CA2343360A1 - Human cytoskeleton associated proteins - Google Patents

Abstract

The invention provides human cytoskeleton associated proteins (CYSKP) and polynucleotides which identify and encode CYSKP. 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 expression of CYSKP.

Description

HUMAN CYTOSKELETON ASSOCIATED PROTEINS
TECHNICAL FIELD
This invention relates to nucleic acid and amino acid sequences of human cyoskeleton , associated proteins and to the use of these sequences in the diagnosis, treatment, and prevention of cell proliferative, autoimmune/inflammatory. vesicle trafficking, neurological, cell motility, reproductive, and muscle disorders.
BACKGROUND OF THE INVENTION
The cytoskeleton, a cytoplasmic system of protein fibers, mediates cell shape, structure, and movement. The cytoskeleton supports the cell membrane and forms tracks along which organelles and other elements move in the cytosol. The cytoskeleton is a dynamic structure that allows cells to adopt various shapes and to carry out directed movements. Major cytoskeletal fibers are the microfilaments, the microtubules, and the intermediate filaments. Motor proteins, including myosin, dynein, and kinesin, drive movement of, or along, the fibers. The motor protein dynamin drives the formation of membrane vesicles. Accessory or associated proteins modify the structure or activity of the fibers white cytoskeletal membrane anchors connect the fibers to the cell membrane. (The cytoskeleton is reviewed in Lodish, H. et al. ( 1995) Molecular Cell Bioio~v Scientific American Books, New York NY.) Microtubules and Associated Proteins Tubulins Microtubules, cytoskeletal fibers with a diameter of 24 nm, have multiple roles in the cell.
Bundles of microtubuies form cilia and flagella, which are whip-like extensions of the cell membrane that are necessary for sweeping materials across an epithelium and for swimming of sperm, respectively. Marginal bands of microtubules in red blood cells and platelets are important for these cells' pliability. Organelles, membrane vesicles, and proteins are transported in the cell along tracks of microtubules. For example, microtubules run through nerve cell axons, allowing bi-directional transport of materials and membrane vesicles between the cell body and the nerve terminal. Failure to supply the nerve terminal with these vesicles blocks the transmission of neural sienals.
Microtubules, in the form of the spindle, are also critical to chromosomal movement during cell division. Both stable and short-lived populations of microtubules exist in the cell.
Microtubules are a polymer of GTP-binding tubulin protein subunits. Each subunit is a heterodimer of a- and Vii- tubulin, multiple isoforms of which exist. The hydrolysis of GTP is linked to the addition of tubulin subunits at the end of a microtubule. The subunits interact head to tail to form protofilaments; the protofilaments interact side to side to form a microtubule. A microtubule is polarized, one end ringed with a-tubulin and the other with ~i-tubulin, and the two ends differ in their rates of assembly. Generally each microtubule is composed of l3 protofilaments although 11 or IS
protofilament-microtubules are sometimes found. Cilia and flagella contain doublet microtubules.
Microtubules grow from specialized structures known as centrosomes or microtubule-organizing centers (MTOCs). MTOCs may contain one or two centrioles, which are pinwheel arrays of triplet microtubules. The basal body, the organizing center located at the base of a cilium or flagellum, contains one centriole. y- tubulin present in the MTOC is important for nucleating the polymerization of a- and ~- tubuiin heterodimers but does not polymerize into microtubules.
The protein pericentrin is found in the MTOC and has a role in microtubule assembly.
Microtubule-Associated Proteins Microtubule-associated proteins (MAPs) have rotes in the assembly and stabilization of microtubules. One major family of MAPs, assembly MAPS, can be identified in neurons as well as non-neuronal cells. Assembly MAPS are responsible for cross-linking microtubutes in the cytosol.
These MAPS are organized into two domains: a basic microtubule-binding domain and an acidic projection domain. The projection domain is the binding site for membranes, intermediate filaments, or other microtubules. Based on sequence analysis, assembly MAPS can be further grouped into two types: Type I and Type II.
Type I MAPS, which include MAP1A and MAPIB, are large, Blamentous molecules that co-purify with microtubules and are abundantly expressed in brain and testis.
They contain several repeats of a positively-charged amino acid sequence motif that binds and neutralizes negatively charged tubulin, leading to stabilization of microtubules. MAP1A and MAP1B are each derived from a single precursor polypeptide that is subsequently proteolytically processed to generate one heavy chain and one light chain.
Another light chain, LC3, is a I 6.4 kDa molecule that binds MAP I A, MAP 1 B, and microtubules. It is suggested that LC3 is synthesized from a source other than the MAP1A or MAP1B transcripts, and the expression of LC3 may be important in regulating the microtubule binding activity of MAP1 A and MAPI B during cell proliferation (Mann, S. S.
et al. ( 1994) 1. Biol.
Chem.269:11492-11497).
Type II MAPs, which include MAP2a, MAP2b, MAP2c, MAP4, and Tau, are characterized by three to four copies of an 18-residue sequence in the microtubule-binding domain. MAP2a, MAP2b, and MAP2c are found only in dendrites, MAP4 is found in non-neuronal cells, and Tau is found in axons and dendrites of nerve cells. Alternative splicing of the Tau mRNA leads to the existence of multiple forms of Tau protein. Tau phosphorylation is altered in neurodegenerative disorders such as Alzheimer's disease, Pick's disease, progressive supranuclear palsy, corticobasal degeneration, and familial frontotemporal dementia and Parkinsonism linked to chromosome 17. The altered Tau phosphorylation leads to a collapse of the microtubule network and the formation of S intraneuronal Tau aggregates (Spillantini, M.G. and Goedert, M. ( 1998) Trends Neurosci. 21:428-433).
Tektins are filamentous proteins that were originally discovered in association with axonemal microtubules of sea urchin sperm. Subsequent work has shown that tektins are also found in association with spindle microtubules in clams and in mammals. (Steffen, W.
and Linck, R.W.
( 1992) J. Cell Sci. 101:809-822.) Tektins may form rod-like alpha-helical structures similar to those of intermediate filament proteins (Norrander, J.M. et al. (1996) J. Mol. Biol.
29:385-397).
Microtubular aggregates are associated with several disorders. An extraskeletal myxoid chondrosarcoma tumor from human contained parallel arrays of microtubules within the rough endoplasmic reticulum (Suzuki, T. et al. (1988) J. Pathol. 156:51-57).
Microtubular aggregates were also found in hepatocytes from chimpanzees infected with hepatitis C virus.
Monoclonal antibodies prepared to these aggregates detect a protein called p44 (or microtubular aggregates protein) (Maeda, T. et al. (1989) J. Gen. Virol. 70:1401-1407). A human homolog of p44 is inducible by interferon-a and interferon-Vii, but not by interferon-7. p44 protein may be a mediator in the antiviral action of interferon (Kitamura, A. et al. ( 1994) Eur. J. Biochem. 224:877-883).
Dvnein-related Motor Proteins Dyneins are (-) end-directed motor proteins which act on microtubules. Two classes of dyneins exist, cytosolic and axonemai. Cytosolic dyneins are responsible for translocation of materials along cytoplasmic microtubules, for example, transport from the nerve terminal to the cell body and transport of endocytic vesicles to lysosomes. Cytoplasmic dyneins are also reported to play a role in mitosis. Axonemal dyneins are responsible for the beating of flagella and cilia. Dynein on one microtubule doublet walks along the adjacent microtubule doublet. This sliding force produces bending forces that cause the flagellum or cilium to beat. Dyneins have a native mass between 1000 and 2000 kDa and contain either two or three force-producing heads driven by the hydrolysis of ATP.
The heads are linked via stalks to a basal domain which is composed of a highly variable number of accessory intermediate and light chains.
Microfilaments and Associated Proteins Actins Microfilaments, cytoskeletal filaments with a diameter of 7-9 nm, are vital to cell locomotion, cell shape, cell adhesion, cell division, and muscle contraction. Assembly and disassembly of the microfilaments allow cells to change their morphology. Microfilaments are the polymerized form of actin, the most abundant intracellular protein in the eukaryotic cell. Human cells contain six isoforms of actin. The three a-actins are found in different kinds of muscle, nonmuscle ~-actin and nonmuscle y-actin are found in nonmuscle cells, and another y-actin is found in intestinal smooth muscle cells.
G-actin, the monomeric form of actin, polymerizes into polarized, helical F-actin filaments, accompanied by the hydrolysis of ATP to ADP. Actin filaments associate to form bundles and networks, providing a framework to support the plasma membrane and determine cell shape. These bundles and networks are connected to the cell membrane. In muscle cells, thin filaments containing actin slide past thick filaments containing the motor protein myosin during contraction. A family of actin-related proteins exist that are not part of the actin cytoskeleton, but rather associate with microtubules and dynein.
Actin-Associated Proteins Actin-associated proteins have roles in cross-linking, severing, and stabilization of actin filaments and in sequestering actin monomers. Several of the actin-associated proteins have multiple functions. Bundles and networks of actin filaments are held together by actin cross-linking proteins.
These proteins have two actin-binding sites, one for each filament. Short cross-linking proteins promote bundle formation while longer, more flexible cross-linking proteins promote network formation. Calmodulin-like calcium-binding domains in actin cross-linking proteins allow calcium regulation of cross-linking. Group I cross-linking proteins have unique actin-binding domains and include the 30 Kd protein, EF-la, fascin, and scruin. Group II cross-linking proteins have a 7,000-MW actin-binding domain and include villin and dematin. Group III cross-linking proteins have pairs of a 26,000-MW actin-binding domain and include fimbrin, spectrin. dystrophin, ABP 120, and filamin.
Severing proteins regulate the length of actin filaments by breaking them into short pieces or by blocking their ends. Severing proteins include gCAP39, severin (fragmin), gelsolin, and villin.
Capping proteins can cap the ends of actin filaments, but cannot break filaments. Capping proteins include CapZ, tropomodulin, and tensin.
Tensin, which is found in focal adhesions, also crosslinks actin filaments.
Integrin activation by the extracellular matrix leads to the phosphorylation of tensin on tyrosine, serine, and threonine residues; this phosphorylation also occurs in cells transformed with oncogenes. Tensin has an SH2 domain and may bind to other tyrosine-phosphorylated proteins. (Lo, S.H. et al. ( 1997) J. Cell Biol.
136:1349-1361.) The N-terminus of tensin contains a region homologous to the catalytic domain of a putative tyrosine phosphatase (PTP) from Saccharomyces cerevisiae. This PTP
domain in tensin may mediate binding interactions with phosphorylated polypeptides (Haynie, D.T.
and Pouting, C.P.
( 1996) Protein Sci. 5:2643-2646). Mice which lack the tensin gene have kidney abnormalities, indicating that the loss of tensin leads to weakening of focal adhesions in the kidney (Lo, supra).
The proteins thymosin and profilin sequester actin monomers in the cytosol, allowing a pool 5, of unpolymerized actin to exist. Profilin may also stimulate F-actin formation by effectively lowering the critical concentration required for actin monomer addition (Gentler, F.B.
et al. ( 1996) Cell 87:227-239).
The EnaNASP (vasodilator-stimulated phosphoprotein) protein family has roles in actin-based motility. These proteins, including Mena, VASP, and Evl (EnaNASP-like), have homology to the Drosoohila Enabled protein which is involved in neural development.
Mammalian Ena/VASP
proteins localize at focal contacts and in regions where actin filaments are highly dynamic. The neural forms of Mena induce F-actin rich outgrowths in fibroblasts. Mena may have roles in microfilament-based extension of filopodia during axonal growth cone migration. In vitro motility assays of the intracellular pathogenic bacterium Listeria monocvto eg_nes in platelet and brain extracts I S show that the EnaNASP proteins play interchangeable roles in the transformation of actin polymerization into active movement and propulsive force. The EnaNASP proteins associate with actin, profilin, the focal adhesion protein zyxin, and vinculin.
Phosphorylation of Mena and VASP
may regulate their activity. (Gentler, supra; Laurent, V. et al. ( 1999) J.
Cell Biol. 144:1245-1258.) The actin-associated proteins tropomyosin, troponin, and caidesmon regulate muscle contraction in response to calcium. The tropomyosin proteins, found in muscle and nonmuscle cells, are a-helical and form coiled-coil dimers. Striated muscle tropomyosin mediates the interactions between the troponin complex and actin, regulating muscle contraction.
(PROSITE PDOC00290 Tropomyosins signature.) The troponin complex is composed of troponin-T, troponin-I, and troponin-C. Troponin-T binds tropomyosin, linking troponin-I and troponin-C to tropomyosin.
Intermediate Filaments and Associated Proteins Intermediate filaments (IFs) are cytoskeletal fibers with a diameter of 10 nm, intermediate between that of microfilaments and microtubules. They serve structural roles in the cell, reinforcing cells and organizing cells into tissues. IFs are particularly, abundant in epidermal cells and in neurons.
IFs are extremely stable, and, in contrast to microfilaments and microtubules, do not function in cell motility. IF proteins include acidic keratins, basic keratins, desmin, glial fibriilary acidic protein, vimentin, peripherin, neurofiiaments, nestin, and lamins.
IFs have a central a-helical rod region interrupted by short nonhelical linker segments. The rod region is bracketed, in most cases, by non-helical head and tail domains.
The rod regions of intermediate filament proteins associate to form a coiled-coil dimer. A highly ordered assembly process leads from the dimers to the IFs. Neither ATP nor GTP is needed for IF
assembly, unlike that of microfilaments and microtubules.
IF-associated proteins (IFAPs) mediate the interactions of IFs with one another and with 5, other cell structures. IFAPs cross-link IFs into a bundle, into a network, or to the plasma membrane, and may cross-link IFs to the microfilament and microtubule cytoskeleton.
Microtubules and IFs in particular are closely associated. IFAPs include BPAG1, plakoglobin, desmoplakin I, desmoplakin II, plectin, ankyrin, filaggrin, and lamin B receptor.
The N-terminal portion of ankyrin consists of a repeated 33-amino acid motif, the ankyrin repeat, which is involved in specific protein-protein interactions. Variable regions within the motif are responsible for specific protein binding, such that different ankyrin repeats are involved in binding to tubulin, anion exchange protein, voltage-gated sodium channel.
Na'/K'-ATPase, and neurofascin. The ankyrin motif is also found in transcription factors, such as NF-x-B, and in the yeast cell cycle proteins CDC10, SW 14, and SW 16. Proteins involved in tissue differentiation, such as Dros_ophila Notch and C. eleg_ans LIN-12 and GLP-1, also contain ankyrin-like repeats. Lux et al.
(1990; Nature 344:36-42) suggest that ankyrin-like repeats function as 'built-in' ankyrins and form binding sites for integral membrane proteins, tubulin, and other proteins.
Other Cvtoskeleton-Associated Proteins Some cytoskeleton-associated proteins contain a conserved, glycine-rich domain of about 42 residues. This domain, called CAP-Gly, is found in restin, a protein associated with intermediate filaments; vertebrate dynactin, which is associated with dynein; and yeast BIKI protein which may be required for the formation or stabilization of microtubules during mitosis and for spindle pole body fusion during conjugation. (PROSITE PDOC00660 CAP-Gly domain signature.) Proteins of the Erythrocyte Membrane Skeleton Distribution of oxygen throughout the vertebrate body is effected by red blood cells (erythrocytes). Oxygen diffuses from surrounding water or from the atmosphere through either gill epithelium or pulmonary epithelial type I cells. Oxygen then diffuses through the blood capillary endothelium directly to the blood circulatory system and through the erythrocyte membrane and is stored as soluble oxyhemoglobin in the cytoplasm. Oxygen is released from hemoglobin at sites throughout the organism and diffuses out from the erythrocyte to other target cells. The structure of the erythrocyte membrane as well as that of other non-erythrocyte cells must be maintained to enable efficient diffusion of oxygen to intracellular compartments.

The erythrocyte membrane is comprised of i) a cholesterol-rich phospholipid bilayer in which many trans-bilayer proteins are embedded, ii) external glycosylphosphatidylinositol-anchored proteins (GPI-proteins), and iii) the erythrocyte oi' membrane skeleton that laminates the inner surface of the bilayer. The trans-bilayer proteins include anion exchangers, glycophorins. glucose transporters. and a variety of cation transporters and pumps. The erythrocyte GPI-proteins include acetylcholinesterase and decay-accelerating factor (CD 55). The skeletal proteins are organized on the cortical, or cytoplasmic, face of the plasma membrane. These proteins include protein 4.1, protein p55, a- and ~i-spectrin, actin, and actin-binding proteins such as dematin, tropomyosin, and tropomodulin. a- and (3-spectrin combine to form a heterotetramer in vivo. The spectrin heterotetramer organizes into a cortical bidimensional network with a hexagonal mesh. The network is linked to trans-bilayer proteins through a protein complex comprising ~i-spectrin, ankyrin, anion exchanger, and protein 4.2 and through the "triangular" interaction between protein 4. l, glycophorin C, and protein p~5. Structural and functional variants of erythrocyte membrane proteins have been have been found in a variety of tissues. Variants may be transcribed from multigene families, e.g., anion exchanger, ankyrin, or spectrin, or from single gene families, e.g., protein 4.1 or protein 4.2.
mRNA transcripts undergo tissue-specific alternative splicing. Many congenital hemolytic anemias result from mutations in the above-mentioned genes encoding erythrocyte membrane proteins. For example, hereditary elliptocytosis stems from an array of mutations in the spectrin genes at or near the head-to-head self association region of the spectrin tetramer, or from mutations in the protein 4.1 gene which reduce levels of protein 4.1. In another example, hereditary spherocytosis is associated with mutations in the ankyrin gene, the anion exchanger gene, the protein 4.2 gene, or the a- and (3-spectrin genes. (Delaunay J. (1995) Transfus. Clin. Biol. 2:207-216.) Protein 4.1 is an 80 kDa erythrocyte membrane protein with four functional domains. These domains include: i) a 30 kDa basic N-terminal domain, homologous to the ERM
(Ezrin/Radixin/Moesin) family of actin- and transmembrane protein-binding proteins (Tsukita, S. et al. (1997) Trends Biochem. Sci. 22:53-58); ii) a 16 kDa hydrophilic domain containing a protein kinase C phosphorylation site; iii) a 10 kDa highly charged domain containing a cAMP-dependent protein kinase phosphorylation site critical for the interaction with spectrin and actin: and iv) a 22/24 kDa acidic domain. Protein 4.1 is a member of a structurally and functionally related protein 4.1 family. The protein 4.1 family is part of an evolutionarily related protein superfamily that includes many tyrosine phosphatases. (Baklouti, F. et al. ( 1997) Genomics 39:289-302.) In contrast to the strictly cortical localization of protein 4.1 in mature enucleate erythrocytes, protein 4.1 epitopes have been observed throughout the cytoplasmic compartment and the nucleoskeleton in nucleated cells. In particular, protein 4.1 is present in the nucleoskeleton during interphase, in the mitotic spindle during mitosis, in perichromatin during telophase, and in the midbody during cytokinesis. (Krauss, S.W. et al. (1997) J. Cell Biol. 137:275-289.) Differential expression of the protein 4.1 gene resulting in a number of mRNA
splice variants has been observed in various human and rodent tissues. Comparison of the gene structure and mRNA
splice variants revealed the extreme genomic sequence conservation of protein 4.1 between different species. The 5' UTR of both the human and rodent mRNA species has not been successfully identified and sequenced, possibly due to GC-rich regions therein which give rise to technical complications during nucleotide sequencing reactions. (Baklouti, supra;
Conboy, J.G. ( 1988) Proc.
Natl. Acad. Sci. 85:9062-9065.) Analysis of proteins included in the ERM family of proteins has indicated that the N-terminal domain interacts with intracellular domains of transmembrane proteins such as CD44 and the C-terminal domain binds actin. Both interactions involve interactions with Rho-GTP protein complex, polyphosphoinositides, and serine/threonine kinase and tyrosine kinase activities. Many of the phosphorylation sites on ERM proteins are conserved. Although expression of ERM proteins in vivo is restricted to tissues such as endothelium, repression of ERM protein gene expression is released under conditions of cell culture. (Tsukita, s. unra.) The cortical actin cytoskeleton participates in various membrane-based processes which necessitate a large amount of functional plasticity in the molecular components involved. A family of proteins homologous to band 4.1 is involved in the reorganization of the actin cytoskeleton in response to various stimuli and probably piays a role in transmembrane signaling. This family includes tyrosine phosphatases, substrates of tyrosine kinases and a candidate for a tumor-suppressor gene. (Arpin M, et al. (1994) Curr. Opin. Cell Biol. 6:136-141.;) Disruptions in cytoskeletal protein interaction have been identified in a number of disease conditions or disorders. Neurofibromatosis type 2 is an autosomal dominant disease of the nervous system. Schwann cells isolated from patients with neurofibromatosis type 2 have characteristic morphology and growth parameters which differ from control Schwann cells. A
gene associated with neurofibromatosis type 2 has been identified and is termed NF2. The NF2 gene product, known as schwannomin or merlin, is a member of the protein 4.1 superfamily, and mutations in the NF2 gene have been shown to be associated with the disease. (Rosenbaum, C. et al.
(1998) Neurobiol. Dis.
5:55-64.) In addition, a form of psoriasis may be due to altered expression or distribution in epidermal epithelium of analogs of erythrocyte protein 4. I . (Shimizu, T. ( 1996) Histol. Histopathol.
11:495-501.) Erythrocytes carrying mutations in spectrin and protein 4.1 showed differing sensitivities to invasion by Plasmodium falciparum. (Facer, C.A. (1995) Parasitol Res. 81:52-57.) Furthermore, antibodies raised against erythrocyte protein 4.1 stained the majority of neurofibrillary tangles in the prefrontal cortex and hippocampus of brain tissue from patients with Alzheimer's disease. A 68 kDa protein was identified as the most likely brain analog of erythrocyte protein 4.1.
(Sihag, R.K. et al. ( 1994) Brain Res. 656:14-26.) The discovery of new human cytoskeleton associated 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 proliferative, autoimmune/inflammatory, vesicle trafficking, neurological, cell motility, reproductive, and muscle disorders.
SUMMARY OF THE INVENTION
The invention features substantially purified polypeptides, human cytoskeleton associated proteins, referred to collectively as "CYSKP" and individually as "CYSKP-1,"
"CYSKP-2,"
"CYSKP-3," "CYSKP-4," "CYSKP-5," "CYSKP-6," "CYSKP-7," "CYSKP-8," "CYSKP-9,"
"CYSKP-10," "CYSKP-I 1," "CYSKP-12." "CYSKP-13," "CYSKP-14," "CYSKP-15,'' and "CYSKP-16." In one aspect, the invention provides a substantially purified polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:1-16 and fragments thereof.
The invention further provides a substantially purified variant having at least 90% amino acid identity to at least one of the amino acid sequences selected from the group consisting of SEQ ID
NO:1-16 and fragments thereof. The invention also provides an isolated and purified polynucleotide encoding the potypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:1-16 and fragments thereof. The invention also includes an isolated and purified polynucleotide variant having at least 90% polynucleotide sequence identity to the polynucleotide encoding the polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:1-16 and fragments thereof.
Additionally, the invention provides an isolated and purified polynucleotide which hybridizes under stringent conditions to the polynucleotide encoding the polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:1-16 and fragments thereof. The invention also provides an isolated and purified polynucleotide having a sequence which is complementary to the poiynucleotide encoding the polypeptide comprising the amino acid sequence selected from the group consisting of SEQ ID NO:1-I6 and fragments thereof.
The invention also provides a method for detecting a polynucleotide in a sample containing nucleic acids, the method comprising the steps of (a) hybridizing the complement of the polynucleotide sequence to at least one of the polynucleotides of the sample, thereby forming a hybridization complex; and (b) detecting the hybridization complex, wherein the presence of the hybridization complex correlates with the presence of a polynucleotide in the sample. In one aspect, the method further comprises amplifying the polynucleotide prior to hybridization.
The invention also provides an isolated and purified polynucleotide comprising a polynucleotide sequence selected from the group consisting of SEQ ID N0:17-32, and fragments 5, thereof. The invention further provides an isolated and purified polynucleotide variant having at least 90% polynucleotide sequence identity to the polynucleotide sequence selected from the group consisting of SEQ ID N0:17-32 and fragments thereof. The invention also provides an isolated and purified polynucleotide having a sequence which is complementary to the polynucleotide comprising a polynucleotide sequence selected from the group consisting of SEQ ID N0:17-32 and fragments thereof.
The invention further provides an expression vector containing at least a fragment of the polynucleotide encoding the polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: l-16 and fragments thereof. In another aspect, the expression vector is contained within a host cell.
The invention also provides a method for producing a polypeptide, the method comprising the steps of: (a) culturing the host cell containing an expression vector containing at least a fragment of a polynucieotide under conditions suitable for the expression of the polypeptide; and (b) recovering the polypeptide from the host cell culture.
The invention also provides a pharmaceutical composition comprising a substantially purified polypeptide having the amino acid sequence selected from the group consisting of SEQ ID NO: I-16 and fragments thereof, in conjunction with a suitable pharmaceutical carrier.
The invention further includes a purified antibody which binds to a polypeptide selected from the group consisting of SEQ ID NO:I-16 and fragments thereof. The invention also provides a purified agonist and a purified antagonist to the polypeptide.
The invention also provides a method for treating or preventing a disorder associated with decreased expression or activity of CYSKP, the method comprising administering to a subject in need of such treatment an effective amount of a pharmaceutical composition comprising a substantially purified polypeptide having the amino acid sequence selected from the group consisting of SEQ ID
NO:1-16 and fragments thereof, in conjunction with a suitable pharmaceutical carrier.
The invention also provides a method for treating or preventing a disorder associated with increased expression or activity of CYSKP, the method comprising administering to a subject in need of such treatment an effective amount of an antagonist of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1-16 and fragments thereof.

