CA2460476A1 - Protein modification and maintenance molecules - Google Patents

Abstract

Various embodiments of the invention provide human protein modification and maintenance molecules(PMMM) and polynucleotides which identify and encode PMMM. Embodiments of the invention also provide expression vectors, host cells, antibodies, agonists, and antagonists. Other embodiments provide methods for diagnosing, treating, or preventing disorders associated with aberrant expression of PMMM.

Description

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PROTEIN MODIFICATION AND MAINTENANCE MOLECULES
TECHNICAL FIELD
The invention relates to novel nucleic acids, protein modification and maintenance molecules encoded by these nucleic acids, and to the use of these nucleic acids and proteins in the diagnosis, treatment, and prevention of gastrointestinal, cardiovascular, autoimmunelinflammatory, cell proliferative, developmental, epithelial, neurological, reproductive, endocrine, pancreatic, adrenal, and metabolic disorders; as well as lipid, copper, and carbohydrate metabolism disorders, disorders associated with gonadal steroid hormones, the immune system, and infections.
The invention also relates to the assessment of the effects of exogenous compounds on the expression of nucleic acid and amino acid sequences of protein modification and maintenance molecules.
BACKGROUND OF THE INVENTION
'The cellular processes regulating modification and maintenance of protein molecules coordinate their function, conformation, stabilization, and degradation. Each of these processes is mediated by key enzymes or proteins such as kinases, phosphatases, proteases, protease inhibitors, isomerases, transferases, and molecular chaperones.
Kinases Kinases catalyze the transfer of high-energy phosphate groups from adenosine triphosphate (ATP) to target proteins on the hydroxyamino acid residues serine, threonine, or tyrosine. Addition of a phosphate group alters the local charge on the acceptor molecule, causing internal conformational changes and potentially influencing intermolecular contacts. Reversible protein phosphorylation is the ubiquitous strategy used to control many of the intracellular events in eukaryotic cells. It is estimated that more than ten percent of proteins active in a typical mammalian cell are phosphorylated.
Extracellular signals including hormones, neurotransmitters, and growth and differentiation factors can activate kinases, which can occur as cell surface receptors or as the activators of the final effector protein, as well as elsewhere along the signal transduction pathway. Kinases are involved in all aspects of a cell's function, from basic metabolic processes, such as glycolysis, to cell-cycle regulation, differentiation, and communication with the extracellular environment through signal transduction cascades. Inappropriate phosphorylation of proteins in cells has been linked to changes in cell cycle progression and cell differentiation. Changes in the cell cycle have been linked to induction of apoptosis or cancer. Changes in cell differentiation have been licked to diseases and disorders of the reproductive system, immune system, and skeletal muscle.

There are two classes of protein kinases. One class, protein tyrosine kinases (PTKs), phosphorylates tyrosine residues, and the other class, protein serine/threonine kinases (STKs), phosphorylates serine and threonine residues. Some PTKs and STKs possess structural characteristics of both families and have dual specificity for both tyrosine and serine/threonine residues. Almost all kinases contain a conserved 250-300 amino acid catalytic domain containing specific residues and sequence motifs characteristic of the kinase family.
(Reviewed in Hardie, G.
and S. Hanks (1995) The Protein Kinase Facts Book, Vol I, Academic Press, San Diego, CA, pp. 17-20).
Phosphatases Phosphatases hydrolytically remove phosphate groups from proteins.
Phosphatases are essential in determining the extent of phosphorylation in the cell and, together with kinases, regulate key cellular processes such as metabolic enzyme activity, proliferation, cell growth and differentiation, cell adhesion, and cell cycle progression. Protein phosphatases are characterized as either serine/threonine- or tyrosine-specific based on their preferred phospho-amino acid substrate. Some phosphatases (DSPs, for dual specificity phosphatases) can act on phosphorylated tyrosine, serine, or threonine residues. The protein serine/threonine phosphatases (PSPs) are important regulators of many cAMP-mediated hormone responses in cells. Protein tyrosine phosphatases (PTPs) play a significant role in cell cycle and cell signaling processes.
Proteases 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, tissue remodeling during embryonic development, wound healing, and normal growth. Proteases can play a role in regulatory processes by affecting the half life of regulatory proteins. Proteases are involved in the etiology or progression of disease states such as inflammation, angiogenesis, tumor dispersion and metastasis, cardiovascular disease, neurological disease, and bacterial, parasitic, and viral infections.
Proteases can be categorized on the basis of where they cleave their substrates.
Exopeptidases, which include aminopeptidases, dipeptidyl peptidases, tripeptidases, carboxypeptidases, peptidyl-di-peptidases, dipeptidases, and omega peptidases, cleave residues at the termini of their substrates. Endopeptidases, including serine proteases, cysteine proteases, and metalloproteases, cleave at residues within the peptide. Four principal categories of mammalian proteases have been identified based on active site structure, mechanism of action, and overall three-dimensional structure.
(See Beynon, R.J. and J.S. Bond (1994) Proteolytic Enzymes: A Practical Approach, Oxford University Press, New York NY, pp. 1-5.) Serine Proteases The serine proteases (SPs) are a large, widespread family of proteolytic enzymes that include the digestive enzymes trypsin and chymotrypsin, components of the complement and blood-clotting cascades, and enzymes that control the degradation and turnover of macromolecules within the cell and in the extracellular matrix. Most of the more than 20 subfamilies can be grouped into six clans, each with a common ancestor. These six clans are hypothesized to have descended from at least four evolutionarily distinct ancestors. SPs are named for the presence of a serine residue found in the active catalytic site of most families. The active site is defined by the catalytic triad, a set of conserved asparagine, histidine, and serine residues critical for catalysis.
These residues form a charge relay network that facilitates substrate binding. Other residues outside the active site form an oxyanion hole that stabilizes the tetrahedral transition intermediate formed during catalysis. SPs have a wide range of substrates and can be subdivided into subfamilies on the basis of their substrate specificity. The main subfamilies are named for the residues) after which they cleave: trypases (after arginine or lysine), aspases (after aspartate), chymases (after phenylalanine or leucine), metases (methionine), and serases (after serine) (Rawlings, N.D. and A.J.
Barrett (1994) Methods 2o Enzymo1.244:19-61).
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. I~ringle domains are triple-looped, disulfide cross-linked domains found in varying copy number. Kringle domains are thought to play a role in binding mediators such as membranes, other proteins or phospholipids, and in the regulation of proteolytic activity (PROSTTE
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-XII, complement factors B, C, and D, granzymes, kallikrein, and tissue- and urokiuase-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, supra).
SPs have functions in many normal processes and some have been implicated in the etiology or treatment of disease. Enterokinase, the initiator of intestinal digestion, is found in the intestinal brush border, where it cleaves the acidic propeptide from trypsinogen to yield active trypsin (Kitamoto, Y. et al. (1994) Proc. Natl. Acad. Sci. USA 91:7588-7592).
Prolylcarboxypeptidase, a lysosomal serine peptidase that cleaves peptides such as angiotensin II and III and [des-Arg9] bradykinin, shares sequence homology with members of both the serine carboxypeptidase and prolylendopeptidase families (Tan, F. et al. (1993) J. Biol. Chem. 268:16631-16638). The protease neuropsin may influence synapse formation and neuronal connectivity in the hippocampus in response to neural signaling (Chen, Z.-L. et al. (1995) J. Neurosci. 15:5088-5097). Tissue plasminogen activator is useful for acute management of stroke (Zivin, J.A. (1999) Neurology 53:14-19) and myocardial infarction (Ross, A.M. (1999) 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 2o extracellular domain. The major agonists for PARs, thrombin, trypsin, and mast cell tryptase, are released in allergy and inflammatory conditions. Control of PAR activation by proteases has been suggested as a promising therapeutic target (Vergnolle, N. (2000) Aliment.
Pharmacol. Ther. 14:257-266; Rice, K.D. et al. (1998) Curr. Pharm. Des. 4:381-396). Prostate-specific antigen (PSA) is a kallikrein-like serine protease synthesized and secreted exclusively by epithelial 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.H. Lange (1989) Urology 33:11-16).
Signal peptidases The mechanism for the trauslocation process into the endoplasmic reticulum (ER) involves the recognition of an N-terminal signal peptide on the elongating protein. The signal peptide directs the protein and attached ribosome to a receptor on the ER membrane. The polypeptide chain passes through a pore in the ER membrane into the lumen while the N-terminal signal peptide remains attached at the membrane surface. The process is completed when signal peptidase located inside the ER cleaves the signal peptide from the protein and releases the protein into the lumen.
The signal peptidase is a specialized class of SP found in all prokaryotic and eukaryotic cell types that serves in the processing of signal peptides from certain proteins.
Signal peptides are amino-terminal domains of a protein which direct the protein from its ribosomal assembly site to a particular cellular or extracellular location. Once the protein has been exported, removal of the signal sequence by a signal peptidase and posttranslational processing, e.g., glycosylation or phosphorylation, activate the protein. Signal peptidases exist as multi-subunit complexes in both yeast and mammals.
The canine signal peptidase complex is composed of five subunits, all associated with the microsomal membrane and containing hydrophobic regions that span the membrane one or more times (Shelness, l0 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.
Thrombin is a serine protease with an essential role in the process of blood coagulation.
Prothrombin, synthesized in the liver, is converted to active thrombin by Factor Xa. Activated thrombin then cleaves soluble fibrinogen to polymer-forming fibrin, a primary component of blood clots. In addition, thrombin activates Factor XBIa, which plays a role in cross-linking fibrin.
Thrombin also stimulates platelet aggregation through proteolytic processing of a 41-residue amino-terminal peptide from protease-activated receptor 1 (PAR-1), formerly known as the thrombin receptor. The cleavage of the amino-terminal peptide exposes n new amino terminus and may also be associated with PAR-1 internalization (Stubbs, M.T. and W. Bode (1994) Curr.
Opin. Struct. Biol.
4:823-832; and Ofoso, F.A. et al. (1998.) Biochem. J. 336:283-285). In addition to stimulating platelet activation through cleavage of the PAR-1 receptor, thrombin also induces platelet aggregation following cleavage of glycoprotein V, also on the surface of platelets.
Glycoprotein V appears to be the major thrombin substrate on intact platelets. Platelets deficient for glycoprotein V are hypersensitive to thrombin, which is still required to cleave PAR-1. While platelet aggregation is required for normal hemostasis in mammals, excessive platelet aggregation can result in arterial thrombosis, atherosclerotic arteries, acute myocardial infarction, and stroke (Ramakrishnan, V. et al.
(1999) Proc. Natl. Acad. Sci. U.S.A. 96:13336-13341 and references within).
Proteases in another family have a serine in their active site and are dependent on the hydrolysis of ATP for their activity. 'These proteases contain proteolytic core domains and regulatory ATPase domains which can be identified by the presence of the P-loop, an ATP/GTP-binding motif (PROSITE PDOC00803). Members of this family include the eukaryotic mitochondrial matrix proteases, Clp protease and the proteasome. Clp protease was originally found in plant chloroplasts but is believed to be widespread in both prokaryotic and eukaryotic cells. The gene for early-onset torsion dystonia encodes a protein related to Clp protease (Ozelius, L.J. et al. (1998) Adv. Neurol.
78:93-105).
The proteasome is an intracellular protease complex found in some bacteria and in all eukaryotic cells, and plays an important role in cellular physiology. The proteasome is a large (2000 kDa) multisubunit complex composed of a central catalytic core containing a variety of proteases arranged in four seven-membered rings with the active sites facing inwards into the central cavity, and terminal ATPase subunits covering the outer port of the cavity and regulating substrate entry (for review, see Schmidt, M. et al. (1999) Curr. Opin. Chem. Biol. 3:584-591).
Proteasomes are associated with the ubiquitin conjugation system (UCS), a major pathway for the degradation of cellular proteins of all types, including proteins that function to activate or repress cellular processes such as transcription and cell cycle progression (Ciechanover, A. (1994) Cell 79:13-21). In the UCS
pathway, proteins targeted for degradation are conjugated to ubiquitin, a small heat stable protein. The ubiquitinated protein is then recognized and degraded by the proteasome. The resultant ubiquitin-peptide complex is hydrolyzed by a ubiquitin carboxyl terminal hydrolase, and free ubiquitin is released for reutilizationby the UCS. Ubiquitin-proteasome systems are implicated in the degradation of mitotic cyclic kinases, oncoproteins, tumor suppressor genes (p53), cell surface receptors associated with signal transduction, transcriptional regulators, and mutated or damaged proteins (Ciechanover, supra). This pathway has been implicated in a number of diseases, including cystic fibrosis, Angelinan's syndrome, and Liddle syndrome (reviewed in Schwartz, A.L. and A.
Ciechanover (1999) , Annu. Rev. Med. 50:57-74). A murine proto-oncogene, Unp, encodes a nuclear ubiquitin protease whose overexpression leads to oncogenic transformation of NIH3T3 cells. The human homolog of this gene is consistently elevated in small cell tumors and adenocarcinomas of the lung (Gray, D.A.
(1995) Oncogene 10:2179-2183). Ubiquitin carboxyl terminal hydrolase is involved in the differentiation of a lymphoblastic leukemia cell line to a non-dividing mature state (Maki, A. et al.
(1996) Differentiation 60:59-66). In neurons, ubiquitin carboxyl terminal hydrolase (PGP 9.5) expression is strong in the abnormal structures that occur in human neurodegenerative diseases (Lowe, J. et al. (1990) J. Pathol. 161:153-160).
Cysteine Proteases Cysteine proteases (CPs) are involved in diverse cellular processes ranging from the processing of precursor proteins to intracellular degradation. Nearly half of the CPs known are present only in viruses. CPs have a cysteine as the major catalytic residue at the active site where catalysis proceeds via a thioester intermediate and is facilitated by nearby histidine and asparagine residues. A glutamine residue is also important, as it helps to form an oxyanion hole. Two important CP families include the papain-like enzymes (C1) and the calpains (C2). Papain-like family members are generally lysosomal or secreted and therefore are synthesized with signal peptides as well as propeptides. Most members bear a conserved motif in the propeptide that may have structural significance (Karrer, K.M. et al. (1993) Proc. Natl. Acad. Sci. USA 90:3063-3067). Three-dimensional structures of papain family members show a bilobed molecule with the catalytic site located between the two lobes. Papains include cathepsins B, C, H, L, and S, certain plant allergens and dipeptidyl peptidase (for a review, see Rawlings, N.D. and A.J. Barrett (1994) Methods Enzymol.
244:461-486).
Some CPs are expressed ubiquitously, while others are produced only by cells of the immune system. Of particular note, CPs are produced by monocytes, macrophages and other cells which migrate to sites of inflammation and secrete molecules involved in tissue repair. Overabundance of these repair molecules plays a role in certain disorders. In autoimmune diseases such as rheumatoid arthritis, secretion of the cysteine peptidase cathepsin C degrades collagen, laminin, elastin and other structural proteins found in the extracellular matrix of bones. Bone weakened by such degradation is also more susceptible to tumor invasion and metastasis. Cathepsin L expression may also contribute to the influx of mononuclear cells which exacerbates the destruction of the rheumatoid synovium (Keyszer, G.M. (1995) Arthritis Rheum. 38:976-984).
Human cathepsin O is a novel cysteine protease with 94% identity to rabbit OC2 and 50%
identity to both human cathepsin S and cathepsin L. It displays potent endoprotease activity against .
fibrinogen at acid pH. The mature protein contains 215 amino acids with one potential N-glycosylation site. The gene for cathepsin O is expressed in osteoclastoma and ovary. Its extremely high expression levels in osteoclastoma suggests a major role in bone remodelling and bone related diseases (Shi, G. P. et al. (1995) FEBS Lett. 357:129-134; Bromine, D. and Okamoto, K.
(1995) Biol. Chem.
Hoppe Seyler 376:379-384).
Calpains are calcium-dependent cytosolic endopeptidases which contain both an N-terminal catalytic domain and a C-terminal calcium-binding domain. Calpain is expressed as a proenzyme heterodimer consisting of a catalytic subunit unique to each isoform and a regulatory subunit common to different isoforms. Each subunit bears a calcium binding EF hand domain.
The regulatory subunit also contains a hydrophobic glycine-rich domain that allows the enzyme to associate with cell membranes. Calpains are activated by increased intracellular calcium concentration, which induces a change in conformation and limited autolysis. The resultant active molecule requires a lower calcium concentration for its activity (Char, S.L. and M.P. Mattson (1999) J.
Neurosci. Res. 58:167-190).
Calpain expression is predominantly neuronal, although it is present in other tissues. Several chronic neurodegenerative disorders, including ALS, Parkinson's disease and Alzheimer's disease are associated with increased calpain expression (Char and Mattson, supra).
Calpain-mediated breakdown of the cytoskeleton has been proposed to contribute to brain damage resulting from head injury (McCracken, E. et al. (1999) J. Neurotrauma 16:749-761). Calpain-3 is predominantly expressed in skeletal muscle, and is responsible for limb-girdle muscular dystrophy type 2A (Minami, N. et al. (1999) J. Neurol. Sci. 171:31-37).
Another family of thiol proteases is the caspases, which are involved in the initiation and execution phases of apoptosis. A pro-apoptotic signal can activate initiator caspases that trigger a proteolytic caspase cascade, leading to the hydrolysis of target proteins and the classic apoptotic death of the cell. Two active site residues, a cysteine and a histidine, have been implicated in the catalytic mechanism. Caspases are among the most specific endopeptidases, cleaving after aspartate residues.
Caspases are synthesized as inactive zymogens consisting of one large (p20) and one small (p10) subunit separated by a small spacer region, and a variable N-terminal prodomain. This prodomain interacts with cofactors that can positively or negatively affect apoptosis.
An activating signal causes autoproteolytic cleavage of a specific aspartate residue (D297 in the caspase-1 numbering convention) and removal of the spacer and prodomain, leaving a p101p20 heterodimer. Two of these heterodimers interact via their small subunits to form the catalytically active tetramer.
The long prodomains of some caspase family members have been shown to promote dimerization and auto-processing of procaspases. Some caspases contain a "death effector domain" in their prodomain by which they can be recruited into self activating complexes with other caspases and FADD
protein associated death receptors or the TNF receptor complex. In addition, two dimers from different caspase family members can associate, changing the substrate specificity of the resultant tetramer. Endogenous caspase inhibitors (inhibitor of apoptosis proteins, or IAPs) also exist. All these interactions have clear effects on the control of apoptosis (reviewed in Chan and Mattson, supt-a;
Salveson, G.S. and V.M.
Dixit (1999) Proc. Natl. Acad. Sci. USA 96:10964-10967).
Caspases have been implicated in a number of diseases. Mice lacking some caspases have severe nervous system defects due to'failed apoptosis in the neuroepithelium and suffer early lethality.
Others show severe defects in the inflammatory response, as caspases are responsible for processing IL-1b and possibly other inflammatory cytokines (Char and Mattson, supra).
Cowpox virus and baculoviruses target caspases to avoid the death of their host cell and promote successful infection. In addition, increases in inappropriate apoptosis have been reported in AIDS, neurodegenerative diseases and ischemic injury, while a decrease in cell death is associated with cancer (Salveson and Dixit, supf'a; Thompson, C.B. (1995) Science 267:1456-1462).

As~artyl proteases Aspartyl proteases (APs) include the lysosomal proteases cathepsins D and E, as well as chymosin, resin, and the gastric pepsins. Most retroviruses encode au AP, usually as part of the pol polyprotein. APs, also called acid proteases, are monomeric enzymes consisting of two domains, each domain containing one half of the active site with its own catalytic aspartic acid residue. APs are most active in the range of pH 2-3, at which one of the aspartate residues is ionized and the other neutral. The pepsin family of APs contains many secreted enzymes, and all are likely to be synthesized with signal peptides and propeptides. Most family members have three disulfide loops, the first ~5 residue loop following the first aspartate, the second 5-6 residue loop preceding the second aspartate, and the third and largest loop occurring toward the C terminus.
Retropepsins, on the other hand, are analogous to a single domain of pepsin, and become active as homodimers with each retropepsin monomer contributing one half of the active site. Retropepsins are required for processing the viral polyproteins.
APs have roles in various tissues, and some have been associated with disease.
Resin mediates the first step in processing the hormone angiotensin, which is responsible for regulating electrolyte balance and 'blood pressure (reviewed in Crews, D.E. and S.R.
Williams (1999) Hum. Biol.
71:475-503). Abnormal regulation and expression of cathepsins are evident in various inflammatory disease states. Expression of cathepsin D is elevated in synovial tissues from patients with rheumatoid arthritis and osteoarthritis. The increased expression and differential regulation of the cathepsins are linked to the metastatic potential of a variety of cancers (Chambers; A.F. et al. (1993) Crit. Rev.
Oncol. 4:95-114).
Metalloproteases Metalloproteases require a metal ion for activity, usually manganese or zinc.
Examples of manganese metalloenzymes include aminopeptidase P and human proline dipeptidase (PEPD).
Aminopeptidase P can degrade bradykinin, a nonapeptide activated in a variety of inflammatory responses. Aminopeptidase P has been implicated in coronary ischemia/reperfusion injury.
Administration of aminopeptidase P inhibitors has been shown to have a cardioprotective effect in rats (Ersahin, C. et al (1999) J. Cardiovasc. Pharmacol. 34:604-611). Most zinc-dependent metalloproteases share a common sequence in the zinc-binding domain. The active site is made up of two histidines which act as zinc ligands and a catalytic glutamic acid C-terminal to the first histidine.
Proteins containing this signature sequence are known as the metzincins and include aminopeptidase N, angiotensin-converting enzyme, neurolysin, the matrix metalloproteases and the adamalysins (ADAMS). An alternate sequence is found in the zinc carboxypeptidases, in which all three conserved residues - two histidines and a glutamic acid - are involved in zinc binding.
Porcine pancreatic preprocarboxypeptidase A1 (prePCPAI) is the proenzyme of carboxypeptidase A (CPA), a metalloprotease secreted from panceratic juice.
The open reading frame of the prePCPAI nucleotide sequence is 1260 nucleotides in length and encodes a protein of 419 amino acids. The molecular mass of the mature proenzyme is 45561 Da. The amino acid sequence of the enzyme shows 85% identity and 55% identity to that of procarboxypeptidases A1 and A2, respectively (Darnis S. (1999) Eur. J. Biochem. 259:719-725).
A number of the neutral metalloendopeptidases, including angiotensin converting enzyme and the aminopeptidases, are involved in the metabolism of peptide hormones. High aminopeptidase B
activity, for example, is found in the adrenal glands and neurohypophyses of hypertensive rats (Prieto, I. et al. (1998) Horm. Metab. Res. 30:246-248). Oligopeptidase M/neurolysin can hydrolyze bradykinin as well as neurotensin (Serizawa, A. et al. (1995) J. Biol. Chem 270:2092-2098).
Neurotensin is a vasoactive peptide that can act as a neurotransmitter in the brain, where it has been implicated in limiting food intake (Tritos, N.A. et al. (1999) Neuropeptides 33:339-349).
The matrix metalloproteases (MMPs) are a family of at least 23 enzymes that can degrade components of the extracellular matrix (ECM). They are Zn2+ endopeptidases with an N-terminal catalytic domain. Nearly all members of the family have a hinge peptide and a C-terminal domain which can bind to substrate molecules in the ECM or to inhibitors produced by the tissue-(TllVIPs, for ' tissue inhibitor of metalloprotease; Campbell, LL. and A. Pagenstecher (1999) Trends Neurosci.
22:285-287). The presence of fibronectin-like repeats, transmembrane domains, or C-terminal hemopexinase-like domains can be used to separate MMPs into collagenase, gelatinise, stromelysin and membrane-type MMP subfamilies. In the inactive form, the Znz+ ion in the active site interacts with a cysteine in the pro-sequence. Activating factors disrupt the Zn2+-cysteine interaction, or "cysteine switch," exposing the active site. This partially activates the enzyme, which then cleaves off its propeptide and becomes fully active. MMPs are often activated by the serine proteases plasmin and furin. MMPs are often regulated by stoichiometric, noncovalent interactions with inhibitors; the balance of protease to inhibitor, then, is very important in tissue homeostasis (reviewed in Yong, V.W.
et al. (1998) Trends Neurosci. 21:75-80).
MMPs are implicated in a number of diseases including osteoarthritis (Mitchell, P. et al.
(1996) J. Clip. Invest. 97:761-768), atherosclerotic plaque rupture (Sukhova, G.K. et al. (1999) Circulation 99:2503-2509), aortic aneurysm (Schneiderman, J. et al. (1998) Am.
J. Path. 152:703-710), non-healing wounds (Saarialho-Kere, U.K. et al. (1994) J. Clip. Invest. 94:79-88), bone resorption (Blavier, L. and J.M. Delaisse (1995) J. Cell Sci. 108:3649-3659), age-related macular degeneration (Steep, B. et al. (1998) Invest. Ophthalmol. Vis. Sci. 39:2194-2200), emphysema (Finlay, G.A. et al.
(1997) Thorax 52:502-506), myocardial infarction (Rohde, L.E. et al. (1999) Circulation 99:3063-3070) and dilated cardiomyopathy (Thomas, C.V. et al. (1998) Circulation 97:1708-1715). MMP inhibitors prevent metastasis of mammary carcinoma and experimental tumors in rat, and Lewis lung carcinoma, hemangioma, and human ovarian carcinoma xenografts in mice (Eccles, S.A. et al. (1996) Cancer Res. 56:2815-2822; Anderson et al. (1996) Cancer Res. 56:715-718; Volpert, O.V. et al. (1996) J.
Clip. Invest. 98:671-679; Taraboletti, G. et al. (1995) J. Natl. Cancer Inst.
87:293-298; Davies, B. et al. (1993) Cancer Res. 53:2087-2091). MMPs may be active in Alzheimer's disease. A number of MMPs are implicated in multiple sclerosis, and administration of MMP
inhibitors can relieve some of its symptoms (reviewed in Yong et al., supra).
Another family of metalloproteases is the ADAMS, for A Disintegrin and Metalloprotease Domain, which they share with their close relatives the adamalysins, snake venom metalloproteases (SVMPs). ADAMS combine features of both cell surface adhesion molecules and proteases, containing a prodomain, a protease domain, a disintegrin domain, a cysteine rich domain, an epidermal growth factor repeat, a transmembrane domain, and a cytoplasmic tail. The first three domains listed above are also found in the SVMPs. The ADAMS possess four potential functions:
proteolysis, adhesion, signaling and fusion. The ADAMS share the metzincin zinc binding sequence and are inhibited by some MMP antagonists such as TIIuVIP-1.
ADAMS are implicated in such processes as sperm-egg binding and fusion, myoblast fusion, and protein-ectodomain processing or shedding of cytokines, cytokine receptors, adhesion proteins and other extracellular protein domains (Schlondorff, J. and C.P. Blobel (1999) J.
Cell. Sci. 112:3603-3617). The Kuzbanian protein cleaves a substrate in the NOTCH pathway (possibly NOTCH itselt~, activating the program for lateral inhibition in I~r-osophila neural development. Two ADAMS, TACE
(ADAM 17) and ADAM 10, are proposed to have analogous roles in the processing of amyloid precursor protein in the brain (SchlondorFf and Blobel, supra). TALE has also been identified as the TNF activating enzyme (Black, R.A. et al. (1997) Nature 385:729-733). TNF is a pleiotropic cytokine that is important in mobilizing host defenses in response to infection or trauma, but can cause severe damage in excess and is often overproduced in autoimmune disease. TACE cleaves membrane-bound pro-TNF to release a soluble form. Other ADAMS may be involved in a similar type of processing of other membrane-bound molecules.
Proteins of the ADAMTS sub-family have all of the features of ADAM family metalloproteases and contain an additional thrombospondin domain (TS). The prototypic ADAMTS

was identified in mouse, and found to be expressed in heart and kidney and upregulated by proinflammatory stimuli (Kuno, K. et al. (1997) J. Biol. Chem. 272:556-562).
To date eleven members are recognized by the Human Genome Organization (HUGO;
http://www.gene.ucl.ac.uk/users/hester/adamts.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 284:1664-1666;
Abbaszade, I. (1999) J. Biol. Chem. 274:23443-23450). Other members are reported to have antiangiogenic potential (Kuno et al., supra) and/or procollagen processing (Colige, A. et al. (1997) Proc. Natl. Acad. Sci. USA 94:2374-2379).
Proteaseinhibitors Protease inhibitors and other regulators of protease activity control the activity and effects of proteases. Protease inhibitors have been shown to control pathogenesis in animal models of proteolytic disorders (Murphy, G. (1991) Agents Actions Suppl. 35:69-76). Low levels of the cystatins, low molecular weight inhibitors of the cysteine proteases, correlate with malignant progression of tumors (Catkins, C. et al. (1995) Biol. Biochem. Hoppe Seyler 376:71-80). The cystatin superfamily of protease inhibitors is characterized by a particular pattern of linearly arranged and tandemly repeated disulfide loops (Kellermann, J. et al. (1989) J. Biol. Chem.
264:14121-14128).
Serpins are inhibitors of mammalian plasma serine proteases. Many serpins serve to regulate the blood clotting cascade and/or the complement cascade in mammals. Sp32 is a positive regulator of the mammalian acrosomal protease, acrosin, that binds the proenzyme, proacrosin, and thereby aides in packaging the enzyme into the acrosomal matrix (Baba, T. et al. (1994) J.
Biol. Chem. 269:10133-10140). The Kunitz family of serine protease inhibitors are characterized by one or more "Kunitz domains" containing a series of cysteine residues that are regularly spaced over approximately 50 amino acid residues and form three intrachain disulfide bonds. Members of this family include aprotinin, tissue factor pathway inhibitor (TFPI-1 and TFPI-2), inter-a-trypsin inhibitor, and bikunin (Marlor, C.W. et al. (1997) J. Biol. Chem. 272:12202-12208). Members of this family are potent inhibitors (in the nanomolar range) against serine proteases such as kallikrein and plasmin. Aprotinin has clinical utility in reduction of perioperative blood loss.
A major portion of all proteins synthesized in eukaryotic cells are synthesized on the cytosolic surface of the endoplasmic reticulum (ER). Before these immature proteins are distributed to other organelles in the cell or are secreted, they must be transported into the interior lumen of the ER where post-translational modifications are performed. These modifications include protein folding and the formation of disulfide bonds, and N-linked glycosylations.
Protein Isomerases Protein folding in the ER is aided by two principal types of protein isomerases, protein disulfide isomerase (PDI), and peptidyl-prolyl isomerase (PPI). PDI catalyzes the oxidation of free sulfhydryl groups in cysteine residues to form intramolecular disulfide bonds in proteins. PPI, au enzyme that catalyzes the isomerization of certain proline imidic bonds in oligopeptides and proteins, is considered to govern one of the rate limiting steps in the folding of many proteins to their final functional conformation. The cyclophilins represent a major class of PPI that was originally identified as the to major receptor for the immunosuppressive drug cyclosporin A
(Handschumacher, R.E. et al. (1984) Science 226: 544-547).
Protein Glycosylation The glycosylation of most soluble secreted and membrane bound proteins by oligosaccharides linked to asparagine residues in proteins is also performed in the ER. This reaction is catalyzed by a membrane-bound enzyme, oligosaccharyl transferase. Although the exact purpose of this "N-linked"
glycosylation is unknown, the presence of oligosaccharides tends to make a glycoprotein resistant to protease digestion. In addition, oligosaccharides attached to cell-surface proteins called selectins are known to function in cell-cell adhesion processes (Alberts, B. et al. (1994) Molecular Biology of the Cell Garland Publishing Co., New York, NY, p. 608). "O-linked" glycosylation of proteins also occurs in the ER by the addition of N-acetylgalactosamine to the hydroxyl group of a serine or threonine residue followed by the sequential addition of other sugar residues to the first. This process is catalyzed by a series of glycosyltrausferases, each specific for a particular donor sugar nucleotide and acceptor molecule (Lodish, H. et al. (1995) Molecular Cell Biolo~y, W. H.
Freeman and Co., New York, NY, pp. 700-708). In many cases, both N- and O-linked oligosaccharides appear to be required for the secretion of proteins or the movement of plasma membrane glycoproteins to the cell surface.
An additional glycosylation mechanism operates in the ER specifically to target lysosomal enzymes to lysosomes and prevent their secretion. Lysosomal enzymes in the ER
receive an N-linked oligosaccharide, like plasma membrane and secreted proteins, but are then phosphorylated on one or two mannose residues. The phosphorylation of mannose residues occurs in two steps, the first step being the addition of an N-acetylglucosamine phosphate residue by N-acetylglucosamine phosphotransferase, and the second the removal of the N-acetylglucosamine group by phosphodiesterase. The phosphorylated mannose residue then targets the lysosomal enzyme to a mannose 6-phosphate receptor which transports it to a lysosome vesicle (Lodish et al. supra, pp. 708-711).
Chaperones Molecular chaperones are proteins that aid in the proper folding of immature proteins and refolding of improperly folded ones, the assembly of protein subunits, and in the transport of unfolded proteins across membranes. Chaperones are also called heat-shock proteins (hsp) because of their tendency to be expressed in dramatically increased amounts following brief exposure of cells to elevated temperatures. This latter property most likely reflects their need in the refolding of proteins that have become denatured by the high temperatures. Chaperones may be divided into several classes according to their location, function, and molecular weight, and include hsp60, TCP1, hsp70, hsp40 (also called DnaJ), and hsp90. For example, hsp90 binds to steroid hormone receptors, represses transcription in the absence of the ligand, and provides proper folding of the ligand binding domain of the receptor in the presence of the hormone (Burston, S.G. and A.R.
Clarke (1995) Essays Biochem. 29:125-136). Hsp60 and hsp70 chaperones aid in the transport and folding of newly .
synthesized proteins. Hsp70 acts early in protein folding, binding a newly synthesized protein before it leaves the ribosome and transporting the protein to the mitochondria or ER
before releasing the folded protein. Hsp60, along with hspl0, binds misfolded proteins and gives them the opportunity to refold correctly. All chaperones share an affinity for hydrophobic patches on incompletely folded proteins and the ability to hydrolyze ATP. The energy of ATP hydrolysis is used to release the lisp bound 2o protein in its properly folded state (Alberts et al., supra;: pp. 214, 571-572).
The putative human lysyl oxidase-like 3 (humanLOXL3) contains 754 amino acids.
The protein contains a copper-binding site with four histidyl residues, the lysyl and tyrosyl residues known to be involved in LOX enzyme in the formation of the quinone cofactor and surrounding sequences, and a cytokine receptor-like domain. It also contains four scavenger receptor cysteine-rich (SRCR) domains in its N-terminal region with the second and fourth of these SRCR
domains truncated.
Further the potential BMP-1 cleavage site is not present. The gene encoding the cDNA has been mapped to chromosome 2p13.3, overlapping at its 3' end the HtrA2 serine protease gene. The central nervous system, neurons, and leukocytes express humanLOXL3. The gene contains 14 exons and there are at least two alternative splice variants of LOXL3, that lack exon 5 and exon 8 (Jourdan-Le Saux, C. et al. (2001) Genomics 74:211-218).
E~ression profiling Microarrays are analytical tools used in bioanalysis. A microarray has a plurality of molecules spatially distributed over, and stably associated with, the surface of a solid support. Microarrays of polypeptides, polynucleotides, and/or antibodies have been developed and find use in a variety of applications, such as gene sequencing, monitoring gene expression, gene mapping, bacterial identification, drug discovery, and combinatorial chemistry.
One area in particular in which microarrays end use is in gene expression analysis. Array technology can provide a simple way to explore the expression of a single polymorphic gene or the expression profile of a large number of related or unrelated genes. When the expression of a single gene is examined, arrays are employed to detect the expression of a specific gene or its variants.
When an expression profile is examined, arrays provide a platform for identifying genes that are tissue specific, are affected by a substance being tested in a toxicology assay, are part of a signaling cascade, carry out housekeeping functions, or are specifically related to a particular genetic predisposition, condition, disease, or disorder. The potential application of gene expression profiling is particularly relevant to improving diagnosis, prognosis, and treatment of disease. For example, both the levels and sequences expressed in tissues from subjects with diabetes may be compared with the levels and sequences expressed in normal tissue.
Lung cancer The potential application of gene. expression profiling is particularly relevant to improving diagnosis, prognosis, and treatment of cancers such as lung cancer. Lung cancer is the leading cause of cancer death for men and the second leading cause of cancer death for women in the U.S. The vast majority of lung cancer cases are attributed to smoking tobacco, and increased use of tobacco products in third world countries is projected to lead to an epidemic of lung cancer in these countries.
Exposure of the bronchial epithelium to tobacco smoke appears to result in changes in tissue morphology, which are thought to be precursors of cancer. Lung cancers are divided into four histopathologically distinct groups. Three groups (squamous cell carcinoma, adenocarcinoma, and large cell carcinoma) are classified as non-small cell lung cancers (NSCLCs).
The fourth group of cancers is referred to as small cell lung cancer (SCLC). Collectively, NSCLCs account for ~70% of cases while SCLCs account for ~18% of cases. The molecular and cellular biology underlying the development and progression of lung cancer are incompletely understood.
Deletions on chromosome 3 are common in this disease and are thought to indicate the presence of a tumor suppressor gene in this region. Activating mutations in K-ras are commonly found in lung cancer and are the basis of one of the mouse models for the disease.
Preadipoc~te Cells The potential application of gene expression profiling is particularly relevant to improving diagnosis, prognosis, and treatment of obesity. The most important function of adipose tissue is its ability to store and release fat during periods of feeding and fasting. White adipose tissue is the major energy reserve in periods of excess energy use, and its primary purpose is mobilization during energy deprivation. Understanding how the various molecules regulate adiposity and energy balance in physiological and pathophysiological situations may lead to the development of novel therapeutics for human obesity. Adipose tissue is also one of the important target tissues for insulin. Adipogenesis and insulin resistance in type II diabetes are linked and present intriguing relations. Most patients with type II diabetes are obese and obesity in turn causes insulin resistance.
The majority of research in adipocyte biology to date has been done using transformed mouse preadi-pocyte cell lines. The culture condition, which stimulates mouse preadipocyte differentiation is different from that for inducing human primary preadipocyte differentiation.
In addition, primary cells are diploid and may therefore reflect the in vivo context better than aneuploid cell lines.
Understanding the gene expression profile during adipogenesis in human will lead to understanding the fundamental mechanism of adiposity regulation. Furthermore, through comparing the gene expression profiles of adipogenesis between donor with normal weight and donor with obesity, identification of crucial genes, potential drug targets for obesity and type II diabetes, will be possible.
Peroxisome Proliferator-activated Receptor Gamma A~onist Thiazolidinediones (TZDs) act as agonists for the peroxisome-proliferator-activated receptor gamma (PPARy), a member of the nuclear hormone receptor superfamily. TZDs reduce hyperglycemia, hyperinsulinemia, and hypertension, in part by promoting glucose metabolism and inhibiting gluconeogenesis. Roles for PPARy and its agonists have been demonstrated in a wide range of pathological conditions including diabetes, obesity, hypertension, atherosclerosis, polycystic ovarian syndrome, and cancers such as breast, prostate, liposarcoma, and colon cancer.
The mechanism by which TZDs and other PPAR~y agonists enhance insulin sensitivity is not fully understood, but may involve the ability of PPARy to promote adipogenesis. When ectopically expressed in cultured preadipocytes, PPARy is a potent inducer of adipocyte differentiation. TZDs, in combination with insulin and other factors, can also enhance differentiation of human preadipocytes in culture (Adams et al. (1997) J. Clip. Invest. 100:3149-3153). The relative potency of different TZDs in promoting adipogenesis in vitro is proportional to both their insulin sensitizing effects in vivo, and their ability to bind and activate PPARy in vitro. Interestingly, adipocytes derived from omental adipose depots are refractory to the effects of TZDs. It has therefore been suggested that the insulin sensitizing effects of TZDs may result from their ability to promote adipogenesis in subcutaneous adipose depots (Adams et al., ibid). Further, dominant negative mutations in the PPARY gene have been identified in two non-obese subjects with severe insulin resistance, hypertension, and overt non-insulin dependent diabetes mellitus (NIDDM) (Barroso et al. (1998) Nature 402:880-883).
NIDDM is the most common form of diabetes mellitus, a chronic metabolic disease that affects 143 million people worldwide. NIDDM is characterized by abnormal glucose and lipid metabolism that result from a combination of peripheral insulin resistance and defective insulin secretion. NIDDM has a complex, progressive etiology and a high degree of heritability. Numerous complications of diabetes including heart disease, stroke, renal failure, retinopathy, and peripheral neuropathy contribute to the high rate of morbidity and mortality.
At the molecular level, PPARy functions as a ligand activated transcription factor. In the presence of ligand, PPARy forms a heterodimer with the retinoid X receptor (RXR) which then activates transcription of target genes containing one or more copies of a PPARy response element (PPRE). Many genes important in lipid storage and metabolism contain PPREs and have been identified as PPARy targets, including PEPCK, aP2, LPL, ACS, and FAT-P
(Auwerx, J. (1999) .
Diabetologia 42:1033-1049. Multiple ligands for PPARy have been identified.
These include a .
variety of fatty acid metabolites; synthetic drugs belonging to the TZD class, such as Pioglitazone and Rosiglitazone (BRL49653); and certain non-glitazone tyrosine analogs such as GI262570 and GW1929. The prostaglandin derivative 15-dPGJ2 is a potent endogenous ligand for PPARY.
Expression of PPARy is very high in adipose but barely detectable in skeletal muscle, the primary site for insulin stimulated glucose disposal in the body. PPARy is also moderately expressed.
2o in large intestine, kidney, liver, vascular smooth muscle, hematopoietic cells, and macrophages. The high expression of PPARy in adipose suggests that the insulin sensitizing effects of TZDs may result from alterations in the expression of one or more PPARy regulated genes in adipose tissue.
Identification of PPAR~y target genes will contribute to better drug design and the development of novel therapeutic strategies for diabetes, obesity, and other conditions.
Systematic attempts to identify PPARy target genes have been made in several rodent models of obesity and diabetes (Suzuki et al. (2000) Jpn. J. Pharmacol. 84:113-123;
Way et al. (2001) Endocrinology 142:1269-1277). However, a serious drawback of the rodent gene expression studies is that significant differences exist between human and rodent models of adipogenesis, diabetes, and obesity (Taylor (1999) Cell 97:9-12; Gregoire et al. (1998) Physiol. Reviews 78:783-809). Therefore, an unbiased approach to identifying TZD regulated genes in primary cultures of human tissues is necessary to fully elucidate the molecular basis for diseases associated with PPARY activity.
Colon Cancer 'The potential application of gene expression profiling is particularly relevant to improving diagnosis, prognosis, and treatment of cancer, such as colon cancer.
Colorectal cancer is the second leading cause of cancer deaths in the United States. Colon cancer is associated with aging, since 90%
of the total cases occur in individuals over the age of 55. A widely accepted hypothesis is that several contributing genetic mutations must accumulate over time in an individual who develops the disease.
To understand the nature of genetic alterations in colorectal cancer, a number of studies have focused on the inherited syndromes. The first known inherited syndrome, Familial Adenomatous Polyposis (FAP), is caused by mutations in the Adenomatous Polyposis Coli gene (APC), resulting in truncated or inactive forms of the protein. This tumor suppressor gene has been mapped to chromosome 5q.
The second known inherited syndrome is hereditary nonpolyposis colorectal cancer (HNPCC), which is caused by mutations in mismatch repair genes.
Although hereditary colon cancer syndromes occur in a small percentage of the population and most colorectal cancers are considered sporadic, knowledge from studies of the hereditary syndromes can be generally applied. For instance, somatic mutations in APC
occur in at least 80% of indiscriminate colon tumors. APC mutations are thought to be the initiating event in the disease:
Other mutations occur subsequently. Approximately 50% of colorectal cancers contain activating mutations in ras, while 85 % contain inactivating mutations in p53. Changes in these genes lead to gene expression changes in colon cancer. Less is understood about downstream targets of these mutations and the role they may play in cancer.development and progression.
Ovarian cancer The potential application of gene expression profiling is particularly relevant to improving diagnosis, prognosis, and treatment of cancer, such as ovarian cancer. Ovarian cancer is the leading cause of death from a gynecologic cancer. The majority of ovarian cancers are derived from epithelial cells, and 70% of patients with epithelial ovarian cancers present with late-stage disease. As a result the long term survival rates for this disease are very low.
Identification of early stage markers for ovarian cancer would significantly increase the survival rate. The molecular events that lead to ovarian cancer are poorly understood. Some of the known aberrations include mutation of p53 and microsatellite instability.
Breast cancer The potential application of gene expression profiling is particularly relevant to improving diagnosis, prognosis, and treatment of cancer, such as breast cancer. More than 180,000 new cases of breast cancer are diagnosed each year, and the mortality rate for breast cancer approaches 10% of all deaths in females between the ages of 45-54 (Gish, K. (1999) ASS Magazine 28:7-10). However the survival rate based on early diagnosis of localized breast cancer is extremely high (97 %), compared with the advanced stage of the disease in which the tumor has spread beyond the breast (22%). Current procedures for clinical breast examination are lacking in sensitivity and specificity, and efforts are underway to develop comprehensive gene expression profiles for breast cancer that may be used in conjunction with conventional screening methods to improve diagnosis and prognosis of this disease (Perou, C.M. et al. (2000) Nature 406:747-752).
Mutations in two genes, BRCA1 and BRCA2, are known to greatly predispose a woman to breast cancer and may be passed on from parents to children (Gish, supra).
However, this type of hereditary breast cancer accounts for only about 5% to 9% of breast cancers, while the vast majority of breast cancer is due to non-inherited mutations that occur in breast epithelial cells.
The relationship between expression of epidermal growth factor (EGF) and its receptor, EGFR, to human mammary carcinoma has been particularly well studied. (See Khazaie, K. et al.
(1993) Cancer and Metastasis Rev. 12:255-274, and references cited therein for a review of this area.) Overexpression of EGFR, particularly coupled with down-regulation of the estrogen receptor, is a marker of poor prognosis in breast cancer patients. In addition, EGFR
expression in breast tumor metastases is frequently elevated relative to the primary tumor, suggesting that EGFR is involved in tumor progression and metastasis. This is supported by accumulating evidence that EGF has effects on cell functions related to metastatic potential, such as cell motility, chemotaxis, secretion and differentiation. Changes in expression of other members of the erbB receptor family, of which EGFR
is one, have also been implicated in breast cancer. The abundance of erbB
receptors, such as HER-2/neu, HER-3, and HER-4, and their ligands in breast cancer points to their functional importance in the pathogenesis of the disease, and may therefore provide targets for therapy of the disease (Bacus, S.S. et al. (1994) Am. J. Clin. Pathol. 102:513-S24). Other known markers of breast cancer include a human secreted frizzled protein mRNA that is downregulated in breast tumors;
the matrix G1a protein which is overexpressed in human breast carcinoma cells; Drg1 or RTP, a gene whose expression is diminished in colon, breast, and prostate tumors; maspin, a tumor suppressor gene downregulated in invasive breast carcinomas; and CaNl9, a member of the S 100 protein family, all of which are down-regulated in mammary carcinoma cells relative to normal mammary epithelial cells (Zhou, Z. et al.
(1998) Int. J. Cancer 78:95-99; Chen, L. et al. (1990) Oncogene 5:1391-1395;
Ulrix, W. et al (1999) FEBS Lett 455:23-26; Sager, R. et al. (1996) Curr. Top. Microbiol. Tmmunol.
213:51-64; and Lee, S.W. et al. (1992) Proc. Natl. Acad. Sci. USA 89:2504-2508).
Cell lines derived from human mammary epithelial cells at various stages of breast cancer provide a useful model to study the process of malignant transformation and tumor progression as it has been shown that these cell lines retain many of the properties of their parental tumors for lengthy culture periods (Wistuba, LI. et al. (1998) Clip. Cancer Res. 4:2931-2938).
Such a model is particularly useful for comparing phenotypic and molecular characteristics of human mammary epithelial cells at various stages of malignant transformation.
There is a need in the art for new compositions, including nucleic acids and proteins, for the diagnosis, prevention, and treatment of gastrointestinal, cardiovascular, autoimmune/inflammatory, cell proliferative, developmental, epithelial, neurological, reproductive, endocrine, pancreatic, adrenal, and metabolic disorders; as well as lipid, copper, and carbohydrate metabolism disorders, disorders associated with gonadal steroid hormones, the immune system, and infections.
SUMMARY OF THE INVENTION
Various embodiments of the invention provide purified polypeptides, protein modification and maintenance molecules, referred to collectively as 'PNIIvIM' and individually as 'PNIIVIM-1,' <P~_2~> <P_3~> <P_4~> <P~_5~> <P_6~> <P~_7~> <P~_8~> 'P_ 9,' <P~~llVIM_10,> 'PNIIVIM-11,' 'PMMM-12,' <P~~VIM_13,> <P~_14,> <PM~~I_15,>
'PNIMM-16,' 'PNNIIVVIM-17,' <P1~~VIM_18,> 'PMMM-19,' 'PMMM-20,' 'PMMM-21,' 'PIVInMM-22,' 'Pn~VIM_ 23,' 'PMIvVIIVVI-24,' <P_25,> <P~~VIM_26,' 'PMMM-27,' <PMIVIT~I_28,> 'PNllVIM-29,' <PN1~4M_ 30,> <P~_31~> <P_32,> <P~_33~> <P~_34,> <P_35~> <P~_36,> <P_ 37,' 'PNIMM-38,' 'P-39,' and 'Pl~wIM-40' and methods for using these proteins and their encoding polynucleotides for the detection, diagnosis, and treatment of diseases and medical conditions. Embodiments also provide methods for utilizing the purified protein modification and maintenance molecules and/or their encoding polynucleotides for facilitating the drug discovery process, including determination of efficacy, dosage, toxicity, and pharmacology. Related embodiments provide methods for utilizing the purified protein modification and maintenance molecules and/or their encoding polynucleotides for investigating the pathogenesis of diseases and medical conditions.
An embodiment provides an isolated polypeptide selected from the group consisting of a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID N0:1-40, b) a polypeptide comprising a naturally occurring amino acid sequence at least 90% identical or at least about 90% identical to an amino acid sequence selected from the group consisting of SEQ ~
NO:1-40, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ 117 N0:1-40, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ m N0:1-40. Another embodiment provides an isolated polypeptide comprising an amino acid sequence of SEQ m N0:1-40.
Still another embodiment provides an isolated polynucleotide encoding a polypeptide selected from the group consisting of a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ m N0:1-40, b) a polypeptide comprising a naturally occurring amino acid sequence at least 90% identical or at least about 90% identical to an amino acid sequence selected from the group consisting of SEQ DJ N0:1-40, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ m N0:1-40, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ m N0:1-40. In another embodiment, the polynucleotide encodes a polypeptide selected from the group consisting of SEQ m N0:1-40. In an alternative embodiment, the polynucleotide is selected from the group consisting of SEQ m N0:41-80.
Still another embodiment provides a recombinant polynucleotide comprising a promoter sequence operably linked to a polynucleotide encoding a polypeptide selected from the group consisting of a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ m N0:1-40, b) a polypeptide comprising a naturally occurring amino acid sequence at least 90% identical or at least about 90% identical to an amino acid sequence selected from the group consisting of SEQ m N0:1-40, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ m N0:1-40, and d) an immunogenic fragment of .
a polypeptide having an amino acid sequence selected from the group consisting of SEQ ~ N0:1-40.
Another embodiment provides a cell transformed with the recombinant polynucleotide. Yet another embodiment provides a transgenic organism comprising the recombinant polynucleotide.
Another embodiment 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 m N0:1-40, b) a polypeptide comprising a naturally occurring amino acid sequence at least 90% identical or at least about 90% identical to an amino acid sequence selected from the group consisting of SEQ m NO:1-40, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ m NO:1-40, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ~ NO:1-40.
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.

