CA2425829A1 - Proteases - Google Patents

Abstract

The invention provides human proteases (PRTS) and polynucleotides which identify and encode PRTS. The invention also provides expression vectors, ho st cells, antibodies, agonists, and antagonists. The invention also provides methods for diagnosing, treating, or preventing disorders associated with aberrant expression of PRTS.

Description

PROTEASES
TECHNICAL FIELD
This invention relates to nucleic acid and amino acid sequences of proteases and to the use of these sequences in the diagnosis, treatment, and prevention of gastrointestinal, cardiovascular, autoimmune/inflarmnatory, cell proliferative, developmental, epithelial, neurological, and reproductive disorders, and in the assessment of the effects of exogenous compounds on the expression of nucleic acid and amino acid sequences of proteases.
BACKGROUND OF THE INVENTION
Proteases cleave proteins and peptides at the peptide bond that forms the backbone of the protein or peptide chain. Proteolysis is one of the most important and frequent enzymatic reactions that occurs both within and outside of cells. Proteolysis is responsible for the activation and maturation of nascent polypeptides, the degradation of misfolded and damaged proteins, and the controlled turnover of peptides within the cell. Proteases participate in digestion, endocrine function, and tissue remodeling during embryonic development, wound healing, and normal growth. Proteases can play a role in regulatory processes by affecting the half life of regulatory proteins. Proteases are involved in the etiology or progression of disease states such as inflammation, angiogenesis, tumor dispersion and metastasis, cardiovascular disease, neurological disease, and bacterial, parasitic, and viral infections.
Proteases can be categorized on the basis of where they cleave their substrates.
Exopeptidases, which include aminopeptidases, dipeptidyl peptidases, tripeptidases, carboxypeptidases, peptidyl-di-peptidases, dipeptidases, and omega peptidases, cleave residues at the termini of their substrates. Endopeptidases, including serine proteases, cysteine proteases, and metalloproteases, cleave at residues within the peptide. Four principal categories of mammalian proteases have been identified based on active site structure, mechanism of action, and overall three-dimensional structure. (See Beynon, R.J. and J.S. Bond (1994) Proteolytic Enzymes: A Practical Approach, Oxford University Press, New York NY, pp. 1-5.) Serine Proteases The serine proteases (SPs) are a large, widespread family of proteolytic enzymes that include the digestive enzymes trypsin and chymotrypsin, components of the complement and blood-clotting cascades, and enzymes that control the degradation and turnover of macromolecules within the cell and in the extracellular matrix. Most of the more than 20 subfamilies can be grouped into six clans, each with a common ancestor. These six clans are hypothesized to have descended from at least four evolutionarily distinct ancestors. SPs are named for the presence of a serine residue found in the active catalytic site of most families. The active site is defined by the catalytic triad, a set of conserved asparagine, histidine, and serine residues critical for catalysis.
These residues form a charge relay network that facilitates substrate binding. Other residues outside the active site form an oxyanion hole that stabilizes the tetrahedral transition intermediate formed during catalysis. SPs have a wide range of substrates and can be subdivided into subfamilies on the basis of their substrate specificity. The main subfamilies are named for the residues) after which they cleave: trypases (after arginine or lysine), aspases (after aspartate), chymases (after phenylalanine or leucine), metases (methionine), and serases (after serine) (Rawlings, N.D, and A.J. Barrett (1994) Methods Enzymol.
244:19-61).
Most mammalian serine proteases are synthesized as zymogens, inactive precursors that are activated by proteolysis. For example, trypsinogen is converted to its active form, trypsin, by enteropeptidase. Enteropeptidase is an intestinal protease that removes an N-terminal fragment from trypsinogen. The remaining active fragment is trypsin, which in turn activates the precursors of the other pancreatic enzymes. Likewise, proteolysis of prothrombin, the precursor of thrombin, generates three separate polypeptide fragments. The N-terminal fragment is released while the other two fragments, which comprise active thrombin, remain associated through disulfide bonds.
The two largest SP subfamilies are the chymotrypsin (S1) and subtilisin (S8) families. Some members of the chymotrypsin family contain two structural domains unique to this family. Kringle domains are triple-looped, disulfide cross-linked domains found in varying copy number. Kringles are thought to play a role in binding mediators such as membranes, other proteins or phospholipids, and in the regulation of proteolytic activity (PROSITE PDOC00020). Apple domains are 90 amino-acid repeated domains, each containing six conserved cysteines. Three disulfide bonds link the first and sixth, second and fifth, and third and fourth cysteines (PROSITE
PDOC00376). Apple domains are involved in protein-protein interactions. S 1 family members include trypsin, chymotrypsin, coagulation factors IX-III, complement factors B, C, and D, granzymes, kallikrein, and tissue- and urokinase-plasminogen activators. The subtilisin family has members found in the eubacteria, archaebacteria, eukaryotes, and viruses. Subtilisins include the proprotein-processing endopeptidases kexin and furin and the pituitary prohormone convertases PC1, PC2, PC3, PC6, and PACE4 (Rawlings and Barrett, supra).
SPs have functions in many normal processes and some have been implicated in the etiology or treatment of disease. Enterokinase, the initiator of intestinal digestion, is found in the intestinal brush border, where it cleaves the acidic propeptide from trypsinogen to yield active trypsin (Kitamoto, Y. et al. (1994) Proc. Natl. Acad. Sci. USA 91:7588-7592).
Prolylcarboxypeptidase, a lysosomal serine peptidase that cleaves peptides such as angiotensin II and III and [des-Arg9]
bradykinin, shares sequence homology with members of both the serine carboxypeptidase and prolylendopeptidase families (Tan, F. et al. (1993) J. Biol. Chem. 268:16631-16638). The protease neuropsin may influence synapse formation and neuronal connectivity in the hippocampus in response to neural signaling (Chen, Z.-L. et al. (1995) J. Neurosci. 15:5088-5097). Tissue plasminogen activator is useful for acute management of stroke (Zivin, J.A.
(1999) Neurology 53:14-19) and myocardial infarction (Ross, A.M. (1999) Clin: Cardiol. 22:165-171).
Some receptors (PAR, for proteinase-activated receptor), highly expressed throughout the digestive tract, are activated by proteolytic cleavage of an extracellular domain. The major agonists for PARs, thrombin, trypsin, and mast Bell tryptase, are released in allergy and inflammatory conditions.
Control of PAR activation by proteases has been suggested as a promising therapeutic target (Vergnolle, N.
(2000) Aliment.
Pharmacol. Ther. 14:257-266; Rice, K.D. et al. (1998) Curr. Pharm. Des. 4:381-396). Prostate-specific antigen (PSA) is a kallikrein-like serine protease synthesized and secreted exclusively by epithelial Bells in the prostate gland. Serum PSA is elevated in prostate cancer and is the most sensitive physiological marker for monitoring cancer progression and response to therapy. PSA can also identify the prostate as the origin of a metastatic tumor (Brawer, M.K.
and P.H. Lange (1989) Urology 33:11-16).
The signal peptidase is a specialized class of SP found in all prokaryotic and eukaryotic cell types that serves in the processing of signal peptides from certain proteins.
Signal peptides are amino-terminal domains of a protein which direct the protein from its ribosomal assembly site to a particular cellular or extracellular location. Once the protein has been exported, removal of the signal sequence by a signal peptidase and posttranslational processing, e.g., glycosylation or phosphorylation, activate the protein. Signal peptidases exist as mufti-subunit complexes in both yeast and mammals. The canine signal peptidase complex is composed of five subunits, all associated with the microsomal membrane and containing hydrophobic regions that span the membrane one or more times (Shelness, G.S. and G. Blobel (1990) J. Biol. Chem.
265:9512-9519).
Some of these subunits serve to fix the complex in its proper position on the membrane while others contain the actual catalytic activity.
Another family of proteases which have a serine in their active site are dependent on the hydrolysis of ATP for their activity. These proteases contain proteolytic core domains and regulatory ATPase domains which can be identified by the presence of the P-loop, an ATP/GTP-binding motif (PROSITE PDOC00803). Members of this family include the eukaryotic mitochondrial matrix proteases, Clp protease and the proteasome. Clp protease was originally found in plant chloroplasts but is believed to be widespread in both prokaryotic and eukaryotic cells. The gene for early-onset torsion dystonia encodes a protein related to Clp protease (Ozelius, L.J. et al. (1998) Adv. Neurol.
78:93-105).
The proteasome is an intracellular protease complex found in some bacteria and in all eukaryotic cells, and plays an important role in cellular physiology.
Proteasomes are associated with the ubiquitin conjugation system (UCS), a major pathway for the degradation of cellular proteins of all types, including proteins that function to activate or repress cellular processes such as transcription and cell cycle progression (Ciechanover, A. (1994) Cell 79:13-21). In the UCS
pathway, proteins targeted for degradation are conjugated to ubiquitin, a small heat stable protein. The ubiquitinated protein is then recognized and degraded by the proteasome. The resultant ubiquitin-peptide complex is hydrolyzed by a ubiquitin carboxyl terminal hydrolase, and free ubiquitin is released for reutilization by the UCS. Ubiquitin-proteasome systems are implicated in the degradation of mitotic cyclic kinases, oncoproteins, tumor suppressor genes (p53), cell surface receptors associated with signal transduction, transcriptional regulators, and mutated or damaged proteins (Ciechanover, supra).
This pathway has been implicated in a number of diseases, including cystic fibrosis, Angelman's syndrome, and Liddle syndrome (reviewed in Schwartz, A.L. and A. Ciechanover (1999} Annu. Rev.
Med. 50:57-74). A murine proto-oncogene, Unp, encodes a nuclear ubiquitin protease whose overexpression leads to oncogenic transformation of NIH3T3 cells. The human homologue of this gene is consistently elevated in small cell tumors and adenocarcinomas of the lung (Gray, D.A.
(1995) Oncogene 10:2179-2183). Ubiquitin carboxyl terminal hydrolase is involved in the differentiation of a lymphoblastic leukemia cell line to a non-dividing mature state (Maki, A. et al.
(1996) Differentiation 60:59-66). In neurons, ubiquitin carboxyl terminal hydrolase (PGP 9.5) expression is strong in the abnormal structures that occur in human neurodegenerative diseases (Lowe, J. et al. (1990) J. Pathol. 161:153-160). The proteasome is a large (2000 kDa) multisubunit complex composed of a central catalytic core containing a variety of proteases arranged in four seven-membered rings with the active sites facing inwards into the central cavity, and terminal ATPase subunits covering the outer port of the cavity and regulating substrate entry (for review, see Schmidt, M. et al. (1999) Curr. Opin. Chem. Biol. 3:584-591).
Cysteine Proteases Cysteine proteases (CPs) are involved in diverse cellular processes ranging from the processing of precursor proteins to intracellular degradation. Nearly half of the CPs known are present only in viruses. CPs have a cysteine as the major catalytic residue at the active site where catalysis proceeds via a thioester intermediate and is facilitated by nearby histidine and asparagine residues. A glutamine residue is also important, as it helps to form an oxyanion hole. Two important CP families include the papain-like enzymes (C1) and the calpains (C2). Papain-like family members are generally lysosomal or secreted and therefore are synthesized with signal peptides as well as propeptides. Most members bear a conserved motif in the propeptide that may have structural significance (Karrer, K.M. et al. (1993) Proc. Natl. Acad. Sci. USA 90:3063-3067). Three-dimensional structures of papain family members show a bilobed molecule with the catalytic site located between the two lobes. Papains include cathepsins B, C, H, L, and S, certain plant allergens and dipeptidyl peptidase (for a review, see Rawlings, N.D. and A.J. Barrett (1994) Methods Enzymol.
244:461-486).
Some CPs are expressed ubiquitously, while others are produced only by cells of the immune system. Of particular note, CPs are produced by monocytes, macrophages and other cells which migrate to sites of inflammation and secrete molecules involved in tissue repair. Overabundance of these repair molecules plays a role in certain disorders. In autoimmune diseases such as rheumatoid arthritis, secretion of the cysteine peptidase cathepsin C degrades collagen, laminin, elastin and other structural proteins found in the extracellular matrix of bones. Bone weakened by such degradation is also more susceptible to tumor invasion and metastasis. Cathepsin L expression may also contribute to the influx of mononuclear cells which exacerbates the destruction of the rheumatoid synovium (Keyszer, G.M. (1995) Arthritis Rheum. 38:976-984).
Calpains are calcium-dependent cytosolic endopeptidases which contain both an N-terminal catalytic domain and a C-terminal calcium-binding domain. Calpain is expressed as a proenzyme heterodimer consisting of a catalytic subunit unique to each isoform and a regulatory subunit common to different isoforms. Each subunit bears a calcium-binding EF-hand domain.
The regulatory subunit also contains a hydrophobic glycine-rich domain that allows the enzyme to associate with cell membranes. Calpains are activated by increased intracellular calcium concentration, which induces a change in conformation and limited autolysis. The resultant active molecule requires a lower calcium concentration for its activity (Chan, S.L. and M.P. Mattson (1999) J.
Neurosci. Res. 58:167-190).
Calpain expression is predominantly neuronal, although it is present in other tissues. Several chronic neurodegenerative disorders, including ALS, Parkinson's disease and Alzheimer's disease are associated with increased calpain expression (Chan and Mattson, supra).
Calpain-mediated breakdown of the cytoskeleton has been proposed to contribute to brain damage resulting from head injury (McCracken, E. et al. (1999) J. Neurotrauma 16:749-761). Calpain-3 is predominantly expressed in skeletal muscle, and is responsible for limb-girdle muscular dystrophy type 2A (Minami, N. et al. (1999) J. Neurol. Sci. 171:31-37).
Another family of thiol proteases is the caspases, which are involved in the initiation and execution phases of apoptosis. A pro-apoptotic signal can activate initiator caspases that trigger a proteolytic caspase cascade, leading to the hydrolysis of target proteins and the classic apoptotic death of the cell. Two active site residues, a cysteine and a histidine, have been implicated in the catalytic mechanism. Caspases acre among the most specific endopeptidases, cleaving after aspartate residues. Caspases are synthesized as inactive zymogens consisting of one large (p20) and one small (p10) subunit separated by a small spacer region, and a variable N-terminal prodomain. This prodomain interacts with cofactors that can positively or negatively affect apoptosis. An activating signal causes autoproteolytic cleavage of a specific aspartate residue (D297 in the caspase-1 numbering convention) and removal of the spacer and prodomain, leaving a p101p20 heterodimer.
Two of these heterodimers interact via their small subunits to form the catalytically active tetramer.
The long prodomains of some caspase family members have been shown to promote dimerization and auto-processing of procaspases. Some caspases contain a "death effector domain" in their prodomain by which they can be recruited into self activating complexes with other caspases and FADD protein associated death receptors or the TNF receptor complex. In addition, two dimers from different caspase family members can associate, changing the substrate specificity of the resultant tetramer.
Endogenous caspase inhibitors (inhibitor of apoptosis proteins, or IAPs) also exist. All these interactions have clear effects on the control of apoptosis (reviewed in Chan and Mattson, supra;
Salveson, G.S. and V.M. Dixit (1999) Proc. Natl. Acad. Sci. USA 96:10964-10967).
Caspases have been implicated in a number of diseases. Mice lacking some caspases have severe nervous system defects due to failed apoptosis in the neuroepithelium and suffer early lethality. Others show severe defects in the inflammatory response, as caspases are responsible for processing IL-lb and possibly other inflammatory cytokines (Chap and Mattson, supra). Cowpox virus and baculoviruses target caspases to avoid the death of their host cell and promote successful infection. In addition, increases in inappropriate apoptosis have been reported in AIDS, neurodegenerative diseases and ischemic injury, while a decrease in cell death is associated with cancer (Salveson and Dixit, supra; Thompson, C.B. (1995) Science 267:1456-1462).
Aspartyl proteases Aspartyl proteases (APs) include the lysosomal proteases cathepsins D and E, as well as chymosin, renin, and the gastric pepsins. Most retroviruses encode an AP, usually as part of the Col polyprotein. APs, also called acid proteases, are monomeric enzymes consisting of two domains, each domain containing one half of the active site with its own catalytic aspartic acid residue. APs are most active in the range of pH 2-3, at which one of the aspartate residues is ionized and the other neutral. The pepsin family of APs contains many secreted enzymes, and all are likely to be synthesized with signal peptides and propeptides. Most family members have three disulfide loops, the first ~5 residue loop following the first aspartate, the second 5-6 residue loop preceding the second aspartate, and the third and largest loop occurring toward the C
terminus. Retropepsins, on the other hand, are analogous to a single domain of pepsin, and become active as homodimers with each retropepsin monomer contributing one half of the active site.
Retropepsins are required for processing the viral polyproteins.
APs have roles in various tissues, and some have been associated with disease.
Renin mediates the first step in processing the hormone angiotensin, which is responsible for regulating electrolyte balance and blood pressure (reviewed in Crews, D.E. and S.R.
Williams (1999) Hum.

Biol. 71:475-503). Abnormal regulation and expression of cathepsins are evident in various inflammatory disease states. Expression of cathepsin D is elevated in synovial tissues from patients with rheumatoid arthritis and osteoarthritis. The increased expression and differential regulation of the cathepsins are linked to the metastatic potential of a variety of cancers (Chambers, A.F. et al.
(1993) Crit. Rev. Oncol. 4:95-114).
Metalloproteases Metalloproteases require a metal ion for activity, usually manganese or zinc.
Examples of manganese metalloenzymes include aminopeptidase P and human proline dipeptidase (PEPD).
Aminopeptidase P can degrade bradykinin, a nonapeptide activated in a variety of inflammatory responses. Aminopeptidase P has been implicated in coronary ischemia/reperfusion injury.
Administration of aminopeptidase P inhibitors has been shown to have a cardioprotective effect in rats (Ersahin, C. et al (1999) J. Cardiovasc. Pharmacol. 34:604-611).
Most zinc-dependent metalloproteases share a common sequence in the zinc-binding domain.
The active site is made up of two histidines which act as zinc ligands and a catalytic glutamic acid C
terminal to the first histidine. Proteins containing this signature sequence are known as the metzincins and include aminopeptidase N, angiotensin-converting enzyme, neurolysin, the matrix metalloproteases and the adamalysins (ADAMS). An alternate sequence is found in the zinc carboxypeptidases, in which all three conserved residues - two histidines and a glutamic acid - are involved in zinc binding.
A number of the neutral metalloendopeptidases, including angiotensin converting enzyme and the aminopeptidases, are involved in the metabolism of peptide hormones. High aminopeptidase B
activity, for example, is found in the adrenal glands and neurohypophyses of hypertensive rats (Prieto, I. et al. (1998) Horm. Metab. Res. 30:246-248). Oligopeptidase M/neurolysin can hydrolyze bradykinin as well as neurotensin (Serizawa, A. et al. (1995) J. Biol. Chem 270:2092-2098).
Neurotensin is a vasoactive peptide that can act as a neurotransmitter in the brain, where it has been implicated in limiting food intake (Tritos, N.A. et al. (1999) Neuropeptides 33:339-349).
The matrix metalloproteases (MMPs) are a family of at least 23 enzymes that can degrade components of the extracellular matrix (ECM). They are Zn+2 endopeptidases with an N-terminal catalytic domain. Nearly all members of the family have a hinge peptide and C-terminal domain which can bind to substrate molecules in the ECM or to inhibitors produced by the tissue (TIIVVIPs, for tissue inhibitor of metalloprotease; Campbell, LL. et al. (1999) Trends Neurosci. 22:285). The presence of fibronectin-like repeats, transmembrane domains, or C-terminal hemopexinase-like domains can be used to separate MMPs into collagenase, gelatinase, stromelysin and membrane-type MMP subfamilies. In the inactive form, the Zn+z ion in the active site interacts with a cysteine in the pro-sequence. Activating factors disrupt the Zn+Z-cysteine interaction, or "cysteine switch," exposing the active site. This partially activates the enzyme, which then cleaves off its propeptide and becomes fully active. MMPs are often activated by the serine proteases plasmin and furin. MMPs are often regulated by stoichiometric, noncovalent interactions with inhibitors; the balance of protease to inhibitor, then, is very important in tissue homeostasis (reviewed in Yong, V.W. et al. (1998) Trends Neurosci.21:75).
MMPs are implicated in a number of diseases including osteoarthritis (Mitchell, P. et al.
(1996) J. Clin. Invest. 97:761), atherosclerotic plaque rupture (Sukhova, G.K.
et al. (1999) Circulation 99:2503), aortic aneurysm (Schneiderman, J. et al. (1998) Am. J.
Path. 152:703), non-healing wounds (Saarialho-Kere, U.K. et al. (1994) J. Clin. Invest.
94:79), bone resorption (Blavier, L. and J.M. Delaisse (1995) J. Cell Sci. 108:3649), age-related macular degeneration (Stem, B. et al. (1998) Invest. Ophthalmol. Vis. Sci. 39:2194), emphysema (Finlay, G.A. et al. (1997) Thorax 52:502), myocardial infarction (Rohde, L.E. et al. (1999) Circulation 99:3063) and dilated cardiomyopathy (Thomas, C.V. et al. (1998) Circulation 97:1708). MMP
inhibitors prevent metastasis of mammary carcinoma and experimental tumors in rat, and Lewis lung carcinoma, hemangioma, and human ovarian carcinoma xenografts in mice (Eccles, S.A. et al. (1996) Cancer Res. 56:2815; Anderson et al. (1996) Cancer Res. 56:715-718; Volpert, O.V. et al. (1996) J. Clin.
Invest. 98:671; Taraboletti, G. et al. (1995) J. NCI 87:293; Davies, B. et al.
(1993) Cancer Res.
53:2087). MMPs may be active in Alzheimer's disease. A number of MMPs are implicated in multiple sclerosis, and administration of MMP inhibitors can relieve some of its symptoms (reviewed in Yong, supra).
Another family of metalloproteases is the ADAMS, for A Disintegrin and Metalloprotease Domain, which they share with their close relatives the adamalysins, snake venom metalloproteases (SVMPs). ADAMS combine features of both cell surface adhesion molecules and proteases, containing a prodomain, a protease domain, a disintegrin domain, a cysteine rich domain, an epidermal growth factor repeat, a transmembrane domain, and a cytoplasmic tail. The first three domains listed above are also found in the SVMPs. The ADAMs possess four potential functions:
proteolysis, adhesion, signaling and fusion. The ADAMs share the metzincin zinc binding sequence and are inhibited by some MMP antagonists such as TllVIP-1.
ADAMs are implicated in such processes as sperm-egg binding and fusion, myoblast fusion, and protein-ectodomain processing or shedding of cytokines, cytokine receptors, adhesion proteins and other extracellular protein domains (Schlondorff, J. and C.P. Blobel (1999) J. Cell. Sci.
112:3603-3617). The Kuzbanian protein cleaves a substrate in the NOTCH pathway (possibly NOTCH itself), activating the program for lateral inhibition in Droso~hila neural development. Two ADAMS, TACE (ADAM 17) and ADAM 10, are proposed to have analogous roles in the processing of amyloid precursor protein in the brain (Schlondorff and Blobel, supra).
TALE has also been identified as the TNF activating enzyme (Black, R.A. et al. (1997) Nature 385:729). TNF is a pleiotropic cytokine that is important in mobilizing host defenses in response to infection or trauma, but can cause severe damage in excess and is often overproduced in autoimmune disease. TACE
cleaves membrane-bound pro-TNF to release a soluble form. Other ADAMs may be involved in a similar type of processing of other membrane-bound molecules.
The ADAMTS sub-family has all of the features of ADAM family metalloproteases and contain an additional thrombospondin domain (TS). The prototypic ADAMTS was identified in mouse, found to be expressed in heart and kidney and upregulated by proinflaxnrnatory stimuli (Kuno, K. et al. (1997) J. Biol. Chem. 272:556-562). To date eleven members are recognized by the Human Genome Organization (HUGO;
http://www.gene.ucl.ac.uk/users/hesterladamts.html#Approved).
Members of this family have the ability to degrade aggrecan, a high molecular weight proteoglycan which provides cartilage with important mechanical properties including compressibility, and which is lost during the development of arthritis. Enzymes which degrade aggrecan are thus considered attractive targets to prevent and slow the degradation of articular cartilage (See, e.g., Tortorella, M.D.
(1999) Science 284:1664; Abbaszade, I. (1999) J. Biol. Chem. 274:23443). Other members are reported to have antiangiogenic potential (Kuno et al., supra) and/or procollagen processing (Colige, A. et al. (1997) Proc. Natl. Acad. Sei. USA 94:2374).
Protease inhibitors Protease inhibitors and other regulators of protease activity control the activity and effects of proteases. Protease inhibitors have been shown to control pathogenesis in animal models of proteolytic disorders (Murphy, G. (1991) Agents Actions Suppl. 35:69-76). Low levels of the cystatins, low molecular weight inhibitors of the cysteine proteases, correlate with malignant progression of tumors (Catkins, C. et al. (1995) Biol. Biochem. Hoppe Seyler 376:71-80). Serpins are inhibitors of nnammalian plasma serine proteases. Many serpins serve to regulate the blood clotting cascade and/or the complement cascade in mammals. Sp32 is a positive regulator of the mammalian acrosomal protease, acrosin, that binds the proenzyme, proaerosin, and thereby aides in packaging the enzyme into the acrosomal matrix (Baba, T. et al. (1994) J. Biol. Chem.
269:10133-10140). The Kunitz family of serine protease inhibitors are characterized by one or more "Kunitz domains"
containing a series of cysteine residues that are regularly spaced over approximately 50 amino acid residues and form three intrachain disulfide bonds. Members of this family include aprotinin, tissue factor pathway inhibitor (TFPI-1 and TFPI-2), inter-oc-trypsin inhibitor, and bikunin. (Manor, C.W. et al. (1997) J. Biol. Chem. 272:12202-12208.) Members of this family are potent inhibitors (in the nanomolar range) against serine proteases such as kallikrein and plasmin.
Aprotinin has clinical utility in reduction of perioperative blood loss.

The discovery of new proteases, and the polynucleotides encoding them, satisfies a need in the art by providing new compositions which are useful in the diagnosis, prevention, and treatment of gastrointestinal, cardiovascular, autoimmune/inflammatory, cell proliferative, developmental, epithelial, neurological, and reproductive disorders, and in the assessment of the effects of exogenous compounds on the expression of nucleic acid and amino acid sequences of proteases.
SUMMARY OF THE INVENTION
The invention features purified polypeptides, proteases, referred to collectively as "PRTS"
and individually as "PRTS-l," "PRTS-2," "PRTS-3," "PRTS-4," "PRTS-5," "PRTS-6," "PRTS-7,"
"PRTS-8," "PRTS-9," "PRTS-10," "PRTS-11," "PRTS-12," "PRTS-13," "PRTS-14," and "PRTS-15." In one aspect, the invention provides an isolated polypeptide selected from the group consisting of a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ll~
N0:1-15, b) a polypeptide comprising a naturally occurring amino acid sequence at least 90%
identical to an amino acid sequence selected from the group consisting of SEQ
ID NO:1-15, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID N0:1-15, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1-15. In one alternative, the invention provides an isolated polypeptide comprising the amino acid sequence of SEQ ID NO:1-15.
The invention further provides an isolated polynucleotide encoding a polypeptide selected from the group consisting of a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ~ NO:1-15, b) a polypeptide comprising a naturally occurring amino acid sequence at least 90% identical to an amino acid sequence selected from the group consisting of SEQ
ID NO:1-15, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1-15, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ m N0:1-15.
In one alternative, the polynucleotide encodes a polypeptide selected from the group consisting of SEQ ll~ NO:1-15. In another alternative, the polynucleotide is selected from the group consisting of SEQ m N0:16-30.
Additionally, the invention provides a recombinant polynucleotide comprising a promoter sequence operably linked to a polynucleotide encoding a polypeptide selected from the group consisting of a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:1-15, b) a polypeptide comprising a naturally occurring amino acid sequence at least 90% identical to an amino acid sequence selected from the group consisting of SEQ m NO:1-15, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ )D NO:1-15, and d) an immunogenic fragment of a polypeptide having an amino t0 l0 acid sequence selected from the group consisting of SEQ ID NO:1-15. In one alternative, the invention provides a cell transformed with the recombinant polynucleotide. In another alternative, the invention provides a transgenic organism comprising the recombinant polynucleotide.
The invention also provides a method for producing a polypeptide selected from the group consisting of a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID N0:1-15, b) a polypeptide comprising a naturally occurring amino acid sequence at least 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NO: l-15, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ll~ NO:1-15, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ll~ NO:1-15. The method comprises a) culturing a cell under conditions suitable for expression of the polypeptide, wherein said cell is transformed with a recombinant polynucleotide comprising a promoter sequence operably linked to a polynucleotide encoding the polypeptide, and b) recovering the polypeptide so expressed.
Additionally, the invention provides an isolated antibody which specifically binds to a polypeptide selected from the group consisting of a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID N0:1-15, b) a polypeptide comprising a naturally occurring amino acid sequence at least 90% identical to an amino acid sequence selected from the group consisting of SEQ 1D N0:1-15, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID
NO:1-15, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1-15.
The invention further provides an isolated polynucleotide selected from the group consisting of a) a polynucleotide comprising a polynucleotide sequence selected from the group consisting of SEQ >D N0:16-30, b) a polynucleotide comprising a naturally occurring polynucleotide sequence at least 90% identical to a polynucleotide sequence selected from the group consisting of SEQ ~
N0:16-30, c) a polynucleotide complementary to the polynucleotide of a), d) a polynucleotide complementary to the polynucleotide of b), and e) an RNA equivalent of a)-d).
In one alternative, the polynucleotide comprises at least 60 contiguous nucleotides.
Additionally, the invention provides a method for detecting a target polynucleotide in a sample, said target polynucleotide having a sequence of a polynucleotide selected from the group consisting of a) a polynucleotide comprising a polynucleotide sequence selected from the group consisting of SEQ ID N0:16-30, b) a polynucleotide comprising a naturally occurring polynucleotide sequence at least 90% identical to a polynucleotide sequence selected from the group consisting of SEQ m NO:16-30, c) a polynucleotide complementary to the polynucleotide of a), d) a polynucleotide complementary to the polynucleotide of b), and e) an RNA
equivalent of a)-d). The method comprises a) hybridizing the sample with a probe comprising at least 20 contiguous nucleotides comprising a sequence complementary to said target polynucleotide in the sample, and which probe specifically hybridizes to said target polynucleotide, under conditions whereby a hybridization complex is formed between said probe and said target polynucleotide or fragments thereof, and b) detecting the presence or absence of said hybridization complex, and optionally, if present, the amount thereof. In one alternative, the probe comprises at least 60 contiguous nucleotides.
The invention further provides a method for detecting a target polynucleotide in a sample, said target polynucleotide having a sequence of a polynucleotide selected from the group consisting of a) a polynucleotide comprising a polynucleotide sequence selected from the group consisting of SEQ m N0:16-30, b) a polynucleotide comprising a naturally occurring polynucleotide sequence at least 90% identical to a polynucleotide sequence selected from the group consisting of SEQ >D
N0:16-30, c) a polynucleotide complementary to the polynucleotide of a), d) a polynucleotide complementary to the polynucleotide of b), and e) an RNA equivalent of a)-d).
The method comprises a) amplifying said target polynucleotide or fragment thereof using polymerase chain reaction amplification, and b) detecting the presence or absence of said amplified target polynucleotide or fragment thereof, and, optionally, if present, the amount thereof.
The invention further provides a composition comprising an effective amount of a polypeptide selected from the group consisting of a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:1-15, b) a polypeptide comprising a naturally occurring amino acid sequence at least 90% identical to an amino acid sequence selected from the group consisting of SEQ m N0:1-15, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ m NO:1-15, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ )~ NO:1-15, and a pharmaceutically acceptable excipient. In one embodiment, the composition comprises an amino acid sequence selected from the group consisting of SEQ >I7 NO: l-15. The invention additionally provides a method of treating a disease or condition associated with decreased expression of functional PRTS, comprising administering to a patient in need of such treatment the composition.
The invention also provides a method for screening a compound for effectiveness as an agonist of a polypeptide selected from the group consisting of a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ m N0:1-15, b) a polypeptide comprising a naturally occurring amino acid sequence at least 90% identical to an amino acid sequence selected from the group consisting of SEQ m NO:1-15, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ m NO:1-15, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1-15. The method comprises a) exposing a sample comprising the polypeptide to a compound, and b) detecting agonist activity in the sample. In one alternative, the invention provides a composition comprising an agonist compound identified by the method and a pharmaceutically acceptable excipient. In another alternative, the invention provides a method of treating a disease or condition associated with decreased expression of functional PRTS, comprising administering to a patient in need of such treatment the composition.
Additionally, the invention provides a method for screening a compound for effectiveness as an antagonist of a polypeptide selected from the group consisting of a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID N0:1-15, b) a polypeptide comprising a naturally occurring amino acid sequence at least 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NO:1-15, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: l-15, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1-15. The method comprises a) exposing a sample comprising the polypeptide to a compound, and b) detecting antagonist activity in the sample.
In one alternative, the invention provides a composition comprising an antagonist compound identified by the method and a pharmaceutically acceptable excipient. In another alternative, the invention provides a method of treating a disease or condition associated with overexpression of functional PRTS, comprising administering to a patient in need of such treatment the composition.
The invention further provides a method of screening for a compound that specifically binds to a polypeptide selected from the group consisting of a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:1-15, b) a polypeptide comprising a naturally occurnng amino acid sequence at least 90% identical to an amino acid sequence selected from the group consisting of SEQ ~ NO:1-15, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ )D
N0:1-15, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1-15. The method comprises a) combining the polypeptide with at least one test compound under suitable conditions, and b) detecting binding of the polypeptide to the test compound, thereby identifying a compound that specifically binds to the polypeptide.
The invention further provides a method of screening for a compound that modulates the activity of a polypeptide selected from the group consisting of a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:1-15, b) a polypeptide comprising a naturally occurring amino acid sequence at least 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NO:1-15, c) a biologically active fragment of a polypeptide ~.3 having an amino acid sequence selected from the group consisting of SEQ >D
NO:1-15, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID N0:1-15. The method comprises a) combining the polypeptide with at least one test compound under conditions permissive for the activity of the polypeptide, b) assessing the activity of the polypeptide in the presence of the test compound, and c) comparing the activity of the polypeptide in the presence of the test compound with the activity of the polypeptide in the absence of the test compound, wherein a change in the activity of the polypeptide in the presence of the test compound is indicative of a compound that modulates the activity of the polypeptide.
The invention further provides a method for screening a compound for effectiveness in altering expression of a target polynucleotide, wherein said target polynucleotide comprises a polynucleotide sequence selected from the group consisting of SEQ ID N0:16-30, the method comprising a) exposing a sample comprising the target polynucleotide to a compound, and b) detecting altered expression of the target polynucleotide.
The invention further provides a method for assessing toxicity of a test compound, said method comprising a) treating a biological sample containing nucleic acids with the test compound;
b) hybridizing the nucleic acids of the treated biological sample with a probe comprising at least 20 contiguous nucleotides of a polynucleotide selected from the group consisting of i) a polynucleotide comprising a polynucleotide sequence selected from the group consisting of SEQ
)D N0:16-30, ii) a polynucleotide comprising a naturally occurring polynucleotide sequence at least 90% identical to a polynucleotide sequence selected from the group consisting of SEQ m N0:16-30, iii) a polynucleotide having a sequence complementary to i), iv) a polynucleotide complementary to the polynucleotide of ii), and v) an RNA equivalent of i)-iv). Hybridization occurs under conditions whereby a specific hybridization complex is formed between said probe and a target polynucleotide in the biological sample, said target polynucleotide selected from the group consisting of i) a polynucleotide comprising a polynucleotide sequence selected from the group consisting of SEQ ID
N0:16-30, ii) a polynucleotide comprising a naturally occurring polynucleotide sequence at least 90% identical to a polynucleotide sequence selected from the group consisting of SEQ ID N0:16-30, iii) a polynucleotide complementary to the polynucleotide of i), iv) a polynucleotide complementary to the polynucleotide of ii), and v) an RNA equivalent of i)-iv).
Alternatively, the target polynucleotide comprises a fragment of a polynucleotide sequence selected from the group consisting of i)-v) above; c) quantifying the amount of hybridization complex; and d) comparing the amount of hybridization complex in the treated biological sample with the amount of hybridization complex in an untreated biological sample, wherein a difference in the amount of hybridization complex in the treated biological sample is indicative of toxicity of the test compound.

BRIEF DESCRIPTION OF THE TABLES
Table 1 summarizes the nomenclature for the full length polynucleotide and polypeptide sequences of the presentinvention.
Table 2 shows the GenBank identification number and annotation of the nearest GenBank homolog for polypeptides of the invention. The probability scores for the matches between each polypeptide and its homalog(s) are also shown.
Table 3 shows structural features of polypeptide sequences of the invention, including predicted motifs and domains, along with the methods, algorithms, and searchable databases used for analysis of the polypeptides.
Table 4 lists the cDNA and/or genomic DNA fragments which were used to assemble polynucleotide sequences of the invention, along with selected fragments of the polynucleotide sequences.
Table 5 shows the representative cDNA library for polynucleotides of the invention.
Table 6 provides an appendix which describes the tissues and vectors used for construction of the cDNA libraries shown in Table 5.
Table 7 shows the tools, programs, and algorithms used to analyze the polynucleotides and polypeptides of the invention, along with applicable descriptions, references, and threshold parameters.
DESCRIPTION OF THE INVENTION
Before the present proteins, nucleotide sequences, and methods are described, it is understood that this invention is not limited to the particular machines, materials and methods described, as these may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention which will be limited only by the appended claims.
It must be noted that as used herein and in the appended claims, the singular forms "a," "an,"
and "the" include plural reference unless the context clearly dictates otherwise. Thus, for example, a reference to "a host cell" includes a plurality of such host cells, and a reference to "an antibody" is a reference to one or more antibodies and equivalents thereof known to those skilled in the art, and so forth.
Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art to which this invention belongs.
Although any machines, materials, and methods similar or equivalent to those described herein can be used to practice or test the present invention, the preferred machines, materials and methods are now described. All publications mentioned herein are cited for the purpose of describing and disclosing l5 the cell lines, protocols, reagents and vectors which are reported in the publications and which might be used in connection with the invention. Nothing herein is to be construed as an admission that the invention is not entitled to antedate such disclosure by virtue of prior invention.
DEFINITIONS
"PRTS" refers to the amino acid sequences of substantially purified PRTS
obtained from any species, particularly a mammalian species, including bovine, ovine, porcine, marine, equine, and human, and from any source, whether natural, synthetic, semi-synthetic, or recombinant.
The term "agonist" refers to a molecule which intensifies or mimics the biological activity of PRTS. Agonists may include proteins, nucleic acids, carbohydrates, small molecules, or any other compound or composition which modulates the activity of PRTS either by directly interacting with PRTS or by acting on components of the biological pathway in which PRTS
participates.
An "allelic variant" is an alternative form of the gene encoding PRTS. Allelic variants may result from at least one mutation in the nucleic acid sequence and may result in altered mRNAs or in polypeptides whose structure or function may or may not be altered. A gene may have none, one, or many allelic variants of its naturally occurring form. Common mutational changes which give rise to allelic variants are generally ascribed to natural deletions, additions, or substitutions of nucleotides.
Each of these types of changes may occur alone, or in combination with the others, one or more times in a given sequence.
"Altered" nucleic acid sequences encoding PRTS include those sequences with deletions, insertions, or substitutions of different nucleotides, resulting in a polypeptide the same as PRTS or a polypeptide with at least one functional characteristic of PRTS. Included within this definition are polymorphisms which may or may not be readily detectable using a particular oligonucleotide probe of the polynucleotide encoding PRTS, and improper or unexpected hybridization to allelic variants, with a locus other than the normal chromosomal locus for the polynucleotide sequence encoding PRTS. The encoded protein may also be "altered," and may contain deletions, insertions, or substitutions of amino acid residues which produce a silent change and result in a functionally equivalent PRTS. 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 or immunological activity of PRTS is retained. For example, negatively charged amino acids may include aspartic acid and glutamic acid, and positively charged amino acids may include lysine and arginine. Amino acids with uncharged polar side chains having similar hydrophilicity values may include: asparagine and glutamine; and serine and threonine.
Amino acids with uncharged side chains having similar hydrophilicity values may include: leucine, isoleucine, and valine; glycine and alanine; and phenylalanine and tyrosine.

The terms "amino acid" and'"amino acid sequence" refer to an oligopeptide, peptide, polypeptide, or protein sequence, or a fragment of any of these, and to naturally occurring or synthetic molecules. Where "amino acid sequence" is recited to refer to a sequence of a naturally occurring protein molecule, "amino acid sequence" and like terms are not meant to limit the amino acid sequence to the complete native amino acid sequence associated with the recited protein molecule.
"Amplification" relates to the production of additional copies of a nucleic acid sequence.
Amplification is generally carried out using polymerase chain reaction (PCR) technologies well known in the art.
The term "antagonist" refers to a molecule which inhibits or attenuates the biological activity of PRTS. Antagonists may include proteins such as antibodies, nucleic acids, carbohydrates, small molecules, or any other compound or composition which modulates the activity of PRTS either by directly interacting with PRTS or by acting on components of the biological pathway in which PRTS
participates.
The term "antibody" refers to intact immunoglobulin molecules as well as to fragments thereof, such as Fab, F(ab')Z, and Fv fragments, which are capable of binding an epitopic determinant.
Antibodies that bind PRTS polypeptides can be prepared using intact polypeptides or using fragments containing small peptides of interest as the immunizing antigen. The polypeptide or oligopeptide used to immunize an animal (e.g., a mouse, a rat, or a rabbit) can be derived from the translation of RNA, or synthesized chemically, and can be conjugated to a carrier protein if desired. Commonly used carriers that are chemically coupled to peptides include bovine serum albumin, thyroglobulin, and keyhole limpet hemocyanin (KI,H). The coupled peptide is then used to immunize the animal.
The term "antigenic determinant" refers to that region of a molecule (i.e., an epitope) that makes contact with a particular antibody. When a protein or a fragment of a protein is used to immunize a host animal, numerous regions of the protein may induce the production of antibodies which bind specifically to antigenic determinants (particular regions or three-dimensional structures on the protein). An antigenic determinant may compete with the intact antigen (i.e., the immunogen used to elicit the immune response) for binding to an antibody.
The term "aptamer" refers to a nucleic acid or oligonucleotide molecule that binds to a specific molecular target. Aptamers are derived from an in vitro evolutionary process (e.g., SELEX
(Systematic Evolution of Ligands by EXponential Enrichment), described in U.S.
Patent No.
5,270,163), which selects for target-specific aptamer sequences from large combinatorial libraries.
Aptamer compositions may be double-stranded or single-stranded, and may include deoxyribonucleotides, ribonucleotides, nucleotide derivatives, or other nucleotide-like molecules.
The nucleotide components of an aptamer may have modified sugar groups (e.g., the 2'-OH group of a ribonucleotide may be replaced by 2'-F or 2'-NHZ), which may improve a desired property, e.g., resistance to nucleases or longer lifetime in blood. Aptamers may be conjugated to other molecules, e.g., a high molecular weight carrier to slow clearance of the aptamer from the circulatory system.
Aptamers may be specifically cross-linked to their cognate ligands, e.g., by photo-activation of a cross-linker. (See, e.g., Brody, E.N. and L. Gold (2000) J. Biotechnol. 74:5-13.) The term "intramer" refers to an aptamer which is expressed in vivo. For example, a vaccinia virus-based RNA expression system has been used to express specific RNA
aptamers at high levels in the cytoplasm of leukocytes (Blind, M. et al. (1999) Proc. Natl Acad. Sci. USA
96:3606-3610).
The term "spiegelmer" refers to an aptamer which includes L-DNA, L-RNA, or other left-handed nucleotide derivatives or nucleotide-like molecules. Aptamers containing left-handed nucleotides are resistant to degradation by naturally occurring enzymes, which normally act on substrates containing right-handed nucleotides.
The term "antisense" refers to any composition capable of base-pairing with the "sense"
(coding) strand of a specific nucleic acid sequence. Antisense compositions may include DNA;
RNA; peptide nucleic acid (PNA); oligonucleotides having modified backbone linkages such as phosphorothioates, methylphosphonates, or benzylphosphonates; oligonucleotides having modified sugar groups such as 2'-methoxyethyl sugars or 2'-methoxyethoxy sugars; or oligonucleotides having modified bases such as 5-methyl cytosine, 2'-deoxyuracil, or 7-deaza-2'-deoxyguanosine. Antisense molecules may be produced by any method including chemical synthesis or transcription. Once introduced into a cell, the complementary antisense molecule base-pairs with a naturally occurring nucleic acid sequence produced by the cell to form duplexes which block either transcription or translation. The designation "negative" or "minus" can refer to the antisense strand, and the designation "positive" or "plus" can refer to the sense strand of a reference DNA molecule.
The term "biologically active" refers to a protein having structural, regulatory, or biochemical functions of a naturally occurring molecule. Likewise, "immunologically active" or "immunogenic"
refers to the capability of the natural, recombinant, or synthetic PRTS, or of any oligopeptide thereof, to induce a specific immune response in appropriate animals or cells and to bind with specific antibodies.
"Complementary" describes the relationship between two single-stranded nucleic acid sequences that anneal by base-pairing. For example, 5'-AGT-3' pairs with its complement, 3'-TCA-5'.
A "composition comprising a given polynucleotide sequence" and a "composition comprising a given amino acid sequence" refer broadly to any composition containing the given polynucleotide or amino acid sequence. The composition may comprise a dry formulation or an aqueous solution.
Compositions comprising polynucleotide sequences encoding PRTS or fragments of PRTS may be employed as hybridization probes. The probes may be stored in freeze-dried form and may be associated with a stabilizing agent such as a carbohydrate. In hybridizations, the probe may be deployed in an aqueous solution containing salts (e.g., NaCI), detergents (e.g., sodium dodecyl sulfate; SDS), and other components (e.g., Denhardt's solution, dry milk, salmon sperm DNA, etc.).
"Consensus sequence" refers to a nucleic acid sequence which has been subjected to repeated DNA sequence analysis to resolve uncalled bases, extended using the XL-PCR kit (Applied Biosystems, Foster City CA) in the 5' and/or the 3' direction, and resequenced, or wluch has been assembled from one or more overlapping cDNA, EST, or genomic DNA fragments using a computer program for fragment assembly, such as the GELVIEW fragment assembly system (GCG, Madison Wn or Phrap (University of Washington, Seattle WA). Some sequences have been both extended and assembled to produce the consensus sequence.
"Conservative amino acid substitutions" are those substitutions that are predicted to least interfere with the properties of the original protein, i.e., the structure and especially the function of the protein is conserved and not significantly changed by such substitutions.
The table below shows amino acids which may be substituted for an original amino acid in a protein and which are regarded as conservative amino acid substitutions.
Original Residue Conservative Substitution Ala Gly, Ser Arg His, Lys Asn Asp, Gln, His Asp ~ Asn, Glu Cys Ala, Ser Gln Asn, Glu, His Glu Asp, Gln, His Gly Ala His Asn, Arg, Gln, Glu Ile Leu, Val Leu Ile, Val Lys Arg, Gln, Glu Met Leu, Ile Phe His, Met, Leu, Trp, Tyr Ser Cys, Thr Thr Ser, Val Trp Phe, Tyr Tyr His, Phe, Trp Val lle, Leu, Thr Conservative amino acid substitutions generally maintain (a) the structure of the polypeptide backbone in the area of the substitution, for example, as a beta sheet or alpha helical conformation, (b) the charge or hydrophobicity of the molecule at the site of the substitution, and/or (c) the bulk of the side chain.
A "deletion" refers to a change in the amino acid or nucleotide sequence that results in the absence of one or more amino acid residues or nucleotides.