BRIEF DESCRIPTION OF THE TABLES
Table 1 shows polypeptide and nucleotide sequence identification numbers (SEQ
ID NOs), clone identification numbers (clone IDs), cDNA libraries. and cDNA fragments used to assemble full-length sequences encoding CYSKP.
Table 2 shows features of each polypeptide sequence, including potential motifs, homologous sequences, and methods and algorithms used for identification of CYSKP.
Table 3 shows selected fragments of each nucleic acid sequence; the tissue-specific expression patterns of each nucleic acid sequence as determined by northern analysis; diseases, disorders, or conditions associated with these tissues; and the vector into which each cDNA was cloned.
Table 4 describes the tissues used to construct the cDNA libraries from which cDNA clones encoding CYSKP were isolated.
Table 5 shows the tools, programs, and algorithms used to analyze CYSKP, 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
"CYSKP" refers to the amino acid sequences of substantially purified CYSKP
obtained from any species, particularly a mammalian species, including bovine, ovine, porcine, murine, equine, and preferably the human species, from any source, whether natural, synthetic, semi-synthetic, or recombinant.
The term ''agonist" refers to a molecule which, when bound to CYSKP, increases or prolongs the duration of the effect of CYSKP. Agonists may include proteins, nucleic acids, carbohydrates, or any other molecules which bind to and modulate the effect of CYSKP.
An "allelic variant" is an alternative form of the gene encoding CYSKP.
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. Any given natural or recombinant gene may have none, one, or many allelic forms. 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 CYSKP include those sequences with deletions, insertions, or substitutions of different nucleotides, resulting in a polynucleotide the same as CYSKP
or a polypeptide with at least one functional characteristic of CYSKP.
Included within this definition are polymorphisms which may or may not be readily detectable using a particular oligonucleotide probe of the polynucleotide encoding CYSKP, and improper or unexpected hybridization to allelic variants, with a locus other than the normal chromosomal locus for the polynucleotide sequence encoding CYSKP. 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 CYSKP. Deliberate amino acid substitutions may be made on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and/or the amphipathic nature of the residues, as long as the biological or immunological activity of CYSKP is retained. For example, negatively charged amino acids may include aspartic acid and glutamic acid, positively charged amino acids may include lysine and arginine, and amino acids with uncharged polar head groups having similar hydrophilicity values may include leucine, isoleucine, and valine; glycine and alanine;
asparagine and glutamine; serine and threonine; 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. In this context, "fragments," "immunogenic fragments," or "antigenic fragments" refer to fragments of CYSKP which are preferably at least 5 to about 1 S amino acids in length, most preferably at least 14 amino acids, and which retain some biological activity or immunological activity of CYSKP. Where "amino acid sequence" is recited to refer to an amino acid sequence of a naturally occurring protein molecule, "amino acid sequence" and like terms are not meant to limit the amino acid sequence to the complete native amino acid sequence associated with the recited protein molecule.
''Amplification" relates to the production of additional copies of a nucleic acid sequence.
Amplification is generally carried out using polymerase chain reaction (PCR) technologies well known in the art.
The term "antagonist" refers to a molecule which, when bound to CYSKP, decreases the amount or the duration of the effect of the biological or immunological activity of CYSKP.
Antagonists may include proteins, nucleic acids, carbohydrates, antibodies, or any other molecules which decrease the effect of CYSKP. .
The term "antibody' refers to intact molecules as well as to fragments thereof, such as Fab, F(ab')Z, and Fv fragments, which are capable of binding the epitopic determinant. Antibodies that bind CYSKP 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 fragment 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 animah numerous regions of the protein may induce the production of antibodies which bind specifically to antigenic determinants {given 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 "antisense" refers to any composition containing a nucleic acid sequence which is complementary to the "sense" strand of a specific nucleic acid sequence.
Antisense molecules may be produced by any method including synthesis or transcription. Once introduced into a cell, the complementary nucleotides combine with natural sequences produced by the cell to form duplexes and to block either transcription or translation. The designation "negative"
can refer to the antisense strand, and the designation "positive" can refer to the sense strand.
The term "biologically active" refers to a protein having structural, regulatory, or biochemical functions of a naturally occurring molecule. Likewise, "immunologically active" refers to the WO 00/17355 PCTlUS99/21565 capability of the natural, recombinant, or synthetic CYSKP, or of any oligopeptide thereof, to induce a specific immune response in appropriate animals or cells and to bind with specific antibodies.
The terms "complementary" and "complementarity" refer to the natural binding of polynucleotides by base pairing. For example, the sequence "5' A-G-T 3"' bonds to the complementary sequence "3' T-C-A 5'." Complementarity between two single-stranded molecules may be ''partial," such that only some of the nucleic acids bind, or it may be "complete," such that' total complementarity exists between the single stranded molecules. The degree of complementarity between nucleic acid strands has significant effects on the efficiency and strength of the hybridization between the nucleic acid strands. This is of particular importance in amplification reactions, which depend upon binding between nucleic acids strands, and in the design and use of peptide nucleic acid (PNA) molecules.
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 CYSKP or fragments of CYSKP 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 dodecy) sulfate; SDS), and other components (e.g., Denhardt's solution, dry milk, salmon sperm DNA, etc.).
"Consensus sequence" refers to a nucleic acid sequence which has been resequenced to resolve uncalled bases, extended using the XL-PCR kit (Perkin-Elmer, Norwalk CT) in the 5' and/or the 3' direction, and resequenced, or which has been assembled from the overlapping sequences of more than one Incyte Clone using a computer program for fragment assembly, such as the GELVIEW
fragment assembly system (GCG, Madison WI). Some sequences have been both extended and assembled to produce the consensus sequence.
The term "correlates with expression of a polynucleotide" indicates that the detection of the presence of nucleic acids, the same or related to a nucleic acid sequence encoding CYSKP, by northern analysis is indicative of the presence of nucleic acids encoding CYSKP in a sample, and thereby correlates with expression of the transcript from the polynucleotide encoding CYSKP.
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 the chemical modification of a polypeptide sequence, or a polynucieotide sequence. Chemical modifications of a polynucleotide sequence can include, for example, replacemem of hydrogen by an alkyl, acyl, or amino group. A
derivative polynucleotide encodes a poiypeptide 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.
The term "similarity" refers to a degree of complementarity. There may be partial similarity or complete similarity. The word "identity" may substitute for the word "similarity." A partially complementary sequence that at least partially inhibits an identical sequence from hybridizing to a target nucleic acid is referred to as "substantially similar." The inhibition of hybridization of the completely complementary sequence to the target sequence may be examined using a hybridization assay (Southern or northern blot, solution hybridization, and the like) under conditions of reduced stringency. A substantially similar sequence or hybridization probe will compete for and inhibit the binding of a completely similar (identical) sequence to the target sequence under conditions of reduced stringency. This is not to say that conditions of reduced stringency are such that non-specific binding is permitted, as reduced stringency conditions require that the binding of two sequences to one another be a specific (i.e., a selective) interaction. The absence of non-specific binding may be tested by the use of a second target sequence which lacks even a partial degree of complementarity (e.g., less than about 30% similarity or identity). In the absence of non-specific binding, the substantially similar sequence or probe will not hybridize to the second non-complementary target sequence.
The phrases "percent identity" and "% identity" refer to the percentage of sequence similarity found in a comparison of two or more amino acid or nucleic acid sequences.
Percent identity can be determined electronically, e.g., by using the MEGALIGN program (DNASTAR, Madison WI) which creates alignments between two or more sequences according to methods selected by the user, e.g., the clustal method. {See, e.g., Higgins, D.G. and P.M. Sharp ( 1988) Gene 73:237-244.) Parameters for each method may be the default parameters provided by MEGALIGN or may be specified by the user. The clustal algorithm groups sequences into clusters by examining the distances between all pairs. The clusters are aligned pairwise and then in groups. The percentage similarity between two amino acid sequences, e.g., sequence A and sequence B, is calculated by dividing the length of sequence A, minus the number of gap residues in sequence, A, minus the number of gap residues in sequence B, into the sum of the residue matches between sequence A and sequence B, times one hundred. Gaps of low or of no similarity between the two amino acid sequences are not included in determining percentage similarity. Percent identity between nucleic acid sequences can also be counted or calculated by other methods known in the art, e.g., the Jotun Hein method. (See, e.g., Hein, J. (1990) Methods Enzymol. 183:626-645.) Identity between sequences can also be determined by other methods known in the art, e.g., by varying hybridization conditions.
"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 stable mitotic chromosome segregation and maintenance.
The term "humanized antibody" refers to antibody molecules 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 any process by which a strand of nucleic acid binds with a complementary strand through base pairing.
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 R.ot 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 mare amino acid residues or nucleotides, respectively, to the sequence found in the naturally occurring molecule.
"Immune response" can refer to conditions associated with inflammation, trauma, immune disorders, or infectious or genetic disease, etc. These conditions can be characterized by expression of various factors, e.g., cytokines, chemokines, and other signaling molecules, which may affect cellular and systemic defense systems.
The term "microarray" refers to an arrangement of distinct polynucleotides on a substrate.
The terms "element" and "array element" in a microarray context, refer to hybridizable polynucleotides arranged on the surface of a substrate.
The term "modulate" refers to a change in the activity of CYSKP. For example, modulation may cause an increase or a decrease in protein activity, binding characteristics, or any other biological, functional, or immunological properties of CYSKP.
The phrases ''nucleic acid" or "nucleic acid sequence," as used herein, 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. In this context, "fragments" refers to those nucleic acid sequences which comprise a region of unique polynucleotide sequence that specifically identifies SEQ ID N0:17-32, for example, as distinct from any other sequence in the same genome. For example, a fragment of SEQ ID N0:17-32 is useful in hybridization and amplification technologies and in analogous methods that distinguish SEQ ID N0:17-32 from related polynucleotide sequences. A fragment of SEQ ID
N0:17-32 is at least about 15-20 nucleotides in length. The precise length of the fragment of SEQ ID N0:17-32 and the region of SEQ ID N0:17-32 to which the fragment corresponds are routinely determinable by one of ordinary skill in the art based on the intended purpose for the fragment.
In some cases, a fragment, when translated, would produce polypeptides retaining some functional characteristic, e.g., antigenicity, or structural domain characteristic, e.g., ATP-binding site, of the full-length polypeptide.
The teens "operably associated" and "operably linked" refer to functionally related nucleic acid sequences. A promoter is operably associated or operably linked with a coding sequence if the promoter controls the translation of the encoded polypeptide. While operably associated or operably linked nucleic acid sequences can be contiguous and in the same reading frame, certain genetic elements, e.g., repressor genes, are not contiguously linked to the sequence encoding the polypeptide but still bind to operator sequences that control expression of the polypeptide.
The teen "oligonucleotide" refers to a nucleic acid sequence of at least about 6 nucleotides to 60 nucleotides, preferably about 15 to 30 nucleotides, and most preferably about 20 to 25 nucleotides, which can be used in PCR amplification or in a hybridization assay or microarray. "Oligonucleotide"
is substantially equivalent to the teens "amplimer," "primer," "oligomer," and "probe," as these teens are commonly defined in the art.
"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.
The teen "sample" is used in its broadest sense. A sample suspected of containing nucleic acids encoding CYSKP, or fragments thereof, or CYSKP itself, 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 teens "specific binding" and "specifically binding" refer to that interaction between a protein or peptide and an agonist, an antibody, or an antagonist. The interaction is dependent upon the presence of a particular structure of the protein, e.g., the antigenic detenninant or epitope, recognized by the binding molecule. For example, if an antibody is specific for epitope "A," the presence of a polypeptide containing 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 "stringent conditions" refers to conditions which permit hybridization between polynucleotides and the claimed polynucleotides. Stringent conditions can be defined by salt concentration, the concentration of organic solvent, e.g., formamide, temperature, and other conditions well known in the art. In particular, stringency can be increased by reducing the concentration of salt, increasing the concentration of formamide, or raising the hybridization temperature.
The term "substantially purified" refers to nucleic acid or amino acid sequences that are removed from their natural environment and are isolated or separated, and are at least about 60% free, preferably about 75% free, and most preferably about 90% free from other components with which they are naturally associated.
A "substitution" refers to the replacement of one or more amino acids or nucleotides by different amino acids or nucleotides, respectively.
"Substrate" refers to any suitable rigid or semi-rigid support including membranes, filters, chips, slides, wafers, fibers, magnetic or nonmagnetic beads, gels, tubing, plates, polymers, microparticles and capillaries. The substrate can have a variety of surface forms, such as wells, trenches, pins, channels and pores, to which polynucleotides or polypeptides are bound.
"Transformation" describes a process by which exogenous DNA enters and changes 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, 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 "variant'' of CYSKP polypeptides refers to an amino acid sequence that is altered by one or more amino acid residues. The variant may have "conservative" changes, wherein a substituted amino acid has similar structural or chemical properties (e.g., replacement of leucine with isoleucine).
More rarely, a variant may have "nonconservative" changes (e.g., replacement of glycine with tryptophan). Analogous minor variations may also include amino acid deletions or insertions, or both. Guidance in determining which amino acid residues may be substituted, inserted, or deleted without abolishing biological or immunological activity may be found using computer programs well known in the art, for example, LASERGENE software (DNASTAR).

The term "variant," when used in the context of a polynucleotide sequence. may encompass a polynucleotide sequence related to CYSKP. This definition may also include, for example, "allelic"
(as defned above), "splice," "species," or "polymorphic" variants. A splice variant may have significant identity to a reference molecule, but will generally have a greater or lesser number of polynucleotides due to alternate splicing of exons during mRNA processing. The corresponding polypeptide may possess additional functional domains or an absence of domains. Species variants are polynucleotide sequences that vary from one species to another. The resulting polypeptides generally will 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 base. The presence of SNPs may be indicative of, for example, a certain population, a disease state, or a propensity for a disease state.
THE INVENTION
The invention is based on the discovery of new human cytoskeleton associated proteins (CYSKP), the polynucleotides encoding CYSKP, and the use of these compositions for the diagnosis, treatment, or prevention of cell proliferative, autoimmune/inflammatory, vesicle trafficking, neurological, cell motility, reproductive, and muscle disorders.
Table I lists the Incyte clones used to assemble full length nucleotide sequences encoding CYSKP. Columns 1 and 2 show the sequence identification numbers (SEQ ID NOs) of the polypeptide and nucleotide sequences, respectively. Column 3 shows the clone IDs of the Incyte clones in which nucleic acids encoding each CYSKP were identified, and column 4 shows the cDNA
libraries from which these clones were isolated. Column 5 shows Incyte clones and their corresponding cDNA libraries. Clones for which cDNA libraries are not indicated were derived from pooled cDNA libraries. The clones in column 5 were used to assemble the consensus nucleotide sequence of each CYSKP and are useful as fragments in hybridization technologies.
The columns of Table 2 show various properties of each of the polypeptides of the invention:
column 1 references the SEQ ID NO; column 2 shows the number of amino acid residues in each polypeptide; column 3 shows potential phosphorylation sites; column 4 shows potential glycosylation sites; column 5 shows the amino acid residues comprising signature sequences and motifs; column 6 30 shows homologous sequences as identified by BLAST analysis; and column 7 shows analytical methods used to characterize each polypeptide through sequence homology and protein motifs.
The columns of Table 3 show the tissue-specificity and diseases, disorders, or conditions associated with nucleotide sequences encoding CYSKP. The first column of Table 3 lists the nucleotide SEQ ID NOs. Column 2 lists fragments of the nucleotide sequences of column 1. These fragments are useful, for example, in hybridization or amplification technologies to identify SEQ ID
N0:17-32 and to distinguish between SEQ ID N0:17-32 and related polynucleotide sequences. The polypeptides encoded by these fragments are useful, for example, as immunogenic peptides. Column 3 lists tissue categories which express CYSKP as a fraction of total tissues expressing CYSKP.
Column 4 lists diseases, disorders, or conditions associated with those tissues expressing CYSKP as a fraction of total tissues expressing CYSKP. Column 5 lists the vectors used to subclone each cDNA
library.
Of particular note is the expression of SEQ ID N0:31 in nervous tissues and the expression of SEQ ID N0:32 in musculoskeletal tissues.
l0 The columns of Table 4 show descriptions of the tissues used to construct the cDNA libraries from which cDNA clones encoding CYSKP were isolated. Column 1 references the nucleotide SEQ
ID NOs, column 2 shows the cDNA libraries from which these clones were isolated, and column 3 shows the tissue origins and other descriptive information relevant to the cDNA libraries in column 2.
The invention also encompasses CYSKP variants. A preferred CYSKP variant is one which has at least about 80%, more preferably at least about 90%, and most preferably at least about 95%
amino acid sequence identity to the CYSKP amino acid sequence, and which contains at least one functional or structural characteristic of CYSKP.
The invention also encompasses polynucleotides which encode CYSKP. In a particular embodiment, the invention encompasses a polynucleotide sequence comprising a sequence selected from the group consisting of SEQ ID N0:17-32, which encodes CYSKP.
The invention also encompasses a variant of a polynucleotide sequence encoding CYSKP. In particular, such a variant polynucleotide sequence will have at least about 70%, more preferably at least about 90%, and most preferably at least about 95% polynucleotide sequence identity to the polynucleotide sequence encoding CYSKP. A particular aspect of the invention encompasses a variant of a polynucleotide sequence comprising a sequence selected from the group consisting of SEQ ID NO:1?-32 which has at least about 70%, more preferably at least about 90%, and most preferably at least about 95% polynucleotide sequence identity to a nucleic acid sequence selected from the group consisting of SEQ ID NO: i 7-32. Any one of the polynucleotide variants described above can encode an amino acid sequence which contains at least one functional or structural characteristic of CYSKP.
It will be appreciated by those skilled in the art that as a result of the degeneracy of the genetic code, a multitude of polynucleotide sequences encoding CYSKP, 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 WO 00/17355 PCTlUS99/21565 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 CYSKP, and all such variations are to be considered as being specifically disclosed.
Although nucleotide sequences which encode CYSKP and its variants are preferably capable of hybridizing to the nucleotide sequence of the naturally occurring CYSKP
under appropriately selected conditions of stringency, it may be advantageous to produce nucleotide sequences encoding CYSKP 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 l0 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 CYSKP and its derivatives without altering the encoded amino acid sequences include the production of RNA transcripts having more desirable properties, such as a greater half life, than transcripts produced from the naturally occurring sequence.
15 The invention also encompasses production of DNA sequences which encode CYSKP and CYSKP 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 CYSKP or any fragment thereof.
20 Also encompassed by the invention are polynucieotide sequences that are capable of hybridizing to the claimed polynucleotide sequences, and, in particular, to those shown in SEQ ID
NO: l 7-32 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.) For example, stringent salt concentration will ordinarily be less than about 750 mM
25 NaCI and 75 mM trisodium citrate, preferably less than about 500 mM NaCI
and 50 mM trisodium citrate, and most preferably less than about 250 mM NaCI and 25 mM trisodium citrate. Low stringency hybridization can be obtained in the absence of organic solvent, e.g., formamide, while high stringency hybridization can be obtained in the presence of at least about 35% formamide, and most preferably at least about 50% formamide. Stringent temperature conditions will ordinarily 30 include temperatures of at feast about 30°C, more preferably of at least about 37°C, and most preferably of at least about 42°C. Varying additional parameters, such as hybridization time, the concentration of detergent, e.g., sodium dodecyi sulfate (SDS), and the inclusion or exclusion of carrier DNA, are well known to those skilled in the art. Various levels of stringency are accomplished by combining these various conditions as needed. In a preferred embodiment, hybridization will occur at 30°C in 750 mM NaCI, 75 mM trisodium citrate, and 1% SDS. In a more preferred embodiment, hybridization will occur at 37°C in 500 mM NaCI, 50 mM trisodium citrate, 1% SDS, 35% formamide, and 100 ug/ml denatured salmon sperm DNA (ssDNA). In a most preferred embodiment, hybridization will occur at 42°C in 250 mM NaCI, 25 mM trisodium citrate, I% SDS, 50 % formamide, and 200 /cg/ml ssDNA. Useful variations on these conditions will be readily apparent to those skilled in the art.
The washing steps which follow hybridization can also vary in stringency. Wash stringency conditions can be defined by salt concentration and by temperature. As above, wash stringency can be increased by decreasing salt concentration or by increasing temperature.
For example, stringent salt concentration for the wash steps will preferably be less than about 30 mM
NaCI and 3 mM
trisodium citrate, and most preferably less than about 15 mM NaCI and 1.5 mM
trisodium citrate.
Stringent temperature conditions for the wash steps will ordinarily include temperature of at least about 25°C, more preferably of at least about 42°C, and most preferably of at least about 68°C. In a preferred embodiment, wash steps will occur at 25°C in 30 mM NaCI, 3 mM
trisodium citrate, and 0.1% SDS. 1n a more preferred embodiment, wash steps will occur at 42°C
in IS mM NaCI, 1.5 mM
trisodium citrate, and 0.1 % SDS. In a most preferred embodiment, wash steps will occur at 68°C in 15 mM NaCI, 1.5 mM trisodium citrate, and 0.1% SDS. Additional variations on these conditions will be readily apparent to those skilled in the art.
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 (Perkin Elmer), thermostable T7 polymerase (Amersham Pharmacia Biotech, Piscataway NJ), or combinations of polymerases and proofreading exonucleases such as those found in the ELONGASE
amplification system (Life Technologies, Gaithersburg MD). Preferably, sequence preparation is automated with machines such as the MICROLAB 2200 liquid transfer system (Hamilton, Reno NV), PTC200 thermal cycler (MJ Research, Watertown MA) and ABI CATALYST 800 thermal cycler (Perkin-Elmer). Sequencing is then carried out using either the ABI 373 or 377 DNA sequencing system (Perkin-Elmer), 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~v, John Wiley & Sons, New York NY, unit 7.7;
Meyers, R.A. ( 1995) Molecular Bioloev and Biotechnoloev, Wiley VCH, New York NY, pp. 856-853.) The nucleic acid sequences encoding CYSKP 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 unknown S 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. 1:1 I 1-119.) In this method, multiple restriction enzyme digestions and ligations may be used to insert an engineered double-stranded sequence into a region of unknown sequence before performing PCR. Other methods which may be used to retrieve unknown sequences are known in the art. (See, e.g., Parker, J.D. et al. ( 1991 ) Nucleic Acids Res.
19:30SS-306). Additionally, one rnay 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 SO% or more, and to anneal to the template at temperatures of about 68°C to 72°C.
When screening for full-length cDNAs, it is preferable to use libraries that have been size-selected to include larger cDNAs. In addition, random-primed libraries, which often include sequences containing the S' 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 S' non-transcribed regulatory regions.
Capillary electrophoresis systems which are commercially available may be used to analyze the size or confirm the nucleotide sequence of sequencing or PCR products. In particular, capillary sequencing may employ flowable polymers for electrophoretic separation, four different nucleotide-specific, laser-stimulated fluorescent dyes, and a charge coupled device camera for detection of the emitted wavelengths. Output/light intensity may be converted to electrical signal using appropriate software (e.g., GENOTYPER and SEQUENCE NAVIGATOR, Perkin-Elmer), and the entire process from loading of samples to computer analysis and electronic data display may be computer controlled. Capillary electrophoresis is especially preferable for sequencing small DNA fragments which may be present in limited amounts in a particular sample.
In another embodiment of the invention, polynucleotide sequences or fragments thereof which encode CYSKP may be cloned in recombinant DNA molecules that direct expression of CYSKP, 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 CYSKP.
S , The nucleotide sequences of the present invention can be engineered using methods generally known in the art in order to alter CYSKP-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 oiigonucleotides 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.
In another embodiment, sequences encoding CYSKP 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) Nucl. Acids Res. Symp. Ser. 7:215-223, and Horn, T. et al. ( 1980) Nucl. Acids Res. Symp.
Ser. 7:225-232.) Alternatively, CYSKP itself or a fragment thereof may be synthesized using chemical methods. For example, peptide synthesis can be performed using various solid-phase techniques. (See, e.g., Roberge, J.Y. et al. ( 1995) Science 269:202-204.) Automated synthesis may be achieved using the ABI 431A peptide synthesizer (Perkin-Elmer). Additionally, the amino acid sequence of CYSKP, 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.
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, T. ( 1984) Proteins. Structures and Molecular Properties, WH
Freeman, New York NY.) In order to express a biologically active CYSKP, the nucleotide sequences encoding CYSKP
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 CYSKP. Such elements may vary in their strength and specificity. Specific initiation signals may also be used to achieve more efficient translation of sequences encoding CYSKP. Such signals include the ATG initiation codon and adjacent sequences, e.g. the Kozak sequence. In cases where sequences encoding CYSKP 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 welt known to those skilled in the art may be used to construct expression vectors containing sequences encoding CYSKP and appropriate transcriptionai 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 Bioloev, 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 CYSKP. 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. 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 poiynucleotide sequences encoding CYSKP. For example, routine cloning, subcioning, and propagation of polynucleotide sequences encoding CYSKP can be achieved using a multifunctional E. coli vector such as PBLUESCRIPT (Stratagene, La Jolla CA) or pSPORTI
plasmid (Life Technologies). Ligation of sequences encoding CYSKP into the vector's multiple cloning site disrupts the IacZ 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 CYSKP are needed, e.g. for the production of antibodies, vectors which direct high level expression of CYSKP may be used.
For example, vectors containing the strong, inducible TS or T7 bacteriophage promoter may be used.
Yeast expression systems may be used for production of CYSKP. A number of vectors containing constitutive or inducible promoters, such as alpha factor, alcohol oxidase, and PGH
promoters, may be used in the yeast Saccharomvces cerevisiae or Pichia pastoris. In addition, such vectors direct either the secretion or intracellular retention of expressed proteins and enable integration of foreign sequences into the host genome for stable propagation.
(See, e.g., Ausubel, 1995, supra; Grant et al. ( 1987) Methods Enzymol. 153:516-54; and Scorer, C.
A. et al. ( 1994) Bio/Technology 12:181-184.) Plant systems may also be used for expression of CYSKP. Transcription of sequences encoding CYSKP may be driven 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.
6:307-3 I 1 ). 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 Technol~
(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 CYSKP
may be ligated into an adenovirus transcription/translation complex consisting of the late promoter and tripartite leader sequence. Insertion in a non-essential E 1 or E3 region of the viral genome may be used to obtain infective virus which expresses CYSKP in host cells. (See, e.g., Logan, J. and T. Shenk (1984) Proc.
Natl. Acad. Sci. 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, poiycationic 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 CYSKP in cell lines is preferred. For example, sequences encoding CYSKP 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 or apr cells, respectively.
(See, e.g., Wigler, M. et al.
(1977) Cell 11:223-232: Lowy, I. et al. (1980) Cell 22:817-823.) Also, antimetabolite, antibiotic, or herbicide resistance can be used as the basis for selection. For example, dhfr confers resistance to methotrexate; neo confers resistance to the aminoglycosides neomycin and G-418; and als or pat confer resistance to chlorsulfuron and phosphinotricin acetyltransferase, respectively. (See, e.g., Wigler, M. et al. (1980) Proc. Natl. Acad. Sci. 77:3567-3570; Colbere-Garapin, F. et al. (1981) J.
Mol. Biol. 150:1-14.) Additional selectable genes have been described, e.g., lrpB and hisD, which alter cellular requirements for metabolites. (See, e.g., Hartrnan, S.C. and R.C. Mulligan ( 1988) Proc.
Natl. Acad. Sci. 85:8047-8051.) Visible markers, e.g., anthocyanins, green fluorescent proteins (GFP; Clontech),13 glucuronidase and its substrate 13-giucuronide, or luciferase and its substrate luciferin may be used. These markers can be used not only to identify transformants, but also to quantify the amount of transient or stable protein expression attributable to a specific vector system.
(See, e.g., Rhodes, C.A. (1995) Methods Mol. Biol. 55:121-131.) Although the presence/absence of marker gene expression suggests that the gene of interest is also present, the presence and expression of the gene may need to be confirmed. For example, if the sequence encoding CYSKP is inserted within a marker gene sequence, transformed cells containing sequences encoding CYSKP can be identified by the absence of marker gene function. Alternatively, a marker gene can be placed in tandem with a sequence encoding CYSKP 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 CYSKP
and that express CYSKP may be identified by a variety of procedures known to those of skill in the art. These procedures include, but are not limited to, DNA-DNA or DNA-RNA hybridizations, PCR
amplification, and protein bioassay or immunoassay techniques which include membrane, solution, or chip based technologies for the detection and/or quantification of nucleic acid or protein sequences.
Immunological methods for detecting and measuring the expression of CYSICP
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 CYSKP 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 Immunoloay, 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 CYSKP
include oligoiabeling, nick translation, end-labeling, or PCR amplification using a labeled nucleotide.
Alternatively, the sequences encoding CYSKP, 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 poiymerase such as T7, T3, or SP6 and labeled nucleotides. These procedures may be conducted using a variety IS of commercially available kits, such as those provided by Amersham Pharmacia Biotech, Promega (Madison WI), and US Biochemical. Suitable reporter molecules or labels which may be used for ease of detection include radionuclides, enzymes, fluorescent, chemiluminescent, or chromogenic agents, as well as substrates, cofactors, inhibitors, magnetic particles, and the like.
Host cells transformed with nucleotide sequences encoding CYSKP 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 CYSKP may be designed to contain signal sequences which direct secretion of CYSKP 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"
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 CYSKP 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 CYSKP protein containing a heterologous moiety that can be recognized by a commercially available antibody may facilitate the screening of peptide libraries for inhibitors of CYSKP
activity. Heterologous protein and peptide moieties may also facilitate purification of fusion proteins using commercially available affinity matrices. Such moieties include, but are not limited to, glutathione S-transferase (GST), maltose binding protein (MBP), thioredoxin (Trx), calmodulin binding peptide (CBP), 6-His, FLAG, c-myc, and hemagglutinin (HA). GST, MBP, Trx, CBP, and 6-His enable purification of their cognate fusion proteins on immobilized glutathione, maltose, phenylarsine oxide, calmodulin, and metal-chelate resins, respectively. FLAG, c-myc, and hemagglutinin (HA) enable immunoaffinity purification of fusion proteins using commercially available monoclonal and polyclonal antibodies that specifically recognize these epitope tags. A fusion protein may also be engineered to contain a proteolytic cleavage site located between the CYSKP encoding sequence and the heterologous protein sequence, so that CYSKP may be cleaved away from the heterologous moiety following purification.
IS Methods for fusion protein expression and purification are discussed in Ausubel (1995, supra, ch 10).
A variety of commercially available kits may also be used to facilitate expression and purification of fusion proteins.
In a further embodiment of the invention, synthesis of radiolabeled CYSKP may be achieved in vitro using the TNT rabbit reticulocyte lysate or wheat germ extract systems (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, preferably '3S-methionine.
Fragments of CYSKP may be produced not only by recombinant production, but also by direct peptide synthesis using solid-phase techniques. (See, e.g., Creighton, supra, pp. 55-60.) Protein synthesis may be performed by manual techniques or by automation.
Automated synthesis may be achieved, for example, using the ABI 431 A peptide synthesizer (Perkin-Elmer). Various fragments of CYSKP may be synthesized separately and then combined to produce the full length molecule.
THERAPEUTICS
Chemical and structural similarity, e.g., in the context of sequences and motifs, exists between regions of CYSKP and human cytoskeleton associated proteins. In addition, the expression of CYSKP is closely associated with cancer, cell proliferation, inflammation, immune response, musculoskeletal, nervous, reproductive, cardiovascular, and gastrointestinal tissues. Therefore, CYSKP appears to play a role in cell proliferative, autoimmune/inflammatory, vesicle trafficking, neurological, cell motility, reproductive, and muscle disorders. In the treatment of disorders associated with increased CYSKP expression or activity, it is desirable to decrease the expression or activity of CYSKP. In the treatment of disorders associated with decreased CYSKP expression or activity, it is desirable to increase the expression or activity of CYSKP.
Therefore, in one embodiment, CYSKP 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 CYSKP. 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, cancers of the adrenal gland, bladder, bone, bone marrow, brain, breast, cervix, gall bladder, ganglia, gastrointestinal tract, heart, kidney, liver, lung, muscle, ovary, pancreas, parathyroid, penis, prostate, salivary glands, skin, spleen, testis, thymus, thyroid, and uterus; an autoimmune/inflammatory IS disorder such as acquired immunodeficiency syndrome (AIDS), actinic keratosis, Addison's disease, adult respiratory distress syndrome, allergies, ankylosing spondylitis, amyloidosis, anemia, arteriosclerosis, asthma, atherosclerosis, autoimmune hemolytic anemia, autoimmune thyroiditis, autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy {APECED), bronchitis, bursitis, cholecystitis, cirrhosis, 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, paroxysmal nocturnal hemoglobinuria, hepatitis, hypereosinophilia, irritable bowel syndrome, mixed connective tissue disease (MCTD), myelofibrosis, multiple sclerosis, myasthenia gravis, myocardial or pericardial inflammation, osteoarthritis, osteoporosis, pancreatitis, polycythemia vera, polymyositis, primary thrombocythemia, 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, protozoai, and helminthic infections, and trauma; a vesicle trafficking disorder such as cystic fibrosis, glucose-galactose malabsorption syndrome, hypercholesterolemia, diabetes mellitus, diabetes insipidus, hyper- and hypoglycemia, Grave's disease, goiter, Cushing's disease, and Addison's disease, gastrointestinal disorders including ulcerative colitis, gastric and duodenal ulcers, other conditions associated with abnormal vesicle trafficking, including acquired immunodeficiency syndrome (AIDS), allergies including hay fever, asthma, and urticaria (hives), autoimmune hemolytic anemia, proliferative glomerulonephritis, inflammatory bowel disease, multiple sclerosis, myasthenia gravis, rheumatoid and osteoarthritis, scleroderma, Chediak-Higashi and Sjogren's syndromes, systemic lupus erythematosus, toxic shock syndrome, traumatic tissue damage, and viral, bacterial, fungal, helminthic, and protoxoal infections; a neurological disorder such as epilepsy, ischemic cerebrovascular disease, stroke, cerebral neoplasms, Alzheimer's disease, Pick's disease, Huntington's disease, dementia, Parkinson's disease and other extrapyramidal disorders, amyotrophic lateral sclerosis and other motor neuron disorders, progressive neural muscular atrophy, retinitis pigmentosa, hereditary ataxias, multiple sclerosis and other demyelinating diseases, bacterial and viral meningitis, brain abscess, subdural empyema, epidural abscess, suppurative intracranial thrombophlebitis, myelitis and radiculitis, viral central nervous system disease, prion diseases including kuru, Creutzfeldt-Jakob disease, and Gerstmann-Straussler-Scheinker syndrome, fatal familial insomnia, nutritional and metabolic diseases of the nervous system, neurofibromatosis, tuberous sclerosis, cerebelloretinal hemangioblastomatosis, encephalotrigeminal syndrome, mental retardation and other developmental disorders of the central nervous system, 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; a cell motility disorder such as ankylosing spondylitis, Chediak-Higashi syndrome, Duchenne and Becker muscular dystrophy, intrahepatic cholestasis, myocardial hyperplasia, cardiomyopathy, early onset peridontitis, cancers such as adenocarcinoma, ovarian carcinoma, and chronic myelogenous leukemia, and bacterial and helminthic infections; and a heart and skeletal muscle disorder such as cardiomyopathy, myocarditis, Duchenne's muscular dystrophy, Becker's muscular dystrophy, myotonic dystrophy, central core disease, nemaline myopathy, centronuclear myopathy, lipid myopathy, mitochondria) myopathy, infectious myositis, polymyositis, dermatomyositis, inclusion body myositis, thyrotoxic myopathy, and ethanol myopathy.
In another embodiment, a vector capable of expressing CYSKP 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 CYSKP including, but not limited to, those described above.
In a further embodiment, a pharmaceutical composition comprising a substantially purified CYSKP 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 CYSKP including, but not limited to, those provided above.
In still another embodiment, an agonist which modulates the activity of CYSKP
may be administered to a subject to treat or prevent a disorder associated with decreased expression or activity of CYSKP including, but not limited to, those listed above.
In a further embodiment, an antagonist of CYSKP may be administered to a subject to treat or prevent a disorder associated with increased expression or activity of CYSKP.
Examples of such disorders include, but are not limited to, those cell proliferative, autoimmune/inflammatory, vesicle trafficking, neurological, cell motility, and heart and skeletal muscle disorders described above; a reproductive disorder such as a disorder of prolactin production, infertility, including tubal disease, ovulatory defects, and endometriosis, a disruption of the estrous cycle, a disruption of the menstrual cycle, polycystic ovary syndrome, ovarian hyperstimulation syndrome, endometrial and ovarian tumors, uterine fibroids, autoimmune disorders, ectopic pregnancies, and teratogenesis, cancer of the breast, fibrocystic breast disease, and galactorrhea, a disruption of spermatogenesis, abnormal sperm physiology, cancer of the testis, cancer of the prostate, benign prostatic hyperplasia, prostatitis, Peyronie's disease, impotence, carcinoma of the male breast, and gynecomastia;
and a smooth muscle disorder. A smooth muscle disorder is defined as any impairment or alteration in the normal action of smooth muscle and may include, but is not limited to, angina, anaphylactic shock, arrhythmias, asthma, cardiovascular shock, Cushing's syndrome, hypertension, hypoglycemia, myocardial infarction, migraine, and pheochromocytoma, and myopathies including cardiomyopathy, encephalopathy, epilepsy, Kearns-Sayre syndrome, lactic acidosis, myoclonic disorder, and ophthalmoplegia. Smooth muscle includes, but is not limited to, that of the blood vessels, gastrointestinal tract, heart, and uterus. In one aspect, an antibody which specifically binds CYSKP
may be used directly as an antagonist or indirectly as a targeting or delivery mechanism for bringing a pharmaceutical agent to cells or tissue which express CYSKP.
In an additional embodiment, a vector expressing the complement of the polynucleotide encoding CYSKP may be administered to a subject to treat or prevent a disorder associated with increased expression or activity of CYSKP 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 CYSKP may be produced using methods which are generally known in the art. In particular, purified CYSKP may be used to produce antibodies or to screen libraries of pharmaceutical agents to identify those which specifically bind CYSKP.
Antibodies to CYSKP 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 especially preferred for therapeutic use.
For the production of antibodies, various hosts including goats, rabbits, rats, mice, humans, and others may be immunized by injection with CYSKP 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 Cor~nebacterium parvum are especially preferable.
It is preferred that the oligopeptides, peptides, or fragments used to induce antibodies to CYSKP have an amino acid sequence consisting of at least about 5 amino acids, and, more preferably, of at least about 10 amino acids. It is also preferable that these ofigopeptides, peptides, or fragments are identical to a portion of the amino acid sequence of the natural protein and contain the entire amino acid sequence of a smail, naturally occurring molecule. Short stretches of CYSKP
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 CYSKP 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.
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. 81:6851-6855; Neuberger, M.S. et al. ( 1984) Nature 312:604-608; and Takeda, S. et ai. ( 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 CYSKP-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. 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. 86: 3833-3837;
Winter, G. et al. (1991) Nature 349:293-299.) Antibody fragments which contain specific binding sites for CYSKP may also be generated.
For example, such fragments include, but are not limited to, F(ab')2 fragments produced by pepsin digestion of the antibody molecule and Fab fragments generated by reducing the disulfide bridges of the F(ab')2 fragments. Alternatively, Fab expression libraries may be constructed to allow rapid and easy identification of monoclonal Fab fragments with the desired specificity.
(See, e.g., Huse, W.D.
et al. (1989) Science 246:1275-1281.) Various immunoassays may be used for screening to identify antibodies having the desired specificity. 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 CYSKP and its specific antibody. A two-site, monoclonal-based immunoassay utilizing monoclonal antibodies reactive to two non-interfering CYSKP epitopes is preferred, but a competitive binding assay may also be employed (Pound, supra).
Various methods such as Scatchard analysis in conjunction with radioimmunoassay techniques may be used to assess the affinity of antibodies for CYSKP.
Affinity is expressed as an association constant, Ke, which is defined as the molar concentration of CYSKP-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 CYSKP epitopes, represents the average affinity, or avidity, of the antibodies for CYSKP. The Ka determined for a preparation of monoclonal antibodies, which are monospecific for a particular CYSKP epitope, represents a true measure of affinity. High-affinity antibody preparations with Ke ranging from about 109 to 10'2 L/mole are preferred for use in immunoassays in which the CYSKP-antibody complex must withstand rigorous manipulations. Low-affinity antibody preparations with Ke ranging from about 106 to 10' L/mole are preferred for use in immunopurification and similar procedures which ultimately require dissociation of CYSKP, preferably in active form, from the antibody (Catty, D. (1988) Antibodies.
Volume 1: A Practical Approach, IRL Press, Washington, DC; Liddell, J. E. and Cryer, A. (1991) A
Practical Guide to Monoclonal Antibodies, John Wiley & Sons, New York NY).
The titer and avidity of poiyclonal 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 I-2 mg specific antibody/ml, preferably S-10 mg specific antibody/ml, is preferred for use in procedures requiring precipitation of CYSKP-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 CYSKP, or any fragment or complement thereof, may be used for therapeutic purposes. In one aspect, the complement of the polynucleotide encoding CYSKP may be used in situations in which it would be desirable to block the transcription of the mRNA. In particular, cells may be transformed with sequences complementary to polynucleotides encoding CYSKP. Thus, complementary molecules or fragments may be used to modulate CYSKP activity, or to achieve regulation of gene function. Such technology is now well known in the art, and sense or antisense oligonucleotides or larger fragments can be designed from various locations along the coding or control regions of sequences encoding CYSKP.
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. Methods which are well known to those skilled in the art can be used to construct vectors to express nucleic acid sequences complementary to the polynucleotides encoding CYSKP. (See, e.g., Sambrook, supra; Ausubel, 1995, supra.) Genes encoding CYSKP can be turned off by transforming a cell or tissue with expression vectors which express high levels of a polynucleotide, or fragment thereof, encoding CYSKP. Such constructs may be used to introduce untranslatable sense or antisense sequences into a cell. Even in the absence of integration into the DNA, such vectors may continue to transcribe RNA molecules until they are disabled by endogenous nucleases. Transient expression may last for a month or more with a non-replicating vector, and may last even longer if appropriate replication elements are part of the vector system.
As mentioned above, modifications of gene expression can be obtained by designing complementary sequences or antisense molecules (DNA, RNA, or PNA) to the control, S', or regulatory regions of the gene encoding CYSKP. Oligonucleotides derived from the transcription initiation site. e.g., between about positions -10 and +10 from the start site, are preferred. 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.1. Can, Molecular and Immunolo-y'c 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 riboryme action involves sequence-specific hybridization of the ribozyme molecule to complementary target RNA, followed by endonucleolytic cleavage.
For example, engineered hammerhead motif ribozyme molecules may specifically and efficiently catalyze endonucieolytic cleavage of sequences encoding CYSKP.
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 ribonuciease 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 CYSKP. Such DNA sequences may be incorporated into a wide variety of vectors with suitable RNA polymerase promoters such as T7 or SP6.
Alternatively, these eDNA
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 phosphodiesierase 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.