Yet another embodiment 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 )D N0:1-40, b) a polypeptide comprising a naturally occurring amino acid sequence at least 90% identical or at least about 90%
identical to an amino acid sequence selected from the group consisting of SEQ )D N0:1-40, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ )D NO:1-40, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ m N0:1-40.
Still yet another embodiment provides an isolated polynucleotide selected from the group consisting of a) a polynucleotide comprising a polynucleotide sequence selected from the group consisting of SEQ ~ N0:41-80, b) a polynucleotide comprising a naturally occurring polynucleotide sequence at least 90% identical or at least about 90% identical to a polynucleotide sequence selected from the group consisting of SEQ m N0:41-80, 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 other embodiments, the polynucleotide can comprise at least about 20, 30, 40, 60, 80, or 100 contiguous nucleotides.
Yet another embodiment provides a method for detecting a target polynucleotide in a sample, said target polynucleotide being selected from,the group consisting of a) a polynucleotide comprising a polynucleotide sequence selected from the group consisting of SEQ ID N0:41-80, b) a polynucleotide comprising a naturally occurring polynucleotide sequence at least 90%
identical or at least about 90%
identical to a polynucleotide sequence selected from the group consisting of SEQ )D N0:41-80, c) a polynucleotide complementary to the polynucleotide of a), d) a polynucleotide complementary to the polynucleotide of b), and e) an RNA equivalent of a)-d). 'The method comprises a) hybridizing the sample with a probe comprising at least 20 contiguous nucleotides comprising a sequence complementary to said target polynucleotide in the sample, and which probe specifically hybridizes to said target polynucleotide, under conditions whereby a hybridization complex is formed between said probe and said target polynucleotide or fragments thereof, and b) detecting the presence or absence of said hybridization complex. In a related embodiment, the method can include detecting the amount of the hybridization complex. In still other embodiments, the probe can comprise at least about 20, 30, 40, 60, 80, or 100 contiguous nucleotides.
Still yet another embodiment provides a method for detecting a target polynucleotide in a sample, said target polynucleotide being selected from the group consisting of a) a polynucleotide comprising a polynucleotide sequence selected from the group consisting of SEQ
)D N0:41-80, b) a polynucleotide comprising a naturally occurring polynucleotide sequence at least 90% identical or at least about 90% identical to a polynucleotide sequence selected from the group consisting of SEQ D7 NO:41-80, 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. In a related embodiment, the method can include detecting the amount of the amplified target polynucleotide or fragment thereof.
Another embodiment 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 ~ N0:1-40, b) a polypeptide comprising a naturally occurring amino acid sequence at least 90% identical or at least about 90% identical to an amino acid sequence selected from the group consisting of SEQ D7 N0:1-40, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ~ N0:1-40, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ m N0:1-40, and a pharmaceutically acceptable excipient. In one embodiment, the composition can comprise an amino acid sequence selected from the group consisting of SEQ m N0:1-40. Other embodiments provide a method of treating a disease or condition associated with decreased or abnormal expression of functional PNNnVVIM, comprising administering to a patient in need of such treatment the composition.
Yet another embodiment 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 N0:1-40, b) a polypeptide comprising a naturally occurring amino acid sequence at least 90% identical or at least about 90% identical to an amino acid sequence selected from the group consisting of SEQ D7 N0:1-40, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ
B7 N0:1-40, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ m NO:1-40. The method comprises a) exposing a sample comprising the polypeptide to a compound, and b) detecting agonist activity in the sample. Another embodiment provides a composition comprising an agonist compound identified by the method and a pharmaceutically acceptable excipient. Yet another embodiment provides a method of treating a disease or condition associated with decreased expression of functional PNJIVIM, comprising administering to a patient in need of such treatment the composition.
Still yet another embodiment provides a method for screening a compound for effectiveness as an antagonist of a polypeptide selected from the group consisting of a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ >D N0:1-40, b) a polypeptide comprising a naturally occurring amino acid sequence at least 90% identical or at least about 90%
identical to an amino acid sequence selected from the group consisting of SEQ
)D N0:1-40, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ m N0:1-40, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1-40. The method comprises a) exposing a sample comprising the polypeptide to a compound, and b) detecting antagonist activity in the sample. Another embodiment provides a composition comprising an antagonist compound identified by the method and a pharmaceutically acceptable excipient. Yet another embodiment provides a method of treating a disease or condition associated with overexpression of functional Pte, comprising administering to a patient in need of such treatment the composition.
Another embodiment provides a method of screening for a compound that specifically binds to a polypeptide selected from the group consisting of a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID N0:1-40,: b) a polypeptide comprising a naturally occurring amino acid sequence at least 90% identical or at least about 90% identical to an amino acid sequence selected from the group consisting of SEQ ID N0:1-40, c) a biologically active , fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ
m N0:1-40, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ 117 N0:1-40. 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.
Yet another embodiment 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 m N0:1-40, b) a polypeptide comprising a naturally occurring amino acid sequence at least 90% identical or at least about 90% identical to an a_rrLno acid sequence selected from the group consisting of SEQ ID N0:1-40, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ

>D N0:1-40, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID N0:1-40. The method comprises a) combining the polypeptide with at least one test compound under conditions permissive for the activity of the polypeptide, b) assessing the activity of the polypeptide in the presence of the test compound, and c) comparing the activity of the polypeptide in the presence of the test compound with the activity of the polypeptide in the absence of the test compound, wherein a change in the activity of the polypeptide in the presence of the test compound is indicative of a compound that modulates the activity of the polypeptide.
Still yet another embodiment 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 )D N0:41-80, the method comprising a) exposing a sample comprising the target polynucleotide to a compound, 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.
Another embodiment provides a method for assessing toxicity of a test compound, said method comprising a) treating a biological sample containing nucleic acids with the test compound; b) hybridizing the nucleic acids of the treated biological sample with a probe comprising at least 20 contiguous nucleotides of a polynucleotide selected from the group consisting of i) a polynucleotide comprising a polynucleotide sequence selected from the group consisting of SEQ
177 N0:41-80, ii) a polynucleotide comprising a naturally occurring polynucleotide sequence at least 90% identical or at least about 90% identical to a polynucleotide sequence selected from the group consisting of SEQ ID
N0:41-80, 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
>D N0:41-80, ii) a polynucleotide comprising a naturally occurring polynucleotide sequence at least 90% identical or at least about 90% identical to a polynucleotide sequence selected from the group consisting of SEQ >D N0:41-80, iii) a polynucleotide complementary to the polynucleotide of i), iv) a polynucleotide complementary to the polynucleotide of ii), and v) an RNA
equivalent of i)-iv).
Alternatively, the target polynucleotide can comprise a fragment of a polynucleotide selected from the group consisting of i)-v) above; c) quantifying the amount of hybridization complex; and d) comparing the amount of hybridization complex in the treated biological sample with the amount of hybridization complex in an untreated biological sample, wherein a difference in the amount of hybridization complex in the treated biological sample is indicative of toxicity of the test compound.
BRIEF DESCRIPTION OF THE TABLES
Table 1 summarizes the nomenclature for full length polynucleotide and polypeptide embodiments of the invention.
Table 2 shows the GenBank identification number and annotation of the nearest GenBank homolog, and the PROTEOME database identification numbers and annotations of PROTBOME
database homologs, for polypeptide embodiments of the invention. 'The probability scores.for the matches between each polypeptide and its homolog(s) are also shown.
Table 3 shows structural features of polypeptide embodiments, including predicted motifs and domains, along with the methods, algorithms, and searchable databases used for analysis of the polypeptides.
Table 4 lists the cDNA andlor genomic DNA fragments which were used to assemble polynucleotide embodiments, along with selected fragments of the polynucleotides.
Table 5 shows representative cDNA libraries for polynucleotide embodiments.
Table 6 provides an appendix which describes the tissues and vectors used for construction of the cDNA libraries shown in Table 5.
Table 7 shows the tools, programs, and algorithms used to analyze polynucleotides and polypeptides, along with applicable descriptions, references, and threshold parameters.
DESCRIPTION OF THE INVENTION
Before the present proteins, nucleic acids, and methods are described, it is understood that embodiments of the invention are not limited to the particular machines, instruments, 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 invention.
As used herein and in the appended claims, the singular forms "a," "an," and "the" include plural reference unless the context clearly dictates otherwise. Thus, for example, a reference to "a host cell" includes a plurality of such host cells, and a reference to "an antibody" is a reference to one or more antibodies and equivalents thereof known to those skilled in the art, and so forth.
Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art to which this invention belongs.
Although any machines, materials, and methods similar or equivalent to those described herein can be used to practice or test the present invention, the preferred machines, materials and methods are now described. All publications mentioned herein are cited for the purpose of describing and disclosing the cell lines, protocols, reagents and vectors which are reported in the publications and which might be used in connection with various embodiments of the invention. Nothing herein is to be construed as an admission that the invention is not entitled to antedate such disclosure by virtue of prior invention.
DEFINITIONS
"PNPVIM" refers to the amino acid sequences of substantially purified Pl~~VIM
obtained from any species, particularly a mammalian species, including bovine, ovine, porcine, murine, equine, and .
human, and from any source, whether natural, synthetic, semi-synthetic, or recombinant.
The term "agonist" refers to a molecule which intensifies or mimics the biological activity of PNEVIM. Agonists may include proteins, nucleic acids, carbohydrates, small molecules, or any other compound or composition which modulates the activity of PMNINI either by directly interacting with PIV11VIM or by acting on components of the biological pathway in which PM1VINI
participates.
An "allelic variant" is an alternative form of the gene encoding P1VBVIM.
Allelic variants may result from at least one mutation in the nucleic acid sequence and may result in altered mRNAs or in polypeptides whose structure or function may or may not be altered. A gene may have none, one, or many allelic variants of its naturally occurring form. Common mutational changes which give rise to allelic variants are generally ascribed to natural deletions, additions, or substitutions of nucleotides.
Each of these types of changes may occur alone, or in combination with the others, one or more times in a given sequence.
"Altered" nucleic acid sequences encoding PNNIIVVIM include those sequences with deletions, insertions, or substitutions of different nucleotides, resulting in a polypeptide the same as PM1VIT~I or a polypeptide with at least one functional characteristic of PNIMM. Included within this definition are polymorphisms which may or may not be readily detectable using a particular oligonucleotide probe of the polynucleotide encoding Pn~llVIM, and improper or unexpected hybridization to allelic variants, with a locus other than the normal chromosomal locus for the polynucleotide encoding P1~~VIM. 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 PIVllVIM.
Deliberate amino acid substitutions may be made on the basis of one or more similarities in polarity, charge, solubility, hydrophobicity, hydrophilicity, and/or the amphipathic nature of the residues, as long as the biological or immunological activity of Pis retained. For example, negatively charged amino acids may include aspartic acid and glutamic acid, and positively charged amino acids may include lysine and arginine. Amino acids with uncharged polar side chains having similar hydrophilicity values may include: asparagine and glutamine; and serine and threonine. Amino acids with uncharged side chains having similar hydrophilicity values may include: leucine, isoleucine, and valine; glycine and alanine; and phenylalanine and tyrosine.
The terms "amino acid" and "amino acid sequence" can refer to an oligopeptide, a peptide, a polypeptide, or a 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. Amplification may be carried out using polymerase chain reaction (PCR) technologies or other nucleic acid amplification technologies well known in the art.
The term "antagonist" refers to a molecule which inhibits or attenuates the biological activity of PM1VIM. Antagonists may include proteins such as antibodies, anticalins, nucleic acids, carbohydrates, small molecules, or any other compound or composition which modulates the activity of pN~~M either by directly interacting with PNhVIM or by acting on components of the biological pathway in which PMIVIM participates.
The term "antibody" refers to intact immunoglobulin molecules as well as to fragments thereof, such as Fab, F(ab')Z, and Fv fragments, which are capable of binding an epitopic determinant.
Antibodies that bind P~ polypeptides can be prepared using intact polypeptides or using fragments containing small peptides of interest as the immunizing antigen. The polypeptide or oligopeptide used to immunize an animal (e.g., a mouse, a rat, or a rabbit) can be derived from the translation of RNA, or synthesized chemically, and can be conjugated to a carrier protein if desired.
Commonly used Garners that are chemically coupled to peptides include bovine serum albumin, thyroglobulin, and keyhole limpet hemocyanin (KLI~. 'The coupled peptide is then used to immunize the animal.

The term "antigenic determinant" refers to that region of a molecule (i.e., an epitope) that makes contact with a particular antibody. When a protein or a fragment of a protein is used to immunize a host animal, numerous regions of the protein may induce the production of antibodies which bind specifically to antigenic determinants (particular regions or three-dimensional structures on the protein). An antigenic determinant may compete with the intact antigen (i.e., the immunogen used to elicit the immune response) for binding to an antibody.
The term "aptamer" refers to a nucleic acid or oligonucleotide molecule that binds to a specific molecular target. Aptamers are derived from an in vitro evolutionary process (e.g., SELEX
(Systematic Evolution of Ligands by EXponential Enrichment), described in U.S.
Patent No.
5,270,163), which selects for target-specific aptamer sequences from large combinatorial libraries.
Aptamer compositions maybe double-stranded or single-stranded, and may include deoxyribonucleotides, ribonucleotides, nucleotide derivatives, or other nucleotide-like molecules. The nucleotide components of an aptamer may have rriodified sugar groups (e.g., the 2'-OH group of a ribonucleotide may be replaced by 2'-F or 2'-NHZ), wluch may improve a desired property, e.g., resistance to nucleases or longer lifetime in blood. Aptamers may be conjugated to other molecules, e.g., a high molecular weight carrier to slow clearance of the aptamer from the circulatory system.
Aptamers may be specifically cross-licked to their cognate ligands, e.g., by photo-activation of a cross-linker (Brody, E.N. and L. Gold (2000) J. Biotechnol. 74:5-13).
The term "intramer" refers to an aptamer which is expressed in vivo. For example, a vaccinia virus-based RNA expression system has been used to express specific RNA aptamers at high levels in the cytoplasm of leukocytes (Blind, M. et al. (1999) Proc.
Natl. Acad. Sci. USA
96:3606-3610).
The term "spiegeliner" refers to an aptamer which includes L-DNA, L-RNA, or other left-handed nucleotide derivatives or nucleotide-like molecules. Aptamers containing left-handed nucleotides are resistant to degradation by naturally occurring enzymes, which normally act on substrates containing right-handed nucleotides.
The term "antisense" refers to any composition capable of base-pairing with the "sense"
(coding) strand of a polynucleotide having a specific nucleic acid sequence.
Antisense compositions may include DNA; RNA; peptide nucleic acid (PNA); oligonucleotides having modified backbone linkages such as phosphorothioates, methylphosphonates, or benzylphosphonates;
oligonucleotides having modified sugar groups such as 2'-methoxyethyl sugars or 2'-methoxyethoxy sugars; or oligonucleotides having modified bases such as 5-methyl cytosine, 2'-deoxyuracil, or 7-deaza-2'-deoxyguanosine. Antisense molecules may be produced by any method including chemical synthesis or transcription. Once introduced into a cell, the complementary antisense molecule base-pairs with a naturally occurring nucleic acid sequence produced by the cell to form duplexes which block either transcription or translation. The designation "negative" or "minus" can refer to the antisense strand, and the designation "positive" or "plus" can refer to the sense strand of a reference DNA molecule.
The term "biologically active" refers to a protein having structural, regulatory, or biochemical functions of a naturally occurring molecule. Likewise, "immunologically active" or "immunogenic"
refers to the capability of the natural, recombinant, or synthetic PMMM, or of any oligopeptide thereof, to induce a specific immune response in appropriate animals or cells and to bind with specific antibodies.
"Complementary" describes the relationship between two single-stranded nucleic acid sequences that anneal by base-pairing. For example, 5'-AGT-3' pairs with its complement, 3'-TCA-5'.
A "composition comprising a given polynucleotide" and a "composition comprising a given polypeptide" can refer to any composition containing the given polynucleotide or polypeptide. The composition may comprise a dry formulation or an aqueous solution.
Compositions comprising polynucleotides encoding PMMM or fragments of PMMM may be employed as hybridization probes.
The probes may be stored in freeze-dried form and may be associated with a stabilizing agent such as a carbohydrate. In. hybridizations, the probe may be deployed in an aqueous solution containing salts (e.g., 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 CA) in the 5' and/or the 3' direction, and resequenced, or which has been assembled from one or more overlapping cDNA, EST, or genornic DNA fragments using a computer program for fragment assembly, such as the GELV1EW fragment assembly system (Accelrys, Burlington MA) or Phrap (University of Washington, Seattle WA). Some sequences have been both extended and assembled to produce the consensus sequence.
"Conservative amino acid substitutions" are those substitutions that are predicted to least interfere with the properties of the original protein, i.e., the structure and especially the function of the protein is conserved and not significantly changed by such substitutions. The table below shows amino acids which may be substituted for an original amino acid in a protein and wluch are regarded as conservative amino acid substitutions.
Original Residue Conservative Substitution Ala Gly, Ser Arg His, Lys Asn Asp, Gln, His Asp Asn, Glu Cys Ala, Ser Gln Asn, Glu, His l0 Glu Asp, Gln, His Gly Ala His Asn, Arg, Gln, Glu Ile Leu, Val Leu Ile, Val Lys Arg, Gln, Glu Met Leu, Ile Phe His, Met, Leu, Trp, Tyr Ser Cys, Thr Thr Ser, Val Trp Phe, Tyr Tyr IIis, 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, andlor (c) the bulk of the side chain.
A "deletion" refers to a change in the amino acid or nucleotide sequence that results in the absence of one or more amino acid residues or nucleotides.
The term "derivative" refers to a chemically modified polynucleotide or polypeptide.
Chemical modifications of a polynucleotide can include, for example, replacement of hydrogen by an alkyl, acyl, hydroxyl, or amino group. A derivative polynucleotide encodes a polypeptide which retains at least one biological or immunological function of the natural molecule. A
derivative polypeptide is one modified by glycosylation, pegylation, or any similar process that retains at least one biological or immunological function of the polypeptide from which it was derived.
A "detectable label" refers to a reporter molecule or enzyme that is capable of generating a measurable signal and is covalently or noncovalently joined to a polynucleotide or polypeptide.
"Differential expression" refers to increased or upregulated; or decreased, downregulated, or absent gene or protein expression, determined by comparing at least two different samples. Such comparisons may be carried out between, for example, a treated and an untreated sample, or a diseased and a normal sample.
"Exon shuffling" refers to the recombination of different coding regions (exons). Since an exon may represent a structural or functional domain of the encoded protein, new proteins may be assembled through the novel reassortment of stable substructures, thus allowing acceleration of the evolution of new protein functions.
A "fragment" is a unique portion of PMMM or a polynucleotide encoding Pwhich can be identical i_n sequence to, but shorter in length than, the parent sequence.
A fragment may comprise up to the entire length of the defined sequence, minus one nucleotide/amino acid residue. For example, a fragment may comprise from about 5 to about 1000 contiguous nucleotides or amino acid residues. A fragment used as a probe, primer, antigen, therapeutic molecule, or for other purposes, may be at least 5, 10, 15, 16, 20, 25, 30, 40, 50, 60, 75, 100, 150, 250 or at least 500 contiguous nucleotides or amino acid residues in length. Fragments may be preferentially selected from certain regions of a molecule. For example, a polypeptide fragment may comprise a certain length of contiguous amino acids selected from the first 250 or 500 amino acids (or first 25% or 50%) of a polypeptide as shown in a certain defined sequence. Clearly these lengths are exemplary, and any length that is supported by the specification, including the Sequence Listing, tables, and figures, may be encompassed by the present embodiments.
A fragment of SEQ B7 N0:41-80 can comprise a region of unique polynucleotide sequence that specifically identifies SEQ m NO:41-80, for example, as distinct from any other sequence in the genome from which the fragment was obtained. A fragment of SEQ m N0:41-80 can be employed in one or more embodiments of methods of the invention, for example, in hybridization and amplification technologies and in analogous methods that distinguish SEQ m N0:41-80 from related polynucleotides. The precise length of a fragment of SEQ m NO:41-80 and the region of SEQ m NO:41-80 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 m N0:1-40 is encoded by a fragment of SEQ m NO:41-80. A
fragment of SEQ m N0:1-40 can comprise a region of unique amino acid sequence that specifically identifies SEQ D7 N0:1-40. For example, a fragment of SEQ m N0:1-40 can be used as an immunogenic peptide for the development of antibodies that specifically recognize SEQ m N0:1-40.
The precise length of a fragment of SEQ m N0:1-40 and the region of SEQ m N0:1-40 to which the fragment corresponds can be determined based on the intended purpose for the fragment using one or more analytical methods described herein or otherwise known in the art.
A "full length" polynucleotide is one containing at least a translation initiation codon (e.g., methionine) followed by an open reading frame and a translation termination codon. A "full length"
polynucleotide sequence encodes a "full length" polypeptide sequence.
"Homology" refers to sequence similarity or, alternatively, sequence identity, between two or more polynucleotide sequences or two or more polypeptide sequences.
The terms "percent identity" and "% identity," as applied to polynucleotide sequences, refer to the percentage of identical residue matches between at least two polynucleotide sequences aligned using a standardized algorithm. Such an algorithm may insert, in a standardized and reproducible way, gaps in the sequences being compared in order to optimize alignment between two sequences, and therefore achieve a more meaningful comparison of the two sequences.
Percent identity between polynucleotide sequences may be determined using one or more computer algorithms or programs known in the art or described herein. For example, percent identity can be determined using the default parameters of the CLUSTAL V algorithm as incorporated into the MEGALIGN version 3.12e sequence alignment program. This program is part of the LASERGENE software package, a suite of molecular biological analysis programs (DNASTAR, Madison WI). CLUSTAL V is described in Higgins, D.G. and P.M. Sharp (1989;
CABIOS 5:151-153) and iwHiggins, D.G. et al. (1992; CABIOS 8:189-191). For pairwise 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.
Alternatively, a suite of commonly used and freely available sequence comparison algorithms which can be used is provided by the National Center for Biotechnology Information (NCBI) Basic Local Alignment Search Tool (BLAST) (Altschul, S.F. et al. (1990) J. Mol.
Biol. 215:403-410), which is available from several sources, including the NCBI, Bethesda, MD, and on the Internet at http://www.ncbi.nlm.nih.gov/BLAST/. The BLAST software suite includes various sequence analysis programs including "blastn," that is. used to align a known polynucleotide sequence with other polynucleotide sequences from a variety of databases. Also available is a tool called "BLAST 2 Sequences" that is used for direct pairwise comparison of two nucleotide sequences. "BLAST 2 Sequences" can be accessed and used interactively at http://www.ncbi.nlm.nih.gov/gorf/bl2.html. The "BLAST 2 Sequences" tool can be used for both blastn and blastp (discussed below). BLAST
programs are commonly used with gap and other parameters set to default settings. For example, to compare two nucleotide sequences, one may use blastn with the "BLAST 2 Sequences" tool Version 2Ø12 (April-21-2000) set at default parameters. Such default parameters may be, for example:
Matrix: BLOSUM62 Reward for match: 1 Penalty for' rnisrnatch: -2 Open Gap: 5 arid Extension Gap: 2 penalties Gap x drop-off. 50 Expect: 10 Word Size: 11 Filter: on Percent identity may be measured over the length of an entire defined sequence, for example, as defined by a particular SEQ ID number, or may be measured over a shorter length, for example, over the length of a fragment taken from a larger, defined sequence, for instance, a fragment of at least 20, at least 30, at least 40, at least 50, at least 70, at least 100, or at least 200 contiguous nucleotides. Such lengths are exemplary only, and it is understood that any fragment length supported by the sequences shown herein, in the tables, figures, or Sequence Listing, may be used to describe a length over which percentage identity may be measured.
Nucleic acid sequences that do not show a high degree of identity may nevertheless encode similar amino acid sequences due to the degeneracy of the genetic code. It is understood that changes in a nucleic acid sequence can be made using this degeneracy to produce multiple nucleic acid sequences that all encode substantially the same protein.
The phrases "percent identity" and "% identity," as applied to polypeptide sequences, refer to the percentage of identical 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. The phrases "percent similarity' and "% similarity," as applied to polypeptide sequences, refer to the percentage of residue matches, including identical residue matches and conservative substitutions, between at least two polypeptide sequences aligned using a standardized algorithm. In contrast, conservative substitutions are not included in the calculation of percent identity between polypeptide sequences.

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.
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:
Matt~ix: BLOSUM62 Open Gap: 11 and Exterzsio~ Gap: 1 penalties Gap x dt-op-off. ~'0 Expect: 10 Word Size: 3 Fi~tet-: on 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 2o instance, a fragment of at least 15, at least 20, at least 30, at least 40, at least 50, at least 70 or at least 150 contiguous residues. Such lengths are exemplary only, and it is understood that any fragment length supported by the sequences shownherein, in the tables, figures or Sequence Listing, may be used to describe a length over which percentage identity may be measured.
"Human artificial chromosomes" (HACs) are linear microchromosomes which may contain DNA sequences of about 6 kb to 10 Mb in size and which contain all of the elements required for chromosome replication, segregation and maintenance.
The term "humanized antibody" refers to an antibody molecule in which the amino acid sequence in the non-antigen binding regions has been altered so that the antibody more closely resembles a human antibody, and still retains its original binding ability.
"Hybridization" refers to the process by which a polynucleotide strand anneals with a complementary strand through base pairing under defined hybridization conditions. Specific hybridization is an indication that two nucleic acid sequences share a high degree of complementarity.

Specific hybridization complexes form under permissive annealing conditions and remain hybridized after the "washing" step(s). The washing steps) is particularly important in determining the stringency of the hybridization process, with more stringent conditions allowing less non-specific binding, i.e., binding between pairs of nucleic acid strands that are not perfectly matched. Permissive conditions for annealing of nucleic acid sequences are routinely determinable by one of ordinary skill in the art and may be consistent among hybridization experiments, whereas wash conditions may be varied among experiments to achieve the desired stringency, and therefore hybridization specificity.
Permissive annealing conditions occur, for example, at 68°C in the presence of about 6 x SSC, about 1% (w/v) SDS, and about 100 p,g/ml sheared, denatured salmon sperm DNA.
Generally, stringency of hybridization is expressed, in part, with reference to the temperature under which the wash step is carried out. Such wash temperatures are typically selected to be about 5°C to 20°C lower than the thermal melting point (T"~ for the specific sequence at a defined ionic strength and pH. The Tm is the temperature (under defined ionic strength and pH) at which 50% of the target sequence hybridizes to a perfectly matched probe. An equation for calculating Tm and conditions for nucleic acid hybridization are well known and can be found in Sambrook, J. and D.W.
Russell (2001; Molecular Cloning: A Laboratory Manual, 3rd ed., vol. 1-3, Cold Spring Harbor Press, Cold Spring Harbor NY, ch. 9).
High stringency conditions for hybridization between polynucleotides of the present invention include wash conditions of 68°C in the presence of about 0.2 x SSC and about 0.1% SDS, for 1 hour:
Alternatively, temperatures of about 65°C, 60°C, 55°C, or 42°C 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 ~,g/ml. Organic solvent, such as formamide at a concentration of about 35-50% v/v, may also be used under particular circumstances, such as for RNA:DNA hybridizations. Useful variations on these wash conditions will be readily apparent to those of ordinary skill in the art. Hybridization, particularly under high stringency conditions, may be suggestive of evolutionary similarity between the nucleotides. Such similarity is strongly indicative of a similar role for the nucleotides and their encoded polypeptides.
The term "hybridization complex" refers to a complex formed between two nucleic acids by virtue of the formation of hydrogen bonds between complementary bases. A
hybridization complex may be formed in solution (e.g., Cot or Rot analysis) or formed between one nucleic acid present in solution and another nucleic acid 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 polynucleotide 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 "immunogenic fragment" is a polypeptide or oligopeptide fragment of P~
which is to 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 PM1VIM 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, 15 polypeptides, antibodies, or other chemical compounds on a substrate.
The terms "element" and "array element" refer to a polynucleotide, polypeptide, antibody, or other chemical compound having a unique and defined position on a microarray.
' The term "modulate" refers to a change in the activity of PMIVINI. For example, modulation may cause an increase or a decrease in protein activity, binding characteristics, or any other biological, 20 functional, or immunological properties of PMMM.
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 genoxnic 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.
25 "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.
30 "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 PMMM 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 P1VIIVIM.
"Probe" refers to nucleic acids encoding Pl~~VIM, their complements, or fragments thereof, which are used to detect identical, allelic or related nucleic acids. 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, e.g., by the polymerase chain reaction (PCR).
Probes and primers as used in the present invention typically comprise at least 15 contiguous nucleotides of a known sequence. In order to enhance specificity, longer probes and primers may also be employed, such as probes and primers that comprise at least 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, or at least 150 consecutive nucleotides of the disclosed nucleic acid sequences. Probes and primers may be considerably longer than these examples, and it is understood that any length supported by the specification, including the tables, figures, and Sequence Listing, may be used.
Methods for preparing and using probes and primers are described in, for example, Sambrook, J. and D.W. Russell (2001; Molecular Cloninw A Laboratory Manual, 3rd ed., vol. 1-3, Cold Spring Harbor Press, Cold Spring Harbor NY),. Ausubel, F.M. et al. (1999; Short Protocols in Molecular Biolo , 4t'' ed., John Wiley & Sons, New York NY), and Innis, M. et al. (1990;
PCR Protocols, A
Guide to Methods and Applications, Academic Press, San Diego CA). PCR primer pairs can be .derived from a known sequence, for example, by using computer programs intended for that purpose such as Primer (Version 0.5, 1991, Whitehead Institute for Biomedical Research, Cambridge MA).
Oligonucleotides for use as primers are selected using software known in the art for such purpose. For example, OLIGO 4.06 software is useful for the selection of PCR
primer pairs of up to 100 nucleotides each, and for the analysis of oligonucleotides and larger polynucleotides of up to 5,000 nucleotides from an input polynucleotide sequence of up to 32 kilobases.
Similar primer selection programs have incorporated additional features for expanded capabilities. For example, the PrimOU

primer selection program (available to the public from the Genome Center at University of Texas South West Medical Center, Dallas TX) is capable of choosing specific primers from megabase sequences and is thus useful for designing primers on a genome-wide scope. The Primer3 primer selection program (available to the public from the Whitehead Institute/1VRT
Center for Genome Research, Cambridge MA) allows the user to input a "mispriming library," in which sequences to avoid as primer binding sites are user-specified. Primer3 is useful, in particular, for the selection of oligonucleotides for microarrays. (The source code for the latter two primer selection programs may also be obtained from their respective sources and modified to meet the user's specific needs.) The PrimeGen program (available to the public from the UK Human Genome Mapping Project Resource Centre, Cambridge UI~) designs primers. based on multiple sequence alignments, thereby allowing selection of primers that hybridize to either the most conserved or least conserved regions of aligned nucleic acid sequences. Hence, this program is useful for identification of both unique and conserved oligonucleotides and polynucleotide fragments. The oligonucleotides and polynucleotide fragments identified by any of the above selection methods are useful in hybridization technologies, for example, as PCR or sequencing primers, microarray elements, or specific probes to identify fully or partially complementary polynucleotides in a sample of nucleic acids. Methods of oligonucleotide selection are not limited to those described above.
A "recombinant nucleic acid" is a nucleic acid that is not naturally occurring or has a sequence that is made by an artificial combination of two or more otherwise separated segments of sequence. Tlus artificial combination is often accomplished by chemical synthesis or, more commonly, by the artificial manipulation of isolated segments of nucleic acids, e.g., by genetic engineering techniques such as those described in Sambrook and Russell (supra). The term recombinant includes nucleic acids that have been altered solely by addition, substitution, or deletion of a portion of the nucleic acid. Frequently, a recombinant nucleic acid may include a nucleic acid sequence operably linked to a promoter sequence. Such a recombinant nucleic acid may be part of a vector that is used, for example, to transform a cell.
Alternatively, such recombinant nucleic acids may be part of a viral vector, e.g., based on a vaccinia virus, that could be use to vaccinate a mammal wherein the recombinant nucleic acid is expressed, inducing a protective immunological response in the mammal.
A "regulatory element" refers to a nucleic acid sequence usually derived from untranslated regions of a gene and includes enhancers, promoters, introns, and 5' and 3' untranslated regions (UTRs). Regulatory elements interact with host or viral proteins which control transcription, translation, or RNA stability.
"Reporter molecules" are chemical or biochemical moieties used for labeling a nucleic acid, amino acid, or antibody. Reporter molecules include radionuclides; enzymes;
fluorescent, chemiluminescent, or chromogenic agents; substrates; cofactors; inhibitors;
magnetic particles; and other moieties known in the art.
An "RNA equivalent," in reference to a DNA molecule, is composed of the same linear sequence of nucleotides as the reference DNA molecule with the exception that all occurrences of the nitrogenous base thymine are replaced with uracil, and the sugar backbone is composed of ribose instead of deoxyribose.
The term "sample" is used in its broadest sense. A sample suspected of containing PI~~VIM, nucleic acids encoding Pte, or fragments thereof may comprise a bodily fluid;
an extract from a cell, chromosome, organelle, or membrane isolated from a cell; a cell; genomic DNA, RNA, or cDNA, in solution or bound to a substrate; a tissue; a tissue print; etc.
The terms "specific binding" and "specifically binding" refer to that interaction between a protein or peptide and an agonist, an antibody, an antagonist, a small molecule, or any natural or synthetic binding composition. The interaction is dependent upon the presence of a particular structure of the protein, e.g., the antigenic determinant or epitope, recognized by the binding molecule. For example, if an antibody is specific for epitope "A," the presence of a polypeptide comprising the epitope A, or the presence of free unlabeled A, in a reaction containing free labeled A and the antibody will reduce the amount of labeled A that binds to the antibody.
The term "substantially purified" refers to nucleic acid or amino acid sequences that are removed from their natural environment and are isolated or separated, and are at least about 60°7o free, preferably at least about 75% free, and most preferably at least about 90% free from other components with which they are naturally associated.
A "substitution" refers to the replacement of one or more amino acid residues or nucleotides by different amino acid residues or nucleotides, respectively.
"Substrate" refers to any suitable rigid or semi-rigid support including membranes, filters, chips, slides, wafers, fibers, magnetic or nonmagnetic beads, gels, tubing, plates, polymers, microparticles and capillaries. The substrate can have a variety of surface forms, such as wells, trenches, pins, channels and pores, to which polynucleotides or polypeptides are bound.

A "transcript image" or "expression profile" refers to the collective pattern of gene expression by a particular cell type or tissue under given conditions at a given time.
"Transformation" describes a process by which exogenous DNA is introduced into a recipient cell. Transformation may occur under natural or artificial conditions according to various methods well known in the art, and may rely on any known method for the insertion of foreign nucleic acid sequences into a prokaryotic or eukaryotic host 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, lipofection, and particle bombardment.
The term "transformed cells" includes stably transformed cells in which the inserted DNA is capable of replication either as an autonomously replicating plasmid or as part of the host chromosome, as well as transiently transformed cells which express the inserted DNA or RNA for limited periods of time.
A "transgenic organism," as used herein, is any organism, including but not limited to animals and plants, in which one or more of the cells of the organism contains heterologous nucleic acid introduced by way of human intervention, such as by transgenic techniques well known in the art. The nucleic acid is introduced into the cell, directly or indirectly by introduction into a precursor of the cell, by way of deliberate genetic manipulation, such as by microinjection or by infection with a recombinant virus. In another embodiment, the nucleic acid can be introduced by infection with a recombinant viral vector, such as a lentiviral vector (Lois, C. et al. (2002) Science 295:868-872). The term genetic manipulation does not include classical cross-breeding, or in vitro fertilization, but rather is directed to the introduction of a recombinant DNA molecule. The transgenic organisms contemplated in accordance with the present invention include bacteria, cyanobacteria, fungi, plants and animals. The isolated DNA of the present invention can be introduced into the host by methods known in the art, for example infection, transfection, transformation or transconjugation. Techniques for transferring the DNA of the present invention into such organisms are widely known and provided in references such as Sambrook and Russell (supra).
A "variant" of a particular nucleic acid sequence is defined as a nucleic acid sequence having at least 40% sequence identity to the particular nucleic acid sequence over a certain length of one of the nucleic acid sequences using blastn with the "BLAST 2 Sequences" tool Version 2Ø9 (May-07-1999) set at default parameters. Such a pair of nucleic acids may show, for example, at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% or greater sequence identity over a certain defined length. A variant maybe described as, for example, an "allelic" (as defined above), "splice," "species," or "polymorphic" variant. A
splice variant may have significant identity to a reference molecule, but will generally have a greater or lesser number of polynucleotides due to alternate splicing of exons during mRNA processing. The corresponding polypeptide may possess additional functional domains or lack domains that are present in the reference molecule. Species variants are polynucleotides 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 l0 presence of SNPs may be indicative of, for example, a certain population, a disease state, or a propensity for a disease state.
A "variant" of a particular polypeptide sequence is defined as a polypeptide sequence having at least 40% sequence identity or sequence similarity to the particular polypeptide sequence over a certain length of one of the polypeptide sequences using blastp with the "BLAST 2 Sequences" tool Version 2Ø9 (May-07-1999) set at default parameters. Such a pair of polypeptides may show,~for example, at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%,. at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% or greater sequence identity or sequence similarity over a certain defined length of one of the polypeptides.
THE INVENTION
Various embodiments of the invention include new human protein modification and maintenance molecules (PMMM), the polynucleotides encoding PMMM, and the use of these compositions for the diagnosis, treatment, or prevention of gastrointestinal, cardiovascular, autoimmune/inflammatory, cell proliferative, developmental, epithelial, neurological, reproductive, endocrine, pancreatic, adrenal, and metabolic disorders; as well as lipid, copper, and carbohydrate metabolism disorders, disorders associated with gonadal steroid hormones, the immune system, and infections.
Table 1 summarizes the nomenclature for the full length polynucleotide and polypeptide embodiments of the invention. Each polynucleotide and its corresponding polypeptide are correlated to a single Incyte project identification number (Incyte Project ID). Each polypeptide sequence is denoted by both a polypeptide sequence identification number (Polypeptide SEQ
)D NO:) and an Incyte polypeptide sequence number (Incyte Polypeptide 117) as shown. Each polynucleotide sequence is denoted by both a polynucleotide sequence identification number (Polynucleotide SEQ ID
NO:) and an Incyte polynucleotide consensus sequence number (Incyte Polynucleotide m) as shown.
Column 6 shows the Incyte ID numbers of physical, full length clones corresponding to the polypeptide and polynucleotide sequences of the invention. The full length clones encode polypeptides which have at least 95oIo sequence identity to the polypeptide sequences shown in column 3.
Table 2 shows sequences with homology to polypeptide embodiments of the invention as identified by BLAST analysis against the GenBank protein (genpept) database and the PROTEOME
database. Columns 1 and 2 show the polypeptide sequence identification number (Polypeptide SEQ
ID NO:) and the corresponding Incyte polypeptide sequence number (Incyte Polypeptide ID) for polypeptides of the invention. Column 3 shows the GenBank identification number (GenBank ID NO:) of the nearest GenBank homolog and the PROTEOME database identification numbers (PROTEOME ID NO:) of the nearest PROTEOME database homologs. Column 4 shows the probability scores for the matches between each polypeptide and its homolog(s). Column 5 shows the ~ annotation of the GenBank and PROTEOME database homolog(s) along with relevant citations where , applicable, all of which are expressly incorporated by reference herein.
Table 3 shows various structural features of the polypeptides of the invention. Columns 1 and -2 show the polypeptide sequence identification number (SEQ ID NO:) and the corresponding Incyte polypeptide sequence number (Incyte Polypeptide ID) for each polypeptide of the invention. Column 3 shows the number of amino acid residues in each polypeptide. Column 4 shows potential phosphorylation sites, and column 5 shows potential glycosylation sites, as determined by the MOTIFS
program of the GCG sequence analysis software package (Accekys, Burlington MA). Column 6 shows amino acid residues comprising signature sequences, domains, and motifs.
Column 7 shows analytical methods for protein structure/function analysis and in some cases, searchable databases to which the analytical methods were applied.
Together, Tables 2 and 3 summarize the properties of polypeptides of the invention, and these properties establish that the claimed polypeptides are protein modification and maintenance molecules.
For example, SEQ ID N0:2 is 100% identical from residue T41 to residue M288, and 95% identical from residue M1 to residue E43, to cathepsin 02 (GenBank m 81195556) as determined by the Basic Local Alignment Search Tool (BLAST). (See Table 2.) The BLAST probability scores are both 1.8e-158, which indicates the probability of obtaining the observed polypeptide sequence alignment by chance. SEQ ID N0:2 is localized to the cytoplasmic lysosome/vacuole, has a protease function, and is a human cathepsin O (cathepsin K), as determined by BLAST analysis using the PROTEOME
database. SEQ ID N0:2 also contains a papain family cysteine protease domain as determined by searching for statistically significant matches in the hidden Markov model (IEVVIM)-based PFAM
database of conserved protein family domains. (See Table 3.) Data from BLllVll'S, MOTIFS, and PROFILESCAN, and other BLAST analyses provide further corroborative evidence that SEQ ID
N0:2 is a cathepsin O. In an alternative example, SEQ ID N0:4 is 100%
identical, from residue M1 to residue R68, and 99% identical, from residue K69 to residue P218, to human prostate-specific antigen (GenBank ID g618464) as determined by the Basic Local Alignment Search Tool (BLAST).
(See Table 2.) The BLAST probability score is 2.0e-118, which indicates the probability of obtaining 1o the observed polypeptide sequence alignment by chance. SEQ ID NO:4 has serine protease activity, and is a kallikrein serine protease, as determined by BLAST analysis using the PROTEOME .
database. SEQ ID N0:4 also contains a trypsin 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 BLIIVVIPS, MOTIFS, BLAST and PROFILESCAN
analyses provide further corroborative evidence that SEQ ~ N0:4 is a serine protease in the trypsin family. In an alternative example, SEQ ID N0:10 is 99% identical, from residue F123 to residue I824, to human dipeptidyl peptidse (GenBank ID g11095188) as determined by the Basic Local Alignment, Search Tool (BLAST). (See Table 2.) The BLAST probability score is 0.0, which indicates the probability of obtaining the observed polypeptide sequence alignment by chance. SEQ ID N0:10 is localized to the subcellular region, has peptidase function, and is a dipeptidyl aminopeptidase 8, as determined by BLAST analysis using the PROTEOME database. SEQ ID N0:10 also contains a prolyl oligopeptidase domain as determined by searching for statistically significant matches in the hidden Markov model (I~~IM)-based PFAM database of conserved protein family domains. (See Table 3.) Data from BLllVIPS, MOTIFS, and PROFILESCAN analyses provide further corroborative evidence that SEQ ~ NO:10 is a peptidase. In an alternative example, SEQ ID N0:14 is 96% identical, from residue M1 to residue I429, and 100% identical, from residue V412 to residue N945, to human UnpEL, a ubiquitin specific protease (GenBank ~ g2656141) as determined by the Basic Local Alignment Search Tool (BLAST). (See Table 2.) The BLAST
probability score is 0.0, which indicates the probability of obtaining the observed polypeptide sequence alignment by chance.
SEQ ID N0:14 also has homology to proteins that are ubiquitin specific proteases and have altered expression in small cell lung carcinoma cell lines, as determined by BLAST
analysis using the PROTEOME database. SEQ D7 N0:14 also contains a ubiquitin carboxyl-terminal hydrolases family 44.

domain as determined by searching for statistically significant matches in the ludden Markov model (I~VIM) based PFAM database of conserved protein family domains. (See Table 3.) Data from BLIIVVIPS and MOTIFS analyses provide further corroborative evidence that SEQ
ID N0:14 is a ubiquitin specific protease. In an alternative example, SEQ LD NO:27 is 99%
identical, from residue I49 to residue H521 and 93% identical, from residue M1 to residue V58, to human chaperonin containing t-complex polypeptide 1, eta subunit (GenBank ID g2559010) as determined by the Basic Local Alignment Search Tool (BLAST). (See Table 2.) The BLAST probability scores are both 1.4e-272, which indicates the probability of obtaining the observed polypeptide sequence alignment by chance. SEQ ID N0:27 also has homology to proteins that are localized to the cytoskeleton, function as chaperones, and are the eta subunit of the cytosolic chaperonin containing TCP-1 (CCT), as determined by BLAST analysis using the PROTEOME database. SEQ ID N0:27 also contains a TCP-1/cpn60 chaperonin family domain as determined by searching for statistically significant matches in the hidden Markov model (I~~IM)-based PFAM database of conserved.
protein family domains. (See Table 3.) Data from BLILVVIPS, MOT1FS, and additional BLAST
analyses provide further corroborative evidence that SEQ ID N0:27 is a chaperonin containing t-complex polypeptide 1, eta subunit. In an alternative example, SEQ D7 NO:34 is 97% identical, from residue M1 to residue N83, and 100% identical, from residue A82 to residue M146, to human cyclophilin (GenBank ID
g3647230, from M1 to D83 and from A113 to M177, respectively) as determined by the Basic Local:
Alignment Search Tool (BLAST). (See Table 2.) The BLAST probability score is 1.8e-75, which indicates the probability of obtaining the observed polypeptide sequence alignment by chance. SEQ
ID N0:34 also has homology to proteins that are localized to the nucleus, have peptidyl-prolyl isomerase activity and are cyclophilins, as determined by BLAST analysis using the PROTEOME
database. SEQ LD NO:34 also contains a cyclophilin-type peptidyl-prolyl cis-traps isomerase domain as determined by searching for statistically significant matches in the hidden Markov model (I~VVIM)-based PFAM database of conserved protein family domains. (See Table 3.) Data from BLM'S, BLAST, MOTIFS, and PROFILESCAN analyses provide further corroborative evidence that SEQ
DJ N0:34 is a member of the class of cyclophilin peptidyl-prolyl isomerases.
SEQ LD N0:1, SEQ ID
NO:3, SEQ ~ N0:5-9, SEQ )D N0:11-13, SEQ ID N0:15-26, SEQ ID NO:28-33, and SEQ
~
N0:35-40 were analyzed and annotated in a similar manner. The algorithms and parameters for the analysis of SEQ ID N0:1-40 are described in Table 7.
As shown in Table 4, the full length polynucleotide embodiments were assembled using cDNA
sequences or coding (exon) sequences derived from genomic DNA, or any combination of these two types of sequences. Column 1 lists the polynucleotide sequence identification number (Polynucleotide SEQ ID NO:), the corresponding Incyte polynucleotide consensus sequence number (Incyte ID) for each polynucleotide of the invention, and the length of each polynucleotide sequence in basepairs.
Column 2 shows the nucleotide start (5') and stop (3') positions of the cDNA
and/or genomic sequences used to assemble the full length polynucleotide embodiments, and of fragments of the polynucleotides which are useful, for example, in hybridization or amplification technologies that identify SEQ ID N0:41-80 or that distinguish between SEQ ID N0:41-80 and related polynucleotides.
'The polynucleotide fragments described in Column 2 of Table 4 may refer specifically, for example, to Iucyte cDNAs derived from tissue-specific cDNA libraries or from pooled cDNA
libraries. Alternatively, the polynucleotide fragments described in column 2 may refer to GenBank cDNAs or ESTs which contributed to the assembly of the full length polynucleotides. In addition, the polynucleotide fragments described in column 2 may identify sequences derived from the ENSEMBL
(The Sanger Centre, Cambridge, UK) database (i.e., those sequences including the designation "ENST"). Alternatively, the polynucleotide fragments described in column 2 may be derived from the NCBI RefSeq Nucleotide Sequence Records Database (i.e., those sequences including the designation "NM" or "NT") or the NCBI RefSeq Protein Sequence Records (i.e., those sequences including the designation "NP"). Alternatively, the polynucleotide fragments described in column 2 may refer to assemblages of both cDNA and Genscan-predicted exons brought together by an "exon stitching" algorithm. For example, a polynucleotide sequence identified as FL XXXXXX-Nj NZ YYYYY_N3 Na represents a "stitched" sequence in which XXXXXX
is the identification number of the cluster of sequences to which the algorithm was applied, and YYYYY is the number of the prediction generated by the algorithm, and NI,z~3...~ if present, represent specific exons that may have been manually edited during analysis (See Example V).
Alternatively, the polynucleotide fragments in column 2 may refer to assemblages of exons brought together by an "exon-stretching" algorithm. For example, a polynucleotide sequence identified as FZ,~'~~YX'XI~ gAAAAA~BBBBB_1 N is 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 GenBank 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 prefix identifies component sequences that were hand-edited, predicted from genomic DNA sequences, or derived from a combination of sequence analysis methods. The following Table lists examples of component sequence prefixes and corresponding sequence analysis methods associated with the prefixes (see Example IV and Example V).
Prefix Type of analysis and/or examples of programs GNN, GFG, Exon prediction from genomic sequences using, for example, ENST GENSCAN (Stanford University, CA, USA) or FGENES

(Computer Genomics Group, The Sanger Centre, Cambridge, UK) GBI Hand-edited analysis of genomic sequences.

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

INCY Full length transcript and exon prediction from mapping of EST

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

In some cases, Incyte cDNA coverage redundant with the sequence coverage shown in Table 4 was obtained to confirm the final consensus polynucleotide sequence, but the relevant Incyte cDNA identification numbers are not shown.
Table 5 shows the representative cDNA libraries for those full length polynucleotides which were assembled using Incyte cDNA sequences. The representative cDNA library is the Incyte cDNA library which is most frequently represented by the Incyte cDNA sequences which were used to assemble and confirm the above polynucleotides. The tissues and vectors which were used to construct the cDNA libraries shown in Table 5 are described in Table 6.
2o The invention also encompasses PNIIV1M variants. Various embodiments of PI~~VIM variants can have at least about 80%, at least about 90%, or at least about 95% amino acid sequence identity to the PNnVIM amino acid sequence, and can contain at least one functional or structural characteristic of P1~~VIM.
Various embodiments also encompass polynucleotides which encode Pte. In a particular embodiment, the invention encompasses a polynucleotide sequence comprising a sequence selected from the group consisting of SEQ ID N0:41-80, which encodes Pn~VIM. The polynucleotide sequences of SEQ D7 N0:41-80, as presented in the Sequence Listing, embrace the equivalent RNA
sequences, wherein occurrences of the nitrogenous base thymine are replaced with uracil, and the sugar backbone is composed of ribose instead of deoxyribose.

