CA2434944A1 - Secreted proteins - Google Patents

Abstract

The invention provides human secreted proteins (SECP) and polynucleotides which identify and encode SECP. The invention also provides expression vectors, host cells, antibodies, agonists, and antagonists. The invention also provides methods for diagnosing, treating, or preventing disorders associated with aberrant expression of SECP.

Description

SECRETED PROTEINS
TECHNICAL FIELD
This invention relates to nucleic acid and amino acid sequences of secreted proteins and to the use of these sequences in the diagnosis, treatment, and prevention of cell proliferative, autoimmune/inflammatory, cardiovascular, neurological, and developmental disorders, and in the assessment of the effects of exogenous compounds on the expression of nucleic acid and amino acid sequences of secreted proteins.
BACKGROUND OF THE INVENTION
Protein transport and secretion are essential for cellular function. Protein transport is mediated by a signal peptide located at the amino terminus of the protein to be transported or secreted. The signal peptide is comprised of about ten to twenty hydrophobic amino acids which target the nascent protein from the ribosome to a particular membrane bound compartment such as the endoplasmic reticulum (ER). Proteins targeted to the ER may either proceed through the secretory pathway or remain in any of the secretory organelles such as the ER, Golgi apparatus, or Iysosomes.
Proteins that transit through the secretory pathway are either secreted into the extracellular space or retained in the plasma membrane. Proteins that are retained in the plasma membrane contain one or more transmembrane domains, each comprised of about 20 hydrophobic amino acid residues.
Secreted proteins are generally synthesized as inactive precursors that are activated by post-translational processing events during transit through the secretory pathway.
Such events include glycosylation, proteolysis, and removal of the signal peptide by a signal peptidase. Other events that may occur during ,protein transport include chaperone-dependent unfolding and folding of the nascent protein and interaction of the protein with a receptor or pore complex.
Examples of secreted proteins with amino terminal signal peptides are discussed below and include proteins with important roles in cell-to-cell signaling. Such proteins include transmembrane receptors and cell surface markers, extracellular matrix molecules, cytokines, hormones, growth and differentiation factors, enzymes, neuropeptides, vasomediators, cell surface markers, and antigen recognition molecules. (Reviewed in Alberts, B. et al. (1994) Molecular Biology of The Cell, Garland Publishing, New York, NY, pp. 557-560, 582-592.) Cell surface markers include cell surface antigens identified on leukocytic cells of the immune system. These antigens have been identified using systematic, monoclonal antibody (mAb)-based "shot gun" techniques. These techniques have resulted in the production of hundreds of mAbs directed against unknown cell surface leukocytic antigens. These antigens have been grouped into "clusters of differentiation" based on common immunocytochemical localization patterns in various differentiated and undifferentiated leukocytic cell types. Antigens in a given cluster are presumed to identify a single cell surface protein and are assigned a "cluster of differentiation" or "CD"
designation. Some of the genes encoding proteins identified by CD antigens have been cloned and verified by standard molecular biology techniques. CD antigens have been characterized as both transmembrane proteins and cell surface proteins anchored to the plasma membrane via covalent attachment to fatty acid-containing glycolipids such as glycosylphosphatidylinositol (GPI).
(Reviewed in Barclay, A.N. et al. (1995) The Leucocyte Antiuen Facts Book, Academic Press, San Diego, CA, pp. 17-20.) Matrix proteins (MPs) are transmembrane and extracellular proteins which function in formation, growth, remodeling, and maintenance of tissues and as important mediators and regulators of the inflammatory response. The expression and balance of MPs may be perturbed by biochemical changes that result from congenital, epigenetic, or infectious diseases. In addition, MPs affect leukocyte migration, proliferation, differentiation, and activation in the immune response. MPs are frequently characterized by the presence of one or more domains which may include collagen-like domains, EGF-like domains, immunoglobulin-like domains, and fibronectin-like domains. In addition, MPs may be heavily glycosylated and may contain an Arginine-Glycine-Aspartate (RGD) tripeptide motif which may play a role in adhesive interactions. MPs include extracellular proteins such as fibronectin, collagen, galectin, vitronectin and its proteolytic derivative somatomedin B; and cell adhesion receptors such as cell adhesion molecules (CAMS), cadherins, and integrins. (Reviewed in Ayad, S. et al. (1994) The Extracellular Matrix Facts Book, Academic Press, San Diego, CA, pp. 2-16; Ruoslahti, E. (1997) Kidney Int. 51:1413-1417; Sjaastad, M.D. and Nelson, W.J. (1997) BioEssays 19:47-55.) Mucins are highly glycosylated glycoproteins that are the major structural component of the mucus gel. The physiological functions of mucins are cytoprotection, mechanical protection, maintenance of viscosity in secretions, and cellular recognition. MUC6 is a human gastric mucin that is also found in gall bladder, pancreas, seminal vesicles, and female reproductive tract (Toribara, N.W. et al. (1997) J. Biol. Chem. 272:16398-16403). The MUC6 gene has been mapped to human chromosome 11 (Toribara, N.W. et al. (1993) J. Biol. Chem. 268:5879-5885).
Hemomucin is a novel Droso~hila surface mucin that may be involved in the induction of antibacterial effector molecules (Theopold, U. et al. (1996) J. Biol. Chem. 217:12708-12715).
Tuftelins are one of four different enamel matrix proteins that have been identified so far.
The other three known enamel matrix proteins are the amelogenins, enamelin and ameloblastin.
Assembly of the enamel extracellular matrix from these component proteins is believed to be critical in producing a matrix competent to undergo mineral replacement. (Paine, C.T.
et al. (1998) Connect Tissue Res. 38:257-267). Tuftelin mRNA has been found to be expressed in human ameloblastoma tumor, a non-mineralized odontogenic tumor (Deutsch, D. et aI. (I998) Connect.
Tissue Res.
39:177-184).
Olfactomedin-related proteins are extracellular matrix, secreted glycoproteins with conserved C-terminal motifs. They are expressed in a wide variety of tissues and in broad range of species, from Caenorhabditis elegans to Homo Sapiens. Olfactomedin-related proteins comprise a gene family with at least 5 family members in humans. One of the five, TIGRlmyocilin protein, is expressed in the eye and is associated with the pathogenesis of glaucoma (Kulkarni, N.H. et al.
(2000) Genet. Res. 76:41-50). Research by Yokoyama et aI. (I996) found a 135-amino acid protein, termed AMY, having 96%
sequence identity with rat neuronal olfactomedin-releated ER localized protein in a neuroblastoma cell line cDNA library, suggesting an essential role for AMY in nerve tissue (Yokoyama, M. et al.
(1996) DNA Res. 3:311-320). Neuron-specific olfactomedin-related glycoproteins isolated from rat brain cDNA libraries show strong sequence similarity with olfactomedin. This similarity is suggestive of a matrix-related function of these glycoproteins in neurons and neurosecretory cells (Danielson, P.E. et al. (1994) J. Neurosci. Res. 38:468-478).
Mac-2 binding protein is a 90-kD serum protein (90K), a secreted glycoprotein isolated from both the human breast carcinoma cell line SK-BR-3, and human breast milk. It specifically binds to a human macrophage-associated lectin, Mac-2. Structurally, the mature protein is 567 amino acids in length and is proceeded by an 18-amino acid leader. There are 16 cysteines and seven potential N-linked glycosylation sites. The first 106 amino acids represent a domain very similar to an ancient protein superfamily defined by a macrophage scavenger receptor cysteine-rich domain (Koths, K. et al. (1993) J. Biol. Chem. 268:14245-14249). 90K is elevated in the serum of subpopulations of ATDS
patients and is expressed at varying levels in primary tumor samples and tumor cell Iines. UIIrich et al. (1994) have demonstrated that 90K stimulates host defense systems and can induce interleukin-2 secretion. This immune stimulation is proposed to be a result of oncogenic transformation, viral infection or pathogenic invasion (Ullrich, A. et al. (1994) J. Biol. Chem.
269:18401-18407).
Semaphorins are a large group of axonal guidance molecules consisting of at least 30 different members and are found in vertebrates, invertebrates, and even certain viruses. All semaphorins contain the sema domain which is approximately 500 amino acids in length. Neuropilin, a semaphorin receptor, has been shown to promote neurite outgrowth in vitro.
The extracellular region of neuropilins consists of three different domains: CUB, discoidin, and MAM domains. The CUB and the MAM motifs of neuropilin have been suggested to have roles in protein-protein interactions and are thought to be involved in the binding of semaphorins through the sema and the C-terminal domains (reviewed in Raper, J.A. (2000) Curr. Opin. Neurobiol.
10:88-94). Plexins are neuronal cell surface molecules that mediate cell adhesion via a hemophilic binding mechanism in the presence of calcium ions. Plexins have been shown to be expressed in the receptors and neurons of particular sensory systems (Ohta, K, et al. (1995) Cell 14:1189-1199). There is evidence that suggests that some plexins function to control motor and CNS axon guidance in the developing nervous system. Plexins, which themselves contain complete semaphorin domains, may be both the ancestors of classical semaphorins and binding partners for semaphorins (Winberg, M.L. et al (1998) Ce1195:903-916).
Human pregnancy-specific beta 1-glycoprotein (PSG) is a family of closely related glycoproteins of molecular weights of 72 KDa, 64KDa, 62KDa, and 54KDa.
Together with the carcinoembryonic antigen, they comprise a subfamily within the immunoglobulin superfamily (Plouzek, C.A. and Chou, J.Y. (1991) Endocrinology 129:950-958) Different subpopulations of PSG
have been found to be produced by the trophoblasts of the human placenta, and the amnionic and chorionic membranes (Plouzek, C.A. et al. (1993) Placenta 14:277-285).
The interaction between a sperm cell and an ovum, which ultimately results in the fusion of two gametes into a single zygote, also involves a number of secreted proteins.
The process of fusion can be divided into four steps: (i) sperm with intact acrosomes (apical secretory vesicles, see below) interact with the extracellular matrix (zona pellucida, ZP) of the ovum (Eddy, E. and O'brian, D.
(1994) in The Physiolo y of Reproduction, Ed. Knobil, E. and Neill, J. New York, NY Raven Press pp. 29-78), (ii) sperm undergo the acrosome reaction and penetrate the ZP, (iii) sperm bind to the plasma membrane of the ovum, and (iv) sperm fuse with the ovum to form the zygote. Much of the seminal work involved in the dissection of these processes has been performed using mice as a model system for mammalian fertilization. The ZP in mice is composed of three N-glycosylated and O-glycosylated secreted proteins, ZP1 (a 200 kDa dimeric protein), ZP2 (120 kDa), and ZP3 (83 kDa) (Wassarman, P. (1988) Anna. Rev. Biochem. 57:415-442). ZP2 and ZP3 polypeptides form filaments consisting of structural repeats. ZP2/ZP3 filaments are crosslinked by ZP1 to form the ZP matrix (Wassarman, P. (1999) Cell 96:175-183).
Mice defective for N-linked glycosylation are fertile, suggesting that N-linked glycosylation is not essential for ZP function (Rosiere, T. and Wassarman, P. (1992) Dev.
Biol. 154:309-317;
reviewed in Snell, W. and White, J. (1996) Cell 85:629-637; and Wassarman, P.
su ra). However, the targeted disruption of both ZP3 alleles in female mice results in decreased ZP thickness and infertility (Liu, C. et al. (1996) Proc. Natl. Acad. Sci. USA 93:5431-5436;
Rankin, T. et al. (1996) Development 122:2903-2910). ZP1 and ZP2 have been less extensively studied than ZP3. Based on results obtained from (i) limited-proteolysis experiments (Rosiere, T. and Wassarman, P. supra), (ii) inactivation experiments using antibodies directed against defined regions of the ZP3 glycoprotein (Millar, S. (1989) Science 246:935-938), and exon-swapping experiments (Kinloch, R. et al. (1995) Proc. Natl. Acad. Sci. USA 92:263-267), putative O-linked glycosylation sites encoded by the 7"' of 8 exons in the marine ZP3 gene were implicated in acrosome binding. Crude site-directed mutagenesis experiments have further delimited the essential O-linked glycosylation site to five serine residues (and no threonine residues) between positions 328-343 (inclusive) of the 424-residue ZP3 polypeptide (Kinloch, R. et al. supra). Interestingly, marine and human ZP3 polypeptides are only 28% identical in this critical region, while being 67% identical overall and sharing all recognized domains. These phylogenetic data suggest that ZP3 protein may function primarily as a scaffold for the presentation of the correct carbohydrate moieties rather than participate in biological interactions as a polypeptide molecule. (Rosiere, T. supra; Snell, W. and White, J. su ra; and Wassarman, P.
supra). The primary amino acid sequence of the ZP3 polypeptide is less important to acrosome binding that the specific glycosylation that occurs in the exon-7 region (Kinloch, R. et al. supra).
Autocrine motility factor (AMF) is one of the motility cytokines regulating tumor cell migration; therefore identification of the signaling pathway coupled with it has critical importance.
Autocrine motility factor receptor (AMF'R) expression has been found to be associated with tumor progression in thymoma (Ohta Y. et al. (2000) Int. J. Oncol. 17:259-264). AMFR
is a cell surface glycoprotein of molecular weight 78KDa.
Hormones are secreted molecules that travel through the circulation and bind to specific receptors on the surface of, or within, target cells. Although they have diverse biochemical compositions and mechanisms of action, hormones can be grouped into two categories. One category includes small lipophilic hormones that diffuse through the plasma membrane of target cells, bind to cytosolic or nuclear receptors, and form a complex that alters gene expression. Examples of these molecules include retinoic acid, thyroxine, and the cholesterol-derived steroid hormones such as progesterone, estrogen, testosterone, cortisol, and aldosterone. The second category includes hydrophilic hormones that function by binding to cell surface receptors that transduce signals across the plasma membrane. Examples of such hormones include amino acid derivatives such as catecholamines (epinephrine, norepinephrine) and histamine, and peptide hormones such as glucagon, insulin, gastrin, secretin, cholecystokinin, adrenocorticotropic hormone, follicle stimulating hormone, luteinizing hormone, thyroid stimulating hormone, and vasopressin. (See, for example, Lodish et aI.
(1995) Molecular Cell Biolo~y, Scientific American Books Inc., New York, NY, pp. 856-864.) Pro-opiomelanocortin (POMC) is the precursor polypeptide of corticotropin (ACTH), a hormone synthesized by the anterior pituitary gland, which functions in the stimulation of the adrenal cortex. POMC is also the precursor polypeptide of the hormone beta-lipotropin (beta-LPH). Each hormone includes smaller peptides with distinct biological activities: alpha-melanotropin (alpha-MSH) and corticotropin-like intermediate lobe peptide (CLIP) are formed from ACTH; gamma-lipotropin (gamma-LPH) and beta-endorphin are peptide components of beta-LPH;
while beta-MSH
is contained within gamma-LPH. Adrenal insufficiency due to ACTH deficiency, resulting from a genetic mutation in exons 2 and 3 of POMC results in an endocrine disorder characterized by early-onset obesity, adrenal insufficiency, and red hair pigmentation (Chretien, M.
et al. (1979) Can. J.
Biochem. 57:1111-1121; Krude, H. et al. (1998) Nat. Genet. 19:155-157; Online Mendelian Inheritance in Man (O1VIIM) 176830).
Growth and differentiation factors are secreted proteins which function in intercellular communication. Some factors require oligomerization or association with membrane proteins for activity. Complex interactions among these factors and their receptors trigger intracellular signal transduction pathways that stimulate or inhibit cell division, cell differentiation, cell signaling, and cell motility. Most growth and differentiation factors act on cells in their local environment (paracrine signaling). There are three broad classes of growth and differentiation factors. The first class includes the large polypeptide growth factors such as epidermal growth factor, fibroblast growth factor, transforming growth factor, insulin-like growth factor, and platelet-derived growth factor. The second class includes the hematopoietic growth factors such as the colony stimulating factors (CSFs).
Hematopoietic growth factors stimulate the proliferation and differentiation of blood cells such as B-lymphocytes, T-lymphocytes, erythrocytes, platelets, eosinophils, basophils, neutrophils, macrophages, and their stem cell precursors. The third class includes small peptide factors such as bombesin, vasopressin, oxytocin, endothelia, transferrin, angiotensin II, vasoactive intestinal peptide, and bradykinin, which function as hormones to regulate cellular functions other than proliferation.
Growth and differentiation factors play critical roles in neoplastic transformation of cells in vitro and in tumor progression in vivo. Inappropriate expression of growth factors by tumor cells may contribute to vascularization and metastasis of tumors. During hematopoiesis, growth factor misregulation can result in anemi.as, leukemias, and lymphomas. Certain growth factors such as interferon are cytotoxic to tumor cells both in vivo and in vitro. Moreover, some growth factors and growth factor receptors are related both structurally and functionally to oncoproteins. In addition, growth factors affect transcriptional regulation of both proto-oncogenes and oncosuppressor genes.
(Reviewed in Pimentel, E. (1994) Handbook of Growth Factors, CRC Press, Aan Arbor, MI, pp. 1-9.) The Slit protein, first identified in Drosophila, is critical in central nervous system midline formation and potentially in nervous tissue histogenesis and axonal pathfmding. Itoh et al. ((1998) Brain Res. Mol. Brain Res. 62:175-186) have identified mammalian homologues of the slit gene (human Slit-1, Slit-2, Slit-3 and rat Slit-1). The encoded proteins are putative secreted proteins containing EGF-like motifs and leucine-rich repeats, both of which are conserved protein-protein interaction domains. Slit-1, -2, and -3 nnRIVAs are expressed in the brain, spinal cord, and thyroid, respectively (Itoh, A. et al., supra). The Slit family of proteins axe indicated to be functional ligands of glypican-1 in nervous tissue and it is suggested that their interactions may be critical in certain stages during central nervous system histogenesis (Liang, Y. et al. (1999) J.
Biol. Chem. 274:17885-17892).

Neuropeptides and vasomediators (NP/VM) comprise a large family of endogenous signaling molecules. Included in this family are neuropeptides and neuropeptide hormones such as bombesin, neuropeptide Y, neurotensin, neuromedin N, melanocortins, opioids, galanin, somatostatin, tachykinins, urotensin II and related peptides involved in smooth muscle stimulation, vasopressin, vasoactive intestinal peptide, and circulatory system-borne signaling molecules such as angiotensin, complement, calcitonin, endothelins, formyl-methionyl peptides, glucagon, cholecystokinin and gastrin. NP/VMs can transduce signals directly, modulate the activity or xelease of other neurotransmitters and hormones, and act as catalytic enzymes in cascades. The effects of NP/VMs range from extremely brief to long-lasting. (Reviewed in Martin, C.R. et al.
(1985) Endocrine Physiolog_y, Oxford University Press, New York, NY, pp. 57-62.) NP/VMs are involved in numerous neurological and cardiovascular disorders. For example, neuropeptide Y is involved in hypertension, congestive heart failure, affective disorders, and appetite regulation. Somatostatin inhibits secretion of growth hormone and prolactin in the anterior pituitary, as well as inhibiting secretion in intestine, pancreatic acinar cells, and pancreatic beta-cells. A
reduction in somatostatin levels has been reported in Alzheimer's disease and Parkinson's disease.
Vasopressin acts in the kidney to increase water and sodium absorption, and in higher concentrations stimulates contraction of vascular smooth muscle, platelet activation, and glycogen breakdown in the liver. Vasopressin and its analogues are used clinically to treat diabetes insipidus. Endothelin and angiotensin are involved in hypertension, and drugs, such as captopril, which reduce plasma levels of angiotensin, are used to reduce blood pressure (Watson, S. and S. Arkinstall (1994) The G-protein Linked Rector Facts Book, Academic Press, San Diego CA, pp. 194; 252; 284; 55;
111).
Neuropeptides have also been shown to have roles in nociception (pain).
Vasoactive intestinal peptide appears to play an important role in chronic neuropathic pain. Nociceptin, an endogenous ligand fox fox the opioid receptor-like 1 receptor, is thought to have a predominantly anti-nociceptive effect, and has been shown to have analgesic properties in different animal models of tonic or chronic pain (Dickinson, T. and Fleetwood-Walker, S.M. (1998) Trends Pharmacol. Sci.
19:346-348).
Other proteins that contain signal peptides include secreted proteins with enzymatic activity.
Such activity includes, for example, oxidoreductase/dehydrogenase activity, transferase activity, hydrolase activity, lyase activity, isomerase activity, or ligase activity.
For example, matrix metalloproteinases are secreted hydrolytic enzymes that degrade the extracellular matrix and thus play an important role in tumor metastasis, tissue morphogenesis, and arthritis (Reponen, P. et al.
(1995) Dev. Dyn. 202:388-396; Firestein, G.S. (1992) Curr. Opin. Rheurnatol.
4:348-354; Ray, J.M.
and Stetler-Stevenson, W.G. (1994) Eur. Respir. J. 7:2062-2072; and Mignatti, P. and Rifkin, D.B.
(1993) Physiol. Rev. 73:161-195). Additional examples are the acetyl-CoA
synthetases which activate acetate for use in Iipid synthesis or energy generation (Luong, A. et al. (2000) J. Biol. Chem.
275:26458-26466). The result of acetyl-CoA synthetase activity is the formation of acetyl-CoA from acetate and CoA. Acetyl-CoA sythetases share a region of sequence similarity identified as the AMP-binding domain signature. Acetyl-CoA synthetase has been shown to be associated with hypertension (Toh, H. (1991) Protein Seq. Data Anal. 4:111-117; and Iwai, N. et al. (I994) Hypertension 23:375-380).
A number of isomerases catalyze steps in protein folding, phototransduction, and various anabolic and catabolic pathways. One class of isomerases is known as peptidyl-prolyl cis-trams isomerases (PPIases). PPIases catalyze the cis to traps isomerization of certain proline imidic bonds in proteins. Two families of PPIases are the FK506 binding proteins (FKBPs), and cyclophilins (CyPs). FKBPs bind the potent immunosuppressants FK506 and rapamycin, thereby inhibiting signaling pathways in T-cells. Specifically, the PPTase activity of FKBPs is inhibited by binding of FK506 or rapamycin. There are five members of the FKBP family which are named according to .
their calculated molecular masses (FKBP12, FKBP13, FKBP25, FKBP52, and FKBP65), and I5 localized to different regions of the cell where they associate with different protein complexes (Coss, M. et al. (1995) J. Biol. Chem. 270:29336-29341; Schreiber, S.L. (1991) Science 251:283-287).
The peptidyl-prolyl isomerase activity of CyP may be part of the signaling pathway that leads to T-cell activation. CyP isomerase activity is associated with protein folding and protein trafficking, and may also be involved in assembly/disassembly of protein complexes and regulation of protein activity. For example, in Drosophila, the CyP NinaA is required for correct localization of rhodopsins, while a mammalian CyP (Cyp40) is part of the Hsp90/Hsc70 complex that binds steroid receptors. The mammalian CypA has been shown to bind the gag protein from human immunodeficiency virus 1 (HIV-1), an interaction that can be inhibited by cyclosporin. Since cyclosporin has potent anti-HIV-1 activity, CypA may play an essential function in HIV-1 replication.
Finally, Cyp40 has been shown to bind and inactivate the transcription factor c-Myb, an effect that is reversed by cyclosporin. This effect implicates CyPs in the regulation of transcription, transformation, and differentiation (Bergsma, D.J. et al (1991) J. Biol. Chem.
266:23204-23214;
Hunter, T. (1998) Cel192:141-143; and Leverson, J.D. and Ness, S.A. (1998) Mol. Cell. 1:203-211).
Gamma-carboxyglutamic acid (Gla) proteins rich in proline (PRGPs) are members of a family of vitamin K-dependent single-pass integral membrane proteins. These proteins are characterized by an extracellular amino terminal domain of approximately 45 amino acids rich in Gla. The intracellular carboxyl terminal region contains one or two copies of the sequence PPXY, a motif present in a variety of proteins involved in such diverse cellular functions as signal transduction, cell cycle progression, and protein turnover (Kulman, J.D. et al. (2001) Proc.
Natl. Acad. Sci. USA
98:1370-1375). The process of post-translational modification of glutamic residues to form Gla is Vitamin I~-dependent carboxylation. Proteins which contain Gla include plasma proteins involved in blood coagulation. These proteins are prothrombin, proteins C, S, and Z, and coagulation factors VII, IX, and X. Osteocalcin (bone-Gla protein, BGP) and matrix Gla-protein (MGP) also contain Gla (Friedman, P.A. and C.T. Przysiecki (1987) Int. J. Biochem. 19:1-7; C. Vermeer (1990) Biochem. J.
266:625-636).
The Drosophila sp. gene crossveinless 2 is characterized as having a putative signal or txansmembrane sequence, a partial Von Willebrand Factor D domain similar to those domains known to regulate the formation of intramolecular and intermolecular bonds, and five cysteine-rich domains, known to bind BMP-like (bone morphogenetic proteins) ligands. These features suggest that crossveinless 2 may act extracelluarly or in the secretory pathway to directly potentiate ligand signaling and hence, involvement in the BMP-like signaling pathway known to play a role in vein specification (Conley, C.A. et al., (2000) Development 127:3947-3959). The dorsal-ventral patterning in both vertebrate and Drosophila embryos requires a conserved system of extracellular proteins to generate a positional informational gradient.
Tmmunoglobulins Antigen recognition molecules are key players in the sophisticated and complex immune systems which all vertebrates have developed to provide protection from viral, bacterial, fungal, and parasitic infections. A key feature of the immune system is its ability to distinguish foreign molecules, or antigens, from "self' molecules. Most cell surface and soluble molecules that mediate functions such as recognition, adhesion or binding have evolved from a common evolutionary precursor (i.e., these proteins have structural homology). A number of molecules outside the immune system that have similar functions are also derived from this same evolutionary precursor. This ability is mediated primarily by secreted and transmembrane proteins expressed by leukocytes (white blood cells) such as lymphocytes, granulocytes, and monocytes. Most of these proteins belong to the immunoglobulin (Ig) superfamily, members of which contain one or more repeats of a conserved structural domain. This Ig domain is comprised of antiparallel (3 sheets joined by a disulfide bond in an arrangement called the Ig fold. The criteria for a protein to be a member of the Ig superfamily is to have one or more Ig domains, which are regions of 70-110 amino acid residues in length homologous to either Ig variable-like (V) or Ig constant-like (C) domains. Members of the Tg superfamily include antibodies (Ab), T cell receptors (TCRs), class I and II majox histocompatibility (MHC) proteins and immune cell-specific surface markers such as the "cluster of differentiation"
or CD antigens, CD2, CD3, CD4, CDB, poly-Ig receptors, Fc receptors, neural cell-adhesion molecule (NCAM) and platelet-derived growth factor receptor (PDGFR). These antigens have been identified using systematic, monoclonal antibody (mAb)-based "shot gun" techniques. These techniques have resulted in the production of hundreds of mAbs directed against unlrnown cell surface leukocytic antigens. These antigens have been grouped into "clusters of differentiation"
based on common immunocytochemical localization patterns in various differentiated and undifferentiated leukocytic cell types. Antigens in a given cluster are presumed to identify a single cell surface protein and are assigned a "cluster of differentiation" or "CD" designation. Some of the genes encoding proteins identified by CD antigens have been cloned and verified by standard molecular biology techniques.
CD antigens have been characterized as both transmembrane proteins and cell surface pxoteins anchored to the plasma membrane via covalent attachment to fatty acid-containing glycolipids such as glycosylphosphatidylinositol (GPI). (Reviewed in Barclay, A. N. et al. (1995) The Leucocyte Antigen Facts Book, Academic Press, San Diego, CA, pp. 17-20.) Ig domains (V and C) are regions of conserved amino acid residues that give a polypeptide a globular tertiary structure called an immunoglobulin (or antibody) fold, which consists of two approximately parallel layers of (3-sheets. Conserved cysteine residues form an intrachain disulfide-bonded loop, 55-75 amino acid residues in length, which connects the two layers of the (3-sheets.
Each (3-sheet has three or four anti-parallel (3-strands of 5-10 amino acid residues. Hydrophobic and hydrophilic interactions of amino acid residues within the (3-strands stabilize the Ig fold (hydrophobic on inward facing amino acid residues and hydrophilic on the amino acid residues in the outward facing portion of the strands). A V domain consists of a longer polypeptide Than a C domain, with an additional pair of ~i-strands in the Ig fold.
A consistent feature of Ig superfamily genes is that each sequence of an Ig domain is encoded by a single exon. It is possible that the superfamily evolved from a gene coding for a single Ig domain involved in mediating cell-cell interactions. New members of the superfamily then arose by exon and gene duplications. Modern Ig superfamily proteins contain different numbers of V and/or C
domains. Another evolutionary feature of this superfamily is the ability to undergo DNA
rearrangements, a unique feature retained by the antigen receptor members of the family.
Many members of the Ig superfarnily are integral plasma membrane proteins with extracellular Ig domains. The hydrophobic amino acid residues of their transmembrane domains and their cytoplasmic tails axe very diverse, with little or no homology among Ig family members or to known signal-transducing structures. There are exceptions to this general superfamily description.
For example, the cytoplasmic tail of PDGFR has tyrosine kinase activity. ~ In addition Thy-1 is a glycoprotein found on thymocytes and T cells. This protein has no cytoplasmic tail, but is instead attached to the plasma membrane by a covalent glycophosphatidylinositol linkage.
Another common feature of many Ig superfamily proteins is the interactions between Ig domains which are essential for the function of these molecules. Intexactions between Ig domains of a multimeric protein can be either hemophilic or heterophilic (i.e., between the same or different Ig domains). Antibodies are multirneric proteins which have both hemophilic and heterophilic interactions between Ig domains. Pairing of constant regions of heavy chains forms the Fc region of an antibody and pairing of variable regions of light and heavy chains form the antigen binding site of an antibody. Heterophilic interactions also occur between Ig dbmains of different molecules. These interactions provide adhesion between cells for significant cell-cell interactions in the immune system and in the developing and mature nervous system. (Reviewed in Abbas, A.K. et al. (1991) Cellular and Molecular Immunolo~y, W.B. Saunders Company, Philadelphia, PA, pp.142-145.) Antibodies MHC proteins are cell surface markers that bind to and present foreign antigens to T cells.
MHC molecules are classified as either class I or class II. Class I MHC
molecules (MHC I) are expressed on the surface of almost all cells and are involved in the presentation of antigen to cytotoxic T cells. For example, a cell infected with virus will degrade intracellular viral proteins and express the protein fragments bound to MHC I molecules on the cell surface.
The MHC I/antigen complex is recognized by cytotoxic T-cells which destroy the infected cell and the virus within.
Class II MHC molecules are expressed primarily on specialized antigen-presenting cells of the immune system, such as B-cells and macrophages. These cells ingest foreign proteins from the extracellular fluid and express MHC II/antigen complex on the cell surface.
This complex activates helper T-cells, which then secrete cytokines and other factors that stimulate the immune response.
MHC molecules also play an important role in organ rejection following transplantation. Rejection occurs when the recipient's T-cells respond to foreign MHC molecules on the transplanted organ in the same way as to self MHC molecules bound to foreign antigen. (Reviewed in Alberts, B. et al.
(1994) Molecular Biology of the Cell, Garland Publishing, New York, NY, pp.
1229-1246.) Antibodies are multimeric members of the Ig superfamily which are either expressed on the surface of B-cells or secreted by B-cells into the circulation. Antibodies bind and neutralize foreign antigens in the blood and other extracellular fluids. The prototypical antibody is a tetramer consisting of two identical heavy polypeptide chains (H=chains) and two identical light polypeptide chains (L-chains) interlinked by disulfide bonds. This arrangement confers the characteristic Y-shape to antibody molecules. Antibodies are classified based on their H-chain composition. The five antibody classes, IgA, IgD, IgE, IgG and IgM, are defined by the a, 8, E, 'y, and ,u H-chain types. There are two types of L-chains, x and ~., either of which may associate as a pair with any H-chain pair. IgG, the most common class of antibody found in the circulation, is tetrameric, while the other classes of antibodies are generally variants ox multimers of this basic structure.
H-chains and L-chains each contain an N-terminal variable region and a C-terminal constant region. The constant region consists of about 110 amino acids in L-chains and about 330 or 440 amino acids in H-chains. The amino acid sequence of the constant region is nearly identical among H- or L-chains of a particular class. The variable region consists of about 110 amino acids in both H-and L-chains. However, the amino acid sequence of the variable region differs among H- or L-chains of a particular class. Within each H- or L-chain variable region are three hypervariable regions of extensive sequence diversity, each consisting of about 5 to 10 amino acids. In the antibody molecule, the H- and L-chain hypervariable regions come together to form the antigext recognition site.
(Reviewed in Alberts, 8. et al. supra, pp. 1206-1213 and 1216-1217.) Both H-chains and L-chains contain the repeated Ig domains of members of the Ig superfaznily. Fox example, a typical H-chain contains four Ig domains, three of which occur within the constant region and one of which occurs within the variable region and contributes to the formation of the antigen recognition site. Likewise, a typical L-chain contains two Ig domains, one of which occurs within the constant region and one of which occurs within the variable region.
The irnrnune system is capable of recognizing and responding to any foreign molecule that enters the body. Therefore, the immune system must be armed with a full repertoire of antibodies against all potential antigens. Such antibody diversity is generated by somatic rearrangement of gene segments encoding variable and constant regions. These gene segments are joined together by site-specific recombination which occurs between highly conserved DNA sequences that flank each gene segment. Because there are hundreds of different gene segments, millions of unique genes can be generated combinatorially. In addition, imprecise joining of these segments and an unusually high rate of somatic mutation within these segments further contribute to the generation of a diverse antibody population.
The discovery of new secreted proteins, and the polynucleotides encoding them, satisfies a need in the art by providing new compositions which are useful in the diagnosis, prevention, and treatment of cell pxoliferative, autoimmune/inflammatory, cardiovascular, neurological, and developmental disorders, and in the assessment of the effects of exogenous compounds on the expression of nucleic acid and amino acid sequences of secreted proteins.
SUMMARY OF THE INVENTION
The invention features purified polypeptides, secreted proteins, referred to collectively as "SECP" and individually as "SECP-l," "SECP-2," "SECP-3," "SECP-4," "SECP-5,"
"SECP-6,"
"SECP-7," "SECP-8," "SECP-9," "SECP-10," "SECP-11," "SECP-I2," "SECP-I3,"
"SECP-14,"
"SECP-15," "SECP-16," "SECP-17," "SECP-18," "SECP-19," "SECP-20," "SECP-21,"
"SECP-22,"
"SECP-23," and "SECP-24." In one aspect, the invention provides an isolated polypeptide selected from the group consisting of a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:1-24, b) a polypeptide comprising a naturally occurring amino acid sequence at least 90% identical to an amino acid sequence selected from the group consisting of SEQ
ID NO:l-24, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID N0:1-24, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID N0:1-24.
In one alternative, the invention provides an isolated polypeptide comprising the amino acid sequence of SEQ m N0:1-24.
The invention further provides an isolated polynucleotide encoding a polypeptide selected from the group consisting of a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID N0:1-24, 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:1-24, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1-24, and d) an imrnunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID N0:1-24.
In one alternative, the polynucleotide encodes a polypeptide selected from the group consisting of SEQ ll~ NO:1-24. In another alternative, the polynucleotide is selected from the group consisting of SEQ m N0:25-4g.
Additionally, the invention provides a recombinant polynucleotide comprising a promoter sequence operably linked to a polynucleotide encoding a polypeptide selected from the group consisting of a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ~ NO:1-24, b) a polypeptide comprising a naturally occurring amino acid sequence at least 90% identical to an amino acid sequence selected from the group consisting of SEQ ID N0:1-24, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ m NO:1-24, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1-24. In one alternative, the invention provides a cell transformed with the recombinant polynucleotide. In another alternative, the invention provides a transgenic organism comprising the recombinant polynucleotide.
The invention also provides a method for producing a polypeptide selected from the group consisting of a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ m NO: l-24, b) a polypeptide comprising a naturally occurring amino acid sequence at least 90% identical to an amino acid sequence selected from the group consisting of SEQ 1D NO:1-24, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ m NO:l-24, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ll~ NO:1-24. 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 pxomoter sequence operably linked to a polynucleotide encoding the polypeptide, and b) recovering the polypeptide so expressed.
Additionally, the invention provides an isolated antibody which specifically binds to a polypeptide selected from the group consisting of a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ m NO:1-24, b) a polypeptide comprising a naturally occurring amino acid sequence at least 90% identical to an amino acid sequence selected from the group consisting of SEQ m NO:1-24, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID
NO:1-24, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ m NO:1-24.
The invention further provides an isolated polynucleotide selected from the group consisting of a) a polynucleotide comprising a polynucleotide sequence selected from the group consisting of SEQ ID N0:25-48, b) a polynucleotide comprising a naturally occurring polynucleotide sequence at least 90% identical to a polynucleotide sequence selected from the group consisting of SEQ m N0:25-48, c) a polynucleotide complementary to the polynucleotide of a), d) a polynucleotide complementary to the polynucleotide of b), and e) an RNA equivalent of a)-d).
In one alternative, the polynucleotide comprises at least 60 contiguous nucleotides.
Additionally, the invention provides a method for detecting a target polynucleotide in a sample, said target polynucleotide having a sequence of a polynucleotide selected from the group consisting of a) a polynucleotide comprising a polynucleotide sequence selected from the group consisting of SEQ m N0:25-48, b) a polynucleotide comprising a naturally occurring polynucleotide sequence at least 90% identical to a polynucleotide sequence selected from the group consisting of SEQ ID N0:25-48, c) a polynucleotide complementary to the polynucleotide of a), d) a polynucleotide complementary to the polynucleotide of b), and e) an RNA
equivalent of a)-d). The method comprises a) hybridizing the sample with a probe comprising at least 20 contiguous nucleotides comprising a sequence complementary to said target polynucleotide in the sample, and which probe specifically hybridizes to said target polynucleotide, under conditions whereby a hybridization complex is formed between said probe and said target polynucleotide or fragments thereof, and b) detecting the presence or absence of said hybridization complex, and optionally, if present, the amount thereof. In one alternative, the probe comprises at least 60 contiguous nucleotides.
The invention further provides a method for detecting a target polynucleotide in a sample, said target polynucleotide having a sequence of a polynucleotide selected from the group consisting of a) a polynucleotide comprising a polynucleotide sequence selected from the group consisting of SEQ )D N0:25-4.8, b) a polynucleotide comprising a naturally occurring polynucleotide sequence at least 90% identical to a polynucleotide sequence selected from the group consisting of SEQ )D
N0:25-48, c) a polynucleotide complementary to the polynucleotide of a), d) a polynucleotide complementary to the polynucleotide of b), and e) an RNA equivalent of a)-d).
The method comprises a) amplifying said target polynucleotide or fragment thereof using polymerase chain reaction amplification, and b) detecting the presence or absence of said amplified target polynucleotide or fragment thereof, and, optionally, if present, the amount thereof.
The invention further provides a composition comprising an effective amount of a polypeptide selected from the group consisting of a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ m NO:1-24, b) a polypeptide comprising a naturally occurring amino acid sequence at least 90% identical to an amino acid sequence selected from the group consisting of SEQ ID N0:1-24, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID
NO:1-24, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ )D N0:1-24, and a pharmaceutically acceptable excipient. In one embodiment, the composition comprises an amino acid sequence selected from the group consisting of SEQ m NO:1-24. The invention additionally provides a method of treating a disease or condition associated with decreased expression of functional SECP, comprising administering to a patient in need of such treatment the composition.
The invention also provides a method for screening a compound for effectiveness as an agonist of a polypeptide selected from the group consisting of a) a polypeptide comprising au amino acid sequence selected from the group consisting of SEQ m NO:1-24, b) a polypeptide comprising a naturally occurring amino acid sequence at least 90% identical to an amino acid sequence selected from the group consisting of SEQ m N0:1-24, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the gxoup consisting of SEQ m NO:1-24, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ m N0:1-24. The method comprises a) exposing a sample comprising the polypeptide to a compound, and b) detecting agonist activity in the sample. In one alternative, the invention provides a composition comprising an agonist compound identified by the method and a pharmaceutically acceptable excipient. In another alternative, the invention provides a method of treating a disease or condition associated with decreased expression of functional SECP, comprising administering to a patient in need of such treatment the composition.
Additionally, the invention provides a method for screening a compound for effectiveness as an antagonist of a polypeptide selected from the group consisting of a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ m NO:1-24, 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:1-24, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ >I? N0:1-24, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ m N0:1-24. The method comprises a) exposing a sample comprising the polypeptide to a compound, and b) detecting antagonist activity in the sample.
In one alternative, the invention provides a composition comprising an antagonist compound identified by the method and a pharmaceutically acceptable excipient. In another alternative, the invention provides a method of treating a disease or condition associated with overexpression of functional SECP, comprising administering to a patient in need of such treatment the composition.
The invention further provides a method of screening for a compound that specifically binds to a polypeptide selected from the group consisting of a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ m NO:1-24, b) a polypeptide comprising a naturally occurring amino acid sequence at least 90% identical to an amino acid sequence selected from the group consisting of SEQ m NO:1-24, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ m N0:1-24, and d) an irnmunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ m NO:1-24. The method comprises a) combining the polypeptide with at least one test compound under suitable conditions, and b) detecting binding of the polypeptide to the test compound, thereby identifying a compound that specifically binds to the polypeptide.
The invention further provides a method of screening for a compound that modulates the activity of a polypeptide selected from the group consisting of a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ m NO:1-24, b) a polypeptide comprising a naturally occurring amino acid sequence at least 90% identical to an amino acid sequence selected from the group consisting of SEQ m N0:1-24, c) a biologically active fragment of a polypeptide having an amtino acid sequence selected from the group consisting of SEQ m N0:1-24, arid d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ m NO:1-24. The method comprises a) combining the polypeptide with at least one test compound under conditions permissive for the activity of the polypeptide, b) assessing the activity of the polypeptide in the presence of the test compound, and c) comparing the activity of the polypeptide in the presence of the test compound with the activity of the polypeptide in the absence of the test compound, wherein a change in the activity of the polypeptide in the presence of the test compound is indicative of a compound that modulates the activity of the polypeptide.
The invention further provides a method for screening a compound for effectiveness in altering expression of a target polynucleotide, wherein said target polynucleotide comprises a polynucleotide sequence selected from the group consisting of SEQ m N0:25-48, 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.
The invention further provides a method for assessing toxicity of a test compound, said method comprising a) treating a biological sample containing nucleic acids with the°test compound;
b) hybridizing the nucleic acids of the treated biological sample with a probe comprising at least 20 contiguous nucleotides of a polynucleotide selected from the group consisting of i) a polynucleotide comprising a polynucleotide sequence selected from the group consisting of SEQ
ID N0:25-48, ii) a polynucleotide comprising a naturally occurring polynucleotide sequence at least 90% identical to a polynucleotide sequence selected from the group consisting of SEQ JD N0:25-48, iii) a polynucleotide having a sequence complementary to i), iv) a polynucleotide complementary to the polynucleotide of ii), and v) an RNA equivalent of i)-iv). Hybridization occurs under conditions whereby a specific hybridization complex is formed between said probe and a target polynucleotide in the biological sample, said target polynucleotide selected from the group consisting of i) a polynucleotide comprising a polynucleotide sequence selected from the group consisting of SEQ ID
N0:25-48, ii) a polynucleotide comprising a naturally occurring polynucleotide sequence at least 90% identical to a polynucleotide sequence selected from the group consisting of SEQ ID N0:25-48, iii) a polynucleotide complementary to the polynucleotide of i), iv) a polynucleotide complementary to the polynucleotide of ii), and v) an RNA equivalent of i)-iv).
Alternatively, the target polynucleotide comprises a fragment of a polynucleotide sequence selected from the group consisting of i)-v) above; c) quantifying the amount of hybridization complex; and d) comparing the amount of hybridization complex in the treated biological sample with the amount of hybridization complex in an untreated biological sample, wherein a difference in the amount of hybridization complex in the treated biological sample is indicative of toxicity of the test compound.
BRIEF DESCRIPTION OF THE TABLES
Table 1 summarizes the nomenclature for the full length polynucleotide and polypeptide sequences of the present invention.
Table 2 shows the GenBank identification number and annotation of the nearest GenBank homolog for polypeptides of the invention. The probability scores for the matches between each polypeptide and its homolog(s) are also shown.
Table 3 shows structural features of polypeptide sequences of the invention, including predicted motifs and domains, along with the methods, algorithms, and seaxchable databases used for analysis of the polypeptides.
Table 4 lists the cDNA and/or genomic DNA fragments which were used to assemble polynucleotide sequences of the invention, along with selected fragments of the polynucleotide sequences.

