saturation mutagenesis, and the resultant mutants can be screened for NOVX biological activity to identify mutants that retain activity. Following mutagenesis SEQ ID NOS:1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91 and 93, the encoded protein can be expressed by any recombinant technology known in the art and the activity of the protein can be determined.
The relatedness of amino acid families may also be determined based on side chain interactions. Substituted amino acids may be fully conserved “strong” residues or fully conserved “weak” residues. The “strong” group of conserved amino acid residues may be any one of the following groups: STA, NEQK, NHQK, NDEQ, QHRK, MILV, MILF, HY, FYW, wherein the single letter amino acid codes are grouped by those amino acids that may be substituted for each other. Likewise, the “weak” group of conserved residues may be any one of the following: CSA, ATV, SAG, STNK, STPA, SGND, SNDEQK, NDEQHK, NEQHRK, HFY, wherein the letters within each group represent the single letter amino acid code.
In one embodiment, a mutant NOVX protein can be assayed for (i) the ability to form protein:protein interactions with other NOVX proteins, other cell-surface proteins, or biologically-active portions thereof, (ii) complex formation between a mutant NOVX protein and an NOVX ligand; or (iii) the ability of a mutant NOVX protein to bind to an intracellular target protein or biologically-active portion thereof; (e.g. avidin proteins).
In yet another embodiment, a mutant NOVX protein can be assayed for the ability to regulate a specific biological function (e.g., regulation of insulin release).
Antisense Nucleic Acids
Another aspect of the invention pertains to isolated antisense nucleic acid molecules that are hybridizable to or complementary to the nucleic acid molecule comprising the nucleotide sequence of SEQ ID NOS:1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91 and 93, or fragments, analogs or derivatives thereof. An “antisense” nucleic acid comprises a nucleotide sequence that is complementary to a “sense” nucleic acid encoding a protein (e.g., complementary to the coding strand of a double-stranded cDNA molecule or complementary to an mRNA sequence). In specific aspects, antisense nucleic acid molecules are provided that comprise a sequence complementary to at least about 10, 25, 50, 100, 250 or 500 nucleotides or an entire NOVX coding strand, or to only a portion thereof. Nucleic acid molecules encoding fragments, homologs, derivatives and analogs of an NOVX protein of SEQ ID NOS:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92 and 94, or antisense nucleic acids complementary to an NOVX nucleic acid sequence of SEQ ID NOS:1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91 and 93, are additionally provided.
In one embodiment, an antisense nucleic acid molecule is antisense to a “coding region” of the coding strand of a nucleotide sequence encoding an NOVX protein. The term “coding region” refers to the region of the nucleotide sequence comprising codons which are translated into amino acid residues. In another embodiment, the antisense nucleic acid molecule is antisense to a “noncoding region” of the coding strand of a nucleotide sequence encoding the NOVX protein. The term “noncoding region” refers to 5′ and 3′ sequences which flank the coding region that are not translated into amino acids (i.e., also referred to as 5′ and 3′ untranslated regions).
Given the coding strand sequences encoding the NOVX protein disclosed herein, antisense nucleic acids of the invention can be designed according to the rules of Watson and Crick or Hoogsteen base pairing. The antisense nucleic acid molecule can be complementary to the entire coding region of NOVX mRNA, but more preferably is an oligonucleotide that is antisense to only a portion of the coding or noncoding region of NOVX mRNA. For example, the antisense oligonucleotide can be complementary to the region surrounding the translation start site of NOVX mRNA. An antisense oligonucleotide can be, for example, about 5, 10, 15, 20, 25, 30, 35, 40, 45 or 50 nucleotides in length. An antisense nucleic acid of the invention can be constructed using chemical synthesis or enzymatic ligation reactions using procedures known in the art. For example, an antisense nucleic acid (e.g., an antisense oligonucleotide) can be chemically synthesized using naturally-occurring nucleotides or variously modified nucleotides designed to increase the biological stability of the molecules or to increase the physical stability of the duplex formed between the antisense and sense nucleic acids (e.g., phosphorothioate derivatives and acridine substituted nucleotides can be used).
Examples of modified nucleotides that can be used to generate the antisense nucleic acid include: 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine, 5-(carboxyhydroxylmethyl) uracil, 5-carboxymethylaminomethyl-2-thiouridine, 5-carboxymethylaminomethyluracil, dihydrouracil, beta-D-galactosylqueosine, inosine, N6-isopentenyladenine, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine, 7-methylguanine, 5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine, 5′-methoxycarboxymethyluracil, 5-methoxyuracil, 2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid (v), wybutoxosine, pseudouracil, queosine, 2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid (v), 5-methyl-2-thiouracil, 3-(3-amino-3-N-2-carboxypropyl) uracil, (acp3)w, and 2,6-diaminopurine. Alternatively, the antisense nucleic acid can be produced biologically using an expression vector into which a nucleic acid has been subdloned in an antisense orientation (i.e., RNA transcribed from the inserted nucleic acid will be of an antisense orientation to a target nucleic acid of interest, described further in the following subsection).
The antisense nucleic acid molecules of the invention are typically administered to a subject or generated in situ such that they hybridize with or bind to cellular mRNA and/or genomic DNA encoding an NOVX protein to thereby inhibit expression of the protein (e.g., by inhibiting transcription and/or translation). The hybridization can be by conventional nucleotide complementarity to form a stable duplex, or, for example, in the case of an antisense nucleic acid molecule that binds to DNA duplexes, through specific interactions in the major groove of the double helix. An example of a route of administration of antisense nucleic acid molecules of the invention includes direct injection at a tissue site. Alternatively, antisense nucleic acid molecules can be modified to target selected cells and then administered systemically. For example, for systemic administration, antisense molecules can be modified such that they specifically bind to receptors or antigens expressed on a selected cell surface (e.g., by linking the antisense nucleic acid molecules to peptides or antibodies that bind to cell surface receptors or antigens). The antisense nucleic acid molecules can also be delivered to cells using the vectors described herein. To achieve sufficient nucleic acid molecules, vector constructs in which the antisense nucleic acid molecule is placed under the control of a strong pol II or pol III promoter are preferred.
In yet another embodiment, the antisense nucleic acid molecule of the invention is an α-anomeric nucleic acid molecule. An α-anomeric nucleic acid molecule forms specific double-stranded hybrids with complementary RNA in which, contrary to the usual β-units, the strands run parallel to each other. See, e.g., Gaultier, et al., 1987. Nucl. Acids Res. 15: 6625-6641. The antisense nucleic acid molecule can also comprise a 2′-o-methylribonucleotide (See, e.g., Inoue, et al. 1987. Nucl. Acids Res. 15: 6131-6148) or a chimeric RNA-DNA analogue (See, e.g., Inoue, et al., 1987. FEBS Lett. 215: 327-330.
Ribozymes and PNA Moieties
Nucleic acid modifications include, by way of non-limiting example, modified bases, and nucleic acids whose sugar phosphate backbones are modified or derivatized. These modifications are carried out at least in part to enhance the chemical stability of the modified nucleic acid, such that they may be used, for example, as antisense binding nucleic acids in therapeutic applications in a subject.
In one embodiment, an antisense nucleic acid of the invention is a ribozyme. Ribozymes are catalytic RNA molecules with ribonuclease activity that are capable of cleaving a single-stranded nucleic acid, such as an mRNA, to which they have a complementary region. Thus, ribozymes (e.g., hammerhead ribozymes as described in Haselhoff and Gerlach 1988. Nature 334: 585-591) can be used to catalytically cleave NOVX mRNA transcripts to thereby inhibit translation of NOVX mRNA. A ribozyme having specificity for an NOVX-encoding nucleic acid can be designed based upon the nucleotide sequence of an NOVX cDNA disclosed herein (i.e., SEQ ID NOS:1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91 and 93). For example, a derivative of a Tetrahymena L-19 IVS RNA can be constructed in which the nucleotide sequence of the active site is complementary to the nucleotide sequence to be cleaved in an NOVX-encoding mRNA. See, e.g., U.S. Pat. No. 4,987,071 to Cech, et al. and U.S. Pat. No. 5,116,742 to Cech, et al. NOVX mRNA can also be used to select a catalytic RNA having a specific ribonuclease activity from a pool of RNA molecules. See, e.g., Bartel et al., (1993) Science 261:1411-1418.
Alternatively, NOVX gene expression can be inhibited by targeting nucleotide sequences complementary to the regulatory region of the NOVX nucleic acid (e.g., the NOVX promoter and/or enhancers) to form triple helical structures that prevent transcription of the NOVX gene in target cells. See, e.g., Helene, 1991. Anticancer Drug Des. 6: 569-84; Helene, et al. 1992. Ann. N.Y. Acad. Sci. 660: 27-36; Maher, 1992. Bioassays 14: 807-15.
In various embodiments, the NOVX nucleic acids can be modified at the base moiety, sugar moiety or phosphate backbone to improve, e.g., the stability, hybridization, or solubility of the molecule. For example, the deoxyribose phosphate backbone of the nucleic acids can be modified to generate peptide nucleic acids. See, e.g., Hyrup, et al., 1996. Bioorg Med Chem 4: 5-23. As used herein, the terms “peptide nucleic acids” or “PNAs” refer to nucleic acid mimics (e.g., DNA mimics) in which the deoxyribose phosphate backbone is replaced by a pseudopeptide backbone and only the four natural nucleobases are retained. The neutral backbone of PNAs has been shown to allow for specific hybridization to DNA and RNA under conditions of low ionic strength. The synthesis of PNA oligomers can be performed using standard solid phase peptide synthesis protocols as described in Hyrup, et al., 1996. supra; Perry-O'Keefe, et al., 1996. Proc. Natl. Acad. Sci. USA 93: 14670-14675.
PNAs of NOVX can be used in therapeutic and diagnostic applications. For example, PNAs can be used as antisense or antigene agents for sequence-specific modulation of gene expression by, e.g., inducing transcription or translation arrest or inhibiting replication. PNAs of NOVX can also be used, for example, in the analysis of single base pair mutations in a gene (e.g., PNA directed PCR clamping; as artificial restriction enzymes when used in combination with other enzymes, e.g., S1 nucleases (See, Hyrup, et al., 1996. supra); or as probes or primers for DNA sequence and hybridization (See, Hyrup, et al., 1996, supra; Perry-O'Keefe, et al., 1996. supra).
In another embodiment, PNAs of NOVX can be modified, e.g., to enhance their stability or cellular uptake, by attaching lipophilic or other helper groups to PNA, by the formation of PNA-DNA chimeras, or by the use of liposomes or other techniques of drug delivery known in the art. For example, PNA-DNA chimeras of NOVX can be generated that may combine the advantageous properties of PNA and DNA. Such chimeras allow DNA recognition enzymes (e.g., RNase H and DNA polymerases) to interact with the DNA portion while the PNA portion would provide high binding affinity and specificity. PNA-DNA chimeras can be linked using linkers of appropriate lengths selected in terms of base stacking, number of bonds between the nucleobases, and orientation (see, Hyrup, et al., 1996. supra). The synthesis of PNA-DNA chimeras can be performed as described in Hyrup, et al., 1996. supra and Finn, et al., 1996. Nucl Acids Res 24: 3357-3363. For example, a DNA chain can be synthesized on a solid support using standard phosphoramidite coupling chemistry, and modified nucleoside analogs, e.g., 5′-(4-methoxytrityl)amino-5′-deoxy-thymidine phosphoramidite, can be used between the PNA and the 5′ end of DNA. See, e.g., Mag, et al., 1989. Nucl Acid Res 17: 5973-5988. PNA monomers are then coupled in a stepwise manner to produce a chimeric molecule with a 5′ PNA segment and a 3′ DNA segment. See, e.g., Finn, et al., 1996. supra. Alternatively, chimeric molecules can be synthesized with a 5′ DNA segment and a 3′ PNA segment. See, e.g., Petersen, et al., 1975. Bioorg. Med. Chem. Lett. 5: 1119-11124.
In other embodiments, the oligonucleotide may include other appended groups such as peptides (e.g., for targeting host cell receptors in vivo), or agents facilitating transport across the cell membrane (see, e.g., Letsinger, et al., 1989. Proc. Natl. Acad. Sci. U.S.A. 86: 6553-6556; Lemaitre, et al., 1987. Proc. Natl. Acad. Sci. 84: 648-652; PCT Publication No. WO88/09810) or the blood-brain barrier (see, e.g., PCT Publication No. WO 89/10134). In addition, oligonucleotides can be modified with hybridization triggered cleavage agents (see, e.g., Krol, et al., 1988. BioTechniques 6:958-976) or intercalating agents (see, e.g., Zon, 1988. Pharm. Res. 5: 539-549). To this end, the oligonucleotide may be conjugated to another molecule, e.g., a peptide, a hybridization triggered cross-linking agent, a transport agent, a hybridization-triggered cleavage agent, and the like.
NOVX Polypeptides
A polypeptide according to the invention includes a polypeptide including the amino acid sequence of NOVX polypeptides whose sequences are provided in SEQ ID NOS:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92 and 94. The invention also includes a mutant or variant protein any of whose residues may be changed from the corresponding residues shown in SEQ ID NOS:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92 and 94 while still encoding a protein that maintains its NOVX activities and physiological functions, or a functional fragment thereof.
In general, an NOVX variant that preserves NOVX-like function includes any variant in which residues at a particular position in the sequence have been substituted by other amino acids, and further include the possibility of inserting an additional residue or residues between two residues of the parent protein as well as the possibility of deleting one or more residues from the parent sequence. Any amino acid substitution, insertion, or deletion is encompassed by the invention. In favorable circumstances, the substitution is a conservative substitution as defined above.
One aspect of the invention pertains to isolated NOVX proteins, and biologically-active portions thereof, or derivatives, fragments, analogs or homologs thereof. Also provided are polypeptide fragments suitable for use as immunogens to raise anti-NOVX antibodies. In one embodiment, native NOVX proteins can be isolated from cells or tissue sources by an appropriate purification scheme using standard protein purification techniques. In another embodiment, NOVX proteins are produced by recombinant DNA techniques. Alternative to recombinant expression, an NOVX protein or polypeptide can be synthesized chemically using standard peptide synthesis techniques.
An “isolated” or “purified” polypeptide or protein or biologically-active portion thereof is substantially free of cellular material or other contaminating proteins from the cell or tissue source from which the NOVX protein is derived, or substantially free from chemical precursors or other chemicals when chemically synthesized. The language “substantially free of cellular material” includes preparations of NOVX proteins in which the protein is separated from cellular components of the cells from which it is isolated or recombinantly-produced. In one embodiment, the language “substantially free of cellular material” includes preparations of NOVX proteins having less than about 30% (by dry weight) of non-NOVX proteins (also referred to herein as a “contaminating protein”), more preferably less than about 20% of non-NOVX proteins, still more preferably less than about 10% of non-NOVX proteins, and most preferably less than about 5% of non-NOVX proteins. When the NOVX protein or biologically-active portion thereof is recombinantly-produced, it is also preferably substantially free of culture medium, i.e., culture medium represents less than about 20%, more preferably less than about 10%, and most preferably less than about 5% of the volume of the NOVX protein preparation.
The language “substantially free of chemical precursors or other chemicals” includes preparations of NOVX proteins in which the protein is separated from chemical precursors or other chemicals that are involved in the synthesis of the protein. In one embodiment, the language “substantially free of chemical precursors or other chemicals” includes preparations of NOVX proteins having less than about 30% (by dry weight) of chemical precursors or non-NOVX chemicals, more preferably less than about 20% chemical precursors or non-NOVX chemicals, still more preferably less than about 10% chemical precursors or non-NOVX chemicals, and most preferably less than about 5% chemical precursors or non-NOVX chemicals.
Biologically-active portions of NOVX proteins include peptides comprising amino acid sequences sufficiently homologous to or derived from the amino acid sequences of the NOVX proteins (e.g., the amino acid sequence shown in SEQ ID NOS:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92 and 94) that include fewer amino acids than the full-length NOVX proteins, and exhibit at least one activity of an NOVX protein. Typically, biologically-active portions comprise a domain or motif with at least one activity of the NOVX protein. A biologically-active portion of an NOVX protein can be a polypeptide which is, for example, 10, 25, 50, 100 or more amino acid residues in length.
Moreover, other biologically-active portions, in which other regions of the protein are deleted, can be prepared by recombinant techniques and evaluated for one or more of the functional activities of a native NOVX protein.
In an embodiment, the NOVX protein has an amino acid sequence shown SEQ ID NOS:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92 and 94. In other embodiments, the NOVX protein is substantially homologous to SEQ ID NOS:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92 and 94, and retains the functional activity of the protein of SEQ ID NOS:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92 and 94, yet differs in amino acid sequence due to natural allelic variation or mutagenesis, as described in detail, below. Accordingly, in another embodiment, the NOVX protein is a protein that comprises an amino acid sequence at least about 45% homologous to the amino acid sequence SEQ ID NOS:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92 and 94, and retains the functional activity of the NOVX proteins of SEQ ID NOS:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92 and 94.
Determining Homology Between Two or More Sequences
To determine the percent homology of two amino acid sequences or of two nucleic acids, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in the sequence of a first amino acid or nucleic acid sequence for optimal alignment with a second amino or nucleic acid sequence). The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are homologous at that position (i.e., as used herein amino acid or nucleic acid “homology” is equivalent to amino acid or nucleic acid “identity”).
The nucleic acid sequence homology may be determined as the degree of identity between two sequences. The homology may be determined using computer programs known in the art, such as GAP software provided in the GCG program package. See, Needleman and Wunsch, 1970. J Mol Biol 48: 443-453. Using GCG GAP software with the following settings for nucleic acid sequence comparison: GAP creation penalty of 5.0 and GAP extension penalty of 0.3, the coding region of the analogous nucleic acid sequences referred to above exhibits a degree of identity preferably of at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99%, with the CDS (encoding) part of the DNA sequence shown in SEQ ID NOS:1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91 and 93.
The term “sequence identity” refers to the degree to which two polynucleotide or polypeptide sequences are identical on a residue-by-residue basis over a particular region of comparison. The term “percentage of sequence identity” is calculated by comparing two optimally aligned sequences over that region of comparison, determining the number of positions at which the identical nucleic acid base (e.g., A, T, C, G, U, or I, in the case of nucleic acids) occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the region of comparison (i.e., the window size), and multiplying the result by 100 to yield the percentage of sequence identity. The term “substantial identity” as used herein denotes a characteristic of a polynucleotide sequence, wherein the polynucleotide comprises a sequence that has at least 80 percent sequence identity, preferably at least 85 percent identity and often 90 to 95 percent sequence identity, more usually at least 99 percent sequence identity as compared to a reference sequence over a comparison region.
Chimeric and Fusion Proteins
The invention also provides NOVX chimeric or fusion proteins. As used herein, an NOVX “chimeric protein” or “fusion protein” comprises an NOVX polypeptide operatively-linked to a non-NOVX polypeptide. An “NOVX polypeptide” refers to a polypeptide having an amino acid sequence corresponding to an NOVX protein SEQ ID NOS:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92 and 94, whereas a “non-NOVX polypeptide” refers to a polypeptide having an amino acid sequence corresponding to a protein that is not substantially homologous to the NOVX protein, e.g., a protein that is different from the NOVX protein and that is derived from the same or a different organism. Within an NOVX fusion protein the NOVX polypeptide can correspond to all or a portion of an NOVX protein. In one embodiment, an NOVX fusion protein comprises at least one biologically-active portion of an NOVX protein. In another embodiment, an NOVX fusion protein comprises at least two biologically-active portions of an NOVX protein. In yet another embodiment, an NOVX fusion protein comprises at least three biologically-active portions of an NOVX protein. Within the fusion protein, the term “operatively-linked” is intended to indicate that the NOVX polypeptide and the non-NOVX polypeptide are fused in-frame with one another. The non-NOVX polypeptide can be fused to the N-terminus or C-terminus of the NOVX polypeptide.
In one embodiment, the fusion protein is a GST-NOVX fusion protein in which the NOVX sequences are fused to the C-terminus of the GST (glutathione S-transferase) sequences. Such fusion proteins can facilitate the purification of recombinant NOVX polypeptides.
In another embodiment, the fusion protein is an NOVX protein containing a heterologous signal sequence at its N-terminus. In certain host cells (e.g., mammalian host cells), expression and/or secretion of NOVX can be increased through use of a heterologous signal sequence.
In yet another embodiment, the fusion protein is an NOVX-immunoglobulin fusion protein in which the NOVX sequences are fused to sequences derived from a member of the immunoglobulin protein family. The NOVX-immunoglobulin fusion proteins of the invention can be incorporated into pharmaceutical compositions and administered to a subject to inhibit an interaction between an NOVX ligand and an NOVX protein on the surface of a cell, to thereby suppress NOVX-mediated signal transduction in vivo. The NOVX-immunoglobulin fusion proteins can be used to affect the bioavailability of an NOVX cognate ligand. Inhibition of the NOVX ligand/NOVX interaction may be useful therapeutically for both the treatment of proliferative and differentiative disorders, as well as modulating (e.g. promoting or inhibiting) cell survival. Moreover, the NOVX-immunoglobulin fusion proteins of the invention can be used as immunogens to produce anti-NOVX antibodies in a subject, to purify NOVX ligands, and in screening assays to identify molecules that inhibit the interaction of NOVX with an NOVX ligand.
An NOVX chimeric or fusion protein of the invention can be produced by standard recombinant DNA techniques. For example, DNA fragments coding for the different polypeptide sequences are ligated together in-frame in accordance with conventional techniques, e.g., by employing blunt-ended or stagger-ended termini for ligation, restriction enzyme digestion to provide for appropriate termini, filling-in of cohesive ends as appropriate, alkaline phosphatase treatment to avoid undesirable joining, and enzymatic ligation. In another embodiment, the fusion gene can be synthesized by conventional techniques including automated DNA synthesizers. Alternatively, PCR amplification of gene fragments can be carried out using anchor primers that give rise to complementary overhangs between two consecutive gene fragments that can subsequently be annealed and reamplified to generate a chimeric gene sequence (see, e.g., Ausubel, et al. (eds.) CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, 1992). Moreover, many expression vectors are commercially available that already encode a fusion moiety (e.g., a GST polypeptide). An NOVX-encoding nucleic acid can be cloned into such an expression vector such that the fusion moiety is linked in-frame to the NOVX protein.
NOVX Agonists and Antagonists
The invention also pertains to variants of the NOVX proteins that function as either NOVX agonists (i.e., mimetics) or as NOVX antagonists. Variants of the NOVX protein can be generated by mutagenesis (e.g., discrete point mutation or truncation of the NOVX protein). An agonist of the NOVX protein can retain substantially the same, or a subset of, the biological activities of the naturally occurring form of the NOVX protein. An antagonist of the NOVX protein can inhibit one or more of the activities of the naturally occurring form of the NOVX protein by, for example, competitively binding to a downstream or upstream member of a cellular signaling cascade which includes the NOVX protein. Thus, specific biological effects can be elicited by treatment with a variant of limited function. In one embodiment, treatment of a subject with a variant having a subset of the biological activities of the naturally occurring form of the protein has fewer side effects in a subject relative to treatment with the naturally occurring form of the NOVX proteins.
Variants of the NOVX proteins that function as either NOVX agonists (i.e., mimetics) or as NOVX antagonists can be identified by screening combinatorial libraries of mutants (e.g., truncation mutants) of the NOVX proteins for NOVX protein agonist or antagonist activity. In one embodiment, a variegated library of NOVX variants is generated by combinatorial mutagenesis at the nucleic acid level and is encoded by a variegated gene library. A variegated library of NOVX variants can be produced by, for example, enzymatically ligating a mixture of synthetic oligonucleotides into gene sequences such that a degenerate set of potential NOVX sequences is expressible as individual polypeptides, or alternatively, as a set of larger fusion proteins (e.g., for phage display) containing the set of NOVX sequences therein. There are a variety of methods which can be used to produce libraries of potential NOVX variants from a degenerate oligonucleotide sequence. Chemical synthesis of a degenerate gene sequence can be performed in an automatic DNA synthesizer, and the synthetic gene then ligated into an appropriate expression vector. Use of a degenerate set of genes allows for the provision, in one mixture, of all of the sequences encoding the desired set of potential NOVX sequences. Methods for synthesizing degenerate oligonucleotides are well-known within the art. See, e.g., Narang, 1983. Tetrahedron 39: 3; Itakura, et al., 1984. Annu. Rev. Biochem. 53: 323; Itakura, et al., 1984. Science 198: 1056; Ike, et al., 1983. Nucl. Acids Res. 11: 477.
Polypeptide Libraries
In addition, libraries of fragments of the NOVX protein coding sequences can be used to generate a variegated population of NOVX fragments for screening and subsequent selection of variants of an NOVX protein. In one embodiment, a library of coding sequence fragments can be generated by treating a double stranded PCR fragment of an NOVX coding sequence with a nuclease under conditions wherein nicking occurs only about once per molecule, denaturing the double stranded DNA, renaturing the DNA to form double-stranded DNA that can include sense/antisense pairs from different nicked products, removing single stranded portions from reformed duplexes by treatment with S1 nuclease, and ligating the resulting fragment library into an expression vector. By this method, expression libraries can be derived which encodes N-terminal and internal fragments of various sizes of the NOVX proteins.
Various techniques are known in the art for screening gene products of combinatorial libraries made by point mutations or truncation, and for screening cDNA libraries for gene products having a selected property. Such techniques are adaptable for rapid screening of the gene libraries generated by the combinatorial mutagenesis of NOVX proteins. The most widely used techniques, which are amenable to high throughput analysis, for screening large gene libraries typically include cloning the gene library into replicable expression vectors, transforming appropriate cells with the resulting library of vectors, and expressing the combinatorial genes under conditions in which detection of a desired activity facilitates isolation of the vector encoding the gene whose product was detected. Recursive ensemble mutagenesis (REM), a new technique that enhances the frequency of functional mutants in the libraries, can be used in combination with the screening assays to identify NOVX variants. See, e.g., Arkin and Yourvan, 1992. Proc. Natl. Acad. Sci. USA 89: 7811-7815; Delgrave, et al., 1993. Protein Engineering 6:327-331.
Antibodies
The term “antibody” as used herein refers to immunoglobulin molecules and immunologically active portions of immunoglobulin (Ig) molecules, i.e., molecules that contain an antigen binding site that specifically binds (immunoreacts with) an antigen. Such antibodies include, but are not limited to, polyclonal, monoclonal, chimeric, single chain, Fab, Fab′ and F(ab′)2 fragments, and an Fab expression library. In general, antibody molecules obtained from humans relates to any of the classes IgG, IgM, IgA, IgE and IgD, which differ from one another by the nature of the heavy chain present in the molecule. Certain classes have subclasses as well, such as IgG1, IgG2, and others. Furthermore, in humans, the light chain may be a kappa chain or a lambda chain. Reference herein to antibodies includes a reference to all such classes, subclasses and types of human antibody species.
An isolated protein of the invention intended to serve as an antigen, or a portion or fragment thereof, can be used as an immunogen to generate antibodies that immunospecifically bind the antigen, using standard techniques for polyclonal and monoclonal antibody preparation. The full-length protein can be used or, alternatively, the invention provides antigenic peptide fragments of the antigen for use as immunogens. An antigenic peptide fragment comprises at least 6 amino acid residues of the amino acid sequence of the full length protein, such as an amino acid sequence shown in SEQ ID NOS:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92 and 94, and encompasses an epitope thereof such that an antibody raised against the peptide forms a specific immune complex with the full length protein or with any fragment that contains the epitope. Preferably, the antigenic peptide comprises at least 10 amino acid residues, or at least 15 amino acid residues, or at least 20 amino acid residues, or at least 30 amino acid residues. Preferred epitopes encompassed by the antigenic peptide are regions of the protein that are located on its surface; commonly these are hydrophilic regions.
In certain embodiments of the invention, at least one epitope encompassed by the antigenic peptide is a region of SECX that is located on the surface of the protein, e.g., a hydrophilic region. A hydrophobicity analysis of the human SECX protein sequence will indicate which regions of a SECX polypeptide are particularly hydrophilic and, therefore, are likely to encode surface residues useful for targeting antibody production. As a means for targeting antibody production, hydropathy plots showing regions of hydrophilicity and hydrophobicity may be generated by any method well known in the art, including, for example, the Kyte Doolittle or the Hopp Woods methods, either with or without Fourier transformation. See, e.g., Hopp and Woods, 1981, Proc. Nat. Acad. Sci. USA 78: 3824-3828; Kyte and Doolittle 1982, J. Mol. Biol. 157: 105-142, each incorporated herein by reference in their entirety. Antibodies that are specific for one or more domains within an antigenic protein, or derivatives, fragments, analogs or homologs thereof, are also provided herein.
A protein of the invention, or a derivative, fragment, analog, homolog or ortholog thereof, may be utilized as an immunogen in the generation of antibodies that immunospecifically bind these protein components.
Various procedures known within the art may be used for the production of polyclonal or monoclonal antibodies directed against a protein of the invention, or against derivatives, fragments, analogs homologs or orthologs thereof (see, for example, Antibodies: A Laboratory Manual, Harlow E, and Lane D, 1988, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., incorporated herein by reference). Some of these antibodies are discussed below.
Polyclonal Antibodies
For the production of polyclonal antibodies, various suitable host animals (e.g., rabbit, goat, mouse or other mammal) may be immunized by one or more injections with the native protein, a synthetic variant thereof, or a derivative of the foregoing. An appropriate immunogenic preparation can contain, for example, the naturally occurring immunogenic protein, a chemically synthesized polypeptide representing the immunogenic protein, or a recombinantly expressed immunogenic protein. Furthermore, the protein may be conjugated to a second protein known to be immunogenic in the mammal being immunized. Examples of such immunogenic proteins include but are not limited to keyhole limpet hemocyanin, serum albumin, bovine thyroglobulin, and soybean trypsin inhibitor. The preparation can further include an adjuvant. Various adjuvants used to increase the immunological response include, but are not limited to, Freund's (complete and incomplete), mineral gels (e.g., aluminum hydroxide), surface active substances (e.g., lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, dinitrophenol, etc.), adjuvants usable in humans such as Bacille Calmette-Guerin and Corynebacterium parvum, or similar immunostimulatory agents. Additional examples of adjuvants which can be employed include MPL-TDM adjuvant (monophosphoryl Lipid A, synthetic trehalose dicorynomycolate).
The polyclonal antibody molecules directed against the immunogenic protein can be isolated from the mammal (e.g., from the blood) and further purified by well known techniques, such as affinity chromatography using protein A or protein G, which provide primarily the IgG fraction of immune serum. Subsequently, or alternatively, the specific antigen which is the target of the immunoglobulin sought, or an epitope thereof, may be immobilized on a column to purify the immune specific antibody by immunoaffinity chromatography. Purification of immunoglobulins is discussed, for example, by D. Wilkinson (The Scientist, published by The Scientist, Inc., Philadelphia Pa., Vol. 14, No. 8 (Apr. 17, 2000), pp. 25-28).
Monoclonal Antibodies
The term “monoclonal antibody” (MAb) or “monoclonal antibody composition”, as used herein, refers to a population of antibody molecules that contain only one molecular species of antibody molecule consisting of a unique light chain gene product and a unique heavy chain gene product. In particular, the complementarity determining regions (CDRs) of the monoclonal antibody are identical in all the molecules of the population. MAbs thus contain an antigen binding site capable of immunoreacting with a particular epitope of the antigen characterized by a unique binding affinity for it.
Monoclonal antibodies can be prepared using hybridoma methods, such as those described by Kohler and Milstein, Nature, 256:495 (1975). In a hybridoma method, a mouse, hamster, or other appropriate host animal, is typically immunized with an immunizing agent to elicit lymphocytes that produce or are capable of producing antibodies that will specifically bind to the immunizing agent. Alternatively, the lymphocytes can be immunized in vitro.
The immunizing agent will typically include the protein antigen, a fragment thereof or a fusion protein thereof. Generally, either peripheral blood lymphocytes are used if cells of human origin are desired, or spleen cells or lymph node cells are used if non-human mammalian sources are desired. The lymphocytes are then fused with an immortalized cell line using a suitable fusing agent, such as polyethylene glycol, to form a hybridoma cell [Goding, Monoclonal Antibodies: Principles and Practice, Academic Press, (1986) pp. 59-103]. Immortalized cell lines are usually transformed mammalian cells, particularly myeloma cells of rodent, bovine and human origin. Usually, rat or mouse myeloma cell lines are employed. The hybridoma cells can be cultured in a suitable culture medium that preferably contains one or more substances that inhibit the growth or survival of the unfused, immortalized cells. For example, if the parental cells lack the enzyme hypoxanthine guanine phosphoribosyl transferase (HGPRT or HPRT), the culture medium for the hybridomas typically will include hypoxanthine, aminopterin, and thymidine (“HAT medium”), which substances prevent the growth of HGPRT-deficient cells.
Preferred immortalized cell lines are those that fuse efficiently, support stable high level expression of antibody by the selected antibody-producing cells, and are sensitive to a medium such as HAT medium. More preferred immortalized cell lines are murine myeloma lines, which can be obtained, for instance, from the Salk Institute Cell Distribution Center, San Diego, Calif. and the American Type Culture Collection, Manassas, Va. Human myeloma and mouse-human heteromyeloma cell lines also have been described for the production of human monoclonal antibodies [Kozbor, J. Immunol., 133:3001 (1984); Brodeur et al., Monoclonal Antibody Production Techniques and Applications, Marcel Dekker, Inc., New York, (1987) pp. 51-63].
The culture medium in which the hybridoma cells are cultured can then be assayed for the presence of monoclonal antibodies directed against the antigen. Preferably, the binding specificity of monoclonal antibodies produced by the hybridoma cells is determined by immunoprecipitation or by an in vitro binding assay, such as radioimmunoassay (RIA) or enzyme-linked immunoabsorbent assay (ELISA). Such techniques and assays are known in the art. The binding affinity of the monoclonal antibody can, for example, be determined by the Scatchard analysis of Munson and Pollard, Anal. Biochem., 107:220 (1980). It is an objective, especially important in therapeutic applications of monoclonal antibodies, to identify antibodies having a high degree of specificity and a high binding affinity for the target antigen.
After the desired hybridoma cells are identified, the clones can be subdloned by limiting dilution procedures and grown by standard methods (Goding, 1986). Suitable culture media for this purpose include, for example, Dulbecco's Modified Eagle's Medium and RPMI-1640 medium. Alternatively, the hybridoma cells can be grown in vivo as ascites in a mammal.
The monoclonal antibodies secreted by the subdlones can be isolated or purified from the culture medium or ascites fluid by conventional immunoglobulin purification procedures such as, for example, protein A-Sepharose, hydroxylapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography.
The monoclonal antibodies can also be made by recombinant DNA methods, such as those described in U.S. Pat. No. 4,816,567. DNA encoding the monoclonal antibodies of the invention can be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of murine antibodies). The hybridoma cells of the invention serve as a preferred source of such DNA. Once isolated, the DNA can be placed into expression vectors, which are then transfected into host cells such as simian COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells that do not otherwise produce immunoglobulin protein, to obtain the synthesis of monoclonal antibodies in the recombinant host cells. The DNA also can be modified, for example, by substituting the coding sequence for human heavy and light chain constant domains in place of the homologous murine sequences (U.S. Pat. No. 4,816,567; Morrison, Nature 368, 812-13 (1994)) or by covalently joining to the immunoglobulin coding sequence all or part of the coding sequence for a non-immunoglobulin polypeptide. Such a non-immunoglobulin polypeptide can be substituted for the constant domains of an antibody of the invention, or can be substituted for the variable domains of one antigen-combining site of an antibody of the invention to create a chimeric bivalent antibody.
Humanized Antibodies
The antibodies directed against the protein antigens of the invention can further comprise humanized antibodies or human antibodies. These antibodies are suitable for administration to humans without engendering an immune response by the human against the administered immunoglobulin. Humanized forms of antibodies are chimeric immunoglobulins, immunoglobulin chains or fragments thereof (such as Fv, Fab, Fab′, F(ab′)2 or other antigen-binding subsequences of antibodies) that are principally comprised of the sequence of a human immunoglobulin, and contain minimal sequence derived from a non-human immunoglobulin. Humanization can be performed following the method of Winter and co-workers (Jones et al., Nature, 321:522-525 (1986); Riechmann et al., Nature, 332:323-327 (1988); Verhoeyen et al., Science, 239:1534-1536 (1988)), by substituting rodent CDRs or CDR sequences for the corresponding sequences of a human antibody. (See also U.S. Pat. No. 5,225,539.) In some instances, Fv framework residues of the human immunoglobulin are replaced by corresponding non-human residues. Humanized antibodies can also comprise residues which are found neither in the recipient antibody nor in the imported CDR or framework sequences. In general, the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or substantially all of the framework regions are those of a human immunoglobulin consensus sequence. The humanized antibody optimally also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin (Jones et al., 1986; Riechmann et al., 1988; and Presta, Curr. Op. Struct. Biol., 2:593-596 (1992)).
Human Antibodies
Fully human antibodies essentially relate to antibody molecules in which the entire sequence of both the light chain and the heavy chain, including the CDRs, arise from human genes. Such antibodies are termed “human antibodies”, or “fully human antibodies” herein. Human monoclonal antibodies can be prepared by the trioma technique; the human B-cell hybridoma technique (see Kozbor, et al., 1983 Immunol Today 4: 72) and the EBV hybridoma technique to produce human monoclonal antibodies (see Cole, et al., 1985 In: MONOCLONAL ANTIBODIES AND CANCER THERAPY, Alan R. Liss, Inc., pp. 77-96). Human monoclonal antibodies may be utilized in the practice of the present invention and may be produced by using human hybridomas (see Cote, et al., 1983. Proc Natl Acad Sci USA 80: 2026-2030) or by transforming human B-cells with Epstein Barr Virus in vitro (see Cole, et al., 1985 In: MONOCLONAL ANTIBODIES AND CANCER THERAPY, Alan R. Liss, Inc., pp. 77-96).
In addition, human antibodies can also be produced using additional techniques, including phage display libraries (Hoogenboom and Winter, J. Mol. Biol., 227:381 (1991); Marks et al., J. Mol. Biol. 222:581 (1991)). Similarly, human antibodies can be made by introducing human immunoglobulin loci into transgenic animals, e.g., mice in which the endogenous immunoglobulin genes have been partially or completely inactivated. Upon challenge, human antibody production is observed, which closely resembles that seen in humans in all respects, including gene rearrangement, assembly, and antibody repertoire. This approach is described, for example, in U.S. Pat. Nos. 5,545,807; 5,545,806; 5,569,825; 5,625,126; 5,633,425; 5,661,016, and in Marks et al. (Bio/Technology 10, 779-783 (1992)); Lonberg et al. (Nature 368 856-859 (1994)); Morrison (Nature 368, 812-13 (1994)); Fishwild et al, (Nature Biotechnology 14, 845-51 (1996)); Neuberger (Nature Biotechnology 14, 826 (1996)); and Lonberg and Huszar (Intern. Rev. Immunol. 13 65-93 (1995)).
Human antibodies may additionally be produced using transgenic nonhuman animals which are modified so as to produce fully human antibodies rather than the animal's endogenous antibodies in response to challenge by an antigen. (See PCT publication WO94/02602). The endogenous genes encoding the heavy and light immunoglobulin chains in the nonhuman host have been incapacitated, and active loci encoding human heavy and light chain immunoglobulins are inserted into the host's genome. The human genes are incorporated, for example, using yeast artificial chromosomes containing the requisite human DNA segments. An animal which provides all the desired modifications is then obtained as progeny by crossbreeding intermediate transgenic animals containing fewer than the full complement of the modifications. The preferred embodiment of such a nonhuman animal is a mouse, and is termed the XenomouseTM as disclosed in PCT publications WO 96/33735 and WO 96/34096. This animal produces B cells which secrete fully human immunoglobulins. The antibodies can be obtained directly from the animal after immunization with an immunogen of interest, as, for example, a preparation of a polyclonal antibody, or alternatively from immortalized B cells derived from the animal, such as hybridomas producing monoclonal antibodies. Additionally, the genes encoding the immunoglobulins with human variable regions can be recovered and expressed to obtain the antibodies directly, or can be further modified to obtain analogs of antibodies such as, for example, single chain Fv molecules.
An example of a method of producing a nonhuman host, exemplified as a mouse, lacking expression of an endogenous immunoglobulin heavy chain is disclosed in U.S. Pat. No. 5,939,598. It can be obtained by a method including deleting the J segment genes from at least one endogenous heavy chain locus in an embryonic stem cell to prevent rearrangement of the locus and to prevent formation of a transcript of a rearranged immunoglobulin heavy chain locus, the deletion being effected by a targeting vector containing a gene encoding a selectable marker; and producing from the embryonic stem cell a transgenic mouse whose somatic and germ cells contain the gene encoding the selectable marker.
A method for producing an antibody of interest, such as a human antibody, is disclosed in U.S. Pat. No. 5,916,771. It includes introducing an expression vector that contains a nucleotide sequence encoding a heavy chain into one mammalian host cell in culture, introducing an expression vector containing a nucleotide sequence encoding a light chain into another mammalian host cell, and fusing the two cells to form a hybrid cell. The hybrid cell expresses an antibody containing the heavy chain and the light chain.
In a further improvement on this procedure, a method for identifying a clinically relevant epitope on an immunogen, and a correlative method for selecting an antibody that binds immunospecifically to the relevant epitope with high affinity, are disclosed in PCT publication WO 99/53049.
Fab Fragments and Single Chain Antibodies
According to the invention, techniques can be adapted for the production of single-chain antibodies specific to an antigenic protein of the invention (see e.g., U.S. Pat. No. 4,946,778). In addition, methods can be adapted for the construction of Fab expression libraries (see e.g., Huse, et al., 1989 Science 246: 1275-1281) to allow rapid and effective identification of monoclonal Fab fragments with the desired specificity for a protein or derivatives, fragments, analogs or homologs thereof. Antibody fragments that contain the idiotypes to a protein antigen may be produced by techniques known in the art including, but not limited to: (i) an F(ab′)2 fragment produced by pepsin digestion of an antibody molecule; (ii) an Fab fragment generated by reducing the disulfide bridges of an F(ab′)2 fragment; (iii) an Fab fragment generated by the treatment of the antibody molecule with papain and a reducing agent and (iv) Fv fragments.
Bispecific Antibodies
Bispecific antibodies are monoclonal, preferably human or humanized, antibodies that have binding specificities for at least two different antigens. In the present case, one of the binding specificities is for an antigenic protein of the invention. The second binding target is any other antigen, and advantageously is a cell-surface protein or receptor or receptor subunit.
Methods for making bispecific antibodies are known in the art. Traditionally, the recombinant production of bispecific antibodies is based on the co-expression of two immunoglobulin heavy-chain/light-chain pairs, where the two heavy chains have different specificities (Milstein and Cuello, Nature, 305:537-539 (1983)). Because of the random assortment of immunoglobulin heavy and light chains, these hybridomas (quadromas) produce a potential mixture of ten different antibody molecules, of which only one has the correct bispecific structure. The purification of the correct molecule is usually accomplished by affinity chromatography steps. Similar procedures are disclosed in WO 93/08829, published 13 May 1993, and in Traunecker et al., EMBO J., 10:3655-3659 (1991).
Antibody variable domains with the desired binding specificities (antibody-antigen combining sites) can be fused to immunoglobulin constant domain sequences. The fusion preferably is with an immunoglobulin heavy-chain constant domain, comprising at least part of the hinge, CH2, and CH3 regions. It is preferred to have the first heavy-chain constant region (CH1) containing the site necessary for light-chain binding present in at least one of the fusions. DNAs encoding the immunoglobulin heavy-chain fusions and, if desired, the immunoglobulin light chain, are inserted into separate expression vectors, and are co-transfected into a suitable host organism. For further details of generating bispecific antibodies see, for example, Suresh et al., Methods in Enzymology, 121:210 (1986).
According to another approach described in WO 96/27011, the interface between a pair of antibody molecules can be engineered to maximize the percentage of heterodimers which are recovered from recombinant cell culture. The preferred interface comprises at least a part of the CH3 region of an antibody constant domain. In this method, one or more small amino acid side chains from the interface of the first antibody molecule are replaced with larger side chains (e.g. tyrosine or tryptophan). Compensatory “cavities” of identical or similar size to the large side chain(s) are created on the interface of the second antibody molecule by replacing large amino acid side chains with smaller ones (e.g. alanine or threonine). This provides a mechanism for increasing the yield of the heterodimer over other unwanted end-products such as homodimers.
Bispecific antibodies can be prepared as full length antibodies or antibody fragments (e.g. F(ab′)2 bispecific antibodies). Techniques for generating bispecific antibodies from antibody fragments have been described in the literature. For example, bispecific antibodies can be prepared using chemical linkage. Brennan et al., Science 229:81 (1985) describe a procedure wherein intact antibodies are proteolytically cleaved to generate F(ab′)2 fragments. These fragments are reduced in the presence of the dithiol complexing agent sodium arsenite to stabilize vicinal dithiols and prevent intermolecular disulfide formation. The Fab′ fragments generated are then converted to thionitrobenzoate (TNB) derivatives. One of the Fab′-TNB derivatives is then reconverted to the Fab′-thiol by reduction with mercaptoethylamine and is mixed with an equimolar amount of the other Fab′-TNB derivative to form the bispecific antibody. The bispecific antibodies produced can be used as agents for the selective immobilization of enzymes.
Additionally, Fab′ fragments can be directly recovered from E. coli and chemically coupled to form bispecific antibodies. Shalaby et al., J. Exp. Med. 175:217-225 (1992) describe the production of a fully humanized bispecific antibody F(ab′)2 molecule. Each Fab′ fragment was separately secreted from E. coli and subjected to directed chemical coupling in vitro to form the bispecific antibody. The bispecific antibody thus formed was able to bind to cells overexpressing the ErbB2 receptor and normal human T cells, as well as trigger the lytic activity of human cytotoxic lymphocytes against human breast tumor targets.
Various techniques for making and isolating bispecific antibody fragments directly from recombinant cell culture have also been described. For example, bispecific antibodies have been produced using leucine zippers. Kostelny et al., J. Immunol. 148(5): 1547-1553 (1992). The leucine zipper peptides from the Fos and Jun proteins were linked to the Fab′ portions of two different antibodies by gene fusion. The antibody homodimers were reduced at the hinge region to form monomers and then re-oxidized to form the antibody heterodimers. This method can also be utilized for the production of antibody homodimers. The “diabody” technology described by Hollinger et al., Proc. Natl. Acad. Sci. USA 90:6444-6448 (1993) has provided an alternative mechanism for making bispecific antibody fragments. The fragments comprise a heavy-chain variable domain (VH) connected to a light-chain variable domain (VL) by a linker which is too short to allow pairing between the two domains on the same chain. Accordingly, the VH and VL domains of one fragment are forced to pair with the complementary VL and VH domains of another fragment, thereby forming two antigen-binding sites. Another strategy for making bispecific antibody fragments by the use of single-chain Fv (sFv) dimers has also been reported. See, Gruber et al., J. Immunol. 152:5368 (1994).
Antibodies with more than two valencies are contemplated. For example, trispecific antibodies can be prepared. Tutt et al., J. Immunol. 147:60 (1991).
Exemplary bispecific antibodies can bind to two different epitopes, at least one of which originates in the protein antigen of the invention. Alternatively, an anti-antigenic arm of an immunoglobulin molecule can be combined with an arm which binds to a triggering molecule on a leukocyte such as a T-cell receptor molecule (e.g. CD2, CD3, CD28, or B7), or Fc receptors for IgG (FcγR), such as FcγRI (CD64), FcγRII (CD32) and FcγRIII (CD16) so as to focus cellular defense mechanisms to the cell expressing the particular antigen. Bispecific antibodies can also be used to direct cytotoxic agents to cells which express a particular antigen. These antibodies possess an antigen-binding arm and an arm which binds a cytotoxic agent or a radionuclide chelator, such as EOTUBE, DPTA, DOTA, or TETA. Another bispecific antibody of interest binds the protein antigen described herein and further binds tissue factor (TF).
Heteroconjugate Antibodies
Heteroconjugate antibodies are also within the scope of the present invention. Heteroconjugate antibodies are composed of two covalently joined antibodies. Such antibodies have, for example, been proposed to target immune system cells to unwanted cells (U.S. Pat. No. 4,676,980), and for treatment of HIV infection (WO 91/00360; WO 92/200373; EP 03089). It is contemplated that the antibodies can be prepared in vitro using known methods in synthetic protein chemistry, including those involving crosslinking agents. For example, immunotoxins can be constructed using a disulfide exchange reaction or by forming a thioether bond. Examples of suitable reagents for this purpose include iminothiolate and methyl-4-mercaptobutyrimidate and those disclosed, for example, in U.S. Pat. No. 4,676,980.
Effector Function Engineering
It can be desirable to modify the antibody of the invention with respect to effector function, so as to enhance, e.g., the effectiveness of the antibody in treating cancer. For example, cysteine residue(s) can be introduced into the Fc region, thereby allowing interchain disulfide bond formation in this region. The homodimeric antibody thus generated can have improved internalization capability and/or increased complement-mediated cell killing and antibody-dependent cellular cytotoxicity (ADCC). See Caron et al., J. Exp Med., 176: 1191-1195 (1992) and Shopes, J. Immunol., 148: 2918-2922 (1992). Homodimeric antibodies with enhanced anti-tumor activity can also be prepared using heterobifunctional cross-linkers as described in Wolff et al. Cancer Research, 53: 2560-2565 (1993). Alternatively, an antibody can be engineered that has dual Fc regions and can thereby have enhanced complement lysis and ADCC capabilities. See Stevenson et al., Anti-Cancer Drug Design, 3: 219-230 (1989).
Immunoconjugates
The invention also pertains to immunoconjugates comprising an antibody conjugated to a cytotoxic agent such as a chemotherapeutic agent, toxin (e.g., an enzymatically active toxin of bacterial, fungal, plant, or animal origin, or fragments thereof), or a radioactive isotope (i.e., a radioconjugate).
Chemotherapeutic agents useful in the generation of such immunoconjugates have been described above. Enzymatically active toxins and fragments thereof that can be used include diphtheria A chain, nonbinding active fragments of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordii proteins, dianthin proteins, Phytolaca americana proteins (PAPI, PAPII, and PAP-S), momordica charantia inhibitor, curcin, crotin, sapaonaria officinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin, enomycin, and the tricothecenes. A variety of radionuclides are available for the production of radioconjugated antibodies. Examples include 212Bi, 131I, 131In, 90Y, and 186Re.
Conjugates of the antibody and cytotoxic agent are made using a variety of bifunctional protein-coupling agents such as N-succinimidyl-3-(2-pyridyldithiol) propionate (SPDP), iminothiolane (IT), bifunctional derivatives of imidoesters (such as dimethyl adipimidate HCL), active esters (such as disuccinimidyl suberate), aldehydes (such as glutareldehyde), bis-azido compounds (such as bis (p-azidobenzoyl) hexanediamine), bis-diazonium derivatives (such as bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such as tolyene 2,6-diisocyanate), and bis-active fluorine compounds (such as 1,5-difluoro-2,4-dinitrobenzene). For example, a ricin immunotoxin can be prepared as described in Vitetta et al., Science, 238: 1098 (1987). Carbon-14-labeled 1-isothiocyanatobenzyl-3-methyldiethylene triaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent for conjugation of radionucleotide to the antibody. See WO94/11026.
In another embodiment, the antibody can be conjugated to a “receptor” (such streptavidin) for utilization in tumor pretargeting wherein the antibody-receptor conjugate is administered to the patient, followed by removal of unbound conjugate from the circulation using a clearing agent and then administration of a “ligand” (e.g., avidin) that is in turn conjugated to a cytotoxic agent.
Immunoliposomes
The antibodies disclosed herein can also be formulated as immunoliposomes. Liposomes containing the antibody are prepared by methods known in the art, such as described in Epstein et al., Proc. Natl. Acad. Sci. USA, 82: 3688 (1985); Hwang et al., Proc. Natl Acad. Sci. USA, 77: 4030 (1980); and U.S. Pat. Nos. 4,485,045 and 4,544,545. Liposomes with enhanced circulation time are disclosed in U.S. Pat. No. 5,013,556.
Particularly useful liposomes can be generated by the reverse-phase evaporation method with a lipid composition comprising phosphatidylcholine, cholesterol, and PEG-derivatized phosphatidylethanolamine (PEG-PE). Liposomes are extruded through filters of defined pore size to yield liposomes with the desired diameter. Fab′ fragments of the antibody of the present invention can be conjugated to the liposomes as described in Martin et al., J. Biol. Chem., 257: 286-288 (1982) via a disulfide-interchange reaction. A chemotherapeutic agent (such as Doxorubicin) is optionally contained within the liposome. See Gabizon et al., J. National Cancer Inst., 81(19): 1484 (1989).
Diagnostic Applications of Antibodies Directed Against the Proteins of the Invention
Antibodies directed against a protein of the invention may be used in methods known within the art relating to the localization and/or quantitation of the protein (e.g., for use in measuring levels of the protein within appropriate physiological samples, for use in diagnostic methods, for use in imaging the protein, and the like). In a given embodiment, antibodies against the proteins, or derivatives, fragments, analogs or homologs thereof, that contain the antigen binding domain, are utilized as pharmacologically-active compounds (see below).
An antibody specific for a protein of the invention can be used to isolate the protein by standard techniques, such as immunoaffinity chromatography or immunoprecipitation. Such an antibody can facilitate the purification of the natural protein antigen from cells and of recombinantly produced antigen expressed in host cells. Moreover, such an antibody can be used to detect the antigenic protein (e.g., in a cellular lysate or cell supernatant) in order to evaluate the abundance and pattern of expression of the antigenic protein. Antibodies directed against the protein can be used diagnostically to monitor protein levels in tissue as part of a clinical testing procedure, e.g., to, for example, determine the efficacy of a given treatment regimen. Detection can be facilitated by coupling (i.e., physically linking) the antibody to a detectable substance. Examples of detectable substances include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, and radioactive materials. Examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, β-galactosidase, or acetylcholinesterase; examples of suitable prosthetic group complexes include streptavidin/biotin and avidin/biotin; examples of suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; an example of a luminescent material includes luminol; examples of bioluminescent materials include luciferase, luciferin, and aequorin, and examples of suitable radioactive material include 125I, 131I, 35S or 3H.
Antibody Therapeutics
Antibodies of the invention, including polyclonal, monoclonal, humanized and fully human antibodies, may used as therapeutic agents. Such agents will generally be employed to treat or prevent a disease or pathology in a subject. An antibody preparation, preferably one having high specificity and high affinity for its target antigen, is administered to the subject and will generally have an effect due to its binding with the target. Such an effect may be one of two kinds, depending on the specific nature of the interaction between the given antibody molecule and the target antigen in question. In the first instance, administration of the antibody may abrogate or inhibit the binding of the target with an endogenous ligand to which it naturally binds. In this case, the antibody binds to the target and masks a binding site of the naturally occurring ligand, wherein the ligand serves as an effector molecule. Thus the receptor mediates a signal transduction pathway for which ligand is responsible.
Alternatively, the effect may be one in which the antibody elicits a physiological result by virtue of binding to an effector binding site on the target molecule. In this case the target, a receptor having an endogenous ligand which may be absent or defective in the disease or pathology, binds the antibody as a surrogate effector ligand, initiating a receptor-based signal transduction event by the receptor.
A therapeutically effective amount of an antibody of the invention relates generally to the amount needed to achieve a therapeutic objective. As noted above, this may be a binding interaction between the antibody and its target antigen that, in certain cases, interferes with the functioning of the target, and in other cases, promotes a physiological response. The amount required to be administered will furthermore depend on the binding affinity of the antibody for its specific antigen, and will also depend on the rate at which an administered antibody is depleted from the free volume other subject to which it is administered. Common ranges for therapeutically effective dosing of an antibody or antibody fragment of the invention may be, by way of nonlimiting example, from about 0.1 mg/kg body weight to about 50 mg/kg body weight. Common dosing frequencies may range, for example, from twice daily to once a week.
Pharmaceutical Compositions of Antibodies
Antibodies specifically binding a protein of the invention, as well as other molecules identified by the screening assays disclosed herein, can be administered for the treatment of various disorders in the form of pharmaceutical compositions. Principles and considerations involved in preparing such compositions, as well as guidance in the choice of components are provided, for example, in Remington: The Science And Practice Of Pharmacy 19th ed. (Alfonso R. Gennaro, et al., editors) Mack Pub. Co., Easton, Pa.: 1995; Drug Absorption Enhancement: Concepts, Possibilities, Limitations, And Trends, Harwood Academic Publishers, Langhorne, Pa., 1994; and Peptide And Protein Drug Delivery (Advances In Parenteral Sciences, Vol. 4), 1991, M. Dekker, New York.
If the antigenic protein is intracellular and whole antibodies are used as inhibitors, internalizing antibodies are preferred. However, liposomes can also be used to deliver the antibody, or an antibody fragment, into cells. Where antibody fragments are used, the smallest inhibitory fragment that specifically binds to the binding domain of the target protein is preferred. For example, based upon the variable-region sequences of an antibody, peptide molecules can be designed that retain the ability to bind the target protein sequence. Such peptides can be synthesized chemically and/or produced by recombinant DNA technology. See, e.g., Marasco et al., Proc. Natl. Acad. Sci. USA, 90: 7889-7893 (1993). The formulation herein can also contain more than one active compound as necessary for the particular indication being treated, preferably those with complementary activities that do not adversely affect each other. Alternatively, or in addition, the composition can comprise an agent that enhances its function, such as, for example, a cytotoxic agent, cytokine, chemotherapeutic agent, or growth-inhibitory agent. Such molecules are suitably present in combination in amounts that are effective for the purpose intended.
The active ingredients can also be entrapped in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization, for example, hydroxymethylcellulose or gelatin-microcapsules and poly-(methylmethacrylate) microcapsules, respectively, in colloidal drug delivery systems (for example, liposomes, albumin microspheres, microemulsions, nano-particles, and nanocapsules) or in macroemulsions.
The formulations to be used for in vivo administration must be sterile. This is readily accomplished by filtration through sterile filtration membranes.
Sustained-release preparations can be prepared. Suitable examples of sustained-release preparations include semipermeable matrices of solid hydrophobic polymers containing the antibody, which matrices are in the form of shaped articles, e.g., films, or microcapsules. Examples of sustained-release matrices include polyesters, hydrogels (for example, poly(2-hydroxyethyl-methacrylate), or poly(vinylalcohol)), polylactides (U.S. Pat. No. 3,773,919), copolymers of L-glutamic acid and γ ethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymers such as the LUPRON DEPOTTM (injectable microspheres composed of lactic acid-glycolic acid copolymer and leuprolide acetate), and poly-D-(−)-3-hydroxybutyric acid. While polymers such as ethylene-vinyl acetate and lactic acid-glycolic acid enable release of molecules for over 100 days, certain hydrogels release proteins for shorter time periods.
ELISA Assay
An agent for detecting an analyte protein is an antibody capable of binding to an analyte protein, preferably an antibody with a detectable label. Antibodies can be polyclonal, or more preferably, monoclonal. An intact antibody, or a fragment thereof (e.g., Fab or F(ab)2) can be used. The term “labeled”, with regard to the probe or antibody, is intended to encompass direct labeling of the probe or antibody by coupling (i.e., physically linking) a detectable substance to the probe or antibody, as well as indirect labeling of the probe or antibody by reactivity with another reagent that is directly labeled. Examples of indirect labeling include detection of a primary antibody using a fluorescently-labeled secondary antibody and end-labeling of a DNA probe with biotin such that it can be detected with fluorescently-labeled streptavidin. The term “biological sample” is intended to include tissues, cells and biological fluids isolated from a subject, as well as tissues, cells and fluids present within a subject. Included within the usage of the term “biological sample”, therefore, is blood and a fraction or component of blood including blood serum, blood plasma, or lymph. That is, the detection method of the invention can be used to detect an analyte mRNA, protein, or genomic DNA in a biological sample in vitro as well as in vivo. For example, in vitro techniques for detection of an analyte mRNA include Northern hybridizations and in situ hybridizations. In vitro techniques for detection of an analyte protein include enzyme linked immunosorbent assays (ELISAs), Western blots, immunoprecipitations, and immunofluorescence. In vitro techniques for detection of an analyte genomic DNA include Southern hybridizations. Procedures for conducting immunoassays are described, for example in “ELISA: Theory and Practice: Methods in Molecular Biology”, Vol. 42, J. R. Crowther (Ed.) Human Press, Totowa, N.J., 1995; “Immunoassay”, E. Diamandis and T. Christopoulus, Academic Press, Inc., San Diego, Calif., 1996; and “Practice and Thory of Enzyme Immunoassays”, P. Tijssen, Elsevier Science Publishers, Amsterdam, 1985. Furthermore, in vivo techniques for detection of an analyte protein include introducing into a subject a labeled anti-an analyte protein antibody. For example, the antibody can be labeled with a radioactive marker whose presence and location in a subject can be detected by standard imaging techniques.
NOVX Recombinant Expression Vectors and Host Cells
Another aspect of the invention pertains to vectors, preferably expression vectors, containing a nucleic acid encoding an NOVX protein, or derivatives, fragments, analogs or homologs thereof. As used herein, the term “vector” refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. One type of vector is a “plasmid”, which refers to a circular double stranded DNA loop into which additional DNA segments can be ligated. Another type of vector is a viral vector, wherein additional DNA segments can be ligated into the viral genome. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g. bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) are integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome. Moreover, certain vectors are capable of directing the expression of genes to which they are operatively-linked. Such vectors are referred to herein as “expression vectors”. In general, expression vectors of utility in recombinant DNA techniques are often in the form of plasmids. In the present specification, “plasmid” and “vector” can be used interchangeably as the plasmid is the most commonly used form of vector. However, the invention is intended to include such other forms of expression vectors, such as viral vectors (e.g. replication defective retroviruses, adenoviruses and adeno-associated viruses), which serve equivalent functions.
The recombinant expression vectors of the invention comprise a nucleic acid of the invention in a form suitable for expression of the nucleic acid in a host cell, which means that the recombinant expression vectors include one or more regulatory sequences, selected on the basis of the host cells to be used for expression, that is operatively-linked to the nucleic acid sequence to be expressed. Within a recombinant expression vector, “operably-linked” is intended to mean that the nucleotide sequence of interest is linked to the regulatory sequence(s) in a manner that allows for expression of the nucleotide sequence (e.g., in an in vitro transcription/translation system or in a host cell when the vector is introduced into the host cell).
The term “regulatory sequence” is intended to includes promoters, enhancers and other expression control elements (e.g., polyadenylation signals). Such regulatory sequences are described, for example, in Goeddel, GENE EXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY 185, Academic Press, San Diego, Calif. (1990). Regulatory sequences include those that direct constitutive expression of a nucleotide sequence in many types of host cell and those that direct expression of the nucleotide sequence only in certain host cells (e.g., tissue-specific regulatory sequences). It will be appreciated by those skilled in the art that the design of the expression vector can depend on such factors as the choice of the host cell to be transformed, the level of expression of protein desired, etc. The expression vectors of the invention can be introduced into host cells to thereby produce proteins or peptides, including fusion proteins or peptides, encoded by nucleic acids as described herein (e.g. NOVX proteins, mutant forms of NOVX proteins, fusion proteins, etc.).
The recombinant expression vectors of the invention can be designed for expression of NOVX proteins in prokaryotic or eukaryotic cells. For example, NOVX proteins can be expressed in bacterial cells such as Escherichia coli, insect cells (using baculovirus expression vectors) yeast cells or mammalian cells. Suitable host cells are discussed further in Goeddel, GENE EXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY 185, Academic Press, San Diego, Calif. (1990). Alternatively, the recombinant expression vector can be transcribed and translated in vitro, for example using T7 promoter regulatory sequences and T7 polymerase.
Expression of proteins in prokaryotes is most often carried out in Escherichia coli with vectors containing constitutive or inducible promoters directing the expression of either fusion or non-fusion proteins. Fusion vectors add a number of amino acids to a protein encoded therein, usually to the amino terminus of the recombinant protein. Such fusion vectors typically serve three purposes: (i) to increase expression of recombinant protein; (ii) to increase the solubility of the recombinant protein; and (iii) to aid in the purification of the recombinant protein by acting as a ligand in affinity purification. Often, in fusion expression vectors, a proteolytic cleavage site is introduced at the junction of the fusion moiety and the recombinant protein to enable separation of the recombinant protein from the fusion moiety subsequent to purification of the fusion protein. Such enzymes, and their cognate recognition sequences, include Factor Xa, thrombin and enterokinase. Typical fusion expression vectors include pGEX (Pharmacia Biotech Inc; Smith and Johnson, 1988. Gene 67: 31-40), pMAL (New England Biolabs, Beverly, Mass.) and pRIT5 (Pharmacia, Piscataway, N.J.) that fuse glutathione S-transferase (GST), maltose E binding protein, or protein A, respectively, to the target recombinant protein.
Examples of suitable inducible non-fusion E. coli expression vectors include pTrc (Amrann et al., (1988) Gene 69:301-315) and pET 11d (Studier et al., GENE EXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY 185, Academic Press, San Diego, Calif. (1990) 60-89).
One strategy to maximize recombinant protein expression in E. coli is to express the protein in a host bacteria with an impaired capacity to proteolytically cleave the recombinant protein. See, e.g., Gottesman, GENE EXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY 185, Academic Press, San Diego, Calif. (1990) 119-128. Another strategy is to alter the nucleic acid sequence of the nucleic acid to be inserted into an expression vector so that the individual codons for each amino acid are those preferentially utilized in E. coli (see, e.g., Wada, et al., 1992. Nucl. Acids Res. 20: 2111-2118). Such alteration of nucleic acid sequences of the invention can be carried out by standard DNA synthesis techniques.
In another embodiment, the NOVX expression vector is a yeast expression vector. Examples of vectors for expression in yeast Saccharomyces cerivisae include pYepSec1 (Baldari, et al., 1987. EMBO J. 6: 229-234), pMFa (Kurjan and Herskowitz, 1982. Cell 30: 933-943), pJRY88 (Schultz et al., 1987. Gene 54: 113-123), pYES2 (Invitrogen Corporation, San Diego, Calif.), and picZ (InVitrogen Corp, San Diego, Calif.).
Alternatively, NOVX can be expressed in insect cells using baculovirus expression vectors. Baculovirus vectors available for expression of proteins in cultured insect cells (e.g., SF9 cells) include the pAc series (Smith, et al., 1983. Mol. Cell. Biol. 3: 2156-2165) and the pVL series (Lucklow and Summers, 1989. Virology 170: 31-39).
In yet another embodiment, a nucleic acid of the invention is expressed in mammalian cells using a mammalian expression vector. Examples of mammalian expression vectors include pCDM8 (Seed, 1987. Nature 329: 840) and pMT2PC (Kaufman, et al., 1987. EMBO J. 6: 187-195). When used in mammalian cells, the expression vector's control functions are often provided by viral regulatory elements. For example, commonly used promoters are derived from polyoma, adenovirus 2, cytomegalovirus, and simian virus 40. For other suitable expression systems for both prokaryotic and eukaryotic cells see, e.g., Chapters 16 and 17 of Sambrook, et al., MOLECULAR CLONING: A LABORATORY MANUAL. 2nd ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989.
In another embodiment, the recombinant mammalian expression vector is capable of directing expression of the nucleic acid preferentially in a particular cell type (e.g., tissue-specific regulatory elements are used to express the nucleic acid). Tissue-specific regulatory elements are known in the art. Non-limiting examples of suitable tissue-specific promoters include the albumin promoter (liver-specific; Pinkert, et al., 1987. Genes Dev. 1: 268-277), lymphoid-specific promoters (Calame and Eaton, 1988. Adv. Immunol. 43: 235-275), in particular promoters of T cell receptors (Winoto and Baltimore, 1989. EMBO J. 8: 729-733) and immunoglobulins (Banerji, et al., 1983. Cell 33: 729-740; Queen and Baltimore, 1983. Cell 33: 741-748), neuron-specific promoters (e.g., the neurofilament promoter; Byrne and Ruddle, 1989. Proc. Natl. Acad. Sci. USA 86: 5473-5477), pancreas-specific promoters (Edlund, et al., 1985. Science 230: 912-916), and mammary gland-specific promoters (e.g., milk whey promoter; U.S. Pat. No. 4,873,316 and European Application Publication No. 264,166). Developmentally-regulated promoters are also encompassed, e.g., the murine hox promoters (Kessel and Gruss, 1990. Science 249: 374-379) and the α-fetoprotein promoter (Campes and Tilghman, 1989. Genes Dev. 3: 537-546).
The invention further provides a recombinant expression vector comprising a DNA molecule of the invention cloned into the expression vector in an antisense orientation. That is, the DNA molecule is operatively-linked to a regulatory sequence in a manner that allows for expression (by transcription of the DNA molecule) of an RNA molecule that is antisense to NOVX mRNA. Regulatory sequences operatively linked to a nucleic acid cloned in the antisense orientation can be chosen that direct the continuous expression of the antisense RNA molecule in a variety of cell types, for instance viral promoters and/or enhancers, or regulatory sequences can be chosen that direct constitutive, tissue specific or cell type specific expression of antisense RNA. The antisense expression vector can be in the form of a recombinant plasmid, phagemid or attenuated virus in which antisense nucleic acids are produced under the control of a high efficiency regulatory region, the activity of which can be determined by the cell type into which the vector is introduced. For a discussion of the regulation of gene expression using antisense genes see, e.g., Weintraub, et al., “Antisense RNA as a molecular tool for genetic analysis,” Reviews-Trends in Genetics, Vol. 1(1) 1986.
Another aspect of the invention pertains to host cells into which a recombinant expression vector of the invention has been introduced. The terms “host cell” and “recombinant host cell” are used interchangeably herein. It is understood that such terms refer not only to the particular subject cell but also to the progeny or potential progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term as used herein.
A host cell can be any prokaryotic or eukaryotic cell. For example, NOVX protein can be expressed in bacterial cells such as E. coli, insect cells, yeast or mammalian cells (such as Chinese hamster ovary cells (CHO) or COS cells). Other suitable host cells are known to those skilled in the art.
Vector DNA can be introduced into prokaryotic or eukaryotic cells via conventional transformation or transfection techniques. As used herein, the terms “transformation” and “transfection” are intended to refer to a variety of art-recognized techniques for introducing foreign nucleic acid (e.g., DNA) into a host cell, including calcium phosphate or calcium chloride co-precipitation, DEAE-dextran-mediated transfection, lipofection, or electroporation. Suitable methods for transforming or transfecting host cells can be found in Sambrook, et al. (MOLECULAR CLONING: A LABORATORY MANUAL. 2nd ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989), and other laboratory manuals.
For stable transfection of mammalian cells, it is known that, depending upon the expression vector and transfection technique used, only a small fraction of cells may integrate the foreign DNA into their genome. In order to identify and select these integrants, a gene that encodes a selectable marker (e.g., resistance to antibiotics) is generally introduced into the host cells along with the gene of interest. Various selectable markers include those that confer resistance to drugs, such as G418, hygromycin and methotrexate. Nucleic acid encoding a selectable marker can be introduced into a host cell on the same vector as that encoding NOVX or can be introduced on a separate vector. Cells stably transfected with the introduced nucleic acid can be identified by drug selection (e.g., cells that have incorporated the selectable marker gene will survive, while the other cells die).
A host cell of the invention, such as a prokaryotic or eukaryotic host cell in culture, can be used to produce (i.e., express) NOVX protein. Accordingly, the invention further provides methods for producing NOVX protein using the host cells of the invention. In one embodiment, the method comprises culturing the host cell of invention (into which a recombinant expression vector encoding NOVX protein has been introduced) in a suitable medium such that NOVX protein is produced. In another embodiment, the method further comprises isolating NOVX protein from the medium or the host cell.
Transgenic NOVX Animals
The host cells of the invention can also be used to produce non-human transgenic animals. For example, in one embodiment, a host cell of the invention is a fertilized oocyte or an embryonic stem cell into which NOVX protein-coding sequences have been introduced. Such host cells can then be used to create non-human transgenic animals in which exogenous NOVX sequences have been introduced into their genome or homologous recombinant animals in which endogenous NOVX sequences have been altered. Such animals are useful for studying the function and/or activity of NOVX protein and for identifying and/or evaluating modulators of NOVX protein activity. As used herein, a “transgenic animal” is a non-human animal, preferably a mammal, more preferably a rodent such as a rat or mouse, in which one or more of the cells of the animal includes a transgene. Other examples of transgenic animals include non-human primates, sheep, dogs, cows, goats, chickens, amphibians, etc. A transgene is exogenous DNA that is integrated into the genome of a cell from which a transgenic animal develops and that remains in the genome of the mature animal, thereby directing the expression of an encoded gene product in one or more cell types or tissues of the transgenic animal. As used herein, a “homologous recombinant animal” is a non-human animal, preferably a mammal, more preferably a mouse, in which an endogenous NOVX gene has been altered by homologous recombination between the endogenous gene and an exogenous DNA molecule introduced into a cell of the animal, e.g., an embryonic cell of the animal, prior to development of the animal.
A transgenic animal of the invention can be created by introducing NOVX-encoding nucleic acid into the male pronuclei of a fertilized oocyte (e.g., by microinjection, retroviral infection) and allowing the oocyte to develop in a pseudopregnant female foster animal. The human NOVX cDNA sequences SEQ ID NOS:1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91 and 93 can be introduced as a transgene into the genome of a non-human animal. Alternatively, a non-human homologue of the human NOVX gene, such as a mouse NOVX gene, can be isolated based on hybridization to the human NOVX cDNA (described further supra) and used as a transgene. Intronic sequences and polyadenylation signals can also be included in the transgene to increase the efficiency of expression of the transgene. A tissue-specific regulatory sequence(s) can be operably-linked to the NOVX transgene to direct expression of NOVX protein to particular cells. Methods for generating transgenic animals via embryo manipulation and microinjection, particularly animals such as mice, have become conventional in the art and are described, for example, in U.S. Pat. Nos. 4,736,866; 4,870,009; and 4,873,191; and Hogan, 1986. In: MANIPULATING THE MOUSE EMBRYO, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. Similar methods are used for production of other transgenic animals. A transgenic founder animal can be identified based upon the presence of the NOVX transgene in its genome and/or expression of NOVX mRNA in tissues or cells of the animals. A transgenic founder animal can then be used to breed additional animals carrying the transgene. Moreover, transgenic animals carrying a transgene-encoding NOVX protein can further be bred to other transgenic animals carrying other transgenes.
To create a homologous recombinant animal, a vector is prepared which contains at least a portion of an NOVX gene into which a deletion, addition or substitution has been introduced to thereby alter, e.g., functionally disrupt, the NOVX gene. The NOVX gene can be a human gene (e.g., the cDNA of SEQ ID NOS:1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91 and 93), but more preferably, is a non-human homologue of a human NOVX gene. For example, a mouse homologue of human NOVX gene of SEQ ID NOS:1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91 and 93 can be used to construct a homologous recombination vector suitable for altering an endogenous NOVX gene in the mouse genome. In one embodiment, the vector is designed such that, upon homologous recombination, the endogenous NOVX gene is functionally disrupted (i.e., no longer encodes a functional protein; also referred to as a “knock out” vector).
Alternatively, the vector can be designed such that, upon homologous recombination, the endogenous NOVX gene is mutated or otherwise altered but still encodes functional protein (e.g., the upstream regulatory region can be altered to thereby alter the expression of the endogenous NOVX protein). In the homologous recombination vector, the altered portion of the NOVX gene is flanked at its 5′- and 3′-termini by additional nucleic acid of the NOVX gene to allow for homologous recombination to occur between the exogenous NOVX gene carried by the vector and an endogenous NOVX gene in an embryonic stem cell. The additional flanking NOVX nucleic acid is of sufficient length for successful homologous recombination with the endogenous gene. Typically, several kilobases of flanking DNA (both at the 5′- and 3′-termini) are included in the vector. See, e.g., Thomas, et al., 1987. Cell 51: 503 for a description of homologous recombination vectors. The vector is ten introduced into an embryonic stem cell line (e.g., by electroporation) and cells in which the introduced NOVX gene has homologously-recombined with the endogenous NOVX gene are selected. See, e.g., Li, et al., 1992. Cell 69: 915.
The selected cells are then injected into a blastocyst of an animal (e.g., a mouse) to form aggregation chimeras. See, e.g., Bradley, 1987. In: TERATOCARCINOMAS AND EMBRYONIC STEM CELLS: A PRACTICAL APPROACH, Robertson, ed. IRL, Oxford, pp. 113-152. A chimeric embryo can then be implanted into a suitable pseudopregnant female foster animal and the embryo brought to term. Progeny harboring the homologously-recombined DNA in their germ cells can be used to breed animals in which all cells of the animal contain the homologously-recombined DNA by germline transmission of the transgene. Methods for constructing homologous recombination vectors and homologous recombinant animals are described further in Bradley, 1991. Curr. Opin. Biotechnol. 2: 823-829; PCT International Publication Nos.: WO 90/11354; WO 91/01140; WO 92/0968; and WO 93/04169.
In another embodiment, transgenic non-humans animals can be produced that contain selected systems that allow for regulated expression of the transgene. One example of such a system is the cre/loxP recombinase system of bacteriophage P1. For a description of the cre/loxP recombinase system, See, e.g., Lakso, et al., 1992. Proc. Natl. Acad. Sci. USA 89: 6232-6236. Another example of a recombinase system is the FLP recombinase system of Saccharomyces cerevisiae. See, O'Gorman, et al., 1991. Science 251:1351-1355. If a cre/loxP recombinase system is used to regulate expression of the transgene, animals containing transgenes encoding both the Cre recombinase and a selected protein are required. Such animals can be provided through the construction of “double” transgenic animals, e.g., by mating two transgenic animals, one containing a transgene encoding a selected protein and the other containing a transgene encoding a recombinase.
Clones of the non-human transgenic animals described herein can also be produced according to the methods described in Wilmut, et al., 1997. Nature 385: 810-813. In brief, a cell (e.g., a somatic cell) from the transgenic animal can be isolated and induced to exit the growth cycle and enter G0 phase. The quiescent cell can then be fused, e.g., through the use of electrical pulses, to an enucleated oocyte from an animal of the same species from which the quiescent cell is isolated. The reconstructed oocyte is then cultured such that it develops to morula or blastocyte and then transferred to pseudopregnant female foster animal. The offspring borne of this female foster animal will be a clone of the animal from which the cell (e.g., the somatic cell) is isolated.
Pharmaceutical Compositions
The NOVX nucleic acid molecules, NOVX proteins, and anti-NOVX antibodies (also referred to herein as “active compounds”) of the invention, and derivatives, fragments, analogs and homologs thereof, can be incorporated into pharmaceutical compositions suitable for administration. Such compositions typically comprise the nucleic acid molecule, protein, or antibody and a pharmaceutically acceptable carrier. As used herein, “pharmaceutically acceptable carrier” is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration. Suitable carriers are described in the most recent edition of Remington's Pharmaceutical Sciences, a standard reference text in the field, which is incorporated herein by reference. Preferred examples of such carriers or diluents include, but are not limited to, water, saline, finger's solutions, dextrose solution, and 5% human serum albumin. Liposomes and non-aqueous vehicles such as fixed oils may also be used. The use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active compound, use thereof in the compositions is contemplated. Supplementary active compounds can also be incorporated into the compositions.
A pharmaceutical composition of the invention is formulated to be compatible with its intended route of administration. Examples of routes of administration include parenteral, e.g., intravenous, intradermal, subcutaneous, oral (e.g., inhalation), transdermal (i.e., topical), transmucosal, and rectal administration. Solutions or suspensions used for parenteral, intradermal, or subcutaneous application can include the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid (EDTA); buffers such as acetates, citrates or phosphates, and agents for the adjustment of tonicity such as sodium chloride or dextrose. The pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide. The parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.
Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor ELTM (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). In all cases, the composition must be sterile and should be fluid to the extent that easy syringeability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as manitol, sorbitol, sodium chloride in the composition. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate and gelatin.
Sterile injectable solutions can be prepared by incorporating the active compound (e.g., an NOVX protein or anti-NOVX antibody) in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle that contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, methods of preparation are vacuum drying and freeze-drying that yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.
Oral compositions generally include an inert diluent or an edible carrier. They can be enclosed in gelatin capsules or compressed into tablets. For the purpose of oral therapeutic administration, the active compound can be incorporated with excipients and used in the form of tablets, troches, or capsules. Oral compositions can also be prepared using a fluid carrier for use as a mouthwash, wherein the compound in the fluid carrier is applied orally and swished and expectorated or swallowed. Pharmaceutically compatible binding agents, and/or adjuvant materials can be included as part of the composition. The tablets, pills, capsules, troches and the like can contain any of the following ingredients, or compounds of a similar nature: a binder such as microcrystalline cellulose, gum tragacanth or gelatin; an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel, or corn starch; a lubricant such as magnesium stearate or Sterotes; a glidant such as colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; or a flavoring agent such as peppermint, methyl salicylate, or orange flavoring.
For administration by inhalation, the compounds are delivered in the form of an aerosol spray from pressured container or dispenser which contains a suitable propellant, e.g., a gas such as carbon dioxide, or a nebulizer.
Systemic administration can also be by transmucosal or transdermal means. For transmucosal or transdermal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art, and include, for example, for transmucosal administration, detergents, bile salts, and fusidic acid derivatives. Transmucosal administration can be accomplished through the use of nasal sprays or suppositories. For transdermal administration, the active compounds are formulated into ointments, salves, gels, or creams as generally known in the art.
The compounds can also be prepared in the form of suppositories (e.g., with conventional suppository bases such as cocoa butter and other glycerides) or retention enemas for rectal delivery.
In one embodiment, the active compounds are prepared with carriers that will protect the compound against rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Methods for preparation of such formulations will be apparent to those skilled in the art. The materials can also be obtained commercially from Alza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions (including liposomes targeted to infected cells with monoclonal antibodies to viral antigens) can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art, for example, as described in U.S. Pat. No. 4,522,811.
