CA2421922A1 - Proteases - Google Patents

Abstract

The invention provides human proteases (PRTS) and polynucleotides which identify and encode PRTS. The invention also provides expression vectors, host 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, autoinlmunelinflammatory, cell proliferative, developmental, epithelial, neurological, and reproductive disorders, and in the assessment of the effects of exogenous compounds on the expression of ~lucleic acid and amino acid sequences of proteases.
so 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 profession 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 anlinopeptidases, 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 mamnaalian 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. 2-S.) 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 comnrion ancestor. These six clans are hypothesized to have descended from at least four WO 02/20736 CA 02421922 2003-03-07 PCT/USOl/28161 evolutionarily distinct ancestors. SPs are named for the presence of a seriue 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. Barxett (1994) Methods Enzymol so 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 ZO domains are triple-looped, disulfide cross-licked 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-XCI, complement factors B, C, and D, grauzymes, 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 PCl, PC2, PC3, PC6, and PACE4 (Rawlings and Barrett, s, upra). .
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 IysosomaI

WO 02/20736 CA 02421922 2003-03-07 PCT/USOl/28161 serine peptidase that cleaves peptides such as angiotensin II and 1~ and [des-Arg9] bradyhinin, 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. thehippocampus in xesponse to neural signaling (Chen, Z. L. et a1. {1995) r. Neurosci.15:5088-5097) Tissue plasminogen activator is useful for acute management of stroke (Zivin, J.A. (1999) Neurology 53:1A.-19) and myocardial infarction (Ross, A.M. (1999) Clip. 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 cell tryptase, are released in allergy and inflammatoxy 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) Curx. Pharm. Des. 4:381-396). Prostate-specific antigen (PSA) is a kallikrein-like serine protease synthesized and secreted exclusively by epithelial cells 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.kT. 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 e~.-ported, removal of the signal sequence by a signal peptidase and posttranslational processing, e.g., glycosylation or phosphorylation, activate the protein. Signal peptidases exist as multi-subunit complexes in both yeast and mammals.
The catvlte 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 coxe domains and regulatory ATPase domains which canbe identified by the presence of the P loop, an ATP/GTP binding motif (PROSTTE 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 xelated to Clp protease (Ozeliu's, L.J. et al. (1998) Adv. Neurol.
78:93-105).

WO 02/20736 CA 02421922 2003-03-07 PCT/USOl/28161 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 pxotein. 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, transcriptaonal 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) Anna. 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:? 179-2183). 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 inhuman neurodegenerative diseases (Lowe, J. et aI.
(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.
Bio1.3:584-591).
Cysteine Proteases Cysteine proteases (CPs) are involved in diverse cellular processes ranging from the processing of precursor proteins to intracellular de~-adation. Nearly half of the CPs known are present only in viruses. CPs have a cysteine as the major catalytic xesidue 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 li'tce 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 WO 02/20736 CA 02421922 2003-03-07 PCT/USOl/28161 significance (Karrer, K.M. et al. (1993) Proc. Natl. Acad. Sci. USA 90:3063-3067). Thxee-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. Barren (1994) Methods Enzymol.
244:461-486).
Some CPs are expressed ubiquitously, while others are produced onlyby 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).
Calpaius are calcium dependent cytosolic endopeptidases which containboth 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 snbunit 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 (Char, S.L. and M.P. Manson (1999) T. 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 (Char and Manson, su ra). Calpain mediated breakdown of the cytoskeleton has been proposed to contn'bute 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 fox liurb-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 iu the catalytic mechanism. Caspases are among the most specific endopeptidases, cleaving after aspartate residues.

WO 02/20736 CA 02421922 2003-03-07 PCT/USOl/28161 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 agoptosis.
An activating signal causes autoproteolytic cleavage of a spec'~f'tc aspartate residue (D297 in the caspase-1 numbering convention) and removal of the spacer and prodomain, leaving a p10/p20 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 prodomainby which they can be recruited into self activating complexes with other caspases and FADD
protein associated death receptors ox 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 IAl's~ 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 1L-1b and possibly other inflammatory cytob~nes (Char and Mattson, supra).
Cowpox vit~xs 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, su ra; Thompson, C.B. (1995) Science 267:1456-1462).
As~artyl Qroteases Aspartyl proteases (APs) include the Iysosomal proteases cathepsins D and E, as well as chymosin, reniu, 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. Al's 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 occurnng toward the C terminus.
Retropepsins, on the other hand, are analogous to a single domain of pepsin, and become active as homodimers with each WO 02/20736 CA 02421922 2003-03-07 PCT/USOl/28161 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 fox regulating electrolyte balance and blood pressure (revitewed in Crews, D.E. and S.R.
Williams (1999) Hum. Biol.
71:475-503). Abnormal regulation and expression of cathepsins are evident in various inflarri~matory 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.
to Oncol.4:95-114).
Metallo~roteases 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 15 responses. Aminopeptidase P has been implicated in coronary ischemia/reperfusion injury.
Administration of aminopeptidase P inh~'bitors 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 Iigands and a catalytic glutamie acid C-20 terminal to the first histidine. Proteins containing this signature sequence are known as the metzincins and include aminopeptidase N, angi_.otensin-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 angiotensiu converting enzyme and 25 the an~iuopeptidases, 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/neuxolysin can. hydrolyze bradyl~nin as well as neuxotensin (Serizawa, A_ et al. (1995) J. Biol. Chem 270:2092-2098).
Neurotensin. is a v~:-oactive peptide that can act as a neurotransmitter in the brain, where it has been 30 implicated in limiting food intake (Tritos, N.A. et al. (1999) Neuropeptides 33:339-349).
The matrix metalloproteases (1V11VIYs) are a family of at least 23 enzymes that can decade components of the extracelIuIar 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 (TIIVIPs, for tissue WO 02/20736 CA 02421922 2003-03-07 PCT/USOl/28161 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~2 ion in the active site interacts with a cysteine in the pro-s sequence. Activating factors disrupt the Zn'~a-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 Turin. 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 1o Neurosci.21:75).
MMPs are implicated in a number of diseases including osteoarthritis (Mitchell, P. et al.
(1996) J. Clip.. Iuvest. 97:761), atherosclerotic plaque rupture (Sukhova, G.T~. et aL (1999) Circulation 99:2503), aortic aneurysm (Schneiderman, J. et al. (1998) Am. J. Path.
152:703), non healing wounds (Saarialho-Kere, U.I~. et al. (1994) J. Clip. Invest. 94:79), bone resorption (Blavier, L. and J.M.
15 Delaisse (1995) J. Cell Sci. 108:3649), age-related macular degeneration (Steep, B. et al. (1998) Invest. Ophthahnol. 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 ofmammary carcinoma and experimental tumors in rat, and Lewis lung carcirxoma, hemangioma, and human 20 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 a1 (1996) J. Clip. Invest.
98:671; Taraboletti, G.
et aI. (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 inlu'bitors can xelieve soma of its symptoms (reviewed in Yong, su ra .
25 Another family of metalloproteases is the ADAMS, fox A Disintegrin and Metalloprbtease Domain, which they share with their close relatives the adamalysins, snake venom metalloproteases (SVMPs). ADAMS combine featuxes 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 30 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 inIu'liited by some MMP antagonists such as T>ZVIP-2.
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 WO 02/20736 CA 02421922 2003-03-07 PCT/USOl/28161 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 inht'bition in Drosophila 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 (Schlondoxff and Blobel, su ra . TACE 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 foam. 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 prointlammatory stimuli (Kuno, K. et al.
(1997) J. Biol. Chem. 272556-562). To date eleven members are recognized by the Human Genome Organization (HUGO; http://www.gene.ucl.ac.uk/users/hester/ada-tnts.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. 274:2 3443). Other members are reported to have autiangiogenic potential (Kuno et al., su ra andlor pxocollagen processiug (Colige, A. et al. (1997) Proc. Natl. Acid. Sci. USA 94:2374).
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/inflammatoxy, 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 indivitdually as "PRTS-1," "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,"
"PRTS-15,"
"PRTS-16," and "PRTS-17:' In one aspect, the invention provides an isolated polypeptide selected from the group consisting of a) a polypeptide comprising an amiuo acid sequence selected from the group consisting of SEQ >D N0:1-17, b) a.polypeptide comprising a naturally occurring amino acid sequence at least 90% identical to au amino acid sequence selected from the group consisting of SEQ
>D N0:1-17, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:I-17, and d) au immunogenic fra~nent of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID
N0:1-17. In one alternative, the invention provides an isolated polypeptide comprising the amino acid sequence of SEQ
ID NO:I-17.
The invention further provides an isolated polynucleotide encoding a polypeptide selected from the group consistitzg of a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ m N0:1-17, 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 >D N0:1-I7, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ >D N0:1-I7, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1-I7. In one alternative, the polynucleotide encodes a polypeptide selected from the group consisting of SEQ
m N0:1-17. In another alternative, the polynucleotide is selected from the group consisting of SEQ ID NO:18-34.
Additionally, the invention provides a recombinant polynueleotide 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 m N0:1-17, 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 )D N0:1-17, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:I-I7, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ 1D NO:I-17. 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 NO:I-17, b) a polypeptide comprising a naturally occurring amino acid sequence at least 90%o identical to au amino acid sequence selected from the group consisting of SEQ )D N0:1-17, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ lD N0:1-17, and d) au immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID N0:1-17. 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 m NO:1-17, 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 N0:1-17, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ 1D N0:1-17, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ
ID N0:1-17.
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
D7 N0:18-34, 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 N0:18-34, 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 pol3~ucleotide in a sample, said target polynucleotide having a sequence of a polynucleotide selected from the group consisting of.
a) a polynueleotide comprising a polynucleotide sequence selected from the group consisting of SEQ
ID N0:18-34, b) a polynucleotide comprising a naturally occurnng polynucleotide sequence at least 90% identical to a polynucleotide sequence selected from the group consisting of SEQ >D N0:18-34, 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 pxobe 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 N0:18-34, 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 N0:18-34, 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 1D N0:1-17, 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 N0:1-17, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ >D N0:1-17, and d) au immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID N0:1-17, and a pharmaceutically acceptable excipient. Iu one embodiment, the composition comprises an amino acid sequence selected from the group consisting of SEQ m N0:1-17. 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 N0:1-17, 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 U~ NO:1-17, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID
NO:1-I7, and d) an immunogenic fragment of a polypeptade having an amibo acid sequence selected from the group consisting of SEQ ?D N0:1-17. The method comprises a) exposing a sample comprising the polypeptide to a compound, and b) detecting agonist activity iu 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 fox 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 >D N0:1-17, 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 N0:1-17, c) a biologically active fragment of a polypeptide having an amino acid sequence selected froril the group consisting of SEQ ID N0:1-17, and d) au immunogenic fragment of a polypeptide having an anliuo acid sequence selected from the group consisting of SEQ ID N0:1-17. 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 au 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 E? N0:1-17, b) a polypeptide comprising a naturally occurzing amino acid sequence at least 90% identical to an amino acid sequence selected from the group consisting of SEQ ID N0:1-17, c) a biologically active fragment of a polypept'tde having an amino acid sequence selected from the group consisting of SEQ ID
NO:1-17, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ 1D NO:1-17. 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 1A N0:1-17, b) a polypeptide comprising a naturally occurring amino acid sequence at least 90% identical to an amine acid sequence selected.
from the group consisting of SEQ 1D N0:1-17, c) a biologically active fragment of a palypeptide ' 25 having an amino acid sequence selected from the group consisting of SEQ m N0:1-17, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ D7 N0:1-17. The method comprises a) combining the polypeptide with at Ieast 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 1D N0:18-34, 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 pxobe 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
I!D NO:18-34, ii) a polynucleotide comprising a naturally occurring polynucleotide sequence at least 90% identical to a polynucleotide sequence selected from the group consisting of SEQ )17 N0:18-34, 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 ll~ NO:18-34, 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 N0:18-34, iii) a polynucleotide complementary to the polynucleotade of i), iv) a polynucleotide complementary to the polynucIeotide 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 TILE TABLES
Table 1 summarizes the nomenclature for the full length polynucleotide and polypeptide sequences of the present invention.
Table 2 shows the GenBank identification number and annotation of the nearest GenBank homolog for polypeptides of the invention. The probability score for the match between each polypeptide and its GenBankhomolog is 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 eDNA 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.
1o 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 15 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 lmown to those skilled in the art, and so 2o 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 descn'bed herein. can be used to practice or test the present invention, the preferred machines, materials and methods are now 25 described. All publications mentioned herein are cited for the purpose of describing and disclosing 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 constnzed as an admission that the invention is not entitled to antedate such disclosure by virtue of prior invention.
DEFINITIONS
30 "PATS" refers to the amino acid sequences of substantially purified PATS
obtained from any species, particularly a mammalian species, including bovine, ovine, porcine, murine, equine, and human and from any source, whether nataraI, synthetic, semi-synthetic, or recombinant.
The term "agonist" refers to a molecule which intensifies or mimics the biological activity of PATS. 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 .nay occur alone, or in combination with the others, one or more tunes 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 xetained. For example, negatively chaxged 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 polymerise chain reaction (PCR) technologies well known in the art.

The tern. "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')2, 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 oIigopeptide 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 (KLIT). The coupled peptide is then used to immunize the animal.
The term "antigenic determinant" refexs 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 "autisense" refers to any composition capable of base-paining 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 pxoduced by any method including chemical synthesis or transcription. Once introduced into a cell, the complementary antisense molecule base-pairs with a naturally occu3ring 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 autisense 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, "immunologicaIly 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 speci~tc 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" xefer 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 poiynucleotide 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 prnbe may be .
deployed in an aqueous solution containing salts (e.g., NaCl), 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 GA) in the 5' and/or the 3' direction, and resequenced, or which has been assembled from one or more overlapping cDNA, EST, or genomic DNA fra~.ents using a computer program for fragment assembly, such as the GELVIEW fragment assembly system (GCG, Madison WI) or Phrap (University of Washington, Seattle WAS. 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 Hts, Lys Asn Asp, Gln, His Asp Asn, Glu Cys Ala, Ser Gln Asn, Glu, His Glu Asp, Gln, His Gly Ala I3is Asn, Arg, GIn, GIu lle Leu, Val Leu lle, Val ' Lys Arg, Glu, Glu i8 Met Leu, Ile Phe His, Met, Leu, Trp, Tyr Sex Cys, Thr Thr Ser, dal Trp Phe, Tyr Tyr His, Phe, Trp Val Ile, 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 bulb 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 rr~easurable signal and is covalently or noncovalently joined to a polynucleotide or polygeptide.
"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 a~a 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 3o 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 nucleotidelamino acid residue. For example, a fragment may comprise from 5 to 1000 contiguous nucleotides or amino acid residues. A fra~nent used as a probe, primer, antigen, therapeutic molecule, or for other purposes, maybe 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%a) 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, n~ay'be encompassed by the present embodiments.
A fragment of SEQ ID N0:18-34 comprises a region of unique polynucleotide sequence that specifically identifies SEQ ID N0:18-34, far example, as distinct from any other sequence in the genome from which the fragment was obtained. A fragment of SEQ ID N0:18-34 is useful, for example, in hybridization and amplification technologies and in aualogous methods that distinguish SEA
ID NO:18-34 from xelated polynucleotide sequences. The precise length of a fragment of SEQ ll?
N0:18-34 and the region of SEQ ID N0:18-34 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 IIJ N0:1-17 is encoded by a fragment of SEQ ID N0:18-34. A
fragment of SEQ 7D N0:1-17 comprises a region of unique amino acid sequence that specifically identifies SEQ ll~ N0:1-17. For example, a fragment of SEQ ID N0:1-17 is useful as au inlmunogenic peptide for the development of antibodies that specifically recognize SEQ 1D N0:1-17.
The pxecise length of a fragment of SEQ >I7 N0:1-17 and the region of SEQ ID
NO:1-17 to which the fragment corresponds are routinely determinable by one of ordinary sIdli in the art based on the . intended purpose far the fragment.
A "full length" polynucleotide sequence is one containing at least a translation initiation eodon (e.g., metbionine) 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 moxe polynucleotide sequences or two or mare 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 mare 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 softwaxe package, a suite of molecular biological analysis programs (DNASTAR, Madison 'Wn. CLUSTAL V is descn'bed 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 paixwise alignments of polynucleotide sequences, the default parameters are set as follows: Ktuple=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 polynucIeotide. sequences.
Alternatively, a suite of conunonly used.and freely available sequence comparison algorithms is provided by the National Center for Biotechnology Information (NCBl~ B asic 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:l/www.ncbi.nhn.n>~.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" canbe accessed and used interactively at http:llwww.ncbinlm.nih.gov/gorf/bl2~tml. The "BLAST 2 Sequences" tool can be used far 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 Re~var d for rnateh: 1 Penalty for rrtis»zatch: -2 Opert Gap: S arzd Extertsiorz Gap: 2 pertalties Gap x drop-off SO
Expect: 10 Word Size: 11 .Filter: orz Percent identity may be measured over the length of au 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 sirtiilar 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 "~o 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:
lVlatr~ix: BLOSUM62 Open Gap: 11 arzd Extensiotz Gap: 1 petzalties Gap x drop-off. 50 Expect: l0 Word Size: 3 Filtet~: oiz 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 ate exemplary only, and it is understood that any fragment length supported by the sequences shownherein, in the tables, figures or Sequence Listltlg, maybe used to describe a length over which percentage identity may be measured.

"Humau at~,tf' tcial chromosomes" (HACs) are linear microchromosomes which may contain.
DNA sequences of about 6 kb to 10 Mb in size and which contain alI of the elements required fox 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 humau 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 deterrni"ing the stringency of the hybridization process, with more stringent conditions allowing Iess 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 ,uglml 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 (T",) for the specific sequence at a defined ionic strength aud~ 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 canbe found in Sambrook, J. et aI. (1989) Molecular Clonin~y A Laboratory Manual, 2n~ 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 2 hour.
Alternatively, temperatures of about 65°C, 60°C, 55°C, or 42°C maybe 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 ~Cglml. Organic solvent, such as formamide at a concentration of about 35-50% vlv, 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 tern? 'hybridization complex" refers to a complex formed between two nucleic acid sequences by virtue of the formation of hydrogen bands between complementary bases. A
hybridization complex may be formed in solution (e.g., Cot or Rot analysis) or fornled 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 amino 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 "imtnunogenic 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 chenucal 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 tern. "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, maybe 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~
ed., vol. 1-3, Coid Spring Harbor Press, Plainview NY; Ausubel, F.M. et al. (1987) Current Protocols in Molecular Biolo , Greene Publ. Assoc. & Wiley Iutersciences, New York N'Y; 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, fox example, by using computer programs intended for that purpose such as Primer (Version 0.5,1991, Whitehead Institute for Biomedical Research, Cambridge MA).

Oligonucleotides fox use as primers axe selected using software lmown 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 Institute/MIT
Center for Genorne 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 fox 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 pxogram (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 usefal for identification of both unique and conserved oligonucleotides and polynucIeotide fragments. The oligonucleotides and poIynucleotide fragments identified by any of the above selection methods are useful in hybridization technologies, for example, as PCR or sequencing primers, microarxay 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 seguients 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 genefiic engineering techniques such as those descn'bed in Sambrook, supra. The term recombinant includes nucleic acids that have been altered solely by addition, substitutton, 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,chromogenic 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 thyaline 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 bindialg molecule. For example, if an antibody is specific for epitope "A," the presence of a poIypeptide comprising the epitope A, or the presence of free unlabeled A, in a reaction containing free labeled A and the antn~body 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%o free, preferably at least 7S % 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, ma~letic 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" 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 cell. 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, lipofectian, and particle bombardment.
The term "transformed cells" includes stably transformed cells in which the inserted DNA is capable of replication either as to 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 lmown in the art. The nucleic acid is introduced into the cell, directly ox 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 transgenie 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), s-ultra.
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, far 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 9b%, 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, far 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 variauts 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 pxesence 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 polypeptade 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, autoimmunelinflammatory, 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 Iucyte project identification number (Incyte Project D7). Each polypeptide sequence is denoted by both a polypeptide sequence identification number (Polypeptide SEQ 1D NO:) and an Incyte polypeptide sequence number (Iucyte Polypeptide ID) as shown. Each polynucleotide sequence is denoted by both a polynucleotide sequence identification number (Polynucleotide SEQ ll~ NO:) and an Iucyte 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 ID NO:) and the corresponding Incyte polypeptide sequence number (Incyte Polypeptide 7D) for polypeptides of the invention. Column 3 shows the GenBank. identification number (Genbank 3D NO:) of the nearest GenBauk homolog.
Column 4 shows the probability score for the match between each polypeptide and its GenBanh homolog. Column 5 shows the annotation of the GenBank homolog 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 poIypeptide sequence identification number (SEQ ll~ 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 phospholylation sites, and column S 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 anal 3 summarize the properties of polypeptides of the invention, and these properties establish that the claimed polypeptides are proteases. For example, SEQ m N0:1 is 89°l0 identical to a human preprocathepsin L precursor (GenBank m 8190418) as determined by the Basic Local Alignment Search Tool (BLAST). (See Table 2.) The BLAST probability score is 4.5e-169, which indicates the probability of obtaining the observed polypeptide sequence alignment by chance.
SEQ ID NO:1 also contains a papain family cysteine protease active site domain as determined by searching fox statistically significant matches in the hidden Markov model (F~1VI)-based PFAM
database of conserved protein family domains. (See Table 3.) The presence of this motif is conhrnied by BLIIVlhS, MOTIF'S, and PROFILESCAN analyses, providing further corroborative evidence that SEQ ID N0:1 is a cysteine protease of the papainfamily. In an alternative example, SEQ ID N0:6' has 44% local identity.to Xenopus ovochymase, a polyprotease of the trypsin family (GenBank ID
82981641), as determined by the Basic Local Alignment Search Tool (BLAST).
(See Table 2.) The BLAST probability score is 6.4e-201, which indicates the probability of obtaining the observed polypeptide sequence alignment by chance. SEQ D7 N0:6 contains a number of protease active site domains as determined by searching for statistically significant matches in the hidden Markov model (~)-based PRAM database of conserved protein family domains. (See Table 3.) The presence of these motifs is confirmed by BLZIvIPS, MOTIFS, and PROF1LESCAN analyses.
These analyses also reveal the pxesence of kringle and CUB domains, as well as a signal peptide. Together, these data provide further corroborative evidence that SEQ m N0:6 is a serine protease of the trypsin family. In an alternative example, SEQ lD N0:10 is 50% identical to a human ubiquitin-specific processing protease (GenBank ID 86942888) as determined by the Basic Local Alignment Search Tool (BLAST). (See Table 2.) The BLAST probability score is 7.5e-273, which indicates the probability of obtaining the observed polypeptide sequence alignment by chance. SEQ ID NO:10 is also 51 % identical to a murine ubiquitin-specific processing protease (GenBank >D 86941890) as determined by the BLAST analysis with a probability score of 4.0e-271. SEQ ff~
NO:IO also contains ubiquitin carboxyl-terminal hydrolase (i.e., ubiquitin-specific protease) domains as determined by searching for statistically significant matches in the hidden Markov model (FMS)-based PFAM
database of conserved protein family domains. (See Table 3.) Data from BLM'S
and MOTIFS
analyses provide further corroborative evidence that SEQ ID N0:10 is a ubiquitin-specific protease.
In. an alternative example, SEQ ID N0:16 has 52% local identity to Xenopus ADAM13 (GenBank 1D
81916617) as determined by the Basic Local Alignment Search Tool (BLAST). (See Table 2.) The BLAST probability score is 1.4e-198, which indicates the probability of obtaining the observed polypeptide sequence alignment by chance. SEQ ID N0:16 contains a reprolysin fanuly neutral zinc protease active site domain, a reprolysin family propeptide, and a disintegrin domain signature as determined by searching for statistically significant matches in the hidden Markov model (HIvIM)-based PFAM database of conserved protein family domains. (See Table 3.) The presence of these domains is confirmed by BLllVIF'S, MOTIFS, and PROF1LESCAN analyses, providing further corroborative evidence that SEQ ID N0:16 is a metalloprotease of the ADAM
family. In an alternative example, SEQ m N0:17 is 30% identical to the human zinc metalloprotease ADAMTS6 (GenBank ID 85923786) as determined by CLUSTAL ~ analysis, and 44% local identity, as determined by the Basic Local Alignment Search Tool (BLAST). (See Table 2.) The BLAST
probability score is 9.1e-164, which indicates the probability of obtaining the observed polypeptide sequence alignment by chance. SEQ m N0:17 also contains a zinc metalloprotease active site domain, a reprolysin family metalloprotease propeptide, and a type I
thrombospondin domain as determined by searching for statistically significant matches in the hidden Markov model (I~VIM)-based PFAM database of conserved protein family domains. (See Table 3.) Data from BLM'S
analysis provide further corroborative evidence that SEQ ID N0:17 is a metalloprotease of the ADAMTS family. SEQ ID N0:2-S, SEQ lD NO:7-9, and SEQ 117 NO: 11-15 were analyzed and annotated in a similar manner. The algorithms and parameters for the analysis of SEQ ID NO:1-17 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 aild 2 list the polynucleotide sequence identification number (Polynucleotide SEQ ID NO:) and the corresponding Incyte polynucleotide consensus sequence number (Incyte Polynucleotide D~} 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 )D N0:18-34 or that distinguish between SEQ ID
N0:18-34 and related polynucleotide sequences. Column 5 shows identification numbers corresponding to cDNA
sequences, coding sequences (exons) predicted from genomic DNA, andlor 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, 6917460H1 is the identification number of an Incyte cDNA sequence, and PLACFER06 is the cDNA
library from which it is derived. Incyte cDNAs for which cDNA h.'braries are not indicated were derived from pooled cDNA libraries (e.g., 72004319V1). Alternatively, the identification numbers ineolumn 5 may refer to GenB~auk cDNAs or ESTs (e.g., g1365166) 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 Sauger Centre, Cambridge, UK) database (i.e., those sequences including the designation "ENST"). Alternatively, the identification numbers in column 5 maybe 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 XX~~X NI NZ YYYI~'_N3 N4 represents a "stitched" sequence in which Xz'~X~is the identification.number of the cluster of sequences to which the algorithm was applied, and YffYl'is the number of the prediction generated by the algorithm, and N1,2,3..., 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, FT .X~Xx~'X~A_A_A_A_A__gBBBBB 1 1V is the identification number of a "stretched" sequence, with XXXXXX 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 GenB au(~ protein homolog, and N referring 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 pxefix identifies component sequences that were hand-edited, predicted from genomic DNA sequences, or derived from a combination of sequence analysis methods. The following Table Iists examples of component sequence prefixes and corresponding sequence aualysis methods associated with the prefixes (see Example IV and Example V).
Prefix Type of analysis andlor 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 Sauger Centre, Cambridge, UK) GBI Hand-edited analysis of genomic sequences.

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

l0 1NCY 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 eDNA coverage redundant with the sequence coverage shown in column 5 was obtained to confirm the final consensus polynucleotide sequence, but the relevant Iucyte cDNA identification numbers are not shown.
25 Table 5 shows the representative cDNA libraries for those full length polynucleotide sequences which were assembled using Incyte cDNA sequences. The representative cDNA library is the Incyte cDNA library which is most frequently represented by the Iucyte cDNA sequences which were used to assemble and conhrrn the above polynucleotide sequences.
The tissues and vectors which were used to construct the cDNA fbraries shown in Table 5 are described in Table 6.
20 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. Iu a particular 25 embodiment, the invention encompasses a polynucIeotide sequence comprising a sequence selected from the group consisting of SEQ >D NO:18-34, which encodes PRTS. The polynucleotide sequences of SEQ )D N0:18-34, 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 deoxynbose.

