CA2450969A1 - Nucleic acid-associated proteins - Google Patents

Abstract

Various embodiments of the invention provide human nucleic acid-associated proteins (NAAP) and polynucleotides which identify and encode NAAP.
Embodiments of the invention also provide expression vectors, host cells, antibodies, agonists, and antagonists. Other embodiments provide methods for diagnosing, treating, or preventing disorders associated with aberrant expression of NAAP.

Description

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NUCLEIC ACID-ASSOCIATED PROTEINS
TECHNICAL FIELD
The invention relates to novel nucleic acids, nucleic acid-associated proteins encoded by these nucleic acids, and to the use of these nucleic acids and proteins in the diagnosis, treatment, and prevention of cell proliferative, neurological, reproductive, developmental, autoimmunelinflammatory, and DNA repair disorders, and infections. The invention also relates to the assessment of the effects of exogenous compounds on the expression of nucleic acids and nucleic acid-associated proteins.
1o BACKGROUND OF THE INVENTION
Multicellular organisms are comprised of diverse cell types that differ dramatically both in structure and function. The identity of a cell is determined by its characteristic pattern of gene expression, and different cell type express overlapping but distinctive sets of genes throughout development. Spatial and temporal regulation of gene expression is critical for the control of cell proliferation, cell differentiation,,. apoptosis, and other processes that contribute to organismal development. Furthermore, gene expression is regulated in response to extracellular signals that mediate cell-cell communication and coordinate the activities of different cell types. Appropriate gene regulation also ensures that cells function efficiently by expressing only those genes whose functions are required at a given time.
The cell nucleus contains all of the genetic information of the cell in the form of DNA, and the components and machinery necessary for replication of DNA and for transcription of DNA into RNA. (See Alberts, B. et al. (1994) Molecular Biolo~y of the Cell, Garland Publishing Inc. New York NY, pp. 335-399.) DNA is organized into compact structures in the nucleus by intexactions with various DNA-binding proteins such as histories and non-histone chromosomal proteins.
DNA-specific nucleases, DNAses, partially degrade these compacted structures prior to DNA
replication or transcription. DNA replication takes place with the aid of DNA
helicases which unwind the double-stranded DNA helix, and DNA polymerases that duplicate the separated DNA strands.
Transcription Factors Transcriptional regulatory proteins are essential for the control of gene expression. Some of these proteins function as transcription factors that initiate, activate, repress, or terminate gene transcription. Transcription factors generally bind to the promoter, enhancer, and upstream regulatory regions of a gene in a sequence-specific manner, although some factors bind regulatory elements within or downstream of a gene coding region. Transcription factors may bind to a specific region of DNA singly or as a complex with other accessory factors (reviewed in Lewin, B.
(1990) Genes IV, Oxford University Press, New York NY, and Cell Press, Cambridge MA, pp. 554-570).
The double helix stricture and repeated sequences of DNA create topological and chemical features which can be recognized by transcription factors. These features are hydrogen bond donor and acceptor groups, hydrophobic patches, major and minor grooves, and regular, repeated stretches of sequence which induce distinct bends in the helix. Typically, transcription factors recognize specific DNA sequence motifs of about 20 nucleotides in length. Multiple, adjacent transcription factor-binding motifs may be required for gene regulation.
Many transcription factors incorporate DNA binding structural motifs which comprise either a Io helices or !3 sheets that bind to the major groove of DNA. Four well-characterized structural motifs are helix-turn helix, zinc finger, leucine zipper, and helix-loop helix.
Proteins containing these motifs may act alone as monomers, or they may form homo- or heterodimers that interact with DNA.
The helix-turn-helix motif consists of two oc helices connected at a fixed angle by a short chain of amino acids. One of the helices binds to the major groove. Helix-turn helix motifs are exemplified by the homeobox motif which is present in homeodomain proteins.
These proteins are critical for specifying the anterior-posterior body axis during development and are conserved throughout the animal kingdom. The Antennapedia and Ultrabithorax proteins of Dr-osophila melanogaster~ are prototypical homeodomain proteins (Patio, C.O. and R.T.
Saner (1992) Annu. Rev.
Biochem. 61:1053-1095).
2o Homeobox genes are a family of highly conserved regulatory genes that encode transcription factors. They are essential during embryonic development. They are important in limb formation and reproductive tract development. They function in uterine receptivity and implantation in mice and probably serve a similar role in humans (Daftary, G. S. and Taylor, H. S.
(2000) Semin. Reprod. Med.
18311-320). Homeobox gene mutations play a role in susceptibility to autism (Ingrain, J. L. et al.
(2000) Teratology 62:393-405) and are implicated in human diseases, such as diabetes to cancer (Cillo, C. et al. (2001) J. Cell Physiol. 188:161-169).
The helix-loop helix motif (HLH) consists of a short a helix connected by a loop to a longer a helix. The loop is flexible and allows the two helices to fold back against each other and to bind to DNA. The protooncogene Myc, a transcription factor that activates genes required for cellular proliferation, contains a prototypical HLH motif.
The zinc finger motif, which binds zinc ions, generally contains tandem repeats of about 30 amino acids consisting of periodically spaced cysteine and histidine residues.
Examples of this sequence pattern, designated C2H2 and C3HC4 ("RING" finger), have been described (Lewin, supra). Zinc finger proteins each contain an oc helix and an antiparallel 13 sheet whose proximity and conformation are maintained by the zinc ion. Contact with DNA is made by the arginine preceding the a helix and by the second, third, and sixth residues of the a helix.
Variants of the zinc finger motif include poorly defined cysteine-rich motifs which bind zinc or other metal ions. These motifs may not contain histidine residues and are generally nonrepetitive. The zinc finger motif may be repeated in a tandem array within a protein, such that the of helix of each zinc finger in the protein makes contact with the major groove of the DNA double helix. This repeated contact between the protein and the DNA produces a strong and specific DNA-protein interaction. The strength and specificity of the interaction can be regulated by the number of zinc finger motifs within the protein. Though originally identified in DNA-binding proteins as regions that interact directly with DNA, zinc fingers occur in a variety of proteins that do not bind DNA (Lodish, H. et al. (1995) Molecular Cell Biolo~y, Scientific American Books, New York NY, pp. 447-451). Fox example, Galcheva-Gargova et aI. (1996;
Science 272:1797-1802) have identified zinc finger proteins that interact with various cytokine receptors.
The C2H2-type zinc finger signature motif contains a 28 amino acid sequence, including 2 conserved Cys and 2 conserved His residues in a C-2-C-12-H-3-H type motif. The motif generally occurs in multiple tandem repeats. A cysteine-rich domain including the motif Asp-His-His-Cys (DHHC-CRD) has been identified as a distinct subgroup of zinc finger proteins.
The DHHC-CRD
region has been implicated in growth and development. One DHHC-CRD mutant shows defective function of Ras, a small membrane-associated GTP-binding protein that regulates cell growth and differentiation, while other DHHC-CRD proteins probably function in pathways not involving Ras (Bartels, D.J. et al. (1999) Mol. Cell Biol. 19:6775-6787).
Zinc-forger transcription factors are often accompanied by modular sequence motifs such as the Kruppel-associated box (KRAB) and the SCAN domain. For example, the hypoalphalipoproteinemia susceptibility gene ZNF202 encodes a SCAN box and a KRAB domain followed by eight C2H2 zinc-finger motifs (Hover, C. et al. (2001) Biochim.
Biophys. Acta 1S 17:441-448). The SCAN domain is a highly conserved, leucine-rich motif of approximately 60 amino acids found at the amino-terminal end of zinc finger transcription factors. SCAN domains are most often linked to C2H2 zinc finger motifs through their carboxyl-terminal end. Biochemical binding studies have established the SCAN domain as a selective hetero- and homotypic oligomerization domain. SCAN domain-mediated protein complexes may function to modulate the biological function of transcription factors (Schumacher, C. et al. (2000) J. Biol. Chem.
275:17173-17179).
The KRAB (Kruppel-associated box) domain is a conserved amino acid sequence spanning approximately 75 amino acids and is found in almost one-third of the 300 to 700 genes encoding C2H2 zinc fingers. The KRAB domain is found N-terminally with respect to the finger repeats. The KRAB
domain is generally encoded by two axons; the KRAB-A region or box is encoded by one axon and the KRAB-B region or box is encoded by a second axon. The function of the KRAB
domain is the S repression of transcription. Transcription repression is accomplished by recruitment of either the KR AR-associated protein-1, a transcriptional coreprescor, or the KRAB-A
interacting protein.
Proteins containing the KRAB domain are likely to play a regulatory role during development (Williams, A.J. et al. (1999) Mol. Cell Biol. 19:8526-8535). A subgroup of highly related human KRAB zinc finger proteins detectable in all human tissues is highly expressed in human T lymphoid cells (Bellefroid, E.J. et al. (1993) EMBO J. 12:1363-1374). The ZNF85 IKRAR
zinc finger gene, a member of the human ZNF91 family, is highly expressed in normal adult testis, in seminomas, and in the NT2/D1 teratocarcinoma cell line (Poncelet, D.A. et al. (1998) DNA Cell Biol.17:931-943).
The C4 motif is found in hormone-regulated proteins. The C4 motif generally includes only 2 repeats. A number of eukaryotic and viral proteins contain a conserved cysteine-rich domain of 40 to 60 residues (called C3HC4 zinc-finger or RING fmgex) that binds two atoms of zinc, and is probably involved in mediating protein-protein interactions. The 3D "cross-brace" structure of the zinc ligation system is unique to the RING domain. The spacing of the cysteines in such a domain is C-x(2)-C-x(9 to 39)-C-x(1 to 3)-H-x(2 to3)-C-x(2)-C-x(4 to 48)-C-x(2)-C. The PHD finger is a C4HC3 zinc-finger-like motif found in nuclear proteins thought to be involved in chromatin-mediated 2o transcriptional regulation.
GATA-type transcription factors contain one or two zinc finger domains which bind specifically to a region of DNA that contains the consecutive nucleotide sequence GATA. NMR
studies indicate that the zinc finger comprises two irregular anti-parallel (3 sheets and an a helix, followed by a long loop to the C-terminal end of the finger (Ominchinski, J.G.
(1993) Science 261:43 8-446). The helix and the loop connecting the two (3-sheets contact the major groove of the DNA, while the C-terminal part, which determines the specificity of binding, wraps around into the minor groove.
The L1M motif consists of about 60 amino acid residues and contains seven conserved cysteine residues and a histidine within a consensus sequence (Schmeichel, K.L. and M.C. Beckerle (1994) Cell 79:211-219). The LIM family includes transcription factors and cytoskeletal proteins which may be involved in development, differentiation, and cell growth. One example is actin-binding LIM protein, which may play roles in regulation of the cytoskeleton and cellular morphogenesis (Roof, D.J. et al. (1997) J. Cell Biol. 138:575-588). The N-terminal domain of actin-binding LIM protein has four double zinc finger motifs with the LIM consensus sequence. The C-terminal domain of actin-binding LIM protein shows sequence similarity to known actin-binding proteins such as dematin and villin. Actin-binding L1M protein binds to F-actin through its dematiu-like C-terminal domain. The LIM domain may mediate protein-protein interactions with other LIM binding proteins.
Myeloid cell development is controlled by tissue-specific transcription factors. Myeloid zinc finger proteins (MZF) include MZF-1 anal MZF-2. MZF-1 functions in regulation of the development of neutrophilic granulocytes. A murine homolog MZF-2 is expressed in myeloid cells, particularly in the cells cornlnitted to the neutrophilic lineage. MZF-2 is down-regulated by G-CSF and appears to have a unique function in neutrophil development (Murai, K. et al. (1997) Genes Cells 2:581-591).
The leucine zipper motif comprises a stretch of amino acids rich in leucine which can form an amphipathic oc helix. This structure provides the basis for dimerization of two leucine zipper proteins.
The region adjacent to the leucine zipper is usually basic, and upon protein dimerization, is optimally positioned for binding to the major groove. Proteins containing such motifs are generally referred to as bZIP transcription factors. The leucine zipper motif is found in the proto-oncogenes Fos and Jun, which comprise the heterodimeric transcription factor AP1 involved in cell growth and the determination of cell lineage (Papavassiliou, A.G. (1995) N. Engl. J. Med.
332:45-47).
The helix-loop helix motif (HLH) consists of a short a helix connected by a loop to a longer oc helix. The loop is flexible and allows the two helices to fold back against each other and to bind to DNA. The transcription factor Myc contains a prototypical HLH motif.
The NF-kappa-B/Rel signature defines a family of eukaryotic transcription factors involved in oncogenesis, embryonic development, differentiation and immune response. Most transcription factors containing the Rel homology domain (RHD) bind as dimers to a consensus DNA
sequence motif termed kappa-B. Members of the Rel family share a highly conserved 300 amino acid domain termed the Rel homology domain. The characteristic Rel C-terminal domain is involved in gene activation and cytoplasmic anchoring functions. Proteins known to contain the RHD domain include vertebrate nuclear factor NF-kappa-B, which is a heterodimer of a DNA binding subunit and the transcription factor p65, mammalian transcription factor RelB, and vertebrate proto-oncogene c-rel, a protein associated with differentiation and lymphopoiesis (Kabrun, N. and P.J.
Enrietto (1994) Semin. Cancer Biol. 5:103-112).
3o A DNA binding motif termed AR)D (AT-rich interactive domain) distinguishes an evolutionarily conserved family of proteins. The approximately 100-residue ARm sequence is present in a series of proteins strongly implicated in the regulation of cell growth, development, and tissue-specific gene expression. AR)D proteins include Bright (a regulator of B-cell-specific gene expression), dead ringer (involved in development), and MRF-2 (which represses expression from the cytomegalovirus enhancer) (Dallas, P.B. et al. (2000) Mol. Cell. Biol. 20:3137-3146).
The ELM2 (Egl-27 and MTA1 homology 2) domain is found in metastasis-associated protein MTA1 and protein ER1. The Caetvor~habelitis elegans gene egl-27 is required for embryonic patterning MTA1, a human gene with elevated expression in metastatic carcinomas, is a component of a protein complex with histone deacetylase and nucleosome remodelling activities (Solari, F. et al.
(1999) Development 126:2483-2494). The ELM2 domain is usually found to the N
terminus of a myb-like DNA binding domain. ELM2 is also found associated with an ARID DNA.
The Iroquois (Irx) family of genes are found in nematodes, insects and vertebrates. Irx genes usually occur in one or two genomic clusters of three genes each and encode transcriptional controllers that possess a characteristic homeodomain. The Irx genes function early in development to specify the identity of diverse territories of the body. Later in development in both Dr-osophila and vertebrates, the Irx genes function again to subdivide those territories into smaller domains (reviewed in Cavodeassi, F. et al. (2001) Development 128:2847-2855). For example, mouse and human Irx4 proteins are 83 % conserved and their 63-as homeodomain is more than 93 °Io identical to that of the Dr-osophila Iroquois patterning genes. Irx4 transcripts are predominantly expressed in the cardiac ventricles. The homeobox gene Irx4 mediates ventricular differentiation during cardiac development (Bruneau, B.G. et al. (2000) Dev. Biol. 217:266-77).
Histidine triad (HTT) proteins share-residues in distinctive dimeric, 10-stranded half barrel structures that form two identical purine nucleotide-binding sites. Hint (histidine triad nucleotide binding protein)-related proteins, found in all forms of life, and fragile histidine triad (Fhit)-related proteins, found in animals and fungi, represent the two main branches of the HIT
superfamily. Fhit homologs bind and cleave diadenosine polyphosphates. Fhit-Ap(n)A complexes appear to function in a proapoptotic tumor suppression pathway in epithelial tissues (Brenner C. et aI.
(1999) J. Cell Physio1.181:179-187).
Most transcription factors contain characteristic DNA binding motifs, and variations on the above motifs and new motifs have been and are currently being characterized (Faisst, S. and S.
Meyer (1992) Nucleic Acids Res. 20:3-26). These include the forkhead motif, found in transcription factors involved in development and oncogenesis (Hacker et al. (1995) EMBO J
14:5306-5317), and the T-box protein T-domain, which forms a novel major and minor groove DNA
contact. T box genes such as Bt-achyufy (T) are essential for tissue specification in development.
(Muller (1997) Nature 389:884-888.) Chromatin Associated Proteins In the nucleus, DNA is packaged into chromatin, the compact organization of which limits the accessibility of DNA to transcription factors and plays a key role in gene regulation (Lewin, supra, pp. 409-410). The compact structure of chromatin is determined and influenced by chromatin-associated proteins such as the histones, the high mobility group (HMG) proteins, and the chromodomain proteins. There are five classes of lustones, H1, H2A, H2B, H3, and H4, all of which.
are highly basic, low molecular weight proteins. The fundamental unit of chromatin, the nucleosome, consists of 200 base pairs of DNA associated with two copies each of H2A, H2B, H3, and H4. H1 links adjacent nucleosomes. HMG proteins are low molecular weight, non-histone proteins that may play a role in unwinding DNA and stabilizing single-stranded DNA. Chromodomain proteins play a key role in the formation of highly compacted heterochromatin, which is transcriptionally silent.
Diseases and Disorders Related to Gene Regulation Mutations in transcription factors contribute to oncogenesis. This is likely due to the role of transcription factors in the expression of genes involved in cell proliferation. For example, mutations in transcription factors encoded by proto-oncogenes, such as Fos, Jun, Myc, Rel, and Spil, may be oncogenic due to increased stimulation of cell proliferation. Conversely, mutations in transcription factors encoded by tumor suppressor genes, such as p53, RB 1, and WT1, may be oncogenic due to decreased inlubition of cell proliferation. (Latchman, D. (1995) Gene Regulation: A Eukaryotic Perspective, Chapman and Hall, London, UI~, pp 242-255.) Many neoplastic disorders in humans can be attributed to inappropriate gene expression.
2o Malignant cell growth may result from either excessive expression of tumor promoting genes or insufficient expression of tumor suppressor genes (Cleary, M.L. (1992) Cancer Surv. 15:89-104). 'The zinc finger-type transcriptional regulator WT1 is a tumor-suppressor protein that is inactivated in children with Wihn's tumor. Deletions of the WT1 gene, or point mutations which destroy the DNA-binding activity of the protein, are associated with development of the pediatric nephroblastoma, Wihns tumor, and Denys-Drash syndrome. (Rauscher, F.J. (1993) FASEB J. 7:896-903.) The 'oncogene bcl-6, which plays an important role in large-cell lymphoma, is also a zinc-finger protein (Papavassiliou, A.G. (1995) N. Engl. J. Med. 332:45-47). Chromosomal translocations may also produce chimeric loci that fuse the coding sequence of one gene with the regulatory regions of a second unrelated gene. Such an arrangement likely results in inappropriate gene transcription, 3o potentially contributing to malignancy. In Burkitt's lymphoma, for example, the transcription factor Myc is translocated to the immunoglobulin heavy chain locus, greatly enhancing Myc expression and resulting in rapid cell growth leading to leukemia (Latchman, D.S. (1996) N.
Engl. J. Med. 334:28-33).
Certain proteins enriched in glutamine are associated with various neurological disorders including spinocerebellar ataxia, bipolar effective disorder, schizophrenia, and autism. (Margolis, R.L.
et al. (1997) Human Genetics 100:114-122.) These proteins contain regions with as many as 15 or more consecutive glutatnine residues and may function as transcription factors with a potential role in regulation of neurodevelopment or neuroplasticity.
Iu addition, the immune system responds to infection or trauma by activating a cascade of events that coordinate the progressive selection, amplification, and mobilization of cellular defense mechanisms. A complex and balanced program of gene activation and repression is involved in this process. However, hyperactivity of the immune system as a result of improper or insufficient regulation of gene expression may result in considerable tissue or organ damage. This damage is well-documented in immunological responses associated with arthritis, allergens, heart attack, stroke, and infections (Isselbacher, K.J. et al. Harrison's Principles of Internal Medicine, 13/e, McGraw Hill, Inc.
and Teton Data Systems Software, 1996). In particular, a zinc finger protein termed Staf50 (for Stimulated trans-acting factor of 50 kDa) is a transcriptional regulator and is induced in various cell lines by interferon-I and -II. Staf50 appears to mediate the antiviral activity of interferon by down-regulating the viral transcription directed by the long terminal repeat promoter region of human immunodeficiency virus type-1 in transfected cells. (Tissot, C. (1,995) J.
Biol. Chem.
270:14891-14898.) Also, the causative gene for autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED) was recently isolated and found to encode a protein with two PHD-type zinc finger motifs (Bjorses, P. et al. (1998) Hum. Mol. Genet. 7:1547-1553).
Furthermore, the generation of multicellular organisms is based upon the induction and coordination of cell differentiation at the appropriate stages of development.
Central to this process is differential gene expression, which confers the distinct identities of cells and tissues throughout the body. Failure to regulate gene expression during development could result in developmental disorders.
Human developmental disorders caused by mutations in zinc finger-type transcriptional regulators include: urogenital developmental abnormalities associated with WT1; Greig cephalopolysyndactyly, Pallister-Hall syndrome, and postaxial polydactyly type A (GLI3), and Townes-Brocks syndrome, characterized by anal, renal, limb, and ear abnormalities (SALL1) (Engelkamp, D. and V. van Heyningen (1996) Curr. Opin. Genet. Dev. 6:334-342; Kohlhase, J. et al. (1999) Am. J. Hum. Genet.
64:435-445).
Human acute leukemias involve reciprocal chromosome translocations that fuse the ALL-1 gene located at chromosome region 11q23 to a series of partner genes positioned on a variety of human chromosomes. The fused genes encode chimeric proteins. The AF17 gene encodes a protein of 1093 amino acids, containing a leucine-zipper dimerization motif located 3' of the fusion point and a cysteine-rich domain at the N terminus that shows homology to a domain within the protein Br140 (peregrin) (Prasad R. et al. (1994) Proc. Natl. Acad. Sci. USA 91:8107-8111).
Impaired transcriptional regulation may lead to Alzheimer's disease, a progressive neurodegenerative disorder that is characterized by the formation of senile plaques and neurofxbrillary tangles containing amyloid beta peptide. These plaques are found in limbic and association cortices of the brain, including hippocampus, temporal cortices, cingulate cortex, amygdala, nucleus basalis and locus caeruleus. Early in Alzheimer's pathology, physiological changes are visible in the cingulate cortex (Minoshima, S. et al. (1997) Ann. Neurol. 42:85-94). In subjects with advanced Alzheimer's disease, accumulating plaques damage the neuronal architecture in limbic areas and eventually cripple the memory process.
SYNTHESIS OF NUCLEIC ACIDS
Polymerases DNA and RNA replication are critical processes for cell replication and function. DNA and RNA replication are mediated by the enzymes DNA and RNA polymerase, respectively, by a "templating" process in which the nucleotide sequence of a DNA or RNA strand is copied by complementary base-pairing into a complementary nucleic acid sequence of either DNA or RNA.
However, there are fundamental differences between the two processes.
DNA polymerase catalyzes the stepwise addition of a deoxyribonucleotide to the 3'-OH end of a polynucleotide strand (the primer strand) that is paired to a second (template) strand. The new DNA strand therefore grows in the 5' to 3' direction (Alberts, B. et a1.
(1994) The Molecular Biolo~y of the Cell, Garlaud Publishing Inc., New York NY, pp 251-254). The substrates for the polymerization reaction are the corresponding deoxynucleotide triphosphates which must base-pair with the correct nucleotide on the template strand in order to be recognized by the polymerase.
Because DNA exists as a double-stranded helix, each of the two strands may serve as a template for the formation of a new complementary strand. Each of the two daughter cells of a dividing cell therefore inherits a new DNA double helix containing one old and one new strand. Thus, DNA is said to be replicated "semiconservatively" by DNA polymerase. In addition to the synthesis of new DNA, DNA polymerase is also involved in the repair of damaged DNA as discussed below under "Ligases."
In contrast to DNA polymerase, RNA polymerase uses a DNA template strand to "transcribe" DNA into RNA using ribonucleotide triphosphates as substrates.
Like DNA
polymerization, RNA polymerization proceeds in a 5' to 3' direction by addition of a ribonucleoside monophosphate to the 3'-OH end of a growing RNA chain. DNA transcription generates messenger RNAs (mRNA) that carry information for protein synthesis, as well as the transfer, ribosomal, and other RNAs that have structural or catalytic functions. In eukaryotes, three discrete RNA
polymerases synthesize the three different types of RNA (Alberts, supra, pp.
367-36~). RNA
polymerase I makes the large ribosomal RNAs, RNA polymerase II makes the mRNAs that will be translated into proteins, and RNA polymerase III makes a variety of small, stable RNAs, including 5S
ribosomal RNA and the transfer RNAs (tRNA). In all cases, RNA synthesis is initiated by binding of the RNA polymerase to a promoter region on the DNA and synthesis begins at a start site within the promoter. Synthesis is completed at a stop (termination) signal in the DNA
whereupon both the polymerase and the completed RNA chain are released.
L~_ DNA repair is the process by which accidental base changes, such as those produced by oxidative damage, hydrolytic attack, or uncontrolled methylation of DNA, are corrected before replication or transcription of the DNA can occur. Because of the efficiency of the DNA repair process, fewer than one in a thousand accidental base changes causes a mutation (Alberts, supf-a, pp.
245-249). The three steps common to most types of DNA repair are (1) excision o~ the damaged or altered base or nucleotide by DNA nucleases,.(2) insertion of the correct nucleotide in the gap left by the excised nucleotide by DNA polymerase using the complementary strand as the template and, (3) sealing the break left between the inserted nucleotides) and the existing DNA
strand by DNA ligase.
In the last reaction, DNA ligase uses the energy from ATP hydrolysis to activate the 5' end of the broken phosphodiester bond before forming the new bond with the 3'-OH of the DNA strand. In Bloom's syndrome, an inherited human disease, individuals are partially deficient in DNA ligation and consequently have an increased incidence of cancer (Alberts, supf~a, p. 247).
Nucleases Nucleases comprise enzymes that hydrolyze both DNA (DNase) and RNA (Rnase).
They serve different purposes in nucleic acid metabolism. Nucleases hydrolyze the phosphodiester bonds between adjacent nucleotides either at internal positions (endonucleases) or at the terminal 3' or 5' nucleotide positions (exonucleases). A DNA exonuclease activity in DNA
polymerase, for example, serves to remove improperly paired nucleotides attached to the 3'-OH end of the growing DNA strand by the polymerase and thereby serves a "proofreading" function. As mentioned above, DNA
endonuclease activity is involved in the excision step of the DNA repair process.
RNases also serve a variety of functions. For example, RNase P is a ribonucleoprotein enzyme which cleaves the 5' end of pre-tRNAs as part of their maturation process. RNase H digests the RNA strand of an RNA/DNA hybrid. Such hybrids occur in cells invaded by retroviruses, and RNase H is an important enzyme in the retroviral replication cycle. Pancreatic RNase secreted by the pancreas into the intestine hydrolyzes RNA present in ingested foods.
RNase activity in serum and cell extracts is elevated in a variety of cancers and infectious diseases (Schein, C.H. (1997) Nat.
Biotechnol. 15:529-536). Regulation of RNase activity is being investigated as a means to control tumor angiogenesis, allergic reactions, viral infection and replication, and fungal infections.
MODIFICATION OF NUCLEIC ACIDS
DNA Repair Cells are constantly faced with replication errors and environmental assault (such as ultraviolet irradiation) that can produce DNA damage. Damage to DNA consists of any change that modifies the structure of the molecule. Changes to DNA can be divided into two general classes, single base changes and structural distortions. Single base changes affect the sequence but not the overall structure of the DNA. Since single base changes do not affect transcription or replication, they exert their effect on future generations. Structural distortions affect the structure of the DNA.
A single stxand nick or removal of a base may prevent a strand from acting as a viable template for synthesis of DNA or RNA. Intrastrand or interstrand covalent linkage between bases, or the addition of a bulky adduct to a base, may distort the structure of the double helix and interfere with transcription and replication. Any damage to DNA can produce a mutation, and the mutation may produce a disorder, such as cancer.
Changes in DNA are recognized by repair systems within the cell. These repair systems act to correct the damage and thus prevent any deleterious affects of a mutational event. Repair systems can be divided into three general types, direct repair, excision repair, and retrieval systems. When the repair systems are eliminated, cells become exceedingly sensitive to environmental mutagens, such as ultraviolet irradiation. Disorders associated with a loss in DNA repair systems often exhibit a high sensitivity to environmental mutagens. Examples of such disorders include xeroderma pigmentosum, Bloom's syndrome, and Werner's syndrome. Xeroderma pigmentosum results in a hypersensitivity to sunlight, especially ultraviolet, and produces skin defects. Bloom's syndrome results in an increased frequency of chromosomal aberrations, including sister chromosome exchanges (Yamagata, K. et al.
(1998) Proc. Natl. Aced. Sci. USA 95:8733-8738).
Direct repair involves the reversal or simple removal of the damaged region of DNA.
Mismatches involving normal bases are repaired based on certain biases within the repair system.
For example, mismatched GT base pairs are frequently caused by deamination of 5-methyl-cytosine to form thymine. Therefore, repair systems convert mismatched GT pairs to GC, instead of AT. Repair also favors the non-methylated strand in hemimethylated DNA, since this strand represents the newly synthesized daughter strand. The recognition of hemimethylated DNA and repair of mismatches on the non-methylated strand involve the products of the genes mutes, mutt, mutS
(which specifically recognizes mismatched base pairs), the helicase encoded by the uvrD gene, and the methylase encoded by the darn gene. C-5 cytosine-specific DNA methylases are enzymes that specifically methylate the C-5 carbon of cytosines in DNA (Kumar, S. et al. (1994) Nucleic Acids Res. 22:1-10).
Excision repair is a system in which mispaired or damaged bases are removed from DNA and a new stretch of DNA is synthesized to replace them. In the incision step, the damaged structuxe is recognized by an endonuclease that cleaves the DNA strand on both sides of the damage. In the excision step, a 5'-3' exonuclease removes a stretch of the damaged DNA
strand. In the synthesis step, the resulting single-stranded region serves as a template for a DNA
polymerase to synthesize a replacement for the excised sequence. Finally, DNA ligase covalently links the 3' end of the new material to the old material. Iu mammals, DNA polymerase beta serves as the DNA repair polymerase. Mutations in the human DNA polymerase beta gene are associated with several types of cancer (Bhattacharyya, N. et al. (1999) DNA Cell Biol. 18:549-554; Matsuzaki, J. et al. (1996) Mol.
Carcinog. 15 :3 8-43 ).
Methylases Methylation of specific nucleotides occurs in both DNA and RNA, and serves different functions in the two macromolecules. Methylation of cytosine residues to form 5-methyl cytosine in DNA occurs specifically in CG sequences which are base-paired with one another in the DNA
double-helix. The pattern of methylation is passed from generation to generation during DNA
replication by au enzyme called "maintenance methylase" that acts preferentially on those CG
sequences that are base-paired with a CG sequence that is already methylated.
Such methylation appears to distinguish active from inactive genes by preventing the binding of regulatory proteins that "turn on" the gene, but permiting the binding of proteins that inactivate the gene (Alberts, supra, pp.
448-451). In RNA metabolism, "tRNA methylase" produces one of several nucleotide modifications in tRNA that affect the conformation and base-pairing of the molecule and facilitate the recognition of the appropriate mRNA codons by specific tRNAs. The primary methylation pattern is the dimethylation of guanine residues to form N,N-dimethyl guanine.
Helicases and Single-stranded Binding Proteins Helicases are enzymes that destabilize and unwind double helix structures in both DNA and RNA. Since DNA replication occurs more or less simultaneously on both strands, the two strands must first separate to generate a replication "fork" for DNA polymerase to act on. Two types of replication proteins contribute to this process, DNA helicases and single-stranded binding proteins.
DNA helicases hydrolyze ATP and use the energy of hydrolysis to separate the DNA strands.

