Human homolog of the rat G protein gamma-5 subunit

The present invention provides a nucleic acid sequence which identifies and encodes a G protein gamma subunit (gpg) which was isolated from human pituitary gland. The invention provides for genetically engineered expression vectors and host cells comprising nucleic acid sequence encoding GPG. The invention also provides for purified GPG.

FIELD OF THE INVENTION 
The present invention is in the field of molecular biology; more 
particularly, the present invention describes the nucleic acid and amino 
acid sequences of a human homolog of the rat G protein gamma-5. 
BACKGROUND OF THE INVENTION 
The heterotrimeric G proteins, a family of GTPases, are present in all 
cells. They control a variety of functions (metabolic, humoral, neural and 
developmental) by transducing hormonal, neurotransmitter and sensory 
signals into an array of cellular responses. Triggered by cell surface 
receptors, each G protein regulates the activity of a specific effector. 
The effectors include adenylate cyclase, phospholipase C, and ion channel 
proteins which initiate appropriate biochemical responses. G proteins can 
exhibit strict subcellular localization and can included in endocytic 
vesicles (Alberts B. et al (1994) Molecular Biology of the Cell, Garland 
Publishing, Hamden Conn.). 
Each G protein is composed of alpha (.alpha.), beta (.beta.) and gamma 
(.gamma.) subunits associated as a complex in the inactive, GDP-bound 
form. Activation of a transmembrane receptor by a hormone results in 
activation of the GTPase and replacement of GDP by GTP. The activated 
heterotrimer, the activated .alpha. subunit, or the .beta.-.gamma. subunit 
may have specific activity. Generally, the .alpha. subunit of a G protein 
disassociates from the .beta. and .gamma. complex, interacts with 
receptors and carries the message to the effector. 
There are at least 20 genes for G.alpha. subunits which encode four major 
classes of proteins with at least 56-95% amino acid identity. The 
stimulatory, Gs class, is sensitive to pertussis toxin which uncouples the 
receptor:G protein interaction. This uncoupling blocks signal transduction 
to those receptors that decrease the cAMP which regulates ion channels and 
activates phospholipases. The inhibitory, Gi class, is also susceptible to 
modification by pertussis toxin which prevents Gi from lowering cAMP 
levels. Two novel classes refractory to pertussis toxin modification, are 
Gq which activates phospholipase C and G.sub.12 which has sequence 
homology with the Drosophila gene concertina which may contribute to the 
regulation of embryonic development. The G.alpha. subunits range in 
molecular weight from 39-52 kDa and include some splice variants. Multiple 
genes also encode at least four .beta. and six .gamma. subunits which 
range in molecular weight from 35-36 kDa and 6-10 kDa, respectively 
(Watson S. and S. Arkinstall (1994) The G protein Linked Receptor Facts 
Book, Academic Press, San Diego Calif.). 
The .beta.-.gamma. dimer promotes the association of the GDP-bound .alpha. 
subunit with ligand-bound receptor. The dimer both orients and stabilizes 
the association so that signal transduction does not occur in the absence 
of agonist. Neer E. J. (1995; Cell 80:249-257) reported that 
.beta.-.gamma. dimers interact with adenylyl cyclase, phospholipase C 
.beta., calmodulin, .beta. adrenergic receptor kinase, phospholipase A2, 
phosducin, phosphoinositide 3-kinase, transducin, etc. In addition, the 
dimer may regulate potassium channels, mediate mitogen-activated protein 
kinase pathways and activate or increase phosphoinositide hydrolysis. In 
yeast, the dimer mediates a G protein-dependent mating response. The five 
.beta. subunit isotypes share 53-90% amino acid identity and are expressed 
ubiquitously although it must be noted that .beta.-4 is more abundant in 
brain and lung than in other tissues (Clapman D. E. and E. J. Neer (1993) 
Nature 365:403-6). 
The known .gamma. subunits from bovine, rat and mouse tissues are most 
divergent in their N-terminal sequence. The .gamma. subunits generally 
display at least one cysteine residue in approximately the middle of their 
amino acid sequence (between residues 35 and 45) which is important for 
dimer formation, ie, the cysteine in the .gamma. subunit cross links with 
a cysteine in the .beta. subunit. Many of the sequences show a C-terminal 
consensus sequence CAAX (where A represents aliphatic residues and X is 
unspecified) which resembles the ras oncogene terminal sequence and is a 
site for post-translational modification. The modification involves 
cleavage of the 3' terminal residues and subsequent carboxymethylation, 
farnesylation, geranylgeranylation or isoprenylation. Post-translational 
modification increases subunit diversity and hydrophobicity and is 
important for membrane association and functional activity. In contrast, 
the rat .beta.-5 sequence which terminates in CSFL is widely expressed and 
was highly expressed in kidney, heart, lung, and brain. 
Although the different G proteins subunits could form some 600 different 
combinations, not all combinations are possible or functional. In the case 
of dimers, the .beta.1-.gamma.1 is only active in retina. Furthermore, the 
pattern of effector regulation may be highly specific. For example, 
whereas one type of adenylyl cyclase is activated by the G.alpha. subunit 
and unaffected by the B-.beta. subunit, a second type is activated by a 
subunit and inhibited by .beta.-.gamma. subunit. In another example 
involving the pituitary-derived GH3 cell line, the somatostatin receptor 
and the muscarinic receptor both regulate calcium channels, but each uses 
an alternatively spliced form of the .alpha..sub.s/o and different 
.beta.-.gamma. subunits. A final example addresses specificity and 
efficiency; in reconstituted vesicles, the .beta.-adrenergic receptor 
activates Gs as much as 3-fold better than Gi and the .beta.-.gamma. 
subunits from either heterotrimer should activate the potassium channel, 
however, only adenylyl cyclase is activated. 
Neer (supra) suggests that G protein regulation depends on a combination of 
factors including the kinetics of ATP hydrolysis, stoichiometry, covalent 
modification, accessory proteins and compartmentalization, and that the 
number of receptors exceeds the number of G proteins. The molecular and 
functional diversity of Gs-stimulated adenylyl cyclases was recently 
reviewed by Iyengar R. (1993; FASEB Jour 7:768-75), and different tissues 
were shown to express a variety of adenylyl cyclases which were 
differentially regulated by the .beta.-.gamma. dimers and other molecules. 
Diseases Associated with Cell Signaling Molecules and Pathways 
Mutations in the molecules and alterations in the expression pattern of the 
components of the cell signaling cascade may result in abnormal activation 
of leukocytes or lymphocytes or cellular proliferation which affects 
growth and development. Inappropriate activation of leukocytes or 
lymphocytes may result in the tissue damage and destruction seen in 
autoimmune diseases such as rheumatoid arthritis, biliary cirrhosis, 
hemolytic anemia, lupus erythematosus, and thyroiditis. For example, 
Aussel C. et al. (1988; J Immunol 140-215) reported that T cell activation 
is a G protein regulated process. Work in Jurkat cells with pertussis 
toxin showed that G protein serves as a transducer for signals via the T 
cell receptor-CD3 complex. In addition, the fact that fluoride ions 
stimulate the release of diacylglycerol but not inositol phosphate 3 
further suggests that G proteins control the activity of phospholipase C. 
Abnormal proliferation of cells can cause endometriosis or tumors, adenomas 
or carcinomas. Cyclic AMP stimulation of brain, thyroid, adrenal, and 
gonadal tissue proliferation is regulated by G proteins. In fact, about 50 
percent of growth hormone-producing pituitary adenomas contain a mutated 
G.alpha..sub.s allele, and similar mutations have been associated with 
thyroid carcinomas and the neoplastic lesions of McCune-Albright syndrome. 
A known mutation in the G.alpha..sub.2i gene is found in tumors derived 
from adrenal cortex and ovary. Persistent extracellular stimulation and 
expression of those receptors coupled to Gq and phospholipase C can also 
result in tumor formation (Isselbacher K. J. et al (1994) Harrison's 
Principles of Internal Medicine, McGraw-Hill, New York N.Y.). In addition, 
multiple endocrine hyperfunction may be due to defects in the G 
protein-cyclic AMP-protein kinase A-dependent pathway. 
Phosphoinositide 3 kinase is a key signaling enzyme implicated in receptor 
stimulated mitogenesis, oxidative bursting in neutrophils, membrane 
ruffling and glucose uptake. Stephens L. et al. (1994; Cell 77:83-93) 
report that phosphoinositide 3 kinase activation in myeloid derived cells 
is regulated by .beta.-.gamma. dimers as well as phosphotyrosine kinase. 
Furthermore, it appears that tissue specificity may be governed by 
concentration of .beta.-.gamma. dimer molecules and that activation is 
more rapid and transient than that regulated by phosphotyrosine kinase. 
Although it was not suggested, it appears that the ability to control 
expression of either .beta. or .gamma. subunits provides a means to 
regulate cell signaling and mitogenesis. 
The diversity of G subunit proteins, their functional combinations and 
their interactions with receptors present opportunities to intercede in 
abnormal cell processes. The activation of G proteins and the rate of GTP 
hydrolysis can be altered by controlling subunit production and 
association. Preventing dimer and heterotrimer formation can diminish cell 
signalling in GTP regulated pathways, reducing the activation of second 
messengers and controlling activation of leukocytes and lymphocytes and 
cell proliferation associated with endometriosis and tumor formation. 
SUMMARY 
The present invention relates to a novel G protein gamma subunit, GPG, 
whose nucleic acid sequence, gpg, was identified among the polynucleotides 
of a human pituitary library and to the use of the nucleic acid and amino 
acid sequences in the study, diagnosis, prevention and treatment of 
disease. 
The novel polynucleotide encoding GPG was first identified in Incyte Clone 
No. 112530 through a computer generated search for nucleotide sequence 
alignments. The clone was resequenced, and the coding region determined. 
The nucleotide sequence (SEQ ID NO:1) encodes a protein of 68 amino acids 
(SEQ ID NO:2). Significant features of the novel GPG are the presence of 
the C.sub.38 linking residue and the presence of the C-terminal CAAX 
motif. Other G protein sequences (SEQ ID NOs:9-31) presented in the 
Sequence Listing are exact matches, related sequences or variants of SEQ 
ID NO:1. 
The present invention and its use is based, in part, on the fact that GPG 
is most closely related to the rat G protein .gamma.-5 subunit. It is also 
based on the tissue distribution of the exact matches, related sequences 
or variants of SEQ ID NO:1 which were found in uterus, thyroid, T cell, 
stomach, spleen, keratinocyte, eosinophil, cardiac, bladder, and 
stimulated macrophage, neuronal, and neutrophil libraries. 
The use of GPG, and of the nucleic acid sequences which encode it, is also 
based on the amino acid and structural homologies between GPG and the 
other known G protein .gamma. subunits as well as on the known 
associations and functions of heterotrimeric and dimeric G proteins. The 
timing of and amount of expression of GPG is implicated in activation of 
leukocytes or lymphocytes in autoimmune diseases such as rheumatoid 
arthritis, biliary cirrhosis, hemolytic anemia, lupus erythematosus, and 
thyroiditis and in cell proliferation associated with endometriosis or 
with the formation of tumors of brain, thyroid, adrenal, and gonadal 
tissues. 
The gpg polynucleotide sequence, oligonucleotides, fragments, portions or 
antisense molecules thereof, may be used in diagnostic assays to detect 
and quantify levels of gpg mRNA in cells and tissues. For example, gpg 
polynucleotides, or fragments thereof, may be used in hybridization assays 
of body fluids or biopsied tissues to detect the level of gpg expression. 
The present invention also relates, in part, to an expression vector and 
host cells comprising nucleic acids encoding GPG. Such transfected host 
cells are useful for the production and recovery of GPG. The present 
invention also encompasses purified GPG. 
The invention further provides diagnostic kits for the detection of 
naturally occurring GPG and provides for the use of purified GPG as a 
positive control and to produce anti-GPG antibodies. These antibodies may 
be used to monitor GPG expression conditions or diseases associated with 
activation of leukocytes or lymphocytes or with cell proliferation in 
endometriosis or tumor formation. 
The invention further provides for methods for treatment of conditions or 
diseases associated with overexpression of GPG by the delivery of 
effective amounts of antisense molecules, including peptide nucleic acids, 
or inhibitors of GPG for the purpose of diminishing leukocyte or 
lymphocyte activation, particularly in autoimmune diseases, or preventing 
cell proliferation in endometriosis or growing tumors. 
The invention also provides pharmaceutical compositions comprising vectors 
containing antisense sequences or inhibitors of GPG which can be used in 
the prevention or treatment of conditions or diseases including, but not 
limited to, excessive leukocyte or lymphocyte activation or cell 
proliferation. For example, specific GPG inhibitors can be used to prevent 
dimer and/or heterotrimer formation thus moderating leukocyte or 
lymphocyte activation in the joints of individuals subject to rheumatoid 
arthritis or slowing cell proliferation associated with tumor formation in 
endocrine tissues.

DETAILED DESCRIPTION OF THE INVENTION 
The present invention relates to a novel G protein gamma subunit whose 
nucleic acid sequence was identified among the polynucleotides of a human 
pituitary library (PITUNOT01) and to the use of the nucleic acid and amino 
acid sequences in the study, diagnosis, prevention and treatment of 
disease. As used herein, the abbreviation for the novel G protein gamma 
subunit in lower case (gpg) refers to a nucleic acid sequence, while the 
upper case (GPG) refers to an amino acid sequence. 
The polynucleotide sequence (FIG. 1) encoding GPG was first identified 
within Incyte Clone No. 112530. A BLAST search (Basic Local Alignment 
Search Tool; Altschul S. F. (1993) J Mol Evol 36:290-300; Altschul S. F. 
et al (1990) J Mol Biol 215:403-10) comparing the cDNAs of the PITUNOT01 
library against the rodent database of GenBank 90 identified Incyte Clone 
112530 as a nonexact homolog of rat G protein .gamma.-5 subunit from rat 
liver tissue (GI 204240; Fisher K. J. and N. N. Aronson (1992) Mol Cell 
Biol 12:1585-91). The nucleotide sequence of Incyte Clone 112530 was 
resequenced to confirm the nucleotide sequence and recompared with the 
sequence of rat G protein .gamma.-5 subunit with which it shows 85% 
identity (FIG. 2). 
The relationships among the G protein subunits identified in Watson and 
Arkinstall (supra), one to another, is shown by alignment of their amino 
acid sequences in FIG. 2: GPG (SEQ ID:2), rat G protein .gamma.-5 subunit 
(GI 204241; SEQ ID NO:3), bovine G protein .gamma.-1 subunit (GI 163787; 
SEQ ID NO:4), bovine G protein .gamma.-2 subunit (GI 163117; SEQ ID NO:5), 
bovine G protein .gamma.-3 subunit (GI 163084; SEQ ID NO:6), and bovine G 
protein .gamma.-7 subunit (GI 163118 (translation); SEQ ID NO:7). As can 
be seen in FIG. 2, the amino acid residues are conserved between GPG and 
the bovine G protein .gamma.-5 subunit and the residues M.sub.1, V.sub.14, 
Q.sub.16, L.sub.17, E.sub.20, R.sub.25, V.sub.28, S.sub.29, D.sub.46, 
P.sub.47, L.sub.48, N.sub.57, P.sub.58, F.sub.59, K.sub.63, and C.sub.65 
are conserved among G proteins known in the art. Other G protein sequences 
(SEQ ID NOs:9-31) presented in the Sequence Listing and in FIG. 4 are 
exact matches, related sequences or variants of SEQ ID NO:1. 