The term "derivative" refers to a chemically modified polynucleotide or polypeptide.
Chemical modifications of a polynucleotide can include, for example, replacement of hydrogen by an alkyl, acyl, hydroxyl, or amino group. A derivative polynucleotide encodes a polypeptide which retains at least one biological or immunological function of the natural molecule. A derivative polypeptide is one modified by glycosylation, pegylation, or any similar process that retains at least one biological or immunological function of the polypeptide from which it was derived.
A "detectable label" refers to a reporter molecule or enzyme that is capable of generating a measurable signal and is covalently or noncovalently joined to a polynucleotide or polypeptide.
"Differential expression" refers to increased or upregulated; or decreased, downregulated, or absent gene or protein expression, determined by comparing at least two different samples. Such comparisons may be carried out between, for example, a treated and an untreated sample, or a diseased and a normal sample.
"Exon shuffling" refers to the recombination of different coding regions (exons). Since an exon may represent a structural or functional domain of the encoded protein, new proteins may be assembled through the novel reassortment of stable substructures, thus allowing acceleration of the evolution of new protein functions.
A "fragment" is a unique portion of PRTS or the polynucleotide encoding PRTS
which is identical in sequence to but shorter in length than the parent sequence. A
fragment may comprise up to the entire length of the defined sequence, minus one nucleotide/amino acid residue. For example, a fragment may comprise from 5 to 1000 contiguous nucleotides or amino acid residues. A fragment used as a probe, primer, antigen, therapeutic molecule, or for other purposes, may be at least 5, 10, 15, 16, 20, 25, 30, 40, 50, 60, 75, 100, 150, 250 or at least 500 contiguous nucleotides or amino acid residues in length. Fragments may be preferentially selected from certain regions of a molecule. For example, a polypeptide fragment may comprise a certain length of contiguous amino acids selected from the first 250 or 500 amino acids (or first 25% or 50%) of a polypeptide as shown in a certain defined sequence. Clearly these lengths are exemplary, and any length that is supported by the specification, including the Sequence Listing, tables, and figures, may be encompassed by the present embodiments.
A fragment of SEQ m N0:16-30 comprises a region of unique polynucleotide sequence that specifically identifies SEQ ID N0:16-30, for example, as distinct from any other sequence in the genome from which the fragment was obtained. A fragment of SEQ ID N0:16-30 is useful, for example, in hybridization and amplification technologies and in analogous methods that distinguish SEQ ID N0:16-30 from related polynucleotide sequences. The precise length of a fragment of SEQ
m N0:16-30 and the region of SEQ m NO:16-30 to which the fragment corresponds are routinely determinable by one of ordinary skill in the art based on the intended purpose for the fragment.

A fragment of SEQ ID NO:1-15 is encoded by a fragment of SEQ ID N0:16-30. A
fragment of SEQ ID NO:1-15 comprises a region of unique amino acid sequence that specifically identifies SEQ ID NO:1-15. For example, a fragment of SEQ ID NO:1-15 is useful as an immunogenic peptide for the development of antibodies that specifically recognize SEQ ID NO:1-15.
The precise length of a fragment of SEQ ID NO: l-15 and the region of SEQ ID N0:1-15 to which the fragment corresponds are routinely determinable by one of ordinary skill in the art based on the intended purpose for the fragment.
A "full length" polynucleotide sequence is one containing at least a translation initiation codon (e.g., methionine) followed by an open reading frame and a translation termination codon. A
"full length" polynucleotide sequence encodes a "full length" polypeptide sequence.
"Homology" refers to sequence similarity or, interchangeably, sequence identity, between two or more polynucleotide sequences or two or more polypeptide sequences.
The terms "percent identity" and "% identity," as applied to polynucleotide sequences, refer to the percentage of residue matches between at least two polynucleotide sequences aligned using a standardized algorithm. Such an algorithm may insert, in a standardized and reproducible way, gaps in the sequences being compared in order to optimize alignment between two sequences, and therefore achieve a more meaningful comparison of the two sequences.
Percent identity between polynucleotide sequences may be determined using the default parameters of the CLUSTAL V algorithm as incorporated into the MEGALIGN
version 3.12e sequence alignment program. This program is part of the LASERGENE software package, a suite of molecular biological analysis programs (DNASTAR, Madison WI). CLUSTAL V is described in Higgins, D.G. and P.M. Sharp (1989) CABIOS 5:151-153 and in Higgins, D.G. et al. (1992) CABIOS
8:189-191. For pairwise al banments of polynucleotide sequences, the default parameters are set as follows: I~tuple=2, gap penalty=5, window=4, and "diagonals saved"=4. The "weighted" residue weight table is selected as the default. Percent identity is reported by CLUSTAL V as the "percent similarity" between aligned polynucleotide sequences.
Alternatively, a suite of commonly used and freely available sequence comparison algorithms is provided by the National Center for Biotechnology Information (NCBI) Basic Local Alignment Search Tool (BLAST) (Altschul, S.F. et al. (1990) J. Mol. Biol. 215:403-410), which is available from several sources, including the NCBI, Bethesda, MD, and on the Internet at http://www.ncbi.nlm.nih.gov/BLAST/. The BLAST software suite includes various sequence analysis programs including "blastn," that is used to align a known polynucleotide sequence with other polynucleotide sequences from a variety of databases. Also available is a tool called "BLAST 2 Sequences" that is used for direct pairwise comparison of two nucleotide sequences. "BLAST 2 Sequences" can be accessed and used interactively at http://www.ncbi.nlm.nih.gov/gorf/bl2.html.

The "BLAST 2 Sequences" tool can be used for both blastn and blastp (discussed below). BLAST
programs are commonly used with gap and other parameters set to default settings. For example, to compare two nucleotide sequences, one may use blastn with the "BLAST 2 Sequences" tool Version 2Ø12 (April-21-2000) set at default parameters. Such default parameters may be, for example:
Matrix: BLOSUM62 Reward for match: 1 Penalty for mismatch: -2 Open Gap: S and Extension Gap: 2 penalties Gap x drop-off.' S0 Expect: l0 Word Size: 11 Filter: orz Percent identity may be measured over the length of an entire defined sequence, for example, as defined by a particular SEQ ID number, or may be measured over a shorter length, for example, over the length of a fragment taken from a larger, defined sequence, for instance, a fragment of at least 20, at least 30, at least 40, at least 50, at least 70, at least 100, or at least 200 contiguous nucleotides. Such lengths are exemplary only, and it is understood that any fragment length supported by the sequences shown herein, in the tables, figures, or Sequence Listing, may be used to describe a length over which percentage identity may be measured.
Nucleic acid sequences that do not show a high degree of identity may nevertheless encode similar amino acid sequences due to the degeneracy of the genetic code. It is understood that changes in a nucleic acid sequence can be made using this degeneracy to produce multiple nucleic acid sequences that all encode substantially the same protein.
The phrases "percent identity" and "°lo identity," as applied to polypeptide sequences, refer to the percentage of residue matches between at least two polypeptide sequences aligned using a standardized algorithm. Methods of polypeptide sequence alignment are well-known. Some alignment methods take into account conservative amino acid substitutions.
Such conservative substitutions, explained in more detail above, generally preserve the charge and hydrophobicity at the site of substitution, thus preserving the structure (and therefore function) of the polypeptide.
Percent identity between polypeptide sequences may be determined using the default parameters of the CLUSTAL V algorithm as incorporated into the MEGALIGN
version 3.12e sequence alignment program (described and referenced above). For pairwise alignments of polypeptide sequences using CLUSTAL V, the default parameters are set as follows: Ktuple=1, gap penalty=3, window=5, and "diagonals saved"=5. The PAM250 matrix is selected as the default residue weight table. As with polynucleotide alignments, the percent identity is reported by CLUSTAL V as the "percent similarity" between aligned polypeptide sequence pairs.
Alternatively the NCBI BLAST software suite may be used. For example, for a pairwise comparison of two polypeptide sequences, one may use the "BLAST 2 Sequences"
tool Version 2Ø12 (April-21-2000) with blastp set at default parameters. Such default parameters may be, for example:
Matrix: BLOSUM62 Open Gap: 11 and Exterzsiorz Gap: 1 penalties Gap x drop-off.' SO
Expect: l0 Word Size: 3 Filter: orz Percent identity may be measured over the length of an entire defined polypeptide sequence, for example, as defined by a particular SEQ ID number, or may be measured over a shorter length, for example, over the length of a fragment taken from a larger, defined polypeptide sequence, for instance, a fragment of at least 15, at least 20, at least 30, at least 40, at least 50, at least 70 or at least 150 contiguous residues. Such lengths are exemplary only, and it is understood that any fragment length supported by the sequences shown herein, in the tables, figures or Sequence Listing, may be used to describe a length over which percentage identity may be measured.
"Human artificial chromosomes" (HACs) are linear microchromosomes which may contain DNA sequences of about 6 kb to 10 Mb in size and which contain all of the elements required for chromosome replication, segregation and maintenance.
The term "humanized antibody" refers to an antibody molecule in which the amino acid sequence in the non-antigen binding regions has been altered so that the antibody more closely resembles a human antibody, and still retains its original binding ability.
"Hybridization" refers to the process by which a polynucleotide strand anneals with a complementary strand through base pairing under defined hybridization conditions. Specific hybridization is an indication that two nucleic acid sequences share a high degree of complementarity.
Specific hybridization complexes form under permissive annealing conditions and remain hybridized after the "washing" step(s). The washing steps) is particularly important in determining the stringency of the hybridization process, with more stringent conditions allowing less non-specific binding, i.e., binding between pairs of nucleic acid strands that are not perfectly matched. Permissive conditions for annealing of nucleic acid sequences are routinely determinable by one of ordinary skill in the art and may be consistent among hybridization experiments, whereas wash conditions may be varied among experiments to achieve the desired stringency, and therefore hybridization specificity.

Permissive annealing conditions occur, for example, at 68°C in the presence of about 6 x SSC, about 1% (w/v) SDS, and about 100 ~tg/ml sheared, denatured salmon sperm DNA.
Generally, stringency of hybridization is expressed, in part, with reference to the temperature under which the wash step is carried out. Such wash temperatures are typically selected to be about 5°C to 20°C lower than the thermal melting point (Tm) for the specific sequence at a defined ionic strength and pH. The Tm is the temperature (under defined ionic strength and pH) at which 50% of the target sequence hybridizes to a perfectly matched probe. An equation for calculating Tm and conditions for nucleic acid hybridization are well known and can be found in Sambrook, J. et al.
(1989) Molecular Clonin~Y A Laboratory Manual, 2"d ed., vol. 1-3, Cold Spring Harbor Press, Plainview NY; specifically see volume 2, chapter 9.
High stringency conditions for hybridization between polynucleotides of the present invention include wash conditions of 68°C in the presence of about 0.2 x SSC and about 0.1% SDS, for 1 hour. Alternatively, temperatures of about 65°C, 60°C, 55°C, or 42°C may be used. SSC
concentration may be varied from about 0.1 to 2 x SSC, with SDS being present at about 0.1%.
Typically, blocking reagents are used to block non-specific hybridization.
Such blocking reagents include, for instance, sheared and denatured salmon sperm DNA at about 100-200 ,ug/ml. Organic solvent, such as formamide at a concentration of about 35-50% v/v, may also be used under particular circumstances, such as for RNA:DNA hybridizations. Useful variations on these wash conditions will be readily apparent to those of ordinary skill in the art. Hybridization, particularly under high stringency conditions, may be suggestive of evolutionary similarity between the nucleotides. Such similarity is strongly indicative of a similar role for the nucleotides and their encoded polypeptides.
The term "hybridization complex" refers to a complex formed between two nucleic acid sequences by virtue of the formation of hydrogen bonds between complementary bases. A
hybridization complex may be formed in solution (e.g., Cot or Rot analysis) or formed between one nucleic acid sequence present in solution and another nucleic acid sequence immobilized on a solid support (e.g., paper, membranes, filters, chips, pins or glass slides, or any other appropriate substrate to which cells or their nucleic acids have been fixed).
The words "insertion" and "addition" refer to changes in an amino acid or nucleotide sequence resulting in the addition of one or more amind acid residues or nucleotides, respectively.
"Immune response" can refer to conditions associated with inflammation, trauma, immune disorders, or infectious or genetic disease, etc. These conditions can be characterized by expression of various factors, e.g., cytokines, chemokines, and other signaling molecules, which may affect cellular and systemic defense systems.
An "immunogenic fragment" is a polypeptide or oligopeptide fragment of PRTS
which is capable of eliciting an immune response when introduced into a living organism, for example, a mammal. The term "immunogenic fragment" also includes any polypeptide or oligopeptide fragment of PRTS which is useful in any of the antibody production methods disclosed herein or known in the art.
The term "microarray" refers to an arrangement of a plurality of polynucleotides, polypeptides, or other chemical compounds on a substrate.
The terms "element" and "array element" refer to a polynucleotide, polypeptide, or other chemical compound having a unique and defined position on a microarray.
The term "modulate" refers to a change in the activity of PRTS. For example, modulation may cause an increase or a decrease in protein activity, binding characteristics, or any other biological, functional, or immunological properties of PRTS.
The phrases "nucleic acid" and "nucleic acid sequence" refer to a nucleotide, oligonucleotide, polynucleotide, or any fragment thereof. These phrases also refer to DNA or RNA of genomic or synthetic origin which may be single-stranded or double-stranded and may represent the sense or the antisense strand, to peptide nucleic acid (PNA), or to any DNA-like or RNA-like material.
"Operably linked" refers to the situation in which a first nucleic acid sequence is placed in a functional relationship with a second nucleic acid sequence. For instance, a promoter is operably linked to a coding sequence if the promoter affects the transcription or expression of the coding sequence. Operably linked DNA sequences may be in close proximity or contiguous and, where necessary to join two protein coding regions, in the same reading frame.
"Peptide nucleic acid" (PNA) refers to an antisense molecule or anti-gene agent which comprises an oligonucleotide of at least about 5 nucleotides in length linked to a peptide backbone of amino acid residues ending in lysine. The terminal lysine confers solubility to the composition.
PNAs preferentially bind complementary single stranded DNA or RNA and stop transcript elongation, and may be pegylated to extend their lifespan in the cell.
"Post-translational modification" of an PRTS may involve lipidation, glycosylation, phosphorylation, acetylation, racemization, proteolytic cleavage, and other modifications known in the art. These processes may occur synthetically or biochemically. Biochemical modifications will vary by cell type depending on the enzymatic milieu of PRTS.
"Probe" refers to nucleic acid sequences encoding PRTS, their complements, or fragments thereof, which are used to detect identical, allelic or related nucleic acid sequences. Probes are isolated oligonucleotides or polynucleotides attached to a detectable label or reporter molecule.
Typical labels include radioactive isotopes, ligands, chemiluminescent agents, and enzymes.
"Primers" are short nucleic acids, usually DNA oligonucleotides, which may be annealed to a target polynucleotide by complementary base-pairing. The primer may then be extended along the target DNA strand by a DNA polymerase enzyme. Primer pairs can be used for amplification (and identification) of a nucleic acid sequence, e.g., by the polymerase chain reaction (PCR).
Probes and primers as used in the present invention typically comprise at least 15 contiguous nucleotides of a known sequence. In order to enhance specificity, longer probes and primers may also be employed, such as probes and primers that comprise at least 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, or at least 150 consecutive nucleotides of the disclosed nucleic acid sequences. Probes and primers may be considerably longer than these examples, and it is understood that any length supported by the specification, including the tables, figures, and Sequence Listing, may be used.
Methods for preparing and using probes and primers are described in the references, for example Sambrook, J. et al. (1989) Molecular Cloning: A Laboratory Manual, 2"d ed., vol. 1-3, Cold Spring Harbor Press, Plainview NY; Ausubel, F.M. et al. (1987) Current Protocols in Molecular Biolo~y, Greene Publ. Assoc. & Wiley-Intersciences, New York NY; Innis, M. et al. (1990) PCR
Protocols, A Guide to Methods and Applications, Academic Press, San Diego CA.
PCR primer pairs can be derived from a known sequence, for example, by using computer programs intended for that purpose such as Primer (Version 0.5, 1991, Whitehead Institute for Biomedical Research, Cambridge MA).
Oligonucleotides for use as primers are selected using software known in the art for such purpose. For example, OLIGO 4.06 software is useful for the selection of PCR
primer pairs of up to 100 nucleotides each, and for the analysis of oligonucleotides and larger polynucleotides of up to 5,000 nucleotides from an input polynucleotide sequence of up to 32 kilobases.
Similar primer selection programs have incorporated additional features for expanded capabilities. For example, the PrimOU primer selection program (available to the public from the Genome Center at University of Texas South West Medical Center, Dallas TX) is capable of choosing specific primers from megabase sequences and is thus useful for designing primers on a genome-wide scope. The Primer3 primer selection program (available to the public from the Whitehead InstitutelMTT Center for Genome Research, Cambridge MA) allows the user to input a "mispriming library," in which sequences to avoid as primer binding sites are user-specified. Primer3 is useful, in particular, for the selection of oligonucleotides for microarrays. (The source code for the latter two primer selection programs may also be obtained from their respective sources and modified to meet the user's specific needs.) The PrimeGen program (available to the public from the UK Human Genome Mapping Project Resource Centre, Cambridge UI~) designs primers based on multiple sequence alignments, thereby allowing selection of primers that hybridize to either the most conserved or least conserved regions of aligned nucleic acid sequences. Hence, this program is useful for identification of both unique and conserved oligonucleotides and polynucleotide fragments. The oligonucleotides and polynucleotide fragments identified by any of the above selection methods are useful in hybridization technologies, for example, as PCR or sequencing primers, microarray elements, or specific probes to identify fully or partially complementary polynucleotides in a sample of nucleic acids. Methods of oligonucleotide selection are not limited to those described above.
A "recombinant nucleic acid" is a sequence that is not naturally occurring or has a sequence that is made by an artificial combination of two or more otherwise separated segments of sequence.
This artificial combination is often accomplished by chemical synthesis or, more commonly, by the artificial manipulation of isolated segments of nucleic acids, e.g., by genetic engineering techniques such as those described in Sambrook, supra. The term recombinant includes nucleic acids that have been altered solely by addition, substitution, or deletion of a portion of the nucleic acid. Frequently, a recombinant nucleic acid may include a nucleic acid sequence operably linked to a promoter sequence. Such a recombinant nucleic acid may be part of a vector that is used, for example, to transform a cell.
Alternatively, such recombinant nucleic acids may be part of a viral vector, e.g., based on a vaccinia virus, that could be use to vaccinate a mammal wherein the recombinant nucleic acid is expressed, inducing a protective immunological response in the mammal.
A "regulatory element" refers to a nucleic acid sequence usually derived from untranslated regions of a gene and includes enhancers, promoters, introns, and 5' and 3' untranslated regions (UTRs). Regulatory elements interact with host or viral proteins which control transcription, translation, or RNA stability.
"Reporter molecules" are chemical or biochemical moieties used for labeling a nucleic acid, amino acid, or antibody. Reporter molecules include radionuclides; enzymes;
fluorescent, chemiluminescent, or ehromogenic agents; substrates; cofactors; inhibitors;
magnetic particles; and other moieties known in the art.
An "RNA equivalent," in reference to a DNA sequence, is composed of the same linear sequence of nucleotides as the reference DNA sequence with the exception that all occurrences of the nitrogenous base thymine are replaced with uracil, and the sugar backbone is composed of ribose instead of deoxyribose.
The term "sample" is used in its broadest sense. A sample suspected of containing PRTS, nucleic acids encoding PRTS, or fragments thereof may comprise a bodily fluid;
an extract from a cell, chromosome, organelle, or membrane isolated from a cell; a cell; genomic DNA, RNA, or cDNA, in solution or bound to a substrate; a tissue; a tissue print; etc.
The terms "specific binding" and "specifically binding" refer to that interaction between a protein or peptide and an agonist, an antibody, an antagonist, a small molecule, or any natural or synthetic binding composition. The interaction is dependent upon the presence of a particular structure of the protein, e.g., the antigenic determinant or epitope, recognized by the binding ~7 molecule. For example, if an antibody is specific for epitope "A," the presence of a polypeptide comprising the epitope A, or the presence of free unlabeled A, in a reaction containing free labeled A
and the antibody will reduce the amount of labeled A that binds to the antibody.
The term "substantially purified" refers to nucleic acid or amino acid sequences that are removed from their natural environment and are isolated or separated, and are at least 60% free, preferably at least 75% free, and most preferably at least 90% free from other components with which they are naturally associated.
A "substitution" refers to the replacement of one or more amino acid residues or nucleotides by different amino acid residues or nucleotides, respectively.
"Substrate" refers to any suitable rigid or semi-rigid support including membranes, filters, chips, slides, wafers, fibers, magnetic or nonmagnetic beads, gels, tubing, plates, polymers, microparticles and capillaries. The substrate can have a variety of surface forms, such as wells, trenches, pins, channels and pores, to which polynucleotides or polypeptides are bound.
A "transcript image" or "expression profile" refers to the collective pattern of gene expression by a particular cell type or tissue under given conditions at a given time.
"Transformation" describes a process by which exogenous DNA is introduced into a recipient cell. Transformation may occur under natural or artificial conditions according to various methods well known in the art, and may rely on any known method for the insertion of foreign nucleic acid sequences into a prokaryotic or eukaryotic host Bell. The method for transformation is selected based on the type of host cell being transformed and may include, but is not limited to, bacteriophage or viral infection, electroporation, heat shock, lipofection, and particle bombardment. The term "transformed cells" includes stably transformed cells in which the inserted DNA is capable of replication either as an autonomously replicating plasmid or as part of the host chromosome, as well as transiently transformed cells which express the inserted DNA or RNA for limited periods of time.
A "transgenic organism," as used herein, is any organism, including but not limited to animals and plants, in which one or more of the cells of the organism contains heterologous nucleic acid introduced by way of human intervention, such as by transgenic techniques well known in the art. The nucleic acid is introduced into the cell, directly or indirectly by introduction into a precursor of the cell, by way of deliberate genetic manipulation, such as by microinjection or by infection with a recombinant virus. The term genetic manipulation does not include classical cross-breeding, or in vitro fertilization, but rather is directed to the introduction of a recombinant DNA molecule. The transgenic organisms contemplated in accordance with the present invention include bacteria, cyanobacteria, fungi, plants and animals. The isolated DNA of the present invention can be introduced into the host by methods known in the art, for example infection, transfection, transformation or transconjugation. Techniques for transferring the DNA of the present invention into such organisms are widely known and provided in references such as Sambrook et al. (1989), su ra.
A "variant" of a particular nucleic acid sequence is defined as a nucleic acid sequence having at least 40% sequence identity to the particular nucleic acid sequence over a certain length of one of the nucleic acid sequences using blastn with the "BLAST 2 Sequences" tool Version 2Ø9 (May-07-1999) set at default parameters. Such a pair of nucleic acids may show, for example, at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% or greater sequence identity over a certain defined length. A variant may be described as, for example, an "allelic" (as defined above), "splice," "species," or "polymorphic" variant. A
splice variant may have significant identity to a reference molecule, but will generally have a greater or lesser number of polynucleotides due to alternate splicing of exons during mRNA processing. The corresponding polypeptide may possess additional functional domains or lack domains that are present in the reference molecule. Species variants are polynucleotide sequences that vary from one species to another. The resulting polypeptides will generally have significant amino acid identity relative to each other. A polymorphic variant is a variation in the polynucleotide sequence of a particular gene between individuals of a given species. Polymorphic variants also may encompass "single nucleotide polymorphisms" (SNPs) in which the polynucleotide sequence varies by one nucleotide base. The presence of SNPs may be indicative of, for example, a certain population, a disease state, or a propensity for a disease state.
A "variant" of a particular polypeptide sequence is defined as a polypeptide sequence having at least 40% sequence identity to the particular polypeptide sequence over a certain length of one of the polypeptide sequences using blastp with the "BLAST 2 Sequences" tool Version 2Ø9 (May-07-1999) set at default parameters. Such a pair of polypeptides may show, for example, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%
or greater sequence identity over a certain defined length of one of the polypeptides.
THE INVENTION
The invention is based on the discovery of new human proteases (PRTS), the polynucleotides encoding PRTS, and the use of these compositions for the diagnosis, treatment, or prevention of gastrointestinal, cardiovascular, autoimmune/inflammatory, cell proliferative, developmental, epithelial, neurological, and reproductive disorders.
Table 1 summarizes the nomenclature for the full length polynucleotide and polypeptide sequences of the invention. Each polynucleotide and its corresponding polypeptide are correlated to a single Incyte project identification number (Incyte Project )D). Each polypeptide sequence is denoted by both a polypeptide sequence identification number (Polypeptide SEQ ID NO:) and an Incyte polypeptide sequence number (Incyte Polypeptide ID) as shown. Each polynucleotide sequence is denoted by both a polynucleotide sequence identification number (Polynucleotide SEQ m NO:) and an Incyte polynucleotide consensus sequence number (Incyte Polynucleotide ID) as shown.
Table 2 shows sequences with homology to the polypeptides of the invention as identified by BLAST analysis against the GenBank protein (genpept) database. Columns 1 and 2 show the polypeptide sequence identification number (Polypeptide SEQ DJ NO:) and the corresponding Incyte polypeptide sequence number (Incyte Polypeptide >D) for polypeptides of the invention. Column 3 shows the GenBank identification number (GenBank ID NO:) of the nearest GenBank homolog.
Column 4 shows the probability scores for the matches between each polypeptide and its homolog(s).
Column 5 shows the annotation of the GenBank homolog(s) along with relevant citations where applicable, all of which are expressly incorporated by reference herein.
Table 3 shows various structural features of the polypeptides of the invention. Columns 1 and 2 show the polypeptide sequence identification number (SEQ ID NO:) and the corresponding Incyte polypeptide sequence number (Incyte Polypeptide ID) for each polypeptide of the invention.
Column 3 shows the number of amino acid residues in each polypeptide. Column 4 shows potential phosphorylation sites, and column 5 shows potential glycosylation sites, as determined by the MOTIFS program of the GCG sequence analysis software package (Genetics Computer Group, Madison WI). Column 6 shows amino acid residues comprising signature sequences, domains, and motifs. Column 7 shows analytical methods for protein structure/function analysis and in some cases, searchable databases to which the analytical methods were applied.
Together, Tables 2 and 3 summarize the properties of polypeptides of the invention, and these properties establish that the claimed polypeptides are proteases. For example, SEQ ll~ N0:3 is 50%
identical to Xenopus ADAM 13 metalloprotease (GenBank ID g1916617) as determined by the Basic Local Alignment Search Tool (BLAST). (See Table 2.) The BLAST probability score is 2.1e-208, which indicates the probability of obtaining the observed polypeptide sequence alignment by chance.
SEQ ID N0:3 also contains a neutral zinc metalloprotease active site domain and a disintegrin domain, as determined by searching for statistically significant matches in the hidden Markov model (HMM)-based PFAM database of conserved protein family domains. (See Table 3.) The presence of these motifs is confirmed by BLM'S, MOTIFS, and PROF1LESCAN analyses, providing further corroborative evidence that SEQ ID N0:3 is a protease of the ADAM family. In an alternate example, SEQ ID N0:4 is 44% identical to human zinc metalloprotease ADAMTS7 (GenBank m g5923788) as determined by the Basic Local Alignment Search Tool (BLAST). (See Table 2.) The BLAST probability score is 2.2e-143, which indicates the probability of obtaining the observed polypeptide sequence alignment by chance. SEQ ID N0:4 also contains a Reprolysin (M12B) family zinc metalloprotease site and a Thrombospondin type 1 domain as determined by searching for statistically significant matches in the hidden Markov model (I-BVIM)-based PFAM database of conserved protein family domains. (See Table 3.) Data from BLIMPS and MOTIFS
analyses provide further corroborative evidence that SEQ ID N0:4 is a metalloprotease (note that the "Thrombospondin type 1 domains" are found at the carboxy-terminal end, and are characteristic of the ADAMTS metalloprotease protein family). In an alternate example, SEQ ID
NO:S is 62%
identical to mouse distal intestinal serine protease (GenBank ID 85921501) as determined by the Basic Local Alignment Search Tool (BLAST). (See Table 2.) The BLAST
probability score is 5.3e-99, which indicates the probability of obtaining the observed polypeptide sequence alignment by chance. SEQ ID N0:5 also contains a trypsin family serine protease active site domain as determined by searching for statistically significant matches in the hidden Markov model (HMM)-based PFAM
database of conserved protein family domains. (See Table 3.) The presence of this motif is confirmed by BLM'S, MOTIFS, and PROFILESCAN analyses. BLIMPS analysis also reveals the presence of kringle and type I fibronectin domains. Together, these data provide further corroborative evidence that SEQ ID N0:5 is a trypsin family serine protease.
In an alternate example, SEQ ID N0:8 is 45% identical to human membrane-type serine protease 1 (GenBank 117 86002714) as determined by the Basic Local Alignment Search Tool (BLAST). (See Table 2.) The BLAST
probability score is 6.1e-69, which indicates the probability of obtaining the observed polypeptide sequence alignment by chance. SEQ 1D N0:8 also contains a trypsin domain as determined by searching for statistically significant matches in the hidden Markov model (IllVIM)-based PFAM
database of conserved protein family domains. (See Table 3.) Data from BLIMPS, MOTIFS, and PROFIL,ESCAN analyses provide further corroborative evidence that SEQ ID N0:8 is a serine protease. In an alternate example, SEQ ID N0:11 is 49% identical to mouse ADAM
4 protein precursor (GenBank ID 8965014) as determined by the Basic Local Alignment Search Tool (BLAST). (See Table 2.) The BLAST probability score is 4.1e-117, which indicates the probability of obtaining the observed polypeptide sequence alignment by chance. SEQ ID
N0:11 also contains a reprolysin family propeptide domain and a disintegrin domain as determined by searching for statistically significant matches in the hidden Markov model (HMM)-based PFAM
database of conserved protein family domains. (See Table 3.) Data from BLllVIPS and PROF1LESCAN analyses provide further corroborative evidence that SEQ ID NO:11 is an ADAM family metalloprotease. In an alternate example, SEQ ll~ N0:12 is 42% identical to bovine enteropeptidase (GenBank ID
8416132) as determined by the Basic Local Alignment Search Tool (BLAST). (See Table 2.) The BLAST probability score is 2.2e-47, which indicates the probability of obtaining the observed polypeptide sequence alignment by chance. SEQ ID N0:12 also contains a trypsin domain as determined by searching for statistically significant matches in the hidden Markov model (HMM)-based PFAM database of conserved protein family domains. (See Table 3.) Data from BLIIUVIPS, MOTIFS, and PROFILESCAN analyses provide further corroborative evidence that SEQ m N0:12 is a trypsin family serine protease. In an alternate example, SEQ ID N0:13 is 52%
identical from residues 110 to 4g2 to Saccharomyces cerevisiae Maplp methionine aminopeptidase (GenBank ID
g662342) as determined by the Basic Local Alignment Search Tool (BLAST), with a probability score of 1.6e-99. (See Table 2.) SEQ ID N0:13 also contains a metallopeptidase family M24 domain as determined by searching for statistically significant matches in the hidden Markov model (I~ZM)-based PFAM database of conserved protein family domains. (See Table 3.) Data from BLI1VVIPS and PROFII,ESCAN analyses provide further corroborative evidence that SEQ )D N0:13 is a methionine aminopeptidase. In an alternate example, SEQ ID N0:15 is 36%
identical to Xenopus epidermis-specific serine protease (GenBank ID g6009515) as determined by the Basic Local Alignment Search Tool (BLAST). (See Table 2.) The BLAST probability score is 7.7e-52, which indicates the probability of obtaining the observed polypeptide sequence alignment by chance. SEQ
ID N0:15 also contains a trypsin family protease active site domain as determined by searching for statistically significant matches in the hidden Markov model (HNI1~~I)-based PFAM database of conserved protein family domains. (See Table 3.) The presence of this motif is confirmed by BLM'S, MOT1FS, and PROFILESCAN analyses. BLM'S analysis also reveals that SEQ
~
NO:15 contains a kringle domain, providing further corroborative evidence that SEQ ID NO:15 is a protease of the trypsin family. SEQ ID N0:2-3, SEQ ID N0:6-7, SEQ ID N0:9-10 and SEQ )D
N0:14 were analyzed and annotated in a similar manner. The algorithms and parameters for the analysis of SEQ ID NO: l-15 are described in Table 7.
As shown in Table 4, the full length polynucleotide sequences of the present invention were assembled using cDNA sequences or coding (exon) sequences derived from genomic DNA, or any combination of these two types of sequences. Columns 1 and 2 list the polynucleotide sequence identification number (Polynucleotide SEQ ID NO:) and the corresponding Incyte polynucleotide consensus sequence number (Incyte Polynucleotide ID) for each polynucleotide of the invention.
Column 3 shows the length of each polynucleotide sequence in basepairs. Column 4 lists fragments of the polynucleotide sequences which are useful, for example, in hybridization or amplification technologies that identify SEQ ll~ N0:16-30 or that distinguish between SEQ ID
N0:16-30 and related polynucleotide sequences. Column 5 shows identification numbers corresponding to cDNA
sequences, coding sequences (exons) predicted from genomic DNA, and/or sequence assemblages comprised of both cDNA and genomic DNA. These sequences were used to assemble the full length polynucleotide sequences of the invention. Columns 6 and 7 of Table 4 show the nucleotide start (5') and stop (3') positions of the cDNA and/or genomic sequences in column 5 relative to their respective full length sequences.
The identification numbers in Column 5 of Table 4 may refer specifically, for example, to Incyte cDNAs along with their corresponding cDNA libraries. For example, 7635792H1 is the identification number of an Incyte cDNA sequence, and SINTD1E01 is the cDNA
library from which it is derived. Incyte cDNAs for which cDNA libraries are not indicated were derived from pooled cDNA libraries (e.g., 55147856J1). Alternatively, the identification numbers in column 5 may refer to GenBank cDNAs or ESTs (e.g., g876900) Which contributed to the assembly of the full length polynucleotide sequences. In addition, the identification numbers in column 5 may identify sequences derived from the ENSEMBL (The Sanger Centre, Cambridge, UK) database (i.e., those sequences including the designation ".ENST"). Alternatively, the identification numbers in column 5 may be derived from the NCBI RefSeq Nucleotide Sequence Records Database (i.e., those sequences including the designation "NM" or "NT") or the NCBI RefSeq Protein Sequence Records (i.e., those sequences including the designation "NP"). Alternatively, the identification numbers in column 5 may refer to assemblages of both cDNA and Genscan-predicted exons brought together by an "exon stitching" algorithm. For example, FL 1~'.d~XXXX_Nl 1Vz_YYYYY_N3 IV4 represents a "stitched"
sequence in which X~XXX is the identification number of the cluster of sequences to which the algorithm was applied, and YYYYY is the number of the prediction generated by the algorithm, and N1,2, j.,a, if present, represent specific exons that may have been manually edited during analysis (See Example V). Alternatively, the identification numbers in column 5 may refer to assemblages of exons brought together by an "exon-stretching" algorithm. For example, FLXXXX~X gAAAAA~BBBBB_1 lV is the identification number of a "stretched"
sequence, with XXX~PI~X being the Incyte project identification number, gAAAAA being the GenBank identification number of the human genomic sequence to which the "exon-stretching" algorithm was applied, gBBBBB being the GenBank identification number or NCBI RefSeq identification number of the nearest GenBank protein homolog, and N refernng to specific exons (See Example V). In instances where a RefSeq sequence was used as a protein homolog for the "exon-stretching" algorithm, a RefSeq identifier (denoted by "NM," "NP,"' or "NT") may be used in place of the GenBank identifier (i.e., gBBBBB).
Alternatively, a prefix identifies component sequences that were hand-edited, predicted from genomic DNA sequences, or derived from a combination of sequence analysis methods. The following Table lists examples of component sequence prefixes and corresponding sequence analysis methods associated with the prefixes (see Example IV and Example V).
Prefix Type of analysis and/or examples of programs GNN, GFG,Exon prediction from genomic sequences using, for example, ENST GENSCAN (Stanford University, CA, USA) or FGENES

(Computer Genomics Group, The Banger Centre, Cambridge, UK).

GBI Hand-edited analysis of genomic sequences.

FL Stitched or stretched genomic sequences (see Example V).

INCY Full length transcript and exon prediction from mapping of EST

sequences to the genome. Genomic location and EST composition data are combined to predict the exons and resulting transcript.

In some cases, Incyte cDNA coverage redundant with the sequence coverage shown in column 5 was obtained to confirm the final consensus polynucleotide sequence, but the relevant Incyte cDNA identification numbers are not shown.
Table 5 shows the representative cDNA libraries for those full length polynucleotide sequences which were assembled using Incyte eDNA sequences. The representative cDNA library is the Incyte cDNA library which is most frequently represented by the Incyte cDNA sequences which were used to assemble and confirm the above polynucleotide sequences. The tissues and vectors which were used to construct the cDNA libraries shown in Table 5 are described in Table 6.
The invention also encompasses PRTS variants. A preferred PRTS variant is one which has at least about 80%, or alternatively at least about 90%, or even at least about 95% amino acid sequence identity to the PRTS amino acid sequence, and which contains at least one functional or structural characteristic of PRTS.
The invention also encompasses polynucleotides which encode PRTS. In a particular embodiment, the invention encompasses a polynucleotide sequence comprising a sequence selected from the group consisting of SEQ ID N0:16-30, which encodes PRTS. The polynucleotide sequences of SEQ ID NO:16-30, as presented in the Sequence Listing, embrace the equivalent RNA
sequences, wherein occurrences of the nitrogenous base thymine are replaced with uracil, and the sugar backbone is composed of ribose instead of deoxyribose.
The invention also encompasses a variant of a polynucleotide sequence encoding PRTS. In particular, such a variant polynucleotide sequence will have at least about 70%, or alternatively at least about 85 %, or even at least about 95 % polynucleotide sequence identity to the polynucleotide sequence encoding PRTS. A particular aspect of the invention encompasses a variant of a polynucleotide sequence comprising a sequence selected from the group consisting of SEQ 117 N0:16-30 which has at least about 70%, or alternatively at least about 85%, or even at least about 95% polynucleotide sequence identity to a nucleic acid sequence selected from the group consisting 34.

of SEQ )D N0:16-30. Any one of the polynucleotide variants described above can encode an amino acid sequence which contains at least one functional or structural characteristic of PRTS.
In addition, or in the alternative, a polynucleotide variant of the invention is a splice variant of a polynucleotide sequence encoding PRTS. A splice variant may have portions which have significant sequence identity to the polynucleotide sequence encoding PRTS, but will generally have a greater or lesser number of polynucleotides due to additions or deletions of blocks of sequence arising from alternate splicing of exons during mRNA processing. A splice variant may have less than about 70%, or alternatively less than about 60%, or alternatively less than about 50%
polynucleotide sequence identity to the polynucleotide sequence encoding PRTS
over its entire length; however, portions of the splice variant will have at least about 70%, or alternatively at least about 85%, or alternatively at least about 95%, or alternatively 100%
polynucleotide sequence identity to portions of the polynucleotide sequence encoding PRTS. Any one of the splice variants described above can encode an amino acid sequence which contains at least one functional or structural characteristic of PRTS.
It will be appreciated by those skilled in the art that as a result of the degeneracy of the genetic code, a multitude of polynucleotide sequences encoding PRTS, some bearing minimal similarity to the polynucleotide sequences of any known and naturally occurring gene, may be produced. Thus, the invention contemplates each and every possible variation of polynucleotide sequence that could be made by selecting combinations based on possible codon choices. These combinations are made in accordance with the standard triplet genetic code as applied to the polynucleotide sequence of naturally occurring PRTS, and all such variations are to be considered as being specifically disclosed.
Although nucleotide sequences which encode PRTS and its variants are generally capable of hybridizing to the nucleotide sequence of the naturally occurnng PRTS under appropriately selected conditions of stringency, it may be advantageous to produce nucleotide sequences encoding PRTS or its derivatives possessing a substantially different codon usage, e.g., inclusion of non-naturally occurring codons. Codons may be selected to increase the rate at which expression of the peptide occurs in a particular prokaryotic or eukaryotic host in accordance with the frequency with which particular codons are utilized by the host. Other reasons for substantially altering the nucleotide sequence encoding PRTS and its derivatives without altering the encoded amino acid sequences include the production of RNA transcripts having more desirable properties, such as a greater half life, than transcripts produced from the naturally occurring sequence.
The invention also encompasses production of DNA sequences which encode PRTS
and PRTS derivatives, or fragments thereof, entirely by synthetic chemistry. After production, the synthetic sequence may be inserted into any of the many available expression vectors and cell systems using reagents well known in the art. Moreover, synthetic chemistry may be used to introduce mutations into a sequence encoding PRTS or any fragment thereof.
Also encompassed by the invention are polynucleotide sequences that are capable of hybridizing to the claimed polynucleotide sequences, and, in particular, to those shown in SEQ ID
N0:16-30 and fragments thereof under various conditions of stringency. (See, e.g., Wahl, G.M. and S.L. Berger (1987) Methods Enzymol. 152:399-407; Kimmel, A.R. (1987) Methods Enzymol.
152:507-511.) Hybridization conditions, including annealing and wash conditions, are described in "Definitions."
Methods for DNA sequencing are well known in the art and may be used to practice any of the embodiments of the invention. The methods may employ such enzymes as the Klenow fragment of DNA polymerase I, SEQUENASE (US Biochemical, Cleveland OH), Taq polymerase (Applied Biosystems), thermostable T7 polymerase (Amersham Pharmacia Biotech, Piscataway NJ), or combinations of polymerases and proofreading exonucleases such as those found in the ELONGASE
amplification system (Life Technologies, Gaithersburg MD). Preferably, sequence preparation is automated with machines such as the MICROLAB 2200 liquid transfer system (Hamilton, Reno NV), PTC200 thermal cycler (MJ Research, Watertown MA) and ABI CATALYST 800 thermal cycler (Applied Biosystems). Sequencing is then carried out using either the ABI 373 or 377 DNA
sequencing system (Applied Biosystems), the MEGABACE 1000 DNA sequencing system (Molecular Dynamics, Sunnyvale CA), or other systems known in the art. The resulting sequences are analyzed using a variety of algorithms which are well known in the art.
(See, e.g., Ausubel, F.M.
(1997) Short Protocols in Molecular Bioloay, John Wiley ~ Sons, New York NY, unit 7.7; Meyers, R.A. (1995) Molecular Biology and Biotechnology, Wiley VCH, New York NY, pp.
856-853.) The nucleic acid sequences encoding PRTS may be extended utilizing a partial nucleotide sequence and employing various PCR-based methods known in the art to detect upstream sequences, such as promoters and regulatory elements. For example, one method which may be employed, restriction-site PCR, uses universal and nested primers to amplify unlalown sequence from genomic DNA within a cloning vector. (See, e.g., Sarkar, G. (1993) PCR Methods Applic.

2:318-322.) Another method, inverse PCR, uses primers that extend in divergent directions to amplify unknown sequence from a circularized template. The template is derived from restriction fragments comprising a known genomic locus and surrounding sequences. (See, e.g., Triglia, T. et al. (1988) Nucleic Acids Res. 16:8186.) A third method, capture PCR, involves PCR amplification of DNA
fragments adjacent to known sequences in human and yeast artificial chromosome DNA.
(See, e.g., Lagerstrom, M. et al. (1991) PCR Methods Applic. 1:111-119.) In this method, multiple restriction enzyme digestions and ligations may be used to insert an engineered double-stranded sequence into a region of unknown sequence before performing PCR. Other methods which may be used to retrieve unknown sequences are known in the art. (See, e.g., Parker, J.D. et al. (1991) Nucleic Acids Res.
19:3055-3060). Additionally, one may use PCR, nested primers, and PROMOTERFINDER libraries (Clontech, Palo Alto CA) to walk genomic DNA. This procedure avoids the need to screen libraries and is useful in finding intron/exon junctions. For all PCR-based methods, primers may be designed using commercially available software, such as OLIGO 4.06 primer analysis software (National Biosciences, Plymouth MN) or another appropriate program, to be about 22 to 30 nucleotides in length, to have a GC content of about 50% or more, and to anneal to the template at temperatures of about 6~°C to 72°C.
When screening for full length cDNAs, it is preferable to use libraries that have been size-selected to include larger cDNAs. In addition, random-primed libraries, which often include sequences containing the 5' regions of genes, are preferable for situations in which an oligo d(T) library does not yield a full-length cDNA. Genomic libraries may be useful for extension of sequence into 5' non-transcribed regulatory regions.
Capillary electrophoresis systems which are commercially available may be used to analyze the size or confirm the nucleotide sequence of sequencing or PCR products. In particular, capillary sequencing may employ flowable polymers for electrophoretic separation, four different nucleotide specific, laser-stimulated fluorescent dyes, and a charge coupled device camera for detection of the emitted wavelengths. Output/light intensity may be converted to electrical signal using appropriate software (e.g., GENOTYPER and SEQUENCE NAVIGATOR, Applied Biosystems), and the entire process from loading of samples to computer analysis and electronic data display may be computer controlled. Capillary electrophoresis is especially preferable for sequencing small DNA fragments which may be present in limited amounts in a particular sample.
In another embodiment of the invention, polynucleotide sequences or fragments thereof which encode PRTS may be cloned in recombinant DNA molecules that direct expression of PRTS, or fragments or functional equivalents thereof, 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 produced and used to express PRTS.
The nucleotide sequences of the present invention can be engineered using methods generally laiown in the art in order to alter PRTS-encoding sequences for a variety of purposes including, but not limited to, modification of the cloning, processing, and/or expression of the gene product. DNA
shuffling by random fragmentation and PCR reassembly of gene fragments and synthetic oligonucleotides may be used to engineer the nucleotide sequences. For example, oligonucleotide-mediated site-directed mutagenesis may be used to introduce mutations that create new restriction sites, alter glycosylation patterns, change codon preference, produce splice variants, and so forth.

The nucleotides of the present invention may be subjected to DNA shuffling techniques such as MOLECULARBREEDING (Maxygen Inc., Santa Clara CA; described in U.S. Patent No.
5,837,458; Chang, C.-C. et al. (1999) Nat. Biotechnol. 17:793-797; Christians, F.C. et al. (1999) Nat.
Biotechnol. 17:259-264; and Crameri, A. et al. (1996) Nat. Biotechnol. 14:315-319) to alter or improve the biological properties of PRTS, such as its biological or enzymatic activity or its ability to bind to other molecules or compounds. DNA shuffling is a process by which a library of gene variants is produced using PCR-mediated recombination of gene fragments. The library is then subjected to selection or screening procedures that identify those gene variants with the desired properties. These preferred variants may then be pooled and further subjected to recursive rounds of DNA shuffling and selection/screening. Thus, genetic diversity is created through "artificial"
breeding and rapid molecular evolution. For example, fragments of a single gene containing random point mutations may be recombined, screened, and then reshuffled until the desired properties are optimized. Alternatively, fragments of a given gene may be recombined with fragments of homologous genes in the same gene family, either from the same or different species, thereby maximizing the genetic diversity of multiple naturally occurring genes in a directed and controllable manner.
In another embodiment, sequences encoding PRTS may be synthesized, in whole or in part, using chemical methods well known in the art. '(See, e.g., Caruthers, M.H. et al. (1980) Nucleic Acids Symp. Ser. 7:215-223; and Horn, T. et al. (1980) Nucleic Acids Symp. Ser.
7:225-232.) Alternatively, PRTS itself or a fragment thereof may be synthesized using chemical methods. For example, peptide synthesis can be performed using various solution-phase or solid-phase techniques.
(See, e.g., Creighton, T. (1984) Proteins, Structures and Molecular Pro cu roes, WH Freeman, New York NY, pp. 55-60; and Roberge, J.Y. et al. (1995) Science 269:202-204.) Automated synthesis may be achieved using the ABI 431A peptide synthesizer (Applied Biosystems).
Additionally, the amino acid sequence of PRTS, or any part thereof, may be altered during direct synthesis and/or combined with sequences from other proteins, or any part thereof, to produce a variant polypeptide or a polypeptide having a sequence of a naturally occurring polypeptide.
The peptide may be substantially purified by preparative high performance liquid chromatography. (See, e.g., Chiez, R.M. and F.Z. Regnier (1990) Methods Enzymol. 182:392-421.) The composition of the synthetic peptides may be confirmed by amino acid analysis or by sequencing. (See, e.g., Creighton, su ra, pp. 28-53.) In order to express a biologically active PRTS, the nucleotide sequences encoding PRTS or derivatives thereof may be inserted into an appropriate expression vector, i.e., a vector which contains the necessary elements for transcriptional and translational control of the inserted coding sequence in a suitable host. These elements include regulatory sequences, such as enhancers, constitutive and 'i8 inducible promoters, and 5' and 3' untranslated regions in the vector and in polynucleotide sequences encoding PRTS. Such elements may vary in their strength and specificity.
Specific initiation signals may also be used to achieve more efficient translation of sequences encoding PRTS. Such signals include the ATG initiation codon and adjacent sequences, e.g. the Kozak sequence. In cases where sequences encoding PRTS and its initiation codon and upstream regulatory sequences are inserted into the appropriate expression vector, no additional transcriptional or translational control signals may be needed. However, in cases where only coding sequence, or a fragment thereof, is inserted, exogenous translational control signals including an in-frame ATG initiation codon should be provided by the vector. Exogenous translational elements and initiation codons may be of various origins, both natural and synthetic. The efficiency of expression may be enhanced by the inclusion of enhancers appropriate for the particular host cell system used. (See, e.g., Scharf, D. et al. (1994) Results Probl. Cell Differ. 20:125-162.) Methods which are well known to those skilled in the art may be used to construct expression vectors containing sequences encoding PRTS and appropriate transcriptional and translational control elements. These methods include in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination. (See, e.g., Sambrook, J. et al. (1989) Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Press, Plainview NY, ch. 4, 8, and 16-17; Ausubel, F.M. et al. (1995) Current Protocols in Molecular Biolo~y, John Wiley & Sons, New York NY, ch. 9, 13, and 16.) A variety of expression vector/host systems may be utilized to contain and express sequences encoding PRTS. 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 viral expression vectors (e.g., baculovirus);
plant cell systems transformed with viral expression vectors (e.g., cauliflower mosaic virus, CaMV, or tobacco mosaic virus, TMV) or with bacterial expression vectors (e.g., Ti or pBR322 plasmids); or animal cell systems. (See, e.g., Sambrook, s, upra; Ausubel, supra; Van Heeke, G. and S.M. Schuster (1989) J. Biol. Chem. 264:5503-5509; Engelhard, E.K. et al. (1994) Proc. Natl.
Acad. Sci. USA
91:3224-3227; Sandig, V. et al. (1996) Hum. Gene Ther. 7:1937-1945; Takamatsu, N. (1987) EMBO
J. 6:307-311; The McGraw Hill Yearbook of Science and Technolo~v (1992) McGraw Hill, New York NY, pp. 191-196; Logan, J. and T. Shenk (1984) Proc. Natl. Acad. Sci. USA
81:3655-3659; and Harnngton, J.J. et al. (1997) Nat. Genet. 15:345-355.) Expression vectors derived from retroviruses, adenoviruses, or herpes or vaccinia viruses, or from various bacterial plasmids, may be used for delivery of nucleotide sequences to the targeted organ, tissue, or cell population. (See, e.g., Di Nicola, M. et al. (1998) Cancer Gen. Ther. 5(6):350-356; Yu, M. et al. (1993) Proc. Natl. Acad. Sci.
USA 90(13):6340-6344; Buller, R.M. et al. (1985) Nature 317(6040):813-815;
McGregor, D.P. et al.