Single-stranded binding proteins (SSBs) then bind to the exposed DNA strands, without covering the bases, thereby temporarily stabilizing them for templating by the DNA
polymerase (Alberts, supra, pp.
255-256).
RNA helicases also alter and regulate RNA conformation and secondary structure. Like the DNA helicases, RNA helicases utilize energy derived from ATP hydrolysis to destabilize and unwind RNA duplexes. The most well-characterized and ubiquitous family of RNA
helicases is the DEAD-box family, so named for the conserved B-type ATP binding motif which is diagnostic of proteins in this family. Over 40 DEAD-box helicases have been identified in organisms as diverse as bacteria, insects, yeast, amphibians, mammals, and plants. DEAD-box helicases function in diverse processes such as translation initiation, splicing, ribosome assembly, and RNA editing, transport, and stability.
Examples of these RNA helicases include yeast Drs1 protein, which is involved in ribosomal RNA
processing; yeast TIF1 and T1F2 and mammalian eIF-4A, which are essential to the initiation of RNA
translation; and human p68 antigen, which regulates cell growth and division (Ripmaster, T.L. et al.
(1992) Proc. Natl. Acad. Sci. USA 89:11131-11135; Chang, T.-H. et al. (1990) Proc. Natl. Acad. Sci.
USA 87:1571-1575). These RNA helicases demonstrate strong sequence homology over a stretch of some 420 amino acids. Included among these conserved sequences are the consensus sequence for the A motif of an ATP binding protein; the "DEAD box" sequence, associated with ATPase activity;
the sequence SAT, associated with the actual helicase unwinding region; and an octapeptide consensus sequence, required for RNA binding and ATP hydrolysis (Pause, A. et al. (1993) Mol. Cell Biol. 13:6789-6798). Differences outside of these conserved regions are believed to reflect differences in the functional roles of individual proteins (Chang et al., supra).
Some DEAD-box helicases play tissue- and stage-specific roles in spermatogenesis and embryogenesis. Overexpression of the DEAD-box 1 protein (DDX1) may play a role in the progression of neuroblastoma (Nb) and retinoblastoma (Rb) tumors (Godbout, R.
et al. (1998) J. Biol.
Chem. 273:21161-21168). These observations suggest that DDX1 may promote or enhance tumor progression by altering the normal secondary structure and expression levels of RNA in cancer cells.
Other DEAD box helicases have been implicated either directly or indirectly in tumorigenesis (Godbout et al., supra). For example, murine p68 is mutated in ultraviolet light-induced tumors, and human DDX6 is located at a chromosomal breakpoint associated with B-cell lymphoma. Similarly, a 3o chimeric protein comprised of DDX10 and NUP9 8, a nucleoporin protein, may be involved in the pathogenesis of certain myeloid malignancies.
To~oisomerases Besides the need to separate DNA strands prior to replication, the two strands must be 'unwound" from one another prior to their separation by DNA helicases. This function is performed by proteins known as DNA topoisomerases. DNA topoisomerase effectively acts as a reversible nuclease that hydrolyzes a phosphodiesterase bond in a DNA strand, permits the two strands to rotate freely about one another to remove the strain of the helix, and then rejoins the original phosphodiester bond between the two strands. Topoisomerases are essential enzymes responsible for the topological rearrangement of DNA brought about by transcription, replication, chromatin formation, recombination, and chromosome segregation. Superhelical coils are introduced into DNA by the passage of processive enzymes such as RNA polymerase, or by the separation of DNA strands by a helicase prior to replication. Knotting and concatenation can occur in the process of DNA synthesis, storage, and repair. Au topoisomerases work by breaking a phosphodiester bond in the ribose-phosphate backbone of DNA. A catalytic tyrosine residue on the enzyme makes a nucleophilic attack on the scissile phosphodiester bond, resulting in a reaction intermediate in which a covalent bond is formed between the enzyme and one end of the broken strand. A tyrosine-DNA
phosphodiesterase functions in DNA repair by hydrolyzing this bond in occasional dead-end topoisomerase I-DNA
intermediates (Pouliot, J.J. et al. (1999) Science 286:552-555).
Two types of DNA topoisomerase exist, types I and II. Type I topoisomerases work as monomers, making a break in a single strand of DNA while type 7I
topoisomerases, working as homodimers, cleave both strands. DNA Topoisomerase I causes a single-strand break in a DNA
helix to allow the rotation of the two strands of the helix about the remaining phosphodiester bond in the opposite strand. DNA topoisomerase II causes a transient break in both strands of a DNA helix where two double helices cross over one another. This type of topoisomerase can efficiently separate two interlocked DNA circles (Alberts, supy~a, pp. 260-262). Type II
topoisomerases are largely conned to proliferating cells in eukaryotes, such as cancer cells. For this reason they are targets for anticancer drugs. Topoisomerase 1I has been implicated in multi-drug resistance (MDR) as it appears to aid in the repair of DNA damage inflicted by DNA binding agents such as doxorubicin and vincristine.
The topoisomerase I family includes topoisomerases I and III (topo I and topo 1~). The crystal structure of human topoisomerase I suggests that rotation about the intact DNA strand is partially controlled by the enzyme. In this "controlled rotation" model, protein-DNA interactions limit the rotation, which is driven by torsional strain in the DNA (Stewart, L. et al. (1998) Science 379:1534-1541). Structurally, topo I can be recognized by its catalytic tyrosine residue and a number of other conserved residues in the active site region. Topo I is thought to function during transcription.
Two topo IIIs are known in humans, and they axe homologous to prokaryotic topoisomerase I, with a conserved tyrosine and active site signature specific to this family. Topo III
has been suggested to play a role in meiotic recombination. A mouse topo llI is highly expressed in testis tissue and its expression increases with the increase in the number of cells in pachytene (Seki, T. et al. (1998) J.
Biol. Chem. 273:28553-28556).
The topoisomerase II family includes two isozymes (Ifa and 1I(3) encoded by different genes.
Topo II cleaves double stranded DNA in a reproducible, nonrandom fashion, preferentially in an AT
rich region, but the basis of cleavage site selectivity is not known.
Structurally, topo 1I is made up of four domains, the first two of which are structurally similar and probably distantly homologous to similar domains in eukaryotic topo I. The second domain bears the catalytic tyrosine, as well as a highly conserved pentapeptide. The IIoc isoform appears to be responsible fox unlinking DNA during chromosome segregation. Cell lines expressing lIoc but not II(3 suggest that 1I(3 is dispensable in cellular processes; however, II(3 knockout mice died perinatally due to a failure in neural development.
That the major abnormalities occurred in predominantly late developmental events (neurogenesis) suggests that II(3 is needed not at mitosis, but rather during DNA repair (Yang, X. et al. (2000) Science 287:131-134).
Topoisomerases have been implicated in a number of disease states, and topoisomerase poisons have proven to be effective anti-tumor drugs for some human malignancies. Topo I is mislocalized in Fanconi's anemia, and may be involved in the chromosomal breakage seen in this disorder (blunder, E. (1984) Hum. Genet. 68:276-281). Overexpression of a truncated topo III in ataxia-telangiectasia (A-T) cells partially suppresses the A-T phenotype, probably through a dominant negative mechanism. This suggests that topo III is deregulated in A-T (Fritz, E. et al. (1997) Proc.
Natl. Acad. Sci. USA 94:4538-4542). Topo III also interacts with the Bloom's Syndrome gene product, and has been suggested to have a role as a tumor suppressor (Wu, L.
et al. (2000) J. Biol.
Chem. 275:9636-9644). Aberrant topo lI activity is often associated with cancer or increased cancer risk. Greatly lowexed topo 1I activity has been found in some, but not all A-T
cell lines (Mohamed, R.
et al. (1987) Biochem. Biophys. Res. Commun. 149:233-238). On the other hand, topo II cab. break DNA in the region of the A-T gene (ATM), which controls all DNA damage-responsive cell cycle checkpoints (Kaufmann, W.K. (1998) Proc. Soc. Exp. Biol. Med. 217:327-334).
The ability of topoisomerases to break DNA has been used as the basis of antitumor drugs.
Topoisomerase poisons act by increasing the number of dead-end covalent DNA-enzyme complexes in the call, ultimately triggering cell death pathways (Fortune, J.M. and N. Osheroff (2000) Prog.
Nucleic Acid Res. Mol.
Biol. 64:221-253; Guichard, S.M. and M.K. Danks (1999) Curr. Opin. Oncol.
11:482-489). Antibodies against topo I are found in the serum of systemic sclerosis patients, and the levels of the antibody may be used as a marker of pulmonary involvement in the disease (Diot, E. et al.
(1999) Chest 116:715-720). Finally, the DNA binding region of human topo I has been used as a DNA
delivery vehicle for gene therapy (Chen, T.Y. et al. (2000) Appl. Microbiol. Biotechnol. 53:558-567).
Recombinases Genetic recombination is the process of rearranging DNA sequences within an organism's genome to provide genetic variation for the organism in response to changes in the environment.
DNA recombination allows variation in the particular combination of genes present in an individual's genome, as well as the timing and level of expression of these genes (Alberts, supra, pp. 263-273).
Two broad classes of genetic recombination are commonly recognized, general recombination and site-specific recombination. General recombination involves genetic exchange between any homologous pair of DNA sequences usually located on two copies of the same chromosome. The process is aided by enzymes, recombiuases, that "nick" one strand of a DNA
duplex more or less randomly and~permit exchange with a complementary strand on another duplex.
The process does not normally change the arrangement of genes in a chromosome. In site-specific recombination, the recombinase recognizes specific nucleotide sequences present in one or both of the recombining molecules. Base-pairing is not involved in this form of recombination and therefore it does not require DNA homology between the recombining molecules. Unlike general recombination, this form of recombination can alter the relative positions of nucleotide sequences in chromosomes.
RNA METABOLISM
Much of the regulation of gene expression in eucaryotic cells occurs at the posttranscriptional level. Messenger RNAs (mRNA), which are produced in the cell nucleus from primary transcripts of protein-encoding genes, are processed and transported to the cytoplasm where the protein synthesis machinery is located. RNAbinding proteins are a group of proteins that participate in the processing, editing, transport, localization, and posttranscriptional regulation of mRNAs, and comprise the protein component of ribosomes as well. The RNA binding activity of many of these proteins is mediated by a series of RNA-binding motifs identified within them. These domains include the RNP motif, the arginine-rich motif, the RGG box, and the I~H motif. (Reviewed in Bard, C. G.
and Dreyfuss, G.
(1994) Science 265:615-621.) The RNP motif is the most widely found and best characterized of these motifs. 'The RNP motif is composed of 90-100 amino acids which form an RNA binding domain and is found in one or more copies in proteins that bind pre-mRNA, mRNA, pre-ribosomal RNA, and small nuclear RNAs. The RNP motif is composed of two short sequences (RNP-1 and RNP-2) and a number of other mostly hydrophobic, conserved amino acids interspersed throughout the motif.
(Burd, su ra; ExPASy PROSITE document PDOC0030.) Ribonucleic acid (RNA) is a linear single-stranded polymer of four nucleotides, ATP, CTP, U'TP, and GTP. In most organisms, RNA is transcribed as a copy of deoxyribonucleic acid (DNA), the genetic material of the organism. In retroviruses RNA rather than DNA
serves as the genetic material. RNA copies of the genetic material encode proteins or serve various structural, catalytic, or regulatory roles in organisms. RNA is classified according to its cellular localization and function.
Messenger RNAs (mRNAs) encode polypeptides. Ribosomal RNAs (rRNAs) are assembled, along with ribosomal proteins, into ribosomes, which are cytoplasmic particles that translate mRNA into polypeptides. Transfer RNAs (tRNAs) are cytosolic adaptor molecules that function in mRNA
translation by recognizing both an mRNA codon and the amino acid that matches that codon.
Heterogeneous nuclear RNAs (hnRNAs) include mRNA precursors and other nuclear RNAs of various sizes. Small nuclear RNAs (snRNAs) are a part of the nuclear spliceosome complex that removes intervening, non-coding sequences (introns) and rejoins exons in pre-mRNAs.
Proteins are associated with RNA during its transcription from DNA, RNA
processing, and translation of mRNA into protein. Proteins are also associated with RNA as it is used for structural, catalytic, and regulatory purposes.
RNA Processing Ribosomal RNAs (rRNAs) are assembled, along with ribosomal proteins, into ribosomes, which are cytoplasmic particles that translate messenger RNA (mRNA) into polypeptides. The eukaryotic ribosome is composed of a 605 (large) subunit and a 405 (small) subunit, which together form the 805 ribosome. In addition to the 185, 285, 5S, and 5:85 rRNAs, ribosornes contain from 50 to over 80 different ribosomal proteins, depending on the organism. Ribosomal proteins are classified according to which subunit they belong (i.e., L, if associated with the large 605 large subunit or S if associated with the small 40S subunit). E. coli ribosomes have been the most thoroughly studied and contain 50 proteins, many of which are conserved in all life forms. The structures of nine ribosomal proteins have been solved to less than 3.0D resolution (i.e., S5, S6, 517, L1, L6, L9, L12, L14, L30), revealing common motifs, such as b-a b protein folds in addition to acidic and basic RNA-binding motifs positioned between b-strands. Most ribosomal proteins are believed to contact rRNA directly (reviewed in Liljas, A. and M. Garber (1995) Curr. Opin. Struct. Biol. 5:721-727; see also Woodson, S.A. and N.B. Leontis (1998) C~rr. Opin. Struct. Biol. 8:294-300;
Ramakrishnan, V. and S.W. White (1998) Trends Biochem. Sci. 23:208-212).
Ribosomal proteins may undergo post-translational modifications or intexact with other ribosome-associated proteins to regulate translation. For example, the highly homologous 405 ribosomal protein S6 kinases (S6K1 and S6K2) play a key role in the regulation of cell growth by controlling the biosynthesis of translational components which make up the protein synthetic apparatus {including the ribosomal proteins). In the case of S6K1, at least eight phosphorylation sites are believed to mediate kinase activation in a hierarchical fashion (Dufner and Thomas {1999) Exp. Cell.
Res. 253:100-109). Some of the ribosomal proteins, including L1, also function as translational repressors by binding to polycistronic mRNAs encoding ribosomal proteins (reviewed in Liljas and Garner, supf~a).
Recent evidence suggests that a number of ribosomal proteins have secondary functions independent of their involvement in protein biosynthesis. These proteins function as regulators of cell proliferation and, in some instances, as inducers of cell death. For example, the expression of human ribosomal protein Ll3a has been shown to induce apoptosis by arresting cell growth in the G2/M
phase of the cell cycle. Inhibition of expression of Ll3a induces apoptosis in target cells, which suggests that this protein is necessary, in the appropriate amount, for cell survival. Similar results have been obtained in yeast where inactivation of yeast homologues of Ll3a, rp22 and rp23, results in severe growth retardation and death. A closely related ribosomal protein, L7, arrests cells in G1 and also induces apoptosis. Thus, it appears that a subset of ribosomal proteins may function as cell cycle checkpoints and compose a new family of cell proliferation regulators.
Mapping of individual ribosomal proteins on the surface of intact ribosomes is accomplished using 3D immunocryoelectronmicroscopy, whereby antibodies raised against specific ribosomal proteins are visualized. Progress has been made toward the mapping of Ll, L7, and Ll2 while the structure of the intact ribosome has been solved to only 20-25D resolution and inconsistencies exist among different crude structures (Frank, J. (1997) C~rr. Opin. Struct. Biol.
7:266-272).
Three distinct sites have been identified on the ribosome. The aminoacyl-tRNA
acceptor site (A site) receives charged tRNAs (with the exception of the initiator-tRNA).
The peptidyl-tRNA site (P site) binds the nascent polypeptide as the amino acid from the A site is added to the elongating chain. Deacylated tRNAs bind in the exit site (E site) prior to their release from the ribosome. (The structure of the ribosome is reviewed in Stryer, L. (1995) Biochemistry, W.H.
Freeman and Company, New York NY, pp. 888-908; Lodish, supra, pp. 119-138; and Lewin, B. {1997) Genes VI, Oxford University Press, Inc. New York NY).
Various proteins are necessary for processing of transcribed RNAs in the nucleus. Pre-mRNA processing steps include capping at the 5' end with methylguanosine, polyadenylating the 3' end, and splicing to remove introns. The primary RNA transript from DNA is a faithful copy of the gene containing both exon and intron sequences, and the latter sequences must be cut out of the RNA
transcript to produce a mRNA that codes for a protein. This "splicing" of the mRNA sequence takes place in the nucleus with the aid of a large, multicomponent ribonucleoproteiu complex known as a spliceosome. The spliceosomal complex is comprised of five small nuclear ribonucleoprotein particles (snRNPs) designated U1, U2, U4, U5, and U6. Each snRNP contains a single species of. snRNA and about ten proteins. The RNA components of some snRNPs recognize and base-pair with intron consensus sequences. The protein components mediate spliceosome assembly and the splicing reaction. Autoantibodies to snRNP proteins are found in the blood of patients with systemic lupus erythematosus (Stryer, supra, p. 863).
Heterogeneous nuclear ribonucleoproteins (buRNPs) have been identified that have roles in splicing, exporting of the mature RNAs to the cytoplasm, and mRNA translation (Biamonti, G. et al.
(1998) Clin. Exp. Rheumatol. 16:317-326). Some examples of hnRNPs include the yeast proteins Hrplp, involved in cleavage and polyadenylation at the 3' end of the RNA;
Cbp80p, involved in capping the 5' end of the RNA; and Npl3p, a homolog of mammalian hnRNP A1, involved in export of mRNA from the nucleus (Shen, E.C. et al. (1998) Genes Dev. 12:679-691). HnRNPs have been shown'to be important targets of the autoimmune response in rheumatic diseases (Biamonti et al., supra).
Many snRNP and hnRNP proteins are characterized by an RNA recognition motif (RRM) (reviewed in Birney, E. et al. (1993) Nucleic Acids Res. 21:5803-5816). The RRM is about 80 amino acids in length and forms four (3-strands and two a-helices arranged in an a /(3 sandwich. The RRM
contains a core RNP-1 octapeptide motif along with surrounding conserved sequences. In addition to snRNP proteins, examples of RNA-binding proteins which contain the above motifs include heteronuclear ribonucleoproteins which stabilize nascent RNA and factors which regulate alternative splicing. Alternative splicing factors include developmentally regulated proteins, specific examples of which have been identified in lower eukaryotes such as Dr-osophila rnelartogaster~ and Caehorl~cabditis elegaris. These proteins play key roles in developmental processes such as pattern formation and sex determination, respectively (Hodgkin, J. et al. (1994) Development 120:3681-3689).
The 3' ends of most eukaryote mRNAs are also posttrauscriptionally modified by polyadenylation. Polyadenylation proceeds through two enzymatically distinct steps: (i) the endonucleolytic cleavage of nascent mRNAs at cis-acting polyadenylation signals in the 3'-untranslated (non-coding) region and (ii) the addition of a poly(A) tract to the 5' mRNA fragment.
The presence of eis-acting RNA sequences is necessary for both steps. These sequences include 5 =
AAUAAA-3' located 10-30 nucleotides upstream of the cleavage site and a less well-conserved GU-or U-rich sequence element located 10-30 nucleotides downstream of the cleavage site. Cleavage stimulation factor (CstF), cleavage factor I (CF I), and cleavage factor II
(CF II) are involved in the cleavage reaction while cleavage and polyadenylation specificity factor (CPSF) and poly(A) polymerase (PAP) are necessary for both cleavage and polyadenylation. An additional enzyme, poly(A) binding protein II (PAB II), promotes poly(A) tract elongation (Riiegsegger, U. et al. (1996) J. Biol. Chem. 271:6107-6113; and references within).
TRANSLATION
Correct translation of the genetic code depends upon each amino acid forming a linkage with the appropriate transfer RNA (tRNA). The aminoacyl-tRNA synthetases (aaRSs) are essential proteins found in all living organisms. The aaRSs are responsible for the activation and correct attachment of an amino acid with its cognate tRNA, as the first step in protein biosynthesis.
. 10 Prokaryotic organisms have at least twenty different types of aaRSs, one for each different amino acid, while eukaryotes usually have two aaRSs, a cytosolic form and a mitochondrial form, for each different amino acid. The 20 aaRS enzymes can be divided into two structural classes. Class I
enzymes add amino acids to the 2' hydroxyl at the 3' end of tRNAs while Class II enzymes add amino acids to the 3' hydroxyl at the 3' end of tRNAs. Each class is characterized by a distinctive topology of the catalytic domain. Class I enzymes contain a catalytic domain based on the nucleotide-binding Rossman 'fold'. In particular, a consensus tetrapeptide motif is highly conserved (Prosite Document PDOC00161, Aminoacyl-transfer RNA synthetases class-I signature). Class I
enzymes are specific for arginine, cysteine, glutamic acid, glutamine, isoleucine, leucine, methionine, tyrosine, tryptophan, and valine. Class II enzymes contain a central catalytic domain, which consists of a seven-stranded antiparallel !3-sheet domain, as well as N- and C- terminal regulatory domains. Class II enzymes are separated into two groups based on the heterodimeric or homodimeric structure of the enzyme; the latter group is further subdivided by the structure of the N- and C-terminal regulatory domains (Hartlein, M. and S. Cusack (1995) J. Mol. Evol. 40:519-530). Class II enzymes are specific for alanine, asparagine, aspartic acid, glycine, histidine, lysine, phenylalanine, proline, serine, and threonine.
Certain aaRSs also have editing functions. IIeRS, for example, can misactivate valine to form Val-tRNAn~, but this product is cleared by a hydrolytic activity that destroys the mischarged product.
This editing activity is located within a second catalytic site found in the connective polypeptide 1 region (CP1), a long insertion sequence within the Rossman fold domain of Class I enzymes (Schimmel, P. et al. (1998) FASEB J. 12:1599-1609). AaRSs also play a role in tRNA processing. It has been shown that mature tRNAs are charged with their respective amino acids in the nucleus before export to the cytoplasm, and charging may serve as a quality control mechanism to insure the tRNAs are functional (Martinis, S.A. et al. (1999) EMBO J. 18:4591-4596).
Under optimal conditions, polypeptide synthesis proceeds at a rate of approximately 40 amino acid residues per second. The rate of misincorporation during translation in on the order of 10~ and is primarily the result of aminoacyl-t-RNAs being charged with the incorrect amino acid. Incorrectly charged tRNA are toxic to cells as they result in the incorporation of incorrect amino acid residues into an elongating polypeptide. The rate of translation is presumed to be a compromise between the optimal rate of elongation and the need for translational fidelity.
Mathematical calculations predict that 10~ is indeed the maximum acceptable error rate for protein synthesis in a biological system (reviewed in Stryer, supra; and Watson, J. et al. (1987) The Benjamin/Cummings Publishing Co., Inc. Memo Park, CA). A particularly error prone aminoacyl-tRNA charging event is the charging of tRNAo~
with Gln. A mechanism exits for the correction of this mischarging event which likely has its origins in evolution. Gln was among the last of the 20 naturally occurring amino acids used in polypeptide synthesis to appear in nature. Gram positive eubacteria, cyanobacteria, Archeae, and eukaryotic organelles possess a rloncanonical pathway for the synthesis of Gln-tRNAG~' based on the transformation of Glu-tRNAG~' (synthesized by Glu-tRNA synthetase, GIuRS) using the enzyme Glu-tRNAG~' amidotransferase (Glu-AdT). The reactions involved in the transamidation pathway are as follows (Curnow, A.W. et al. (1997) Nucleic Acids Symposium 36:2-4):
GluRS
tRNAG'~ + Glu + ATP ~ Glu-tRNAG~ + AMP + PPi Glu-AdT
Glu-tRNAG'" + Gln + ATP ~ Gln-tRNAG~" + Glu + ADP + P
A similar enzyme, Asp-tRNA'~" amidotransferase, exists in Archaea, which trausforms Asp-tRNA~" to Asn-tRNA~n. Formylase, the enzyme that transforms Met-tRNA~Met to fMet-tRNA~Met in eubacteria, is likely to be a related enzyme. A hydrolytic activity has also been identified that destroys mischarged Val-tRNAne (Schimmel, P. et al. (1998) FASBB J. 12:1599-1609). One likely scenario for the evolution of Glu-AdT in primitive life forms is the absence of a specific glutarninyl-tRNA
synthetase (GlnRS), requiring an alternative pathway for the synthesis of Gln-tRNAG~'. In fact, deletion of the Glu-AdT operon in Gram positive bacteria is lethal (Curnow, A.W. et al. (1997) Proc.
Natl. Acad. Sci. USA 94:11819-11826). The existence of GluRS activity in other organisms has been inferred by the high degree of conservation in translation machinery in nature; however, GluRS has not been identified in all organisms, including Horno Sapiens. Such an enzyme would be responsible for ensuring translational fidelity and reducing the synthesis of defective polypeptides.
In addition to their function in protein synthesis, specific aminoacyl tRNA
synthetases also play roles in cellular ~.delity, RNA splicing, RNA trafficking, apoptosis, and transcriptional and translational regulation. For example, human tyrosyl-tRNA synthetase can be proteolytically cleaved into two fragments with distinct cytokine activities. The carboxy-terminal domain exhibits monocyte and leukocyte chemotaxis activity as well as stimulating production of myeloperoxidase, tumor necrosis factor-o~, and tissue factor. The N-terminal domain binds to the interleukin-8 type A receptor and functions as au interleukin-8-like cytokine. Human tyrosyl-tRNA synthetase is secreted from apoptotic tumor cells and may accelerate apoptosis (Wakasugi, K., and Schimmel, P. (1999) Science 284:147-151). Mitochondrial Neurospora crassa TyrRS and S. cerevisiae LeuRS
are essential factors for certain group I intron splicing activities, and human mitochondrial LeuRS can substitute for the yeast LeuRS in a yeast null strain. Certain bacterial aaRSs are involved in xegulating their own transcription or translation (Martinis et al., supt'a). Several aaRSs are able to synthesize diadenosine oligophosphates, a class of signalling molecules with roles in cell proliferation, differentiation, and apoptosis (Kisselev, L.L et al. (1998) FEBS Lett. 427:157-163; Vartanian, A.
et al. (1999) FEBS Lett.
456:175-180).
Autoantibodies against aminoacyl-tRNAs are generated by patients with autoimmune diseases such as rheumatic arthritis, dermatomyositis and polymyositis, and correlate strongly with complicating interstitial lung disease (ILD) (Freist, W. et al. (1999) Biol. Chem. 380:623-646; Freist, W. et al.
(1996) Biol. Chem. Hoppe Seyler 377:343-356). These antibodies appear to be generated in response to viral infection, and coxsackie virus has been used to induce experimental viral myositis iu animals.
Comparison of aaRS structures between humans and pathogens has been useful in the design of novel antibiotics (Schimmel et al., supr a). Genetically engineered aaRSs have been utilized to allow site-specific incorporation of unnatural amino acids into proteins itt vivo (Liu, D.R. et al. (1997) Proc. Natl. Acid. Sci. USA 94:10092-10097).
tRNA Modifications The modified ribonucleoside, pseudouridine (yf), is present ubiquitously in the anticodon regions of transfer RNAs (tRNAs), large and small ribosomal RNAs (rRNAs), and small nuclear RNAs (snRNAs). y is the most common of the modified nucleosides (i.e., other than G, A, U, and C) present in tRNAs. Only a few yeast tRNAs that are not involved in protein synthesis do not contain yr (Cortese, R. et al. (1974) J. Biol. Chem. 249:1103-1108). The enzyme responsible for the conversion of uridine to W, pseudouridine synthase (pseudouridylate synthase), was first isolated from Salmoftella typhimuf'iunt (Arena, F. et al. (1978) Nucleic Acids Res. 5:4523-4536). 'The enzyme has since been isolated from a number of mammals, including steer and mice (Green, C.J. et al. (1982) J. Biol. Chem.
257:3045-52; and Chen, J. and J.R. Patton (1999) RNA 5:409-419). tRNA
pseudouridine synthases have been the most extensively studied members of the family. They require a thiol donor (e.g., cysteine) and a monovalent canon (e.g., ammonia or potassium) for optimal activity. Additional cofactors or high energy molecules (e.g., ATP or GTP) are not required (Green et al., supt-a). Other eukaryotic pseudouridine syntheses have been identified that appear to be specific for rRNA
(reviewed in Smith, C.M. and J.A. Steitz (1997) Cell 89:669-672) and a dual-specificity enzyme has been identified that uses both tRNA and rRNA substrates (Wrzesinski, J. et al.
(1995) RNA 1:
437-448). The absence of y! in the anticodon loop of tRNAs results in reduced growth in both bacteria (Singer, C.E. et al. (1972) Nature New Biol. 238:72-74) and yeast (Lecointe, F. (1998) J.
Biol. Chem. 273:1316-1323), although the genetic defect is not lethal.
Another ribonucleoside modification that occurs primarily in eukaryotic cells is the conversion of guanosine to NZ,NZ-dimethylguanosine (m22G) at position 26 or 10 at the base of the D-stem of cytosolic and mitochondrial tRNAs. This posttranscriptional modification is believed to stabilize tRNA
structure by preventing the formation of alternative tRNA secondary and tertiary structures. Yeast tRNA'~P is unusual in that it does not contain this modification. The modification does not occur in eubacteria, presumably because the structure of tRNAs in these cells and organelles is sequence constrained and does not require posttxanscriptional modification to prevent the formation of alternative structures (Steinberg, S. and R. Cedergren (1995) RNA 1:886-891, and references within).
The enzyme responsible for the conversion of guanosine to m22G is a 63 kDa S-adenosylmethionine (SAM)-dependent tRNA NZ,NZ-dimethyl-guanosine methyltransferase (also referred to as the TRllll gene product and herein referred to as TRM) (Edqvist, J. (1995) Biochimie 77:54-61). The enzyme localizes to both the nucleus and the mitochondria (Li, J-M. et al. (1989) J.
Cell Biol. 109:1411-1419).
Based on studies with TRM from ~Yenopus laevis, there appears to be a requirement for base pairing at positions C11-G24 and G10-C25 immediately preceding the G26 to be modified, with other structural features of the tRNA also being required for the proper presentation of the G26 substrate (Edqvist. J. et al. (1992) Nucleic Acids Res. 20:6575-6581). Studies in yeast suggest that cells carrying a weak ochre tRNA suppressor (sup3-i) are unable to suppress translation termination in the absence of TRM activity, suggesting a role for TRM in modifying the frequency of suppression in eukaryotic cells (Niederberger, C. et al. (1999) FEBS Lett. 464:67-70), in addition to the more general function of ensuring the proper three-dimensional structures for tRNA.
3o Translation Initiation Initiation of translation can be divided into three stages. The first stage brings an initiator transfer RNA (Met-tRNAf) together with the 40S ribosomal subunit to form the 43S preinitiation complex. The second stage binds the 43S preinitiation complex to the mRNA, followed by migration of the complex to the correct AUG initiation codon. The third stage brings the 60S ribosomal subunit to the 40S subunit to generate an 80S ribosome at the inititation codon.
Regulation of translation primarily involves the first and second stage in the initiation process (Pain, V.M. (1996) Eur. J.
Biochem. 236:747-771).
Several initiation factors, many of which contain multiple subunits, are involved in bringing an initiator tRNA and the 40S ribosomal subunit together. elF2, a guanine nucleotide binding protein, recruits the initiator tRNA to the 40S ribosomal subunit. Only when elF2 is bound to GTP does it associate with the initiator tRNA. elF2B, a guanine nucleotide exchange protein, is responsible for converting eIF'2 from the GDP bound inactive form to the GTP-bound active form. Two other factors, eIFlA and elF3 bind and stabilize the 40S subunit by interacting with the 18S ribosomal RNA
and specific ribosomal structural proteins. eIF3 is also involved in association of the 40S ribosomal subunit with mRNA. The Met-tRNAf, eIFlA, elF3, and 40S ribosomal subunit together make up the 43S preinitiation complex (Pain, supra).
Additional factors are required for binding of the 43S preinitiation complex to an mRNA
molecule, and the process is regulated at several levels. eIF4F is a complex consisting of three proteins: eIF4E, eIF4A, and elF4G. eIF4E recognizes and binds to the mRNA 5 =terminal m'GTP
cap, eIF4A is a bidirectional RNA-dependent helicase, and eIF4G is a scaffolding polypeptide. eIF4G
has three binding domains. The N-terminal third of elF4G interacts with elF4E, the central third interacts with eIF4A, and the C-terminal third interacts with eIF3 bound to the 43S preinitiation complex. Thus, eIF4G acts as a bridge between the 40S ribosomal subunit and the mRNA (Hentze, M.W. (1997) Science 275:500-501).
The ability of eIF4F to initiate binding of the 43S preinitiation complex is regulated by structural features of the mRNA. The mRNA molecule has an untranslated region (UTR) between the 5' cap and the AUG start codon. In some mRNAs this region forms secondary structures that impede binding of the 43S preinitiation complex. The helicase activity of elF4A is thought to function in removing this secondary structure to facilitate binding of the 43S
preinitiation complex (Pain, supra).
Translation Elongation Elongation is the process whereby additional amino acids are joined to the initiator methionine to form the complete polypeptide chain. The elongation factors EFla, EF1(3y, and EF2 are involved in elongating the polypeptide chain following initiation. EFla is a GTP
binding protein. In EFla's GTP-bound form, it brings an aminoacyl-tRNA to the ribosome's A site. The amino acid attached to the newly arrived aminoacyl-tRNA forms a peptide bond with the initiation metluonine. The GTP on EFla is hydrolyzed to GDP, and EFla-GDP dissociates from the ribosome. EFl~3y binds EFla-GDP
and induces the dissociation of GDP from EFla, allowing EFla to bind GTP and a new cycle to begin.
As subsequent aminoacyl-tRNAs are brought to the ribosome, EF-G, another GTP
binding protein, catalyzes the translocation of tRNAs from the A site to the P site and finally to the E site of the ribosome. This allows the ribosome and the mRNA to remain attached during translation.
Translation Termination The release factor eRF carries out termination of translation. eRF recognizes stop codons in the mRNA, leading to the release of the polypeptide chain from the ribosome.
E~ression profiling Microarrays are analytical tools used in bioanalysis. A microarray has a plurality of molecules spatially distributed over, and stably associated with, the surface of a solid support. Microarrays of polypeptides, polynucleotides, and/or antibodies have been developed and hnd use in a variety of applications, such as gene sequencing, monitoring gene expression, gene mapping, bacterial identification, drug discovery, and combinatorial chemistry.
One area in particular in which microarrays find use is in gene expression analysis. Array technology can provide a simple way to explore the expression of a single polymorphic gene or the expression profile of a large number of related or unrelated genes. When the expression of a single gene is examined, arrays are employed to detect the expression of a specific gene or its variants.
When an expression profile is examined, arrays provide a platform for identifying genes that are tissue specific, are affected by a substance being tested in a toxicology assay, are part of a signaling cascade, carry out housekeeping functions, or are specifically related to a particular genetic predisposition, condition, disease, or disorder.
Breast Cancer There are more than 180,000 new cases of breast cancer diagnosed each year, and the mortality rate for breast cancer approaches 10°Io of all deaths in females between the ages of 45-54 (K. Gish (1999) AWIS Magazine 28:7-10). However the survival rate based on early diagnosis of localized breast cancer is extremely high (97%), compared with the advanced stage of the disease in which the tumor has spread beyond the breast (22%). Current procedures for clinical breast examination are lacking in sensitivity and specificity, and efforts are underway to develop comprehensive gene expression profiles for breast cancer that may be used in conjunction with conventional screening methods to improve diagnosis and prognosis of this disease (Perou, C.M. et al.
(2000) Nature 406:747-752).
Mutations in two genes, BRCA1 and BRCA2, are known to greatly predispose a woman to breast cancer and may be passed on from parents to children (Gish, K. (1999) AWIS Magazine 28:7-10). However, this type of hereditary breast cancer accounts for only about 5 % to 9 % of breast cancers, while the vast majority of breast cancer is due to non-inherited mutations that occur in breast epithelial cells.
The relationship between expression of epidermal growth factor (EGF) and its receptor, EGFR, to human mammary carcinoma has been particularly well studied (see Khazaie, K. et al.
(1993) Cancer and Metastasis Rev. 12:255-274, and references cited therein for a review of this area). Overexpression of EGFR, particularly coupled with down-regulation of the estrogen receptor, is a marker of poor prognosis in breast cancer patients. In addition, EGFR
expression in breast tumor metastases is frequently elevated relative to the primary tumor, suggesting that EGFR is involved in tumor progression and metastasis. This is supported by accumulating evidence that EGF has effects on cell functions related to metastatic potential, such as cell motility, chemotaxis, secretion and.
differentiation. Changes in expression of other members of the erbB receptor family, of which EGFR
is one, have also been implicated in breast cancer. The abundance of erbB
receptors, such as HER-2lneu, HER-3, and HER-4, and their ligands in breast cancer points to their functional importance in the pathogenesis of the disease, and may therefore provide targets for therapy of the disease (Bacus, S. S. et al. (1994) Am. J. Clip. Pathol. 102:513-S24). Other known markers of breast cancer include a human secreted frizzled protein mRNA that is downregulated in breast tumors;
the matrix G1 a protein which is overexpressed is human breast carcinoma cells; Drg1 or RTP, a gene whose expression is diminished in colon, breast, and prostate tumors; maspin, a tumor suppressor gene downregulated in invasive breast carcinomas; and CaNl9, a member of the 5100 protein family, all of which are down regulated in mammary carcinoma cells relative to normal mammary epithelial cells (Zhou, Z, et al. (1998) Int. J. Cancer 78:95-99; Chen, L. et al. (1990) Oncogene 5:1391-1395; LTJrix, W. et al (1999) FEBS Lett. 455:23-26; Sager, R. et al. (1996) Curr. Top.
Microbiol. Tmmunol. 213:51-64; and Lee, S. W. et al. (1992) Proc. Natl. Acad. Sci. USA 89:2504-2508).
Cell lines derived from human mammary epithelial cells at various stages of breast cancer provide a useful model to study the process of malignant transformation and tumor progression as it has been shown that these cell lines retain many of the properties of their parental tumors for lengthy culture periods (Wistuba, LI. et al. (1998) Clip. Cancer Res. 4:2931-2938).
Such a model is particularly useful for comparing phenotypic and molecular characteristics of human mammary epithelial cells at various stages of malignant transformation.
The immune system responds to infection or trauma by activating a cascade of events that coordinate the progressive selection, amplification, and mobilization of cellular defense mechanisms.