The present invention and the use of GPG, and of the nucleic acid sequences 
which encode it, is based, in part, on the amino acid homology between GPG 
and the rat G protein .gamma. subunit from rat liver tissue (Fisher K. J. 
and N. N. Aronson (1992) Mol Cell Biol 12:1585-91). It is also based on 
the tissue distribution of variant, closely related or exact cDNA 
sequences in uterus, neonatal keratinocytes, T cells, neutrophils, and 
stimulated macrophages, and on the known associations and functions of 
heterotrimeric and dimeric G proteins. Given the tissue distribution 
(.alpha.t2 in lymphocytes and tumor cell lines; .alpha.11, in brain, and 
.alpha.16, in hematopoietic cells; .beta.4, in brain and reproductive 
tissues) and functions of known G protein subunits, the GPG of this 
application is surely involved in activation of leukocytes or lymphocytes 
and in cell proliferation associated with endometriosis or tumor formation 
in endocrine hormone stimulated or producing brain, thyroid, adrenal, or 
gonadal tissues. 
The gpg polynucleotide sequence, oligonucleotides, fragments, portions or 
antisense thereof, may be used in diagnostic assays to detect and quantify 
levels of gpg mRNA in cells and tissues. For example, gpg polynucleotides, 
or fragments thereof, may be used in hybridization assays of body fluids 
or biopsied tissues to detect the level of gpg expression. 
The present invention also relates, in part, to an expression vector and 
host cells comprising nucleic acids encoding GPG. Such transfected host 
cells are useful for the production and recovery of GPG. The present 
invention also encompasses purified GPG. 
The invention further provides diagnostic kits for the detection of 
naturally occurring GPG and provides for the use of purified GPG as a 
positive control and to produce anti-GPG antibodies. These antibodies may 
be used to monitor GPG expression in conditions or diseases associated 
with activation of leukocytes or lymphocytes or with cell proliferation in 
endometriosis or tumor formation. 
The invention further provides for methods for treatment of conditions or 
diseases associated with overexpression of GPG by the delivery of 
effective amounts of antisense molecules, including peptide nucleic acids, 
or inhibitors of GPG for the purpose of diminishing leukocyte or 
lymphocyte activation, particularly in autoimmune diseases such as 
rheumatoid arthritis, biliary cirrhosis, hemolytic anemia, lupus 
erythematosus, and thyroiditis, or preventing cell proliferation in 
endometriosis or growing tumors of endocrine tissues. 
The invention also provides pharmaceutical compositions comprising vectors 
containing antisense molecules or inhibitors of GPG which can be used in 
the prevention or treatment of conditions or diseases including, but not 
limited to, excessive leukocyte or lymphocyte activation or cell 
proliferation. For example, specific GPG inhibitors can be used to prevent 
dimer and/or heterotrimer formation thus moderating leukocyte or 
lymphocyte activation in the joints of individuals subject to rheumatoid 
arthritis or slowing cell proliferation associated with tumor formation in 
endocrine tissues. "Nucleic acid sequence" as used herein refers to an 
oligonucleotide, nucleotide or polynucleotide sequence, and fragments or 
portions thereof, and to DNA or RNA of genomic or synthetic origin which 
may be single- or double-stranded, and represent the sense or antisense 
strand. Similarly, amino acid sequence as used herein refers to an 
oligopeptide, peptide, polypeptide or protein sequence. "Peptide nucleic 
acid" as used herein refers to a molecule which comprises an antisense 
oligomer to which an amino acid residue, such as lysine, and an amino 
group have been added. These small molecules, also designated anti-gene 
agents, stop transcript elongation by binding to their complementary 
(template) strand of DNA (Nielsen P. E. et al (1993) Anticancer Drug Des 
8:53-63). 
As used herein, GPG refers to the amino acid sequence of GPG from any 
species, including, bovine, ovine, porcine, equine, murine and preferably 
human, in a naturally occurring form or from any source, whether natural, 
synthetic, semi-synthetic or recombinant. As used herein, "naturally 
occurring" refers to a molecule, nucleic acid or amino acid sequence, 
found in nature. 
The present invention also encompasses GPG variants. A preferred GPG 
variant is one having at least 80% amino acid sequence similarity, a more 
preferred GPG variant is one having at least 90% amino acid sequence 
similarity and a most preferred GPG variant is one having at least 95% 
amino acid sequence similarity to the GPG amino acid sequence (SEQ ID 
NO:2). A "variant" of GPG may have an amino acid sequence that is 
different by one or more amino acid "substitutions". The variant may have 
"conservative" changes, wherein a substituted amino acid has similar 
structural or chemical properties, eg, replacement of leucine with 
isoleucine. More rarely, a variant may have "nonconservative" changes, eg, 
replacement of a glycine with a tryptophan. Similar minor variations may 
also include amino acid deletions or insertions, or both. Guidance in 
determining which and how many amino acid residues may be substituted, 
inserted or deleted without abolishing biological or immunological 
activity may be found using computer programs well known in the art, for 
example, DNASTAR software. 
The term "biologically active" refers to a gpg having structural, 
regulatory or biochemical functions of the naturally occurring GPG. 
Likewise, "immunologically active" defines the capability of the natural, 
recombinant or synthetic GPG, or any oligopeptide thereof, to induce a 
specific immune response in appropriate animals or cells and to bind with 
specific antibodies. The term "derivative" as used herein refers to the 
chemical modification of a gpg or the encoded GPG. Illustrative of such 
modifications would be replacement of hydrogen by an alkyl, acyl, or amino 
group. A gpg derivative would encode a polypeptide which retains essential 
biological characteristics of a G protein g subunit such as, for example, 
association with a .beta. subunit to form of a functional dimer. 
As used herein, the term "purified" refers to molecules, either nucleic or 
amino acid sequences, that are removed from their natural environment and 
isolated or separated from at least one other component with which they 
are naturally associated. 
The GPG Coding Sequences 
The nucleic and deduced amino acid sequences of GPG are shown in FIG. 1. In 
accordance with the invention, any nucleotide sequence which encodes the 
amino acid sequence of GPG can be used to generate recombinant molecules 
which express GPG. In a specific embodiment described herein, gpg was 
first isolated and identified within Incyte Clone 112530 from the human 
pituitary library (PITUNOT01), patent application Ser. No. 08/320,011, 
"Novel Human Pituitary Cell Derived Polynucleotides and Polypeptides", by 
Seilhamer et al, filed Oct. 10, 1994, and hereby incorporated by 
reference. 
Methods for DNA sequencing are well known in the art and employ such 
enzymes as the Klenow fragment of DNA polymerase I Sequenase.RTM. (US 
Biochemical Corp, Cleveland Ohio)), Taq polymerase (Perkin Elmer, Norwalk 
Conn.), thermostable T7 polymerase (Amersham, Chicago Ill.), or 
combinations of recombinant polymerases and proofreading exonucleases such 
as the ELONGASE Amplification System marketed by Gibco BRL (Gaithersburg 
Md.) Methods to extend the DNA from an oligonucleotide primer annealed to 
the DNA template of interest have been developed for both single-stranded 
and double-stranded templates. Chain termination reaction products were 
separated using electrophoresis and detected via their incorporated, 
labelled precursors. Recent improvements in mechanized reaction 
preparation, sequencing and analysis have permitted expansion in the 
number of sequences that can be determined per day. Preferably, the 
process is automated with machines such as the Hamilton Micro Lab 2200 
(Hamilton, Reno Nev.), Peltier Thermal Cycler (PTC200; MJ Research, 
Watertown Mass.) and the ABI Catalyst 800 and 377 and 373 DNA sequencers 
(Perkin Elmer). 
The quality of any particular cDNA library may be determined by performing 
a pilot scale analysis of the cDNAs and checking for percentages of clones 
containing vector, lambda or E. coli DNA, mitochondrial or repetitive DNA, 
and clones with exact or homologous matches to public databases. 
Extending the Polynucleotide Sequence 
The polynucleotide sequence of gpg may be extended utilizing partial 
nucleotide sequence and various methods known in the art to detect 
upstream sequences such as promoters and regulatory elements. Gobinda et 
al (1993; PCR Methods Applic 2:318-22) disclose "restriction-site 
polymerase chain reaction (PCR)" as a direct method which uses universal 
primers to retrieve unknown sequence adjacent to a known locus. First, 
genomic DNA is amplified in the presence of primer to a linker sequence 
and a primer specific to the known region. The amplified sequences are 
subjected to a second round of PCR with the same linker primer and another 
specific primer internal to the first one. Products of each round of PCR 
are transcribed with an appropriate RNA polymerase and sequenced using 
reverse transcriptase. 
Inverse PCR can be used to amplify or extend sequences using divergent 
primers based on a known region (Triglia T. et al(1988) Nucleic Acids Res 
16:8186). The primers may be designed using Oligo 4.0 (National 
Biosciences Inc, Plymouth Minn.), or another appropriate program, to be 
22-30 nucleotides in length, to have a GC content of 50% or more, and to 
anneal to the target sequence at temperatures about 68.degree.-72.degree. 
C. The method uses several restriction enzymes to generate a suitable 
fragment in the known region of a gene. The fragment is then circularized 
by intramolecular ligation and used as a PCR template. 
Capture PCR (Lagerstrom M. et al (1991) PCR Methods Applic 1:111-19) is a 
method for PCR amplification of DNA fragments adjacent to a known sequence 
in human and yeast artificial chromosome (YAC) DNA. Capture PCR also 
requires multiple restriction enzyme digestions and ligations to place an 
engineered double-stranded sequence into an unknown portion of the DNA 
molecule before PCR. 
Parker J. D. et al (1991; Nucleic Acids Res 19:3055-60), teach walking PCR, 
a method for targeted gene walking which permits retrieval of unknown 
sequence. PromoterFinder.TM. a new kit available from Clontech (Palo Alto 
Calif.) uses PCR, nested primers and PromoterFinder libraries to walk in 
genomic DNA. This process avoids the need to screen libraries and is 
useful in finding intron/exon junctions. 
Another PCR method, "Improved Method for Obtaining Full Length cDNA 
Sequences" by Guegler et al, patent application Ser. No. 08/487,112, filed 
Jun. 7, 1995 and hereby incorporated by reference, employs XL-PCR.TM. 
(Perkin-Elmer) to amplify and/or extend nucleotide sequences. 
Preferred libraries for screening for full length cDNAs are ones that have 
been size-selected to include larger cDNAs. Also, random primed libraries 
are preferred in that they will contain more sequences which contain the 
5' and upstream regions of genes. A randomly primed library may be 
particularly useful if an oligo d(T) library does not yield a full-length 
cDNA. Genomic libraries are useful for extension 5' of the promoter 
binding region. 
A new method for analyzing either the size or confirming the nucleotide 
sequence of sequencing or PCR products is capillary electrophoresis. 
Systems for rapid sequencing are available from Perkin Elmer, Beckman 
Instruments (Fullerton Calif.), and other companies. Capillary sequencing 
employs flowable polymers for electrophoretic separation, four different 
fluorescent dyes (one for each nucleotide) which are laser activated, and 
detection of the emitted wavelengths by a charge coupled devise camera. 
Output/light intensity is converted to electrical signal using appropriate 
software (eg. Genotyper.TM. and Sequence Navigator.TM. from Perkin Elmer) 
and the entire process from loading of samples to computer analysis and 
electronic data display is computer controlled. Capillary electrophoresis 
is particularly suited to the sequencing of small pieces of DNA which 
might be present in limited amounts in a particular sample. The 
reproducible sequencing of up to 350 bp of M13 phage DNA in 30 min has 
been reported (Ruiz-Martinez M. C. et al (1993) Anal Chem 65:2851-8). 
Expression of the Nucleotide Sequence 
In accordance with the present invention, gpg polynucleotide sequences 
which encode GPG, fragments of the polypeptide, fusion proteins or 
functional equivalents thereof, may be used to generate recombinant DNA 
molecules that direct the expression of GPG in appropriate host cells. Due 
to the inherent degeneracy of the genetic code, other DNA sequences which 
encode substantially the same or a functionally equivalent amino acid 
sequence, may be used to clone and express GPG. As will be understood by 
those of skill in the art, it may be advantageous to produce GPG-encoding 
nucleotide sequences possessing non-naturally occurring codons. Codons 
preferred by a particular prokaryotic or eukaryotic host (Murray E. et al 
(1989) Nuc Acids Res 17:477-508) can be selected, for example, to increase 
the rate of GPG expression or to produce recombinant RNA transcripts 
having desirable properties, such as a longer half-life, than transcripts 
produced from naturally occurring sequence. 
Also included within the scope of the present invention are polynucleotide 
sequences that are capable of hybridizing to the nucleotide sequence of 
FIG. 1 under conditions of intermediate to maximal stringency. 
Hybridization conditions are based on the melting temperature (Tm) of the 
nucleic acid binding complex, as taught in Berger and Kimmel (1987, Guide 
to Molecular Cloning Techniques, Methods in Enzymology, Vol 152, Academic 
Press, San Diego Calif.) incorporated herein by reference, and confer a 
defined "stringency" as explained below. 
"Maximum stringency" typically occurs at about Tm-5.degree. C. (5.degree. 
C. below the Tm of the probe); "high stringency"at about 5.degree. C. to 
10.degree. C. below Tm; "intermediate stringency" at about 10.degree. C. 
to 20.degree. C. below Tm; and "low stringency" at about 20.degree. C. to 
25.degree. C. below Tm. As will be understood by those of skill in the 
art, a maximum stringency hybridization can be used to identify or detect 
identical polynucleotide sequences while an intermediate (or low) 
stringency hybridization can be used to identify or detect similar or 
related polynucleotide sequences. The term "hybridization" as used herein 
shall include "the process by which a strand of nucleic acid joins with a 
complementary strand through base pairing" (Coombs J. (1994) Dictionary of 
Biotechnology, Stockton Press, New York N.Y.) as well as the process of 
amplification has carried out in polymerase chain reaction technologies as 
described in Dieffenbach C. W. and G. S. Dveksler (1995, PCR Primer, a 
Laboratory Manual, Cold Spring Harbor Press, Plainview N.Y.) and 
incorporated herein by reference. 
As used herein a "deletion" is defined as a change in either nucleotide or 
amino acid sequence in which one or more nucleotides or amino acid 
residues, respectively, are absent. 
As used herein an "insertion" or "addition" is that change in a nucleotide 
or amino acid sequence which has resulted in the addition of one or more 
nucleotides or amino acid residues, respectively, as compared to the 
naturally occurring gpg. 
As used herein "substitution" results from the replacement of one or more 
nucleotides or amino acids by different nucleotides or amino acids, 
respectively. 
Altered gpg polynucleotide sequences which may be used in accordance with 
the invention include deletions, insertions or substitutions of different 
nucleotide residues resulting in a polynucleotide that encodes the same or 
a functionally equivalent GPG. The protein may also show deletions, 
insertions or substitutions of amino acid residues which produce a silent 
change and result in a functionally equivalent GPG. Deliberate amino acid 
substitutions may be made on the basis of similarity in polarity, charge, 
solubility, hydrophobicity, hydrophilicity, and/or the amphipathic nature 
of the residues as long as the biological activity of GPG is retained. For 
example, negatively charged amino acids include aspartic acid and glutamic 
acid; positively charged amino acids include lysine and arginine; and 
amino acids with uncharged polar head groups having similar hydrophilicity 
values include leucine, isoleucine, valine; glycine, alanine; asparagine, 
glutamine; serine, threonine phenylalanine, and tyrosine. 
