# Patent

## Title
Methods of diagnosis of ovarian cancer, compositions and methods of screening for modulators of ovarian cancer

## Abstract

Described herein are genes whose expression are up-regulated or down-regulated in ovarian cancer. Related methods and compositions that can be used for diagnosis and treatment of ovarian cancer are disclosed. Also described herein are methods that can be used to identify modulators of ovarian cancer.

## Background
CROSS-REFERENCES TO RELATED APPLICATIONS
This application is a continuation of U.S. Ser. No. 10/173,999, filed Jun. 17, 2002, now U.S. Pat. No. 7,189,507 which is incorporated herein by reference, and which claims priority from U.S. provisional applications U.S. Ser. No. 60/299,234, filed Jun. 18, 2001; U.S. Ser. No. 60/315,287, filed Aug. 27, 2001; U.S. Ser. No. 60/350,666, filed Nov. 13, 2001; and U.S. Ser. No. 60/372,246, filed Apr. 12, 2002, each of which is incorporated herein by reference for all purposes.


FIELD OF THE INVENTION
The invention relates to the identification of nucleic acid and protein expression profiles and nucleic acids, products, and antibodies thereto that are involved in ovarian cancer; and to the use of such expression profiles and compositions in the diagnosis, prognosis, and therapy of ovarian cancer. The invention further relates to methods for identifying and using agents and/or targets that inhibit ovarian cancer.
BACKGROUND OF THE INVENTION
Ovarian cancer is the sixth most common cancer in women, accounting for 6% of all female cancers. It ranks fifth as the cause of cancer death in women. The American Cancer Society predicts that there will be about 23,100 new cases of ovarian cancer in this country in the year 2000 and about 14,000 women will die of the disease. Because many ovarian cancers cannot be detected early in their development, they account for a disproportionate number of fatal cancers, being responsible for almost half the deaths from cancer of the female genital tract; more deaths than any other reproductive organ cancer.
Most patients with epithelial ovarian cancer, the predominant form, are asymptomatic in early-stage disease and usually present with stage III or IV disease. Their five-year survival is less than 25%, with lower survival among African-American women. The minority of patients discovered with early-stage disease have a five-year survival rate of 80%-90%. See, Parker, et. al. (1997) “Cancer Statistics, 1997” CA Cancer J. Clin. 47:5-27.
In the absence of a family history of ovarian cancer, lifetime risk of ovarian cancer is 1/70. Risk factors include familial cancer syndromes (risk of up to 82% by age 70 in women with hereditary breast/ovarian syndrome); family history (1.4% lifetime risk with no affected relatives, 5% with one affected relative, 7% with two affected relatives; Kerlikowske, et al. (1992) Obstet. Gynecol. 80:700-707); nulliparity; advancing age; obesity; personal history of breast, endometrial, or colorectal cancer; fewer pregnancies; or older age (>35 years) at first pregnancy. However, 95% of all ovarian cancers occur in women without risk factors. Use of hormonal contraceptives, oophorectomy, and tubal sterilization reduce risk of ovarian cancer (Kerlikowske, et al. (1992) Obstet. Gynecol. 80:700-707; Grimes (1992) Am J. Obstet. Gynecol. 166:1950-1954; Hankinson, et. al. (1993) JAMA 270:2813-2818); however, even bilateral oophorectomy may not be completely effective in preventing ovarian cancer.
Treatment of ovarian cancer consists largely of surgical oophorectomy, anti-hormone therapy, and/or chemotherapy. Although many ovarian cancer patients are effectively treated, the current therapies can all induce serious side effects which diminish quality of life. Deciding on a particular course of treatment is typically based on a variety of prognostic parameters and markers (Fitzgibbons, et al. (2000) Arch. Pathol. Lab. Med. 124:966-978; Hamilton and Piccart (2000) Ann. Oncol. 11:647-663), including genetic predisposition markers BRCA-1 and BRCA-2 (Robson (2000) J. Clin. Oncol. 18:113sup-118sup).
The identification of novel therapeutic targets and diagnostic markers is essential for improving the current treatment of ovarian cancer patients. Recent advances in molecular medicine have increased the interest in tumor-specific cell surface antigens that could serve as targets for various immunotherapeutic or small molecule strategies. Antigens suitable for immunotherapeutic strategies should be highly expressed in cancer tissues and ideally not expressed in normal adult tissues. Expression in tissues that are dispensable for life, however, may be tolerated. Examples of such antigens include Her2/neu and the B-cell antigen CD20. Humanized monoclonal antibodies directed to Her2/neu (HERCEPTIN®/trastuzumab) are currently in use for the treatment of metastatic breast cancer. Ross and Fletcher (1998) Stem Cells 16:413-428. Similarly, anti-CD20 monoclonal antibodies (RITUXIN®/rituximab) are used to effectively treat non-Hodgkin's lymphoma. Maloney, et al. (1997) Blood 90:2188-2195; Leget and Czuczman (1998) Curr. Opin. Oncol. 10:548-551.
Potential immunotherapeutic targets have been identified for ovarian cancer. One such target is polymorphic epithelial mucin (MUC1). MUC1 is a transmembrane protein, present at the apical surface of glandular epithelial cells. It is often overexpressed in ovarian cancer, and typically exhibits an altered glycosylation pattern, resulting in an antigenically distinct molecule, and is in early clinical trials as a vaccine target. Gilewski, et al. (2000) Clin. Cancer Res. 6:1693-1701; Scholl, et al. (2000) J. Immunother. 23:570-580. The tumor-expressed protein is often cleaved into the circulation, where it is detectable as the tumor marker, CA 15-3. See, e.g., Bon, et al. (1997) Clin. Chem. 43:585-593. However, many patients have tumors that express neither HER2 nor MUC-1; therefore, it is clear that other targets need to be identified to manage localized and metastatic disease.
Mutations in both BRCA1 and BRCA2 are associated with increased susceptibility to ovarian cancer. Mutations in BRCA1 occur in approximately 5 percent (95 percent confidence interval, 3 to 8 percent) of women in whom ovarian cancer is diagnosed before the age of 70 years. See Stratton, et al. (1997) N.E.J. Med. 336:1125-1130. And, in BRCA1 gene carriers, the risk for developing ovarian cancer is 0.63. See Easton (1995) Am. J. Hum. Genet. 56:267-xxx; and Elit (2001) Can. Fam. Physician 47:778-84.
Other biochemical markers such as CA125 have been reported to be associated with ovarian cancer, but they are not absolute indicators of disease. Although roughly 85% of women with clinically apparent ovarian cancer have increased levels of CA125, CA125 is also increased during the first trimester of pregnancy, during menstruation, in the presence of non-cancerous illnesses, and in cancers of other sites.
While industry and academia have identified novel gene sequences, there has not been an equal effort exerted to identify the function of these novel sequences. The elucidation of a role for novel proteins and compounds in disease states for identification of therapeutic targets and diagnostic markers is essential for improving the current treatment of ovarian cancer patients. Accordingly, provided herein are molecular targets for therapeutic intervention in ovarian and other cancers. Additionally, provided herein are methods that can be used in diagnosis and prognosis of ovarian cancer. Further provided are methods that can be used to screen candidate bioactive agents for the ability to modulate ovarian cancer.
SUMMARY OF THE INVENTION
The present invention therefore provides nucleotide sequences of genes that are up- and down-regulated in ovarian cancer cells. Such genes are useful for diagnostic purposes, and also as targets for screening for therapeutic compounds that modulate ovarian cancer, such as hormones or antibodies. The methods of detecting nucleic acids of the invention or their encoded proteins can be used for many purposes, e.g., early detection of ovarian cancers, monitoring and early detection of relapse following treatment, monitoring response to therapy, selecting patients for postoperative chemotherapy or radiation therapy, selecting therapy, determining tumor prognosis, treatment, or response to treatment (of primary or metastatic tumors), and early detection of pre-cancerous lesions. Other aspects of the invention will become apparent to the skilled artisan by the following description of the invention.
In one aspect, the present invention provides a method of detecting an ovarian cancer-associated transcript in a cell from a patient, the method comprising contacting a biological sample from the patient with a polynucleotide that selectively hybridizes to a sequence at least 80% identical to a sequence as shown in Table 20.
In one embodiment, the present invention provides a method of determining the level of an ovarian cancer associated transcript in a cell from a patient.
In one embodiment, the present invention provides a method of detecting an ovarian cancer-associated transcript in a cell from a patient, the method comprising contacting a biological sample from the patient with a polynucleotide that selectively hybridizes to a sequence at least 80% identical to a sequence as shown in Table 20.
In one embodiment, the polynucleotide selectively hybridizes to a sequence at least 95% identical to a sequence as shown in Table 20.
In one embodiment, the biological sample is a tissue sample. In another embodiment, the biological sample comprises isolated nucleic acids, e.g., mRNA.
In one embodiment, the polynucleotide is labeled, e.g., with a fluorescent label.
In one embodiment, the polynucleotide is immobilized on a solid surface.
In one embodiment, the patient is undergoing a therapeutic regimen to treat ovarian cancer. In another embodiment, the patient is suspected of having metastatic ovarian cancer.
In one embodiment, the patient is a human.
In one embodiment, the ovarian cancer associated transcript is mRNA.
In one embodiment, the method further comprises the step of amplifying nucleic acids before the step of contacting the biological sample with the polynucleotide.
In another aspect, the present invention provides a method of monitoring the efficacy of a therapeutic treatment of ovarian cancer, the method comprising the steps of: (i) providing a biological sample from a patient undergoing the therapeutic treatment; and (ii) determining the level of an ovarian cancer-associated transcript in the biological sample by contacting the biological sample with a polynucleotide that selectively hybridizes to a sequence at least 80% identical to a sequence as shown in Table 20, thereby monitoring the efficacy of the therapy. In a further embodiment, the patient has metastatic ovarian cancer. In a further embodiment, the patient has a drug resistant form of ovarian cancer.
In one embodiment, the method further comprises the step of: (iii) comparing the level of the ovarian cancer-associated transcript to a level of the ovarian cancer-associated transcript in a biological sample from the patient prior to, or earlier in, the therapeutic treatment.
Additionally, provided herein is a method of evaluating the effect of a candidate ovarian cancer drug comprising administering the drug to a patient and removing a cell sample from the patient. The expression profile of the cell is then determined. This method may further comprise comparing the expression profile to an expression profile of a healthy individual. In a preferred embodiment, said expression profile includes a gene of Table 19A.
In one aspect, the present invention provides an isolated nucleic acid molecule consisting of a polynucleotide sequence as shown in Table 20.
In one embodiment, an expression vector or cell comprises the isolated nucleic acid.
In one aspect, the present invention provides an isolated polypeptide which is encoded by a nucleic acid molecule having polynucleotide sequence as shown in Table 20.
In another aspect, the present invention provides an antibody that specifically binds to an isolated polypeptide which is encoded by a nucleic acid molecule having polynucleotide sequence as shown in Table 20.
In one embodiment, the antibody is conjugated to an effector component, e.g., a fluorescent label, a radioisotope or a cytotoxic chemical.
In one embodiment, the antibody is an antibody fragment. In another embodiment, the antibody is humanized.
In one aspect, the present invention provides a method of detecting an ovarian cancer cell in a biological sample from a patient, the method comprising contacting the biological sample with an antibody as described herein.
In another aspect, the present invention provides a method of detecting antibodies specific to ovarian cancer in a patient, the method comprising contacting a biological sample from the patient with a polypeptide encoded by a nucleic acid comprising a sequence from Table 20.
In another aspect, the present invention provides a method for identifying a compound that modulates an ovarian cancer-associated polypeptide, the method comprising the steps of: (i) contacting the compound with an ovarian cancer-associated polypeptide, the polypeptide encoded by a polynucleotide that selectively hybridizes to a sequence at least 80% identical to a sequence as shown in Table 20; and (ii) determining the functional effect of the compound upon the polypeptide.
In one embodiment, the functional effect is a physical effect, an enzymatic effect, or a chemical effect.
In one embodiment, the polypeptide is expressed in a eukaryotic host cell or cell membrane. In another embodiment, the polypeptide is recombinant.
In one embodiment, the functional effect is determined by measuring ligand binding to the polypeptide.
In another aspect, the present invention provides a method of inhibiting proliferation of an ovarian cancer-associated cell to treat ovarian cancer in a patient, the method comprising the step of administering to the subject a therapeutically effective amount of a compound identified as described herein.
In one embodiment, the compound is an antibody.
In another aspect, the present invention provides a drug screening assay comprising the steps of: (i) administering a test compound to a mammal having ovarian cancer or to a cell sample isolated from; (ii) comparing the level of gene expression of a polynucleotide that selectively hybridizes to a sequence at least 80% identical to a sequence as shown in Table 20 in a treated cell or mammal with the level of gene expression of the polynucleotide in a control cell sample or mammal, wherein a test compound that modulates the level of expression of the polynucleotide is a candidate for the treatment of ovarian cancer.
In one embodiment, the control is a mammal with ovarian cancer or a cell sample that has not been treated with the test compound. In another embodiment, the control is a normal cell or mammal, or is non-malignant tissue.
In one embodiment, the test compound is administered in varying amounts or concentrations. In another embodiment, the test compound is administered for varying time periods. In another embodiment, the comparison can occur after addition or removal of the drug candidate.
In one embodiment, the levels of a plurality of polynucleotides that selectively hybridize to a sequence at least 80% identical to a sequence as shown in Table 20 are individually compared to their respective levels in a control cell sample or mammal. In a preferred embodiment the plurality of polynucleotides is from three to ten.
In another aspect, the present invention provides a method for treating a mammal having ovarian cancer comprising administering a compound identified by the assay described herein.
In another aspect, the present invention provides a pharmaceutical composition for treating a mammal having ovarian cancer, the composition comprising a compound identified by the assay described herein and a physiologically acceptable excipient.
In one aspect, the present invention provides a method of screening drug candidates by providing a cell expressing a gene that is up- and down-regulated as in an ovarian cancer. In one embodiment, a gene is selected from Table 19A. The method further includes adding a drug candidate to the cell and determining the effect of the drug candidate on the expression of the expression profile gene.
In one embodiment, the method of screening drug candidates includes comparing the level of expression in the absence of the drug candidate to the level of expression in the presence of the drug candidate, wherein the concentration of the drug candidate can vary when present, and wherein the comparison can occur after addition or removal of the drug candidate. In a preferred embodiment, the cell expresses at least two expression profile genes. The profile genes may show an increase or decrease.
Also provided is a method of evaluating the effect of a candidate ovarian cancer drug comprising administering the drug to a transgenic animal expressing or over-expressing the ovarian cancer modulatory protein, or an animal lacking the ovarian cancer modulatory protein, for example as a result of a gene knockout.
Moreover, provided herein is a biochip comprising one or more nucleic acid segments of Table 20, wherein the biochip comprises fewer than 1000 nucleic acid probes. Preferably, at least two nucleic acid segments are included. More preferably, at least three nucleic acid segments are included.
Furthermore, a method of diagnosing a disorder associated with ovarian cancer is provided. The method comprises determining the expression of a gene of Table 19A in a first tissue type of a first individual, and comparing the distribution to the expression of the gene from a second normal tissue type from the first individual or a second unaffected individual. A difference in the expression indicates that the first individual has a disorder associated with ovarian cancer.
In a further embodiment, the biochip also includes a polynucleotide sequence of a gene that is not up- and down-regulated in ovarian cancer.
In one embodiment a method for screening for a bioactive agent capable of interfering with the binding of an ovarian cancer modulating protein (ovarian cancer modulatory protein) or a fragment thereof and an antibody which binds to said ovarian cancer modulatory protein or fragment thereof. In a preferred embodiment, the method comprises combining an ovarian cancer modulatory protein or fragment thereof, a candidate bioactive agent and an antibody which binds to said ovarian cancer modulatory protein or fragment thereof. The method further includes determining the binding of said ovarian cancer modulatory protein or fragment thereof and said antibody. Wherein there is a change in binding, an agent is identified as an interfering agent. The interfering agent can be an agonist or an antagonist. Preferably, the agent inhibits ovarian cancer.
Also provided herein are methods of eliciting an immune response in an individual. In one embodiment a method provided herein comprises administering to an individual a composition comprising an ovarian cancer modulating protein, or a fragment thereof. In another embodiment, the protein is encoded by a nucleic acid selected from those of Table 20.
Further provided herein are compositions capable of eliciting an immune response in an individual. In one embodiment, a composition provided herein comprises an ovarian cancer modulating protein, preferably encoded by a nucleic acid of Table 20 or a fragment thereof, and a pharmaceutically acceptable carrier. In another embodiment, said composition comprises a nucleic acid comprising a sequence encoding an ovarian cancer modulating protein, preferably selected from the nucleic acids of Table 20, and a pharmaceutically acceptable carrier.
Also provided are methods of neutralizing the effect of an ovarian cancer protein, or a fragment thereof, comprising contacting an agent specific for said protein with said protein in an amount sufficient to effect neutralization. In another embodiment, the protein is encoded by a nucleic acid selected from those of Table 20.
In another aspect of the invention, a method of treating an individual for ovarian cancer is provided. In one embodiment, the method comprises administering to said individual an inhibitor of an ovarian cancer modulating protein. In another embodiment, the method comprises administering to a patient having ovarian cancer an antibody to an ovarian cancer modulating protein conjugated to a therapeutic moiety. Such a therapeutic moiety can be a cytotoxic agent or a radioisotope.