A complex and balanced program of gene activation and repression is involved in this process.
However, hyperactivity of the immune system as a result of improper or insufficient regulation of gene expression may result in considerable tissue or organ damage. This damage is well documented in immunological responses associated with arthritis, allergens, heart attack, stroke, and infections (Harrison's Principles of Internal Medicine, 13/e, McGraw Hill, Inc. and Teton Data Systems Software, 1996). In particular, a zinc finger protein termed Staf50 (for Stimulated traps-acting factor of 50 kDa) is a transcriptional regulator and is induced in various cell lines by interferon-I and -1I.
Staf50 appears to mediate the antiviral activity of interferon by down-regulating the viral transcription directed by the long terminal repeat promoter region of human immunodeficiency virus type-1 in transfected cells (Tissot, C. (1995) J. Biol. Chem. 270:14891-14898).
Dendritic cells (DC) are antigen presenting cells (APC) that play a key role in the primary immune response because of their unique ability to present antigens to naive T-cells. In addition, DC
differentiate into separate subsets of mature immune 'cells that sustain and regulate immune responses following initial contact with antigen. DC subsets include those that preferentially induce particular T
helper 1 (Th1) or T helper 2 (Th2) responses and those that regulate B cell responses. Moreover, DC
are being used with increasing frequency to manipulate immune responses, either to downregulate aberrant autoimmune response or to enhance vaccination or tumor-specific response.
DC are functionally specialized in correlation with their particular differentiation state. CD34+
myeloid cells found in the bone marrow mature in response to signals into CD14+ CDllc+ monocytes.
An innate or antigen non-specific response takes place initially when monocytes circulate to nonlymphoid tissues and respond to lipopolysaccharide (LPS), a bacterially-derived mitogen, and viruses. Such direct encounters with antigen cause secretion of pro-inflammatory cytokines that attract and regulate natural killer cells, macrophages, and eosinophils in the first line of defense against invading pathogens. Monocytes then mature into DC, which efficiently capture antigen through endocytosis and antigen-receptor uptake. Antigen processing and presentation trigger activation and differentiation into mature DC that express MHC class 1T molecules on the cell surface and efficiently activate T-cells, initiating antigen-specific T-cell and B-cell responses. In turn, T-cells activate DC
through CD40 ligand - CD40 interactions, which stimulate expression of the costimulatory molecules CD80 and CD86, the latter most potent in amplifying T-cell responses. DC
interaction via CD40 with T cells also stimulates the production of inflammatory cytokines such as TNF
alpha and IL-1.
Engagement of RANK, a member of the TNF receptor family by its ligand, TRANCE, which is expressed on activated T cells, enhances the survival of DC through inhibition of apoptosis, thereby enhancing T cell activation. The maturation and differentiation of monocytes into mature DC links the antigen non-specific innate immune response to the antigen-specific adaptive immune response.
Human peripheral blood mononuclear cells (PBMCs) can be classified into discrete cellular populations representing the major components of the immune system. PBMCs contain about 52%
lymphocytes (12% B lymphocytes, 40% T lymphocytes {25% CD4+ and 15% CD8+]), 20% NK
cells, 25% monocytes, and 3% various cells that include dendritic cells and progenitor cells. The proportions, as well as the biology of these cellular components tend to vary slightly between healthy individuals, depending on factors such as age, past medical history, and genetic backgrounds.
Steroid Hormones Steroids are a class of lipid-soluble molecules, including cholesterol, bile acids, vitamin D, and hormones, that share a common four-ring structure based on cyclopentanoperhydrophenanthrene and that carrry out a wide variety of functions. Cholesterol, for example, is a component of cell membranes that controls membrane fluidity. It is also a precursor for bile acids which solubilize lipids and facilitate absorption in the small intestine during digestion. Vitamin D
regulates the absorption of calcium in the small intestine and controls the concentration of calcium iu plasma. Steroid hormones, produced by the adrenal cortex, ovaries, and testes, include glucocorticoids, mineralocorticoids, androgens, and estrogens. They control various biological processes by binding to intracellular receptors that regulate transcription of specific genes in the nucleus.
Glucocorticoids, for example, increase blood glucose concentrations by regulation of gluconeogenesis in the liver, increase blood concentrations of fatty acids by promoting lipolysis in adipose tissues, modulate sensitivity to catcholamines in the central nervous system, and reduce inflammation. The principal mineralocorticoid, aldosterone, is produced by the adrenal cortex and acts on cells of the distal tubules of the kidney to enhance sodium ion reabsorption. Androgens, produced by the interstitial cells of Leydig in the testis, include the male sex hormone testosterone, which triggers changes at puberty, the production of sperm and maintenance of secondary sexual characteristics.
Female sex hormones, estrogen and progesterone, are produced by the ovaries and also by the placenta and adrenal cortex of the fetus during pregnancy. Estrogen regulates female reproductive processes and secondary sexual characteristics. Progesterone regulates changes iu the endometrium during the menstrual cycle and pregnancy.
Steroid hormones are widely used for fertility control and in anti-inflammatory treatments for physical injuries and diseases such as arthritis, asthma, and auto-immune disorders. Progesterone, a naturally occurring progestin, is primarily used to treat amenorrhea, abnormal uterine bleeding, or as a contraceptive. Endogenous progesterone is responsible for inducing secretory activity in the endometrium of the estrogen-primed uterus in preparation for the implantation of a fertilized egg and for the maintenance of pregnancy. It is secreted from the corpus luteum in response to luteinizing hormone (LH). The primary contraceptive effect of exogenous progestins involves the suppression of the midcycle surge of LH. At the cellular level, progestins diffuse freely into target cells and bind to the progesterone receptor. Target cells include the female reproductive tract, the mammary gland, the hypothalamus, and the pituitary. Once bound to the receptor, progestins slow the frequency of release of gonadotropin releasing hormone from the hypothalamus and blunt the pre-ovulatory LH
surge, thereby preventing follicular maturation and ovulation. Progesterone has minimal estrogenic and androgenic activity. Progesterone is metabolized hepatically to pregnanediol and conjugated with glucuronic acid.
Medroxyprogesterone (MAH), also known as 6a-methyl-17-hydroxyprogesterone, is a synthetic progestin with a pharmacological activity about 15 times greater than progesterone. MAH is used for the treatment of renal and endometrial carcinomas, amenorrhea, abnormal uterine bleeding, and endometriosis associated with hormonal imbalance. MAH has a stimulatory effect on respiratory centers and has been used in cases of low blood oxygenation caused by sleep apnea, chronic obstructive pulmonary disease, or hypercapnia.
Mifepristone, also known as RU-486, is an antiprogesterone drug that blocks receptors of progesterone. It counteracts the effects of progesterone, which is needed to sustain pregnancy.
Mifepristone induces spontaneous abortion when administered in early pregnancy followed by treatment with the prostaglandin, misoprostol. Further, studies show that mifepristone at a substantially lower dose can be lughly effective as a postcoital contraceptive when administered within five days after unprotected intercourse, thus providing women with a "morning-after pill" in case of contraceptive failure or sexual assault. Mifepristone also has potential uses in the treatment of breast and ovarian cancers in cases in which tumors are progesterone-dependent. It interferes with steroid-dependent growth of brain meningiomas, and may be useful in treatment of endometriosis where it blocks the estrogen-dependent growth of endometrial tissues. It may also be useful in treatment of uterine fibroid tumors and C~shing's Syndrome. Mifepristone binds to glucocorticoid receptors and interferes with cortisol binding. Mifepristone also may act as an anti-glucocorticoid and be effective for treating conditions where cortisol levels are elevated such as AIDS, anorexia nervosa, ulcers, diabetes, Parkinson's disease, multiple sclerosis, and Alzheimer's disease.
Danazol is a synthetic steroid derived from ethinyl testosterone. Danazol indirectly reduces estrogen production by lowering pituitary synthesis of follicle-stimulating hormone and LH. Danazol also binds to sex hormone receptors in target tissues, thereby exhibiting anabolic, antiestrognic, and weakly androgenic activity. Dauazol does not possess any progestogenic activity, and does not suppress normal pituitary release of corticotropin or release of cortisol by the adrenal glands. Danazol is used in the treatment of endometriosis to relieve pain and inhibit endometrial cell growth. It is also used to treat fibrocystic breast disease and hereditary angioedema.
Corticosteroids are used to relieve inflammation and to suppress the immune response. They inhibit eosinophil, basophil, and airway epithelial cell function by regulation of cytokines that mediate the inflammatory response. They inhibit leukocyte infiltration at the site of inflammation, interfere in the function of mediators of the inflammatory response, and suppress the humoral immune response.
Corticosteroids are used to treat allergies, asthma, arthritis, and skin conditions. Beclomethasone is a synthetic glucocorticoid that is used to treat steroid-dependent asthma, to relieve symptoms associated with allergic or nonallergic (vasomotor) rhinitis, or to prevent recurrent nasal polyps following surgical removal. The anti-inflammatory and vasoconstrictive effects of intranasal beclomethasone are 5000 times greater than those produced by hydrocortisone. Budesonide is a corticosteroid used to control symptoms associated with allergic rhinitis or asthma. Budesonide has high topical anti-inflammatory activity but Iow systemic activity. Dexamethasone is a synthetic glucocorticoid used in anti-inflammatory or immunosuppressive compositions. It is also used in inhalants to prevent symptoms of asthma. Due to its greater ability to reach the central nervous system, dexamethasone is usually the treatment of choice to control cerebral edema. Dexamethasone is approximately 20-30 times more potent than hydrocortisone and 5-7 times more potent than prednisone.
Prednisone is metabolized in the liver to its active form, prednisolone, a glucocorticoid with anti-inflammatory properties.
Prednisone is approximately 4 times more potent than hydrocortisone and the duration of action of prednisone is intermediate between hydrocortisone and dexamethasone.
Prednisone is used to treat allograft rejection, asthma, systemic lupus erythematosus, arthritis, ulcerative colitis, and other inflammatory conditions. Betamethasone is a synthetic glucocorticoid with antiinflammatory and immunosuppressive activity and is used to treat psoriasis and fungal infections, such as athlete's foot and ringworm.
The anti-inflammatory actions of corticosteroids are thought to involve phospholipase AZ
inhibitory proteins, collectively called lipocortins. Lipocortins, in turn, control the biosynthesis of potent mediators of inflammation such as prostaglandins and leukotrienes by inhibiting the release of the precursor molecule arachidonic acid. Proposed mechanisms of action include decreased IgE
synthesis, increased number of (3-adrenergic receptors on leukocytes, and decreased arachidonic acid metabolism. During an immediate allergic reaction, such as in chronic bronchial asthma, allergens bridge the IgE antibodies on the surface of mast cells, which triggers these cells to release chemotactic substances. Mast cell influx and activation, therefore, is partially responsible for the inflammation and hyperirritability of the oral mucosa in asthmatic patients.
This inflammation can be retarded by administration of corticosteroids.
There is a need in the art for new compositions, including nucleic acids and proteins, for the diagnosis, prevention, and treatment of cell proliferative, neurological, reproductive, developmental, autoimmune/inflammatory, and DNA repair disorders, and infections.
SUMMARY OF THE INVENTION
Various embodiments of the invention provide purified polypeptides, nucleic acid-associated proteins, referred to collectively as "NAAP" and individually as "NAAP-1,"
"NAAP-2," "1VAAP-3,"
"NAAP-4," "NAAP-5," "NAAP-6," "NAAP-7," "NAAP-8," "NAAP-9," "NAAP-10," "NAAP-11,". "NAAP-12," "NAAP-13," "NAAP-14," "NAAP-15," "NAAP-16," "NAAP-17, "NAAP-18,"
"NAAP-19," "NAAP-20," "NAAP-21," "NAAP-22," "NAAP-23," "NAAP-24," "NAAP-25,"
"NAAP-26," "NAAP-27," "NAAP-28," "NAAP-29," "NAAP-30," "NAAP-31," "NAAP-32,"
"NAAP-33," "NAAP-34," "NAAP-35," and "NAAP-36," and methods for using these proteins and their encoding polynucleotides for the detection, diagnosis, and treatment of diseases and medical conditions. Embodiments also provide methods for utilizing the purified nucleic acid-associated proteins and/or their encoding polynucleotides for facilitating the drug discovery process, including determination of efficacy, dosage, toxicity, and pharmacology. Related embodiments provide methods for utilizing the purified nucleic acid-associated proteins and/or their encoding polynucleotides for investigating the pathogenesis of diseases and medical conditions.
An embodiment provides an isolated polypeptide selected from the group consisting of a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:1-36, b) a polypeptide comprising a naturally occurring amino acid sequence at least 90% identical or at least about 90% identical to an amino acid sequence selected from the group consisting of SEQ ID
N0:1-36, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID N0:1-36, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID
N0:1-36. Another embodiment provides an isolated polypeptide comprising an amino acid sequence of SEQ ID N0:1-36.
Still another embodiment provides an isolated polynucleotide encoding a polypeptide selected from the group consisting of a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ D7 N0:1-36, b) a polypeptide comprising a naturally occurring amino acid sequence at least 90% identical or at least about 90% identical to an amino acid sequence selected from the group consisting of SEQ ll~ N0:1-36, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ m N0:1-36, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ m N0:1-36. In another embodiment, the polynucleotide encodes a polypeptide selected from the group consisting of SEQ m NO:1-36. In an alternative embodiment, the polynucleotide is selected from the group consisting of SEQ m N0:37-72.
Still another embodiment provides a recombinant polynucleotide comprising a promoter sequence operably linked to a polynucleotide encoding a polypeptide selected from the group consisting of a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID N0:1-36, b) a polypeptide comprising a naturally occurring amino acid sequence at least 90% identical or at least about 90% identical to an amino acid sequence selected from the group consisting of SEQ m N0:1-36, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1-36, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID N0:1-36.
Another embodiment provides a cell transformed with the recombinant polynucleotide. Yet another embodiment provides a transgenic organism comprising the recombinant polynucleotide.
Another embodiment provides a method for producing a polypeptide selected from the group consisting of a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ B? N0:1-36, b) a polypeptide comprising a naturally occurring amino acid sequence at least 90% identical or at least about 90% identical to an amino acid sequence selected from the group consisting of SEQ m N0:1-36, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ m N0:1-36, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ 117 N0:1-36.
The method comprises a) culturing a cell under conditions suitable for expression of the polypep'tide, wherein said cell is transformed with a recombinant polynucleotide comprising a promoter sequence operably linked to a polynucleotide encoding the polypeptide, and b) recovering the polypeptide so expressed.
Yet another embodiment provides an isolated antibody which specifically binds to a polypeptide selected from the group consisting of a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ m NO:1-36, b) a polypeptide comprising a naturally occurring amino acid sequence at least 90% identical or at least about 90%
identical to an amino acid sequence selected from the group consisting of SEQ ID N0:1-36, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ m N0:1-36, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ m NO:1-36.
Still yet another embodiment provides an isolated polynucleotide selected from the group consisting of a) a polynucleotide comprising a polynucleotide sequence selected from the group consisting of SEQ m NO:37-72, b) a polynucleotide comprising a naturally occurring polynucleotide sequence at least 90% identical or at least about 90% identical to a polynucleotide sequence selected from the group consisting of SEQ m N0:37-72, c) a polynucleotide complementary to the polynucleotide of a), d) a polynucleotide complementary to the polynucleotide of b), and e) an RNA
equivalent of a)-d). In other embodiments, the polynucleotide can comprise at least about 20, 30, 40, 60, 80, or 100 contiguous nucleotides.
Yet another embodiment provides a method for detecting a target polynucleotide in a sample, said target polynucleotide being selected from the group consisting of a) a polynucleotide comprising a polynucleotide sequence selected from the group consisting of SEQ m NO:37-72, b) a polynucleotide comprising a naturally occurring polynucleotide sequence at least 90%
identical or at least about 90%
identical to a polynucleotide sequence selected from the group consisting of SEQ m N0:37-72, c) a polynucleotide complementary to the polynucleotide of a), d) a polynucleotide complementary to the polynucleotide of b), and e) an RNA equivalent of a)-d). The method comprises a) hybridizing the sample with a probe comprising at least 20 contiguous nucleotides comprising a sequence complementary to said target polynucleotide in the sample, and which probe specifically hybridizes to said target polynucleotide, under conditions whereby a hybridization complex is formed between said probe and said target polynucleotide or fragments thereof, and b) detecting the presence or absence of said hybridization complex. In a related embodiment, the method can include detecting the amount of the hybridization complex. In still other embodiments, the probe can comprise at least about 20, 30, 40, 60, 80, or 100 contiguous nucleotides.
Still yet another embodiment provides a method for detecting a target polynucleotide in a sample, said target polynucleotide being selected from the group consisting of a) a polynucleotide comprising a polynucleotide sequence selected from the group consisting of SEQ
m NO:37-72, b) a polynucleotide comprising a naturally occurring polynucleotide sequence at least 90% identical or at least about 90% identical to a polynucleotide sequence selected from the group consisting of SEQ m N0:37-72, c) a polynucleotide complementary to the polynucleotide of a), d) a polynucleotide complementary to the polynucleotide of b), and e) an RNA equivalent of a)-d).
The method comprises a) amplifying said target polynucleotide or fragment thereof using polymerase chain reaction amplification, and b) detecting the presence or absence of said amplified target polynucleotide or fragment thereof. In a related embodiment, the method can include detecting the amount of the amplified target polynucleotide or fragment thereof.
Another embodiment provides a composition comprising an effective amount of a polypeptide selected from the group consisting of a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ll~ N0:1-36, b) a polypeptide comprising a naturally occurring amino acid sequence at least 90% identical or at least about 90% identical to an amino acid sequence selected from the group consisting of SEQ m N0:1-36, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ m N0:1-36, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ll~ N0:1-36, and a pharmaceutically acceptable excipient. In one embodiment, the composition can comprise an amino acid sequence selected from the group consisting of SEQ m N0:1-36. Other embodiments provide a method of treating a disease or condition associated with decreased or abnormal expression of functional NAAP, comprising administering to a patient in need of such treatment the composition.
Yet another embodiment provides a method for screening a compound for effectiveness as an agonist of a polypeptide selected from the group consisting of a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:1-36, b) a polypeptide comprising a naturally occurring amino acid sequence at least 90% identical or at least about 90% identical to an amino acid sequence selected from the group consisting of SEQ ll~ N0:1-36, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ
~ N0:1-36, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ m N0:1-36. The method comprises a) exposing a sample comprising the polypeptide to a compound, and b) detecting agonist activity in the sample. Another embodiment provides a composition comprising an agonist compound identified by the method and a pharmaceutically acceptable excipient. Yet another embodiment provides a method of treating a disease or condition associated with decreased expression of functional NAAP, comprising administering to a patient in need of such treatment the composition.
Still yet another embodiment provides a method for screening a compound for effectiveness as an antagonist of a polypeptide selected from the group consisting of a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ m N0:1-36, b) a polypeptide comprising a naturally occurring amino acid sequence at least 90% identical or at least about 90%
identical to an amino acid sequence selected from the group consisting of SEQ
m N0:1-36, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ 1D N0:1-36, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ D? N0:1-36. The method comprises a) exposing a sample comprising the polypeptide to a compound, and b) detecting antagonist activity in the sample. Another embodiment provides a composition comprising an antagonist compound identified by the method and a pharmaceutically acceptable excipient. Yet another embodiment provides a method of treating a disease or condition associated with overexpression of functional NAAP, comprising administering to a patient in need of such treatment the composition.
Another embodiment provides a method of screening for a compound that specifically binds to a polypeptide selected from the group consisting of a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ m N0:1-36, b) a polypeptide comprising a naturally occurring amino acid sequence at least 90% identical or at least about 90% identical to an amino acid sequence selected from the group consisting of SEQ 1D N0:1-36, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ
m NO:1-36, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ m N0:1-36. The method comprises a) combining the polypeptide with at least one test compound under suitable conditions, and b) detecting binding of the polypeptide to the test compouxld, thereby identifying a compound that specifically binds to the polypeptide.
2o Yet another embodiment provides a method of screening for a compound that modulates the activity of a polypeptide selected from the group consisting of a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ m N0:1-36, b) a polypeptide comprising a naturally occurring amino acid sequence at least 90% identical or at least about 90% identical to an amino acid sequence selected from the group consisting of SEQ B7 N0:1-3 6, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ
>D N0:1-36, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ll~ N0:1-36. The method comprises a) combining the polypeptide with at least one test compound under conditions permissive for the activity of the polypeptide, b) assessing the activity of the polypeptide in the presence of the test compound, and c) comparing the activity of the polypeptide in the presence of the test compound with the activity of the polypeptide in the absence of the test compound, wherein a change in the activity of the polypeptide in the presence of the test compound is indicative of a compound that modulates the activity of the polypeptide.

Still yet another embodiment provides a method for screening a compound for effectiveness in altering expression of a target polynucleotide, wherein said target polynucleotide comprises a polynucleotide sequence selected from the group consisting of SEQ ID N0:37-72, the method comprising a) exposing a sample comprising the target polynucleotide to a compound, b) detecting altered expression of the target polynucleotide, and c) comparing the expression of the target polynucleotide in the presence of varying amounts of the compound and in the absence of the compound.
Another embodiment provides a method for assessing toxicity of a test compound, said method comprising a) treating a biological sample containing nucleic acids with the test compound; b) hybridizing the nucleic acids of the treated biological sample with a probe comprising at least 20 contiguous nucleotides of a polynucleotide selected from the group consisting of i) a polynucleotide comprising a polynucleotide sequence selected from the group consisting of SEQ
ID N0:37-72, ii) a polynucleotide comprising a naturally occurring polynucleotide sequence at least 90% identical or at least about 90% identical to a polynucleotide sequence selected from the group consisting of SEQ ll~
N0:37-72, iii) a polynucleotide having a sequence complementary to i), iv) a polynucleotide complementary to the polynucleotide of ii), and v) an RNA equivalent of i)-iv). Hybridization occurs under conditions whereby a specific hybridization complex is formed between said probe and a target polynucleotide in the biological sample, said target polynucleotide selected from the group consisting of i) a polynucleotide comprising a polynucleotide sequence selected from the group consisting of SEQ
ll~ N0:37-72, ii) a polynucleotide comprising a naturally occurring polynucleotide sequence at least 90% identical or at least about 90% identical to a polynucleotide sequence selected from the group consisting of SEQ ID N0:37-72, iii) a polynucleotide complementary to the polynucleotide of i), iv) a polynucleotide complementary to the polynucleotide of ii), and v) an RNA
equivalent of i)-iv).
Alternatively, the target polynucleotide can comprise a fragment of a polynucleotide selected from the group consisting of i)-v) above; c) quantifying the amount of hybridization complex; and d) comparing the amount of hybridization complex in the treated biological sample with the amount of hybridization complex in an untreated biological sample, wherein a difference in the amount of hybridization complex in the treated biological sample is indicative of toxicity of the test compound.
3o BRIEF DESCRIPTION OF THE TABLES
Table 1 summarizes the nomenclature for full length polynucleotide and polypeptide embodiments of the invention.
Table 2 shows the GenBank identification number and annotation of the nearest GenBank homolog, and the PROTEOME database identification numbers and annotations of PROTEOME
database horriologs, for polypeptide embodiments of the invention. The probability scores for the matches between each polypeptide and its homolog(s) are also shown.
Table 3 shows structural features of polypeptide embodiments, including predicted motifs and domains, along with the methods, algorithms, and searchable databases used for analysis of the polypeptides.
Table 4 lists the cDNA and/or genomic DNA fragments which were used to assemble polynucleotide embodiments, along with selected fragments of the polynucleotides.
Table 5 shows representative cDNA libraries for polynucleotide embodiments.
Table 6 provides an appendix which describes the tissues and vectors used for construction of the cDNA libraries shown in Table 5.
Table 7 shows the tools, programs, and algorithms used to analyze polynucleotides and polypeptides, along with applicable descriptions, references, and threshold parameters.
Table 8 shows single nucleotide polymorphisms found in polynucleotide embodiments, along with allele frequencies in different human populations.
DESCRIPTION OF THE INVENTION
Before the present proteins, nucleic acids, and methods are described, it is understood that embodiments of the invention are not limited to the particular machines, instruments, materials, and methods described, as these may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the invention.
As used herein and in the appended claims, the singular forms "a," "an," and "the" include plural reference unless the context clearly dictates otherwise. Thus, for example, a reference to "a host cell" includes a plurality of such host cells, and a reference to "an antibody" is a reference to one or more antibodies and equivalents thereof known to those skilled in the art, and so forth.
Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art to which this invention belongs.
Although any machines, materials, and methods similar or equivalent to those described herein can be used to practice or test the present invention, the preferred machines, materials and methods are now described. All publications mentioned herein are cited for the purpose of describing and disclosing the cell lines, protocols, reagents and vectors which are reported in the publications and which might be used in connection with various embodiments of the invention. Nothing herein is to be construed as an admission that the invention is not entitled to antedate such disclosure by virtue of prior invention.
DEFINITIONS
"NAAP" refers to the amino acid sequences of substantially purified NAAP
obtained from any species, particularly a mammalian species, including bovine, ovine, porcine, murine, equine, and human, and from any source, whether natural, synthetic, semi-synthetic, or recombinant.
The term "agonist" refers to a molecule which intensifies or mimics the biological activity of NAAP. Agonists may include proteins, nucleic acids, carbohydrates, small molecules, or any other compound or composition which modulates the activity of NAAP either by directly interacting with NAAP or by acting on components of the biological pathway in which NAAP
participates.
An "allelic variant" is an alternative form of the gene encoding NAAP. Allelic variants may result from at least one mutation in the nucleic acid sequence and may result in altered mRNAs or in polypeptides whose structure or function may or may not be altered. A gene may have none, one, or many allelic variants of its naturally occurring form. Common mutational changes which give rise to allelic variants are generally ascribed to natural deletions, additions, or substitutions of nucleotides.
Each of these types of changes may occur alone, or in combination with the others, one or more times in a given sequence.
"Altered" nucleic acid sequences encoding NAAP include those sequences with deletions, insertions, or substitutions of different nucleotides, resulting in a polypeptide the same as NAAP or a polypeptide with at least one functional characteristic of NAAP. Included within this definition are polymorphisms which may or may not be readily detectable using a particular oligonucleotide probe of the polynucleotide encoding NAAP, and improper or unexpected hybridization to allelic variants, with a locus other than the normal chromosomal locus for the polynucleotide encoding NAAP. 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 NAAP. Deliberate amino acid substitutions may be made on the basis of one or more similarities in polarity, charge, solubility, hydrophobicity, hydrophilicity, and/or the amphipathic nature of the residues, as long as the biological or immunological activity of NAAP is retained. For example, negatively charged amino acids may include aspartic acid and glutanuc acid, and positively charged amino acids may include lysine and arginine. Amino acids with uncharged polar side chains having similar hydrophilicity values may include: asparagine and glutamine; and serine and threonine. Amino acids with uncharged side chains having similar hydrophilicity values may include: leucine, isoleucine, and valine; glycine and alanine; and phenylalanine and tyrosine.
The terms "amino acid" and "amino acid sequence" can refer to an oligopeptide, a peptide, a 3~

polypeptide, or a protein sequence, or a fragment of any of these, and to naturally occurring or synthetic molecules. Where "amino acid sequence" is recited to refer to a sequence of a naturally occurring protein molecule, "amino acid sequence" and like terms are not meant to limit the amino acid sequence to the complete native amino acid sequence associated with the recited protein molecule.
"Amplification" relates to the production of additional copies of a nucleic acid. Amplification may be carried out using polymerase chain reaction (PCR) technologies or other nucleic acid amplification technologies well known in the art.
The term "antagonist" refers to a molecule wluch inhibits or attenuates the biological activity of NAAP. Antagonists may include proteins such as antibodies, anticalins, nucleic acids, carbohydrates, small molecules, or any other compound or composition which modulates the activity of NAAP either by directly interacting with NAAP or by acting on components of the biological pathway in which NAAP participates.
The term "antibody' refers to intact immunoglobulin molecules as well as to fragments thereof, such as Fab, F(ab')2, and Fv fragments, which are capable of binding an epitopic determinant.
Antibodies that bind NAAP polypeptides can be prepared using intact polypeptides or using fragments containing small peptides of interest as the immunizing antigen. The polypeptide or oligopeptide used to immunize an animal (e.g., a mouse, a rat, or a rabbit) can be derived from the translation of RNA, or synthesized chemically, and can be conjugated to a carrier protein if desired. Commonly used carriers that are chemically coupled to peptides include bovine serum albumin, thyroglobulin, and keyhole limpet hemocyanin (KLH). The coupled peptide is then used to immunize the animal.
The term "antigenic determinant" refers to that region of a molecule (i.e., an epitope) that makes contact with a particular antibody. When a protein or a fragment of a protein is used to immunize a host animal, numerous regions of the protein may induce the production of antibodies which bind specifically to antigenic determinants (particular regions or three-dimensional structures on the protein). An antigenic determinant may compete with the intact antigen (i.e., the immunogen used to elicit the immune response) for binding to an antibody.
The term "aptamer" refers to a nucleic acid or oligonucleotide molecule that binds to a specific molecular target. Aptamers are derived from an i~t vitro evolutionary process (e.g., SELEX
(Systematic Evolution of Ligands by EXponential Enrichment), described in U.S.
Patent No.
5,270,163), which selects for target-specific aptamer sequences from large combinatorial libraries.
Aptamer compositions may be double-stranded or single-stranded, and may include deoxyribonucleotides, ribonucleotides, nucleotide derivatives, or other nucleotide-like molecules. The nucleotide components of an aptamer may have modified sugar groups (e.g., the 2'-OH group of a ribonucleotide may be replaced by 2'-F or 2'-NI32), which may improve a desired property, e.g., resistance to nucleases or longer lifetime in blood. Aptamers may be conjugated to other molecules, e.g., a high molecular weight carrier to slow clearance of the aptamer from the circulatory system.
Aptamers may be specifically cross-linked to their cognate ligands, e.g., by photo-activation of a cross-linker (Brody, E.N. and L. Gold (2000) J. Biotechnol. 74:5-13).
The term "intramer" refers to an aptamer which is expressed i~c vivo. Fox example, a vaccinia virus-based RNA expression system has been used to egress specific RNA aptamers at high levels in the cytoplasm of leukocytes (Blind, M. et al. (1999) Proc.
Natl. Acad. Sci. USA
96:3606-3610).
The term "spiegelmer" refers to an aptamer which includes L-DNA, L-RNA, or other left-handed nucleotide derivatives or nucleotide-like molecules. Aptamers containing left-handed nucleotides are resistant to degradation by naturally occurring enzymes, which normally act on substrates containing right-handed nucleotides.
The term "antisense" refers to any composition capable of base-pairing with the "sense"
(coding) strand of a polynucleotide having a specific nucleic acid sequence.
Antisense compositions may include DNA; RNA; peptide nucleic acid (PNA); oligonucleotides having modified backbone linkages such as phosphorothioates, methylphosphonates, or benzylphosphonates;
oligonucleotides having modified sugar groups such as 2'-methoxyethyl sugars or 2'-methoxyethoxy sugars; or oligonucleotides having modified bases such as 5-methyl cytosine, 2'-deoxyuracil, or 7-deaza-2'-deoxyguanosine. Antisense molecules may be produced by any method including chemical synthesis or transcription. Once introduced into a cell, the complementary antisense molecule base-pairs with a naturally occurring nucleic acid sequence produced by the cell to form duplexes which block either transcription or translation. The designation "negative" or "minus" can refer to the antisense strand, and the designation "positive" or "plus" can refer to the sense strand of a reference DNA molecule.
The term "biologically active" refers to a protein having structural, regulatory, or biochemical functions of a naturally occurring molecule. Likewise, "immunologically active" or "immunogenic"
refers to the capability of the natural, recombinant, or synthetic NAAP, or of any oligopeptide thereof, to induce a specific immune response in appropriate animals or cells and to bind with specific antibodies.
"Complementary" describes the relationship between two single-stranded nucleic acid sequences that anneal by base-pairing. For example, 5'-AGT-3' pairs with its complement, 3'-TCA-5'.
A "composition comprising a given polynucleotide" and a "composition comprising a given polypeptide" can refer to any composition containing the given polynucleotide or polypeptide. The composition may comprise a dry formulation or an aqueous solution.
Compositions comprising polynucleotides encoding NAAP or fragments of NAAP may be employed as hybridization probes.
The probes may be stored in freeze-dried form and may be associated with a stabilizing agent such as a carbohydrate. In hybridizations, the probe may be deployed in an aqueous solution containing salts (e.g., NaCl), detergents (e.g., sodium dodecyl sulfate; SDS), and other components (e.g., Denhardt's solution, dry milk, salmon sperm DNA, etc.).
"Consensus sequence" refers to a nucleic acid sequence which has been subjected to repeated DNA sequence analysis to resolve uncalled bases, extended using the XL-PCR kit (Applied 1o Biosystems, Foster City CA) in the 5' and/or the 3' direction, and resequenced, or which has been assembled from one or more overlapping cDNA, EST, or genomic DNA fragments using a computer program for fragment assembly, such as the GELV1EW fragment assembly system (GCG, Madison WI) or Phrap (University of Washington, Seattle WA). Some sequences have been both extended and assembled to produce the consensus sequence.
"Conservative amino acid substitutions" are those substitutions that are predicted to least interfere with the properties of the original protein, i.e., the structure and especially the function of the protein is conserved and not significantly changed by such substitutions. The table below shows amino acids which may be substituted for an original amino acid in a protein and which are regarded as conservative amino acid substitutions.
Original Residue Conservative Substitution Ala Gly, Ser Arg His, Lys Asn Asp, Gln, His Asp Asn, Glu Cys Ala, S er Gln Asn, Glu, His Glu Asp, Gln, His Gly Ala His Asn, Arg, Gln, Glu Ile Leu, Val Leu Ile, Val Lys Arg, Gln, Glu Met Leu, Ile Phe His, Met, Leu, Trp, Tyr Ser Cys, Thr Thr Ser, Val Trp Phe, Tyr Tyr His, Phe, Trp Val Ile, Leu, Thr Conservative amino acid substitutions generally maintain (a) the structure of the polypeptide backbone in the area of the substitution, for example, as a beta sheet or alpha helical conformation, (b) the charge or hydrophobicity of the molecule at the site of the substitution, and/or (c) the bulk of the side chain.
A "deletion" refers to a change in the amino acid or nucleotide sequence that results in the absence of one or more amino acid residues or nucleotides.
The term "derivative" refers to a chemically modified polynucleotide or polypeptide.
Chemical modifications of a polynucleotide can include, for example, replacement of hydrogen by an alkyl, acyl, hydroxyl, or amino group. A derivative polynucleotide encodes a polypeptide which retains at least one biological or immunological function of the natural molecule. A
derivative polypeptide is one modified by glycosylation, pegylation, or any similar process that retains at least one biological or immunological function of the polypeptide from which it was derived.
A "detectable label" refers to a reporter molecule or enzyme that is capable of generating a measurable signal and is covalently or noncovalently joined to a polynucleotide or polypeptide.
"Differential expression" refers to increased or upregulated; or decreased, downregulated, or absent gene or protein expression, determined by comparing at least two different samples. Such comparisons may be carried out between, for example, a treated and an untreated sample, or a diseased and a normal sample.
"Exon shuffling" refers to the recombination of different coding regions (exons). Since an exon may represent a structural or functional domain of the encoded protein, new proteins may be assembled through the novel reassortment of stable substructures, thus allowing acceleration of the evolution of new protein functions.
A "fragment" is a unique portion of NAAP or a polynucleotide encoding NAAP
which can be identical in sequence to, but shorter in length than, the parent sequence. A
fragment may comprise up to the entire length of the defined sequence, minus one nucleotide/amino acid residue. For example, a fragment may comprise from about 5 to about 1000 contiguous nucleotides or amino acid residues. A
fragment used as a probe, primer, antigen, therapeutic molecule, or for other purposes, may be at least 5, 10, 15, 16, 20, 25, 30, 40, 50, 60, 75, 100, 150, 250 or at least 500 contiguous nucleotides or amino acid residues in length. Fragments may be preferentially selected from certain regions of a molecule.
For example, a polypeptide fragment may comprise a certain length of contiguous amino acids selected from the first 250 or 500 amino acids (or first 25% or 50%) of a polypeptide as shown in a certain defined sequence. Clearly these lengths are exemplary, and any length that is supported by the specification, including the Sequence Listing, tables, and figures, may be encompassed by the present embodiments.
A fragment of SEQ ID NO:37-72 can comprise a region of unique polynucleotide sequence that specifically identifies SEQ ID N0:37-72, for example, as distinct from any other sequence in the genome from which the fragment was obtained. A fragment of SEQ ID N0:37-72 can be employed in one or more embodiments of methods of the invention, for example, in hybridization and amplification technologies and in analogous methods that distinguish SEQ ID
N0:37-72 from related polynucleotides. The precise length of a fragment of SEQ ll~ N0:37-72 and the region of SEQ ID
N0:37-72 to which the fragment corresponds are routinely determinable by one of ordinary skill in the art based on the intended purpose for the fragment.
A fragment of SEQ ID N0:1-36 is encoded by a fragment of SEQ ID N0:37-72. A
fragment of SEQ ID NO:1-36 can comprise a region of unique amino acid sequence that specifically identifies SEQ ID N0:1-36. For example, a fragment of SEQ ID NO:1-36 can be used as an immunogenic peptide for the development of antibodies that specifically recognize SEQ ID N0:1-36.
The precise length of a fragment of SEQ D7 N0:1-36 and the region of SEQ ID
N0:1-36 to wluch the fragment corresponds can be determined based on the intended purpose for the fragment using one or more analytical methods described herein or otherwise known in the art.
A "full length" polynucleotide is one containing at least a translation initiation codon (e.g., methionine) followed by an open reading frame and a translation termination codon. A "full length"
polynucleotide sequence encodes a "full length" polypeptide sequence.
"Homology' refers to sequence similarity or, interchangeably, sequence identity, between two or more polynucleotide sequences or two or more polypeptide sequences.
The terms "percent identity' and "% identity," as applied to polynucleotide sequences, refer to the percentage of residue matches between at least two polynucleotide sequences aligned using a standardized algorithm. Such an algorithm may insert, in a standardized and reproducible way, gaps in the sequences being compared in order to optimize alignment between two sequences, and therefore achieve a more meaningful comparison of the two sequences.
Percent identity between polynucleotide sequences may be determined using one or more computer algorithms or programs known in the art or described herein. For example, percent identity canbe determined using the default parameters of the CLUSTAL V algorithm as incorporated into the MEGALIGN version 3.12e sequence alignment program. This program is part of the LASERGENE software package, a suite of molecular biological analysis programs (DNASTAR, Madison WI). CLUSTAL V is described in Higgins and Sharp (1989; CABIOS 5:151-153) and in.
Higgins et al. (1992; CABIOS 8:189-191). For pairwise alignments of polynucleotide sequences, the default parameters are set as follows: Ktuple=2, gap penalty=5, window=4, and "diagonals saved"=4.
The "weighted" residue weight table is selected as the default. Percent identity is reported by CLUSTAL V as the "percent similarity" between aligned polynucleotide sequences.
Alternatively, a suite of commonly used and freely available sequence comparison algorithms which can be used is provided by the National Center for Biotechnology Information (NCBI) Basic Local Alignment Search Tool (BLAST) (Altschul, S.F. et al. (1990) J. Mol.
Biol. 215:403-410), which is available from several sources, including the NCBI, Bethesda, MD, and on the Internet at http://www.ncbi.nhn.nih.govBLAST/. The BLAST software suite includes various sequence analysis programs including "blastn," that is used to align a known polynucleotide sequence with other 1o polynucleotide sequences from a variety of databases. Also available is a tool called "BLAST 2 Sequences" that is used for direct pairwise comparison of two nucleotide sequences. "BLAST 2 Sequences" can be accessed and used interactively at http://www.ncbi.nlm.nih.gov/gorf/bl2.html. The "BLAST 2 Sequences" tool can be used for both blastn and blastp (discussed below). BLAST
programs are commonly used with gap and other parameters set to default settings. For example, to compare two nucleotide sequences, one may use blastn with the "BLAST 2 Sequences" tool Version 2Ø12 (April-21-2000) set at default parameters. Such default parameters may be, for example:
Matrix: BLOS UM62 Reward for match: 1 Penalt,~ for mismatch: -2 2o Open Gap: S and Extension Gap: 2 penalties Gap x drop-off. 50 Expect: 10 Word Size: 1l Filter: on Percent identity may be measured over the length of an entire defined sequence, for example, as defined by a particular SBQ ID number, or may be measured over a shorter length, for example, over the length of a fragment taken from a larger, defined sequence, for instance, a fragment of at least 20, at least 30, at least 40, at least 50, at least 70, at least 100, or at least 200 contiguous nucleotides. Such lengths are exemplary only, and it is understood that any fragment length supported by the sequences shown herein, in the tables, figures, or Sequence Listing, may be used to describe a length over which percentage identity may be measured.
Nucleic acid sequences that do not show a high degree of identity may nevertheless encode similar amino acid sequences due to the degeneracy of the genetic code. It is understood that changes in a nucleic acid sequence can be made using this degeneracy to produce multiple nucleic acid sequences that all encode substantially the same protein.
The phrases "percent identity" and "°!o identity," as applied to polypeptide sequences, refer to the percentage of residue matches between at least two polypeptide sequences aligned using a standardized algorithm. Methods of polypeptide sequence alignment are well-known. Some alignment methods take into account conservative amino acid substitutions. Such conservative substitutions, explained in more detail above, generally preserve the charge and hydrophobicity at the site of substitution, thus preserving the structure (and therefore function) of the polypeptide.
Percent identity between polypeptide sequences may be determined using the default 1o parameters of the CLUSTAL V algorithm as incorporated into the MEGALIGN
version 3.12e sequence alignment program (described and referenced above). For pairwise alignments of polypeptide sequences using CLUSTAL V, the default parameters are set as follows: Ktuple=1, gap penalty=3, window=5, and "diagonals saved"=5. The PAM250 matrix is selected as the default residue weight table. As with polynucleotide alignments, the percent identity is reported by CLUSTAL V as the "percent similarity" between aligned polypeptide sequence pairs.
Alternatively the NCBI BLAST software suite may be used. For example, for a pairwise comparison of two polypeptide sequences, one may use the "BLAST 2 Sequences"
tool Version 2Ø12 (April-21-2000) with blastp set at default parameters. Such default parameters may be, for example:
2o Matrix: BLOSUM62 Opera Gap: 11 arcd ExteTasion Gap: 1 penalties Gap x drop-off. 50 Expect: 10 Word Size: 3 Filter: oy~c Percent identity may be measured over the length of an entire defined polypeptide sequence, for example, as defined by a particular SEQ ID number, or may be measured over a shorter length, for example, over the length of a fragment taken from a larger, defined polypeptide sequence, for instance, a fragment of at least 15, at least 20, at least 30, at least 40, at least 50, at least 70 or at least 150 contiguous residues. Such lengths are exemplary only, and it is understood that any fragment length supported by the sequences shown herein, in the tables, figures or Sequence Listing, may be used to describe a length over which percentage identity may be measured.
"Human artificial chromosomes" (HACs) are linear microchromosomes which may contain DNA sequences of about 6 kb to 10 Mb in size and which contain all of the elements required for chromosome replication, segregation and maintenance.
The term "humanized antibody" refers to an antibody molecule in which the amino acid sequence in the non-antigen binding regions has been altered so that the antibody more closely resembles a human antibody, and still retains its original binding ability.
"Hybridization" refers to the process by which a polynucleotide strand anneals with a complementary strand through base pairing under defined hybridization conditions. Specific hybridization is an indication that two nucleic acid sequences share a high degree of complementarity.
Specific hybridization complexes form under permissive annealing conditions and remain hybridized after the "washing" step(s). The washing steps) is particularly important in determining the stringency of the hybridization process, with more stringent conditions allowing less non-specific binding, i.e., binding between pairs of nucleic acid strands that are not perfectly matched. Permissive conditions for annealing of nucleic acid sequences are routinely determinable by one of ordinary skill in the art and may be consistent among hybridization experiments, whereas wash conditions may be varied among experiments to achieve the desired stringency, and therefore hybridization specificity.
Permissive annealing conditions occur, for example, at 68°C in the presence of about 6 x SSC, about 1% (w/v) SDS, and about 100 ~.g/ml sheared, denatured salmon sperm DNA.
Generally, stringency of hybridization is expressed, in part, with reference to the temperature under which the wash step is carried out. Such wash temperatures are typically selected to be about 5°C to 20°C lower than the thermal melting point (T"~ for the specific sequence at a defined ionic strength and pH. The Tm is the temperature (under defined ionic strength and pH) at which 50% of the target sequence hybridizes to a perfectly matched probe. An equation for calculating Tm and conditions for nucleic acid hybridization are well known and can be found in Sambrook et al. (1989;
Molecular Cloning: A Laboratory Manual, 2"d ed., vol. 1-3, Cold Spring Harbor Press, Plainview NY;
specifically see volume 2, chapter 9).
High stringency conditions for hybridization between polynucleotides of the present invention include wash conditions of 68°C in the presence of about 0.2 x SSC and about 0.1% SDS, for 1 hour.
Alternatively, temperatures of about 65°C, 60°C, 55°C, or 42°C may be used. SSC concentration may be varied from about 0.1 to 2 x SSC, with SDS being present at about 0.1%.
Typically, blocking reagents are used to block non-specific hybridization. Such blocking reagents include, for instance, sheared and denatured salmon sperm DNA at about 100-200 ~Cg/ml. Organic solvent, such as formamide at a concentration of about 35-50% v/v, may also be used under particular circumstances, such as for RNA:DNA hybridizations. Useful variations on these wash conditions will be readily apparent to those of ordinary skill in the art. Hybridization, particularly under high stringency conditions, may be suggestive of evolutionary similarity between the nucleotides. Such similarity is strongly indicative of a similar role for the nucleotides and their encoded polypeptides.
The term "hybridization complex" refers to a complex formed between two nucleic acids by virtue of the formation of hydrogen bonds between complementary bases. A
hybridization complex may be formed in solution (e.g., Cot or Rot analysis) or formed between one nucleic acid present in solution and another nucleic acid immobilized on a solid support (e.g., paper, membranes, filters, chips, pins or glass slides, or any other appropriate substrate to which cells or their nucleic acids have been fixed).
1o The words "insertion" and "addition" refer to changes in an amino acid or polynucleotide sequence resulting in the addition of one or more amino acid residues or nucleotides, respectively.
"Immune response" can refer to conditions associated with inflammation, trauma, immune disorders, or infectious or genetic disease, etc. These conditions can be characterized by expression of various factors, e.g., cytokines, chemokines, and other signaling molecules, which may affect .15 cellular and systemic defense systems.
An "immunogenic fragment" is a polypeptide or oligopeptide fragment of NAAP
which is capable of eliciting an immune response when introduced into a living organism, for example, a mammal. The term "immunogenic fragment" also includes any polypeptide or oligopeptide fragment of NAAP which is useful in any of the antibody production methods disclosed herein or known in the 20 art.
The term "microarray" refers to an arrangement of a plurality of polynucleotides, polypeptides, antibodies, or other chemical compounds on a substrate.
The terms "element" and "array element" refer to a polynucleotide, polypeptide, antibody, or other chemical compound having a unique and defined position on a microarray.
25 The term "modulate" refers to a change in the activity of NAAP. For example, modulation may cause an increase or a decrease in protein activity, binding characteristics, or any other biological, functional, or immunological properties of NAAP.
The phrases "nucleic acid" and "nucleic acid sequence" refer to a nucleotide, oligonucleoti.de, polynucleotide, or any fragment thereof. These phrases also refer to DNA or RNA of genomic or 30 synthetic origin which may be single-stranded or double-stranded and may represent the sense or the antisense strand, to peptide nucleic acid (PNA), or to any DNA-like or RNA-like material.
"Operably linked" refers to the situation in which a first nucleic acid sequence, is placed in a functional relationship with a second nucleic acid sequence. For instance, a promoter is operably linked to a coding sequence if the promoter affects the transcription or expression of the coding sequence. Operably linked DNA sequences may be in close proximity or contiguous and, where necessary to join two protein coding regions, in the same reading frame.
"Peptide nucleic acid" (PNA) refers to an antisense molecule or anti-gene agent which comprises an oligonucleotide of at least about 5 nucleotides in length licked to a peptide backbone of amino acid residues ending in lysine. The terminal lysine confers solubility to the composition. PNAs preferentially bind complementary single stranded DNA or RNA and stop transcript elongation, and may be pegylated to extend their lifespan in the cell.
"Post-translational modification" of an NAAP may involve lipidation, glycosylation, phosphorylation, acetylation, racemization, proteolytic cleavage, and other modifications known in the art. These processes may occur synthetically or biochemically. Biochemical modifications will vary by cell type depending on the enzymatic milieu of NAAP.
"Probe" refers to nucleic acids encoding NAAP, their complements, or fragments thereof, which are used to detect identical, allelic or related nucleic acids. Probes are isolated oligonucleotides or polynucleotides attached to a detectable label or reporter molecule.
Typical labels include radioactive isotopes, ligands, chemiluminescent agents, and enzymes. "Primers"
are short nucleic acids, usually DNA oligonucleotides, which may be annealed to a target polynucleotide by complementary base-pairing. The primer may then be extended along the target DNA strand by a DNA polymerise enzyme. Primer pairs can be used for amplification (and identification) of a nucleic acid, e.g., by the polymerise chain reaction (PCR).
Probes and primers is used in the present invention typically comprise at least 15 contiguous nucleotides of a known sequence. In order to enhance specificity, longer probes and primers may also be employed, such as probes and primers that comprise at least 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, or at least 150 consecutive nucleotides of the disclosed nucleic acid sequences. Probes and primers may be considerably longer than these examples, and it is understood that any length supported by the specification, including the tables, figures, and Sequence Listing, may be used.
Methods for preparing and using probes and primers are described in the references, for example Sambrook et i1. (supra); Ausubel, F.M. et al. (1987; Current Protocols in Molecular Biology, Greene Publ. Assoc. & Wiley-Intersciences, New York NY); and Innis, M. et al.
(1990; PCR
Protocols, A Guide to Methods and Applications, Academic Press, San Diego CA).
PCR primer pairs can be derived from a known sequence, for example, by using computer programs intended for that purpose such is Primer (Version 0.5, 1991, Whitehead Institute for Biomedical Research, Cambridge MA).