Included within the scope of the present invention are alleles of gpg. As 
used herein, an "allele" or "allelic sequence" is an alternative form of 
gpg. Alleles result from a mutation, ie, a change in the nucleic acid 
sequence, and generally produce altered mRNAs or polypeptides whose 
structure or function may or may not be altered. Any given gene may have 
none, one or many allelic forms. Common mutational changes which give rise 
to alleles are generally ascribed to deletions, additions or substitutions 
of amino acids. Each of these types of changes may occur alone, or in 
combination with the others, one or more times in a given sequence. 
The nucleotide sequences of the present invention may be engineered in 
order to alter a gpg coding sequence for a variety of reasons, including 
but not limited to, alterations which modify the cloning, processing 
and/or expression of the gene product. For example, mutations may be 
introduced using techniques which are well known in the art, eg, 
site-directed mutagenesis to insert new restriction sites, to alter 
glycosylation patterns, to change codon preference, etc. 
In another embodiment of the invention, a gpg natural, modified or 
recombinant sequence may be ligated to a heterologous sequence to encode a 
fusion protein. For example, for screening of peptide libraries for 
inhibitors of GPG activity, it may be useful to encode a chimeric GPG 
protein expressing a heterologous epitope that is recognized by a 
commercially available antibody. A fusion protein may also be engineered 
to contain a cleavage site located between a gpg sequence and the 
heterologous protein sequence, so that the GPG may be cleaved and purified 
away from the heterologous moiety. 
In an alternate embodiment of the invention, the coding sequence of gpg 
could be synthesized, whole or in part, using chemical methods well known 
in the art (see Caruthers M. H. et al (1980) Nuc Acids Res Symp Ser 
215-23, Horn T. et al(1980) Nuc Acids Res Symp Ser 225-32, etc). 
Alternatively, the protein itself could be produced using chemical methods 
to synthesize a gpg amino acid sequence, whole or in part. For example, 
peptides can be synthesized by solid phase techniques, cleaved from the 
resin, and purified by preparative high performance liquid chromatography 
(eg, Creighton (1983) Proteins Structures And Molecular Principles, W. H. 
Freeman and Co, New York N.Y.). The composition of the synthetic peptides 
may be confirmed by amino acid analysis or sequencing (eg, the Edman 
degradation procedure; Creighton, supra) 
Direct peptide synthesis can be performed using various solid-phase 
techniques (Roberge J. Y. et al (1995) Science 269:202-204) and automated 
synthesis may be achieved, for example, using the ABI 431A Peptide 
Synthesizer (Perkin Elmer) in accordance with the instructions provided by 
the manufacturer. Additionally the amino acid sequence of GPG, or any part 
thereof, may be altered during direct synthesis and/or combined using 
chemical methods with sequence from other .gamma. subunits, or any part 
thereof, to produce a variant polypeptide. 
Expression Systems 
In order to express a biologically active GPG, the nucleotide sequence 
coding for GPG, or a functional equivalent, is inserted into an 
appropriate expression vector, ie, a vector which contains the necessary 
elements for the transcription and translation of the inserted coding 
sequence. 
Methods which are well known to those skilled in the art can be used to 
construct expression vectors containing a gpg coding sequence and 
appropriate transcriptional or translational controls. These methods 
include in vitro recombinant DNA techniques, synthetic techniques and in 
vivo recombination or genetic recombination. Such techniques are described 
in Maniatis et al (1989) Molecular Cloning, A Laboratory Manual, Cold 
Spring Harbor Press, Plainview N.Y. and Ausubel F. M. et al. (1989) 
Current Protocols in Molecular Biology, John Wiley & Sons, New York N.Y. 
A variety of expression vector/host systems may be utilized to contain and 
express a gpg coding sequence. These include but are not limited to 
microorganisms such as bacteria transformed with recombinant 
bacteriophage, plasmid or cosmid DNA expression vectors; yeast transformed 
with yeast expression vectors; insect cell systems infected with virus 
expression vectors (eg, baculovirus); plant cell systems transfected with 
virus expression vectors (eg, cauliflower mosaic virus, CaMV; tobacco 
mosaic virus, TMV) or transformed with bacterial expression vectors (eg, 
Ti or pBR322 plasmid); or animal cell systems. 
The "control elements" or "regulatory sequences" of these systems vary in 
their strength and specificities and are those nontranslated regions of 
the vector, enhancers, promoters, and 3' untranslated regions, which 
interact with host cellular proteins to carry out transcription and 
translation. Depending on the vector system and host utilized, any number 
of suitable transcription and translation elements, including constitutive 
and inducible promoters, may be used. For example, when cloning in 
bacterial systems, inducible promoters such as the hybrid lacZ promoter of 
the Bluescript.RTM. phagemid (Stratagene, LaJolla Calif.) and ptrp-lac 
hybrids and the like may be used. The baculovirus polyhedrin promoter may 
be used in insect cells. Promoters or enhancers derived from the genomes 
of plant cells (eg, heat shock, RUBISCO; and storage protein genes) or 
from plant viruses (eg, viral promoters or leader sequences) may be cloned 
into the vector. In mammalian cell systems, promoters from the mammalian 
genes or from mammalian viruses are most appropriate. If it is necessary 
to generate a cell line that contains multiple copies of gpg, vectors 
based on SV40 or EBV may be used with an appropriate selectable marker. 
In bacterial systems, a number of expression vectors may be selected 
depending upon the use intended for GPG. For example, when large 
quantities of GPG are needed for the induction of antibodies, vectors 
which direct high level expression of fusion proteins that are readily 
purified may be desirable. Such vectors include, but are not limited to, 
the E. coli cloning and expression vector Bluescript.RTM. (Stratagene), in 
which the gpg coding sequence may be ligated into the vector in frame with 
sequences for the amino-terminal Met and the subsequent 7 residues of 
.beta.-galactosidase so that a hybrid protein is produced; pIN vectors 
(Van Heeke G. & S. M. Schuster (1989) J Biol Chem 264:5503-5509); and the 
like. pGEX vectors (Promega, Madison Wis.) may also be used to express 
foreign polypeptides as fusion proteins with glutathione S-transferase 
(GST). In general, such fusion proteins are soluble and can easily be 
purified from lysed cells by adsorption to glutathione-agarose beads 
followed by elution in the presence of free glutathione. Proteins made in 
such systems are designed to include heparin, thrombin or factor XA 
protease cleavage sites so that the cloned polypeptide of interest can be 
released from the GST moiety at will. 
In the yeast, Saccharomyces cerevisiae, a number of vectors containing 
constitutive or inducible promoters such as alpha factor, alcohol oxidase 
and PGH may be used. For a review of the vectors and promoters, see 
Ausubel et al (supra). 
In cases where plant expression vectors are used, the expression of a gpg 
coding sequence may be driven by any of a number of promoters. For 
example, viral promoters such as the 35S or 19S promoters of CaMV (Rhodes 
C. A. et al (1988) Science 240:204-207) may be used alone or in 
combination with the omega leader sequence from TMV (Takamatsu N. et al 
(1987) EMBO J 6:307-311). Alternatively, plant promoters such as the small 
subunit of RUBISCO (Coruzzi G. et al (1984) EMBO J 3:1671-79; Broglie R. 
et al (1984) Science 224:838-43); or heat shock promoters (Winter J. and 
Sinibaldi R. M. (1991) Results Probl Cell Differ 17:85-105) may be used. 
These constructs can be introduced into plant cells by direct DNA 
transformation or pathogen-mediated transfection. For reviews of such 
techniques, see Hobbs S or Murry L E in McGraw Yearbook of Science and 
Technology (1992) McGraw Hill New York N.Y., pp 191-196. 
An alternative expression system which could be used to express gpg is an 
insect system. In one such system, Autographa californica nuclear 
polyhedrosis virus (AcNPV) is used as a vector to express foreign genes in 
Spodoptera frugiperda cells or in Trichoplusia larvae. The gpg coding 
sequence may be cloned into a nonessential region of the virus, such as 
the polyhedrin gene, and placed under control of the polyhedrin promoter. 
Successful insertion of gpg will render the polyhedrin gene inactive and 
produce recombinant virus lacking coat protein coat. The recombinant 
viruses are then used to infect S. frugiperda cells or Trichoplusia larvae 
in which GPG is expressed (Smith G. et al (1983) J Virol 46:584; Engelhard 
E. K. et al (1994) Proc Nat Acad Sci 91:3224-7). 
In mammalian host cells, a number of viral-based expression systems may be 
utilized. In cases where an adenovirus is used as an expression vector, a 
gpg coding sequence may be ligated into an adenovirus 
transcription/translation complex consisting of the late promoter and 
tripartite leader sequence. Insertion in a nonessential E1 or E3 region of 
the viral genome will result in a viable virus capable of expressing GPG 
in infected host cells. (Logan and Shenk (1984) Proc Natl Acad Sci 
81:3655-59). In addition, transcription enhancers, such as the rous 
sarcoma virus (RSV) enhancer, may be used to increase expression in 
mammalian host cells. 
Specific initiation signals may also be required for efficient translation 
of an inserted gpg sequence. These signals include the ATG initiation 
codon and adjacent sequences. In cases where gpg, its initiation codon and 
upstream sequences are inserted into the appropriate expression vector, no 
additional translational control signals may be needed. However, in cases 
where only coding sequence, or a portion thereof, is inserted, exogenous 
transcriptional control signals including the ATG initiation codon must be 
provided. Furthermore, the initiation codon must be in the correct reading 
frame to ensure transcription of the entire insert. Exogenous 
transcriptional elements and initiation codons can be of various origins, 
both natural and synthetic. The efficiency of expression may be enhanced 
by the inclusion of enhancers appropriate to the cell system in use 
(Scharf D. et al (1994) Results Probl Cell Differ 20:125-62; Bittner M. et 
al (1987) Methods in Enzymol 1 53:51 6-544). 
In addition, a host cell strain may be chosen for its ability to modulate 
the expression of the inserted sequences or to process the expressed 
protein in the desired fashion. Such modifications of the polypeptide 
include, but are not limited to, acetylation, carboxylation, 
glycosylation, phosphorylation, lipidation and acylation. 
Post-translational processing which cleaves a "prepro" form of the protein 
may also be important for correct insertion, folding and/or function. 
Different host cells such as CHO, HeLa, MDCK, 293, WI38, etc have specific 
cellular machinery and characteristic mechanisms for such 
post-translational activities and may be chosen to ensure the correct 
modification and processing of the introduced, foreign protein. 
For long-term, high-yield production of recombinant proteins, stable 
expression is preferred. For example, cell lines which stably express GPG 
may be transformed using expression vectors which contain viral origins of 
replication or endogenous expression elements and a selectable marker 
gene. Following the introduction of the vector, cells may be allowed to 
grow for 1-2 days in an enriched media before they are switched to 
selective media. The purpose of the selectable marker is to confer 
resistance to selection and its presence allows growth and recovery of 
cells which successfully express the introduced sequences. Resistant 
clumps of stably transformed cells can be proliferated using tissue 
culture techniques appropriate to the cell type. 
Any number of selection systems may be used to recover transformed cell 
lines. These include, but are not limited to, the herpes simplex virus 
thymidine kinase (Wigler M. et al (1977) Cell 11:223-32) and adenine 
phosphoribosyltransferase (Lowy I. et al (1980) Cell 22:817-23) genes 
which can be employed in tk.sup.- or aprt.sup.- cells, respectively. 
Also, antimetabolite, antibiotic or herbicide resistance can be used as 
the basis for selection; for example, dhfr which confers resistance to 
methotrexate (Wigler M. et al (1980) Proc Natl Acad Sci 77:3567-70); npt, 
which confers resistance to the aminoglycosides neomycin and G-418 
(Colbere-Garapin F. et al (1981) J Mol Biol 150:1-14) and als or pat, 
which confer resistance to chlorsulfuron and phosphinotricin 
acetyltransferase, respectively (Murry, supra). Additional selectable 
genes have been described, for example, trpB, which allows cells to 
utilize indole in place of tryptophan, or hisD, which allows cells to 
utilize histinol in place of histidine (Hartman S. C. and R. C. Mulligan 
(1988) Proc Natl Acad Sci 85:8047-51). Recently, the use of visible 
markers has gained popularity with such markers as anthocyanins, .beta. 
glucuronidase and its substrate, GUS, and luciferase and its substrate, 
luciferin, being widely used not only to identify transformants, but also 
to quantify the amount of transient or stable protein expression 
attributable to a specific vector system (Rhodes C. A. et al (1995) 
Methods Mol Biol 55:121-131). 
Identification of Transformants Containing the Polynucleotide Sequence 
Although the presence/absence of marker gene expression suggests that the 
gene of interest is also present, its presence and expression should be 
confirmed. For example, if the gpg is inserted within a marker gene 
sequence, recombinant cells containing gpg can be identified by the 
absence of marker gene function. Alternatively, a marker gene can be 
placed in tandem with a gpg sequence under the control of a single 
promoter. Expression of the marker gene in response to induction or 
selection usually indicates expression of gpg as well. 
Alternatively, host cells which contain the coding sequence for gpg and 
express GPG may be identified by a variety of procedures known to those of 
skill in the art. These procedures include, but are not limited to, 
DNA-DNA or DNA-RNA hybridization and protein bioassay or immunoassay 
techniques which include membrane, solution, or chip based technologies 
for the detection and/or quantification of the nucleic acid or protein. 
The presence of the gpg polynucleotide sequence can be detected by DNA-DNA 
or DNA-RNA hybridization or amplification using probes, portions or 
fragments of gpg. Nucleic acid amplification based assays involve the use 
of oligonucleotides or oligomers based on the gpg sequence to detect 
transformants containing gpg DNA or RNA. As used herein "oligonucleotides" 
or "oligomers" refer to a nucleic acid sequence of at least about 10 
nucleotides and as many as about 60 nucleotides, preferably about 15 to 30 
nucleotides, and more preferably about 20-25 nucleotides which can be used 
as a probe or amplimer. 
The role of GPG in the mobilization of Ca.sup.++ as part of the signal 
transduction pathway can be assayed in vitro. It requires preloading 
neutrophils or T cells with a fluorescent dye such as FURA-2 or BCECF 
(Universal Imaging Corp, Westchester Pa.) whose emission characteristics 
have been altered by Ca.sup.++ binding. When the cells are exposed to one 
or more activating stimuli artificially (ie, anti-CD3 antibody ligation of 
the T cell receptor) or physiologically (ie, by allogeneic stimulation), 
Ca.sup.++ flux takes place. This flux can be observed and quantified by 
assaying the cells in a fluorometer or fluorescent activated cell sorter. 
The measurement of Ca.sup.++ mobilization in neutrophils has been 
described in Grynkievicz G. et al (1985) J Biol Chem 260:3440, and McColl 
S. et al (1993) J Immunol 150:4550-4555, and in T cells, in Aussel C. et 
al. (supra), incorporated herein by reference. 
A variety of protocols for detecting and measuring the expression of GPG, 
using either polyclonal or monoclonal antibodies specific for the protein 
are known in the art. Examples include enzyme-linked immunosorbent assay 
(ELISA), radioimmunoassay (RIA) and fluorescent activated cell sorting 
(FACS). A two-site, monoclonal-based immunoassay utilizing monoclonal 
antibodies reactive to two non-interfering epitopes on GPG is preferred, 
but a competitive binding assay may be employed. These and other assays 
are described, among other places, in Hampton R. et al (1990, Serological 
Methods, a Laboratory Manual, APS Press, St Paul Minn.) and Maddox D. E. 
et al (1983, J Exp Med 158:1211). 