The invention also encompasses variants of a polynucleotide encoding PMMM. In particular, such a variant polynucleotide will have at least about 70%, or alternatively at least about 85%, or even at least about 95% polynucleotide sequence identity to a polynucleotide encoding PNIIVIM. A
particular aspect of the invention encompasses a variant of a polynucleotide comprising a sequence selected from the group consisting of SEQ ID N0:41-80 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 ID NO:41-80. Any one of the polynucleotide variants described above can encode a polypeptide which contains at least one functional or structural characteristic of PD~VIM.
In addition, or in the alternative, a polynucleotide variant of the invention is a splice variant of a polynucleotide encoding P1~~VIM. A splice variant may have portions which have significant sequence identity to a polynucleotide encoding PI~~VIM, but will generally have a greater or lesser number of polynucleotides due to additions or deletions of blocks of sequence arising from alternate splicing of exons during mRNA processing. A splice variant may have less than about 70%, or alternatively less than about 60%, or alternatively less than about 50% polynucleotide sequence identity to a polynucleotide encoding P1~~V1M over its entire length; however, portions of the splice variant will have at least about 70%, or alternatively at least about 85%, or alternatively at least about 95%, or alternatively 100% polynucleotide sequence identity to portions of the polynucleotide encoding PMn~VI. For example, a polynucleotide comprising a sequence of SEQ ID NO:43 and a polynucleotide comprising a sequence of SEQ ID N0:52 are splice variants of each other; a polynucleotide comprising a sequence of SEQ,ID NO:44 and a polynucleotide comprising a sequence of SEQ ll~ NO:69 are splice variants of each other; and a polynucleotide comprising a sequence of SEQ ID NO:46, a polynucleotide comprising a sequence of SEQ ID N0:49, and a polynucleotide comprising a sequence of SEQ ID N0:50 are splice variants of each other. Any one of the splice variants described above can encode a polypeptide which contains at least one functional or structural characteristic of PNNI1VVIM.
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 P1~~VIM, 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 PMMM, and all such variations are to be considered as being specifically disclosed.
Although polynucleotides which encode PMMM and its variants are generally capable of hybridizing to polynucleotides encoding naturally occurring PNN11VVIM under appropriately selected conditions of stringency, it may be advantageous to produce polynucleotides encoding PMMM or its derivatives possessing a substantially different codon usage, e.g., inclusion of non-naturally occurring codons. Codons may be selected to increase the rate at which expression of the peptide occurs in a particular prokaryotic or eukaryotic host in accordance with the frequency with which particular codons are utilized by the host. Other reasons for substantially altering the nucleotide sequence encoding PMMM 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 polynucleotides which encode PMMM
and PMMM derivatives, or fragments thereof, entirely by synthetic chemistry. After production, the synthetic polynucleotide 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 polynucleotide encoding PMMM or any fragment thereof.
Embodiments of the invention can also include polynucleotides that are capable of hybridizing to the claimed polynucleotides, and, in particular, to those having the sequences shown in SEQ ID
N0:41-80 and fragments thereof, under various conditions of stringency (Wahl, G.M. and S.L. Berger (1987) Methods Enzymol. 152:399-407; IKimmel, A.R. (1987) Methods Enzymol.
152:507-511).
Hybridization conditions, including annealing and wash conditions, are described in "Definitions."
Methods for DNA sequencing are well known in the art and may be used to practice any of the embodiments of the invention. The methods may employ such enzymes as the I~lenow fragment of DNA polymerase I, SEQUENASE (US Biochemical, Cleveland OH), Taq polymerase (Applied Biosystems), thermostable T7 polymerase (Amersham Biosciences, Piscataway NJ), or combinations of polymerases and proofreading exonucleases such as those found in the ELONGASE amplification system (Invitrogen, Carlsbad CA). Preferably, sequence preparation is automated with machines such as the MICROLAB 2200 liquid transfer system (Hamilton, Reno NV), PTC200 thermal cycler (MJ Research, Watertown MA) and ABI CATALYST 800 thermal cycler (Applied Biosystems).
Sequencing is then carried out using either the ABI 373 or 377 DNA sequencing system (Applied Biosystems), the MEGABACE 1000 DNA sequencing system (Amersham Biosciences), or other systems known in the art. The resulting sequences are analyzed using a variety of algorithms which are well known in the art (Ausubel et al., supf~a, ch. 7; Meyers, R.A. (1995) Molecular Biology and Biotechnology, Wiley VCH, New York NY, pp. 856-853).
The nucleic acids encoding PMIVINI may be extended utilizing a partial nucleotide sequence and employing various PCR-based methods known in the art to detect upstream sequences, such as promoters and regulatory elements. For example, one method which may be employed, restriction-site PCR, uses universal and nested primers to amplify unknown sequence from genomic DNA within a cloning vector (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 (Triglia, T. et al. (1988) Nucleic Acids Res. 16:8186).
A third method, capture PCR, involves PCR amplification of DNA fragments adjacent to known sequences in human and yeast artificial chromosome DNA (Lagerstrom, M. et al. (1991) PCR Methods Applic. 1:111-119). In this method, multiple restriction enzyme digestions and ligations may be used to insert an engineered double-stranded sequence into a region of unknown sequence before performing PCR. Other methods which may be used to retrieve unknown sequences are known in the art (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 libraries and is useful in finding intron/exon junctions. For all PCR based methods, primers may be designed using commercially available software, such as OLIGO 4.06 primer analysis software (National Biosciences, Plymouth MN) or another appropriate program, to be about 22 to 30 nucleotides in length, to have a GC content of about 50% or more, and to anneal to the template at temperatures of about 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. Genomic libraries may be useful for extension of sequence into 5' non-transcribed regulatory regions.
Capillary electrophoresis systems which are commercially available may be used to analyze the size or confirm the nucleotide sequence of sequencing or PCR products. In particular, capillary sequencing may employ flowable polymers for electrophoretic separation, four different nucleotide-specific, laser-stimulated fluorescent dyes, and a charge coupled device camera for detection of the emitted wavelengths. Output/light intensity may be converted to electrical signal using appropriate software (e.g., GENOTYPER and SEQUENCE NAVIGATOR, Applied Biosystems), and the entire process from loading of samples to computer analysis and electronic data display may be computer controlled. Capillary electrophoresis is especially preferable for sequencing small DNA fragments which may be present in limited amounts in a particular sample.
In another embodiment of the invention, polynucleotides or fragments thereof which encode PMMM may be cloned in recombinant DNA molecules that direct expression of PMMM, or fragments or functional equivalents thereof, in appropriate host cells. Due to the inherent degeneracy of the genetic code, other polynucleotides which encode substantially the same or a functionally equivalent polypeptides may be produced and 'used to express PMMM.
The polynucleotides of the invention can be engineered using methods generally known in the art in order. to alter PMMM-encoding sequences for a variety of purposes including, but not limited to, modification of the cloning, processing, and/or expression of the gene product. DNA shuffling by random fragmentation and PCR reassembly of gene fragments and synthetic oligonucleotides may be used to engineer the nucleotide sequences. For examples oligonucleotide-mediated site-directed mutagenesis may be used to introduce mutations that create new restriction sites, alter glycosylation patterns, change codon preference, produce splice variants, and so forth.
The nucleotides of the present invention may be subjected to DNA shuffling techniques such as MOLECULARBREED1NG (Maxygen Inc., Santa Clara CA; described in U.S. Patent No.
5,837,458; Chang, C.-C. et al. (1999) Nat. Biotechnol. 17:793-797; Christians, F.C. et al. (1999) Nat.
Biotechnol. 17:259-264; and Crameri, A. et al. (1996) Nat. Biotechnol. 14:315-319) to alter or improve the biological properties of PMMM, such as its biological or enzymatic activity or its ability to bind to other molecules or compounds. DNA shuffling is a process by which a library of gene variants is produced using PCR-mediated recombination of gene fragments. The library is then subjected to selection or screening procedures that identify those gene variants with the desired properties. These preferred variants may then be pooled and further subjected to recursive rounds of DNA shuffling and selection/screening. Thus, genetic diversity is created through "artificial"
breeding and rapid molecular evolution. For example, fragments of a single gene containing random point mutations may be recombined, screened, and then reshuffled until the desired properties are optimized. Alternatively, fragments of a given gene may be recombined with fragments of homologous genes in the same gene family, either from the same or different species, thereby maximizing the genetic diversity of multiple naturally occurring genes in a directed and controllable manner.

In another embodiment, polynucleotides encoding PI~~VIM may be synthesized, in whole or in part, using one or more chemical methods well known in the art (Caruthers, M.H. et al. (1980) Nucleic Acids Symp. Ser. 7:215-223; Horn, T. et al. (1980) Nucleic Acids Symp.
Ser. 7:225-232).
Alternatively, PMMM itself or a fragment thereof may be synthesized using chemical methods known in the art. For example, peptide synthesis can be performed using various solution-phase or solid-phase techniques (Creighton, T. (1984) Proteins Structures and Molecular Properties, WH
Freeman, New York NY, pp. 55-60; Roberge, J.Y. et al. (1995) Science 269:202-204). Automated synthesis may be achieved using the ABI 431A peptide synthesizer (Applied Biosystems).
Additionally, the amino acid sequence of PMMM, 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 (Chiez, R.M. and F.Z. Regnier (1990) Methods Enzymol. 182:392-421). .The composition of the synthetic peptides may be confirmed by amino acid analysis or by sequencing (Creighton, supra, pp. 28-53).
In order to express a biologically active PMMM, the polynucleotides encoding P1~~VIM or derivatives thereof may be inserted into an appropriate expression vector, i.e., a vector which contains the necessary elements for transcriptional and translational control of the inserted coding sequence in a suitable host. These elements include regulatory sequences, such as enhancers, constitutive and inducible promoters, and 5' and 3' untranslated regions in the vector and in polynucleotides encoding PI~~VIM. Such elements may vary in their strength and specificity. Specific initiation signals may also be used to achieve more efficient translation of polynucleotides encoding PMMM. Such signals include the ATG initiation codon and adjacent sequences, e.g. the Kozak sequence. In cases where a polynucleotide sequence encoding PMMM and its initiation codon and upstream regulatory sequences are inserted into the appropriate expression vector, no additional transcriptional or translational control signals may be needed. However, in cases where only coding sequence, or a fragment thereof, is inserted, exogenous translational control signals including an in-frame ATG
initiation codon should be provided by the vector. Exogenous translational elements and initiation codons may be of various origins, both natural and synthetic. The efficiency of expression may be enhanced by the inclusion of enhancers appropriate for the particular host cell system used (Scharf, D. et al. (1994) Results Probl.
Cell Differ. 20:125-162).

Methods which are well known to those skilled in the art may be used to construct expression vectors containing polynucleotides encoding PMMM and appropriate transcriptional and translational control elements. These methods include itv vitt~o recombinant DNA techniques, synthetic techniques, and ira vivo genetic recombination (Sambrook and Russell, supra, ch. 1-4, and 8; Ausubel et al., supra, ch. 1, 3, and 15).
A variety of expression vector/host systems may be utilized to contain and express polynucleotides encoding PMMM. These include, but are not limited to, microorganisms such as bacteria transformed with recombinant bacteriophage, plasmid, or cosmid DNA
expression vectors;
yeast transformed with yeast expression vectors; insect cell systems infected with viral expression vectors (e.g.~ baculovirus); plant cell systems transformed with viral expression vectors (e.g., cauliflower mosaic virus, CaMV, or tobacco mosaic virus, TMV) or with bacterial expression vectors (e.g., Ti or pBR322 plasmids); or animal cell systems (Sambrook and Russell, supt~a; Ausubel et al., supra; Van Heeke, G. and S.M. Schuster (1989) J. Biol. Chem. 264:5503-5509;
Engelhard, E.K. et al.
(1994) Proc..Natl. Acad. Sci. USA 91:3224-3227; Sandig, V. et al. (1996) Hum.
Gene Ther. 7:1937-1945; Takamatsu, N. (1987) EMBO J. 6:307-311; The McGraw Hill Yearbook of Science and Technola~y (1992) McGraw Hill, New York NY, pp. 191-196; Logan, J. and T.
Shenk (1984) Proc.:
Natl. Acad. Sci. USA 81:3655-3659; Harrington, J.J. et al. (1997) Nat. Genet.
15:345-355).
Expression vectors derived from retroviruses, adenoviruses, or herpes or vaccinia viruses, or from various bacterial plasmids, may be used for delivery of polynucleotides to the targeted organ, tissue, or cell population (Di Nicola, M. et al. (1998) Cancer Gen. Ther. 5:350-356; Yu, M. et al. (1993) Proc:
Natl. Acad. Sci. USA 90:6340-6344; Buller, R.M. et al. (1985) Nature 317:813-815; McGregor, D.P.
et al. (1994) Mol. Trmmunol. 31:219-226; Verma, LM. and N. Somia (1997) Nature 389:239-242). The invention is not limited by the host cell employed.
In bacterial systems, a number of cloning and expression vectors may be selected depending upon the use intended for polynucleotides encoding PMMM. For example, routine cloning, subcloning, and propagation of polynucleotides encoding PMMM can be achieved using a multifunctional E. eoli vector such as PBLUESCRIPT (Stratagene, La Jolla CA) or PSPORT1 plasmid (Invitrogen).
Ligation of polynucleotides encoding PMMM 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 ifi vitro transcription, dideoxy sequencing, single strand rescue with helper phage, and creation of nested deletions in the cloned sequence (Van Heeke, G. and S.M. Schuster (1989) J. Biol. Chem. 264:5503-5509). When large quantities of P1~~VIM are needed, e.g. for the production of antibodies, vectors which direct high level expression of Pl~~VIM may be used. For example, vectors containing the strong, iuducible SP6 or T7 bacteriophage promoter may be used.
Yeast expression systems may be used for production of PMMM. A number of vectors containing constitutive or inducible promoters, such as alpha factor, alcohol oxidase, and PGH
promoters, may be used in the yeast Sacchar'omyces cer~evisiae or Pichia pastoris. In addition, such vectors direct either the secretion or intracellular retention of expressed proteins and enable integration of foreign polynucleotide sequences into the host genome for stable propagation (Ausubel et al., supf'a; Bitter, G.A. et al. (1987) Methods Enzymol. 153:516-544;
Scorer, C.A. et al. (1994) to Bio/Technology 12:181-184).
Plant systems may also be used for expression of PNNIIVVIM. Transcription of polynucleotides encoding PMNIM may be driven by viral promoters, e.g., the 35S and 19S
promoters of CaMV used alone or in combination with the omega leader sequence from TMV (Takamatsu, N.
(1987) EMBO J.
6:307-311). Alternatively, plant promoters such as the small subunit of RUBISC~ or heat shock 15 promoters may be used (Coruzzi, G. et al. (1984) EMB~ J. 3:1671-1680;
Brogue, R. et al. (1984) Science 224:838-843; 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 (The McGraw Hill Yearbook of Science and Technolo~y (1992) McGraw Hill, New York NY, pp.
191-196).
20 In mammalian cells, a number of viral-based expression systems may be utilized. In cases where an adenovirus is used as an expression vector, polynucleotides encoding PMMM may be ligated into an adenovirus transcription/trauslation complex consisting of the late promoter and tripartite leader sequence. Insertion in a non-essential E1 or E3 region of the viral genome may be used to obtain infective virus which expresses PMMM in host cells (Logan, J. and T.
Shenk (1984) Proc.
25 Natl. Acad. Sci. USA 81:3655-3659). In addition, transcription enhancers, such as the Rous sarcoma virus (RSV) enhancer, may be used to increase expression in mammalian host cells. SV40 or EBV-based vectors may also be used for high-level protein expression.
Human artificial chromosomes (HACs) may also be employed to deliver larger fragments of DNA than can be contained in and expressed from a plasmid. HACs of about 6 kb to 10 Mb are 30 constructed and delivered via conventional delivery methods (liposomes, polycationic amino polymers, or vesicles) for therapeutic purposes (Harrington, J.J. et al. (1997) Nat.
Genet. 15:345-355).

For long term production of recombinant proteins in mammalian systems, stable expression of PMMM in cell lines is preferred. For example, polynucleotides encoding PMMM
can be transformed into cell lines using expression vectors which may contain viral origins of replication and/or endogenous expression elements and a selectable marker gene on the same or on a separate vector.
Following the introduction of the vector, cells may be allowed to grow for about 1 to 2 days in enriched media before being switched to selective media. The purpose of the selectable marker is to confer resistance to a selective agent, and its presence allows growth and recovery of cells which successfully express the introduced sequences. Resistant clones of stably transformed cells may be propagated using tissue culture techniques appropriate to the cell type.
Any number of selection systems may be used to recover transformed cell lines.
These include, but are not limited to, the herpes simplex virus thymidine kinase and adenine phosphoribosyltransferase genes, for use in tk and apf~ cells, respectively (Wigler, M. et al. (1977) Cell 11:223-232; Lowy, I. et al. (1980) Cell 22:817-823). Also, antimetabolite, antibiotic, or herbicide resistance can be used as the basis for selection. For example, dh, frv confers resistance to methotrexate; ~ceo confers resistance to the aminoglycosides neomycin and G-418; and als and pat confer resistance to chlorsulfuron and phosphinotricin acetyltransferase, respectively (Wigler, M. et al. .
(1980) Proc. Natl: Acad. Sci. USA 77:3567-3570; Colbere-Garapin, F. et al.
(1981) J. Mol. Biol.
150:1-14). Additional selectable genes have been described, e.g., tfpB and hisD, which alter cellular requirements for metabolites (Hartxnan, S.C. and R.C. Mulligan (1988) Proc.
Natl. Acad. Sci. USA
85:8047-8051). Visible markers, e.g., anthocyanins, green fluorescent proteins (GFP; Clontech), ~3-glucuronidase and its substrate (3-glucuronide, or luciferase and its substrate luciferin may be used.
These markers can be used not only to identify transformants, but also to quantify the amount of transient or stable protein expression attributable to a specific vector system (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 PMNIM is inserted within a marker gene sequence, transformed cells containing polynucleotides encoding PMMM can be identified by the absence of marker gene function. Alternatively, a marker gene can be placed in tandem with a sequence encoding PMMM
under the control of a single promoter. Expression of the marker gene in response to induction or selection usually indicates expression of the tandem gene as well.

In general, host cells that contain the polynucleotide encoding PMMM and that express PMMM may be identified by a variety of procedures known to those of skill in the art. These procedures include, but are not limited to, DNA-DNA or DNA-RNA hybridizations, PCR
amplification, and protein bioassay or immunoassay techniques which include membrane, solution, or chip based technologies for the detection and/or quantification of nucleic acid or protein sequences.
T_mmunological methods for detecting and measuring the expression of PMMM
using either specific polyclonal or monoclonal autibodies are known in the art. Examples of such techniques include enzyme-linked immunosorbent assays (ELISAs), radioimmunoassays (RIAs), and fluorescence activated cell sorting (FACS). A two-site, monoclonal-based immunoassay utilizing monoclonal antibodies reactive to two non-interfering epitopes on PMMM is preferred, but a competitive binding assay may be employed. These and other assays are well known in the art (Hampton, R. et al. (1990) Serological Methods, a Laboratory Manual, APS
Press, St. Paul MN, Sect.
IV; Coligan, J.E. et al. (1997) Current Protocols in Immunology, ~Greene Pub.
Associates and Wiley-Interscience, New York NY; Pound, J.D. (1998) Tmmunochemical Protocols, Humana Press, Totowa NJ).
A wide variety of labels and conjugation techniques are known by those skilled in the art and may be used in various nucleic acid and amino acid assays. Means for producing labeled hybridization or PCR probes for detecting sequences related to polynticleotides encoding PMMM include oligolabeling, nick translation, end-labeling, or PCR amplification using a labeled nucleotide.
Alternatively, polynucleotides encoding PMMM, or any fragments thereof, may be cloned into a vector for the production of an mRNA probe. Such vectors are known in the art, are commercially available, and may be used to synthesize RNA probes i~c vitro by addition of an appropriate RNA
polymerase such as T7, T3, or SP6 and labeled nucleotides. These procedures may be conducted using a variety of commercially available kits, such as those provided by Amersham Biosciences, Promega (Madison WI), and US Biochemical. Suitable reporter molecules or labels which may be used for ease of detection include radionuclides, enzymes, fluorescent, chemiluminescent, or chromogenic agents, as well as substrates, cofactors, inhibitors, magnetic particles, and the like.
Host cells transformed with polynucleotides encoding PM1VBVI 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 PM1V>T~I may be designed to contain signal sequences which direct secretion of PMMM through a prokaryotic or eukaryotic cell membrane.
In addition, a host cell strain may be chosen for its ability to modulate expression of the inserted polynucleotides or to process the expressed protein in the desired fashion. Such modifications of the polypeptide include, but are not limited to, acetylation, carboxylation, glycosylation, phosphorylation, lipidation, and acylation. Post-translational processing which cleaves a "prepro" or "pro" form of the protein may also be used to specify protein targeting, folding, and/or activity.
Different host cells which have specific cellular machinery and characteristic mechanisms for post-translational activities (e.g., CHO, HeLa, MDCK, HEK293, and WI38) are available from the American Type Culture Collection (ATCC, Manassas VA) and may be chosen to ensure the correct modification and processing of the foreign protein.
In another embodiment of the invention, natural, modified, or recombinant polynucleotides encoding PMMM 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 PMMM
protein containing a heterologous moiety that can be recognized by a commercially available antibody may facilitate the screening of peptide libraries for inhibitors of PMMM 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-trausferase (GST), maltose binding protein (MBP), thioredoxin (Trx), caltnodulin binding peptide (CBP), 6-His, FLAG, c-myc, and hemagglutinin (HA). GST, MBP, Trx, CBP, and 6-His enable purification of their cognate fusion proteins on immobilized glutathione, maltose, phenylarsine oxide, calmodulin, and metal-chelate resins, respectively. FLAG, c-myc, and hemagglutinin (HA) enable immunoaffinity purification of fusion proteins using commercially available monoclonal and polyclonal antibodies that specifically recognize these epitope tags. A fusion protein may also be engineered to contain a proteolytic cleavage site located between the P11/IIVBVI encoding sequence and the heterologous protein sequence, so that PMMM may be cleaved away from the heterologous moiety following purification.
Methods for fusion protein expression and purification are discussed in Ausubel et al.
(supf-a, ch. 10 and 16). A
variety of commercially available kits may also be used to facilitate expression and purification of fusion proteins.
In another embodiment, synthesis of radiolabeled PMIVITiI may be achieved in vitt-o using the TN'T rabbit reticulocyte lysate or wheat germ extract system (Promega). These systems couple transcription and translation of protein-coding sequences operably associated with the T7, T3, or SP6 promoters. Translation takes place in the presence of a radiolabeled amino acid precursor, for example, 35S-methionine.
pNnVIM, fragments of PNBVIM, or variants of PMMM may be used to screen for compounds that specifically bind to PI~~VIM. One or more test compounds may be screened for specific binding to PMMM. In various embodiments, 1, 2, 3, 4, 5, 10, 20, 50, 100, or 200 test compounds can be screened for specific binding to PNMVI. Examples of test compounds can include antibodies, anticalins, oligonucleotides, proteins (e.g., ligands or receptors), or small molecules.
In related embodiments, variants of PI~~VIM can be used to screen for binding of test compounds, such as antibodies, to PI~~VIM, a variant of PMMM, or a combination of PMNIM andlor one or more variants PM1VINI. In an embodiment, a variant of PMMM can be used to screen for compounds that bind to a variant of PMMM, but not to PMMM having the exact sequence of a sequence of SEQ ID N0:1-40. PI~~VIM variants used to perform such screening can have a range of about SO% to about 99% sequence identity to PMIVIIVI, with various embodiments having 60%, 70%, 75%, 80%, 85%, 90%, and 95% sequence identity.
In an embodiment, a compound identified in a screen for specific binding to P1~~VIM can be closely related to the natural ligand of PNEVIM, e.g., a ligand or fragment thereof, a natural substrate, a structural or functional mimetic, or a natural binding partner (Coligan, J.E.
et al. (1991) Current Protocols in hnmunolo~y 1(2):Chapter 5). In another embodiment, the compound thus identified can be a natural ligand of a receptor PNNIIVVIM (Howard, A.D. et al. (2001) Trends Pharmacol. Sci.22:132-140; Wise, A. et al: (2002) Drug Discovery Today 7:235-246). ' In other embodiments, a compound identified in a screen for specific binding to PNIIVIM can be closely related to the natural receptor to which PNINIM binds, at least a fragment of the receptor, or a fragment of the receptor including all or a portion of the ligand binding site or binding pocket. For example, the compound may be a receptor for PMMM which is capable of propagating a signal, or a decoy receptor for P1~~VIM which is not capable of propagating a signal (Ashkenazi, A. and V.M.
Divit (1999) Curr. Opin. Cell Biol. 11:255-260; Mantovani, A. et al. (2001) Trends Tm_m__unol. 22:328-336). The compound can be rationally designed using known techniques. Examples of such techniques include those used to construct the compound etanercept (ENBREL;
Amgen Inc., 'Thousand Oaks CA), which is efficacious for treating rheumatoid arthritis in humans. Etanercept is an engineered p75 tumor necrosis factor (TNF) receptor dimer linked to the Fc portion of human IgGl (Taylor, P.C. et al. (2001) Curr. Opin. T_mmunol. 13:611-616).
5s In one embodiment, two or more antibodies having similar or, alternatively, different specificities can be screened for specific binding to PM1VW, fragments of PMMM, or variants of per. The binding specificity of the antibodies thus screened can thereby be selected to identify particular fragments or variants of P1~~VIM. In one embodiment, an antibody can be selected such that its binding specificity allows for preferential identification of specific fragments or variants of pT~VIM. In another embodiment, an antibody can be selected such that its binding specificity allows for preferential diagnosis of a specific disease or condition having increased, decreased, or otherwise abnormal production of PNNFvvIM.
In an embodiment, anticalins can be screened for specific binding to PMMM, fragments of PMMM, or variants of PT~VIM. Anticalins are ligand-binding proteins that have been constructed based on a lipocalin scaffold (Weiss, G.A. and H.B. Lowman (2000) Chem. Biol.
7:8177-8184;
Skerra, A. (2001) J. Biotechnol. 74:257-275). The protein architecture of lipocalins can include a beta-barrel having eight antiparallel beta-strands, which supports four loops at its open end. These loops form the natural ligand-binding site of the lipocalins, a site which can be re-engineered ih vitro by amino acid substitutions to impart novel binding specificities. The amino acid substitutions can be .
made using methods known in the art or described herein, and can include conservative substitutions (e.g., substitutions that do not alter binding specificity) or substitutions that modestly, moderately, or significantly alter binding specificity.
In one embodiment, screening for compounds which specifically bind to, stimulate; or inhibit PMMMM involves producing appropriate cells which express PMMMM, either as a secreted protein or on the cell membrane. Preferred cells can include cells from mammals, yeast, Dt-osopltila, or E. coli.
Cells expressing PNFVIM or cell membrane fractions which contain Pl~ are then contacted with a test compound and binding, stimulation, or inhibition of activity of either PM1VPVI or the compound is analyzed.
An assay may simply test binding of a test compound to the polypeptide, wherein binding is detected by a fluorophore, radioisotope, enzyme conjugate, or other detectable label. For example, the assay may comprise the steps of combining at least one test compound with PI~~VIM, either in solution or affixed to a solid support, and detecting the binding of PMMM to the compound. Alternatively, the assay may detect or measure binding of a test compound in the presence of a labeled competitor.
Additionally, the assay may be carried out using cell-free preparations, chemical libraries, or natural product mixtures, and the test compounds) may be free in solution or affixed to a solid support.

An assay can be used to assess the ability of a compound to bind to its natural ligand and/or to inhibit the binding of its natural ligand to its natural receptors. Examples of such assays include radio-labeling assays such as those described in U.S. Patent No. 5,914,236 and U.S.
Patent No. 6,372,724.
In a related embodiment, one or more amino acid substitutions can be introduced into a polypeptide compound (such as a receptor) to improve or alter its ability to bind to its natural ligands (Matthews, D.J. and J.A. Wells. (1994) Chem. Biol. 1:25-30). In another related embodiment, one or more amino acid substitutions can be introduced into a polypeptide compound (such as a ligand) to improve or alter its ability to bind to its natural receptors (Cumingham, B.C. and J.A. Wells (1991) Proc. Natl. Acad.
Sci. USA 88:3407-3411; Lowman, H.B. et al. (1991) J. Biol. Chem. 266:10982-10988).
Pte, fragments of PMMM, or variants of PMMM may be used to screen for compounds that modulate the activity of PMMM. Such compounds may include agonists, antagonists, or partial or inverse agonists. In one embodiment, an assay is performed under conditions permissive for PMMM
activity, wherein PMMM is combined with at least one test compound, and the activity of PT~VIM in the presence of a test compound is compared with the activity of PMMM in the absence of the test compound. A change in the activity of PMMM in the presence of the test compound is indicative of a compound that modulates the activity of PMMM. Alternatively, a test compound is combined with an i>1 vitro or cell-free system comprising PNNEVVIM under conditions suitable for PMMM activity, and the assay is performed. In either of these assays, a test compound which modulates the activity of PM1VIM may do so indirectly and need not come in direct contact with the test compound. At least one and up to a plurality of test compounds may be screened.
In another embodiment, polynucleotides encoding PMMM or their mammalian homologs may be "knocked out" in an animal model system using homologous recombination in embryonic stem (ES) cells. Such techniques are well known in the art and are useful for the generation of animal models of human disease (see, e.g., U.S. Patent No. 5,175,383 and U.S. Patent No.
5,767,337). For example, mouse ES cells, such as the mouse 129/SvJ cell line, are derived from the early mouse embryo and grown in culture. The ES cells are transformed with a vector containing the gene of interest disrupted by a marker gene, e.g., the neomycin phosphotransferase gene (tteo; Capecchi, M.R. (1989) Science 244:1288-1292). The vector integrates into the corresponding region of the host genome by homologous recombination. Alternatively, homologous recombination takes place using the Cre-loxP
system to knockout a gene of interest in a tissue- or developmental stage-specific manner (Marth, J.D.
(1996) Clip. Invest. 97:1999-2002; Wagner, K.U. et al. (1997) Nucleic Acids Res. 25:4323-4330).
Transformed ES cells are identified and microinjected into mouse cell blastocysts such as those from the C57BL/6 mouse strain. The blastocysts are surgically transferred to pseudopregnant dams, and the resulting chimeric progeny are genotyped and bred to produce heterozygous or homozygous strains. Transgenic animals thus generated may be tested with potential therapeutic or toxic agents.
Polynucleotides encoding PMMM may also be manipulated it2 vitf~o in ES cells derived from human blastocysts. Human ES cells have the potential to differentiate into at least eight separate cell lineages including endoderm, mesoderm, and ectodermal cell types. These cell lineages differentiate into, for example, neural cells, hematopoietic lineages, and cardiomyocytes (Thomson, J.A. et al.
(1998) Science 282:1145-1147).
Polynucleotides encoding PMMM can also be used to create "knockin" humanized animals (pigs) or transgenic animals (mice or rats) to model human disease. With knockin technology, a region of a polynucleotide encoding P~ 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 PNIMM, e.g., by secreting Pin its milk, may also serve as a convenient source of that protein (Janne, J. et al. (1998) Biotechnol. Annu.
Rev. 4:55-74).
THERAPEUTICS
Chemical and structural similarity, e.g., in the context of sequences and motifs, exists between-.
regions of PM1VI1~~I and protein modification and maintenance molecules. In addition, examples of tissues expressing PNNIIVVIM can be found in Table 6 and can also be found in Example XI. Therefore, pI~~VIM appears to play a role in gastrointestinal, cardiovascular, autoimmune/inflammatory, cell proliferative, developmental, epithelial, neurological, reproductive, endocrine, pancreatic, adrenal, and metabolic disorders; as well as lipid, copper, and carbohydrate metabolism disorders, disorders associated with gonadal steroid hormones, the immune system, and infections.
In. the treatment of disorders associated with increased PMNEVI expression or activity, it is desirable to decrease the expression or activity of PI~~VIM. In the treatment of disorders associated with decreased P1~~VIM
expression or activity, it is desirable to increase the expression or activity of PMIV>TZ.
Therefore, in one embodiment, PNll~~IM 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 Pte. Examples of such disorders include, but are not limited to, a gastrointestinal disorder, such as dysphagia, peptic esophagitis, esophageal spasm, esophageal stricture, esophageal carcinoma, dyspepsia, indigestion, gastritis, gastric carcinoma, anorexia, nausea, emesis, gastroparesis, antral or pyloric edema, abdominal angina, pyrosis, gastroenteritis, intestinal obstruction, infections of the intestinal tract, peptic ulcer, cholelithiasis, cholecystitis, cholestasis, pancreatitis, pancreatic carcinoma, biliary tract disease, hepatitis, hyperbilirubinemia, cirrhosis, passive congestion of the liver, hepatoma, infectious colitis, ulcerative colitis, ulcerative proctitis, Crohn's disease, Whipple's disease, Mallory-Weiss syndrome, colonic carcinoma, colonic obstruction, irritable bowel syndrome, short bowel syndrome, diarrhea, constipation, gastrointestinal hemorrhage, acquired immunodeficiency syndrome (AIDS) enteropathy, jaundice, hepatic encephalopathy, hepatorenal syndrome, hepatic steatosis, hemochromatosis, Wilson's disease, alphai antitrypsin deficiency, Reye's syndrome, primary sclerosing cholangitis, liver infarction, portal vein obstruction and thrombosis, centrilobular necrosis, peliosis hepatis, hepatic vein thrombosis, veno-occlusive disease, preeclampsia, eclampsia, acute fatty liver of pregnancy, intrahepatic cholestasis of pregnancy, and hepatic tumors including nodular hyperplasias, adenomas, and carcinomas; an endocrine disorder such as a disorder of the hypothalamus andlor pituitary resulting from lesions such as a primary brain tumor, adenoma, infarction associated with pregnancy, hypophysectomy, aneurysm, vascular malformation, thrombosis, infection, immunological disorder, and complication due to head trauma; a disorder associated with hypopituitarism including hypogonadism, Sheehan syndrome, diabetes insipidus, Kallman's disease, Hand-Schuller-Christian disease, Letterer-Siwe disease, sarcoidosis, empty sella syndrome, and dwarfism; a disorder associated with hyperpituitarism including acromegaly, giantism, and syndrome of inappropriate antidiuretic hormone (ADH) secretion (SIADH) often caused by benign adenoma; a disorder associated with hypothyroidism including goiter, myxedema, acute thyroiditis associated with bacterial infection, subacute thyroiditis associated with viral infection, autoimmune thyroiditis (Hashimoto's disease), and cretinism; a disorder associated with hyperthyroidism including thyrotoxicosis and its various forms, Grave's disease, pretibial myxedema, toxic multinodular goiter, thyroid carcinoma, and Plummer's disease; a disorder associated with hyperparathyroidism including Coon disease (chronic hypercalemia); a pancreatic disorder such as Type I or Type lI diabetes mellitus and associated complications; a disorder associated with the adrenals such as hyperplasia, carcinoma, or adenoma of the adrenal cortex, hypertension associated with alkalosis, amyloidosis, hypokalemia, Cushing's disease, Liddle's syndrome, and Arnold-Healy-Gordon syndrome, pheochromocytoma tumors, and Addison's disease; a disorder associated with gonadal steroid 3o hormones such as: in women, abnormal prolactin production, infertility, endometriosis, perturbation of the menstrual cycle, polycystic ovarian disease, hyperprolactinemia, isolated gonadotropin deficiency, amenorrhea, galactorrhea, hermaphroditism, hirsutism and virilization, breast cancer, and, in post-menopausal women, osteoporosis; and, in men, Leydig cell deficiency, male climacteric phase, and germinal cell aplasia, a hypergonadal disorder associated with Leydig cell tumors, androgen resistance associated with absence of androgen receptors, syndrome of 5 a-reductase, and gynecomastia; a disorder of the immune system such as inflammation, actinic keratosis, acquired immunodeficiency syndrome (AmS), Addison's disease, adult respiratory distress syndrome, allergies, ankylosing spondylitis, amyloidosis, anemia, arteriosclerosis, asthma, atherosclerosis, autoimmune hemolytic anemia, autoimmune thyroiditis, bronchitis, bursitis, cholecystitis, cirrhosis, contact dermatitis, Crohn's disease, atopic dermatitis, dermatomyositis, diabetes mellitus, emphysema, erythroblastosis fetalis, erythema nodosum, atrophic gastritis, glomerulonephritis, Goodpasture's syndrome, gout, Graves' disease, Hashimoto's thyroiditis, paroxysmal nocturnal hemoglobinuria, hepatitis, hypereosinophilia, irritable bowel syndrome, episodic lymphopenia with lymphocytotoxins, mixed connective tissue disease (MCTD), multiple sclerosis, myasthenia gravis, myocardial or pericardial inflammation, myelofibrosis, osteoarthritis, osteoporosis, pancreatitis, polycythenua vera, polymyositis, psoriasis, Reiter's syndrome, rheumatoid arthritis, scleroderma,.Sjogren's syndrome, systemic anaphylaxis, systemic lupus erythematosus, systemic sclerosis, primary thrombocythemia, thrombocytopenic purpura, ulcerative colitis, uveitis, Werner, syndrome, complications of cancer, hemodialysis; and extracorporeal circulation, trauma, and hematopoietic cancer including lymphoma, leukemia, and myeloma; an infection caused by a viral agent classified as adenovirus, arenavirus, bunyavirus, calicivirus, coronavirus, filovirus, hepadnavirus, herpesvirus, flavivirus, orthomyxovirus, parvovirus, papovavirus, paramyxovirus, picornavirus, poxvirus, reovirus, retrovirus, rhabdovirus, or togavirus; an infection caused by a bacterial agent classified as pneumococcus, staphylococcus, streptococcus, bacillus, corynebacterium, clostridium, meningococcus, gonococcus, listeria, moraxella, kingella, haemophilus, legionella, bordetella, gram-negative enterobacterium including shigella, salmonella, or campylobacter, pseudomonas, vibrio, brucella, francisella, yersinia, bartonella, norcardium, actinomyces, mycobacterium, spirochaetale, rickettsia, chlamydia, or mycoplasma; an infection caused by a fungal agent classified as aspergillus, blastomyces, dermatophytes, cryptococcus, coccidioides, malasezzia, histoplasma, or other mycosis-causing fungal agent; and an infection caused by a parasite classified as plasmodium or malaria-causing, parasitic entamoeba, leishmania, trypanosoma, toxoplasma, pneumocystis carinii, intestinal protozoa such as giardia, trichomonas, tissue nematode such as trichinella, intestinal nematode such as ascaris, lymphatic filarial nematode, trematode such as schistosoma, and cestrode such as tapeworm; a metabolic disorder such as Addison's disease, cerebrotendinous xanthomatosis, congenital adrenal hyperplasia, coumarin resistance, cystic fibrosis, diabetes, fatty hepatocirrhosis, fructose-1,6-diphosphatase deficiency, galactosemia, goiter, glucagonoma, glycogen storage diseases, hereditary fructose intolerance, hyperadrenalism, hypoadrenalism, hyperparathyroidism, hypoparathyroidism, hypercholesterolemia, hyperthyroidism, hypoglycemia, hypothyroidism, hyperlipidemia, hyperlipemia, lipid myopathies, lipodystrophies, lysosomal storage diseases, mannosidosis, neuraminidase deficiency, obesity, pentosuria phenylketonuria, pseudovitamin_ D-deficiency rickets; a disorder of carbohydrate metabolism such as congenital type II dyserythropoietic anemia, diabetes, insulin-dependent diabetes mellitus, non-insulin-dependent diabetes mellitus, fructose-1,6-diphosphatase deficiency, galactosemia, glucagonoma, hereditary fructose intolerance, hypoglycemia, mannosidosis, neuraminidase deficiency, obesity, galactose epimerase deficiency, glycogen storage diseases, lysosomal storage diseases, fructosuria, pentosuria, and inherited abnormalities of pyruvate metabolism; a disorder of lipid metabolism such as fatty liver, cholestasis, primary biliary cirrhosis, carnitine deficiency, carnitine .
palmitoyltransferase deficiency, myoadenylate deaminase deficiency, hypertriglyceridemia, lipid storage disorders such Fabry's disease, Gaucher's disease, Niemann-Pick's disease, metachromatic leukodystrophy, adrenoleukodystrophy, GMZ gangliosidosis, and ceroid lipofuscinosis, abetalipoproteinemia, Tangier disease, hyperlipoproteinemia, diabetes mellitus, lipodystrophy, lipomatoses, acute panniculitis, disseminated fat necrosis, adiposis dolorosa, lipoid adrenal hyperplasia, minimal change disease, lipomas, atherosclerosis, hypercholesterolemia, hypercholesterolemia with hypertriglyceridemia, primary hypoalphalipoproteinemia, hypothyroidism, renal disease, liver disease, lecithin:cholesterol acyltransferase deficiency, cerebrotendinous xanthomatosis, sitosterolemia, hypocholesterolemia, Tay-Sachs disease, Sandhoff's disease, hyperlipidemia, hyperlipemia, lipid myopathies, and obesity; a disorder of copper metabolism such as Menke's disease, Wilson's disease, and Ehlers-Danlos syndrome type IX.; a cardiovascular disorder, such as arteriovenous fistula, atherosclerosis, hypertension, vasculitis, Raynaud's disease, aneurysms, arterial dissections, varicose veins, thrombophlebitis and phlebothrombosis, vascular tumors, and complications of thrombolysis, balloon angioplasty, vascular replacement, and coronary artery bypass graft surgery, congestive heart failure, ischemic heart disease, angina pectoris, myocardial infarction, hypertensive heart disease, degenerative valvular heart disease, calcific aortic valve stenosis, congenitally bicuspid aortic valve, mitral annular calcification, mural valve prolapse, rheumatic fever and rheumatic heart disease, infective endocarditis, nonbacterial thrombotic endocarditis, endocarditis of systemic lupus erythematosus, carcinoid heart disease, cardiomyopathy, myocarditis, pericarditis, neoplastic heart disease, congenital heart disease, and complications of cardiac transplantation; an autoimmune/inflammatory disease, such as acquired immunodeficiency syndrome (AIDS), Addison's disease, adult respiratory distress syndrome, allergies, ankylosing spondylitis, amyloidosis, anemia, asthma, atherosclerosis, atherosclerotic plaque rupture, autoimmune hemolytic anemia, autoimmune thyroiditis, autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED), bronchitis, cholecystitis, contact dermatitis, Crohn's disease, atopic dermatitis, dermatomyositis, diabetes mellitus, emphysema, episodic lymphopenia with lymphocytotoxins, erythroblastosis fetalis, erythema nodosum, atrophic gastritis, glomerulonephritis, Goodpasture's syndrome, gout, Graves' disease, Hashimoto's thyroiditis, hypereosinophilia, irritable bowel syndrome, multiple sclerosis, myasthenia gravis, myocardial or pericardial inflammation, osteoarthritis, degradation of articular cartilage, osteoporosis, pancreatitis, polymyositis, psoriasis, Reiter's syndrome, rheumatoid arthritis, scleroderma, Sjogren's syndrome, systemic anaphylaxis, systemic lupus erythematosus, systemic sclerosis, thrombocytopenic purpura, ulcerative colitis, uveitis, Werner syndrome, complications of cancer, hemodialysis, and extracorporeal circulation, viral, bacterial, fungal, parasitic, protozoal, and hehninthic infections, and trauma; a cell proliferative disorder such as actinic keratosis, arteriosclerosis, atherosclerosis, bursitis cirrhosis, hepatitis, mixed connective tissue disease (MCTD), myelofibrosis, paroxysmal nocturnal hemoglobinuria, polycythemia vera, psoriasis, primary thrombocythemia, and cancers including adenocarcinoma, leukemia, lymphoma, melanoma, myeloma, sarcoma, teratocarcinoma, and, in particular, cancers of the adrenal gland, bladder, bone, bone marrow, brain, breast, cervix, gall bladder ganglia, gastrointestinal tract, heart, kidney, liver, lung, muscle, ovary, pancreas, parathyroid; penis, prostate, salivary glands, skin, spleen, testis, thymus, thyroid, and uterus;
a developmental disorder, such as renal tubular acidosis, anemia, C~xshing's syndrome, achondroplastic dwarfism, Duchenne and Becker muscular dystrophy, bone resorption, epilepsy, gonadal dysgenesis, WAGR
syndrome (Wilms' tumor, aniridia, genitourinary abnormalities, and mental retardation), Smith-Magenis syndrome, myelodysplastic syndrome, hereditary mucoepithelial dysplasia, hereditary keratodermas, hereditary neuropathies such as Charcot-Marie-Tooth disease and neurofibromatosis, hypothyroidism, hydrocephalus, seizure disorders such as Syndenham's chorea and cerebral palsy, spina bifida, anencephaly, craniorachischisis, congenital glaucoma, cataract, age-related macular degeneration, and sensorineural hearing loss; an epithelial disorder, such as dyshidrotic eczema, allergic contact dermatitis, keratosis pilaris~ melasma, vitiligo, actinic keratosis, basal cell carcinoma, squamous cell carcinoma, seborrheic keratosis, folliculitis, herpes simplex, herpes zoster, varicella, candidiasis, dermatophytosis, scabies, insect bites, cherry angioma, keloid, dermatofibroma, acrochordons, urticaria, transient acantholytic dermatosis, xerosis, eczema, atopic dermatitis, contact dermatitis, hand eczema, nurnmular eczema, lichen simplex chronicus, asteatotic eczema, stasis dermatitis and stasis ulceration, seborrheic dermatitis, psoriasis, lichen planus, pityriasis rosea, impetigo, ecthyma, dermatophytosis, tinea versicolor, warts, acne vulgaris, acne rosacea, pemphigus vulgaris, pemphigus foliaceus, paraneoplastic pemphigus, bullous pemphigoid, herpes gestationis, dermatitis herpetiformis, linear IgA disease, epidermolysis bullosa acquisita, dermatomyositis, lupus erythematosus, scleroderma and morphea, erythroderma, alopecia, figurate skin lesions, telangiectasias, hypopigmentation, hyperpigmentation, vesicles/bullae, exanthems, cutaneous drug reactions, papulonodular skin lesions, chronic non-healing wounds, photosensitivity diseases, epidermolysis bullosa simplex, epidermolytic hyperkeratosis, epidermolytic and nonepidermolytic palmoplantar keratoderma, ichthyosis bullosa of Siemens, ichthyosis exfoliativa, keratosis palmaris et plantaris, keratosis palinoplantaris, 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 neoplasrns, Alzheimer's disease, Pick's disease, Huntington's disease, dementia, Parkinson's disease and other extrapyramidal .
disorders, amyotrophic lateral sclerosis and other motor neuron disorders, progressive neural muscular atrophy, retinitis pigmentosa, hereditary ataxias, multiple sclerosis and other demyelinating diseases, bacterial and viral meningitis, brain abscess, subdural empyema, epidural abscess, suppurative intracranial thrombophlebitis, myelitis and radiculitis, viral central nervous system disease, priori diseases including kuru, Creutzfeldt-Jakob disease, and Gerstmann-Straussler-Scheiuker syndrome, fatal familial insomnia, nutritional and metabolic diseases of the nervous system, neurofibromatosis, tuberous sclerosis, cerebelloretinal hemangioblastomatosis, encephalotrigeminal syndrome, mental retardation and other developmental disorders of the central nervous system including Down syndrome, cerebral palsy, neuroskeletal disorders, autonomic nervous system disorders, cranial nerve disorders, spinal cord diseases, muscular dystrophy and other neuromuscular disorders, peripheral nervous system disorders, dermatomyositis and polymyositis, inherited, metabolic, endocrine, and toxic myopathies, myasthenia gravis, periodic paralysis, mental disorders including mood, anxiety, and schizophrenic disorders, seasonal affective disorder (SAD), akathesia, amnesia, catatonia, diabetic neuropathy, tardive dyskinesia, dystonias, paranoid psychoses, postherpetic neuralgia, Tourette's disorder, progressive supranuclear palsy, corticobasal degeneration, and familial frontotemporal dementia; and a reproductive disorder, such as infertility, including tubal disease, ovulatory defects, and endometriosis, a disorder of prolactin production, a disruption of the estrous cycle, a disruption of the menstrual cycle, polycystic ovary syndrome, ovarian hyperstimulation syndrome, an endometrial or ovarian tumor, a uterine fibroid, autoimmune disorders, au ectopic pregnancy, and teratogenesis;
cancer of the breast, fibrocystic breast disease, and galactorrhea; a disruption of spermatogenesis, abnormal sperm physiology, cancer of the testis, cancer of the prostate, benign prostatic hyperplasia, prostatitis, Peyronie's disease, impotence, carcinoma of the male breast, and gynecomastia.
In another embodiment, a vector capable of expressing PN)ZVJ:M 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 PNBVIM including, but not limited to, those described above.
In a further embodiment, a composition comprising a substantially purified P~
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 P1~~V1M
including, but not limited to, those provided above.
In still another embodiment, an agonist which modulates the activity of PI\~VIM may be administered to a,subject to treat or prevent a disorder associated with decreased expression or activity of PMMM including, but not limited to, those listed above.
In a further embodiment, an antagonist of PMMM maybe administered to a subject to treat or prevent a disorder associated with increased expression or activity of Pl~~VIM. Examples of such disorders include, but are not limited to, those gastrointestinal, cardiovascular, autoimmune/inflammatory, cell proliferative, developmental, epithelial, neurological, reproductive, endocrine, pancreatic, adrenal, and metabolic disorders; as well as lipid, copper, and carbohydrate metabolism disorders, disorders associated with gonadal steroid hormones, the immune system, and infections described above. In one aspect, an antibody which specifically binds PMMM may be used directly as an antagonist or indirectly as a targeting or delivery mechanism for bringing a pharmaceutical agent to cells or tissues which express PMMM.
In an additional embodiment, a vector expressing the complement of the polynucleotide encoding PI~~VIM may be administered to a subject to treat or prevent a disorder associated with increased expression or activity of Pn~VIM including, but not limited to, those described above.
In other embodiments, any protein, agonist, antagonist, antibody, complementary sequence, or vector embodiments may be administered in combination with other appropriate therapeutic agents.
Selection of the appropriate agents for use in combination therapy may be made by one of ordinary skill in the art, according to conventional pharmaceutical principles. The combination of therapeutic agents may act synergistically to effect the treatment or prevention of the various disorders described above. Using this approach, one may be able to achieve therapeutic efficacy with lower dosages of each agent, thus reducing the potential for adverse side effects.
An antagonist of PMMM may be produced using methods which are generally known in the art. In particular, purified PMMM may be used to produce antibodies or to screen libraries of pharmaceutical agents to identify those which specifically bind PMMM.
Antibodies to PMMM may also be generated using methods that are well known in the art. Such antibodies may include, but are not limited to, polyclonal, monoclonal, chimeric, and single chain antibodies, Fab fragments, and fragments produced by a Fab expression library. In an embodiment, neutralizing antibodies (i.e., those which inhibit dimer formation) can be used therapeutically. Single chain antibodies (e.g., from camels or llamas) may be potent enzyme inhibitors and may have application in the design of peptide mim__etics, and in the development of immuno-adsorbents and biosensors (Muyldermans, S.
(2001) J. Biotechnol.
74:277-302).
For the production of autibodies, various hosts including goats, rabbits, rats, mice, camels dromedaries, llamas, humans, and others may be immunized by injection with PMMM or with any fragment or oligopeptide thereof which has immunogenic properties. Depending on the host species, various adjuvauts may be used to increase immunological response. Such adjuvants include, but are not.limited to, Freund's, mineral gels such as aluminum hydroxide, and surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, KLH, and dinitrophenol. Among adjuvants used in humans, BCG (bacilli Calmette-Guerin) and Corynebacterium parvum are especially preferable.
It is preferred that the oligopeptides, peptides, or fragments used to induce antibodies to PNnVlM have an amino acid sequence consisting of at least about 5 amino acids, and generally will consist of at least about 10 amino acids. It is also preferable that these oligopeptides, peptides, or fragments are substantially identical to a portion of the amino acid sequence of the natural protein.
Short stretches of PMMM amino acids may be fused with those of another protein, such as I~LH, and antibodies to the chimeric molecule may be produced.
Monoclonal antibodies to PMMM 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 (Kohler, G. et al. (1975) Nature 256:495-497; Kozbor, D. et al.
(1985) J. Tm_m__unol. Methods 81:31-42; Cote, R.J. et al. (1983) Proc. Natl. Acad. Sci. USA 80:2026-2030;
Cole, S.P. et al. (1984) Mol. Cell Biol. 62:109-120).

In addition, techniques developed for the production of "chimeric antibodies,"
such as the splicing of mouse antibody genes to human antibody genes to obtain a molecule with appropriate antigen specificity and biological activity, can be used (Morrison, S.L. et al. (1984) Proc. Natl. Acad.
Sci. USA 81:6851-6855; Neuberger, M.S. et al. (1984) Nature 312:604-608;
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 PMMM-specific single chain antibodies.
Antibodies with related specificity, but of distinct idiotypic composition, may be generated by chain shuffling from random combinatorial immunoglobulin libraries (Burton, D.R.
(1991) Proc. Natl. Acad.
Sci. USA 88:10134-10137).
Antibodies may also be produced by inducing in vivo production in the lymphocyte population or by screening immunoglobulin libraries or panels of highly specific binding reagents as disclosed in the literature (Orlandi, R. et al. (1989) Proc. Natl. Acad. Sci. USA 86:3833-3837; Winter, G. et al.
(1991) Nature 349:293-299).
Antibody fragments which contain specific binding sites for P~ 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 (Huse, W.D. et al. (1989) Science 246:1275-1281).
Various immunoassays may be 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 formation between PMMM and its specific antibody. A two-site, monoclonal-based immunoassay utilizing monoclonal antibodies reactive to two non-interfering PMMM 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 Ph~VIM. Affinity is expressed as an association constant, Ka, which is defined as the molar concentration of PMIVINI-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 PMMM epitopes, represents the average affinity, or avidity, of the antibodies for Pte.