Oligonucleotides for use as primers are selected using software known in the art for such purpose. For example, OLIGO 4.06 software is useful for the selection of PCR
primer pairs of up to 100 nucleotides each, and for the analysis of oligonucleotides and larger polynucleotides of up to 5,000 nucleotides from an input polynucleotide sequence of up to 32 kilobases.
Similar primer selection programs have incorporated additional features for expanded capabilities. For example, the PrimOU
primer selection program (available to the public from the Genome Center at University of Texas South West Medical Center, Dallas TX) is capable of choosing specific primers from megabase sequences and is thus useful for designing primers on a genome-wide scope. The Primer3 primer selection program (available to the public from the Whitehead Institute/MTT
Center for Genome Research, Cambridge MA) allows the user to input a "mispriming library," in which sequences to avoid as primer binding sites are user-specified. Primer3 is useful, in particular, for the selection of oligonucleotides for microarrays. (The source code for the latter two primer selection programs may also be obtained from their respective sources and modified to meet the user's specific needs.) The PrimeGen program (available to the public from the UK Human Genome Mapping Project Resource Centre, Cambridge UI~) designs primers based on multiple sequence alignments, thereby allowing selection of primers that hybridize to either the most conserved or least conserved regions of aligned nucleic acid sequences. Hence, this program is useful for identification of both unique and conserved oligonucleotides and polynucleotide fragments. The oligonucleotides and polynucleotide fragments identified by any of the above selection methods are useful in hybridization technologies, for example, as PCR or sequencing primers, microarray elements, or specific probes to identify fully or partially complementary polynucleotides in a sample of nucleic acids. Methods of oligonucleotide selection are not limited to those described above.
A "recombinant nucleic acid" is a nucleic acid that is not naturally occurring or has a sequence that is made by an artificial combination of two or more otherwise separated segments of sequence. This artificial combination is often accomplished by chemical synthesis or, more commonly, by the artificial manipulation of isolated segments of nucleic acids, e.g., by genetic engineering techniques such as those described in Sambrook, supf-a. The term recombinant includes nucleic acids that have been altered solely by addition, substitution, or deletion of a portion of the nucleic acid.
Frequently, a recombinant nucleic acid may include a nucleic acid sequence operably linked to a promoter sequence. Such a recombinant nucleic acid may be part of a vector that is used, for example, to transform a cell.
Alternatively, such recombinant nucleic acids may be part of a viral vector, e.g., based on a vaccinia virus, that could be use to vaccinate a mammal wherein the recombinant nucleic acid is expressed, inducing a protective immunological response in the mammal.
A "regulatory element" refers to a nucleic acid sequence usually derived from untranslated regions of a gene and includes enhancers, promoters, introns, and 5' and 3' untranslated regions (UTRs). Regulatory elements interact with host or viral proteins which control transcription, translation, or RNA stability.
"Reporter molecules" are chemical or biochemical moieties used for labeling a nucleic acid, amino acid, or antibody. Reporter molecules include radionuclides; enzymes;
fluorescent, chemiluminescent, or chromogenic agents; substrates; cofactors; inhibitors;
magnetic particles; and other moieties known in the art.
An "RNA equivalent," in reference to a DNA molecule, is composed of the same linear sequence of nucleotides as the reference DNA molecule with the exception that all occurrences of the nitrogenous base thymine are replaced with uracil, and the sugar backbone is composed of ribose instead of deoxyribose.
The term "sample" is used in its broadest sense. A sample suspected of containing NAAP, nucleic acids encoding NAAP, or fragments thereof may comprise a bodily fluid;
an extract from a cell, chromosome, organelle, or membrane isolated from a cell; a cell; genomic DNA, RNA, or cDNA, in solution or bound to a substrate; a tissue; a tissue print; etc.
The terms "specific binding" and "specifically binding" refer to that interaction between a protein or peptide and an agonist, an antibody, an antagonist, a small molecule, or any natural or synthetic binding composition. The interaction is dependent upon the presence of a particular structure of the protein, e.g., the antigenic determinant or epitope, recognized by the binding molecule. For example, if an antibody is specific for epitope "A," the presence of a polypeptide comprising the epitope A, or the presence of free unlabeled A, in a reaction containing free labeled A and the antibody will reduce the amount of labeled A that binds to the antibody.
The term "substantially purified" refers to nucleic acid or amino acid sequences that are removed from their natural environment and are isolated or separated, and are at least about 60%
free, preferably at least about 75% free, and most preferably at least about 90% free from other components with which they are naturally associated.
A "substitution" refers to the replacement of one or more amino acid residues or nucleotides by different amino acid residues or nucleotides, respectively.
"Substrate" refers to any suitable rigid or semi-rigid support including membranes, filters, chips, slides, wafers, fibers, magnetic or nonmagnetic beads, gels, tubing, plates, polymers, microparticles and capillaries. The substrate can have a variety of surface forms, such as wells, trenches, pins, channels and pores, to which polynucleotides or polypeptides are bound.
A "transcript image" or "expression profile" refers to the collective pattern of gene expression by a particular cell type or tissue under given conditions at a given time.
"Transformation" describes a process by which exogenous DNA is introduced into a recipient cell. Transformation may occux under natural or artificial conditions according to various methods well known in the art, and may rely on any known method for the insertion of foreign nucleic acid sequences into a prokaryotic or eukaryotic host cell. The method for transformation is selected based on the type of host cell being transformed and may include, but is not limited to, bacteriophage or viral infection, electroporation, heat shock, lipofection, and particle bombardment.
The term "transformed cells" includes stably transformed cells in which the inserted DNA is capable of replication either as an autonomously replicating plasmid or as part of the host chromosome, as well as transiently transformed cells which express the inserted DNA or RNA for limited periods of time.
A "transgenic organism," as used herein, is any organism, including but not limited to animals and plants, in which one or more of the cells of the organism contains heterologous nucleic acid introduced by way of human intervention, such as by transgenic techniques well known in the art. The nucleic acid is introduced into the cell, directly or indirectly by introduction into a precursor of the cell, by way of deliberate genetic manipulation, such as by microinjection or by infection with a recombinant virus. In another embodiment, the nucleic acid can be introduced by infection with a recombinant viral vector, such as a lentiviral vector (Lois, C. et al. (2002) Science 295:868-872). The term genetic manipulation does not include classical cross breeding, or ih vitro fertilization, but rather is directed to the introduction of a recombinant DNA molecule. The transgenic organisms contemplated in accordance with the present invention include bacteria, cyanobacteria, fungi, plants and animals. The isolated DNA of the present invention can be introduced into the host by methods known in the art, fox example infection, trausfection, transformation or transconjugation. Techniques for transferring the DNA of the present invention into such organisms are widely known and provided in references such as Sambrook et al., supra.
A "variant" of a particular nucleic acid sequence is defined as a nucleic acid sequence having at least 40% sequence identity to the particular nucleic acid sequence over a certain length of one of the nucleic acid sequences using blastn with the "BLAST 2 Sequences" tool Version 2Ø9 (May-07-1999) set at default parameters. Such a pair of nucleic acids may show, for example, at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% or greater sequence identity over a certain defined length. A variant may be described as, for example, an "allelic" (as defined above), "splice," "species," or "polymorphic" variant. A
splice variant may have significant identity to a reference molecule, but will generally have a greater or lesser number of polynucleotides due to alternate splicing of exons during mRNA processing. The corresponding polypeptide may possess additional functional domains or lack domains that are present in the reference molecule. Species variants are polynucleotides that vary from one species to another. The resulting polypeptides will generally have significant amino acid identity relative to each other. A
polymorphic variant is a variation in the polynucleotide sequence of a particular gene between individuals of a given species. Polymorphic variants also may encompass "single nucleotide polymorphisms" (SNPs) in which the polynucleotide sequence varies by one nucleotide base. The presence of SNPs may be indicative of, for example, a certain population, a disease state, or a propensity for a disease state.
A "variant" of a particular polypeptide sequence is defined as a polypeptide sequence having at least 40% sequence identity to the particular polypeptide sequence over a certain length of one of the polypeptide sequences using blastp with the "BLAST 2 Sequences" tool Version 2Ø9 (May-07-1999) set at default parameters. Such a pair of polypeptides may show, for example, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%
or greater sequence identity over a certain defined length of one of the polypeptides.
2o THE INVENTION
Various embodiments of the invention include new human nucleic acid-associated proteins (NAAP), the polynucleotides encoding NAAP, and the use of these compositions for the diagnosis, treatment, or prevention of cell proliferative, neurological, reproductive, developmental, autoimmune/inflammatory, and DNA repair disorders, and infections.
Table 1 summarizes the nomenclature for the full length polynucleotide and polypeptide embodiments of the invention. Each polynucleotide and its corresponding polypeptide are correlated to a single Incyte project identification number (Incyte Project ID). Each polypeptide sequence is denoted by both a polypeptide sequence identification number (Polypeptide SEQ
)D NO:) and an Incyte polypeptide sequence number (Incyte Polypeptide m) as shown. Each polynucleotide sequence is denoted by both a polynucleotide sequence identification number (Polynucleotide SEQ )D
NO:) and an Incyte polynucleotide consensus sequence number (Incyte Polynucleotide )D) as shown.
Column 6 shows the Incyte ID numbers of physical, full length clones corresponding to polypeptide and polynucleotide embodiments. The full length clones encode polypeptides which have at least 95%

sequence identity to the polypeptides shown in column 3.
Table 2 shows sequences with homology to the polypeptides of the invention as identified by BLAST analysis against the GenBank protein (genpept) database and the PROTEOME
database.
Columns 1 and 2 show the polypeptide sequence identification number (Polypeptide SEQ ID NO:) and the corresponding Incyte polypeptide sequence number (Incyte Polypeptide ID) for polypeptides of the invention. Column 3 shows the GenBank identification number (GenBank ID NO:) of the nearest GenEank homolog and the PROTEOME database identification numbers (PROTEOME ID
NO:) of the nearest PROTEOME database homologs. Column 4 shows the probability scores for the matches between each polypeptide and its homolog(s). Column 5 shows the annotation of the GenBank and PROTEOME database homolog(s) along with relevant citations where applicable, all of which are expressly incorporated by reference herein.
Table 3 shows various structural features of the polypeptides of the invention. Columns 1 and 2 show the polypeptide sequence identification number (SEQ 117 NO:) and the corresponding Incyte polypeptide sequence number (Incyte Polypeptide ID) for each polypeptide of the invention. Column 3 shows the number of amino acid residues in each polypeptide. Column 4 shows potential phosphorylation sites, and column 5 shows potential glycosylation sites, as determined by the MOTIFS
program of the GCG sequence analysis software package (Genetics Computer Group, Madison WI).
Column 6 shows amino acid residues comprising signature sequences, domains, and motifs. Column 7 shows analytical methods for protein structure/function analysis and in some cases, searchable databases to which the analytical methods were applied.
Together, Tables 2 and 3 summarize the properties of polypeptides of the invention, and these properties establish that the claimed polypeptides are nucleic acid-associated proteins. For example, SEQ ll~ NO:1 is 88% identical, from residue M1 to residue L304, to mouse genomic screen homeobox protein 2 (GenBank ID 81042009) as determined by the Basic Local Alignment Search Tool (BLAST). The BLAST probability score is 2.1e-146, which indicates the probability of obtaining the observed polypeptide sequence alignment by chance. SEQ ID N0:1 also contains a homeobox domain as determined by searching for statistically significant matches in the hidden Markov model (I~VIM)-based PFAM database of conserved protein family domains. (See Table 3.) Data from BLllVIPS, MOTIFS, PROFILESCAN, and additional BLAST analyses provide further corroborative evidence that SEQ ll? N0:1 is a homeobox protein.
In an alternative example, SEQ ID NO:8 is 99% identical, from residue G24 to residue E384, to human DNA-binding protein B (GenBank ID 8181486) as determined by the Basic Local Alignment Search Tool (BLAST). (See Table 2.) The BLAST probability score is 5.8e-199, which indicates the probability of obtaining the observed polypeptide sequence alignment by chance. SEQ
ID N0:8 also contains 'cold-shock' DNA binding domain as determined by searching for statistically significant matches in the hidden Markov model (I~~IM)-based PFAM database of conserved protein family domains. (See Table 3.) Data from BLM'S, MOTIFS, and PROFILESCAN
analyses provide further corroborative evidence that SEQ ID N0:8 is a DNA binding protein.
In another example, SEQ ID N0:13 is 94% identical, from residue M780 to residue E1598, to human centriole associated protein CEP110 (GenBank ID g3435244) as determined by the Basic Local Alignment Search Tool (BLAST). (See Table 2.) The BLAST probability score is 0.0, which indicates the probability of obtaining the observed polypeptide sequence alignment by chance. Data from MOTIFS and further BLAST analyses provide corroborative evidence that SEQ
ID N0:13 is a centriole associated protein.
In yet another example, SEQ ID NO:15 is 87% identical, from residue E435 to residue L2523, to human bromodomain PHD finger transcription factor (GenBank D7 g6683492) as determined by the Basic Local Alignment Search Tool (BLAST). (See Table 2.) The BLAST
probability score is 0.0, which indicates the probability of obtaining the observed polypeptide sequence alignment by chance.
SEQ ID NO:15 also contains a PHD ftuger domain and a bromodomain as determined by searching for statistically significant matches in the hidden Markov model (I llVIM)-based PFAM database of conserved protein family domains. (See Table 3.) Data from BLllVIPS, MOTIFS, and PROFILESCAN analyses provide further corroborative evidence that SEQ ll~ N0:15 is a bromodomain PHD finger transcription factor.
In an alternative example, SEQ ID NO:21 is 41% identical, from residue T141 to residue E370, to human Kurppel-like zinc finger protein HZF2 (GenBank ID g8163824) as determined by the Basic Local Alignment Search Tool (BLAST). (See Table 2.) The BLAST
probability score is 6.7e-71, which indicates the probability of obtaining the observed polypeptide sequence alignment by chance. SEQ ID N0:21 also contains Zinc finger C2H2 type domains as determined by searching for statistically significant matches in the hidden Markov model (FhVIM)-based PFAM database of conserved protein family domains. (See Table 3.) Data from BLILVVIPS and MOTIFS analyses provide further corroborative evidence that SEQ ID N0:21 is a C2H2 type zinc finger protein.
In yet another example, SEQ ID NO:30 is 33% identical, from residue T556 to residue E1699, and 32% identical, from residue S 10 to Y211, to Schizosacchar-omyces pombe putative helicase (GenBank ID g6901197) as determined by the Basic Local Alignment Search Tool (BLAST). (See Table 2.) The BLAST probability score is 2.9e-137, which indicates the probability of obtaining the observed polypeptide sequence alignment by chance. SEQ ID NO:30 also contains a DEAD/DEAH

box helicase domain as determined by searching for statistically significant matches in the hidden Markov model (I~VVIM)-based PFAM database of conserved protein family domains.
(See Table 3.) Data from BLAST analyses of the PRODOM and DOMO databases provide further corroborative evidence that SEQ ID N0:30 is a helicase.
In yet another example, SEQ m N0:33 is 97% identical, from residue M1 to residue V602, a murine T-box transcription factor (GenBank ID g3169261) as determined by the Basic Local Alignment Search Tool (BLAST). (See Table 2.) The BLAST probability score is 0.0, which indicates the probability of obtaining the observed polypeptide sequence alignment by chance. SEQ
m N0:33 also contains a T box domain as determined by searching for statistically significant matches in the ludden Markov model (I~VIM)-based PFAM database of conserved protein family domains. (See Table 3.) Data from BLIMPS, PRODOM and DOMO BLAST, and MOTIFS
analyses provide further corroborative evidence that SEQ ID N0:33 is a transcription factor molecule.
Taken together, the foregoing provides evidence that SEQ ID N0:1, SEQ ID N0:8, SEQ ID
NO:13, SEQ ll~ N0:15, SEQ ID N0:21, SEQ ID N0:30, and SEQ ID N0:33 are all molecules associated with nucleic acids. SEQ ID NO:2-7, SEQ ID NO:9-12, SEQ ID N0:14, SEQ l17 N0:16-20, SEQ ID N0:22-29, SEQ ID N0:31-32, and SEQ ID N0:34-36 were analyzed and annotated in a similar manner. The algorithms and parameters for the analysis of SEQ ID N0:1-36 are described in Table 7.
As shown in Table 4, the full length polynucleotide embodiments were assembled using cDNA
sequences or coding (exon) sequences derived from genomic DNA, or any combination of these two types of sequences. Column 1 lists the polynucleotide sequence identification number (Polynucleotide SEQ ll~ NO:), the corresponding Incyte polynucleotide consensus sequence number (Incyte ID) for each polynucleotide of the invention, and the length of each polynucleotide sequence in basepairs.
Column 2 shows the nucleotide start (5') and stop (3') positions of the cDNA
and/or genomic sequences used to assemble the full length polynucleotide embodiments, and of fragments of the polynucleotides which are useful, for example, in hybridization or amplification technologies that identify SEQ ID N0:37-72 or that distinguish between SEQ ID N0:37-72 and related polynucleotides.
The polynucleotide fragments described in Column 2 of Table 4 may refer specifically, for example, to Incyte cDNAs derived from tissue-specific cDNA libraries or from pooled cDNA
libraries. Alternatively, the polynucleotide fragments described in column 2 may refer to GenBank cDNAs or ESTs which contributed to the assembly of the full length polynucleotides. In addition, the polynucleotide fragments described in column 2 may identify sequences derived from the ENSEMBL
(The Sanger Centre, Cambridge, UK) database (i.e., those sequences including the designation "ENST"). Alternatively, the polynucleotide fragments described in column 2 may be derived from the NCBI RefSeq Nucleotide Sequence Records Database (i.e., those sequences including the designation "NM" or "NT") or the NCBI RefSeq Protein Sequence Records (i.e., those sequences including the designation "NP"). Alternatively, the polynucleotide fragments described in column 2 may refer to assemblages of both cDNA and Genscan-predicted exons brought together by an "exon stitching" algorithm. For example, a polynucleotide sequence identified as FL_~'~~xXXX NI NZ YYYYY N3 N4 represents a "stitched" sequence in which XXX~XX
is the identification number of the cluster of sequences to which the algorithm was applied, and YYYI'Y is the number of the prediction generated by the algorithm, and Nl,z~3..., if present, represent specific exons that may have been manually edited during analysis (See Example V).
Alternatively, the polynucleotide fragments in column 2 may refer to assemblages of exons brought together by an "exon-stretching" algorithm. For example, a polynucleotide sequence identified as FZ,~~X~:XXX_gAAAAA~BBBBB_1 N is a "stretched" sequence, with ~:~~~ being the Incyte project identification number, gAAA~4 being the GenBank identification number of the human genomic sequence to which the "exon-stretching" algorithm was applied, gBBBBB
being the GenBank identification number or NCBI RefSeq identification number of the nearest GenBank protein homolog, and N referring to specific exons (See Example V). In instances where a RefSeq sequence was used as a protein homolog for the "exon-stretching" algorithm, a RefSeq identifier (denoted by "NM,"
"NP," or "NT") may be used in place of the GenBank identifier (i.e., gBBBBB).
Alternatively, a prefix identifies component sequences that were hand-edited, predicted from genomic DNA sequences, or derived from a combination of sequence analysis methods. The following Table lists examples of component sequence prefixes and corresponding sequence analysis methods associated with the prefixes (see Example IV and Example V).
Prefix Type of analysis andlor examples of programs GNN, GFG, Exon prediction from genomic sequences using, for example, ENST GENSCAN (Stanford University, CA, USA) or FGENES

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

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

INCY Full length transcript and exon prediction from mapping of EST

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

In some cases, Incyte cDNA coverage redundant with the sequence coverage shown in Table 4 was obtained to confirm the final consensus polynucleotide sequence, but the relevant Incyte cDNA identification numbers are not shown.
Table 5 shows the represenEative cDNA libraries for those fall length polynucleotides which were assembled using Incyte cDNA sequences. The representative cDNA library is the Incyte cDNA library which is most frequently represented by the Incyte cDNA sequences which were used to assemble and confirm the above polynucleotides. The tissues and vectors which were used to construct the cDNA libraries shown in Table 5 are described in Table 6.
Table 8 shows single nucleotide polymorphisms (SNPs) found in polynucleotide embodiments, along with allele frequencies in different human populations. Columns 1 and 2 show the polynucleotide sequence identification number (SEQ ID NO:) and the corresponding Incyte project identification number (P117) for polynucleotides of the invention. Column 3 shows the Incyte identification number for the EST in which the SNP was detected (EST D.7), and column 4 shows the identification number for the SNP (SNP ID). Column 5 shows the position within the EST sequence at which the SNP is located (EST SNP), and column 6 shows the position of the SNP within the full-length polynucleotide sequence (CB 1 SNP). Column 7 shows the allele found in the EST sequence.
Columns 8 and 9 show the two alleles found at the SNP site. Column 10 shows the amiuo acid encoded by the codon including the SNP site, based,upon the allele found in the EST. Columns 11-14 show the frequency of allele 1 in four different human populations. ' An entry of n/d (not detected) indicates that the frequency of allele 1 in the population was too low to be detected, while nla (not available) indicates that the allele frequency was not determined for the population.
The invention also encompasses NAAP variants. A preferred NAAP variant is one which has at least about 80%, or alternatively at least about 90%, or even at least about 95% amino acid sequence identity to the NAAP amino acid sequence, and which contains at least one functional or structural characteristic of NAAP.
Various embodiments also encompass polynucleotides which encode NAAP. In a particular embodiment, the invention encompasses a polynucleotide sequence comprising a sequence selected from the group consisting of SEQ ID N0:37-72, which encodes NAAP. The polynucleotide sequences of SEQ ID N0:37-72, as presented in the Sequence Listing, embrace the equivalent RNA
sequences, wherein occurrences of the nitrogenous base thymine are replaced with uracil, and the sugar backbone is composed of ribose instead of deoxyribose.
The invention also encompasses variants of a polynucleotide encoding NAAP. In particular, such a variant polynucleotide will have at least about 70%, or alternatively at least about 85%, or even at least about 9S% polynucleotide sequence identity to a polynucleotide encoding NAAP. A particular aspect of the invention encompasses a variant of a polynucleotide comprising a sequence selected from the group consisting of SEQ ID N0:37-72 which has at least about 70%, or alternatively at least about 8S%, or even at least about 9S% polynucleotide sequence identity to a nucleic acid sequence selected from the group consisting of SEQ ID N0:37-72. Any one of the polynucleotide variants described above can encode a polypeptide which contains at least one functional or structural characteristic of NAAP.
In addition, or in the alternative, a polynucleotide variant of the invention is a splice variant of a polynucleotide encoding NAAP. A splice variant may have portions which have significant sequence identity to a polynucleotide encoding NAAP, but will generally have a greater or lesser number of polynucleotides due to additions or deletions of blocks of sequence arising from alternate splicing of exons during mRNA processing. A splice variant may have less than about 70%, or alternatively less than about 60%, or alternatively less than about SO% polynucleotide sequence identity to a polynucleotide encoding NAAP over its entire length; however, portions of the splice variant will have at least about 70%, or alternatively at least about 8S%, or alternatively at least about 9S%, or alternatively 100% polynucleotide sequence identity to portions of the polynucleotide encoding NAAP.
For example, a polynucleotide comprising a sequence of SEQ ID N0:72 is a splice variant of a polynucleotide comprising a sequence of SEQ ID NO:SO. Any one of the splice variants described above can encode a polypeptide which contains at least one functional or structural characteristic of 2o NAAP.
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 NAAP, some bearing minimal similarity to the polynucleotide sequences of any known and naturally occurring gene, may be produced. Thus, the invention contemplates each and every possible variation of polynucleotide sequence that could be made by selecting combinations based on possible codon choices. These combinations are made in accordance with the standard triplet genetic code as applied to the polynucleotide sequence of naturally occurring NAAP, and all such variations are to be considered as being specifically disclosed.
Although polynucleotides which encode NAAP and its variants are generally capable of hybridizing to polynucleotides encoding naturally occurring NAAP under appropriately selected conditions of stringency, it may be advantageous to produce polynucleotides encoding NAAP or its derivatives possessing a substantially different codon usage, e.g., inclusion of non-naturally occurring codons. Codons may be selected to increase the rate at which expression of the peptide occurs in a particular prokaryotic or eukaryotic host in accordance with the frequency with wluch particular codons are utilized by the host. Other reasons for substantially altering the nucleotide sequence encoding NAAP and its derivatives without altering the encoded amino acid sequences include the production of RNA transcripts having more desirable properties, such as a greater half life, than transcripts produced from the naturally occurring sequence.
The invention also encompasses production of polynucleotides which encode NAAP
and NAAP derivatives, or fragments thereof, entirely by synthetic chemistry. After production, the synthetic polynucleotide may be inserted into any of the many available expression vectors and cell systems using reagents well known in the art. Moreover, synthetic chemistry may be used to introduce mutations into a polynucleotide encoding NAAP or any fragment thereof.
Embodiments of the invention can also include polynucleotides that are capable of hybridizing to the claimed polynucleotides, and, in particular, to those having the sequences shown in SEQ ID
N0:37-72 and fragments thereof, under various conditions of stringency (Wahl, G.M. and S.L. Berger (1987) Methods Enzymol. 152:399-407; Iel, A.R. (1987) Methods Enzymol. 152:507-511).
Hybridization conditions, including annealing and wash conditions, are described in "Definitions."
Methods for DNA sequencing are well known in the art and may be used to practice any of the embodiments of the invention. The methods may employ such enzymes as the Klenow fragment of DNA polymerise I, SEQUENASE (US Biochemical, Cleveland OH), Taq polymerise (Applied Biosystems), thermostable T7 polymerise (Amersham Biosciences, Piscataway NJ), or combinations of polymerises and proofreading exonucleases such as those found in the ELONGASE amplification system (Invitrogen, Carlsbad CA). Preferably, sequence preparation is automated with machines such as the MICROLAB 2200 liquid transfer system (Hamilton, Reno NV), PTC200 thermal cycler (MJ Research, Watertown MA) and ABI CATALYST $00 thermal cycler (Applied Biosystems).
Sequencing is then carried out using either the ABI 373 or 377 DNA sequencing system (Applied Biosystems), the MEGABACE 1000 DNA sequencing system (Amersham Biosciences), or other systems known in the art. The resulting sequences are analyzed using a variety of algorithms which are well known in the art (Ausubel, F.M. (1997) Short Protocols in Molecular Biology, John Wiley &
Sons, New York NY, unit 7.7; Meyers, R.A. (1995) Molecular Biology and Biotechnology, Wiley VCH, New York NY, pp. 856-853).
The nucleic acids encoding NAAP may be extended utilizing a partial nucleotide sequence and employing various PCR-based methods known in the art to detect upstream sequences, such as promoters and regulatory elements. For example, one method which may be employed, restriction-site PCR, uses universal and nested primers to amplify unknown sequence from genomic DNA within a cloning vector (Sarkar, G. (1993) PCR Methods Applic. 2:318-322). Another method, inverse PCR, uses primers that extend in divergent directions to amplify unknown sequence from a circularized template. The template is derived from restriction fragments comprising a known genomic locus and surrounding sequences (Triglia, T. et al. (1988) Nucleic Acids Res. 16:8186).
A third method, capture PCR, involves PCR amplification of DNA fragments adjacent to known sequences in human and yeast artificial chromosome DNA (Lagerstrom, M. et al. (1991) PCR Methods Applic. 1:111-119). In this method, multiple restriction enzyme digestions and legations may be used to insert an engineered double-stranded sequence into a region of unknown sequence before performing PCR. Other methods which may be used to retrieve unknown sequences are known in the art (Parker, J.D. et al.
(1991) Nucleic Acids Res. 19:3055-3060). Additionally, one may use PCR, nested primers, and 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 5' regions of genes, are preferable for situations in which an oligo d(T) library does not yield a full-length cDNA. Genomic libraries may be useful for extension of sequence into 5' non-transcribed regulatory regions. .
Capillary electrophoresis systems which are commercially available may be used to analyze the size or confirm the nucleotide sequence of sequencing or PCR products. In particular, capillary sequencing may employ flowable polymers for electrophoretic separation, four different nucleotide-specific, laser-stimulated fluorescent dyes, and a charge coupled device camera for detection of the emitted wavelengths. Output/light intensity may be converted to electrical signal using appropriate software (e.g., GENOTYPER and SEQUENCE NAVIGATOR, Applied Biosystems), and the entire process from loading of samples to computer analysis and electronic data display may be computer controlled. Capillary electrophoresis is especially preferable for sequencing small DNA fragments which may be present in limited amounts in a particular sample.
In another embodiment of the invention, polynucleotides or fragments thereof which encode NAAP may be cloned in recombinant DNA molecules that direct expression of NAAP, or fragments or functional equivalents thereof, in appropriate host cells. Due to the inherent degeneracy of the genetic code, other polynucleotides which encode substantially the same or a functionally equivalent polypeptides may be produced and used to express NAAP.
The polynucleotides of the invention can be engineered using methods generally known in the art in order to alter NAAP-encoding sequences for a variety of purposes including, but not limited to, modification of the cloning, processing, and/or expression of the gene product. DNA shuffling by random fragmentation and PCR reassembly of gene fragments and synthetic oligonucleotides may be used to engineer the nucleotide sequences. For example, oligonucleotide-mediated site-directed mutagenesis may be used to introduce mutations that create new restriction sites, alter glycosylation patterns, change codon preference, produce splice variants, and so forth.
1o The nucleotides of the present invention may be subjected to DNA shuffling techniques such as MOLECULARBREEDING (Maxygen Inc., Santa Clara CA; described in U.S. Patent No.
5,837,458; Chang, C.-C. et aI. (1999) Nat. Biotechnol. 17:793-797; Christians, F.C. et aI. (1999) Nat.
Biotechnol. 17:259-264; and Crameri, A. et al. (1996) Nat. Biotechnol. 14:315-319) to alter or improve the biological properties of NAAP, such as its biological or enzymatic activity or its ability to bind to other molecules or compounds. DNA shuffling is a process by which a library of gene variants is produced using PCR-mediated recombination of gene fragments. The library is then subjected to selection or screening procedures that identify those gene variants with the desired properties. These preferred variants may then be pooled and further subjected to recursive rounds of DNA shuffling and selection/screening. Thus, genetic diversity is created through "artificial"
breeding and rapid molecular 2o evolution. For example, fragments of a single gene containing random point mutations may be recombined, screened, and then reshuffled until the desired properties are optimized. Alternatively, fragments of a given gene may be recombined with fragments of homologous genes in the same gene family, either from the same or different species, thereby maximizing the genetic diversity of multiple naturally occurring genes in a directed and controllable manner.
In another embodiment, polynucleotides encoding NAAP may be synthesized, in whole or in part, using one or more chemical methods well known in the art (Caruthers, M.H. et al. (1980) Nucleic Acids Symp. Ser. 7:215-223; and Horn, T. et al. (1980) Nucleic Acids Symp. Ser. 7:225-232).
Alternatively, NAAP itself or a fragment thereof may be synthesized using chemical methods known in the art. For example, peptide synthesis can be performed using various solution-phase or solid-phase techniques (Creighton, T. (1984) Proteins, Structures and Molecular Properties, WH
Freeman, New York NY, pp. 55-60; Roberge, J.Y. et al. (1995) Science 269:202-204). Automated synthesis may be achieved using the ABI 431A peptide synthesizer (Applied Biosystems).
Additionally, the amino acid sequence of NAAP, or any part thereof, may be altered during direct synthesis and/or combined with sequences from other proteins, or any part thereof, to produce a variant polypeptide or a polypeptide having a sequence of a naturally occurring polypeptide.
The peptide may be substantially purified by preparative high performance liquid chromatography (Chiez, R.M. and F.Z. Regnier (1990) Methods Enzymol. 182:392-421). The composition of the synthetic peptides may be confirmed by amino acid analysis or by sequencing (Creighton, supra, pp. 28-53).
In order to express a biologically active NAAP, the polynucleotides encoding NAAP or derivatives thereof may be inserted into an appropriate expression vector, i.e., a vector which contains the necessary elements for transcriptional and translational control of the inserted coding sequence in a suitable host. These elements include regulatory sequences, such as enhancers, constitutive and inducible promoters, and 5' and 3' untranslated regions in the vector and in polynucleotides encoding NAAP. Such elements may vary in their strength and specificity. Specific initiation signals may also be used to achieve more efficient translation of polynucleotides encoding NAAP. Such signals include the ATG initiation codon and adjacent sequences, e.g. the Kozak sequence. In cases where a polynucleotide sequence encoding NAAP and its initiation codon and upstream regulatory sequences are inserted into the appropriate expression vector, no additional transcriptional or translational control signals may be needed. However, in cases where only coding sequence, or a fragment thereof, is inserted, exogenous translational control signals including an in-frame ATG
initiation codon should be provided by the vector. Exogenous translational elements and initiation codons may be of various origins, both natural and synthetic. The efficiency of expression may be enhanced by the inclusion of enhancers appropriate for the particular host cell system used (Scharf, D. et al. (1994) Results Probl.
Cell Differ. 20:125-162.) Methods which are well known to those skilled in the art may be used to construct expression vectors containing polynucleotides encoding NAAP and appropriate transcriptional and translational control elements. These methods include iti vitf-o recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination (Sambrook et al., supra, ch. 4, 8, and 16-17; Ausubel, F.M. et al.
(1995) Current Protocols in Molecular Biology, 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 polynucleotides encoding NAAP. These include, but are not limited to, microorganisms such as = bacteria transformed with recombinant bacteriophage, plasmid, or cosmid DNA
expression vectors;
yeast transformed with yeast expression vectors; insect cell systems infected with viral expression vectors (e.g., baculovirus); plant cell systems transformed with viral expression vectors (e.g., cauliflower mosaic virus, CaMV, or tobacco mosaic virus, TMV) or with bacterial expression vectors (e.g., Ti or pBR322 plasmids); or animal cell systems (Sambrook et al., supra;
Ausubel, supra; Van Heeke, G. and S.M. Schuster (1989) J. Biol. Chem. 264:5503-5509; Engelhard, E.K. et al. (1994) Proc. Natl. Acad. Sci. USA 91:3224-3227; Sandig, V, et al. (1996) Hum. Gene Ther. 7:1937-1945;
Takamatsu, N. (1987) EMBO J. 6:307-311; The McGraw Hill Yearbook of Science and Technolo~y (1992) McGraw Hill, New York NY, pp. 191-196; Logan, J. and T. Shenk (1984) Proc. Natl. Acad.
Sci. USA 81:3655-3659; and Harrington, J.J. et al. (1997) Nat. Genet. 15:345-355). Expression vectors derived from retroviruses, adenoviruses, or herpes or vaccinia viruses, or from various bacterial plasmids, may be used for delivery of polynucleotides to the targeted organ, tissue, or cell population (Di Nicola, M. et al. (1998) Cancer Gen. Ther. 5(6):350-356; Yu, M.
et al. (1993) Proc.
to Natl. Acad. Sci. USA 90(13):6340-6344; Buller, R.M. et al. (1985) Nature 317(6040):813-815;
McGregor, D.P. et al. (1994) Mol. Itnmunol. 31(3):219-226; and Verma, LM. and N. Somia (1997) Nature 389:239-242). The invention is not limited by the host cell employed.
In bacterial systems, a number of cloning and expression vectors may be selected depending upon the use intended for polynucleotides encoding NAAP. For example, routine cloning, subcloning, and propagation of polynucleotides encoding NAAP can be achieved using a multifunctional E. coli vector such as PBLUESCRIPT (Stratagene, La Jolla CA) or PSPORT1 plasmid (Invitrogen).
Ligation of polynucleotides encoding NAAP into the vector's multiple cloning site disrupts the lacZ
gene, allowing a colorimetric screening procedure for identification of transformed bacteria containing recombinant molecules. In addition, these vectors may be useful for i~c vitro transcription, dideoxy sequencing, single strand rescue with helper phage, and creation of nested deletions in the cloned sequence (Van Heeke, G. and S.M. Schuster (1989) J. Biol. Chem. 264:5503-5509). When large quantities of NAAP are needed, e.g. for the production of antibodies, vectors which direct high level expression of NAAP may be used. For example, vectors containing the strong, inducible SP6 or T7 bacteriophage promoter may be used.
Yeast expression systems may be used for production of NAAP. A number of vectors containing constitutive or inducible promoters, such as alpha factor, alcohol oxidase, and PGH
promoters, may be used in the yeast Saccharomyces cef~evisiae or Pichia pastoris. In addition, such vectors direct either the secretion or intracellular retention of expressed proteins and enable integration of foreign polynucleotide sequences into the host genome for stable propagation (Ausubel, 1995, 3o supra; Bitter, G.A. et al. (1987) Methods Enzymol. 153:516-544; and Scorer, C.A. et al. (1994) Bio/Technology 12:181-184).
Plant systems may also be used for expression of NAAP. Transcription of polynucleotides encoding NAAP may be driven by viral promoters, e.g., the 35S and 19S
promoters of CaMV used alone or in combination with the omega leader sequence from TMV (Takamatsu, N.
(1987) EMBO J.