WO 00/17355 PCT/US99/2t565 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) Nature Biotechnology 15:462-466.) Any of the therapeutic methods described above may be applied to any subject in need of such therapy, including, for example, mammals such as dogs, cats, cows, horses, rabbits, monkeys, and most preferably, humans.
An additional embodiment of the invention relates to the administration of a pharmaceutical or sterile composition, in conjunction with a pharmaceutically acceptable carrier, for any of the therapeutic effects discussed above. Such pharmaceutical compositions may consist of CYSKP, antibodies to CYSKP, and mimetics, agonists, antagonists, or inhibitors of CYSKP. The compositions may be administered alone or in combination with at least one other agent, such as a stabilizing compound, which may be administered in any sterile, biocompatible pharmaceutical carrier including, but not limited to, saline, buffered saline, dextrose, and water. The compositions may be administered to a patient alone, or in combination with other agents, drugs, or hormones.
The pharmaceutical compositions utilized in this invention may be administered by any number of routes including, but not limited to, oral, intravenous, intramuscular, infra-arterial, intramedullary, intrathecal, intraventricular, transdermal, subcutaneous, intraperitoneal, intranasal, enteral, topical, sublingual, or rectal means.
In addition to the active ingredients, these pharmaceutical compositions may contain suitable pharmaceutically-acceptable carriers comprising excipients and auxiliaries which facilitate processing of the active compounds into preparations which can be used pharmaceutically.
Further details on techniques for formulation and administration may be found in the latest edition of Remin t~s Pharmaceutical Sciences (Maack Publishing, Easton PA).
Pharmaceutical compositions for oral administration can be formulated using pharmaceutically acceptable carriers well known in the art in dosages suitable for oral administration.
Such carriers enable the pharmaceutical compositions to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions, and the like, for ingestion by the patient.
Pharmaceutical preparations for oral use can be obtained through combining active compounds with solid excipient and processing the resultant mixture of granules (optionally, after grinding) to obtain tablets or dragee cores. Suitable auxiliaries can be added, if desired. Suitable excipients include carbohydrate or protein fillers, such as sugars, including lactose, sucrose, mannitol, and sorbitol; starch from corn, wheat, rice, potato, or other plants;
cellulose, such as methyl cellulose, hydroxypropylmethyl-cellulose, or sodium carboxymethylcellulose; gums, including arabic and tragacanth; and proteins, such as gelatin and collagen. If desired, disintegrating or solubilizing agents may be added, such as the cross-linked polyvinyl pyrrolidone, agar, and alginic acid or a salt thereof, such as sodium alginate.
Dragee cores may be used in conjunction with suitable coatings, such as concentrated sugar solutions, which may also contain gum arabic, talc, polyvinylpyrrolidone, carbopol gel, polyethylene glycol, and/or titanium dioxide, lacquer solutions, and suitable organic solvents or solvent mixtures.
Dyestuffs or pigments may be added to the tablets or dragee coatings for product identification or to characterize the quantity of active compound, i.e., dosage.
Pharmaceutical preparations which can be used orally include push-fit capsules made of gelatin. as well as soft, sealed capsules made of gelatin and a coating, such as glycerol or sorbitol.
Push-fit capsules can contain active ingredients mixed with fillers or binders, such as lactose or starches, lubricants, such as talc or magnesium stearate, and, optionally, stabilizers. In soft capsules, the active compounds may be dissolved or suspended in suitable liquids, such as fatty oils, liquid, or liquid polyethylene glycol with or without stabilizers.
Pharmaceutical formulations suitable for parenteral administration may be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hanks' solution, Ringer's solution, or physiologically buffered saline. Aqueous injection suspensions may contain substances which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran. Additionally, suspensions of the active compounds may be prepared as appropriate oily injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils, such as sesame oil, or synthetic fatty acid esters, such as ethyl oieate, triglycerides, or liposomes. Non-lipid polycationic amino polymers may also be used for delivery. Optionally, the suspension may also contain suitable stabilizers or agents to increase the solubility of the compounds and allow for the preparation of highly concentrated solutions.
For topical or nasal administration, penetrants appropriate to the particular barrier to be permeated are used in the formulation. Such penetrants are generally known in the art.
The pharmaceutical compositions of the present invention may be manufactured in a manner that is known in the art, e.g., by means of conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping, or lyophilizing processes.
The pharmaceutical composition may be provided as a salt and can be formed with many acids, including but not limited to, hydrochloric, sulfuric, acetic, lactic, tartaric, malic, and succinic acids. Salts tend to be more soluble in aqueous or other protonic solvents than are the corresponding free base forms. In other cases, the preferred preparation may be a lyophilized powder which may contain any or all ofthe following: 1 mM to SO mM histidine, 0.1% to 2%
sucrose. and 2% to 7%
mannitol, at a pH range of 4.5 to 5.5, that is combined with buffer prior to use.
After pharmaceutical compositions have been prepared, they can be placed in an appropriate container and labeled for treatment of an indicated condition. F'or administration of CYSKP, such labeling would include amount, frequency, and method of administration.
Pharmaceutical 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.
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, 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 CYSKP or fragments thereof, antibodies of CYSKP, and agonists, antagonists or inhibitors of CYSKP, 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 EDS° (the dose therapeutically effective in SO%
of the population) or LDS° (the dose lethal to 50% of the population) statistics. The dose ratio of toxic to therapeutic effects is the therapeutic index, which can be expressed as the LDS°/ED,°
ratio. Pharmaceutical 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 EDs° 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 pharmaceutical 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.

WO 00/17355 PCT/US99/215b5 Normal dosage amounts may vary from about 0.1 ~cg to 100,000 beg, 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 CYSKP may be used for the diagnosis of disorders characterized by expression of CYSKP, or in assays to monitor patients being treated with CYSKP or agonists, antagonists, or inhibitors of CYSKP.
Antibodies useful for diagnostic purposes may be prepared in the same manner as described above for therapeutics.
Diagnostic assays for CYSKP include methods which utilize the antibody and a label to detect CYSKP in human body fluids or in extracts of cells or tissues. 'The antibodies may be used with or without modification, and may be labeled by covalent or non-covalent attachment of a reporter molecule. A wide variety of reporter molecules, several of which are described above, are known in the art and may be used.
A variety of protocols for measuring CYSKP, including ELISAs, RIAs, and FACS, are known in the art and provide a basis for diagnosing altered or abnormal levels of CYSKP expression.
Normal or standard values for CYSKP expression are established by combining body fluids or cell extracts taken from normal mammalian subjects, preferably human, with antibody to CYSKP under conditions suitable for complex formation. The amount of standard complex formation may be quantitated by various methods, preferably by photometric means. Quantities of CYSKP 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 poiynucleotides encoding CYSKP 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 quantitate gene expression in biopsied tissues in which expression of CYSKP may be correlated with disease. The diagnostic assay may be used to determine absence, presence, and excess expression of CYSKP, and to monitor regulation of CYSKP levels during therapeutic intervention.
In one aspect, hybridization with PCR probes which are capable of detecting polynucleotide sequences, including genomic sequences, encoding CYSKP or closely related molecules may be used to identify nucleic acid sequences which encode CYSKP. 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 (maximal, high, intermediate. or low), will determine whether the probe identifies only naturally occurring sequences encoding CYSKP, allelic variants, or related sequences.
Probes may also be used for the detection of related sequences, and should preferably have at least 50% sequence identity to any of the CYSKP encoding sequences. The hybridization probes of the subject invention may be DNA or RNA and may be derived from the sequence of SEQ ID N0:17-32 or from genomic sequences including promoters, enhancers, and introns of the CYSKP gene.
Means for producing specific hybridization probes for DNAs encoding CYSKP
include the cloning of polynucleotide sequences encoding CYSKP or CYSKP 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 3zP or'SS, or by enzymatic labels, such as alkaline phosphatase coupled to the probe via avidin/biotin coupling systems, and the like.
Polynucleotide sequences encoding CYSKP may be used for the diagnosis of disorders associated with expression of CYSKP. 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 adenocarcinorna, leukemia, lymphoma, melanoma, myeloma, sarcoma, teratocarcinoma, and, in particular, cancers of the adrenal gland, bladder, bone, bone marrow, brain, breast, cervix, gall bladder, ganglia, gastrointestinal tract, heart, kidney, liver, lung, muscle, ovary, pancreas, parathyroid, penis, prostate, salivary glands, skin, spleen. testis, thymus, thyroid, and uterus; an autoimmune/inflammatory disorder such as acquired immunodeficiency syndrome (AIDS), actinic keratosis, Addison's disease, adult respiratory distress syndrome, allergies, ankylosing spondylitis, amyloidosis, anemia, arteriosclerosis, asthma, atherosclerosis, autoimmune hemolytic anemia, autoimmune thyroiditis, autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy {APECED), bronchitis, bursitis, cholecystitis, cirrhosis, 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, paroxysmal nocturnal hemoglobinuria, hepatitis, hypereosinophilia, irritable bowel syndrome, mixed connective tissue disease (MCTD), myelofibrosis, multiple sclerosis, myasthenia gravis, myocardial or pericardial inflammation, osteoarthritis, osteoporosis, pancreatitis, polycythemia vera, polymyositis, primary thrombocythemia, 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 extracorporeai circulation, viral, bacterial, fungal, parasitic, protozoal, and helminthic infections, and trauma; a vesicle trafficking disorder such as cystic fibrosis, glucose-galactose malabsorption syndrome, hypercholesterolemia, diabetes mellitus, diabetes insipidus, hyper-and hypoglycemia, Grave's disease, goiter, Cushing's disease, and Addison's disease, gastrointestinal disorders including ulcerative colitis, gastric and duodenal ulcers, other conditions associated with abnormal vesicle trafficking, including acquired immunodeficiency syndrome (AIDS), allergies including hay fever, asthma, and urticaria (hives), autoimmune hemolytic anemia, proliferative glomerulonephritis, inflammatory bowel disease, multiple sclerosis, myasthenia gravis, rheumatoid and osteoarthritis, scleroderma, Chediak-Higashi and Sjogren's syndromes, systemic lupus erythematosus, toxic shock syndrome, traumatic tissue damage, and viral, bacterial, fungal, helminthic, and protozoal infections;
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, Creutzfeidt-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, cerebral palsy, neuroskeletai 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 supranuciear palsy, corticobasal degeneration, and familial frontotemporal dementia; a cell motility disorder such as ankylosing spondylitis, Chediak-Higashi syndrome, Duchenne and Becker muscular dystrophy, intrahepatic cholestasis, myocardial hyperplasia, cardiomyopathy, early onset peridontitis, cancers such as adenocarcinoma, ovarian carcinoma, and chronic myelogenous leukemia, and bacterial and helminthic infections; a heart and skeletal muscle disorder such as cardiomyopathy, myocarditis, Duchenne's muscular dystrophy, Becker's muscular dystrophy, myotonic dystrophy, central core disease, nemaline myopathy, centronuclear myopathy, lipid myopathy, mitochondria) myopathy, infectious myositis, polymyositis, dermatomyositis, inclusion body myositis, thyrotoxic myopathy, and ethanol myopathy; a reproductive disorder such as a disorder of prolactin production, infertility, including tuba) disease, ovulatory defects, and endometriosis, a disruption of the estrous cycle, a disruption of the menstrual cycle, poiycystic ovary syndrome, ovarian hyperstimulation syndrome, endometrial and ovarian tumors, uterine fibroids, autoimmune disorders, ectopic pregnancies, and teratogenesis; cancer of the breast, fibrocystic breast disease, and galactorrhea, a disruption of spermatogenesis, abnormal sperm physiology, cancer of the testis, cancer of the prostate, benign prostatic hyperplasia, prostatitis, Peyronie's disease, impotence, carcinoma of the male breast, and gynecomastia;
and a smooth muscle disorder. A smooth muscle disorder is defined as any impairment or alteration in the normal action of smooth muscle and may include, but is not limited to, angina, anaphylactic shock, arrhythmias, asthma, cardiovascular shock, Cushing's syndrome, hypertension, hypoglycemia, myocardial infarction, migraine, and pheochromocytoma, and myopathies including cardiomyopathy, encephalopathy, epilepsy, Kearns-Sayre syndrome, lactic acidosis, myoclonic disorder, and ophthalmoplegia. Smooth muscle includes, but is not limited to, that of the blood vessels, gastrointestinal tract, heart, and uterus. The polynucleotide sequences encoding CYSKP 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 CYSKP expression. Such qualitative or quantitative methods are well known in the art.
In a particular aspect, the nucleotide sequences encoding CYSKP may be useful in assays that detect the presence of associated disorders, particularly those mentioned above. The nucleotide sequences encoding CYSKP 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 quantitated 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 CYSKP 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 CYSKP, 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 CYSKP, under conditions suitable for hybridization or amplification. Standard hybridization may be quantified by comparing the values obtained from normal subjects with values from an experiment in which a known amount of a substantially purified polynucleotide is used. Standard values obtained in this manner may be compared with values obtained from samples from patients who are symptomatic for a disorder.
Deviation from standard values is used to establish the presence of a disorder.
Once the presence of a disorder is established and a treatment protocol is initiated, hybridization assays may be repeated on a regular basis to determine if the level of expression in the patient begins to approximate that which is observed in the normal subject.
The results obtained from successive assays may be used to show the efficacy of treatment over a period ranging from several days to months.
With respect to cancer, the presence of an abnormal amount of transcript (either under- or overexpressed) in biopsied tissue from an individual may indicate a predisposition for the I S 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 CYSKP may involve the use of PCR. These oligomers may be chemically synthesized, generated enzymatically, or produced in vitro. Olieomers will preferably contain a fragment of a polynucleotide encoding CYSKP, or a fragment of a polynucleotide complementary to the polynucleotide encoding CYSKP, 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 quantitation of closely related DNA or RNA sequences.
Methods which may also be used to quantify the expression of CYSKP 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 al. ( 1993) Anal. Biochem. 212:229-236.) The speed of quantitation of multiple samples may be accelerated by running the assay in an ELISA format where the oligomer of interest is presented in various dilutions and a spectrophotometric or colorimetric response gives rapid quantitation.
In further embodiments, oligonucleotides or longer fragments derived from any of the polynucleotide sequences described herein may be used as targets in a microarray. The microarray can be used to monitor the expression level of large numbers of genes simultaneously and 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, and to develop and monitor the activities of therapeutic agents.
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.
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. 94:2150-2155;
and Heller, M.J. et al. (1997) U.S. Patent No. 5,605,662.) In another embodiment of the invention, nucleic acid sequences encoding CYSKP
may be used to generate hybridization probes useful in mapping the naturally occurring genomic sequence.
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 ai. ( 1997) Nat Genet. 15:345-355; Price, C.M. (1993) Blood Rev. 7:127-134; and Trask, B.J. (1991) Trends Genet. 7:149-154.) Fluorescent in situ hybridization (FISH) may be correlated with other physical chromosome mapping techniques and genetic map data. (See, e.g., Heinz-Ulrich, et al.
(1995) in Meyers, supra, pp. 965-968.) Examples of genetic map data can be found in various scientific journals or at the Online Mendelian Inheritance in Man (OMIM) site. Correlation between the location of the gene encoding CYSKP on a physical chromosomal map and a specific disorder, or a predisposition to a specific disorder, may help define the region of DNA associated with that disorder. The nucleotide sequences of the invention may be used to detect differences in gene sequences among normal, carrier, and affected individuals.
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 number or arm of a particular human chromosome is not known. New sequences can be assigned to chromosomal arms by physical mapping.
This provides valuable information to investigators searching for disease genes using positional cloning or other gene discovery techniques. Once the disease or syndrome has been crudely localized by genetic linkage to a particular genomic region, e.g., ataxia-telangiectasia to 11 q22-23, 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 subject 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, CYSKP, 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 CYSKP 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.) 1n this method, large numbers of different small test compounds are synthesized on a solid substrate. The test compounds are reacted with CYSKP, or fragments thereof, and washed. Bound CYSKP is then detected by methods well known in the art.
Purified CYSKP 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 CYSKP specifically compete with a test compound for binding CYSKP. In this manner, antibodies can be used to detect the presence of any peptide which shares one or more antigenic determinants with CYSKP.
In additional embodiments, the nucleotide sequences which encode CYSKP may be used in any molecular biology techniques that have yet to be developed, provided the new techniques rely on properties of nucleotide sequences that are currently known, including, but not limited to, such properties as the triplet genetic code and specific base pair interactions.
Without further elaboration, it is believed that one skilled in the art can, using the preceding description, utilize the present invention to its fullest extent. The following preferred specific embodiments are, therefore, to be construed as merely illustrative, and not limitative of the remainder of the disclosure in any way whatsoever.
The disclosures of all patents, applications, and publications mentioned above and below, in particular U.S. Ser. No. 60/131,321, and [Atty Docket No. PF-0594 P, filed September 18, 1998] are hereby expressly incorporated by reference.