(1994) Mol. Immunol. 31(3):219-226; and Verma, LM. and N. Somia (1997) Nature 389:239-242.) The invention is not limited by the host cell employed.
In bacterial systems, a number of cloning and expression vectors may be selected depending upon the use intended for polynucleotide sequences encoding PRTS. For example, routine cloning, subcloning, and propagation of polynucleotide sequences encoding PRTS can be achieved using a multifunctional E. coli vector such as PBLLTESCRIPT (Stratagene, La Jolla CA) or PSPORT1 plasmid (Life Technologies). Ligation of sequences encoding PRTS into the vector's multiple cloning site disrupts the lacZ gene, allowing a colorimetric screening procedure for identification of transformed bacteria containing recombinant molecules. In addition, these vectors may be useful for in vitro transcription, dideoxy sequencing, single strand rescue with helper phage, and creation of nested deletions in the cloned sequence. (See, e.g., Van Heeke, G. and S.M.
Schuster (1989) J. Biol.
Chem. 264:5503-5509.) When large quantities of PRTS are needed, e.g. for the production of antibodies, vectors which direct high level expression of PRTS may be used.
For example, vectors containing the strong, inducible SP6 or T7 bacteriophage promoter may be used.
Yeast expression systems may be used for production of PRTS. A number of vectors containing constitutive or inducible promoters, such as alpha factor, alcohol oxidase, and PGH
promoters, may be used in the yeast Saccharomyces cerevisiae or Pichia pastoris. In addition, such vectors direct either the secretion or intracellular retention of expressed proteins and enable integration of foreign sequences into the host genome for stable propagation.
(See, e.g., Ausubel, 1995, su ra; Bitter, G.A. et al. (1987) Methods Enzymol. 153:516-544; and Scorer, C.A. et al. (1994) Bio/Technology 12:181-184.) Plant systems may also be used for expression of PRTS. Transcription of sequences encoding PRTS may be driven by viral promoters, e.g., the 35S and 19S
promoters of CaMV used alone or in combination with the omega leader sequence from TMV (Takamatsu, N.
(1987) EMBO J.
6:307-311). Alternatively, plant promoters such as the small subunit of RUBISCO or heat shock promoters may be used. (See, e.g., Coruzzi, G. et al. (1984) EMBO J. 3:1671-1680; Broglie, R. et al.
(1984) Science 224:838-843; and Winter, J. et al. (1991) Results Probl. Cell Differ. 17:85-105.) These constructs can be introduced into plant cells by direct DNA
transformation or pathogen-mediated transfection. (See, e.g., The McGraw Hill Yearbook of Science and Technolo~y (1992) McGraw Hill, New York NY, pp. 191-196.) In mammalian cells, a number of viral-based expression systems may be utilized. In cases where an adenovirus is used as an expression vector, sequences encoding PRTS
may be ligated into an adenovirus transcription/translation complex consisting of the late promoter and tripartite leader sequence. Insertion in a non-essential E1 or E3 region of the viral genome may be used to obtain infective virus which expresses PRTS in host cells. (See, e.g., Logan, J. and T. Shenk (1984) Proc.

Natl. Acad. Sci. USA 81:3655-3659.) In addition, transcription enhancers, such as the Rous sarcoma virus (RSV) enhancer, may be used to increase expression in mammalian host cells. SV40 or EBV-based vectors may also be used for high-level protein expression.
Human artificial chromosomes (HACs) may also be employed to deliver larger fragments of DNA than can be contained in and expressed from a plasmid. HACs of about 6 kb to 10 Mb are constructed and delivered via conventional delivery methods (liposomes, polycationic amino polymers, or vesicles) for therapeutic purposes. (See, e.g., Harrington, J.J.
et al. (1997) Nat. Genet.
15:345-355.) For long term production of recombinant proteins in mammalian systems, stable expression of PRTS in cell lines is preferred. For example, sequences encoding PRTS can be transformed into cell lines using expression vectors which may contain viral origins of replication and/or endogenous expression elements and a selectable marker gene on the same or on a separate vector. Following the introduction of the vector, cells may be allowed to grow for about 1 to 2 days in enriched media before being switched to selective media. The purpose of the selectable marker is to confer resistance to a selective agent, and its presence allows growth and recovery of cells which successfully express the introduced sequences. Resistant clones of stably transformed cells may be propagated 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 and adenine phosphoribosyltransferase genes, for use in tk- and apr cells, respectively.
(See, e.g., Wigler, M. et al. (1977) Cell 11:223-232; Lowy, I. et al. (1980) Cell 22:817-823.) Also, antimetabolite, antibiotic, or herbicide resistance can be used as the basis for selection. For example, dhfr confers resistance to methotrexate; neo confers resistance to the aminoglycosides neomycin and G-418; and als and pat confer resistance to chlorsulfuron and phosphinotricin acetyltransferase, respectively. (See, e.g., Wigler, M. et al. (1980) Proc. Natl. Acad. Sci. USA 77:3567-3570; Colbere-Garapin, F. et al. (1981) J. Mol. Biol. 150:1-14.) Additional selectable genes have been described, e.g., trpB and hvsD, which alter cellular requirements for metabolites. (See, e.g., Hartman, S.C. and R.C. Mulligan (1988) Proc.
Natl. Acad. Sci. USA 85:8047-8051.) Visible markers, e.g., anthocyanins, green fluorescent proteins (GFP; Clontech),13 glucuronidase and its substrate 13-glucuronide, or luciferase and its substrate luciferin may be used. These markers can be used not only to identify transformants, but also to quantify the amount of transient or stable protein expression attributable to a specific vector system.
(See, e.g., Rhodes, C.A. (1995) Methods Mol. Biol. 55:121-131.) Although the presence/absence of marker gene expression suggests that the gene of interest is also present, the presence and expression of the gene may need to be confirmed. For example, if the sequence encoding PRTS is inserted within a marker gene sequence, transformed cells containing sequences encoding PRTS can be identified by the absence of marker gene function. Alternatively, a marker gene can be placed in tandem with a sequence encoding PRTS under the control of a single promoter. Expression of the marker gene in response to induction or selection usually indicates expression of the tandem gene as well.
In general, host cells that contain the nucleic acid sequence encoding PRTS
and that express PRTS 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 hybridizations, PCR
amplification, and protein bioassay or immunoassay techniques which include membrane, solution, or chip based technologies for the detection and/or quantification of nucleic acid or protein sequences.
Immunological methods for detecting and measuring the expression of PRTS using either specific polyclonal or monoclonal antibodies are known in the art. Examples of such techniques include enzyme-linked immunosorbent assays (ELISAs), radioimmunoassays (RIAs), and fluorescence activated cell sorting (FACS). A two-site, monoclonal-based immunoassay utilizing monoclonal antibodies reactive to two non-interfering epitopes on PRTS is preferred, but a competitive binding assay may be employed. These and other assays are well known in the art. (See, e.g., Hampton, R. et al. (1990) Serological Methods, a Laboratory Manual, APS
Press, St. Paul MN, Sect. IV; Coligan, J.E. et al. (1997) Current Protocols in Immunology, Greene Pub. Associates and Wiley-Interscience, New York NY; and Pound, J.D. (1998) Immunochemical Protocols, Humana Press, Totowa NJ.) A wide variety of labels and conjugation techniques are known by those skilled in the art and may be used in various nucleic acid and amino acid assays. Means for producing labeled hybridization or PCR probes for detecting sequences related to polynucleotides encoding PRTS
include oligolabeling, nick translation, end-labeling, or PCR amplification using a labeled nucleotide.
Alternatively, the sequences encoding PRTS, or any fragments thereof, 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 labeled nucleotides. These procedures may be conducted using a variety of commercially available kits, such as those provided by Amersham Pharmacia Biotech, Promega (Madison WI), and US Biochemical. Suitable reporter molecules or labels which may be used for ease of detection include radionuclides, enzymes, fluorescent, chemiluminescent, or chromogenic agents, as well as substrates, cofactors, inhibitors, magnetic particles, and the like.
Host cells transformed with nucleotide sequences encoding PRTS may be cultured under conditions suitable for the expression and recovery of the protein from cell culture. The protein produced by a transformed cell may be secreted or retained intracellularly depending on the sequence and/or the vector used. As will be understood by those of skill in the art, expression vectors containing polynucleotides which encode PRTS may be designed to contain signal sequences which direct secretion of PRTS through a prokaryotic or eukaryotic cell membrane.
In addition, a host cell strain may be chosen for its ability to modulate 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" or "pro" form of the protein may also be used to specify protein targeting, folding, andlor activity.
Different host cells which have specific cellular machinery and characteristic mechanisms for post-translational activities (e.g., CHO, HeLa, MDCK, HEK293, and WI38) are available from the American Type Culture Collection (ATCC, Manassas VA) and may be chosen to ensure the correct modification and processing of the foreign protein.
In another embodiment of the invention, natural, modified, or recombinant nucleic acid sequences encoding PRTS may be ligated to a heterologous sequence resulting in translation of a fusion protein in any of the aforementioned host systems. For example, a chimeric PRTS protein containing a heterologous moiety that can be recognized by a commercially available antibody may facilitate the screening of peptide libraries for inhibitors of PRTS activity.
Heterologous protein and peptide moieties may also facilitate purification of fusion proteins using commercially available affinity matrices. Such moieties include, but are not limited to, glutathione S-transferase (GST), maltose binding protein (MBP), thioredoxin (Trx), calmodulin binding peptide (CBP), 6-His, FLAG, c-myc, and hemagglutinin (HA). GST, MBP, Trx, CBP, and 6-His enable purification of their cognate fusion proteins on immobilized glutathione, maltose, phenylarsine oxide, calmodulin, and metal-chelate resins, respectively. FLAG, c-myc, and hemagglutinin (HA) enable immunoaffinity purification of fusion proteins using commercially available monoclonal and polyclonal antibodies that specifically recognize these epitope tags. A fusion protein may also be engineered to contain a proteolytic cleavage site located between the PRTS encoding sequence and the heterologous protein sequence, so that PRTS may be cleaved away from the heterologous moiety following purification.
Methods for fusion protein expression and purification are discussed in Ausubel (1995, supra, ch. 10).
A variety of commercially available kits may also be used to facilitate expression and purification of fusion proteins.
In a further embodiment of the invention, synthesis of radiolabeled PRTS may be achieved in vitro using the TNT rabbit reticulocyte lysate or wheat germ extract system (Promega). These systems couple transcription and translation of protein-coding sequences operably associated with the T7, T3, or SP6 promoters. Translation takes place in the presence of a radiolabeled amino acid precursor, for example, 35S-methionine.

PRTS of the present invention or fragments thereof may be used to screen for compounds that specifically bind to PRTS. At least one and up to a plurality of test compounds may be screened for specific binding to PRTS. Examples of test compounds include antibodies, oligonucleotides, proteins (e.g., receptors), or small molecules.
In one embodiment, the compound thus identified is closely related to the natural ligand of PRTS, e.g., a ligand or fragment thereof, a natural substrate, a structural or functional mimetic, or a natural binding partner. (See, e.g., Coligan, J.E. et al. (1991) Current Protocols in Immunolo~y 1(2):
Chapter 5.) Similarly, the compound can be closely related to the natural receptor to which PRTS
binds, or to at least a fragment of the receptor, e.g., the ligand binding site. In either case, the compound can be rationally designed using known techniques. In one embodiment, screening for these compounds involves producing appropriate cells which express PRTS, either as a secreted protein or on the cell membrane. Preferred cells include cells from mammals, yeast, Drosophila, or E. coli. Cells expressing PRTS or cell membrane fractions which contain PRTS
are then contacted with a test compound and binding, stimulation, or inhibition of activity of either PRTS or the compound is analyzed.
An assay may simply test binding of a test compound to the polypeptide, wherein binding is detected by a fluorophore, radioisotope, enzyme conjugate, or other detectable label. For example, the assay may comprise the steps of combining at least one test compound with PRTS, either in solution or affixed to a solid support, and detecting the binding of PRTS to the compound.
Alternatively, the assay may detect or measure binding of a test compound in the presence of a labeled competitor. Additionally, the assay may be carried out using cell-free preparations, chemical libraries, or natural product mixtures, and the test compounds) may be free in solution or affixed to a solid support.
PRTS of the present invention or fragments thereof may be used to screen for compounds that modulate the activity of PRTS. Such compounds may include agonists, antagonists, or partial or inverse agonists. In one embodiment, an assay is performed under conditions permissive for PRTS
activity, wherein PRTS is combined with at least one test compound, and the activity of PRTS in the presence of a test compound is compared with the activity of PRTS in the absence of the test compound. A change in the activity of PRTS in the presence of the test compound is indicative of a compound that modulates the activity of PRTS. Alternatively, a test compound is combined with an in vitro or cell-free system comprising PRTS under conditions suitable for PRTS activity, and the assay is performed. In either of these assays, a test compound which modulates the activity of PRTS
may do so indirectly and need not come in direct contact with the test compound. At least one and up to a plurality of test compounds may be screened.

In another embodiment, polynucleotides encoding PRTS or their mammalian homologs may be "knocked out" in an animal model system using homologous recombination in embryonic stem (ES) cells. Such techniques are well known in the art and are useful for the generation of animal models of human disease. (See, e.g., U.S. Patent No. 5,175,383 and U.S. Patent No. 5,767,337.) For example, mouse ES cells, such as the mouse 129/SvJ cell line, are derived from the early mouse embryo and grown in culture. The ES cells are transformed with a vector containing the gene of interest disrupted by a marker gene, e.g., the neomycin phosphotransferase gene (neo; Capecchi, M.R.
(1989) Science 244:1288-1292). The vector integrates into the corresponding region of the host genome by homologous recombination. Alternatively, homologous recombination takes place using the Cre-loxP system to knockout a gene of interest in a tissue- or developmental stage-specific manner (Marth, J.D. (1996) Clin. Invest. 97:1999-2002; Wagner, K.U. et al.
(1997) Nucleic Acids Res. 25:4323-4330). Transformed ES cells are identified and microinjected into mouse cell blastocysts such as those from the C57BL/6 mouse strain. The blastocysts are surgically transferred to pseudopregnant dams, and the resulting chimeric progeny are genotyped and bred to produce heterozygous or homozygous strains. Transgenic animals thus generated may be tested with potential therapeutic or toxic agents.
Polynucleotides encoding PRTS may also be manipulated in vitro in ES cells derived from human blastocysts. Human ES cells have the potential to differentiate into at least eight separate cell lineages including endoderm, mesoderm, and ectodermal cell types. These cell lineages differentiate into, for example, neural cells, hematopoietic lineages, and cardiomyocytes (Thomson, J.A. et al.
(1998) Science 282:1145-1147).
Polynucleotides encoding PRTS can also be used to create "knockin" humanized animals (pigs) or transgenic animals (mice or rats) to model human disease. With knockin technology, a region of a polynucleotide encoding PRTS is injected into animal ES cells, and the injected sequence integrates into the animal cell genome. Transformed cells are injected into blastulae, and the blastulae are implanted as described above. Transgenic progeny or inbred lines are studied and treated with potential pharmaceutical agents to obtain information on treatment of a human disease.
Alternatively, a mammal inbred to overexpress PRTS, e.g., by secreting PRTS in its milk, may also serve as a convenient source of that protein (Janne, J. et al. (1998) Biotechnol. Annu. Rev. 4:55-74).
THERAPEUTICS
Chemical and structural similarity, e.g., in the context of sequences and motifs, exists between regions of PRTS and proteases. In addition, the expression of PRTS is closely associated with reproductive, normal and tumorous gastrointestinal, urogenital, bone tumor, breast, brain, testis, and adrenal tumor tissues, as well as with adherent mononuclear cells.
Therefore, PRTS appears to play a role in gastrointestinal, cardiovascular, autoimmune/inflammatory, cell proliferative, developmental, epithelial, neurological, and reproductive disorders. In the treatment of disorders associated with increased PRTS expression or activity, it is desirable to decrease the expression 'or activity of PRTS. In the treatment of disorders associated with decreased PRTS
expression or activity, it is desirable to increase the expression or activity of PRTS.
Therefore, in one embodiment, PRTS or a fragment or derivative thereof may be administered to a subject to treat or prevent a disorder associated with decreased expression or activity of PRTS. Examples of such disorders include, but are not limited to, a gastrointestinal disorder, such as dysphagia, peptic esophagitis, esophageal spasm, esophageal stricture, esophageal carcinoma, dyspepsia, indigestion, gastritis, gastric carcinoma, anorexia, nausea, emesis, gastroparesis, antral or pyloric edema, abdominal angina, pyrosis, gastroenteritis, intestinal obstruction, infections of the intestinal tract, peptic ulcer, cholelithiasis, cholecystitis, cholestasis, pancreatitis, pancreatic carcinoma, biliary tract disease, hepatitis, hyperbilirubinemia, cirrhosis, passive congestion of the liver, hepatoma, infectious colitis, ulcerative colitis, ulcerative proctitis, Crohn's disease, Whipple's disease, Mallory-Weiss syndrome, colonic carcinoma, colonic obstruction, irritable bowel syndrome, short bowel syndrome, diarrhea, constipation, gastrointestinal hemorrhage, acquired immunodeficiency syndrome (A1177S) enteropathy, jaundice, hepatic encephalopathy, hepatorenal syndrome, hepatic steatosis, hemochromatosis, Wilson's disease, alpha~-antitrypsin deficiency, Reye's syndrome, primary sclerosing cholangitis, liver infarction, portal vein obstruction and thrombosis, centrilobular necrosis, peliosis hepatis, hepatic vein thrombosis, veno-occlusive disease, preeclampsia, eclampsia, acute fatty liver of pregnancy, intrahepatic cholestasis of pregnancy, and hepatic tumors including nodular hyperplasias, adenomas, and carcinomas; a cardiovascular disorder, such as arteriovenous fistula, atherosclerosis, hypertension, vasculitis, Raynaud's disease, aneurysms, arterial dissections, varicose veins, thrombophlebitis and phlebothrombosis, vascular tumors, and complications of thrombolysis, balloon angioplasty, vascular c replacement, and coronary artery bypass graft surgery, congestive heart failure, ischemic heart disease, angina pectoris, myocardial infarction, hypertensive heart disease, degenerative valvular heart disease, calcific aortic valve stenosis, congenitally bicuspid aortic valve, mitral annular calcification, mitral valve prolapse, rheumatic fever and rheumatic heart disease, infective endocarditis, nonbacterial thrombotic endocarditis, endocarditis of systemic lupus erythematosus, carcinoid heart disease, cardiomyopathy, myocarditis, pericarditis, neoplastic heart disease, congenital heart disease, and complications of cardiac transplantation; an autoimmune/inflammatory disorder, such as acquired immunodeficiency syndrome (AIDS), Addison's disease, adult respiratory distress syndrome, allergies, ankylosing spondylitis, amyloidosis, anemia, asthma, atherosclerosis, atherosclerotic plaque rupture, autoimmune hemolytic anemia, autoimmune thyroiditis, autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED), bronchitis, cholecystitis, contact dermatitis, Crohn's disease, atopic dermatitis, dermatomyositis, diabetes mellitus, emphysema, episodic lymphopenia with lymphocytotoxins, erythroblastosis fetalis, erythema nodosum, atrophic gastritis, glomerulonephritis, Goodpasture's syndrome, gout, Graves' disease, Haslumoto's thyroiditis, hypereosinophilia, irritable bowel syndrome, multiple sclerosis, myasthenia gravis, myocardial or pericardial inflammation, osteoarthritis, degradation of articular cartilage, osteoporosis, pancreatitis, polymyositis, psoriasis, Reiter's syndrome, rheumatoid arthritis, scleroderma, Sjogren's syndrome, systemic anaphylaxis, systemic lupus erythematosus, systemic sclerosis, thrombocytopenic purpura, ulcerative colitis, uveitis, Werner syndrome, complications of cancer, hemodialysis, and extracorporeal circulation, viral, bacterial, fungal, parasitic, protozoal, and helminthic infections, and trauma; a cell proliferative disorder such as actinic keratosis, arteriosclerosis, atherosclerosis, bursitis, cirrhosis, hepatitis, mixed connective tissue disease (MCTD), myelofibrosis, paroxysmal nocturnal hemoglobinuria, polycythemia vera, psoriasis, primary thrombocythemia, and cancers including adenocarcinoma, leukemia, lymphoma, melanoma, myeloma, sarcoma, teratocarcinoma, and, in particular, cancers of the adrenal gland, bladder, bone, bone marrow, brain, breast, cervix, gall bladder, ganglia, gastrointestinal tract, heart, kidney, liver, lung, muscle, ovary, pancreas, parathyroid, penis, prostate, salivary glands, skin, spleen, testis, thymus, thyroid, and uterus; a developmental disorder, such as renal tubular acidosis, anemia, Cushing's syndrome, achondroplastic dwa~sm, Duchenne and Becker muscular dystrophy, bone resorption, epilepsy, gonadal dysgenesis, WAGR
syndrome (Wilms' tumor, aniridia, genitourinary abnormalities, and mental retardation), Smith-Magenis syndrome, myelodysplastic syndrome, hereditary mucoepithelial dysplasia, hereditary keratodermas, hereditary neuropathies such as Charcot-Marie-Tooth disease and neurofibromatosis, hypothyroidism, hydrocephalus, seizure disorders such as Syndenham's chorea and cerebral palsy, spina bifida, anencephaly, craniorachischisis, congenital glaucoma, cataract, age-related macular degeneration, and sensorineural hearing loss; an epithelial disorder, such as dyshidrotic eczema, allergic contact dermatitis, keratosis pilaris, melasma, vitiligo, actinic keratosis, basal cell carcinoma, squamous cell carcinoma, seborrheic keratosis, folliculitis, herpes simplex, herpes zoster, varicella, candidiasis, dermatophytosis, scabies, insect bites, cherry angioma, keloid, dermatofibroma, acrochordons, urticaria, transient acantholytic dermatosis, xerosis, eczema, atopic dermatitis, contact dermatitis, hand eczema, nummular eczema, lichen simplex chronicus, asteatotic eczema, stasis dermatitis and stasis ulceration, seborrheic dermatitis, psoriasis, lichen planus, pityriasis rosea, impetigo, ecthyma, dermatophytosis, tinea versicolor, warts, acne vulgaris, acne rosacea, pemphigus vulgaris, pemphigus foliaceus, paraneoplastic pemphigus, bullous pemphigoid, herpes gestationis, dermatitis herpetiformis, linear IgA disease, epidermolysis bullosa acquisita, dermatomyositis, lupus erythematosus, scleroderma and morphea, erythroderma, alopecia, figurate skin lesions, telangiectasias, hypopigmentation, hyperpigmentation, vesicles/bullae, exanthems, cutaneous drug reactions, papulonodular skin lesions, chronic non-healing wounds, photosensitivity diseases, epidermolysis bullosa simplex, epidermolytic hyperkeratosis, epidermolytic and nonepidermolytic palmoplantar keratoderma, ichthyosis bullosa of Siemens, ichthyosis exfoliativa, keratosis palmaris et plantaris, keratosis palmoplantaris, palmoplantar keratoderma, keratosis punctata, Meesmann's corneal dystrophy, pachyonychia congenita, white sponge nevus, steatocystoma multiplex, epidermal nevi/epidermolytic hyperkeratosis type, monilethrix, trichothiodystrophy, chronic hepatitis/cryptogenic cirrhosis, and colorectal hyperplasia; a neurological disorder, such as epilepsy, ischemic cerebrovascular disease, stroke, cerebral neoplasms, Alzheimer's disease, Pick's disease, Huntington's disease, dementia, Parkinson's disease and other extrapyramidal disorders, amyotrophic lateral sclerosis and other motor neuron disorders, progressive neural muscular atrophy, retinitis pigmentosa, hereditary ataxias, multiple sclerosis and other demyelinating diseases, bacterial and viral meningitis, brain abscess, subdural empyema, epidural abscess, suppurative intracranial thrombophlebitis, myelitis and radiculitis, viral central nervous system disease, prion diseases including kuru, Creutzfeldt-Jakob disease, and Gerstmann-Straussler-Scheinker syndrome, fatal familial insomnia, nutritional and metabolic diseases of the nervous system, neurofibromatosis, tuberous sclerosis, cerebelloretinal hemangioblastomatosis, encephalotrigeminal syndrome, mental retardation and other developmental disorders of the central nervous system including Down syndrome, cerebral palsy, neuroskeletal disorders, autonomic nervous system disorders, cranial nerve disorders, spinal cord diseases, muscular dystrophy and other neuromuscular disorders, peripheral nervous system disorders, dermatomyositis and polymyositis, inherited, metabolic, endocrine, and toxic myopathies, myasthenia gravis, periodic paralysis, mental disorders including mood, anxiety, and schizophrenic disorders, seasonal affective disorder (SAD), akathesia, amnesia, catatonia, diabetic neuropathy, tardive dyskinesia, dystonias, paranoid psychoses, postherpetic neuralgia, Tourette's disorder, progressive supranuclear palsy, corticobasal degeneration, and familial frontotemporal dementia; and a reproductive disorder, such as infertility, including tubal disease, ovulatory defects, and endometriosis, a disorder of prolactin production, a disruption of the estrous cycle, a disruption of the menstrual cycle, polycystic ovary syndrome, ovarian hyperstimulation syndrome, an endometrial or ovarian tumor, a uterine fibroid, autoimmune disorders, an ectopic pregnancy, and teratogenesis; cancer of the breast, fibrocystic breast disease, and galactorrhea; a disruption of spermatogenesis, abnormal sperm physiology, cancer of the testis, cancer of the prostate, benign prostatic hyperplasia, prostatitis, Peyronie's disease, impotence, carcinoma of the male breast, and gynecomastia.
In another embodiment, a vector capable of expressing PRTS or a fragment or derivative thereof may be administered to a subject to treat or prevent a disorder associated with decreased expression or activity of PRTS including, but not limited to, those described above.

In a further embodiment, a composition comprising a substantially purified PRTS in conjunction with a suitable pharmaceutical carrier may be administered to a subject to treat or prevent a disorder associated with decreased expression or activity of PRTS including, but not limited to, those provided above.
In still another embodiment, an agonist which modulates the activity of PRTS
may be administered to a subject to treat or prevent a disorder associated with decreased expression or activity of PRTS including, but not limited to, those listed above.
In a further embodiment, an antagonist of PRTS may be administered to a subject to treat or prevent a disorder associated with increased expression or activity of PRTS.
Examples of such disorders include, but are not limited to, those gastrointestinal, cardiovascular, autoimmune/inflammatory, cell proliferative, developmental, epithelial, neurological, and reproductive disorders described above. In one aspect, an antibody which specifically binds PRTS
may be used directly as an antagonist or indirectly as a targeting or delivery mechanism for bringing a pharmaceutical agent to cells or tissues which express PRTS.
In an additional embodiment, a vector expressing the complement of the polynucleotide encoding PRTS may be administered to a subject to treat or prevent a disorder associated with increased expression or activity of PRTS including, but not limited to, those described above.
In other embodiments, any of the proteins, antagonists, antibodies, agonists, complementary sequences, or vectors of the invention may be administered in combination with other appropriate therapeutic agents. Selection of the appropriate agents for use in combination therapy may be made by one of ordinary skill in the art, according to conventional pharmaceutical principles. The combination of therapeutic agents may act synergistically to effect the treatment or prevention of the various disorders described above. Using this approach, one may be able to achieve therapeutic efficacy with lower dosages of each agent, thus reducing the potential for adverse side effects.
An antagonist of PRTS may be produced using methods which are generally known in the art.
In particular, purified PRTS may be used to produce antibodies or to screen libraries of pharmaceutical agents to identify those which specifically bind PRTS.
Antibodies to PRTS may also ~be generated using methods that are well known in the art. Such antibodies may include, but are not limited to, polyclonal, monoclonal, chimeric, and single chain antibodies, Fab fragments, and fragments produced by a Fab expression library. Neutralizing antibodies (i.e., those which inhibit dimer formation) are generally preferred for therapeutic use.
For the production of antibodies, various hosts including goats, rabbits, rats, mice, humans, and others may be immunized by injection with PRTS or with any fragment or oligopeptide thereof which has 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, KLH, and dinitrophenol. Among adjuvants used in humans, BCG (bacilli Calmette-Guerin) and Corynebacterium parvum are especially preferable.
It is preferred that the oligopeptides, peptides, or (fragments used to induce antibodies to PRTS have an amino acid sequence consisting of at least about 5 amino acids, and generally will consist of at least about 10 amino acids. It is also preferable that these oligopeptides, peptides, or fragments are identical to a portion of the amino acid sequence of the natural protein. Short stretches of PRTS amino acids may be fused with those of another protein, such as KLH, and antibodies to the chimeric molecule may be produced.
Monoclonal antibodies to PRTS 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, the human B-cell hybridoma technique, and the EBV-hybridoma technique. (See, e.g., Kohler, G. et al. (1975) Nature 256:495-497; Kozbor, D.
et al. (1985) J.
hnmunol. Methods 81:31-42; Cote, R.J. et al. (1983) Proc. Natl. Acad. Sci. USA
80:2026-2030; and Cole, S.P. et al. (1984) Mol. Cell Biol. 62:109-120.) In addition, techniques developed for the production of "chimeric antibodies,"
such as the splicing of mouse antibody genes to human antibody genes to obtain a molecule with appropriate antigen specificity and biological activity, can be used. (See, e.g., Morrison, S.L. et al. (1984) Proc.
Natl. Acad. Sci. USA 81:6851-6855; Neuberger, M.S. et al. (1984) Nature 312:604-608; and Takeda, S. et al. (1985) Nature 314:452-454.) Alternatively, techniques described for the production of single chain antibodies may be adapted, using methods known in the art, to produce PRTS-specific single chain antibodies. Antibodies with related specificity, but of distinct idiotypic composition, may be generated by chain shuffling from random combinatorial immunoglobulin libraries. (See, e.g., Burton, D.R. (1991) Proc. Natl. Acad. Sci. USA 88:10134-10137.) Antibodies may also be produced by inducing in vivo production in the lymphocyte population or by screening immunoglobulin libraries or panels of highly specific binding reagents as disclosed in the literature. (See, e.g., Orlandi, R. et al. (1989) Proc. Natl.
Acad. Sci. USA
86:3833-3837; Winter, G. et al. (1991) Nature 349:293-299.) Antibody fragments which contain specific binding sites for PRTS may also be generated.
For example, such fragments include, but are not limited to, F(ab')2 fragments produced by pepsin digestion of the antibody molecule and Fab fragments generated by reducing the disulfide bridges of the F(ab')2 fragments. Alternatively, Fab expression libraries may be constructed to allow rapid and easy identification of monoclonal Fab fragments with the desired specificity.
(See, e.g., Huse, W.D.
et al. (1989) Science 246:1275-1281.) Various immunoassays may be used for screening to identify antibodies having the desired specificity. Nwnerous 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 measurement of complex formation between PRTS and its specific antibody. A two-site, monoclonal-based immunoassay utilizing monoclonal antibodies reactive to two non-interfering PRTS epitopes is generally used, but a competitive binding assay may also be employed (Pound, supra).
Various methods such as Scatchard analysis in conjunction with radioimmunoassay techniques may be used to assess the affinity of antibodies for PRTS. Affinity is expressed as an association constant, Ka, which is defined as the molar concentration of PRTS-antibody complex divided by the molar concentrations of free antigen and free antibody under equilibrium conditions.
The Ka determined for a preparation of polyclonal antibodies, which are heterogeneous in their affinities for multiple PRTS epitopes, represents the average affinity, or avidity, of the antibodies for PRTS. The Ka determined for a preparation of monoclonal antibodies, which are monospecific for a particular PRTS epitope, represents a true measure of affinity. High-affinity antibody preparations with Ka ranging from about 109 to 1012 Llmole are preferred for use in immunoassays in which the PRTS-antibody complex must withstand rigorous manipulations. Low-affinity antibody preparations with Ka ranging from about 106 to 10' L/mole are preferred for use in immunopurification and similar procedures which ultimately require dissociation of PRTS, preferably in active form, from the antibody (Catty, D. (1988) Antibodies, Volume I: A Practical Ap rp oath, IRL
Press, Washington DC;
Liddell, J.E. and A. Cryer (1991) A Practical Guide to Monoclonal Antibodies, John Wiley & Sons, New York NY).
The titer and avidity of polyclonal antibody preparations may be further evaluated to determine the quality and suitability of such preparations for certain downstream applications. For example, a polyclonal antibody preparation containing at least 1-2 mg specific antibody/ml, preferably 5-10 mg specific antibody/ml, is generally employed in procedures requiring precipitation of PRTS-antibody complexes. Procedures for evaluating antibody specificity, titer, and avidity, and guidelines for antibody quality and usage in various applications, are generally available. (See, e.g., Catty, supra, and Coligan et al. supra.) In another embodiment of the invention, the polynucleotides encoding PRTS, or any fragment or complement thereof, may be used for therapeutic purposes. In one aspect, modifications of gene expression can be achieved by designing complementary sequences or antisense molecules (DNA, RNA, PNA, or modified oligonucleotides) to the coding or regulatory regions of the gene encoding PRTS. Such technology is well known in the art, and antisense oligonucleotides or larger fragments can be designed from various locations along the coding or control regions of sequences encoding PRTS. (See, e.g., Agrawal, S., ed. (1996) Antisense Thera ep utics, Humana Press Inc., Totawa NJ.) In therapeutic use, any gene delivery system suitable for introduction of the antisense sequences into appropriate target cells can be used. Antisense sequences can be delivered intracellularly in the form of an expression plasmid which, upon transcription, produces a sequence complementary to at least a portion of the cellular sequence encoding the target protein. (See, e.g., Slater, J.E. et al. (1998) J. Allergy Clin. Immunol. 102(3):469-475; and Scanlon, K.J. et al. (1995) 9(13):1288-1296.) Antisense sequences can also be introduced intracellularly through the use of viral vectors, such as retrovirus and adeno-associated virus vectors. (See, e.g., Miller, A.D. (1990) Blood 76:271; Ausubel, supra; Uckert, W. and W. Walther (1994) Pharmacol. Ther.
63(3):323-347.) Other gene delivery mechanisms include liposome-derived systems, artificial viral envelopes, and other systems known in the art. (See, e.g., Rossi, J.J. (1995) Br. Med. Bull.
51(1):217-225; Boado, R.J. et al. (1998) J. Pharm. Sci. 87(11):1308-1315; and Morris, M.C. et al. (1997) Nucleic Acids Res.
25(14):2730-2736.) In another embodiment of the invention, polynucleotides encoding PRTS may be used for somatic or germline gene therapy. Gene therapy may be performed to (i) correct a genetic deficiency (e.g., in the cases of severe combined immunodeBciency (SCID)-X1 disease characterized by X-linked inheritance (Cavazzana-Calvo, M. et al. (2000) Science 288:669-672), severe combined immunodeficiency syndrome associated with an inherited adenosine deaminase (ADA) deficiency (Blaese, R.M. et al. (1995) Science 270:475-480; Bordignon, C. et al. (1995) Science 270:470-475), cystic fibrosis (Zabner, J. et al. (1993) Cell 75:207-216; Crystal, R.G. et al. (1995) Hum. Gene Therapy 6:643-666; Crystal, R.G. et al. (1995) Hum. Gene Therapy 6:667-703), thalassamias, familial hypercholesterolemia, and hemophilia resulting from Factor VIII or Factor IX
deficiencies (Crystal, R.G. (1995) Science 270:404-410; Verma, LM. and N. Somia (1997) Nature 389:239-242)), (ii) express a conditionally lethal gene product (e.g., in the case of cancers which result from unregulated cell proliferation), or (iii) express a protein which affords protection against intracellular parasites (e.g., against human retroviruses, such as human immunodeficiency virus (HIV) (Baltimore, D.
(1988) Nature 335:395-396; Poeschla, E. et al. (1996) Proc. Natl. Acad. Sci.
USA. 93:11395-11399), hepatitis B or C virus (HBV, HCV); fungal parasites, such as Candida albicans and Paracoccidioides brasiliensis; and protozoan parasites such as Plasmodium falciparum and Trypanosoma cruzi). In the case where a genetic deficiency in PRTS expression or regulation causes disease, the expression of PRTS from an appropriate population of transduced cells may alleviate the clinical manifestations caused by the genetic deficiency.
In a further embodiment of the invention, diseases or disorders caused by deficiencies in PRTS are treated by constructing mammalian expression vectors encoding PRTS
and introducing these vectors by mechanical means into PRTS-deficient cells. Mechanical transfer technologies for use with cells in vivo or ex vitro include (i) direct DNA microinjection into individual cells, (ii) ballistic gold particle delivery, (iii) liposome-mediated transfection, (iv) receptor-mediated gene transfer, and (v) the use of DNA transposons (Morgan, R.A. and W.F. Anderson (1993) Annu. Rev.
Biochem. 62:191-217; Ivics, Z. (1997) Cell 91:501-510; Boulay, J-L. and H.
Recipon (1998) Curr.
Opin. Biotechnol. 9:445-450).
Expression vectors that may be effective for the expression of PRTS include, but are not limited to, the PCDNA 3.1, EPITAG, PRCCMV2, PREP, PVAX, PCR2-TOPOTA vectors (Invitrogen, Carlsbad CA), PCMV-SCRIPT, PCMV-TAG, PEGSH/PERV (Stratagene, La Jolla CA), and PTET-OFF, PTET-ON, PTRE2, PTRE2-LUC, PTK-HYG (Clontech, Palo Alto CA).
PRTS may be expressed using (i) a constitutively active promoter, (e.g., from cytomegalovirus (CMV), Rous sarcoma virus (RSV), SV40 virus, thymidine kinase (TK), or ~3-actin genes), (ii) an inducible promoter (e.g., the tetracycline-regulated promoter (Gossen, M. and H. Bujard (1992) Proc. Natl.
Acad. Sci. USA 89:5547-5551; Gossen, M. et al. (1995) Science 268:1766-1769;
Rossi, F.M.V. and H.M. Blau (1998) Curr. Opin. Biotechnol. 9:451-456), commercially available in the T-REX plasmid (Invitrogen)); the ecdysone-inducible promoter (available in the plasmids PVGRXR and PIIVD;
Invitrogen);,the FK506/rapamycin inducible promoter; or the RU486/mifepristone inducible promoter (Rossi, F.M.V. and H.M. Blau, su ra)), or (iii) a tissue-specific promoter or the native promoter of the endogenous gene encoding PRTS from a normal individual.
Commercially available liposome transformation kits (e.g., the PERFECT LIPID
TRANSFECTION KIT, available from Invitrogen) allow one with ordinary skill in the art to deliver polynucleotides to target cells in culture and require minimal effort to optimize experimental parameters. In the alternative, transformation is performed using the calcium phosphate method (Graham, F.L. and A.J. Eb (1973) Virology 52:456-467), ox by electroporation (Neumann, E. et al.
(1982) EMBO J. 1:841-845). The introduction of DNA to primary cells requires modification of these standardized mammalian transfection protocols.
In another embodiment of the invention, diseases or disorders caused by genetic defects with respect to PRTS expression are treated by constructing a retrovirus vector consisting of (i) the polynucleotide encoding PRTS under the control of an independent promoter or the retrovirus long terminal repeat (LTR) promoter, (ii) appropriate RNA packaging signals, and (iii) a Rev-responsive element (RRE) along with additional retrovirus cis-acting RNA sequences and coding sequences required for efficient vector propagation. Retrovirns vectors (e.g., PFB and PFBNEO) are commercially available (Stratagene) and are based on published data (Riviere, I. et al. (1995) Proc.
Natl. Acad. Sci. USA 92:6733-6737), incorporated by reference herein. The vector is propagated in an appropriate vector producing cell line (VPCL) that expresses an envelope gene with a tropism for receptors on the target cells or a promiscuous envelope protein such as VSVg (Armentano, D. et al.
(1987) J. Virol. 61:1647-1650; Bender, M.A. et al. (1987) J. Virol. 61:1639-1646; Adam, M.A. and A.D. Miller (1988) J. Virol. 62:3802-3806; Dull, T. et al. (1998) J. Virol.
72:8463-8471; Zufferey, R.
et al. (1998) J. Virol. 72:9873-9880). U.S. Patent No. 5,910,434 to Rigg ("Method for obtaining retrovirus packaging cell lines producing high transducing efficiency retroviral supernatant") discloses a method for obtaining retrovirus packaging cell lines and is hereby incorporated by reference. Propagation of retrovirus vectors, transduction of a population of cells (e.g., CD4+ T-cells), and the return of transduced cells to a patient are procedures well known to persons skilled in the art of gene therapy and have been well documented (Ranga, U. et al. (1997) J. Virol. 71:7020-7029; Bauer, G. et al. (1997) Blood 89:2259-2267; Bonyhadi, M.L. (1997) J.
Virol. 71:4707-4716;
Ranga, U. et al. (1998) Proc. Natl. Acad. Sci. USA 95:1201-1206; Su, L. (1997) Blood 89:2283-2290).
In the alternative, an adenovirus-based gene therapy delivery system is used to deliver polynucleotides encoding PRTS to cells which have one or more genetic abnormalities with respect to the expression of PRTS. The construction and packaging of adenovirus-based vectors are well known to those with ordinary skill in the art. Replication defective adenovirus vectors have proven to be versatile for importing genes encoding immunoregulatory proteins into intact islets in the pancreas (Csete, M.E. et al. (1995) Transplantation 27:263-268). Potentially useful adenoviral vectors are described in U.S. Patent No. 5,707,618 to Armentano ("Adenovirus vectors for gene therapy"), hereby incorporated by reference. For adenoviral vectors, see also Antinozzi, P.A. et al. (1999) Annu. Rev.
Nutr. 19:511-544 and Verma, LM. and N. Somia (1997) Nature 18:389:239-242, both incorporated by reference herein.
In another alternative, a herpes-based, gene therapy delivery system is used to deliver polynucleotides encoding PRTS to target cells which have one or more genetic abnormalities with respect to the expression of PRTS. The use of herpes simplex virus (HSV)-based vectors may be especially valuable for introducing PRTS to cells of the central nervous system, for which HSV has a tropism. The construction and packaging of herpes-based vectors are well known to those with ordinary skill in the art. A replication-competent herpes simplex virus (HSV) type 1-based vector has been used to deliver a reporter gene to the eyes of primates (Liu, X. et al.
(1999) Exp. Eye Res.
169:385-395). The construction of a HSV-1 virus vector has also been disclosed in detail in U.S.
Patent No. 5,804,413 to DeLuca ("Herpes simplex virus strains for gene transfer"), which is hereby incorporated by reference. U:S. Patent No. 5,804,413 teaches the use of recombinant HSV d92 which consists of a genome containing at least one exogenous gene to be transferred to a cell under the control of the appropriate promoter for purposes including human gene therapy.
Also taught by this patent are the construction and use of recombinant HSV strains deleted for ICP4, ICP27 and ICP22.

For HSV vectors, see also Goins, W.F. et al. (1999) J. Virol. 73:519-532 and Xu, H. et al. (1994) Dev. Biol. 163:152-161, hereby incorporated by reference. The manipulation of cloned heipesvirus sequences, the generation of recombinant virus following the transfection of multiple plasmids containing different segments of the large herpesvirus genomes, the growth and propagation of herpesvirus, and the infection of cells with herpesvirus are techniques well lrnown to those of ordinary skill in the art.
In another alternative, an alphavirus (positive, single-stranded RNA vixus) vector is used to deliver polynucleotides encoding PRTS to target cells. The biology of the prototypic alphavirus, Semliki Forest Virus (SFV), has been studied extensively and gene transfer vectors have been based on the SFV genome (Garoff, H. and K.-J. Li (1998) Curr. Opin. Biotechnol.
9:464-469). During alphavirus RNA replication, a subgenomic RNA is generated that normally encodes the viral capsid proteins. This subgenomic RNA replicates to higher levels than the full length genomic RNA, resulting in the overproduction of capsid proteins relative to the viral proteins with enzymatic activity (e.g., protease and polymerase). Similarly, inserting the coding sequence for PRTS into the alphavirus genome in place of the capsid-coding region results in the production of a large number of PRTS-coding RNAs and the synthesis of high levels of PRTS in vector transduced cells. While alphavirus infection is typically associated with cell lysis within a few days, the ability to establish a persistent infection in hamster normal kidney cells (BHK-21) with a variant of Sindbis virus (SIN) indicates that the lytic replication of alphaviruses can be altered to suit the needs of the gene therapy application (Dryga, S.A. et al. (1997) Virology 228:74-83). The wide host range of alphaviruses will allow the introduction of PRTS into a variety of cell types. The specific transduction of a subset of cells in a population may require the sorting of cells prior to transduction.
The methods of manipulating infectious cDNA clones of alphaviruses, performing alphavirus cDNA and RNA
transfections, and performing alphavirus infections, are well known to those with ordinary skill in the art.
Oligonucleotides derived from the transcription initiation site, e.g., between about positions -10 and +10 from the start site, may also be employed to inhibit gene expression. Similarly, inhibition can be achieved using triple helix base-pairing methodology. Triple helix pairing is useful because it causes inhibition of 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 have been described in the literature. (See, e.g., Gee, J.E. et al. (1994) in Huber, B.E.
and B.I. Carr, Molecular and Immunolo icg-Approaches, Futura Publishing, Mt.
Kisco NY, pp. 163-177.) A complementary sequence or antisense molecule may also be designed to block translation of mRNA by preventing the transcript from binding to ribosomes.