Table 5 shows the representative cDNA library for polynucleotides of the invention.
Table 6 provides an appendix which describes the tissues and vectors used for construction of the cDNA libraries shown in Table 5.
Table 7 shows the tools, programs, and algorithms used to analyze the polynucleotides and polypeptides of the invention, along with applicable descriptions, references, and threshold parameters.
DESCRIPTION OF THE INVENTION
Before the present proteins, nucleotide sequences, and methods are described, it is understood that this invention is not limited to the particular machines, materials and methods described, as these may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention which will be limited only by the appended claims.
It must be noted that as used herein and in the appended claims, the singular forms "a," "an,"
and "the" include plural reference unless the context clearly dictates otherwise. Thus, for example, a reference to "a host cell" includes a plurality of such host cells, and a reference to "an antibody" is a reference to one or more antibodies and equivalents thereof known to those skilled in the art, and so forth.
Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art to which this invention belongs.
Although any machines, materials, and methods similar or equivalent to those described herein can be used to practice or test the present invention, the preferred machines, materials and methods are now described. All publications mentioned herein are cited for the purpose of describing and disclosing the cell lines, protocols, reagents and vectors which are reported in the publications and which might be used in connection with the invention. Nothing herein is to be construed as an admission that the invention is not entitled to antedate such disclosure by virtue of prior invention.
DEFINITIONS
"SECP" refers to the amino acid sequences of substantially purified SECP
obtained from any species, particularly a mammalian species, including bovine, ovine, porcine, marine, equine; and human, and from any source, whether natural, synthetic, semi-synthetic, or recombinant.
The term "agonist" refers to a molecule which intensifies or mimics the biological activity of SECP. Agonists may include proteins, nucleic acids, carbohydrates, small molecules, or any other compound or composition which modulates the activity of SECP either by directly interacting with SECP or by acting on components of the biological pathway in which SECP
participates.
An "allelic variant" is an alternative form of the gene encoding SECP. 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 SECP include those sequences with deletions, insertions, or substitutions of different nucleotides, resulting in a polypeptide the same as SECP or a polypeptide with at least one functional characteristic of SECP. Included within this definition are polymorphisms which may or may not be readily detectable using a particular oligonucleotide probe of the polynucleotide encoding SECP, and improper or unexpected hybridization to allelic variants, with a locus other than the normal chromosomal locus for the polynucleotide sequence encoding SECP. 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 SECP. Deliberate amino acid substitutions may be made on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, andlor the amphipathic nature of the residues, as long as the biological or immunological activity of SECP is retained. For example, negatively charged amino acids may include aspartic acid and glutamic acid, and positively charged amino acids may include lysine and arginine. Amino acids with uncharged polar side chains having similar hydrophilicity values may include: asparagine and glutamine; and serine and threonine.
Amino acids with uncharged side chains having similar hydrophilicity values may include: leucine, isoleucine, and valine; glycine and alanine; and phenylalanine and tyrosine.
The terms "amino acid" and "amino acid sequence" refer to an oligopeptide, peptide, polypeptide, or protein sequence, or a fragment of any of these, and to naturally occurring or synthetic molecules. Where "amino acid sequence" is recited to refer to a sequence of a naturally occurring protein molecule, "amino acid sequence" and life terms are not meant to limit the amino acid sequence to the complete native amino acid sequence associated with the recited protein molecule.
"Amplification" relates to the production of additional copies of a nucleic acid sequence.
Amplification is generally carried out using polymerase chain reaction (PCR) technologies well known in the art.
The term "antagonist" refers to a molecule which inhibits or attenuates the biological activity of SECP. Antagonists may include proteins such as antibodies, nucleic acids, carbohydrates, small molecules, or any other compound or composition which modulates the activity of SECP either by directly interacting with SECP or by acting on components of the biological pathway in which SECP
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 SECP polypeptides can be prepared using intact polypeptides or using fragments containing small peptides of interest as the immunizing antigen. The polypeptide or oligopeptide used to immunize an animal (e.g., a mouse, a rat, or a rabbit) can be derived from the translation of RNA, or synthesized chemically, and can be conjugated to a carrier protein if desired. Commonly used carriers that are chemically coupled to peptides include bovine serum albumin, thyroglobulin, and keyhole limpet hemocyanin (KLH). 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. Aptarners are derived from an in vitro evolutionary process (e.g., SELEX
(Systematic Evolution of Ligands by EXponential Enrichment), described in U.S.
Patent No.
5,270,163), which selects for target-specific aptamer sequences from large combinatorial libraries.
Aptamer compositions may be double-stranded or single-stranded, and may include deoxyribonucleotides, ribonucleotides, nucleotide derivatives, or other nucleotide-like molecules.
The nucleotide components of an aptamer may have modified sugar groups (e.g., the 2'-OH group of a ribonucleotide may be replaced by 2'-F or 2'-NHZ), which may improve a desired property, e.g., resistance to nucleases or longer lifetime in blood. Aptamers may be conjugated to other molecules, e.g., a high molecular weight carrier to slow clearance of the aptamer from the circulatory system.
Aptamers may be specifically cross-linked to their cognate ligands, e.g., by photo-activation of a cross-linker. (See, e.g., Brody, E.N. and L. Gold (2000) J. Bioteclmol. 74:5-13.) The term "intramer" refers to an aptamer which is expressed in vivo. For example, a vaccinia virus-based RNA expression system has been used to express specific RNA
aptamers at high levels in the cytoplasm of leukocytes (Blind, M. et al. (1999) Proc. Natl Acad. Sci. USA
96:3606-3610).
The term "spiegelmer" refers to an aptamer which includes L-DNA, L-RNA, or other left-handed nucleotide derivatives or nucleotide-like molecules. Aptamers containing Ieft-handed nucleotides are resistant to degradation by naturally occurring enzymes, which normally act on substrates containing right-handed nucleotides.
The term "antisense" refers to any composition capable of base-pairing with the "sense"
(coding) strand of a specific nucleic acid sequence. Antisense compositions may include DNA;

RNA; peptide nucleic acid (PNA); oligonucleotides having modified backbone linkages such as phosphorothioates, methylphosphonates, or benzylphosphonates; oligonucleotides having modified sugar groups such as 2 =methoxyethyl sugars or 2'-methoxyethoxy sugars; or oligonucleotides having modified bases such as 5-methyl cytosine, 2'-deoxyuracil, ox 7-deaza-2'-deoxyguanosine. Antisense molecules may be produced by any method including chemical synthesis ox 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 SECP, or of any oligopeptide thereof, to induce a specific immune response in appropriate animals or cells and to bind with specific antibodies.
"Complementary" describes the relationship between two single-stranded nucleic acid sequences that anneal by base-pairing. For example, 5'-AGT-3' pairs with its complement, 3'-TCA-5'.
A "composition comprising a given polynucleotide sequence" and a "composition comprising a given amino acid sequence" refer broadly to any composition containing the given polynucleotide or amino acid sequence. The composition may comprise a dry formulation or an aqueous solution.
Compositions comprising polynucleotide sequences encoding SECP or fragments of SECP may be employed as hybridization probes. The probes may be stored in freeze-dried form and may be associated with a stabilizing agent such as a carbohydrate. In hybridizations, the probe may be deployed in an aqueous solution containing salts (e.g., NaCI), detergents (e.g., sodium dodecyl sulfate; SDS), and other components (e.g., Denhardt's solution, dry milk, salmon sperm DNA, etc.).
"Consensus sequence" xefers 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 genomic DNA fragments using a computer program for fragment assembly, such as the GELVIEW fragment assembly system (GCG, Madison WI] or Phrap (University of Washington, Seattle WA). Some sequences have been both extended and assembled to produce the consensus sequence.
"Conservative amino acid substitutions" are those substitutions that are predicted to least interfere with the properties of the original protein, i.e., the structure and especially the function of the protein is conserved and not significantly changed by such substitutions.
The table below shows amino acids which may be substituted for an original amino acid in a protein and which are regarded as conservative amino acid substitutions.
Original Residua Conservative Substitution Ala Gly, Ser Arg His, Lys Asn Asp, Gln, His Asp Asn, Glu Cys Ala, Ser Gln ~ Asn, GIu, His Glu Asp, GIn, His Gly Ala His Asn, Arg, Gln, Glu IIe Leu, Val Leu Ile, Val Lys Arg, Gln, Glu Met Leu, Tle Phe His, Met, Leu, Trp, Tyr Ser Cys, Thr ' Thr Ser, Val Trp Phe, Tyr Tyr His, Phe, Trp Val Ile, Leu, Thr Conservative amino acid substitutions generally maintain (a) the structure of the polypeptide backbone in the area of the substitution, for example, as a beta sheet or alpha helical conformation, (b) the charge or hydrophobicity of the molecule at the site of the substitution, and/or (c) the 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 c 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 xepresent a structural or functional domain of the encoded protein, new proteins rnay 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 SECP or the polynucleotide encoding SECP
which is identical in sequence to but shorter in length than the parent sequence. A
fragment may comprise up to the entire length of the defined sequence, minus one nucleotide/amino acid residue. For example, a fragment may comprise from 5 to 1000 contiguous nucleotides or amino acid residues. A fragment used as a probe, primer, antigen, therapeutic molecule, or for other purposes, may be at least 5, 10, 15, 16, 20, 25, 30, 40, 50, 60, 75, 100, 150, 250 or at least 500 contiguous nucleotides or amino acid 20 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 ID N0:25-48 comprises a region of unique polynucleotide sequence that specifically identifies SEQ m N0:25-48, for example, as distinct from any other sequence in the genome from which the fragment was obtained. A fragment of SEQ ID N0:25-48 is useful, for example, in hybridization and amplification technologies and in analogous methods that distinguish SEQ m N0:25-48 from related polynucleotide sequences. The precise length of a fragment of SEQ
ID N0:25-48 and the region of SEQ ID N0:25-48 to which the fragment corresponds are routinely determinable by one of ordinary skill in the art based on the intended purpose for the fragment.
A fragment of SEQ ID NO:1-24 is encoded by a fragment of SEQ ID N0:25-48. A
fragment of SEQ ID N0:1-24 comprises a region of unique amino acid sequence that specifically identifies SEQ TD NO:1-24. For example, a fragment of SEQ m NO:1-24 is useful as an immunogenic peptide for the development of antibodies that specifically recognize SEQ ll~ NO:1-24.
The precise length of a fragment of SEQ m NO:1-24 and the region of SEQ 1D NO:1-24 to which the fragment corresponds are routinely determinable by one of ordinary skill in the art based on the intended purpose for the fragment.
A "full length" polynucleotide sequence is one containing at least a translation initiation codon (e.g., methionine) followed by an open reading frame and a translation termination codon. A
"full length" polynucleotide sequence encodes a "full length" polypeptide sequence.
"Homology" refers to sequence similarity or, interchangeably, sequence identity, between two or more polynucleotide sequences or two or more polypeptide sequences.
The terms "percent identity" and "% identity," as applied to polynucleotide sequences, refer to the percentage of residue matches between at least two polynucleotide sequences aligned using a standardized algorithm. Such an algorithm may insert, in a standardized and reproducible way, gaps in the sequences being compared in order to optimize alignment between two sequences, and therefore achieve a more~meaningful comparison of the two sequences.
Percent identity between polynucleotide sequences may be determined using the default parameters of the CLUSTAL V algorithm as incorporated into the MEGALIGN
version 3.12e sequence alignment program. This program is part of the LASERGENE software package, a suite of molecular biological analysis programs (DNASTAR, Madison WI). CLUSTAL V is described in Higgins, D.G. and P.M. Sharp (1989) CABIOS 5:151-153 and in Higgins, D.G. et al. (1992) CABIOS
8:189-191. For pairwise alignments of polynucleotide sequences, the default parameters are set as follows: Ktuple=2, gap penalty=5, window=4, and "diagonals saved"=4. The "weighted" residue weight table is selected as the default. Percent identity is reported by CLUSTAL V as the "percent similarity" between aligned polynucleotide sequences.
Alternatively, a suite of commonly used and freely available sequence comparison algorithms is provided by the National Center for Biotechnology Information (NCBl> 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.govlBLAST/. 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 rnisrrcatch: -2 Open Gap: 5 and Extension Gap: 2 penalties Gap x drop-off.' SO
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, fox 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 residue matches between at least two polypeptide sequences aligned using a standardized algorithm. Methods of polypeptide sequence alignment are well-known. Some alignment methods take into account conservative amino acid substitutions.
Such conservative substitutions, explained in more detail above, generally preserve the charge and-hydrophobicity at the site of substitution, thus preserving the structure (and therefore function) of the polypeptide.
Percent identity between polypeptide sequences may be determined using the default parameters of the CLUSTAL V algorithm as incorporated into the MEGALIGN
version 3.12e sequence alignment program (described and referenced above). For pairwise alignments of polypeptide sequences using CLUSTAL V, the default parameters are set as follows: Ktuple=l, gap penalty=3, window=5, and "diagonals saved"=5. The PAM250 matrix is selected as the default residue weight table. As with polynueleotide alignments, the percent identity is reported by CLUSTAL V as the "percent similarity" between aligned polypeptide sequence pairs.
Alternatively the NCBI BLAST software suite may be used. For example, for a pairwise comparison of two polypeptide sequences, one may use the "BLAST 2 Sequences"
tool Version 2Ø12 (April-21-2000) with blastp set at default parameters. Such default parameters may be, for example:
Matrix: BLOSUM62 Open Gap: 11 arid Extension Gap: 1 penalties Gap x drop-off. SO
Expect: 10 .
Word Size: 3 Filter: 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 instance, a fragment of at least 15, at least 20, at least 30, at least 40, at least 50, at least 70 or at least 150 contiguous residues. Such lengths are exemplary only, and it is understood that any fragment length supported by the sequences shown herein, in the tables, figures or Sequence Listing, may be used to describe a length over which percentage identity may be measured.
"Human artificial chromosomes" (HACs) are linear microchromosomes which may contain DNA sequences of about 6 kb to 10 Mb in size and which contain all of the elements required for chromosome replication, segregation and maintenance.
The term "humanized antibody" refers to an antibody molecule in which the amino acid sequence in the non-antigen binding regions has been altered so that the antibody more closely resembles a human antibody, and still retains its original binding ability.
"Hybridization" refers to the process by which a polynucleotide strand anneals with a complementary strand through base pairing under defined hybridization conditions. Specific hybridization is an indication that two nucleic acid sequences share a high degree of complementarity.
Specific hybridization complexes form under permissive annealing conditions and remain hybridized after the "washing" step(s). The washing steps) is particularly important in determining the stringency of the hybridization process, with more stringent conditions allowing less non-specific binding, i.e., binding between pairs of nucleic acid strands that are not perfectly matched. Pernussive 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 ~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 (Tin) fox 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, T. et al.
(1989) Molecular Clonin ~: A Laboratory Manual, 2"d ed., vol. 1-3, Cold Spring Harbor Press, Plainview NY; specifically see volume 2, chapter 9.
High stringency conditions for hybridization between polynucleotides of the present invention include wash conditions of 68°C in the presence of about 0.2 x SSC and about 0.1 % SDS, for 1 hour. Alternatively, temperatures of about 65°C, 60°C, 55°C, or 42°C may be used. SSC
concentration may be varied from about 0.1 to 2 x SSC, with SDS being present at about 0.1%.

Typically, blocking reagents are used to block non-specific hybridization.
Such blocking reagents include, for instance, sheared and denatured salmon sperm DNA at about 100-200 ,ug/znl. Organic solvent, such as formamide at a concentration of about 35-50% v/v, may also be used under particular circumstances, such as for RNA:DNA hybridizations. Useful variations on these wash conditions will be readily apparent to those of ordinary skill in the art. Hybridization, particularly under high stringency conditions, may be suggestive of evolutionary similarity between the nucleotides. Such similarity is strongly indicative of a similar role for the nucleotides and their encoded polypeptides.
The term "hybridization complex" refers to a complex formed between two nucleic acid sequences by virtue of the formation of hydrogen bonds between complementary bases. A
hybridization complex may be formed in solution (e.g., Cot or Rot analysis) or formed between one nucleic acid sequence present in solution and another nucleic acid sequence immobilized on a solid support (e.g., paper, membranes, filters, chips, pins or glass slides, or any other appropriate substrate to which cells or their nucleic acids have been fixed).
The words "insertion" and "addition" refer to changes in an amino acid or nucleotide sequence resulting in the addition of one or more amino acid residues or nucleotides, respectively.
"hnmune 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 SECP
which is capable of eliciting an immune response when introduced into a living organism, for example, a mammal. The term "immunogenic fragment" also includes any polypeptide or oligopeptide fragment of SECP which is useful in any of the antibody production methods disclosed herein or known in the art.
The term "microarray" refers to an arrangement of a plurality of polynucleotides, polypeptides, or other chemical compounds on a substrate.
The terms "element" and "array element" refer to a polynucleotide, polypeptide, or other chemical compound having a unique and defined position on a microarray.
The term "modulate" refers to a change in the activity of SECP. For example, modulation may cause an increase or a decrease in protein activity, binding characteristics, or any other biological, functional, or immunological properties of SECP.
The phrases "nucleic acid" and "nucleic acid sequence" refer to a nucleotide, oligonucleotide, polynucleotide, or any fragment thereof. These phrases also refer to DNA or RNA of genomic or synthetic origin which may be single-stranded or double-stranded and may represent the sense or the antisense strand, to peptide nucleic acid (PNA), or to any DNA-like or RNA-like material.

"Operably linked" refers to the situation in which a first nucleic acid sequence is placed in a functional relationship with a second nucleic acid sequence. For instance, a promoter is operably linked to a coding sequence if the promoter affects the transcription or expression of the coding sequence. Operably linked DNA sequences rnay be in close proximity or contiguous and, where necessary to join two protein coding regions, in the same reading frame.
"Peptide nucleic acid" (PNA) refers to an antisense molecule or anti-gene agent which comprises an oligonucleotide of at least about 5 nucleotides in length linked to a peptide backbone of amino acid residues ending in lysine. The terminal lysine confers solubility to the composition.
PNAs preferentially bind complementary single stranded DNA or RNA and stop transcript elongation, and may be pegylated to extend their lifespan in the cell.
"Post-translational modification" of an SECP 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 SECP.
"Probe" refers to nucleic acid sequences encoding SECP, their complements, or fragments thereof, which are used to detect identical, allelic or related nucleic acid sequences. Probes are isolated oligonucleotides or polynucleotides attached to a detectable label or reporter molecule.
Typical labels include radioactive isotopes, ligands, chemiluminescent agents, and enzymes.
"Primers" are short nucleic acids, usually DNA oligonucleotides, which may be annealed to a target polynucleotide by complementary base-pairing. The primer may then be extended along the target DNA strand by a DNA polymerase enzyme. Primer pairs can be used for amplification (and identification) of a nucleic acid sequence, e.g., by the polymerase chain reaction (PCR).
Probes and primers as used in the present invention typically comprise at least 15 contiguous nucleotides of a known sequence. In order to enhance specificity, longer probes and primers may also be employed, such as probes and primers that comprise at least 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, or at least 150 consecutive nucleotides of the disclosed nucleic acid sequences. Probes and primers may be considerably longer than these examples, and it is understood that any length supported by the specification, including the tables, figures, and Sequence Listing, may be used.
Methods for preparing and using probes and primers are described in the references, for example Sambrook, J. et al. (1989) Molecular Cloning: A Laborator~Manual, 2"d ed., vol. 1-3, Cold Spring Harbor Press, Plainview NY; Ausubel, F.M. et al. (1987) Current Protocols in Molecular Biolo~y, Greene Publ. Assoc. & Wiley-Intersciences, New York NY; Innis, M. et al. (1990) PCR
Protocols, A Guide to Methods and Applications, Academic Press, San Diego CA.
PCR primer pairs can be derived from a known sequence, fox example, by using computer programs intended for that purpose such as Primer (Version 0.5, 1992, 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/MIT 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 UK) 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 bath unique and conserved oligonucleotides and polynucleotide fragments. The oligonucleotides and polynucleotide fragments identified by any of the above selection methods are useful in hybridization technologies, for example, as PCR or sequencing primers, microarray elements, or specific probes to identify fully or partially complementary polynucleotides in a sample of nucleic acids. Methods of oligonucleotide selection are not limited to those described above.
A "recombinant nucleic acid" is a sequence that is not naturally occurring or has a sequence that is made by an artificial combination of two or more otherwise separated segments of sequence.
This artificial combination is often accomplished by chemical synthesis or, more commonly, by the artificial manipulation of isolated segments of nucleic acids, e.g., by genetic engineering techniques such as those described in Sambrook, supra. The term recombinant includes nucleic acids that have been altered solely by addition, substitution, or deletion of a portion of the nucleic acid. Frequently, a recombinant nucleic acid may include a nucleic acid sequence operably linked to a promoter sequence. Such a recombinant nucleic acid may be part of a vector that is used, for example, to transform a cell.
Alternatively, such recombinant nucleic acids may be part of a viral vector, e.g., based on a vaccinia virus, that could be use to vaccinate a mammal wherein the recombinant nucleic acid is expressed, inducing a protective immunological response in the mammal.

A "regulatory element" refers to a nucleic acid sequence usually derived from untranslated regions of a gene and includes enhancers, promoters, introns, and 5' and 3' untxanslated regions (UTRs). Regulatory elements interact with host or viral proteins which control transcription, translation, or RNA stability.
"Reporter molecules" are chemical or biochemical moieties used for labeling a nucleic acid, amino acid, or antibody. Reporter molecules include radionuclides; enzymes;
fluorescent, chemiluminescent, or chromogenic agents; substrates; cofactors; inhibitors;
magnetic particles; and other moieties known in the art.
An "RNA equivalent," in reference to a DNA sequence, is composed of the same linear sequence of nucleotides as the reference DNA sequence with the exception that all occurrences of the nitrogenous base 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 SECP, nucleic acids encoding SECP, 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 60% free, preferably at least 75% free, and most preferably at least 90% free from other components with which they are naturally associated.
A "substitution" refers to the replacement of one or more amino acid residues or nucleotides by different amino acid residues or nucleotides, respectively.
"Substrate" refers to any suitable rigid or semi-rigid support including membranes, filters, chips, slides, wafers, fibers, magnetic or nonmagnetic beads, gels, tubing, plates, polymers, microparticles and capillaries. The substrate can have a variety of surface forms, such as wells, trenches, pins, chamiels 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 undex 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. The term genetic manipulation does not include classical cross-breeding, or in vitro fertilization, but rather is directed to the introduction of a recombinant DNA molecule. The transgenic organisms contemplated in accordance with the present invention include bacteria, cyanobacteria, fungi, plants and animals. The isolated DNA of the present invention can be introduced into the host by methods known in the art, for example infection, transfection, transformation or transconjugation. Techniques for transferring the DNA of the present invention into such organisms are widely known and provided in references such as Sambrook et al. (1989), su ra.
A."variant" of a particular nucleic acid sequence is defined as a nucleic acid sequence having at least 40% sequence identity to the particular nucleic acid sequence over a certain length of one of the nucleic acid sequences using blastn with the "BLAST 2 Sequences" tool Version 2Ø9 (May-07-1999) set at default parameters. Such a pair of nucleic acids may show, for example, at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% or greater sequence identity over a certain defined length. A variant may be described as, for example, an "allelic" (as defined above), "splice," "species," or "polymorphic" variant. A
splice variant rnay have significant identity to a reference molecule, but will generally have a greater or lesser number of polynucleotides due to alternate splicing of exons during mRNA processing. The corresponding polypeptide may possess additional functional domains or lack domains that are present in the reference molecule. Species variants are polynucleotide sequences that vary 'from one species to another. The xesulting polypeptides will generally have significant amino acid identity relative to each other. A polymorphic variant is a variation in the polynucleotide sequence of a particular gene between individuals of a given species. Polymorphic variants also may encompass "single nucleotide polymorphisms" (SNPs) in which the polynucleotide sequence varies by one nucleotide base. The presence of SNPs may be indicative of, for example, a certain population, a disease state, or a propensity for a disease state.
A "variant" of a particular polypeptide sequence is defined as a polypeptide sequence having at least 40% sequence identity to the particular polypeptide sequence over a certain length of one of the polypeptide sequences using blastp with the "BLAST 2 Sequences" tool Version 2Ø9 (May-07-1999) set at default parameters. Such a pair of polypeptides may show, for example, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%
or greater sequence identity over a certain defined length of one of the polypeptides.
THE INVENTION
The invention is based on the discovery of new human secreted proteins (SECP), the polynucleotides encoding SECP, and the use of these compositions for the diagnosis, treatment, or prevention of cell proliferative, autoimmune/inflammatory, cardiovascular, neurological, and developmental disorders.
Table 1 summarizes the nomenclature for the full length polynucleotide and polypeptide sequences of the invention. Each polynucleotide and its corresponding polypeptide are correlated to a single Incyte project identification number (Incyte Project )D). Each polypeptide sequence is denoted by both a polypeptide sequence identification number (Polypeptide SEQ m NO:) and an Incyte polypeptide sequence number (Incyte Polypeptide )D) 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 ID) as shown.
Table 2 shows sequences with homology to the polypeptides of SEQ ID NO: I-2, SEQ )D
N0:10-13, and SEQ >D N0:17-22, as identified by BLAST analysis against the GenBank protein (genpept) database. Columns 1 and 2 show polypeptides of SEQ m NO:1-2, SEQ ID
NO:10-13, and SEQ m N0:17-22 and their corresponding Incyte polypeptide sequence numbers (Incyte Polypeptide ll~). Column 3 shows the GenBank identification number (Genbank ID NO:) of the nearest GenBank homolog. Column 4 shows the probability score for the matches between each polypeptide and its homolog. Column 5 shows the annotation of the GenBank database homologs along with relevant citations where applicable, all of which are expressly incorporated by reference herein.
Table 3 shows various structural features of each of the polypeptides of the invention.
Columns 1 and 2 show the polypeptide sequence identification number (SEQ m NO:) and the corresponding Incyte polypeptide sequence number (Incyte Polypeptide ID) for each polypeptide of the invention. Column 3 shows the number of amino acid residues in each polypeptide. Column 4 shows potential phosphorylation sites, and column 5 shows potential glycosylation sites, as determined by the MOTIFS program of the GCG sequence analysis software package (Genetics Computer Group, Madison WI). Column 6 shows amino acid residues comprising signature sequences, domains, and motifs, including the locations of signal peptides (as indicated by "Signal Peptide" and/or "signal_cleavage".) 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 arid 3 summarize the properties of polypeptides of the invention, and these properties establish that the claimed polypeptides are secreted proteins. For example, SEQ ID NO:1 is 30% identical, from residue C101 to residue C449, to Arabidopsis thaliana pectinacetylestexase (GenBank 113 g6478931) as determined by the Basic Local Alignment Search Tool (BLAST). (See Table 2.) The BLAST probability score is 1.1e-22, which indicates the probability of obtaining the observed polypeptide sequence alignment by chance. SEQ ID NO:1 also contains a signal peptide as determined by searching for statistically significant matches in the HMMER
database of conserved protein family domains. (See Table 3.) In an alternative example, SEQ ID N0:2 is 76% identical, from residue Ml to residue D185, to human pancreatitis-associated protein (PAP) (GenBank ID
g482909) as determined by the Basic Local Alignment Search Tool (BLAST). (See Table 2.) The BLAST probability score is 7.0e-76, which indicates the probability of obtaining the observed .
polypeptide sequence alignment by chance. SEQ ID N0:2 also contains a lectin C-type domain as determined by searching for statistically significant matches in the hidden Markov model (HMM)-based PFAM database of conserved protein family domains. (See Table 3.) Data from BLIMPS, MOTIFS, and PROFILESCAN analyses provide further corroborative evidence that SEQ ID NO:2 is a pancreatitis-associated protein. Note that the C-type lectin domain is found in PAP. In an alternative example, SEQ ll~ N0:7-9 contain signal peptides as determined by the I~MER algorithm used for searching for statistically significant matches in the hidden Markov model (HMM)-based database of conserved protein family domains. Data from Spscan provides further corroborative evidence that SEQ 117 N0:7-9 are secreted proteins. In an alternative example, SEQ DJ N0:13 is 99% identical, from residue M1 to residue F331, to human pregnancy-specific beta-1-glycoprotein (GenBank ID g190647) as determined by the Basic Local Alignment Search Tool (BLAST). (See Table 2.) The BLAST probability score is 4.1e-181, which indicates the probability of obtaining the observed polypeptide sequence alignment by chance. SEQ lD N0:13 also contains immunoglobulin domains as determined by searching for statistically significant matches in the hidden Markov model (HMM)-based PFAM database of conserved protein family domains. (See Table 3.) In an alternative example, SEQ )D N0:19 is 40% identical, from residue S 177 to residue P317, to the marine secreted polypeptide, ZSIG37 (GenBank )D g6274477), as determined by the Basic Local Alignment Search Tool (BLAST). (See Table 2.) The BLAST probability score is 6.6e-18, which indicates the probability of obtaining the observed polypeptide sequence alignment by chance. SEQ m N0:19 r also contains C1q domains as determined by searching for statistically significant matches in the hidden Markov model (IBvIM)-based PFAM database of conserved protein family domains. (See Table 3.) Data from BLIMPS and MOTIFS analyses provide further corroborative evidence that SEQ
m N0:19 is a secreted polypeptide with.Clq domains. In an alternative example, SEQ ID N0:20 is 68% identical, from residue W9 to residue A631, to the rat zona pellucida (ZP) 1 glycoprotein (GenBank ID g2804566), as determined by BLAST analysis, with a probability score of 3.7e-230.
SEQ ID N0:20 also contains a ZP domain as determined by searching for statistically significant matches in the hidden Markov model (HMM)-based PFAM database of conserved protein family domains. Data from BLIMPS and PROFILESCAN analyses provide further corroborative evidence that SEQ 117 N0:20 is a ZP-related protein. In an alternative example, SEQ )D
N0:21 is 42%
identical, from residue L11 to residue S320, to a human immunoglobulin superfamily member protein (GenBank )D g7767239), as determined by BLAST analysis, with a probability score of 5.9e-77.
SEQ ID N0:21 also contains immunoglobulin domains as determined by searching for statistically significant matches in the hidden Markov model (HMM)-based PFAM database of conserved protein family domains. Data from BLIMPS analysis provides further corroborative evidence that SEQ ~
N0:21 is a member of the immunoglobulin superfamily. In an alternative example, SEQ ID N0:22 is 96% identical, from residue Ml to residue D225, to marine gliacolin, a C1q-like protein expressed in Glial cells (GenBank )D g10566471), as determined by BLAST analysis, with a probability score of 1.2e-137. SEQ )D N0:22 also contains a Clq domain, and a collagen repeat motif characteristic of Clq proteins, as determined by searching for statistically significant matches in the hidden Markov model (HMM)-based PFAM database of conserved protein family domains. Data from BLIMPS and MOTIFS analyses provide further corroborative evidence that SEQ )D N0:22 is a C1q-related polypeptide. The algorithms and parameters for the analysis of SEQ m N0:3-6, SEQ TD NO:10-12, SEQ ID N0:14-18, and SEQ ID N0:23-24, were analyzed and annotated in a similar manner. The algorithms and parameters for the analysis of SEQ m N0:1-24 are described in Table 7.
As shown in Table 4, the full length polynucleotide sequences of the present invention were assembled using cDNA sequences or coding (exon) sequences derived from genomic DNA, or any combination of these two types of sequences. Column 1 lists the polynucleotide sequence identification number (Polynucleotide SEQ )D NO:), the corresponding Incyte polynucleotide consensus sequence number (Incyte m) for each polynucleotide of the invention, and the length of each polynucleotide sequence in basepairs. Column 2 shows the nucleotide start (S') and stop (3') positions of the cDNA andJor genomic sequences used to assemble the full length polynucleotide sequences of the invention, and of fragments of the polynucleotide sequences which are useful, for example, in hybridization or amplification technologies that identify SEQ m N0:25-48 or that distinguish between SEQ ID N0:25-48 and related polynucleotide sequences.
The polynucleotide fragments described in Column 2 of Table 4 may refer specifically, for example, to Incyte 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 polynucleotide sequences. 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_NI 1V2 YYYYY N3 N4 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 Nl,a,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 FLXXXXXX_gAAAAA_gBBBBB_1 1V 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 polynucleotide sequences which were assembled using Incyte cDNA sequences. The representative cDNA library is the Incyte eDNA library which is most frequently represented by the Incyte cDNA sequences which were used to assemble and confirm the above polynucleotide sequences. The tissues and vectors which were used to construct the cDNA libraries shown in Table 5 are described in Table 6.
The invention also encompasses SECP variants. A preferred SECP variant is one which has at least about 80%, or alternatively at least about 90%, or even at least about 95% amino acid sequence identity to the SECP amino acid sequence, and which contains at least one functional or structural characteristic of SECP.
The invention also encompasses polynucleotides which encode SECP. In a particular embodiment, the invention encompasses a polynucleotide sequence comprising a sequence selected from the group consisting of SEQ TD N0:25-48, which encodes SECP. The polynucleotide sequences of SEQ ID N0:25-48, as presented in the Sequence Listing, embrace the equivalent RNA sequences, wherein occurrences of the nitrogenous base thymine are replaced with uracil, and the sugar backbone is composed of ribose instead of deoxyribose.
The invention also encompasses a variant of a polynucleotide sequence encoding SECP. In particular, such a variant polynucleotide sequence will have at least about 70%, or alternatively at least about 85%, or even at least about 95% polynucleotide sequence identity to the polynucleotide sequence encoding SECP. A particular aspect of the invention encompasses a variant of a polynucleotide sequence comprising a sequence selected from the group consisting of SEQ ID
N0:25-48 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 N0:25-48. Any one of the polynucleotide variants described above can encode an amino acid sequence which contains at least one functional or structural characteristic of SECP.
In addition, or in the alternative, a polynucleotide variant of the invention is a splice variant of a polynucleotide sequence encoding SECP. A splice variant may have portions which have significant sequence identity to the polynucleotide sequence encoding SECP, but will generally have a greater or lesser number of polynucleotides due to additions or deletions of blocks of sequence arising from alternate splicing of exons during mRNA processing. A splice variant may have less than about 70%, or alternatively less than about 60%, or alternatively less than about 50%
polynucleotide sequence identity to the polynucleotide sequence encoding SECP
over its entire length; however, portions of the splice variant will have at least about 70%, or alternatively at least about 85%, or alternatively at least about 95%, or alternatively 100%
polynucleotide sequence identity to portions of the polynucleotide sequence encoding SECP. For example, a polynucleotide comprising a sequence of SEQ DJ N0:36 is a splice variant of a polynucleotide comprising a sequence of SEQ m N0:47, and a polynucleotide comprising a sequence of SEQ m N0:42 is a splice variant of a polynucleotide comprising a sequence of SEQ m N0:48. Any one of the splice variants described above can encode an amino acid sequence which contains at least one functional or structural characteristic of SECP.
Tt 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 SECP, 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 SECP, and all such variations are to be considered as being specifically disclosed.
Although nucleotide sequences which encode SECP and its variants are generally capable of hybridizing to the nucleotide sequence of the naturally occurring SECP under appropriately selected conditions of stringency, it may be advantageous to produce nucleotide sequences encoding SECP 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 SECP 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 Iife, than transcripts produced from the naturally occurring sequence.
The invention also encompasses production of DNA sequences which encode SECP
and SECP derivatives, or fragments thereof, entirely by synthetic chemistry. After production, the synthetic sequence may be inserted into any of the many available expression vectors and cell systems using reagents well known in the art. Moreover, synthetic chemistry may be used to introduce mutations into a sequence encoding SECP or any fragment thereof.
Also encompassed by the invention are polynucleotide sequences that are capable of hybridizing to the claimed polynucleotide sequences, and, in particular, to those shown in SEQ ID
N0:25-48 and fragments thereof under various conditions of stringency. (See, e.g., Wahl, G.M. and S.L. Berger (1987) Methods Enzymol. 152:399-407; Kimmel, A.R. (1987) Methods Enzymol.
152:507-511.) Hybridization conditions, including annealing and wash conditions, are described in "Definitions."
Methods for DNA sequencing are well known in the art and may be used to practice any of the embodiments of the invention. The methods may employ such enzymes as the Klenow fragment of DNA polymerase I, SEQUENASE (US Biochemical, Cleveland OH), Taq polymerase (Applied Biosystems), thermostable T7 polymerase (Amersham Pharmacia Biotech, Piscataway NJ), or combinations of polymerases and proofreading exonucleases such as those found in the ELONGASE
amplification system (Life Technologies, Gaithersburg MD). Preferably, sequence preparation is automated with machines such as the MICROLAB 2200 liquid transfer system (Hanulton, Reno NV), PTC200 thermal cycler (MJ Research, Watertown MA) and ABI CATALYST 800 thermal cycler (Applied Biosystems). Sequencing is then carried out using either the ABI 373 or 377 DNA
sequencing system (Applied Biosystems), the MEGABACE 1000 DNA sequencing system (Molecular Dynamics, Sunnyvale CA), or other systems known in the art. The resulting sequences are analyzed using a variety of algorithms which are well known in the art.
(See, e.g., Ausubel, F.M.
(1997) Short Protocols in Molecular Biolo~y, John Wiley & Sons, New York NY, unit 7.7; Meyers, R.A. (1995) Molecular Biology and Biotechnolo~y, Wiley VCH, New York NY, pp.
856-853.) The nucleic acid sequences encoding SECP 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. (See, e.g., Sarkar, G. (1993) PCR Methods Applic.
2:318-322.) Another method, inverse PCR, uses primers that extend in divergent directions to amplify unlrnown sequence from a circularized template. The template is derived from restriction fragments comprising a known genomic locus and surrounding sequences. (See, e.g., Triglia, T. et al. (1988) Nucleic Acids Res. 16:8186.) A third method, capture PCR, involves PCR amplification of DNA
fragments adjacent to known sequences in human and yeast artificial chromosome DNA.
(See, e.g., Lagerstrom, M. et al. (1991) PCR Methods Applic. 1:111-119.) In this method, multiple restriction enzyme digestions and ligations may be used to insert an engineered double-stranded sequence into a region of unknown sequence before performing PCR. Other methods which may be used to retrieve unlrnown sequences are la~own in the art. (See, e.g., Parker, J.D. et al.
(1991) Nucleic Acids Res.
19:3055-3060). Additionally, one may use PCR, nested primers, and PROMOTERFINDER libraries (Clontech, Palo Alto CA) to walk genomic DNA. This procedure avoids the need to screen libraries and is useful in finding intron/exon junctions. For all PCR-based methods, primers may be designed using commercially available software, such as OLIGO 4.06 primer analysis software (National Biosciences, Plymouth MN) or another appropriate program, to be about 22 to 30 nucleotides in length, to have a GC content of about 50% or more, and to anneal to the template at temperatures of about G8°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-transcxibed 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 SEQITENCE 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 fox sequencing small DNA fragments which may be present in limited amounts in a particular sample.
In another embodiment of the invention, polynucleotide sequences or fragments thereof which encode SECP may be cloned in recombinant DNA molecules that direct expression of SECP, or fragments or functional equivalents thereof, in appropriate host cells. Due to the inherent degeneracy of the genetic code, other DNA sequences which encode substantially the same or a functionally equivalent amino acid sequence may be produced and used to express SECP.
The nucleotide sequences of the present invention can be engineered using methods generally known in the art in order to alter SECP-encoding sequences for a variety of purposes including, but not limited to, modification of the cloning, processing, and/or expression of the gene product. DNA
shuffling by random fragmentation and PCR reassembly of gene fragments and synthetic oligonucleotides may be used to engineer the nucleotide sequences. For example, oligonucleotide-mediated site-directed mutagenesis may be used to introduce mutations that create new restriction sites, alter glycosylation patterns, change codon preference, produce splice variants, and so forth.
The nucleotides of the present invention may be subjected to DNA shuffling techniques such as MOLECULARBREEDING (Maxygen Inc., Santa Clara CA; described in U.S. Patent No.