DETAILED DESCRIPTION OF THE INVENTION
In accordance with the objects outlined above, the present invention provides novel methods for diagnosis and prognosis evaluation for ovarian cancer (OC), including metastatic ovarian cancer, as well as methods for screening for compositions which modulate ovarian cancer. Also provided are methods for treating ovarian cancer and related conditions, e.g., ovarian carcinoma (e.g., epithelial (including malignant serous tumors, malignant mucinous tumors, and malignant endometrioid tumors), germ cell (including teratomas, choriocarcinomas, polyembryomas, embryonal carcinoma, endodermal sinus tumor, dysgerminoma, and gonadoblastoma), and stromal carcinomas (e.g., granulosal stromal cell tumors)), fallopian tube carcinoma, and peritoneal carcinoma.
Table 19A provides unigene cluster identification numbers for the nucleotide sequence of genes that exhibit increased or decreased expression in ovarian cancer samples. Table 19A also provides an exemplar accession number that provides a nucleotide sequence that is part of the unigene cluster.
Definitions
The term “ovarian cancer protein” or “ovarian cancer polynucleotide” or “ovarian cancer-associated transcript” refers to nucleic acid and polypeptide polymorphic variants, alleles, mutants, and interspecies homologues that: (1) have a nucleotide sequence that has greater than about 60% nucleotide sequence identity, 65%, 70%, 75%, 80%, 85%, 90%, preferably 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or greater nucleotide sequence identity, preferably over a region of over a region of at least about 25, 50, 100, 200, 500, 1000, or more nucleotides, to a nucleotide sequence of or associated with a gene of Table 19A; (2) bind to antibodies, e.g., polyclonal antibodies, raised against an immunogen comprising an amino acid sequence encoded by a nucleotide sequence of or associated with a gene of Table 19A, and conservatively modified variants thereof; (3) specifically hybridize under stringent hybridization conditions to a nucleic acid sequence, or the complement thereof of Table 19A and conservatively modified variants thereof; or (4) have an amino acid sequence that has greater than about 60% amino acid sequence identity, 65%, 70%, 75%, 80%, 85%, 90%, preferably 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or greater amino sequence identity, preferably over a region of over a region of at least about 25, 50, 100, 200, 500, 1000, or more amino acid, to an amino acid sequence encoded by a nucleotide sequence of or associated with a gene of Table 19A. A polynucleotide or polypeptide sequence is typically from a mammal including, but not limited to, primate, e.g., human; rodent, e.g., rat, mouse, hamster; cow, pig, horse, sheep, or other mammal. An “ovarian cancer polypeptide” and an “ovarian cancer polynucleotide,” include both naturally occurring or recombinant forms.
A “full length” ovarian cancer protein or nucleic acid refers to an ovarian cancer polypeptide or polynucleotide sequence, or a variant thereof, that contains all of the elements normally contained in one or more naturally occurring, wild type ovarian cancer polynucleotide or polypeptide sequences. The “full length” may be prior to, or after, various stages of post-translation processing or splicing, including alternative splicing.
“Biological sample” as used herein is a sample of biological tissue or fluid that contains nucleic acids or polypeptides, e.g., of an ovarian cancer protein, polynucleotide or transcript. Such samples include, but are not limited to, tissue isolated from primates, e.g., humans, or rodents, e.g., mice, and rats. Biological samples may also include sections of tissues such as biopsy and autopsy samples, frozen sections taken for histologic purposes, blood, plasma, serum, sputum, stool, tears, mucus, hair, skin, etc. Biological samples also include explants and primary and/or transformed cell cultures derived from patient tissues. A biological sample is typically obtained from a eukaryotic organism, most preferably a mammal such as a primate e.g., chimpanzee or human; cow; dog; cat; a rodent, e.g., guinea pig, rat, mouse; rabbit; or a bird; reptile; or fish. Livestock and domestic animals are of particular interest.
“Providing a biological sample” means to obtain a biological sample for use in methods described in this invention. Most often, this will be done by removing a sample of cells from an animal, but can also be accomplished by using previously isolated cells (e.g., isolated by another person, at another time, and/or for another purpose), or by performing the methods of the invention in vivo. Archival tissues, having treatment or outcome history, will be particularly useful.
The terms “identical” or percent “identity,” in the context of two or more nucleic acids or polypeptide sequences, refer to two or more sequences or subsequences that are the same or have a specified percentage of amino acid residues or nucleotides that are the same (e.g., about 60% identity, preferably 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher identity over a specified region, when compared and aligned for maximum correspondence over a comparison window or designated region) as measured using a BLAST or BLAST 2.0 sequence comparison algorithms with default parameters described below, or by manual alignment and visual inspection (see, e.g., NCBI web site www.ncbi.nlm.nih.gov/BLAST/ or the like). Such sequences are then said to be “substantially identical.” This definition also refers to, or may be applied to, the compliment of a test sequence. The definition also includes sequences that have deletions and/or additions, as well as those that have substitutions, as well as naturally occurring, e.g., polymorphic or allelic variants, and man-made variants. As described below, the preferred algorithms can account for gaps and the like. Preferably, identity exists over a region that is at least about 25 amino acids or nucleotides in length, or more preferably over a region that is 50-100 amino acids or nucleotides in length.
For sequence comparison, typically one sequence acts as a reference sequence, to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are entered into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. Preferably, default program parameters can be used, or alternative parameters can be designated. The sequence comparison algorithm then calculates the percent sequence identities for the test sequences relative to the reference sequence, based on the program parameters.
A “comparison window”, as used herein, includes reference to a segment of one of the number of contiguous positions selected from the group consisting typically of from 20 to 600, usually about 50 to about 200, more usually about 100 to about 150 in which a sequence may be compared to a reference sequence of the same number of contiguous positions after the two sequences are optimally aligned. Methods of alignment of sequences for comparison are well-known in the art. Optimal alignment of sequences for comparison can be conducted, e.g., by the local homology algorithm of Smith and Waterman (1981) Adv. Appl. Math. 2:482-489, by the homology alignment algorithm of Needleman and Wunsch (1970) J. Mol. Biol. 48:443-453, by the search for similarity method of Pearson and Lipman (1988) Proc. Nat'l. Acad. Sci. USA 85:2444-2448, by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, Wis.), or by manual alignment and visual inspection (see, e.g., Ausubel, et al. (eds. 1995 and supplements) Current Protocols in Molecular Biology Lippincott.
Preferred examples of algorithms that are suitable for determining percent sequence identity and sequence similarity include the BLAST and BLAST 2.0 algorithms, which are described in Altschul, et al. (1977) Nuc. Acids Res. 25:3389-3402 and Altschul, et al. (1990) J. Mol. Biol. 215:403-410. BLAST and BLAST 2.0 are used, with the parameters described herein, to determine percent sequence identity for the nucleic acids and proteins of the invention. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (www.ncbi.nlm.nih.gov). This algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence, which either match or satisfy some positive-valued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as the neighborhood word score threshold (Altschul, et al., supra). These initial neighborhood word hits act as seeds for initiating searches to find longer HSPs containing them. The word hits are extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Cumulative scores are calculated using, e.g., for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always >0) and N (penalty score for mismatching residues; always <0). For amino acid sequences, a scoring matrix is used to calculate the cumulative score. Extension of the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses as defaults a wordlength (W) of 11, an expectation (E) of 10, M=5, N=−4 and a comparison of both strands. For amino acid sequences, the BLASTP program uses as defaults a word length of 3, and expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff and Henikoff (1989) Proc. Nat'l Acad. Sci. USA 89:10915-919) alignments (B) of 50, expectation (E) of 10, M=5, N=−4, and a comparison of both strands.
The BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g., Karlin and Altschul (1993) Proc. Nat'l Acad. Sci. USA 90:5873-5887). One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance. For example, a nucleic acid is considered similar to a reference sequence if the smallest sum probability in a comparison of the test nucleic acid to the reference nucleic acid is less than about 0.2, more preferably less than about 0.01, and most preferably less than about 0.001. Log values may be large negative numbers, e.g., 5, 10, 20, 30, 40, 40, 70, 90, 110, 150, 170, etc.
An indication that two nucleic acid sequences or polypeptides are substantially identical is that the polypeptide encoded by the first nucleic acid is immunologically cross reactive with the antibodies raised against the polypeptide encoded by the second nucleic acid, as described below. Thus, a polypeptide is typically substantially identical to a second polypeptide, e.g., where the two peptides differ only by conservative substitutions. Another indication that two nucleic acid sequences are substantially identical is that the two molecules or their complements hybridize to each other under stringent conditions, as described below. Yet another indication that two nucleic acid sequences are substantially identical is that the same primers can be used to amplify the sequences.
A “host cell” is a naturally occurring cell or a transformed cell that contains an expression vector and supports the replication or expression of the expression vector. Host cells may be cultured cells, explants, cells in vivo, and the like. Host cells may be prokaryotic cells such as E. coli, or eukaryotic cells such as yeast, insect, amphibian, or mammalian cells, such as CHO, HeLa, and the like (see, e.g., the American Type Culture Collection catalog or web site, www.atcc.org).
The terms “isolated,” “purified,” or “biologically pure” refer to material that is substantially or essentially free from components that normally accompany it as found in its native state. Purity and homogeneity are typically determined using analytical chemistry techniques such as polyacrylamide gel electrophoresis or high performance liquid chromatography. A protein or nucleic acid that is the predominant species present in a preparation is substantially purified. In particular, an isolated nucleic acid is separated from some open reading frames that naturally flank the gene and encode proteins other than protein encoded by the gene. The term “purified” in some embodiments denotes that a nucleic acid or protein gives rise to essentially one band in an electrophoretic gel. Preferably, it means that the nucleic acid or protein is at least 85% pure, more preferably at least 95% pure, and most preferably at least 99% pure. “Purify” or “purification” in other embodiments means removing at least one contaminant from the composition to be purified. In this sense, purification does not require that the purified compound be homogenous, e.g., 100% pure.
The terms “polypeptide,” “peptide” and “protein” are used interchangeably herein to refer to a polymer of amino acid residues. The terms apply to amino acid polymers in which one or more amino acid residue is an artificial chemical mimetic of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers, those containing modified residues, and non-naturally occurring amino acid polymers.
The term “amino acid” refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function similarly to the naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code, as well as those amino acids that are later modified, e.g., hydroxyproline, γ-carboxyglutamate, and O-phosphoserine. Amino acid analogs refers to compounds that have the same basic chemical structure as a naturally occurring amino acid, e.g., an a carbon that is bound to a hydrogen, a carboxyl group, an amino group, and an R group, e.g., homoserine, norleucine, methionine sulfoxide, methionine methyl sulfonium. Such analogs may have modified R groups (e.g., norleucine) or modified peptide backbones, but retain the same basic chemical structure as a naturally occurring amino acid. Amino acid mimetics refers to chemical compounds that have a structure that is different from the general chemical structure of an amino acid, but that functions similarly to a naturally occurring amino acid.
Amino acids may be referred to herein by either their commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Nucleotides, likewise, may be referred to by their commonly accepted single-letter codes.
“Conservatively modified variants” applies to both amino acid and nucleic acid sequences. With respect to particular nucleic acid sequences, conservatively modified variants refers to those nucleic acids which encode identical or essentially identical amino acid sequences, or where the nucleic acid does not encode an amino acid sequence, to essentially identical or associated, e.g., naturally contiguous, sequences. Because of the degeneracy of the genetic code, a large number of functionally identical nucleic acids encode most proteins. For instance, the codons GCA, GCC, GCG, and GCU all encode the amino acid alanine. Thus, at every position where an alanine is specified by a codon, the codon can be altered to another of the corresponding codons described without altering the encoded polypeptide. Such nucleic acid variations are “silent variations,” which are one species of conservatively modified variations. Every nucleic acid sequence herein which encodes a polypeptide also describes silent variations of the nucleic acid. In certain contexts each codon in a nucleic acid (except AUG, which is ordinarily the only codon for methionine, and TGG, which is ordinarily the only codon for tryptophan) can be modified to yield a functionally identical molecule. Accordingly, a silent variation of a nucleic acid which encodes a polypeptide is implicit in a described sequence with respect to the expression product, but not necessarily with respect to actual probe sequences.
As to amino acid sequences, one of skill will recognize that individual substitutions, deletions, or additions to a nucleic acid, peptide, polypeptide, or protein sequence which alters, adds, or deletes a single amino acid or a small percentage of amino acids in the encoded sequence is a “conservatively modified variant” where the alteration results in the substitution of an amino acid with a chemically similar amino acid. Conservative substitution tables providing functionally similar amino acids are well known in the art. Such conservatively modified variants are in addition to and do not exclude polymorphic variants, interspecies homologs, and alleles of the invention. Typically conservative substitutions for one another: 1) Alanine (A), Glycine (G); 2) Aspartic acid (D), Glutamic acid (E); 3) Asparagine (N), Glutamine (Q); 4) Arginine (R), Lysine (K); 5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); 6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W); 7) Serine (S), Threonine (T); and 8) Cysteine (C), Methionine (M) (see, e.g., Creighton (1984) Proteins Freeman).
Macromolecular structures such as polypeptide structures can be described in terms of various levels of organization. For a general discussion of this organization, see, e.g., Alberts, et al. (2001) Molecular Biology of the Cell (4th ed.) Garland Pub.; and Cantor and Schimmel (1980) Biophysical Chemistry Part I: The Conformation of Biological Macromolecules Freeman. “Primary structure” refers to the amino acid sequence of a particular peptide. “Secondary structure” refers to locally ordered, three dimensional structures within a polypeptide. These structures are commonly known as domains. Domains are portions of a polypeptide that often form a compact unit of the polypeptide and are typically 25 to approximately 500 amino acids long. Typical domains are made up of sections of lesser organization such as stretches of β-sheet and α-helices. “Tertiary structure” refers to the complete three dimensional structure of a polypeptide monomer. “Quaternary structure” refers to the three dimensional structure formed, usually by the non-covalent association of independent tertiary units. Anisotropic terms are also known as energy terms.
“Nucleic acid” or “oligonucleotide” or “polynucleotide” or grammatical equivalents used herein means at least two nucleotides covalently linked together. Oligonucleotides are typically from about 5, 6, 7, 8, 9, 10, 12, 15, 25, 30, 40, 50, or more nucleotides in length, up to about 100 nucleotides in length. Nucleic acids and polynucleotides are a polymers of any length, including longer lengths, e.g., 200, 300, 500, 1000, 2000, 3000, 5000, 7000, 10,000, etc. A nucleic acid of the present invention will generally contain phosphodiester bonds, although in some cases, nucleic acid analogs are included that may have at least one different linkage, e.g., phosphoramidate, phosphorothioate, phosphorodithioate, or O-methylphosphoroamidite linkages (see Eckstein (1992) Oligonucleotides and Analogues: A Practical Approach Oxford University Press); and peptide nucleic acid backbones and linkages. Other analog nucleic acids include those with positive backbones; non-ionic backbones, and non-ribose backbones, including those described in U.S. Pat. Nos. 5,235,033 and 5,034,506, and Chapters 6 and 7 of Sanghvi and Cook (eds. 1994) Carbohydrate Modifications in Antisense Research ASC Symposium Series 580. Nucleic acids containing one or more carbocyclic sugars are also included within one definition of nucleic acids. Modifications of the ribose-phosphate backbone may be done for a variety of reasons, e.g., to increase the stability and half-life of such molecules in physiological environments or as probes on a biochip. Mixtures of naturally occurring nucleic acids and analogs can be made; alternatively, mixtures of different nucleic acid analogs, and mixtures of naturally occurring nucleic acids and analogs may be made.
A variety of references disclose such nucleic acid analogs, including, e.g., phosphoramidate (Beaucage, et al. (1993) Tetrahedron 49:1925-1963 and references therein; Letsinger (1970) J. Org. Chem. 35:3800-3803; Sprinzl, et al. (1977) Eur. J. Biochem. 81:579-589; Letsinger, et al. (1986) Nucl. Acids Res. 14:3487-499; Sawai, et al. (1984) Chem. Lett. 805, Letsinger, et al. (1988) J. Am. Chem. Soc. 110:4470-4471; and Pauwels, et al. (1986), Chemica Scripta 26:141-149), phosphorothioate (Mag, et al. (1991) Nucl. Acids Res. 19:1437-441; and U.S. Pat. No. 5,644,048), phosphorodithioate (Brill, et al. (1989) J. Am. Chem. Soc. 111:2321-2322), O-methylphosphoroamidite linkages (see Eckstein (1992) Oligonucleotides and Analogues: A Practical Approach Oxford Univ. Press), and peptide nucleic acid backbones and linkages (see Egholm (1992) J. Am. Chem. Soc. 114:1895-897; Meier, et al. (1992) Angew. Chem. Int. Ed. Engl. 31:1008-1010; Nielsen (1993) Nature, 365:566-568; Carlsson, et al. (1996) Nature 380:207, each of which is incorporated by reference). Other analog nucleic acids include those with positive backbones (Denpcy, et al. (1995) Proc. Nat'l Acad. Sci. USA 92:6097-101; non-ionic backbones (U.S. Pat. Nos. 5,386,023, 5,637,684, 5,602,240, 5,216,141 and 4,469,863; Kiedrowshi, et al. (1991) Angew. Chem. Intl. Ed. English 30:423-426; Letsinger, et al. (1988) J. Am. Chem. Soc. 110:4470-4471; Jung, et al. (1994) Nucleoside and Nucleotide 13:1597; Chapters 2 and 3, in Sanghvi and Cook (eds. 1994) Carbohydrate Modifications in Antisense Research ASC Symposium Series 580; Mesmaeker, et al. (1994) Bioorganic and Medicinal Chem. Lett. 4:395-398; Jeffs, et al. (1994) J. Biomolecular NMR 34:17-xx; Horn, et al. (1996) Tetrahedron Lett. 37:743-xxx) and non-ribose backbones, including those described in U.S. Pat. Nos. 5,235,033 and 5,034,506, and Chapters 6 and 7, in Sanghvi and Cook (eds. 1994) Carbohydrate Modifications in Antisense Research ASC Symposium Series 580. Nucleic acids containing one or more carbocyclic sugars are also included within one definition of nucleic acids (see Jenkins, et al. (1995) Chem. Soc. Rev. pp 169-176). Several nucleic acid analogs are described in Rawls (p. 35 Jun. 2, 1997) C&E News. Each of these references is hereby expressly incorporated by reference.
Particularly preferred are peptide nucleic acids (PNA) which includes peptide nucleic acid analogs. These backbones are substantially non-ionic under neutral conditions, in contrast to the highly charged phosphodiester backbone of naturally occurring nucleic acids. This results in two advantages. First, the PNA backbone exhibits improved hybridization kinetics. PNAs have larger changes in the melting temperature (Tm) for mismatched versus perfectly matched base pairs. DNA and RNA typically exhibit a 2-4° C. drop in Tm for an internal mismatch. With the non-ionic PNA backbone, the drop is closer to 7-9° C. Similarly, due to their non-ionic nature, hybridization of the bases attached to these backbones is relatively insensitive to salt concentration. In addition, PNAs are not degraded by cellular enzymes, and thus can be more stable.
The nucleic acids may be single stranded or double stranded, as specified, or contain portions of both double stranded or single stranded sequence. As will be appreciated by those in the art, the depiction of a single strand also defines the sequence of the complementary strand; thus the sequences described herein also provide the complement of the sequence. The nucleic acid may be DNA, both genomic and cDNA, RNA, or a hybrid, where the nucleic acid may contain combinations of deoxyribo- and ribo-nucleotides, and combinations of bases, including uracil, adenine, thymine, cytosine, guanine, inosine, xanthine hypoxanthine, isocytosine, isoguanine, etc. “Transcript” typically refers to a naturally occurring RNA, e.g., a pre-mRNA, hnRNA, or mRNA. As used herein, the term “nucleoside” includes nucleotides and nucleoside and nucleotide analogs, and modified nucleosides such as amino modified nucleosides. In addition, “nucleoside” includes non-naturally occurring analog structures. Thus, e.g., the individual units of a peptide nucleic acid, each containing a base, are referred to herein as a nucleoside.
A “label” or a “detectable moiety” is a composition detectable by spectroscopic, photochemical, biochemical, immunochemical, chemical, or other physical means. For example, useful labels include 32P, fluorescent dyes, electron-dense reagents, enzymes (e.g., as commonly used in an ELISA), biotin, digoxigenin, or haptens and proteins or other entities which can be made detectable, e.g., by incorporating a radiolabel into the peptide or used to detect antibodies specifically reactive with the peptide. The labels may be incorporated into the ovarian cancer nucleic acids, proteins and antibodies at any position. Any method known in the art for conjugating the antibody to the label may be employed, including those methods described by Hunter, et al. (1962) Nature 144:945-xxx; David, et al. (1974) Biochemistry 13:1014-1021; Pain, et al. (1981) J. Immunol. Meth. 40:219-230; and Nygren (1982) J. Histochem. and Cytochem. 30:407-412.
An “effector” or “effector moiety” or “effector component” is a molecule that is bound (or linked, or conjugated), either covalently, through a linker or a chemical bond, or non-covalently, through ionic, van der Waals, electrostatic, or hydrogen bonds, to an antibody. The “effector” can be a variety of molecules including, e.g., detection moieties including radioactive compounds, fluorescent compounds, an enzyme or substrate, tags such as epitope tags, a toxin; activatable moieties, a chemotherapeutic agent; a lipase; an antibiotic; or a radioisotope emitting “hard” e.g., beta radiation.
A “labeled nucleic acid probe or oligonucleotide” is one that is bound, either covalently, through a linker or a chemical bond, or non-covalently, through ionic, van der Waals, electrostatic, or hydrogen bonds to a label such that the presence of the probe may be detected by detecting the presence of the label bound to the probe. Alternatively, method using high affinity interactions may achieve the same results where one of a pair of binding partners binds to the other, e.g., biotin, streptavidin.
As used herein a “nucleic acid probe or oligonucleotide” is a nucleic acid capable of binding to a target nucleic acid of complementary sequence through one or more types of chemical bonds, usually through complementary base pairing, usually through hydrogen bond formation. As used herein, a probe may include natural (e.g., A, G, C, or T) or modified bases (7-deazaguanosine, inosine, etc.). In addition, the bases in a probe may be joined by a linkage other than a phosphodiester bond, so long as it does not functionally interfere with hybridization. Thus, e.g., probes may be peptide nucleic acids in which the constituent bases are joined by peptide bonds rather than phosphodiester linkages. Probes may bind target sequences lacking complete complementarity with the probe sequence depending upon the stringency of the hybridization conditions. The probes are preferably directly labeled, e.g., with isotopes, chromophores, lumiphores, chromogens, or indirectly labeled such as with biotin to which a streptavidin complex may later bind. By assaying for the presence or absence of the probe, one can detect the presence or absence of the select sequence or subsequence. Diagnosis or prognosis may be based at the genomic level, or at the level of RNA or protein expression.
The term “recombinant” when used with reference, e.g., to a cell, or nucleic acid, protein, or vector, indicates that the cell, nucleic acid, protein or vector, has been modified by the introduction of a heterologous nucleic acid or protein or the alteration of a native nucleic acid or protein, or that the cell is derived from a cell so modified. Thus, e.g., recombinant cells express genes that are not found within the native (non-recombinant) form of the cell or express native genes that are otherwise abnormally expressed, under expressed or not expressed at all. By the term “recombinant nucleic acid” herein is meant nucleic acid, originally formed in vitro, in general, by the manipulation of nucleic acid, e.g., using polymerases and endonucleases, in a form not normally found in nature. In this manner, operably linkage of different sequences is achieved. Thus an isolated nucleic acid, in a linear form, or an expression vector formed in vitro by ligating DNA molecules that are not normally joined, are both considered recombinant for the purposes of this invention. It is understood that once a recombinant nucleic acid is made and reintroduced into a host cell or organism, it will replicate non-recombinantly, e.g., using the in vivo cellular machinery of the host cell rather than in vitro manipulations; however, such nucleic acids, once produced recombinantly, although subsequently replicated non-recombinantly, are still considered recombinant for the purposes of the invention. Similarly, a “recombinant protein” is a protein made using recombinant techniques, e.g., through the expression of a recombinant nucleic acid as depicted above.
The term “heterologous” when used with reference to portions of a nucleic acid indicates that the nucleic acid comprises two or more subsequences that are not normally found in the same relationship to each other in nature. For instance, the nucleic acid is typically recombinantly produced, having two or more sequences, e.g., from unrelated genes arranged to make a new functional nucleic acid, e.g., a promoter from one source and a coding region from another source. Similarly, a heterologous protein will often refer to two or more subsequences that are not found in the same relationship to each other in nature (e.g., a fusion protein).
A “promoter” is defined as an array of nucleic acid control sequences that direct transcription of a nucleic acid. As used herein, a promoter includes necessary nucleic acid sequences near the start site of transcription, such as, in the case of a polymerase II type promoter, a TATA element. A promoter also optionally includes distal enhancer or repressor elements, which can be located as much as several thousand base pairs from the start site of transcription. A “constitutive” promoter is a promoter that is active under most environmental and developmental conditions. An “inducible” promoter is a promoter that is active under environmental or developmental regulation. The term “operably linked” refers to a functional linkage between a nucleic acid expression control sequence (such as a promoter, or array of transcription factor binding sites) and a second nucleic acid sequence, e.g., wherein the expression control sequence directs transcription of the nucleic acid corresponding to the second sequence.
An “expression vector” is a nucleic acid construct, generated recombinantly or synthetically, with a series of specified nucleic acid elements that permit transcription of a particular nucleic acid in a host cell. The expression vector can be part of a plasmid, virus, or nucleic acid fragment. Typically, the expression vector includes a nucleic acid to be transcribed operably linked to a promoter.
The phrase “selectively (or specifically) hybridizes to” refers to the binding, duplexing, or hybridizing of a molecule only to a particular nucleotide sequence under stringent hybridization conditions when that sequence is present in a complex mixture (e.g., total cellular or library DNA or RNA).
The phrase “stringent hybridization conditions” refers to conditions under which a probe will hybridize to its target subsequence, typically in a complex mixture of nucleic acids, but to no other sequences. Stringent conditions are sequence-dependent and will be different in different circumstances. Longer sequences hybridize specifically at higher temperatures. An extensive guide to the hybridization of nucleic acids is found in “Overview of principles of hybridization and the strategy of nucleic acid assays” in Tijssen (1993) Hybridization with Nucleic Probes (Laboratory Techniques in Biochemistry and Molecular Biology) (vol. 24) Elsevier. Generally, stringent conditions are selected to be about 5-10° C. lower than the thermal melting point (Tm) for the specific sequence at a defined ionic strength pH. The Tm is the temperature (under defined ionic strength, pH, and nucleic concentration) at which 50% of the probes complementary to the target hybridize to the target sequence at equilibrium (as the target sequences are present in excess, at Tm, 50% of the probes are occupied at equilibrium). 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 concentration (or other salts) at pH 7.0 to 8.3 and the temperature is at least about 30° C. for short probes (e.g., 10 to 50 nucleotides) and at least about 60° C. for long probes (e.g., greater than 50 nucleotides). Stringent conditions may also be achieved with the addition of destabilizing agents such as formamide. For selective or specific hybridization, a positive signal is typically at least two times background, preferably 10 times background hybridization. Exemplary stringent hybridization conditions can be as following: 50% formamide, 5×SSC, and 1% SDS, incubating at 42° C., or, 5×SSC, 1% SDS, incubating at 65° C., with wash in 0.2×SSC, and 0.1% SDS at 65° C. For PCR, a temperature of about 36° C. is typical for low stringency amplification, although annealing temperatures may vary between about 32-48° C. depending on primer length. For high stringency PCR amplification, a temperature of about 62° C. is typical, although high stringency annealing temperatures can range from about 50° C. to about 65° C., depending on the primer length and specificity. Typical cycle conditions for both high and low stringency amplifications include a denaturation phase of 90-95° C. for 30-120 sec, an annealing phase lasting 30-120 sec, and an extension phase of about 72° C. for 1-2 min. Protocols and guidelines for low and high stringency amplification reactions are available, e.g., in Innis, et al. (1990) PCR Protocols: A Guide to Methods and Applications Academic Press, N.Y.
Nucleic acids that do not hybridize to each other under stringent conditions are still substantially identical if the polypeptides which they encode are substantially identical. This occurs, e.g., when a copy of a nucleic acid is created using the maximum codon degeneracy permitted by the genetic code. In such cases, the nucleic acids typically hybridize under moderately stringent hybridization conditions. Exemplary “moderately stringent hybridization conditions” include a hybridization in a buffer of 40% formamide, 1 M NaCl, 1% SDS at 37° C., and a wash in 1×SSC at 45° C. A positive hybridization is at least twice background. Alternative hybridization and wash conditions can be utilized to provide conditions of similar stringency. Additional guidelines for determining hybridization parameters are provided, e.g., Ausubel, et al. (ed. 1991 and supplements) Current Protocols in Molecular Biology Lippincott.
The phrase “functional effects” in the context of assays for testing compounds that modulate activity of an ovarian cancer protein includes the determination of a parameter that is indirectly or directly under the influence of the ovarian cancer protein or nucleic acid, e.g., a functional, physical, physiological, or chemical effect, such as the ability to decrease ovarian cancer. It includes ligand binding activity; cell growth on soft agar; anchorage dependence; contact inhibition and density limitation of growth; cellular proliferation; cellular transformation; growth factor or serum dependence; tumor specific marker levels; invasiveness into Matrigel; tumor growth and metastasis in vivo; mRNA and protein expression in cells undergoing metastasis, and other characteristics of ovarian cancer cells. “Functional effects” include in vitro, in vivo, and ex vivo activities.
By “determining the functional effect” is meant assaying for a compound that increases or decreases a parameter that is indirectly or directly under the influence of an ovarian cancer protein sequence, e.g., functional, enzymatic, physical, physiological, and chemical effects. Such functional effects can be measured by any means known to those skilled in the art, e.g., changes in spectroscopic characteristics (e.g., fluorescence, absorbance, refractive index), hydrodynamic (e.g., shape), chromatographic, or solubility properties for the protein, measuring inducible markers or transcriptional activation of the ovarian cancer protein; measuring binding activity or binding assays, e.g., binding to antibodies or other ligands, and measuring cellular proliferation. Determination of the functional effect of a compound on ovarian cancer can also be performed using ovarian cancer assays known to those of skill in the art such as an in vitro assays, e.g., cell growth on soft agar; anchorage dependence; contact inhibition and density limitation of growth; cellular proliferation; cellular transformation; growth factor or serum dependence; tumor specific marker levels; invasiveness into Matrigel; tumor growth and metastasis in vivo; mRNA and protein expression in cells undergoing metastasis, and other characteristics of ovarian cancer cells. The functional effects can be evaluated by means known to those skilled in the art, e.g., microscopy for quantitative or qualitative measures of alterations in morphological features, measurement of changes in RNA or protein levels for ovarian cancer-associated sequences, measurement of RNA stability, or identification of downstream or reporter gene expression (CAT, luciferase, β-gal, GFP, and the like), e.g., via chemiluminescence, fluorescence, colorimetric reactions, antibody binding, inducible markers, and ligand binding assays.
“Inhibitors”, “activators”, and “modulators” of ovarian cancer polynucleotide and polypeptide sequences are used to refer to activating, inhibitory, or modulating molecules or compounds identified using in vitro and in vivo assays of ovarian cancer polynucleotide and polypeptide sequences. Inhibitors are compounds that, e.g., bind to, partially or totally block activity, decrease, prevent, delay activation, inactivate, desensitize, or down regulate the activity or expression of ovarian cancer proteins, e.g., antagonists. Antisense or inhibitory nucleic acids may inhibit expression and subsequent function of the protein. “Activators” are compounds that increase, open, activate, facilitate, enhance activation, sensitize, agonize, or up regulate ovarian cancer protein activity. Inhibitors, activators, or modulators also include genetically modified versions of ovarian cancer proteins, e.g., versions with altered activity, as well as naturally occurring and synthetic ligands, antagonists, agonists, antibodies, small chemical molecules, and the like. Assays for inhibitors and activators include, e.g., expressing the ovarian cancer protein in vitro, in cells, or cell membranes, applying putative modulator compounds, and then determining the functional effects on activity, as described above. Activators and inhibitors of ovarian cancer can also be identified by incubating ovarian cancer cells with the test compound and determining increases or decreases in the expression of one or more ovarian cancer proteins, e.g., 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 40, 50, or more ovarian cancer proteins, such as ovarian cancer proteins encoded by the sequences set out in Table 19A.
Samples or assays comprising ovarian cancer proteins that are treated with a potential activator, inhibitor, or modulator are compared to control samples without the inhibitor, activator, or modulator to examine the extent of inhibition. Control samples (untreated with inhibitors) are assigned a relative protein activity value of 100%. Inhibition of a polypeptide is achieved when the activity value relative to the control is about 80%, preferably 50%, more preferably 25% or less. Activation of an ovarian cancer polypeptide is achieved when the activity value relative to the control (untreated with activators) is 110%, more preferably 150%, more preferably 200-500% (e.g., 2-5 fold higher relative to the control), more preferably 1000-3000% higher.
The phrase “changes in cell growth” refers to a change in cell growth and proliferation characteristics in vitro or in vivo, e.g., cell viability, formation of foci, anchorage independence, semi-solid or soft agar growth, change in contact inhibition or density limitation of growth, loss of growth factor or serum requirements, change in cell morphology, gain or loss of immortalization, gain or loss of tumor specific markers, ability to form or suppress tumors when injected into suitable animal hosts, and/or immortalization of the cell. See, e.g., pp. 231-241 in Freshney (1994) Culture of Animal Cells: A Manual of Basic Technique (3d ed.) Wiley-Liss.
“Tumor cell” refers to pre-cancerous, cancerous, and normal cells in a tumor.
“Cancer cells,” “transformed” cells or “transformation” in tissue culture, refers to spontaneous or induced phenotypic changes that do not necessarily involve the uptake of new genetic material. Although transformation can arise from infection with a transforming virus and incorporation of new genomic DNA, or uptake of exogenous DNA, it can also arise spontaneously or following exposure to a carcinogen, thereby mutating an endogenous gene. Transformation is typically associated with phenotypic changes, such as immortalization of cells, aberrant growth control, non-morphological changes, and/or malignancy. See, Freshney (1994) Culture of Animal Cells. 
“Antibody” refers to a polypeptide comprising a framework region from an immunoglobulin gene or fragments thereof that specifically binds and recognizes an antigen. The recognized immunoglobulin genes include the kappa, lambda, alpha, gamma, delta, epsilon, and mu constant region genes, as well as the myriad immunoglobulin variable region genes. Light chains are classified as either kappa or lambda. Heavy chains are classified as gamma, mu, alpha, delta, or epsilon, which in turn define the immunoglobulin classes, IgG, IgM, IgA, IgD, and IgE, respectively. Typically, the antigen-binding region of an antibody or its functional equivalent will be most critical in specificity and affinity of binding. See, e.g., Paul (ed. 1999) Fundamental Immunology (4th ed.) Raven.
An exemplary immunoglobulin (antibody) structural unit comprises a tetramer. Each tetramer is composed of two identical pairs of polypeptide chains, each pair having one “light” (about 25 kD) and one “heavy” chain (about 50-70 kD). The N-terminus of each chain defines a variable region of about 100 to 110 or more amino acids primarily responsible for antigen recognition. The terms variable light chain (VL) and variable heavy chain (VH) refer to these light and heavy chains respectively.
Antibodies exist, e.g., as intact immunoglobulins or as a number of well-characterized fragments produced by digestion with various peptidases. Thus, e.g., pepsin digests an antibody below the disulfide linkages in the hinge region to produce F(ab)′2, a dimer of Fab which itself is a light chain joined to VH-CH1 by a disulfide bond. The F(ab)′2 may be reduced under mild conditions to break the disulfide linkage in the hinge region, thereby converting the F(ab)′2 dimer into an Fab′ monomer. The Fab′ monomer is essentially Fab with part of the hinge region. See Paul (ed. 1999) Fundamental Immunology (4th ed.) Raven. While various antibody fragments are defined in terms of the digestion of an intact antibody, one of skill will appreciate that such fragments may be synthesized de novo either chemically or by using recombinant DNA methodology. Thus, the term antibody, as used herein, also includes antibody fragments either produced by the modification of whole antibodies, or those synthesized de novo using recombinant DNA methodologies (e.g., single chain Fv) or those identified using phage display libraries. See, e.g., McCafferty, et al. (1990) Nature 348:552-554.
For preparation of antibodies, e.g., recombinant, monoclonal, or polyclonal antibodies, many techniques known in the art can be used (see, e.g., Kohler and Milstein (1975) Nature 256:495-497; Kozbor, et al. (1983) Immunology Today 4:72; Cole, et al., pp. 77-96 in Reisfeld and Sell (1985) Monoclonal Antibodies and Cancer Therapy Liss; Coligan (1991) Current Protocols in Immunology Lippincott; Harlow and Lane (1988) Antibodies: A Laboratory Manual CSH Press; and Goding (1986) Monoclonal Antibodies: Principles and Practice (2d ed.) Academic Press. Techniques for the production of single chain antibodies (U.S. Pat. No. 4,946,778) can be adapted to produce antibodies to polypeptides of this invention. Transgenic mice, or other organisms, e.g., other mammals, may be used to express humanized antibodies. Alternatively, phage display technology can be used to identify antibodies and heteromeric Fab fragments that specifically bind to selected antigens. See, e.g., McCafferty, et al. (1990) Nature 348:552-554; and Marks, et al. (1992) Biotechnology 10:779-783.
A “chimeric antibody” is an antibody molecule in which (a) the constant region, or a portion thereof, is altered, replaced or exchanged so that the antigen binding site (variable region) is linked to a constant region of a different or altered class, effector function and/or species, or an entirely different molecule which confers new properties to the chimeric antibody, e.g., an enzyme, toxin, hormone, growth factor, drug, etc.; or (b) the variable region, or a portion thereof, is altered, replaced or exchanged with a variable region having a different or altered antigen specificity.
Identification of Ovarian Cancer-associated Sequences
In one aspect, the expression levels of genes are determined in different patient samples for which diagnosis information is desired, to provide expression profiles. An expression profile of a particular sample is essentially a “fingerprint” of the state of the sample; while two states may have any particular gene similarly expressed, the evaluation of a number of genes simultaneously allows the generation of a gene expression profile that is characteristic of the state of the cell. That is, normal tissue (e.g., normal ovarian or other tissue) may be distinguished from cancerous or metastatic cancerous tissue of the ovarian, or ovarian cancer tissue or metastatic ovarian cancerous tissue can be compared with tissue samples of ovarian and other tissues from surviving cancer patients. By comparing expression profiles of tissue in known different ovarian cancer states, information regarding which genes are important (including both up- and down-regulation of genes) in each of these states is obtained. Molecular profiling may distinguish subtypes of a currently collective disease designation, e.g., different forms of a cancer.
The identification of sequences that are differentially expressed in ovarian cancer versus non-ovarian cancer tissue allows the use of this information in a number of ways. For example, a particular treatment regime may be evaluated: does a chemotherapeutic drug act to down-regulate ovarian cancer, and thus tumor growth or recurrence, in a particular patient. Alternatively, does existing treatment induce expression of a target. Similarly, diagnosis and treatment outcomes may be done or confirmed by comparing patient samples with the known expression profiles. Metastatic tissue can also be analyzed to determine the stage of ovarian cancer in the tissue or origin of the primary tumor. Furthermore, these gene expression profiles (or individual genes) allow screening of drug candidates with an eye to mimicking or altering a particular expression profile; e.g., screening can be done for drugs that suppress the ovarian cancer expression profile. This may be done by making biochips comprising sets of the important ovarian cancer genes, which can then be used in these screens. These methods can also be based on evaluating protein expression; that is, protein expression levels of the ovarian cancer proteins can be evaluated for diagnostic purposes or to screen candidate agents. In addition, the ovarian cancer nucleic acid sequences can be administered for gene therapy purposes, including the administration of antisense or RNAi nucleic acids, or the ovarian cancer proteins (including antibodies and other modulators thereof) administered as therapeutic drugs.
Thus the present invention provides nucleic acid and protein sequences that are differentially expressed in ovarian cancer relative to normal tissues and/or non-malignant tissues, herein termed “ovarian cancer sequences.” As outlined below, ovarian cancer sequences include those that are up-regulated (e.g., expressed at a higher level) in ovarian cancer, as well as those that are down-regulated (e.g., expressed at a lower level). In a preferred embodiment, the ovarian cancer sequences are from humans; however, as will be appreciated by those in the art, ovarian cancer sequences from other organisms may be useful in animal models of disease and drug evaluation; thus, other ovarian cancer sequences are provided, from vertebrates, including mammals, including rodents (rats, mice, hamsters, guinea pigs, etc.), primates, farm animals (including sheep, goats, pigs, cows, horses, etc.) and pets (e.g., dogs, cats, etc.). Ovarian cancer sequences, e.g., counterpart genes, from other organisms may be obtained using the techniques outlined below.
Ovarian cancer sequences can include both nucleic acid and amino acid sequences. Ovarian cancer nucleic acid sequences are useful in a variety of applications, including diagnostic applications, which will detect naturally occurring nucleic acids. Screening applications; e.g., biochips comprising nucleic acid probes or PCR microtiter plates with selected probes to the ovarian cancer sequences, are also provided.
An ovarian cancer sequence can be initially identified by substantial nucleic acid and/or amino acid sequence homology to the ovarian cancer sequences outlined herein. Such homology can be based upon the overall nucleic acid or amino acid sequence, and is generally determined as outlined below, using either homology programs or hybridization conditions.
For identifying ovarian cancer-associated sequences, the ovarian cancer screen typically includes comparing genes identified in different tissues, e.g., normal and cancerous tissues, or tumor tissue samples from patients who have metastatic disease vs. non metastatic tissue. Other suitable tissue comparisons include comparing ovarian cancer samples with metastatic cancer samples from other cancers, such as lung, ovarian, gastrointestinal cancers, etc. Samples of different stages of ovarian cancer, e.g., survivor tissue, drug resistant states, and tissue undergoing metastasis, are applied to biochips comprising nucleic acid probes. The samples are first microdissected, if applicable, and treated for the preparation of mRNA. Suitable biochips are commercially available, e.g., from Affymetrix. Gene expression profiles as described herein are generated and the data analyzed.
In one embodiment, the genes showing changes in expression as between normal and disease states are compared to genes expressed in other normal tissues, preferably normal ovarian, but also including, and not limited to, lung, heart, brain, liver, ovarian, kidney, muscle, colon, small intestine, large intestine, spleen, bone, and/or placenta. In a preferred embodiment, those genes identified during the ovarian cancer screen that are expressed in any significant amount in other tissues are removed from the profile, although in some embodiments, expression in non-essential tissues may be tolerated. That is, when screening for drugs, it is usually preferable that the target be disease specific, to minimize possible side effects by interaction with target present in other organs.
In a preferred embodiment, ovarian cancer sequences are those that are up-regulated in ovarian cancer; that is, the expression of these genes is higher in the ovarian cancer tissue as compared to non-cancerous tissue. “Up-regulation” as used herein often means at least about a two-fold change, preferably at least about a three fold change, with at least about five-fold or higher being preferred. Other embodiments are directed to sequences up regulated in non-malignant conditions relative to normal.
Unigene cluster identification numbers and accession numbers herein refer to the GenBank sequence database and the sequences of the accession numbers are hereby expressly incorporated by reference. GenBank is known in the art, see, e.g., Benson, et al. (1998) Nucl. Acids Res. 26:1-7; and www.ncbi.nlm.nih.gov. Sequences are also available in other databases, e.g., European Molecular Biology Laboratory (EMBL) and DNA Database of Japan (DDBJ). In some situations, the sequences may be derived from assembly of available sequences or be predicted from genomic DNA using exon prediction algorithms, e.g., FGENESH. See Salamov and Solovyev (2000) Genome Res. 10:516-522. In other situations, sequences have been derived from cloning and sequencing of isolated nucleic acids.
In another preferred embodiment, ovarian cancer sequences are those that are down-regulated in ovarian cancer; that is, the expression of these genes is lower in ovarian cancer tissue as compared to non-cancerous tissue. “Down-regulation” as used herein often means at least about a two-fold change, preferably at least about a three-fold change, with at least about five-fold or higher being preferred.
Informatics
The ability to identify genes that are over or under expressed in ovarian cancer can additionally provide high-resolution, high-sensitivity datasets which can be used in the areas of diagnostics, therapeutics, drug development, pharmacogenetics, protein structure, biosensor development, and other related areas. Expression profiles can be used in diagnostic or prognostic evaluation of patients with ovarian cancer. Subcellular toxicological information can be generated to better direct drug structure and activity correlation (see Anderson (Jun. 11-12, 1998) Pharmaceutical Proteomics: Targets, Mechanism, and Function, paper presented at the IBC Proteomics conference, Coronado, Calif.) or in a biological sensor device to predict the likely toxicological effect of chemical exposures and likely tolerable exposure thresholds (see U.S. Pat. No. 5,811,231). Similar advantages accrue from datasets relevant to other biomolecules and bioactive agents (e.g., nucleic acids, saccharides, lipids, drugs, and the like).
Thus, in another embodiment, the present invention provides a database that includes at least one set of assay data. The data contained in the database is acquired, e.g., using array analysis either singly or in a library format. The database can be in a form in which data can be maintained and transmitted, but is preferably an electronic database, and can be maintained on any electronic device allowing for the storage of and access to the database, such as a personal computer, but is preferably distributed on a wide area network, such as the World Wide Web.
The focus of the present section on databases that include peptide sequence data is for clarity of illustration only. It will be apparent to those of skill in the art that similar databases can be assembled for any assay data acquired using an assay of the invention.
The compositions and methods for identifying and/or quantitating the relative and/or absolute abundance of a variety of molecular and macromolecular species from a biological sample undergoing ovarian cancer, e.g., the identification of ovarian cancer-associated sequences described herein, provide an abundance of information which can be correlated with pathological conditions, predisposition to disease, drug testing, therapeutic monitoring, gene-disease causal linkages, identification of correlates of immunity and physiological status, and outcome data, among others. Although data generated from the assays of the invention is suited for manual review and analysis, in a preferred embodiment, data processing using high-speed computers is utilized.
An array of methods for indexing and retrieving biomolecular information is known in the art. For example, U.S. Pat. Nos. 6,023,659 and 5,966,712 disclose a relational database system for storing biomolecular sequence information in a manner that allows sequences to be catalogued and searched according to one or more protein function hierarchies. U.S. Pat. No. 5,953,727 discloses a relational database having sequence records containing information in a format that allows a collection of partial-length DNA sequences to be catalogued and searched according to association with one or more sequencing projects for obtaining full-length sequences from the collection of partial length sequences. U.S. Pat. No. 5,706,498 discloses a gene database retrieval system for making a retrieval of a gene sequence similar to a sequence data item in a gene database based on the degree of similarity between a key sequence and a target sequence. U.S. Pat. No. 5,538,897 discloses a method using mass spectroscopy fragmentation patterns of peptides to identify amino acid sequences in computer databases by comparison of predicted mass spectra with experimentally-derived mass spectra using a closeness-of-fit measure. U.S. Pat. No. 5,926,818 discloses a multi-dimensional database comprising a functionality for multi-dimensional data analysis described as on-line analytical processing (OLAP), which entails the consolidation of projected and actual data according to more than one consolidation path or dimension. U.S. Pat. No. 5,295,261 reports a hybrid database structure in which the fields of each database record are divided into two classes, navigational and informational data, with navigational fields stored in a hierarchical topological map which can be viewed as a tree structure or as the merger of two or more such tree structures.
Fundamentals of bioinformatics are provided, e.g., in Mount, et al. (2001) Bioinformatics: Sequence and Genome Analysis CSH Press, NY; Durbin, et al. (eds. 1999) Biological Sequence Analysis: Probabilistic Models of Proteins and Nucleic Acids Cambridge Univ. Press; Baxevanis and Oeullette (eds. 1998) Bioinformatics: A Practical Guide to the Analysis of Genes and Proteins (2d ed.) Wiley-Liss; Rashidi and Buehler (1999) Bioinformatics: Basic Applications in Biological Science and Medicine CRC Press; Setubal, et al. (eds 1997) Introduction to Computational Molecular Biology Brooks/Cole; Misener and Krawetz (eds. 2000) Bioinformatics: Methods and Protocols Humana Press; Higgins and Taylor (eds. 2000) Bioinformatics: Sequence, Structure, and Databanks: A Practical Approach Oxford Univ. Press; Brown (2001) Bioinformatics: A Biologist's Guide to Biocomputing and the Internet Eaton Pub.; Han and Kamber (2000) Data Mining: Concepts and Techniques Kaufmann Pub.; and Waterman (1995) Introduction to Computational Biology: Maps, Sequences, and Genomes Chap and Hall.
The present invention provides a computer database comprising a computer and software for storing in computer-retrievable form assay data records cross-tabulated, e.g., with data specifying the source of the target-containing sample from which each sequence specificity record was obtained.
In an exemplary embodiment, at least one of the sources of target-containing sample is from a control tissue sample known to be free of pathological disorders. In a variation, at least one of the sources is a known pathological tissue specimen, e.g., a neoplastic lesion or another tissue specimen to be analyzed for ovarian cancer. In another variation, assay records cross-tabulate one or more of the following parameters for a target species in a sample: (1) a unique identification code, which can include, e.g., a target molecular structure and/or characteristic separation coordinate (e.g., electrophoretic or genomic position coordinates); (2) sample source; and (3) absolute and/or relative quantity of target species present in the sample.
The invention also provides for the storage and retrieval of a collection of target data in a computer data storage apparatus, which can include magnetic disks, optical disks, magneto-optical disks, DRAM, SRAM, SGRAM, SDRAM, RDRAM, DDR RAM, magnetic bubble memory devices, and other data storage devices, including CPU registers and on-CPU data storage arrays. Typically, the target data records are stored as a bit pattern in an array of magnetic domains on a magnetizable medium or as an array of charge states or transistor gate states, such as an array of cells in a DRAM device (e.g., each cell comprised of a transistor and a charge storage area, which may be on the transistor). In one embodiment, the invention provides such storage devices, and computer systems built therewith, comprising a bit pattern encoding a protein expression fingerprint record comprising unique identifiers for at least 10 target data records cross-tabulated with target source.
When the target is a peptide or nucleic acid, the invention preferably provides a method for identifying related peptide or nucleic acid sequences, comprising performing a computerized comparison between a peptide or nucleic acid sequence assay record stored in or retrieved from a computer storage device or database and at least one other sequence. The comparison can include a sequence analysis or comparison algorithm or computer program embodiment thereof (e.g., FASTA, TFASTA, GAP, BESTFIT) and/or the comparison may be of the relative amount of a peptide or nucleic acid sequence in a pool of sequences determined from a polypeptide or nucleic acid sample of a specimen.
The invention also preferably provides a magnetic disk, such as an IBM-compatible (DOS, Windows, Windows95/98/2000, Windows NT, OS/2) or other format (e.g., Linux, SunOS, Solaris, AIX, SCO Unix, VMS, MV, Macintosh, etc.) floppy diskette or hard (fixed, Winchester) disk drive, comprising a bit pattern encoding data from an assay of the invention in a file format suitable for retrieval and processing in a computerized sequence analysis, comparison, or relative quantitation method.
The invention also provides a network, comprising a plurality of computing devices linked via a data link, such as an Ethernet cable (coax or 10BaseT), telephone line, ISDN line, wireless network, optical fiber, or other suitable signal transmission medium, whereby at least one network device (e.g., computer, disk array, etc.) comprises a pattern of magnetic domains (e.g., magnetic disk) and/or charge domains (e.g., an array of DRAM cells) composing a bit pattern encoding data acquired from an assay of the invention.
The invention also provides a method for transmitting assay data that includes generating an electronic signal on an electronic communications device, such as a modem, ISDN terminal adapter, DSL, cable modem, ATM switch, or the like, wherein the signal includes (in native or encrypted format) a bit pattern encoding data from an assay or a database comprising a plurality of assay results obtained by the method of the invention.
In a preferred embodiment, the invention provides a computer system for comparing a query target to a database containing an array of data structures, such as an assay result obtained by the method of the invention, and ranking database targets based on the degree of identity and gap weight to the target data. A central processor is preferably initialized to load and execute the computer program for alignment and/or comparison of the assay results. Data for a query target is entered into the central processor via an I/O device. Execution of the computer program results in the central processor retrieving the assay data from the data file, which comprises a binary description of an assay result.
The target data or record and the computer program can be transferred to secondary memory, which is typically random access memory (e.g., DRAM, SRAM, SGRAM, or SDRAM). Targets are ranked according to the degree of correspondence between a selected assay characteristic (e.g., binding to a selected affinity moiety) and the same characteristic of the query target and results are output via an I/O device. For example, a central processor can be a conventional computer (e.g., Intel Pentium, PowerPC, Alpha, PA-8000, SPARC, MIPS 4400, MIPS 10000, VAX, etc.); a program can be a commercial or public domain molecular biology software package (e.g., UWGCG Sequence Analysis Software, Darwin); a data file can be an optical or magnetic disk, a data server, a memory device (e.g., DRAM, SRAM, SGRAM, SDRAM, EPROM, bubble memory, flash memory, etc.); an I/O device can be a terminal comprising a video display and a keyboard, a modem, an ISDN terminal adapter, an Ethernet port, a punched card reader, a magnetic strip reader, or other suitable I/O device.
The invention also preferably provides the use of a computer system, e.g., which typically comprises one or more of: (1) a computer; (2) a stored bit pattern encoding a collection of peptide sequence specificity records obtained by methods of the inventions, which may be stored in the computer; (3) a comparison target, such as a query target; and (4) a program for alignment and comparison, typically with rank-ordering of comparison results on the basis of computed similarity values.
Characteristics of Ovarian Cancer-Associated Proteins
Ovarian cancer proteins of the present invention may be categorized as secreted proteins, transmembrane proteins, or intracellular proteins. In one embodiment, the ovarian cancer protein is an intracellular protein. Intracellular proteins may be found in the cytoplasm and/or in the nucleus. Intracellular proteins are involved in all aspects of cellular function and replication (including, e.g., signaling pathways); aberrant expression of such proteins often results in unregulated or disregulated cellular processes. See, e.g., Alberts, et al. (eds. 1994) Molecular Biology of the Cell (3d ed.) Garland. For example, many intracellular proteins have enzymatic activity such as protein kinase activity, protein phosphatase activity, protease activity, nucleotide cyclase activity, polymerase activity, and the like. Intracellular proteins can also serve as docking proteins that are involved in organizing complexes of proteins, or targeting proteins to various subcellular localizations, and are often involved in maintaining the structural integrity of organelles.
An increasingly appreciated concept in characterizing proteins is the presence in the proteins of one or more structural motifs for which defined functions have been attributed. In addition to the highly conserved sequences found in the enzymatic domain of proteins, highly conserved sequences have been identified in proteins that are involved in protein-protein interaction. For example, Src-homology-2 (SH2) domains bind tyrosine-phosphorylated targets in a sequence dependent manner. PTB domains, which are distinct from SH2 domains, also bind tyrosine phosphorylated targets. SH3 domains bind to proline-rich targets. In addition, PH domains, tetratricopeptide repeats and WD domains to name only a few, have been shown to mediate protein-protein interactions. Some of these may also be involved in binding to phospholipids or other second messengers. As will be appreciated by one of ordinary skill in the art, these motifs can be identified on the basis of amino acid sequence; thus, an analysis of the sequence of proteins may provide insight into both the enzymatic potential of the molecule and/or molecules with which the protein may associate. One useful database is Pfam (protein families), which is a large collection of multiple sequence alignments and hidden Markov models covering many common protein domains. Versions are available via the internet from Washington University in St. Louis, the Sanger Center in England, and the Karolinska Institute in Sweden. See, e.g., Bateman, et al. (2000) Nuc. Acids Res. 28:263-266; Sonnhammer, et al. (1997) Proteins 28:405-420; Bateman, et al. (1999) Nuc. Acids Res. 27:260-262; and Sonnhammer, et al. (1998) Nuc. Acids Res. 26:320-322.
In another preferred embodiment, the ovarian cancer sequences are transmembrane proteins. Transmembrane proteins are molecules that span a phospholipid bilayer of a cell. They may have an intracellular domain, an extracellular domain, or both. The intracellular domains of such proteins may have a number of functions including those already described for intracellular proteins. For example, the intracellular domain may have enzymatic activity and/or may serve as a binding site for additional proteins. Frequently the intracellular domain of transmembrane proteins serves both roles. For example certain receptor tyrosine kinases have both protein kinase activity and SH2 domains. In addition, autophosphorylation of tyrosines on the receptor molecule itself, creates binding sites for additional SH2 domain containing proteins.