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 )D N0:18-34 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 of SEQ
D7 N0:18-34. Any one of the polynucleotide variants descn'bed 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 occurring PRTS under appropriately selected conditions of stringency, it may be advantageous to produce nucleotide sequences encoding PRTS or its derivatives pOSSesSlng 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, entirelyby 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
N0:18-34 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 Enzytnol. 152:507-511.) Hybridization conditions, including annealing and wash conditions, are descn'bed 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 S 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), to 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 ystem (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.
15 (1997) Short Protocols in Molecular Biology, John Wiley & Sons, New York NY, unit 7.7; Meyers, R.A. (1995) Molecular Biology and Biotechnolo~y, 'Wiley VCH, New 'York NY, pp.
856-$53.) The nucleic acid sequences encoding PRTS may be extended utilizing a partial nucleotide sequence and employing various PCR based methods Imown in the art to detect upstream sequences, such as promoters and regulatory elements. For example, one method which may be employed, 20 restriction-site PCR, uses universal and nested primers to amplify unlolown 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 25 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 per.~orrning PCR. Other methods which may be used to xetrieve unlmown sequences 30 are lmowri 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 PROMOTERF1NDER libraries (Clontech, Palo Alto CA) to walk genomic DNA. This procedure avoids the need to screen h'braries and is useful in finding intronlexon 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) ox another appropriate program, to be about 22 to 30 nucleotides in length, to have a GC content of about SO~fo or more, and to anneal to the template at temperatures of about 68°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. Genonlic libraries maybe 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 ,0 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 known 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 fra,~aments and synthetic oligonucleotides may be used to engineer the nucleotide sequences. For example, oligonucleotide-mediated site-directed mutagenesis maybe used to introduce mutations that create new restriction sites, alter glycosylation patterns, change eodon 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 Clare CA; descn'bed in U.S. Patent Number 5,837,458; Chang, C.-C. et al. (1999) Nat. Biotechnol. 17:793-797; Christians, F.C. et a1 (1999) Nat.
Biotechnol. 17:259-264; and Crameri, A. et al. (1996) Nat. Biotechnol. 14:315-319) to alter or improve the biological properta.es 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 selectionlscreening. 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 l0 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., Carathers, 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 sofid-phase techniques. (See, e.g., Creighton, T. (1984) Proteins, Structures and Molecular Properties, WH
Freeman, New York NY, pp.
55-60; and Roberge, J.Y. et al. (1995) Science 269:202 204.) Automated synthesis maybe 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 EnzymoI. 182:392-42L) The composition of the synthetic peptides may be confirmed by amino acid analysis or by sequencing.
(See, e.g., Creighton, supra, 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 trauslational control of the inserted coding sequence in a suitable host. These elements include regulatory sequences, such as enhancers, constitutive and 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. 1u 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 colon should be provided by the vector. Exogenous translational elements anal initiation colons 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. (I994) 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, 3ohn Wiley & Sons, New York NY, ch. 9, I3, 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, CaM V, 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, s_ upra; Van Heeke, G. and S.M. Schuster (1989) J. Biol. Chem. 264:5503-5509; Engelhard, E.I~ et al. (1994) Proc. Nail.
Acad. Sci. USA
91:3224-3227; Sandig, V. et a1. (1996) Hum. Gene Ther. 7:1937-1945; Takamatsu, N. (1987) EMBO
J. 6:307-311; The McGraw Hill Yearbook of Science and TechnoloQy (1992) MeGraw Hill, New York NY, pp. 191-196; Logan, J. and T. Shenk (1984) Proc. Natl. Acad. Sci. USA
813655-3659; and Hatx~tngton, J.J. et aI. (1997) Nat. Genet. 15:345-355.) Expression vectors derived from retroviruses, adenoviruses, or hezpes 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. (199$) 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. 1_tnmunol. 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.
Zn 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 PBLUESCRIPT (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, supra;
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 sequer<ce from TMV (Takamatsu, N. (1987) EMBO J.
6:307-311). Alternatively, plant promoters such as the small subunit of RUBISCO or heat shock promoters maybe used. (See, e.g., Coruzzi, G. et al. (1984) EMBO J. 3:1671-16$0; Brogue, R. et a1.
(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) MeGraw 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. lusertion in a non-essential E1 or E3 region of the viral genome maybe used to obtain 3o in;fective virus which expresses PRTS in host cells. (See, e.g., Logan, J.
and T. Shenk (1984) Proc.
Nail. 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 M6 are constructed and delivered via conventional delivery methods (liposomes, polycationic amino polymers, or vesicles) for therapeutic purposes. (See, e.g., Hanrington, 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. Fox 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 Bells 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 apz' cells, respectively.
(See, e.g., Wigler, M. et al. (1977) Cell 11:223-232; Lowy, T. et al. (2980) Cell 22:817-823.) Also, antimetabolite, antibiotic, or herbicide resistance can be used as the basis for selection. For example, dlzfi- confers resistance to methotrexate; ~aeo 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. MoI. Biol. 150:1-14.) Additional selectable genes have been described, e.g., ttpB and ItisD, which alter cellular requirements for metabolites. (See, e.g., Hartman, S.C. and.RC.
Mulligan (1988) Proc.
Natl. Acad. Sci. USA 85:8047-8051.) Visible markers, e.g., authocyanins, green fluorescent proteins (GFP; Clontech), !3 glucuronidase and its substrate B-glucuronide, or lueiferase and its substraxe luciferin may be used. These markers coat 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 ex.-pression 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 lmown to those of skill int 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.
Iznmunological methods for detecting and measuring the expression of PRTS
using either specific polyclonal or monoclonal antibodies are lmown 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 Inununolo~y, Greene Pub.
Associates and Wiley-Interscience, New York NY; and Pound, J.D. (1998) Tm_m_unochenucal Protocols, Humana Press, Totowa NJ.) A wide variety of labels and conjugation techniques are Imown 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, maybe cloned into a vector for the production of an mRNA probe. Such. vectors are Icaown in the art, are commercially available, anal may be used to synthesize RNA probes in vitro by addition of an appropriate RNA polymerase such as T7, T3, ox 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 xadionuclides, enzymes, fluorescent, chemiluminescent, or chromogenic agents, as well as substrates, cofactors, inlu-bitors, 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 prok~yotic 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, phosphorylatior lipidation, and acylation. Post-translational processing which cleaves a "prepro" ox "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,1VB~CI~, I3EK293, 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.
Tn 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-transfexase (GST), maltose binding protein (MBP), thioredoxin (Txx), calmodulinbinding peptide (CBP), 6-His, FLAG, c-tnyc, and hemagglutinin (HA). GST, MBP, Txx, CBP, and 6-His enable purification of their cognate fusion proteins.on imrnobili~ed glutathione, maltose, phenylarsine oxide, calmodulin, and metal-chelate resins, respectively. FLAG, c-nzyc, 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.
Tn 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, ox SP6 promoters. Translation takes place in. the presence of a radiolabeled amino acid precursor, for example, 35S niethionine.

.. .. ,._. ~, :w.~ .~",. .. ...." :W.~:..,::" .......
PRTS of the present invention or fragments thereof may be used to screen for compounds that specifically bind to PRTS. At Ieast 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 Itnmunolo~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, Drosonhila, 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 fluorophoxe, 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 0 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.

WO 02/20736 CA 02421922 2003-03-07 PCT/USOl/28161 Iu another embodiment, polynucleotides encoding PRTS or their mammalian homologs may be "knocked out" in au animal model system using homologous recombination in embryonic stem (ES) cells. Such techniques are well kaown in the art and are useful for the generation of animal models of human disease. (See, e.g., U.S. Patent Number 5,175,383 anal U.S. Patent Number 5,767,,337.) Fox example, mouse ES calls, such as the mouse 129lSvJ cell liue, 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-IoxP 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 celtblastocysts 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 ectodertnal cell types. These cell lineages differentiate into, for example, neural cells, hematopoietic lineages, and cardiomyocytes (Thomson, J.A. et al.
{I998) Science 282:I145-1147).
Polynucleotides encoding PRTS can also be used to create '~uockin" 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., iu the conte~.-t of sequences and motifs, exists between regions of PRTS anal proteases. In addition, the expression of PRTS is closely associated with digestive, lung, neurological, gastrointestinal, cardiovascular, urinary, reproductive, fibroblastic, developmental, and endothelial tissues, and also prostate cancer and other tumorous tissue. Therefore, WO 02/20736 CA 02421922 2003-03-07 PCT/USOl/28161 PRTS appears to play a role in gastrointestinal, cardiovascular, autoinunune/inflammatory, cell prolifexative, developmental, epithelial, neurological, and reproductive disorders. In the treatment of disorders associated with increased PRTS eh-pression or activity, it is desirable to decrease the expression or activity of PRTS. In the treatment of disorders associated with decreased PRTS
S 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, l0 dyspepsia, iundigestion, 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-15 Weiss syndrome, colonic carcinoma, colonic obstruction; irritable bowel syndrome, short bowel syndrome, diarrhea, constipation, gastrointestinal hemorrhage, acquired immunodeficiency syndrome (AIDS) enteropathy, jaundice, hepatic encephalopathy, hepatorenal syndrome, hepatic steatosis, hemochromatosis, Wilson's disease, alphai antitrypsin deficiency, Reye's s5~drome, primary sclerosing cholangitis, liver infarction, portal vein obstruction and thrombosis, centrilobular necrosis, 20 peliosis hepatis, hepatic vein thrombosis, verso-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, 25 balloon angioplasty, vascular replacement, and coronary artery bypass daft 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, nutral annular calciri.cation, mural valve prolapse, rheumatic fever and rheumatic heart disease, infective endocarditis, nonbacterial thrombotic endocarditis, endocarditis of systemic lupus 30 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 spondyJitis, amyloidosis, anemia, asthma, atherosclerosis, athexosclerotic plaque rapture, autoirnmune hemolytic anemia, autoimmune A.5 thyroiditis, autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED), bronchitis, cholecystitis, contact dermatitis, Crohn's disease, atopic dermatitis, dermatomyositis, diabetes mellitus, emphysema, episoclic Iymphopenia with Iymphocytotoxit~s, erytlZroblastosis fetalis, erythema nodosum, atrophic gastritis, glomerulonephritis, Goodpasture's syndrome, gout, Graves' disease, Hashimoto's thyroiditis, hypereosinophilia, irntable bowel syndrome, multiple sclerosis, myasthenia gravis, myocardial or pericardial inflammation, osteoarthritis, degzadation of ai-ticular cartilage, osteoporosis, pancreatitis, polymyositis, psoriasis, Reiter's syndrome, xheumatoid arthritis, scleroderma, Sjogren's syndrome, systemic anaphylaxis, systemic lupus erythematosus, systemic sclerosis, thrombocytopenic purpura, ulcerative colitis, uveitis, 'V~erner syndrome, complications of cancer, hemodialysis, and extracorporeal cixculation, viral, bacterial, fungal, parasitic, protozoal, and helrniathic infections, and trauma; a cell proliferative disorder such as actinic keratosis, arteriosclerosis, atherosclerosis, bursitis, cirrhosis, hepatitis, mixed connective tissue disease (MCTD), myeIofibrosis, paroxysmal nocturnal hemoglobinuria, polyeythemia vera, psoriasis, primary throw bocythemia, 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 (Wihns' tumor, auiridia, genitourinary abnormalities, and mental retardation), Smith-Magenis syndrome, myelodysplastic syndrome, hereditary mucoepithelial dysplasia, hereditary keratodermas, hereditary neuropathies such as Charcot Marie-Tdoth disease and neurofibromatosis, hypothyroidism, hydrocephalus, seizure disorders such as Syndenham's chorea and cerebral palsy, spins bifida, auencephaIy, craniorachischisis, congenital glaucoma, cataract, age-related macular degeneration, and sensorineural hearing loss; an epithelial disorder, such as dyshidrotie eczema, allergic contact dermatitis, kexatosis pilaris, melasma, vitiligo, actinic keratosis, basal cell carcinoma, squamous cell carcinoma, sebort3~eic keratosis, folliculitis, herpes simplex, herpes zoster, varicella, candidiasis, dermatophytosis, scabies, insect bites, cherry angioma, keloid, dermatofibroma, acrochordons, urtiearia, 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, pemphib s foliaceus, paraneoplastic pemphigus, bullous pemphigoid, herpes gestationis, dermatitis herpetiformis, linear IgA disease, epidermolysis bullosa acquisita, dermatomyositis, lupus erythematosus, scleroderma and morphea, erythroderma, alopecia, figuxate skin lesions, teIangiectasias, hypopigmentation, hyperpi~nentation, vesicles/bullae, exauthems, cutaneous drug reactions;
papulonodular skin lesions, chronic non healing wounds, photosensitivity diseases, epidermolysis bullosa simplex, epidermolytic hyperkeratosis, epidermolytic and nonepidermolytic palmoplautar keratoderma, ichthyosis bullosa of Siemens, ichthyosis exfoliativa, keratosi.s 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 extxapyramidal disorders, amyotrophic lateral sclerosis and other motor neuron disorders, progressive neural muscular atrophy, xetinitis 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 fanulial insomnia, nutritional and metabolic diseases.of the nervous system, neurofibromatosis, tuberous sclerosis, cerebelloretinai hemaugioblastomatosis, encephalotrigeminaT syndrome, mental retardation and other developmental disorders of the central nervous system including l.~own 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 suprauuclear 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 disraption of the menstrual cycle, polycystic ovary syndrome, ovarian hyperstimulation syndrome, au 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 hyperplasita, prostatitis, Peyronie's disease, impotence, carcinoma of the male breast, and gynecomastia.

Iu 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 disordex 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 Iinuted 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, au 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, au antibody which specifically binds PRTS may be used directly as au antagonist or indirectly as a targeting or delivery mechanism for bringing a pharmaceutical agent to cells or tissues which express PRTS.
In au 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 descnbed 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 ~5 combination of therapeutic agents may act synergistically to effect the treatment or prevention of the various disorders described above. Using this approach, one maybe 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 auti'bodies 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 lmown in the art. Such antibodies may include, but are not limited to, poIyclonal, 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.
4s 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, polyanious, peptides, oil emulsions, KI T3, and dinitrophenol. Among adjuvauts used in humans, BCG
(bacilli Calmette-Guerin) and Cor~nebacterium uarvum 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 S amino acids, and genexally 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 Eased with those of another protein, such as I~T H- 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.
T-mmunol. Methods 81:31-42; Cote, R.J. et al. (1983) Proc. Nail. Acid. Sci.
USA 80:2026-2030; and Cole, S.P. et a1. (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, canbe used. (See, e.g., Morrison, S.L. et al. (1984) Proc.
Natl. Acid. Sci. USA 81:6851-6855; Neuberger, M.S. et al. (I984) 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 h'braries. (See,.e.g., Burton, D.R. (1991) Proc. Natl. Acid. 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 a1. (1989) Proc. Natl. Acid. Sci.
USA 86;3833-3837; Winter, G. et al. (1991) Nature 349:293-299.) Aufbody 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 aI. (1989) Science 246:1275-1281.) Various immunoassays maybe used for screening to identify antibodies having the desired specificity. Numerous 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 formationbetween 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 I~
ranging from about 109 to 101a.Llmole are preferred for use in immunoassays in which the PRTS-antibody complex nmst withstand rigorous manipulations. Low-affinity antibody preparations with K, ranging from about 106 to 10' L/mole are preferred for use in imulunopurification and similar procedures which ultimately require dissociation of PRTS, preferably in active form, from the antibody (Catty, D. (1988) Antibodies, Volume I: A Practical Approach, IRI. 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 anixbody 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 iti 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, s. upra, and Coligan et a1. suura.) 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 xegions of sequences encoding PRTS. (See, e.9., Agrawal, S., ed. (1996) Antisense Therapeutics, Humaua Press Tnc., Totawa NJ.) In therapeutic use, any gene delivery system suitable for introduction of the antisense sequences itzto appropriate target cells can be used. Antisense sequences can be delivered intracellularly in the form of au expression plasmid which, upon transcription, produces a sequence complementary to at least a portion of the cellular sequence encoding the target protein. (See, e.9., Slater, J.B. et al. (1998) J. Allergy Clip. Immunol.102(3):469-4.75; and Scaulon, K.J. et aI. (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.9., Miller. A.D. (1990) Blood 76:271; Ausubel, supra; Uckert, W. and W. Walther (1994) Pharmacol.. Ther.
63(3):323-347.) Other gene delivery nnechanisms include liposome-derived systems, artificial viral envelopes, and other systems known in the art. (See, e.9., Rossi, J.T. (1995) Br. Med. Bull.
51(1):217-225; Boado, R.J. et aI. (1998) J. Pharm. Sci. 87(11):1308-1315; and Morris, M.C. et aI. (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.9., in the cases of severe combined immunodehciency (SCll~)-X1 disease characterized by X-liuked inheritance (Cavazzaua-Calvo, M. et al. (2000) Science 288:669-672), severe combined immunodeficiency syndrome associated with au 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 aI. (T995) Hum. Gene Therapy 6:667-703), thaIassamias, 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.9., in the case of cancers which result from unregulated cell proliferation), or (iii) express a pxotein. which affords protection against intracellular parasites (e.9., 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 Caudida 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 farther 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, EP1TAG, PRCCMV2, PREP, PVAX, PCR2-TOPOTA vectors (Invitrogen, Carlsbad CA), PCMV-SCRIPT, PCMV-TAG, PEGSH/i'ERV (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.
2o 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. 9:452-456), commercially available in the T-REX
plasmid (Invitrogen));
the ecdysone-inducible promoter (available in the plasmids PVGRXR and PIND;
Invitrogen); the FK506/rapamycin inducible promoter; or the RU486/mifepristone induci'ble promoter (Rossi, F.M.V.
and Blau, H.M. supra)), or (iii) a tissue-specific promoter or the native promoter of the endogenous gene encoding PRTS from a normal indi~~idual.
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 miuimal effort to optimize experimental parameters. Iu the alternative, transformation is performed using the calcium phosphate method (Graham, F.L. and A.J. Eb (1973) Virology 52:456-9.67), or by electroporation (Neumann, E. et a1 (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 eis-acting RNA sequences and coding sequences required for efficient vector propagation. Retrovirus vectors (e.g., PFB and PFBNEO) axe 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 a1.
(1987) J. Vitol. 61:1647-1650; Bender, M.A. et al. (1987) J. Virol. 61:1639-1646; Adam, M.A. and to 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 Number 5,910,434 to Rigg ("Method for obtaining.
retravirus packaging cell lines producing high transducing efficiency retroviral supernatant") discloses a method for obtaining retrovirus packaging cell Iines 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 axe procedures well known to persons skilled in the art of gene therapy and have been well documented (Ranga, U. et a1. (1997) J. Virol.
71:7020-7029; Bauer, G. et al. (1997) Blood 89:2259-2267; Bonyhadi, M.L. (1997) J. Virol. 71:4707-4.716;
Ranga, U. et al. (1998) Proc. Natl. Acad. Sci. USA 95:1201-1206; Su,~L. (I997) Blood 89:2283-2290).
In the alternative, au 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 lmown 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). Potentiallyuseful adenoviral vectors are described inU.S. Patent Number 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) Aunu. 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 (IiSV)-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 att. 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 vitas vectorhas also been disclosed in detail in U.S.
Patent Number 5,804,413 o DeLuca ("Herpes simplex virus strains for gene transfer"), which is hereby incorporated by reference. U.S. Patent Number 5,804,413 teaches the use of recombinant ~5 HSV d92 which consists of a genome containing at least one exogenous gene to be trausfezxed to a cell under the control of the appropriate promoter for puzposes 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 herpesvirus 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 lmown to those of ordinary skill in the art. .
In another alternative, au alphavirus (positive, single-stranded RNA virus}
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. T. Li (1998) G~rr. 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 trausduced 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 (SIIL~ indicates that the lytic replication of alphaviuuses 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 fox the binding of polymerises, transcription factors, or regulatory molecules. Recent therapeutic advances using triplex DNA have been described in the literature. (See, e.g., Gee, 3.E. et al. (1994) in Huber, B.E. and B.I. Carr, Molecular and Imn~unolo~ic Approaches, Futura Publishing, Mt. Kisco NY, pp.163-177.) A
complementary sequence or autisense molecule may also be designed to block translation of mRNA
by preventing the transcript from binding to ribosomes.
Riboz~mes, 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 endonueleolytic cleavage.
For example, engineered hauunerhead 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, GULT, 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 lmown in the art fox 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 polymerise promoters such as T7 or SP6. Alternatively, these cDNA constricts that synthesize complementary RNA, constitutively or inducibly, canbe introduced into cell Iines, 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 S' and/or 3' ends of the molecule, or the use of phosphorothioate ox 2' O-methyl rather than phosphodiesterase linkages within the backbone of the molecule. This concept is inherent in the production of PNAs and can be extended in all of these molecules by the inclusion of nontraditional bases such as inosine, queosine, and wybutosine, as well as acetyl-, methyl-, thio_, arid 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 expxession of a specific polynucleotide may include, but are not limited to, oligonucleotides, antisense oligonucleotides, triple helix-forming oligonucleotides, transcription factors and other polypeptide tr~scriptional regulators, and non-macromolecular chemical entities which are capable of interacting with specific polynucleotide sequences. Effective compounds may alter polynucleotide expression by acting as eithex 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 maybe 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 lmown to be effective in altering polynucleotide expxession; 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 polynueleotide 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 polynueleotide coin be carxied out, for example, using a. Schizosaccharomyces bombe 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 deoxyrt~bonucleotides, nbonucleotides, peptide nucleic acids, and modified oligonucleotides) for antisense activity against a specific polynucleotide sequence (Bruice, T.W et al.
(2997) U.S. Patent No. 5,686,24.2; Bruiee, T.W. et a1. (2000) U.S. Patent No.
6,022,69x).
Many methods for introducing vectors into cells or tissues are available and equally suitable for use in: viyo, in vitro, and ex vivo. For ex vivo therapy, vectors may be introduced into stem cells taken from the patient and clonally propagated far 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. 15:462-4.66.) 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 Remington's Pharmaceutical Sciences (Maack Publishing, Easton P.A.). 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, intra-arterial, intramedullary, intrathecal, intraventricular, pulmonary, trausdermal, subcutaneous, intraperztoneal, 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 patie~. In the case of small molecules (e.g. traditional low molecular weight organic drugs), aerosol delivery of fast-acting formulations is well lmown in the art. Iu the case of macromolecules (e.g. larger peptides and proteins), recent developments in the field of pulinonaty delivery via the alveolar region of the lung have enabled the practical delivery of drags 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 for 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 shoat cationic N-terminal portion from the HIV Tat-I 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 285:1569-2572).
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 deternlined by standard pharmaceutical procedures in cell cultures or with experimental animals, such as by 25 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, drag combination(s), reaction sensitivities, and xesponse to therapy. Long-acting compositions maybe administered every 3 to 4 days, every week, or biweekly depending on the half life and clearance rate of the partitcular formulation.
Normal dosage amounts may vary from about 0.1 pg 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 fox 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 S diagnosis of disorders characterized by expression of PRTS, or in assays to monitor patients being treated with PRTS 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 20 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 he 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 15 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 PRTS
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.
20 In. another embodiment of the invention, the polynucleotides encoding PRTS
maybe 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 25 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 maybe 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 S'reb~ulatory region, or from a less specific region, e.g., a 30 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
N0:18-34. or from genomic sequences including promoters, enhancers, and iutrons 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 mown in the art, are commercially available, and may be used to synthesize RNA probes in vitro by rneaus of the addition of the appropriate RNA
polyn~exases and the appropxiate labeled nucleotides. Hybridization probes may be labeled by a variety of reporter groups, for example, by radionuclides such as 3aP 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, autral or pyloric edema, abdominal angina, pyrosis, gastroenteritis, intestinal obstruction, infections of the intestinal tract, peptic ulcer, cholelithiasis, cholecystitis, cholestasis, pancreatatis, pancreatic carcinoma, biliary tract disease, hepatitis, hyperbilixubinemia, cirrhosis, passive congestion of the liver, hepatoma, infectious colitis, ulcerative colitis, ulcerative proctitis, Crohn's disease, Whipple's disease, Mallory-Weiss syndrome, colonic carcinoma, eolonic obstruction, irritable bowel syndrome, short bowel syndrome, diarrhea, constipation, gastrointestinal hemorrhage, acquired itnmunodeficiency syndrome (AIDS enteropathy, jaundice, hepatic encephalopathy, hepatorenal syndrome, hepatic steatosis, hemochromatosis; Wilson's disease, alphas 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 choIestasis. of pregnancy, and hepatic tamors includiung 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 augioplasty, 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, nutral annular calcification, :mitral valve prolapse, rheumatic fever and rheumatic heart disease, infective endocarditis, nonbacterial thrombotic endocarditis, endocarditis of systemic Iupus erythematosus, carcinoid heart disease, cardiomyopathy, myocarditis, pericarditis, neoplastic heart disease, congenitalheart disease, and complications of cardiac transplantation; an autoimmune/irJflammatory disorder, such as acquired immunodeficiency syndrome (AIDS), Addison's disease, adult respiratory distress syndrome, aller~tes, ankylosing spondylitis, amyloidosis, anemia, asthma, atherosclerosis; atherosclerotic plaque rupture, autoimmune hemolytic anemia, autoimmune thyroiditis, autoimmune polyendocrinopathy canclidiasis-ectodermal dystrophy (APECED), bronchitis, cholecystitis, contact dermatitis, Crohn's disease, atopic dermatitis, dermatomyositis, diabetes mellitus, emphysema, episodic Iymphopenia 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, paucreatitis, polymyositis, psoriasis, Reiter's syndrome, rheumatoid arthritis, scleroderma, Sjogren's syndrome, systemic anaphylaxis, systemic Iupus 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, myeioma, 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 xenal tubular acidosis anemia, Cushing's syndrome, aehondroplastic dwarfism, Duchenne and Becker muscular dystrophy, bone resorption, epilepsy, gonadal dysgenesis, WAGR
syndrome (Wihns' tumor, aniridia, genitourinary abnormaIities~ and mental retardation), Smith-Magenis syndrome, myelodysplastic syndrome, hereditary mucoepithelial dysplasia, hereditarykeratodermas, hereditary neuropathies such as Charcot-Maria-Tooth disease and neuxofibromatosis, hypothyroidism, hydrocephalus, seizure disorders such as Syndenham's chorea and cerebral palsy, spina bifida, auencephaly, craniorachischisis, congenital glaucoma, cataract, age xelated macular degeneration, and sensorineural hearing loss; au 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 dern3atosis, xerosis, eczema, atopic dermatitis, contact dermatitis, hand eczema, nummular eczema, lichen simplex chronicus, asteatotic eczema, stasis dermatitis and stasis ulceration, seborrheic dermatitis, psoriasis, lichen planes, pityriasis rosea, impetigo, ecthyma, dermatophytosis, tinea versicolor, warts, acne vulgaris, acne rosacea, pemphigus vulgaxis, pemphigus foliaceus, paraneoplastac pemphigus, bullous pemphigoid, herpes gestationis, dermatitis herpetiformis, linear TgA disease, epidermolysis bullosa acquisita, derxnatomyositis, lupus erythematosus, scleroderma and morphea, erythroderma, alopecia, figurate skin lesions, telaugiectasias, hypopigmentation, hyperpigmentation, vesicles/bullae, exantherns, cutaneous drug reactions, papulonodular skin lesiops, 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 plautaris, keratosis palinoplantaris, palmvplantar !a keratoderma, keratosis punctata, Meesmann's corneal dystrophy, pachyonychia congenita, white sponge nevus, steatocystoma multiplex, epidermal neviYepidermolytic hyperkeratosis type, monilethrix, trichotbiodystrophy, 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 extrapyranudal 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, priors diseases including l.'unt, Creutzfeldt-Jakob disease, and Gerstmann-Straussler-Scheinker syndrome, fatal fan5i1ia1 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 tnbal 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, ovarianhyperstimulation syndrome, an endometrial or ovarian tumor, a uterine fibroid, autoimmune disorders, an ectopic pregnancy, and teratogenesis;
cancer of the bre~t, fibrocystic breast disease, and galactorrhea; a disruption of spermatogenesis, abnormal sperv~. 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 maybe used in Southern or northern analysis, dotblot, 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 l0 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 fox expression is established. This may be accomplished by combining body fluids or cell extracts taken from narmal 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 ox condition. Oligomers may also be employed under Iess 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 frag~aents which assemble znto 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. Imrriunol. Methods 159:235 244; Duplaa, C.
et aI. (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 maybe 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 ants'bodies specific for PRTS may be used as elements on a nlicroarray. The microarray may be used to monitor or measure protein protein 25 interactions, drug-target interactions, and-gene expressionpro~les, 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 Number 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 Iines, biopsies, or other biological samples. The transcript image maythus 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-occurrjng 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. Caxcinog. 24:153-159; Steiner, S. and N.L.
Anderson (2000) Toxicol. Lett. 1.22-113:467--X1.71, 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 laxge numbex 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 eh-pression 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.xu'h.gov/oc/news/toxchip.htm.) Therefore, it is important and desirable in toxicological screening using toxicant signatuxes to include all expressed 15. 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 polynueleotides 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 quantifyi~ag 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, su ra), The proteins are visualized in the gel as discrete anal 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 chenucal or enzymatic cleavage followed by mass spectrometry. The identity of the protein in a spot may be determined by comparing its paxtial 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.
l0 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 saW ple and detecting the levels of protein bound to each array element (Lueking, A. et aI. (1999) Anal: Biochem. 270:103-111; Mendoze, L.G. et al. (1999) Biotechniques 27:778-788). Detection maybe performed by a variety of methods lmown 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 i~Iuids is difficult, due to rapid degradation of mRNA, so pxoteomic 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. Pxoteins 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 reco~ized 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.
Microanays may be prepared, used, and analyzed using methods known in the art.
(See, e.g., Brennau, T.M. et a1. (1995) U.S. Patent No. 5,474,796; Schena, M. et al.
(1996) Proc. Natl. Aced.
Sci. USA 93:10614-10619; Baldesehweiler et al. (1995) PCT application W095/251116; Shalon, D. et al. (1995) PCT application W095135505; Heller, R.A. et aI. (1997) Proc. Natl.
Aced. 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 Univexsity 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 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 multi-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., Harrington, 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 orrestriction fragment length polymorphism. (RFLP).
(See, far example, Lender, E.S. and D. Botstein (1986) Proc. Natl. Aced. Sci.
USA 83:7353-7357.) Fluorescent itz situ hybridization (FISH) may be correlated with other physical and genetic map data. (See, e.g., Heinz-Ulrich, et al. (1995) in Meyers, ~, pp. 965-968.) Examples of genetic map data can be found in various scientific journals or at the Online Mendelian Inheritance in Man (OM11VI) 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 farther positional cloning efforts.
In 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 mammalian 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 reb latory genes for further investigation.
(See, e.g., Gatti, R.A. et aI. (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 fra~ient 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 af~uity to the protein. of interest. (See, e.g., Geysers, et al. (1984) PCT
application W084103564.) 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 thus 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 are currently Ioaown, including, but not limited to, such properties as the triplet genetic code and specifte base pair interactions.
Without fuzfiher 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 Iimitative 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. 5er. No. 60!231,039, U.S. Ser. No. 60!232,812, U.S. Ser. No.
60!234,850, U.S. Ser.
No. 60/236,500, U.S. Ser. No. 601238,773, and U.S. Ser. No. 60/239,658, are hereby expressly incorporated by reference.
EXAMPLES
I. Construction of cDNA Libraries Incyte cDNAs were derived from cDNA libraries described in the LTFESEQ GOLD
database (Iucyte Genomics, Palo Alto CA) and shown in Table 4, column 5. Some tissues were homogenized and lysed in b anidinium isothiocyanate, while others were homogenized and lysed in phenol or in a suitable mixture of denaturants, such as TRI20L (Life Technologies), a monophasic solution of phenol and guanidine isothiocyanate. The resulting lysates were centrifuged over CsCl 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 Iysates using other RNA isolation kits, e.g., the POLY(A)PURE mRNA purification kit (Ambion, Austin TX). .
Iu some cases, Stratagene was provided with RNA and constructed the corresponding cDNA
libraries. Otherwise, eDNA 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, supra, 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 IZ'braries, the cDNA was size-selected (300-1000 bp) using SEPHACRYL 51000, SEPHAROSE CL2B, or SEPI3.~AROSE CL4B column chromatography (Amersham Pharmacia Biotech) or preparative agarose geI
electrophoresis. cDNAs were ligated into compatible restriction enzyme sites of the polylinker of a suitable plasnud, e.g., PBLUESCRIPT plasmid (Stratagene), PSPORT1 plasmid (Life Technologies), PCDNA2.1 plasmid (Invitrogen, Carlsbad CA), PBK-CMV plasmid (Stratagene), PCR2-TOPOTA
(Invitrogen), PCMV-ICIS (Stratagene), or pINCY (Iucyte Genomics, Palo Alto CA), or derivatives thereof. Recombinant plasmids were transformed into competent E. coli cells including XL1 Blue, XL1 BlueMRF, or SOLR
from Stratagene or DH5a, DH10B, or ElectroMA~~ DH10B 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 Mniprep purification kit (Edge Biosystems, Gaithersburg MD); and QIAWELL