5,837,458; Chang, C.-C. et al. (I999) 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 SECP, such as its biological or enzymatic activity or its ability to bind to other molecules or compounds. DNA shuffling is a process by which a library of gene variants is produced using PCR-mediated recombination of gene fragments. The library is then subjected to selection or screening procedures that identify those gene variants with the desired properties. These preferred variants may then be pooled and further subjected to recursive rounds of DNA shuffling and selection/screening. Thus, genetic diversity is created through "artificial"
breeding and rapid molecular evolution. For example, fragments of a single gene containing random point mutations may be recombined, screened, and then reshuffled until the desired properties are optimized. Alternatively, fragments of a given gene may be recombined with fragments of homologous genes in the same gene family, either from the same or different species, thereby maximizing the genetic diversity of multiple naturally occurring genes in a directed and controllable manner.
In another embodiment, sequences encoding SECP may be synthesized, in whole or in part, using chemical methods well known in the art. (See, e.g., Caruthers, M.H. et al. (1980) Nucleic Acids Symp. Ser. 7:215-223; and Horn, T. et al. (1980) Nucleic Acids Symp. Ser.
7:225-232.) Alternatively, SECP itself or a fragment thereof may be synthesized using chemical methods. For example, peptide synthesis can be performed using various solution-phase or solid-phase techniques.
(See, e.g., Creighton, T. (1984) Proteins, Structures and Molecular Properties, WH Freeman, New York NY, pp. 55-60; and Roberge, J.Y. et al. (1995) Science 269:202-204.) Automated synthesis may be achieved using the ABI 431A peptide synthesizer (Applied Biosystems).
Additionally, the amino acid sequence of SECP, or any part thereof, may be altered during direct synthesis and/or combined with sequences from other proteins, or any part thereof, to produce a variant polypeptide or a polypeptide having a sequence of a naturally occurring polypeptide.
The peptide may be substantially purified by preparative high performance liquid chromatography. (See, e.g., Chiez, R.M. and F.Z. Regnier (1990) Methods Enzymol. 182:392-421.) The composition of the synthetic peptides may be confirmed by amino acid analysis or by sequencing. (See, e.g., Creighton, supra, pp. 28-53.) In order to express a biologically active SECP, the nucleotide sequences encoding SECP 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 polynucleotide sequences encoding SECP. Such elements may vary in their strength and specificity.
Specific initiation signals may also be used to achieve more efficient translation of sequences encoding SECP. Such signals include the ATG initiation codon and adjacent sequences, e.g. the Kozak sequence. In cases where sequences encoding SECP and its initiation codon and upstream regulatory sequences are inserted into the appropriate expression vector, no additional transcriptional or translational control signals may be needed. However, in cases where only coding sequence, or a fragment thereof, is inserted, exogenous translational control signals including an in-frame ATG initiation codon should be provided by the vector. Exogenous translational elements and initiation codons may be of various origins, both natural and synthetic. The efficiency of expression may be enhanced by the inclusion of enhancers appropriate for the particular host cell system used. (See, e.g., Scharf, D. et al. (1994) Results Probl. Cell Differ. 20:125-162.) Methods which are well known to those skilled in the art may be used to construct expression vectors containing sequences encoding SECP and appropriate transcriptional and translational control elements. These methods include in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination. (See, e.g., Sambrook, J. et al. (1989) Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Press, Plainview NY, ch. 4, 8, and 16-17; Ausubel, F.M. et al. (1995) Current Protocols in Molecular Biolo~y, John Wiley & Sons, New York NY, ch. 9, 13, and 16.) A variety of expression vector/host systems may be utilized to contain and express sequences encoding SECP. These include, but are not limited to, microorganisms such as bacteria transformed with recombinant bacteriophage, plasmid, or cosmid DNA expression vectors;
yeast transformed with yeast expression vectors; insect cell systems infected with viral expression vectors (e.g., baculovirus);
plant cell systems transformed with viral expression vectors (e.g., cauliflower mosaic virus, CaMV, or tobacco mosaic virus, TMV) or with bacterial expression vectors (e.g., Ti or pBR322 plasmids); or animal cell systems. (See, e.g., Sambrook, su ra; Ausubel, su ra; 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 aI. (1996) Hum. Gene Ther. 7:1937-1945; Takamatsu, N. (I987) EMBO
J. 6:307-311; The McGraw Hill Yearbook of Science and Technolo~y (1992) McGraw Hill, New York NY, pp. 191-196; Logan, J. and T. Shenk (1984) Proc. Natl. Acad. Sci. USA
81:3655-3659; and 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 nucleotide sequences to the targeted organ, tissue, or cell population. (See, e.g., Di Nicola, M. et al. (1998) Cancer Gen. Ther. 5(6):350-356; Yu, M. et al. (1993) Proc. Natl. Acad. Sci.
USA 90(13):6340-6344; Buller, R.M. et al. (1985) Nature 317(6040):813-815;
McGregor, D.P. et al.

(1994) Mol. Immunol. 31(3):219-226; and Verma, LM. and N. Somia (1997) Nature 389:239-242.) The invention is not limited by the host cell employed.
In bacterial systems, a number of cloning and expression vectors may be selected depending upon the use intended for polynucleotide sequences encoding SECP. For example, routine cloning, subcloning, and propagation of polynucleotide sequences encoding SECP can be achieved using a multifunctional E. coli vector such as PBLLTESCRIPT (Stratagene, La Jolla CA) or PSPORT1 plasmid (Life Technologies). Ligation of sequences encoding SECP into the vector's multiple cloning site disrupts the lacZ gene, allowing a colorimetric screening procedure for identification of transformed bacteria containing recombinant molecules. In addition, these vectors may be useful for in vitro transcription, dideoxy sequencing, single strand rescue with helper phage, and creation of nested deletions in the cloned sequence. (See, e.g., Van Heeke, G. and S.M.
Schuster (1989) J. Biol.
Chem. 264:5503-5509.) When large quantities of SECP are needed, e.g. for the production of antibodies, vectors which direct high level expression of SECP may be used.
For example, vectors containing the strong, inducible SP6 or T7 bacteriophage promoter may be used.
Yeast expression systems may be used for production of SECP. A number of vectors containing constitutive or inducible promoters, such as alpha factor, alcohol oxidase, and PGH
promoters, may be used in the yeast Saccharom~ces cerevisiae or Pichia asp toris. In addition, such vectors direct either the secretion or intracellular retention of expressed proteins and enable integration of foreign sequences into the host genome for stable propagation.
(See, e.g., Ausubel, 1995, supra; Bitter, G.A. et al. ( 1987) Methods Enzymol. 153:516-544; and Scorer, C.A. et al. ( 1994) Bio/Technology 12:181-I84.) Plant systems may also be used for expression of SECP. Transcription of sequences encoding SECP may be driven by viral promoters, e.g., the 35S and 19S
promoters of CaMV used alone or in combination with the omega leader sequence from TMV (Takamatsu, N.
(1987) EMBO J.

6:307-311). Alternatively, plant promoters such as the small subunit of RUBISCO or heat shock promoters may be used. (See, e.g., Coruzzi, G. et al. (1984) EMBO J. 3:1671-1680; Brogue, R, et al.
(1984) Science 224:838-843; and Winter, J. et al. (1991) Results Probl. Cell Differ. 17:85-105.) These constructs can be introduced into plant cells by direct DNA
transformation or , pathogen-mediated transfection. (See, e.g., The McGraw Hill Yearbook of Science and Technolo~y (1992) McGraw Hill, New York NY, pp. 191-196.) In mammalian cells, a number of viral-based expression systems may be utilized. In cases where an adenovirus is used as an expression vector, sequences encoding SECP
may be ligated into an adenovirus transcription/translation complex consisting of the late promoter and tripartite leader sequence. Insertion in a non-essential El or E3 region of the viral genome may be used to obtain infective virus which expresses SECP in host cells. (See, e.g., Logan, J. and T. Shenk (1984) Proc.

Natl. Acad. Sci. USA 81:3655-3659.) In addition, transcription enhancers, such as the Rous sarcoma virus (RSV) enhancer, may be used to increase expression in mammalian host cells. SV40 or EBV-based vectors may also be used for high-level protein expression.
Human artificial chromosomes (HACs) may also be employed to deliver larger fragments of DNA than can be contained in and expressed from a plasmid. HACs of about 6 kb to 10 Mb are constructed and delivered via conventional delivery methods (liposomes, polycationic amino polymers, or vesicles) for therapeutic purposes. (See, e.g., Harrington, J.J.
et al. (1997) Nat. Genet.
15:345-355.) For long term production of recombinant proteins in mammalian systems, stable expression of SECP in cell lines is preferred. For example, sequences encoding SECP can be transformed into cell lines using expression vectors which may contain viral origins of replication and/or endogenous expression elements and a selectable marker gene on the same or on a separate vector. Following the introduction of the vector, cells may be allowed to grow for about 1 to 2 days in enriched media before being switched to selective media. The purpose of the selectable marker is to confer resistance to a. selective agent, and its presence allows growth and recovery of cells which successfully express the introduced sequences. Resistant clones of stably transformed cells may be propagated using tissue culture techniques appropriate to the cell type.
Any number of selection systems may be used to recover transformed cell lines.
These include, but are not limited to, the herpes simplex virus thymidine kinase and adenine phosphoribosyltransferase genes, for use in tk- and apr cells, respectively.
(See, e.g., Wigler, M. et al. (1977) Cell 11:223-232; Lowy, I. et al. (1980) Cell 22:817-823.) Also, antimetabolite, antibiotic, or herbicide resistance can be used as the basis for selection. For example, dlzfr confers resistance to methotrexate; neo confers resistance to the aminoglycosides neomycin and G-418; and als and pat confer resistance to chlorsulfuron and phosphinotricin acetyltransferase, respectively. (See, e.g., Wigler, M. et al. (1980) Proc. Natl. Acad. Sci. USA 77:3567-3570; Colbere-Garapin, F. et al. (1981) J. Mol. Biol. 150:1-14.) Additional selectable genes have been described, e.g., trpB and hisD, which alter cellular requirements for metabolites. (See, e.g., Hartman, S.C. and R.C. Mulligan (1988) Proc.
Natl. Acad. Sci. USA 85:8047-8051.) Visible markers, e.g., anthocyanins, green fluorescent proteins (GFP; Clontech),13 glucuronidase and its substrate 13-glucuronide, ox luciferase and its substrate luciferin may be used. These markers can be used not only to identify transformants, but also to quantify the amount of transient or stable protein expression attributable to a specific vector system.
(See, e.g., Rhodes, C.A. (1995) Methods Mol. Biol. 55:121-131.) Although the presence/absence of marker gene expression suggests that the gene of interest is also present, the presence and expression of the gene may need to be confirmed. For example, if the sequence encoding SECP is inserted within a marker gene sequence, transformed cells containing sequences encoding SECP can be identified by the absence of marker gene function. Alternatively, a marker gene can be placed in tandem with a sequence encoding SECP under the control of a single promoter: Expression of the marker gene in response to induction or selection usually indicates expression of the tandem gene as well.
In general, host cells that contain the nucleic acid sequence encoding SECP
and that express SECP 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.
hnmunological methods for detecting and measuring the expression of SECP using either specific polyclonal or monoclonal antibodies are known in the art. Examples of such techniques include enzyme-linked immunosorbent assays (ELISAs), radioimmunoassays (RIAs), and fluorescence activated cell sorting (FACS). A two-site, monoclonal-based immunoassay utilizing monoclonal antibodies reactive to two non-interfering epitopes on SECP is preferred, but a competitive binding assay may be employed. These and other assays are well known in the art. (See, e.g., Hampton, R. et al. (1990) Serological Methods, a Laboratory Manual, APS
Press, St. Paul MN, Sect. IV; Coligan, J.E. et al. (1997) Current Protocols in Immunolo~y, Greene Pub. Associates and Wiley-Interscience, New York NY; and Pound, J.D. (1998) Immunochemical Protocols, Humana Press, Totowa NJ.) A wide variety of labels and conjugation techniques are known by those skilled in the art and may be used in various nucleic acid and amino acid assays. Means for producing labeled hybridization or PCR probes for detecting sequences related to polynucleotides encoding SECP
include oligolabeling, nick translation, end-labeling, or PCR amplification using a labeled nucleotide.
Alternatively, the sequences encoding SECP, or any fragments thereof, may be cloned into a vector for the production of an mRNA probe. Such vectors are known in the art, are commercially available, and may be used to synthesize RNA probes in vitro by addition of an appropriate RNA polymerase such as T7, T3, or SP6 and labeled nucleotides. These procedures may be conducted using a variety of commercially available kits, such as those provided by Amersham Pharmacia Biotech, Promega (Madison Wn, and US Biochemical. Suitable reporter molecules or labels which may be used for ease of detection include radionuclides, enzymes, fluorescent, chemiluminescent, or chromogenic agents, as well as substrates, cofactors, inhibitors, magnetic particles, and the like.
Host cells transformed with nucleotide sequences encoding SECP 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 SECP may be designed to contain signal sequences which direct secretion of SECP through a prokaryotic or eukaryotic cell membrane.
In addition, a host cell strain may be chosen for its ability to modulate expression of the inserted sequences or to process the expressed protein in the desired fashion.
Such modifications of the polypeptide include, but are not limited to, acetylation, carboxylation, glycosylation, phosphorylation, lipidation, and acylation. Post-translational processing which cleaves a "prepro" or "pro" form of the protein may also be used to specify protein targeting, folding, 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 nucleic acid sequences encoding SECP 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 SECP protein containing a heterologous moiety that can be recognized by a commercially available antibody may facilitate the screening of peptide libraries for inhibitors of SECP activity.
Heterologous protein and peptide moieties may also facilitate purification of fusion proteins using commercially available affinity matrices. Such moieties include, but are not limited to, glutathione S-transferase (GST), maltose binding protein (MBP), thioredoxin (Trx), calmodulin binding peptide (CBP), 6-His, FLAG, c-myc, and hemagglutinin (HA). GST, MBP, Trx, CBP, and 6-His enable purification of their cognate fusion proteins on immobilized glutathione, maltose, phenylarsine oxide, calmodulin, and metal-chelate resins, respectively. FLAG, c-~ryc, 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 SECP encoding sequence and the heterologous protein sequence, so that SECP may be cleaved away from the heterologous moiety following purification.
Methods fox fusion protein expression and purification are discussed in Ausubel (1995, supra, ch. 10).
A variety of commercially available kits may also be used to facilitate expression and purification of fusion proteins.
In a further embodiment of the invention, synthesis of radiolabeled SECP may be achieved in vitro using the TNT rabbit reticulocyte lysate or wheat germ extract system (Promega). These systems couple transcription and translation of protein-coding sequences operably associated with the T7, T3, or SP6 promoters. Translation takes place in the presence of a radiolabeled amino acid precursor, for example, 35S-methionine.
SECP of the present invention or fragments thereof may be used to screen for compounds that specifically bind to SECP. At Ieast one and up to a plurality of test compounds may be screened fox specific binding to SECP. Examples of test compounds include antibodies, oligonucleotides, proteins (e.g., receptors), or small molecules.
In one embodiment, the compound thus identified is closely related to the natural ligand of SECP, e.g., a ligand or fragment thereof, a natural substrate, a structural or functional mimetic, or a natural binding partner. (See, e.g., Coligan, J.E. et al. (1991) Current Protocols in Immunolo~y 1(2):
Chapter 5.) Similarly, the compound can be closely related to the natural receptor to which SECP
binds, or to at least a fragment of the receptor, e.g., the ligand binding site. In either case, the compound can be rationally designed using known techniques. In one embodiment, screening for these compounds involves producing appropriate cells which express SECP, either as a secreted protein or on the cell membrane. Preferred cells include cells from mammals, yeast, Drosophila, or E. coli. Cells expressing SECP or cell membrane fractions which contain SECP
are then contacted with a test compound and binding, stimulation, or inhibition of activity of either SECP 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 SECP, either in solution or affixed to a solid support, and detecting the binding of SECP 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.
SECP of the present invention or fragments thereof may be used to screen for compounds that modulate the activity of SECP. Such compounds may include agonists, antagonists, or partial or inverse agonists. In one embodiment, an assay is performed under conditions permissive for SECP
activity, wherein SECP is combined with at least one test compound, and the activity of SECP in the presence of a test compound is compared with the activity of SECP in the absence of the test compound. A change in the activity of SECP in the presence of the test compound is indicative of a compound that modulates the activity of SECP. Alternatively, a test compound is combined with an in vitro or cell-free system comprising SECP under conditions suitable for SECP activity, and the assay is performed. In either of these assays, a test compound which modulates the activity of SECP
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 SECP or their mammalian homologs may be "knocked out" in an animal model system using homologous recombination in embryonic stem (ES) cells. Such techniques are well known in the art and are useful for the generation of animal models of human disease. (See, e.g., U.S. Patent No. 5,175,383 and U.S. Patent No. 5,767,337.) For example, mouse ES cells, such as the mouse 129/SvJ cell line, are derived from the early mouse embryo and grown in culture. The ES cells are transformed with a vector containing the gene of interest disrupted by a marker gene, e.g., the neomycin phosphotransferase gene (neo; Capecchi, M.R.
(1989) Science 244:1288-1292). The vector integrates into the corresponding region of the host genome by homologous recombination. Alternatively, homologous recombination takes place using the Cre-loxP system to knockout a gene of interest in a tissue- or developmental stage-specific manner (Marth, J.D. (1996) Clin. Invest. 97:1999-2002; Wagner, I~.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 SECP may also be manipulated in vitro in ES cells derived from human blastocysts. Human ES cells have the potential to differentiate into at least eight separate cell lineages including endoderm, mesoderm, and ectodermal cell types. These cell lineages differentiate into, for example, neural cells, hematopoietic lineages, and cardiomyocytes (Thomson, J.A. et al.
(1998) Science 282:1145-1147).
Polynucleotides encoding SECP 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 SECP 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 SECP, e.g., by secreting SECP in its milk, may also serve as a convenient source of that protein (Janne, J. et al. (1998) Biotechnol. Annu. Rev. 4:55-74).
THERAPEUTICS
Chemical and structural similarity, e.g., in the context of sequences and motifs, exists between regions of SECP and secreted proteins. In addition, examples of tissues expressing SECP
can be found in Table 6. Therefore, SECP appears to play a role in cell proliferative, autoimmune/inflammatory, cardiovascular, neurological, and developmental disorders. In the treatment of disorders associated with increased SECP expression or activity, it is desirable to decrease the expression or activity of SECP. In the treatment of disorders associated with decreased SECP expression or activity, it is desirable to increase the expression or activity of SECP.

Therefore, in one embodiment, SECP 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 SECP. Examples of such disorders include, but are not limited to, 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, a cancer 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; an autoimmune/inflammatory disorder such as acquired immunodeficiency syndrome (A)DS), Addison's disease, adult respiratory distress syndrome, allergies, ankylosing spondylitis, amyloidosis, anemia, asthma, atheroselerosis, 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, osteoporosis, pancreatitis, polymyositis, psoriasis, Reiter's syndrome, rheumatoid arthritis, scleroderma, Sjogren's syndrome, systemic anaphylaxis, systemic lupus erythematosus, systemic sclerosis, thrombocytopenic purpura, ulcerative colitis, uveitis, Werner syndrome, complications of cancer, hemodialysis, and extracorporeal circulation, viral, bacterial, fungal, parasitic, protozoal, and helminthic infections, and trauma; a cardiovasculax disorder such as 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 rhewnatic heart disease, infective endocarditis, nonbacterial thrombotic endocarditis, endocarditis of systemic lupus erythematosus, carcinoid heart disease, cardiomyopathy, myocarditis, pericarditis, neoplastic heart disease, congenital heart disease, complications of cardiac transplantation, arteriovenous fistula, atherosclerosis, hypertension, vasculitis, Raynaud's disease, aneurysms, arterial dissections, varicose veins, thrombophlebitis and phlebothrombosis, vascular tumors, and complications of throxribolysis, balloon angioplasty, vascular replacement, and coronary artery bypass graft surgery; a neurological disorder such as epilepsy, ischemic cerebrovascular disease, stroke, cerebral neoplasms, Alzheimer's disease, Pick's disease, Huntington's disease, dementia, Parkinson's disease and other extrapyramidal disorders, amyotrophic lateral sclerosis and other motor neuron disorders, progressive neural muscular atrophy, retinitis pigmentosa, hereditary ataxias, multiple sclerosis and other demyelinating diseases, bacterial and viral meningitis, brain abscess, subdural empyema, epidural abscess, suppurative intracranial thrombophlebitis, myelitis and radiculitis, viral central nervous system disease, prion diseases including kuru, Creutzfeldt-Jakob disease, and Gerstmann-Stxaussler-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 developmental disorder such as renal tubular acidosis, anemia, Cushing's syndrome, achondroplastic dwarfism, Duchenne and Becker muscular dystrophy, 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, and sensorineural hearing loss.
In another embodiment, a vector capable of expressing SECP 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 SECP including, but not limited to, those described above.
In a further embodiment, a composition comprising a substantially purified SECP 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 SECP including, but not limited to, those provided above.
In still another embodiment, an agonist which modulates the activity of SECP
may be administered to a subject to treat or prevent a disorder associated with decreased expression or activity of SECP including, but not limited to, those listed above.
In a further embodiment, an antagonist of SECP may be administered to a subject to treat or prevent a disorder associated with increased expression or activity of SECP.
Examples of such disorders include, but are not limited to, those cell proliferative, autoimmune/inflammatory, cardiovascular, neurological, and developmental disorders described above. In one aspect, an antibody which specifically binds SECP 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 SECP.
In an additional embodiment, a vector expressing the complement of the polynucleotide encoding SECP may be administered to a subject to treat or prevent a disorder associated with increased expression or activity of SECP including, but not limited to, those described above.
In other embodiments, any of the proteins, antagonists, antibodies, agonists, complementary sequences, or vectors of the invention may be administered in combination with other appropriate therapeutic agents. Selection of the appropriate agents for use in combination therapy may be made by one of ordinary skill in the art, according to conventional pharmaceutical principles. The combination of therapeutic agents may act synergistically to effect the treatment or prevention of the various disorders described above. Using this approach, one may be able to achieve therapeutic efficacy with lower dosages of each agent, thus reducing the potential for adverse side effects.
An antagonist of SECP may be produced using methods which are generally known in the art.
In particular, purified SECP may be used to produce antibodies or to screen libraries of pharmaceutical agents to identify those which specifically bind SECP.
Antibodies to SECP may also be generated using methods that are well known in the art. Such antibodies may include, but are not limited to, polyclonal, monoclonal, chimeric, and single chain antibodies, Fab fragments, and fragments produced by a Fab expression library. Neutralizing antibodies (i.e., those which inhibit dimer formation) are generally preferred for therapeutic use. Single chain antibodies (e.g., from camels or llamas) may be potent enzyme inhibitors and may have advantages in the design of peptide mimetics, and in the development of immuno-adsorbents and biosensors (Muyldermans, S. (2001) J.
Biotechnol. 74:277-302).
For the production of antibodies, various hosts including goats, rabbits, rats, mice, camels, dromedaries, llamas, humans, and others may be immunized by injection with SECP or with any fragment or oligopeptide thereof which has immunogenic properties. Depending on the host species, various adjuvants may be used to increase immunological response. Such adjuvants include, but are not limited to, Freund's, mineral gels such as aluminum hydroxide, and surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, KLH, and dinitrophenol.
Among adjuvants used in humans, BCG (bacilli Calmette-Guerin) and Corynebacterium parvum are especially preferable.
It is preferred that the oligopeptides, peptides, or fragments used to induce antibodies to SECP have an amino acid sequence consisting of at least about 5 amino acids, and generally will consist of at least about 10 amino acids. It is also preferable that these oligopeptides, peptides, or fragments are identical to a portion of the amino acid sequence of the natural protein. Short stretches of SECP amino acids may be fused with those of another protein, such as KLH, and antibodies to the chimeric molecule may be produced.
Monoclonal antibodies to SECP may be prepared using any technique which provides for the production of antibody molecules by continuous cell lines in culture. These include, but are not limited to, the hybridoma technique, the human B-cell hybridoma technique, and the EBV-hybridoma technique. (See, e.g., Kohler, G. et al. (1975) Nature 256:495-497; Kozbor, D.
et al. (1985) J.
Immunol. Methods 81:31-42; Cote, R.J. et al. (1983) Proc. Natl. Acad. Sci. USA
80:2026-2030; and Cole, S.P. et al. (1984) Mol. Cell Biol. 62:109-120.) In addition, techniques developed for the production of "chimeric antibodies,"
such as the splicing of mouse antibody genes to human antibody genes to obtain a molecule with appropriate antigen specificity and biological activity, can be used. (See, e.g., Morrison, S.L. et al. (1984) Proc.
Natl. Acad. Sci. USA 81:6851-6855; Neuberger, M.S. et al. (1984) Nature 312:604-608; and Takeda, S. et al. (1985) Nature 314:452-454.) Alternatively, techniques described for the production of single chain antibodies may be adapted, using methods known in the art, to produce SECP-specific single chain antibodies. Antibodies with related specificity, but of distinct idiotypic composition, may be generated by chain shuffling from random combinatorial immunoglobulin libraries. (See, e.g., Burton, D.R. (1991) Proc. Natl. Acad. Sci. USA 88:10134-10137.) Antibodies may also be produced by inducing in vivo production in the lymphocyte population or by screening immunoglobulin libraries or panels of highly specific binding reagents as disclosed in the literature. (See, e.g., Orlandi, R. et al. (1989) Proc. Natl.
Acad. Sci. USA
86:3833-3837; Winter, G. et al. (1991) Nature 349:293-299.) Antibody fragments which contain specific binding sites for SECP may also be generated.
For example, such fragments include, but are not limited to, F(ab')y fragments produced by pepsin digestion of the antibody molecule and Fab fragments generated by reducing the disulfide bridges of the F(ab')2 fragments. Alternatively, Fab expression libraries may be constructed to allow rapid and easy identification of monoclonal Fab fragments with the desired specificity.
(See, e.g., Huse, W.D.
et al. (1989) Science 246:1275-1281.) Various immunoassays may be used for screening to identify antibodies having the desired specificity. 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 SECP and its specific antibody: A two-site, monoclonal-based immunoassay utilizing monoclonal antibodies reactive to two non-interfering SECP epitopes is generally used, but a competitive binding assay may also be employed (Pound, su ra .