Ribozymes, enzymatic RNA molecules, may also be used to catalyze 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.
For example, engineered hammerhead motif ribozyme molecules may specifically and efficiently catalyze endonucleolytic cleavage of sequences encoding PRTS.
Specific ribozyme cleavage sites within any potential RNA target are initially identified by scanning the target molecule for ribozyme cleavage sites, including 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 inoperable.
The suitability of candidate targets may also be evaluated by testing accessibility to hybridization with complementary oligonucleotides using ribonuclease protection assays.
Complementary ribonucleic acid molecules and ribozymes of the invention may be prepared by any method known in the art for the synthesis of nucleic acid 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 PRTS. Such DNA sequences may be incorporated into a wide variety of vectors with suitable RNA polymerase promoters such as T7 or SP6. Alternatively, these cDNA constructs that synthesize complementary 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-, thin-, and similarly modified forms of adenine, cytidine, guanine, thymine, and uridine which are not as easily recognized by endogenous endonucleases.
An additional embodiment of the invention encompasses a method for screening for a compound which is effective in altering expression of a polynucleotide encoding PRTS. Compounds which may be effective in altering expression of a specific polynucleotide may include, but are not limited to, oligonucleotides, antisense oligonucleotides, triple helix-forming oligonucleotides, transcription factors and other polypeptide transcriptional regulators, and non-macromolecular chemical entities which are capable of interacting with specific polynucleotide sequences. Effective compounds may alter polynucleotide expression by acting as either inhibitors or promoters of polynucleotide expression. Thus, in the treatment of disorders associated with increased PRTS
expression or activity, a compound which specifically inhibits expression of the polynucleotide encoding PRTS may be therapeutically useful, and in the treatment of disorders associated with decreased PRTS expression or activity, a compound which specifically promotes expression of the polynucleotide encoding PRTS may be therapeutically useful.
At least one, and up to a plurality, of test compounds may be screened for effectiveness in altering expression of a specific polynucleotide. A test compound may be obtained by any method commonly known in the art, including chemical modification of a compound known to be effective in altering polynucleotide expression; selection from an existing, commercially-available or proprietary library of naturally-occurring or non-natural chemical compounds; rational design of a compound based on chemical andlor structural properties of the target polynucleotide;
and selection from a library of chemical compounds created combinatorially or randomly. A sample comprising a polynucleotide encoding PRTS is exposed to at least one test compound thus obtained. The sample may comprise, for example, an intact or permeabilized cell, or an in vitro cell-free or reconstituted biochemical system. Alterations in the expression of a polynucleotide encoding PRTS are assayed by any method commonly known in the art. Typically, the expression of a specific nucleotide is detected by hybridization with a probe having a nucleotide sequence complementary to the sequence of the polynucleotide encoding PRTS. The amount of hybridization may be quantified, thus forming the basis for a comparison of the expression of the polynucleotide both with and without exposure to one or more test compounds. Detection of a change in the expression of a polynucleotide exposed to a test compound indicates that the test compound is effective in altering the expression of the polynucleotide. A screen for a compound effective in altering expression of a specific polynucleotide can be carried out, for example, using a Schizosaccharomyces pombe gene expression system (Atkins, D. et al. (1999) U.S. Patent No. 5,932,435; Arndt, G.M. et al. (2000) Nucleic Acids Res.
28:E15) or a human cell line such as HeLa cell (Clarke, M.L. et al. (2000) Biochem. Biophys. Res.
Commun. 268:8-13). A particular embodiment of the present invention involves screening a combinatorial library of oligonucleotides (such as deoxyribonucleotides, ribonucleotides, peptide nucleic acids, and modified oligonucleotides) for antisense activity against a specific polynucleotide sequence (Bruice, T.W. et al. (1997) U.S. Patent No. 5,686,242; Bruice, T.W.
et al. (2000) U.S.
Patent No. 6,022,691).
Many methods for introducing vectors into cells or tissues are available and equally suitable for use in vivo, in vitro, and ex vivo. For ex vivo therapy, vectors may be introduced into stem cells taken from the patient and clonally propagated for autologous transplant back into that same patient.
Delivery by transfection, by liposome injections, or by polycationic amino polymers may be achieved using methods which are well known in the art. (See, e.g., Goldman, C.K. et al. (1997) Nat.
Biotechnol. 15:462-466.) Any of the therapeutic methods described above may be applied to any subject in need of such therapy, including, for example, mammals such as humans, dogs, cats, cows, horses, rabbits, and monkeys.
An additional embodiment of the invention relates to the administration of a composition which generally comprises an active ingredient formulated with a pharmaceutically acceptable excipient. Excipients may include, for example, sugars, starches, celluloses, gums, and proteins.
Various formulations are commonly known and are thoroughly discussed in the latest edition of Remin~,ton's Pharmaceutical Sciences (Maack Publishing, Easton PA). Such compositions may consist of PRTS, antibodies to PRTS, and mimetics, agonists, antagonists, or inhibitors of PRTS.
The compositions utilized in this invention may be administered by any number of routes including, but not limited to, oral, intravenous, intramuscular, infra-arterial, intramedullary, intrathecal, intraventricular, pulmonary, transdermal, subcutaneous, intraperitoneal, intranasal, enteral, topical, sublingual, or rectal means.
Compositions for pulmonary administration may be prepared in liquid or dry powder form.
These compositions are generally aerosolized immediately prior to inhalation by the patient. In the case of small molecules (e.g. traditional low molecular weight organic drugs), aerosol delivery of fast-acting formulations is well-known in the art. In the case of macromolecules (e.g. larger peptides and proteins), recent developments in the field of pulmonary delivery via the alveolar region of the lung have enabled the practical delivery of drugs such as insulin to blood circulation (see, e.g., Patton, J.S. et al., U.S. Patent No. 5,997,848). Pulmonary delivery has the advantage of administration without needle injection, and obviates the need fox potentially toxic penetration enhancers.
Compositions suitable for use in the 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.
Specialized forms of compositions may be prepared for direct intracellular delivery of macromolecules comprising PRTS or fragments thereof. For example, liposome preparations containing a cell-impermeable macromolecule may promote cell fusion and intracellular delivery of the macromolecule. Alternatively, PRTS or a fragment thereof may be joined to a short cationic N-terminal portion from the HIV Tat-1 protein. Fusion proteins thus generated have been found to transduce into the cells of all tissues, including the brain, in a mouse model system (Schwarze, S.R. et al. (1999) Science 285:1569-1572).
For any compound, the therapeutically effective dose can be estimated initially either in cell culture assays, e.g., of neoplastic cells, or in animal models such as mice, rats, rabbits, dogs, monkeys, or pigs. An animal model may also be used to determine the appropriate concentration range and route of administration. Such information can then be used to determine useful doses and routes for administration in humans.
A therapeutically effective dose refers to that amount of active ingredient, for example PRTS
or fragments thereof, antibodies of PRTS, and agonists, antagonists or inhibitors of PRTS, which ameliorates the symptoms or condition. Therapeutic efficacy and toxicity may be determined by standard pharmaceutical procedures in cell cultures or with experimental animals, such as by calculating the EDSO (the dose therapeutically effective in 50% of the population) or LDso (the dose lethal to 50% of the population) statistics. The dose ratio of toxic to therapeutic effects is the therapeutic index, which can be expressed as the LDSO/EDSO ratio. Compositions which exhibit large therapeutic indices are preferred. The data obtained from cell culture assays and animal studies are used to formulate a range of dosage for human use. The dosage contained in such compositions is preferably within a range of circulating concentrations that includes the EDSO
with little or no toxicity.
The dosage varies within this range depending upon the dosage form employed, the sensitivity of the patient, and the route of administration.
The exact dosage will be determined by the practitioner, in light of factors related to the subject requiring treatment. Dosage and administration are adjusted to provide sufficient levels of the active moiety or to maintain the desired effect. Factors which may be taken into account include the severity of the disease state, the general health of the subject, the age, weight, and gender of the subject, time and frequency of administration, drug combination(s), reaction sensitivities, and response to therapy. Long-acting compositions may be administered every 3 to 4 days, every week, or biweekly depending on the half life and clearance rate of the particular formulation.
Normal dosage amounts may vary from about O.l ,ug to 100,000 ~.g, up to a total dose of about 1 gram, depending upon the route of administration. Guidance as to particular dosages and methods of delivery is provided in the literature and generally available to practitioners in the art.
Those skilled in the art will employ different formulations for nucleotides than for proteins or their inhibitors. Similarly, delivery of polynucleotides or polypeptides will be specific to particular cells, conditions, locations, etc.
DIAGNOSTICS
In another embodiment, antibodies which specifically bind PRTS may be used for the diagnosis of disorders characterized by expression of PRTS, or in assays to monitor patients being treated with PRT5 or agonists, antagonists, or inhibitors of PRTS. Antibodies useful for diagnostic purposes may be prepared in the same manner as described above for therapeutics. Diagnostic assays for PRTS include methods which utilize the antibody and a label to detect PRTS
in human body fluids or in extracts of cells or tissues. The antibodies may be used with or without modification, and may be labeled by covalent or non-covalent attachment of a reporter molecule.
A wide variety of reporter molecules, several of which are described above, are known in the art and may be used.
A variety of protocols for measuring PRTS, including ELISAs, RIAs, and FAGS, are known in the art and provide a basis for diagnosing altered or abnormal levels of PRTS expression. Normal or standard values for PRTS expression are established by combining body fluids or cell extracts taken from normal mammalian subjects, for example, human subjects, with antibodies to PRTS under conditions suitable for complex formation. The amount of standard complex formation may be quantitated by various methods, such as photometric means. Quantities of FRTS
expressed in subject, control, and disease samples from biopsied tissues are compared with the standard values.
Deviation between standard and subject values establishes the parameters for diagnosing disease.
In another embodiment of the invention, the polynucleotides encoding PRTS may be used for diagnostic purposes. The polynucleotides which may be used include oligonucleotide sequences, complementary RNA and DNA molecules, and PNAs. The polynucleotides may be used to detect and quantify gene expression in biopsied tissues in which expression of PRTS
may be correlated with disease. The diagnostic assay may be used to determine absence, presence, and excess expression of PRTS, and to monitor regulation of PRTS levels during therapeutic intervention.
In one aspect, hybridization with PCR probes which are capable of detecting polynucleotide sequences, including genomic sequences, encoding PRTS or closely related molecules may be used to identify nucleic acid sequences which encode PRTS. The specificity of the probe, whether it is made from a highly specific region, e.g., the 5'regulatory region, or from a less specific region, e.g., a conserved motif, and the stringency of the hybridization or amplification will determine whether the probe identifies only naturally occurring sequences encoding PRTS, allelic variants, or related sequences.
Probes may also be used for the detection of related sequences, and may have at least 50%
sequence identity to any of the PRTS encoding sequences. The hybridization probes of the subject invention may be DNA or RNA and may be derived from the sequence of SEQ ID
N0:16-30 or from genomic sequences including promoters, enhancers, and introns of the PRTS
gene.
Means for producing specific hybridization probes for DNAs encoding PRTS
include the cloning of polynucleotide sequences encoding PRTS or PRTS derivatives into vectors for the production of mRNA probes. Such vectors are known in the art, are commercially available, and may be used to synthesize RNA probes in vitro by means of the addition of the appropriate RNA
polymerases and the appropriate labeled nucleotides. Hybridization probes may be labeled by a variety of reporter groups, for example, by radionuclides such as 32P or 355, or by enzymatic labels, such as alkaline phosphatase coupled to the probe via avidin/biotin coupling systems, and the like.

Polynucleotide sequences encoding PRTS may be used for the diagnosis of disorders associated with expression of PRTS. Examples of such disorders include, but are not limited to, a gastrointestinal disorder, such as dysphagia, peptic esophagitis, esophageal spasm, esophageal stricture, esophageal carcinoma, dyspepsia, indigestion, gastritis, gastric carcinoma, anorexia, nausea, emesis, gastroparesis, antral or pyloric edema, abdominal angina, pyrosis, gastroenteritis, intestinal obstruction, infections of the intestinal tract, peptic ulcer, cholelithiasis, cholecystitis, cholestasis, pancreatitis, pancreatic carcinoma, biliary tract disease, hepatitis, hyperbilirubinemia, cirrhosis, passive congestion of the liver, hepatoma, infectious colitis, ulcerative colitis, ulcerative proctitis, Crohn's disease, Whipple's disease, Mallory-Weiss syndrome, colonic carcinoma, colonic obstruction, irntable bowel syndrome, short bowel syndrome, diarrhea, constipation, gastrointestinal hemorrhage, acquired immunodeficiency syndrome (A)DS) enteropathy, jaundice, hepatic encephalopathy, hepatorenal syndrome, hepatic steatosis, hemochromatosis, Wilson's disease, alphal-antitrypsin deficiency, Reye's syndrome, primary sclerosing cholangitis, liver infarction, portal vein obstruction and thrombosis, centrilobular necrosis, peliosis hepatis, hepatic vein thrombosis, veno-occlusive disease, preeclampsia, eclampsia, acute fatty liver of pregnancy, intrahepatic cholestasis of pregnancy, and hepatic tumors including nodular hyperplasias, adenomas, and carcinomas; a cardiovascular disorder, such as arteriovenous fistula, atherosclerosis, hypertension, vasculitis, Raynaud's disease, aneurysms, arterial dissections, varicose veins, thrombophlebitis and phlebothrombosis, vascular tumors, and complications of thrombolysis, balloon angioplasty, vascular replacement, and coronary artery bypass graft surgery, congestive heart failure, ischemic heart disease, angina pectoris, myocardial infarction, hypertensive heart disease, degenerative valvular heart disease, calcific aortic valve stenosis, congenitally bicuspid aortic valve, mitral annular calcification, mitral valve prolapse, rheumatic fever and rheumatic heart disease, infective endocarditis, nonbacterial thrombotic endocarditis, endocarditis of systemic lupus erythematosus, carcinoid heart disease, cardiomyopathy, myocarditis, pericarditis, neoplastic heart disease, congenital heart disease, and complications of cardiac transplantation; an autoimmune/inflammatory disorder, such as acquired immunodeficiency syndrome (A)DS), Addison's disease, adult respiratory distress syndrome, allergies, ankylosing spondylitis, amyloidosis, anemia, asthma, atherosclerosis, atherosclerotic plaque rupture, autoimmune hemolytic anemia, autoimmune thyroiditis, autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED), bronchitis, cholecystitis, contact dermatitis, Crohn's disease, atopic dermatitis, dermatomyositis, diabetes mellitus, emphysema, episodic lymphopenia with lymphocytotoxins, erythroblastosis fetalis, erythema nodosum, atrophic gastritis, glomerulonephritis, Goodpasture's syndrome, gout, Graves' disease, Hashimoto's thyroiditis, hypereosinophilia, irritable bowel syndrome, multiple sclerosis, myasthenia gravis, myocardial or pericardial inflammation, osteoarthritis, degradation of articular cartilage, osteoporosis, pancreatitis, polymyositis, psoriasis, Reiter's syndrome, rheumatoid arthritis, scleroderma, Sjogren's syndrome, systemic anaphylaxis, systemic lupus erythematosus, systemic sclerosis, thrombocytopenic purpura, ulcerative colitis, uveitis, Werner syndrome, complications of cancer, hemodialysis, and extracorporeal circulation, viral, bacterial, fungal, parasitic, protozoal, and helminthic infections, and trauma; a cell proliferative disorder such as actinic keratosis, arteriosclerosis, atherosclerosis, bursitis, cirrhosis, hepatitis, mixed connective tissue disease (MCTD), myelofibrosis, paroxysmal nocturnal hemoglobinuria, polycythemia vera, psoriasis, primary thrombocythemia, and cancers including adenocarcinoma, leukemia, lymphoma, melanoma, myeloma, sarcoma, teratocarcinoma, and, in particular, cancers of the adrenal gland, bladder, bone, bone marrow, brain, breast, cervix, gall bladder, ganglia, gastrointestinal tract, heart, kidney, liver, lung, muscle, ovary, pancreas, parathyroid, penis, prostate, salivary glands, skin, spleen, testis, thymus, thyroid, and uterus; a developmental disorder, such as renal tubular acidosis, anemia, Cushing's syndrome, achondroplastic dwarfism, Duchenne and Becker muscular dystrophy, bone resorption, epilepsy, gonadal dysgenesis, WAGR
syndrome (Wilms' tumor, aniridia, genitourinary abnormalities, and mental retardation), Smith-Magenis syndrome, myelodysplastic syndrome, hereditary mucoepithelial dysplasia, hereditary keratodermas, hereditary neuropathies such as Charcot-Marie-Tooth disease and neurofibromatosis, hypothyroidism, hydrocephalus, seizure disorders such as Syndenham's chorea and cerebral palsy, spins bifida, anencephaly, craniorachischisis, congenital glaucoma, cataract, age-related macular degeneration, and sensorineural hearing loss; an epithelial disorder, such as dyshidrotic eczema, allergic contact dermatitis, keratosis pilaris, melasma, vitiligo, actinic keratosis, basal cell carcinoma, squamous cell carcinoma, seborrheic keratosis, folliculitis, herpes simplex, herpes zoster, varicella, candidiasis, dermatophytosis, scabies, insect bites, cherry angioma, keloid, dermatofibroma, acrochordons, urticaria, transient acantholytic dermatosis, xerosis, eczema, atopic dermatitis, contact dermatitis, hand eczema, nummular eczema, lichen simplex chronicus, asteatotic eczema, stasis dermatitis and stasis ulceration, seborrheic dermatitis, psoriasis, lichen planus, pityriasis roses, impetigo, ecthyma, dermatophytosis, tines versicolor, warts, acne vulgaris, acne rosacea, pemphigus vulgaris, pemphigus foliaceus, paraneoplastic pemphigus, bullous pemphigoid, herpes gestationis, dermatitis herpetiformis, linear IgA disease, epidermolysis bullosa acquisita, dermatomyositis, lupus erythematosus, scleroderma and morphea, erythroderma, alopecia, figurate skin lesions, telangiectasias, hypopigmentation, hyperpigmentation, vesicleslbullae, exanthems, cutaneous drug reactions, papulonodular skin lesions, chronic non-healing wounds, photosensitivity diseases, epidermolysis bullosa simplex, epidermolytic hyperkeratosis, epidermolytic and nonepidermolytic palmoplantar keratoderma, ichthyosis bullosa of Siemens, ichthyosis exfoliativa, keratosis palmaris et plantaris, keratosis palmoplantaris, palmoplantar keratoderma, keratosis punctata, Meesmann's corneal dystrophy, pachyonychia congenita, white sponge nevus, steatocystoma multiplex, epidermal nevi/epidermolytic hyperkeratosis type, monilethrix, trichothiodystrophy, chronic hepatitis/cryptogenic cirrhosis, and colorectal hypezplasia; a neurological disorder, such as epilepsy, ischemic cerebrovascular disease, stroke, cerebral neoplasms, Alzheimer's disease, Pick's disease, Huntington's disease, dementia, Parkinson's disease and other extrapyramidal disorders, amyotrophic lateral sclerosis and other motor neuron disorders, progressive neural muscular atrophy, retinitis pigmentosa, hereditary ataxias, multiple sclerosis and other demyelinating diseases, bacterial and viral meningitis, brain abscess, subdural empyema, epidural abscess, suppurative intracranial thrombophlebitis, myelitis and radiculitis, viral central nervous system disease, prion diseases including kuru, Creutzfeldt-Jakob disease, and Gerstmann-Straussler-Scheinker syndrome, fatal familial insomnia, nutritional and metabolic diseases of the nervous system, neurofibromatosis, tuberous sclerosis, cerebelloretinal hemangioblastomatosis, encephalotrigeminal syndrome, mental retardation and other developmental disorders of the central nervous system including Down syndrome, cerebral palsy, neuroskeletal disorders, autonomic nervous system disorders, cranial nerve disorders, spinal cord diseases, muscular dystrophy and other neuromuscular disorders, peripheral nervous system disorders, dermatomyositis and polymyositis, inherited, metabolic, endocrine, and toxic myopathies, myasthenia gravis, periodic paralysis, mental disorders including mood, anxiety, and schizophrenic disorders, seasonal affective disorder (SAD), akathesia, amnesia, catatonia, diabetic neuropathy, tardive dyskinesia, dystonias, paranoid psychoses, postherpetic neuralgia, Tourette's disorder, progressive supranuclear palsy, corticobasal degeneration, and familial frontotemporal dementia; and a reproductive disorder, such as infertility, including tubal disease, ovulatory defects, and endometriosis, a disorder of prolactin production, a disruption of the estrous cycle, a disruption of the menstrual cycle, polycystic ovary syndrome, ovarian hyperstimulation syndrome, an endometrial or ovarian tumor, a uterine fibroid, autoimmune disorders, an ectopic pregnancy, and teratogenesis; cancer of the breast, fibrocystic breast disease, and galactorrhea; a disruption of spermatogenesis, abnormal sperm physiology, cancer of the testis, cancer of the prostate, benign prostatic hyperplasia, prostatitis, Peyronie's disease, impotence, carcinoma of the male breast, and gynecomastia. The polynucleotide sequences encoding PRTS may be used in Southern or northern analysis, dot blot, or other membrane-based technologies;
in PCR technologies;
in dipstick, pin, and multiformat ELISA-like assays; and in microarrays utilizing fluids or tissues from patients to detect altered PRTS expression. Such qualitative or quantitative methods are well known in the art.
In a particular aspect, the nucleotide sequences encoding PRTS may be useful in assays that detect the presence of associated disorders, particularly those mentioned above. The nucleotide sequences encoding PRTS may be labeled by standard methods and added to a fluid or tissue sample from a patient under conditions suitable for the formation of hybridization complexes. After a suitable incubation period, the sample is washed and the signal is quantified and compared with a standard value. If the amount of signal in the patient sample is significantly altered in comparison to a control sample then the presence of altered levels of nucleotide sequences encoding PRTS in the sample indicates the presence of the associated disorder. Such assays may also be used to evaluate the efficacy of a particular therapeutic treatment regimen in animal studies, in clinical trials, or to monitor the treatment of an individual patient.
In order to provide a basis for the diagnosis of a disorder associated with expression of PRTS, a normal or standard profile for expression is established. This may be accomplished by combining body fluids or cell extracts taken from normal subjects, either animal or human, with a sequence, or a fragment thereof, encoding PRTS, under conditions suitable for hybridization or amplification.
Standard hybridization may be quantified by comparing the values obtained from normal subjects with values from an experiment in which a known amount of a substantially purified polynucleotide is used. Standard values obtained in this manner may be compared with values obtained from samples from patients who are symptomatic for a disorder. Deviation from standard values is used to establish the presence of a disorder.
Once the presence of a disorder is established and a treatment protocol is initiated, hybridization assays may be repeated on a regular basis to determine if the level of expression in the patient begins to approximate that which is observed in the normal subject.
The results obtained from successive assays may be used to show the efficacy of treatment over a period ranging from several days to months.
With respect to cancer, the presence of an abnormal amount of transcript (either under- or overexpressed) in biopsied tissue from an individual may indicate a predisposition for the development of the disease, or may provide a means for detecting the disease prior to the appearance of actual clinical symptoms. A more definitive diagnosis of this type may allow health professionals to employ preventative measures or aggressive treatment earlier thereby preventing the development or further progression of the cancer.
Additional diagnostic uses for oligonucleotides designed from the sequences encoding PRTS
may involve the use of PCR. These oligomers may be chemically synthesized, generated enzymatically, or produced in vitro. Oligomers will preferably contain a fragment of a polynucleotide encoding PRTS, or a fragment of a polynucleotide complementary to the polynucleotide encoding PRTS, and will be employed under optimized conditions for identification of a specific gene or condition. Oligomers may also be employed under less stringent conditions for detection or quantification of closely related DNA or RNA sequences.
In a particular aspect, oligonucleotide primers derived from the polynucleotide sequences encoding PRTS may be used to detect single nucleotide polymorphisms (SNPs).
SNPs are substitutions, insertions and deletions that are a frequent cause of inherited or acquired genetic disease in humans. Methods of SNP detection include, but are not limited to, single-stranded conformation polymorphism (SSCP) and fluorescent SSCP (fSSCP) methods. In SSCP, oligonucleotide primers derived from the polynucleotide sequences encoding PRTS are used to amplify DNA using the polymerase chain reaction (PCR). The DNA may be derived, for example, from diseased or normal tissue, biopsy samples, bodily fluids, and the like.
SNPs in the DNA cause differences in the secondary and tertiary structures of PCR products in single-stranded form, and these differences are detectable using gel electrophoresis in non-denaturing gels. In fSCCP, the oligonucleotide primers are fluorescently labeled, which allows detection of the amplimers in high-throughput equipment such as DNA sequencing machines. Additionally, sequence database analysis methods, termed in silico SNP (isSNP), are capable of identifying polymorphisms by comparing the sequence of individual overlapping DNA fragments which assemble into a common consensus sequence. These computer-based methods filter out sequence variations due to laboratory preparation of DNA and sequencing errors using statistical models and automated analyses of DNA sequence chromatograms. In the alternative, SNPs may be detected and characterized by mass spectrometry using, for example, the high throughput MASSARRAY system (Sequenom, Inc., San Diego CA).
Methods which may also be used to quantify the expression of PRTS include radiolabeling or biotinylating nucleotides, coamplification of a control nucleic acid, and interpolating results from standard curves. (See, e.g., Melby, P.C. et al. (1993) J. >inmunol. Methods 159:235-244; Duplaa, C.
et al. (1993) Anal. Biochem. 212:229-236.) The speed of quantitation of multiple samples may be accelerated by running the assay in a high-throughput format where the oligomer or polynucleotide of interest is presented in various dilutions and a spectrophotometric or colorimetric response gives rapid quantitation.
In further embodiments, oligonucleotides or longer fragments derived from any of the polynucleotide sequences described herein may be used as elements on a microarray. The microarray can be used in transcript imaging techniques which monitor the relative expression levels of large numbers of genes simultaneously as described below. The microarray may also be used to identify genetic variants, mutations, and polymorphisms. This information may be used to determine gene function, to understand the genetic basis of a disorder, to diagnose a disorder, to monitor progression/regression of disease as.a function of gene expression, and to develop and monitor the activities of therapeutic agents in the treatment of disease. In particular, this information may be used to develop a pharmacogenomic profile of a patient in order to select the most appropriate and effective treatment regimen for that patient. For example, therapeutic agents which are highly effective and display the fewest side effects may be selected for a,patient based on his/her pharmacogenomic profile.

In another embodiment, PRTS, fragments of PRTS, or antibodies specific for PRTS may be used as elements on a microarray. The microarray may be used to monitor or measure protein-protein interactions, drug-target interactions, and gene expression profiles, as described above.
A particular embodiment relates to the use of the polynucleotides of the present invention to generate a transcript image of a tissue or cell type. A transcript image represents the global pattern of gene expression by a particular tissue or cell type. Global gene expression patterns are analyzed by quantifying the number of expressed genes and their relative abundance under given conditions and at a given time. (See Seilhamer et al., "Comparative Gene Transcript Analysis,"
U.S. Patent No.
5,840,484, expressly incorporated by reference herein.) Thus a transcript image may be generated by hybridizing the polynucleotides of the present invention or their complements to the totality of transcripts or reverse transcripts of a particular tissue or cell type. In one embodiment, the hybridization takes place in high-throughput format, wherein the polynucleotides of the present invention or their complements comprise a subset of a plurality of elements on a microarray. The resultant transcript image would provide a profile of gene activity.
Transcript images may be generated using transcripts isolated from tissues, cell lines, biopsies, or other biological samples. The transcript image may thus reflect gene expression in vivo, as in the case of a tissue or biopsy sample, or in vitro, as in the case of a cell line.
Transcript images which profile the expression of the polynucleotides of the present invention may also be used in conjunction with in vitro model systems and preclinical evaluation of pharmaceuticals, as well as toxicological testing of industrial and naturally-occurring environmental' compounds. All compounds induce characteristic gene expression patterns, frequently termed molecular fingerprints or toxicant signatures, which are indicative of mechanisms of action and toxicity (Nuwaysir, E.F. et al. (1999) Mol. Carcinog. 24:153-159; Steiner, S.
and N.L. Anderson (2000) Toxicol. Lett. 112-113:467-471, expressly incorporated by reference herein). If a test compound has a signature similar to that of a compound with known toxicity, it is likely to share those toxic properties. These fingerprints or signatures are most useful and refined when they contain expression information from a large number of genes and gene families.
Ideally, a genome-wide measurement of expression provides the highest quality signature. Even genes whose expression is not altered by any tested compounds are important as well, as the levels of expression of these genes are used to normalize the rest of the expression data. The normalization procedure is useful for comparison of expression data after treatment with different compounds. While the assignment of gene function to elements of a toxicant signature aids in interpretation of toxicity mechanisms, knowledge of gene function is not necessary for the statistical matching of signatures which leads to prediction of toxicity. (See, for example, Press Release 00-02 from the National Institute of Environmental Health Sciences, released February 29, 2000, available at http://www.niehs.nih.gov/oc/news/toxchip.htm.) Therefore, it is important and desirable in toxicological screening using toxicant signatures to include all expressed gene sequences.
In one embodiment, the toxicity of a test compound is assessed by treating a biological sample containing nucleic acids with the test compound. Nucleic acids that are expressed in the treated biological sample are hybridized with one or more probes specific to the polynucleotides of the present invention, so that transcript levels corresponding to the polynucleotides of the present invention may be quantified. The transcript levels in the treated biological sample are compared with levels in an untreated biological sample. Differences in the transcript levels between the two samples are indicative of a toxic response caused by the test compound in the treated sample.
Another particular embodiment relates to the use of the polypeptide sequences of the present invention to analyze the proteome of a tissue or cell type. The term proteome refers to the global pattern of protein expression in a particular tissue or cell type. Each protein component of a proteome can be subjected individually to further analysis. Proteome expression patterns, or profiles, are analyzed by quantifying the number of expressed proteins and their relative abundance under given conditions and at a given time. A profile of a cell's proteome may thus be generated by separating and analyzing the polypeptides of a particular tissue or cell type.
In one embodiment, the separation is achieved using two-dimensional gel electrophoresis, in which proteins from a sample are separated by isoelectric focusing in the first dimension, and then according to molecular weight by sodium dodecyl sulfate slab gel electrophoresis.in the second dimension (Steiner and Anderson, supra). The proteins are visualized in the gel as discrete and uniquely positioned spots, typically by staining the gel with an agent such as Coomassie Blue or silver or fluorescent stains. The optical density of each protein spot is generally proportional to the level of the protein in the sample. The optical densities of equivalently positioned protein spots from different samples, for example, from biological samples either treated or untreated with a test compound or therapeutic agent, are compared to identify any changes in protein spot density related to the treatment. The proteins in the spots are partially sequenced using, for example, standard methods employing chemical or enzymatic cleavage followed by mass spectrometry. The identity of the protein in a spot may be determined by comparing its partial sequence, preferably of at least 5 contiguous amino acid residues, to the polypeptide sequences of the present invention. In some cases, further sequence data may be obtained for definitive protein identification.
A proteomic profile may also be generated using antibodies specific for PRTS
to quantify the levels of PRTS expression. In one embodiment, the antibodies are used as elements on a microarray, and protein expression levels are quantified by exposing the microarray to the sample and detecting the levels of protein bound to each array element (Lueking, A. et al. (1999) Anal. Biochem. 270:103-111; Mendoze, L.G. et al. (1999) Biotechniques 27:778-788). Detection may be performed by a variety of methods known in the art, for example, by reacting the proteins in the sample with a thiol-or amino-reactive fluorescent compound and detecting the amount of fluorescence bound at each array element.
Toxicant signatures at the proteome level are also useful for toxicological screening, and should be analyzed in parallel with toxicant signatures at the transcript level. There is a poor correlation between transcript and protein abundances for some proteins in some tissues (Anderson, N.L. and J. Seilhamer (1997) Electrophoresis 18:533-537), so proteome toxicant signatures may be useful in the analysis of compounds which do not significantly affect the transcript image, but which alter the proteomic profile. In addition, the analysis of transcripts in body fluids is difficult, due to rapid degradation of mRNA, so proteomic profiling may be more reliable and informative in such cases.
In another embodiment, the toxicity of a test compound is assessed by treating a biological sample containing proteins with the test compound. Proteins that are expressed in the treated biological sample are separated so that the amount of each protein can be quantified. The amount of each protein is compared to the amount of the corresponding protein in an untreated biological sample. A difference in the amount of protein between the two samples is indicative of a toxic response to the test compound in the treated sample. Individual proteins are identified by sequencing the amino acid residues of the individual proteins and comparing these partial sequences to the polypeptides of the present invention.
In another embodiment, the toxicity of a test compound is assessed by treating a biological sample containing proteins with the test compound. Proteins from the biological' sample are incubated with antibodies specific to the polypeptides of the present invention. The amount of protein recognized by the antibodies is quantified. The amount of protein in the treated biological sample is compared with the amount in an untreated biological sample. A
difference in the amount of protein between the two samples is indicative of a toxic response to the test compound in the treated sample.
Microarrays may be prepared, used, and analyzed using methods known in the art. (See, e.g., Brennan, T.M. et al. (1995) U.S. Patent No. 5,474,796; Schena, M. et al.
(1996) Proc. Natl. Acad. Sci.
USA 93:10614-10619; Baldeschweiler et al. (1995) PCT application W095/251116;
Shalom D. et al.
(1995) PCT application W095/35505; Heller, R.A. et al. (1997) Proc. Natl.
Acad. Sci. USA 94:2150-2155; and Heller, M.J. et al. (1997) U.S. Patent No. 5,605,662.) Various types of microarrays are well known and thoroughly described in DNA Microarrays: A Practical Approach, M. Schena, ed.
(1999) Oxford University Press, London, hereby expressly incorporated by reference.
In another embodiment of the invention, nucleic acid sequences encoding PRTS
may be used to generate hybridization probes useful in mapping the naturally occurring genomic sequence. Either ~8 coding or noncoding sequences may be used, and in some instances, noncoding sequences may be preferable over coding sequences. For example, conservation of a coding sequence among members of a mufti-gene family may potentially cause undesired cross hybridization during chromosomal mapping. The sequences may be mapped to a particular chromosome, to a specific region of a chromosome, or to artificial chromosome constructions, e.g., human artificial chromosomes (HACs), yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs), bacterial P1 constructions, or single chromosome cDNA libraries. (See, e.g., Haxrington, J.J. et al. (1997) Nat.
Genet. 15:345-355; Price, C.M. (1993) Blood Rev. 7:127-134; and Trask, B.J.
(1991) Trends Genet.
7:149-154.) Once mapped, the nucleic acid sequences of the invention may be used to develop genetic linkage maps, for example, which correlate the inheritance of a disease state with the inheritance of a particular chromosome region or restriction fragment length polymorphism (RFLP).
(See, for example, Lander, E.S. and D. Botstein (1986) Proc. Natl. Acad. Sci.
USA 83:7353-7357.) Fluorescent in situ hybridization (FISH) may be correlated with other physical and genetic map data. (See, e.g., Heinz-Ulrich, et al. (1995) in Meyers, supra, pp. 965-968.) Examples of genetic map data can be found in various scientific journals or at the Online Mendelian Inheritance in Man (OMIM) World Wide Web site. Correlation between the location of the gene encoding PRTS on a physical map and a specific disorder, or a predisposition to a specific disorder, may help define the region of DNA associated with that disorder and thus may further positional cloning efforts.
Tn situ hybridization of chromosomal preparations and physical mapping techniques, such as linkage analysis using established chromosomal markers, may be used for extending genetic maps.
Often the placement of a gene on the chromosome of another marninalian species, such as mouse, may reveal associated markers even if the exact chromosomal locus is not known. This information is valuable to investigators searching for disease genes using positional cloning or other gene discovery techniques. Once the gene or genes responsible for a disease or syndrome have been crudely localized by genetic linkage to a particular genomic region, e.g., ataxia-telangiectasia to 11q22-23, any sequences mapping to that area may represent associated or regulatory genes for further investigation. (See, e.g., Gatti, R.A. et al. (1988) Nature 336:577-580.) The nucleotide sequence of the instant invention may also be used to detect differences in the chromosomal location due to translocation, inversion, etc., among normal, carrier, or affected individuals.
In another embodiment of the invention, PRTS, its catalytic or immunogenic fragments, or oligopeptides thereof can be used for screening libraries of compounds in any of a variety of drug screening techniques. The fragment employed in such screening may be free in solution, affixed to a solid support, borne on a cell surface, or located intracellularly. The formation of binding complexes between PRTS and the agent being tested may be measured.

Another technique for drug screening provides for high throughput screening of compounds having suitable binding affinity to the protein of interest. (See, e.g., Geysen, et al. (1984) PCT
application W084/03564.) In this method, large numbers of different small test compounds are synthesized on a solid substrate. The test compounds are reacted with PRTS, or fragments thereof, and washed. Bound PRTS is then detected by methods well known in the art.
Purified PRTS 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.
In another embodiment, one may use competitive drug screening assays in which neutralizing antibodies capable of binding PRTS specifically compete with a test compound for binding PRTS. In this manner, antibodies can be used to detect the presence of any peptide which shares one or more antigenic determinants with PRTS.
In additional embodiments, the nucleotide sequences which encode PRTS may be used in any molecular biology techniques that have yet to be developed, provided the new techniques rely on properties of nucleotide sequences that axe currently known, including, but not limited to, such properties as the triplet genetic code and specific base pair interactions.
Without further elaboration, it is believed that one skilled in the art can, using the preceding description, utilize the present invention to its fullest extent. The following embodiments are, therefore, to be construed as merely illustrative, and not limitative of the remainder of the disclosure in any way whatsoever.
The disclosures of all patents, applications and publications, mentioned above and below including U.S. Ser. No. 60/241,573, U.S. Ser. No. 60/243,643, U.S. Ser. No.
60/245,256, U.S. Ser.
No. 60/248,395, U.S. Ser. No. 60/249,826, U.S. Ser. No. 60/252,303, U.S. Ser.
No. 60/250,981, are expressly incorporated by reference herein.
EXAMPLES
I. Construction of cDNA Libraries Incyte cDNAs were derived from cDNA libraries described in the LIFESEQ GOLD
database (Incyte Genomics, Palo Alto CA) and shown in Table 4, column 5. Some tissues were homogenized and lysed in guanidinium isothiocyanate, while others were homogenized and lysed in phenol or in a suitable mixture of denaturants, such as TRIZOL (Life Technologies), a monophasic solution of phenol and guanidine isothiocyanate. The resulting lysates were centrifuged over CsCI cushions or extracted with chloroform. RNA was precipitated from the lysates with either isopropanol or sodium acetate and ethanol, or by other routine methods.

Phenol extraction and precipitation of RNA were repeated as necessary to increase RNA
purity. In some cases, RNA was treated with DNase. For most libraries, poly(A)+ RNA was isolated using oligo d(T)-coupled paramagnetic particles (Promega), OLIGOTEX latex particles (QIAGEN, Chatsworth CA), or an OLIGOTEX mRNA purification kit (QIAGEN). Alternatively, RNA was isolated directly from tissue lysates using other RNA isolation kits, e.g., the POLY(A)PURE mRNA
purification kit (Ambion, Austin TX).
In some cases, Stratagene was provided with RNA and constructed the corresponding cDNA
libraries. Otherwise, cDNA was synthesized and cDNA libraries were constructed with the UNIZAP
vector system (Stratagene) or SUPERSCRIPT plasmid system (Life Technologies), using the recommended procedures or similar methods known in the art. (See, e.g., Ausubel, 1997, su ra, units 5.1-6.6.) Reverse transcription was initiated using oligo d(T) or random primers. Synthetic oligonucleotide adapters were ligated to double stranded cDNA, and the cDNA
was digested with the appropriate restriction enzyme or enzymes. For most libraries, the cDNA was size-selected (300-1000 bp) using SEPHACRYL S 1000, SEPHAROSE CL2B, or SEPHAROSE CL4B column chromatography (Amersham Pharmacia Biotech) or preparative agarose gel electrophoresis. cDNAs were ligated into compatible restriction enzyme sites of the polylinker of a suitable plasmid, e.g., PBLUESCRIPT plasmid (Stratagene), PSPORT1 plasmid (Life Technologies), PCDNA2.1 plasmid (Invitrogen, Carlsbad CA), PBI~-CMV plasmid (Stratagene), PCR2-TOPOTA plasmid (Invitrogen), PCMV-ICIS plasmid (Stratagene), pIGEN (Incyte Genomics, Palo Alto CA), or pINCY (Incyte Genomics), or derivatives thereof. Recombinant plasmids were transformed into competent E. coli cells including XLl-Blue, XL1-BIueMRF, or SOLR from Stratagene or DHSa, DH10B, or ElectroMAX DHlOB from Life Technologies.
II. Isolation of cDNA Clones Plasmids obtained as described in Example I were recovered from host cells by in vivo excision using the UNIZAP vector system (Stratagene) or by cell lysis.
Plasmids were purified using at least one of the following: a Magic or WIZARD Minipreps DNA purification system (Promega); an AGTC Miniprep purification kit (Edge Biosystems, Gaithersburg MD); and QIAWELL
8 Plasmid, QIAWELL 8 Plus Plasmid, QIAWELL 8 Ultra Plasmid purification systems or the R.E.A.L. PREP 96 plasmid purification kit from QIAGEN. Following precipitation, plasmids were resuspended in 0.1 ml of distilled water and stored, with or without lyophilization, at 4°C.
Alternatively, plasmid DNA was amplified from host cell lysates using direct link PCR in a high-throughput format (Rao, V.B. (1994) Anal. Biochem. 216:1-14). Host cell lysis and thermal cycling steps were carried out in a single reaction mixture. Samples were processed and stored in 384-well plates, and the concentration of amplified plasmid DNA was quantified fluorometrically using PICOGREEN dye (Molecular Probes, Eugene OR) and a FLUOROSKAN II
fluorescence scanner (Labsystems Oy, Helsinki, Finland).
III. Sequencing and Analysis Incyte cDNA recovered in plasmids as described in Example 1I were sequenced as follows.
Sequencing reactions were processed using standard methods or high-throughput instrumentation such as the ABI CATALYST 800 (Applied Biosystems) thermal cycler or the PTC-200 thermal cycler (MJ Research) in conjunction with the HYDRA microdispenser (Robbins Scientific) or the MICROLAB 2200 (Hamilton) liquid transfer system. cDNA sequencing reactions were prepared using reagents provided by Amersham Pharmacia Biotech or supplied in ABI
sequencing kits such as the ABI PRISM BIGDYE Terminator cycle sequencing ready reaction kit (Applied Biosystems).
Electrophoretic separation of cDNA sequencing reactions and detection of labeled polynucleotides were carried out using the MEGABACE 1000 DNA sequencing system (Molecular Dynamics); the ABI PRISM 373 or 377 sequencing system (Applied Biosystems) in conjunction with standard ABI
protocols and base calling software; or other sequence analysis systems known in the art. Reading frames within the cDNA sequences were identified using standard methods (reviewed in Ausubel, 1997, supra, unit 7.7). Some of the cDNA sequences were selected for extension using the techniques disclosed in Example VIII.
The polynucleotide sequences derived from Incyte cDNAs were validated by removing vector, linker, and poly(A) sequences and by masking ambiguous bases, using algorithms and programs based on BLAST, dynamic programming, and dinucleotide nearest neighbor analysis. The Incyte cDNA sequences or translations thereof were then queried against a selection of public databases such as the GenBank primate, rodent, mammalian, vertebrate, and eukaryote databases, and BLOCKS, PRINTS, DOMO, PRODOM; PROTEOME databases with sequences from Homo sa iens, Rattus norve i~ cus, Mus musculus, Caenorhabditis ele,g~anS, Saccharomyces cerevisiae, Schizosaccharomyces bombe, and Candida albicans (Incyte Genomics, Palo Alto CA); and hidden Markov model (HMM)-based protein family databases such as PFAM. (HMM is a probabilistic approach which analyzes consensus primary structures of gene families. See, for example, Eddy, S.R. (1996) Curr. Opin. Struct. Biol. 6:361-365.) The queries were performed using programs based on BLAST, FASTA, BLIMPS, and IIMMER. The Incyte cDNA sequences were assembled to produce full length polynucleotide sequences. Alternatively, GenBank cDNAs, GenBank ESTs, stitched sequences, stretched sequences, or Genscan-predicted coding sequences (see Examples IV
and V) were used to extend Incyte cDNA assemblages to full length. Assembly was performed using programs based on Phred, Phrap, and Consed, and cDNA assemblages were screened for open reading frames using programs based on GeneMark, BLAST, and FASTA. The full length polynucleotide sequences were translated to derive the corresponding full length polypeptide sequences. Alternatively, a polypeptide of the invention may begin at any of the methionine residues of the full length translated polypeptide. Full length polypeptide sequences were subsequently analyzed by querying against databases such as the GenBank protein databases (genpept), SwissProt, the PROTEOME databases, BLOCKS, PRINTS, DOMO, PRODOM, Prosite, and hidden Markov model (ITVIM)-based protein family databases such as PFAM. Full length polynucleotide sequences are also analyzed using MACDNASIS PRO software (Hitachi Software Engineering, South San Francisco CA) and LASERGENE software (DNASTAR). Polynucleotide and polypeptide sequence alignments are generated using default parameters specified by the CLUSTAL
algorithm as incorporated into the MEGALIGN multisequence alignment program (DNASTAR), which also calculates the percent identity between aligned sequences.
Table 7 summarizes the tools, programs, and algorithms used for the analysis and assembly of Incyte cDNA and full length sequences and provides applicable descriptions, references, and threshold parameters. The first column of Table 7 shows the tools, programs, and algorithms used, the second column provides brief descriptions thereof, the third column presents appropriate references, all of which are incorporated by reference herein in their entirety, and the fourth column presents, where applicable, the scores, probability values, and other parameters used to evaluate the strength of a match between two sequences (the higher the score or the lower the probability value, the greater the identity between two sequences).
The programs described above for the assembly and analysis of full length polynucleotide and polypeptide sequences were also used to identify polynucleotide sequence fragments from SEQ
ID N0:16-30. Fragments from about 20 to about 4000 nucleotides which are useful in hybridization and amplification technologies are described in Table 4, column 4.
IV. Identification and Editing of Coding Sequences from Genomic DNA
Putative proteases were initially identified by running the Genscan gene identification program against public genomic sequence databases (e.g., gbpri and gbhtg).
Genscan is a general-purpose gene identification program which analyzes genomic DNA sequences from a variety of organisms (See Burge, C. and S. Karlin (1997) J. Mol. Biol. 268:78-94, and Burge, C. and S. Karlin (1998) Curr. Opin. Struct. Biol. 8:346-354). The program concatenates predicted exons to form an assembled cDNA sequence extending from a methionine to a stop codon. The output of Genscan is a FASTA database of polynucleotide and polypeptide sequences. The maximum range of sequence for Genscan to analyze ~at once was set to 30 kb. To determine which of these Genscan predicted cDNA
sequences encode proteases, the encoded polypeptides were analyzed by querying against PFAM
models for proteases. Potential proteases were also identified by homology to Incyte cDNA
sequences that had been annotated as proteases. These selected Genscan-predicted sequences were then compared by BLAST analysis to the genpept and gbpri public databases.
Where necessary, the Genscan-predicted sequences were then edited by comparison to the top BLAST
hit from genpept to correct errors in the sequence predicted by Genscan, such as extra or omitted exons. BLAST analysis was also used to fmd any Incyte cDNA or public cDNA coverage of the Genscan-predicted sequences, thus providing evidence for transcription. When Incyte cDNA
coverage was available, this information was used to correct or confirm the Genscan predicted sequence. Full length polynucleotide sequences were obtained by assembling Genscan-predicted coding sequences with Incyte cDNA sequences and/or public cDNA sequences using the assembly process described in Example IIC. Alternatively, full length polynucleotide sequences were derived entirely from edited or unedited Genscan-predicted coding sequences.
V. Assembly of Genomic Sequence Data with cDNA Sequence Data "Stitched" Sequences Partial cDNA sequences were extended with exons predicted by the Genscan gene identification program described in Example IV. Partial cDNAs assembled as described in Example III were mapped to genomic DNA and parsed into clusters containing related cDNAs and Genscan exon predictions from one or more genomic sequences. Each cluster was analyzed using an algorithm based on graph theory and dynamic programming to integrate cDNA and genomic information, generating possible splice variants that were subsequently confirmed, edited, or extended to create a full length sequence. Sequence intervals in which the entire length of the interval was present on more than one sequence in the cluster Were identified, and intervals thus identified were considered to be equivalent by transitivity. For example, if an interval was present on a cDNA and two genomic sequences, then all three intervals were considered to be equivalent. This process allows unrelated but consecutive genomic sequences to be brought together, bridged by cDNA
sequence. Intervals thus identified were then "stitched" together by the stitching algorithm in the order that they appear along their parent sequences to generate the longest possible sequence, as well as sequence variants.
Linkages between intervals which proceed along one type of parent sequence (cDNA to cDNA or genomic sequence to genomic sequence) were given preference over linkages which change parent type (cDNA to genomic sequence). The resultant stitched sequences were translated and compared by BLAST analysis to the genpept and gbpri public databases. Incorrect exons predicted by Genscan were corrected by comparison to the top BLAST hit from genpept. Sequences were further extended with additional cDNA sequences, or by inspection of genomic DNA, when necessary.
"Stretched" Sequences Partial DNA sequences were extended to full length with an algorithm based on BLAST
analysis. First, partial cDNAs assembled as described in Example III were queried against public databases such as the GenBank primate, rodent, mammalian, vertebrate, and eukaryote databases using the BLAST program. The nearest GenBank protein homolog was then compared by BLAST

analysis to either Incyte cDNA sequences or GenScan exon predicted sequences described in Example 1V. A chimeric protein was generated by using the resultant high-scoring segment pairs (HSPs) to map the translated sequences onto the GenBank protein homolog.
Insertions or deletions may occur in the chimeric protein with respect to the original GenBank protein homolog. The GenBank protein homolog, the chimeric protein, or both were used as probes to search for homologous genomic sequences from the public human genome databases. Partial DNA sequences were therefore "stretched" or extended by the addition of homologous genomic sequences. The resultant stretched sequences were examined to determine whether it contained a complete gene.
VI. Chromosomal Mapping of PRTS Encoding Polynucleotides The sequences which were used to assemble SEQ ID N0:16-30 were compared with sequences from the Incyte LIFESEQ database and public domain databases using BLAST and other implementations of the Smith-Waterman algorithm. Sequences from these databases that matched SEQ ID N0:16-30 were assembled into clusters of contiguous and overlapping sequences using assembly algorithms such as Phrap (Table 7). Radiation hybrid and genetic mapping data available from public resources such as the Stanford Human Genome Center (SHGC), Whitehead Institute for Genome Research (WIGR), and Genethon were used to determine if any of the clustered sequences had been previously mapped. Inclusion of a mapped sequence in a cluster resulted in the assignment of all sequences of that cluster, including its particular SEQ 1D NO:, to that map location.
Map locations are represented by ranges, or intervals, of human chromosomes.
The map position of an interval, in centiMorgans, is measured relative to the terminus of the chromosome's p-arm. (The centiMorgan (cM) is a unit of measurement based on recombination frequencies between chromosomal markers. On average, 1 cM is roughly equivalent to 1 megabase (Mb) of DNA in humans, although this can vary widely due to hot and cold spots of recombination.) The cM
distances are based on genetic markers mapped by Genethon which provide boundaries for radiation hybrid markers whose sequences were included in each of the clusters. Human genome maps and other resources available to the public, such as the NCBI "GeneMap'99" World Wide Web site (http://www.ncbi.nlin.nih.govlgenemap/), can be employed to determine if previously identified disease genes map within or in proximity to the intervals indicated above.
VII. Analysis of Polynucleotide Expression Northern analysis is a laboratory technique used to detect the presence of a transcript of a gene and involves the hybridization of a labeled nucleotide sequence to a membrane on which RNAs from a particular cell type or tissue have been bound. (See, e.g., Sambrook, supra, ch. 7; Ausubel (1995) supra, ch. 4 and 16.) Analogous computer techniques applying BLAST were used to search for identical or related molecules in cDNA databases such as GenBank or LIFESEQ (Incyte Genomics). This analysis is much faster than multiple membrane-based hybridizations. In addition, the sensitivity of the computer search can be modified to determine whether any particular match is categorized as exact or similar. The basis of the search is the product score, which is defined as:
BLAST Score x Percent Identity x minimum {length(Seq. 1), length(Seq. 2)}
The product score takes into account both the degree of similarity between two sequences and the length of the sequence match. The product score is a normalized value between 0 and 100, and is calculated as follows: the BLAST score is multiplied by the percent nucleotide identity and the product is divided by (5 times the length of the shorter of the two sequences). The BLAST score is calculated by assigning a score of +5 for every base that matches in a high-scoring segment pair (HSP), and -4 for every mismatch. Two sequences may share more than one HSP
(separated by gaps). If there is more than one HSP, then the pair with the highest BLAST
score is used to calculate the product score. The product score represents a balance between fractional overlap and quality in a BLAST alignment. For example, a product score of 100 is produced only for 100%
identity over the entire length of the shorter of the two sequences being compared. A product score of 70 is produced either by 100% identity and 70% overlap at one end, or by 88% identity and 100% overlap at the other. A product score of 50 is produced either by 100% identity and 50%
overlap at one end, or 79%
identity and 100% overlap.
Alternatively, polynucleotide sequences encoding PRTS are analyzed with respect to the tissue sources from which they were derived. For example, some full length sequences are assembled, at least in part, with overlapping Incyte cDNA sequences (see Example )~. Each cDNA
sequence is derived from a cDNA library constructed from a human tissue. Each human tissue is classified into one of the following organ/tissue categories: cardiovascular system; connective tissue;
digestive system; embryonic structures; endocrine system; exocrine glands;
genitalia, female;
genitalia, male; germ cells; hemic and immune system; liver; musculoskeletal system; nervous system; pancreas; respiratory system; sense organs; skin; stomatognathic system; unclassified/mixed;
or urinary tract. The number of libraries in each category is counted and divided by the total number of libraries across all categories. Similarly, each human tissue is classified into one of the following disease/condition categories: cancer, cell line, developmental, inflammation, neurological, trauma, cardiovascular, pooled, and other, and the number of libraries in each category is counted and divided by the total number of libraries across all categories. The resulting percentages reflect the tissue- and disease-specific expression of cDNA encoding PRTS. cDNA sequences and cDNA
library/tissue information are found in the L1FESEQ GOLD database (Incyte Genomics, Palo Alto CA).