A wide variety of labels and conjugation techniques are known by those 
skilled in the art and can be used in various nucleic and amino acid 
assays. Means for producing labelled hybridization or PCR probes for 
detecting sequences related to gpg include oligolabelling, nick 
translation, end-labelling or PCR amplification using a labelled 
nucleotide. Alternatively, the gpg sequence, or any portion of it, may be 
cloned into a vector for the production of an mRNA probe. Such vectors are 
known in the art, are commercially available, and may be used to 
synthesize RNA probes in vitro by addition of an appropriate RNA 
polymerase such as T7, T3 or SP6 and labelled nucleotides. 
A number of companies such as Pharmacia Biotech (Piscataway N.J.), Promega 
(Madison Wis.), and US Biochemical Corp (Cleveland Ohio) supply commercial 
kits and protocols for these procedures. Suitable reporter molecules or 
labels include those radionuclides, enzymes, fluorescent, 
chemiluminescent, or chromogenic agents as well as substrates, cofactors, 
inhibitors, magnetic particles and the like. Patents teaching the use of 
such labels include U.S. Pat. Nos. 3,817,837; 3,850,752; 3,939,350; 
3,996,345; 4,277,437; 4,275,149 and 4,366,241. Also, recombinant 
immunoglobulins may be produced as shown in U.S. Pat. No. 4,816,567 
incorporated herein by reference. 
Purification of GPG 
Host cells transformed with a gpg nucleotide sequence may be cultured under 
conditions suitable for the expression and recovery of the encoded protein 
from cell culture. The protein produced by a recombinant cell may be 
secreted or may be contained intracellularly depending on the sequence 
and/or the vector used. As will be understood by those of skill in the 
art, expression vectors containing gpg can be designed with signal 
sequences which direct secretion of GPG through a particular prokaryotic 
or eukaryotic cell membrane. Other recombinant constructions may join gpg 
to nucleotide sequence encoding a polypeptide domain which will facilitate 
purification of soluble proteins (Kroll D. J. et al (1993) DNA Cell Biol 
12:441-53; see also above discussion of vectors containing fusion 
proteins). 
GPG may also be expressed as a recombinant protein with one or more 
additional polypeptide domains added to facilitate protein purification. 
Such purification facilitating domains include, but are not limited to, 
metal chelating peptides such as histidine-tryptophan modules that allow 
purification on immobilized metals, protein A domains that allow 
purification on immobilized immunoglobulin, and the domain utilized in the 
FLAGS extension/affinity purification system (Immunex Corp, Seattle Wash). 
The inclusion of a cleavable linker sequences such as Factor XA or 
enterokinase (Invitrogen, San Diego Calif.) between the purification 
domain and GPG is useful to facilitate purification. 
Uses of GPG 
The rationale for diagnostic and therapeutic uses of GPG sequences is based 
on the nucleotide and amino acid sequences, their homology to the rat G 
protein .gamma. subunit, their tissue distribution in uterus, neonatal 
keratinocytes, T cells, neutrophils, and stimulated macrophages, and the 
known associations and functions of heterotrimeric and dimeric G proteins. 
The nucleic acid sequence presented in FIG. 1, its complement, fragments 
or oligomers, and anti-GPG antibodies may be used as diagnostic 
compositions in assays of cells, tissues or their extracts. Purified gpg 
or GPG can be used as the positive controls in their respective nucleic 
acid or protein based assays for conditions or diseases characterized by 
the excess expression of GPG. Antisense molecules, antagonists or 
inhibitors capable of specifically binding gpg or GPG can be used as 
pharmaceutical compositions for conditions or diseases characterized by 
the excess expression of GPG. Such conditions include activation of 
leukocytes and lymphocytes in autoimmune diseases and cell proliferation 
associated with endometriosis or tumor formation. 
The regulation of .gamma. subunit expression or of dimer formation and 
activity provides an opportunity for early intervention in conditions 
based on cell proliferation. In endometriosis, menstrual tissue is found 
outside the uterus (in the fallopian tubes, around the ovaries and in the 
abdomen). These endometrial cells as well as those located in the uterus 
respond to the monthly hormone cycle by swelling and bleeding. This 
condition, which can be quite painful, is seen in 10-15% of women between 
25 and 44 and shows a familial inheritance pattern (The Merck Manual of 
Diagnosis and Therapy (1992) Merck Research Laboratories, Rahway N.J.). 
Since estrogen is the stimulus for the cell cycle cascades which result in 
endometrial proliferation, pharmaceutical intervention in this condition 
has been based on suppressing estrogen. 
In less severe cases of endometriosis, especially in women beyond their 
child bearing years, contraceptives are the prescribed therapy. In more 
recalcitrant cases, administration of Danocrine.RTM. (Sanofi Winthrop, New 
York N.Y.) which suppresses the pituitary-ovarian axis has been more 
successful, but has caused virilizing side effects in some women. Although 
surgery cannot guarantee the removal of all endometrial cells, it has 
provided temporary relief for women with extreme abdominal proliferation 
and pain. 
For extra-uterine endometriosis, a vector containing and capable of 
expressing gpg antisense sequences, peptide nucleic acids (PNA), or 
inhibitors of GPG can be introduced in liposomes via abdominal lavage, 
particularly after surgery. Suppression of estrogen induced signal can 
prevent endometrial proliferation while not interfering with the regular 
uterine menstrual cycle. This treatment would be particularly effective 
following surgical removal of endometrial tissue form the abdomen. 
In an analogous manner, appropriate delivery of vectors expressing 
antisense sequences, peptide nucleic acids (PNA), or inhibitors of GPG can 
be used to prevent cell proliferation producing tumors in endocrine 
hormone-stimulated tissues such as the pituitary gland, thyroid, adrenal 
glands, testes or ovaries. Delivery of these therapies, further described 
below under Pharmaceutical Compositions, is necessarily tissue/tumor 
specific and depend on the diagnosis, size and status of neoplasm or 
tumor. 
The regulation of .gamma. subunit expression or of dimer formation and 
activity provides an opportunity to intervene in the activation of 
leukocytes and lymphocytes. Activation of T cells requires at least two 
signals, one cell surface, for example, the T cell receptor, and one 
soluble, for example, IL-2. The soluble factors are secreted by accessory 
cells and interact with specific receptors on the surface of T cells. 
Specific examples of T cell activation as a G protein regulated process 
are presented in Aussel C. et al. (supra). 
Inappropriate activation of leukocytes or lymphocytes may result in the 
tissue damage and destruction seen in autoimmune diseases such as 
rheumatoid arthritis, biliary cirrhosis, hemolytic anemia, lupus 
erythematosus, and thyroiditis. For example, transfection of the 
leukocytes or lymphocytes of the rheumatoid synovium with vectors 
expressing antisense sequences or with liposomes bearing PNAs or 
inhibitors of GPG can be used to avoid the formation of functional G 
protein dimers and subsequent activation of the leukocytes and lymphocytes 
which perpetuate tissue destruction. 
GPG Antibodies 
Procedures well known in the art can be used for the production of 
antibodies to GPG Such antibodies include, but are not limited to, 
polyclonal, monoclonal, chimeric, single chain, Fab fragments and 
fragments produced by a Fab expression library. Neutralizing antibodies, 
ie, those which inhibit dimer formation, are especially preferred for 
diagnostics and therapeutics. 
For the production of antibodies, various hosts including goats, rabbits, 
rats, mice, etc may be immunized by injection with GPG or any portion, 
fragment or oligopeptide which retains immunogenic properties. Depending 
on the host species, various adjuvants may be used to increase 
immunological response. Such adjuvants include but are not limited to 
Freund's, mineral gels such as aluminum hydroxide, and surface active 
substances such as lysolecithin, pluronic polyols, polyanions, peptides, 
oil emulsions, keyhole limpet hemocyanin, and dinitrophenol. BCG (bacilli 
Calmette-Guerin) and Corynebacterium parvum are potentially useful human 
adjuvants. 
Monoclonal antibodies to GPG may be prepared using any technique which 
provides for the production of antibody molecules by continuous cell lines 
in culture. These include but are not limited to the hybridoma technique 
originally described by Koehler and Milstein (1975 Nature 25 256:495-497), 
the human B-cell hybridoma technique (Kosbor et al (1983) Immunol Today 
4:72; Cote et al (1983) Proc Natl Acad Sci 80:2026-2030) and the 
EBV-hybridoma technique (Cole et al (1985) Monoclonal Antibodies and 
Cancer Therapy, Alan R. Liss Inc, New York N.Y., pp 77-96). In addition, 
techniques developed for the production of "chimeric antibodies", the 
splicing of mouse antibody genes to human antibody genes to obtain a 
molecule with appropriate antigen specificity and biological activity can 
be used (Morrison et al (1984) Proc Natl Acad Sci 81:6851-6855; Neuberger 
et al (1984) Nature 312:604-608; Takeda et al (1985) Nature 314:452-454). 
Alternatively, techniques described for the production of single chain 
antibodies (U.S. Pat. No. 4,946,778) can be adapted to produce 
GPG-specific single chain antibodies. 
Antibodies may also be produced by inducing in vivo production in the 
lymphocyte population or by screening recombinant immunoglobulin libraries 
or panels of highly specific binding reagents as disclosed in Orlandi et 
al (1989, Proc Natl Acad Sci 86: 3833-3837), and Winter G and Milstein C. 
(1991; Nature 349:293-299). 
Antibody fragments which contain specific binding sites for GPG may also be 
generated. For example, such fragments include, but are not limited to, 
the F(ab').sub.2 fragments which can be produced by pepsin digestion of 
the antibody molecule and the Fab fragments which can be generated by 
reducing the disulfide bridges of the F(ab').sub.2 fragments. 
Alternatively, Fab expression libraries may be constructed to allow rapid 
and easy identification of monoclonal Fab fragments with the desired 
specificity (Huse W. D. et al (1989) Science 256:1275-1281). 
GPG-specific antibodies are useful for the diagnosis of conditions and 
diseases associated with excessive expression of GPG. A variety of 
protocols for competitive binding or immunoradiometric assays using either 
polyclonal or monoclonal antibodies with established specificities are 
well known in the art. Such immunoassays typically involve the formation 
of complexes between GPG and its specific antibody and the measurement of 
complex formation. A two-site, monoclonal-based immunoassay utilizing 
monoclonal antibodies reactive to two noninterfering epitopes on a 
specific GPG protein is preferred, but a competitive binding assay may 
also be employed. These assays are described in Maddox D. E. et al (1983, 
J Exp Med 158:1211). 
Diagnostic Assays Using GPG Specific Antibodies 
Particular GPG antibodies are useful for the diagnosis of conditions or 
diseases characterized by excessive expression of GPG. Diagnostic assays 
for GPG include methods utilizing the antibody and a label to detect GPG 
in human body fluids, cells, tissues or extracts of such tissues. The 
polypeptides and antibodies of the present invention may be used with or 
without modification. Frequently, the polypeptides and antibodies will be 
labeled by joining them, either covalently or noncovalently, with a 
reporter molecule. A wide variety of reporter molecules are known, several 
of which were described above. 
A variety of protocols for measuring GPG, using either polyclonal or 
monoclonal antibodies specific for the respective protein are known in the 
art. Examples include enzyme-linked immunosorbent assay (ELISA), 
radioimmunoassay (RIA) and fluorescent activated cell sorting (FACS). A 
two-site, monoclonal-based immunoassay utilizing monoclonal antibodies 
reactive to two non-interfering epitopes on GPG is preferred, but a 
competitive binding assay may be employed. These assays are described, 
among other places, in Maddox, D. E. et al (1983, J Exp Med 158:1211). 
In order to provide a basis for the diagnosis of disease, normal or 
standard values for GPG expression must be established. This is 
accomplished by combining body fluids or cell extracts taken from normal 
subjects, either animal or human, with antibody to GPG under conditions 
suitable for complex formation which are well known in the art. The amount 
of standard complex formation may be quantified by comparing it with a 
dilution series of positive controls where a known amount of antibody is 
combined with known concentrations of purified GPG. Then, standard values 
obtained from normal samples may be compared with values obtained from 
samples from subjects potentially affected by a disorder or disease 
related to GPG expression. Deviation between standard and subject values 
establishes the presence of disease state. 
Drug Screening 
GPG, its catalytic or immunogenic fragments or oligopeptides can be used 
for screening therapeutic compounds in any of a variety of drug screening 
techniques. The fragment employed in such a test may be free in solution, 
affixed to a solid support, borne on a cell surface, or located 
intracellularly. The abolition of catalytic activity or the formation of 
binding complexes, between GPG and the agent being tested, may be 
measured. 
Another technique for drug screening which provides for high throughput 
screening of compounds having suitable binding affinity to the GPG is 
described in detail in "Determination of Amino Acid Sequence Antigenicity" 
by Geysen HN, WO Application 84/03564, published on Sep. 13, 1984, and 
incorporated herein by reference. In summary, large numbers of different 
small peptide test compounds are synthesized on a solid substrate, such as 
plastic pins or some other surface. The peptide test compounds are reacted 
with fragments of GPG and washed. Bound GPG is then detected by methods 
well known in the art. Purified GPG can also be coated directly onto 
plates for use in the aforementioned drug screening techniques. 
Alternatively, non-neutralizing antibodies can be used to capture the 
peptide and immobilize it on a solid support. 
This invention also contemplates the use of competitive drug screening 
assays in which neutralizing antibodies capable of binding GPG 
specifically compete with a test compound for binding GPG. In this manner, 
the antibodies can be used to detect the presence of any peptide which 
shares one or more antigenic determinants with GPG. 
Uses of the Polynucleotide Encoding GPG 
A polynucleotide, gpg, or any part thereof, may be used for diagnostic 
and/or therapeutic purposes. For diagnostic purposes, the gpg of this 
invention may be used to detect and quantitate gene expression in 
conditions or diseases in which GPG activity may be implicated. These 
specifically include, but are not limited to, activation of leukocytes and 
lymphocytes in autoimmune diseases, and cell proliferation in 
endometriosis and tumors, particularly of tissues with endocrine 
functions. Included in the scope of the invention are oligonucleotide 
sequences, antisense RNA and DNA molecules, PNAs and ribozymes, which 
function to inhibit translation of a GPG. 
Another aspect of the subject invention is to provide for hybridization or 
PCR probes which are capable of detecting polynucleotide sequences, 
including genomic sequences, encoding GPG or closely related molecules. 
The specificity of the probe, whether it is made from a highly conserved 
region, eg, 10 unique nucleotides in the 5' regulatory region, or a less 
conserved region, eg, between cysteine residues especially in the 3' 
region, and the stringency of the hybridization or amplification (high, 
intermediate or low) will determine whether the probe identifies only 
naturally occurring GPG or related sequences. 
Diagnostics 
Polynucleotide sequences encoding GPG may be used for the diagnosis of 
diseases resulting from excessive expression of gpg. For example, 
polynucleotide sequences encoding GPG may be used in hybridization or PCR 
assays of tissues from biopsies or autopsies to detect abnormalities in 
gpg expression. The form of such qualitative or quantitative methods may 
include Southern or northern analysis, dot blot or other membrane-based 
technologies; PCR technologies; dip stick, pin, chip and ELISA 
technologies. All of these techniques are well known in the art, and are 
in fact the basis of many commercially available diagnostic kits. 
Such assays may be tailored to evaluate the efficacy of a particular 
therapeutic treatment regime and may be used in animal studies, in 
clinical trials, or in monitoring the treatment of an individual patient. 