Various methods such as Scatchard analysis in conjunction with radioimmunoassay techniques may be used to assess the affinity of antibodies for SECP. Affinity is expressed as an association constant, Ka, which is defined as the molar concentration of SECP-antibody complex divided by the molar concentrations of free antigen and free antibody under equilibrium conditions.
The K~ determined for a preparation of polyclonal antibodies, which are heterogeneous in their affinities for multiple SECP epitopes, represents the average affinity, or avidity, of the antibodies for SECP. The Ka determined for a preparation of monoclonal antibodies, which axe monospecific for a particular SECP 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 SECP-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 SECP, 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. Cryex (1991) A Practical Guide to Monoclonal Antibodies, John Wiley & Sons, New York NY).
The titer and avidity of polyclonal antibody preparations may be further evaluated to determine the quality and suitability of such preparations for certain downstream applications. For example, a polyclonal antibody preparation containing at least 1-2 mg specific antibody/ml, preferably 5-10 mg specific antibody/mI, is generally employed in procedures requiring precipitation of SECP-antibody complexes. Procedures for evaluating antibody specificity, titer, and avidity, and guidelines for antibody quality and usage in various applications, are generally available. (See, e.g., Catty, supra, and Coligan et al. supra.) In another embodiment of the invention, the polynucleotides encoding SECP, 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 SECP. 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 SECP. (See, e.g., Agrawal, S., ed. (1996) Antisense Therapeutics, Humana Press Inc., Totawa NJ.) In therapeutic use, any gene delivery system suitable for introduction of the antisense sequences into appropriate target cells can be used. Antisense sequences can be delivered intracellularly in the form of an expression plasmid which, upon transcription, produces a sequence complementary to at least a portion of the cellular sequence encoding the target protein. (See, e.g., Slater, J.E. et al. (1998) J. Allergy Clin. Immunol. 102(3):469-475; and Scanlon, K.J. et al. (1995) 9(13):1288-1296.) Antisense sequences can also be introduced intracellularly through the use of viral vectors, such as retrovirus and adeno-associated virus vectors. (See, e.g., Miller, A.D. (1990) Blood 76:271; Ausubel, supra; Uckert, W. and W. Walther (1994) Pharmacol. Ther.
63(3):323-347.) Other gene delivery mechanisms include liposome-derived systems, artificial viral envelopes, and othex systems known in the art. (See, e.g., Rossi, J.J. (1995) Br. Med. Bull.
51(1):217-225; Boado, R.J. et al. (1998) J. Pharxn. Sci. 87(11):1308-1315; and Morris, M.C. et al. (1997) Nucleic Acids Res.
25 ( 14):2730-2736.) In another embodiment of the invention, polynucleotides encoding SECP 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 (SLID)-X1 disease characterized by X-linked inheritance (Cavazzana-Calvo, M. et al. (2000) Science 288:669-672), severe combined immunodeficiency syndrome associated with an inherited adenosine deaminase (ADA) deficiency (Blaese, R.M. et al. (1995) Science 270:475-480; Bordignon, C. et al. (1995) Science 270:470-475), cystic fibrosis (Zabner, J. et al. (1993) Cell 75:207-216; Crystal, R.G. et al. (1995) Hum. Gene Therapy 6:643-666; Crystal, R.G. et al. (1995) Hum. Gene Therapy 6:667-703), thalassamias, familial hypercholesterolemia, and hemophilia resulting from Factor VIII or Factor IX
deficiencies (Crystal, R.G. (1995) Science 270:404-410; Verma, LM. and N. Somia (1997) Nature 389:239-242)), (ii) express a conditionally lethal gene product (e.g., in the case of cancers which result from unregulated cell proliferation), or (iii) express a protein which affords protection against intracellular parasites (e.g., against human retroviruses, such as human immunodeficiency virus (HIV) (Baltimore, D.
(1988) Nature 335:395-396; Poeschla, E. et al. (1996) Proc, Natl. Acad. Sci.
USA 93:11395-11399), hepatitis B or C virus (HBV, HCV); fungal parasites, such as Candida albicans and Paracoccidioides brasiliensis; and protozoan parasites such as Plasmodium falciparum and Trynanosoma cruzi). In the case where a genetic deficiency in SECP expression or regulation causes disease, the expression of SECP 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 SECP are treated by constructing mammalian expression vectors encoding SECP
and introducing these vectors by mechanical means into SECP-deficient cells. Mechanical transfer technologies for use with cells in vivo or ex vitro include (i) direct DNA microinjection into individual cells, (ii) ballistic gold particle delivery, (iii) liposome-mediated transfection, (iv) receptor-mediated gene transfer, and (v) the use of DNA transposons (Morgan, R.A. and W.F. Anderson (1993) Annu. Rev.
Biochem. 62:191-217; Ivics, Z. (1997) Cell 91:501-510; Boulay, J-L. and H.
Recipon (1998) Curr.
Opin. Biotechnol. 9:445-450).
Expression vectors that may be effective for the expression of SECP include, but are not limited to, the PCDNA 3.1, EPITAG, PRCCMV2, PREP, PVAX, PCR2-TOPOTA vectors (Invitrogen, Carlsbad CA), PCMV-SCRIPT, PCMV-TAG, PEGSH/PERV (Stratagene, La Jolla CA), and PTET-OFF, PTET-ON, PTRE2, PTRE2-LUC, PTK-HYG (Clontech, Palo Alto CA).
SECP may be expressed using (i) a constitutively active promoter, (e.g., from cytomegalovirus (CMV), Rous sarcoma virus (RSV), SV40 virus, thymidine kinase (TK), or (3-actin genes), (ii) an inducible promoter (e.g., the tetracycline-regulated promoter (Gossen, M. and H. Bujard (1992) Proc. Natl.
Acad. Sci. USA 89:5547-5551; Gossen, M. et al. (1995) Science 268:1766-1769;
Rossi, F.M.V. and H.M. Blau (1998) Curr. Opin. Biotechnol. 9:451-456), commercially available in the T-REX plasmid (Invitrogen)); the ecdysone-inducible promoter (available in the plasmids PVGRXR and 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 SECP 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 SECP expression are treated by constructing a retrovirus vector consisting of (i) the polynucleotide encoding SECP 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 retroviral supernatant") discloses a method for obtaining retrovirus packaging cell lines and is hereby incorporated by reference. Propagation of retrovirus vectors, transduction of a population of cells (e.g., CD4+ T-cells), and the return of transduced cells to a patient are procedures well known to persons skilled in the art of gene therapy and have been well documented (Ranga, U. et al. (1997) J. Virol. 71:7020-7029; Bauer, G. et al. '(1997) Blood 89:2259-2267; Bonyhadi, M.L. (1997) J.
Virol. 71:4707-4716;
Ranga, U. et al. (1998) Proc. Natl. Acad. Sci. USA 95:1201-1206; Su, L. (I997) Blood 89:2283-2290).
In the alternative, an adenovirus-based gene therapy delivery system is used to deliver polynucleotides encoding SECP to cells which have one or more genetic abnormalities with respect to the expression of SECP. The construction and packaging of adenovirus-based vectors are well known to those with ordinary skill in the art. Replication defective adenovirus vectors have proven to be versatile for importing genes encoding immunoregulatory proteins into intact islets in the pancreas (Csete, M.E. et al. (1995) Transplantation 27:263-268). Potentially useful adenoviral vectors are described in U.S. Patent No. 5,707,618 to Armentano ("Adenovirus vectors fox gene therapy"), hereby incorporated by reference. For adenoviral vectors, see also Antinozzi, P.A. et al. (1999) Annu. Rev. Nutr. 19:511-544 and Verma, LM. and N. Somia (1997) Nature 18:389:239-242, both ' incorporated by reference herein.
In another alternative, a herpes-based, gene therapy delivery system is used to deliver polynucleotides encoding SECP to target cells which have one or more genetic abnormalities with respect to the expression of SECP. The use of herpes simplex virus (HSV)-based vectors may be especially valuable for introducing SECP 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.
269:385-395). The construction of a HSV-1 virus vector has also been disclosed in detail in U.S.
Patent No. 5,804,413 to DeLuca ("Herpes simplex virus strains for gene transfer"), which is hereby incorporated by reference. U.S. Patent No. 5,804,413 teaches the use of recombinant HSV d92 which consists of a genome containing at least one exogenous gene to be transferred to a cell under the control of the appropriate promoter for purposes including human gene therapy.
Also taught by this patent are the construction and use of recombinant HSV strains deleted for ICP4, ICP27 and ICP22.
For HSV vectors, see also Goins, W.F. et al. (1999) J. Virol. 73:519-532 and Xu, H. et al. (1994) Dev. Biol. 163:152-161, hereby incorporated by reference. The manipulation of cloned 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 hezpesvirus are techniques well known to those of ordinary skill in the art. ' In another alternative, an alphavirus (positive, single-stranded RNA virus) vector is used to deliver polynucleotides encoding SECP to target cells. The biology of the prototypic alphavirus, Semliki Forest Virus (SFV), has been studied extensively and gene transfer vectors have been based on the SFV genome (Garoff, H. and K.-J. Li (1998) Curr. Opin. Biotechnol.
9:464-469). During alphavirus RNA replication, a subgenomic RNA is generated that normally encodes the viral capsid proteins. This subgenomic RNA replicates to higher levels than the full length genomic RNA, resulting in the overproduction of capsid proteins relative to the viral proteins with enzymatic activity (e.g., protease and polymerase). Similarly, inserting the coding sequence for SECP into the alphavirus genome in place of the capsid-coding region results in the production of a'large number of SECP-coding RNAs and the synthesis of high levels of SECP 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 (Dxyga, S.A. et al. (1997) Virology 228:74-83). The wide host range of alphaviruses will allow the introduction of SECP into a variety of cell types. The specific transduction of a subset of cells in a population may require the sorting of cells prior to transduction.
The methods of manipulating infectious cDNA clones of alphaviruses, performing alphavirus cDNA and RNA
transfections, and performing alphavirus infections, are well known to those with ordinary skill in the art.
Oligonucleotides derived from the transcription initiation site, e.g., between about positions -10 and +10 from the start site, may also be employed to inhibit gene expression. Similarly, inhibition can be achieved using triple helix base-pairing methodology. Triple helix pairing is useful because it causes inhibition of the ability of the double helix to open sufficiently for the binding of polymerases, transcription factors, or regulatory molecules. Recent therapeutic advances using triplex DNA have been described in the literature. (See, e.g., Gee, J.E. et al. (1994) in Huber, B.E.
and B.I. Carr, Molecular and hnmunologic 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 xibozyme molecules may specifically and efficiently catalyze endonucleolytic cleavage of sequences encoding SECP.
Specific ribozyme cleavage sites within any potential RNA target are initially identified by scanning the target molecule for ribozyme cleavage sites, including the following sequences: GUA, GUU, and GUC. Once identified, short RNA sequences of between 15 and 20 ribonucleotides, corresponding to the region of the target gene containing the cleavage site, may be evaluated for secondary structural features which may render the oligonucleotide inoperable.
The suitability of candidate targets may also be evaluated by testing accessibility to hybridization with complementary oligonucleotides using ribonuclease protection assays.
Complementary ribonucleic acid molecules and ribozymes of the invention may be prepared by any method known in the art for the synthesis of nucleic acid molecules.
These include techniques for chemically synthesizing oligonucleotides such as solid phase phosphoramidite chemical synthesis.
Alternatively, RNA molecules may be generated by in vitro and in vivo transcription of DNA
sequences encoding SECP. Such DNA sequences may be incorporated into a wide variety of vectors with suitable RNA polymerase promoters such as T7 or SP6. Alternatively, these cDNA constructs that synthesize complementary RNA, constitutively or inducibly, can be introduced into cell lines, cells, or tissues.
RNA molecules may be modified to increase intracellular stability and half-life. Possible modifications include, but are not limited to, the addition of flanking sequences at the 5' and/or 3' ends of the molecule, or the use of phosphorothioate or 2' O-methyl rather than phosphodiesterase linkages within the backbone of the molecule. This concept is inherent in the production of PNAs and can be extended in all of these molecules by the inclusion of nontraditional bases such as inosine, queosine, and wybutosine, as well as acetyl-, methyl-, thio-, and similarly modified forms of adenine, cytidine, guanine, thymine, and uridine which are not as easily recognized by endogenous endonucleases.
An additional embodiment of the invention encompasses a method for screening for a compound which is effective in altering expression of a polynucleotide encoding SECP. 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 SECP
expression or activity, a compound which specifically inhibits expression of the polynucleotide encoding SECP may be therapeutically useful, and in the treatment of disorders associated with decreased SECP expression or activity, a compound which specifically promotes expression of the polynucleotide encoding SECP may be therapeutically useful.
At least one, and up to a plurality, of test compounds may be screened for effectiveness in altering expression of a specific polynucleotide. A test compound may be obtained by any method commonly known in the art, including chemical modification of a compound known to be effective in altering polynucleotide expression; selection from an existing, commercially-available or proprietary library of naturally-occurring or non-natural chemical compounds; rational design of a compound based on chemical 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 SECP is exposed to at least one test compound thus obtained. The sample may comprise, for example, an intact or permeabilized cell, or an in vitro cell-free or reconstituted biochemical system. Alterations in the expression of a polynucleotide encoding SECP 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 SECP. The amount of hybridization may be quantified, thus forming the basis for a comparison of the expression of the polynucleotide both with and without exposure to one or more test compounds. Detection of a change in the expression of a polynucleotide exposed to a test compound indicates that the test compound is effective in altering the expression of the polynucleotide. A screen for a compound effective in altering expression of a specific polynucleotide can be carried out, for example, using a Schizosaccharomyces pombe gene expression system (Atkins, D. et al. (1999) U.S. Patent No. 5,932,435; Arndt, G.M. et al. (2000) Nucleic Acids Res.
28:EI5) or a human cell line such as HeLa cell (Clarke, M.L. et aI. (2000) Biochem. Biophys. Res.
Commun. 268:8-13). A particular embodiment of the present invention involves screening a combinatorial library of oligonucleotides (such as deoxyribonucleotides, ribonucleotides, peptide nucleic acids, and modified oligonucleotides) for antisense activity against a specific polynucleotide sequence (Bruice, T.W. et al. (1997) U.S. Patent No. 5,686,242; Bruice, T.W.
et al. (2000) U.S.
Patent No. 6,022,691).
Many methods for introducing vectors into cells or tissues are available and equally suitable for use in vivo, in vitro, and ex vivo. For ex vivo therapy, vectors may be introduced into stem cells taken from the patient and clonally propagated for autologous transplant back into that same patient.
Delivery by transfection, by liposome injections, or by polycationic amino polymers may be achieved using methods which are well known in the art. (See, e.g., Goldman, C.K. et al. (1997) Nat.
Biotechnol. I5:462-466.
Any of the therapeutic methods described above may be applied to any subject in need of such therapy, including, for example, mammals such as humans, dogs, cats, cows, horses, rabbits, and monkeys.
An additional embodiment of the invention relates to the administration of a composition which generally comprises an active ingredient formulated with a pharmaceutically acceptable excipient. Excipients may include, for example, sugars, starches, celluloses, gums, and proteins.
Various formulations are commonly known and are thoroughly discussed in the latest edition of Remin~ton's Pharmaceutical Sciences (Maack Publishing, Easton PA). Such compositions may consist of SECP, antibodies to SECP, and mimetics, agonists, antagonists, or inhibitors of SECP.
The compositions utilized in this invention may be administered by any number of routes including, but not limited to, oral, intravenous, intramuscular, intra-arterial, intramedullary, intrathecal, intraventricular, pulmonary, transdermal, subcutaneous, intraperitoneal, intranasal, enteral, topical, sublingual, or rectal means Compositions for pulmonary administration may be prepared in liquid or dry powder form.
These compositions are generally aerosolized immediately prior to inhalation by the patient. In the case of small molecules (e.g. traditional low molecular weight organic drugs), aerosol delivery of fast-acting formulations is well-known in the art. In the case of macromolecules (e.g. larger peptides and proteins), recent developments in the field of pulmonary delivery via the alveolar region of the lung have enabled the practical delivery of drugs such as insulin to blood circulation (see, e.g., Patton, J.S. et al., U.S. Patent No. 5,997,848). Pulmonary delivery has the advantage of administration without needle injection, and obviates the need 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 SECP or fragments thereof. For example, liposome preparations containing a cell-impermeable macromolecule may promote cell fusion and intracellular delivery of the macromolecule. Alternatively, SECP or a fragment thereof may be joined to a short cationic N-terminal portion from the HIV Tat-1 protein. Fusion proteins thus generated have been found to transduce into the cells of all tissues, including the brain, in a mouse model system (Schwarze, S.R. et al. (1999) Science 285:1569-1572).
For any compound, the therapeutically effective dose can be estimated initially either in cell culture assays, e.g., of neoplastic cells, or in animal models such as mice, rats, rabbits, dogs, monkeys, or pigs. An animal model may also be used to determine the appropriate concentration range and route of administration. Such information can then be used to determine useful doses and routes for administration in humans.
A therapeutically effective dose refers to that amount of active ingredient, for example SECP
or fragments thereof, antibodies of SECP, and agonists, antagonists or inhibitors of SECP, 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 eaepressed as the LDSO/EDSO ratio.
Compositions which exhibit large therapeutic indices are preferred. The data obtained from cell culture assays and animal studies are used to formulate a range of dosage for human use. The dosage contained in such compositions is preferably within a range of circulating concentrations that includes the EDSO
with little or no toxicity.
The dosage varies within this range depending upon the dosage form employed, the sensitivity of the patient, and the route of administration.
The exact dosage will be determined by the practitioner, in light of factors related to the subject requiring treatment. Dosage and administration are adjusted to provide sufficient levels of the active moiety or to maintain the desired effect. Factors which may be taken into account include the severity of the disease state, the general health of the subject, the age, weight, and gender of the subject, time and frequency of administration, drug combination(s), reaction sensitivities, and response to therapy. Long-acting compositions may be administered every 3 to 4 days, every week, or biweekly depending on the half life and clearance rate of the particular formulation.
Normal dosage amounts may vary from about O.l ,ug to 100,000 fig, 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 SECP may be used for the diagnosis of disorders characterized by expression of SECP, or in assays to monitor patients being treated with SECP or agonists, antagonists, or inhibitors of SECP. Antibodies useful for diagnostic purposes may be prepared in the same manner as described above for therapeutics. Diagnostic assays for SECP include methods which utilize the antibody and a label to detect SECP
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, axe known in the art and may be used.
A variety of protocols for measuring SECP, including ELISAs, RIAs, and FACS, are known in the art and provide a basis for diagnosing altered or abnormal levels of SECP expression. Normal or standard values for SECP expression are established by combining body fluids or cell extracts taken from normal mammalian subjects, for example, human subjects, with antibodies to SECP under conditions suitable for complex formation. The amount of standard complex formation may be quantitated by various methods, such as photometric means. Quantities of SECP
expressed in subject, control, and disease samples from biopsied tissues are compared with the standard values.
Deviation between standard and subject values establishes the parameters for diagnosing disease.

In another embodiment of the invention, the polynucleotides encoding SECP may be used for diagnostic purposes. The polynucleotides which may be used include oligonucleotide sequences, complementary RNA and DNA molecules, and PNAs. The polynucleotides may be used to detect and quantify gene expression in biopsied tissues in which expression of SECP
may be correlated with disease. The diagnostic assay may be used to determine absence, presence, and excess expression of SECP, and to monitor regulation of SECP levels during therapeutic intervention.
In one aspect, hybridization with PCR probes which are capable of detecting polynucleotide sequences, including genomic sequences, encoding SECP or closely related molecules may be used to identify nucleic acid sequences which encode SECP. The specificity of the probe, whether it is made from a highly specific region, e.g., the 5'regulatory region, or from a less specific region, e.g., a conserved motif, and the stringency of the hybridization or amplification will determine whether the probe identifies only naturally occurring sequences encoding SECP, 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 SECP encoding sequences. The hybridization probes of the subject invention may be DNA or RNA and may be derived from the sequence of SEQ m N0:25-48 or from genomic sequences including promoters, enhancers, and introns of the SECP
gene.
Means for producing specific hybridization probes for DNAs encoding SECP
include the cloning of polynucleotide sequences encoding SECP or SECP derivatives into vectors for the production of mRNA probes. Such vectors are known in the art, are commercially available, and may be used to synthesize RNA probes in vitro by means of the addition of the appropriate RNA
polymerases and the appropriate labeled nucleotides. Hybridization probes may be labeled by a variety of reporter groups, for example, by radionuclides such as 32P or 355, or by enzymatic labels, such as alkaline phosphatase coupled to the probe via avidinlbiotin coupling systems, and the like.
Polynucleotide sequences encoding SECP may be used for the diagnosis of disorders associated with expression of SECP. Examples of such disorders include, but are not limited to, 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, a cancer 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; an autoimmunelinflammatory disorder such as acquired immunodeficiency syndrome (AmS), Addison's disease, adult respiratory distress syndrome, allergies, ankylosing spondyhtis, amyloidosis, anemia, asthma, atherosclerosis, 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 ox pericardial inflammation, osteoarthritis, osteoporosis, pancreatitis, polymyositis, psoriasis, Reiter's syndrome, rheumatoid arthritis, scleroderma, Sjogren's syndrome, systemic anaphylaxis, systemic lupus erythematosus, systemic sclerosis, thrombocytopenic purpura, ulcerative colitis, uveitis, Werner syndrome, complications of cancer, hemodialysis, and extracorporeal circulation, viral, bacterial, fungal, parasitic, protozoal, and helminthic infections, and trauma; a cardiovascular disorder such as congestive heart failure, ischemic heart disease, angina pectoris, myocardial infarction, hypertensive heart disease, degenerative valvular heart disease, calcific aortic valve stenosis, congenitally bicuspid aortic valve, mitral annular calcification, mitral valve prolapse, rheumatic fever and rheumatic heart disease, infective endocarditis, nonbacterial thrombotic endocarditis, endocarditis of systemic lupus erythematosus, carcinoid heart disease, cardiomyopathy, myocarditis, pericarditis, neoplastic heart disease, congenital heart disease, complications of cardiac transplantation, arteriovenous fistula, atherosclerosis, hypertension, vasculitis, Raynaud's disease, aneurysms, arterial dissections, varicose veins, thrombophlebitis and phlebothrombosis, vascular tumors, and complications of thxombolysis, balloon angioplasty, vascular replacement, and coronary artery bypass graft surgery; 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 neuxal muscular atrophy, retinitis pigmentosa, hereditary ataxias, multiple sclerosis and other demyelinating diseases, bacterial and viral meningitis, brain abscess, subdural empyema, epidural abscess, suppurative intracranial thrombophlebitis, myelitis and radiculitis, viral central nervous system disease, prion diseases including kuru, Creutzfeldt-Jakob disease, and Gerstmann-Straussler-Scheinker syndrome, fatal familial insomnia, nutritional and metabolic diseases of the nervous system, neurofibromatosis, tuberous sclerosis, cerebelloretinal hemangioblastomatosis, encephalotrigeminal syndrome, mental retardation and other developmental disorders of the central nervous system including Down syndrome, cerebral palsy, neuroskeletal disorders, autonomic nervous system disorders, cranial nerve disorders, spinal cord diseases, muscular dystrophy and other neuromuscular disorders, peripheral nervous system disorders, dermatomyositis and polymyositis, inherited, metabolic, endocrine, and toxic myopathies, myasthenia gravis, periodic paralysis, mental disorders including mood, anxiety, and schizophrenic disorders, seasonal affective disorder (SAD), akathesia, amnesia, catatonia, diabetic neuropathy, taxdive dyskinesia, dystonias, paranoid psychoses, postherpetic neuralgia, Tourette's disorder, progressive supranuclear palsy, corticobasal degeneration, and familial frontotemporal dementia; and a developmental disorder such as renal tubular acidosis, anemia, Cushing's syndrome, achondroplastic dwarfism, Duchenne and Becker muscular dystrophy, epilepsy, gonadal dysgenesis, WAGR
syndrome (Wilms' tumor, aniridia, genitourinary abnornialities, 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, and sensorineural hearing loss. The polynucleotide sequences encoding SECP 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 SECP expression. Such qualitative or quantitative methods are well known in the art.
In a particular aspect, the nucleotide sequences encoding SECP may be useful in assays that detect the presence of associated disorders, particularly those mentioned above. The nucleotide sequences encoding SECP may be labeled by standard methods and added to a fluid or tissue sample from a patient under conditions suitable for the formation of hybridization complexes. After a suitable incubation period, the sample is washed and the signal is quantified and compared with a standard value. If the amount of signal in the patient sample is significantly altered in comparison to a control sample then the presence of altered levels of nucleotide sequences encoding SECP 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 SECP, 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 SECP, 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 lrnown amount of a substantially purified polynucleotide is used. Standard values obtained in this manner may be compared with values obtained from samples from patients who are symptomatic for a disorder. Deviation from standard values is used to establish the presence of a disorder.
Once the presence of a disorder is established and a treatment protocol is initiated, hybridization assays may be repeated on a regular basis to determine if the level of expression in the patient begins to approximate that which is observed in the normal subject.
The results obtained from successive assays may be used to show the efficacy of treatment over a period ranging from several days to months.
With respect to cancer, the presence of an abnormal amount of transcript (either under- or overexpressed) in biopsied tissue from an individual may indicate a predisposition for the development of the disease, or may provide a means for detecting the disease prior to the appearance of actual clinical symptoms. A more definitive diagnosis of this type may allow health professionals to employ preventative measures or aggressive treatment earlier thereby preventing the development or further progression of the cancer.
Additional diagnostic uses for oligonucleotides designed from the sequences encoding SECP
may involve the use of PCR. These oligomers may be chemically synthesized, generated enzymatically, or produced in vitro. Oligomers will preferably contain a fragment of a polynucleotide encoding SECP, or a fragment of a polynucleotide complementary to the polynucleotide encoding SECP, and will be employed under optimized conditions for identification of a specific gene or condition. Oligomers may also be employed under less stringent conditions for detection or quantification of closely related DNA or RNA sequences.
In a particular aspect, oligonucleotide primers derived from the polynucleotide sequences encoding SECP may be used to detect single nucleotide polymorphisms (SNPs).
SNPs are substitutions, insertions and deletions that are a frequent cause of inherited or acquired genetic disease in humans. Methods of SNP detection include, but are not limited to, single-stranded conformation polymorphism (SSCP) and fluorescent SSCP (fSSCP) methods. In SSCP, oligonucleotide primers derived from the polynucleotide sequences encoding SECP 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).
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 ALOX5 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 5:538-543; Nowotny, P. et al. (2001) Curr. Opin.
Neurobiol. 11:637-641.) Methods which may also be used to quantify the expression of SECP include radiolabeling or biotinylating nucleotides, coamplification of a control nucleic acid, and interpolating results from standard curves. (See, e.g., Melby, P.C. et al. (1993) J. Immunol. Methods 159:235-244; Duplaa, C.
et aI. (1993) Anal. Biochem. 212:229-236.) The speed of quantitation of multiple samples may be accelerated by running the assay in a high-throughput format where the oligomer or polynucleotide of interest is presented in various dilutions and a spectrophotometric or colorimetric response gives rapid quantitation.
In further embodiments, oligonucleotides or longer fragments derived from any of the polynucleotide sequences described herein 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, SECP, fragments of SECP, or antibodies specific for SECP may be used as elements on a microarray. The microarray may be used to monitor or measure protein-protein interactions, drug-target interactions, and gene expression profiles, as described above.

A particular embodiment relates to the use of the polynucleotides of the present invention to generate a transcript image of a tissue or cell type. A transcript image represents the global pattern of gene expression by a particular tissue or cell type. Global gene expression patterns are analyzed by quantifying the number of expressed genes and their relative abundance under given conditions and at a given time. (See Seilhamer et al., "Comparative Gene Transcript Analysis,"
U.S. Patent No.
5,840,484, expressly incorporated by reference herein.) Thus a transcript image may be generated by hybridizing the polynucleotides of the present invention or their complements to the totality of transcripts or reverse transcripts of a particular tissue or cell type. In one embodiment, the hybridization takes place in high-throughput format, wherein the polynucleotides of the present invention or their complements comprise a subset of a plurality of elements on a microarray. The resultant transcxipt image would provide a profile of gene activity.
Transcript images may be generated using transcripts isolated from tissues, cell lines, biopsies, or other biological samples. The transcript image may thus reflect gene expression in vivo, as in the case of a tissue or biopsy sample, or in vitro, as in the case of a cell line.
Transcript images which profile the expression of the polynucleotides of the present invention may also be used in conjunction with in vitro model systems and preclinical evaluation of pharmaceuticals, as well as toxicological testing of industrial and naturally-occurring environmental compounds. All compounds induce characteristic gene expression patterns, frequently termed molecular fingerprints or toxicant signatures, which are indicative of mechanisms of action and toxicity (Nuwaysir, E.F. et al. (1999) Mol. Carcinog. 24:153-159; Steiner, S.
and N.L. Anderson (2000) Toxicol. Lett. 112-113:467-471, expressly incorporated by reference herein). If a test compound has a signature similar to that of a compound with known toxicity, it is likely to share those toxic properties. These fingerprints or signatures are most useful and refined when they contain expression information from a large number of genes and gene families.
Ideally, a genorne-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:/lwww.niehs.nih.gov/oclnews/toxchip.htm.) Therefore, it is important and desirable in toxicological screening using toxicant signatures to include all expressed gene sequences.
In one embodiment, the toxicity of a test compound is assessed by treating a biological sample containing nucleic acids with the test compound. Nucleic acids that are expressed in the treated biological sample are hybridized with one or more probes specific to the polynucleotides of the present invention, so that transcript levels corresponding to the polynucleotides of the present invention may be quantified. The transcript levels in the treated biological sample are compared with levels in an untreated biological sample. Differences in the transcript levels between the two samples are indicative of a toxic response caused by the test compound in the treated sample.
Another particular embodiment relates to the use of the polypeptide sequences of the present invention to analyze the proteome of a tissue or cell type. The term proteome refers to the global pattern of protein expression in a particular tissue or cell type. Each protein component of a proteome can be subjected individually to further analysis. Proteome expression patterns, or profiles, are analyzed by quantifying the number of expressed proteins and their relative abundance under given conditions and at a given time. A profile of a cell's proteome may thus be generated by separating and analyzing the polypeptides of a particular tissue or cell type.
In one embodiment, the separation is achieved using two-dimensional gel electrophoresis, in which proteins from a sample are separated by isoelectric focusing in the first dimension, and then according to molecular weight by sodium dodecyl sulfate slab gel electrophoresis in the second dimension (Steiner and Anderson, supra). The proteins are visualized in the gel as discrete and uniquely positioned spots, typically by staining the gel with an agent such as Coomassie Blue or silver or fluorescent stains. The optical density of each protein spot is generally proportional to the level of the protein in the sample. The optical densities of equivalently positioned protein spots from different samples, for example, from biological samples either treated or untreated With a test compound or therapeutic agent, are compared to identify any changes in protein spot density related to the treatment. The proteins in the spots are partially sequenced using, for example, standard methods employing chemical or enzymatic cleavage followed by mass spectrometry. The identity of the protein in a spot may be determined by comparing its partial sequence, preferably of at least 5 contiguous amino acid residues, to the polypeptide sequences of the present invention. In some cases, further sequence data may be obtained for definitive protein identification.
A proteomic profile may also be generated using antibodies specific for SECP
to quantify the levels of SECP 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 laiown in the art, for example, by reacting the proteins in the sample with a thiol-or amino-reactive fluorescent compound and detecting the amount of fluorescence bound at each array element.

Toxicant signatures at the proteome level are also useful for toxicological screening, and should be analyzed in parallel with toxicant signatures at the transcript level. There is a poor correlation between transcript and protein abundances for some proteins in some tissues (Anderson, N.L. and J. Seilhamer (1997) Electrophoresis 18:533-537), so proteome toxicant signatures may be useful in the analysis of compounds which do not significantly affect the transcript image, but which alter the proteomic profile. Tn addition, the analysis of transcripts in body fluids is difficult, due to rapid degradation of mRNA, so proteomic profiling may be more reliable and informative in such cases.
In another embodiment, the toxicity of a test compound is assessed by treating a biological sample containing proteins with the test compound. Proteins that are expressed in the treated biological sample are separated so that the amount of each protein can be quantified. The amount of each protein is compared to the amount of the corresponding protein in an untreated biological sample. A difference in the amount of protein between the two samples is indicative of a toxic response to the test compound in the treated sample. Individual proteins are identified by sequencing the amino acid residues of the individual proteins and comparing these partial sequences to the polypeptides of the present invention.
In another embodiment, the toxicity of a test compound is assessed by treating a biological sample containing proteins with the test compound. Proteins from the biological sample are incubated with antibodies specific to the polypeptides of the present invention. The amount of protein recognized by the antibodies is quantified. The amount of protein in the treated biological sample is compared with the amount in an untreated biological sample. A
difference in the amount of protein between the two samples is indicative of a toxic response to the test compound in the treated sample.
Microarrays may be prepared, used, and analyzed using methods known in the art. (See, e.g., Brennan, T.M. et al. (1995) U.S. Patent No. 5,474,796; Schena, M. et al.
(1996) Proc. Natl. Acad. Sci.
USA 93:10614-10619; Baldeschweiler et al. (1995) PCT application W095/251116;
Shalom D. et al.
(1995) PCT application W095/35505; Heller, R.A. et al. (1997) Proc. Natl.
Acad. Sci. USA 94:2150-2155; and Heller, M.J. et al. (1997) U.S. Patent No. 5,605,662.) Various types of microarrays are well known and thoroughly described in DNA Microarrays: A Practical Ap rp oach, M. Schena, ed.
(1999) Oxford University Press, London, hereby expressly incorporated by reference.
In another embodiment of the invention, nucleic acid sequences encoding SECP
may be used to generate hybridization probes useful in mapping the naturally occurring genomic sequence. Either coding or noncoding sequences may be used, and in some instances, noncoding sequences may be preferable over coding sequences. For example, conservation of a coding sequence among members of a mufti-gene family may potentially cause undesired cross hybridization during chromosomal mapping. The sequences may be mapped to a particular chromosome, to a specific region of a chromosome, or to artificial chromosome constructions, e.g., human artificial chromosomes (HACs), yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs), bacterial P1 constructions, or single chromosome cDNA libraries. (See, e.g., Harrington, J.J. et al. (1997) Nat.
Genet. 15:345-355; Price, C.M. (1993) Blood Rev. 7:127-134; and Trask, B.J.
(1991) Trends Genet.