8 Plasmid, QIA.WELL 8 Plus Plasniid, QIAWELL 8 Ultra PIasmid purification systems or the R.E.A.L. PREP
96 plasnud purification kit from QIAGEN. Following precipitation, plasnuds wexe resuspended in 0.1 1o ml of distilled water and stored, with or without lyophilization, at 4°C.
Alternatively, plasmid DNA was amplified from host cell lysates using direct Iink 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 It fluorescence scanner (Labsystems Oy, Helsinki, Finland).
III. Sequencing and Analysis Iucyte cDNA recovered in plasmids as described in Example II 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 (Bobbins Scientific) or the MICROLAB 2200 (Hamilton) liquid transfer system. eDNA sequencing reactions were prepared using reagents provided by Amersham Pharmacia Biotech or supplied in ABI
sequencing kits such. as the ABI FRISM 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 Dynarsiics); the ABI PRTSM 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, s-uyra, unit 7.7). Some of the cDNA sequences were selected for extension using the techniques disclosed in Example VIII.
The poIynucleotide 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, and hidden Markov model (I~1~1M)-based protein family databases such as PFAM. (HNCM 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, BLM'S, and HMMER.
The Incyte eDNA 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 '~ 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 metbionine 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, BLOCKS, PRINTS, DOMO, PRODOM, Prosite, and hidden Markov model (FllVIM) based protein family databases such as PFAM. Full Ient,~th polynucleotide sequences are also analyzed using MACDNASIS PRO software (Iiitachi Software Engineering, South San Francisco CA) and LASERGENE software (DNASTAR). Polynucleotide and polypeptide sequence aliments 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 fra~nents from SEQ 1D
N0:18-34. Fragments from about 20 to about 4000 nucleotides which are useful inhybridization 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). Genscau 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) Curx.
Opin. Struct. Biol. 8:346-354). The program concatenates predicted exons to form au 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 fox 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 Genscau-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 ox omitted exons. BLAST
analysis was also used to find any Incyte cDNA or public cDNA coverage of the Genscan-predicted sequences, thus providing evidence for transcription. When Iucyte cDNA
coverage was available, this information was used to correct or conftrm the Genscan predicted sequence. Full length polynucleotide sequences were obtained by assembling Genscan-predicted coding sequences with lncyte cDNA sequences audlor public cDNA sequences using the assembly process described in Example )a. 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 Genscau gene identification program described in Example N. Partial cDNAs assembled as described zn Example III were mapped to genomic DNA and parsed into clusters containing related eDNAs and Genscan exon predictions from one or more genomic sequences. Each cluster was analyzed using an algorithm based on graph theory and dynamic programaning 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 genornic 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 B~,AST hit from genpept. Sequences were farther extended with additional cDNA sequences, or by inspection of genomic DNA, when necessary.
"Stretched" Sequences Partial DNA sequences were extended to fall 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 IV. A chimeric protein was generated by using the resultant high-scoring segment pairs (TISPs) to map the translated sequences onto the GenBank protein homolog.
Insertions or deletions may occur in the chinieric 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 PoIynucIeotides The sequences which were used to assemble SEQ ID N0:18-34 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 ll~ NO:18-34 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 pa~icular SEQ ID NO:, to that map location.
Map locations are represented by ranges, or intervals, of human chromosomes.
The map position of an interval, in centiMorgaus, is measured relative to the terminus of the chromosome's p-arnl. (The centilVlorgan (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.nhu.nih.gov/genemapn, can be employed to determine if previously identified disease genes map within or in proximity to the intervals indicated above.
In this manner, SEQ Il? N0:18 was mapped to chromosome 16 within the interval from 33.4 1o to 42.7 centiMorgans. In this manner, SEQ 7D N0:22 was mapped to chromosome 1 within the interval from 219.2 to 222.7 centiMorgans.
VII. Analysis of Polynucleotide Expression Northern analysis is a laboratory technique used to detect the pxesence 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 i6.) Analogous computer techniques applying BLAST were used to search fox 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 detexmine 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 5 x minimum {length(Seq.1), Iength(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 moxe than one HSP
(separated by gaps). If there is more than one IiSP, 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 III). 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; heroic and immune system; liver; musculoskeletal system;
nervous system;
pancreas; respiratory system; sense organs; skin; stomatognathic system;
unclassified/mixed; or urinary tract. The number of h'braries 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 hbraries 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 LIFESEQ GOLD database (Ineyte Genomics, Palo Alto CA).
2o VIII. Extension of PRTS Encoding Polynucleotides Full length polynucleotide sequences were also produced by extension of an appropriate fra~nent of the full length molecule using oligonucleotide primers designed from this fragment. One primer was synthesized to initiate 5' extension of the Imown fragment, and the other primer was synthesized to initiate 3' extension of the lmown 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 an~.plification 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, Iuc.). The reaction mix contained DNA template, 200 nmol of each primer, reaction buffer containing lVlgz~, (NH~)zS04, and 2-mercaptoethanol, Taq DNA polymerase (Amersham Pharmacia Biotech), ELONGASE

enzyme (Life Technologies), and Pfu DNA poiymerase (Stratagene), with the following parameters for primer pair PCI A and PCI B: Step 1: 94 °C, 3 min; Step 2: 94 °C,15 sec; Step 3: 60 °C,1 min;
Step 4: 68°G, 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 SI~+
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 niiu; 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 determiued by dispensing 100 ~tl PICOGREEN
quantitation reagent (0.25 % (v/v) PICOGREEN; Molecular Probes, Eugene OR) dissolved in 1~ TE
and 0.5 ~.d 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 ~l to 10 ~xl aliquot of the reaction mixture was aualyzed 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 relegation into pUC 18 vector (Amersham Pharmacia Biotech). For shotgun sequencing, the digested nucleotide's were separated on Iow concentration (0.6 to 0.8%) agarose gels, fragments were excised, and agar digested with Agar ACE
(Promega). Extended clones were relegated using T4 Iigase (New Englaud Biolabs, Beverly MA) into pUC 18 vector (Amersham Pharmacia Biotech), treated with Pfu DNA polymerase (Stratagene) to fill-in restriction site overhaugs, 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 curb 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 Tex~ninator cycle sequencing ready reaction. kit (Applied Biosystems).

Iu 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 oligonucIeotides designed for such extension, and an appropriate genomic library.
IX. Labeling and Use of Tndividual Hybridization Probes Hybridization probes derived from SEQ T17 N0:18-34 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 fra~nents. 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 ~xCi of [y-32P] adenosine triphosphate (Amersham Pharmacia Biotech), and T4 polynucleotide kinase (DuPont NEN, Boston MA). The labeled ohigonucleotides 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 IF, Eco RI, Pst I, Xba l, or Pvu II (DuPont NEN).
The DNA from each digest is fractionated on a 0.7% agarose gel and transferred to nylon membranes (Nytxan Plus, Schleicher & Schuell, Durham NH). Hybridization is carried out for 16 hours at 40 °C. To remove nonspec'h"xc signals, blots are sequentially washed at room temperature under. conditions of up to, for example, 0.1 x saline sodium citrate and 0.5%o 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 microspotting technologies, and derivatives thereof. The substrate in each of the aforementioned technologies should be uniform and solid with a non porous surface (Schena (1999), su ra .
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, W, chemical, or mechanical bonding procedures. A
typical array may be produced using available methods and fnaclvnes 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; Shalon, D. et al. (1996) Genome Res. 6:639-645; Marshall, A. and J. Hodgson (1998) Nat. Bioteclmol. 16:27-31.) 7~

Full length cDNAs, Expressed Sequence Tags (SSTs), or frab~ents oroligomers thereof may comprise the elements of the microazray. Fragments or oligomers suitable for hybridization can be selected using software well lnlown 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 are 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 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 Gell 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/~Cl oligo-(dT) primer (2lmer), 1X first strand buffer, 0.03 unitslpl RNase inhibitor, 500 ,uM dATP, 500 ~tM dGTP, 500 p,M dTTP, 40 p,M
dCTP, 40 ~tM dCTP-Cy3 (BDS) or dCTP-Cy5 (Amersham Pharm.acia Biotech). The reverse transcription reaction is performed in a 25 ml volume containing 200 ng poly(A)* RNA with GEMBRIGHT kits (Iucyte). 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 Gy5 labeling) is treated with 2.5 ml of 0.5M sodium hydroxide and incubated fox 20 minutes at 85° C to the stop the reaction and degrade the RNA. Samples are purified using two successive CHROMA SPIN 30 geI filtration spin columns (CLONTECHLaboratories, lnc.
(CLONTECH), Palo Alto CA) and after combining, both reaction samples are ethanol precipitated using 1 ml of glycogen (1 mglml), 60 ml sodium acetate, and 300 ml of 100%
ethanol. The sample is then dried to completion using a SpeedVAC (Savant Instruments Iuc., Holbrook N~ and resuspended in 14 j.~l 5X 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 ~Cg.
Amplified array elements are then purified using SEPHACRYL-400 (Amersham.
Pharmacia Biotech).

Purified array elements are inunobilized 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 (V~WR
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. US
Patent No. 5,807,522, incorporated herein by reference. 1 ~ul 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 ~0 apparatus. The apparatus then deposits about 5 n1 of array element sample per slide.
Microarrays are UV-crosslinked using a STRATR W-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
followedby washes in 0.2%
SDS and distilled water as before.
Hybridization Hybridization reactions contain 9 ~tl 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 anal covered with an 1.8 ~ cma 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 ,u1 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 (IX 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 um for excitation of Cy3 and at 632 um 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 ill 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 um for Cy3 and 650 um. 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 au 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 ran~.ng from blue (low signal} to red (high signal). The data is also analyzed quantitatively Where two different ftuorophores are excited and measured simultaneously, the data are first corrected for optical crosstalk.(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).
~~I. 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 frag-tnents. Appropriate oligonucleotides are designed using OLIGO
4.06 software (National Biosciences) and the coding sequence of PRTS. To inhiibit 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. Fox expression of PRTS inbacteria, cDNA is subcioned into an appropriate vector containing an autl'biotic resistance gene and an inducbIe 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 isopxopyl beta-D-thiogalactopyranoside (1PTG). Expression of PRTS in eukaryotic cells is achieved by infecting insect or mammalian cell lines with recombinant Auto~raphica californica nuclear polyhedrosis virus (AcMNPV), commonly known 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 fruuiperda (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. (2994) Proc.
Natl. Acad. Sci.'USA 91:3224-3227; Sandig, V. et al. (1996) Hum. Gene Ther.
7:1937-1945.) 1u 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 iaponicum, enables the purification of fusion proteins on immobilized glutathione under conditions that maintain protein activity and antigenicity (Amersham Pharmacia Biotech). Following pun~cation, the GST .moiety can be proteolytically cleaved from PRTS at specifically engineered sites. FLAG, au 8-amino acid peptide, enables immunoaffiuitypuxification using commercially available monoclonal and poIyclonal anti-FLAG antibodies (Eastman Kodak). 6-His, a stretch of six consecutive histidine residues, enables purif cation on metal-chelate resins (~IAGEN). Methods for protein expression and purification are discussed in Ausubel (1995, s. upra, ch. 10 and 16). Purified PRTS obtained by these methods can be used directly in the assays shown in Examples XVI, XVII, XVJII, and XIX where applicable.
XIII. Functional Assays 3Q ~ PRTS function is assessed by expressing the sequences encoding PRTS at physiologically elevated levels in mammalian cell culture systems. cDNA is subcloned into a man~nnaliau expression vector containing a strong pxomoter 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 ~.tg of recombinant vector are transiently transfected into a human cell line, fox example, an endothelial or hematopoietic cell line, using either liposome formulations or electroporation. 1-2 ~,tg of an additional plasmid containing sequences encoding a marker protein are co-transfected. Expression of a marker protein provides a mesas to distinguish transfected cells from nontrausfected cells and is a reliable predictor of eDNA 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 cytometzy (FCM), au 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 iu flout 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 trausfected 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 puribed using polyacrylamide gel electrophoresis (PAGE;
see, e.g., Harxington, M.G. (1990) Methods Enzymol. 18:488-4.95), or other purification teahuiques, is used to imrilulltZe 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 hydrophi3ic regions are well described in the art. (See, e.g., Ausubel,1995, supra, ch. 11.) Typically, oligopeptides of about T5 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-hydxoxysuccinixnide ester (MBS) to increase immunogenicity: (See, e.g., Ausubel,1995, su xa.) Rabbits are immunized with the . oligopeptide-KLH complex in complete Freund's adjuvant. Resulting antisera are tested fox 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 Io Naturally occurring or recombinant PRTS is substantially purified by immunoaffinity - chromatography using antibodies specific for PRTS. An immunoaffinity column is constructed by covalently coupling anti-PRTS antibody to an activated chromatographic resin, such as CNBr-activated SEPHA.ROSE (Amersham Fharmacia 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 disrapt 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.
2o XVI. Identification of Molecules Which Interact with PRTS
PRTS, or biologically active fragments thereof, are labeled with ~I 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 multi-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 axe 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 twohybrid system as descn'bed 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 r.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, pxocollagen 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 increase/decrease in absorbance of the chromogen released during hydrolysis of the peptide substrate is measured: The change in absorbance 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 BFP5 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 (Mitra, 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 canbe 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 ICI in a hlamentous phage library. Gene DI codes for a coat protein, and the epitope will be displayed on the surface of each phage pa~cle. The h'brary is incubated with PRTS under ss t proteolytac 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 au 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 WI) or yeast cells (pYD1 yeast display vector kit, Tnvitrogen, Carlsbad CA). In this case, entire cDNAs are fused between Gene la and the appropriate epitope.
n i n_. Identification of PRTS Inhibitors Compounds to be tested are arrayed in the wells of a multi-well plate in varying concentrations along with an appropriate buffer and substrate, as described in the assays in Example XVB. PRTS activity is measured for each well and the ability of each compound to inhibit PRTS
activity canbe 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, multi-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 are tested for PRTS inhibitory activity using an assay described in Example XV11.
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.
Iudeed; 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.

WO 02/20736 CA 02421922 2003-03-07 PCT/USOl/28161 A

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I ~l 2 <110> INCYTE GENOMICS, INC.
TODD, Stephen DELEGEANE, Angelo M.
GANDHI, Ameena R.
NGUYEN, Danniel B.
HAFALIA, April J.A.
KEARNEY, Liam LU, Yan LEE, Ernestine A.
WALIA, Narinder K.
DAS, Debopriya PATTERSON, Chandra YAO, Monique G.
KALLICK, Deborah A.
ELLIOTT, Vicki S.
DING, Li YUE, Henry REDDY, Roopa BURFORD, Neil BAUGHN, Mariah R.
LAL, Preeti BOROWSKY, Mark L.
LU, Dyung Aina M.
RAMKUMAR, Jayala~ani YANG, Junming TRTBOULEY, Catherine M.
KHAN, Farrah A.
GURURAJAN, Rajagopal TANG, Y. Tom AU-YOUNG, Janice WARREN, Bridget A.
HERNANDEZ, Roberto DUGGAN, Brendan M.
<120> PROTEASES
<130> PI-0212 PCT
<140> To Be Assigned <141> Herewith <150> 60/231,039; 60/232,812; 60/234,850; 60/236,500; 60/238,773; 60/239,658 <151> 2000-09-08; 2000-09-15; 2000-09-22; 2000-09-29; 2000-10-05; 2000-10-11 <160> 34 <170> PERL Program <210> 1 <211> 333 <212> PRT
<213> Homo sapiens <220>
<221> misc feature <223> Incyte ID No: 6930294CD1 Ser Ala Thr Leu Thr Phe Asp His Ser Leu Glu Ala Gln Trp Thr Lys Trp Lys Ala Met His Asn Arg Leu Tyr Gly Met Asn Glu Glu Gly Trp Arg Arg Ala Val Trp Glu Lys Asn Met Lys Met Ile Glu Leu His Asn Gln Glu Tyr Arg Glu Gly Lys His Ser Phe Thr Met Ala Met Asn Ala Phe Gly Asp Met Thr Ser Glu Glu Phe Arg Gln Val Met Asn Gly Phe Gln Asn Arg Lys Pro Arg Lys Gly Lys Val Phe Gln Glu Pro Leu Phe Tyr Glu Ala Pro Arg Ser Val Asp Trp Arg Glu Lys Gly Tyr Val Thr Pro Val Lys Asn Gln Gly Gln Cys Gly Ser Cys Trp A1a Phe Ser Ala Thr Gly Ala Leu Glu Gly Gln Met Phe Arg Lys Thr Gly Arg Leu Ile Ser Leu Ser Glu Gln Asn Leu Val Asp Cys Ser Gly Pro Gln Gly Asn Glu Gly Cys Asn Gly Gly Leu Met Asp Tyr Ala Phe Gln Tyr Val Gln Asp Thr Gly Gly Leu Asp Ser Glu Glu Ser Tyr Pro Tyr Glu Ala Thr Glu Glu Ser Cys Arg Tyr Asn Pro Lys Tyr Ser Ala Ala Asn Asp Thr Gly Phe Val Asp Ile Pro Ser Gln Glu Lys Asp Leu Ala Lys Ala Va1 Ala Thr Val Gly Pro Ile Ser Val Ala Ala Gly Ala Ser His Val Ser Phe Gln Phe Tyr Lys Lys Gly Ile Tyr Phe Glu Pro Arg Cys Asp Pro Glu Gly Leu Asp His Ala Met Leu Leu Val Gly Tyr Ser Tyr Glu Gly Ala Asp Ser Asp Asn Asn Lys Tyr Trp Leu Val Lys Asn Arg Tyr Gly Lys Asn Trp Gly Met Asp Gly Tyr Ile Lys Met Ala Lys Asp Gln Arg Asn Asn Cys Gly Ile Ala Thr Ala Ala Ser Tyr 320 ' 325 330 Pro Thr Val <210> 2 <211> 90 <212> PRT
<213> Homo sapiens <220>
<221> misc_feature <223> Incyte ID No: 7473018CD1 <400> 2 Met Ala Asp Gln Leu Leu Arg Lys Lys Arg Arg Ile Phe Ile His Ser Val Gly Ala Gly Thr Ile Asn Ala Leu Leu Asp Cys Leu Leu Glu Asp Glu Val Ile Ser Gln Glu Asp Met Asn Lys Val Arg Asp Glu Asn Asp Thr Val Met Asp Lys Ala Arg Val Leu Ile Asp Leu Val Thr Gly Lys Gly Pro Lys Ser Cys Cys Lys Phe Ile Lys His Leu Cys Glu Glu Asp Pro Gln Leu Ala Ser Lys Met Gly Leu His <210> 3 <211> 605 <212> PRT
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte ID No: 7479221CD2 <400> 3 Met Ser Gln Leu Ser Ser Thr Leu Lys Arg Tyr Thr Glu Ser Ala Arg Tyr Thr Asp Ala His Tyr Ala Lys Ser Gly Tyr Gly Ala Tyr Thr Pro Ser Ser Tyr Gly Ala Asn Leu A1a Ala Ser Leu Leu Glu 35 . 40 45 Lys Glu Lys Leu Gly Phe Lys Pro Val Pro Thr Ser Ser Phe Leu Thr Arg Pro Arg Thr Tyr Gly Pro Ser Ser Leu Leu Asp Tyr Asp Arg Gly Arg Pro Leu Leu Arg Pro Asp Ile Thr Gly Gly Gly Lys Arg A1a Glu Ser Gln Thr Arg Gly Thr Glu Arg Pro Leu Gly Ser Gly Leu Ser Gly Gly Ser Gly Phe Pro Tyr Gly Val Thr Asn Asn Cys Leu Ser Tyr Leu Pro Ile Asn Ala Tyr Asp Gln Gly Val Thr Leu Thr Gln Lys Leu Asp Ser G1n Ser Asp Leu Ala Arg Asp Phe Ser Ser Leu Arg Thr Ser Asp Ser Tyr Arg Ile Asp Pro Arg Asn Leu G1y Arg Ser Pro Met Leu Ala Arg Thr Arg Lys Glu Leu Cys Thr Leu Gln Gly Leu Tyr Gln Thr Ala Ser Cys Pro Glu Tyr Leu Val Asp Tyr Leu Glu Asn Tyr Gly Arg Lys Gly Ser Ala Ser Gln Val Pro Ser Gln Ala Pro Pro Ser Arg Val Pro Glu Ile Ile Ser Pro Thr Tyr Arg Pro Ile Gly Arg Tyr Thr Leu Trp Glu Thr Gly Lys Gly Gln Ala Pro Gly Pro Ser Arg Ser Ser Ser Pro Gly Arg Asp Gly Met Asn Ser Lys Ser Ala Gln Gly Leu Ala Gly Leu Arg Asn Leu Gly Asn Thr Cys Phe Met Asn Ser Ile Leu Gln Cys Leu Ser Asn Thr Arg Glu Leu Arg Asp Tyr Cys Leu Gln Arg Leu Tyr Met Arg Asp Leu His His Gly Ser Asn Ala His Thr Ala Leu Val Glu Glu Phe Ala Lys Leu Ile Gln Thr Ile Trp Thr Ser Ser Pro Asn Asp Val Va1 Ser Pro Ser Glu Phe Lys Thr Gln Ile Gln Arg Tyr Ala Pro Arg Phe Val Gly Tyr Asn Gln Gln Asp Ala Gln Glu Phe Leu Arg Phe Leu Leu Asp Gly Leu His Asn Glu Val Asn Arg Val Thr Leu Arg Pro Lys Ser Asn Pro Glu Asn Leu Asp His Leu Pro Asp Asp Glu Lys Gly Arg Gln Met Trp Arg Lys Tyr Leu Glu Arg Glu Asp Ser Arg Ile Gly Asp Leu Phe Val Gly Gln Leu Lys Ser Ser Leu Thr Cys Thr Asp Cys Gly Tyr Cys Ser Thr Val Phe Asp Pro Phe Trp Asp Leu Ser Leu Pro Ile Ala Lys Arg Gly Tyr Pro Glu Val Thr Leu Met Asp Cys Met Arg Leu Phe Thr Lys Glu Asp Val Leu Asp Gly Asp Glu Lys Pro Thr Cys Cys Arg Cys Arg Gly Arg Lys Arg Cys Ile Lys Lys Phe Ser Ile Gln Arg Phe Pro Lys I1e Leu Val Leu His Leu Lys Arg Phe Ser Glu Ser Arg Tle Arg Thr Ser Lys Leu Thr Thr Phe Val Asn Phe Pro Leu Arg Asp Leu Asp Leu Arg Glu Phe Ala Ser Glu Asn Thr Asn His Ala Val Tyr Asn Leu Tyr Ala Val Ser Asn His Ser Gly Thr Thr Met Gly Gly His Tyr Thr Ala Tyr Cys Arg Ser Pro Gly Thr Gly Glu Trp His Thr Phe Asn Asp Ser Ser Va1 Thr Pro Met Ser Ser Ser Gln Val Arg Thr Ser Asp Ala Tyr Leu Leu Phe Tyr Glu Leu Ala Ser Pro Pro Ser Arg Met <210> 4 <211> 743 <212> PRT
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte ID No: 2923874CD1 <400> 4 Met Leu Ile Ser Gly Ile Leu Trp Thr Phe Met His Gln Lys Pro Thr Ala Ser His Tyr Leu Gln Val Lys Ser Gln Asp Gly Ile Leu Ser Pro Gly Lys Gly Leu Glu Asp Thr Asp Val Val Tyr Lys Ser Glu Asn Gly His Val Ile Lys Leu Asn Ile Glu Thr Asn Ala Thr 50 ' 55 60 Thr Leu Leu Leu Glu Asn Thr Thr Phe Val Thr Phe Lys Ala Ser Arg His Ser Val Ser Pro Asp Leu Lys Tyr Val Leu Leu Ala Tyr Asp Val Lys Gln Ile Phe His Tyr Ser Tyr Thr Ala Ser Tyr Va1 Ile Tyr Asn Ile His Thr Arg Glu Val Trp Glu Leu Asn Pro Pro Glu Val Glu Asp Ser Val Leu Gln Tyr Ala Ala Trp Gly Val Gln Gly Gln Gln Leu Ile Tyr Ile Phe Glu Asn Asn Ile Tyr Tyr G1n Pro Asp Ile Lys Ser Ser Ser Leu Arg Leu Thr Ser Ser Gly Lys Glu Glu Ile Ile Phe Asn Gly Ile Ala Asp Trp Leu Tyr Glu Glu Glu Leu Leu His Ser His Ile Ala His Trp Trp Ser Pro Asp Gly Glu Arg Leu Ala Phe Leu Met Ile Asn Asp Ser Leu Val Pro Thr Met Val Ile Pro Arg Phe Thr Gly Ala Leu Tyr Pro Lys Gly Lys Gln Tyr Pro Tyr Pro Lys Ala Gly Gln Val Asn Pro Thr Ile Lys Leu Tyr Val Val Asn Leu Tyr Gly Pro Thr His Thr Leu Glu Leu Met Pro Pro Asp Ser Phe Lys Ser Arg Glu Tyr Tyr Ile Thr Met Val Lys Trp Val Ser Asn Thr Lys Thr Val Val Arg Trp Leu Asn Arg Pro Gln Asn Ile Ser Ile Leu Thr Val Cys Glu Thr Thr Thr Gly Ala Cys Ser Lys Lys Tyr Glu Met Thr Ser Asp Thr Trp Leu Ser Gln Gln Asn Glu Glu Pro Val Phe Ser Arg Asp Gly Ser Lys Phe Phe Met Thr Val Pro Val Lys Gln Gly Gly Arg Gly Glu Phe His His Ile Ala Met Phe Leu Ile Gln Ser Lys Ser Glu Gln Ile Thr Val Arg His Leu Thr Ser Gly Asn Trp Glu Val Ile Lys Ile Leu Ala Tyr Asp Glu Thr Thr Gln Lys Ile Tyr Phe Leu Ser Thr Glu Ser Ser Pro Arg Gly Arg Gln Leu Tyr Ser Ala Ser Thr Glu Gly Leu Leu Asn Arg Gln Cys Ile Ser Cys Asn Phe Met Lys Glu Gln Cys Thr Tyr Phe Asp Ala Ser Phe Ser Pro Met Asn Gln His Phe Leu Leu Phe Cys Glu Gly Pro Arg Va1 Pro Val Val Ser Leu His Ser Thr Asp Asn Pro Ala Lys Tyr Phe Ile Leu Glu Ser Asn Ser Met Leu Lys Glu Ala Ile Leu Lys Lys Lys Ile Gly Lys Pro Glu Ile Lys Ile Leu His Ile Asp Asp Tyr Glu Leu Pro Leu Gln Leu Ser Leu Pro Lys Asp Phe Met Asp Arg Asn Gln Tyr Ala Leu Leu Leu Ile Met Asp Glu Glu Pro Gly Gly Gln Leu Val Thr Asp Lys Phe His Ile Asp Trp Asp Ser Val Leu Ile Asp Met Asp Asn Val Ile Val Ala Arg Phe Asp Gly Arg Gly Ser Gl~r Phe Gln Gly Leu Lys Ile Leu Gln Glu Ile His Arg Arg Leu G1y Ser Val G1u Val Lys Asp G1n Ile Thr Ala Val Lys Phe Leu Leu Lys Leu Pro Tyr Ile Asp Ser Lys Arg Leu Ser Ile Phe Gly Lys Gly Tyr Gly Gly Tyr Ile Ala Ser Met Ile Leu Lys Ser Asp Glu Lys Leu Phe Lys Cys Gly Ser Val Val Ala Pro Ile Thr Asp Leu Lys Leu Tyr Ala Ser Ala Phe Ser Glu Arg Tyr Leu G1y Met Pro Ser Lys Glu Glu Ser Thr Tyr Gln Ala Ala Ser Val Leu His Asn Val His Gly Leu Lys Glu Glu Asn Ile Leu Ile Ile His Gly Thr Ala Asp Thr Lys Val His Phe Gln His Ser Ala Glu Leu Ile Lys His Leu Ile 680 ~ 685 690 Lys Ala Gly Val Asn Tyr Thr Met Gln Va1 Tyr Pro Asp G1u Gly His Asn Val Ser Glu Lys Ser Lys Tyr His Leu Tyr Ser Thr I1e Leu Lys Phe Phe Ser Asp Cys Leu Lys Glu Glu Ile Ser Val Leu Pro Gln Glu Pro Glu Glu Asp Glu <210> 5 <211> 650 <212> PRT
<213> Homo Sapiens <220>