EXAMPLES
I. Construction of cDNA Libraries RNA was purchased from Clontech or isolated from tissues described in Table 4.
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
l0 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).
IS 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., Ausubet, 1997, supra, units S. I-6.6.) Reverse transcription was initiated using oligo d(T) or random primers. Synthetic 20 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., 25 PBLUESCRIPT plasmid (Stratagene), pSPORTI plasmid (Life Technologies), or pINCY (Incyte Pharmaceuticals, Palo Alto CA). Recombinant plasmids were transformed into competent E. coli cells including XLI-Blue, XL1-BIueMRF, or SOLR from Stratagene or DHSa, DHIOB, or ElectroMAX DHIOB from Life Technologies.
30 II. Isolation of cDNA Clones Plasmids were recovered from host cells by in vivo excision using the UNIZAP
vector system (Stratagene) or by cell lysis. Piasmids 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, Ultra Plasmid purification systems or the R.E.A.L. PREP 96 piasmid purification kit from QIAGEN.
Following precipitation, piasmids 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 PCB 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 fluorometricaily using PICOGREEN dye (Molecular Probes, Eugene OR) and a FLUOROSKAN II
fluorescence scanner (Labsystems Oy, Helsinki, Finland).
III. Sequencing and Analysis eDNA sequencing reactions were processed using standard methods or high-throughput instrumentation such as the ABI CATALYST 800 (Perkin-Eimer) thermal cycler or the PTC-200 thermal cycler (MJ Research) in conjunction with the HYDRA microdispenser (Bobbins Scientific) or the MICROLAB 2200 (Hamilton) liquid transfer system. 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 (Perkin-Elmer).
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 (Perkin-Elmer) 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 V.
The polynucleotide sequences derived from cDNA sequencing were assembled and analyzed using a combination of software programs which utilize algorithms well known to those skilled in the art. Table 5 summarizes the tools, programs, and algorithms used and provides applicable descriptions, references, and threshold parameters. The first column of Table 5 shows the tools, programs, and algorithms used. the second column provides brief descriptions thereof, the third column presents appropriate references, all of which are incorporated by reference herein in their entirety, and the fourth column presents, where applicable, the scores, probability values, and other parameters used to evaluate the strength of a match between two sequences (the higher the score, the greater the homology between two sequences). Sequences were analyzed using MACDNASIS PRO
software (Hitachi Software Engineering, South San Francisco CA) and LASERGENE
software (DNASTAR). Polynucleotide and polypeptide sequence alignments were generated using the 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.
The polynucleotide sequences were validated by removing vector, linker, and polyA
sequences and by masking ambiguous bases, using algorithms and programs based on BLAST, dynamic programing, and dinucleotide nearest neighbor analysis. The sequences were then queried against a selection of public databases such as the GenBank primate, rodent, mammalian, vertebrate, and eukaryote databases, and BLOCKS to acquire annotation using programs based on BLAST, FASTA, and BLIMPS. The sequences were assembled into full length polynucleotide sequences using programs based on Phred, Phrap, and Consed, and 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 amino acid sequences, and these full length sequences were subsequently analyzed by querying against databases such as the GenBank databases (described above), SwissProt, BLOCKS, PRINTS, Prosite, and Hidden Markov Model (HMM)-based protein family databases such as PFAM. HMM is a probabilistic approach which analyzes consensus primary structures of gene families. (See, e.g., Eddy, S.R. ( 1996) Curr.
Opin. Str. Biol. 6:361-365.) The programs described above for the assembly and analysis of full length polynucleotide and amino acid sequences were also used to identify polynucleotide sequence fragments from SEQ
ID N0:17-32. Fragments from about 20 to about 4000 nucleotides which are useful in hybridization and amplification technologies were described in The Invention section above.
IV. Northern Analysis Northern analysis is a laboratory technique used to detect the presence of a transcript of a gene and involves the hybridization of a labeled nucleotide sequence to a membrane on which RNAs from a particular cell type or tissue have been bound. (See, e.g., Sambrook, supra, ch. 7; Ausubel, 1995, supra, ch. 4 and 16.) Analogous computer techniques applying BLAST were used to search for identical or related molecules in nucleotide databases such as GenBank or LIFESEQ (Incyte Pharmaceuticals). 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:
seauence identity x % maximum BLAST score The product score takes into account both the degree of similarity between two sequences and the length of the sequence match. For example, with a product score of 40, the match will be exact within a 1% to 2% error, and, with a product score of 70, the match will be exact. Similar molecules are usually identified by selecting those which show product scores between 1 S and 40, although lower scores may identify related molecules.
The results of northern analyses are reported as a percentage distribution of libraries in which the transcript encoding CYSKP occurred. Analysis involved the categorization of cDNA libraries by organ/tissue and disease. The organ/tissue categories included cardiovascular, dermatologic, developmental, endocrine, gastrointestinal, hematopoietic/immune, musculoskeletal. nervous, reproductive, and urologic. The disease/condition categories included cancer, inflammation/trauma, cell proliferation, neurological, and pooled. For each category, the number of libraries expressing the sequence of interest was counted and divided by the total number of libraries across all categories.
Percentage values of tissue-specific and disease- or condition-specific expression are reported in Table 3.
V. Extension of CYSKP Encoding Polynucleotides The full length nucleic acid sequences of SEQ ID N0:17-32 were produced by extension of an appropriate fragment of the full length molecule using oligonucleotide primers designed from this I S fragment. One primer was synthesized to initiate 5' extension of the known fragment, and the other primer, to initiate 3' extension of the known fragment. The initial primers were designed using OLIGO 4.06 software (National Biosciences), or another appropriate program, to be about 22 to 30 nucleotides in length, to have a GC content of about 50% or more, and to anneal to the target sequence at temperatures of about 68°C to about 72°C. Any stretch of nucleotides which would result in hairpin structures and primer-primer dimerizations was avoided.
Selected human cDNA libraries were used to extend the sequence. If more than one extension was necessary or desired, additional or nested sets of primers were designed.
High fidelity amplification was obtained by PCR using methods well known in the art. PCR
was performed in 96-well plates using the PTC-200 thermal cycler (MJ Research, Inc.). The reaction mix contained DNA template, 200 nmol of each primer, reaction buffer containing Mg2', (NH4)ZSO,, and (i-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 ul PICOGREEN
quantitation reagent (0.25% (v/v) PICOGREEN; Molecular Probes, Eugene OR) dissolved in 1 X TE
and 0.5 pl 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 ~ ul to 10 ul aliquot of the reaction mixture was analyzed by electrophoresis on a 1 % agarose mini-gel to determine which reactions were successful in extending the sequence.
The extended nucleotides were desalted and concentrated. transferred to 384-well plates, digested with CviJI cholera virus endonuclease (Molecular Biology Research, Madison WI), and sonicated or sheared prior to relegation into pUC 18 vector (Amersham Pharmacia Biotech). For shotgun sequencing, the digested 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 relegated using T4 lipase (New England Biolabs, Beverly MA) into pUC 18 vector (Amersham Pharmacia Biotech), treated with Pfu DNA polymerise (Stratagene) to fill-in restriction site overhangs. and transfected into competent E. coli cells. Transformed cells were selected on antibiotic-containing media, individual colonies were picked and cultured overnight at 37°C in 384-welt plates in LB/2x carb liquid media.
The cells were lysed, and DNA was amplified by PCR using Taq DNA polymerise (Amersham Pharmacia Biotech) and Pfu DNA polymerise (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% dimethysulphoxide ( 1:2, v/v), and sequenced using DYENAMIC energy transfer sequencing primers and the DYENAMIC DIRECT kit (Amersham Pharmacia Biotech) or the ABI
PRISM BIGDYE Terminator cycle sequencing ready reaction kit (Perkin-Elmer).
In like manner, the nucleotide sequences of SEQ ID N0:17-32 are used to obtain 5' regulatory sequences using the procedure above, oligonucleotides designed for such extension, and an appropriate genomic library.
VI. Labeling and Use of Individual Hybridization Probes Hybridization probes derived from SEQ ID N0:17-32 are employed to screen cDNAs, genomic DNAs, or mRNAs. Although the labeling of oligonucleotides, consisting of about 20 base pairs, is specifically described, essentially the same procedure is used with larger nucleotide fragments. Oligonucleotides are designed using state-of the-art software such as OLIGO 4.06 software (National Biosciences) and labeled by combining 50 pmol of each oligomer, 250 uCi of [y-''-P] adenosine triphosphate (Amersham Pharmacia Biotech), and T4 polynucleotide kinase (DuPont NEN, Boston MA1. 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, Xbal, or Pvu II (DuPont NEN).
The DNA from each digest is fractionated on a 0.7% agarose gel and transferred to nylon membranes (Nytran Plus, Schleicher & Schuell. Durham NH). Hybridization is carried out for 16 hours at 40°C. To remove nonspecific signals, blots are sequentially washed at room temperature under increasingly stringent conditions up to 0.1 x saline sodium citrate and 0.5% sodium dodecyl sulfate. Hybridization patterns are visualized using autoradiography and compared.
VII. Microarrays A chemical coupling procedure and an ink jet device can be used to synthesize array elements on the surface of a substrate. (See, e.g., Baldeschweiler, s-u~ra.) An array 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 by hand or using available methods and machines and contain any appropriate number of elements. After hybridization, nonhybridized probes are removed and a scanner used to determine the levels and patterns of fluorescence. The degree of compiementarity and the relative abundance of each probe which hybridizes to an element on the microarray may be assessed through analysis of the scanned images.
Full-length cDNAs, Expressed Sequence Tags (ESTs), or fragments thereof may comprise the elements of the microarray. Fragments suitable for hybridization can be selected using software well known in the art such as LASERGENE software (DNASTAR). Full-length cDNAs, ESTs, or fragments thereof corresponding to one of the nucleotide sequences of the present invention, or selected at random from a cDNA library relevant to the present invention, are arranged on an appropriate substrate, e.g., a glass slide. The cDNA is fixed to the slide using, e.g., UV cross-linking followed by thermal and chemical treatments and subsequent drying. (See, e.g., Schena, M. et al.
(1995) Science 270:467-470; Shalon, D. et al. (1996) Genome Res. 6:639-645.) Fluorescent probes are prepared and used for hybridization to the elements on the substrate. The substrate is analyzed by procedures described above.
VIII. Complementary Polynucleotides Sequences complementary to the CYSKP-encoding sequences, or any parts thereof, are used to detect, decrease, or inhibit expression of naturally occurring CYSKP.
Although use of oligonucleotides comprising from about I~ 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 CYSKP. To inhibit transcription. a complementary oligonucleotide is designed from the most unique S' 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 CYSKP-encoding transcript.
IX. Expression of CYSKP
Expression and purification of CYSKP is achieved using bacterial or virus-based expression systems. For expression of CYSKP in bacteria, cDNA is subcioned 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 TS or T7 bacteriophage promoter in conjunction with the lac operator regulatory element. Recombinant vectors are transformed into suitable bacterial hosts, e.g., BL21(DE3).
Antibiotic resistant bacteria express CYSKP upon induction with isopropyl beta-D-thiogalactopyranoside (IPTG). Expression of CYSKP in eukaryotic cells is achieved by infecting insect or mammalian cell lines with recombinant Autoera~hica californica nuclear polyhedrosis virus (AcMNPV), commonly known as baculovirus. The nonessential polyhedrin gene of baculovirus is replaced with cDNA encoding CYSKP by either homologous recombination or bacterial-mediated transposition involving transfer plasmid intermediates. Viral infectivity is maintained and the strong polyhedrin promoter drives high levels of cDNA transcription. Recombinant baculovirus is used to infect Spodoptera fru inerda (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.
7:193 7-1945.) In most expression systems, CYSKP 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-kilodaiton 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 CYSKP at specifically engineered sites. FLAG, an $-amino acid peptide, enables immunoaffinity purification using commercially available monoclonal and polyclonal anti-FLAG
antibodies (Eastman Kodak). 6-His. a stretch of six consecutive histidine residues, enables purification on metal-chelate resins (QIAGEN). Methods for protein expression and purification are discussed in Ausubel ( 1995, supra, ch 10 and 16). Purified CYSKP obtained by these methods can be used directly in the following activity assay.
X. Demonstration of CYSKP Activity A microtubule motility assay for CYSKP activity measures motor domain function. In this assay, recombinant CYSKP is immobilized onto a glass slide or similar substrate. Taxol-stabilized bovine brain microtubules (commercially available) in a solution containing ATP and cytosolic extract are perfused onto the slide. Movement of microtubules as driven by CYSKP motor activity can be visualized and quantified using video-enhanced light microscopy and image analysis techniques. CYSKP activity is directly proportional to the frequency and velocity of microtubule movement.
In the alternative, an assay for CYSKP measures the binding affinity of CYSKP
for actin as described by Hammell, R.L. and Hitchcock-DeGregori, S.E. (1997, J. Biol. Chem.
272:22409-22416). CYSKP and actin are prepared from in vitro recombinant cDNA expression systems and the N-terminus of CYSKP is acetylated using methods well known in the art. Binding of N-terminal acetyl-CYSKP to actin is measured by cosedimentation at 25 °C in a Beckman model TL-100 centrifuge as described. The bound and free CYSKP are determined by quantitative densitometry of SDS-polyacrylamide gels stained with Coomassie Blue. Apparent binding constants (K,pP) and Hill coefficients (H) are determined by using methods well known in the art to fit the data to the equation as described by Hammell and Hitchcock-DeGregori (1997, supra). The CYSKP:actin ratio, determined using densitometry, is normalized. Hammell and Hitchcock-DeGregori (1997, supra) have shown that saturation of binding corresponds to a CYSKP:actin molar ratio of 0.14, a stoichiometry of 1 CYSKP:7 actin. The binding of CYSKP to actin is proportional to the CYSKP
activity.
In the alternative, CYSKP are assayed by their ability to bind to F-actin using a blot overlay system similar to that described by Luna, E.J. et al. ( 1997, Soc. Gen.
Physiol. Ser. 52:3-18). Proteins in plasma membrane-enriched cell extracts containing CYSKP are separated using SDS
polyacrylamide gel electrophoresis (10% acrylamide). The gel-separated proteins are transferred to nitrocellulose using methods well known in the art and the blot is washed and pretreated with non-specific blocking agents. [''-SI]-labeled F-actin is prepared and suspended in overlay buffer, then incubated with the blot for at least 16 hours at 4°C. Unbound label is washed with washing buffer, the blot is air dried and subjected to autoradiography for at least one hour.
The autoradiograph band corresponding to the expected molecular mass of CYSKP is identified. The amount of observed ['ZSI)-labeled F-actin which binds to CYSKP is proportional to the amount of CYSKP present in the sample.
In the alternative, CYSKP activity is associated with its ability to form protein-protein complexes and is measured by its ability to regulate growth characteristics of NIH3T3 mouse fibroblast cells. A cDNA encoding CYSKP is subcloned into an appropriate eukaryotic expression vector. This vector is transfected into NIH3T3 cells using methods known in the art. Transfected cells are compared with non-transfected cells for the following quantifiable properties: growth in culture to high density, reduced attachment of cells to the substrate, altered cell morphology, and ability to induce tumors when injected into immunodeficient mice. The activity of CYSKP is proportional to the extent of increased growth or frequency of altered cell morphology in NIH3T3 cells transfected with CYSKP.
In the alternative, CYSKP activity is measured as ability to bind to microtubules.
Microtubules are purified from adult rat brain by reversible assembly (Vallee, R. B. (1982) Methods Enzymol. 134:89-104) or the taxol method (Vallee, R. B. ( 1982) J. Cell Biol.
92:435-442) using PEM
buffer ( 100 mM PIPES, pH 6.6, 1 mM EGTA, 1 mM MgS04). To separate the MAPS
from tubulin, the pellets from twice-cycled microtubules are resuspended in PEM buffer and applied to a 0.1 M
MgSO~ saturated phosphocellulose column as described by Sloboda, R. D. and Rosenbaum, J. L.
((1982) Methods Enzymol. 85:409-416). The fractions containing protein are applied to a second phosphocellulose column. In a total volume of 100 ml, 20 ml of CYSKP (250 mg/ml) is added to 80 ml of whole microtubules (450 mg/ml) or tubulin (300 mg/ml) and incubated at 37 °C for 10 minutes I S in the presence of 1 mM GTP and 50 mM taxol. The suspension is centrifuged, the supernatant is removed, and the microtubule pellet is resuspended to the original reaction volume in PEM buffer.
To assess the partitioning of CYSKP between the supernatant and pellet fractions, equal amounts of supernatant and resuspended pellet are placed in SDS sample buffer and assayed on a ~-20% gradient SDS polyacrylamide gel stained with Coomassie Brilliant Blue. The amount of CYSKP in the pellet fraction is proportional to the binding of CYSKP to microtubules.
In the alternative, CYSKP, or biologically active fragments thereof, are labeled with 'ZSI
Bolton-Hunter reagent. (See, e.g., Bolton et al. ( 1973) Biochem. J. 133:529.) Candidate molecules previously arrayed in the wells of a multi-well plate are incubated with the labeled CYSKP, washed, and any wells with labeled CYSKP complex are assayed. Data obtained using different concentrations of CYSKP are used to calculate values for the number, affinity, and association of CYSKP with the candidate molecules.
XI. Functional Assays CYSKP function is assessed by expressing the sequences encoding CYSKP 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, Carisbad CA), both of which contain the cytomegalovirus promoter. 5-10 ~g of recombinant vector are transiently transfected into a human cell line, preferably of endothelial or hematopoietic origin, using either liposome formulations or electroporation. 1-2 ~g of an additional plasmid containing sequences encoding a marker protein are co-transfected. Expression of a marker protein provides a means to distinguish transfected cells from nontransfected cells and is a reliable predictor of cDNA
expression from the recombinant vector. Marker proteins of choice include, e.g., Green Fluorescent Protein (GFP; Clontech), CD64. or a CD64-GFP fusion protein. Flow cytometry (FCM), an automated, laser optics-based technique, is used to identify transfected cells expressing GFP or CD64-GFP and to evaluate the apoptotic state of the cells and other cellular properties. FCM detects and quantifies the uptake of fluorescent molecules that diagnose events preceding or coincident with cell death. These events include changes in nuclear DNA content as measured by staining of DNA
with propidium iodide; changes in cell size and granularity as measured by forward light scatter and 90 degree side light scatter; down-regulation of DNA synthesis as measured by decrease in bromodeoxyuridine uptake; alterations in expression of cell surface and intracellular proteins as measured by reactivity with specific antibodies; and alterations in plasma membrane composition as measured by the binding of fluorescein-conjugated Annexin V protein to the cell surface. Methods in flow cytometry are discussed in Ormerod, M. G. ( 1994) Flow Cytometry, Oxford, New York NY.
The influence of CYSKP on gene expression can be assessed using highly purified populations of cells transfected with sequences encoding CYSKP 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 CYSKP and other genes of interest can be analyzed by northern analysis or microarray techniques.
XII. Production of CYSKP Specific Antibodies CYSKP substantially purified using polyacrylamide gel electrophoresis (PAGE;
see, e.g., Harrington, M.G. ( 1990) Methods Enzymol. I 82:488-495), or other purification techniques, is used to immunize rabbits and to produce antibodies using standard protocols.
Alternatively, the CYSKP 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. 1 I.) Typically, oligopeptides 15 residues in length are synthesized using an ABI
431A peptide synthesizer (Perkin-Elmer) 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 amisera are tested for antipeptide activity by, for example, binding the peptide to plastic, blocking with t % BSA, reacting with rabbit antisera, washing, and reacting with radio-iodinated goat anti-rabbit IgG.
XIII. Purification of Naturally Occurring CYSKP Using Specific Antibodies Naturally occurring or recombinant CYSKP is substantially purified by immunoaffinity chromatography using antibodies specific for CYSKP. An immunoaffinity column is constructed by covalently coupling anti-CYSKP antibody to an activated chromatographic resin, such as CNBr-activated SEPHAROSE (Amersham Phatmacia Biotech). After the coupling, the resin is blocked and washed according to the manufacturer's instructions.
Media containing CYSKP are passed over the immunoaffinity column, and the column is washed under conditions that allow the preferential absorbance of CYSKP (e.g., high ionic strength buffers in the presence of detergent). The column is eluted under conditions that disrupt antibody/CYSKP binding (e.g., a buffer of pH 2 to pH 3, or a high concentration of a chaotrope, such as urea or thiocyanate ion), and CYSKP is collected.
Various modifications and variations of the described methods and systems of the invention IS will be apparent to those skilled in the art without departing from the scope and spirit of the invention.
Although the invention has been described in connection with specific preferred embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention which are obvious to those skilled in molecular biology or related fields are intended to be within the scope of the following claims.