6:307-311). Alternatively, plant promoters such as the small subunit of RUBISC~ or heat shock promoters maybe used (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 (The McGraw Hill Yearbook of Science and Technology (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, polynucleotides encoding NAAP may be Iigated into an adenovirus transcription/translation complex consisting of the late promoter and tripartite leader sequence. Insertion in a non-essential E1 or E3 region of the viral genome may be used to obtain infective virus which expresses NAAP in host cells (Logan, J. and T. Shenk (1984) Proc. Natl. Acad.
Sci. USA 81:3655-3659). In addition, transcription enhancers, such as the Rous sarcoma virus (RSV) enhancer, may be used to increase expression in mammalian host cells. SV40 or EBV-based vectors may also be used for high-level protein expression.
Human artificial chromosomes (HACs) may also be employed to deliver larger fragments of DNA than can be contained in and expressed from a plasmid. HACs of about 6 kb to 10 Mb are constructed and delivered via conventional delivery methods (liposomes, polycationic amino polymers, or vesicles) for therapeutic purposes (Harrington, J.J. et al. (1997) Nat.
Genet. 15:345-355).
For long term production of recombinant proteins in mammalian systems, stable expression of NAAP in cell lines is preferred. For example, polynucleotides encoding NAAP
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 3o include, but are not limited to, the herpes simplex virus thymidine kinase and adenine phosphoribosyltransferase genes, for use in t7~ and apf~ cells, respectively (Wigler, M. et al. (1977) Cell 11:223-232; Lowy, I. et al. (1980) Cell 22:817-823). Also, antimetabolite, antibiotic, or herbicide resistance can be used as the basis for selection. For example, dhft~ confers resistance to methotrexate; neo confers resistance to the aminoglycosides neomycin and G-418; and als and pat confer resistance to chlorsulfuron and phosphinotricin acetyltransferase, respectively (Wigler, M. et al.
(1980) Proc. Natl. Acad. Sci. USA 77:3567-3570; Colbere-Garapin, F. et al.
(1981) J. Mol. Biol.
150:1-14). Additional selectable genes have been described, e.g., trpB and hisD, which alter cellular requirements for metabolites (Hartman, S.C. and R.C. Mulligan (1988) Proc.
Natl. Acad. Sci. USA
85:8047-8051). Visible markers, e.g., anthocyanins, green fluorescent proteins (GFP; Clontech),13 glucuronidase and its substrate l3-glucuronide, or luciferase and its substrate luciferin may be used.
These markers can be used not only to identify transformants, but also to quantify the amount of transient or stable protein expression attributable to a specific vector system (Rhodes, C.A. (1995) Methods Mol. Biol. 55:121-131).
Although the presencelabsence 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 NAAP is inserted within a marker gene sequence, transformed cells containing polynucleotides encoding NAAP can be identified by the absence of marker gene function.
Alternatively, a marker gene can be placed in tandem with a sequence encoding NAAP under the control of a single promoter. Expression of the marker gene in response to induction or selection usually indicates expression of the tandem gene as well.
In general, host cells that contain the polynucleotide encoding NAAP and that express NAAP
may be identified by a variety of procedures known to those of skill in the art. These procedures 2o 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 andlor quantification of nucleic acid or protein sequences.
T_m_m__unological methods for detecting and measuring the expression of NAAP
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 NAAP is preferred, but a competitive binding assay may be employed. These and other assays are well known in the art (Hampton, R. et al. (1990) Serological Methods, a Laboratory Manual, APS
Press, St. Paul MN, Sect.
IV; Coligan, J.E. et al. (1997) Current Protocols in Immunolo~y, Greene Pub.
Associates and Wiley-Interscience, New York NY; and Pound, J.D. (1998) Tm_m__unochemical 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 NAAP
include oligolabeling, nick translation, end-labeling, or PCR amplification using a labeled nucleotide.
Alternatively, polynucleotides encoding NAAP, or any fragments thereof, may be cloned into a vector for the production of an mRNA probe. Such vectors are known in the art, are commercially available, and may be used to synthesize RNA probes i~t vitt~o by addition of an appropriate RNA polymerase such as T7, T3, or SP6 and labeled nucleotides. These procedures may be conducted using a variety of commercially available kits, such as those provided by Amersham Biosciences, Promega (Madison WI), and US Biochemical. Suitable reporter molecules or labels which may be used for ease of detection include radionuclides, enzymes, fluorescent, chemiluminescent, or chromogenic agents, as well as substrates, cofactors, inhibitors, magnetic particles, and the like.
Host cells transformed with polynucleotides encoding NAAP 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 NAAP may be designed to contain signal sequences which direct secretion of NAAP through a prokaryotic or eukaryotic cell membrane. .
In addition, a host cell strain may be chosen for its ability to modulate expression of the inserted polynucleotides or to process the expressed protein in the desired fashion. Such modifications of the polypeptide include, but are not limited to, acetylation, carboxylation, glycosylation, phosphorylation, lipidation, and acylation. Post-translational processing which cleaves a "prepro" or "pro" form of the protein may also be used to specify protein targeting, folding, and/or activity.
Different host cells which have specific cellular machinery and characteristic mechanisms for post-translational activities (e.g., CHO, HeLa, MDCK, HEK293, and WI3~) are available from the American Type Culture Collection (ATCC, Manassas VA) and may be chosen to ensure the correct modification and processing of the foreign protein.
In another embodiment of the invention, natural, modified, or recombinant polynucleotides encoding NAAP 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 NAAP
protein containing a heterologous moiety that can be recognized by a commercially available antibody may facilitate the screening of peptide libraries for inhibitors of NAAP 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-rnyc, 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 NAAP encoding sequence and the heterologous protein sequence, so that NAAP
may be cleaved away from the heterologous moiety following purification.
Methods for fusion protein expression and purification are discussed in Ausubel (1995, supra, ch. 10). A
variety of commercially available kits may also be used to facilitate expression and purification of fusion proteins.
In another embodiment, synthesis of radiolabeled NAAP may be achieved in vitro using the TNT rabbit reticulocyte lysate or wheat gexm extract system (Promega). These systems couple transcription and translation of protein-coding sequences operably associated with the T7, T3, or SP6 promoters. Translation takes place in the presence of a radiolabeled amino acid precursor, for example, 35S-methionine.
NAAP, fragments of NAAP, or variants of NAAP may be used to screen for compounds that specifically bind to NAAP. One or more test compounds may be screened for specific binding to NAAP. In various embodiments, 1, 2, 3, 4, 5, 10, 20, 50, 100, or 200 test compounds can be screened for specific binding to NAAP. Examples of test compounds can include antibodies, anticalins, oligonucleotides, proteins (e.g., ligands or receptors), or small molecules.
In related embodiments, variants of NAAP can be used to screen for binding of test compounds, such as antibodies, to NAAP, a variant of NAAP, or a combination of NAAP and/or one or more variants NAAP. In an embodiment, a variant of NAAP can be used to screen for compounds that bind to a variant of NAAP, but not to NAAP having the exact sequence of a sequence of SEQ m N0:1-36. NAAP variants used to perform such screening can have a range of about 50% to about 99% sequence identity to NAAP, with various embodiments having 60%, 70%, 75%, 80%, 85%, 90%, and 95% sequence identity.
In an embodiment, a compound identified in a screen fox specific binding to NAAP can be closely related to the natural ligand of NAAP, e.g., a ligand or fragment thereof, a natural substrate, a structural or functional mimetic, or a natural binding partner (Coligan, J.E.
et al. (1991) Current Protocols in Immunology 1(2):Chapter 5). In another embodiment, the compound thus identified can be a natural ligand of a receptor NAAP (Howard, A.D. et al. (2001) Trends Pharmacol. Sci.22:132-140; Wise, A. et al. (2002) Drug Discovery Today 7:235-246).

In other embodiments, a compound identified in a screen for specific binding to NAAP can be closely related to the natural receptor to which NAAP binds, at least a fragment of the receptor, or a fragment of the receptor including all or a portion of the ligand binding site or binding pocket. For example, the compound may be a receptor for NAAP which is capable of propagating a signal, or a decoy receptor for NAAP which is not capable of propagating a signal (Ashkenazi, A. and V.M. Divit (1999) Curr. Opin. Cell Biol. 11:255-260; Mantovani, A. et al. (2001) Trends T_m_m__unol. 22:328-336).
The compound can be rationally designed using known techniques. Examples of such techniques include those used to construct the compound etanercept (ENBREL; Tmmunex Corp., Seattle WA), which is efficacious for treating rheumatoid artlwitis in humans. Etanercept is an engineered p75 l0 tumor necrosis factor (TNF) receptor dimer linked to the Fc portion of human IgG1 (Taylor, P.C. et al.
(2001) Curr. Opin. Immunol. 13:611-616).
In one embodiment, two or more antibodies having similar or, alternatively, different specificities can be screened for specific binding to NAAP, fragments of NAAP, or variants of NAAP. The binding specificity of the antibodies thus screened can thereby be selected to identify particular fragments or variants of NAAP. In one embodiment, an antibody can be selected such that its binding specificity allows for preferential identification of specific fragments or variants of NAAP.
In another embodiment, an antibody can be selected such that its binding specificity allows for preferential diagnosis of a specific disease or condition having increased, decreased, or otherwise -abnormal production of NAAP.
In an. embodiment, anticalins can be screened for specific binding to NAAP, fragments of NAAP, or variants of NAAP. Anticalins are ligand-binding proteins that have been constructed based on a Iipocalin scaffold (Weirs, G.A. and H.B. Lowman (2000) Chem. Biol. 7:8177-8184; Skerra, A.
(2001) J. Biotechnol. 74:257-275). The protein architecture of lipocalins can include a beta-barrel having eight antiparallel beta-strands, which supports four loops at its open end. These loops form the natural ligand-binding site of the lipocalins, a site which can be re-engineered irt vitro by amino acid substitutions to impart novel binding specificities. The amino acid substitutions can be made using methods known in the art or described herein, and can include conservative substitutions (e.g., substitutions that do not alter binding specificity) or substitutions that modestly, moderately, or significantly alter binding specificity.
In one embodiment, screening for compounds which specifically bind to, stimulate, or inhibit NAAP involves producing appropriate cells which express NAAP, either as a secreted protein or on the cell membrane. Preferred cells include cells from mammals, yeast, Dr-osophila, or E. coli. Cells expressing NAAP or cell membrane fractions which contain NAAP are then contacted with a test compound and binding, stimulation, or inhibition of activity of either NAAP or the compound is analyzed.
An assay may simply test binding of a test compound to the polypeptide, wherein binding is detected by a fluorophore, radioisotope, enzyme conjugate, or other detectable label. For example, the assay may comprise the steps of combining at least one test compound with NAAP, either in solution or affixed to a solid support, and detecting the binding of NAAP to the compound. Alternatively, the assay may detect or measure binding of a test compound in the presence of a labeled competitor.
Additionally, the assay may be carried out using cell-free preparations, chemical libraries, or natural product mixtures, and the test compounds) may be free in solution or affixed to a solid support.
An assay can be used to assess the ability of a compound to bind to its natural ligand andlor to inhibit the binding of its natural ligand to its natural receptors. Examples of such assays include radio-labeling assays such as those described in U.S. Patent No. 5,914,236 and U.S.
Patent No. 6,372,724. , In a related embodiment, one or more amino acid substitutions can be introduced into a polypeptide compound (such as a receptor) to improve or alter its ability to bind to its natural ligands (Matthews, D.J. and J.A. Wells. (1994) Chem. Biol. 1:25-30). In another related embodiment, one or more amino acid substitutions can be introduced into a polypeptide compound (such as a ligand) to improve or alter its ability to bind to its natural receptors (Cunningham, B.C. and J.A. Wells (1991) Proc. Natl. Acad.
Sci. USA 88:3407-3411; Lowman, H.B. et al. (1991) J. Biol. Chem. 266:10982-10988).
NAAP, fragments of NAAP, or variants of NAAP may be used to screen for compounds that modulate the activity of NAAP. Such compounds may include agonists, antagonists, or partial or inverse agonists. In one embodiment, an assay is performed under conditions permissive for NAAP
activity, wherein NAAP is combined with at least one test compound, and the activity of NAAP in the presence of a test compound is compared with the activity of NAAP in the absence of the test compound. A change in the activity of NAAP in the presence of the test compound is indicative of a compound that modulates the activity of NAAP. Alternatively, a test compound is combined with an i~ vity~o or cell-free system comprising NAAP under conditions suitable for NAAP activity, and the assay is performed. In either of these assays, a test compound which modulates the activity of NAAP may do so indirectly and need not come in direct contact with the test compound. At least one and up'to a plurality of test compounds may be screened.
In another embodiment, polynucleotides encoding NAAP or their mammalian homologs may be "knocked out" in an animal model system using homologous recombination in embryonic stem (ES) cells. Such techniques are well known in the art and are useful for the generation of animal models of human disease (U.S. Patent No. 5,175,383 and U.S. Patent No. 5,767,337). For example, mouse ES

cells, such as the mouse 129/SvJ cell line, are derived from the early mouse embryo and grown in culture. The ES cells are transformed with a vector containing the gene of interest disrupted by a marker gene, e.g., the neomycin phosphotransferase gene (neo; Capecchi, M.R.
(1989) Science 244:1288-1292). The vector integrates into the corresponding region of the host genome by homologous recombination. Alternatively, homologous recombination takes place using the Cre-loxP °
system to knockout a gene of interest in a tissue- or developmental stage-specific manner (March, J.D.
(1996) Clip. Invest. 97:1999-2002; Wagner, K.U. et al. (1997) Nucleic Acids Res. 25:4323-4330).
Transformed ES cells are identified and microinjected into mouse cell blastocysts such as those from the C57BL/6 mouse strain. The blastocysts are surgically trausferred to pseudopregnant dams, and the resulting chimeric progeny are genotyped and bred to produce heterozygous or homozygous strains. Trausgenic animals thus generated may be tested with potential therapeutic or toxic agents.
Polynucleotides encoding NAAP may also be manipulated in vitro in ES cells derived from human blastocysts. Human ES cells have the potential to differentiate into at least eight separate cell lineages including endoderm, mesoderm, and ectodermal cell types. These cell lineages differentiate into, for example, neural cells, hematopoietic lineages, and cardiomyocytes (Thomson, J.A. et al.
(1998) Science 282:1145-1147).
Polynucleotides encoding NAAP can also be used to create "knockin" humanized animals (pigs) or transgenic animals (mice or rats) to model human disease. With knockin technology, a region of a polynucleotide encoding NAAP is injected into animal ES cells, and the injected sequence integrates into the animal cell genome. Transformed cells are injected into blastulae, and the blastulae are implanted as described above. Transgenic progeny or inbred lines are studied and treated with potential pharmaceutical agents to obtain information on treatment of a human disease. Alternatively, a mammal inbred to overexpress NAAP, e.g., by secreting NAAP in its milk, may also serve as a convenient source of that protein (Janne, J. et al. (1998) Biotechnol. Annu.
Rev. 4:55-74).
THERAPEUTICS
Chemical and structural similarity, e.g., in the context of sequences and motifs, exists between regions of NAAP and nucleic acid-associated proteins. In addition, examples of tissues expressing NAAP can. be found in Table 6 and can also be found in Example XI. Therefore, NAAP appears to play a role in cell proliferative, neurological, reproductive, developmental, autoimmune/inflammatory, and DNA repair disorders, and infections. In the treatment of disorders associated with increased NAAP expression or activity, it is desirable to decrease the expression or activity of NAAP. In the treatment of disorders associated with decreased NAAP expression or activity, it is desirable to increase the expression or activity of NAAP.

Therefore, in one embodiment, NAAP 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 NAAP.. Examples of such disorders include, but are not limited to, a cell proliferative disorder such as actinic keratosis, arteriosclerosis, atherosclerosis, bursitis, cirrhosis, hepatitis, mixed connective tissue disease (MCTD), myelofibrosis, paroxysmal nocturnal hemoglobinuria, polycythemia vera, psoriasis, primary thrombocythemia, and cancers including adenocarcinoma, leukemia, lymphoma, melanoma, myeloma, sarcoma, teratocarcinoma, and, in particular, a cancer of the adrenal gland, bladder, bone, bone marrow, brain, breast, cervix, gall bladder, ganglia, gastrointestinal tract, heart, kidney, liver, lung, muscle, ovary, pancreas, parathyroid, penis, prostate, salivary glands, skin, spleen, testis, thymus, thyroid, and uterus; a neurological disorder such as progressive supranuclear palsy, corticobasal degeneration, familial frontotemporal dementia, 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 disorder of the central nervous system, cerebral palsy, a neuroskeletal disorder, an autonomic nervous system disorder, a cranial nerve disorder, a spinal cord disease, muscular dystrophy and other neuromuscular disorder, a peripheral nervous system disorder, dermatomyositis and polymyositis, inherited, metabolic, endocrine, and toxic myopathy, myasthenia gravis, periodic paralysis, a mental disorder including mood, anxiety, and schizophrenic disorder, seasonal affective disorder (SAD), akathesia, amnesia, catatonia, diabetic neuropathy, tardive dyskinesia, dystonias, paranoid psychoses, postherpetic neuralgia, and Tourette's disorder; 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; a developmental disorder such as renal tubular acidosis, anemia, Cushing's syndrome, achondroplastic dwarfism, Duchenne and Becker muscular dystrophy, epilepsy, gonadal dysgenesis, WAGR syndrome (Wilms' tumor, aniridia, genitourinary abnormalities,, and mental retardation), Smith-Magenis syndrome, myelodysplastic syndrome, hereditary mucoepithelial dysplasia, hereditary keratodermas, hereditary neuropathies such as Charcot-Marie-Tooth disease and neurofibromatosis, hypothyroidism, hydrocephalus, seizure disorders such as Syndenham's chorea and cerebral palsy, spine bifida, anencephaly, craniorachischisis, congenital glaucoma, cataract, and sensorineural hearing loss; an autoimmunelinflammatory disorder such as acquired immunodeficiency syndrome (AIDS), Addison's disease, adult respiratory distress syndrome, allergies, ankylosing spondylitis, amyloidosis, anemia, asthma, atherosclerosis, autoimmune hemolytic anemia, autoimtnune thyroiditis, autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED), bronchitis, cholecystitis, contact dermatitis, Crohn's disease, atopic dermatitis, dermatomyositis, diabetes mellitus, emphysema, episodic lymphopenia with lymphocytotoxins, erythroblastosis,fetalis, erythema nodosum, atrophic gastritis, glomerulonephritis, Goodpasture's syndrome, gout, Graves' disease, Hashimoto's thyroiditis, hypereosinophilia, irritable bowel syndrome, multiple sclerosis, myasthenia gravis, myocardial or pericardial inflammation, osteoartluitis, osteoporosis, pancreatitis, polymyositis, psoriasis, Reiter's syndrome, rheumatoid at~tbritis, scleroderma, Sjogren's syndrome, systemic anaphylaxis, systemic lupus erythematosus, systemic sclerosis, thrombocytopenic purpura, ulcerative colitis, uveitis, Werner syndrome, complications of cancer, hemodialysis, and extracorporeal circulation, viral, bacterial, fungal, parasitic, protozoal, and hehninthic infections, and trauma; an infection, such as those caused by a viral agent classified as adenovirus, arenavirus, bunyavirus, calicivirus, coronavirus, filovirus, hepadnavirus, herpesvirus, flavivirus, orthomyxovirus, parvovirus, papovavirus, paramyxovirus, picornavirus, poxvirus, reovirus, retrovirus, rhabdovirus, or togavirus; an infection caused by a bacterial agent classified as pneumococcus, staphylococcus, streptococcus, bacillus, corynebacterium, clostridium, meningococcus, gonococcus, listeria, moraxella, kingella, haemophilus, legionella, bordetella, gram-negative enterobacterium including shigella, salmonella, or campylobacter, pseudomonas, vibrio, brucella, francisella, yersinia, bartonella, norcardium, actinomyces, mycobacterium, spirochaetale, rickettsia, chlamydia, or mycoplasma; an infection caused by a fungal agent classified as aspergillus, blastomyces, dermatophytes, cryptococcus, coccidioides, malasezzia, histoplasma, or other mycosis-causing fungal agent; an infection caused by a parasite classified as plasmodium or malaria-causing, parasitic entamoeba, leishmania, trypanosome, toxoplasma, pneumocystis carinii, intestinal protozoa such as giardia, trichomonas, tissue nematode such as trichinella, intestinal nematode such as ascaris, lymphatic filarial nematode, trematode such as schistosoma, and cestode such as tapeworm; and a DNA repair disorder such as xeroderma pigmentosum, Bloom's syndrome, and Werner's syndrome.
In another embodiment, a vector capable of expressing NAAP 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 NAAP including, but not limited to, those described above.
In a further embodiment, a composition comprising a substantially purified NAAP 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 NAAP
including, but not limited to, those provided above.
In still another embodiment, an agonist which modulates the activity of NAAP
may be administered to a subject to treat or prevent a disorder associated with decreased expression or activity of NAAP including, but not limited to, those listed above.
In a further embodiment, an antagonist of NAAP may be administered to a subject to treat or prevent a disorder associated with increased expression or activity of NAAP.
Examples of such disorders include, but are not limited to, those cell proliferative, neurological, reproductive, developmental, autoimmune/inflammatory, and DNA repair disorders, and infections, described above.
In one aspect, an antibody which specifically binds NAAP may be used directly as an antagonist or indirectly as a targeting or delivery mechanism for bringing a pharmaceutical agent to cells or tissues which express NAAP.
20. In an additional embodiment, a vector expressing the complement of the polynucleotide encoding NAAP may be administered to a subject to treat or prevent a disorder associated with increased expression or activity of NAAP including, but not limited to, those described above.
In other embodiments, any protein, agonist, antagonist, antibody, complementary sequence, or vector embodiments may be administered in combination with other appropriate therapeutic agents.
Selection of the appropriate agents for use in combination therapy may be made by one of ordinary skill in the art, according to conventional pharmaceutical principles. The combination of therapeutic agents may act synergistically to effect the treatment or prevention of the various disorders described above. Using this approach, one may be able to achieve therapeutic efFicacy with lower dosages of each agent, thus reducing the potential for adverse side effects.
An antagonist of NAAP may be produced using methods which are generally known in the art. In particular, purified NAAP may be used to produce antibodies or to screen libraries of pharmaceutical agents to identify those which specifically bind NAAP.
Antibodies to NAAP may also be generated using methods that are well known in the art. Such antibodies may include, but are not limited to, polyclonal, monoclonal, chimeric, and single chain antibodies, Fab fragments, and fragments produced by a Fab expression library. Neutralizing antibodies (i.e., those which inhibit dimer formation) are generally preferred for therapeutic use. Single chain antibodies (e.g., from camels or llamas) may be potent enzyme inhibitors and may have advantages in the design of peptide mimetics, and in the development of immuno-adsorbents and biosensors (Muyldermans, S. (2001) J.
Biotechnol. 74:277-302).
For the production of antibodies, various hosts including goats, rabbits, xats, mice, camels, dromedaries, llamas, humans, and others may be immunized by injection with NAAP or with any fragment or oligopeptide thereof which has immunogenic properties. Depending on the host species, various adjuvants may be used to increase i_mmunological 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 Coryvebacter~iurn parvur~z are especially preferable.
It is preferred that the oligopeptides, peptides, or fragments used to induce antibodies to NAAP have an amino acid sequence consisting of at least about 5 amino acids, and generally will consist of at least about 10 amino acids. It is also preferable that these oligopeptides, peptides, or fragments are identical to a portion of the amino acid sequence of the natural protein. Short stretches of NAAP 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 NAAP may be prepared using any technique which provides for the production of antibody molecules by continuous cell lines in culture. These include, but are not limited to, the hybridoma technique, the human B-cell hybridoma technique, and the EBV-hybridoma technique (Kohler, G. et al. (1975) Nature 256:495-497; Kozbor, D. et al.
(1985) J. Tmmunol. Methods 81:31-42; Cote, R.J. et al. (1983) Proc. Natl. Acad. Sci. USA 80:2026-2030;
and Cole, S.P. et al.
(1984) Mol. Cell Biol. 62:109-120).
In addition, techniques developed for the production of "chimeric antibodies,"
such as the splicing of mouse antibody genes to human antibody genes to obtain a molecule with appropriate antigen specificity and biological activity, can be used (Morrison, S.L. et al. (1984) Proc. Natl. Acad.
Sci. USA 81:6851-6855; Neuberger, M.S. et al. (1984) Nature 312:604-608; and Takeda, S. et al.
(1985) Nature 314:452-454). Alternatively, techniques described for the production of single chain antibodies may be adapted, using methods known in the art, to produce NAAP-specific single chain antibodies. Antibodies with related specificity, but of distinct idiotypic composition, may be generated by chain shuffling from random combinatorial immunoglobulin libraries (Burton, D.R. (1991) Proc.
Natl. Acad. Sci. USA 88:10134-10137).
Antibodies may also be produced by inducing in vivo production in the lymphocyte population or by screening immunoglobulin libraries or panels of highly specific binding reagents as disclosed in the literature (Orlaudi, R. et al. (1989) Proc. Natl. Acad. Sci. USA 86:3833-3837; Winter, G. et al.
(1991) Nature 349:293-299).
Antibody fragments which contain specific binding sites for NAAP may also be generated.
For example, such fragments include, but are not limited to, F(ab~2 fragments produced by pepsin digestion of the antibody molecule and Fab fragments generated by reducing the disulfide bridges of the F(ab~2 fragments. Alternatively, Fab expression libraries may be constructed to allow rapid and easy identification of monoclonal Fab fragments with the desired specificity (Huse, W.D. et al. (1989) Science 246:1275-1281.) Various immunoassays may be used for screening to identify antibodies having the desired specificity. Numerous protocols for competitive binding or immunoradiometric assays using either polyclonal or monoclonal antibodies with established specificities are well known in the art. Such immunoassays typically involve the measurement of complex formation between NAAP and its specific antibody. A two-site, monoclonal-based immunoassay utilizing monoclonal antibodies reactive to two non-interfering NAAP epitopes is generally used, but a competitive binding assay may also be employed (Pound, supra).
Various methods such as Scatchard analysis in conjunction with radioimmunoassay techniques may be used to assess the affinity of antibodies for NAAP. Affinity is expressed as an association constant, K~, which is defined as the molar concentration of NAAP-antibody complex divided by the molar concentrations of free antigen and free antibody under equilibrium conditions. The Ka determined for a preparation of polyclonal antibodies, which are heterogeneous in their affinities for multiple NAAP epitopes, represents the average affinity, or avidity, of the antibodies for NAAP. The K~ determined for a preparation of monoclonal antibodies, which are monospecific for a particular NAAP epitope, represents a true measure of affinity. High-affinity antibody preparations with Ka ranging from about 109 to 1012 L/mole are preferred for use in immunoassays in which the NAAP-antibody complex must withstand rigorous manipulations. Low-affinity antibody preparations with Ka ranging from about 10~ to 10' Llmole are preferred for use in i_m_m__unopurification and similar procedures which ultimately require dissociation of NAAP, preferably in active form, from the antibody (Catty, D. (1988) Antibodies, Volume I: A Practical Approach, IRL
Press, Washington DC;
Liddell, J.E. and A. Cryer (1991) A Practical Guide to Monoclonal Antibodies, John Wiley & Sons, New York NY).
The titer and avidity of polyclonal antibody preparations may be further evaluated to determine the quality and suitability of such preparations for certain downstream applications. For example, a polyclonal antibody preparation containing at least 1-2 mg specific antibody/ml, preferably 5-10 mg specific antibody/ml, is generally employed in procedures requiring precipitation of NAAP-antibody complexes. Procedures for evaluating antibody specificity, titer, and avidity, and guidelines for antibody quality and usage in various applications, are generally available (Catty, supra, and Coligan et al., supra).
In another embodiment of the invention, polynucleotides encoding NAAP, or any fragment or complement thereof, may be used for therapeutic purposes. In one aspect, modifications of gene expression can be achieved by designing complementary sequences or antisense molecules (DNA, RNA, PNA, or modified oligonucleotides) to the coding or regulatory regions of the gene encoding NAAP. Such technology is well known in the art, and antisense oligonucleotides or larger fragments can be designed from various locations along the coding or control regions of sequences encoding NAAP (Agrawal, S., ed. (1996) Antisense Therapeutics, Humana Press Inc., Totawa NJ).
In therapeutic use, any gene delivery system suitable for introduction of the antisense sequences into appropriate target cells can be used. Antisense sequences can be delivered intracellularly in the form of an expression plasmid which, upon transcription, produces a sequence complementary to at least a portion of the cellular sequence encoding the target protein (Slater, J.E. et 2o al. (1998) J. Allergy Clip. Tmmunol. 102(3):469-475; and Scanlon, I~.J. et al. (1995) 9(13):1288-1296).
Antisense sequences can also be introduced intracellularly through the use of viral vectors, such as retrovirus and adeno-associated virus vectors (Miller, A.D. (1990) Blood 76:271; Ausubel, supra;
Uckert, W. and W. Walther (1994) Pharmacol. Ther. 63(3):323-347). Other gene delivery mechanisms include liposome-derived systems, artificial viral envelopes, and other systems known in the art (Rossi, J.J. (1995) Br. Med. Bull. 51(1):217-225; Boado, R.J. et al.
(1998) J. Pharm. Sci.
87(11):1308-1315; and Morris, M.C. et al. (1997) Nucleic Acids Res.
25(14):2730-2736).
In another embodiment of the invention, polynucleotides encoding NAAP may be used for somatic or germline gene therapy. Gene therapy may be performed to (i) correct a genetic deficiency (e.g., in the cases of severe combined immunodeficiency (SCll~)-X1 disease characterized by X-linked inheritance (Cavazzana-Calvo, M. et al. (2000) Science 288:669-672), severe combined immunodeficiency syndrome associated with an inherited adenosine deaminase (ADA) deficiency (Blaese, R.M. et al. (1995) Science 270:475-480; Bordignon, C. et al. (1995) Science 270:470-475), cystic fibrosis (Zabner, J. et al. (1993) Cell 75:207-216; Crystal, R.G. et al. (1995) Hum. Gene Therapy 6:643-666; Crystal, R.G. et al. (1995) Hum. Gene Therapy 6:667-703), thalassamias, familial hypercholesterolemia, and hemophilia resulting from Factor VIII or Factor IX
deficiencies (Crystal, R.G. (1995) Science 270:404-410; Verma, LM, and N. Somia (1997) Nature 389:239-242)), (ii) express a conditionally lethal gene product (e.g., in the case of cancers which result from unregulated cell proliferation), or (iii) express a protein which affords protection against intracellular parasites (e.g., against human retroviruses, such as human immunodeficiency virus (HIV) (Baltimore, D. (1988) Nature 335:395-396; Poeschla, E. et al. (1996) Proc. Natl. Acad. Sci. USA
93:11395-11399), hepatitis B or C virus (HBV, HCV); fungal parasites, such as Candida albicahs and Par~acoccidioides br-asiliensis; and protozoan parasites such as Plas»ZOdiurn falcipar-um and Trypanosoma cf~uzi). In the case where a genetic deficiency in NAAP expression or regulation causes disease, the expression of NAAP from an appropriate population of trausduced cells may alleviate the clinical manifestations caused by the genetic deficiency.
In a further embodiment of the invention, diseases or disorders caused by deficiencies in NAAP are treated by constructing mammalian expression vectors encoding NAAP
and introducing these vectors by mechanical means into NAAP-deficient cells. Mechanical transfer technologies for use with cells in vivo or ex vitro include (i) direct DNA microinjection into individual cells, (ii) ballistic gold particle delivery, (iii) liposome-mediated transfection, (iv) receptor-mediated gene transfer, and (v) the use of DNA transposons (Morgau, R.A: and W.F. Anderson (1993) Annu.
Rev. Biochem.
62:191-217; Ivics, Z. (1997) Cell 91:501-510; Boulay, J-L: and H. Recipon (1998) C~rr. Opin.
Biotechno1.9:445-450).
Expression vectors that may be effective for the expression of NAAP include, but are not limited to, the PCDNA 3.1, EPITAG, PRCCMV2, PREP, PVAX, PCR2-TOPOTA vectors (Invitrogen, Carlsbad CA), PCMV-SCR1PT, PCMV-TAG, PEGSH/PERV (Stratagene, La Jolla CA), and PTET-OFF, PTET-ON, PTRE2, PTRE2-LUC, PTK-HYG (Clontech, Palo Alto CA).
NAAP
may be expressed using (i) a constitutively active promoter, (e.g., from cytomegalovirus (CMV), Rous sarcoma virus (RSV), SV40 virus, thytnidine kiuase (TK), or (3-actin genes), (ii) an inducible promoter (e.g., the tetracycline-regulated promoter (Gossen, M. and H. Bujard (1992) Proc. Natl. Acad. Sci.
USA 89:5547-5551; Gossen, M. et a1. (1995) Science 268:1766-1769; Rossi, F.M.V. and H.M. Blau (1998) Curr. Opin. Biotechnol. 9:451-456), commercially available in the T-REX
plasmid (Invitrogen));
the ecdysone-inducible promoter (available in the plasmids PVGRXR and PIND;
Invitrogen); the FK506/rapamycin inducible promoter; or the RU486lmifepristone inducible promoter (Rossi, F.M.V.
and H.M. Blau, supra)), or (iii) a tissue-specific promoter or the native promoter of the endogenous gene encoding NAAP from a normal individual.