It is especially advantageous to formulate oral or parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the subject to be treated; each unit containing a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The specification for the dosage unit forms of the invention are dictated by and directly dependent on the unique characteristics of the active compound and the particular therapeutic effect to be achieved, and the limitations inherent in the art of compounding such an active compound for the treatment of individuals.
The nucleic acid molecules of the invention can be inserted into vectors and used as gene therapy vectors. Gene therapy vectors can be delivered to a subject by, for example, intravenous injection, local administration (see, e.g., U.S. Pat. No. 5,328,470) or by stereotactic injection (see, e.g., Chen, et al., 1994. Proc. Natl. Acad. Sci. USA 91: 3054-3057). The pharmaceutical preparation of the gene therapy vector can include the gene therapy vector in an acceptable diluent, or can comprise a slow release matrix in which the gene delivery vehicle is imbedded. Alternatively, where the complete gene delivery vector can be produced intact from recombinant cells, e.g., retroviral vectors, the pharmaceutical preparation can include one or more cells that produce the gene delivery system.
The pharmaceutical compositions can be included in a container, pack, or dispenser together with instructions for administration.
Screening and Detection Methods
The isolated nucleic acid molecules of the invention can be used to express NOVX protein (e.g., via a recombinant expression vector in a host cell in gene therapy applications), to detect NOVX mRNA (e.g., in a biological sample) or a genetic lesion in an NOVX gene, and to modulate NOVX activity, as described further, below. In addition, the NOVX proteins can be used to screen drugs or compounds that modulate the NOVX protein activity or expression as well as to treat disorders characterized by insufficient or excessive production of NOVX protein or production of NOVX protein forms that have decreased or aberrant activity compared to NOVX wild-type protein (e.g.; diabetes (regulates insulin release); obesity (binds and transport lipids); metabolic disturbances associated with obesity, the metabolic syndrome X as well as anorexia and wasting disorders associated with chronic diseases and various cancers, and infectious disease(possesses anti-microbial activity) and the various dyslipidemias. In addition, the anti-NOVX antibodies of the invention can be used to detect and isolate NOVX proteins and modulate NOVX activity. In yet a further aspect, the invention can be used in methods to influence appetite, absorption of nutrients and the disposition of metabolic substrates in both a positive and negative fashion.
The invention further pertains to novel agents identified by the screening assays described herein and uses thereof for treatments as described, supra.
Screening Assays
The invention provides a method (also referred to herein as a “screening assay”) for identifying modulators, i.e., candidate or test compounds or agents (e.g., peptides, peptidomimetics, small molecules or other drugs) that bind to NOVX proteins or have a stimulatory or inhibitory effect on, e.g., NOVX protein expression or NOVX protein activity. The invention also includes compounds identified in the screening assays described herein.
In one embodiment, the invention provides assays for screening candidate or test compounds which bind to or modulate the activity of the membrane-bound form of an NOVX protein or polypeptide or biologically-active portion thereof. The test compounds of the invention can be obtained using any of the numerous approaches in combinatorial library methods known in the art, including: biological libraries; spatially addressable parallel solid phase or solution phase libraries; synthetic library methods requiring deconvolution; the “one-bead one-compound” library method; and synthetic library methods using affinity chromatography selection. The biological library approach is limited to peptide libraries, while the other four approaches are applicable to peptide, non-peptide oligomer or small molecule libraries of compounds. See, e.g., Lam, 1997. Anticancer Drug Design 12: 145.
A “small molecule” as used herein, is meant to refer to a composition that has a molecular weight of less than about 5 kD and most preferably less than about 4 kD. Small molecules can be, e.g., nucleic acids, peptides, polypeptides, peptidomimetics, carbohydrates, lipids or other organic or inorganic molecules. Libraries of chemical and/or biological mixtures, such as fungal, bacterial, or algal extracts, are known in the art and can be screened with any of the assays of the invention.
Examples of methods for the synthesis of molecular libraries can be found in the art, for example in: DeWitt, et al., 1993. Proc. Natl. Acad. Sci. U.S.A. 90: 6909; Erb, et al., 1994. Proc. Natl. Acad. Sci. U.S.A. 91: 11422; Zuckermann, et al., 1994. J. Med. Chem. 37: 2678; Cho, et al., 1993. Science 261: 1303; Carrell, et al., 1994. Angew. Chem. Int. Ed. Engl. 33: 2059; Carell, et al., 1994. Angew. Chem. Int. Ed. Engl. 33: 2061; and Gallop, et al., 1994. J. Med. Chem. 37:1233.
Libraries of compounds may be presented in solution (e.g., Houghten, 1992. Biotechniques 13: 412-421), or on beads (Lam, 1991. Nature 354: 82-84), on chips (Fodor, 1993. Nature 364: 555-556), bacteria (Ladner, U.S. Pat. No. 5,223,409), spores (Ladner, U.S. Pat. No. 5,233,409), plasmids (Cull, et al., 1992. Proc. Natl. Acad. Sci. USA 89: 1865-1869) or on phage (Scott and Smith, 1990. Science 249: 386-390; Devlin, 1990. Science 249: 404-406; Cwirla, et al., 1990. Proc. Natl. Acad. Sci. U.S.A. 87: 6378-6382; Felici, 1991. J. Mol. Biol. 222: 301-310; Ladner, U.S. Pat. No. 5,233,409.).
In one embodiment, an assay is a cell-based assay in which a cell which expresses a membrane-bound form of NOVX protein, or a biologically-active portion thereof, on the cell surface is contacted with a test compound and the ability of the test compound to bind to an NOVX protein determined. The cell, for example, can of mammalian origin or a yeast cell. Determining the ability of the test compound to bind to the NOVX protein can be accomplished, for example, by coupling the test compound with a radioisotope or enzymatic label such that binding of the test compound to the NOVX protein or biologically-active portion thereof can be determined by detecting the labeled compound in a complex. For example, test compounds can be labeled with 125I, 35S, 14C, or 3H, either directly or indirectly, and the radioisotope detected by direct counting of radioemission or by scintillation counting. Alternatively, test compounds can be enzymatically-labeled with, for example, horseradish peroxidase, alkaline phosphatase, or luciferase, and the enzymatic label detected by determination of conversion of an appropriate substrate to product. In one embodiment, the assay comprises contacting a cell which expresses a membrane-bound form of NOVX protein, or a biologically-active portion thereof, on the cell surface with a known compound which binds NOVX to form an assay mixture, contacting the assay mixture with a test compound, and determining the ability of the test compound to interact with an NOVX protein, wherein determining the ability of the test compound to interact with an NOVX protein comprises determining the ability of the test compound to preferentially bind t o NOVX protein or a biologically-active portion thereof as compared to the known compound.
In another embodiment, an assay is a cell-based assay comprising contacting a cell expressing a membrane-bound form of NOVX protein, or a biologically-active portion thereof, on the cell surface with a test compound and determining the ability of the test compound to modulate (e.g., stimulate or inhibit) the activity of the NOVX protein or biologically-active portion thereof. Determining the ability of the test compound to modulate the activity of NOVX or a biologically-active portion thereof can be accomplished, for example, by determining the ability of the NOVX protein to bind to or interact with an NOVX target molecule. As used herein, a “target molecule” is a molecule with which an NOVX protein binds or interacts in nature, for example, a molecule on the surface of a cell which expresses an NOVX interacting protein, a molecule on the surface of a second cell, a molecule in the extracellular milieu, a molecule associated with the internal surface of a cell membrane or a cytoplasmic molecule. An NOVX target molecule can be a non-NOVX molecule or an NOVX protein or polypeptide of the invention. In one embodiment, an NOVX target molecule is a component of a signal transduction pathway that facilitates transduction of an extracellular signal (e.g. a signal generated by binding of a compound to a membrane-bound NOVX molecule) through the cell membrane and into the cell. The target, for example, can be a second intercellular protein that has catalytic activity or a protein that facilitates the association of downstream signaling molecules with NOVX.
Determining the ability of the NOVX protein to bind to or interact with an NOVX target molecule can be accomplished by one of the methods described above for determining direct binding. In one embodiment, determining the ability of the NOVX protein to bind to or interact with an NOVX target molecule can be accomplished by determining the activity of the target molecule. For example, the activity of the target molecule can be determined by detecting induction of a cellular second messenger of the target (i.e. intracellular Ca2+, diacylglycerol, IP3, etc.), detecting catalytic/enzymatic activity of the target an appropriate substrate, detecting the induction of a reporter gene (comprising an NOVX-responsive regulatory element operatively linked to a nucleic acid encoding a detectable marker, e.g., luciferase), or detecting a cellular response, for example, cell survival, cellular differentiation, or cell proliferation.
In yet another embodiment, an assay of the invention is a cell-free assay comprising contacting an NOVX protein or biologically-active portion thereof with a test compound and determining the ability of the test compound to bind to the NOVX protein or biologically-active portion thereof. Binding of the test compound to the NOVX protein can be determined either directly or indirectly as described above. In one such embodiment, the assay comprises contacting the NOVX protein or biologically-active portion thereof with a known compound which binds NOVX to form an assay mixture, contacting the assay mixture with a test compound, and determining the ability of the test compound to interact with an NOVX protein, wherein determining the ability of the test compound to interact with an NOVX protein comprises determining the ability of the test compound to preferentially bind to NOVX or biologically-active portion thereof as compared to the known compound.
In still another embodiment, an assay is a cell-free assay comprising contacting NOVX protein or biologically-active portion thereof with a test compound and determining the ability of the test compound to modulate (e.g. stimulate or inhibit) the activity of the NOVX protein or biologically-active portion thereof. Determining the ability of the test compound to modulate the activity of NOVX can be accomplished, for example, by determining the ability of the NOVX protein to bind to an NOVX target molecule by one of the methods described above for determining direct binding. In an alternative embodiment, determining the ability of the test compound to modulate the activity of NOVX protein can be accomplished by determining the ability of the NOVX protein further modulate an NOVX target molecule. For example, the catalytic/enzymatic activity of the target molecule on an appropriate substrate can be determined as described, supra.
In yet another embodiment, the cell-free assay comprises contacting the NOVX protein or biologically-active portion thereof with a known compound which binds NOVX protein to form an assay mixture, contacting the assay mixture with a test compound, and determining the ability of the test compound to interact with an NOVX protein, wherein determining the ability of the test compound to interact with an NOVX protein comprises determining the ability of the NOVX protein to preferentially bind to or modulate the activity of an NOVX target molecule.
The cell-free assays of the invention are amenable to use of both the soluble form or the membrane-bound form of NOVX protein. In the case of cell-free assays comprising the membrane-bound form of NOVX protein, it may be desirable to utilize a solubilizing agent such that the membrane-bound form of NOVX protein is maintained in solution. Examples of such solubilizing agents include non-ionic detergents such as n-octylglucoside, n-dodecylglucoside, n-dodecylmaltoside, octanoyl-N-methylglucamide, decanoyl-N-methylglucamide, Triton® X-100, Triton® X-114, Thesit®, Isotridecypoly(ethylene glycol ether)n, N-dodecyl-N,N-dimethyl-3-ammonio-1-propane sulfonate, 3-(3-cholamidopropyl) dimethylamminiol-1-propane sulfonate (CHAPS), or 3-(3-cholamidopropyl)dimethylamminiol-2-hydroxy-1-propane sulfonate (CHAPSO).
In more than one embodiment of the above assay methods of the invention, it may be desirable to immobilize either NOVX protein or its target molecule to facilitate separation of complexed from uncomplexed forms of one or both of the proteins, as well as to accommodate automation of the assay. Binding of a test compound to NOVX protein, or interaction of NOVX protein with a target molecule in the presence and absence of a candidate compound, can be accomplished in any vessel suitable for containing the reactants. Examples of such vessels include microtiter plates, test tubes, and micro-centrifuge tubes. In one embodiment, a fusion protein can be provided that adds a domain that allows one or both of the proteins to be bound to a matrix. For example, GST-NOVX fusion proteins or GST-target fusion proteins can be adsorbed onto glutathione sepharose beads (Sigma Chemical, St. Louis, Mo.) or glutathione derivatized microtiter plates, that are then combined with the test compound or the test compound and either the non-adsorbed target protein or NOVX protein, and the mixture is incubated under conditions conducive to complex formation (e.g., at physiological conditions for salt and pH). Following incubation, the beads or microtiter plate wells are washed to remove any unbound components, the matrix immobilized in the case of beads, complex determined either directly or indirectly, for example, as described, supra. Alternatively, the complexes can be dissociated from the matrix, and the level of NOVX protein binding or activity determined using standard techniques.
Other techniques for immobilizing proteins on matrices can also be used in the screening assays of the invention. For example, either the NOVX protein or its target molecule can be immobilized utilizing conjugation of biotin and streptavidin. Biotinylated NOVX protein or target molecules can be prepared from biotin-NHS (N-hydroxy-succinimide) using techniques well-known within the art (e.g., biotinylation kit, Pierce Chemicals, Rockford, Ill.), and immobilized in the wells of streptavidin-coated 96 well plates (Pierce Chemical). Alternatively, antibodies reactive with NOVX protein or target molecules, but which do not interfere with binding of the NOVX protein to its target molecule, can be derivatized to the wells of the plate, and unbound target or NOVX protein trapped in the wells by antibody conjugation. Methods for detecting such complexes, in addition to those described above for the GST-immobilized complexes, include immunodetection of complexes using antibodies reactive with the NOVX protein or target molecule, as well as enzyme-linked assays that rely on detecting an enzymatic activity associated with the NOVX protein or target molecule.
In another embodiment, modulators of NOVX protein expression are identified in a method wherein a cell is contacted with a candidate compound and the expression of NOVX mRNA or protein in the cell is determined. The level of expression of NOVX mRNA or protein in the presence of the candidate compound is compared to the level of expression of NOVX mRNA or protein in the absence of the candidate compound. The candidate compound can then be identified as a modulator of NOVX mRNA or protein expression based upon this comparison. For example, when expression of NOVX mRNA or protein is greater (i.e., statistically significantly greater) in the presence of the candidate compound than in its absence, the candidate compound is identified as a stimulator of NOVX mRNA or protein expression. Alternatively, when expression of NOVX mRNA or protein is less (statistically significantly less) in the presence of the candidate compound than in its absence, the candidate compound is identified as an inhibitor of NOVX mRNA or protein expression. The level of NOVX mRNA or protein expression in the cells can be determined by methods described herein for detecting NOVX mRNA or protein.
In yet another aspect of the invention, the NOVX proteins can be used as “bait proteins” in a two-hybrid assay or three hybrid assay (see, e.g., U.S. Pat. No. 5,283,317; Zervos, et al., 1993. Cell 72: 223-232; Madura, et al., 1993. J. Biol. Chem. 268: 12046-12054; Bartel, et al., 1993. Biotechniques 14: 920-924; Iwabuchi, et al., 1993. Oncogene 8: 1693-1696; and Brent WO 94/10300), to identify other proteins that bind to or interact with NOVX (“NOVX-binding proteins” or “NOVX-bp”) and modulate NOVX activity. Such NOVX-binding proteins are also likely to be involved in the propagation of signals by the NOVX proteins as, for example, upstream or downstream elements of the NOVX pathway.
The two-hybrid system is based on the modular nature of most transcription factors, which consist of separable DNA-binding and activation domains. Briefly, the assay utilizes two different DNA constructs. In one construct, the gene that codes for NOVX is fused to a gene encoding the DNA binding domain of a known transcription factor (e.g., GAL-4). In the other construct, a DNA sequence, from a library of DNA sequences, that encodes an unidentified protein (“prey” or “sample”) is fused to a gene that codes for the activation domain of the known transcription factor. If the “bait” and the “prey” proteins are able to interact, in vivo, forming an NOVX-dependent complex, the DNA-binding and activation domains of the transcription factor are brought into close proximity. This proximity allows transcription of a reporter gene (e.g., LacZ) that is operably linked to a transcriptional regulatory site responsive to the transcription factor. Expression of the reporter gene can be detected and cell colonies containing the functional transcription factor can be isolated and used to obtain the cloned gene that encodes the protein which interacts with NOVX.
The invention further pertains to novel agents identified by the aforementioned screening assays and uses thereof for treatments as described herein.
Detection Assays
Portions or fragments of the cDNA sequences identified herein (and the corresponding complete gene sequences) can be used in numerous ways as polynucleotide reagents. By way of example, and not of limitation, these sequences can be used to: (i) map their respective genes on a chromosome; and, thus, locate gene regions associated with genetic disease; (ii) identify an individual from a minute biological sample (tissue typing); and (iii) aid in forensic identification of a biological sample. Some of these applications are described in the subsections, below.
Chromosome Mapping
Once the sequence (or a portion of the sequence) of a gene has been isolated, this sequence can be used to map the location of the gene on a chromosome. This process is called chromosome mapping. Accordingly, portions or fragments of the NOVX sequences, SEQ ID NOS:1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91 and 93, or fragments or derivatives thereof, can be used to map the location of the NOVX genes, respectively, on a chromosome. The mapping of the NOVX sequences to chromosomes is an important first step in correlating these sequences with genes associated with disease.
Briefly, NOVX genes can be mapped to chromosomes by preparing PCR primers (preferably 15-25 bp in length) from the NOVX sequences. Computer analysis of the NOVX, sequences can be used to rapidly select primers that do not span more than one exon in the genomic DNA, thus complicating the amplification process. These primers can then be used for PCR screening of somatic cell hybrids containing individual human chromosomes. Only those hybrids containing the human gene corresponding to the NOVX sequences will yield an amplified fragment.
Somatic cell hybrids are prepared by fusing somatic cells from different mammals (e.g., human and mouse cells). As hybrids of human and mouse cells grow and divide, they gradually lose human chromosomes in random order, but retain the mouse chromosomes. By using media in which mouse cells cannot grow, because they lack a particular enzyme, but in which human cells can, the one human chromosome that contains the gene encoding the needed enzyme will be retained. By using various media, panels of hybrid cell lines can be established. Each cell line in a panel contains either a single human chromosome or a small number of human chromosomes, and a full set of mouse chromosomes, allowing easy mapping of individual genes to specific human chromosomes. See, e.g., D'Eustachio, et al., 1983. Science 220: 919-924. Somatic cell hybrids containing only fragments of human chromosomes can also be produced by using human chromosomes with translocations and deletions.
PCR mapping of somatic cell hybrids is a rapid procedure for assigning a particular sequence to a particular chromosome. Three or more sequences can be assigned per day using a single thermal cycler. Using the NOVX sequences to design oligonucleotide primers, sub-localization can be achieved with panels of fragments from specific chromosomes.
Fluorescence in situ hybridization (FISH) of a DNA sequence to a metaphase chromosomal spread can further be used to provide a precise chromosomal location in one step. Chromosome spreads can be made using cells whose division has been blocked in metaphase by a chemical like colcemid that disrupts the mitotic spindle. The chromosomes can be treated briefly with trypsin, and then stained with Giemsa. A pattern of light and dark bands develops on each chromosome, so that the chromosomes can be identified individually. The FISH technique can be used with a DNA sequence as short as 500 or 600 bases. However, clones larger than 1,000 bases have a higher likelihood of binding to a unique chromosomal location with sufficient signal intensity for simple detection. Preferably 1,000 bases, and more preferably 2,000 bases, will suffice to get good results at a reasonable amount of time. For a review of this technique, see, Verma, et al., HUMAN CHROMOSOMES: A MANUAL OF BASIC TECHNIQUES (Pergamon Press, New York 1988).
Reagents for chromosome mapping can be used individually to mark a single chromosome or a single site on that chromosome, or panels of reagents can be used for marking multiple sites and/or multiple chromosomes. Reagents corresponding to noncoding regions of the genes actually are preferred for mapping purposes. Coding sequences are more likely to be conserved within gene families, thus increasing the chance of cross hybridizations during chromosomal mapping.
Once a sequence has been mapped to a precise chromosomal location, the physical position of the sequence on the chromosome can be correlated with genetic map data. Such data are found, e.g., in McKusick, MENDELIAN INHERITANCE IN MAN, available on-line through Johns Hopkins University Welch Medical Library). The relationship between genes and disease, mapped to the same chromosomal region, can then be identified through linkage analysis (co-inheritance of physically adjacent genes), described in, e.g., Egeland, et al., 1987. Nature, 325: 783-787.
Moreover, differences in the DNA sequences between individuals affected and unaffected with a disease associated with the NOVX gene, can be determined. If a mutation is observed in some or all of the affected individuals but not in any unaffected individuals, then the mutation is likely to be the causative agent of the particular disease. Comparison of affected and unaffected individuals generally involves first looking for structural alterations in the chromosomes, such as deletions or translocations that are visible from chromosome spreads or detectable using PCR based on that DNA sequence. Ultimately, complete sequencing of genes from several individuals can be performed to confirm the presence of a mutation and to distinguish mutations from polymorphisms.
Tissue Typing
The NOVX sequences of the invention can also be used to identify individuals from minute biological samples. In this technique, an individual's genomic DNA is digested with one or more restriction enzymes, and probed on a Southern blot to yield unique bands for identification. The sequences of the invention are useful as additional DNA markers for RFLP (“restriction fragment length polymorphisms,” described in U.S. Pat. No. 5,272,057).
Furthermore, the sequences of the invention can be used to provide an alternative technique that determines the actual base-by-base DNA sequence of selected portions of an individual's genome. Thus, the NOVX sequences described herein can be used to prepare two PCR primers from the 5′- and 3′-termini of the sequences. These primers can then be used to amplify an individual's DNA and subsequently sequence it.
Panels of corresponding DNA sequences from individuals, prepared in this manner, can provide unique individual identifications, as each individual will have a unique set of such DNA sequences due to allelic differences. The sequences of the invention can be used to obtain such identification sequences from individuals and from tissue. The NOVX sequences of the invention uniquely represent portions of the human genome. Allelic variation occurs to some degree in the coding regions of these sequences, and to a greater degree in the noncoding regions. It is estimated that allelic variation between individual humans occurs with a frequency of about once per each 500 bases. Much of the allelic variation is due to single nucleotide polymorphisms (SNPs), which include restriction fragment length polymorphisms (RFLPs).
Each of the sequences described herein can, to some degree, be used as a standard against which DNA from an individual can be compared for identification purposes. Because greater numbers of polymorphisms occur in the noncoding regions, fewer sequences are necessary to differentiate individuals. The noncoding sequences can comfortably provide positive individual identification with a panel of perhaps 10 to 1,000 primers that each yield a noncoding amplified sequence of 100 bases. If predicted coding sequences, such as those in SEQ ID NOS:1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91 and 93 are used, a more appropriate number of primers for positive individual identification would be 500-2,000.
Predictive Medicine
The invention also pertains to the field of predictive medicine in which diagnostic assays, prognostic assays, pharmacogenomics, and monitoring clinical trials are used for prognostic (predictive) purposes to thereby treat an individual prophylactically. Accordingly, one aspect of the invention relates to diagnostic assays for determining NOVX protein and/or nucleic acid expression as well as NOVX activity, in the context of a biological sample (e.g., blood, serum, cells, tissue) to thereby determine whether an individual is afflicted with a disease or disorder, or is at risk of developing a disorder, associated with aberrant NOVX expression or activity. The disorders include metabolic disorders, diabetes, obesity, infectious disease, anorexia, cancer-associated cachexia, cancer, neurodegenerative disorders, Alzheimer's Disease, Parkinson's Disorder, immune disorders, and hematopoietic disorders, and the various dyslipidemias, metabolic disturbances associated with obesity, the metabolic syndrome X and wasting disorders associated with chronic diseases and various cancers. The invention also provides for prognostic (or predictive) assays for determining whether an individual is at risk of developing a disorder associated with NOVX protein, nucleic acid expression or activity. For example, mutations in an NOVX gene can be assayed in a biological sample. Such assays can be used for prognostic or predictive purpose to thereby prophylactically treat an individual prior to the onset of a disorder characterized by or associated with NOVX protein, nucleic acid expression, or biological activity.
Another aspect of the invention provides methods for determining NOVX protein, nucleic acid expression or activity in an individual to thereby select appropriate therapeutic or prophylactic agents for that individual (referred to herein as “pharmacogenomics”). Pharmacogenomics allows for the selection of agents (e.g. drugs) for therapeutic or prophylactic treatment of an individual based on the genotype of the individual (e.g., the genotype of the individual examined to determine the ability of the individual to respond to a particular agent.)
Yet another aspect of the invention pertains to monitoring the influence of agents (e.g., drugs, compounds) on the expression or activity of NOVX in clinical trials.
These and other agents are described in further detail in the following sections.
Diagnostic Assays
An exemplary method for detecting the presence or absence of NOVX in a biological sample involves obtaining a biological sample from a test subject and contacting the biological sample with a compound or an agent capable of detecting NOVX protein or nucleic acid (e.g., mRNA, genomic DNA) that encodes NOVX protein such that the presence of NOVX is detected in the biological sample. An agent for detecting NOVX mRNA or genomic DNA is a labeled nucleic acid probe capable of hybridizing to NOVX mRNA or genomic DNA. The nucleic acid probe can be, for example, a full-length NOVX nucleic acid, such as the nucleic acid of SEQ ID NOS:1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91 and 93, or a portion thereof, such as an oligonucleotide of at least 15, 30, 50, 100, 250 or 500 nucleotides in length and sufficient to specifically hybridize under stringent conditions to NOVX mRNA or genomic DNA. Other suitable probes for use in the diagnostic assays of the invention are described herein.
An agent for detecting NOVX protein is an antibody capable of binding to NOVX protein, preferably an antibody with a detectable label. Antibodies can be polyclonal, or more preferably, monoclonal. An intact antibody, or a fragment thereof (e.g., Fab or F(ab′)2) can be used. The term “labeled”, with regard to the probe or antibody, is intended to encompass direct labeling of the probe or antibody by coupling (i.e., physically linking) a detectable substance to the probe or antibody, as well as indirect labeling of the probe or antibody by reactivity with another reagent that is directly labeled. Examples of indirect labeling include detection of a primary antibody using a fluorescently-labeled secondary antibody and end-labeling of a DNA probe with biotin such that it can be detected with fluorescently-labeled streptavidin. The term “biological sample” is intended to include tissues, cells and biological fluids isolated from a subject, as well as tissues, cells and fluids present within a subject. That is, the detection method of the invention can be used to detect NOVX mRNA, protein, or genomic DNA in a biological sample in vitro as well as in vivo. For example, in vitro techniques for detection of NOVX mRNA include Northern hybridizations and in situ hybridizations. In vitro techniques for detection of NOVX protein include enzyme linked immunosorbent assays (ELISAs), Western blots, immunoprecipitations, and immunofluorescence. In vitro techniques for detection of NOVX genomic DNA include Southern hybridizations. Furthermore, in vivo techniques for detection of NOVX protein include introducing into a subject a labeled anti-NOVX antibody. For example, the antibody can be labeled with a radioactive marker whose presence and location in a subject can be detected by standard imaging techniques.
In one embodiment, the biological sample contains protein molecules from the test subject. Alternatively, the biological sample can contain mRNA molecules from the test subject or genomic DNA molecules from the test subject. A preferred biological sample is a peripheral blood leukocyte sample isolated by conventional means from a subject.
In another embodiment, the methods further involve obtaining a control biological sample from a control subject, contacting the control sample with a compound or agent capable of detecting NOVX protein, mRNA, or genomic DNA, such that the presence of NOVX protein, mRNA or genomic DNA is detected in the biological sample, and comparing the presence of NOVX protein, mRNA or genomic DNA in the control sample with the presence of NOVX protein, mRNA or genomic DNA in the test sample.
The invention also encompasses kits for detecting the presence of NOVX in a biological sample. For example, the kit can comprise: a labeled compound or agent capable of detecting NOVX protein or mRNA in a biological sample; means for determining the amount of NOVX in the sample; and means for comparing the amount of NOVX in the sample with a standard. The compound or agent can be packaged in a suitable container. The kit can further comprise instructions for using the kit to detect NOVX protein or nucleic acid.
Prognostic Assays
The diagnostic methods described herein can furthermore be utilized to identify subjects having or at risk of developing a disease or disorder associated with aberrant NOVX expression or activity. For example, the assays described herein, such as the preceding diagnostic assays or the following assays, can be utilized to identify a subject having or at risk of developing a disorder associated with NOVX protein, nucleic acid expression or activity. Alternatively, the prognostic assays can be utilized to identify a subject having or at risk for developing a disease or disorder. Thus, the invention provides a method for identifying a disease or disorder associated with aberrant NOVX expression or activity in which a test sample is obtained from a subject and NOVX protein or nucleic acid (e.g., mRNA, genomic DNA) is detected, wherein the presence of NOVX protein or nucleic acid is diagnostic for a subject having or at risk of developing a disease or disorder associated with aberrant NOVX expression or activity. As used herein, a “test sample” refers to a biological sample obtained from a subject of interest. For example, a test sample can be a biological fluid (e.g., serum), cell sample, or tissue.
Furthermore, the prognostic assays described herein can be used to determine whether a subject can be administered an agent (e.g., an agonist, antagonist, peptidomimetic, protein, peptide, nucleic acid, small molecule, or other drug candidate) to treat a disease or disorder associated with aberrant NOVX expression or activity. For example, such methods can be used to determine whether a subject can be effectively treated with an agent for a disorder. Thus, the invention provides methods for determining whether a subject can be effectively treated with an agent for a disorder associated with aberrant NOVX expression or activity in which a test sample is obtained and NOVX protein or nucleic acid is detected (e.g., wherein the presence of NOVX protein or nucleic acid is diagnostic for a subject that can be administered the agent to treat a disorder associated with aberrant NOVX expression or activity).
The methods of the invention can also be used to detect genetic lesions in an NOVX gene, thereby determining if a subject with the lesioned gene is at risk for a disorder characterized by aberrant cell proliferation and/or differentiation. In various embodiments, the methods include detecting, in a sample of cells from the subject, the presence or absence of a genetic lesion characterized by at least one of an alteration affecting the integrity of a gene encoding an NOVX-protein, or the misexpression of the NOVX gene. For example, such genetic lesions can be detected by ascertaining the existence of at least one of: (i) a deletion of one or more nucleotides from an NOVX gene; (ii) an addition of one or more nucleotides to an NOVX gene; (iii) a substitution of one or more nucleotides of an NOVX gene, (iv) a chromosomal rearrangement of an NOVX gene; (v) an alteration in the level of a messenger RNA transcript of an NOVX gene, (vi) aberrant modification of an NOVX gene, such as of the methylation pattern of the genomic DNA, (vii) the presence of a non-wild-type splicing pattern of a messenger RNA transcript of an NOVX gene, (viii) a non-wild-type level of an NOVX protein, (ix) allelic loss of an NOVX gene, and (x) inappropriate post-translational modification of an NOVX protein. As described herein, there are a large number of assay techniques known in the art which can be used for detecting lesions in an NOVX gene. A preferred biological sample is a peripheral blood leukocyte sample isolated by conventional means from a subject. However, any biological sample containing nucleated cells may be used, including, for example, buccal mucosal cells.
In certain embodiments, detection of the lesion involves the use of a probe/primer in a polymerase chain reaction (PCR) (see, e.g., U.S. Pat. Nos. 4,683,195 and 4,683,202), such as anchor PCR or RACE PCR, or, alternatively, in a ligation chain reaction (LCR) (see, e.g., Landegran, et al., 1988. Science 241: 1077-1080; and Nakazawa, et al., 1994. Proc. Natl. Acad. Sci. USA 91: 360-364), the latter of which can be particularly useful for detecting point mutations in the NOVX-gene (see, Abravaya, et al., 1995. Nucl. Acids Res. 23: 675-682). This method can include the steps of collecting a sample of cells from a patient, isolating nucleic acid (e.g., genomic, mRNA or both) from the cells of the sample, contacting the nucleic acid sample with one or more primers that specifically hybridize to an NOVX gene under conditions such that hybridization and amplification of the NOVX gene (if present) occurs, and detecting the presence or absence of an amplification product, or detecting the size of the amplification product and comparing the length to a control sample. It is anticipated that PCR and/or LCR may be desirable to use as a preliminary amplification step in conjunction with any of the techniques used for detecting mutations described herein.
Alternative amplification methods include: self sustained sequence replication (see, Guatelli, et al., 1990. Proc. Natl. Acad. Sci. USA 87: 1874-1878), transcriptional amplification system (see, Kwoh, et al., 1989. Proc. Natl. Acad. Sci. USA 86: 1173-1177); Qβ Replicase (see, Lizardi, et al, 1988. BioTechnology 6: 1197), or any other nucleic acid amplification method, followed by the detection of the amplified molecules using techniques well known to those of skill in the art. These detection schemes are especially useful for the detection of nucleic acid molecules if such molecules are present in very low numbers.
In an alternative embodiment, mutations in an NOVX gene from a sample cell can be identified by alterations in restriction enzyme cleavage patterns. For example, sample and control DNA is isolated, amplified (optionally), digested with one or more restriction endonucleases, and fragment length sizes are determined by gel electrophoresis and compared. Differences in fragment length sizes between sample and control DNA indicates mutations in the sample DNA. Moreover, the use of sequence specific ribozymes (see, e.g., U.S. Pat. No. 5,493,531) can be used to score for the presence of specific mutations by development or loss of a ribozyme cleavage site.
In other embodiments, genetic mutations in NOVX can be identified by hybridizing a sample and control nucleic acids, e.g., DNA or RNA, to high-density arrays containing hundreds or thousands of oligonucleotides probes. See, e.g., Cronin, et al., 1996. Human Mutation 7: 244-255; Kozal, et al., 1996. Nat. Med. 2: 753-759. For example, genetic mutations in NOVX can be identified in two dimensional arrays containing light-generated DNA probes as described in Cronin, et al., supra. Briefly, a first hybridization array of probes can be used to scan through long stretches of DNA in a sample and control to identify base changes between the sequences by making linear arrays of sequential overlapping probes. This step allows the identification of point mutations. This is followed by a second hybridization array that allows the characterization of specific mutations by using smaller, specialized probe arrays complementary to all variants or mutations detected. Each mutation array is composed of parallel probe sets, one complementary to the wild-type gene and the other complementary to the mutant gene.
In yet another embodiment, any of a variety of sequencing reactions known in the art can be used to directly sequence the NOVX gene and detect mutations by comparing the sequence of the sample NOVX with the corresponding wild-type (control) sequence. Examples of sequencing reactions include those based on techniques developed by Maxim and Gilbert, 1977. Proc. Natl. Acad. Sci. USA 74: 560 or Sanger, 1977. Proc. Natl. Acad. Sci. USA 74: 5463. It is also contemplated that any of a variety of automated sequencing procedures can be utilized when performing the diagnostic assays (see, e.g., Naeve, et al., 1995. Biotechniques 19: 448), including sequencing by mass spectrometry (see, e.g., PCT International Publication No. WO 94/16101; Cohen, et al., 1996. Adv. Chromatography 36: 127-162; and Griffin, et al., 1993. Appl. Biochem. Biotechnol. 38: 147-159).
Other methods for detecting mutations in the NOVX gene include methods in which protection from cleavage agents is used to detect mismatched bases in RNA/RNA or RNA/DNA heteroduplexes. See, e.g., Myers, et al., 1985. Science 230: 1242. In general, the art technique of “mismatch cleavage” starts by providing heteroduplexes of formed by hybridizing (labeled) RNA or DNA containing the wild-type NOVX sequence with potentially mutant RNA or DNA obtained from a tissue sample. The double-stranded duplexes are treated with an agent that cleaves single-stranded regions of the duplex such as which will exist due to basepair mismatches between the control and sample strands. For instance, RNA/DNA duplexes can be treated with RNase and DNA/DNA hybrids treated with S1 nuclease to enzymatically digesting the mismatched regions. In other embodiments, either DNA/DNA or RNA/DNA duplexes can be treated with hydroxylamine or osmium tetroxide and with piperidine in order to digest mismatched regions. After digestion of the mismatched regions, the resulting material is then separated by size on denaturing polyacrylamide gels to determine the site of mutation. See, e.g., Cotton, et al., 1988. Proc. Natl. Acad. Sci. USA 85: 4397; Saleeba, et al., 1992. Methods Enzymol. 217: 286-295. In an embodiment, the control DNA or RNA can be labeled for detection.
In still another embodiment, the mismatch cleavage reaction employs one or more proteins that recognize mismatched base pairs in double-stranded DNA (so called “DNA mismatch repair” enzymes) in defined systems for detecting and mapping point mutations in NOVX cDNAs obtained from samples of cells. For example, the mutY enzyme of E. coli cleaves A at G/A mismatches and the thymidine DNA glycosylase from HeLa cells cleaves T at G/T mismatches. See, e.g., Hsu, et al., 1994. Carcinogenesis 15: 1657-1662. According to an exemplary embodiment, a probe based on an NOVX sequence, e.g. a wild-type NOVX sequence, is hybridized to a cDNA or other DNA product from a test cell(s). The duplex is treated with a DNA mismatch repair enzyme, and the cleavage products, if any, can be detected from electrophoresis protocols or the like. See, e.g., U.S. Pat. No. 5,459,039.
In other embodiments, alterations in electrophoretic mobility will be used to identify mutations in NOVX genes. For example, single strand conformation polymorphism (SSCP) may be used to detect differences in electrophoretic mobility between mutant and wild type nucleic acids. See, e.g., Orita, et al., 1989. Proc. Natl. Acad. Sci. USA: 86: 2766; Cotton, 1993. Mutat. Res. 285: 125-144; Hayashi, 1992. Genet. Anal. Tech. Appl. 9: 73-79. Single-stranded DNA fragments of sample and control NOVX nucleic acids will be denatured and allowed to renature. The secondary structure of single-stranded nucleic acids varies according to sequence, the resulting alteration in electrophoretic mobility enables the detection of even a single base change. The DNA fragments may be labeled or detected with labeled probes. The sensitivity of the assay may be enhanced by using RNA (rather than DNA), in which the secondary structure is more sensitive to a change in sequence. In one embodiment, the subject method utilizes heteroduplex analysis to separate double stranded heteroduplex molecules on the basis of changes in electrophoretic mobility. See, e.g., Keen, et al., 1991. Trends Genet. 7: 5.
In yet another embodiment, the movement of mutant or wild-type fragments in polyacrylamide gels containing a gradient of denaturant is assayed using denaturing gradient gel electrophoresis (DGGE). See, e.g., Myers, et al., 1985. Nature 313: 495. When DGGE is used as the method of analysis, DNA will be modified to insure that it does not completely denature, for example by adding a GC clamp of approximately 40 bp of high-melting GC-rich DNA by PCR. In a further embodiment, a temperature gradient is used in place of a denaturing gradient to identify differences in the mobility of control and sample DNA. See, e.g., Rosenbaum and Reissner, 1987. Biophys. Chem. 265: 12753.
Examples of other techniques for detecting point mutations include, but are not limited to, selective oligonucleotide hybridization, selective amplification, or selective primer extension. For example, oligonucleotide primers may be prepared in which the known mutation is placed centrally and then hybridized to target DNA under conditions that permit hybridization only if a perfect match is found. See, e.g., Saiki, et al., 1986. Nature 324: 163; Saiki, et al., 1989. Proc. Natl. Acad. Sci. USA 86: 6230. Such allele specific oligonucleotides are hybridized to PCR amplified target DNA or a number of different mutations when the oligonucleotides are attached to the hybridizing membrane and hybridized with labeled target DNA.
Alternatively, allele specific amplification technology that depends on selective PCR amplification may be used in conjunction with the instant invention. Oligonucleotides used as primers for specific amplification may carry the mutation of interest in the center of the molecule (so that amplification depends on differential hybridization; see, e.g., Gibbs, et al., 1989. Nucl. Acids Res. 17: 2437-2448) or at the extreme 3′-terminus of one primer where, under appropriate conditions, mismatch can prevent, or reduce polymerase extension (see, e.g., Prossner, 1993. Tibtech. 11: 238). In addition it may be desirable to introduce a novel restriction site in the region of the mutation to create cleavage-based detection. See, e.g., Gasparini, et al., 1992. Mol. Cell Probes 6: 1. It is anticipated that in certain embodiments amplification may also be performed using Taq ligase for amplification. See, e.g., Barany, 1991. Proc. Natl. Acad. Sci. USA 88: 189. In such cases, ligation will occur only if there is a perfect match at the 3′-terminus of the 5′ sequence, making it possible to detect the presence of a known mutation at a specific site by looking for the presence or absence of amplification.
The methods described herein may be performed, for example, by utilizing pre-packaged diagnostic kits comprising at least one probe nucleic acid or antibody reagent described herein, which may be conveniently used, e.g., in clinical settings to diagnose patients exhibiting symptoms or family history of a disease or illness involving an NOVX gene.
Furthermore, any cell type or tissue, preferably peripheral blood leukocytes, in which NOVX is expressed may be utilized in the prognostic assays described herein. However, any biological sample containing nucleated cells may be used, including, for example, buccal mucosal cells.
Pharmacogenomics
Agents, or modulators that have a stimulatory or inhibitory effect on NOVX activity (e.g., NOVX gene expression), as identified by a screening assay described herein can be administered to individuals to treat (prophylactically or therapeutically) disorders (The disorders include metabolic disorders, diabetes, obesity, infectious disease, anorexia, cancer-associated cachexia, cancer, neurodegenerative disorders, Alzheimer's Disease, Parkinson's Disorder, immune disorders, and hematopoietic disorders, and the various dyslipidemias, metabolic disturbances associated with obesity, the metabolic syndrome X and wasting disorders associated with chronic diseases and various cancers.) In conjunction with such treatment, the pharmacogenomics (i.e., the study of the relationship between an individual's genotype and that individual's response to a foreign compound or drug) of the individual may be considered. Differences in metabolism of therapeutics can lead to severe toxicity or therapeutic failure by altering the relation between dose and blood concentration of the pharmacologically active drug. Thus, the pharmacogenomics of the individual permits the selection of effective agents (e.g., drugs) for prophylactic or therapeutic treatments based on a consideration of the individual's genotype. Such pharmacogenomics can further be used to determine appropriate dosages and therapeutic regimens. Accordingly, the activity of NOVX protein, expression of NOVX nucleic acid, or mutation content of NOVX genes in an individual can be determined to thereby select appropriate agent(s) for therapeutic or prophylactic treatment of the individual.
Pharmacogenomics deals with clinically significant hereditary variations in the response to drugs due to altered drug disposition and abnormal action in affected persons. See e.g., Eichelbaum, 1996. Clin. Exp. Pharmacol. Physiol., 23: 983-985; Linder, 1997. Clin. Chem., 43: 254-266. In general, two types of pharmacogenetic conditions can be differentiated. Genetic conditions transmitted as a single factor altering the way drugs act on the body (altered drug action) or genetic conditions transmitted as single factors altering the way the body acts on drugs (altered drug metabolism). These pharmacogenetic conditions can occur either as rare defects or as polymorphisms. For example, glucose-6-phosphate dehydrogenase (G6PD) deficiency is a common inherited enzymopathy in which the main clinical complication is hemolysis after ingestion of oxidant drugs (anti-malarials, sulfonamides, analgesics, nitrofurans) and consumption of fava beans.
As an illustrative embodiment, the activity of drug metabolizing enzymes is a major determinant of both the intensity and duration of drug action. The discovery of genetic polymorphisms of drug metabolizing enzymes (e.g., N-acetyltransferase 2 (NAT 2) and cytochrome P450 enzymes CYP2D6 and CYP2C19) has provided an explanation as to why some patients do not obtain the expected drug effects or show exaggerated drug response and serious toxicity after taking the standard and safe dose of a drug. These polymorphisms are expressed in two phenotypes in the population, the extensive metabolizer (EM) and poor metabolizer (PM). The prevalence of PM is different among different populations. For example, the gene coding for CYP2D6 is highly polymorphic and several mutations have been identified in PM, which all lead to the absence of functional CYP2D6. Poor metabolizers of CYP2D6 and CYP2C19 quite frequently experience exaggerated drug response and side effects when they receive standard doses. If a metabolite is the active therapeutic moiety, PM show no therapeutic response, as demonstrated for the analgesic effect of codeine mediated by its CYP2D6-formed metabolite morphine. At the other extreme are the so called ultra-rapid metabolizers who do not respond to standard doses. Recently, the molecular basis of ultra-rapid metabolism has been identified to be due to CYP2D6 gene amplification.
Thus, the activity of NOVX protein, expression of NOVX nucleic acid, or mutation content of NOVX genes in an individual can be determined to thereby select appropriate agent(s) for therapeutic or prophylactic treatment of the individual. In addition, pharmacogenetic studies can be used to apply genotyping of polymorphic alleles encoding drug-metabolizing enzymes to the identification of an individual's drug responsiveness phenotype. This knowledge, when applied to dosing or drug selection, can avoid adverse reactions or therapeutic failure and thus enhance therapeutic or prophylactic efficiency when treating a subject with an NOVX modulator, such as a modulator identified by one of the exemplary screening assays described herein.
Monitoring of Effects During Clinical Trials
Monitoring the influence of agents (e.g., drugs, compounds) on the expression or activity of NOVX (e.g., the ability to modulate aberrant cell proliferation and/or differentiation) can be applied not only in basic drug screening, but also in clinical trials. For example, the effectiveness of an agent determined by a screening assay as described herein to increase NOVX gene expression, protein levels, or upregulate NOVX activity, can be monitored in clinical trails of subjects exhibiting decreased NOVX gene expression, protein levels, or downregulated NOVX activity. Alternatively, the effectiveness of an agent determined by a screening assay to decrease NOVX gene expression, protein levels, or downregulate NOVX activity, can be monitored in clinical trails of subjects exhibiting increased NOVX gene expression, protein levels, or upregulated NOVX activity. In such clinical trials, the expression or activity of NOVX and, preferably, other genes that have been implicated in, for example, a cellular proliferation or immune disorder can be used as a “read out” or markers of the immune responsiveness of a particular cell.
By way of example, and not of limitation, genes, including NOVX, that are modulated in cells by treatment with an agent (e.g., compound, drug or small molecule) that modulates NOVX activity (e.g., identified in a screening assay as described herein) can be identified. Thus, to study the effect of agents on cellular proliferation disorders, for example, in a clinical trial, cells can be isolated and RNA prepared and analyzed for the levels of expression of NOVX and other genes implicated in the disorder. The levels of gene expression (i.e., a gene expression pattern) can be quantified by Northern blot analysis or RT-PCR, as described herein, or alternatively by measuring the amount of protein produced, by one of the methods as described herein, or by measuring the levels of activity of NOVX or other genes. In this manner, the gene expression pattern can serve as a marker, indicative of the physiological response of the cells to the agent. Accordingly, this response state may be determined before, and at various points during, treatment of the individual with the agent.
In one embodiment, the invention provides a method for monitoring the effectiveness of treatment of a subject with an agent (e.g., an agonist, antagonist, protein, peptide, peptidomimetic, nucleic acid, small molecule, or other drug candidate identified by the screening assays described herein) comprising the steps of (i) obtaining a pre-administration sample from a subject prior to administration of the agent; (ii) detecting the level of expression of an NOVX protein, mRNA, or genomic DNA in the preadministration sample; (iii) obtaining one or more post-administration samples from the subject; (iv) detecting the level of expression or activity of the NOVX protein, mRNA, or genomic DNA in the post-administration samples; (v) comparing the level of expression or activity of the NOVX protein, mRNA, or genomic DNA in the pre-administration sample with the NOVX protein, mRNA, or genomic DNA in the post administration sample or samples; and (vi) altering the administration of the agent to the subject accordingly. For example, increased administration of the agent may be desirable to increase the expression or activity of NOVX to higher levels than detected, i.e., to increase the effectiveness of the agent. Alternatively, decreased administration of the agent may be desirable to decrease expression or activity of NOVX to lower levels than detected, i.e., to decrease the effectiveness of the agent.
Methods of Treatment
The invention provides for both prophylactic and therapeutic methods of treating a subject at risk of (or susceptible to) a disorder or having a disorder associated with aberrant NOVX expression or activity. The disorders include cardiomyopathy, atherosclerosis, hypertension, congenital heart defects, aortic stenosis, atrial septal defect (ASD), atrioventricular (A-V) canal defect, ductus arteriosus, pulmonary stenosis, subaortic stenosis, ventricular septal defect (VSD), valve diseases, tuberous sclerosis, scleroderma, obesity, transplantation, adrenoleukodystrophy, congenital adrenal hyperplasia, prostate cancer, neoplasm; adenocarcinoma, lymphoma, uterus cancer, fertility, hemophilia, hypercoagulation, idiopathic thrombocytopenic purpura, immunodeficiencies, graft versus host disease, AIDS, bronchial asthma, Crohn's disease; multiple sclerosis, treatment of Albright Hereditary Ostoeodystrophy, and other diseases, disorders and conditions of the like.
These methods of treatment will be discussed more fully, below.
Disease and Disorders
Diseases and disorders that are characterized by increased (relative to a subject not suffering from the disease or disorder) levels or biological activity may be treated with Therapeutics that antagonize (i.e., reduce or inhibit) activity. Therapeutics that antagonize activity may be administered in a therapeutic or prophylactic manner. Therapeutics that may be utilized include, but are not limited to: (i) an aforementioned peptide, or analogs, derivatives, fragments or homologs thereof; (ii) antibodies to an aforementioned peptide; (iii) nucleic acids encoding an aforementioned peptide; (iv) administration of antisense nucleic acid and nucleic acids that are “dysfunctional” (i.e., due to a heterologous insertion within the coding sequences of coding sequences to an aforementioned peptide) that are utilized to “knockout” endogenous function of an aforementioned peptide by homologous recombination (see, e.g., Capecchi, 1989. Science 244: 1288-1292); or (v) modulators (i.e., inhibitors, agonists and antagonists, including additional peptide mimetic of the invention or antibodies specific to a peptide of the invention) that alter the interaction between an aforementioned peptide and its binding partner.
Diseases and disorders that are characterized by decreased (relative to a subject not suffering from the disease or disorder) levels or biological activity may be treated with Therapeutics that increase (i.e., are agonists to) activity. Therapeutics that upregulate activity may be administered in a therapeutic or prophylactic manner. Therapeutics that may be utilized include, but are not limited to, an aforementioned peptide, or analogs, derivatives, fragments or homologs thereof; or an agonist that increases bioavailability.
Increased or decreased levels can be readily detected by quantifying peptide and/or RNA, by obtaining a patient tissue sample (e.g., from biopsy tissue) and assaying it in vitro for RNA or peptide levels, structure and/or activity of the expressed peptides (or mRNAs of an aforementioned peptide). Methods that are well-known within the art include, but are not limited to, immunoassays (e.g., by Western blot analysis, immunoprecipitation followed by sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis, immunocytochemistry, etc.) and/or hybridization assays to detect expression of mRNAs (e.g., Northern assays, dot blots, in situ hybridization, and the like).
Prophylactic Methods
In one aspect, the invention provides a method for preventing, in a subject, a disease or condition associated with an aberrant NOVX expression or activity, by administering to the subject an agent that modulates NOVX expression or at least one NOVX activity. Subjects at risk for a disease that is caused or contributed to by aberrant NOVX expression or activity can be identified by, for example, any or a combination of diagnostic or prognostic assays as described herein. Administration of a prophylactic agent can occur prior to the manifestation of symptoms characteristic of the NOVX aberrancy, such that a disease or disorder is prevented or, alternatively, delayed in its progression. Depending upon the type of NOVX aberrancy, for example, an NOVX agonist or NOVX antagonist agent can be used for treating the subject. The appropriate agent can be determined based on screening assays described herein. The prophylactic methods of the invention are further discussed in the following subsections.
Therapeutic Methods
Another aspect of the invention pertains to methods of modulating NOVX expression or activity for therapeutic purposes. The modulatory method of the invention involves contacting a cell with an agent that modulates one or more of the activities of NOVX protein activity associated with the cell. An agent that modulates NOVX protein activity can be an agent as described herein, such as a nucleic acid or a protein, a naturally-occurring cognate ligand of an NOVX protein, a peptide, an NOVX peptidomimetic, or other small molecule. In one embodiment, the agent stimulates one or more NOVX protein activity. Examples of such stimulatory agents include active NOVX protein and a nucleic acid molecule encoding NOVX that has been introduced into the cell. In another embodiment, the agent inhibits one or more NOVX protein activity. Examples of such inhibitory agents include antisense NOVX nucleic acid molecules and anti-NOVX antibodies. These modulatory methods can be performed in vitro (e.g., by culturing the cell with the agent) or, alternatively, in vivo (e.g. by administering the agent to a subject). As such, the invention provides methods of treating an individual afflicted with a disease or disorder characterized by aberrant expression or activity of an NOVX protein or nucleic acid molecule. In one embodiment, the method involves administering an agent (e.g., an agent identified by a screening assay described herein), or combination of agents that modulates (e.g., up-regulates or down-regulates) NOVX expression or activity. In another embodiment, the method involves administering an NOVX protein or nucleic acid molecule as therapy to compensate for reduced or aberrant NOVX expression or activity.
Stimulation of NOVX activity is desirable in situations in which NOVX is abnormally downregulated and/or in which increased NOVX activity is likely to have a beneficial effect. One example of such a situation is where a subject has a disorder characterized by aberrant cell proliferation and/or differentiation (e.g., cancer or immune associated disorders). Another example of such a situation is where the subject has a gestational disease (e.g., preclampsia).
Determination of the Biological Effect of the Therapeutic
In various embodiments of the invention, suitable in vitro or in vivo assays are performed to determine the effect of a specific Therapeutic and whether its administration is indicated for treatment of the affected tissue.
In various specific embodiments, in vitro assays may be performed with representative cells of the type(s) involved in the patient's disorder, to determine if a given Therapeutic exerts the desired effect upon the cell type(s). Compounds for use in therapy may be tested in suitable animal model systems including, but not limited to rats, mice, chicken, cows, monkeys, rabbits, and the like, prior to testing in human subjects. Similarly, for in vivo testing, any of the animal model system known in the art may be used prior to administration to human subjects.
Prophylactic and Therapeutic Uses of the Compositions of the Invention
The NOVX nucleic acids and proteins of the invention are useful in potential prophylactic and therapeutic applications implicated in a variety of disorders including, but not limited to: metabolic disorders, diabetes, obesity, infectious disease, anorexia, cancer-associated cancer, neurodegenerative disorders, Alzheimer's Disease, Parkinson's Disorder, immune disorders, hematopoietic disorders, and the various dyslipidemias, metabolic disturbances associated with obesity, the metabolic syndrome X and wasting disorders associated with chronic diseases and various cancers.
As an example, a cDNA encoding the NOVX protein of the invention may be useful in gene therapy, and the protein may be useful when administered to a subject in need thereof. By way of non-limiting example, the compositions of the invention will have efficacy for treatment of patients suffering from: metabolic disorders, diabetes, obesity, infectious disease, anorexia, cancer-associated cachexia, cancer, neurodegenerative disorders, Alzheimer's Disease, Parkinson's Disorder, immune disorders, hematopoietic disorders, and the various dyslipidemias.
Both the novel nucleic acid encoding the NOVX protein, and the NOVX protein of the invention, or fragments thereof, may also be useful in diagnostic applications, wherein the presence or amount of the nucleic acid or the protein are to be assessed. A further use could be as an anti-bacterial molecule (i.e., some peptides have been found to possess anti-bacterial properties). These materials are further useful in the generation of antibodies, which immunospecifically-bind to the novel substances of the invention for use in therapeutic or diagnostic methods.
The invention will be further described in the following examples, which do not limit the scope of the invention described in the claims.
EXAMPLES
Example 1
Identification of NOVX Nucleic Acids
TblastN using CuraGen Corporation's sequence file for polypeptides or homologs was run against the Genomic Daily Files made available by GenBank or from files downloaded from the individual sequencing centers. Exons were predicted by homology and the intron/exon boundaries were determined using standard genetic rules. Exons were further selected and refined by means of similarity determination using multiple BLAST (for example, tBlastN, BlastX, and BlastN) searches, and, in some instances, GeneScan and Grail. Expressed sequences from both public and proprietary databases were also added when available to further define and complete the gene sequence. The DNA sequence was then manually corrected for apparent inconsistencies thereby obtaining the sequences encoding the full-length protein.
The novel NOVX target sequences identified in the present invention were subjected to the exon linking process to confirm the sequence. PCR primers were designed by starting at the most upstream sequence available, for the forward primer, and at the most downstream sequence available for the reverse primer. PCR primer sequences were used for obtaining different clones. In each case, the sequence was examined, walking inward from the respective termini toward the coding sequence, until a suitable sequence that is either unique or highly selective was encountered, or, in the case of the reverse primer, until the stop codon was reached. Such primers were designed based on in silico predictions for the full length cDNA, part (one or more exons) of the DNA or protein sequence of the target sequence, or by translated homology of the predicted exons to closely related human sequences from other species. These primers were then employed in PCR amplification based on the following pool of human cDNAs: adrenal gland, bone marrow, brain—amygdala, brain—cerebellum, brain—hippocampus, brain—substantia nigra, brain—thalamus, brain—whole, fetal brain, fetal kidney, fetal liver, fetal lung, heart, kidney, lymphoma—Raji, mammary gland, pancreas, pituitary gland, placenta, prostate, salivary gland, skeletal muscle, small intestine, spinal cord, spleen, stomach, testis, thyroid, trachea, uterus. Usually the resulting amplicons were gel purified, cloned and sequenced to high redundancy. The PCR product derived from exon linking was cloned into the pCR2.1 vector from Invitrogen. The resulting bacterial clone has an insert covering the entire open reading frame cloned into the pCR2.1 vector. The resulting sequences from all clones were assembled with themselves, with other fragments in CuraGen Corporation's database and with public ESTs. Fragments and ESTs were included as components for an assembly when the extent of their identity with another component of the assembly was at least 95% over 50 bp. In addition, sequence traces were evaluated manually and edited for corrections if appropriate. These procedures provide the sequence reported herein.
Physical clone: Exons were predicted by homology and the intron/exon boundaries were determined using standard genetic rules. Exons were further selected and refined by means of similarity determination using multiple BLAST (for example, tBlastN, BlastX, and BlastN) searches, and, in some instances, GeneScan and Grail. Expressed sequences from both public and proprietary databases were also added when available to further define and complete the gene sequence. The DNA sequence was then manually corrected for apparent inconsistencies thereby obtaining the sequences encoding the full-length protein.
Example 2
Identification of Single Nucleotide Polymorphisms in NOVX Nucleic Acid Sequences
Variant sequences are also included in this application. A variant sequence can include a single nucleotide polymorphism (SNP). A SNP can, in some instances, be referred to as a “cSNP” to denote that the nucleotide sequence containing the SNP originates as a cDNA. A SNP can arise in several ways. For example, a SNP may be due to a substitution of one nucleotide for another at the polymorphic site. Such a substitution can be either a transition or a transversion. A SNP can also arise from a deletion of a nucleotide or an insertion of a nucleotide, relative to a reference allele. In this case, the polymorphic site is a site at which one allele bears a gap with respect to a particular nucleotide in another allele. SNPs occurring within genes may result in an alteration of the amino acid encoded by the gene at the position of the SNP. Intragenic SNPs may also be silent, when a codon including a SNP encodes the same amino acid as a result of the redundancy of the genetic code. SNPs occurring outside the region of a gene, or in an intron within a gene, do not result in changes in any amino acid sequence of a protein but may result in altered regulation of the expression pattern. Examples include alteration in temporal expression, physiological response regulation, cell type expression regulation, intensity of expression, and stability of transcribed message.
SeqCalling assemblies produced by the exon linking process were selected and extended using the following criteria. Genomic clones having regions with 98% identity to all or part of the initial or extended sequence were identified by BLASTN searches using the relevant sequence to query human genomic databases. The genomic clones that resulted were selected for further analysis because this identity indicates that these clones contain the genomic locus for these SeqCalling assemblies. These sequences were analyzed for putative coding regions as well as for similarity to the known DNA and protein sequences. Programs used for these analyses include Grail, Genscan, BLAST, HMMER, FASTA, Hybrid and other relevant programs.
Some additional genomic regions may have also been identified because selected SeqCalling assemblies map to those regions. Such SeqCalling sequences may have overlapped with regions defined by homology or exon prediction. They may also be included because the location of the fragment was in the vicinity of genomic regions identified by similarity or exon prediction that had been included in the original predicted sequence. The sequence so identified was manually assembled and then may have been extended using one or more additional sequences taken from CuraGen Corporation's human SeqCalling database. SeqCalling fragments suitable for inclusion were identified by the CuraToolsTM program SeqExtend or by identifying SeqCalling fragments mapping to the appropriate regions of the genomic clones analyzed.
The regions defined by the procedures described above were then manually integrated and corrected for apparent inconsistencies that may have arisen, for example, from miscalled bases in the original fragments or from discrepancies between predicted exon junctions, EST locations and regions of sequence similarity, to derive the final sequence disclosed herein. When necessary, the process to identify and analyze SeqCalling assemblies and genomic clones was reiterated to derive the full length sequence (Alderborn et al., Determination of Single Nucleotide Polymorphisms by Real-time Pyrophosphate DNA Sequencing. Genome Research. 10 (8) 1249-1265, 2000).
Example 3
Quantitative Expression Analysis of Clones in Various Cells and Tissues
The quantitative expression of various clones was assessed using microtiter plates containing RNA samples from a variety of normal and pathology-derived cells, cell lines and tissues using real time quantitative PCR (RTQ PCR). RTQ PCR was performed on an Applied Biosystems ABI PRISM® 7700 or an ABI PRISM® 7900 HT Sequence Detection System. Various collections of samples are assembled on the plates, and referred to as Panel 1 (containing normal tissues and cancer cell lines), Panel 2 (containing samples derived from tissues from normal and cancer sources), Panel 3 (containing cancer cell lines), Panel 4 (containing cells and cell lines from normal tissues and cells related to inflammatory conditions), Panel 5D/5I (containing human tissues and cell lines with an emphasis on metabolic diseases), AI_comprehensive_panel (containing normal tissue and samples from autoimmune diseases), Panel CNSD0.01 (containing central nervous system samples from normal and diseased brains) and CNS_neurodegeneration_panel (containing samples from normal and Alzheimer's diseased brains).
RNA integrity from all samples is controlled for quality by visual assessment of agarose gel electropherograms using 28S and 18S ribosomal RNA staining intensity ratio as a guide (2:1 to 2.5:1 28s:18s) and the absence of low molecular weight RNAs that would be indicative of degradation products. Samples are controlled against genomic DNA contamination by RTQ PCR reactions run in the absence of reverse transcriptase using probe and primer sets designed to amplify across the span of a single exon.
First, the RNA samples were normalized to reference nucleic acids such as constitutively expressed genes (for example, β-actin and GAPDH). Normalized RNA (5 ul) was converted to cDNA and analyzed by RTQ-PCR using One Step RT-PCR Master Mix Reagents (Applied Biosystems; Catalog No. 4309169) and gene-specific primers according to the manufacturer's instructions.
In other cases, non-normalized RNA samples were converted to single strand cDNA (sscDNA) using Superscript II (Invitrogen Corporation; Catalog No. 18064-147) and random hexamers according to the manufacturer's instructions. Reactions containing up to 10 μg of total RNA were performed in a volume of 20 μl and incubated for 60 minutes at 42° C. This reaction can be scaled up to 50 μg of total RNA in a final volume of 100 μl. sscDNA samples are then normalized to reference nucleic acids as described previously, using 1×TaqMan® Universal Master mix (Applied Biosystems; catalog No. 4324020), following the manufacturer's instructions.
Probes and primers were designed for each assay according to Applied Biosystems Primer Express Software package (version I for Apple Computer's Macintosh Power PC) or a similar algorithm using the target sequence as input. Default settings were used for reaction conditions and the following parameters were set before selecting primers: primer concentration=250 nM, primer melting temperature (Tm) range=58°-60° C., primer optimal Tm=59° C., maximum primer difference=2° C., probe does not have 5′G, probe Tm must be 10° C. greater than primer Tm, amplicon size 75 bp to 100 bp. The probes and primers selected (see below) were synthesized by Synthegen (Houston, Tex., USA). Probes were double purified by HPLC to remove uncoupled dye and evaluated by mass spectroscopy to verify coupling of reporter and quencher dyes to the 5′ and 3′ ends of the probe, respectively. Their final concentrations were: forward and reverse primers, 900 nM each, and probe, 200 nM.
PCR conditions: When working with RNA samples, normalized RNA from each tissue and each cell line was spotted in each well of either a 96 well or a 384-well PCR plate (Applied Biosystems). PCR cocktails included either a single gene specific probe and primers set, or two multiplexed probe and primers sets (a set specific for the target clone and another gene-specific set multiplexed with the target probe). PCR reactions were set up using TaqMan® One-Step RT-PCR Master Mix (Applied Biosystems, Catalog No. 4313803) following manufacturer's instructions. Reverse transcription was performed at 48° C. for 30 minutes followed by amplification/PCR cycles as follows: 95° C. 10 min, then 40 cycles of 95° C. for 15 seconds, 60° C. for 1 minute. Results were recorded as CT values (cycle at which a given sample crosses a threshold level of fluorescence) using a log scale, with the difference in RNA concentration between a given sample and the sample with the lowest CT value being represented as 2 to the power of delta CT. The percent relative expression is then obtained by taking the reciprocal of this RNA difference and multiplying by 100.
When working with sscDNA samples, normalized sscDNA was used as described previously for RNA samples. PCR reactions containing one or two sets of probe and primers were set up as described previously, using 1×TaqMan® Universal Master mix (Applied Biosystems; catalog No. 4324020), following the manufacturer's instructions. PCR amplification was performed as follows: 95° C. 10 min, then 40 cycles of 95° C. for 15 seconds, 60° C. for 1 minute. Results were analyzed and processed as described previously.
Panels 1, 1.1, 1.2, and 1.3D
The plates for Panels 1, 1.1, 1.2 and 1.3D include 2 control wells (genomic DNA control and chemistry control) and 94 wells containing cDNA from various samples. The samples in these panels are broken into 2 classes: samples derived from cultured cell lines and samples derived from primary normal tissues. The cell lines are derived from cancers of the following types: lung cancer, breast cancer, melanoma, colon cancer, prostate cancer, CNS cancer, squamous cell carcinoma, ovarian cancer, liver cancer, renal cancer, gastric cancer and pancreatic cancer. Cell lines used in these panels are widely available through the American Type Culture Collection (ATCC), a repository for cultured cell lines, and were cultured using the conditions recommended by the ATCC. The normal tissues found on these panels are comprised of samples derived from all major organ systems from single adult individuals or fetuses. These samples are derived from the following organs: adult skeletal muscle, fetal skeletal muscle, adult heart, fetal heart, adult kidney, fetal kidney, adult liver, fetal liver, adult lung, fetal lung, various regions of the brain, the spleen, bone marrow, lymph node, pancreas, salivary gland, pituitary gland, adrenal gland, spinal cord, thymus, stomach, small intestine, colon, bladder, trachea, breast, ovary, uterus, placenta, prostate, testis and adipose.
In the results for Panels 1, 1.1, 1.2 and 1.3D, the following abbreviations are used:
ca.=carcinoma,
*=established from metastasis,
met=metastasis,
s cell var=small cell variant,
non-s=non-sm=non-small,
squam=squamous,
pl. eff=pl effusion=pleural effusion,
glio=glioma,
astro=astrocytoma, and
neuro=neuroblastoma.
General_screening_panel_v1.4
The plates for Panel 1.4 include 2 control wells (genomic DNA control and chemistry control) and 94 wells containing cDNA from various samples. The samples in Panel 1.4 are broken into 2 classes: samples derived from cultured cell lines and samples derived from primary normal tissues. The cell lines are derived from cancers of the following types: lung cancer, breast cancer, melanoma, colon cancer, prostate cancer, CNS cancer, squamous cell carcinoma, ovarian cancer, liver cancer, renal cancer, gastric cancer and pancreatic cancer. Cell lines used in Panel 1.4 are widely available through the American Type Culture Collection (ATCC), a repository for cultured cell lines, and were cultured using the conditions recommended by the ATCC. The normal tissues found on Panel 1.4 are comprised of pools of samples derived from all major organ systems from 2 to 5 different adult individuals or fetuses. These samples are derived from the following organs: adult skeletal muscle, fetal skeletal muscle, adult heart, fetal heart, adult kidney, fetal kidney, adult liver, fetal liver, adult lung, fetal lung, various regions of the brain, the spleen, bone marrow, lymph node, pancreas, salivary gland, pituitary gland, adrenal gland, spinal cord, thymus, stomach, small intestine, colon, bladder, trachea, breast, ovary, uterus, placenta, prostate, testis and adipose. Abbreviations are as described for Panels 1, 1.1, 1.2, and 1.3D.
Panels 2D and 2.2
The plates for Panels 2D and 2.2 generally include 2 control wells and 94 test samples composed of RNA or cDNA isolated from human tissue procured by surgeons working in close cooperation with the National Cancer Institute's Cooperative Human Tissue Network (CHTN) or the National Disease Research Initiative (NDRI). The tissues are derived from human malignancies and in cases where indicated many malignant tissues have “matched margins” obtained from noncancerous tissue just adjacent to the tumor. These are termed normal adjacent tissues and are denoted “NAT” in the results below. The tumor tissue and the “matched margins” are evaluated by two independent pathologists (the surgical pathologists and again by a pathologist at NDRI or CHTN). This analysis provides a gross histopathological assessment of tumor differentiation grade. Moreover, most samples include the original surgical pathology report that provides information regarding the clinical stage of the patient. These matched margins are taken from the tissue surrounding (i.e. immediately proximal) to the zone of surgery (designated “NAT”, for normal adjacent tissue, in Table RR). In addition, RNA and cDNA samples were obtained from various human tissues derived from autopsies performed on elderly people or sudden death victims (accidents, etc.). These tissues were ascertained to be free of disease and were purchased from various commercial sources such as Clontech (Palo Alto, Calif.), Research Genetics, and Invitrogen.
Panel 3D
The plates of Panel 3D are comprised of 94 cDNA samples and two control samples. Specifically, 92 of these samples are derived from cultured human cancer cell lines, 2 samples of human primary cerebellar tissue and 2 controls. The human cell lines are generally obtained from ATCC (American Type Culture Collection), NCI or the German tumor cell bank and fall into the following tissue groups: Squamous cell carcinoma of the tongue, breast cancer, prostate cancer, melanoma, epidermoid carcinoma, sarcomas, bladder carcinomas, pancreatic cancers, kidney cancers, leukemias/lymphomas, ovarian/uterine/cervical, gastric, colon, lung and CNS cancer cell lines. In addition, there are two independent samples of cerebellum. These cells are all cultured under standard recommended conditions and RNA extracted using the standard procedures. The cell lines in panel 3D and 1.3D are of the most common cell lines used in the scientific literature.
Panels 4D, 4R, and 4.1D
Panel 4 includes samples on a 96 well plate (2 control wells, 94 test samples) composed of RNA (Panel 4R) or cDNA (Panels 4D/4.1D) isolated from various human cell lines or tissues related to inflammatory conditions. Total RNA from control normal tissues such as colon and lung (Stratagene, La Jolla, Calif.) and thymus and kidney (Clontech) was employed. Total RNA from liver tissue from cirrhosis patients and kidney from lupus patients was obtained from BioChain (Biochain Institute, Inc., Hayward, Calif.). Intestinal tissue for RNA preparation from patients diagnosed as having Crohn's disease and ulcerative colitis was obtained from the National Disease Research Interchange (NDRI) (Philadelphia, Pa.).
Astrocytes, lung fibroblasts, dermal fibroblasts, coronary artery smooth muscle cells, small airway epithelium, bronchial epithelium, microvascular dermal endothelial cells, microvascular lung endothelial cells, human pulmonary aortic endothelial cells, human umbilical vein endothelial cells were all purchased from Clonetics (Walkersville, Md.) and grown in the media supplied for these cell types by Clonetics. These primary cell types were activated with various cytokines or combinations of cytokines for 6 and/or 12-14 hours, as indicated. The following cytokines were used; IL-1 beta at approximately 1-5 ng/ml, TNF alpha at approximately 5-10 ng/ml, IFN gamma at approximately 20-50 ng/ml, IL-4 at approximately 5-10 ng/ml, IL-9 at approximately 5-10 ng/ml, IL-13 at approximately 5-10 ng/ml. Endothelial cells were sometimes starved for various times by culture in the basal media from Clonetics with 0.1% serum.
Mononuclear cells were prepared from blood of employees at CuraGen Corporation, using Ficoll. LAK cells were prepared from these cells by culture in DMEM 5% FCS (Hyclone), 100 μM non essential amino acids (Gibco/Life Technologies, Rockville, Md.), 1 mM sodium pyruvate (Gibco), mercaptoethanol 5.5×10−5M (Gibco), and 10 mM Hepes (Gibco) and Interleukin 2 for 4-6 days. Cells were then either activated with 10-20 ng/ml PMA and 1-2 μg/ml ionomycin, IL-12 at 5-10 ng/ml, IFN gamma at 20-50 ng/ml and IL-18 at 5-10 ng/ml for 6 hours. In some cases, mononuclear cells were cultured for 4-5 days in DMEM 5% FCS (Hyclone), 100 μM non essential amino acids (Gibco), 1 mM sodium pyruvate (Gibco), mercaptoethanol 5.5×10−5M (Gibco), and 10 mM Hepes (Gibco) with PHA (phytohemagglutinin) or PWM (pokeweed mitogen) at approximately 5 μg/ml. Samples were taken at 24, 48 and 72 hours for RNA preparation. MLR (mixed lymphocyte reaction) samples were obtained by taking blood from two donors, isolating the mononuclear cells using Ficoll and mixing the isolated mononuclear cells 1:1 at a final concentration of approximately 2×106 cells/ml in DMEM 5% FCS (Hyclone), 100 μM non essential amino acids (Gibco), 1 mM sodium pyruvate (Gibco), mercaptoethanol (5.5×10−5M) (Gibco), and 10 mM Hepes (Gibco). The MLR was cultured and samples taken at various time points ranging from 1-7 days for RNA preparation.
Monocytes were isolated from mononuclear cells using CD14 Miltenyi Beads, +ve VS selection columns and a Vario Magnet according to the manufacturer's instructions. Monocytes were differentiated into dendritic cells by culture in DMEM 5% fetal calf serum (FCS) (Hyclone, Logan, Utah), 100 μM non essential amino acids (Gibco), 1 mM sodium pyruvate (Gibco), mercaptoethanol 5.5×10−5M (Gibco), and 10 mM Hepes (Gibco), 50 ng/ml GMCSF and 5 ng/ml IL-4 for 5-7 days. Macrophages were prepared by culture of monocytes for 5-7 days in DMEM 5% FCS (Hyclone), 100 μM non essential amino acids (Gibco), 1 mM sodium pyruvate (Gibco), mercaptoethanol 5.5×10−5M (Gibco), 10 mM Hepes (Gibco) and 10% AB Human Serum or MCSF at approximately 50 ng/ml. Monocytes, macrophages and dendritic cells were stimulated for 6 and 12-14 hours with lipopolysaccharide (LPS) at 100 ng/ml. Dendritic cells were also stimulated with anti-CD40 monoclonal antibody (Pharmingen) at 10 μg/ml for 6 and 12-14 hours.
CD4 lymphocytes, CD8 lymphocytes and NK cells were also isolated from mononuclear cells using CD4, CD8 and CD56 Miltenyi beads, positive VS selection columns and a Vario Magnet according to the manufacturer's instructions. CD45RA and CD45RO CD4 lymphocytes were isolated by depleting mononuclear cells of CD8, CD56, CD14 and CD19 cells using CD8, CD56, CD14 and CD19 Miltenyi beads and positive selection. CD45RO beads were then used to isolate the CD45RO CD4 lymphocytes with the remaining cells being CD45RA CD4 lymphocytes. CD45RA CD4, CD45RO CD4 and CD8 lymphocytes were placed in DMEM 5% FCS (Hyclone), 100 μM non essential amino acids (Gibco), 1 mM sodium pyruvate (Gibco), mercaptoethanol 5.5×10−5M (Gibco), and 10 mM Hepes (Gibco) and plated at 106 cells/ml onto Falcon 6 well tissue culture plates that had been coated overnight with 0.5 μg/ml anti-CD28 (Pharmingen) and 3 ug/ml anti-CD3 (OKT3, ATCC) in PBS. After 6 and 24 hours, the cells were harvested for RNA preparation. To prepare chronically activated CD8 lymphocytes, we activated the isolated CD8 lymphocytes for 4 days on anti-CD28 and anti-CD3 coated plates and then harvested the cells and expanded them in DMEM 5% FCS (Hyclone), 100 μM non essential amino acids (Gibco), 1 mM sodium pyruvate (Gibco), mercaptoethanol 5.5×10−5M (Gibco), and 10 mM Hepes (Gibco) and IL-2. The expanded CD8 cells were then activated again with plate bound anti-CD3 and anti-CD28 for 4 days and expanded as before. RNA was isolated 6 and 24 hours after the second activation and after 4 days of the second expansion culture. The isolated NK cells were cultured in DMEM 5% FCS (Hyclone), 100 μM non essential amino acids (Gibco), 1 mM sodium pyruvate (Gibco), mercaptoethanol 5.5×10−5M (Gibco), and 10 mM Hepes (Gibco) and IL-2 for 4-6 days before RNA was prepared.
To obtain B cells, tonsils were procured from NDRI. The tonsil was cut up with sterile dissecting scissors and then passed through a sieve. Tonsil cells were then spun down and resupended at 106 cells/ml in DMEM 5% FCS (Hyclone), 100 μM non essential amino acids (Gibco), 1 mM sodium pyruvate (Gibco), mercaptoethanol 5.5×10−5M (Gibco), and 10 mM Hepes (Gibco). To activate the cells, we used PWM at 5 μg/ml or anti-CD40 (Pharmingen) at approximately 10 μg/ml and IL-4 at 5-10 ng/ml. Cells were harvested for RNA preparation at 24, 48 and 72 hours.
To prepare the primary and secondary Th1/Th2 and Tr1 cells, six-well Falcon plates were coated overnight with 10 μg/ml anti-CD28 (Pharmingen) and 2 μg/ml OKT3 (ATCC), and then washed twice with PBS. Umbilical cord blood CD4 lymphocytes (Poietic Systems, German Town, Md.) were cultured at 105-106 cells/ml in DMEM 5% FCS (Hyclone), 100 μM non essential amino acids (Gibco), 1 mM sodium pyruvate (Gibco), mercaptoethanol 5.5×10−5M (Gibco), 10 mM Hepes (Gibco) and IL-2 (4 ng/ml). IL-12 (5 ng/ml) and anti-IL4 (1 μg/ml) were used to direct to Th1, while IL-4 (5 ng/ml) and anti-IFN gamma (1 μg/ml) were used to direct to Th2 and IL-10 at 5 ng/ml was used to direct to Tr1. After 4-5 days, the activated Th1, Th2 and Tr1 lymphocytes were washed once in DMEM and expanded for 4-7 days in DMEM 5% FCS (Hyclone), 100 μM non essential amino acids (Gibco), 1 mM sodium pyruvate (Gibco), mercaptoethanol 5.5×10−5M (Gibco), 10 mM Hepes (Gibco) and IL-2 (1 ng/ml). Following this, the activated Th1, Th2 and Tr1 lymphocytes were re-stimulated for 5 days with anti-CD28/OKT3 and cytokines as described above, but with the addition of anti-CD95L (1 μg/ml) to prevent apoptosis. After 4-5 days, the Th1, Th2 and Tr1 lymphocytes were washed and then expanded again with IL-2 for 4-7 days. Activated Th1 and Th2 lymphocytes were maintained in this way for a maximum of three cycles. RNA was prepared from primary and secondary Th1, Th2 and Tr1 after 6 and 24 hours following the second and third activations with plate bound anti-CD3 and anti-CD28 mAbs and 4 days into the second and third expansion cultures in Interleukin 2.
The following leukocyte cells lines were obtained from the ATCC: Ramos, EOL-1, KU-812. EOL cells were further differentiated by culture in 0.1 mM dbcAMP at 5×105 cells/ml for 8 days, changing the media every 3 days and adjusting the cell concentration to 5×105 cells/ml. For the culture of these cells, we used DMEM or RPMI (as recommended by the ATCC), with the addition of 5% FCS (Hyclone), 100 μM non essential amino acids (Gibco), 1 mM sodium pyruvate (Gibco), mercaptoethanol 5.5×10−5M (Gibco), 10 mM Hepes (Gibco). RNA was either prepared from resting cells or cells activated with PMA at 10 ng/ml and ionomycin at 1 μg/ml for 6 and 14 hours. Keratinocyte line CCD106 and an airway epithelial tumor line NCI-H292 were also obtained from the ATCC. Both were cultured in DMEM 5% FCS (Hyclone), 100 μM non essential amino acids (Gibco), 1 mM sodium pyruvate (Gibco), mercaptoethanol 5.5×10−5M (Gibco), and 10 mM Hepes (Gibco). CCD1106 cells were activated for 6 and 14 hours with approximately 5 ng/ml TNF alpha and 1 ng/ml IL-1 beta, while NCI-H292 cells were activated for 6 and 14 hours with the following cytokines: 5 ng/ml IL-4, 5 ng/ml IL-9, 5 ng/ml IL-13 and 25 ng/ml IFN gamma.
For these cell lines and blood cells, RNA was prepared by lysing approximately 107 cells/ml using Trizol (Gibco BRL). Briefly, 1/10 volume of bromochloropropane (Molecular Research Corporation) was added to the RNA sample, vortexed and after 10 minutes at room temperature, the tubes were spun at 14,000 rpm in a Sorvall SS34 rotor. The aqueous phase was removed and placed in a 15 ml Falcon Tube. An equal volume of isopropanol was added and left at −20° C. overnight. The precipitated RNA was spun down at 9,000 rpm for 15 min in a Sorvall SS34 rotor and washed in 70% ethanol. The pellet was redissolved in 300 μl of RNAse-free water and 35 μl buffer (Promega) 5 μl DTT, 7 μl RNAsin and 8 μl DNAse were added. The tube was incubated at 37° C. for 30 minutes to remove contaminating genomic DNA, extracted once with phenol chloroform and re-precipitated with 1/10 volume of 3M sodium acetate and 2 volumes of 100% ethanol. The RNA was spun down and placed in RNAse free water. RNA was stored at −80° C.
AI_comprehensive panel_v1.0
The plates for AI_comprehensive panel_v1.0 include two control wells and 89 test samples comprised of cDNA isolated from surgical and postmortem human tissues obtained from the Backus Hospital and Clinomics (Frederick, Md.). Total RNA was extracted from tissue samples from the Backus Hospital in the Facility at CuraGen. Total RNA from other tissues was obtained from Clinomics.
Joint tissues including synovial fluid, synovium, bone and cartilage were obtained from patients undergoing total knee or hip replacement surgery at the Backus Hospital. Tissue samples were immediately snap frozen in liquid nitrogen to ensure that isolated RNA was of optimal quality and not degraded. Additional samples of osteoarthritis and rheumatoid arthritis joint tissues were obtained from Clinomics. Normal control tissues were supplied by Clinomics and were obtained during autopsy of trauma victims.
Surgical specimens of psoriatic tissues and adjacent matched tissues were provided as total RNA by Clinomics. Two male and two female patients were selected between the ages of 25 and 47. None of the patients were taking prescription drugs at the time samples were isolated.
Surgical specimens of diseased colon from patients with ulcerative colitis and Crohns disease and adjacent matched tissues were obtained from Clinomics. Bowel tissue from three female and three male Crohn's patients between the ages of 41-69 were used. Two patients were not on prescription medication while the others were taking dexamethasone, phenobarbital, or tylenol. Ulcerative colitis tissue was from three male and four female patients. Four of the patients were taking lebvid and two were on phenobarbital.
Total RNA from post mortem lung tissue from trauma victims with no disease or with emphysema, asthma or COPD was purchased from Clinomics. Emphysema patients ranged in age from 40-70 and all were smokers, this age range was chosen to focus on patients with cigarette-linked emphysema and to avoid those patients with alpha-1 anti-trypsin deficiencies. Asthma patients ranged in age from 36-75, and excluded smokers to prevent those patients that could also have COPD. COPD patients ranged in age from 35-80 and included both smokers and non-smokers. Most patients were taking corticosteroids, and bronchodilators.
In the labels employed to identify tissues in the AI_comprehensive panel_v1.0 panel, the following abbreviations are used:
AI=Autoimmunity
Syn=Synovial
Normal=No apparent disease
Rep22 /Rep20=individual patients
RA=Rheumatoid arthritis
Backus=From Backus Hospital
OA=Osteoarthritis
(SS) (BA) (MF)=Individual patients
Adj=Adjacent tissue
Match control=adjacent tissues
-M=Male
-F=Female
COPD=Chronic obstructive pulmonary disease
Panels 5D and 5I
The plates for Panel 5D and 5I include two control wells and a variety of cDNAs isolated from human tissues and cell lines with an emphasis on metabolic diseases. Metabolic tissues were obtained from patients enrolled in the Gestational Diabetes study. Cells were obtained during different stages in the differentiation of adipocytes from human mesenchymal stem cells. Human pancreatic islets were also obtained.
In the Gestational Diabetes study subjects are young (18-40 years), otherwise healthy women with and without gestational diabetes undergoing routine (elective) Caesarean section. After delivery of the infant, when the surgical incisions were being repaired/closed, the obstetrician removed a small sample (<1 cc) of the exposed metabolic tissues during the closure of each surgical level. The biopsy material was rinsed in sterile saline, blotted and fast frozen within 5 minutes from the time of removal. The tissue was then flash frozen in liquid nitrogen and stored, individually, in sterile screw-top tubes and kept on dry ice for shipment to or to be picked up by CuraGen. The metabolic tissues of interest include uterine wall (smooth muscle), visceral adipose, skeletal muscle (rectus) and subcutaneous adipose. Patient descriptions are as follows:
