7:149-I54.) Once mapped, the nucleic acid sequences of the invention may be used to develop genetic linkage maps, for example, which correlate the inheritance of a disease state with the inheritance of a particular chromosome region or restriction fragment length polymorphism (RFLP).
(See, for example, Lander, E.S. and D. Botstein (1986) Proc. Natl. Acad. Sci.
USA 83:7353-7357.) Fluorescent in situ hybridization (FISH) may be correlated with other physical and genetic map data. (See, e.g., Heinz-Ulrich, et al. (1995) in Meyers, supra, pp. 965-968.) Examples of genetic map data can be found in various scientific journals or at the Online Mendelian Inheritance in Man (OM1M) World Wide Web site. Correlation between the location of the gene encoding SECP 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 positionah 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-tehangiectasia to 11q22-23, any sequences mapping to that area may represent associated or regulatory genes for further investigation. (See, e.g., Gatti, R.A. et al. (1988) Nature 336:577-580.) The nucleotide sequence of the instant invention may also be used to detect differences in the chromosomal location due to translocation, inversion, etc., among normal, carrier, or affected individuals.
In another embodiment of the invention, SECP, 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 SECP and the agent being tested may be measured.
Another technique for drug screening provides for high throughput screening of compounds having suitable binding affinity to the protein of interest. (See, e.g., Geysen, et al. (1984) PCT
application W084/03564.) In this method, large numbers of different small test compounds are synthesized on a solid substrate. The test compounds are reacted with SECP, or fragments thereof, arid washed. Bound SECP is then detected by methods well known in the art.
Purified SECP 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 SECP specifically compete with a test compound for binding SECP. In this manner, antibodies can be used to detect the presence of any peptide which shares one or more antigenic determinants with SECP.
In additional embodiments, the nucleotide sequences which encode SECP 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 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. 601267,924, U.S. Ser. No. 60/266,195, U.S. Ser. No.
60/268,112, U.S. Ser.
No. 60/267,816, U.S. Ser. No. 60/271,639, U.S. Ser. No. 60/317,818, and U.S.
Ser. No. 60/343,553, 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 (Life Technologies), a monophasic solution of phenol and guanidine isothiocyanate. The resulting lysates were centrifuged over CsCI cushions or extracted with chloroform. RNA was precipitated from the lysates with either isopropanol or sodium acetate and ethanol, or by other routine methods.
Phenol extraction and precipitation of RNA were repeated as necessary to increase RNA
purity. In some cases, RNA was treated with DNase. For most libraries, poly(A)+ RNA was isolated using oligo d(T)-coupled paramagnetic particles (Promega), OLIGOTEX latex particles (QIAGEN, Chatsworth CA), or an OLIGOTEX mRNA purification kit (QIAGEN). Alternatively, RNA was isolated directly from tissue lysates using other RNA isolation kits, e.g., the POLY(A)PURE mRNA

purification kit (Ambion, Austin TX).
In some cases, Stratagene was provided with RNA and constructed the corresponding cDNA
libraries. Otherwise, cDNA was synthesized and cDNA libraries were constructed with the UNIZAP
vector system (Stratagene) or SUPERSCRIPT plasmid system (Life Technologies), using the recommended procedures or similar methods known in the art. (See, e.g., Ausubel, 1997, s, unra, units 5.1-6.6.) Reverse transcription was initiated using oligo d(T) or random primers. Synthetic oligonucleotide adapters were ligated to double stranded cDNA, and the cDNA
was digested with the appropriate restriction enzyme or enzymes. For most libraries, the cDNA was size-selected (300-1000 bp) using SEPHACRYL S 1000, SEPHAROSE CL2B, or SEPHAROSE CL4B column chromatography (Amersham Pharmacia Biotech) or preparative agarose gel electrophoresis. cDNAs were ligated into compatible restriction enzyme sites of the polylinker of a suitable plasmid, e.g., PBLUESCRIl'T phasnnid (Stratagene), PSPORT1 plasmid (Life Technologies), PCDNA2.1 phasmid (Invitrogen, Carlsbad CA), PBI~-CMV plasmid (Stratagene), PCR2-TOPOTA plasmid (Invitrogen), PCMV-ICIS plasmid (Stratagene), pIGEN (Incyte Genomics, Palo Alto CA), pRARE
(Incyte Genomics), or pINCY (Incyte Genomics), or derivatives thereof. Recombinant plasmids were transformed into competent E. coli cells including XL1-Blue, XL1-BhueMRF, or SOLR from Stratagene or DHSa, DHlOB, or ElectroMAX DHlOB from Life Technologies.
II. Isolation of cDNA Clones Plasmids obtained as described in Example I were recovered from host cells by in vivo excision using the UNIZAP vector system (Stratagene) or by cell lysis.
Plasmids were purified using at least one of the following: a Magic or WIZARD Minipreps DNA purification system (Promega); an AGTC Miniprep purification kit (Edge Biosystems, Gaithersburg MD); and QIAWELL

8 Plasmid, QIAWELL 8 Plus Plasmid, QIAWELL 8 Ultra Plasmid purification systems or the R.E.A.L. PREP 96 phasmid 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 fluorometricalhy using PICOGREEN dye (Molecular Probes, Eugene OR) and a FLUOROSKAN II
fluorescence scanner (Labsystems Oy, Helsinki, Finhand).
III. 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 Pharmacia Biotech or supplied in ABI
sequencing kits such as the ABI PRISM BIGDYE Terminator cycle sequencing ready reaction kit (Applied Biosystems).
Electrophoretic separation of cDNA sequencing reactions and detection of labeled polynucleotides were carried out using the MEGABACE 1000 DNA sequencing system (Molecular Dynamics); the ABI PRISM 373 or 377 sequencing system (Applied Biosystems) in conjunction with standard ABI
protocols and base calling software; or other sequence analysis systems known in the art. Reading frames within the eDNA sequences were identified using standard methods (reviewed in Ausubel, 1997, supra,, unit 7.7). Some of the cDNA sequences were selected for extension using the techniques disclosed in Example VIII.
The polynucleotide sequences derived from Incyte cDNAs were validated by removing vector, linker, and poly(A) sequences and by masking ambiguous bases, using algorithms and programs based on BLAST, dynamic programming, and dinucleotide nearest neighbor analysis. The Incyte cDNA sequences or translations thereof were then queried against a selection of public databases such as the GenBank primate, rodent, mammalian, vertebrate, and eukaryote databases, and BLOCKS, PRINTS, DOMO, PRODOM; PROTEOME databases with sequences from Homo sapiens, Rattus norve icus, Mus musculus, Caenorhabditis ele_gans, Saccharomyces cerevisiae, Schizosaccharo~ces pombe, and Candida albicans (Incyte Genomics, Palo Alto CA); hidden Markov model (HMM)-based protein family databases such as PFAM; and HMM-based protein domain databases such as SMART (Schultz et al. (1998) Proc. Natl. Acad. Sci.
USA 95:5857-5864;
Letunic, I. et al. (2002) Nucleic Acids Res. 30:242-244). (HMM is a probabilistic approach which analyzes consensus primary structures of gene families. See, for example, Eddy, S.R. (1996) Curr.
Opin. Struct. Biol. 6:361-365.) The queries were performed using programs based on BLAST, FASTA, BLIMPS, and HMMER. The Incyte cDNA sequences were assembled to produce full length polynucleotide sequences. Alternatively, GenBank eDNAs, GenBank ESTs, stitched sequences, stretched sequences, or Genscan-predicted coding sequences (see Examples IV
and V) were used to extend Incyte cDNA assemblages to full length. Assembly was performed using programs based on Phred, Phrap, and Consed, and cDNA assemblages were screened for open reading frames using programs based on GeneMark, BLAST, and FASTA. The full length polynucleotide sequences were translated to derive the corresponding full length polypeptide sequences.
Alternatively, a polypeptide of the invention may begin at any of the methionine residues of the full length translated polypeptide.
Full length polypeptide sequences were subsequently analyzed by querying against databases such as the GenBank protein databases (genpept), SwissProt, the PROTEOME databases, BLOCKS, PRINTS, DOMO, PRODOM, Prosite, hidden Markov model (HMM)-based protein family databases such as PFAM; and HMM-based protein domain databases such as SMART. Full length polynucleotide sequences are also analyzed using MACDNASIS PRO software (Hitachi Software Engineering, South San Francisco CA) and LASERGENE software (DNASTAR).
Polynucleotide and polypeptide sequence alignments are generated using default parameters specified by the CLUSTAL algorithm as incorporated into the MEGALIGN multisequence alignment program (DNASTAR), which also calculates the percent identity between aligned sequences.
Table 7 summarizes the tools, programs, and algorithms used for the analysis and assembly of Incyte cDNA and full length sequences and provides applicable descriptions, references, and threshold parameters. The first column of Table 7 shows the tools, programs, and algorithms used, the second colmnn 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 N0:25-48. 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 secreted proteins were initially identified by running the Genscan gene identification program against public genomic sequence databases (e.g., gbpri and gbhtg).
Genscan is a general-purpose gene identification program which analyzes genomic DNA sequences from a variety of organisms (See Burge, C. and S. Karlin (1997) J. Mol. Biol. 268:78-94, and Burge, C. and S. Karlin (1998) Curr. Opin. Struct. Biol. 8:346-354). The program concatenates predicted exons to form an assembled cDNA sequence extending from a methionine to a stop codon. The output of Genscan is a FASTA database of polynucleotide and polypeptide sequences. The maximum range of sequence for Genscan to analyze at once was set to 30 kb. To determine which of these Genscan predicted cDNA
sequences encode secreted proteins, the encoded polypeptides were analyzed by querying against PFAM models for secreted proteins. Potential secreted proteins were also identified by homology to Incyte cDNA sequences that had been annotated as secreted proteins. 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 eDNA sequences using the assembly process described in Example III. Alternatively, full length polynucleotide sequences were dexived entirely from edited or unedited Genscan-predicted coding sequences.
V. Assembly of Genomic Sequence Data with cDNA Sequence Data "Stitched" Sequences Partial cDNA sequences were extended with axons 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 eDNAs and Genscan axon 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 eDNA 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 axons 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 axon 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 SECP Encoding Polynucleotides The sequences which were used to assemble SEQ ID ~V0:25-48 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 N0:25-48 were assembled into clusters of contiguous and overlapping sequences using assembly algorithms such as Phrap (Table 7). Radiation hybrid and genetic mapping data available from public resources such as the Stanford Human Genome Center (SHGC), Whitehead Institute for Genome Research (WIGR), and Genethon were used to determine if any of the clustered sequences .
had been previously mapped. Inclusion of a mapped sequence in a cluster resulted in the assignment of all sequences of that cluster, including its particular SEQ 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-ann. (The centiMorgan (cM) is a unit of measurement based on recombination frequencies between chromosomal markers. On average, 1 cM is roughly equivalent to 1 megabase (Mb) of DNA in humans, although this can vary widely due to hot and cold spots of recombination.) The cM
distances are based on genetic markers mapped by Genethon which provide boundaries for radiation hybrid markers whose sequences were included in each of the clusters. Human genome maps and other resources available to the public, such as the NCBI "GeneMap'99" World Wide Web site (http://www.ncbi.nlin.nih.gov/genemap/), can be employed to determine if previously identified disease genes map within or in proximity to the intervals indicated above.
In this manner, SEQ ID N0:36 was mapped to chromosome 3 within the interval from 142.20 to 148.70 centiMorgans. SEQ ID N0:37 was mapped to chromosome 19 within the interval from 51.00 to 51.70 centiMorgans, and within the interval from 62.00 to 69.90 centiMorgans, and to chromosome 5 within the interval from 141.40 to 142.60 centiMorgans. More than one map location is reported for SEQ ID N0:37, indicating that sequences having different map locations were assembled into a single cluster. This situation occurs, for example, when sequences having strong similarity, but not complete identity, are assembled into a single cluster.
VII. Analysis of Polynucleotide Expression Northern analysis is a laboratory technique used to detect the presence of a transcript of a gene and involves the hybridization of a labeled nucleotide sequence to a membrane on which RNAs from a particular cell type or tissue have been bound. (See, e.g., Sambrook, su ra, ch. 7; Ausubel (1995) su ra, ch. 4 and 16.) Analogous computer techniques applying BLAST were used to search for identical or related molecules in cDNA databases such as GenBank or LIFESEQ (Incyte Genomics). This analysis is much faster than multiple membrane-based hybridizations. In addition, the sensitivity of the computer search can be modified to determine whether any particular match is categorized as exact or similar. The basis of the search is the product score, which is defined as:
BLAST Score x Percent Identity 5 x minimum {length(Seq. 1), length(Seq. 2,)}
The product score takes into account both the degree of similarity between two sequences and the length of the sequence match. The product score is a normalized value between 0 and 100, and is calculated as follows: the BLAST score is multiplied by the percent nucleotide identity and the product is divided by (5 times the length of the shorter of the two sequences). The BLAST score is calculated by assigning a score of +5 for every base that matches in a high-scoring segment pair (HSP), and -4 for every mismatch. Two sequences may share more than one HSP
(separated by gaps). If there is more than one HSP, then the pair with the highest BLAST
score is used to calculate the product score. The product score represents a balance between fractional overlap and quality in a BLAST alignment. For example, a product score of 100 is produced only for 100%
identity over the entire length of the shorter of ethe two sequences being compared. A product score of 70 is produced either by 100% identity and 70% overlap at one end, or by 88% identity and 100% overlap at the other. A product score of 50 is produced either by 100% identity and 50%
overlap at one end, or 79%
identity and 100% overlap.
Alternatively, polynucleotide sequences encoding SECP are analyzed with respect to the tissue sources from which they were derived. For example, some full length sequences are assembled, at least in part, with overlapping Incyte cDNA sequences (see Example III). Each cDNA
sequence is derived from a cDNA library constructed from a human tissue. Each human tissue is classified into one of the following organ/tissue categories: cardiovascular system; connective tissue;
digestive system; embryonic structures; endocrine system; exocrine glands;
genitalia, female;
genitalia, male; germ cells; heroic and immune system; liver; musculoskeletal system; nervous system; pancreas; respiratory system; sense organs; skin; stomatognathic system; unclassified/mixed;
or urinary tract. The number of 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 SECP. cDNA sequences and cDNA
libraryltissue information are found in the LIFESEQ GOLD database (Incyte Genomics, Palo Alto CA).
VIII. Extension of SECP Encoding Polynucleotides Full length polynucleotide sequences were also produced by extension of an appropriate fragment of the full length molecule using oligonucleotide primers designed from this fragment. One primer was synthesized to initiate 5' extension of the known fragment, and the other primer was synthesized to initiate 3' extension of the known fragment. The initial primers were designed using OLIGO 4.06 software (National Biosciences), or another appropriate program, to be about 22 to 30 nucleotides in length, to have a GC content of about 50% or more, and to anneal to the target sequence at temperatures of about 68°C to about 72°C. Any stretch of nucleotides which would result in hairpin structures and primer-primer dimerizations was avoided.
Selected human cDNA libraries were used to extend the sequence. If more than one extension was necessary or desired, additional or nested sets of primers were designed.
High fidelity amplification was obtained by PCR using methods well known in the art. PCR
was performed in 96-well plates using the PTC-200 thermal cycler (MJ Research, Tnc.). The reaction mix contained DNA template, 200 nmol of each primer, reaction buffer containing Mg2+, (NHø)ZS04, and 2-mercaptoethanol, Taq DNA polymerase (Amersham Pharmacia Biotech), ELONGASE enzyme (Life Technologies), and Pfu DNA polymerase (Stratagene), with the following parameters for primer pair PCI A and PCI B: Step 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 ~,1 PICOGREEN
quantitation reagent (0.25% (v/v) PICOGREEN; Molecular Probes, Eugene OR) dissolved in 1X TE
and 0.5 p.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 ~1 to 10 ,u1 aliquot of the reaction mixture was analyzed by electrophoresis on a 1 % agarose gel to determine which reactions were successful in extending the sequence.
The extended nucleotides were desalted and concentrated, transferred to 384-well plates, digested with CviJI cholera virus endonuclease (Molecular Biology Research, Madison WI), and sonicated or sheared prior to religation into pUC 18 vector (Amersham Pharmacia Biotech). For shotgun sequencing, the digested nucleotides were separated on low concentration (0.6 to 0.8%) agarose gels, fragments were excised, and agar digested with Agar ACE
(Promega). Extended clones were religated using T4 ligase (New England Biolabs, Beverly MA) into pUC I8 vector (Amersham Pharmacia Biotech), treated With Pfu DNA polymerase (Stratagene) to fill-in restriction site overhangs, and transfected into competent E. coli cells. Transformed cells were selected on antibiotic-containing media, and individual colonies were picked and cultured overnight at 37 ° C in 384-well plates in LB/2x Garb liquid media.
The cells were lysed, and DNA was amplified by PCR using Taq DNA polymerase (Amersham Pharmacia Biotech) and Pfu DNA polymerase (Stratagene) with the following parameters: Step 1: 94 ° C, 3 min; Step 2: 94 ° C, 15 sec; Step 3 : 60 ° C, 1 min; Step 4: 72 ° C, 2 min;
Step 5: steps 2, 3, and 4 repeated 29 times; Step 6: 72°C, 5 min; Step 7: storage at 4°C. DNA was quantified by PICOGREEN reagent (Molecular Probes) as described above. Samples with low DNA
recoveries were reamplified using the same conditions as described above.
Samples were diluted with 20% dimethysulfoxide (1:2, v/v), and sequenced using DYENAMIC energy transfer sequencing primers and the DYENAMIC DIRECT kit (Amersham Pharmacia Biotech) or the ABI
PRTSM
BTGDYE Terminator cycle sequencing ready reaction kit (Applied Biosystems).
In like manner, full length polynucleotide sequences are vexified 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 SECP Encoding Polynucleotides Common DNA sequence variants known as single nucleotide polymorphisms (SNPs) were identified in SEQ m N0:25-48 using the LIFESEQ database (Incyte 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 exrors and errors resulting from impropex 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 algoritluns 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 Use of Individual Hybridization Probes Hybridization probes derived from SEQ ll~ N0:25-48 are employed to screen cDNAs, genonnic 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 ~Ci of [y-3zP] adenosine triphosphate (Amersham Pharmacia Biotech), and T4 polynucleotide kinase (DuPont NEN, Boston MA). The labeled oligonucleotides are substantially purified using a SEPHADEX G-25 superfine size exclusion dextran bead column (Amersham Pharmacia Biotech).
An aliquot containing 10' counts per minute of the labeled probe is used in a typical membrane-based hybridization analysis of human genomic DNA digested with one of the following endonucleases:
Ase I, Bgl II, Eco RI, Pst I, Xba I, ox 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 undex 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, supra.), mechanical microspotting technologies, and derivatives thereof. The substrate in each of the aforementioned technologies should be uniform and solid with a non-porous surface (Schena (1999), sutra). Suggested substrates include silicon, silica, glass slides, glass chips, and silicon wafers.
Alternatively, a procedure analogous to a dot or slot blot may also be used to arrange and link elements to the surface of a substrate using thermal, UV, chemical, or mechanical bonding procedures. A typical array may be produced using available methods and machines well known to those of ordinary skill in the art and may contain any appropriate number of elements. (See, e.g., Schena, M. et al. (1995) Science 270:467-470; Shalon, D. et al. (1996) Genome Res. 6:639-645;
Marshall, A. and J. Hodgson (1998) Nat. Biotechnol. 16:27-31.) Full length cDNAs, Expressed Sequence Tags (ESTs), ox 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 pgl/tl oligo-(dT) primer (2lmer), 1X
. first strand buffer, 0.03 units/~,I RNase inhibitor, 500 ~.M dATP, 500 ~M
dGTP, 500 ~M dTTP, 40 ~.M dCTP, 40 ~.M dCTP-Cy3 (BDS) or dCTP-Cy5 (Amersham Pharmacia Biotech). The reverse transcription reaction is performed in a 25 ml volume containing 200 ng poly(A)+ RNA with GEMBRIGHT kits (Incyte). Specific control poly(A)+ RNAs are synthesized by in vitro transcription from non-coding yeast genomic DNA. After incubation at 37° C for 2 hr, each reaction sample (one with Cy3 and another With Cy5 labeling) is treated with 2.5 ml of 0.5M sodium hydroxide and incubated for 20 minutes at 85°C to the stop the reaction and degrade the RNA. Samples are purified using two successive CHROMA SPIN 30 gel filtration spin columns (CLONTECH
Laboratories, Inc.
(CLONTECH), Palo Alto CA) and after combining, both reaction samples are ethanol precipitated using 1 rnl of glycogen (1 mg/ml), 60 ml sodium acetate, and 300 ml of 100%
ethanol. The sample is then dried to completion using a SpeedVAC (Savant Instruments Inc., Holbrook NY) and resuspended in 14 ~,15X SSC/0.2% SDS.
Microarra~reparation Sequences of the present invention are used to generate array elements. Each array element is amplified from bacterial cells containing vectors with cloned cDNA inserts.
PCR amplification uses primers complementary to the vector sequences flanking the cDNA insert.
Array elements are amplified in thirty cycles of PCR from an initial quantity of 1-2 ng to a final quantity greater than 5 ,ug. Amplified array elements are then purified using SEPHACRYL-400 (Amersham Pharmacia Biotech).
Purified array elements are immobilized on polymer-coated glass slides. Glass microscope slides (Corning) axe cleaned by ultrasound in 0.1 % SDS and acetone, with extensive distilled water washes between and after treatments. Glass slides axe etched in 4%
hydrofluoric acid (VWR
Scientific Products Corporation (VWR), West Chester PA), washed extensively in distilled water, and coated with 0.05% aminopropyl silane (Sigma) in 95% ethanol. Coated slides are cured in a 110°C oven.
Array elements are applied to the coated glass substrate using a procedure described in U.S.
Patent No. 5,807,522, incorporated herein by reference. 1 p1 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 STRATALINI~ER UV-crosslinker (Stratagene).
Microarrays are washed at room temperature once in 0.2% SDS and three times in distilled water.
Non-specific binding sites are blocked by incubation of microarrays in 0.2%
casein in phosphate buffered saline (PBS) (Tropix, Inc., Bedford MA) for 30 minutes at 60°
C followed by washes in 0.2% SDS and distilled water as before.
Hybridization Hybridization reactions contain 9 p1 of sample mixture consisting of 0.2 ~.g each of Cy3 and Cy5 labeled cDNA synthesis products in 5X SSC, 0.2% SDS hybridization buffer.
The sample mixture is heated to 65° C for 5 minutes and is aliquoted onto the microarray surface and covered with an 1.8 cm2 coverslip. The arrays are transferred to a waterproof chamber having a cavity just slightly larger than a microscope slide. The chamber is kept at 100% humidity internally by the addition of 140 ,u1 of 5X SSC in a corner of the chamber. The chamber containing the arrays is incubated for about 6.5 hours at 60° C. The arrays are washed for 10 min at 45° C in a first wash buffer ( 1X SSC, 0.1 % SDS), three times for 10 minutes each at 45 ° C
in a second wash buffer (0.1X
SSC), and dried.
Detection Reporter-labeled hybridization complexes are detected with a microscope equipped with an Innova 70 mixed gas 10 W laser (Coherent, Inc., Santa Clara CA) capable of generating spectral lines at 488 nm for excitation of Cy3 and at 632 nm for excitation of CyS. The excitation laser light is focused on the array using a 20X microscope objective (Nikon, Inc., Melville NY). The slide containing the array is placed on a computer-controlled X-Y stage on the microscope and raster-scanned past the objective. The 1.8 cm x 1.8 cm array used in the present example is scanned with a resolution of 20 micrometers.
In two separate scans, a mixed gas multiline laser excites the two fluorophores sequentially.
Emitted light is split, based on wavelength, into two photomultiplier tube detectors (PMT 81477, Hamamatsu Photonics Systems, Bridgewater NJ) corresponding to the two fluorophores. Appropriate filters positioned between the array and the photomultiplier tubes are used to filter the signals. The emission maxima of the fluorophores used are 565 nm for Cy3 and 650 nm for CyS. Each array is typically scanned twice, one scan per fluorophore using the appropriate filters at the laser source, although the apparatus is capable of recording the spectra from both fluorophores simultaneously.
The sensitivity of the scans is typically calibrated using the signal intensity generated by a cDNA control species added to the sample mixture at a known concentration. A
specific location on the array contains a complementary DNA sequence, allowing the intensity of the signal at that location to be correlated with a weight ratio of hybridizing species of 1:100,000. When two samples from different sources (e.g., representing test and control cells), each labeled with a different fluorophore, are hybridized to a single array for the purpose of identifying genes that are differentially expressed, the calibration is done by labeling samples of the calibrating cDNA with the two fluorophores and adding identical amounts of each to the hybridization mixture.
The output of the photomultiplier tube is digitized using a 12-bit RTI-~35H
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).
XII. Complementary Polynucleotides Sequences complementary to the SECP-encoding sequences, or any parts thereof, are used to detect, decrease, or inhibit expression of naturally occurring SECP. 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 SECP. 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 SECP-encoding transcript.
XIII. Expression of SECP
Expression and purification of SECP is achieved using bacterial or virus-based expression systems. For expression of SECP in bacteria, cDNA is subcloned into an appropriate vector containing an antibiotic resistance gene and an inducible promoter that directs high levels of cDNA
transcription. Examples of such promoters include, but are not limited to, the trp-lac (tac) hybrid promoter and the T5 or T7 bacteriophage promoter in conjunction with the lac operator regulatory element. Recombinant vectors axe transformed into suitable bacterial hosts, e.g., BL21(DE3).
Antibiotic resistant bacteria express SECP upon induction with isopropyl beta-D-thiogalactopyranoside (IPTG). Expression of SECP in eukaryotic cells is achieved by infecting insect or mammalian cell lines with recombinant Auto~raphica californica nuclear polyhedrosis virus (AcMNPV), commonly known as baculovirus. The nonessential polyhedrin gene of baculovirus is replaced with cDNA encoding SECP by either homologous recombination or bacterial-mediated transposition involving transfer plasmid intermediates. Viral infectivity is maintained and the strong polyhedxin promoter drives high levels of cDNA transcription. Recombinant baculovirus is used to infect Spodoptera fru~iperda (Sf9) insect cells in most cases, or human hepatocytes, in some cases.
Infection of the latter requires additional genetic modifications to baculovirus. (See Engelhard, E.K.
et al. (1994) Proc. Natl. Acad. Sci. USA 91:3224-3227; Sandig, V. et al.
(1996) Hum. Gene Ther.
7:1937-1945.) In most expression systems, SECP is synthesized as a fusion protein with, e.g., glutathione S-transferase (GST) or a peptide epitope tag, such as FLAG or 6-His, permitting rapid, single-step, affinity-based purification of recombinant fusion protein from crude cell lysates. GST, a 26-kilodalton enzyme from Schistosoma~japonicum, enables the purification of fusion proteins on immobilized glutathione under conditions that maintain protein activity and antigenicity (Amersham Pharmacia Biotech). Following purification, the GST moiety can be proteolytically cleaved from SECP at specifically engineered sites. FLAG, an 8-amino acid peptide, enables immunoaffinity purification using commercially available monoclonal and polyclonal anti-FLAG
antibodies (Eastman Kodak). 6-His, a stretch of six consecutive histidine residues, enables purification on metal-chelate resins (QIAGEN). Methods for protein expression and purification axe discussed in Ausubel (1995, supra,, ch. 10 and 16). Purified SECP obtained by these methods can be used directly in the assays shown in Examples XVII, XVIII, and XIX, where applicable.
XIV. Functional Assays SECP function is assessed by expressing the sequences encoding SECP at physiologically elevated levels in mammalian cell culture systems. cDNA is subcloned into a mammalian expression vector containing a strong promoter that drives high levels of cDNA
expression. Vectors of choice include PCMV SPORT (Life Technologies) and PCR3.1 (Invitrogen, Carlsbad CA), both of which contain the cytomegalovirus promoter. 5-10 ,ug of recombinant vector are transiently transfected into a human cell line, for example, an endothelial or hematopoietic cell line, using either liposome formulations or electroporation. 1-2 ~cg 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 CytometrX, Oxford, New York NY.
The influence of SECP on gene expression can be assessed using highly purified populations of cells transfected with sequences encoding SECP 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 SECP and other genes of interest can be analyzed by northern analysis or microarray techniques.
XV. Production of SECP Specific Antibodies SECP 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 SECP amino acid sequence is analyzed using LASERGENE
software (DNASTAR) to determine regions of high immunogenicity, and a corresponding oligopeptide is synthesized and used to raise antibodies by means known to those of skill in the art. Methods for selection of appropriate epitopes, such as those near the C-terminus or in hydrophilic regions are well described in the art. (See, e.g., Ausubel, 1995, 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. (See, e.g., Ausubel, 1995, supra.) Rabbits are immunized with the oligopeptide-KLH complex in complete Freund's adjuvant. Resulting antisera axe tested for antipeptide and anti-SECP activity by, for example, binding the peptide or SECP 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 SECP Using Specific Antibodies Naturally occurring or recombinant SECP is substantially purified by immunoaffmity chromatography using antibodies specific for SECP. An immunoaffinity column is constructed by covalently coupling anti-SECP antibody to an activated chromatographic resin, such as CNBr-activated SEPHAROSE (Amersham Pharmacia Biotech). After the coupling, the resin is blocked and washed according to the manufacturer's instructions.
Media containing SECP are passed over the immunoaffinity column, and the column is washed under conditions that allow the preferential absorbance of SECP (e.g., high ionic strength buffers in the presence of detergent). The column is eluted under conditions that disrupt antibody/SECP binding (e.g., a buffer of pH 2 to pH 3, or a high concentration of a chaotrope, such as urea ox thiocyanate ion), and SECP is collected.
XVII. Identification of Molecules Which Interact with SECP
SECP, or biologically active fragments thereof, are labeled with'ZSI Bolton-Hunter reagent.
(See, e.g., Bolton, A.E. and W.M. Hunter (1973) Biochem. J. 133:529-539.) Candidate molecules previously arrayed in the wells of a mufti-well plate axe incubated with the labeled SECP, washed, and any wells with labeled SECP complex axe assayed. Data obtained using different concentrations of SECP are used to calculate values for the number, affinity, and association of SECP With the candidate molecules.
Alternatively, molecules interacting with SECP 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).
SECP 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 SECP Activity An assay for growth stimulating or inhibiting activity of SECP measures the amount of DNA

synthesis in Swiss mouse 3T3 cells (McKay, I. and Leigh, L, eds. (1993) Growth Factors: A Practical Approach, Oxford University Press, New York, NY). In this assay, varying amounts of SECP are added to quiescent 3T3 cultured cells in the presence of [3H]thymidine, a radioactive DNA precursor.
SECP for this assay can be obtained by recombinant means or from biochemical preparations.
Incorporation of [3H]thymidine into acid-precipitable DNA is measured over an appropriate time interval, and the amount incorporated is directly proportional to the amount of newly synthesized DNA. A linear dose-response curve over at least a hundred-fold SECP
concentration range is indicative of growth modulating activity. One unit of activity per milliliter is defined as the concentration of SECP producing a 50% response level, where 100% represents maximal incorporation of [3H]thymidine into acid-precipitable DNA .
Alternatively, an assay for SECP activity measures the stimulation or inhibition of neurotransmission in cultured cells. Cultured CHO fibroblasts are exposed to SECP. Following endocytic uptake of SECP, the cells are washed with fresh culture medium, and a whole cell voltage-clamped Xenopus myocyte is manipulated into contact with one of the fibroblasts in SECP-free medium. Membrane currents are recorded from the myocyte. Increased or decreased current relative to control values are indicative of neuromodulatory effects of SECP (Morimoto, T. et al. (1995) Neuron 15:689-696).
Alternatively, an assay for SECP activity measures the amount of SECP in secretory, membrane-bound organelles. Transfected cells as described above are harvested and lysed. The lysate is fractionated using methods known to those of skill in the art, for example, sucrose gradient ultracentrifugation. Such methods allow the isolation of subcellular components such as the Golgi apparatus, ER, small membrane-bound vesicles, and other secretory organelles.
hnmunoprecipitations from fractionated and total cell lysates are performed using SECP-specific antibodies, and immunoprecipitated samples are analyzed using SDS-PAGE and immunoblotting techniques. The concentration of SECP in secretory organelles relative to SECP
in total cell lysate is proportional to the amount of SECP in transit through the secretory pathway.
Alternatively, AMP binding activity is measured by combining SECP with32P-labeled AMP.
The reaction is incubated at 37°C and terminated by addition of trichloroacetic acid. The acid extract is neutralized and subjected to gel electrophoresis to remove unbound label.
The radioactivity retained in the gel is proportional to SECP activity.
XIX. Demonstration of Immunoglobulin Activity An assay for SECP activity measures the ability of SECP to recognize and precipitate antigens from serum. This activity can be measured by the quantitative precipitin reaction. (Golub, E.S. et al. (1987) Immunology: A Synthesis, Sinauer Associates, Sunderland, MA, pages 113-115.) SECP is isotopically labeled using methods known in the art. Various serum concentrations are added to constant amounts of labeled SECP. SECP-antigen complexes precipitate out of solution and are collected by centrifugation. The amount of precipitable SECP-antigen complex is proportional to the amount of radioisotope detected in the precipitate. The amount of precipitable SECP-antigen complex is plotted against the serum concentration. For various serum concentrations, a characteristic precipitin curve is obtained, in which the amount of precipitable SECP-antigen complex initially increases proportionately with increasing serum concentration, peaks at the equivalence point, and then decreases proportionately with further increases in serum concentration. Thus, the amount of precipitable SECP-antigen complex is a measure of SECP activity which is characterized by sensitivity to both limiting and excess quantities of antigen.
Alternatively, an assay for SECP activity measures the expression of SECP on the cell surface, cDNA encoding SECP is transfected into a non-leukocytic cell line.
Cell surface proteins are labeled with biotin (de la Fuente, M.A. et al. (1997) Blood 90:2398-2405).
Immunoprecipitations are performed using SECP-specific antibodies, and immunoprecipitated samples are analyzed using SDS-PAGE and immunoblotting techniques. The ratio of labeled immunoprecipitant to unlabeled immunoprecipitant is proportional to the amount of SECP expressed on the cell surface.
Alternatively, an assay for SECP activity measures the amount of cell aggregation induced by overexpression of SECP. In this assay, cultured cells such as NIH3T3 are transfected with cDNA
encoding SECP contained within a suitable mammalian expression vector under control of a strong promoter. Cotransfection with cDNA encoding a fluorescent marker protein, such as Green Fluorescent Protein (CLONTECH), is useful for identifying stable transfectants. The amount of cell agglutination, or clumping, associated with transfected cells is compared with that associated with untransfected cells. The amount of cell agglutination is a direct measure of SECP activity.
Various modifications and variations of the described methods and systems of the invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described in connection with certain embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention which are obvious to those skilled in molecular biology or related fields are intended to be within the scope of the following claims.

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P; P~ P~-. P~. U ~ ~ E-<110> INCYTE GENOMICS, INC.
TANG, Y. Tom YUE, Henry GANDHI, Ameena R.
YAO, Monique G.
WARREN, Bridget A.
DING, Li DUGGAN, Brendan M.
XU, Yuming YANG, Junming THANGAVELU, Kavitha LAL, Preeti G.
HONCHELL, Cynthia D.
WALIA, Narinder K.
LEE, Sally LEE, Ernestine A.
RICHARDSON, Thomas BAUGHN, Mariah R.
ELLIOTT, Vicki S.
<120> SECRETED PROTEINS
<130> PI-0359 PCT
<140> To Be Assigned <141> Herewith <150> 60/266,195; 60/267,924; 60/268,112; 60/267,816; 60/271,639 60/317,818; 60/343,553 <151> 2001-02-02; 2001-02-O8; 2001-02-09; 2001-02-09; 2001-02-26;
2001-09-07; 2001-12-21 <160> 48 <170> PERL Program <210> 1 <211> 496 <212> PRT
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte ID No: 1849820CD1 <400> 1 Met Gly Arg Gly Val Arg Val Leu Leu Leu Leu Ser Leu Leu His Cys Ala Gly Gly Ser G1u Gly Arg Lys Thr Trp Arg Arg Arg Gly G1n Gln Pro Pro Pro Pro Pro Arg Thr Glu Ala Ala Pro Ala Ala Gly Gln Pro Val Glu Ser Phe Pro Leu Asp Phe Thr Ala Val Glu Gly Asn Met Asp Ser Phe Met Ala Gln Val Lys Ser Leu Ala Gln Ser Leu Tyr Pro Cys Ser Ala Gln Gln Leu Asn Glu Asp Leu Arg 80 85 ' 90 Leu His Leu Leu Leu Asn Thr Ser Val Thr Cys Asn Asp Gly Ser Pro Ala Gly Tyr Tyr Leu Lys Glu Ser Arg Gly Ser Arg Arg Trp Leu Leu Phe Leu Glu Gly Gly Trp Tyr Cys Phe Asn Arg Glu Asn Cys Asp Ser Arg Tyr Asp Thr Met Arg Arg Leu Met Ser Ser Arg Asp Trp Pro Arg Thr Arg Thr Gly Thr Gly Ile Leu Ser Ser Gln Pro Glu Glu Asn Pro Tyr Trp Trp Asn Ala Asn Met Val Phe Ile Pro Tyr Cys Ser Ser Asp Val Trp Ser Gly Ala Ser Ser Lys Ser Glu Lys Asn Glu Tyr Ala Phe Met Gly Ala Leu Ile Ile Gln Glu 200 205 2~.0 Val Val Arg Glu Leu Leu Gly Arg Gly Leu Ser Gly Ala Lys Val Leu Leu Leu Ala Gly Ser Ser AIa Gly GIy Thr Gly Val Leu Leu Asn Val Asp Arg Val Ala Glu Gln Leu Glu Lys Leu Gly Tyr Pro Ala Ile Gln Va1 Arg Gly Leu Ala Asp Ser Gly Trp Phe Leu Asp Asn Lys Gln Tyr Arg His Thr Asp Cys Val Asp Thr Ile Thr Cys Ala Pro Thr Glu Ala Ile Arg Arg Gly Ile Arg Tyr Trp Asn Gly Va1 Val Pro G1u Arg Cys Arg Arg Gln Phe Gln Glu Gly Glu Glu 305 ~ 310 315 Trp Asn Cys Phe Phe G1y Tyr Lys Val Tyr Pro Thr Leu Arg Cys Pro Val Phe Val Val Gln Trp Leu Phe Asp Glu A1a Gln Leu Thr Val Asp Asn Val His Leu Thr Gly Gln Pro Val Gln Glu Gly Leu Arg Leu Tyr Ile Gln Asn Leu Gly Arg Glu Leu Arg His Thr Leu Lys Asp Val Pro Ala Ser Phe AIa Pro Ala Cys Leu Ser His Glu Ile Ile Ile Arg Ser His Trp Thr Asp Val GIn Val Lys Gly Thr Ser Leu Pro Arg Ala Leu His Cys Trp Asp Arg Ser Leu His Asp Ser His Lys Ala Ser Lys Thr Pro Leu Lys Gly Cys Pro Val His Leu Val Asp Ser Cys Pro Trp Pro His Cys Asn Pro Ser Cys Pro Thr Val Arg Asp Gln Phe Thr G1y Gln GIu Met Asn Val Ala Gln Phe Leu Met His Met Gly Phe Asp Met Gln Thr Val Ala Gln Pro Gln Gly Leu Glu Pro Ser Glu Leu Leu Gly Met Leu Ser Asn Gly Ser <210> 2 <212> 185 <212> PRT
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte ID No: 70610307CD1 <400> 2 Met Leu Pro Pro Met Ala Leu Pro Ser Val Ser Trp Leu Leu Phe Ile Leu Leu Ser Pro Phe Ser Ser Thr Pro Gly Glu Glu Thr Gln Lys Glu Leu Pro Ser Pro Arg Ile Ser Cys Pro Lys Gly Ser Lys 35 40 ,45 Ala Tyr Gly Ser Pro Cys Tyr Ala Leu Phe Leu Ser Pro Lys Ser Trp Met Asp Ala Asp Leu Ala Cys Gln Lys Arg Pro Ser Gly Lys Leu Val Ser Val Leu Ser Gly Ala Glu G1y Ser Phe Val Ser Ser Leu Val Arg Ser Ile Ser Asn Ser Tyr Ser Tyr Ile Trp Ile Gly Leu His Asp Pro Thr Gln Gly Ser Glu Pro Asp Gly Asp Gly Trp Glu Trp Ser Ser Thr Asp Val Met Asn Tyr Phe A1a Trp Glu Lys Asn Pro Ser Thr Ile Leu Asn Pro Gly His Cys Gly Ser Leu Ser Arg Ser Thr A1a Pro Cys Leu Leu Tyr Ser Va1 Ser Leu Gly Phe Leu Lys Trp Lys Asp Tyr Asn Cys Asp Ala Lys Leu Pro Tyr Val Cys Lys Phe Lys Asp <210> 3 i <211> 73 <212> PRT
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte ID No: 8137559CD2 <400> 3 Met Pro Met Lys Lys Ser Trp Met Pro Lys Thr Cys His Ile Phe Leu Leu Leu Ala Ala Phe Phe G1n Asn Arg Leu Thr Asp Pro Phe Pro Cys Ser Ile Cys Gly Glu Cys Gln Tyr Gly Phe Ser Phe Pro Ser Phe Phe Phe Leu Ile Ser Ser AIa Pro Val Lys A1a Phe Pro Leu Ser Thr Asp His Ser Cys Gly Leu Cys Trp Ala Val <210> 4 <211> 75 <212> PRT
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte ID No: 4801255CD1 <400> 4 Met Arg Glu Asn Val Ser Leu Leu Ala Ala Leu Val Ile Ala Thr Cys Leu Pro Gln Asn Arg Ser Ser.Met Asp Glu Gln Thr Glu Lys Cys Val Arg Arg Leu Val Thr Glu Thr Asp Gln Gly Arg His Leu Lys Lys His Leu Ser Cys Asp Leu Leu Ala Gly Thr Leu Lys Ser Cys Tyr Ala Asn Gly Pro Ala Thr Leu Ala Gly Arg Asn Tyr Cys <210> 5 <211> 96 <212> PRT
<213> Homo Sapiens <220> , <221> misc_feature <223> Incyte ID No: 6160719CD1 <400> 5 Met His Glu Ser Pro Leu Ala Trp Ala Ser Val His Leu Ser Ser Leu Pro Leu Leu Cys Thr Ala Cys Ser Ser Leu Leu Met Gly Asn Ser Val Leu Cys Arg Ala Pro Ala Asp Met Gly Leu Ala Trp Met Leu Leu Leu Ser Glu Pro Arg Arg Val Val Pro Gly Ile Ala Ala Gln Val Leu Thr Ala Leu Arg Arg Arg Leu Leu Ser Gly Thr Leu Pro Ser Phe Pro Arg Arg Lys Asn Pro Leu His G1u His Leu Leu Ala Phe Ile Val Arg Leu <210> 6 <211> 87 <212> PRT
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte ID No: 3524602CD1 <400> 6 Met Ser Met Trp Asp Val Gln Ser Arg Gly Thr Ser Tyr Gln Glu Trp Pro Phe Ile Ser Thr Val Pro Thr Met Ala Lys Ser Cys Gln Ser Leu Met Leu Gly Ser His Leu Leu Ser Asn Pro Pro Gln Thr Gln Lys Pro Gly Thr Leu Ile Leu His Gln Pro Glu Val Met Ser Val Asp Asp Met Asn Asp Ser Ile Gly Gln Val Gln Leu Arg Val Arg Gln Ile Leu Ala Asn His Ser Asp Gly Glu Gly ' <210> 7 <211> 96 <212> PRT
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte ID No: 513718CD1 <400> 7 Met Ser Pro His Leu Leu Ala Gln Ile Leu Leu Cys Val Leu Val Ser Ser Glu Lys Gly Cys Ser Phe Pro Leu Ser Met Arg Ala Ser Leu Thr Pro Gly Ser Asn Val Leu Leu Gln Gly Lys Val Arg Lys Ser Phe Leu Gly Leu Met Thr G1y Leu Arg Glu Lys Gly Arg Thr Arg Glu Gly Glu Ser Gly Leu Ser Ala Phe Ala Val Phe Ser Asn Ala Asn Phe Leu Phe Ser Gly Ala Ile Cys Pro Glu Pro His Gln Asp Glu Ala Pro Ala Pro <210> 8 <211> 101 <212> PRT
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte ID No: 896308CD1 <400> 8 Met Met Ser Val Arg Met Lys Arg Gly His Val Pro Arg Arg His Leu His Gly Pro A1a Trp Leu Val Leu Thr Ser Ser Ala His Pro Cys Pro Pro Ala Pro Thr Thr His Ser Ala Trp Phe Thr Val Pro His Ala Pro Tyr Thr Leu Pro His Leu Gly Val Ser Ala Arg Leu Val Pro Val Pro Gly Lys Ser Ser Arg Leu Thr Pro Lys Cys Leu Pro Pro Pro Phe Leu Ser Gly Val Cys Pro Asn Val Ala Leu Ser Val Arg Pro Phe Leu Thr Thr Arg Leu Lys I1e <210> 9 <211> 93 <212> PRT
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte ID No: 2105862CD1 <400> 9 Met His Ile Leu His Leu Val Leu Ile Met Ile Ser Thr Phe His Leu G1n Leu Ala Tyr Ser Thr Val Leu Arg Lys His Arg Phe Leu Pro Ile Leu Tyr Lys Ser Ala Phe Lys Ile Lys Gln Thr Ser Phe Cys Lys Ile Ile Tyr Lys Asp Thr Trp Pro Cys His Leu Ser Phe~