VIII. Extension of PRTS Encoding Polynucleotides Full length polynucleotide sequences were also produced by extension of an appropriate fragment of the full length molecule using oligonucleotide primers designed from this fragment. One primer was synthesized to initiate 5' extension of the known fragment, and the other primer was synthesized to initiate 3' extension of the known fragment. The initial primers were designed using OLIGO 4.06 software (National Biosciences), or another appropriate program, to be about 22 to 30 nucleotides in length, to have a GC content of about 50% or more, and to anneal to the target sequence at temperatures of about 68°C to about 72°C. Any stretch of nucleotides which would result in hairpin structures and primer-primer dimerizations was avoided.
Selected human cDNA libraries were used to extend the sequence. If more than one extension was necessary or desired, additional or nested sets of primers were designed.
High fidelity amplification was obtained by PCR using methods well known in the art. PCR
was performed in 96-well plates using the PTC-200 thermal cycler (MJ Research, Inc.). The reaction mix contained DNA template, 200 nmol of each primer, reaction buffer containing Mgz+, (NH4)ZS04, and 2-mercaptoethanol, Taq DNA polymerase (Amersham Pharmacia Biotech), ELONGASE enzyme (Life Technologies), and Pfu DNA polymerase (Stratagene), with the following parameters for primer pair PCI A and PCI B: Step l: 94°C, 3 min; Step 2: 94°C, 15 sec;
Step 3: 60°C, 1 min; Step 4: 68°C, 2 min; Step 5: Steps 2, 3, and 4 repeated 20 times; Step 6: 68°C, 5 min; Step 7: storage at 4°C. In the alternative, the parameters for primer pair T7 and SKI- were as follows: Step 1: 94°C, 3 min; Step 2:
94°C, 15 sec; Step 3: 57°C, 1 min; Step 4: 68°C, 2 min;
Step 5: Steps 2, 3, and 4 repeated 20 times;
Step 6: 68°C, 5 min; Step 7: storage at 4°C.
The concentration of DNA in each well was determined by dispensing 100 ,u1 PICOGREEN
quantitation reagent (0.25% (v/v) PICOGREEN; Molecular Probes, Eugene OR) dissolved in 1X TE
and 0.5 ~l of undiluted PCR product into each well of an opaque fluorimeter plate (Corning Costar, Acton MA), allowing the DNA to bind to the reagent. The plate was scanned in a Fluoroskan II
(Labsystems Oy, Helsinki, Finland) to measure the fluorescence of the sample and to quantify the concentration of DNA. A 5 ,u1 to 10 ,u1 aliquot of the reaction mixture was analyzed by electrophoresis on a 1 % agarose gel to determine which reactions were successful in extending the sequence.
The extended nucleotides were desalted and concentrated, transferred to 384-well plates, digested with CviJI cholera virus endonuclease (Molecular Biology Research, Madison WI), and sonicated or sheared prior to religation into pUC 18 vector (Amersham Pharmacia Biotech). For shotgun sequencing, the digested nucleotides were separated on low concentration (0.6 to 0.8%) agarose gels, fragments were excised, and agar digested with Agar ACE
(Promega). Extended clones were religated using T4 ligase (New England Biolabs, Beverly MA) into pUC 18 vector (Amersham Pharmacia Biotech), treated with Pfu DNA polymerase (Stratagene) to fill-in restriction site overhangs, and transfected into competent E. coli cells. Transformed cells were selected on antibiotic-containing media, and individual colonies were picked and cultured overnight at 37°C in 384-well plates in LB/2x carb liquid media.
The cells were lysed, and DNA was amplified by PCR using Taq DNA polymerase , (Amersham Pharmacia Biotech) and Pfu DNA polymerase (Stratagene) with the following parameters: Step 1: 94°C, 3 min; Step 2: 94°C, 15 sec; Step 3:
60°C, 1 min; Step 4: 72°C, 2 min;
Step 5: steps 2, 3, and 4 repeated 29 times; Step 6: 72°C, 5 min; Step 7: storage at 4°C. DNA was quantified by PICOGREEN reagent (Molecular Probes) as described above. Samples with low DNA
recoveries were reamplified using the same conditions as described above.
Samples were diluted with 20% dimethysulfoxide (1:2, v/v), and sequenced using DYENAMIC energy transfer sequencing primers and the DYENAMIC DIRECT kit (Amersham Pharmacia Biotech) or the ABI
PRISM
BIGDYE Terminator cycle sequencing ready reaction kit (Applied Biosystems).
In like manner, full length polynucleotide sequences are verified using the above procedure or are used to obtain 5'regulatory sequences using the above procedure along with oligonucleotides designed for such extension, and an appropriate genomic library.
IX. Labeling and Use of Individual Hybridization Probes Hybridization probes derived from SEQ DD N0:16-30 are employed to screen cDNAs, genomic DNAs, or mRNAs. Although the labeling of oligonucleotides, consisting of about 20 base pairs, is specifically described, essentially the same procedure is used with larger nucleotide fragments. Oligonucleotides are designed using state-of the-art software such as OLIGO 4.06 software (National Biosciences) and labeled by combining 50 pmol of each oligomer, 250 ~cCi of ~Y 32P~ adenosine- triphosphate (Amersham Pharmacia Biotech), and T4 polynucleotide kinase (DuPont NEN, Boston MA). The labeled oligonucleotides are substantially purified using a SEPHADEX G-25 superfine size exclusion dextran bead column (Amersham Pharmacia Biotech).
An aliquot containing 10' counts per minute of the labeled probe is used in a typical membrane-based hybridization analysis of human genomic DNA digested with one of the following endonucleases:
Ase I, Bgl II, Eco RT, Pst I, Xba I, or Pvu II (DuPont NEN).
The DNA from each digest is fractionated on a 0.7% agarose gel and transferred to nylon membranes (Nytran Plus, Schleicher & Schuell, Durham NH). Hybridization is carried out for 16 hours at 40°C. To remove nonspecific signals, blots are sequentially washed at room temperature under conditions of up to, for example, 0.1 x saline sodium citrate and 0.5%
sodium dodecyl sulfate.
Hybridization patterns are visualized using autoradiography or an alternative imaging means and compared.
X. Microarrays The linkage or synthesis of array elements upon a microarray can be achieved utilizing photolithography, piezoelectric printing (ink jet printing, See, e.g., Baldeschweiler, supra.), mechanical nucrospotting technologies, and derivatives thereof. The substrate in each of the aforementioned technologies should be uniform and solid with a non-porous surface (Schena (1999), supra). Suggested substrates include silicon, silica, glass slides, glass chips, and silicon wafers.
Alternatively, a procedure analogous to a dot or slot blot may also be used to arrange and link elements to the surface of a substrate using thermal, UV, chemical, or mechanical bonding procedures. A typical array may be produced using available methods and machines well known to those of ordinary skill in the art and may contain any appropriate number of elements. (See, e.g., Schena, M. et al. (1995) Science 270:467-470; Shalom D. et al. (1996) Genome Res. 6:639-645;
Marshall, A. and J. Hodgson (1998) Nat. Biotechnol. 16:27-31.) Full length cDNAs, Expressed Sequence Tags (ESTs), or fragments or oligomers thereof may comprise the elements of the microarray. Fragments or oligomers suitable for hybridization can be selected using software well known in the art such as LASERGENE software (DNASTAR). The array elements are hybridized with polynucleotides in a biological sample. The polynucleotides in the biological sample axe conjugated to a fluorescent label or other molecular tag for ease of detection.
After hybridization, nonhybridized nucleotides from the biological sample are removed, and a fluorescence scanner is used to detect hybridization at each array element.
Alternatively, laser desorbtion and mass spectrometry may be used for detection of hybridization.
The degree of 2,0 complementarity and the relative abundance of each polynucleotide which hybridizes to an element on the microarray may be assessed. In one embodiment, microarray preparation and usage is described in detail below.
Tissue or Cell Sample Preparation Total RNA is isolated from tissue samples using the guanidinium thiocyanate method and poly(A)~ RNA is purified using the oligo-(dT) cellulose method. Each poly(A)~
RNA sample is reverse transcribed using MMLV reverse-transcriptase, 0.05 pg/~,1 oligo-(dT) primer (2lmer), 1X
first strand buffer, 0.03 units/~tl RNase inhibitor, 500 ~,M dATP, 500 ,uM
dGTP, 500 ~M dTTP, 40 ,uM dCTP, 40 ~.M dCTP-Cy3 (BDS) or dCTP-Cy5 (Amersham Pharmacia Biotech). The reverse transcription reaction is performed in a 25 ml volume containing 200 ng poly(A)+ RNA with GEMBRIGHT kits (Incyte). Specific control poly(A)+ RNAs are synthesized by in vitro transcription from non-coding yeast genomic DNA. After incubation at 37° C for 2 hr, each reaction sample (one with Cy3 and another with Cy5 labeling) is treated with 2.5 ml of 0.5M sodium hydroxide and incubated for 20 minutes at 85° C to the stop the reaction and degrade the RNA. Samples are purified using two successive CHROMA SPIN 30 gel filtration spin columns (CLONTECH
Laboratories, Inc.
(CLONTECH), Palo Alto CA) and after combining, both reaction samples are ethanol precipitated using 1 ml of glycogen (1 mg/ml), 60 ml sodium acetate, and 300 ml of 100%
ethanol. The sample is then dried to completion using a SpeedVAC (Savant Instruments Inc., Holbrook NY) and resuspended in 14 ~,15X SSC/0.2% SDS.
Microarray Preparation Sequences of the present invention are used to generate array elements. Each array element is amplified from bacterial cells containing vectors with cloned cDNA inserts.
PCR amplification uses primers complementary to the vector sequences flanking the cDNA insert.
Array elements are amplified in thirty cycles of PCR from an initial quantity of 1-2 ng to a final quantity greater than 5 ~.g. Amplified array elements are then purified using SEPHACRYL-400 (Amersham Pharmacia Biotech).
Purified array elements are immobilized on polymer-coated glass slides. Glass microscope slides (Corning) are cleaned by ultrasound in 0.1 % SDS and acetone, with extensive distilled water washes between and after treatments. Glass slides are etched in 4%
hydrofluoric acid (VWR
Scientific Products Corporation (VWR), West Chester PA), washed extensively in distilled water, and coated with 0.05% aminopropyl silane (Sigma) in 95% ethanol. Coated slides are cured in a 110°C oven.
Array elements are applied to the coated glass substrate using a procedure described in U.S.
Patent No. 5,807,522, incorporated herein by reference. 1 ~.1 of the array element DNA, at an average concentration of 100 ng/~,1, is loaded into the open capillary printing element by a high-speed robotic apparatus. The apparatus then deposits about 5 n1 of array element sample per slide.
Microarrays are UV-crosslinked using a STRATALINKER UV-crosslinker (Stratagene).
Microarrays are washed at room temperature once in 0.2% SDS and three times in distilled water.
Non-specific binding sites are blocked by incubation of microarrays in 0.2%
casein in phosphate buffered saline (PBS) (Tropix, Inc., Bedford MA) for 30 minutes at 60°
C followed by washes in 0.2% SDS and distilled water as before.
Hybridization Hybridization reactions contain 9 ~,1 of sample mixture consisting of 0.2 ~,g each of Cy3 and Cy5 labeled cDNA synthesis products in 5X SSC, 0.2% SDS hybridization buffer.
The sample mixture is heated to 65° C for 5 minutes and is aliquoted onto the microarray surface and covered with an 1.8 cm2 coverslip. The arrays are transferred to a waterproof chamber having a cavity just slightly larger than a microscope slide. The chamber is kept at 100% humidity internally by the addition of 140 p1 of 5X SSC in a corner of the chamber. The chamber containing the arrays is incubated for about 6.5 hours at 60° C. The arrays are washed for 10 min at 45° C in a first wash buffer (1X SSC, 0.1% SDS), three times for 10 minutes each at 45°C in a second wash buffer (0.1X
SSC), and dried.

Detection Reporter-labeled hybridization complexes are detected with a microscope equipped with an Innova 70 mixed gas 10 W laser (Coherent, Inc., Santa Clara CA) capable of generating spectral lines at 488 nm for excitation of Cy3 and at 632 nm for excitation of CyS. The excitation laser light is focused on the array using a 20X microscope objective (Nikon, Inc., Melville NY). The slide containing the array is placed on a computer-controlled X-Y stage on the microscope and raster-scanned past the objective. The 1.8 cm x 1.8 cm array used in the present example is scanned with a resolution of 20 micrometers.
In two separate scans, a mixed gas multiline laser excites the two fluorophores sequentially.
Emitted light is split, based on wavelength, into two photomultiplier tube detectors (PMT 81477, Hamamatsu Photonics Systems, Bridgewater NJ) corresponding to the two fluorophores. Appropriate filters positioned between the array and the photomultiplier tubes are used to filter the signals. The emission maxima of the fluorophores used are 565 nm for Cy3 and 650 nm for CyS. Each array is typically scanned twice, one scan per fluorophore using the appropriate filters at the laser source, although the apparatus is capable of recording the spectra from both fluorophores simultaneously.
The sensitivity of the scans is typically calibrated using the signal intensity generated by a cDNA control species added to the sample mixture at a known concentration. A
specific location on the array contains a complementary DNA sequence, allowing the intensity of the signal at that location to be correlated with a weight ratio of hybridizing species of 1:100,000. When two samples from different sources (e.g., representing test and control cells), each labeled with a different fluorophore, are hybridized to a single array for the purpose of identifying genes that are differentially expressed, the calibration is done by labeling samples of the calibrating cDNA with the two fluorophores and adding identical amounts of each to the hybridization mixture.
The output of the photomultiplier tube is digitized using a 12-bit RTI-835H
analog-to-digital (A!D) conversion board (Analog Devices, Inc., Norwood MA) installed in an IBM-compatible PC
computer. The digitized data are displayed as an image where the signal intensity is mapped using a linear 20-color transformation to a pseudocolor scale ranging from blue (low signal) to red (high signal). The data is also analyzed quantitatively. Where two different fluorophores are excited and measured simultaneously, the data are first corrected for optical crosstallc (due to overlapping emission spectra) between the fluorophores using each fluorophore's emission spectrum.
A grid is superimposed over the fluorescence signal image such that the signal from each spot is centered in each element of the grid. The fluorescence signal within each element is then integrated to obtain a numerical value corresponding to the average intensity of the signal. The software used for signal analysis is the GEMTOOLS gene expression analysis program (Incyte).

XI. Complementary Polynucleotides Sequences complementary to the PRTS-encoding sequences, or any parts thereof, are used to detect, decrease, or inhibit expression of naturally occurring PRTS. Although use of oligonucleotides comprising from about 15 to 30 base pairs is described, essentially the same procedure is used with smaller or with larger sequence fragments. Appropriate oligonucleotides are designed using OLIGO
4.06 software (National Biosciences) and the coding sequence of PRTS. To inhibit transcription, a complementary oligonucleotide is designed from the most unique 5' sequence and used to prevent promoter binding to the coding sequence. To inhibit translation, a complementary oligonucleotide is designed to prevent ribosomal binding to the PRTS-encoding transcript.
XII. Expression of PRTS
Expression and purification of PRTS is achieved using bacterial or virus-based expression systems. For expression of PRTS in bacteria, cDNA is subcloned into an appropriate vector containing an antibiotic resistance gene and an inducible promoter that directs high levels of cDNA
transcription. Examples of such promoters include, but are not limited to, the trp-lac (tac) hybrid promoter and the T5 or T7 bacteriophage promoter in conjunction with the lac operator regulatory element. Recombinant vectors are transformed into suitable bacterial hosts, e.g., BL21(DE3).
Antibiotic resistant bacteria express PRTS upon induction with isopropyl beta-D-thiogalactopyranoside (IPTG). Expression of PRTS in eulcaryotic cells is achieved by infecting insect or mammalian cell lines with recombinant Autographica californica nuclear polyhedrosis virus (AcMNPV), commonly lmown as baculovirus. The nonessential polyhedrin gene of baculovirus is replaced with cDNA encoding PRTS by either homologous recombination or bacterial-mediated transposition involving transfer plasmid intermediates. Viral infectivity is maintained and the strong polyhedrin promoter drives high levels of cDNA transcription. Recombinant baculovirus is used to infect Spodoptera fruginerda (Sf9) insect cells in most cases, or human hepatocytes, in some cases.
Infection of the latter requires additional genetic modifications to baculovirus. (See Engelhard, E.K.
et al. (1994) Proc. Natl. Acad. Sci. USA 91:3224-3227; Sandig, V. et al.
(1996) Hum. Gene Ther.
7:1937-1945.) In most expression systems, PRTS is synthesized as a fusion protein with, e.g., glutathione S-transferase (GST) or a peptide epitope tag, such as FLAG or 6-His, permitting rapid, single-step, affinity-based purification of recombinant fusion protein from crude cell lysates. GST, a 26-kilodalton enzyme from Schistosoma j~onicum, enables the purification of fusion proteins on immobilized glutathione under conditions that maintain protein activity and antigenicity (Amersham Pharmacia Biotech). Following purification, the GST moiety can be proteolytically cleaved from PRTS at specifically engineered sites. FLAG, an 8-amino acid peptide, enables immunoaffinity purification using commercially available monoclonal and polyclonal anti-FLAG
antibodies (Eastman Kodak). 6-His, a stretch of six consecutive histidine residues, enables purification on metal-chelate resins (QIAGEN). Methods for protein expression and purification are discussed in Ausubel (1995, supra, ch. 10 and 16). Purified PRTS obtained by these methods can be used directly in the assays shown in Examples XVI, XVII, XVIII, and XlX where applicable.
XIII. Functional Assays PRTS function is assessed by expressing the sequences encoding PRTS at physiologically elevated levels in mammalian cell culture systems. cDNA is subcloned into a mammalian expression vector containing a strong promoter that drives high levels of cDNA
expression. Vectors of choice include PCMV SPORT (Life Technologies) and PCR3.1 (Invitrogen, Carlsbad CA), both of which contain the cytomegalovirus promoter. 5-10 ,ug of recombinant vector are transiently transfected into a human cell line, for example, an endothelial or hematopoietic cell line, using either liposome formulations or electroporation. 1-2 ,ug of an additional plasmid containing sequences encoding a marker protein are co-transfected. Expression of a marker protein provides a means to distinguish transfected cells from nontransfected cells and is a reliable predictor of cDNA expression from the recombinant vector. Marker proteins of choice include, e.g., Green Fluorescent Protein (GFP;
Clontech), CD64, or a CD64-GFP fusion protein. Flow cytometry (FCM), an automated, laser optics-based technique, is used to identify transfected cells expressing GFP or CD64-GFP and to evaluate the apoptotic state of the cells and other cellular properties. FCM detects and quantifies the uptake of fluorescent molecules that diagnose events preceding or coincident with cell death. These events include changes in nuclear DNA content as measured by staining of DNA with propidium iodide;
changes in cell size and granularity as measured by forward light scatter and 90 degree side light scatter; down-regulation of DNA synthesis as measured by decrease in bromodeoxyuridine uptake;
alterations in expression of cell surface and intracellular proteins as measured by reactivity with specific antibodies; and alterations in plasma membrane composition as measured by the binding of fluorescein-conjugated Annexin V protein to the cell surface. Methods in flow cytometry are discussed in Ormerod, M.G. (1994) Flow Cytometry, Oxford, New York NY.
The influence of PRTS on gene expression can be assessed using highly purified populations of cells transfected with sequences encoding PRTS and either CD64 or CD64-GFP.
CD64 and CD64-GFP are expressed on the surface of transfected cells and bind to conserved regions of human immunoglobulin G (IgG). Transfected cells are efficiently separated from nontransfected cells using magnetic beads coated with either human IgG or antibody against CD64 (DYNAL, Lake Success NY). mRNA can be purified from the cells using methods well known by those of skill in the art.
Expression of mRNA encoding PRTS and other genes of interest can be analyzed by northern analysis or microarray techniques.
XIV. Production of PRTS Specific Antibodies PRTS substantially purified using polyacrylamide gel electrophoresis (PAGE;
see, e.g., Harrington, M.G. (1990) Methods Enzymol. 182:488-495), or other purification techniques, is used to immunize rabbits and to produce antibodies using standard protocols.
Alternatively, the PRTS amino acid sequence is analyzed using LASERGENE
software (DNASTAR) 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. Methods for selection of appropriate epitopes, such as those near the C-terminus or in hydrophilic regions are well described in the art. (See, e.g., Ausubel, 1995, su__Pra,, ch. 11.) Typically, oligopeptides of about 15 residues in length are synthesized using an ABI 431A
peptide synthesizer (Applied Biosystems) using FMOC chemistry and coupled to KLH (Sigma-Aldrich, St. Louis MO) by reaction with N-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS) to increase immunogenicity. (See, e.g., Ausubel, 1995, supra.) Rabbits are immunized with the oligopeptide-KL,H complex in complete Freund's adjuvant. Resulting antisera are tested for antipeptide and anti-PRTS activity by, for example, binding the peptide or PRTS to a substrate, blocking with 1 % BSA, reacting with rabbit antisera, washing, and reacting with radio-iodinated goat anti-rabbit IgG.
XV. Purification of Naturally Occurring PRTS Using Specific Antibodies Naturally occurring or recombinant PRTS is substantially purified by immunoaffinity chromatography using antibodies specific fox PRTS. An immunoaffinity column is constructed by covalently coupling anti-PRTS antibody to an activated chromatographic resin, such as CNBr-activated SEPHAROSE (Amersham Pharmacia Biotech). After the coupling, the resin is blocked and washed according to the manufacturer's instructions.
Media containing PRTS are passed over the immunoaffinity column, and the column is washed under conditions that allow the preferential absorbance of PRTS (e.g., high ionic strength buffers in the presence of detergent). The column is eluted under conditions that disrupt antibody/PRTS binding (e.g., a buffer of pH 2 to pH 3, or a high concentration of a chaotrope, such as urea or thiocyanate ion), and PRTS is collected.
XVI. Identification of Molecules Which Interact with PRTS
PRTS, or biologically active fragments thereof, are labeled with'z5I Bolton-Hunter reagent.
(See, e.g., Bolton, A.E. and W.M. Hunter (1973) Biochem. J. 133:529-539.) Candidate molecules previously arrayed in the wells of a mufti-well plate are incubated with the labeled PRTS, washed, and any wells with labeled PRTS complex are assayed. Data obtained using different concentrations of PRTS are used to calculate values for the number, affinity, and association of PRTS with the candidate molecules.

Alternatively, molecules interacting with PRTS are analyzed using the yeast two-hybrid system as described in Fields, S. and O. Song (1989) Nature 340:245-246, or using commercially available kits based on the two-hybrid system, such as the MATCHMAKER system (Clontech).
PRTS may also be used in the PATHCALLING process (CuraGen Corp., New Haven CT) which employs the yeast two-hybrid system in a high-throughput manner to determine all interactions between the proteins encoded by two large libraries of genes (Nandabalan, K.
et al. (2000) U.S.
Patent No. 6,057,101).
XVII. Demonstration of PRTS Activity Protease activity is measured by the hydrolysis of appropriate synthetic peptide substrates conjugated with various chromogenic molecules in which the degree of hydrolysis is quantified by spectrophotometric (or.fluorometric) absorption of the released chromophore (Beynon, R.J. and J.S.
Bond (1994) Proteolytic Enzymes: A Practical Approach, Oxford University Press, New York NY, pp.25-55). Peptide substrates are designed according to the category of protease activity as endopeptidase (serine, cysteine, aspartic proteases, or metalloproteases), aminopeptidase (leucine aminopeptidase), or carboxypeptidase (carboxypeptidases A and B, procollagen C-proteinase).
Commonly used chromogens are 2-naphthylamine, 4-nitroaniline, and furylacrylic acid. Assays are .
performed at ambient temperature and contain an aliquot of the enzyme and the appropriate substrate in a suitable buffer. Reactions are carried out in an optical cuvette, and the increaseldecrease in absorbents of the chromogen released during hydrolysis of the peptide substrate is measured. The change in absorbents is proportional to the enzyme activity in the assay.
An alternate assay for ubiquitin hydrolase activity measures the hydrolysis of a ubiquitin precursor. The assay is performed at ambient temperature and contains an aliquot of PRTS and the appropriate substrate in a suitable buffer. Chemically synthesized human ubiquitin-valine may be used as substrate. Cleavage of the C-terminal valine residue from the substrate is monitored by capillary electrophoresis (Franklin, K. et al. (1997) Anal. Biochem. 247:305-309).
In the alternative, an assay for protease activity takes advantage of fluorescence resonance energy transfer (FRET) that occurs when one donor and one acceptor fluorophore with an appropriate spectral overlap are in close proximity. A flexible peptide linker containing a cleavage site specific for PRTS is fused between a red-shifted variant (RSGFP4) and a blue variant (BFPS) of Green Fluorescent Protein. This fusion protein has spectral properties that suggest energy transfer is occurring from BFPS to RSGFP4. When the fusion protein is incubated with PRTS, the substrate is cleaved; and the two fluorescent proteins dissociate. This is accompanied by a marked decrease in energy transfer which is quantified by comparing the emission spectra before and after the addition of PRTS (Mitre, R.D. et al. (1996) Gene 173:13-17). This assay can also be performed in living cells.
In this case the fluorescent substrate protein is expressed constitutively in cells and PRTS is introduced on an inducible vector so that FRET can be monitored in the presence and absence of PRTS {Sagot, I. et al. (1999) FEBS Lett. 447:53-57).
XVIII. Identification of PRTS Substrates Phage display libraries can be used to identify optimal substrate sequences for PRTS. A
random hexamer followed by a linker and a known antibody epitope is cloned as an N-terminal extension of gene III in a filamentous phage library. Gene III codes for a coat protein, and the epitope will be displayed on the surface of each phage particle. The library is incubated with PRTS under proteolytic conditions so that the epitope will be removed if the hexamer codes for a PRTS cleavage site. An antibody that recognizes the epitope is added along with immobilized protein A. Uncleaved phage, which still bear the epitope, are removed by centrifugation. Phage in the supernatant are then amplified and undergo several more rounds of screening. Individual phage clones are then isolated and sequenced. Reaction kinetics for these peptide substrates can be studied using an assay in Example XVII, and an optimal cleavage sequence can be derived (Ke, S.H. et al.
(1997) J. Biol.
Chem. 272:16603-16609).
To screen for in vivo PRTS substrates, this method can be expanded to screen a cDNA
expression library displayed on the surface of phage particles (T7SELECT 10-3 Phage display vector, Novagen, Madison Wn or yeast cells (pYDl yeast display vector kit, Invitrogen, Carlsbad CA). In this case, entire cDNAs are fused between Gene III and the appropriate epitope.
XIX. Identification of PRTS Inhibitors Compounds to be tested are arrayed in the wells of a mufti-well plate in varying concentrations along with an appropriate buffer and substrate, as described in the assays in Example XVII. PRTS activity is measured for each well and the ability of each compound to inhibit PRTS
activity can be determined, as well as the dose-response kinetics. This assay could also be used to identify molecules which enhance PRTS activity.
In the alternative, phage display libraries can be used to screen for peptide PRTS inhibitors.
Candidates are found among peptides which bind tightly to a protease. In this case, mufti-well plate wells are coated with PRTS and incubated with a random peptide phage display library or a cyclic peptide library (Koivunen, E. et al. (1999) Nat. Biotechnol. 17:768-774).
Unbound phage are washed away and selected phage amplified and rescreened for several more rounds.
Candidates axe tested for PRTS inhibitory activity using an assay described in Example XV1I.
Various modifications and variations of the described methods and systems 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 certain 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.