The Ka determined for a preparation of monoclonal antibodies, which are monospecific for a particular PMMM epitope, represents a true measure of affinity. High-affinity antibody preparations with Ka ranging from about 109 to 1012 L/mole are preferred for use in immunoassays in which the PMIVIIVI-antibody complex must withstand rigorous manipulations. Low-affinity antibody preparations with Ka ranging from about 106 to 10' L/mole are preferred for use in immunopurification and similar procedures which ultimately require dissociation of PNNEVVIM, preferably in active form, from the antibody (Catty, D. (1988) Antibodies Volume I: A Practical Approach, IRL
Press, Washington DC;
Liddell, J.E. and A. Cryer (1991) A Practical Guide to Monoclonal Antibodies, John Wiley & Sons, New York NY).
1o The titer and avidity of polyclonal antibody preparations may be further evaluated to determine the quality and suitability of such preparations for certain downstream applications. For example, a polyclonal antibody preparation containing at least 1-2 mg specific antibody/ml, preferably 5-10 mg specific antibody/ml, is generally employed in procedures requiring precipitation of Pl~~VIM-antibody complexes. Procedures for evaluating antibody specificity, titer, and avidity, and guidelines for antibody quality and usage in various applications, are generally available (Catty, supt-a; Coligan et al., supra) In another embodiment of the invention, polynucleotides encoding PI~~VIM, 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 PMMM. Such technology is well known in the art, and antisense oligonucleotides or larger fragments can be designed from various locations along the coding or control regions of sequences encoding Pl~~VIM (Agrawal, S., ed. (1996) Antisense Therapeutics, Humaua Press, Totawa NJ).
In therapeutic use, any gene delivery system suitable for introduction of the antisense sequences into appropriate target cells can be used. Antisense sequences can be delivered intracellularly in the form of an expression plasmid which, upon transcription, produces a sequence complementary to at least a portion of the cellular sequence encoding the target protein (Slater, J.E. et al. (1998) J. Allergy Clin. Tmmunol. 102:469-475; Scanlon, K.J. et al. (1995) 9:1288-1296). Antisense sequences can also be introduced intracellularly through the use of viral vectors, such as retrovirus and adeno-associated virus vectors (Miller, A.D. (1990) Blood 76:271; Ausubel et al., sup~~a; Uckert, W.
and W. Walther (1994) Pharmacol. Ther. 63:323-347). Other gene delivery mechanisms include liposome-derived systems, artificial viral envelopes, and other systems known in the art (Rossi, J.J.
7o (1995) Br. Med. Bull. 51:217-225; Boado, R.J. et al. (1998) J. Pharm. Sci.
87:1308-1315; Morris, M.C. et al. (1997) Nucleic Acids Res. 25:2730-2736).
In another embodiment of the invention, polynucleotides encoding PMMM may be used for somatic or germline gene therapy. Gene therapy may be performed to (i) correct a genetic deficiency (e.g., in the cases of severe combined immunodeficiency (SCID)-X1 disease characterized by X-linked inheritance (Cavazzana-Calvo, M. et al. (2000) Science 288:669-672), severe combined immunodeficiency syndrome associated with an inherited adenosine deaminase (ADA) deficiency (Blaese, R.M. et al. (1995) Science 270:475-480; Bordignon, C. et al. (1995) Science 270:470-475), cystic fibrosis (Zabner, J. et al. (1993) Cell 75:207-216; Crystal, R.G. et al. (1995) Hum. Gene Therapy 6:643-666; Crystal, R.G. et al. (1995) Hum. Gene Therapy 6:667-703), thalassamias, familial hypercholesterolemia, and hemophilia resulting from Factor VIII or Factor IX
deficiencies (Crystal, R.G. (1995) Science 270:404-410; Verma, LM. and N. Somia (1997) Nature 389:239-242)), (ii) express a conditionally lethal gene product (e.g., in the case of cancers which result from unregulated cell proliferation), or (iii) express a protein which affords protection against intracellular parasites (e.g., against human retroviruses, such as human immunodeficiency virus (HIV) (Baltimore, D. (1988) Nature 335:395-396; Poeschla, E. et al. (1996) Proc. Natl. Acad. Sci. USA
93:11395-11399), hepatitis B or C virus (HBV, HCV); fungal parasites, such as Candida albicans and Par-acoccidioides br-asiliensis; and protozoan parasites such as Plasmodium falcipaf~um and Tfypanosoma cruzi). In the case where a genetic deficiency in PMMM expression or regulation causes disease, the expression of PM1VII~~I from an appropriate population of transduced cells may alleviate the clinical manifestations caused by the genetic deficiency.
In a further embodiment of the invention, diseases or disorders caused by deficiencies in PMMM are treated by constructing mammalian expression vectors encoding PMMM
and introducing these vectors by mechanical means into PMMM-deficient cells. Mechanical transfer technologies for use with cells iyi 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 PMMM include, but are not limited to, the PCDNA 3.1, EPTTAG, PRCCMV2, PREP, PVAX, PCR2-TOPOTA vectors (Invitrogen, Carlsbad CA), PCMV-SCRIPT, PCMV-TAG, PEGSH/PERV (Stratagene, La Jolla CA), and PTET-OFF, PTET-ON, PTRE2, PTRE2-LUC, PTK-HYG (Clontech, Palo Alto CA).
PMMM
may be expressed using (i) a constitutively active promoter, (e.g., from cytomegalovirus (CMV), Rous sarcoma virus (RSV), SV40 virus, thymidine kiuase (TK), or (3-actin. genes), (ii) an inducible promoter (e.g., the tetracycline-regulated promoter (Gossen, M. and H. Bujard (1992) Proc. Natl. Acad. Sci.
USA 89:5547-5551; Gossen, M. et al. (1995) Science 268:1766-1769; Rossi, F.M.V. and H.M. Blau (1998) Curr. Opin. Biotechnol. 9:451-456), commercially available in the T-REX
plasmid (Invitrogen));
the ecdysone-inducible promoter (available in the plasmids PVGRXR and PIND;
Invitrogen); the FK506/rapamycin inducible promoter; or the RU486/mifepristone inducible promoter (Rossi, F.M.V.
and H.M. Blau, supra)), or (iii) a tissue-specific promoter or the native promoter of the endogenous gene encoding PMMM from a normal individual.
Commercially available liposome transformation kits (e.g., the PERFECT LIPID
TRANSFECTION KIT, available from Invitrogen) allow one with ordinary skill in the art to deliver polynucleotides to target cells in culture and require minimal effort to optimize experimental parameters. In the alternative, transformation is performed using the calcium phosphate method (Graham, F.L. and A.J. Eb (1973) Virology 52:456-467), or by electroporation (Neumann, E. et al.
(1982) EMBO J. 1:841-845). The introduction of DNA to primary cells requires modification of these standardized mammalian transfection protocols.
In another embodiment of the invention, diseases or disorders caused by genetic defects with respect to PMMM expression are treated by constructing a retrovirus vector consisting of (i) the 2o polynucleotide encoding PMIVIn~I under the control of an independent promoter or the retrovirus long terminal repeat (LTR) promoter, (ii) appropriate RNA packaging signals, and (iii) a Rev-responsive element (RRE) along with additional retrovirus cis-acting RNA sequences and coding sequences required for efficient vector propagation. Retrovirus vectors (e.g., PFB and PFBNEO) are commercially available (Stratagene) and are based on published data (Riviere, I. et al. (1995) Proc.
Natl. Acad. Sci. USA 92:6733-6737), incorporated by reference herein. The vector is propagated in an appropriate vector producing cell line (VPCL) that expresses an envelope gene with a tropism for receptors on the target cells or a promiscuous envelope protein such as VSVg (Armentano, D. et al.
(1987) J. Virol. 61:1647-1650; Bender, M.A. et al. (1987) J. Virol. 61:1639-1646; Adam, M.A. and A.D. Miller (1988) J. Virol. 62:3802-3806; Dull, T. et al. (1998) J. Virol.
72:8463-8471; Zufferey, R. et al. (1998) J. Virol. 72:9873-9880). U.S. Patent No. 5,910,434 to Rigg ("Method for obtaining retrovirus packaging cell lines producing high transducing efficiency retxoviral supernatant") discloses a method for obtaining retrovirus packaging cell lines and is hereby incorporated by reference.

Propagation of retrovirus vectors, transduction of a population of cells (e.g., CD4+ T-cells), and the return of transduced cells to a patient are procedures well known to persons skilled in the art of gene therapy and have been well documented (Ranga, U. et al. (1997) J. Virol.
71:7020-7029; Bauer, G. et al. (1997) Blood 89:2259-2267; Bonyhadi, M.L. (1997) J. Virol. 71:4707-4716;
Ranga, U. et al. (1998) Proc. Natl. Acad. Sci. USA 95:1201-1206; Su, L. (1997) Blood 89:2283-2290).
In an embodiment, an adenovirus-based gene therapy delivery system is used to deliver polynucleotides encoding PMMM to cells which have one or more genetic abnormalities with respect to the expression of PMMM. The construction and packaging of adenovirus based vectors are well known to those with ordinary skill in the art. Replication defective adenovirus vectors have proven to to be versatile for importing genes encoding immunoregulatory proteins into intact islets in the pancreas (Csete, M.E. et al. (1995) Transplantation 27:263-268). Potentially useful adenoviral vectors are described in U.S. Patent No. 5,707,618 to Armentano ("Adenovirus vectors for gene therapy"), hereby incorporated by reference. Fox adenoviral vectors, see also Antinozzi, P.A. et al. (1999; Annu.
Rev. Nutr. 19:511-544) and Verma, LM. and N. Somia (1997; Nature 18:389:239-242).
In another embodiment, a herpes-based; gene therapy delivery system is used to deliver polynucleotides encoding PMMM to target cells which have one or more genetic abnormalities with respect to the expression of PMMM. The use of herpes simplex virus (HSV)-based vectors may be especially valuable for introducing PNIn~IM to cells of the central nervous system, for which HSV has a tropism. The construction and packaging of herpes-based vectors are well known to those with ordinary skill in the art. A replication-competent herpes simplex virus (HSV) type 1-based vector has been used to deliver a reporter gene to the eyes of primates (Liu, X. et al.
(1999) Exp. Eye Res.
169:385-395). The construction of a HSV-1 virus vector has also been disclosed in detail in U.S.
Patent No. 5,804,413 to DeLuca ("Herpes simplex virus strains for gene transfer"), which is hereby incorporated by reference. U.S. Patent No. 5,804,413 teaches the use of recombinant HSV d92 which consists of a genome containing at least one exogenous gene to be transferred to a cell under the control of the appropriate promoter for purposes including human gene therapy. Also taught by tlus 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). 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 known to those of ordinary skill in the art.

In another embodiment, an alphavirus (positive, single-stranded RNA virus) vector is used to deliver polynucleotides encoding PMMM to target cells. The biology of the prototypic alphavil-us, Semlik_i Forest Virus (SFV), has been studied extensively and gene transfer vectors have been based on the SFV genome (Garoff, H. and I~.-J. Li (1998) Curr. Opin. Biotechnol.
9:464-469). During alphavirus RNA replication, a subgenomic RNA is generated that normally encodes the viral capsid proteins. This subgenomic RNA replicates to higher levels than the full length genomic RNA, resulting in the overproduction of capsid proteins relative to the viral proteins with enzymatic activity (e.g., protease and polymerase). Similarly, inserting the coding sequence for PMMM into the alphavirus genome in place of the capsid-coding region results in the production of a large number of PNIIVIM-coding RNAs and the synthesis of high levels of PMMM in vector transduced cells. While alphavirus infection is typically associated with cell lysis within a few days, the ability to establish a persistent infection in hamster normal kidney cells (BHK-21) with a variant of Sindbis virus (SIN) indicates that the lytic replication of alphaviruses can be altered to suit the needs of the gene therapy application (Dryga, S.A. et al. (1997) Virology 228:74-83). The wide host range of alphaviruses will allow the introduction of PMMM into a variety of cell types. The specific transduction of a subset of cells in a population may require the sorting of cells prior to transduction.
The methods of manipulating infectious cDNA clones of alphaviruses, performing alphavirus cDNA and RNA
transfections, and performing alphavirus infections, are well known to those with ordinary skill in the art.
Oligonucleotides derived, from the transcription initiation site, e.g., between about positions -10 and +10 from the start site, may also be employed to inhibit gene expression.
Similarly, inhibition can be achieved using triple helix base-pairing methodology. Triple helix pairing is useful because it causes inhibition of the ability of the double helix to open sufficiently for the binding of polymerases, transcription factors, or regulatory molecules. Recent therapeutic advances using triplex DNA have been described in the literature (Gee, J.E. et al. (1994) in Huber, B.E. and B.I. Carr, Molecular and T_mmunolo~ic Approaches, Futura Publishing, Mt. Kisco NY, pp. 163-177). A
complementary sequence or antisense molecule may also be designed to block translation of mRNA by preventing the transcript from binding to ribosomes.
Ribozymes, enzymatic RNA molecules, may also be used to catalyze the specific cleavage of RNA. The mechanism of ribozyme action involves sequence-specific hybridization of the ribozyme molecule to complementary target RNA, followed by endonucleolytic cleavage.
For example, engineered hammerhead motif ribozyme molecules may specifically and efficiently catalyze endonucleolytic cleavage of RNA molecules encoding P1~~VIM.
Specific ribozyme cleavage sites within any potential RNA target are initially identified by scanning the target molecule for ribozyme cleavage sites, including the following sequences: GUA, GUU, and GUC. Once identified, short RNA sequences of between 15 and 20 ribonucleotides, corresponding to the region of the target gene containing the cleavage site, may be evaluated for secondary structural features which may render the oligonucleotide inoperable.
The suitability of candidate targets may also be evaluated by testing accessibility to hybridization with complementary oligonucleotides using ribonuclease protection assays.
Complementary ribonucleic acid molecules and ribozymes may be prepared by any method known in the art for the synthesis of nucleic acid molecules. These include techniques for chemically synthesizing oligonucleotides such as solid phase phosphoramidite chemical synthesis. Alternatively, RNA molecules may be generated by in vitf-o and ire vivo transcription of DNA
molecules encoding Pn~VIM. Such DNA sequences may be incorporated into a wide variety of vectors with suitable RNA polynierase promoters such as T7 or SP6. Alternatively, these cDNA
constructs that synthesize complementary RNA, constitutively or inducibly, can be introduced into cell lines, cells, or tissues.
RNA molecules may be modified to increase intracellular stability and half life. Possible modifications include, but are not limited to, the addition of flanking sequences at the 5' and/or 3' ends of the molecule, or the use of phosphorothioate or 2' O-methyl rather than phosphodiesterase linkages within the backbone of the molecule. This concept is inherent in the production of PNAs and can be extended in all of these molecules by the inclusion of nontraditional bases such as inosine, queosine, and wybutosine, as well as acetyl-, methyl-, thio-, and similarly modified forms of adenine, cytidine, guanine, thymine, and uridine which are not as easily recognized by endogenous endonucleases.
In other embodiments of the invention, the expression of one or more selected polynucleotides of the present invention can be altered, inhibited, decreased, or silenced using RNA interference (RNAi) or post-transcriptional gene silencing (PTGS) methods known in the art.
RNAi is a post-transcriptional mode of gene silencing in which double-stranded RNA (dsRNA) introduced into a targeted cell specifically suppresses the expression of the homologous gene (i.e., the gene bearing the sequence complementary to the dsRNA). This effectively knocks out or substantially reduces the expression of the targeted gene. PTGS can also be accomplished by use of DNA
or DNA fragments as well. RNAi methods are described by Fire, A. et al. (1998; Nature 391:806-811) and Gura, T.

(2000; Nature 404:804-808). PTGS can also be initiated by introduction of a complementary segment of DNA into the selected tissue using gene delivery and/or viral vector delivery methods described herein or known in the art.
RNAi can be induced in mammalian cells by the use of small interfering RNA
also known as siRNA. SiRNA are shorter segments of dsRNA (typically about 21 to 23 nucleotides in length) that result i~. vivo from cleavage of introduced dsRNA by the action of an endogenous ribonuclease.
SiRNA appear to be the mediators of the RNAi effect in mammals. The most effective siRNAs appear to be 21 nucleotide dsRNAs with 2 nucleotide 3' overhangs. The use of siRNA for inducing RNAi in mammalian cells is described by Elbashir, S.M. et al. (2001; Nature 411:494-498).
SiRNA can either be generated indirectly by introduction of dsRNA into the targeted cell, or directly by mammalian transfection methods and agents described herein or known in the art (such as liposome-mediated transfection, viral vector methods, or other polynucleotide delivery/introductory methods). Suitable Si_RNAs can be selected by examining a transcript of the target polynucleotide (e.g., mRNA) for nucleotide sequences downstream from the AUG start codon and recording the occurrence of each nucleotide and the 3' adjacent 19 to 23 nucleotides as potential siRNA target sites, with sequences having a 21 nucleotide length being preferred. Regions to be avoided for target siRNA sites include the 5' and 3' untranslated regions (U'TRs) and regions near the start codon (within 75 bases), as these may be richer in regulatory protein binding sites. UTR-binding proteins andlor translation initiation complexes may interfere with binding of the siRNP
endonuclease complex. The selected target sites for siRNA can then be compared to the appropriate genome database (e.g., human, etc.) using BLAST or other sequence comparison algorithms known in the art. Target sequences with significant homology to other coding sequences can be eliminated from consideration.
The selected SiRNAs can be produced by chemical synthesis methods known in the art or by iii vitro transcription using commercially available methods and kits such as the SILENCER siRNA
construction kit (Ambion, Austin TX).
In alternative embodiments, long-term gene silencing and/or RNAi effects can be induced in selected tissue using expression vectors that continuously express siRNA. This can be accomplished using expression vectors that are engineered to express hairpin RNAs (shRNAs) using methods known in the art (see, e.g., Brummelkamp, T.R. et al. (2002) Science 296:550-553; and Paddison, P.J.
et al. (2002) Genes Dev. 16:948-958). In these and related embodiments, shRNAs can be delivered to target cells using expression vectors known in the art. An example of a suitable expression vector for delivery of siRNA is the PSILENCER1.0-U6 (circular) plasmid (Ambion). Once delivered to the target tissue, shRNAs are processed in vivo into siRNA-like molecules capable of carrying out gene-specific silencing.
In various embodiments, the expression levels of genes targeted by RNAi or PTGS methods can be determined by assays for mRNA and/or protein analysis. Expression levels of the mRNA of a targeted gene, can be determined by northern analysis methods using, for example, the NORTHERNMAX-GLY kit (Ambion); by microarray methods; by PCR methods; by real time PCR
methods; and by other RNA/polynucleotide assays known in the art or described herein. Expression levels of the protein encoded by the targeted gene can be determined by Western analysis using standard techniques known in the art.
An additional embodiment of the invention encompasses a method for screening for a compound which is effective in altering expression of a polynucleotide encoding PNIIVIM. Compounds which may be effective in altering expression of a specific polynucleotide may include, but are not limited to, oligonucleotides, antisense oligonucleotides, triple helix-forming oligonucleotides, transcription factors and other polypeptide transcriptional regulators, and non-macromolecular chemical entities which are capable of interacting with specific polynucleotide sequences. Effective . , compounds may alter polynucleotide expression by acting as either inhibitors or promoters of polynucleotide expression. Thus, in the treatment of disorders associated with increased PMIVI1~~I
expression or activity, a compound which specifically inhibits expression of the polynucleotide encoding PMIvITiI may be therapeutically useful, and in the treatment of disorders associated with decreased PM1VINI expression or activity, a compound which specifically promotes expression of the polynucleotide encoding PMIV~~I may be therapeutically useful.
In various embodiments, one or more test compounds may be screened for effectiveness in altering expression of a specific polynucleotide. A test compound may be obtained by any method commonly known in the art, including chemical modification of a compound known to be effective in altering polynucleotide expression; selection from an existing, commercially-available or proprietary library of naturally-occurring or non-natural chemical compounds; rational design of a compound based on chemical and/or structural properties of the target polynucleotide;
and selection from a library of chemical compounds created combinatorially or randomly. A sample comprising a polynucleotide encoding PMIVW is exposed to at least one test compound thus obtained. The sample 3o 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 Pl~~VIM 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 PMMM. 'The amount of hybridization may be quantified, thus forming the basis for a comparison of the expression of the polynucleotide both with and without exposure to one or more test compounds. Detection of a change in the expression of a polynucleotide exposed to a test compound indicates that the test compound is effective in altering the expression of the polynucleotide. A screen for a compound effective in altering expression of a specific polynucleotide can be carried out, for example, using a Schizosacchar-omyces pornbe gene expression system (Atkins, D. et al. (1999) U.S. Patent No. 5,932,435; Arndt, G.M. et al. (2000) Nucleic Acids Res.
28:E15) or a human cell line such as HeLa cell (Clarke, M.L. et al. (2000) Biochem. Biophys. Res.
Commun. 268:8-13). A particular embodiment of the present invention involves screening a combinatorial library of oligonucleotides (such as deoxyribonucleotides, ribonucleotides, peptide nucleic acids, and modified oligonucleotides) for antisense activity against a specific polynucleotide sequence (Bruice, T.W. et al. (1997) U.S. Patent No. 5,686,242; Bruice, T.W. et al.
(2000) U.S. Patent No.
6,022,691).
~ Many methods for introducing vectors into cells or tissues are available and equally suitable for use in vivo, in vitro, and ex vivo. For ex vivo therapy, vectors may be introduced into stem cells taken from the patient and clonally propagated for autologous transplant back into that same patient.
Delivery by transfection, by liposome injections, or by polycationic amino polymers may be achieved using methods which are well known in the art (Goldman, C.K. et al. (1997) Nat. Biotechnol..15:462-466).
Any of the therapeutic methods described above may be applied to any subject in need of such therapy, including, for example, mammals such as humans, dogs, cats, cows, horses, rabbits, and monkeys.
An additional embodiment of the invention relates to the administration of a composition which generally comprises an active ingredient formulated with a pharmaceutically acceptable excipient.
Excipients may include, for example, sugars, starches, celluloses, gums, and proteins. Various formulations are commonly known and are thoroughly discussed in the latest edition of Remin won's Pharmaceutical Sciences (Maack Publishing, Easton PA). Such compositions may consist of PMMM, antibodies to PMMM, and mimetics, agonists, antagonists, or inhibitors of PMMM.
In various embodiments, the compositions described herein, such as pharmaceutical compositions, may be administered by any number of routes including, but not limited to, oral, intravenous, intramuscular, intra-arterial, intramedullary, intrathecal, intraventricular, pulmonary, transdermal, subcutaneous, intraperitoneal, intranasal, enteral, topical, sublingual, or rectal means.
Compositions for pulmonary administration may be prepared in liquid or dry powder form.
These compositions are generally aerosolized immediately prior to inhalation by the patient. In the case of small molecules (e.g. traditional low molecular weight organic drugs), aerosol delivery of fast-acting formulations is well-known in the art. In the case of macromolecules (e.g. larger peptides and proteins), recent developments in the field of pulmonary delivery via the alveolar region of the lung have enabled the practical delivery of drugs such as insulin to blood circulation (see, e.g., Patton, J.S.
et al., U.S. Patent No. 5,997,848). Pulmonary delivery allows 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 PMT~VI or fragments thereof. For example, liposome preparations containi_ug a cell-impermeable macromolecule may promote cell fusion and intracellular delivery of the macromolecule. Alternatively, PMMMM or a fragment thereof may be joined to a short cationic N- .
terminal portion from the HIV Tat-1 protein. Fusion proteins thus generated have been found to transduce into the cells of all tissues, including the brain, in a mouse model system (Schwarze, S.R. et 2o al. (1999) Science 285:1569-1572).
For any compound, the therapeutically effective dose can be estimated initially either in cell culture assays, e.g., of neoplastic cells, or in animal models such as mice, rats, rabbits, dogs, monkeys, or pigs. An animal model may also be used to determine the appropriate concentration range and route of administration. Such information can then be used to determine useful doses and routes for administration in humans.
A therapeutically effective dose refers to that amount of active ingredient, for example PI~~VIM or fragments thereof, antibodies of Pn~VIM, and agonists, antagonists or inhibitors of PM1V~~I, which ameliorates the symptoms or condition. Therapeutic efficacy and toxicity may be determined by standard pharmaceutical procedures in cell cultures or with experimental animals, such as by calculating the EDSO (the dose therapeutically effective in 50% of the population) or LDSO (the dose lethal to 50% of the population) statistics. The dose ratio of toxic to therapeutic effects is the therapeutic index, which can be expressed as the LDso/EDSO ratio. Compositions which exhibit large therapeutic indices are preferred. The data obtained from cell culture assays and animal studies are used to formulate a range of dosage for human use. The dosage contained in such compositions is preferably within a range of circulating concentrations that includes the EDso with little or no toxicity.
The dosage varies within this range depending upon the dosage form employed, the sensitivity of the patient, and the route of administration.
The exact dosage will be determined by the practitioner, in light of factors related to the subject requiring treatment. Dosage and administration are adjusted to provide sufficient levels of the active moiety or to maintain the desired effect. Factors which may be taken into account include the severity of the disease state, the general health of the subject, the age, weight, and gender of the subject, time and frequency of administration, drug combination(s), reaction sensitivities, and response to therapy. Long-acting compositions may be administered every 3 to 4 days, every week, or biweekly depending on the half life and clearance rate of the particular formulation.
Normal dosage amounts may vary from about 0.1,ug to 100,000 ,ug, up to a total dose of about 1 gram, depending upon the route of administration. Guidance as to particular dosages and methods of delivery is provided in the literature and generally available to practitioners in the art.
Those skilled in the art will employ different formulations for nucleotides than for proteins or their inhibitors. Similarly, delivery of polynucleotides or polypeptides will be specific to particular cells, conditions, locations, etc.
DIAGNOSTICS
In another embodiment, antibodies which specifically bind PI~~VIM may be used for the diagnosis of disorders characterized by expression of P1~~VIM, or in assays to monitor patients being treated with PNI1VIM or agonists, antagonists, or inhibitors of Pn~VIM.
Antibodies useful for diagnostic purposes may be prepared in the same manner as described above for therapeutics. Diagnostic assays for Pinclude methods which utilize the antibody and a label to detect PM1VIM in human body fluids or in extracts of cells or tissues. The antibodies may be used with or without modification, and may be labeled by covalent or non-covalent attachment of a reporter molecule. A wide variety of reporter molecules, several of which are described above, are known in the art and may be used.
A variety of protocols for measuring PM1VIM, including ELISAs, RIAs, and FACS, are known in the art and provide a basis for diagnosing altered or abnormal levels of PMIVIn~I expression. Normal or standard values for PNIIVIM expression are established by combining body fluids or cell extracts taken from normal mammalian subjects, for example, human subjects, with antibodies to PI~MMM
under conditions suitable for complex formation. The amount of standard complex formation may be so quantitated by various methods, such as photometric means. Quantities of PMMM
expressed in subject, control, and disease samples from biopsied tissues are compared with the standard values.
Deviation between standard and subject values establishes the parameters for diagnosing disease.
In another embodiment of the invention, polynucleotides encoding PMMM may be used for diagnostic purposes. The polynucleotides which may be used include oligonucleotides, 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 Pmay be correlated with disease. The diagnostic assay may be used to determine absence, presence, and excess expression of PMMM, and to monitor regulation of PMMM levels during therapeutic intervention.
In one aspect, hybridization with PCR probes which are capable of detecting polynucleotides, including genomic sequences, encoding PNnVIM or closely related molecules may be used to identify nucleic acid sequences which encode PMMM. The specificity of the probe, whether it is made from a highly specific region, e.g., the 'regulatory region, or from a less specific region, e.g., a conserved motif, and the stringency of the hybridization or amplification will determine whether the probe identifies only naturally occurring sequences encoding PNNnVVIM, 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 PMMM encoding sequences. The hybridization probes of the subject invention may be DNA or RNA and may be derived from the sequence of SEQ m N0:41-80 or from genomic sequences including promoters, enhancers, and introns of the PMLVEVI
gene.
Means for producing specific hybridization probes for polynucleotides encoding P
include the cloning of polynucleotides encoding PMMIVI or PNEVIM derivatives into vectors for the production of mRNA probes. Such vectors are known in the art, are commercially available, and may be used to synthesize RNA probes in vitt~o by means of the addition of the appropriate RNA
polymerases and the appropriate labeled nucleotides. Hybridization probes may be labeled by a variety of reporter groups, for example, by radionuclides such as 32P or 355, or by enzymatic labels, such as alkaline phosphatase coupled to the probe via avidin/biotin coupling systems, and the like.
Polynucleotides encoding PMMMM may be used for the diagnosis of disorders associated with expression of PNEVIM. Examples of such disorders include, but are not limited to, a gastrointestinal disorder, such as dysphagia, peptic esophagitis, esophageal spasm, esophageal stricture, esophageal carcinoma, dyspepsia, indigestion, gastritis, gastric carcinoma, anorexia, nausea, emesis, gastroparesis, antral or pyloric edema, abdominal angina, pyrosis, gastroenteritis, intestinal obstruction, infections of the intestinal tract, peptic ulcer, cholelithiasis, cholecystitis, cholestasis, pancreatitis, pancreatic carcinoma, biliary tract disease, hepatitis, hyperbilirubinemia, cirrhosis, passive congestion of the liver, hepatoma, infectious colitis, ulcerative colitis, ulcerative proctitis, Crohn's disease, Whipple's disease, Mallory-Weiss syndrome, colonic carcinoma, colonic obstruction, irritable bowel syndrome, short bowel syndrome, diarrhea, constipation, gastrointestinal hemorrhage, acquired immunodeficiency syndrome (AIDS) enteropathy, jaundice, hepatic encephalopathy, hepatorenal syndrome, hepatic steatosis, hemochromatosis, Wilson's disease, alphai antitrypsin deficiency, Reye's syndrome, primary sclerosing cholangitis, liver infarction, portal vein obstruction and thrombosis, centrilobular necrosis, peliosis hepatis, hepatic vein thrombosis, veno-occlusive disease, preeclampsia, eclampsia, acute fatty liver of pregnancy, intrahepatic cholestasis of pregnancy, and hepatic tumors including nodular hyperplasias, adenomas, and carcinomas; an endocrine disorder such as a disorder of the hypothalamus and/or pituitary resulting from lesions such as a primary brain tumor, adenoma, infarction associated with pregnancy, hypophysectomy, aneurysm, vascular malformation, thrombosis, infection, imtnunological disorder, and complication due to head trauma; a disorder associated with hypopituitarism including hypogonadism, Sheehan syndrome, diabetes insipidus, Kallman's disease, Hand-Schuller-Christian disease, Letterer-Siwe disease, sarcoidosis, empty sella syndrome,' and dwarfism; a disorder associated with hyperpituitarism includilig acromegaly, giantism, and syndrome of inappropriate antidiuretic hormone (ADH) secretion (SIADH) often caused by benign adenoma; a.
disorder associated with hypothyroidism including goiter, myxedema, acute thyroiditis associated with bacterial infection, subacute thyroiditis associated with viral infection, autoimmune thyroiditis (Hashimoto's disease), and cretinism; a disorder associated with hyperthyroidism including thyrotoxicosis and its various forms, Grave's disease, pretibial myxedema, toxic multinodular goiter, thyroid carcinoma, and Plummer's disease; a disorder associated with hyperparathyroidism including Conn disease (chronic hypercalemia); a pancreatic disorder such as Type I or Type II diabetes mellitus and associated complications; a disorder associated with the adrenals such as hyperplasia, carcinoma, or adenoma of the adrenal cortex, hypertension associated with alkalosis, amyloidosis, hypokalemia, C~xshing's disease, Liddle's syndrome, and Arnold-Healy-Gordon syndrome, pheochromocytoma tumors, and Addison's disease; a disorder associated with gonadal steroid hormones such as: in women, abnormal prolactin production, infertility, endometriosis, perturbation of the menstrual cycle, polycystic ovarian disease, hyperprolactinemia, isolated gonadotropin deficiency, amenorrhea, galactorrhea, hermaphroditism, hirsutism and virilization, breast cancer, and, in post-menopausal women, osteoporosis; and, in men, Leydig cell deficiency, male climacteric phase, and germinal cell aplasia, a hypergonadal disorder associated with Leydig cell tumors, androgen resistance associated with absence of androgen receptors, syndrome of 5 a-reductase, and gynecomastia; a disorder of the immune system such as inflammation, actinic keratosis, acquired immunodeficiency syndrome (AmS), Addison's disease, adult respiratory distress syndrome, allergies, ankylosing spondylitis, amyloidosis, anemia, arteriosclerosis, asthma, atherosclerosis, autoimmune hemolytic anemia, autoimmune thyroiditis, bronchitis, bursitis, cholecystitis, cirrhosis, contact dermatitis, Crohn's disease, atopic dermatitis, dermatomyositis, diabetes mellitus, emphysema, erythroblastosis fetalis, erythema nodosum, atrophic gastritis, glomerulonephritis, Goodpasture's syndrome, gout, Graves' disease, Hashimoto's thyroiditis, paroxysmal nocturnal hemoglobinuria, hepatitis, hypereosinophilia, irritable bowel syndrome, episodic lymphopenia with lymphocytotoxins, mixed connective tissue disease (MCTD), multiple sclerosis, myasthenia gravis, myocardial or pericardial inflammation, myelofibrosis, osteoartluitis, osteoporosis, pancreatitis, polycythemia vera, polymyositis, psoriasis, Reiter's syndrome, rheumatoid arrlu7.tis, scleroderma, Sjogren's syndrome, systemic anaphylaxis, systemic lupus erythematosus, systemic sclerosis, primary thrombocythemia, thrombocytopenic purpura~ ulcerative colitis, uveitis, Werner syndrome, complications of cancer, hemodialysis, and extracorporeal circulation, trauma, and hematopoietic cancer including lymphoma, leukemia, and myeloma; an infection caused by a viral agent classified as adenovirus, arenavirus, bunyavirus, calicivirus, coronavirus, filovirus, hepadnavirus, herpesvirus, flavivirus, orthomyxovirus; parvovirus, .
papovavirus, paramyxovirus, picornavirus, poxvirus, reovirus, retrovirus, rhabdovirus, or togavirus; an..
infection caused by a bacterial agent classified as pneumococcus, staphylococcus, streptococcus bacillus, corynebacterium, clostridium, meningococcus, gonococcus, listeria, moraxella, kingella, haemophilus, legionella, bordetella, gram-negative enterobacterium including shigella, salmonella, or campylobacter, pseudomonas, vibrio, brucella, francisella, yersinia, bartonella, norcardium, actinomyces, mycobacterium, spirochaetale, rickettsia, chlamydia, or mycoplasma; an infection caused by a fungal agent classified as aspergillus, blastomyces, dermatophytes, cryptococcus, coccidioides, malasezzia, histoplasma, or other mycosis-causing fungal agent; an infection caused by a parasite classified as plasmodium or malaria-causing, parasitic entamoeba, leishmania, trypanosome, toxoplasma, pneumocystis carinii, intestinal protozoa such as giardia, trichomonas, tissue nematode such as trichinella, intestinal nematode such as ascaris, lymphatic filarial nematode, trematode such as schistosoma, and cestrode such as tapeworm; a metabolic disorder such as Addison's disease, cerebrotendinous xanthomatosis, congenital adrenal hyperplasia, coumarin resistance, cystic fibrosis, diabetes, fatty hepatocirrhosis, fructose-1,6-diphosphatase deficiency, galactosemia, goiter, glucagonoma, glycogen storage diseases, hereditary fructose intolerance, hyperadrenalism, hypoadrenalism, hyperparathyroidisrri, hypoparathyroidism, hypercholesterolemia, hyperthyroidism, hypoglycemia, hypothyroidism, hyperlipidemia, hyperlipemia, lipid myopathies, lipodystrophies, lysosomal storage diseases, mannosidosis, neuraminidase deficiency, obesity, pentosuria phenylketonuria, pseudovitamin D-deficiency rickets; a disorder of carbohydrate metabolism such as congenital type II dyserythropoietic anemia, diabetes, insulin-dependent diabetes mellitus, non-insulin-dependent diabetes mellitus, fructose-1,6-diphosphatase deficiency, galactosemia, glucagonoma, hereditary fructose intolerance, hypoglycemia, mannosidosis, neuraminidase deficiency, obesity, galactose epimerase deficiency, glycogen storage diseases, lysosomal storage diseases, fructosuria, pentosuria, and inherited abnormalities of pyruvate metabolism; a disorder of lipid metabolism such as fatty liver, cholestasis, primary biliary cirrhosis, carnitine deficiency, carnitine palinitoyltransferase deficiency, myoadenylate deaminase deficiency, hypertriglyceridemia,.lipid storage disorders such Fabry's disease, Gaucher's disease, Niemann-Pick's disease, metachromatic leukodystrophy, adrenoleukodystrophy, GMa gangliosidosis, and ceroid lipofuscinosis, abetalipoproteinemia, Tangier disease, hyperlipoproteinemia, diabetes mellitus, lipodystrophy, lipomatoses, acute panniculitis, disseminated fat necrosis, adiposis dolorosa, lipoid adrenal hyperplasia, minimal change disease, lipomas, atherosclerosis, hypercholesterolemia, hypercholesterolemia with hypertriglyceridemia~ primary hypoalphalipoproteinemia, hypothyroidism, renal disease, liver disease, ~ .
lecithin:cholesterol acyltransferase deficiency, cerebrotendinous xanthomatosis, sitosterolemia, hypocholesterolemia, Tay-Sachs disease, Sandhoff's disease, hyperlipidemia, hyperlipemia, lipid myopathies, and obesity; a disorder of copper metabolism such as Menke's disease, Wilson's disease, and Ehlers-Danlos syndrome type IX.; a cardiovascular disorder, such as arteriovenous fistula, atherosclerosis, hypertension, vasculitis, Raynaud's disease, aneurysms, arterial dissections, varicose veins, thrombophlebitis and phlebothrombosis, vascular tumors, and complications of thrombolysis, balloon angioplasty, vascular replacement, and coronary artery bypass graft surgery, congestive heart failure, ischemic heart disease, angina pectoris, myocardial infarction, hypertensive heart disease, degenerative valvular heart disease, calcific aortic valve stenosis, congenitally bicuspid aortic valve, mural annular calcification, mitral valve prolapse, rheumatic fever and rheumatic heart disease, infective endocarditis, nonbacterial thrombotic endocarditis, endocarditis of systemic lupus erythematosus, carcinoid heart disease, cardiomyopathy, myocarditis, pericarditis, neoplastic heart disease, congenital heart disease, and complications of cardiac transplantation; an autoimmune/inflammatory disease, such as acquired immunodeficiency syndrome (AIDS), Addison's disease, adult respiratory distress syndrome, allergies, ankylosing spondylitis, amyloidosis, anemia, asthma, atherosclerosis, atherosclerotic plaque rupture, autoimmune hemolytic anemia, autoimmune thyroiditis, autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED), bronchitis, cholecystitis, contact dermatitis, Crohn's disease, atopic dermatitis, dermatomyositis, diabetes mellitus, emphysema, episodic lymphopenia with lymphocytotoxins, erythroblastosis fetalis, erythema nodosum, atrophic gastritis, glomerulonephritis, Goodpasture's syndrome, gout, Graves' disease, Hashimoto's thyroiditis, hypereosinophilia, irritable bowel syndrome, multiple sclerosis, myasthenia gravis, myocardial or pericardial inflammation, osteoarthritis, degradation of articular cartilage, osteoporosis, pancreatitis, polymyositis, psoriasis, Reiter's syndrome, rheumatoid arthritis, scleroderma, Sjogren's syndrome, systemic anaphylaxis, systemic lupus erythematosus, systemic sclerosis, thrombocytopenic purpura, ulcerative colitis, uveitis, Werner syndrome, complications of cancer, hemodialysis, and extracorporeal circulation, viral, bacterial, fungal, parasitic, protozoal, and heltninthic 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 adenocarcindma, 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 (Wilins' tumor, aniridia, genitourinary abnormalities, and mental retardation), Smith-Magenis syndrome, myelodysplastic syndrome, hereditary mucoepithelial dysplasia, hereditary keratodermas, hereditary neuropathies such as Charcot-Marie-Tooth disease and neurofibromatosis, hypothyroidism, hydrocephalus, seizure disorders such as Syndenham's chorea and cerebral palsy, spina bifida, anencephaly, craniorachischisis, congenital glaucoma, cataract, age-related macular degeneration, and sensorineural hearing loss; an epithelial disorder, such as dyshidrotic eczema, allergic contact dermatitis, keratosis pilaris, melasma, vitiligo, actinic keratosis, basal cell carcinoma, squamous cell carcinoma, seborrheic keratosis, folliculitis, herpes simplex, herpes zoster, varicella, candidiasis, dermatophytosis, scabies, insect bites, cherry angioma, keloid, dermatofibroma, acrochordons, urticaria, transient acantholytic dermatosis, xerosis, eczema, atopic dermatitis, contact dermatitis, hand eczema, nummular eczema, lichen simplex chronicus, asteatotic eczema, stasis dermatitis and stasis ulceration, seborrheic dermatitis, psoriasis, lichen planus, pityriasis rosea, impetigo, ecthyma, dermatophytosis, tinea versicolor, warts, acne vulgaris, acne rosacea, pemphigus vulgaris, pemphigus foliaceus, paraneoplastic pemphigus, bullous pemphigoid, herpes gestationis, dermatitis herpetiformis, linear IgA disease, epidermolysis bullosa acquisita, dermatomyositis, lupus erythematosus, scleroderma and morphea, erythroderma, alopecia, hgurate skin lesions, telangiectasias, hypopigmentation, hyperpigmentation, vesicles/bullae, exanthems, cutaneous drug reactions, papulonodular skin lesions, chronic non-healing wounds, photosensitivity diseases, epidermolysis bullosa simplex, epidermolytic hyperkeratosis, epidermolytic and nonepidermolytic palmoplantar keratoderma, ichthyosis bullosa of Siemens, ichthyosis exfoliativa, keratosis palmaris et plantaris, keratosis palmoplantaris, palmoplantar keratoderma, keratosis punctata, Meesmann's corneal dystrophy, pachyonychia congenita, white sponge nevus, steatocystoma multiplex, epidermal nevi/epidermolytic hyperkeratosis type, monilethrix, trichothiodystrophy, chronic hepatitis/cryptogenic cirrhosis, and colorectal hyperplasia; a neurological disorder, such as epilepsy, ischemic cerebrovascular disease, stroke, cerebral neoplasms, Alzheimer's disease, Pick's disease, Huntington's disease, dementia, Parkinson's disease and other extrapyramidal disorders, amyotrophic lateral sclerosis and other motor neuron disorders, progressive neural muscular atrophy, retinitis pigmentosa, hereditary ataxias, multiple sclerosis and other demyelinating diseases, bacterial and viral meningitis, brain abscess, subdural empyema, epidural abscess, suppurative intracranial thrombophlebitis, myelitis and radiculitis, viral central nervous system disease, priors diseases including kuru, Creutzfeldt-Jakob disease, and Gerstmann-Straussler-Scheinker syndrome, fatal familial insomnia, nutritional and metabolic diseases of the nervous system, neurofibromatosis, tuberous sclerosis, cerebelloretinal hemangioblastomatosis, encephalotrigeminal syndrome, mental retardation and other developmental disorders of the central nervous system including Down syndrome, cerebral palsy, neuroskeletal disorders, autonomic nervous system disorders, cranial nerve disorders, spinal cord diseases, muscular dystrophy and other neuromuscular disorders, peripheral nervous system disorders, dermatomyositis and polymyositis, inherited, metabolic, endocrine, and toxic myopathies, myasthenia gravis, periodic paralysis, mental disorders including mood, anxiety, and schizophrenic disorders, seasonal affective disorder (SAD), akathesia, amnesia, catatonia, diabetic neuropathy, tardive dyskinesia, dystonias, paranoid psychoses, postherpetic neuralgia, Tourette's disorder, progressive supranuclear palsy, corticobasal degeneration, and familial frontotemporal dementia; and a reproductive disorder, such as infertility, including tubal disease, ovulatory defects, and endometriosis, a disorder of prolactin production, a disruption of the estrous cycle, a disruption of the menstrual cycle, polycystic ovary syndrome, ovarian hyperstimulation syndrome, an endometrial or ovarian tumor, a uterine fibroid, autoimmune disorders, an ectopic pregnancy, and teratogenesis;

cancer of the breast, fibrocystic breast disease, and galactorrhea; a disruption of spermatogenesis, abnormal sperm physiology, cancer of the testis, cancer of the prostate, benign prostatic hyperplasia, prostatitis, Peyronie's disease, impotence, carcinoma of the male breast, and gynecomastia.
Polynucleotides encoding P1~~VIM may be used in Southern or northern analysis, dot blot, or other membrane-based technologies; in PCR technologies; in dipstick, pin, and multiformat ELISA-like assays; and in microarrays utilizing fluids or tissues from patients to detect altered P1~~VIM expression.
Such qualitative or quantitative methods are well known in the art.
In a particular embodiment, polynucleotides encoding PNIIVIM may be used in assays that detect the presence of associated disorders, particularly those mentioned above. Polynucleotides complementary to sequences encoding PMIVI1~Z may be labeled by standard methods and added to a fluid or tissue sample from a patient under conditions suitable for the formation of hybridization complexes. After a suitable incubation period, the sample is washed and the signal is quantified and compared with a standard value. If the amount of signal in the patient sample is significantly altered in comparison to a control sample then the presence of altered levels of polynucleotides encoding Pl~~VIM 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 PT~VIM, a normal or standard profile for expression is established. This may be accomplished by combining body fluids or cell extracts taken from normal subjects, either animal or human, with a sequence, or a fragment thereof, encoding PNIIV1M, 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.
s~

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 PMMM
may involve the use of PCR. These oligomers may be chemically synthesized, generated enzymatically, or produced iti vitro. Oligomers will preferably contain a fragment of a polynucleotide encoding PMMM, or a fragment of a polynucleotide complementary to the polynucleotide encoding PMMM, and will be employed under optimized conditions for identification of a specific gene or condition. Oligomers may also be employed under less stringent conditions for detection or quantification of closely related DNA or RNA sequences.
In a particular aspect, oligonucleotide primers derived from polynucleotides encoding PNRVIM
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 polynucleotides encoding PMMM are used to amplify DNA using the polymerase chain reaction (PCR). The DNA
may be derived, for example, from diseased or normal tissue, biopsy samples, bodily fluids, and the like. SNPs in the DNA cause differences in the secondary and tertiary structures of PCR products in single-stranded form, and these differences are detectable using gel electrophoresis in non-denaturing gels. In fSCCP, the oligonucleotide primers are fluorescently labeled, which allows detection of the amplimers in high-throughput equipment such as DNA sequencing machines.
Additionally, sequence database analysis methods, termed in silico SNP (isSNP), are capable of identifying polymorphisms by comparing the sequence of individual overlapping DNA fragments which assemble into a common consensus sequence. These computer-based methods filter out sequence variations due to laboratory preparation of DNA and sequencing errors using statistical models and automated analyses of DNA
sequence chromatograms. In the alternative, SNPs may be detected and characterized by mass spectrometry using, for example, the high throughput MASSARRAY system (Sequenom, Inc., San Diego CA).
s$

SNPs may be used to study the genetic basis of human disease. For example, at least 16 common SNPs have been associated with non-insulin-dependent diabetes mellitus.
SNPs are also useful for examining differences in disease outcomes in monogenic disorders, such as cystic fibrosis, sickle cell anemia, or chronic granulomatous disease. For example, variants in the mannose-binding lectin, MBL2, have been shown to be correlated with deleterious pulmonary outcomes in cystic fibrosis. SNPs also have utility in pharmacogenomics, the identification of genetic variants that influence a patient's response to a drug, such as life-threatening toxicity.
For example, a variation in N-acetyl transferase is associated with a high incidence of peripheral neuropathy in response to the anti-tuberculosis drug isoniazid, while a variation in the core promoter of the ALOXS gene results in diminished clinical response to treatment with an anti-asthma drug that targets the 5-lipoxygenase pathway. Analysis of the distribution of SNPs in different populations is useful for investigating genetic drift, mutation, recombination, and selection, as well as for tracing the origins of populations and their migrations (Taylor, J.G. et al. (2001) Trends Mol. Med. 7:507-512;
Kwok, P.-Y. and Z. Gu (1999) Mol. Med. Today 5:53-543; Nowotny, P. et al. (2001) Curr. Opin.
Neurobiol. 11:637-641).
Methods which may also be used to quantify the expression of PMMM include radiolabeling or biotinylating nucleotides, coamplification of a control nucleic acid, and interpolating results from standard curves (Melby, P.C. et al. (1993) J. Tmmunol. Methods 159:235-244;
Duplaa, C. et al. (1993) Anal. Biochem. 212:229-236). The speed of quantitation of multiple samples may be accelerated by running the assay in a high-throughput format where the oligomer or polynucleotide of interest is presented in various dilutions and a spectrophotometric or colorimetric response gives rapid quantitation.
In further embodiments, oligonucleotides or longer fragments derived from any of the polynucleotides described herein may be used as elements on a microarray. The microarray can be used in transcript imaging techniques which monitor the relative expression levels of large numbers of genes simultaneously as described below. The microarray may also be used to identify genetic variants, mutations, and polymorphisms. This information may be used to determine gene function, to understand the genetic basis of a disorder, to diagnose a disorder, to monitor progression/regression of disease as a function of gene expression, and to develop and monitor the activities of therapeutic agents in the treatment of disease. In particular, this information may be used to develop a pharmacogenomic profile of a patient in order to select the most appropriate and effective treatment regimen for that patient. For example, therapeutic agents which are highly effective and display the fewest side effects may be selected for a patient based on his/her pharmacogenomic profile.

In another embodiment, PMMM, fragments of PMMMM, or antibodies specific for PMMM
may be used as elements on a microarray. The microarray may be used to monitor or measure protein-protein interactions, drug-target interactions, and gene expression profiles, as described above.
A particular embodiment relates to the use of the polynucleotides of the present invention to generate a transcript image of a tissue or cell type. A transcript image represents the global pattern of gene expression by a particular tissue or cell type. Global gene expression patterns are analyzed by quantifying the number of expressed genes and their relative abundance under given conditions and at a given time (Seilhamer et al., "Comparative Gene Transcript Analysis," U.S.
Patent No. 5,840,484;
hereby expressly incorporated by reference herein). Thus a transcript image may be generated by hybridizing the polynucleotides of the present invention or their complements to the totality of transcripts or reverse transcripts of a particular tissue or cell type. In one embodiment, the hybridization takes place in high-throughput format, wherein the polynucleotides of the present invention or their complements comprise a subset of a plurality of elements on a microarray. The resultant transcript image would provide a profile of gene activity.
Transcript images may be generated using transcripts isolated from tissues, cell lines, biopsies, ;
or other biological samples. The transcript image may thus reflect gene expression in vivo, as in the case of a tissue or biopsy sample, or iii vitro, as in the case of a cell line.
Transcript images which proftle the expression of the polynucleotides of the present invention may also be used in conjunction with in vitro model systems and preclinical evaluation of pharmaceuticals, as well as toxicological testing of industrial and naturally-occurring environmental compounds. All compounds induce characteristic gene expression patterns, frequently termed molecular fingerprints or toxicant signatures, which are indicative of mechanisms of action and toxicity (Nuwaysir, E.F. et al. (1999) Mol. Carcinog. 24:153-159; Steiner, S. and N.L.
Anderson (2000) Toxicol. Lett. 112-113:467-471). If a test compound has a signature similar to that of a compound with known toxicity, it is likely to share those toxic properties. These fingerprints or signatures are most useful and refined when they contain expression information from a large number of genes and gene families. Ideally, a genome-wide measurement of expression provides the highest quality signature. Even genes whose expression is not altered by any tested compounds are important as well, as the levels of expression of these genes are used to normalize the rest of the expression data.
The normalization procedure is useful for comparison of expression data after treatment with different compounds. While the assignment of gene function to elements of a toxicant signature aids in interpretation of toxicity mechanisms, knowledge of gene function is not necessary for the statistical matching of signatures which leads to prediction of toxicity (see, for example, Press Release 00-02 from the National Institute of Environmental Health Sciences, released February 29, 2000, available at http://www.niehs.nih.gov/oc/news/toxchip.htm). Therefore, it is important and desirable in toxicological screening using toxicant signatures to include all expressed gene sequences.
In an embodiment, the toxicity of a test compound can be assessed by treating a biological sample containing nucleic acids with the test compound. Nucleic acids that are expressed in the treated biological sample are hybridized with one or more probes specific to the polynucleotides of the present invention, so that transcript levels corresponding to the polynucleotides of the present invention may be quantified. The transcript levels in the treated biological sample are compared with levels in an untreated biological sample. Differences in the transcript levels between the two samples are indicative of a toxic response caused by the test compound in the treated sample.
Another embodiment relates to the use of the polypeptides disclosed herein to analyze the proteome of a tissue or cell type. The term proteome refers to the global pattern of protein expression in a particular tissue or cell type. Each protein component of a proteome can be subjected individually to further analysis. Proteome expression patterns, or profiles, are analyzed by quantifying the number of expressed proteins and their relative abundance under given conditions and at a given time. A
profile of a cell's proteome may thus be generated by separating and analyzing the polypeptides of a particular tissue or cell type. In one embodiment, the separation is achieved using two-dimensional gel electrophoresis, in which proteins from a sample are separated by isoelectric focusing in the first dimension, and then according to molecular weight by sodium dodecyl sulfate slab gel electrophoresis in the second dimension (Steiner and Anderson, supra). The proteins are visualized in the gel as discrete and uniquely positioned spots, typically by staining the gel with an agent such as Coomassie Blue or silver or fluorescent stains. The optical density of each protein spot is generally proportional to the level of the protein in the sample. The optical densities of equivalently positioned protein spots from different samples, for example, from biological samples either treated or untreated with a test compound or therapeutic agent, are compared to identify any changes in protein spot density related to the treatment. The proteins in the spots are partially sequenced using, for example, standard methods employing chemical or enzymatic cleavage followed by mass spectrometry. The identity of the protein in a spot may be determined by comparing its partial sequence, preferably of at least 5 contiguous amino acid residues, to the polypeptide sequences of interest. In some cases, further sequence data may be obtained for definitive protein identification.