Commercially available liposome transformation kits (e.g., the PERFECT LIPID
TRANSFECTION KTT~ available from Iuvitrogen) allow one with ordinary skill in the art to deliver polynucleotides to target cells in culture and require minimal effort to optimize experimental parameters. In the alternative, transformation is performed using the calcium phosphate method (Graham, F.L. and A.J. Eb (1973) Virology 52:456-467), or by electroporation (Neumann, E. et al.
(1982) EMBO J. 1:841-845). The introduction of DNA to primary cells requires modification of these standardized mammalian transfection protocols.
In another embodiment of the invention, diseases or disorders caused by genetic defects with respect to NAAP expression are treated by constructing a retrovirus vector consisting of (i) the polynucleotide encoding NAAP under the control of an independent promoter or the retrovirus long terminal repeat (LTR) promoter, (ii) appropriate RNA packaging signals, and (iii) a Rev-responsive element (RRE) along with additional retrovirus cis-acting RNA sequences and coding sequences required for efficient vector propagation. Retrovirus vectors (e.g., PFB and PFBNEO) are commercially available (Stratagene) and are based on published data (Riviere, I. et al. (1995) Proc.
Natl. Acad. Sci. USA 92:6733-6737), incorporated by reference herein. The vector is propagated in an appropriate vector producing cell line (VPCL) that expresses an envelope gene with a tropism for receptors on the target cells or a promiscuous envelope protein such as VSVg (Armentano, D. et al.
(1987) J. Virol. 61:1647-1650; Bender, M.A. et al. (1987) J. Virol. 61:1639-1646; Adam, M.A. and A.D. Miller (1988) J. Virol. 62:3802-3806; Dull, T. et al. (1998) J. Virol.
72:8463-8471; Zufferey, R. et al. (1998) J. Virol. 72:9873-9880). U.S. Patent No. 5,910,434 to Rigg ("Method for obtaining retrovirus packaging cell lines producing high transducing efficiency retroviral supernatant") discloses a method for obtaining retrovirus packaging cell lines and is hereby incorporated by reference.
Propagation of retrovirus vectors, transduction of a population of cells (e.g., CD4+ T-cells), and the return of transduced cells to a patient are procedures well known to persons skilled in the art of gene therapy and have been well documented (Ranga, U. et al. (1997) J. Virol.
71:7020-7029; Bauer, G. et al. (1997) Blood 89:2259-2267; Bonyhadi, M.L. (1997) J. Virol. 71:4707-4716;
Ranga, U. et al. (1998) Proc. Natl. Acad. Sci. USA 95:1201-1206; Su, L. (1997) Blood 89:2283-2290).
In an embodiment, an adenovirus-based gene therapy delivery system is used to deliver polynucleotides encoding NAAP to cells which have one or more genetic abnormalities with respect to the expression of NAAP. The construction and packaging of adenovirus-based vectors are well known to those with ordinary skill in the art. Replication defective adenovirus vectors have proven to be versatile for importing genes encoding immunoregulatory proteins into intact islets in the pancreas (Csete, M.E. et al. (1995) Transplantation 27:263-268). Potentially useful adenoviral vectors are described in U.S. Patent No. 5,707,618 to Armentano ("Adenovirus vectors for gene therapy'), hereby incorporated by reference. For adenoviral vectors, see also Antinozzi et al. (1999; Annu. Rev.
Nutr. 19:511-544) and Verma and Somia (1997; Nature 18:389:239-242), both incorporated by reference herein.
In another embodiment, a herpes-based, gene therapy delivery system is used to deliver polynucleotides encoding NAAP to target cells which have one or more genetic abnormalities with respect to the expression of NAAP. The use of herpes simplex virus (HSV) based vectors may be especially valuable for introducing NAAP to cells of the central nervous system, for which HSV has a tropism. The construction and packaging of herpes-based vectors are well known to those with ordinary skill in the art. A replication-competent herpes simplex virus (HSV) type 1-based vector has been used to deliver a reporter gene to the eyes of primates (Liu, X. et al.
(1999) Exp. Eye Res.
169:385-395). The construction of a HSV-1 virus vector has also been disclosed in detail in U.S.
Patent No. 5,804,413 to DeLuca ("Herpes simplex virus strains for gene transfer"), which is hereby incorporated by reference. U.S. Patent No. 5,804,413 teaches the use of recombinant HSV d92 which consists of a genome containing at least one exogenous gene to be transferred to a cell under the control of the appropriate promoter for purposes including human gene therapy. Also taught by this patent are the construction and use of recombinant HSV strains deleted for ICP4, ICP27 and ICP22. For HSV vectors, see also Goins, et al. (1999; J. Virol. 73:519-532) and Xu et al. (1994; Dev.
Biol. 163:152-161), hereby incorporated by reference. The manipulation of cloned herpesvirus sequences, the generation of recombinant virus following the transfection of multiple plasmids containing different segments of the large herpesvirus genomes, the growth and propagation of herpesvirus, and the infection of cells with herpesvirus are techniques well known to those of ordinary skill in the art.
In another embodiment, an alphavirus (positive, single-stranded RNA virus) vector is used to deliver polynucleotides encoding NAAP to target cells. The biology of the prototypic alphavirus, Semliki Forest Virus (SFV), has been studied extensively and gene transfer vectors have been based on the SFV genome (Garoff, H. and K.-J. Li (1998) Curr. Opin. Biotechnol.
9:464-469). During alphavirus RNA replication, a subgenomic RNA is generated that normally encodes the viral capsid proteins. This subgenomic RNA replicates to higher levels than the full length genomic RNA, resulting in the overproduction of capsid proteins relative to the viral proteins with enzymatic activity (e.g., protease and polymerase). Similarly, inserting the coding sequence for NAAP into the alphavirus genome in place of the capsid-coding region results in the production of a large number of NAAP-coding RNAs and the synthesis of high levels of NAAP in vector transduced cells. While alphavirus infection is typically associated with cell lysis within a few days, the ability to establish a persistent infection in hamster normal kidney cells (BHI~-21) with a variant of Sindbis virus (SIN) indicates that the lytic replication of alphaviruses can be altered to suit the needs of the gene therapy application (Dryga, S.A. et al. (1997) Virology 228:74-83). The wide host range of alphaviruses will allow the introduction of NAAP into a variety of cell types. The specific transduction of a subset of cells in a population may require the sorting of cells prior to transduction.
The methods of manipulating infectious cDNA clones of alphaviruses, performing alphavirus cDNA and RNA
transfections, and performing alphavirus infections, are well known to those with ordinary skill in the art.
Oligonucleotides derived from the transcription initiation site, e.g., between about positions -10 and +10 from the start site, may also be employed to inhibit gene expression.
Similarly, inhibition can be achieved using triple helix base-pairing methodology. Triple helix pairing is useful because it causes inhibition of the ability of the double helix to open sufficiently for the binding of polymerases, transcription factors, or regulatory molecules. Recent therapeutic advances using triplex DNA have been described in the literature (Gee, J.E. et al. (1994) in Huber, B.E. and B.I. Carr, Molecular and Tmmunolo~ic Approaches, Futura Publishing, Mt. Kisco NY, pp. 163-177). A
complementary sequence or antisense molecule may also be designed to block translation of mRNA by preventing the transcript from binding to ribosomes.
Ribozymes, enzymatic RNA molecules, may also be used to catalyze the specific cleavage of RNA. The mechanism of ribozyme action involves sequence-specific hybridization of the ribozyme molecule to complementary target RNA, followed by endonucleolytic cleavage.
For example, engineered hammerhead motif ribozyme molecules may specifically and efficiently catalyze endonucleolytic cleavage of RNA molecules encoding NAAP.
Specific ribozyme cleavage sites within any potential RNA target are initially identified by scarmiug the target molecule for ribozyme cleavage sites, including the following sequences: GUA, GUU, and GUC. Once identified, short RNA sequences of between 15 and 20 ribonucleotides, corresponding to the region of the target gene containing the cleavage site, may be evaluated for secondary structural features which may render the oligonucleotide inoperable.
The suitability of candidate targets may also be evaluated by testing accessibility to hybridization with complementary oligonucleotides using ribonuclease protection assays.
Complementary ribonucleic acid molecules and ribozymes may be prepared by any method known in the art for the synthesis of nucleic acid molecules. These include techniques for chemically synthesizing oligonucleotides such as solid phase phosphoramidite chemical synthesis. Alternatively, RNA molecules may be generated by in vitt~o and in vivo transcription of DNA
molecules encoding NAAP. Such DNA sequences may be incorporated into a wide variety of vectors with suitable RNA
polymerase promoters such as T7 or SP6. Alternatively, these cDNA constructs that synthesize complementary RNA, constitutively or inducibly, can be introduced into cell lines, cells, or tissues.
RNA molecules may be modified to increase intracellular stability and half life. Possible modifications include, but are not limited to, the addition of flanking sequences at the 5' and/or 3' ends of the molecule, or the use of phosphorothioate or 2' O-methyl rather than phosphodiesterase linkages within the backbone of the molecule. This concept is inherent in the production of PNAs and can be extended in all of these molecules by the inclusion of nontraditional bases such as inosine, queosine, and wybutosine, as well as acetyl-, methyl-, thio-, and similarly modified forms of adenine, cytidine, guanine, thiamine, and uridine which are not as easily recognized by endogenous endonucleases.
An additional embodiment of the invention encompasses a method for screening for a compound which is effective in altering expression of a polynucleotide encoding NAAP. Compounds which may be effective in altering expression of a specific polynucleotide may include, but are not limited to, oligonucleotides, antisense oligonucleotides, triple helix-forming oligonucleotides, transcription factors and other polypeptide transcriptional regulators, and non-macromolecular chemical entities which are capable of interacting with specific polynucleotide sequences. Effective compounds may alter polynucleotide expression by acting as either inhibitors or promoters of polynucleotide expression. Thus, in the treatment of disorders associated with increased NAAP
expression or activity, a compound which specifically inhibits expression of the polynucleotide encoding NAAP may be therapeutically useful, and in the treatment of disorders associated with decreased NAAP expression or activity, a compound which specifically promotes expression of the polynucleotide encoding NAAP may be therapeutically useful.
At least one, and up to a plurality, of test compounds may be screened for effectiveness in altering expression of a specific polynucleotide. A test compound may be obtained by any method commonly known in the art, including chemical modification of a compound known to be effective in altexing polynucleotide expression; selection from an existing, commercially-available or proprietary library of naturally-occurring or non-natural chemical compounds; rational design of a compound based on chemical and/or structural properties of the target polynucleotide;
and selection from a library of chemical compounds created combinatorially or randomly. A sample comprising. a polynucleotide encoding NAAP is exposed to at least one test compound thus obtained. The sample may comprise, for example, an intact or permeabilized cell, or an irt vitro cell-free or reconstituted biochemical system. Alterations in the expression of a polynucleotide encoding NAAP are assayed by any method commonly known in the art. Typically, the expression of a specific nucleotide is detected by hybridization with a probe having a nucleotide sequence complementary to the sequence of the polynucleotide encoding NAAP. The amount of hybridization may be quantified, thus forming the basis for a comparison of the expression of the polynucleotide both with and without exposure to one or more test compounds. Detection of a change in the expression of a polynucleotide exposed to a test compound indicates that the test compound is effective in altering the expression of the polynucleotide. A screen for a compound effective in altering expression of a specific polynucleotide can be carried out, for example, using a Schizosacchar-omyces pombe gene expression system (Atkins, D. et al. (1999) U.S. Patent No. 5,932,435; Arndt, G.M. et al. (2000) Nucleic Acids Res.
28:E15) or a human cell line such as HeLa cell (Clarke, M.L. et al. (2000) Biochem. Biophys. Res.
Common. 268:8-13). A particular embodiment of the present invention involves screening a combinatorial library of oligonucleotides (such as deoxyribonucleotides, ribonucleotides, peptide nucleic acids, and modified oligonucleotides) for antisense activity against a specific polynucleotide sequence (Bruice, T.W, et al. (1997) U.S. Patent No. 5,686,242; Bruice, T.W. et al.
(2000) U.S. Patent No.
6,022,691).
Many methods for introducing vectors into cells or tissues are available and equally suitable for use in vivo, ira vitro, and ex vivo. For ex vivo therapy, vectors may be introduced into stem cells taken from the patient and clonally propagated for autologous transplant back into that same patient.
Delivery by transfection, by liposome injections, or by polycationic amino polymers may be achieved using methods which are well known in the art (Goldman, C.K. et al. (1997) Nat. Biotechnol. 15:462-466).
Any of the therapeutic methods described above may be applied to any subject in need of such therapy, including, for example, mammals such as humans, dogs, cats, cows, horses, rabbits, and monkeys.
An additional embodiment of the invention relates to the administration of a composition which generally comprises an active ingredient formulated with a pharmaceutically acceptable excipient.
Excipients may include, for example, sugars, starches, celluloses, gums, and proteins. Various formulations are commonly known and are thoroughly discussed in the latest edition of Remin~ton's Pharmaceutical Sciences (Maack Publishing, Easton PA). Such compositions may consist of NAAP, antibodies to NAAP, and mimetics, agonists, antagonists, or inhibitors of NAAP.
The compositions utilized in this invention may be administered by any number of routes including, but not limited to, oral, intravenous, intramuscular, infra-arterial, intramedullary, intrathecal, intraventricular, pulmonary, transdermal, subcutaneous, intraperitoneal, intranasal, enteral, topical, sublingual, or rectal means.
Compositions for pulmonary administration may be prepared in liquid or dry powder form.
These compositions are generally aerosolized immediately prior to inhalation by the patient. In the case of small molecules (e.g. traditional low molecular weight organic drugs), aerosol delivery of fast-s acting formulations is well-known in the art. In the case of macromolecules (e.g. larger peptides and proteins), recent developments in the field of pulinonary delivery via the alveolar region of the lung have enabled the practical delivery of drugs such as insulin to blood circulation (see, e.g., Patton, J.S.
et al., U.S. Patent No. 5,997,848). Pulmonary delivery has the advantage of administration without needle injection, and obviates the need for potentially toxic penetration enhancers.
Compositions suitable for use in the invention include compositions wherein the active ingredients are contained in an effective amount to achieve the intended purpose. The determination of an effective dose is well within the capability of those skilled in the art.
Specialized forms of compositions may be prepared for direct intracellular delivery of macromolecules comprising NAAP or fragments thereof. For example, liposome preparations containing a cell-impermeable macromolecule may promote cell fusion and intracellular delivery of the macromolecule. Alternatively, NAAP or a fragment thereof may be joined to a short cationic N-terminal portion from the HIV Tat-1 protein. Fusion proteins thus generated have been found to transduce into the cells of all tissues, including the brain, in a mouse model system (Schwarze, S.R. et al. (1999) Science 285:1569-1572).
For any compound, the therapeutically effective dose can be estimated initially either in cell culture assays, e.g., of neoplastic cells, or in animal models such as mice, rats, rabbits, dogs, monkeys, or pigs. An animal model may also be used to determine the appropriate concentration range and route of administration. Such information can then be used to determine useful doses and routes for administration in humans.
A therapeutically effective dose refers to that amount of active ingredient, for example NAAP or fragments thereof, antibodies of NAAP, and agonists, antagonists or inhibitors of NAAP, which ameliorates the symptoms or condition. Therapeutic efficacy and toxicity may be determined by standard pharmaceutical procedures in cell cultures or with experimental animals, such as by calculating the EDSO (the dose therapeutically effective in 50% of the population) or LDSO (the dose lethal to 50% of the population) statistics. The dose ratio of toxic to therapeutic effects is the therapeutic index, which can be expressed as the LDSO/EDso ratio. Compositions which exhibit large therapeutic indices are preferred. The data obtained from cell culture assays and animal studies are used to formulate a range of dosage for human use. The dosage contained in such compositions is preferably within a range of circulating concentrations that includes the EDSO
with little or no toxicity.
The dosage varies within this range depending upon the dosage form employed, the sensitivity of the patient, and the route of administration.
The exact dosage will be determined by the practitioner, in light of factors related to the subject requiring treatment. Dosage and administration are adjusted to provide sufficient levels of the active moiety or to maintain the desired effect. Factors which may be taken into account include the severity of the disease state, the general health of the subject, the age, weight, and gender of the subject, time and frequency of administration, drug combination(s), reaction sensitivities, and response to therapy. Long-acting compositions may be administered every 3 to 4 days, every week, or 1o biweekly depending on the half life and clearance rate of the particular formulation.
Normal dosage amounts may vary from about 0.1 ~g to 100,000 fig, up to a total dose of about 1 gram, depending upon the route of administration. Guidance as to particular dosages and methods of delivery is provided in the literature and generally available to practitioners in the art.
Those skilled in the art will employ different formulations for nucleotides than for proteins or their inhibitors. Similarly, delivery of polynucleotides or polypeptides will be specific to particular cells, conditions, locations, etc.
DIAGNOSTICS
In another embodiment, antibodies which specifically bind NAAP may be used for the diagnosis of disorders characterized by expression of NAAP, or in assays to monitor patients being treated with NAAP or agonists, antagonists, or inhibitors of NAAP. Antibodies useful for diagnostic purposes may be prepared in the same manner as described above for therapeutics. Diagnostic assays for NAAP include methods which utilize the antibody and a label to detect NAAP 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 NAAP, including ELISAs, RIAs, and FACS, are known in the art and provide a basis for diagnosing altered or abnormal levels of NAAP expression. Normal or standard values for NAAP expression are established by combining body fluids or cell extracts taken from normal mammalian subjects, for example, human subjects, with antibodies to NAAP under conditions suitable for complex formation. The amount of standard complex formation may be quantitated by various methods, such as photometric means. Quantities of NAAP
expressed in subject, control, and disease samples from biopsied tissues are compared with the standard values.
Deviation between standard and subject values establishes the parameters for diagnosing disease.

In another embodiment of the invention, polynucleotides encoding NAAP may be used for diagnostic purposes. The polynucleotides which may be used include oligonucleotides, complementary RNA and DNA molecules, and PNAs. The polynucleotides may be used to detect and quantify gene expression in biopsied tissues in which expression of NAAP may be correlated with disease. The diagnostic assay maybe used to determine absence, presence, and excess expression of NAAP, and to monitor regulation of NAAP levels during therapeutic intervention.
In one aspect, hybridization with PCR probes which are capable of detecting polynucleotides, including genomic sequences, encoding NAAP or closely related molecules may be used to identify nucleic acid sequences which encode NAAP. The specificity of the probe, whether it is made from a highly specific region, e.g., the 5'regulatory region, or from a less specific region, e.g., a conserved motif, and the stringency of the hybridization or amplification will determine whether the probe identifies only naturally occurring sequences encoding NAAP, allelic variants, or related sequences.
Probes may also be used for the detection of related sequences, and may have at least 50%
sequence identity to any of the NAAP encoding sequences. The hybridization probes of the subject invention may be DNA or RNA and may be derived from the sequence of SEQ >D
N0:37-72 or from genomic sequences including promoters, enhancers, and introns of the NAAP
gene.
Means for producing specific hybridization probes for polynucleotides encoding NAAP include the cloning of polynucleotides encoding NAAP or NAAP 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 ih vitro by means of the addition of the appropriate RNA
polymerases and the appropriate labeled nucleotides. Hybridization probes may be labeled by a variety of reporter groups, for example, by radionuclides such as 32P or 355, or by enzymatic labels, such as alkaline phosphatase coupled to the probe via avidin/biotin coupling systems, and the like.
Polynucleotides encoding NAAP may be used for the diagnosis of disorders associated with expression of NAAP. Examples of such disorders include, but are not limited to, a cell proliferative disorder such as actinic keratosis, arteriosclerosis, atherosclerosis, bursitis, cirrhosis, hepatitis, mixed connective tissue disease (MCTD), myelofibrosis, paroxysmal nocturnal hemoglobinuria, polycythemia vera, psoriasis, primary thrombocythemia, and cancers including adenocarcinoma, leukemia, lymphoma, melanoma, myeloma, sarcoma, teratocarcinoma, and, in particular, a cancer of the adrenal gland, bladder, bone, bone marrow, brain, breast, cervix, gall bladder, ganglia, gastrointestinal tract, heart, kidney, liver, lung, muscle, ovary, pancreas, parathyroid, penis, prostate, salivary glands, skin, spleen, testis, thymus, thyroid, and uterus; a neurological disorder such as progressive supranuclear palsy, corticobasal degeneration, familial frontotemporal dementia, epilepsy, ischemic cerebrovascular disease, stroke, cerebral neoplasms, Alzheimer's disease, Pick's disease, Huntington's disease, dementia, Parkinson's disease and other extrapyramidal disorders, amyotrophic lateral sclerosis and other motor neuron disorders, progressive neural muscular atrophy, retinitis pigmentosa, hereditary ataxias, multiple sclerosis and other demyelinating diseases, bacterial and viral meningitis, brain abscess, subdural empyema, epidural abscess, suppurative intracranial thrombophlebitis, myelitis and radiculitis, viral central nervous system disease, priors diseases including kuru, Creutzfeldt-Jakob disease, and Gerstmann-Straussler-Scheinker syndrome, fatal familial insomnia, nutritional and metabolic diseases of the nervous system, neurofibromatosis, tuberous sclerosis, cerebelloretinal hemangioblastomatosis, encephalotrigeminal syndrome, mental retardation and other developmental disorder of the central nervous system, cerebral palsy, a neuroskeletal disorder, an autonomic nervous system disorder, a cranial nerve disorder, a spinal cord disease, muscular dystrophy and other neuromuscular disorder, a peripheral nervous system disorder, dermatomyositis and polymyositis, inherited, metabolic, endocrine, and toxic myopathy, myasthenia gravis, periodic paralysis, a mental disorder including mood, anxiety, and schizophrenic disorder, seasonal affective disorder (SAD), akathesia, amnesia, catatonia, diabetic neuropathy, tardive dyskinesia, dystonias, paranoid psychoses, postherpetic neuralgia, and Tourette's disorder; 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; a developmental disorder such as renal tubular acidosis, anemia, Cushing's syndrome, achondroplastic dwarfism, Duchenne and Becker muscular dystrophy, epilepsy, gonadal dysgenesis, WAGR syndrome (Wilms' tumor, aniridia, genitourinary abnormalities, and mental retardation), Smith-Magenis syndrome, myelodysplastic syndrome, hereditary mucoepithelial dysplasia, hereditary keratodermas, hereditary neuropathies such as Charcot-Marie-Tooth disease and neurofibromatosis, hypothyroidism, hydrocephalus, seizure disorders such as Syndenham's chorea and cerebral palsy, spina bifida, anencephaly, craniorachischisis, congenital glaucoma, cataract, and 3o sensorineural hearing loss; an autoimmune/inflammatory disorder such as acquired immunodeficiency syndrome (AIDS), Addison's disease, adult respiratory distress syndrome, allergies, ankylosing spondylitis, amyloidosis, anemia, asthma, atherosclerosis, autoimmune hemolytic anemia, autoimmune thyroiditis, autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED), bronchitis, cholecystitis, contact dermatitis, Crohn's disease, atopic dermatitis, dermatomyositis, diabetes mellitus, emphysema, episodic lymphopenia with lymphocytotoxins, erythroblastosis fetalis, erythema nodosum, atrophic gastritis, glomerulonephritis, Goodpasture's syndrome, gout, Graves' disease, Hashimoto's thyroiditis, hypereosinophilia, irritable bowel syndrome, multiple sclerosis, myasthenia gravis, . myocardial or pericardial inflammation, osteoartluitis, osteoporosis, pancreatitis, polymyositis, psoriasis, Reiter's syndrome, rheumatoid arthritis, scleroderma, Sjogren's syndrome, systemic anaphylaxis, systemic lupus erythematosus, systemic sclerosis, thrombocytopenic purpura, ulcerative colitis, uveitis, Werner syndrome, complications of cancer, hemodialysis, and extracorporeal circulation, viral, bacterial, fungal, parasitic, protozoal, and helininthic infections, and trauma; an infection, such as those caused by a viral agent classified as adenovirus, arenavirus, bunyavirus, calicivirus, coronavirus, filovirus, hepadnavirus, herpesvirus, flavivirus, orthomyxovirus, parvovirus, papovavirus, paramyxovirus, picornavirus, poxvirus, reovirus, retrovirus, rhabdovirus, or togavirus; an infection caused by a bacterial agent classified as pneumococcus, staphylococcus, streptococcus, bacillus, corynebacterium, clostridium, meningococcus, gonococcus, listeria, moraxella, kingella, haemophilus, legionella, bordetella, gram-negative enterobacterium including shigella, salmonella, or campylobacter, pseudomonas, vibrio, brucella, francisella, yersinia, bartonella, norcardium, actinomyces, mycobacterium, spirochaetale, rickettsia, chlamydia,.or mycoplasma; an infection caused by a fungal agent classified as aspergillus, blastomyces, dermatophytes, cryptococcus, coccidioides, malasezzia, histoplasma, or other mycosis-causing fungal agent; an infection caused by a parasite classified as plasmodium or malaria-causing, parasitic entamoeba, leishmania, trypanosoma, toxoplasma, pneumocystis carinii, intestinal protozoa such as giardia, trichomonas, tissue nematode such as trichinella, intestinal nematode such as ascaris, lymphatic filarial nematode, trematode such as schistosoma, and cestode such as tapeworm; and a DNA repair disorder such as xeroderma pigmentosum, Bloom's syndrome, and Werner's syndrome. Polynucleotides encoding NAAP 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 NAAP expression. Such qualitative or quantitative methods are well known in the art.
In a particular aspect, polynucleotides encoding NAAP may be used in assays that detect the presence of associated disorders, particularly those mentioned above.
Polynucleotides complementary to sequences encoding NAAP 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 quantifted and compared with a ~7 standard value. If the amount of signal in the patient sample is significantly altered in comparison to a control sample then the presence of altered levels of polynucleotides encoding NAAP 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 NAAP, 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 NAAP, 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.
2o With respect to cancer, the presence of an abnormal amount of transcript (either under- or overexpressed) in biopsied tissue from an individual may indicate a predisposition for the development of the disease, or may provide a means for detecting the disease prior to the appearance of actual clinical symptoms. A more definitive diagnosis of this type may allow health professionals to employ preventative measures or aggressive treatment earlier, thereby preventing the development or further progression of the cancer.
Additional diagnostic uses for oligonucleotides designed from the sequences encoding NAAP
may involve the use of PCR. These oligomers may be chemically synthesized, generated enzymatically, or produced iv vitro. Oligomers will preferably contain a fragment of a polynucleotide encoding NAAP, or a fragment of a polynucleotide complementary to the polynucleotide encoding 3o NAAP, and will be employed under optimized conditions for identification of a specific gene or condition. Oligomers may also be employed under less stringent conditions for detection or quantification of closely related DNA or RNA sequences.
In a particular aspect, oligonucleotide primers derived from polynucleotides encoding NAAP

may be used to detect single nucleotide polymorphisms (SNPs). SNPs are substitutions, insertions and deletions that are a frequent cause of inherited or acquired genetic disease in humans. Methods of SNP detection include, but are not limited to, single-stranded conformation polymorphism (SSCP) and fluorescent SSCP (fSSCP) methods. In SSCP, oligonucleotide primers derived from polynucleotides encoding NAAP are used to amplify DNA using the polymerase chain reaction (PCR). The DNA
may be derived, for example, from diseased or normal tissue, biopsy samples, bodily fluids, and the like. SNPs in the DNA cause differences in the secondary and tertiary structures of PCR products in single-stranded form, and these differences are detectable using gel electrophoresis in non-denaturing gels. In fSCCP, the oligonucleotide primers are fluorescently labeled, which allows detection of the amplimers in high-throughput equipment such as DNA sequencing machines.
Additionally, sequence database analysis methods, termed in silico SNP (isSNP), are capable of identifying polymorphisms by comparing the sequence of individual overlapping DNA fragments which assemble into a common consensus sequence. These computer-based methods filter out sequence variations due to laboratory preparation of DNA and sequencing errors using statistical models and automated analyses of DNA
sequence chromatograms. In the alternative, SNPs may be detected and characterized by mass spectrometry~using, for example, the high throughput MASSARRAY system (Sequenom, Inc., San Diego CA).
SNPs may be used to study the genetic basis of human. disease. For example, at least 16 common SNPs have been associated with non-insulin-dependent diabetes mellitus.
SNPs are also useful for examining differences in disease outcomes in monogenic disorders, such as cystic fibrosis, sickle cell anemia, or chronic granulomatous disease. For example, variants in the mannose-binding lectin, MBL2, have been shown to be correlated with deleterious pulmonary outcomes in cystic fibrosis. SNPs also have utility in pharmacogenomics, the identification of genetic variants that influence a patient's response to a drug, such as life-threatening toxicity.
For example, a variation in N-acetyl transferase is associated with a high incidence of peripheral neuropathy in response to the anti-tuberculosis drug isoniazid, while a variation in the core promoter of the ALOXS gene results in diminished clinical response to treatment with an anti-asthma drug that targets the 5-lipoxygenase pathway. Analysis of the distribution of SNPs in different populations is useful for investigating genetic drift, mutation, recombination, and selection, as well as for tracing the origins of populations and their migrations (Taylor, J.G. et al. (2001) Trends Mol. Med. 7:507-512;
Kwok, P.-Y. and Z. Gu (1999) Mol. Med. Today 5:538-543; Nowotny, P. et al. (2001) Curr. Opin.
Neurobiol. 11:637-641).
Methods which may also be used to quantify the expression of NAAP include radiolabeling or biotinylating nucleotides, coamplification of a control nucleic acid, and interpolating results from standard curves (Melby, P.C. et al. (1993) J. Tmmunol. Methods 159:235-244;
Duplaa, C. et al. (1993) Anal. Biochem. 212:229-236). The speed of quantitation of multiple samples may be accelerated by running the assay in a high-throughput format where the oligomer or polynucleotide of interest is presented in various dilutions and a spectrophotometric or colorimetric response gives rapid quantitation.
In further embodiments, oligonucleotides or longer fragments derived from any of the polynucleotides described herein may be used as elements on a microarray. The microarray can be used in transcript imaging techniques which monitor the relative expression levels of large numbers of genes simultaneously as described below. The microarray may also be used to identify genetic variants, mutations, and polymorphisms. This information may be used to determine gene function, to understand the genetic basis of a disorder, to diagnose a disorder, to monitor progression/regression of disease as a function of gene expression, and to develop and monitor the activities of therapeutic agents in the treatment of disease. In particular, this information may be used to develop a pharmacogenomic profile of a patient in order to select the most appropriate and effective treatment regimen for that patient. For example, therapeutic agents which are highly effective and display the fewest side effects may be selected for a patient based on his/her pharmacogenomic profile.
In another embodiment, NAAP, fragments of NAAP, or antibodies specific for NAAP may be used as elements on a microarray. The microarray may be used to monitor or measure protein-protein interactions, drug-target interactions, and gene expression profiles, as described above.
A particular embodiment relates to the use of the polynucleotides of the present invention to generate a transcript image of a tissue or cell type. A transcript image represents the global pattern of gene expression by a particular tissue or cell type. Global gene expression patterns are analyzed by quantifying the number of expressed genes and their relative abundance under given conditions and at a given time (See Seilhamer et al., "Comparative Gene Transcript Analysis,"
U.S. Patent No.
5,840,484, expressly incorporated by reference herein). Thus a transcript image may be generated by hybridizing the polynucleotides of the present invention or their complements to the totality of transcripts or reverse transcripts of a particular tissue or cell type. In one embodiment, the hybridization takes place in high-throughput format, wherein the polynucleotides of the present invention or their complements comprise a subset of a plurality of elements on a microarray. The 3o resultant transcript image would provide a profile of gene activity.
Transcript images may be generated using transcripts isolated from tissues, cell lines, biopsies, or other biological samples. The transcript image may thus reflect gene expression in vivo, as in the case of a tissue or biopsy sample, or in vitro, as in the case of a cell line.