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-d b p ~ c w o d P.~ U can E-~ E-lls <110> INCYTE GENOMICS, INC.
LEE, Ernestine A.
HAFALIA, April YUE, Henry LAL, Preeti G.
YAO, Monique G.
LU, Yan WALIA, Narinder K.
WARREN, Bridget A.
LU, Dyung Aina M.
BAUGHN, Mariah R.
DELEGEANE, Angelo M.
BURFORD, Neil BOROWSKY, Mark L.
LEE, Sally XU, Yuming GRIFFIN, Jennifer A.
KALLICK, Deborah A.
GANDHI, Ameena R.
ARVIZU, Chandra ISON, Craig H.
TANG, Y. Tom AZIMZAI, Yalda ELLIOTT, Vicki S.
SWARNAKAR, Anita RAMKUMAR, Jayalaxini NGUYEN, Danniel B.
TRIBOULEY, Catherine M.
L0, Terence P.
AU-YOUNG, Janice THANGAVELU, Kavitha KEARNEY, Liam <120> PROTEASES
<130> PI-0263 PCT
<140> To Be Assigned <141> Herewith <150> 60/241,573; 60/243,643; 60/245,256; 60!248,395; 60!249,826 60/252,303; 60/250,981 <151> 2000-10-18; 2000-10-25; 2000-11-02; 2000-11-13; 2000-11-16 2000-11-20; 2000-12-01 <160> 32 <170> PERL Program <210> 1 <211> 334 <212> PRT
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte ID No: 6926819CD1 <400> 1 Met Asn Pro Ser Leu Leu Leu A1a Ala Phe Phe Leu Gly Ile Ala 1 5 . 10 15 Ser Ala A1a Leu Thr Arg Asp His Ser Leu Asp Ala Gln Trp Thr Lys Trp Lys Ala Lys His Lys Arg Leu Tyr Gly Met Asn Arg Asn His Trp Ile Arg Val Leu Trp Glu Lys Asp Val Lys Met Ile Glu Gln His Asn Gln Glu Tyr Ser Gln Gly Lys His Ser Phe Thr Met Ala Met Asn Ala Phe Gly Asp Met Val Ser Glu Glu Phe Arg Gln Val Met Asn G1y Phe G1n Tyr Gln Lys His Arg Lys Gly Lys Gln Phe Gln Glu Arg Leu Leu Leu Glu Ile Pro Thr Ser Val Asp Trp Arg G1u Lys Gly Tyr Met Thr Pro Val Lys Asp Gln Gln Gly Gln Cys Gly Ser Cys Trp Ala Phe Ser A1a Thr Gly Ala Leu Glu G1y Gln Met Phe Trp Lys Thr Gly Lys Leu Ile Ser Leu Asn Glu Gln 155 160 165 ff Asn Leu Val Asp Cys Ser Gly Pro G1n Gly Asn Glu Gly Cys Asn Gly Asp Phe Met Asp Asn Pro Phe Arg Tyr Val Gln Glu Asn Gly Gly Leu Asp Ser Glu Ala Ser Tyr Pro Tyr Glu Gly Lys Val Lys Thr Cys Arg Tyr Asn Pro Lys Tyr Ser Ala A1a Asn Asp Thr Gly Phe Val Asp I1e Pro Ser Arg Glu Lys Asp Leu Ala Lys Ala Val Ala Thr Val Gly Pro Ile Ser Val A1a Val G1y Ala Ser His Val 245 250 ' 255 Phe Phe Gln Phe Tyr Lys Lys Gly Ile Tyr Phe Glu Pro Arg Cys Asp Pro Glu Gly Leu Asp His Ala Met Leu Val Val Gly Tyr Ser Tyr Glu Gly Ala Asp Ser Asp Asn Asn Lys Tyr Trp Leu Va1 Lys Asn Ser Trp Gly Lys Asn Trp G1y Met Asp Gly Tyr Ile Lys Met Ala Lys Asp Arg Arg Asn Asn Cys Gly Ile Ala Thr Ala Ala Ser Tyr Pro Thr Val <210> 2 <211> 511 <212> PRT
<213> Homo sapiens <220>
<221> misc_feature <223> Incyte ID No: 7473526CD1 <400> 2 Met Ser Leu Trp Pro Pro Phe Arg Cys Arg Trp Lys Leu Ala Pro Arg Tyr Ser Arg Arg Ala Ser Pro Gln Gln Pro Gln Gln Asp Phe Glu Ala Leu Leu Ala Glu Cys Leu Arg Asn Gly Cys Leu Phe Glu Asp Thr Ser Phe Pro Ala Thr Leu Ser Ser I1e Gly Ser Gly Ser Leu Leu Gln Lys Leu Pro Pro Arg Leu Gln Trp Lys Arg Pro Pro Glu Leu His Ser Asn Pro Gln Phe Tyr Phe Ala Lys Ala Lys Arg Leu Asp Leu Cys Gln G1y Ile Val Gly Asp Cys Trp Phe Leu Ala Ala Leu Gln Ala Leu Ala Leu His Gln Asp Ile Leu Ser Arg Va1 Val Pro Leu Asn Gln Ser Phe Thr Glu Lys Tyr Ala Gly Ile Phe Arg Phe Trp Phe Trp His Tyr Gly Asn Trp Val Pro Val Val T1e Asp Asp Arg Leu Pro Val Asn Glu Ala Gly Gln Leu Val Phe Val Ser Ser Thr Tyr Lys Asn Leu Phe Trp Gly Ala Leu Leu Glu Lys Ala Tyr Ala Lys Leu Ser Gly Ser Tyr Glu Asp Leu Gln Ser Gly Gln Val Ser Glu Ala Leu Val Asp Phe Thr Gly Gly Val Thr Met Thr Ile Asn Leu Ala Glu Ala His Gly Asn Leu Trp Asp Ile Leu Ile Glu Ala Thr Tyr Asn Arg Thr Leu Ile Gly Cys Gln Thr His Ser Gly Glu Lys Ile Leu Glu Asn Gly Leu Val Glu Gly His Ala Tyr Thr Leu Thr Gly Ile Arg Lys Val Thr Cys Lys His Arg Pro Glu Tyr Leu Val Lys Leu Arg Asn Pro Trp Gly Lys Val Glu Trp Lys Gly Asp Trp Ser Asp Ser Ser Ser Lys Trp Glu Leu Leu Ser Pro Lys Glu Lys Ile Leu Leu Leu Arg Lys Asp Asn Asp G1y Glu Phe Trp Met Thr Leu G1n Asp Phe Lys Thr His Phe Val Leu Leu Val Ile Cys Lys Leu Thr Pro Gly Leu Leu Ser Gln G1u Ala Ala Gln Lys Trp Thr Tyr Thr Met Arg Glu Gly Arg Trp Glu Lys Arg Ser Thr Ala G1y Gly Gln Arg Gln Leu Leu Gln Asp Thr Phe Trp Lys Asn Pro Gln Phe Leu Leu Ser Val Trp Arg Pro Glu Glu Gly Arg Arg Ser Leu Arg Pro Cys Ser Val Leu Val Ser Leu Leu G1n Lys Pro Arg His Arg Cys Arg Lys Arg Lys Pro Leu Leu Ala Ile Gly Phe Tyr Leu Tyr Arg Met Asn Lys Tyr His Asp Asp Gln Arg Arg Leu Pro Pro Glu Phe Phe Gln Arg Asn Thr Pro Leu Ser G1n Pro Asp Arg Phe Leu Lys Glu Lys Glu Va1 Ser Gln Glu Leu Cys Leu Glu Pro Gly Thr Tyr Leu Ile Val Pro Ala Tyr Trp Arg Pro Thr Arg Ser Gln Ser Ser Ser Ser Gly Ser Ser Pro G1y Ser Thr Ser Phe Met Lys Leu Ala Ala Ile Leu Val Ser Ser Ser Gln Arg Arg <210> 3 <211> 812 <212> PRT
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte ID No: 7478443CD1 <400> 3 Met Gly Trp Arg Pro Arg Arg Ala Arg Gly Thr Pro Leu Leu Leu Leu Leu Leu Leu Leu Leu Leu Trp Pro Va1 Pro Gly Ala Gly Val Leu Gln Gly His Ile Pro Gly Gln Pro Val Thr Pro His Trp Val Leu Asp Gly Gln Pro Trp Arg Thr Val Ser Leu Glu Glu Pro Val Ser Lys Pro Asp Met Gly Leu Val Ala Leu Glu Ala Glu Gly Gln Glu Leu Leu Leu Glu Leu Glu Lys Asn His Arg Leu Leu Ala Pro Gly Tyr Ile Glu Thr His Tyr Gly Pro Asp Gly Gln Pro Val Val Leu Ala Pro Asn His Thr Asp His Cys His Tyr Gln Gly Arg Val Arg Gly Phe Pro Asp Ser Trp Val Val Leu Cys Thr Cys Ser G1y Met Ser Gly Leu Ile Thr Leu Ser Arg Asn Ala Ser Tyr Tyr Leu Arg Pro Trp Pro Pro Arg Gly Ser Lys Asp Phe Ser Thr His Glu Ile Phe Arg Met Glu Gln Leu Leu Thr Trp Lys Gly Thr Cys Gly His Arg Asp Pro Gly Asn Lys Ala Gly Met Thr Ser Leu Pro Gly Gly Pro Gln Ser Arg Gly Arg Arg Glu Ala Arg Arg Thr Arg Lys Tyr Leu G1u Leu Tyr Ile Val Ala Asp His Thr Leu Phe Leu Thr Arg His Arg Asn Leu Asn His Thr Lys Gln Arg Leu Leu Glu Val Ala Asn Tyr Val Asp Gln Leu Leu Arg Thr Leu Asp Ile Gln Val Ala Leu Thr Gly Leu Glu Val Trp Thr Glu Arg Asp Arg Ser Arg Va1 Thr Gln Asp Ala Asn Ala Thr Leu Trp Ala Phe Leu Gln Trp Arg Arg Gly Leu Trp A1a Gln Arg Pro His Asp Ser Ala Gln Leu Leu Thr Gly Arg Ala Phe Gln Gly Ala Thr Val Gly Leu Ala Pro Val Glu Gly Met Cys Arg A1a G1u Ser Ser Gly Gly Val Ser Thr Asp His Ser Glu Leu Pro I1e Gly Ala Ala Ala Thr Met Ala His Glu Ile Gly His Ser Leu Gly Leu Ser His Asp Pro Asp Gly Cys Cys Val Glu Ala Ala Ala Glu Ser Gly Gly Cys Val Met A1a Ala Ala Thr Gly His Pro Phe Pro Arg Val Phe Ser Ala Cys Ser Arg Arg Gln Leu Arg A1a Phe Phe Arg Lys Gly Gly Gly Ala Cys Leu Ser Asn Ala Pro Asp Pro Gly Leu Pro Va1 Pro Pro Ala Leu Cys G1y Asn Gly Phe Val Glu Ala Gly Glu Glu Cys Asp Cys Gly Pro Gly Gln Glu Cys Arg Asp Leu Cys Cys Phe A1a His Asn Cys Ser Leu Arg Pro Gly Ala Gln Cys Ala His Gly Asp Cys Cys Val Arg Cys Leu Leu Lys Pro Ala G1y Ala Leu Cys Arg Gln Ala Met Gly Asp Cys Asp Leu Pro Glu Phe Cys Thr Gly Thr Ser Ser His Cys Pro Pro Asp Val Tyr Leu Leu Asp Gly Ser Pro Cys Ala Arg Gly Ser Gly Tyr Cys Trp Asp Gly Ala Cys Pro Thr Leu Glu Gln Gln Cys Gln Gln Leu Trp Gly Pro Gly Ser His Pro Ala Pro Glu Ala Cys Phe Gln Va1 Val Asn Ser Ala G1y Asp Ala His Gly Asn Cys Gly Gln Asp Ser Glu Gly His Phe Leu Pro Cys Ala Gly Arg Asp Ala Leu Cys G1y Lys Leu Gln Cys Gln Gly Gly Lys Pro Ser Leu Leu Ala Pro His Met Val Pro Val Asp Ser Thr Val His Leu Asp Gly Gln Glu Val Thr Cys Arg Gly Ala Leu Ala Leu Pro Ser Ala Gln Leu Asp Leu Leu Gly Leu Gly Leu Val Glu Pro Gly Thr Gln Cys G1y Pro Arg Met Val Cys Gln Ser Arg Arg Cys Arg Lys Asn Ala Phe Gln Glu Leu Gln Arg Cys Leu Thr A1a Cys His Ser His Gly Val Cys Asn Ser Asn His Asn Cys His Cys Ala Pro Gly Trp Ala Pro Pro Phe Cys Asp Lys Pro Gly Phe Gly Gly Ser Met Asp Ser G1y Pro Val Gln Ala Glu Asn His Asp Thr Phe Leu Leu Ala Met Leu Leu Ser Val Leu Leu Pro Leu Leu Pro Gly Ala Gly Leu Ala Trp Cys Cys Tyr Arg Leu Pro Gly Ala His Leu Gln Arg Cys Ser Trp Gly Cys Arg Arg Asp Pro Ala Cys Ser Gly Pro Lys Asp Gly Pro His Arg Asp His Pro Leu Gly Gly Val His Pro Met Glu Leu Gly Pro Thr Ala Thr Gly Gln Pro Trp Pro Leu Asp Pro Glu Asn Ser His Glu Pro Ser Ser His Pro Glu Lys Pro Leu Pro Ala Val Ser Pro Asp Pro Gln Asp Gln Val Gln Met Pro Arg Ser Cys Leu Trp <210> 4 <211> 1236 <212> PRT
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte ID No: 3533147CD1 <400> 4 Met Thr Gly Thr Gly Gly Arg Lys Pro Thr Gly Asp Lys Gln Glu Val His Pro Trp Glu Lys Gln Glu Val Arg Glu Gln Thr Glu Ser Pro Gln Glu Leu Thr Arg Ser Pro Gln Gly Thr Asp Arg Asn Asp Thr Val Thr Ile Tyr Thr Asp Thr Gln Ser Arg Lys Ala Gly Ala 50 55 ' 60 Ser Arg Lys Ile Arg Asn Met Leu Asn Ile Tyr Leu Val Trp Leu Val Lys Ile Asn Gln Ile Ile Ile Asn Val Phe Tyr Gln Asn Pro Glu Pro Thr Ile Trp Asn Ser Ala Phe Ile Va1 Asp Ile Thr Ala Ile Va1 Pro Thr Ala Leu Phe Pro Phe Asn Va1 Ala Lys Pro Lys Met Leu Val Glu Asn Leu G1n Glu Gly Asp Phe Arg Glu Leu Arg Gly Asn Ser His His Cys Leu Thr Lys Lys Gly Leu Gly Asn A1a Pro Pro Gly Leu Gln Phe Thr Leu Tyr Lys Cys Leu Asp Ser Ser Arg Thr Ala Gln Pro His Ala Gly Leu His Tyr Val Asp Ile Asn Ser Gly Met Ile Arg Thr Glu Glu Ala Asp Tyr Phe Leu Arg Pro Leu Pro Ser His Leu Ser Trp Lys Leu Gly Arg Ala Ala Gln Gly Ser Ser Pro Ser His Val Leu Tyr Lys Arg Ser Thr G1u Pro His Ala Pro Gly A1a Ser Glu Val Leu Val Thr Ser Arg Thr Trp Glu Leu Ala His G1n Pro Leu His Ser Ser Asp Leu Arg Leu Gly Leu Pro Gln Lys G1n His Phe Cys Gly Arg Arg Lys Lys Tyr Met Pro Gln Pro Pro Lys Glu Asp Leu Phe Ile Leu Pro Asp Glu Tyr Lys Ser Cys Leu Arg His Lys Arg Ser Leu Leu Arg Ser His Arg Asn Glu Glu Leu Asn Val Glu Thr Leu Val Val Val Asp Lys Lys Met Met G1n Asn His Gly His Glu Asn Ile Thr Thr Tyr Val Leu Thr Ile Leu Asn Met Val Ser Ala Leu Phe Lys Asp Gly Thr Ile Gly G1y Asn Ile Asn Ile A1a Ile Val Gly Leu I1e Leu Leu Glu Asp Glu G1n Pro Gly Leu Va1 Ile Ser His His Ala Asp His Thr Leu Ser Ser Phe Cys Gln Trp Gln Ser Gly Leu Met Gly Lys Asp Gly 380 385. 390 Thr Arg His Asp His Ala Ile Leu Leu Thr Gly Leu Asp Ile Cys Ser Trp Lys Asn Glu Pro Cys Asp Thr Leu Gly Phe Ala Pro Ile Ser Gly Met Cys Ser Lys Tyr Arg Ser Cys Thr Ile Asn Glu Asp Thr Gly Leu Gly Leu Ala Phe Thr I1e Ala His Glu Ser Gly His Asn Phe Gly Met Ile His Asp Gly Glu Gly Asn Met Cys Lys Lys Ser Glu Gly Asn Ile Met Ser Pro Thr Leu Ala Gly Arg Asn Gly Val Phe Ser Trp Ser Pro Cys Ser Arg Gln Tyr Leu His Lys Phe Leu Ser Thr Ala Gln Ala Ile Cys Leu Ala Asp Gln Pro Lys Pro Val Lys G1u Tyr Lys Tyr Pro Glu Lys Leu Pro Gly Glu Leu Tyr Asp Ala Asn Thr Gln Cys Lys Trp G1n Phe Gly Glu Lys A1a Lys Leu Cys Met Leu Asp Phe Lys Lys Asp Ile Cys Lys Ala Leu Trp Cys His Arg Ile Gly Arg Lys Cys Glu Thr Lys Phe Met Pro Ala Ala Glu Gly Thr Ile Cys Gly His Asp Met Trp Cys Arg Gly Gly Gln Cys Val Lys Tyr Gly Asp Glu G1y Pro Lys Pro Thr His Gly His Trp Ser Asp Trp Ser Ser Trp Ser Pro Cys Ser Arg Thr Cys Gly Gly Gly Val Ser His Arg Ser Arg Leu Cys Thr Asn Pro Lys Pro Ser His Gly Gly Lys Phe Cys Glu G1y Ser Thr Arg Thr Leu Lys Leu Cys Asn Ser Gln Lys Cys Pro Arg Asp Ser Val Asp Phe Arg Ala Ala Gln Cys Ala Glu His Asn Ser Arg Arg Phe Arg Gly Arg His Tyr Lys Trp Lys Pro Tyr Thr Gln Val Glu Asp Gln Asp Leu Cys Lys Leu Tyr Cys Ile Ala Glu Gly Phe Asp Phe Phe Phe Ser Leu Ser Asn Lys Val Lys Asp Gly Thr Pro Cys Ser Glu Asp Ser Arg Asn Val Cys Ile Asp Gly Ile Cys G1u Arg Val Gly Cys Asp Asn Val Leu Gly Ser Asp Ala Val Glu Asp Val Cys Gly Val Cys Asn Gly Asn Asn Ser Ala Cys Thr Ile His Arg Gly Leu Tyr Thr Lys His His His Thr Asn Gln Tyr Tyr His Met Val Thr Ile Pro Ser Gly Ala Arg Ser Ile Arg Ile Tyr Glu Met Asn Val Ser Thr Ser Tyr Ile Ser Val Arg Asn Ala Leu Arg Arg Tyr Tyr Leu Asn Gly His Trp Thr Val Asp Trp Pro Gly Arg Tyr Lys Phe Ser G1y Thr Thr Phe Asp Tyr Arg Arg Ser Tyr Asn Glu Pro Glu Asn 830 ~ 835 840 Leu Ile Ala Thr Gly Pro Thr Asn Glu Thr Leu Ile Val Glu Leu Leu Phe Gln Gly Arg Asn Pro Gly Va1 Ala Trp Glu Tyr Ser Met Pro Arg Leu Gly Thr Glu Lys Gln Pro Pro Ala Gln Pro Ser Tyr Thr Trp Ala Ile Val Arg Ser Glu Cys Ser Val Ser Cys Gly Gly Gly Gln Met Thr Val Arg Glu Gly Cys Tyr Arg Asp Leu Lys Phe Gln Val Asn Met Ser Phe Cys Asn Pro Lys Thr Arg Pro Val Thr 920 ' 925 930 Gly Leu Val Pro Cys Lys Val Ser Ala Cys Pro Pro Ser Trp Ser Val Gly Asn Trp Ser Ala Cys Ser Arg Thr Cys Gly Gly Gly Ala Gln Ser Arg Pro Va1 Gln Cys Thr Arg Arg Val His Tyr Asp Ser Glu Pro Val Pro Ala Gly Leu Cys Pro Gln Leu Val Pro Pro Ala Gly Arg Pro Ala Thr Leu Arg Ala Ala His Leu His Gly Ala Pro Gly Pro Gly Gln Ser Ala His Thr Pro Val Gly Arg Val Glu G1u Arg Ala Va1 Ala Cys Lys Ser Thr Asn Pro Ser Ala Arg Ala Gln Leu Leu Pro Asp Ala Val Cys Thr Ser Glu Pro Lys Pro Arg Met His Glu Ala Cys Leu Leu Gln Arg Cys His Lys Pro Lys Lys Leu Gln Trp Leu Val Ser A1a Trp Ser G1n Cys Ser Val Thr Cys Glu Arg Gly Thr Gln Lys Arg Phe Leu Lys Cys Ala Glu Lys Tyr Val Ser Gly Lys Tyr Arg Glu Leu Ala Ser Lys Lys Cys Ser His Leu Pro Lys Pro Ser Leu Glu Leu Glu Arg Ala Cys Ala Pro Leu Pro Cys Pro Arg His Pro Pro Phe Ala Ala Ala Gly Pro Ser Arg Gly Ser Trp Phe Ala Ser Pro Trp Ser Gln Cys Thr Ala Ser Cys Gly Gly Gly Val Gln Thr Arg Ser Val Gln Cys Leu Ala Gly Gly Arg Pro Ala Ser Gly Cys Leu Leu His Gln Lys Pro Ser Ala Ser Leu Ala Cys Asn Thr His Phe Cys Pro Ile Ala Glu Lys Lys Asp Ala Phe Cys Lys Asp Tyr Phe His Trp Cys Tyr Leu Val Pro Gln His Gly Met Cys Ser His Lys Phe Tyr Gly Lys Gln Cys Cys Lys Thr Cys Ser Lys Ser Asn Leu <210> 5 <211> 304 <212> PRT
<21.3> Homo sapiens <220>
<221> misc_feature <223> Incyte ID No: 7483438CD1 <400> 5 Met Gly Leu Arg Ala Gly Pro Ile Leu Leu Leu Leu Leu Trp Leu Leu Pro Gly A1a His Trp Asp Val Leu Pro her G1u Cys Gly His Ser Lys Glu Ala Gly Arg Ile Val Gly Gly Gln Asp Thr Gln Glu Gly Arg Trp Pro Trp Gln Val Gly Leu Trp Leu Thr Ser Val G1y His Val Cys Gly Gly Ser Leu Ile His Pro Arg Trp Val Leu Thr Ala Ala His Cys Phe Leu Arg Ser Glu Asp Pro Gly Leu Tyr His Val Lys Val Gly Gly Leu Thr Pro Ser Leu Ser G1u Pro His Ser Ala Leu Val Ala Val Arg Arg Leu Leu Val His Ser Ser Tyr His Gly Thr Thr Thr Ser G1y Asp Ile Ala Leu Met G1u Leu Asp Ser Pro Leu Gln A1a Ser Gln Phe Ser Pro I1e Cys Leu Pro G1y Pro Gln Thr Pro Leu Ala I1e G1y Thr Val Cys Trp Val Asn Gly Leu Gly Glu Val A1a Val Pro Leu Leu Asp Ser Asn Met Cys G1u Leu Met Tyr His Leu G1y Glu Pro Ser Leu Ala Gly Gln Arg Leu Ile Gln Asp Asp Met Leu Cys A1a Gly Ser Val Gln Gly Lys Lys Asp Ser Cys Gln G1y Asp Ser Gly Gly Pro Leu Val Cys Pro Ile Asn Asp Thr Trp Ile Gln Ala Gly Ile Val Ser Trp Gly Phe Gly Cys Ala Arg Pro Phe Arg Pro Gly Val Tyr Thr Gln Val Leu Ser Tyr Thr Asp Trp Ile Gln Arg Thr Leu Ala Glu Ser His Ser Gly Met Ser Gly Ala Arg Pro Gly Ala Pro Gly Ser His Ser Gly Thr Ser Arg Ser His Pro Val Leu Leu Leu Glu Leu Leu Thr Val Cys Leu Leu Gly Ser Leu <210> 6 <211> 980 <212> PRT
<213> Homo sapiens <220>
<221> misc_feature <223> Incyte ID No: 7246467CD1 <400> 6 Met Ser Pro Leu Lys Ile His Gly Pro Ile Arg I1e Arg Ser Met Gln Thr Gly Ile Thr Lys Trp Lys Glu Gly Ser Phe Glu Ile Val Glu Lys Glu Asn Lys Val Ser Leu Val Val His Tyr Asn Thr Gly Gly Ile Pro Arg I1e Phe Gln Leu Ser His Asn Ile Lys Asn Val Val Leu Arg Pro Ser Gly Ala Lys Gln Ser Arg Leu Met Leu Thr Leu Gln Asp Asn Ser Phe Leu Ser Ile Asp Lys Val Pro Ser Lys Asp Ala Glu Glu Met Arg Leu Phe Leu Asp Ala Va1 His Gln Asn Arg Leu Pro Ala Ala Met Lys Pro Ser G1n Gly Ser Gly Ser Phe Gly Ala Ile Leu Gly Ser Arg Thr Ser Gln Lys Glu Thr Ser Arg Gln Leu Ser Tyr Ser Asp Asn Gln Ala Ser Ala Lys Arg Gly Ser Leu G1u Thr Lys Asp Asp Ile Pro Phe Arg Lys Val Leu G1y Asn Pro Gly Arg Gly Ser Ile Lys Thr Val Ala Gly Ser Gly Ile Ala Arg Thr Ile Pro Ser Leu Thr Ser Thr Ser Thr Pro Leu Arg Ser G1y Leu Leu Glu Asn Arg Thr Glu Lys Arg Lys Arg Met Ile Ser Thr Gly Ser Glu Leu Asn Glu Asp Tyr Pro Lys Glu Asn Asp Ser Ser Ser Asn Asn Lys Ala Met Thr Asp Pro Ser Arg Lys Tyr Leu Thr Ser Ser Arg Glu Lys Gln Leu Ser Leu Lys Gln Ser Glu Glu Asn Arg Thr Ser Gly Gly Leu Leu Pro Leu Gln Ser Ser Ser Phe Tyr Gly Ser Arg Ala Gly Ser Lys Glu His Ser Ser Gly Gly Thr Asn Leu Asp Arg Thr Asn Val Ser Ser Gln Thr Pro Ser Ala Lys Arg Ser Leu Gly Phe Leu Pro Gln Pro Val Pro Leu Ser Val Lys Lys Leu Arg Cys Asn Gln Asp Tyr Thr G1y Trp Asn Lys Pro Arg Val Pro Leu Ser Ser His Gln Gln Gln Gln Leu Gln Gly Phe Ser Asn Leu Gly Asn Thr Cys Tyr Met Asn A1a Ile Leu Gln Ser Leu Phe Ser Leu Gln Ser Phe Ala Asn Asp Leu Leu Lys Gln Gly Ile Pro Trp Lys Lys Ile Pro Leu Asn Ala Leu Ile Arg Arg Phe Ala His Leu Leu Val Lys Lys Asp Ile Cys Asn Ser Glu Thr Lys Lys Asp Leu Leu Lys Lys Val Lys Asn Ala Ile Ser Ala Thr Ala Glu Arg Phe Ser Gly Tyr Met Gln Asn Asp Ala His G1u Phe Leu Ser Gln Cys Leu Asp Gln Leu Lys Glu Asp Met Glu Lys Leu Asn Lys Thr Trp Lys Thr Glu Pro Val Ser Gly Glu Glu Asn Ser Pro Asp Ile Ser Ala Thr Arg Ala Tyr Thr Cys Pro Val I1e Thr Asn Leu Glu Phe Glu Val Gln His Ser Ile Ile Cys Lys Ala Cys Gly Glu Ile Ile Pro Lys Arg Glu Gln Phe Asn Asp Leu Ser Ile Asp Leu Pro Arg Arg Lys Lys Pro Leu Pro Pro Arg Ser Ile G1n Asp Ser Leu Asp Leu Phe Phe Arg Ala Glu Glu Leu Glu Tyr Ser Cys Glu Lys Cys Gly Gly Lys Cys Ala Leu Val Arg His Lys Phe Asn Arg Leu Pro Arg Val Leu Ile Leu His Leu Lys Arg Tyr Ser Phe Asn Val Ala Leu Ser Leu Asn Asn Lys Ile Gly Gln Gln Val I1e Ile Pro Arg Tyr Leu Thr Leu Ser Ser His Cys Thr Glu Asn Thr Lys Pro Pro Phe Thr Leu Gly Trp Ser Ala His Met Ala Met Ser Arg Pro Leu Lys Ala Ser Gln Met Va1 Asn Ser Cys Ile Thr Ser Pro Ser Thr Pro Ser Lys Lys Phe Thr Phe Lys Ser Lys Ser Ser Leu Ala Leu Cys Leu Asp Ser Asp Ser G1u Asp Glu Leu Lys Arg Ser Val Ala Leu Ser Gln Arg Leu Cys Glu Met Leu G1y Asn Glu Gln Gln G1n Glu Asp Leu Glu Lys Asp Ser Lys Leu Cys Pro I1e Glu Pro Asp Lys Ser Glu Leu Glu Asn Ser Gly Phe Asp Arg Met Ser G1u Glu Glu Leu Leu Ala Ala Va1 Leu Glu Ile Ser Lys Arg Asp Ala Ser Pro Ser Leu Ser His Glu Asp Asp Asp Lys Pro Thr Ser Ser Pro Asp Thr Gly Phe Ala Glu Asp Asp Ile Gln Glu Met Pro Glu Asn Pro Asp Thr Met Glu Thr Glu Lys Pro Lys Thr Ile Thr Glu Leu Asp Pro Ala Ser Phe Thr Glu Ile Thr Lys Asp Cys Asp Glu Asn Lys G1u Asn Lys Thr Pro Glu Gly Ser Gln Gly Glu Val Asp Trp Leu Gln Gln Tyr Asp Met Glu Arg Glu Arg Glu Glu Gln Glu Leu Gln Gln Ala Leu Ala Gln Ser Leu G1n Glu Gln Glu Ala Trp Glu Gln Lys Glu Asp Asp Asp Leu Lys Arg Ala Thr Glu Leu Ser Leu Gln Glu Phe Asn Asn Ser Phe Val Asp Ala Leu Gly Ser Asp Glu Asp Ser Gly Asn Glu Asp Val Phe Asp Met Glu Tyr Thr Glu Ala Glu Ala Glu Glu Leu Lys Arg Asn Ala G1u Thr Gly Asn Leu Pro His Ser Tyr Arg Leu Ile Ser Va1 Val Ser His Ile Gly Ser Thr Ser Ser Ser G1y His Tyr Ile Ser Asp Val Tyr Asp Ile Lys Lys Gln Ala Trp Phe Thr Tyr Asn Asp Leu Glu Val Ser Lys I1e Gln Glu Ala Ala Val Gln Ser Asp Arg Asp Arg Ser Gly Tyr Ile Phe Phe Tyr Met His Lys Glu Ile Phe Asp Glu Leu Leu Glu Thr Glu Lys Asn Ser Gln Ser Leu Ser Thr Glu Val Gly Lys Thr Thr Arg Gln Ala Ser <210> 7 <211> 1251 <212> PRT
<213> Homo sapiens <220>
<221> misc_feature <223> Incyte ID No: 7997881CD1 <400> 7 Met Thr Ile Val Asp Lys Ala Ser Glu Ser Ser Asp Pro Ser Ala Tyr Gln Asn Gln Pro Gly Ser Ser Glu Ala Val Ser Pro Gly Asp Met Asp Ala Gly Ser Ala Ser Trp Gly Ala Val Ser Ser Leu Asn Asp Va1 Ser Asn His Thr Leu Ser Leu Gly Pro Val Pro Gly Ala Val Va1 Tyr Ser Ser Ser Ser Val Pro Asp Lys Ser Lys Pro Ser Pro Gln Lys Asp G1n Ala Leu Gly Asp Gly Ile Ala Pro Pro Gz,n 80 85 ~ ~0 Lys Val Leu Phe Pro Ser Glu Lys Ile Cys Leu Lys Trp Gln Gln Thr His Arg Val Gly Ala Gly Leu Gln Asn Leu Gly Asn Thr Cys Phe Ala Asn Ala Ala Leu Gln Cys Leu Thr Tyr Thr Pro Pro Leu Ala Asn Tyr Met Leu Ser His Glu His Ser Lys Thr Cys His Ala Glu Gly Phe Cys Met Met Cys Thr Met Gln Ala His Ile Thr G1n Ala Leu Ser Asn Pro G1y Asp Val I1e Lys Pro Met Phe Val Ile Asn Glu Met Arg Arg Ile Ala Arg His Phe Arg Phe Gly Asn Gln Glu Asp Ala His Glu Phe Leu Gln Tyr Thr Val Asp Ala Met Gln Lys Ala Cys Leu Asn G1y Ser Asn Lys Leu Asp Arg His Thr G1n Ala Thr Thr Leu Val Cys Gln Ile Phe Gly Gly Tyr Leu Arg Ser Arg Val Lys Cys Leu Asn Cys Lys Gly Val Ser Asp Thr Phe Asp Pro Tyr Leu Asp Ile Thr Leu Glu Ile Lys Ala Ala Gln Ser Val Asn Lys Ala Leu Glu Gln Phe Val Lys Pro Glu Gln Leu Asp Gly Glu Asn Ser Tyr Lys Cys Ser Lys Cys Lys Lys Met Va1 Pro Ala Ser Lys Arg Phe Thr Ile His Arg Ser Ser Asn Val Leu Thr Leu Ser Leu Lys Arg Phe Ala Asn Phe Thr Gly G1y Lys I1e Ala Lys Asp Val Lys Tyr Pro Glu Tyr Leu Asp Ile Arg Pro Tyr Met Ser Gln Pro Asn Gly Glu Pro I1e Val Tyr Val Leu Tyr Ala Val Leu Val His Thr Gly Phe Asn Cys His Ala Gly His Tyr Phe Cys Tyr Ile Lys Ala Ser Asn Gly Leu Trp Tyr G1n Met Asn Asp Ser Ile Val Ser Thr Ser Asp Ile Arg Ser Val Leu Ser Gln Gln Ala Tyr Val Leu Phe Tyr Ile Arg Ser His Asp Va1 Lys Asn Gly Gly Glu Leu Thr His Pro Thr His Ser Pro Gly Gln Ser Ser Pro Arg Pro Val Ile Ser Gln Arg Val Val Thr Asn Lys Gln Ala Ala Pro Gly Phe Ile Gly Pro Gln Leu Pro Ser His Met Ile Lys Asn Pro Pro His Leu Asn Gly Thr Gly Pro Leu Lys Asp Thr Pro Ser Ser Ser Met Ser Ser Pro Asn Gly Asn Ser Ser Val Asn Arg Ala Ser Pro Val Asn Ala Ser Ala Ser Val Gln Asn Trp Ser Val Asn Arg Ser Ser Val Ile Pro Glu His Pro Lys Lys Gln Lys Ile Thr Ile Ser Ile His Asn Lys Leu Pro Val Arg Gln Cys Gln Ser Gln Pro Asn Leu His Ser Asn Ser Leu Glu Asn Pro Thr Lys Pro Val Pro Ser Ser Thr Ile Thr Asn Ser Ala Va1 Gln Ser Thr Ser Asn Ala Ser Thr Met Ser Val Ser Ser Lys Val Thr Lys Pro Ile Pro Arg Ser Glu Ser Cys Ser Gln Pro Val Met Asn Gly Lys Ser Lys Leu Asn Ser Ser Val Leu Val Pro Tyr Gly Ala Glu Ser Ser G1u Asp Ser Asp Glu Glu Ser Lys Gly Leu Gly Lys Glu Asn Gly Ile Gly Thr I1e Val Ser Ser His Ser Pro Gly Gln Asp Ala G1u Asp Glu Glu A1a Thr Pro His Glu Leu Gln Glu Pro Met Thr Leu Asn Gly Ala Asn Ser Ala Asp Ser Asp Ser Asp Pro Lys G1u Asn Gly Leu Ala Pro Asp Gly Ala Ser Cys Gln Gly Gln Pro Ala Leu His Ser Glu Asn Pro Phe Ala Lys Ala Asn Gly Leu Pro Gly Lys Leu Met Pro Ala Pro Leu Leu Ser Leu Pro Glu Asp Lys Ile Leu Glu Thr Phe Arg Leu Ser Asn Lys Leu Lys Gly Ser Thr Asp Glu Met Ser Ala Pro Gly Ala Glu Arg Gly Pro Pro Glu Asp Arg Asp Ala Glu Pro Gln Pro Gly Ser Pro Ala Ala Glu Ser Leu Glu Glu Pro Asp Ala Ala Ala Gly Leu Ser Ser Thr Lys Lys Ala Pro Pro Pro Arg Asp Pro Gly Thr Pro A1a Thr Lys Glu Gly Ala Trp Glu A1a Met Ala Val Ala Pro Glu Glu Pro Pro Pro Ser Ala Gly Glu Asp Ile Va1 Gly Asp Thr Ala Pro Pro Asp Leu Cys Asp Pro Gly Ser Leu Thr Gly Asp Ala Ser Pro Leu Ser Gln Asp Ala Lys Gly Met Ile Ala Glu Gly Pro Arg Asp Ser Ala Leu Ala Glu Ala Pro Glu Gly Leu Ser Pro Ala Pro Pro Ala Arg Ser Glu G1u Pro Cys Glu Gln Pro Leu Leu Val His Pro Ser Gly Asp His Ala Arg Asp Ala Gln Asp Pro Ser Gln Ser Leu Gly Ala Pro Glu Ala Ala Glu Arg Pro Pro Ala Pro Val Leu Asp Met Ala Pro Ala Gly His Pro Glu Gly Asp Ala G1u Pro Ser Pro G1y Glu Arg Val Glu Asp Ala Ala Ala Pro Lys A1a Pro Gly Pro Ser Pro Ala Lys Glu Lys Ile G1y Ser Leu Arg Lys Val Asp Arg Gly His Tyr Arg Ser Arg Arg Glu Arg Ser Ser Ser Gly Glu Pro Ala Arg Glu Ser Arg Ser Lys Thr Glu Gly His Arg His Arg Arg Arg Arg Thr Cys Pro Arg Glu Arg Asp Arg Gln Asp Arg His Ala Pro Glu His His Pro Gly His Gly Asp Arg Leu Ser Pro Gly Glu Arg Arg Ser Leu Gly Arg Cys Ser His His His Ser Arg His Arg Ser Gly Val Glu Leu Asp Trp Val Arg His 1025 x.030 1035 His Tyr Thr Glu Gly Glu Arg Gly Trp Gly Arg Glu Lys Phe Tyr Pro Asp Arg Pro Arg Trp Asp Arg Cys Arg Tyr Tyr His Asp Arg Tyr Ala Leu Tyr Ala Ala Arg Asp Trp Lys Pro Phe His Gly Gly Arg Glu His Glu Arg Ala Gly Leu His Glu Arg Pro His Lys Asp His Asn Arg Gly Arg Arg Gly Cys Glu Pro Ala Arg Glu Arg Glu Arg His Arg Pro Ser Ser Pro Arg Ala Gly Ala Pro His Ala Leu Ala Pro His Pro Asp Arg Phe Ser His Asp Arg Thr Ala Leu Val Ala Gly Asp Asn Cys Asn Leu Ser Asp Arg Phe His Glu His Glu Asn Gly Lys Ser Arg Lys Arg Arg His Asp Ser Val Glu Asn Ser Asp Ser His Val Glu Lys Lys A1a Arg Arg Ser Glu Gln Lys Asp Pro Leu Glu Glu Pro Lys A1a Lys Lys His Lys Lys Ser Lys Lys Lys Lys Lys Ser Lys Asp Lys His Arg Asp Arg Asp Ser Arg His Gln Gln Asp Ser Asp Leu Ser A1a Ala Cys Ser Asp Ala Asp Leu His Arg His Lys Lys Lys Glu G1u Glu Lys Glu Glu Thr Phe Lys Lys Ile Arg Gly Leu Cys <210> 8 <211> 1128 <212> PRT
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte ID No: 7484378CD1 <400> 8 Met Glu Pro Thr Val Ala Asp Val His Leu Val Pro Arg Thr Thr Lys Glu Val Pro Ala Leu Asp Ala Ala Cys Cys Arg Ala Ala Ser Ile Gly Val Val Ala Thr Ser Leu Val Val Leu Thr Leu Gly Val Leu Leu Gly Gly Met Asn Asn Ser Arg His Ala Ala Leu Arg Ala 50 5r5 60 Ala Thr Leu Pro Gly Lys Val Tyr Ser Val Thr Pro Glu Ala Ser Lys Thr Thr Asn Pro Pro Glu Gly Arg Asn Ser Glu His Ile Arg Thr Ser Ala Arg Thr Asn Ser Gly His Thr I1e Phe Lys Lys Cys Asn Thr Gln Pro Phe Leu Ser Thr Gln G1y Phe His Val Asp His Thr Ala Glu Leu Arg Gly I1e Arg Trp Thr Ser Ser Leu Arg Arg Glu Thr Ser Asp Tyr His Arg Thr Leu Thr Pro Thr Leu G1u Ala Leu Leu His Phe Leu Leu Arg Pro Leu Gln Thr Leu Ser Leu Gly Leu Glu Glu Glu Leu Leu Gln Arg Gly Ile Arg Ala Arg Leu Arg Glu His Gly Ile Ser Leu Ala Ala Tyr Gly Thr Ile Val Ser Ala Glu Leu Thr Gly Arg His Lys Gly Pro Leu Ala Glu Arg Asp Phe Lys Ser Gly Arg Cys Pro Gly Asn Ser Phe Ser Cys Gly Asn Ser 215 . 220 225 Gln Cys Val Thr Lys Val Asn Pro Glu Cys Asp Asp Gln Glu Asp Cys Ser Asp Gly Ser Asp Glu Ala His Cys Glu Cys Gly Leu Gln Pro Ala Trp Arg Met Ala Gly Arg I1e Val Gly Gly Met Glu Ala 260 265 ~ 270 Ser Pro Gly Glu Phe Pro Trp Gln A1a Ser Leu Arg Glu Asn Lys Glu His Phe Cys Gly Ala Ala Ile Ile Asn Ala Arg Trp Leu Val Ser Ala A1a His Cys Phe Asn Glu Phe Gln Asp Pro Thr Lys Trp Val Ala Tyr Val Gly Ala Thr Tyr Leu Ser Gly Ser Glu Ala Ser Thr Val Arg Ala Gln Val Val Gln Ile Val Lys His Pro Leu Tyr Asn Ala Asp Thr Ala Asp Phe Asp Val Ala Val Leu Glu Leu Thr Ser Pro Leu Pro Phe Gly Arg His I1e Gln Pro Val Cys Leu Pro Ala Ala Thr His Ile Phe Pro Pro Ser Lys Lys Cys Leu Ile Ser Gly Trp Gly Tyr Leu Lys Glu Asp Phe Arg Lys His Leu Pro Arg Pro Ala Met Val Lys Pro Glu Val Leu Gln Lys Ala Thr Val Glu Leu Leu Asp Gln Ala Leu Cys Ala Ser Leu Tyr G1y His Ser Leu Thr Asp Arg Met Val Cys Ala Gly Tyr Leu Asp Gly Lys Val Asp Ser Cys Gln Gly Asp Ser Gly Gly Pro Leu Val Cys G1u Glu Pro Ser Gly Arg Phe Phe Leu Ala Gly Ile Val Ser Trp Gly Ile Gly Cys Ala Glu Ala Arg Arg Pro Gly Val Tyr Ala Arg Val Thr Arg Leu Arg Asp Trp Ile Leu Glu Ala Thr Thr Lys Ala Ser Met Pro Leu Ala Pro Thr Met Ala Pro Ala Pro Ala Ala Pro Ser Thr Ala Trp Pro Thr Ser Pro Glu Ser Pro Val Val Ser Thr Pro Thr Lys Ser Met Gln Ala Leu Ser Thr Val Pro Leu Asp Trp Val Thr Val Pro Lys Leu G1n Glu Cys Gly Ala Arg Pro Ala Met Glu Lys Pro Thr Arg Val Val Gly Gly Phe Gly Ala Ala Ser Gly Glu Val Pro Trp G1n Val Ser Leu Lys Glu Gly Ser Arg His Phe Cys Gly Ala Thr Val Val Gly Asp Arg Trp Leu Leu Ser Ala A1a His Cys Phe Asn His Thr Lys Val Glu Gln Val Arg Ala His Leu Gly Thr Ala Ser Leu Leu Gly Leu Gly Gly Ser Pro Val Lys Ile Gly Leu Arg Arg Val Val Leu His Pro Leu Tyr Asn Pro Gly Ile Leu Asp Phe Asp Leu Ala Val Leu Glu Leu Ala Ser Pro Leu Ala Phe Asn Lys Tyr Ile Gln Pro Val Cys Leu Pro Leu Ala Ile G1n Lys Phe Pro Val G1y Arg Lys Cys Met Ile Ser Gly Trp Gly Asn Thr Gln Glu Gly Asn Ala Thr Lys Pro Glu Leu Leu Gln Lys Ala Ser Val Gly Tle Ile Asp Gln Lys Thr Cys Ser Val Leu Tyr Asn Phe Ser Leu Thr Asp Arg Met Ile Cys Ala Gly Phe Leu Glu Gly Lys Val Asp Ser Cys Gln Gly Asp Ser Gly Gly Pro Leu Ala Cys Glu Glu Ala Pro Gly Val Phe Tyr Leu Ala Gly Ile Val Ser Trp Gly Ile Gly Cys A1a Gln Val Lys Lys Pro Gly Val Tyr Thr Arg Ile Thr Arg Leu Lys Gly Trp Ile Leu Glu Ile Met Ser Ser Gln Pro Leu Pro Met Ser Pro Pro Ser Thr Thr Arg Met Leu Ala Thr Thr Ser Pro Arg Thr Thr Ala Gly Leu Thr Val Pro Gly Ala Thr Pro Ser Arg Pro Thr Pro Gly Ala A1a Ser Arg Va1 Thr Gly Gln Pro Ala Asn Ser Thr Leu Ser Ala Val Ser Thr Thr Ala Arg Gly Gln Thr Pro Phe Pro Asp A1a Pro Glu Ala Thr Thr His Thr Gln Leu Pro Asp Cys Gly Leu Ala Pro Ala Ala Leu Thr Arg Ile Val Gly Gly Ser Ala Ala Gly Arg Gly Glu Trp Pro Trp Gln Val Ser Leu Trp Leu Arg Arg Arg Glu His Arg Cys Gly Ala Val Leu Val Ala Glu Arg Trp Leu Leu Ser Ala Ala His Cys Phe Asp Va1 Tyr Gly Asp Pro Lys Gln Trp Ala Ala Phe Leu Gly Thr Pro Phe Leu Ser Gly Ala Glu Gly Gln Leu Glu Arg Val Ala Arg Ile Tyr Lys His Pro Phe Tyr Asn Leu Tyr Thr Leu Asp Tyr Asp Val Ala Leu Leu Glu Leu Ala Gly Pro Val Arg Arg Ser Arg Leu Val Arg Pro Ile Cys Leu Pro Glu Pro Ala Pro Arg Pro Pro Asp Gly Thr Arg Cys Val Ile Thr Gly Trp Gly Ser Val Arg Glu G1y Gly Ser Met Ala Arg Gln Leu Gln Lys Ala Ala Val Arg Leu Leu Ser Glu Gln Thr Cys Arg Arg Phe Tyr Pro Val Gln I1e Ser Ser Arg Met Leu Cys Ala Gly Phe Pro Gln Gly Gly Val Asp Ser Cys Ser Gly Asp Ala Gly Gly Pro Leu Ala Cys Arg Glu Pro Ser Gly Arg Trp Val Leu Thr Gly Val Thr Ser Trp Gly Tyr Gly Cys Gly Arg Pro His Phe Pro Gly Val Tyr Thr Arg Val Ala Ala Val Arg Gly Trp Ile Gly Gln His Ile Gln Glu <210> 9 <211> 462 <212> PRT
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte ID No: 7473143CD1 <400> 9 Met Ile Pro Phe Thr Glu Leu Gly Gly Arg Gln Gln Lys Arg Arg Glu Trp Val Gly Gly His Arg Glu His Pro Lys Gly Val Met Gly Leu Ala His Arg Gly Met Ala Gly Leu Asp His Asp Val Val Ser Asn Gln Cys Thr Ser Gly Lys Ser Pro Lys Ser Glu Arg Gly Ala Glu Ala Leu Ala Arg Arg Leu Lys Gly Gly Arg Glu Arg Ala Gly Ala Gly Lys Glu Tyr Gly Ile Val Gly Gly Ser Ser Gly His Cys Cys Ser Lys Cys Gly Pro Thr Glu Gly Ile Ile Thr Ser Pro Gly Ser Met Val Gly Arg Gln Ser Leu Gln Leu His Pro Gly Val Asp Leu Asn Leu His Leu Arg Gln Ile Pro Gln Val Met Arg Val His Ser Gln Asn Cys Thr Phe Gln Leu His Gly Pro Asn Gly Thr Val G1u Ser Pro Gly Phe Pro Tyr Gly Tyr Pro Asn Tyr Ala Asn Cys Thr Trp Thr Ile Thr Ala Glu Glu Gln His Arg Ile Gln Leu Val Phe G1n Ser Phe Ala Leu Glu Glu Asp Phe Asp Val Leu Ser Val Phe Asp G1y Pro Pro Gln Pro Glu Asn Leu Arg Thr Arg Leu Thr Gly Phe G1n Leu Pro Ala Thr Ile Val Ser Ala Ala Thr Thr Leu Ser Leu Arg Leu Ile Ser Asp Tyr Ala Val Ser Ala Gln Gly Phe His Ala Thr Tyr Glu Val Leu Pro Ser His Thr Cys Gly Asn Pro Gly Arg Leu Pro Asn Gly Ile Gln Gln Gly Ser Thr Phe Asn Leu Gly Asp Lys Val Arg Tyr Ser Cys Asn Leu Gly Phe Phe Leu Glu Gly His Ala Val Leu Thr Cys His Ala Gly Ser Glu Asn Ser Ala Thr Trp Asp Phe Pro Leu Pro Ser Cys Arg Ala Asp Asp Ala Cys Gly Gly Thr Leu Arg Gly Gln Ser Gly Ile Ile Ser Ser Pro His Phe Pro Ser Glu Tyr His Asn Asn Ala Asp Cys Thr Trp Thr I1e Leu Ala Glu Leu Gly Asp Thr Ile Ala Leu Val Phe Ile Asp Phe Gln Leu Glu Asp Gly Tyr Asp Phe Leu Glu Va1 Thr Gly Thr Glu Gly Ser Ser Leu Trp Phe Thr Gly Ala Ser Leu Pro Ala Pro Val Ile Ser Ser Lys Asn Trp Leu Arg Leu His Phe Thr Ser Asp Gly Asn His Arg Gln Arg Gly Phe Ser Ala Gln Tyr Gln Val Lys Lys Gln Ile Glu Leu Lys Ser Arg Gly Val Lys Leu Met Pro Ser Lys Asp Asn Ser Gln Lys Thr Ser Val Cys Phe His Leu Thr Pro Arg Ala Cys Leu Ser Leu Ser Ser Leu Leu Pro Cys Val <210> 10 <211> 659 <212> PRT
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte ID No: 4382838CD1 <400> 10 Met Leu Trp Ser Glu Arg Val Arg Pro Ser Tyr Ser Cys I1e Ala Asn Asn Asn Val Gly Asn Pro Ala Lys Lys Ser Thr Asn Ile Ile Val Arg Ala Leu Lys Lys Gly Arg Phe Trp Ile Thr Pro Asp Pro Tyr His Lys Asp Asp Asn Ile Gln Ile Gly Arg Glu Val Lys Ile Ser Cys Gln Val Glu Ala Val Pro Ser Glu Glu Val Thr Phe Ser Trp Phe Lys Asn Gly Arg Pro Leu Arg Ser Ser Glu Arg Met Val Ile Thr Gln Thr Asp Pro Asp Val Ser Pro Gly Thr Thr Asn Leu Asp Ile Ile Asp Leu Lys Phe Thr Asp Phe Gly Thr Tyr Thr Cys Val Ala Ser Leu Lys Gly Gly Gly Ile Ser Asp Ile Ser Ile Asp Val Asn Ile Ser Ser Ser Thr Val Pro Pro Asn Leu Thr Val Pro Gln Glu Lys Ser Pro Leu Val Thr Arg Glu Gly Asp Thr Ile Glu Leu Gln Cys Gln Val Thr Gly Lys Pro Lys Pro Ile Ile Leu Trp Ser Arg Ala Asp Lys Glu Val Ala Met Pro Asp Gly Ser Met Gln Met Glu Ser Tyr Asp Gly Thr Leu Arg Ile Val Asn Val Ser Arg Glu Met Ser Gly Met Tyr Arg Cys Gln Thr Ser Gln Tyr Asn Gly Phe Asn Val Lys Pro Arg Glu Ala Leu Val Gln Leu Ile Val Gln Tyr Pro Pro Ala Val Glu Pro Ala Phe Leu Glu Ile Arg Gln Gly Gln Asp Arg Ser Val Thr Met Ser Cys Arg Val Leu Arg Ala Tyr Pro Ile Arg Val Leu Thr Tyr Glu Trp Arg Leu Gly Asn Lys Leu Leu Arg Thr Gly Gln Phe Asp Ser Gln Glu Tyr Thr Glu Tyr Ala Val Lys Ser Leu Ser Asn Glu Asn Tyr Gly Val Tyr Asn Cys Ser Ile Ile Asn Glu Ala Gly Ala Gly Arg Cys Ser Phe Leu Val Thr G1y Lys Ala Tyr Ala Pro Glu Phe Tyr Tyr Asp Thr Tyr Asn Pro Val Trp Gln Asn Arg His Arg Val Tyr Ser Tyr Ser Leu Gln Trp Thr G1n Met Asn Pro Asp Ala Val Asp Arg Ile Val Ala Tyr Arg Leu Gly Ile Arg Gln Ala Gly G1n Gln Arg Trp Trp Glu Gln Glu Ile Lys Ile Asn Gly Asn Ile G1n Lys Gly Glu Leu Ile Thr Tyr Asn Leu Thr Glu Leu Ile Lys Pro Glu Ala Tyr Glu Val Arg Leu Thr Pro Leu Thr Lys Phe Gly G1u Gly Asp Ser Thr Ile Arg Val Ile Lys Tyr Ser Ala Pro Val Asn Pro His Leu Arg Glu Phe His Arg Gly Phe Glu Asp Gly Asn Ile Cys Leu Phe Thr Gln Asp Asp Thr Asp Asn Phe Asp Trp Thr Lys Gln Ser Thr Ala Thr Arg Asn Thr Lys Tyr Thr Pro Asn Thr Gly Pro Asn Ala Asp Arg Ser Gly Ser Lys Glu Gly Phe Tyr Met Tyr Ile Glu Thr Ser Arg Pro Arg Leu Glu Gly Glu Lys Ala Arg Leu Pro Ser Pro Val Phe Ser I1e Ala Pro Lys Asn Pro Tyr Gly Pro Thr Asn Thr Ala Tyr Cys Phe Ser Phe Phe Tyr His Met Tyr Gly Gln His Ile Gly Val Leu Asn Val Tyr Leu Arg Leu Lys Gly Gln Thr Thr Ile G1u Asn Pro Leu Trp Ser Ser Ser Gly Asn Lys Gly Gln Arg Trp Asn Glu Ala His Val Asn Ile Tyr Pro Ile Thr Ser Phe G1n Leu Ile Phe Glu Gly Ile Arg Gly Pro Gly Ile Glu G1y Asp Ile Ala Ile Asp Asp Va1 Ser Ile Ala G1u Gly Glu Cys A1a Lys Gln Asp Leu Ala Thr Lys Asn Ser Val Asp Gly Ala Val G1y Ile Leu Val His Ile Trp Leu Phe Pro Ile Ile Val Leu Ile Ser Ile Leu Ser Pro Arg Arg <210> 11 <211> 626 <212> PRT
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte ID No: 6717888CD1 <400> 11 Met Gly Pro Ala Trp Val Gln Asp Pro Leu Thr Gly Ala Leu Trp Leu Pro Val Leu Trp Ala Leu Leu Ser G1n Val~Tyr Cys Phe His ' 20 25 30 Asp Pro Pro Gly Trp Arg Phe Thr Ser Ser Glu Ile Val Ile Pro Arg Lys Val Pro His Arg Arg Gly Gly Val Glu Met Pro Asp Gln Leu Ser Tyr Ser Met His Phe Arg Gly Gln Arg His Val Ile His Met Lys Leu Lys Lys Asn Met Met Pro Arg His Leu Pro Val Phe Thr Asn Asn Asp Gln Gly Ala Met Gln Glu Asn Tyr Pro Phe Va1 Pro Arg Asp Cys Tyr Tyr Asp Cys Tyr Leu Glu Gly Val Pro Gly Ser Val Ala Thr Leu Asp Thr Cys Arg Gly Gly Leu Arg Gly Met Leu Gln Val Asp Asp Leu Thr Tyr Glu Ile Lys Pro Leu Glu Ala Phe Ser Lys Phe Glu Tyr Val Val Ser Leu Leu Val Ser Glu Glu Arg Pro Gly Glu Val Ser Arg Cys Lys Thr Glu Gly Glu Glu Ile Asp Gln Glu Ser Glu Lys Val Lys Leu Ala Glu Thr Pro Arg Glu Gly His Val Tyr Leu Trp Arg His His Arg Lys Asn Leu Lys Leu His Tyr Thr Val Thr Asn Gly Leu Phe Met Gln Asn Pro Asn Met Ser His Ile I1e Glu Asn Val Val Ile Ile Asn Ser Ile Ile His Thr Ile Phe Lys Pro Val Tyr Leu Asn Val Tyr Val Arg Val Leu Cys Ile Trp Asn Asp Met Asp Ile Val Met Tyr Asn Met Pro Ala Asp Leu Va1 Val Gly Glu Phe Gly Ser Trp Lys Tyr Tyr Glu Trp Phe Ser Gln Ile Pro His Asp Thr Ser Val Val Phe Thr Ser Asn Arg Leu Gly Asn Thr Pro Arg Cys Gly Asp Lys Ile Lys Asn Gln Arg G1u Glu Cys Asp Cys Gly Ser Leu Lys Asp Cys Ala Ser Asp Arg Cys Cys Glu Thr Ser Cys Thr Leu Ser Leu Gly Ser Val Cys Asn Thr Gly Leu Cys Cys His Lys Cys Lys Tyr Ala Ala Pro Gly Val Val Cys Arg Asp Leu Gly Gly Ile Cys Asp Leu Pro Glu Tyr Cys Asp G1y Lys Lys Glu Glu Cys Pro Asn Asp Ile Tyr Ile Gln 380 385' 390 Asp Gly Thr Pro Cys Ser Ala Val Ser Val Cys Ile Arg Gly Asn Cys Ser Asp Arg Asp Met Gln Cys Gln Ala Leu Phe Gly Tyr Gln Val Lys Asp Gly Ser Pro Ala Cys Tyr Arg Lys Leu Asn Arg Ile Gly Asn Arg Phe Gly Asn Cys Gly Val Ile Leu Arg Arg Gly Gly Ser Arg Pro Phe Pro Cys Glu Glu Asp Asp Val Phe Cys Gly Met Leu His Cys Ser Arg Val Ser His I1e Pro Gly Gly Gly Glu His Thr Thr ~Phe Cys Asn Ile Leu Val His Asp Ile Lys G1u Glu Lys Cys Phe Gly Tyr Glu Ala His Gln Gly Thr Asp Leu Pro Glu Met Gly Leu Val Val Asp Gly Ala Thr Cys Gly Pro Gly Ser Tyr Cys Leu Lys Arg Asn Cys Thr Phe Tyr Gln Asp Leu His Phe Glu Cys Asp Leu Lys Thr Cys Asn Tyr Lys Gly Val Cys Asn Asn Lys Lys His Cys His Cys Leu His Glu Trp Gln Pro Pro Thr Cys Glu Leu Arg Gly Lys Gly Gly Ser Ile Asp Ser Gly Pro Leu Pro Asp Lys Gln Tyr Arg Ile Ala Gly Ser Ile Leu Val Asn Thr Asn Arg Ala Leu Val Leu Ile Cys Ile Arg Tyr Ile Leu Phe Val Val Ser Leu Leu Phe G1y Gly Phe Ser Gln Ala Ile Gln Cys <210> 12 <211> 557 <212> PRT
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte ID No: 7472044CD1 <400> 12 Met Leu Leu Ala Val Leu Leu Leu Leu Pro Leu Pro Ser Ser Trp Phe Ala His Gly His Pro Leu Tyr Thr Arg Leu Pro Pro Ser Ala Leu Gln Val Phe Thr Leu Leu Leu Gly Ala Glu Thr Val Leu Gly Arg Asn Leu Asp Tyr Val Cys Glu Gly Pro Cys Gly Glu Arg Arg Pro Ser Thr Ala Asn Val Thr Arg Ala His Gly Arg I1e Val Gly Gly Ser Ala Ala Pro Pro Gly Ala Trp Pro Trp Leu Val Arg Leu Gln Leu G1y Gly Gln Pro Leu Cys Gly Gly Val Leu Val Ala Ala Ser Trp Val Leu Thr Ala Ala His Cys Phe Val Gly Cys Arg Ser Thr Arg Ser Ala Pro Asn Glu Leu Leu Trp Thr Val Thr Leu Ala Glu Gly Ser Arg Gly Glu Gln Ala Glu Glu Val Pro Val Asn Arg Ile Leu Pro His Pro Lys Phe Asp Pro Arg Thr Phe His Asn Asp Leu Ala Leu Val Gln Leu Trp Thr Pro Val Ser Pro Gly Gly Ser Ala Arg Pro Val Cys Leu Pro Gln Glu Pro Gln Glu Pro Pro Ala Gly Thr Ala Cys Ala Ile Ala Gly Trp Gly Ala.Leu Phe Glu Asp Gly Pro Glu Ala Glu Ala Val Arg Glu Ala Arg Val Pro Leu Leu Ser Thr Asp Thr Cys Arg Arg Ala Leu Gly Pro Gly Leu Arg Pro Ser Thr Met Leu Cys A1a Gly Tyr Leu Ala Gly G1y Val Asp Ser Cys Gln G1y Asp Ser Gly Gly Pro Leu Thr Cys Ser Glu Pro Gly Pro Arg Pro Arg Glu Val Leu Phe Gly Val Thr Ser Trp Gly Asp Gly Cys Gly Glu Pro Gly Lys Pro Gly Val Tyr Thr Arg Val Ala Val Phe Lys Asp Trp Leu Gln Glu Gln Met Ser Ala Ser Ser Ser Ser Arg Glu Pro Ser Cys Arg Glu Leu Leu Ala Trp Asp Pro Pro Gln Glu Leu Gln Ala Asp Ala Ala Arg Leu Cys Ala Phe Tyr Ala Arg Leu Cys Pro Gly Ser Gln Gly Ala Cys Ala Arg Leu Ala His Gln Gln Cys Leu Gln Arg Arg Arg Arg Cys Glu Leu Arg Ser Leu Ala His Thr Leu Leu Gly Leu Leu Arg Asn Ala Gln Glu Leu Leu Gly Pro Arg Pro Gly Leu Arg Arg Leu A1a Pro A1a Leu Ala Leu Pro Ala Pro Ala Leu Arg Glu Ser Pro Leu His Pro Ala Arg Glu Leu Arg Leu His Ser Gly Cys Pro Gly Leu Glu Pro Leu Arg Gln Lys Leu Ala Ala Leu Gln Gly Ala His Ala Trp Ile Leu Gln Val Pro Ser Glu His Leu Ala Met Asn Phe His Glu Val Leu Ala Asp Leu Gly Ser Lys Thr Leu Thr Gly Leu Phe Arg Ala Trp Val Arg Ala Gly Leu G1y Gly Arg His Val Ala Phe Ser Gly Leu Val Gly Leu Glu Pro Ala Thr Leu Ala Arg Ser Leu Pro Arg Leu Leu Val Gln Ala Leu Gln Ala Phe Arg Val Ala Ala Leu Ala Glu Gly Glu Pro Glu Gly Pro Trp Met Asp Val Gly Gln Gly Pro Gly Leu G1u Arg Lys Gly His His Pro Leu Asn Pro G1n Val Pro Pro Ala Arg Gln Pro <210> 13 <211> 494 <212> PRT
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte ID No: 7477384CD1 <400> 13 Met Gly Gly Pro Cys Arg Ala Pro Leu Gln Pro Gln Cys A1a Arg Arg Arg Glu Ala Trp A1a Arg Arg His Arg Arg Arg Gly Ala Gly Arg Arg Arg Arg Gly Gly A1a Pro A1a Ala Arg Ala Gly Arg Gly Arg Gly Arg Gly Arg Gly Ala Leu Arg Gly Pro Gly Arg Pro Trp Ala Pro Pro Pro Pro Ala Pro Arg Pro Ala A1a Gly Pro Ala Pro Pro Pro Thr Arg Ser Leu Ser Pro Pro Leu Arg Pro Ala Val Pro Pro Ser Arg Arg Arg Leu Phe Leu Gly Glu Ala Leu Phe Gln Arg Ala Gly Ser Met Ala Ala Val Glu Thr Arg Val Cys Glu Thr Asp Gly Cys Ser Ser Glu Ala Lys Leu Gln Cys Pro Thr Cys Ile Lys Leu Gly Tle Gln Gly Ser Tyr Phe Cys Ser Gln Glu Cys Phe Lys Gly Ser Trp Ala Thr His Lys Leu Leu His Lys Lys Ala Lys Asp Glu Lys Ala Lys Arg Glu Val Ser Ser Trp Thr Val Glu Gly Asp Ile Asn Thr Asp Pro Trp Ala Gly Tyr Arg Tyr Thr Gly Lys Leu Arg Pro His Tyr Pro Leu Met Pro Thr Arg Pro Val Pro Ser Tyr Ile Gln Arg Pro Asp Tyr Ala Asp His Pro Leu Gly Met Ser Glu Ser Glu Gln Ala Leu Lys Gly Thr Ser Gln I1e Lys Leu Leu Ser Ser Glu Asp Ile Glu Gly Met Arg Leu Val Cys Arg Leu Ala Arg Glu Val Leu Asp Val Ala Ala G1y Met Ile Lys Pro Gly Val Thr Thr G1u Glu Ile Asp His Ala Val His Leu Ala Cys Ile Ala Arg Asn Cys Tyr Pro Ser Pro Leu Asn Tyr Tyr Asn Phe Pro Lys Ser Cys Cys Thr Ser Val Asn Glu Val Ile Cys His Gly Ile Pro Asp Arg Arg Pro Leu Gln Glu Gly Asp Ile Val Asn Val Asp Ile Thr Leu Tyr Arg Asn Gly Tyr His Gly Asp Leu Asn Glu Thr Phe Phe Val G1y Glu Val Asp Asp Gly Ala Arg Lys Leu Val Gln Thr Thr Tyr Glu Cys Leu Met Gln Ala Ile Asp Ala Val Lys Pro Gly Val Arg Tyr Arg Glu Leu Gly Asn Ile Ile Gln Lys His Ala Gln Ala Asn Gly Phe Ser Val Val Arg Ser Tyr Cys Gly His Gly Ile His Lys Leu Phe His Thr Ala Pro Asn Val Pro His Tyr Ala Lys Asn Lys Ala Val Gly Val Met Lys Ser Gly His Va1 Phe Thr Ile Glu Pro Met Ile Cys Glu Gly Gly Trp Gln Asp Glu Thr Trp Pro Asp Gly Trp Thr Ala Val Thr Arg Asp Gly Lys Arg Ser Ala Gln Phe Glu His Thr Leu Leu Val Thr Asp Thr Gly Cys Glu I1e Leu Thr Arg Arg Leu Asp Ser Ala Arg Pro His Phe Met Ser Gln Phe <210> 14 <211> 593 <212> PRT
<213> Homo sapiens <220>
<221> misc_feature <223> Incyte ID No: 7077175CD1 <400> 15 Met Asn Val Leu Lys Leu Asp Thr Leu Val Val A1a Gln Leu Trp Arg Tyr Glu Asn Ala Lys Pro Thr Gly Glu Leu Gly Glu Pro Tyr Glu Ala Gly Ile Asn Cys Ser Gly Ser Gly Ala Glu Glu Lys Glu Asp Arg Arg Met Ala Ile Ile Trp Ala Val Pro Ser Thr Ser Val Ser Trp Glu Gln Thr Ser Arg Lys Thr Gln Ile Arg Lys Lys Arg Pro Ala Pro Arg Cys Lys Gln Leu Gly Thr Arg Gln Arg Val Leu Pro Val Val Lys Pro Glu Val Leu Gln Lys Ala Thr Va1 G1u Leu Leu Asp Gln Ala Leu Cys Ala Ser Leu Tyr Gly His Ser Leu Thr Asp Arg Met Val Cys Ala Gly Tyr Leu Asp Gly Lys Val Asp Ser Cys Gln Gly Asp Ser Gly Gly Pro Leu Val Cys Glu Glu Pro Ser Gly Arg Phe Phe Leu Ala Gly Ile Val Ser Trp Gly Ile Gly Cys Ala Glu Ala Arg Arg Pro Gly Val Tyr Ala Arg Val Thr Arg Leu Arg Asp Trp Ile Leu Glu Ala Thr Thr Lys Ala Ser Met Pro Leu Ala Pro Thr Met Ala Pro Ala Pro Ala Ala Pro Ser Thr Ala Trp Pro Thr Ser Pro Glu Ser Pro Val Val Ser Thr Pro Thr Lys Ser Met Gln Ala Leu Ser Thr Val Pro Leu Asp Trp Va1 Thr Val Pro Lys Leu Gln Glu Cys Gly Ala Arg Pro Ala Met Glu Lys Pro Thr Arg Val Val Gly Gly Phe G1y Ala A1a Ser Gly Glu Va1 Pro Trp Gln Val Ser Leu Lys Glu Gly Ser Arg His Phe Cys Gly Ala Thr Val Ala Gly Asp Arg Trp Leu Leu Ser Ala Ala His Cys Phe Asn His Thr Lys Val Glu Gln Va1 Arg Ala His Leu Gly Thr Ala Ser Leu Leu Gly Leu Gly Gly Ser Pro Val Lys Ile Gly Leu Arg Arg Val Val Leu His Pro Leu Tyr Asn Pro Gly Ile Leu Asp Phe Asp Leu Ala Val Leu Glu Leu Ala Ser Pro Leu Ala Phe Asn Lys Tyr Ile Gln Pro Val Cys Leu Pro Leu A1a Ile Gln Lys Phe Pro Val Gly Arg Lys Cys Met Ile Ser Gly Trp Gly Asn Thr Gln Glu Gly Asn Ala Thr Lys Pro Glu Leu Leu G1n Lys Ala Ser Val Gly Ile Ile Asp Gln Lys Thr Cys Ser Val Leu Tyr Asn Phe Ser Leu Thr Asp Arg Met Ile Cys Ala G1y Phe Leu Glu Gly Lys Val Asp Ser Cys Gln Gly Asp Ser Gly Gly Pro Leu Ala Cys Glu Glu Ala Pro G1y Val Phe Tyr Leu Ala Gly Ile Val Ser Trp Gly Ile Gly Cys Ala Gln Val Lys Lys Pro Gly Val Tyr Thr Arg Ile Thr Arg Leu Lys Gly Trp Ile Leu Glu I1e Met Ser Ser Gln Pro Leu Pro Met Ser Pro Pro Ser Thr Thr Arg Met Leu Ala Thr Thr Ser Pro Arg Thr Thr Ala Gly Leu Thr Val Pro Gly Ala Thr Pro Ser Arg Pro Thr Pro Gly Ala Ala Ser Arg Val Thr Gly Gln Pro Ala Asn Ser Thr Leu Ser Ala Val Ser Thr Thr Ala Arg Gly Gln Thr Pro Phe Pro Asp Ala Pro Glu Ala Thr Thr His Thr Gln Leu Pro Gly Thr Gly Arg Asp Gly Gly Ile Pro Gly Ser Gly Gly Ser His Val Asn Gln Pro Gly Leu Pro Asn Lys Thr <210> 15 <211> 319 <212> PRT
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte ID No: 7480124CD1 <400> 16 Met Gly Pro Leu Gly Pro Ser Ala Leu Gly Leu Leu Leu Leu Leu Leu Val Val Ala Pro Pro Arg Val Ala Ala Leu Val His Arg Gln Pro Glu Asn Gln Gly Ile Ser Leu Thr Gly Ser Val Ala Cys Gly Arg Pro Ser Met Glu Gly Lys Ile Leu Gly Gly Val Pro Ala Pro Glu Arg Lys Trp Pro Trp Gln Val Ser Val His Tyr Ala Gly Leu His Val Cys Gly Gly Ser Ile Leu Asn Glu Tyr Trp Va1 Leu Ser Ala Ala His Cys Phe His Arg Asp Lys Asn Ile Lys Ile Tyr Asp Met Tyr Val Gly Leu Va1 Asn Leu Arg Val Ala Gly Asn His Thr Gln Trp Tyr Gly Val Asn Arg Val Ile Leu His Pro Thr Tyr Gly Met Tyr His Pro Ile Gly Gly Asp Val Ala Leu Val Gln Leu Lys Thr Arg Ile Val Phe Ser Glu Ser Val Leu Pro Val Cys Leu Ala Thr Pro G1u Val Asn Leu Thr Ser Ala Asn Cys Trp Ala Thr Gly Trp Gly Leu Val Ser Lys Gln Gly G1u Thr Ser Asp Glu Leu Gln Glu Val Gln Leu Pro Leu Ile Leu G1u Pro Trp Cys His Leu Leu Tyr Gly His Met Ser Tyr Ile Met Pro Asp Met Leu Cys Ala Gly Asp Ile Leu Asn Ala Lys Thr Val Cys Glu Gly Asp Ser Gly Gly Pro Leu Val Cys G1u Phe Asn Arg Ser Trp Leu Gln Ile Gly Ile Val Ser Trp Gly Arg Gly Cys Ser Asn Pro Leu Tyr Pro Gly Va1 Tyr Ala Ser Val Ser Tyr Phe Ser Lys Trp Ile Cys Asp Asn Ile Glu Ile Thr Pro Thr Pro Ala Gln Pro Ala Pro Ala Leu Ser Pro Ala Leu Gly Pro Thr Leu Ser Val Leu Met Ala Met Leu Ala Gly Trp Ser Val Leu <210> 16 <211> 2406 <212> DNA
<213> Homo sapiens <220>
<221> misc_feature <223> Incyte ID No: 6926819CB1 <400> 17 gttaagctga aaatgcacac agggctcctg taaatttctt ttcataaacc acccgcccag 60 ggcattaaat agggtactta gttgatccga accctccagg gagacctccg acccttctct 120 tCgtagCCCC CagCtCCCCt cccccggttc cactgaggca aggggactga gctgctccac 1.80 atgccaggag tcagcacgcc ggaaggcccc gcccagcggc tggcgcagcc aatcgcagag 240 cgggcaagtg gtgggggcgg gcctgcctgg gcggcaaggg ggcagcgggg tctaggggct 300 ttacaggtca attagctgct ttcgggcggc cttaggcgac aggagactcc tggacccagc 360 acctgcccac tgtgcctgtc cacctgtggc tacagcagCt gagaccccag tgggctaaag 420 attggacagg ggcccaccag ggacccagca agtccttcag ctctgtgagt gagggatttt 480 ccggagtgcc aggccgcagt attcccaggg ccgtggggtg ggacagggag gctcgacccc 540 ggcaaatcag gcagaggcgc cccttgctcc ctgcaacatc gcccacgtcc tggggccaca 600 gtgagcatga gcggagggcg ggagcaagag ccaggggacc tggcctgggt ccccagccca 660 aagcctggga agctgcctac ccacccctgt gtgggcgcgg acactgggga ctctggcttc 720 cggtggttcg gccacctgat tcagtttatg ctctgtgagg ggagctggag tgttggcagg 780 actggcccac ctgcaggact gcaggactgc gggaacggcg gtagatgggt gctctccttc 840 ecagtttgtc ctgggaagac attcaataac tgtttcatta caaggggcat ttggaaaaca 900 tacttcacct tctgttgtgt attagccaag aacaaggtgt gatgtgactt cccaattatt 960 ggggatccct ttgtcccttc ttgaaattag atgtcttcat tcttgaggtt ttgcctggat 1020 gacctcagca caattggtac aaaacctggg ccaatggttt cctagtttcc cggttgttgc 1080 cttaagcttc tcgcccatca ggtaccttcc tgtccttgtt catagcctgt catcatcatt 1140 ccagaaaact gtttcaactc ctacagctgt ggacaggctg cttttcattt tggtgggtcc 1200 ctccaatacc tCCaCttgCC CtgtttttCt ccagccacat ccttggcctc ttccacagtc 1260 cttaggtaaa tgcttggaag aataatttaa atatttttat tctaccatgg tggccctagt 1320 ttctcagggg gtagtaaaat ggctttttag gatcggtcta atcagatcct catttctttt 1380 cccttcctag atttttgaaa catgaatcct tcactcctcc tggctgcctt tttcctggga 1440 attgcctcag ctgctctaac acgtgaccac agtttagacg cacaatggac caagtggaag 1500 gcaaagcaca agagattata tggcatgaac aggaaccact ggattagagt cctctgggag 1560 aaggacgtga agatgattga gcagcacaat caggaataca gccaagggaa acacagcttc 1620 acaatggcca tgaacgcctt tggagacatg gtaagtgaag aattcaggca ggtgatgaat 1680 ggttttcaat accagaagca caggaagggg aaacagttcc aggaacgcct gcttcttgag 1740 atccccacat ctgtggactg gagagagaaa ggctacatga ctcctgtgaa ggatcagcag 1800 ggtcagtgtg gctcttgttg ggcttttagt gcaactggtg ctctggaagg gcagatgttc 1860 tggaaaacag gcaaacttat ctcactgaat gagcagaatc tggtagactg ctctgggcct 1920 caaggcaatg agggctgcaa tggtgact~tc atggataatc ccttccggta tgttcaggag 1980 aacggaggcc tggactctga ggcatcctat ccatatgaag gaaaggttaa aacctgtagg 2040 tacaatccca agtattctgc tgctaatgac actggttttg tggacatccc ttcacgggag 2100 aaggacctgg cgaaggcagt ggcaactgtg gggcccatct ctgttgctgt tggtgcaagc 2160 catgtcttct tccagttcta taaaaaagga atttattttg agccacgctg tgaccctgaa 2220 ggcctggatc atgctatgct ggtggttggc tacagctatg aaggagcaga ctcagataac 2280 aataaatatt ggctggtgaa gaacagctgg ggtaaaaact ggggcatgga tggctacata 2340 aagatggcca aagaccggag gaacaactgt ggaattgcca cagcagccag ctaccccact 2400 gtgtga 2406 <210> 17 <211> 1967 <212> DNA
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte ID No: 7473526CB1 <400> 18 cggacgcgtg ggcggacgcg tgggtgccca ggcgcttaaa gaagcaaaat ctcttgtgca 60 ggagcagcag agactcctca ggaagactca ctggactgta cccaccacct gccatgtctc.120 tgtggccacc tttccgatgc agatggaagc tggcgccaag gtactctagg agggcgtctc 180 cacagcaacc ccaacaggac tttgaggccc tgctggcaga gtgcctgagg aatggctgcc 240 tctttgaaga caccagcttc ccggccaccc tgagctccat cggcagtggc tccctgctgc 300 agaagctgcc accccgcctg cagtggaaga ggcccccgga gctgcacagc aatccccagt 360 tttattttgc caaggccaaa aggctggatc tgtgccaggg gatagtagga gactgctggt 420 tcttggctgc tttgcaagct ctggccttgc accaggacat cctgagccgg gttgttcccc 480 tgaatcagag tttcactgag aagtatgctg gcatcttccg gttctggttc tggcactatg 540 ggaactgggt tcctgtggtg atcgatgacc gtctgcctgt gaatgaggct ggccagctgg 600 tctttgtctc ctccacctat aagaacttgt tctggggagc acttctggaa aaggcctatg 660 ccaagctctc tggttcctat gaagacttgc agtcaggaca ggtgtctgaa gcccttgtag 720 acttcactgg aggggtgaca atgaccatca acctggcaga agcccatggc aacctctggg 780 acatcctcat cgaagccacc tacaacagaa ccctcattgg ctgccagacc cactcagggg 840 agaagattct ggagaatggg ctggtggaag gccatgccta tactctcaca ggaatcagga 900 aggtgacctg caaacataga cctgaatatc tcgtcaagct acggaacccc tggggaaagg 960 tggaatggaa aggagactgg agtgacagtt caagtaaatg ggagctgctg agccccaagg 1020 agaagattct gcttctgagg aaagacaatg acggagaatt ctggatgacg ctgcaggact 1080 ttaaaacaca tttcgtgctc ctggttatct gtaaactgac cccaggcctg ttgagccagg 1140 aggcggccca gaagtggacg tacaccatgc gggaggggag atgggagaag cggagcacag 1200 ctggtggcca gaggcagttg ctgcaggaca cattttggaa gaacccgcag ttcctgctgt 1260 ctgtctggag gcccgaggag ggcaggagat ccctgaggcc ctgcagcgtg ctggtgtccc 1320 tgctccagaa gcccaggcac aggtgccgca agcggaagcc tctcctcgcc attggcttct 1380 acctctatag gatgaacaag taccatgatg accagaggag actgccccct gagttcttcc 1440 agagaaacac tcctctgagc cagcctgata ggtttctcaa ggagaaagaa gtgagtcagg 1500 agctgtgtct ggaaccaggg acgtacctca tcgtgcctgc atattggagg cccaccagaa 1560 gtcagagttc gtcctcaggg tcttctccag gaagcacatc ttttatgaaa ttggcagcaa 1620 ttctggtgtc gtcttctcaa aggagataga agaccaaaat gaaaggcagg atgaattctt 1680 caccaaattc ttttgaaaag catccagaga ttaatgcagt tcaacttcag aacctcctga 1740 accagatgac ctggtcaagt ctggggagca gacagccctt tctttagcct ggaagcctgc 1800 aggggatcct ggccttactg accttaatgc atcaggtact atgagcatcc caggaatcag 1860 gcacctgttg gaaggagtga agtctctcag aaggtctcca caagcaacac cgtgggtcag 1920 gaactgaact ggagcaatgg acgtgcagaa ggagcagaac acgccag 1967 <210> 18 <211> 3446 <212> DNA
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte ID No: 7478443CB1 <400> 19 tgcctagagg ccgaggagct cacagctatg ggctggaggc cccggagagc tcgggggacc 60 ccgttgctgc tgctgctact actgctgctg ctctggccag tgccaggcgc cggggtgctt 120 caaggacata tccctgggca gccagtcacc ccgcactggg tcctggatgg acaaccctgg 180 cgcaccgtca gcctggagga gccggtctcg aagccagaca tggggctggt ggccctggag 240 gctgaaggcc aggagctcct gcttgagctg gagaagaacc acaggctgct ggccccagga 300 tacatagaaa cccactacgg cccagatggg cagccagtgg tgctggcccc caaccacacg 360 gatcattgcc actaccaagg gcgagtaagg ggcttccccg actcctgggt agtcctctgc 420 acctgctctg ggatgagtgg cctgatcacc ctcagcagga atgccagcta ttatctgcgt 480 ccctggccac cccggggctc caaggacttc tcaacccacg agatctttcg gatggagcag 540 ctgctcacct ggaaaggaac ctgtggccac agggatcctg ggaacaaagc gggcatgacc 600 agccttcctg gtggtcccca gagcaggggc aggcgagaag cgcgcaggac ccggaagtac 660 ctggaactgt acattgtggc agaccacacc ctgttcttga ctcggcaccg aaacttgaac 720 cacaccaaac agcgtctcct ggaagtcgcc aactacgtgg accagcttct caggactctg 780 gacattcagg tggcgctgac cggcctggag gtgtggaccg agcgggaccg cagccgcgtc 840 acgcaggacg ccaacgccac gctctgggcc ttcctgcagt ggcgccgggg gctgtgggcg 900 cagcggcccc acgactccgc gcagctgctc acgggccgcg ccttccaggg cgccacagtg 960 ggcctggcgc ccgtcgaggg catgtgccgc gccgagagct cgggaggcgt gagcacggac 1020 cactcggagc tccccatcgg cgccgcagcc accatggccc atgagatcgg ccacagcctc 1080 ggcctcagcc acgaccccga cggctgctgc gtggaggctg cggccgagtc cggaggctgc 1140 gtcatggctg cggccaccgg gcacccgttt ccgcgcgtgt tcagcgcctg cagccgccgc 1200 cagctgcgcg ccttcttccg caaggggggc ggcgcttgcc tctccaatgc cccggacccc 1260 ggactcccgg tgccgccggc gctctgcggg aacggcttcg tggaagcggg cgaggagtgt 1320 gactgcggcc ctggccagga gtgccgcgac ctctgctgct ttgctcacaa ctgctcgctg 1380 cgcccggggg cccagtgcgc ccacggggac tgctgcgtgc gctgcctgct gaagccggct 1440 ggagcgctgt gccgccaggc catgggtgac tgtgacctcc ctgagttttg cacgggcacc 1500 tcctcccact gtcccccaga cgtttaccta ctggacggct caccctgtgc caggggcagt 1560 ggctactgct gggatggcgc atgtcccacg ctggagcagc agtgccagca gctctggggg 1620 cctggctccc acccagctcc cgaggcctgt ttccaggtgg tgaactctgc gggagatgct 1680 catggaaact gcggccagga cagcgagggc cacttcctgc cctgtgcagg gagggatgcc 1740 ctgtgtggga agctgcagtg ccagggtgga aagcccagcc tgctcgcacc gcacatggtg 1800 ccagtggact ctaccgttca cctagatggc caggaagtga cttgtcgggg agccttggca 1860 ctccccagtg cccagctgga cctgcttggc ctgggcctgg tagagccagg cacccagtgt 1920 ggacctagaa tggtgtgcca gagcaggcgc tgcaggaaga atgccttcca ggagcttcag 1980 cgctgcctga ctgcctgcca cagccacggg gtttgcaata gcaaccataa ctgccactgt 2040 gctccaggct gggctccacc cttctgtgac aagccaggct ttggtggcag catggacagt 2100 ggccctgtgc aggctgaaaa ccatgacacc ttcctgctgg ccatgctcct cagcgtcctg 2160 ctgcctctgc tcccaggggc cggcctggcc tggtgttgct accgactccc aggagcccat 2220 ctgcagcgat gcagctgggg ctgcagaagg gaccctgcgt gcagtggccc caaagatggc 2280 ccacacaggg accaccccct gggcggcgtt caccccatgg agttgggccc cacagccact 2340 ggacagccct ggcccctgga ccctgagaac tctcatgagc ccagcagcca ccctgagaag 2400 cctctgccag cagtctcgcc tgacccccaa gatcaagtcc agatgccaag atcctgcctc 2460 tggtgagagg tagctcctaa aatgaacaga tttaaagaca ggtggccact gacagccact 2520 ccaggaactt gaactgcagg ggcagagcca gtgaatcacc ggacctccag cacctgcagg 2580 cagcttggaa gtttcttccc cgagtggagc ttcgacccac ccactccagg aacccagagc 2640 cacattagaa gttcctgagg gctggagaac actgctgggc acactctcca gctcaataaa 2700 ccatcagtcc cagaagcaaa ggtcacacag cccctgacct ccctcaccag tggaggctgg 2760 gtagtgctgg ccatcccaaa agggctctgt cctgggagtc tggtgtgtct cctacatgca 2820 atttccacgg acccagctct gtggagggca tgactgctgg ccagaagcta gtggtcctgg 2880 ggccctatgg ttcgactgag tccacactcc cctggagcct ggctggcctc tgcaaacaaa 2940 cataattttg gggaccttcc~ttcctgtttc ttcccaccct gtcttctccc ctaggtggtt 3000 cctgagcccc cacccccaat cccagtgcta cacctgaggt tctggagctc agaatctgac 3060 agcctctccc ccattctgtg tgtgtcgggg ggacagaggg aaccatttaa gaaaagatac 3120 caaagtagaa gtcaaaagaa agacatgttg gctataggcg tggtggctca tgcctataat 3180 cccagcactt tgggaagccg gggtaggagg atcaccagag gccagcaggt ccacaccagc 3240 ctgggcaaca cagcaagaca ccgcatctac agaaaaattt taaaattagc tgggcgtggt 3300 ggtgtgtacc tgtaggccta gctgctcagg aggctgaagc aggaggatca cttgagcctg 3360 agttcaacac tgcagtgagc tatggtggca ccactgcact ccagcctggg tgacagagca 3420 agaccctgtc tctaaaataa atttta 3446 <210> 19 <211> 4888 <212> DNA
<213> Homo sapiens <220>
<221> misc_feature <223> Incyte ID No: 3533147CB1 <400> 20 atgacaggaa caggaggcag gaagcccact ggggacaaac aggaagtcca cccctgggaa 60 aaacaggaag tgagggaaca gacagaaagt ccacaggagc tgacaagaag tccacagggg 120 acagacagga atgatacagt gaccatctat actgacaccc aaagccgaaa ggctggcgct 180 tctcgtaaaa tcagaaacat gctcaacatt taccttgttt ggttagttaa gataaaccag 240 ataataatca atgtctttta tcaaaatcca gaaccaacta tctggaattc tgcatttatt 300 gtggacataa cagcaatagt tccaacagca ttatttccat ttaatgtggc caagccaaaa 360 atgctcgtgg agaatttaca ggaaggtgac ttcagggagc ttcgtggtaa cagccaccac 420 tgcctgacca aaaagggtct aggaaatgct cctccaggcc tgcagttcac actgtacaaa 480 tgtctggact catccaggac agcccagccc catgcagggc ttcactacgt ggacattaat 540 tcaggcatga tacgaacaga agaggcagat tacttcctaa ggccacttcc ttcacacctc 600 tcatggaaac tcggcagagc tgcccaaggc agctcgccat cccacgtact gtacaagaga 660 tccacagagc cccatgctcc tggggccagt gaggtcctgg tgacctcaag gacatgggag 720 ctggcacatc aacccctgca cagcagcgac cttcgcctgg gactgccaca aaagcagcat 780 ttctgtggaa gacgcaagaa atacatgccc cagcctccca aggaagacct cttcatcttg 840 ccagatgagt ataagtcttg cttacggcat aagcgctctc ttctgaggtc ccatagaaat 900 gaagaactga acgtggagac cttggtggtg gtcgacaaaa agatgatgca aaaccatggc 960 catgaaaata tcaccaccta cgtgctcacg atactcaaca tggtatctgc tttattcaaa 1020 gatggaacaa taggaggaaa catcaacatt gcaattgtag gtctgattct tctagaagat 1080 gaacagccag gactggtgat aagtcaccac gcagaccaca ccttaagtag cttctgccag 1140 tggcagtctg gattgatggg gaaagatggg actcgtcatg accacgccat cttactgact 1200 ggtctggata tatgttcctg gaagaatgag ccctgtgaca ctttgggatt tgcacccata 1260 agtggaatgt gtagtaaata tcgcagctgc acgattaatg aagatacagg tcttggactg 1320 gccttcacca ttgcccatga gtctggacac aactttggca tgattcatga tggagaaggg 1380 aacatgtgta aaaagtccga gggcaacatc atgtccccta cattggcagg acgcaatgga 1440 gtcttctcct ggtcaccctg cagccgccag tatctacaca aatttctaag caccgctcaa 1500 gctatctgcc ttgctgatca gccaaagcct gtgaaggaat acaagtatcc tgagaaattg 1560 ccaggagaat tatatgatgc aaacacacag tgcaagtggc agttcggaga gaaagccaag 1620 ctctgcatgc tggactttaa aaaggacatc tgtaaagccc tgtggtgcca tcgtattgga 1680 aggaaatgtg agactaaatt tatgccagca gcagaaggca caatttgtgg gcatgacatg 1740 tggtgccggg gaggacagtg tgtgaaatat ggtgatgaag gccccaagcc cacccatggc 1800 cactggtcgg actggtcttc ttggtcccca tgctccagga cctgcggagg gggagtatct 1860 cataggagtc gcctctgcac caaccccaag ccatcgcatg gagggaagtt ctgtgagggc 1920 tccactcgca ctctgaagct ctgcaacagt cagaaatgtc cccgggacag tgttgacttc 1980 cgtgctgctc agtgtgccga gcacaacagc agacgattca gagggcggca ctacaagtgg 2040 aagccttaca ctcaagtaga agatcaggac ttatgcaaac tctactgtat cgcagaagga 2100 tttgatttct tcttttcttt gtcaaataaa gtcaaagatg ggactccatg ctcggaggat 2160 agccgtaatg tttgtataga tgggatatgt gagagagttg gatgtgacaa tgtccttgga 2220 tctgatgctg ttgaagacgt ctgtggggtg tgtaacggga ataactcagc ctgcacgatt 2280 cacaggggtc tctacaccaa gcaccaccac accaaccagt attatcacat ggtcaccatt 2340 ccttctggag cccggagtat ccgcatctat gaaatgaacg tctctacctc ctacatttct 2400 gtgcgcaatg ccctcagaag gtactacctg aatgggcact ggaccgtgga ctggcccggc 2460 cggtacaaat tttcgggcac tactttcgac tacagacggt cctataatga gcccgagaac 2520 ttaatcgcta ctggaccaac caacgagaca ctgattgtgg agctgctgtt tcagggaagg 2580 aacccgggtg ttgcctggga atactccatg cctcgcttgg ggaccgagaa gcagccccct 2640 gcccagccca gctacacttg ggccatcgtg cgctctgagt gctccgtgtc ctgcggaggg 2700 ggacagatga ccgtgagaga gggctgctac agagacctga agtttcaagt aaatatgtcc 2760 ttctgcaatc ccaagacacg acctgtcacg gggctggtgc cttgcaaagt atctgcctgt 2820 cctcccagct ggtccgtggg gaactggagt gcctgcagtc ggacgtgtgg cgggggtgcc 2880 cagagccgcc ccgtgcagtg cacacggcgg gtgcactatg actcggagcc agtcccggca 2940 ggcctgtgcc ctcagctggt ccctccagca ggcaggcctg caactctcag agctgcccac 3000 ctgcatggag cgccgggccc tgggcagagt gctcacacac ctgtgggaag ggtggaggaa 3060 cgggcagtgg cctgtaagag caccaacccc tcggccagag cgcagctgct gcccgacgct 3120 gtctgcacct ccgagcccaa gcccaggatg catgaagcct gtctgcttca gcgctgccac 3180 aagcccaaga agctgcagtg gctggtgtcc gcctggtccc agtgctctgt gacatgtgaa 3240 agaggaacac agaaaagatt cttaaaatgt gctgaaaagt atgtttctgg aaagtatcga 3300 gagctggcct caaagaagtg ctcacatttg ccgaagccca gcctggagct ggaacgtgcc 3360 tgcgccccgc ttccatgccc caggcacccc ccatttgctg ctgcgggacc ctcgaggggc 3420 agctggtttg cctcaccctg gtctcagtgc acggccagct gtgggggagg cgttcagacg 3480 aggtccgtgc agtgcctggc tgggggccgg ccggcctcag gctgcctcct gcaccagaag 3540 ccttcggcct ccctggcctg caacactcac ttctgcccca ttgcagagaa gaaagatgcc 3600 ttctgcaaag actacttcca ctggtgctac ctggtacccc agcacgggat gtgcagccac 3660 aagttctacg gcaagcagtg ctgcaagact tgctctaagt ccaacttgtg agttgggacc 3720 gctctccgta gcagagaaag tgcctgcgtg gcacagaaat ttcccacaaa tgagctgtgc 3780 aatctacgtc ggaatacatc caaggaagag caaagccaaa agaagaaaac cgtgttaggc 3840 tctttgacca ggagtgtatg tatgtggttc actgtgagcc tgggtgcaga cctgtgtccc 3900 catgcacaca gtgtctcctg tcaggctgaa atgtggcacc ctggcagaca gagctgtggc 3960 tcgtgaggca gaaggcaggc accacaacgg gagaggcagc actcacccct gcctgttgca 4020 gctaaatcaa gtcaaaaaga caggcgaggc tgaacttgct aaatgtctgg tgccttagaa 4080 aaagaaggaa aggccatgaa ataaggaaaa catacaaaat atgtaccccc tagttcacca 4140 gcctcccctc ccactaggag ggcccctcga gccatcagga gtgaccaact tcctgggtgg 4200 aggtcagggg agctccagga ggctgcccag gctcctcctc ctcctcccca gcggccgagc 4260 atctcttacc aggaacctgg agccaccgcc ggagccagcg tcatctctag ggtcactggc 4320 caggggactg cattctggtt tgggactttg cctatggaaa tgggaaaaat gaaattcctg 4380 ctaaggtgct tctatctctt tcagattcat gcattgaagg agagattttt tatactttat 4440 gttttatctt tctcagttat ttgcaagtga gtgtcctttt aaaaacacac ttcttcatgc 4500 ttttctttgt aaatgacaga tcgaagtata ggttacatca aaaccctacc atcctgagaa 4560 gagttatggt tctattatag cagacgtcag ccacacagcc tatgtgacaa taaccttaga 4620 gtcctgtgtt ttgtttttgt gtgttgtgag attttaatct tttttttttt cggtgagtct 4680 ggccatttct ataatgccag gtgggaagcc aggctgcggg tgttagggtg ggaatctgcc 4740 cggcgtctct ggcaccctcc ctgccatcct cagtgcggct gctgttctcc tgtccggtgc 4800 tgtggctcca ttccaaaggg gcacctggat atttatattt gctgaagttt tataataaag 4860 tttatatggt acagtgaaaa aaaaaaaa 4888 <210> 20 <211> 1074 <212> DNA
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte ID No: 7483438CB1 <400> 21 gccgtgcaca acaccaagcg catggactct gaccccagtg cagtgtctgt agggaagagg 60 agccatgggg cttcgggcag gccccatcct gcttctgctg ctgtggctgc tgccaggggc 120 ccattgggat gtgctgcctt cagaatgcgg ccactccaag gaggccggga ggattgtggg 180 aggccaagac acccaggaag gacgctggcc gtggcaggtt ggcctgtggt tgacctcagt 240 ggggcatgta tgtgggggct ccctcatcca cccacgctgg gtgctcacag ccgcccactg 300 cttcctgagg tctgaggatc ccgggctcta ccatgttaaa gtcggagggc tgacaccctc 360 actttcagag ccccactcgg ccttggtggc tgtgaggagg ctcctggtcc actcctcata 420 ccatgggacc accaccagcg gggacattgc cctgatggag ctggactccc ccttgcaggc 480 ctcccagttc agccccatct gcctcccagg accccagacc cccctcgcca ttgggaccgt 540 gtgctgggta aacgggctgg gggaggtggc tgtgcccctc ctggactcga acatgtgtga 600 gctgatgtac cacctaggag agcccagcct ggctggccag cgcctcatcc aggacgacat 660 gctctgtgct ggctctgtcc agggcaagaa agactcctgc cagggtgact ccggggggcc 720 gctggtctgc cccatcaatg atacgtggat ccaggccggc attgtgagct ggggattcgg 780 ctgtgcccgg cctttccggc ctggtgtcta cacccaggtg ctaagctaca cagactggat 840 tcagagaacc ctggctgaat ctcactcagg catgtctggg gcccgcccag gtgccccagg 900 atcccactca ggcacctcca gatcccaccc agtgctgctg cttgagctgt tgaccgtatg 960 cttgcttggg tccctgtgaa ccatgagcca tggagtccgg gatccccttt ctggtaggat 1020 tgatggaatc taataataaa aactgtaggt tttttatgtg taaaaaaaaa aaaa 1074 <210> 21 <211> 3573 <212> DNA
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte ID No: 7246467CB1 <400> 22 caagaattcg gcacaggggt tctgtatccc tacttccatt accctcggct cctcccactc 60 ctcgggggct ccgtgctttc cgcgggtctg tccgggggct ccggaccctc ggcgcacgtg 120 agttgatggc ttccggagaa ctggcatagc tgcagaatat gagtagtgtc ccaagaagag 180 tgctttgcct ttgcgacaag gatcagaata aagttatttg ccatacagta accagagacc 240 tccaactagg ggcccccaaa ctgtatcctg cctgtagaac ctgccaggta aaggtatatt 300 tttgcttttt aatttagcca gaagcaattt ttaaagaaaa tatgtctcct ctgaagatac 360 atggtcctat cagaattcga agtatgcaga ctgggattac aaagtggaaa gaaggatcct 420 ttgaaattgt agaaaaagag aataaagtca gcctagtagt tcactacaat actggaggaa 480 ttccaaggat atttcagcta agtcataaca ttaaaaatgt ggtgcttcga cccagtggag 540 cgaaacaaag ccgcctaatg ttaactctgc aagataacag cttcttgtct attgacaaag 600 taccaagtaa ggatgcagag gaaatgaggt tgtttctaga tgcagtccat caaaacagac 660 ttcctgcagc catgaaaccg tctcaggggt ctggtagttt tggagccatt ctgggcagca 720 ggacctcaca gaaggaaacc agcaggcagc tttcttactc agacaatcag gcttctgcaa 780 aaagaggaag tttggaaact aaagatgata ttccatttcg aaaagttctt ggtaatccgg 840 gtagaggatc gattaagact gtagcaggaa gtggaatagc tcggacgatt ccttctttga 900 catctacttc aacacctctt agatcagggt tgctagaaaa tcgtactgaa aagaggaaaa 960 gaatgatatc aactggctca gaattgaatg aagattaccc taaggaaaat gattcatcat 1020 cgaacaacaa ggccatgaca gatccctcca gaaagtattt aaccagcagt agagaaaagc 1080 agctgagttt gaaacagtca gaagagaata ggacatcagg tgggctttta cctttacagt 1140 catcatcctt ttatggtagc agagctggat ccaaggaaca ctcttctggt ggcactaact 1200 tagacaggac taatgtttca agccagactc cctctgccaa aagaagtttg ggatttcttc 1260 ctcagccagt tcctctttct gttaaaaaac tgaggtgtaa ccaggattac actggctgga 1320 ataaaccaag agtgcccctt tcctctcacc aacagcagca actgcagggc ttctccaatt 1380 tgggaaatac ctgctatatg aatgctattc tacaatctct attttcactc cagtcatttg 1440 caaatgactt gcttaaacaa ggtatcccat ggaagaaaat tccactcaat gcacttatca 1500 gacgctttgc acacttgctt gttaaaaaag atatctgtaa ttcagagacc aaaaaggatt 1560 tactcaagaa ggttaaaaat gccatttcag ctacagcaga gagattctct ggttatatgc 1620 agaatgatgc tcatgaattt ttaagtcagt gtttggacca gctgaaagaa gatatggaaa 1680 aattaaataa aacttggaag actgaacctg tttctggaga agaaaattca ccagatattt 1740 cagctaccag agcatacact tgccctgtta ttactaattt ggagtttgag gttcagcact 1800 ccatcatttg taaagcatgt ggagagatta tccccaaaag agaacagttt aatgacctct 1860 ctattgacct tcctcgtagg aaaaaaccac tccctcctcg ttcaattcaa gattctcttg 1920 atcttttctt tagggccgaa gaactggagt attcttgtga gaagtgtggt gggaagtgtg 1980 ctcttgtcag gcacaaattt aacaggcttc ctagggtcct cattctccat ttgaaacgat 2040 atagcttcaa tgtggctctc tcgcttaaca ataagattgg gcagcaagtc atcattccaa 2100 gatacctgac cctgtcatct cattgcactg aaaatacaaa accacctttt acccttggtt 2160 ggagtgcaca tatggcaatg tctagaccat tgaaagcctc tcaaatggtg aattcctgca 2220 tcaccagccc ttctacacct tcaaagaaat tcaccttcaa atccaagagc tccttggctt 2280 tatgccttga ttcagacagt gaggatgagc taaaacgttc tgtggccctc agccagagac 2340 tttgtgaaat gttaggcaac gaacagcagc aggaagacct ggaaaaagat tcaaaattat 2400 gcccaataga gcctgacaag tctgaattgg aaaactcagg atttgacaga atgagcgaag 2460 aagagcttct agcagctgtc ttggagataa gtaagagaga tgcttcacca tctctgagtc 2520 atgaagatga tgataagcca actagcagcc cagataccgg atttgcagaa gatgatattc 2580 aagaaatgcc agaaaatcca gacactatgg aaactgagaa gcccaaaaca atcacagagc 2640 tggatcctgc cagttttact gagataacta aagactgtga tgagaataaa gaaaacaaaa 2700 ctccagaagg atctcaggga gaagttgatt ggctccagca gtatgatatg gagcgtgaaa 2760 gggaagagca agagcttcag caggcactgg ctcagagcct tcaagagcaa gaggcttggg 2820 aacagaaaga agatgatgac ctcaaaagag ctaccgagtt aagtcttcaa gagtttaaca 2880 actcctttgt ggatgcattg ggttctgatg aggactctgg aaatgaggat gtttttgata 2940 tggagtacac agaagctgaa gctgaggaac tgaaaagaaa tgctgagaca ggaaatctgc 3000 ctcattcgta ccggctcatc agtgttgtca gtcacattgg tagcacttct tcttcaggtc 3060 attacattag tgatgtatat gacattaaga agcaagcgtg gtttacttac aatgacctgg 3120 aggtatcaaa aatccaagag gctgccgtgc agagtgatcg agatcggagt ggctacatct 3180 tcttttatat gcacaaggag atctttgatg agctgctgga aacagaaaag aactctcagt 3240 cacttagcac ggaagtgggg aagactaccc gtcaggcctc gtgaggaaca aactcctggg 3300 ttggcagcat gcactgcata tttgttactg ctgcccacct cacctttcct ctgctgaagg 33&0 agaatttgga attctacttg atgcgggagc aacaaacagc tcagggccaa accaaaagac 3420 aaaaattgga gtaacgtaga atgctccatg ctattttatg gaaactttgg tctcacatcc 3480 gtagctgatt~atcctctttt tctcctatga gtggcacttc ttttgtctta ggaatacctg 3540 ttgtacatct gtctccgtgt tgtgtttttt ccc 3573 <210> 22 <211> 4659 <212> DNA
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte ID No: 7997881CB1 <400> 23 ggcggcgggc gcggcgctga cccggaggcg gcggcggcgg tgcccggatg gaggcacgtc 60 attgtacccc cgccgggggg ctgggctgtg tgcggcggcg gcggcggcgg ccgaggggga 120 tggagcgagc gccgagccgg gtcagagttg aacaatgacc atagttgaca aagcttctga 180 atcttcagac ccatcagcct atcagaatca gcctggcagc tccgaggcag tctcacctgg 240 agacatggat gcaggttctg ccagctgggg tgctgtgtct tcattgaatg atgtgtcaaa 300 tcacacactt tctttaggac cagtacctgg tgctgtagtt tattcgagtt catctgtacc 360 tgataaatca aaaccatcac cacaaaagga tcaagcccta ggtgatggca tcgctcctcc 420 acagaaagtt cttttcccat ctgagaagat ttgtcttaag tggcaacaaa ctcatagagt 480 tggagctggg ctccagaatt tgggcaatac ctgttttgcc aatgcagcac tgcagtgttt 540 aacctacaca ccacctcttg ccaattacat gctatcacat gaacactcca aaacatgtca 600 tgcagaaggc ttttgtatga tgtgtacaat gcaagcacat attacccagg cactcagtaa 660 tcctggggac gttattaaac caatgtttgt catcaatgag atgcggcgta tagctaggca 720 cttccgtttt ggaaaccaag aagatgccca tgaattcctt caatacactg ttgatgctat 780 gcagaaagca tgcttgaatg gcagcaataa attagacaga cacacccagg CCaccactCt 84O
tgtttgtcag atatttggag gatacctaag atctagagtc aaatgtttaa attgcaaggg 900 cgtttcagat acttttgatc catatcttga tataacattg gagataaagg ctgctcagag 960 tgtcaacaag gcattggagc agtttgtgaa gccggaacag cttgatggag aaaactcgta 1020 caagtgcagc aagtgtaaaa agatggttcc agcttcaaag aggttcacta tccatagatc 1080 ctctaatgtt cttacacttt ctctgaaacg ttttgcaaat tttaccggtg gaaaaattgc 1140 taaggatgtg aaataccctg agtatcttga tattcggcca tatatgtctc aacccaacgg 1200 agagccaatt gtctacgtct tgtatgcagt gctggtccac actggtttta attgccatgc 1260 tggccattac ttctgctaca taaaagctag caatggcctc tggtatcaaa tgaatgactc 1320 cattgtatct accagtgata ttagatcggt actcagccaa caagcctatg tgctctttta 1380 tatcaggtcc catgatgtga aaaatggagg tgaacttact catcccaccc atagccccgg 1440 ccagtcctct ccccgccccg tcatcagtca gcgggttgtc accaacaaac aggctgcgcc 1500 aggctttatc ggaccacagc ttccctctca catgataaag aatccacctc acttaaatgg 1560 gactggacca ttgaaagaca cgccaagcag ttccatgtcg agtcctaacg ggaattccag 1620 tgtcaacagg gctagtcctg ttaatgcttc agcttctgtc caaaactggt cagttaatag 1680 gtcctcagtg atcccagaac atcctaagaa acaaaaaatt acaatcagta ttcacaacaa 1740 gttgcctgtt cgccagtgtc agtctcaacc taaccttcat agtaattctt tggagaaccc 1800 taccaagccc gttccctctt ctaccattac caattctgca gtacagtcta cctcgaacgc 1860 atctacgatg tcagtttcta gtaaagtaac aaaaccgatc ccccgcagtg aatcctgctc 1920 ccagcccgtg atgaatggca aatccaagct gaactccagc gtgctggtgc cctatggcgc 1980 cgagtcctct gaggactctg acgaggagtc aaaggggctg ggcaaggaga atgggattgg 2040 tacgattgtg agctcccact ctcccggcca agatgccgaa gatgaggagg ccactccgca 2100 cgagcttcaa gaacccatga ccctaaacgg tgctaatagt gcagacagcg acagtgaccc 2160 gaaagaaaac ggcctagcgc ctgatggtgc cagctgccaa ggccagcctg ccctgcactc 2220 agaaaatccc tttgctaagg caaacggtct tcctggaaag ttgatgcctg ctcctttgct 2280 gtCtCtCCCa gaagacaaaa tcttagagac cttcaggctt agcaacaaac tgaaaggctc 2340 gacggatgaa atgagtgcac ctggagcaga gaggggccct cccgaggacc gcgacgccga 2400 gCCtCagCCt ggCagCCCCg ccgccgaatc cctggaggag ccagatgcgg ccgccggcct 2460 cagcagcacc aagaaggctc cgccgccccg cgatcccggc acccccgcta ccaaagaagg 2520 cgcctgggag gccatggccg tcgcccccga ggagcctccg cccagcgccg gcgaggacat 2580 cgtgggggac acagcacccc ctgacctgtg tgatcccggg agcttaacag gcgatgcgag 2640 cccgttgtcc caggacgcaa aggggatgat cgcggagggc ccgcgggact cggcgttggc 2700 ggaagccccg gaagggttga gtccggctcc gcctgcgcgg tcggaggagc cctgcgagca 2760 gccactcctt gttcacccca gcggggacca cgcccgggac gctcaggacc catcccagag 2820 cttgggcgca cccgaggccg cagagcggcc gccagctcct gtgctggaca tggccccggc 2880 cggtcacccg gaaggggacg ctgagcctag ccccggcgag agggtcgagg acgccgcggc 2940 gccgaaagcc ccaggccctt ccccagcgaa ggagaaaatc ggcagcctca gaaaggtgga 3000 ccgaggccac taccgcagcc ggagagagcg ctcgtccagc ggggagcccg ccagagagag 3060 caggagcaag actgagggcc accgtcaccg gcggcgccgc acctgccccc gggagcgcga 3120 ccgccaggac cgccacgccc cggagcacca ccccggccac ggcgacaggc tcagccctgg 3180 cgagcgccgc tctctgggca ggtgcagtca ccaccactcc cgacaccgga gcggggtgga 3240 gctggactgg gtcagacacc actacaccga gggcgagcgt ggctggggcc gggagaagtt 3300 ctaccccgac aggccgcgct gggacaggtg ccggtactac catgacaggt acgccctgta 3360 cgctgcccgg gactggaagc ccttccacgg cggccgcgag cacgagcggg ccgggctgca 3420 cgagcggccg cacaaggacc acaaccgggg ccgtaggggc tgcgagccgg cccgggagag 3480 ggagcggcac cgccccagca gcccccgcgc aggcgcgccc cacgccctcg ccccgcaccc 3540 cgaccgcttc tcccacgaca gaactgcact tgtagccgga gacaactgta acctctctga 3600 tcggtttcac gaacacgaaa atggaaagtc ccggaaacgg agacacgaca gtgtggagaa 3660 cagtgacagt catgttgaaa agaaagcccg gaggagcgaa cagaaggatc ctctagaaga 3720 gcctaaagca aagaagcaca aaaaatcaaa gaagaaaaag aaatccaaag acaaacaccg 3780 agaccgcgac tccaggcatc agcaggactc agacctctca gcagcgtgct ctgacgctga 384 cctccacaga cacaaaaaaa aagaagaaga aaaagaagag acattcaaga aaatcagagg 3900 actttgttaa agattcagaa ctgcacttac ccagggtcac cagcttggag actgtcgccc 3960 agttccggag agcccagggt ggctttcctc tctctggtgg cccgcctctg gaaggcgtcg 4020 gacctttccg tgagaaaacg aaacacttac ggatggaaag cagggatgac aggtgtcgtc 4080 tctttgagta tggccagggt gattgaaaac tcagcctcaa aacaaaaaat tcactagtta 4140 tgattcaacg cgttcaacag aagccatccc cagcccagct taaattataa agatagacaa 4200 taactctgtt ccaatctgcg tggtgcttct ttagtaaata ctgtacagat tttaccatgg 4260 agaacttttt ttttagtttt taccttttct taattaccct tattccgaat ggacgaacac 4320 tttctaccac tgctgaccat tgtaaaatac cgtgtatata aatcccattg aaataatgcc 4380 ctggaataga acatctcaaa tgctgcttaa ttacagactc aggtcgatta cttgtatttc 4440 atgtaatgtt cctccaagtt agacatctgg tgcaagacca accgggagac catggaattg 4500 tcaaaagtac aaactgacag tgtgtatatt taatttaaag acttatttaa aaactcacaa 4560 gctctcacct agactttgga gagcagtctg ttttctgtaa tgtctgatac tagaaactaa 4620 tttgcttatt ttagttgtat tcaagatttg aagatgtat 4659 <210> 23 <211> 3711 <212> DNA
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte TD No: 7484378CB1 <400> 24 atggagccca ctgtggctga cgtacacctc gtgcccagga caaccaagga agtccccgct 60 ctggatgccg cgtgctgtcg agcggccagc attggcgtgg tggccaccag ccttgtcgtc 120 ctcaccctgg gagtcctttt gggaggaatg aacaactcca gacacgctgc cttaagagct 180 gcaacactcc ctgggaaggt ctacagcgtc actcctgaag caagcaagac cacgaaccca 240 ccagaaggaa gaaattccga acacatccga acatcagcaa gaacaaactc cggacacacc 300 atctttaaga aatgtaacac tcagcccttc ctctctacac agggcttcca cgtggaccac 360 acggccgagc tgcggggaat ccggtggacc agcagtttgc ggcgggagac ctcggactat 420 caccgcacgc tgacgcccac cctggaggca ctgctgcact ttctgctgcg acccctccag 480 acgctgagcc tgggcctgga ggaggagcta ttgcagcgag ggatccgggc aaggctgcgg 540 gagcacggca tctccctggc tgcctatggc acaattgtgt cggctgagct cacagggaga 600 cataagggac ccttggcaga aagagacttc aaatcaggcc gctgtccagg gaactccttt 660 tcctgcggga acagccagtg tgtgaccaag gtgaacccgg agtgtgacga ccaggaggac 720 tgctccgatg ggtccgacga ggcgcactgc gagtgtggct tgcagcctgc ctggaggatg 780 gccggcagga tcgtgggcgg catggaagca tccccggggg agtttccgtg gcaagccagc 840 cttcgagaga acaaggagca cttctgtggg gccgccatca tcaacgccag gtggctggtg 900 tctgctgctc actgcttcaa tgagttccaa gacccgacga agtgggtggc ctacgtgggt 960 gcgacctacc tcagcggctc ggaggccagc accgtgcggg cccaggtggt ccagatcgtc 1020 aagcaccccc tgtacaacgc ggacacggcc gactttgacg tggctgtgct ggagctgacc 1080 agccctctgc ctttcggccg gcacatccag cccgtgtgcc tcccggctgc cacacacatc 1140 ttcccaccca gcaagaagtg cctgatctca ggctggggct acctcaagga ggacttccgt 1200 aagcatcttc ctcggcctgc aatggtcaag ccagaggtgc tgcagaaagc cactgtggag 1260 ctgctggacc aggcactgtg tgccagcttg tacggccatt cactcactga caggatggtg 1320 tgcgctggct acctggacgg gaaggtggac tcctgccagg gtgactcagg aggacccctg 1380 gtctgcgagg agccctctgg ccggttcttt ctggctggca tcgtgagctg gggaatcggg 1440 tgtgcggaag cccggcgtcc aggggtctat gcccgagtca ccaggctacg tgactggatc 1500 ctggaggcca ccaccaaagc cagcatgcct CtggCCCCCa CCatggCtCC tgcccctgcc 1560 gcccccagca cagcctggcc caccagtcct gagagccctg tggtcagcac ccccaccaaa 1620 tcgatgcagg ccctcagtac cgtgcctctt gactgggtca ccgttcctaa gctacaagaa 1680 tgtggggcca ggcctgcaat ggagaagccc acccgggtcg tgggcgggtt cggagctgcc 1740 tccggggagg tgccctggca ggtcagcctg aaggaagggt cccggcactt ctgcggagca 1800 actgtggtgg gggaccgctg gctgctgtct gccgcccact gcttcaacca cacgaaggtg 1860 gagcaggttc gggcccacct gggcactgcg tccctcctgg gcctgggcgg gagcccggtg 1920 aagatcgggc tgcggcgggt agtgctgcac cccctctaca accctggcat cctggacttc 1980 gacctggctg tcctggagct ggccagcccc ctggccttca acaaatacat ccagcctgtc 2040 tgcctgcccc tggccatcca gaagttccct gtgggccgga agtgcatgat ctccggatgg 2100 ggaaatacgc aggaaggaaa tgccaccaag cccgagctcc tgcagaaggc gtccgtgggc 2160 atcatagacc agaaaacctg tagtgtgctc tacaacttct ccctcacaga ccgcatgatc 2220 tgcgcaggct tcctggaagg caaagtcgac tcctgccagg gtgactctgg gggccccctg 2280 gcctgcgagg aggcccctgg cgtgttttat ctggcaggga tcgtgagctg gggtattggc 2340 tgcgctcagg ttaagaagcc gggcgtgtac acgcgcatca ccaggctaaa gggctggatc 2400 ctggagatca tgtcctccca gccccttccc atgtctcccc cctcgaccac aaggatgctg 2460 gccaccacca gccccaggac gacagctggc ctcacagtcc cgggggccac acccagcaga 2520 cccacccctg gggctgccag cagggtgacg ggccaacctg ccaactcaac cttatctgcc 2580 gtgagcacca ctgctagggg acagacgcca tttccagacg ccccggaggc caccacacac 2640 acccagctac cagactgtgg cctggcgccg gccgcgctca ccaggattgt gggcggcagc 2700 gcagcgggcc gtggggagtg gccgtggcag gtgagcctgt ggctgcggcg ccgggaacac 2760 cgttgcgggg ccgtgctggt ggcagagagg tggctgctgt cggcggcgca ctgcttcgac 2820 gtctacgggg accccaagca gtgggcggcc ttcctaggca cgccgttcct gagcggcgcg 2880 gaggggcagc tggagcgcgt ggcgcgcatc tacaagcacc cgttctacaa tctctacacg 2940 ctcgactacg acgtggcgct gctggagctg gcggggccgg tgcgtcgcag ccgcctggtg 3000 cgtcccatct gcctgcccga gcccgcgccg cgacccccgg acggcacgcg ctgcgtcatc 3060 accggctggg gctcggtgcg cgaaggaggc tccatggcgc ggcagctgca gaaggcggcc 3120 gtgcgcctcc tcagcgagca gacctgccgc cgcttctacc cagtgcagat cagcagccgc 3180 atgctgtgtg ccggcttccc gcagggtggc gtggacagct gctcgggtga cgctggggga 3240 cccctggcct gcagggagcc ctctggacgg tgggtgctaa ctggggtcac tagctggggc 3300 tatggctgtg gccggcccca cttcccaggt gtctataccc gggtggcagc tgtgagaggc 3360 tggataggac agcacatcca ggagtgacca ccacgtgact gcccaggccg agactctacg 3420 tgaaagcaac aggagcagca ggccacccaa caccccacgc gccaccgtac cctacccaag 3480 gacgggtgtg ggggggctgt gggtcatggg gatgcatctt tgggtaccac cctttagttc 3540 caataaacac agcccctcca ccctagctca ctggctcagc acctcagtgt cacagcgaag 3600 gaccacatgc atggtgctcc accaggaccc ggggtggcac taaggggaaa gatggacttc 3660 tcccaaccca ggggaggctg agaccctccg agctggggtt ccagggacac g 3711 <210> 24 <211> 2017 <212> DNA
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte 2D No: 7473143CB1 <400> 25 tgcagtgcaa gagtgtggca gatacaaagg acagaaacag gcaggatttg gctggaaagc 60 tggtggatat gaagctggga ggtcactaag ggccaggcca tgtggaaagc cttgtatact 120 ttaattttcc ttattgaaga gcaaggagga gccattgaat gtttggggca tttgggaggt 180 tggcatgacc tcaccacctt ctgcgtgcag tgtgaagaac agattggcaa ggaccagagc 240 aaatgtggct gaccagttag gagttaatac ggcagtttag gaatgagctt ggtgtagggt 300 ggggacagac ggagatagag atagagtggt agattagcca tggggttgtg aagaagagga 360 agcttctagg tgagccttac ttagataaag agatggaggc atgattccat tcactgagtt 420 ggggggtagg caacagaaga ggagagagtg ggtgggggga catcgagagc atcccaaagg 480 ggtgatgggc ctggcccaca gagggatggc tggcctggat catgacgttg tgagtaacca 540 atgcacaagt gggaagtccc ccaaatcgga gagaggagca gaggccttgg cacggagact 600 gaaaggaggc agagaaagag caggagcagg aaaggagtat ggtattgtgg gaggaagctc 660 agggcattgc tgctcaaagt gtggtcccac agagggcatc atcacatcac cagggagcat 720 ggtgggaagg cagtccctcc agctccaccc cggtgtcgat ctgaatctcc atttgagaca 780 gattccccag gtgatgcgtg tgcacagcca gaactgcacg ttccaactgc acggtcccaa 840 tgggacagtt gagagcccag ggttcccata tggctacccc aattacgcca actgcacgtg 900 gaccatcacc gcggaagagc agcacagaat ccagcttgtg ttccagtcct ttgccctgga 960 agaggacttt gatgtcctgt cggtgtttga tggtccaccc cagccagaga atctgcgtac 1020 gaggctcaca ggctttcagc tgccagccac cattgttagt gcagccacca ccctctctct 1080 gcgcctcatc agcgactatg cagtcagtgc ccaaggcttc cacgccacct atgaagttct 1140 ccccagccac acatgtggga acccagggag gctgcccaat ggcatccagc agggttcaac 1200 cttcaacctc ggtgacaagg tccgctacag ctgcaacctt ggcttcttcc tggagggcca 1260 cgccgtgctc acctgccacg ctggctctga gaacagcgcc acgtgggact tccccctgcc 1320 ttcctgcaga gctgatgatg cctgtggtgg gaccctgcgg ggccagagtg gcatcatctc 1380 cagcccccac ttcccctcgg agtaccataa caatgccgac tgcacatgga ccatcctggc 1440 tgagctgggg gacaccatcg ccctggtgtt tattgacttc cagctggagg atggttacga 1500 ctttctggaa gtcactggga cagaaggctc ctccctctgg ttcaccggag ccagcctccc 1560 agcccccgtt atcagcagca agaactggct gcgactgcac ttcacatcgg atggcaacca 1620 ccggcagcgc ggattcagtg cccaatacca agtcaagaag caaattgagt tgaagtctcg 1680 aggtgtgaag ctgatgccca gcaaagacaa cagccagaag acgtctgtgt gtttccacct 1740 cactcctcgt gcctgtctat ctttgtcatc tctgttgccg tgtgtctaaa tcctattagc 1800 tcagaaggtc catgttcgat gccacctctt ccaggcagcc tcacatgcgg gtgcatcctt 1860 catccctccc cactgtggtc ccacagtccg cttccgtggt ttatgtcctc actcaactgg 1920 aaactccttg aggacagtgg tcttatctga ctacctttcg catttccatg gtatccaaat 1980 aaagccttgt acacagtaaa aaaaaaaaaa aaaaaaa 2017 <210> 25 <211> 2646 <212> DNA
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte ID No: 4382838CB1 <400> 26 tccttctgga tgttgtggtc agaaagagta cggccatctt acagctgcat tgccaataat 60 aatgtgggaa accctgcaaa aaagtccacc aacatcattg tgagagcatt aaaaaaagga 120 cgattttgga tcacaccaga tccttatcac aaagatgaca acatccagat tggccgtgag 180 gtgaaaatat cttgccaagt agaagctgtt ccttctgagg aggtaacatt tagttggttt 240 aaaaatggtc gtccattaag aagttctgag cggatggtca ttacacagac tgatcctgat 300 gtctctccgg gaacaacaaa cttggacatc attgatttaa aattcacgga ttttgggacg 360 tacacatgtg tagcatctct gaagggagga ggaatatctg atatcagtat cgatgttaat 420 atatccagca gcacagttcc acccaatctg actgttccac aggaaaaatc accattggtc 480 accagagaag gagacacaat agaactgcaa tgtcaagtaa ctggcaaacc taaaccaatc 540 atcctttggt ctagagcgga taaagaagtt gcaatgcctg atggatcaat gcaaatggag 600 agttatgatg gaacactgag gattgtgaat gtatctaggg aaatgtcagg aatgtacaga 660 tgtcagacca gccaatacaa tggatttaac gtgaaaccaa gggaagcctt ggtgcagctc 720 atcgttcagt atccccctgc agtggaacca gcattcttgg aaatccggca aggacaggat 780 cgaagtgtca ctatgagttg cagagtactg agagcctatc caatacgggt gctgacctat 840 gagtggcgct tgggcaataa attattacgg acgggtcaat ttgactctca ggaatacaca 900 gagtacgctg tgaagagtct ttccaatgaa aactatgggg tttataactg tagcatcata 960 aatgaagctg gagctgggag atgcagcttt cttgttacag gaaaggccta tgctccagaa 1020 ttctattatg atacctacaa tccagtatgg cagaacagac accgtgttta ttcttacagt 1080 ctacagtgga cacagatgaa tcctgatgca gtggatcgga ttgttgcata ccggttgggc 1140 atcaggcagg ctggacagca gcgctggtgg gagcaggaga ttaaaataaa tgggaatatt 1200 caaaagggag aattaattac atataacttg acagagctaa ttaaaccaga agcttatgaa 1260 gtccgactga ctcctctcac caaatttggt gaaggagatt caacaattcg ggtgatcaaa 1320 tatagtgctc ctgtaaatcc tcatttgaga gaatttcatc gtggatttga agatggtaat 1380 atttgtttgt tcactcaaga tgatacagat aattttgact ggacaaagca aagtacagca 1440 acaagaaata caaaatatac tcctaataca ggacctaatg ctgaccgtag tggctccaaa 1500 gaaggttttt atatgtacat tgagacatca cgacccagat tggaaggcga aaaggctcga 1560 cttcccagcc ctgttttcag catagctccc aaaaaccctt atggacccac aaacactgca 1620 tattgtttca gcttctttta tcacatgtat ggacaacata taggtgtctt aaatgtttat 1680 ctacgtttga aagggcaaac aacaatagag aatccactgt ggtcttcaag tgggaataaa 1740 ggacaaagat ggaatgaggc tcatgttaat atatacccaa ttacttcatt tcagctcatt 1800 tttgaaggta tccgaggtcc tggaatagaa ggtgacattg ctattgatga tgtatcaatt 1860 gcagaaggag aatgtgcaaa acaagaccta gcaactaaga attccgttga tggtgctgtt 1920 gggattttgg ttcatatatg gctttttccc attatcgtcc tcatctctat cttaagtcct 1980 cgaaggtgac cttatcctgg cagaggctat aaaagattca ccaggcactg gcatgaagaa 2040 agagtctttg taaatggaca ttgaacaaac aaactaccaa agattcctcc actgactact 2100 gactcaaaaa taaaataata aaaacaaatt tttttaagcg ctggggataa aaagacatca 2160 tggaagtata acttattcca gactaaacat aaaagataat cttgacctga gtagagaaga 2220 gaccttcagg tgcttttgtg gctaaaaaga ttacagcgtc atctggttga actctggaaa 2280 aaaaaaaaaa aaaatgaaaa aaagaaaaaa aaaagagcta tagaaatcct tgtcaaagca 2340 caaagtcatg gctggttttg tttcaaatga atagtttgct tgttaccatg gaaacctaat 2400 ggcctgccaa caaaaacctc actgtaaaca gggtacgtga agagctggca tttattttcc 2460 ttacgagaag gttttcgtag agaattaaat aaatgtaggc ccttttacct ttggctgtta 2520 cccttccttg aaaataaacc cgacttcgat ttttttaaag cttcctgttt tttacccacc 2580 tttttcccca tccccccctt attattatta ttattaatac cctggggtaa ggttgagtaa 2640 cataac 2646 <210> 26 <211> 2088 <212> DNA
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte ID No: 6717888CB1 <400> 27 atgggacctg cctgggtcca ggaccccttg acaggtgctc tctggctgcc tgtcctctgg 60 gcactcttgt cccaggtcta ttgttttcat gacccaccag gatggcgctt cacttcctca 120 gaaattgtga tccccaggaa agtgccccac aggaggggtg gagttgagat gccagaccag 180 ctctcttaca gcatgcattt ccggggccaa agacacgtga ttcacatgaa gctcaagaag 240 aacatgatgc ccagacattt acctgttttt actaataatg accaaggggc catgcaggag 300 aactaccctt ttgtcccacg agactgttac tacgattgct acctggaagg ggttcctggg 360 tctgtggcca cattggacac ctgccgtgga ggtctgcgtg gcatgctgca ggtggatgac 420 ctgacttatg aaatcaaacc cctggaggct ttttccaaat ttgagtatgt agtatctctg 480 cttgtgtcag aagaaagacc aggagaggtc agtagatgta agactgaagg ggaagagata 540 gatcaagaat ctgaaaaggt aaaactggct gaaactccca gagaaggcca cgtttatttg 600 tggaggcatc atagaaaaaa cttgaaactt cactacacag ttactaatgg attattcatg 660 cagaacccta atatgtcaca cataatagag aatgtagtga ttattaacag catcatacat 720 accattttca aaccagttta tttaaatgtc tatgtacgtg ttttgtgcat atggaatgat 780 atggatatag taatgtataa catgcctgcc gacctggttg taggagagtt tggttcgtgg 840 aaatattatg aatggttttc acaaattcca catgatacct cagttgtttt tacatcaaat 900 cgacttggaa acactcctcg ttgtggagac aagatcaaaa atcagaggga agaatgtgac 960 tgtggctccc ttaaagattg tgccagtgat agatgttgtg agacctcttg taccctttct 1020 cttggcagtg tttgcaatac aggactttgc tgccataagt gtaaatatgc tgcccctgga 1080 gtggtttgca gagacttggg tggtatatgt gatctaccgg aatactgtga tgggaaaaag 1140 gaagagtgtc caaatgacat ctacatccag gatggaaccc catgttcagc agtatctgtt 1200 tgtataagag gaaactgcag tgaccgtgat atgcagtgtc aagccctttt tggctaccaa 1260 gtgaaagacg gttccccagc gtgctatcga aaattgaata ggattggtaa ccgatttgga 1320 aactgtgggg ttattctacg gcgaggggga agtagacctt ttccatgtga agaagatgat 1380 gttttttgtg gaatgttgca ctgtagccgt gtcagccaca ttcccggtgg aggtgagcac 1440 actacatttt gtaatatatt agtacacgac ataaaagaag aaaaatgctt tggctatgaa 1500 gcacaccagg ggacagactt gccagaaatg gggctggtag tggatggtgc aacctgtggc 1560 ccagggagct actgtcttaa acgcaattgt actttttatc aagacetgca ttttgagtgt 1620 gatcttaaaa catgcaatta caaaggagta tgtaacaaca aaaaacattg tcattgtctg 1680 catgagtggc aaccaccaac atgtgaactg agaggaaaag gaggtagtat agatagtggc 1740 cctctacctg acaaacaata tcgtattgca ggcagcatac ttgtaaatac aaaccgagca 1800 ctagttttaa tatgtattcg ttacatcctt tttgtggttt cgcttctctt tggtggcttt 1860 tcacaagcaa tacaatgtta gggaagagaa aggaaaagag cccacacatg gagtaaatta 1920 cattgacact tactgggaga tataatcaat agtcactctg acaattacat catcttttag 1980 caattctgat gtcatcttga aataaaatcc cttggcaatt taaaaaggtc tgtgtgttta 2040 aatttactta acatttcatg tctggtcaca ttctcaatac ttctatag 2088 <210> 27 <211> 1890 <212> DNA