A proteomic profile may also be generated using antibodies specific for PMMM
to quantify the levels of P~ expression. In one embodiment, the antibodies are used as elements on a microarray, and protein expression levels are quantified by exposing the microarray to the sample and detecting the levels of protein bound to each array element (Lueking, A. et al. (1999) Anal. Biochem.
270:103-111; Mendoze, L.G. et al. (1999) Biotechniques 27:778-788). Detection may be performed by a variety of methods known in the art, for example, by reacting the proteins in the sample with a thiol-or amino-reactive fluorescent compound and detecting the amount of fluorescence bound at each array element.
Toxicant signatures at the proteome level are also useful for toxicological screening, and should be analyzed in parallel with toxicant signatures at the transcript level. There is a poor correlation between transcript and protein abundances for some proteins in some tissues (Anderson, N:L. and J. Seilhamer (1997) Electrophoresis 18:533-537), so proteome toxicant signatures maybe useful in the analysis of compounds which do not significantly affect the transcript image, but which alter the proteomic profile. In addition, the analysis of transcripts in body fluids is difficult, due to rapid degradation of mRNA, so proteomic profiling may be more reliable and informative in such cases.
In another embodiment, the toxicity of a test compound is assessed by treating a biological sample containing proteins with the test compound. Proteins that are expressed in the treated biological sample are separated so that the amount of each protein can be quantified. The amount of each protein is compared to the amount of the corresponding protein in an untreated biological sample:
A difference in the amount of protein between the two samples is indicative of a toxic response to the test compound in the treated sample. Individual proteins are identified by sequencing the amino acid residues of the individual proteins and comparing these partial sequences to the polypeptides of the present invention.
In another embodiment, the toxicity of a test compound is assessed by treating a biological sample containing proteins with the test compound. Proteins from the biological sample are incubated with antibodies specific to the polypeptides of the present invention. The amount of protein recognized by the antibodies is quantified. The amount of protein in the treated biological sample is compared with the amount in an untreated biological sample. A difference in the amount of protein between the two samples is indicative of a toxic response to the test compound in the treated sample.
Microarrays may be prepared, used, and analyzed using methods known in the art (Brennan, T.M. et al. (1995) U.S. Patent No. 5,474,796; Schena, M. et al. (1996) Proc.
Natl. Acad. Sci. USA
93:10614-10619; Baldeschweiler et al. (1995) PCT application W095/251116;
Shalom D. et al. (1995) PCT application W095/35505; Heller, R.A. et al. (1997) Proc. Natl. Aced. Sci.
USA 94:2150-2155;
Heller, M.J. et al. (1997) U.S. Patent No. 5,605,662). Various types of microarrays are well known and thoroughly described in Schena, M., Bd. (1999; DNA Microarrays: A
Practical Approach, Oxford University Press, London).
In another embodiment of the invention, nucleic acid sequences encoding PMMM
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 Boding sequences. For example, conservation of a coding sequence among members of a multi-gene family may potentially cause undesired cross hybridization during 1o 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 (PACs), bacterial artificial chromosomes (BACs), bacterial P1 constructions; or single chromosome cDNA libraries (Harrington, J.J. et al.
(1997) Nat. Genet. 15:345-355; Price, C.M. (1993) Blood Rev. 7:127-134; Trask, B.J. (1991) Trends Genet.
7:149-154). Once mapped, the nucleic acid sequences may be used to develop genetic linkage maps, for example, which correlate the inheritance of a disease state with the inheritance of a particular chromosome region or restriction fragment length polymorphism (RFLP) (Lender, E.S. and D. Botstein (1986) Proc. Natl.
Aced. Sci. USA 83:7353-7357).
Fluorescent i~ situ hybridization (FISH) may be correlated with other physical and genetic map data (Heinz-Ulrich, et al. (1995) in Meyers, supr-~c, pp. 965-968).
Examples of genetic map data can be found in various scientific journals or at the Online Mendelian Inheritance in Man (OMIM) World Wide Web site. Correlation between the location of the gene encoding PMMM on a physical map and a specific disorder, or a predisposition to a specific disorder, may help define the region of DNA associated with that disorder and thus may further positional cloning efforts.
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 regulatory genes for further investigation (Gatti, R.A. et al. (1988) Nature 336:577-580). The nucleotide sequence of the instant invention may also be used to detect differences in the chromosomal location due to translocation, inversion, etc., among normal, carrier, or affected individuals.
In another embodiment of the invention, P1~MMM, its catalytic or immunogenic fragments, or oligopeptides thereof can be used for screening libraries of compounds in any of a variety of drug screening techniques. The fragment employed in such screening may be free in solution, affixed to a solid support, borne on a cell surface, or located intracellularly. The formation of binding complexes between Pn~VIM and the agent being tested may be measured.
Another technique for drug screening provides for high throughput screening of compounds having suitable binding affinity to the protein of interest (Geysen, et al.
(1984) PCT application W084/03564). In this method, large numbers of different small test compounds are synthesized on a solid substrate. The test compounds are reacted with PA~VIM, or fragments thereof, and washed.
Bound PMMMM is then detected by methods well known in the art. Purified PNNnVVIM 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 PM1VIT~I specifically compete with a test compound for binding PNnVIM. -' In this manner, antibodies can be used to detect the presence of any peptide which shares one or more .
antigenic determinants with PNnVIM.
In additional embodiments, the nucleotide sequences which encode PM1VIM 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 known, including, but not limited to, such properties as the triplet genetic code and specific base pair interactions.
Without further elaboration, it is believed that one skilled in the art can, using the preceding description, utilize the present invention to its fullest extent. The following preferred specific embodiments are, therefore, to be construed as merely illustrative, and not limitative of the remainder of the disclosure in any way whatsoever.
The disclosures of all patents, applications, and publications mentioned above and below, including U.S. Ser. No. 60/329,689, U.S. Ser. No. 60/335,703, U.S. Ser. No.
60/348,887, U.S. Ser.
No. 60/334,145, U.S. Ser. No. 60/337,451, and U.S. Ser. No. 60/340,584 are hereby expressly incorporated by reference.

EXAMPLES
I. Construction of cDNA Libraries Incyte cDNAs were derived from cDNA libraries described in the LIFESEQ GOLD
database (Incyte Genomics, Palo Alto CA). Some tissues were homogenized and lysed in guanidinium isothiocyanate, while others were homogenized and lysed in phenol or in a suitable mixture of denaturants, such as TRIZOL (Invitrogen), 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 lysates using other RNA isolation kits, e.g., the POLY(A)PURE mRNA purification kit (Ambion, Austin TX).
In some cases, Stratagene was provided with RNA and constructed the corresponding cDNA
libraries. Otherwise, cDNA was synthesized and cDNA libraries were constructed with the UNIZAP vector system (Stratagene) or SUPERSCRIPT plasmid system (Invitrogen), using the recommended procedures or similar methods known in the art (Ausubel et al., supra, ch. 5). Reverse transcription was initiated using oligo d(T) or random primers. Synthetic oligonucleotide adapters were ligated to double stranded cDNA, and the cDNA was digested with the appropriate restriction enzyme or enzymes. For most libraries, the cDNA was.size-selected (300-1000 bp) using SEPHACRYL
S 1000, SEPHAROSE CL2B, or SEPHAROSE CL4B column chromatography (Amersham Biosciences) or preparative agarose gel electrophoresis. cDNAs were ligated into compatible restriction enzyme sites of the polylinker of a suitable plasmid, e.g., PBLUESCRIPT plasmid (Stratagene), PSPORT1 plasmid (Invitrogen, Carlsbad CA), PCDNA2.1 plasmid (Invitrogen), PBI~-CMV plasmid (Stratagene), PCR2-TOPOTA plasmid (Invitrogen), PCMV-ICIS plasmid (Stratagene), pIGEN (Incyte Genomics, Palo Alto CA), pRARE (Incyte Genomics), or pllVCY
(Incyte Genomics), 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 ElectroMAX

from Invitrogen.
II. Isolation of cDNA Clones Plasmids obtained as described in Example I were recovered from host cells by in vivo excision using the UNIZAP vector system (Stratagene) or by cell lysis.
Plasmids were purified using at least one of the following: a Magic or WIZARD Minipreps DNA purification system (Promega); an AGTC Miniprep purification kit (Edge Biosystems, Gaithersburg MD); and QIAWELL
8 Plasmid, QIAWELL 8 Plus Plasmid, QIAWELL 8 Ultra Plasmid purification systems or the R.E.A.L. PREP
96 plasmid purification kit from QIAGEN. Following precipitation, plasmids were resuspended in 0.1 ml of distilled water and stored, with or without lyophilization, at 4°C.
Alternatively, plasmid DNA was amplified from host cell lysates using direct link PCR in a high-throughput format (Rao, V.B. (1994) Anal. Biochem. 216:1-14). Host cell lysis and thermal cycling steps were carried out in a single reaction mixture. Samples were processed and stored in 384-well plates, and the concentration of amplified plasmid DNA was quantified fluorometrically using PICOGREEN dye (Molecular Probes, Eugene OR) and a FLUOROSKAN II fluorescence scanner (Labsystems Oy, Helsinki, Finland).
IIL Sequencing and Analysis Incyte 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 (Robbins Scientific) or the MICROLAB 2200 (Hamilton) liquid transfer system. cDNA sequencing reactions were prepared using reagents provided by Amersham Biosciences or supplied in ABI sequencing kits such as the ABI PRISM BIGDYE Terminator cycle sequencing ready reaction kit (Applied Biosystems).
Electrophoretic separation of cDNA sequencing reactions and detection of labeled polynucleotides were carried out using the MEGABACE 1000 DNA sequencing system (Amersham Biosciences);
the ABI PRISM 373 or 377 sequencing system (Applied Biosystems) in conjunction with standard ABI protocols and base calling software; or other sequence analysis systems known in the art.
Reading frames within the cDNA sequences were identified using standard methods (Ausubel et al., supra, ch. 7). Some of the cDNA sequences were selected for extension using the techniques disclosed in Example VlIT.
The polynucleotide sequences derived from Incyte cDNAs were validated by removing vector, linker, and poly(A) sequences and by masking ambiguous bases, using algorithms and programs based on BLAST, dynamic prograrxnning, and dinucleotide nearest neighbor analysis. The Incyte cDNA sequences or translations thereof were then queried against a selection of public databases such as the GenBank primate, rodent, mammalian, vertebrate, and eukaryote databases, and BLOCKS, PRINTS, DOMO, PRODOM; PROTEOME databases with sequences from Hotno Sapiens, Rattus norvegicus, Mus rnusculus, Caenor~habditis elegans, Sacclaarornyces cer~evisiae, Schizosacchar-ornyces pombe, and Candida albicans (Incyte Genomics, Palo Alto CA); hidden Markov model (HMM)-based protein family databases such as PFAM, llVCY, and TIGRFAM (Haft, D.H. et al. (2001) Nucleic Acids Res. 29:41-43); and HMM-based protein domain databases such as SMART (Schultz, J. et al. (1998) Proc. Natl. Acad. Sci. USA 95:5857-5864;
Letunic, I. et al. (2002) Nucleic Acids Res. 30:242-244). (I~VIM 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, BLIIVVIPS, and ~R. The Incyte cDNA sequences were assembled to produce full length polynucleotide sequences. Alternatively, GenBank cDNAs, GenBank ESTs, stitched sequences, stretched sequences, or Genscan-predicted coding sequences (see Examples IV and V) were used to extend Incyte cDNA assemblages to full length. Assembly was performed using programs based on Phred, Phrap, and Consed, and cDNA assemblages were screened for open reading frames using programs based on GeneMark, BLAST, and FASTA. The full length polynucleotide sequences were translated to derive the corresponding full length polypeptide sequences. Alternatively, a polypeptide may begin at any of the methionine residues of the full length translated polypeptide.
Full length polypeptide sequences. were subsequently analyzed by querying against databases such as the GenBank protein 2o databases (genpept), SwissProt, the PROTEOME databases, BLOCKS, PRINTS, DOMO, PRODOM, Prosite, hidden Markov model (I~VIM)-based protein family databases such as PFAM, INCY, and TIGRFAM; and I3T~IM-based protein domain databases such as SMART.
Full length polynucleotide sequences are also analyzed using MACDNASIS PRO software (MiraiBio, Alameda CA) and LASERGENE software (DNASTAR). Polynucleotide and polypeptide sequence alignments are generated using default parameters specified by the CLUSTAL algorithm as incorporated into the MEGALIGN multisequence alignment program (DNASTAR), which also calculates the percent Identity between aligned sequences.
Table 7 summarizes the tools, programs, and algorithms used for the analysis and assembly of Incyte cDNA and full length sequences and provides applicable descriptions, references, and threshold parameters. The first column of Table 7 shows the tools, programs, and algorithms used, the second column provides brief descriptions thereof, the third column presents appropriate references, all of which are incorporated by reference herein in their entirety, and the fourth column presents, where applicable, the scores, probability values, and other parameters used to evaluate the strength of a match between two sequences (the higher the score or the lower the probability value, the greater the identity between two sequences).
The programs described above for the assembly and analysis of full length polynucleotide and polypeptide sequences were also used to identify polynucleotide sequence fragments from SEQ ID
N0:41-80. Fragments from about 20 to about 4000 nucleotides which are useful in hybridization and amplification technologies are described in Table 4, column 2.
IV. Identification and Editing of Coding Sequences from Genomic DNA
Putative protein modification and maintenance molecules were initially identified by running the Genscan gene identification program against public genomic sequence databases (e.g., gbpri and gbhtg). Genscan is a general-purpose gene identification program which analyzes genomic DNA
sequences from a variety of organisms (Burge, C. and S. Karlin (1997) J. Mol.
Biol. 268:78-94; Burge, C. and S. Karlin (1998) Curr. Opin. Struct. Biol. 8:346-354). The program concatenates predicted exons to form an assembled cDNA sequence extending from a methionine to a stop codon. The output of Genscan is a FASTA database of polynucleotide and polypeptide sequences. The maximum .
range of sequence for Genscan to analyze at once was set to 30 kb. To determine which of these Genscan predicted cDNA sequences encode protein modification and maintenance molecules, the encoded polypeptides were analyzed by querying against PFAM models for protein modification and, maintenance molecules. Potential protein modification and maintenance molecules were also identified by homology to Incyte cDNA sequences that had been annotated as protein modification and maintenance molecules. These selected Genscan-predicted sequences were then compared by BLAST analysis to the genpept and gbpri public databases. Where necessary, the Genscan-predicted sequences were then edited by comparison to the top BLAST hit from genpept to correct errors in the sequence predicted by Genscan, such as extra or omitted exons. BLAST analysis was also used to find any Incyte cDNA or public cDNA coverage of the Genscan-predicted sequences, thus providing evidence for transcription. When Incyte cDNA coverage was available, this information was used to correct or confirm the Genscan predicted sequence. Full length polynucleotide sequences were obtained by assembling Genscan-predicted coding sequences with Incyte cDNA
sequences and/or public cDNA sequences using the assembly process described in Example III.
Alternatively, full length polynucleotide sequences were derived entirely from edited or unedited Genscau-predicted coding sequences.
V. Assembly of Genomic Sequence Data with cDNA Sequence Data "Stitched" Sequences Partial cDNA sequences were extended with exons predicted by the Genscan gene identification program described in Example IV. Partial cDNAs assembled as described in Example III were mapped to genomic DNA and parsed into clusters containing related cDNAs and Genscan exon predictions from one or more genomic sequences. Each cluster was analyzed using an algorithm based on graph theory and dynamic programming to integrate cDNA and genomic information, generating possible splice variants that were subsequently confirmed, edited, or extended to create a full length'sequence. Sequence intervals in which the entire length of the interval was present on more than one sequence in the cluster were identified, and intervals thus identified were considered to be equivalent by transitivity. For example, if an interval was present on a cDNA and two genomic sequences, then all three intervals were considered to be equivalent. This process allows unrelated but consecutive genomic sequences to be brought together, bridged by cDNA
sequence. Intervals thus identified were then "stitched" together by the stitching algorithm in the order that they appear along their parent sequences to generate the longest possible sequence, as well as sequence variants.
Linkages between intervals which proceed along one type of parent sequence (cDNA to cDNA or genomic sequence to genomic sequence) were given preference over linkages which change parent type (cDNA to genomic sequence). The resultant stitched sequences were translated and compared by BLAST analysis to the genpept and gbpri public databases. Incorrect exons predicted by Genscan were corrected by comparison to the top BLAST hit from genpept. Sequences were further extended with additional cDNA sequences, or by inspection of genomic DNA, when necessary.
"Stretched" Sequences Partial DNA sequences were extended to full length with an algorithm based on BLAST
analysis. First, partial cDNAs assembled as described in Example III were queried against public databases such as the GenBank primate, rodent, mammalian, vertebrate, and eukaryote databases using the BLAST program. The nearest GenBank protein homolog was then compared by BLAST
analysis to either Incyte cDNA sequences or GenScan exon predicted sequences described in Example IV. A chimeric protein was generated by using the resultant high-scoring segment pairs (HSPs) to map the translated sequences onto the GenBank protein homolog.
Insertions or deletions may occur in the chimeric protein with respect to the original GenBank protein homolog. The GenBank protein homolog, the chimeric protein, or both were used as probes to search for homologous genomic sequences from the public human genome databases. Partial DNA
sequences were therefore "stretched" or extended by the addition of homologous genomic sequences. The resultant stretched sequences were examined to determine whether it contained a complete gene.
VI. Chromosomal Mapping of PMMM Encoding Polynucleotides The sequences which were used to assemble SEQ ID N0:41-80 were compared with sequences from the Incyte LIFESEQ database and public domain databases using BLAST and other implementations of the Smith-Waterman algorithm. Sequences from these databases that matched SEQ ID N0:41-80 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 Iustitute for Genome Research (WIGR), and Genethon were used to determine if any of the clustered sequences had been previously mapped. Inclusion of a mapped sequence in a cluster resulted in the assignment of all sequences of that cluster, including its particular SEQ ID NO:, to that map location.
Map locations are represented by ranges, or intervals, of human chromosomes.
The map position of an interval, in centiMorgans, is measured relative to the terminus of the chromosome's p-arm. (The centiMorgan (cM) is a unit of measurement based on recombination frequencies between chromosomal markers. On average, 1 cM is roughly equivalent to 1 megabase (Mb) of DNA in humans, although this can vary widely due to hot and cold spots of recombination.) The cM distances are based on genetic markers mapped by Genethon which provide boundaries for radiation hybrid markers whose sequences were includedin 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.nlm.nih.gov/genemapn, can be employed to determine if previously identified disease genes map within or in proximity to the intervals indicated above.
Association of PMMM polynucleotides with Parkinson's Disease Several genes have been identified as showing linkage to autosomal dominant forms of Parkinson's Disease (PD). PD is a common neurodegenerative disorder causing bradykinesia, resting tremor, muscular rigidity, and postural instability. Cytoplasmic eosinophilic inclusions called Lewy bodies, and neuronal loss especially in the substantia nigra pats compacta, are pathological hallmarks of PD (Valente, E.M. et al (2001) Am. J. Hum. Genet. 68:895-900). Lewy body Parkinson disease has been thought to be a specific autosomal dominant disorder (Wakabayashi, K.
et al. (1998) Acta Neuropath. 96:207-210). Juvenile parkinsonism may be a specific autosomal recessive disorder (Matsumine, H. et al. (1997) Am. J. Hum. Genet. 60: 588-596, 1997). (Online Mendelian Inheritance in Man, OMIM. Johns Hopkins University, Baltimore, MD. MIM Number: 168600:
Sept. 9, 2002: .
World Wide Web URL: http://www.ncbi.nlm.nih.gov/omim/) Association of a disease with a chromosomal locus can be determined by lod score. Lod score is a statistical method used to test the linkage of two or more loci within families having a genetic disease. The lod score is the logarithm to base 10 of the odds in favor of linkage. Linkage is defined as the tendency of two genes located on the same chromosome to be inherited together through meiosis (Gefaetics in Medicine, Fifth Edition, (1991) Thompson, M.W.
et al., W.B. Saunders Co. Philadelphia). A lod score of +3 or greater (1000:1 odds in favor of linkage) indicates a probability of 1 in 1000 that a particular marker was found solely by chance in affected individuals, which is strong evidence that two genetic loci are linked.
One such gene implicated in PD is PARK3, which maps to 2p13 (Gasser, T. et al.
(1998) Nature Genet. 18:262-265). A marker at chromosomal position D2S441 was found to have a lod score of 3.2 in the region of PARK3. This marker supported the disease association of PARK3 in the chromosomal interval from D2S 134 to D2S286 (Gasser et al., supra).
Markers located within chromosomal intervals D2S134 and D2S286, which map.between 83.88 to 94.05 centiMorgans on the, short arm of chromosome 2, were used to identify genes that map in the region between D2S 134 and D2S286.
PMMM polynucleotides were found to map within the chromosomal region in which markers' associated with disease or other physiological processes of interest were located. Genomic contigs available from NCBI were used to identidy PMMM polynucleotides which map to a disease locus.
Contigs longer than 1Mb were broken into subcontigs of 1Mb in length with overlapping sections of 100 kb. A preliminary step used an algorithm, similar to MEGABLAST (NCBI), to identify mRNA
sequence/masked genomic DNA contig pairings. SIM4 (Florea, L. et al. (1998) Genome Res. 8:967-74, version May 2000 was optimized for high throughput and strand assignment confidence, and used to further select cDNA/genomic pairings. The SIM4-selected mRNA
sequence/genomic contig pairs were further processed to determine the correct location of the P1~~VIM
polynucleotides on the genomic contig and their strand identity.
SEQ >D NO:43 mapped to GBI:NT_005428_001.7 from the February 2002 Genbank release covering a 9.65 Mb region of the genome that also contains PD-associated genetic markers D2S134 and D2S286. The maximum distance between SEQ ID NO:43 and markers D2S134 and D2S286, therefore, is 9.65 Mb. Thus, SEQ ID NO:43 is in proximity with genetic markers shown to consistently associate with PD. In various embodiments, SEQ )D N0:43 can be used for one or more of the following: i) linkage analysis of persons andlor families to the PD
disease region at 2q12-q22, ii) diagnostic assays for osteoarthritis and interleukin expression abnormalities, and iii) developing therapeutics andlor other treatments for PD.
VII. Analysis of Polynucleotide Expression Northern analysis is a laboratory technique used to detect the presence of a transcript of a gene and involves the hybridization of a labeled nucleotide sequence to a membrane on which RNAs from a particular cell type or tissue have been bound (Sambrook and Russell, supra, ch. 7; Ausubel et al., supra, ch. 4).
Analogous computer techniques applying BLAST were used to search for identical or related molecules in databases such as GenBank or LIFESBQ (Incyte Genomics). This analysis is much faster than multiple membrane-based hybridizations. In addition, the sensitivity of the computer search can be modified to determine whether any particular match is categorized as exact or similar. The basis of the search is the product score, which is defined as:
BLAST Score x Percent Identity 5 x minimum f length(Seq. 1), length(Seq. 2)}
The product score takes into account both the degree of similarity between two sequences 'and the length of the sequence match. The product score is a normalized value between 0 and 100, and is .
calculated as follows: the BLAST score is multiplied by the percent nucleotide identity and the product is divided by (5 times the length of the shorter of the two sequences). The BLAST score is calculated by assigning a score of +5 for every base that matches in a high-scoring segment pair (HSP), and -4 for every mismatch. Two sequences may share more than one HSP
(separated by gaps). If there is more than one HSP, then the pair with the highest BLAST
score is used to calculate the product score. The product score represents a balance between fractional overlap and quality in a BLAST alignment. For example, a product score of 100 is produced only for 100%
identity over the entire length of the shorter of the two sequences being compared. A product score of 70 is produced either by 100% identity and 70% overlap at one end, or by 88% identity and 100% overlap at the other. A product score of 50 is produced either by 100% identity and 50%
overlap at one end, or 79%
identity and 100% overlap.
Alternatively, polynucleotides encoding PMMM 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; hernic and immune system; liver; musculoskeletal system; nervous system; pancreas;
respiratory system; sense organs; skin; stomatognathic system;
unclassified/mixed; or urinary tract.
The number of libraries in each category is counted and divided by the total number of libraries across all categories. Similarly, each human tissue is classified into one of the following disease/condition categories: cancer, cell line, developmental, inflammation, neurological, trauma, cardiovascular, pooled, and other, and the number of libraries in each category is counted and divided by the total number of libraries across all categories. The resulting percentages reflect the tissue-and disease-specific expression of cDNA encoding PMMM. cDNA sequences and cDNA library/tissue information are found in the LIFESEQ GOLD database (Incyte Genomics, Palo Alto CA).
VIII. Extension of PMMM Encoding Polynucleotides Full length polynucleotides are produced by extension of an appropriate fragment of the full length molecule using oligonucleotide primers designed from this fragment. One primer was synthesized to initiate 5' extension of the known fragment, and the other primer was synthesized to initiate 3' extension of the known fragment. The initial primers were designed using OLIGO 4.06 software (National Biosciences), or another appropriate program, to be about 22 to 30 nucleotides in .
length, to have a GC content of about 50% or more, and to anneal to the target sequence at temperatures of about 68 °C to about 72 °C. Any stretch of nucleotides which would result in hairpin structures and primer-primer dimeri2ations was avoided.
Selected human cDNA libraries were used to extend the sequence. If more than one extension was necessary or desired, additional or nested sets of primers were designed.
High fidelity amplification was obtained by PCR using methods well known in the art. PCR
was performed in 96-well plates using the PTC-200 thermal cycler (MJ Research, Inc.). The reaction mix contained DNA template, 200 nmol of each primer, reaction buffer containing Mg2+, (NH~)ZSO4, and 2-mercaptoethanol, Taq DNA polymerase (Amersham Biosciences), ELONGASE
enzyme (Invitrogen), and Pfu DNA polymerase (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°C, 2 min; Step 5: Steps 2, 3, and 4 repeated 20 times; Step 6: 68°C, 5 min;
Step 7: storage at 4°C. In the alternative, the parameters for primer pair T7 and SK+ were as follows: Step 1: 94°C, 3 min; Step 2:

94°C, 15 sec; Step 3: 57°C, 1 min; Step 4: 68°C, 2 min;
Step 5: Steps 2, 3, and 4 repeated 20 times;
Step 6: 68°C, 5 min; Step 7: storage at 4°C.
The concentration of DNA in each well was determined by dispensing 100 ~,l PICOGREEN
quantitation reagent (0.25% (v/v) PICOGREEN; Molecular Probes, Eugene OR) dissolved in 1X TE
and 0.5 ~,1 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 /.d to 10 ~1 aliquot of the reaction mixture was analyzed by electrophoresis on a 1 % agarose gel to determine which reactions were successful in extending the sequence.
The extended nucleotides were desalted and concentrated, transferred to 384-well plates, digested with CviJI cholera virus endonuclease (Molecular Biology Research, Madison WI), and sonicated or sheared prior to religation into pUC 18 vector (Amersham Biosciences). For shotgun sequencing, the digested nucleotides were separated on low concentration (0.6 to 0.8%) agarose gels, fragments were excised, and agar digested with Agar ACE (Promega). Extended clones were religated using T4 ligase (New England Biolabs, Beverly MA) into pUC 18 vector (Amersham Biosciences), treated with Pfu DNA polymerase (Stratagene) to fill in restriction site overhangs, and transfected into competent E. coli cells: Transformed cells were selected on antibiotic-containing media, and individual colonies were picked and cultured overnight at 37 °C in 384-well plates in LB/2x carb liquid media.
The cells were lysed, and DNA was amplified by PCR using Taq DNA polymerase (Amersham Biosciences) 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, vlv), and sequenced using DYENAMIC energy transfer sequencing primers and the DYENAMIC DIRECT kit (Amersham Biosciences) or the ABI PRISM BIGDYE
Terminator cycle sequencing ready reaction kit (Applied Biosystems).
In like manner, full length polynucleotides are verified using the above procedure or are used to obtain 5' regulatory sequences using the above procedure along with oligonucleotides designed for such extension, and an appropriate genomic library.

IX. Identification of Single Nucleotide Polymorphisms in PMMM Encoding Polynucleotides Common DNA sequence variants known as single nucleotide polymorphisms (SNPs) were identified in SEQ ID N0:41-80 using the L1FESEQ database (Iucyte Genomics).
Sequences from the same gene were clustered together and assembled as described in Example III, allowing the identification of all sequence variants in the gene. An algorithm consisting of a series of filters was used to distinguish SNPs from other sequence variants. Preliminary filters removed the majority of basecall errors by requiring a minimum Phred quality score of 15, and removed sequence alignment errors and errors resulting from improper trimming of vector sequences, chimeras, and splice variants.
An automated procedure of advanced chromosome analysis analysed the original chromatogram files in the vicinity of the putative SNP. Clone error filters used statistically generated algorithms to identify errors introduced during laboratory processing, such as those caused by reverse transcriptase, polymerase, or somatic mutation. Clustering error filters used statistically generated algorithms to identify errors resulting from clustering of close homologs or pseudogenes, or due to contamination by non-human sequences. A final set of filters removed duplicates and SNPs found in immunoglobulins or T-cell receptors.
Certain SNPs were selected for further characterization by mass spectrometry using the high throughput MASSARRAY system (Sequenom, Inc.) to analyze allele frequencies at the SNP sites in four different human populations. The Caucasian population comprised 92 individuals (46 male, 46 female), including 83 from Utah, four French, three Venezualan, and two Amish individuals. The African population comprised 194 individuals (97 male, 97 female), all African Americans. The Hispanic population comprised 324 individuals (162 male, 162 female), all Mexican Hispanic. The Asian population comprised 126 individuals (64 male, 62 female) with a reported parental breakdown of 43% Chinese, 31% Japanese, 13% Korean, 5% Vietnamese, and 8% other Asian.
Allele frequencies were first analyzed in the Caucasian population; in some cases those SNPs which showed no allelic variance in this population were not further tested in the other three populations.
X. Labeling and LTse of Individual Hybridization Probes Hybridization probes derived from SEQ ID NO:41-80 are employed to screen cDNAs, genomic DNAs, or mRNAs. Although the labeling of oligonucleotides, consisting of about 20 base pairs, is specifically described, essentially the same procedure is used with larger nucleotide fragments. Oligonucleotides are designed using state-of the-art software such as OLIGO 4.06 software (National Biosciences) and labeled by combining 50 pmol of each oligomer, 250 ,uCi of ~~ 32P, adenosine triphosphate (Amersham Biosciences), and T4 polynucleotide kinase (DuPont NEN, BostoJn MA). The labeled oligonucleotides are substantially purified using a superfine size exclusion dextran bead column (Amersham Biosciences). An aliquot containing 10' counts per minute of the labeled probe is used in a typical membrane-based hybridization analysis of human genomic DNA digested with one of the following endonucleases: Ase I, Bgl II, Eco RI, Pst I, Xba I, or Pvu II (DuPont NEN).
The DNA from each digest is fractionated on a 0.7% agarose gel and transferred to nylon membranes (Nytran Plus, Schleicher & Schuell, Durham NH). Hybridization is carried out for 16 hours at 40 °C. To remove nonspecific signals, blots are sequentially washed at room temperature under conditions of up to, for example, 0.1 x saline sodium citrate and 0.5%
sodium dodecyl sulfate.
Hybridization patterns are visualized using autoradiography or an alternative imaging means and compared.
XI. 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 et al., 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, M., ed.
(1999) DNA Microarrays: A Practical Apt~roach, Oxford University Press, London). Suggested substrates include silicon, silica, glass sfides, glass chips, and silicon wafers. Alternatively, a procedure analogous to a dot or slot blot may also be used to arrange and link elements to the surface of a substrate using thermal, UV, chemical, or mechanical bonding procedures. A
typical array may be produced using available methods and machines well known to those of ordinary skill in the art and may contain any appropriate number of elements (Schena, M. et al. (1995) Science 270:467-470;
Shalom D. et al. (1996) Genome Res. 6:639-645; Marshall, A. and J. Hodgson (1998) Nat. Biotechuol.
16:27-31 ).
Full length cDNAs, Expressed Sequence Tags (ESTs), or fragments or oligomers thereof may comprise the elements of the microarray. Fragments or oligomers suitable for hybridization can be selected using software well known in the art such as LASERGENE software (DNASTAR). The array elements are hybridized with polynucleotides in a biological sample. The polynucleotides in the biological sample 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 Cell Sample Preparation Total RNA is isolated from tissue samples using the guanidinium thiocyanate method and poly(A)+ RNA is purified using the oligo-(dT) cellulose method. Each poly(A)+
RNA sample is reverse transcribed using MMLV reverse-transcriptase, 0.05 pg/~.1 oligo-(dT) primer (2lmer), 1X first strand buffer, 0.03 units/~.1 RNase inhibitor, 500 ACM dATP, 500 ~.M dGTP, 500 ~,M dTTP, 40 ixM
dCTP, 40 ~,M dCTP-Cy3 (BDS) or dCTP-Cy5 (Amersham Biosciences). The reverse transcription .
reaction is performed in a 25 ml volume containing 200 ng poly(A)+ RNA with GEMBRIGHT kits (Incyte Genomics). Specific control poly(A)+ RNAs are synthesized by in vitf-o transcription from non-coding yeast genomic DNA. After incubation at 37° C for 2 hr, each reaction sample (one with Cy3 and another with Cy5 labeling) is treated with 2.5 ml of 0.5M sodium hydroxide and incubated for 20 minutes at 85° C to the stop the reaction and degrade the RNA.
Samples are purified using two successive CHRQMA SPIN 30 gel filtration spin columns (Clontech, Palo Alto CA) and after combining, both reaction samples are ethanol precipitated using 1 inl of glycogen (1 mg/ml), 60 ml sodium acetate, and 300 ml of 100% ethanol. The sample is then dried to completion using a SpeedVAC (Savant Instruments Inc., Holbrook NY) and resuspended in 14 ~,l 5X
SSC/0.2% SDS.
2o 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 ampliftcation uses primers complementary to the vector sequences flanking the cDNA insert. Array elements are amplified in thirty cycles of PCR from an initial quantity of 1-2 ng to a final quantity greater than 5 ~,g.
Amplified array elements are then purified using SEPHACRYL-400 (Amersham Biosciences).
Purified array elements are immobilized on polymer-coated glass slides. Glass microscope slides (Corning) are cleaned by ultrasound in 0.1 % SDS and acetone, with extensive distilled water washes between and after treatments. Glass slides are etched in 4%
hydrofluoric acid (VWR
Scientific Products Corporation (VWR), West Chester PA), washed extensively in distilled water, and coated with 0.05% aminopropyl silane (Sigma). in 95% ethanol. Coated slides are cured in a 110°C
oven.
l07 Array elements are applied to the coated glass substrate using a procedure described in U.S.
Patent No. 5,807,522, incorporated herein by reference. 1 ~.1 of the array element DNA, at an average concentration of 100 ng/~,1, is loaded into the open capillary printing element by a high-speed robotic apparatus. The apparatus then deposits about 5 n1 of array element sample per slide.
Microarrays are UV-crosslinked using a STRATALINKER UV-crosslinker (Stratagene).
Microarrays are washed at room temperature once in 0.2% SDS and three times in distilled water.
Non-specific binding sites are blocked by incubation of microarrays in 0.2%
casein in phosphate buffered saline (PBS) (Tropix, Inc., Bedford MA) for 30 minutes at 60°
C followed by washes in 0.2%
SDS and distilled water as before.
1o Hybridization Hybridization reactions contain 9 ~Cl of sample mixture consisting of 0.2 p.g each of Cy3 and Cy5 labeled cDNA synthesis products in 5X SSC, 0.2% SDS hybridization buffer.
The sample mixture is heated to 65° C for 5 minutes and is aliquoted onto the microarray surface and covered with an 1.8 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 .. ~1 of 5X SSC in a corner of the chamber. The chamber containing the arrays is incubated for about 6.5 hours at 60° C. The arrays are washed for 10 min at 45° C in a first wash buffer (1X SSC, 0.1 %
SDS), three times for 10 minutes each at 45° C in a second wash buffer (0.1X SSC), and dried.
Detection Reporter-labeled hybridization complexes are detected with a microscope equipped with an Iunova 70 mixed gas 10 W laser (Coherent, Inc., Santa Clara CA) capable of generating spectral lines at 488 nm for excitation of Cy3 and at 632 mn for excitation of CyS. The excitation laser light is focused on the array using a 20X microscope objective (Nikon, Inc., Melville NY). The slide containing the array is placed on a computer-controlled X-Y stage on the microscope and raster-scanned past the objective. The 1.8 cm x 1.8 cm array used in the present example is scanned with a resolution of 20 micrometers.
In two separate scans, a mixed gas multiline laser excites the two fiuorophores 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 fiuorophores used are 565 nn1 for Cy3 and 650 nm for CyS. Eaeh 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 1o adding identical amounts of each to the hybridization mixture.
The output of the photomultiplier tube is digitized using a 12-bit RTI-835H
analog-to-digital (A/D) conversion board (Analog Devices Inc., Norwood MA) installed in an IBM-compatible PC
computer. The digitized data are displayed as an image where the signal intensity is mapped using a .
linear 20-color transformation to a pseudocolor scale ranging from blue (low signal) to red (high signal). The data is also analyzed quantitatively. Where two different fluorophores are excited and measured simultaneously, the data are first corrected for optical 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 Genomics). Array elements that exhibit at least about a two-fold change in expression, a signal-to-background ratio of at least about 2.5, and an element spot size of at least about 40%, are considered to be differentially expressed.
Expression For example, SEQ ll~ N0:41, SEQ D? NO:55, and SEQ ID NO:57 showed differential expression in association with breast cancer, as determined by microarray analysis. The expression of SEQ LD N0:41 was decreased by at least 2.8-fold in breast tumor when matched with normal tissue from the same donor. The tumorous breast tissue was obtained from tumor from the right breast of a 43-year-old female with invasive lobular carcinoma in situ. The tumor was well differentiated and metastatic to two out of 13 lymph nodes. Normal tissue was obtained from grossly uninvolved breast tissue from the same donor. Matched normal and tumorigenic breast tissue samples were provided by the Huntsman Cancer Institute (Salt Lake City, ITT). In addition, the expression of SEQ ID N0:55 in several tumor cell lines representing various stages of breast tumor progression was compared with that in the non-malignant mammary epithelial cell line, MCF-10A. The expression of SEQ )D N0:55 from six tumor cell lines (MCF7, T-47D, Sk-BR-3, BT20, MDA-mb-231, and MDA-mb-435S) was compared with that in MCF-10A cells grown in the supplier's recommended medium or grown in defined serum-free H14 medium to 70-80°~o confluence prior to comparison. MCF-10A is a breast mammary gland (luminal ductal characteristics) cell line that was isolated from a 36-year-old woman with fibrocystic breast disease. MCF-10A expresses cytoplasmic keratins, epithelial sialomucins, and milkfat globule antigens. This cell line exhibits three-dimensional growth in collagen and forms domes in confluent culture. MCF7 is a nonmalignant breast adenocarcinoma cell line isolated from the pleural effusion of a 69-year-old female. MCF7 has retained characteristics of the mammary epithelium such as the ability to process estradiol via cytoplasmic estrogen receptors and the capacity to form domes in culture. T-47D is a breast carcinoma cell line isolated from a pleural effusion obtained from a 54-year-old female with an infiltrating ductal carcinoma of the breast. Sk-BR-3 is a breast adenocarcinoma cell line isolated from a malignant pleural effusion. of a 43-year-old female. It forms poorly differentiated adenocarcinoma when injected into nude mice. BT-20 is a breast carcinoma cell line derived in vitro from cells emigrating out of thin slices of the tumor mass isolated from a 74-year-old female. MDA-mb-231 is a breast tumor cell line isolated from the pleural effusion of a 51-year old female. It forms poorly differentiated adenocarcinoma in nude mice and ALS treated BALB/c mice. It also expresses the Wnt3 oncogene, EGF, and TGF-a. MDA-mb-435S
is a spindle shaped strain that evolved from the parent line (435) as isolated in 1976 by R. Cailleau from the pleural effusion of a 31-year-old female with metastatic, ductal adenocarcinoma of the breast. The expression of SEQ ID N0:55 was increased by at least two-fold in T-47D and BT-20 cells as compared to the nonmalignant MCF-10A cells. In addition, the expression of SEQ
ID NO:57 in the six breast cell tumor lines described above was compared to that in a primary breast epithelial cell line derived from a normal donor, HMEC. The expression of SEQ ll~ N0:57 was increased by at least two-fold in MCF7, T-47D, Sk-BR-3, BT-20, and MDA-mb-435S cells as compared to HMEC cells.
Therefore, in various embodiments, SEQ ID N0:41, SEQ ID NO:55, and SEQ ID
N0:57 can be used for one or more of the following: i) monitoring treatment of breast cancer, ii) diagnostic assays for breast cancer, and iii) developing therapeutics and/or other treatments for breast cancer.
In another example, SEQ D7 N0:41-42, SEQ ID N0:46, SEQ ID N0:49 and SEQ ID
NO:78 showed differential expression in association with lung cancer, as determined by microarray analysis.

The expression of SEQ m N0:41 was decreased at least 2.4-fold in one of five lung squamous cell carcinoma when matched with normal tissue from the same donor. The tumorous lung tissue was obtained from lung squamous cells of a 66-year-old male. Normal tissue was obtained from grossly uninvolved lung tissue from the same donor. Matched normal and tumorigenic lung tissue samples were provided by the Roy Castle Lung Cancer Foundation (Liverpool, UK). In addition, the expression of SEQ ~ N0:42 was increased at least 2.9-fold in one of four lung adenocarcinoma when matched with normal tissue from the same donor. The expression of SEQ D7 N0:42 also was increased at least two-fold in one of five lung squamous cell carcinoma when matched with normal tissue from the same donor. The tumorous lung tissue was obtained from lung squamous cells of a 73-year-old male. Normal tissue was obtained from grossly uninvolved lung tissue from the same donor.
In a separate matched tissue experiment, the expression of SEQ ID N0:46 was decreased by at least two-fold in lung squamous cell carcinoma tissue, comprising 60% overt tumor cells. In addition, the expression of SEQ m N0:49 was decreased by at least two-fold in the lung tumor tissue as compared to normal lung tissue from the same donor. -In a separate matched tissue experiment, the expression of SEQ ID N0:78 was increased' more than two-fold in lung squamous cell carcinoma tissue, with 70% overt tumor cells, from a 75-year-old female, as compared to grossly uninvolved tissue from the same donor. Therefore, in various embodiments, SEQ ID N0:41-42, SEQ D7 NO:46, SEQ ID N0:49 and SEQ D7 N0:78 can be used for one or more of the following: i) monitoring treatment of lung cancer, ii) diagnostic assays for lung cancer, and iii) developing therapeutics and/or other treatments for lung cancer.
In a further example, SEQ ID NO: 41 showed differential expression in association with obesity, as determined by microarray analysis. The expression of SEQ m N0:41 was decreased at least 2.3-fold, to as much as 11-fold, in treated human adipocytes from obese and normal donors when compared to non-treated adipocytes from the same donors. The normal human primary subcutaneous preadipocytes were isolated from adipose tissue of a 28-year-old healthy female with a body mass index (BMI) of 23.59. The obese human primary subcutaneous preadipocytes were isolated from adipose tissue of a 40-year-old healthy female with a body mass index (BMI) of 32.47. The preadipocytes were cultured and induced to differentiate into adipocytes by culturing them in the differentiation medium containing the active components, PPAR-'y agonist and human insulin. Human preadipocytes were treated with human insulin and PPAR-'y agonist for three days and subsequently were switched to medium containing insulin for 24 hours, 48 hours, four days, 1.1, 2.1, or 2.6 weeks before the cells were collected for analysis. Differentiated adipocytes were compared to untreated preadipocytes maintained in culture in the absence of inducing agents. Between 80% and 90% of the preadipocytes finally differentiated to adipocytes as observed under phase contrast microscope.
In the alternative, obese human primary subcutaneous preadipocytes were isolated from adipose tissue of a 36-year-old healthy female with a body mass index (BMI) of 27.7. The preadipocytes were cultured and induced to differentiate into adipocytes by culturing them in a proprietary differentiation medium containing active components such as PPAR-y agonist and human insulin. Human preadipocytes were treated with human insulin and PPAP-'y agonist for 3 days and subsequently switched to medium containing insulin only for 5, 9, and 12 more days. Differentiated adipocytes were compared to untreated preadipocytes maintained in culture in the absence of inducing agents. An overall differentiation rate of more than 60% was observed after 15 days in culture.
Therefore, in various embodiments, SEQ ID N0:41 can be used for one or more of the following: i) monitoring treatment of diabetes mellitus and other disorders, such as obesity, hypertension, and atherosclerosis, ii) diagnostic assays for diabetes mellitus and other disorders, such as obesity, hypertension, and atherosclerosis, and iii) developing therapeutics and/or other treatments for diabetes mellitus and other disorders, such as obesity, hypertension, and atherosclerosis.
Matched normal and obese preadipocyte samples were provided by the (Zen-Bio, Research Triangle Park NC).
In an alternative example, SEQ m NO:63 showed differential expression in association with colon cancer, as determined by microarray analysis. The expression of SEQ ID
N0:63 was decreased by at least two-fold in sigmoid colon tumor tissue when matched with normal tissue from the same donor. Tumorous tissue was obtained from a 48-year-old female with sigmoid colon tumor originating from a metastatic gastric sarcoma (stromal tumor). Normal tissue was obtained from grossly uninvolved sigmoid colon tissue from the same donor. Matched normal and tumorigenic sigmoid colon tissue samples are provided by the Huntsman Cancer Institute, (Salt Lake City, UT).
Therefore, in various embodiments, SEQ ID N0:63 can be used for one or more of the following: i) monitoring treatment of colon cancer, ii) diagnostic assays for colon cancer, and iii) developing therapeutics and/or other treatments for colon cancer.
In another example, SEQ ID NO:78 showed differential expression in association with ovarian cancer, as determined by microarray analysis. The expression of SEQ ID N0:78 was increased by at least 2-fold in ovarian tumor tissue as compared to normal tissue from the same donor, a 79-year-old female donor with ovarian adenocarcinoma. Matched normal and tumorigenic ovarian tissue samples were provided by the Huntsman Cancer Institute, (Salt Lake City, UT).
Therefore, in various embodiments, SEQ ID N0:78 can be used for one or more of the following: i) monitoring treatment of ovarian cancer, ii) diagnostic assays for ovarian cancer, and iii) developing therapeutics and/or other treatments for ovarian cancer.
XII. Complementary Polynucleotides Sequences complementary to the PNNIIVVIM-encoding sequences, or any parts thereof, are used to detect, decrease, or inhibit expression of naturally occurring PN>ZVIM.
Although use of oligonucleotides comprising from about 15 to 30 base pairs is described, essentially the same procedure is used with smaller or with larger sequence fragments. Appropriate oligonucleotides are designed using OLIGO 4.06 software (National Biosciences) and the coding sequence of PIV>ZVIM.
To inhibit transcription, a complementary oligonucleotide is designed from the most unique 5' sequence and used to prevent promoter binding to the coding sequence. To inhibit translation, a complementary oligonucleotide is designed to prevent ribosomal binding to the PM1V>IVI-encoding transcript.
XIII. Expression of PMMM
Expression and purification of PNBVIM is achieved using bacterial or virus-based expression systems. For expression of PMIVIA~I in bacteria, cDNA is subcloned into an appropriate vector containing an antibiotic resistance gene and an inducible promoter that directs high levels of cDNA
transcription. Examples of such promoters include, but are not limited to, the trp-hac (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 PM1V>NI upon induction with isopropyl beta-D-thiogalactopyranoside (IPTG). Expression of PA~VIM in eukaryotic cells is achieved by infecting insect or mammalian cell lines with recombinant Autogr-aphica cal iforwica nuclear polyhedrosis virus (AcMNPV), commonly known as baculovirus. The nonessential polyhedrin gene of baculovirus is replaced with cDNA encoding Pl~~VIM 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 Spoeloptera frugiper-da (Sf9) insect cells in most cases, or human hepatocytes, in some cases.
Infection of the latter requires additional genetic modifications to baculovirus (Engelhard, E.K. et al.
(1994) Proc. Natl. Acad. Sci. USA 91:3224-3227; Sandig, V. et al. (1996) Hum.
Gene Ther. 7:1937-1945).
In most expression systems, PI~~llVIM 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 Schistosorna japonicum, enables the purification of fusion proteins on immobilized glutathione under conditions that maintain protein activity and antigenicity (Amersham Biosciences).
Following purification, the GST moiety can be proteolytically cleaved from PMIVhVI at specifically engineered sites. FLAG, an 8-amino acid peptide, enables immunoaf~nity purification using commercially available monoclonal and polyclonal anti-FLAG antibodies (Eastman Kodak). 6-His, a stretch of six consecutive histidine residues, enables purification on metal-chelate resins (QIAGEN).
Methods for protein expression and purification are discussed in Ausubel et al. (supra, ch. 10 and 16).
Purified PMMM obtained by these methods can be used directly in the assays shown in Examples XVII, XVIJI, XIX, and XX, where applicable.
XIV. Functional Assays PMMM function is assessed by expressing the sequences encoding PMMM at physiologically elevated levels in mammalian cell culture systems. cDNA is subcloned into a mammalian expression vector containing a strong promoter that drives.high levels of cDNA
expression. Vectors of choice include PCMV SPORT plasmid (Invitrogen, Carlsbad CA) and PCR3.1 plasmid (Invitrogen), both of which contain the cytomegalovirus promoter. 5-10 ~cg of recombinant vector are transiently transfected into a human cell line, for example, an endothelial or hematopoietic cell line, using either liposome .formulations or electroporation. 1-2 ~g of an additional plasmid containing sequences encoding a marker protein are co-transfected. Expression. of a marker protein provides a means to distinguish transfected cells from nontransfected cells and, is a reliable predictor of cDNA expression from the recombinant vector. Marker proteins of choice include, e.g., Green Fluorescent Protein (GFP; Clontech), CD64, or a CD64-GFP fusion protein. Flow cytometry (FCM), an automated, laser optics based technique, is used to identify transfected cells expressing GFP
or CD64-GFP and to evaluate the apoptotic state of the cells and other cellular properties. FCM
detects and quantifies the uptake of fluorescent molecules that diagnose events preceding or coincident with cell death. These events include changes in nuclear DNA content as measured by staining of DNA
with propidium iodide; changes in cell size and granularity as measured by forward light scatter and 90 degree side light scatter; down-regulation of DNA synthesis as measured by decrease in bromodeoxyuridine uptake; alterations in expression of cell surface and intracellular proteins as measured by reactivity with specific antibodies; and alterations in plasma membrane composition as measured by the binding of fluorescein-conjugated Annexin V protein to the cell surface. Methods in flow cytometry are discussed in Ormerod, M.G. (1994; Flow Cytometry, Oxford, New York NY).