<221> misc_feature <223> Incyte ID No: 55122335CD1 <400> 5 Met Ala Ser Gly Glu His Ser Pro Gly Ser Gly Ala Ala Arg Arg Pro Leu His Ser Ala Gln Ala Val Asp Val Ala Ser Ala Ser Asn Phe Arg Ala Phe Glu Leu Leu His Leu His Leu Asp Leu Arg Ala Glu Phe Gly Pro Pro Gly Pro Gly Ala Gly Ser Arg Gly Leu Ser Gly Thr Ala Val Leu Asp Leu Arg Cys Leu Glu Pro Glu Gly Ala Ala Glu Leu Arg Leu Asp Ser His Pro Cys Leu Glu Val Thr Ala Ala Ala Leu Arg Arg Glu Arg Pro Gly Ser Glu Glu Pro Pro Ala Glu Pro Val Ser Phe Tyr Thr Gln Pro Phe Ser His Tyr Gly Gln Ala Leu Cys Val Ser Phe Pro Gln Pro Cys Arg Ala Ala Glu Arg Leu Gln Val Leu Leu Thr Tyr Arg Val Gly Glu Gly Pro Gly Val Cys Trp Leu Ala Pro Glu Gln Thr Ala Gly Lys Lys Lys Pro Phe Val Tyr Thr Gln Gly Gln Ala Val Leu Asn Arg Ala Phe Phe Pro Cys Phe Asp Thr Pro Ala Val Lys Tyr Lys Tyr Ser Ala Leu Ile Glu Val Pro Asp G1y Phe Thr Ala Val Met Ser Ala Ser Thr Trp Glu Lys Arg Gly Pro Asn Lys Phe Phe Phe Gln Met Cys Gln Pro Ile Pro Ser Tyr Leu Ile Ala Leu Ala Ile Gly Asp Leu Val Ser Ala Glu Val Gly Pro Arg Ser Arg Val Trp Ala Glu Pro Cys Leu Ile Asp Ala Ala Lys Glu Glu Tyr Asn Gly Val I1e Glu Glu Phe Leu Ala Thr Gly Glu Lys Leu Phe Gly Pro Tyr Val Trp Gly Arg Tyr Asp Leu Leu Phe Met Pro Pro Ser Phe Pro Phe Gly Gly Met Glu Asn Pro Cys Leu Thr Phe Val Thr Pro Cys Leu Leu Ala Gly Asp Arg Ser Leu Ala Asp Val Ile Ile His Glu Tle Ser His Ser Trp Phe Gly Asn Leu Val Thr Asn Ala Asn Trp G1y Glu Phe Trp Leu Asn Glu Gly Phe Thr Met Tyr Ala Gln Arg Arg Ile Ser Thr Ile Leu Phe Gly Ala Ala Tyr Thr Cys Leu Glu Ala Ala Thr G1y Arg Ala Leu Leu Arg Gln His Met Asp Ile Thr Gly Glu Glu Asn Pro Leu Asn Lys Leu Arg Val Lys Ile Glu Pro Gly Val Asp Pro Asp Asp Thr,Tyr Asn Glu Thr Pro Tyr Glu Lys Gly Phe Cys Phe Val Ser Tyr Leu Ala His Leu Val Gly Asp Gln Asp Gln Phe Asp Ser Phe Leu Lys Ala Tyr Val His Glu Phe Lys Phe Arg Ser Ile Leu Ala Asp Asp Phe Leu Asp Phe Tyr Leu Glu Tyr Phe Pro Glu Leu Lys Lys Lys Arg Val Asp Ile Ile Pro Gly Phe Glu Phe Asp Arg Trp Leu Asn Thr Pro Gly Trp Pro Pro Tyr Leu Pro Asp Leu Ser Pro Gly Asp Ser Leu Met Lys Pro Ala Glu Glu Leu Ala Gln Leu Trp Ala Ala Glu Glu Leu Asp Met Lys Ala Ile Glu Ala Val A1a Ile Ser Pro Trp Lys Thr Tyr Gln Leu Val Tyr Phe Leu Asp Lys Ile Leu Gln Lys Ser Pro Leu Pro Pro G1y Asn Val Lys Lys 545 ' 550 555 Leu Gly Asp Thr Tyr Pro Ser IIe Ser Asn Ala Arg Asn Ala Glu Leu Arg Leu Arg Trp Gly G1n Ile Ile Leu Lys Asn Asp His Gln Glu Asp Phe Trp Lys Val Lys Glu Phe Leu His Asn Gln Gly Lys Gln Lys Tyr Thr Leu Pro Leu Tyr His Ala Met Met Gly Gly Ser Glu Val Ala Gln Thr Leu Ala Lys Glu Thr Phe Ala Ser Thr Ala Ser Gln Leu His Ser Asn Val Val Asn Tyr Val Gln Gln Ile Val Ala Pro Lys Gly Ser <210> 6 <211> 932 <212> PRT
<213> Homo Sapiens <220>
<221> misc~feature <223> Incyte ID No: 7473550CD1 <400> 6 Met Gly Leu Leu Ala Ser Ala Gly Leu Leu Leu Leu Leu Val Ile Gly His Pro Arg Ser Leu Gly Leu Lys Cys Gly Ile Arg Met Val Asn Met Lys Ser Lys Glu Pro Ala Val Gly Ser Arg Phe Phe Ser Arg Ile Ser Ser Trp Arg Asn Ser Thr Val Thr Gly His Pro Trp Gln Val Tyr Leu Lys Ser Asp G1u His His Phe Cys Gly Gly Ser Leu Ile Gln Glu Asp Arg Val Val Thr Ala Ala His Cys Leu His Ser Leu Ser Glu Lys Gln Leu Lys Asn Ile Thr Val Thr Ser Gly Glu Tyr Ser Leu Phe Gln Lys Asp Lys Gln Glu Gln Asn Ile Pro Val Ser Lys Ile Ile Thr His Pro Glu Tyr Asn Ser Arg Glu Tyr Met Ser Pro Asp Ile Ala Leu Leu Tyr Leu Lys His Lys Val Lys Phe Gly Asn Ala Val Gln Pro Ile Cys Leu Pro Asp Ser Asp Asp Lys Val Glu Pro-Gly Ile Leu Cys Leu Ser Ser Gly Trp Gly Lys Ile Ser Lys Thr Ser Glu Tyr Ser Asn Val Leu Gln Glu Met Glu Leu Pro Ile Met Asp Asp Arg Ala Cys Asn Thr Val Leu Lys Ser Met Asn Leu Pro Pro Leu Gly Arg Thr Met Leu Cys Ala Gly Phe Pro Asp Trp Gly Met Asp Ala Cys Gln Gly Asp Ser Gly Gly Pro, Leu Val Cys Arg Arg Gly Gly Gly Ile Trp Ile Leu Ala Gly Ile Thr Ser Trp Val Ala Gly Cys Ala Gly Gly Ser Val Pro Val Arg Asn Asn His Val Lys Ala Ser Leu Gly Ile Phe Ser Lys Val Ser Glu Leu Met Asp Phe Ile Thr Gln Asn Leu Phe Thr Gly Leu Asp Arg Gly G1n Pro Leu Ser Lys Val Gly Ser Arg Tyr Ile Thr Lys Ala Leu Ser Ser Val Gln Glu Va1 Asn Gly Ser G1n Arg Asp Lys Ile Ile Leu Ile Lys Phe Thr Ser Leu Asp Met Glu Lys Gln Val Gly Cys Asp His Asp Tyr Val Ser Leu Arg Ser Ser Ser Gly Val Leu Phe Ser Lys Va1 Cys Gly Lys Tle Leu Pro Ser Pro Leu Leu Ala Glu Thr Ser Glu Ala Met Val Pro Phe Val Ser Asp Thr Glu Asp Ser G1y Ser G1y Phe Glu Leu Thr Val Thr Ala Val Gln Lys Ser G1u Ala Gly Ser Gly Cys Gly Ser Leu A1a I1e Leu Val Glu Glu Gly Thr Asn His Ser Ala Lys Tyr Pro Asp Leu Tyr Pro Ser Asn Thr Arg Cys His Trp Phe Ile Cys Ala Pro Glu Lys His Ile Ile Lys Leu Thr Phe Glu Asp Phe Ala Val Lys Phe Ser Pro Asn Cys Ile Tyr Asp Ala Val Val Ile Tyr G1y Asp Ser Glu Glu Lys His Lys Leu Ala Lys Leu Cys Gly Met Leu Thr Ile Thr Ser Ile Phe Ser Ser Ser Asn Met Thr Val Ile Tyr Phe Lys Ser Asp G1y Lys Asn Arg Leu Gln Gly Phe Lys Ala Arg Phe Thr Ile Leu Pro Ser Glu Ser Leu Asn Lys Phe Glu Pro Lys Leu Pro Pro Gln Asn Asn Pro Val Ser Thr Val Lys Ala Ile Leu His Asp Val Cys Gly Ile Pro Pro Phe Ser Pro Gln Trp Leu Ser Arg Arg Ile Ala Gly Gly Glu Glu Ala Cys Pro His Cys Trp Pro Trp Gln Val Gly Leu Arg Phe Leu Gly Asp Tyr Gln Cys G1y Gly Ala Ile Ile Asn Pro Val Trp Ile Leu Thr A1a Ala His Cys Val Gln Leu Lys Asn Asn Pro Leu Ser Trp Thr Ile Ile Ala Gly Asp His Asp Arg Asn Leu Lys Glu Ser Thr Glu Gln Val Arg Arg Ala Lys His Ile Ile Val His Glu Asp Phe Asn Thr Leu Ser Tyr Asp Ser Asp Ile Ala Leu Ile Gln Leu Ser Ser Pro Leu Glu Tyr Asn Ser Val Val Arg Pro Val Cys Leu Pro His Ser Ala Glu Pro Leu Phe Ser Ser Glu Ile Cys Ala Val Thr Gly Trp Gly Ser Ile Ser Ala Glu Leu Ser Leu Asn Val Ser Ser Leu Asp Gly Gly Leu Ala Ser Arg Leu Gln Gln Ile Gln Val His Val Leu Glu Arg Glu Val Cys Glu His Thr Tyr Tyr Se_r A1a His Pro Gly Gly Ile Thr Glu Lys Met Ile Cys Ala Gly Phe Ala Ala Ser Gly Glu Lys Asp Phe Cys Gln Gly Asp Ser Gly Gly Pro Leu Val Cys Arg His Glu Asn Gly Pro Phe Va1 Leu Tyr Gly Ile Val Ser Trp Gly Ala Gly Cys Val Gln Pro Trp Lys Pro Gly Val Phe Ala Arg Va1 Met Ile Phe Leu Asp Trp Ile Gln Ser Lys Ile Asn Gly Lys Leu Phe Ser Asn Va1 Ile Lys Thr Ile Thr Ser Phe Phe Arg Val Gly Leu Gly Thr Val Ser Cys Cys Ser Glu Ala Glu Leu Glu Lys Pro Arg Gly Phe Phe Pro Thr Pro Arg Tyr Leu Leu Asp Tyr Arg Gly Arg Leu Glu Cys Ser Trp Val Leu Arg Val Ser Ala Ser Ser Met Ala Lys Phe Thr Ile Glu Tyr Leu Ser Leu Leu Gly Ser Pro Val Cys Gln Asp Ser Val Leu Ile Ile 890 ' 895 900 Tyr Glu G1u Arg His Ser Lys Arg Lys Thr Ala Gly Asn Pro Ser Ser Phe Leu Lys Ala Tyr Val His Glu Phe Ly Trp His Leu Pro Met Glu Ile Ser Ser Pro Phe Lys Ser His His Ser Ala <210> 7 <211> 990 <212> PRT
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte ID No: 7478108CD1 <400> 7 Met Gly Pro Pro Ser Ser Ser Gly Phe Tyr Val Ser Arg Ala Val Ala Leu Leu Leu Ala Gly Leu Val Ala Ala Leu Leu Leu Ala Leu Ala Val Leu Ala Ala Leu Tyr Gly His Cys Glu Arg Val Pro Pro Ser Glu Leu Pro Gly Leu Arg Asp Ser Glu Ala Glu Ser Ser Pro Pro Leu Arg Gln Lys Pro Thr Pro Thr Pro Lys Pro Ser Ser Ala Arg Glu Leu Ala Val Thr Thr Thr Pro Ser Asn Trp Arg Pro Pro Gly Pro Trp Asp Gln Leu Arg Leu Pro Pro Trp Leu Val Pro Leu His Tyr Asp Leu Glu Leu Trp Pro Gln Leu Arg Pro Asp Glu Leu Pro Ala Gly Ser Leu Pro Phe Thr Gly Arg Val Asn Ile Thr Val Arg Cys Thr Val Ala Thr Ser Arg Leu Leu Leu His Ser Leu Phe Gln Asp Cys Glu Arg Ala Glu Val Arg Gly Pro Leu Ser Pro Gly Thr Gly Asn Ala Thr Val Gly Arg Val Pro Val Asp Asp Val Trp Phe Ala Leu Asp Thr Glu Tyr Met Val Leu Glu Leu Ser Glu Pro Leu Lys Pro Gly Ser Ser Tyr Glu Leu Gln Leu Ser Phe Ser Gly Leu Val Lys Glu Asp Leu Arg Glu Gly Leu Phe Leu Asn Val Tyr Thr Asp Gln Gly Glu Arg Arg Ala Leu Leu Ala Ser Gln Leu G1u Pro Thr Phe Ala Arg Tyr Val Phe Pro Cys Phe Asp Glu Pro Ala Leu Lys Ala Thr Phe Asn Tle Thr Met Ile His His Pro Ser Tyr Val Ala Leu Ser Asn Met Pro Lys Leu Gly Gln Ser Glu Lys Glu Asp Val Asn Gly Ser Lys Trp Thr Val Thr Thr Phe Ser Thr Thr 290 2g5 300 Pro His Met Pro Thr Tyr Leu Val Ala Phe Val Ile Cys Asp Tyr Asp His Val Asn Arg Thr Glu Arg Gly Lys Glu Ile Arg Ile Trp Ala Arg Lys Asp Ala Ile Ala Asn Gly Ser Ala Asp Phe Ala Leu Asn Ile Thr Gly Pro Ile Phe Ser Phe Leu Glu Asp Leu Phe Asn Ile Ser Tyr Ser Leu Pro Lys Thr Asp Ile Ile Ala Leu Pro Ser Phe Asp Asn His Ala Met Glu Asn Trp Gly Leu Met Ile Phe Asp Glu Ser Gly Leu Leu Leu Glu Pro Lys Asp Gln Leu Thr Glu Lys Lys Thr Leu Ile Ser Tyr Val Val Ser His Glu Ile Gly His Gln Trp Phe Gly Asn Leu Val Thr Met Asn Trp Trp Asn Asn Ile Trp Leu Asn Glu Gly Phe Ala Ser Tyr Phe Glu Phe Glu Val Ile Asn Tyr Phe Asn Pro Lys Leu Pro Arg Asn Glu Ile Phe Phe Ser Asn Ile Leu His Asn Ile Leu Arg Glu Asp His Ala Leu Val Thr Arg Ala Val Ala Met Lys Val Glu Asn Phe Lys Thr Ser Glu Ile G1n G1u Leu Phe Asp Ile Phe Thr Tyr Ser Lys Gly Ala Ser Met Ala Arg Met Leu Ser Cys Phe Leu Asn Glu His Leu Phe Val Ser Ala Leu Lys Ser Tyr Leu Lys Thr Phe Ser Tyr Ser Asn Ala Glu Gln Asp Asp Leu Trp Arg His Phe Gln Met Ala Ile Asp Asp Gln Ser Thr Val Ile Leu Pro Ala Thr Ile Lys Asn Tle Met Asp Ser Trp Thr His Gln 5er Gly Phe Pro Val Ile Thr Leu Asn Val Ser Thr Gly Val Met Lys Gln Glu Pro Phe Tyr Leu Glu Asn Ile Lys Asn Arg Thr Leu Leu Thr Ser Asn Asp Thr Trp Ile Val Pro Ile Leu Trp Ile Lys Asn Gly Thr Thr Gln Pro Leu Val Trp Leu Asp Gln Ser Ser Lys Val Phe Pro Glu Met Gln Val Ser Asp Ser Asp His Asp Trp Val Ile Leu Asn Leu Asn Met Thr Gly Tyr Tyr Arg Val Asn Tyr Asp Lys Leu Gly Trp Lys Lys Leu Asn Gln G1n Leu Glu Lys Asp Pro Lys Ala Ile Pro Val Ile His Arg Leu Gln Phe Ile Asp Asp Ala Phe Ser Leu Ser Lys Asn Asn Tyr Ile G1u Ile Glu Thr Ala Leu Glu Leu Thr Lys Tyr Leu Ala Glu Glu Asp Glu Ile Tle Val Trp His Thr Val Leu Val Asn Leu Val Thr Arg Asp Leu Val Ser Glu Val Asn Ile Tyr Asp Ile Tyr Ser Leu Leu Lys Arg Tyr Leu Leu Lys Arg Leu Asn Leu Ile Trp Asn Ile Tyr Ser Thr Ile Ile Arg Glu Asn Va1 Leu Ala Leu Gln Asp Asp Tyr Leu Ala Leu Ile Ser Leu Glu Lys Leu Phe Val Thr Ala Cys Trp Leu Gly Leu Glu Asp Cys Leu Gln Leu Ser Lys Glu Leu Phe Ala Lys Trp Val Asp His Pro Glu Asn Glu Ile Pro Tyr Pro Ile Lys Asp Val Val Leu Cys Tyr Gly Ile Ala Leu Gly Ser Asp Lys Glu Trp Asp I1e Leu Leu Asn Thr Tyr Thr Asn Thr Thr Asn Lys Glu Glu Lys Ile Gln Leu Ala Tyr Ala Met Ser Cys Ser Lys Asp Pro Trp Ile Leu Asn Arg Tyr Met Glu Tyr Ala Ile Ser Thr Ser Pro Phe Thr Ser Asn Glu Thr Asn Ile Ile Glu Val Va1 Ala Ser Ser Glu Val Gly Arg Tyr Val Ala Lys Asp Phe Leu Val Asn Asn Trp Gln Ala Val Ser Lys Arg Tyr Gly Thr Gln Ser Leu Ile Asn Leu Ile Tyr Thr Ile Gly Arg Thr Val Thr Thr Asp Leu Gln Ile Va1 Glu Leu G1n Gln Phe Phe Ser Asn Met Leu Glu Glu His Gln Arg Ile Arg Val His Ala Asn Leu Gln Thr Ile Lys Asn Glu Asn Leu Lys Asn Lys Lys Leu Ser Ala Arg Ile Ala Ala Trp Leu Arg Arg Asn Thr <210> 8 <211> 396 <212> PRT
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte ID No: 7482021CD1 <400> 8 Met Arg Thr Ser Tyr Thr Val Thr Leu Pro Glu Asp Pro Pro A1a Ala Pro Phe Pro Ala Leu Ala Lys Glu Leu Arg Pro Arg Ser Pro Leu Ser Pro Ser Leu Leu Leu Ser Thr Phe Va1 Gly Leu Leu Leu Asn Lys Ala Lys Asn Ser Lys Ser Ala Gln Gly Leu Ala Gly Leu Arg Asn Leu Gly Asn Thr Cys Phe Met Asn Ser Ile Leu Gln Cys Leu Ser Asn Thr Arg Glu Leu Arg Asp Tyr Cys Leu Gln Arg Leu Tyr Met Arg Asp Leu His His Gly Ser Asn Ala His Thr Ala Leu Val Glu Glu Phe Ala Lys Leu Ile Gln Thr Ile Trp Thr Ser Ser Pro Asn Asp Val Val Ser Pro Ser Glu Phe Lys Thr Gln Ile Gln Arg Tyr Ala Pro Arg Phe Val Gly Tyr Asn Gln Gln Asp Ala Gln Glu Phe Leu Arg Phe Leu Leu Asp Gly Leu His Asn Glu Val Asn Arg Val Thr Leu Arg Pro Lys Ser Asn Pro Glu Asn Leu Asp His Leu Pro Asp Asp Glu Lys Gly Arg Gln Met Trp Arg Lys Tyr Leu 185 190 l95 Glu Arg Glu Asp Sex Arg Ile Gly Asp Leu Phe Val Gly G1n Leu 200 205 27.0 Lys Ser Ser Leu Thr Cys Thr Asp Cys G1y Tyr Cys Ser Thr Val Phe Asp Pro Phe Trp Asp Leu Ser Leu Pro Ile A1a Lys Arg Gly Tyr Pro Glu Val Thr Leu Met Asp Cys Met Arg Leu Phe Thr Lys Glu Asp Val Leu Asp Gly Asp Glu Lys Pro Thr Cys Cys Arg Cys Arg Gly Arg Lys Arg Cys Ile Lys Lys Phe Ser Ile Gln Arg Phe Pro Lys Ile Leu Val Leu His Leu Lys Arg Phe Ser Glu Ser Arg Ile Arg Thr Ser Lys Leu Thr Thr Phe Va1 Asn Phe Pro Leu Arg Asp Leu Asp Leu'Arg Glu Phe Ala Ser Glu Asn Thr Asn His Ala Val Tyr Asn Leu Tyr Ala Val Ser Asn His Ser Gly Thr Thr Met Gly Gly His Tyr Thr Ala Tyr Cys Arg Ser Pro Gly Thr Gly G1u Trp His Thr Phe Asn Asp Ser Ser Val Thr Pro Met Ser Ser Ser Gln Va1 Arg Thr Ser Asp Ala Tyr Leu Leu Phe Tyr Glu Leu Ala Ser Pro Pro Ser Arg Met <210> 9 <211> 250 <212> PRT
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte ID No: 7482145CD1 <400> 9 Met A1a Ser Arg Tyr Asp Arg Ala Ile Thr Val Phe Ser Pro Asp Gly His Leu Phe Gln Val Glu Tyr Ala Gln Glu Ala Val Lys Lys Gly Ser Thr Ala Val Gly Ile Arg Gly Thr Asn Ile Val Val Leu Gly Val Glu Lys Lys Ser Val Ala Lys Leu Gln Asp Glu Arg Thr Val Arg Lys Ile Cys Ala Leu Asp Asp His Val Cys Met Ala Phe 65 ,70 75 Ala G1y Leu Thr Ala Asp Ala Arg Val Val Ile Asn Arg Ala Arg Val Glu Cys Gln Ser His Lys Leu Thr Val Glu Asp Pro Val Thr Val Glu Tyr Ile Thr Arg Phe Ile Ala Thr Leu Lys Gln Lys Tyr Thr Gln Ser Asn Gly Arg Arg Pro Phe G1y Ile Ser Ala Leu Ile Val Gly Phe Asp Asp Asp Gly Ile Ser Arg Leu Tyr Gln Thr Asp Pro Ser Gly Thr Tyr His Ala Trp Lys Ala Asn Ala Ile Gly Arg Ser Ala Lys Thr Val Arg Glu Phe Leu Glu Lys Asn Tyr Thr Glu Asp Ala Ile Ala Ser Asp Ser GIu Ala IIe Lys Leu Ala Ile Lys Ala Leu Leu Glu Va1 Val G1n Ser Gly Gly Lys Asn Tle Glu Leu Ala Ile Ile Arg Arg Asn Gln Pro Leu Lys Met Phe Ser Ala Lys Glu Val Glu Leu Tyr Val Thr Glu Ile Glu Lys Glu Lys Glu Glu 230 . 235 240 Ala Glu Lys Lys Lys Ser Lys Lys Ser Va1 <210> 10 <211> 1045 <212> PRT
<213> Homo sapiens <220>
<221> misc_feature <223> Incyte ID No: 5502258&CD1 <400> 10 Met Thr Ala Glu Leu Gln Gln Asp Asp Ala Ala Gly Ala Ala Asp Gly His Gly Ser Ser Cys Gln Met Leu Leu Asn Gln Leu Arg Glu Ile Thr Gly Ile Gln Asp Pro Ser Phe Leu His Glu A1a Leu Lys Ala Ser Asn GIy Asp Ile Thr Gln Ala Val Ser Leu Leu Thr Asp Glu Arg Val Lys Glu Pro Sex Gln Asp Thr Val Ala Thr Glu Pro Ser Glu Val Glu Gly Ser Ala Ala Asn Lys Glu Val Leu Ala Lys Val Ile Asp Leu Thr His Asp Asn Lys Asp Asp Leu Gln Ala Ala Ile Ala Leu Ser Leu Leu Glu Ser Pro Lys Ile Gln Ala Asp Gly 110 l15 120 Arg Asp Leu Asn Arg Met His Glu Ala Thr Ser Ala Glu Thr Lys Arg Ser Lys Arg Lys Arg Cys Glu Val Trp Gly Glu Asn Pro Asn Pro Asn Asp Trp Arg Arg Val Asp Gly Trp Pro Val Gly Leu Lys Asn Val Gly Asn Thr Cys Trp Phe Ser Ala Val Ile Gln Ser Leu 170 175 l80 Phe Gln Leu Pro Glu Phe Arg Arg Leu Val Leu Ser Tyr Ser Leu Pro Gln Asn Val Leu Glu Asn Cys Arg Ser His Thr Glu Lys Arg 200 205 2l0 Asn Ile Met Phe Met Gln Glu Leu Gln Tyr Leu Phe Ala Leu Met Met Gly Ser Asn Arg Lys Phe Val Asp Pro Ser Ala Ala Leu Asp Leu Leu Lys Gly Ala Phe Arg Ser Ser Glu Glu Gln Gln Gln Asp Val Ser Glu Phe Thr His Lys Leu Leu Asp Trp Leu Glu Asp Ala Phe Gln Leu Ala Val Asn Val Asn Ser Pro Arg Asn Lys Ser Glu Asn Pro Met Val Gln Leu Phe Tyr Gly Thr Phe Leu Thr Glu Gly Val Arg Glu Gly Lys Pro Phe Cys Asn Asn Glu Thr Phe Gly G1n Tyr Pro Leu Gln Val Asn Gly Tyr Arg Asn Leu Asp Glu Cys Leu Glu Gly Ala Met Val Glu Gly Asp Val Glu Leu Leu Pro Ser Asp His Ser Val Lys Tyr Gly G1n Glu Arg Trp Phe Thr Lys Leu Pro Pro Val Leu Thr Phe Glu Leu Ser Arg Phe Glu Phe Asn Gln Ser Leu Gly Gln Pro Glu Lys Ile His Asn Lys Leu Glu Phe Pro Gln Ile Ile Tyr Met Asp Arg Tyr Met Tyr Arg Ser Lys Glu Leu Ile Arg Asn Lys Arg Glu Cys Ile Arg Lys Leu Lys Glu Glu Ile Lys Ile Leu Gln Gln Lys Leu Glu Arg Tyr Val Lys Tyr Gly Ser Gly Pro Ala Arg Phe Pro Leu Pro Asp Met Leu Lys Tyr Val Ile Glu Phe Ala Ser Thr Lys Pro Ala Ser Glu Ser Cys Pro Pro Glu Ser Asp Thr His Met Thr Leu Pro Leu Ser Ser Val His Cys Ser Val Ser Asp G1n Thr Ser Lys Glu Ser Thr Ser Thr G1u Ser Ser Ser Gln Asp Val Glu Ser Thr Phe Ser Ser Pro Glu Asp Ser Leu Pro Lys Ser Lys Pro Leu Thr Ser Ser Arg Ser Ser Met Glu Met Pro Ser Gln Pro Ala Pro Arg Thr Val Thr Asp Glu Glu Ile Asn Phe Val Lys Thr Cys Leu Gln Arg Trp Arg Ser Glu Ile Glu Gln Asp Ile Gln Asp Leu Lys Thr Cys Ile Ala Ser Thr Thr Gln Thr Ile Glu Gln Met Tyr Cys Asp Pro Leu Leu Arg Gln Val Pro Tyr Arg Leu His Ala Val Leu Val His Glu Gly Gln Ala Asn Ala Gly His Tyr Trp Ala Tyr Ile Tyr Asn Gln Pro Arg Gln Ser Trp Leu Lys Tyr Asn Asp Ile Ser Val Thr Glu Ser Ser Trp Glu G1u Val Glu Arg Asp Ser Tyr Gly Gly Leu Arg Asn Val Ser Ala Tyr Cys Leu Met Tyr Ile Asn Asp Lys Leu Pro Tyr Phe Asn Ala Glu Ala Ala Pro Thr G1u Ser Asp Gln Met Ser Glu Val Glu Ala Leu Ser Val Glu Leu Lys His Tyr Ile Gln Glu Asp Asn Trp Arg Phe Glu Gln Glu Val Glu Glu Trp G1u Glu Glu Gln Ser Cys Lys Ile Pro Gln Met Glu Ser Ser Thr Asn Ser Ser Ser Gln Asp Tyr Ser Thr Ser Gln G1u Pro Ser Val Ala Ser Ser His Gly Val Arg Cys Leu Ser Ser Glu His A1a Val Ile Val Lys Glu Gln Thr Ala Gln Ala I1e Ala Asn Thr Ala Arg Ala Tyr Glu Lys Ser Gly Val Glu Ala Ala Leu Ser Glu Ala Phe His Glu G1u Tyr Ser Arg Leu Tyr Gln Leu Ala Lys Glu Thr Pro Thr Ser His Ser Asp Pro Arg Leu Gln His Val Leu Val Tyr Phe Phe Gln Asn Glu A1a Pro Lys Arg Val Val Glu Arg Thr Leu Leu Glu Gln Phe Ala Asp Lys Asn Leu Ser Tyr Asp Glu Arg Ser Ile Ser Ile Met Lys Val Ala Gln Ala Lys Leu Lys Glu Ile Gly Pro Asp Asp Met Asn Met Glu Glu Tyr Lys Lys Trp His Glu Asp Tyr Ser Leu Phe Arg Lys Val Ser Va1 Tyr Leu Leu Thr Gly Leu Glu Leu Tyr Gln Lys Gly Lys Tyr Gln Glu Ala Leu Ser Tyr Leu Val Tyr A1a Tyr Gln Ser Asn Ala Ala Leu Leu Met Lys Gly Pro Arg Arg Gly Val Lys Glu Ser Val Ile Ala Leu Tyr Arg Arg Lys Cys Leu Leu Glu Leu Asn Ala Lys,Ala Ala Ser Leu Phe Glu Thr Asn Asp Asp His Ser Val Thr Glu Gly Ile Asn Val Met Asn Glu Leu Ile Ile Pro Cys Ile His Leu Ile Ile Asn Asn Asp Ile Ser Lys Asp Asp Leu Asp Ala Ile Glu Val Met Arg Asn His Trp Cys Ser Tyr Leu Gly Gln Asp Ile Ala Glu Asn Leu Gln Leu Cys Leu G1y Glu Phe Leu Pro Arg Leu Leu Asp Pro Ser Ala Glu Ile Ile Va1 Leu Lys Glu Pro Pro Thr Ile Arg Pro Asn Ser Pro Tyr Asp Leu Cys Ser Arg Phe Ala Ala Val Met Glu Ser Ile Gln Gly Val Ser Thr Val Thr Val Lys <210> 1l <211> 622 <212> PRT
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte ID No: 3238072CD1 <400> 11 Met Glu Val Arg Asp Leu Tyr Val Phe Cys Tyr Leu Cys Lys Asp 1 5 l0 15 Tyr Val Leu Asn Asp Asn Pro Glu Gly Asp Leu Lys Leu Leu Arg Ser Ser Leu Leu Ala Val Arg Gly Gln Lys Gln Asp Thr Pro Val Arg Arg Gly Arg Thr Leu Arg Ser Met Ala Ser Gly Glu Asp Val Val Leu Pro Gln Arg Ala Pro Gln Gly Gln Pro Gln Met Leu Thr 65 ' 70 75 Ala Leu Trp Tyr Arg Arg G1n Arg Leu Leu Ala Arg Thr Leu Arg Leu Trp Phe Glu Lys Ser Ser Arg Gly G1n Ala Lys Leu Glu Gln Arg Arg Gln G1u Glu Ala Leu G1u Arg Lys Lys Glu Glu Ala Arg Arg Arg Arg Arg G1u Val Lys Arg Arg Leu Leu Glu Glu Leu Ala Ser Thr Pro Pro Arg Lys Ser Ala Arg Leu Leu Leu His Thr Pro Arg Asp Ala Gly Pro Ala Ala Ser Arg Pro Ala Ala Leu Pro Thr Ser Arg Arg Val Pro Ala Ala Thr Leu Lys Leu Arg Arg Gln Pro Ala Met Ala Pro Gly Val Thr Gly Leu Arg Asn Leu Gly Asn Thr Cys Tyr Met Asn Ser Ile Leu Gln Val Leu Ser His Leu Gln Lys Phe Arg Glu Cys Phe Leu Asn Leu Asp Pro Ser Lys Thr Glu His 215 220 ' 225 Leu Phe Pro Lys Ala Thr Asn Gly Lys Thr Gln Leu Ser Gly Lys Pro Thr Asn Ser Ser Ala Thr Glu Leu Ser Leu Arg Asn Asp Arg Ala Glu Ala Cys Glu Arg Glu Gly Phe Cys Trp Asn Gly Arg Ala Ser Ile Ser Arg Ser Leu Glu Leu Ile Gln Asn Lys Glu Pro Ser Ser Lys His Ile Ser Leu Cys Arg Glu Leu His Thr Leu Phe Arg Val Met Trp Ser Gly Lys Trp Ala Leu Val Ser Pro Phe Ala Met Leu His Ser Val Trp Ser Leu Ile Pro~Ala Phe Arg Gly Tyr Asp Gln Gln Asp Ala Gln Glu Phe Leu Cys Glu Leu Leu His Lys Val Gln Gln Glu Leu Glu Ser Glu Gly Thr Thr Arg Arg Ile Leu Ile Pro Phe Ser Gln Arg Lys Leu Thr Lys Gln Val Leu Lys Val Val Asn Thr Ile Phe His Gly Gln Leu Leu Ser Gln Val Thr Cys Ile Ser Cys Asn Tyr Lys Ser Asn Thr Ile G1u Pro Phe Trp Asp Leu Ser Leu Glu Phe Pro Glu Arg Tyr His Cys Ile Glu Lys Gly Phe Val Pro Leu Asn Gln Thr Glu Cys Leu Leu Thr G1u Met Leu Ala Lys Phe Thr Glu Thr Glu Ala Leu Glu Gly Arg Ile Tyr Ala Cys Asp Gln Cys Asn Ser Lys Arg Arg Lys Ser Asn Pro Lys Pro Leu Val Leu Ser Glu Ala Arg Lys Gln Leu Met Ile Tyr Arg Leu Pro Gln Val Leu Arg Leu His Leu Lys Arg Phe Arg Trp Ser Gly Arg Asn His Arg Glu Lys Ile Gly Val His Val Val Phe Asp Gln Val Leu Thr Met Glu Pro Tyr Cys Cys Arg Asp Met Leu Ser Ser Leu Asp Lys Glu Thr Phe Ala Tyr Asp Leu Ser Ala Val Val Met His His Gly Lys Gly Phe Gly Ser Gly His Tyr Thr Ala Tyr Cys Tyr Asn Thr Glu Gly Gly Phe Trp Val His Cys Asn Asp Ser Lys Leu 560 565 ' 570 Asn Val Cys Ser Va1 Glu Glu Val Cys Lys Thr Gln Ala Tyr Ile Leu Phe Tyr Thr Gln Arg Thr Val Gln Gly Asn Ala Arg Ile Ser Glu Thr His Leu Gln Ala G1n Val Gln Ser Ser Asn Asn Asp Glu Gly Arg Pro Gln Thr Phe Ser <210> 12 <211> 345 <212> PRT