In order to provide a basis for the diagnosis of disease, a normal or 
standard profile for gpg expression must be established. This is 
accomplished by combining body fluids or cell extracts taken from normal 
subjects, either animal or human, with gpg or a portion thereof, under 
conditions suitable for hybridization or amplification. Standard 
hybridization may be quantified by comparing the values obtained for 
normal subjects with a dilution series of positive controls run in the 
same experiment where a known amount of purified gpg is used. Standard 
values obtained from normal samples may be compared with values obtained 
from samples from subjects potentially affected by a disorder or disease 
related to gpg expression. Deviation between standard and subject values 
establishes the presence of the disease state. 
If disease is established, an existing therapeutic agent is administered, 
and a treatment profile may be generated. Finally, the assay may be 
repeated on a regular basis to evaluate whether the values in the profile 
progress toward or return to the normal or standard pattern. Successive 
treatment profiles may be used to show the efficacy of treatment over a 
period of several days or several months. 
PCR as described in U.S. Pat. Nos. 4,683,195; 4,800,195; and 4,965,188 
provides additional uses for oligonucleotides based upon the gpg sequence. 
Such oligomers are generally chemically synthesized, but they may be 
generated enzymatically or produced from a recombinant source. Oligomers 
generally comprise two nucleotide sequences, one with sense orientation 
(5'.fwdarw.3') and one with antisense (3'.fwdarw.5') employed under 
optimized conditions for identification of a specific gene or condition. 
The same two oligomers, nested sets of oligomers, or even a degenerate 
pool of oligomers may be employed under less stringent conditions for 
detection and/or quantitation of closely related DNA or RNA sequences. 
Additionally methods to quantitate the expression of a particular molecule 
include radiolabeling (Melby P. C. et al 1993 J Immunol Methods 
159:235-44) or biotinylating (Duplaa C. et al 1993 Anal Biochem 229-36) 
nucleotides, coamplification of a control nucleic acid, and standard 
curves onto which the experimental results are interpolated. Quantitation 
of multiple samples may be speeded up by running the assay in an ELISA 
format where the oligomer-of-interest is presented in various dilutions 
and a spectrophotometric or colorimetric response gives rapid 
quantitation. For example, increased gpg in fluid removed from a 
rheumatoid synovium may indicate leukocyte and lymphocyte activation and 
progressive tissue destruction. A definitive diagnosis of this type may 
allow health professionals to treat the patient and prevent further 
worsening of the condition. Similarly, assays known to those of skill in 
the art can be used to monitor the progress of a patient displaying a gpg 
associated disease state during therapy. 
Therapeutics 
An antisense sequence based on the gpg sequence of this application may be 
useful in the treatment of various conditions or diseases. By introducing 
antisense sequence into cells, gene therapy can be used to treat 
conditions or diseases characterized by overexpression of GPG. In such 
instances, the antisense sequence binds with the complementary DNA strand 
and either prevents transcription or stops transcript elongation (Hardman 
J. G. et al. (1996) Goodman and Gilson's The Pharmacological Basis of 
Therapeutics. McGraw Hill, New York N.Y.) 
Expression vectors derived retroviruses, adenovirus, herpes or vaccinia 
viruses, or from various bacterial plasmids, may be used for delivery of 
antisense sequences to the targeted cell population. Methods which are 
well known to those skilled in the art can be used to construct 
recombinant vectors which will express the antisense sequence. See, for 
example, the techniques described in Maniatis et al (supra) and Ausubel et 
al (supra). Alternatively, antisense molecules such as PNAs can be 
produced and delivered to target cells or tissues in liposomes. 
The full length cDNA sequence and/or its regulatory elements enable 
researchers to use gpg as a tool in sense (Youssoufian H. and H. F. Lodish 
1993 Mol Cell Biol 13:98-104) or antisense (Eguchi et al (1991) Annu Rev 
Biochem 60:631-652) investigations or regulation of gene function. Such 
technology is now well known in the art, and sense or antisense oligomers, 
or larger fragments, can be designed from various locations along the 
coding or control regions. 
Sequences encoding GPG can be turned off by transfecting a cell or tissue 
with expression vectors which express high levels of a gpg fragment. Such 
constructs can flood cells with untranslatable sense or antisense 
sequences. Even in the absence of integration into the DNA, such vectors 
may continue to transcribe RNA molecules until all copies are disabled by 
endogenous nucleases. Such transient expression may last for a month or 
more with a non-replicating vector (Mettler I, personal communication) and 
even longer if appropriate replication elements are part of the vector 
system. 
On the other hand, stable transformation of appropriate germ line cells, or 
preferably a zygote, with a vector containing gpg fragments may produce a 
transgenic organism (U.S. Pat. No. 4,736,866, 12 Apr. 1988), which 
produces enough copies of the sense or antisense sequence to significantly 
compromise or entirely eliminate activity of the naturally occurring gpg 
gene. 
As mentioned above, modifications of gene expression can be obtained by 
designing antisense DNA or RNA molecules or PNAs to the control regions of 
gpg, ie, the promoters, enhancers, and introns. Oligonucleotides derived 
from the transcription initiation site, eg, between -10 and +10 regions of 
the leader sequence, are preferred. The antisense molecules may also be 
designed to block translation of mRNA by preventing the transcript from 
binding to ribosomes. Similarly, inhibition can be achieved using "triple 
helix" base-pairing methodology. Triple helix pairing compromises the 
ability of the double helix to open sufficiently for the binding of 
polymerases, transcription factors, or regulatory molecules. Recent 
therapeutic advances using triplex DNA were reviewed by Gee J. E. et al. 
(In Huber B. E. and B. I. Carr (1994) Molecular and Immunologic 
Approaches. Futura Publ. Co, Mt Kisco N.Y.). 
Ribozymes are enzymatic RNA molecules capable of catalyzing the specific 
cleavage of RNA. The mechanism of ribozyme action involves sequence 
specific hybridization of the ribozyme molecule to complementary target 
RNA, followed by endonucleolytic cleavage. Within the scope of the 
invention are engineered hammerhead motif ribozyme molecules that can 
specifically and efficiently catalyze endonucleolytic cleavage of gpg. 
Specific ribozyme cleavage sites within any potential RNA target are 
initially identified by scanning the target molecule for ribozyme cleavage 
sites which include the following sequences, GUA, GUU and GUC. Once 
identified, short RNA sequences of between 15 and 20 ribonucleotides 
corresponding to the region of the target gene containing the cleavage 
site may be evaluated for secondary structural features which may render 
the oligonucleotide sequence inoperable. The suitability of candidate 
targets may also be evaluated by testing accessibility to hybridization 
with complementary oligonucleotides using ribonuclease protection assays. 
Antisense molecules and ribozymes of the invention may be prepared by any 
method known in the art for the synthesis of RNA molecules. These include 
techniques for chemically synthesizing oligonucleotides such as solid 
phase phosphoramidite chemical synthesis. Alternatively, RNA molecules may 
be generated by in vitro and in vivo transcription of DNA sequences 
encoding GPG. Such DNA sequences may be incorporated into a wide variety 
of vectors with suitable RNA polymerase promoters such as T7 or SP6. 
Alternatively, antisense cDNA constructs that synthesize antisense RNA 
constitutively or inducibly can be introduced into cell lines, cells or 
tissues. 
RNA molecules may be modified to increase intracellular stability and 
half-life. Possible modifications include, but are not limited to, the 
addition of flanking sequences at the 5' and/or 3' ends of the molecule or 
the use of phosphorothioate or 2' O-methyl rather than phosphodiesterase 
linkages within the backbone of the molecule. This concept is inherent in 
the production of PNAs and can be extended in all of these molecules by 
the inclusion of nontraditional bases such as inosine, queosine and 
wybutosine as well as acetyl-, methyl-, thio- and similarly modified forms 
of adenine, cytidine, guanine, thymine, and uridine which are not as 
easily recognized by endogenous endonucleases. 
Methods for introducing vectors into cells or tissues include those methods 
discussed infra and which are equally suitable for in vivo, in vitro and 
ex vivo therapy. Ex vivo therapy, the introduction of vectors into stem 
cells taken from the patient and clonally propagated for autologous 
transplant back into that same patient is presented in U.S. Pat. Nos. 
5,399,493 and 5,437,994, disclosed herein by reference. 
Furthermore, the nucleotide sequences for gpg disclosed herein may be used 
in molecular biology techniques that have not yet been developed, provided 
the new techniques rely on properties of nucleotide sequences that are 
currently known, including but not limited to such properties as the 
triplet genetic code and specific base pair interactions. 
Detection and Mapping of Related Polynucleotide Sequences 
The nucleic acid sequence for gpg can also be used to generate 
hybridization probes for mapping the naturally occurring genomic sequence. 
The sequence may be mapped to a particular chromosome or to a specific 
region of the chromosome using well known techniques. These include in 
situ hybridization to chromosomal spreads, flow-sorted chromosomal 
preparations, or artificial chromosome constructions such as YACs, 
bacterial artificial chromosomes (BACs), bacterial P1 constructions or 
single chromosome cDNA libraries (reviewed in Price C. M. (1993) Blood Rev 
7:127-34 and Trask B. J. (1991) Trends Genet 7:149-54). 
In situ hybridization of chromosomal preparations and physical mapping 
techniques such as linkage analysis using established chromosomal markers 
are invaluable in extending genetic maps. Examples of genetic maps can be 
found in Science (1995; 270:410f and 1994; 265:1981f). Often the placement 
of a gene on the chromosome of another mammalian species may reveal 
associated markers even if the number or arm of a particular human 
chromosome is not known. New sequences can be assigned to chromosomal 
arms, or parts thereof, by physical mapping. This provides valuable 
information to investigators searching for disease genes using positional 
cloning or other gene discovery techniques. Once a disease or syndrome, 
such as ataxia telangiectasia (AT), has been crudely localized by genetic 
linkage to a particular genomic region, for example, AT to 11q22-23 (Gatti 
et al (1988) Nature 336:577-580), any sequences mapping to that area may 
represent associated or regulatory genes for further investigation. The 
nucleotide sequence of the subject invention may also be used to detect 
differences in the chromosomal location due to translocation, inversion, 
etc. between normal, carrier or affected individuals. 
Pharmaceutical Compositions 
The present invention comprises pharmaceutical compositions which may 
comprise antibodies, antagonists, or inhibitors of GPG, alone or in 
combination with at least one other agent, such as stabilizing compound, 
which may be administered in any sterile, biocompatible pharmaceutical 
carrier, including, but not limited to, saline, buffered saline, dextrose, 
and water. 
Antagonists, or inhibitors of GPG can be administered to a patient alone, 
or in combination with other agents, drugs or hormones, in pharmaceutical 
compositions where it is mixed with excipient(s) or pharmaceutically 
acceptable carriers. In one embodiment of the present invention, the 
pharmaceutically acceptable carrier is pharmaceutically inert. Since GPG 
is a cytoplasmic subunit which associates with other subunits in order to 
carry out cell signaling, the preferred route for administration these 
antagonists or inhibitors by vesicular including liposome technology. 
Antagonists or inhibitors of GPG may be used alone or in combination with 
other chemotherapeutic molecules to prevent activation of leukocytes and 
lymphocytes or cell proliferation associated with endometriosus or growth 
and development of tumors of endocrine tissues. For example, liposomes 
carrying antagonists or inhibitors may be injected into inflamed 
rheumatoid synovia. The fusion of these liposomes with leukocytes or 
lymphocytes in the synovium compromises the activation process and reduces 
inflammation. In the case of Antagonists and inhibitors destined for 
endocrine tissue are administered locally in vesicles targeted to the 
tissue of interest. 
An effective amount of GPG inhibitor, alone or in combination with 
antisense molecules, may be administered in liposomes via abdominal lavage 
to females with endometriosis. The treatment parallels the antibiotic 
lavage administered in cases of burst appendix and may be repeated for two 
to three months to eliminate all endometrial cells. This lavage of 
therapeutic nucleotide and ligand molecules would work by inhibiting dimer 
and heterotrimer formation and by suppressing gpg expression. 
Further details on techniques for formulation and administration may be 
found in the latest edition of "Remington's Pharmaceutical Sciences" (Mack 
Publishing Co, Easton Pa.). Although local delivery is desirable, there 
are other means, for example, oral; parenteral delivery, including 
intra-arterial (directly to the tumor), intramuscular, subcutaneous, 
intramedullary, intrathecal, intraventricular, intravenous, 
intraperitoneal, or intranasal administration. 
For injection, the pharmaceutical compositions of the invention may be 
formulated in aqueous solutions, preferably in physiologically compatible 
buffers such as Hanks's solution, Ringer's solution, or physiologically 
buffered saline. For tissue or cellular administration, penetrants 
appropriate to the particular barrier to be permeated are used in the 
formulation. Such penetrants are generally known in the art. 
The pharmaceutical compositions can be formulated using pharmaceutically 
acceptable carriers well known in the art in dosages suitable for oral 
administration. Such carriers enable the pharmaceutical compositions to be 
formulated as tablets, pills, capsules, liquids, gels, syrups, slurries, 
suspensions and the like, for oral or nasal ingestion by a patient to be 
treated. 
Pharmaceutical compositions suitable for use in the present invention 
include compositions wherein the active ingredients are contained in an 
effective amount to achieve the intended purpose. The determination of an 
effective dose is well within the capability of those skilled in the art, 
especially in light of the disclosure provided below. 
In addition to the active ingredients these pharmaceutical compositions may 
contain suitable pharmaceutically acceptable carriers comprising 
excipients and auxiliaries which facilitate processing of the active 
compounds into preparations which can be used pharmaceutically. The 
preparations formulated for oral administration may be in the form of 
tablets, dragees, capsules, or solutions. 
The pharmaceutical compositions of the present invention may be 
manufactured in a manner that is itself known, eg, by means of 
conventional mixing, dissolving, granulating, dragee-making, levigating, 
emulsifying, encapsulating, entrapping or lyophilizing processes. 
Pharmaceutical formulations for parenteral administration include aqueous 
solutions of the active compounds in water-soluble form. Additionally, 
suspensions of the active compounds may be prepared as appropriate oily 
injection suspensions. Suitable lipophilic solvents or vehicles include 
fatty oils such as sesame oil, or synthetic fatty acid esters, such as 
ethyl oleate or triglycerides, or liposomes. Aqueous injection suspensions 
may contain substances which increase the viscosity of the suspension, 
such as sodium carboxymethyl cellulose, sorbitol, or dextran. Optionally, 
the suspension may also contain suitable stabilizers or agents which 
increase the solubility of the compounds to allow for the preparation of 
highly concentrated solutions. 
Pharmaceutical preparations for oral use can be obtained by combining the 
active compounds with solid excipient, optionally grinding a resulting 
mixture, and processing the mixture of granules, after adding suitable 
auxiliaries, if desired, to obtain tablets or dragee cores. Suitable 
excipients are carbohydrate or protein fillers such as sugars, including 
lactose, sucrose, mannitol, or sorbitol; starch from corn, wheat, rice, 
potato, etc; cellulose such as methyl cellulose, 
hydroxypropylmethyl-cellulose, or sodium carboxymethylcellulose; and gums 
including arabic and tragacanth; and proteins such as gelatin and 
collagen. If desired, disintegrating or solubilizing agents may be added, 
such as the cross-linked polyvinyl pyrrolidone, agar, alginic acid or a 
salt thereof such as sodium alginate. 