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<110> INCYTE PHARMACEUTICALS, INC.
LAL, Preeti TANG, Y. Tom YUE, Henry HILLMAN, Jennifer L.
BANDMAN, Olga CORLEY, Neil C.
GUEGLER, Karl J.
PATTERSON, Chandra AZIMZAI, Yalda BAUGHN, Mariah R.
<120> HUMAN CYTOSKELETON ASSOCIATED PROTEINS
<130> PF-0594 PCT
<140> To Be Assigned <141> Herewith <150> 09/156,470; unassigned; 60/131,321 <151> 1998-09-18; 1998-09-18; 1999-04-27 <160> 32 <170> PERL Program <210> 1 <211> 1005 <212> PRT
<213> Homo sapiens <220>
<221> misc_feature <223> Incyte ID No: 1285395 <400> 1 Met Thr Thr Glu Val Gly Ser Val Ser Glu Val Lys Lys Asp Ser Ser Gln Leu Gly Thr Asp Ala Thr Lys Glu Lys Pro Lys Glu Val Ala Glu Asn Gln Gln Asn Gln Ser Ser Asp Pro Glu Glu Glu Lys Gly Ser Gln Pro Pro Pro Ala Ala Glu Ser Gln Ser Ser Leu Arg Arg Gln Lys Arg Glu Lys Glu Thr Ser Glu Ser Arg Gly Ile Ser Arg Phe Ile Pro Pro Trp Leu Lys Lys Gln Lys Ser Tyr Thr Leu Val Val Ala Lys Asp Gly Gly Asp Lys Lys Glu Pro Thr Gln Ala Val Val Glu Glu Gln Val Leu Asp Lys Glu Glu Pro Leu Pro Glu Glu Gln Arg Gln Ala Lys Gly Asp Ala Glu Glu Met Ala Gln Lys Lys Gln Glu Ile Lys Val Glu Val Lys Glu Glu Lys Pro Ser Val Ser Lys Glu Glu Lys Pro Ser Val Ser Lys Val Glu Met Gln Pro Thr Glu Leu Val Ser Lys Glu Arg Glu Glu Lys Val Lys Glu Thr Gln Glu Asp Lys Leu Glu Gly Gly Ala Ala Lys Arg Glu Thr Lys Glu Val Gln Thr Asn Glu Leu Lys Ala Glu Lys Ala Ser Gln Lys Val Thr Lys Lys Thr Lys Thr Val Gln Cys Lys Val Thr Leu Leu Asp Gly Thr Glu Tyr Ser Cys Asp Leu Glu Lys His Ala Lys Gly Gln Val Leu Phe Asp Lys Val Cys Glu His Leu Asn Leu Leu Glu Lys Asp Tyr Phe Gly Leu Leu Phe Gln Glu Ser Pro Glu Gln Lys Asn Trp Leu Asp Pro Ala Lys Glu Ile Lys Arg Gln Leu Arg Asn Leu Pro Trp Leu Phe Thr Phe Asn Val Lys Phe Tyr Pro Pro Asp Pro Ser Gln Leu Thr Glu Asp Ile Thr Arg Tyr Phe Leu Cys Leu Gln Leu Arg Gln Asp Ile Ala Ser Gly Arg Leu Pro Cys Ser Phe Val Thr His Ala Leu Leu Gly Ser Tyr Thr Leu Gln Ala Glu Leu Gly Asp Tyr Asp Pro Glu Glu His Gly Ser Ile Asp Leu Ser Glu Phe Gln Phe Ala Pro Thr Gln Thr Lys Glu Leu Glu Glu Lys Val Ala Glu Leu His Lys Thr His Arg Gly Leu Ser Pro Ala Gln Ala Asp Ser Gln Phe Leu Glu Asn Ala Lys Arg Leu Ser Met Tyr Gly Val Asp Leu His His Ala Lys Asp Ser Glu Gly Val Asp Ile Lys Leu Gly Val Cys Ala Asn Gly Leu Leu Ile Tyr Lys Asp Arg Leu Arg Ile Asn Arg Phe Ala Trp Pro Lys Ile Leu Lys Ile Ser Tyr Lys Arg Ser Asn Phe Tyr Ile Lys Val Arg Pro Ala Glu Leu Glu Gln Phe Glu Ser Thr Ile Gly Phe Lys Leu Pro Asn His Arg Ala Ala Lys Arg Leu Trp Lys Val Cys Val Glu His His Thr Phe Tyr Arg Leu Val Ser Pro Glu Gln Pro Pro Lys Ala Lys Phe Leu Thr Leu Gly Ser Lys Phe Arg Tyr Ser Gly Arg Thr Gln Ala Gln Thr Arg Gln Ala Ser Thr Leu Ile Asp Arg Pro Ala Pro His Phe Glu Arg Thr Ser Ser Lys Arg Val Ser Arg Ser Leu Asp Gly Ala Pro Ile Gly Val Met Asp Gln Ser Leu Met Lys Asp Phe Pro Gly Ala Ala Gly Glu Ile Ser Ala Tyr Gly Pro Gly Leu Val Ser Ile Ala Val Val Gln Asp Gly Asp Gly Arg Arg Glu Val Arg Ser Pro Thr Lys Ala Pro His Leu Gln Leu Ile Glu Gly Lys Lys Asn Ser Leu Arg Val Glu Gly Asp Asn Ile Tyr Val Arg His Ser Asn Leu Met Leu Glu Glu Leu Asp Lys Ala Gln Glu Asp Ile Leu Lys His Gln Ala Ser Ile Ser Glu Leu Lys Arg Asn Phe Met Glu Ser Thr Pro Glu Pro Arg Pro Asn Glu Trp Glu Lys Arg Arg Ile Thr Pro Leu Ser Leu Gln Thr Gln Gly Sex Ser His Glu Thr Leu Asn Ile Val Glu Glu Lys Lys Arg Ala Glu Val Gly Lys Asp Glu Arg Val Ile Thr Glu Glu Met Asn Gly Lys Glu Ile Ser Pro Gly Ser Gly Pro Gly Glu Ile Arg Lys Val Glu Pro Val Thr Gln Lys Asp Ser Thr Ser Leu Ser Ser Glu Ser Ser Ser Ser Ser Ser Glu Ser Glu Glu Glu Asp Val Gly Glu Tyr Arg Pro His His Arg Val Thr Glu Gly Thr Ile Arg Glu Glu Gln Glu Tyr Glu Glu Glu Val Glu Glu Glu Pro Arg Pro Ala Ala Lys Val Val Glu Arg Glu Glu Ala Val Pro Glu Ala Ser Pro Val Thr Gln Ala Gly Ala Ser Val Ile Thr Val Glu Thr Val Ile Gln Glu Asn Val Gly Ala Gln Lys Ile Pro Gly Glu Lys Ser Val His Glu Gly Ala Leu Lys Gln Asp Met Gly Glu Glu Ala Glu Glu Glu Pro Gln Lys Val Asn Gly Glu Val Ser His Val Asp Ile Asp Val Leu Pro Gln Ile Ile Cys Cys Ser Glu Pro Pro Val Val Lys Thr Glu Met Val Thr Ile Ser Asp Ala Ser Gln Arg Thr Glu Ile Ser Thr Lys Glu Val Pro Ile Val Gln Thr Glu Thr Lys Thr Ile Thr Tyr Glu Ser Pro Gln Ile Asp Gly Gly Ala Gly Gly Asp Sex Gly Thr Leu Leu Thr A1a Gln Thr Ile Thr Ser Glu Ser Val Ser Thr Thr Thr Thr Thr His Ile Thr Lys Thr Val Lys Gly Gly Ile Ser Glu Thr Arg Ile Glu Lys Arg Ile Val Ile Thr Gly Asp Gly Asp Ile Asp His Asp Gln Ala Leu Ala Gln Ala Ile Arg Glu Ala Arg Glu Gln His Pro Asp Met Ser Val Thr Arg Val Val Val His Lys Glu Thr Glu Leu Ala Glu Glu Gly Glu Asp _ <210> 2 <211> 1045 <212> PRT
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte ID No: 1320252 <400> 2 Met Gly Glu Ile Glu Gln Arg Pro Thr Pro Gly Ser Arg Leu Gly Ala Pro Glu Asn Ser Gly Ile Ser Thr Leu Glu Arg Gly Gln Lys Pro Pro Pro Thr Pro Ser Gly Lys Leu Val Ser Ile Lys Ile Gln Met Leu Asp Asp Thr Gln Glu Ala Phe Glu Val Pro Gln Arg Ala Pro Gly Lys Val Leu Leu Asp Ala Val Cys Asn His Leu Asn Leu Val Glu Gly Asp Tyr Phe Gly Leu Glu Phe Pro Asp His Lys Lys Ile Thr Val Trp Leu Asp Leu Leu Lys Pro Ile Val Lys Gln Ile Arg Arg Pro Lys His Val Val Val Lys Phe Val Val Lys Phe Phe Pro Pro Asp His Thr Gln Leu Gln Glu Glu Leu Thr Arg Tyr Leu Phe Ala Leu Gln Val Lys Gln Asp Leu Ala Gln Gly Arg Leu Thr Cys Asn Asp Thr Ser Ala Ala Leu Leu Ile Ser His Ile Val Gln Ser Glu Ile Gly Asp Phe Asp Glu Ala Leu Asp Arg Glu His Leu Ala Lys Asn Lys Tyr Ile Pro Gln Gln Asp Ala Leu Glu Asp Lys Ile Val Glu Phe His His Asn His Ile Gly Gln Thr Pro Ala Glu Ser Asp Phe Gln Leu Leu Glu Ile Ala Arg Arg Leu Glu Met Tyr Gly Ile Arg Leu His Pro Ala Lys Asp Arg Glu Gly Thr Lys Ile Asn Leu Ala Val Ala Asn Thr Gly Ile Leu Val Phe Gln Gly Phe Thr Lys Ile Asn Ala Phe Asn Trp Ala Lys Val Arg Lys Leu Ser Phe Lys Arg Lys Arg Phe Phe Ile Lys Leu Arg Pro Asp Ala Asn Ser Ala Tyr Gln Asp Thr Leu Glu Phe Leu Met Ala Ser Arg Asp Phe Cys Lys Ser Phe Trp Lys Ile Cys Val Glu His His Ala Phe Phe Arg Leu Phe Glu Glu Pro Lys Pro Lys Pro Lys Pro Val Leu Phe Ser Arg Gly Ser Ser Phe Arg Phe Ser Gly Arg Thr Gln Lys Gln Val Leu Asp Tyr Val Lys Glu Gly Gly His Lys Lys Val Gln Phe Glu Arg Lys His Ser Lys Ile His Ser Ile Arg Ser Leu Ala Ser Gln Pro Thr Glu Leu Asn Ser Glu Val Leu Glu Gln Ser Gln Gln Ser Thr Ser Leu Thr Phe Gly Glu Gly Ala Glu Ser Pro Gly Gly Gln Ser Cys Arg Arg Gly Lys Glu Pro Lys Val Ser Ala Gly Glu Pro Gly Ser His Pro Ser Pro Ala Pro Arg Arg Ser Pro Ala Giy Asn Lys Gln Ala Asp Gly Ala Ala Sex Ala Pro Thr Glu Glu Glu Glu Glu Val Val Lys Asp Arg Thr Gln Gln Ser Lys Pro Gln Pro Pro Gln Pro Ser Thr Gly Ser Leu Thr Gly Ser Pro His Leu Ser Glu Leu Ser Val Asn Ser Gln Gly Gly Val Ala Pro Ala Asn Val Thr Leu Ser Pro Asn Leu Ser Pro Asp Thr Lys Gln Ala Ser Pro Leu Ile Ser Pro Leu Leu Asn Asp Gln Ala Cys Pro Arg Thr Asp Asp Glu Asp Glu Gly Arg Arg Lys Arg Phe Pro Thr Asp Lys Ala Tyr Phe Ile Ala Lys Glu Val Ser Thr Thr Glu Arg Thr Tyr Leu Lys Asp Leu Glu Val Ile Thr Ser Trp Phe Gln Ser Thr Val Ser Lys Glu Asp Ala Met Pro Glu Ala Leu Lys Ser Leu Ile Phe Pro Asn Phe Glu Pro Leu His Lys Phe His Thr Asn Phe Leu Lys Glu T_le Glu Gln Arg Leu Ala Leu Trp Glu Gly Arg Ser Asn Ala Gln Ile Arg Asp Tyr Gln Arg Ile Gly Asp Val Met Leu Lys Asn Ile Gln Gly Met Lys His Leu Ala Ala His Leu Trp Lys His Ser Glu Ala Leu Glu Ala Leu Glu Asn Gly Ile Lys Ser Ser Arg Arg Leu Glu Asn Phe Cys Arg Asp Phe Glu Leu Gln Lys Val Cys Tyr Leu Pro Leu Asn Thr Phe Leu Leu Arg Pro Leu His Arg Leu Met His Tyr Lys Gln Val Leu Glu Arg Leu Cys Lys His His Pro Pro Ser His Ala Asp Phe Arg Asp Cys Arg Ala Ala Leu Ala Glu Ile Thr Glu Met Val Ala Gln Leu His Gly Thr Met Iie Lys Met Glu _ Asn Phe Gln Lys Leu His Glu Leu Lys Lys Asp Leu Ile Gly Ile Asp Asn Leu Val Val Pro Gly Arg Glu Phe Ile Arg Leu Gly Ser Leu Ser Lys Leu Ser Gly Lys Gly Leu Gln Gln Arg Met Phe Phe Leu Phe Asn Asp Val Leu Leu Tyr Thr Ser Arg Gly Leu Thr Ala Ser Asn Gln Phe Lys Val His Gly Gln Leu Pro Leu Tyr Gly Met Thr Ile Glu Glu Ser Glu Asp Glu Trp Gly Val Pro His Cys Leu Thr Leu Arg Gly Gln Arg Gln Ser Ile Ile Val Ala Ala Ser Ser Arg Ser Glu Met Glu Lys Trp Val Glu Asp Ile Gln Met Ala Ile Asp Leu Ala Glu Lys Ser Ser Ser Pro Ala Pro Glu Phe Leu Ala Ser Ser Pro Pro Asp Asn Lys Ser Pro Asp Glu Ala Thr Ala Ala Asp Gln Glu Ser Glu Asp Asp Leu Ser Ala Ser Arg Thr Ser Leu Glu Arg Gln Ala Pro His Arg Gly Asn Thr Met Val His Val Cys Trp His Arg Asn Thr Ser Val Ser Met Val Asp Phe Ser Ile Ala Val Glu Asn Gln Leu Ser G1y Asn Leu Leu Arg Lys Phe Lys Asn Ser Asn Gly Trp Gln Lys Leu Trp Val Val Phe Thr Asn Phe Cys Leu Phe Phe Tyr Lys Ser His Gln Asp Asn His Pro Leu Ala Ser Leu Pro Leu Leu Gly Tyr Ser Leu Thr Ile Pro Ser Glu Ser Glu Asn Ile Gln Lys Asp Tyr Val Phe Lys Leu His Phe Lys Ser His Val Tyr Tyr Phe Arg Ala Glu Ser Glu Tyr Thr Phe Glu Arg Trp Met Glu Val Ile Arg Ser Ala Thr Ser Ser Ala Ser Arg Pro His Val Leu Ser His Lys Glu Ser Leu Val Tyr <210> 3 <211> 324 <212> PRT

<213> Homo sapiens <220>
<221> misc_feature <223> Incyte ID No: 1259001 <400> 3 _ Met Cys Phe Gln Ala Pro Glu Glu Leu Val Leu His Leu Ala Val Lys Val Ala Asn Gln Ala Ser Leu Pro Leu Val Asp Phe Ile Ile Gln Asn Gly Gly His Leu Asp Ala Lys Ala Ala Asp Gly Asn Thr Ala Leu His Tyr Ala Ala Leu Tyr Asn Gln Pro Asp Cys Leu Lys Leu Leu Leu Lys Gly Arg Ala Leu Val Gly Thr Val Asn Glu Ala Gly Glu Thr Ala Leu Asp Ile Ala Arg Lys Lys His His Lys Glu Cys Glu Glu Leu Leu Glu Gln Ala Gln Ala Gly Thr Phe Ala Phe Pro Leu His Val Asp Tyr Ser Trp Val Ile Ser Thr Glu Pro Gly Ser Asp Ser Glu Glu Asp Glu Glu Glu Lys Arg Cys Leu Leu Lys Leu Pro Ala Gln Ala His Trp Ala Ser Gly Arg Leu Asp Ile Ser Asn Lys Thr Tyr Glu Thr Val Ala Ser Leu Gly Ala Ala Thr Pro Gln Gly Glu Ser Glu Asp Cys Pro Pro Pro Leu Pro Val Lys Asn Ser Ser Arg Thr Leu Val Gln Gly Cys Ala Arg His Ala Ser Gly Asp Arg Ser Glu Val Ser Ser Leu Ser Ser Glu Ala Pro Glu Thr Pro Glu Ser Leu Gly Ser Pro Ala Ser Ser Ser Ser Leu Met Ser Pro Leu Glu Pro Gly Asp Pro Ser Gln Ala Pro Pro Asn Ser Glu Glu Gly Leu Arg Glu Pro Pro Gly Thr Ser Arg Pro Ser Leu Thr Ser Gly Thr Thr Pro Ser Glu Met Tyr Leu Pro Val Arg Phe Ser Ser Glu Ser Thr Arg Ser Tyr Arg Arg Gly Ala Arg Ser Pro Glu Asp Gly Pro Ser Ala Arg Gln Pro Leu Pro Arg Arg Asn Val Pro Val Gly Ile Thr Glu Gly Asp Gly Ser Arg Thr Gly Ser Leu Pro Ala Ser Ser Val Gln Leu Leu Gln Asp <210> 4 <211> 385 <212> PRT
<213> Homo Sapiens <220>
<221> misc_feature -<223> Incyte ID No: 1627027 <400> 4 Met Ser Val Ser Arg Thr Met Glu Asp Ser Cys Glu Leu Asp Leu Val Tyr Val Thr Glu Arg Ile Ile Ala Val Ser Phe Pro Ser Thr Ala Asn Glu Glu Asn Phe Arg Ser Asn Leu Arg Glu Val Ala Gln Met Leu Lys Ser Lys His Gly Gly Asn Tyr Leu Leu Phe Asn Leu Ser Glu Arg Arg Pro Asp Ile Thr Lys Leu His Ala Lys Val Leu Glu Phe Gly Trp Pro Asp Leu His Thr Pro Ala Leu Glu Lys Ile Cys Ser Ile Cys Lys Ala Met Asp Thr Trp Leu Asn Ala Asp Pro His Asn Val Val Val Leu His Asn Lys Gly Asn Arg Gly Arg Ile Gly Val Val Ile Ala Ala Tyr Met His Tyr Ser Asn Ile Ser Ala Ser Ala Asp Gln Ala Leu Asp Arg Phe Ala Met Lys Arg Phe Tyr Glu Asp Lys Ile Val Pro Ile Gly Gln Pro Ser Gln Arg Arg Tyr Vai His Tyr Phe Ser Gly Leu Leu Ser Gly Ser Ile Lys Met Asn Asn Lys Pro Leu Phe Leu His His Val Ile Met His Gly Ile Pro Asn Phe Glu Ser Lys Gly Gly Cys Arg Pro Phe Leu Arg Ile Tyr Gln Ala Met Gln Pro Val Tyr Thr Ser Gly Ile Tyr Asn Ile Pro Gly Asp Ser Gln Thr Ser Val Cys Ile Thr Ile Glu Pro Gly Leu Leu Leu Lys Gly Asp Ile Leu Leu Lys Cys Tyr His Lys Lys Phe Arg Ser Pro Ala Arg Asp Val Ile Phe Arg Val Gln Phe His Thr Cys Ala Ile His Asp Leu Gly Val Val Phe Gly Lys Glu Asp Leu Asp Asp Ala Phe Lys Asp Asp Arg Phe Pro Glu Tyr Gly Lys Val Glu Phe Val Phe Ser Tyr Gly Pro Glu Lys Ile Gln Gly Met Glu 305 310 3i5 His Leu Glu Asn Gly Pro Ser Val Ser Val Asp Tyr Asn Thr Ser Asp Pro Leu Ile Arg Trp Asp Ser Tyr Asp Asn Phe Ser Gly His Arg Asp Asp Gly Met Glu Asp Gly Asn Lys Gln Asn Thr Asn Ser Gln Ser Ile Gly Ser Ile Ser Gly Gly Leu Glu Asp Gln Tyr Thr Trp Pro Asp Thr His Trp Pro Ser Gln Ser <210> 5 <211> 364 <212> PRT
<213> Homo sapiens <220>
<221> misc_feature <223> Incyte ID No: 1905315 <400> 5 Met Ser Ser Ala Pro Arg Ser Pro Thr Pro Arg Pro Arg Arg Met Lys Lys Asp Glu Ser Phe Leu Gly Lys Leu Gly Gly Thr Leu Ala Arg Lys Arg Arg Ala Arg Glu Val Ser Asp Leu Gln Glu Glu Gly Lys Asn Ala Ile Asn Ser Pro Met Ser Pro Ala Leu Ala Asp Val His Pro Glu Asp Thr Gln Leu Glu Glu Asn Glu Glu Arg Thr Met Ile Asp Pro Thr Ser Lys Glu Asp Pro Lys Phe Lys G1u Leu Val Lys Val Leu Leu Asp Trp Ile Asn Asp Val Leu Val Glu Glu Arg Ile Ile Val Lys Gln Leu Glu Glu Asp Leu Tyr Asp Gly Gln Val Leu Gln Lys Leu Leu Glu Lys Leu Ala Gly Cys Lys Leu Asn Val Ala Glu Val Thr Gln Ser Glu Ile Gly Gln Lys Gln Lys Leu Gln Thr val Leu Glu Ala Val His Asp Leu Leu Arg Pro Arg Gly Trp Ala Leu Arg Trp Ser Val Asp Ser Ile His Gly Lys Asn Leu Val AIa Ile Leu His Leu Leu Val Ser Leu Ala Met His Phe Arg Ala Pro Ile Arg Leu Pro Glu His Val Thr Val Gln Val Val Val VaI