Transcript images which profile the expression of the polynucleotides of the present invention may also be used in conjunction with irc vitro model systems and preclinical evaluation of pharmaceuticals, as well as toxicological testing of industrial and naturally occurring environmental compounds. All compounds induce characteristic gene expression patterns, frequently termed molecular fingerprints or toxicant signatures, which are indicative of mechanisms of action and toxicity (Nuwaysir, E.F. et al. (1999) Mol. Carcinog. 24:153-159; Steiner, S. and N.L.
Auderson (2000) Toxicol. Lett. 112-113:467-471). If a test compound has a signature similar to that of a compound with known toxicity, it is likely to share those toxic properties. These fingerprints or signatures are most useful and refined when they contain expression information from a large number of genes and gene families. Ideally, a genome-wide measurement of expression provides the highest quality signature. Even genes whose expression is not altered by any tested compounds are important as well, as the levels of expression of these genes are used to normalize the rest of the expression data.
The normalization procedure is useful for comparison of expression data after treatment with different compounds. While the assignment of gene function to elements of a toxicant signature aids in interpretation of toxicity mechanisms, knowledge of gene function is not necessary for the statistical matching of signatures which leads to prediction of toxicity (See, for example, Press Release 00-02 from the National.Institute of Environmental Health Sciences, released February 29, 2000, available at http://www.niehs.nih.gov/oc/news/toxclup.htm.) Therefore, it is important and desirable in toxicological screening using toxicant signatures to include all expressed gene sequences.
In an embodiment, the toxicity of a test compound can be assessed by treating a biological sample containing nucleic acids with the test compound. Nucleic acids that are expressed in the treated biological sample are hybridized with one or more probes specific to the polynucleotides of the present invention, so that transcript levels corresponding to the polynucleotides of the present invention may be quantified. 'The transcript levels in the treated biological sample are compared with levels in an untreated biological sample. Differences in the transcript levels between the two samples are indicative of a toxic response caused by the test compound in the treated sample.
Another embodiment relates to the use of the polypeptides disclosed herein to analyze the proteome of a tissue or cell type. The term proteome refers to the global pattern of protein expression in a particular tissue or cell type. Each protein component of a proteome can be subjected individually to further analysis. Proteome expression patterns, or profiles, are analyzed by quantifying the number of expressed proteins and their relative abundance under given conditions and at a given time. A
profile of a cell's proteome may thus be generated by separating and analyzing the polypeptides of a particular tissue or cell type. In one embodiment, the separation is achieved using two-dimensional gel electrophoresis, in which proteins from a sample are separated by isoelectric focusing in the first dimension, and then according to molecular weight by sodium dodecyl sulfate slab gel electrophoresis in the second dimension (Steiner and Anderson, supf~a). The proteins are visualized in the gel as discrete and uniquely positioned spots, typically by staining the gel with an agent such as Coomassie Blue or silver or fluorescent stains. The optical density of each protein spot is generally proportional to the level of the protein in the sample. The optical densities of equivalently positioned protein spots from different samples, for example, from biological samples either treated or untreated with a test compound or therapeutic agent, are compared to identify any changes in protein spot density related to the treatment. The proteins in the spots are partially sequenced using, for example, standard methods employing chemical or enzymatic cleavage followed by mass spectrometry. The identity of the protein in a spot may be determined by comparing its partial sequence, preferably of at least 5 contiguous amino acid residues, to the polypeptide sequences of interest. In some cases, further sequence data may be obtained for definitive protein identification.
A proteomic profile may also be generated using antibodies specific for NAAP
to quantify the levels of NAAP expression. In one embodiment, the antibodies are used as elements on a microarray, and protein expression levels are quantified by exposing the microarray to the sample and detecting the levels of protein bound to each array element (Lueking, A. et al. (1999) Anal. Biochem. 270:103-111; Mendoze, L.G. et al. (1999) Biotechniques 27:778-788). Detection may be performed by a variety of methods known in the art, for example, by reacting the proteins in the sample with a thiol- or amino-reactive fluorescent compound and detecting the amount of fluorescence bound at each array element.
Toxicant signatures at the proteome level are also useful for toxicological screening, and should be analyzed in parallel with toxicant signatures at the transcript level. There is a poor correlation between transcript and protein abundances for some proteins in some tissues (Anderson, N.L. and J. Seilhamer (1997) Electrophoresis 18:533-537), so proteome toxicant signatures may be useful in the analysis of compounds which do not significantly affect the transcript image, but which alter the proteomic profile. In addition, the analysis of transcripts in body fluids is difficult, due to rapid degradation of mRNA, so proteomic pxofiling may be more reliable and informative in such cases.
In another embodiment, the toxicity of a test compound is assessed by treating a biological sample containing proteins with the test compound. Proteins that are expressed in the treated biological sample are separated so that the amount of each protein can be quantified. The amount of each protein is compared to the amount of the corresponding protein in an untreated biological sample.
A difference in the amount of protein between the two samples is indicative of a toxic response to the test compound in the treated sample. Individual proteins are identified by sequencing the amino acid residues of the individual proteins and comparing these partial sequences to the polypeptides of the present invention.
In another embodiment, the toxicity of a test compound is assessed by treating a biological sample containing proteins with the test compound. Proteins from the biological sample are incubated with antibodies specific to the polypeptides of the present invention. The amount of protein recognized by the antibodies is quantified. The amount of protein in the treated biological sample is compared with the amount in an untreated biological sample. A difference in the amount of protein between the two samples is indicative of a toxic response to the test compound in the treated sample.
1o Microarrays may be prepared, used, and analyzed using methods known in the art (Brennan, T.M. et al. (1995) U.S. Patent No. 5,474,796; Schena, M. et al. (1996) Proc.
Natl. Acad. Sci. USA
93:10614-10619; Baldeschweiler et al. (1995) PCT application W095/251116;
Shalon, D. et al. (1995) PCT application WO95/35505; Heller, R.A. et al. (1997) Proc. Natl. Acad. Sci.
USA 94:2150-2155;
and Heller, M.J. et al. (1997) U.S. Patent No. 5,605,662): Various types of microarrays are well known and thoroughly described in DNA Microarrays: A, Practical Approach, M.
Schena, ed. (1999) Oxford University Press, London.
In another embodiment of the invention, nucleic acid sequences encoding NAAP
may be used to generate hybridization probes useful in mapping the naturally occurring genomic sequence. Either coding or noncoding sequences may be used, and in some instances, noncoding sequences may be preferable over coding sequences. For example, conservation of a coding sequence among members of a multi-gene family may potentially cause undesired cross hybridization during chromosomal mapping. The sequences may be mapped to a particular chromosome, to a specific region of a chromosome, or to artificial chromosome constructions, e.g., human artificial chromosomes (HACs), yeast artificial chromosomes (PACs), bacterial artificial chromosomes (BACs), bacterial P1 constructions, or single chromosome cDNA libraries (Harrington, J.J. et al.
(1997) Nat. Genet. 15:345-355; Price, C.M. (1993) Blood Rev. 7:127-134; and Trask, B.J. (1991) Trends Genet. 7:149-154).
Once mapped, the nucleic acid sequences may be used to develop genetic linkage maps, for example, wluch correlate the inheritance of a disease state with the inheritance of a particular chromosome region or restriction fragment length polymorphism (RFLP) (Larder, E.S. and D.
Botstein (1986) Proc. Natl. Acad. Sci. USA 83:7353-7357).
Fluorescent if2 situ hybridization (FISH) may be correlated with other physical and genetic map data (Heinz-Ulrich, et al. (1995) in Meyers, supra, pp. 965-968). Examples of genetic map data can be found in various scientific journals or at the Online Mendelian Inheritance in Man (OMIM) World Wide Web site. Correlation between the location of the gene encoding NAAP on a physical map and a specific disorder, or a predisposition to a specific disorder, may help define the region of DNA associated with that disorder and thus may further positional cloning efforts.
Ifi situ hybridization of chromosomal preparations and physical mapping techniques, such as linkage analysis using established chromosomal markers, may be used for extending genetic maps.
Often the placement of a gene on the chromosome of another mammalian species, such as mouse, may reveal associated markers even if the exact chromosomal locus is not known. This information is valuable to investigators searching for disease genes using positional cloning or other gene discovery techniques. Once the gene or genes responsible for a disease or syndrome have been crudely localized by genetic linkage to a particular genomic region, e.g., ataxia-telangiectasia to 11q22-23, any sequences mapping to that area may represent associated or regulatory genes for further investigation (Gatti, R.A. et al. (1988) Nature 336:577-580): The nucleotide sequence of the instant invention may also be used to detect differences in the chromosomal location due to translocation, inversion, etc., among normal, carrier, or affected individuals.
In another embodiment of the invention, NAAP, 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 NAAP and the agent being tested may be measured.
Another technique for drug screening provides for high throughput screening of compounds having suitable binding affinity to the protein of interest (Geysen, et al.
(1984) PCT application W084/03564). In this method, large numbers of different small test compounds are synthesized on a solid substrate. 'The test compounds are reacted with NAAP, or fragments thereof, and washed.
Bound NAAP is then detected by methods well known in the art. Purified NAAP
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 NAAP specifically compete with a test compound for binding NAAP.
In this manner, antibodies can be used to detect the presence of any peptide which shares one or more 3o antigenic determinants with NAAP.
In additional embodiments, the nucleotide sequences which encode NAAP may be used in any molecular biology techniques that have yet to be developed, provided the new techniques rely on properties of nucleotide sequences that are currently known, including, but not limited to, such properties as the triplet genetic code and specific base pair interactions.
Without further elaboration, it is believed that one skilled in the art can, using the preceding description, utilize the present invention to its fullest extent. The following embodiments are, therefore, to be construed as merely illustrative, and not limitative of the remainder of the disclosure in any way whatsoever.
The disclosures of all patents, applications and publications, mentioned above and below, including U.S. Ser. No. 60/301,893, U.S. Ser. No. 60/300,518, U.S. Ser. No.
60/301,787, U.S. Ser.
No. 60/301,892, U.S. Ser. No. 60/301,792, U.S. Ser. No. 60/303,442, U.S. Ser.
No. 60/303,405, and U.S. Ser. No. 60/364,438, are expressly incorporated by reference herein.
EXAMPLES
I. Construction of cDNA Libraries Iucyte cDNAs were derived from cDNA libraries described in the L1FESEQ GOLD
database (Incyte Genomics, Palo Alto CA). Some tissues were homogenized and lysed in guanidinium isothiocyanate, while others were homogenized and lysed in phenol or in a suitable mixture of denaturants, such as TRIZOL (Invitrogen), a monophasic solution of phenol and guanidine isothiocyanate. The resulting lysates were centrifuged over CsCl cushions or extracted with chloroform. RNA was precipitated from the lysates with either isopropanol or sodium acetate and ethanol, or by other routine methods.
Phenol extraction and precipitation of RNA were repeated as necessary to increase RNA
purity. In some cases, RNA was treated with DNase. For most libraries, poly(A)+ RNA was isolated using oligo d(T)-coupled paramagnetic particles (Promega), OLIGOTEX
latex particles (QIAGEN, Chatsworth CA), or an OLIGOTEX mRNA purification kit (QIAGEN).
Alternatively, RNA was isolated directly from tissue lysates using other RNA isolation kits, e.g., the POLY(A)PURE mRNA purification kit (Ambion, Austin TX).
In some cases, Stratagene was provided with RNA and constructed the corresponding cDNA
libraries. Otherwise, cDNA was synthesized and cDNA libraries were constructed with the UNIZAP vector system (Stratagene) or SUPERSCRIPT plasmid system (Iuvitrogen), using the recommended procedures or similar methods known in the art (Ausubel, 1997, supra, units 5.1-6.6).
Reverse transcription was initiated using oligo d(T) or random primers.
Synthetic oligonucleotide adapters were ligated to double stranded cDNA, and the cDNA was digested with the appropriate restriction enzyme or enzymes. For most libraries, the cDNA was size-selected (300-1000 bp) using SEPHACRYL S 1000, SEPHAROSE CL2B, or SEPHAROSE CL4B column chromatography (Amersham Biosciences) or preparative agarose gel electrophoresis. cDNAs were ligated into compatible restriction enzyme sites of the polylinker of a suitable plasmid, e.g., PBLUESCRIPT
plasmid (Stratagene), PSPORT1 plasmid (Invitrogen), PCDNA2.1 plasmid (Invitrogen, Carlsbad CA), PBI~-CMV plasmid (Stratagene), PCR2-TOPOTA plasmid (Invitrogen), PCMV-ICIS
plasmid (Stratagene), pIGEN (Incyte Genomics, Palo Alto CA), pRARE (Incyte Genomics), or pINCY
(Incyte Genomics), or derivatives thereof. Recombinant plasmids were transformed into competent E.
coli cells including XL1-Blue, XL1-BlueMRF, or SOLR from Stratagene or DHSa, DH10B, or ElectroMAX DH10B from Invitrogen.
II. Isolation of cDNA Clones Plasmids obtained as described in Example I were recovered from host cells by iti vivo excision using the UNIZAP vector system (Stratagene) or by cell lysis.
Plasmids were purified using at least one of the following: a Magic or WIZARD Minipreps DNA purification system (Promega); an AGTC Miniprep purification kit (Edge Biosystems, Gaithersburg MD); and QIAWELL
8 Plasmid, QIAWELL 8 Plus Plasmid, QIAWELL 8 Ultra Plasmid purification systems or the R.E.A.L. PREP
96 plasmid purification kit from QIAGEN. Following precipitation, plasmids were resuspended in 0.1 m1 of distilled water and stored, with or without lyophilization, at 4°C.
Alternatively, plasmid DNA was amplified from host cell lysates using direct link PCR in a high-throughput format (Rao, V.B. (1994) Anal. Biochem. 216:1-14). Host cell lysis and thermal cycling steps were carried out in a single reaction mixture. Samples were processed and stored in 3 84-well plates, and the concentration of amplified plasmid DNA was quantified fluorometrically using PICOGREEN dye (Molecular Probes, Eugene OR) and a FLUOROSKAN II fluorescence scanner (Labsystems Oy, Helsinki, Finland).
III. Sequencing and Analysis Incyte cDNA recovered in plasmids as described in Example lI were sequenced as follows.
Sequencing reactions were processed using standard methods or high-throughput instrumentation such as the ABI,CATALYST 800 (Applied Biosystems) thermal cycler or the PTC-200 thermal cyclex (MJ Research) in conjunction with the HYDRA microdispenser (Robbins Scientific) or the MICROLAB 2200 (Hamilton) liquid transfer system. cDNA sequencing reactions were prepared using reagents provided by Amersham Biosciences or supplied in ABI sequencing kits such as the ABI PRISM BIGDYE Terminator cycle sequencing ready reaction kit (Applied Biosystems).
Electrophoretic separation of cDNA sequencing reactions and detection of labeled polynucleotides were carried out using the MEGABACE 1000 DNA sequencing system (Amersham Biosciences);
the ABI PRISM 373 or 377 sequencing system (Applied Biosystems) in conjunction with standard ABI protocols and base calling software; or other sequence analysis systems known in the art.
Reading frames within the cDNA sequences were identi~xed using standard methods (reviewed in Ausubel, 1997, supra, unit 7.7). Some of the cDNA sequences were selected for extension using the techniques disclosed in Example VIII.
The polynucleotide sequences derived from Incyte cDNAs were validated by removing vector, linker, and poly(A) sequences and by masking ambiguous bases, using algorithms and programs based on BLAST, dynamic programming, and dinucleotide nearest neighbor analysis. The Incyte cDNA sequences or translations thereof were then queried against a selection of public databases such as the GenBank primate, rodent, mammalian, vertebrate, and eukaryote databases, and BLOCKS, PRINTS, DOMO, PRODOM; PROTEOME databases with sequences from Homo Sapiens, Rattus norvegicus, Mus musculus, Caenorhabditis elegans, Sacclaar~omyces cerevisiae, Sclaizosacchaf-omyces pombe, and Candida albican.s (Incyte Genomics, Palo Alto CA); hidden Markov model (HIVEVI) based protein family databases such as PFAM,1NCY, and TIGRFAM (Haft, D.H. et al. (2001) Nucleic Acids Res. 29:41-43); and HIVEVI based protein domain databases such as SMART (Schultz et al. (1998) Proc. Natl. Acad. Sci. USA 95:5857-5864; Letunic, I. et al. (2002) Nucleic Acids Res. 30:242-244). (H1VI~~I is a probabilistic approach which analyzes consensus primary structures of gene families. See, for example, Eddy, S.R. (1996) Curr.
Opin. Struct. Biol.
6:361-365). The queries were performed using programs based on BLAST, FASTA, BLllVIPS, anal I~VIER. The Incyte cDNA sequences were assembled to produce full length polynucleotide sequences. Alternatively, GenBank cDNAs, GenBank ESTs, stitched sequences, stretched sequences, or Genscan-predicted coding sequences (see Examples IV and V) were used to extend Incyte cDNA assemblages to full length. Assembly was performed using programs based on Phred, Phrap, and Consed, and cDNA assemblages were screened for open reading frames using programs based on GeneMark, BLAST, and FASTA. The full length polynucleotide sequences were translated to derive the corresponding full length polypeptide sequences. Alternatively, a polypeptide may begin at any of the methionine residues of the full length translated polypeptide.
Full length polypeptide sequences were subsequently analyzed by querying against databases such as the GenBank protein databases (genpept), SwissProt, the PROTEOME databases, BLOCKS, PR1NTS, DOMO, PRODOM, Prosite, hidden Markov model (HMM)-based protein family databases such as PFAM, INCY, and TIGRFAM; and HMM-based protein domain databases such as SMART. Full length polynucleotide sequences are also analyzed using MACDNASIS PRO software (Hitaclu Software Engineering, South San Francisco CA) and LASERGENE software (DNASTAR).
Polynucleotide and polypeptide sequence alignments are generated using default parameters specified by the CLUSTAL algorithm as incorporated into the MEGALIGN multisequence alignment program (DNASTAR), which also calculates the percent identity between aligned sequences.
Table 7 summarizes the tools, programs, and algorithms used for the analysis and assembly of Incyte cDNA and full length sequences and provides applicable descriptions, references, and threshold parameters. The first column of Table 7 shows the tools, programs, and algorithms used, the second column provides brief descriptions thereof, the third column presents appropriate references, all of which are incorporated by reference herein in their entirety, and the fourth column presents, whexe applicable, the scores, probability values, and other parameters used to evaluate the strength of a match between two sequences (the higher the score or the lower the probability value, the greater the Identity between two sequences).
The programs described above for the assembly and analysis of full length polynucleotide and polypeptide sequences were also used to identify polynucleotide sequence fragments from SEQ ID
N0:37-72. Fragments from about 20 to about 4000 nucleotides which are useful in hybridization and amplification technologies are described in Table 4, column 2.
IV. Identification and Editing of Coding Sequences from Genomic DNA
Putative nucleic acid-associated proteins were initially identified by running the Genscan gene identification program against public genomic sequence databases (e.g., gbpri and gbhtg). Genscan is a general-purpose gene identification program which analyzes genomic DNA
sequences from a variety of organisms (Burge, C. and S. Karlin (1997) J. Mol. Biol. 268:78-94;
Burge, C. and S. Karlin (1998) Curr. Opin. Struct. Biol. 8:346-354). The program concatenates predicted exons to form an assembled cDNA sequence extending from a methionine to a stop codon. The output of Genscan is a FASTA database of polynucleotide and polypeptide sequences. The maximum range of sequence for Genscan to analyze at once was set to 30 kb. To determine which of these Genscan predicted cDNA
sequences encode nucleic acid-associated proteins, the encoded polypeptides were analyzed by querying against PFAM models for nucleic acid-associated proteins. Potential nucleic acid-associated proteins were also identified by homology to Incyte cDNA sequences that had been annotated as nucleic acid-associated proteins. These selected Genscan-predicted sequences were then compared by BLAST analysis to the genpept and gbpri public databases. Where necessary, the Genscan-predicted sequences were then edited by comparison to the top BLAST hit from genpept to correct errors in the sequence predicted by Genscan, such as extra or omitted exons.
BLAST analysis was also used to find any Iucyte cDNA or public cDNA coverage of the Genscan-predicted sequences, thus providing evidence for transcription. When Incyte cDNA coverage was available, this information was used to correct or confirm the Genscan predicted sequence.
Full length polynucleotide sequences were obtained by assembling Genscau-predicted coding sequences with Incyte cDNA sequences and/or public cDNA sequences using the assembly process described in Example III. Alternatively, full length polynucleotide sequences were derived entirely from edited or unedited Genscan-predicted coding sequences.
V. Assembly of Genomic Sequence Data with cDNA Sequence Data "Stitched" Seauences Partial cDNA sequences were extended with exons predicted by the Genscan gene identification program described in Example IV. Partial cDNAs assembled as described in Example llI were mapped to genomic DNA and parsed into clusters containing related cDNAs and Genscan 20 exon predictions from one or more genomic sequences. Each cluster was analyzed using an algorithm based on graph theory and dynamic prograrrLmi~g to integrate cDNA and genomic information, generating possible splice variants that were subsequently confirmed, edited, or extended to create a full length sequence. Sequence intervals in which the entire length of the interval was present on more than one sequence in the cluster were identified, and intervals thus identified were considered to 15 be equivalent by transitivity. For example, if an interval was present on a cDNA and two genomic sequences, then all three intervals were considered to be equivalent. This process allows unrelated but consecutive genomic sequences to be brought together, bridged by cDNA
sequence. Intervals thus identified were then "stitched" together by the stitching algorithm in the order that they appear along their parent sequences to generate the longest possible sequence, as well as sequence variants.
20 Linkages between intervals which proceed along one type of parent sequence (cDNA to cDNA or genomic sequence to genomic sequence) were given preference over linkages wluch change parent type (cDNA to genomic sequence). The resultant stitched sequences were translated and compared by BLAST analysis to the genpept and gbpri public databases. Incorrect exons predicted by Genscan were corrected by comparison to the top BLAST hit from genpept. Sequences were further extended 25 with additional cDNA sequences, or by inspection of genomic DNA, when necessary.
"Stretched" Sequences Partial DNA sequences were extended to full length with an algorithm based on BLAST
analysis. First, partial cDNAs assembled as described in Example III were queried against public databases such as the GenBank primate, rodent, mammalian, vertebrate, and eukaryote databases 30 using the BLAST program. The nearest GenBank pxotein homolog was then compared by BLAST
analysis to either Incyte cDNA sequences or GenScan exon predicted sequences described in Example IV. A chimeric protein was generated by using the resultant high-scoring segment pairs (HSPs) to map the translated sequences onto the GenBank protein homolog.
Iusertions or deletions may occur in the chimeric protein with respect to the original GenBank protein homolog. The GenBank protein homolog, the chimeric protein, or both were used as probes to search for homologous genomic sequences from the public human genome databases. Partial DNA
sequences were therefore "stretched" or extended by the addition of homologous genomic sequences. The resultant stretched sequences were examined to determine whether it contained a complete gene.
VI. Chromosomal Mapping of NAAP Encoding Polynucleotides The sequences which were used to assemble SEQ ID NO:37-72 were compared with sequences from the Incyte LIFESEQ database and public domain databases using BLAST and other implementations of the Smith-Waterman algorithm. Sequences from these databases that matched SEQ ll~ N0:37-72 were assembled into clusters of contiguous and overlapping sequences using assembly algorithms such as Phrap (Table 7). Radiation hybrid and genetic mapping data available from public resources such as the Stanford Human Genome Center (SHGC), Whitehead Institute for Genome Research (WIGR), and Genethon were used to determine if any of the clustered sequences had been previously mapped. Inclusion of a mapped sequence in a cluster resulted in the assignment of all sequences of that cluster, including its particular SEQ ID NO:, to that map location.
Map locations are represented by ranges, or intervals, of human chromosomes.
The map position of an interval, in centiMorgans, is measured relative to the terminus of the chromosome's p-arm. (The centiMorgan (cM) is a unit of measurement based on recombination frequencies between chromosomal markers. On average, 1 cM is roughly equivalent to 1 megabase (Mb) of DNA in humans, although this can vary widely due to hot and cold spots of recombination.) 'The cM distances are based on genetic markers mapped by Genethon which provide boundaries for radiation hybrid markers whose sequences were included in each of the clusters. Human genome maps and other resources available to the public, such as the NCBI "GeneMap'99" World Wide Web site (http://www.ncbi.nlm.nih.govlgenemapn, can be employed to determine if previously identified disease genes map within or in proximity to the intervals indicated above.
VII. Analysis of Polynucleotide Expression Northern analysis is a laboratory technique used to detect the presence of a transcript of a gene and involves the hybridization of a labeled nucleotide sequence to a membrane on which RNAs from a particular cell type or tissue have been bound (Sambrook et al., supra, ch. 7; Ausubel (1995) 3o supra, ch. 4 and 16).
Analogous computer techniques applying BLAST were used to search for identical or related molecules in cDNA databases such as GenBank or LIFESEQ (Incyte Genomics). This analysis is much faster than multiple membrane-based hybridizations. In addition, the sensitivity of the computer search can be modified to determine whether any particular match is categorized as exact or similar.
The basis of the search is the product score, which is defined as:
BLAST Score x Percent Identity x minimum {length(Seq. 1), length(Seq. 2)}
The product score takes into account both the degree of similarity between two sequences and the length of the sequence match. The product score is a normalized value between 0 and 100, and is calculated as follows: the BLAST score is multiplied by the percent nucleotide identity and the product is divided by (5 times the.length of the shorter of the two sequences). The BLAST score is calculated by assigning a score of +5 for every base that matches in a high-scoring segment pair (HSP), and -4 for every mismatch. Two sequences may share more than one HSP
(separated by gaps). If there is more than one HSP, then the pair with the highest BLAST
score is used to calculate the product score. The product score represents a balance between fractional overlap and quality in a BLAST alignment. For example, a product score of 100 is produced only for 100%
identity over the entire length of the shorter of the two sequences being compared. A product score of 70 is produced either by 100% identity and 70% overlap at one end, or by 88% identity and 100% overlap at the other. A product score of 50 is produced either by 100% identity and 50%
overlap at one end, or 79%
identity and 100% overlap.
Alternatively, polynucleotides encoding NAAP are analyzed with respect to the tissue sources from which they were derived. For example, some full length sequences are assembled, at least in part, with overlapping Incyte cDNA sequences (see Example III). Each cDNA
sequence is derived from a cDNA library constructed from a human tissue. Each human tissue is classified into one of the following organ/tissue categories: cardiovascular system; connective tissue;
digestive system;
embryonic structures; endocrine system; exocrine glands; genitalia, female;
genitalia, male; germ cells;
heroic and immune system; liver; musculoskeletal system; nervous system;
pancreas; respiratory system; sense organs; skin; stomatognathic system; unclassified/mixed; or urinary tract. The number of libraries in each category is counted and divided by the total number of libraries across all categories. Similarly, each human tissue is classified into one of the following diseaselcondition categories: cancer, cell line, developmental, inflammation, neurological, trauma, cardiovascular, pooled, and other, and the number of libraries in each category is counted and divided by the total number of libraries across all categories. The resulting percentages reflect the tissue-and disease-specific expression of cDNA encoding NAAP.

VIII. Extension of NAAP Encoding Polynucleotides Full length polynucleotides are produced by extension of an appropriate fragment of the full length molecule using oligonucleotide primers designed from this fragment. One primer was synthesized to initiate 5' extension of the known fragment, and the other primer was synthesized to initiate 3' extension of the known fragment. The initial primers were designed using OLIGO 4.06 software (National Biosciences), or another appropriate program, to be about 22 to 30 nucleotides in length, to have a GC content of about 50% or more, and to anneal to the target sequence at temperatures of about 68°C to about 72°C. Any stretch of nucleotides which would result in hairpin structures and primer-primer 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 Mga+, (NIi.~)ZS04, and 2-mercaptoethanol, Taq DNA polymerase (Amersham Biosciences), ELONGASE
enzyme (Invitrogen), and Pfu DNA polymerase (Stratagene), with the following parameters for primer pair PCI A and PCIB: Step 1: 94°C, 3 min; Step 2: 94°C, 15 sec; Step 3: 60°C, 1 min; Step 4: 68°C, 2 min; Step 5: Steps 2, 3, and 4 repeated 20 times; Step 6: 68°C, 5 min;
Step 7: storage at 4°C. In the alternative, the parameters for primer pair T7 and SK+ were as follows: Step 1: 94°C, 3 min; Step 2:
94°C, 15 sec; Step 3: 57°C, 1 min; Step 4: 68°C, 2 min;
Step 5: Steps 2, 3, and 4 repeated 20 times;
Step 6: 68°C, 5 min; Step 7: storage at 4°C.
The concentration of DNA in each well was determined by dispensing 100 ~.1 PICOGREEN
quantitation reagent (0.25% (v/v) PICOGREEN; Molecular Probes, Eugene OR) dissolved in 1X TE
and 0.5 p.1 of undiluted PCR product into each well of an opaque fluorimeter plate (Corning Costar, Acton MA), allowing the DNA to bind to the reagent. The plate was scanned in a Fluoroskan lI
(Labsystems Oy, Helsinki, Finland) to measure the fluorescence of the sample and to quantify the concentration of DNA. A 5 ~1 to 10 ,u1 aliquot of the reaction mixture was analyzed by electrophoresis on a 1 % agarose gel to determine which reactions were successful in extending the sequence.
The extended nucleotides were desalted and concentrated, transferred to 384-well plates, digested with CviJI cholera virus endonuclease (Molecular Biology Research, Madison WI), and sonicated ar sheared prior to religation into pUC 18 vector (Amersham Biosciences). For shotgun sequencing, the digested nucleotides were separated on low concentration (0.6 to 0.8%) agarose gels, fragments were excised, and agar digested with Agar ACE (Promega). Extended clones were religated using T4 ligase (New England Biolabs, Beverly MA) into pUC 18 vector (Amersham Biosciences), treated with Pfu DNA polymerise (Stratagene) to fill-in restriction site overhangs, and transfected into competent E. coli cells. Transformed cells were selected on antibiotic-containing media, and individual colonies were picked and cultured overnight at 37 °C in 384-well plates in LB/2x carb liquid media.
The cells were lysed, and DNA was amplified by PCR using Taq DNA polymerise (Amersham Biosciences) 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%
dimethysulfoxide (1:2, vlv), and sequenced using DYENAMIC energy trausfer sequencing primers and the DYENAMIC DIRECT kit (Amersham Biosciences) or the ABI PRISM BIGDYE
Terminator cycle sequencing ready reaction kit (Applied Biosystems).
In like manner, full length polynucleotides are verified using the above procedure or are used to obtain 5'regulatory sequences using the above procedure along with oligonucleotides designed for such extension, and an appropriate genomic library.
IX. Identification of Single Nucleotide Polymorphisms in NAAP Encoding 2o Polynucleotides Common DNA sequence variants known as single nucleotide polymorphisms (SNPs) were identified in SEQ 117 NO:37-72 using the LIFESEQ database (Incyte Genomics).
Sequences from the same gene were clustered together and assembled as described in Example DI, allowing the identification of all sequence variants in the gene. An algorithm consisting of a series of filters was used to distinguish SNPs from other sequence variants. Preliminary filters removed the majority of basecall errors by requiring a inimum Phred quality score of 15, and removed sequence alignment errors and errors resulting from improper trim ing of vector sequences, chimeras, and splice variants.
An automated procedure of advanced chromosome analysis analysed the original chromatogram files in the vicinity of the putative SNP. Clone error filters used statistically generated algorithms to identify errors introduced during laboratory processing, such as those caused by reverse transcriptase, polymerise, or somatic mutation. Clustering error filters used statistically generated algorithms to identify errors resulting from clustering of close homologs or pseudogenes, or due to contamination by non-human sequences. A final set of filters removed duplicates and SNPs found in immunoglobulins or T-cell receptors.
Certain SNPs were selected for further characterization by mass spectrometry using the high throughput MASSARRAY system (Sequenom, Inc.) to analyze allele frequencies at the SNP sites in four different human populations. The Caucasian population comprised 92 individuals (46 male, 46 female), including 83 from Utah, four French, three Venezualan, and two Amish individuals. The African population comprised 194 individuals (97 male, 97 female), all African Americans. The Hispanic population comprised 324 individuals (162 male, 162 female), all Mexican Hispanic. The Asian population comprised 126 individuals (64 male, 62 female) with a reported parental breakdown of 43 % Chinese, 31 % Japanese, 13 % Korean, 5% Vietnamese, anal 8% other Asian. Allele frequencies were first analyzed in the Caucasian population; in some cases those SNPs which showed no allelic variance in this population were not further tested in the other three populations.
X. Labeling and Use of Individual Hybridization Probes Hybridization probes derived from SEQ ID N0:37-72 are employed to screen cDNAs, genomic DNAs, or rnRNAs. 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 32P~ adenosine triphosphate (Amersham Biosciences), and T4 polynucleotide kinase (DuPont NEN, Boston MA). The labeled oligonucleotides are substantially purified using a superfine size exclusion dextran bead column (Amersham Biosciences). An aliquot containing 10' counts per minute of the labeled probe is used in a typical membrane-based hybridization analysis of human genomic DNA digested with one of the following endonucleases: Ase I, Bgl 1I, Eco RI, Pst I, Xba I, or Pvu lI (DuPont NEN).
The DNA from each digest is fractionated on a 0.7% agarose gel and transferred to nylon membranes (Nytran Plus, Schleicher & Schuell, Durham NH). Hybridization is carried out for 16 hours at 40 °C. To remove nonspecific signals, blots are sequentially washed at room temperature under conditions of up to, for example, 0.1 x saline sodium citrate and 0.5%
sodium dodecyl sulfate.
Hybridization patterns are visualized using autoradiography or an alternative imaging means and compared.
3o XI. Microarrays The linkage or synthesis of array elements upon a microarray can be achieved utilizing photolithography, piezoelectric printing (ink jet printing; see, e.g., Baldeschweiler, supra), mechanical microspotting technologies, and derivatives thereof. The substrate in each of the aforementioned technologies should be uniform and solid with a non-porous surface (Schena (1999), supt-a).
Suggested substrates include silicon, silica, glass slides, glass chips, and silicon wafers. Alternatively, a procedure analogous to a dot or slot blot may also be used to arrange and link elements to the surface of a substrate using thermal, UV, chemical, or mechanical bonding procedures.
A typical array may be produced using available methods and machines well known to those of ordinary skill in the art and may contain any appropriate number of elements (Schena, M. et al. (1995) Science 270:467-470;
Shalon, D. et al. (1996) Genome Res. 6:639-645; Marshall, A. and J. Hodgson (1998) Nat. Biotechnol.
16:27-31).
Full length cDNAs, Expressed Sequence Tags (ESTs), or fragments or oligomers thereof may comprise the elements of the microarray. Fragments or oligomers suitable for hybridization canbe selected using software well known in the art such as LASERGENE software (DNASTAR). The array elements are hybridized with polynucleotides in a biological sample. The polynucleotides in the biological sample are conjugated to a fluorescent label or other molecular tag for ease of detection.
After hybridization, nonhybridized nucleotides from the biological sample are removed, and a fluorescence scanner is used to detect hybridization at each array element.
Alternatively, laser desorbtion and mass spectrometry may be used for detection of hybridization.
The degree of complementarity and the relative abundance of each polynucleotide which hybridizes to an element on the microarray may be assessed. In one embodiment, microarray preparation and usage is described in detail below.
2o Tissue or Cell Sample Preparation Total RNA is isolated from tissue samples using the guanidinium thiocyanate method and poly(A)+ RNA is purified using the oligo-(dT) cellulose method. Each poly(A)+
RNA sample is reverse transcribed using MMLV reverse-transcriptase, 0.05 pgl~,l oligo-(dT) primer (2lmer), 1X first strand buffer, 0.03 units/~,l RNase inhibitor, 500 ~.M dATP, 500 p.M dGTP, 500 ~.M dTTP, 40 ~.M
dCTP, 40 ACM dCTP-Cy3 (BDS) or dCTP-Cy5 (Amersham Biosciences). The reverse transcription reaction is performed in a 25 ml volume containing 200 ng poly(A)+ RNA with GEMBRIGHT kits (Incyte). Specific control poly(A)+ RNAs are synthesized by ih vitf-o transcription from non-coding yeast genomic DNA. After incubation at 37° C for 2 hr, each reaction sample (one with Cy3 and another with Cy5 labeling) is treated with 2.5 ml of 0.5M sodium hydroxide and incubated for 20 minutes at 85° C to the stop the reaction and degrade the RNA. Samples are purified using two successive CHROMA SPIN 30 gel filtration spin columns (CLONTECH Laboratories, Inc.
(CLONTECH), Palo Alto CA) and after combining, both reaction samples are ethanol precipitated using 1 ml of glycogen (1 mg/ml), 60 ml sodium acetate, and 300 ml of 100%
ethanol. The sample is then dried to completion using a SpeedVAC (Savant Instruments Inc., Holbrook NY) and resuspended in 14 ~,l 5X SSC/0.2% SDS.
Microarray Preparation Sequences of the present invention are used to generate array elements. Each array element is amplified from bacterial cells containing vectors with cloned cDNA inserts.
PCR amplification uses primers complementary to the vector sequences flanking the cDNA insert. Array elements are amplified in tlvrty cycles of PCR from an initial quantity of 1-2 ng to a final quantity greater than 5 ~,g.
Amplified array elements are then purified using SEPHACRYL-400 (Amersham Biosciences).
Purified array elements are immobilized on polymer-coated glass slides. Glass microscope slides (Corning) are cleaned by ultrasound in 0.1% SDS and acetone, with extensive distilled water washes between and after treatments. Glass slides are etched in 4%
hydrofluoric acid (VWR
Scientific Products Corporation (VWR), West Chester PA), washed extensively in distilled water, and coated with 0.05% aminopropyl silane (Sigma) in 95% ethanol. Coated slides are cured in a 110°C
oven.
Array elements are applied to the coated glass substrate using a procedure described in U.S.
Patent No. 5,807,522, incorporated herein by reference. 1 ~,1 of the array element DNA, at an average concentration of 100 ng/~l, is loaded into the open capillary printing element by a high-speed robotic apparatus. The apparatus then deposits about 5 n1 of array element sample per slide.
Microarrays are UV-crosslinked using a STRATALINKER UV-crosslinker (Stratagene).
Microarrays are washed at room temperature once in 0.2% SDS and three times in distilled water.
Non-specific binding sites are blocked by incubation of microarrays in 0.2%
casein in phosphate buffered saline (PBS) (Tropix, Inc., Bedford MA) for 30 minutes at 60°
C followed by washes in 0.2%
SDS and distilled water as before.
Hybridization Hybridization reactions contain 9 ~,l of sample mixture consisting of 0.2 p,g each of Cy3 and Cy5 labeled cDNA synthesis products in 5X SSC, 0.2% SDS hybridization buffer.
The sample mixture is heated to 65° C for 5 minutes and is aliquoted onto the microarray surface and covered with an 1.8 cm2 coverslip. The arrays are transferred to a waterproof chamber having a cavity just slightly larger than a microscope slide. The chamber is kept at 100% humidity internally by the addition of 140 p.1 of 5X SSC in a corner of the chamber. The chamber containing the arrays is incubated for about 6.5 hours at 60° C. The arrays are washed for 10 min at 45° C in a first wash buffer (1X SSC, 0.1%
SDS), three times for 10 minutes each at 45° C in a second wash buffer (0.1X SSC), and dried.
Detection Reporter-labeled hybridization complexes are detected with a microscope equipped with an Innova 70 mixed gas 10 W laser (Coherent, Inc., Santa Clara CA) capable of generating spectral lines at 488 nm for excitation of Cy3 and at 632 nm for excitation of CyS. The excitation laser light is focused on the array using a 20X microscope objective (Nikon, Inc., Melville NY). The slide containing the array is placed on a computer-controlled X-Y stage on the microscope and raster-scanned past the objective. The 1.8 cm x 1.8 cm array used in the present example is scanned with a resolution of 20 micrometers.
In two separate scans, a mixed gas multiline laser excites the two fluorophores sequentially.
Emitted light is split, based on wavelength, into two photomultiplier tube detectors (PMT 81477, Hamamatsu Photonics Systems, Bridgewater NJ) corresponding to the two fluorophores. Appropriate filters positioned between the array and the photomultiplier tubes are used to filter the signals. The emission maxima of the fluorophores used are 565 nm for Cy3 and 650 nm for CyS. Each array is typically scanned twice, one scan per fluorophore using the appropriate filters at the laser source, although the apparatus is capable of recording the spectra from both fluorophores simultaneously.
The sensitivity of the scans is typically calibrated using the signal intensity generated by a cDNA control species added to the sample mixture at a known concentration. A
specific location on the array contains a complementary DNA sequence, allowing the intensity of the signal at that location to be correlated with a weight ratio of hybridizing species of 1:100,000. When two samples from different sources (e.g" representing test and control cells), each labeled with a different fluorophore, are hybridized to a single array for the purpose of identifying genes that are differentially expressed, the calibration is done by labeling samples of the calibrating cDNA with the two fluorophores anal adding identical amounts of each to the hybridization mixture.
The output of the photomultiplier tube is digitized using a 12-bit RTI-835H
analog-to-digital (A/D) conversion board (Analog Devices, Iuc., Norwood MA) installed in an IBM-compatible PC
computer. The digitized data are displayed as an image where the signal intensity is mapped using a linear 20-color transformation to ~ pseudocolor scale ranging from blue (low signal) to red (lugh signal). The data is also analyzed quantitatively. Where two different fluorophores are excited and measured simultaneously, the data are first corrected for optical crosstalk (due to overlapping emission spectra) between the fluorophores using each fluorophore's emission spectrum.
A grid is superimposed over the fluorescence signal image such that the signal from each spot is centered in each element of the grid. The fluorescence signal within each element is then integrated to obtain a numerical value corresponding to the average intensity of the signal. The software used for signal analysis is the GEMTOOLS gene expression analysis program (Incyte).
Array elements that exhibited at least about a two-fold change in expression, a signal-to-background ratio of at least 2.5, and an element spot size of at least 40% were identified as differentially expressed using the GEMTOOLS program (Incyte Genomics).
Expression In one example, SEQ >D N0:41 showed differential expression in several human breast cancer cell lines, as determined by microarray analysis. H1VIEC is a human primary mammary epithelial cell strain derived from normal mammary tissue (Clonetics, San Diego, CA). The following cell lines were tested on microarrays: MCF-10A is a human breast mammary gland cell line isolated from a 36-year-old female with fibrocystic breast disease; SkBR3 is a breast adenocarcinoma cell line isolated from a malignant pleural effusion of a 43-year-old female; MCF7 is a breast adenocarcinoma cell line derived from the pleural effusion of a 69-year-old female; T47D is a breast carcinoma cell line derived from a pleural effusion from a 54-year-old female with an infiltrating ductal carcinoma of the breast; BT20 is a breast carcinoma cell line derived in vitf-o from cells emigrating out of thin slices of a tumor mass isolated from a 74-year-old female; MDA-mb-231 is a metastatic breast tumor cell line derived from the pleural effusion of a 51-year-old female with metastatic breast carcinoma. All cell cultures were propagated in media according to the supplier's recommendations and grown to 70-80% confluence prior~to RNA isolation.
'The expression of cDNAs from the five tumor cell lines representing various stages of breast tumor progression (BT20, MCF7, MDA-mb-231, SKBr3, and T47D) were compared with that of the 2o non-malignant mammary epithelial cell lines, IMC or MCF-10A.
SEQ ll~ N0:41 showed at least two-fold differential expression when comparing H1V>EC cells versus Sk-BR-3 and T-47D cells. Additionally, SEQ )D N0:41 expression was decreased at least two-fold when comparing breast cells from fibrocystic breast tissue versus BT-20, MCF-7, MDA-mb-231, Sk-BR-3, and T-47D cancerous cell lines. These experiments indicate that SEQ )D NO:41 was significantly under-expressed in the breast tumor cell lines tested, further establishing the utility of SEQ
>D N0:41 as a diagnostic marker or as a potential therapeutic target for breast cancer.
In another example, SEQ >D NO:44 is upregulated 3.9 fold in DC as compared to monocytes, suggesting that SEQ )D N0:44, encoding SEQ )D NO:B, could be used for example, to understand the process by which monocytes differentiate into immature dendritic cells and eventually allow manipulation of the immune system leading to potential i_m_m__unotherapies for diseases such as cancer, A)175, and infectious diseases; and enhancing vaccine efficacy.
In. another example, the expression of SEQ JD N0:48 is upregulated in six out of seven PBMC populations (each of which was obtained from a different donor) treated with Staphylococcal exotoxins (SEB). The PBMCs were stimulated irv vitro with SEB for 24 and 72 hours. The expression of SEQ ID N0:48 was higher after 24 hours and dropped after 72 hours. Therefore, SEQ
ID N0:48 is useful in diagnostic assays for immune responses.
In yet another example, SEQ 117 NO:64 showed differential expression in the C3A cell line, a well-established ih vitf~o model of the mature human liver (Mickelson, J.K. et al. (1995) Hepatology 22:866-875; Nagendra, A.R. et al. (1997) Am. J. Physiol. 272:6408-6416), as determined by microarray analysis. The effects upon liver metabolism and hormone clearance mechanisms are important to understand the pharmacodynamics of a drug. For example, the human C3A cell line is a clonal derivative of HepG2/C3 (hepatoma cell line, isolated from a 15-year-old male with liver tumor), which was selected for strong contact inhibition of growth. The use of a clonal population enhances the reproducibility of the cells. C3A cells have many characteristics of primary human hepatocytes in culture: i) expression of insulin receptor and insulin-like growth factor II
receptor; ii) secretion of a high ratio of serum albumin compared with a-fetoprotein; iii) conversion of ammonia to urea and glutamine; iv) abilitiy to metabolize aromatic amino acids; and v) proliferation in glucose-free and insulin-free medium. SEQ ll~ NO:64 showed differential expression in C3A cells treated with a variety of steroids including beclomethasone, medroxyprogesterone, budesonide, prednisone, dexamethasone, and progesterone, versus untreated C3A cells, as determined by microarray analysis.
Therefore, SEQ II? N0:64 is useful for the diagnosis and monitoring of liver, endocrine, and reproductive diseases and in the diagnosis of and as a therapeutic target for inflammatory diseases and 2o humoral immune response.
XII. Complementary Polynucleotides Sequences complementary to the NAAP-encoding sequences, or any parts thereof, are used to detect, decrease, or inhibit expression of naturally occurring NAAP.
Although use of oligonucleotides comprising from about 15 to 30 base pairs is described, essentially the same procedure is used with smaller or with larger sequence fragments. Appropriate oligonucleotides are designed using OLIGO 4.06 software (National Biosciences) and the coding sequence of NAAP. To inhibit transcription, a complementary oligonucleotide is designed from the most unique 5' sequence and used to prevent promoter binding to the coding sequence. To inhibit translation, a complementary oligonucleotide is designed to prevent ribosomal binding to the NAAP-encoding transcript.
3o XIII. Expression of NAAP
Expression and purification of NAAP is achieved using bacterial or virus-based expression systems. For expression of NAAP in bacteria, cDNA is subcloned into an appropriate vector containing an antibiotic resistance gene and an inducible promoter that directs high levels of cDNA