Dragee cores are provided with suitable coatings such as concentrated sugar 
solutions, which may also contain gum arabic, talc, polyvinylpyrrolidone, 
carbopol gel, polyethylene glycol, and/or titanium dioxide, lacquer 
solutions, and suitable organic solvents or solvent mixtures. Dyestuffs or 
pigments may be added to the tablets or dragee coatings for product 
identification or to characterize the quantity of active compound, ie, 
dosage. 
Pharmaceutical preparations which can be used orally include push-fit 
capsules made of gelatin, as well as soft, sealed capsules made of gelatin 
and a coating such as glycerol or sorbitol. The push-fit capsules can 
contain the active ingredients mixed with a filler or binders such as 
lactose or starches, lubricants such as talc or magnesium stearate, and, 
optionally, stabilizers. In soft capsules, the active compounds may be 
dissolved or suspended in suitable liquids, such as fatty oils, liquid 
paraffin, or liquid polyethylene glycol with or without stabilizers. 
Compositions comprising a compound of the invention formulated in a 
pharmaceutical acceptable carrier may be prepared, placed in an 
appropriate container, and labeled for treatment of an indicated 
condition. For administration of GPG, such labeling would include amount, 
frequency and method of administration. 
The pharmaceutical composition may be provided as a salt and can be formed 
with many acids, including but not limited to hydrochloric, sulfuric, 
acetic, lactic, tartaric, malic, succinic, etc. Salts tend to be more 
soluble in aqueous or other protonic solvents that are the corresponding 
free base forms. In other cases, the preferred preparation may be a 
lyophilized powder in 1 mM-50 mM histidine, 0.1%-2% sucrose, 2%-7% 
mannitol at a pH range of 4.5 to 5.5 that is combined with buffer prior to 
use. 
For any compound used in the method of the invention, the therapeutically 
effective dose can be estimated initially from cell culture assays, eg, of 
neoplastic cells. Then, preferably, dosage can be formulated in animal 
models affected with the neoplasm to achieve a desirable concentration 
range and route of administration that inhibits MMPs. Such information can 
be used to determine useful doses and route of administration in humans. 
A therapeutically effective dose refers to that amount of antisense 
molecules or inhibitors of GPG which ameliorates symptoms, eg, prevents 
cell proliferation. Toxicity and therapeutic efficacy of such compounds 
can be determined by standard pharmaceutical procedures in cell cultures 
or experimental animals, eg, for determining the LD50 (the dose lethal to 
50% of the population) and the ED50 (the dose therapeutically effective in 
50% of the population). The dose ratio between toxic and therapeutic 
effects is the therapeutic index, and it can be expressed as the ratio 
LD50/ED50. Compounds, GPG variants or fragments, which exhibit large 
therapeutic indices are preferred. The data obtained from these cell 
culture assays and additional animal studies can be used in formulating a 
range of dosage for human use. The dosage of such compounds lies 
preferably within a range of circulating concentrations that include the 
ED50 with little or no toxicity. The dosage varies within this range 
depending upon the dosage form employed, sensitivity of the patient, and 
the route of administration. 
The exact dosage is chosen by the individual physician in view of the 
patient to be treated. Dosage and administration are adjusted to provide 
sufficient levels of the active moiety or to maintain the desired effect. 
Additional factors which may be taken into account include the severity of 
the disease state, eg, tumor size and location; age, weight, and gender of 
the patient; diet, time and frequency of administration, drug 
combination(s), reaction sensitivities, and tolerance/response to therapy. 
Long acting pharmaceutical compositions might be administered every 3 to 4 
days, every week, or once every two weeks depending on half-life and 
clearance rate of the particular formulation. 
Normal dosage amounts may vary from 0.1 to 100,000 micrograms, up to a 
total dose of about 1 g, depending upon the route of administration. 
Guidance as to particular dosages and methods of delivery is provided in 
the literature. See U.S. Pat. No. 4,657,760; 5,206,344; or 5,225,212. 
Those skilled in the art will employ different formulations for antisense 
molecules than for the inhibitors of GPG. Similarly, administration of gpg 
to a T cell will necessitate delivery in a manner different from that of a 
GPG inhibitor being delivered to endometrial cells. 
It is contemplated that conditions associated with known activities of GPG 
are treatable with either antisense or PNA molecules of gpg or antagonists 
or inhibitors of GPG. The timing of and amount of expression of GPG is 
implicated in activation of leukocytes or lymphocytes in autoimmune 
diseases such as rheumatoid arthritis, biliary cirrhosis, hemolytic 
anemia, lupus erythematosus, and thyroiditis and in cell proliferation 
associated with endometriosis or with the formation of tumors of brain, 
thyroid, adrenal, and gonadal tissues. The assays previously discussed may 
be used to diagnose or treat these conditions and to monitor such 
treatment. 
The examples below are provided to illustrate the subject invention. These 
examples are provided by way of illustration and are not included for the 
purpose of limiting the invention. 
EXAMPLES 
I Pituitary cDNA Library Construction 
The human pituitary library was constructed from a pooled sample of 21 
whole, pituitary glands from brains of Caucasian males and females with a 
range of ages from 16-70 years. The poly A+ RNA was obtained from Clontech 
Laboratories Inc. (Catalogue #6584-1 and #6584-2, Palo Alto Calif.), and 
Stratagene (La Jolla Calif.) used the poly A+ RNA to construct the 
library. cDNA synthesis was primed using both oligo d(T) and random 
hexamers, and the two cDNA libraries were handled separately. Synthetic 
adapter oligonucleotides were ligated onto the ends of the cDNA molecules 
enabling insertion into the Stratagene Uni-ZAP.TM. vector system. Phagemid 
forms of individual cDNA clones were obtained by the in vivo excision 
process, in which the host bacterial strain, XL1-Blue.RTM. (Stratagene), 
was co-infected with both the library phage and an f1 helper phage. Equal 
numbers of bacteriophage from the two cDNA libraries were mixed and used 
to reinfect fresh host cells (SOLR, Stratagene) where double-stranded 
phagemid DNA was produced. The newly transformed bacteria were selected on 
medium containing ampicillin. 
II Isolation and Sequencing of cDNA Clones 
Phagemid DNA was purified using the QIAWELL-8 Plasmid Purification System 
(QIAGEN Inc, Chatsworth Calif.) including the recommended protocols and 
prepared for sequencing. The cDNA inserts from random isolates of the 
pituitary library were sequenced by the method of Sanger F. and A. R. 
Coulson (1975; J Mol Biol 94:441f). Methods for DNA sequencing are well 
known in the art and use DNA polymerase Klenow fragment, SEQUENASE.TM. (US 
Biochemical Corp, Cleveland Ohio) or Taq polymerase to extend DNA chains 
from an oligonucleotide primer annealed to the DNA template of interest. 
Methods have been developed to sequence both single- and double-stranded 
templates. 
The chain termination reaction products were electrophoresed on 
urea-polyacrylamide gels and detected by fluorescence. The pituitary cDNAs 
were prepared and sequenced using the ABI Catalyst 800 and 377 or 373 DNA 
sequencers (Perkin Elmer, Norwalk Conn.). 
III Homology Searching of CDNA Clones and Their Deduced Proteins 
Each cDNA was compared to sequences in GenBank using a search algorithm 
incorporated into the INHERIT.TM. Sequence Analysis System (Perkin Elmer). 
In this algorithm, Pattern Specification Language (TRW Inc, Los Angeles 
Calif.) was used to determine regions of homology. The three parameters 
that determine how the sequence comparisons run were window size, window 
offset, and error tolerance. Using a combination of these three 
parameters, the DNA database was searched for sequences containing regions 
of homology to the query sequence, and the appropriate sequences were 
scored with an initial value. Subsequently, these homologous regions were 
examined using dot matrix homology plots to distinguish regions of 
homology from chance matches. Smith-Waterman alignments were used to 
display the results of the homology search. 
Peptide and protein sequence homologies were ascertained using the 
INHERIT.TM. Sequence Analysis System in a way similar to that used in DNA 
sequence homologies. Pattern Specification Language and parameter windows 
were used to search protein databases for sequences containing regions of 
homology which were scored with an initial value. Dot-matrix homology 
plots were examined to distinguish regions of significant homology from 
chance matches. 
BLAST, which stands for Basic Local Alignment Search Tool (Altschul S. F. 
(1993) J Mol Evol 36:290-300; Altschul, S. F. et al (1990) J Mol Biol 
215:403-10), was used to search for local sequence alignments. BLAST 
produces alignments of both nucleotide and amino acid sequences to 
determine sequence similarity. Because of the local nature of the 
alignments, BLAST is especially useful in determining exact matches or in 
identifying homologs. BLAST is useful for matches which do not contain 
gaps. The fundamental unit of BLAST algorithm output is the High-scoring 
Segment Pair (HSP). 
An HSP consists of two sequence fragments of arbitrary but equal lengths 
whose alignment is locally maximal and for which the alignment score meets 
or exceeds a threshold or cutoff score set by the user. The BLAST approach 
is to look for HSPs between a query sequence and a database sequence, to 
evaluate the statistical significance of any matches found, and to report 
only those matches which satisfy the user-selected threshold of 
significance. The parameter E establishes the statistically significant 
threshold for reporting database sequence matches. E is interpreted as the 
upper bound of the expected frequency of chance occurrence of an HSP (or 
set of HSPs) within the context of the entire database search. Any 
database sequence whose match satisfies E is reported in the program 
output. 
IV Extension of the Polynucleotide Sequence to Recover Regulatory Elements 
The nucleic acid sequence of full length gpg (SEQ ID NO:1) or any of the 
related or variant molecules (SEQ ID NOs: 9-31)may be used to design 
oligonucleotide primers for obtaining control sequences from genomic 
libraries. One primer is synthesized to initiate extension in the 
antisense direction (XLR) and the other is synthesized to extend sequence 
in the sense direction (XLF). The primers allowed the known gpg sequence 
to be extended "outward" generating amplicons containing new, unknown 
nucleotide sequence for the control region of interest. The initial 
primers may be designed from the cDNA using Oligo 4.0 (National 
Biosciences Inc, Plymouth Minn.), or another appropriate program, to be 
22-30 nucleotides in length, to have a GC content of 50% or more, and to 
anneal to the target sequence at temperatures about 68.degree.-72.degree. 
C. Any stretch of nucleotides which would result in hairpin structures and 
primer-primer dimerizations is avoided. 
A human genomic library is used to extend and amplify 5' upstream sequence. 
If necessary, a second set of primers is designed to further extend the 
known region. 
By following the instructions for the XL-PCR kit (Perkin Elmer) and 
thoroughly mixing the enzyme and reaction mix, high fidelity amplification 
is obtained. Beginning with 40 pmol of each primer and the recommended 
concentrations of all other components of the kit, PCR is performed using 
the Peltier Thermal Cycler (PTC200; MJ Research, Watertown Mass.) and the 
following parameters: 
______________________________________ 
Step 1 94.degree. C. for 1 min (initial denaturation) 
Step 2 65.degree. C. for 1 min 
Step 3 68.degree. C. for 6 min 
Step 4 94.degree. C. for 15 sec 
Step 5 65.degree. C. for 1 min 
Step 6 68.degree. C. for 7 min 
Step 7 Repeat step 4-6 for 15 additional cycles 
Step 8 94.degree. C. for 15 sec 
Step 9 65.degree. C. for 1 min 
Step 10 68.degree. C. for 7:15 min 
Step 11 Repeat step 8-10 for 12 cycles 
Step 12 72.degree. C. for 8 min 
Step 13 4.degree. C. (and holding) 
______________________________________ 
A 5-10 .mu.aliquot of the reaction mixture is analyzed by electrophoresis 
on a low concentration (about 0.6-0.8%) agarose mini-gel to determine 
which reactions were successful in extending the sequence. The largest 
products or bands were selected and cut out of the gel. Further 
purification involves using a commercial gel extraction method such as 
QIAQuick.TM. (QIAGEN Inc). After recovery of the DNA, Klenow enzyme was 
used to trim single-stranded, nucleotide overhangs creating blunt ends 
which facilitate religation and cloning. 
After ethanol precipitation, the products are redissolved in 13 .mu.l of 
ligation buffer, 1 .mu.l T4-DNA ligase (15 units) and 1 .mu.l T4 
polynucleotide kinase are added, and the mixture is incubated at room 
temperature for 2-3 hours or overnight at 16.degree. C. Competent E. coli 
cells (in 40 .mu.l of appropriate media) are transformed with 3 .mu.l of 
ligation mixture and cultured in 80 .mu.l of SOC medium (Sambrook J. et 
al, supra). After incubation for one hour at 37.degree. C., the whole 
transformation mixture is plated on Luria Bertani (LB)-agar (Sambrook J. 
et al, supra) containing 2.times. Carb. The following day, several 
colonies are randomly picked from each plate and cultured in 150 .mu.l of 
liquid LB/2.times. carb medium placed in an individual well of an 
appropriate, commercially-available, sterile 96-well microtiter plate. The 
following day, 5 .mu.l of each overnight culture is transferred into a 
non-sterile 96-well plate and after dilution 1:10 with water, 5 .mu.l of 
each sample is transferred into a PCR array. 
For PCR amplification, 18 .mu.l of concentrated PCR reaction mix 
(3.3.times.) containing 4 units of rTth DNA polymerase, a vector primer 
and one or both of the gene specific primers used for the extension 
reaction are added to each well. Amplification is performed using the 
following conditions: 
______________________________________ 
Step 1 94.degree. C. for 60 sec 
Step 2 94.degree. C. for 20 sec 
Step 3 55.degree. C. for 30 sec 
Step 4 72.degree. C. for 90 sec 
Step 5 Repeat steps 2-4 for an additional 29 cycles 
Step 6 72.degree. C. for 180 sec 
Step 7 4.degree. C. (and holding) 
______________________________________ 
Aliquots of the PCR reactions are run on agarose gels together with 
molecular weight markers. The sizes of the PCR products are compared to 
the original partial cDNAs, and appropriate clones are selected, ligated 
into plasmid and sequenced. 
V Labeling of Hybridization Probes 
Hybridization probes derived from SEQ ID NO:1 may be employed to screen 
cDNAs, mRNAs or genomic DNAs. Although the labeling of oligonucleotides, 
consisting of about 20 base-pairs, is specifically described, essentially 
the same procedure may be used with larger cDNA fragments. 
Oligonucleotides are labeled by combining 50 pmol of each oligomer and 250 
mCi of .gamma.-.sup.32 P! adenosine triphosphate (Amersham, Chicago Ill.) 
and T4 polynucleotide kinase (DuPont NEN.RTM. Boston Mass.). The labeled 
oligonucleotides are purified with Sephadex G-25 super fine resin column 
(Pharmacia). A portion containing 10.sup.7 counts per minute of each is 
used in a typical membrane based hybridization analysis of human genomic 
DNA digested with one of the following endonucleases (Ase I, Bgl II, EcoR 
I, Pst I, Xba 1, or Pvu II; DuPont NEN.RTM.). 
The DNA from each digest is fractionated on a 0.7 percent agarose gel and 
transferred to nylon membranes (Nytran Plus, Schleicher & Schuell, Durham 
N.H.). Hybridization is carried out for 16 hours at 40.degree. C. To 
remove nonspecific signals, blots are sequentially washed at room 
temperature under increasingly stringent conditions up to 0.1.times.saline 
sodium citrate and 0.5% sodium dodecyl sulfate. After XOMAT AR.TM. film 
(Kodak, Rochester N.Y.) is exposed to the blots in a Phosphoimager 
cassette (Molecular Dynamics, Sunnyvale Calif.) for several hours, 
hybridization patterns are compared visually. 