<213> Homo sapiens <220>
<221> misc_feature <223> Incyte ID No: 7472044CB1 <400> 28 atgctgctgg ctgtgctgct gctgctaccc ctcccaagct catggtttgc ccacgggcac 60 ccactgtaca cacgcctgcc ccccagcgcc ctgcaagtct tcactctcct cttgggggca 120 gagactgtgt tgggccgcaa cctagactac gtttgtgaag ggccgtgcgg cgagaggcgt 180 ccgagcactg ccaatgtgac gcgggcccac ggccgcatcg tggggggcag cgcggcgccg 240 cccggggcct ggccctggct ggtgaggctg cagctcggcg ggcagcctct gtgcggcggc 300 gtcctggtag cggcctcctg ggtgctcacg gcagcgcact gctttgtagg ctgccgctcg 360 acccgcagcg ccccgaatga gcttctgtgg actgtgacgc tggcagaggg gtcccggggg 420 gagcaagcgg aggaggtgcc agtgaaccgc atcctgcccc accccaagtt tgacccgcgg 480 accttccaca acgacctggc cctggtgcag ctgtggacgc cggtgagccc ggggggatcg 540 gcgcgccccg tgtgcctgcc ccaggagccc caggagcccc ctgccggaac cgcctgcgcc 600 atcgcgggct ggggcgccct cttcgaagac gggcctgagg ctgaagcagt gagagaggcc 660 CgtgttCCCC tgctcagcac cgacacctgc cgaagagccc tggggcccgg gctgcgcccc 720 agcaccatgc tctgcgccgg gtacctggcg gggggcgttg actcgtgcca gggtgactcg 780 ggaggccccc tgacctgttc tgagcctggc ccccgcccta gagaggtcct gttcggagtc 840 acctcctggg gggacggctg cggggagcca gggaagcccg gggtctacac ccgcgtggca 900 gtgttcaagg actggctcca ggagcagatg agcgcctcct cctccagccg cgagcccagc 960 tgcagggagc ttctggcctg ggaccccccc caggagctgc aggcagacgc cgcccggctc 1020 tgcgccttct atgcccgcct gtgcccgggg tcccagggcg cctgtgcgcg cctggcgcac 1080 cagcagtgcc tgcagcgccg gcggcgatgc gagctgcgct cgctggcgca cacgctgctg 1140 ggcctgctgc ggaacgcgca ggagctgctc gggcctcgtc cgggactgcg gcgcctggcc 1200 cccgccctgg ctctccccgc tccagcgctc agggagtctc ctctgcaccc cgcccgggag 1260 ctgcggcttc actcaggctg ccctgggctg gagcccctgc gacagaagtt ggctgccctg 1320 cagggggccc atgcctggat cctgcaggtc ccctcggagc acctggccat gaactttcat 1380 gaggtcctgg cagatctggg ctccaagaca ctgaccgggc ttttcagagc ctgggtgcgg 1440 gcaggcttgg ggggccggca tgtggccttc agcggcctgg tgggcctgga gccggccaca 1500 ctggctcgca gcctcccccg gctgctggtg caggccctgc aggccttccg cgtggctgcc 1560 ctggcagaag gggagcccga gggaccctgg atggatgtag ggcaggggcc cgggctggag 1620 aggaaggggc accacccact caaccctcag gtaccccccg ccaggcaacc ctgagccatg 1680 tctgggcccc cagcccctgg ggaggaccta ctgctcccag gggctgagag gggttcggga 1740 gcataatgac aaactgtcgc tgccccagtg gctgggtgtg tgtgggtggg atggggtggg 1800 ggtcctgggc cccccgtgtc ttcccaggtt tacaatcaga gaatcacagc tgctttaata 1860 aatgttattt ataataaaaa aaaaaaaaaa 1890 <210> 28 <211> 2984 <212> DNA
<213> Homo sapiens <220>
<221> misc_feature <223> Incyte ID No: 7477384CB1 <400> 29 agtgaagacg actgtcttat ctgactgtag gcacagagag tgcgccgcga gagggcggct 60 cctcaccgtc aggcgccggc aggtcgcgtt ctctgctggc cgacgcccga aggcgccgaa 120 tgggggggcc ctgccgagct cccttacagc cccaatgtgc gcgccgccgg gaggcttggg 180 cacgcaggca ccgccggcgg ggggcggggc gaaggcggcg gggcggggca ccagctgcgc 240 gcgcggggcg ggggcggggg cgggggcggg gcgcgctgcg tggtcccggc cggccctggg 300 ctcctccccc tcccgcgccc aggccagcgg cgggcccagc tcctcccccg actcggtctc 360 tCtCCCCtCC CCtCCgCCCg gCagttCCtC CCtCCCgCCg ccgcctcttc ctcggtgagg 420 cgctcttcca gcgggcaggc agcatggcgg ccgtggagac gcgggtgtgc gagacagacg 480 gctgcagcag tgaggccaag ctccagtgtc ccacttgcat caagctgggc atccagggct 540 cgtacttctg ctcgcaggaa tgttttaaag gaagttgggc tactcacaag ttactacata 600 agaaagcaaa agatgaaaag gcgaagcgag aagtgtcttc ctggactgtg gaaggtgata 660 ttaatactga cccatgggca ggttatcgat atactggtaa actcagacca cattatccac 720 tgatgccaac aaggccagtg ccaagttata ttcaaagacc agattatgct gatcatccct 780 taggaatgtc tgaatctgaa caggctctta aaggtacttc tcagattaaa ttactctcat 840 ctgaagatat agaagggatg cgacttgtat gtaggcttgc tagagaagtt ttggatgttg 900 ctgccggcat gattaaacca ggtgtaacta ctgaagaaat agatcacgct gtacacttag 960 catgtattgc aagaaattgc tacccttctc ccctgaatta ttataatttc ccaaagtctt 1020 gttgtacctc agtgaatgaa gtcatttgcc atggaatacc agacagaagg cccttacaag 1080 aaggtgacat tgttaatgtg gatatcactc tttatcgcaa tggttatcat ggggacctga 1140 atgagacatt ttttgttgga gaagtggatg atggagcacg gaaacttgtt cagaccacat 1200 atgagtgcct gatgcaagcc attgatgcag tgaagcctgg tgttcggtac agagaattgg 1260 gaaacattat ccagaagcat gcccaagcaa atgggttttc agttgttcga agctattgtg 1320 ggcatggaat ccacaagctt tttcatacag ctcccaatgt accccactat gctaaaaata 1380 aagcagttgg agtgatgaag tcgggccatg tatttacaat tgagccaatg atttgtgaag 1440 gcggatggca ggatgaaacc tggccagatg gttggactgc ggtgacaaga gacggaaagc 1500 ggtctgctca gtttgagcac accctcctgg tcacagacac tggctgtgaa atcctaaccc 1560 ggcgacttga cagtgcacgg cctcacttca tgtctcaatt ttaatttctc ccaagatggc 1620 acatctcagt accttcttac tgtgctatgc attttattga gagtacagaa aggaagagga 1680 accttttttt aatcacttgt tttgttttga ctatagataa gaaaggacta cagcatttga 1740 tgtgtgtcct caagaacttg tcttgggtct gaaaaagctg agaagaataa aggaaacatt 1800 gctcaactct tcagccccct ccccctgcac acctgttttc tcatttgccc tttgagcact 1860 tttacttaaa cttgcttgta gttgctttta tcactgccgc aaaacagcca tcaagagcca 1920 tctgctttcc aggtgaacat tggaaatgag aatctttgaa acttagcaat atgtgttgca 1980 ccagattttt taaattatat atatggaaat atatatgtat acattttaag ttctgtatac 2040 ataattacca aacactatgt gacctggagt ttgtgttgtt tctgctctga caggtttata 2100 tgttcttaca aatggatcca tagtttgcag tgatttaatt cctggttggg atttggcctc 2160 ccctctcccc catgctaatt atttaccctt gtaattgtgc atagggaagc actcacccaa 2220 tgagactttc tccaatgtgg actctgtgtg tcagtgaatg aatgtagtaa aattcacttt 2280 ggaaggttat caggctttta aaaatctagt ttatggcaaa aatagccatt ttccaagtgg 2340 tggctgactg ttgcagggaa tgagaatttc ataatacact gctatttcag acctctgttt 2400 ggtcagaaat ggaaaagaaa aagccccctt tcttcccttt tctgttttac ttcaagggca 2460 taccttggag gtgctcagag aagcgtgaag tttgcactat ggtggaggat ggggaaagag 2520 ttctaaagtg tctccagctg tgaacccagg aggtcaagtg ggctattaaa atctaacgtt 2580 gagtaaatgt gatagtgatg agaaaggaat tttgtgtact gtaaccttgc agtagagatg 2640 cagctgtcct tcgtgtgtgg aaacacacct ctcctttaca tagttgggaa cctcattaga 2700 aatgacctca gctgccccat atctacgttc ctttcagcag ttgtccaagt aggagtgtat 2760 ccagtgaaga catatcaaat cacaaagtca ttgtcattag agtgtacttg attactgggc 2820 atccttgtaa tataatttca taccactgac acattatact tgtaagagaa catctttccc 2880 agagtgcctc agaccttatt gctttaaaat ataataatgt tttcattact tttattattt 2940 gaatgattta gtaaagttga ctgaatctgg tatagacttt ggga 2984 <210> 29 <211> 2255 <212> DNA
<213> Homo sapiens <220>
<221> misc_feature <223> Incyte ID No: 7077175CB1 <400> 31 ccacagtgtg gatgcccctt gaggatgtca cactcatgag acgccagaca caaaacgcca 60 cacagtgtgt aatcccattt ccatgaaatg tccaggtcag gccagtgcac agacacaggc 120 agcgggtgtg tgggcagcgg ggctggagaa ggggacgggg agtgaccgct gagggggaca 180 ggcttctttt agtggggatg aacgttctaa aattggacac attggtggtg gcacagctgt 240 ggagatatga aaacgcgaaa cctacgggtg agctgggtga accttatgag gcgggaatta 300 actgctccgg ctctggcgct gaggaaaagg aggaccggag gatggcgatc atctgggccg 360 tgccctccac atctgtgtcc tgggaacaga cttctagaaa aacccaaatc aggaaaaagc 420 ggccagctcc acgctgcaaa cagctgggca ccaggcagag agtgttacca gtggtcaagc 480 cagaggtgct gcagaaagcc actgtggagc tgctggacca ggcactgtgt gccagcttgt 540 acggccattc actcactgac aggatggtgt gcgctggcta cctggacggg aaggtggact 600 cctgccaggg tgactcagga ggacccctgg tctgcgagga gccctctggc cggttctttc 660 tggctggcat cgtgagctgg ggaatcgggt gtgcggaagc ccggcgtcca ggggtctatg 720 cccgagtcac caggctacgt gactggatcc tggaggccac caccaaagcc agcatgcctc 780 tggcccccac catggctcct gcccctgccg cccccagcac agcctggccc accagtcctg 840 agagccctgt ggtcagcacc cccaccaaat cgatgcaggc cctcagtacc gtgcctcttg 900 actgggtcac cgttcctaag ctacaagaat gtggggccag gcctgcaatg gagaagccca 960 cccgggtcgt gggcgggttc ggagctgcct ccggggaggt gccctggcag gtcagcctga 1020 aggaagggtc ccggcacttc tgcggagcaa ctgtggcggg ggaccgctgg ctgctgtctg 1080 ccgcccactg cttcaaccac acgaaggtgg agcaggttcg ggcccacctg ggcactgcgt 1140 ccctcctggg cctgggcggg agcccggtga agatcgggct gcggcgggta gtgctgcacc 1200 ccctctacaa ccctggcatc ctggacttcg acctggctgt cctggagctg gccagccccc 1260 tggccttcaa caaatacatc cagcctgtct gcctgcccct ggccatccag aagttccctg 1320 tgggccggaa gtgcatgatc tccggatggg gaaatacgca ggaaggaaat gccaccaagc 1380 ccgagctcct gcagaaggcg tccgtgggca tcatagacca gaaaacctgt agtgtgctct 1440 acaacttctc cctcacagac cgcatgatct gcgcaggctt cctggaaggc aaagtcgact 1500 cctgccaggg tgactctggg ggccccctgg cctgcgagga ggcccctggc gtgttttatc 1560 tggcagggat cgtgagctgg ggtattggct gcgctcaggt taagaagccg ggcgtgtaca 1620 cgcgcatcac caggctaaag ggctggatcc tggagatcat gtcctcccag ccccttccca 1680 tgtctccccc ctcgaccaca aggatgctgg ccaccaccag ccccaggacg acagctggcc 1740 tcacagtccc gggggccaca cccagcagac ccacccctgg ggctgccagc agggtgacgg 1800 gccaacctgc caactcaacc ttatctgccg tgagcaccac tgctagggga cagacgccat 1860 ttccagacgc cccggaggcc accacacaca cccagctacc aggtaccggg agagacggag 1920 ggatccctgg gagtggaggg tcccatgtta atcagcctgg gctgcctaac aagacataac 1980 gtcgtccact ttgggaggcc gaggcgggcg gatcaagagg tcaggagatc gagaccatcc 2040 tggcgaacac ggtgaaacct tgtctctact aaaaaaatac aaaaaattag ccaggcgtgg 2100 tggtgggcgc ctgtagtccc aactacgcgg gaggctaagg caggagaatg gcatgaagcc 2160 gggaggcgga gcttgcagtg agctgcatgc cactgcactc cagcctggca acaagcgaaa 2220 ctccgtctca aaaaagaaaa agacataacg gcctc 2255 <210> 30 <211> 1250 <212> DNA
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte ID No: 7480124CB1 <400> 32 ccgtcatggg cccactcggg ccctctgccc tgggccttct gctgctgctc ctggtggtgg 60 cccctccccg ggtcgcagca ttggtccaca gacagccaga gaaccaggga atctccctaa 120 ctggcagcgt ggcctgtggt cggcccagca tggaggggaa aatcctgggc ggcgtccctg 180 cgcccgagag gaagtggccg tggcaggtca gcgtgcacta cgcaggcctc cacgtctgcg 240 gcggctccat cctcaatgag tactgggtgc tgtcagctgc gcactgcttt cacagggaca 300 agaatatcaa aatctatgac atgtacgtag gcctcgtaaa cctcagggtg gccggcaacc 360 acacccagtg gtatggggtg aacagggtga tcctgcaccc cacatatggg atgtaccacc 420 ccatcggagg tgacgtggcc ctggtgcagc tgaagacccg cattgtgttt tctgagtccg 480 tgctcccggt ttgccttgca actccagaag tgaaccttac cagtgccaat tgctgggcta 540 cgggatgggg actagtctca aaacaaggtg agacctcaga cgagctgcag gaggtgcagc 600 tcccgctgat cctggagccc tggtgccacc tgctctacgg acacatgtcc tacatcatgc 660 ccgacatgct gtgtgctggg gacatcctga atgctaagac cgtgtgtgag ggcgactccg 720 ggggcccact tgtctgtgaa ttcaaccgca gctggttgca gattggaatt gtgagctggg 780 gccgaggctg ctccaaccct ctgtaccctg gagtgtatgc cagtgtttcc tatttctcaa 840 aatggatatg tgataacata gaaatcacgc ccactcctgc tcagccagcc cctgctctct 900 ctccagctct ggggcccact ctcagcgtcc taatggccat gctggctggc tggtcagtgc 960 tgtgaggtca ggatacccac tctaggattc tcatggctgc acaccctgcc ccagcccagc 1020 tgcctccaga cccctaagca tctcctgtcc tggcctctct gaagcagaca agggccacct 1080 atcccggggg tggatgctga gtccaggagg tgatgagcaa gtgtacaaaa gaaaaaaggg 1140 aagggggaga ggggctggtc agggagaacc cagcttgggc agagtgcacc tgagatttga 1200 taagatcatt aaatatttac aaagcaaaaa aaaaaaaaaa aaaaaaattg 1250