Transmembrane proteins may contain from one to many transmembrane domains. For example, receptor tyrosine kinases, certain cytokine receptors, receptor guanylyl cyclases and receptor serine/threonine protein kinases contain a single transmembrane domain. However, various other proteins including channels and adenylyl cyclases contain numerous transmembrane domains. Many important cell surface receptors such as G protein coupled receptors (GPCRs) are classified as “seven transmembrane domain” proteins, as they contain 7 membrane spanning regions. Characteristics of transmembrane domains include approximately 17 consecutive hydrophobic amino acids that may be followed by charged amino acids. Therefore, upon analysis of the amino acid sequence of a particular protein, the localization and number of transmembrane domains within the protein may be predicted (see, e.g., PSORT web site located at psort.nibb.ac.jp). Important transmembrane protein receptors include, but are not limited to the insulin receptor, insulin-like growth factor receptor, human growth hormone receptor, glucose transporters, transferrin receptor, epidermal growth factor receptor, low density lipoprotein receptor, epidermal growth factor receptor, leptin receptor, interleukin receptors, e.g., IL-1 receptor, IL-2 receptor, etc.
The extracellular domains of transmembrane proteins are diverse; however, conserved motifs are found repeatedly among various extracellular domains. Conserved structure and/or functions have been ascribed to different extracellular motifs. Many extracellular domains are involved in binding to other molecules. In one aspect, extracellular domains are found on receptors. Factors that bind the receptor domain include circulating ligands, which may be peptides, proteins, or small molecules such as adenosine and the like. For example, growth factors such as EGF, FGF, and PDGF are circulating growth factors that bind to their cognate receptors to initiate a variety of cellular responses. Other factors include cytokines, mitogenic factors, neurotrophic factors and the like. Extracellular domains also bind to cell-associated molecules, or may be processed or shed to the blood stream. In this respect, they can mediate cell-cell interactions. Cell-associated ligands can be tethered to the cell, e.g., via a glycosylphosphatidylinositol (GPI) anchor, or may themselves be transmembrane proteins. Extracellular domains also associate with the extracellular matrix and contribute to the maintenance of the cell structure.
Ovarian cancer proteins that are transmembrane are particularly preferred in the present invention as they are readily accessible targets for immunotherapeutics, as are described herein. In addition, as outlined below, transmembrane proteins can be also useful in imaging modalities. Antibodies may be used to label such readily accessible proteins in situ. Alternatively, antibodies can also label intracellular proteins, in which case samples are typically permeablized to provide access to intracellular proteins. In addition, some membrane proteins can be processed to release a soluble protein, or to expose a residual fragment. Released soluble proteins may be useful diagnostic markers, processed residual protein fragments may be useful ovarian markers of disease.
It will also be appreciated by those in the art that a transmembrane protein can be made soluble by removing transmembrane sequences, e.g., through recombinant methods. Furthermore, transmembrane proteins that have been made soluble can be made to be secreted through recombinant means by adding an appropriate signal sequence.
In another embodiment, the ovarian cancer proteins are secreted proteins; the secretion of which can be either constitutive or regulated. These proteins may have a signal peptide or signal sequence that targets the molecule to the secretory pathway. Secreted proteins are involved in numerous physiological events; e.g., if circulating, they often serve to transmit signals to various other cell types. The secreted protein may function in an autocrine manner (acting on the cell that secreted the factor), a paracrine manner (acting on cells in close proximity to the cell that secreted the factor), an endocrine manner (acting on cells at a distance, e.g., secretion into the blood stream), or exocrine (secretion, e.g., through a duct or to an adjacent epithelial surface as sweat glands, sebaceous glands, pancreatic ducts, lacrimal glands, mammary glands, wax producing glands of the ear, etc.). Thus, secreted molecules often find use in modulating or altering numerous aspects of physiology. Ovarian cancer proteins that are secreted proteins are particularly preferred as good diagnostic markers, e.g., for blood, plasma, serum, or stool tests. Those which are enzymes may be antibody or small molecule therapeutic targets. Others may be useful as vaccine targets, e.g., via CTL mechanisms, as protein or DNA vaccines.
Use of Ovarian Cancer Nucleic Acids
As described above, ovarian cancer sequence is initially identified by substantial nucleic acid and/or amino acid sequence homology or linkage to the ovarian cancer sequences outlined herein. Such homology can be based upon the overall nucleic acid or amino acid sequence, and is generally determined as outlined below, using either homology programs or hybridization conditions. Typically, linked sequences on a mRNA are found on the same molecule.
The ovarian cancer nucleic acid sequences of the invention, e.g., in Table 19A, can be fragments of larger genes, e.g., they are nucleic acid segments. “Genes” in this context includes coding regions, non-coding regions, and mixtures of coding and non-coding regions. Accordingly, as will be appreciated by those in the art, using the sequences provided herein, extended sequences, in either direction, of the ovarian cancer genes can be obtained, using techniques well known in the art for cloning either longer sequences or the full length sequences; see Ausubel, et al., supra. Much can be done by informatics and many sequences can be clustered to include multiple sequences corresponding to a single gene, e.g., systems such as UniGene (see, www.ncbi.nlm.nih.gov/UniGene).
Once the ovarian cancer nucleic acid is identified, it can be cloned and, if necessary, its constituent parts recombined to form the entire ovarian cancer nucleic acid coding regions or the entire mRNA sequence. Once isolated from its natural source, e.g., contained within a plasmid or other vector or excised as a linear nucleic acid segment, the recombinant ovarian cancer nucleic acid can be further-used as a probe to identify and isolate other ovarian cancer nucleic acids, e.g., extended coding regions. It can also be used as a “precursor” nucleic acid to make modified or variant ovarian cancer nucleic acids and proteins.
The ovarian cancer nucleic acids of the present invention are useful in several ways. In a first embodiment, nucleic acid probes to the ovarian cancer nucleic acids are made and attached to biochips to be used in screening and diagnostic methods, as outlined below, or for administration, e.g., for gene therapy, vaccine, RNAi, and/or antisense applications. Alternatively, the ovarian cancer nucleic acids that include coding regions of ovarian cancer proteins can be put into expression vectors for the expression of ovarian cancer proteins, again for screening purposes or for administration to a patient.
In a preferred embodiment, nucleic acid probes to ovarian cancer nucleic acids (both the nucleic acid sequences outlined in the figures and/or the complements thereof) are made. The nucleic acid probes attached to the biochip are designed to be substantially complementary to the ovarian cancer nucleic acids, e.g., the target sequence (either the target sequence of the sample or to other probe sequences, e.g., in sandwich assays), such that hybridization of the target sequence and the probes of the present invention occurs. As outlined below, this complementarity need not be perfect; there may be any number of base pair mismatches which will interfere with hybridization between the target sequence and the single stranded nucleic acids of the present invention. However, if the number of mutations is so great that no hybridization can occur under even the least stringent of hybridization conditions, the sequence is not a complementary target sequence. Thus, by “substantially complementary” herein is meant that the probes are sufficiently complementary to the target sequences to hybridize under normal reaction conditions, particularly high stringency conditions, as outlined herein.
A nucleic acid probe is generally single stranded but can be partially single and partially double stranded. The strandedness of the probe is dictated by the structure, composition, and properties of the target sequence. In general, the nucleic acid probes range from about 8 to about 100 bases long, with from about 10 to about 80 bases being preferred, and from about 30 to about 50 bases being particularly preferred. That is, generally whole genes are not used. In some embodiments, much longer nucleic acids can be used, up to hundreds of bases.
In a preferred embodiment, more than one probe per sequence is used, with either overlapping probes or probes to different sections of the target being used. That is, two, three, four or more probes, with three being preferred, are used to build in a redundancy for a particular target. The probes can be overlapping (e.g., have some sequence in common), or separate. In some cases, PCR primers may be used to amplify signal for higher sensitivity.
As will be appreciated by those in the art, nucleic acids can be attached or immobilized to a solid support in a wide variety of ways. By “immobilized” and grammatical equivalents herein is meant the association or binding between the nucleic acid probe and the solid support is sufficient to be stable under the conditions of binding, washing, analysis, and removal as outlined below. The binding can typically be covalent or non-covalent. By “non-covalent binding” and grammatical equivalents herein is meant one or more of electrostatic, hydrophilic, and hydrophobic interactions. Included in non-covalent binding is the covalent attachment of a molecule, such as, streptavidin to the support and the non-covalent binding of the biotinylated probe to the streptavidin. By “covalent binding” and grammatical equivalents herein is meant that the two moieties, the solid support and the probe, are attached by at least one bond, including sigma bonds, pi bonds and coordination bonds. Covalent bonds can be formed directly between the probe and the solid support or can be formed by a cross linker or by inclusion of a specific reactive group on either the solid support or the probe or both molecules. Immobilization may also involve a combination of covalent and non-covalent interactions.
In general, the probes are attached to the biochip in a wide variety of ways, as will be appreciated by those in the art. As described herein, the nucleic acids can either be synthesized first, with subsequent attachment to the biochip, or can be directly synthesized on the biochip.
The biochip comprises a suitable solid substrate. By “substrate” or “solid support” or other grammatical equivalents herein is meant a material that can be modified to contain discrete individual sites appropriate for the attachment or association of the nucleic acid probes and is amenable to at least one detection method. As will be appreciated by those in the art, the number of possible substrates are very large, and include, but are not limited to, glass and modified or functionalized glass, plastics (including acrylics, polystyrene and copolymers of styrene and other materials, polypropylene, polyethylene, polybutylene, polyurethanes, TeflonJ, etc.), polysaccharides, nylon or nitrocellulose, resins, silica or silica-based materials including silicon and modified silicon, carbon, metals, inorganic glasses, plastics, etc. In general, the substrates allow optical detection and do not appreciably fluoresce. See, e.g., WO0055627 Reusable Low Fluorescent Plastic Biochip.
Generally the substrate is planar, although as will be appreciated by those in the art, other configurations of substrates may be used as well. For example, the probes may be placed on the inside surface of a tube, for flow-through sample analysis to minimize sample volume. Similarly, the substrate may be flexible, such as a flexible foam, including closed cell foams made of particular plastics.
In a preferred embodiment, the surface of the biochip and the probe may be derivatized with chemical functional groups for subsequent attachment of the two. Thus, e.g., the biochip is derivatized with a chemical functional group including, but not limited to, amino groups, carboxyl groups, oxo groups and thiol groups, with amino groups being particularly preferred. Using these functional groups, the probes can be attached using functional groups on the probes. For example, nucleic acids containing amino groups can be attached to surfaces comprising amino groups, e.g., using linkers as are known in the art; e.g., homo-or hetero-bifunctional linkers as are well known (see 1994 Pierce Chemical Company catalog, technical section on cross-linkers, pages 155-200). In addition, in some cases, additional linkers, such as alkyl groups (including substituted and heteroalkyl groups) may be used.
In this embodiment, oligonucleotides are synthesized as is known in the art, and then attached to the surface of the solid support. As will be appreciated by those skilled in the art, either the 5′ or 3′ terminus may be attached to the solid support, or attachment may be via an internal nucleoside.
In another embodiment, the immobilization to the solid support may be very strong, yet non-covalent. For example, biotinylated oligonucleotides can be made, which bind to surfaces covalently coated with streptavidin, resulting in attachment.
Alternatively, the oligonucleotides may be synthesized on the surface, as is known in the art. For example, photoactivation techniques utilizing photopolymerization compounds and techniques are used. In a preferred embodiment, the nucleic acids can be synthesized in situ, using well known photolithographic techniques, such as those described in WO 95/25116; WO 95/35505; U.S. Pat. Nos. 5,700,637 and 5,445,934; and references cited within, all of which are expressly incorporated by reference; these methods of attachment form the basis of the Affymetrix GeneChipTM technology.
Often, amplification-based assays are performed to measure the expression level of ovarian cancer-associated sequences. These assays are typically performed in conjunction with reverse transcription. In such assays, an ovarian cancer-associated nucleic acid sequence acts as a template in an amplification reaction (e.g., Polymerase Chain Reaction, or PCR). In a quantitative amplification, the amount of amplification product will be proportional to the amount of template in the original sample. Comparison to appropriate controls provides a measure of the amount of ovarian cancer-associated RNA. Methods of quantitative amplification are well known to those of skill in the art. Detailed protocols for quantitative PCR are available. See, e.g., Innis, et al. (1990) PCR Protocols: A Guide to Methods and Applications Academic Press.
In some embodiments, a TaqMan based assay is used to measure expression. TaqMan based assays use a fluorogenic oligonucleotide probe that contains a 5′ fluorescent dye and a 3′ quenching agent. The probe hybridizes to a PCR product, but cannot itself be extended due to a blocking agent at the 3′ end. When the PCR product is amplified in subsequent cycles, the 5′ nuclease activity of the polymerase, e.g., AmpliTaq, results in the cleavage of the TaqMan probe. This cleavage separates the 5′ fluorescent dye and the 3′ quenching agent, thereby resulting in an increase in fluorescence as a function of amplification (see, e.g., literature provided by Perkin-Elmer, e.g., www2.perkin-elmer.com).
Other suitable amplification methods include, but are not limited to, ligase chain reaction (LCR; see Wu and Wallace (1989) Genomics 4:560-569; Landegren, et al. (1988) Science 241:1077-1980; and Barringer, et al. (1990) Gene 89:117-122), transcription amplification (Kwoh, et al. (1989) Proc. Nat'l Acad. Sci. USA 86:1173-1177), self-sustained sequence replication (Guatelli, et al. (1990) Proc. Nat'l Acad. Sci. USA 87:1874-1878), dot PCR, linker adapter PCR, etc.
Expression of Ovarian Cancer Proteins from Nucleic Acids
In a preferred embodiment, ovarian cancer nucleic acids, e.g., encoding ovarian cancer proteins are used to make a variety of expression vectors to express ovarian cancer proteins which can then be used in screening assays, as described below. Expression vectors and recombinant DNA technology are well known and are used to express proteins. See, e.g., Ausubel, supra; and Fernandez and Hoeffler (eds. 1999) Gene Expression Systems Academic Press. The expression vectors may be either self-replicating extrachromosomal vectors or vectors which integrate into a host genome. Generally, these expression vectors include transcriptional and translational regulatory nucleic acid operably linked to the nucleic acid encoding the ovarian cancer protein. The term “control sequences” refers to DNA sequences used for the expression of an operably linked coding sequence in a particular host organism. Control sequences that are suitable for prokaryotes, e.g., include a promoter, optionally an operator sequence, and a ribosome binding site. Eukaryotic cells are known to utilize promoters, polyadenylation signals, and enhancers.
Nucleic acid is “operably linked” when it is placed into a functional relationship with another nucleic acid sequence. For example, DNA for a pre-sequence or secretory leader is operably linked to DNA for a polypeptide if it is expressed as a pre-protein that participates in the secretion of the polypeptide; a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence; a ribosome binding site is operably linked to a coding sequence if it is positioned so as to facilitate translation; and two sequences may be operably linked when they are physically part of the same polymer. Generally, “operably linked” means that the DNA sequences being linked are contiguous, and, in the case of a secretory leader, contiguous and in reading phase. However, enhancers do not have to be contiguous. Linking is typically accomplished by ligation at convenient restriction sites. If such sites do not exist, synthetic oligonucleotide adaptors or linkers are used in accordance with conventional practice. Transcriptional and translational regulatory nucleic acid will generally be appropriate to the host cell used to express the ovarian cancer protein. Numerous types of appropriate expression vectors, and suitable regulatory sequences are known in the art for a variety of host cells.
In general, transcriptional and translational regulatory sequences may include, but are not limited to, promoter sequences, ribosomal binding sites, transcriptional start and stop sequences, translational start and stop sequences, and enhancer or activator sequences. In a preferred embodiment, the regulatory sequences include a promoter and transcriptional start and stop sequences.
Promoter sequences typically encode constitutive or inducible promoters. The promoters may be naturally occurring promoters or hybrid promoters. Hybrid promoters, which combine elements of more than one promoter, are also known in the art, and are useful in the present invention.
In addition, an expression vector may comprise additional elements. For example, the expression vector may have two replication systems, thus allowing it to be maintained in two organisms, e.g., in mammalian or insect cells for expression and in a procaryotic host for cloning and amplification. Furthermore, for integrating expression vectors, the expression vector contains at least one sequence homologous to the host cell genome, and preferably two homologous sequences which flank the expression construct. The integrating vector may be directed to a specific locus in the host cell by selecting the appropriate homologous sequence for inclusion in the vector. Constructs for integrating vectors are available. See, e.g., Fernandez and Hoeffler, supra.
In addition, in a preferred embodiment, the expression vector contains a selectable marker gene to allow the selection of transformed host cells. Selection genes are well known in the art and will vary with the host cell used.
The ovarian cancer proteins of the present invention are produced by culturing a host cell transformed with an expression vector containing nucleic acid encoding an ovarian cancer protein, under the appropriate conditions to induce or cause expression of the ovarian cancer protein. Conditions appropriate for ovarian cancer protein expression will vary with the choice of the expression vector and the host cell, and will be easily ascertained by one skilled in the art through routine experimentation or optimization. For example, the use of constitutive promoters in the expression vector will require optimizing the growth and proliferation of the host cell, while the use of an inducible promoter requires the appropriate growth conditions for induction. In addition, in some embodiments, the timing of the harvest is important. For example, the baculovirus systems used in insect cell expression are lytic viruses, and thus harvest time selection can be crucial for product yield.
Appropriate host cells include yeast, bacteria, archaebacteria, fungi, and insect and animal cells, including mammalian cells. Of particular interest are Saccharomyces cerevisiae and other yeasts, E. coli, Bacillus subtilis, Sf9 cells, C129 cells, 293 cells, Neurospora, BHK, CHO, COS, HeLa cells, HUVEC (human umbilical vein endothelial cells), THP1 cells (a macrophage cell line) and various other human cells and cell lines.
In a preferred embodiment, the ovarian cancer proteins are expressed in mammalian cells. Mammalian expression systems are also known in the art, and include retroviral and adenoviral systems. One expression vector system is a retroviral vector system such as is generally described in PCT/US97/01019 and PCT/US97/01048, both of which are hereby expressly incorporated by reference. Of particular use as mammalian promoters are the promoters from mammalian viral genes, since the viral genes are often highly expressed and have a broad host range. Examples include the SV40 early promoter, mouse mammary tumor virus LTR promoter, adenovirus major late promoter, herpes simplex virus promoter, and the CMV promoter. See, e.g., Fernandez and Hoeffler, supra. Typically, transcription termination and polyadenylation sequences recognized by mammalian cells are regulatory regions located 3′ to the translation stop codon and thus, together with the promoter elements, flank the coding sequence. Examples of transcription terminator and polyadenylation signals include those derived form SV40.
The methods of introducing exogenous nucleic acid into mammalian hosts, as well as other hosts, is well known in the art, and will vary with the host cell used. Techniques include dextran-mediated transfection, calcium phosphate precipitation, polybrene mediated transfection, protoplast fusion, electroporation, viral infection, encapsulation of the polynucleotide(s) in liposomes, and direct microinjection of the DNA into nuclei.
In a preferred embodiment, ovarian cancer proteins are expressed in bacterial systems. Bacterial expression systems are well known in the art. Promoters from bacteriophage may also be used and are known in the art. In addition, synthetic promoters and hybrid promoters are also useful; e.g., the tac promoter is a hybrid of the trp and lac promoter sequences. Furthermore, a bacterial promoter can include naturally occurring promoters of non-bacterial origin that have the ability to bind bacterial RNA polymerase and initiate transcription. In addition to a functioning promoter sequence, an efficient ribosome binding site is desirable. The expression vector may also include a signal peptide sequence that provides for secretion of the ovarian cancer protein in bacteria. The protein is either secreted into the growth media (gram-positive bacteria) or into the periplasmic space, located between the inner and outer membrane of the cell (gram-negative bacteria). The bacterial expression vector may also include a selectable marker gene to allow for the selection of bacterial strains that have been transformed. Suitable selection genes include genes which render the bacteria resistant to drugs such as ampicillin, chloramphenicol, erythromycin, kanamycin, neomycin, and tetracycline. Selectable markers also include biosynthetic genes, such as those in the histidine, tryptophan, and leucine biosynthetic pathways. These components are assembled into expression vectors. Expression vectors for bacteria are well known in the art, and include vectors for Bacillus subtilis, E. coli, Streptococcus cremoris, and Streptococcus lividans, among others. See Fernandez and Hoeffler, supra. The bacterial expression vectors are transformed into bacterial host cells using techniques well known in the art, such as calcium chloride treatment, electroporation, and others.
In one embodiment, ovarian cancer proteins are produced in insect cells. Expression vectors for the transformation of insect cells, and in particular, baculovirus-based expression vectors, are well known in the art.
In a preferred embodiment, an ovarian cancer protein is produced in yeast cells. Yeast expression systems are well known in the art, and include expression vectors for Saccharomyces cerevisiae, Candida albicans and C. maltosa, Hansenula polymorpha, Kluyveromyces fragilis and K. lactis, Pichia guillerimondii and P. pastoris, Schizosaccharomyces pombe, and Yarrowia lipolytica. 
The ovarian cancer protein may also be made as a fusion protein, using techniques well known in the art. Thus, e.g., for the creation of monoclonal antibodies, if the desired epitope is small, the ovarian cancer protein may be fused to a carrier protein to form an immunogen. Alternatively, the ovarian cancer protein may be made as a fusion protein to increase expression, or for other reasons. For example, when the ovarian cancer protein is an ovarian cancer peptide, the nucleic acid encoding the peptide may be linked to other nucleic acid for expression purposes.
In a preferred embodiment, the ovarian cancer protein is purified or isolated after expression. Ovarian cancer proteins may be isolated or purified in a variety of ways known to those skilled in the art depending on what other components are present in the sample. Standard purification methods include electrophoretic, molecular, immunological and chromatographic techniques, including ion exchange, hydrophobic, affinity, and reverse-phase HPLC chromatography, and chromatofocusing. For example, the ovarian cancer protein may be purified using a standard anti-ovarian cancer protein antibody column. Ultrafiltration and diafiltration techniques, in conjunction with protein concentration, are also useful. For general guidance in suitable purification techniques, see Scopes (1982) Protein Purification Springer-Verlag. The degree of purification necessary will vary depending on the use of the ovarian cancer protein. In some instances no purification will be necessary.
Once expressed and purified if necessary, the ovarian cancer proteins and nucleic acids are useful in a number of applications. They may be used as immunoselection reagents, as vaccine reagents, as screening agents, etc.
Variants of Ovarian Cancer Proteins
In one embodiment, the ovarian cancer proteins are derivative or variant ovarian cancer proteins as compared to the wild-type sequence. That is, as outlined more fully below, the derivative ovarian cancer peptide will often contain at least one amino acid substitution, deletion or insertion, with amino acid substitutions being particularly preferred. The amino acid substitution, insertion, or deletion may occur at most any residue within the ovarian cancer peptide.
Also included within one embodiment of ovarian cancer proteins of the present invention are amino acid sequence variants. These variants typically fall into one or more of three classes: substitutional, insertional or deletional variants. These variants ordinarily are prepared by site specific mutagenesis of nucleotides in the DNA encoding the ovarian cancer protein, using cassette or PCR mutagenesis or other techniques well known in the art, to produce DNA encoding the variant, and thereafter expressing the DNA in recombinant cell culture as outlined above. However, variant ovarian cancer protein fragments having up to about 100-150 residues may be prepared by in vitro synthesis using established techniques. Amino acid sequence variants are characterized by the predetermined nature of the variation, a feature that sets them apart from naturally occurring allelic or interspecies variation of the ovarian cancer protein amino acid sequence. The variants typically exhibit the same qualitative biological activity as the naturally occurring analogue, although variants can also be selected which have modified characteristics as will be more fully outlined below.
While the site or region for introducing an amino acid sequence variation is predetermined, the mutation per se need not be predetermined. For example, in order to optimize the performance of a mutation at a given site, random mutagenesis may be conducted at the target codon or region and the expressed ovarian cancer variants screened for the optimal combination of desired activity. Techniques for making substitution mutations at predetermined sites in DNA having a known sequence are well known, e.g., M13 primer mutagenesis and PCR mutagenesis. Screening of the mutants is done using assays of ovarian cancer protein activities.
Amino acid substitutions are typically of single residues; insertions usually will be on the order of from about 1 to 20 amino acids, although considerably larger insertions may be tolerated. Deletions range from about 1 to about 20 residues, although in some cases deletions may be much larger.
Substitutions, deletions, insertions or any combination thereof may be used to arrive at a final derivative. Generally these changes are done on a few amino acids to minimize the alteration of the molecule. However, larger changes may be tolerated in certain circumstances. When small alterations in the characteristics of the ovarian cancer protein are desired, substitutions are generally made in accordance with the amino acid substitution relationships provided in the definition section.
The variants typically exhibit the same qualitative biological activity and will elicit the same immune response as the naturally-occurring analog, although variants also are selected to modify the characteristics of the ovarian cancer proteins as needed. Alternatively, the variant may be designed such that the biological activity of the ovarian cancer protein is altered. For example, glycosylation sites may be altered or removed.
Substantial changes in function or immunological identity are made by selecting substitutions that are less conservative than those described above. For example, substitutions may be made which more significantly affect: the structure of the polypeptide backbone in the area of the alteration, for example the alpha-helical or beta-sheet structure; the charge or hydrophobicity of the molecule at the target site; or the bulk of the side chain. The substitutions which in general are expected to produce the greatest changes in the polypeptide's properties are those in which (a) a hydrophilic residue, e.g., serine or threonine is substituted for (or by) a hydrophobic residue, e.g., leucine, isoleucine, phenylalanine, valine, or alanine; (b) a cysteine or proline is substituted for (or by) any other residue; (c) a residue having an electropositive side chain, e.g., lysine, arginine, or histidine, is substituted for (or by) an electronegative residue, e.g., glutamic or aspartic acid; (d) a residue having a bulky side chain, e.g., phenylalanine, is substituted for (or by) one not having a side chain, e.g., glycine; or (e) a proline residue is incorporated or substituted, which changes the degree of rotational freedom of the peptidyl bond.
Covalent modifications of ovarian cancer polypeptides are included within the scope of this invention. One type of covalent modification includes reacting targeted amino acid residues of an ovarian cancer polypeptide with an organic derivatizing agent that is capable of reacting with selected side chains or the N-or C-terminal residues of an ovarian cancer polypeptide. Derivatization with bifunctional agents is useful, for instance, for crosslinking ovarian cancer polypeptides to a water-insoluble support matrix or surface for use in the method for purifying anti-ovarian cancer polypeptide antibodies or screening assays, as is more fully described below. Commonly used crosslinking agents include, e.g., 1,1-bis(diazoacetyl)-2-phenylethane, glutaraldehyde, N-hydroxysuccinimide esters, e.g., esters with 4-azidosalicylic acid, homobifunctional imidoesters, including disuccinimidyl esters such as 3,3′-dithiobis(succinimidylpropionate), bifunctional maleimides such as bis-N-maleimido-1,8-octane and agents such as methyl-3-((p-azidophenyl)dithio)propioimidate.
Other modifications include deamidation of glutamine and asparagine residues to the corresponding glutamic and aspartic acid residues, respectively, hydroxylation of proline and lysine, phosphorylation of hydroxyl groups of serine, threonine, or tyrosine residues, methylation of the amino groups of the lysine, arginine, and histidine side chains (e.g., pp. 79-86, Creighton (1983) Proteins: Structure and Molecular Properties Freeman), acetylation of the N-terminal amine, and amidation of a C-terminal carboxyl group.
Another type of covalent modification of the ovarian cancer polypeptide included within the scope of this invention comprises altering the native glycosylation pattern of the polypeptide. “Altering the native glycosylation pattern” is intended for purposes herein to mean deleting one or more carbohydrate moieties found in native sequence ovarian cancer polypeptide, and/or adding one or more glycosylation sites that are not present in the native sequence ovarian cancer polypeptide. Glycosylation patterns can be altered in many ways. For example the use of different cell types to express ovarian cancer-associated sequences can result in different glycosylation patterns.
Addition of glycosylation sites to ovarian cancer polypeptides may also be accomplished by altering the amino acid sequence thereof. The alteration may be made, e.g., by the addition of, or substitution by, one or more serine or threonine residues to the native sequence ovarian cancer polypeptide (for O-linked glycosylation sites). The ovarian cancer amino acid sequence may optionally be altered through changes at the DNA level, particularly by mutating the DNA encoding the ovarian cancer polypeptide at pre-selected bases such that codons are generated that will translate into the desired amino acids.
Another means of increasing the number of carbohydrate moieties on the ovarian cancer polypeptide is by chemical or enzymatic coupling of glycosides to the polypeptide. See, e.g., WO 87/05330, and pp. 259-306 in Aplin and Wriston (1981) CRC Crit. Rev. Biochem. CRC Press.
Removal of carbohydrate moieties present on the ovarian cancer polypeptide may be accomplished chemically or enzymatically or by mutational substitution of codons encoding for amino acid residues that serve as targets for glycosylation. Chemical deglycosylation techniques are applicable. See, e.g., Sojar and Bahl (1987) Arch. Biochem. Biophys. 259:52-57; and Edge, et al. (1981) Anal. Biochem. 118:131-137. Enzymatic cleavage of carbohydrate moieties on polypeptides can be achieved by the use of a variety of endo-and exo-glycosidases. See, e.g., Thotakura, et al. (1987) Meth. Enzymol., 138:350-359.
Another type of covalent modification of ovarian cancer comprises linking the ovarian cancer polypeptide to one of a variety of non-proteinaceous polymers, e.g., polyethylene glycol, polypropylene glycol, or polyoxyalkylene. See, e.g., U.S. Pat. No. 4,640,835; 4,496,689; 4,301,144; 4,670,417; 4,791,192; or 4,179,337.
Ovarian cancer polypeptides of the present invention may also be modified in a way to form chimeric molecules, e.g., comprising an ovarian cancer polypeptide fused to another heterologous polypeptide or amino acid sequence. In one embodiment, such a chimeric molecule comprises a fusion of an ovarian cancer polypeptide with a tag polypeptide which provides an epitope to which an anti-tag antibody can selectively bind. The epitope tag is generally placed at the amino-or carboxyl-terminus of the ovarian cancer polypeptide. The presence of such epitope-tagged forms of an ovarian cancer polypeptide can be detected using an antibody against the tag polypeptide. Also, provision of the epitope tag enables the ovarian cancer polypeptide to be readily purified by affinity purification using an anti-tag antibody or another type of affinity matrix that binds to the epitope tag. In an alternative embodiment, the chimeric molecule may comprise a fusion of an ovarian cancer polypeptide with an immunoglobulin or a particular region of an immunoglobulin. For a bivalent form of the chimeric molecule, such a fusion could be to the Fc region of an IgG molecule.
Various tag polypeptides and their respective antibodies are well known in the art. Examples include poly-histidine (poly-his) or poly-histidine-glycine (poly-his-gly) tags; His6 and metal chelation tags, the flu HA tag polypeptide and its antibody 12CA5 (Field, et al. (1988) Mol. Cell. Biol. 8:2159-2165); the c-myc tag and the 8F9, 3C7, 6E10, G4, B7, and 9E10 antibodies thereto (Evan, et al. (1985) Mol. Cell. Biol. 5:3610-3616); and the Herpes Simplex virus glycoprotein D (gD) tag and its antibody (Paborsky, et al. (1990) Protein Engineering 3:547-553). Other tag polypeptides include, e.g., the Flag-peptide (Hopp, et al. (1988) BioTechnology 6:1204-1210); the KT3 epitope peptide (Martin, et al. (1992) Science 255:192-194); tubulin epitope peptide (Skinner, et al. (1991) J. Biol. Chem. 266:15163-15166); and the T7 gene 10 protein peptide tag (Lutz-Freyermuth et al. (1990) Proc. Nat'l Acad. Sci. USA 87:6393-6397).
Also included are other ovarian cancer proteins of the ovarian cancer family, and ovarian cancer proteins from other organisms, which are cloned and expressed as outlined below. Thus, probe or degenerate polymerase chain reaction (PCR) primer sequences may be used to find other related ovarian cancer proteins from humans or other organisms. As will be appreciated by those in the art, particularly useful probe and/or PCR primer sequences include the unique areas of the ovarian cancer nucleic acid sequence. As is generally known in the art, preferred PCR primers are from about 15 to about 35 nucleotides in length, with from about 20 to about 30 being preferred, and may contain inosine as needed. The conditions for the PCR reaction are well known in the art (e.g., Innis, PCR Protocols, supra).
Antibodies to Ovarian Cancer Proteins
In a preferred embodiment, when the ovarian cancer protein is to be used to generate antibodies, e.g., for immunotherapy or immunodiagnosis, the ovarian cancer protein should share at least one epitope or determinant with the full length protein. By “epitope” or “determinant” herein is typically meant a portion of a protein which will generate and/or bind an antibody or T-cell receptor in the context of MHC. Thus, in most instances, antibodies made to a smaller ovarian cancer protein will be able to bind to the full-length protein, particularly linear epitopes. In a preferred embodiment, the epitope is unique; that is, antibodies generated to a unique epitope show little or no cross-reactivity.
Methods of preparing polyclonal antibodies are known to the skilled artisan (e.g., Coligan, supra; and Harlow and Lane, supra). Polyclonal antibodies can be raised in a mammal, e.g., by one or more injections of an immunizing agent and, if desired, an adjuvant. Typically, the immunizing agent and/or adjuvant will be injected in the mammal by multiple subcutaneous or intraperitoneal injections. The immunizing agent may include a protein encoded by a nucleic acid of the figures or fragment thereof or a fusion protein thereof. It may be useful to conjugate the immunizing agent to a 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. Examples of adjuvants which may be employed include Freund's complete adjuvant and MPL-TDM adjuvant (monophosphoryl Lipid A, synthetic trehalose dicorynomycolate). The immunization protocol may be selected by one skilled in the art without undue experimentation.
The antibodies may, alternatively, be monoclonal antibodies. Monoclonal antibodies may be prepared using hybridoma methods, such as those described by Kohler and Milstein (1975) Nature 256:495-497. 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 may be immunized in vitro. The immunizing agent will typically include a polypeptide encoded by a nucleic acid of Table 19A or fragment thereof, or a fusion protein thereof. Generally, either peripheral blood lymphocytes (“PBLs”) 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 (e.g., pp. 59-103 in Goding (1986) Monoclonal Antibodies: Principles and Practice Academic Press). 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 may 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.
In one embodiment, the antibodies are bispecific antibodies. Bispecific antibodies are monoclonal, preferably human or humanized, antibodies that have binding specificities for at least two different antigens or that have binding specificities for two epitopes on the same antigen. In one embodiment, one of the binding specificities is for a protein encoded by a nucleic acid Table 1-20 or a fragment thereof, the other one is for any other antigen, and preferably for a cell-surface protein or receptor or receptor subunit, preferably one that is tumor specific. Alternatively, tetramer-type technology may create multivalent reagents.
In a preferred embodiment, the antibodies to ovarian cancer protein are capable of reducing or eliminating a biological function of an ovarian cancer protein, as is described below. That is, the addition of anti-ovarian cancer protein antibodies (either polyclonal or preferably monoclonal) to ovarian cancer tissue (or cells containing ovarian cancer) may reduce or eliminate the ovarian cancer. Generally, at least a 25% decrease in activity, growth, size or the like is preferred, with at least about 50% being particularly preferred and about a 95-100% decrease being especially preferred.
In a preferred embodiment the antibodies to the ovarian cancer proteins are humanized antibodies (e.g., Xenerex Biosciences; Medarex, Inc.; Abgenix, Inc.; Protein Design Labs, Inc.) Humanized forms of non-human (e.g., murine) antibodies are chimeric molecules of immunoglobulins, immunoglobulin chains or fragments thereof (such as Fv, Fab, Fab′, F(ab′)2 or other antigen-binding subsequences of antibodies) which contain minimal sequence derived from non-human immunoglobulin. Humanized antibodies include human immunoglobulins (recipient antibody) in which residues from a complementary determining region (CDR) of the recipient are replaced by residues from a CDR of a non-human species (donor antibody) such as mouse, rat or rabbit having the desired specificity, affinity and capacity. In some instances, Fv framework residues of the human immunoglobulin are replaced by corresponding non-human residues. Humanized antibodies may also comprise residues which are found neither in the recipient antibody nor in the imported CDR or framework sequences. In general, a 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 (FR) 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. Humanization can be essentially performed following the method of Winter and co-workers, e.g., by substituting rodent CDRs or CDR sequences for the corresponding sequences of a human antibody. See, e.g., Jones, et al. (1986) Nature 321:522-525; Riechmann, et al. (1988) Nature 332:323-329; Presta (1992) Curr. Op. Struct. Biol. 2:593-596; and Verhoeyen, et al. (1988) Science 239:1534-1536). Accordingly, such humanized antibodies are chimeric antibodies (U.S. Pat. No. 4,816,567), wherein substantially less than an intact human variable domain has been substituted by the corresponding sequence from a non-human species.
Human antibodies can also be produced using various techniques known in the art, including phage display libraries (see, e.g., Hoogenboom and Winter (1991) J. Mol. Biol. 227:381-388; and Marks, et al. (1991) J. Mol. Biol. 222:581-597) or human monoclonal antibodies (see, e.g., p. 77, Cole, et al. in Reisfeld and Sell (1985) Monoclonal Antibodies and Cancer Therapy Liss; and Boerner, et al. (1991) J. Immunol. 147:86-95). Similarly, human antibodies can be made by introducing of 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. See, e.g., U.S. Pat. Nos. 5,545,807; 5,545,806; 5,569,825; 5,625,126; 5,633,425; 5,661,016; Marks, et al. (1992) Bio/Technology 10:779-783; Lonberg, et al. (1994) Nature 368:856-859; Morrison (1994) Nature 368:812-13; Neuberger (1996) Nature Biotechnology 14:826 commenting on Fishwild, et al. (1996) Nature Biotechnology 14:845-51; and Lonberg and Huszar (1995) Intern. Rev. Immunol. 13:65-93.
By immunotherapy is meant treatment of ovarian cancer, e.g., with an antibody raised against ovarian cancer proteins. As used herein, immunotherapy can be passive or active. Passive immunotherapy as defined herein is the passive transfer of antibody to a recipient (patient). Active immunization is the induction of antibody and/or T-cell responses in a recipient (patient). Induction of an immune response is the result of providing the recipient with an antigen to which antibodies are raised. The antigen may be provided by injecting a polypeptide against which antibodies are desired to be raised into a recipient, or contacting the recipient with a nucleic acid capable of expressing the antigen and under conditions for expression of the antigen, leading to an immune response.
In a preferred embodiment the ovarian cancer proteins against which antibodies are raised are secreted proteins as described above. Without being bound by theory, antibodies used for treatment, bind and prevent the secreted protein from binding to its receptor, thereby inactivating the secreted ovarian cancer protein.
In another preferred embodiment, the ovarian cancer protein to which antibodies are raised is a transmembrane protein. Without being bound by theory, antibodies used for treatment, bind the extracellular domain of the ovarian cancer protein and prevent it from binding to other proteins, such as circulating ligands or cell-associated molecules. The antibody may cause down-regulation of the transmembrane ovarian cancer protein. As will be appreciated by one of ordinary skill in the art, the antibody may be a competitive, non-competitive or uncompetitive inhibitor of protein binding to the extracellular domain of the ovarian cancer protein. The antibody is also an antagonist of the ovarian cancer protein. Further, the antibody prevents activation of the transmembrane ovarian cancer protein. In one aspect, when the antibody prevents the binding of other molecules to the ovarian cancer protein, the antibody prevents growth of the cell. The antibody may also be used to target or sensitize the cell to cytotoxic agents, including, but not limited to TNF-α, TNF-β, IL-1, INF-7, and IL-2, or chemotherapeutic agents including 5FU, vinblastine, actinomycin D, cisplatin, methotrexate, and the like. In some instances the antibody belongs to a sub-type that activates serum complement when complexed with the transmembrane protein thereby mediating cytotoxicity or antigen-dependent cytotoxicity (ADCC). Thus, ovarian cancer is treated by administering to a patient antibodies directed against the transmembrane ovarian cancer protein. Antibody-labeling may activate a co-toxin, localize a toxin payload, or otherwise provide means to locally ablate cells.
In another preferred embodiment, the antibody is conjugated to an effector moiety. The effector moiety can be any number of molecules, including labeling moieties such as radioactive labels or fluorescent labels, or can be a therapeutic moiety. In one aspect the therapeutic moiety is a small molecule that modulates the activity of the ovarian cancer protein. In another aspect the therapeutic moiety modulates the activity of molecules associated with or in close proximity to the ovarian cancer protein. The therapeutic moiety may inhibit enzymatic activity such as protease or collagenase or protein kinase activity associated with ovarian cancer.
In a preferred embodiment, the therapeutic moiety can also be a cytotoxic agent. In this method, targeting the cytotoxic agent to ovarian cancer tissue or cells, results in a reduction in the number of afflicted cells, thereby reducing symptoms associated with ovarian cancer. Cytotoxic agents are numerous and varied and include, but are not limited to, cytotoxic drugs or toxins or active fragments of such toxins. Suitable toxins and their corresponding fragments include diphtheria A chain, exotoxin A chain, ricin A chain, abrin A chain, curcin, crotin, phenomycin, enomycin and the like. Cytotoxic agents also include radiochemicals made by conjugating radioisotopes to antibodies raised against ovarian cancer proteins, or binding of a radionuclide to a chelating agent that has been covalently attached to the antibody. Targeting the therapeutic moiety to transmembrane ovarian cancer proteins not only serves to increase the local concentration of therapeutic moiety in the ovarian cancer afflicted area, but also serves to reduce deleterious side effects that may be associated with the untargeted therapeutic moiety.
In another preferred embodiment, the ovarian cancer protein against which the antibodies are raised is an intracellular protein. In this case, the antibody may be conjugated to a protein which facilitates entry into the cell. In one case, the antibody enters the cell by endocytosis. In another embodiment, a nucleic acid encoding the antibody is administered to the individual or cell. Moreover, wherein the ovarian cancer protein can be targeted within a cell, e.g., the nucleus, an antibody thereto contains a signal for that target localization, e.g., a nuclear localization signal.
The ovarian cancer antibodies of the invention specifically bind to ovarian cancer proteins. By “specifically bind” herein is meant that the antibodies bind to the protein with a Kd of at least about 0.1 mM, more usually at least about 1 μM, preferably at least about 0.1 μM or better, and most preferably, 0.01 μM or better. Selectivity of binding is also important.
Detection of Ovarian Cancer Sequence for Diagnostic and Therapeutic Applications
In one aspect, the RNA expression levels of genes are determined for different cellular states in the ovarian cancer phenotype. Expression levels of genes in normal tissue (e.g., not undergoing ovarian cancer) and in ovarian cancer tissue (and in some cases, for varying severities of ovarian cancer that relate to prognosis, as outlined below, or in non-malignant disease are evaluated to provide expression profiles. An expression profile of a particular cell state or point of development is essentially a “fingerprint” of the state of the cell. While two states may have any particular gene similarly expressed, the evaluation of a number of genes simultaneously allows the generation of a gene expression profile that is reflective of the state of the cell. By comparing expression profiles of cells in different states, information regarding which genes are important (including both up- and down-regulation of genes) in each of these states is obtained. Then, diagnosis may be performed or confirmed to determine whether a tissue sample has the gene expression profile of normal or cancerous tissue. This will provide for molecular diagnosis of related conditions.
“Differential expression,” or grammatical equivalents as used herein, refers to qualitative or quantitative differences in the temporal and/or cellular gene expression patterns within and among cells and tissue. Thus, a differentially expressed gene can qualitatively have its expression altered, including an activation or inactivation, in, e.g., normal versus ovarian cancer tissue. Genes may be turned on or turned off in a particular state, relative to another state thus permitting comparison of two or more states. A qualitatively regulated gene will exhibit an expression pattern within a state or cell type which is detectable by standard techniques. Some genes will be expressed in one state or cell type, but not in both. Alternatively, the difference in expression may be quantitative, e.g., in that expression is modulated, either up-regulated, resulting in an increased amount of transcript, or down-regulated, resulting in a decreased amount of transcript. The degree to which expression differs need only be large enough to quantify via standard characterization techniques as outlined below, such as by use of Affymetrix GeneChipTM expression arrays. See, e.g., Lockhart (1996) Nature Biotechnology 14:1675-1680. Other techniques include, but are not limited to, quantitative reverse transcriptase PCR, northern analysis, and RNase protection. As outlined above, preferably the change in expression (e.g., up-regulation or down-regulation) is at least about 50%, more preferably at least about 100%, more preferably at least about 150%, more preferably at least about 200%, with from 300 to at least 1000% being especially preferred.
Evaluation may be at the gene transcript, or the protein level. The amount of gene expression may be monitored using nucleic acid probes to the DNA or RNA equivalent of the gene transcript, and the quantification of gene expression levels, or, alternatively, the final gene product itself (protein) can be monitored, e.g., with antibodies to the ovarian cancer protein and standard immunoassays (ELISAs, etc.) or other techniques, including mass spectroscopy assays, 2D gel electrophoresis assays, etc. Proteins corresponding to ovarian cancer genes, e.g., those identified as being important in an ovarian cancer or disease phenotype, can be evaluated in an ovarian disease diagnostic test. In a preferred embodiment, gene expression monitoring is performed simultaneously on a number of genes. Multiple protein expression monitoring can be performed, or on an individual basis.
In this embodiment, the ovarian cancer nucleic acid probes are attached to biochips as outlined herein for the detection and quantification of ovarian cancer sequences in a particular sample. The assays are further described below in the example. PCR techniques can be used to provide greater sensitivity.
In a preferred embodiment nucleic acids encoding the ovarian cancer protein are detected. Although DNA or RNA encoding the ovarian cancer protein may be detected, of particular interest are methods wherein an mRNA encoding an ovarian cancer protein is detected. Probes to detect mRNA can be a nucleotide/deoxynucleotide probe that is complementary to and hybridizes with the mRNA and includes, but is not limited to, oligonucleotides, cDNA or RNA. Probes also should contain a detectable label, as defined herein. In one method the mRNA is detected after immobilizing the nucleic acid to be examined on a solid support such as nylon membranes and hybridizing the probe with the sample. Following washing to remove the non-specifically bound probe, the label is detected. In another method detection of the mRNA is performed in situ. In this method permeabilized cells or tissue samples are contacted with a detectably labeled nucleic acid probe for sufficient time to allow the probe to hybridize with the target mRNA. Following washing to remove the non-specifically bound probe, the label is detected. For example a digoxygenin labeled riboprobe (RNA probe) that is complementary to the mRNA encoding an ovarian cancer protein is detected by binding the digoxygenin with an anti-digoxygenin secondary antibody and developed with nitro blue tetrazolium and 5-bromo-4-chloro-3-indoyl phosphate.
In a preferred embodiment, various proteins from the three classes of proteins as described herein (secreted, transmembrane or intracellular proteins) are used in diagnostic assays. The ovarian cancer proteins, antibodies, nucleic acids, modified proteins and cells containing ovarian cancer sequences are used in diagnostic assays. This can be performed on an individual gene or corresponding polypeptide level. In a preferred embodiment, the expression profiles are used, preferably in conjunction with high throughput screening techniques to allow monitoring for expression profile genes and/or corresponding polypeptides.
As described and defined herein, ovarian cancer proteins, including intracellular, transmembrane, or secreted proteins, find use as prognostic or diagnostic markers of ovarian disease. Detection of these proteins in putative ovarian cancer tissue allows for detection, diagnosis, or prognosis of ovarian disease, and for selection of therapeutic strategy. In one embodiment, antibodies are used to detect ovarian cancer proteins. A preferred method separates proteins from a sample by electrophoresis on a gel (typically a denaturing and reducing protein gel, but may be another type of gel, including isoelectric focusing gels and the like). Following separation of proteins, the ovarian cancer protein is detected, e.g., by immunoblotting with antibodies raised against the ovarian cancer protein. Methods of immunoblotting are well known to those of ordinary skill in the art.
In another preferred method, antibodies to the ovarian cancer protein find use in in situ imaging techniques, e.g., in histology. See, e.g., Asai (ed. 1993) Methods in Cell Biology: Antibodies in Cell Biology (vol. 37) Academic Press. Cells are contacted with from one to many antibodies to the ovarian cancer protein(s). Following washing to remove non-specific antibody binding, the presence of the antibody or antibodies is detected. In one embodiment the antibody is detected by incubating with a secondary antibody that contains a detectable label. In another method the primary antibody to the ovarian cancer protein(s) contains a detectable label, e.g., an enzyme marker that can act on a substrate. In another preferred embodiment each one of multiple primary antibodies contains a distinct and detectable label. This method finds particular use in simultaneous screening for a plurality of ovarian cancer proteins. As will be appreciated by one of ordinary skill in the art, many other histological imaging techniques are also provided by the invention.
In a preferred embodiment the label is detected in a fluorometer which has the ability to detect and distinguish emissions of different wavelengths. In addition, a fluorescence activated cell sorter (FACS) can be used in the method.
In another preferred embodiment, antibodies find use in diagnosing ovarian cancer from blood, serum, plasma, stool, and other samples. Such samples, therefore, are useful as samples to be probed or tested for the presence of ovarian cancer proteins. Antibodies can be used to detect an ovarian cancer protein by previously described immunoassay techniques including ELISA, immunoblotting (western blotting), immunoprecipitation, BIACORE technology, and the like. Conversely, the presence of antibodies may indicate an immune response against an endogenous ovarian cancer protein.
In a preferred embodiment, in situ hybridization of labeled ovarian cancer nucleic acid probes to tissue arrays is done. For example, arrays of tissue samples, including ovarian cancer tissue and/or normal tissue, are made. In situ hybridization (see, e.g., Ausubel, supra) is then performed. When comparing the fingerprints between an individual and a standard, the skilled artisan can make a diagnosis, a prognosis, or a prediction based on the findings. It is further understood that the genes which indicate the diagnosis may differ from those which indicate the prognosis and molecular profiling of the condition of the cells may lead to distinctions between responsive or refractory conditions or may be predictive of outcomes.
In a preferred embodiment, the ovarian cancer proteins, antibodies, nucleic acids, modified proteins and cells containing ovarian cancer sequences are used in prognosis assays. As above, gene expression profiles can be generated that correlate to ovarian cancer, clinical, pathological, or other information, in terms of long term prognosis. Again, this may be done on either a protein or gene level, with the use of a plurality of genes being preferred. As above, ovarian cancer probes may be attached to biochips for the detection and quantification of ovarian cancer sequences in a tissue or patient. The assays proceed as outlined above for diagnosis. PCR method may provide more sensitive and accurate quantification.
Assays for Therapeutic Compounds
In a preferred embodiment members of the proteins, nucleic acids, and antibodies as described herein are used in drug screening assays. The ovarian cancer proteins, antibodies, nucleic acids, modified proteins and cells containing ovarian cancer sequences are used in drug screening assays or by evaluating the effect of drug candidates on a “gene expression profile” or expression profile of polypeptides. In a preferred embodiment, the expression profiles are used, preferably in conjunction with high throughput screening techniques to allow monitoring for expression profile genes after treatment with a candidate agent. See, e.g., Zlokarnik, et al. (1998) Science 279:84-88; and Heid (1996) Genome Res. 6:986-994.
In a preferred embodiment, the ovarian cancer proteins, antibodies, nucleic acids, modified proteins and cells containing the native or modified ovarian cancer proteins are used in screening assays. That is, the present invention provides novel methods for screening for compositions which modulate the ovarian cancer phenotype or an identified physiological function of an ovarian cancer protein. As above, this can be done on an individual gene level or by evaluating the effect of drug candidates on a “gene expression profile”. In a preferred embodiment, the expression profiles are used, preferably in conjunction with high throughput screening techniques to allow monitoring for expression profile genes after treatment with a candidate agent. See, e.g., Zlokarnik, supra.
Having identified the differentially expressed genes herein, a variety of assays may be executed. In a preferred embodiment, assays may be run on an individual gene or protein level. That is, having identified a particular gene as up regulated in ovarian cancer, test compounds can be screened for the ability to modulate gene expression or for binding to the ovarian cancer protein. “Modulation” thus includes both an increase and a decrease in gene expression. The preferred amount of modulation will depend on the original change of the gene expression in normal versus tissue undergoing ovarian cancer, with changes of at least 10%, preferably 50%, more preferably 100-300%, and in some embodiments 300-1000% or greater. Thus, if a gene exhibits a 4-fold increase in ovarian cancer tissue compared to normal tissue, a decrease of about four-fold is often desired; similarly, a 10-fold decrease in ovarian cancer tissue compared to normal tissue often provides a target value of a 10-fold increase in expression to be induced by the test compound.
The amount of gene expression may be monitored using nucleic acid probes and the quantification of gene expression levels, or, alternatively, the gene product itself can be monitored, e.g., through the use of antibodies to the ovarian cancer protein and standard immunoassays. Proteomics and separation techniques may also allow quantification of expression.
In a preferred embodiment, gene expression or protein monitoring of a number of entities, e.g., an expression profile, is monitored simultaneously. Such profiles will typically involve a plurality of those entities described herein.
In this embodiment, the ovarian cancer nucleic acid probes are attached to biochips as outlined herein for the detection and quantification of ovarian cancer sequences in a particular cell. Alternatively, PCR may be used. Thus, a series, e.g., of microtiter plate, may be used with dispensed primers in desired wells. A PCR reaction can then be performed and analyzed for each well.
Expression monitoring can be performed to identify compounds that modify the expression of one or more ovarian cancer-associated sequences, e.g., a polynucleotide sequence set out in Tables 1-20. Generally, in a preferred embodiment, a test modulator is added to the cells prior to analysis. Moreover, screens are also provided to identify agents that modulate ovarian cancer, modulate ovarian cancer proteins, bind to an ovarian cancer protein, or interfere with the binding of an ovarian cancer protein and an antibody or other binding partner.
The term “test compound” or “drug candidate” or “modulator” or grammatical equivalents as used herein describes any molecule, e.g., protein, oligopeptide, small organic molecule, polysaccharide, polynucleotide, etc., to be tested for the capacity to directly or indirectly alter the ovarian cancer phenotype or the expression of an ovarian cancer sequence, e.g., a nucleic acid or protein sequence. In preferred embodiments, modulators alter expression profiles, or expression profile nucleic acids or proteins provided herein. In one embodiment, the modulator suppresses an ovarian cancer phenotype, e.g., to a normal or non-malignant tissue fingerprint. In another embodiment, a modulator induced an ovarian cancer phenotype. Generally, a plurality of assay mixtures are run in parallel with different agent concentrations to obtain a differential response to the various concentrations. Typically, one of these concentrations serves as a negative control, e.g., at zero concentration or below the level of detection.
Drug candidates encompass numerous chemical classes, though typically they are organic molecules, preferably small organic compounds having a molecular weight of more than 100 and less than about 2,500 daltons. Preferred small molecules are less than 2000, or less than 1500 or less than 1000 or less than 500 D. Candidate agents comprise functional groups necessary for structural interaction with proteins, particularly hydrogen bonding, and typically include at least an amine, carbonyl, hydroxyl or carboxyl group, preferably at least two of the functional chemical groups. The candidate agents often comprise cyclical carbon or heterocyclic structures and/or aromatic or polyaromatic structures substituted with one or more of the above functional groups. Candidate agents are also found among biomolecules including peptides, saccharides, fatty acids, steroids, purines, pyrimidines, derivatives, structural analogs or combinations thereof. Particularly preferred are peptides.
In one aspect, a modulator will neutralize the effect of an ovarian cancer protein. By “neutralize” is meant that activity of a protein is inhibited or blocked and the consequent effect on the cell.
In certain embodiments, combinatorial libraries of potential modulators will be screened for an ability to bind to an ovarian cancer polypeptide or to modulate activity. Conventionally, new chemical entities with useful properties are generated by identifying a chemical compound (called a “lead compound”) with some desirable property or activity, e.g., inhibiting activity, creating variants of the lead compound, and evaluating the property and activity of those variant compounds. Often, high throughput screening (HTS) methods are employed for such an analysis.
In one preferred embodiment, high throughput screening methods involve providing a library containing a large number of potential therapeutic compounds (candidate compounds). Such “combinatorial chemical libraries” are then screened in one or more assays to identify those library members (particular chemical species or subclasses) that display a desired characteristic activity. The compounds thus identified can serve as conventional “lead compounds” or can themselves be used as potential or actual therapeutics.