VI Antisense Molecules 
The gpg sequence, or any part thereof, provides the basis for the design of 
antisense molecules which may be used to inhibit in vivo expression of 
naturally occurring gpg (see Hardman J. G. et al. supra) Although use of 
antisense oligomers, consisting of approximately 20 base-pairs, is 
specifically described, essentially the same procedure may be used with 
larger or smaller nucleic acid fragments. A complementary oligonucleotide 
based on the 5' untranslated region of gpg may be used to inhibit 
expression of naturally occurring gpg. The complementary oligonucleotide 
can be designed to inhibit transcription by preventing promoter binding or 
translation of a gpg transcript by preventing the ribosomal binding. 
VII Expression of GPG 
Expression of the GPG may be accomplished by subcloning the cDNAs into 
appropriate vectors and transfecting the vectors into host cells. The 
vector, pBluescript, is used to express GPG in E. coli, strain 
XL1-BlueMRF.TM. (Stratagene). Upstream of the cloning site, this vector 
contains a promoter for .beta.-galactosidase, followed by sequence 
containing the amino-terminal Met and the subsequent 7 residues of 
.beta.-galactosidase. Immediately following these eight residues is a 
bacteriophage promoter useful for transcription and a linker containing a 
number of unique restriction sites. 
Induction of an isolated, transfected bacterial strain with IPTG using 
standard methods produces a fusion protein which consists of the first 
seven residues of .beta.-galactosidase, about 5 to 15 residues of linker, 
and the full length gpg. A signal sequence may be added to direct the 
secretion of GPG into the bacterial growth media for easier purification. 
VIII GPG Activity 
GPG can readily be assayed in vitro by monitoring the mobilization of 
Ca.sup.++ in neutrophil signal transduction pathways. Neutrophils are 
preloaded with purified GPG and with a fluorescent dye such as FURA-2 or 
BCECF (Universal Imaging Corp) whose emission characteristics have been 
altered by Ca.sup.++ binding. Then, the cells are exposed to allogeneic 
stimulation and Ca.sup.++ flux is observed and quantified using a 
fluorescent activated cell sorter. Measurements of CA.sup.++ flux are 
compared between cells in their normal state and those preloaded with GPG. 
Increased mobilization attributable to increased GPG availability results 
in increased emission. 
IX Production of GPG Specific Antibodies 
Although GPG purified using PAGE electrophoresis (Maniatis, supra) can be 
used to immunize rabbits using standard protocols, a monoclonal approach 
is more easily employed. The amino acid sequence translated from GPG is 
analyzed using DNASTAR software (DNASTAR Inc) to determine regions of high 
immunogenicity and a corresponding oligopeptide is synthesized and used to 
raise antibodies by means known to those of skill in the art. Analysis to 
select appropriate epitopes, such as those near the C-terminus or in 
adjacent hydrophilic regions is described by Ausubel F. M. et al (supra). 
Typically, the oligopeptides are 15 residues in length, synthesized using 
an ABI Peptide Synthesizer Model 431A (Perkin Elmer) using fmoc-chemistry, 
and coupled to keyhole limpet hemocyanin (KLH, Sigma) by reaction with 
M-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS; Ausubel F. M. et al, 
supra). Rabbits are immunized with the oligopeptide-KLH complex in 
complete Freund's adjuvant. The resulting antisera are tested for 
antipeptide activity, for example, by binding the peptide to plastic, 
blocking with 1% BSA, reacting with rabbit antisera, washing, and reacting 
with radioiodinated, goat anti-rabbit IgG. 
X Purification of Naturally occurring GPG Using Specific Antibodies 
Naturally occurring or recombinant GPG can be purified by immunoaffinity 
chromatography using antibodies specific for GPG. An immunoaffinity column 
is constructed by covalently coupling GPG antibody to an activated 
chromatographic resin such as CnBr-activated Sepharose (Pharmacia 
Biotech). After the coupling, the resin is blocked and washed according to 
the manufacturer's instructions. 
Media containing GPG is passed over the immunoaffinity column, and the 
column is washed under conditions that allow the preferential absorbance 
of GPG (eg, high ionic strength buffers in the presence of detergent). The 
column is eluted under conditions that disrupt antibody/GPG binding (eg, a 
buffer of pH 2-3 or a high concentration of a chaotrope such as urea or 
thiocyanate ion), and GPG is collected. 
XI Identification of Molecules Which Interact with GPG 
GPG, or biologically active fragments thereof, is labeled with .sup.125 I 
Bolton-Hunter reagent (Bolton, A. E. and Hunter, W. M. (1973) Biochem J 
133: 529). Candidate molecules previously arrayed in the wells of a 96 
well plate are incubated with the labeled GPG, washed and any wells with 
labeled GPG complex are assayed. Data obtained using different 
concentrations of GPG are used to calculate values for the number, 
affinity, and association of GPG with the candidate inhibitory molecules. 
All publications and patents mentioned in the above specification are 
herein incorporated by reference. Various modifications and variations of 
the described method and system of the invention will be apparent to those 
skilled in the art without departing from the scope and spirit of the 
invention. Although the invention has been described in connection with 
specific preferred embodiments, it should be understood that the invention 
as claimed should not be unduly limited to such specific embodiments. 
Indeed, various modifications of the described modes for carrying out the 
invention which are obvious to those skilled in molecular biology or 
related fields are intended to be within the scope of the following 
claims. 
__________________________________________________________________________ 
SEQUENCE LISTING 
(1) GENERAL INFORMATION: 
(iii) NUMBER OF SEQUENCES: 31 
(2) INFORMATION FOR SEQ ID NO:1: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 393 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: cDNA 
(vii) IMMEDIATE SOURCE: 
(A) LIBRARY: PITUITARY 
(B) CLONE: 112530 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:1: 
AAAGTTCGGAGCCCTGCCCCNGCCGCGCGCCGCTGAGTTGTCTGGCCCCGCCGACCCACG60 
GCCCACGACCCACCGACCCACGAATCGGCCCGGCCGTCGCGTGCACCATGTCTGGCTCCT120 
CCAGCGTCGCCGCTATGAAGAAAGTGGTTCAACAGCTCCGGCTGGAGGCCGGACTCAACC180 
GCGTAAAAGTTTCCCAGGCAGCTGCAGACTTGAAACAGTTCTGTCTGCAGAATGCTCAAC240 
ATGACCCTCTGCTGACTGGAGTATCTTCAAGTACAAATCCCTTCAGACCCCAGAAAGTCT300 
GTTCCTTTTTGTAGTAAAATGAATCTTTCAAAGGTTTCCCAAACCACTCCTTATGATCCA360 
GTGAATATTCAAGAGGAGCTACATTTGAAGCCT393 
(2) INFORMATION FOR SEQ ID NO:2: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 68 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(vii) IMMEDIATE SOURCE: 
(A) LIBRARY: PITUITARY 
(B) CLONE: 112530 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: 
MetSerGlySerSerSerValAlaAlaMetLysLysValValGlnGln 
151015 
LeuArgLeuGluAlaGlyLeuAsnArgValLysValSerGlnAlaAla 
202530 
AlaAspLeuLysGlnPheCysLeuGlnAsnAlaGlnHisAspProLeu 
354045 
LeuThrGlyValSerSerSerThrAsnProPheArgProGlnLysVal 
505560 
CysSerPheLeu 
65 
(2) INFORMATION FOR SEQ ID NO:3: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 1392 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: cDNA 
(vii) IMMEDIATE SOURCE: 
(A) LIBRARY: GenBank 
(B) CLONE: 204241 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: 
AGTTGGCCCGGCTGTCCCGAGCGCCATGTCGGGTTCTTCTAGCGTCGCCGCCATGAAGAA60 
GGTGGTTCAGCAACTCCGGCTGGAGGCCGGGCTCAACCGCGTGAAGGTTTCCCAGGCAGC120 
TGCAGACTTGAAACAGTTCTGTCTGCAGAATGCTCAACATGACCCTCTGCTGACTGGAGT180 
GTCTTCAAGTACAAATCCCTTCAGACCCCAGAAAGTCTGCTCCTTTTTGTAGTCATATAT240 
CTCGAGGTTTCTCAAACTACTTTTCATGAACCAGTGAATATTCACGAGAACTAAGTTCGA300 
AGTCACTAAGTTTGAAGTCTGTACAGAAGCTTCTCTTTAACACGTGCCATACACATAATC360 
TTCTACTCGTCAGTCCTTAACATCTACCTCTCTGGATTTTCATGGATCTCTGTTTCACAA420 
GGTTTAACTGTTTTATATACACTGGCTGTAGCATACAATAAAGCAGCATACAAACTTTTG480 
GCCTTGTTATTGATATGAAATGTGCTGTATACTAATTTTTTCAACATCAGGACTCACTGC540 
CTTATTGGCAAGGCTTCTAGGAATTTACAGAACAACTGCAAATCTTTGTTCAAAGGCCGG600 
AAGACTTAAGAGTTTCTAATCCTTCAGTCAGTTATGGGAATTATCTTAAATATCCCAAAT660 
ATAGGTAGGGAGATGGCTCGGTGGCTAAGAGCACTTGCTCTGCAGTTAGTTATGCTGAGT720 
TCAGATCCTGCCACCCATGTAAAAAGCTGGGCGTGGCTGTACATGCCCGTGGCCACAGCT780 
TCGGGGAGATGGTTTGTTGGCTGCCAGCGAGGGTAAGGTTGTAATTAGCTCCGTGAGAAC840 
GAGGCAGAAAGGGATACAGGTGCCTGACACTGCCATGTGGGCTCACACAGGCAACAAACA900 
ACTCTAGTGGCGTCAGCAGTTAGTGCTACCAAGAAGGTGGCTGCTTCCATCTGGAAAAAG960 
AGTTAAAGATTCACAGAATCAAGACCTTGAGGACTTACGACAATGCCTCAAGTAGGCAAG1020 
TGGAGGTAATTAGGTAGAAAGGAACAAGAAAACAGGTTAACCTCTGTGACCTGTAACTTT1080 
GCTCCAAGTCCCAATAACCTGTCCTTTAGAACTGGTATATTAAATCAGGGTCATACACTA1140 
TCTACCAACAAGCCTTTTTTCTAGCCTACAAGTTCTTTGGGAATGAAAATTATAAAGTTT1200 
GAATCGTCATTCCTAAGAAATTATTACAACTAATCCAAAATGACAACAGCTTTTATGACT1260 
TTCATACATAATTTTTCAGACAAAAATAAAATTATATTTATTTATATTTACTATATGCAG1320 
TGGAAACTCATAGCACTTGGTCATTTCTTCAAACACAGGATTTATAAAATAAAATCCCAT1380 
TTTGAAAAGTAA1392 
(2) INFORMATION FOR SEQ ID NO:4: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 68 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(vii) IMMEDIATE SOURCE: 
(A) LIBRARY: GenBank 
(B) CLONE: 204241 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:4: 
MetSerGlySerSerSerValAlaAlaMetLysLysValValGlnGln 
151015 
LeuArgLeuGluAlaGlyLeuAsnArgValLysValSerGlnAlaAla 
202530 
AlaAspLeuLysGlnPheCysLeuGlnAsnAlaGlnHisAspProLeu 
354045 
LeuThrGlyValSerSerSerThrAsnProPheArgProGlnLysVal 
505560 
CysSerPheLeu 
65 
(2) INFORMATION FOR SEQ ID NO:5: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 74 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(vii) IMMEDIATE SOURCE: 
(A) LIBRARY: GenBank 
(B) CLONE: 163787 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:5: 
MetProValIleAsnIleGluAspLeuThrGluLysAspLysLeuLys 
151015 
MetGluValAspGlnLeuLysLysGluValThrLeuGluArgMetLeu 
202530 
ValSerLysCysCysGluGluPheArgAspTyrValGluGluArgSer 
354045 
GlyGluAspProLeuValLysGlyIleProGluAspLysAsnProPhe 
505560 
LysGluLeuLysGlyGlyCysValIleSer 
6570 
(2) INFORMATION FOR SEQ ID NO:6: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 71 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(vii) IMMEDIATE SOURCE: 
(A) LIBRARY: GenBank 
(B) CLONE: 163117 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:6: 
MetAlaSerAsnAsnThrAlaSerIleAlaGlnAlaArgLysLeuVal 
151015 
GluGlnLeuLysMetGluAlaAsnIleAspArgIleLysValSerLys 
202530 
AlaAlaAlaAspLeuMetAlaTyrCysGluAlaHisAlaLysGluAsp 
354045 
ProLeuLeuThrProValProAlaSerGluAsnProPheArgGluLys 
505560 
LysPhePheCysAlaIleLeu 
6570 
(2) INFORMATION FOR SEQ ID NO:7: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 75 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(vii) IMMEDIATE SOURCE: 
(A) LIBRARY: GenBank 
(B) CLONE: 163084 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:7: 
MetLysGlyGluThrProValAsnSerThrMetSerIleGlyGlnAla 
151015 
ArgLysMetValGluGlnLeuLysIleGluAlaSerLeuCysArgIle 
202530 
LysValSerLysAlaAlaAlaAspLeuMetThrTyrCysAspAlaHis 
354045 
AlaCysGluAspProLeuIleThrProValProThrSerGluAsnPro 
505560 
PheArgGluLysLysPhePheCysAlaLeuLeu 
657075 
(2) INFORMATION FOR SEQ ID NO:8: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 68 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(vii) IMMEDIATE SOURCE: 
(A) LIBRARY: GenBank 
(B) CLONE: 163118 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:8: 
MetSerAlaThrAsnAsnIleAlaGlnAlaArgLysLeuValGluGln 
151015 
LeuArgIleGluAlaGlyIleGluArgIleLysValSerLysAlaSer 
202530 
SerGluLeuMetSerTyrCysGluGlnHisAlaArgAsnAspProLeu 
354045 
LeuValGlyValProAlaSerGluAsnProPheLysAspLysLysPro 
505560 
CysIleIleLeu 
65 
(2) INFORMATION FOR SEQ ID NO:9: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 136 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: cDNA 