Patient 2
Diabetic Hispanic, overweight, not on insulin



Patient 7-9
Nondiabetic Caucasian and obese (BMI > 30)



Patient 10
Diabetic Hispanic, overweight, on insulin



Patient 11
Nondiabetic African American and overweight



Patient 12
Diabetic Hispanic on insulin










Adipocyte differentiation was induced in donor progenitor cells obtained from Osirus (a division of Clonetics/BioWhittaker) in triplicate, except for Donor 3U which had only two replicates. Scientists at Clonetics isolated, grew and differentiated human mesenchymal stem cells (HuMSCs) for CuraGen based on the published protocol found in Mark F. Pittenger, et al., Multilineage Potential of Adult Human Mesenchymal Stem Cells Science Apr. 2, 1999: 143-147. Clonetics provided Trizol lysates or frozen pellets suitable for mRNA isolation and ds cDNA production. A general description of each donor is as follows:
Donor 2 and 3 U: Mesenchymal Stem cells, Undifferentiated Adipose
Donor 2 and 3 AM: Adipose, AdiposeMidway Differentiated
Donor 2 and 3 AD: Adipose, Adipose Differentiated
Human cell lines were generally obtained from ATCC (American Type Culture Collection), NCI or the German tumor cell bank and fall into the following tissue groups: kidney proximal convoluted tubule, uterine smooth muscle cells, small intestine, liver HepG2 cancer cells, heart primary stromal cells, and adrenal cortical adenoma cells. These cells are all cultured under standard recommended conditions and RNA extracted using the standard procedures. All samples were processed at CuraGen to produce single stranded cDNA.
Panel 5I contains all samples previously described with the addition of pancreatic islets from a 58 year old female patient obtained from the Diabetes Research Institute at the University of Miami School of Medicine. Islet tissue was processed to total RNA at an outside source and delivered to CuraGen for addition to panel 5I.
In the labels employed to identify tissues in the 5D and 5I panels, the following abbreviations are used:
GO Adipose=Greater Omentum Adipose
SK=Skeletal Muscle
UT=Uterus
PL=Placenta
AD=Adipose Differentiated
AM=Adipose Midway Differentiated
U=Undifferentiated Stem Cells
Panel CNSD0.01
The plates for Panel CNSD0.01 include two control wells and 94 test samples comprised of cDNA isolated from postmortem human brain tissue obtained from the Harvard Brain Tissue Resource Center. Brains are removed from calvaria of donors between 4 and 24 hours after death, sectioned by neuroanatomists, and frozen at −80° C. in liquid nitrogen vapor. All brains are sectioned and examined by neuropathologists to confirm diagnoses with clear associated neuropathology.
Disease diagnoses are taken from patient records. The panel contains two brains from each of the following diagnoses: Alzheimer's disease, Parkinson's disease, Huntington's disease, Progressive Supemuclear Palsy, Depression, and “Normal controls”. Within each of these brains, the following regions are represented: cingulate gyrus, temporal pole, globus palladus, substantia nigra, Brodman Area 4 (primary motor strip), Brodman Area 7 (parietal cortex), Brodman Area 9 (prefrontal cortex), and Brodman area 17 (occipital cortex). Not all brain regions are represented in all cases; e.g., Huntington's disease is characterized in part by neurodegeneration in the globus palladus, thus this region is impossible to obtain from confirmed Huntington's cases. Likewise Parkinson's disease is characterized by degeneration of the substantia nigra making this region more difficult to obtain. Normal control brains were examined for neuropathology and found to be free of any pathology consistent with neurodegeneration.
In the labels employed to identify tissues in the CNS panel, the following abbreviations are used:
PSP=Progressive supranuclear palsy
Sub Nigra=Substantia nigra
Glob Palladus=Globus palladus
Temp Pole=Temporal pole
Cing Gyr=Cingulate gyrus
BA 4=Brodman Area 4
Panel CNS_Neurodegeneration_V1.0
The plates for Panel CNS_Neurodegeneration_V1.0 include two control wells and 47 test samples comprised of cDNA isolated from postmortem human brain tissue obtained from the Harvard Brain Tissue Resource Center (McLean Hospital) and the Human Brain and Spinal Fluid Resource Center (VA Greater Los Angeles Healthcare System). Brains are removed from calvaria of donors between 4 and 24 hours after death, sectioned by neuroanatomists, and frozen at −80° C. in liquid nitrogen vapor. All brains are sectioned and examined by neuropathologists to confirm diagnoses with clear associated neuropathology.
Disease diagnoses are taken from patient records. The panel contains six brains from Alzheimer's disease (AD) patients, and eight brains from “Normal controls” who showed no evidence of dementia prior to death. The eight normal control brains are divided into two categories: Controls with no dementia and no Alzheimer's like pathology (Controls) and controls with no dementia but evidence of severe Alzheimer's like pathology, (specifically senile plaque load rated as level 3 on a scale of 0-3; 0=no evidence of plaques, 3=severe AD senile plaque load). Within each of these brains, the following regions are represented: hippocampus, temporal cortex (Brodman Area 21), parietal cortex (Brodman area 7), and occipital cortex (Brodman area 17). These regions were chosen to encompass all levels of neurodegeneration in AD. The hippocampus is a region of early and severe neuronal loss in AD; the temporal cortex is known to show neurodegeneration in AD after the hippocampus; the parietal cortex shows moderate neuronal death in the late stages of the disease; the occipital cortex is spared in AD and therefore acts as a “control” region within AD patients. Not all brain regions are represented in all cases.
In the labels employed to identify tissues in the CNS_Neurodegeneration_V1.0 panel, the following abbreviations are used:

    
    
        AD=Alzheimer's disease brain; patient was demented and showed AD-like pathology upon autopsy
        Control=Control brains; patient not demented, showing no neuropathology
        Control (Path)=Control brains; pateint not demented but showing sever AD-like pathology
        SupTemporal Ctx=Superior Temporal Cortex
        Inf Temporal Ctx=Inferior Temporal Cortex

A. NOV1: Androgen-Regulated Short-Chain Dehydrogenase/Reductase

    
    


Expression of gene CG57413-01 was assessed using the primer-probe set Ag3230, described in Table AA. Results of the RTQ-PCR runs are shown in Tables AB, AC, AD and AE.







TABLE AA







Probe Name Ag3230













Start


Primers
Sequences
Length  Position














Forward
5′-gatggtacgtgatgctcttgtt-3′
(SEQ ID NO:293)
22
10


Probe
TET-5′-cccttgccgataggaagttctttgct-3′-TAMRA
(SEQ ID NO:294)
26
33


Reverse
5′-aggaagctgcacatttgttcta-3′
(SEQ ID NO:295)
22
70
















TABLE AB







CNS_neurodegeneration_v1.0











Rel. Exp. (%) Ag3230, Run

Rel. Exp. (%) Ag3230, Run


Tissue Name
206533576
Tissue Name
206533576













AD 1 Hippo
0.0
Control (Path) 3
0.0




Temporal Ctx


AD 2 Hippo
0.0
Control (Path) 4
54.7




Temporal Ctx


AD 3 Hippo
8.7
AD 1 Occipital Ctx
18.2


AD 4 Hippo
0.0
AD 2 Occipital Ctx
0.0




(Missing)


AD 5 hippo
32.1
AD 3 Occipital Ctx
0.0


AD 6 Hippo
95.3
AD 4 Occipital Ctx
8.5


Control 2 Hippo
0.0
AD 5 Occipital Ctx
6.6


Control 4 Hippo
9.4
AD 6 Occipital Ctx
22.8


Control (Path) 3 Hippo
21.6
Control 1 Occipital Ctx
0.0


AD 1 Temporal Ctx
8.2
Control 2 Occipital Ctx
36.3


AD 2 Temporal Ctx
4.3
Control 3 Occipital Ctx
0.0


AD 3 Temporal Ctx
0.0
Control 4 Occipital Ctx
0.0


AD 4 Temporal Ctx
8.2
Control (Path) 1
100.0




Occipital Ctx


AD 5 Inf Temporal Ctx
32.5
Control (Path) 2
15.3




Occipital Ctx


AD 5 SupTemporal Ctx
15.2
Control (Path) 3
0.0




Occipital Ctx


AD 6 Inf Temporal Ctx
66.9
Control (Path) 4
25.5




Occipital Ctx


AD 6 Sup Temporal Ctx
67.8
Control 1 Parietal Ctx
0.0


Control 1 Temporal Ctx
0.0
Control 2 Parietal Ctx
5.6


Control 2 Temporal Ctx
18.0
Control 3 Parietal Ctx
20.4


Control 3 Temporal Ctx
8.1
Control (Path) 1
37.9




Parietal Ctx


Control 4 Temporal Ctx
17.7
Control (Path) 2
7.5




Parietal Ctx


Control (Path) 1
14.8
Control (Path) 3
0.0


Temporal Ctx

Parietal Ctx


Control (Path) 2
34.2
Control (Path) 4
43.8


Temporal Ctx

Parietal Ctx
















TABLE AC







Panel 1.3D











Rel. Exp. (%) Ag3230, Run

Rel. Exp. (%) Ag3230, Run


Tissue Name
165524130
Tissue Name
165524130













Liver adenocarcinoma
0.0
Kidney (fetal)
0.0


Pancreas
0.0
Renal ca. 786-0
9.8


Pancreatic ca. CAPAN 2
0.0
Renal ca. A498
0.0


Adrenal gland
0.0
Renal ca. RXF 393
0.0


Thyroid
0.0
Renal ca. ACHN
0.0


Salivary gland
0.0
Renal ca. UO-31
0.0


Pituitary gland
11.1
Renal ca. TK-10
0.0


Brain (fetal)
0.0
Liver
0.0


Brain (whole)
20.0
Liver (fetal)
0.0


Brain (amygdala)
33.9
Liver ca. (hepatoblast)
18.0




HepG2


Brain (cerebellum)
0.0
Lung
0.0


Brain (hippocampus)
16.4
Lung (fetal)
0.0


Brain (substantia nigra)
14.1
Lung ca. (small cell)
17.9




LX-1


Brain (thalamus)
0.0
Lung ca. (small cell)
0.0




NCI-H69


Cerebral Cortex
22.2
Lung ca. (s. cell var.)
0.0




SHP-77


Spinal cord
0.0
Lung ca. (large cell)
0.0




NCI-H460


glio/astro U87-MG
0.0
Lung ca. (non-sm. cell)
0.0




A549


glio/astro U-118-MG
0.0
Lung ca. (non-s. cell)
0.0




NCI-H23


astrocytoma SW1783
0.0
Lung ca. (non-s. cell)
0.0




HOP-62


neuro*; met SK-N-AS
0.0
Lung ca. (non-s. cl) NCI-
0.0




H522


astrocytoma SF-539
0.0
Lung ca. (squam.) SW
0.0




900


astrocytoma SNB-75
13.8
Lung ca. (squam.) NCI-
0.0




H596


glioma SNB-19
0.0
Mammary gland
0.0


glioma U251
100.0
Breast ca.* (pl. ef)
12.7




MCF-7


glioma SF-295
0.0
Breast ca.* (pl. ef)
0.0




MDA-MB-231


Heart (fetal)
0.0
Breast ca.* (pl. ef) T47D
0.0


Heart
0.0
Breast ca. BT-549
0.0


Skeletal muscle (fetal)
0.0
Breast ca. MDA-N
0.0


Skeletal muscle
0.0
Ovary
0.0


Bone marrow
0.0
Ovarian ca. OVCAR-3
0.0


Thymus
0.0
Ovarian ca. OVCAR-4
0.0


Spleen
0.0
Ovarian ca. OVCAR-5
0.0


Lymph node
39.5
Ovarian ca. OVCAR-8
0.0


Colorectal
43.2
Ovarian ca. IGROV-1
0.0


Stomach
43.5
Ovarian ca.* (ascites)
36.6




SK-OV-3


Small intestine
38.7
Uterus
57.8


Colon ca. SW480
0.0
Placenta
0.0


Colon ca.* SW620 (SW480
0.0
Prostate
38.2


met)


Colon ca. HT29
0.0
Prostate ca.* (bone
0.0




met) PC-3


Colon ca. HCT-116
0.0
Testis
17.9


Colon ca. CaCo-2
13.4
Melanoma Hs688 (A).T
0.0


Colon ca.
0.0
Melanoma* (met)
0.0


tissue (ODO3866)

Hs688 (B).T


Colon ca. HCC-2998
14.3
Melanoma UACC-62
0.0


Gastric ca.* (liver met)
79.6
Melanoma M14
18.6


NCI-N87


Bladder
26.8
Melanoma LOX IMVI
0.0


Trachea
0.0
Melanoma* (met) SK-
0.0




MEL-5


Kidney
0.0
Adipose
0.0
















TABLE AD







Panel 2.2











Rel. Exp. (%) Ag3230,

Rel. Exp. (%) Ag3230,


Tissue Name
Run 174442812
Tissue Name
Run 174442812













Normal Colon
18.2
Kidney Margin (OD04348)
38.4


Colon cancer (OD06064)
0.0
Kidney malignant cancer
23.2




(OD06204B)


Colon Margin (OD06064)
0.0
Kidney normal adjacent
0.0




tissue (OD06204E)


Colon cancer (OD06159)
0.0
Kidney Cancer (OD04450-
0.0




01)


Colon Margin (OD06159)
0.0
Kidney Margin (OD04450-
0.0




03)


Colon cancer (OD06297-04)
0.0
Kidney Cancer 8120613
22.7


Colon Margin (OD06297-05)
17.9
Kidney Margin 8120614
0.0


CC Gr. 2 ascend colon
0.0
Kidney Cancer 9010320
0.0


(ODO3921)


CC Margin (ODO3921)
9.0
Kidney Margin 9010321
0.0


Colon cancer metastasis
0.0
Kidney Cancer 8120607
0.0


(OD06104)


Lung Margin (OD06104)
0.0
Kidney Margin 8120608
20.3


Colon mets to lung
41.5
Normal Uterus
12.6


(OD04451-01)


Lung Margin (OD04451-02)
19.9
Uterine Cancer 064011
0.0


Normal Prostate
0.0
Normal Thyroid
0.0


Prostate Cancer (OD04410)
12.6
Thyroid Cancer 064010
0.0


Prostate Margin (OD04410)
20.0
Thyroid Cancer A302152
0.0


Normal Ovary
0.0
Thyroid Margin A302153
0.0


Ovarian cancer (OD06283-
0.0
Normal Breast
44.4


03)


Ovarian Margin (OD06283-
0.0
Breast Cancer (OD04566)
0.0


07)


Ovarian Cancer 064008
41.8
Breast Cancer 1024
18.9


Ovarian cancer (OD06145)
0.0
Breast Cancer (OD04590-
0.0




01)


Ovarian Margin (OD06145)
4.7
Breast Cancer Mets
0.0




(OD04590-03)


Ovarian cancer (OD06455-
0.0
Breast Cancer Metastasis
38.2


03)

(OD04655-05)


Ovarian Margin (OD06455-
0.0
Breast Cancer 064006
15.7


07)


Normal Lung
32.1
Breast Cancer 9100266
0.0


Invasive poor diff. lung
0.0
Breast Margin 9100265
0.0


adeno (ODO4945-01


Lung Margin (ODO4945-03)
0.0
Breast Cancer A209073
0.0


Lung Malignant Cancer
0.0
Breast Margin A2090734
20.4


(OD03126)


Lung Margin (OD03126)
0.0
Breast cancer (OD06083)
39.0


Lung Cancer (OD05014A)
0.0
Breast cancer node
45.1




metastasis (OD06083)


Lung Margin (OD05014B)
100.0
Normal Liver
6.0


Lung cancer (OD06081)
0.0
Liver Cancer 1026
0.0


Lung Margin (OD06081)
0.0
Liver Cancer 1025
27.9


Lung Cancer (OD04237-01)
0.0
Liver Cancer 6004-T
20.0


Lung Margin (OD04237-02)
0.0
Liver Tissue 6004-N
0.0


Ocular Melanoma Metastasis
12.1
Liver Cancer 6005-T
0.0


Ocular Melanoma Margin
0.0
Liver Tissue 6005-N
0.0


(Liver)


Melanoma Metastasis
18.8
Liver Cancer 064003
19.2


Melanoma Margin (Lung)
0.0
Normal Bladder
0.0


Normal Kidney
20.4
Bladder Cancer 1023
0.0


Kidney Ca, Nuclear grade 2
82.4
Bladder Cancer A302173
0.0


(OD04338)


Kidney Margin (OD04338)
9.0
Normal Stomach
44.4


Kidney Ca Nuclear grade 1/2
53.2
Gastric Cancer 9060397
0.0


(OD04339)


Kidney Margin (OD04339)
18.3
Stomach Margin 9060396
18.7


Kidney Ca, Clear cell type
38.2
Gastric Cancer 9060395
33.0


(OD04340)


Kidney Margin (OD04340)
0.0
Stomach Margin 9060394
21.0


Kidney Ca, Nuclear grade 3
0.0
Gastric Cancer 064005
0.0


(OD04348)
















TABLE AE







Panel 4D











Rel. Exp. (%) Ag3230,

Rel. Exp. (%) Ag3230,


Tissue Name
Run 164532020
Tissue Name
Run 164532020













Secondary Th1 act
0.0
HUVEC IL-1beta
7.9


Secondary Th2 act
0.0
HUVEC IFN gamma
9.2


Secondary Tr1 act
11.1
HUVEC TNF alpha + IFN
0.0




gamma


Secondary Th1 rest
0.0
HUVEC TNF alpha + IL4
0.0


Secondary Th2 rest
13.9
HUVEC IL-11
4.0


Secondary Tr1 rest
12.2
Lung Microvascular EC none
15.2


Primary Th1 act
7.1
Lung Microvascular EC
15.2




TNF alpha + IL-1beta


Primary Th2 act
17.7
Microvascular Dermal EC none
9.2


Primary Tr1 act
0.0
Microsvasular Dermal EC
7.2




TNF alpha + IL-1beta


Primary Th1 rest
57.0
Bronchial epithelium
15.9




TNF alpha + IL1beta


Primary Th2 rest
11.5
Small airway epithelium none
22.4


Primary Tr1 rest
7.9
Small airway epithelium
100.0




TNF alpha + IL-1beta


CD45RA CD4 lymphocyte
1.9
Coronery artery SMC rest
0.0


act


CD45RO CD4 lymphocyte
25.2
Coronery artery SMC
0.0


act

TNF alpha + IL-1beta


CD8 lymphocyte act
7.1
Astrocytes rest
0.0


Secondary CD8
53.6
Astrocytes TNF alpha + IL-1beta
0.0


lymphocyte rest


Secondary CD8
0.0
KU-812 (Basophil) rest
0.0


lymphocyte act


CD4 lymphocyte none
41.8
KU-812 (Basophil)
58.2




PMA/ionomycin


2ry Th1/Th2/Tr1_anti-
7.0
CCD1106 (Keratinocytes) none
0.0


CD95 CH11


LAK cells rest
24.8
CCD1106 (Keratinocytes)
0.0




TNF alpha + IL-1beta


LAK cells IL-2
32.5
Liver cirrhosis
51.1


LAK cells IL-2 + IL-12
22.2
Lupus kidney
0.0


LAK cells IL-2 + IFN
27.4
NCI-H292 none
0.0


gamma


LAK cells IL-2 + IL-18
15.3
NCI-H292 IL-4
31.9


LAK cells
0.0
NCI-H292 IL-9
12.3


PMA/ionomycin


NK Cells IL-2 rest
12.8
NCI-H292 IL-13
0.0


Two Way MLR 3 day
28.9
NCI-H292 IFN gamma
0.0


Two Way MLR 5 day
0.0
HPAEC none
0.0


Two Way MLR 7 day
27.5
HPAEC TNF alpha + IL-1beta
3.2


PBMC rest
9.4
Lung fibroblast none
20.9


PBMC PWM
21.8
Lung fibroblast TNF alpha + IL-
14.9




1beta


PBMC PHA-L
7.9
Lung fibroblast IL-4
13.2


Ramos (B cell) none
0.0
Lung fibroblast IL-9
0.0


Ramos (B cell) ionomycin
0.0
Lung fibroblast IL-13
0.0


B lymphocytes PWM
25.9
Lung fibroblast IFN gamma
0.0


B lymphocytes CD40L
30.8
Dermal fibroblast CCD1070 rest
0.0


and IL-4


EOL-1 dbcAMP
0.0
Dermal fibroblast CCD1070
0.0




TNF alpha


EOL-1 dbcAMP
0.0
Dermal fibroblast CCD1070 IL-
0.0


PMA/ionomycin

1beta


Dendritic cells none
59.5
Dermal fibroblast IFN gamma
0.0


Dendritic cells LPS
0.0
Dermal fibroblast IL-4
7.7


Dendritic cells anti-CD40
54.3
IBD Colitis 2
0.0


Monocytes rest
49.7
IBD Crohn's
7.1


Monocytes LPS
8.7
Colon
14.4


Macrophages rest
21.6
Lung
16.2


Macrophages LPS
8.0
Thymus
92.7


HUVEC none
0.0
Kidney
0.0


HUVEC starved
0.0









CNS_neurodegeneration_v1.0 Summary: Ag3230 This panel does not show differential expression of the CG57413-01 gene in Alzheimer's disease. However, this expression profile confirms the presence of this gene in the brain. This gene encodes a homolog of an androgen regulated short chain dehydrogenase. Members of this family are known to function in the processing of hormones in the brain. Brain hormone regulation mediates numerous clinically significant conditions, including psychiatric disorders such as anxiety, overeating and memory disorders. Therefore, agents that modulate the activity of this gene product have potential utility in the treatment of these disorders. In addition, steroid treatment is used in a number of clinical conditions including Alzheimer's disease (estrogen), menopause associated symptoms (estrogen), multiple sclerosis (glucocorticoids), and spinal cord injury (methylprednisolone). Treatment with an antagonist of this gene product, or reduction of the levels of this gene product could slow steroid degradation and lower the necessary amount given for therapeutic effect, thus reducing peripheral side effects (Biswas and Russell, J Biol Chem 1997 June 20;272(25):15959-66; Matsumoto et al., Spine 2001 February 15;26(4):426-30; Holinka, Ann N Y Acad Sci 2001 September;943:89-108; Burkman et al., Am J Obstet Gynecol 2001 August;185(2 Suppl):S13-23; Gaillard et al., Neuroreport 2001 July 20;12(10):2189-93; Penning et al., Biochem J 2000 October 1;351(Pt 1):67-77).
Panel 1.3D Summary: Ag3230 Expression of the CG57413-01 gene is restricted to a sample derived from a brain cancer cell line (CT=34.8). Thus, expression of this gene could be used to differentiate between this sample and other samples on this panel and as a marker to detect the presence of brain cancer. Furthermore, therapeutic modulation of the expression or function of this gene may be effective in the treatment of brain cancer.
Panel 2.2 Summary: Ag3230 Expression of the CG57413-01 gene is restricted to a sample derived from a normal lung tissue (CT=34.95). Thus, expression of this gene could be used to differentiate between this sample and other samples on this panel and as a marker of normal lung tissue.
Panel 4D Summary: Ag3230 Highest expression of the CG57413-01 gene is seen in Small airway epithelium stimulated with TNFalpha+IL-1 beta (CT=33.4). Significant expression is also seen in the basophil cell line KU-812 stimulated with PMA/ionomycin. This cell line constitutes a reasonable model for the inflammatory cells that take part in various inflammatory lung diseases. This expression profile in combination with the exclusive expression in normal lung seen in Panel 2D suggests that the protein encoded by this gene may be involved in the proliferation or activation of airway epithelium. Therefore, therapuetics designed with the protein encoded by this transcript could be important in the treatment of pathological and inflammatory lung disorders, including emphysema, allergies, asthma and COPD.
Panel 5 Islet Summary: Ag3230 Expression of the CG57413-01 gene is low/undetectable in all samples on this panel (CTs>35).
B. NOV2a and NOV2b: Aryl-Acylamidase
Expression of gene CG57391-01 and variant CG57391-02 was assessed using the primer-probe sets Ag3053 and Ag3224, described in Tables BA and BB. Results of the RTQ-PCR runs are shown in Table BC.