A combinatorial chemical library is a collection of diverse chemical compounds generated by either chemical synthesis or biological synthesis by combining a number of chemical “building blocks” such as reagents. For example, a linear combinatorial chemical library, such as a polypeptide (e.g., mutein) library, is formed by combining a set of chemical building blocks called amino acids in every possible way for a given compound length (e.g., the number of amino acids in a polypeptide compound). Millions of chemical compounds can be synthesized through such combinatorial mixing of chemical building blocks. See, e.g., Gallop, et al. (1994) J. Med. Chem. 37:1233-1251.
Preparation and screening of combinatorial chemical libraries is well known to those of skill in the art. Such combinatorial chemical libraries include, but are not limited to, peptide libraries (see, e.g., U.S. Pat. No. 5,010,175; Furka (1991) Pept. Prot. Res. 37:487-493; and Houghton, et al. (1991) Nature 354:84-88), peptoids (PCT Publication No WO 91/19735), encoded peptides (PCT Publication WO 93/20242), random bio-oligomers (PCT Publication WO 92/00091), benzodiazepines (U.S. Pat. No. 5,288,514), diversomers such as hydantoins, benzodiazepines and dipeptides (Hobbs, et al. (1993) Proc. Nat'l Acad. Sci. USA 90:6909-913), vinylogous polypeptides (Hagihara, et al. (1992) J. Amer. Chem. Soc. 114:6568-570), non-peptidal peptidomimetics with a Beta-D-Glucose scaffolding (Hirschmann, et al. (1992) J. Amer. Chem. Soc. 114:9217-218), analogous organic syntheses of small compound libraries (Chen, et al. (1994) J. Amer. Chem. Soc. 116:2661-662), oligocarbamates (Cho, et al. (1993) Science 261:1303-305), and/or peptidyl phosphonates (Campbell, et al. (1994) J. Org. Chem. 59:658-xxx). See, generally, Gordon, et al. (1994) J. Med. Chem. 37:1385-401, nucleic acid libraries (see, e.g., Stratagene, Corp.), peptide nucleic acid libraries (see, e.g., U.S. Pat. No. 5,539,083), antibody libraries (see, e.g., Vaughn, et al. (1996) Nature Biotechnology 14:309-314; and PCT/US96/10287), carbohydrate libraries (see, e.g., Liang, et al. (1996) Science 274:1520-1522; and U.S. Pat. No. 5,593,853), and small organic molecule libraries (see, e.g., benzodiazepines, page 33, Baum (Jan. 18, 1993) C&E News; isoprenoids, U.S. Pat. No. 5,569,588; thiazolidinones and metathiazanones, U.S. Pat. No. 5,549,974; pyrrolidines, U.S. Pat. Nos. 5,525,735 and 5,519,134; morpholino compounds, U.S. Pat. No. 5,506,337; benzodiazepines, U.S. Pat. No. 5,288,514; and the like).
Devices for the preparation of combinatorial libraries are commercially available. See, e.g., 357 MPS, 390 MPS, Advanced Chem Tech, Louisville Ky.; Symphony, Rainin, Woburn, Mass.; 433A Applied Biosystems, Foster City, Calif.; 9050 Plus, Millipore, Bedford, Mass.
A number of well known robotic systems have also been developed for solution phase chemistries. These systems include automated workstations like the automated synthesis apparatus developed by Takeda Chemical Industries, LTD. (Osaka, Japan) and many robotic systems utilizing robotic arms (Zymate II, Zymark Corporation, Hopkinton, Mass.; Orca, Hewlett-Packard, Palo Alto, Calif.), which mimic the manual synthetic operations performed by a chemist. Any of the above devices are suitable for use with the present invention. The nature and implementation of modifications to these devices (if any) so that they can operate as discussed herein will be apparent to persons skilled in the relevant art. In addition, numerous combinatorial libraries are themselves commercially available (see, e.g., ComGenex, Princeton, N.J.; Asinex, Moscow, RU; Tripos, Inc., St. Louis, Mo.; ChemStar, Ltd, Moscow, RU; 3D Pharmaceuticals, Exton, Pa.; Martek Biosciences, Columbia, Md.; etc.).
The assays to identify modulators are amenable to high throughput screening. Preferred assays thus detect enhancement or inhibition of ovarian cancer gene transcription, inhibition or enhancement of polypeptide expression, and inhibition or enhancement of polypeptide activity.
High throughput assays for the presence, absence, quantification, or other properties of particular nucleic acids or protein products are well known to those of skill in the art. Similarly, binding assays and reporter gene assays are similarly well known. Thus, e.g., U.S. Pat. No. 5,559,410 discloses high throughput screening methods for proteins, U.S. Pat. No. 5,585,639 discloses high throughput screening methods for nucleic acid binding (e.g., in arrays), while U.S. Pat. Nos. 5,576,220 and 5,541,061 disclose high throughput methods of screening for ligand/antibody binding.
In addition, high throughput screening systems are commercially available (see, e.g., Zymark Corp., Hopkinton, Mass.; Air Technical Industries, Mentor, Ohio; Beckman Instruments, Inc. Fullerton, Calif.; Precision Systems, Inc., Natick, Mass., etc.). These systems typically automate entire procedures, including all sample and reagent pipetting, liquid dispensing, timed incubations, and final readings of the microplate in detector(s) appropriate for the assay. These configurable systems provide high throughput and rapid start up as well as a high degree of flexibility and customization. The manufacturers of such systems provide detailed protocols for various high throughput systems. Thus, e.g., Zymark Corp. provides technical bulletins describing screening systems for detecting the modulation of gene transcription, ligand binding, and the like.
In one embodiment, modulators are proteins, often naturally occurring proteins or fragments of naturally occurring proteins. Thus, e.g., cellular extracts containing proteins, or random or directed digests of proteinaceous cellular extracts, may be used. In this way libraries of proteins may be made for screening in the methods of the invention. Particularly preferred in this embodiment are libraries of bacterial, fungal, viral, and mammalian proteins, with the latter being preferred, and human proteins being especially preferred. Particularly useful test compound will be directed to the class of proteins to which the target belongs, e.g., substrates for enzymes or ligands and receptors.
In a preferred embodiment, modulators are peptides of from about 5 to about 30 amino acids, with from about 5 to about 20 amino acids being preferred, and from about 7 to about 15 being particularly preferred. The peptides may be digests of naturally occurring proteins as is outlined above, random peptides, or “biased” random peptides. By “randomized” or grammatical equivalents herein is meant that each nucleic acid and peptide consists of essentially random nucleotides and amino acids, respectively. Since generally these random peptides (or nucleic acids, discussed below) are chemically synthesized, they may incorporate any nucleotide or amino acid at any position. The synthetic process can be designed to generate randomized proteins or nucleic acids, to allow the formation of all or most of the possible combinations over the length of the sequence, thus forming a library of randomized candidate bioactive proteinaceous agents.
In one embodiment, the library is fully randomized, with no sequence preferences or constants at any position. In a preferred embodiment, the library is biased. That is, some positions within the sequence are either held constant, or are selected from a limited number of possibilities. For example, in a preferred embodiment, the nucleotides or amino acid residues are randomized within a defined class, e.g., of hydrophobic amino acids, hydrophilic residues, sterically biased (either small or large) residues, towards the creation of nucleic acid binding domains, the creation of cysteines, for cross-linking, prolines for SH-3 domains, serines, threonines, tyrosines or histidines for phosphorylation sites, etc., or to purines, etc.
Modulators of ovarian cancer can also be nucleic acids, as defined above.
As described above generally for proteins, nucleic acid modulating agents may be naturally occurring nucleic acids, random nucleic acids, or “biased” random nucleic acids. For example, digests of procaryotic or eucaryotic genomes may be used as is outlined above for proteins.
In a preferred embodiment, the candidate compounds are organic chemical moieties, a wide variety of which are available in the literature.
After the candidate agent has been added and the cells allowed to incubate for some period of time, the sample containing a target sequence to be analyzed is added to the biochip. If required, the target sequence is prepared using known techniques. For example, the sample may be treated to lyse the cells, using known lysis buffers, electroporation, etc., with purification and/or amplification such as PCR performed as appropriate. For example, an in vitro transcription with labels covalently attached to the nucleotides is performed. Generally, the nucleic acids are labeled with biotin-FITC or PE, or with cy3 or cy5.
In a preferred embodiment, the target sequence is labeled with, e.g., a fluorescent, a chemiluminescent, a chemical, or a radioactive signal, to provide a means of detecting the target sequence's specific binding to a probe. The label also can be an enzyme, such as, alkaline phosphatase or horseradish peroxidase, which when provided with an appropriate substrate produces a product that can be detected. Alternatively, the label can be a labeled compound or small molecule, such as an enzyme inhibitor, that binds but is not catalyzed or altered by the enzyme. The label also can be a moiety or compound, such as, an epitope tag or biotin which specifically binds to streptavidin. For the example of biotin, the streptavidin is labeled as described above, thereby, providing a detectable signal for the bound target sequence. Unbound labeled streptavidin is typically removed prior to analysis.
As will be appreciated by those in the art, these assays can be direct hybridization assays or can comprise “sandwich assays”, which include the use of multiple probes, as is generally outlined in U.S. Pat. Nos. 5,681,702; 5,597,909; 5,545,730; 5,594,117; 5,591,584; 5,571,670; 5,580,731; 5,571,670; 5,591,584; 5,624,802; 5,635,352; 5,594,118; 5,359,100; 5,124,246; and 5,681,697, each of which is hereby incorporated by reference. In this embodiment, in general, the target nucleic acid is prepared as outlined above, and then added to the biochip comprising a plurality of nucleic acid probes, under conditions that allow the formation of a hybridization complex.
A variety of hybridization conditions may be used in the present invention, including high, moderate and low stringency conditions as outlined above. The assays are generally run under stringency conditions which allows formation of the label probe hybridization complex only in the presence of target. Stringency can be controlled by altering a step parameter that is a thermodynamic variable, including, but not limited to, temperature, formamide concentration, salt concentration, chaotropic salt concentration pH, organic solvent concentration, etc.
These parameters may also be used to control non-specific binding, as is generally outlined in U.S. Pat. No. 5,681,697. Thus it may be desirable to perform certain steps at higher stringency conditions to reduce non-specific binding.
The reactions outlined herein may be accomplished in a variety of ways. Components of the reaction may be added simultaneously, or sequentially, in different orders, with preferred embodiments outlined below. In addition, the reaction may include a variety of other reagents. These include salts, buffers, neutral proteins, e.g., albumin, detergents, etc. which may be used to facilitate optimal hybridization and detection, and/or reduce non-specific or background interactions. Reagents that otherwise improve the efficiency of the assay, such as protease inhibitors, nuclease inhibitors, anti-microbial agents, etc., may also be used as appropriate, depending on the sample preparation methods and purity of the target.
The assay data are analyzed to determine the expression levels, and changes in expression levels as between states, of individual genes, forming a gene expression profile.
Screens are performed to identify modulators of the ovarian cancer phenotype. In one embodiment, screening is performed to identify modulators that can induce or suppress a particular expression profile, thus preferably generating the associated phenotype. In another embodiment, e.g., for diagnostic applications, having identified differentially expressed genes important in a particular state, screens can be performed to identify modulators that alter expression of individual genes. In an another embodiment, screening is performed to identify modulators that alter a biological function of the expression product of a differentially expressed gene. Again, having identified the importance of a gene in a particular state, screens are performed to identify agents that bind and/or modulate the biological activity of the gene product.
In addition screens can be done for genes that are induced in response to a candidate agent. After identifying a modulator based upon its ability to suppress an ovarian cancer expression pattern leading to a normal expression pattern, or to modulate a single ovarian cancer gene expression profile so as to mimic the expression of the gene from normal tissue, a screen as described above can be performed to identify genes that are specifically modulated in response to the agent. Comparing expression profiles between normal tissue and agent treated ovarian cancer tissue reveals genes that are not expressed in normal tissue or ovarian cancer tissue, but are expressed in agent treated tissue. These agent-specific sequences can be identified and used by methods described herein for ovarian cancer genes or proteins. In particular these sequences and the proteins they encode find use in marking or identifying agent treated cells. In addition, antibodies can be raised against the agent induced proteins and used to target novel therapeutics to the treated ovarian cancer tissue sample.
Thus, in one embodiment, a test compound is administered to a population of ovarian cancer cells, that have an associated ovarian cancer expression profile. By “administration” or “contacting” herein is meant that the candidate agent is added to the cells in such a manner as to allow the agent to act upon the cell, whether by uptake and intracellular action, or by action at the cell surface. In some embodiments, nucleic acid encoding a proteinaceous candidate agent (e.g., a peptide) may be put into a viral construct such as an adenoviral or retroviral construct, and added to the cell, such that expression of the peptide agent is accomplished, e.g., PCT US97/01019. Regulatable gene therapy systems can also be used.
Once the test compound has been administered to the cells, the cells can be washed if desired and are allowed to incubate under preferably physiological conditions for some period of time. The cells are then harvested and a new gene expression profile is generated, as outlined herein.
Thus, e.g., ovarian cancer or non-malignant tissue may be screened for agents that modulate, e.g., induce or suppress the ovarian cancer phenotype. A change in at least one gene, preferably many, of the expression profile indicates that the agent has an effect on ovarian cancer activity. By defining such a signature for the ovarian cancer phenotype, screens for new drugs that alter the phenotype can be devised. With this approach, the drug target need not be known and need not be represented in the original expression screening platform, nor does the level of transcript for the target protein need to change.
In a preferred embodiment, as outlined above, screens may be done on individual genes and gene products (proteins). That is, having identified a particular differentially expressed gene as important in a particular state, screening of modulators of either the expression of the gene or the gene product itself can be done. The gene products of differentially expressed genes are sometimes referred to herein as “ovarian cancer proteins” or a “ovarian cancer modulatory protein”. The ovarian cancer modulatory protein may be a fragment, or alternatively, be the full length protein to the fragment encoded by the nucleic acids of the Tables. Preferably, the ovarian cancer modulatory protein is a fragment. In a preferred embodiment, the ovarian cancer amino acid sequence which is used to determine sequence identity or similarity is encoded by a nucleic acid of the Tables. In another embodiment, the sequences are naturally occurring allelic variants of a protein encoded by a nucleic acid of the Tables. In another embodiment, the sequences are sequence variants as further described herein.
Preferably, the ovarian cancer modulatory protein is a fragment of approximately 14 to 24 amino acids long. More preferably the fragment is a soluble fragment. Preferably, the fragment includes a non-transmembrane region. In a preferred embodiment, the fragment has an N-terminal Cys to aid in solubility. In another embodiment, the C-terminus of the fragment is kept as a free acid and the N-terminus is a free amine to aid in coupling, e.g., to cysteine. Or, the ovarian cancer proteins are conjugated to an immunogenic agent, e.g., to BSA.
Measurements of ovarian cancer polypeptide activity, or of ovarian cancer or the ovarian cancer phenotype can be performed using a variety of assays. For example, the effects of the test compounds upon the function of the ovarian cancer polypeptides can be measured by examining parameters described above. A suitable physiological change that affects activity can be used to assess the influence of a test compound on the polypeptides of this invention. When the functional consequences are determined using intact cells or animals, one can also measure a variety of effects such as, in the case of ovarian cancer associated with tumors, tumor growth, tumor metastasis, neovascularization, hormone release, transcriptional changes to both known and uncharacterized genetic markers (e.g., northern blots), changes in cell metabolism such as cell growth or pH changes, and changes in intracellular second messengers such as cGMP. In the assays of the invention, mammalian ovarian cancer polypeptide is typically used, e.g., mouse, preferably human.
Assays to identify compounds with modulating activity can be performed in vitro. For example, an ovarian cancer polypeptide is first contacted with a potential modulator and incubated for a suitable amount of time, e.g., from 0.5 to 48 hours. In one embodiment, the ovarian cancer polypeptide levels are determined in vitro by measuring the level of protein or mRNA. The level of protein is measured using immunoassays such as western blotting, ELISA and the like with an antibody that selectively binds to the ovarian cancer polypeptide or a fragment thereof. For measurement of mRNA, amplification, e.g., using PCR, LCR, or hybridization assays, e.g., northern hybridization, RNAse protection, dot blotting, are preferred. The level of protein or mRNA is detected using directly or indirectly labeled detection agents, e.g., fluorescently or radioactively labeled nucleic acids, radioactively or enzymatically labeled antibodies, and the like, as described herein.
Alternatively, a reporter gene system can be devised using the ovarian cancer protein promoter operably linked to a reporter gene such as luciferase, green fluorescent protein, CAT, or β-gal. The reporter construct is typically transfected into a cell. After treatment with a potential modulator, the amount of reporter gene transcription, translation, or activity is measured according to standard techniques known to those of skill in the art.
In a preferred embodiment, as outlined above, screens may be done on individual genes and gene products (proteins). That is, having identified a particular differentially expressed gene as important in a particular state, screening of modulators of the expression of the gene or the gene product itself can be done. The gene products of differentially expressed genes are sometimes referred to herein as “ovarian cancer proteins.” The ovarian cancer protein may be a fragment, or alternatively, be the full length protein to a fragment shown herein.
In one embodiment, screening for modulators of expression of specific genes is performed. Typically, the expression of only one or a few genes are evaluated. In another embodiment, screens are designed to first find compounds that bind to differentially expressed proteins. These compounds are then evaluated for the ability to modulate differentially expressed activity. Moreover, once initial candidate compounds are identified, variants can be further screened to better evaluate structure activity relationships.
In a preferred embodiment, binding assays are done. In general, purified or isolated gene product is used; that is, the gene products of one or more differentially expressed nucleic acids are made. For example, antibodies are generated to the protein gene products, and standard immunoassays are run to determine the amount of protein present. Alternatively, cells comprising the ovarian cancer proteins can be used in the assays.
Thus, in a preferred embodiment, the methods comprise combining an ovarian cancer protein and a candidate compound, and determining the binding of the compound to the ovarian cancer protein. Preferred embodiments utilize the human ovarian cancer protein, although other mammalian proteins, e.g., counterparts, may also be used, e.g., for the development of animal models of human disease. In some embodiments, as outlined herein, variant or derivative ovarian cancer proteins may be used.
Generally, in a preferred embodiment of the methods herein, the ovarian cancer protein or the candidate agent is non-diffusably bound to an insoluble support having isolated sample receiving areas (e.g., a microtiter plate, an array, etc.). The insoluble supports may be made of any composition to which the compositions can be bound, is readily separated from soluble material, and is otherwise compatible with the overall method of screening. The surface of such supports may be solid or porous and of any convenient shape. Examples of suitable insoluble supports include microtiter plates, arrays, membranes and beads. These are typically made of glass, plastic (e.g., polystyrene), polysaccharides, nylon or nitrocellulose, teflonTM, etc. Microtiter plates and arrays are especially convenient because a large number of assays can be carried out simultaneously, using small amounts of reagents and samples. The particular manner of binding of the composition is not crucial so long as it is compatible with the reagents and overall methods of the invention, maintains the activity of the composition and is non-diffusible. Preferred methods of binding include the use of antibodies (which do not sterically block either the ligand binding site or activation sequence when the protein is bound to the support), direct binding to “sticky” or ionic supports, chemical crosslinking, the synthesis of the protein or agent on the surface, etc. Following binding of the protein or agent, excess unbound material is removed by washing. The sample receiving areas may then be blocked through incubation with bovine serum albumin (BSA), casein or other innocuous protein or other moiety.
In a preferred embodiment, the ovarian cancer protein is bound to the support, and a test compound is added to the assay. Alternatively, the candidate agent is bound to the support and the ovarian cancer protein is added. Novel binding agents include specific antibodies, non-natural binding agents identified in screens of chemical libraries, peptide analogs, etc. Of particular interest are screening assays for agents that have a low toxicity for human cells. A wide variety of assays may be used for this purpose, including labeled in vitro protein-protein binding assays, electrophoretic mobility shift assays, immunoassays for protein binding, functional assays (phosphorylation assays, etc.) and the like.
The determination of the binding of the test modulating compound to the ovarian cancer protein may be done in a number of ways. In a preferred embodiment, the compound is labeled, and binding determined directly, e.g., by attaching all or a portion of the ovarian cancer protein to a solid support, adding a labeled candidate agent (e.g., a fluorescent label), washing off excess reagent, and determining whether the label is present on the solid support. Various blocking and washing steps may be utilized as appropriate.
In some embodiments, only one of the components is labeled, e.g., the proteins (or proteinaceous candidate compounds) can be labeled. Alternatively, more than one component can be labeled with different labels, e.g., 125I for the proteins and a fluorophor for the compound. Proximity reagents, e.g., quenching or energy transfer reagents are also useful.
In one embodiment, the binding of the test compound is determined by competitive binding assay. The competitor is a binding moiety known to bind to the target molecule (e.g., an ovarian cancer protein), such as an antibody, peptide, binding partner, ligand, etc. Under certain circumstances, there may be competitive binding between the compound and the binding moiety, with the binding moiety displacing the compound. In one embodiment, the test compound is labeled. Either the compound, or the competitor, or both, is added first to the protein for a time sufficient to allow binding, if present. Incubations may be performed at a temperature which facilitates optimal activity, typically 4-40° C. Incubation periods are typically optimized, e.g., to facilitate rapid high throughput screening. Typically between 0.1 and 1 hr will be sufficient. Excess reagent is generally removed or washed away. The second component is then added, and the presence or absence of the labeled component is followed, to indicate binding.
In a preferred embodiment, the competitor is added first, followed by the test compound. Displacement of the competitor is an indication that the test compound is binding to the ovarian cancer protein and thus is capable of binding to, and potentially modulating, the activity of the ovarian cancer protein. In this embodiment, either component can be labeled. Thus, e.g., if the competitor is labeled, the presence of label in the wash solution indicates displacement by the agent. Alternatively, if the test compound is labeled, the presence of the label on the support indicates displacement.
In an alternative embodiment, the test compound is added first, with incubation and washing, followed by the competitor. The absence of binding by the competitor may indicate that the test compound is bound to the ovarian cancer protein with a higher affinity. Thus, if the test compound is labeled, the presence of the label on the support, coupled with a lack of competitor binding, may indicate that the test compound is capable of binding to the ovarian cancer protein.
In a preferred embodiment, the methods comprise differential screening to identity agents that are capable of modulating the activity of the ovarian cancer proteins. In this embodiment, the methods comprise combining an ovarian cancer protein and a competitor in a first sample. A second sample comprises a test compound, an ovarian cancer protein, and a competitor. The binding of the competitor is determined for both samples, and a change, or difference in binding between the two samples indicates the presence of an agent capable of binding to the ovarian cancer protein and potentially modulating its activity. That is, if the binding of the competitor is different in the second sample relative to the first sample, the agent is capable of binding to the ovarian cancer protein.
Alternatively, differential screening is used to identify drug candidates that bind to the native ovarian cancer protein, but cannot bind to modified ovarian cancer proteins. The structure of the ovarian cancer protein may be modeled, and used in rational drug design to synthesize agents that interact with that site. Drug candidates that affect the activity of an ovarian cancer protein are also identified by screening drugs for the ability to either enhance or reduce the activity of the protein.
Positive controls and negative controls may be used in the assays. Preferably control and test samples are performed in at least triplicate to obtain statistically significant results. Incubation of all samples is for a time sufficient for the binding of the agent to the protein. Following incubation, samples are washed free of non-specifically bound material and the amount of bound, generally labeled agent determined. For example, where a radiolabel is employed, the samples may be counted in a scintillation counter to determine the amount of bound compound.
A variety of other reagents may be included in the screening assays. These include reagents like salts, neutral proteins, e.g., albumin, detergents, etc. which may be used to facilitate optimal protein-protein binding and/or reduce non-specific or background interactions. Also reagents that otherwise improve the efficiency of the assay, such as protease inhibitors, nuclease inhibitors, anti-microbial agents, etc., may be used. The mixture of components may be added in an order that provides for the requisite binding.
In a preferred embodiment, the invention provides methods for screening for a compound capable of modulating the activity of an ovarian cancer protein. The methods comprise adding a test compound, as defined above, to a cell comprising ovarian cancer proteins. Preferred cell types include almost any cell. The cells contain a recombinant nucleic acid that encodes an ovarian cancer protein. In a preferred embodiment, a library of candidate agents are tested on a plurality of cells.
In one aspect, the assays are evaluated in the presence or absence or previous or subsequent exposure of physiological signals, e.g., hormones, antibodies, peptides, antigens, cytokines, growth factors, action potentials, pharmacological agents including chemotherapeutics, radiation, carcinogenics, or other cells (e.g., cell-cell contacts). In another example, the determinations are determined at different stages of the cell cycle process.
In this way, compounds that modulate ovarian cancer agents are identified. Compounds with pharmacological activity are able to enhance or interfere with the activity of the ovarian cancer protein. Once identified, similar structures are evaluated to identify critical structural feature of the compound.
In one embodiment, a method of inhibiting ovarian cancer cell division is provided. The method comprises administration of an ovarian cancer inhibitor. In another embodiment, a method of inhibiting ovarian cancer is provided. The method comprises administration of an ovarian cancer inhibitor. In a further embodiment, methods of treating cells or individuals with ovarian cancer are provided. The method comprises administration of an ovarian cancer inhibitor.
In one embodiment, an ovarian cancer inhibitor is an antibody as discussed above. In another embodiment, the ovarian cancer inhibitor is an antisense or RNAi molecule.
A variety of cell viability, growth, proliferation, and metastasis assays are known to those of skill in the art, as described below.
Soft Agar Growth or Colony Formation in Suspension
Normal cells require a solid substrate to attach and grow. When the cells are transformed, they lose this phenotype and grow detached from the substrate. For example, transformed cells can grow in stirred suspension culture or suspended in semi-solid media, such as semi-solid or soft agar. The transformed cells, when transfected with tumor suppressor genes, regenerate normal phenotype and require a solid substrate to attach and grow. Soft agar growth or colony formation in suspension assays can be used to identify modulators of ovarian cancer sequences, which when expressed in host cells, inhibit abnormal cellular proliferation and transformation. A therapeutic compound would reduce or eliminate the host cells' ability to grow in stirred suspension culture or suspended in semi-solid media, such as semi-solid or soft.
Techniques for soft agar growth or colony formation in suspension assays are described in Freshney (1994) Culture of Animal Cells: A Manual of Basic Technique (3d ed.) Wiley-Liss, herein incorporated by reference. See also, the methods section of Garkavtsev, et al. (1996), supra, herein incorporated by reference.
Contact Inhibition and Density Limitation of Growth
Normal cells typically grow in a flat and organized pattern in a petri dish until they touch other cells. When the cells touch one another, they are contact inhibited and stop growing. When cells are transformed, however, the cells are not contact inhibited and continue to grow to high densities in disorganized foci. Thus, the transformed cells grow to a higher saturation density than normal cells. This can be detected morphologically by the formation of a disoriented monolayer of cells or rounded cells in foci within the regular pattern of normal surrounding cells. Alternatively, labeling index with (3H)-thymidine at saturation density can be used to measure density limitation of growth. See, e.g., Freshney (1994), supra. The transformed cells, when transfected with tumor suppressor genes, regenerate a normal phenotype and become contact inhibited and would grow to a lower density.
In this assay, labeling index with (3H)-thymidine at saturation density is a preferred method of measuring density limitation of growth. Transformed host cells are transfected with an ovarian cancer-associated sequence and are grown for 24 hr at saturation density in non-limiting medium conditions. The percentage of cells labeling with (3H)-thymidine is determined autoradiographically. See, e.g., Freshney (1994), supra.
Growth Factor or Serum Dependence
Transformed cells typically have a lower serum dependence than their normal counterparts. See, e.g., Temin (1966) J. Nat'l Cancer Inst. 37:167-175; Eagle, et al. (1970) J. Exp. Med. 131:836-879; and Freshney, supra. This is in part due to release of various growth factors by the transformed cells. Growth factor or serum dependence of transformed host cells can be compared with that of control.
Tumor Specific Markers Levels
Tumor cells release an increased amount of certain factors (hereinafter “tumor specific markers”) than their normal counterparts. For example, plasminogen activator (PA) is released from human glioma at a higher level than from normal brain cells (see, e.g., Gullino, pp. 178-184 “Angiogenesis, tumor vascularization, and potential interference with tumor growth” in Mihich (ed. 1985) Biological Responses in Cancer Plenum. Similarly, tumor angiogenesis factor (TAF) is released at a higher level in tumor cells than their normal counterparts. See, e.g., Folkman (1992) Sem Cancer Biol. 3:89-96.
Various techniques which measure the release of these factors are described in Freshney (1994), supra. Also, see, Unkeless, et al. (1974) J. Biol. Chem. 249:4295-4305; Strickland and Beers (1976) J. Biol. Chem. 251:5694-5702; Whur, et al. (1980) Br. J. Cancer 42:305-312; Gullino, pp. 178-184 “Angiogenesis, tumor vascularization, and potential interference with tumor growth” in Mihich (ed. 1985) Biological Responses in Cancer Plenum; and Freshney (1985) Anticancer Res. 5:111-130.
Invasiveness into Matrigel
The degree of invasiveness into Matrigel or some other extracellular matrix constituent can be used as an assay to identify compounds that modulate ovarian cancer-associated sequences. Tumor cells exhibit a good correlation between malignancy and invasiveness of cells into Matrigel or some other extracellular matrix constituent. In this assay, tumorigenic cells are typically used as host cells. Expression of a tumor suppressor gene in these host cells would decrease invasiveness of the host cells.
Alternatively, the level of invasion of host cells can be measured by using filters coated with Matrigel or some other extracellular matrix constituent. Penetration into the gel, or through to the distal side of the filter, is rated as invasiveness, and rated histologically by number of cells and distance moved, or by pre-labeling the cells with 125I and counting the radioactivity on the distal side of the filter or bottom of the dish. See, e.g., Freshney (1984), supra.
Tumor Growth In Vivo
Effects of ovarian cancer-associated sequences on cell growth can be tested in transgenic or immune-suppressed mice. Knock-out transgenic mice can be made, in which the ovarian cancer gene is disrupted or in which an ovarian cancer gene is inserted. Knock-out transgenic mice can be made by insertion of a marker gene or other heterologous gene into the endogenous ovarian cancer gene site in the mouse genome via homologous recombination. Such mice can also be made by substituting the endogenous ovarian cancer gene with a mutated version of the ovarian cancer gene, or by mutating the endogenous ovarian cancer gene, e.g., by exposure to carcinogens.
A DNA construct is introduced into the nuclei of embryonic stem cells. Cells containing the newly engineered genetic lesion are injected into a host mouse embryo, which is re-implanted into a recipient female. Some of these embryos develop into chimeric mice that possess germ cells partially derived from the mutant cell line. By breeding the chimeric mice it is possible to obtain a new line of mice containing the introduced genetic lesion. See, e.g., Capecchi, et al. (1989) Science 244:1288-1292. Chimeric targeted mice can be derived according to Hogan, et al. (1988) Manipulating the Mouse Embryo: A Laboratory Manual CSH Press; and Robertson (ed. 1987) Teratocarcinomas and Embryonic Stem Cells: A Practical Approach IRL Press, Washington, D.C.
Alternatively, various immune-suppressed or immune-deficient host animals can be used. For example, genetically athymic “nude” mouse (see, e.g., Giovanella, et al. (1974) J. Nat'l Cancer Inst. 52:921-930), a SCID mouse, a thymectomized mouse, or an irradiated mouse (see, e.g., Bradley, et al. (1978) Br. J. Cancer 38:263-272; Selby, et al. (1980) Br. J. Cancer 41:52-61) can be used as a host. Transplantable tumor cells (typically about 106 cells) injected into isogenic hosts will produce invasive tumors in a high proportions of cases, while normal cells of similar origin will not. In hosts which developed invasive tumors, cells expressing an ovarian cancer-associated sequences are injected subcutaneously. After a suitable length of time, preferably 4-8 weeks, tumor growth is measured (e.g., by volume or by its two largest dimensions) and compared to the control. Tumors that have statistically significant reduction (using, e.g., Student's T test) are said to have inhibited growth.
Polynucleotide Modulators of Ovarian Cancer
Antisense and RNAi Polynucleotides
In certain embodiments, the activity of an ovarian cancer-associated protein is down-regulated, or entirely inhibited, by the use of antisense polynucleotide, e.g., a nucleic acid complementary to, and which can preferably hybridize specifically to, a coding mRNA nucleic acid sequence, e.g., an ovarian cancer protein mRNA, or a subsequence thereof. Binding of the antisense polynucleotide to the mRNA reduces the translation and/or stability of the mRNA.
In the context of this invention, antisense polynucleotides can comprise naturally-occurring nucleotides, or synthetic species formed from naturally-occurring subunits or their close homologs. Antisense polynucleotides may also have altered sugar moieties or inter-sugar linkages. Exemplary among these are the phosphorothioate and other sulfur containing species which are known for use in the art. Analogs are comprehended by this invention so long as they function effectively to hybridize with the ovarian cancer protein mRNA. See, e.g., Isis Pharmaceuticals, Carlsbad, Calif.; Sequitor, Inc., Natick, Mass.
Such antisense polynucleotides can readily be synthesized using recombinant means, or can be synthesized in vitro. Equipment for such synthesis is sold by several vendors, including Applied Biosystems. The preparation of other oligonucleotides such as phosphorothioates and alkylated derivatives is also well known to those of skill in the art.
Antisense molecules as used herein include antisense or sense oligonucleotides. Sense oligonucleotides can, e.g., be employed to block transcription by binding to the anti-sense strand. The antisense and sense oligonucleotide comprise a single-stranded nucleic acid sequence (either RNA or DNA) capable of binding to target mRNA (sense) or DNA (antisense) sequences for ovarian cancer molecules. A preferred antisense molecule is for an ovarian cancer sequences in Table 19A, or for a ligand or activator thereof. Antisense or sense oligonucleotides, according to the present invention, comprise a fragment generally at least about 14 nucleotides, preferably from about 14 to 30 nucleotides. An antisense or a sense oligonucleotide can be developed based upon a cDNA sequence encoding a given protein. See, e.g., Stein and Cohen (1988) Cancer Res. 48:2659-2668; and van der Krol, et al. (1988) BioTechniques 6:958-976.
RNA interference is a mechanism to suppress gene expression in a sequence specific manner. See, e.g., Brumelkamp, et al. (2002) Sciencexpress (21 Mar. 2002); Sharp (1999) Genes Dev. 13:139-141; and Cathew (2001) Curr. Op. Cell Biol. 13:244-248. In mammalian cells, short, e.g., 21 nt, double stranded small interfering RNAs (siRNA) have been shown to be effective at inducing an RNAi response. See, e.g., Elbashir, et al. (2001) Nature 411:494-498. The mechanism may be used to down-regulate expression levels of identified genes, e.g., treatment of or validation of relevance to disease.
Ribozymes
In addition to antisense polynucleotides, ribozymes can be used to target and inhibit transcription of ovarian cancer-associated nucleotide sequences. A ribozyme is an RNA molecule that catalytically cleaves other RNA molecules. Different kinds of ribozymes have been described, including group I ribozymes, hammerhead ribozymes, hairpin ribozymes, RNase P, and axhead ribozymes (see, e.g., Castanotto, et al. (1994) Adv. Pharmacol. 25: 289-317 for a general review of the properties of different ribozymes).
The general features of hairpin ribozymes are described, e.g., in Hampel, et al. (1990) Nucl. Acids Res. 18:299-304; European Patent Publication No. 0 360 257; U.S. Pat. No. 5,254,678. Methods of preparing them are well known to those of skill in the art. See, e.g., WO 94/26877; Ojwang, et al. (1993) Proc. Nat'l Acad. Sci. USA 90:6340-6344; Yamada, et al. (1994) Hum. Gene Ther. 1:39-45; Leavitt, et al. (1995) Proc. Nat'l Acad. Sci. USA 92:699-703; Leavitt, et al. (1994) Hum. Gene Ther. 5:1151-120; and Yamada, et al. (1994) Virology 205:121-126.
Polynucleotide modulators of ovarian cancer may be introduced into a cell containing the target nucleotide sequence by formation of a conjugate with a ligand binding molecule, as described in WO 91/04753. Suitable ligand binding molecules include, but are not limited to, cell surface receptors, growth factors, other cytokines, or other ligands that bind to cell surface receptors. Preferably, conjugation of the ligand binding molecule does not substantially interfere with the ability of the ligand binding molecule to bind to its corresponding molecule or receptor, or block entry of the sense or antisense oligonucleotide or its conjugated version into the cell. Alternatively, a polynucleotide modulator of ovarian cancer may be introduced into a cell containing the target nucleic acid sequence, e.g., by formation of an polynucleotide-lipid complex, as described in WO 90/10448. It is understood that the use of antisense molecules or knock out and knock in models may also be used in screening assays as discussed above, in addition to methods of treatment.
Thus, in one embodiment, methods of modulating ovarian cancer in cells or organisms are provided. In one embodiment, the methods comprise administering to a cell an anti-ovarian cancer antibody that reduces or eliminates the biological activity of an endogenous ovarian cancer protein. Alternatively, the methods comprise administering to a cell or organism a recombinant nucleic acid encoding an ovarian cancer protein. This may be accomplished in any number of ways. In a preferred embodiment, e.g., when the ovarian cancer sequence is down-regulated in ovarian cancer, such state may be reversed by increasing the amount of ovarian cancer gene product in the cell. This can be accomplished, e.g., by over-expressing the endogenous ovarian cancer gene or administering a gene encoding the ovarian cancer sequence, using known gene-therapy techniques, e.g. In a preferred embodiment, the gene therapy techniques include the incorporation of the exogenous gene using enhanced homologous recombination (EHR), e.g., as described in PCT/US93/03868, hereby incorporated by reference in its entirety. Alternatively, e.g., when the ovarian cancer sequence is up-regulated in ovarian cancer, the activity of the endogenous ovarian cancer gene is decreased, e.g., by the administration of an ovarian cancer antisense or RNAi nucleic acid.
In one embodiment, the ovarian cancer proteins of the present invention may be used to generate polyclonal and monoclonal antibodies to ovarian cancer proteins. Similarly, the ovarian cancer proteins can be coupled, using standard technology, to affinity chromatography columns. These columns may then be used to purify ovarian cancer antibodies useful for production, diagnostic, or therapeutic purposes. In a preferred embodiment, the antibodies are generated to epitopes unique to an ovarian cancer protein; that is, the antibodies show little or no cross-reactivity to other proteins. The ovarian cancer antibodies may be coupled to standard affinity chromatography columns and used to purify ovarian cancer proteins. The antibodies may also be used as blocking polypeptides, as outlined above, since they will specifically bind to the ovarian cancer protein.
Methods of Identifying Variant Ovarian Cancer-associated Sequences
Without being bound by theory, expression of various ovarian cancer sequences is correlated with ovarian cancer. Accordingly, disorders based on mutant or variant ovarian cancer genes may be determined. In one embodiment, the invention provides methods for identifying cells containing variant ovarian cancer genes, e.g., determining all or part of the sequence of at least one endogenous ovarian cancer genes in a cell. This may be accomplished using any number of sequencing techniques. In a preferred embodiment, the invention provides methods of identifying the ovarian cancer genotype of an individual, e.g., determining all or part of the sequence of at least one ovarian cancer gene of the individual. This is generally done in at least one tissue of the individual, and may include the evaluation of a number of tissues or different samples of the same tissue. The method may include comparing the sequence of the sequenced ovarian cancer gene to a known ovarian cancer gene, e.g., a wild-type gene.
The sequence of all or part of the ovarian cancer gene can then be compared to the sequence of a known ovarian cancer gene to determine if any differences exist. This can be done using any number of known homology programs, such as Bestfit, etc. In a preferred embodiment, the presence of a difference in the sequence between the ovarian cancer gene of the patient and the known ovarian cancer gene correlates with a disease state or a propensity for a disease state, as outlined herein.
In a preferred embodiment, the ovarian cancer genes are used as probes to determine the number of copies of the ovarian cancer gene in the genome.
In another preferred embodiment, the ovarian cancer genes are used as probes to determine the chromosomal localization of the ovarian cancer genes. Information such as chromosomal localization finds use in providing a diagnosis or prognosis in particular when chromosomal abnormalities such as translocations, and the like are identified in the ovarian cancer gene locus.
Administration of Pharmaceutical and Vaccine Compositions
In one embodiment, a therapeutically effective dose of an ovarian cancer protein or modulator thereof, is administered to a patient. By “therapeutically effective dose” herein is meant a dose that produces effects for which it is administered. The exact dose will depend on the purpose of the treatment, and will be ascertainable by one skilled in the art using known techniques. See, e.g., Ansel, et al. (1999) Pharmaceutical Dosage Forms and Drug Delivery Systems Lippincott; Lieberman. (1992) Pharmaceutical Dosage Forms (vols. 1-3) Dekker, ISBN 0824770846, 082476918X, 0824712692, 0824716981; Lloyd (1999) The Art, Science and Technology of Pharmaceutical Compounding Amer. Pharmaceutical Assn.; and Pickar (1999) Dosage Calculations Thomson. Adjustments for ovarian cancer degradation, systemic versus localized delivery, and rate of new protease synthesis, as well as the age, body weight, general health, sex, diet, time of administration, drug interaction, and the severity of the condition may be necessary, and will be ascertainable with routine experimentation by those skilled in the art. U.S. patent application Ser. No. 09/687,576, further discloses the use of compositions and methods of diagnosis and treatment in ovarian cancer is hereby expressly incorporated by reference.
A “patient” for the purposes of the present invention includes both humans and other animals, particularly mammals. Thus the methods are applicable to both human therapy and veterinary applications. In the preferred embodiment the patient is a mammal, preferably a primate, and in the most preferred embodiment the patient is human.
The administration of the ovarian cancer proteins and modulators thereof of the present invention can be done in a variety of ways as discussed above, including, but not limited to, orally, subcutaneously, intravenously, intra-nasally, transdermally, intraperitoneally, intramuscularly, intrapulmonary, vaginally, rectally, or intraocularly. In some instances, e.g., in the treatment of wounds and inflammation, the ovarian cancer proteins and modulators may be directly applied as a solution or spray.
The pharmaceutical compositions of the present invention comprise an ovarian cancer protein in a form suitable for administration to a patient. In the preferred embodiment, the pharmaceutical compositions are in a water soluble form, such as being present as pharmaceutically acceptable salts, which is meant to include both acid and base addition salts. “Pharmaceutically acceptable acid addition salt” refers to those salts that retain the biological effectiveness of the free bases and that are not biologically or otherwise undesirable, formed with inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid and the like, and organic acids such as acetic acid, propionic acid, glycolic acid, pyruvic acid, oxalic acid, maleic acid, malonic acid, succinic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, salicylic acid, and the like. “Pharmaceutically acceptable base addition salts” include those derived from inorganic bases such as sodium, potassium, lithium, ammonium, calcium, magnesium, iron, zinc, copper, manganese, aluminum salts and the like. Particularly preferred are the ammonium, potassium, sodium, calcium, and magnesium salts. Salts derived from pharmaceutically acceptable organic non-toxic bases include salts of primary, secondary, and tertiary amines, substituted amines including naturally occurring substituted amines, cyclic amines and basic ion exchange resins, such as isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, and ethanolamine.
The pharmaceutical compositions may also include one or more of the following: carrier proteins such as serum albumin; buffers; fillers such as microcrystalline cellulose, lactose, corn and other starches; binding agents; sweeteners and other flavoring agents; coloring agents; and polyethylene glycol.
The pharmaceutical compositions can be administered in a variety of unit dosage forms depending upon the method of administration. For example, unit dosage forms suitable for oral administration include, but are not limited to, powder, tablets, pills, capsules, and lozenges. It is recognized that ovarian cancer protein modulators (e.g., antibodies, antisense constructs, ribozymes, small organic molecules, etc.) when administered orally, should be protected from digestion. This is typically accomplished either by complexing the molecule(s) with a composition to render it resistant to acidic and enzymatic hydrolysis, or by packaging the molecule(s) in an appropriately resistant carrier, such as a liposome or a protection barrier. Means of protecting agents from digestion are well known in the art.
The compositions for administration will commonly comprise an ovarian cancer protein modulator dissolved in a pharmaceutically acceptable carrier, preferably an aqueous carrier. A variety of aqueous carriers can be used, e.g., buffered saline and the like. These solutions are sterile and generally free of undesirable matter. These compositions may be sterilized by conventional, well known sterilization techniques. The compositions may contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions such as pH adjusting and buffering agents, toxicity adjusting agents and the like, e.g., sodium acetate, sodium chloride, potassium chloride, calcium chloride, sodium lactate and the like. The concentration of active agent in these formulations can vary widely, and will be selected primarily based on fluid volumes, viscosities, body weight, and the like in accordance with the particular mode of administration selected and the patient's needs. See, e.g., Remington's Pharmaceutical Science (15th ed., 1980) and Hardman and Limbird (eds. 2001) Goodman and Gillman: The Pharmacological Basis of Therapeutics (10th ed.) McGraw-Hill. Thus, a typical pharmaceutical composition for intravenous administration would be about 0.1 to 10 mg per patient per day. Dosages from 0.1 up to about 100 mg per patient per day may be used, particularly when the drug is administered to a secluded site and not into the blood stream, such as into a body cavity or into a lumen of an organ. Substantially higher dosages are possible in topical administration. Actual methods for preparing parenterally administrable compositions are readily available.
The compositions containing modulators of ovarian cancer proteins can be administered for therapeutic or prophylactic treatments. In therapeutic applications, compositions are administered to a patient suffering from a disease (e.g., a cancer) in an amount sufficient to cure or at least partially arrest the disease and/or its complications. An amount adequate to accomplish this is defined as a “therapeutically effective dose.” Amounts effective for this use will depend upon the severity of the disease and the general state of the patient's health. Single or multiple administrations of the compositions may be administered depending on the dosage and frequency as required and tolerated by the patient. In any event, the composition should provide a sufficient quantity of the agents of this invention to effectively treat the patient. An amount of modulator that is capable of preventing or slowing the development of cancer in a mammal is referred to as a “prophylactically effective dose.” The particular dose required for a prophylactic treatment will depend upon the medical condition and history of the mammal, the particular cancer being prevented, as well as other factors such as age, weight, gender, administration route, efficiency, etc. Such prophylactic treatments may be used, e.g., in a mammal who has previously had cancer to prevent a recurrence of the cancer, or in a mammal who is suspected of having a significant likelihood of developing cancer based, e.g., in part, upon gene expression profiles. Vaccine strategies may be used, in either a DNA vaccine form, or protein vaccine.
It will be appreciated that the present ovarian cancer protein-modulating compounds can be administered alone or in combination with additional ovarian cancer modulating compounds or with other therapeutic agent, e.g., other anti-cancer agents or treatments.
In numerous embodiments, one or more nucleic acids, e.g., polynucleotides comprising nucleic acid sequences set forth in Table 19A, such as RNAi, antisense polynucleotides or ribozymes, will be introduced into cells, in vitro or in vivo. The present invention provides methods, reagents, vectors, and cells useful for expression of ovarian cancer-associated polypeptides and nucleic acids using in vitro (cell-free), ex vivo or in vivo (cell or organism-based) recombinant expression systems.
The particular procedure used to introduce the nucleic acids into a host cell for expression of a protein or nucleic acid is application specific. Many procedures for introducing foreign nucleotide sequences into host cells may be used. These include the use of calcium phosphate transfection, spheroplasts, electroporation, liposomes, microinjection, plasma vectors, viral vectors and any of the other well known methods for introducing cloned genomic DNA, cDNA, synthetic DNA or other foreign genetic material into a host cell. See, e.g., Berger and Kimmel (1987) Guide to Molecular Cloning Techniques from Methods in Enzymology (vol. 152) Academic Press; Ausubel, et al. (eds. 1999 and supplements) Current Protocols Lippincott; and Sambrook, et al. (2001) Molecular Cloning: A Laboratory Manual (3d ed., Vol. 1-3) CSH Press.
In a preferred embodiment, ovarian cancer proteins and modulators are administered as therapeutic agents, and can be formulated as outlined above. Similarly, ovarian cancer genes (including both the full-length sequence, partial sequences, or regulatory sequences of the ovarian cancer coding regions) can be administered in a gene therapy application. These ovarian cancer genes can include antisense applications, either as gene therapy (e.g., for incorporation into the genome) or as antisense compositions, as will be appreciated by those in the art.
Ovarian cancer polypeptides and polynucleotides can also be administered as vaccine compositions to stimulate HTL, CTL, and antibody responses. Such vaccine compositions can include, e.g., lipidated peptides (see, e.g., Vitiello, et al. (1995) J. Clin. Invest. 95:341-349), peptide compositions encapsulated in poly(D,L-lactide-co-glycolide, “PLG”) microspheres (see, e.g., Eldridge, et al. (1991) Molec. Immunol. 28:287-294; Alonso, et al. (1994) Vaccine 12:299-306; Jones, et al. (1995) Vaccine 13:675-681), peptide compositions contained in immune stimulating complexes (ISCOMS; see, e.g., Takahashi, et al. (1990) Nature 344:873-875; Hu, et al. (1998) Clin. Exp. Immunol. 113:235-243), multiple antigen peptide systems (MAPs; see, e.g., Tam (1988) Proc. Nat'l Acad. Sci. USA 85:5409-5413; Tam (1996) J. Immunol. Methods 196:17-32), peptides formulated as multivalent peptides; peptides for use in ballistic delivery systems, typically crystallized peptides, viral delivery vectors (Perkus, et al., p. 379, in Kaufmann (ed. 1996) Concepts in Vaccine Development de Gruyter; Chakrabarti, et al. (1986) Nature 320:535-537; Hu, et al. (1986) Nature 320:537-540; Kieny, et al. (1986) AIDS Bio/Technology 4:790-795; Top, et al. (1971) J. Infect. Dis. 124:148-154; Chanda, et al. (1990) Virology 175:535-547), particles of viral or synthetic origin (see, e.g., Kofler, et al. (1996) J. Immunol. Methods 192:25-35; Eldridge, et al. (1993) Sem. Hematol. 30:16-24; Falo, et al. (1995) Nature Med. 7:649-653), adjuvants (Warren, et al. (1986) Ann. Rev. Immunol. 4:369-388; Gupta, et al. (1993) Vaccine 11:293-306), liposomes (Reddy, et al. (1992) J. Immunol. 148:1585-1589; Rock (1996) Immunol. Today 17:131-137), or, naked or particle absorbed cDNA (Ulmer, et al. (1993) Science 259:1745-1749; Robinson, et al. (1993) Vaccine 11:957-960; Shiver, et al., p. 423, in Kaufmann (ed. 1996) Concepts in Vaccine Development de Gruyter; Cease and Berzofsky (1994) Ann. Rev. Immunol. 12:923-989; and Eldridge, et al. (1993) Sem. Hematol. 30:16-24). Toxin-targeted delivery technologies, also known as receptor mediated targeting, such as those of Avant Immunotherapeutics, Inc. (Needham, Mass.) may also be used.
Vaccine compositions often include adjuvants. Many adjuvants contain a substance designed to protect the antigen from rapid catabolism, such as aluminum hydroxide or mineral oil, and a stimulator of immune responses, such as lipid A, Bortadella pertussis, or Mycobacterium tuberculosis derived proteins. Certain adjuvants are commercially available as, e.g., Freund's Incomplete Adjuvant and Complete Adjuvant (Difco Laboratories, Detroit, Mich.); Merck Adjuvant 65 (Merck and Company, Inc., Rahway, N.J.); AS-2 (SmithKline Beecham, Philadelphia, Pa.); aluminum salts such as aluminum hydroxide gel (alum) or aluminum phosphate; salts of calcium, iron or zinc; an insoluble suspension of acylated tyrosine; acylated sugars; cationically or anionically derivatized polysaccharides; polyphosphazenes; biodegradable microspheres; monophosphoryl lipid A and quil A. Cytokines, such as GM-CSF, interleukin-2, -7, -12, and other like growth factors, may also be used as adjuvants.
Vaccines can be administered as nucleic acid compositions wherein DNA or RNA encoding one or more of the polypeptides, or a fragment thereof, is administered to a patient. See, e.g., Wolff et. al. (1990) Science 247:1465-1468; U.S. Pat. Nos. 5,580,859; 5,589,466; 5,804,566; 5,739,118; 5,736,524; 5,679,647; and WO 98/04720. Examples of DNA-based delivery technologies include “naked DNA”, facilitated (bupivicaine, polymers, peptide-mediated) delivery, cationic lipid complexes, and particle-mediated (“gene gun”) or pressure-mediated delivery (see, e.g., U.S. Pat. No. 5,922,687).
For therapeutic or prophylactic immunization purposes, the peptides of the invention can be expressed by viral or bacterial vectors. Examples of expression vectors include attenuated viral hosts, such as vaccinia or fowlpox. This approach involves the use of vaccinia virus, e.g., as a vector to express nucleotide sequences that encode ovarian cancer polypeptides or polypeptide fragments. Upon introduction into a host, the recombinant vaccinia virus expresses the immunogenic peptide, and thereby elicits an immune response. Vaccinia vectors and methods useful in immunization protocols are described in, e.g., U.S. Pat. No. 4,722,848. Another vector is BCG (Bacille Calmette Guerin). BCG vectors are described in Stover, et al. (1991) Nature 351:456-460. A wide variety of other vectors useful for therapeutic administration or immunization e.g., adeno and adeno-associated virus vectors, retroviral vectors, Salmonella typhi vectors, detoxified anthrax toxin vectors, and the like, will be apparent. See, e.g., Shata, et al. (2000) Mol. Med. Today 6:66-71; Shedlock, et al. (2000) J. Leukoc. Biol. 68:793-806; and Hipp, et al. (2000) In Vivo 14:571-85.
Methods for the use of genes as DNA vaccines are well known, and include placing an ovarian cancer gene or portion of an ovarian cancer gene under the control of a regulatable promoter or a tissue-specific promoter for expression in an ovarian cancer patient. The ovarian cancer gene used for DNA vaccines can encode full-length ovarian cancer proteins, but more preferably encodes portions of the ovarian cancer proteins including peptides derived from the ovarian cancer protein. In one embodiment, a patient is immunized with a DNA vaccine comprising a plurality of nucleotide sequences derived from an ovarian cancer gene. For example, ovarian cancer-associated genes or sequence encoding subfragments of an ovarian cancer protein are introduced into expression vectors and tested for their immunogenicity in the context of Class I MHC and an ability to generate cytotoxic T cell responses. This procedure provides for production of cytotoxic T cell responses against cells which present antigen, including intracellular epitopes.
In a preferred embodiment, the DNA vaccines include a gene encoding an adjuvant molecule with the DNA vaccine. Such adjuvant molecules include cytokines that increase the immunogenic response to the ovarian cancer polypeptide encoded by the DNA vaccine. Additional or alternative adjuvants are available.
In another preferred embodiment ovarian cancer genes find use in generating animal models of ovarian cancer. When the ovarian cancer gene identified is repressed or diminished in cancer tissue, gene therapy technology, e.g., wherein antisense RNA directed to the ovarian cancer gene will also diminish or repress expression of the gene. Animal models of ovarian cancer find use in screening for modulators of an ovarian cancer-associated sequence or modulators of ovarian cancer. Similarly, transgenic animal technology including gene knockout technology, e.g., as a result of homologous recombination with an appropriate gene targeting vector, will result in the absence or increased expression of the ovarian cancer protein. When desired, tissue-specific expression or knockout of the ovarian cancer protein may be necessary.
It is also possible that the ovarian cancer protein is overexpressed in ovarian cancer. As such, transgenic animals can be generated that overexpress the ovarian cancer protein. Depending on the desired expression level, promoters of various strengths can be employed to express the transgene. Also, the number of copies of the integrated transgene can be determined and compared for a determination of the expression level of the transgene. Animals generated by such methods find use as animal models of ovarian cancer and are additionally useful in screening for modulators to treat ovarian cancer.
Kits for Use in Diagnostic and/or Prognostic Applications
For use in diagnostic, research, and therapeutic applications suggested above, kits are also provided by the invention. In the diagnostic and research applications such kits may include any or all of the following: assay reagents, buffers, ovarian cancer-specific nucleic acids or antibodies, hybridization probes and/or primers, siRNA or antisense polynucleotides, ribozymes, dominant negative ovarian cancer polypeptides or polynucleotides, small molecules inhibitors of ovarian cancer-associated sequences etc. A therapeutic product may include sterile saline or another pharmaceutically acceptable emulsion and suspension base.
In addition, the kits may include instructional materials containing directions (e.g., protocols) for the practice of the methods of this invention. While the instructional materials typically comprise written or printed materials they are not limited to such. Any medium capable of storing such instructions and communicating them to an end user is contemplated by this invention. Such media include, but are not limited to electronic storage media (e.g., magnetic discs, tapes, cartridges, chips), optical media (e.g., CD ROM), and the like. Such media may include addresses to internet sites that provide such instructional materials.
The present invention also provides for kits for screening for modulators of ovarian cancer-associated sequences. Such kits can be prepared from readily available materials and reagents. For example, such kits can comprise one or more of the following materials: an ovarian cancer-associated polypeptide or polynucleotide, reaction tubes, and instructions for testing ovarian cancer-associated activity. Optionally, the kit contains biologically active ovarian cancer protein. A wide variety of kits and components can be prepared according to the present invention, depending upon the intended user of the kit and the particular needs of the user. Diagnosis would typically involve evaluation of a plurality of genes or products. The genes will be selected based on correlations with important parameters in disease which may be identified in historical or outcome data.
EXAMPLES
Example 1
Gene Chip Analysis
Molecular profiles of various normal and cancerous tissues were determined and analyzed using gene chips. RNA was isolated and gene chip analysis was performed as described (Glynne, et al. (2000) Nature 403:672-676; Zhao, et al. (2000) Genes Dev. 14:981-993).