The influence of PNIIVIM on gene expression can be assessed using highly purified populations of cells transfected with sequences encoding PMMM 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 PMMM and other genes of interest can be analyzed by northern analysis or microarray techniques.
XV. Production of PMMM Specific Antibodies PNNIIVVIM substantially purified using polyacrylamide gel electrophoresis (PAGE; see, e.g., Harrington, M.G. (1990) Methods Enzymol. 182:488-495), or other purification techniques, is used to immunize animals (e.g., rabbits, mice, etc.) and to produce antibodies using standard protocols.
Alternatively, the PMMM amino acid sequence is analyzed using LASERGENE
software (DNASTAR) to determine regions of high immunogenicity, and a corresponding oligopeptide is synthesized and used to raise antibodies by means known to those of: skill in the art. Methods for selection of appropriate epitopes, such as those near the C-terminus or in hydrophilic regions are well described in the art (Ausubel et al., supra, ch. 11).
Typically, oligopeptides of about 15 residues in length are synthesized using an ABI 431A
peptide synthesizer (Applied Biosystems) using FMOC chemistry and coupled to KLH (Sigma-Aldrich, St. Louis MO) by reaction with N-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS) to increase immunogenicity (Ausubel et al., supra). Rabbits are immunized with the oligopeptide-KLH
complex in complete Freund's adjuvant. Resulting antisera are tested for antipeptide and anti-PNIIVIM
activity by, for example, binding the peptide or PMMM to a substrate, blocking with 1 % BSA, reacting with rabbit antisera, washing, and reacting with radio-iodinated goat anti-rabbit IgG.
XVI. Purification of Naturally Occurring PMMM Using Specific Antibodies Naturally occurring or recombinant PMMM is substantially purified by immunoaffinity chromatography using antibodies specific for PNIMM. An immunoaffinity column is constructed by covalently coupling anti-PI\~VIM antibody to an activated chromatographic resin, such as CNBr-activated SEPHAROSE (Amersham Biosciences). After the coupling, the resin is blocked and washed according to the manufacturer's instructions.
Media containing PMMMM are passed over the imrnunoaffinity column, and the column is washed under conditions that allow the preferential absorbance of PMMM (e.g., high ionic strength buffers in the presence of detergent). The column is eluted under conditions that disrupt antibody/PMMM 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 PMMM is collected.
XVII. Identification of Molecules Which Interact with PMMM
PMMM, or biologically active fragments thereof, are labeled with 1'~I Bolton-Hunter reagent (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 PMMM, washed, and any wells with labeled PMMM complex are assayed. Data obtained using different concentrations of PMMM are used to calculate values for the number, affinity, and association of PMMM with the candidate molecules.
Alternatively, molecules interacting with PMMM are analyzed using the yeast two hybrid system as described in Fields, S. and O. Song (1989; Nature 340:245-246), or using commercially available kits based on the two-hybrid system, such as the MATCHMAKER system (Clontech).
PMMM 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).
XVIII. Demonstration of PMMM Activity PMMM activity can be demonstrated using a generic immunoblotting strategy or through a variety of specific activity assays, some of which are outlined below. As a general approach, cell lines or tissues transformed with a vector containing PMMM coding sequences can be assayed for PNNnVVIM
activity by immunoblotting. Transformed cells are denatured in SDS in the presence of b-mercaptoethanol, nucleic acids are removed by ethanol precipitation, and proteins are purified by acetone precipitation. Pellets are resuspended in 20 mM Tris buffer at pH 7.5 and incubated with Protein G-Sepharose pre-coated with an antibody specific for PM1VINI. After washing, the Sepharose beads are boiled in electrophoresis sample buffer, and the eluted proteins subjected to SDS-PAGE.
The SDS-PAGE is transferred to a membrane for immunoblotting, and the PMMM
activity is assessed by visualizing and quantifying bands on the blot using the antibody specific for P1~RVIM as the primary antibody and 1~I-labeled IgG specific for the primary antibody as the secondary antibody.
PMMM kinase activity is measured by quantifying the phosphorylation of a protein substrate by PMMM in the presence of gamma-labeled 3aP-ATP. PMIVINI is incubated with the protein substrate, 32P-ATP, and an appropriate kinase buffer. The 32P incorporated into the substrate is separated from free 32P-ATP by electrophoresis and the incorporated 32P is counted using a radioisotope counter. The amount of incorporated 3aP is proportional to the activity of PMMM. A
determination of the specific amino acid residue phosphorylated is made by phosphoamino acid analysis of the hydrolyzed protein.
PMMM phosphatase activity is measured by the hydrolysis of p-nitrophenyl phosphate (PNPP). PMMM is incubated together with PNPP in HEPES buffer, pH 7.5, in the presence of 0.1 % (i-mercaptoethanol at 37 °C for 60 min. The reaction is stopped by the addition of 6 ml of 10 N
NaOH and the increase in light absorbance at 410 nm resulting from the hydrolysis of PNPP is measured using a spectrophotometer. The increase in light absorbance is proportional to the activity of PMMM in the assay (Diamond, R.H. et al. (1994) Mol. Cell. Biol. 14:3752-3762).
In the alternative, PMMM phosphatase activity is determined by measuring the amount of phosphate removed from a phosphorylated protein substrate. Reactions are performed. with 2 or 4 nM
enzyme in a final volume of 30 ~.1 containing 60 mM Tris, pH 7.6, 1 mM EDTA, 1 mM EGTA, 0.1 %
2-mercaptoethanol and 10 ~,M substrate, 32P-labeled on serine/threonine or tyrosine, as appropriate.
Reactions are initiated with substrate and incubated at 30° C for 10-15 min. Reactions are quenched with 450 p,1 of 4% (w/v) activated charcoal in 0.6 M HCl, 90 mM Na4P20~, and 2 mM NaH2P04, then centrifuged at 12,000 x g for 5 min. Acid-soluble 32Pi is quantified by liquid scintillation counting (Sinclair, C. et al. (1999) J. Biol. Chem. 274:23666-23672).
PMMM protease activity is measured by the hydrolysis of appropriate synthetic peptide substrates conjugated with various chromogenic molecules in which the degree of hydrolysis is quantified by spectrophotometric (or fluorometric) absorption of the released chromophore (Beynon, R.J. and J.S. Bond (1994) Proteol 'c Enzymes: A Practical Approach, Oxford University Press, New York, NY, pp. 25-55). Peptide substrates are designed according to the category of protease activity as endopeptidase (serine, cysteine, aspartic proteases, or metalloproteases), aminopeptidase (leucine aminopeptidase), or carboxypeptidase (carboxypeptidases A and B, procollagen C-proteinase).
Commonly used chromogens are 2-naphthylamine, 4-nitroaniline, and furylacrylic acid. Assays are performed at ambient temperature and contain an aliquot of the enzyme and the appropriate substrate in a suitable buffer. Reactions are carried out in an optical cuvette, and the 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.
In the alternative, an assay for PMMM 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 PMMM 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 PMMM, the substrate is cleaved, and the two fluorescent proteins dissociate. This is accompanied by a marked decrease in energy transfer which is quantified by compering the emission spectra before and after the addition of PI~~VIM (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 PMMM is introduced on an inducible vector so that FRET can be monitored in the presence and absence of l0 PM)VIM (Sagot, I. et al (1999) FEBS Letters 447:53-57).
An assay for ubiquitin hydrolase activity measures the hydrolysis of a ubiquitin precursor. The assay is performed at ambient temperature and contains an aliquot of Pn~VIM
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, I~. et al. (1997) Anal. Biochem. 247:305-309).
PMMM protease inhibitor activity for alpha 2-HS-glycoprotein (AHSG) can be measured as a decrease in osteogenic activity in dexamethasone-treated rat bone marrow cell cultures (dex-RBMC). .
Assays are carried out in 96-well culture plates containing minimal essential medium supplemented with 15°7o fetal bovine serum, ascorbic acid (50 mg/ml), antibiotics (100 mg/ml penicillin G, 50 mg/ml gentamicin, 0.3 mg/ml fungjzone), 10 mM B-glycerophosphate, dexamethasone (10-8 M) and various concentrations of PMMM for 12-14 days. Mineralized tissue formation in the cultures is quantified by measuring the absorbance at 525 rim using a 96-well plate reader (Binkert, C.
et al. (1999) J. Biol.
Chem. 274:28514-28520}.
PMMM protease inhibitor activity for inter-alpha-trypsin inhibitor (ITI) can be measured by a continuous spectrophotometric rate determination of trypsin activity. The assay is performed at ambient temperature in a quartz cuvette in pH 7.6 assay buffer containing 63 mM sodium phosphate, 0.23 mM N a-benzoyle-L-arginine ethyl ester, 0.06 mM hydrochloric acid, 100 units trypsin, and various concentrations of Pte. Tmmediately after mixing by inversion, the increase in Aa53 "", is recorded for approximately 5 minutes and the enzyme activity is calculated (Bergmeyer, H.U. et al.
(1974) Meth. Enzym. Anal. 1:515-516).
Pl~~VIM isomerase activity such as peptidyl prolyl cisltr-atvs isomerase activity can be assayed by au enzyme assay described by Rahfeld, J.U., et al. (1994; FEBS Lett.
352:180-184). The assay is performed at 10°C in 35 mM HEPES buffer, pH 7.8, containing chymotrypsin (0.5 mg/ml) and PMMM at a variety of concentrations. Under these assay conditions, the substrate, Suc-Ala-Xaa-Pro-Phe-4-NA, is in equilibrium with respect to the prolyl bond, with 80-95%
in tr~ans and 5-20% in cis conformation. An aliquot (2 ml) of the substrate dissolved in dimethyl sulfoxide (10 mg/ml) is added to the reaction mixture described above. Only the cis isomer of the substrate is a substrate for cleavage by chymotrypsin. Thus, as the substrate is isomerized by PMMM, the product is cleaved by chymotrypsin to produce 4-nitroanilide, which is detected by it's absorbance at 390 rim. 4-nitroanilide appears in a time-dependent and a PMMM concentration-dependent manner.
PMMM galactosyltransferase activity can be determined by measuring the trausfer of to radiolabeled galactose from UDP-galactose to a GlcNAc-terminated oligosaccharide chain (Kolbinger, F. et al. (1998) J. Biol. Chem. 273:58-65). The sample is incubated with 14 ~.l of assay stock solution (180 mM sodium cacodylate, pH 6.5, 1 mg/ml bovine serum albumin, 0.26 mM UDP-galactose, 2 ~.l of UDP-[3H]galactose), 1 ~,1 of MnCl2 (500 mM), and 2.5 p1 of GlcNAc(3O-(CHz)$
COZMe (37 mg/ml in dimethyl sulfoxide) for 60 minutes at 37 °C. The reaction is quenched by the addition of 1 ml of water and loaded on a C18 Sep-Pak cartridge (Waters), and the column is washed twice with 5 ml of water to remove unreacted UDP-['H]galactose. The [3H]galactosylated GlcNAc(30-(CHZ)8 COzMe remains bound to the column during the water washes and is eluted with 5 ml of methanol. Radioactivity in the eluted material is measured by liquid scintillation counting and is proportional to galactosyltxansferase activity in the starting sample.
PMLVBVI induction by heat or toxins may be demonstrated using primary cultures of human fibroblasts or human cell lines such as CCL-13, HEK293, or HEP G2 (ATCC). To heat induce PMMM expression, aliquots of cells are incubated at 42°C for 15, 30, or 60 minutes. Control aliquots are incubated at 37°C for the same time periods. To induce PMMM
expression by toxins, aliquots of cells are treated with 100 ~.M arsenite or 20 mM azetidine-2-carboxylic acid for 0, 3, 6, or 12 hours.
After exposure to heat, arsenite, or the amino acid analogue, samples of the treated cells are harvested and cell lysates prepared for analysis by western blot. Cells are lysed in lysis buffer containing 1% Nonidet P-40, 0.15 M NaCl, 50 mM Tris-HCl, 5 mM EDTA, 2 mM N-ethylmaleimide, 2 mM phenylmethylsulfonyl fluoride, 1 mg/ml leupeptin, and 1 mg/ml pepstatin.
Twenty micrograms of the cell lysate is separated on an 8% SDS-PAGE gel and transferred to a membrane. After blocking with 5% nonfat dry milk/phosphate buffered saline for 1 h, the membrane is incubated overnight at 4°C or at room temperature for 2-4 hours with an appropriate dilution of anti-PMMM serum in 2%
nonfat dry milk/phosphate-buffered saline. The membrane is then washed and incubated with a 1:1000 dilution of horseradish peroxidase-conjugated goat anti-rabbit IgG in 2~/o dry milk/phosphate-buffered saline. After washing with 0.1% Tween 20 in phosphate-buffered saline, the PMMM protein is detected and compared to controls using chemiluminescence.
PNIIVIM lysyl hydroxylase activity is determined by measuring the production of hydroxy[14C]lysine from [14C]lysine. Radiolabeled protocollagen is incubated with PMMM in buffer containing ascorbic acid, iron sulfate, dithiothreitol, bovine serum albumin, and catalase. Following a 30 minute incubation, the reaction is stopped by the addition of acetone, and centrifuged. The sedimented material is dried, and the hydroxy[14C]lysine is converted to [14C]formaldehyde by oxidation with periodate, and then extracted into toluene. The amount of 14C
extracted into toluene is quantified by scintillation counting, and is proportional to the activity of PMMM in the sample (Kivirikko, K., and R. Myllyla (1982) Methods Enzymol. 82:245-304).
XIX. Identification of PMMM Substrates Phage display libraries can be used to identify optimal substrate sequences for PMIVIIbI. A
random hexamer followed by a linker and a known antibody epitope is cloned as an N-terminal extension of gene III in a filamentous phage library. Gene III codes for a coat protein, and the epitope will be displayed on the surface of each phage particle. The library is incubated with PMMM under proteolytic conditions so that the epitope will be removed if the hexamer codes for a PMMM cleavage site. An antibody that recognizes the epitope is added along with immobilized protein A. TJncleaved phage, which still bear the epitope, are removed by centrifugation: Phage in the supernatant are then amplified and undergo several more rounds of screening. Individual phage clones are then isolated and sequenced. Reaction kinetics for these peptide substrates can be studied using an assay in Example X~, and an optimal cleavage sequence can be derived (Ke, S.H. et al.
(1997) J. Biol.
Chem. 272:16603-16609).
To screen for itt vivo PMMM substrates, this method can be expanded to screen a cDNA
expression library displayed on the surface of phage particles (T7SELECT10-3 Phage display vector, Novagen, Madison, WI) or yeast cells (pYD1 yeast display vector kit, Invitrogen, Carlsbad, CA). In this case, entire cDNAs are fused between Gene DI and the appropriate epitope.
XX. Identification of PMMM 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 XVIII. PMMM activity is measured for each well and the ability of each compound to inhibit PMMM

activity can be determined, as well as the dose-response kinetics. This assay could also be used to identify molecules which enhance PMIVEVI activity.
In the alternative, phage display libraries can be used to screen for peptide PMMM inhibitors.
Candidates are found among peptides which bind tightly to a protease. In this case, multi-well plate wells are coated with PMIVI~~I and incubated with a random peptide phage display library or a cyclic peptide library (Koivunen, E. et al. (1999) Nature Biotech 17:768-774).
Unbound phage are washed away and selected phage amplified and rescreened for several more rounds.
Candidates are tested for PMMM inhibitory activity using an assay described in Example XVIII.
Various modifications and variations of the described compositions, 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. It will be appreciated that the invention provides novel and useful proteins, and their encoding polynucleotides, which can be used in the drug discovery process, as well as methods for using these compositions for the detection, diagnosis, and treatment of diseases and conditions.
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.
Nor should the description of such embodiments be considered exhaustive or limit the invention to the precise forms disclosed. Furthermore, elements from one embodiment can be readily recombined with elements from one or more other embodiments. Such combinations can form a number of embodiments within the scope of the invention. It is intended that the scope of the invention be defined by the following claims and their equivalents.

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N ~ N vo d~ ~ ~, ~n ~p ~ ~ ~ ~
M ~
V

~ ~ ~O ~ O ~ ~
~ O ~ V1 ~ d' N d' ~ ~
0 ~ ~ ~ ~ ~ ~ O ~
N ' ~
N N
N -.r O
N v0 ~ r .
,-y ,-i N M ~ ,-~ ,-i ,-y ~O Ov O~ N
N M ~l. ~p l~ , N N N N N , , , O N d' Vi v0 Vi cW O "'' ~ O due. t~ ~ ~ v~
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~O OW ~ O oo N
Ov O Ov N

N ,-~ N ,~ N ,--rN N yt ~r7 ~n ~ ,-; O ~
N ~Y O ~n 0 \p ,~.~ 00 ~ pp r 0 ~ Vl ~ ~ ~O pp V~ O M 1n ~ l0 M ~
' ue V7 M ~O t~ d 00 ~ ~t ~ M ~ \O ~O ~ O~ ~
d~ N V7 O O r r r r ~ d ~ O ' ~ N
O ~

N . N oo Ov N v0 p N r oo -, O ~ N ~ N O M ,-i ~ W O
N N N N N N '-' N ~ r, ,~ ~ ~ ~ ~ t~ ~ ~
N dw0 N
' O ~t v0 OW O Ov p~ vO oo ~ ~ d- t~
N oo ,~ O wO M ~t d' d 00 t~ M ~ M ~--i M ~ V'1 ~ O~ M N ~ O N
V'7 O ~O V~ l~ M d' V~ ~ ~ ON1 ~ ~
0 ~

N ~ M ,w0 00 0o in M
N n l .-r N
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~ N
N ~NOO N
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O
N

i!~ o ~ ~~M~ o O~ ~ ~' ~ N c~1 ~n ~
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N
N

N N N ~ Cy Ov ~ . N r., dy0 t~ t~
~ ~ oo Ov N ~ M
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00 00 M v0 V'7 in ~t ~ ~''~ V7 v0 O V7 V7 N l~ ~ N M l~ ~ ~ cn V'7 ~ N Ov lp ' ~ 00 M O~
'd' ~ M ~ V7 O -W M
V~ V7 V'1 ~ -~O OW ~ N 00 N O . ~-~ N O~ CV N f1 N 01 t~ ~D ~
V~ d' N O 00 ,...., .~ "' O~ 0 N ~ N ~ N ~ N r ~ O ~ r r r r M_ ~ O~ ~O 'd' ~ ~ N .~
r r r r r O ~ ~O 00 ~ 0 M

N ~ ~ ~ ~ cmn N ~ ~ ~

U ~~ ~ N ~ N ~ ~ N -r ~ 01 M M Oi .-i O V) ~ N ~ ~ ~O N
'"' ' ' '' ' N N r ONNOOVOVD ~~~"''N
N~ 0 N ~ "-' "' O ~ r N" ~n d' Ov Ov ,-i ~n r N"
N"
d i N
N
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W O W v0 ~ oo O ~ v0 .-r N
N 0o N ~ N Ov N ~--i d- N o0 N N wt N W O ~ M ~ ~ ~ ~
N ~ N ~-~ N -v N v0 t~ N
N

N
U

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w ~ ~ ' o a 2os Table 5 PolynucleotideIncyte ProjectRepresentative Library SEQ ID:
ID NO:

42 7500511CB BONEUNROl 56 7504774CB LIVRNOTOl 57 7503166CB BRACNOI~02 64 7506567CB I~IDNNOT 19 67 7505765CB OVARTUTOl cC ~ ~ ~ 'C3 'C O ~ .U h '~" "° r' °' ~ ''-~' v '~ '> ~ a~ _:
.b o v ~ '~. ~ 'b ' ~~ ~ ~ a~ b ~ ~.
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cd ~ .ty U v.. ~ '~ ~ O '-~ ~ .
,.'~o ~ b '~ o ° ~ ~ ~ ~ w .o ~ ~ ~ E.., o ~ c~ ~
> ~ ~ ~s 3 ° ~w ° ~ ~~,~~a ~ ~~~ ~z ~ ,~ .o U o°0 0 0 ~ o .~ ~ o ~ ~ ~ o ~ ~
O ~ O O '~' a3 .fl b ~'' N ~ O ~ .;~ y'' 'd ~ C~
b N pp Z' ~ ~ >c~' ~~ ~ ~'' c~ ~ '~ ~ 'b ~O N
b-0 .~»~r w~U NU
a~ ~ o ° a~ '~ .~ ~ ° ~ 4. ~ a, ~ v~
o a~
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~N ~' U N ~ O ~ M ~ t1 .O ~ U O .b~L1 P-~ .C N O
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d cn O ~ ~ ~~ .~ N .~-.'' ~ N O ~ ~ ~ .~~ ~" .cC O c~
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OO ~ ,.~n, N ~ N ~ ~ ~ ~O d- N ~ U ~ 'C V ~ ' N N
w .rte. w .f, b0 ~.,' ~ ~ t"' ~ ~' ~p d. ~ N ~ vwn O cUn cG ~ _O .d. O O O ~ N b N c~C
p> .~ '+i ;~ U U ."~~, U ~" O ~ at >> cc3 ~ c~G ~ ~ ~ "..' I~- '.C.", .U~-a~ on ,.d p. ~ o '~ o~,n -d > °' '~' ° d" ~ ~ ~ U >
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<110> INCYTE GENOMICS, INC.
RAMKUMAR, Jayalaxmi GORVAD, Ann E.
BAUGHN, Mariah R.
EMERLING, Brooke M.
YANG, Junming LEE, Soo Yeun TRAN, Uyen K.
BECHA, Shanya D.
DUGGAN, Brendan M.
LEE, Ernestine A.
GRIFFIN, Jennifer A.
LI, Joana X.
SPRAGUE, William W.
HAFALIA, April J.A.
CHAWLA, Narinder K.
LEHR-MASON, Patricia M.
KABLE, Amy E.
YUE, Henry MARQUIS Joseph P.
YAO, Monique G.
RICHARDSON, Thomas W.
TANG, Y. Tom JIN, Pei CHIEN, David BHATIA, Umesh G.
BURRILL, John D.
LEE, Sally BLAKE, Julie J.
HO, Anne ZHENG, Wenjin <120> PROTEIN MODIFICATION AND MAINTENANCE PROTEINS
<130> PF-1237 PCT
<140> To Be Assigned <141> Herewith <150> US 60/329,689 <151> 2001-10-12 <150> US 60/335,703 <151> 2001-10-25 <150> US 60/348,887 <151> 2001-11-09 <150> US 60/334,145 <151> 2001-11-28 <150> US 60/337,451 <151> 2001-12-06 <150> US 60/340,584 <151> 2001-12-14 <160> 80 <170> PERL Program <210> 1 <211> 270 <212> PRT
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte ID No: 7500287CD1 <400> 1 Met His Gly Ser Cys Ser Phe Leu Met Leu Leu Leu Pro Leu Leu Leu Leu Leu Val Ala Thr Thr Gly Pro Val Gly Ala Leu Thr Asp Glu Glu Lys Arg Leu Met Val Glu Leu His Asn Leu Tyr Arg Ala Gln Val Ser Pro Pro Ala Ser Asp Met Leu His Met Arg Trp Asp Glu Glu Leu Ala Ala Phe Ala Lys Ala Tyr Ala Arg Gln Cys Val Trp Gly His Asn Lys Glu Arg Gly Arg Arg Gly Glu Asn Leu Phe Ala Ile Thr Asp Glu Gly Met Asp Val Pro Leu Ala Met Glu Glu Trp His His Glu Arg Glu His Tyr Asn Leu Ser Ala Ala Thr Cys Ser Pro Gly Gln Met Cys Gly His Tyr Thr Gln Val Val Trp Ala Lys Thr Glu Arg Ile Gly Cys Gly Ser His Phe Cys Glu Lys Leu Gln Gly Val Glu Glu Thr Asn Ile Glu Leu Leu Val Cys Asn Tyr Glu Pro Pro Gly Asn Val Lys Gly Lys Arg Pro Tyr Gln Glu Gly Thr Pro Cys Ser Gln Cys Pro Ser Gly Tyr His Cys Lys Asn Ser Leu Cys Glu Pro Ile Gly Ser Pro Glu Asp Ala Gln Asp Leu Pro Tyr Leu Val Thr Glu Ala Pro Ser Phe Arg Ala Thr Glu Ala Ser Asp Ser Arg Lys Met Gly Ala Glu Gly Pro Asp Lys Pro Ser Val Val Ser Gly Leu Asn Ser Gly Pro Gly His Val Trp Gly Pro Leu Leu Gly Leu Leu Leu Leu Pro Pro Leu Val Leu Ala Gly Ile Phe <210> 2 <211> 288 <212> PRT
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte ID No: 7500511CD1 <400> 2 Met Trp Gly Leu Lys Val Leu Leu Leu Pro Val Val Ser Phe Ala Leu Tyr Pro Glu Glu Ile Leu Asp Thr His Trp Glu Leu Trp Lys Lys Thr His Arg Lys Gln Tyr Asn Asn Lys Thr Ser Glu Glu Val Val Gln Lys Met Thr Gly Leu Lys Val Pro Leu Ser His Ser Arg Ser Asn Asp Thr Leu Tyr Ile Pro Glu Trp Glu Gly Arg Ala Pro Asp Ser Val Asp Tyr Arg Lys Lys Gly Tyr Val Thr Pro Val Lys Asn Gln Gly Gln Cys Gly Ser Cys Trp Ala Phe Ser Ser Val Gly Ala Leu Glu Gly Gln Leu Lys Lys Lys Thr Gly Lys Leu Leu Asn Leu Ser Pro Gln Asn Leu Val Asp Cys Val Ser Glu Asn Asp Gly Cys Gly Gly Gly Tyr Met Thr Asn Ala Phe Gln Tyr Val Gln Lys Asn Arg Gly Ile Asp Ser Glu Asp Ala Tyr Pro Tyr Val Gly Gln Glu Glu Ser Cys Met Tyr Asn Pro Thr Gly Lys Ala Ala Lys Cys Arg Gly Tyr Arg Glu Ile Pro Glu Gly Asn Glu Lys Ala Leu Lys Arg Ala Val Ala Arg Val Gly Pro Val Ser Val Ala Ile Asp Ala Ser Leu Thr Ser Phe Gln Phe Tyr Ser Lys Gly Val Tyr Tyr Asp Glu Ser Cys Asn Ser Asp Asn Leu Asn His Ala Val Leu Ala Val Gly Tyr Gly Ile Gln Lys Gly Asn Lys His Trp Ile Ile Lys Asn Ser Trp Gly Glu Asn Trp Gly Asn Lys Gly Tyr Ile Leu Met Ala Arg Asn Lys Asn Asn Ala Cys Gly Ile Ala Asn Leu Ala Ser Phe Pro Lys Met <210> 3 <211> 608 <212> PRT
<213> Homo Sapiens <220>
<221> mist-feature <223> Incyte ID No: 7500273CD1 <400> 3 Met Arg Pro Val Ser Val Trp Gln Trp Ser Pro Trp Gly Leu Leu Leu Cys Leu Leu Cys Ser Ser Cys Leu Gly Ser Pro Ser Pro Ser Thr Gly Pro Glu Lys Lys Ala Gly Ser Gln Gly Leu Arg Phe Arg Leu Ala Gly Phe Pro Arg Lys Pro Tyr Glu Gly Arg Val Glu I1e Gln Arg Ala Gly Glu Trp Gly Thr Ile Cys Asp Asp Asp Phe Thr Leu Gln Ala Ala His Ile Leu Cys Arg Glu Leu Gly Phe Thr Glu Ala Thr Gly Trp Thr His Ser Ala Lys Tyr Gly Pro Gly Thr Gly Arg Ile Trp Leu Asp Asn Leu Ser Cys Ser Gly Thr Glu Gln Ser Val Thr Glu Cys Ala Ser Arg Gly Trp Gly Asn Ser Asp Cys Thr His Asp Glu Asp Ala Gly Val Ile Cys Lys Asp Gln Arg Leu Pro Gly Phe Ser Asp Ser Asn Val Ile Glu Ala Arg Val Arg Leu Lys Gly Gly Ala His Pro Gly Glu Gly Arg Val Glu Val Leu Lys Ala Ser Thr Trp Gly Thr Val Cys Asp Arg Lys Trp Asp Leu His Ala Ala Ser Val Val Cys Arg Glu Leu Gly Phe Gly Ser Ala Arg Glu Ala Leu Ser Gly Ala Arg Met Gly Gln Gly Met Gly Ala Ile His Leu Ser Glu Val Arg Cys Ser Gly Gln Glu Leu Ser Leu Trp Lys Cys Pro His Lys Asn Ile Thr Ala Glu Asp Cys Ser His Ser Gln Asp Ala G1y Val Arg Cys Asn Leu Pro Tyr Thr Gly Ala Glu Thr Arg Ile Arg Leu Ser Gly Gly Arg Ser Gln His Glu Gly Arg Val Glu Val Gln Ile Gly Gly Pro Gly Pro Leu Arg Trp Gly Leu Ile Cys Gly Asp Asp Trp Gly Thr Leu Glu Ala Met Val Ala Cys Arg Gln Leu Gly Leu Gly Tyr Ala Asn His Gly Leu Gln Glu Thr Trp Tyr Trp Asp Ser Gly Asn Ile Thr Glu Va1 Val Met Ser Gly Val Arg Cys Thr Gly Thr Glu Leu Ser Leu Asp Gln Cys Ala His His Gly Thr His Ile Thr Cys Lys Arg Thr Gly Thr Arg Phe Thr Ala Gly Val Ile Cys Ser G1u Thr Ala Ser Asp Leu Leu Leu His Ser Ala Leu Val Gln G1u Thr Ala Tyr Ile Glu Asp Arg Pro Leu His Met Leu Tyr Cys Ala Ala Glu Glu Asn Cys Leu Ala Ser Ser Ala Arg Ser Ala Asn Trp Pro Tyr Gly His Arg Arg Leu Leu Arg Phe Ser Ser Gln Ile His Asn Leu Gly Arg Ala Asp Phe Arg Pro Lys Ala Gly Arg His Ser Trp Val Trp His Glu Cys His Gly His Tyr His Ser Met Asp Ile Phe Thr His Tyr Asp Ile Leu Thr Pro Asn Gly Thr Lys Val Ala Glu Gly His Lys Ala Ser Phe Cys Leu Glu 485 . 490 ~ 495 Asp Thr Glu Cys Gln Glu Asp Val Ser Lys Arg Tyr Glu Cys Ala Asn Phe Gly Glu Gln Gly Ile Thr Val Gly Cys Trp Asp Leu Tyr Arg His Asp Ile Asp Cys Gln Trp Ile Asp Ile Thr Asp Val Lys Pro Gly Asn Tyr Ile Leu Gln Val Val Ile Asn Pro Asn Phe Glu Val Ala Glu Ser Asp Phe Thr Asn Asn Ala Met Lys Cys Asn Cys Lys Tyr Asp Gly His Arg Ile Trp Val His Asn Cys His Ile Gly Asp Ala Phe Ser Glu Glu Ala Asn Arg Arg Phe Glu Arg Tyr Pro Gly Gln Thr Ser Asn Gln Ile I'le <210> 4 <211> 218 <212> PRT
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte ID No: 7500183CD1 <400> 4 Met Trp Val Pro Val Val Phe Leu Thr Leu Ser Val Thr Trp Ile Gly Ala Ala Pro Leu Ile Leu Ser Arg Ile Val Gly Gly Trp Glu Cys Glu Lys His Ser Gln Pro Trp Gln Val Leu Val Ala Ser Arg Gly Arg Ala Val Cys Gly Gly Val Leu Val His Pro Gln Trp Val Leu Thr Ala Ala His Cys Ile Arg Lys Pro Gly Asp Asp Ser Ser His Asp Leu Met Leu Leu Arg Leu Ser Glu Pro Ala Glu Leu Thr Asp Ala Val Lys Val Met Asp Leu Pro Thr Gln Glu Pro Ala Leu g5 100 105 Gly Thr Thr Cys Tyr Ala Ser Gly Trp Gly Ser Ile Glu Pro Glu 110 115 ' 120 Glu Phe Leu Thr Pro Lys Lys Leu Gln Cys Val Asp Leu His Val Ile Ser Asn Asp Val Cys Ala Gln Val His Pro Gln Lys Val Thr Lys Phe Met Leu Cys Ala Gly Arg Trp Thr Gly Gly Lys Ser Thr Cys Ser Gly Asp Ser Gly Gly Pro Leu Val Cys Asn Gly Val Leu Gln Gly Ile Thr Ser Trp Gly Ser Glu Pro Cys Ala Leu Pro Glu Arg Pro Ser Leu Tyr Thr Lys Val Val His Tyr Arg Lys Trp Ile Lys Asp Thr Ile Val Ala Asn Pro <210> 5 <211> 172 <212> PRT
<213> Homo sapiens <220>
<221> misc_feature <223> Incyte ID No: 7499957CD1 <400> 5 Met Ala Glu Leu Thr Ala Leu Glu Ser Leu Ile Glu Met Gly Phe Pro Arg Gly Arg Ala Glu Lys Ala Leu Ala Leu Thr Gly Asn Gln Gly Ile Glu Ala Ala Met Asp Trp Leu Met Glu His Glu Asp Asp Pro Asp Val Asp Glu Pro Leu Glu Thr Pro Leu Gly His Ile Leu Gly Arg Glu Pro Thr Ser Ser Glu Pro Gly Pro Val Pro Ser Ser Pro Ser Gln Glu Pro Pro Thr Lys Arg Glu Tyr Asp Gln Cys Arg Ile Gln Val Arg Leu Pro Asp Gly Thr Ser Leu Thr Gln Thr Phe Arg Ala Arg Glu Gln Leu Ala Ala Val Arg Leu Tyr Val Glu Leu His Arg Gly Glu Glu Leu Gly Gly Gly Gln Asp Pro Val Gln Leu Leu Ser Gly Phe Pro Arg Arg Ala Phe Ser Glu Ala Asp Met Glu Arg Pro Leu Gln Glu Leu Gly Leu Val Pro Ser Ala Val Leu Ile Val Ala Lys Lys Cys Pro Ser <210> 6 <211> 831 <212> PRT
<213> Homo sapiens <220>
<221> misc_feature <223> Incyte ID No: 7500001CD1 ~400> 6 Met Ala Ala Ala Met Glu Thr Glu Gln Leu Gly Val Glu Ile Phe 1 ~ 5 10 15 Glu Thr Ala Asp Cys Glu Glu Asn Ile Glu Ser Gln Asp Arg Pro Lys Leu Glu Pro Phe Tyr Val Glu Arg Tyr Ser Trp Ser Gln Leu Lys Lys Leu Leu Ala Asp Thr Arg Lys Tyr His Gly Tyr Met Met Ala Lys Ala Pro His Asp Phe Met Phe Val Lys Arg Asn Asp Pro Asp Gly Pro His Ser Asp Arg Ile Tyr Tyr Leu Ala Met Ser Gly Glu Asn Arg Glu Asn Thr Leu Phe Tyr Ser Glu Ile Pro Lys Thr 95 l00 105 Ile Asn Arg Ala Ala Val Leu Met Leu Ser Trp Lys Pro Leu Leu Asp Leu Phe Gln Ala Thr Leu Asp Tyr Gly Met Tyr Ser Arg Glu Glu Glu Leu Leu Arg Glu Arg Lys Arg Ile Gly Thr Val Gly Ile Ala Ser Tyr Asp Tyr His Gln Gly Ser Gly Thr Phe Leu Phe Gln Ala Gly Ser Gly Ile Tyr His Val Lys Asp Gly Gly Pro Gln Gly Phe Thr Gln Gln Pro Leu Arg Pro Asn Leu Val Glu Thr Ser Cys Pro Asn Ile Arg Met Asp Pro Lys Leu Cys Pro Ala Asp Pro Asp Trp Ile Ala Phe Ile His Ser Asn Asp Ile Trp Ile Ser Asn Ile Val Thr Arg Glu Glu Arg Arg Leu Thr Tyr Val His Asn Glu Leu Ala Asn Met Glu Glu Asp Ala Arg Ser Ala Gly Val Ala Thr Phe Val Leu Gln Glu Glu Phe Asp Arg Tyr Ser Gly Tyr Trp Trp Cys Pro Lys Ala Glu Thr Thr Pro 5er Gly Gly Lys Ile Leu Arg Ile Leu Tyr Glu Glu Asn Asp Glu Ser Glu Val Glu Ile Ile His Val Thr Ser Pro Met Leu Glu Thr Arg Arg Ala Asp Ser Phe Arg Tyr Pro Lys Thr Gly Thr Ala Asn Pro Lys Val Thr Phe Lys Met Ser G1u Ile Met Ile Asp Ala Glu Gly Arg Ile Ile Asp Val Ile Asp Lys Glu Leu Ile Gln Pro Phe Glu Ile Leu Phe Glu Gly Val Glu Tyr Ile Ala Arg Ala Gly Trp Thr Pro Glu Gly Lys Tyr Ala Trp Ser Ile Leu Leu Asp Arg Ser Gln Thr Arg Leu Gln Ile Val Leu Ile Ser Pro Glu Leu Phe Ile Pro Val Glu Asp Asp Val Met Glu Arg Gln Arg Leu Ile Glu Ser Val Pro Asp Ser Val Thr Pro Leu Ile Ile Tyr Glu Glu Thr Thr Asp Ile Trp Ile Asn Ile His Asp Ile Phe His Val Phe Pro Gln Ser His Glu G1u Glu Ile Glu Phe Ile Phe Ala Ser Glu Cys Lys Thr Gly Phe Arg His Leu Tyr Lys Ile Thr Ser Ile Leu Lys Glu Ser Lys Tyr Lys Arg Ser Ser Gly Gly Leu Pro Ala Pro Ser Asp Phe Lys Cys Pro Ile Lys Glu Glu Ile Ala Ile Thr Ser Gly Glu Trp Glu Val Leu Gly Arg His Gly Ser Asn Ile Gln Val Asp Glu Val Arg Arg Leu Val Tyr Phe Glu Gly Thr Lys Asp Ser Pro Leu GTu His His Leu Tyr Val Val Ser Tyr Val Asn Pro Gly Glu Val Thr Arg Leu Thr Asp Arg Gly Tyr Ser His Ser Cys Cys Ile Ser Gln His Cys Asp Phe Phe Ile Ser Lys Tyr Ser Asn Gln Lys Asn Pro His Cys Val Ser Leu Tyr Lys Leu Ser Ser Pro Glu Asp Asp Pro Thr Cys Lys Thr Lys Glu Phe Trp Ala Thr Ile Leu Asp Ser Ala Gly Pro Leu Pro Asp Tyr Thr Pro Pro Glu Ile Phe Ser Phe Glu Ser Thr Thr Gly Phe Thr Leu Tyr Gly Met Leu Tyr Lys Pro His Asp Leu Gln Pro Gly Lys Lys Tyr Pro Thr Val Leu Phe Ile Tyr Gly Gly Pro Gln Val Gln Leu Val Asn Asn Arg Phe Lys Gly Val Lys Tyr Phe Arg Leu Asn Thr Leu Ala Ser Leu Gly Tyr Val Val Val Val Ile Asp Asn Arg Gly Ser Cys His Arg Gly Leu Lys Phe Glu Gly Ala Phe Lys Tyr Lys Met Val Ala Ile Ala Gly Ala Pro Val Thr Leu Trp Ile Phe Tyr Asp Thr Gly Tyr Thr Glu Arg Tyr Met Gly His Pro Asp Gln Asn Glu Gln Gly Tyr Tyr Leu Gly Ser Val Ala Met Gln Ala Glu Lys Phe Pro Ser Glu Pro Asn Arg Leu Leu Leu Leu His Gly Phe Leu Asp Glu Asn Val His Phe Ala His Thr Ser Ile Leu Leu Ser Phe Leu Val Arg Ala Gly Lys Pro Tyr Asp Leu Gln Ile Tyr Pro Gln Glu Arg His Ser Ile Arg Val Pro Glu Ser Gly Glu His Tyr Glu Leu His Leu Leu His Tyr Leu Gln Glu Asn Leu Gly Ser Arg Ile g15 820 825 Ala Ala Leu Lys Val Ile <210> 7 <211> 936 <212> PRT
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte ID No: 7503245CD1 <400> 7 Met Thr Leu Leu Ala Pro Trp Tyr Thr Gly Pro Met Ile Pro Met Asp Val Asn Glu Pro Ser Ser Val Thr Thr Ala Pro Thr Leu Ser Ser Ser Leu Gln His Ile Ser Ser Phe Leu Ala Thr Gly Lys Lys Leu Ser'Leu His Phe Gly His Pro Arg Glu Cys Glu Val Thr Arg Ile Asp Asp Lys Asn Arg Arg Gly Leu Glu Asp Ser Glu Pro Gly Ala Lys Leu Phe Asn Asn Asp Gly Val Cys Cys Cys Leu Gln Lys Arg Gly Pro Val Asn Ile Thr Ser Val Cys Val Ser Pro Arg Thr g5 100 105 Leu Gln Ile Ser Val Phe Val Leu Ser Glu Lys Tyr Glu Gly Ile Val Lys Phe Glu Ser Asp Glu Leu Pro Phe Gly Val Ile Gly Ser Asn Ile Gly Asp Ala His Phe Gln Glu Phe Arg Ala Gly Ile Ser Trp Lys Pro Val Val Asp Pro Asp Asp Pro Ile Pro Gln Phe Pro Asp Cys Cys Ser Ser Ser Ser Ser Arg Ile Pro Ser Val Ser Val Leu Val Ala Val Pro Leu Val Ala Gly His Lys Gly Gln Ala Phe Ile Glu Arg Met Leu Gly Cys Phe Lys Glu Leu Lys Gln Glu Leu Thr Gln Glu Gly Pro Gly Gly Gly His Pro Arg Ser Ala Trp Pro Pro Arg Arg His Ala Gln Trp Pro Pro Glu Pro Cys Glu Gln Gly Glu Glu Pro Pro Pro Val Glu Ala Glu Glu Val Glu Glu Ala Glu Thr Ala Glu Lys Ala Glu Arg Lys Val Glu Ala Glu Ala Lys Val Glu Gly Lys Ala Glu Ala Ala Gly Lys Ala Glu Ala Ala Gly Lys Val Asp Ala Thr Glu Lys Val Glu Thr Ala Gly Lys Val Asp Ala Ala Gly Lys Val Glu Thr Ala Glu Gly Pro Gly Arg Arg Ala Glu Leu Lys Leu Glu Pro Glu Pro Glu Pro Va1 Arg Glu Ala Glu Gln Glu Pro Lys Gln Glu Leu Glu Asp Glu Asn Pro Ala Arg Ser Gly Gly Gly Gly Asn Ser Asp Glu Val Pro Pro Pro Thr Leu Pro Ser Asp Pro Pro Arg Pro Pro Asp Pro Ser Pro Arg Arg Ser Arg Ala Pro Arg Arg Arg Pro Arg Pro Arg Pro Gln Thr Arg Leu Arg Thr Pro Pro Gln Pro Arg Pro Arg Pro Pro Pro Arg Pro Arg Pro Arg 395 ~ 400 405 Arg Gly Pro Gly Gly Gly Cys Leu Asp Val Asp Phe Ala Val Gly Pro Pro Gly Cys Ser His Val Asn Ser Phe Lys Val Gly Glu Asn Trp Arg Gln Glu Leu Arg Val Ile Tyr Gln Cys Phe Val Trp Cys Gly Thr Pro Glu Thr Arg Lys Ser Lys Ala Lys Ser Cys Ile Cys His Val Cys Gly Thr His Leu Asn Arg Leu His Ser Cys Leu Ser Cys Val Phe Phe Gly Cys Phe Thr Glu Lys His Ile His Glu His Ala Glu Thr Lys Gln His Asn Leu Ala Val Asp Leu Tyr Tyr Gly Gly Ile Tyr Cys Phe Met Cys Lys Asp Tyr Val Tyr Asp Lys Asp Ile Glu Gln Ile Ala Lys Glu Glu Gln Gly Glu Ala Leu Lys Leu Gln Ala Ser Thr Ser Thr Glu Val Ser His Gln Gln Cys Ser Val 545 550 ~ 555 Pro Gly Leu Gly Glu Lys Phe Pro Thr Trp Glu Thr Thr Lys Pro Glu Leu Glu Leu Leu Gly His Asn Pro Arg Arg Arg Arg Ile Thr Ser Ser Phe Thr Ile Gly Leu Arg Gly Leu Ile Asn Leu Gly Asn Thr Cys Phe Met Asn Cys Ile Val Gln Ala Leu Thr His Thr Pro Ile Leu Arg Asp Phe Phe Leu Ser Asp Arg His Arg Cys Glu Met Pro Ser Pro Glu Leu Cys Leu Val Cys Glu Met Ser Lys Leu Leu His Leu Val Trp Ile His Ala Arg His Leu Ala Gly Tyr Arg Gln Gln Asp Ala His Glu Phe Leu Ile Ala Ala Leu Asp Val Leu His Arg His Cys Lys Gly Asp Asp Val Gly Lys Ala Ala Asn Asn Pro Asn His Cys Asn Cys Ile Ile Asp Gln Ile Phe Thr Gly Gly Leu Gln Ser Asp Val Thr Cys Gln Ala Cys His Gly Val Ser Thr Thr Ile Asp Pro Cys Trp Asp Ile Ser Leu Asp Leu Pro Gly Ser Cys Thr Ser Phe Trp Pro Met Ser Pro Gly Arg Glu Ser Ser Val Asn Gly Glu Ser His Ile Pro Gly Ile Thr Thr Leu Thr Asp Cys Leu Arg Arg Phe Thr Arg Pro Glu His Leu Gly Ser Ser Ala Lys Ile Lys Cys Gly Ser Cys Gln Ser Tyr Gln Glu Ser Thr Lys Gln Leu Thr Met Asn Lys Leu Pro Val Val Ala Cys Phe His Phe Lys Arg Phe Glu His Ser Ala Lys Gln Arg Arg Lys Ile Thr Thr Tyr Ile Ser Phe Pro Leu Glu Leu Asp Met Thr Pro Phe Met Ala Ser Ser Lys Glu Ser Arg Met Asn Gly Gln Leu Gln Leu Pro Thr Asn Ser Gly Asn Asn Glu Asn Lys Tyr Ser Leu Phe Ala Val Val Asn His Gln Gly Thr Leu Glu Ser Gly His Tyr Thr Ser Phe Ile Arg His His Lys Asp Gln Trp Phe Lys Cys Asp Asp A1a Val Ile Thr Lys Ala Ser Ile Lys Asp Val Leu Asp Ser Glu Gly Tyr Leu Leu Phe Tyr His Lys Gln Val Leu Glu His Glu Ser Glu Lys Val Lys Glu Met Asn Thr Gln Ala Tyr <2l0> 8 <211> 456 <212> PRT
<213> Homo sapiens <220>
<221> misc_feature <223> Incyte ID No: 7503237CD1 <400> 8 Met Leu Ser Ser Arg Ala Glu Ala Ala Met Thr Ala Ala Asp Arg Ala Ile Gln Arg Phe Leu Arg Thr Gly Ala Ala Val Arg Tyr Lys Val Met Lys Asn Trp Gly Val Ile Gly Gly Ile Ala Ala Ala Leu Ala Ala Gly Ile Tyr Val Ile Trp Gly Pro Ile Thr Glu Arg Lys Lys Arg Arg Lys Ala Leu Ser Cys Gln Glu Val Thr Asp Asp Glu Val Leu Asp Ala Ser Cys Leu Leu Asp Val Leu Arg Met Tyr Arg Trp Gln Ile Ser Ser Phe Glu Glu Gln Asp Ala His Glu Leu Phe g5 100 105 His Val Ile Thr Ser Ser Leu Glu Asp Glu Arg Asp Arg Gln Pro 110 115 ~.2 0 Arg Val Thr His Leu Phe Asp Val His Ser Leu Glu Gln Gln Ser Glu Ile Thr Pro Lys Gln Ile Thr Cys Arg Thr Arg Gly Ser Pro 1l/76 His Pro Thr Ser Asn His Trp Lys Ser Gln His Leu Phe His Gly Arg Leu Thr Ser Asn Met Val Cys Lys His Cys Glu His Gln Ser Pro Val Arg Phe Asp Thr Phe Asp Ser Leu Ser Leu Ser Ile Pro Ala Ala Thr Trp Gly His Pro Leu Thr Leu Asp His Cys Leu His His Phe Ile Ser Ser Glu Ser Val Arg Asp Val Val Cys Asp Asn Cys Thr Lys Ile Glu Ala Lys Gly Thr Leu Asn Gly Glu Lys Val Glu His Gln Arg Thr Thr Phe Val Lys Gln Leu Lys Leu Gly Lys Leu Pro Gln Cys Leu Cys Ile His Leu Gln Arg Leu Ser Trp Ser Ser His Gly Thr Pro Leu Lys Arg His Glu His Va1 Gln Phe Asn Glu Phe Leu Met Met Asp Ile Tyr Lys Tyr His Leu Leu Gly His Lys Pro Ser Gln His Asn Pro Lys Leu Asn Lys Asn Pro Gly Pro Thr Leu Glu Leu Gln Asp Gly Pro Gly Ala Pro Thr Pro Val Leu Asn Gln Pro Gly Ala Pro Lys Thr Gln Ile Phe Met Asn Gly Ala Cys Ser Pro Ser Leu Leu Pro Thr Leu Ser Ala Pro Met Pro Phe Pro Leu Pro Val Val Pro Asp Tyr Ser Ser Ser Thr Tyr Leu Phe Arg Leu Met Ala Val Val Val His His Gly Asp Met His Ser Gly His Phe Val Thr Tyr Arg Arg Ser Pro Pro Ser Ala Arg Asn Pro Leu Ser Thr Ser Asn Gln Trp Leu Trp Val Ser Asp Asp Thr Val Arg Lys Ala Ser Leu Gln Glu Val Leu Ser Ser Ser Ala Tyr Leu Leu Phe Tyr Glu Arg Val Leu Ser Arg Met Gln His Gln Ser Gln Glu Cys Lys Ser Glu Glu <210> 9 <211> 516 <212> PRT
<213> Homo sapiens <220>
<221> misc_feature <223> Incyte ID No: 7503230CD1 <400> 9 Met Pro Asp Gln Leu Glu Ser Leu Pro Leu Phe Ser Lys Lys Asn Leu Tle Asp Ile Leu A1a Ile Gly Gly Val Gln Lys Leu Lys Gln Ile Ile Asp Val Ile Asp Lys Glu Leu Ile Gln Pro Phe Glu Ile Leu Phe Glu Gly Val Glu Tyr Ile Ala Arg Ala Gly Trp Thr Pro Glu Gly Lys Tyr Ala Trp Ser Ile Leu Leu Asp Arg Ser Gln Thr Arg Leu Gln Ile Val Leu Ile Ser Pro Glu Leu Phe Ile Pro Val Glu Asp Asp Val Met Glu Arg Gln Arg Leu Ile Glu Ser Val Pro Asp Ser Val Thr Pro Leu Ile Ile Tyr Glu Glu Thr Thr Asp Ile Trp Ile Asn Ile His Asp Ile Phe His Val Phe Pro Gln Ser His Glu Glu Glu Ile Glu Phe Ile Phe Ala Ser Glu Cys Lys Thr Gly Phe Arg His Leu Tyr Lys Ile Thr 5er Ile Leu Lys Glu Ser Lys 155 160 l65 Tyr Lys Arg Ser Ser Gly Gly Leu Pro Ala Pro Thr Val Thr Trp 170 l75 180 Met Ile Thr Phe Met Arg Ser Leu Gly Thr Pro Ser Cys Met Cys Val Thr His Ile Val Glu Ile Gln Val Asp Glu Val Arg Arg Leu Val Tyr Phe Glu Gly Thr Lys Asp Ser Pro Leu Glu His His Leu Tyr Val Val Ser Tyr Val Asn Pro Gly Glu Val Thr Arg Leu Thr Asp Arg Gly Tyr Ser His Ser Cys Cys Ile Ser Gln His Cys Asp Phe Phe Ile Ser Lys Tyr Ser Asn Gln Lys Asn Pro His Cys Val Ser Leu Tyr Lys Leu Ser Ser Pro Glu Asp Asp Pro Thr Cys Lys Thr Lys Glu Phe Trp Ala Thr Ile Leu Asp Ser Ala Gly Pro Leu Pro Asp Tyr Thr Pro Pro Glu Ile Phe Ser Phe Glu Ser Thr Thr Gly Phe Thr Leu Tyr Gly Met Leu Tyr Lys Pro His Asp Leu Gln Pro Gly Lys Lys Tyr Pro Thr Val Leu Phe Ile Tyr Gly Gly Pro Gln Val Gln Leu Val Asn Asn Arg Phe Lys Gly Val Lys Tyr Phe Arg Leu Asn Thr Leu Ala Ser Leu Gly Tyr Val Val Val Val Ile Asp Asn Arg Gly Ser Cys His Arg Gly Leu Lys Phe Glu Gly Ala Phe Lys Tyr Lys Met Val Ala Ile Ala Gly Ala Pro Val Thr Leu Trp Ile Phe Tyr Asp Thr Gly Tyr Thr Glu Arg Tyr Met Gly His Pro Asp Gln Asn Glu Gln Gly Tyr Tyr Leu Gly Ser Val Ala Met Gln Ala Glu Lys Phe Pro Ser Glu Pro Asn Arg Leu Leu Leu Leu His Gly Phe Leu Asp Glu Asn Val His Phe Ala His Thr Ser Ile Leu Leu Ser Phe Leu Val Arg Ala Gly Lys Pro Tyr Asp Leu Gln Glu Arg His Ser Ile Arg Val Pro Glu Ser Gly Glu His Tyr Glu Leu His Leu Leu His Tyr Leu Gln Glu Asn Leu Gly Ser Arg Ile Ala Ala Leu Lys Val Ile <210> 10 <211> 824 <212> PRT
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte ID No: 7503231CD1 <400> 10 Met Ala Ala Ala Met Glu Thr Glu Gln Leu Gly Val Glu Ile Phe Glu Thr Ala Asp Cys Glu Glu Asn Ile Glu Ser Gln Asp Arg Pro Lys Leu Glu Pro Phe Tyr Val Glu Arg Tyr Ser Trp Ser Gln Leu Lys Lys Leu Leu Ala Asp Thr Arg Lys Tyr His Gly Tyr Met Met Ala Lys Ala Pro His Asp Phe Met Phe Val Lys Arg Asn Asp Pro Asp Gly Pro His Ser Asp Arg Ile Tyr Tyr Leu Ala Met Ser Gly Glu Asn Arg Glu Asn Thr Leu Phe Tyr Ser Glu Ile Pro Lys Thr Ile Asn Arg Ala Ala Val Leu Met Leu Ser Trp Lys Pro Leu Leu Asp Leu Phe Gln Gln Gln Pro Leu Arg Pro Asn Leu Val Glu Thr Ser Cys Pro Asn Ile Arg Met Asp Pro Lys Leu Cys Pro Ala Asp Pro Asp Trp Ile Ala Phe Ile His Ser Asn Asp Ile Trp Ile Ser Asn Ile Val Thr Arg Glu Glu Arg Arg Leu Thr Tyr Val His Asn Glu Leu Ala Asn Met Glu Glu Asp Ala Arg Ser Ala Gly Val Ala Thr Phe Val Leu Gln Glu Glu Phe Asp Arg Tyr Ser G1y Tyr Trp Trp Cys Pro Lys Ala Glu Thr Thr Pro Ser Gly Gly Lys Ile Leu Arg Ile Leu Tyr Glu Glu Asn Asp Glu Ser Glu Val Glu Ile Ile His Val Thr Ser Pro Met Leu Glu Thr Arg Arg Ala Asp Ser Phe Arg Tyr Pro Lys Thr Gly Thr Ala Asn Pro Lys Val Thr Phe Lys Met Ser Glu Ile Met Ile Asp Ala Glu Gly Arg Ile Ile Asp Val Ile Asp Lys Glu Leu Ile Gln Pro Phe Glu Ile Leu Phe Glu Gly Val Glu Tyr Ile Ala Arg Ala Gly Trp Thr Pro Glu Gly Lys Tyr Ala Trp Ser Ile Leu Leu Asp Arg Ser Gln Thr Arg Leu Gln Ile Val Leu Ile Ser Pro Glu Leu Phe Ile Pro Val Glu Asp Asp Val Met Glu Arg Gln Arg Leu Ile Glu Ser Val Pro Asp Ser Val Thr Pro Leu Ile Ile Tyr Glu Glu Thr Thr Asp Ile Trp Ile Asn Ile His Asp Ile Phe His Val Phe Pro Gln Ser His Glu Glu Glu Ile Glu Phe Ile Phe Ala Ser Glu Cys Lys Thr Gly Phe Arg His Leu Tyr Lys Ile Thr Ser Ile Leu Lys Glu Ser Lys Tyr Lys Arg Ser Ser Gly Gly Leu Pro Ala Pro Ser Asp Phe Lys Cys Pro Ile Lys Glu Glu Ile Ala Ile Thr Ser Gly Glu Trp Glu Val Leu Gly Arg His Gly Ser Asn Ile Gln Val Asp Glu Val Arg Arg Leu Val Tyr Phe Glu Gly Thr Lys Asp Ser Pro Leu Glu His His Leu Tyr Val Va1 Ser Tyr Val Asn Pro Gly Glu Val Thr Arg Leu Thr Asp Arg G1y Tyr Ser His Ser Cys Cys Ile Ser Gln His Cys Asp Phe Phe Ile Ser Lys Tyr Ser Asn Gln Lys Asn Pro His Cys Val Ser Leu Tyr Lys Leu Ser Ser Pro Glu Asp Asp Pro Thr Cys Lys Thr Lys Glu Phe Trp Ala Thr Ile Leu Asp Ser Ala Gly Pro Leu Pro Asp Tyr Thr Pro Pro Glu Ile Phe Ser Phe Glu Ser Thr Thr Gly Phe Thr Leu Tyr Gly Met Leu Tyr Lys Pro His Asp Leu Gln Pro Gly Lys Lys Tyr Pro Thr Val Leu Phe Ile Tyr Gly Gly Pro Gln Val Gln Leu Val Asn Asn Arg Phe Lys Gly Val Lys Tyr Phe Arg Leu Asn Thr Leu Ala Ser Leu Gly Tyr Val Val Val Val Ile Asp Asn Arg Gly Ser Cys His Arg Gly Leu Lys Phe Glu Gly Ala Phe Lys Tyr Lys Met Gly Gln Ile Glu Ile Asp Asp Gln Val Glu Gly Leu Gln Tyr Leu Ala Ser Arg Tyr Asp Phe Ile Asp Leu Asp Arg Val Gly Ile His Gly Trp Ser Tyr Gly Gly Tyr Leu Ser Leu Met Ala Leu Met Gln Arg Ser Asp Ile Phe Arg Val Ala Ile Ala Gly Ala Pro Val Thr Leu Trp Ile Phe Tyr Asp Thr Gly Tyr Thr Glu Arg Tyr Met Gly His Pro Asp Gln Asn Glu Gln Gly Tyr Tyr Leu Gly Ser Val Ala Met Gln Ala Glu Lys Phe Pro Ser Glu Pro Asn Arg Leu Leu Leu Leu His Gly Phe Leu Asp Glu Asn Val His Phe Ala His Thr Ser Ile Leu Leu Ser Phe Leu Val Arg Ala Gly Lys Pro Tyr Asp Leu Gln Ile Tyr Pro Gln Glu Arg His Ser Ile Arg Val Pro Glu Ser Gly Glu His Tyr Glu Leu His Leu Leu His Tyr Leu Gln Glu Asn Leu Gly Ser Arg Ile Ala Ala Leu Lys Val Ile 8l5 820 <210> 11 <211> 188 <212> PRT
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte ID No: 71592234CD1 <400> 11 Met Thr Thr Pro Phe Phe Leu Leu Arg Thr Ala Gly Phe Ala Asp Ala Thr Thr Thr Gly Ser Pro Ile Leu Ser Ala Thr Val Asn Leu Thr Met Phe Phe Asp Ile Ala Val Asp Gly Glu Pro Leu Gly His Val Ser Phe Gly Ile Leu Ser Ala Arg Ile Pro Lys Thr Ala Glu Asn Leu Trp Cys Thr Gly Glu Lys Gly Phe Gly Tyr Lys Gly Ser Cys Phe His Arg Tle Ile Pro Gly Phe Met Cys Gln Cys Gly Lys Phe Thr Arg His Asn Gly Thr Gly Gly Lys Ser Ile Cys Gly Glu g5 100 105 Lys Phe Asp Asp Lys Asn Val Ile Leu Lys His Thr Gly Arg Gly Ile Leu Ser Met Glu Thr Gly Gly Pro Asn Thr Asn Gly Ser Gln Phe Phe Ile Cys Thr Gly Lys Thr Glu Trp Leu Asp Gly Lys Tyr Met Val Phe Ser Lys Val Lys Glu Gly Lys Asn Ile Val Glu Ala Met Glu Arg Phe Gly Ser Arg Asn Gly Lys Ile Ser Lys Lys Ile Thr Ile Ala Asp Cys Gly Gln Leu <210> 12 <211>=552 <212> PRT
<213> Homo sapiens <220>
<221> misc_feature <223> Incyte ID No: 90035161CD1 <400> 12 Met Arg Pro Val Ser Val Trp Gln Trp Ser Pro Trp Gly Leu Leu Leu Cys Leu Leu Cys Ser Ser Cys Leu Gly Ser Pro Ser Pro Ser Thr Gly Pro Glu Lys Lys Ala Gly Ser Gln Gly Leu Arg Phe Arg Leu Ala Gly Phe Pro Arg Lys Pro Tyr Glu Gly Arg Val Glu Ile Gln Arg Ala Gly Glu Trp Gly Thr Ile Cys Asp Asp Asp Phe Thr Leu Gln Ala Ala His Ile Leu Cys Arg Glu Leu Gly Phe Thr Glu 80 ' 85 90 Ala Thr Gly Trp Thr His Ser Ala Lys Tyr Gly Pro Gly Thr Gly Arg Ile Trp Leu Asp Asn Leu Ser Cys Ser Gly Thr Glu Gln Ser Val Thr Glu Cys Ala Ser Arg Gly Trp Gly Asn Ser Asp Cys Thr His Asp Glu Asp Ala Gly Val Ile Cys Lys Asp Gln Arg Leu Pro Gly Phe Ser Asp Ser Asn Val Ile Glu Ala Arg Val Arg Leu Lys Gly Gly Ala His Pro Gly Glu Gly Arg Val Glu Val Leu Lys Ala Ser Thr Trp Gly Thr Val Cys Asp Arg Lys Trp Asp Leu His Ala Ala Ser Val Val Cys Arg Glu Leu Gly Phe Gly Ser Ala Arg Glu Ala Leu Ser Gly Ala Arg Met Gly Gln Gly Met Gly Ala Ile His Leu Ser Glu Val Arg Cys Ser Gly Gln Glu Leu Ser Leu Trp Lys Cys Pro His Lys Asn Ile Thr Ala Glu Asp Cys Ser His Ser Gln Asp Ala Gly Val Arg Cys Asn Leu Pro Tyr Thr Gly Ala Glu Thr Arg Glu Thr Trp Tyr Trp Asp Ser Gly Asn Ile Thr Glu Val Val Met Ser Gly Val Arg Cys Thr Gly Thr Glu Leu Ser Leu Asp Gln Cys Ala His His Gly Thr His Ile Thr Cys Lys Arg Thr Gly Thr Arg Phe Thr Ala Gly Val Ile Cys Ser Glu Thr Ala Ser Asp Leu Leu Leu His Ser Ala Leu Val Gln Glu Thr Ala Tyr Ile Glu Asp Arg Pro Leu His Met Leu Tyr Arg Ala Ala Glu Glu Asn Cys Leu Ala Ser Ser Ala Arg Ser Ala Asn Trp Pro Tyr Gly His Arg Arg Leu Leu Arg Phe Ser Ser Gln Ile His Asn Leu Gly Arg Ala Asp Phe Arg Pro Lys Ala Gly Arg His Ser Trp Val Trp His Glu Cys His Gly His Tyr His Ser Met Asp Ile Phe Thr His Tyr Asp Ile ~Leu Thr Pro Asn Gly Thr Lys Val Ala Glu Gly His Lys Ala Ser Phe Cys Leu Glu Asp Thr Glu Cys Gln Glu Asp Val Ser Lys Arg Tyr Glu Cys Ala Asn Phe Gly Glu Gln Gly Ile Thr Val Gly Cys Trp Asp Leu Tyr Arg His Asp Ile Asp Cys Gln Trp Ile Asp Ile Thr Asp Val Lys Pro Gly Asn Tyr Ile Leu Gln Val Val Ile Asn Pro Asn Phe Glu Val Ala Glu Ser Asp Phe Thr Asn Asn Ala Met Lys Cys Asn Cys Lys Tyr Asp Gly His Arg Ile Trp Val His Asn Cys His Ile Gly Asp Ala Phe Ser Glu Glu Ala Asn Arg Arg Phe Glu Arg Tyr Pro Gly Gln Thr Ser Asn Gln Ile Ile <210> 13 <211> 303 <212> PRT
<213> Homo Sapiens <220>
<221> mist-feature <223> Incyte ID No: 55141453CD1 <400> 13 Met Gly Ser Ala Gly Arg Leu His Tyr Leu Ala Met Thr Ala Glu Asn Pro Thr Pro Gly Asp Leu Ala Pro Ala Pro Leu Ile Thr Cys 20 25 ~ 30 Lys Leu Cys Leu Cys Glu Gln Ser Leu Asp Lys Met Thr Thr Leu Gln Glu Cys Gln Cys Ile Phe Cys Thr Ala Cys Leu Lys Gln Tyr Met Gln Leu Ala Ile Arg Glu Gly Cys Gly Ser Pro Ile Thr Cys Pro Asp Met Val Cys Leu Asn His Gly Thr Leu G1n Glu Ala Glu Ile Ala Cys Leu Val Pro Val Asp Gln Phe Gln Leu Tyr Gln Arg Leu Lys Phe Glu Arg Glu Val His Leu Asp Pro Tyr Arg Thr Trp Cys Pro Val Ala Asp Cys Gln Thr Val Cys Pro Val Ala Ser Ser Asp Pro Gly Gln Pro Val Leu Val Glu Cys Pro Ser Cys His Leu Lys Phe Cys Ser Cys Cys Lys Asp Ala Trp His Ala Glu Val Ser Cys Arg Asp Ser Gln Pro Ile Val Leu Pro Thr Glu His Arg Ala Leu Phe Gly Thr Asp Ala Glu Ala Pro Ile Lys Gln Cys Pro Val Cys Arg Val Tyr Ile Glu Arg Asn Glu Gly Cys Ala Gln Met Met Cys Lys Asn Cys Lys His Thr Phe Cys Trp Tyr Cys,Leu Gln Asn Leu Asp Asn Asp Ile Phe Leu Arg His Tyr Asp Lys Gly Pro Cys Arg Asn Lys Leu Gly His Ser Arg Ala Ser Val Met Trp Asn Arg Thr Gln Val Val Gly Ile Leu Val Gly Leu Gly Ile Ile Ala Leu 260 265 ~ 270 Val Thr Ser Pro Leu Leu Leu Leu Ala Ser Pro Cys Ile Ile Cys Cys Val Cys Lys Ser Cys Arg Gly Lys Lys Lys Lys His Asp Pro Ser Thr Thr <210> 14 <211> 945 <212> PRT
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte ID No: 7503135CD1 <400> 14 Met Ala Glu Gly Gly Gly Cys Arg Glu Arg Pro Asp Ala Glu Thr Gln Lys Ser Glu Leu Gly Pro Leu Met Arg Thr Thr Leu Gln Arg Gly Ala Gln Trp Tyr Leu Ile Asp Ser Arg Trp Phe Lys Gln Trp Lys Lys Tyr Val Gly Phe Asp Ser Trp Gly Met Tyr Asn Val Gly Glu His Asn Leu Phe Pro Gly Pro Ile Asp Asn Ser Gly Leu Phe Ser Asp Pro Glu Ser Gln Thr Leu Lys Glu His Leu Ile Asp Glu g0 85 90 Leu Asp Tyr Val Leu Val Pro Thr Glu Ala Trp Asn Lys Leu Leu g5 100 105 Asn Trp Tyr Gly Cys Val Glu Gly Gln Gln Pro Ile Val Arg Lys Val Val Glu His Gly Leu Phe Val Lys His Cys Lys Val Glu Val Tyr Leu Leu Glu Leu Lys Leu Cys Glu Asn Ser Asp Pro Thr Asn Val Leu Ser Cys His Phe Ser Lys Ala Asp Thr Ile Ala Thr Ile Glu Lys Glu Met Arg Lys Leu Phe Asn Ile Pro Ala Glu Arg Glu Thr Arg Leu Trp Asn Lys Tyr Met Ser Asn Thr Tyr Glu Gln Leu Ser Lys Leu Asp Asn Thr Val Gln Asp Ala Gly Leu Tyr Gln Gly Gln Val Leu Val Ile Glu Pro Gln Asn Glu Asp Gly Thr Trp Pro Arg Gln Thr Leu Gln Ser Lys Ser Ser Thr Ala Pro Ser Arg Asn Phe Thr Thr Ser Pro Lys Ser Ser Ala Ser Pro Tyr Ser Ser Val Ser Ala Ser Leu Ile Ala Asn Gly Asp Ser Thr Ser Thr Cys Gly Met His Ser Ser Gly Val Ser Arg Gly Gly Ser Gly Phe Ser Ala Ser Tyr Asn Cys Gln Glu Pro Pro Ser Ser His Ile Gln Pro Gly Leu Cys Gly Leu Gly Asn Leu Gly Asn Thr Cys Phe Met Asn Ser Ala Leu Gln Cys Leu Ser Asn Thr Ala Pro Leu Thr Asp Tyr Phe Leu Lys Asp Glu Tyr Glu Ala Glu Ile Asn Arg Asp Asn Pro Leu Gly Met Lys Gly Glu Ile Ala Glu Ala Tyr Ala Glu Leu Ile Lys Gln Met Trp Ser Gly Arg Asp Ala His Val Ala Pro Arg Met Phe Lys Thr Gln Val Gly Arg Phe Ala Pro Gln Phe Ser Gly Tyr Gln Gln Gln Asp Ser Gln Glu Leu Leu Ala Phe Leu Leu Asp Gly Leu His Glu Asp Leu Asn Arg Val Val Ala Lys Glu Ala Trp Glu Asn His Arg Leu Arg Asn Asp Ser Val Ile Val Asp Thr Phe His Gly Leu Phe Lys Ser Thr Leu Val Cys Pro Glu Cys Ala Lys Val Ser Val Thr Phe Asp Pro Phe Cys Tyr Leu Thr Leu Pro Leu Pro Leu Lys Lys Asp Arg Val Met Glu Val Phe Leu Val Pro Ala Asp Pro His Cys Arg Pro Thr Gln Tyr Arg Val Thr Val Pro Leu Met Gly Ala Val Ser Asp Leu Cys Glu Ala Leu Ser Arg Leu Ser Gly Ile Ala Ala Glu Asn Met Val Val Ala Asp Val Tyr Asn His Arg Phe His Lys Ile Phe Gln Met Asp Glu Gly Leu Asn His Ile Met Pro Arg Asp Asp Ile Phe Val Tyr Glu Val Cys Ser Thr Ser Val Asp Gly Ser Glu Cys Val Thr Leu Pro Val Tyr Phe Arg Glu Arg Lys Ser Arg Pro Ser Ser Thr Ser Ser Ala Ser Ala Leu Tyr Gly Gln Pro Leu Leu Leu Ser Val Pro Lys His Lys Leu Thr Leu Glu Ser Leu Tyr Gln Ala Val Cys Asp Arg Ile Ser Arg Tyr Val Lys Gln Pro Leu Pro Asp Glu Phe Gly Ser Ser Pro Leu Glu Pro Gly Ala Cys Asn Gly Ser Arg Asn Ser Cys Glu Gly Glu Asp Glu Glu Glu Met Glu His Gln Glu Glu Gly Lys Glu Gln Leu Ser Glu Thr Glu Gly Ser Gly Glu Asp Glu Pro Gly Asn Asp Pro Ser Glu Thr Thr Gln Lys Lys Ile Lys Gly Gln Pro Cys Pro Lys Arg Leu Phe Thr Phe Ser Leu Val Asn Ser Tyr C~~ly Thr Ala Asp Ile Asn Ser Leu Ala Ala Asp Gly Lys Leu Leu Lys Leu Asn Ser Arg Ser Thr Leu Ala Met Asp Trp Asp Ser Glu Thr Arg Arg Leu Tyr Tyr Asp Glu Gln Glu Ser Glu Ala Tyr Glu Lys His Val Ser Met Leu Gln Pro Gln Lys Lys Lys Lys Thr Thr Val Ala Leu Arg Asp Cys Ile Glu Leu Phe Thr Thr Met Glu Thr Leu Gly Glu His Asp Pro Trp Tyr Cys Pro Asn Cys Lys Lys His Gln Gln Ala Thr Lys Lys Phe Asp Leu Trp Ser Leu Pro Lys Ile Leu Val Val His Leu Lys Arg Phe Ser Tyr Asn Arg Tyr Trp Arg Asp Lys Leu Asp Thr Val Val Glu Phe Pro Ile Arg Gly Leu Asn Met Ser Glu Phe Val Cys Asn Leu Ser Ala Arg Pro Tyr Val Tyr Asp Leu Ile Ala Val Ser Asn His Tyr Gly Ala Met Gly Val Gly His Tyr Thr Ala Tyr Ala Lys Asn Lys Leu Asn Gly Lys Trp Tyr Tyr Phe Asp Asp Ser Asn Val Ser Leu Ala Ser Glu Asp Gln Ile Val Thr Lys Ala Ala Tyr Val Leu Phe Tyr Gln Arg Arg Asp Asp Glu Phe Tyr Lys Thr Pro Ser Leu Ser Ser Ser Gly Ser Ser Asp Gly Gly Thr Arg Pro Ser Ser Ser Gln Gln Gly Phe Gly Asp Asp Glu Ala Cys Ser Met Asp Thr Asn <210> 15 <211> 315 <212> PRT
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte ID No: 7503242CD1 <400> 15 Met Lys Arg Ala Ala Met Ala Leu His Ser Pro Gln Tyr Ile Phe Gly Asp Phe Ser Pro Asp Glu Phe Asn Gln Phe Phe Val Thr Pro Arg Ser Ser Val Glu Gly Arg Gln Glu Asp Ala Glu Glu Tyr Leu 35 40 ~ 45 Gly Phe Ile Leu Asn Gly Leu His Glu Glu Met Leu Asn Leu Lys Lys Leu Leu Ser Pro Ser Asn Glu Lys Leu Thr Ile Ser Asn Gly Pro Lys Asn His Ser Val Asn Glu Glu Glu Gln Glu Glu Gln Gly Glu Gly Ser Glu Asp Glu Trp Glu Gln Val Gly Pro Arg Asn Lys Thr Ser Val Thr Arg Gln Ala Asp Phe Val Gln Thr Pro Ile Thr Gly Ile Phe Gly Gly His Ile Arg Ser Val Val Tyr Gln Gln Ser Ser Lys Glu Ser Ala Thr Leu Gln Pro Phe Phe Thr Leu Gln Leu Asp Ile Gln Ser Asp Lys Ile Arg Thr Val Gln Asp Ala Leu Glu Ser Leu Val Ala Arg Glu Ser Val Gln Gly Tyr Thr Thr Lys Thr Lys Gln Glu Val Glu Ile Ser Arg Arg Val Thr Leu Glu Lys Leu Pro Pro Val Leu Val Leu His Leu Lys Arg Phe Val Tyr Glu Lys Thr Gly Gly Cys Gln Lys Leu Ile Lys Asn Ile Glu Tyr Pro Val Asp Leu Glu Ile Ser Lys Glu Leu Leu Ser Pro Gly Val Lys Asn Lys Asn Phe Lys Cys His Arg Thr Tyr Arg Leu Phe Ala Val Val Tyr His His Gly Asn Ser Ala Thr Gly Gly His Tyr Thr Thr Asp Val Phe Gln Ile Gly Leu Asn Gly Trp Leu Arg Ile Asp Asp Gln Thr Val Lys Val Ile Asn Gln Tyr Gln Val Val Lys Pro Thr Ala Glu Arg Thr Ala Tyr Leu Leu Tyr Tyr Arg Arg Val Asp Leu Leu <210> 16 <211> 204 <212> PRT