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte ID No: 7482034CD1 <400> 12 Met Lys Arg Gln Leu Thr His Leu Pro Gly Arg Phe Trp Leu Trp Pro Ser Phe Ser Val Ala Ser Leu Leu Ser His Gln Thr Pro Ala Thr Asn Ser Trp Leu Ala Ser Ser Lys Leu His Ser Ala Pro Gly Met Ala Leu Gln Asp Va1 Cys Lys Trp Gln Ser Pro Asp Thr Gln Gly Pro Ser Pro His Leu Pro Arg Ala Gly Gly Trp Ala Val Pro Arg Gly Cys Asp Pro Gln Thr Phe Leu Gln Ile His Gly Pro Arg Leu Ala His Gly Thr Thr Thr Leu Ala Phe Arg Phe Arg His Gly Val Ile Ala Ala Ala Asp Thr Arg Ser Ser Cys Gly Ser Tyr Val Ala Cys Pro Ala Ser Cys Lys Val Ile Pro Val His Gln His Leu Leu Gly Thr Thr Ser Gly Thr Ser Ala Asp Cys Ala Thr Trp Tyr Arg Val Leu Gln Arg Glu Leu Arg Leu Arg Glu Leu Arg Glu Gly Gln Leu Pro Ser Val Ala Ser Ala Ala Lys Leu Leu Ser Ala Met Met Ser Gln Tyr Arg Gly Leu Asp Leu Cys Val Ala Thr Ala Leu Cys Gly Trp Asp Arg Ser Gly Pro Glu Leu Phe Tyr Val Tyr Ser Asp Gly Thr Arg Leu G1n Gly Asp Ile Phe Ser Val Gly Ser Gly Ser Pro Tyr Ala Tyr Gly Val Leu Asp Arg G1y Tyr Arg Tyr Asp Met Ser Thr Gln Glu Ala Tyr Ala Leu Ala Arg Cys Ala Val Ala His Ala Thr His Arg Asp Ala Tyr Ser Gly Gly Ser Val Asp Leu Phe His Val Arg Glu Ser Gly Trp Glu His Val Ser Arg Ser Asp Ala Cys Val Leu Tyr Val Glu Leu Gln Lys Leu Leu Glu Pro Glu Pro Glu G1u Asp Ala Ser His Ala His Pro Glu Pro Ala Thr Ala His Arg Ala Ala Glu Asp Arg Glu Leu Ser Val G1y Pro Gly Glu Val Thr Pro Gly Asp Ser Arg Met Pro Ala Gly Thr Glu Thr Val <210> 13 <211> 948 <212> PRT
<213> Homo sapiens <220>
<221> misc_feature <223> Incyte ID No: 7474351CD1 <400> 13 Met Val Ser Lys Gly Gly Val Ala Ala Glu Pro Glu Pro His Tyr Cys Glu Asp Ser Glu Arg Gly Pro Asn Thr Leu Thr Gly Pro Gly Ser Leu Pro Arg Gly Gly Gly Ile Glu Val G1y Met Glu Phe Pro Gly Cys Ser Gly Glu Gly Cys Val Lys Pro His Glu Glu Ala Ala 50 55 60 , Arg G1u Gly Ala Gly Arg Gly Lys Arg Ala Val Pro Gly Pro Lys Arg Arg Gln Gln Gly Ser Ala Glu Gly Pro Ala A1a Gly Trp Thr Leu Glu Gln Glu Thr Arg Gly Asp Val Leu Glu Asp Lys Asn Glu Arg Ala Asp Glu Glu Ile Leu Arg Leu Ala Pro Gly Lys Gly Arg Leu Pro Ile Asp Ser Lys His Leu Lys Pro Val Ile Ser Ser Phe Pro Val Arg Ser Gln Glu Leu Gly Glu Gly Ala Gly A1a Gly Thr Leu Arg Gly Lys Met Ala Glu Phe Asn Trp Ser Met Ala Phe Lys Gly Pro Ala Ala Gly His Glu Glu Arg Leu Asn Ser Val Ser Ser Arg Ala Lys Lys Gly Ile Gly Trp Asp Va1 Ala Ala Ala Ser Leu Arg Gly Val Asp His Phe Ser Asp Leu Pro Pro Pro Leu Gln Va1 Arg Glu Glu Leu Glu Ala Cys A1a Phe Arg Val Gln Val Gly Gln Leu Arg Leu Tyr Glu Asp Asp Gln Arg Thr Lys Val Val Glu Ile Val Arg His Pro Gln Tyr Asn Glu Ser Leu Ser Ala Gln Gly Gly Ala Asp Ile Ala Leu Leu Lys Leu Glu A1a Pro Val Pro Leu Ser Glu Leu Ile His Pro Val Ser Leu Pro Ser Ala Ser Leu Asp Val Pro Ser Gly Lys Thr Cys Trp Val Thr G1y Trp Gly Val Ile Gly Arg Gly Glu Leu Leu Pro Trp Pro Leu Ser Leu Trp Glu Ala Thr Val Lys Val Arg Ser Asn Val Leu Cys Asn Gln Thr Cys Arg Arg Arg Phe Pro Ser Asn His Thr Glu Arg Phe Glu Arg Leu Ile Lys Asp Asp Met Leu Cys Ala Gly Asp Gly Asn His Gly Ser Trp Pro Gly Asp Asn Gly Gly Pro Leu Leu Cys Arg Arg Asn Cys Thr Trp Val Gln Val Glu Val Val Ser Trp Gly Lys Leu Cys Gly Leu Arg Gly Tyr Pro Gly~Met Tyr Thr Arg Val Thr Ser Tyr Val Ser Trp Ile Arg Gln Pro Cys Pro Ser Ala Gln Thr Pro Ala Val Va1 Arg Arg Phe Val Leu Pro Pro Asn Pro Asp Val Glu Ala Leu Thr Pro Ser Val Met Gly Ser Gly Ala Pro Leu Pro Pro Ala Pro Asp Leu Gln Glu Ala Glu Val Pro Ile Met Arg Thr Arg Ala Cys Glu Arg Met Tyr His Lys Gly Pro Thr Ala His Gly Gln Val Thr Ile Ile Lys Ala Ala Met Pro Cys Ala Gly Arg Lys Gly Gln G1y Ser Cys 485 490 ~ 495 Gln Ala Ala Leu Arg Thr Glu Asp Leu Thr Pro Thr Thr Pro Asn Thr Glu Val Ser Pro Arg Ala Asp Pro Arg Leu Ser Gln Pro Glu Asp Ile Trp Pro Glu Trp Ala Trp Pro Val Val Val Gly Thr Thr Met Leu Leu Leu Leu Leu Phe Leu Ala Val Ser Ser Leu Gly Ser Cys Ser Thr Gly Ser Pro Ala Pro Val Pro Glu Asn Asp Leu Val Gly Ile Val Gly Gly His Asn Thr Pro Gly Glu Val Va1 Val Ala Va1 Gly Ala Asp Arg Arg Ser Leu His Phe Pro Glu Gly His Arg Pro Val His Leu Pro Asp Ser His Gln Gly Cys Va1 Ser Val Arg Gly Pro G1y Ala Ala G1u Cys Gln Pro Asp Arg Arg Pro Pro Asn Tyr Ser Val Phe Phe Leu G1y Ala Asp Ile Ala Leu Leu Lys Leu Ala Thr Ser Ser Leu Glu Phe Thr Asp Ser Asp Asn Cys Trp Asn Thr Gly Trp Gly Met Val Gly Leu Leu Asp Met Leu Pro Pro Pro Tyr Arg Pro Gln Gln Val Lys Val Leu Thr Leu Ser Asn Ala Asp Cys Glu Arg G1n Thr Tyr Asp Ala Phe Pro Gly Ala Gly Asp Arg Lys Phe Ile Gln Asp Asp Met Ile Cys Ala Gly Arg Thr Gly Arg Arg Thr Trp Lys Gly Asp Ser Gly Gly Pro Leu Val Cys Lys Lys Lys Gly Thr Trp Leu Gln Ala Gly Val Val Ser Trp Gly Phe Tyr Ser Asp Arg Pro Ser Ile Gly Val Tyr Thr Arg Pro Glu Thr Ser 755 760 7&5 Trp Gln Gly Ala Asn His Ala Asp Ala Gln Arg Pro Ala Gly Arg Val Pro Thr Met Gln Arg Pro Arg Asp Met Gly Gln Gly Gln Glu Trp Val Cys Arg Pro Phe Thr His Val Thr Cys Tyr Pro Thr Ala Ile Pro Arg Pro Phe Thr His Val Thr Cys Tyr Leu Met Ala Val Pro Ser Thr Leu Thr His Val Thr Cys Tyr Pro Thr Ala Val Pro Arg Pro Phe Thr His Val Thr Cys Tyr Leu Met A1a Val Pro Ser Thr Leu Thr His Ile Thr Cys Tyr Met Met Ala Val Pro Arg Pro Phe Thr His Ile Thr Cys Tyr Pro Met Ala Val Pro Ser Thr Leu Thr His Val Thr Cys His Pro Thr Ala Ile Pro Arg Pro Phe Thr His Ile Thr Cys Tyr Thr Met Ala Ile Pro Arg Pro Ser Thr Thr Pro Pro Ala Thr Arg Arg Pro Ser Pro Ala Pro Ser Pro Thr Ser Pro Ala Thr Arg Trp Pro Ser Pro Gly Pro Ser Pro Met Ser Pro Ala Thr Arg <210> 14 <21l> 444 <212> PRT
<213> Homo sapiens <220>
<221> misc_feature <223> Incyte ID No: 2232483CD1 <400> 14 Met Gly Ser Ala Pro Trp Ala Pro Val Leu Leu Leu Ala Leu Gly Leu Arg Gly Leu Gln Ala Gly Ala Arg Arg Ala Pro Asp Pro Gly Phe Gln Glu Arg Phe Phe Gln Gln Arg Leu Asp His Phe Asn Phe Glu Arg Phe Gly Asn Arg Thr Phe Pro Gln Arg Phe Leu Val Ser Asp Arg Phe Trp Val Arg Gly Glu Gly Pro Ile Phe Phe Tyr Thr Gly Asn Glu Gly Asp Val Trp Ala Phe Ala Asn Asn Sex Ala Phe Val Ala Glu Leu Ala A1a Glu Arg Gly Ala Leu Leu Val Phe Ala Glu His Arg Tyr Tyr Gly Lys Ser Leu Pro Phe Gly Ala Gln Ser Thr Gln Arg Gly His Thr Glu Leu Leu Thr Val Glu Gln Ala Leu Ala Asp Phe Ala Glu Leu Leu Arg Ala Leu Arg Arg Asp Leu Gly Ala Gln Asp Ala Pro Ala Ile Ala Phe Gly Gly Ser Tyr Gly Gly Met Leu Ser Ala Tyr Leu Arg Met Lys Tyr Pro His Leu Val Ala Gly A1a Leu Ala Ala Ser Ala Pro Val Leu Ala Val Ala Gly Leu Gly Asp Ser Asn Gln Phe Phe Arg Asp Val Thr Ala Gly Ala Tyr Asp Thr Val Arg Trp Glu Phe Gly Thr Cys Gln Pro Leu Ser Asp Glu Lys Asp Leu Thr Gln Leu Phe Met Phe Ala Arg Asn Ala Phe Thr Val Leu Ala Met Met Asp Tyr Pro Tyr Pro Thr Asp Phe Leu Gly Pro Leu 'Pro Ala Asn Pro Val Lys Val Gly Cys Asp Arg Leu Leu Ser Glu Ala Gln Arg Ile Thr Gly Leu Arg Ala Leu Ala Gly Leu Val Tyr Asn Ala Ser Gly Ser Glu His Cys Tyr Asp Ile Tyr Arg Leu Tyr His Ser Cys Ala Asp Pro Thr Gly Cys Gly Thr Gly Pro Asp Ala Arg Ala Trp Asp Tyr Gln Ala Cys Thr Glu Ile Asn Leu Thr Phe Ala Ser Asn Asn Val Thr Asp Met Phe Pro Asp Leu Pro Phe Thr Asp Glu Leu Arg Pro Ser Asp Leu Arg Ala Ala Ser Asn Ile Ile Phe Ser Asn Gly Asn Leu Asp Pro Cys Gly Arg Gly G1y Ile Arg Arg Asn Leu Ser Ala Ser Val Ile Ala Val Thr Ile Gln G1y Gly Ala His His Leu Asp Leu Arg Ala Ser His Pro Glu Asp Pro Ala Ser Val Val Glu Ala Arg Lys Leu Glu A1a Thr Ile Ile Gly Glu Cys Val Lys Ala Ala Arg Arg Glu Gln G1n Pro A1a Leu Arg Trp Gly Ala Gln Ile Ser Leu <210> 15 <211> 514 <212> PRT
<213> Homo sapiens <220>
<221> misc_feature <223> Incyte ID No: 7481712CD1 <400> 15 Met Arg Val Pro Phe Ser Glu Leu Lys Asp Ile Lys Ala Tyr Leu Glu Ser His Gly Leu Ala Tyr Ser Ile Met Ile Lys Asp Ile Gln Val Lys Pro Cys Pro Ser Trp Asp Pro Ala Phe Arg Leu Pro Phe Trp Leu Gly Pro Asn Met Glu Glu Met Phe Ser Gly Leu Lys Val Asp Met Trp Phe Leu Gly Leu His Gln Arg Val Cys Glu His Ala Val Glu Gly Thr Gly Cys Pro Pro Pro His Phe Thr Lys Ala Ser Leu Asp Asn Val Thr Arg Asn Phe Gln Ile Gln Pro Asp Gly Arg Leu Ser Met Phe Leu Phe Gln Gln His Asn Trp Ser Leu Ser Pro Ser Trp Ser Leu Ser Leu Pro Leu Ala Ser Arg Thr Ser Val Phe Cys Leu Gln Pro Ala Pro Pro Leu Leu Asp Pro Thr Ala Tyr Ser Val Phe Pro Pro Gly Gly Ala Met Gly Ile Ser Asn Phe Pro Ala Pro Gly Met Glu Gln Thr Leu Val His Phe Pro Gly Gln Gly Arg Phe Leu Phe Leu Glu Val Gly Pro Ala Val Leu Leu Asp Glu Glu Arg Gln Ala Met Ala Lys Ser Arg Arg Leu Glu Arg Ser Thr Asn Ser Phe Ser Tyr Ser Ser Tyr His Thr Leu Glu Glu Ile Tyr Ser Trp Ile Asp Asn Phe Val Met Glu His Ser Asp Ile Val Ser Lys Ile Gln Ile Gly Asn Ser Phe Glu Asn Gln Ser Ile Leu Val Leu Lys Phe Ser Thr Gly Gly Ser Arg His Pro Ala Ile Trp Ile Asp Thr Gly Ile His Ser Arg Glu Trp Ile Thr His Ala Thr Gly Ile Trp Thr Ala Asn Lys Ile Va1 Ser Asp Tyr Gly Lys Asp Arg Va1 Leu Thr Asp Ile Leu Asn Ala Met Asp Ile Phe Ile Glu Leu Val Thr Asn Pro Asp Gly Phe Ala Phe Thr His Ser Met Asn Arg Leu Trp Arg Lys Asn Lys Ser Ile Arg Pro Gly Ile Phe Cys Ile Gly Val Asp Leu Asn Arg Asn Trp Lys Ser G1y Phe Gly Gly Asn Gly Ser Asn Ser Asn Pro Cys Ser Glu Thr Tyr His Gly Pro Ser Pro Gln Ser Glu Pro G1u Val Ala Ala Ile Val Asn Phe Tle Thr Ala His Gly Asn Phe Lys Ala Leu Ile Ser Ile His Ser Tyr Ser Gln Met Leu Met Tyr Pro Tyr Gly Arg Leu Leu Glu Pro Val Ser Asn Gln Arg Glu Leu Tyr Asp Leu Ala Lys Asp Ala Val Glu Ala Leu Tyr Lys Val His Gly Ile Glu Tyr Ile Phe Gly Ser Ile Ser Thr Thr Leu Tyr Val Ala Ser Gly Ile Thr Val Asp Trp Ala Tyr Asp Ser Gly Ile Lys Tyr Ala Phe Ser Phe Glu Leu Arg Asp Thr Gly Gln Tyr Gly Phe Leu Leu Pro Ala Thr Gln I1e Ile Pro Thr Ala Gln Glu Thr Trp Met Ala Leu Arg Thr Ile Met Glu His Thr Leu Asn His Pro Tyr <210> 16 <211> 787 <212> PRT
<213> Homo Sapiens <220>
<221> misc feature <223> Incyte ID No: 8213480CD1 <400> 16 Met Gly Trp Arg Pro Arg Arg Ala Arg Gly Thr Pro Leu Leu Leu Leu Leu Leu Leu Leu Leu Leu Trp Pro Val 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 G1n Glu Leu Leu Leu Glu Leu Glu Lys Asn His Arg Leu Leu A1a 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 Gly 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 G1y Gly Pro Gln Ser Arg G1y Arg Arg Glu Ala Arg Arg Thr Arg Lys Tyr Leu Glu 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 Val Thr Gln Asp Ala Asn Ala Thr Leu Trp Ala Phe Leu Gln Trp Arg Arg Gly Leu Trp Ala 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 Ala Glu Ser Ser Gly Gly Val Ser Thr Asp His Ser Glu Leu Pro Ile Gly Ala Ala Ala Thr Met Ala His Glu Ile Gly His Ser Leu Gly Leu Ser His Asp Pro Asp Gly Cys Cys Va1 Glu Ala Ala Ala Glu Ser Gly Gly Cys Val Met Ala Ala Ala Thr Gly His Pro Phe Pro Arg Val Phe Ser Ala Cys Ser Arg Arg Gln Leu Arg Ala Phe Phe Arg Lys Gly Gly Gly Ala Cys Leu Ser Asn Ala Pro Asp Pro Gly Leu Pro Val Pro Pro Ala Leu Cys Gly Asn Gly Phe Val Glu Ala Gly Glu Glu Cys Asp Cys G1y Pro Gly Gln Glu Cys Arg Asp Leu Cys Cys Phe Ala His Asn Cys Ser Leu Arg Pro GIy Ala Gln Cys Ala His Gly Asp Cys Cys Val Arg Cys Leu Leu Lys Pro Ala Gly 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 A1a 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 Val Val Asn Ser Ala Gly Asp Ala His Gly Asn Cys Gly Gln Asp Ser Glu Gly His Phe Leu Pro Cys Ala Gly Arg Asp Ala Leu Cys Gly 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 Gly Pro Arg Met Val Cys Asn Ser Asn His Asn Cys His Cys 635 640 645"~
Ala Pro Gly Trp Ala Pro Pro Phe Cys Asp Lys Pro Gly Phe Gly Gly Ser Met Asp Ser Gly 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 A1a Cys Ser Gly Pro Lys Asp Gly Pro His Arg Asp His Pro Leu Gly Gly Val His Pro Thr Glu Leu Gly Pro Thr Ala Thr Gly Gln Ser Trp Pro Leu Asp Pro Glu Asn Ser His Glu Pro Ser Ser His Pro Glu Lys Pro Leu Pro A1a Val Ser Pro Asp Pro G1n Ala Asp Gln Val Gln Met Pro Arg Ser Cys Leu Trp <210> 17 <211> 1082 <212> PRT
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte ID No: 7478405CD1 <400> 17 Met Glu Cys Ala Leu Leu Leu Ala Cys Ala Phe Pro Ala Ala Gly Ser Gly Pro Pro Arg Gly Leu Ala Gly Leu Gly Arg Val Ala Lys Ala Leu G1n Leu Cys Cys Leu Cys Cys Ala Ser Val Ala Ala Ala Leu Ala Ser Asp Ser Ser Ser Gly Ala Ser Gly Leu Asn Asp Asp Tyr Val Phe Val Thr Pro Val Glu Val Asp Ser Ala Gly Ser Tyr Ile Ser His Asp Ile Leu His Asn Gly Arg Lys Lys Arg Ser Ala Gln Asn Ala Arg Ser Ser Leu His Tyr Arg Phe Ser Ala Phe Gly Gln Glu Leu His Leu Glu Leu Lys Pro Ser A1a Ile Leu Ser Ser His Phe Ile Val Gln Val Leu Gly Lys Asp Gly Ala Ser Glu Thr Gln Lys Pro Glu Val Gln Gln Cys Phe Tyr GIn Gly Phe IIe Arg Asn Asp Ser Ser Ser Ser Val Ala Val Ser Thr Cys Ala Gly Leu Ser Gly Leu Ile Arg Thr Arg Lys Asn Glu Phe Leu Ile Ser Pro Leu Pro GIn Leu Leu Ala Gln Glu His Asn Tyr Ser Ser Pro Ala Gly His His Pro His Val Leu Tyr Lys Arg Thr Ala Glu Glu Lys Ile Gln Arg Tyr Arg Gly Tyr Pro Gly Ser Gly Arg Asn Tyr Pro 2l5 220 225 Gly Tyr Ser Pro Ser His Ile Pro His Ala Ser Gln Ser Arg Glu Thr Glu Tyr His His Arg Arg Leu Gln Lys Gln His Phe Cys Gly Arg Arg Lys Lys Tyr Ala Pro Lys Pro Pro Thr Glu Asp Thr Tyr Leu Arg Phe Asp Glu Tyr Gly Ser Ser Gly Arg Pro Arg Arg Ser Ala Gly Lys Ser Gln Lys Gly Leu Asn Val Glu Thr Leu Val Val Ala Asp Lys Lys Met Val Glu Lys His Gly Lys Gly Asn Val Thr 305 310 3l5 Thr Tyr Ile Leu Thr Val Met Asn Met Val Ser Gly Leu Phe Lys Asp Gly Thr Ile Gly Ser Asp Ile Asn Val Val Val Val Ser Leu Ile Leu Leu Glu Gln Glu Pro Gly Gly Leu Leu Ile Asn His His Ala Asp Gln Ser Leu Asn Ser Phe Cys Gln Trp Gln Ser Ala Leu Ile Gly Lys Asn Gly Lys Arg His Asp His Ala Ile Leu Leu Thr Gly Phe 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 Ile Ala His Glu Ser Gly His Asn Phe Gly Met Ile His Asp Gly Glu Gly Asn Pro Cys Arg Lys Ala Glu Gly Asn Ile Met Ser Pro Thr Leu Thr Gly Asn Asn Gly Val Phe Ser Trp Ser Ser Cys Ser Arg Gln Tyr Leu Lys Lys Phe Leu Ser Thr Pro Gln Ala Gly Cys Leu Val Asp Glu Pro Lys Gln A1a Gly Gln Tyr Lys Tyr Pro Asp Lys Leu Pro Gly Gln Ile Tyr Asp Ala~Asp Thr Gln Cys Lys Trp Gln Phe Gly Ala Lys Ala Lys Leu Cys Ser Leu Gly Phe Val Lys Asp Ile Cys Lys Ser Leu Trp Cys His Arg Val Gly His Arg Cys Glu Thr Lys Phe Met Pro Ala Ala Glu Gly Thr Val Cys Gly Leu Ser Met Trp Cys Arg Gln Gly Gln Cys Val Lys Phe Gly Glu Leu Gly Pro Arg Pro Ile His Gly Gln Trp Ser Ala Trp Ser Lys Trp Ser Glu Cys Ser Arg Thr Cys Gly Gly Gly Val Lys Phe Gln Glu Arg His Cys Asn Asn Pro Lys Pro G1n Tyr Gly Gly Ile Phe Cys Pro Gly Ser Ser Arg Ile Tyr Gln Leu Cys Asn Ile Asn Pro Cys Asn Glu Asn Ser Leu Asp Phe Arg Ala Gln Gln Cys Ala Glu Tyr Asn Ser Lys Pro Phe Arg Gly Trp Phe Tyr Gln Trp Lys Pro Tyr Thr Lys Val Glu Glu Glu Asp Arg Cys Lys Leu Tyr Cys Lys Ala Glu Asn 680 685 ~ 690 Phe Glu Phe Phe Phe Ala Met Ser Gly Lys Val Lys Asp Gly Thr Pro Cys Ser Pro Asn Lys Asn Asp Val Cys Ile Asp Gly Val Cys Glu Leu Val Gly Cys Asp His Glu Leu Gly Ser Lys Ala Val Ser Asp Ala Cys Gly Val Cys Lys GIy Asp Asn Ser Thr Cys Lys Phe Tyr Lys Gly Leu Tyr Leu Asn Gln His Lys Ala Asn Glu Tyr Tyr Pro Val Val Ile Ile Pro Ala Gly Ala Arg Ser Ile Glu Ile Gln Glu Leu Gln Val Ser Ser Ser Tyr Leu Ala Val Arg Ser Leu Ser Gln Lys Tyr Tyr Leu Thr Gly Gly Trp Ser Ile Asp Trp Pro Gly Glu Phe Pro Phe Ala Gly Thr Thr Phe Glu Tyr Gln Arg Ser Phe Asn Arg Pro Glu Arg Leu Tyr Ala Pro Gly Pro Thr Asn Glu Thr Leu Val Phe Glu Ile Leu Met Gln Gly Lys Asn Pro Gly Ile Ala Trp Lys Tyr Ala Leu Pro Lys Val Met Asn Gly Thr Pro Pro Ala Thr Lys Arg Pro Ala Tyr Thr Trp Ser Ile Val Gln Ser Glu Cys Ser Val Ser Cys Gly Gly Gly Tyr Ile Asn Val Lys Ala Ile Cys Leu Arg Asp Gln Asn Thr Gln Val Asn Ser Ser Phe Cys Ser Ala Lys Thr Lys Pro Val Thr Glu Pro Lys Ile Cys Asn Ala Phe Ser Cys Pro Ala Tyr Trp Met Pro Gly Glu Trp Ser Thr Cys Ser Lys Ala Cys Ala G1y Gly Gln Gln Ser Arg Lys Ile Gln Cys Val Gln Lys Lys Pro Phe Gln Lys Glu Glu Ala Val Leu His Ser Leu Cys 965 970 g75 Pro Val Ser Thr Pro Thr Gln Val Gln Ala Cys Asn Ser His Ala Cys Pro Pro Gln Trp Ser Leu Gly Pro Trp Ser Gln Cys Ser Lys 995 1000 ~ 1005 Thr Cys Gly Arg Gly Val Arg Lys Arg Glu Leu Leu Cys Lys Gly Ser Ala Ala Glu Thr Leu Pro Glu Ser Gln Cys Thr Ser Leu Pro Arg Pro Glu Leu Gln Glu Gly Cys Val Leu Gly Arg Cys Pro Lys Asn Ser Arg Leu Gln Trp Val Ala Ser Ser Trp Ser Glu Va1 Leu Ile Arg Ser His Cys Trp Val Arg Arg Leu Arg Pro Ser Trp Leu Thr Gln <210> 18 <211> 1187 <212> DNA
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte ID No: 6930294CB1 <400> 18 ccgcaacctt gaagcggcat ccgtggagtg cgcctgcgca gctacgaccg cagcaggaaa 60 gcgccgccgg ccaggcccag ctgtggccgg acagggactg gaagagagga cgcggtcgag 120 taggtgtgca ccagccctgg caacgagagc gtctaccccg aactctgctg gccttgaggt 180 tttaaaacat gaatcctaca ctcatccttg ctgccttttg cctgggaatt gcctcagcta 240 ctctaacatt tgatcacagt ttagaggcac agtggaccaa gtggaaggcg atgcacaaca 300 gattatacgg catgaatgaa gaaggatgga ggagagcagt gtgggagaag aacatgaaga 360 tgattgaact gcacaatcag gaatacaggg aagggaaaca cagcttcaca atggccatga 420 acgcctttgg agacatgacc agtgaagaat tcaggcaggt gatgaatggc tttcaaaacc 480 gtaagcccag gaaggggaaa gtgttccagg aacetctgtt ttatgaggcc cccagatctg 540 tggattggag agagaaaggc tacgtgactc ctgtgaagaa tcagggtcag tgtggttctt 600 gttgggcttt tagtgctact ggtgctcttg aaggacagat gttccggaaa actgggaggc 660 ttatctcact gagtgagcag aatctggtag actgctctgg gcctcaaggc aatgaaggct 720 gcaatggtgg cctaatggat tatgctttcc agtatgttca ggatactgga ggcctggact 780 ctgaggaatc ctatccatat gaggcaacag aagaatcctg taggtacaat cccaagtatt 840 ctgctgctaa tgacactggc tttgtggaca tcccttcaca ggagaaggac ctggcgaagg 900 cagtggcaac tgtggggccc atctctgttg ctgctggtgc aagccatgtc tccttccagt 960 tctataaaaa aggtatttat tttgagccac gctgtgaccc cgaaggtctg gatcatgcta 1020 tgctgctggt tggctacagc tatgaaggag cagactcaga taacaataaa tattggctgg 1080 tgaagaacag gtatggtaaa aactggggca tggatggcta cataaagatg gccaaagacc 1140 agaggaacaa ctgtggaatt gccacagcag ccagctaccc cactgtg 1187 <210> 19 <211> 461 <212> DNA
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte ID No: 7473018CB1 <400> 19 aagaattcgg cacgagggcc atggctgacc aactcttgcg taaaaagaga agaattttta 60 tccattcagt gggtgcaggc acaataaatg ccttgctgga ttgcctatta gaggatgaag 120 ttattagcca ggaagacatg aacaaagtga gagatgaaaa tgacactgtc atggataagg 180 ctcgagtctt gattgacctt gttactggaa aaggacccaa gtcttgctgc aaatttatca 240 agcatctctg tgaagaagac cctcaacttg cctcaaagat gggtttgcac taagagagaa 300 gatggaactc tggagcactt cagagacttc ccagagcttc ttccaaggga gaagatattc 360 tcgtgaaaga aaaaaacaaa acaaaacaac agtgcttttt tcaaacctga ttaatttcat 420 caatttccaa taaatctttc attctctcaa aaaaaaaaaa a 461 <210> 20 <211> 1884 <212> DNA
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte ID No: 7479221CB1 <400> 20 atgtcccagc tctcctccac cctgaagcgc tacacagaat cggcccgcta cacagatgcc 60 cactatgcca agtcgggcta tggtgcctac accccatcct cctatggggc caatctggct 120 gcctccttac tggagaagga gaaacttggt ttcaagccgg tccccaccag cagcttcctc 180 acccgtcccc gtacctatgg cccctcctcc ctcctggact atgaccgggg ccgccccctg 240 ctgagacccg acatcactgg gggtggtaag cgggcagaga gccagacccg gggtactgag 300 cggcctttag gcagtggcct cagcgggggc agcggattcc cttatggagt gaccaacaac 360 tgcctcagct acctgcccat caatgcctat gaccaggggg tgaccctaac ccagaagctg 420 gacagccaat cagacctggc ccgggatttc tccagcctcc ggacctcaga tagctaccgg 480 atagacccca ggaacctggg ccgcagcccc atgctggccc ggacgcgcaa ggagctctgc 540 accctgcagg ggctctacca gacagccagc tgccctgaat acctggtcga ctacctggag 600 aactatggtc gcaagggcag tgcatctcag gtgccctccc aggcccctcc ctcacgagtc 660 cctgaaatca tcagcccaac ctaccgaccc attggccgct acacgctgtg ggagacggga 720 aagggtcagg cccctgggcc cagccgctcc agctccccgg gaagagacgg catgaattct 780 aagagtgccc agggtctggc tggtcttcga aaccttggga acacgtgctt catgaactca 840 attctgcagt gcctgagcaa cactcgggag ttgagagatt actgcctcca gaggctctac 900 atgcgggacc tgcaccacgg cagcaatgca cacacagccc tcgtggaaga gtttgcaaaa 960 ctaattcaga ccatatggac ttcatccccc aatgatgtgg tgagcccatc tgagttcaag 1020 acccagatcc agagatatgc accgcgcttt gttggctata atcagcagga tgctcaggag 1080 ttccttcgct ttcttctgga tgggctccat aacgaggtga accgagtgac actgagacct 1140 aagtccaacc ctgagaacct cgatcatctt cctgatgacg agaaaggccg acagatgtgg 1200 agaaaatatc tagaacggga agacagtagg atcggggatc tctttgttgg gcagctaaag 1260 agctcgctga cgtgtacaga ttgtggttac tgttctacgg tcttcgaccc cttctgggac 1320 ctctcactgc ccattgctaa gcgaggttat cctgaggtga cattaatgga ctgcatgagg 1380 ctcttcacca aagaggatgt gcttgatgga gatgaaaagc caacatgctg tcgctgccga 1440 ggcagaaaac ggtgtataaa gaagttctcc atccagaggt tcccaaagat cttggtgctc 1500 catctgaagc ggttctcaga atccaggatc cgaaccagca agctcacaac atttgtgaac 1560 ttccccctaa gagaectgga cttaagagaa tttgcctcag aaaacaccaa ccatgctgtt 1620 tacaacctgt acgctgtgtc caatcactcc ggaaccacca tgggtggcca ctatacagcc 1680 tactgtcgca gtccagggac aggagaatgg cacactttca acgactccag cgtcactccc 1740 atgtcctcca gccaagtgcg caccagcgac gcctacctgc tcttctacga actggccagc 1800 ccgccctccc gaatgtagcg ccaggagcca cgtcccttct cccttccccg tggtggcccc 1860 gctccctaaa ttttttaaaa aaac 1884 <210> 21 <211> 2576 <212> DNA