TABLE 8B





Pkey
CAT Number
Accession







101249
2520_1
L18964 NM_002740 L33881 AA095249 BE080871 AW605320 M85571 AA179776 AA160650 AW117327 BE467131 AW088338 AW937631




AW087514 AI480090 AI873147 T57875 AI217404 AA827196 AI279471 AA969093 AA815168 AA988896 AI754623 T28044 AW950302 AW950294




AI032193 AI953696 AI630583 AA062633 BE541355 AA180493 AW015748 AA255651


100661
23182_1
BE623001 L05096 AA383604 AW966416 N53295 AA460213 AW571519 AA603655


116401
95855_1
AW893940 AW978851 AA034240 AI686323 AI767653 AA829515 AA053933 AA737691 W92607 AW261869 AA835698 AA447216 AI623248




Z21891 AA835700 AA599963 T20152 AA533167


116416
373989_1
AW753676 R11789 AW001886 AA609219 AW780420 AI860557 AI280331 AI334300 AI288870 AA669343 N29918 BE537790 AA934687 H79075




N42970 R63752


132191
54683_4
AA507576 AI610269 AI380079 R40309 AI203932 AI342128 AI342578 R43110 AW583269 AI375234 AI092708 R52802 AI028462 AI016062




AI189144 AI016691 W45515 AA551452 AA449431 T10046 AA424059 N62822 AW197701 AA465242 AI418989 AI942433 AI891115 BE302316




AI743979 AI283341 AW340338 AA774643 AW104778 AI078020 N21487 H97562 AA970063 BE327945 F03880 F03885 AA970699 AI298468




AI380330 AI247787 AA770467 AI200154 AI089863 AI089890 AI695738 W88524 AI471010 AA700191 AA778937 BE440182 R79225 AA338236




AA548984 AA907692 N21250 AW904736 AI909337 AA987772 AW959228 AI149372 N29644 AI039967 AA677529 AI694291 R85811 N28672




AA465598 AA321185 AW130492 AI824479 AI682992


130941
2774_1
NM_000869 D49394 BE252349 AW249320 AW249140 AW250535 S82612 AJ003079 AJ005205 AW178407 AA811360 AW976407 AW976408




AW248903 AA731733 AA804189 AA703169 AI435492 AI076288 AA912176 AW248713 AA743457 R08170 C06167 R02351


115909
47548_1
AW872527 AA453863 AA442475 AF086541 AA365801 AI692575 AW131631 AA732993 W96131 AA436666 AA453779 AA365504 AW959717




AA975337 AA365503 AI632902 AA659686 AA665087 C00396 AA988869


108778
18565_1
AF133123 NM_012086 AA128292 S81493 AL137453 BE614053 AA307628 BE009521 BE085659 BE085542 BE085598 AL120654 R13165




AA429306 R13465 R55236 AW994182 W00838 AW994417 AW994404 AW994426 AW994321 AA516147 AA345603 AW953009 BE315104




AI126654 AA626457 AA291327 H67983 H66271 H67976 AW270955 AA758221 AI023487 AI921811 AI953370 AF085850 R70992 N25129




AW295143 AI433661 AW608361 AA873402 AI217453 AI953358 AA262143 AA928495 AI475268 AI167211 AW385961 AA259045 AI762630




AA428238 AI001932 AI735550 AI951370 AA766807 S81492 AA918976 AI040967 R70939 AA469065 T70340 AA477615 AA478070 AI017743




AI608833 AI635824 AI186039 AA741312 AI040184 H67656 AA258221 AA731316 AI381293 AW298473 R55237 R37375 AI768014 AA128548 AI206773 AI879827 R64193


102136
17647_1
AA300576 U15552 NM_014597 AA223318 AA171806 BE269461 AW578439 AW604388 AW953513 AA772816 AW604383 AW577851 BE169672




AW117711 AW366303 AW366302 AW366308 AW366304 AW366300 AI908432 AW591937 AI358420 AW272622 C75067 AI926471 AW002266




BE064947 BE064722 T10372 AW838681 AI811119 AW262098 AA588547 AI916666 AI440083 AI078150 F24260 AA512919 AI953413 AI064798




AI420425 AA191324 BE503222 AI632721 AA180035 AA558329 W44843 T10610 W38442 BE542869 AI125024 BE279566 AW747936 AI589491




AA559096 AI090265 AA548959 AA223220 AW515936 AW368395 AW368407 BE540776 AI039762 AI584020 AA171691


108857
61_1
AK001468 AA190315 AA374980 AW961179 AA307782 AA315295 AA347194 AW953073 AW368190 AW368192 AA280772 AA251247 N85676




AI215522 AI216389 N87835 R12261 R57094 AI660045 AA347193 R16712 AW119006 N55905 N87768 AW900167 AI341261 AI818674 D20285




AI475165 AA300756 R40626 AI122827 AA133250 AI952488 AA970372 AA889845 AW069517 AI524385 AA190314 AI673359 AA971105 AI351088




AI872789 AI919056 AI611216 AK001472 BE568761 AA581004


102305
18424_1
AL043202 U33286 NM_001316 AF053641 AL048759 N99830 AA263091 AW408174 N90467 R84306 AA317882 BE613644 AA307378 T10722




AA207207 AA315560 AA113938 AW386317 AW386316 L44546 AW386335 AA243317 AA713588 AA192541 AA649035 BE300737 AW752491




AW902334 AW993922 BE003403 AA251521 AA382754 AA339152 AA382619 H58600 H67810 T70379 T82109 D81644 D60375 H59003 BE075732




AA471242 H17790 F11801 T84903 R78076 BE614356 R16380 R16395 AA876127 W95535 AA164768 AI279876 H02142 C18698 AA365866




AW954410 AI539769 T39128 AL121103 AA192466 AA213367 AI963800 BE090601 Z20096 BE566508 AI969470 BE044090 T65536 AA837311




AA075484 AA075621 AA778294 AA587266 T69722 AA446118 X85624 AI334209 AI587101 AI281280 AA568602 AA946837 C75603 AA236997




AA459274 AI150191 AA165156 AI198839 AA789258 AI139373 AA236574 AI127770 AA678954 AI140786 AA113939 AI187231 AI754062 AI753243




AA934719 AW439362 H02038 C17463 AI400951 AA227539 N66040 R89384 AA872668 AI344110 W95420 AA164700 C05669 BE094097




AI826398 H58956 T17222 AW139044 AI271344 T16445 R42323 C75565 AA165228 AI025443 AA165229 C21496 AI826239 AI868711 AA582354




AA524392 R01549 R01641 Z21083 AA528463 T39127 AA989472 F09450 AA084485 BE004378 AW974353 AA137250 AI278406 AW609291




AA137249 AA142866 AA639198 AW609271 AW149760 AI025112 AA236620 AA937248 U90736 AW005487 AW674427 BE397971 AW609285




T65602 T99684 T97378 AW609366 T85647 AI572235 T99083 AA199583 AW303874 T35523 AA586445 Z39669 AA459503 N95643 AW821210




AW813461 AW582064 AW609293 AW609320 AW609270 AW582085 AW582071 AW609318 AW813451 AW813456 AW582079 AW609276




AW609280 AW609290 AW582101 AW582102 AW609263 AW609317 AW609256 AW609305 AW582063 F06655 AW605343 AA446426 BE090595




AW969578 T79852 AI082505 N63239 AI973168 AI086182 AA846711 AI874213 AA730605 AI927257 AA912624 D60376 T10180 AA705847




AI018123 AA493197 T67083 R77739 AA953087 R00885 AI370606 R01642 AA862914 N57843 AW023353 H77483 H68082 R42337 H58601 T97267


109648
708849_1
H17800 AI362549 AI671064 T23526 F03426 F04694 F04600 AI635856 Z38715 F02039 AW022635


132528
11027_8
T78736 AA284422 AA283006


101804
26687_1
M86699 NM_003318 AL133475 AA122377 Z21415 R57092 AA806569 AA811904 BE538323 R41558 AA421620 AI337292 AA470077 AW795371




AA543024 AI677941 AI472200 AI215042 AA732384 AA837143 AA804229 AI907533 AI742701 AA121159 AI973225 AI620839


132572
31281_1
AI929659 AA227827 AF069765 AW408768 NM_006947 AF077019 AA220974 H07969 C14621 D52294 BE512960 BE614138 BE258539 BE251981




AA355433 AA481126 AW403053 BE542282 AI929818 AL120605 AW753079 AW391834 BE018603 BE395262 W21406 AW663259 AW975690




W93905 W96519 AI863832 AA443177 AA730942 T99558 T86581 W19444 N55583 AI701020 AI928986 AI857864 AI590849 AW081819 AA714970




AI122630 W04887 AW662427 AA602680 W93645 AA582946 AW008812 AA311187 AA463631 AI421918 AI400518 AI921404 AA143770 AA587675




BE302192 AA813080 AI493386 AW327435 AW340871 AI143616 AA687231 AA218961 AI362249 AI378345 N74716 AW969249 AA468581




AA516399 AI274726 AI131244 AI572604 AI929236 AW327971 R65637 N90309 H07877 W96486 AI358806 T90801 AI383246 AI740957 T86758




AI471248 AI864233 AA910590 AI079094 AW805781 AA709025 AW196707 AW327436 AI903790 AI873956 T99348 AI924643 AW103910 AI802993




AI080390 T99098 D19794 AW327972 AI935904 AI288575 AW360875 AA779784 N93574 AW769295 W32639 AA363094 N89012 W39751




AI291329 AI291371 AA829411 AI985219 AI422775 AA918940 AA363108 AA192633 AF086131 AA373679 AA165043 AA355705 AI243507




AI027796 AA573461 AA757260 AI370979 AA574149 AA558276 N70650 AI478948 R35393 AA448435 AA334659 AW879356 AA436527 AW972044




W25165 AA521219 AI094141 AI302096 AW578551 AW578534 AW390535 AI131472 N50381 AA736938 AI089112 AA863053 AI359793 AA962268




T27353 D82590 AA448297 AI277168 AI368457 AA872737 AA330346 AA308346 AA342341 AA355159 T85701 BE162893 T99703


131985
113870_1
AA503020 AI858190 AI686571 AW615203 AW073686 AW172459 AI828762 AW150534 AI859795 AA411046 AI539195 AA404609 AI638559




AA434329 AA171844 AI684143 AA953518 AW470108 AI870700 AA706376 AI539668 AI683712 AA075579 AI682137 AA291512 AA554431




H51315 AA404225 AA075632 AA172293 H51911


132624
42095_1
AA326108 W74020 AW612698 AI750909 AA487800 AJ270695 AA044941 H20708 AA296750 AA018401 AA378581 AW964159 AA018887 R68533




AA525338 AA526640 H84308 AA278942 AA164818 AA847110 T82335 N25519 AA021474 N31381 N36297 AA838191 AA318932 AA961206




N41430 N41439 AW630477 W37595 BE394538 AA365256 N47771 N34873 AA988105 AI242138 AW148523 AI978761 N50882 AA527448




AW086200 AI750910 N50868 AA709437 N51946 AI222179 AA732883 H96742 AW615360 N53720 W37490 R87362 AA613273 H98999 AI469022




AI368442 AI460122 N20486 N24087 AA164819 N24878 AW471270 AW590458 R68240 AA594434 N20400 AI419626 AW500664 AI033658




AA593215 AA907408 AA713508 AI422627 H85551 AA923571 D62680 AW627456 H96206 AA016289 AA485896 N25691


132632
4312_1
AU076916 BE298110 AW239395 AW672700 NM_003875 U10860 AW651755 BE297958 C03806 AI795876 AA644165 T36030 AW392852




AA446421 AW881866 AI469428 BE548103 T96204 R94457 N78225 AI564549 AW004984 AW780423 AW675448 AW087890 AA971454 AA305698




AA879433 AA535069 AI394371 AA928053 AI378367 N59764 AI364000 AI431285 T81090 AW674657 AW674987 AA897396 AW673412 BE063175




AW674408 AI202011 R00723 AI753769 AI460161 AW079585 AW275744 AI873729 D25791 BE537646 T81139 R00722


102610
9336_1
U65011 NM_006115 AW182053 BE383930 BE407839 BE409930 BE408826 AW370292 AA312859 AA136204 AW365852 AW365735 BE622732




AW939295 AA781195 AI017284 AW375329 AW375366 AW178384 AW178333 AW178424 AW365726 AF025440 AW172852 AI570998 AW117792




AI885499 BE465516 BE207427 AW130942 AW513316 AW770892 BE207426 AW173563 AW168292 AW173565 AI810101 AI744983 AI861974




BE207404 AI744982 AI613210 AW591505 AW169285 AI521444 AI745044 AI627904 AI690634 AI289305 AA861253 AI612799 BE207425 AI149694




AA902662 AI082468 AI014752 AA613844 AA725693 AA136089 AI290092 AA565489 AI689083 AI859014 AW051225 AA665758 AA496991




AA564738 T19428 AI567170 AW166726 AW084200 AW188723 AA617626 AI918664 AW381473 AW381543 AA598817 AW088942 AW050423




AI362502 AI680308 AI687500 BE327836 D20455 BE410282 BE254766 BE256014 AA357423 BE618208 AA489577 AW182114 BE379147


102627
25245_1
AL021918 AA160639 U66561 AA321623 U52098 AL119453 AA455712 N80080 N46550 W07223 N75923 W05057 AI811577 AA455657 AI275409




AI139121 AI927568 AI927562 AI139471 AA160473 N78795 AI719983 AI718928 AA723097 AI335776 N39140 N59184 AI587600 AI864812




AA732097 N74667 AA832398 H89600 D19825 AI554833


132725
29101_1
NM_006276 NM_006276 L41887 L22253 BE379909 BE567870 BE274265 BE539518 AW239523 AW239271 AI093618 BE504485 AA580279




AA494481 BE440161 AW780428 BE543960 D55986 AA852399 AI630020 W77996 AA278193 R10505 AI963201 AI739336 BE174301 AA662222




AA664912 AA244152 AW611553 BE503285 AA211023 AA383016 AI698174 AW195381 AA948229 AI768495 AI690437 N30025 AI718952




AI953572 BE464509 AA777315 AI337221 AW070910 AI953848 AW674561 H54177 AW510890 AW078699 AI436178 AA630759 BE502074




AA278769 AI499038 AW469072 AA778071 AW236753 AI933033 AI690458 AI276691 AW768235 AI952118 AA425156 AA610579 W73953




AA244153 W86034 AI948872 AI952678 AW087811 AI333591 AI869883 AI926911 W48865 AL048024 AA214485 AI972522 AI151368 W48738




AA214467 AA334640 AI678170 AA927525 AA581588 W96283 AA365470 AI471919 AW611488 AA211834 AI365198 AI698365 AW002238




AA507624 W96150 AA446490 AL048025 AA852400 AA362221 AI338376 R35083 AA290812 R10397 AA975988 AW236462 Z43032 H16969




F13487 D19858 AA452207 BE085942 AA344396 AW949533 AA279472 AW902406 AW070440 BE395195 H00835 AA300750 AA729303




AA420591 AA385025 AA420542 R69155 AA420592 AA281747 N88502 AI458206 AI700996 AW418607 AW341202 AI825692 W00640 AA214405




AA044744 AI950617 BE467493 AW474113 AA446310 BE328705 AI911573 AW243968 AI628622 AW173020 AW079958 AI140387 AW051969




AW299438 AI127170 AW769164 AI422435 AI307116 BE549519 AI371116 AA281748 AA701073 AA679948 BE551197 N50345 AW338776




BE326601 AI142892 AW470687 AI989568 AA911241 AW294822 AI174414 AA804366 AI004725 AW271994 AI559313 AI270102 AI351542




AW768904 AA765964 AI961708 AI149231 AA995907 AI094280 AI185753 H01219 AW768846 AA747500 AA970106 AI601238 AA513452




AW612802 BE075163 R39171 AI565328 AI375559 F10356 AA284625 BE241509 AI702889 AW193010 AA649847 AW439150 AA721407




AA810333 AA706384 AI049887 BE569015 BE622280 BE566618 AW967342 R69269


111234
83711_2
AA902656 AI185915 R43705 H15150 H09794 AA832464 AI697438 AI354538 AI436354 AA948272 AA928143 AI091263 R41658 AI352580




AI122948 AA946670 AI340088 AI275007 N70255 AA721176 AI934162 AA827098 AA935934 AA827088 AI081207 AA992399 AW130757 AI805667




AA035556 AI379266 AI093901 AI095234 AA909079 AA516079 AI572357 AA205969 AI432383 AA905290 AW628920 AW182996 AI266084 N49879




AW024457 AI246246 AI934031 AI369270 AI003836 AA010063 AA494361 AI284151 AI919536 N34884 N69287 AW510465 AI358609 AW081421




AA706205 AI085317 AI140633 AI347104 AA602547 AI686707 AA872686 AA694028 AI094546 Z40832 AI382838 AA610132 AA501433 H84120




AI140722 AW674839 BE503822 AW663895 BE327472 AW393494 AI340087 W04189 AW393499 H56506 BE089878 BE301950 AI025475




AA724446 BE275324 H15210 AW957667


134161
16074_1
AA634543 AI682259 AF117108 BE396917 NM_006547 U97188 U76705 BE560799 BE396918 BE269531 BE560268 BE560346 AA836048




AL023775 BE545535 AA427803 C18804 D58801 AA303353 U46218 BE539704 AA187966 AA252545 AA261821 D63197 AI824109 AI088047




AI424833 AI807368 AI250857 AA741476 AI146832 AA169615 AI809821 AI274288 AW136704 AI206172 AA917039 AA243584 AI808611




AW674709 AA935733 AW450092 AA905172 AA471196 AA302256 AW673348 AI352044 AW511295 AA247134 W81035 AA722962 AW662471




R64432 AW044616 AI086619 AW628546 AW043682 AA425750 AI743038 AI368723 AA187143 AI376987 AI803976 AI275537 AW471358




AW104877 AA195464 W81072 AW197351 AA932674 AI393420 AI434998 R63822 AW085083 AI240272 W87006 AA011347 H58428 AI497895 R23223


132939
11857_1
AB009284 AF000416 AA022636 BE082610 AW367997 AA491410 AA337477 AA336421 W38526 AA625283 AA773685 AA490078 T66134




AA847838 AA022647 AW054726 AI918001 AA431966 AI263596 AI804298 AW469314 U76189 AA779001 AA625945 AL042357 AI674730




AA410350 AK001450 AV654353 AA058443 D81618 AA853665 W31930 AA334445 AW955767 N47777 AA883784 AA428916 AI652062 AA329703




AI417923 AI435031 AA708791 AI989636 AI220345 AI239913 AI220102 AI435875 AI076731 AI377049 AI039173 AW972638 N90076 AW263652




BE440048 BE440013 AA577463 AI038774 AW204992 AA846580 AA501952 AA342058 AA508525 D61670 W31725 AI689499 AI955969 AA526628 AI282717 T66198


111345
6692_1
AW263155 AA314512 AW408152 AA360413 BE206274 AK001402 AA307665 AW954678 W39078 AW369236 AW369115 AW369096 AW753235




AW369072 AW361194 AW369125 AW364187 H94225 T79502 AA131908 BE071359 AW368503 AW801517 AW801322 AI674163 AA861077




AI738568 AI830199 AW118577 AI478895 AI688497 H92996 AW083479 AW970625 AW613124 AI632234 AI654210 AI696847 AI923423 AA131909




AA806630 AA806158 AW592520 R96509 AA813923 AA502823 AW467889 AA960972 AA994566 AI280346 AA884588 AA653563 AI200023 N89820 AW768792 C000145 R96554




T79416


104301
145380_1
AA768491 AA476251 AA809748 AI186268 AA621244 AI379029 BE550341 AA651915 AI216376 AI215585 AI471780 AA772159 AW181980




AI151169 AA759270 AI675769 AI018776 AA757335 AA148511 AI138378 AA504167 AI420617 AW261930 AW872797 N51769 AW614403




AI860533 AW573108 N64830 AI693732 AI436159 BE501089 AI436163 AA971485 AI269364 AI935358 AI222050 AW303978 AW573247 AI871154




T16758 AI765893 AW969016 AA744720 BE094085 AA743769 AI476407 AA156619 AI768535 R81435 D45332 N75682 N51177 AW207406




AA425184 R20997 AA504168 Z43298


134520
13358_3
BE091005 BE541579 AW387738 AW386083 H13769 AW377820 AW369180 AW753239 AW672695 BE379572 AW021732 AW891450 AW891416




BE091358 N50375 BE091354 BE091365 BE091361 BE091363 BE091350 AA353863 AA845510 N21407 AW770981 AI361577 AA526557




AA525443 AW893622 AA630898 AI418983 BE172016 AA550754 AA664574 C16147 AA355902 AW958586 N51590 C20995 BE544186 AI337578




AA090549 AI807374 AW450654 BE067578 AA446781 AA447058 T10807 AA457082 AI267703 AI880220 BE568979 AW380506 BE150744




AW380468 AW380546 BE150713 D60029 H88099 BE546301 BE150731 AW368467 BE091348


135242
5782_1
AI583187 M73812 AW339829 M74093 BE252510 BE252518 BE536901 U40788 W95578 BE018493 BE544205 N83637 AI671049 AW439693




AW300786 AW374970 AA592960 AI215885 AI215884 BE302101 AI186210 AW771831 T54213 AW452924 AA834019 W95471 AA628312




AW304866 AA570076 AI559873 T54121


134621
27351_1
AA037145 L02547 NM_001324 AW411516 AA314711 AA143605 BE394455 AA325731 AA093227 AA083307 BE299438 BE295669 AA370886




AA338272 AA166862 BE304837 BE298306 R60507 AW238966 N72750 AW505406 AW994153 AA309742 AA309929 BE090721 AW904189




AW904214 AA363564 R94127 AA352101 R28249 AA206337 AW577208 AA385473 AA355463 AA400696 AA075587 W72815 AA554033




AA075530 AA620555 AA554034 T27804 AW950014 AI570740 AI268538 AA704423 AW411517 AI278646 AW339924 AI668917 AI796034




AA994601 R94082 AW027137 AA400652 AW299746 W72816 AA988494 AA146582 AW087489 AA992763 AW516454 AA992759 AW270444




AA227188 AI208929 AA167720 AI052527 AA865660 AA569368 AI888464 AI670003 AA827620 AA507543 AI888385 R88418 AW959083 AI341077




AI825719 BE552285 AA738076 AW085903 R28242


126802
116467_4
AW805510 AW805503 AW805500 AW805819 AW517040 AW473670 AW516701 T30141 AA894497 BE349504 AI272007 AI985274 BE501962




AW102975 AI801727 AW197918 T24046 AA947601 AW900958


126892
38252_1
AF121856 BE242657 U83194 AA226732 AI160190 AA948725 AI079958 AW513369 W39443 AW408479 W06854 AA094683 AI985095 AA316647




H96313 N78438 R81582 H95034 R79674 AA488552 W25292 W31697 W19918 T30640 R08686 H78637 AA165100 Z41909 AA165080 R34212




AA150886 T82168 N77082 W56864 R19848 AA888217 AA314539 AW750293 N62714 R58039 AA845453 N63268 W03474 N41923 AI264123




AI808533 AA824288 AW198143 N99916 AA902465 AA775397 AA772387 AI567675 AA227473 AI082614 R58334 N78878 R34329 AW438902




AA164685 AA558790 AI590102 AA863422 BE002625 AA934039 AI298102 W15351 H93997 AA725325 BE180993 W05350 AI510771 W06941




AA488414 R79863 N91264 R76884 AA312948 T71267 AW959659 AI086695 N90421 AI278098 AA164538 AI300271 AA854381 R81331 AA700449




H06174 AW518427 AA876634 AA150778 N32393 H78585 T85364 BE002808 AW663196 N90337 BE252097 T71401 H06438 N40268 N31015




R77046 T99588 T85462 W25298 T59815 H09416 T85403 W32150 N79109 R78812


105298
8689_1
BE387790 BE276849 BE246825 BE246900 AA380487 AA332996 AW408727 AK000294 AI636887 AW197272 AW590657 AW594006 AI768979




AI751632 AA580098 AA313261 AA300475 AA133237 AA233499 BE242126 BE242597 BE242254 AA314374 W961210 AW939345 AA173535




AA305779 R99373 AA128304 AA447246 AA476365 T34973 BE264878 N25149 Z24939 BE263038 AA128317 Z46082 H23504 AW378551




AA088533 AA442625 H15235 H79172 AA344697 AA344742 AL040280 BE173256 BE173129 T59749 AA088410 BE242311 AA173576 AA455337




AA129715 AA582953 AW612637 AA917038 AW340019 AW006315 AW593024 H05915 AW294592 AI146814 AW195659 AW440271 AI209090




AI288689 H98630 H15174 AI214454 AI569471 AA085808 AW188361 AI751631 AW440477 AW300860 AA516088 AI365108 AI305805 AI264064




AI246276 AA442611 AA807059 AA233459 AA875987 AI374653 AA972665 AA947515 AA436867 AI216423 AI657181 AI680306 AA436854




AI537153 AA883723 Z28659 AA705973 Z40741 AA463884 AI216025 AI564986 AA476316 AI432566 AI571662 AA447126 AW293675 AI675617




AW009004 H23498


120438
166102_1
AW015242 AA831493 AI292346 AI076966 AI191561 AA243441 AI183309 AA252613 AI038422 AI306531 AW051480 AI348605 AA195119




AI817119 AI091896 AA738440 AA195013 AA976687 AA459659 AI246250 BE219252 AI703457 AA243291 AA243401 AA989100 AA931640 AA459782


105516
9334_1
AK001269 AL354613 AA147472 AA490803 BE207628 AW816113 AA085574 AW503392 AA299910 AW750305 BE079539 BE079484 BE512838




AK001593 AW968772 AW967440 AW206280 AA251270 AI627886 AA303599 AA147473 BE206616 AA490611 AA715039 AW590866 AW590447




AI864512 AA204731 AA894490 BE001136 AA612785 AA237035 AA149960 Z44257 R12986 AA448446 AI734041 AA422167 BE220551 R66041




R32927 R32942 AA258773 AW386142 R53730 N54624 AW880296 AA253485 AW954441 H98989 AW614348 AI654838 AA779793 AW237213




N66635 AI186812 AA947479 BE158011 AI859480 AW805579 N52010 AA806305 AI628445 AW270990 AA778165 AA149949 AI650728 AA749108




AA687257 AI261661 AA747442 AA481351 AA206339 AA903407 AW473306 AI688930 AA262281 AA448310 AA748820 AI347430 BE465692




R32839 AW510564 AA436408 AA257971 AA253362 AA938330 AA513150 AA976840 AA687117 AI281547 AA046243 R32825 AI631554




AW139818 AI244536 R52946 AW235443 R40183 AA299909 AA811958 AI302918 Z40213 BE158047 BE158060 AA767245 AW748159 AW500735




AA094074


129097
25953_1
BE243933 AA355449 T29766 F08396 N83324 NM_006963 S50223 AI207648 AA258092 AA113952 AI311718 AI128612 AW607449 M77172




AI951311 X52346 AA903307 AI569810 N55421 W77876 R37223 R83788 AA031666 H47092 AA133451 AA311095 AA906963 H87667 N56058




AA393593 W24864 H10710 F06925 F07239 AW386140 AA325018 AA235950 AW373176 N57158 AA258093 N39467 R21609 BE089979 R34173




AW889005 AA745644 AI693852 AA424914 AA744771 W72632 AI291213 AA524318 AI472134 AI911230 AA528418 AA115745 AA775720




AI671134 AA975044 AW298117 AA321015 N26288 AW952194 AI743379 AI204233 AI801026 AA830690 AI146980 AW104611 AI338576 R21507




AI367623 BE244484 AI269308 AA031667 AI884346 AA731989 AA988943 AA235951 AA807887 AA642645 AI246489 N29739 AI216718 AI383349




AI038618 AI351476 AA806031 AI914178 H10711 AI095573 H89220 AW470854 AA729015 R83353 AA782239 R34295 H87165 AW419059




AI653689 Z40349 H89114 AW074506 AA397785 AA888377 AI911228 F03193 AI468783 AA702615 AI830829 AA748323 R37224 AA424915




AA731647 H47183


120619
169895_1
AW965339 AL045632 AA333229 AI806195 AA284372 AA206108 AA682533 AW449514 AA804785 AI215473 AI357263 AI651208 AI651753


129229
20927_1
AF013758 NM_006451 AI538709 AA209236 AA300293 AA367274 AA126598 AA324825 AW955225 F11436 AW374740 AW374714 AW374774




AW751514 W74780 AI909015 AW997079 AW997067 AW379344 AW363397 W38589 AA043823 BE169280 AI909016 AW994851 AI740638




AW148560 AW368339 AI858333 AA314718 AW954872 AW468734 AI681980 AW519045 AW055171 AA579286 AW069164 AW615004 AA345052




AI446735 AI142106 AA662683 AW002813 AI418280 AW613203 AI613333 AI354480 AI929755 AI146977 W74674 AW799610 AI798529 AI589422




AA043957 AI223043 AA157016 AI446759 D56729 AI587471 F30716 AA812125 AI537301 AA653347 D11966 AI434383 AA598533 AI287254




AW139140 AW051033 AA601911 AI702506 AA737460 T30221 AI129081 N90213 AA805225 AI798518 BE001071 T10841 W20199 AW664594




AW195667 D60123 D61496 AW468018 AI720097 N90553 AA829375 AW513266 H92758 AA585324 C14767 AI922391 D60124 D60666 AW071558 BE044120 AA728821 AA211941


120821
19274_2
Y19062 NM_014393 AW296801 AK001576 AL079288 W16667 Z45664 AI768561 AL079286 R12736 AW080147 AW136530 AI202958 AW241579




R21013 AA347419 AI929333 AW196689 AI040867 F13437 AA918240 AI869798 AI365176 AW440030 AW440072 N80892 AW242030 Z44807




R12417 AA436784 AA442041 AA046503 AL157526 AI929265 AA055542 AA045462 AA683542 N51374 AW193508 AI873524 AW473151




AW004719 AI810504 AI581093 AA493977 Z40600 F04553 R46130 F09321


106459
3897_1
AA789081 AW408328 NM_006530 U61384 AA449641 AW138216 AA448598 AJ245746 AI365301 N44728 AA255743 AA360783 BE550380




AW593925 AI962309 AA322097 AW964625 AI695988 AW672827 BE543256 AK001413 AW603395 AA651700 AA449053 AA465540 AW083185




T62128 Z78373 AW673713 AW468061 BE350755 AW673958 AW675504 AA995709 AW574841 AA835883 AI248439 AA548364 T62072 N33193




AA814046 AI376210 AI340020 AA449766 AA703407 AA427613 AI470108 AI298757 AA507602 AI658941 AA449478 AA633165 AA449741 AA831821 AA903673 AA682588




AW673075


115094
190995_1
AA255920 AI817197 AA255921 AI612925 AW874669 AA493440


129571
1726_1
X51630 M80232 X61631 S75264 AA172249 AA134066 AA130278 AA130187 AA130291 AA031554 AI246677 Z21455 AI745434 AW273544




AW088613 AW471307 AI745483 AI399854 AI683952 AA031555 AA298075 AI935945 T29809 AA172099 AA356120


121779
287665_1
AW513143 AA422036 AI821669 AW514232 AA477828 AW772009 AW439799 AW089884


106738
174703_1
AW149266 R49246 AW237401 AA938113 AW665871 AI969698 AI950812 AW874276 AI571939 AA741222 AI869822 AW104061 AI569994




AW972559 AA506012 AI553704 AA470145 AI332421 AA218990 AW131361 AI709076 AW392488 AW392477 AI970981 AW392476 D61949 H44981 BE172698


123005
75629_1
AW369771 AW748174 AA290801 AA419198 AA044331 AA127909 AW995442 AI480343 AA044582 AW956159 AA373451 AA127965 AL134913




AW994956 BE622314 BE006298 BE006312 BE006305 BE006317 BE006303 AA043906 AA234175 AA479726


108055
100690_1
AJ404672 AJ289819 AW976000 AA043561 AW450885 AW452879 AA043562 AA788832 AI564338 AI564330 AI368875 AA643607 AA994375 AA810342 AI367704


115291
22325_1
BE545072 AI540751 AA301103 AI916675 N85422 BE563965 AA327978 AI816094 AK001515 BE501319 AA279943 BE138895 AA343765




AW963051 AW082308 AI823992 AI653752 AI589007 AI816135 AI566535 BE501307 AW272765 AW242239 AA766315 AI014927 AA578848




AI354483 AI476548 AI038579 AA973322 AA992180 AW472921 BE504789 AI392988 AA506076 AA769228 AI370562 AL137710 BE005656 AW965920


130376
24827_−4
R40873


115536
61_1
AK001468 AA190315 AA374980 AW961179 AA307782 AA315295 AA347194 AW953073 AW368190 AW368192 AA280772 AA251247 N85676




AI215522 AI216389 N87835 R12261 R57094 AI660045 AA347193 R16712 AW119006 N55905 N87768 AW900167 AI341261 AI818674 D20285




AI475165 AA300756 R40626 AI122827 AA133250 AI952488 AA970372 AA889845 AW069517 AI524385 AA190314 AI673359 AA971105 AI351088 AI872789 AI919056 AI611216




AK001472 BE568761 AA581004


114965
153955_1
AI733881 AA165164 AI826437 AI972791 AA165165 BE219575 AI732586 AI821571 AA250737 AW136875 AI984273 AI249271


131228
8262_1
AW207469 AL079814 AA354351 AF020043 AW291396 BE550484 NM_005445 BE046917 AW594249 AI651554 AI631515 AW771344 AI969758




AI699982 AA247175 AI244676 D44780 AW593978 AI638479 AI373676 AW089547 AL121432 AA554698 AI016991 AI087260 AW449939




AF067163 W40482 AW316558 AI537184 AW381979 W40150 AI810562 AA573151 AI630288 AI675561 AI674420 AW840733 AW022653




AA114219 AJ005015 AL046587 AA878141 AW271896 AW085287 AA150465 BE536295 AA463412 BE093222 AA213739 AA485586 AI825913




AA706307 AI337348 R31995 AI819641 R32095 AW976653 AA742375 AA142957 AI808214 AW468303 AI205987 AI206347 AI769095 BE501640




AA113866 AI093931 AI752855 AA612743 AA463411 AA279157 AI123791 AA213570 AI207305 AW627814 R31945 R32040


116238
10772_1
AV660717 NM_015437 AL050285 R95774 AI867094 AA443833 AI367670 AA609046 AI440298 AI613139 AI291826 AW028954 AI123242 AI824715




AW079750 AA479362 AW150151 AI952267 AA814094 AI168431 AI566595 AI521422 AI920793 AW051241 N70051 AI689429 AI783813 AI769315




AI743691 AI915645 AA479473 C21435 N50944 N50902 AW978102 H23837 BE087538 AA316516


122802
287993_1
AI687303 AW571681 AI554465 AI684252 AI581056 AA604098 AI628160 AI859843 AA424021 AA460530 BE042778 AW273200 AW273223




AW167288 AW083347 AI654306 AW517496 AW104706 AW273214 BE139512 AW189487 AW130822 AW167419 AI289485 AW150010 H88004 AI743745 AW088710


123494
21202_1
AW179019 AW179011 AF135160 NM_014050 AF078860 BE018005 AK000285 AF151038 BE245156 AW179007 AA345114 BE619758 BE619209




W25509 AA314339 AA336674 AA337956 AW954843 AW390412 N46796 AA316235 AA314286 R15686 BE535633 N57134 N46483 AW368462




AA923517 AA665223 AI418513 AA837523 AI359320 AI309273 AI522278 N40939 AA904977 AA938272 N30240 AA887965 AI671972 AI028109




AA094652 AA883262 AA887781 AI744447 AW592944 AI077790 AW860883 AW148667 N89861 AA557195 AI191824 AI433166 AI719760




AA453089 AA630656 AA300976 AA639620 AW675033 AA284393 AW886987 AI476335 AI332939 BE301513 AA452920 AW674302 AI925483




AW170412 AI698717 AI375985 BE220535 AI688151 AW514809 AW062346 AA599786 BE350848 AI560848 AI023075 AA864875 AA166871




AI807947 AW514579 AI978602 AI860340 AA830886 AI374788 AI283592 AA683152 AA743159 AI379932 AI432056 AI128904 AW150433 N38909


116296
11967_2
AW149502 Z43342 AW002826 AL049382 AA442545 AW971471 BE220243 AW968952 AA043607 AW299245 AA659892 AI038768 H26330




BE463534 AI628252 AA836139 AI277291 AA489033 AA741239 AI209064 AI300253 AI275761 Z39417 C01835





Pkey: Unique Eos probeset identifier number


CAT number: Gene cluster number


Accession: Genbank accession numbers






Table 10A lists about 733 genes up-regulated in ovarian cancer compared to normal adult tissues. These were selected from 59680 probesets on the Affymetrix/Eos-Hu03 GeneChip array such that the ratio of “average” ovarian cancer to “average” normal adult tissues was greater than or equal to 3.0. The “average” ovarian cancer level was set to the about the 80th percentile amongst various ovarian cancers. The “average” normal adult tissue level was set to the 90th percentile value amongst various non-malignant tissues. In order to remove gene-specific background levels of non-specific hybridization, the 15th percentile value amongst the non-malignant tissues was subtracted from both the numerator and the denominator before the ratio was evaluated.