Claims (85)

What is claimed is:

1. An isolated polypeptide selected from the group consisting of:
a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:1-15, b) a polypeptide comprising a naturally occurring amino acid sequence at least 90%
identical to an amino acid sequence selected from the group consisting of SEQ
ID
NO:1-15, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1-15, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1-15.

2. An isolated polypeptide of claim 1 comprising an amino acid sequence selected from the group consisting of SEQ ID NO:1-15.

3. An isolated polynucleotide encoding a polypeptide of claim 1.

4. An isolated polynucleotide encoding a polypeptide of claim 2.

5. An isolated polynucleotide of claim 4 comprising a polynucleotide sequence selected from the group consisting of SEQ ID NO:16-30.

6. A recombinant polynucleotide comprising a promoter sequence operably linked to a polynucleotide of claim 3.

7. A cell transformed with a recombinant polynucleotide of claim 6.

8. A transgenic organism comprising a recombinant polynucleotide of claim 6.

9. A method of producing a polypeptide of claim 1, the method comprising:
a) culturing a cell under conditions suitable for expression of the polypeptide, wherein said cell is transformed with a recombinant polynucleotide, and said recombinant polynucleotide comprises a promoter sequence operably linked to a polynucleotide encoding the polypeptide of claim 1, and b) recovering the polypeptide so expressed.

10. A method of claim 9, wherein the polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NO:1-15.

11. An isolated antibody which specifically binds to a polypeptide of claim 1.

12. An isolated polynucleotide selected from the group consisting of:
a) a polynucleotide comprising a polynucleotide sequence selected from the group consisting of SEQ ID NO:16-30, b) a polynucleotide comprising a naturally occurring polynucleotide sequence at least 90% identical to a polynucleotide sequence selected from the group consisting of SEQ ID NO:16-30, c) a polynucleotide complementary to a polynucleotide of a), d) a polynucleotide complementary to a polynucleotide of b), and e) an RNA equivalent of a)-d).

13. An isolated polynucleotide comprising at least 60 contiguous nucleotides of a polynucleotide of claim 12.

14. A method of detecting a target polynucleotide in a sample, said target polynucleotide having a sequence of a polynucleotide of claim 12, the method comprising:
a) hybridizing the sample with a probe comprising at least 20 contiguous nucleotides comprising a sequence complementary to said target polynucleotide in the sample, and which probe specifically hybridizes to said target polynucleotide, under conditions whereby a hybridization complex is formed between said probe and said target polynucleotide or fragments thereof, and b) detecting the presence or absence of said hybridization complex, and, optionally, if present, the amount thereof.

15. A method of claim 14, wherein the probe comprises at least 60 contiguous nucleotides.

16. A method of detecting a target polynucleotide in a sample, said target polynucleotide having a sequence of a polynucleotide of claim 12, the method comprising:

a) amplifying said target polynucleotide or fragment thereof using polymerase chain reaction amplification, and b) detecting the presence or absence of said amplified target polynucleotide or fragment thereof, and, optionally, if present, the amount thereof.

17. A composition comprising a polypeptide of claim 1 and a pharmaceutically acceptable excipient.

18. A composition of claim 17, wherein the polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NO:1-15.

19. A method for treating a disease or condition associated with decreased expression of functional PRTS, comprising administering to a patient in need of such treatment the composition of claim 17.

20. A method of screening a compound for effectiveness as an agonist of a polypeptide of claim 1, the method comprising:
a) exposing a sample comprising a polypeptide of claim 1 to a compound, and b) detecting agonist activity in the sample.

21. A composition comprising an agonist compound identified by a method of claim 20 and a pharmaceutically acceptable excipient.

22. A method for treating a disease or condition associated with decreased expression of functional PRTS, comprising administering to a patient in need of such treatment a composition of claim 21.

23. A method of screening a compound for effectiveness as an antagonist of a polypeptide of claim 1, the method comprising:
a) exposing a sample comprising a polypeptide of claim 1 to a compound, and b) detecting antagonist activity in the sample.

24. A composition comprising an antagonist compound identified by a method of claim 23 and a pharmaceutically acceptable excipient.

25. A method for treating a disease or condition associated with overexpression of functional PRTS, comprising administering to a patient in need of such treatment a composition of claim 24.

26. A method of screening for a compound that specifically binds to the polypeptide of claim 1, the method comprising:
a) combining the polypeptide of claim 1 with at least one test compound under suitable conditions, and b) detecting binding of the polypeptide of claim 1 to the test compound, thereby identifying a compound that specifically binds to the polypeptide of claim 1.

27. A method of screening for a compound that modulates the activity of the polypeptide of claim 1, the method comprising:
a) combining the polypeptide of claim 1 with at least one test compound under conditions permissive for the activity of the polypeptide of claim 1, b) assessing the activity of the polypeptide of claim 1 in the presence of the test compound, and c) comparing the activity of the polypeptide of claim 1 in the presence of the test compound with the activity of the polypeptide of claim 1 in the absence of the test compound, wherein a change in the activity of the polypeptide of claim 1 in the presence of the test compound is indicative of a compound that modulates the activity of the polypeptide of claim 1.

28. A method of screening a compound for effectiveness in altering expression of a target polynucleotide, wherein said target polynucleotide comprises a sequence of claim 5, the method comprising:
a) exposing a sample comprising the target polynucleotide to a compound, under conditions suitable for the expression of the target polynucleotide, b) detecting altered expression of the target polynucleotide, and c) comparing the expression of the target polynucleotide in the presence of varying amounts of the compound and in the absence of the compound.

29. A method of assessing toxicity of a test compound, the method comprising:
a) treating a biological sample containing nucleic acids with the test compound, b) hybridizing the nucleic acids of the treated biological sample with a probe comprising at least 20 contiguous nucleotides of a polynucleotide of claim 12 under conditions whereby a specific hybridization complex is formed between said probe and a target polynucleotide in the biological sample, said target polynucleotide comprising a polynucleotide sequence of a polynucleotide of claim 12 or fragment thereof, c) quantifying the amount of hybridization complex, and d) comparing the amount of hybridization complex in the treated biological sample with the amount of hybridization complex in an untreated biological sample, wherein a difference in the amount of hybridization complex in the treated biological sample is indicative of toxicity of the test compound.

30. A diagnostic test for a condition or disease associated with the expression of PRTS in a biological sample, the method comprising:
a) combining the biological sample with an antibody of claim 11, under conditions suitable for the antibody to bind the polypeptide and form an antibody:polypeptide complex, and b) detecting the complex, wherein the presence of the complex correlates with the presence of the polypeptide in the biological sample.

31. The antibody of claim 11, wherein the antibody is:
a) a chimeric antibody, b) a single chain antibody, c) a Fab fragment, d) a F(ab')2 fragment, or e) a humanized antibody.

32. A composition comprising an antibody of claim 11 and an acceptable excipient.

33. A method of diagnosing a condition or disease associated with the expression of PRTS in a subject, comprising administering to said subject an effective amount of the composition of claim 32.

34. A composition of claim 32, wherein the antibody is labeled.

35. A method of diagnosing a condition or disease associated with the expression of PRTS in a subject, comprising administering to said subject an effective amount of the composition of claim 34.

36. A method of preparing a polyclonal antibody with the specificity of the antibody of claim 11, the method comprising:
a) immunizing an animal with a polypeptide consisting of an amino acid sequence selected from the group consisting of SEQ ID NO:1-15, or an immunogenic fragment thereof, under conditions to elicit an antibody response, b) isolating antibodies from said animal, and c) screening the isolated antibodies with the polypeptide, thereby identifying a polyclonal antibody which binds specifically to a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:1-15.

37. A polyclonal antibody produced by a method of claim 36.

38. A composition comprising the polyclonal antibody of claim 37 and a suitable carrier.

39. A method of making a monoclonal antibody with the specificity of the antibody of claim 11, the method comprising:
a) immunizing an animal with a polypeptide consisting of an amino acid sequence selected from the group consisting of SEQ ID NO:1-15, or an immunogenic fragment thereof, under conditions to elicit an antibody response, b) isolating antibody producing cells from the animal, c) fusing the antibody producing cells with immortalized cells to form monoclonal antibody-producing hybridoma cells, d) culturing the hybridoma cells, and e) isolating from the culture monoclonal antibody which binds specifically to a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:1-15.

40. A monoclonal antibody produced by a method of claim 39.

41. A composition comprising the monoclonal antibody of claim 40 and a suitable carrier.

42. The antibody of claim 11, wherein the antibody is produced by screening a Fab expression library.

43. The antibody of claim 11, wherein the antibody is produced by screening a recombinant immunoglobulin library.

44. A method of detecting a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:1-15 in a sample, the method comprising:
a) incubating the antibody of claim 11 with a sample under conditions to allow specific binding of the antibody and the polypeptide, and b) detecting specific binding, wherein specific binding indicates the presence of a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:1-15 in the sample.

45. A method of purifying a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:1-15 from a sample, the method comprising:
a) incubating the antibody of claim 11 with a sample under conditions to allow specific binding of the antibody and the polypeptide, and b) separating the antibody from the sample and obtaining the purified polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID
NO:1-15.

46. A microarray wherein at least one element of the microarray is a polynucleotide of claim 13.

47. A method of generating an expression profile of a sample which contains polynucleotides, the method comprising:
a) labeling the polynucleotides of the sample, b) contacting the elements of the microarray of claim 46 with the labeled polynucleotides of the sample under conditions suitable for the formation of a hybridization complex, and c) quantifying the expression of the polynucleotides in the sample.

48. An array comprising different nucleotide molecules affixed in distinct physical locations on a solid substrate, wherein at least one of said nucleotide molecules comprises a first oligonucleotide or polynucleotide sequence specifically hybridizable with at least 30 contiguous nucleotides of a target polynucleotide, and wherein said target polynucleotide is a polynucleotide of claim 12.

49. An array of claim 48, wherein said first oligonucleotide or polynucleotide sequence is completely complementary to at least 30 contiguous nucleotides of said target polynucleotide.

50. An array of claim 48, wherein said first oligonucleotide or polynucleotide sequence is completely complementary to at least 60 contiguous nucleotides of said target polynucleotide.

51. An array of claim 48, wherein said first oligonucleotide or polynucleotide sequence is completely complementary to said target polynucleotide.

52. An array of claim 48, which is a microarray.

53. An array of claim 48, further comprising said target polynucleotide hybridized to a nucleotide molecule comprising said first oligonucleotide or polynucleotide sequence.

54. An array of claim 48, wherein a linker joins at least one of said nucleotide molecules to said solid substrate.

55. An array of claim 48, wherein each distinct physical location on the substrate contains multiple nucleotide molecules, and the multiple nucleotide molecules at any single distinct physical location have the same sequence, and each distinct physical location on the substrate contains nucleotide molecules having a sequence which differs from the sequence of nucleotide molecules at another distinct physical location on the substrate.

56. A polypeptide of claim 1, comprising the amino acid sequence of SEQ ID
NO:1.

57. A polypeptide of claim 1, comprising the amino acid sequence of SEQ ID
NO:2.

58. A polypeptide of claim 1, comprising the amino acid sequence of SEQ ID
NO:3.

59. A polypeptide of claim 1, comprising the amino acid sequence of SEQ ID
NO:4.

60. A polypeptide of claim 1, comprising the amino acid sequence of SEQ ID
NO:5.

61. A polypeptide of claim 1, comprising the amino acid sequence of SEQ ID
NO:6.

62. A polypeptide of claim 1, comprising the amino acid sequence of SEQ ID
NO:7.

63. A polypeptide of claim 1, comprising the amino acid sequence of SEQ ID
NO:8.

64. A polypeptide of claim 1, comprising the amino acid sequence of SEQ ID
NO:9.

65. A polypeptide of claim 1, comprising the amino acid sequence of SEQ ID
NO:10.

66. A polypeptide of claim 1, comprising the amino acid sequence of SEQ ID
NO:11.

67. A polypeptide of claim 1, comprising the amino acid sequence of SEQ ID
NO:12.

68. A polypeptide of claim l, comprising the amino acid sequence of SEQ ID
NO:13.

69. A polypeptide of claim l, comprising the amino acid sequence of SEQ ID
NO:14.

70. A polypeptide of claim 1, comprising the amino acid sequence of SEQ ID
NO:15.

71. A polynucleotide of claim 12, comprising the polynucleotide sequence of SEQ ID
NO:16.

72. A polynucleotide of claim 12, comprising the polynucleotide sequence of SEQ ID
NO:17.

73. A polynucleotide of claim 12, comprising the polynucleotide sequence of SEQ ID
NO:18.

74. A polynucleotide of claim 12, comprising the polynucleotide sequence of SEQ ID
NO:19.

75. A polynucleotide of claim 12, comprising the polynucleotide sequence of SEQ ID
NO:20.

76. A polynucleotide of claim 12, comprising the polynucleotide sequence of SEQ ID
NO:21.

77. A polynucleotide of claim 12, comprising the polynucleotide sequence of SEQ ID
NO:22.

78. A polynucleotide of claim 12, comprising the polynucleotide sequence of SEQ ID
NO:23.

79. A polynucleotide of claim 12, comprising the polynucleotide sequence of SEQ ID
NO:24.

80. A polynucleotide of claim 12, comprising the polynucleotide sequence of SEQ ID
NO:25.

81. A polynucleotide of claim 12, comprising the polynucleotide sequence of SEQ ID
NO:26.

82. A polynucleotide of claim 12, comprising the polynucleotide sequence of SEQ ID
NO:27.

83. A polynucleotide of claim 12, comprising the polynucleotide sequence of SEQ ID
NO:28.

84. A polynucleotide of claim 12, comprising the polynucleotide sequence of SEQ ID
NO:29.

85. A polynucleotide of claim 12, comprising the polynucleotide sequence of SEQ ID
NO:30.