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte ID No: 2923874CB1 <400> 21 tagacaaaag aaaaatccaa agggaaaatg ctgatctctg gaatactttg gacattcatg 60 catcaaaagc caactgcaag ccactatttg caagtcaagt ctcaagatgg tatactatct 120 ccaggaaaag gcttggaaga tacagatgtg gtgtataaaa gcgagaatgg acatgtcatt 180 aaactgaata tagaaacaaa tgctaccaca ttattattgg aaaacacaac ttttgtaacc 240 ttcaaagcat caagacattc agtttcacca gatttaaaat atgtccttct ggcatatgat 300 gtcaaacaga tttttcatta ttcgtatact gcttcatatg tgatttacaa catacacact 360 agggaagttt gggagttaaa tcctccagaa gtagaggact ccgtcttgca gtacgcggcc 420 tggggtgtcc aagggcagca gctgatttat atttttgaaa ataatatcta ctatcaacct 480 gatataaaga gcagttcatt gcgactgaca tcttctggaa aagaagaaat aatttttaat 540 gggattgctg actggttata tgaagaggaa ctcctgcatt ctcacatcgc ccactggtgg 600 tcaccagatg gagaaagact tgccttcctg atgataaatg actctttggt acccaccatg 660 gttatccctc ggtttactgg agcgttgtat cccaaaggaa agcagtatcc gtatcctaag 720 gcaggtcaag tgaacccaac aataaaatta tatgttgtaa acctgtatgg accaactcac 780 actttggagc tcatgccacc tgacagcttt aaatcaagag aatactatat cactatggtt 840 aaatgggtaa gcaataccaa gactgtggta agatggttaa accgacctca gaacatctcc 900 atcctcacag tctgtgagac cactacaggt gcttgtagta aaaaatatga gatgacatca 960 gatacgtggc tctctcagca gaatgaggag cccgtgtttt ctagagacgg cagcaaattc 1020 tttatgacag tgcctgttaa gcaaggggga cgtggagaat ttcaccacat agctatgttc 1080 ctcatccaga gtaaaagtga gcaaattacc gtgcggcatc tgacatcagg aaactgggaa 2140 gtgataaaga tcttggcata cgatgaaact actcaaaaaa tttactttct gagcactgaa 1200 tcttctccca gaggaaggca gctgtacagt gcttctactg aaggattatt gaatcgccaa 1260 tgcatttcat gtaatttcat gaaagaacaa tgtacatatt ttgatgccag ttttagtccc 1320 atgaatcaac atttcttatt attctgtgaa ggtccaaggg tcccagtggt cagcctacat 1380 agtacggaca acccagcaaa atattttata ttggaaagca attctatgct gaaggaagct 1440 atcctgaaga agaagatagg aaagccagaa attaaaatcc ttcatattga cgactatgaa 1500 cttcctttac agttgtccct tcccaaagat tttatggacc gaaaccagta tgctcttctg 1560 ttaataatgg atgaagaacc aggaggccag ctggttacag ataagttcca tattgactgg 1620 gattccgtac tcattgacat ggataatgtc attgtagcaa gatttgatgg cagaggaagt 1680 ggattccagg gtctgaaaat tttgcaggag attcatcgaa gattaggttc agtagaagta 1740 aaggaccaaa taacagctgt gaaatttttg ctgaaactgc cttacattga ctccaaaaga 1800 ttaagcattt ttggaaaggg ttatggtggc tatattgcat caatgatctt aaaatcagat 1860 gaaaagcttt ttaaatgtgg atccgtggtt gcacctatca cagacttgaa attgtatgcc 1920 tcagctttct ctgaaagata ccttgggatg ccatctaagg aagaaagcac ttaccaggca 1980 gccagtgtgc tacataatgt tcatggcttg aaagaagaaa atatattaat aattcatgga 2040 actgctgaca caaaagttca tttccaacac tcagcagaat taatcaagca cctaataaaa 2100 gctggagtga attatactat gcaggtctac ccagatgaag gtcataacgt atctgagaag 2160 agcaagtatc atctctacag cacaatcctc aaattcttca gtgattgttt gaaggaagaa 2220 atatctgtgc taccacagga accagaagaa gatgaataat ggaccgtatt tatacagaac 2280 tgaagggaat attgaggctc aatgaaacct gacaagagac tgtaatattg tagttgctcc 2340 agaatgtcaa gggcagctta cggagatgtc actggagcag cacgctcaga gacagtgaac 2400 tagcatttga atacacaagt ccaagtctac tgtgttgcta ggggtgcaga acccgtttct 2460 ttgtatgaga gaggtcaagg gttggtttcc tgggagaaat tagttttgca ttaaagtagg 2520 agtagtgcat gttttcttct gttatccccc tgtttgttct gtaactagtt ctctca 2576 <210> 22 <211> 2000 <212> DNA.
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte ID No: 55122335CB1 <400> 22 gctctgcggc catggcgagc ggcgagcatt cccccggcag cggcgcggcc cggcggccgc 60 tgcactccgc gcaggctgtg gacgtggcct cggcctccaa cttccgggcc tttgagctgc 120 tgcacttgca cctggacctg cgggctgagt tcgggcctcc agggcccggc gcagggagcc 180 gggggctgag cggcaccgcg gtcctggacc tgcgctgcct ggagcccgag ggcgccgccg 240 agctgcggct ggactcgcac ccgtgcctgg aggtgacggc ggcggcgctg cggcgggagc 300 ggcccggctc ggaggagccg cctgcggagc ccgtgagctt ctacacgcag cccttctcgc 360 actatggcca ggccctgtgc gtgtccttcc cgcagccctg ccgcgccgcc gagcgcctcc 420 aggtgctgct cacctaccgc gtcggggagg gacccggggt ttgctggttg gctcccgagc 480 agacagcagg aaagaagaag cccttcgtgt acacccaggg ccaggctgtc ctaaaccggg 540 ccttcttccc ttgcttcgac acgcctgctg ttaaatacaa gtattcagct cttattgagg 600 tcccagatgg cttcacagct gtgatgagtg ctagcacctg ggagaagaga ggtccaaata 660 agttcttctt ccagatgtgt cagcccatcc cctcctatct gatagctttg gccatcggag 720 atctggtttc ggctgaagtt ggacccagga gccgggtgtg ggctgagccc tgcctgattg 780 atgctgccaa ggaggagtac aacggggtga tagaagaatt tttggcaaca ggagagaagc 840 tttttggacc ttatgtttgg ggaaggtatg acttgctctt catgccaccg tcctttccat 900 ttggaggaat ggagaaccct tgtctgacct ttgtcacccc ctgcctgcta gctggggacc 960 gctccttggc agatgtcatc atccatgaga tctcccacag ttggtttggg aacctggtca 1020 ccaacgccaa ctggggtgaa ttctggctca atgaaggttt caccatgtac gcccagagga 1080 ggatctccac catcctcttt ggcgctgcgt acacctgctt ggaggctgca acggggcggg 1140 ctctgctgcg tcagcacatg gacatcactg gagaggaaaa cccactcaac aagctccgcg 1200 tgaagattga accaggcgtt gacccggacg acacctataa tgagaccccc tacgagaaag 1260 gtttctgctt tgtctcatac ctggcccact tggtgggtga tcaggatcag tttgacagtt 1320 ttctcaaggc ctatgtgcat gaattcaaat tccgaagcat cttagccgat gactttctgg 1380 acttctactt ggaatatttc cctgagctta agaaaaagag agtggatatc attccaggtt 1440 ttgagtttga tcgatggctg aatacccccg gctggccccc gtacctccct gatctctccc 1500 ctggggactc actcatgaag cctgctgaag agctagccca actgtgggca gccgaggagc 1560 tggacatgaa ggccattgaa gccgtggcca tctctccctg gaagacctac cagctggtct 1620 acttcctgga taagatcctc cagaaatccc ctctccctcc tgggaatgtg aaaaaacttg 1680 gagacacata cccaagtatc tcaaatgccc ggaatgcaga gctccggctg cgatggggcc 1740 aaatcatcct taagaacgac caccaggaag atttctggaa agtgaaggag ttcctgcata 1800 accaggggaa gcagaagtat acacttccgc tgtaccacgc aatgatgggt ggcagtgagg 1860 tggcccagac cctcgccaag gagacttttg catccaccgc ctcccagctc cacagcaatg 1920 ttgtcaacta tgtccagcag atcgtggcac ccaagggcag ttagaggctc gtgtgcatgg 1980 cccctgcctc ttcaggctct 2000 <210> 23 <211> 3522 <212> DNA
<213> Homo sapiens~-<220>
<221> misc feature <223> Incyte ID No: 7473550CB1 <400> 23 ggagggagga cgtgcgaggc cggtgcgtgg aggctatggg cctgctggcc agtgctggtt 60 tgttgctgtt gctggtcatc ggccacccca gaagcctagg actgaagtgt ggaattcgca 120 tggtcaacat gaaaagtaag gaacctgccg tgggatctag attcttctct agaattagta 180 gttggagaaa ttcaacagtg actggacatc catggcaggt ctacctaaaa tcagatgagc 240 accacttctg tggaggaagc ttgattcaag aagatcgggt tgttacagca gcacactgcc 300 tgcacagcct cagtgagaag cagctgaaga atataactgt gacttctggg gagtacagcc 360 tctttcagaa ggataagcaa gaacagaata ttcctgtctc aaaaattatt acccatcctg 420 aatacaacag ccgtgaatat atgagtcctg atattgcact gctgtatcta aaacacaaag 480 tcaagtttgg aaatgctgtt cagccaatct gtcttcctga cagcgatgat aaagttgaac 540 caggaattct ttgcttatcc agtggatggg gcaagatttc caaaacatca gaatattcaa 600 atgtcctaca agaaatggaa cttcccatca tggatgacag agcgtgtaat actgtgctca 660 agagcatgaa cctccctccc ctgggaagga ccatgctgtg tgctggcttc cctgattggg 720 gaatggacgc ctgccagggg gactctggag gaccactggt ttgtagaaga ggtggtggaa 780 tctggattct tgctgggata acttcctggg tagctggttg tgctggaggt tcagttcccg 840 taagaaacaa ccatgtgaag gcatcacttg gcattttctc caaagtgtct gagttgatgg 900 attttatcac tcaaaacctg ttcacaggtt tggatcgggg ccaacccctc tcaaaagtgg 960 gctcaaggta tataacaaag gccctgagtt ctgtccaaga agtgaatgga agccagagag 1020 ataaaataat cctgataaaa tttacaagtt tagacatgga aaagcaagtt ggatgtgatc 1080 atgactatgt atctttacga tcaagcagtg gagtgctttt tagtaaggtc tgtggaaaaa 1140 tattgccttc accattgctg gcagagacca gtgaggccat ggttccattt gtttctgata 1200 cagaagacag tggcagtggc tttgagctta ccgttactgc tgtacagaag tcagaagcag 1260 ggtcaggttg tgggagtctg gctatattgg tagaagaagg gacaaatcac tctgccaagt 1320 atcctgattt gtatcccagt aacacaaggt gtcattggtt catttgtgct ccagagaagc 1380 acattataaa gttgacattt gaggactttg ctgtcaaatt tagtccaaac tgtatttatg 1440 atgctgttgt gatttacggt gattctgaag aaaagcacaa gttagctaaa ctttgtggaa 1500 tgttgaccat cacttcaata ttcagttcta gtaacatgac ggtgatatac tttaaaagtg'1560 atggtaaaaa tcgtttacaa ggcttcaagg ccagatttac cattttgccc tcagagtctt 1620 taaacaaatt tgaaccaaag ttacctcccc aaaacaatcc tgtatctacc gtaaaagcta 1680 ttctgcatga tgtctgtggc atccctccat ttagtcccca gtggctttcc agaagaatcg 1740 caggagggga agaagcctgc ccccactgtt ggccatggca ggtgggtctg aggtttctag 1800 gcgattacca atgtggaggt gccatcatca acccagtgtg gattctgacc gcagcccact 1860 gtgtgcaatt gaagaataat ccactctcct ggactattat tgctggggac catgacagaa 1920 acctgaagga atcaacagag caggtgagaa gggccaaaca cataatagtg catgaagact 1980 ttaacacact aagttatgac tctgacattg ccctaataca actaagctct cctctggagt 2040 acaactcggt ggtgaggcca gtatgtctcc cacacagcgc agagcctcta ttttcctcgg 2100 agatctgtgc tgtgaccgga tggggaagca tcagtgcaga gctctctctg aatgtttctt 2160 cattagatgg tggcctagca agtcgcctac agcagattca agtgcatgtg ttagaaagag 2220 aggtctgtga acacacttac tattctgccc atccaggagg gatcacagag aagatgatct 2280 gtgctggctt tgcagcatct ggagagaaag atttctgcca gggagactct ggtgggccac 2340 .
tagtatgtag acatgaaaat ggtccctttg tcctctatgg cattgtcagc tggggagctg 2400 gctgtgtcca gccatggaag ccgggtgtat ttgccagagt gatgatcttc ttggactgga 2460 tccaatcaaa aatcaatggt aaattgtttt caaatgttat taaaacaata acctctttct 2520 ttagagtggg tttgggaaca gtgagttgtt gctctgaagc agagctagaa aagcctagag 2580 gcttttttcc cacaccacgg tatctactgg attatagagg aagactggaa tgttcttggg 2640 tgctcagagt ttcagcaagc agtatggcaa aatttaccat tgagtatctg tcactcctgg 2700 ggtctcctgt gtgtcaagac tcagttctaa ttatttatga agaaagacac agtaagagaa 2760 agacggcagg taatccttcc tggcatttgc caatggagat tagtagcccc tttaaatcac 2820 atcattcagc ttaatattat taacttcccg atgaagccaa caacttttgt ctgtcatggt 2880 catctgcgtg tttacgaagg atttggacca ggaaaaaaat taataggttt ctcaaggatg 2940 tccagtattg gatttgattc cagtgacttc tgttgagatc acatctcttg attatcctaa 3000 cagttaactc aacatgctga attacacttg gactttttat tccacaacag taaataaaat 3060 gaaggccatg attaaagact ttataacaga agaatctttg aactgtgatt gggattatat 3120 aatatttatg atagaccata tcagcaatca attctacttt ttatgtcatt gcacagaaaa 3180 agtactttaa ttatcgaata tgtctttcat tgcagctcat ttacatggct ccaagaaaga 3240 gtttatcttg atatccagtg ctgcttacct gactgtgcat tttaagactg atgagtctaa 3300 ggttggttgg tagctgtgct gcatctgcaa tgtcatggcc ctggctagtt agtctgcagc 3360 acgggagatt ctggaagacc accgcaatgt gcccggcatg ggaagtacaa gcttgttgac 3420 attgtgagct gaggcagcag tcactgccat cccactgcac caccttcaca agaatttctg 3480 cctgcaggga ttggatcaca tctgccacta gaggagaagt ga 3522 <210> 24 <211> 3277 <212> DNA
<213> Homo sapiens <220>
<221> misc_feature <223> Incyte ID No: 7478108CB1 <400> 24 ccggtccctg ccatggggcc cccttccagc tcaggcttct atgtgagccg cgcagtggcc 60 ctgctgctgg ctgggttggt agccgccctc ctgctggcgc tggccgtact cgccgccttg 120 tacggccact gcgagcgcgt cccaccgtcg gagctgcctg gactcaggga ctcggaagcc 180 gagtcttccc ctcccctcag gcagaagccg acgccgaccc cgaaacccag cagtgcacgc 240 gagctagcgg tgacgaccac cccgagcaac tggcgacccc cggggccctg ggaccagcta 300 cgcctgccgc cctggctcgt gccgctgcac tacgatctgg agctgtggcc gcagctgagg 360 cccgacgagc ttccggccgg gtctttgccc ttcactggcc gcgtgaacat cacggtgcgc 420 tgcacggtgg ccacctctcg actgctgctg catagcctct tccaggactg cgagcgcgcc 480 gaggtgcggg gacccctttc cccgggcact gggaacgcca cagtgggccg egtgcccgtg 540 gacgacgtgt ggttcgcgct ggacacggaa tacatggtgc tggagctcag tgagcccctg 600 aaacctggta gcagctacga gctgcagctt agcttctcgg gcctggtgaa ggaagacctc 660 agggagggac tcttcctcaa cgtctacacc gaccagggcg agcgcagggc cctgttagcg 720 tcccagctgg aaccaacatt tgccaggtat gttttccctt gttttgatga gccagctctg 780 aaggcaactt ttaatattac aatgattcat catccaagtt atgtggccct ttccaacatg 840 ccaaagctag gtcagtctga aaaagaagat gtgaatggaa gcaaatggac tgttacaacc 900 ttttccacta cgccccacat gccaacttac ttagtcgcat ttgttatatg tgactatgac 960 cacgtcaaca gaacagaaag gggcaaggag atacgcatct gggcccggaa agatgcaatt 1020 gcaaatggaa gtgcagactt tgctttgaac atcacaggtc ccatcttctc ttttctggag 1080 gatttgttta atatcagtta ctctcttcca aaaacagata taattgcctt gcctagtttt 1140 gacaaccatg caatggaaaa ctggggacta atgatatttg atgaatcagg attgttgttg 1200 gaaccaaaag atcaactgac agaaaaaaag actctgatct cctatgttgt ctcccacgag 1260 attggacacc agtggtttgg aaacttggtt accatgaatt ggtggaacaa tatctggctc 1320 aacgagggtt ttgcatctta ttttgagttt gaagtaatta actactttaa tcctaaactc 1380 ccaagaaatg agatcttttt ttctaacatt ttacataata tcctcagaga agatcacgcc 1440 ctggtgacta gagctgtggc catgaaggtg gaaaatttca aaacaagtga aatacaggaa 1500 ctctttgaca tatttactta cagcaaggga gcgtctatgg cccggatgct ttcttgtttc 1560 ttgaatgagc atttatttgt cagtgcactc aagtcatatt tgaagacatt ttcctactca 1620 aacgctgagc aagatgatct atggaggcat tttcaaatgg ccatagatga ccagagtaca 1680 gttattttgc cagcaacaat aaaaaacata atggacagtt ggacacacca gagtggtttt 1740 ccagtgatca ctttaaacgt gtctactggc gtcatgaaac aggagccatt ttatcttgaa 1800.
aacattaaaa atcggactct tctaaccagc aatgacacat ggattgtccc tattctttgg 1860 ataaaaaatg gaactacaca acctttagtc tggctagatc aaagcagcaa agtattccca 1920 gaaatgcaag tttcagattc tgaccatgac tgggtgattt tgaatttgaa tatgactgga 1980 tattatagag ttaattatga taaattaggt tggaagaaac taaatcaaca acttgaaaag 2040 gatcctaagg ctattcctgt tattcacaga ctgcagttca ttgatgatgc cttttccttg 2100 tctaaaaaca attatattga gattgaaaca gcacttgagt taaccaagta ccttgctgaa 2160 gaagatgaaa ttatagtatg gcatacagtc ttggtaaact tggtaaccag ggatcttgtt 2220 tctgaggtga acatctatga tatatactca ttattaaaga ggtacctatt aaagagactt 2280 aatttaatat ggaatattta ttcaactata attcgtgaaa atgtgttggc attacaagat 2340 gactacttag ctctaatatc actggaaaaa ctttttgtaa ctgcgtgttg gttgggcctt 2400 gaagactgcc ttcagctgtc aaaagaactt ttcgcaaaat gggtggatca tccagaaaat 2460 gaaatacctt atccaattaa agatgtggtt ttatgttatg gcattgcctt gggaagtgat 2520 aaagagtggg acatcttgtt aaatacttac actaatacaa caaacaaaga agaaaagatt 2580 caacttgctt atgcaatgag ctgcagcaaa gacccatgga tacttaacag atatatggag 2640 tatgccatca gcacatctcc attcacttct aatgaaacaa atataattga ggttgtggct 2700 tcatctgaag ttggccggta tgtcgcaaaa gacttcttag tcaacaactg gcaagctgtg 2760 agtaaaaggt atggaacaca atcattgatt aatctaatat atacaatagg gagaaccgta 2820 actacagatt tacagattgt ggagctgcag cagtttttca gtaacatgtt ggaggaacac 2880 cagaggatca gagttcatgc caacttacag acaataaaga atgaaaatct gaaaaacaag 2940 aagctaagtg ccaggatagc tgcgtggcta aggagaaaca catagcttgt ggctatcttt 3000 cagcactcct cttgcatatt ataatgtagt ttgttcacag ttttgtcttc caatactttg 3060 tgagtctgga aaaccacaca ttttatttgt atttcagtca catttattac tcagagtgcc 3120 attcttctca tattgtcatg tttggccctg agggtgggtg attgctgaca attttgccaa 3180 tgctgctgta tttctgggaa agatgtcact tcatgttggg ttataatccc acagaattta 3240 ctttaaatgt cacgtaaaaa caaattcaaa aaaaaaa 3277 <210> 25 <211> 1254 <212> DNA
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte ID No: 7482021CB1 <400> 25 atgcgcacct cgtacaccgt gaccctgccc gaggaccccc ccgccgcccc ctttcccgcc 60 ctcgccaagg agctgcggcc gcgctcccct ctctccccgt ccctgctgct ctccaccttc 120 gtggggctcc tgctcaacaa agccaagaat tctaagagtg cccagggtct ggctggtctt 180 cgaaaccttg ggaacacgtg cttcatgaac tcaattctgc agtgcctgag caacactcgg 240 gagttgagag attactgcct ccagaggctc tacatgcggg acctgcacca cggcagcaat 300 gcacacacag ccctcgtgga agagtttgca aaactaattc agaccatatg gacttcatcc 360 cccaatgatg tggtgagccc atctgagttc aagacccaga tccagagata tgcaccgcgc 420 tttgttggct ataatcagca ggatgctcag gagttccttc gctttcttct ggatgggctc 480 cataacgagg tgaaccgagt gacactgaga cctaagtcca accctgagaa cctcgatcat 540 cttcctgatg acgagaaagg ccgacagatg tggagaaaat atctagaacg ggaagacagt 600 aggatcgggg atctctttgt tgggcagcta aagagctcgc tgacgtgtac agattgtggt 660 tactgttcta cggtcttcga coccttctgg gacctctcac tgcccattgc taagcgaggt 720 tatcctgagg tgacattaat ggactgcatg aggctcttca ccaaagagga tgtgcttgat 780 ggagatgaaa agccaacatg ctgtcgctgc cgaggcagaa aacggtgtat aaagaagttc 840 tccatccaga ggttcccaaa gatcttggtg ctocatctga agcggttctc agaatccagg 900 atccgaacca gcaagctcac aacatttgtg aacttccccc taagagacct ggacttaaga 960 gaatttgcct cagaaaacac caaccatgct gtttacaacc tgtacgctgt gtccaatcac 1020 tccggaacca ccatgggtgg ccactataca gcctactgtc gcagtccagg gacaggagaa 1080 tggcacactt tcaacgactc cagcgtcact cccatgtcct ccagccaagt gcgcaccagc 1140 gacgcctacc tgctcttcta cgaaetggcc agcccgccct cccgaatgta gcgccaggag 1200 ccacgtccct tctcccttcc ccgtggtggc cccgctccct aaatttttta aaaa 1254 <210> 26 <211> 1120 <212> DNA
<213> Homo sapiens <220>
<221> misc_feature <223> Incyte TD No: 7482145CB1 <400> 26 cgcgtgtgga agcgettccg ggcggtagca cgctgtgttg gcggcggctc cccgcttgcc 60 tcagctgcag cagcgggaag ctcggtggca agcccttgta gtcctgtgcg atggcgtctc 120 gatatgacag ggcgatcact gtcttctccc cagacggaca cctttttcaa gttgaatatg 180 cccaggaagc ggtgaagaaa ggatccaccg cggtcggaat tcgaggtacc aatatagttg 240 ttcttggggt agaaaaaaaa tctgttgcca agcttcaaga tgaaagaact gtgaggaaaa 300 aaaaatcaaa gaaatctgtc taattcttag gatgaccact gggaggtctt aatgttttgt 900 tttattgtac tgcctgaggt tgtttagtga aattttagag gaaaacagtt attttgcagc 960 attacatgca gtacttgtgt gatgttttga gaatgccaga tctgtggctg tcttcattct 1020 attacatagt caaacatagg tttatgtgaa gattttcttt gaaaggggat ttcagtaatt 1080 gttgagagca gtcataattc cacataagcc tgagactcta 1120 <210> 27 <211> 4577 <212> DNA
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte ID No: 55022586CB1 <400> 27 agtgatcact atagggcctg gttatctaat gctgctcgag cgcgcgcagt gtgctggaaa 60 gcgcggctgg gcgcctcggc catgactgcg gagctgcagc aggacgacgc ggccggcgcg 120 gcagacggcc acggctcgag ctgccaaatg ctgttaaatc aactgagaga aatcacaggc 180 attcaggacc cttcctttct ccatgaagct ctgaaggcca gtaatggtga cattactcag 240 gcagtcagcc ttctcactga tgagagagtt aaggagccca gtcaagacac tgttgctaca 300 gaaccatctg aagtagaggg gagtgctgcc aacaaggaag tattagcaaa agttatagac 360 cttactcatg ataacaaaga tgatcttcag gctgccattg ctttgagtct actggagtct 420 cccaaaattc aagctgatgg aagagatctt aacaggatgc atgaagcaac ctctgcagaa 480 actaaacgct caaagagaaa acgctgtgaa gtctggggag aaaaccccaa tcccaatgac 540 tggaggagag ttgatggttg gccagttggg ctgaaaaatg ttggcaatac atgttggttt 600 agtgctgtta ttcagtctct ctttcaattg cctgaatttc gaagacttgt tctcagttat 660 agtctgccac aaaatgtact tgaaaattgt cgaagtcata cagaaaagag aaatatcatg 720 tttatgcaag agcttcagta tttgtttgct ctaatgatgg gatcaaatag aaaatttgta 780 gacccgtctg cagccctgga tctattaaag ggagcattcc gatcatctga ggaacagcag 840 caagatgtga gtgaattcac acacaagctc ctggattggc tagaggacgc attccagcta 900 gctgttaatg ttaacagtcc caggaacaaa tctgaaaatc caatggtgca gctgttctat 960 ggtactttcc tgactgaagg ggttcgtgaa ggaaaaccct tttgtaacaa tgagaccttc 1020 ggccagtatc ctcttcaggt aaacggttat cgcaacttag acgagtgttt ggaaggggcc 1080 atggtggagg gtgatgttga gcttcttccc tccgatcact cggtgaagta tggacaagag 1140 cgttggttta caaagctacc tccagtgttg acctttgaac tctcaagatt tgagtttaat 1200 cagtcccttg ggcagccaga gaaaattcac aataagctgg aatttcctca gattatttat 1260 atggacaggt acatgtacag gagcaaggag cttattcgaa ataagagaga gtgtattcga 1320 aagttgaagg aggaaataaa aattctgcag caaaaattgg aaaggtatgt gaaatatggc 1380 tcaggcccag ctcggttccc gctcccggac atgctgaaat atgttattga atttgctagt 1440 acaaaacctg cctcagaaag ctgtccacct gaaagtgaca cacatatgac attaccactt 1500 tcttcagtgc actgctcggt ttctgaccag acatccaagg aaagtacaag tacagaaagc 1560 tcttctcagg atgttgaaag taccttttct tctcctgaag attctttacc caagtctaaa 1620 ccactgacat cttctcggtc ttccatggaa atgccttcac agccagctcc acgaacagtc 1680 acagatgagg agataaattt tgttaagacc tgtcttcaga gatggaggag tgagattgaa 1740 caagatatac aagatttaaa gacttgtatt gcaagtacta ctcagactat tgaacagatg 1800 tactgcgatc ctctccttcg tcaggtgcct tatcgcttgc atgcagttct tgttcatgaa 1860 ggacaagcaa atgctggaca ctattgggcc tatatctata atcaaccccg acagagctgg 1920 ctcaagtaca atgacatctc tgttactgaa tcttcctggg aagaagttga aagagattcc 1980 tatggaggcc tgagaaatgt tagtgcttac tgtctgatgt acattaatga caaactaccc 2040 tacttcaatg cagaggcagc cccaactgaa tcagatcaaa tgtcagaagt ggaagcccta 2100 tctgtggaac tcaagcatta cattcaggag gataactggc ggtttgagca ggaagtagag 2160 gagtgggaag aagagcagtc ttgcaaaatc cctcaaatgg agtcctccac caactcctca 2220 tcacaggact actctacatc acaagagcct tcagtagcct cttctcatgg ggttcgctgc 2280 ttgtcatctg agcatgctgt gattgtaaag gagcaaactg cccaggctat tgcaaacaca 2340 gcccgtgcct atgagaagag cggtgtagaa gcggcactga gtgaggcatt ccatgaagaa 2400 tactccaggc tctatcagct tgccaaagag acccccacct ctcacagtga tcctcgactt 2460 cagcatgtcc ttgtctactt tttccaaaat gaagcaccca aaagggtagt agaacgaacc 2520 cttctggaac agtttgcaga taaaaatctt agctatgatg aaagatcaat cagcattatg 2580 aaggtggctc aagcgaaact gaaggaaatt ggtccagatg acatgaatat ggaagagtac 2640 aagaagtggc atgaagatta tagtttgttc cgaaaagtgt ctgtgtatct cctaacaggc 2700 ctagaactct atcaaaaagg aaagtaccaa gaggcacttt cctacctggt atatgcctac 2760 cagagcaatg ctgccctgct gatgaagggg ccccgccggg gggtcaaaga atccgtgatt 2820 gctttatacc gaagaaaatg ccttctggag ctgaatgcca aagcagcttc tctttttgaa 2880 acaaatgatg atcactccgt aactgagggc attaatgtga tgaatgaact gatcatcccc 2940 tgcattcacc ttatcattaa taatgacatt tccaaggatg atctggatgc cattgaggtc 3000 atgagaaacc attggtgctc ttaccttggg caagatattg cagaaaatct gcagctgtgc 3060 ctaggggagt ttctacccag acttctagat ccttctgcag aaatcatcgt cttgaaagag 3120 cctccaacta ttcgacccaa ttctccctat gacctatgta gccgatttgc agctgtcatg 3180 gagtcaattc agggagtttc aactgtgaca gtgaaataag ctcccacatg ttcaaggccc 3240 attctggttc ctggctgcct gcctcttgca cagaagttcg ttgtcatagt gctcaccttg 3300 ggaaaaggat taggtgggca cataagattc cgatcagacc ccaaccatgc tgcatgtgta 3360 aagaaggatt gaaaataaaa ttgcactttt taggtacaaa atcataaaag ctgtttcact 3420 agaaaaggca gaaagcagtg tattaaggtg ttgaattacg ccagaagacc tgaaatgcct 3480 tgtacctaca acaatgctta ggcttttcta agcctcttgc cacttttaaa attatccttc 3540 aggcataaat atttttgaca gcagaataga agaatgattc atgagaacct gaaccagatg 3600 aacagctact agttatttta tcaaatacag atgacattta aaaattctta actacaagag 3660 attagaaata taaaccttgc ctggctcttg ccaggagata acaaaatggg ttgctgatga 3720 actgcaccct tttacatgtg ggtagaatat aagctcacat ggcagtgaga tgttgaaaag 3780 tcaaaagaga cctgtctctc tcctttcttt tctatcttta aaccagaaaa cctcatactc 3840 agtcctcagt gaaagaaagt aaagtattaa ggactttaga cagaagagca ttgtgtaact 3900 tgactgaaga tcatccatta atagttatta ggcatttagg taaaattttc taatacctaa 3960 aaattgtcaa aaacagtcaa tagggctact gctggcccaa agaccattta ggtccacctc 4020:
ctcttttttg ctcttttttt ttttctgtga cagtttcact gtgtcgccca ggctggcgtt 4080 cagtggtgca atctcagctc actgcaaact ctgtctcctg ggctcaagtg attctcgtgc 4140 ctcagcctcc cgaatagctg gaattacggg catgcaccac cacacctggc taatttttgt 4200 atttttaata gagatggggt ttcaccatat tggccaggct gatctctaac tcctggcctc 4260 aagtgatcta tctgcctccc tcagcctccc aaagtctggg attgcagaca agtcatcgta 4320 cccggccttc ttttttgccc ttaaaagtaa gggatgtggg tttgtacaaa aaaaaaacaa 4380 aaaaaaaaag aggggcggcc gcgcgattat tgagtctctt gcaacccgcg aatttatttc 4440 cgaaccggtt acctgagggc gttcccagtt tcctaatggt gagtcgtttt acagcttgta 4500 gtaatcatga acaagctgtc ctgtgtgaat tgtttcgttc catccacata tcacacacac 4560 aacacggacg gaagacg 4577 <210> 28 <211> 1952 <212> DNA
<213> Homo sapiens <220>
<221> misc_feature <223> Incyte ID No: 3238072CB1 <400> 28 aagtgctccc acgtggcctg cggccgctat attgaggacc acgccctgaa acactttgag 60 gagacgggac acccgctagc catggaagtc cgggatctct acgtgttctg ttacctgtgc 120 aaggactacg tgctcaatga taacccagag ggggacctga agctgctaag aagctccctc 180 ctggcggtcc ggggccagaa acaggacacg ccggtgagac gtgggcggac gctgcggtcc 240 atggcttcgg gtgaggacgt ggtcctgccg cagcgcgctc ctcagggaca gccgcagatg 300 ctcacggctc tgtggtaccg gcgtcagcgc ctgctggcca ggacgctgcg gctgtggttc 360 gagaagagct cccggggcca ggcgaagctg gagcagcggc ggcaggagga ggccctggag 420 cgcaagaagg aggaggcgcg gaggcggcgg cgcgaggtga aacggcggct gctggaggag 480 ctggccagca cccctccgcg caagagtgca cggctgctcc tgcacacgcc ccgcgacgcg 540 ggcccggctg cctcgcgccc cgccgccctc cctacctcac gcagagtgcc cgccgccaca 600 ctcaagctgc gtcgccagcc ggccatggcc ccaggcgtca cgggcctgcg caacctgggc 660 a.acacctgct~acatgaactc catcctccag gtgctcagcc acctccagaa gttccgagaa 720 tgtttcctca accttgaccc ttccaaaacg gaacatctgt ttcccaaagc caccaacggg 780 aagactcagc tttctggcaa gccaaccaac agctcggcca cggagctgtc cttgagaaat 840 gacagggccg aggcatgcga gcgggagggc ttctgctgga acggcagggc ctccattagt 900 cggagtctgg agctcatcca gaacaaggag ccgagttcaa agcacatttc cctctgccgt 960 gaactgcaca ccctcttccg agtcatgtgg tccgggaagt gggccctagt gtcgcccttc 1020 gccatgctgc actcagtgtg gagcctgatc cctgccttcc gcggctacga ccaacaggac 1080 gcgcaggaat ttctctgcga gctgctgcac aaggtgcagc aggaactcga gtctgagggc 1140 accacacgcc ggatcctcat ccccttctcc cagaggaagc tcaccaaaca ggtcttaaag 1200 gtggtgaata ccatatttca tgggcagctg ctcagtcagg tcacatgtat atcatgcaat 1260 tacaaatcca ataccattga gcccttttgg gacctatccc tggaattccc tgaacgctat 1320 cactgcatag aaaaggggtt tgtccctttg aatcaaacag agtgcttgct cactgagatg 1380 ctggccaaat tcacagagac agaggccctg gaagggagaa tctacgcttg tgaccagtgt 1440 aacagcaaac gacgaaaatc caatcccaaa ccccttgttc tgagtgaagc tagaaagcag 1500 ttaatgatct acagactacc tcaggttctc cggctgcacc ttaaaagatt caggtggtct 1560 ggccgtaatc atcgagagaa gattggggtc catgtcgtct ttgaccaggt attaaccatg 1620 gaaccttact gctgcaggga catgctctcc tctcttgaca aagagacctt tgcctatgat 1680 ctctccgcag tggtcatgca tcacgggaaa gggtttggct caggacacta cacagcctat 1740 tgctacaaca cagagggagg tttttgggtc cactgcaatg actcaaagct gaatgtatgc 1800 agtgtcgagg aagtgtgcaa aacccaggcc tacatccttt tttacactca aagaacagtg 1860 cagggcaatg caagaatctc agaaacccat ctccaagctc aggtgcagtc cagcaacaat 1920 gatgaaggca gaccacagac attttcctga at 1952 <220> 29 <211> 1092 <212> DNA
<213> Homo Sapiens <220>
<221> misc feature <223> Incyte ID No: 7482034CB1 <400> 29 aagggggctg ctccatcagc caatcccaaa gcctgaattg ggggttgagg agaaatgaag 60 cgtcagctca cacaccttcc tggccggttc tggctgtggc ccagcttctc tgtagcgtcc 120 ctcctatccc accagacccc agccacaaat tcctggcttg cttcttccaa acttcattca 180 gccccaggga tggctctgca ggatgtgtgc aagtggcagt cccctgacac ccagggacca 240 tcacctcacc tgcctcgggc tggcggctgg gctgtgcccc ggggttgtga ccctcaaacc 300 ttcctgcaga tccatggccc cagactggcc cacggcacca ccactctggc cttccgcttc 360 cgtcatggag tcattgctgc agctgacacg cgttcctcct gtggcagcta tgtggcgtgt 420 ccagcctcat gcaaggtcat ccctgtgcac cagcacctcc tgggtaccac ctctggcacc 480 tctgccgact gtgctacctg gtatcgggta ttacagcggg agctgcggct tcgggaactg 540 agggagggtc agctgcccag tgtggccagt gctgccaagc tcttgtcagc catgatgtct 600 caataccggg gactggatct ctgtgtggcc actgccctct gcggctggga ccgctctggc 660 cctgagctct tctacgtcta tagcgacggc acccgcctgc agggggacat cttctctgtg 720 ggctctggat ctccctatgc ctacggcgtg ctagaccgtg gctatcgcta cgacatgagc 780 acccaggaag cctacgccct ggctcgctgc gccgtggccc acgccaccca ccgtgatgcc 840 tattcagggg gctctgtaga ccttttccac gtgcgggaga gtggatggga gcatgtgtca 900 cgcagtgatg cctgtgtgct gtacgtggag ttacagaagc tcctggagcc ggagccagag 960 gaggatgcca gccatgccca tcctgagcct gccactgccc acagagctgc agaagataga 1020 gagctctctg tggggccagg ggaggtgaca ccaggagact ccaggatgcc agcagggact 2080 gagacggtgt ga 1092 <210> 30 <211> 2847 <212> DNA
<213> Homo sapiens <220>
<221> misc_feature <223> Incyte ID No: 7474351CB1 <400> 30 atggtcagca aggggggagt tgctgcagag ccagagccac actattgtga ggacagtgaa 60 agaggcccca acaccctcac aggtccgggc agccttccta gaggaggtgg cattgaggtg 120 ggcatggagt ttccgggatg cagcggtgaa gggtgcgtga agccccatga ggaggcggcc 180 cgggaggggg cgggcagagg caagagggct gtgccgggac ccaagcgacg gcagcagggg 240 tcagcagagg ggcctgcggc ggggtggacg ctggagcagg agaccagggg agatgtctta 300 gaggataaaa atgagcgggc agatgaagag atactcaggc tggcaccagg gaaaggcagg 360 ctcccaatag acagcaaaca cctgaaaccg gtgatcagca gcttcccggt aagatctcag 420 gagctgggcg agggggctgg agcaggcaca ctaagaggca aaatggcaga gtttaactgg 480 tctatggcct tcaagggacc tgcggctggt catgaagagc gcctcaactc tgtgtccagc 540 agggccaaga agggcattgg ctgggatgtc gctgctgctt ctcttcgtgg tgttgaccat 600 ttctcagacc tccccccgcc cctgcaggtc agggaggagt tggaggcttg cgcgtttaga 660 gtgcaggtgg ggcagctgag gctctatgag gacgaccagc ggacgaaggt ggttgagatc 720 gtccgtcacc cccagtacaa cgagagcctg tctgcccagg gcggtgcgga catcgccctg 780 ctgaagctgg aggccccggt gccgctgtct gagctcatcc acccggtctc gctcccgtct 840 gcctccctgg acgtgccctc ggggaagacc tgctgggtga ccggctgggg tgtcattgga 900 .
cgtggagaac tactgccctg gcccctcagc ttgtgggagg cgacggtgaa ggtcaggagc 960 aacgtcctct gtaaccagac ctgtcgccgc cgctttcctt ccaaccacac tgagcggttt 1020:
gagcggctca tcaaggacga catgctgtgt gccggggacg ggaaccacgg ctcctggcca 1080 ggcgacaacg ggggccccct cctgtgcagg cggaattgca cctgggtcca ggtggaggtg 1140 gtgagctggg gcaaactctg cggccttcgc ggctatcccg gcatgtacac ccgcgtgacg 1200 agctacgtgt cctggatccg ccagccatgc ccctcagctc agacccctgc tgtggtccga 1260 agatttgtgc tccccccaaa tccagatgtt gaagccctaa ctcccagtgt gatgggatca 1320 ggagcgccgc tgcccccggc ccccgacctg caagaggccg aggtccccat catgaggacc 1380 cgagcttgcg agaggatgta tcacaaaggc cccactgccc acggccaggt caccatcatc 1440 aaggctgcca tgccgtgtgc agggaggaag gggcagggtt cctgccaggc cgctctgagg 1500 acggaggacc tcaccccaac cacacccaac acggaggtgt ctccacgtgc agaccccagg 1560 ctgagccagc cggaggacat ctggccagag tgggcttggc cagttgtggt gggcaccacc 1620 atgctgctgc tgctgctgtt cctggctgtc tcctccctgg ggagctgtag cactgggagt 1680 ccagctcccg tccccgagaa tgacctggtg ggcattgtgg ggggccacaa caccccaggg 1740 gaagtggtcg tggcagtggg tgctgaccgc cgctcactgc attttccgga aggacaccga 1800 cccgtccacc taccggattc acaccaggga tgtgtatctg tacgggggcc gggggctgct 1860 gaatgtcagc cagatcgtcg tccacccaac tactctgtct tcttcctggg ggcagacatc 1920 gccctgctga agctggccac cagttccctg gagttcactg acagtgacaa ctgctggaac 1980 acaggctggg gcatggtcgg cttgttggat atgctgccgc ctccttaccg cccgcagcag 2040 gtgaaggtcc tcacactgag caatgcagac tgtgagcggc agacctacga tgcttttcct 2100 ggtgctggag acagaaagtt catccaggat gacatgatct gtgccggccg cacgggccgc 2160 cgcacctgga agggtgactc aggcggcccc ctggtctgca agaagaaggg tacctggctc 2220 caggcgggag tagtgagctg gggattttac agtgatcggc ccagcattgg cgtctacaca 2280 cgcccagaga,ccagctggca gggtgccaac catgcagacg cccagagacc agctggcagg 2340 gtgccaacca tgcagaggcc cagagacatg ggccagggcc aggagtgggt ctgcaggccc 2400 ttcacccacg tcacctgcta cccgacggcc atccccaggc ccttcaccca tgtcacctgc 2460 tacctgatgg ctgtccccag caccctcacc cacgtcacct gctacccgac ggccgtcccc 2520 aggcccttca cccatgtcac ctgctacctg atggctgtcc ccagcaccct cacccacatc 2580 acctgctaca tgatggccgt ccccaggccc tttacccaca tcacctgcta cccaatggct 2640 gtccccagca cccttaccca cgtcacctgc cacccgacgg ccatccccag gcccttcacc 2700 cacatcacct gctacacgat ggccatcccc aggccttcaa ccacgccacc tgctacacga 2760 cggccatccc cagcaccctc acccacgtca cctgctacac gatggccgtc cccaggccca 2820 tcacccatgt cacctgctac acgatag 2847 <210> 31 <211> 1396 <212> DNA
<213> Homo sapiens <220>
<221> misc_feature <223> Incyte ID No: 2232483CB1 <400> 31 gcccacgtga cgggcgcccg cggaaggcga catgggctcc gctccctggg ccccggtcct 60 gctgctggcg ctcgggctgc gcggcctcca ggcgggggcc cgcagggccc cggaccccgg 120 cttccaggag cgcttcttcc agcagcgtct ggaccacttc aacttcgagc gcttcggcaa 180 caggaccttc cctcagcgct tcctggtgtc ggacaggttc tgggtccggg gcgaggggcc 240 catcttcttc tacactggga acgagggcga cgtgtgggcc ttcgccaaca actcggcctt 300 cgtcgcggag ctggcggccg agcggggggc tctactggtc ttcgcggagc accgctacta 360 cgggaagtcg ctgccgttcg gtgcgcagtc cacgcagcgc gggcacacgg agctgctgac 420 ggtggagcag gccctggccg acttcgcaga gctgctccgc gcgctacgac gcgacctcgg 480 ggcccaggat gcccccgcca tcgccttcgg tggaagttat ggggggatgc tcagtgccta 540 cctgaggatg aagtatcccc acctggtggc gggggcgctg gcggccagcg cgcccgttct 600 agctgtggca ggcctcggcg actccaacca gttcttccgg gacgtcacgg cgggagccta 660 cgacacggtc cgctgggagt tcggcacctg ccagccgctg tcagacgaga aggacctgac 720 ccagctcttc atgttcgccc ggaatgcctt caccgtgctg gccatgatgg actaccccta 780 ccccactgac ttcctgggtc ccctccctgc caaccccgtc aaggtgggct gtgatcggct 840 gctgagtgag gcccagagga tcacggggct gcgagcactg gcagggctgg tctacaacgc 900 ctcgggctcc gagcactgct acgacatcta ccggctctac cacagctgtg ctgaccccac 960 tggctgcggc accggccccg acgccagggc ctgggactac caggcctgca ccgagatcaa 1020 cctgaccttc gccagcaaca atgtgaccga tatgttcccc gacctgccct tcactgacga 1080 gctccggcca agcgatctca gagccgccag caacatcatc ttctccaacg ggaacctgga 1140 cccctgtggc aggggcggga ttcggaggaa cctgagtgcc tcagtcatcg ccgtcaccat 1200 ccagggggga gcgcaccacc tcgacctcag agcctcccac ccagaagatc ctgcttccgt 1260 ggttgaggcg cggaagctgg aggccaccat catcggcgag tgcgtaaagg cagccaggcg 1320 tgagcagcag ccagctctgc gttggggggc ccagatcagc ctctgagcac aggactggag 1380 gggtctcagg gctcta ~ 1396 <210> 32 <211> 1853 <212> DNA
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte ID No: 7481712CB1 <400> 32 tcagagctgc ctgcgagtcc tgcctgctca gggctccttc agcctccact catgcggtgc 60 ctcctggcct ccccgcctgg ccctcagctg ggcctctgct ccactctgcc ccttccaagt 120 gacacagcct tccagggatc cttctccgca gcttcctccg cccctccctg tgtttttcta 180 gggaccaggt tcttcgagtc ctggccaaag atgagaagca gctttcactt ctcggggatc 240 tggagggcct gaaaccccag aaggtggact tctggcgtgg cccagccagg cccagcctcc 300 ctgtggatat gagagttcct ttctccgaac tgaaagacat caaagcttat ctggagtctc 360 atggacttgc ttacagcatc atgataaagg acatccaggt gaagccctgc cccagctggg 420 accctgcctt ccgccttcct ttctggttgg ggcccaacat ggaggagatg ttctcggggc 480 taaaagtgga catgtggttt ctgggtctcc atcagcgtgt ttgtgaacat gctgtggaag 540 gaacaggctg cccaccccct cacttcacca aagcttccct cgacaatgtc acacgcaact 600 tccagatcca acccgatggc cgactctcaa tgttcctctt ccaacagcac aactggtcac 660 tctctccttc ctggagcctg tctcttcccc tggcatccag gacttctgtg ttctgtctcc 720 agccagcacc tcctctcctg gatccaaccg cctactcagt gtttccacct gggggtgcaa 780 tgggcatctc caactttcca gccccaggaa tggagcaaac gctggtgcat tttccaggcc 840 aaggcagatt cctgttcctg gaagtggggc cagctgtgct gctggatgag gaaagacagg 900 ccatggcgaa atcccgccgg ctggagcgca gcaccaacag cttcagttac tcatcatacc 960 acaccctgga ggagatatat agctggattg acaactttgt aatggagcat tccgatattg 1020 tctcaaaaat tcagattggc aacagctttg aaaaccagtc cattcttgtc ctgaagttca 1080 gcactggagg ttctcggcac ccagccatct ggattgacac tggaattcac tcccgggagt 1140 ggatcaccca tgccaccggc atctggactg ccaataagat tgtcagtgat tatggcaaag 1200 accgtgtcct gacagacata ctgaatgcca tggacatctt catagagctc gtcacaaacc 1260 ctgatgggtt tgcttttacc cacagcatga accgcttatg gcggaagaac aagtccatca 1320 gacctggaat cttctgcatc ggcgtggatc tcaacaggaa ctggaagtcg ggttttggag 1380 gaaatggttc taacagcaac ccctgctcag aaacttatca cgggccctcc cctcagtcgg 1440 agccggaggt ggctgccata gtgaacttca tcacagccca tggcaacttc aaggctctga 1500 tctccatcca cagctactct cagatgctta tgtaccctta cggccgattg ctggagcccg 1560 tttcaaatca gagggagttg tacgatcttg ccaaggatgc ggtggaggcc ttgtataagg 1620 tccatgggat cgagtacatt tttggcagca tcagcaccac cctctatgtg gccagtggga 1680 tcaccgtcga ctgggcctat gacagtggca tcaagtacgc cttcagcttt gagctccggg 1740 acactgggca gtatggcttc ctgctgccgg ccacacagat catccccacg gcccaggaga 1800 cgtggatggc gcttcggacc atcatggagc acaccctgaa tcacccctac tag 1853 <210> 33 <211> 3344 <212> DNA
<213> Homo Sapiens <220>
<221> misc feature <223> Incyte ID No: 8213480CB1 <400> 33 atgggctgga ggccccggag agctcggggg accccgttgc tgctgctgct actactgctg 60 ctgctctggc cagtgccagg cgccggggtg cttcaaggac atatccctgg gcagccagtc 120 accccgcact gggtcctgga tggacaaccc tggcgcaccg tcagcctgga ggagccggtc 180 tcgaagccag acatggggct ggtggccctg gaggctgaag gccaggagct cctgcttgag 240 ctggagaaga accacaggct gctggcccca ggatacatag aaacccacta cggcccagat 300 gggcagccag tggtgctggc ccccaaccac acggatcatt gccactacca agggcgagta 360 aggggcttcc ccgactcctg ggtagtcctc tgcacctgct ctgggatgag tggcctgatc 420 accctcagca ggaatgccag ctattatctg cgtccctggc caccccgggg ctccaaggac 480 ttctcaaccc acgagatctt tcggatggag cagctgctca cctggaaagg aacctgtggc 540 cacagggatc ctgggaacaa agcgggcatg accagccttc ctggtggtcc ccagagcagg 600 ggcaggcgag aagcgcgcag gacccggaag tacctggaac tgtacattgt ggcagaccac 660 accctgttct tgactcggca ccgaaacttg aaccacacca aacagcgtct cctggaagtc 720 gccaactacg tggaccagct tctcaggact ctggacattc aggtggcgct gaccggcctg 780 gaggtgtgga ccgagcggga ccgcagccgc gtcacgcagg acgccaacgc cacgctctgg 840 gccttcctgc agtggcgccg gggactgtgg gcgcagcggc cccacgactc cgcgcagctg 900 ctcacgggcc gcgccttcca gggcgccaca gtgggcctgg cgcccgtcga gggcatgtgc 960 cgcgccgaga gctcgggagg cgtgagcacg gaccactcgg agctccccat cggcgccgca 1020 gccaccatgg cccatgagat cggccacagc ctcggcctca gccacgaccc cgacggctgc 1080 tgcgtggagg ctgcggccga gtccggaggc tgcgtcatgg ctgcggccac cgggcacccg 1140 tttccgcgcg tgttcagcgc ctgcagccgc cgccagctgc gcgccttctt ccgcaagggg 1200 ggcggcgctt gcctctccaa tgccccggac cccggactcc cggtgccgcc ggcgctctgc 1260 gggaacggct tcgtggaagc gggcgaggag tgtgactgcg gccctggcca ggagtgccgc 1320 gacctctgct gctttgctca caactgctcg ctgcgcccgg gggcccagtg cgcccacggg 1380 gactgctgcg tgcgctgcct gctgaagccg gctggagcgc tgtgccgcca ggccatgggt .1440 gactgtgacc tccctgagtt ttgcacgggc acctcctccc actgtccccc agacgtttac 1500 ctactggacg gctcaccctg tgccaggggc agtggctact gctgggatgg cgcatgtccc 1560 acgctggagc agcagtgcca gcagctctgg gggcctggct cccacccagc tcccgaggcc 1620 tgtttccagg tggtgaactc tgcgggagat gctcatggaa actgcggcca ggacagcgag 1680 ggccacttcc tgccctgtgc agggagggat gccctgtgtg ggaagctgca gtgccagggt 1740 ggaaagccca gcctgctcgc accgcacatg gtgccagtgg actctaccgt tcacctagat 1800 ggccaggaag tgacttgtcg gggagccttg gcactcccca gtgcccagct ggacctgctt 1860 ggcctgggcc tggtagagcc aggcacccag tgtggaccta gaatggtttg caatagcaac 1920 cataactgcc actgtgctcc aggctgggct ccacccttct gtgacaagcc aggctttggt 1980 ggcagcatgg acagtggccc tgtgcaggct gaaaaccatg acaccttcct gctggccatg 2040 ctcctcagcg tcctgctgcc tctgctccca ggcgccggcc tggcctggtg ttgctaccga 2100 ctcccaggag cccatctgca gcgatgcagc tggggctgca gaagggaccc tgcgtgcagt 2160 ggccccaaag atggcccaca cagggaccac cccctgggcg gcgttcaccc cacggagttg 2220 ggccccacag ccactggaca gtcctggccc ctggaccctg agaactctca tgagcccagc 2280 agccaccctg agaagcctct gccagcagtc tcgcctgacc cccaagcaga tcaagtccag 2340 atgccaagat cctgcctctg gtgagaggta gctcctaaaa tgaacagatt taaagacagg 2400 tggccactga cagccactcc aggaacttga actgcagggg cagagccagt gaatcaccgg 2460 acctccagca cctgcaggca gcttggaagt ttcttccccg agtggagctt cgacccaccc 2520 actccaggaa cccagagcca cattagaagt tcctgagggc tggagaacac tgctgggcac 2580 actctccagc tcaataaacc atcagtccca gaagcaaagg tcacacagcc cctgacctcc 2640 ctcaccagtg gaggctgggt agtgctggcc atcccaaaag ggctctgtcc tgggagtctg 2700 gtgtgtctcc tacatgcaat ttccacggac ccagctctgt ggagggcatg actgctggcc 2760 agaagctagt ggtcctgggg ccctatggtt cgactgagtc cacactcccc tgcagcctgg 2820 ctggcctctg caaacaaaca taattttggg gaccttcctt cctgtttctt cccaccctgt 2880 , cttctcccct aggtggttcc tgagccccca cccccaatcc cagtgctaca cctgaggttc 2940 tggagctcag aatctgacag cctctccccc attctgtgtg tgtcgggggg acagagggaa 3000, ccatttaaga aaagatacca aagtagaagt caaaagaaag acatgttggc tataggcgtg 3060 gtggctcatg cctataatcc cagcactttg ggaagccggg gtaggaggat caccagaggc 3120 cagcaggtcc acaccagcct gggcaacaca gcaagacacc gcatctacag aaaaatttta 3180 aaattagctg ggcgtggtgg tgtgtacctg taggcctagc tgctcaggag gctgaagcag 3240 gaggatcact tgagcctgag ttcaacactg cagtgagcta tggtggcacc actgcactcc 3300 agcctgggtg acagagcaag accatgtctc taaaataaat ttta 3344 <210> 34 <211> 3389 <212> DNA
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte ID No: 7478405CB1 <400> 34 cggccgcgga aagaatgcgc gccgcccgtg cgctccgcct gccgcgtctg gccacccgca 60 gccgccgcgt ccgcacctga ccatggagtg cgccctcctg ctcgcgtgtg ccttcccggc 120 tgcgggttcg ggcccgccga ggggcctggc gggactgggg cgcgtggcca aggcgctcca 180 gctgtgctgc ctctgctgtg cgtcggtcgc cgcggcctta gccagtgaca gcagcagcgg 240 cgccagcgga ttaaatgatg attacgtctt tgtcacgcca gtagaagtag actcagccgg 300 gtcatatatt tcacacgaca ttttgcacaa cggcaggaaa aagcgatcgg cgcagaatgc 360 cagaagctcc ctgcactacc gattttcagc atttggacag gaactgcact tagaacttaa 420 gccctcggcg attttgagca gtcactttat tgtccaggta cttggaaaag atggtgcttc 480 agagactcag aaacccgagg tgcagcaatg cttctatcag ggatttatca gaaatgacag 540 ctcctcctct gtcgctgtgt ctacgtgtgc tggcttgtca ggtttaataa ggacacgaaa 600 aaatgaattc ctcatctcgc cattacctca gcttctggcc caggaacaca actacagctc 660 ccctgcgggt caccatcctc acgtactgta caaaaggaca gcagaggaga agatccagcg 720 gtaccgtggc taccccggct ctggccggaa ttatcctggt tactccccaa gtcacattcc 780 ccatgcatct cagagtcgag agacagagta tcaccatcga aggttgcaaa agcagcattt 840 ttgtggacga cgcaagaaat atgctcccaa gcctcccaca gaggacacct atctaaggtt 900 tgatgaatat gggagctctg ggcgacccag aagatcagct ggaaaatcac aaaagggcct 960 caatgtggaa accctcgtgg tggcagacaa gaaaatggtg~gaaaagcatg gcaagggaaa 1020 tgtcaccaca tacattctca cagtaatgaa catggtttct ggcctattta aagatgggac 1080 tattggaagt gacataaacg tggttgtggt gagcctaatt cttctggaac aagaacctgg 1140 aggattattg atcaaccatc atgcagacca gtctctgaat agtttttgtc aatggcagtc 1200 tgccctcatt ggaaagaatg gcaagagaca tgatcatgcc atcttactaa caggatttga 1260 tatttgttct tggaagaatg aaccatgtga cactctaggg tttgccccca tcagtggaat 1320 gtgctctaag taccgaagtt gtaccatcaa tgaggacaca ggacttggcc ttgccttcac 1380 catcgctcat gagtcagggc acaactttgg tatgattcac gacggagaag ggaatccctg 1440 cagaaaggct gaaggcaata tcatgtctcc cacactgacc ggaaacaatg gagtgttttc 1500 atggtcttcc tgcagccgcc agtatctcaa gaaattcctc agcacacctc aggcggggtg 1560 tctagtggat gagcccaagc aagcaggaca gtataaatat ccggacaaac taccaggaca 1620 gatttatgat gctgacacac agtgtaaatg gcaatttgga gcaaaagcca agttatgcag 1680 ccttggtttt gtgaaggata tttgcaaatc actttggtgc caccgagtag gccacaggtg 1740 tgagaccaag tttatgcccg cagcagaagg gaccgtttgt ggcttgagta tgtggtgtcg 1800 gcaaggccag tgcgtaaagt ttggggagct cgggccccgg cccatccacg gccagtggtc 1860 cgcctggtcg aagtggtcag aatgttcccg gacatgtggt ggaggagtca agttccagga 1920 gagacactgc aataacccca agcctcagta tggtggcata ttctgtccag gttctagccg 1980 tatttatcag ctgtgcaata ttaacccttg caatgaaaat agcttggatt ttcgggctca 2040 acagtgtgca gaatataaca gcaaaccttt ccgtggatgg ttctaccagt ggaaacccta 2100 tacaaaagtg gaagaggaag atcgatgcaa actgtactgc aaggctgaga actttgaatt 2160 tttttttgca atgtccggca aagtgaaaga tggaactccc tgctccccaa acaaaaatga 2220 tgtttgtatt gacggggttt gtgaactagt gggatgtgat catgaactag gctctaaagc 2280, agtttcagat gcttgtggcg tttgcaaagg tgataattca acttgcaagt tttataaagg 2340 cctgtacctc aaccagcata aagcaaatga atattatccg gtggtcatca ttccagctgg 2400 cgcccgaagc atcgaaatcc aggagctgca ggtttcctcc agttacctcg cagttcgaag 2460 cctcagtcaa aagtattacc tcaccggggg ctggagcatc gactggcctg gggagttccc 2520 cttcgctggg accacgtttg aataccagcg ctctttcaac cgcccggaac gtctgtacgc 2580 gccagggccc acaaatgaga cgctggtctt tgaaattctg atgcaaggca aaaatccagg 2640 gatagcttgg aagtatgcac ttcccaaggt catgaatgga actccaccag ccacaaaaag 2700 acctgcctat acctggagta tcgtgcagtc agagtgctcc gtctcctgtg gtggaggtta 2760 cataaatgta aaggccattt gcttgcgaga tcaaaatact caagtcaatt cctcattctg 2820 cagtgcaaaa accaagccag taactgagcc caaaatctgc aacgctttct cctgcccggc 2880 ttactggatg ccaggtgaat ggagtacatg cagcaaggcc tgtgctggag gccagcagag 2940 ccgaaagatc cagtgtgtgc aaaagaagcc cttccaaaag gaggaagcag tgttgcattc 3000 tctctgtcca gtgagcacac ccactcaggt ccaagcctgc aacagccatg cctgccctcc 3060 acaatggagc cttggaccct ggtctcagtg ttccaagacc tgtggacgag gggtgaggaa 3120 gcgtgaactc ctctgcaagg gctctgccgc agaaaccctc cccgagagcc agtgtaccag 3180 tctccccaga cctgagctgc aggagggctg tgtgcttgga cgatgcccca agaacagccg 3240 gctacagtgg gtcgcttctt cgtggagcga ggtattgatt agaagtcact gctgggtcag 3300 gagattgaga ccatcctggc taacacagtg aaaccctgtc tctactaaaa atacaaaaaa 3360 ttagccaggc aaggtggcag gcgcctgta 3389