TABLE BA







Probe Name Ag3053













Start


Primers
Sequences
Length
Position














Forward
5′-accagggatgtagccataaaac-3′
(SEQ ID NO:296)
22
547


Probe
TET-5′-tttcaccaaggatgaagcacttccct-3′-TAMRA
(SEQ ID NO:297)
26
508


Reverse
5′-gcatgtgttggtttcttctcat-3′
(SEQ ID NO:298)
22
481
















TABLE BB







Probe Name Ag3224













Start


Primers
Sequences
Length
Position














Forward
5′-accagggatgtagccataaaac-3′
(SEQ ID NO:299)
22
547


Probe
TET-5′-tttcaccaaggatgaagcacttccct-3′-TAMRA
(SEQ ID NO:300)
26
508


Reverse
5′-gcatgtgttggtttcttctcat-3′
(SEQ ID NO:301)
221
481
















TABLE BC







Panel 1.3D










Rel. Exp.(%)
Rel. Exp.(%)



Ag3053, Run
Ag3224, Run


Tissue Name
167985386
165524086












Liver
0.0
0.0


adenocarcinoma


Pancreas
0.0
0.0


Pancreatic ca.
0.0
0.0


CAPAN 2


Adrenal gland
0.0
0.0


Thyroid
11.3
0.0


Salivary gland
0.0
0.0


Pituitary gland
66.0
12.4


Brain (fetal)
0.0
0.0


Brain (whole)
0.0
0.0


Brain (amygdala)
0.0
0.0


Brain (cerebellum)
0.0
0.0


Brain (hippocampus)
0.0
0.0


Brain (substantia
0.0
0.0


nigra)


Brain (thalamus)
0.0
0.0


Cerebral Cortex
0.0
0.0


Spinal Cord
0.0
4.8


glio/astro U87-MG
0.0
0.0


glio/astro U-118
0.0
0.0


MG


astrocytoma
0.0
0.0


SW1783
0.0
0.0


neuro*; met SK-N-
0.0
0.0


AS


astrocytoma SF-539
0.0
4.6


astrocytoma SNB-57
57.0
0.0


glioma SNB-19
0.0
0.0


glioma U251
0.0
11.8


glioma SF-295
0.0
0.0


Heart (fetal)
0.0
0.0


Heart
0.0
0.0


Skeletal muscle
0.0
0.0


(fetal)


Skeletal muscle
0.0
0.0


Bone marrow
0.0
0.0


Thymus
7.7
0.0


Spleen
0.0
0.0


Lymph node
8.4
13.0


Colorectal
0.0
0.0


Stomach
0.0
6.8


Small intestine
42.9
100.0


Colon ca. SW480
0.0
0.0


Colon ca*
0.0
0.0


SW620 (SW480 met)


Colon ca. HT29
0.0
0.0


Colon ca. HCT-116
0.0
0.0


Colon ca. CaCo-2
7.9
0.0


Colon ca.
5.9
0.0


tissue (ODO3866)


Colon ca. HCC-2998
0.0
5.9


Gastric ca.* (liver
11.4
4.9


met) NCI-N87


Bladder
0.0
0.0


Trachea
24.8
9.6


Kidney
0.0
0.0


Kidney (fetal)
24.8
0.0


Renal ca. 786-0
0.0
0.0


Renal ca. A498
22.4
6.1


Renal ca. RXF
0.0
0.0


Renal ca. ACHN
0.0
0.0


Renal ca. UO-31
0.0
0.0


Renal ca. TK-10
0.0
0.0


Liver
0.0
0.0


Liver (fetal)
0.0
0.0


Liver ca.
0.0
0.0


(hepatoblast)


HepG2


Lung
0.0
0.0


Lung (fetal)
27.5
0.0


Lung ca. (small
9.5
0.0


cell) LX-1


Lung ca. (small
0.0
0.0


cell) NCI-H69


Lung ca (s. cell
0.0
0.0


var.) SHP-77


Lung ca. (large
0.0
0.0


cell)NCI-H460


Lung ca. (non-
100.0
5.8


sm. cell) A549


Lung ca. (non-
9.3
0.0


s. cell) NCI-H23


Lung ca. (non
17.0
0.0


s. cell) HOP-62


Lung ca. (non-
10.7
0.0


s. cl) NCI-H522


Lung ca.
36.3
21.9


(squam.) SW 900


Lung ca.
0.0
0.0


(squam.) NCI-


H596


Mammary gland
0.0
0.0


Breast ca.*
0.0
0.0


(pl. ef) MCF-7


Breast ca.*
0.0
0.0


(pl. ef) MDA-


MB-231


Breast ca.*
0.0
0.0


(pl. ef) T47D


Breast ca. BT-
0.0
4.0


549


Breast ca. MDA-
0.0
0.0


N


Ovary
0.0
0.0


Ovarian ca.
12.8
10.4


OVCAR-3


Ovarian ca.
0.0
0.0


OVCAR-4


Ovarian ca.
20.4
0.0


Ovarian ca.
0.0
0.0


OVCAR-8


Ovarian ca.
0.0
0.0


IGROV-1


Ovarian ca.*
0.0
0.0


(ascites) SK-OV-
0.0
0.0


3


Uterus
0.0
10.7


Placenta
9.7
18.2


Prostate
0.0
0.0


Prostate ca.*
0.0
0.0


(bone met) PC-3


Testis
9.5
4.7


Melanoma
0.0
0.0


Hs688 (A).T


Melanoma*
0.0
0.0


(met)


Hs688 (B).T


Melanoma
0.0
0.0


UACC-62


Melanoma
0.0
0.0


Melanoma LOX
0.0
0.0


IMVI


Melanoma*
0.0
0.0


(met) SK-MEL-5


Adipose
57.8
20.9









Panel 1.3D Summary: Ag3053/Ag3224 Two experiments with the same probe and primer set show expression of the CG57391-01 gene restricted to samples derived from the small intestine and a lung cancer cell line (CTs=33-35). Thus, expression of this gene could be used to differentiate between these samples and other samples on this panel and as a marker for intestinal tissue and lung cancer.
Panel 4D Summary: Ag3053 Expression of the CG57391-01 gene is low/undetectable in all samples on this panel (CTs>35). (Data not shown.)
C. NOV3a: Insulysin
Expression of gene CG57433-01 was assessed using the primer-probe set Ag3225, described in Table CA. Results of the RTQ-PCR runs are shown in Tables CB, CC and CD.







TABLE CA







Probe Name Ag3225













Start


Primers
Sequences
Length
Position














Forward
5′-gggtgtttaggattccctgtag-3′
(SEQ ID NO:302)
22
677


Probe
TET-5′-ccaattgaaagagtctccaggcatca-3′-TAMRA
(SEQ ID NO:303)
26
645


Reverse
5′-tgcagttgattcagaacatgag-3′
(SEQ ID NO:304)
22
609
















TABLE CB







Panel 1.3D











Rel. Exp. (%) Ag3225, Run

Rel. Exp. (%) Ag3225, Run


Tissue Name
165524115
Tissue Name
165524115













Liver adenocarcinoma
29.9
Kidney (fetal)
6.7


Pancreas
7.2
Renal ca. 786-0
27.2


Pancreatic ca. CAPAN 2
31.0
Renal ca. A498
23.5


Adrenal gland
8.7
Renal ca. RXF 393
23.3


Thyroid
10.2
Renal ca. ACHN
20.9


Salivary gland
7.1
Renal ca. UO-31
28.5


Pituitary gland
10.3
Renal ca. TK-10
7.1


Brain (fetal)
12.2
Liver
3.3


Brain (whole)
24.1
Liver (fetal)
22.2


Brain (amygdala)
7.7
Liver ca. (hepatoblast)
47.0




HepG2


Brain (cerebellum)
21.5
Lung
8.2


Brain (hippocampus)
11.0
Lung (fetal)
2.7


Brain (substantia nigra)
7.2
Lung ca. (small cell) LX-1
37.1


Brain (thalamus)
20.6
Lung ca. (small cell)
5.0




NCI-H69


Cerebral Cortex
13.5
Lung ca. (s. cell var.)
16.6




SHP-77


Spinal cord
14.3
Lung ca. (large cell) NCI-
100.0




H460


glio/astro U87-MG
30.1
Lung ca. (non-sm. cell)
35.6




A549


glio/astro U-118-MG
50.3
Lung ca. (non-s. cell)
23.3




NCI-H23


astrocytoma SW1783
31.0
Lung ca. (non-s. cell)
30.1




HOP-62


neuro*; met SK-N-AS
31.2
Lung ca. (non-s. cl) NCI-
13.1




H522


astrocytoma SF-539
25.5
Lung ca. (squam.) SW
19.9




900


astrocytoma SNB-75
34.6
Lung ca. (squam.) NCI-
14.2




H596


glioma SNB-19
28.7
Mammary gland
12.7


glioma U251
92.7
Breast ca.* (pl. ef) MCF-7
31.0


glioma SF-295
24.7
Breast ca.* (pl. ef) MDA-
62.9




MB-231


Heart (fetal)
5.1
Breast ca.* (pl. ef) T47D
13.9


Heart
14.2
Breast ca. BT-549
61.1


Skeletal muscle (fetal)
2.2
Breast ca. MDA-N
5.7


Skeletal muscle
90.1
Ovary
2.8


Bone marrow
7.5
Ovarian ca. OVCAR-3
27.2


Thymus
7.0
Ovarian ca. OVCAR-4
23.8


Spleen
12.2
Ovarian ca. OVCAR-5
18.0


Lymph node
20.3
Ovarian ca. OVCAR-8
8.6


Colorectal
7.5
Ovarian ca. IGROV-1
10.2


Stomach
17.6
Ovarian ca.* (ascites)
20.0




SK-OV-3


Small intestine
17.4
Uterus
17.8


Colon ca. SW480
18.9
Placenta
7.1


Colon ca.* SW620 (SW480
25.7
Prostate
7.9


met)


Colon ca. HT29
9.7
Prostate ca.* (bone
16.6




met) PC-3


Colon ca. HCT-116
30.6
Testis
10.2


Colon ca. CaCo-2
29.9
Melanoma Hs688 (A).T
9.6


Colon ca.
24.5
Melanoma* (met)
10.9


tissue (ODO3866)

Hs688 (B).T


Colon ca. HCC-2998
14.3
Melanoma UACC-62
8.0


Gastric ca.* (liver met)
36.1
Melanoma M14
47.0


NCI-N87


Bladder
17.4
Melanoma LOX IMVI
3.4


Trachea
7.5
Melanoma* (met) SK-
9.2




MEL-5


Kidney
7.0
Adipose
9.2
















TABLE CC







Panel 5 Islet











Rel. Exp. (%)

Rel. Exp. (%)



Ag3225, Run

Ag3225, Run


Tissue Name
223846465
Tissue Name
223846465













97457_Patient-
12.2
94709_Donor 2 AM - A_adipose
32.5


02go_adipose


97476_Patient-07sk_skeletal
13.6
94710_Donor 2 AM - B_adipose
16.5


muscle


97477_Patient-07ut_uterus
10.5
94711_Donor 2 AM - C_adipose
15.8


97478_Patient-
15.5
94712_Donor 2 AD - A_adipose
37.9


07pl_placenta


99167_Bayer Patient 1
9.7
94713_Donor 2 AD - B_adipose
37.4


97482_Patient-08ut_uterus
7.8
94714_Donor 2 AD - C_adipose
41.5


97483_Patient-
13.3
94742_Donor 3 U - A_Mesenchymal
5.7


08pl_placenta

Stem Cells


97486_Patient-09sk_skeletal
4.0
94743_Donor 3 U - B_Mesenchymal
25.7


muscle

Stem Cells


97487_Patient-09ut_uterus
18.4
94730_Donor 3 AM - A_adipose
42.0


97488_Patient-
6.9
94731_Donor 3 AM - B_adipose
16.4


09pl_placenta


97492_Patient-10ut_uterus
28.5
94732_Donor 3 AM - C_adipose
26.4


97493_Patient-
23.0
94733_Donor 3 AD - A_adipose
59.5


10pl_placenta


97495_Patient-
8.9
94734_Donor 3 AD - B_adipose
11.7


11go_adipose


97496_Patient-11sk_skeletal
20.7
94735_Donor 3 AD - C_adipose
35.4


muscle


97497_Patient-11ut_uterus
23.5
77138_Liver_HepG2untreated
100.0


97498_Patient-
17.0
73556_Heart_Cardiac stromal cells
10.8


11pl_placenta

(primary)


97500 Patient-
18.3
81735_Small Intestine
26.8


12go_adipose


97501_Patient-12sk_skeletal
49.3
72409_Kidney_Proximal Convoluted
12.1


muscle

Tubule


97502_Patient-12ut_uterus
29.7
82685_Small intestine_Duodenum
10.4


97503_Patient-
12.9
90650_Adrenal_Adrenocortical
9.8


12pl_placenta

adenoma


94721_Donor 2 U -
14.3
72410_Kidney_HRCE
38.4


A_Mesenchymal Stem Cells


94722_Donor 2 U -
12.3
72411_Kidney_HRE
32.3


B_Mesenchymal Stem Cells


94723_Donor 2 U -
23.5
73139_Uterus_Uterine smooth
11.0


C_Mesenchymal Stem Cells

muscle cells
















TABLE CD







Panel 5D











Rel. Exp. (%)

Rel. Exp. (%)



Ag3225, Run

Ag3225, Run


Tissue Name
169271478
Tissue Name
169271478













97457_Patient-
9.7
94709_Donor 2 AM - A_adipose
26.2


02go_adipose


97476_Patient-07sk_skeletal
7.1
94710_Donor 2 AM - B_adipose
19.2


muscle


97477_Patient-07ut_uterus
9.7
94711_Donor 2 AM - C_adipose
18.4


97478_Patient-
13.9
94712_Donor 2 AD - A_adipose
35.1


07pl_placenta


97481_Patient-08sk_skeletal
7.7
94713_Donor 2 AD - B_adipose
37.9


muscle


97482_Patient-08ut_uterus
10.2
94714_Donor 2 AD - C_adipose
38.4


97483_Patient-
7.9
94742_Donor 3 U - A_Mesenchymal
18.9


08pl_placenta

Stem Cells


97486_Patient-09sk_skeletal
7.7
94743_Donor 3 U - B_Mesenchymal
25.7


muscle

Stem Cells


97487_Patient-09ut_uterus
12.4
94730_Donor 3 AM - A_adipose
40.1


97488_Patient-
8.4
94731_Donor 3 AM - B_adipose
20.7


09pl_placenta


97492_Patient-10ut_uterus
16.0
94732_Donor 3 AM - C_adipose 
30.1


97493_Patient-
24.5
94733_Donor 3 AD - A_adipose
43.2


10pl_placenta


97495_Patient-
6.7
94734_Donor 3 AD - B_adipose
27.2


11go_adipose


97496_Patient-11sk_skeletal
22.5
94735_Donor 3 AD - C_adipose
37.9


muscle


97497_Patient-11ut_uterus
22.4
77138_Liver_HepG2untreated
100.0


97498_Patient-
8.2
73556_Heart_Cardiac stromal cells
9.7


11pl_placenta

(primary)


97500_Patient-
9.3
81735_Small Intestine
16.5


12go_adipose


97501_Patient-12sk_skeletal
43.2
72409_Kidney_Proximal Convoluted
10.2


muscle

Tubule


97502_Patient-12ut_uterus
25.2
82685_Small intestine_Duodenum
13.6


97503_Patient-
9.6
90650_Adrenal_Adrenocortical
8.5


12pl_placenta

adenoma


94721_Donor 2 U -
15.6
72410_Kidney_HRCE
26.8


A_Mesenchymal Stem Cells


94722_Donor 2 U -
12.6
72411_Kidney_HRE
27.2


B_Mesenchymal Stem Cells


94723_Donor 2 U -
13.3
73139_Uterus_Uterine smooth
11.0


C_Mesenchymal Stem Cells

muscle cells









Panel 1.3D Summary: 3225 The expression of the CG57433-01 gene appears to be highest in a sample derived from a lung cancer cell line (H460 (CT=29.5). In addition, there appears to be substantial expression in other samples derived from lung cancer cell lines, brain cancer cell lines, kidney cancer cell lines, colon cancer cell lines and breast cancer cell lines. Of note, is the apparent overexpression of this gene in skeletal muscle (CT=30) in comparison to fetal skeletal muscle (CT=35). Thus, the expression of this gene could be used to distinguish H460 cells from other samples in this panel. In addition, the expression of this gene could be used to distinguish adult skeletal muscle and fetal skeletal muscle. Moreover, therapeutic modulation of this gene, through the use of small molecule drugs, protein therapeutics, or antibodies could be of benefit in the treatment of lung, brain, kidney, colon or breast cancer.
Among metabolic tissues, this gene has low levels of expression in pancreas, adrenal, thyroid, pituitary, heart, liver and adipose. Inhibition of this insulin-degrading enzyme may increase insulin signalling and action and thus be a treatment for the prevention and treatment of Type 2 diabetes. This gene product may also be a small molecule target for the treatment of metabolic and endocrine disease, including thyroidopathies, Type 1 diabetes, and obesity.
This gene is also expressed throughout the brain at low levels, indicating a role in CNS processes. Insulysin (also known as Insulin-degrading enyzme) has been shown to rapidly remove the beta-amyloid precursor protein intracellular domain (AICD) and eliminate proteins with amyloidogenic potential, which are thought to mediate of the pathological features of Alzheimer's disease. Potentiation of this process is a likely means of addressing Alzneimer's disease pathology. Therefore, agents that increase the physiological activity of this gene product are likely to have utility as clinically therapeutic treatments for Alzheimer's disease, and potentially other neurodegenerative diseases involving protein aggregation (Edbauer et al., Biol Chem 2002 Jan. 23; [epub ahead of print]; Kurochkin, Trends Biochem Sci 2001 July;26(7):421-5).
Panel 5 Islet and 5D Summary: Ag3225 The CG57433-01 gene is expressed at low levels in islets of Langerhans and mesenchymal stem cells that differentiate in vitro into adipocytes, chondrocytes and osteocytes. Therefore, therapeutic modulation of this enzyme may be a treatment for diseases of bone and cartilage as well as Types 1 and 2 diabetes.
D. NOV5: Membrane Protein
Expression of gene CG57360-01 was assessed using the primer-probe set Ag3215, described in Table DA. Results of the RTQ-PCR runs are shown in Tables DB, DC, DD and DE.







TABLE DA







Probe Name Ag3215













Start


Primers
Sequences
Length
Position














Forward
5′-ttgctctcatgaggtcagagat-3′
(SEQ ID NO:305)
22
43


Probe
TET-5′-ccataggatgagtcttctgttcttaaaccg-3′-TAMRA
(SEQ ID NO:306)
30
73


Reverse
5′-ccgacagccttatccttacact-3′
(SEQ ID NO:307)
22
103
















TABLE DB







CNS_neurodegeneration_v1.0











Rel. Exp. (%) Ag3215, Run

Rel. Exp. (%) Ag3215, Run


Tissue Name
209861782
Tissue Name
209861782













AD 1 Hippo
5.3
Control (Path) 3
3.7




Temporal Ctx


AD 2 Hippo
21.8
Control (Path) 4
37.6




Temporal Ctx


AD 3 Hippo
0.0
AD 1 Occipital Ctx
8.1


AD 4 Hippo
15.5
AD 2 Occipital Ctx
0.0




(Missing)


AD 5 hippo
42.0
AD 3 Occipital Ctx
2.0


AD 6 Hippo
51.8
AD 4 Occipital Ctx
20.3


Control 2 Hippo
25.2
AD 5 Occipital Ctx
4.1


Control 4 Hippo
11.8
AD 6 Occipital Ctx
33.4


Control (Path) 3 Hippo
1.3
Control 1 Occipital Ctx
5.8


AD 1 Temporal Ctx
7.5
Control 2 Occipital Ctx
62.9


AD 2 Temporal Ctx
33.0
Control 3 Occipital Ctx
14.2


AD 3 Temporal Ctx
2.7
Control 4 Occipital Ctx
6.9


AD 4 Temporal Ctx
27.2
Control (Path) 1
100.0




Occipital Ctx


AD 5 Inf Temporal Ctx
41.8
Control (Path) 2
22.8




Occipital Ctx


AD 5 SupTemporal Ctx
36.3
Control (Path) 3
3.3




Occipital Ctx


AD 6 Inf Temporal Ctx
56.6
Control (Path) 4
20.3




Occipital Ctx


AD 6 Sup Temporal Ctx
73.2
Control 1 Parietal Ctx
4.2


Control 1 Temporal Ctx
1.5
Control 2 Parietal Ctx
25.9


Control 2 Temporal Ctx
8.7
Control 3 Parietal Ctx
20.6


Control 3 Temporal Ctx
19.5
Control (Path) 1
60.7




Parietal Ctx


Control 4 Temporal Ctx
14.5
Control (Path) 2
17.3




Parietal Ctx


Control (Path) 1
59.5
Control (Path) 3
1.5


Temporal Ctx

Parietal Ctx


Control (Path) 2
54.3
Control (Path) 4
33.0


Temporal Ctx

Parietal Ctx
















TABLE DC







Panel 1.3D











Rel. Exp. (%) Ag3215, Run

Rel. Exp. (%) Ag3215, Run


Tissue Name
168012859
Tissue Name
168012859













Liver adenocarcinoma
64.6
Kidney (fetal)
80.7


Pancreas
22.4
Renal ca. 786-0
0.0


Pancreatic ca. CAPAN 2
79.6
Renal ca. A498
4.3


Adrenal gland
24.0
Renal ca. RXF 393
20.7


Thyroid
17.3
Renal ca. ACHN
15.8


Salivary gland
1.6
Renal ca. UO-31
1.6


Pituitary gland
26.1
Renal ca. TK-10
4.3


Brain (fetal)
86.5
Liver
26.1


Brain (whole)
55.5
Liver (fetal)
20.7


Brain (amygdala)
69.7
Liver ca. (hepatoblast)
18.9




HepG2


Brain (cerebellum)
37.9
Lung
15.1


Brain (hippocampus)
20.7
Lung (fetal)
38.7


Brain (substantia nigra)
46.3
Lung ca. (small cell) LX-1
6.0


Brain (thalamus)
23.8
Lung ca. (small cell)
5.5




NCI-H69


Cerebral Cortex
17.6
Lung ca. (s. cell var.)
35.4




SHP-77


Spinal cord
19.3
Lung ca. (large cell) NCI-
4.5




H460


glio/astro U87-MG
20.0
Lung ca. (non-sm. cell)
7.6




A549


glio/astro U-118-MG
27.7
Lung ca. (non-s. cell)
39.0




NCI-H23


astrocytoma SW1783
28.5
Lung ca. (non-s. cell)
29.7




HOP-62


neuro*; met SK-N-AS
15.2
Lung ca. (non-s. cl) NCI-
46.3




H522


astrocytoma SF-539
13.8
Lung ca. (squam.) SW
17.7




900


astrocytoma SNB-75
13.5
Lung ca. (squam.) NCI-
12.7




H596


glioma SNB-19
2.3
Mammary gland
42.0


glioma U251
18.4
Breast ca.* (pl. ef) MCF-7
0.0


glioma SF-295
36.6
Breast ca.* (pl. ef) MDA-
18.7




MB-231


Heart (fetal)
2.2
Breast ca.* (pl. ef) T47D
15.2


Heart
2.3
Breast ca. BT-549
13.6


Skeletal muscle (fetal)
12.8
Breast ca. MDA-N
4.2


Skeletal muscle
4.2
Ovary
15.0


Bone marrow
11.7
Ovarian ca. OVCAR-3
6.0


Thymus
14.5
Ovarian ca. OVCAR-4
12.2


Spleen
19.2
Ovarian ca. OVCAR-5
39.5


Lymph node
17.2
Ovarian ca. OVCAR-8
3.8


Colorectal
4.4
Ovarian ca. IGROV-1
41.2


Stomach
26.2
Ovarian ca.* (ascites)
14.7




SK-OV-3


Small intestine
8.8
Uterus
24.7


Colon ca. SW480
19.3
Placenta
100.0


Colon ca.* SW620 (SW480
19.8
Prostate
8.0


met)


Colon ca. HT29
4.1
Prostate ca.* (bone
11.1




met) PC-3


Colon ca. HCT-116
7.9
Testis
49.3


Colon ca. CaCo-2
29.7
Melanoma Hs688 (A).T
5.7


Colon ca.
9.1
Melanoma* (met)
7.9


tissue (ODO3866)

Hs688 (B).T


Colon ca. HCC-2998
9.9
Melanoma UACC-62
4.0


Gastric ca.* (liver met)
28.5
Melanoma M14
2.9


NCI-N87


Bladder
14.0
Melanoma LOX IMVI
15.8


Trachea
9.3
Melanoma* (met) SK-
2.0




MEL-5


Kidney
43.8
Adipose
8.4
















TABLE DD







Panel 2.2











Rel. Exp. (%) Ag3215,

Rel. Exp. (%) Ag3215,


Tissue Name
Run 174416485
Tissue Name
Run 174416485













Normal Colon
4.4
Kidney Margin (OD04348)
100.0


Colon cancer (OD06064)
10.4
Kidney malignant cancer
47.6




(OD06204B)


Colon Margin (OD06064)
2.9
Kidney normal adjacent
29.9




tissue (OD06204E)


Colon cancer (OD06159)
0.0
Kidney Cancer (OD04450-
27.7




01)


Colon Margin (OD06159)
4.7
Kidney Margin (OD04450-
31.2




03)


Colon cancer (OD06297-04)
0.0
Kidney Cancer 8120613
0.0


Colon Margin (OD06297-05)
4.6
Kidney Margin 8120614
36.6


CC Gr. 2 ascend colon
21.8
Kidney Cancer 9010320
10.7


(ODO3921)


CC Margin (ODO3921)
0.0
Kidney Margin 9010321
12.3


Colon cancer metastasis
0.0
Kidney Cancer 8120607
27.9


(OD06104)


Lung Margin (OD06104)
0.0
Kidney Margin 8120608
4.5


Colon mets to lung
7.9
Normal Uterus
21.0


(OD04451-01)


Lung Margin (OD04451-02)
19.2
Uterine Cancer 064011
18.8


Normal Prostate
14.6
Normal Thyroid
0.0


Prostate Cancer (OD04410)
2.1
Thyroid Cancer 064010
0.0


Prostate Margin (OD04410)
9.6
Thyroid Cancer A302152
49.7


Normal Ovary
18.4
Thyroid Margin A302153
0.0


Ovarian cancer (OD06283-
18.9
Normal Breast
0.0


03)


Ovarian Margin (OD06283-
23.5
Breast Cancer (OD04566)
0.0


07)


Ovarian Cancer 064008
14.4
Breast Cancer 1024
79.0


Ovarian cancer (OD06145)
5.8
Breast Cancer (OD04590-
33.7




01)


Ovarian Margin (OD06145)
39.5
Breast Cancer Mets
17.2




(OD04590-03)


Ovarian cancer (OD06455-
59.9
Breast Cancer Metastasis
49.0


03)

(OD04655-05)


Ovarian Margin (OD06455-
0.0
Breast Cancer 064006
27.4


07)


Normal Lung
14.8
Breast Cancer 9100266
3.8


Invasive poor diff. lung
3.5
Breast Margin 9100265
10.4


adeno (ODO4945-01


Lung Margin (ODO4945-03)
3.6
Breast Cancer A209073
0.0


Lung Malignant Cancer
0.0
Breast Margin A2090734
54.7


(OD03126)


Lung Margin (OD03126)
10.8
Breast cancer (OD06083)
17.7


Lung Cancer (OD05014A)
15.7
Breast cancer node
76.3




metastasis (OD06083)


Lung Margin (OD05014B)
2.0
Normal Liver
18.3


Lung cancer (OD06081)
12.4
Liver Cancer 1026
0.0


Lung Margin (OD06081)
4.4
Liver Cancer 1025
19.6


Lung Cancer (OD04237-01)
5.6
Liver Cancer 6004-T
0.0


Lung Margin (OD04237-02)
0.0
Liver Tissue 6004-N
0.0


Ocular Melanoma Metastasis
8.4
Liver Cancer 6005-T
0.0


Ocular Melanoma Margin
0.0
Liver Tissue 6005-N
0.0


(Liver)


Melanoma Metastasis
0.0
Liver Cancer 064003
18.9


Melanoma Margin (Lung)
0.0
Normal Bladder
33.7


Normal Kidney
37.4
Bladder Cancer 1023
4.0


Kidney Ca, Nuclear grade 2
82.9
Bladder Cancer A302173
20.0


(OD04338)


Kidney Margin (OD04338)
7.6
Normal Stomach
20.7


Kidney Ca Nuclear grade 1/2
94.6
Gastric Cancer 9060397
0.0


(OD04339)


Kidney Margin (OD04339)
19.1
Stomach Margin 9060396
6.0


Kidney Ca, Clear cell type
0.0
Gastric Cancer 9060395
6.0


(OD04340)


Kidney Margin (OD04340)
28.7
Stomach Margin 9060394
4.0


Kidney Ca, Nuclear grade 3
0.0
Gastric Cancer 064005
11.9


(OD04348)
















TABLE DE







Panel 4D











Rel. Exp. (%) Ag3215,

Rel. Exp. (%) Ag3215,


Tissue Name
Run 164682514
Tissue Name
Run 164682514













Secondary Th1 act
6.8
HUVEC IL-1beta
5.8


Secondary Th2 act
7.4
HUVEC IFN gamma
29.5


Secondary Tr1 act
8.7
HUVEC TNF alpha + IFN
6.0




gamma


Secondary Th1 rest
2.8
HUVEC TNF alpha + IL4
17.6


Secondary Th2 rest
4.0
HUVEC IL-11
11.5


Secondary Tr1 rest
6.3
Lung Microvascular EC none
34.4


Primary Th1 act
12.7
Lung Microvascular EC
42.3




TNF alpha + IL-1beta


Primary Th2 act
12.5
Microvascular Dermal EC none
14.1


Primary Tr1 act
11.1
Microsvasular Dermal EC
13.9




TNF alpha + IL-1beta


Primary Th1 rest
12.8
Bronchial epithelium
100.0




TNF alpha + IL1beta


Primary Th2 rest
5.7
Small airway epithelium none
28.1


Primary Tr1 rest
2.4
Small airway epithelium
74.2




TNF alpha + IL-1beta


CD45RA CD4 lymphocyte
7.9
Coronery artery SMC rest
26.8


act


CD45RO CD4 lymphocyte
12.0
Coronery artery SMC
2.8


act

TNF alpha + IL-1beta


CD8 lymphocyte act
19.3
Astrocytes rest
17.3


Secondary CD8
13.5
Astrocytes TNF alpha + IL-1beta
2.9


lymphocyte rest


Secondary CD8
10.1
KU-812 (Basophil) rest
9.0


lymphocyte act


CD4 lymphocyte none
1.6
KU-812 (Basophil)
6.3




PMA/ionomycin


2ry Th1/Th2/Tr1_anti-
5.1
CCD1106 (Keratinocytes) none
25.2


CD95 CH11


LAK cells rest
1.0
CCD1106 (Keratinocytes)
26.2




TNF alpha + IL-1beta


LAK cells IL-2
12.3
Liver cirrhosis
13.2


LAK cells IL-2 + IL-12
5.7
Lupus kidney
8.4


LAK cells IL-2 + IFN
5.8
NCI-H292 none
31.6


gamma


LAK cells IL-2 + IL-18
7.2
NCI-H292 IL-4
22.2


LAK cells
2.9
NCI-H292 IL-9
57.4


PMA/ionomycin


NK Cells IL-2 rest
9.2
NCI-H292 IL-13
26.4


Two Way MLR 3 day
14.4
NCI-H292 IFN gamma
35.1


Two Way MLR 5 day
1.9
HPAEC none
15.0


Two Way MLR 7 day
12.9
HPAEC TNF alpha + IL-1beta
6.9


PBMC rest
7.1
Lung fibroblast none
17.6


PBMC PWM
12.5
Lung fibroblast TNF alpha + IL-
12.2




1beta


PBMC PHA-L
18.9
Lung fibroblast IL-4
21.9


Ramos (B cell) none
2.3
Lung fibroblast IL-9
17.0


Ramos (B cell) ionomycin
0.0
Lung fibroblast IL-13
16.7


B lymphocytes PWM
20.3
Lung fibroblast IFN gamma
18.8


B lymphocytes CD40L
8.7
Dermal fibroblast CCD1070 rest
44.8


and IL-4


EOL-1 dbcAMP
0.0
Dermal fibroblast CCD1070
30.1




TNF alpha


EOL-1 dbcAMP
0.0
Dermal fibroblast CCD1070 IL-
25.3


PMA/ionomycin

1beta


Dendritic cells none
8.8
Dermal fibroblast IFN gamma
13.3


Dendritic cells LPS
0.0
Dermal fibroblast IL-4
14.1


Dendritic cells anti-CD40
6.0
IBD Colitis 2
2.6


Monocytes rest
25.2
IBD Crohn's
11.1


Monocytes LPS
3.3
Colon
26.2


Macrophages rest
11.1
Lung
19.3


Macrophages LPS
6.7
Thymus
56.3


HUVEC none
37.9
Kidney
18.9


HUVEC starved
11.3









CNS_neurodegeneration_v1.0 Summary: Ag3215 This panel does not show differential expression of the CG57360-01 gene in Alzheimer's disease. However, this expression profile confirms the presence of this gene in the brain. Please see Panel 1.3D for discussion of utility of this gene in the central nervous system.
Panel 1.3D Summary: Ag3215 Highest expression of the CG57360-01 gene is seen in the placenta (CT=31.7). Significant expression is also seen in samples derived from pancreatic cancer, liver cancer, lung cancer, ovarian cancer, brain cancer, and colon cancer. Thus, expression of this gene could be used to differentiate these samples from other samples on this panel and as a marker to detect the presence of these cancers. Furthermore, therapeutic modulation of the expression or function of this gene may be effective in the treatment of these cancers.
Among tissues with metabolic function, this gene is expressed in fetal and adult liver, pancreas, and the adrenal and thyroid glands. This widespread expression of the gene in these tissues suggests that its protein product may play a role in normal metabolic function. This gene product may also be useful in the treatment of metabolic disorders, including obesity and diabetes.
In addition, this molecule is expressed at moderate to low levels in the CNS and may be useful for the treatment of neurologic diseases.
Panel 2.2 Summary: Ag 3215 The expression of the CG57360-01 gene appears to be highest in a sample derived from a normal kidney tissue (CT=33.8). In addition, there appears to be substantial expression in other samples derived from kidney cancers and breast cancers. Thus, the expression of this gene could be used to distinguish normal kidney tissue from other samples in the panel. Moreover, therapeutic modulation of this gene, through the use of small molecule drugs, protein therapeutics, or antibodies could be of benefit in the treatment of kidney or breast cancer.
Panel 4D Summary: Ag3215 Highest expression of the CG57360-01 gene is seen in bronchial epithelium treated with TNF-alpha+IL-1 beta (CT=31.2). Significant expression is also seen in TNF-alpha/IL-1 beta stimulated small airway epithelium, IL-4, IL-9, IL-13, IFN gamma activated-NCI-H292 mucoepidermoid cells as well as untreated NCI-H292 cells, IL-4, IL-9, IL-13 and IFN gamma activated lung fibroblasts, and both treated and untreated lung microvascular endothelial cells The expression of this gene in cells derived from or within the lung suggests that this gene may be involved in normal conditions as well as pathological and inflammatory lung disorders, including chronic obstructive pulmonary disease, asthma, allergy and emphysema.
E. NOV6: BCSC-1
Expression of gene CG57362-01 was assessed using the primer-probe set Ag3217, described in Table EA. Results of the RTQ-PCR runs are shown in Tables EB, EC, ED, EE and EF.







TABLE EA







Probe Name Ag3217













Start


Primers
Sequences
Length
Position














Forward
5′-actgggaacaggcactgtct-3′
(SEQ ID NO:308)
20
1327


Probe
TET-5′-cagcagaactgtccagcccatgg-3′-TAMRA
(SEQ ID NO:309)
23
1347


Reverse
5′-gggtctgtcagagcatcagtac-3′
(SEQ ID NO:310)
22
1395
















TABLE EB







CNS_neurodegeneration_v1.0













Rel. Exp. (%)
Rel. Exp. (%)

Rel. Exp. (%)
Rel. Exp. (%)



Ag3217, Run
Ag3217, Run

Ag3217, Run
Ag3217, Run


Tissue Name
209861783
224079666
Tissue Name
209861783
224079666















AD 1 Hippo
18.3
24.8
Control (Path)
15.6
17.7





3 Temporal Ctx


AD 2 Hippo
30.4
42.6
Control (Path)
72.2
81.8





4 Temporal Ctx


AD 3 Hippo
20.2
18.3
AD 1 Occipital
25.2
32.5





Ctx


AD 4 Hippo
21.3
22.2
AD 2 Occipital
0.0
0.0





Ctx (Missing)


AD 5 Hippo
82.9
78.5
AD 3 Occipital
7.2
7.4





Ctx


AD 6 Hippo
29.9
33.2
AD 4 Occipital
52.1
36.6





Ctx


Control 2
43.2
45.1
AD 5 Occipital
46.3
38.4


Hippo


Ctx


Control 4
26.2
17.7
AD 6 Occipital
17.2
17.9


Hippo


Ctx


Control (Path)
24.3
12.8
Control 1
5.1
8.0


3 Hippo


Occipital Ctx


AD 1
12.6
15.3
Control 2
51.8
75.8


Temporal Ctx


Occipital Ctx


AD2
51.8
33.4
Control 3
71.7
85.9


Temporal Ctx


Occipital Ctx


AD 3
12.3
13.3
Control 4
14.4
17.6


Temporal Ctx


Occipital Ctx


AD 4
45.4
46.7
Control (Path)
100.0
100.0


Temporal Ctx


1 Occipital Ctx


AD 5 Inf
56.6
51.4
Control (Path)
46.3
53.2


Temporal Ctx


2 Occipital Ctx


AD 5 Sup
43.8
33.2
Control (Path)
5.9
6.3


Temporal Ctx


3 Occipital Ctx


AD 6 Inf
40.3
33.7
Control (Path)
50.7
58.6


Temporal Ctx


4 Occipital Ctx


AD 6 Sup
38.4
46.3
Control 1
10.6
10.4


Temporal Ctx


Parietal Ctx


Control 1
12.9
11.0
Control 2
34.4
43.2


Temporal Ctx


Parietal Ctx


Control 2
45.7
41.8
Control 3
49.7
37.1


Temporal Ctx


Parietal Ctx


Control 3
57.4
71.7
Control (Path)
82.4
86.5


Temporal Ctx


1 Parietal Ctx


Control 3
42.3
37.1
Control (Path)
55.5
63.7


Temporal Ctx


2 Parietal Ctx


Control 3
87.1
99.3
Control (Path)
12.9
9.3


1 Temporal


3 Parietal Ctx


Ctx


Control (Path)
92.0
97.3
Control (Path)
90.1
95.9


2 Temporal


4 Parietal Ctx


Ctx
















TABLE EC







Panel 1.3D











Rel. Exp. (%) Ag3217, Run

Rel. Exp. (%) Ag3217, Run


Tissue Name
168012860
Tissue Name
168012860













Liver adenocarcinoma
3.9
Kidney (fetal)
2.6


Pancreas
8.1
Renal ca. 786-0
0.4


Pancreatic ca. CAPAN 2
0.0
Renal ca. A498
0.0


Adrenal gland
24.3
Renal ca. RXF 393
0.0


Thyroid
0.0
Renal ca. ACHN
0.0


Salivary gland
5.4
Renal ca. UO-31
0.1


Pituitary gland
12.9
Renal ca. TK-10
1.2


Brain (fetal)
10.2
Liver
0.9


Brain (whole)
31.6
Liver (fetal)
0.0


Brain (amygdala)
72.2
Liver ca. (hepatoblast)
0.0




HepG2


Brain (cerebellum)
21.5
Lung
0.0


Brain (hippocampus)
39.5
Lung (fetal)
1.4


Brain (substantia nigra)
11.3
Lung ca. (small cell) LX-1
1.7


Brain (thalamus)
31.6
Lung ca. (small cell)
6.6




NCI-H69


Cerebral Cortex
39.8
Lung ca. (s. cell var.)
27.7




SHP-77


Spinal cord
1.4
Lung ca. (large cell) NCI-
0.0




H460


glio/astro U87-MG
0.0
Lung ca. (non-sm. cell)
4.4




A549


glio/astro U-118-MG
0.0
Lung ca. (non-s. cell)
0.4




NCI-H23


astrocytoma SW1783
0.0
Lung ca. (non-s. cell)
0.0




HOP-62


neuro*; met SK-N-AS
0.3
Lung ca. (non-s. cl) NCI-
2.9




H522


astrocytoma SF-539
0.1
Lung ca. (squam.) SW
1.0




900


astrocytoma SNB-75
0.0
Lung ca. (squam.) NCI-
100.0




H596


glioma SNB-19
2.4
Mammary gland
0.0


glioma U251
3.4
Breast ca.* (pl. ef) MCF-7
0.0


glioma SF-295
0.8
Breast ca.* (pl. ef)MDA-
0.0




MB-231


Heart (fetal)
0.0
Breast ca.* (pl. ef) T47D
4.6


Heart
0.0
Breast ca. BT-549
0.0


Skeletal muscle (fetal)
1.1
Breast ca. MDA-N
0.2


Skeletal muscle
0.0
Ovary
1.4


Bone marrow
1.4
Ovarian ca. OVCAR-3
0.0


Thymus
0.0
Ovarian ca. OVCAR-4
0.8


Spleen
0.4
Ovarian ca. OVCAR-5
3.2


Lymph node
0.0
Ovarian ca. OVCAR-8
0.8


Colorectal
2.6
Ovarian ca. IGROV-1
0.0


Stomach
3.2
Ovarian ca.* (ascites)
0.3




SK-OV-3


Small intestine
1.3
Uterus
0.0


Colon ca. SW480
1.5
Placenta
0.0


Colon ca.* SW620(SW480
11.9
Prostate
1.6


met)


Colon ca. HT29
0.0
Prostate ca.* (bone
0.4




met) PC-3


Colon ca. HCT-116
2.9
Testis
2.6


Colon ca. CaCo-2
0.0
Melanoma Hs688(A).T
0.0


Colon ca.
0.0
Melanoma* (met)
0.0


tissue (ODO3866)

Hs688(B).T


Colon ca. HCC-2998
0.0
Melanoma UACC-62
0.0


Gastric ca.* (liver met)
0.0
Melanoma M14
0.0


NCI-N87


Bladder
0.8
Melanoma LOX IMVI
0.0


Trachea
0.0
Melanoma* (met) SK-
0.0




MEL-5


Kidney
0.0
Adipose
0.0
















TABLE ED







Panel 2.2











Rel. Exp. (%) Ag3217,

Rel. Exp. (%) Ag3217,


Tissue Name
Run 174416493
Tissue Name
Run 174416493













Normal Colon
11.9
Kidney Margin (OD04348)
9.9


Colon cancer (OD06064)
0.0
Kidney malignant cancer
0.0




(OD06204B)


Colon Margin (OD06064)
0.0
Kidney normal adjacent
0.0




tissue (OD06204E)


Colon cancer (OD06159)
0.0
Kidney Cancer (OD04450-
3.9




01)


Colon Margin (OD06159)
6.3
Kidney Margin (OD04450-
0.0




03)


Colon cancer (OD06297-04)
0.0
Kidney Cancer 8120613
0.0


Colon Margin (OD06297-05)
0.0
Kidney Margin 8120614
0.0


CC Gr. 2 ascend colon
0.0
Kidney Cancer 9010320
47.6


(ODO3921)


CC Margin (ODO3921)
0.0
Kidney Margin 9010321
0.0


Colon cancer metastasis
0.0
Kidney Cancer 8120607
3.7


(OD06104)


Lung Margin (OD06104)
0.0
Kidney Margin 8120608
0.0


Colon mets to lung
0.0
Normal Uterus
0.0


(OD04451-01)


Lung Margin (OD04451-02)
0.0
Uterine Cancer 064011
12.7


Normal Prostate
22.4
Normal Thyroid
13.6


Prostate Cancer (OD04410)
8.4
Thyroid Cancer 064010
0.0


Prostate Margin (OD04410)
0.0
Thyroid Cancer A30152
0.0


Normal Ovary
0.0
Thyroid Margin A302153
7.6


Ovarian cancer (OD06283-
0.0
Normal Breast
0.0


03)


Ovarian Margin (OD06283-
0.0
Breast Cancer (OD04566)
0.0


07)


Ovarian Cancer 064008
9.5
Breast Cancer 1024
0.0


Ovarian cancer (OD06145)
0.0
Breast Cancer (OD04590-
19.3




01)


Ovarian Margin (OD06145)
100.0
Breast Cancer Mets
35.8




(OD04590-03)


Ovarian cancer (OD06455-
0.0
Breast Cancer Metastasis
24.5


03)

(OD04655-05)


Ovarian Margin (OD06455-
0.0
Breast Cancer 064006
0.0


07)


Normal Lung
0.0
Breast Cancer 9100266
10.0


Invasive poor diff. lung
0.0
Breast Margin 9100265
0.0


adeno (ODO4945-01


Lung Margin (ODO4945-03)
0.0
Breast Cancer A209073
0.0


Lung Malignant Cancer
17.4
Breast Margin A2090734
9.1


(OD03126)


Lung Margin (OD03126)
8.8
Breast cancer (OD06083)
0.0


Lung Cancer (OD05014A)
0.0
Breast cancer node
5.1




metastasis (OD06083)


Lung Margin (OD05014B)
0.0
Normal Liver
6.7


Lung cancer (OD06081)
12.9
Liver Cancer 1026
5.6


Lung Margin (OD06081)
0.0
Liver Cancer 1025
8.5


Lung Cancer (OD04237-01)
0.0
Liver Cancer 6004-T
0.0


Lung Margin (OD04237-02)
0.0
Liver Tissue 6004-N
6.0


Ocular Melanoma Metastasis
0.0
Liver Cancer 6005-T
0.0


Ocular Melanoma Margin
0.0
Liver Tissue 6005-N
0.0


(Liver)


Melanoma Metastasis
0.0
Liver Cancer 064003
0.0


Melanoma Margin (Lung)
0.0
Normal Bladder
8.4


Normal Kidney
0.0
Bladder Cancer 1023
0.0


Kidney Ca, Nuclear grade 2
22.1
Bladder Cancer A302173
8.7


(OD04338)


Kidney Margin (OD04338)
0.0
Normal Stomach
0.0


Kidney Ca Nuclear grade 1/2
40.9
Gastric Cancer 9060397
0.0


(OD04339)


Kidney Margin (OD04339)
0.0
Stomach Margin 9060396
12.4


Kidney Ca, Clear cell type
0.0
Gastric Cancer 9060395
6.4


(OD04340)


Kidney Margin (OD04340)
0.0
Stomach Margin 9060394
9.3


Kidney Ca, Nuclear grade 3
0.0
Gastric Cancer 064005
0.0


(OD04348)
















TABLE EE







Panel 4D











Rel. Exp. (%) Ag3217,

Rel. Exp. (%) Ag3217,


Tissue Name
Run 164682517
Tissue Name
Run 164682517













Secondary Th1 act 
0.0
HUVEC IL-1beta
0.0


Secondary Th2 act
0.0
HUVEC IFN gamma
0.0


Secondary Tr1 act
0.0
HUVEC TNF alpha + IFN
0.0




gamma


Secondary Th1 rest
0.0
HUVEC TNF alpha + IL4
0.0


Secondary Th2 rest
0.0
HUVEC IL-11
0.0


Secondary Tr1 rest
0.0
Lung Microvascular EC none
0.0


Primary Th1 act
0.0
Lung Microvascular EC
0.0




TNF alpha + IL-1beta


Primary Th2 act
0.0
Microvascular Dermal EC none
0.0


Primary Tr1 act
0.0
Microsvasular Dermal EC
0.0




TNF alpha + IL-1beta


Primary Th1 rest
6.9
Bronchial epithelium TNF alpha +
4.1




IL1beta


Primary Th2 rest
0.0
Small airway epithelium none
8.2


Primary Tr1 rest
0.0
Small airway epithelium
0.0




TNF alpha + IL-1beta


CD45RA CD4 lymphocyte
0.0
Coronery artery SMC rest
0.0


act


CD45RO CD4 lymphocyte
0.0
Coronery artery SMC TNF alpha +
0.0


act

IL-1beta


CD8 lymphocyte act
3.2
Astrocytes rest
0.0


Secondary CD8
0.0
Astrocytes TNF alpha + IL-1beta
0.0


lymphocyte rest


Secondary CD8
0.0
KU-812 (Basophil) rest
0.0


lymphocyte act


CD4 lymphocyte none
0.0
KU-812 (Basophil)
0.0




PMA/ionomycin


2ry Th1/Th2/Tr1_anti-
0.0
CCD1106 (Keratinocytes) none
0.0


CD95 CH11


LAK cells rest
0.0
CCD1106 (Keratinocytes)
0.0




TNF alpha + IL-1beta


LAK cells IL-2
0.0
Liver cirrhosis
6.9


LAK cells IL-2 + IL-12
0.0
Lupus kidney
0.0


LAK cells IL-2 + IFN
0.0
NCI-H292 none
5.3


gamma


LAK cells IL-2 + IL-18
0.0
NCI-H292 IL-4
0.0


LAK cells
22.7
NCI-H292 IL-9
16.8


PMA/ionomycin


NK Cells IL-2 rest
0.0
NCI-H292 IL-13
0.0


Two Way MLR 3 day
5.6
NCI-H292 IFN gamma
0.0


Two Way MLR 5 day
0.0
HPAEC none
8.5


Two Way MLR 7 day
0.0
HPAEC TNF alpha + IL-1beta
0.0


PBMC rest
0.0
Lung fibroblast none
0.0


PBMC PWM
3.8
Lung fibroblast TNF alpha + IL-
0.0




1beta


PBMC PHA-L
0.0
Lung fibroblast IL-4
0.0


Ramos (B cell) none
0.0
Lung fibroblast IL-9
0.0


Ramos (B cell) ionomycin
0.0
Lung fibroblast IL-13
0.0


B lymphocytes PWM
7.2
Lung fibroblast IFN gamma
0.0


B lymphocytes CD40L
0.0
Dermal fibroblast CCD1070 rest
5.4


and IL-4


EOL-1 dbcAMP
5.3
Dermal fibroblast CCD1070
0.0




TNF alpha


EOL-1 dbcAMP
0.0
Dermal fibroblast CCD1070 IL-
0.0


PMA/ionomycin

1beta


Dendritic cells none
0.0
Dermal fibroblast IFN gamma
0.0


Dendritic cells LPS
0.0
Dermal fibroblast IL-4
0.0


Dendritic cells anti-CD40
0.0
IBD Colitis 2
8.3


Monocytes rest
0.0
IBD Crohn's
20.6


Monocytes LPS
0.0
Colon
100.0


Macrophages rest
0.0
Lung
47.6


Macrophages LPS
0.0
Thymus
19.3


HUVEC none
0.0
Kidney
0.0


HUVEC starved
0.0
















TABLE EF







Panel CNS_1











Rel. Exp. (%) Ag3217, Run

Rel. Exp. (%) Ag3217, Run


Tissue Name
171694584
Tissue Name
171694584













BA4 Control
21.0
BA17 PSP
14.3


BA4 Control2
23.3
BA17 PSP2
27.2


BA4 Alzheimer's2
9.9
Sub Nigra Control
16.6


BA4 Parkinson's
57.4
Sub Nigra Control2
8.1


BA4 Parkinson's2
52.5
Sub Nigra Alzheimer's2
9.2


BA4 Huntington's
11.9
Sub Nigra Parkinson's2
10.9


BA4
24.1
Sub Nigra Huntington's
5.3


Huntington's2


BA4 PSP
3.6
Sub Nigra
17.9




Huntington's2


BA4 PSP2
8.5
Sub Nigra PSP2
1.9


BA4 Depression
20.0
Sub Nigra Depression
0.0


BA4 Depression2
9.7
Sub Nigra Depression2
4.1


BA7 Control
28.9
Glob Palladus Control
5.6


BA7 Control2
31.0
Glob Palladus Control2
4.6


BA7 Alzheimer's2
15.7
Glob Palladus
5.3




Alzheimer's


BA7 Parkinson's
31.0
Glob Palladus
4.7




Alzheimer's2


BA7 Parkinson's2
26.2
Glob Palladus
29.5




Parkinson's


BA7 Huntington's
19.9
Glob Palladus
8.5




Parkinson's2


BA7
55.9
Glob Palladus PSP
2.6


Huntington's2


BA7 PSP
15.5
Glob Palladus PSP2
4.0


BA7 PSP2
12.3
Glob Palladus
4.0




Depression


BA7 Depression
13.5
Temp Pole Control
13.1


BA9 Control
15.3
Temp Pole Control2
31.4


BA9 Control2
31.6
Temp Pole Alzheimer's
8.0


BA9 Alzheimer's
9.4
Temp Pole Alzheimer's2
8.0


BA9 Alzheimer's2
10.4
Temp Pole Parkinson's
33.2


BA9 Parkinson's
42.6
Temp Pole Parkinson's2
26.1


BA9 Parkinson's2
25.9
Temp Pole Huntington's
22.2


BA9 Huntington's
11.6
Temp Pole PSP
6.0


BA9
29.9
Temp Pole PSP2
2.3


Huntington's2


BA9 PSP
11.0
Temp Pole Depression2
15.1


BA9 PSP2
9.5
Cing Gyr Control
34.2


BA9 Depression
16.4
Cing Gyr Control2
13.2


BA9 Depression2
12.8
Cing Gyr Alzheimer's
3.2


BA17 Control
68.3
Cing Gyr Alzheimer's2
6.9


BA17 Control2
28.3
Cing Gyr Parkinson's
17.9


BA17
18.7
Cing Gyr Parkinson's2
14.8


Alzheimer's2


BA17 Parkinson's
48.3
Cing Gyr Huntington's
6.6


BA17
100.0
Cing Gyr Huntington's2
9.7


Parkinson's2


BA17
29.5
Cing Gyr PSP
2.2


Huntington's


BA17
32.3
Cing Gyr PSP2
6.1


Huntington's2


BA17 Depression
21.0
Cing Gyr Depression
5.2


BA17 Depression2
53.2
Cing Gyr Depression2
8.8









CNS_neurodegeneration_v1.0 Summary: Ag3217 Two experiments with the same probe and primer set show that the CG57362-01 gene exhibits decreased expression in the temporal cortex of brains suffering from Alzheimer's disease (p
Panel 1.3D Summary: Ag3217 Highest expression of the CG57362-01 gene is seen in a lung cancer cell line (CT=30.1). Significant expression is also seen in a cluster of lung cancer cell lines and samples derived from the brain. Thus, expression of this gene could be used to differentiate between brain-derived samples, the lung cancer cell lines and the other samples on this panel. Furthermore, therapeutic modulation of the expression or function of this gene may be effective in the treatment of lung cancer.
Among tissues with metabolic function, this gene is expressed in the adrenal and pituitary glands, and the pancreas. Thus, this gene product may be useful in the diagnosis and/or treatment of metabolic disease, including obesity and diabetes.
In addition, brain-preferential expression of thsi gene indicates that drugs that target this potential mediator of Alzheimer's disease, as seen in CNS_neurodegeneration_V1.0, are likely to have brain-specific action, a desireable feature of effective drugs.
Panel 2.2 Summary: Ag3217 Expression of the CG57362-01 gene is restricted to a sample derived from ovarian tissue (CT=34.9). Thus, expression of this gene could be used to differentiate between this sample and other samples on this panel.
Panel 4D Summary: Ag3217 Expression of the CG57362-01 gene is restricted to a sample derived from the colon (CT=34.8). Therefore, expression of this gene may be used to distinguish colon from the other tissues on this panel. Furthermore, expression of this gene is decreased in colon samples from patients with IBD colitis and Crohn's disease relative to normal colon. Therefore, therapeutic modulation of the activity of the GPCR encoded by this gene may be useful in the treatment of inflammatory bowel disease
Panel CNS—1 Summary: Ag3217 Expression in this panel confirms the presence of the CG57362-01 gene in the brain. Please see Panels 1.3D and CNS_neurodegeneration_v1.0 for discussion of utility of this gene in the central nervous system.
F. NOV7a and NOV7b: Amino Acid Transporter
Expression of gene CG57387-01 and variant CG57387-02 was assessed using the primer-probe sets Ag3222 and Ag4965, described in Tables FA and FB.







TABLE FA







Probe Name Ag3222













Start


Primers
Sequences
Length
Position














Forward
5′-tgcaatttggagctaatatcca-3′
(SEQ ID NO:311)
22
1020


Probe
TET-5′-taaccctcaacctgcccttgtaccag-3′-TAMRA
(SEQ ID NO:312)
26
1050


Reverse
5′-cgatggagtacagcagcttaac-3′
(SEQ ID NO:313)
22
1079
















TABLE FB







Probe Name Ag4965













Start


Primers
Sequences
Length
Position














Forward
5′-ggaaaacaaaatgaaggatcct-3′
(SEQ ID NO:314)
22
919


Probe
TET-5′-cggaagttcccactcatcctgtacct-3′-TAMRA
(SEQ ID NO:315)
26
941


Reverse
5′-atgtagaggatggtgacgatga-3′
(SEQ ID NO:316)
22
975









CNS_neurodegeneration_v1.0 Summary: Ag3222 Expression of the CG57387-01 gene is low/undetectable in all samples on this panel (CTs>35). (Data not shown.)
General_screening_panel_v1.4 Summary: Ag4965 Expression of the CG57387-01 gene is low/undetectable in all samples on this panel (CTs>35). (Data not shown.) The amp plot indicates that there is a high probability of a probe failure.
Panel 1.3D Summary: Ag3222 Expression of the CG57387-01 gene is low/undetectable in all samples on this panel (CTs>35). (Data not shown.)
Panel 2.2 Summary: Ag3222 Results from one experiment with the CG57387-01 gene are not included. The amp plot indicates that there were experimental difficulties with this run.
Panel 4D Summary: Ag3222 Expression of the CG57387-01 gene is low/undetectable in all samples on this panel (CTs>35). (Data not shown.)
G. NOV7c: Amino Acid Transporter Like
Expression of gene CG57387-03 was assessed using the primer-probe set Ag5127, described in Table GA. Results of the RTQ-PCR runs are shown in Tables GB, GC and GD. This probe/primer set recognizes a distinct portion of this gene and shows a distinctive expression pattern when compared to expression seen using probe and primer sets Ag3222 and Ag4965 that recognize variants CG57387-01 and CG57387-02. This observation may indicate that the probe/primer sets can distinguish splice variants of this gene. In contrast to the results obtained with Ag3222 and Ag4965 which show no expression of the CG57387-01 and CG57387-02 genes, the CG57387-03 gene produces a distinct expression profile. Please see below for presentation of the results and more complete discussion of the expression profile.