(vii) IMMEDIATE SOURCE: 
(A) LIBRARY: CARDNOT01 
(B) CLONE: 183288 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:9: 
CCGGGGCTAGCGCGAGGTCCTGCAGCGCTTGGTAGAGCAGCTCAAGTTGGAGGCTGGCGT60 
GGAGAGGNTCAAGGTCTCTCAGGCAGCTGCAGAGCTTCAACAGTACTGTATGCAGAATGC120 
CTGCAAGGATGCCCTG136 
(2) INFORMATION FOR SEQ ID NO:10: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 222 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: cDNA 
(vii) IMMEDIATE SOURCE: 
(A) LIBRARY: SPLNNOT02 
(B) CLONE: 206842 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:10: 
AATATTCAAGAGAGCTACATTTGAAGCCTGTACAAAAGCTTATCCCTGTAACACATGTGC60 
CATAATATACAAACTTCTACTTTCGTCAGTCCTTAACATCTACCTCTCTGAATTTTCATG120 
AATTTCTATTTCACAAGGGTAATTGTTTTATATACACTGGCAGCAGCNTNCAATAAAACT180 
TNGNNTGNAAACTTTNAAAANTAAAAANTAAAAAACTCGGGG222 
(2) INFORMATION FOR SEQ ID NO:11: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 194 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: cDNA 
(vii) IMMEDIATE SOURCE: 
(A) LIBRARY: SPLNNOT02 
(B) CLONE: 211765 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:11: 
ATTATTCAAGAGAGCTACATTTGAGGCCTGTACAAAAGCTTATCCCTGTAACACATGTGC60 
CATAATATACAAACTTCTACTTTNGTCAGTCCTTAACATCTACCTCTNTGANTTTNCATG120 
ANTNTNTATTTCACAAGGGTAATNGTTTTATATACACTGGCAGCAGCATACAATAAAACT180 
TAGTATGAAACTTT194 
(2) INFORMATION FOR SEQ ID NO:12: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 221 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: cDNA 
(vii) IMMEDIATE SOURCE: 
(A) LIBRARY: STOMNOT01 
(B) CLONE: 215213 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:12: 
GTTGCAGCAGGATAGTAATGATGACACTGAAGATGTTTCACTGTTTGATGCGGAAGAGGA60 
GACGACTAATAGACCAAGAAAAGCCAAAATCAGACATCCAGTAGCATCGTTTTTCCACTT120 
ATTCTTTCGAGTCAGTGCAATCATCGTCTATCTTCTCTGTNAGTTGCTCAGCAGCAGCTT180 
TATTACCTGTATGGTGACAATTATCTTGTTGTTGTCGTGTT221 
(2) INFORMATION FOR SEQ ID NO:13: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 126 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: cDNA 
(vii) IMMEDIATE SOURCE: 
(A) LIBRARY: STOMNOT01 
(B) CLONE: 215233 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:13: 
GTTGCAGCAGGATAGTAATGATGACACTGAAGATGTTTCAGCATTNCCATGANTNCCTAT60 
TTCACAAGGGTAATTGTTTTATATACACTGGCAGCAGCATANAATAAAANTTAGTATGAA120 
ANTTTT126 
(2) INFORMATION FOR SEQ ID NO:14: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 292 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: cDNA 
(vii) IMMEDIATE SOURCE: 
(A) LIBRARY: EOSIHET02 
(B) CLONE: 286874 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:14: 
TCAAGTACAAATCCCTTCAGACCCCAGGAAAGTCTGTNCCTTTTTGTAGTAAAATGAATC60 
TTTCAAAGGTTTCCCAAACCACTCCTTATGATCCANTGAATATTCAAGAGAGNTACATTT120 
GANGCCTGTACAAAAGCTTATCCCTGTAACANATGTGCCATAATATACAAACTTCTACTT180 
TNGTCAGTCCTTAACATCTACCTCTCTGANTTTNCATGANTTTNTATTTCACAAGGGTAA240 
TTGTTTTATATACACTGGCAGCAGCATACAATAAAACTTAGTATGAAACTTT292 
(2) INFORMATION FOR SEQ ID NO:15: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 356 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: cDNA 
(vii) IMMEDIATE SOURCE: 
(A) LIBRARY: TMLR3DT1 
(B) CLONE: 292714 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:15: 
GGNCTCCTCCAGACGTCGANCGACTATGAAGNAAAGTGGTTCAACAGCTCCGGCTGGAGG60 
CCGGACTCAACCGCGTAAAANTTTCCCAGGCAGCTGCAGACTTGAAACAGTTCTGTCTGC120 
AGAATGCTCAACATGACCCTCTGCTGACTGGAGTATNTTCAAGTACAAATCCCTTCAGAC180 
CCCAGAAAGTCTGTNCCTTTTTGTAGTAAAATGAATCTTTCAAAGGTTTCCCAAACCACT240 
CCTTATGATCCAGTGAATATTCAAGAGAGCTACATTTGAAGCCTGTACAAAAGCTTATCC300 
CTGTAACACATGTGCCATAATATACAAACTTCTTCTTTCGTCAGTCCTTAACATCT356 
(2) INFORMATION FOR SEQ ID NO:16: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 230 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: cDNA 
(vii) IMMEDIATE SOURCE: 
(A) LIBRARY: LVENNOT01 
(B) CLONE: 352443 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:16: 
GTTCAACAGCTCCGGCTGGAGGCCGGACTCAACCGCGTAAAAGTTTCCCAGGCAGCTGCA60 
GACTTGAAACAGTTCTGTCTGCAGAATGCTCAACATGACCCTCTGCTGACTGGAGTATCT120 
TCAAGTACAAATCCCTTCAGACCCCAGAAAGTCTGTTCCTTTTTNTAGTAAAATGAATCT180 
TTCAAAGGTTTCCCAAACCACTCCTTATGATCCAGTGAATATTCAAGAGG230 
(2) INFORMATION FOR SEQ ID NO:17: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 248 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: cDNA 
(vii) IMMEDIATE SOURCE: 
(A) LIBRARY: TMLR3DT1 
(B) CLONE: 404483 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:17: 
GGCTNCTTCAGCGTCGGCGNTATGAAGAAAGTGGTTCAACANCTTCGGCTNGAGGCCCGA60 
CTTAACCGCGTAAAAGTTTCCCAGGGAACTNCAGACTTGAAACAGTCTGTCTGCAGAATG120 
CTCAACATGACCCTCTGCTGACTNGGGTATCTTCAAGTACAAATCCCTTCAGACCCCAGA180 
AAGTCTGTTCCNTTTTGTAGTAAAATGAATCTTTCAAAGGTTTTCCAAACCATTCTTATG240 
ATCCCGTG248 
(2) INFORMATION FOR SEQ ID NO:18: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 184 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: cDNA 
(vii) IMMEDIATE SOURCE: 
(A) LIBRARY: BLADNOT01 
(B) CLONE: 427016 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:18: 
TCAAGGTCTCTCAGGCAGCTGCAGAGCTTCAACAGTACTGTATGCAGAATGCCTGCAAGG60 
ATGCCCTGCTGGTGGGTGTTCCAGCTGGAAGTAACCCCTTCCGGGAGCCTAGATCCTGTG120 
CTTTACTCTGAAGACTCTAGGAGAGAAGTTTGCTGAGGAATGCCTTCAAGCACAAAGTGA180 
TGGG184 
(2) INFORMATION FOR SEQ ID NO:19: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 233 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: cDNA 
(vii) IMMEDIATE SOURCE: 
(A) LIBRARY: THYRNOT01 
(B) CLONE: 433742 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:19: 
CGCCGCCGCCATGTCCTCCGGGGCTAGCGCGAGGCCCTGCAGCGCTTGGTAGAGCAGCTC60 
AAGTTGGAGGCTGGCGTGGAGAGGATCAAGGTCTCTCAGGCAGCTGCAGAGCTTCAACAG120 
TACTGTATGCAGAATGCCTGCAAGGATGCCCTGCTGGTGGGTGTTCCAGCTGGAAGTAAC180 
CCCTTCCGGGAGCCTAGATCCTGTGCTTTACTCTGAAGACTCTAGGAGAGAAG233 
(2) INFORMATION FOR SEQ ID NO:20: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 230 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: cDNA 
(vii) IMMEDIATE SOURCE: 
(A) LIBRARY: THYRNOT01 
(B) CLONE: 439616 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:20: 
TGGAGGCCGGACTCAACCGCGTAAAAGTTTCCCAGGCAGCTGCAGACTTGAAACAGTTCT60 
GTCTGCAGAATGCTCAACATGACCCTCTGCTGACTGGAGTATCTTCAAGTACAAATCCCT120 
TCAGACCCCAGAAAGTCTGTTCCTTTTTGTAGTAAAATGAATCTTTCAAAGGTTTCCCAA180 
ACCACTCCTTATGATCCAGTGAATATTCAAGAGAGCTACATTTGAAGCCT230 
(2) INFORMATION FOR SEQ ID NO:21: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 219 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: cDNA 
(vii) IMMEDIATE SOURCE: 
(A) LIBRARY: TLYMNOT2 
(B) CLONE: 453899 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:21: 
AAAGTTCGGAGCCCTGCCCCNGCCGCGCGCCGCTGAGTTGTCTGGCCCCGCCGACCCACG60 
GCCCACGACCCACCGACCCACGAATCGGCCCGGNCGTCGNGTGCANNATGTCTGGNTCCT120 
NCAGCGTCGCCGCTATGAAGAAAGTGGTTCAACAGCTCCGNNTGGNGGCCGGACTGAANC180 
GCGTAAAAGTTTGCCAGGGAGCTGCAGACTTGNAACAGT219 
(2) INFORMATION FOR SEQ ID NO:22: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 241 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: cDNA 
(vii) IMMEDIATE SOURCE: 
(A) LIBRARY: KERANOT01 
(B) CLONE: 460437 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:22: 
CGCCGACCCACGGNCCACGNCCCACCGACCCACGNATCGGCCCGGCCGTCGCGTGCANCA60 
TGTCTGGCTCCTCCAGCGTCGCCGNTATGAAGAAAGTGGTTCAANAGCTCCGGCTGGAGG120 
CCGGACTCAACCGCGTAAAAGTTTCCCAGGNAGCTGCAGACTTGAAACAGTTCTGTCTGC180 
AGAATGNTCAACATGACCCTCTGNTGACTGGAGTATCTTCAAGTACAAATCCCTTCAGAC240 
C241 
(2) INFORMATION FOR SEQ ID NO:23: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 275 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: cDNA 
(vii) IMMEDIATE SOURCE: 
(A) LIBRARY: MMLR2DT1 
(B) CLONE: 475026 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:23: 
GGCCCGGCCGTCGCGTGCACCATGTCTGGGTCCTNCAGCGTCGCCGCTATGAAGAAAGTG60 
GTTCAACAGCTCCGGCTGGAGGCCGGACTNAACCGCGTAAAAGTTTNCCAGGNAGNTGCA120 
GACTTGAAACAGTTCTGTNTGCAGAATGCTCAACATGACCCTCTGCTGACTGGAGTATCT180 
TCAAGTACAAATCCCTTCAGACCCCAGAAAGTNTGTTCCTTTTTGTAGTAAAATGAATCT240 
TTCAAAGGTTTTCCAAACCANTNCTTATGATCCAG275 
(2) INFORMATION FOR SEQ ID NO:24: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 216 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: cDNA 
(vii) IMMEDIATE SOURCE: 
(A) LIBRARY: HNT2RAT01 
(B) CLONE: 482881 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:24: 
AGGAGCCCAGCGGCCGNNGGCCATTTCTCCNGGGTTAANGGGGGGNCCCNNAAAAATTTT60 
TGGGGAGATCAAAATTGAGGGTTGNGTNGAGAGGATCAAGGTCTCTCAGGCAGCTGCAGA120 
GCTTCAANAGTANTGTATGCAGAATGCCTGCAAGGATGCCCTGCTGGTGGGTGTTCCAGC180 
TGGAAGTAANCCNTTNCGGGAGCCTAGATNCTGTGC216 
(2) INFORMATION FOR SEQ ID NO:25: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 179 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: cDNA 
(vii) IMMEDIATE SOURCE: 
(A) LIBRARY: HNT2RAT01 
(B) CLONE: 484339 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:25: 
CGNCGCCGCCATGTCCTCNGGGGNTAGCGNGAGGCCCTGCAGCGCTTGGTAGAGCAGCTC60 
AAGTTGGAGGNTGGCGTGGAGAGGATCAAGGTCTCTCAGGCAGCTGCAGAGCTTCAACAG120 
TANTGTATGCAGAATGCCTGCAAGGNTGCCCTGCTGGTGGGTGTTCCAGCTGGAAGTAA179 
(2) INFORMATION FOR SEQ ID NO:26: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 261 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: cDNA 
(vii) IMMEDIATE SOURCE: 
(A) LIBRARY: NEUTLPT01 
(B) CLONE: 498118 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:26: 
CTGGCCCCGCCGACCCACGGCCCACGACCCACCGACCCACGAATNGGCCCGGCCGTCGCG60 
TGCACCATGTCTGGCTCCTCCAGCGTCGNCGNTATGAAGAAAGTGGTTCAACAGCTCCGG120 
CTGGAGGCCGGACTCAACCGCGTAAAAGTTTCCCAGGCAGCTGCAGACTTGAAACAGTTN180 
TGTNTGCAGAATGNTCAACATGACCNTGTGCTGACTGGAGTATCTTCAAGTACAAATCCC240 
TTCAGACCCCAGAAAGTNTGT261 
(2) INFORMATION FOR SEQ ID NO:27: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 193 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: cDNA 
(vii) IMMEDIATE SOURCE: 
(A) LIBRARY: NEUTLPT01 
(B) CLONE: 498822 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:27: 
CCCGGCCGTCGCGTGCACCATGTCTGGCTCCTCCAGCGTCGCCGCTATGAAGAAAGTGGT60 
TCAACAGCTCCGGCTGGAGGCCGGACTCAACCGCGTAAAAGTTTCCCAGGCAGCTGCAGA120 
CTTGAAACAGTTCTGTCTGCAGAATGCTCAACATGACCCTCTGCTGACTGGAGTATCTTC180 
AAGTACAAATCCC193 
(2) INFORMATION FOR SEQ ID NO:28: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 66 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: cDNA 
(vii) IMMEDIATE SOURCE: 
(A) LIBRARY: NEUTLPT01 
(B) CLONE: 499687 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:28: 
CTATTTCACAAGGGTAATTGTTTTATATACACTGGCAGCAGCATACAATAAAACTTAGTA60 
TGAAAC66 
(2) INFORMATION FOR SEQ ID NO:29: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 206 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: cDNA 
(vii) IMMEDIATE SOURCE: 
(A) LIBRARY: NEUTLPT01 
(B) CLONE: 500281 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:29: 
GCCGGACTTAACCGCGTAAAAGTTTNCCAGGGAGCTNCAGACTTGAAACAGTTCTGTCTN60 
CAGAATGCTCAACATGACCCTCTGCTGACTGGAGTATCTTCAAGTACAAATCCCTTCAGA120 
CCCCAGAAAGTCTGTTCCNTTTTGTAGTAAAATGAATCTTTCAAAGGTTTTCCAAACCAC180 
TCTTATGATCCNGTGGATATTNAANG206 
(2) INFORMATION FOR SEQ ID NO:30: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 277 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: cDNA 
(vii) IMMEDIATE SOURCE: 
(A) LIBRARY: MMLR3DT01 
(B) CLONE: 567503 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:30: 
CGCGCCGCTGAGTTGTCTGGCCCCGNCGACCCACGGCCCACGACCCACCGACCCACGAAT60 
CGGCCCGGCCGTNGCGTGCACCATGTCTGGNTNCTNCAGCGTCGCCGGTATGAAGAAAGT120 
GGTTCAACAGCTCCGGCTGGAGGCCGGACTNAACCGCGTAAAAGTTTCCCAGGCAGCTGC180 
AGACTTGAAACAGTTCTGTCTGCAGAATGNTCAACATGACCCTNTGNTGACTGGAGTATC240 
TTCAAGTACAAATCCCTTCAGACCCCAGAAAGTNTGT277 
(2) INFORMATION FOR SEQ ID NO:31: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 260 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: cDNA 
(vii) IMMEDIATE SOURCE: 
(A) LIBRARY: UTRSNOT01 
(B) CLONE: 586994 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:31: 
GCGCCGCTGAGTTGTCTGGCCCGGCCGACCCACGGNTCACGACCCACCGACCCACGAATC60 
GGCCCGGCCGTCGNGTGCACCATGTCTGGNTNCTTCAGCGTCGGCGGTATGAAGAAAGTG120 
GTTCAACAGCTTCGGNTGGAGGCCGGACTTAACCGCGTAAAAGTTTTCCAGGGAGCTGCA180 
GACTTGAAACAGTTCTGTCTGCAGAATGTTAACATGACCCTNTGNTGANTTGAGTATTTN240 
AAGTACAAATCCTTNAGANC260 
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