Glu Asn Asn Tyr Gly Thr Cys Phe Leu Asn Leu Leu Arg Gly Ile Ser Phe Cys Cys Lys Ile Leu Leu Leu Ser Glu Val Lys Leu Tyr Phe Lys Lys <210> 10 <211> 466 <212> PRT
<213> Homo sapiens <220>
<221> misc_feature <223> Incyte ID No: 7939381CD1 <400> 10 Met Leu Arg Ser Thr Ser Thr Val Thr Leu Leu Ser Gly Gly Ala Ala Arg Thr Pro Gly Ala Pro Ser Arg Arg Ala Asn Val Cys Arg Leu Arg Leu Thr Val Pro Pro Glu Ser Pro Val Pro Glu Gln Cys Glu Lys Lys Ile Glu Arg Lys Glu Gln Leu Leu Asp Leu Ser Asn Gly Glu Pro Thr Arg Lys Leu Pro Gln Gly Val Val Tyr Gly Val Val Arg Arg Ser Asp Gln Asn G1n Gln Lys Glu Met Val Val Tyr Gly Trp Ser Thr Ser Gln Leu Lys Glu Glu Met Asn Tyr Ile Lys Asp Val Arg Ala Thr Leu Glu Lys Val Arg Lys Arg Met Tyr Gly Asp Tyr Asp Glu Met Arg G1n Lys Ile Arg Gln Leu Thr Gln Glu Leu Ser Va1 Ser His A1a Gln Gln Glu Tyr Leu Glu Asn His Ile Gln Thr Gln Ser Ser Ala Leu Asp Arg Phe Asn Ala Met Asn Ser Ala Leu Ala Ser Asp Ser Ile Gly Leu Gln Lys Thr Leu Val Asp Val Thr Leu Glu Asn Ser Asn Ile Lys Asp Gln Ile Arg Asn Leu Gln Gln Thr Tyr Glu Ala Ser Met Asp Lys Leu Arg Glu Lys Gln Arg Gln Leu Glu Val Ala Gln Val Glu Asn Gln Zeu Leu Lys Met Lys Val Glu Ser Ser Gln Glu Ala Asn Ala Glu Val Met Arg Glu Met Thr Lys Lys Leu Tyr Ser Gln Tyr Glu Glu Lys Leu Gln Glu Glu Gln Arg Lys His Ser Ala Glu Lys Glu Ala Leu Leu Glu Glu Thr Asn Ser Phe Leu Lys Ala Ile Glu Glu Ala Asn Lys Lys Met Gln Ala Ala Glu Ile Ser Leu Glu Glu Lys Asp Gln Arg Ile Gly Glu Leu Asp Arg Leu Ile Glu Arg Met Glu Lys Glu Arg His Gln Leu Gln Leu Gln Leu Leu Glu His Glu Thr Glu Met Ser G1y Glu Leu Thr Asp Ser Asp Lys Glu Arg Tyr Gln Gln Leu Glu Glu Ala Ser Ala Ser Leu Arg Glu Arg Ile Arg His Leu Asp Asp Met Val His Cys Gln Gln Lys Lys Va1 Lys Gln Met Val Glu Glu Ile Glu Ser Leu Lys Lys Lys Leu Gln Gln Lys Gln Leu Leu Ile Leu Gln Leu Leu Glu Lys Ile Ser Phe Leu Glu Gly Glu Asn Asn Glu Leu Gln Ser Arg Leu Asp Tyr Leu Thr Glu Thr Gln Ala Lys Thr Glu Val Glu Thr Arg Glu Ile Gly VaI Gly Cys Asp Leu Leu Pro Ser Gln Thr Gly Arg Thr Arg Glu Ile Va1 Met Pro Ser Arg Asn Tyr Thr Pro Tyr Thr Arg Val Leu Glu Leu Thr Met Lys Lys Thr Leu Thr <210> 11 <211> 730 <212> PRT
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte TD No: 7487507CD1 <400> 11 Met Ala Leu Pro Ala Leu Gly Leu Asp Pro Trp Ser Leu Leu Gly Leu Phe Leu Phe Gln Leu Leu Gln Leu Leu Leu Pro Thr Thr Thr Ala Gly Gly Gly Gly Gln Gly Pro Met Pro Arg Val Arg Tyr Tyr Ala Gly Asp Glu Arg Arg Ala Leu Ser Phe Phe His Gln Lys Gly Leu Gln Asp Phe Asp Thr Leu Leu Leu Ser Gly Asp Gly Asn Thr Leu Tyr Val Gly Ala Arg Glu Ala Ile Leu Ala Leu Asp Ile Gln Asp Pro Gly Val Pro Arg Leu Lys Asn Met Ile Pro Trp Pro Ala Ser Asp Arg Lys Lys Ser Glu Cys Ala Phe Lys Lys Lys Ser Asn Glu Thr Gln Cys Phe Asn Phe Ile Arg Val Leu Val Ser Tyr Asn Val Thr His Leu Tyr Thr Cys Gly Thr Phe Ala Phe Ser Pro Ala Cys Thr Phe 21e Glu Leu Gln Asp Ser Tyr Leu Leu Pro I1e Ser Glu Asp Lys Va1 Met Glu Gly Lys Gly Gln Ser Pro Phe Asp Pro.

Ala His Lys His Thr Ala Val Leu Val Asp Gly Met Leu Tyr Ser Gly Thr Met Asn Asn Phe Leu Gly Ser Glu Pro Tle Leu Met Arg Thr Leu Gly Ser Gln Pro Val Leu Lys Thr Asp Asn Phe Leu Arg Trp Leu His His Asp Ala Ser Phe Val Ala Ala Ile Pro Ser Thr Gln Val Val Tyr Phe Phe Phe Glu Glu Thr Ala Ser Glu Phe Asp Phe Phe Glu Arg Leu His Thr Ser Arg Val Ala Arg Val Cys Lys Asn Asp Val Gly Gly Glu Lys Leu Leu Gln Lys Lys Trp Thr Thr Phe Leu Lys Ala Gln Leu Leu Cys Thr Gln Pro Gly Gln Leu Pro Phe Asn Val Ile Arg His Ala Val Leu Leu Pro Ala Asp Ser Pro Thr Ala Pro His Ile Tyr Ala Val Phe Thr Ser Gln Trp Gln Val Gly Gly Thr Arg Ser Ser Ala Val Cys Ala Phe Ser Leu Leu Asp Ile Glu Arg Va1 Phe Lys Gly Lys Tyr Lys Glu Leu Asn Lys Glu Thr Ser Arg Trp Thr Thr Tyr Arg Gly Pro Glu Thr Asn Pro Arg Pro Gly Ser Cys Ser Val Gly Pro Ser Ser Asp Lys A1a Leu Thr Phe Met Lys Asp His Phe Leu Met Asp Glu Gln Val Val Gly Thr Pro Leu Leu Val Lys Ser Gly Val Glu Tyr Thr Arg Leu Ala Val 410 . 415 420 Glu Thr Ala Gln Gly Leu Asp Gly His Ser His Leu Val Met Tyr Leu Gly Thr Thr Thr Gly Ser Leu His Lys Ala Val Gly Ala Val Phe Val Gly Phe Ser Gly Gly Val Trp Arg Val Pro Arg Ala Asn Cys Ser Val Tyr Glu Ser Cys Val Asp Cys Val Leu Ala Arg Asp Pro His Cys Ala Trp Asp Pro Glu Ser Arg Thr Cys Cys Leu Leu Ser Ala Pro Asn Leu Asn Ser Trp Lys Gln Asp Met Glu Arg Gly Asn Pro Glu Trp Ala Cys Ala Ser Gly Pro Met Ser Arg Ser Leu 515 520 , 525 Arg Pro Gln Ser Arg Pro Gln Ile I1e Lys Glu Val Leu Ala Val Pro Asn Ser Ile Leu G1u Leu Pro Cys Pro His Leu Ser Ala Leu Ala Ser Tyr Tyr Trp Ser His Gly Pro Ala Ala Val Pro Glu Ala Ser Ser Thr Val Tyr Asn Gly Ser Leu Leu Leu Ile Val Gln Asp Gly Val Gly Gly Leu Tyr Gln Cys Trp Ala Thr Glu Asn Gly Phe Ser Tyr Pro Val Ile Ser Tyr Trp Val Asp Ser Gln Asp Gln Thr Leu Ala Leu Asp Pro Glu Leu Ala Gly Ile Pro Arg Glu His Val Lys Val Pro Leu Thr Arg Val Ser Gly Gly Ala Ala Leu Ala Ala Gln Gln Ser Tyr Trp Pro His Phe Val Thr Val Thr Val Leu Phe Ala Leu Val Leu Ser Gly Ala Leu Ile Ile Leu Va1 A1a Ser Pro Leu Arg Ala Leu Arg Ala Arg Gly Lys Val Gln Gly Cys Glu Thr Leu Arg Pro Gly Glu Lys A1a Pro Leu Ser Arg Glu Gln His Leu Gln Ser Pro Lys Glu Cys Arg Thr Ser Ala Ser Asp Val Asp Ala Asp Asn Asn Cys Leu Gly Thr Glu Val Ala <210> 12 <211> 575 <212> PRT
<213> Homo Sapiens <220>
<221> misc_feature <2~3> Incyte ID No: 1483931CD1 <400> 12 Met Lys Arg Ser Leu Gln Ala Leu Tyr Cys Gln Leu Leu Ser Phe Leu Leu Ile Leu Ala Leu Thr Glu Ala Leu Ala Phe Ala Ile Gln Glu Pro Ser Pro Arg Glu Ser Leu Gln Val Leu Pro Ser Gly Thr Pro Pro Gly Thr Met Val Thr Ala Pro His Ser Ser Thr Arg His Thr Ser Val Val Met Leu Thr Pro Asn Pro Asp Gly Pro Pro Ser Gln Ala Ala Ala Pro Met Ala Thr Pro Thr Pro Arg Ala Glu Gly His Pro Pro Thr His Thr Ile Ser Thr Ile Ala Ala Thr Va1 Thr Ala Pro His Ser Glu Ser Ser Leu Ser Thr Gly Pro Ala Pro Ala Ala Met Ala Thr Thr Ser Ser Lys Pro Glu Gly Arg Pro Arg Gly Gln A1a Ala Pro Thr Ile Leu Leu Thr Lys Pro Pro Gly Ala Thr Ser Arg Pro Thr Thr Ala Pro Pro Arg Thr Thr Thr Arg Arg Pro Pro Arg Pro Pro Gly Ser Ser Arg Lys Gly Ala Gly Asn Ser Ser Arg Pro Val Pro Pro Ala Pro Gly Gly His Ser Arg Ser Lys Glu Gly Gln Arg Gly Arg Asn Pro Ser Ser Thr Pro Leu Gly Gln Lys Arg Pro Leu Gly Lys Ile Phe Gln Ile Tyr Lys Gly Asn Phe Thr Gly Ser Val Glu Pro Glu Pro Ser Thr Leu Thr Pro Arg Thr Pro Leu Trp Gly Tyr Ser Ser Ser Pro Gln Pro Gln Thr Val Ala Ala Thr Thr Val Pro Ser Asn Thr Ser Trp Ala Pro Thr Thr Thr Ser Leu G1y Pro Ala Lys Asp Lys Pro Gly Leu Arg Arg Ala Ala Gln Gly Gly Gly Ser Thr Phe Thr Ser Gln Gly Gly Thr Pro Asp Ala Thr Ala Ala Ser Gly Ala Pro Val Ser Pro Gln Ala Ala Pro Val Pro Ser Gln Arg Pro His His Gly Asp Pro Gln Asp Gly Pro Ser His Ser Asp Ser Trp Leu Thr Val Thr Pro Gly Thr Ser Arg Pro Leu Ser Thr Ser Ser Gly Val Phe Thr Ala Ala Thr Gly Pro Thr Pro Ala A1a Phe Asp Thr Ser Val Ser Ala Pro Ser Gln G1y Ile Pro Gln Gly A1a Ser Thr Thr Pro Gln Ala Pro Thr His Pro Ser Arg Val Ser Glu Ser Thr Ile Ser Gly Ala Lys Glu Glu Thr Val Ala Thr Leu Thr Met Thr Asp Arg Val Pro Ser Pro Leu Ser Thr Val Val Ser Thr Ala Thr Gly Asn Phe Leu Asn Arg Leu Val Pro Ala Gly Thr Trp Lys Pro Gly Thr Ala Gly Asn Ile Ser His Val Ala Glu Gly Asp Lys Pro.Gln His Arg Ala Thr Ile Cys Leu Ser Lys Met Asp Ile Ala Trp Val Ile Leu Ala Ile Ser Val Pro Ile Ser Ser Cys Ser Val Leu Leu Thr Val Cys Cys Met Lys Arg Lys Lys Lys Thr Ala Asn Pro Glu Asn Asn Leu Ser Tyr Trp Asn Asn Thr Ile Thr Met Asp Tyr Phe Asn Arg His Ala Val Glu Leu Pro Arg Glu Ile Gln Ser Leu Glu Thr Ser Glu Asp Gln Leu Ser Glu Pro Arg Ser Pro Ala Asn Gly Asp Tyr Arg Asp Thr Gly Met Val Leu Val Asn Pro Phe Cys Gln Glu Thr Leu Phe Val Gly Asn Asp Gln Val Ser Glu Ile <210> 13 <211> 335 <212> PRT
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte ID No: 8175223CD1 <400> 13 Met Gly Pro Leu Ser Ala Pro Pro Cys Thr Glu His Ile Lys Trp Lys Gly Leu Leu Leu Thr Ala Leu Leu Leu Asn Phe Trp Asn Leu Pro Thr Thr Ala Gln Val Met Ile Glu Ala Gln Pro Pro Lys Va1 Ser Glu Gly Lys Asp Val Leu Leu Leu Val His Asn Leu Pro Gln Asn Leu Thr Gly Tyr Ile Trp Tyr Lys Gly Gln Ile Arg Asp Leu Tyr His Tyr Ile Thr Ser Tyr Val Val Asp Gly Gln Ile Ile Ile Tyr G1y Pro Ala Tyr Ser Gly Arg Glu Thr Val Tyr Ser Asn Ala Ser Leu Leu Ile Gln Asn Val Thr Arg Glu Asp Ala Gly Ser Tyr Thr Leu His Ile Ile Lys Arg G1y Asp Gly Thr Arg Gly Val Thr Gly Tyr Phe Thr Phe Thr Leu Tyr Leu Glu Thr Pro Lys Pro Ser Ile Ser Ser Ser Asn Leu Asn Pro Arg Glu Ala Met Glu Thr Val Ile Leu Thr Cys Asn Pro Glu Thr Pro Asp Ala Ser Tyr Leu Trp Trp Met Asn Gly Gln Ser Leu Pro Met Thr His Arg Met Gln Leu Ser Glu Thr Asn Arg Thr Leu Phe Leu Phe Gly Val Thr Lys Tyr Thr Ala Gly Pro Tyr Glu Cys Glu Ile Trp Asn Ser Gly Ser Ala Ser Arg Ser Asp Pro Val Thr Leu Asn Leu Leu His G1y Pro Asp 230 ~ 235 240 Leu Pro Arg Ile Phe Pro Ser Val Thr Ser Tyr Tyr Ser Gly Glu Asn Leu Asp Leu Ser Cys Phe Ala Asn Ser Asn Pro Pro Ala Gln Tyr Ser Trp Thr Ile Asn Gly Lys Phe Gln Leu Ser Gly Gln Lys Leu Phe Ile Pro Gln Ile Thr Pro Lys His Asn Gly Leu Tyr A1a Cys Ser Ala Arg Asn Ser Ala Thr Gly Glu Glu Ser Ser Thr Ser Leu Thr Ile Arg Val Ile Ala Pro Pro Gly Leu Gly Thr Phe Ala Phe Asn Asn Pro Thr <210> 14 <211> 120 <212> PRT
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte ID No: 4173218CD1 <400> 14 Met Ala Asn Pro Gly Leu Gly Leu Leu Leu Ala Leu Gly Leu Pro Phe Leu Leu Ala Arg Trp Gly Arg Ala Trp Gly G1n Ile Gln Thr Thr Ser Ala Asn Glu Asn Ser Thr Val Leu Pro Ser Ser Thr Ser Ser Ser Ser Asp Gly Asn Leu Arg Pro Glu A1a Ile Thr Ala Tle Ile Val Val Phe Ser Leu Leu Ala Ala Leu Leu Leu Ala Val Gly Leu Ala Leu Leu Val Arg Lys Leu Arg Glu Lys Arg Gln Thr Glu Gly Thr Tyr Arg Pro Ser Ser Glu Glu Gln Val Gly Ala Arg Val 95 ~ 100 105 Pro Pro Thr Pro Asn Leu Lys Leu Pro Pro Glu Glu Arg Leu I1e <210> 15 <211> 103 <212> PRT
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte TD No: 5014679CD1 <400> 15 Met Phe Arg Phe Phe Ser Ser Leu Pro Gly Leu Cys Arg Cys Cys Cys Ser Leu Cys Leu Glu Met Phe Ile Trp Pro Glu Ser Lys Ser Leu Ser Lys Leu Leu Leu Phe Ser Met Thr Glu Leu Gln Leu Phe Val Cys Pro Pro Ser Pro Leu G1u Tyr Met Val Pro Lys Gly Lys Pro His Ala Ile His Ser Pro Pro Asn His Pro Gln Gln Leu Val Gly Lys His Glu Thr Leu Leu Leu Asn Asn Val Phe Pro Leu Ala Gln Gly Leu Gln Ser Gln Arg Leu Thr Arg Gly Lys Asp <210> 16 <211> 170 <212> PRT
<213> Homo sapiens <220>
<221> misc_feature <223> Incyte ID No: 7487510CD1 <400> 16 Met Thr Arg Asp Leu Trp Glu Ala Thr Gln Leu Lys Val Leu Gly His Val Met Glu Ala Pro Glu Ala Ser Leu Thr Leu Ser Leu Ser Ala Ser Ser Ser Ser Ala Ser Phe Lys Asn Gln Ala Leu Phe Ser Ser Ser Asp His Trp Val Ala Pro Gln Asn Trp Phe Cys Asp Tyr Arg Ala Leu Lys Gly Gly Leu Gly Val Trp Val Asn Ser Met Ile Met Leu Val Cys Arg Arg Ser Lys Thr Ala Asn Tyr Leu Gln Cys His Val Val Leu Pro Asn Ala Cys Gly Val Pro Ala Leu Gly Cys Phe Pro Ser Ala Ser Ser G1n Arg Ile Thr Asn Thr Phe His Gly Leu Thr Ser Leu Glu Ala Phe Trp Ile Leu Cys A1a Ala Gln Ala Ala Arg Asp Leu Gly Gly Gln Ala Glu Ser Met Ala Pro Glu Pro Ala Arg Thr Cys His Trp Arg Pro Gly Ala Lys Gly Pro Ser Glu Leu Gly Arg Glu Gly <210> 17 <211> 617 <212> PRT
<213> Homo Sapiens -<220>
<221> misc_feature <223> Incyte ID No: 2682619CD1 <400> 17 Met Phe Arg Thr Ala Val Met Met Ala A1a Ser Leu Ala Leu Thr Gly Ala Val Val Ala His Ala Tyr Tyr Leu Lys His Gln Phe Tyr Pro Thr Val Val Tyr Leu Thr Lys Ser Ser Pro Ser Met Ala Va1 Leu Tyr Ile Gln Ala Phe Val Leu Val Phe Leu Leu Gly Lys Val Met Gly Lys Val Phe Phe Gly Gln Leu Arg Ala Ala Glu Met Glu His Leu Leu Glu Arg Ser Trp Tyr Ala Val Thr Glu Thr Cys Leu A1a Phe Thr Val Phe Arg Asp Asp Phe Ser Pro Arg Phe Val Ala Leu Phe Thr Leu Leu Leu Phe Leu Lys Cys Phe His Trp Leu Ala Glu Asp Arg Val Asp Phe Met Glu Arg Ser Pro Asn Ile Ser Trp Leu Phe His Cys Arg Ile Val Ser Leu Met Phe Leu Leu Gly Ile Leu Asp Phe Leu Phe Val Ser His Ala Tyr His Ser Ile Leu Thr Arg Gly Ala Ser Val Gln Leu Val Phe Gly Phe Glu Tyr Ala Ile Leu Met Thr Met Val Leu Thr Ile Phe Ile Lys Tyr Val Leu His Ser Val Asp Leu Gln Ser Glu Asn Pro Trp Asp Asn Lys Ala Val Tyr Met Leu Tyr Thr Glu Leu Phe Thr Gly Phe Ile Lys Val Leu Leu Tyr Met Ala Phe Met Thr Ile Met Ile Lys Val His Thr Phe Pro Leu Phe Ala Ile Arg Pro Met Tyr Leu Ala Met Arg Gln Phe Lys Lys Ala Val Thr Asp A1a Ile Met Ser Arg Arg Ala Ile Arg Asn Met Asn Thr Leu Tyr Pro Asp Ala Thr Pro Glu Glu Leu Gln Ala Met Asp Asn Val Cys Ile Ile Cys Arg Glu Glu Met Val Thr Gly Ala Lys Arg Leu Pro Cys Asn His Ile Phe His Thr Ser Cys Leu Arg Ser Trp Phe G1n Arg G1n Gln Thr Cys Pro Thr Cys Arg Met Asp Val Leu Arg Ala Ser Leu Pro A1a G1n Ser Pro Pro Pro ' 335 340 345 Pro Glu Pro Ala Asp Gln Gly Pro Pro Pro Ala Pro His Pro Pro Pro Leu Leu Pro Gln Pro Pro Asn Phe Pro Gln Gly Leu Leu Pro Pro Phe Pro Pro Gly Met Phe Pro Leu Trp Pro Pro Met Gly Pro Phe Pro Pro Val Pro Pro Pro Pro Ser Ser Gly Glu A1a Val A1a Pro Pro Ser Thr Ser Ala Ala Ala Leu Ser Arg Pro Ser Gly Ala Ala Thr Thr Thr Ala Ala Gly Thr Ser Ala Thr Ala Ala Ser A1a Thr Ala Ser Gly Pro Gly Ser Gly Ser Ala Pro Glu Ala Gly Pro Ala Pro Gly Phe Pro Phe Pro Pro Pro Trp Met G1y Met Pro Leu Pro Pro Pro Phe Ala Phe Pro Pro Met Pro Val Pro Pro Ala Gly Phe Ala Gly Leu Thr Pro Glu Glu Leu Arg Ala Leu Glu G1y His Glu Arg Gln His Leu Glu Ala Arg Leu Gln Ser Leu Arg Asn Ile His Thr Leu Leu Asp Ala Ala Met Leu Gln Ile Asn Gln Tyr Leu Thr Val Leu Ala Ser Leu Gly Pro Pro Arg Pro Ala Thr Ser Val Asn Ser Thr Glu Glu Thr Ala Thr Thr Val Val Ala Ala Ala Ser Ser Thr Ser I1e Pro Ser Ser Glu Ala Thr Thr Pro Thr Pro Gly Ala Ser Pro Pro Ala Pro Glu Met Glu Arg Pro Pro Ala Pro Glu Ser Val Gly Thr Glu Glu Met Pro Glu Asp Gly Glu Pro Asp Ala Ala Glu Leu Arg Arg Arg Arg Leu G1n Lys Leu Glu Ser Pro Val Ala His <210> 18 <211> 221 <212> PRT
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte ID No: 4582105CD1 <400> 18 Met Ala Gly Thr Gly Leu Leu Ala Leu Arg Thr Leu Pro Gly Pro 1 ~ 5 10 15 Ser Trp Val Arg Gly Ser Gly Pro Ser Va1 Leu Ser Arg Leu Gln Asp Ala Ala Val Val Arg Pro Gly Phe Leu Ser Thr Ala Glu Glu Glu Thr Leu Ser Arg Glu Leu Glu Pro Glu Leu Arg Arg Arg Arg Tyr Glu Tyr Asp His Trp Asp Ala Ala Ile His Gly Phe Arg Glu Thr Glu Lys Ser Arg Trp Ser Glu Ala Ser Arg Ala Ile Leu Gln Arg Val Gln Ala Ala Ala Phe Gly Pro Gly Gln Thr Leu Leu Ser Ser Val His Val Leu Asp Leu Glu Ala Arg Gly Tyr Ile Lys Pro His Val Asp Sex Ile Lys Phe Cys Gly Ala Thr Ile Ala Gly Leu Ser Leu Leu Sex Pro Ser Val Met Arg Leu Val His Thr Gln Glu Pro Gly Glu Trp Leu Glu Leu Leu Leu Glu Pro Gly Ser Leu Tyr Ile Leu Arg Gly Ser Ala Arg Tyr Asp Phe Ser His Glu Ile Leu Arg Asp Glu Glu Ser Phe Phe Gly Glu Arg Arg Ile Pro Arg Gly Arg Arg Ile Ser Va1 Ile Cys Arg Ser Leu Pro Glu Gly Met Gly Pro Gly Glu Ser Gly Gln Pro Pro Pro Ala Cys <210> 19 <211> 329 <212> PRT
<223> Homo Sapiens <220>
<221> misc_feature <223> Incyte ID No: 931619CD1 <400> 19 Met Leu Pro Leu Leu Leu Gly Leu Leu Gly Pro Ala Ala Cys Trp Ala Leu Gly Pro Thr Pro Gly Pro Gly Ser Ser Glu Leu Arg Ser Ala Phe Ser Ala Ala Arg Thr Thr Pro Leu Glu Gly Thr Ser Glu Met Ala Val Thr Phe Asp Lys Val Tyr Val Asn Ile Gly G1y Asp Phe Asp Val Ala Thr Gly Gln Phe Arg Cys Arg Val Pro Gly Ala Tyr Phe Phe Ser Phe Thr Ala Gly Lys Ala Pro His Lys Ser Leu Ser Val Met Leu Val Arg Asn Arg Asp Glu Val Gln Ala Leu Ala Phe Asp Glu Gln Arg Arg Pro Gly Ala Arg Arg Ala Ala Ser Gln Ser Ala Met Leu Gln Leu Asp Tyr Gly Asp Thr Val Trp Leu Arg Leu Leu Gly Ala Pro Gln Tyr Ala Leu Gly Ala Pro Gly Ala Thr Phe Ser Gly Tyr Leu Val Tyr Ala Asp Ala Asp Ala Asp Ala Pro Ala Arg Gly Pro Pro Ala Pro Pro Glu Pro Arg Ser Ala Phe Ser Ala Ala Arg Thr Arg Ser Leu Val Gly Ser Asp Ala Gly 'Pro Gly Pro Arg His G1n Pro Leu Ala Phe Asp Thr Glu Phe Val Asn Ile Gly Gly Asp Phe Asp Ala A1a Ala Gly Val Phe Arg Cys Arg Leu Pro Gly Ala Tyr Phe Phe Ser Phe Thr Leu Gly Lys Leu Pro Arg Lys Thr Leu Ser Val Lys Leu Met Lys Asn Arg Asp Glu Val Gln Ala Met Tle Tyr Asp Asp Gly Ala Ser Arg Arg Arg Glu Met Gln Ser Gln Ser Val Met Leu A1a Leu Arg Arg Gly Asp Ala Val Trp Leu Leu Ser His Asp His Asp Gly Tyr Gly Ala Tyr Ser Asn His Gly Lys Tyr Ile Thr Phe Ser Gly Phe Leu Val Tyr Pro Asp Leu Ala Pro Ala Ala Pro Pro Gly Leu Gly Ala Ser G1u Leu Leu <210> 20 <211> 640 <212> PRT
<213> Homo sapiens <220>
<221> misc_feature <223> Incyte ID No: 2155025CD1 <400> 20 Met Ala G1y Gly Ser Ala Thr Thr Trp Gly Tyr Pro Val Ala Leu Leu Leu Leu Va1 Ala Thr Leu Gly Leu Gly Arg Trp Leu Gln Pro Asp Pro Gly Leu Pro Gly Leu Arg His Ser Tyr Asp Cys Gly Ile Lys Gly Met Gln Leu Leu Va1 Phe Pro Arg Pro Gly Gln Thr Leu Arg Phe Lys Val Val Asp Glu Phe Gly Asn Arg Phe Asp Val Asn Asn Cys Ser Ile Cys Tyr His Trp Val Thr Ser Arg Pro Gln Glu Pro Ala Val Phe Ser Ala Asp Tyr Arg Gly Cys His Val Leu Glu Lys Val Gly Asp Gly Arg Phe His Leu Arg Val Phe Met Glu Ala Val Leu Pro Asn Gly Arg Val Asp Val Ala Gln Asp Ala Thr Leu Ile Cys Pro Lys Pro Asp Pro Ser Arg Thr Leu Asp Ser Gln Leu Ala Pro Pro Ala Met Phe Ser Val Ser Ile Pro Gln Thr Leu Ser 155 ' 160 165 Phe Leu Pro Thr Ser Gly His Thr Ser Gln Gly Ser~.Gly His Ala Phe Pro Ser Pro Leu Asp Pro Gly His Ser Ser Val His Pro Thr Pro Ala Leu Pro Ser Pro Gly Pro Gly Pro Thr Leu Ala Thr Leu Ala Gln Pro His Trp Gly Thr Leu Glu His Trp Asp Val Asn Lys Arg Asp Tyr Ile Gly Thr His Leu Ser Gln Glu G1n Cys G1n Val Ala Ser Gly His Leu Pro Cys Ile Val Arg Arg Thr Ser Lys Glu Ala Cys Gln Gln Ala Gly Cys Cys Tyr Asp Asn Thr Arg Glu Val Pro Cys Tyr Tyr Gly Asn Thr Ala,Thr Val Gln Cys Phe Arg Asp Gly Tyr Phe Val Leu Val Val Ser Gln Glu Met Ala Leu Thr His Arg Ile Thr Leu Ala Asn Ile His Leu Ala Tyr Ala Pro Thr Ser Cys Ser Pro Thr Gln His Thr Glu Ala Phe Val Val Phe Tyr Phe Pro Leu Thr His Cys G1y Thr Thr Met Gln Val Ala Gly Asp Gln Leu 21e Tyr Glu Asn Trp Leu Val Ser Gly Ile His Ile Gln Lys Gly Pro Gln Gly Ser I1e Thr Arg Asp Ser Thr Phe Gln Leu His Val Arg Cys Val Phe Asn Ala Ser Asp Phe Leu Pro Ile Gln Ala Ser Ile Phe Pro Pro Pro Ser Pro A1a Pro Met Thr Gln Pro Gly Pro Leu Arg Leu Glu Leu Arg Ile Ala Lys Asp Glu Thr Phe Ser Ser Tyr Tyr Gly Glu Asp Asp Tyr Pro Ile Val Arg Leu Leu Arg Glu Pro Val His VaI Glu Val Arg Leu Leu Gln Arg Thr Asp Pro Asn Leu Val Leu Leu Leu His Gln Cys Trp Gly Ala Pro Ser Ala Asn Pro Phe Gln Gln Pro Gln Trp Pro Ile Leu Ser Asp Gly Cys Pro Phe Lys Gly Asp Ser Tyr Arg Thr Gln Met Val Ala Leu Asp Gly Ala Thr Pro Phe Gln Ser His Tyr Gln Arg Phe Thr Val Ala Thr Phe Ala Leu Leu Asp Ser Gly Ser Gln Arg Ala Leu Arg Gly Leu Val Tyr Leu Phe Cys Ser Thr Ser Ala Cys His Thr Ser Gly Leu Glu Thr Cys Ser Thr Ala Cys Ser Thr Gly Thr Thr Arg Gln Arg Arg Ser Ser Gly His Arg Asn Asp Thr Ala Arg Pro Gln Asp Ile Val Ser Ser Pro Gly Pro Val Gly Phe Glu Asp Ser Tyr Gly Gln Glu Pro Thr Leu Gly Pro Thr Asp Ser Asn Gly Asn Ser Ser Leu Arg Pro Leu Leu Trp Ala Val Leu Leu Leu Pro Ala Val Ala Leu Val Leu Gly Phe Gly Val Phe Val Gly Leu Ser Gln Thr Trp Ala Gln Lys Leu Trp Glu Ser Asn Arg Gln <210> 21 <211> 388 <212> PRT
<213> Homo Sapiens <220>
<221> misc_feature <223> InCyte ID No: 7640495CD2 <400> 2l Met Gly Arg Ala Arg Arg Phe Gln Trp Pro Leu Leu Leu Leu.Trp Ala A1a Ala Ala Gly Pro G1y Ala Gly Gln Glu Val Gln Thr G1u Asn Val Thr Val Ala Glu Gly Gly Val Ala Glu Ile Thr Cys Arg Leu His Gln Tyr Asp Gly Ser Ile Val Val Tle Gln Asn Pro Ala Arg Gln Thr Leu Phe Phe Asn Gly Thr Arg Ala Leu Lys Asp Glu Arg Phe G1n Leu Glu Glu Phe Ser Pro Arg Arg Val Arg Tle Arg Leu Ser Asp AIa Arg Leu Glu Asp Glu Gly Gly Tyr Phe Cys Gln Leu Tyr Thr Glu Asp Thr His His Gln Ile A1a Thr Leu Thr Va1 Leu Va1 Ala Pro Glu Asn Pro Val Val Glu Val Arg G1u Gln Ala Val Glu Gly Gly G1u Val Glu Leu Ser Cys Leu Val Pro Arg Ser Arg Pro Ala Ala Thr Leu Arg Trp Tyr Arg Asp Arg Lys Glu Leu Lys Gly Val Ser Ser Ser Gln Glu Asn Gly Lys Val Trp Ser Val Ala Ser Thr Val Arg Phe Arg Val Asp Arg Lys Asp Asp Gly Gly Ile Ile Ile Cys Glu A1a Gln Asn G1n Ala Leu Pro Ser Gly His Ser Lys Gln Thr Gln Tyr Val Leu Asp Val Gln Tyr Ser Pro Thr Ala Arg Ile His Ala Ser Gln Ala Val Val Arg Glu Gly Asp Thr Leu Val Leu Thr'Cys Ala Val Thr Gly Asn Pro Arg Pro Asn Gln Ile Arg Trp Asn Arg Gly Asn Glu Ser Leu Pro Glu Arg Ala Glu Ala Val Gly Glu Thr Leu Thr Leu Pro Gly Leu Val Ser Ala Asp, 17!33 Asn Gly Thr Tyr Thr Cys Glu Ala Ser Asn Lys His Gly His Ala Arg Ala Leu Tyr Val Leu Val Val Tyr Asp Pro Gly Ala Val Val Glu Ala G1n Thr Ser Val Pro Tyr Ala Ile Val Gly Gly Ile Leu Ala Leu Leu Val Phe Leu Ile Ile Cys Val Leu Val Gly Met Val Trp Cys Ser Va1 Arg Gln Lys Gly Ser Tyr Leu Thr His Glu Ala Ser Gly Leu Asp Glu Gln Gly Glu Ala Arg Glu Ala Phe Leu Asn Gly Ser Asp Gly His Lys Arg Lys Glu Glu Phe Phe Ile <210> ~2 <211> 255 <212> PRT
<213> Homo sapiens <220>
<221> misc_feature <223> Incyte ID No: 5960219CD1 <400> 22 Met Leu Leu Val Leu Val Val Leu Ile Pro Val Leu Val Ser Ser G1y Gly Pro Glu Gly His Tyr Glu Met Leu Gly Thr Cys Arg Met Val Cys Asp Pro Tyr Gly Gly Thr Lys Ala Pro Ser Thr Ala Ala Thr Pro Asp Arg Gly Leu Met Gln Ser Leu Pro Thr Phe Ile Gln Gly Pro Lys Gly Glu Ala Gly Arg Pro Gly Lys Ala G1y Pro Arg Gly Pro Pro Gly Glu Pro Gly Pro Pro Gly Pro Met Gly Pro Pro Gly Glu Lys Gly Glu Pro Gly Arg Gln Gly Leu Pro Gly Pro Pro Gly Ala Pro Gly Leu Asn Ala Ala Gly Ala Ile Ser Ala Ala Thr Tyr Ser Thr Val Pro Lys Ile Ala Phe Tyr Ala Gly Leu Lys Arg Gln His Glu Gly Tyr Glu Val Leu Lys Phe Asp Asp Va1 Val Thr Asn Leu Gly Asn His Tyr Asp Pro Thr Thr Gly Lys Phe Thr Cys Ser Ile Pro Gly T1e Tyr Phe Phe Thr Tyr His Val Leu Met Arg Gly Gly Asp Gly Thr Ser Met Trp Ala Asp Leu Cys Lys Asn Asn G1n Val Arg Ala Ser Ala Ile Ala Gln Asp Ala Asp Gln Asn Tyr Asp Tyr A1a Ser Asn Ser Va1 Val Leu His Leu Glu Pro Gly Asp Glu Val Tyr Ile Lys Leu Asp Gly Gly Lys Ala His Gly Gly Asn Asn Asn Lys Tyr Ser Thr Phe Ser Gly Phe Ile Ile Tyr Ala Asp <210> 23 <211> 105 <Z12> PRT
<213> Homo sapiens <~20>
<221> misc_feature <223> Incyte ID No: 7500143CD1 <400> 23 Met Lys Arg Ser Leu Gln Ala Leu Tyr Cys Gln Leu Leu Thr Val Leu Leu Thr Val Cys Cys Met Lys Arg Lys Lys Lys Thr Ala Asn Pro Glu Asn Asn Leu Ser Tyr Trp Asn Asn Thr Ile Thr Met Asp Tyr Phe Asn Arg His Ala Val Glu Leu Pro Arg Glu Ile Gln Ser Leu Glu Thr Ser Glu Asp Gln Leu Ser Glu Pro Arg Ser Pro Ala Asn Gly Asp Tyr Arg Asp Thr Gly Met Val Leu Val Asn Pro Phe Cys Gln Glu Thr Leu Phe Val Gly Asn Asp Gln Val Ser Glu Ile <210> 24 <211> 190 <212> PRT
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte ID No: 7503605CD1 <400> 24 Met Ala Gly Thr Gly Leu Leu Ala Leu Arg Thr Leu Pro Gly Pro Ser Trp Val Arg Gly Ser Gly Pro Ser Val Leu Ser Arg Arg Tyr Glu Tyr Asp His Trp Asp Ala Ala Ile His Gly Phe Arg G1u Thr Glu Lys Ser Arg Trp Ser Glu Ala Ser Arg A1a Ile Leu G1n Arg Val Gln Ala Ala Ala Phe Gly Pro G1y Gln Thr Leu Leu Ser Ser Va1 His Val Leu Asp Leu Glu Ala Arg Gly Tyr I1e Lys Pro His Val Asp Ser Ile Lys Phe Cys Gly Ala Thr Ile Ala Gly Leu Ser Leu Leu Ser Pro Ser Val Met Arg Leu Val His Thr Gln Glu Pro Gly Glu Trp Leu Glu Leu Leu Leu Glu Pro Gly Ser Leu Tyr Ile Leu Arg Gly Ser Ala Arg Tyr Asp Phe Ser His Glu Ile Leu Arg.