TABLE GA







Probe Name Ag5127













Start


Primers
Sequences
Length
Position














Forward
5′-cagaccaaggtcttccaca-3′
(SEQ ID NO:317)
19
815


Probe
TET-5′-agagcaggttcctctctgtcttcagca-3′-TAMRA
(SEQ ID NO:318)
27
835


Reverse
5′-actgttccatttgagactcca-3′
(SEQ ID NO:319)
21
864
















TABLE GB







CNS_neurodegeneration_v1.0











Rel. Exp. (%) Ag5127, Run

Rel. Exp. (%) Ag5127, Run


Tissue Name
226203927
Tissue Name
226203927













AD 1 Hippo
4.1
Control (Path) 3
4.0




Temporal Ctx


AD 2 Hippo
13.4
Control (Path) 4
22.1




Temporal Ctx



AD 3 Hippo
1.4
AD 1 Occipital Ctx
6.0


AD 4 Hippo
3.1
AD 2 Occipital Ctx
0.0




(Missing)


AD 5 Hippo
69.3
AD 3 Occipital Ctx
6.0


AD 6 Hippo
33.0
AD 4 Occipital Ctx
11.9


Control 2 Hippo
22.4
AD 5 Occipital Ctx
25.7


Control 4 Hippo
3.5
AD 6 Occipital Ctx
9.5


Control (Path) 3 Hippo
1.8
Control 1 Occipital Ctx
1.8


AD 1 Temporal Ctx
4.7
Control 2 Occipital Ctx
69.3


AD 2 Temporal Ctx
14.5
Control 3 Occipital Ctx
12.1


AD 3 Temporal Ctx
1.6
Control 4 Occipital Ctx
2.2


AD 4 Temporal Ctx
11.6
Control (Path) 1
100.0




Occipital Ctx


AD 5 Inf Temporal Ctx
75.8
Control (Path) 2
17.8




Occipital Ctx


AD 5 Sup Temporal
21.5
Control (Path) 3
1.0


Ctx

Occipital Ctx 


AD 6 Inf Temporal Ctx
33.4
Control (Path) 4
12.3




Occipital Ctx


AD 6 Sup Temporal
38.2
Control 1 Parietal Ctx
1.6


Ctx


Control 1 Temporal Ctx
0.5
Control 2 Parietal Ctx
22.2


Control 2 Temporal Ctx
31.0
Control 3 Parietal Ctx
6.5


Control 3 Temporal Ctx
2.4
Control (Path) 1
64.6




Parietal Ctx


Control 3 Temporal Ctx
3.8
Control (Path) 2
48.6




Parietal Ctx


Control (Path) 1
55.9
Control (Path) 3
1.5


Temporal Ctx

Parietal Ctx


Control (Path) 2
46.7
Control (Path) 4
37.9


Temporal Ctx

Parietal Ctx
















TABLE GC







General_screening_panel_v1.5











Rel. Exp. (%) Ag5127, Run

Rel. Exp. (%) Ag5127, Run


Tissue Name
228783308
Tissue Name
228783308













Adipose
5.2
Renal ca. TK-10
8.1


Melanoma* Hs688 (A).T
2.5
Bladder
12.6


Melanoma* Hs688 (B).T
2.0
Gastric ca. (liver met.)
9.9




NCI-N87


Melanoma* M14
7.4
Gastric ca. KATO III
3.6


Melanoma* LOXIMVI
3.4
Colon ca. SW-948
2.6


Melanoma* SK-MEL-5
6.1
Colon ca. SW480
8.0


Squamous Cell
4.4
Colon ca.* (SW480 met)
3.6


carcinoma SCC-4

SW620


Testis Pool
2.3
Colon ca. HT29
1.7


Prostate ca.* (bone met)
0.6
Colon ca. HCT-116
7.3


PC-3


Prostate Pool
6.8
Colon ca. CaCo-2
42.0


Placenta
1.0
Colon cancer tissue
1.9


Uterus Pool
4.0
Colon ca. SW1116
2.4


Ovarian ca. OVCAR-3
6.2
Colon ca. Colo-205
0.7


Ovarian ca. SK-OV-3
17.2
Colon ca. SW-48
0.9


Ovarian ca. OVCAR-4
4.7
Colon Pool
6.1


Ovarian ca. OVCAR-5
15.9
Small Intestine Pool
2.5


Ovarian ca. IGROV-1
1.2
Stomach Pool
2.8


Ovarian ca. OVCAR-8
3.0
Bone Marrow Pool
5.3


Ovary
1.6
Fetal Heart
1.6


Breast ca. MCF-7
9.6
Heart Pool
0.6


Breast ca. MDA-MB-
10.6
Lymph Node Pool
9.9


231


Breast ca. BT 549
9.8
Fetal Skeletal Muscle
2.5


Breast ca. T47D
2.6
Skeletal Muscle Pool
12.3


Breast ca. MDA-N
4.5
Spleen Pool
3.2


Breast Pool
5.6
Thymus Pool
9.5


Trachea
3.5
CNS cancer (glio/astro)
13.7




U87-MG


Lung
1.4
CNS cancer (glio/astro) U-
9.9




118-MG


Fetal Lung
4.9
CNS cancer (neuro;met)
9.7




SK-N-AS


Lung ca. NCI-N417
0.3
CNS cancer (astro) SF-539
1.5


Lung ca. LX-1
4.0
CNS cancer (astro) SNB-75
28.7


Lung ca. NCI-H146
0.2
CNS cancer (glio) SNB-19
1.4


Lung ca. SHP-77
3.8
CNS cancer (glio) SF-295
11.3


Lung ca. A549
8.4
Brain (Amygdala) Pool
3.9


Lung ca. NCI-H526
1.0
Brain (cerebellum)
100.0


Lung ca. NCI-H23
15.9
Brain (fetal)
17.9


Lung ca. NCI-H460
10.3
Brain (Hippocampus) Pool
3.2


Lung ca. HOP-62
3.9
Cerebral Cortex Pool
4.2


Lung ca. NCI-H522
9.5
Brain (Substantia nigra)
2.4




Pool


Liver
0.2
Brain (Thalamus) Pool
6.0


Fetal Liver
41.8
Brain (whole)
8.4


Liver ca. HepG2
3.3
Spinal Cord Pool
3.3


Kidney Pool
6.3
Adrenal Gland
5.2


Fetal Kidney
1.0
Pituitary gland Pool
0.9


Renal ca. 786-0
5.0
Salivary Gland
2.0


Renal ca. A498
1.6
Thyroid (female)
0.2


Renal ca. ACHN
5.0
Pancreatic ca. CAPAN2
2.8


Renal ca. UO-31
7.0
Pancreas Pool
5.3
















TABLE GD







Panel 4.1D











Rel. Exp. (%) Ag5127,

Rel. Exp. (%) Ag5127,


Tissue Name
Run 225784356
Tissue Name
Run 225784356













Secondary Th1 act
5.4
HUVEC IL-1beta
3.6


Secondary Th2 act
18.8
HUVEC IFN gamma
12.8


Secondary Tr1 act
19.1
HUVEC TNF alpha + IFN
7.8




gamma


Secondary Th1 rest
5.4
HUVEC TNF alpha + IL4
5.0


Secondary Th2 rest
4.2
HUVEC IL-11
4.5


Secondary Tr1 rest
6.0
Lung Microvascular EC none
19.6


Primary Th1 act
8.2
Lung Microvascular EC
12.8




TNF alpha + IL-1beta


Primary Th2 act
8.7
Microvascular Dermal EC none
10.8


Primary Tr1 act
13.4
Microsvasular Dermal EC
4.2




TNF alpha + IL-1beta


Primary Th1 rest
9.5
Bronchial epithelium TNF alpha +
5.7




IL1beta


Primary Th2 rest
9.0
Small airway epithelium none
7.3


Primary Tr1 rest
22.7
Small airway epithelium
8.5




TNF alpha + IL-1beta


CD45RA CD4 lymphocyte
10.4
Coronery artery SMC rest
5.1


act


CD45RO CD4 lymphocyte
21.8
Coronery artery SMC TNF alpha +
1.5


act

IL-1beta


CD8 lymphocyte act
17.2
Astrocytes rest
2.6


Secondary CD8
16.8
Astrocytes TNF alpha + IL-1beta
1.2


lymphocyte rest


Secondary CD8
5.5
KU-812 (Basophil) rest
16.7


lymphocyte act


CD4 lymphocyte none
5.2
KU-812 (Basophil)
33.4




PMA/ionomycin


2ry Th1/Th2/Tr1_anti-
21.9
CCD1106 (Keratinocytes) none
14.9


CD95 CH11


LAK cells rest
42.0
CCD1106 (Keratinocytes)
22.1




TNF alpha + IL-1beta


LAK cells IL-2
14.5
Liver cirrhosis
2.8


LAK cells IL-2 + IL-12
10.4
NCI-H292 none
6.5


LAK cells IL-2 + IFN
10.0
NCI-H292 IL-4
10.1


gamma


LAK cells IL-2 + IL-18
15.6
NCI-H292 IL-9
13.7


LAK cells
19.3
NCI-H292 IL-13
11.2


PMA/ionomycin


NK Cells IL-2 rest
24.8
NCI-H292 IFN gamma
8.4


Two Way MLR 3 day
25.9
HPAEC none
3.6


Two Way MLR 5 day
13.0
HPAEC TNF alpha + IL-1beta
12.8


Two Way MLR 7 day
10.7
Lung fibroblast none
19.1


PBMC rest
6.8
Lung fibroblast TNF alpha + IL-
11.3




1beta


PBMC PWM
3.6
Lung fibroblast IL-4
27.0


PBMC PHA-L
10.3
Lung fibroblast IL-9
48.3


Ramos (B cell) none
11.3
Lung fibroblast IL-13
22.7


Ramos (B cell) ionomycin
8.7
Lung fibroblast IFN gamma
22.1


B lymphocytes PWM
8.9
Dermal fibroblast CCD1070 rest
17.3


B lymphocytes CD40L
18.9
Dermal fibroblast CCD1070
20.7


and IL-4

TNF alpha


EOL-1 dbcAMP
84.1
Dermal fibroblast CCD1070 IL-
5.4




1beta


EOL-1 dbcAMP
30.6
Dermal fibroblast IFN gamma
6.4


PMA/ionomycin


Dendritic cells none
36.6
Dermal fibroblast IL-4
9.8


Dendritic cells LPS
21.3
Dermal Fibroblasts rest
15.1


Dendritic cells anti-CD40
45.1
Neutrophils TNFa + LPS
14.7


Monocytes rest
84.1
Neutrophils rest
62.0


Monocytes LPS
66.4
Colon
11.7


Macrophages rest
73.7
Lung
4.9


Macrophages LPS
4.8
Thymus
51.4


HUVEC none
5.7
Kidney
100.0


HUVEC starved
7.0









CNS_neurodegeneration_v1.0 Summary: Ag5127 This panel does not show differential expression of the CG57387-03 gene in Alzheimer's disease. However, this expression profile confirms the presence of this gene in the brain. Please see Panel 1.3D for discussion of utility of this gene in the central nervous system.
General_screening_panel_v1.5 Summary: Ag5127 Highest expression of the CG57387-03 gene is seen in the cerebellum (CT=27.7). This high level of expression in the cerebellum among all tissues examined indicates a specific role for this gene product in this brain region. Given the role of amino acid transporters in lysosomal storage disorders and the role of lysosomal disorders in CNS disease, this gene product may play an important role in CNS disease. Therefore, pharmacologic modulators of the activity of this gene product may have utility in treatment of neurodegenerative disorders such as Niemann-Pick type C disease and other diseases such as Alzheimer's disease, Parkinson's disease, and huntington's disease. In particular, spinocerebellar ataxias may benefit from the modulation of this gene, since they typically involve cerebellar dysfunction.
Significant expression is also seen in a colon cancer cell line and two ovarian cancer cell lines. Thus, expression of this gene could be used to differentiate between these samples and other samples on this panel and as a marker of cerebellar tissue and ovarian and colon cancer. Furthermore, therapeutic modulation of the expression or function of this gene may be effective in the treatment of colon or ovarian cancer.
Among tissues with metabolic function, this gene is expressed at moderate to low levels in adipose, adrenal gland, pancreas, fetal heart, and adult and fetal skeletal muscle and liver. This widespread expression among these tissues suggests that this gene product may be useful for the diagnosis and/or treatment of metabolic disease, including obesity and diabetes.
In addition, this gene is expressed at much higher levels in fetal liver(CT=29) than adult liver (CT=37). Thus, expression of this gene could be used to differentiate between the two sources of this tissue (German et al., Neuroscience 2002 February 14;109(3):437-50; Sagne et al., Proc Natl Acad Sci U S A 2001 June 19;98(13):7206-11).
Panel 4.1D Summary: Ag5127 Highest expression of the CG57387-03 gene is seen in the kidney (CT=29.9). This gene is also expressed at moderate levels in a wide range of cell types of significance in the immune response in health and disease. These cells include members of the T-cell, B-cell, endothelial cell, macrophage/monocyte, and peripheral blood mononuclear cell family, as well as epithelial and fibroblast cell types from lung and skin, and normal tissues represented by colon, lung, thymus and kidney. This ubiquitous pattern of expression suggests that this gene product may be involved in homeostatic processes for these and other cell types and tissues. This pattern is in agreement with the expression profile in General_screening_panel_v1.5 and also suggests a role for the gene product in cell survival and proliferation. Therefore, modulation of the gene product with a functional therapeutic may lead to the alteration of functions associated with these cell types and lead to improvement of the symptoms of patients suffering from autoimmune and inflammatory diseases such as asthma, allergies, inflammatory bowel disease, lupus erythematosus, psoriasis, rheumatoid arthritis, and osteoarthritis.
H. NOV8a and NOV8c: Lymphocyte Antigen Precursor
Expression of gene CG56417-01 and variant CG56417-03 was assessed using the primer-probe set Ag2917, described in Table HA. Results of the RTQ-PCR runs are shown in Tables HB, HC, HD, and HE.







TABLE HA







Probe Name Ag2917













Start


Primers
Sequences
Length
Position














Forward
5′-cagtctccaggccatgaag-3′
(SEQ ID NO:320)
19
36


Probe
TET-5′-accttgtccctggtcctgctggt-3′-TAMRA
(SEQ ID NO:321)
23
55


Reverse
5′-agaccctgagctctctccat-3′
(SEQ ID NO:322)
20
91
















TABLE HB







CNS_neurodegeneration_v1.0











Rel. Exp. (%) Ag2917, Run

Rel. Exp. (%) Ag2917, Run


Tissue Name
209735957
Tissue Name
209735957













AD 1 Hippo
1.8
Control (Path) 3
0.0




Temporal Ctx


AD 2 Hippo
12.7
Control (Path) 4
6.6




Temporal Ctx


AD 3 Hippo
0.0
AD 1 Occipital Ctx
6.6


AD 4 Hippo
0.0
AD 2 Occipital Ctx
0.0




(Missing)


AD 5 hippo
51.8
AD 3 Occipital Ctx
5.3


AD 6 Hippo
3.7
AD 4 Occipital Ctx
3.6


Control 2 Hippo
0.0
AD 5 Occipital Ctx
36.3


Control 4 Hippo
6.9
AD 6 Occipital Ctx
29.9


Control (Path) 3 Hippo
5.4
Control 1 Occipital Ctx
0.0


AD 1 Temporal Ctx
1.1
Control 2 Occipital Ctx
12.7


AD 2 Temporal Ctx
7.3
Control 3 Occipital Ctx
4.5


AD 3 Temporal Ctx
0.0
Control 4 Occipital Ctx
0.0


AD 4 Temporal Ctx
6.7
Control (Path) 1
15.5




Occipital Ctx


AD 5 Inf Temporal Ctx
100.0
Control (Path) 2
0.0




Occipital Ctx


AD 5 Sup Temporal Ctx
7.7
Control (Path) 3
4.5




Occipital Ctx


AD 6 Inf Temporal Ctx
9.4
Control (Path) 4
16.6




Occipital Ctx


AD 6 Sup Temporal Ctx
0.0
Control 1 Parietal Ctx
0.0


Control 1 Temporal Ctx
0.0
Control 2 Parietal Ctx
17.0


Control 2 Temporal Ctx
8.9
Control 3 Parietal Ctx
9.5


Control 3 Temporal Ctx
19.8
Control (Path) 1
19.5




Parietal Ctx


Control 4 Temporal Ctx
0.0
Control (Path) 2
1.6




Parietal Ctx


Control (Path) 1
31.2
Control (Path) 3
4.5


Temporal Ctx

Parietal Ctx


Control (Path) 2
13.5
Control (Path) 4
19.5


Temporal Ctx

Parietal Ctx
















TABLE HC







Panel 1.3D













Rel. Exp. (%)
Rel. Exp. (%)

Rel. Exp. (%)
Rel. Exp. (%)



Ag2917, Run
Ag2917, Run

Ag2917, Run
Ag2917, Run


Tissue Name
161411861
165721698
Tissue Name
161411861
165721698















Liver
0.0
0.0
Kidney (fetal)
0.0
0.0


adenocarcinoma


Pancreas
0.0
0.0
Renal ca. 786-0
0.0
0.0


Pancreatic ca.
2.5
25.2
Renal ca. A498
6.5
18.7


CAPAN 2


Adrenal gland
0.0
0.0
Renal ca. RXF
0.0
0.0





393


Thyroid
0.0
0.0
Renal ca. ACHN
0.4
0.0


Salivary gland
0.0
0.0
Renal ca. UO-31
0.0
0.0


Pituitary gland
0.0
0.0
Renal ca. TK-10
0.0
0.0


Brain (fetal)
0.0
0.0
Liver
0.0
0.0


Brain (whole)
0.0
100.0
Liver (fetal)
0.0
0.0


Brain (amygdala)
5.6
10.9
Liver ca.
0.0
0.0





(hepatoblast)





HepG2


Brain (cerebellum)
6.5
2.5
Lung
0.0
0.0


Brain (hippocampus)
0.6
41.2
Lung (fetal)
0.0
6.7


Brain (substantia
0.0
0.0
Lung ca. (small
0.0
0.0


nigra)


cell) LX-1


Brain (thalamus)
6.6
0.0
Lung ca. (small
0.0
0.0





cell) NCI-H69


Cerebral Cortex
100.0
17.1
Lung ca. (s. cell
0.0
0.0





var.) SHP-77


Spinal cord
12.9
26.2
Lung ca. (large
0.0
0.0





cell) NCI-H460


glio/astro U87-MG
1.7
0.0
Lung ca. (non-
0.0
0.0





sm. cell) A549


glio/astro U-118-
0.0
0.0
Lung ca. (non-
0.0
0.0


MG


s. cell) NCI-H23


astrocytoma
6.4
0.0
Lung ca. (non-
0.0
0.0


SW1783


s. cell) HOP-62


neuro*; met SK-N-
0.5
13.4
Lung ca. (non-
0.3
0.0


AS


s. cl) NCI-H522


astrocytoma SF-539
0.0
0.0
Lung ca.
0.0
0.0





(squam.) SW 900


astrocytoma SNB-75
0.0
0.0
Lung ca.
0.0
0.0





(squam.) NCI-





H596


glioma SNB-19
0.0
0.0
Mammary gland
0.0
0.0


glioma U251
0.0
0.0
Breast ca.*
0.0
0.0





(pl. ef) MCF-7


glioma SF-295
0.0
0.0
Breast ca.*
0.0
0.0





(pl. ef) MDA-





MB-231


Heart (fetal)
2.1
0.0
Breast ca.*
0.0
0.0





(pl. ef) T47D


Heart
0.0
12.6
Breast ca. BT-
0.0
0.0





549


Skeletal muscle
9.0
0.0
Breast ca. MDA-N
5.5
0.0


(fetal)


Skeletal muscle
0.4
38.4
Ovary
9.8
0.0


Bone marrow
0.0
0.0
Ovarian ca.
0.0
0.0





OVCAR-3


Thymus
7.4
0.0
Ovarian ca.
0.0
0.0





OVCAR-4


Spleen
13.4
37.6
Ovarian ca.
0.4
10.3





OVCAR-5


Lymph node
0.0
0.0
Ovarian ca.
0.8
17.0





OVCAR-8


Colorectal
0.0
0.0
Ovarian ca.
6.3
15.0





IGROV-1


Stomach
0.0
0.0
Ovarian ca.*
0.0
0.0





(ascites) SK-OV-3


Small intestine
0.0
0.0
Uterus
0.0
0.0


Colon ca. SW480
0.0
7.7
Placenta
0.0
0.0


Colon ca.*
0.0
0.0
Prostate
0.0
0.0


SW620 (SW480 met)


Colon ca. HT29
0.0
0.0
Prostate ca.*
0.0
0.0





(bone met)PC-3


Colon ca. HCT-116
0.0
0.0
Testis
18.0
0.0


Colon ca. CaCo-2
7.8
0.0
Melanoma
0.0
0.0





Hs688 (A).T


Colon ca.
0.0
0.0
Melanoma*
1.2
0.0


tissue (ODO3866)


(met)





Hs688 (B).T


Colon ca. HCC-2998
0.0
0.0
Melanoma
0.0
0.0





UACC-62


Gastric ca.* (liver
44.1
71.7
Melanoma M14
0.0
0.0


met) NCI-N87


Bladder
2.0
0.0
Melanoma LOX
0.0
0.0





IMVI


Trachea
10.9
4.8
Melanoma*
0.0
0.0





(met) SK-MEL-5


Kidney
0.0
0.0
Adipose
9.2
0.0
















TABLE HD







Panel 2D











Rel. Exp. (%) Ag2917,

Rel. Exp. (%) Ag2917,


Tissue Name
Run 161463558
Tissue Name
Run 161463558













Normal Colon
3.2
Kidney Margin 8120608
0.0


CC Well to Mod Diff
0.0
Kidney Cancer 8120613
0.0


(ODO3866)


CC Margin (ODO3866)
0.0
Kidney Margin 8120614
0.0


CC Gr. 2 rectosigmoid
0.0
Kidney Cancer 9010320
0.0


(ODO3868)


CC Margin (ODO3868)
0.8
Kidney Margin 9010321
0.9


CC Mod Diff (ODO3920)
0.0
Normal Uterus
0.0


CC Margin (ODO3920)
2.0
Uterus Cancer 064011
1.7


CC Gr. 2 ascend colon
0.2
Normal Thyroid
3.8


(ODO3921)


CC Margin (ODO3921)
0.0
Thyroid Cancer 064010
0.1


CC from Partial Hepatectomy
0.0
Thyroid Cancer A302152
0.0


(ODO4309) Mets


Liver Margin (ODO4309)
0.0
Thyroid Margin A302153
6.0


Colon mets to lung (OD04451-
0.0
Normal Breast
0.0


01)


Lung Margin (OD04451-02)
0.0
Breast Cancer (OD04566)
0.0


Normal Prostate 6546-1
1.6
Breast Cancer (OD04590-
0.0




01)


Prostate Cancer (OD04410)
0.2
Breast Cancer Mets
0.0




(OD04590-03)


Prostate Margin (OD04410)
0.8
Breast Cancer Metastasis
0.0




(OD04655-05)


Prostate Cancer (OD04720-01)
0.5
Breast Cancer 064006
0.9


Prostate Margin (OD04720-02)
0.8
Breast Cancer 1024
1.4


Normal Lung 061010
0.0
Breast Cancer 9100266
0.1


Lung Met to Muscle
1.7
Breast Margin 9100265
0.0


(ODO4286)


Muscle Margin (ODO4286)
2.0
Breast Cancer A209073
1.8


Lung Malignant Cancer
4.2
Breast Margin A209073
1.2


(OD03126)


Lung Margin (OD03126)
1.7
Normal Liver
0.0


Lung Cancer (OD04404)
0.0
Liver Cancer 064003
0.0


Lung Margin (OD04404)
0.1
Liver Cancer 1025
0.0


Lung Cancer (OD04565)
0.9
Liver Cancer 1026
0.0


Lung Margin (OD04565)
0.0
Liver Cancer 6004-T
0.4


Lung Cancer (OD04237-01)
0.0
Liver Tissue 6004-N
1.4


Lung Margin (OD04237-02)
0.0
Liver Cancer 6005-T
0.0


Ocular Mel Met to Liver
0.0
Liver Tissue 6005-N
0.0


(ODO4310)


Liver Margin (ODO4310)
0.0
Normal Bladder
0.0


Melanoma Mets to Lung
0.0
Bladder Cancer 1023
0.0


(OD04321)


Lung Margin (OD04321)
0.9
Bladder Cancer A302173
0.0


Normal Kidney
0.5
Bladder Cancer
100.0




(OD04718-01)


Kidney Ca, Nuclear grade 2
0.0
Bladder Normal Adjacent
0.0


(OD04338)

(OD04718-03)


Kidney Margin (OD04338)
0.0
Normal Ovary
0.0


Kidney Ca Nuclear grade 1/2
0.0
Ovarian Cancer 064008
0.8


(OD04339)


Kidney Margin (OD04339)
1.2
Ovarian Cancer
0.0




(OD04768-07)


Kidney Ca, Clear cell type
0.0
Ovary Margin (OD04768-
1.6


(OD04340)

08)


Kidney Margin (OD04340)
0.1
Normal Stomach
0.0


Kidney Ca, Nuclear grade 3
0.0
Gastric Cancer 9060358
0.0


(OD04348)


Kidney Margin (OD04348)
0.3
Stomach Margin 9060359
0.0


Kidney Cancer (OD04622-01)
0.0
Gastric Cancer 9060395
1.8


Kidney Margin (OD04622-03)
0.4
Stomach Margin 9060394
14.5


Kidney Cancer (OD04450-01)
0.0
Gastric Cancer 9060397
0.0


Kidney Margin (OD04450-03)
0.0
Stomach Margin 9060396
0.0


Kidney Cancer 8120607
0.0
Gastric Cancer 064005
31.6
















TABLE HE







Panel 3D











Rel. Exp. (%)

Rel. Exp. (%)



Ag2917, Run

Ag2917, Run


Tissue Name
164629842
Tissue Name
164629842













Daoy-Medulloblastoma
0.0
Ca Ski-Cervical epidermoid
0.0




carcinoma (metastasis)


TE671-Medulloblastoma
0.0
ES-2-Ovarian clear cell carcinoma
0.0


D283 Med-Medulloblastoma
0.0
Ramos-Stimulated with
0.0




PMA/ionomycin 6h


PFSK-1-Primitive
0.0
Ramos-Stimulated with
0.0


Neuroectodermal

PMA/ionomycin 14h


XF-498-CNS
0.0
MEG-01-Chronic myelogenous
0.0




leukemia (megokaryoblast)


SNB-78-Glioma
0.0
Raji-Burkitt's lymphoma
0.0


SF-268-Glioblastoma
0.0
Daudi-Burkitt's lymphoma
0.0


T98G-Glioblastoma
0.0
U266-B-cell plasmacytoma
0.0


SK-N-SH-Neuroblastoma
0.0
CA46-Burkitt's lymphoma
0.0


(metastasis)


SF-295-Glioblastoma
0.0
RL-non-Hodgkin's B-cell
0.0




lymphoma


Cerebellum
7.5
JM1-pre-B-cell lymphoma
0.0


Cerebellum
0.0
Jurkat-T cell leukemia
0.0


NCI-H292-Mucoepidermoid
0.0
TF-1-Erythroleukemia
0.0


lung carcinoma


DMS-114-Small cell lung
0.0
HUT 78-T-cell lymphoma
0.0


cancer


DMS-79-Small cell lung
10.3
U937-Histiocytic lymphoma
4.3


cancer


NCI-H146-Small cell lung
5.7
KU-812-Myelogenous leukemia
0.0


cancer


NCI-H526-Small cell lung
0.0
769-P-Clear cell renal carcinoma
0.0


cancer


NCI-N417-Small cell lung
0.0
Caki-2-Clear cell renal carcinoma
2.5


cancer


NCI-H82-Small cell lung
0.0
SW 839-Clear cell renal carcinoma
0.0


cancer


NCI-H157-Squamous cell
0.0
G401-Wilms' tumor
0.0


lung cancer (metastasis)


NCI-H1155-Large cell lung
0.0
Hs766T-Pancreatic carcinoma (LN
42.9


cancer

metastasis)


NCI-H1299-Large cell lung
0.0
CAPAN-1-Pancreatic
0.0


cancer

adenocarcinoma (liver metastasis)


NCI-H727-Lung carcinoid
0.0
SU86.86-Pancreatic carcinoma
0.6




(liver metastasis)


NCI-UMC-11-Lung
0.0
BxPC-3-Pancreatic
18.7


carcinoid

adenocarcinoma


LX-1-Small cell lung cancer
0.0
HPAC-Pancreatic adenocarcinoma
92.0


Colo-205-Colon cancer
0.0
MIA PaCa-2-Pancreatic carcinoma
0.0


KM12-Colon cancer
6.3
CFPAC-1-Pancreatic ductal
3.3




adenocarcinoma


KM20L2-Colon cancer
0.0
PANC-1-Pancreatic epithelioid
0.0




ductal carcinoma


NCI-H716-Colon cancer
0.0
T24-Bladder carcinma (transitional
0.0




cell)


SW-48-Colon
0.0
5637-Bladder carcinoma
0.0


adenocarcinoma


SW1116-Colon
0.0
HT-1197-Bladder carcinoma
13.6


adenocarcinoma


LS 174T-Colon
0.0
UM-UC-3-Bladder carcinma
0.0


adenocarcinoma

(transitional cell)


SW-948-Colon
0.0
A204-Rhabdomyosarcoma
0.0


adenocarcinoma


SW-480-Colon
0.0
HT-1080-Fibrosarcoma
0.0


adenocarcinoma


NCI-SNU-5-Gastric
0.0
MG-63-Osteosarcoma
0.0


carcinoma


KATO III-Gastric carcinoma
100.0
SK-LMS-1-Leiomyosarcoma
0.0




(vulva)


NCI-SNU-16-Gastric
0.0
SJRH30-Rhabdomyosarcoma (met
0.0


carcinoma

to bone marrow)


NCI-SNU-1-Gastric
0.0
A431-Epidermoid carcinoma
0.0


carcinoma


RF-1-Gastric
0.0
WM266-4-Melanoma
0.0


adenocarcinoma


RF-48-Gastric
0.0
DU 145-Prostate carcinoma (brain
0.0


adenocarcinoma

metastasis)


MKN-45-Gastric carcinoma
0.0
MDA-MB-468-Breast
12.2




adenocarcinoma


NCI-N87-Gastric carcinoma
4.0
SCC-4-Squamous cell carcinoma
0.0




of tongue


OVCAR-5-Ovarian
3.4
SCC-9-Squamous cell carcinoma
0.0


carcinoma

of tongue


RL95-2-Uterine carcinoma
3.0
SCC-15-Squamous cell carcinoma
0.0




of tongue


HelaS3-Cervical
0.0
CAL 27-Squamous cell carcinoma
0.0


adenocarcinoma

of tongue









CNS_neurodegeneration_v1.0 Summary: Ag2917 This panel does not show differential expression of the CG56417-01 gene in Alzheimer's disease. However, this expression profile confirms the presence of this gene in the brain. Please see Panel 1.3D for discussion of utility of this gene in the central nervous system.
Panel 1.3D Summary: Ag2917 Two experiments with the same probe and primer set both show highest expression of the CG56417-01 gene, a putative lymphocyte antigen, in regions of the brain (CTs=32-34). Preferential expression in the cerebral cortex indicates a role for this gene product in CNS processes. Lymphocytes are involved in the inflammatory response to brain pathologies such as trauma. Therefore, targeting this gene product with drugs or directing toxins to cells that specifically express this gene product may have utility in countering the inflammation that is thought to be part of the pathologic etiology of numerous CNS pathologies, such as stroke, trauma, and neurodegenerative disorders such as Alzheimer's, Parkinson's and Huntington's diseases.
Significant expression is also seen in a gastric cancer cell line. Thus, expression of this gene could be used to differentiate samples from the brain and the cancer cell line from other samples on this panel and as a marker for brain tissue and gastric cancer (Holmin et al., Neurosurgery 1998 February;42(2):291-8).
Panel 2D Summary: Ag2917 Highest expression of the CG56417-01 gene is seen in a bladder cancer (CT=29.4). Significant expression is also seen in a gastric cancer and normal thyroid. Thus, expression of this gene could be used to differentiate between these samples and other samples on this panel and as a marker for the presence of these cancers. Furthermore, therapeutic modulation of the expression or function of this gene may be effective in the treatment of bladder, gastric, or thyroid cancer.
Panel 3D Summary: Ag2917 Expression of the CG56417-01 gene is limited to cell lines derived from gastric cancer, pancreatic cancer, and bladder cancer (CTs=32-35). This expression is consistent with expression in Panel 2D. Thus, expression of this gene could be used to differentiate between these samples and other samples on this panel.
Panel 4.1D Summary: Ag2917 Expression of the CG56417-01 gene is low/undetectable in all samples on this panel (CTs>35). (Data not shown.)
Panel 4D Summary: Ag2917 Results from one experiment with the CG56417-01 gene are not included. The amp plot indicates that there were experimental difficulties with this run.
I. NOV8b and NOV8d: Lymphocyte Antigen Like
Expression of gene CG56417-02 and variant CG56417-04 was assessed using the primer-probe set Ag4251, described in Table IA.







TABLE IA







Probe Name Ag4251













Start





Posi- 


Primers
Sequences
Length
tion














Forward
5′-gtgttgctaggaagcatgttct-3′
(SEQ ID NO:323)
22
87


Probe
TET-5′-acacgtgcagtagctgcacctgctt-3′-TAMRA
(SEQ ID NO:324)
25
128


Reverse
5′-atgacacatgcgacgtatgag-3′
(SEQ ID NO:325)
21
154









CNS_neurodegeneration_v1.0 Summary: Ag4251 Expression of the CG56417-02 gene is low/undetectable in all samples on this panel (CTs>35). (Data not shown.) The amp plot indicates that there is a high probability of a probe failure.
General_screening_panel_v1.4 Summary: Ag4251 Expression of the CG56417-02 gene is low/undetectable in all samples on this panel (CTs>35). (Data not shown.) The amp plot indicates that there is a high probability of a probe failure.
Panel 4.1D Summary: Ag4251 Expression of the CG56417-02 gene is low/undetectable in all samples on this panel (CTs>35). (Data not shown.) The amp plot indicates that there is a high probability of a probe failure.
J. NOV9a and NOV9b: Early B-Cell Factor
Expression of gene CG57480-01 and variant CG57480-02 was assessed using the primer-probe set Ag3253, described in Table JA. Results of the RTQ-PCR runs are shown in Tables JB, JC and JD.







TABLE JA







Probe Name Ag3253













Start


Primers
Sequences
Length
Position














Forward
5′-ctttgtctacaccgcccttaat-3′
(SEQ ID NO:326)
22
1050


Probe
TET-5′-ccaaccatagattacggctttcagagg-3′-TAMRA
(SEQ ID NO:327)
27
1075


Reverse
5′-gatgtcttgggatcactttctg-3′
(SEQ ID NO:328)
22
1105
















TABLE JB







CNS_neurodegeneration_v1.0











Rel. Exp. (%) Ag3253, Run

Rel. Exp. (%) Ag3253, Run


Tissue Name
206533590
Tissue Name
206533590













AD 1 Hippo
0.0
Control (Path) 3
0.0




Temporal Ctx


AD 2 Hippo
7.9
Control (Path) 4
100.0




Temporal Ctx


AD 3 Hippo
0.0
AD 1 Occipital Ctx
0.0


AD 4 Hippo
0.0
AD 2 Occipital Ctx
0.0




(Missing)


AD 5 hippo
45.7
AD 3 Occipital Ctx
0.0


AD 6 Hippo
94.6
AD 4 Occipital Ctx
23.0


Control 2 Hippo
23.5
AD 5 Occipital Ctx
15.6


Control 4 Hippo
0.0
AD 6 Occipital Ctx
0.0


Control (Path) 3 Hippo
0.0
Control 1 Occipital Ctx
37.9


AD 1 Temporal Ctx
51.4
Control 2 occipital Ctx
0.0


AD 2 Temporal Ctx
0.0
Control 3 Occipital Ctx
0.0


AD 3 Temporal Ctx
39.8
Control 4 Occipital Ctx
55.1


AD 4 Temporal Ctx
21.2
Control (Path) 1
42.0




Occipital Ctx


AD 5 Inf Temporal Ctx
37.1
Control (Path) 2
22.4




Occipital Ctx


AD 5 SupTemporal Ctx
87.1
Control (Path) 3
0.0




Occipital Ctx


AD 6 Inf Temporal Ctx
76.8
Control (Path) 4
70.7




Occipital Ctx


AD 6 Sup Temporal Ctx
41.2
Control 1 Parietal Ctx
0.0


Control 1 Temporal Ctx
11.0
Control 2 Parietal Ctx
45.1


Control 2 Temporal Ctx
21.8
Control 3 Parietal Ctx
15.2


Control 3 Temporal Ctx
50.0
Control (Path) 1
19.8




Parietal Ctx


Control 4 Temporal Ctx
0.0
Control (Path) 2
34.6




Parietal Ctx


Control (Path) 1
54.0
Control (Path) 3
17.7


Temporal Ctx

Parietal Ctx


Control (Path) 2
40.6
Control (Path) 4
0.0


Temporal Ctx

Parietal Ctx
















TABLE JC







Panel 1.3D











Rel. Exp. (%) Ag3253, Run

Rel. Exp. (%) Ag3253, Run


Tissue Name
165524927
Tissue Name
165524927













Liver adenocarcinoma
0.0
Kidney (fetal)
0.7


Pancreas
3.3
Renal ca. 786-0
0.0


Pancreatic ca. CAPAN 2
0.0
Renal ca. A498
0.8


Adrenal gland
1.2
Renal ca. RXF 393
0.0


Thyroid
5.1
Renal ca. ACHN
20.9


Salivary gland
7.9
Renal ca. UO-31
13.6


Pituitary gland
1.0
Renal ca. TK-10
0.0


Brain (fetal)
0.0
Liver
0.7


Brain (whole)
3.8
Liver (fetal)
0.6


Brain (amygdala)
0.0
Liver ca. (hepatoblast)
0.0




HepG2


Brain (cerebellum)
8.8
Lung
0.0


Brain (hippocampus)
1.8
Lung (fetal)
0.8


Brain (substantia nigra)
81.8
Lung ca. (small cell) LX-1
0.0


Brain (thalamus)
0.0
Lung ca. (small cell)
5.9




NCI-H69


Cerebral Cortex
0.0
Lung ca. (s. cell var.)
0.0




SHP-77


Spinal cord
17.4
Lung ca. (large cell)NCI-
0.0




H460


glio/astro U87-MG
0.0
Lung ca. (non-sm. cell)
2.1




A549


glio/astro U-118-MG
0.0
Lung ca. (non-s. cell)
0.0




NCI-H23


astrocytoma SW1783
0.0
Lung ca. (non-s. cell)
0.0




HOP-62


neuro*; met SK-N-AS
75.8
Lung ca. (non-s. cl) NCI-
0.0




H522


astrocytoma SF-539
0.0
Lung ca. (squam.) SW
2.4




900


astrocytoma SNB-75
0.0
Lung ca. (squam.) NCI-
67.4




H596


glioma SNB-19
0.0
Mammary gland
29.1


glioma U251
0.0
Breast ca.* (pl. ef) MCF-7
0.0


glioma SF-295
0.0
Breast ca.* (pl. ef) MDA-
0.0




MB-231


Heart (fetal)
22.5
Breast ca.* (pl. ef) T47D
0.0


Heart
23.3
Breast ca. BT-549
0.0


Skeletal muscle (fetal)
57.0
Breast ca. MDA-N
0.0


Skeletal muscle
100.0
Ovary
3.3


Bone marrow
9.5
Ovarian ca. OVCAR-3
0.0


Thymus
6.0
Ovarian ca. OVCAR-4
0.0


Spleen
0.0
Ovarian ca. OVCAR-5
1.0


Lymph node
98.6
Ovarian ca. OVCAR-8
0.0


Colorectal
4.3
Ovarian ca. IGROV-1
0.0


Stomach
4.7
Ovarian ca.* (ascites)
0.0




SK-OV-3


Small intestine
6.4
Uterus
25.0


Colon ca. SW480
1.6
Placenta
11.0


Colon ca.* SW620 (SW480
0.0
Prostate
2.5


met)


Colon ca. HT29
0.0
Prostate ca.* (bone
0.0




met)PC-3


Colon ca. HCT-116
0.7
Testis
11.0


Colon ca. CaCo-2
2.8
Melanoma Hs688 (A).T
0.7


Colon ca.
9.3
Melanoma* (met)
24.0


tissue (ODO3866)

Hs688 (B).T


Colon ca. HCC-2998
0.0
Melanoma UACC-62
47.6


Gastric ca.* (liver met)
0.0
Melanoma M14
2.0


NCI-N87


Bladder
2.3
Melanoma LOX IMVI
0.0


Trachea
14.8
Melanoma* (met) SK-
17.9




MEL-5


Kidney
0.0
Adipose
58.6
















TABLE JD







Panel 4D











Rel. Exp. (%) Ag3253,

Rel. Exp. (%) Ag3253,


Tissue Name
Run 164391363
Tissue Name
Run 164391363













Secondary Th1 act
0.0
HUVEC IL-1beta
0.2


Secondary Th2 act
0.0
HUVEC IFN gamma
2.2


Secondary Tr1 act
0.0
HUVEC TNF alpha + IFN
0.6




gamma


Secondary Th1 rest
0.0
HUVEC TNF alpha + IL4
1.7


Secondary Th2 rest
0.0
HUVEC IL-11
2.6


Secondary Tr1 rest
0.0
Lung Microvascular EC none
0.8


Primary Th1 act
0.0
Lung Microvascular EC
0.1




TNF alpha + IL-1beta


Primary Th2 act
0.0
Microvascular Dermal EC none
0.9


Primary Tr1 act
0.0
Microsvasular Dermal EC
0.5




TNF alpha + IL-1beta


Primary Th1 rest
0.0
Bronchial epithelium TNF alpha +
0.0




IL1beta


Primary Th2 rest
0.0
Small airway epithelium none
0.0


Primary Tr1 rest
0.0
Small airway epithelium
0.0




TNF alpha + IL-1beta


CD45RA CD4 lymphocyte
3.0
Coronery artery SMC rest
36.1


act


CD45RO CD4 lymphocyte
0.0
Coronery artery SMC TNF alpha +
17.6


act

IL-1beta


CD8 lymphocyte act
0.0
Astrocytes rest
0.0


Secondary CD8
0.0
Astrocytes TNF alpha + IL-1beta
0.1


lymphocyte rest


Secondary CD8
0.0
KU-812 (Basophil) rest
0.0


lymphocyte act


CD4 lymphocyte none
0.0
KU-812 (Basophil)
0.0




PMA/ionomycin


2ry Th1/Th2/Tr1_anti-
0.2
CCD1106 (Keratinocytes) none
0.0


CD95 CH11


LAK cells rest
0.0
CCD1106 (Keratinocytes)
0.0




TNF alpha + IL-1beta


LAK cells IL-2
0.0
Liver cirrhosis
0.2


LAK cells IL-2 + IL-12
0.0
Lupus kidney
0.3


LAK cells IL-2 + IFN
0.0
NCI-H292 none
0.0


gamma


LAK cells IL-2 + IL-18
0.0
NCI-H292 IL-4
0.0


LAK cells
0.0
NCI-H292 IL-9
0.0


PMA/ionomycin


NK Cells IL-2 rest
0.0
NCI-H292 IL-13
0.0


Two Way MLR 3 day
0.0
NCI-H292 IFN gamma
0.0


Two Way MLR 5 day
0.0
HPAEC none
0.0


Two Way MLR 7 day
0.0
HPAEC TNF alpha + IL-1beta
0.0


PBMC rest
0.0
Lung fibroblast none
0.5


PBMC PWM
0.0
Lung fibroblast TNF alpha + IL-
0.5




1beta


PBMC PHA-L
0.0
Lung fibroblast IL-4
1.0


Ramos (B cell) none
0.0
Lung fibroblast IL-9
0.3


Ramos (B cell) ionomycin
0.0
Lung fibroblast IL-13
0.8


B lymphocytes PWM
0.4
Lung fibroblast IFN gamma
2.2


B lymphocytes CD40L
0.0
Dermal fibroblast CCD1070 rest
15.3


and IL-4


EOL-1 dbcAMP
40.1
Dermal fibroblast CCD1070
8.5




TNF alpha


EOL-1 dbcAMP
7.6
Dermal fibroblast CCD1070 IL-
6.4


PMA/ionomycin

1beta


Dendritic cells none
0.0
Dermal fibroblast IFN gamma
100.0


Dendritic cells LPS
0.0
Dermal fibroblast IL-4
36.1


Dendritic cells anti-CD40
0.0
IBD Colitis 2
0.2


Monocytes rest
0.0
IBD Crohn's
0.3


Monocytes LPS
0.1
Colon
2.2


Macrophages rest
0.0
Lung
3.5


Macrophages LPS
0.0
Thymus
0.9


HUVEC none
3.3
Kidney
3.3


HUVEC starved
8.8









CNS_neurodegeneration_v1.0 Summary: Ag3253 Expression of the CG57480-01 gene is low/undetectable in all samples on this panel (CTs>35). (Data not shown.)
Panel 1.3D Summary: Ag3253 Highest expression of the CG57480-01 gene is seen in skeletal muscle (CT=29). Significant expression is also seen in fetal skeletal muscle, adipose, fetal and adult heart, pancreas, and adrenal gland. This widespread expression among tissues with metabolic function suggests that this gene product may be important for the pathogenesis and/or treatment of disease involving these tissues, including obesity and diabetes.
Significant expression is also seen in cell lines derived from melanoma, lung cancer, renal cancer and brain cancer. Thus, expression of this gene could be used as a marker for the presence of these cancers.
Low but significant expression of this putative neuronal transcription factor is also seen in the substantia nigra, indicating a specific role for this gene product in this brain region. The substantia nigra is specifically vulnerable to neurodegeneration in Parkinson's disease. Neurodegeneration is known to involve gene regulation that is under the control of transcription factors. Therefore, drugs or treatments that modulate the activity of this gene product are potentially useful for the treatment of Parkinson's disease.
Panel 4D Summary: Ag3253 Highest expression of the CG57480-01 gene is seen in dermal fibroblasts treated with IFN-gamma (CT=28.6). Significant expression is also seen in a cluster of treated dermal fibroblast samples. This expression profile suggests that this gene product may be involved in skin disorders, including psoriasis.
The transcript is also expressed in differentiated EOL-1 cells, and is downregulated after activation suggesting that it may be important in eosinophil differentiation. Regulating the transcript with antisense strategies or the protein product with small molecule therapeutics may be usefue in the treatment of hematopoietic disorders involving eosinphils, parasitic infections and asthma.
K. NOV10: High-Affinity CAMP-Specific and IBMX-Insensitive
Expression of gene CG57389-01 was assessed using the primer-probe set Ag3223, described in Table KA. Results of the RTQ-PCR runs are shown in Table KB.







TABLE KA







Probe Name Ag3223













Start


Primers
Sequences
Length
Position














Forward
5′-attcttacactgctccgagtca-3′
(SEQ ID NO:329)
22
1723


Probe
TET-5′-tcatggttacaaattatcgaagccaa-3′-TAMRA
(SEQ ID NO:330)
26
1754


Reverse
5′-ggtagggattggaggaatgata-3′
(SEQ ID NO:331)
22
1781
















TABLE KB







Panel 1.3D











Rel. Exp. (%) Ag3223, Run

Rel. Exp. (%) Ag3223, Run


Tissue Name
165527086
Tissue Name
165527086













Liver adenocarcinoma
44.4
Kidney (fetal)
21.8


Pancreas
14.1
Renal ca. 786-0
13.2


Pancreatic ca. CAPAN 2
33.7
Renal ca. A498
23.7


Adrenal gland
42.9
Renal ca. RXF 393
35.8


Thyroid
11.0
Renal ca. ACHN
6.6


Salivary gland
8.0
Renal ca. UO-31
8.8


Pituitary gland
18.2
Renal ca. TK-10
6.7


Brain (fetal)
7.9
Liver
14.0


Brain (whole)
100.0
Liver (fetal)
13.8


Brain (amygdala)
32.5
Liver ca. (hepatoblast)
44.8




HepG2


Brain (cerebellum)
21.2
Lung
13.8


Brain (hippocampus)
55.9
Lung (fetal)
9.8


Brain (substantia nigra)
80.1
Lung ca. (small cell) LX-1
26.4


Brain (thalamus)
98.6
Lung ca. (small cell)
4.0




NCI-H69


Cerebral Cortex
67.4
Lung ca. (s. cell var.)
17.7




SHP-77


Spinal cord
58.6
Lung ca. (large cell)NCI-
64.2




H460


glio/astro U87-MG
32.5
Lung ca. (non-sm. cell)
47.6




A549


glio/astro U-118-MG
63.3
Lung ca. (non-s. cell)
31.2




NCI-H23


astrocytoma SW1783
18.6
Lung ca. (non-s. cell)
15.5




HOP-62


neuro*; met SK-N-AS
13.1
Lung ca. (non-s. cl) NCI-
6.7




H522


astrocytoma SF-539
34.4
Lung ca. (squam.) SW
11.1




900


astrocytoma SNB-75
19.3
Lung ca. (squam.) NCI-
7.7




H596


glioma SNB-19
32.3
Mammary gland
18.2


glioma U251
64.2
Breast ca.* (pl. ef) MCF-7
30.4


glioma SF-295
9.3
Breast ca.* (pl. ef) MDA-
37.1




MB-231


Heart (fetal)
26.6
Breast ca.* (pl. ef) T47D
7.4


Heart
10.8
Breast ca. BT-549
28.5


Skeletal muscle (fetal)
16.2
Breast ca. MDA-N
10.8


Skeletal muscle
18.3
Ovary
18.9


Bone marrow
4.2
Ovarian ca. OVCAR-3
15.7


Thymus
7.3
Ovarian ca. OVCAR-4
6.8


Spleen
14.9
Ovarian ca. OVCAR-5
33.7


Lymph node
35.1
Ovarian ca. OVCAR-8
3.6


Colorectal
43.5
Ovarian ca. IGROV-1
7.7


Stomach
19.5
Ovarian ca.* (ascites)
18.2




SK-OV-3


Small intestine
49.0
Uterus
26.8


Colon ca. SW480
29.7
Placenta
7.5


Colon ca.* SW620 (SW480
31.6
Prostate
6.5


met)


Colon ca. HT29
8.1
Prostate ca.* (bone
19.2




met) PC-3


Colon ca. HCT-116
22.8
Testis
25.3


Colon ca. CaCo-2
10.1
Melanoma Hs688 (A).T
4.8


Colon ca.
20.2
Melanoma* (met)
9.0


tissue (ODO3866)

Hs688 (B).T


Colon ca. HCC-2998
13.1
Melanoma UACC-62
12.4


Gastric ca.* (liver met)
74.7
Melanoma M14
74.2


NCI-N87


Bladder
19.9
Melanoma LOX IMVI
3.3


Trachea
7.3
Melanoma* (met) SK-
12.3




MEL-5


Kidney
25.2
Adipose
17.1









Panel 1.3D Summary: Ag3223 The expression of the CG57389-01 gene, a nucleotide phosphodiesterase homolog, appears to be highest in a sample derived from a normal brain tissue. In addition, this gene is expressed in all CNS regions examined. Nucleotide phosphodiesterases are involved in a wide range of processes in the CNS. Therefore, this gene may be useful as a drug target in neurodegenerative diseases such as Alzheimer's, Parkinson's, Huntington's, stroke, head or spinal cord trauma, or neuropsychiatric disease.
In addition, there appears to be substantial expression in other samples derived from brain cancer cell lines, lung cancer cell lines and colon cancer cell lines. Thus, the expression of this gene could be used to distinguish normal brain tissue from other samples in the panel. Moreover, therapeutic modulation of this gene, through the use of small molecule drugs, protein therapeutics, or antibodies could be of benefit in the treatment of brain, lung or colon cancer.
Among metabolic tissues, this gene is expressed at low levels in pancreas, adrenal, thyroid, pituitary, heart, liver, adipose and skeletal muscle. Regulation of cyclic nucleotide levels is believed to be important for insulin secretion, which is dysregulated or impaired in diabetes. Thus, this gene product may be a small molecule target for the treatment of metabolic and endocrine disease, specifically Types 1 and 2 diabetes and obesity.
L. NOV12a, NOV12b, NOV12c and NOV13: TWIK-3 and TASK-4
Expression of gene CG57220-01 and variants CG57220-02, CG57220-03 and CG57220-04 was assessed using the primer-probe sets Ag4293 and Ag937, described in Tables LA and LB. Results of the RTQ-PCR runs are shown in Tables LC, LD and LE. Please note that only variants CG57220-02 and CG57220-04 match the probe and primer set Ag937.







TABLE LA







Probe Name Ag4293













Start


Primers
Sequences
Length
Position














Forward
5′-gaggtacccactgtggtacaag-3′
(SEQ ID NO:332)
22
733


Probe
TET-5′-acatggtgtccctgtggatcctcttt-3′-TAMRA
(SEQ ID NO:333)
26
756


Reverse
5′-gggagaggatgagtttgatgat-3′
(SEQ ID NO:334)
22
803
















TABLE LB







Probe Name Ag937













Start


Primers
Sequences
Length
Position














Forward
5′-ggctccttcttcttttctgtgt-3′
(SEQ ID NO:335)
22
341


Probe
TET-5′-atcaccaccattggctatggcaacct-3′-TAMRA
(SEQ ID NO:336)
26
368


Reverse
5′-ggcaaagaagatgcagaagag-3′
(SEQ ID NO:337)
21
419
















TABLE LC







CNS_neurodegeneration_v1.0











Rel. Exp. (%) Ag4293, Run

Rel. Exp. (%) Ag4293, Run


Tissue Name
224073647
Tissue Name
224073647













AD 1 Hippo
19.5
Control (Path) 3
0.0




Temporal Ctx


AD 2 Hippo
52.9
Control (Path) 4
24.0




Temporal Ctx


AD 3 Hippo
0.0
AD 1 Occipital Ctx
18.8


AD 4 Hippo
10.6
AD 2 Occipital Ctx
0.0




(Missing)


AD 5 Hippo
26.4
AD 3 Occipital Ctx
0.0


AD 6 Hippo
82.4
AD 4 Occipital Ctx
11.4


Control 2 Hippo
50.7
AD 5 Occipital Ctx
33.4


Control 4 Hippo
49.0
AD 6 Occipital Ctx
23.3


Control (Path) 3 Hippo
11.7
Control 1 Occipital Ctx
8.5


AD 1 Temporal Ctx
20.9
Control 2 Occipital Ctx
52.5


AD 2 Temporal Ctx
47.6
Control 3 Occipital Ctx
0.0


AD 3 Temporal Ctx
0.0
Control 4 Occipital Ctx
0.0


AD 4 Temporal Ctx
3.2
Control (Path) 1
27.9




Occipital Ctx


AD 5 Inf Temporal Ctx
49.3
Control (Path) 2
42.3




Occipital Ctx


AD 5 Sup Temporal
27.5
Control (Path) 3
17.9


Ctx

Occipital Ctx


AD 6 Inf Temporal Ctx
12.4
Control (Path) 4
35.6




Occipital Ctx


AD 6 Sup Temporal
13.6
Control 1 Parietal Ctx
10.0


Ctx


Control 1 Temporal Ctx
12.7
Control 2 Parietal Ctx
0.0


Control 2 Temporal Ctx
3.1
Control 3 Parietal Ctx
0.0


Control 3 Temporal Ctx
0.0
Control (Path) 1
24.7




Parietal Ctx


Control 3 Temporal Ctx
0.0
Control (Path) 2
53.2




Parietal Ctx


Control (Path) 1
100.0
Control (Path) 3
10.8


Temporal Ctx

Parietal Ctx


Control (Path) 2
52.5
Control (Path) 4
56.6


Temporal Ctx

Parietal Ctx
















TABLE LD







General_screening_panel_v1.4











Rel. Exp. (%) Ag4293, Run

Rel. Exp. (%) Ag4293, Run


Tissue Name
222182574
Tissue Name
222182574













Adipose
3.0
Renal ca. TK-10
0.0


Melanoma* Hs688 (A).T
0.0
Bladder
5.9


Melanoma* Hs688 (B).T
0.0
Gastric ca. (liver met.)
0.0




NCI-N87


Melanoma* M14
0.0
Gastric ca. KATO III
0.0


Melanoma* LOXIMVI
0.0
Colon ca. SW-948
0.0


Melanoma* SK-MEL-5
0.0
Colon ca. SW480
0.0


Squamous cell
0.0
Colon ca.* (SW480 met)
0.0


carcinoma SCC-4

SW620


Testis Pool
3.3
Colon ca. HT29
0.0


Prostate ca.* (bone met)
0.0
Colon ca. HCT-116
0.0


PC-3


Prostate Pool
1.5
Colon ca. CaCo-2
0.0


Placenta
7.5
Colon cancer tissue
2.1


Uterus Pool
2.5
Colon ca. SW1116
0.0


Ovarian ca. OVCAR-3
0.2
Colon ca. Colo-205
0.0


Ovarian ca. SK-OV-3
0.1
Colon ca. SW-48
0.0


Ovarian ca. OVCAR-4
0.2
Colon Pool
7.6


Ovarian ca. OVCAR-5
0.5
Small Intestine Pool
3.1


Ovarian ca. IGROV-1
0.0
Stomach Pool
4.2


Ovarian ca. OVCAR-8
0.0
Bone Marrow Pool
1.2


Ovary
0.5
Fetal Heart
3.2


Breast ca. MCF-7
0.2
Heart Pool
5.8


Breast ca. MDA-MB-
0.0
Lymph Node Pool
4.0


231


Breast ca. BT 549
0.0
Fetal Skeletal Muscle
4.6


Breast ca. T47D
0.6
Skeletal Muscle Pool
0.0


Breast ca. MDA-N
0.4
Spleen Pool
0.7


Breast Pool
3.1
Thymus Pool
7.4


Trachea
1.2
CNS cancer (glio/astro)
0.0




U87-MG


Lung
2.0
CNS cancer (glio/astro) U-
0.0




118-MG


Fetal Lung
100.0
CNS cancer (neuro; met)
0.0




SK-N-AS


Lung ca. NCI-N417
0.0
CNS cancer (astro) SF-539
0.0


Lung ca. LX-1
0.0
CNS cancer (astro) SNB-75
0.0


Lung ca. NCI-H146
71.2
CNS cancer (glio) SNB-19
0.2


Lung ca. SHP-77
0.0
CNS cancer (glio) SF-295
0.0


Lung ca. A549
0.0
Brain (Amygdala) Pool
1.5


Lung ca. NCI-H526
0.0
Brain (cerebellum)
1.7


Lung ca. NCI-H23
0.2
Brain (fetal)
0.2


Lung ca. NCI-H460
0.0
Brain (Hippocampus) Pool
1.6


Lung ca. HOP-62
0.0
Cerebral Cortex Pool
1.1


Lung ca. NCI-H522
0.0
Brain (Substantia nigra)
0.8




Pool


Liver
1.0
Brain (Thalamus) Pool
0.5


Fetal Liver
1.3
Brain (whole)
1.4


Liver ca. HepG2
0.0
Spinal Cord Pool
2.0


Kidney Pool
10.2
Adrenal Gland
0.6


Fetal Kidney
1.9
Pituitary gland Pool
1.2


Renal ca. 786-0
0.0
Salivary Gland
0.4


Renal ca. A498
0.0
Thyroid (female)
6.0


Renal ca. ACHN
0.0
Pancreatic ca. CAPAN2
0.0


Renal ca. UO-31
0.0
Pancreas Pool
3.1
















TABLE LE







Panel 4.1D











Rel. Exp. (%) Ag4293,

Rel. Exp. (%) Ag4293,


Tissue Name
Run 181981932
Tissue Name
Run 181981932













Secondary Th1 act
0.0
HUVEC IL-1beta
0.0


Secondary Th2 act
0.0
HUVEC IFN gamma
0.0


Secondary Tr1 act
0.0
HUVEC TNF alpha + IFN
0.0




gamma


Secondary Th1 rest
0.0
HUVEC TNF alpha + IL4
0.0


Secondary Th2 rest
2.8
HUVEC IL-11
0.0


Secondary Tr1 rest
0.0
Lung Microvascular EC none
0.0


Primary Th1 act
0.0
Lung Microvascular EC
0.0




TNF alpha + IL-1beta


Primary Th2 act
0.0
Microvascular Dermal EC none
0.0


Primary Tr1 act
0.0
Microsvasular Dermal EC
0.0




TNF alpha + IL-beta


Primary Th1 rest
0.0
Bronchial epithelium TNF alpha +
0.0




IL1beta


Primary Th2 rest
1.8
Small airway epithelium none
0.0


Primary Tr1 rest
0.0
Small airway epithelium
0.0




TNF alpha + IL-1beta


CD45RA CD4 lymphocyte
0.0
Coronery artery SMC rest
0.0


act


CD45RO CD4 lymphocyte
0.0
Coronery artery SMC TNF alpha +
0.0


act

IL-1beta


CD8 lymphocyte act
0.0
Astrocytes rest
0.0


Secondary CD8
0.0
Astrocytes TNF alpha + IL-1beta
0.0


lymphocyte rest


Secondary CD8
0.0
KU-812 (Basophil) rest
1.0


lymphocyte act


CD4 lymphocyte none
2.0
KU-812 (Basophil)
0.0




PMA/ionomycin


2ry Th1/Th2/Tr1_anti-
0.0
CCD1106 (Keratinocytes) none
0.0


CD95 CH11


LAK cells rest
0.0
CCD1106 (Keratinocytes)
0.0




TNF alpha + IL-1beta


LAK cells IL-2
0.0
Liver cirrhosis
0.9


LAK cells IL-2 + IL-12
0.0
NCI-H292 none
6.7


LAK cells IL-2 + IFN
0.0
NCI-H292 IL-4
20.2


gamma


LAK cells IL-2 + IL-18
0.0
NCI-H292 IL-9
10.7


LAK cells
0.0
NCI-H292 IL-13
29.1


PMA/ionomycin


NK Cells IL-2 rest
4.9
NCI-H292 IFN gamma
12.9


Two Way MLR 3 day
0.8
HPAEC none
0.0


Two Way MLR 5 day
0.0
HPAEC TNF alpha + IL-1beta
0.0


Two Way MLR 7 day
0.0
Lung fibroblast none
0.0


PBMC rest
2.1
Lung fibroblast TNF alpha + IL-
0.0




1beta


PBMC PWM
0.0
Lung fibroblast IL-4
0.0


PBMC PHA-L
0.0
Lung fibroblast IL-9
0.0


Ramos (B cell) none
0.0
Lung fibroblast IL-13
0.0


Ramos (B cell) ionomycin
0.0
Lung fibroblast IFN gamma
0.0


B lymphocytes PWM
0.0
Dermal fibroblast CCD1070 rest
0.0


B lymphocytes CD40L
0.0
Dermal fibroblast CCD1070
0.7


and IL-4

TNF alpha


EOL-1 dbcAMP
8.1
Dermal fibroblast CCD1070 IL-
1.0




1beta


EOL-1 dbcAMP
0.0
Dermal fibroblast IFN gamma
0.0


PMA/ionomycin


Dendritic cells none
0.0
Dermal fibroblast IL-4
0.0


Dendritic cells LPS
0.0
Dermal Fibroblasts rest
0.0


Dendritic cells anti-CD40
0.0
Neutrophils TNFa + LPS
0.0


Monocytes rest
0.0
Neutrophils rest
4.0


Monocytes LPS
0.0
Colon
4.8


Macrophages rest
0.0
Lung
100.0


Macrophages LPS
0.0
Thymus
24.0


HUVEC none
0.0
Kidney
77.9


HUVEC starved
0.0









CNS_neurodegeneration_v1.0 Summary: Ag4293 No change is detected in the expression of the CG57220-01 gene in the postmortem Alzheimer's diseased brain when compared to controls. However this panel confirms the expression of this gene in the CNS in an independent group of patients. Thus, therapeutic modulation of the expression or function of this gene product may be useful in the treatment of neurologic disease. A second experiment with the probe primer set Ag937 showed low/undetectable levels of expression in all the samples on this panel (CTs>35). (Data not shown.)
General_screening_panel_v1.4 Summary: Ag4293 Highest expression of the CG57220-01 gene is seen in the fetal lung (CT=29)and a lung cancer cell line. In addition, this gene appears to be expressed at much higher levels in the fetal tissue than in the adult (CT=34.7). Higher levels of expression are also seen in fetal skeletal muscle C(T=33) than in adult skeletal muscle. Thus, expression of this gene could be used to differentiate between the two lung derived samples and other samples on this panel and as a marker to detect the presence of lung cancer. In addition, expression of this gene could be used to differentiate between the two sources of lung and skeletal muscle tissue. Furthermore, therapeutic modulation of the expression or function of this gene may be effective in the treatment of lung cancer.
Among tissues with metabolic function, this gene is expressed at low but significant levels in adipose, thyroid, pancreas, and adult and fetal heart. This widespread expression among these tissues suggests that this gene product may be useful for the diagnosis and/or treatment of metabolic disease, including obesity and diabetes.
Panel 4.1D Summary: Ag4293 Highest expression of the CG57220-01 gene is seen in the lung (CT=31). Significant levels of expression are also seen in a cluster of treated and untreated samples derived from the muco-epidermoid cell line NCI-H292, often used as a model for airway epithelium. The prominent expression in lung derived samples, both in this panel and General_screening_panel v1.4 suggests that this gene may be involved in normal lung homeostasis as well as pathological and inflammatory lung conditions. Therefore, therapeutic modulation of this gene product may resuce symptoms associated with chronic obstructive pulmonary disease, asthma, allergy and emphysema.
Panel CNS—1 Summary: Ag937 Expression of the CG57220-02 gene is low/undetectable in all samples on this panel (CTs>35). (Data not shown.)
M. NOV15: Cytokeratin
Expression of gene CG57454-01 was assessed using the primer-probe set Ag3244, described in Table MA. Results of the RTQ-PCR runs are shown in Tables MB, MC, MD and ME.