Claims (89)

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:1-17, 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:1-17, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1-17, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1-17.

2. An isolated polypeptide of claim 1 selected from the group consisting of SEQ ID NO:1-17.

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 selected from the group consisting of SEQ ID
NO:18-34.

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 has an amino acid sequence selected from the group consisting of SEQ ID NO:1-17.

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:18-34, 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:18-34, 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 has an amino acid sequence selected from the group consisting of SEQ ID NO:1-17.

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 2 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 12, 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 12, 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 12 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 12, the method comprising:
a) immunizing an animal with a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1-17, 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 having an amino acid sequence selected from the group consisting of SEQ ID NO:1-17.

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 12, the method comprising:
a) immunizing an animal with a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1-17, 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 having an amino acid sequence selected from the group consisting of SEQ
ID NO:1-17.

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 12, wherein the antibody is produced by screening a Fab expression library.

43. The antibody of claim 12, wherein the antibody is produced by screening a recombinant immunoglobulin library.

44. A method of detecting a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1-17 in a sample, the method comprising:

a) incubating the antibody of claim 12 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 having an amino acid sequence selected from the group consisting of SEQ
ID NO:1-17 in the sample.

45. A method of purifying a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1-17 from a sample, the method comprising:

a) incubating the antibody of claim 12 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 having an amino acid sequence selected from the group consisting of SEQ ID NO:1-17.

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

47. A method of generating a transcript image 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 1D
N0:6.

62. A polypeptide of claim 1, comprising the amino acid sequence of SEQ ID
N0:7.

63. A polypeptide of claim 1, comprising the amino acid sequence of SEQ >ID
N0:8.

64. A polypeptide of claim 1, comprising the amino acid sequence of SEQ ID
N0:9.

65. A polypeptide of claim 1, comprising the amino acid sequence of SEQ ID
N0:10.

66. A polypeptide of claim 1, comprising the amino acid sequence of SEQ 1D
N0:11.

67. A polypeptide of claim 1, comprising the amino acid sequence of SEQ ID
N0:12.

68. A polypeptide of claim 1, comprising the amino acid sequence of SEQ ID
NO:13.

69. A polypeptide of claim 1, comprising the amino acid sequence of SEQ 1D
N0:14.

70. A polypeptide of claim 1, comprising the amino acid sequence of SEQ ID
N0:15.

71. A polypeptide of claim 1, comprising the amino acid sequence of SEQ ID
N0:16.

72. A polypeptide of claim 1, comprising the amino acid sequence of SEQ ID
NO:17.

73. A polynucleotide of claim 12, comprising the polynucleotide sequence of SEQ ID N0:18.

74. A polynucleotide of claim 12, comprising the polynucleotide sequence of SEQ ID N0:19.

75. A polynucleotide of claim 12, comprising the polynucleotide sequence of SEQ ID N0:20

76. A polynncleotide of claim 12, comprising the polynucleotide sequence of SEQ ID N0:21.

77. A polynucleotide of claim 12, comprising the polynucleotide sequence of SEQ ID N0:22.

78. A polynucleotide of claim 12, comprising the polynucleotide sequence of SEQ ID N0:23.

79. A polynucleotide of claim 12, comprising the polynucleotide sequence of SEQ ID N0:24.

80. A polynucleotide of claim 12, comprising the polynucleotide sequence of SEQ ID N0:25.

81. A polynucleotide of claim 12, comprising the polynucleotide sequence of SEQ ID N0:26.

82. A polynucleotide of claim 12, comprising the polynucleotide sequence of SEQ 1D N0:27.

83. A polynucleotide of claim 12, comprising the polynucleotide sequence of SEQ ID N0:28.

84. A polynucleotide of claim 12, comprising the polynucleotide sequence of SEQ 1D N0:29.

85. A polynucleotide of claim 12, comprising the polynucleotide sequence of SEQ ID N0:30.

86. A polynucleotide of claim 12, comprising the polynucleotide sequence of SEQ ID N0:31.

87. A polynucleotide of claim 12, comprising the polynucleotide sequence of SEQ ID N0:32.

88. A polynucleotide of claim 22, comprising the polynucleotide sequence of SEQ ID N0:33.

89. A polynucleotide of claim 22, comprising the polynucleotide sequence of SEQ ID N0:34.