<213> Homo sapiens <220>
<221> misc_feature <223> Incyte ID No: 7504774CD1 <400> 16 Met Lys Arg Leu Thr Cys Phe Phe Ile Cys Phe Phe Leu Ser Glu Val Ser Gly Phe Glu Ile Pro Ile Asn Gly Leu Ser Glu Phe Val Asp Tyr Glu Asp Leu Val Glu Leu Ala Pro Gly Lys Phe Gln Leu Val Ala Glu Asn Arg Arg Tyr Gln Arg Ser Leu Pro Gly Glu Ser Glu Glu Met Met Glu Glu Val Asp Gln Val Thr Leu Tyr Ser Tyr Lys Val Gln Ser Thr Ile Thr Ser Arg Met Ala Thr Thr Met Ile Gln Ser Lys Val Val Asn Asn Ser Pro Gln Pro Gln Asn Val Val Phe Asp Val Gln Ile Pro Lys Gly Ala Phe Ile Ser Asn Phe Ser 110 ~ 115 120 Met Thr Val Asp Gly Lys Thr Phe Arg Ser Ser Ile Lys Glu Lys Thr Val Gly Arg Ala Leu Tyr Ala Gln Ala Arg Ala Lys Gly Lys Thr Ala Gly Leu Val Arg Gly Leu Gln Lys Asp Tyr Arg Thr Asp Leu Val Phe Gly Thr Asp Val Thr Cys Trp Phe Val His Asn Ser Gly Lys Gly Phe Tle Asp Gly His Tyr Lys Asp Tyr Phe Val Pro Gln Leu Tyr Ser Phe Leu Lys Arg Pro <210> 17 <211> 228 <212> PRT
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte ID No: 7503166CD1 <400> 17 Met Phe Leu Val Asn Ser Phe Leu Lys Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Arg Ile Leu Gly Gly Val Ile Ser Ala Ile Ser Glu Ala Ala Ala Gln Tyr Asn Pro Glu Pro Pro Pro Pro Arg Thr His Tyr Ser Asn Ile Glu Ala Asn Glu Ser Glu Glu Val Arg Gln Phe Arg Arg Leu Phe Ala Gln Leu Ala Gly Asp Asp Met Glu Val Ser Ala Thr Glu Leu Met Asn Ile Leu Asn Lys Val Val Thr Arg His Pro Asp Leu Lys Thr Asp Gly Phe Gly Ile Asp Thr Cys Arg Ser Met Val Ala Val Met Asp Ser Asp Thr Thr Gly Lys Leu Gly Phe Glu Glu Phe Lys Tyr Leu Trp Asn Asn Ile Lys Arg Trp Gln Ala Ile Tyr Lys Gln Phe Asp Thr Asp Arg Ser Gly Thr Ile Cys Ser Ser Glu Leu Pro Gly Ala Phe Glu Ala Ala Gly Phe His Leu Asn Glu His Leu Tyr Asn Met Ile Ile Arg Arg Tyr Ser Asp Glu Ser Gly Asn Met Asp Phe Asp Asn Phe Ile Ser Cys Leu Val Arg Leu Asp Ala Met Phe Arg Ala Phe Lys Ser Leu Asp Lys Asp Gly Thr Gly Gln Ile Gln Val Asn Ile Gln Glu Trp Leu Gln Leu Thr Met Tyr Ser <210> 18 <211> 160 <212> PRT
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte ID No: 7503171CD1 <400> 18 Met Glu Tyr Leu Ile Gly Ile Gln Gly Pro Asp Tyr Val Leu Val Ala Ser Asp Arg Val Ala Ala Ser Asn Ile Val Gln Met Lys Asp Gly Tyr Glu Leu Ser Pro Thr Ala Ala Ala Asn Phe Thr Arg Arg Asn Leu Ala Asp Cys Leu Arg Ser Arg Thr Pro Tyr His Val Asn Leu Leu Leu Ala Gly Tyr Asp Glu His Glu Gly Pro Ala Leu Tyr Tyr Met Asp Tyr Leu Ala Ala Leu Ala Lys Ala Pro Phe A1a Ala His Gly Tyr Gly Ala Phe Leu Thr Leu Ser Ile Leu Asp Arg Tyr Tyr Thr Pro Thr Ile Ser Arg Glu Arg Ala Val Glu Leu Leu Arg Lys Cys Leu Glu Glu Leu Gln Lys Arg Phe Ile Leu Asn Leu Pro Thr Phe Ser Val Arg Ile Ile Asp Lys Asn Gly Ile His Asp Leu Asp Asn Ile Ser Phe Pro Lys Gln Gly Ser <210> 19 <211> 139 <212> PRT
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte ID No: 7503220CD1 <400> 19 Met Leu Ser Leu Asp Phe Leu Asp Asp Val Arg Arg Met Asn Lys Arg Gln Leu Tyr Tyr Gln Val Leu Asn Phe Gly Met Ile Val Ser Ser Ala Leu Met Ile Trp Lys Gly Leu Met Val Ile Thr Gly Ser Glu Ser Pro Ile Val Val Val Leu Ser Gly Ser Met Glu Pro Ala Phe His Arg Gly Asp Leu Leu Phe Leu Thr Asn Arg Val Glu Asp Pro Ile Arg Val Gly Glu Ile Val Val Phe Arg Ile Glu Gly Arg Glu Ile Pro Ile Val His Arg Val Leu Lys Ile His Glu Lys Phe Val Pro Tyr Ile Gly Ile Val Thr Ile Leu Met Asn Asp Tyr Pro Lys Phe Lys Tyr Ala Va1 Leu Phe Leu Leu Gly Leu Phe Val Leu Val His Arg Glu <210> 20 <211> 216 <212> PRT
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte ID No: 6145631CD1 <400> 20 Met Lys Gly Lys Glu Glu Lys Glu Gly Gly Ala Arg Leu Gly Ala Gly Gly Gly Ser Pro Glu Lys Ser Pro Ser Ala Gln Glu Leu Lys Glu Gln Gly Asn Arg Leu Phe Val Gly Arg Lys Tyr Pro Glu Ala Ala Ala Cys Tyr Gly Arg Ala Ile Cys Gln Leu Glu Met Glu Ser Tyr Asp Glu Ala Ile Ala Asn Leu Gln Arg Ala Tyr Ser Leu Ala Lys Glu Gln Arg Leu Asn Phe Gly Asp Asp Ile Pro Ser Ala Leu Arg Ile Ala Lys Lys Lys Arg Trp Asn Ser Ile Glu Glu Arg Arg Ile His Gln Glu Ser Glu Leu His Ser Tyr Leu Ser Arg Leu Ile Ala Ala Glu Arg Glu Arg Glu Leu Glu Glu Cys Gln Arg Asn His Glu Gly Asp Glu Asp Asp Ser His Val Arg Ala Gln Gln Ala Cys Ile Glu Ala Lys His Val Arg Val Pro Pro Thr His Met Trp Val Cys Val Cys Ala Arg Gly Val Gly Ala Ser Pro Pro Cys Val Gly Ser Val Pro His Gly Gly Gly Arg Trp Gly Val Ser Pro Lys His Ser Thr Gln Leu Phe Thr Gly Gln Val His Gly Gly His Gly Arg Ala Phe Phe Ser Gly Gly <210> 21 <211> 194 <212> PRT
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte ID No: 7504980CD1 <400> 21 Met Val Ser Arg Met Val Ser Thr Met Leu Ser Gly Leu Leu Phe Trp Leu Ala Ser Gly Trp Thr Pro Ala Phe Ala Tyr Ser Pro Arg Thr Pro Asp Arg Val Ser Glu Ala Asp Ile Gln Arg Leu Leu His Gly Val Met Glu Gln Leu Gly Ile Ala Arg Pro Arg Val Glu Tyr Pro Ala His Gln Ala Met Asn Leu Val Gly Pro Gln Ser Ile Glu Gly Gly Ala His Glu Gly Leu Gln His Leu Gly Pro Phe Gly Asn Ile Pro Asn Ile Val Ala Glu Leu Thr Gly Asp Asn Ile Pro Lys Asp Phe Ser Glu Asp Gln Gly Tyr Pro Asp Pro Pro Asn Pro Cys Pro Val Gly Lys Thr Ala Asp Asp Gly Cys Leu Glu Asn Thr Pro Asp Thr Ala Glu Phe Ser Arg Glu Phe Gln Leu His Gln His Leu Phe Asp Pro G1u His Asp Tyr Pro Gly Leu Gly Lys Trp Ser Val Asn Pro Tyr Leu Gln Gly Gln Arg Leu Asp Asn Val Val Ala Lys Lys Ser Val Pro His Phe Ser Asp Glu Asp Lys Asp Pro Glu <210> 22 <211> 361 <212> PRT