TABLE MA







Probe Name Ag3244













Start


Primers
Sequences
Length
Position














Forward
5′-gctatgcaggtggtctgagtt-3′
(SEQ ID NO:338)
21
1267


Probe
TET-5′-ctcacaagccctggcctcagctat-3′-TAMRA
(SEQ ID NO:339)
24
1302


Reverse
5′-agagccaaagctggagctta-3′
(SEQ ID NO:340)
20
1330
















TABLE MB







CNS_neurodegeneration_v1.0











Rel. Exp. (%) Ag3244, Run

Rel. Exp. (%) Ag3244, Run


Tissue Name
206533582
Tissue Name
206533582













AD 1 Hippo
18.7
Control (Path) 3
0.0




Temporal Ctx


AD 2 Hippo
23.0
Control (Path) 4
19.6




Temporal Ctx


AD 3 Hippo
2.3
AD 1 Occipital Ctx
9.6


AD 4 Hippo
9.9
AD 2 Occipital Ctx
0.0




(Missing)


AD 5 hippo
71.7
AD 3 Occipital Ctx
6.0


AD 6 Hippo
42.0
AD 4 Occipital Ctx
31.6


Control 2 Hippo
29.1
AD 5 Occipital Ctx
4.2


Control 4 Hippo
22.8
AD 6 Occipital Ctx
34.4


Control (Path) 3 Hippo
0.4
Control 1 Occipital Ctx
2.7


AD 1 Temporal Ctx
69.3
Control 2 Occipital Ctx
30.6


AD 2 Temporal Ctx
23.0
Control 3 Occipital Ctx
33.0


AD 3 Temporal Ctx
4.4
Control 4 Occipital Ctx
12.9


AD 4 Temporal Ctx
33.4
Control (Path) 1
88.3




Occipital Ctx


AD 5 InfTemporal Ctx
100.0
Control (Path) 2
9.0




Occipital Ctx


AD 5 SupTemporal Ctx
23.8
Control (Path) 3
0.0




Occipital Ctx


AD 6 InfTemporal Ctx
57.0
Control (Path) 4
11.7




Occipital Ctx


AD 6 Sup Temporal Ctx
42.0
Control 1 Parietal Ctx
24.5


Control 1 Temporal Ctx
4.4
Control 2 Parietal Ctx
59.5


Control 2 Temporal Ctx
28.7
Contro1 3 Parietal Ctx
4.5


Control 3 Temporal Ctx
15.5
Control (Path) 1
59.0




Parietal Ctx


Control 4 Temporal Ctx
5.3
Control (Path) 2
23.2




Parietal Ctx


Control (Path) 1
28.7
Control (Path) 3
0.0


Temporal Ctx

Parietal Ctx


Control (Path) 2
32.5
Control (Path) 4
37.6


Temporal Ctx

Parietal Ctx
















TABLE MC







Panel 1.3D











Rel. Exp. (%) Ag3244, Run

Rel. Exp. (%) Ag3244, Run


Tissue Name
165524419
Tissue Name
165524419













Liver adenocarcinoma
91.4
Kidney (fetal)
10.6


Pancreas
3.4
Renal ca. 786-0
8.3


Pancreatic ca. CAPAN 2
23.7
Renal ca. A498
29.1


Adrenal gland
0.0
Renal ca. RXF 393
1.7


Thyroid
13.4
Renal ca. ACHN
10.9


Salivary gland
4.1
Renal ca. UO-31
42.3


Pituitary gland
57.0
Renal ca. TK-10
9.4


Brain (fetal)
0.0
Liver
5.4


Brain (whole)
48.3
Liver (fetal)
12.5


Brain (amygdala)
82.9
Liver ca. (hepatoblast)
18.3




HepG2


Brain (cerebellum)
36.3
Lung
9.4


Brain (hippocampus)
88.3
Lung (fetal)
1.7


Brain (substantia nigra)
63.3
Lung ca. (small cell) LX-1
52.5


Brain (thalamus)
41.8
Lung ca. (small cell)
1.7




NCI-H69


Cerebral Cortex
7.1
Lung ca. (s. cell var.)
19.3




SHP-77


Spinal cord
60.7
Lung ca. (large cell) NCI-
9.5




H460


glio/astro U87-MG
0.0
Lung ca. (non-sm. cell)
20.6




A549


glio/astro U-118-MG
0.0
Lung ca. (non-s. cell)
0.0




NCI-H23


astrocytoma SW1783
0.0
Lung ca. (non-s. cell)
10.2




HOP-62


neuro*; met SK-N-AS
7.4
Lung ca. (non-s. cl) NCI-
0.0




H522


astrocytoma SF-539
0.0
Lung ca. (squam.) SW
7.4




900


astrocytoma SNB-75
17.1
Lung ca. (squam.) NCI-
10.6




H596


glioma SNB-19
2.4
Mammary gland
7.0


glioma U251
2.6
Breast ca.* (pl. ef) MCF-7
80.7


glioma SF-295
0.0
Breast ca.* (pl. ef) MDA-
34.9




MB-231


Heart (fetal)
3.1
Breast ca.* (pl. ef) T47D
21.8


Heart
2.7
Breast ca. BT-549
0.0


Skeletal muscle (fetal)
0.0
Breast ca. MDA-N
0.0


Skeletal muscle
0.0
Ovary
6.0


Bone marrow
0.0
Ovarian ca. OVCAR-3
37.6


Thymus
0.0
Ovarian ca. OVCAR-4
22.5


Spleen
0.0
Ovarian ca. OVCAR-5
98.6


Lymph node
0.0
Ovarian ca. OVCAR-8
5.2


Colorectal
9.3
Ovarian ca. IGROV-1
6.0


Stomach
15.1
Ovarian ca.* (ascites)
5.2




SK-OV-3


Small intestine
39.8
Uterus
0.0


Colon ca. SW480
70.7
Placenta
52.1


Colon ca.* SW620 (SW480
9.2
Prostate
19.1


met)


Colon ca. HT29
27.2
Prostate ca.* (bone
0.0




met) PC-3


Colon ca. HCT-116
23.5
Testis
20.4


Colon ca. CaCo-2
22.7
Melanoma Hs688 (A).T
0.0


Colon ca.
85.9
Melanoma* (met)
0.0


tissue (ODO3866)

Hs688 (B).T


Colon ca. HCC-2998
17.8
Melanoma UACC-62
0.0


Gastric ca.* (liver met)
100.0
Melanoma M14
0.0


NCI-N87


Bladder
9.5
Melanoma LOX IMVI
0.0


Trachea
19.9
Melanoma* (met) SK-
0.0




MEL-5


Kidney
8.1
Adipose
3.8
















TABLE MD







Panel 2.2











Rel. Exp. (%) Ag3244,

Rel. Exp. (%) Ag3244,


Tissue Name
Run 174441100
Tissue Name
Run 174441100













Normal Colon
31.0
Kidney Margin (OD04348)
12.8


Colon cancer (OD06064)
59.0
Kidney malignant cancer
25.0




(OD06204B)


Colon Margin (OD06064)
8.0
Kidney normal adjacent
6.5




tissue (OD06204E)


Colon cancer (OD06159)
100.0
Kidney Cancer (OD04450-
39.5




01)


Colon Margin (OD06159)
43.2
Kidney Margin (OD04450-
2.2




03)


Colon cancer (OD06297-04)
16.0
Kidney Cancer 8120613
6.3


Colon Margin (OD06297-05)
38.7
Kidney Margin 8120614
11.3


CC Gr. 2 ascend colon
23.5
Kidney Cancer 9010320
2.0


(ODO3921)


CC Margin (ODO3921)
20.7
Kidney Margin 9010321
20.9


Colon cancer metastasis
12.9
Kidney Cancer 8120607
17.1


(OD06104)


Lung Margin (OD06104)
30.1
Kidney Margin 8120608
4.4


Colon mets to lung
45.4
Normal Uterus
0.0


(OD04451-01)


Lung Margin (OD04451-02)
3.3
Uterine Cancer 064011
0.0


Normal Prostate
9.4
Normal Thyroid
8.1


Prostate Cancer (OD04410)
3.8
Thyroid Cancer 064010
16.4


Prostate Margin (OD04410)
1.5
Thyroid Cancer A302152
9.3


Normal Ovary
2.6
Thyroid Margin A302153
4.5


Ovarian cancer (OD06283-
7.5
Normal Breast
58.6


03)


Ovarian Margin (OD06283-
1.3
Breast Cancer (OD04566)
16.0


07)


Ovarian Cancer 064008
3.0
Breast Cancer 1024
17.4


Ovarian cancer (OD06145)
8.0
Breast Cancer (OD04590-
59.0




01)


Ovarian Margin (OD06145)
3.2
Breast Cancer Mets
24.7




(OD04590-03)


Ovarian cancer (OD06455-
10.4
Breast Cancer Metastasis
21.6


03)

(OD04655-05)


Ovarian Margin (OD06455-
0.0
Breast Cancer 064006
12.8


07)


Normal Lung
1.4
Breast Cancer 9100266
10.0


Invasive poor diff. lung
14.2
Breast Margin 9100265
1.4


adeno (ODO4945-01


Lung Margin (ODO4945-03)
2.5
Breast Cancer A209073
7.6


Lung Malignant Cancer
25.3
Breast Margin A2090734
24.7


(OD03126)


Lung Margin (OD03126)
3.7
Breast cancer (OD06083)
46.0


Lung Cancer (OD05014A)
6.7
Breast cancer node
22.7




metastasis (OD06083)


Lung Margin (OD05014B)
1.3
Normal Liver
5.5


Lung cancer (OD06081)
1.0
Liver Cancer 1026
22.7


Lung Margin (OD06081)
1.1
Liver Cancer 1025
68.3


Lung Cancer (OD04237-01)
18.6
Liver Cancer 6004-T
19.8


Lung Margin (OD04237-02)
16.3
Liver Tissue 6004-N
11.6


Ocular Melanoma Metastasis
0.0
Liver Cancer 6005-T
62.4


Ocular Melanoma Margin
10.0
Liver Tissue 6005-N
31.4


(Liver)


Melanoma Metastasis
11.3
Liver Cancer 064003
7.4


Melanoma Margin (Lung)
4.1
Normal Bladder
31.2


Normal Kidney
7.3
Bladder Cancer 1023
20.2


Kidney Ca, Nuclear grade 2
12.0
Bladder Cancer A302173
20.6


(OD04338)


Kidney Margin (OD04338)
16.2
Normal Stomach
51.1


Kidney Ca Nuclear grade 1/2
22.5
Gastric Cancer 9060397
49.0


(OD04339)


Kidney Margin (OD04339)
3.6
Stomach Margin 9060396
50.3


Kidney Ca, Clear cell type
11.7
Gastric Cancer 9060395
52.1


(OD04340)


Kidney Margin (OD04340)
3.7
Stomach Margin 9060394
39.5


Kidney Ca, Nuclear grade 3
1.1
Gastric Cancer 064005
46.3


(OD04348)
















TABLE ME







Panel 4D











Rel. Exp. (%) Ag3244,

Rel. Exp. (%) Ag3244,


Tissue Name
Run 164390753
Tissue Name
Run 164390753













Secondary Th1 act
0.0
HUVEC IL-1beta
0.0


Secondary Th2 act
0.0
HUVEC IFN gamma
3.9


Secondary Tr1 act
0.0
HUVEC TNF alpha + IFN
3.1




gamma


Secondary Th1 rest
0.0
HUVEC TNF alpha + IL4
0.0


Secondary Th2 rest
0.0
HUVEC IL-11
5.1


Secondary Tr1 rest
0.0
Lung Microvascular EC none
27.5


Primary Th1 act
0.0
Lung Microvascular EC
39.5




TNF alpha + IL-1beta


Primary Th2 act
0.0
Microvascular Dermal EC none
0.0


Primary Tr1 act
0.0
Microsvasular Dermal EC
6.3




TNF alpha + IL-1beta


Primary Th1 rest
0.0
Bronchial epithelium TNF alpha +
18.3




IL1beta


Primary Th2 rest
0.0
Small airway epithelium none
7.6


Primary Tr1 rest
0.0
Small airway epithelium
23.7




TNF alpha + IL-1beta


CD45RA CD4 lymphocyte
2.8
Coronery artery SMC rest
0.0


act


CD45RO CD4 lymphocyte
0.0
Coronery artery SMC TNF alpha +
0.0


act

IL-1beta


CD8 lymphocyte act
3.1
Astrocytes rest
0.6


Secondary CD8
0.0
Astrocytes TNF alpha + IL-1beta
1.3


lymphocyte rest


Secondary CD8
0.0
KU-812 (Basophil) rest
0.0


lymphocyte act


CD4 lymphocyte none
0.0
KU-812 (Basophil)
0.0




PMA/ionomycin


2ry Th1/Th2/Tr1_anti-
0.0
CCD1106 (Keratinocytes) none
16.2


CD95 CH11


LAK cells rest
0.0
CCD1106 (Keratinocytes)
25.0




TNF alpha + IL-1beta


LAK cells IL-2
0.7
Liver cirrhosis
40.1


LAK cells IL-2 + IL-12
0.0
Lupus kidney
2.5


LAK cells IL-2 + IFN
0.0
NCI-H292 none
38.4


gamma


LAK cells IL-2 + IL-18
0.0
NCI-H292 IL-4
100.0


LAK cells
0.0
NCI-H292 IL-9
58.2


PMA/ionomycin


NK Cells IL-2 rest
3.3
NCI-H292 IL-13
59.0


Two Way MLR 3 day
0.0
NCI-H292 IFN gamma
87.7


Two Way MLR 5 day
0.0
HPAEC none
3.7


Two Way MLR 7 day
0.0
HPAEC TNF alpha + IL-1beta
0.0


PBMC rest
0.0
Lung fibroblast none
2.4


PBMC PWM
0.0
Lung fibroblast TNF alpha + IL-
0.0




1beta


PBMC PHA-L
0.0
Lung fibroblast IL-4
27.7


Ramos (B cell) none
0.0
Lung fibroblast IL-9
13.8


Ramos (B cell) ionomycin
0.0
Lung fibroblast IL-13
11.1


B lymphocytes PWM
0.0
Lung fibroblast IFN gamma
15.4


B lymphocytes CD40L
0.0
Dermal fibroblast CCD1070 rest
1.0


and IL-4


EOL-1 dbcAMP
0.0
Dermal fibroblast CCD1070
7.8




TNF alpha


EOL-1 dbcAMP
0.0
Dermal fibroblast CCD1070 IL-
0.0


PMA/ionomycin

1beta


Dendritic cells none
3.2
Dermal fibroblast IFN gamma
80.1


Dendritic cells LPS
0.0
Dermal fibroblast IL-4
98.6


Dendritic cells anti-CD40
0.8
IBD Colitis 2
3.5


Monocytes rest
0.0
IBD Crohn's
3.8


Monocytes LPS
0.0
Colon
51.4


Macrophages rest
0.0
Lung
17.9


Macrophages LPS
0.0
Thymus
9.3


HUVEC none
10.0
Kidney
1.5


HUVEC starved
4.7









CNS_neurodegeneration_v1.0 Summary: Ag3244 No change is detected in the expression of the CG57454-01 gene in the postmortem Alzheimer's diseased brain when compared to controls. See panel 1.3 for discussion of utility.
Panel 1.3D Summary: Ag3244 The expression of the CG57454-01 gene appears to be highest in a sample derived from a gastric cancer cell line (NCI-N87)(CT=33.9). In addition, there appears to be substantial expression in samples derived from ovarian cancer cell lines and colon cancer cell lines. Thus, the expression of this gene could be used to distinguish NCI-N87 cells from other samples in the panel. Moreover, therapeutic modulation of this gene, through the use of small molecule drugs, protein therapeutics or antibodies could be of benefit in the treatment of gastric cancer, colon cancer or ovarian cancer.
Panel 2.2 Summary: Ag3244 The expression of the CG57454-01 gene appears to be highest in a sample derived from a colon cancer sample (CT=33.1). In addition there appears to be substantial expression in other samples derived from colon cancers. Thus, the expression of this gene could be used to distinguish colon cancer from other samples in the panel. Moreover, therapeutic modulation of this gene, through the use of small molecule drugs, protein therapeutics or antibodies could be of benefit in the treatment of colon cancer.
Panel 4D Summary: Ag3244 The CG57454-01 transcript is expressed in dermal fibroblasts, lung fibroblasts, NCI-H292 cells and lung microvascular cells. The transcript is also expressed in normal colon but not in colitis samples. Thus, the transcript or the protein it encodes could be used to differentiate these cells and tissues from other samples on this panel that do not express this gene. Therapeutically, the protein encoded by this transcript could be used to design treatments for colitis, psoriasis, arthritis, asthma and emphysema.
N. NOV16a: Protocadherin 10
Expression of gene CG57448-01 was assessed using the primer-probe sets Ag1096, Ag3242 and Ag704, described in Tables NA, NB and NC. Results of the RTQ-PCR runs are shown in Tables ND, NE, NF, NG and NH.







TABLE NA







Probe Name Ag1096













Start


Primers
Sequences
Length
Position














Forward
5′-actttggaagaggcattgct-3′
(SEQ ID NO:341)
20
2455


Probe
TET-5′-cagacagattatgggctccatcgctt-3′-TAMRA
(SEQ ID NO:342)
26
2476


Reverse
5′-ctcgggataaccatgatcact-3′
(SEQ ID NO:343)
21
2515
















TABLE NB







Probe Name Ag3242













Start


Primers
Sequences
Length
Position














Forward
5′-gggaccaatgctcaaattactt-3′
(SEQ ID NO:344)
22
1090


Probe
TET-5′-tcagaaagttccacaagcatctaagga-3′-TAMRA
(SEQ ID NO:345)
27
1122


Reverse
5′-atgactccagtgttttcatcca-3′
(SEQ ID NO:346)
22
1160
















TABLE NC







Probe Name Ag704













Start


Primers
Sequences
Length
Position














Forward
5′-agacttggggaccaatgct-3′
(SEQ ID NO:347)
19
1083


Probe
TET-5′-tcagaaagttccacaagcatctaagga-3′-TAMRA
(SEQ ID NO:348)
27
1122


Reverse
5′-tgactccagtgttttcatcca-3′
(SEQ ID NO:349)
21
1160
















TABLE ND







CNS_neurodegeneration_v1.0













Rel. Exp. (%)
Rel. Exp. (%)

Rel. Exp. (%)
Rel. Exp. (%)



Ag1096, Run
Ag3242, Run

Ag1096, Run
Ag3242, Run


Tissue Name
206231465
206533581
Tissue Name
206231465
206533581















AD 1 Hippo
27.0
16.5
Control (Path)
0.9
1.4





3 Temporal Ctx


AD 2 Hippo
11.5
16.8
Control (Path)
11.8
4.9





4 Temporal Ctx


AD 3 Hippo
8.8
5.3
AD 1 Occipital
6.2
2.9





Ctx


AD 4 Hippo
2.0
1.9
AD 2 Occipital
0.0
0.0





Ctx (Missing)


AD 5 Hippo
46.7
36.6
AD 3 Occipital
5.6
2.8





Ctx


AD 6 Hippo
100.0
100.0
AD 4 Occipital
6.3
4.1





Ctx


Control 2
51.1
35.1
AD 5 Occipital
25.0
16.8


Hippo


Ctx


Control 4
5.3
4.0
AD 6 Occipital
5.3
1.0


Hippo


Ctx


Control (Path)
4.6
1.0
Control 1
0.8
0.3


3 Hippo


Occipital Ctx


AD 1
3.6
2.7
Control 2
17.1
19.5


Temporal Ctx


Occipital Ctx


AD 2
12.5
7.5
Control 3
3.1
3.5


Temporal Ctx


Occipital Ctx


AD 3
2.5
1.0
Control 4
1.4
0.8


Temporal Ctx


Occipital Ctx


AD 4
6.3
3.3
Control (Path)
34.6
30.1


Temporal Ctx


1 Occipital Ctx


AD 5 Inf
74.7
59.5
Control (Path)
2.2
0.6


Temporal Ctx


2 Occipital Ctx


AD 5 Sup
66.0
61.6
Control (Path)
0.7
0.4


Temporal Ctx


3 Occipital Ctx


AD 6 Inf
12.2
12.2
Control (Path)
3.2
1.4


Temporal Ctx


4 Occipital Ctx


AD 6 Sup
14.7
12.3
Control 1
1.3
1.4


Temporal Ctx


Parietal Ctx


Control 1
1.9
2.2
Control 2
21.8
19.6


Temporal Ctx


Parietal Ctx


Control 2
21.8
22.2
Control 3
5.9
5.7


Temporal Ctx


Parietal Ctx


Control 3
5.9
4.6
Control (Path)
38.7
38.7


Temporal Ctx


1 Parietal Ctx


Control 3
1.7
0.9
Control (Path)
9.5
4.3


Temporal Ctx


2 Parietal Ctx


Control (Path)
33.9
28.7
Control (Path)
1.4
0.5


1 Temporal


3 Parietal Ctx


Ctx


Control (Path)
17.3
12.3
Control (Path)
16.8
15.5


2 Temporal


4 Parietal Ctx


Ctx
















TABLE NE







Panel 1.2













Rel. Exp. (%)
Rel. Exp. (%)

Rel. Exp. (%)
Rel. Exp. (%)



Ag1096, Run
Ag1096, Run

Ag1096, Run
Ag1096, Run


Tissue Name
125094875
134204525
Tissue Name
125094875
134204525















Endothelial cells
0.0
0.0
Renal ca. 786-0
0.0
0.0


Heart (Fetal)
4.5
5.6
Renal ca. A498
0.0
0.0


Pancreas
0.2
0.3
Renal ca. RXF
0.1
0.1





393


Pancreatic ca.
0.0
0.0
Renal ca. ACHN
31.4
80.1


CAPAN 2


Adrenal Gland
1.6
2.8
Renal ca. UO-31
0.0
0.0


Thyroid
0.1
0.4
Renal ca.TK-10
1.0
1.3


Salivary gland
3.0
4.8
Liver
1.9
5.3


Pituitary gland
2.7
6.7
Liver (fetal)
0.8
1.7


Brain (fetal)
2.1
7.9
Liver ca.
0.0
0.0





(hepatoblast)





HepG2


Brain (whole)
9.9
39.8
Lung
1.7
5.9


Brain (amygdala)
15.0
34.2
Lung (fetal)
1.8
9.9


Brain (cerebellum)
0.8
1.9
Lung ca. (small
0.4
1.2





cell) LX-1


Brain
39.0
73.2
Lung ca. (small
0.2
0.5


(hippocampus)


cell) NCI-H69


Brain (thalamus)
2.5
9.2
Lung ca. (s. cell
2.3
2.6





var.) SHP-77


Cerebral Cortex
27.0
56.3
Lung ca. (large
50.7
100.0





cell) NCI-H460


Spinal cord
2.2
4.8
Lung ca. (non-
0.1
0.0





sm. cell) A549


glio/astro U87-MG
0.1
0.2
Lung ca. (non-
5.4
4.0





s. cell) NCI-H23


glio/astro U-118-
0.1
0.2
Lung ca. (non-
15.3
33.2


MG


s. cell) HOP-62


astrocytoma
0.0
0.0
Lung ca. (non-
0.0
0.0


SW1783


s. cl) NCI-H522


neuro*; met SK-N-
6.9
7.3
Lung ca.
5.4
12.5


AS


(squam.) SW 900


astrocytoma SF-
0.0
0.0
Lung ca.
0.0
0.1


539


(squam.) NCI-





H596


astrocytoma SNB-
7.0
6.8
Mammary gland
0.6
2.7


75


glioma SNB-19
2.4
2.2
Breast ca.* (pl. ef)
0.0
0.0





MCF-7


glioma U251
5.5
11.6
Breast ca.* (pl. ef)
0.0
0.0





MDA-MB-231


glioma SF-295
0.0
0.0
Breast ca.* (pl.
0.9
1.1





ef) T47D


Heart
13.2
15.5
Breast ca. BT-
0.7
0.7





549


Skeletal Muscle
1.1
1.2
Breast ca. MDA-N
0.0
0.0


Bone marrow
0.0
0.1
Ovary
0.6
0.4


Thymus
0.5
1.7
Ovarian ca.
0.5
0.3





OVCAR-3


Spleen
0.4
0.8
Ovarian ca.
0.0
0.1





OVCAR-4


Lymph node
0.2
0.2
Ovarian ca.
15.4
15.2





OVCAR-5


Colorectal Tissue
4.3
8.1
Ovarian ca.
0.4
1.2





OVCAR-8


Stomach
3.9
8.8
Ovarian ca.
0.1
0.0





IGROV-1


Small intestine
1.9
3.7
Ovarian ca.
1.9
1.3





(ascites) SK-OV-3


Colon ca. SW480
0.0
0.1
Uterus
2.2
10.6


Colon ca.* SW620
0.1
0.2
Placenta
8.2
10.1


(SW480 met)


Colon ca. HT29
0.0
0.1
Prostate
3.4
3.5


Colon ca. HCT-
0.0
0.0
Prostate ca.*
2.6
3.5


116


(bone met) PC-3


Colon ca. CaCo-2
0.0
0.0
Testis
1.3
2.1


Colon ca. Tissue
0.3
1.1
Melanoma
0.0
0.0


(ODO3866)


Hs688(A).T


Colon ca. HCC-
2.9
4.3
Melanoma* (met)
0.0
0.1


2998


Hs688(B).T


Gastric ca.* (liver
0.0
0.0
Melanoma
0.0
0.0


met) NCI-N87


UACC-62


Bladder
0.4
0.7
Melanoma M14
0.5
0.1


Trachea
3.6
16.5
Melanoma LOX
0.1
0.0





IMVI


Kidney
13.2
2.7
Melanoma* (met)
100.0
86.5





SK-MEL-5


Kidney (fetal)
7.2
10.3
















TABLE NF







Panel 1.3D











Rel. Exp. (%) Ag3242, Run

Rel. Exp. (%) Ag3242, Run


Tissue Name
165524415
Tissue Name
165524415













Liver adenocarcinoma
0.0
Kidney (fetal)
2.7


Pancreas
1.0
Renal ca. 786-0
0.0


Pancreatic ca. CAPAN 2
0.0
Renal ca. A498
0.0


Adrenal gland
0.3
Renal ca. RXF 393
0.4


Thyroid
0.0
Renal ca. ACHN
100.0


Salivary gland
0.6
Renal ca. UO-31
0.0


Pituitary gland
4.8
Renal ca. TK-10
1.1


Brain (fetal)
11.8
Liver
1.0


Brain (whole)
40.6
Liver (fetal)
0.0


Brain (amygdala)
31.6
Liver ca. (hepatoblast)
0.0




HepG2


Brain (cerebellum)
3.0
Lung
4.8


Brain (hippocampus)
76.3
Lung (fetal)
2.2


Brain (substantia nigra)
2.1
Lung ca. (small cell) LX-1
3.7


Brain (thalamus)
6.3
Lung ca. (small cell)
0.0




NCI-H69


Cerebral Cortex
27.7
Lung ca. (s. cell var.)
16.0




SHP-77


Spinal cord
7.7
Lung ca. (large cell) NCI-
46.0




H460


glio/astro U87-MG
0.6
Lung ca (non-sm. cell)
0.0




A549


glio/astro U-118-MG
0.5
Lung ca. (non-s. cell)
8.0




NCI-H23


astrocytoma SW1783
0.0
Lung ca. (non-s. cell)
13.6




HOP-62


neuro*; met SK-N-AS
7.5
Lung ca. (non-s. cl) NCI-
0.0




H522


astrocytoma SF-539
0.0
Lung ca. (squam.) SW
18.8




900


astrocytoma SNB-75
8.4
Lung ca. (squam.) NCI-
0.0




H596


glioma SNB-19
3.0
Mammary gland
1.4


glioma U251
66.4
Breast ca.* (pl. ef) MCF-7
0.0


glioma SF-295
0.6
Breast ca.* (pl. ef) MDA-
0.0




MB-231


Heart (fetal)
3.7
Breast ca.* (pl. ef) T47D
1.4


Heart
1.7
Breast ca. BT-549
1.6


Skeletal muscle (fetal)
6.9
Breast ca. MDA-N
0.0


Skeletal muscle
1.9
Ovary
1.5


Bone marrow
0.0
Ovarian ca. OVCAR-3
1.4


Thymus
0.4
Ovarian ca. OVCAR-4
0.0


Spleen
0.0
Ovarian ca. OVCAR-5
24.8


Lymph node
0.0
Ovarian ca. OVCAR-8
0.6


Colorectal
3.5
Ovarian ca. IGROV-1
0.0


Stomach
2.5
Ovarian ca.* (ascites)
5.4




SK-OV-3


Small intestine
7.2
Uterus
15.5


Colon ca. SW480
0.0
Placenta
1.1


Colon ca.* SW620 (SW480
0.0
Prostate
1.4


met)


Colon ca. HT29
0.0
Prostate ca.* (bone
0.4




met) PC-3


Colon ca. HCT-116
0.0
Testis
2.3


Colon ca. CaCo-2
0.0
Melanoma Hs688 (A).T
0.0


Colon ca.
0.0
Melanoma* (met)
0.0


tissue (ODO3866)

Hs688 (B).T


Colon ca. HCC-2998
2.1
Melanoma UACC-62
0.0


Gastric ca.* (liver met)
0.0
Melanoma M14
1.6


NCI-N87


Bladder
0.0
Melanoma LOX IMVI
0.0


Trachea
9.0
Melanoma* (met) SK-
58.6




MEL-5


Kidney
1.5
Adipose
1.5
















TABLE NG







Panel 2.2











Rel. Exp. (%) Ag3242,

Rel. Exp. (%) Ag3242,


Tissue Name
Run 174443348
Tissue Name
Run 174443348













Normal Colon
4.4
Kidney Margin (OD04348)
1.0


Colon cancer (OD06064)
1.8
Kidney malignant cancer
0.4




(OD06204B)


Colon Margin (OD06064)
8.9
Kidney normal adjacent
1.8




tissue (OD06204E)


Colon cancer (OD06159)
2.0
Kidney Cancer (OD04450-
2.3




01)


Colon Margin (OD06159)
7.1
Kidney Margin (OD04450-
0.9




03)


Colon cancer (OD06297-04)
2.4
Kidney Cancer 8120613
0.0


Colon Margin (OD06297-05)
2.2
Kidney Margin 8120614
0.4


CC Gr. 2 ascend colon
0.9
Kidney Cancer 9010320
1.4


(ODO3921)


CC Margin (ODO3921)
3.8
Kidney Margin 9010321
1.3


Colon cancer metastasis
0.7
Kidney Cancer 8120607
0.8


(OD06104)


Lung Margin (OD06104)
0.0
Kidney Margin 8120608
0.2


Colon mets to lung
1.7
Normal Uterus
5.3


(OD04451-01)


Lung Margin (OD04451-02)
10.1
Uterine Cancer 064011
0.5


Normal Prostate
0.4
Normal Thyroid
0.0


Prostate Cancer (OD04410)
0.3
Thyroid Cancer 064010
0.7


Prostate Margin (OD04410)
0.0
Thyroid Cancer A302152
0.9


Normal Ovary
0.0
Thyroid Margin A302153
0.0


Ovarian cancer (OD06283-
0.0
Normal Breast
0.6


03


Ovarian Margin (OD06283-
0.0
Breast Cancer (OD04566)
0.0


07)


Ovarian Cancer 064008
1.9
Breast Cancer 1024
2.1


Ovarian cancer (OD06145)
0.0
Breast Cancer (OD04590-
0.0




01)


Ovarian Margin (OD06145)
0.0
Breast Cancer Mets
0.0




(OD04590-03)


Ovarian cancer (OD06455-
0.0
Breast Cancer Metastasis
0.4


03)

(OD04655-05)


Ovarian Margin (OD06455-
1.2
Breast Cancer 064006
0.7


07)


Normal Lung
2.3
Breast Cancer 9100266
0.7


Invasive poor diff. lung
1.1
Breast Margin 9100265
0.2


adeno (ODO4945-01)


Lung Margin (ODO4945-03)
1.6
Breast Cancer A209073
0.0


Lung Malignant Cancer
0.7
Breast Margin A2090734
0.0


(OD03126)


Lung Margin (OD03126)
1.2
Breast cancer (OD06083)
0.4


Lung Cancer (OD05014A)
1.2
Breast cancer node
0.9




metastasis (OD06083)


Lung Margin (OD05014B)
4.4
Normal Liver
1.3


Lung cancer (OD06081)
0.8
Liver Cancer 1026
0.3


Lung Margin (OD06081)
2.8
Liver Cancer 1025
0.0


Lung Cancer (OD04237-01)
0.0
Liver Cancer 6004-T
0.0


Lung Margin (OD04237-02)
6.0
Liver Tissue 6004-N
0.0


Ocular Melanoma Metastasis
100.0
Liver Cancer 6005-T
2.7


Ocular Melanoma Margin
0.7
Liver Tissue 6005-N
3.3


(Liver)


Melanoma Metastasis
1.2
Liver Cancer 064003
7.1


Melanoma Margin (Lung)
4.9
Normal Bladder
0.0


Normal Kidney
0.0
Bladder Cancer 1023
0.0


Kidney Ca, Nuclear grade 2
1.3
Bladder Cancer A302173
0.3


(OD04338)


Kidney Margin (OD04338)
0.4
Normal Stomach
5.8


Kidney Ca Nuclear grade 1/2
0.6
Gastric Cancer 9060397
0.0


(OD04339)


Kidney Margin (OD04339)
1.6
Stomach Margin 9060396
2.6


Kidney Ca, Clear cell type
0.0
Gastric Cancer 9060395
2.2


(OD04340)


Kidney Margin (OD04340)
1.1
Stomach Margin 9060394
3.4


Kidney Ca, Nuclear grade 3
0.0
Gastric Cancer 064005
0.3


(OD04348)
















TABLE NH







Panel 4D













Rel. Exp. (%)
Rel. Exp. (%)

Rel. Exp. (%)
Rel. Exp. (%)



Ag1096, Run
Ag3242, Run

Ag1096, Run
Ag3242, Run


Tissue Name
160353275
164390548
Tissue Name
160353275
164390548















Secondary Th1 act
0.0
0.0
HUVEC IL-1beta
0.0
0.0


Secondary Th2 act
0.0
0.0
HUVEC IFN gamma
0.4
0.0


Secondary Tr1 act
0.0
0.0
HUVEC TNF alpha +
0.0
0.0





IFN gamma


Secondary Th1 rest
0.0
0.0
HUVEC TNF alpha +
2.0
0.0





IL4


Secondary Th2 rest
0.0
0.0
HUVEC IL-11
0.0
0.0


Secondary Tr1 rest
0.0
0.0
Lung Microvascular
0.0
0.0





EC none


Primary Th1 act
0.0
0.0
Lung Microvascular
0.0
0.0





EC TNFalpha + IL-





1beta


Primary Th2 act
0.0
0.0
Microvascular
0.0
0.0





Dermal EC none


Primary Tr1 act
0.0
0.0
Microsvasular Dermal
0.0
0.0





EC TNFalpha + IL-





1beta


Primary Th1 rest
0.0
0.0
Bronchial epithelium
0.0
0.0





TNFalpha+ IL1beta


Primary Th2 rest
0.0
0.0
Small airway
0.0
0.0





epithelium none


Primary Tr1 rest
0.0
0.0
Small airway
6.5
11.7





epithelium





TNFalpha + IL-1beta


CD45RA CD4
0.0
0.0
Coronery artery SMC
0.7
0.0


lymphocyte act


rest


CD45RO CD4
0.0
0.0
Coronery artery SMC
0.0
0.0


lymphocyte act


TNFalpha + IL-1beta


CD8 lymphocyte act
1.3
0.0
Astrocytes rest
13.9
16.8


Secondary CD8
0.7
0.0
Astrocytes
3.6
5.3


lymphocyte rest


TNFalpha + IL-1beta


Secondary CD8
0.0
0.0
KU-812 (Basophil)
0.9
1.0


lymphocyte act


rest


CD4 lymphocyte
0.0
0.0
KU-812 (Basophil)
100.0
100.0


none


PMA/ionomycin


2ry
0.0
0.0
CCD1106
0.0
0.0


Th1/Th2/Tr1_anti-


(Keratinocytes) none


CD95 CH11


LAK cells rest
0.0
0.0
CCD1106
0.0
0.0





(Keratinocytes)





TNFalpha + IL-1beta


LAK cells IL-2
0.0
0.0
Liver cirrhosis
1.9
3.8


LAK cells IL-2 + IL-
0.0
0.0
Lupus kidney
1.3
1.4


12


LAK cells IL-2 + IFN
0.8
0.0
NCI-H292 none
42.9
48.0


gamma


LAK cells IL-2 + IL-
0.0
0.0
NCI-H292 IL-4
14.7
14.8


18


LAK cells
0.0
0.0
NCI-H292 IL-9
40.6
36.6


PMA/ionomycin


NK Cells IL-2 rest
0.8
0.0
NCI-H292 IL-13
12.2
7.4


Two Way MLR 3
0.0
0.0
NCI-H292 IFN
16.8
10.9


day


gamma


Two Way MLR 5
0.0
0.0
HPAEC none
0.0
0.0


day


Two Way MLR 7
1.5
0.0
HPAEC TNF alpha +
0.0
0.0


day


IL-1 beta


PBMC rest
0.0
0.0
Lung fibroblast none
0.0
1.7


PBMC PWM
0.0
0.0
Lung fibroblast TNF
0.0
0.0





alpha + IL-1 beta


PBMC PHA-L
0.0
0.0
Lung fibroblast IL-4
1.7
0.0


Romos (B cell) none
0.0
0.0
Lung fibroblast IL-9
0.0
0.0


Romos (B cell)
0.0
0.0
Lung fibroblast IL-13
0.7
0.0


ionomycin


B lymphocytes PWM
0.0
0.0
Lung fibroblast IFN
0.0
0.0





gamma


B lymphocytes
0.0
0.0
Dermal fibroblast
0.0
0.0


CD40L and IL-4


CCD1070 rest


EOL-1 dbcAMP
0.0
0.0
Dermal fibroblast
0.0
0.0





CCD1070 TNF alpha


EOL-1 dbcAMP
0.0
0.0
Dermal fibroblast
0.9
0.0


PMA/ionomycin


CCD1070 IL-1 beta


Dendritic cells none
0.0
0.0
Dermal fibroblast IFN
0.0
0.0





gamma


Dendritic cells LPS
0.0
0.0
Dermal fibroblast IL-4
0.0
0.0


Dendritic cells anti-
0.0
0.0
IBD Colitis 2
0.8
2.6


CD40


Monocytes rest
0.0
0.0
IBD Crohn's
0.0
2.0


Monocytes LPS
1.7
0.0
Colon
28.1
23.2


Macrophages rest
0.0
0.0
Lung
34.9
51.4


Macrophages LPS
0.0
0.0
Thymus
35.1
28.3


HUVEC none
0.0
0.0
Kidney
8.2
7.4


HUVEC starved
0.0
0.0









CNS_neurodegeneration_v1.0 Summary: Ag1096/Ag3242 Two experiments with two different probe and primer sets produce results that are in excellent agreement, with highest expression of the CG57448-01 gene in the hippocampus of a patient with Alzheimer's disease. No change is detected in the expression of this gene in the postmortem Alzheimer's diseased brain when compared to controls; however this panel confirms the expression of this gene in the CNS in an independent group of patients. See panel 1.2 for a discussion of utility. A third experiment with the probe and primer set Ag704 shows low/undetectable levels of expression (CTs>35). (Data not shown.) The data suggest that there is a probability of a probe failure.
Panel 1.2 Summary: Ag1096/Ag3242 Two experiments with two different probe and primer sets produce results that are in excellent agreement, with highest expression of the CG57448-01 gene in cancer cell lines derived from lung cancer and melanoma (CTs=24-25). Significant levels of expression are also seen in a renal cancer cell line, ovarian cancer cell lines and brain cancer cell lines. Thus, expression of this gene could be used to differentiate between these samples and other samples on this panel and as a diagnostic marker for the presence of these cancers. This gene encodes a protein that is homologous to cadherin, a cell-adhesion molecule. Therefore, therapeutic modulation of the expression or function of this gene may be effective in the treatment of lung, renal and melanoma cancers.
Expression of this gene is also high in many regions of the brain, including the amygdala, thalamus, cerebellum, and cerebral cortex, with highest expression in the hippocampus. Expression is also detected in the spinal cord. Cadherins can act as axon guidance and cell adhesion proteins, specifically during development and in the response to injury (ref 1). Therefore, manipulation of levels of this protein may be of use in inducing a compensatory synaptogenic response to neuronal death in Alzheimer's disease, Parkinson's disease, Huntington's disease, spinocerebellar ataxia, progressive supranuclear palsy, ALS, head trauma, stroke, or any other disease/condition associated with neuronal loss.
Among tissues with metabolic function, this gene is moderately expressed in pituitary gland, adrenal gland, thyroid, pancreas, skeletal muscle, and liver, reflecting the widespread role of cadherins in cell-cell adhesion. This expression suggests that this gene product may play a role in normal metabolic and neuroendocrine function and that dysregulated expression of this gene may contribute to metabolic diseases (such as obesity and diabetes) or neuroendocrine disorders (Ranscht, Int. J. Dev. Neurosci. 18: 643-651, 2000).
Panel 1.3D Summary: Ag3242 Highest expression of the CG57448-01 gene is seen in a renal cancer cell line (CT=31.1). Significant expression is also seen in cell lines derived from ovarian cancer, lung cancer, brain cancer and melanoma. This is in concordance with the results in the previous panel. Please see Panel 1.2 for discussion of utility of this gene in the treatment of cancer.
As in the previous panel, this gene is also highly expressed in the central nervous system. Please see Panel 1.2 for a fuller discussion of utility of this gene in the central nervous system.
Results from a second experiment with the probe primer set Ag704 are not included. The amp plot indicates that there is a high probability of a probe failure.
Panel 2.2 Summary: Ag3242 Highest expression of the CG57448-01 gene is seen in a sample derived from an ocular melanoma metastasis (CT=29). Thus, expression of this gene could be used to differentiate between this sample and other samples on this pane.
Panel 2D Summary: Ag704 Results from one experiment with the CG57448-01 gene are not included. The amp plot indicates that there were experimental difficulties with this run.
Panel 4D Summary: Ag1096/Ag3242 Two experiments with two different probe and primer sets produce results that are in excellent agreement, with highest expression of the CG57448-01 gene in the basophil cell line (KU-812) treated with PMA/ionomycin (CTs=30-32). Significant expression is also seen in a cluster of treated and untreated samples derived from the muco-epidermoid cell line NCI-H292. Thus, this gene, which encodes a cadherin homolog, is expressed in both a cell line that is often used as a model of airway epithelium (NCI-H292) and a cell line that is a reasonable model for the inflammatory cells that contribute to various inflammatory lung diseases. This suggests that suggest that therapeutic modulation of this gene product may reduce or eliminate the symptoms of patients suffering from pathological and inflammatory lung disorders, including chronic obstructive pulmonary disease, asthma, allergy and emphysema.
Low but significant levels of expression are also seen in the samples derived from normal colon, kidney, lung and thymus. This suggests that this gene product may play a role in the homeostasis of these tissues. Therefore, therapeutic modulation of the expression or function of this gene product may be important for maintaining or restoring normal function to these organs during inflammation.Results from a third experiment with the probe primer set Ag704 are not included. The amp plot indicates that there is a high probability of a probe failure.
O. NOV16b: Protocadherin
Expression of gene CG57446-01 was assessed using the primer-probe set Ag3241, described in Table OA. Results of the RTQ-PCR runs are shown in Table OB.







TABLE OA







Probe Name Ag3241













Start


Primers
Sequences
Length
Position














Forward
5′-cttgtgctagtgatcgacatca-3′
(SEQ ID NO:350)
22
1251


Probe
TET-5′-ccccttattctcaatcctcctactacg-3′-TAMRA
(SEQ ID NO:351)
27
1285


Reverse
5′-ccgttgttttcgtttacgtaaa-3′
(SEQ ID NO:352)
22
1312
















TABLE OB







Panel 1.3D











Rel. Exp. (%) Ag3241, Run

Rel. Exp. (%) Ag3241, Run


Tissue Name
165524414
Tissue Name
165524414













Liver adenocarcinoma
0.0
Kidney (fetal)
0.0


Pancreas
0.0
Renal ca. 786-0
0.0


Pancreatic ca. CAPAN 2
0.0
Renal ca. A498
0.0


Adrenal gland
0.0
Renal ca. RXF 393
0.0


Thyroid
0.0
Renal ca. ACHN
0.0


Salivary gland
0.0
Renal ca. UO-31
0.0


Pituitary gland
0.0
Renal ca. TK-10
0.0


Brain (fetal)
0.0
Liver
0.0


Brain (whole)
0.0
Liver (fetal)
0.0


Brain (amygdala)
0.0
Liver ca. (hepatoblast)
0.0




HepG2


Brain (cerebellum)
0.0
Lung
0.0


Brain (hippocampus)
0.0
Lung (fetal)
0.0


Brain (substantia nigra)
0.0
Lung ca. (small cell) LX-1
0.0


Brain (thalamus)
0.0
Lung ca. (small cell)
0.0




NCI-H69


Cerebral Cortex
0.0
Lung ca. (s. cell var.)
0.0




SHP-77


Spinal cord
0.0
Lung ca. (large cell)NCI-
0.0




H460


glio/astro U87-MG
0.0
Lung ca. (non-sm. cell)
0.0




A549


glio/astro U-118-MG
0.0
Lung ca. (non-s. cell)
0.0




NCI-H23


astrocytoma SW1783
0.0
Lung ca. (non-s. cell)
0.0




HOP-62


neuro*; met SK-N-AS
0.0
Lung ca. (non-s. cl) NCI-
0.0




H522


astrocytoma SF-539
0.0
Lung ca. (squam.) SW
0.0




900


astrocytoma SNB-75
0.0
Lung ca. (squam.) NCI-
0.0




H596


glioma SNB-19
0.0
Mammary gland
0.0


glioma U251
0.0
Breast ca.* (pl. ef) MCF-7
0.0


glioma SF-295
0.0
Breast ca.* (pl. ef) MDA-
0.0




MB-231


Heart (fetal)
0.0
Breast ca.* (pl. ef) T47D
0.0


Heart
0.0
Breast ca. BT-549
0.0


Skeletal muscle (fetal)
100.0
Breast ca. MDA-N
0.0


Skeletal muscle
0.0
Ovary
0.0


Bone marrow
0.0
Ovarian ca. OVCAR-3
0.0


Thymus
0.0
Ovarian ca. OVCAR-4
0.0


Spleen
0.0
Ovarian ca. OVCAR-5
0.0


Lymph node
0.0
Ovarian ca. OVCAR-8
0.0


Colorectal
0.0
Ovarian ca. IGROV-1
0.0


Stomach
0.0
Ovarian ca.* (ascites)
0.0




SK-OV-3


Small intestine
0.0
Uterus
0.0


Colon ca. SW480
0.0
Placenta
0.0


Colon ca.* SW620 (SW480
0.0
Prostate
0.0


met)


Colon ca. HT29
0.0
Prostate ca.* (bone
0.0




met)PC-3


Colon ca. HCT-116
0.0
Testis
0.0


Colon ca. CaCo-2
0.0
Melanoma Hs688 (A).T
0.0


Colon ca.
0.0
Melanoma* (met)
0.0


tissue (ODO3866)

Hs688 (B).T


Colon ca. HCC-2998
0.0
Melanoma UACC-62
0.0


Gastric ca.* (liver met)
0.0
Melanoma M14
0.0


NCI-N87


Bladder
0.0
Melanoma LOX IMVI
0.0


Trachea
0.0
Melanoma* (met) SK-
0.0




MEL-5


Kidney
0.0
Adipose
0.0









Panel 1.3D Summary: Ag3241 Expression of the CG57446-01 gene is restricted to fetal skeletal muscle (CT=33.8). Thus, expression of this gene could be used to differentiate between fetal and adult skeletal muscle (CT=40). This gene encodes a protein that is homologous to cadherin, a cell adhesion molecule that has been implicated in muscle differentiation. Thus, the relative overexpression of this gene in fetal skeletal muscle suggests that the protein product may enhance muscular growth or development in the fetus and may also act in a regenerative capacity in the adult. Therefore, therapeutic modulation of the protein encoded by this gene could be useful in treatment of muscle related diseases. More specifically, treatment of weak or dystrophic muscle with the protein encoded by this gene could restore muscle mass or function (Goichberg et al., J Cell Sci 2001 April;114(Pt 7):1309-19).
Panel 2.2 Summary: Ag3241 Expression of the CG57446-01 gene is low/undetectable in all samples on this panel (CTs>35). (Data not shown.) The amp plot indicates that there is a possibility of a probe failure.
Panel 4D Summary: Ag3241 Expression of the CG57446-01 gene is low/undetectable in all samples on this panel (CTs>35). (Data not shown.) The amp plot indicates that there is a possibility of a probe failure.
Panel CNS—1 Summary: Ag3241 Expression of the CG57446-01 gene is low/undetectable in all samples on this panel (CTs>35). (Data not shown.) The amp plot indicates that there is a possibility of a probe failure.
P. NOV16c: Protocadherin
Expression of gene CG57444-01 was assessed using the primer-probe set Ag3240, described in Table PA. Results of the RTQ-PCR runs are shown in Tables PB, PC, PD, PE and PF.







TABLE PA







Probe Name Ag3240













Start


Primers
Sequences
Length
Position














Forward
5′-ggagactgctttgagtcagttc-3′
(SEQ ID NO:353)
22
2184


Probe
TET-5′-tctgctccaagtccggacctgtg-3′-TAMRA
(SEQ ID NO:354)
23
2206


Reverse
5′-aattataggcatagggcaacgt-3′
(SEQ ID NO:355)
22
2253
















TABLE PB







CNS_neurodegeneration_v1.0











Rel. Exp. (%) Ag3240, Run

Rel. Exp. (%) Ag3240, Run


Tissue Name
210037849
Tissue Name
210037849













AD 1 Hippo
17.7
Control (Path) 3
6.9




Temporal Ctx


AD 2 Hippo
37.9
Control (Path) 4
28.1




Temporal Ctx


AD 3 Hippo
6.8
AD 1 Occipital Ctx
13.7


AD 4 Hippo
8.3
AD 2 Occipital Ctx
0.0




(Missing)


AD 5 Hippo
55.1
AD 3 Occipital Ctx
8.7


AD 6 Hippo
100.0
AD 4 Occipital Ctx
21.8


Control 2 Hippo
23.5
AD 5 Occipital Ctx
8.4


Control 4 Hippo
23.5
AD 6 Occipital Ctx
17.6


Control (Path) 3 Hippo
8.4
Control 1 Occipital Ctx
4.3


AD 1 Temporal Ctx
18.3
Control 2 Occipital Ctx
24.0


AD 2 Temporal Ctx
32.5
Control 3 Occipital Ctx
14.2


AD 3 Temporal Ctx
6.3
Control 4 Occipital Ctx
13.0


AD 4 Temporal Ctx
24.7
Control (Path) 1
51.4




Occipital Ctx


AD 5 Inf Temporal Ctx
59.0
Control (Path) 2
8.7




Occipital Ctx


AD 5 Sup Temporal
53.6
Control (Path) 3
4.9


Ctx

Occipital Ctx


AD 6 Inf Temporal Ctx
43.8
Control (Path) 4
15.4




Occipital Ctx


AD 6 Sup Temporal
56.6
Control 1 Parietal Ctx
11.6


Ctx


Control 1 Temporal Ctx
9.6
Control 2 Parietal Ctx
37.1


Control 2 Temporal Ctx
14.6
Control 3 Parietal Ctx
15.5


Control 3 Temporal Ctx
8.9
Control (Path) 1
36.3




Parietal Ctx


Control 3 Temporal Ctx
9.0
Control (Path) 2
22.7




Parietal Ctx


Control (Path) 1
34.9
Control (Path) 3
5.8


Temporal Ctx

Parietal Ctx


Control (Path) 2
19.3
Control (Path) 4
37.1


Temporal Ctx

Parietal Ctx
















TABLE PC







Panel 1.3D











Rel. Exp. (%) Ag3240, Run

Rel. Exp. (%) Ag3240, Run


Tissue Name
165524271
Tissue Name
165524271













Liver adenocarcinoma
2.5
Kidney (fetal)
6.0


Pancreas
1.7
Renal ca. 786-0
3.2


Pancreatic ca. CAPAN 2
3.5
Renal ca. A498
3.7


Adrenal gland
6.6
Renal ca. RXF 393
6.7


Thyroid
3.9
Renal ca. ACHN
2.0


Salivary gland
3.7
Renal ca. UO-31
0.4


Pituitary gland
14.0
Renal ca. TK-10
0.2


Brain (fetal)
23.3
Liver
1.9


Brain (whole)
19.3
Liver (fetal)
2.8


Brain (amygdala)
21.9
Liver ca. (hepatoblast)
0.4




HepG2


Brain (cerebellum)
48.6
Lung
5.9


Brain (hippocampus)
23.3
Lung (fetal)
2.3


Brain (substantia nigra)
9.5
Lung ca. (small cell) LX-1
1.0


Brain (thalamus)
17.8
Lung ca. (small cell)
0.4




NCI-H69


Cerebral Cortex
10.2
Lung ca. (s. cell var.)
0.6




SHP-77


Spinal cord
29.7
Lung ca. (large cell) NCI-
3.5




H460


glio/astro U87-MG
1.0
Lung ca. (non-sm. cell)
0.1




A549


glio/astro U-118-MG
100.0
Lung ca. (non-s. cell)
11.5




NCI-H23


astrocytoma SW1783
4.2
Lung ca. (non-s. cell)
5.2




HOP-62


neuro*; met SK-N-AS
34.6
Lung ca. (non-s. cl) NCI-
2.4




H522


astrocytoma SF-539
3.9
Lung ca. (squam.) SW
5.8




900


astrocytoma SNB-75
6.6
Lung ca. (squam.) NCI-
0.9




H596


glioma SNB-19
8.1
Mammary gland
14.7


glioma U251
9.4
Breast ca.* (pl. ef) MCF-7
4.8


glioma SF-295
3.2
Breast ca.* (pl. ef) MDA-
4.9




MB-231


Heart (fetal)
3.6
Breast ca.* (pl. ef) T47D
3.9


Heart
8.7
Breast ca. BT-549
5.8


Skeletal muscle (fetal)
11.0
Breast ca. MDA-N
2.5


Skeletal muscle
8.1
Ovary
31.2


Bone marrow
1.4
Ovarian ca. OVCAR-3
1.5


Thymus
1.5
Ovarian ca. OVCAR-4
0.5


Spleen
5.8
Ovarian ca. OVCAR-5
11.2


Lymph node
7.5
Ovarian ca. OVCAR-8
0.5


Colorectal
6.2
Ovarian ca. IGROV-1
5.7


Stomach
3.3
Ovarian ca.* (ascites)
6.9




SK-OV-3


Small intestine
12.4
Uterus
15.8


Colon ca. SW480
0.1
Placenta
4.3


Colon ca.* SW620 (SW480
0.2
Prostate
5.3


met)


Colon ca. HT29
0.2
Prostate ca.* (bone
4.1




met) PC-3


Colon ca. HCT-116
1.0
Testis
4.2


Colon ca. CaCo-2
0.8
Melanoma Hs688 (A).T
6.3


Colon ca.
3.2
Melanoma* (met)
8.0


tissue (ODO3866)

Hs688 (B).T


Colon ca. HCC-2998
5.1
Melanoma UACC-62
2.8


Gastric ca.* (liver met)
8.9
Melanoma M14
5.1


NCI-N87


Bladder
13.3
Melanoma LOX IMVI
0.4


Trachea
5.5
Melanoma* (met) SK-
3.0




MEL-5


Kidney
6.7
Adipose
3.8
















TABLE PD







Panel 2.2











Rel. Exp. (%) Ag3240,

Rel. Exp. (%) Ag3240,


Tissue Name
Run 174443262
Tissue Name
Run 174443262













Normal Colon
13.7
Kidney Margin (OD04348)
40.9


Colon cancer (OD06064)
12.9
Kidney malignant cancer
1.2




(OD06204B)


Colon Margin (OD06064)
20.3
Kidney normal adjacent
11.7




tissue (OD06204E)


Colon cancer (OD06159)
0.4
Kidney Cancer (OD04450-
9.0




01)


Colon Margin (OD06159)
18.7
Kidney Margin (OD04450-
9.5




03)


Colon cancer (OD06297-04)
5.6
Kidney Cancer 8120613
1.4


Colon Margin (OD06297-05)
34.2
Kidney Margin 8120614
14.1


CC Gr. 2 ascend colon
0.9
Kidney Cancer 9010320
3.4


(ODO3921)


CC Margin (ODO3921)
4.6
Kidney Margin 9010321
5.5


Colon cancer metastasis
0.8
Kidney Cancer 8120607
7.0


(OD06104)


Lung Margin (OD06104)
2.5
Kidney Margin 8120608
9.5


Colon mets to lung
1.8
Normal Uterus
52.9


(OD04451-01)


Lung Margin (OD04451-02)
10.9
Uterine Cancer 064011
17.9


Normal Prostate
15.7
Normal Thyroid
2.7


Prostate Cancer (OD04410)
17.3
Thyroid Cancer 064010
13.7


Prostate Margin (OD04410)
4.9
Thyroid Cancer A302152
17.0


Normal Ovary
48.3
Thyroid Margin A302153
8.4


Ovarian cancer (OD06283-
1.9
Normal Breast
42.6


03)


Ovarian Margin (OD06283-
14.1
Breast Cancer (OD04566)
1.7


07)


Ovarian Cancer 064008
10.1
Breast Cancer 1024
40.3


Ovarian cancer (OD06145)
41.2
Breast Cancer (OD04590-
8.4




01)


Ovarian Margin (OD06145)
37.1
Breast Cancer Mets
9.4




(OD04590-03)


Ovarian cancer (OD06455-
2.6
Breast Cancer Metastasis
8.9


03)

(OD04655-05)


Ovarian Margin (OD06455-
41.8
Breast Cancer 064006
4.3


07)


Normal Lung
23.8
Breast Cancer 9100266
16.4


Invasive poor diff. lung
1.8
Breast Margin 9100265
18.4


adeno (ODO4945-01


Lung Margin (ODO4945-03)
21.3
Breast Cancer A209073
4.1


Lung Malignant Cancer
10.4
Breast Margin A2090734
16.4


(OD03126)


Lung Margin (OD03126)
13.7
Breast cancer (OD06083)
34.2


Lung Cancer (OD05014A)
3.7
Breast cancer node
26.6




metastasis (OD06083)


Lung Margin (OD05014B)
33.0
Normal Liver
9.7


Lung cancer (OD06081)
0.0
Liver Cancer 1026
1.6


Lung Margin (OD06081)
15.3
Liver Cancer 1025
9.2


Lung Cancer (OD04237-01)
1.1
Liver Cancer 6004-T
7.5


Lung Margin (OD04237-02)
31.6
Liver Tissue 6004-N
1.6


Ocular Melanoma Metastasis
0.6
Liver Cancer 6005-T
5.8


Ocular Melanoma Margin
4.5
Liver Tissue 6005-N
17.1


(Liver)


Melanoma Metastasis
0.5
Liver Cancer 064003
7.4


Melanoma Margin (Lung)
33.9
Normal Bladder
8.0


Normal Kidney
8.0
Bladder Cancer 1023
4.6


Kidney Ca, Nuclear grade 2
19.3
Bladder Cancer A302173
3.3


(OD04338)


Kidney Margin (OD04338)
54.7
Normal Stomach
32.5


Kidney Ca Nuclear grade 1/2
100.0
Gastric Cancer 9060397
0.0


(OD04339)


Kidney Margin (OD04339)
18.3
Stomach Margin 9060396
6.1


Kidney Ca, Clear cell type
6.7
Gastric Cancer 9060395
6.9


(OD04340)


Kidney Margin (OD04340)
9.5
Stomach Margin 9060394
22.2


Kidney Ca, Nuclear grade 3
8.7
Gastric Cancer 064005
6.1


(OD04348)
















TABLE PE







Panel 4D











Rel. Exp. (%) Ag3240,

Rel. Exp. (%) Ag3240,


Tissue Name
Run 164389761
Tissue Name
Run 164389761













Secondary Th1 act
0.0
HUVEC IL-1beta
16.7


Secondary Th2 act
0.0
HUVEC IFN gamma
33.2


Secondary Tr1 act
0.0
HUVEC TNF alpha + IFN
13.4




gamma


Secondary Th1 rest
0.0
HUVEC TNF alpha + IL4
17.1


Secondary Th2 rest
0.0
HUVEC IL-11
13.5


Secondary Tr1 rest
0.0
Lung Microvascular EC none
25.9


Primary Th1 act
0.0
Lung Microvascular EC
23.5




TNF alpha + IL-1beta


Primary Th2 act
0.0
Microvascular Dermal EC none
35.6


Primary Tr1 act
0.0
Microsvasular Dermal EC
21.6




TNF alpha + IL-1beta


Primary Th1 rest
0.0
Bronchial epithelium TNF alpha +
27.7




IL1beta


Primary Th2 rest
0.4
Small airway epithelium none
9.4


Primary Tr1 rest
0.0
Small airway epithelium
27.0




TNF alpha + IL-1beta


CD45RA CD4 lymphocyte
8.5
Coronery artery SMC rest
31.6


act


CD45RO CD4 lymphocyte
0.2
Coronery artery SMC TNF alpha +
11.0


act

IL-1beta


CD8 lymphocyte act
0.0
Astrocytes rest
35.4


Secondary CD8
0.2
Astrocytes TNF alpha + IL-1beta
19.9


lymphocyte rest


Secondary CD8
0.0
KU-812 (Basophil) rest
2.9


lymphocyte act


CD4 lymphocyte none
0.1
KU-812 (Basophil)
8.2




PMA/ionomycin


2ry Th1/Th2/Tr1_anti-
0.0
CCD1106 (Keratinocytes) none
3.5


CD95 CH11


LAK cells rest
1.5
CCD1106 (Keratinocytes)
0.9




TNF alpha + IL-1beta


LAK cells IL-2
0.9
Liver cirrhosis
6.5


LAK cells IL-2 + IL-12
0.4
Lupus kidney
6.6


LAK cells IL-2 + IFN
1.9
NCI-H292 none
71.7


gamma


LAK cells IL-2 + IL-18
1.1
NCI-H292 IL-4
55.9


LAK cells
0.6
NCI-H292 IL-9
87.7


PMA/ionomycin


NK Cells IL-2 rest
0.0
NCI-H292 IL-13
28.7


Two Way MLR 3 day
1.6
NCI-H292 IFN gamma
43.2


Two Way MLR 5 day
0.4
HPAEC none
19.6


Two Way MLR 7 day
0.0
HPAEC TNF alpha + IL-1beta
15.4


PBMC rest
0.4
Lung fibroblast none
49.7


PBMC PWM
1.4
Lung fibroblast TNF alpha + IL-
16.8




1beta


PBMC PHA-L
0.2
Lung fibroblast IL-4
84.7


Ramos (B cell) none
7.6
Lung fibroblast IL-9
59.0


Ramos (B cell) ionomycin
26.6
Lung fibroblast IL-13
55.9


B lymphocytes PWM
3.7
Lung fibroblast IFN gamma
100.0


B lymphocytes CD40L
3.9
Dermal fibroblast CCD1070 rest
51.4


and IL-4


EOL-1 dbcAMP
5.1
Dermal fibroblast CCD1070
33.2




TNF alpha


EOL-1 dbcAMP
5.7
Dermal fibroblast CCD1070 IL-
20.0


PMA/ionomycin

1beta


Dendritic cells none
0.7
Dermal fibroblast IFN gamma
36.6


Dendritic cells LPS
1.2
Dermal fibroblast IL-4
52.5


Dendritic cells anti-CD40
0.7
IBD Colitis 2
2.8


Monocytes rest
0.0
IBD Crohn's
2.6


Monocytes LPS
0.2
Colon
24.8


Macrophages rest
0.2
Lung
59.5


Macrophages LPS
1.2
Thymus
42.9


HUVEC none
20.4
Kidney
17.3


HUVEC starved
51.4
















TABLE PF







Panel CNS_1











Rel. Exp. (%) Ag3240, Run

Rel. Exp. (%) Ag3240, Run


Tissue Name
171694589
Tissue Name
171694589













BA4 Control
13.0
BA17 PSP
28.7


BA4 Control2
21.6
BA17 PSP2
6.7


BA4 Alzheimer's2
4.8
Sub Nigra Control
41.8


BA4 Parkinson's
59.9
Sub Nigra Control2
36.9


BA4 Parkinson's2
34.6
Sub Nigra Alzheimer's2
18.7


BA4 Huntington's
18.8
Sub Nigra Parkinson's2
58.2


BA4
5.0
Sub Nigra Huntington's
85.3


Huntington's2


BA4 PSP
2.7
Sub Nigra
23.8




Huntington's2


BA4 PSP2
23.3
Sub Nigra PSP2
11.6


BA4 Depression
19.2
Sub Nigra Depression
13.9


BA4 Depression2
17.4
Sub Nigra Depression2
11.4


BA7 Control
27.0
Glob Palladus Control
22.8


BA7 Control2
15.9
Glob Palladus Control2
17.9


BA7 Alzheimer's2
13.5
Glob Palladus
17.2




Alzheimer's


BA7 Parkinson's
34.2
Glob Palladus
9.8




Alzheimer's2


BA7 Parkinson's2
26.8
Glob Palladus
100.0




Parkinson's


BA7 Huntington's
43.5
Glob Palladus
10.5




Parkinson's2


BA7
40.9
Glob Palladus PSP
17.2


Huntington's2


BA7 PSP
48.3
Glob Palladus PSP2
7.4


BA7 PSP2
17.1
Glob Palladus
9.3




Depression


BA7 Depression
27.0
Temp Pole Control
11.8


BA9 Control
11.1
Temp Pole Control2
33.4


BA9 Control2
39.5
Temp Pole Alzheimer's
1.5


BA9 Alzheimer's
4.7
Temp Pole Alzheimer's2
5.7


BA9 Alzheimer's2
18.7
Temp Pole Parkinson's
39.8


BA9 Parkinson's
26.8
Temp Pole Parkinson's2
12.9


BA9 Parkinson's2
41.5
Temp Pole Huntington's
19.6


BA9 Huntington's
54.7
Temp Pole PSP
6.8


BA9
8.8
Temp Pole PSP2
0.7


Huntington's2


BA9 PSP
24.5
Temp Pole Depression2
10.9


BA9 PSP2
7.1
Cing Gyr Control
42.3


BA9 Depression
6.1
Cing Gyr Control2
20.9


BA9 Depression2
5.0
Cing Gyr Alzheimer's
23.3


BA17 Control
40.3
Cing Gyr Alzheimer's2
7.9


BA17 Control2
26.1
Cing Gyr Parkinson's
38.2


BA17
6.0
Cing Gyr Parkinson's2
51.1


Alzheimer's2


BA17 Parkinson's
52.1
Cing Gyr Huntington's
59.0


BA17
32.1
Cing Gyr Huntington's2
19.6


Parkinson's2


BA17
21.0
Cing Gyr PSP
51.1


Huntington's


BA17
15.4
Cing Gyr PSP2
13.6


Huntington's2


BA17 Depression
25.7
Cing Gyr Depression
18.3


BA17 Depression2
10.2
Cing Gyr Depression2
15.0









CNS_neurodegeneration_v1.0 Summary: Ag3240 Highest expression of the CG57444-01 gene is seen in the hippocampus of a patient with Alzheimer's disease (CT=27.6). This panel suggests a slight upregulation of this gene in the AD temporal cortex that falls short of significance (p=0.051). Thus, this gene product may be a drug target for the treatment of Alzheimer's disease; specifically, it may be involved in compensatory synaptogenesis in response to neuronal loss.
Panel 1.3D Summary: Ag3240 The CG57444-01 gene, a cadherin homolog, is expressed in many samples in this panel, reflecting the widespread role of cadherins in cell-cell adhesion. Highest expression is seen in a brain cancer cell line (CT=28.6). Thus, expression of this gene could be used to differentiate between this sample and other samples on this panel. Overall, however, expression appears to be associated with normal tissues rather than cancer cell lines. This can be seen in the samples from normal ovarian and breast tissue, which show higher levels of expression than the cell lines derived from breast and ovarian cancers. Loss of function of the related E-cadherin protein has been described in many tumors, along with an increased invasiveness and a decreased prognosis of many carcinomas, including tumors of endocrine glands and their target systems (ref 1). Thus, this gene product might similarly be useful as a protein therapeutic to treat a variety of tumors.
In addition, this gene is moderately expressed in pituitary gland, adrenal gland, thyroid, pancreas, and fetal and adult skeletal muscle, heart and liver, again reflecting the widespread role of this cadherin homolog. This observation may suggest that this gene product plays a role in normal metabolic and neuroendocrine function and that disregulated expression of this gene may contribute to metabolic diseases (such as obesity and diabetes) or neuroendocrine disorders.
Expression of this gene is also high in many regions of the brain, including the amygdala, thalamus, cerebellum, cerebral cortex, hippocampus, and substantia nigra. Expression is also detected in the spinal cord. Cadherins can act as axon guidance and cell adhesion proteins, specifically during development and in the response to injury (ref 2). Manipulation of levels of this protein may be of use in inducing a compensatory synaptogenic response to neuronal death in Alzheimer's disease, Parkinson's disease, Huntington's disease, spinocerebellar ataxia, progressive supranuclear palsy, ALS, head trauma, stroke, or any other disease/condition associated with neuronal loss (Potter et al., Endocr. Rev. 20: 207-239, 1999; Ranscht, Int. J. Dev. Neurosci. 18: 643-651, 2000).
Panel 2.2 Summary: Ag3240 Highest expression of the CG57444-01 gene is seen in a kidney cancer (CT=29.7). Overall, however, expression appears to be associated with normal tissues, as seen in Panel 1.3D. Thus, higher levels of expression are seen in normal colon, ovary, lung and stomach, when compared to adjacent tumors. Thus, absence of expression of this gene could be used as a diagnostic marker of these cancers. Furthermore, therapeutic modulation of the expression or function of this gene could be effective in the treatment of colon, ovarian, lung and stomach cancers.
Panel 4D Summary: Ag3240 The CG57444-01 transcript is expressed in endothelium, fibroblasts and NCI-H292 cells. This gene is also expressed in normal colon, kidney, thymus, and lung. This transcript encodes a putative protocadherin family member. Protocadherins mediate cell:cell interactions and signalling. Since this gene product is expressed in endothelium, it may also be involved in normal blood cell recirculation through the tissues. Alternatively, the protein encoded by this gene may be important for regulating tissue or organ organization and normal function since it is expressed on fibroblasts and epithelium.
Panel CNS—1 Summary: Ag3240 Results from this experiment confirm expression of the CG57444-01 gene in the brain. Please see Panel 1.3D for discussion of utility of this gene in the central nervous system.
Q. NOV17a and NOV17b: Cadherin 23
Expression of gene CG57429-01 and variant CG57429-02 was assessed using the primer-probe sets Ag3234, Ag3279 and Ag616, described in Tables QA, QB and QC. Results of the RTQ-PCR runs are shown in Tables QD, QE, QF, QG, QH, QI, QJ and QK.