Arg Lys Arg Glu Gly Leu Leu His Ser Ser His Ile Ser Glu Glu Leu Thr Thr Thr Thr Glu Met Met Met Gly Arg Phe Glu Arg Asp Ala Phe Asp Thr Leu Phe Asp His Ala Pro Asp Lys Leu Ser Val Val Lys Lys Ser Leu Ile Thr Phe Val Asn Lys His Leu Asn Lys Leu Asn Leu Glu Val Thr Glu Leu Glu Thr Gln Phe Ala Asp Gly Val Tyr Leu Val Leu Leu Met Gly Leu Leu Glu Asp Tyr Phe Val Pro Leu His His Phe Tyr Leu Thr Pro Glu Ser Phe Asp Gln Lys Val His Asn Val Ser Phe Ala Phe Glu Leu Met Leu Asp Gly Gly Leu Lys Lys Pro Lys Ala Arg Pro Glu Asp Val Val Asn Leu Asp Leu Lys Ser Thr Leu Arg Val Leu Tyr Asn Leu Phe Thr Lys Tyr Lys Asn Val Glu <210> 6 <211> 395 <212> PRT
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte ID No: 1997789 <400> 6 Met Ser Glu Gln Ser Ile Cys Gln Ala Arg Ala Ser Val Met Val Tyr Asp Asp Thr Ser Lys Lys Trp Val Pro Ile Lys Pro Gly Gln Gln Gly Phe Ser Arg Ile Asn Ile Tyr His Asn Thr Ala Ser Asn Thr Phe Arg Val Val Gly Val Lys Leu Gln Asp Gln Gln Val Val Ile Asn Tyr Ser Ile Val Lys Gly Leu Lys Tyr Asn Gln Ala Thr Pro Thr Phe His Gln Trp Arg Asp Ala Arg Gln Val Tyr Gly Leu Asn Phe Ala Ser Lys Glu Glu Ala Thr Thr Phe Ser Asn Ala Met Leu Phe Ala Leu Asn Ile Met Asn Ser Gln Glu Gly Gly Pro Ser Ser Gln Arg Gln Val Gln Asn Gly Pro Ser Pro Asp Glu Met Asp Ile Gln Arg Arg Gln Val Met Glu Gln His Gln Gln Gln Arg Gln Glu Ser Leu Glu Arg Arg Thr Ser Ala Thr Gly Pro Ile Leu Pro Pro Gly His Pro Ser Ser Ala Ala Ser Ala Pro Val Ser Cys Ser Gly Pro Pro Pro Pro Pro Pro Pro Leu Val Pro Pro Pro Pro Thr Gly Ala Thr Pro Pro Pro Pro Pro Pro Leu Pro Ala Gly Gly Ala Gln Gly Ser Ser His Asp Glu Ser Ser Met Ser Gly Leu Ala Ala Ala Ile Ala Gly Ala Lys Leu Arg Arg Val Gln Arg Pro Glu Asp Ala Ser Gly Gly Ser Ser Pro Ser Gly Thr Ser Lys Ser Asp Ala Asn Arg Ala Ser Ser Gly Gly Gly Gly Gly Gly Leu Met Glu Glu Met Asn Lys Leu Leu Ala Lys Arg Arg Lys Ala Ala Ser Gln Ser Asp Lys Pro Ala Glu Lys Lys Glu Asp Glu Ser Gln Met Glu Asp Pro Ser Thr Ser Pro Ser Pro Gly Thr Arg Ala Ala Ser Gln Pro Pro Asn Ser Ser Glu Ala Gly Arg Lys Pro Trp Glu Arg Ser Asn Ser Val Glu Lys Pro Val Ser Ser Ile Leu Ser Arg Met Lys Pro Ala Gly Ser Val Asn Asp Met Ala Leu Asp Ala Phe Asp Leu Asp Arg Met Lys Gln Glu Ile Leu Glu Glu Val Val Arg Glu Leu His Lys Val Lys Glu Glu Ile Ile Asp Ala Ile Arg Gln Glu Leu Ser Gly Ile Ser Thr Thr <210> 7 <211> 523 <212> PRT
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte ID No: 2303465 <400> 7 Met Ala Ala Val Gly Arg Val Gly Ser Phe Gly Ser Ser Pro Pro Gly Leu Ser Ser Thr Tyr Thr Gly Gly Pro Leu Gly Asn Glu Ile Ala Ser Gly Asn Gly Gly Ala Ala Ala Gly Asp Asp Glu Asp Gly Gln Asn Leu Trp Ser Cys Ile Leu Ser Glu Val Sex Thr Arg Ser Arg Ser Lys Leu Pro Ala Gly Lys Asn Val Leu Leu Leu Gly Glu Asp Gly Ala Gly Lys Thr Ser Leu Ile Arg Lys Ile Gln Gly Ile Glu Glu Tyr Lys Lys Gly Arg Gly Leu Glu Tyr Leu Tyr Leu Asn Val His Asp Glu Asp Arg Asp Asp Gln Thr Arg Cys Asn Val Trp Ile Leu Asp Gly Asp Leu Tyr His Lys Gly Leu Leu Lys Phe Ser Leu Asp Ala Val Ser Leu Lys Asp Thr Leu Val Met Leu Val Val Asp Met Ser Lys Pro Trp Thr Ala Leu Asp Ser Leu Gln Lys Trp Ala Ser Val Val Arg Glu His Val Asp Lys Leu Lys Ile Pro Pro Glu Glu Met Lys Gln Met Giu Gln Lys Leu Ile Arg Asp Phe Gln 185 190 195 _ Glu Tyr Val Glu Pro Gly Glu Asp Phe Pro Ala Ser Pro Gln Arg Arg Asn Thr Ala Ser Gln Glu Asp Lys Asp Asp Ser Val Val Leu Pro Leu Gly Ala Asp Thr Leu Thr His Asn Leu Gly Ile Pro Val Leu Val Val Cys Thr Lys Cys Asp Ala Ile Ser Val Leu Glu Lys Glu His Asp Tyr Arg Asp Glu His Phe Asp Phe Ile Gln Ser His Ile Arg Lys Phe Cys Leu Gln Tyr Gly Ala Ala Leu Ile Tyr Thr Ser Val Lys Glu Asn Lys Asn Ile Asp Leu Val Tyr Lys Tyr Ile Val Gln Lys Leu Tyr Gly Phe Pro Tyr Lys Ile Pro Ala Val Val Val Glu Lys Asp Ala Val Phe Ile Pro Ala Gly Trp Asp Asn Asp Lys Lys Ile Gly Ile Leu His Glu Asn Phe Gln Thr Leu Lys Ala Glu Asp Asn Phe Glu Asp Ile Ile Thr Lys Pro Pro Val Arg Lys Phe Val His Glu Lys Glu Ile Met Ala Glu Asp Asp Gln Val Phe Leu Met Lys Leu Gln Ser Leu Leu Ala Lys Gln Pro Pro Thr Ala Ala Gly Arg Pro Val Asp Ala Ser Pro Arg Val Pro Gly Gly Ser Pro Arg Thr Pro Asn Arg Ser Val Ser Ser Asn Val Ala Ser Val Ser Pro Ile Pro Ala Gly Ser Lys Lys Ile Asp Pro Asn Met Lys Ala Gly Ala Thr Ser Glu Gly Val Leu Ala Asn Phe Phe Asn Ser Leu Leu Ser Lys Lys Thr Gly Ser Pro Gly Gly Pro Gly Val Ser Gly Gly Ser Pro Ala Gly Gly Ala Gly Gly Gly Ser Ser Gly Leu Pro Pro Ser Thr Lys Lys Ser Gly Gln Lys Pro Val Leu Asp Val His Ala Glu Leu Asp Arg I:Le Thr Arg Lys Pro Val Thr Val Ser Pro Thr Thr Pro Thr Ser Pro Thr Glu Gly Glu Ala Ser <210> 8 <211> 348 <212> PRT
<213> Homo Sapiens <220>
<221> misc_feature ._ <223> Incyte ID No: 236317$
<400> 8 Met Ala Lys Leu Leu Gln Pro Pro Pro Lys Phe Leu Pro Ser Glu Trp His Ile Ala Asn Lys Asn Gln Tyr His Arg Ala Asp Ala Gln Arg Ser Arg Ser Glu Arg Leu Val Aia Glu Ser Gln Arg Leu Val Asp Glu Ile Glu Lys Thr Thr Arg Lys Ser Gln Ser Asp Val Asn Lys Lys Leu Glu Gln Arg Leu Glu Glu Val Gln Phe Trp Lys Lys Glu Leu Asp Asp Lys Leu Glu Gln Leu Val Asn Val Thr Asp Asp Leu Leu Ile Tyr Lys Ile Arg Leu Glu Lys Ala Leu Glu Thr Leu Lys GIu Pro Leu His Ile Thr Glu Thr Cys Leu Ala Tyr Arg Glu Lys Arg Ile Gly Ile Asp Leu Val His Asp Thr Val Glu His Glu Leu Ile Lys Glu Ala Glu Ile Ile Gln Gly Ile Met Ala Leu Leu Thr Arg Thr Leu Glu Glu Ala Ser Glu Gln Ile Arg Met Asn Arg Ser Ala Lys Tyr Asn Leu Glu Lys Asp Leu Lys Asp Lys Phe Val Ala Leu Thr Ile Asp Asp Ile Cys Phe Ser Leu Asn Asn Asn Ser Pro Asn Ile Arg Tyr Ser Glu Asn Ala Val Arg Ile Glu Pro Asn Sex Val Ser Leu Glu Asp Trp Leu Asp Phe Ser Ser Thr Asn Val Glu Lys Ala Asp Lys Gln Arg Asn Asn Ser Leu Met Leu Lys Ala Leu Val Asp Arg Ile Leu Ser Gln Thr Ala Asn Asp Leu Arg Lys Gln Cys Asp Val Val Asp Thr Ala Phe Lys Asn Gly Leu Lys Asp Thr Lys Asp Ala Arg Asp Lys Leu Ala Asp His Leu Ala Lys Ile Glu Gly Asn Phe Ser Pro Ser Ser Gly Arg Ala Glu Arg Ala Ala Ser Gln Thr Ala Cys Pro Ala Gly Gly Asp Pro Gly Gln Arg Glu His His Leu Tyr Arg Arg Ser Ala Val Tyr Ala Asp Glu Glu Ile His Pro Thr Ser Gly Trp Gly Arg Pro Trp Gly Leu Gly Trp Gly Pro Pro Pro <210> 9 <211> 731 <212> PRT _ <213> Homo sapiens <220>
<221> misc_feature <223> Incyte ID No: 2363327 <400> 9 Met Gln Val Glu Leu Pro Pro Leu Glu Ile Asn Ser Arg Val Ser Leu Lys Val Gly Glu Thr Ile Glu Ser Gly Thr Val Ile Phe Cys Asp Val Leu Pro Gly Lys Glu Ser Leu Gly Tyr Phe Val Gly Val Asp Met Asp Asn Pro Ile Gly Asn Trp Asp Gly Arg Phe Asp Gly Val Gln Leu Cys Ser Phe Ala Cys Val Glu Ser Thr Ile Leu Leu His Ile Asn Asp Ile Ile Pro Glu Ser Val Thr Gln Glu Arg Arg Pro Pro Lys Leu Ala Phe Met Ser Arg Gly Val Gly Asp Lys Gly Ser Sex Ser His Asn Lys Pro Lys Ala Thr Gly Ser Thr Ser Asp Pro Gly Asn Arg Asn Arg Ser Glu Leu Phe Tyr Thr Leu Asn Gly Ser Ser Val Asp Ser Gln Pro Gln Ser Lys Ser Lys Asn Thr Trp Tyr Ile Asp Glu Val Ala Glu Asp Pro Ala Lys Ser Leu Thr Glu Ile Ser Thr Asp Phe Asp Arg Ser Ser Pro Pro Leu Gln Pro Pro Pro Val Asn Ser Leu Thr Thr Glu Asn Arg Phe His Ser Leu Pro Phe Ser Leu Thr Lys Met Pro Asn Thr Asn Gly Ser Ile Gly His Ser Pro Leu Ser Leu Ser Ala Gln Ser Val Met Glu Glu Leu Asn Thr Ala Pro Val Gln Glu Ser Pro Pro Leu Ala Met Pro Pro Gly Asn Ser His Gly Leu Glu Val Gly Ser Leu Ala Glu Val Lys Glu Asn Pro Pro Phe Tyr Gly Val Ile Arg Trp Ile Gly Gln Pro Pro Gly Leu Asn Glu Val Leu Ala Gly Leu Glu Leu Glu Asp Glu Cys Ala Gly Cys Thr Asp Gly Thr Phe Arg Gly Thr Arg Tyr Phe Thr Cys Ala Leu Lys Lys Ala Leu Phe Val Lys Leu Lys Ser Cys Arg Pro Asp Ser Arg Phe Ala Ser Leu Gln Pro Val Ser Asn Gln Ile Glu Arg Cys Asn Ser Leu Ala Phe Gly Gly Tyr Leu Ser Glu Val Val Glu Glu Asn Thr Pro Pro Lys Met Glu Lys Glu Gly Leu Glu Ile Met Ile Gly Lys Lys Lys Gly Ile Gln Gly His Tyr Asn Ser Cys Tyr Leu Asp Ser Thr Leu Phe Cys Leu Phe Ala Phe Sex Ser Val Leu Asp Thr Val Leu Leu Arg Pro Lys Glu Lys Asn Asp Val Glu Tyr Tyr Ser Glu Thr Gln Glu Leu Leu Arg Thr Glu Ile Val Asn Pro Leu Arg Ile Tyr Gly Tyr Val Cys Ala Thr Lys Ile Met Lys Leu Arg Lys Ile Leu Glu Lys Val Glu Ala Ala Ser Gly Phe Thr Ser Glu Glu Lys Asp Pro Glu Glu Phe Leu Asn Ile Leu Phe His His Ile Leu Arg Val Glu Pro Leu Leu Lys Ile Arg Ser Ala Gly Gln Lys Val Gln Asp Cys Tyr Phe Tyr Gln Ile Phe Met Glu 4$5 490 495 Lys Asn Glu Lys Val Gly Val Pro Thr Ile Gln Gln Leu Leu Glu Trp Ser Phe Ile Asn Ser Asn Leu Lys Phe Ala Glu Ala Pro Ser Cys Leu Ile Ile Gln Met Pro Arg Phe Gly Lys Asp Phe Lys Leu Phe Lys Lys Ile Phe Pro Ser Leu Glu Leu Asn Ile Thr Asp Leu Leu Glu Asp Thr Pro Arg Gln Cys Arg Ile Cys Gly Gly Leu Ala Met Tyr Glu Cys Arg Glu Cys Tyr Asp Asp Pro Asp Ile Ser Ala Gly Lys Ile Lys Gln Phe Cys Lys Thr Cys Asn Thr Gln Val His Leu His Pro Lys Arg Leu Asn His Lys Tyr Asn Pro Val Ser Leu Pro Lys Asp Leu Pro Asp Trp Asp Trp Arg His Gly Cys Ile Pro Cys Gln Asn Met Glu Leu Phe Ala Val Leu Cys Ile Glu Thr Ser His Tyr Val Ala Phe Val Lys Tyr Gly Lys Asp Asp Ser Ala Trp Leu Phe Phe Asp Ser Met Ala Asp Arg Asp Gly Gly Gln Asn Gly Phe Asn Ile Pro Gln Val Thr Pro Cys Pro Glu Val Gly Glu Tyr Leu Lys Met Ser Leu Glu Asp Leu His Ser Leu Asp Ser Arg Arg Ile Gln Gly Cys Ala Arg Arg Leu Leu Cys Asp Ala Tyr Met Cys Met Tyr Gln Ser Pro Thr Met Ser Leu Tyr Lys <210> 10 <211> 147 <212> PRT
<213> Homo sapiens <220>
<221> misc_feature <223> Incyte ID No: 2508327 <400> 10 Met Pro Pro Pro Gln Lys Ile Pro Ser Val Arg Pro Phe Lys Gln Arg Lys Ser Leu Ala Ile Arg Gln Glu Glu Val Ala Gly Ile Arg Ala Lys Phe Pro Asn Lys Ile Pro Val Val Val Glu Arg Tyr Pro Arg Glu Thr Phe Leu Pro Pro Leu Asp Lys Thr Lys Phe Leu Val Pro Gln Glu Leu Thr Met Thr Gln Phe Leu Ser Ile Ile Arg Ser Arg Met Val Leu Arg Ala Thr Glu Ala Phe Tyr Leu Leu Val Asn Asn Lys Ser Leu Val Ser Met Ser Ala Thr Met Ala Glu Ile Tyr Arg Asp Tyr Lys Asp Glu Asp Gly Phe Val Tyr Met Thr Tyr Ala Ser Gln Glu Thr Phe Gly Cys Leu Glu Ser Ala Ala Pro Arg Asp Gly Ser Ser Leu Glu Asp Arg Pro Cys Asn Pro Leu <210> 11 <211> 57 <212> PRT
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte ID No: 2524555 <400> 11 Met Pro Asn Cys Arg Glu Ser Ser Phe Ser Ser Ala Thr Met Ser Asp Lys Pro Asp Met Ala Glu Ile Glu Lys Phe Asp Lys Ser Lys Leu Lys Lys Thr Glu Thr Gln Glu Lys Asn Pro Leu Pro Ser Lys Glu Thr Ile Glu Gln Glu Lys Gln Ala Gly Glu Ser <210> 12 <211> 452 <212> PRT
<213> Homo Sapiens <220>
<221> misc_feature _ <223> Incyte ID No: 2900717 <400> 12 Met Glu Val Thr Thr Arg Leu Thr Trp Asn Asp Glu Asn His Leu Arg Lys Leu Leu Gly Asn Val Ser Leu Ser Leu Leu Tyr Lys Ser Ser Val His Gly Gly Ser Ile Glu Asp Met Val Glu Arg Cys Ser Arg Gln Gly Cys Thr Ile Thr Met Ala Tyr Ile Asp Tyr Asn Met Ile Val Ala Phe Met Leu Gly Asn Tyr Ile Asn Leu Arg Glu Ser Ser Thr Glu Pro Asn Asp Ser Leu Trp Phe Ser Leu Gln Lys Lys Asn Asp Thr Thr Glu Ile Glu Thr Leu Leu Leu Asn Thr Ala Pro Lys Ile Ile Asp Glu Gln Leu Val Cys Arg Leu Ser Lys Thr Asp Ile Phe Ile Ile Cys Arg Asp Asn Lys Ile Tyr Leu Asp Lys Met Ile Thr Arg Asn Leu Lys Leu Arg Phe Tyr Gly His Arg Gln Tyr Leu Glu Cys Glu Val Phe Arg Val Glu Gly Ile Lys Asp Asn Leu Asp Asp Ile Lys Arg Ile Ile Lys Ala Arg Glu His Arg Asn Arg Leu Leu Ala Asp Ile Arg Asp Tyr Arg Pro Tyr Ala Asp Leu Val Ser Glu Ile Arg Ile Leu Leu Val Gly Pro Val Gly Ser Gly Lys Ser Ser Phe Phe Asn Ser Val Lys Ser Ile Phe His Gly His Val Thr Gly Gln Ala Val Val Gly Ser Asp Thr Thr Ser Ile Thr Glu Arg Tyr Arg Ile Tyr Ser Val Lys Asp Gly Lys Asn Gly Lys Ser Leu Pro Phe Met Leu Cys Asp Thr Met Gly Leu Asp Gly Ala Glu Gly Ala Gly Leu Cys Met Asp Asp Ile Pro His Ile Leu Lys Gly Cys Met Pro Asp Arg Tyr Gln Phe Asn Ser Arg Lys Pro Ile Thr Pro Glu His Ser Thr Phe Ile Thr Ser Pro Ser Leu Lys Asp Arg Ile His Cys Val Ala Tyr Val Leu Asp Ile Asn Ser Ile Asp Asn Leu Tyr Ser Lys Met Leu Ala Lys Val Lys Gln Val His Lys Glu Val Leu Asn Cys Gly Ile Ala Tyr Val Ala Leu Leu Thr Lys Val Asp Asp Cys Ser Glu Val Leu Gln Asp Asn Phe Leu Asn Met Ser Arg Ser Met Thr Ser Gln Ser Arg Val Met Asn Val His Lys Met 380 385 390 _ Leu Gly Ile Pro Ile Ser Asn Ile Leu Met Val Gly Asn Tyr Ala Ser Asp Leu Glu Leu Asp Pro Met Lys Asp Ile Leu Ile Leu Ser Ala Leu Arg Gln Met Leu Arg Ala Ala Asp Asp Phe Leu Glu Asp Leu Pro Leu Glu Glu Thr Gly Ala Ile Glu Arg Ala Leu Gln Pro Cys Ile <210> 13 <211> 281 <212> PRT
<213> Homo sapiens <220>
<221> misc_feature <223> Incyte ID No: 3088904 <400> 13 Met Asp Ala Ile Lys Lys Lys Met Gln Met Leu Lys Glu Asn Ala Ile Asp Arg Ala Glu Gln Ala Glu Ala Asp Lys Lys Gln Ala Glu Asp Arg Cys Lys Gln Leu Glu Glu Glu Gln Gln Ala Leu Gln Lys Lys Leu Lys Gly Thr Glu Asp Glu Val Glu Lys Tyr Ser Glu Ser Val Lys Glu Ala Gln Glu Lys Leu Glu Gln Ala Glu Lys Lys Ala Thr Asp Ala Glu Ala Asp Val Ala Ser Leu Asn Arg Arg Ile Gln Leu Val Glu Glu Glu Leu Asp Arg Ala Gln Glu Arg Leu Ala Thr Ala Leu Gln Lys Leu Glu Glu Ala Glu Lys Ala Ala Asp Glu Ser Glu Arg Gly Met Lys Val Ile Glu Asn Arg Ala Met Lys Asp Glu Glu Lys Met Glu Leu Gln Glu Met Gln Leu Lys Glu Ala Lys His Ile Ala Glu Asp Ser Asp Arg Lys Tyr Glu Glu Val Ala Arg Lys Leu Val IIe Leu Glu Gly Glu Leu Glu Arg Ser Glu Glu Arg Ala Glu Val Ala Glu Ser Arg Ala Arg Gln Leu Glu Glu Glu Leu Arg Thr Met Asp Gln Ala Leu Lys Sex Leu Met Ala Ser Glu Glu Glu Tyr Ser Thr Lys Glu Asp Lys Tyr Glu Glu Glu Ile Lys Leu Leu Glu Glu Lys Leu Lys Glu Ala Glu Thr Arg Ala Glu Phe Ala Glu Arg Ser Val Ala Lys Leu Glu Lys Thr Ile Asp Asp Leu Glu Glu Thr Leu Ala Ser Ala Lys Glu Glu Asn Val Glu Ile His Gln Thr Leu Asp Gln Thr Leu Leu Glu Leu Asn Asn Leu <210> 14 <211> 92 <212> PRT
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte ID No: 3745193 <400> 14 Met Asp Glu Gln Ile Arg Leu Met Asp Gln Asn Leu Lys Cys Leu Ser Ala Ala Glu Glu Lys Tyr Ser Gln Lys Glu Asp Lys Cys Glu Glu Glu Met Lys Ile Leu Thr Asp Asn Leu Lys Glu Ala Glu Thr His Ala Glu Leu Ala Glu Arg Ser Val Ala Lys Leu Glu Lys Thr Ile Asp Asp Leu Glu Asp Lys Leu Lys Cys Thr Lys Glu Glu His Leu Cys Thr Gln Arg Met Leu Asp Gln Thr Leu Leu Asp Leu Asn Glu Met <210> 15 <211> 448 <212> PRT
<213> Homo sapiens <220>
<221> misc_feature <223> Incyte ID No: 3822123 <400> 15 Met Arg Glu Cys Ile Ser Val His Val Gly Gln Ala Gly Val Gln Ile Gly Asn Ala Cys Trp Glu Leu Phe Cys Leu Glu His Gly Ile Gln Ala Asp Gly Thr Phe Asp Ala Gln Ala Ser Lys Ile Asn Asp Asp Asp Ser Phe Thr Thr Phe Phe Ser Glu Thr Gly Asn Gly Lys His Val Pro Arg Ala Val Met Ile Asp Leu Glu Pro Thr Val Val Asp Glu Vai Arg Ala Gly Thr Tyr Arg Gln Leu Phe His Pro Glu Gln Leu Ile Thr Gly Lys Glu Asp Ala Ala Asn Asn Tyr Ala Arg Gly His Tyr Thr Val Gly Lys Glu Ser Ile Asp Leu Val Leu Asp Arg Ile Arg Lys Leu Thr Asp Ala Cys Sex Gly Leu Gln Gly Phe Leu Ile Phe His Ser Phe Gly Gly Gly Thr Gly Ser Gly Phe Thr Ser Leu Leu Met Glu Arg Leu Ser Leu Asp Tyr Gly Lys Lys Ser Lys Leu Glu Phe Ser Ile Tyr Pro Ala Pro Gln Val Ser Thr Ala Val Val Glu Pro Tyr Asn Ser Tyr Leu Thr Thr His Thr Thr Leu Glu His Ser Asp Cys Ala Phe Met Val Asp Asn Glu Ala Ile Tyr Asp Ile Cys Arg Arg Asn Leu Asp Ile Glu Arg Pro Thr Tyr Thr Asn Leu Asn Arg Leu Ile Ser Gln Ile Val Ser Ser Ile Thr Ala Ser Leu Arg Phe Asp Gly Ala Leu Asn Val Asp Leu Thr Glu Phe Gln Thr Asn Leu Val Pro Tyr Pro Arg Ile His Phe Pro Leu Ala Thr Tyr Ala Pro Val Ile Ser Ala Glu Lys Ala Tyr His Glu Gln Leu Ser Val Ala Glu Ile Thr Asn Ala Cys Phe Glu Pro Ala Asn Gln Met Val Lys Cys Asp Pro Arg His Gly Lys Tyr Met Ala Cys Cys Leu Leu Tyr Arg Gly Asp Val Val Pro Lys Asp Val Asn Ala Ala Ile Ala Ala Ile Lys Thr Lys Arg Ser Ile Gln Phe Val Asp Trp Cys Pro Thr Gly Phe Lys Val Gly Ile Asn Tyr Gln Pro Pro Thr Val Val Pro Gly Giy Asp Leu Ala Lys Val Gln Arg Ala Val Cys Met Leu Ser Asn Thr Thr Ala Ile Ala Glu Ala Trp Ala Arg Leu Asp His Lys Phe Asp Leu Met Tyr Ala Lys Arg Ala Phe Val His Trp Tyr Val Gly Glu Gly Met Glu Glu Gly Glu Phe Ser Glu Ala Arg Glu Asp Met Ala Ala Leu Glu Lys Asp Tyr Glu Glu Val Gly Ile Asp Ser Tyr Glu Asp Glu Asp Glu Gly Glu Glu <210> 16 <211> 269 <212> PRT
<213> Homo Sapiens <220>
<221> misc_feature _ <223> Incyte ID No: 4217506 <400> 16 Met Ser Asp Glu Glu Val Glu Gln Val Glu Glu Gln Tyr Glu Glu Glu Glu Glu Ala His Glu Glu Ala Ala Glu Val His Glu Glu Val His Glu Pro Glu Glu Val Gln Glu Asp Thr Ala Glu Glu Asp Ala Glu Glu Glu Lys Pro Arg Pro Lys Leu Thr Ala Pro Lys Ile Pro Glu Gly Glu Lys Val Asp Phe Asp Asp Ile Gln Lys Lys Arg Gln Asn Lys Asp Leu Met Glu Leu Gln Ala Leu Ile Asp Ser His Phe Glu Ala Arg Lys Lys Glu Glu Glu Glu Leu Val Ala Leu Lys Glu Arg Ile Glu Lys Arg Arg Ala Glu Arg Ala Glu Gln Gln Arg Ile Arg Ala Glu Lys Glu Arg Glu Arg Gln Asn Arg Leu Ala Glu Glu Lys Ala Arg Arg Glu Glu Glu Asp Ala Lys Arg Arg Ala Glu Asp Asp Leu Lys Lys Lys Lys Ala Leu Ser Ser Met Gly Ala Asn Tyr Ser Ser Tyr Leu Ala Lys Ala Asp Gln Lys Arg Gly Lys Lys Gln Thr Ala Arg Glu Met Lys Lys Lys Ile Leu Ala Glu Arg Arg Lys Pro Leu Asn Ile Asp His Leu Gly Glu Asp Lys Leu Arg Asp Lys Ala Lys Glu Leu Trp Glu Thr Leu His Gln Leu Glu Ile Asp Lys Phe Glu Phe Gly Glu Lys Leu Lys Arg Gln Lys Tyr Asp Ile Thr Thr Leu Arg Ser Arg Ile Asp Gln Ala Gln Lys His Ser Lys Lys Ala Gly Thr Pro Ala Lys Gly Lys Val Gly Gly Arg Trp Lys <210> 17 <211> 3620 <212> DNA
<213> Homo Sapiens <220>
<221> misc feature <223> Incyte ID No: 1285395 <400> 17 tgccggcggc tgggactgaa gagggacggg tcccgcggcg agcgagctcc tgagcataag 60 ctgtggccat gactactgaa gtaggctctg tgtctgaagt gaagaaggac tctagccagt 120 taggaacaga tgcaaccaag gaaaaaccta aagaagtagc agaaaatcag cagaatcagt 180 cttccgatcc agaggaggaa aaaggttccc agccacctcc tgcagctgaa agccaaagta 240 gtctacgccg ccagaagaga gagaaggaaa catcggagag caggggtatt tctcggttca 300 taccgccatg gcttaagaag caaaagtcat ataccttagt agtggccaaa gatggaggag 360 ataaaaaaga gcctacccaa gctgttgttg aagaacaggt cttagataaa gaggaacccc 420 ttccagaaga acagagacag gctaagggtg atgctgaaga aatggctcag aagaaacaag 480 agattaaagt tgaagtcaag gaagaaaaac cctcagtgag caaggaagaa aaaccctcag 540 tgagcaaagt ggagatgcag cctactgaat tagtaagtaa ggagagagaa gagaaggtaa 600 aagaaacaca ggaagacaaa ttagaaggag gagcagcaaa aagggagacc aaggaagtgc 660 agaccaatga gctgaaagca gagaaggcat ctcaaaaagt caccaagaag accaaaactg 720 tccagtgtaa agtgaccctc ttagatggca ccgaatacag ctgtgacctg gagaaacatg 780 ccaagggaca agtgttattt gacaaagtgt gtgaacacct caatctcttg gagaaagact 840 actttggact tttgtttcag gaaagccctg agcagaaaaa ctggttagat cctgctaaag 900 aaataaagag acaactgaga aaccttccat ggctattcac ttttaatgtg aagttttatc 960 ctcctgatcc ttctcaattg actgaagata tcaccagata cttcttgtgc cttcagctcc 1020 ggcaggacat tgcctctggc cgcctgccct gctcttttgt gactcatgct ctcctgggat 1080 cctacaccct gcaggctgaa cttggtgact atgacccaga agaacatggc agcatcgacc 1140 tcagtgaatt ccagtttgcc cctactcaga ctaaggagct ggaagagaag gtggcagagc 1200 tgcacaaaac ccacaggggc ttatcgccag cacaagctga ttcccagttc ttagaaaatg 1260 caaagaggct ttccatgtat ggtgttgacc tacatcatgc caaggactca gaaggtgtgg 1320 aeatcaagct gggcgtgtgt gctaatggac ttctcattta caaagacaga ctgcgaatca 1380 atcgttttgc ttggccgaaa atcttaaaaa tttcctataa acgcagtaac ttctacatta 1440 aagtcagacc ggcagagctg gaacagtttg agagtaccat tggattcaaa ctgccaaacc 1500 accgggcagc gaaaagacta tggaaagtgt gcgtggagca tcatactttc tacaggcttg 1560 tttctccaga gcagccacca aaagccaagt tcctgacctt ggggtccaaa tttcgctata 1620 gtggccgcac ccaagcacag acccgccagg ccagcaccct catagatagg ccagcaccac 1680 actttgagcg cacttctagt aaacgggtct ccaggagtct agatggagct ccgattggtg 1740 tcatggacca aagtcttatg aaggattttc ctggcgctgc tggggagatt tcagcctatg 1800 gacctggact tgtcagcatt gccgtggtac aagatgggga cggcaggagg gaagtgagaa 1860 gcccaactaa agccccacat ttgcagctca ttgaaggaaa gaaaaattcc ttgagagtag 1920 aaggggataa tatttatgtc agacatagca atttaatgtt ggaggaactg gataaggccc 1980 aggaggacat actgaaacat caggctagca ttagtgaact caagcgcaat tttatggaat 2040 ccacacctga gccgcgccct aatgaatggg aaaaacgacg tatcacacct ctgtccctac 2100 agacacaagg gagttcacat gagactctga atatagtgga ggagaagaag cgggcagagg 2160 ttgggaaaga cgaaagagta atcacagaag aaatgaatgg taaagagata tcacctggga 2220 gtggtcctgg ggagattcgt aaggtggagc ctgtgacaca aaaagactcc acctccctgt 2280 cttctgagag cagcagcagc agcagtgaga gtgaggagga agacgtggga gagtaccgtc 2340 cccaccaccg agtgaccgag ggcaccatca gggaggaaca ggagtatgaa gaagaggtgg 2400 aggaagaacc ccgcccggca gccaaggtag tagagaggga ggaagcagtg cccgaagcca 2460 gcccagtcac acaagcaggt gccagtgtaa tcacagtaga aacagtgatc caggaaaatg 2520 taggtgccca aaagataccc ggagagaaga gtgtacacga aggcgctctt aagcaagaca 2580 tgggagaaga agcagaggaa gagccacaga aagttaacgg agaggtgtcc catgttgaca 2640 ttgatgtttt gccacaaatt atttgttgtt cagagccacc agtggtaaaa acagagatgg 2700 taacaatttc tgatgcctca caaaggacag aaatctccac caaggaagtc cccattgtcc 2760 aaactgagac caaaaccatc acatatgagt ctccacagat tgatggcggg gctggtggtg 2820 attcgggcac gttactgacc gcacaaacca tcacatctga gtccgtgtca acaacgacaa 2880 ccacacacat caccaagact gtaaaaggtg gaatttctga aacaagaatt gagaaacgca 2940 ttgtgatcac aggagatgga gatattgatc atgaccaggc actggctcag gcgatcaggg 3000 aagccagaga gcagcaccct gacatgtcgg tcacaagagt ggtggtacac aaagaaacag 3060 agttggctga ggaaggggaa gattaagtaa gaaagtcatt ttttaaacaa cactcaactt 3120 tgtgaacccc tgaagatttt ttgaccgttc caagtcttaa tgccacacca ctattccagc 3180 gaatttatgc tacaactggt aacaatgacc agaagcctga agaattaaaa tgccaacacc 3240 aaacctttcc ttaccagctc tggtctatat tgctcccatg catttaatat attattttgt 3300 tttataacca cttctaaata ttctcagttc tttctttttg ttgttgttaa ttaaggggtt 3360 ttggttttgt tttctgttta ctttgtgtgc aactacctgc ttttaatgac tcactttgat 3420 caaatgacag tgaacaaagc cagcccaagc tggtaaggtg ctgttcactt gaacaggtgc 3480 tgttgcgcag aaaggaaact ctgtgactaa tttagatagt ggctttcctt cttctggatt 3540 -cttttcattg aattctcaca gtaaatattt acggagtttt caaattgcag caaatatact 3600 gtatgagaaa atattaatac 3620 <210> 18 <211> 4687 <212> DNA
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte ID No: 1320252 <400> 18 cactccagct tgggtgacag agtgagaccg ggagagggag ccgccgcagc cgccggcgct 60 gtggagatat tctctaagcc gctttcatca tgggagaaat agagcagagg ccgaccccag 120 gatcacgact gggggccccg gaaaattcgg ggatcagtac cttggaacgt ggacagaagc 180 cgcccccaac accttcagga aaactcgtgt ccatcaaaat ccagatgctg gatgacaccc 240 aggaggcatt tgaagttcca caaagagctc ctgggaaggt gctgctggat gcagtttgca 300 accacctcaa cctcgtggaa ggtgactatt ttggcctcga gtttcctgat cacaaaaaga 360 tcacggtgtg gctggatctc ctaaaaccca ttgtgaaaca gattagaagg ccaaagcacg 420 ttgttgttaa gtttgtggtg aaattctttc cgcctgacca cacacaactc caagaagaac 480 tcacaaggta cctgttcgcg ctgcaggtga agcaggactt ggctcaaggc aggttgacgt 540 gtaatgacac cagcgcagct ctcttgattt cacacattgt gcaatctgag attggggatt 600 ttgatgaagc cttggacaga gagcacttag caaaaaataa atacatacct cagcaagacg 660 cactagagga caaaatcgtg gaatttcacc ataaccacat tggacaaaca ccagcagaat 720 cagatttcca gctcctagag attgcccgtc ggctagagat gtatggaatc cggttgcacc 780 cggccaagga cagggaaggc acgaagatca atctggccgt tgccaacacg ggaattctag 840 tgtttcaggg tttcactaag atcaatgcct tcaactgggc caaggtgcgg aagctgagct 900 tcaagaggaa gcgctttttc atcaagctcc ggccagatgc caatagtgcg taccaggata 960 ccttggaatt cctgatggcc agtcgggatt tctgcaagtc cttctggaaa atctgtgttg 1020 aacatcatgc cttctttaga ctttttgaag agcccaaacc aaagcccaag cccgtcctct 1080 ttagccgggg gtcatcattt cggttcagtg gtcggactca gaagcaggtt ctcgactatg 1140 ttaaagaagg aggacataag aaggtgcagt ttgaaaggaa gcacagcaag attcattcta 1200 tccggagcct tgcttcacag cctacagaac tgaattcgga agtgctggag cagtctcagc 1260 agagcaccag ccttacattt ggagaaggtg ccgaatctcc agggggccag agctgccggc 1320 gaggaaagga accgaaggtt tccgccgggg agccggggtc gcacccgagc cctgcgccga 1380 ggagaagccc cgcgggtaac aagcaggcgg acggagccgc ctcggcgccc acggaggaag 1440 aggaggaggt cgttaaggat aggacccagc agagtaaacc tcagcccccg cagccaagca 1500 caggctccct gactggcagt cctcaccttt ccgagctgtc tgtgaactcg caggggggag 1560 tggcccctgc caacgtgacc ttgtctccca acctgagccc cgacaccaag caggcctctc 1620 ccttgatcag cccgctgctg aatgaccagg cctgcccccg gacggacgat gaggatgagg 1680 gccggaggaa gagattccca actgataaag cgtacttcat agctaaggaa gtgtctacca 1740 ccgagcgaac atatctgaag gatctcgaag ttatcacttc gtggtttcag agcacagtga 1800 gcaaagagga cgccatgccg gaagcactga aaagtctcat attcccgaat tttgaacctt 1860 tgcacaaatt tcatactaat tttctcaagg aaattgagca acgacttgcc ctgtgggaag 1920 gccgctcaaa tgcccaaatc agagattacc aaagaatcgg cgatgtcatg ctgaagaaca 1980 ttcagggcat gaagcacctg gcggctcacc tgtggaagca cagcgaggcc ttggaggccc 2040 tggagaatgg aatcaagagc tcccggcggc tggagaactt ctgcagagac tttgagctgc 2100 agaaggtgtg ttacctaccg ctcaacacct tcctcctgcg gccactgcac cggctcatgc 2160 actacaagca ggtcctggag cggctgtgca aacaccaccc gccgagccac gccgacttca 2220 gggactgccg agccgctttg gcagagatca cggagatggt ggcacagctc cacggtacga 2280 tgatcaagat ggagaatttc cagaagctgc acgaactcaa gaaagatttg attggcattg 2340 acaatcttgt ggttccggga agggagttca tccgtctggg cagcctcagc aagctctcgg 2400 ggaaggggct ccagcagcgc atgttcttcc tgttcaacga cgtcctgcta tacacgagcc 2460 gggggctgac ggcctccaat cagtttaaag tccacgggca gctcccgctc tatggcatga 2520 cgattgagga gagcgaagac gagtgggggg tgccccactg cctgaccctc cggggccagc 2580 ggcagtccat catcgtggcc gccagttctc ggtccgagat ggagaagtgg gttgaggaca 2640 tccagatggc cattgacctg gcggagaaga gcagcagccc cgcccctgag ttcctggcca 2700 gcagcccccc tgacaacaag tcccctgatg aagccaccgc ggctgaccag gagtcagagg 2760 atgacctgag cgcctcgcgc acatcgctgg agcgccaggc cccgcaccgc ggcaacacaa 2820 tggtgcacgt gtgctggcac cgcaacacca gcgtctccat ggtggacttc agcatcgcag 2880 tggagaatca gttgtctgga aacctgctga ggaaattcaa aaacagcaac gggtggcaga 2940 agctgtgggt ggtgttcaca aacttctgcc tgttcttcta caaatcacac caggacaatc 3000 atccccttgc cagcctgcct ctgctcggct actcgctcac catcccctct gagtccgaga 3060 acatccagaa agactacgtg ttcaagctgc acttcaagtc ccacgtctac tacttcaggg 3120 cggaaagcga gtacacgttc gaaaggtgga tggaagtgat ccgcagtgcc accagctctg 3180 cctcgcgacc ccacgtgttg agtcacaaag agtctcttgt gtattgatgg ccggacacac 3240 tcgtttccgc agtggctgct ttcctggaag acgtttcctt tcttctgtat taatgaagcc 3300 tggtaaaatt aacacctgtc tgaaaatcaa aaacatggct tcccagcagc tctcctgtct 3360 ccacagccgc gttttttaac cccgacctct cagcgtctga atgaacagcg ctcccacctc 3420 cagtcctggc atccgctggg ggcgctgttc tttagctagt gccagtatta aaacattgtc 3480 attacgagag tgccaaatga catcttccct ccaccctgcc cctgaaaaac agtacacaca 3540 catccgttca acacaagaca gggcaagtgt ttttcttcct aaaaaaagtt ctttctttta 3600 ttattttcac ctattggctg ctgcatttta cgaagtggac ttcccggtgt ttgtttgttt 3660 gtttgcaata cactcagtgc agccttaagc aaatgagatc attttcagat ttcatttttt 3720 ttttcagtct ttctactttt gtaataatag gaagttagta ggactcactt ctctgattaa 3780 taagcaattt gcagcacaca gcgttccact gcggggtttc acgctcacct gaaaacacct 3840 gttcccaacc tacttcttgg tgcaagttga ccaaatcgtt ttaagtggta actctttcca 3900 accgtagcag ggttgttttc tgttaagcaa agccgagatc cagtgcaata cctggactgt 3960 caccgtcctg tgagtggtgt acacaatggg aagataataa gccgtggtgt tttgctgtct 4020 gtctgtgtca caagcatgaa aacccgtgtg tcattgatca gcaccatttg tggtatgttc 4080 cctgatgagc gtttagtgag cctgctggct gcagagcact atgaaatcat ggtacgtagt 4140 ccccggcacc tgtcgttatt cctatatcct cctgcaactg tggtttgaaa ctgcgcattc 4200 tctagtagta tatatcgtgc ctgtcttcaa aaacatttcc ctttttatac tcattccccc 4260 caggcatggg gtagtgtcag tcggactgca cagggaacac ggtttccagt ggctttggcc 4320 cctactcggg aaacgtctgc ctgttctcga tggtgatggg gtggctgcca ttcccttggt 4380 tttcctaagc cctttctaac gagagtctca aacaagcgga ggcgagggcc aattcaaccc 4440 cattctttcc agcgccccgc accatagcac ctgcccacct gagaaccagg aacgcaccct 4500 ctctgtggag ctctgactgg tgtacctgga aacaaacagc aacttgcaaa cggacgaaga 4560 gcctgccgtg tgttaatcat ttgccttaca agatgtacca gacggtttcc agtactaaca 4620 aagggaataa aaatacctca cgccacaatc cagcatattg atgttttaag gcaaaacaaa 4680 aaaaaaa 4687 <210> 19 <211> 2331 <212> DNA
<213> Homo sapiens <220>
<221> misc_feature <223> Incyte ID No: 1259001 <400> 19 gggaaatact gggttggagg caggtgcact ggggggggaa tctcagaggt gtggatgtgt 60 tttcaggcac ctgaagaact cgtcttgcat ttggctgtca aagtcgccaa ccaggcttcc 120 ctgcctctgg tggatttcat catccagaac ggtggtcacc tggatgccaa ggctgctgac 180 gggaacacgg ctctgcacta cgcagcactc tacaaccagc ccgactgcct caagctgctg 240 ctgaagggga gagctttggt tggcacagta aatgaagcag gcgagacagc tctggacata 300 gccaggaaga agcaccacaa ggagtgtgag gagctgctgg agcaggccca ggcggggacc 360 tttgccttcc ctctacatgt ggactactcc tgggtaattt ccacagagcc tggctctgac 420 agtgaggagg atgaggaaga gaagcgctgc ttgctgaagc tcccggccca ggctcactgg 480 gccagtggga ggctggacat cagcaacaag acctatgaga ctgtcgccag cctgggagca 540 gccacccctc agggcgagag tgaggactgt cccccgccct tgccagtcaa aaactcttct 600 cggactttgg tccaagggtg tgcaagacat gccagtggag atcgttctga agtctccagc 660 ctgagttcag aggcccctga gacccctgag agcctgggca gtccagcctc ctcctccagt 720 ctgatgagcc ccttggaacc tggggatccc agccaagccc cacccaactc tgaagagggc 780 ctccgagagc ccccaggcac ctccagaccc agcctgacat ccgggaccac cccttcggag 840 atgtacctcc ccgtcagatt cagctccgag agcactcgct cctatcggcg gggggcgcgg 900 agccctgaag atggtccctc agccaggcag cctctgccca gaaggaacgt gccggttggc 960 atcactgaag gagatggctc aaggactggg agtctcccag caagttctgt gcaacttttg 1020 caagactagc tccttgctgg cccccacatg ccccatgcta ggccccaatg ttcagagctg 1080 ggacttgagc tcacaaaact ggggagctga gacatttgtt ctcttggatc tcactctctc 1140 tgtcccttgt gcctctgtag ctggccttct tcctgccaca ggccatgcct ctaccaagga 1200 cacatggcct ttccctgtta gggctgatgg cggttctttc ctatctcatt acccgctagg 1260 ggcctgggag ccctgtggct ggatctgagt gctcctgagc tggcttcagc tgcagaactc 1320 tcagtccctc atcagatcga gactctattt cccccgtcag tctgggggct tcacaagggc 1380 aggagagccc tccatcactg acttccagat cagggaccct gccaagtagg gactgtcttc 1440 tcagccagcc atttattagt ctaatattcc ttcactaaat tccaactcta tgtctggacc 1500 tgtgttaggc acttcagata ccacacgagt aagacaaggg ccctgcaggg gtggtccttt 1560 ggtggaaagc tggtcttaag ggttgggctt gggaataggc agggtcagat tccagggcat 1620 ggctctggac tcagctggtt tatacctata tgaccattac agttgtctac agatcacatc 1680 cattctggct ggtcaacatg catgctgtac tggctgttaa ataaaaatat tctgaatgtc 1740 actccttttg agggacagca cagccttccc taggcattct cctatattcc ccagccaaat 1800 tgtagagtca gatgcaccca catttgcctg tgtccttgat ttagcaggaa ggaaaggaat 1860 agtcggggtt gatggatgcc cacttctctt ctctttctct tggtcaactc aggagccttt 1920 tagtctgagg gaatggagag gcaaagaaag aagggagagt aatagaattg ggagggcaga 1980 gacttaaggg ttctgcttcc cagccctaga aattctatca ttgctcagcc ccaatgagaa 2040 agcagataca cctaagccat catcaaccac taacatctca acttgccagt tgctgggtgc 2100 tgggccctgg caggaatggg ccaagccaag caggggagac tagagagcac caatggccaa 2160 cacagctgcc tggctgggga ggctgtgctg tttcccctgg agacctgact ggtctgtggt 2220 tcccacagga acagggttgt cttttgagcc cccagtgtct ggtttcattc atctcagact 2280 tgttatttca ctcatctcta ataaaggatt ggggggtcaa aaaaaaaaaa a 2331 <210> 20 <211> 1918 <212> DNA
<213> Homo sapiens <220>
<221> misc_feature <223> Incyte ID No: 1627027 <400> 20 cagtggccct gggaccagct ctgctccttg caccccgctc cctgcctgga cacaggctca 60 ctcgctgcct tcttctgggg gaaaccagct tcttgccagc cacagctgct gcctccgcca 120 ctggccaccg cccctgtcct gggagtccct tggcccagac acccacctga cttagtggct 180 cctctgcagg aaaggtggct gccccctgcg ttcctccatc caaccatgag ctggtgccca 240 tcaccactga gaatgcacca aagaatgtag tggacaaggg agaaggagcc tcccggggtg 300 gaaacacacg gaaaagcctc gaggacaacg gctccaccag ggtcaccccg agtgtccagc 360 cccacctcca gcccatcaga aacatgagtg tgagccggac catggaggac agctgtgagc 420 tggacctggt gtacgtcaca gagaggatca tcgctgtctc cttccccagc acagccaatg 480 aggagaactt ccggagcaac ctccgtgagg tggcgcagat gctcaagtcc aaacatggag 540 gcaactacct gctgttcaac ctctctgagc ggagacctga catcacgaag ctccatgcca 600 aggtactgga atttggctgg cccgacctcc acaccccagc cctggagaag atctgcagca 660 tctgtaaggc catggacaca tggctcaatg cagaccctca caatgtcgtt gttctacaca 720 acaagggaaa ccgaggcagg ataggagttg tcatcgcggc ttacatgcac tacagcaaca 780 tttctgccag tgcggaccag gctctggacc ggtttgcaat gaagcggttc tatgaggata 840 agattgtgcc cattggccag ccatcccaaa gaaggtacgt gcattacttc agtggcctgc 900 tctccggctc catcaaaatg aacaacaagc ccttgtttct gcaccacgtg atcatgcacg 960 gcatccccaa ctttgagtct aaaggaggat gtcggccatt tctccgcatc taccaggcca 1020 tgcaacctgt gtacacatct ggcatctaca acatcccagg agacagccag actagcgtct 1080 gcatcaccat cgagccagga ctgctcttga agggagacat cttgctgaag tgctaccaca 1140 agaagttccg aagcccagcc cgagacgtca tcttccgtgt gcagttccac acctgtgcca 1200 tccatgacct gggggttgtc tttgggaagg aggaccttga tgatgctttc aaagatgatc 1260 gatttccaga gtatggcaaa gtggagtttg tattttctta tgggccagag aaaattcaag 1320 gcatggagca cctggagaac gggccgagcg tgtctgtgga ctataacacc tccgaccccc 1380 tcatccgctg ggactcctac gacaacttca gtgggcatcg agatgacggc atggaggatg 1440 ggaacaaaca gaataccaat agccagtcaa ttggaagcat ctcaggtggc ctggaggatc 1500 aatatacctg gccagacaca cactggcctt cccaaagtta ggccctccaa aatgtcaagt 1560 tgccaagcca gaatccaaag ctttgcactg agaacttcat gttggggctc ctgtcccctc 1620 ctggagccct gcctgggtga caccccagtc cttctattca ggtcctgtgt tcacctctgg 1680 gaagtgcagg accctgtggg gttttttctt tttccttttc caagccaagg aagtgttctg 1740 gctcctggac tctgccctgt ggaggggaaa gggggagaga gagggaaaaa aagatttcca 1800 ccttattgtt cagcagctgg aaaattgttg tcatctgttc tgccttccat ttgctgagaa 1860 tggaaaaaaa atgtggaaaa tttggggaaa taaatctcaa taccctcaaa aaaaaaaa 1918 <210> 21 <211> 1386 <212> DNA
<213> Homo sapiens <220>
<221> misc_feature <223> Incyte ID No: 1905315 <400> 21 cgggcggctc cacacgcgct gcgcccgccg ccggccccac gcgcggccca tgtcctccgc 60 gccgcgctcg cccaccccgc ggccccgcag gatgaagaag gacgagtcgt tcctgggcaa 120 gctgggcggc accctggcca ggaagcggag ggcgcgcgag gtgagtgacc tgcaggaaga 180 aggcaagaat gccatcaact caccgatgtc ccccgccctg gcggatgttc accctgaaga 240 cacccagctc gaggagaacg aggagcgcac gatgattgac cccacttcca aggaagaccc 300 caagttcaag gaactggtca aggtcctcct cgactggatt aatgacgtgc tggtggagga 360 gaggatcatt gtgaagcagc tggaggaaga cctgtatgac ggccaggtgc tgcagaagct 420 cttggaaaaa ctggcagggt gcaagctgaa tgtggctgag gtgacacagt ccgaaatagg 480 gcagaaacag aagctgcaga cggtgctgga agcagtacat gacctgctgc ggccccgagg 540 ctgggcgctc cggtggagcg tggactcaat tcacgggaag aacctggtgg ccatcctcca 600 cctgctggtc tctctggcca tgcacttcag ggcccccatc cgccttcctg agcatgtaac 660 ggtgcaggtg gtggtcgtgc ggaaacggga aggcctgctg cattccagcc acatctcgga 720 ggagctgacc acaactacag agatgatgat gggccggttc gagcgggatg ccttcgacac 780 gctgttcgac cacgccccgg ataagctcag cgtggtgaag aagtctctca tcacttttgt 840 gaacaagcac ctgaacaagc tgaatttgga ggtgacggaa ctggagaccc agtttgcaga 900 tggcgtgtac ctggttctgc tcatgggcct tctggaagac tactttgttc ctctccacca 960 cttctacctg actccggaaa gcttcgatca gaaggtccac aatgtgtcct tcgcctttga 1020 gctgatgctg gacggaggcc tcaagaaacc caaggctcgt cctgaagacg tggttaactt 1080 ggacctcaaa tccaccctga gggttcttta caacctgttc accaagtaca agaacgtgga 1140 gtgacggggg agctgtggat ggtggcagga gtgtcccagc aagaaaggcg gcatccgtct 1200 gtgccctgtg cctttccagg gagccaggcg ccatgggctt ctggtccaag ctgtgttgac 1260 _ tgtcatcccc accccacccc tacctcacgc ctgccccacc ccctgcctct tttggttgtt 1320 gttcttaatc tcctctccat gtagttccca gtgggcaaga gcctttgaaa atgcaggatt 1380 ctaaaa 1386 <210> 22 <211> 1745 <212> DNA
<213> Homo sapiens <220>
<221> misc_feature <223> Incyte ID No: 1997789 <400> 22 ccgggtcgcc cctggcccgg cgcgcccgtc cccggcagcg acaatgagtg aacagagtat 60 ctgccaagcc cgggcttccg tgatggtcta cgatgacacc agtaagaaat gggtaccaat 120 caaacctggc cagcagggat tcagccggat caacatctac cacaacactg ccagcaacac 180 cttcagagtc gttggagtca agttgcagga tcagcaggtt gtgatcaatt attcaatcgt 240 gaaagggctg aagtacaatc aggccacgcc aaccttccac cagtggcgag atgcccgcca 300 ggtctacggc ttaaactttg caagtaaaga agaggcaacc acgttctcca atgcaatgct 360 gtttgccctg aacatcatga attcccaaga aggaggcccc tccagccagc gtcaggtgca 420 gaatggcccc tctcctgatg agatggacat ccagagaaga caagtgatgg agcagcacca 480 gcagcagcgt caggaatctc tagaaagaag aacctcggcc acagggccca tcctcccacc 540 aggacatcct tcatctgcag ccagcgcccc cgtctcatgt agtgggcctc caccgccccc 600 cccacctcta gtcccacctc cacccactgg ggctacccca cctcccccac ccccactgcc 660 agccggagga gcccaggggt ccagccacga cgagagctcc atgtcaggac tggccgctgc 720 catagctggg gccaagctga gaagagtcca acggccagaa gacgcatctg gaggctccag 780 tcccagtggg acctcaaagt ccgatgccaa ccgggcaagc agcgggggtg gcggaggagg 840 cctcatggag gaaatgaaca aactgctggc caagaggaga aaagcagcct cccagtcaga 900 caagccagcc gagaagaagg aagatgaaag ccaaatggaa gatcctagta cctccccctc 960 tccggggacc cgagcagcca gccagccacc taactcctca gaggctggcc ggaagccctg 1020 ggagcggagc aactcggtgg agaagcctgt gtcctcgatt ctgtccagga tgaagcctgc 1080 tgggagcgtg aatgacatgg ccctggatgc cttcgacttg gaccggatga agcaggagat 1140 cctagaggag gtggtgagag agctccacaa ggtgaaggag gagatcatcg acgccatcag 1200 gcaggagctg agtgggatca gcaccacgta aggggccggc ctcgctgcgc tgattcgtcg 1260 agcccatccg gcgacagagg acagccagaa gcccagccag ccccagactc cagtgcacca 1320 gagcacgcac aggagcctgg gcgcgctgct gtgaaacgtc ctgacctgtg atcacacatg 1380 acagtgagga aaccaagtgc aactcctggg tttttttaga ttctgcctga cacggaacac 1440 caggtctgct cgtctttttt gtgttttata tttgcttatt taaggtacat ttctttgggt 1500 ttctagagac gcccctaagt cacctgcttc attagacggt ttccaggttt tctcccaggt 1560 gacgctgtta gcgcctcagc tggcggtgac agccggccca gcgtggcgcc accacacacc 1620 gcagagctgt ccaggcacag ctccgtcccc agcgctcatg gtgttgaaac tgtctgtcat 1680 gcaccacggt gtctgtgtcc acacagtaat aaacggttta ctgtccgcaa aaaaaaaaaa 1740 aaagg 1745 <210> 23 <211> 2451 <212> DNA
<213> Homo sapiens <220>
<221> misc_feature _ <223> Incyte ID No: 2303465 <400> 23 cgcctcagcc gcctcgcaca tttagtcttg ccgggagtgg tgtgattccc gaccaagatg 60 gcggccgtgg ggcgagtcgg ctccttcggt tcttctccgc cgggattatc ctcgacttac 120 actggcggcc ccttgggcaa cgagatagcg tcgggcaacg gtggcgccgc ggcaggcgac 180 gacgaggacg ggcagaacct ttggtcctgc atcctcagcg aggtctccac ccgctcgcgc 240 tccaagctcc ctgcggggaa gaacgtgcta ctgctgggtg aagatggagc tggaaaaaca 300 agcttaataa gaaaaattca gggaatagag gagtataaga aaggaagagg attggaatat 360 ttgtacttaa atgtgcatga tgaagacagg gatgatcaaa caagatgtaa tgtttggatc 420 ttagatggag acctatatca caaaggcctc cttaaatttt cactggatgc cgtatctctg 480 aaggatactc tagttatgct ggttgttgac atgtcaaagc cttggactgc tttggattct 540 ttacagaaat gggcaagtgt tgttagagaa catgttgaca aactgaaaat ccctcctgaa 600 gaaatgaaac aaatggaaca aaagttgatt agagacttcc aagaatatgt agagccagga 660 gaagacttcc cggcttctcc ccagagaaga aatactgcgt cacaagaaga caaagatgac 720 agtgtagttt tacctctggg tgcggataca cttacacata acttgggcat tccagtacta 780 gtagtttgca caaagtgtga tgccattagt gtattggaga aagaacatga ctacagagat 840 gaacattttg attttattca gtcacatatc cggaagtttt gtttacagta tggtgcagca 900 cttatttaca cttcagtaaa agaaaacaaa aatatagact tagtatataa atacatcgtt 960 cagaaactat atggatttcc ctataagatt cctgctgttg ttgtggaaaa ggatgcagta 1020 tttattccag cagggtggga taatgataag aaaataggaa tattacatga aaattttcaa 1080 acattaaaag cagaagataa ttttgaagac atcataacta aaccacctgt tcgaaagttt 1140 gtacatgaga aggaaattat ggcagaagat gatcaggtgt ttcttatgaa gctacagtcc 1200 cttttagcaa agcaaccacc aactgcagct ggaaggcctg tggatgcctc accaagagtc 1260 ccaggaggct ccccacgaac accaaataga tctgtatcat ctaatgttgc cagcgtgtca 1320 cccattcctg ctgggtcaaa aaaaattgat ccaaacatga aagctggagc tacaagtgaa 1380 ggcgttctgg caaatttctt caacagtttg ttgagtaaaa agactggctc tccaggaggc 1440 cctggtgtga gtggtggtag ccctgcaggt ggggctggag gtggaagcag tggtttacca 1500 ccatccacca aaaagtcagg ccagaagcct gtcttagatg ttcatgcaga actagacaga 1560 attacacgaa aaccagttac agtttctccc acaacaccta catctcctac ggaaggagaa 1620 gcttcttgaa gataccaaat aaagccattt attctgtttt ctgggataat gtaaacatgc 1680 ctctgccttt tccttcaaaa gtggaattag aaagctggag tgcttcttca gatggactaa 1740 atttatgtcg tgtgtgtgtg tgtgtgtgtg tgtggttacc cattttttag aaggagccgt 1800 acagaagaaa aattattcta cattatgtag gattgctgtt tgcattgcca ttttgcataa 1860 gaaagtaatt tttgattttg aaaatctcaa aacttttaga tctgaaatac agccatgtga 1920 tcgatcatat tctaaaggct atttaaaaca tgtaaaagga tttggggaac ggcagaaaac 1980 atgcagtttg ggcatttgac tgacttggaa gtctaagctt attttagtta taactattaa 2040 aatcattttt aaaaatttgt tagttttgct gacagagaaa aatcgtcagt tgtcagattt 2100 ', tgcacaccac aataaatgta ccaccacaca cggaatatgc taagaaaact atcagatagc 2160 gtttgataca ctagtcattg tctcaatcac tgatcctgta agttgtcatc aaaatatgat 2220 ttagaaatat tggccaaggt gttgctttaa ctgaggagaa aagaaagcac actgcctaaa 2280 tgtgtaaaag aaaaatgcag aggttattaa aatgtaaaga agtaacaatc tttggatttg 2340 tctatacata tatatatata tatatggctt tgccttaata tacccccttt tttgtttgtg 2400 actttcaact gtaatcagtt aataaagtat ttattctctg caaaaaaaaa a 2451 <210> 24 <211> 1162 <212> DNA
<213> Homo sapiens <220>
<221> misc_feature <223> Incyte ID No: 2363178 <400> 24 ggaggctccc atcccgagct tgcgtagtgg ttctgtgtgg cgctgaggaa tttggaaacc 60 tcaaatggct aaactattac aacctccgcc caagttcctg ccctcagagt ggcacattgc 120 taacaagaac cagtaccaca gagcagacgc tcaaaggtcc cgatcagaac gcctggtcgc 180 agaaagccag aggcttgtgg atgaaattga aaagaccaca agaaaatctc aaagcgatgt 240 gaacaagaaa ctagaacaga gactcgagga agtccagttc tggaagaagg agttagatga 300 caaacttgag cagcttgtga atgtaactga tgatctactc atatataaga tcagattgga 360 aaaagccctg gagaccttga aagagccctt gcacatcact gagacatgcc tggcatacag 420 ggagaagcgc attggcattg acctggtgca cgacacagtg gagcatgagc tgataaagga 480 ggctgagatc atccagggca ttatggctct gctgacccgt accttggagg aggcttccga 540 gcagattcgg atgaaccgct ctgccaagta caatcttgag aaggatttga aggacaagtt 600 tgtggccctg accatagatg atatctgctt ctcgctcaac aacaactcac caaacatcag 660 atattctgag aacgccgtga ggattgagcc aaactccgtg agtctggaag actggttgga 720 cttctccagc accaatgtgg agaaggctga caagcagcgg aacaactccc tgatgctgaa 780 agccctggtg gatcgaatcc tgtcccagac agccaatgat ctgcgcaagc agtgtgatgt 840 ggtggacacg gcattcaaga atgggctgaa ggatacaaag gatgccaggg acaagctggc 900 tgatcatctg gccaagattg aaggaaactt tagcccaagc tcaggcagag ctgaaagggc 960 tgcatcgcag acagcttgcc ctgcaggagg agatccaggt caaagagaac accatttata 1020 tcgacgaagt gctgtgtatg cagatgagga aatccatccc acttcgggat ggggaagacc 1080 atggggtctg ggctgggggc ctccgccctg atgctgtctg ctaatagtag ggctagttcc 1140 aattctcatt aaaccgggat ga 1162 <210> 25 <211> 4527 <212> DNA
<213> Homo sapiens <220>
<221> misc_feature <223> Incyte ID No: 2363327 <400> 25 agcccgggac gcgggctggg gagccggggc gaggggcgac gccccgccgc ccgagtttcc 60 ccctttctag ggtgaggatg gttctacaca gccacccgga gttccttagt tgaaaggtgc 120 gccctgctgt gacagcatgg acaccacgtt gctgaaaaca tgctttggga ctgccactga 180 atttatcttt tgcggtttta tgacaaagtt attagtagtt tccctttttt gaattagtat 240 tttgaagtta atatcacaat gagtttccag gcttatggag ccaagaaaaa gtcacttcac 300 cctactggga agagcggatt ttttacttgc ttcttcaaga atgcagcgtt acagacaaac 360 aaacacaaaa gctccttaaa gtaccgaagg aagtatagga cagtatattc aagatcgttc 420 tgtggggcat tcaaggattc cttctgcaaa aggcaagaaa aatcagattg gattaaaaat 480 tctagagcaa cctcatgcag ttctctttgt tgatgaaaag gatgttgtag agataaatga 540 aaagttcaca gagttacttt tggcaattac caattgtgag gagaggttca gcctgtttaa 600 aaacagaaac agactaagta aaggcctcca aatagacgtg ggctgtcctg tgaaagtaca 660 gctgagatct ggggaagaaa aatttcctgg agttgtacgc ttcagaggac ccctgttagc 720 agagaggaca gtctccggaa tattctttgg agttgaattg ctggaagaag gtcgtggtca 780 aggtttcact gacggggtgt accaagggaa acagcttttt cagtgtgatg aagattgtgg 840 cgtgtttgtt gcattggaca agctagaact catagaagat gatgacactg cattggaaag 900 tgattacgca ggtcctgggg acacaatgca ggtcgaactt cctcctttgg aaataaactc 960 cagagtttct ttgaaggttg gagaaacaat agaatctgga acagttatat tctgtgatgt 1020 tttgccagga aaagaaagct taggatattt tgttggtgtg gacatggata accctattgg 1080 caactgggat ggaagatttg atggagtgca gctttgtagt tttgcgtgtg ttgaaagtac 1140 aattctattg cacatcaatg atatcatccc agagagtgtg acgcaggaaa ggaggcctcc 1200 caaacttgcc tttatgtcaa gaggtgttgg ggacaaaggt tcatccagtc ataataaacc 1260 aaaggctaca ggatctacct cagaccctgg aaatagaaac agatctgaat tattttatac 1320 _ cttaaatggg tcttctgttg actcacaacc acaatccaaa tcaaaaaata catggtacat 1380 tgatgaagtt gcagaagacc ctgcaaaatc tcttacagag atatctacag actttgaccg 1440 ttcttcacca ccactccagc ctcctcctgt gaactcactg accaccgaga acagattcca 1500 ctctttacca ttcagtctca ccaagatgcc caataccaat ggaagtattg gccacagtcc 1560 actttctctg tcagcccagt ctgtaatgga agagctaaac actgcacccg tccaagagag 1620 tccacccttg gccatgcctc ctgggaactc acatggtcta gaagtgggct cattggctga 1680 agttaaggag aaccctcctt tctatggggt aatccgttgg atcggtcagc caccaggact 1740 gaatgaagtg ctcgctggac tggaactgga agatgagtgt gcaggctgta cggatggaac 1800 cttcagaggc actcggtatt tcacctgtgc cctgaagaag gcgctgtttg tgaaactgaa 1860 gagctgcagg cctgactcta ggtttgcatc attgcagccg gtttccaatc agattgagcg 1920 ctgtaactct ttagcatttg gaggctactt aagtgaagta gtagaagaaa atactccacc 1980 aaaaatggaa aaagaaggct tggagataat gattgggaag aagaaaggca tccagggtca 2040 ttacaattct tgttacttag actcaacctt attctgctta tttgctttta gttctgttct 2100 ggacactgtg ttacttagac ccaaagaaaa gaacgatgta gaatattata gtgaaaccca 2160 agagctactg aggacagaaa ttgttaatcc tctgagaata tatggatatg tgtgtgccac 2220 aaaaattatg aaactgagga aaatacttga aaaggtggag gctgcatcag gatttacctc 2280 tgaagaaaaa gatcctgagg aattcttgaa tattctgttt catcatattt taagggtaga 2340 acctttgcta aaaataagat cagcaggtca aaaggtacaa gattgttact tctatcaaat 2400 ttttatggaa aaaaatgaga aagttggcgt tcccacaatt cagcagttgt tagaatggtc 2460 ttttatcaac agtaacctga aatttgcaga ggcaccatca tgtctgatta ttcagatgcc 2520 tcgatttgga aaagacttta aactatttaa aaaaattttt ccttctctgg aattaaatat 2580 aacagattta cttgaagaca ctcccagaca gtgccggata tgtggagggc ttgcaatgta 2640 tgagtgtaga gaatgctacg acgatccgga catctcagct ggaaaaatca agcagttttg 2700 taaaacctgc aacactcaag tccaccttca tccgaagagg ctgaatcata aatataaccc 2760 agtgtcactt cccaaagact tacccgactg ggactggaga cacggctgca tcccttgcca 2820 gaatatggag ttatttgctg ttctctgcat agaaacaagc cactatgttg cttttgtgaa 2880 gtatgggaag gacgattctg cctggctctt ctttgacagc atggccgatc gggatggtgg 2940 tcagaatggc ttcaacattc ctcaagtcac cccatgccca gaagtaggag agtacttgaa 3000 gatgtctctg gaagacctgc attccttgga ctccaggaga atccaaggct gtgcacgaag 3060 actgctttgt gatgcatata tgtgcatgta ccagagtcca acaatgagtt tgtacaaata 3120 actggggtca tcgggaaagg caaagaaact gaaggcagag tcctaacgtt gcatcttatt 3180 cgagctggca gttctgttca cgtccattgc cggcaatgga tgtctttgtg gtgatgatcc 3240 ttcagaaaag gatgcctctg tttaaaaaca aattgctttt gtgtccctga agtatttaat 3300 aagaagcatt ttgcactcta gaaagtatgt ttgtgttggt tttttaagaa gtctaaatga 3360 agttattaat acctgaagct ttaagttaag tgcattgatc atatgatatt tttggaagca 3420 tacaatttta attgtggaag tttaaagcct cttttagtcc attgagaatg taaataaatg 3480 tgtcttcttt atggaccaag gatatgaaat catttttctt ttgtagctaa cggttgcctt 3540 gaggaagaaa taatttggtt ttattaagag tctactctca atccagttat tagagatgta 3600 ctgagtttga tttgttaatc ctttctatat actgctgatc ttgcatgtct acaatctgct 3660 cagtttttct gtgtttctgc aatagtggtc agaaaaatac ttaaattccc ttaatggtgt 3720 tgttttctat ttgttctggt tttgagataa atgagtgatt ctgtccccaa atgtccattt 3780 ttgaagtgat tttcctggag gattagggta tttagcagtt gaagctcttc attcatagta 3840 gttactgtca gctaacaggt tttttaaggc ttttaactat taatatttta tggaatgggg 3900 caaagtaaat tgatgaaaga attggagtga taatagtcct ttacaaacat acagtccata 3960 agaaaatgaa tttggcatat agaattatta caatttcctg ggagagatgg atatttaaac 4020 ctctattatt ttagacaaga ctgtctagaa cttaagtttg atctgtcagc cagtactccc 4080 attaaattca gtgtagtttc acttgataga atcagatatg ttatcgaaat gttagcagca 4140 gcttcatcct ccttctgatt aaagtaagta gaaatgggat gttttgttta ataacagcca 4200 tagtgtgtgt ttagaccaca gcggatgttg tagaccagga ccatagatga tacatgtcag 4260 tgctgtggaa tgtgcattct ctgagtgttg ttttgtggta tcattgtctt tcctgaatga 4320 ctttctaact gtgcagaaag gcagaaaagt catcatatgt atatgtcata tgactttata 4380 aaatatttaa tgtgacaaaa agtggaaaga atctttacaa accctgcaat tactttttta 4440 aaggcacttt tactctttgg ttttatcatt ccattttgct aatatttact agctttataa 4500 attacagtaa ggtacaaaaa aaaaaaa 4527 <210> 26 <211> 870 <212> DNA
<213> Homo sapiens <220>
<221> misc_feature <223> Incyte ID No: 2508327 <400> 26 ggaggaatga gttaggttcc cggttgcggg acagtttttt tttctttttt aaaacagaca 60 cagctactga gtgcaatgcc gcctccacag aaaatcccaa gcgtcagacc cttcaagcag 120 aggaaaagct tggcaatcag acaagaggaa gttgctggaa tccgggcaaa gttccccaac 180 aaaatcccgg tggtagtgga gcgctacccc agggagacgt tcctgccccc gctggacaaa 240 accaagttcc tggtcccgca ggagctgacc atgacccagt tcctcagcat catccggagc 300 cgcatggtcc tgagagccac ggaagccttt tacttgctgg tgaacaacaa gagcctggtc 360 agcatgagcg caaccatggc agagatctac agagactaca aggatgagga tggcttcgtg 420 tacatgacct acgcctccca ggagacattt ggctgcctgg agtcagcagc ccccagggat 480 gggagcagcc ttgaggacag accctgcaat cctctctagc ccatgtcggg aaggatgtgt 540 gctctgacag acgtgtcaga tgctggcaga agggattggt tttctcctgt gtatacagtg 600 gaggagtctg agaagcaggg atgcctgggg tgatcagctc caaccagtgg cagcagagtg 660 gtggctcttt cctagtttag tttttttttc cgtttttttg tgtccttcta agaaaaatgt 720 gggccgcccc tgaaaggtat ataattatca tttttttttt tttgaaacag ggtcttgtct 780 tactgccttg gttggagcac agtgttgcct tggattgtac ctactgtact tccatcctgt 840 gcttttaatg tgactcgctg tacttgaaga 870 <210> 27 <211> 729 <212> DNA
<213> Homo sapiens <220>
<221> misc_feature <223> Incyte ID No: 2524555 <400> 27 gtggccactg cgcagaccag acttcgctcg tactcgtgcg cctcgcttcg gtgagcccca 60 gggcccctgc ctccttcctc ctgccgtcct gcctccgtcc ccgccctttc atcatccgcg 120 tccctgtgaa ggcattccct aaatccgagc ccgagtggtt ctccccggga aggctacttt 180 ggggagctgg ggggatgcga aacaccctag atactggata atggggtggg gaaatcgatg 240 atttaagaac aaaaccgaaa aactggcgtt ttgtcgtgcc gctcggaggg gacattaaaa 300 aatttcttag tgtttgcccg caaaggtatt gtgcgttgcc ttggaggctg agatatgggg 360 gaatagacaa gtcctttgtt ctgaggttca tcttccgagc cccgagcctc ctcccagcct 420 cggacggctg cgcgggctgc atctgtgcag cctggcggcg gcggggctgt gctatgacat 480 ctttacagtc cttcttgcag agacatgtgt gccagggatg ccgaattgcc gggagagcag 540 cttttcctcc gcaaccatgt ctgacaaacc cgatatggct gagatcgaga aattcgataa 600 gtcgaaactg aagaagacag agacgcaaga gaaaaatcca ctgccttcca aagaaacgat 660 tgaacaggag aagcaagcag gcgaatcgta atgaggcgtg cgccgccaat atgcactgta 720 cattccaca 729 <210> 28 <211> 2062 _ <212> DNA
<213> Homo sapiens <220>
<221> misc_feature <223> Incyte ID No: 2900717 <400> 28 gagtctctga agccacagat ctcttaagaa ctttctgtct ccaaaccgtg gctgctcgat 60 aaatcagaca gaacagttaa tcctcaattt aagcctgatc taacccctag aaacagatat 120 agaacaatgg aagtgacaac aagattgaca tggaatgatg aaaatcatct gcgcaagctg 180 cttggaaatg tttctttgag tcttctctat aagtctagtg ttcatggagg tagcattgaa 240 gatatggttg aaagatgcag ccgtcaggga tgtactataa caatggctta cattgattac 300 aatatgattg tagcctttat gcttggaaat tatattaatt tacgtgaaag ttctacagag 360 ccaaatgatt ccctatggtt ttcacttcaa aagaaaaatg acaccactga aatagaaact 420 ttactcttaa atacagcacc aaaaattatt gatgagcaac tggtgtgtcg tttatcgaaa 480 acggatattt tcattatatg tcgagataat aaaatttatc tagataaaat gataacaaga 540 aacttgaaac taaggtttta tggccaccgt cagtatttgg aatgtgaagt ttttcgagtt 600 gaaggaatta aggataacct agacgacata aagaggataa ttaaagccag agagcacaga 660 aataggcttc tagcagacat cagagactat aggccctatg cagacttggt ttcagaaatt 720 cgtattcttt tggtgggtcc agttgggtct ggaaagtcca gttttttcaa ttcagtcaag 780 tctatttttc atggccatgt gactggccaa gccgtagtgg ggtctgatac caccagcata 840 accgagcggt ataggatata ttctgttaaa gatggaaaaa atggaaaatc tctgccattt 900 atgttgtgtg acactatggg gctagatggg gcagaaggag caggactgtg catggatgac 960 attccccaca tcttaaaagg ttgtatgcca gacagatatc agtttaattc ccgtaaacca 1020 attacacctg agcattctac ttttatcacc tctccatctc tgaaggacag gattcactgt 1080 gtggcttatg tcttagacat caactctatt gacaatctct actctaaaat gttggcaaaa 1140 gtgaagcaag ttcacaaaga agtattaaac tgtggtatag catatgtggc cttgcttact 1200 aaagtggatg attgcagtga ggttcttcaa gacaactttt taaacatgag tagatctatg 1260 acttctcaaa gccgggtcat gaatgtccat aaaatgctag gcattcctat ttccaatatt 1320 ttgatggttg gaaattatgc ttcagatttg gaactggacc ccatgaagga tattctcatc 1380 ctctctgcac tgaggcagat gctgcgggct gcagatgatt ttttagaaga tttgcctctt 1440 gaggaaactg gtgcaattga gagagcgtta cagccctgca tttgagataa gttgccttga 1500 ttctgacatt tggcccagcc tgtactggtg tgccgcaatg agagtcaatc tctattgaca 1560 gcctgcttca gattttgctt ttgttcgttt tgccttctgt ccttggaaca gtcatatctc 1620 aagttcaaag gccaaaacct gagaagcggt gggctaagat aggtcctact gcaaaccacc 1680 cctccatatt tccgtaccat ttacaattca gtttctgtga catcttttta aaccactgga 1740 ggaaaaatga gatattctct gagatattct ctaatttatt cttctataac actctatata 1800 gagctatgtg agtactaatc acattgaata atagttataa aattattgta tagacatctg 1860 cttcttaaac agattgtgag ttctttgaga aacagcgtgg attttactta tctgtgtatt 1920 cacagagctt agcacagtgc ctggtaatga gcaagcatac ttgccattac ttttccttcc 1980 cactctctcc aacatcacat tcactttaaa tttttctgta tatagaaagg aaaactagcc 2040 tgggcaacat gatgaaaccc ct 2062 <210> 29 <211> 1020 <212> DNA