<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte ID No: 3118830CD1 <400> 22 Met Ala Gln Arg Cys Val Cys Val Leu Ala Leu Val Ala Met Leu Leu Leu Val Phe Pro Thr Val Ser Arg Ser Met Gly Pro Arg Ser Gly Glu His Gln Arg Ala Ser Arg Tle Pro Ser Gln Phe Ser Lys Glu Glu Arg Val Ala Met Lys Glu Ala Leu Lys Gly Ala Ile Gln Ile Pro Thr Val Thr Phe Ser Ser Glu Lys 5er Asn Thr Thr Ala Leu Ala Glu Phe Gly Lys Tyr Ile His Lys Val Phe Pro Thr Val Val Ser Thr Ser Phe Ile Gln His Glu Val Val Glu Glu Tyr Ser His Leu Phe Thr Ile Gln Gly Ser Asp Pro Ser Leu Gln Pro Tyr Leu Leu Met Ala His Phe Asp Val Val Pro Ala Pro Glu Glu Gly Trp Glu Val Pro Pro Phe Ser Gly Leu Glu Arg Asp Gly Val Ile Tyr Gly Arg Gly Thr Leu Asp Asp Lys Asn Ser Va1 Met Ala Leu Leu Gln Ala Leu Glu Leu Leu Leu Ile Arg Lys Tyr Ile Pro Arg x Arg Ser Phe Phe Ile Ser Leu Gly His Asp Glu Glu Ser Ser Gly Thr Gly Ala Gln Arg Ile Ser Ala Leu Leu Gln Ser Arg Gly Val Gln Leu Ala Phe Ile Val Asp Glu Gly Gly Phe Ile Leu Asp Asp Phe Ile Pro Asn Phe Lys Lys Pro Ile Ala Leu Ile Ala Val Ser Glu Lys Gly Ser Met Asn Leu Met Leu Gln Val Asn Met Thr Ser Gly His Ser Ser Ala Pro Pro Lys Glu Thr Ser Ile Gly Ile Leu Ala Ala Ala Val Ser Arg Leu Glu Gln Thr Pro Met Pro Ile Ile Phe Gly Ser Gly Thr Val Val Thr Val Leu Gln Gln Leu Ala Asn Glu Val Tyr Gly Glu Lys Ser Leu Asn Gln Cys Asn Asn Gln Asp His His Gly Thr His His Ile Gln Ser Arg Gly Gln Val Gln Cys His Pro Pro Ser G1y Pro Gly His Ser Gln Leu Pro Asp Ser Pro Trp Thr Asp Ser Pro Arg Gly Pro Arg Thr His Glu Glu His Cys Gly <210> 23 <211> 487 <212> PRT
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte ID No: 7505187CD1 <400> 23 Met Ala Pro Ala Arg Thr Met Ala Arg Ala Arg Leu Ala Pro Ala Gly Ile Pro Ala Val Ala Leu Trp Leu Leu Cys Thr Leu Gly Leu Gln Gly Thr Gln Ala Gly Pro Pro Pro Ala Pro Pro Gly Leu Pro Ala Gly Ala Asp Cys Leu Asn Ser Phe Thr Ala Gly Val Pro Gly Phe Val Leu Asp Thr Asn Ala Ser Val Ser Asn Gly Ala Thr Phe Leu Glu Ser Pro Thr Val Arg Arg Gly Trp Asp Cys Val Arg Ala gp g5 90 Cys Cys Thr Thr Gln Asn Cys Asn Leu Ala Leu Val Glu Leu Gln Pro Asp Arg Gly Glu Asp Ala Ile Ala Ala Cys Phe Leu Ile Asn Cys Leu Tyr Glu Gln Asn Phe Val Cys Lys Phe Ala Pro Arg Glu Gly Phe Ile Asn Tyr Leu Thr Arg Glu Val Tyr Arg Ser Tyr Arg Gln Leu Arg Thr Gln Gly Phe Gly Gly Ser Gly Ile Pro Lys Ala Trp Ala Gly Ile Asp Leu Lys Val Gln Pro Gln Glu Pro Leu Val Leu Lys Asp Val Glu Asn Thr Asp Trp Arg Leu Leu Arg Gly Asp Thr Asp Val Arg Val Glu Arg Lys Asp Pro Asn Gln Val Glu Leu Trp Gly Leu Lys Glu Gly Thr Tyr Leu Phe Gln Leu Thr Val Thr Ser Ser Asp His Pro Glu Asp Thr Ala Asn Val Thr Val Thr Val Leu Ser Thr Lys Gln Thr Glu Asp Tyr Cys Leu Ala Ser Asn Lys Val Gly Arg Cys Arg Gly Ser Phe Pro Arg Trp Tyr Tyr Asp Pro Thr Glu Gln Ile Cys Lys Ser Phe Val Tyr Gly Gly Cys Leu Gly Asn Lys Asn Asn Tyr Leu Arg Glu Glu Glu Cys Ile Leu Ala Cys Arg Gly Val Gln Gly Gly Pro Leu Arg Gly Ser Ser Gly Ala Gln Ala Thr Phe Pro Gln Gly Pro Ser Met Glu Arg Arg His Pro Asp Thr Ser Gly Phe Asp Glu Leu Gln Arg Ile His Phe Pro Ser Asp Lys Gly His Cys Val Asp Leu Pro Asp Thr Gly Leu Cys Lys Glu Ser Ile Pro Arg Trp Tyr Tyr Asn Pro Phe Ser Glu His Cys Ala Arg Phe Thr Tyr Gly Gly Cys Tyr Gly Asn Lys Asn Asn Phe Glu Glu Glu Gln Gln Cys Leu Glu Ser Cys Arg Gly Ile Ser Lys Lys Asp Val Phe Gly Leu Arg Arg Glu Ile Pro Ile Pro Ser Thr Gly Ser Val Glu Met Ala Val Ala Val Phe Leu Val Ile Cys Ile Val Val Val Val Ala Ile Leu Gly Tyr Cys Phe Phe Lys Asn Gln Arg Lys Asp Phe His Gly His His His His Pro Pro Pro Thr Pro Ala Ser Ser Thr Val Ser Thr Thr Glu Asp Thr Glu His Leu Val Tyr Asn His Thr Thr Arg Pro Leu <210> 24 <211> 329 <212> PRT
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte ID No: 7506567CD1 <400> 24 Met Lys Leu Ile Thr Ile Leu Phe Leu Cys Ser Arg Leu Leu Leu Ser Leu Thr Gln Glu Ser Gln Ser Glu Glu Ile Asp Cys Asn Asp Lys Asp Leu Phe Lys Ala Val Val Thr Ala Gln Tyr Asp Cys Leu Gly Cys Val His Pro Ile Ser Thr Gln Ser Pro Asp Leu Glu Pro Ile Leu Arg His Gly Ile Gln Tyr Phe Asn Asn Asn Thr Gln His Ser Ser Leu Phe Met Leu Asn Glu Val Lys Arg Ala Gln Arg Gln Val Val Ala Gly Leu Asn Phe Arg Ile Thr Tyr Ser Ile Val Gln Thr Asn Cys Ser Lys Glu Asn Phe Leu Phe Leu Thr Pro Asp Cys Lys Ser Leu Trp Asn Gly Asp Thr Gly Glu Cys Thr Asp Asn Ala Tyr Ile Asp Ile Gln Leu Arg Ile Ala Ser Phe Ser Gln Asn Cys Asp Ile Tyr Pro Gly Lys Asp Phe Val Gln Pro Pro Thr Lys Ile Cys Val Gly Cys Pro Arg Asp Ile Pro Thr Asn Ser Pro Glu Leu Glu Glu Thr Leu Thr His Thr Ile Thr Lys Leu Asn Ala Glu Asn Asn Ala Thr Phe Tyr Phe Lys Ile Asp Asn Val Lys Lys Ala Arg Val Gln Val Val Ala Gly Lys Lys Tyr Phe Ile Asp Phe Val Ala Arg Glu Thr Thr Cys Ser Lys Glu Ser Asn Glu Glu Leu Thr Glu Ser Cys Glu Thr Lys Lys Leu Gly Gln Ser Leu Asp Cys Asn Ala Glu Val Tyr Val Val Pro Trp Glu Lys Lys Ile Tyr Pro Thr Val Asn Cys Gln Pro Leu Gly Met Ile Ser Leu Met Lys Arg Pro Pro Gly Phe Ser Pro Phe Arg Ser Ser Arg Ile Gly Glu Tle Lys Glu Glu Thr Thr Ser His Leu Arg Ser Cys Glu Tyr Lys Gly Arg Pro Pro Lys Ala Gly Ala Glu Pro Ala Ser Glu Arg Glu Val Ser <210> f5 <211> 151 <212> PRT
<213> Homo sapiens <220>
<221> misc_feature <223> Incyte ID No: 7503673CD1 <400> 25 Met Pro Gly Gly Ala Gly Ala Ala Arg Leu Cys Leu Leu Ala Phe Ala Leu Gln Pro Leu Arg Pro Arg Ala Ala Arg Glu Pro Gly Trp Thr Arg Gly Ser Glu Glu Gly Ser Pro Lys Leu Gln His Glu Leu Ile Ile Pro G1n Trp Lys Thr Ser Glu Ser Pro Va1 Arg Glu Lys His Pro Leu Lys Ala Glu Leu Arg Val Met Ala Glu Gly Arg Glu Leu Ile Leu Asp Leu Glu Lys Asn Glu Gln Leu Phe Ala Pro Ser Tyr Thr Glu Thr His Tyr Thr Ser Ser Gly Asn Pro Gln Thr Thr Thr Arg Lys Leu Glu Asp His Cys Phe Tyr His Gly Thr Val Arg Glu Thr Glu Leu Ser Ser Val Thr Leu Ser Thr Cys Arg Gly Ile Ser Phe Arg Arg Ile Asp Glu Thr Arg Thr Pro Pro Asn Thr Ser Ser <210> 26 <211> 341 <212> PRT
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte ID No: 90123303CD1 <400> 26 Met Gln Gly Thr Pro Gly Gly Gly Thr Arg Pro Gly Pro Ser Pro Val Asp Arg Arg Thr Leu Leu Val Phe Ser Phe Ile Leu Ala Ala Ala Leu Gly Gln Met Asn Phe Thr Gly Asp Gln Val Leu Arg Val Leu Ala Lys Asp Glu Lys Gln Leu Ser Leu Leu Gly Asp Leu Glu Gly Leu Lys Pro Gln Lys Val Asp Phe Trp Arg Gly Pro Ala Arg Pro Ser Leu Pro Val Asp 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 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 Val Ser Asp Tyr Gly Lys Asp Arg Val 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 Gly Phe Gly Asp 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 Ile Ile Pro Thr Ala Gln Glu Thr Trp Met Ala Leu Arg Thr Ile Met Glu His Thr Leu Asn His Pro Tyr <210> 27 <211> 521 <212> PRT
<213> Homo sapiens <220>
<221> misc_feature <223> Incyte ID No: 7505765CD1 <400> 27 Met Met Pro Thr Pro Val Ile Leu Leu Lys Glu Gly Thr Asp Ser Ser Gln Gly Ile Pro Gln Leu Val Ser Asn Ile Ser Ala Cys Gln Val Ile Ala Glu Ala Val Arg Thr Thr Leu Gly Pro Arg Gly Met Asp Lys Leu Ile Val Asp Gly Arg Ala Lys Thr Leu Val Asp Ile Ala Lys Ser Gln Asp Ala Glu Val Gly Asp Gly Thr Thr Ser Val Thr Leu Leu Ala Ala Glu Phe Leu Lys Gln Val Lys Pro Tyr Val Glu Glu Gly Leu His Pro Gln Ile Ile Ile Arg Ala Phe Arg Thr g5 100 105 Ala Thr Gln Leu Ala Val Asn Lys Ile Lys Glu Ile Ala Val Thr Val Lys Lys Ala Asp Lys Val Glu Gln Arg Lys Leu Leu Glu Lys Cys Ala Met Thr Ala Leu Ser Ser Lys Leu Tle Ser Gln Gln Lys Ala Phe Phe Ala Lys Met Val Val Asp Ala Val Met Met Leu Asp Asp Leu Leu Gln Leu Lys Met Ile Gly Ile Lys Lys Val Gln Gly Gly Ala Leu Glu Asp Ser Gln Leu Val Ala Gly Val Ala Phe Lys Lys Thr Phe Ser Tyr Ala Gly Phe Glu Met Gln Pro Lys Lys Tyr His Asn Pro Lys Ile Ala Leu Leu Asn Val Glu Leu Glu Leu Lys Ala Glu Lys Asp Asn Ala Glu Ile Arg Val His Thr Val Glu Asp Tyr Gln Ala Ile Val Asp Ala Glu Trp Asn Ile Leu Tyr Asp Lys Leu Glu Lys Ile His His Ser Gly Ala Lys Val Val Leu Ser Lys Leu Pro Ile Gly Asp Val Ala Thr Gln Tyr Phe Ala Asp Arg Asp Met Phe Cys Ala Gly Arg Val Pro Glu Glu Asp Leu Lys Arg Thr Met Met Ala Cys Gly Gly Ser Ile Gln Thr Ser Val Asn Ala Leu Ser Ala Asp Val Leu Gly Arg Cys Gln Val Phe Glu Glu Thr Gln Ile Gly Gly Glu Arg Tyr Asn Phe Phe Thr Gly Cys Pro Lys Ala Lys Thr Cys Thr Phe Ile Leu Arg Gly Gly Ala Glu Gln Phe Met Glu Glu Thr Glu Arg Ser Leu His Asp Ala Ile Met Ile Val Arg Arg Ala Ile Lys Asn Asp Ser Val Val Ala Gly Gly Gly Ala Ile Glu Met Glu Leu Ser Lys Tyr Leu Arg Asp Tyr Ser Arg Thr Ile Pro Gly Lys Gln Gln Leu Leu Ile Gly Ala Tyr Ala Lys Ala Leu Glu Ile Ile Pro Arg Gln Leu Cys Asp Asn Ala Gly Phe Asp Ala Thr Asn Ile Leu Asn Lys Leu Arg Ala Arg His Ala Gln Gly Gly Thr Trp Tyr Gly Val Asp Ile Asn Asn Glu Asp Ile Ala Asp Asn Phe Glu Ala Phe Val Trp Glu Pro Ala Met Val Arg Ile Asn Ala Leu Thr Ala Ala Ser Glu Ala Ala Cys Leu Ile Val Ser Val Asp Glu Thr Ile Lys Asn Pro Arg Ser Thr Val Asp Ala Pro Thr Ala Ala Gly Arg Gly Arg Gly Arg Gly Arg Pro His <2l0> 28 <211> 432 <212> PRT
<213> Homo Sapiens <220>
<221> misc-feature <223> Incyte ID No: 7505775CD1 <400> 28 Met Asp Ile Asp Lys Asp Leu Glu Ala Pro Leu Tyr Leu Thr Pro Glu Gly Trp Ser Leu Phe Leu Gln Arg Tyr Tyr Gln Val Val His Glu Gly Ala Glu Leu Arg His Leu Asp Thr Gln Val Gln Arg Cys Glu Asp Ile Leu Gln Gln Leu Gln Ala Val Val Pro Gln Ile Asp Met Glu Gly Asp Arg Asn Ile Trp Ile Val Lys Pro Gly Ala Lys Ser Arg Gly Arg Gly Ile Met Cys Met Asp His Leu Glu Glu Met Leu Lys Leu Val Asn Gly Asn Pro Val Val Met Lys Asp Gly Asn g5 100 105 Ser Val His Leu Cys Asn Asn Ser Ile Gln Lys His Leu Glu Asn 110 115 l20 Ser Cys His Arg His Pro Leu Leu Pro Pro Asp Asn Met Trp Ser Ser Gln Arg Phe Gln Ala His Leu Gln Glu Met Gly Ala Pro Asn Ala Trp Ser Thr Ile Ile Val Pro Gly Met Lys Asp Ala Val Ile His Ala Leu Gln Thr Ser Gln Asp Thr Val Gln Cys Arg Lys Ala Ser Phe Glu Leu Tyr Gly Ala Asp Phe Val Phe Gly Glu Asp Phe Gln Pro Trp Leu Ile Glu Ile Asn Ala Ser Pro Thr Met Ala Pro Ser Thr Ala Val Thr Ala Arg Leu Cys Ala Gly Val Gln Ala Asp Thr Leu Arg Val Val Ile Asp Arg Met Leu Asp Arg Asn Cys Asp Thr Gly Ala Phe Glu Leu Ile Tyr Lys Gln Pro Ala Val Glu Val Pro Gln Tyr Val Gly Ile Arg Leu Leu Val Glu Gly Phe Thr Ile Lys Lys Pro Met Ala Met Cys His Arg Arg Met Gly Val Arg Pro Ala Val Pro Leu Leu Thr Gln Arg Gly Ser Gly Glu Gly Lys Asp Ser Gly Thr Pro Thr His Arg Ser Ala Ser Arg Lys Gly Thr Gly Ala Arg Ser Leu Gly His Ser Glu Lys Pro Val Ser Thr Ala Thr Thr Ser Ala Pro Gly Lys Gly Lys Lys Gly Lys Ala Lys Arg Ala Thr Ala Leu Val Cys Pro Asn Leu Trp Glu Trp Asp Ala Pro Ser Thr Arg Met Gly Cys Ile Phe Thr Met Thr Phe Ser Ser Gly Asp Arg Gln Pro His His Leu Asn Arg Leu Pro Leu Ser Pro Lys Asn Pro Gln Ala Leu Gly Lys Thr Ile Pro Pro Lys His Pro Ser Val Pro Arg Arg Phe Ile Pro Ala Leu Gln Ala Pro Pro Asn His Leu Asp Gln Pro Pro His Gln Arg Ala Thr Ser Ser Lys <210> 29 <211> 220 <212> PRT
<213> Homo Sapiens <220>
<221> misc-feature <223> Incyte ID No: 7500181CD1 <400> 29 Met Trp Val Pro Val Val Phe Leu Thr Leu Ser Val Thr Trp Ile Gly Ala A1a Pro Leu Ile Leu Ser Arg Ile Val Gly Gly Trp Glu Cys Glu Lys His Ser Gln Pro Trp Gln Val Leu Val Ala Ser Arg Gly Arg Ala Val Cys Gly Gly Val Leu Val His Pro Gln Trp Val Leu Thr Ala Ala His Cys Ile Arg Asn Lys Ser Val Ile Leu Leu Gly Arg His Ser Leu Phe His Pro Glu Asp Thr Gly Gln Val Phe Gln Val Ser His Ser Phe Pro His Pro Leu Tyr Asp Met Ser Leu Leu Lys Asn Arg Phe Leu Arg Pro Gly Asp Asp Ser Ser Ile Glu Pro Glu Glu Phe Leu Thr Pro Lys Lys Leu Gln Cys Val Asp Leu His Val Ile Ser Asn Asp Val Cys Ala Gln Val His Pro Gln Lys Val Thr Lys Phe Met Leu Cys Ala Gly Arg Trp Thr Gly Gly Lys Ser Thr Cys Ser Gly Asp Ser Gly Gly Pro Leu Val Cys Asn Gly Val Leu Gln Gly Ile Thr Ser Trp Gly Ser Glu Pro Cys Ala Leu Pro Glu Arg Pro Ser Leu Tyr Thr Lys Val Val His Tyr Arg Lys Trp Ile Lys Asp Thr Ile Val Ala Asn Pro <210> 30 <211> 195 <212> PRT
<213> Homo sapiens <220>
<221> misc_feature <223> Incyte ID No: 7503799CD1 <400> 30 Met Glu Ala Phe Leu Gly Ser Arg Ser Gly Leu Trp Ala Gly Gly Pro Ala Pro Gly Gln Phe Tyr Arg Ile Pro Ser Thr Pro Asp Ser Phe Met Asp Pro Ala Ser Ala Leu Tyr Arg Gly Pro Ile Thr Arg Thr Gln Ile Asp Glu Glu Leu Leu Gly Asp Gly His Ser Tyr Ser Pro Arg Ala Ile His Ser Trp Leu Thr Arg Ala Met Tyr Ser Arg Arg Ser Lys Met Asn Pro Leu Trp Asn Thr Met Val Ile Gly Gly g0 g5 90 Tyr Ala Asp Gly Glu Ser Phe Leu Gly Tyr Val Asp Met Leu Gly Val Ala Tyr Glu Ala Pro Ser Leu Ala Thr Gly Tyr Gly Ala Tyr Leu Ala Gln Pro Leu Leu Arg Glu Val Leu Glu Lys Gln Pro Va1 Leu Ser Gln Thr Glu Ala Arg Asp Leu Val Glu Arg Cys Met Arg Val Leu Tyr Tyr Arg Asp Ala Arg Ser Tyr Asn Arg Phe Gln Thr Ala Thr Val Thr Glu Lys Gly Val Glu Ile Glu Gly Pro Leu Ser Thr Glu Thr Asn Trp Asp Ile Ala His Met Ile Ser Gly Phe Glu <210> 31 <211> 372 <212> PRT
<213> Homo sapiens <220>
<221> misc_feature <223> Incyte ID No: 7504602CD1 <400> 31 Met Val Trp Lys Val Ala Val Phe Leu Ser Val Ala Leu Gly Ile Gly Ala Val Pro Ile Asp Asp Pro Glu Asp Gly Gly Lys His Trp Val Val Ile Val Ala Gly Ser Asn Gly Trp Tyr Asn Tyr Arg His Gln Ala Asp Ala Cys His Ala Tyr Gln Ile Ile His Arg Asn Gly Ile Pro Asp Glu Gln Ile Val'Val Met Met Tyr Asp Asp Ile Ala Tyr Ser Glu Asp Asn Pro Thr Pro Gly Ile Val Ile Asn Arg Pro Asn Gly Thr Asp Val Tyr Gln Gly Val Pro Lys Asp Tyr Thr Gly Glu Asp Val Thr Pro Gln Asn Phe Leu Ala Val Leu Arg Gly Asp Ala Glu Ala Val Lys Gly Ile Gly Ser Gly Lys Val Leu Lys Ser Gly Pro Gln Asp His Val Phe Ile Tyr Phe Thr Asp His Gly Ser Thr Gly Ile Leu Val Phe Pro Asn Glu Asp Leu His Val Lys Asp Leu Asn Glu Thr Ile His Tyr Met Tyr Lys His Lys Met Tyr Arg Lys Met Val Phe Tyr Ile Glu Ala Cys Glu Ser Gly Ser Met Met Asn His Leu Pro Asp Asn Ile Asn Val Tyr Ala Thr Thr Ala Ala Asn Pro Arg Glu Ser Ser Tyr Ala Cys Tyr Tyr Asp Glu Lys Arg Ser Thr Tyr Leu Gly Asp Trp Tyr Ser Val Asn Trp Met Glu Asp Ser Asp Val Glu Asp Leu Thr Lys Glu Thr Leu His Lys G1n Tyr His Leu Val Lys Ser His Thr Asn Thr Ser His Val Met Gln Tyr Gly Asn Lys Thr Ile Ser Thr Met Lys Val Met Gln Phe Gln Gly Met Lys Arg Lys Ala Ser Ser Pro Val Pro Leu Pro Pro Val Thr His Leu Asp Leu Thr Pro Ser Pro Asp Val Pro Leu Thr Ile Met Lys Arg Lys Leu Met Asn Thr Asn Asp Leu Glu Glu Ser Arg Gln Leu Thr Glu Glu Ile Gln Arg His Leu Asp Asp Lys Ile Val His Gly Pro Arg Val Pro Trp Ser Leu Leu Lys Ser Cys Leu Leu Glu Ala Phe Pro Ser Val Ser Ala Pro Pro Thr Val Cys <210> 32 <211> 410 <212> PRT
<213> Homo sapiens <220>
<221> misc_feature <223> Incyte ID No: 5873533CD1 <400> 32 Met His Leu Thr Asn Tyr Ser Ile Asn Lys His Ser Ser Asn Phe Ser Arg Asp Ala His Ser Gly Ser Lys Arg Lys Leu Ser Thr Phe Ser Ala Tyr Leu Glu Asp His Ser Tyr Asn Val Glu Gln Ile Trp Arg Asp Ile Glu Asp Val Ile Ile Lys Thr Leu Ile Ser Ala His Pro Ile Ile Arg His Asn Tyr His Thr Cys Phe Pro Asn His Thr Leu Asn Ser Ala Cys Phe Glu Ile Leu Gly Phe Asp Ile Leu Leu Asp His Lys Leu Lys Pro Trp Leu Leu Glu Val Asn His Ser Pro g5 100 105 Ser Phe Ser Thr Asp Ser Arg Leu Asp Lys Glu Val Lys Asp Gly Leu Leu Tyr Asp Thr Leu Val Leu Ile Asn Leu Glu Ser Cys Asp Lys Lys Lys Val Leu Glu Glu Glu Arg Gln Arg Gly Gln Phe Leu Gln Gln Cys Cys Ser Arg Glu Met Arg Ile Glu Glu Ala Lys Gly Phe Arg Ala Val Gln Leu Lys Lys Thr Glu Thr Tyr Glu Lys Glu Asn Cys Gly Gly Phe Arg Leu Ile Tyr Pro Ser Leu Asn Ser Glu Lys Tyr Glu Lys Phe Phe Gln Asp Asn Asn Ser Leu Phe Gln Asn Thr Val Ala Ser Arg Ala Arg Glu Glu Tyr Ala Arg Gln Leu Ile Gln Glu Leu Arg Leu Lys Arg Glu Lys Lys Pro Phe Gln Met Lys Lys Lys Val Glu Met Gln Gly Glu Ser Ala Gly Glu Gln Val Arg Lys Lys Gly Met Arg Gly Trp Gln Gln Lys Gln Gln Gln Lys Asp Lys Ala Ala Thr Gln Ala Ser Lys Gln Tyr Ile Gln Pro Leu Thr Leu Val Ser Tyr Thr Pro Asp Leu Leu Leu Ser Val Arg Gly Glu Arg Lys Asn Glu Thr Asp Ser Ser Leu Asn Gln Glu Ala Pro Thr Glu Lys Ala Ser Ser Val Phe Pro Lys Leu Thr Ser Ala Lys Pro Phe Ser Ser Leu Pro Asp Leu Arg Asn Ile Asn Leu Ser Ser Ser Lys Leu Glu Pro Ser Lys Pro Asn Phe Ser Ile Lys Glu Ala Lys Ser Ala Ser Ala Val Asn Val Phe Thr Gly Thr Val Lys Thr Pro Pro Cys Leu Gly Ser Ala Thr Pro Ala Val Thr Ala Leu Ala Arg Arg Gly Ser Trp Met Cys Pro Pro Ser Ser Cys Arg Val Leu Arg Ala Ile Met Leu Leu <210> 33 <211> 265 <212> PRT
<213> Homo Sapiens <220>
<221> misc_feature <223> 2ncyte ID No: 71033239CD1 <400> 33 Met Ala Glu Asp Lys Glu Thr Lys His Gly Gly His Lys Asn Gly 1 5 10 . l5 Arg Lys Gly Gly Leu Ser Gly Thr Ser Phe Phe Thr Trp Phe Met Val Ile Ala Leu Leu Gly Val Trp Thr Ser Val Ala Val Val Trp Phe Asp Leu Val Asp Tyr Glu Glu Val Leu Gly Lys Leu Gly Ile Tyr Asp Ala Asp Gly Asp Gly Asp Phe Asp Val Asp Asp Ala Lys Val Leu Leu Gly Leu Lys Glu Arg Ser Thr Ser Glu Pro Ala Val Pro Pro Glu Glu Ala Glu Pro His Thr Glu Pro Glu Glu Gln Val Pro Val Glu Ala Glu Pro Gln Asn Ile Glu Asp Glu Ala Lys Glu Gln Ile Gln Ser Leu Leu His Glu Met Val His Ala Glu His Val Glu Gly Glu Asp Leu Gln Gln Glu Asp Gly Pro Thr Gly Glu Pro Gln Gln Glu Asp Asp Glu Phe Leu Met Ala Thr Asp Val Asp Asp Arg Phe Glu Thr Leu Glu Pro Glu Val Ser His Glu Glu Thr Glu His Ser Tyr His Val Glu Glu Thr Asp 5er Ser Glu Pro Val Val Glu Asp Glu Arg Leu His His Asp Thr Asp Asp Val Thr Tyr Gln Val Tyr Glu Glu Gln Ala Val Tyr Glu Pro Leu Glu Asn Glu Gly Ile Glu Ile Thr Glu Val Thr Ala Pro Pro Glu Asp Asn Pro Val Glu Asp Ser Gln Val Ile Val Glu Glu Val Ser Ile Phe Pro Val Glu Glu Gln Gln Glu Val Pro Pro Asp Thr <210> 34 <211> 146 <212> PRT
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte ID No: 7506001CD1 <400> 34 Met Ala Val Ala Asn Ser Ser Pro Val Asn Pro Val Val Phe Phe Asp Val Ser Ile Gly Gly Gln Glu Val Gly Arg Met Lys Ile Glu Leu ~Phe Ala Asp Val Val Pro Lys Thr Ala Glu Asn Phe Arg Gln Phe Cys Thr Gly Glu Phe Arg Lys Asp Gly Val Pro Ile Gly Tyr Lys Gly Ser Thr Phe His Arg Val Ile Lys Asp Phe Met Ile Gln Gly Gly Asp Phe Val Asn Ala Asn Ser Gly Pro Ser Thr Asn Gly Cys Gln Phe Phe Ile Thr Cys Ser Lys Cys Asp Trp Leu Asp Gly g5 100 105 Lys His Val Val Phe Gly Lys Ile Ile Asp Gly Leu Leu Val Met Arg Lys Ile Glu Asn Val Pro Thr Gly Pro Asn Asn Lys Pro Lys Leu Pro Val Val Ile Ser Gln Cys Gly Glu Met <210> 35 <211> 196 <212> PRT
<213> Homo Sapiens <220>
<221> mist-feature <223> Incyte ID No: 7506026CD1 <400> 35 Met Glu Asp Ser Met Asp Met Asp Met Ser Pro Leu Arg Pro Gln Asn Tyr Leu Phe Ala Val Glu Glu Asp Ala Glu Ser Glu Asp Glu Glu Glu Glu Asp Val Lys Leu Leu Ser Ile Ser Gly Lys Arg Ser Ala Pro Gly Gly Gly Ser Lys Val Pro Gln Lys Lys Val Lys Leu Ala Ala Asp Glu Asp Asp Asp Asp Asp Asp Glu Glu Asp Asp Asp Glu Asp Asp Asp Asp Asp Asp Phe Asp Asp Glu Glu Ala Glu Glu Lys Ala Pro Val Lys Lys Ser Ile Arg Asp Thr Pro Ala Lys Asn Ala Gln Lys Ser Asn Gln Asn Gly Lys Asp Ser Lys Pro Ser Ser Thr Pro Arg Ser Lys Gly Gln Glu Ser Phe Lys Lys Gln Glu Lys Thr Pro Lys Thr Pro Lys Gly Pro Ser Ser Val Glu Asp Ile Lys Ala Lys Met Gln Ala Ser Ile Glu Lys Gly Gly Ser Leu Pro Lys Val Glu Ala Lys Phe Ile Asn Tyr Val Lys Asn Cys Phe Arg Met Thr Asp Gln Glu Ala Ile Gln Asp Leu Trp Gln Trp Arg Lys Ser Leu <210> 36 <211> 431 <212> PRT
<213> Homo sapiens <220>
<221> misc-feature <223> Incyte ID No: 8167924CD1 <400> 36 Met Ser Asp Glu Gly Ser Arg Gly Ser Arg Leu Pro Leu Ala Leu Pro Pro Ala Ser Gln Gly Cys Ser Ser Gly Gly Gly Gly Gly Gly Ser Ser Ala Gly Gly~Ser Gly Asn Ser Arg Pro Pro Arg Asn Leu Gln Gly Leu Leu Gln Met Ala Ile Thr Ala Gly Ser Glu Glu Pro Asp Pro Pro Pro Glu Pro Met Ser Glu Glu Arg Arg Gln Trp Leu Gln Glu Ala Met Ser Ala Ala Phe Arg Gly Gln Arg Glu Glu Val g0 85 90 Glu Gln Met Lys Ser Cys Leu Arg Val Leu Ser Gln Pro Met Pro g5 100 105 Pro Thr Ala Gly Glu Ala Glu Gln Ala Ala Asp Gln Gln Glu Arg Glu Gly Ala Leu Glu Leu Leu Ala Asp Leu Cys Glu Asn Met Asp Asn Ala Ala Asp Phe Cys Gln Leu Ser Gly Met His Leu Leu Val Gly Arg Tyr Leu Glu Ala Gly Ala Ala Gly Leu Arg Trp Arg Ala Ala Gln Leu Ile Gly Thr Cys Ser Gln Asn Val Ala Ala Ile Gln Glu Gln Val Leu Gly Leu Gly Ala Leu Arg Lys Leu Leu Arg Leu Leu Asp Arg Asp Ala Cys Asp Thr Val Arg Val Lys Ala Leu Phe Ala Ile Ser Cys Leu Val Arg Glu Gln Glu Ala Gly Leu Leu Gln Phe Leu Arg Leu Asp Gly Phe Ser Val Leu Met Arg Ala Met Gln Gln Gln Val Gln Lys Leu Lys Val Lys Ser Ala Phe Leu Leu Gln Asn Leu Leu Val Gly His Pro Glu His Lys Gly Thr Leu Cys Ser Met Gly Met Val Gln Gln Leu Val Ala Leu Val Arg Thr Glu His Ser Pro Phe His Glu His Val Leu Gly Ala Leu Cys Arg Val Cys Ala Ser Val Gly Ser Arg Asn Trp Ala Trp Arg Ser Ser Ser Ala Thr Ala Val Ser Cys Cys Ser Ser Met Arg Ser Thr Arg Arg Ser Trp Ser Ser Val Lys Ser Cys Tyr Arg Pro Val Ser Pro Ala Gln Arg Thr Thr Ala Trp Ile Gly Glu Thr Arg Trp Leu Leu Ala Pro Phe Ser Val Gly Thr Pro Gly Leu Leu Pro Pro Ser Pro Pro Thr Arg Pro Ser Pro Lys Gly Ser Gln Gly Leu Gly Ala Trp Thr Gln Gly Val Pro Ala Arg Leu Cys Ala Val Pro Gly Arg Gly Ala Glu Lys Gly Thr Ser Ser Leu Asp Pro Thr Ser His Ala Leu Thr Leu Ile Pro Val Leu Leu Ser Thr Gln Leu Phe Gln <210> 37 <211> 300 <212> PRT
<213> Homo Sapiens <220>
<221> mist-feature <223> Incyte ID No: 2365313CD1 <400> 37 Met Glu Pro Pro Met Glu Pro Ser G1y Gly Glu Gln Glu Pro Gly Ala Val Arg Phe Leu Asp Leu Pro Trp Glu Asp Val Leu Leu Pro His Val Leu Asn Arg Val Pro Leu Arg Gln Leu Leu Arg Leu Gln Arg Val Ser Arg Ala Phe Arg Ser Leu Val Gln Leu His Leu Ala Gly Leu Arg Arg Phe Asp Ala Ala Gln Val Gly Pro Gln Ile Pro Arg Ala Ala Leu Ala Arg Leu Leu Arg Asp Ala Glu Gly Leu Gln Glu Leu Ala Leu Ala Pro Cys His Glu Trp Leu Ser Asp Glu Asp g5 100 105 Leu Val Pro Val Leu Ala Arg Asn Pro Gln Leu Arg Ser Val Ala Leu Gly Gly Cys Gly Gln Leu Ser Arg Arg Ala Leu Gly Ala Leu Ala Glu G1y Cys Pro Arg Leu Gln Arg Leu Ser Leu Ala His Cys Asp Trp Val Asp Gly Leu Ala Leu Arg Gly Leu Ala Asp Arg Cys Pro Ala Leu Glu Glu Leu Asp Leu Thr Ala Cys Arg Gln Leu Lys Asp Glu Ala Ile Val Tyr Leu Ala Gln Arg Arg Gly Ala Gly Leu Arg Ser Leu Ser Leu Ala Val Asn Ala Asn Val Gly Asp Ala Ala Val Gln Glu Leu Ala Arg Asn Cys Pro Glu Leu His His Leu Asp Leu Thr Gly Cys Leu Arg Val Gly Ser Asp Gly Val Arg Thr Leu Ala Glu Tyr Cys Pro Val Leu Arg Ser Leu Arg Val Arg His Cys His His Val Ala Glu Ser Ser Leu Ser Arg Leu Arg Lys Arg Gly Val Asp Ile Asp Val Glu Pro Pro Leu His Gln Ala Leu Val Leu Leu Gln Asp Met Ala Gly Phe Ala Pro Phe Va1 Asn Leu Gln Val 2g0 295 300 <210> 38 <211> 554 <212> PRT
<213> Homo sapiens <220>
<221> misc-feature <223> Incyte ID No: 7503156CD1 <400> 38 Met Ala Glu Leu Ser Glu Glu Ala Leu Leu Ser Val Leu Pro Thr Ile Arg Val Pro Lys Ala Gly Asp Arg Val His Lys Asp Glu Cys Ala Phe Ser Phe Asp Thr Pro Glu Ser Glu Gly Gly'Leu Tyr Ile Cys Met Asn Thr Phe Leu Gly Phe Gly Lys Gln Tyr Val Glu Arg His Phe Asn Lys Thr Gly Gln Arg Val Tyr Leu His Leu Arg Arg Thr Arg Arg Pro Lys Glu Glu Asp Pro Ala Thr Gly Thr Gly Asp Pro Pro Arg Lys Lys Pro Thr Arg Leu Ala Ile Gly Val Glu Gly Gly Phe Asp Leu Ser Glu Glu Lys Phe Glu Leu Asp Glu Asp Val Lys Ile Val Ile Leu Pro Asp Tyr Leu Glu Ile Ala Arg Asp Gly Leu Gly Gly Leu Pro Asp Ile Val Arg Asp Arg Val Thr Ser Ala Val Glu Ala Leu Leu Ser Ala Asp Ser Ala Ser Arg Lys Gln Glu Val Gln Ala Trp Asp Gly Glu Val Arg Gln Val Ser Lys His Ala Phe Ser Leu Lys Gln Leu Asp Asn Pro Ala Arg Ile Pro Pro Cys Gly Trp Lys Cys Ser Lys Cys Asp Tyr Ile Met Gln Leu Pro Val Pro Met Asp Ala Ala Leu Asn Lys Glu Glu Leu Leu Glu Tyr Glu Glu Lys Lys Arg Gln Ala Glu Glu Glu Lys Met Ala Leu Pro Glu Leu Val Arg Ala Gln Val Pro Phe Ser Ser Cys Leu Glu Ala Tyr Gly Ala Pro Glu Gln Val Asp Asp Phe Trp Ser Thr Ala Leu Gln Ala Lys Ser Val Ala Val Lys Thr Thr Arg Phe Ala Ser Phe Pro Asp Tyr Leu Val Tle Gln Ile Lys Lys Phe Thr Phe Gly Leu Asp Trp Val Pro Lys Lys Leu Asp Val Ser Ile Glu Met Pro Glu Glu Leu Asp Ile Ser Gln Leu Arg Gly Thr Gly Leu Gln Pro Gly Glu 320 325 ~ 330 Glu Glu Leu Pro Asp Ile Ala Pro Pro Leu Val Thr Pro Asp Glu Pro Lys Ala Pro Met Leu Asp Glu Ser Val Ile Tle Gln Leu Val Glu Met Gly Phe Pro Met Asp Ala Cys Arg Lys Ala Val Tyr Tyr Thr Gly Asn Ser Gly Ala Glu Ala Ala Met Asn Trp Val Met Ser His Met Asp Asp Pro Asp Phe Ala Asn Pro Leu Ile Leu Pro Gly Ser Ser Gly Pro Gly Ser Thr Ser Ala Ala Ala Asp Pro Pro Pro Glu Asp Cys Val Thr Thr Ile Val Ser Met Gly Phe Ser Arg Asp Gln Ala Leu Lys Ala Leu Arg Ala Thr Asn Asn Ser Leu Glu Arg Ala Val Asp Trp Ile Phe Ser His Ile Asp Asp Leu Asp Ala Glu Ala Ala Met Asp Ile Ser Glu Gly Arg Ser Ala Ala Asp Ser Ile Ser Glu Ser Val Pro Val Gly Pro Lys Val Arg Asp Gly Pro Gly Lys Tyr Gln Leu Phe Ala Phe Ile Ser His Met Gly Thr Ser Thr Met Cys Gly His Tyr Val Cys His Ile Lys Lys Glu G1y Arg Trp Val Ile Tyr Asn Asp Gln Lys Val Cys Ala Ser Glu Lys Pro Pro Lys Asp Leu Gly Tyr Ile Tyr Phe Tyr Gln Arg Val Ala Ser <210> 39 <211> 278 <212> PRT
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte ID No: 7505980CD1 <400> 39 Met Glu Thr Arg Tyr Asn Leu Lys Ser Pro Ala Val Lys Arg Leu Met Lys Glu Ala Ala Glu Leu Lys Asp Pro Thr Asp His Tyr His Ala Gln Pro Leu Glu Asp Asn Leu Phe Glu Trp His Phe Thr Val 35 40 ' 45 Arg Gly Pro Pro Asp Ser Asp Phe Asp Gly Gly Val Tyr His Gly Arg Ile Val Leu Pro Pro Glu Tyr Pro Met Lys Pro Pro Ser Ile Ile Leu Leu Thr Ala Asn Gly Arg Phe Glu Val Gly Lys Lys Ile Cys Leu Ser Ile Ser Gly His His Pro Glu Thr Trp Gln Pro Ser Trp Ser Ile Arg Thr Ala Leu Leu Ala Ile Ile Gly Phe Met Pro Thr Lys Gly Glu Gly Ala Ile Gly Ser Leu Asp Tyr Thr Pro Glu Glu Arg Arg Ala Leu Ala Lys Lys Ser Gln Asp Phe Cys Cys Glu Gly Cys Gly Ser Ala Met Lys Asp Val Leu Leu Pro Leu Lys Ser Gly Ser Asp Ser Ser Gln Ala Asp Gln Glu Ala Lys Glu Leu Ala Arg Gln Ile Ser Phe Lys Tyr Gly Leu Gln Asn Ser Ser Ala Ala Ser Phe His Gln Pro Thr Gln Pro Val Ala Lys Asn Thr Ser Met Ser Pro Arg Gln Arg Arg Ala Gln Gln Gln Ser Gln Arg Arg Leu Ser Thr Ser Pro Asp Val Ile Gln Gly His Gln Pro Arg Asp Asn His Thr Asp His Gly Gly Ser Ala Val Leu Ile Val Ile Leu Thr Leu Ala Leu Ala Ala Leu Ile Phe Arg Arg Ile Tyr Leu Ala Asn Glu Tyr Ile Phe Asp Phe Glu Leu <210> 40 <211> 688 <212> PRT
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte ID No: 7646577CD1 <400> 40 Met Asp Arg Cys Lys His Val Gly Arg Leu Arg Leu Ala Gln Asp His Ser Ile Leu Asn Pro Gln Lys Trp Cys Cys Leu Glu Cys Ala Thr Thr Glu Ser Val Trp Ala Cys Leu Lys Cys Ser His Val Ala Cys Gly Arg Tyr Ile Glu Asp His Ala Leu Lys His Phe Glu Glu Thr Gly His Pro Leu Ala Met Glu Val Arg Asp Leu Tyr Val Phe Cys Tyr Leu Cys Lys Asp Tyr Val Leu Asn Asp Asn Pro Glu Gly Asp Leu Lys Leu Leu Arg Ser Ser Leu Leu Ala Val Arg Gly Gln g5 100 105 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 Ala Leu Trp Tyr Arg Arg Gln Arg Leu Leu Ala Arg Thr Leu Arg Leu Trp Phe Glu Lys Ser Ser Arg Gly Gln Ala Lys Leu Glu Gln Arg Arg Gln Glu Glu Ala Leu Glu Arg Lys Lys Glu Glu Ala Arg Arg Arg Arg Arg Glu 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 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 Tle 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 Glu 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 Glu 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 Asn Val Cys Ser Val 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 Gln Val Gln Ser Ser Asn Asn Asp Glu Gly Arg Pro Gln Thr Phe Ser <210> 41 <211> 1544 <212> DNA
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte ID No: 7500287CB1 <400>, 41 aggaactcac acagcttttg gcctgagccc ccgttaccaa gagaaaggag gtttttgcca 60 aggactccaa ggggagtgca cttgatgctg gtcgggaccc aaagcgccca gccctccctg 120 agacattgtg tgagtcgggc tgggcctcaa acacggcccc cactgcccca ccccagccag 180 ggtggtgctt gtgtgggtag gactttaaat ccagctgcca gacccctgga cgggagaagg 240 agagacggct ggccaccatg cacggctcct gcagtttcct gatgcttctg ctgccgctac 300 tgctactgct ggtggccacc acaggccccg ttggagccct cacagatgag gagaaacgtt 360 tgatggtgga gctgcacaac ctctaccggg cccaggtatc cccgccggcc tcagacatgc 420 tgcacatgag atgggacgag gagctggccg ccttcgccaa ggcctacgca cggcagtgcg 480 tgtggggcca caacaaggag cgcgggcgcc gcggcgagaa tctgttcgcc atcacagacg 540 agggcatgga cgtgccgctg gccatggagg agtggcacca cgagcgtgag cactacaacc 600 tcagcgccgc cacctgcagc ccaggccaga tgtgcggcca ctacacgcag gtggtatggg 66.0 ccaagacaga gaggatcggc tgtggttccc acttctgtga gaagctccag ggtgttgagg 720 agaccaacat cgaattactg gtgtgcaact atgagcctcc ggggaacgtg aaggggaaac 780 ggccctacca ggaggggact ccgtgctccc aatgtccctc tggctaccac tgcaagaact 840 ccctctgtga acccatcgga agcccggaag atgctcagga tttgccttac ctggtaactg 900 aggccccatc cttccgggcg actgaagcat cagactctag gaaaatgggt gcagagggcc 960 ctgacaagcc tagcgtcgtg tcagggctga actcgggccc tggtcatgtg tggggccctc 1020 tcctgggact actgctcctg cctcctctgg tgttggctgg aatcttctga aggggatacc 1080 actcaaaggg tgaagaggtc agctgtcctc ctgtcatctt ccccaccctg tccccagccc 1140 ctaaacaaga tacttcttgg ttaaggccct ccggaaggga aaggctacgg ggcatgtgcc 1200 tcatcacacc atccatcctg gaggcacaag gcctggctgg ctgcgagctc aggaggccgc 1260 ctgaggactg cacaccgggc ccacacctot cctgcccctc cctcctgagt cctgggggtg 1320 ggaggatttg agggagctca ctgcctacct ggcctggggc tgtctgccca cacagcatgt 1380 gcgctctccc tgagtgcctg tgtagctggg gatggggatt cctaggggca gatgaaggac 1440 aagccccact ggagtggggt tctttgagtg ggggaggcag ggacgaggga aggaaagtaa 1500 ctcctgactc tccaataaaa acctgtccaa cctgtgaaaa aaaa 1544 <210> 42 <211> 1586 <212> DNA
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte ID No: 7500511CB1 <400> 42 caaattttcc agccgatcac tggagctgac ttccgcaatc ccgatggaat aaatctagca 60 cccctgatgg tgtgcccaca ctttgctgcc gaaacgaagc cagacaacag atttccatca 120 gcaggatgtg ggggctcaag gttctgctgc tacctgtggt gagctttgct ctgtaccctg 180 aggagatact ggacacccac tgggagctat ggaagaagac ccacaggaag caatataaca 240 acaagaccag tgaagaggtg gttcagaaga tgactggact caaagtaccc ctgtctcatt 300 cccgcagtaa tgacaccctt tatatcccag aatgggaagg tagagcccca gactctgtcg 360 actatcgaaa gaaaggatat gttactcctg tcaaaaatca gggtcagtgt ggttcctgtt 420 gggcttttag ctctgtgggt gccctggagg gccaactcaa gaagaaaact ggcaaactct 480 taaatctgag tccccagaac ctagtggatt gtgtgtctga gaatgatggc tgtggagggg 540 gctacatgac caatgccttc caatatgtgc agaagaaccg gggtattgac tctgaagatg 600 cctacccata tgtgggacag gaagagagtt gtatgtacaa cccaacaggc aaggcagcta 660 aatgcagagg gtacagagag atccccgagg ggaatgagaa agccctgaag agggcagtgg 720 cccgagtggg acctgtctct gtggccattg atgcaagcct gacctccttc cagttttaca 780 gcaaaggtgt gtattatgat gaaagctgca atagcgataa tctgaaccat gcggttttgg 840 cagtgggata tggaatccag aagggaaaca agcactggat aattaaaaac agctggggag 900 aaaactgggg aaacaaagga tatatcctca tggctcgaaa taagaacaac gcctgtggca 960 ttgccaacct ggccagcttc cccaagatgt gactccagcc agccaaatcc atcctgctct 1020 'tccatttctt ccacgatggt gcagtgtaac gatgcacttt ggaagggagt tggtgtgcta 1080 tttttgaagc agatgtggtg atactgagat tgtctgttca gtttccccat ttgtttgtgc 1140 ttcaaatgat ccttcctact ttgcttctct ccacccatga cctttttcac tgtggccatc 1200 aggactttcc ctgacagctg tgtactctta ggctaagaga tgtgactaca gcctgcccct 1260 gactgtgttg tcccagggct gatgctgtac aggtacaggc tggagatttt cacataggtt 1320 agattctcat tcacgggact agttagcttt aagcacccta gaggactagg gtaatctgac 1380 ttctcacttc ctaagttccc ttctatatcc tcaaggtaga aatgtctatg ttttctactc~1440 caattcataa atctattcat aagtctttgg tacaagttta catgataaaa agaaatgtga 1500 tttgtcttcc cttctttgca cttttgaaat aaagtattta tctcctgtct acagtttaat 1560 aaatagcatc tagtacacaa aaaaaa 1586 <210> 43 <211> 2368 <212> DNA
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte ID No: 7500273CB1 <400> 43 ctaggactga tctccaggac cagcactctt ctcccagccc ttagggtcct gctcggccaa 60 ggccttccct gccatgcgac ctgtcagtgt ctggcagtgg agcccctggg ggctgctgct 120 gtgcctgctg tgcagttcgt gcttggggtc tccgtcccct tccacgggcc ctgagaagaa 180 ggccgggagc caggggcttc ggttccggct ggctggcttc cccaggaagc cctacgaggg 240 ccgcgtggag atacagcgag ctggtgaatg gggcaccatc tgcgatgatg acttcacgct 300 gcaggctgcc cacatcctct gccgggagct gggcttcaca gaggccacag gctggaccca 360 cagtgccaaa tatggccctg gaacaggccg catctggctg gacaacttga gctgcagtgg 420 gaccgagcag agtgtgactg aatgtgcctc ccggggctgg gggaacagtg actgtacgca 480 cgatgaggat gctggggtca tctgcaaaga ccagcgcctc cctggcttct cggactccaa 540 tgtcattgag gcccgtgtcc gtctaaaggg cggcgcccac cctggagagg gccgggtaga 600 agtcctgaag gccagcacat ggggcacagt ctgtgaccgc aagtgggacc tgcatgcagc 660 cagcgtggtg tgtcgggagc tgggcttcgg gagtgctcga gaagctctga gtggcgctcg 720 catggggcag ggcatgggtg ctatccacct gagtgaagtt cgctgctctg gacaggagct 780 ctccctctgg aagtgccccc acaagaacat cacagctgag gattgttcac atagccagga 840 tgccggggtc cggtgcaacc taccttacac tggggcagag accaggatcc gactcagtgg 900 gggccgcagc caacatgagg ggcgagtcga ggtgcaaata gggggacctg ggccccttcg 960 ctggggcctc atctgtgggg atgactgggg gaccctggag gccatggtgg cctgtaggca 1020 actgggtctg ggctacgcca accacggcct gcaggagacc tggtactggg actctgggaa 1080 tataacagag gtggtgatga gtggagtgcg ctgcacaggg actgagctgt ccctggatca 1140 gtgtgcccat catggcaccc acatcacctg caagaggaca gggacccgct tcactgctgg 1200-agtcatctgt tctgagactg catcagatct gttgctgcac tcagcactgg tgcaggagac 1260 cgcctacatc gaagaccggc ccctgcatat gttgtactgt gctgcggaag agaactgcct 1320 ggccagctca gcccgctcag ccaactggcc ctatggtcac cggcgtctgc tccgattctc 1380 ctcccagatc cacaacctgg gacgagctga cttcaggccc aaggctgggc gccactcctg 144 0 ggtgtggcac gagtgccatg ggcattacca cagcatggac atcttcactc actatgatat 1500 cctcacccca aatggcacca aggtggctga gggccacaaa gctagtttct gtctcgaaga 1560 cactgagtgt caggaggatg tctccaagcg gtatgagtgt gccaactttg gagagcaagg 1620 catcactgtg ggttgctggg atctctaccg gcatgacatt gactgtcagt ggattgacat 1680 cacggatgtg aagccaggaa actacattct ccaggttgtc atcaacccaa actttgaagt 1740 agcagagagt gactttacca acaatgcaat gaaatgtaac tgcaaatatg atggacatag 1800 aatctgggtg cacaactgcc acattggtga tgccttcagt gaagaggcca acaggaggtt 1860 tgaacgctac cctggccaga ccagcaacca gattatctaa gtgccactgc cctctgcaaa 1920 ccaccactgg cccctaatgg caggggtctg aggctgccat tacctcagga gcttaccaag 1980 aaacccatgt cagcaaccgc actcatcaga ccatgcacta tggatgtgga actgtcaagc 2040 agaagttttc accctccttc agaggccagc tgtcagtatc tgtagccaag catgggaatc 2100 tttgctccca ggcccagcac cgagcagaac agaccagagc ccaccacacc acaaagagca 2160 gcacctgact aactgcccac aaaagatggc agcagctcat tttctttaat aggaggtcag 2220 gatggtcagc tccagtatct cccctaagtt tagggggata cagctttacc tctagccttt 2280 tggtggggga aaagatccag ccctcccacc tcatttttta ctataatatg ttgctaggta 2340 taattttatt ttatataaaa agtgttga 2368 <210> 44 <211> 801 <212> DNA
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte ID No: 7500183CB1 <400> 44 accacctgca cccggagagc tgtgtcacca tgtgggtccc ggttgtcttc ctcaccctgt 60 ccgtgacgtg gattggtgct gcacccctca tcctgtctcg gattgtggga ggctgggagt 120 gcgagaagca ttcccaaccc tggcaggtgc ttgtggcctc tcgtggcagg gcagtctgcg 180 gcggtgttct ggtgcacccc cagtgggtcc tcacagctgc ccactgcatc aggaagccag 240 gtgatgactc cagccacgac ctcatgctgc tccgcctgtc agagcctgcc gagctcacgg 300 atgctgtgaa ggtcatggac ctgcccaccc aggagccagc actggggacc acctgctacg 360 cctcaggctg gggcagcatt gaaccagagg agttcttgac cccaaagaaa cttcagtgtg 420 tggacctcca tgttatttcc aatgacgtgt gtgcgcaagt tcaccctcag aaggtgacca 480 agttcatgct gtgtgctgga cgctggacag ggggcaaaag cacctgctcg ggtgattctg 540 ggggcccact tgtctgtaat ggtgtgcttc aaggtatcac gtcatggggc agtgaaccat 600 gtgccctgcc cgaaaggcct tccctgtaca ccaaggtggt gcattaccgg aagtggatca 660 aggacaccat cgtggccaac ccctgagcac ccctatcaac cccctattgt agtaaacttg 720 gaaccttgga aatgaccagg ccaagactca agcctcccca gttctactga cctttgtcct 780 taggtgtgag gtccagggtt g <210> 45 <211> 756 <212> DNA
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte ID No: 7499957CB1 <400> 45 gctttcttct cgtcggtgtt cccggctgct atagagccgg gtgagagagc gagcgcccgt 60 cggcgggtgt cgagagcggg ttgcctcgcg ctgacccttc ccgccctcct tctcgtcaca 120 caccaggtcc ccgcggaagc cgcggtgtcg gcgccatggc ggagctgacg gctcttgaga 180 gtctcatcga gatgggcttc cccaggggac gcgcggagaa ggctctggcc ctcacaggga 240 accagggcat cgaggctgcg atggactggc tgatggagca cgaagacgac cccgatgtgg 300 acgagccttt agagactccc cttggacata tcctgggacg ggagcccact tcctcagagc 360 caggtcctgt tccctcttct cccagccagg agcctcccac caagcgggag tatgaccagt 420 gtcgcataca ggtcaggctg ccagatggga cctcactgac ccagacgttc cgggcccggg 480 aacagctggc agctgtgagg ctctatgtgg agctccaccg tggggaggaa ctaggtgggg 540 gccaggaccc tgtgcaattg ctcagtggct tccccagacg ggccttctca gaagctgaca 600 tggagcggcc tctgcaggag ctgggactcg tgccttctgc tgttctcat~ gtggccaaga 660 aatgtcccag ctgagggcct ttgtcccatt gtccctctgt gaccccttca tctttgataa 720 agcactgaca tctccttcct aataaataga ccctga 756 <210> 46 <211> 2952 <212> DNA
<213> Homo sapiens <220>
<221> misc_feature <223> Incyte ID No: 7500001CB1 <400> 46 gcctccgagg ccaaggccgc tgctactgcc gccgctgctt cttagtgccg cgttcgccgc 60 ctgggttgtc accggcgccg ccgccgagga agccactgca accaggaccg gagtggaggc 120 ggcgcagcat gaagcggcgc aggcccgctc catagcgcac gtcgggacgg tccgggcggg 180 gccgggggga aggaaaatgc aacatggcag cagcaatgga aacagaacag ctgggtgttg 240 agatatttga aactgcggac tgtgaggaga atattgaatc acaggatcgg cctaaattgg 300 agccttttta tgttgagcgg tattcctgga gtcagcttaa aaagctgctt gccgatacca 360 gaaaatatca tggctacatg atggctaagg caccacatga tttcatgttt gtgaagagga 420 atgatccaga tggacctcat tcagacagaa tctattacct tgccatgtct ggtgagaaca 480 gagaaaatac actgttttat tctgaaattc ccaaaactat caatagagca gcagtcttaa 540 tgctctcttg gaagcctctt ttggatcttt ttcaggcaac actggactat ggaatgtatt 600 ctcgagaaga agaactatta agagaaagaa aacgcattgg aacagtcgga attgcttctt 660 acgattatca ccaaggaagt ggaacatttc tgtttcaagc cggtagtgga atttatcacg 720 taaaagatgg agggccacaa ggatttacgc aacaaccttt aaggcccaat ctagtggaaa 780 ctagttgtcc caacatacgg atggatccaa aattatgccc tgctgatcca gactggattg 840 cttttataca tagcaacgat atttggatat ctaacatcgt aaccagagaa gaaaggagac 900 tcacttatgt gcacaatgag ctagccaaca tggaagaaga tgccagatca gctggagtcg 960 ctacctttgt tctccaagaa gaatttgata gatattctgg ctattggtgg tgtccaaaag 1020 ctgaaacaac tcccagtggt ggtaaaattc ttagaattct atatgaagaa aatgatgaat 108 0 ctgaggtgga aattattcat gttacatccc ctatgttgga aacaaggagg gcagattcat 1140 tccgttatcc taaaacaggt acagcaaatc ctaaagtcac ttttaagatg tcagaaataa 1200 tgattgatgc tgaaggaagg atcatagatg tcatagataa ggaactaatt caaccttttg 1260 agattctatt tgaaggagtt gaatatattg ccagagctgg atggactcct gagggaaaat 1320 atgcttggtc catcctacta gatcgctccc agactcgcct acagatagtg ttgatctcac 1380 ctgaattatt tatcccagta gaagatgatg ttatggaaag gcagagactc attgagtcag 1440' tgcctgattc tgtgacgcca ctaattatct atgaagaaac aacagacatc tggataaata 1500 tccatgacat ctttcatgtt tttccccaaa gtcacgaaga ggaaattgag tttatttttg 1560 cctctgaatg caaaacaggt ttccgtcatt tatacaaaat tacatctatt ttaaaggaaa 1620 gcaaatataa acgatccagt ggtgggctgc ctgctccaag tgatttcaag tgtcctatca 1680 aagaggagat agcaattacc agtggtgaat gggaagttct tggccggcat ggatctaata 174 0 tccaagttga tgaagtcaga aggctggtat attttgaagg caccaaagac tcccctttag 1800 agcatcacct gtacgtagtc agttacgtaa atcctggaga ggtgacaagg ctgactgacc 1860 gtggctactc acattcttgc tgcatcagtc agcactgtga cttctttata agtaagtata 1920 gtaaccagaa gaatccacac tgtgtgtccc tttacaagct atcaagtcct gaagatgacc 1980 caacttgcaa aacaaaggaa ttttgggcca ccattttgga ttcagcaggt cctcttcctg 2040 actatactcc tccagaaatt ttctcttttg aaagtactac tggatttaca ttgtatggga 2100 tgctctacaa gcctcatgat ctacagcctg gaaagaaata tcctactgtg ctgttcatat 2160 atggtggtcc tcaggtgcag ttggtgaata atcggtttaa aggagtcaag tatttccgct 2220 tgaataccct agcctctcta ggttatgtgg ttgtagtgat agacaacagg ggatcctgtc 2280 accgagggct taaatttgaa ggcgccttta aatataaaat ggttgctatt gctggggccc 2340 cagtcactct gtggatcttc tatgatacag gatacacgga acgttatatg ggtcaccctg 2400 accagaatga acagggctat tacttaggat ctgtggccat gcaagcagaa aagttcccct 2460' ctgaaccaaa tcgtttactg ctcttacatg gtttcctgga tgagaatgtc cattttgcac 2520 ataccagtat attactgagt tttttagtga gggctggaaa gccatatgat ttacagatct 2580 atcctcagga gagacacagc ataagagttc ctgaatctgg agaacattat gaactgcatc 2640 ttttgcacta ccttcaagaa aaccttggat cacgtattgc tgctctaaaa gtgatataat 2700 tttgacctgt gtagaactct ctggtataca ctggctattt aaccaaatga ggaggtttaa 2760 tcaacagaaa acacagaatt gatcatcaca ttttgatacc tgccatgtaa catctactcc 2820 tgaaaataaa tgtggtgcca tgcaggggtc tacggtttgt ggtagtaatc taatacctta 2880 DEMANDE OU BREVET VOLUMINEUX
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Claims (135)

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:1-40, 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:1-3, SEQ ID NO:7-13, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:25-26, SEQ
ID NO:28, SEQ ID NO:32, and SEQ ID NO:36, c) a polypeptide comprising a naturally occurring amino acid sequence at least 91%
identical to an amino acid sequence selected from the group consisting of SEQ
ID
NO:35 and SEQ ID NO:40, d) a polypeptide comprising a naturally occurring amino acid sequence at least 92%
identical to the amino acid sequence of SEQ ID NO:31, e) a polypeptide comprising a naturally occurring amino acid sequence at least 93%
identical to the amino acid sequence of SEQ ID NO:30, f) a polypeptide comprising a naturally occurring amino acid sequence at least 97%
identical to the amino acid sequence of SEQ ID NO:23, g) a polypeptide comprising a naturally occurring amino acid sequence at least 99%
identical to an amino acid sequence selected from the group consisting of SEQ
ID
NO:33 and SEQ ID NO:37, h) a polypeptide consisting essentially of a naturally occurring amino acid sequence at least 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NO:14-19, SEQ ID NO:21, SEQ ID NO:24, SEQ ID NO:27, SEQ ID
NO:29, SEQ ID NO:34, and SEQ ID NO:38-39, i) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1-40, and j) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1-40.

2. An isolated polypeptide of claim 1 comprising an amino acid sequence selected from the group consisting of SEQ ID NO:1-40.

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

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

5. An isolated polynucleotide of claim 4 comprising a polynucleotide sequence selected from the group consisting of SEQ ID NO:41-80.

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

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

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

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

10. A method of claim 9, wherein the polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NO:1-40.

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:41-80, 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:41-66, SEQ ID NO:68-75, and SEQ ID NO:77-80, c) a polynucleotide comprising a naturally occurring polynucleotide sequence at least 93Q1o identical to the polynucleotide sequence of SEQ ID NO:76, d) a polynucleotide comprising a naturally occurring polynucleotide sequence at least 96% identical to the polynucleotide sequence of SEQ ID NO:67, e) a polynucleotide complementary to a polynucleotide of a), f) a polynucleotide complementary to a polynucleotide of b), g) a polynucleotide complementary to a polynucleotide of c), h) a polynucleotide complementary to a polynucleotide of d), and i) an RNA equivalent of a)-h).

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

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

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

16. A method of detecting a target polynucleotide in a sample, said target polynucleotide having a sequence of a polynucleotide of claim 12, the method comprising:
a) amplifying said target polynucleotide or fragment thereof using polymerase chain reaction amplification, and b) detecting the presence or absence of said amplified target polynucleotide or fragment thereof, and, optionally, if present, the amount thereof.

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

18. A composition of claim 17, wherein the polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NO:1-40.

19. A method for treating a disease or condition associated with decreased expression of functional PMMM, 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 PMMM, 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 PMMM, comprising administering to a patient in need of such treatment a composition of claim 24.

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

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

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

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

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

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

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

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

34. A composition of claim 32, further comprising a label.

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

36. A method of preparing a polyclonal antibody with the specificity of the antibody of claim 11, the method comprising:
a) immunizing an animal with a polypeptide consisting of an amino acid sequence selected from the group consisting of SEQ ID NO:1-40, or an immunogenic fragment thereof, under conditions to elicit an antibody response, b) isolating antibodies from the animal, and c) screening the isolated antibodies with the polypeptide, thereby identifying a polyclonal antibody which specifically binds to a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:1-40.

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

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

39. A method of making a monoclonal antibody with the specificity of the antibody of claim 11, the method comprising:
a) immunizing an animal with a polypeptide consisting of an amino acid sequence selected from the group consisting of SEQ ID NO:1-40, 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 specifically binds to a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:1-40.

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

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

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

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

44. A method of detecting a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:1-40 in a sample, the method comprising:
a) incubating the antibody of claim 11 with the sample under conditions to allow specific binding of the antibody and the polypeptide, and b) detecting specific binding, wherein specific binding indicates the presence of a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:1-40 in the sample.

45. A method of purifying a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:1-40 from a sample, the method comprising:
a) incubating the antibody of claim 11 with the sample under conditions to allow specific binding of the antibody and the polypeptide, and b) separating the antibody from the sample and obtaining the purified polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID
NO:1-40.

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

69. A polypeptide of claim 1, comprising the amino acid sequence of SEQ ID
NO:14.

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

71. A polypeptide of claim 1, comprising the amino acid sequence of SEQ ID
NO:16.

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

73. A polypeptide of claim 1, comprising the amino acid sequence of SEQ ID
NO:18.

74. A polypeptide of claim 1, comprising the amino acid sequence of SEQ ID
NO:19.

75. A polypeptide of claim 1, comprising the amino acid sequence of SEQ ID
NO:20.

76. A polypeptide of claim 1, comprising the amino acid sequence of SEQ ID
NO:21.

77. A polypeptide of claim 1, comprising the amino acid sequence of SEQ ID
NO:22.

78. A polypeptide of claim 1, comprising the amino acid sequence of SEQ ID
NO:23.

79. A polypeptide of claim 1, comprising the amino acid sequence of SEQ ID
NO:24.

80. A polypeptide of claim 1, comprising the amino acid sequence of SEQ ID
NO:25.

81. A polypeptide of claim 1, comprising the amino acid sequence of SEQ ID
NO:26.

82. A polypeptide of claim 1, comprising the amino acid sequence of SEQ ID
NO:27.

83. A polypeptide of claim 1, comprising the amino acid sequence of SEQ ID
NO:28.

84. A polypeptide of claim 1, comprising the amino acid sequence of SEQ ID
NO:29.

85. A polypeptide of claim 1, comprising the amino acid sequence of SEQ ID
NO:30.

86. A polypeptide of claim 1, comprising the amino acid sequence of SEQ ID
NO:31.

87. A polypeptide of claim 1, comprising the amino acid sequence of SEQ ID
NO:32.

88. A polypeptide of claim 1, comprising the amino acid sequence of SEQ ID
NO:33.

89. A polypeptide of claim 1, comprising the amino acid sequence of SEQ ID
NO:34.

90. A polypeptide of claim 1, comprising the amino acid sequence of SEQ ID
NO:35.

91. A polypeptide of claim 1, comprising the amino acid sequence of SEQ ID
NO:36.

92. A polypeptide of claim 1, comprising the amino acid sequence of SEQ ID
NO:37.

93. A polypeptide of claim 1, comprising the amino acid sequence of SEQ ID
NO:38.

94. A polypeptide of claim 1, comprising the amino acid sequence of SEQ ID
NO:39.

95. A polypeptide of claim 1, comprising the amino acid sequence of SEQ ID
NO:40.

96. A polynucleotide of claim 12, comprising the polynucleotide sequence of SEQ ID NO:41.

97. A polynucleotide of claim 12, comprising the polynucleotide sequence of SEQ ID NO:42.

98. A polynucleotide of claim 12, comprising the polynucleotide sequence of SEQ ID NO:43.

99. A polynucleotide of claim 12, comprising the polynucleotide sequence of SEQ ID NO:44.

100. A polynucleotide of claim 12, comprising the polynucleotide sequence of SEQ ID
NO:45.

101. A polynucleotide of claim 12, comprising the polynucleotide sequence of SEQ ID
NO:46.

102. A polynucleotide of claim 12, comprising the polynucleotide sequence of SEQ ID
NO:47.

103. A polynucleotide of claim 12, comprising the polynucleotide sequence of SEQ ID
NO:48.

104. A polynucleotide of claim 12, comprising the polynucleotide sequence of SEQ ID
NO:49.

105. A polynucleotide of claim 12, comprising the polynucleotide sequence of SEQ ID
NO:50.

106. A polynucleotide of claim 12, comprising the polynucleotide sequence of SEQ ID
NO:51.

107. A polynucleotide of claim 12, comprising the polynucleotide sequence of SEQ ID
NO:52.

108. A polynucleotide of claim 12, comprising the polynucleotide sequence of SEQ ID
NO:53.

109. A polynucleotide of claim 12, comprising the polynucleotide sequence of SEQ ID
NO:54.

110. A polynucleotide of claim 12, comprising the polynucleotide sequence of SEQ ID
NO:55.

111. A polynucleotide of claim 12, comprising the polynucleotide sequence of SEQ ID
NO:56.

112. A polynucleotide of claim 12, comprising the polynucleotide sequence of SEQ ID , NO:57.

113. A polynucleotide of claim 12, comprising the polynucleotide sequence of SEQ ID
NO:58.

114. A polynucleotide of claim 12, comprising the polynucleotide sequence of SEQ ID
NO:59.

115. A polynucleotide of claim 12, comprising the polynucleotide sequence of SEQ ID
NO:60.

116. A polynucleotide of claim 12, comprising the polynucleotide sequence of SEQ ID
NO:61.

117. A polynucleotide of claim 12, comprising the polynucleotide sequence of SEQ ID
NO:62.

118. A polynucleotide of claim 12, comprising the polynucleotide sequence of SEQ ID
NO:63.

119. A polynucleotide of claim 12, comprising the polynucleotide sequence of SEQ ID
NO:64.

120. A polynucleotide of claim 12, comprising the polynucleotide sequence of SEQ ID
NO:65.

121. A polynucleotide of claim 12, comprising the polynucleotide sequence of SEQ ID
NO:66.

122. A polynucleotide of claim 12, comprising the polynucleotide sequence of SEQ ID
NO:67.

123. A polynucleotide of claim 12, comprising the polynucleotide sequence of SEQ ID
NO:68.

124. A polynucleotide of claim 12, comprising the polynucleotide sequence of SEQ ID
NO:69.

125. A polynucleotide of claim 12, comprising the polynucleotide sequence of SEQ ID
NO:70.

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

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

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

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

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

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

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

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

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

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