TABLE 10A







ABOUT 733 UP-REGULATED GENES, OVARIAN CANCER VERSUS NORMAL ADULT TISSUES











Pkey
Ex. Accn
UG ID
Title
ratio














432938
T27013
Hs.3132
steroidogenic acute regulatory protein
56.1


418179
X51630
Hs.1145
Wilms tumor 1
33.5


400292
AA250737
Hs.72472
BMPR-lb; bone morphogenetic protein receptor
30.0


452838
U65011
Hs.30743
Preferentially expressed antigen in melanoma
29.5


415511
AI732617
Hs.182362
ESTs
28.1


422956
BE545072
Hs.122579
ESTs
28.1


410929
H47233
Hs.30643
ESTs
27.4


400289
X07820
Hs.2258
Matrix Metalloproteinase 10 (Stromolysin 2)
25.2


449034
AI624049
Hs.277523
gb: ts41a09.x1 NCI_CGAP_Ut1 Homo sapiens cDNA
23.7


427585
D31152
Hs.179729
collagen; type X; alpha 1 (Schmid metaphyseal
22.7


428392
H10233
Hs.2265
secretory granule, neuroendocrine protein 1
21.9


448243
AW369771
Hs.77496
ESTs
21.3


430691
C14187
Hs.103538
ESTs
21.2


444783
AK001468
Hs.62180
ESTs
20.8


407638
AJ404672
Hs.288693
EST
20.1


423739
AA398155
Hs.97600
ESTs
19.7


436982
AB018305
Hs.5378
spondin 1, (f-spondin) extracellular matrix p
19.0


451110
AI955040
Hs.301584
ESTs
18.8


426427
M86699
Hs.169840
TTK protein kinase
18.7


428227
AA321649
Hs.2248
INTERFERON-GAMMA INDUCED
18.3


419854
AW664873
Hs.87836
Homo sapiens PAC clone RP5-1087M19 from 7q11.
18.3


439706
AW872527
Hs.59761
ESTs
18.3


428579
NM_005756
Hs.184942
G protein-coupled receptor 64
17.4


410247
AF181721
Hs.61345
RU2S
17.0


428153
AW513143
Hs.98367
hypothetical protein FLJ22252 similar to SRY-
16.9


415076
NM_000857
Hs.77890
guanylate cyclase 1, soluble, beta 3
16.6


416209
AA236776
Hs.79078
MAD2 (mitotic arrest deficient, yeast, homolo
16.6


424905
NM_002497
Hs.153704
NIMA (never in mitosis gene a)-related kinase
16.2


423685
BE350494
Hs.49753
Homo sapiens mRNA for KIAA1561 protein, parti
15.9


428187
AI687303
Hs.285529
ESTs
15.9


438817
AI023799
Hs.163242
ESTs
15.9


424906
AI566086
Hs.153716
Homo sapiens mRNA for Hmob33 protein, 3′ untr
15.9


407721
Y12735
Hs.38018
dual-specificity tyrosine-(Y)-phosphorylation
15.7


412723
AA648459
Hs.179912
ESTs
15.3


424717
H03754
Hs.152213
wingless-type MMTV integration site family, m
15.2


443646
AI085198
Hs.298699
ESTs
15.1


424345
AK001380
Hs.145479
Homo sapiens cDNA FLJ10518 fis, clone NT2RP20
14.8


428976
AL037824
Hs.194695
ras homolog gene family, member I
14.6


418738
AW388633
Hs.6682
solute carrier family 7, member 11
14.3


428479
Y00272
Hs.184572
cell division cycle 2, G1 to S and G2 to M
14.2


436209
AW850417
Hs.254020
ESTs, Moderately similar to unnamed protein p
14.1


427356
AW023482
Hs.97849
ESTs
13.9


418601
AA279490
Hs.86368
calmegin
13.8


416661
AA634543
Hs.79440
IGF-II mRNA-binding protein 3
13.7


428532
AF157326
Hs.184786
TBP-interacting protein
13.6


402408


0
13.6


447350
AI375572
Hs.172634
ESTs; HER4 (c-erb-B4)
13.4


451807
W52854
Hs.27099
DKFZP564J0863 protein
13.4


423575
C18863
Hs.163443
ESTs
13.2


443211
AI128388
Hs.143655
ESTs
13.2


437872
AK002015
Hs.5887
RNA binding motif protein 7
13.0


451659
BE379761
Hs.14248
ESTs, Weakly similar to ALU8_HUMAN ALU SUBFAM
12.7


452904
AL157581
Hs.30957
Homo sapiens mRNA; cDNA DKFZp434E0626 (from c
12.7


442655
AW027457
Hs.30323
ESTs
12.5


452096
BE394901
Hs.226785
ESTs
12.4


414972
BE263782
Hs.77695
KIAA0008 gene product
12.3


435039
AW043921
Hs.130526
ESTs
12.3


447033
AI357412
Hs.157601
EST - not in UniGene
12.3


433764
AW753676
Hs.39982
ESTs
12.2


442611
BE077155
Hs.177537
ESTs
12.0


408562
AI436323
Hs.31141
Homo sapiens mRNA for KIAA31568 protein, parti
11.9


427344
NM_000869
Hs.2142
5-hydroxytryptamine (serotonin) receptor 3A
11.8


421478
AI683243
Hs.97258
ESTs
11.8


426635
BE395109
Hs.129327
ESTs
11.8


415989
AI267700
Hs.111128
ESTs
11.7


433159
AB035898
Hs.150587
kinesin-like protein 2
11.5


452249
BE394412
Hs.61252
ESTs
11.4


418506
AA084248
Hs.85339
G protein-coupled receptor 39
11.3


442353
BE379594
Hs.49136
ESTs
11.3


447700
AI420183
Hs.171077
ESTs, Weakly similar to similar to serine/thr
11.3


450480
X82125
Hs.25040
zinc finger protein 239
11.3


425176
AW015644
Hs.301430
ESTs, Moderately similar to TEF1_HUMAN TRANSC
11.2


435496
AW840171
Hs.265398
ESTs, Weakly similar to transformation-relate
11.2


433133
AB027249
Hs.104741
PDZ-binding kinase; T-cell originated protein
11.1


445258
AI635931
Hs.147613
ESTs
11.1


432677
NM_004482
Hs.278611
UDP-N-acetyl-alpha-D-galactosamine:polypeptid
11.0


429782
NM_005754
Hs.220689
Ras-GTPase-activating protein SH3-domain-bind
10.9


404567


0
10.8


423811
AW299598
Hs.50895
homeo box C4
10.7


452891
N75582
Hs.212875
ESTs, Weakly similar to KIAA0357 [H. sapiens]
10.6


441627
AA947552
Hs.58086
ESTs
10.3


443555
N71710
Hs.21398
ESTs, Moderately similar to GNPI_HUMAN GLUCOS
10.3


412140
AA219691
Hs.73625
RAB6 interacting, kinesin-like (rabkinesin6)
10.2


427469
AA403084
Hs.269347
ESTs
10.1


415227
AW821113
Hs.72402
ESTs
10.1


445413
AA151342
Hs.12677
CGI-147 protein
10.0


425734
AF056209
Hs.159396
peptidylglycine alpha-amidating monooxygenase
10.0


421451
AA291377
Hs.50831
ESTs
10.0


410044
BE566742
Hs.58169
highly expressed in cancer, rich in leucine h
9.8


427878
C05766
Hs.181022
CGI-07 protein
9.7


408460
AA054726
Hs.285574
ESTs
9.7


422972
N59319
Hs.145404
ESTs
9.7


443715
AI583187
Hs.9700
cyclin E1
9.7


440901
AA909358
Hs.128612
ESTs
9.6


453160
AI263307
Hs.146228
ESTs
9.6


415211
R64730.comp
Hs.155986
ESTs; Highly similar to SPERM SURFACE PROTEIN
9.5


425282
AW163518
Hs.155485
huntingtin interacting protein 2
9.5


400250


0
9.5


410568
AW162948
Hs.64542
pre-mRNA cleavage factor Im (68 kD)
9.3


442957
AI949952
Hs.49397
ESTs
9.3


453922
AF053306
Hs.36708
budding uninhibited by benzimidazoles 1 (yeas
9.3


434401
AI864131
Hs.71119
Putative prostate cancer tumor suppressor
9.2


453628
AW243307
Hs.170187
ESTs
9.1


452055
AI377431
Hs.293772
ESTs
9.1


424086
AI351010
Hs.102267
lysyl oxidase
9.1


442875
BE623003
Hs.23625
Homo sapiens clone TCCCTA00142 mRNA sequence
9.1


416208
AW291168
Hs.41295
ESTs
9.0


407168
R45175
Hs.117183
gb: yg40f01.s1 Soares infant brain 1NIB Homo s
9.0


445537
AJ245671
Hs.12844
EGF-like-domain; multiple 6
8.9


409269
AA576953
Hs.22972
Homo sapiens cDNA FLJ13352 fis, clone OVARC10
8.9


433527
AW235613
Hs.133020
ESTs
8.9


409928
AL137163
Hs.57549
hypothetical protein dJ473B4
8.8


423020
AA383092
Hs.1608
replication protein A3 (14 kD)
8.7


425665
AK001050
Hs.159066
ESTs
8.6


443204
AW205878
Hs.29643
Homo sapiens cDNA FLJ13103 fis, clone NT2RP30
8.6


449433
AI672096
Hs.9012
ESTs
8.6


453878
AW964440
Hs.19025
ESTs
8.6


450505
NM_004572
Hs.25051
plakophilin 2
8.6


407001
U12471
Hs.247954
Human thrombospondin-1 gene, partial cds
8.5


414315
Z24878

gb: HSB65D052 STRATAGENE Human skeletal muscle
8.5


425492
AL021918
Hs.158174
zinc finger protein 184 (Kruppel-like)
8.5


435181
AA669339
Hs.28838
KIAA1571 protein
8.5


436396
AI683487
Hs.299112
Homo sapiens cDNA FLJ11441 fis, clone HEMBA10
8.5


418384
AW149266
Hs.25130
ESTs
8.4


453370
AI470523
Hs.182356
ESTs, Moderately similar to translation initi
8.4


409041
AB033025
Hs.50081
KIAA1199 protein
8.4


447078
AW885727
Hs.301570
ESTs
8.4


448674
W31178
Hs.154140
ESTs
8.3


433393
AF038564
Hs.98074
atrophin-1 interacting protein 4
8.3


433496
AF064254
Hs.49765
VERY-LONG-CHAIN ACYL-COA SYNTHETASE
8.3


421155
H87879
Hs.102267
lysyl oxidase
8.2


438394
BE379623
Hs.27693
CGI-124 protein
8.2


400298
AA032279
Hs.61635
STEAP1
8.1


409092
AI735283
Hs.172608
ESTs
8.1


440250
AA876179
Hs.134650
ESTs
8.1


409143
AW025980
Hs.138965
ESTs
8.1


407771
AL138272
Hs.62713
ESTs
8.1


419088
AI538323
Hs.77496
ESTs
8.1


431725
X65724
Hs.2839
Norrie disease (pseudoglioma)
7.9


431750
AA514986
Hs.283705
ESTs
7.9


435635
AF220050
Hs.181385
uncharacterized hematopoietic stem/progenitor
7.9


441826
AW503603
Hs.129915
phosphotriesterase related
7.9


417728
AW138437
Hs.24790
KIAA1573 protein
7.8


418845
AA852985
Hs.89232
chromobox homolog 5 (Drosophila HP1 alpha)
7.8


421039
NM_003478
Hs.101299
cullin 5
7.8


446999
AA151520
Hs.279525
hypothetical protein PRO2605
7.8


429609
AF002246
Hs.210863
cell adhesion molecule with homology to L1CAM
7.8


415139
AW975942
Hs.48524
ESTs
7.7


450192
AA263143
Hs.24596
RAD51-interacting protein
7.7


423992
AW898292
Hs.137206
Homo sapiens mRNA; cDNA DKFZp564H1663 (from c
7.7


436211
AK001581
Hs.80961
polymerase (DNA directed), gamma
7.7


450101
AV649989
Hs.24385
Human hbc647 mRNA sequence
7.5


426921
AA037145
Hs.172865
cleavage stimulation factor, 3′ pre-RNA, subu
7.5


433330
AW207084
Hs.132816
ESTs
7.5


439759
AL359055
Hs.67709
Homo sapiens mRNA full length insert cDNA clo
7.5


427660
AI741320
Hs.114121
Homo sapiens cDNA: FLJ23228 fis, clone CAE066
7.5


422095
AI868872
Hs.288966
ceruloplasmin (ferroxidase)
7.5


436476
AA326108
Hs.53631
ESTs
7.5


412170
D16532
Hs.73729
very low density lipoprotein receptor
7.4


428954
AF100781
Hs.194678
WNT1 inducible signaling pathway protein 3
7.4


450221
AA328102
Hs.24641
cytoskeleton associated protein 2
7.4


439262
AA832333
Hs.124399
ESTs
7.4


435420
AI928513
Hs.59203
ESTs
7.3


422892
AA988176
Hs.121553
hypothetical protein FLJ20641
7.3


457030
AI301740
Hs.173381
dihydropyrimidinase-like 2
7.3


411571
AA122393
Hs.70811
hypothetical protein FLJ20516
7.2


409916
BE313625
Hs.57435
solute carrier family 11 (proton-coupled diva
7.2


418007
M13509
Hs.83169
Matrix metalloprotease 1 (interstitial collag
7.2


420900
AL045633
Hs.44269
ESTs
7.2


424001
W67883
Hs.137476
KIAA1051 protein
7.2


400301
X03635
Hs.1657
Estrogen receptor 1
7.1


400238


0
7.1


413573
AI733859
Hs.149089
ESTs
7.1


428071
AF212848
Hs.182339
transcription factor ESE-3B
7.1


447164
AF026941
Hs.17518
Homo sapiens cig5 mRNA, partial sequence
7.1


453062
AW207538
Hs.61603
ESTs
7.1


456965
AW131888
Hs.172792
ESTs, Weakly similar to hypothetical protein
7.1


442500
AI819068
Hs.209122
ESTs
7.1


446142
AI754693
Hs.145968
ESTs
7.0


417791
AW965339
Hs.111471
ESTs
7.0


418524
AA300576
Hs.85769
acidic 82 kDa protein mRNA
7.0


451797
AW663858
Hs.56120
ESTs
7.0


452909
NM_015368
Hs.30985
pannexin 1
7.0


453616
NM_003462
Hs.33846
dynein, axonemal, light intermediate polypept
7.0


436281
AW411194
Hs.120051
ESTs
7.0


449897
AW819642
Hs.24135
transmembrane protein vezatin; hypothetical p
6.9


414142
AW368397
Hs.150042
ESTs
6.9


448776
BE302464
Hs.30057
transporter similar to yeast MRS2
6.9


419423
D26488
Hs.90315
KIAA0007 protein
6.9


420908
AL049974
Hs.100261
Homo sapiens mRNA; cDNA DKFZp564B222 (from cl
6.8


452971
AI873878
Hs.91789
ESTs
6.8


413597
AW302885
Hs.117183
ESTs
6.8


415138
C18356
Hs.78045
tissue factor pathway inhibitor 2 TFPI2
6.8


437478
AL390172
Hs.118811
ESTs
6.7


425292
NM_005824
Hs.155545
37 kDa leucine-rich repeat (LRR) protein
6.7


421184
NM_003616
Hs.102456
survival of motor neuron protein interacting
6.7


410227
AB009284
Hs.61152
exostoses (multiple)-like 2
6.6


446608
N75217
Hs.257846
ESTs
6.6


438167
R28363
Hs.24286
ESTs
6.6


445459
AI478629
Hs.158465
ESTs
6.6


452291
AF015592
Hs.28853
CDC7 (cell division cycle 7, S. cerevisiae, h
6.6


410011
AB020641
Hs.57856
PFTAIRE protein kinase 1
6.6


410292
AA843087
Hs.124194
ESTs
6.5


415716
N59294
Hs.301141
Homo sapiens cDNA FLJ11689 fis, clone HEMBA10
6.5


424770
AA425562

gb: zw46e05.r1 Soares_total_fetus_Nb2HF8_9w Ho
6.5


438122
AI620270
Hs.129837
ESTs
6.5


439820
AL360204
Hs.283853
Homo sapiens mRNA full length insert cDNA clo
6.5


444743
AA045648
Hs.11817
nudix (nucleoside diphosphate linked moiety X
6.5


450638
AK001826
Hs.25245
hypothetical protein FLJ11269
6.5


418203
X54942
Hs.83758
CDC28 protein kinase 2
6.5


439901
N73885
Hs.124169
ESTs
6.5


428758
AA433988
Hs.98502
Homo sapiens cDNA FLJ14303 fis, clone PLACE20
6.4


404552


0
6.4


404599


0
6.4


419503
AA243642
Hs.137422
ESTs
6.4


420149
AA255920
Hs.88095
ESTs
6.4


440411
N30256
Hs.156971
ESTs, Weakly similar to Weak similarity with
6.4


449108
AI140683
Hs.98328
ESTs
6.4


452097
AB002364
Hs.27916
ADAM-TS3; a disintegrin-like and metallopr
6.4


453619
H87648
Hs.33922
H. sapiens novel gene from PAC 117P20, chromos
6.4


410273
BE326877
Hs.281523
ESTs
6.3


434486
AA678816
Hs.117142
ESTs
6.3


454036
AA374756
Hs.93560
ESTs, Weakly similar to unnamed protein produ
6.3


403381


0
6.2


421308
AA687322
Hs.192843
ESTs
6.2


419346
AI830417

gb: wh94d12.x1 NCI_CGAP_CLL1 Homo sapiens cDNA
6.2


446140
AA356170
Hs.26750
Homo sapiens cDNA: FLJ21908 fis, clone HEP038
6.2


453047
AW023798
Hs.286025
ESTs
6.2


442573
H93366
Hs.7567
Branched chain aminotransferase 1, cytosolic,
6.1


410102
AW248508
Hs.279727
ESTs;
6.1


410004
AI298027
Hs.299115
ESTs
6.1


413335
AI613318
Hs.48442
ESTs
6.1


424945
AI221919
Hs.173438
hypothetical protein FLJ10582
6.1


427510
Z47542
Hs.179312
small nuclear RNA activating complex, polypep
6.1


451229
AW967707
Hs.48473
ESTs
6.1


452641
AW952893
Hs.237825
signal recognition particle 72 kD
6.1


433172
AB037841
Hs.102652
hypothetical protein ASH1
6.1


425465
L18964
Hs.1904
protein kinase C; iota
6.1


437117
AL049256
Hs.122593
ESTs
6.0


423440
R25234
Hs.143434
contactin 1
6.0


430510
AW162916
Hs.241576
hypothetical protein PRO2577
6.0


433252
AB040957
Hs.151343
KIAA1524 protein
6.0


434699
AA643687
Hs.149425
Homo sapiens cDNA FLJ11980 fis, clone HEMBB10
6.0


436954
AA740151
Hs.130425
ESTs
5.9


436032
AA150797
Hs.109276
latexin protein
5.9


424590
AW966399
Hs.46821
hypothetical protein FLJ20086
5.9


444078
BE246919
Hs.10290
U5 snRNP-specific 40 kDa protein (hPrp8-bindi
5.9


418379
AA218940
Hs.137516
fidgetin-like 1
5.9


438081
H49546
Hs.298964
ESTs
5.8


443270
NM_004272
Hs.9192
Homer, neuronal immediate early gene, 1B
5.8


450459
AI697193
Hs.299254
ESTs
5.8


433612
AF078164
Hs.61188
Homo sapiens Ku70-binding protein (KUB3) mRNA
5.8


449048
Z45051
Hs.22920
similar to S68401 (cattle) glucose induced ge
5.8


417251
AW015242
Hs.99488
ESTs; Weakly similar to ORF YKR074w [S. cerevi
5.7


429181
AW979104
Hs.294009
ESTs
5.7


454933
BE141714

gb: QV0-HT0101-061099-032-c04 HT0101 Homo sapi
5.7


456553
AA721325
Hs.189058
ESTs, Weakly similar to cAMP-regulated guanin
5.7


430371
D87466
Hs.240112
KIAA0276 protein
5.7


425371
D49441
Hs.155981
mesothelin
5.7


424513
BE385864
Hs.149894
mitochondrial translational initiation factor
5.6


432015
AL157504
Hs.159115
ESTs
5.6


438109
AI076621
Hs.71367
ESTs, Moderately similar to ALU7_HUMAN ALU SU
5.6


407137
T97307
Hs.199067
v-erb-b2 avian erythroblastic leukemia viral
5.6


407945
X69208
Hs.606
ATPase, Cu++ transporting, alpha polypeptide
5.6


416565
AW000960
Hs.44970
ESTs
5.6


417830
AW504786
Hs.132808
epithelial cell transforming sequence 2 oncog
5.5


419752
AA249573
Hs.152618
ESTs
5.5


422093
AF151852
Hs.111449
CGI-94 protein
5.5


424583
AF017445
Hs.150926
fucose-1-phosphate guanylyltransferase
5.5


430388
AA356923
Hs.240770
nuclear cap binding protein subunit 2, 20 kD
5.5


452534
AW083022
Hs.149425
Homo sapiens cDNA FLJ11980 fis, clone HEMBB10
5.5


453279
AW893940
Hs.59698
ESTs
5.5


424188
AW954552
Hs.142634
zinc finger protein
5.5


453884
AA355925
Hs.36232
KIAA0186 gene product
5.5


424641
AB001106
Hs.151413
glia maturation factor, beta
5.5


444478
W07318
Hs.240
M-phase phosphoprotein 1
5.5


427975
AI536065
Hs.122460
ESTs
5.5


424620
AA101043
Hs.151254
kallikrein 7 (chymotryptic; stratum corneum)
5.5


442914
AW188551
Hs.99519
Homo sapiens cDNA FLJ14007 fis, clone Y79AA10
5.5


417995
AW974175
Hs.188751
ESTs
5.4


418946
AI798841
Hs.132103
ESTs
5.4


419963
AA743276
Hs.301052
ESTs
5.4


420362
U79734
Hs.97206
huntingtin interacting protein 1
5.4


422670
AA371612
Hs.115351
ESTs
5.4


432837
AA310693
Hs.279512
HSPC072 protein
5.4


447020
T27308
Hs.16986
hypothetical protein FLJ11046
5.4


458027
L49054
Hs.85195
ESTs, Highly similar to t(3; 5)(q25.1; p34) fus
5.4


425217
AU076696
Hs.155174
CDC5 (cell division cycle 5, S. pombe, homolo
5.4


422938
NM_001809
Hs.1594
centromere protein A (17 kD)
5.4


450434
AA166950
Hs.18645
ESTs, Weakly similar to partial CDS [C. elegan
5.4


438279
AA805166
Hs.165165
ESTs, Moderately similar to ALU8_HUMAN ALU SU
5.4


413384
NM_000401
Hs.75334
exostoses (multiple) 2
5.3


420328
Y19062
Hs.96870
staufen (Drosophila, RNA-binding protein) hom
5.3


436586
AI308862
Hs.167028
ESTs
5.3


435793
AB037734
Hs.4993
ESTs
5.3


422306
BE044325
Hs.227280
Homo sapiens mRNA for Lsm5 protein
5.3


425154
NM_001851
Hs.154850
collagen, type IX, alpha 1
5.2


453293
AA382267
Hs.10653
ESTs
5.2


429944
R13949
Hs.226440
Homo sapiens clone 24881 mRNA sequence
5.2


434891
AA814309
Hs.123583
ESTs
5.2


415263
AA948033
Hs.130853
ESTs
5.2


409506
NM_006153
Hs.54589
NCK adaptor protein 1
5.2


412848
AA121514
Hs.70832
ESTs
5.2


421246
AW582962
Hs.300961
ESTs, Highly similar to AF151805 1 CGI-47 pro
5.2


431548
AI834273
Hs.9711
Homo sapiens cDNA FLJ13018 fis, clone NT2RP30
5.2


412719
AW016610
Hs.129911
ESTs
5.2


411945
AL033527
Hs.92137
v-myc avian myelocytomatosis viral oncogene h
5.1


424078
AB006625
Hs.139033
paternally expressed gene 3
5.1


433558
AA833757
Hs.201769
ESTs
5.1


434265
AA846811
Hs.130554
Homo sapiens cDNA: FLJ23089 fis, clone LNG070
5.1


453911
AW503857
Hs.4007
Sarcolemmal-associated protein
5.1


415539
AI733881
Hs.72472
BMPR-lb; bone morphogenetic protein receptor
5.1


442717
R88362
Hs.180591
ESTs, Weakly similar to R06F6.5b [C. elegans]
5.1


432358
AI093491
Hs.72830
ESTs
5.0


409731
AA125985
Hs.56145
thymosin, beta, identified in neuroblastoma c
5.0


419699
AA248998
Hs.31246
ESTs
5.0


420313
AB023230
Hs.96427
KIAA1013 protein
5.0


422505
AL120862
Hs.124165
ESTs; (HSA)PAP protein (programmed cell deat
5.0


425733
F13287
Hs.159388
Homo sapiens clone 23578 mRNA sequence
5.0


434160
BE551196
Hs.114275
ESTs
5.0


435094
AI560129
Hs.277523
EST
5.0


436812
AW298067

gb: UI-H-BW0-ajp-g-09-0-UI.s1 NCI_CGAP_Sub6 Ho
5.0


432415
T16971
Hs.289014
ESTs
4.9


406117


0
4.9


438018
AK001160
Hs.5999
hypothetical protein FLJ10298
4.9


447505
AL049266
Hs.18724
Homo sapiens mRNA; cDNA DKFZp564F093 (from cl
4.9


448621
AI097144
Hs.5250
ESTs, Weakly similar to BACR37P7.g [D. melanog
4.9


453001
AW131636
Hs.191260
ESTs
4.9


410561
BE540255
Hs.6994
Homo sapiens cDNA: FLJ22044 fis, clone HEP091
4.9


418811
AK001407
Hs.88663
hypothetical protein FLJ10545
4.9


436754
AI061288
Hs.133437
ESTs, Moderately similar to gonadotropin indu
4.8


437212
AI765021
Hs.210775
ESTs
4.8


447312
AI434345
Hs.36908
activating transcription factor 1
4.8


409732
NM_016122
Hs.56148
NY-REN-58 antigen
4.8


434690
AI867679
Hs.148410
ESTs
4.8


444172
BE147740
Hs.104558
ESTs
4.8


424539
L02911
Hs.150402
activin A receptor, type I
4.8


418677
S83308
Hs.87224
SRY (sex determining region Y)-box 5
4.8


406076
AL390179
Hs.137011
Homo sapiens mRNA; cDNA DKFZp547P134 (from cl
4.8


420179
N74530
Hs.21168
ESTs
4.7


450375
AA009647
Hs.8850
a disintegrin and metalloproteinase domain 12
4.7


419247
S65791
Hs.89764
fragile X mental retardation 1
4.7


420850
BE139590
Hs.122406
ESTs
4.7


425420
BE536911
Hs.234545
ESTs
4.7


428664
AK001666
Hs.189095
similar to SALL1 (sal (Drosophila)-like
4.7


419131
AA406293
Hs.301622
ESTs
4.7


422278
AF072873
Hs.114218
ESTs
4.7


451684
AF216751
Hs.26813
CDA14
4.6


400296
AA305627
Hs.139336
ATP-binding cassette; sub-family C (CFTR/MRP)
4.6


408425
AW058674
Hs.44787
Homo sapiens mRNA; cDNA DKFZp434O0227 (from c
4.6


417168
AL133117
Hs.81376
Homo sapiens mRNA; cDNA DKFZp586L1121 (from c
4.6


429486
AF155827
Hs.203963
hypothetical protein FLJ10339
4.6


442917
AA314907
Hs.85950
ESTs
4.6


443268
AI800271
Hs.129445
hypothetical protein FLJ12496
4.6


452795
AW392555
Hs.18878
hypothetical protein FLJ21620
4.6


457300
AW297436
Hs.158849
Homo sapiens cDNA: FLJ21663 fis, clone COL088
4.6


459551
AI472808

gb: tj70e07.x1 Soares_NSF_F8_9W_OT_PA_P_S1 Hom
4.6


421977
W94197
Hs.110165
ribosomal protein L26 homolog
4.6


429441
AJ224172
Hs.204096
lipophilin B (uteroglobin family member), pro
4.6


449722
BE280074
Hs.23960
cyclin B1
4.6


431689
AA305688
Hs.267695
UDP-Gal: betaGlcNAc beta 1,3-galactosyltransfe
4.5


425178
H16097
Hs.161027
ESTs
4.5


429597
NM_003816
Hs.2442
a disintegrin and metalloproteinase domain 9
4.5


436556
AI364997
Hs.7572
ESTs
4.5


400534


0
4.5


417845
AL117461
Hs.82719
Homo sapiens mRNA; cDNA DKFZp586F1822 (from c
4.5


423123
NM_012247
Hs.124027
SELENOPHOSPHATE SYNTHETASE; Human selenium d
4.5


448305
AA625207
Hs.264915
Homo sapiens cDNA FLJ12908 fis, clone NT2RP20
4.5


441006
AW605267
Hs.7627
CGI-60 protein
4.5


414569
AF109298
Hs.118258
Prostate cancer associated protein 1
4.5


447924
AI817226
Hs.170337
ESTs
4.5


425506
NM_003666
Hs.158205
basic leucine zipper nuclear factor 1 (JEM-1)
4.5


411630
U42349
Hs.71119
Putative prostate cancer tumor suppressor
4.4


432842
AW674093
Hs.279525
hypothetical protein PRO2605
4.4


413472
BE242870
Hs.75379
solute carrier family 1 (glial high affinity
4.4


414699
AI815523
Hs.76930
synuclein, alpha (non A4 component of amyloid
4.4


412733
AA984472
Hs.74554
KIAA0080 protein
4.4


419790
U79250
Hs.93201
glycerol-3-phosphate dehydrogenase 2 (mitocho
4.4


433377
AI752713
Hs.43845
ESTs
4.4


449535
W15267
Hs.23672
low density lipoprotein receptor-related prot
4.4


453900
AW003582
Hs.226414
ESTs, Weakly similar to ALU8_HUMAN ALU SUBFAM
4.4


443881
R64512
Hs.237146
Homo sapiens cDNA FLJ14234 fis, clone NT2RP40
4.4


423025
AA831267
Hs.12244
Homo sapiens cDNA: FLJ23581 fis, clone LNG136
4.4


408621
AI970672
Hs.46638
chromosome 11 open reading frame 8; fetal br
4.3


416241
N52639
Hs.32683
ESTs
4.3


432005
AA524190
Hs.120777
ESTs, Weakly similar to ELL2_HUMAN RNA POLYME
4.3


435532
AW291488
Hs.117305
ESTs
4.3


451813
NM_016117
Hs.27182
phospholipase A2-activating protein
4.3


454193
BE141183

gb: MR0-HT0071-191199-001-b04 HT0071 Homo sapi
4.3


418478
U38945
Hs.1174
cyclin-dependent kinase inhibitor 2A (melanom
4.3


406069


0
4.3


419465
AW500239
Hs.21187
Homo sapiens cDNA: FLJ23068 fis, clone LNG055
4.3


418413
R95735
Hs.117753
ESTs, Weakly similar to antigen of the monocl
4.3


452028
AK001859
Hs.27595
hypothetical protein FLJ10997
4.3


418693
AI750878
Hs.87409
thrombospondin 1
4.3


410361
BE391804
Hs.62661
guanylate binding protein 1, interferon-induc
4.2


409763
AL043212

gb: DKFZp434H0623_r1 434 (synonym: htes3) Homo
4.2


455601
AI368680
Hs.816
SRY (sex determining region Y)-box 2, partial
4.2


408908
BE296227
Hs.48915
serine/threonine kinase 15
4.2


413582
AW295647
Hs.71331
Homo sapiens cDNA: FLJ21971 fis, clone HEP057
4.2


423248
AA380177
Hs.125845
ribulose-5-phosphate-3-epimerase
4.2


425024
R39235
Hs.12407
ESTs
4.2


447153
AA805202
Hs.173912
eukaryotic translation initiation factor 4A,
4.2


447406
BE618060
Hs.282882
ESTs
4.2


449347
AV649748
Hs.295901
ESTs
4.2


414279
AW021691
Hs.3804
DKFZP564C1940 protein
4.2


428856
AA436735
Hs.183171
Homo sapiens cDNA: FLJ22002 fis, clone HEP066
4.2


407872
AB039723
Hs.40735
frizzled (Drosophila) homolog 3
4.2


421502
AF111856
Hs.105039
solute carrier family 34 (sodium phosphate),
4.2


436406
AW105723
Hs.125346
ESTs
4.2


438209
AL120659
Hs.6111
KIAA0307 gene product
4.2


443653
AA137043
Hs.9663
programmed cell death 6-interacting protein
4.1


454556
AW807073

gb: MR4-ST0062-031199-018-d06 ST0062 Homo sapi
4.1


424834
AK001432
Hs.153408
Homo sapiens cDNA FLJ10570 fis, clone NT2RP20
4.1


412593
Y07558
Hs.74088
early growth response 3
4.1


416566
NM_003914
Hs.79378
cyclin A1
4.1


426342
AF093419
Hs.169378
multiple PDZ domain protein
4.1


428417
AK001699
Hs.184227
F-box only protein 21
4.1


429317
AA831552
Hs.268016
solute carrier family 5 (inositol transporter
4.1


446880
AI811807
Hs.108646
Homo sapiens cDNA FLJ12534 fis, clone NT2RM40
4.1


422988
AW673847
Hs.97321
ESTs
4.0


434657
AA641876
Hs.191840
ESTs
4.0


412494
AL133900
Hs.792
ADP-ribosylation factor domain protein 1, 64k
4.0


443271
BE568568
Hs.195704
ESTs
4.0


421437
AW821252
Hs.104336
ESTs
4.0


401644


0
4.0


405095


0
4.0


418417
R77182

gb: yi65e02.r1 Soares placenta Nb2HP Homo sapi
4.0


420807
AA280627
Hs.57846
ESTs
4.0


429529
AA454190
Hs.193811
ESTs, Moderately similar to reduced expressio
4.0


457726
AI217477
Hs.194591
ESTs
4.0


431130
NM_006103
Hs.2719
epididymis-specific; whey-acidic protein type
4.0


453403
BE466639
Hs.61779
Homo sapiens cDNA FLJ13591 fis, clone PLACE10
4.0


442768
AL048534
Hs.48458
ESTs, Weakly similar to ALU8_HUMAN ALU SUBFAM
4.0


413430
R22479
Hs.24650
Homo sapiens cDNA FLJ13047 fis, clone NT2RP30
4.0


424081
NM_006413
Hs.139120
ribonuclease P (30 kD)
4.0


425692
D90041
Hs.155956
NAT1; arylamine N-acetyltransferase
4.0


407792
AI077715
Hs.39384
putative secreted ligand homologous to fjx1
4.0


408353
BE439838
Hs.44298
hypothetical protein
4.0


421175
AI879099
Hs.102397
GIOT-3 for gonadotropin inducible transcripti
3.9


420324
AF163474
Hs.96744
DKFZP586D0823 protein, Prostate androgen-regu
3.9


417531
NM_003157
Hs.1087
serine/threonine kinase 2
3.9


458924
BE242158
Hs.24427
DKFZP566O1646 protein
3.9


400195


0
3.9


401480


0
3.9


410360
AW663690

gb: hj21g03.x1 NCI_CGAP_Li8 Homo sapiens cDNA
3.9


410908
AA121686
Hs.10592
ESTs
3.9


420159
AI572490
Hs.99785
ESTs
3.9


422805
AA436989
Hs.121017
H2A histone family; member A
3.9


424639
AI917494
Hs.131329
ESTs
3.9


428555
NM_002214
Hs.184908
integrin, beta 8
3.9


431699
NM_001173
Hs.267831
Homo sapiens cDNA FLJ12952 fis, clone NT2RP20
3.9


433703
AA210863
Hs.3532
nemo-like kinase
3.9


437144
AL049466
Hs.7859
ESTs
3.9


452728
AI915676
Hs.239708
ESTs
3.9


430447
W17064
Hs.241451
SWI/SNF related, matrix associated, actin dep
3.9


440594
AW445167
Hs.126036
ESTs
3.9


408938
AA059013
Hs.22607
ESTs
3.9


427051
BE178110
Hs.173374
ESTs
3.9


447568
AF155655
Hs.18885
CGI-116 protein
3.9


457211
AW972565
Hs.32399
ESTs, Weakly similar to Similar to Ena-VASP I
3.9


443475
AI066470
Hs.134482
ESTs
3.9


433447
U29195
Hs.3281
neuronal pentraxin II
3.9


428093
AW594506
Hs.104830
ESTs
3.8


437938
AI950087

ESTs; Weakly similar to Gag-Pol polyprotein [
3.8


408829
NM_006042
Hs.48384
heparan sulfate (glucosamine) 3-O-sulfotransf
3.8


429250
H56585
Hs.198308
tryptophan rich basic protein
3.8


441859
AW194364
Hs.128022
ESTs, Weakly similar to FIG. 1 MOUSE FIG-1 PROT
3.8


437700
AA766060
Hs.122848
ESTs
3.8


439560
BE565647
Hs.74899
hypothetical protein FLJ12820
3.8


409564
AA045857
Hs.54943
fracture callus 1 (rat) homolog
3.8


429474
AA453441
Hs.31511
ESTs
3.8


431965
BE175190

gb: QV2-HT0577-010500-165-g04 HT0577 Homo sapi
3.8


454018
AW016892
Hs.241652
ESTs
3.8


426320
W47595
Hs.169300
transforming growth factor, beta 2
3.8


439635
AA477288
Hs.94891
Homo sapiens cDNA: FLJ22729 fis, clone HSI156
3.8


417517
AF001176
Hs.82238
POP4 (processing of precursor, S. cerevisiae
3.8


446402
AI681145
Hs.160724
ESTs
3.8


450236
AW162998
Hs.24684
KIAA1376 protein
3.8


410804
U64820
Hs.66521
Machado-Joseph disease (spinocerebellar ataxi
3.8


400268


0
3.8


418217
AI910647
Hs.13442
ESTs
3.8


421928
AF013758
Hs.109643
polyadenylate binding protein-interacting pro
3.8


417300
AI765227
Hs.55610
solute carrier family 30 (zinc transporter),
3.8


414136
AA812434
Hs.178227
ESTs
3.8


453945
NM_005171
Hs.36908
activating transcription factor 1
3.7


400240


0
3.7


407877
AW016811
Hs.234478
Homo sapiens cDNA: FLJ22648 fis, clone HSI073
3.7


450581
AF081513
Hs.25195
endometrial bleeding associated factor (left-
3.7


418223
NM_014733
Hs.83790
KIAA0305 gene product
3.7


411704
AI499220
Hs.71573
hypothetical protein FLJ10074
3.7


432712
AB016247
Hs.288031
sterol-C5-desaturase (fungal ERG3, delta-5-de
3.7


422809
AK001379
Hs.121028
hypothetical protein FLJ10549
3.7


402820


0
3.7


408090
BE173621
Hs.292478
ESTs
3.7


416421
AA134006
Hs.79306
eukaryotic translation initiation factor 4E
3.7


418282
AA215535
Hs.98133
ESTs
3.7


418454
AA315308

gb: EST187095 Colon carcinoma (HCC) cell line
3.7


418668
AW407987
Hs.87150
Human clone A9A2BR11 (CAC)n/(GTG)n repeat-con
3.7


422290
AA495854
Hs.48827
hypothetical protein FLJ12085
3.7


432824
AK001783
Hs.279012
hypothetical protein FLJ10921
3.7


439907
AA853978
Hs.124577
ESTs
3.7


447479
AB037834
Hs.18685
Homo sapiens mRNA for KIAA1413 protein, parti
3.7


451073
AI758905
Hs.206063
ESTs
3.7


450377
AB033091
Hs.24936
ESTs
3.7


414343
AL036166
Hs.75914
coated vesicle membrane protein
3.7


448807
AI571940
Hs.7549
ESTs
3.7


442821
BE391929
Hs.8752
Putative type II membrane protein
3.7


426300
U15979
Hs.169228
delta-like homolog (Drosophila)
3.7


418068
AW971155
Hs.293902
ESTs, Weakly similar to prolyl 4-hydroxylase
3.7


411263
BE297802
Hs.69360
kinesin-like 6 (mitotic centromere-associated
3.7


443054
AI745185
Hs.8939
yes-associated protein 65 kDa
3.7


421154
AA284333
Hs.287631
Homo sapiens cDNA FLJ14269 fis, clone PLACE10
3.7


411402
BE297855
Hs.69855
NRAS-related gene
3.7


450447
AF212223
Hs.25010
hypothetical protein P15-2
3.6


414706
AW340125
Hs.76989
KIAA0097 gene product
3.6


434228
Z42047
Hs.283978
ESTs; KIAA0738 gene product
3.6


434164
AW207019
Hs.148135
ESTs
3.6


409533
AW969543
Hs.21291
mitogen-activated protein kinase kinase kinas
3.6


402222


0
3.6


404915


0
3.6


404996


0
3.6


411560
AW851186

gb: IL3-CT0220-150200-071-H05 CT0220 Homo sapi
3.6


419750
AL079741
Hs.183114
Homo sapiens cDNA FLJ14236 fis, clone NT2RP40
3.6


426010
AA136563
Hs.1975
Homo sapiens cDNA: FLJ21007 fis, clone CAE038
3.6


427038
NM_014633
Hs.173288
KIAA0155 gene product
3.6


439255
BE164500

gb: RC4-HT0469-230300-014-e10 HT0469 Homo sapi
3.6


458242
BE299588
Hs.28465
Homo sapiens cDNA: FLJ21869 fis, clone HEP024
3.6


415115
AA214228
Hs.127751
hypothetical protein
3.6


453468
W00712
Hs.32990
DKFZP566F084 protein
3.6


441205
AW137827
Hs.176904
ESTs
3.6


452693
T79153
Hs.48589
zinc finger protein 228
3.6


417389
BE260964
Hs.82045
Midkine (neurite growth-promoting factor 2)
3.6


448105
AW591433
Hs.170675
ESTs, Weakly similar to TMS2_HUMAN TRANSMEMBR
3.6


451522
BE565817
Hs.26498
hypothetical protein FLJ21657
3.6


440048
AA897461
Hs.158469
ESTs, Weakly similar to envelope protein [H. s
3.5


419359
AL043202
Hs.90073
chromosome segregation 1 (yeast homolog)-like
3.5


452030
AL137578
Hs.27607
Homo sapiens mRNA; cDNA DKFZp564N2464 (from c
3.5


400666


0
3.5


422646
H87863
Hs.151380
ESTs
3.5


407846
AA426202
Hs.40403
Cbp/p300-interacting transactivator, with Glu
3.5


408730
AV660717
Hs.47144
DKFZP586N0819 protein
3.5


401517


0
3.5


413775
AW409934
Hs.75528
nucleolar GTPase
3.5


417177
NM_004458
Hs.81452
fatty-acid-Coenzyme A ligase, long-chain 4
3.5


427943
AW959075

gb: EST371145 MAGE resequences, MAGE Homo sapi
3.5


439107
AL046134
Hs.27895
ESTs
3.5


447268
AI370413
Hs.36563
Homo sapiens cDNA: FLJ22418 fis, clone HRC085
3.5


412604
AW978324
Hs.47144
DKF7P586N0819 protein
3.5


427134
AA398409
Hs.173561
EST
3.5


430273
AI311127
Hs.125522
ESTs
3.5


436671
AW137159
Hs.146151
ESTs
3.5


433037
NM_014158
Hs.279938
HSPC067 protein
3.5


453745
AA952989
Hs.63908
Homo sapiens HSPC316 mRNA, partial cds
3.5


400531
AF151064
Hs.36069
hypothetical protein
3.5


433345
AI681545
Hs.152982
EST cluster (not in UniGene)
3.4


406400
AA343629
Hs.104570
kallikrein 8 (neuropsin/ovasin)
3.4


407596
R86913

gb: yq30f05.r1 Soares fetal liver spleen 1NFLS
3.4


453779
N35187
Hs.43388
ESTs
3.4


444858
AI199738
Hs.208275
ESTs, Weakly similar to unnamed protein produ
3.4


447688
N87079
Hs.19236
NADH dehydrogenase (ubiquinone) 1 beta subcom
3.4


424856
AA347746
Hs.9521
ESTs, Weakly similar to KIAA1015 protein [H. s
3.4


407864
AF069291
Hs.40539
chromosome 8 open reading frame 1
3.4


404108


0
3.4


403729


0
3.4


404232


0
3.4


423687
AA329633
Hs.133011
ESTs, Highly similar to Z117_HUMAN ZINC FINGE
3.4


428372
AK000684
Hs.183887
hypothetical protein FLJ22104
3.4


439741
BE379646
Hs.6904
Homo sapiens mRNA full length insert cDNA clo
3.4


441447
AA934077
Hs.126980
ESTs
3.4


448358
R44433
Hs.106614
Human DNA sequence from clone RP4-534K7 on ch
3.4


450926
AI744361
Hs.205591
ESTs, Weakly similar to zinc finger protein P
3.4


458477
NM_000314
Hs.10712
phosphatase and tensin homolog (mutated in mu
3.4


421379
Y15221
Hs.103982
small inducible cytokine subfamily B (Cys-X-C
3.4


452822
X85689
Hs.288617
Homo sapiens cDNA: FLJ22621 fis, clone HSI056
3.4


441111
AI806867
Hs.126594
ESTs
3.4


447519
U46258
Hs.23448
ESTs
3.4


446913
AA430650
Hs.16529
transmembrane 4 superfamily member (tetraspan
3.4


449581
AI989517
Hs.181605
ESTs
3.4


456132
BE219771
Hs.237146
Homo sapiens cDNA FLJ14234 fis, clone NT2RP40
3.4


448186
AA262105
Hs.4094
Homo sapiens cDNA FLJ14208 fis, clone NT2RP30
3.4


422611
AA158177
Hs.118722
fucosyltransferase 8 (alpha (1,6) fucosyltran
3.4


441433
AA933809
Hs.42746
ESTs
3.4


417837
AL079905
Hs.1103
transforming growth factor, beta 1
3.4


450516
AA902656
Hs.21943
NIF3 (Ngg1 interacting factor 3, S. pombe homo
3.4


407796
AA195509
Hs.272239
lymphocyte activation-associated protein
3.3


419200
AW966405
Hs.288856
prefoldin 5
3.3


423161
AL049227
Hs.124776
Homo sapiens mRNA; cDNA DKFZp564N1116 (from c
3.3


445679
AI343868
Hs.58800
Homo sapiens cDNA FLJ12488 fis, clone NT2RM20
3.3


435014
BE560898
Hs.10026
ribosomal protein L17 isolog
3.3


446619
AU076643
Hs.313
secreted phosphoprotein 1 (osteopontin, bone
3.3


439170
AA332365
Hs.165539
ESTs
3.3


429830
AI537278
Hs.225841
DKFZP434D193 protein
3.3


428943
AW086180
Hs.37636
ESTs, Weakly similar to KIAA1392 protein [H. s
3.3


445817
NM_003642
Hs.13340
histone acetyltransferase 1
3.3


408805
H69912
Hs.48269
vaccinia related kinase 1
3.3


441134
W29092
Hs.7678
cellular retinoic acid-binding protein 1
3.3


408532
AI453137
Hs.63176
ESTs
3.3


409517
X90780
Hs.54668
troponin I, cardiac
3.3


414304
AI621276
Hs.165998
DKFZP564M2423 protein
3.3


436427
AI344378
Hs.143399
ESTs
3.3


436662
AI582393
Hs.126695
ESTs
3.3


440304
BE159984
Hs.125395
ESTs
3.3


447385
F12863

gb: HSC3FE081 normalized infant brain cDNA Hom
3.3


451177
AI969716
Hs.13034
ESTs
3.3


428949
AA442153
Hs.104744
ESTs, Weakly similar to AF208855 1 BM-013 [H.
3.3


451743
AW074266
Hs.23071
ESTs
3.3


421515
Y11339
Hs.105352
GalNAc alpha-2, 6-sialyltransferase I, long f
3.3


446351
AW444551
Hs.258532
ESTs
3.3


435102
AW899053
Hs.76917
F-box only protein 8
3.3


418216
AA662240
Hs.283099
AF15q14 protein
3.3


401508


0
3.3


437108
AA434054
Hs.80624
Homo sapiens cDNA: FLJ23442 fis, clone HSI009
3.3


416530
U62801
Hs.79361
kallikrein 6 (neurosin, zyme)
3.3


443171
BE281128
Hs.9030
TONDU
3.3


458627
AW088642
Hs.97984
ESTs; Weakly similar to WASP-family protein [
3.3


412078
X69699
Hs.73149
paired box gene 8
3.3


414080
AA135257
Hs.47783
ESTs, Weakly similar to T12540 hypothetical p
3.3


401197


0
3.3


422134
AW179019
Hs.112110
ESTs
3.3


409044
AI129586
Hs.33033
ESTs
3.3


416198
H27332
Hs.99598
ESTs
3.2


436481
AA379597
Hs.5199
HSPC150 protein similar to ubiquitin-conjugat
3.2


436525
AA721428
Hs.26145
Homo sapiens cDNA FLJ14127 fis, clone MAMMA10
3.2


409142
AL136877
Hs.50758
chromosome-associated polypeptide C
3.2


428819
AL135623
Hs.193914
KIAA0575 gene product
3.2


428728
NM_016625
Hs.191381
ESTs; Weakly similar to hypothetical protein
3.2


421261
AA600853
Hs.98133
ESTs
3.2


446219
AI287344
Hs.149827
ESTs
3.2


457574
H88717
Hs.27774
ESTs, Highly similar to AF161349 1 HSPC086 [H
3.2


409172
Z99399
Hs.118145
ESTs
3.2


419388
T67012
Hs.75323
prohibitin
3.2


434187
AA627098
Hs.99103
ESTs, Weakly similar to I38428 T-complex prot
3.2


445060
AA830811
Hs.88808
ESTs
3.2


448254
AI829900
Hs.22929
ESTs
3.2


452943
BE247449
Hs.31082
hypothetical protein FLJ10525
3.2


411393
AW797437
Hs.69771
B-factor, properdin
3.2


453775
NM_002916
Hs.35120
replication factor C (activator 1) 4 (37 kD)
3.2


408418
AW963897
Hs.44743
KIAA1435 protein
3.2


442025
AW887434
Hs.11810
ESTs, Weakly similar to CD4.2 [C. elegans]
3.2


417006
AW673606
Hs.80758
aspartyl-tRNA synthetase
3.2


407881
AW072003
Hs.40968
heparan sulfate (glucosamine) 3-O-sulfotransf
3.2


444755
AA431791
Hs.183001
ESTs
3.2


402829


0
3.2


451593
AF151879
Hs.26706
CGI-121 protein
3.2


419926
AW900992
Hs.93796
DKFZP586D2223 protein
3.2


434551
BE387162
Hs.280858
ESTs, Highly similar to XPB_HUMAN DNA-REPAIR
3.2


445929
AI089660
Hs.7838
makorin, ring finger protein, 1
3.2


409365
AA702376
Hs.226440
Homo sapiens clone 24881 mRNA sequence
3.2


418836
AI655499
Hs.161712
ESTs
3.2


441020
W79283
Hs.35962
ESTs
3.1


422363
T55979
Hs.115474
replication factor C (activator 1) 3 (38 kD)
3.1


413010
AA393273
Hs.75133
transcription factor 6-like 1 (mitochondrial
3.1


452092
BE245374
Hs.27842
hypothetical protein FLJ11210
3.1


410486
AW235094
Hs.193424
ESTs, Weakly similar to KIAA1064 protein [H. s
3.1


434540
NM_016045
Hs.5184
TH1 drosophila homolog
3.1


409178
BE393948
Hs.50915
kallikrein 5
3.1


439480
AL038511
Hs.125316
ESTs
3.1


417848
AA206581
Hs.39457
ESTs
3.1


446293
AI420213
Hs.149722
ESTs
3.1


408108
AI580492
Hs.42743
hypothetical protein
3.1


415947
U04045
Hs.78934
mutS (E. coli) homolog 2 (colon cancer, nonpo
3.1


410519
AW612264
Hs.131705
ESTs
3.1


421987
AI133161
Hs.286131
CGI-101 protein
3.1


440046
AW402306
Hs.6877
hypothetical protein FLJ10483
3.1


453931
AL121278
Hs.25144
ESTs
3.1


454423
AW603985
Hs.179662
nucleosome assembly protein 1-like 1
3.1


459089
F13036
Hs.27373
Homo sapiens mRNA; cDNA DKFZp564O1763 (from c
3.1


418735
N48769
Hs.44609
ESTs
3.1


414245
BE148072
Hs.75850
WAS protein family, member 1
3.1


410909
AW898161
Hs.53112
ESTs, Weakly similar to ALU8_HUMAN ALU SUBFAM
3.1


434926
BE543269
Hs.50252
Homo sapiens HSPC283 mRNA, partial cds
3.1


409239
AA740875
Hs.44307
ESTs
3.1


429017
AA463605
Hs.238995
ESTs
3.1


447072
D61594
Hs.17279
tyrosylprotein sulfotransferase 1
3.1


426514
BE616633
Hs.301122
bone morphogenetic protein 7 (osteogenic prot
3.1


448133
AA723157
Hs.73769
folate receptor 1 (adult)
3.1


418792
AB037805
Hs.88442
KIAA1384 protein
3.1


427528
AU077143
Hs.179565
minichromosome maintenance deficient (S. cere
3.1


402077


0
3.1


440671
AW297920
Hs.130054
ESTs
3.1


419890
X17360
Hs.278255
homeo box D4
3.1


406687
M31126
Hs.272620
pregnancy specific beta-1-glycoprotein 9
3.1


409151
AA306105
Hs.50785
SEC22, vesicle trafficking protein (S. cerevi
3.1


431221
AA449015
Hs.286145
SRB7 (suppressor of RNA polymerase B; yeast)
3.1


443584
AI807036
Hs.101619
ESTs
3.1


445525
BE149866
Hs.14831
ESTs
3.1


410441
BE298210

gb: 601118016F1 NIH_MGC_17 Homo sapiens cDNA c
3.1


422634
NM_016010
Hs.118821
CGI-62 protein
3.0


420022
AA256253
Hs.120817
ESTs
3.0


453912
AL121031
Hs.32556
KIAA0379 protein
3.0


456844
AI264155
Hs.152981
CDP-diacylglycerol synthase (phosphatidate cy
3.0


414941
C14865
Hs.182159
ESTs
3.0


407807
AL031427
Hs.40094
Human DNA sequence from clone 167A19 on chrom
3.0


414725
AA769791
Hs.120355
Homo sapiens cDNA FLJ13148 fis, clone NT2RP30
3.0


444420
AI148157
Hs.146766
ESTs
3.0


431742
NM_016652
Hs.268281
CGI-201 protein
3.0


412519
AA196241
Hs.73980
troponin T1, skeletal, slow
3.0


418348
AI537167
Hs.96322
Homo sapiens cDNA: FLJ23560 fis, clone LNG098
3.0


444261
AA298958
Hs.10724
MDS023 protein
3.0


457465
AW301344
Hs.195969
ESTs
3.0


443933
AI091631
Hs.135501
Homo sapiens two pore potassium channel KT3.3
3.0


442150
AI368158
Hs.128864
ESTs
3.0


414883
AA926960
Hs.77550
CDC28 protein kinase 1
3.0


442879
AF032922
Hs.8813
syntaxin binding protein 3
3.0


437949
U78519
Hs.41654
ESTs
3.0


403515


0
3.0


403864


0
3.0


407785
AW207285
Hs.98279
ESTs
3.0


426199
AA371865
Hs.97090
ESTs
3.0


426324
AW291787
Hs.200933
ESTs
3.0


427738
NM_000318
Hs.180612
peroxisomal membrane protein 3 (35 kD, Zellweg
3.0


427837
U87309
Hs.180941
vacuolar protein sorting 41 (yeast homolog)
3.0


439430
AF124250
Hs.6564
breast cancer anti-estrogen resistance 3
3.0


442039
AW276240
Hs.128352
ESTs, Weakly similar to p80 [R. norvegicus]
3.0


446978
NM_001938
Hs.16697
down-regulator of transcription 1, TBP-bindin
3.0


452431
U88879
Hs.29499
toll-like receptor 3
3.0


452841
T17431
Hs.65412
DEAD/H (Asp-Glu-Ala-Asp/His) box polypeptide
3.0


432114
AL036021
Hs.225597
ESTs
3.0


445640
AW969626
Hs.31704
ESTs, Weakly similar to KIAA0227 [H. sapiens]
3.0


442607
AA507576
Hs.288361
KIAA0741 gene product
3.0


453920
AI133148
Hs.36602
I factor (complement)
3.0


430000
AW205931
Hs.99598
ESTs
3.0


429164
AI688663
Hs.116586
ESTs
3.0


453331
AI240665
Hs.8895
ESTs
3.0


448663
BE614599
Hs.106823
H. sapiens gene from PAC 426I6, similar to syn
3.0


425776
U25128
Hs.159499
parathyroid hormone receptor 2
3.0


401714


0
3.0


400903


0
3.0


428428
AL037544
Hs.184298
cyclin-dependent kinase 7 (homolog of Xenopus
3.0


443761
AI525743
Hs.160603
ESTs
3.0


451640
AA195601
Hs.26771
Human DNA sequence from clone 747H23 on chrom
3.0


442580
AI733682
Hs.130239
ESTs
3.0





Pkey: Primekey


Ex. Accn: Exemplar Accession


UG ID: UniGene ID


Title: UniGene title


ratio: ratio tumor vs normal tissues






Table 13A lists about 1086 genes up-regulated in ovarian cancer compared to normal ovaries. These were selected as for Table 10A, except that the ratio was greater than or equal to 10, and the denominator was the median value for various non-malignant ovary specimens.