TABLE QA







Probe Name Ag3234













Start


Primers
Sequences
Length
Position














Forward
5′-gcaaaatcgtcgtctctgttac-3′
(SEQ ID NO:356)
22
668


Probe
TET-5′-ccctctgaaagccaccagcagtg-3′-TAMRA
(SEQ ID NO:357)
23
705


Reverse
5′-ccaagaggttcacaaacactgt-3′
(SEQ ID NO:358)
22
730
















TABLE QB







Probe Name Ag3279













Start


Primers
Sequences
Length
Position














Forward
5′-gcaaaatcgtcgtctctgttac-3′
(SEQ ID NO:359)
22
668


Probe
TET-5′-ccctctgaaagccaccagcagtg-3′-TAMRA
(SEQ ID NO:360)
23
705


Reverse
5′-ccaagaggttcacaaacactgt-3′
(SEQ ID NO:361)
22
730
















TABLE QC







Probe Name Ag616













Start


Primers
Sequences
Length
Position














Forward
5′-tcgttgtccgtgcagttcag-3′
(SEQ ID NO:362)
20
1156


Probe
TET-5′-cagaccacccggaactcgcgtg-3′-TAMRA
(SEQ ID NO:363)
22
1133


Reverse
5′-cggccgtgtacaatgtgtct-3′
(SEQ ID NO:364)
20
1097
















TABLE QD







CNS_neurodegeneration_v1.0













Rel. Exp. (%)
Rel. Exp. (%)

Rel. Exp. (%)
Rel. Exp. (%)



Ag3234, Run
Ag3279, Run

Ag3234, Run
Ag3279, Run


Tissue Name
209862304
210060481
Tissue Name
209862304
210060481















AD 1 Hippo
16.3
21.8
Control (Path)
14.6
16.2





3 Temporal





Ctx


AD 2 Hippo
23.8
32.3
Control (Path)
26.4
31.2





4 Temporal





Ctx


AD 3 Hippo
13.2
14.1
AD 1 Occipital
17.1
19.2





Ctx


AD 4 Hippo
19.8
22.5
AD 2 Occipital
0.0
0.0





Ctx (Missing)


AD 5 hippo
58.2
49.7
AD 3 Occipital
23.5
22.4





Ctx


AD 6 Hippo
58.6
100.0
AD 4 Occipital
18.3
21.2





Ctx


Control 2 Hippo
19.8
24.5
AD 5 Occipital
31.9
38.4





Ctx


Control 4 Hippo
29.1
23.5
AD 6 Occipital
24.1
37.9





Ctx


Control (Path) 3
11.7
7.1
Control 1
19.8
26.1


Hippo


Occipital Ctx


AD 1 Temporal
28.1
28.7
Control 2
31.0
33.7


Ctx


Occipital Ctx


AD 2 Temporal
18.7
28.9
Control 3
21.8
22.7


Ctx


Occipital Ctx


AD 3 Temporal
15.5
15.7
Control 4
13.3
20.7


Ctx


Occipital Ctx


AD 4 Temporal
27.7
25.5
Control (Path)
37.1
32.5


Ctx


1 Occipital Ctx


AD 5 Inf
38.2
52.1
Control (Path)
14.7
12.6


Temporal Ctx


2 Occipital Ctx


AD 5
45.7
49.7
Control (Path)
20.3
19.2


SupTemporal Ctx


3 Occipital Ctx


AD 6 Inf
81.8
67.8
Control (Path)
20.4
26.1


Temporal Ctx


4 Occipital Ctx


AD 6 Sup
100.0
94.6
Control 1
14.2
22.4


Temporal Ctx


Parietal Ctx


Control 1
10.1
14.2
Control 2
35.8
44.4


Temporal Ctx


Parietal Ctx


Control 2
15.0
11.9
Control 3
5.5
12.8


Temporal Ctx


Parietal Ctx


Control 3
8.6
14.7
Control (Path)
17.7
21.9


Temporal Ctx


1 Parietal Ctx


Control 4
8.9
12.1
Control (Path)
19.2
25.3


Temporal Ctx


2 Parietal Ctx


Control (Path) 1
23.7
31.0
Control (Path)
13.0
17.9


Temporal Ctx


3 Parietal Ctx


Control (Path) 2
14.3
16.6
Control (Path)
32.3
33.0


Temporal Ctx


4 Parietal Ctx
















TABLE QE







General_screening_panel_v1.4











Rel. Exp. (%) Ag3279, Run

Rel. Exp. (%) Ag3279, Run


Tissue Name
216512994
Tissue Name
216512994













Adipose
35.8
Renal ca. TK-10
0.0


Melanoma* Hs688 (A).T
0.0
Bladder
9.4


Melanoma* Hs688 (B).T
0.0
Gastric ca. (liver met.)
0.4




NCI-N87


Melanoma* M14
0.0
Gastric ca. KATO III
0.0


Melanoma* LOXIMVI
0.0
Colon ca. SW-948
0.0


Melanoma* SK-MEL-5
0.1
Colon ca. SW480
2.1


Squamous cell
0.0
Colon ca.* (SW480 met)
0.7


carcinoma SCC-4

SW620


Testis Pool
11.0
Colon ca. HT29
0.0


Prostate ca.* (bone met)
0.1
Colon ca. HCT-116
0.2


PC-3


Prostate Pool
3.8
Colon ca. CaCo-2
1.0


Placenta
0.6
Colon cancer tissue
0.8


Uterus Pool
3.1
Colon ca. SW1116
0.0


Ovarian ca. OVCAR-3
10.4
Colon ca. Colo-205
0.0


Ovarian ca. SK-OV-3
1.4
Colon ca. SW-48
0.0


Ovarian ca. OVCAR-4
0.0
Colon Pool
8.0


Ovarian ca. OVCAR-5
4.0
Small Intestine Pool
12.1


Ovarian ca. IGROV-1
2.4
Stomach Pool
6.1


Ovarian ca. OVCAR-8
1.4
Bone Marrow Pool
8.4


Ovary
37.4
Fetal Heart
15.6


Breast ca. MCF-7
0.1
Heart Pool
9.4


Breast ca. MDA-MB-
0.0
Lymph Node Pool
13.7


231


Breast ca. BT 549
0.0
Fetal Skeletal Muscle
4.7


Breast ca. T47D
5.8
Skeletal Muscle Pool
4.9


Breast ca. MDA-N
0.0
Spleen Pool
5.8


Breast Pool
7.0
Thymus Pool
13.0


Trachea
4.3
CNS cancer (glio/astro)
0.2




U87-MG


Lung
16.0
CNS cancer (glio/astro) U-
0.0




118-MG


Fetal Lung
24.5
CNS cancer (neuro;met)
0.0




SK-N-AS


Lung ca. NCI-N417
2.2
CNS cancer (astro) SF-539
0.0


Lung ca. LX-1
0.3
CNS cancer (astro) SNB-75
0.1


Lung ca. NCI-H146
0.3
CNS cancer (glio) SNB-19
1.8


Lung ca. SHP-77
0.0
CNS cancer (glio) SF-295
0.8


Lung ca. A549
4.2
Brain (Amygdala) Pool
9.7


Lung ca. NCI-H526
10.2
Brain (cerebellum)
100.0


Lung ca. NCI-H23
12.0
Brain (fetal)
8.2


Lung ca. NCI-H460
0.1
Brain (Hippocampus) Pool
10.2


Lung ca. HOP-62
0.0
Cerebral Cortex Pool
7.9


Lung ca. NCI-H522
6.0
Brain (Substantia nigra)
6.0




Pool


Liver
9.1
Brain (Thalamus) Pool
10.9


Fetal Liver
3.5
Brain (whole)
16.0


Liver ca. HepG2
0.0
Spinal Cord Pool
8.9


Kidney Pool
34.2
Adrenal Gland
3.9


Fetal Kidney
5.0
Pituitary gland Pool
0.6


Renal ca. 786-0
0.0
Salivary Gland
2.5


Renal ca. A498
1.1
Thyroid (female)
1.4


Renal ca. ACHN
0.0
Pancreatic ca. CAPAN2
0.0


Renal ca. UO-31
0.1
Pancreas Pool
9.5
















TABLE QF







Panel 1.1











Rel. Exp. (%) Ag616, Run

Rel. Exp. (%) Ag616, Run


Tissue Name
111162134
Tissue Name
111162134













Adrenal gland
3.7
Renal ca. UO-31
0.0


Bladder
11.7
Renal ca. RXF 393
0.0


Brain (amygdala)
0.2
Liver
26.2


Brain (cerebellum)
76.8
Liver (fetal)
0.1


Brain (hippocampus)
7.4
Liver ca. (hepatoblast)
0.0




HepG2


Brain (substantia nigra)
76.3
Lung
4.2


Brain (thalamus)
16.2
Lung (fetal)
3.8


Cerebral Cortex
6.0
Lung ca. (non-s. cell)
0.0




HOP-62


Brain (fetal)
2.2
Lung ca. (large cell) NCI-
0.0




H460


Brain (whole)
31.0
Lung ca. (non-s. cell)
6.0




NCI-H23


glio/astro U-118-MG
0.0
Lung ca. (non-s. cl) NCI-
18.6




H522


astrocytoma SF-539
0.0
Lung ca. (non-sm. cell)
6.2




A549


astrocytoma SNB-75
0.0
Lung ca. (s. cell var.)
0.0




SHP-77


astrocytoma SW1783
0.0
Lung ca. (small cell) LX-1
0.0


glioma U251
0.0
Lung ca. (small cell)
1.5




NCI-H69


glioma SF-295
0.0
Lung ca. (squam.) SW
0.0




900


glioma SNB-19
0.0
Lung ca. (squam.) NCI-
18.4




H596


glio/astro U87-MG
0.0
Lymph node
3.4


neuro*; met SK-N-AS
0.0
Spleen
2.9


Mammary gland
25.0
Thymus
5.6


Breast ca. BT-549
0.0
Ovary
47.3


Breast ca. MDA-N
0.0
Ovarian ca. IGROV-1
8.6


Breast ca.* (pl. ef) T47D
0.0
Ovarian ca. OVCAR-3
13.5


Breast ca.* (pl. ef) MCF-7
0.0
Ovarian ca. OVCAR-4
0.0


Breast ca.* (pl. ef) MDA-
0.0
Ovarian ca. OVCAR-5
6.8


MB-231


Small intestine
2.4
Ovarian ca. OVCAR-8
0.2


Colorectal
0.0
Ovarian ca.* (ascites)
0.1




SK-OV-3


Colon ca. HT29
0.0
Pancreas
100.0


Colon ca. CaCo-2
0.0
Pancreatic ca. CAPAN 2
0.0


Colon ca. HCT-15
0.0
Pituitary gland
0.0


Colon ca. HCT-116
0.0
Placenta
0.5


Colon ca. HCC-2998
0.0
Prostate
0.7


Colon ca. SW480
0.0
Prostate ca.* (bone met)
0.0




PC-3


Colon ca.* SW620
0.0
Salivary gland
1.8


(SW480 met)


Stomach
3.4
Trachea
1.0


Gastric ca. (liver met)
0.0
Spinal cord
10.4


NCI-N87


Heart
46.3
Testis
10.2


Skeletal muscle (Fetal)
19.2
Thyroid
0.7


Skeletal muscle
19.2
Uterus
7.6


Endothelial cells
0.0
Melanoma M14
0.0


Heart (Fetal)
4.1
Melanoma LOX IMVI
0.0


Kidney
0.6
Melanoma UACC-62
0.0


Kidney (fetal)
0.2
Melanoma SK-MEL-28
0.0


Renal ca. 786-0
0.0
Melanoma* (met) SK-
0.0




MEL-5


Renal ca. A498
0.0
Melanoma Hs688 (A).T
0.0


Renal ca. ACHN
0.0
Melanoma* (met)
0.0




Hs688 (B).T


Renal ca. TK-10
0.0
















TABLE QG







Panel 1.2











Rel. Exp. (%) Ag616, Run

Rel. Exp. (%) Ag616, Run


Tissue Name
118515000
Tissue Name
118515000













Endothelial cells
0.0
Renal ca. 786-0
0.0


Heart (Fetal)
8.5
Renal ca. A498
0.1


Pancreas
100.0
Renal ca. RXF 393
0.0


Pancreatic ca. CAPAN 2
0.0
Renal ca. ACHN
0.0


Adrenal Gland
25.5
Renal ca. UO-31
0.0


Thyroid
8.5
Renal ca. TK-10
0.0


Salivary gland
8.1
Liver
69.7


Pituitary gland
6.2
Liver (fetal)
6.6


Brain (fetal)
17.8
Liver ca. (hepatoblast)
0.0




HepG2


Brain (whole)
52.9
Lung
21.5


Brain (amygdala)
23.5
Lung (fetal)
12.0


Brain (cerebellum)
66.4
Lung ca. (small cell) LX-1
0.8


Brain (hippocampus)
21.2
Lung ca. (small cell)
8.4




NCI-H69


Brain (thalamus)
23.3
Lung ca. (s. cell var.)
0.0




SHP-77


Cerebral Cortex
0.0
Lung ca. (large cell) NCI-
0.0




H460


Spinal cord
19.3
Lung ca. (non-sm. cell)
12.8




A549


glio/astro U87-MG
0.0
Lung ca. (non-s. cell)
11.2




NCI-H23


glio/astro U-118-MG
0.0
Lung ca. (non-s. cell)
0.1




HOP-62


astrocytoma SW1783
0.0
Lung ca. (non-s. cl) NCI-
36.3




H522


neuro*; met SK-N-AS
0.0
Lung ca. (squam.) SW
1.6




900


astrocytoma SF-539
0.0
Lung ca. (squam.) NCI-
29.5




H596


astrocytoma SNB-75
0.0
Mammary gland
44.1


glioma SNB-19
0.1
Breast ca.* (pl. ef) MCF-7
0.0


glioma U251
0.0
Breast ca.* (pl. ef) MDA-
0.0




MB-231


glioma SF-295
0.0
Breast ca.* (pl. ef) T47D
0.1


Heart
69.7
Breast ca. BT-549
0.0


Skeletal Muscle
32.1
Breast ca. MDA-N
0.0


Bone marrow
6.6
Ovary
53.2


Thymus
17.4
Ovarian ca. OVCAR-3
18.9


Spleen
15.6
Ovarian ca. OVCAR-4
0.4


Lymph node
15.9
Ovarian ca. OVCAR-5
10.5


Colorectal Tissue
0.0
Ovarian ca. OVCAR-8
3.6


Stomach
11.8
Ovarian ca. IGROV-1
18.8


Small intestine
15.9
Ovarian ca. (ascites) SK-
1.9




OV-3


Colon ca. SW480
0.4
Uterus
22.7


Colon ca.* SW620
0.2
Placenta
6.7


(SW480 met)


Colon ca. HT29
1.0
Prostate
6.5


Colon ca. HCT-116
0.0
Prostate ca.* (bone met)
0.0




PC-3


Colon ca. CaCo-2
0.7
Testis
50.3


Colon ca. Tissue
0.3
Melanoma Hs688 (A).T
0.0


(ODO3866)


Colon ca. HCC-2998
0.6
Melanoma* (met)
0.0




Hs688 (B).T


Gastric ca.* (liver met)
0.2
Melanoma UACC-62
0.0


NCI-N87


Bladder
28.3
Melanoma M14
0.0


Trachea
7.0
Melanoma LOX IMVI
0.0


Kidney
2.8
Melanoma* (met) SK-
0.0




MEL-5


Kidney (fetal)
5.3
















TABLE QH







Panel 1.3D











Rel. Exp. (%) Ag3234, Run

Rel. Exp. (%) Ag3234, Run


Tissue Name
165524160
Tissue Name
165524160













Liver adenocarcinoma
5.8
Kidney (fetal)
2.4


Pancreas
43.2
Renal ca. 786-0
0.0


Pancreatic ca. CAPAN 2
0.0
Renal ca. A498
0.0


Adrenal gland
4.5
Renal ca. RXF 393
0.0


Thyroid
0.0
Renal ca. ACHN
0.0


Salivary gland
6.3
Renal ca. UO-31
0.0


Pituitary gland
1.4
Renal ca. TK-10
0.0


Brain (fetal)
5.8
Liver
6.3


Brain (whole)
45.4
Liver (fetal)
2.9


Brain (amygdala)
27.2
Liver ca. (hepatoblast)
0.0




HepG2


Brain (cerebellum)
100.0
Lung
12.2


Brain (hippocampus)
21.3
Lung (fetal)
6.5


Brain (substantia nigra)
21.0
Lung ca. (small cell) LX-1
1.4


Brain (thalamus)
27.2
Lung ca. (small cell)
1.2




NCI-H69


Cerebral Cortex
14.3
Lung ca. (s. cell var.)
0.0




SHP-77


Spinal cord
35.4
Lung ca. (large cell) NCI-
2.0




H460


glio/astro U87-MG
0.0
Lung ca. (non-sm. cell)
4.9




A549


glio/astro U-118-MG
0.0
Lung ca. (non-s. cell)
6.2




NCI-H23


astrocytoma SW1783
0.0
Lung ca. (non-s. cell)
0.0




HOP-62


neuro*; met SK-N-AS
1.6
Lung ca. (non-s. cl) NCI-
0.6




H522


astrocytoma SF-539
0.0
Lung ca. (squam.) SW
0.7




900


astrocytoma SNB-75
0.0
Lung ca. (squam.) NCI-
7.4




H596


glioma SNB-19
0.0
Mammary gland
28.9


glioma U251
0.0
Breast ca.* (pl. ef) MCF-7
0.0


glioma SF-295
0.0
Breast ca.* (pl. ef) MDA-
0.0




MB-231


Heart (fetal)
15.2
Breast ca.* (pl. ef) T47D
0.0


Heart
13.3
Breast ca. BT-549
0.0


Skeletal muscle (fetal)
9.9
Breast ca. MDA-N
0.0


Skeletal muscle
11.2
Ovary
50.7


Bone marrow
6.2
Ovarian ca. OVCAR-3
7.1


Thymus
18.6
Ovarian ca. OVCAR-4
1.3


Spleen
8.0
Ovarian ca. OVCAR-5
6.0


Lymph node
14.4
Ovarian ca. OVCAR-8
3.0


Colorectal
7.4
Ovarian ca. IGROV-1
1.6


Stomach
4.5
Ovarian ca.* (ascites)
1.2




SK-OV-3


Small intestine
16.6
Uterus
42.6


Colon ca. SW480
0.0
P1acenta
1.7


Colon ca.* SW620(SW480
0.0
Prostate
3.4


met)


Colon ca. HT29
0.0
Prostate ca.* (bone
0.0




met) PC-3


Colon ca. HCT-116
0.0
Testis
15.6


Colon ca. CaCo-2
0.0
Melanoma Hs688 (A).T
0.0


Colon ca.
1.4
Melanoma* (met)
0.0


tissue(ODO3866)

Hs688 (B).T


Colon ca. HCC-2998
0.0
Melanoma UACC-62
0.0


Gastric ca.* (liver met)
0.0
Melanoma M14
0.0


NCI-N87


Bladder
5.2
Melanoma LOX IMVI
0.0


Trachea
4.1
Melanoma* (met) SK-
0.0




MEL-5


Kidney
0.0
Adipose
28.1
















TABLE QI







Panel 2.2











Rel. Exp. (%) Ag3234,

Rel. Exp. (%) Ag3234,


Tissue Name
Run 174442923
Tissue Name
Run 174442923













Normal Colon
7.1
Kidney Margin (OD04348)
4.8


Colon cancer (OD06064)
6.7
Kidney malignant cancer
22.2




(OD06204B)


Colon Margin (OD06064)
5.6
Kidney normal adjacent
3.4




tissue (OD06204E)


Colon cancer (OD06159)
0.0
Kidney Cancer (OD04450-
0.0




01)


Colon Margin (OD06159)
5.9
Kidney Margin (OD04450-
1.8




03)


Colon cancer (OD06297-04)
0.0
Kidney Cancer 8120613
0.0


Colon Margin (OD06297-05)
2.9
Kidney Margin 8120614
1.4


CC Gr.2 ascend colon
1.8
Kidney Cancer 9010320
0.0


(ODO3921)


CC Margin (ODO3921)
0.0
Kidney Margin 9010321
1.3


Colon cancer metastasis
1.2
Kidney Cancer 8120607
1.9


(OD06104)


Lung Margin (OD06104)
0.7
Kidney Margin 8120608
1.5


Colon mets to lung
0.0
Normal Uterus
33.9


(OD04451-01)


Lung Margin (OD04451-02)
57.4
Uterine Cancer 064011
6.7


Normal Prostate
2.6
Normal Thyroid
1.3


Prostate Cancer (OD04410)
0.0
Thyroid Cancer 064010
0.0


Prostate Margin (OD04410)
7.7
Thyroid Cancer A302152
1.8


Normal Ovary
100.0
Thyroid Margin A302153
0.8


Ovarian cancer (OD06283-
5.4
Normal Breast
22.5


03)


Ovarian Margin (OD06283-
27.9
Breast Cancer (OD04566)
3.9


07)


Ovarian Cancer 064008
12.9
Breast Cancer 1024
11.4


Ovarian cancer (OD06145)
12.7
Breast Cancer (OD04590-
20.0




01)


Ovarian Margin (OD06145)
19.3
Breast Cancer Mets
17.2




(OD04590-03)


Ovarian cancer (OD06455-
8.1
Breast Cancer Metastasis
31.0


03)

(OD04655-05)


Ovarian Margin (OD06455-
25.9
Breast Cancer 064006
3.9


07)


Normal Lung
14.8
Breast Cancer 9100266
10.0


Invasive poor diff. lung
0.5
Breast Margin 9100265
16.8


adeno (ODO4945-01


Lung Margin (ODO4945-03)
28.7
Breast Cancer A209073
10.4


Lung Malignant Cancer
7.0
Breast Margin A2090734
28.1


(OD03126)


Lung Margin (OD03126)
3.2
Breast cancer (OD06083)
12.6


Lung Cancer (OD05014A)
3.8
Breast cancer node
13.7




metastasis (OD06083)


Lung Margin (OD05014B)
28.5
Normal Liver
41.5


Lung cancer (OD06081)
3.1
Liver Cancer 1026
10.5


Lung Margin (OD06081)
18.2
Liver Cancer 1025
40.1


Lung Cancer (OD04237-01)
2.7
Liver Cancer 6004-T
21.0


Lung Margin (OD04237-02)
12.2
Liver Tissue 6004-N
3.4


Ocular Melanoma Metastasis
0.0
Liver Cancer 6005-T
59.9


Ocular Melanoma Margin
22.2
Liver Tissue 6005-N
59.5


(Liver)


Melanoma Metastasis
0.0
Liver Cancer 064003
10.4


Melanoma Margin (Lung)
18.4
Normal Bladder
7.6


Normal Kidney
1.2
Bladder Cancer 1023
2.5


Kidney Ca, Nuclear grade 2
6.7
Bladder Cancer A302173
1.4


(OD04338)


Kidney Margin (OD04338)
1.7
Normal Stomach
12.3


Kidney Ca Nuclear grade 1/2
22.1
Gastric Cancer 9060397
1.0


(OD04339)


Kidney Margin (OD04339)
5.0
Stomach Margin 9060396
1.6


Kidney Ca, Clear cell type
3.5
Gastric Cancer 9060395
1.3


(OD04340)


Kidney Margin (OD04340)
3.7
Stomach Margin 9060394
3.1


Kidney Ca, Nuclear grade 3
0.8
Gastric Cancer 064005
0.0


(OD04348)
















TABLE QJ







Panel 4D













Rel. Exp. (%)
Rel. Exp. (%)

Rel. Exp. (%)
Rel. Exp. (%)



Ag3234, Run
Ag3279, Run

Ag3234, Run
Ag3279, Run


Tissue Name
164328482
164634320
Tissue Name
164328482
164634320















Secondary Th1 act
0.0
0.0
HUVEC IL-1beta
0.0
0.0


Secondary Th2 act
0.2
0.0
HUVEC IFN gamma
0.0
0.0


Secondary Tr1 act
0.0
0.0
HUVEC TNF alpha +
0.0
0.0





IFN gamma


Secondary Th1 rest
0.6
0.2
HUVEC TNF alpha +
0.0
0.0





IL4


Secondary Th2 rest
0.0
0.0
HUVEC IL-11
0.0
0.0


Secondary Tr1 rest
0.3
0.1
Lung Microvascular
0.0
0.0





EC none


Primary Th1 act
0.2
0.1
Lung Microvascular
0.0
0.0





EC TNFalpha + IL-





1beta


Primary Th2 act
0.0
0.0
Microvascular
0.0
0.0





Dermal EC none


Primary Tr1 act
0.0
0.9
Microsvasular Dermal
0.0
0.0





EC TNFalpha + IL-





1beta


Primary Th1 rest
0.7
0.9
Bronchial epithelium
0.0
0.0





TNFalpha + IL1beta


Primary Th2 rest
0.2
0.2
Small airway
0.0
0.0





epithelium none


Primary Tr1 rest
0.8
0.2
Small airway
0.0
0.0





epithelium





TNFalpha + IL-1beta


CD45RA CD4
0.0
0.0
Coronery artery SMC
0.0
0.0


lymphocyte act


rest


CD45RO CD4
0.0
0.0
Coronery artery SMC
0.0
0.0


lymphocyte act


TNFalpha + IL-1beta


CD8 lymphocyte act
0.0
0.0
Astrocytes rest
0.0
0.0


Secondary CD8
0.0
0.0
Astrocytes 
0.0
0.0


lymphocyte rest


TNFalpha + IL-1beta


Secondary CD8
0.0
0.0
KU-812 (Basophil)
0.5
0.0


lymphocyte act


rest


CD4 lymphocyte
0.2
0.7
KU-812 (Basophil)
0.2
0.3


none


PMA/ionomycin


2ry
0.2
0.0
CCD1106
0.0
0.0


Th1/Th2/Tr1_anti-


(Keratinocytes) none


CD95 CH11


LAK cells rest
6.2
7.6
CCD1106
0.0
0.0





(Keratinocytes)





TNFalpha +





IL-1beta


LAK cells IL-2
0.0
0.0
Liver cirrhosis
1.4
1.2


LAK cells IL-2 + IL-
0.0
0.0
Lupus kidney
0.4
0.2


12


LAK cells IL-2 + IFN
0.1
0.4
NCI-H292 none
0.0
0.2


gamma


LAK cells IL-2 + IL-
0.4
0.0
NCI-H292 IL-4
0.0
0.0


18


LAK cells
1.3
2.7
NCI-H292 IL-9
0.0
0.0


PMA/ionomycin


NK Cells IL-2 rest
0.0
0.0
NCI-H292 IL-13
0.0
0.0


Two Way MLR 3
1.1
1.3
NCI-H292 IFN
0.0
0.2


day


gamma


Two Way MLR 5
1.3
0.6
HPAEC none
0.0
0.0


day


Two Way MLR 7
0.8
0.4
HPAEC TNF alpha +
0.0
0.0


day


IL-1beta


PBMC rest
3.3
3.1
Lung fibroblast none
0.0
0.0


PBMC PWM
0.0
0.1
Lung fibroblast TNF
0.0
0.0





alpha + IL-1 beta


PBMC PHA-L
0.1
0.0
Lung fibroblast IL-4
0.0
0.0


Ramos (B cell) none
0.0
0.0
Lung fibroblast IL-9
0.0
0.0


Ramos (B cell)
0.0
0.0
Lung fibroblast IL-13
0.0
0.0


ionomycin


B lymphocytes PWM
0.0
0.0
Lung fibroblast IFN
0.0
0.0





gamma


B lymphocytes
0.0
0.3
Dermal fibroblast
0.0
0.0


CD40L and IL-4


CCD1070 rest


EOL-1 dbcAMP
0.0
0.2
Dermal fibroblast
0.2
0.6





CCD1070 TNF alpha


EOL-1 dbcAMP
0.1
0.0
Dermal fibroblast
0.0
0.0


PMA/ionomycin


CCD1070 IL-1 beta


Dendritic cells none
25.9
49.0
Dermal fibroblast IFN
0.0
0.2





gamma


Dendritic cells LPS
61.1
92.0
Dermal fibroblast IL-4
0.2
0.0


Dendritic cells anti-
100.0
94.6
IBD Colitis 2
0.0
0.7


CD40


Monocytes rest
12.2
23.0
IBD Crohn's
0.3
0.7


Monocytes LPS
2.6
2.5
Colon
3.0
2.8


Macrophages rest
92.0
100.0
Lung
7.4
9.7


Macrophages LPS
10.4
18.0
Thymus
1.2
3.7


HUVEC none
0.0
0.0
Kidney
35.4
33.4


HUVEC starved
0.0
0.0
















TABLE QK







Panel CNS_1











Rel. Exp. (%) Ag3279, Run

Rel. Exp. (%) Ag3279, Run


Tissue Name
171694591
Tissue Name
171694591













BA4 Control
3.2
BA17 PSP
13.4


BA4 Control2
10.0
BA17 PSP2
5.3


BA4 Alzheimer's2
3.8
Sub Nigra Control
58.6


BA4 Parkinson's
6.5
Sub Nigra Control2
21.3


BA4 Parkinson's2
11.7
Sub Nigra Alzheimer's2
12.2


BA4 Huntington's
7.6
Sub Nigra Parkinson's2
63.3


BA4
4.5
Sub Nigra Huntington's
100.0


Huntington's2


BA4 PSP
5.1
Sub Nigra
87.1




Huntington's2


BA4 PSP2
8.8
Sub Nigra PSP2
16.8


BA4 Depression
6.7
Sub Nigra Depression
7.9


BA4 Depression2
7.4
Sub Nigra Depression2
24.3


BA7 Control
4.5
Glob Palladus Control
24.3


BA7 Control2
1.9
Glob Palladus Control2
8.1


BA7 Alzheimer's2
1.0
Glob Palladus
14.9




Alzheimer's


BA7 Parkinson's
24.1
Glob Palladus
1.2




Alzheimer's2


BA7 Parkinson's2
17.4
Glob Palladus
33.0




Parkinson's


BA7 Huntington's
6.3
Glob Palladus
2.0




Parkinson's2


BA7
28.5
Glob Palladus PSP
3.0


Huntington's2


BA7 PSP
18.9
Glob Palladus PSP2
6.0


BA7 PSP2
3.8
Glob Palladus
8.8




Depression


BA7 Depression
1.7
Temp Pole Control
5.9


BA9 Control
5.3
Temp Pole Control2
11.7


BA9 Control2
4.1
Temp Pole Alzheimer's
7.4


BA9 Alzheimer's
3.6
Temp Pole Alzheimer's2
3.0


BA9 Alzheimer's2
8.8
Temp Pole Parkinson's
11.9


BA9 Parkinson's
18.6
Temp Pole Parkinson's2
7.9


BA9 Parkinson's2
20.4
Temp Pole Huntington's
8.8


BA9 Huntington's
15.0
Temp Pole PSP
4.7


BA9
7.4
Temp Pole PSP2
0.0


Huntington's2


BA9 PSP
5.3
Temp Pole Depression2
12.6


BA9 PSP2
1.6
Cing Gyr Control
18.0


BA9 Depression
4.7
Cing Gyr Control2
20.2


BA9 Depression2
4.5
Cing Gyr Alzheimer's
8.6


BA17 Control
20.2
Cing Gyr Alzheimer's2
4.2


BA17 Control2
7.9
Cing Gyr Parkinson's
12.2


BA17
3.2
Cing Gyr Parkinson's2
15.3


Alzheimer's2


BA17 Parkinson's
14.1
Cing Gyr Huntington's
28.1


BA17
8.7
Cing Gyr Huntington's2
4.7


Parkinson's2


BA17
22.2
Cing Gyr PSP
7.8


Huntington's


BA17
18.2
Cing Gyr PSP2
11.2


Huntington's2


BA17 Depression
4.9
Cing Gyr Depression
2.9


BA17 Depression2
19.9
Cing Gyr Depression2
8.7









CNS_neurodegeneration_v1.0 Summary: Ag3234/Ag3279 Two experiments with the same probe and primer set produce results that are in excellent agreement. Both experiments show a difference in expression of the CG57429-01 gene between Alzheimer's diseased postmortem brains and controls for this gene. Expression is increased in the temporal cortex of patients with AD (p=0.016 for ag3234 and p=0.024 for ag3279) and in the hippocampus. Both the temporal cortex and hippocampus are regions that show severe neurodegeneration in AD. In contrast, expression in the occipital cortex, a region that does not degenerate in Alzheimer's disease, is not disregulated. Together, these data suggest that the CG57429-01 protein product may be involved in the pathology or response to Alzheimer's disease. Therefore, this may be a useful drug target for the treatment of this disease.
General_screening_panel_v1.4 Summary: Ag3279 Highest expression of the CG57429-01 gene is in the cerebellum. Significant levels of expression are also seen in other regions of the brain including the amygdala, hippocampus, cerebral cortex, substantia nigra, and thalamus. Cadherins can act as axon guidance and cell adhesion proteins, specifically during development and in the response to injury (ref 1). Manipulation of levels of this protein may be of use in inducing a compensatory synaptogenic response to neuronal death in Alzheimer's disease, Parkinson's disease, Huntington's disease, spinocerebellar ataxia, progressive supranuclear palsy, ALS, head trauma, stroke, or any other disease/condition associated with neuronal loss.
In addition, this gene is highly expressed in pituitary gland, adrenal gland, thyroid, pancreas, adult and fetal skeletal muscle, heart and liver, reflecting the widespread role of cadherins in cell-cell adhesion. This observation may suggest that this gene plays a role in normal metabolic and neuroendocrine function and that disregulated expression of this gene may contribute to metabolic diseases (such as obesity and diabetes) or neuroendocrine disorders.
Overall, gene expression is associated with normal tissues rather than cancer cell lines. Loss of function of the related E-cadherin protein has been described in many tumors, along with an increased invasiveness and a decreased prognosis of many carcinomas, including tumors of endocrine glands and their target systems (ref 1). Thus, this gene product might similarly be useful as a protein therapeutic to treat a variety of tumors, since it is found in normal cells but missing from cancer cells (Potter et al., Endocr. Rev. 20: 207-239, 1999; Ranscht, Int. J. Dev. Neurosci. 18: 643-651, 2000).
Panel 1.1 and 1.2 Summary: Ag616 Highest expression of the CG57429-01 gene, a cadherin homolog, is seen in pancreas (CT=23.2). Significant expression is also seen in adrenal gland, fetal and adult skeletal muscle, liver and heart. This widespread expression among tissues with metabolic function is consistent with expression seen in General_screening_panel_v1.4. Please see that panel for further discussion of utility of this gene in metabolic disorders.
In addition, there is higher expression in adult liver (CT=27) when compared to experession in fetal liver (CT=34.8). Thus, expression of this gene could be used to differentiate between fetal and adult liver.
Overall, expression in this panel is in agreement with expression in the previous panel. Please see that panel for further discussion of utility of this gene.
Panel 1.3D Summary: Ag3234 Results from this experiment produce a similar profile to Panel 1.4. Please see that Panel for further discussion of expression and utility of this gene.
Panel 2.2 Summary: Ag3234 The expression of the CG57429-01 gene appears to be highest in a sample derived from a normal ovarian tissue (CT=32.3). In addition, there appears to be substantial expression in other samples derived from liver cancers. Furthermore, there appears to be expression specific to normal lung tissue when compared to malignant lung tissue. Thus, the expression of this gene could be used to distinguish normal ovarian tissue from other samples in the panel. Moreover, therapeutic modulation of this gene, through the use of small molecule drugs, protein therapeutics or antibodies could be of benefit in the treatment of liver cancer, ovarian cancer or lung cancer.
Panel 4D Summary: Ag3234/Ag3279 The CG57429-01 gene, a cadherin 23-like molecule, is expressed selectively at moderate levels (CTs=28.1-30.1) in resting and activated dendritic cells, and in resting and activated macrophages. Thus, small molecule antagonists or therapeutic antibodies that block the function of the CG57429-01 gene product may be useful in the reduction or elimination of the symptoms in patients with autoimmune and inflammatory diseases in which dendritic cells and macrophages play an important role in antigen presentation and other functions, such as, but not limited to, including Crohn's disease, ulcerative colitis, multiple sclerosis, chronic obstructive pulmonary disease, asthma, emphysema, rheumatoid arthritis, lupus erythematosus, or psoriasis.
Panel CNS—1 Summary: Ag3279 This panel confirms expression of the CG57429-01 gene in the brain. Please see Panel 1.3D for discussion of utility of this gene in the central nervous system. Results from a second experiment with Ag3234 are not included. The amp plot indicates that there were experimental difficulties with this run.
R. NOV21: Platelet Glycoprotein V
Expression of gene CG57436-01 was assessed using the primer-probe set Ag3236, described in Table SA. Results of the RTQ-PCR runs are shown in Tables RB, RC, RD and RE.







TABLE RA







Probe Name Ag3236













Start


Primers
Sequences
Length
Position














Forward
5′-gaggtgtgactcagacatcctt-3′
(SEQ ID NO:365)
22
1409


Probe
TET-5′-aactggctcctgctcaaccagcct-3′-TAMRA
(SEQ ID NO:366)
24
1440


Reverse
5′-gtacagtgtccgtccctaacct-3′
(SEQ ID NO:367)
22
1464
















TABLE RB







General_screening_panel_v1.4











Rel. Exp. (%) Ag3236, Run

Rel. Exp. (%) Ag3236, Run


Tissue Name
214693630
Tissue Name
214693630













Adipose
0.5
Renal ca. TK-10
0.0


Melanoma* Hs688 (A).T
38.2
Bladder
0.6


Melanoma* Hs688 (B).T
9.4
Gastric ca. (liver met.)
0.0




NCI-N87


Melanoma* M14
0.0
Gastric ca. KATO III
0.0


Melanoma* LOXIMVI
0.7
Colon ca. SW-948
0.0


Melanoma* SK-MEL-5
0.0
Colon ca. SW480
0.0


Squamous cell
0.0
Colon ca.* (SW480 met)
0.0


carcinoma SCC-4

SW620


Testis Pool
0.0
Colon ca. HT29
0.0


Prostate ca.* (bone met)
0.0
Colon ca. HCT-116
0.0


PC-3


Prostate Pool
0.1
Colon ca. CaCo-2
0.0


Placenta
0.6
Colon cancer tissue
1.9


Uterus Pool
0.2
Colon ca. SW1116
0.0


Ovarian ca. OVCAR-3
0.0
Colon ca. Colo-205
0.0


Ovarian ca. SK-OV-3
0.0
Colon ca. SW-48
0.0


Ovarian ca. OVCAR-4
0.0
Colon Pool
0.3


Ovarian ca. OVCAR-5
0.1
Small Intestine Pool
0.0


Ovarian ca. IGROV-1
0.0
Stomach Pool
0.2


Ovarian ca. OVCAR-8
0.0
Bone Marrow Pool
0.3


Ovary
0.2
Fetal Heart
0.0


Breast ca. MCF-7
0.0
Heart Pool
0.1


Breast ca. MDA-MB-
0.5
Lymph Node Pool
0.2


231


Breast ca. BT 549
0.0
Fetal Skeletal Muscle
0.1


Breast ca. T47D
0.3
Skeletal Muscle Pool
0.1


Breast ca. MDA-N
0.0
Spleen Pool
0.1


Breast Pool
0.2
Thymus Pool
0.0


Trachea
0.2
CNS cancer (glio/astro)
14.8




U87-MG


Lung
0.0
CNS cancer (glio/astro) U-
100.0




118-MG


Fetal Lung
0.0
CNS cancer (neuro; met)
0.0




SK-N-AS


Lung ca. NCI-N417
0.0
CNS cancer (astro) SF-539
13.2


Lung ca. LX-1
0.0
CNS cancer (astro) SNB-75
5.3


Lung ca. NCI-H146
0.0
CNS cancer (glio) SNB-19
0.0


Lung ca. SHP-77
0.0
CNS cancer (glio) SF-295
0.3


Lung ca. A549
0.0
Brain (Amygdala) Pool
0.0


Lung ca. NCI-H526
0.0
Brain (cerebellum)
0.0


Lung ca. NCI-H23
0.0
Brain (fetal)
0.0


Lung ca. NCI-H460
0.0
Brain (Hippocampus) Pool
0.0


Lung ca. HOP-62
0.0
Cerebral Cortex Pool
0.0


Lung ca. NCI-H522
0.0
Brain (Substantia nigra)
0.0




Pool


Liver
0.0
Brain (Thalamus) Pool
0.0


Fetal Liver
0.0
Brain (whole)
0.0


Liver ca. HepG2
0.0
Spinal Cord Pool
0.0


Kidney Pool
0.2
Adrenal Gland
0.1


Fetal Kidney
0.0
Pituitary gland Pool
0.0


Renal ca. 786-0
0.1
Salivary Gland
0.0


Renal ca. A498
0.1
Thyroid (female)
0.6


Renal ca. ACHN
0.0
Pancreatic ca. CAPAN2
0.0


Renal ca. UO-31
0.4
Pancreas Pool
0.1
















TABLE RC







Panel 2.2











Rel. Exp. (%) Ag3236,

Rel. Exp. (%) Ag3236,


Tissue Name
Run 174442924
Tissue Name
Run 174442924













Normal Colon
0.2
Kidney Margin (OD04348)
0.5


Colon cancer (OD06064)
7.3
Kidney malignant cancer
0.8




(OD06204B)


Colon Margin (OD06064)
0.2
Kidney normal adjacent
0.0




tissue (OD06204E)


Colon cancer (OD06159)
0.3
Kidney Cancer (OD04450-
0.0




01)


Colon Margin (OD06159)
0.6
Kidney Margin (OD04450-
0.2




03)


Colon cancer (OD06297-04)
1.7
Kidney Cancer 8120613
0.0


Colon Margin (OD06297-05)
2.4
Kidney Margin 8120614
0.0


CC Gr.2 ascend colon
1.6
Kidney Cancer 9010320
1.1


(ODO3921)


CC Margin (ODO3921)
1.9
Kidney Margin 9010321
1.1


Colon cancer metastasis
0.3
Kidney Cancer 8120607
4.7


(OD06104)


Lung Margin (OD06104)
0.6
Kidney Margin 8120608
0.2


Colon mets to lung
0.6
Normal Uterus
1.4


(OD04451-01)


Lung Margin (OD04451-02)
0.3
Uterine Cancer 064011
0.0


Normal Prostate
0.0
Normal Thyroid
0.5


Prostate Cancer (OD04410)
0.3
Thyroid Cancer 064010
1.0


Prostate Margin (OD04410)
0.0
Thyroid Cancer A302152
11.9


Normal Ovary
2.7
Thyroid Margin A302153
0.0


Ovarian cancer (OD06283-
13.9
Normal Breast
0.7


03)


Ovarian Margin (OD06283-
1.0
Breast Cancer (OD04566)
3.7


07)


Ovarian Cancer 064008
27.0
Breast Cancer 1024
3.4


Ovarian cancer (OD06145)
0.7
Breast Cancer (OD04590-
8.4




01)


Ovarian Margin (OD06145)
1.1
Breast Cancer Mets
9.9




(OD04590-03)


Ovarian cancer (OD06455-
0.0
Breast Cancer Metastasis
0.5


03)

(OD04655-05)


Ovarian Margin (OD06455-
0.0
Breast Cancer 064006
31.2


07)


Normal Lung
0.0
Breast Cancer 9100266
14.3


Invasive poor diff. lung
4.6
Breast Margin 9100265
19.1


adeno (ODO4945-01


Lung Margin (ODO4945-03)
2.1
Breast Cancer A209073
5.8


Lung Malignant Cancer
6.4
Breast Margin A2090734
0.4


(OD03126)


Lung Margin (OD03126)
0.5
Breast cancer (OD06083)
15.7


Lung Cancer (OD05014A)
0.5
Breast cancer node
100.0




metastasis (OD06083)


Lung Margin (OD05014B)
0.0
Normal Liver
0.2


Lung cancer (OD06081)
0.8
Liver Cancer 1026
0.3


Lung Margin (OD06081)
0.0
Liver Cancer 1025
1.9


Lung Cancer (OD04237-01)
0.8
Liver Cancer 6004-T
0.0


Lung Margin (OD04237-02)
8.5
Liver Tissue 6004-N
0.0


Ocular Melanoma Metastasis
0.0
Liver Cancer 6005-T
0.8


Ocular Melanoma Margin
0.0
Liver Tissue 6005-N
0.0


(Liver)


Melanoma Metastasis
0.0
Liver Cancer 064003
0.0


Melanoma Margin (Lung)
1.9
Normal Bladder
1.8


Normal Kidney
0.0
Bladder Cancer 1023
8.4


Kidney Ca, Nuclear grade 2
0.2
Bladder Cancer A302173
6.7


(OD04338)


Kidney Margin (OD04338)
0.0
Normal Stomach
1.6


Kidney Ca Nuclear grade 1/2
0.0
Gastric Cancer 9060397
2.4


(OD04339)


Kidney Margin (OD04339)
0.0
Stomach Margin 9060396
0.8


Kidney Ca, Clear cell type
0.0
Gastric Cancer 9060395
0.3


(OD04340)


Kidney Margin (OD04340)
0.0
Stomach Margin 9060394
0.9


Kidney Ca, Nuclear grade 3
0.0
Gastric Cancer 064005
0.0


(OD04348)
















TABLE RD







Panel 3D











Rel. Exp. (%)

Rel. Exp. (%)



Ag3236, Run

Ag3236, Run


Tissue Name
182114365
Tissue Name
182114365













Daoy-Medulloblastoma
0.6
Ca Ski-Cervical epidermoid
0.0




carcinoma (metastasis)


TE671-Medulloblastoma
0.0
ES-2-Ovarian clear cell carcinoma
0.0


D283 Med-Medulloblastoma
0.0
Ramos-Stimulated with
0.0




PMA/ionomycin 6 h


PFSK-1-Primitive
0.2
Ramos-Stimulated with
0.0


Neuroectodermal

PMA/ionomycin 14 h


XF-498-CNS
100.0
MEG-01-Chronic myelogenous
0.0




leukemia (megokaryoblast)


SNB-78-Glioma
32.5
Raji-Burkitt's lymphoma
0.0


SF-268-Glioblastoma
0.3
Daudi-Burkitt's lymphoma
0.0


T98G-Glioblastoma
0.2
U266-B-cell plasmacytoma
0.2


SK-N-SH-Neuroblastoma
0.0
CA46-Burkitt's lymphoma
0.0


(metastasis)


SF-295-Glioblastoma
6.3
RL-non-Hodgkin's B-cell
0.0




lymphoma


Cerebellum
0.0
JM1-pre-B-cell lymphoma
0.0


Cerebellum
0.0
Jurkat-T cell leukemia
0.0


NCI-H292-Mucoepidermoid
0.0
TF-1-Erythroleukemia
0.2


lung carcinoma


DMS-114-Small cell lung
0.0
HUT 78-T-cell lymphoma
0.2


cancer


DMS-79-Small cell lung
0.0
U937-Histiocytic lymphoma
0.0


cancer


NCI-H146-Small cell lung
0.0
KU-812-Myelogenous leukemia
0.0


cancer


NCI-H526-Small cell lung
0.0
769-P-Clear cell renal carcinoma
0.0


cancer


NCI-N417-Small cell lung
0.0
Caki-2-Clear cell renal carcinoma
0.0


cancer


NCI-H82-Small cell lung
0.0
SW 839-Clear cell renal carcinoma
0.0


cancer


NCI-H157-Squamous cell
0.0
G401-Wilms' tumor
0.5


lung cancer (metastasis)


NCI-H1155-Large cell lung
0.0
Hs766T-Pancreatic carcinoma (LN
0.7


cancer

metastasis)


NCI-H1299-Large cell lung
0.0
CAPAN-1-Pancreatic
0.0


cancer

adenocarcinoma (liver metastasis)


NCI-H727-Lung carcinoid
0.0
SU86.86-Pancreatic carcinoma
0.2




(liver metastasis)


NCI-UMC-11-Lung
0.0
BxPC-3-Pancreatic
0.0


carcinoid

adenocarcinoma


LX-1-Small cell lung cancer
0.0
HPAC-Pancreatic adenocarcinoma
0.0


Colo-205-Colon cancer
0.0
MIA PaCa-2-Pancreatic carcinoma
0.0


KM12-Colon cancer
0.0
CFPAC-1-Pancreatic ductal
0.0




adenocarcinoma


KM20L2-Colon cancer
0.0
PANC-1-Pancreatic epithelioid
1.6




ductal carcinoma


NCI-H716-Colon cancer
0.0
T24-Bladder carcinma (transitional
0.0




cell)


SW-48-Colon
0.0
5637-Bladder carcinoma
0.0


adenocarcinoma


SW1116-Colon
0.0
HT-1197-Bladder carcinoma
0.1


adenocarcinoma


LS 174T-Colon
0.0
UM-UC-3-Bladder carcinma
0.0


adenocarcinoma

(transitional cell)


SW-948-Colon
0.0
A204-Rhabdomyosarcoma
0.9


adenocarcinoma


SW-480-Colon
0.0
HT-1080-Fibrosarcoma
0.3


adenocarcinoma


NCI-SNU-5-Gastric
0.0
MG-63-Osteosarcoma
0.0


carcinoma


KATO III-Gastric carcinoma
0.0
SK-LMS-1-Leiomyosarcoma
0.0




(vulva)


NCI-SNU-16-Gastric
4.4
SJRH30-Rhabdomyosarcoma (met
0.0


carcinoma

to bone marrow)


NCI-SNU-1-Gastric
0.0
A431-Epidermoid carcinoma
0.0


carcinoma


RF-1-Gastric
0.0
WM266-4-Melanoma
0.7


adenocarcinoma


RF-48-Gastric
0.0
DU 145-Prostate carcinoma (brain
0.0


adenocarcinoma

metastasis)


MKN-45-Gastric carcinoma
0.0
MDA-MB-468-Breast
0.0




adenocarcinoma


NCI-N87-Gastric carcinoma
0.0
SCC-4-Squamous cell carcinoma
0.0




of tongue


OVCAR-5-Ovarian
0.0
SCC-9-Squamous cell carcinoma
0.0


carcinoma

of tongue


RL95-2-Uterine carcinoma
0.0
SCC-15-Squamous cell carcinoma
0.0




of tongue


HelaS3-Cervical
0.0
CAL 27-Squamous cell carcinoma
0.0


adenocarcinoma

of tongue
















TABLE RE







Panel 4D











Rel. Exp. (%) Ag3236,

Rel. Exp. (%) Ag3236,


Tissue Name
Run 164328488
Tissue Name
Run 164328488













Secondary Th1 act
0.0
HUVEC IL-1 beta
0.0


Secondary Th2 act
0.0
HUVEC IFN gamma
0.0


Secondary Tr1 act
0.0
HUVEC TNF alpha + IFN
0.0




gamma


Secondary Th1 rest
0.0
HUVEC TNF alpha + IL4
0.0


Secondary Th2 rest
0.0
HUVEC IL-11
0.0


Secondary Tr1 rest
0.7
Lung Microvascular EC none
0.0


Primary Th1 act
0.0
Lung Microvascular EC
0.6




TNF alpha + IL-1 beta


Primary Th2 act
0.0
Microvascular Dermal EC none
0.6


Primary Tr1 act
0.0
Microsvasular Dermal EC
0.0




TNF alpha + IL-1 beta


Primary Th1 rest
0.0
Bronchial epithelium TNF alpha +
1.1




IL1 beta


Primary Th2 rest
0.0
Small airway epithelium none
3.2


Primary Tr1 rest
0.0
Small airway epithelium
0.0




TNF alpha + IL-1 beta


CD45RA CD4 lymphocyte
4.6
Coronery artery SMC rest
41.5


act


CD45RO CD4 lymphocyte
1.2
Coronery artery SMC TNF alpha +
54.3


act

IL-1 beta


CD8 lymphocyte act
0.0
Astrocytes rest
17.1


Secondary CD8
0.0
Astrocytes TNF alpha + IL-1 beta
17.4


lymphocyte rest


Secondary CD8
0.0
KU-812 (Basophil) rest
0.5


lymphocyte act


CD4 lymphocyte none
0.0
KU-812 (Basophil)
0.0




PMA/ionomycin


2ry Th1/Th2/Tr1_anti-
0.0
CCD1106 (Keratinocytes) none
0.0


CD95 CH11


LAK cells rest
0.0
CCD1106 (Keratinocytes)
0.7




TNF alpha + IL-1 beta


LAK cells IL-2
0.0
Liver cirrhosis
5.2


LAK cells IL-2 + IL-12
0.0
Lupus kidney
0.0


LAK cells IL-2 + IFN
0.0
NCI-H292 none
0.0


gamma


LAK cells IL-2 + IL-18
0.0
NCI-H292 IL-4
0.7


LAK cells
0.0
NCI-H292 IL-9
0.0


PMA/ionomycin


NK Cells IL-2 rest
0.0
NCI-H292 IL-13
0.0


Two Way MLR 3 day
0.0
NCI-H292 IFN gamma
0.0


Two Way MLR 5 day
0.0
HPAEC none
0.0


Two Way MLR 7 day
0.0
HPAEC TNF alpha + IL-1 beta
1.7


PBMC rest
0.0
Lung fibroblast none
15.9


PBMC PWM
0.0
Lung fibroblast TNF alpha + IL-
100.0




1 beta


PBMC PHA-L
0.0
Lung fibroblast IL-4
24.1


Ramos (B cell) none
0.0
Lung fibroblast IL-9
26.6


Ramos (B cell) ionomycin
0.0
Lung fibroblast IL-13
6.6


B lymphocytes PWM
0.0
Lung fibroblast IFN gamma
28.3


B lymphocytes CD40L
0.7
Dermal fibroblast CCD1070 rest
46.3


and IL-4


EOL-1 dbcAMP
0.0
Dermal fibroblast CCD1070
34.6




TNF alpha


EOL-1 dbcAMP
0.0
Dermal fibroblast CCD1070 IL-
74.2


PMA/ionomycin

1 beta


Dendritic cells none
0.0
Dermal fibroblast IFN gamma
81.2


Dendritic cells LPS
0.0
Dermal fibroblast IL-4
82.9


Dendritic cells anti-CD40
0.0
IBD Colitis 2
0.7


Monocytes rest
0.0
IBD Crohn's
1.3


Monocytes LPS
0.0
Colon
5.0


Macrophages rest
0.0
Lung
35.4


Macrophages LPS
0.0
Thymus
0.0


HUVEC none
0.0
Kidney
4.2


HUVEC starved
0.0









CNS_neurodegeneration_v1.0 Summary: Ag3236 Expression of the CG57436-01 is low/undetectable in all samples on this panel (CTs>35). (Data not shown.)
General_screening_panel_v1.4 Summary: Ag3236 Highest expression of the CG57436-01 gene is seen in a brain cancer cell line (CT=23.6). Significant levels of expression are also seen in a cluster of cell lines derived from brain cancer and melanoma. Thus, expression of this gene could be used to differentiate between these samples and other samples on this panel and as a diagnostic marker for the presence of these cancers. Furthermore, therapeutic modulation of the expression or function of this gene or gene product may be effective in the treatment of brain cancer or melanoma.
Among tissues with metabolic function, this gene is expressed at moderate/low levels in pancreas, adrenal gland, adipose, adult and fetal skeletal muscle and adult heart. This widespread expression suggests that this gene may play a role in normal metabolic function and that dysregulated expression of this gene may contribute to metabolic diseases, such as obesity and diabetes.
Panel 2.2 Summary: Ag3236 Highest expression of the CG57436-01 gene is seen in a breast cancer metastasis (CT=29). Significant expression is also seen in a cluster fo breast cancer samples. This is in concordance with the expression in General_screening_panel_v1.4, where expression is predominantly in cancer cell lines. Thus, expression of this gene could be used to differentiate between these sample and other samples on this panel and as a marker to detect the presence of breast cancer. Furthermore, therapeutic modulation of the expression or function of this gene may be effective in the treatment of breast cancer.
Panel 3D Summary: Ag3236 Highest expression of the CG57436-01 gene is seen in brain cancer cell lines, consistent with General_screening_panel_v1.4. Thus, expression of this gene could be used to differentiate between this sample and other samples on this panel.
Panel 4D Summary: Ag3236 Highest expression of the CG57436-01 gene is seen in a cluster of treated and untreated samples derived from lung and dermal fibroblasts, with highest expression in TNF-alpha and IL-1 beta treated lung fibroblasts (CT=29.8). Therefore, therapeutic antibody antagonists that block the binding of von Willebrand factor or other proteins to the CG57436-01 gene product may be useful in the treatment of disorders that involve activation of the coagulation cascade or binding by other ligands, including clotting disorders, atherosclerosis, cardiovascular disease, and autoimmune and inflammatory diseases, such as Crohn's disease, ulcerative colitis, multiple sclerosis, chronic obstructive pulmonary disease, asthma, emphysema, rheumatoid arthritis, lupus erythematosus, or psoriasis.
S. NOV22: GARPIN-like
Expression of gene CG57529-01 was assessed using the primer-probe set Ag3276, described in Table SA. Results of the RTQ-PCR runs are shown in Tables SB, SC and SD.