Asp Glu Glu Ser Phe Phe Gly Glu Arg Arg Ile Pro Arg Gly Arg Arg Ile Ser Val Ile Cys Arg Ser Leu Pro Glu Gly Met Gly Pro Gly Glu Ser Gly Gln Pro Pro Pro Ala Cys <210> 25 <211> 1992 <212> DNA
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte ID No: 1849820CB1 <400> 25 ggccgcccgc tccccgcagg tggacgcggc catgggccga ggggtgcgcg tgctgctgct 60 gctgagcctg ctgcactgcg ccgggggcag cgagggcagg aagacctggc ggcgccgggg 120 tcagcagccg cctcctcccc cgcggaccga ggcggcgccg gcggccggac agcccgtgga 180 gagcttcccg ctggacttca cggccgtgga gggtaacatg gacagcttca tggcgcaagt 240 caagagcctg gcgcagtccc tgtacccctg ctccgcgcag cagctcaacg aggacctgcg 300 CCtgCaCCtC CtaCtCaaCa CCtCggtgaC ctgcaacgac ggcagccccg ccggctacta 360 cctgaaggag tccaggggca gccggcggtg gctcctcttc ctggaaggcg gctggtactg 420 cttcaaccgc gagaactgcg actccagata cgacaccatg cggegcetca tgagctcccg 480 ggactggccg cgcactcgca caggcacagg gatcctgtcc tcacagccgg aggagaaccc 540 ctactggtgg aacgcaaaca tggtcttcat cccctactgc tccagtgatg tttggagcgg 600 ggcttcatcc aagtctgaga agaacgagta cgccttcatg ggcgccctca tcatccagga 660 ggtggtgcgg gagcttctgg gcagagggct gagcggggcc aaggtgctgc tgctggccgg 720 gagcagcgcg gggggcaccg gggtgctcct gaatgtggac cgtgtggctg agcagctgga 780 gaagctgggc tacccagcca tccaggtgcg aggcctggct gactccggct ggttcctgga 840 caacaagcag tatcgccaca cagactgcgt cgacacgatc acgtgcgcgc ccacggaggc 900 catccgccgt ggcatcaggt actggaacgg ggtggtcccg gagcgctgcc gacgccagtt 960 ccaggagggc gaggagtgga actgcttctt tggctacaag gtctacccga ccctgcgctg 1020 ccctgtgttc gtggtgcagt ggctgtttga cgaggcacag ctgacggtgg acaacgtgca 1080 cctgacgggg cagccggtgc aggagggcct gcggctgtac atccagaacc tcggccgcga 1140 gctgcgccac acactcaagg acgtgccggc cagctttgcc cccgcctgcc tctcccatga 1200 gatcatcatc cggagccact ggacggatgt ccaggtgaag gggacgtcgc tgccccgagc 1260 actgcactgc tgggacagga gcctccatga cagccacaag gccagcaaga cccccctcaa 1320 gggctgcccc gtCCa.CCtgg tggacagctg CCCCtggCCC CdCtgCaaCC cctcatgccc 1380 caccgtccga gaccagttca cggggcaaga gatgaacgtg gcccagttcc tcatgcacat 1440 gggcttcgac atgcagacgg tggcccagcc gcagggactg gagcccagtg agctgctggg 1500 gatgctgagc aacggaagct aggcagactg tctggaggag gagccggcac tgaggggccc 1560 agacacccgc tgccccagtg CCaCCtCaCC CCCCa.CCagC aggCCCtCCC gtctcttcgg 1620 gacagggccc cagccgtccc ccctgtctgg gtctgcccac tgccctcctg ccccggcttt 1680 ccctgcccct ctcccacagc ccagccagag acaagggacc tgctgtcatc cccatctgtg 1740 gcctgggggt ccttcctgac aacgaggggg tagccagaag agaagcactg gattcctcag 1800 tccaccagct cagacagcac ccaccggccc cacccatcaa gcccttttat attattttat 1860 aaagtgactt ttttattact ttaatttttt aaaaaaagga aaataagaat atatgatgaa 1920 tgatattgtt ttgtaacttt ttaaaaatga ttttaaagag acaaaaaaga acctcacaaa 2980 aaaaaaaaaa as 1992 <210> 26 <211> 806 <212> DNA
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte ID No: 70610307CB1 <400> 26 atgctgcctc ccatggccct gcccagtgtg tcctggctac tcttcatttt actctctccc 60 ttttcttcca ccccaggtga agaaacccag aaggaactgc cctctccacg gatcagctgt 120 cccaaaggct ccaaggccta tggctccccc tgctatgcct tgtttttgtc accaaaatcc 180 tggatggatg cagatctggc ttgccagaag cggccctctg gaaaactggt gtctgtgctc 240 agtggggctg agggatcctt cgtgtcctcc ctggtgagga gcattagtaa cagctattca 300 tacatctgga ttgggctcca tgaccccaca cagggctctg agcctgatgg agatggatgg 360 gagtggagta gcactgatgt gatgaattac tttgcatggg agaaaaatcc ctccaccatc 420 ttaaaccctg gccactgtgg gagcctgtca agaagcacag ccccatgcct tttatattct 480 gtctccctag gatttctgaa gtggaaagat tataactgtg atgcaaagtt accctatgtc 540 tgcaagttca aggactaggg ccagtgggaa atcatcagcc tcaacttcgc gtgcagctca 600 tcatggacat gagacccgtg tgaagactca ccctgtggag agaatattct cccccaactg 660 ccctacctga ctaccctgtc atgatcctcc ctctttttcc cttttcttca ccctcatttc 720 aggcttttct tccgtcttcc aagtcttgag atctcagaga attattatta aaatgttact 780 ttatacttta aaaaaaaaaa aaaagg 806 <210> 27 <211> 1004 <212> DNA
<213> Homo Sapiens <220>
<~21> misc_feature <223> Incyte ID No: 8137559031 <400> 27 caatcggccg aggcagtatc tgtagctggg agtgtggtac ttgagtaata acaaaccctg 60 catcaggagt ggggcatcaa gagggacaga gactggggtg tagcacactc agacaccttc 120 ctgcttctcc ttgctgctct ataacaagtg tgccttgagg agacaaaatg gactcatggg 180 ctggtccttg agcccagaga gctgagtacc cacaggagga gggtgggaag gaagagctca 240 ttctctcttt gtatgtgtat gtgtgtgtgt gtttgtgtgt gcatgcagga gagagagaaa 300 gaggggaggg ggagagagag agagagaaaa aaaaaacctg ctgcctgagg gagctggggc 360 tcagtgagcg aaaacactct gcttttcaaa ctctacatgg ttacagctaa acccagcaag 420 tcctaggtca tgacctacag aaatgcccat gaagaaaagt tggatgccca agacttgcca 480 catatttctg ttacttgcag ctttctttca gaacagactg actgaccctt ttccatgttc 540 catctgcggg gaatgccagt atggcttctc atttCCttCt ttCttCttCC tcatcagttc 600 agccccagta aaagcattcc ccctttccac agaccactcc tgtggactct gctgggctgt 660 gtaatgtaat gtgccttctt ctgggctccc tggactctgc agtctctgtg cctgttaatt 720 tctctgtttc cactggttga cattttgttc ctcatggcca tgggactctg tctaatcttt 780 ccttatcttc catcctccac ctccaggtgt ggaacagagc aggagtttag gaactatttg 840 gtgagtgagt gagtgataat agataattag atgctcatat atagataatt aggtgctcat 900 atatCC'tCta ttgCCttaCt tttttCtCtC tCtgCCCttt CdCCtCattg CtttgtCCtC 960 cttttgcttt ccaggttgcc ttcgtgaagg ggggcggtgc ggcg 1004 <210> 28 <211> 1202 <212> DNA
<213> Homo Sapiens <220>
<221> misc_feature <~23> Incyte ID No: 48012550B1 <400> 28 aggctggaga tttcatcaag ctactcacag cagcatgcca tttaaaattc atgaattatt 60 tatttctgga attttccgtt taatattttt ggaccacaat tgacctcaga ttttactttc 120 tactcttgtc agtcatatct gcaaggctga cagttaccag gggctgtggc caggcatgag 180 ggagaatgtg tcattgctgg ctgcgttggt catagcaacg tgtctgcctc agaacagatc 240 cagcatggat gagcaaacag aaaaatgtgt aagaaggctt gtgactgaaa cagaccaagg 300 cagacacttg aagaagcatc tttcttgtga tctcctggct ggtaccctga agtcctgtta 360 tgccaatgga ccagcaaccc tcgcaggtag gaattactgc tagaaagact atcctctctg 420 caggcaaatt tctggaagga tactatttta tggggaaaaa tgtgaacaac tgtaggtaat 480 ttcacagtct gtagggcttg tctcctattt gtaatttctt acatgtttac tcgtggtgga 540 aatgtggggg tggctaagga acaagcaggc ccgctgtgtt tcagtggctt tcagagaagg 600 tggcgggaaa tagtctcttc tcagcagcag ctgaatccca gtgttagcag acggagcctt 660 gtttcttgaa agtacattcc cagggattcc cttgaagaat gtgagagatc agctggaatg 720 aaactggatg ttttagggtc ctgagggaaa tgtaaatcca gcagcttatt ctcaagatgt 780 ggctccagaa ccagcagcac tgattcttct aaagagttga gagtgcattt tagctgtgat 840 atctttgtac tgttaagaaa tcctcctcaa caagacaaaa cttgaccaag agcattcagt 900 tgtcttccag aaggctccct tgaggactgg gactcttcct ggatgcaccc aagtaatcta 960 atgaaacaag gactcgtgat ggcagcccct cacctgacaa catggatttt ccccagagat 1020 catttgtaat gtttttcctg aatcattaaa ttgtgtttcc ctattagcaa tgttatgtat 1080 ttctatatcg catatcataa gtgaccgcca tgtagaacat aacctccatt tatagcatgg 1240 acacacagcc catcacgtat catgaccttg catacgccta gataacaaag atcctcacca 1200 cc 1202 <210> 29 <211> 1416 <212> DNA
<213> Homo sapiens <220>
<221> misc_feature <223> Incyte ID No: 6160719CB1 <400> 29 cttttttttt gtaaaaatgt tttactttta atttttatgg atatatagag atgtttaaat 60 tactagccca aagtcacagt tggtaaaatg ttgagctgga atttgaattg agcaagcagt 120 ctacccctta gcagaatctg ctttttttaa atgaactaag cagcctggcc agagtccatg 280 ccctcagcag ctacgttgtg ttgtccttaa ggaggagcat gttttagaaa atcagttata 240 aaatcaggtg attgatgaag tgcagggtta agttaagaga agagtgaact gccttcagaa 300 tccagtgctg ttaccactca agtggagatc tcagataaat cacttattgg agcttctgta 360 caatcatctg taaaaccatt acttcccact tggagagatt tttgaggatt aaatgagata 420 atgcatgaaa gccctctagc ttgggcatca gtacacctga gttCCCttCC CttgCtCtgC 480 acagcctgct catcactact gatggggaac tctgtcctct gtagggcccc tgcagacatg 540 ggccttgcct ggatgctgct gctgtcggag cctaggagag ttgtgcctgg catcgcagca 600 caggtactca cagctctcag aaggagactc ctgtctggga ccctgccctc attcccacgt 660 aggaaaaatc ctttacatga gcatctcctg gccttcattg ttaggttgta gactacaatg 720 aatgatattc tgtgtttaat tacattatgc acaacactct acagagtggg tggttttgaa 780 tcccaaccac taatttacga agtggagcgg ctctgctggc tctgtgaagt atgtgttgtg 840 gagccagagg tgatgctgtt ggatgtgggt ggtgatttac gggagagcag cataagcaga 900 ggaaggcaca gagacctggg ttcaaatccc actgccagtg ctatctgacg tgagacttcg 960 gacaagttat ttaaccttaa ggcttagtgt ccttgcatgt aaaatacaaa taatgctgac 1020 ctcattgatt ccttgtgagg agcccatggg ataatgtggg gtaccatgca tcagtatcat 1080 ttccctttcc cttgataatg attatattgg acagctagtg tcataggaag ctaatggtta 1140 ggaattcaga ctcacacaag aatagatttt gttttgcagt ggaaacttag tgataacttt 1200 ggactaagac aatgtgaaga cattttggca gacaatcata gagtagttat attcttgtga 1260 ggtctgcaga agtccctagt ggggagagca gacagagcct tggatgctca gacctagttt 1320 ggctgtgaaa ctgaagcttt agtcctttta agtctgtgtt tctgaccctg gaggaaaaaa 1380 gttatacata ccttgaaaga ttttccttga aaaaaa 1416 <210> 30 <211> 1707 <212> DNA
<213> Homo sapiens <220>
<221> misc_feature <223> Incyte ID No: 3524602CB1 <400> 30 cagggcgaag taaaatatat ttgaaataat cgttcaggtc agttttggtt tgtaatggaa 60 atttctcttc caatgtaaat aaagcatttg atatgaaaat tacaacatgc aaagagctgc 120 tgagtacatt ttaggcatct caagtggatt ttaatgtaac attttaatag cttttacatt 180 tctttttaca aaacacagct atttcattag ctattcagat ttccttacca cttccatttg 240 aaccctatga aaatatatga aaaaatatat cccccacccc caaccagcaa ttgtataaga 300 atagcaatta ttttaaaaag aacttgcata gtattctgaa tttatcatgt tgttagaaag 360 gctgcctatg gatttttttg agattttcag aaaatgctgc aaactggaca ctccagttct 420 tccagatatt ggtagccgcg gcgcagaggc aatgctgtgt ggttaggagt gattacacca 480 ccagagactt caggactatg aagaggaaaa agtagtgttt ttaaaaaata aggagatctt 540 actaaaaagg atctgggaca gaggcatgaa ggaagtgaac aggattcaac agctgagaac 600 catcagcCag aacataaagg atagggaaga aaatagatga taacaggctt atggccatgt 660 ggaagtgaac acacactcac cagtgttaat ttacatccaa gactaactga aattatgctg 720 aaaacattat tcactggctg gtgcatatct gtgacaaaat gacagaacac taagagttct 780 tccagttaaa tttattcatt ttttaaaaat tctgagctat gttattgtta acagataaaa 840 taccacttgt tttataaaac tacactgata gtcctcagtt caggttttcc tggaagctga 900 ccttatgatg atgatgtgag tacaagtagt ttatctggga ggcaaaggga accccagtaa 960 gaaaatgtag ccaaaaaatt aagggttaca aagccagcta ccactgtgac ggacagatgc 1020 ttgatctggt gaagaagctc taggcaaagt gtaagtatac atgtaagagt tggcacaccc 1080 caggcaggag cggggaccat atttacagac tctgtgtgtc actgattgaa agctgcttcc 1140 caggtcttaa cttcctggca cttctggctg ccacatactg gggcagggct ggcttttgac 1200 aagagccctc agatatgtca atgtgggatg tacagtctcg gggaacaagt taccaagagt 1260 ggccttttat ttccactgtt cctaccatgg caaaatcatg tcagtcattg atgttgggtt 1320 cccacttact ttccaaccct ccccaaaccc aaaagcctgg gacactgata ctccaccagc 1380 cagaggtcat gtcagttgat gacatgaatg acagtatagg gcaggtgcag ctcagagtga 1440 gacaaattct tgccaatcat tctgacgggg aaggatgact ctggaactag caagagaagc 1500 actgaaggat gcctttcctg accctcacat ccaacaaggg cctccaaatc ctcgccttcc 1560 tgatccattt gttgttgttg ttgttgttga gatagagcct tgcttggtca cccaggctgg 1620 agtgcagtgg tgcaatctcg gctcactgca acctccactt ctcaggttca aacgattctc 1680 ctgcctaggc ctcccgagta gctggga 1707 <210> 31 <211> 1436 <212> DNA
<213> Homo sapiens <220>
<221> misc_feature <223> Incyte ID No: 513718CB1 <400> 31 gaaaaaggcc ggccaccgag cggctgtggg tgaatgagtt aaccaaagag caaatgtctg 60 agttggatca tgaaacagca gatcaagtgg gctcagtgtc tgtaaacatc tacactgccc 120 atgaggacaa gatctgtatg ctccatctac acccagaggc tgaaggaatg cctgtggtgt 180 agacaactca cttttctaga gaaatggaga agggattaga catgttaacc ctgaagaaaa 240 ggaaacttgg ggtagggtag gctcagaaag tcttcagagg ttgggaacgt ggcccatgaa 300 agagcaggga gactggaaag ggaccctgcc tgtggaggca gctctgggcc ccacttgaac 360 acacacgggt gaaccacttg acccacctta cagtgaaaga agctggcacc tgtggaccac 420 tggagaaacc tcagtagcca tctggtgttc cccaatgact gtgttgctca caaccctcaa 480 gagtcctttg ttgcttttgg aattgtaggg ctggcaagat gtcttcaccc actgctagat 540 tcatggctga ggcccctata atgaaggaca gatgaacaag agaagaacat acaaacgcat 600 agaatataag ttttacatga cacagaagcc tttgagggga aatgaagaac cagagaaaca 660 gggaaacctg tgtatttgct atgctcgatt ggatgaagag tggacagtca tggagaggca 720 tgaccgagca aagcgggtga taacctggag ggaatgtcac cacatctgtt ggctcagatt 780 Ctcctctgtg tccttgtgtc ttcagaaaaa ggatgttcct ttcctctgag tatgcgggca 840 tcactcacac cagggtccaa tgtcctactt caggggaaag tcaggaaatc cttcctggga 900 ctgatgaccg gcctcagaga gaagggtaga acaagggaag gtgagagtgg cctttctgct 960 tttgctgttt tctcaaatgc caattttcta ttttcagggg caatatgtcc tgaaccccat 1020 caggatgaag ccccagctcc atgaagcccc.tcttgaactg gtccatccta ctgttccagt 1080 CtCagCtCCC tCtgCtCCag gCCttgaaCt tCaCattCCa gcctggggac tcctggcagt 1140 tccctaaata tgcaaagagc tcagctttct ttcctggtcc tggtcctggt CaCttggCCC 1200 tCtCtgtCtC tCCCttCCtt CttCCCatCt CtCttCCttt ctggctcatg tccctcgcac 1260 atgctgtaaa ctcttcctgg aacatctccc ttccacctgt gaatctcttg ccagctcact 1320 tcttttctcc ctttaagaat ccacttctca aaagaaaatg gcaactctgt gaggtgatag 1380 aaatgctgat tcgcttgatc attgtggcgt ccacaatgta tatataaaac aagttg 1436 <210> 32 <211> 1984 <212> DNA
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte ID No: 896308CB1 <400> 32 cgcccctttt tttttttttt cttttcagag ggtctcgctc tgtcaccctg gcaggagcac 60 agtggtgcaa tcatagctca ctgtagactc aaatttgggc tcaagagatc ctcctgtctc 120 agcctcccga gtagctagta gctgagacca caggcgggag ccactgggcc tgtctcttat 180 atctttatgt cagctctttt ctgatggttt ccttagtctc tacatatttt tactcttaaa 240 cctcaggcat gcacataccc ctctgaggtg cttcccagtc tgtagcctcc tccaatttca 300 catgccccga attaaactca ctattctcct ccggaaaact tcttgctact ctattccaca 360 tttcacttca tggcccacca cctatccagc ctctgaaggc ggcggttggg gagacactta 420 ggactgttca caccaacaga agtggccaga cactctgtct cctaattatt ttaaacatgc 480 attctactat ccgttcccaa aaaatatgta gcactcgtgc aagtttgtgt ctacattatt 540 agaactaatc agcctctacc tccctcgagg cagaggctca tctgtcattg accccgggac 600 acgtctgctc caggcatgct gactctggcc ttgacgttcc ccaagcacat ggtatccttt 660 gacaaggcca catctgcacc ctcagcaacc ccagcagcct octctgctgg caagctcctg 720 caggcgcatt ccaaacagtt caaatgtcac cgcctctcct ccagttctcc ctcacaacaa 780 gttagtgttg ctcctttact gtggatacac agtgcttcgc acaggtctcc attcaaatca 840 agccaaggaa tgtttttaaa cacctgttgt gcaaccagta cactgtgctg ggcactggaa 900 aatatgaaaa taactaagag ctgaactcta tccttatgga tttcaccgtc tagaaggaga 960 cagacatgta aggaaataat gagcatacag cactataaat gcaaagcagc agagaggcca 1020 ggacagctga ggtggaatct gaagagccaa gagttttcat tggaacgatt tgcttagaca 1080 CCCCCttatC CCCtCaCCCt ccaaccagta agctccttaa gagggcatat ttgcttaatc 1140 ctgtttgtag tctcagaatc tagtacactg cccaacacag agcagcgact ctaagcactt 1200 cttgaatgaa tgggcaagcc tgcactgtga catattaagc ctactcatga gtcattccac 1260 aaagcaatct tctccataat tactgccaag taagaattgt gtgaaacgcc tggaaaagat 1320 caagcattta caagtgatga tgtccgtgag gatgaaaaga ggacatgtgo ctcgcaggca 1380 CCtgCatggC cctgcttggt tggtcctgac ctcctccgcc CatCCatgtC CtCCagCCCC 1440 caccacacac agtgcgtggt tcactgttcc tcacgcaccc tacaccctgc ctcacctcgg 1500 ggtctctgca cggctggtcc ctgtgcctgg aaagtcctcc cgcctgacac ccaaatgcct 1560 ccctcctccc ttcctttcag gtgtttgccc aaatgtcgcc ctctcagtga ggcetttctt 1620 gaccacccgt ttaaaaatct aatctccttc actgctttat ttttatttta ttttattttt 1680 tgcttctttt ttttgagata gggtcttgct ctgtcaccca ggctggagtg cagtggtgcg 1740 acctcagcta gctgcaacct ccacctccca ggttcaagag attctcctgc ctcagggaag 1800 gggaggttgt ggtgagecac gattgcgtca attgcactcc agcctgggca acaagagtga 1860 aactccatct cataaataaa taaataaatt aattaataaa tcaaacaaac aaaactcatg 1920 gccatactaa tagtattgag attaataatg tgttggttaa cagcatcact ggctggtctg 1980 aatg 1984 <210> 33 <211> 1131 <212> DNA
<213> Homo Sapiens <220>
<2~2> misc_feature <223> zncyte TD No: 2105862CB1 <400> 33 gaatcaaaat ggagttacta atgttaagga aacccaggga agctgtgtac agagccaggg 60 aaggctgtga agagagtgtt ctcacacttg tatgcctgat aatgaaagag actacaaaga 120 ccatatcctt acacaaaggc catcaccacc ttacacaaaa tagtacttct gcaaggacat 180 ctgcccagca actgcctgtt cagcctccaa ctggtgtcac ccttgttatt gatctttgta 240 gctaaggata tttatttcaa aactattata taacccttgt ttctccttta aaaacctttg 300 tcttccttta cctccctgaa tatgtatggc atagtttact atgttatgca tattcctatt 360 gcaatgctct gttctgaaat aaacatcttt tctttgtgat cctctctctg ttatgtaggt 420 tgacagtaaa aattcaaagt ggtttagtga tttgaacttt gctttatata aacatttcct 480 ttttatatgc tttgtgaaac tagaaggtgt aataattaat ggcatactta gttaaaaaaa 540 tttatatgat atgcacatac tgcatttggt cctaataatg atctcaacat ttcatttaca 600 attagcttat tccacagtat taagaaaaca taggtttctt cctattttat ataaatcagc 660 ctttaaaata aaacaaacca gcttttgcaa aattatttac aaagatacct ggccttgtca 720 tttgtcattt gaaaataatt atgggacctg ctttttaaat ttactgaggg ggatctcttt 780 ctgctgtaaa attttacttc tttctgaagt aaaattgtat tttaaaaaat gatataatga 840 aaggccaggt gcagtggttc atgoctgtaa atcccagtac tttggaggct gaggcaggta 900 gatcacctga ggtgtcagga gttcgagatc agcctggtca acatggcaaa accccgtctc 960 tactaaaaat acaaaaatta gccgggcctg gtggcacatg ectgtaatcc cagctacttg 1020 ggagggtgag gcacgagaat cacttgactt gggaggcaga ggttgcagtg agccgagatc 1080 ctgccactgc actccccagc ctgggcgaca gagtgagact ccaaaaaaaa a 1131 <210> 34 <211> 2417 <212> DNA
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte ID No: 7939381CB1 <400> 34 gggccttcgc ggtgcagctg aggctgcaag tagccggcgc cgtcccgcgt cgcccccgcg 60 cagggcgggc cccgcacgct tatcctgccc gggaggaacg ccggcgtcca gcccgctacc 120 ggccgccgct gcgggatgct gcgctccacg tccacggtca ccctgctctc gggcggcgcc 180 gccaggacgc ccggggcgcc cagcaggagg gcaaatgttt gcagactacg gctgaccgta 240 cctcctgaga gtccagttcc tgagcaatgt gaaaagaaga ttgagagaaa agagcagctt 300 cttgacctga gcaatggaga acctaccagg aaacttcctc agggtgttgt ttatggtgtg 360 gtgcgaagat cagatcaaaa tcagcagaaa gaaatggtgg tgtatgggtg gtccaccagt 420 cagctgaaag aagagatgaa ctacatcaaa gatgtgagag ccactttgga aaaggtgaga 480 aagcgaatgt atggagacta tgatgagatg agacagaaga ttcgacagct cacccaggaa 540 ctatcagttt cccatgctca gcaggagtat ctggagaatc acatccaaac ccagtcgtct 600 gccctggatc gttttaatgc catgaactca gccttggcgt cagattccat tggcctgcag 660 aaaaccctcg tggatgtgac tttggaaaac agcaacatta aggatcaaat cagaaatctg 720 cagcagacgt atgaagcatc catggacaag ctgagggaaa agcagaggca gttggaggta 780 gcgcaagttg aaaaccagct gctaaaaatg aaggtggaat cgtcccaaga agccaatgct 840 gaggtgatgc gagagatgac caagaagctg tacagccagt atgaggagaa gctgcaggaa 900 gaacagagga agcacagtgc tgagaaggag gctcttttgg aagaaaccaa tagttttctg 960 aaagcgattg aagaagccaa taaaaagatg caagcagcag agatcagcct agaggagaaa 1020 gaccagagga tcggggagct ggacaggctg attgagcgca tggaaaagga acgtcatcaa 1080 ctgcaacttc aactcctaga acatgaaaca gaaatgtctg gggagttaac tgattctgac 1140 aaggaaaggt atcagcagtt ggaggaggca tcagccagcc tccgtgagcg gatcagacac 1200 ctagatgaca tggtgcattg ccagcagaag aaagtcaagc agatggtcga ggagattgaa 1260 tcattaaaga aaaagttgca acagaaacag ctcttaatac tgcagctttt agaaaagata 1320 tctttcttag aaggagagaa taatgaacta caaagcaggt tggactattt aacagaaacc 1380 caggccaaga ccgaagtgga aaccagagag ataggagtgg gctgtgatct tctacccagc 1440 caaacaggca ggactcgtga aattgtgatg ccttctagga actacaccca atacacaaga 1500 gtcctggagt taaccatgaa gaaaactctg acttaggcac tcagaggcat acacttttta 1560 cagatggaca aaagctctgg aaccctgtgg cttcaaatcc tttgggaagg gtgactgttg 1620 tttcccctac acacagtgta agccggaatg ggaatcgctg aggctctgat ccacttCtaa 1680 gacaggaagg aaagtgaagg cagagtgagc aggtaagaga gggatataca aggtcacatt 1740 tcagacaccc actcggcata ccctgccgta ctgcatcatc atttgttttc tttgtagaca 1800 ctgaaatcct atcaggagga ttccttcaca atgtatttta tttgctagac tttggttggg 1860 agggaaaagg acattaattt gaagtttcat gttattcatg ccaggattgt ttgatagagc 1920 atgaaggttt tgtttaccca taaaagtatt agaggcagcg tttctctgat acagagaggc 1980 ctgtccacaa gaagcatggg cacccagcca aacttgaacc tggaagggag ggttccaggc 2040 ctgcaggtgc tctttcctct tggtcccaag catctgtgca gggtcgtggg agccacactg 2100 agagacttgt gtgggccaga caagcttcat tctgatgcgc tagtcccttg gtttaatttg 2160 tgccttatgc tttcattgga ccagctgaaa tcactgtatt tattcaactt gtgatttttt 2220 tttctttctc actttaactt aaagagaatt ttatatgtct tggaaattta ataatttagt 2280 gttctcagta tcaattggtg tttttgttaa acgaatgaat catctgttca tgcatgctct 2340 actttgatat tataacctat gtcacatgtg tttaataaat accatatatt ttgttctaaa 2400 aaaaaaaaaa aaaaaaa 2417 <210> 35 <211> 2768 <212> DNA
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte ID No: 7487507CB1 <400> 35 ttggcatgat gggcacctgg agggccgcac tcccgttcca gccaggctga gccttctgtc 60 ccctgcctct ggggcctggg aacccccctt cttctttctc Ctgaatggca Cccccgccct 120 25!33 agaatccaga caccgagttt cccactgtgg ctggttcaag ggtatgtgag agctccctgg 180 tgacagtctg tggctgagca tggccctccc agccctgggc ctggacccct ggagcctcct 240 gggccttttc ctcttccaac tgcttcagct gctgctgccg acgacgaccg cggggggagg 300 cgggcagggg cccatgccca gggtcagata ctatgcaggg gatgaacgta gggcacttag 360 cttcttccac cagaagggcc tccaggattt tgacactctg ctcctgagtg gtgatggaaa 420 tactctctac gtgggggctc gagaagccat tctggccttg gatatccagg atccaggggt 480 ccccaggcta aagaacatga taccgtggcc agccagtgac agaaaaaaga gtgaatgtgc 540 ctttaagaag aagagcaatg agacacagtg tttcaacttc atccgtgtcc tggtttctta 600 caatgtcacc catctctaca cctgcggcac cttcgccttc agccctgctt gtaccttcat 660 tgaacttcaa gattcctacc tgttgcccat ctcggaggac aaggtcatgg agggaaaagg 720 ccaaagcccc tttgaccccg ctcacaagca tacggctgtc ttggtggatg ggatgctcta 780 ttctggtact atgaacaact tectgggcag tgagcccatc ctgatgcgca cactgggatc 840 ccagcctgtc ctcaagaccg acaacttcct ccgctggctg catcatgacg cctcctttgt 900 ggCagCCatC CCttCgaCCC aggtCgtCta cttCttCttC gaggagacag ccagcgagtt 960 tgacttcttt gagaggctcc acacatcgcg ggtggctaga gtetgcaaga atgacgtggg 1020 cggcgaaaag ctgctgcaga agaagtggac caccttcctg aaggcccagc tgctctgcac 1080 ccagccgggg cagctgccct tcaacgtcat ccgccacgcg gtcctgctcc ccgccgattc 1140 tcccacagct ccccacatct acgcagtctt cacctcccag tggcaggttg gcgggaccag 1200 gagctctgcg gtttgtgcct tctctctctt ggacattgaa cgtgtcttta aggggaaata 1260 caaagagttg aacaaagaaa cttcacgctg gactacttat aggggccctg agaccaaccc 1320 ccggccaggc agttgctcag tgggcccctc ctctgataag gccctgacct tcatgaagga 1380 ccatttcctg atggatgagc aagtggtggg gacgcccctg ctggtgaaat ctggcgtgga 1440 gtatacacgg cttgcagtgg agacagccca gggccttgat gggcacagcc atcttgtcat 1500 gtacctggga accaccacag ggtcgctcca caaggctgtg ggtgcagtgt ttgtaggctt 1560 ctcaggaggt gtctggaggg tgccccgagc caactgtagt gtctatgaga gctgtgtgga 1620 ctgtgtcctt gcccgggacc cccactgtgc ctgggaccct gagtcccgaa cctgttgcct 1680 cctgtctgcc cccaacctga actcctggaa gcaggacatg gagcggggga acccagagtg 1740 ggcatgtgcc agtggcccca tgagcaggag ccttcggcct cagagccgcc cgcaaatcat 1800 taaagaagtc ctggctgtcc ccaactccat CCtggagCtC CCCtgCCCCC aCCtgtCagC 1860 cttggcctct tattattgga gtcatggccc agcagcagtc ccagaagcct cttccactgt 1920 ctacaatggc tccctcttgc tgatagtgca ggatggagtt gggggtctct accagtgctg 1980 ggcaactgag aatggctttt cataccctgt gatctcctac tgggtggaca gccaggacca 2040 gaccctggcc ctggatcctg aactggcagg catcccccgg gagcatgtga aggtcccgtt 2100 gaccagggtc agtggtgggg ccgccctggc tgcccagcag tcctactggc cccactttgt 2160 cactgtcact gtcctctttg ccttagtgct ttcaggagcc ctcatcatcc tcgtggcctc 2220 cccattgaga gcactccggg ctcggggcaa ggttcagggc tgtgagaccc tgcgccctgg 2280 ggagaaggcc ccgttaagca gagagcaaca cctccagtct cccaaggaat gcaggacctc 2340 tgccagtgat gtggacgctg acaacaactg cctaggcact gaggtagctt aaactctagg 2400 cacaggccgg ggctgcggtg caggcacccg gccatgctgg ctgggcggcc caagcacagc 2460 cctgactagg atgacagcag cacaaaagac cacctttctc ccctgagagg agcttctgct.2520 actctgcatc actgatgaca ctcagcaggg tgatgcacag cagtctgcct cccctatggg 2580 actcccttct accaagcaca tgagctctct aacagggtgg gggctacccc cagacctgct 2640 cctacactga tattgaagaa cctggagagg atccttcagt tctggccatt ccagggaccc 2700 tccagaaaca cagtgtttca agagacccta aaaaacctgc ctgtcccaga cactcttgtg 2760 cagcactt 2768 <210> 36 <211> 2096 <212> DNA
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte ID No: 1483931CB1 <400> 36 gagggagccg gagcgcttct cccgagttgg tgatagattg gtggtcatcc aacatgcaga 60 aatgaatgag cagtgaaaag cagcagagcc gatgggtcat gaggatgtaa gtgcgtttga 120 aggcttccac accctctact ccaggacaga atcatgaata aactggagga taagcaggac 180 cagatgatac catgaagaga agtttacagg ccctctattg ccaactgtta agtttcctgc 240 tgatcttggc actgaccgaa gcgctggcat ttgccatcca ggaaccatct cccagggaat 300 ctcttcaggt cctcccttca ggcactcccc cgggaaccat ggtgacagca ccccacagct 360 ctaccagaca tacttctgtg gtgatgctga cccccaatcc cgatggaccc ccctcacagg 420 atctttatgt cagctctttt ctgatgg ctgcagctcc catggcaaca ccgacacccc gtgcagaggg gcaccctcct acgcacacca 480 tctccaccat cgctgcgaca gtaaccgccc cccattctga aagctccctg tccacagggc 540 ccgctccagc agccatggca accacatcct ccaagccaga gggccgccct cgagggcagg 600 CtgCCCCCdC CatCCtgCtg acaaagccac cgggggccac cagccgcecc accacagcgc 6&0 ccccccgcac taccacacgc aggcccccca ggcccccagg ctcttcccga aaaggggctg 720 gtaattcatc acgccctgtc ccgcctgcac ctggtggcca ctccaggagt aaagaaggac 780 agcgaggacg aaatccaagc tccacacctc tggggcagaa gcggcccctg gggaaaatct 840 ttcagatcta caagggcaac ttcacagggt ctgtggaacc agagccctct accctcaccc 900 ccaggacccc actctggggc tactcctctt caccacagcc ccagacagtg gctgcgacca 960 cagtgcccag caatacctca tgggcaccca ccaccacctc cctggggcct gcaaaggaca 1020 agccaggcct tcgcagagca gcccaggggg gtggttctac cttcaccagc caaggaggga 1080 caccagatgc cacagcagcc tcaggtgccc ctgtcagtcc acaagctgcc ccagtgcctt 1140 ctcagcgccc ccaccacggt gacccacagg atggccccag ccatagtgac tcttggctta 1200 ctgttacccc tggcaccagc agacctctgt ctaccagctc tggggtcttc acggctgcca 1260 cggggcccac cccagctgcc ttcgatacca gtgtctcagc cccttcccag gggattcctc 1320 agggagcatc cacaacccca caagctccaa cccatccctc cagggtctca gaaagcacta 1380 tttctggagc caaggaggag actgtggcca ccctcaccat gaccgaccgg gtgcccagtc 1440 ctctctccac agtggtatcc acagccacag gcaatttcct caaccgcctg gtccccgccg 1500 ggacctggaa gcctgggaca gcagggaaca tctcccatgt ggccgagggg gacaaaccgc 1560 agcacagagc caccatctgc ctgagcaaga tggatatcgc ctgggtgatc ctggccatca 1620 gcgtgcccat ctcctcctgc tctgtcctgc tgacggtgtg ctgcatgaag aggaagaaga 1680 agaccgccaa cccggagaac aacctgagct actggaacaa caccatcacc atggactact 1740 tcaacaggca tgctgtggag ctgcccaggg agatccagtc ccttgaaacc tctgaggacc 1800 agctctcaga gccccgctcc ccagccaatg gcgactatag agacactggg atggtccttg 1860 ttaacccctt ctgtcaagaa acactgtttg tgggaaacga tcaagtatct gagatctaac 1920 tacagcaggc atcactttgc cattccgtat ttttcgtctc taaattataa atatacaaat 1980 atatatatta taaatataac ctttgtgtaa ccctgactta atgagaaaca ttttcagctt 3040 tttttcctat gaattgtcaa catctttttt acaagtgtgg tttaaaaaaa aaaaaa 2096 <210> 37 <211> 1563 <212> DNA
<313> Homo sapiens <330>
<321> misc_feature <233> Incyte ID No: 8175323CB1 <400> 37 gggcaggtcg ggaagagtct cagcgcagaa ggaggaagga cagcacagct gacagccgtg 60 ctctggaagc ttctggatcc taggctcatc tccacagagg agaacatgca cgcagcagag 120 atcatggggc ccctctcagc ccctccctgc acagagcaca tcaaatggaa ggggctcctg 180 ctcacagcat tacttttaaa cttctggaac ttgcctacca ctgcccaagt catgattgaa 240 gcccagccac ccaaagtgtc cgaggggaag gatgttcttc tacttgtcca caatttgccc 300 cagaatctta ctggctacat ctggtacaaa ggacaaatca gggacctcta ccattacatt 360 acatcatatg tagtagacgg tcaaataatt atatatggac cggcatacag tggacgagaa 420 acagtatatt ccaatgcatc cctgctgatc cagaatgtca cccgggagga cgcaggatcc 480 tacaccttac acatcataaa gcgaggtgat gggactagag gagtaactgg atatttcacc 540 ttcaccttat acctggagac tcccaagccc tccatctcca gcagcaactt aaaccccagg 600 gaggccatgg agactgtgat cttaacctgt aatcctgaga ctccggacgc aagctacctg 660 tggtggatga atggtcagag cctccctatg actcatagga tgcagctgtc tgaaaccaac 720 aggaccctct ttctatttgg tgtcacaaag tatactgcag gaccctatga atgtgaaata 780 tggaactcag ggagtgccag ccgcagtgac ccagtcaccc tgaatctcct ccatggtcca 840 gacctcccca gaattttccc ttcagtcacc tcttactatt caggagagaa cctcgacttg 900 tcctgcttcg caaactctaa cccaccagca cagtattctt ggacaattaa tgggaagttt 960 cagctatcag gacaaaagct ctttatccct cagattactc caaagcataa tgggctctat 1020 gcttgctctg ctcgtaactc agccactggc gaggaaagct ccacatcctt gacaatcaga 1080 gtcattgotc ctccaggatt aggaactttt gctttcaata atccaacgta gcagccgtga 1140 tgtcattttt gtatttcagg aagactggca ggagatttat ggaaaagact atgaaaagga 1200 ctcttgaata caagttcctg ataacttcaa gatcatacca ctggactaag aactttcaaa 1260 attttgatga acaggctgat accttcatga aattcaagac aaagaagaaa agaactccat 1330 ttcattggac taaataacaa aaggataatg ttttcataat tttttattgg aaaatgtgct 1380 gattttttga atgttttatc ctccagattt atgaattttt ttcttcagca attggtaaag 1440 tatacttttg taaacaaaaa ttgaaacatt tgcttttgct ctctgagtgc cccagaatgg 1500 gaatctattc atgaatattc atatgtttat ggtactaaag ttatttgcac aagtttaaaa 1560 as 1562 <210> 38 <211> 1110 <212> DNA
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte ID No: 4173218CB1 <400> 38 gggtggggtc ggacccacag aacgaccgac ggaccgaggg ttcgagggag ggacacggac 60 caggaacctg agctaggtca aagacgcccg ggccaggtgc cccgtcgcag gtgcccctgg 120 ccggagatgc ggtaggaggg gcgagcgcga gaagcccctt CCtCggCgCt gCCaaCCCgC 180 cacccagccc atggcgaacc ccgggctggg gctgcttctg gcgctgggcc tgccgttcct 240 gctggcccgc tggggccgag cctgggggca aatacagacc acttctgcaa atgagaatag 300 cactgttttg ccttcatcca ccagctccag ctccgatggc aacctgcgtc cggaagccat 360 cactgctatc atcgtggtct tctccctctt ggctgccttg ctcctggctg tggggctggc 420 actgttggtg cggaagcttc gggagaagcg gcagacggag ggcacctacc ggcccagtag 480 cgaggagcag gtgggtgccc gcgtgccacc gacccccaac ctcaagttgc cgccggaaga 540 gcggctcatc tgaacgctgg ggcctgctgc agccaccaac actgcccagg actgcgggtt 600 gctggcttgt acaccgcagc tgccaccgag acaccagcct ctgatggctc aggaggactt 660 gtggggagag gctgggggca cccatgtggt gggctctgtg cagcatgttg cctctgcttg 720 gctgtgcctg cagctcaggg tgctggggct cgggacccac ccccctgctt gcggaaccaa 780 cttttctctg tgtgtccagc aggccccaca accccctctc ctttctttca gttctcccat 840 gcagccgagg cccgggcccc tcaggactcc aaggagacgg tgcagggctg cctgcccatc 900 taggtcccct ctcctgcatc tgtctccctt cattgctgtg tgaccttggg gaaaggcagt 960 gccctctctg ggcagtcaga tccacccagt gcttaatagc agggaagaag gtacttcaaa 1020 gactctgccc ctgaggtcaa gagaggatgg ggctattcac ttttatatat ttatataaaa 1080 ttagtagtga gatgtaaaaa aaaaaaaaaa 1110 <210> 39 <211> 1412 <212> DNA
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte ID No: 5014679CB1 <400> 39 ggatttgtgt agattgtatg catatacaac atcattttat tccagagact tgagcatctg 60 cgaatttgga tatccaaagg aagtcctaga accagttccc gacaaatacc aagagatgac 120 tttactttag acctaagatc ttgctagaca gcctttgaga ggacgaagga taactagagc 180 agaagcaaat cgcagcgaag tagactacaa caatcaagtc aaaaaacata aagctcctat 240 caacacctca agattttaga aaagcctctt aacgggcaat tttcttttaa ataaagactg 300 ggtcccacta tgttagccag gctggtcaca aactcctgga ctcaagtgat cctcctgcct 360 tggcctccta aagtgctggg attacaggca tgagccactg tgcccggcct gggcagtcga 420 ttttcaggaa gccacttaat gttcagattc ttttcttctc tccaaggact ttgcagatgc 480 tgctgttccc tctgcctgga gatgttcatc tggcctgaat ccaaaagtct gtccaagtta 540 cttctctttt ccatgacaga gttacaacta tttgtctgtc ccccttcccc attagagtat 600 atggtcccca aaggaaagcc ccatgccatt cactcaccac ctaatcatcc ccaacaacta 660 gtgggcaaac atgaaacact tttgttgaat aatgtgtttc ctttagccca gggattacaa 720 agtcaaaggc ttacaagggg gaaggactag caggtgtgct cggaggggac tctagccaac 780 tggagagtaa ctggtgagaa gacaatgaca tgctcttagg gtgtctgcta ctcagatcct 840 etcagttaag tcccgtgcac aaaggtgggt caggtctact gttgccagat cttcagatca 900 ttcaaaagtt acccgaattc tggattttca tgcaaatctc ccaattttta aaaatgttgg 960 catcaaattc agaatgtttt aaccagccac actgtgtggg ccagggaaaa caggcccaaa 1020 ggccaggtga agctgcagtt ttcaactgct gccttggcct ttcccagagc cccttaagtc 1080 ctaagaaggc tcagtcaaca cactggcact cgtttcatcc cactggttga gaaagcaagt 1140 cttagcaaag taatggggga gtcttaccaa aactgcaaag gaaaaaaata tgataccatg 1200 aaattagaaa ttcactgcat ttgaaatgaa aatgacttat ttctgtactt ctataactca 1260 caaatttagc agacatcatt tagacatttt tgtagtgcaa cagatttaaa tctggaattt 1320 catggtttgt aaatcactat gtgatactag ctataaccat gagcaatatt acgggcagta 1380 aatgttgatt tctatttcgt ttcttgaaaa as 1412 <210> 40 <211> 1060 <212> DNA
<213> Homo sapiens <220>
<221> misc_feature <223> Incyte ID No: 7487510CB1 <400> 40 agcccctaac cgcgagtgat ccgccagcct cggcctcccg aggtgccggg attgcagacg 60 gagtctcgtt cactcagtgc tcaatggtgc ccaggctgga gtgcagtggc gtgatctcgg 120 ctcgctacaa cctccacctc ccagccgcct gccttggcct cccaaagagc cgagattgca 180 gcctctgccc ggccgccacc ccgtctggga agtgaggagc gtctctgcct ggccgcccat 240 cgtctgggat gtgaggagcg tctctgcccg gctgcccagt ctgggaagtg aggagcgcct 300 cctcccggcc gccatcccgt ctaggaagtg aggagcgtct ctgcccggcc gcccatcgtc 360 tgagatgtgg ggagcgccac tgccccgccg ccccgtccgg gaggtgcctc ggcttccgca 420 tctgtcgtat gacccgtgat ctctgggaag ccacacagct caaggtcttg gggcacgtca 480 tggaggctcc ggaagcgtca cttaccctgt ccctgtcggc atcatcatcg tcagcatcgt 540 ttaagaatca agccctgttt tcttcttctg accactgggt ggctccgcag aattggttct 600 gtgattatcg cgctctcaaa ggcggccttg gggtttgggt gaacagtatg ataatgctgg 660 tttgtcgtag gtcaaaaaca gcaaattatc tgcaatgtca tgtggttcta cctaatgctt 720 gcggtgtccc tgccctgggc tgtttcectt cggcttcatc tcagcgaatc acgaacacat 780 tccacggact cacctccttg gaagcctttt ggattctctg cgcagcccaa gctgcccggg 840 atctgggagg ccaggctgag tctatggccc cggagcccgc ccggacttgc cactggagac 900 ctggggccaa gggcccatcc gagctgggaa gagagggcta gaaagagagc attagaatcg 960 aggggctggg tgcggaggct cacgcctgtc atcccagcac tttgggagcc gagggagatg 1020 gatcacctga ggttaggaat tcaagagcag cctggccaac 1060 <210> 41 <211> 2268 <212> DNA
<213> Homo sapiens <220>
<221> misc_feature <223> Incyte ID No: 2682619CB1 <400> 41 ggaagttccg gttcgcaggt ggtggggagt gttgttaacc ggaggggcag ccgcagtcgc 60 gcggattgag cgggctcgcg gcgctgggtt cctggtctcc gggccaggca atgttccgca 120 cggcagtgat gatggcggcc agcctggcgc tgaccggggc tgtggtggct cacgcctact 180 acctcaaaca ccagttctac cccactgtgg tgtacctgac caagtccagc cccagcatgg 240 cagtcctgta catccaggcc tttgtccttg tcttccttct gggcaaggtg atgggcaagg 300 tgttctttgg gcaactgagg gcagcagaga tggagcacct tctggaacgt tectggtacg 360 ccgtcacaga gacttgtctg gccttcaccg tttttcggga tgacttcagc ccccgctttg 420 ttgcactctt cactcttctt ctcttcctca aatgtttcca ctggctggct gaggaccgtg 480 tggactttat ggaacgcagc cccaacatct CCtggCtCtt tCaCtgCCgC attgtCtCtC 540 ttatgttcct cctgggcatc ctggacttcc tcttcgtcag ccacgcctat cacagcatcc 600 tgacccgtgg ggcctctgtg cagctggtgt ttggctttga gtatgccatc ctgatgacga 660 tggtgctcac catcttcatc aagtatgtgc tgcactccgt ggacctccag agtgagaacc 720 cctgggacaa caaggctgtg tacatgctct acacagagct gtttacaggc ttcatcaagg 780 ttctgctgta catggccttc atgaccatca tgatcaaggt gcacaccttc ccactctttg 840 ccatccggcc catgtacctg gccatgagac agttcaagaa agctgtgaca gatgccatca 900 tgtctcgccg agccatccgc aacatgaaca ccctgtatcc agatgccacc,ccagaggagc 960 tccaggcaat ggacaatgtc tgcatcatct gccgagaaga gatggtgact ggtgccaaga 1020 gactgccctg caaccacatt ttccatacca gctgcctgcg ctcctggttc cagcggcagc 1080 agacctgccc cacctgccgt atggatgtcc ttcgtgcatc gctgccagcg cagtcaccac 1140 cacccccgga gcctgcggat cacgggccac cccctgcccc ccacccccca ccactcttgc 1200 ctcagccccc caacttcccc cagggcctcc tgcctccttt tcctccaggc atgttcccac 1260 tgtggccccc catgggcccc tttCCa.CCtg tCCCgCCtCC CCCCagCtCa ggagaggctg 1320 tggctcctcc atccaccagt gcagcagccc tttctcggcc cagtggagca gctacaacca 1380 cagctgctgg caccagtgct actgctgctt ctgccacagc atctggccca ggctctggct 1440 ctgccccaga ggCtggCCCt gCCCCtggtt tCCCCttCCC tcctccctgg atgggtatgc 1500 CCCtgCCtCC aCCCtttgCC ttccccccaa tgcctgtgcc ccctgcgggc tttgctgggc 1560 tgaccccaga ggagctacga gctctggagg gccatgagcg gcagcacctg gaggcccggc 1620 tgcagagcct gcgtaacatc cacacactgc tggacgccgc catgctgcag atcaaccagt 1680 acctcaccgt gctggcctcc ttggggCCCC CCCggCCtgC CaCttCagtC aaCtCCaCtg 1740 aggagactgc cactacagtt gttgctgctg cctcctccac cagcatccct agctcagagg 1800 ccacgacccc aaccccagga gcctccccac cagcccctga aatggaaagg cctccagctc 1860 ctgagtcagt gggcacagag~gagatgcctg aggatggaga gcccgatgca gcagagctcc 1920 gccggcgccg cctgcagaag CtggagtCtC CtgttgCCCa CtgaCaCtgC CCCagCCCag 1980 ccccagcctc tgctcttttg agcagccctc gctggaacat gtcctgccac caagtgccag 2040 CtCCCtCtCt gtCtgCa.CCa gggagtagta cccccagctc tgagaaagag gcggcatccc 2100 ctaggccaag tggaaagagg ctggggttcc catttgactc cagtcccagg cagccatggg 2160 gatctcgggt cagttccagc cttcctctcc aactcatcag ccctgtgttc tgctggggcc 2220 atgaaggcag aaggtttagc ctctgaagag cctcttcttc ccccaacc 2268 <210> 42 <211> 821 <212> DNA
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte ID No: 4582105CB1 <400> 42 gagggaagtc aatcgctgcc gcaggtaccg ccaatggctt ttggcggggg cgttccccaa 60 CCCtgCCCtC tctcatgacc ccgctccggg attatggccg ggactgggct gctggcgctg 120 cggacgctgc cagggcccag ctgggtgcga ggctcgggcc cttccgtgct gagccgcctg 180 caggacgcgg ccgtggtgcg gcctggcttc ctgagcacgg cagaggagga gacgctgagc 240 cgagaactgg agcccgagct gcgccgccgc cgctacgaat acgatcactg ggacgcggcc 300 atccacggct tccgagagac agagaagtcg cgctggtcag aagccagccg ggccatcctg 360 cagcgcgtgc aggcggccgc ctttggcecc ggccagaccc tgctctcctc cgtgcacgtg 420 ctggacctgg aagcccgcgg ctacatcaag ccccacgtgg acagcatcaa gttctgcggg 480 gccaccatcg ccggcctgtc tctcctgtct cccagcgtta tgcggctggt gcacacccag 540 gagccggggg agtggctgga actcttgctg gagccgggct ccctctacat ccttaggggc 600 tcagcccgtt atgacttctc ccatgagatc cttcgggatg aagagtcctt ctttggggaa 660 cgccggattc cccggggccg gcgcatctcc gtgatctgcc gctccctccc tgagggcatg 720 gggccagggg agtctggaca gCCgCCCCCa gcctgctgac ccccagcttt ctacagacac 780 cagatttgtg aataaagttg gggaatggac agcctaaaaa a 821 <210> 43 <~11> 1325 <212> DNA
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte TD No: 931619CB1 <400> 43 ccgggaaaag cagccggagc ccccgccgcc cctgccgcag cgcgggcggt cagcgcgcag 60 cccggcaccc gcagcctgca gcctgcagcc cgcagcccgc agcccggagc cagatcgcgg 120 gctcagaccg aacccgactc gaccgccgcc cccagccagg cgccatgctg ccgcttctgc 180 tgggcctgct gggcccagcg gcctgctggg ccctgggccc gacccccggc ccgggatcct 240 ctgagctgcg ctcggccttc tcggcggcac gcaccacccc cctggagggc acgtcggaga 300 tggcggtgac cttcgacaag gtgtacgtga acatcggggg cgacttcgat gtggccaccg 360 gccagtttcg ctgccgcgtg cccggcgcct acttcttctc cttcacggct ggcaaggccc 420 cgcacaagag cctgtcggtg atgctggtgc gaaaccgcga cgaggtgcag gcgctggcct 480 tcgacgagca gcggcggcca ggcgcgcggc gcgcagccag ccagagcgcc atgctgcagc 540 tcgactacgg cgacacagtg tggctgcggc tgcttggcgc cccgcagtac gcgctaggcg 600 cgcccggcgc caccttcagc ggctacctag tctacgccga cgccgacgct gacgcgcctg 660 cgcgcgggcc gcccgcgccc cccgagccgc gctcggcctt ctcggcggcg cgcacgcgca 720 gcttggtggg ctcggacgct ggccccgggc cgcggcacca accactcgcc ttcgacaccg 780 agttcgtcaa cattggcggc gacttcgacg cggcggccgg cgtgttccgc tgccgtctgc 840 CCggCgCCta CttCttCtCC ttcacgctgg gcaagctgcc gcgtaagacg ctgtcggtta 900 agctgatgaa gaaccgcgac gaggtgcagg ccatgattta cgacgacggc gcgtcgcggc 960 gccgcgagat gcagagccag agcgtgatgc tggccctgcg gcgcggcgac gccgtctggc 1020 tgctcagcca cgaccacgac ggctacggcg cctacagcaa ccacggcaag tacatcacct 1080 tctccggctt cctggtgtac cccgacctcg cccccgccgc cccgccgggc ctcggggcct 1140 cggagctact gtgagccccg ggccagagaa gagcccggga gggccagggg cgtgcatgcc 1200 aggccgggcc cgaggctcga aagtcccgcg cgagcgccac ggcctccggg cgcgcctgga 1260 ctctgccaat aaagcggaaa gcgggcacgc gcagcgcccg gcagcccagg aaaaaaaaaa 1320 aaaaa 1325 <210> 44 <211> 2030 <212> DNA
<213> Homo sapiens <220>
<221> misc_feature <223> Incyte ID No: 2155025CB1 <400> 44 gggtgtgtct gtggcgtctc atggcaggag gctcagccac gacctggggt taccctgtgg 60 ccctgctact gctggtcgcc accctggggc tgggtaggtg gctccagccc gaCCCtggCC 120 tcccaggcct ccggcacagc tacgactgtg ggatcaaggg aatgcagctg ctggtgttcc 180 ccaggccagg ccagactctc cgcttcaagg tggtggatga atttgggaac cgatttgatg 240 tcaacaactg ctccatctgc taccactggg tcacctccag gccgcaggag cctgcagtct 300 tctcggccga ttacagaggc tgccacgtgc tggagaaggt aggggatggg cgtttccacc 360 tgagggtgtt catggaggct gtgctgccca atggtcgtgt ggatgtggca caagacgcta 420 CtCtgatCtg tCCCaaaCCt gaCCCCtCCC ggactctgga ctcccagctg gcaccacccg 480 ccatgttctc tgtctcaatc ccacaaaccc tttccttcct ccccacctct ggccatacct 540 cccaaggctc tggccatgcc tttcccagcc cactggaccc agggcacagc tctgtccacc 600 CaaCCCCtgC tttaCCatCC CCtggaCCtg gacctaccct cgccaccctg gctcaacccc 660 actggggcac cttggaacac tgggatgtga acaaacgaga ttacataggt acccacctga 720 gccaggagca gtgccaggtg gcctcagggc acctcccctg catcgtgaga agaacttcaa 780 aagaagcctg tcagcaggct ggctgctgct atgacaacac cagagaggtt ccctgttact 840 atggcaacac agctactgtc cagtgcttca gagatggcta cttcgtcctc gtggtgtccc 900 aagaaatggc cttgacacac aggatcacac tggccaacat ccacctggcc tatgccccca 960 ccagctgctc cccaacacag cacacggaag ctttcgtggt cttctacttc cctctcaccc 1020 actgtggaac cacaatgcag gtggctggcg accagctcat ctatgagaac tggctggtgt 1080 ctggcatcca catccaaaag gggccacagg gttccatcac gcgggacagc accttccagc 1140 ttcatgtgcg ctgtgtcttc aacgccagtg acttcctgcc cattcaggca tccattttcc 1200 cacccccatc gcctgctcct atgacccagc ccggccecct gcggcttgag ctgcggattg 1260 ccaaagacga gaccttcagc tcgtactatg gggaggatga ctatcccatc gtgaggctgc 1320 tccgagaacc agtccatgtg gaggtccggc ttctgcagag gacagacccc aacctggtcc 1380 tgetgctgca ccagtgctgg ggcgctccca gtgccaaccc cttccagcag ccccagtggc 1440 ccatcctgtc agacggatgc cctttcaagg gcgacagcta cagaacccaa atggtagcct 1500 tggacggggc cacacctttc cagtcgcact accagcgatt cactgttgct accttcgccc 1560 tcctggactc aggctcccag agagccctca gaggactggt ttacttgttc tgcagcacct 1620 ctgcctgcca cacctcaggg ctggagactt gctccactgc atgtagcact ggcactacaa 1680 gacagcgacg atcctcaggt caccgtaatg acactgccag gccccaggac atcgtgagct 1740 ctccggggcc agtgggcttt gaggattctt atgggcagga gcccacactt gggcccacag 1800 actccaatgg gaactccagc ctgagacctc tcctttgggc ggtccttttg ctgccagctg 1860 ttgccctggt ccttgggttt ggtgtctttg tgggcctgag ccagacctgg gcccagaagc 1920 tctgggaaag caacagacag tgaatgggcc caataaacaa tcatttcaaa cctactgaaa 1980 ccaggtgtgg agaagttatt tgtgacgact agaacagaac tatttcttat 2030 <210> 45 <211> 1307 <.212> DNA
<~13> Homo Sapiens <220>
<221> misc_feature <223> Incyte ID No: 7640495CB1 <400> 45 cgggccggga gcgagcggcg gcggcggcgg cggcggcacc atgggccggg cccggcgctt 60 ccagtggccg ctgctgctgc tgtgggcggc cgcggcgggg ccaggggcag gacaggaagt 120 acagacagag aacgtgacag tggctgaggg tggggtggct gagatcacct gccgtctgca 180 ccagtatgat gggtccatag ttgtcatcca gaacccagcc cggcagaccc tcttcttcaa 240 tggcacccgt gccttgaagg atgagcgttt ccagcttgag gagttctccc cacgccgggt 300 gcggatccgg ctctcagatg cccgcctgga ggacgagggg ggctatttct gccagctcta 360 cacagaagac acccaccacc agattgccac gctcacggta ctagtggccc cagagaatcc 420 tgtggtggag gtccgggagc aggcggtaga gggcggcgag gtggagctca gctgcctcgt 480 tccgcggtcc cgtccggctg ccaccctgcg ctggtaccgg gaccgcaagg agctgaaagg 540 agtgagcagc agccaggaaa atggcaaggt ctggagcgtg gcaagcacag tacggtttcg 600 tgtggaccgt aaggacgacg gtggtatcat catctgtgag gcgcagaacc aggcgctgcc 660 ctccggacac agcaagcaga cgcagtacgt gctggatgtg cagtactccc ccacggcccg 720 gattcatgcc tcccaagctg tggtgaggga gggagacacg ctggtgttga cgtgtgctgt 780 cacggggaac cccaggccaa accagatccg ctggaaccgc gggaatgagt ctttgccgga 840 gagggcggag gccgtgggag agacgctcac gctgccgggt ctggtatccg cggataacgg 900 cacctacact tgcgaggcgt ccaataagca cggccatgcg agggcgctct acgtacttgt 960 ggtctacgac cctggtgcgg tggtagaggc tcagacgtcg gttccctatg ccattgtggg 1020 cggcatcctg gcgctgctgg tgtttctgat catatgtgtg ctagtgggca tggtctggtg 1080 ctcggtacgg cagaagggtt cctatctgac ccacgaagcc agtggcttgg atgaacaggg 1140 agaagcaaga gaagccttcc tcaatggcag cgacggacac aagaggaaag aggaattctt 1200 catctgaccc tatccccacc ccaggcctag gcctgggcct gggctggggt CCCCCCCaCt 1260 gccagctgca agggaaccag caaagacatt taccagagtc tgggatg 1307 <220> 46 <211> 768 <212> DNA .
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte ID No: 5960119CB1 <400> 46 atgctgctgg tgctggtggt gctcatcccc gtgctggtga gctcgggcgg cccggaaggc 60 cactatgaga tgctgggcac ctgccgcatg gtgtgcgacc cctacggggg caccaaggcg 120 CCCagCaCCg CtgCCaCgCC CgaCCgCggC CtCatgCagt ccctgcccac cttcatccag 180 ggccccaaag gcgaggccgg caggcccggg aaggcgggtc cgcgcgggcc ccccggagag 240 cccgggccac ccggccccat ggggcccccg ggcgagaagg gcgagccggg ccgccaaggc 300 ctgccgggcc cgcecggggc gcccggcctg aacgcggccg gggccatcag egccgccacc 360 tacagcacgg tgcccaagat cgccttctac gccggcctca agcggcagca tgaaggctac 420 gaggtgctca agttcgacga cgtggtcacc aacctcggaa accactacga ccccaccacc 480 ggcaagttca cctgctccat cccgggcatc tacttcttca cctaccacgt cctgatgcgc 540 ggaggggacg gcaccagcat gtgggctgat ctctgcaaaa acaaccaggt gcgtgctagt 600 gcaattgccc aagatgctga tcagaattac gactatgcca gtaacagtgt ggttcttcat 660 ttggagccgg gagatgaagt ctatatcaaa ttagatggcg ggaaagccca tggaggaaac 720 aacaacaaat acagcacgtt ttctggattt attatttatg ctgactga 768 <210> 47 <211> 782 <212> DNA
<213> Homo sapiens <220>
<221> misc feature <223> Incyte ID No: 7500143CB1 <400> 47 gctttctcct gagccgttgg agggagccgg agcgcttctc ccgagttggt gatagattgg 60 tggtcatcca acatgcagaa atgaatgagc agtgaaaagc agcagagccg atgggtcatg 120 aggatgtaag tgcgtttgaa ggcttccaca ccctctactc caggacagaa tcatgaataa 180 actggaggat aagcaggacc agatgatacc atgaagagaa gtttacaggc cctctattgc 240 caactgttaa ctgtcctgct gacggtgtgc tgcatgaaga ggaagaagaa gaccgccaac 300 ccggagaaca acctgagcta ctggaacaac accatcacca tggactactt caacaggcat 360 gctgtggagc tgcccaggga gatccagtcc cttgaaacct ctgaggacca gctctcagag 420 ccccgctccc cagccaatgg cgactataga gacactggga tggtccttgt taaccccttc 480 tgtcaagaaa cactgtttgt gggaaacgat caagtatctg agatctaact acagcaggca 540 tcactttgcc attccgtatt tttcgtctct aaattataaa tatacaaata tatatattat 600 aaatataacc tttgtgtaac cctgacttaa tgagaaacat tttcagcttt ttttcctatg 660 aattgtcaac atctttttta caagtgtggt ttaaaaaaaa aaa.aacttta cagaatgatc 720 tgtggcttta taaaataaag gtatttctaa gcaaaaaaaa aaaaaaaaaa aaaaaaaaaa 780 tt 782 <210> 48 <211> 893 <212> DNA
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte ID No: 7503605CB1 <400> 48 gagggaagtc aatcgctgcc gcaggtaccg ccaatggctt ttggcggggg cgttccccaa 60 ccctgccctc tctcatgacc ccgctccggg attatggccg ggactgggct gctggcgctg 120 cggacgctgc cagggcocag ctgggtgcga ggctcgggcc cttccgtgct gagccgccgc 180 tacgaatacg atcactggga cgcggccatc cacggcttcc gagagacaga gaagtcgcgc 240 tggtcagaag ccagccgggc catcctgcag cgcgtgcagg cggccgcctt tggccccggc 300 cagaccctgc tctcctccgt gcacgtgctg gacctggaag cccgcggcta catcaagccc 360 cacgtggaca gcatcaagtt ctgcggggcc accatcgccg gcctgtctct cctgtctccc 420 agcgttatgc ggctggtgca cacccaggag ccgggggagt ggctggaact cttgctggag 480 ccgggctccc tctacatcct taggggctca gcccgttatg acttctccca tgagatcctt 540 cgggatgaag agtccttctt tggggaacgc cggattcccc ggggccggcg catctccgtg 600 atctgccgct ccctccctga gggcatgggg ccaggggagt ctggacagcc gcccccagcc 660 tgctgacccc cagctttcta cagacaccag atttgtgaat aaagttgggg aatggacagc 720 ctaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 780 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaagg gggggggcgc ccccaaaagg 840 gagggccgcc ccccgcccgg ggaatttttt cgcccggccg ggcccccgcg gga 893