<213> Homo sapiens <220>
<221> misc_feature <223> Incyte ID No: 3088904 <400> 29 _ ctcgcctgcc accggtgcac ccagtccgct cacccagccc agtccgtccg gtcctcaccg 60 cctgccggcc ggcccacccc ccaccgcagc catggacgcc atcaagaaga agatgcagat 120 gctgaaggag aacgccatcg accgcgccga gcaggccgaa gccgacaaga agcaagctga 180 ggaccgctgc aagcagctgg aggaggagca gcaggccctc cagaagaagc tgaaggggac 240 agaggatgag gtggaaaagt attctgaatc cgtgaaggag gcccaggaga aactggagca 300 ggccgagaag aaggccactg atgctgaggc agatgtggcc tccctgaacc gccgcattca 360 gctggttgag gaggagctgg accgggccca ggagcgcctg gctacagccc tgcagaagct 420 ggaggaggcc gagaaggcgg ctgatgagag cgagagagga atgaaggtca tcgaaaaccg 480 ggccatgaag gatgaggaga agatggaact gcaggagatg cagctgaagg aggccaagca 540 catcgctgag gattcagacc gcaaatatga agaggtggcc aggaagctgg tgatcctgga 600 aggagagctg gagcgctcgg aggagagggc tgaggtggcc gagagccgag ccagacagct 660 ggaggaggaa cttcgaacca tggaccaggc cctcaagtcc ctgatggcct cagaggagga 720 gtattccacc aaagaagata aatatgaaga ggagatcaaa ctgttggagg agaagctgaa 780 ggaggctgag acccgagcag agtttgccga gaggtctgtg gcaaagttgg agaaaaccat 840 cgatgaccta gaagagacct tggccagtgc caaggaggag aacgtcgaga ttcaccagac 900 cttggaccag accctgctgg aactcaacaa cctgtgaggg ccagccccac ccccagccag 960 gctatggttg ccaccccaac ccaataaaac tgatgttact agcctctcaa aaaaaaaaaa 1020 <210> 30 <211> 1120 <212> DNA
<213> Homo sapiens <220>
<221> misc_feature <223> Incyte ID No: 3745193 <400> 30 cggtgccacc gcggcgcaga ggagtctgca atgccgagtg gaggaaggag gaaccggagt 60 gtgagcagta gctgggtggg cagcatggct gggatcacca ccatcgaggc agtgaagcgc 120 aagatccagg ttctgcagca ccaggcagat gatgcagagg agtgagctga gcacctccag 180 tgagaagctg agggaaaaag gtgggcctgg gaacaggcag aggctgaagt ggcctccgtg 240 aacggtagga tccagctggt tgaagaggag ctggactgtg ctcaggagcg cctggccact 300 gccctgcaaa agctggaaga agcgggaaaa gctgctgatg agagtgagag agatacaaag 360 gttattgaaa tctgggcctt aaaagatgaa gaagatggaa ctccaggaaa tccaactcaa 420 agaagctaag cacattgcag atgaggcaga tgggaagtat gaagaggtgg ctcgtaagtt 480 ggtgatcatt gaaggagaca tgggatgcac agaggaacga gctgagctgg cagagtcccg 540 ttgctgagag atggatgagc agatcagact gatggaccag aacctgaagt gtctgagtgc 600 agctgaagaa aagtactctc aaaaagaaga caaatgtgag gaagagatga agattcttac 660 tgataatctc aaggaggcag agacccatgc tgagttggct gagagatcag tagccaagct 720 ggaaaagaca attgatgact tggaagataa actgaaatgc accaaagagg aacacctctg 780 ' tacacaaagg atgctggacc agactttgct tgacctgaat gagatgtaga atgccccagt 840 cccaccctgc tgctgctcct tcctctgacc ctgactctgc ctgaggccag cctgcccgaa 900 gctgaccttt acctgagggc tgatctttaa cgggaaggct gctttctcct tttgccaccc 960 cctccttccc tgcctctttt tcaccaaact gtctctgcct cttcctggag attccagctg 1020 ggctagaggc tgagaacctt tgcaaacaac atttaaggga atgtgagccc aatgcataat 1080 gtctttaaaa atcatgttga gaaaaaaaaa atacataata 1120 <210> 31 <21I> 1471 <212> DNA
<213> Homo Sapiens <220> _ <221> misc_feature <223> Incyte ID No: 3822123 <400> 31 caggcccagc tgagaggtgc gcgggcgagg acagcggcag cgatgcggga atgcatatca 60 gtccacgtgg gccaagcggg agttcagatt ggcaatgcct gctgggagct cttctgcctg 120 gaacacggca tccaggcaga cggcactttt gatgctcaag ctagcaagat caacgatgat 180 gactccttca ccaccttttt cagcgagact ggcaatggga agcatgtgcc ccgggccgtc 240 atgatagatc tggagcctac tgtagtggat gaggttcggg caggaaccta ccgccagctc 300 ttccatccag agcagctgat cacaggaaag gaggatgcag ccaacaacta tgcccggggc 360 cactacacgg tgggcaagga gagcattgac ctggtgctgg accgcatacg gaagctgaca 420 gatgcttgct ctggcctgca gggcttcctg attttccaca gttttggtgg gggcactggc 480 tccggcttca cttctctgct gatggaacgc ctctccctgg attatggcaa gaaatccaag 540 ctggaattct ccatctaccc agccccccag gtgtctacag ccgtggtcga gccctacaac 600 tcttatctga ccacccacac caccctggag cactcagact gtgccttcat ggtggacaac 660 gaagcaatct atgacatctg ccgccgcaac ctagacatcg agcgcccaac ctacaccaac 720 ctcaatcgcc tcattagcca aattgtctcc tccatcacag cttctctgcg ctttgacggg 780 gccctcaatg tggacctgac agagttccag accaacctgg tgccctaccc tcgcatccac 840 ttccccctgg ccacctatgc accagtcatc tctgcagaaa aggcatacca cgagcagctg 900 tcggtggcag agatcaccaa tgcctgcttt gagcctgcca accagatggt aaagtgtgat 960 ccccggcacg gcaagtacat ggcctgctgc ctgctgtacc gtggagatgt ggtgcccaag 1020 gatgtcaacg ctgccattgc cgccatcaag accaagcgca gcattcagtt tgtggactgg 1080 tgccccacag gcttcaaggt tggtatcaac taccagcctc ccactgtggt gcctgggggt 1140 gacctggcca aggtgcagcg tgccgtgtgc atgctgagca acacgaccgc catcgccgag 1200 gcctgggccc gcctggacca caagttcgac ctgatgtatg ccaagagggc gtttgtgcac 1260 tggtatgtgg gtgagggcat ggaggagggt gagttctccg aggcccgtga ggatatggct 1320 gccctggaga aggattatga ggaggtgggc atcgactcct atgaggacga ggatgaggga 1380 gaagaataaa gcagctgcct ggagcctatt cactatgttt attgcaaaat cctttcgaaa 1440 taaacagttt ccttgcaaaa aaaaaaaaaa a 1471 <210> 32 <211> 1079 <212> DNA
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte ID No: 4217506 <400> 32 cagcccttct cacactcgac ccgcagaaac cacccacctt caccatgtct gacgaggaag 60 ttgaacaggt ggaggagcag tacgaagaag aagaggaagc ccatgaggaa gctgcagaag 120 tccatgagga agttcatgaa ccagaggaag ttcaagaaga caccgcagag gaggacgcgg 180 aagaggagaa accgagaccc aaactcactg ctcctaagat cccagaaggg gagaaagtgg 240 acttcgatga catccagaag aagcgtcaga acaaagacct aatggagctc caggccctca 300 tcgacagcca ctttgaagcc cggaagaagg aggaggagga gctggtcgct ctcaaagaga 360 gaatcgagaa gcgccgtgca gagagagcgg agcagcagag gattcgtgca gagaaggaga 420 gggagcgcca gaacagactg gcggaggaaa aggccagaag ggaggaggag gatgccaaga 480 ggagggcaga ggacgacctg aagaagaaga aagctctgtc ttccatggga gccaactaca 540 gcagctacct ggccaaggct gaccagaaga gaggcaagaa gcagacagcc cgggaaatga 600 agaagaagat tctggctgag agacgcaagc cgctcaacat cgatcacctt ggtgaagaca 660 aactgaggga caaggccaag gagctctggg agaccctgca ccagctggag attgacaagt 720 tcgagtttgg ggagaagctg aaacgccaga aatatgacat caccacgctc aggagccgca 780 ttgaccaggc ccagaagcac agcaagaagg ctgggacccc agccaagggc aaagtcggcg 840 ggcgctggaa gtagagaggc cagaaaggcc cctcgaggca gagaccctcc gccctcttgc 900 acaccagggc cgctcgtggg actccacatc ctccagcccc cacaatcctg tcaggggctc 960 cctgacagtc ctgggggtgg agaggccatc ccggggcgtc ccccgcgtct gtgtccttgc 1020 tgccttcatc ccctggggcc tgtgaataaa gctgcagaac ccccaaaaaa aaaaaaaaa 1079