TABLE 13A







About 1086 UP-REGULATED GENES, OVARIAN CANCER VERSUS NORMAL OVARY











Pkey
Ex. Accn
UG ID
Title
ratio














439706
AW872527
Hs.59761
ESTs
109.2


446619
AU076643
Hs.313
secreted phosphoprotein 1 (osteopontin, bone
107.8


422095
AI868872
Hs.288966
ceruloplasmin (ferroxidase)
104.4


447111
AI017574
Hs.17409
cysteine-rich protein 1 (intestinal)
88.3


431130
NM_006103
Hs.2719
epididymis-specific; whey-acidic protein type
82.8


431369
BE184455
Hs.251754
secretory leukocyte protease inhibitor (antil
81.9


413859
AW992356
Hs.8364
ESTs
73.9


446291
BE397753
Hs.14623
interferon, gamma-inducible protein 30
72.7


426050
AF017307
Hs.166096
E74-like factor 3 (ets domain transcription f
68.1


411469
T09997
Hs.70327
cysteine-rich protein 2
66.6


429504
X99133
Hs.204238
lipocalin 2 (oncogene 24p3)
65.7


416971
R34657
Hs.80658
uncoupling protein 2 (mitochondrial, proton c
64.9


450273
AW296454
Hs.24743
hypothetical protein FLJ20171
62.5


446441
AK001782
Hs.15093
hypothetical protein
60.7


428758
AA433988
Hs.98502
Homo sapiens cDNA FLJ14303 fis, clone PLACE20
59.7


441406
Z45957
Hs.7837
Homo sapiens cDNA FLJ10457 fis, clone NT2RP10
57.8


441859
AW194364
Hs.128022
ESTs, Weakly similar to FIG. 1 MOUSE FIG-1 PROT
56.7


448406
AW772298
Hs.21103
Homo sapiens mRNA; cDNA DKFZp564B076
55.7


414602
AW630088
Hs.76550
Homo sapiens mRNA; cDNA DKFZp564B1264
55.2


418068
AW971155
Hs.293902
ESTs, Weakly similar to prolyl 4-hydroxylase
54.8


428330
L22524
Hs.2256
matrix metalloproteinase 7 (matrilysin, uteri
53.4


412636
NM_004415
Hs.74316
desmoplakin (DPI, DPII)
51.4


430634
AI860651
Hs.26685
ESTs
50.7


439318
AW837046
Hs.6527
G protein-coupled receptor 56
50.7


417259
AW903838
Hs.81800
chondroitin sulfate proteoglycan 2 (versican)
50.6


407786
AA687538
Hs.38972
tetraspan 1
50.4


426836
N41720
Hs.172684
vesicle-associated membrane protein 8 (endobr
49.7


417308
H60720
Hs.81892
KIAA0101 gene product
48.9


436876
AI124756
Hs.5337
isocitrate dehydrogenase 2 (NADP+), mitochond
48.4


439180
AI393742
Hs.199067
v-erb-b2 avian erythroblastic leukemia viral
47.1


428289
M26301
Hs.2253
complement component 2
46.3


405484


0
46.1


425371
D49441
Hs.155981
mesothelin
45.7


403912


0
45.0


443021
AA368546
Hs.8904
Ig superfamily protein
44.6


427697
T18997
Hs.180372
BCL2-like 1
44.3


428227
AA321649
Hs.2248
INTERFERON-GAMMA INDUCED PROTEIN
44.0


404678


0
43.9


400289
X07820
Hs.2258
Matrix Metalloproteinase 10 (Stromolysin 2)
43.8


451035
AU076785
Hs.430
plastin 1 (I isoform)
43.8


440848
BE314650
Hs.7476
ATPase, H+ transporting, lysosomal (vacuolar
42.8


436278
BE396290
Hs.5097
synaptogyrin 2
42.4


413936
AF113676
Hs.75621
serine (or cysteine) proteinase inhibitor, cl
42.1


420859
AW468397
Hs.100000
S100 calcium-binding protein A8 (calgranulin
42.1


428411
AW291464
Hs.10338
ESTs
41.8


422166
W72424
Hs.112405
S100 calcium-binding protein A9 (calgranulin
41.5


412477
AA150864
Hs.790
microsomal glutathione S-transferase 1
40.7


417130
AW276858
Hs.81256
S100 calcium-binding protein A4 (calcium prot
40.1


424673
AA345051
Hs.294092
ESTs
39.8


416530
U62801
Hs.79361
kallikrein 6 (neurosin, zyme)
39.7


443162
T49951
Hs.9029
ESTs; Highly similar to KERATIN; TYPE I CYTO
39.5


413719
BE439580
Hs.75498
small inducible cytokine subfamily A (Cys—Cys
39.3


424687
J05070
Hs.151738
matrix metalloproteinase 9 (gelatinase B, 92k
38.9


413063
AL035737
Hs.75184
chitinase 3-like 1 (cartilage glycoprotein-39
38.5


429441
AJ224172
Hs.204096
lipophilin B (uteroglobin family member), pro
38.1


418526
BE019020
Hs.85838
solute carrier family 16 (monocarboxylic acid
37.9


415511
AI732617
Hs.182362
ESTs
37.7


409453
AI885516
Hs.95612
ESTs
37.7


445537
AJ245671
Hs.12844
EGF-like-domain; multiple 6
37.3


442432
BE093589
Hs.38178
Homo sapiens cDNA: FLJ23468 fis, clone HSI116
37.3


408243
Y00787
Hs.624
interleukin 8
37.3


419092
J05581
Hs.89603
mucin 1, transmembrane
36.7


444172
BE147740
Hs.104558
ESTs
36.0


412115
AK001763
Hs.73239
hypothetical protein FLJ10901
35.8


420440
NM_002407
Hs.97644
mammaglobin 2
35.7


414386
X00442
Hs.75990
haptoglobin
35.3


423225
AA852604
Hs.125359
Thy-1 cell surface antigen
35.1


440596
H13032
Hs.103378
ESTs, Weakly similar to DRR1 [H. sapiens]
35.0


413278
BE563085
Hs.833
interferon-stimulated protein, 15 kDa
34.9


418506
AA084248
Hs.85339
G protein-coupled receptor 39
34.8


445919
T53519
Hs.290357
ESTs
34.7


416854
H40164
Hs.80296
Purkinje cell protein 4
34.4


414186
U33446
Hs.75799
protease, serine, 8 (prostasin)
34.2


434371
AA631362

gb: np86b01.s1 NCI_CGAP_Thy1 Homo sapiens cDNA
33.9


421937
AI878857
Hs.109706
HN1 protein
33.9


449722
BE280074
Hs.23960
cyclin B1
33.8


400965


0
33.7


452203
X57522
Hs.158164
ATP-binding cassette, sub-family B (MDR/TAP),
33.5


411945
AL033527
Hs.92137
v-myc avian myelocytomatosis viral oncogene h
33.5


425811
AL039104
Hs.159557
karyopherin alpha 2 (RAG cohort 1, importin a
33.4


408901
AK001330
Hs.48855
hypothetical protein FLJ10468
33.3


438461
AW075485
Hs.286049
phosphoserine aminotransferase
33.3


422963
M79141
Hs.13234
ESTs
33.3


426158
NM_001982
Hs.199067
v-erb-b2 avian erythroblastic leukemia viral
33.2


431836
AF178532
Hs.271411
beta-site APP-cleaving enzyme 2
32.8


421502
AF111856
Hs.105039
solute carrier family 34 (sodium phosphate),
32.5


431211
M86849
Hs.5566
Homo sapiens connexin 26 (GJB2) mRNA, complet
32.5


436552
NM_014038
Hs.5216
HSPC028 protein
32.5


442533
AA161224
Hs.8372
ubiquinol-cytochrome c reductase (6.4 kD) subu
32.5


406400
AA343629
Hs.104570
kallikrein 8 (neuropsin/ovasin)
32.4


450353
AI244661
Hs.103296
ESTs
32.4


422158
L10343
Hs.112341
protease inhibitor 3, skin-derived (SKALP)
32.4


433412
AV653729
Hs.8185
CGI-44 protein; sulfide dehydrogenase like (y
32.3


441020
W79283
Hs.35962
ESTs
32.2


432201
AI538613
Hs.135657
TMPRSS3a mRNA for serine protease (ECHOS1) (T
32.0


424125
M31669
Hs.1735
inhibin, beta B (activin AB beta polypeptide)
31.9


453309
AI791809
Hs.32949
defensin, beta 1
31.8


408380
AF123050
Hs.44532
diubiquitin
31.7


419329
AY007220
Hs.288998
S100-type calcium binding protein A14
31.6


409231
AA446644
Hs.692
GA733-2; epithelial glycoprotein (EGP) (KSA)
31.6


423961
D13666
Hs.136348
Homo sapiens mRNA for osteoblast specific fac
31.2


413840
AI301558
Hs.290801
ESTs
30.8


440943
AW082298
Hs.146161
ESTs, Weakly similar to KIAA0859 protein [H. s
30.8


419239
AA468183
Hs.184598
Homo sapiens cDNA: FLJ23241 fis, clone COL013
30.4


410132
NM_003480
Hs.58882
Microfibril-associated glycoprotein-2
30.2


418203
X54942
Hs.83758
CDC28 protein kinase 2
30.1


412719
AW016610
Hs.129911
ESTs
30.0


407862
BE548267
Hs.50724
Homo sapiens cDNA FLJ10934 fis, clone OVARC10
30.0


431563
AI027643
Hs.120912
ESTs
29.9


431743
AW972642
Hs.293055
ESTs
29.8


443295
AI049783
Hs.241284
ESTs
29.7


413745
AW247252
Hs.75514
nucleoside phosphorylase
29.7


441028
AI333660
Hs.17558
ESTs
29.6


442315
AA173992
Hs.7956
ESTs
29.6


452838
U65011
Hs.30743
Preferentially expressed antigen in melanoma
29.5


428479
Y00272
Hs.184572
cell division cycle 2, G1 to S and G2 to M
29.5


432280
BE440142
Hs.2943
signal recognition particle 19 kD
29.4


420158
AI791905
Hs.95549
hypothetical protein
29.3


445033
AV652402
Hs.155145
ESTs
29.2


452367
U71207
Hs.29279
eyes absent (Drosophila) homolog 2
29.1


432706
NM_013230
Hs.286124
CD24
29.0


422163
AF027208
Hs.297332
prominin (mouse)-like 1
28.7


447035
NM_004753
Hs.17144
short-chain dehydrogenase/reductase 1
28.6


443958
BE241880
Hs.10029
cathepsin C
28.2


422956
BE545072
Hs.122579
ESTs
28.1


450377
AB033091
Hs.24936
ESTs
28.0


447471
AF039843
Hs.18676
sprouty (Drosophila) homolog 2
28.0


444725
AW952022
Hs.234174
Homo sapiens cDNA FLJ13819 fis, clone THYRO10
27.8


430250
NM_016929
Hs.283021
chloride intracellular channel 5
27.7


416305
AU076628
Hs.79187
coxsackie virus and adenovirus receptor
27.6


418174
L20688
Hs.83656
Rho GDP dissociation inhibitor (GDI) beta
27.5


417233
W25005
Hs.24395
small inducible cytokine subfamily B (Cys-X-C
27.4


417866
AW067903
Hs.82772
collagen, type XI, alpha 1
27.3


427344
NM_000869
Hs.2142
5-hydroxytryptamine (serotonin) receptor 3A
27.2


442993
BE018682
Hs.44343
ESTs
27.2


407137
T97307
Hs.199067
v-erb-b2 avian erythroblastic leukemia viral
27.0


419356
AI656166
Hs.7331
ESTs
27.0


433662
W07162
Hs.150826
CATX-8 protein
26.7


422576
BE548555
Hs.118554
CGI-83 protein
26.4


423271
W47225
Hs.126256
interleukin 1, beta
26.3


443715
AI583187
Hs.9700
cyclin E1
26.1


420186
NM_015925
Hs.95697
liver-specific bHLH-Zip transcription factor
26.0


419551
AW582256
Hs.91011
anterior gradient 2 (Xenepus laevis) homolog
25.9


443672
AA323362
Hs.9667
butyrobetaine (gamma), 2-oxoglutarate dioxyge
25.8


416889
AW250318
Hs.80395
mal, T-cell differentiation protein
25.3


408474
AA188823
Hs.83196
Homo sapiens cDNA: FLJ23597 fis, clone LNG152
25.3


411825
AK000334
Hs.72289
hypothetical protein FLJ20327
25.3


400881


0
25.2


440594
AW445167
Hs.126036
ESTs
25.1


414586
AA306160
Hs.76506
lymphocyte cytosolic protein 1 (L-plastin)
25.1


411925
AW014588
Hs.72925
chromosome 11 open reading frame 13
25.1


417869
BE076254
Hs.82793
proteasome (prosome, macropain) subunit, beta
25.0


433447
U29195
Hs.3281
neuronal pentraxin II
25.0


450858
C18458
Hs.25597
elongation of very long chain fatty acids (FE
24.8


410619
BE512730
Hs.65114
keratin 18
24.8


434094
AA305599
Hs.238205
hypothetical protein PRO2013
24.6


421924
BE514514
Hs.109606
coronin, actin-binding protein, 1A
24.6


446859
AI494299
Hs.16297
COX17 (yeast) homolog, cytochrome c oxidase a
24.5


421451
AA291377
Hs.50831
ESTs
24.3


433929
AI375499
Hs.27379
ESTs
24.3


438930
AW843633
Hs.81256
S100 calcium-binding protein A4 (calcium prot
24.2


444212
AW503976
Hs.10649
basement membrane-induced gene
24.2


441633
AW958544
Hs.112242
ESTs
24.2


441134
W29092
Hs.7678
cellular retinoic acid-binding protein 1
24.2


417715
AW969587
Hs.86366
ESTs
24.1


409361
NM_005982
Hs.54416
sine oculis homeobox (Drosophila) homolog 1
24.1


416984
H38765
Hs.80706
diaphorase (NADH/NADPH) (cytochrome b-5 reduc
24.1


430125
U46418
Hs.233950
serine protease inhibitor, Kunitz type 1
23.9


434078
AW880709
Hs.283683
EST
23.8


408669
AI493591
Hs.78146
platelet/endothelial cell adhesion molecule (
23.8


439413
AI598252
Hs.37810
ESTs
23.7


449034
AI624049
Hs.277523
gb: ts41a09.x1 NCI_CGAP_Ut1 Homo sapiens cDNA
23.7


420344
BE463721
Hs.97101
Putative G protein-coupled receptor GPCR150
23.6


431243
U46455
Hs.252189
syndecan 4 (amphiglycan, ryudocan)
23.6


417515
L24203
Hs.82237
ataxia-telangiectasia group D-associated prot
23.5


451267
AI033894
Hs.117865
solute carrier family 17 (anion/sugar transpo
23.4


450101
AV649989
Hs.24385
Human hbc647 mRNA sequence
23.4


419693
AA133749
Hs.92323
FXYD domain-containing ion transport regulato
23.4


431103
M57399
Hs.44
pleiotrophin (heparin binding growth factor 8
23.4


451110
AI955040
Hs.301584
ESTs
23.3


426295
AW367283
Hs.75839
zinc finger protein 6 (CMPX1)
23.2


448517
AA082750
Hs.42194
hypothetical protein FLJ22649 similar to sign
23.1


424670
W61215
Hs.116651
epithelial V-like antigen 1
23.1


417847
AI521558
Hs.288312
Homo sapiens cDNA: FLJ22316 fis, clone HRC052
23.1


449027
AJ271216
Hs.22880
dipeptidylpeptidase III
23.1


424969
AW950928
Hs.153998
creatine kinase, mitochondrial 1 (ubiquitous)
23.1


433159
AB035898
Hs.150587
kinesin-like protein 2
23.0


411393
AW797437
Hs.69771
B-factor, properdin
23.0


434815
AF155582
Hs.46744
core1 UDP-galactose:N-acetylgalactosamine-alp
22.8


427585
D31152
Hs.179729
collagen; type X; alpha 1 (Schmid metaphyseal
22.7


445721
H92136
Hs.13144
HSPC160 protein
22.6


448258
BE386983
Hs.85015
ESTs, Weakly similar to A4P_HUMAN INTESTINAL
22.6


456844
AI264155
Hs.152981
CDP-diacylglycerol synthase (phosphatidate cy
22.6


452698
NM_001295
Hs.301921
ESTs
22.5


418693
AI750878
Hs.87409
thrombospondin 1
22.4


414880
AW247305
Hs.119140
eukaryotic translation initiation factor 5A
22.4


401519


0
22.3


402496


0
22.3


420324
AF163474
Hs.96744
DKFZP586D0823 protein, Prostate androgen-regu
22.3


403022


0
22.2


434042
AI589941
Hs.8254
hypothetical protein PRO0899
22.1


419080
AW150835
Hs.18878
hypothetical protein FLJ21620
22.1


406545
AB018249
Hs.10458
small inducible cytokine subfamily A (Cys—Cys
22.1


447362
AW176120
Hs.9061
ESTs
22.0


429547
AW009166
Hs.99376
ESTs
22.0


427954
J03060
Hs.247551
metaxin 1
22.0


423161
AL049227
Hs.124776
Homo sapiens mRNA; cDNA DKFZp564N1116 (from c
22.0


428392
H10233
Hs.2265
secretory granule, neuroendocrine protein 1 (
21.9


444107
T46839
Hs.10319
UDP glycosyltransferase 2 family, polypeptide
21.7


414421
AI521130
Hs.55567
ESTs, Weakly similar to LAK-4p [H. sapiens]
21.5


412589
R28660
Hs.24305
ESTs
21.5


446525
AW967069
Hs.211556
Homo sapiens cDNA: FLJ23378 fis, clone HEP162
21.5


416847
L43821
Hs.80261
enhancer of filamentation 1 (cas-like docking
21.5


436972
AA284679
Hs.25640
claudin 3
21.5


428698
AA852773
Hs.297939
ESTs; Weakly similar to neogenin [H. sapiens]
21.5


421340
F07783
Hs.1369
decay accelerating factor for complement (CD5
21.4


413966
AA133935
Hs.173704
ESTs
21.4


448243
AW369771
Hs.77496
ESTs
21.3


421928
AF013758
Hs.109643
polyadenylate binding protein-interacting pro
21.3


403399


0
21.3


435793
AB037734
Hs.4993
ESTs
21.3


432629
AW860548
Hs.280658
ESTs
21.2


449057
AB037784
Hs.22941
ESTs
21.2


437575
AW954355
Hs.36529
ESTs
21.2


401131


0
21.0


407207
T03651
Hs.179661
tubulin, beta polypeptide
20.8


444783
AK001468
Hs.62180
ESTs
20.8


426230
AA367019
Hs.241395
protease, serine, 1 (trypsin 1)
20.8


447343
AA256641
Hs.236894
ESTs; Highly similar to LOW-DENSITY LIPOPROTE
20.7


409041
AB033025
Hs.50081
KIAA1199 protein
20.6


421305
BE397354
Hs.289721
diptheria toxin resistance protein required f
20.6


411704
AI499220
Hs.71573
hypothetical protein FLJ10074
20.5


417018
M16038
Hs.80887
v-yes-1 Yamaguchi sarcoma viral related oncog
20.5


432827
Z68128
Hs.3109
Rho GTPase activating protein 4
20.4


410174
AA306007
Hs.59461
DKFZP434C245 protein
20.4


425184
BE278288
Hs.155048
Lutheran blood group (Auberger b antigen incl
20.4


452322
BE566343
Hs.28988
glutaredoxin (thioltransferase)
20.3


447526
AL048753
Hs.340
small inducible cytokine A2 (monocyte chemota
20.2


447335
BE617695
Hs.286192
protein phosphatase 1, regulatory (inhibitor)
20.2


424867
AI024860
Hs.153591
Not56 (D. melanogaster)-like protein
20.1


410275
U85658
Hs.61796
transcription factor AP-2 gamma (activating e
20.1


429083
Y09397
Hs.227817
BCL2-related protein A1
20.0


410173
AA706017
Hs.119944
ESTs
19.8


433047
M86135
Hs.279946
methionine-tRNA synthetase
19.8


419088
AI538323
Hs.77496
ESTs
19.7


403381


0
19.6


409162
H25530
Hs.50868
solute carrier family 22 (organic cation tran
19.5


426150
NM_003658
Hs.167218
BarH-like homeobox 2
19.4


449292
AI990292
Hs.225457
ESTs
19.4


425207
AB014551
Hs.155120
rho/rac guanine nucleotide exchange factor (G
19.4


419950
AK001645
Hs.93871
hypothetical protein FLJ10783
19.3


436481
AA379597
Hs.5199
HSPC150 protein similar to ubiquitin-conjugat
19.3


445930
AF055009
Hs.13456
Homo sapiens clone 24747 mRNA sequence
19.2


446608
N75217
Hs.257846
ESTs
19.1


425222
M85430
Hs.155191
villin 2 (ezrin)
19.1


428309
M97815
Hs.183650
cellular retinoic acid-binding protein 2
19.1


420005
AW271106
Hs.133294
ESTs
19.1


436982
AB018305
Hs.5378
spondin 1, (f-spondin) extracellular matrix p
19.0


407142
AA412535
Hs.55235
sphingomyelin phosphodiesterase 2, neutral me
19.0


430122
NM_013342
Hs.233765
TCF3 (E2A) fusion partner (in childhood Leuke
18.9


446293
AI420213
Hs.149722
ESTs
18.9


444825
AW167613
Hs.248
mitogen-activated protein kinase kinase kinas
18.9


407634
AW016569
Hs.301280
UDP-GlcNAc:betaGal beta-1,3-N-acetylglucosami
18.9


445200
AA084460
Hs.12409
somatostatin
18.9


418917
X02994
Hs.1217
adenosine deaminase
18.8


435777
AW419202
Hs.286192
protein phosphatase 1, regulatory (inhibitor)
18.8


431049
AA846576
Hs.103267
hypothetical protein FLJ22548 similar to gene
18.7


426427
M86699
Hs.169840
TTK protein kinase
18.7


436281
AW411194
Hs.120051
ESTs
18.6


425907
AA365752
Hs.155965
ESTs
18.6


459720


ESTs
18.6


421242
AW161386
Hs.13561
ESTs, Weakly similar to dJ37E16.5 [H. sapiens]
18.5


457715
AA642402
Hs.59142
ESTs
18.5


451668
Z43948
Hs.26789
ASPIC (acidic secreted protein in cartilage)A
18.4


437142
AI791617
Hs.145068
ESTs
18.4


418588
BE387040
Hs.182476
ESTs, Weakly similar to similar to alpha/beta
18.3


433068
NM_006456
Hs.288215
sialyltransferase
18.3


419854
AW664873
Hs.87836
Homo sapiens PAC clone RP5-1087M19 from 7q11.
18.3


444726
NM_006147
Hs.11801
interferon regulatory factor 6
18.3


423011
NM_000683
Hs.299847
ESTs, Highly similar to A2AD_HUMAN ALPHA-2C-2
18.2


451428
AW083384
Hs.11067
ESTs, Weakly similar to K02E10.2 [C. elegans]
18.2


424865
AF011333
Hs.153563
lymphocyte antigen 75
18.2


418742
AW451197
Hs.113418
ESTs
18.1


446627
AI973016
Hs.15725
ESTs; hypothetical protein SBBI48
18.1


424885
AI333771
Hs.82204
ESTs
18.1


402926


0
18.0


405452


0
18.0


428641
AA431367
Hs.234546
GMPR2 for guanosine monophosphate reductase i
18.0


454390
AB020713
Hs.56966
KIAA0906 protein
18.0


441784
AI522132
Hs.28700
ESTs
18.0


418758
AW959311
Hs.87019
ESTs
17.9


408621
AI970672
Hs.46638
chromosome 11 open reading frame 8; fetal br
17.9


426201
AW182614
Hs.128499
ESTs
17.8


410442
X73424
Hs.63788
propionyl Coenzyme A carboxylase, beta polype
17.8


456423
AW748920

gb: CM2-BT0306-171199-034-g02 BT0306 Homo sapi
17.8


422867
L32137
Hs.1584
cartilage oligomeric matrix protein
17.8


448110
AA626937
Hs.181551
ESTs
17.7


421750
AK000768
Hs.107872
hypothetical protein FLJ20761
17.7


405224


0
17.7


447630
AI660149
Hs.44865
lymphoid enhancer-binding factor 1
17.7


407663
NM_016429
Hs.37482
COPZ2 for nonclathrin coat protein zeta-COP
17.7


427490
Z95152
Hs.178695
mitogen-activated protein kinase 13
17.6


414812
X72755
Hs.77367
monokine induced by gamma interferon
17.6


427691
AW194426
Hs.20726
ESTs
17.6


420650
AA455706
Hs.44581
heat shock protein hsp70-related protein
17.5


439841
AF038961
Hs.6710
mannose-P-dolichol utilization defect 1
17.5


425810
AI923627
Hs.31903
ESTs
17.5


425397
J04088
Hs.156346
topoisomerase (DNA) II alpha (170 kD)
17.5


456098
AW747800
Hs.55016
hypothetical protein FLJ21935
17.4


428579
NM_005756
Hs.184942
G protein-coupled receptor 64
17.4


410361
BE391804
Hs.62661
guanylate binding protein 1, interferon-induc
17.4


442402
NM_000954
Hs.8272
prostaglandin D2 synthase (21 kD, brain)
17.4


411734
AW374954
Hs.71779
Homo sapiens DNA from chromosome 19, cosmid F
17.3


405295


0
17.3


408340
AB037762
Hs.44268
myelin gene expression factor 2
17.3


456068
AI677897
Hs.76640
RGC32 protein
17.3


448571
AA486794
Hs.66915
ESTs, Weakly similar to 16.7 Kd protein [H. sap
17.2


441829
AL117482
Hs.7978
DKFZP434C131 protein
17.2


418004
U37519
Hs.87539
aldehyde dehydrogenase 8
17.2


412078
X69699
Hs.73149
paired box gene 8
17.2


414658
X58528
Hs.76781
ATP-binding cassette, sub-family D (ALD), mem
17.1


418478
U38945
Hs.1174
cyclin-dependent kinase inhibitor 2A (melanom
17.0


426805
AB032945
Hs.172506
myosin VB
17.0


410247
AF181721
Hs.61345
RU2S
17.0


434516
AA807814
Hs.70582
ESTs, Moderately similar to AF144056 1 apopto
16.9


428153
AW513143
Hs.98367
hypothetical protein FLJ22252 similar to SRY-
16.9


417793
AW405434
Hs.82575
small nuclear ribonucleoprotein polypeptide B
16.9


454163
AW175997

gb: QV0-BT0078-190899-005-E02 BT0078 Homo sapi
16.9


415402
AA164687
Hs.297889
ESTs
16.9


420309
AW043637
Hs.21766
ESTs
16.9


419201
M22324
Hs.1239
alanyl (membrane) aminopeptidase (aminopeptid
16.9


444391
AL137597
Hs.11114
hypothetical protein dJ1181N3.1
16.9


457705
AW974668

gb: EST386757 MAGE resequences, MAGM Homo sapi
16.8


412723
AA648459
Hs.179912
ESTs
16.8


435774
R88066
Hs.4992
tumor suppressing subtransferable candidate 1
16.8


408753
AI337192
Hs.47438
SH3 domain binding glutamic acid-rich protein
16.8


447783
AF054178
Hs.19561
NADH dehydrogenase (ubiquinone) 1 alpha subco
16.8


418085
R40328
Hs.258822
ESTs
16.7


452472
AW957300
Hs.294142
ESTs, Weakly similar to SP49_HUMAN SPLICEOSOM
16.7


409112
BE243971
Hs.50649
quinone oxidoreductase homolog
16.7


410250
AI082777
Hs.61384
KIAA1445 protein
16.7


446219
AI287344
Hs.149827
ESTs
16.6


428928
BE409838
Hs.194657
cadherin 1, type 1, E-cadherin (epithelial)
16.6


425812
AA364128
Hs.245633
ESTs
16.6


411742
AW247593
Hs.71819
eukaryotic translation initiation factor 4E b
16.6


415076
NM_000857
Hs.77890
guanylate cyclase 1, soluble, beta 3
16.6


416209
AA236776
Hs.79078
MAD2 (mitotic arrest deficient, yeast, homolo
16.6


440667
BE076969
Hs.7337
hypothetical protein FLJ10936
16.6


430375
AW371048
Hs.93758
H4 histone family, member H
16.6


419607
R52557
Hs.91579
Homo sapiens clone 23783 mRNA sequence
16.6


410328
BE080190
Hs.62275
CGI-141 protein
16.5


405426


0
16.5


432636
AA340864
Hs.278562
claudin 7
16.5


434725
AK000796
Hs.4104
hypothetical protein
16.5


414683
S78296
Hs.76888
internexin neuronal intermediate filament pro
16.5


429500
X78565
Hs.289114
hexabrachion (tenascin C, cytotactin)
16.5


449944
AF290512
Hs.58215
Homo sapiens rhotekin mRNA, partial cds
16.4


400666


0
16.4


421536
BE250690
Hs.105509
CTL2 gene
16.4


436032
AA150797
Hs.109276
latexin protein
16.4


418196
AI745649
Hs.26549
ESTs, Weakly similar to T00066 hypothetical p
16.4


452323
W44356
Hs.292812
ESTs, Weakly similar to C43H8.1 [C. elegans]
16.4


407699
AA825974
Hs.32646
Homo sapiens cDNA: FLJ21901 fis, clone HEP034
16.4


414617
AI339520
Hs.20524
ESTs, Moderately similar to hexokinase I [H. s
16.3


408204
AA454501
Hs.43666
protein tyrosine phosphatase type IVA, member
16.3


452650
AW270150
Hs.254516
ESTs
16.3


432906
BE265489
Hs.3123
lethal giant larvae (Drosophila) homolog 2
16.3


402408


0
16.3


408805
H69912
Hs.48269
vaccinia related kinase 1
16.3


447155
AA100605
Hs.121557
ESTs, Weakly similar to AF251041 1 SGC32445 p
16.3


405699


0
16.2


406893
M22406

gb: Human intestinal mucin mRNA, partial cds,
16.2


418629
BE247550
Hs.86859
growth factor receptor-bound protein 7 (GRB7)
16.2


424905
NM_002497
Hs.153704
NIMA (never in mitosis gene a)-related kinase
16.2


424243
AI949359
Hs.301837
ESTs, Highly similar to cis Golgi-localized c
16.2


418462
BE001596
Hs.85266
integrin, beta 4
16.1


457205
AI905780
Hs.198272
NADH dehydrogenase (ubiquinone) 1 beta subcom
16.1


428188
M98447
Hs.22
transglutaminase 1 (K polypeptide epidermal t
16.1


449845
AW971183
Hs.60054
ESTs
16.1


406429


0
16.1


407375
AA091354

gb: II0815.seq.F Human fetal heart, Lambda ZAP
16.1


448377
AI494514
Hs.171380
ESTs
16.1


431156
NM_002220
Hs.2722
inositol 1,4,5-trisphosphate 3-kinase A
16.0


450043
AA885699
Hs.24332
CGI-26 protein
16.0


403121


0
16.0


400214


0
15.9


453252
R02436
Hs.215725
ESTs
15.9


451734
NM_006176
Hs.26944
neurogranin (protein kinase C substrate, RC3)
15.9


416855
AA188763
Hs.36793
Homo sapiens cDNA: FLJ23188 fis, clone LNG120
15.9


424474
AA308883
Hs.148680
calcyon; D1 dopamine receptor-interacting pro
15.9


423685
BE350494
Hs.49753
Homo sapiens mRNA for KIAA1561 protein, parti
15.9


428187
AI687303
Hs.285529
ESTs
15.9


438817
AI023799
Hs.163242
ESTs
15.9


425692
D90041
Hs.155956
NAT1; arylamine N-acetyltransferase
15.9


421674
T10707
Hs.296355
neuronal PAS domain protein 2
15.9


439999
AA115811
Hs.6838
ras homolog gene family, member E
15.9


411351
W02919
Hs.283476
peroxisomal acyl-CoA thioesterase
15.9


413027
NM_002885
Hs.75151
RAP1, GTPase activating protein 1
15.9


453884
AA355925
Hs.36232
KIAA0186 gene product
15.8


407894
AJ278313
Hs.41143
phosphoinositide-specific phospholipase C-bet
15.8


422748
AA316266
Hs.129349
ESTs
15.8


414591
AI888490
Hs.55902
ESTs
15.8


421877
AW250380
Hs.109059
mitochondrial ribosomal protein L12
15.8


404780


0
15.8


401192


0
15.8


447519
U46258
Hs.23448
ESTs
15.8


434262
AF121858
Hs.12169
sorting nexin 8
15.7


451253
H48299
Hs.26126
claudin 10
15.7


435499
R89344
Hs.14148
ESTs
15.7


422424
AI186431
Hs.116577
prostate differentiation factor; placental bo
15.7


424834
AK001432
Hs.153408
Homo sapiens cDNA FLJ10570 fis, clone NT2RP20
15.7


424562
AI420859
Hs.150557
basic transcription element binding protein 1
15.7


443247
BE614387
Hs.47378
ESTs
15.7


430696
AA531276
Hs.59509
ESTs
15.6


437044
AL035864
Hs.69517
ESTs, highly similar to differentially expres
15.6


428237
AF175206
Hs.183125
killer cell lectin-like receptor F1
15.6


440048
AA897461
Hs.158469
ESTs, Weakly similar to envelope protein [H. s
15.6


414922
D00723
Hs.77631
glycine cleavage system protein H (aminomethy
15.6


422030
X51416
Hs.110849
estrogen-related receptor alpha
15.6


408716
AI567839
Hs.151714
ESTs
15.5


410258
X52638
Hs.739
6-phosphofructo-2-kinase/fructose-2,6-biphosp
15.5


410530
M25809
Hs.64173
ESTs, Highly similar to VAB1_HUMAN VACUOLAR A
15.5


447072
D61594
Hs.17279
tyrosylprotein sulfotransferase 1
15.5


409015
BE389387
Hs.49767
NADH dehydrogenase (ubiquinone) Fe—S protein
15.5


447549
AI871120
Hs.231265
ESTs
15.5


449704
AK000733
Hs.23900
GTPase activating protein
15.4


427337
Z46223
Hs.176663
Fc fragment of IgG, low affinity IIIb, recept
15.4


421630
NM_001956
Hs.1407
endothelin 2
15.4


433018
AI669760
Hs.188881
ESTs
15.4


422938
NM_001809
Hs.1594
centromere protein A (17 kD)
15.3


407014
U38268

gb: Human cytochrome b pseudogene, partical cds
15.2


429311
AF080157
Hs.198998
conserved helix-loop-helix ubiquitous kinase
15.2


431842
NM_005764
Hs.271473
epithelial protein up-regulated in carcinoma,
15.2


406907
Z25427

gb: H. sapiens protein-serine/threonine kinase
15.2


458495
AI202029
Hs.148593
ESTs
15.2


420551
AL137692
Hs.98790
Homo sapiens mRNA; cDNA DKFZp434P182 (from cl
15.1


448443
AW167128
Hs.231934
ESTs
15.1


443646
AI085198
Hs.298699
ESTs
15.1


431538
AL137547
Hs.259619
Homo sapiens mRNA; cDNA DKFZp434B1120 (from c
15.1


436687
AA868643
Hs.120461
ESTs
15.1


420917
AW135716
Hs.117330
ESTs
15.0


428575
M19684
Hs.184929
serine (or cysteine) proteinase inhibitor, cl
15.0


403482


0
15.0


421499
AI271438
Hs.105022
Homo sapiens PAC clone RP4-701O16 from 7q33-q
15.0


401047


0
14.9


417749
U09196
Hs.82520
polymerase (DNA-directed), delta 4
14.9


416693
AI373204
Hs.79531
Homo sapiens TTF-I interacting peptide 20 mRN
14.9


428474
AB023182
Hs.184523
KIAA0965 protein
14.9


428862
NM_000346
Hs.2316
SRY (sex-determining region Y)-box 9 (campome
14.9


430271
T06199
Hs.237506
heat shock cognate 40
14.9


414328
Z21666
Hs.75900
aconitase 2, mitochondrial
14.9


415314
N88802
Hs.5422
glycoprotein M6B
14.8


453735
AI066629
Hs.125073
ESTs
14.8


424345
AK001380
Hs.145479
Homo sapiens cDNA FLJ10518 fis, clone NT2RP20
14.8


423575
C18863
Hs.163443
ESTs
14.8


438081
H49546
Hs.298964
ESTs
14.8


403485


0
14.8


452114
N22687
Hs.8236
ESTs
14.8


426559
AB001914
Hs.170414
paired basic amino acid cleaving system 4
14.8


412869
AA290712
Hs.82407
Homo sapiens HSPC296 mRNA, partial cds
14.8


452101
T60298

gb: yb87f12.r1 Stratagene liver (937224) Homo
14.7


420505
AW967984
Hs.291612
ESTs
14.7


426125
X87241
Hs.166994
FAT tumor suppressor (Drosophila) homolog
14.7


433336
AF017986
Hs.31386
ESTs; Highly similar to FRIZZLED PROTEIN PRE
14.7


428977
AK001404
Hs.194698
cyclin B2
14.7


429785
H82114
Hs.301769
ESTs
14.7


402424


0
14.7


424971
AA479005
Hs.154036
tumor suppressing subtransferable candidate 3
14.7


433037
NM_014158
Hs.279938
HSPC067 protein
14.6


421670
BE207318
Hs.106674
BRCA1 associated protein-1 (ubiquitin carboxy
14.6


438598
AI805943
Hs.5723
Homo sapiens cDNA: FLJ23439 fis, clone HSI001
14.6


453370
AI470523
Hs.182356
ESTs, Moderately similar to translation initi
14.6


410561
BE540255
Hs.6994
Homo sapiens cDNA: FLJ22044 fis, clone HEP091
14.6


402287


0
14.6


419741
NM_007019
Hs.93002
ubiquitin carrier protein E2-C
14.6


442047
AA974598
Hs.150324
ESTs
14.5


428582
BE336699
Hs.185055
BENE protein
14.5


440006
AK000517
Hs.6844
hypothetical protein FLJ20510
14.5


406851
AA609784
Hs.180255
major histocompatibility complex, class II, D
14.5


457316
AI123657
Hs.127264
ESTs
14.5


420453
AL157500
Hs.97840
Homo sapiens mRNA; cDNA DKFZp434G015 (from cl
14.5


436406
AW105723
Hs.125346
ESTs
14.5


420736
AI263022
Hs.82204
ESTs
14.5


419743
AW408762
Hs.127478
ESTs
14.5


429113
D28235
Hs.196384
Prostaglandin-endoperoxide synthase 2 (COX-2)
14.5


450256
AA286887
Hs.24724
MFH-amplified sequences with leucine-rich tan
14.5


424906
AI566086
Hs.153716
Homo sapiens mRNA for Hmob33 protein, 3′ untr
14.5


427414
F11750
Hs.6647
Homo sapiens cDNA FLJ13088 fis, clone NT2RP30
14.4


419839
U24577
Hs.93304
phospholipase A2, group VII (platelet-activat
14.4


418738
AW388633
Hs.6682
solute carrier family 7, member 11
14.3


429414
AI783656
Hs.202095
empty spiracles (Drosophila) homolog 2
14.3


424669
AA417181
Hs.120858
Homo sapiens cDNA FLJ13945 fis, clone Y79AA10
14.3


408989
AW361666
Hs.49500
KIAA0746 protein
14.3


406788
AI911841
Hs.5184
TH1 drosophila homolog
14.3


417861
AA334551
Hs.82767
sperm specific antigen 2
14.3


402104


0
14.3


416368
R88849

gb: ym96a06.r1 Soares adult brain N2b4HB55Y Ho
14.2


405802


0
14.2


448357
N20169
Hs.108923
ESTs
14.2


444261
AA298958
Hs.10724
MDS023 protein
14.2


407846
AA426202
Hs.40403
Cbp/p300-interacting transactivator, with Glu
14.2


425163
D10040
Hs.154890
fatty-acid-Coenzyme A ligase, long-chain 2
14.1


402520


0
14.1


429597
NM_003816
Hs.2442
a disintegrin and metalloproteinase domain 9
14.1


430044
AA464510
Hs.152812
EST cluster (not in UniGene)
14.1


429663
M68874
Hs.211587
Human phosphatidylcholine 2-acylhydrolase (cP
14.1


427036
AA397625
Hs.163913
ESTs
14.1


444381
BE387335
Hs.283713
ESTs
14.1


432090
AW972855
Hs.292853
ESTs
14.0


406778
H06273
Hs.101651
Homo sapiens mRNA; cDNA DKFZp434C107 (from cl
14.0


404961
AW972195
Hs.284236
aldo-keto reductase family 7, member A3 (afla
14.0


452313
Y00486
Hs.28914
adenine phosphoribosyltransferase
14.0


452355
N54926
Hs.29202
G protein-coupled receptor 34
14.0


429942
AI338993
Hs.134535
ESTs
14.0


403165


0
13.9


442150
AI368158
Hs.128864
ESTs
13.9


439709
AW401433
Hs.6649
hypothetical protein FLJ20128
13.9


456799
AC004923
Hs.135187
Homo sapiens clone CDABP0025 mRNA sequence
13.9


427356
AW023482
Hs.97849
ESTs
13.9


448982
AI638164
Hs.225520
ESTs
13.9


432025
BE407132
Hs.111286
hypothetical protein FLJ22512
13.8


427505
AA361562
Hs.178761
26S proteasome-associated pad1 homolog
13.8


402965


0
13.8


418601
AA279490
Hs.86368
calmegin
13.8


436954
AA740151
Hs.130425
ESTs
13.8


405024


0
13.8


453976
BE463830
Hs.163714
ESTs
13.8


431921
N46466
Hs.58879
ESTs
13.8


401735


0
13.8


445496
AB007860
Hs.12802
development and differentiation enhancing fac
13.8


425007
AA456483
Hs.172081
phosphodiesterase 4D, cAMP-specific (dunce (D
13.7


409463
AI458165
Hs.17296
ESTs
13.7


430193
AI826653
Hs.102928
Homo sapiens cDNA FLJ13479 fis, clone PLACE10
13.7


458869
AI637934
Hs.224978
ESTs
13.7


426769
AA075596
Hs.172153
glutathione peroxidase 3 (plasma)
13.7


416661
AA634543
Hs.79440
IGF-II mRNA-binding protein 3
13.7


439901
N73885
Hs.124169
ESTs
13.7


431374
BE258532
Hs.251871
CTP synthase
13.7


432861
AA339526
Hs.279593
HSPC171 protein
13.7


441172
AI279652
Hs.132879
ESTs
13.7


410001
AB041036
Hs.57771
kallikrein 11; serine protease (TLSP)
13.7


430315
NM_004293
Hs.239147
guanine deaminase
13.6


422769
AA938905
Hs.289112
CGI-43 protein
13.6


402389


0
13.6


448977
X91809
Hs.22698
regulator of G-protein signalling 19
13.6


459648


gb: IL3-CT0220-150200-070-B02 CT0220 Homo sapi
13.6


452972
M31732
Hs.31210
B-cell CLL/lymphoma 3
13.6


431441
U81961
Hs.2794
sodium channel, nonvoltage-gated 1 alpha
13.6


448585
AB020676
Hs.21543
KIAA0869 protein
13.6


428385
AF112213
Hs.184062
putative Rab5-interacting protein
13.6


434699
AA643687
Hs.149425
Homo sapiens cDNA FLJ11980 fis, clone HEMBB10
13.6


447238
AW451676
Hs.158564
ESTs
13.6


437108
AA434054
Hs.80624
Homo sapiens cDNA: FLJ23442 fis, clone HSI009
13.6


425749
AW328587
Hs.159448
surfeit 2
13.5


425154
NM_001851
Hs.154850
collagen, type IX, alpha 1
13.5


413753
U17760
Hs.301103
Laminin, beta 3 (nicein (125 kD), kalinin (140
13.5


419034
NM_002110
Hs.89555
hemopoietic cell kinase
13.5


448361
H82028
Hs.238707
Homo sapiens cDNA: FLJ22457 fis, clone HRC099
13.5


412754
AW160375
Hs.74565
amyloid beta (A4) precursor-like protein 1
13.5


419081
AI798863
Hs.87191
ESTs
13.5


407732
AW138839
Hs.24210
ESTs
13.5


423329
AF054910
Hs.127111
tektin 2 (testicular)
13.5


422627
BE336857
Hs.118787
transforming growth factor, beta-induced, 68k
13.4


439636
AF086467

gb: Homo sapiens full length insert cDNA clone
13.4


417605
AF006609
Hs.82294
regulator of G-protein signalling 3
13.4


445861
BE293423
Hs.11809
single Ig IL-1R-related molecule
13.4


447350
AI375572
Hs.172634
ESTs; HER4 (c-erb-B4)
13.4


451807
W52854
Hs.27099
DKFZP564J0863 protein
13.4


421515
Y11339
Hs.105352
GalNAc alpha-2, 6-sialyltransferase I, long f
13.4


422443
NM_014707
Hs.116753
histone deacetylase 7B
13.4


412504
Z44496
Hs.26039
Homo sapiens cDNA FLJ13937 fis, clone Y79AA10
13.4


453344
BE349075
Hs.44571
ESTs
13.4


402885


0
13.4


438712
AW978161
Hs.169877
ESTs
13.4


421774
AL050374
Hs.108169
DKFZP586C1619 protein
13.3


425638
NM_012337
Hs.158450
nasopharyngeal epithelium specific protein 1
13.3


401897


0
13.3


425601
AW629485
Hs.293352
ESTs
13.3


450779
AW204145
Hs.156044
ESTs
13.3


444858
AI199738
Hs.208275
ESTs, Weakly similar to unnamed protein produ
13.3


442619
AA447492
Hs.20183
ESTs, Weakly similar to AF164793 1 protein x
13.3


434263
N34895
Hs.44648
ESTs
13.3


426059
BE292842
Hs.166120
interferon regulatory factor 7
13.3


407467
D55638

gb: Human B-cell PABL (pseudoautosomal boundar
13.3


412560
R24601
Hs.108300
CCR4-NOT transcription complex, subunit 3
13.2


442986
AI025990
Hs.285520
ESTs
13.2


420317
AB006628
Hs.96485
KIAA0290 protein
13.2


443211
AI128388
Hs.143655
ESTs
13.2


434361
AF129755
Hs.117772
ESTs
13.2


423493
AI815965
Hs.129683
ubiquitin-conjugating enzyme E2D 1 (homologou
13.2


414183
AW957446
Hs.301711
ESTs
13.2


447778
BE620592
Hs.71190
ESTs
13.2


435106
AA100847
Hs.193380
ESTs, Highly similar to AF174600 1 F-box prot
13.1


439490
AW249197
Hs.100043
ESTs, Weakly similar to PSF_HUMAN PTB-ASSOCIA
13.1


409606
AW444594
Hs.2387
transglutaminase 4 (prostate)
13.1


421308
AA687322
Hs.192843
ESTs
13.1


414950
C15407

gb: C15407 Clontech human aorta polyA+ mRNA (6