Claims (103)

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:1-24, 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:1-20, c) a polypeptide comprising a naturally occurring amino acid sequence at least 97%
identical to an amino acid sequence selected from the group consisting of SEQ
ID
NO:21-22, d) 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:23-24, e) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1-24, and f) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1-24.

2. An isolated polypeptide of claim 1 comprising an amino acid sequence selected from the group consisting of SEQ ID NO:1-24.

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:25-48.

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:1-24.

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:25-48, 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:25-44 and SEQ ID NO:47-48, c) a polynucleotide comprising a naturally occurring polynucleotide sequence at least 97% identical to a polynucleotide sequence selected from the group consisting of SEQ ID NO:45-46, d) a polynucleotide complementary to a polynucleotide of a), e) a polynucleotide complementary to a polynucleotide of b), f) a polynucleotide complementary to a polynucleotide of c), and g) an RNA equivalent of a)-f).

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:1-24.

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

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

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

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

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

30. A diagnostic test for a condition or disease associated with the expression of SECP 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 Flab')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 SECP in a subject, comprising administering to said subject an effective amount of the composition of claim 32.

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

35. A method of diagnosing a condition or disease associated with the expression of SECP 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:1-24, or an immunogenic fragment thereof, under conditions to elicit an antibody response, b) isolating antibodies from said animal, and c) screening the isolated antibodies with the polypeptide, thereby identifying a polyclonal antibody which binds specifically to a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:1-24.

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:1-24, or an immunogenic fragment thereof, under conditions to elicit an antibody response, b) isolating antibody producing cells from the animal, c) fusing the antibody producing cells with immortalized cells to form monoclonal antibody-producing hybridoma cells, d) culturing the hybridoma cells, and e) isolating from the culture monoclonal antibody which binds specifically to a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:1-24.

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:1-24 in a sample, the method comprising:
a) incubating the antibody of claim 11 with a sample under conditions to allow specific binding of the antibody and the polypeptide, and b) detecting specific binding, wherein specific binding indicates the presence of a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:1-24 in the sample.

45. A method of purifying a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:1-24 from a sample, the method comprising:
a) incubating the antibody of claim 11 with a sample under conditions to allow specific binding of the antibody and the polypeptide, and b) separating the antibody from the sample and obtaining the purified polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID
NO:1-24.

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

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

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

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

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

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

86. A polynucleotide of claim 12, comprising the polynucleotide sequence of SEQ ID
NO:31.

87. A polynucleotide of claim 12, comprising the polynucleotide sequence of SEQ ID
NO:32.

88. A polynucleotide of claim 12, comprising the polynucleotide sequence of SEQ ID
NO:33.

89. A polynucleotide of claim 12, comprising the polynucleotide sequence of SEQ ID
N0:34.

90. A polynucleotide of claim 12, comprising the polynucleotide sequence of SEQ ID
N0:35.

91. A polynucleotide of claim 12, comprising the polynucleotide sequence of SEQ ID
N0:36.

92. A polynucleotide of claim 12, comprising the polynucleotide sequence of SEQ ID
N0:37.

93. A polynucleotide of claim 12, comprising the polynucleotide sequence of SEQ ID
N0:38.

94. A polynucleotide of claim 12, comprising the polynucleotide sequence of SEQ ID
N0:39.

95. A polynucleotide of claim 12, comprising the polynucleotide sequence of SEQ ID
N0:40.

96. A polynucleotide of claim 12, comprising the polynucleotide sequence of SEQ ID
N0:41.

97. A polynucleotide of claim 12, comprising the polynucleotide sequence of SEQ ID
N0:42.

98. A polynucleotide of claim 12, comprising the polynucleotide sequence of SEQ ID
N0:43.

99. A polynucleotide of claim 12, comprising the polynucleotide sequence of SEQ ID
N0:44.

100. A polynucleotide of claim 12, comprising the polynucleotide sequence of SEQ ID
N0:45.

101. A polynucleotide of claim 12, comprising the polynucleotide sequence of SEQ ID
N0:46.

102. A polynucleotide of claim 12, comprising the polynucleotide sequence of SEQ ID
N0:47.

103. A polynucleotide of claim 12, comprising the polynucleotide sequence of SEQ ID
N0:48.