Claims (20)

What is claimed is:

1. A substantially purified polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID
NO:5, SEQ ID
NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ
ID NO:12.
SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, and fragments thereof.

2. A substantially purified variant having at least 90% amino acid sequence identity to the amino acid sequence of claim 1.

3. An isolated and purified polynucleotide encoding the polypeptide of claim 1.

4. An isolated and purified polynucleotide variant having at least 90%
polynucleotide sequence identity to the polynucleotide of claim 3.

5. An isolated and purified polynucleotide which hybridizes under stringent conditions to the polynucleotide of claim 3.

6. An isolated and purified polynucleotide having a sequence which is complementary to the polynucleotide of claim 3.

7. A method for detecting a polynucleotide, the method comprising the steps of:
(a) hybridizing the polynucleotide of claim 6 to at least one nucleic acid in a sample, thereby forming a hybridization complex; and (b) detecting the hybridization complex, wherein the presence of the hybridization complex correlates with the presence of the polynucleotide in the sample.

8. The method of claim 7 further comprising amplifying the polynucleotide prior to hybridization.

9. An isolated and purified polynucleotide comprising a polynucleotide sequence selected from the group consisting of SEQ ID NO:18, SEQ ID NO:19, SEQ ID
NO:20, SEQ ID
NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID
NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ 1D NO:32, and fragments thereof.

10. An isolated and purified polynucleotide variant having at least 90%
polynucleotide sequence identity to the polynucleotide of claim 9.

11. An isolated and purified polynucleotide having a sequence which is complementary to the polynucleotide of claim 9.

12. An expression vector comprising at least a fragment of the polynucleotide of claim 3.

13. A host cell comprising the expression vector of claim 12.

14. A method for producing a polypeptide, the method comprising the steps of:
a) culturing the host cell of claim 13 under conditions suitable for the expression of the polypeptide; and b) recovering the polypeptide from the host cell culture.

15. A pharmaceutical composition comprising the polypeptide of claim 1 in conjunction with a suitable pharmaceutical carrier.

16. A purified antibody which specifically binds to the polypeptide of claim 1.

17. A purified agonist of the polypeptide of claim 1.

18. A purified antagonist of the polypeptide of claim 1.

19. A method for treating or preventing a disorder associated with decreased expression or activity of CYSKP, the method comprising administering to a subject in need of such treatment an effective amount of the pharmaceutical composition of claim 15.

20. A method for treating or preventing a disorder associated with increased expression or activity of CYSKP, the method comprising administering to a subject in need of such treatment an effective amount of the antagonist of claim 18.