13.1


416783
AA206186
Hs.79889
monocyte to macrophage differentiation-associ
13.1


415927
AL120168
Hs.78919
Kell blood group precursor (McLeod phenotype)
13.1


422605
H16646
Hs.118666
Human clone 23759 mRNA, partial cds
13.0


430427
AA296701
Hs.241413
opticin
13.0


424620
AA101043
Hs.151254
kallikrein 7 (chymotryptic; stratum corneum)
13.0


421693
X71490
Hs.106876
ATPase, H+ transporting, lysosomal (vacuolar
13.0


407727
AW411148
Hs.38044
DKFZP564M082 protein
13.0


427706
AW971225
Hs.293800
ESTs,Weakly similar to ALU1_HUMAN ALU SUBFAM
13.0


406709
AI355761
Hs.242463
keratin 8
13.0


405353


0
13.0


453060
AW294092
Hs.21594
ESTs
13.0


459299
BE094291
Hs.155651
hepatocyte nuclear factor 3, beta
13.0


447843
AW337186
Hs.224891
ESTs
13.0


446576
AI659477
Hs.51820
ESTs, Moderately similar to ALU7_HUMAN ALU SU
13.0


449700
L02867
Hs.78358
ESTs
13.0


436476
AA326108
Hs.53631
ESTs
13.0


432532
AW058459
Hs.162246
ESTs
13.0


408405
AK001332
Hs.44672
hypothetical protein FLJ10470
13.0


432673
AB028859
Hs.278605
ER-associated DNAJ; ER-associated Hsp40 co-ch
12.9


414684
AW630023
Hs.76893
3-hydroxybutyrate dehydrogenase (heart, mitoc
12.9


447210
AF035269
Hs.17752
phosphatidylserine-specific phospholipase A1a
12.9


427923
AW274357
Hs.268384
Fzr1 protein
12.9


437395
AL365408
Hs.10632
hypothetical protein DKFZp762M136
12.9


441627
AA947552
Hs.58086
ESTs
12.9


419084
AA496539
Hs.179902
transporter-like protein
12.9


423067
AA321355
Hs.285401
ESTs
12.9


423070
R55677
Hs.155569
ESTs
12.8


441344
BE250144
Hs.41514
ESTs
12.8


423527
AI206965
Hs.105861
Homo sapiens cDNA FLJ13824 fis, clone THYRO10
12.8


417006
AW673606
Hs.80758
aspartyl-tRNA synthetase
12.8


453552
AL041941
Hs.154729
3-phosphoinositide dependent protein kinase-1
12.8


453657
W23237
Hs.296162
ESTs
12.8


434414
AI798376

gb: tr34b07.x1 NCI_CGAP_Ov23 Homo sapiens cDNA
12.7


456051
T85626
Hs.76239
hypothetical protein FLJ20608
12.7


451659
BE379761
Hs.14248
ESTs, Weakly similar to ALU8_HUMAN ALU SUBFAM
12.7


418216
AA662240
Hs.283099
AF15q14 protein
12.7


423281
AJ271684
Hs.126355
C-type (calcium dependent, carbohydrate-recog
12.7


424275
AW673173
Hs.144505
DKFZP566F0546 protein
12.7


440062
AI350518
Hs.129692
ESTs
12.7


444371
BE540274
Hs.239
Forkhead box M1
12.7


412520
AA442324
Hs.795
H2A histone family, member O
12.7


413349
BE086692

gb: QV1-BT0678-130400-156-g07 BT0678 Homo sapi
12.7


414500
W24087
Hs.76285
DKFZP564B167 protein
12.6


429261
AW176254
Hs.143475
ESTs
12.6


402238


0
12.6


400280


0
12.6


421246
AW582962
Hs.300961
ESTs, Highly similar to AF151805 1 CGI-47 pro
12.6


442029
AW956698
Hs.14456
neural precursor cell expressed, developmenta
12.6


435502
L13266
Hs.105
glutamate receptor, ionotropic, N-methyl D-as
12.6


409964
AW368226
Hs.67928
ESTs
12.6


418793
AW382987
Hs.88474
prostaglandin-endoperoxide synthase 1 (prosta
12.5


452117
AI421760
Hs.77870
Homo sapiens cDNA FLJ12750 fis, clone NT2RP20
12.5


448074
BE621355
Hs.27160
ESTs
12.5


442655
AW027457
Hs.30323
ESTs
12.5


409928
AL137163
Hs.57549
hypothetical protein dJ473B4
12.5


400240


0
12.5


413048
M93221
Hs.75182
mannose receptor, C type 1
12.5


426215
AW963419
Hs.155223
ESTs
12.5


430024
AI808780
Hs.227730
integrin, alpha 6
12.5


445655
AA873830
Hs.167746
B cell linker protein
12.5


419941
X98654
Hs.93837
phosphatidylinositol transfer protein, membra
12.5


425280
U31519
Hs.1872
phosphoenolpyruvate carboxykinase 1 (soluble)
12.5


427767
AI879283
Hs.180714
cytochrome c oxidase subunit VIa polypeptide
12.4


450243
AW119084
Hs.201037
ESTs
12.4


408930
AA146721
Hs.49005
hypothetical protein
12.4


418783
T41368

gb: ph1d1_19/1TV Outward Alu-primed hncDNA lib
12.4


452096
BE394901
Hs.226785
ESTs
12.4


424513
BE385864
Hs.149894
mitochondrial translational initiation factor
12.4


422306
BE044325
Hs.227280
Homo sapiens mRNA for Lsm5 protein
12.4


409031
AA376836
Hs.76728
ESTs
12.4


435515
N40080
Hs.6879
DC13 protein
12.4


429583
NM_006412
Hs.209119
1-acylglycerol-3-phosphate O-acyltransferase
12.3


449643
R05989
Hs.19603
ESTs
12.3


440313
AL050060
Hs.7158
DKFZP566H073 protein
12.3


425593
AA278921
Hs.1908
proteoglycan 1, secretory granule
12.3


447357
AI375922
Hs.159367
ESTs
12.3


405089


0
12.3


414972
BE263782
Hs.77695
KIAA0008 gene product
12.3


435039
AW043921
Hs.130526
ESTs
12.3


447033
AI357412
Hs.157601
EST - not in UniGene
12.3


427521
AW973352
Hs.299056
ESTs
12.3


409377
AA300274
Hs.115659
Homo sapiens cDNA: FLJ23461 fis, clone HSI077
12.3


400116


0
12.3


445806
AL137516
Hs.13323
hypothetical protein FLJ22059
12.2


457817
AA247751
Hs.79572
cathepsin D (lysosomal aspartyl protease)
12.2


442410
AW996503
Hs.197680
ESTs
12.2


445404
AI261687
Hs.145541
ESTs, Weakly similar to JC4974 sodium iodide
12.2


403372
AW249152
Hs.44017
SIR2 (silent mating type information regulati
12.2


427082
AB037858
Hs.173484
hypothetical protein FLJ10337
12.2


433764
AW753676
Hs.39982
ESTs
12.2


400268


0
12.2


433190
M26901
Hs.3210
renin
12.2


444863
AW384082
Hs.301323
ESTs
12.2


434779
AF153815
Hs.50151
potassium inwardly-rectifying channel, subfam
12.2


451346
NM_006338
Hs.26312
glioma amplified on chromosome 1 protein (leu
12.2


430262
AA218780
Hs.237323
N-acetylglucosamine-phosphate mutase
12.2


421071
AI311238
Hs.104476
ESTs
12.2


426773
NM_015556
Hs.172180
KIAA0440 protein
12.1


409178
BE393948
Hs.50915
kallikrein 5
12.1


400250


0
12.1


428450
NM_014791
Hs.184339
KIAA0175 gene product
12.1


414531
T69387
Hs.76364
allograft inflammatory factor 1
12.1


448210
AW247775
Hs.7393
hypothetical protein from EUROIMAGE 1987170
12.1


440081
AA863389
Hs.135643
ESTs
12.1


413179
N99692
Hs.75227
NADH dehydrogenase (ubiquinone) 1 alpha subco
12.1


447551
BE066634
Hs.929
myosin, heavy polypeptide 7, cardiac muscle,
12.1


400517
AF242388
Hs.149585
lengsin
12.1


401610


0
12.0


454381
AI935093
Hs.193428
ESTs
12.0


443997
AW081465
Hs.299644
ESTs
12.0


402944


0
12.0


430637
BE160081
Hs.256290
S100 calcium-binding protein A11 (calgizzarin
12.0


415099
AI492170
Hs.77917
ubiquitin carboxyl-terminal esterase L3 (ubiq
12.0


445422
AV653731
Hs.282829
ESTs
12.0


416667
AK000526
Hs.79457
hypothetical protein FLJ20519
12.0


442611
BE077155
Hs.177537
ESTs
12.0


443271
BE568568
Hs.195704
ESTs
12.0


415120
N64464
Hs.34950
ESTs
12.0


439574
AI469788
Hs.165190
ESTs
12.0


405804


0
12.0


412519
AA196241
Hs.73980
troponin T1, skeletal, slow
12.0


414135
NM_004419
Hs.2128
dual specificity phosphatase 5
12.0


447075
AV662037
Hs.124740
ESTs
12.0


416841
N33878
Hs.249495
heterogeneous nuclear ribonucleoprotein A1
12.0


402943


0
11.9


416933
BE561850
Hs.80506
small nuclear ribonucleoprotein polypeptide A
11.9


439744
AL389994
Hs.301272
ESTs, Weakly similar to homologue of Drosphil
11.9


405762


0
11.9


408983
NM_000492
Hs.663
cystic fibrosis transmembrane conductance reg
11.9


455102
BE005496

gb: CM1-BN0117-110400-183-b09 BN0117 Homo sapi
11.9


402840


0
11.9


449183
AW445022
Hs.196985
Homo sapiens cDNA: FLJ21135 fis, clone CAS072
11.9


439273
AW139099
Hs.269701
ESTs
11.9


450484
BE220675

gb: ht98f11.x1 NCI_CGAP_Lu24 Homo sapiens cDNA
11.9


445431
AF137386
Hs.12701
plasmolipin
11.9


401888


0
11.9


426037
AW160780
Hs.166071
cyclin-dependent kinase 5
11.9


416742
R38644
Hs.248420
ESTs
11.9


418324
AW246273
Hs.84131
threonyl-tRNA synthetase
11.8


412870
N22788
Hs.82407
Homo sapiens HSPC296 mRNA, partial cds
11.8


432680
T47364
Hs.278613
interferon, alpha-inducible protein 27
11.8


421478
AI683243
Hs.97258
ESTs
11.8


426635
BE395109
Hs.129327
ESTs
11.8


420523
AA262999
Hs.42788
ESTs
11.8


426227
U67058
Hs.168102
Human proteinase activated receptor-2 mRNA; 3
11.8


416658
U03272
Hs.79432
fibrillin 2 (congenital contractural arachnod
11.8


441816
AI401807
Hs.149997
ESTs
11.8


424596
AB020639
Hs.151017
estrogen-related receptor gamma
11.8


400640


0
11.8


448133
AA723157
Hs.73769
folate receptor 1 (adult)
11.8


401532


0
11.8


400161


0
11.8


442556
AL137761
Hs.8379
Homo sapiens mRNA; cDNA DKFZp586L2424 (from c
11.7


451002
AA013299
Hs.8018
ESTs, Weakly similar to ALU3_HUMAN ALU SUBFAM
11.7


401879


0
11.7


415989
AI267700
Hs.111128
ESTs
11.7


416434
AW163045
Hs.79334
nuclear factor, interleukin 3 regulated
11.7


410616
AW873401
Hs.273599
ESTs
11.7


449239
T24653
Hs.23360
likely ortholog of yeast ARV1
11.7


447669
AL049985
Hs.19180
Homo sapiens mRNA; cDNA DKFZp564E122 (from cl
11.7


436877
AA931484
Hs.121255
ESTs, Weakly similar to cDNA EST EMBL: D67419
11.7


434560
R13052
Hs.3964
Homo sapiens clone 24877 mRNA sequence
11.7


448105
AW591433
Hs.170675
ESTs, Weakly similar to TMS2_HUMAN TRANSMEMBR
11.7


400279


0
11.6


440497
AA887266
Hs.144979
ESTs
11.6


451260
AW750773

gb: CM0-CN0044-260100-164-h03 CN0044 Homo sapi
11.6


429175
AI953040
Hs.127714
ESTs, Moderately similar to SOX30 protein [H.
11.6


408209
NM_004454
Hs.43697
ets variant gene 5 (ets-related molecule)
11.6


428856
AA436735
Hs.183171
Homo sapiens cDNA: FLJ22002 fis, clone HEP066
11.6


420153
N22120
Hs.75277
hypothetical protein FLJ13910
11.6


428760
AI351459
Hs.192398
ESTs
11.6


421401
AW410478
Hs.104019
transforming, acidic coiled-coil containing p
11.6


404502


0
11.6


430423
AI190548
Hs.143479
ESTs, Weakly similar to hypothetical protein
11.6


405192


0
11.6


439092
AA830149

gb: oc44f08.s1 NCI_CGAP_GCB1 Homo sapiens cDNA
11.6


401714


0
11.5


439335
AA742697
Hs.62492
ESTs, Weakly similar to S59856 collagen alpha
11.5


406082
S47833
Hs.82927
adenosine monophosphate deaminase 2 (isoform
11.5


401010


0
11.5


412140
AA219691
Hs.73625
RAB6 interacting, kinesin-like (rabkinesin6)
11.5


409339
AB020686
Hs.54037
ectonucleotide pyrophosphatase/phosphodiester
11.5


459684


gb: ao86a08.x1 Schiller meningioma Homo sapien
11.5


451051
BE254309
Hs.125262
DKFZP586G1624 protein
11.5


415323
BE269352
Hs.949
neutrophil cytosolic factor 2 (65 kD, chronic
11.5


412153
R87934

gb: yo47b10.r1 Soares adult brain N2b4HB55Y Ho
11.5


427256
AL042436
Hs.97723
ESTs
11.5


406708
AI282759
Hs.242463
keratin 8
11.4


457644
AA770080
Hs.144962
ESTs, Moderately similar to I59365 ubiquitin
11.4


422848
Z25884
Hs.121483
chloride channel 1, skeletal muscle (Thomsen
11.4


424134
AF070637
Hs.140950
hypothetical protein
11.4


451931
AK000208
Hs.27267
Homo sapiens cDNA FLJ20201 fis, clone COLF121
11.4


400438
AF185611
Hs.115352
growth hormone 1
11.4


412994
D32257
Hs.75113
general transcription factor IIIA
11.4


408124
U89337
Hs.42853
cAMP responsive element binding protein-like
11.4


452249
BE394412
Hs.61252
ESTs
11.4


424627
AA344555

gb: EST50715 Gall bladder I Homo sapiens cDNA
11.4


405626


0
11.4


436690
AA373970
Hs.183096
ESTs
11.4


415862
R51034
Hs.144513
ESTs
11.4


406755
N80129
Hs.94360
metallothionein 1L
11.4


433657
AI244368
Hs.8124
PH domain containing protein in retina 1
11.4


429612
AF062649
Hs.252587
pituitary tumor-transforming 1
11.4


423334
AK000906
Hs.127273
hypothetical protein FLJ10044
11.4


433053
BE301909
Hs.279952
glutathione S-transferase subunit 13 homolog
11.4


428423
AU076517
Hs.184276
solute carrier family 9 (sodium/hydrogen exch
11.3


442353
BE379594
Hs.49136
ESTs
11.3


447700
AI420183
Hs.171077
ESTs, Weakly similar to similar to serine/thr
11.3


402077


0
11.3


409203
AA780473
Hs.687
cytochrome P450, subfamily IVB, polypeptide 1
11.3


405145


0
11.3


428248
AI126772
Hs.40479
ESTs
11.3


425508
AA991551
Hs.97013
ESTs
11.3


428340
AF261088
Hs.154721
aconitase 1, soluble
11.3


431452
AI073641
Hs.152372
ESTs
11.3


446651
AA393907
Hs.97179
ESTs
11.3


443755
C18397
Hs.9730
tachykinin 3 (neuromedin K, neurokinin beta)
11.3


436209
AW850417
Hs.254020
ESTs, Moderately similar to unnamed protein p
11.3


401020


0
11.3


456724
AW247388
Hs.301423
calcium binding protein 1 (calbrain)
11.2


407227
H94949
Hs.171955
trophinin associated protein (tastin)
11.2


402066


0
11.2


442721
AI015892
Hs.101282
Homo sapiens mRNA; cDNA DKFZp434B102 (from cl
11.2


401025


0
11.2


452423
AA991724
Hs.180535
Homo sapiens cDNA: FLJ22711 fis, clone HSI133
11.2


431685
AW296135
Hs.267659
vav 3 oncogene
11.2


425176
AW015644
Hs.301430
ESTs, Moderately similar to TEF1_HUMAN TRANSC
11.2


435496
AW840171
Hs.265398
ESTs, Weakly similar to transformation-relate
11.2


409079
W87707
Hs.82065
interleukin 6 signal transducer (gp130; oncos
11.2


456995
T89832
Hs.170278
ESTs
11.2


419223
X60111
Hs.1244
CD9 antigen (p24)
11.2


407788
BE514982
Hs.38991
S100 calcium-binding protein A2
11.2


407604
AW191962
Hs.288061
actin, beta
11.2


437929
T09353
Hs.106642
ESTs, Weakly similar to hypothetical protein
11.1


415789
H01581

gb: yj33f08.r1 Soares placenta Nb2HP Homo sapi
11.1


424447
AL137376
Hs.147368
Homo sapiens mRNA; cDNA DKFZp434J0226 (from c
11.1


436034
AF282693
Hs.150185
inflammation-related G protein-coupled recept
11.1


404931


0
11.1


445979
AI695047
Hs.202395
ESTs
11.1


446733
AA863360
Hs.26040
ESTs; Highly similar to CYTOCHROME P45 IVA2
11.1


433133
AB027249
Hs.104741
PDZ-binding kinase; T-cell originated protein
11.1


445258
AI635931
Hs.147613
ESTs
11.1


417251
AW015242
Hs.99488
ESTs; Weakly similar to ORF YKR074w [S. cerevi
11.1


421041
N36914
Hs.14691
ESTs
11.1


425537
AB007913
Hs.158291
KIAA0444 protein
11.1


435763
AI243929
Hs.190419
ESTs
11.1


444790
AB030506
Hs.11955
B9 protein
11.1


453857
AL080235
Hs.35861
DKFZP586E1621 protein
11.1


433882
U90441
Hs.3622
procollagen-proline, 2-oxoglutarate 4-dioxyge
11.1


405358


0
11.1


435814
AW615179
Hs.152870
ESTs
11.0


422809
AK001379
Hs.121028
hypothetical protein FLJ10549
11.0


446772
AW294404
Hs.144515
Homo sapiens cDNA FLJ11672 fis, clone HEMBA10
11.0


456694
AW016382
Hs.105642
Homo sapiens cDNA: FLJ23271 fis, clone HEP001
11.0


441128
AA570256
Hs.54628
ESTs
11.0


432677
NM_004482
Hs.278611
UDP-N-acetyl-alpha-D-galactosamine: polypeptid
11.0


412576
AA447718
Hs.107057
ESTs
11.0


411122
F00809
Hs.143696
coactivator-associated arginine methyltransfe
11.0


427225
AA432391
Hs.258903
Homo sapiens mRNA for KIAA1640 protein, parti
11.0


426260
NM_002541
Hs.168669
oxoglutarate dehydrogenase (lipoamide)
11.0


444652
BE513613
Hs.11538
actin related protein 2/3 complex, subunit 1A
11.0


431947
AL359613
Hs.49933
hypothetical protein DKFZp762D1011
11.0


414432
BE378174
Hs.26506
Homo sapiens clone CDABP0005 mRNA sequence
11.0


458627
AW088642
Hs.97984
ESTs; Weakly similar to WASP-family protein [
10.9


409142
AL136877
Hs.50758
chromosome-associated polypeptide C
10.9


447627
AF090922
Hs.285902
CGI-113 protein
10.9


447656
NM_003726
Hs.19126
src kinase-associated phosphoprotein of 55 kD
10.9


454227
AW963897
Hs.44743
KIAA1435 protein
10.9


402927


0
10.9


422380
AA309881
Hs.136246
ESTs
10.9


455986
BE177736

gb: RC1-HT0598-140300-021-g06 HT0598 Homo sapi
10.9


410962
BE273749
Hs.752
FK506-binding protein 1A (12 kD)
10.9


450361
BE327108
Hs.202512
ESTs
10.9


457484
H57645

gb: yr21e01.r1 Soares fetal liver spleen 1NFLS
10.9


407903
AI287341
Hs.154029
bHLH factor Hes4
10.9


403398


0
10.9


401405


0
10.9


405570


0
10.9


421240
R72730
Hs.29283
ESTs, Weakly similar to PLK_HUMAN PROTEOGLYCA
10.9


403649


0
10.9


447824
BE620800

gb: 601483379T1 NIH_MGC_69 Homo sapiens cDNA c
10.9


450935
BE514743
Hs.25664
tumor suppressor deleted in oral cancer-relat
10.9


439853
AL119566
Hs.6721
lysophospholipase-like
10.9


451852
R51928

gb: yj71c05.r1 Soares breast 2NbHBst Homo sapi
10.9


431218
NM_002145
Hs.2733
homeo box B2
10.9


457794
AA689292
Hs.246850
ESTs
10.9


444374
AA009841
Hs.11039
Homo sapiens cDNA FLJ12798 fis, clone NT2RP20
10.9


456566
AW235317
Hs.259214
ESTs
10.8


405552


0
10.8


439436
BE140845
Hs.57868
ESTs
10.8


435310
AA705075
Hs.169536
Rhesus blood group-associated glycoprotein
10.8


411125
AA151647
Hs.68877
cytochrome b-245, alpha polypeptide
10.8


415807
H03139
Hs.24683
ESTs
10.8


409430
R21945
Hs.166975
splicing factor, arginine/serine-rich 5
10.8


417033
H83784
Hs.40532
ESTs, Weakly similar to PEBP MOUSE PHOSPHATID
10.8


418464
R87580

gb: ym89h07.r1 Soares adult brain N2b4HB55Y Ho
10.8


404567


0
10.8


418384
AW149266
Hs.25130
ESTs
10.8


421971
U63127
Hs.110121
SEC7 homolog
10.8


428769
AW207175
Hs.106771
ESTs
10.8


459104
R19238
Hs.282057
ESTs
10.8


410896
AW809637

gb: MR4-ST0124-261099-015-b07 ST0124 Homo sapi
10.8


416969
AI815443
Hs.283404
organic cation transporter
10.8


408796
AA688292
Hs.118553
ESTs
10.8


426298
AW965058
Hs.111583
ESTs
10.8


421595
AB014520
Hs.105958
Homo sapiens cDNA: FLJ22735 fis, clone HUV001
10.8


408007
AW135965
Hs.246783
ESTs
10.8


400167


0
10.7


445243
AI217439
Hs.109854
ESTs, Weakly similar to ALU1_HUMAN ALU SUBFAM
10.7


421733
AL119671
Hs.1420
fibroblast growth factor receptor 3 (achondro
10.7


412241
AW948343

gb: RC0-MT0015-130400-031-c01 MT0015 Homo sapi
10.7


425827
W28316

gb: 45b6 Human retina cDNA randomly primed sub
10.7


420255
NM_007289
Hs.1298
membrane metallo-endopeptidase (neutral endop
10.7


430891
U22492
Hs.248118
G protein-coupled receptor 8
10.7


402883


0
10.7


423811
AW299598
Hs.50895
homeo box C4
10.7


447078
AW885727
Hs.301570
ESTs
10.7


414343
AL036166
Hs.75914
coated vesicle membrane protein
10.7


446913
AA430650
Hs.16529
transmembrane 4 superfamily member (tetraspan
10.7


452279
AA286844
Hs.61260
hypothetical protein FLJ13164
10.7


401220


0
10.7


459259
AJ003294

gb: AJ003294 Selected chromosome 21 cDNA libra
10.7


414171
AA360328
Hs.865
RAP1A, member of RAS oncogene family
10.7


448449
BE314567
Hs.211440
ESTs
10.7


429670
L01087
Hs.211593
protein kinase C, theta
10.7


446759
R61463
Hs.16165
expressed in activated T/LAK lymphocytes
10.7


400776


0
10.7


428093
AW594506
Hs.104830
ESTs
10.7


412801
AA121055

gb: zm22b01.r1 Stratagene pancreas (937208) Ho
10.6


440545
AW183201
Hs.190559
ESTs
10.6


434540
NM_016045
Hs.5184
TH1 drosophila homolog
10.6


414273
BE269057

gb: 601184231F1 NIH_MGC_8 Homo sapiens cDNA cl
10.6


401817


0
10.6


410423
AW402432
Hs.63489
protein tyrosine phosphatase, non-receptor ty
10.6


430590
AW383947
Hs.246381
CD68 antigen
10.6


426680
AA320160
Hs.171811
adenylate kinase 2
10.6


445413
AA151342
Hs.12677
CGI-147 protein
10.6


402947


0
10.6


457426
AW971119

gb: EST383206 MAGE resequences, MAGL Homo sapi
10.6


424148
BE242274
Hs.1741
integrin, beta 7
10.6


404944


0
10.6


405421


0
10.6


416772
AI733872
Hs.79769
protocadherin 1 (cadherin-like 1)
10.6


414191
AW250089
Hs.75807
PDZ and LIM domain 1 (elfin)
10.6


457588
AI571225
Hs.284171
KIAA1535 protein
10.6


406038
Y14443
Hs.88219
zinc finger protein 200
10.6


404790


0
10.6


418922
AW956580
Hs.42699
Thrombospondin-1 (Hs.87409)
10.6


425940
AB023184
Hs.163990
KIAA0967 protein
10.6


448749
AW859679
Hs.21902
Homo sapiens clone 25237 mRNA sequence
10.6


418870
AF147204
Hs.89414
CXCR4; chemokine CXC receptor 4 (fusin)
10.5


417933
X02308
Hs.82962
thymidylate synthetase
10.5


450538
AW297396
Hs.227052
ESTs
10.5


427928
AA417662
Hs.119217
ESTs
10.5


432721
AL121478
Hs.3132
steroidogenic acute regulatory protein
10.5


429267
AA299290
Hs.246857
ESTs, Highly similar to S71100 protein kinase
10.5


439190
AW978693
Hs.293811
ESTs
10.5


408975
AW958693
Hs.49391
hypothetical protein LOC54149
10.5


415130
W85893
Hs.249867
ESTs
10.5


425738
H29630
Hs.159408
Homo sapiens clone 24420 mRNA sequence
10.5


440232
AI766925
Hs.112554
ESTs
10.5


425065
AA371906
Hs.294151
ESTs, Moderately similar to KIAA0544 protein
10.5


420829
AW665612
Hs.221969
ESTs
10.5


430466
AF052573
Hs.241517
polymerase (DNA directed), theta
10.5


407771
AL138272
Hs.62713
ESTs
10.5


444611
AK002180
Hs.11449
DKFZP564O123 protein
10.5


444665
BE613126
Hs.47783
ESTs, Weakly similar to T12540 hypothetical p
10.5


448030
N30714
Hs.20161
HDCME31P protein
10.5


438982
AW979101
Hs.291980
ESTs
10.5


446224
AW450551
Hs.13308
ESTs
10.5


405108


0
10.5


438233
W52448
Hs.56147
ESTs
10.5


401799


0
10.5


454038
X06374
Hs.37040
platelet-derived growth factor alpha polypept
10.5


414222
AL135173
Hs.878
sorbitol dehydrogenase
10.5


421828
AW891965
Hs.289109
dimethylarginine dimethylaminohydrolase 1
10.5


422626
AA344932
Hs.118786
metallothionein 2A
10.5


449261
AI637592
Hs.224958
ESTs
10.4


416218
R21499
Hs.23213
ESTs
10.4


457848
W26524
Hs.125682
ESTs; Weakly similar to D2092.2 [C. elegans]
10.4


442577
AA292998
Hs.163900
ESTs
10.4


406505
AF016272
Hs.115418
cadherin 16, KSP-cadherin
10.4


412258
AA376768
Hs.288977
Homo sapiens cDNA: FLJ22622 fis, clone HSI056
10.4


429224
AI905780
Hs.198272
NADH dehydrogenase (ubiquinone) 1 beta subcom
10.4


447774
BE018118
Hs.19554
chromosome 1 open reading frame 2
10.4


403914


0
10.4


406329


0
10.4


402423


0
10.4


431986
AA536130
Hs.149018
ESTs
10.4


423145
BE264548
Hs.222190
ESTs, Weakly similar to secretory carrier mem
10.4


414402
BE294186

gb: 601172959F1 NIH_MGC_17 Homo sapiens cDNA c
10.4


417079
U65590
Hs.81134
interleukin 1 receptor antagonist
10.4


426095
AI278023
Hs.89986
ESTs
10.4


434577
R37316
Hs.179769
Homo sapiens cDNA: FLJ22487 fis, clone HRC109
10.4


442415
AI005101
Hs.129550
ESTs
10.3


421506
BE302796
Hs.105097
thymidine kinase 1, soluble
10.3


435084
D17516
Hs.301607
adenylate cyclase activating polypeptide 1 (p
10.3


431724
AA514535
Hs.283704
ESTs
10.3


456798
AJ006422
Hs.135183
centaurin-alpha
10.3


417370
T28651
Hs.82030
tryptophanyl-tRNA synthetase
10.3


422596
AF063611
Hs.118633
2′-5′oligoadenylate synthetase-like
10.3


435226
AI248938
Hs.270106
ESTs
10.3


433192
AB040880
Hs.225594
ESTs, Moderately similar to KIAA1447 protein
10.3


419879
Z17805
Hs.93564
Homer, neuronal immediate early gene, 2
10.3


416228
AW505190
Hs.79089
sema domain, immunoglobulin domain (Ig), tran
10.3


453403
BE466639
Hs.61779
Homo sapiens cDNA FLJ13591 fis, clone PLACE10
10.3


447906
AL050062
Hs.19999
DKFZP566K023 protein
10.3


401782
NM_012434
Hs.117865
solute carrier family 17 (anion/sugar transpo
10.3


453927
AA082465
Hs.301751
ESTs, Weakly similar to /prediction
10.3


450737
AW007152
Hs.203330
ESTs
10.3


421633
AF121860
Hs.106260
sorting nexin 10
10.3


409881
AF139799
Hs.202830
ESTs
10.3


432883
U48936
Hs.3112
sodium channel, nonvoltage-gated 1, gamma
10.3


440099
AL080058
Hs.6909
DKFZP564G202 protein
10.3


419024
X56411
Hs.1219
alcohol dehydrogenase 4 (class II), pi polype
10.3


401835


0
10.3


408896
AI610447
Hs.48778
niban protein
10.3


443120
AW402677
Hs.290801
ESTs
10.3


400208


0
10.2


416908
AA333990
Hs.80424
coagulation factor XIII, A1 polypeptide
10.2


400166


0
10.2


434642
W25739
Hs.135287
ESTs
10.2


424837
BE276113
Hs.153436
N-acetyltransferase, homolog of S. cerevisiae
10.2


435075
R51094
Hs.12400
ESTs
10.2


425912
AL137629
Hs.162189
serine/threonine kinase with Dbl- and pleckst
10.2


435080
AI831760
Hs.155111
ESTs
10.2


414998
NM_002543
Hs.77729
oxidised low density lipoprotein (lectin-like
10.2


410020
T86315
Hs.728
ribonuclease, RNase A family, 2 (liver, eosin
10.2


411410
R20693
Hs.69954
laminin, gamma 3
10.2


450294
H42587
Hs.238730
ESTs
10.2


421154
AA284333
Hs.287631
Homo sapiens cDNA FLJ14269 fis, clone PLACE10
10.2


414271
AK000275
Hs.75871
protein kinase C binding protein 1
10.2


400812


0
10.2


425843
BE313280
Hs.159627
death associated protein 3
10.2


449392
Z41698
Hs.26039
Homo sapiens cDNA FLJ13937 fis, clone Y79AA10
10.2


409089
NM_014781
Hs.50421
KIAA0203 gene product
10.2


401383


0
10.2


456855
AF035528
Hs.153863
MAD (mothers against decapentaplegic, Drosoph
10.2


442912
AI088060
Hs.131450
ESTs
10.2


400954
D25969
Hs.76325
Homo sapiens cDNA: FLJ23125 fis, clone LNG082
10.2


401029
BE382701
Hs.25960
v-myc avian myelocytomatosis viral related on
10.2


416602
NM_006159
Hs.79389
nel (chicken)-like 2
10.2


421905
AI660247
Hs.32699
ESTs, Weakly similar to LIV-1 protein [H. sapi
10.2


405094


0
10.2


450832
AW970602
Hs.105421
ESTs
10.2


440076
R32052
Hs.178617
ESTs, Weakly similar to AF151840 1 CGI-82 pro
10.2


447563
BE536115
Hs.160983
ESTs
10.2


421238
AB033101
Hs.102796
KIAA1275 protein
10.2


400882


0
10.2


415738
BE539367
Hs.295953
ESTs, Weakly similar to AF220049 1 uncharacte
10.1


445464
AW172389
Hs.249999
ESTs
10.1


459042
AW272058
Hs.210338
ESTs
10.1


414469
R51952
Hs.32587
steriod receptor RNA activator 1 (complexes w
10.1


434732
AI078443

gb: oz05g05.x1 Soares_fetal_liver_spleen_1NFLS
10.1


441030
AW204139
Hs.174424
ESTs, Weakly similar to p140mDia [M. musculus]
10.1


446855
BE616767
Hs.16269
B-cell CLL/lymphoma 7B
10.1


456785
AF151074
Hs.132744
hypothetical protein
10.1


404182


0
10.1


410358
AW975168
Hs.13337
ESTs, Weakly similar to unnamed protein produ
10.1


430355
NM_006219
Hs.239818
phosphoinositide-3-kinase, catalytic, beta po
10.1


442152
R39246
Hs.239666
Homo sapiens cDNA FLJ13495 fis, clone PLACE10
10.1


436354
AI879252
Hs.5151
Homo sapiens mRNA; cDNA DKFZp564C2163 (from c
10.1


426711
AA383471
Hs.180669
conserved gene amplified in osteosarcoma
10.1


450599
AA460865
Hs.48516
ESTs
10.1


454393
BE153288

gb: PM0-HT0335-180400-008-c08 HT0335 Homo sapi
10.1


403383


0
10.1


415947
U04045
Hs.78934
mutS (E. coli) homolog 2 (colon cancer, nonpo
10.1


411773
NM_006799
Hs.72026
protease, serine, 21 (testisin)
10.1


412116
AW402166
Hs.784
Epstein-Barr virus induced gene 2 (lymphocyte
10.1


413808
J00287
Hs.182183
caldesmon 1
10.0


458572
AI223423
Hs.292794
ESTs
10.0


403295


0
10.0


403910


0
10.0


453400
AI991901
Hs.82590
ESTs, Moderately similar to ALU7_HUMAN ALU SU
10.0


406502


0
10.0


404743


0
10.0


412517
BE271584

gb: 601141065F1 NIH_MGC_9 Homo sapiens cDNA cl
10.0


402679


0
10.0


455864
BE148970

gb: CM0-HT0245-031199-085-h05 HT0245 Homo sapi
10.0


425734
AF056209
Hs.159396
peptidylglycine alpha-amidating monooxygenase
10.0


419280
W07506
Hs.283725
Homo sapiens cDNA FLJ12627 fis, clone NT2RM40
10.0


443503
AV645438
Hs.282927
ESTs
10.0


423165
AI937547
Hs.124915
Human DNA sequence from clone 380A1 on chromo
10.0


450206
AI796450
Hs.201600
ESTs
10.0


459052
AA298812
Hs.98539
ESTs
10.0


456248
AL035786
Hs.82425
actin related protein 2/3 complex, subunit 5
10.0


428438
NM_001955
Hs.2271
Endothelin 1
10.0


456525
AW468397
Hs.100000
S100 calcium-binding protein A8 (calgranulin
10.0


426127
L36983
Hs.167013
dynamin 2
10.0





Pkey: Primekey


Ex. Accn: Exemplay Accession


UG ID: UniGene ID


Title: UniGene title


ratio: ration tumor vs. normal ovary






Table 19A provides UnigenelD, UnigeneTitle, Pkey, and Exemplar Accession for sequences in Table 20. Information in Table 19A is linked by SEQ ID NO: to Table 20.











TABLE 19A









SEQ


Pkey
ExAccn
UG ID
Title
ID NO:







428187
AI687303
Hs.285529
G protein-coupled receptor 49
1 and 2





Pkey: Unique Eos probeset identifier number


ExAccn: Exemplar Accession number, Genbank accession number


UG ID: UniGene number


Title: UniGene gene title


SEQ ID NO: Sequence Identification number for sequences in Table 20













TABLE 20







SEQ ID NO: 1


DNA sequence


Nucleic Acid Accession #: NM_003667.1


Coding sequence: 1..2651


 








 


GGGGGCAGCT CTCCCAGGTC TGGTGTGTTG CTGAGGGGCT GCCCCACACA CTGTCATTGC   120


 


GAGCCCGACG GCAGGATGTT GCTCAGGGTG GACTGCTCCG ACCTGGGGCT CTCGGAGCTG   180


 


CCTTCCAACC TCAGCGTCTT CACCTCCTAC CTAGACCTCA GTATGAACAA CATCAGTCAG   240


 


CTGCTCCCGA ATCCCCTGCC CAGTCTCCGC TTCCTGGAGG AGTTACGTCT TGCGGGAAAC   300


 


GCTCTGACAT ACATTCCCAA GGGAGCATTC ACTGGCCTTT ACAGTCTTAA AGTTCTTATG   360


 


CTGCAGAATA ATCAGCTAAG ACACGTACCC ACAGAAGCTC TGCAGAATTT GCGAAGCCTT   420


 


CAATCCCTGC GTCTGGATGC TAACCACATC AGCTATGTGC CCCCAAGCTG TTTCAGTGGC   480


 


CTGCATTCCC TGAGGCACCT GTGGCTGGAT GACAATGCGT TAACAGAAAT CCCCGTCCAG   540


 


GCTTTTAGAA GTTTATCGGC ATTGCAAGCC ATGACCTTGG CCCTGAACAA AATACACCAC   600


 


ATACCAGACT ATGCCTTTGG AAACCTCTCC AGCTTGGTAG TTCTACATCT CCATAACAAT   660


 


AGAATCCACT CCCTGGGAAA GAAATGCTTT GATGGGCTCC ACAGCCTAGA GACTTTAGAT   720


 


TTAAATTACA ATAACCTTGA TGAATTCCCC ACTGCAATTA GGACACTCTC CAACCTTAAA   780


 


GAACTACATT TCTATGACAA TCCCATCCAA TTTGTTGGGA GATCTGCTTT TCAACATTTA   840


 


CCTGAACTAA GAACACTGAC TCTGAATGGT GCCTCACAAA TAACTGAATT TCCTGATTTA   900


 


ACTGGAACTG CAAACCTGGA GAGTCTGACT TTAACTGGAG CACAGATCTC ATCTCTTCCT   960


 


CAAACCGTCT GCAATCAGTT ACCTAATCTC CAAGTGCTAG ATCTGTCTTA CAACCTATTA  1020


 


GAAGATTTAC CCAGTTTTTC AGTCTGCCAA AAGCTTCAGA AAATTGACCT AAGACATAAT  1080


 


GAAATCTACG AAATTAAAGT TGACACTTTC CAGCAGTTGC TTAGCCTCCG ATCGCTGAAT  1140


 


TTGGCTTGGA ACAAAATTGC TATTATTCAC CCCAATGCAT TTTCCACTTT GCCATCCCTA  1200


 


ATAAAGCTGG ACCTATCGTC CAACCTCCTG TCGTCTTTTC CTATAACTGG GTTACATGGT  1260


 


TTAACTCACT TAAAATTAAC AGGAAATCAT GCCTTACAGA GCTTGATATC ATCTGAAAAC  1320


 


TTTCCAGAAC TCAAGGTTAT AGAAATGCCT TATGCTTACC AGTGCTGTGC ATTTGGAGTG  1380


 


TGTGAGAATG CCTATAAGAT TTCTAATCAA TGGAATAAAG GTGACAACAG CAGTATGGAC  1440


 


GACCTTCATA AGAAAGATGC TGGAATGTTT CAGGCTCAAG ATGAACGTGA CCTTGAAGAT  1500


 


TTCCTGCTTG ACTTTGAGGA AGACCTGAAA GCCCTTCATT CAGTGCAGTG TTCACCTTCC  1560


 


CCAGGCCCCT TCAAACCCTG TGAACACCTG CTTGATGGCT GGCTGATCAG AATTGGAGTG  1620


 


TGGACCATAG CAGTTCTGGC ACTTACTTGT AATGCTTTGG TGACTTCAAC AGTTTTCAGA  1680


 


TCCCCTCTGT ACATTTCCCC CATTAAACTG TTAATTGGGG TCATCGCAGC AGTGAACATG  1740


 


CTCACGGGAG TCTCCAGTGC CGTGCTGGCT GGTGTGGATG CGTTCACTTT TGGCAGCTTT  1800


 


GCACGACATG GTGCCTGGTG GGAGAATGGG GTTGGTTGCC ATGTCATTGG TTTTTTGTCC  1860


 


ATTTTTGCTT CAGAATCATC TGTTTTCCTG CTTACTCTGG CAGCCCTGGA GCGTGGGTTC  1920


 


TCTGTGAAAT ATTCTGCAAA ATTTGAAACG AAAGCTCCAT TTTCTAGCCT GAAAGTAATC  1980


 


ATTTTGCTCT GTGCCCTGCT GGCCTTGACC ATGGCCGCAG TTCCCCTGCT GGGTGGCAGC  2040


 


AAGTATGGCG CCTCCCCTCT CTGCCTGCCT TTGCCTTTTG GGGAGCCCAG CACCATGGGC  2100


 


TACATGGTCG CTCTCATCTT GCTCAATTCC CTTTGCTTCC TCATGATGAC CATTGCCTAC  2160


 


ACCAAGCTCT ACTGCAATTT GGACAAGGGA GACCTGGAGA ATATTTGGGA CTGCTCTATG  2220


 


GTAAAACACA TGGCCCTGTT GCTCTTCACC AACTGCATCC TAAACTGCCC TGTGGCTTTC  2280


 


TTGTCCTTCT CCTCTTTAAT AAACCTTACA TTTATCAGTC CTGAAGTAAT TAAGTTTATC  2340


 


CTTCTGGTGG TAGTCCCACT TCCTGCATGT CTCAATCCCC TTCTCTACAT CTTGTTCAAT  2400


 


CCTCACTTTA AGGAGGATCT GGTGAGCCTG AGAAAGCAAA CCTACGTCTG GACAAGATCA  2460


 


AAACACCCAA GCTTGATGTC AATTAACTCT GATGATGTCG AAAAACAGTC CTGTGACTCA  2520


 


ACTCAAGCCT TGGTAACCTT TACCAGCTCC AGCATCACTT ATGACCTGCC TCCCAGTTCC  2580


 


GTGCCATCAC CAGCTTATCC AGTGACTGAG AGCTGCCATC TTCCCTCTGT GGCATTTGTC  2640


 


CCATGTCTTA A


 


SEQ ID NO: 2


Protein sequence:


Protein Accession #: NP_003658.1


 








 


PSNLSVFTSY LDLSMNNISQ LLPNPLPSLR FLEELRLAGN ALTYIPKGAF TGLYSLKVLM   120


 


LQNNQLRHVP TEALQNLRSL QSLRLDANHI SYVPPSCFSG LHSLRHLWLD DNALTEIPVQ   180


 


AFRSLSALQA MTLALNKIHH IPDYAFGNLS SLVVLHLHNN RIHSLGKKCF DGLHSLETLD   240


 


LNYNNLDEFP TAIRTLSNLK ELHFYDNPIQ FVGRSAFQHL PELRTLTLNG ASQITEFPDL   300


 


TGTANLESLT LTGAQISSLP QTVCNQLPNL QVLDLSYNLL EDLPSFSVCQ KLQKIDLRHN   360


 


EIYEIKVDTF QQLLSLRSLN LAWNKIAIIH PNAFSTLPSL IKLDLSSNLL SSFPITGLHG   420


 


LTHLKLTGNH ALQSLISSEN FPELKVIEMP YAYQCCAFGV CENAYKISNQ WNKGDNSSMD   480


 


DLHKKDAGMF QAQDERDLED FLLDFEEDLK ALHSVQCSPS PGPFKPCEHL LDGWLIRIGV   540


 


WTIAVLALTC NALVTSTVFR SPLYISPIKL LIGVIAAVNM LTGVSSAVLA GVDAFTFGSF   600


 


ARHGAWWENG VGCHVIGFLS IFASESSVFL LTLAALERGF SVKYSAKFET KAPFSSLKVI   660


 


ILLCALLALT MAAVPLLGGS KYGASPLCLP LPFGEPSTMG YMVALILLNS LCFLMMTIAY   720


 


TKLYCNLDKG DLENIWDCSM VKHIALLLFT NCILNCPVAF LSFSSLINLT FISPEVIKFI   780


 


LLVVVPLPAC LNPLLYILFN PHFKEDLVSL RKQTYVWTRS KHPSLMSINS DDVEKQSCDS   840


 


TQALVTFTSS SITYDLPPSS VPSPAYPVTE SCHLSSVAFV PCL









It is understood that the examples described above in no way serve to limit the true scope of this invention, but rather are presented for illustrative purposes. All publications, sequences of accession numbers, and patent applications cited in this specification are herein incorporated by reference as if each individual publication or patent application were specifically and individually indicated to be incorporated by reference.

## Claims

1. A method of detecting ovarian cancer in a patient, the method comprising:
(i) detecting a nucleic acid in a first sample from the patient, wherein the nucleic acid sequence is at least 95% identical to SEQ ID NO:1, and wherein the nucleic acid encodes a G protein.coupled receptor 49; and
(ii) comparing the expression level of the nucleic acid in the-first sample to the expression level in a normal sample;
wherein an increase in the level of the nucleic acid relative to the normal sample is indicative of ovarian cancer.

2. The method of claim 1, wherein the first sample comprises isolated nucleic acids.

3. The method of claim 2, wherein the nucleic acids are mRNA.

4. The method of claim 2, wherein the method further comprises the step of amplifying nucleic acids before the step of detecting the nucleic acid.

5. The method of claim 1, wherein the nucleic acid comprises SEQ ID NO:1.

6. The method of claim 1, wherein the detecting step is carried out by using a nucleic acid probe that hybridizes under stringent conditions to a nucleic acid comprising SEQ ID NO:1.

7. The method of claim 6, wherein the probe is immobilized on a solid surface.

8. The method of claim 1, wherein the patient is suspected of having ovarian cancer.

9. The method of claim 1, wherein the patient is suspected of having ovarian cancer.

10. An isolated nucleic acid molecule, wherein the nucleic acid molecule comprises a sequence at least 99% identical to SEQ ID NO:1.

11. The nucleic acid molecule of claim 10, wherein the nucleic acid molecule is labeled.

12. An expression vector comprising the nucleic acid of claim 10.

13. A host cell comprising the expression vector of claim 12.

14. A method of monitoring ovarian cancer in a human patient, the method comprising:
(i) detecting a nucleic acid in a first sample from the patient, wherein the nucleic acid sequence is at least 95% identical to SEQ ID NO:1, and wherein the nucleic acid encodes a G protein-coupled receptor 49;
(ii) comparing the expression level of the nucleic acid in the first sample to the expression level in a normal sample.

15. The method of claim 14, wherein the sample comprises blood from the patient.

16. The method of claim 14, wherein the nucleic acid sequence comprises SEQ ID NO:1.

17. The method of claim 14, wherein the detecting step is carried out by using a nucleic acid probe that hybridizes under stringent conditions to a sequence comprising SEQ ID NO:1.

18. The method of claim 17, wherein the probe is immobilized on a solid surface.