TABLE SA







Probe Name Ag3276













Start


Primers
Sequences
Length
Position














Forward
5′-acatgagccacaatcagatctc-3′
(SEQ ID NO:368)
22
1274


Probe
TET-5′-actttgtcccctgccagctgcct-3′-TAMRA
(SEQ ID NO:369)
23
1296


Reverse
5′-attcctgaaatccacacagcta-3′
(SEQ ID NO:370)
22
1338
















TABLE SB







CNS_neurodegeneration_v1.0











Rel. Exp. (%) Ag3276, Run

Rel. Exp. (%) Ag3276, Run


Tissue Name
206533680
Tissue Name
206533680













AD 1 Hippo
27.4
Control (Path) 3
6.7




Temporal Ctx


AD 2 Hippo
57.0
Control (Path) 4
18.9




Temporal Ctx


AD 3 Hippo
4.2
AD 1 Occipital Ctx
20.7


AD 4 Hippo
4.4
AD 2 Occipital Ctx
0.0




(Missing)


AD 5 hippo
88.3
AD 3 Occipital Ctx
12.4


AD 6 Hippo
97.9
AD 4 Occipital Ctx
8.7


Control 2 Hippo
38.4
AD 5 Occipital Ctx
10.5


Control 4 Hippo
42.9
AD 6 Occipital Ctx
46.3


Control (Path) 3 Hippo
2.8
Control 1 Occipital Ctx
5.5


AD 1 Temporal Ctx
6.3
Control 2 Occipital Ctx
48.0


AD 2 Temporal Ctx
27.2
Control 3 Occipital Ctx
9.8


AD 3 Temporal Ctx
6.4
Control 4 Occipital Ctx
8.8


AD 4 Temporal Ctx
15.5
Control (Path) 1
66.9




Occipital Ctx


AD 5 Inf Temporal Ctx
66.0
Control (Path) 2
6.0




Occipital Ctx


AD 5 SupTemporal Ctx
100.0
Control (Path) 3
2.6




Occipital Ctx


AD 6 Inf Temporal Ctx
98.6
Control (Path) 4
24.7




Occipital Ctx


AD 6 Sup Temporal Ctx
99.3
Control 1 Parietal Ctx
6.4


Control 1 Temporal Ctx
6.7
Control 2 Parietal Ctx
33.9


Control 2 Temporal Ctx
29.3
Control 3 Parietal Ctx
14.3


Control 3 Temporal Ctx
5.3
Control (Path) 1
60.7




Parietal Ctx


Control 4 Temporal Ctx
14.9
Control (Path) 2
21.6




Parietal Ctx


Control (Path) 1
53.2
Control (Path) 3
2.4


Temporal Ctx

Parietal Ctx


Control (Path) 2
24.0
Control (Path) 4
32.1


Temporal Ctx

Parietal Ctx
















TABLE SC







Panel 1.3D











Rel. Exp. (%) Ag3276, Run

Rel. Exp. (%) Ag3276, Run


Tissue Name
165524215
Tissue Name
165524215













Liver adenocarcinoma
0.5
Kidney (fetal)
2.6


Pancreas
1.6
Renal ca. 786-0
0.0


Pancreatic ca. CAPAN 2
0.4
Renal ca. A498
0.3


Adrenal gland
9.0
Renal ca. RXF 393
0.0


Thyroid
6.0
Renal ca. ACHN
0.4


Salivary gland
4.3
Renal ca. UO-31
0.4


Pituitary gland
4.2
Renal ca. TK-10
0.7


Brain (fetal)
1.7
Liver
4.0


Brain (whole)
8.5
Liver (fetal)
25.7


Brain (amygdala)
9.8
Liver ca. (hepatoblast)
0.0




HepG2


Brain (cerebellum)
7.3
Lung
15.2


Brain (hippocampus)
8.8
Lung (fetal)
7.3


Brain (substantia nigra)
7.3
Lung ca. (small cell) LX-1
0.4


Brain (thalamus)
5.3
Lung ca. (small cell)
0.0




NCI-H69


Cerebral Cortex
4.0
Lung ca. (s. cell var.)
3.5




SHP-77


Spinal cord
16.6
Lung ca. (large cell) NCI-
1.3




H460


glio/astro U87-MG
9.0
Lung ca. (non-sm. cell)
0.5




A549


glio/astro U-118-MG
7.7
Lung ca. (non-s. cell)
1.4




NCI-H23


astrocytoma SW1783
0.7
Lung ca. (non-s. cell)
0.2




HOP-62


neuro*; met SK-N-AS
0.7
Lung ca. (non-s. cl) NCI-
0.0




H522


astrocytoma SF-539
3.1
Lung ca. (squam.) SW
4.4




900


astrocytoma SNB-75
1.8
Lung ca. (squam.) NCI-
0.0




H596


glioma SNB-19
0.8
Mammary gland
17.4


glioma U251
1.6
Breast ca.* (pl. ef) MCF-7
0.0


glioma SF-295
0.0
Breast ca.* (pl. ef) MDA-
4.5




MB-231


Heart (fetal)
3.8
Breast ca.* (pl. ef) T47D
0.0


Heart
5.2
Breast ca. BT-549
15.7


Skeletal muscle (fetal)
7.7
Breast ca. MDA-N
21.6


Skeletal muscle
2.9
Ovary
5.7


Bone marrow
33.9
Ovarian ca. OVCAR-3
2.2


Thymus
7.3
Ovarian ca. OVCAR-4
0.0


Spleen
42.6
Ovarian ca. OVCAR-5
0.8


Lymph node
37.4
Ovarian ca. OVCAR-8
0.0


Colorectal
1.1
Ovarian ca. IGROV-1
1.7


Stomach
7.5
Ovarian ca.* (ascites)
0.4




SK-OV-3


Small intestine
18.9
Uterus
14.3


Colon ca. SW480
1.5
Placenta
8.6


Colon ca.* SW620 (SW480
1.5
Prostate
4.2


met)


Colon ca. HT29
0.4
Prostate ca.* (bone
0.4




met) PC-3


Colon ca. HCT-116
0.0
Testis
3.1


Colon ca. CaCo-2
1.2
Melanoma Hs688 (A).T
0.4


Colon ca.
4.7
Melanoma* (met)
0.0


tissue (ODO3866)

Hs688 (B).T


Colon ca. HCC-2998
0.6
Melanoma UACC-62
25.0


Gastric ca.* (liver met)
0.8
Melanoma M14
100.0


NCI-N87


Bladder
4.0
Melanoma LOX IMVI
0.0


Trachea
5.5
Melanoma* (met) SK-
4.4




MEL-5


Kidney
1.8
Adipose
5.0
















TABLE SD







Panel 4D











Rel. Exp. (%) Ag3276,

Rel. Exp. (%) Ag3276,


Tissue Name
Run 164635082
Tissue Name
Run 164635082













Secondary Th1 act
34.6
HUVEC IL-1 beta
1.9


Secondary Th2 act
26.2
HUVEC IFN gamma
5.3


Secondary Tr1 act
23.3
HUVEC TNF alpha + IFN
0.7




gamma


Secondary Th1 rest
4.9
HUVEC TNF alpha + IL4
0.9


Secondary Th2 rest
12.0
HUVEC IL-11
3.8


Secondary Tr1 rest
15.3
Lung Microvascular EC none
2.3


Primary Th1 act
18.9
Lung Microvascular EC
0.2




TNF alpha + IL-1 beta


Primary Th2 act
17.0
Microsvasular Dermal EC none
1.4


Primary Tr1 act
27.0
Microvascular Dermal EC
0.5




TNF alpha + IL-1 beta


Primary Th1 rest
67.8
Bronchial epithelium TNF alpha +
0.4




IL1 beta


Primary Th2 rest
40.9
Small airway epithelium none
0.2


Primary Tr1 rest
18.8
Small airway epithelium
0.0




TNF alpha + IL-1 beta


CD45RA CD4 lymphocyte
12.7
Coronery artery SMC rest
0.2


act


CD45RO CD4 lymphocyte
26.1
Coronery artery SMC TNF alpha +
0.0


act

IL-1 beta


CD8 lymphocyte act
18.0
Astrocytes rest
0.0


Secondary CD8
19.2
Astrocytes TNF alpha + IL-1 beta
0.1


lymphocyte rest


Secondary CD8
21.9
KU-812 (Basophil) rest
34.2


lymphocyte act


CD4 lymphocyte none
2.6
KU-812 (Basophil)
66.0




PMA/ionomycin


2ry Th1/Th2/Tr1_anti-
10.2
CCD1106 (Keratinocytes) none
0.3


CD95 CH11


LAK cells rest
62.9
CCD1106 (Keratinocytes)
0.2




TNF alpha + IL-1 beta


LAK cells IL-2
30.8
Liver cirrhosis
1.1


LAK cells IL-2 + IL-12
20.2
Lupus kidney
0.6


LAK cells IL-2 + IFN
30.1
NCI-H292 none
0.1


gamma


LAK cells IL-2 + IL-18
18.2
NCI-H292 IL-4
0.0


LAK cells
12.9
NCI-H292 IL-9
0.1


PMA/ionomycin


NK Cells IL-2 rest
24.3
NCI-H292 IL-13
0.2


Two Way MLR 3 day
40.3
NCI-H292 IFN gamma
0.0


Two Way MLR 5 day
21.9
HPAEC none
2.7


Two Way MLR 7 day
17.8
HPAEC TNF alpha + IL-1 beta
0.6


PBMC rest
13.7
Lung fibroblast none
0.3


PBMC PWM
46.7
Lung fibroblast TNF alpha + IL-
1.3




1 beta


PBMC PHA-L
22.1
Lung fibroblast IL-4
1.4


Ramos (B cell) none
0.5
Lung fibroblast IL-9
1.5


Ramos (B cell) ionomycin
5.4
Lung fibroblast IL-13
1.5


B lymphocytes PWM
50.3
Lung fibroblast IFN gamma
1.8


B lymphocytes CD40L
7.4
Dermal fibroblast CCD1070 rest
0.3


and IL-4


EOL-1 dbcAMP
46.0
Dermal fibroblast CCD1070
45.7




TNF alpha


EOL-1 dbcAMP
34.4
Dermal fibroblast CCD1070 IL-
0.0


PMA/ionomycin

1 beta


Dendritic cells none
76.3
Dermal fibroblast IFN gamma
0.8


Dendritic cells LPS
30.6
Dermal fibroblast IL-4
0.8


Dendritic cells anti-CD40
69.7
IBD Colitis 2
0.3


Monocytes rest
60.7
IBD Crohn's
0.2


Monocytes LPS
20.6
Colon
2.9


Macrophages rest
100.0
Lung
7.6


Macrophages LPS
34.9
Thymus
1.0


HUVEC none
3.6
Kidney
10.8


HUVEC starved
7.8









CNS_neurodegeneration_v1.0 Summary: Ag3276 This panel does not show differential expression of the CG57529-01 gene in Alzheimer's disease. However, this expression profile confirms the presence of this gene in the brain. Please see Panel 1.3D for discussion of utility of this gene in the central nervous system.
Panel 1.3D Summary: Ag3276 Highest expression of the CG57529-01 gene is seen in a melanoma cell line (CT=28.5). Significant expression is also seen in a cluster of breast cancer and melanoma cell lines. Thus, expression of this gene could be used to differentiate between these samples and other samples on this panel and as a diagnostic marker for the presence of these cancers. Furthermore, therapeutic modulation of the expression or function of this gene may be effective in the treatment of breast cancer or melanoma.
This gene encodes a type 1 membrane protein with several leucine-rich-repeat domains, indicating that this gene product may be involved in extracellular signalling and/or interactions with the extracellular matrix. Among metabolically relevant tissues, this gene is expressed at low but significant levels in the adrenal gland, thyroid, pancreas, adipose, and fetal and adult heart, skeletal muscle and liver. As a potential extracellular signalling molecule, the this gene product may serve as an antibody target for diseases involving any or all of these tissues, including obesity and diabetes.
This gene is also detected in several regions of the CNS including amygdala, cerebellum, substantia nigra, hippocampus, thalamus, cerebral cortex and spinal cord. In Drosophilia, the LRR region of axon guidance proteins has been shown to be critical for function (especially in axon repulsion) (ref. 1). Since this gene encodes a leucine-rich-repeat protein that is expressed across all brain regions, it is an excellent candidate neuronal guidance protein for axons, dendrites and/or growth cones in general. Therefore, therapeutic modulation of the levels of this protein, or possible signaling via this protein, may be of utility in enhancing/directing compensatory synaptogenesis and fiber growth in the CNS in response to neuronal death (stroke, head trauma), axon lesion (spinal cord injury), or neurodegeneration (Alzheimer's, Parkinson's, Huntington's, vascular dementia or any neurodegenerative disease) (Battye et al., J. Neurosci. 21: 4290-4298, 2001).
Panel 4D Summary: Ag3276 Highest expression of the CG57529—01 transcript is found in resting macrophages and dendritic cells (CTs=25-27), cells that play a crucial role in antigen presentation that acts to activate or tolerize T cells. Significant expression of this transcript is also found in most of the cell types across this panel. This expression in cells that play a role in linking innate immunity to adaptive immunity suggests a role for this gene product in initiating inflammatory reactions. Therefore, modulation of the expression or activity of this gene may reduce or prevent early stages of inflammation and reduce the severity of inflammatory diseases such as psoriasis, asthma, inflammatory bowel disease, rheumatoid arthritis, osteoarthritis and other lung inflammatory diseases.
T. NOV23: Centaurin Beta-2
Expression of gene CG57351-01 was assessed using the primer-probe set Ag3208, described in Table TA. Results of the RTQ-PCR runs are shown in Tables TB, TC, TD and TE.







TABLE TA







Probe Name Ag3208













Start


Primers
Sequences
Length
Position














Forward
5′-cagcaacgctttcaagacat-3′
(SEQ ID NO:371)
20
848


Probe
TET-5′-ttctccattcagaacagccagctggt-3′-TAMRA
(SEQ ID NO:372)
26
882


Reverse
5′-catccttgagcttcttctggta-3′
(SEQ ID NO:373)
22
909
















TABLE TB







CNS_neurodegeneration_v1.0











Rel. Exp. (%) Ag3208, Run

Rel. Exp. (%) Ag3208, Run


Tissue Name
209861777
Tissue Name
209861777













AD 1 Hippo
19.5
Control (Path) 3
3.1




Temporal Ctx


AD 2 Hippo
29.5
Control (Path) 4
34.4




Temporal Ctx


AD 3 Hippo
8.1
AD 1 Occipital Ctx
12.2


AD 4 Hippo
7.3
AD 2 Occipital Ctx
0.0




(Missing)


AD 5 hippo
93.3
AD 3 Occipital Ctx
3.8


AD 6 Hippo
42.9
AD 4 Occipital Ctx
18.2


Control 2 Hippo
32.3
AD 5 Occipital Ctx
16.7


Control 4 Hippo
12.2
AD 6 Occipital Ctx
42.3


Control (Path) 3 Hippo
5.2
Control 1 Occipital Ctx
3.1


AD 1 Temporal Ctx
14.4
Control 2 Occipital Ctx
79.0


AD 2 Temporal Ctx
33.7
Control 3 Occipital Ctx
13.9


AD 3 Temporal Ctx
6.7
Control 4 Occipital Ctx
2.8


AD 4 Temporal Ctx
26.8
Control (Path) 1
90.8




Occipital Ctx


AD 5 Inf Temporal Ctx
100.0
Control (Path) 2
14.3




Occipital Ctx


AD 5 SupTemporal Ctx
58.6
Control (Path) 3
1.5




Occipital Ctx


AD 6 Inf Temporal Ctx
41.5
Control (Path) 4
21.9




Occipital Ctx


AD 6 Sup Temporal Ctx
44.1
Control 1 Parietal Ctx
7.3


Control 1 Temporal Ctx
5.0
Control 2 Parietal Ctx
52.5


Control 2 Temporal Ctx
61.6
Control 3 Parietal Ctx
22.7


Control 3 Temporal Ctx
15.1
Control (Path) 1
87.1




Parietal Ctx


Control 4 Temporal Ctx
11.7
Control (Path) 2
20.6




Parietal Ctx


Control (Path) 1
72.7
Control (Path) 3
3.6


Temporal Ctx

Parietal Ctx


Control (Path) 2
46.0
Control (Path) 4
43.2


Temporal Ctx

Parietal Ctx
















TABLE TC







Panel 1.3D











Rel. Exp. (%) Ag3208, Run

Rel. Exp. (%) Ag3208, Run


Tissue Name
168012842
Tissue Name
168012842













Liver adenocarcinoma
10.2
Kidney (fetal)
15.3


Pancreas
7.2
Renal ca. 786-0
5.0


Pancreatic ca. CAPAN 2
3.9
Renal ca. A498
6.8


Adrenal gland
4.9
Renal ca. RXF 393
10.4


Thyroid
4.7
Renal ca. ACHN
3.8


Salivary gland
11.0
Renal ca. UO-31
5.9


Pituitary gland
20.3
Renal ca. TK-10
2.0


Brain (fetal)
73.2
Liver
5.3


Brain (whole)
99.3
Liver (fetal)
5.2


Brain (amygdala)
35.8
Liver ca. (hepatoblast)
10.2




HepG2


Brain (cerebellum)
100.0
Lung
8.5


Brain (hippocampus)
21.8
Lung (fetal)
14.8


Brain (substantia nigra)
20.6
Lung ca. (small cell) LX-1
18.0


Brain (thalamus)
29.1
Lung ca. (small cell)
5.7




NCI-H69


Cerebral Cortex
50.3
Lung ca. (s. cell var.)
42.3




SHP-77


Spinal cord
14.7
Lung ca. (large cell) NCI-
0.6




H460


glio/astro U87-MG
6.7
Lung ca. (non-sm. cell)
7.1




A549


glio/astro U-118-MG
9.7
Lung ca. (non-s. cell)
3.7




NCI-H23


astrocytoma SW1783
6.7
Lung ca. (non-s. cell)
5.6




HOP-62


neuro*; met SK-N-AS
7.7
Lung ca. (non-s. cl) NCI-
6.0




H522


astrocytoma SF-539
11.7
Lung ca. (squam.) SW
7.2




900


astrocytoma SNB-75
13.9
Lung ca. (squam.) NCI-
11.7




H596


glioma SNB-19
3.1
Mammary gland
16.7


glioma U251
19.6
Breast ca.* (pl. ef) MCF-7
8.5


glioma SF-295
9.8
Breast ca.* (pl. ef) MDA-
8.7




MB-231


Heart (fetal)
37.1
Breast ca.* (pl. ef) T47D
9.7


Heart
9.0
Breast ca. BT-549
5.2


Skeletal muscle (fetal)
12.8
Breast ca. MDA-N
5.5


Skeletal muscle
10.2
Ovary
4.9


Bone marrow
4.7
Ovarian ca. OVCAR-3
4.3


Thymus
16.6
Ovarian ca. OVCAR-4
5.0


Spleen
4.8
Ovarian ca. OVCAR-5
16.5


Lymph node
6.5
Ovarian ca. OVCAR-8
1.1


Colorectal
1.2
Ovarian ca. IGROV-1
1.4


Stomach
6.7
Ovarian ca.* (ascites)
14.3




SK-OV-3


Small intestine
7.7
Uterus
10.5


Colon ca. SW480
5.3
Placenta
2.6


Colon ca.* SW620 (SW480
19.9
Prostate
3.4


met)


Colon ca. HT29
4.1
Prostate ca.* (bone
4.2




met)PC-3


Colon ca. HCT-116
3.9
Testis
3.3


Colon ca. CaCo-2
8.5
Melanoma Hs688 (A).T
3.9


Colon ca.
3.3
Melanoma* (met)
3.7


tissue (ODO3866)

Hs688 (B).T


Colon ca. HCC-2998
21.5
Melanoma UACC-62
9.8


Gastric ca.* (liver met)
4.6
Melanoma M14
3.3


NCI-N87


Bladder
2.0
Melanoma LOX IMVI
4.2


Trachea
4.7
Melanoma* (met) SK-
2.5




MEL-5


Kidney
9.9
Adipose
6.8
















TABLE TD







Panel 4D











Rel. Exp. (%) Ag3208,

Rel. Exp. (%) Ag3208,


Tissue Name
Run 164531754
Tissue Name
Run 164531754













Secondary Th1 act
32.5
HUVEC IL-1beta
3.5


Secondary Th2 act
47.6
HUVEC IFN gamma
22.8


Secondary Tr1 act
53.6
HUVEC TNF alpha + IFN
10.9




gamma


Secondary Th1 rest
14.3
HUVEC TNF alpha + IL4
21.6


Secondary Th2 rest
25.2
HUVEC IL-11
17.9


Secondary Tr1 rest
17.7
Lung Microvascular EC none
33.9


Primary Th1 act
28.5
Lung Microvascular EC
37.1




TNF alpha + IL-1beta


Primary Th2 act
28.9
Microvascular Dermal EC none
48.6


Primary Tr1 act
33.0
Microsvasular Dermal EC
18.8




TNF alpha + IL-1beta


Primary Th1 rest
59.0
Bronchial epithelium TNF alpha +
43.8




IL-1beta


Primary Th2 rest
33.7
Small airway epithelium none
21.5


Primary Tr1 rest
32.3
Small airway epithelium
61.6




TNF alpha + IL-1beta


CD45RA CD4 lymphocyte
33.0
Coronery artery SMC rest
21.6


act


CD45RO CD4 lymphocyte
24.7
Coronery artery SMC TNF alpha +
13.7


act

IL-1beta


CD8 lymphocyte act
30.1
Astrocytes rest
45.7


Secondary CD8
29.7
Astrocytes TNF alpha + IL-1beta
32.8


lymphocyte rest


Secondary CD8
30.6
KU-812 (Basophil) rest
24.5


lymphocyte act


CD4 lymphocyte none
13.5
KU-812 (Basophil)
29.7




PMA/ionomycin


2ry Th1/Th2/Tr1_anti-
36.3
CCD1106 (Keratinocytes) none
39.8


CD95 CH11


LAK cells rest
25.0
CCD1106 (Keratinocytes)
25.0




TNF alpha + IL-1beta


LAK cells IL-2
31.9
Liver cirrhosis
2.6


LAK cells IL-2 + IL-12
19.9
Lupus kidney
5.8


LAK cells IL-2 + IFN
26.2
NCI-H292 none
15.8


gamma


LAK cells IL-2 + IL-18
29.7
NCI-H292 IL-4
16.0


LAK cells
19.8
NCI-H292 IL-9
17.6


PMA/ionomycin


NK Cells IL-2 rest
28.3
NCI-H292 IL-13
18.6


Two Way MLR 3 day
32.5
NCI-H292 IFN gamma
12.9


Two Way MLR 5 day
29.1
HPAEC none
20.0


Two Way MLR 7 day
15.4
HPAEC TNF alpha + IL-1beta
17.1


PBMC rest
23.8
Lung fibroblast none
59.0


PBMC PWM
44.4
Lung fibroblast TNF alpha + IL-
20.3




1beta


PBMC PHA-L
44.8
Lung fibroblast IL-4
73.2


Ramos (B cell) none
29.5
Lung fibroblast IL-9
62.4


Ramos (B cell) ionomycin
67.8
Lung fibroblast IL-13
65.1


B lymphocytes PWM
42.6
Lung fibroblast IFN gamma
55.1


B lymphocytes CD40L
31.9
Dermal fibroblast CCD1070 rest
89.5


and IL-4


EOL-1 dbcAMP
49.3
Dermal fibroblast CCD1070
72.7




TNF alpha


EOL-1 dbcAMP
42.3
Dermal fibroblast CCD1070 IL-
43.2


PMA/ionomycin

1beta


Dendritic cells none
31.6
Dermal fibroblast IFN gamma
39.5


Dendritic cells LPS
21.2
Dermal fibroblast IL-4
73.7


Dendritic cells anti-CD40
33.0
IBD Colitis 2
5.0


Monocytes rest
57.4
IBD Crohn's
3.4


Monocytes LPS
11.0
Colon
36.6


Macrophages rest
51.1
Lung
23.2


Macrophages LPS
5.8
Thymus
59.0


HUVEC none
28.3
Kidney
100.0


HUVEC starved
32.1
















TABLE TE







Panel CNS_1











Rel. Exp. (%) Ag3208, Run

Rel. Exp. (%) Ag3208, Run


Tissue Name
171694583
Tissue Name
171694583













BA4 Control
17.9
BA17 PSP
9.7


BA4 Control2
44.1
BA17 PSP2
11.0


BA4 Alzheimer's2
5.3
Sub Nigra Control
26.6


BA4 Parkinson's
37.6
Sub Nigra Control2
23.5


BA4 Parkinson's2
61.1
Sub Nigra Alzheimer's2
10.7


BA4 Huntington's
32.8
Sub Nigra Parkinson's2
31.2


BA4
14.5
Sub Nigra Huntington's
37.9


Huntington's2


BA4 PSP
7.3
Sub Nigra
24.0




Huntington's2


BA4 PSP2
20.4
Sub Nigra PSP2
0.9


BA4 Depression
17.0
Sub Nigra Depression
3.4


BA4 Depression2
3.8
Sub Nigra Depression2
2.9


BA7 Control
38.2
Glob Palladus Control
23.5


BA7 Control2
69.3
Glob Palladus Control2
17.9


BA7 Alzheimer's2
9.3
Glob Palladus
4.9




Alzheimer's


BA7 Parkinson's
26.8
Glob Palladus
6.9




Alzheimer's2


BA7 Parkinson's2
38.2
Glob Palladus
100.0




Parkinson's


BA7 Huntington's
56.6
Glob Palladus
15.1




Parkinson's2


BA7
50.3
Glob Palladus PSP
2.2


Huntington's2


BA7 PSP
22.4
Glob Palladus PSP2
4.5


BA7 PSP2
25.2
Glob Palladus
7.5




Depression


BA7 Depression
7.7
Temp Pole Control
19.3


BA9 Control
27.7
Temp Pole Control2
66.4


BA9 Control2
99.3
Temp Pole Alzheimer's
8.2


BA9 Alzheimer's
2.2
Temp Pole Alzheimer's2
7.4


BA9 Alzheimer's2
11.0
Temp Pole Parkinson's
36.1


BA9 Parkinson's
37.1
Temp Pole Parkinson's2
42.3


BA9 Parkinson's2
43.2
Temp Pole Huntington's
39.8


BA9 Huntington's
50.3
Temp Pole PSP
2.5


BA9
27.2
Temp Pole PSP2
5.6


Huntington's2


BA9 PSP
8.4
Temp Pole Depression2
7.5


BA9 PSP2
1.7
Cing Gyr Control
50.7


BA9 Depression
6.4
Cing Gyr Control2
41.2


BA9 Depression2
12.3
Cing Gyr Alzheimer's
14.5


BA17 Control
52.1
Cing Gyr Alzheimer's2
10.5


BA17 Control2
57.0
Cing Gyr Parkinson's
30.6


BA17
3.3
Cing Gyr Parkinson's2
33.2


Alzheimer's2


BA17 Parkinson's
37.4
Cing Gyr Huntington's
61.6


BA17
45.1
Cing Gyr Huntington's2
23.8


Parkinson's2


BA17
52.1
Cing Gry PSP
9.4


Huntington's


BA17
22.1
Cing Gyr PSP2
5.0


Huntington's2


BA17 Depression
8.4
Cing Gry Depression
3.6


BA17 Depression2
27.5
Cing Gyr Depression2
15.8









CNS_neurodegeneration_v1.0 Summary: Ag3208 This panel does not show differential expression of the CG57351-01 gene in Alzheimer's disease. However, this expression profile confirms the presence of this gene in the brain. Please see Panel 1.3D for discussion of utility of this gene in the central nervous system.
Panel 1.3D Summary: Ag3208 The CG57351-01 gene, a centaurin homolog, shows a brain-preferential expression pattern. Thus, the expression of this gene could be used to distinguish normal brain tissue from other samples in the panel. Because centaurins play a role in vesicular trafficing, they may be involved in synaptic vesicle movement/release. Therefore, this gene may be a drug target for the treatment of any disorder involving neurotransmission (epilepsy, bipolar disorder, schizophrenia, mania, depression, obsessive-compulsive disorder or any neuropsychiatric condition.
In addition, there appears to be substantial expression in samples derived from lung cancer cell lines and colon cancer cell lines. Moreover, therapeutic modulation of this gene, through the use of small molecule drugs, protein therapeutics or antibodies could be of benefit in the treatment of lung or colon cancer.
Among metabolic tissues, this gene is expressed at moderate levels in pancreas, adrenal, thyroid, pituitary, heart, liver, adipose and skeletal muscle. The role of centaurins in vesicular trafficking suggests that this gene may be involved in endocrine secretory processes. Therefore, this widespread expression in metabolic tissues suggests that this gene product may be important for the pathogenesis, diagnosis and/or treatment of metabolic and endocrine disease, including Types 1 and 2 diabetes and obesity.
Panel 4D Summary: Ag3208 The CG57351-01 transcript is expressed at high to moderate levels in a wide range of cell types of significance in the immune response and tissue response in health and disease. Therefore, targeting of this gene product with a small molecule drug or antibody therapeutic may modulate the functions of cells of the immune system as well as resident tissue cells and lead to improvement of the symptoms of patients suffering from autoimmune and inflammatory diseases such as COPD, emphysema, asthma, allergies, inflammatory bowel disease, lupus erythematosus, and arthritis, including osteoarthritis and rheumatoid arthritis
Panel CNS—1 Summary: Ag3208 This expression profile further confirms the presence of the CG57351-01 gene in the brain. Please see Panel 1.3D for discussion of utility of this gene in the central nervous system.
U. NOV24: Sorting Nexin 9
Expression of gene CG57515-01 was assessed using the primer-probe set Ag3262, described in Table UA. Results of the RTQ-PCR runs are shown in Tables UB and UC.







TABLE UA







Probe Name Ag3262













Start


Primers
Sequences
Length
Position














Forward
5′-tccatcaaattcgagtgagaga-3′
(SEQ ID NO:374)
22
1782


Probe
TET-5′-tcacagatgcagcatttcttacaacaaca-3′-TAMRA
(SEQ ID NO:375)
29
1811


Reverse
5′-gcttcttccaacttctgggtaa-3′
(SEQ ID NO:376)
22
1860
















TABLE UB







CNS_neurodegeneration_v1.0











Rel. Exp. (%) Ag3262, Run

Rel. Exp. (%) Ag3262, Run


Tissue Name
210038237
Tissue Name
210038237













AD 1 Hippo
12.3
Control (Path) 3
7.7




Temporal Ctx


AD 2 Hippo
40.6
Control (Path) 4
34.9




Temporal Ctx


AD 3 Hippo
14.3
AD 1 Occipital Ctx
26.1


AD 4 Hippo
9.5
AD 2 Occipital Ctx
0.0




(Missing)


AD 5 hippo
84.1
AD 3 Occipital Ctx
15.9


AD 6 Hippo
71.2
AD 4 Occipital Ctx
20.3


Control 2 Hippo
30.4
AD 5 Occipital Ctx
21.2


Control 4 Hippo
21.3
AD 6 Occipital Ctx
43.5


Control (Path) 3 Hippo
10.9
Control 1 Occipital Ctx
9.2


AD 1 Temporal Ctx
29.9
Control 2 Occipital Ctx
51.1


AD 2 Temporal Ctx
40.1
Control 3 Occipital Ctx
23.3


AD 3 Temporal Ctx
10.2
Control 4 Occipital Ctx
10.7


AD 4 Temporal Ctx
18.9
Control (Path) 1
86.5




Occipital Ctx


AD 5 Inf Temporal Ctx
100.0
Control (Path) 2
10.7




Occipital Ctx


AD 5 SupTemporal Ctx
74.7
Control (Path) 3
5.1




Occipital Ctx


AD 6 Inf Temporal Ctx
85.3
Control (Path) 4
18.2




Occipital Ctx


AD 6 Sup Temporal Ctx
76.8
Control 1 Parietal Ctx
12.2


Control 1 Temporal Ctx
14.6
Control 2 Parietal Ctx
69.3


Control 2 Temporal Ctx
32.5
Control 3 Parietal Ctx
16.3


Control 3 Temporal Ctx
17.8
Control (Path) 1
72.2




Parietal Ctx


Control 4 Temporal Ctx
7.8
Control (Path) 2
42.3




Parietal Ctx


Control (Path) 1
62.4
Control (Path) 3
12.1


Temporal Ctx

Parietal Ctx


Control (Path) 2
33.9
Control (Path) 4
41.8


Temporal Ctx

Parietal Ctx
















TABLE UC







Panel 4D











Rel. Exp. (%) Ag3262,

Rel. Exp. (%) Ag3262,


Tissue Name
Run164634869
Tissue Name
Run 164634869













Secondary Th1 act
15.2
HUVEC IL-1beta
7.1


Secondary Th2 act
11.7
HUVEC IFN gamma
7.6


Secondary Tr1 act
15.3
HUVEC TNF alpha + IFN
3.6




gamma


Secondary Th1 rest
5.2
HUVEC TNF alpha + IL4
6.6


Secondary Th2 rest
5.4
HUVEC IL-11
3.4


Secondary Tr1 rest
5.4
Lung Microvascular EC none
5.1


Primary Th1 act
2.5
Lung Microvascular EC
3.5




TNF alpha + IL-1beta


Primary Th2 act
2.2
Microvascular Dermal EC none
5.7


Primary Tr1 act
3.3
Microsvasular Dermal EC
2.2




TNF alpha + IL-lbeta


Primary Th1 rest
30.6
Bronchial epithelium TNF alpha  +
14.4




IL1beta


Primary Th2 rest
18.2
Small airway epithelium none
5.7


Primary Tr1 rest
6.7
Small airway epithelium
17.9




TNF alpha + IL-1beta


CD45RA CD4 lymphocyte
15.1
Coronery artery SMC rest
9.8


act


CD45RO CD4 lymphocyte
15.9
Coronery artery SMC TNF alpha +
7.3


act

IL-1beta


CD8 lymphocyte act
11.5
Astrocytes rest
26.6


Secondary CD8
5.2
Astrocytes TNF alpha + IL-1beta
11.0


lymphocyte rest


Secondary CD8
20.0
KU-812 (Basophil) rest
1.4


lymphocyte act


CD4 lymphocyte none
4.3
KU-812 (Basophil)
7.5




PMA/ionomycin


2ry Th1/Th2/Tr1 anti-
13.2
CCD1106 (Keratinocytes) none
13.7


CD95 CH11


LAK cells rest
16.3
CCD1106 (Keratinocytes)
6.0




TNF alpha + IL-1beta


LAK cells IL-2
45.1
Liver cirrhosis
1.7


LAK cells IL-2 + IL-12
13.3
Lupus kidney
1.4


LAK cells IL-2 + IFN
18.9
NCI-H292 none
13.8


gamma


LAK cells IL-2 + IL-18
14.1
NCI-H292 IL-4
15.1


LAK cells
3.5
NCI-H292 IL-9
19.1


PMA/ionomycin


NK Cells IL-2 rest
16.8
NCI-H292 IL-13
11.7


Two Way MLR 3 day
11.5
NCI-H292 IFN gamma
16.7


Two Way MLR 5 day
8.0
HPAEC none
7.3


Two Way MLR 7 day
12.0
HPAEC TNF alpha + IL-1beta
3.7


PBMC rest
7.4
Lung fibroblast none
24.7


PBMC PWM
25.7
Lung fibroblast TNF alpha + IL-
14.1




1beta


PBMC PHA-L
6.6
Lung fibroblast IL-4
100.0


Ramos (B cell) none
0.4
Lung fibroblast IL-9
49.0


Ramos (B cell) ionomycin
2.2
Lung fibroblast IL-13
51.8


B lymphocytes PWM
25.3
Lung fibroblast IFN gamma
93.3


B lymphocytes CD40L
17.2
Dermal fibroblast CCD1070 rest
30.6


and IL-4


EOL-1 dbcAMP
7.1
Dermal fibroblast CCD1070
73.7




TNF alpha


EOL-1 dbcAMP
4.0
Dermal fibroblast CCD1070 IL-
19.3


PMA/ionomycin

1beta


Dendritic cells none
11.5
Dermal fibroblast IFN gamma
28.9


Dendritic cells LPS
5.6
Dermal fibroblast IL-4
26.2


Dendritic cells anti-CD40
7.9
IBD Colitis 2
0.8


Monocytes rest
31.9
IBD Crohn's
1.0


Monocytes LPS
13.8
Colon
10.2


Macrophages rest
14.0
Lung
12.4


Macrophages LPS
10.9
Thymus
15.9


HUVEC none
12.7
Kidney
4.2


HUVEC starved
16.6









CNS_neurodegeneration_v1.0 Summary: Ag3262 This panel does not show differential expression of the CG57515-01 gene in Alzheimer's disease. However, this expression profile confirms the presence of this gene, a sorting nexin homolog, in the brain. Sorting nexins have been implicated in axonal guidance; Therefore, this gene may serve as a useful drug target in diseases/clinical conditions where neurons have degenerated/died and compensatory sprouting and/or synaptogenesis is desireable (spinal cord/head trauma, stroke, Huntington's, Parkinson's, or Alzheimer's diseases) (Worby et al., J Biol Chem 2001)
General_screening_panel_v1.4 Summary: Ag3262 Results from one experiment with the CG57515-01 gene are not included. The amp plot indicates that there were experimental difficulties with this run.
Panel 4D Summary: Ag3262 Highest expression of the CG57515-01 transcript is found in lung fibroblasts treated with either Il-4 or IFNg (CT 25.5). It is also observed at high levels in dermal fibroblasts. In addition, this transcript is expressed at high to moderate levels in a wide range of cell types of significance in the immune response and tissue response in health and disease. This transcript encodes a sorting nexin 9 like (SH3PX1) protein. This protein interacts with metalloprotease disintegrins, cysteine-rich proteins that may possess diverse biological functions such as cytokine and protein processing. This interaction is consistent with the expression profile of this gene product. Therefore, this gene may be a useful target for treatment of diseases such as osteoarthritis and rheumatoid arythritis, psoriasis, asthma and chronic obstructive pulmonary diseases (Howard et al., J Biol Chem 1999 October 29;274(44):31693-9).
V. NOV26: Calpain
Expression of gene CG57509-01 was assessed using the primer-probe set Ag2073, described in Table VA. Results of the RTQ-PCR runs are shown in Tables VB and VC.







TABLE VA







Probe Name Ag2073













Start


Primers
Sequences
Length
Position














Forward
5′-acccaagtggcatctattcag-3′
(SEQ ID NO:377)
21
2234


Probe
TET-5′-tcagccgcaattttcctattatcgga-3′-TAMRA
(SEQ ID NO:378)
26
2202


Reverse
5′-gtgaagttgctcgaatgtcttc-3′
(SEQ ID NO:379)
22
2172
















TABLE VB







Panel 1.3D











Rel. Exp. (%) Ag2073, Run

Rel. Exp. (%) Ag2073, Run


Tissue Name
165627447
Tissue Name
165627447













Liver adenocarcinoma
0.0
Kidney (fetal)
0.1


Pancreas
0.1
Renal ca. 786-0
0.1


Pancreatic ca. CAPAN 2
0.0
Renal ca. A498
0.1


Adrenal gland
0.1
Renal ca. RXF 393
0.0


Thyroid
0.5
Renal ca. ACHN
0.0


Salivary gland
0.3
Renal ca. UO-31
0.0


Pituitary gland
0.3
Renal ca. TK-10
0.1


Brain (fetal)
0.0
Liver
0.3


Brain (whole)
0.7
Liver (fetal)
0.3


Brain (amygdala)
0.4
Liver ca. (hepatoblast)
0.0




HepG2


Brain (cerebellum)
0.5
Lung
0.4


Brain (hippocampus)
0.8
Lung (fetal)
0.4


Brain (substantia nigra)
0.3
Lung ca. (small cell) LX-1
0.2


Brain (thalamus)
0.5
Lung ca. (small cell)
0.0




NCI-H69


Cerebral Cortex
0.2
Lung ca. (s. cell var.)
0.0




SHP-77


Spinal cord
0.6
Lung ca. (large cell) NCI-
0.0




H460


glio/astro U87-MG
0.0
Lung ca. (non-sm. cell)
0.0




A549


glio/astro U-118-MG
0.2
Lung ca. (non-s. cell)
0.1




NCI-H23


astrocytoma SW1783
0.0
Lung ca. (non-s. cell)
0.0




HOP-62


neuro*; met SK-N-AS
0.0
Lung ca. (non-s. cl) NCI-
0.0




H522


astrocytoma SF-539
0.0
Lung ca. (squam.) SW
0.0




900


astrocytoma SNB-75
0.0
Lung ca. (squam.) NCI-
0.0




H596


glioma SNB-19
0.1
Mammary gland
0.3


glioma U251
0.4
Breast ca.* (pl. ef) MCF-7
0.0


glioma SF-295
0.0
Breast ca.* (pl. ef) MDA-
0.1




MB-231


Heart (fetal)
0.1
Breast ca.* (pl. ef) T47D
0.0


Heart
0.3
Breast ca. BT-549
0.1


Skeletal muscle (fetal)
7.7
Breast ca. MDA-N
0.0


Skeletal muscle
100.0
Ovary
0.0


Bone marrow
2.0
Ovarian ca. OVCAR-3
0.1


Thymus
0.2
Ovarian ca. OVCAR-4
0.0


Spleen
0.4
Ovarian ca. OVCAR-5
0.1


Lymph node
1.0
Ovarian ca. OVCAR-8
0.0


Colorectal
0.2
Ovarian ca. IGROV-1
0.0


Stomach
0.4
Ovarian ca.* (ascites)
0.2




SK-OV-3


Small intestine
0.9
Uterus
0.6


Colon ca. SW480
0.0
Placenta
0.1


Colon ca.* SW620 (SW480
0.0
Prostate
0.3


met)


Colon ca. HT29
0.0
Prostate ca.* (bone
0.1




met)PC-3


Colon ca. HCT-116
0.0
Testis
0.8


Colon ca. CaCo-2
0.0
Melanoma Hs688 (A).T
0.0


Colon ca.
0.0
Melanoma* (met)
0.0


tissue (ODO3866)

Hs688 (B).T


Colon ca. HCC-2998
0.0
Melanoma UACC-62
0.1


Gastric ca.* (liver met)
0.8
Melanoma M14
0.1


NCI-N87


Bladder
0.1
Melanoma LOX IMVI
0.0


Trachea
0.3
Melanoma* (met) SK-
0.0




MEL-5


Kidney
0.5
Adipose
0.5
















TABLE VC







Panel 5D











Rel. Exp. (%)

Rel. Exp. (%)



Ag2073. Run

Ag2073. Run


Tissue Name
169269384
Tissue Name
169269384













97457_Patient-
0.7
94709_Donor 2 AM - A_adipose
0.2


02go_adipose


97476_Patient-07sk_skeletal
7.9
94710_Donor 2 AM - B_adipose
0.1


muscle


97477_Patient-07ut_uterus
0.0
94711_Donor 2 AM - C_adipose
0.0


97478_Patient-
0.2
94712_Donor 2 AD - A_adipose
0.0


07pl_placenta


97481_Patient-08sk_skeletal
8.7
94713_Donor 2 AD - B_adipose
0.7


muscle


97482_Patient-08ut_uterus
0.1
94714_Donor 2 AD - C_adipose
0.3


97483_Patient-
0.1
94742_Donor 3 U - A_Mesenchymal
0.0


08pl_placenta

Stem Cells


97486_Patient-09sk_skeletal
14.1
94743_Donor 3 U - B_Mesenchymal
0.0


muscle

Stem Cells


97487_Patient-09ut_uterus
0.1
94730_Donor 3 AM - A_adipose
0.1


97488_Patient-
0.1
94731_Donor 3 AM - B_adipose
0.2


09pl_placenta


97492_Patient-10ut_uterus
0.3
94732_Donor 3 AM - C_adipose
0.2


97493_Patient-
0.1
94733_Donor 3 AD - A_adipose
0.2


10pl_placenta


97495_Patient-
0.2
94734_Donor 3 AD - B_adipose
0.0


11go_adipose


97496_Patient-11sk_skeletal
82.9
94735_Donor 3 AD - C_adipose
0.2


muscle


97497_Patient-11ut_uterus
0.1
77138_Liver_HepG2untreated
0.0


97498_Patient-
0.3
73556_Heart_Cardiac stromal cells
0.2


11pl_placenta

(primary)


97500_Patient-
0.5
81735_Small Intestine
0.4


12go_adipose


97501_Patient-12sk_skeletal
100.0
72409_Kidney_Proximal Convoluted
0.2


muscle

Tubule


97502_Patient-12ut_uterus
0.3
82685_Small intestine_Duodenum
0.4


97503_Patient-
0.1
90650_Adrenal_Adrenocortical
0.1


12pl_placenta

adenoma


94721_Donor 2 U -
0.2
72410_Kidney_HRCE
0.3


A_Mesenchymal Stem Cells


94722_Donor 2 U -
0.0
72411_Kidney_HRE
0.1


B_Mesenchymal Stem Cells


94723_Donor 2 U -
0.4
73139_Uterus_Uterine smooth
0.1


C_Mesenchymal Stem Cells

muscle cells









Panel 1.3D Summary: Ag2073 The CG57909-01 gene, a calpain homolog, has low levels of expression in thyroid, pituitary, heart, adipose and liver. Calpain 10 was recently identified as a susceptibility gene for type 2 diabetes. Thus, this gene product may be a small molecule target for the treatment of endocrine and metabolic disease, including the thyroidopathies, Types 1 and 2 diabetes and obesity. In addition, this gene is highly expressed in skeletal muscle. Mutations in the calpain 3 gene have been proven to be responsible for limb-girdle muscular dystrophy (LGMD) type 2A. Thus, therapeutic modulation of this gene product may be a treatment for LGMD type 2A (Chae et al., Neuromuscul Disord. 2001 September;11(6-7):547-55; Huang and Wang, Trends Mol Med. 2001 August;7(8):355-62).
Panel 4D Summary: Ag2073 Results from one experiment with the CG56003-01 gene are not included. The amp plot indicates that there were experimental difficulties with this run.
Panel 5D Summary: Ag2073 Expression of the CG57509-01 gene is restricted to skeletal muscle, confirming the results from Panel 1.3D. Please see Panel 1.3D for discussion of utility of this gene in metabolic disease.
W. NOV27: Kerratin 18
Expression of gene CG57484-01 was assessed using the primer-probe set Ag1597, described in Table WA. Results of the RTQ-PCR runs are shown in Table WB.







TABLE WA







Probe Name Ag1597













Start


Primers
Sequences
Length
Position














Forward
5′-gactgaggagagcaccacagt-3′
(SEQ ID NO:380)
21
810


Probe
TET-5′-acactctgccaaggtcagagctgct-3′-TAMRA
(SEQ ID NO:381)
25
840


Reverse
5′-gtctcagctccgttgtcatct-3′
(SEQ ID NO:382)
21
866
















TABLE WB







Panel 2D











Rel. Exp. (%) Ag1597,

Rel. Exp. (%) Ag1597,


Tissue Name
Run 164988404
Tissue Name
Run 164988404













Normal Colon
2.3
Kidney Margin 8120608
0.0


CC Well to Mod Diff
0.0
Kidney Cancer 8120613
0.0


(ODO3866)


CC Margin (ODO3866)
0.0
Kidney Margin 8120614
0.0


CC Gr.2 rectosigmoid
0.0
Kidney Cancer 9010320
0.0


(ODO3868)


CC Margin (ODO3868)
0.0
Kidney Margin 9010321
0.0


CC Mod Diff (ODO3920)
0.0
Normal Uterus
0.0


CC Margin (ODO3920)
0.0
Uterus Cancer 064011
9.3


CC Gr.2 ascend colon
0.0
Normal Thyroid
0.0


(ODO3921)


CC Margin (ODO3921)
0.0
Thyroid Cancer 064010
2.5


CC from Partial Hepatectomy
0.0
Thyroid Cancer A302152
0.0


(ODO4309) Mets


Liver Margin (ODO4309)
0.0
Thyroid Margin A302153
0.0


Colon mets to lung (OD04451-
0.0
Normal Breast
2.4


01)


Lung Margin (OD04451-02)
1.4
Breast Cancer (OD04566)
3.5


Normal Prostate 6546-1
10.0
Breast Cancer (OD04590-
0.0




01)


Prostate Cancer (OD04410)
7.7
Breast Cancer Mets
30.4




(OD04590-03)


Prostate Margin (OD04410)
2.6
Breast Cancer Metastasis
74.7




(OD04655-05)


Prostate Cancer (OD04720-01)
22.4
Breast Cancer 064006
14.0


Prostate Margin (OD04720-02)
10.8
Breast Cancer 1024
39.2


Normal Lung 061010
9.3
Breast Cancer 9100266
0.0


Lung Met to Muscle
7.7
Breast Margin 9100265
3.1


(ODO4286)


Muscle Margin (ODO4286)
0.0
Breast Cancer A209073
23.2


Lung Malignant Cancer
21.0
Breast Margin A209073
30.1


(OD03126)


Lung Margin (OD03126)
6.1
Normal Liver
16.7


Lung Cancer (OD04404)
2.9
Liver Cancer 064003
0.0


Lung Margin (OD04404)
0.0
Liver Cancer 1025
27.0


Lung Cancer (OD04565)
0.0
Liver Cancer 1026
0.0


Lung Margin (OD04565)
0.0
Liver Cancer 6004-T
0.0


Lung Cancer (OD04237-01)
100.0
Liver Tissue 6004-N
0.0


Lung Margin (OD04237-02)
10.9
Liver Cancer 6005-T
0.0


Ocular Mel Met to Liver
2.9
Liver Tissue 6005-N
0.0


(ODO4310)


Liver Margin (ODO4310)
0.0
Normal Bladder
0.0


Melanoma Mets to Lung
2.0
Bladder Cancer 1023
0.0


(OD04321)


Lung Margin (OD04321)
0.0
Bladder Cancer A302173
0.0


Normal Kidney
55.1
Bladder Cancer
0.0




(OD04718-01)


Kidney Ca, Nuclear grade 2
14.3
Bladder Normal Adjacent
0.0


(OD04338)

(OD04718-03)


Kidney Margin (OD04338)
0.0
Normal Ovary
0.0


Kidney Ca Nuclear grade 1/2
0.0
Ovarian Cancer 064008
0.0


(OD04339)


Kidney Margin (OD04339)
0.0
Ovarian Cancer
0.0




(OD04768-07)


Kidney Ca, Clear cell type
0.0
Ovary Margin (OD04768-
0.0


(OD04340)

08)


Kidney Margin (OD04340)
0.0
Normal Stomach
13.4


Kidney Ca, Nuclear grade 3
0.0
Gastric Cancer 9060358
0.0


(OD04348)


Kidney Margin (OD04348)
36.9
Stomach Margin 9060359
0.0


Kidney Cancer (OD04622-01)
0.0
Gastric Cancer 9060395
0.0


Kidney Margin (OD04622-03)
4.7
Stomach Margin 9060394
3.0


Kidney Cancer (OD04450-01)
0.0
Gastric Cancer 9060397
0.0


Kidney Margin (OD04450-03)
0.0
Stomach Margin 9060396
0.0


Kidney Cancer 8120607
0.0
Gastric Cancer 064005
0.0









Panel 2D Summary: Ag1597 Expression of the CG57484-01 gene is limited to samples derived from breast cancer and lung cancer (CTs=33). Thus, expression of this gene could be used to differentiate between these samples and other samples on this panel and as a marker to detect the presence of breast and lung cancers. This gene encodes a protein that is homologous to keratin 18. Expression of keratin 18 has been shown in both lung and breast cancers. Therefore, therapeutic modulation of the expression or function of this gene may be effective in the treatment of breast and lung cancers (Chu et al., Selection of invasive and metastatic subpopulations from a human lung adenocarcinoma cell line. Am J Respir Cell Mol Biol 1997 September;17(3):353-60).
X. NOV29: Cholinephosphate Cytidylyltransferase
Expression of gene CG57589-01 was assessed using the primer-probe set Ag4146, described in Table XA. Results of the RTQ-PCR runs are shown in Tables XB and XC.







TABLE XA







Probe Name Ag4146













Start


Primers
Sequences
Length
Position














Forward
5′-catcttccgtcagattgacagt-3′
(SEQ ID NO:383)
22
975


Probe
TET-5′-agcaacctcaccacagacctcatcgt-3′-TAMRA
(SEQ ID NO:384)
26
1000


Reverse
5′-atactccaacctgttggtgatg-3′
(SEQ ID NO:385)
22
1035
















TABLE XB







General_screening_panel_v1.4











Rel. Exp. (%) Ag4146, Run

Rel. Exp. (%) Ag4146, Run


Tissue Name
221290908
Tissue Name
221290908













Adipose
0.8
Renal ca. TK-10
19.3


Melanoma* Hs688 (A).T
4.7
Bladder
4.2


Melanoma* Hs688 (B).T
4.8
Gastric ca. (liver met.)
22.7




NCI-N87


Melanoma* M14
11.1
Gastric ca. KATO III
49.3


Melanoma* LOXIMVI
5.0
Colon ca. SW-948
16.2


Melanoma* SK-MEL-5
21.5
Colon ca. SW480
24.1


Squamous cell
4.7
Colon ca.* (SW480 met)
12.2


carcinoma SCC-4

SW620


Testis Pool
27.9
Colon ca. HT29
13.8


Prostate ca.* (bone met)
3.3
Colon ca. HCT-116
25.0


PC-3


Prostate Pool
2.3
Colon ca. CaCo-2
26.2


Placenta
2.7
Colon cancer tissue
2.2


Uterus Pool
0.4
Colon ca. SW1116
4.9


Ovarian ca. OVCAR-3
15.4
Colon ca. Colo-205
12.0


Ovarian ca. SK-OV-3
11.4
Colon ca. SW-48
6.5


Ovarian ca. OVCAR-4
12.4
Colon Pool
1.8


Ovarian ca. OVCAR-5
32.3
Small Intestine Pool
0.9


Ovarian ca. IGROV-1
11.0
Stomach Pool
1.0


Ovarian ca. OVCAR-8
5.1
Bone Marrow Pool
0.6


Ovary
2.3
Fetal Heart
2.9


Breast ca. MCF-7
14.0
Heart Pool
1.7


Breast ca. MDA-MB-
21.9
Lymph Node Pool
2.1


231


Breast ca. BT 549
11.5
Fetal Skeletal Muscle
0.8


Breast ca. T47D
100.0
Skeletal Muscle Pool
3.7


Breast ca. MDA-N
10.9
Spleen Pool
0.8


Breast Pool
1.6
Thymus Pool
1.6


Trachea
4.1
CNS cancer (glio/astro)
26.8




U87-MG


Lung
0.4
CNS cancer (glio/astro) U-
15.0




118-MG


Fetal Lung
3.9
CNS cancer (neuro; met)
12.5




SK-N-AS


Lung ca. NCI-N417
2.6
CNS cancer (astro) SF-539
22.4


Lung ca. LX-1
13.7
CNS cancer (astro) SNB-75
17.6


Lung ca. NCI-H146
1.8
CNS cancer (glio) SNB-19
12.2


Lung ca. SHP-77
7.4
CNS cancer (glio) SF-295
10.9


Lung ca. A549
5.4
Brain (Amygdala) Pool
6.1


Lung ca. NCI-H526
5.5
Brain (cerebellum)
12.6


Lung ca. NCI-H23
2.2
Brain (fetal)
7.1


Lung ca. NCI-H460
1.2
Brain (Hippocampus) Pool
6.2


Lung ca. HOP-62
5.8
Cerebral Cortex Pool
6.8


Lung ca. NCI-H522
6.3
Brain (Substantia nigra)
10.1




Pool


Liver
7.3
Brain (Thalamus) Pool
10.4


Fetal Liver
14.2
Brain (whole)
8.9


Liver ca. HepG2
29.5
Spinal Cord Pool
5.3


Kidney Pool
2.8
Adrenal Gland
3.1


Fetal Kidney
3.3
Pituitary gland Pool
0.7


Renal ca. 786-0
11.3
Salivary Gland
1.8


Renal ca. A498
4.9
Thyroid (female)
2.0


Renal ca. ACHN
9.9
Pancreatic ca. CAPAN2
12.5


Renal ca. UO-31
4.2
Pancreas Pool
2.8
















TABLE XC







Panel 5 Islet











Rel. Exp. (%)




Ag4146, Run



Tissue Name
242413200














97457_Patient-
4.0



02go_adipose



97476_Patient-07sk_skeletal
1.2



muscle



97477_Patient-07ut_uterus
5.4



97478_Patient-
6.0



07pl_placenta



99167_Bayer Patient 1
10.9



97482_Patient-08ut_uterus
4.0



97483_Patient-
1.3



08pl_placenta



97486_Patient-09sk_skeletal
0.7



muscle



97487_Patient-09ut_uterus
1.1



97488_Patient-
2.4



09pl_placenta



97492_Patient-10ut_uterus
4.4



97493_Patient-
8.0



10pl_placenta



97495_Patient-
1.8



11go_adipose



97496_Patient-11sk_skeletal
3.7



muscle



97497_Patient-11ut uterus
5.3



97498_Patient-
3.7



11pl_placenta



97500_Patient-
9.3



12go_adipose



97501_Patient-12sk_skeletal
11.7



muscle



97502 Patient-12ut_uterus
7.0



97503_Patient-
6.5



12pl_placenta



94721_Donor 2 U-
5.8



A_Mesenchymal Stem Cells



94722_Donor 2 U -
7.5



B_Mesenchymal Stem Cells



94723_Donor 2 U-
14.1



C_Mesenchymal Stem Cells



94709_Donor 2 AM - A_adipose
18.0



94710_Donor 2 AM - B_adipose
8.0



94711_Donor 2 AM - C_adipose
4.7



94712_Donor 2 AD - A_adipose
13.6



94713_Donor 2 AD - B_adipose
15.3



94714_Donor 2 AD - C_adipose
14.4



94742_Donor 3 U - A_Mesenchymal
8.2



Stem Cells



94743_Donor 3 U - B_Mesenchymal
6.0



Stem Cells



94730_Donor 3 AM - A_adipose
8.9



94731_Donor 3 AM - B_adipose
8.6



94732_Donor 3 AM - C_adipose
5.9



94733_Donor 3 AD - A_adipose
17.6



94734_Donor 3 AD - B_adipose
4.8



94735_Donor 3 AD - C_adipose
8.8



77138_Liver_HepG2untreated
100.0



73556_Heart_Cardiac stromal cells
3.7



(primary)



81735_Small Intestine
13.1



72409_Kidney_Proximal Convoluted
4.1



Tubule



82685_Small intestine_Duodenum
3.9



90650_Adrenal_Adrenocortical
2.6



adenoma



72410_Kidney_HRCE
37.1



72411_Kidney_HRE
6.0



73139_Uterus_Uterine smooth
4.5



muscle cells










General_screening_panel_v1.4 Summary: Ag4146 The expression of the CG57589-01 gene appears to be highest in a sample derived from a breast cancer cell line (T47D)(CT=27.2). In addition, there appears to be substantial expression in other samples derived from breast cancer cell lines, ovarian cancer cell lines, brain cancer cell lines and colon cancer cell lines. Thus, the expression of this gene could be used to distinguish T47D cells from other samples in the panel. Moreover, therapeutic modulation of this gene, through the use of small molecule drugs, protein therapeutics or antibodies could be of benefit in the treatment of breast, ovarian, brain or colon cancer.
Among metabolic tissue, this gene has low levels of expression in adipose, heart, skeletal muscle, adrenal, pituitary, thyroid, and pancreas. Dysregulated levels of phosphatidylcholine may be involved in the pathogenesis of disease in these tissues. Therefore, this gene may be a small molecule target for the treatment of metabolic and endocrine disease, including Types 1 and 2 diabetes and obesity.
This gene, a cholinephosphate cytidylyltransferase homolog, is also expressed at moderate levels in all CNS regions examined. This protein has been shown to be upregulated in the brain during triethyltin-induced cerebral edema. Therefore this gene may be a drug target for the treatment of cerebral edema (Mages et al., Pharmacol Toxicol 1989 October;65(4):302-5).
Panel 5 Islet Summary: Ag4146 The CG57589-01 is expressed at a low level in the islets of Langerhans, with highest expression in a liver derived sample (CT=30.5). Thus, expression of this gene could be used to differentiate between this sample and other samples on this panel.
Y. NOV30: mac25/IGFBP-7
Expression of gene CG57558-01 was assessed using the primer-probe set Ag3285, described in Table YA.







TABLE YA







Probe Name Ag3285













Start


Primers
Sequences
Length
Position














Forward
5′-gacaacaaacaagcaaacacaa-3′
(SEQ ID NO:386)
22
11


Probe
TET-5′-accttccacttccttctggtcctgct-3′-TAMRA
(SEQ ID NO:387)
26
62


Reverse
5′-gcaggagaggaggaagaagag-3′
(SEQ ID NO:388)
21
89









CNS_neurodegeneration_v1.0 Summary: Ag3285 Expression of this gene is low/undetectable (CTs>35) across all of the samples on this panel.
Panel 1.3D Summary: Ag3285 Expression of this gene is low/undetectable (CTs>35) across all of the samples on this panel.
Panel 4D Summary: Ag3285 Expression of this gene is low/undetectable (CTs>35) across all of the samples on this panel.
OTHER EMBODIMENTS
Although particular embodiments have been disclosed herein in detail, this has been done by way of example for purposes of illustration only, and is not intended to be limiting with respect to the scope of the appended claims, which follow. In particular, it is contemplated by the inventors that various substitutions, alterations, and modifications may be made to the invention without departing from the spirit and scope of the invention as defined by the claims. The choice of nucleic acid starting material, clone of interest, or library type is believed to be a matter of routine for a person of ordinary skill in the art with knowledge of the embodiments described herein. Other aspects, advantages, and modifications considered to be within the scope of the following claims.

## Claims

1. An isolated nucleic acid molecule comprising a nucleic acid sequence encoding a polypeptide comprising an amino acid sequence, SEQ ID NO: 26.

2. A nucleic acid molecule of claim 1, wherein said nucleic acid molecule comprises SEQ ID NO: 25.

3. A vector comprising the nucleic acid molecule of claim 1.

4. The vector of claim 3, further comprising a promotor operably-linked to said nucleic acid molecule.

5. A cell comprising the vector of claim 3.

6. An isolated nucleic acid molecule comprising the complement of the nucleic acid molecule of claim 1.