transcription. Examples of such promoters include, but are not limited to, the trp-lac (tac) hybrid promoter and the 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 NAAP upon induction with isopropyl beta-D-thiogalactopyranoside (IPTG). Expression of NAAP in eukaryotic cells is achieved by infecting insect or mammalian cell lines with recombinant Autogr-aphica californica nuclear polyhedrosis virus (AcMNPV), commonly known as baculovirus. The nonessential polyhedrin gene of baculovirus is replaced with cDNA encoding NAAP by either homologous recombination or bacterial-mediated transposition involving transfer plasmid intermediates. Viral infectivity is maintained and the strong 1o polyhedrin promoter drives high levels of cDNA transcription. Recombinant baculovirus is used to infect Spodoptef~a frugiperda (Sf9) insect cells in most cases, or human hepatocytes, in some cases.
Infection of the latter requires additional genetic modifications to baculovirus (See Engelhard, E.K. et al. (1994) Proc. Natl. Acad. Sci. USA 91:3224-3227; Sandig, V. et al. (1996) Hum. Gene Ther.

7:1937-1945.) In most expression systems, NAAP is synthesized as a fusion.protein with, e.g., glutatluone S-transferase (GST) or a peptide epitope tag, such as FLAG or 6-His, permitting rapid, single-step, aff'mity-based purification of recombinant fusion protein from crude cell lysates. GST, a 26-kilodalton enzyme from Sclxistosoma japonicum, enables the purification of fusion proteins on immobilized glutathione under conditions that maintain protein activity and antigenicity (Amersham Biosciences).
Following purification, the GST moiety can be proteolytically cleaved from NAAP at specifically engineered sites. FLAG, an 8-amino acid peptide, enables immunoaffinity purification using commercially available monoclonal and polyclonal anti-FLAG antibodies (Eastman Kodak). 6-His, a stretch of six consecutive histidine residues, enables purification on metal-chelate resins (QIAGEN).
Methods for protein expression and purification are discussed in Ausubel (1995, supf-a, ch. 10 and 16).
Purified NAAP obtained by these methods can be used directly in the assays shown in Examples XV1I, XV)ZI, and X1X, where applicable.
XIV. Functional Assays NAAP function is assessed by expressing the sequences encoding NAAP at physiologically elevated levels in mammalian cell culture systems. cDNA is subcloned into a mammalian expression vector containing a strong promoter that drives lugh levels of cDNA
expression. Vectors of choice include PCMV SPORT plasmid (Invitrogen, Carlsbad CA) and PCR3.1 plasmid (Invitrogen), both of which contain the cytomegalovirus promoter. S-10 ,ug of recombinant vector are transiently transfected into a human cell line, for example, an endothelial or hematopoietic cell line, using either liposome formulations or electroporation. 1-2 /.tg 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 Cytometr~, Oxford, New York NY.
The influence of NAAP on gene expression can be assessed using highly purified populations of cells transfeeted with sequences encoding NAAP 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 2o 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 NAAP and other genes of interest can be analyzed by northern analysis or microarray techniques.
XV. Production of NAAP Specific Antibodies NAAP substantially purified using polyacrylamide gel electrophoresis (PAGE;
see, e.g., Harrington, M.G. (1990) Methods Enzymol. 182:488-495), or other purification techniques, is used to immunize animals (e.g., rabbits, mice, etc.) and to produce antibodies using standard protocols.
Alternatively, the NAAP amino acid sequence is analyzed using LASERGENE
software (DNASTAR) to determine regions of high immunogenicity, and a corresponding oligopeptide is synthesized and used to raise antibodies by means known to those of skill in the art. Methods for selection of appropriate epitopes, such as those near the C-terminus or in hydrophilic regions are well described in the art (Ausubel, 1995, supra, ch. 11).
Typically, oligopeptides of about 15 residues in length are synthesized using an ABI 431A ~~

peptide synthesizer (Applied Biosystems) using FMOC chemistry and coupled to KLH (Sigma-Aldrich, St. Louis MO) by reaction with N-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS) to increase immunogenicity (Ausubel, 1995, supra). Rabbits are immunized with the oligopeptide-KLH
complex in complete Freund's adjuvant. Resulting antisera are tested for antipeptide and anti-NAAP
activity by, for example, binding the peptide or NAAP to a substrate, blocking with 1 % BSA, reacting with rabbit antisera, washing, and reacting with radio-iodinated goat anti-rabbit IgG.
XVI. Purification of Naturally Occurring NAAP Using Specific Antibodies Naturally occurring or recombinant NAAP is substantially purified by immunoafhnity chromatography using antibodies specific for NAAP. An immunoaffinity column is constructed by covalently coupling anti-NAAP antibody to an activated chromatographic resin, such as CNBr-activated SEPHAROSE (Amersham Biosciences). After the coupling, the resin is blocked and washed according to the manufacturer's instructions.
Media containing NAAP are passed over the immunoaffinity column, and the column is washed under conditions that allow the preferential absorbance of NAAP (e.g., high ionic strength buffers in the presence of detergent). The column is eluted under conditions that disrupt antibody/NAAP 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 NAAP is collected.
XVII. Identification of Molecules Which Interact with NAAP
NAAP, or biologically active fragments thereof, are labeled with lasl Bolton-Hunter reagent (Bolton, A.E. and W.M. Hunter (1973) Biochem. J. 133:529-539). Candidate molecules previously arrayed in the wells of a multi-well plate are incubated with the labeled NAAP, washed, and any wells with labeled NAAP complex are assayed. Data obtained using different concentrations of NAAP are used to calculate values for the number, affinity, and association of NAAP
with the candidate molecules.
Alternatively, molecules interacting with NAAP are analyzed using the yeast two hybrid system as described in Fields, S. and O. Song (1989) Nature 340:245-246, or using commercially available kits based on the two-hybrid system, such as the MATCHMAKER system (Clontech).
NAAP may also be used in the PATHCALLING process (CuraGen Corp., New Haven CT) which employs the yeast two-hybrid system in a high-throughput manner to determine all interactions between the proteins encoded by two large libraries of genes (Nandabalan, K.
et al. (2000) U.S.
Patent No. 6,057,101).
XVIII. Demonstration of NAAP Activity NAAP activity is measured by its ability to stimulate transcription of a reporter gene (Liu, H.Y, et al. (1997) EMBO J. 16:5289-5298). The assay entails the use of a well characterized reporter gene construct, LexA°p-LacZ, that consists of LexA DNA
transcriptional control elements (LexA°p) fused to sequences encoding the E. calf LacZ enzyme. The methods for constructing and expressing fusion genes, introducing them into cells, and measuring LacZ
enzyme activity, are well known to those skilled in the art. Sequences encoding NAAP are cloned into a plasmid that directs the synthesis of a fusion protein, LexA-NAAP, consisting of NAAP and a DNA
binding domain derived from the LexA transcription factor. The resulting plasmid, encoding a LexA-NAAP fusion protein, is introduced into yeast cells along with a plasmid containing the LexA°p LacZ reporter gene.
The amount of LacZ enzyme activity associated with LexA-NAAP transfected cells, relative to control cells, is proportional to the amount of transcription stimulated by the NAAP.
Alternatively, NAAP activity is measured by its ability to bind zinc. A 5-10 ~.M sample solution in 2.5 mM ammonium acetate solution at pH 7.4 is combined with 0.05 M
zinc sulfate solutio (Aldrich, Milwaukee WI) in the presence of 100 ~M dithiothreitol with 10%
methanol added. The sample and zinc sulfate solutions are allowed to incubate for 20 minutes. The reaction solution is passed through a VYDAC column (Grace Vydac, Hesperia, CA) with approximately 300 Angstrom bore size and 5 ~.M particle size to isolate zinc-sample complex from the solution, and into.a mass spectrometer (PE Sciex, Ontario, Canada). Zinc bound to sample is quantified using the functional atomic mass of 63.5 Da observed by Whittal et al. (2000; Biochemistry 39:8406-8417).
In the alternative, a method to determine nucleic acid binding activity of NAAP involves a polyacrylaxnide gel mobility-shift assay. In preparation for this assay, NAAP
is expressed by transforming a mammalian cell line such as COS7, HeLa or CHO with a eukaryotic expression vector containing NAAP cDNA. The cells are incubated for 48-72 hours after transformation under conditions appropriate for the cell line to allow expression and accumulation of NAAP. Extracts containing solubilized proteins can be prepared from cells expressing NAAP by methods well known in the art. Portions of the extract containing NAAP are added to [32P]-labeled RNA or DNA.
Radioactive nucleic acid can be synthesized in vitro by techniques well known in the art. The mixtures are incubated at 25°C in the presence of RNase- and DNase-inhibitors under buffered conditions for S-10 minutes. After incubation, the samples are analyzed by polyacrylamide gel electrophoresis followed by autoradiography. The presence of a band on the autoradiogram indicates the formation of a complex between NAAP and the radioactive transcript. A band of similar mobility will not be present in samples prepared using control extracts prepared from untransfoxmed cells.
In the alternative, a method to determine methylase activity of NAAP measures transfer of radiolabeled methyl groups between a donor substrate and an acceptor substrate. Reaction mixtures (50 ~.l final volume) contain 15 mM HEPES, pH 7.9,1.5 mM MgClz, 10 mM
dithiothreitol, 3 %
polyvinylalcohol, 1.5 p.Ci [methyl-3H]AdoMet (0.375 p,M AdoMet) (DuPont-NEN), 0.6 ~,g NAAP, and acceptor substrate (e.g., 0.4 ,ug [35S]RNA, or 6-mercaptopurine (6-MP) to 1 mM
final concentration).
Reaction mixtures are incubated at 30°C for 30 minutes, then 65°C for 5 minutes.
Analysis of [methyl-3H]RNA is as follows: (1) 50 ~tl of 2 x loading buffer (20 mM Tris-HCl, pH 7.6, 1 M LiCl, 1 mM EDTA, 1% sodium dodecyl sulphate (SDS)) and 50 p1 oligo d(T)-cellulose (10 mg/ml in 1 x loading buffer) are added to the reaction mixture, and incubated at ambient temperature with shaking for 30 minutes. (2) Reaction mixtures are transferred to a 96-well filtration plate attached to a vacuum apparatus. (3) Each sample is washed sequentially with three 2.4 ml aliquots of 1 x oligo d(T) loading buffer containing 0.5% SDS, 0.1% SDS, or no SDS. (4) RNA is eluted with 300 p1 of water into a 96-well collection plate, transferred to scintillation vials containing liquid scintillant, and radioactivity determined.
Analysis of [methyl-3H]6-MP is as follows: (1) 500 ~.l 0.5 M borate buffer, pH
10.0, and then 2.5 ml of 20% (v/v) isoamyl alcohol in toluene are added to the reaction mixtures. (2) The samples are mixed by vigorous vortexing for ten seconds. (3) After centrifugation at 700g for 10 minutes, 1.5 ml of the organic phase is transferred to scintillation vials containing 0.5 ml absolute ethanol and liquid scintillant, and radioactivity determined. (4) Results are corrected for the extraction of 6-MP into the organic phase (approximately 41%). For both [methyl-3H]RNA and [methyl-3H]6-MP, NAAP
activity is proportional to the measured radioactivity.
Alternatively, DNA repair activity of DNAME is measured as incorporation of [32P]dATP
into a plasmid treated with a DNA damaging agent, such as cisplatin or ultraviolet irradiation, relative to a control, untreated plasmid DNA (Coudore, F. et al. (1997) FEBS Lett.
414:581-584). Cell extracts are purified from mammalian cell lines, E. coli, or S.
cef°evisiae having compromised endogenous repair activities due to mutations in repair enzymes. Cell extracts are prepared by hypotonic lysis of cells followed by centrifugation at 300,000 x g. Extracts are treated with 63 %
ammonium sulfate to minimsze non-specific nuclease activity. The repair synthesis assay is performed in a 50 ~Cl reaction volume containing 200 ~,g protein in cell extract, 300 ng damaged plasmid, 300 ng control plasmid, 4 p,M dATP, 20 ACM each dCTP, dTTP, and dGTP, 0.2 ~tM
[32P]dATP, 20 mM
HEPES-KOH (pH 7.8), 2.5 ~Cg creatine phosphokinase, 7 mM MgClz, and 2 mM EGTA.
Identical reactions are set up with and without purified DNAME. After a 3 h incubation at 30°C, reaction mixtures are treated with 200 p,g/ml proteinase K and 0.5% SDS. Plasmid DNA is purified from reaction mixtures by phenol-chloroform extraction and ethanol precipitation.
Data is quantified by gel electrophoresis of linearized plasmid followed by autoradiography, scintillation counting of excised DNA bands, and densitometry of the photographic negative of the gel to normalize for plasmid DNA
recovery.
In the alternative, type I topoisomerase activity of NAAP can be assayed based on the relaxation of a supercoiled DNA substrate. NAAP is incubated with its substrate in a buffer lacking Mg2+ and ATP, the reaction is terminated, and the products are loaded on an agarose gel. Altered topoisomers can be distinguished from supercoiled substrate electrophoretically. This assay is specific for type I topoisomerase activity because Mg2+ and ATP are necessary cofactors for type II
topoisomerases.
Type II topoisomerase activity of NAAP can be assayed based on the decatenation of a kinetoplast DNA (KDNA) substrate. NAAP is incubated with KDNA, the reaction is terminated, and the products are loaded on an agarose gel. Monomeric circular KDNA can be distinguished from catenated KDNA electrophoretically. Kits for measuring type I and type II
topoisomerase activities are available commercially from Topogen (Columbus OH).
ATP-dependent RNA helicase unwinding activity of NAAP can be measured by the method described by Zhang and Grosse (1994; Biochemistry 33:3906-3912). The substrate for RNA
unwinding consists of 32P-labeled RNA composed of two RNA strands of 194 and 130 nucleotides in -length containing a duplex region of 17 base-pairs. The RNA substrate is incubated together with ATP, Mg2+, and varying amounts of NAAP in a Tris-HCl buffer, pH 7.5, at 37°C for 30 minutes. The single-stranded RNA product is then separated from the double-stranded RNA
substrate by electrophoresis through a 10% SDS-polyacrylamide gel, and quantitated by autoradiography. The amount of single-stranded RNA recovered is proportional to the amount of NAAP
in the preparation.
In the alternative, NAAP function is assessed by expressing the sequences encoding NAAP
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
expxession. Vectors of choice include pCMV SPORT (Life Technologies) and pCR3.1 (Invitrogen Corporation, Carlsbad 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 NAAP on gene expression can be assessed using highly purified populations of cells transfected with sequences encoding NAAP 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, Inc., 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 NAAP and other genes of interest can be analyzed by northern analysis or microarray techniques.
Pseudouridine synthase activity of NAAP is assayed using a tritium (3H) release assay modified from Nurse et al. (1995; RNA 1:102-112), which measures the release of 3H from the CS
position of the pyrimidine component of uridylate (U) when 3H-radiolabeled U
in RNA is isomerized to pseudouridine (yr). A typical 500 ~cl assay mixture contains 50 mM HEPES
buffer (pH 7.5), 100 mM
ammonium acetate, 5 mM dithiothreitol, 1 mM EDTA, 30 units RNase inhibitor, and 0.1-4.2 ~.M
[5 3H]tRNA (approximately 1 ,uCi/nmol tRNA). The reaction is initiated by the addition of <5 ~.1 of a concentrated solution of NAAP (or sample containing NAAP) and incubated for 5 min at 37 °C.
Portions of the reaction mixture are removed at various times (up to 30 min) following the addition of NAAP and quenched by dilution into 1 ml 0.1 M HCl containing Norit-SA3 (12%
wlv). The quenched reaction mixtures are centrifuged for 5 min at maximum speed in a microcentrifuge, and the supernatants are filtered through a plug of glass wool. The pellet is washed twice by resuspension in 1 ml 0.1 M HCl, followed by centrifugation. The supernatants from the washes are separately passed through the glass wool plug and combined with the original filtrate. A portion of the combined filtrate is mixed with scintillation fluid (up to 10 ml) and counted using a scintillation counter. The amount of 3H released from the RNA and present in the soluble ftltrate is proportional to the amount of peudouridine synthase activity in the sample (Ramamurthy, V. (1999) J. Biol.
Chem.
274:22225-22230).
In the alternative, pseudouridine synthase activity of NAAP is assayed at 30 °C to 37 °C in a mixture containing 100 mM Tris-HCl (pH 8.0), 100 mM ammonium acetate, 5 mM
MgClz, 2 mM
dithiothreitol, 0.1 mM EDTA, and 1-2 fmol of [3aP]-radiolabeled runoff transcripts (generated in vitro by an appropriate RNA polymerase, i.e., T7 or SP6) as substrates. NAAP is added to initiate the reaction or omitted from the reaction in control samples. Following incubation, the RNA is extracted with phenol-chloroform, precipitated in ethanol, and hydrolyzed completely to 3-nucleotide monophosphates using RNase T2. The hydrolysates are analyzed by two-dimensional thin layer chromatography, and the amount of 32P radiolabel present in the AMP and UMP
spots are evaluated after exposing the thin layer chromatography plates to film or a Phosphorlmager screen. Taking into account the relative numbex of uridylate residues in the substrate RNA, the relative amount yrMP and UMP are determined and used to calculate the relative amount of yr per tRNA
molecule (expressed in mol y /mol of tRNA or mol t~ /mol of tRNA/minute), which corresponds to the amount of pseudouridine synthase activity in the NAAP sample (Lecointe, supra).
NZ,NZ-dimethylguanosine transferase ((m22G)methyltransferase) activity of NAAP
is measured in a 160 ~Cl reaction mixture containing 100 mM Tris-HCl (pH 7.5)', 0:1 mM EDTA, 10 mM
MgCl2, 20 mM NH4Cl, 1mM dithiothreitol, 6.2 ~,M S-adenosyl-L-[methyl-3H]methioi~ine (30-70 Ci/mM), 8 ~.g m22G-deficient tRNA or wild type tRNA from yeast, and approximately 100 dug of purified NAAP or a sample comprising NAAP. The reactions are incubated at 30 °C for 90 min and chilled on ice. A portion of each reaction is diluted to 1 ml in water containing 100 ~.g BSA. 1 ml of 2 M HCl is added to each sample and the acid insoluble products are allowed to precipitate on ice for 20 min before being collected by filtration through glass fiber filters. The collected material is washed several times with HCl and quantitated using a liquid scintillation counter.
The amount of 3H
incorporated into the m~2G-deficient, acid-insoluble tRNAs is proportional to the amount of N2,Nz-dimethylguanosine transferase activity in the NAAP sample. Reactions comprising no substrate tRNAs, or wild-type tRNAs that have akeady been modified, serve as control reactions which should not yield acid-insoluble 3H-labeled products.
Polyadenylation activity of NAAP is measured using an iti vitro polyadenylation reaction.
The reaction mixture is assembled on ice and comprises 10 ~.1 of 5 mM
dithiothreitol, 0.025% (v/v) NGNmET P-40, SO mM creative phosphate, 6.5°70 (w/v) polyvinyl alcohol, 0.5 unit/~,1 RNAGUARD
(Pharmacia), 0.025 ~,g/~.1 creative kinase, 1.25 mM cordycepin 5'-triphosphate, and 3.75 mM MgCl2, in a total volume of 25 p1. 60 fmol of CstF, 50 fmol of CPSF, 240 fmol of PAP, 4 p1 of crude or partially purified CF IT and various amounts of amounts CF I are then added to the reaction mix. The volume is adjusted to 23.5 ~.l with a buffer containing 50 mM TrisHCl, pH 7.9, 10%
(v/v) glycerol, and 0.1 mN
Na-EDTA. The final ammonium sulfate concentration should be below 20 mM. The reaction is initiated (on ice) by the addition of 15 fmol of 32P-labeled pre-mRNA
template, along with 2.5 pg of unlabeled tRNA, in 1.5 ~tl of water. Reactions are then incubated at 30 °C for 75-90 min and stopped by the addition of 75 ~,l (approximately two-volumes) of proteinase K mix (0.2 M Tris-HCl, pH 7.9, 300 mM NaCl, 25 mM Na-EDTA, 2% (w/v) SDS), 1 ,u1 of 10 mg/xnl proteinase K, 0.25 ~.l of 20 mg/m glycogen, and 23.75 ~.1 of water). Following incubation, the RNA is precipitated with ethanol and analyzed on a 6% (w/v) polyacrylamide, 8.3 M urea sequencing gel. The dried gel is developed by autoradiography or using a phosphoimager. Cleavage activity is determined by comparing the amount of cleavage product to the amount of pre-mRNA template. The omission of any of the polypeptide components of the reaction and substitution of NAAP is useful for identifying the specific biological function of NAAP in pre-mRNA polyadenylation (Riiegsegger, supra; and references within).
tRNA synthetase activity is measured as the aminoacylation of a substrate tRNA
in the presence of [14C]-labeled amino acid. NAAP is incubated with jl4C]-labeled amino acid and the appropriate cognate tRNA (for example, [24C]alanine and tRNAaia) in a buffered solution. 14C-labeled product is separated from free [14C]amino acid by chromatography, and the incorporated 14C
is quantified by scintillation counter. The amount of 14C-labeled product detected is proportional to the activity of NAAP in this assay.
In the alternative, NAAP activity is measured by incubating a sample containing NAAP in a solution containing 1 mM ATP, 5 mM Hepes-KOH (pH 7.0), 2.5 mM KCl, 1.5 mM
magnesium chloride, and 0.5 mM DTT along with misacylated [14C]-Glu-tRNAGln (e.g., 1 ~,M) and a similar concentration of unlabeled L-glutamine. Following the quenching of the reaction with 3 M sodium acetate (pH 5.0), the mixture is extracted with an equal volume of water-saturated phenol, and the aqueous and organic phases are separated by centrifugation at 15,000 x g at room temperature for 1 min. The aqueous phase is removed and precipitated with 3 volumes of ethanol at -70°C for 15 min.
The precipitated aminoacyl-tRNAs are recovered by centrifugation at 15,000 x g at 4°C forl5 min.
The pellet is resuspended in of 25 mM KOH, deacylated at 65°C for 10 min., neutralized with 0.1 M
HCl (to final pH 6-7), and dried under vacuum. The dried pellet is resuspended iu water and spotted onto a cellulose TLC plate. The plate is developed in either isopropanol/formic acid/water or am_m__onia/water/chloroform/ methanol. The image is subjected to densitometric analysis and the relative amounts of Glu and Gln are calculated based on the Rf values and relative intensities of the spots. NAAP activity is calculated based on the amount of Gln resulting from the transformation of Glu while acylated as Glu-tRNAG~' (adapted from Curnow, A.W. et al. (1997) Proc. Natl. Acad. Sci.
USA 94:11819-26).
XIX. Identification of NAAP Agonists and Antagonists Agonists or antagonists of NAAP activation or inhibition may be tested using the assays described in section XVIB. Agonists cause an increase in NAAP activity and antagonists cause a decrease in NAAP activity.
Various modifications and variations of the described compositions, methods, and systems of the invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. It will be appreciated that the invention provides novel and useful proteins, and their encoding polynucleotides, which can be used in the drug discovery process, as well as methods for using these compositions for the detection, diagnosis, and treatment of diseases and conditions.
Although the invention has been described in connection with certain embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments.
Nor should the description of such embodiments be considered exhaustive or limit the invention to the precise forms disclosed. Furthermore, elements from one embodiment can be readily recombined with elements from one or more other embodiments. Such combinations can form a number of embodiments within the scope of the invention. It is intended that the scope of the invention be defined by the following claims and their equivalents.

Table 1 Incyte PolypeptideIncyte PolynucleotideIncyte CA2 Reagents Project SEQ ID PolypeptideSEQ ID Polynucleotide ID NO: ID NO: ID

2383223 3 2383223CD139 2383223CB1 90088564CA2, 7477891 5 7477891CD141 ' 7477891CB1 7487977 17 7487977CD53 7487977CB 90175683CA2, 1553836 27 1553836CD163 1553836CB1 90092916CA2, 90092932CA2, 90097748CA2, ~.
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<110> INCYTE GENOMICS, INC.
GANDHI, Ameena R.
SWARNAKER, Anita HAFALIA, April J.A.
WARREN, Bridget A.
EMERLING, Brooke M.
ARVIZU, Chandra S.
ISON, Craig H.
HONCHELL, Cynthia D.
LEE, Ernestine A.
YUE, Henry FORSYTHE, zan J.
RAMKUMAR, Jayala~an.i GRIFFIN, Jennifer A.
YANG, Junming SANJANWALA, Madhu M.
BAUGHN, Mariah R.
BOROWSKY, Mark L.
YAO, Monique G.
WALIA, Narinder K.
BANDMAN, Olga LAL, PREETI G.
BECHA, Shanya D.
LEE, Soo Yeun RICHARDSON, Thomas W.
ELLIOTT, Vicki S.
LUO, Wen TANG, Y. Tom ZEBARJADIAN, Yeganeh LU, Yan <120> Nucleic Acid-Associated Proteins <130> PF-1031 PCT
<140> To Be Assigned <141> Herewith <150> US 60/300,518 <151> 2001-06-22 <150> US 60/301,787 <151> 2001-06-29 <150> US 60/301,792 <151> 2001-06-29 <150> US 60/301,892 <151> 2001-06-29 <150> US 60/301,893 <151> 2001-06-29 <150> US 60/303,405 <151> 2001-07-06 <150> US 60/303,442 <151> 2001-07-06 <150> US 60/364,438 <151> 2002-03-12 <160> 72 <150> US 60/303,442 <151> 2001-07-06 <150> US 60/364,438 <151> 2002-03-12 <160> 72 <170> PERL Program <210> 1 <211> 304 <212> PRT
<213> Homo sapiens <220>
<221> misc_feature <223> Incyte ID No: 7490148CD1 <400> 1 Met Ser Arg Ser Phe Tyr Val Asp Ser Leu Ile Ile Lys Asp Thr 1 5 10 ~ 15 Ser Arg Pro Ala Pro Ser Leu Pro Glu Pro His Pro Gly Pro Asp Phe Phe Ile Pro Leu Gly Met Pro Pro Pro Leu Val Met Ser Val Ser Gly Pro Gly Cys Pro Ser Arg Lys Ser Gly Ala Phe Cys Val Cys Pro Leu Cys Val Thr Ser His Leu His Ser Ser Arg Gly Ser Val Gly Pro Ala Ser G,ly Gly Ala Gly Pro Gly Phe Pro Gly Pro Gly Asp Ser Gly ~Val Ala Gly Pro Ala Gly Ala Leu Pro Leu Leu Lys Gly Gln Phe Ser Ser Ala Pro Gly Asp Ala Gln Phe Cys Pro Arg Val Asn His Ala His His His His His Pro Pro Gln His His His His His His Gln Pro Gln Gln Pro Gly Ser Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Leu Gly His Pro Gln His His Ala Pro Val Cys Thr Ala Thr Thr Tyr Asn Val Ala Asp Pro Arg Arg Phe His Cys Leu Thr Met Gly G1y Ser Asp Ala Ser Gln Val Pro Asn Gly Lys Arg Met Arg Thr Ala Phe Thr Ser Thr Gln Leu Leu Glu Leu Glu Arg Glu Phe Ser Ser Asn Met Tyr Leu Ser Arg Leu Arg Arg Ile Glu Ile Ala Thr Tyr Leu Asn Leu Ser Glu Lys Gln Val Lys Ile Trp Phe Gln Asn Arg Arg Val Lys His Lys Lys Glu Gly Lys Gly Thr Gln Arg Asn Ser His Ala Gly Cys Lys Cys Val Gly Ser Gln Val His Tyr Ala Arg Ser Glu Asp Glu Asp Ser Leu.Ser Pro Ala Ser Ala Asn Asp Asp Lys Glu Ile Ser Pro Leu <210> 2 <211> 198 <212> PRT
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte ID No: 7490301CD1 <400> 2 Met Glu Thr Gly Arg Gln Ala Gly Val Ser Ala Glu Met Phe Ala Met Pro Arg Asp Leu Lys Gly Ser Asn Lys Asp Gly Ile Pro Glu Asp Leu Asp Gly Asn Leu Glu Glu Pro Arg Asp Gln Glu Gly Glu Leu Arg Ser Glu Asp Val Met Asp Leu Thr Glu Gly Asp Asn Glu Ala Ser Ala Ser Ala Pro Pro Ala Ala Lys Arg Arg Lys Thr Asp Thr Lys Gly Lys Lys Glu Arg Lys Pro Thr Val Asp Ala Glu Glu Ala Gln Arg Met Thr Thr Leu Leu Ser Ala Met Ser Glu Glu Gln Leu Ser Arg Tyr Glu Val Cys Arg Arg Ser A1a Phe Pro Lys Ala Cys Ile Ala Gly Leu Met Arg Ser Ile Thr Gly Arg Ser Val Ser Glu Asn Val Ala Ile Ala Met Ala Gly Ile A1a Lys Val Phe Val Gly Glu Val Val Glu Glu Ala Leu Asp Val Cys Glu Met Trp Gly Glu Met Pro Pro Leu Gln Pro Lys His Leu Arg Glu Ala Val Arg Arg Leu Lys Pro Lys Gly Leu Phe Pro Asn Ser Asn Tyr Lys Lys Ile Met Phe <210> 3 <211> 576 <212> PRT
<213> Homo Sapiens <220>
<221> misc_feature <223> Tncyte ID No: 2383223CD1 <400> 3 Met Asp Ser Val Ala Phe Glu Asp Val Ser Val Ser Phe Ser Gln Glu Glu Trp Ala Leu Leu Ala Pro Ser Gln Lys Lys Leu Tyr Arg Asp Val Met Gln Glu Thr Phe Lys Asn Leu Ala Ser Ile Gly Glu Lys Trp Glu Asp Pro Asn Val Glu Asp Gln His Lys Asn Gln Gly Arg Asn Leu Arg Ser His Thr Gly Glu Arg Leu Cys Glu Gly Lys Glu Gly Ser Gln Cys Ala Glu Asn Phe Ser Pro Asn Leu Ser Val Thr Lys Lys Thr Ala Gly Val Lys Pro Tyr Glu Cys Thr Ile Cys Gly Lys Ala Phe Met Arg Leu Ser Ser Leu Thr Arg His Met Arg Ser His Thr Gly Tyr Glu Leu Phe Glu Lys Pro Tyr Lys Cys Lys Glu Cys Glu Lys Ala Phe Ser Tyr Leu Lys Ser Phe Gln Arg His Glu Arg Ser His Thr Gly Glu Lys Pro Tyr Lys Cys Lys Gln Cys Gly Lys Thr Phe Ile Tyr His Gln Pro Phe Gln Arg His Glu Arg Thr His Ile Gly Glu Lys Pro Tyr Glu Cys Lys Gln Cys Gly Lys Ala Leu Ser Cys Ser Ser Ser Leu Arg Val His Glu Arg Ile His Thr Gly Glu Lys Pro Tyr Glu Cys Lys Gln Cys Gly Lys Ala Phe Ser Cys Ser Ser Ser Ile Arg Val His Glu Arg Thr His Thr Gly Glu Lys Pro Tyr Ala Cys Lys Glu Cys Gly Lys Ala Phe Ile Ser His Thr Ser Val Leu Thr His Met Ile Thr His Asn Gly Asp Arg Pro Tyr Lys Cys Lys Glu Cys Gly Lys Ala Phe Ile Phe Pro Ser Phe Leu Arg Val His Glu Arg Ile His Thr Gly Glu Lys Pro Tyr Lys Cys Lys Gln Cys Gly Lys Ala Phe Arg Cys Ser Thr Ser Ile Gln Ile His Glu Arg Ile His Thr Gly Glu Lys Pro Tyr Lys Cys Lys Glu Cys Gly Lys Ser Phe Ser Ala Arg Pro Ala Phe Arg Val His Val Arg Val His Thr Gly Glu Lys Pro Tyr Lys Cys Lys Glu Cys Gly Lys Ala Phe Ser Arg Ile Ser Tyr Phe Arg Ile His Glu Arg Thr His Thr Gly Glu Lys Pro Tyr Glu Cys Lys Lys Cys Gly Lys Thr Phe Asn Tyr Pro Leu Asp Leu Lys Ile His Lys Arg Asn His Thr Gly Glu Lys Pro Tyr Glu Cys Lys Glu Cys Ala Lys Thr Phe Ile Ser Leu Glu Asn Phe Arg Arg His Met Ile Thr His Thr Gly Asp Gly Pro Tyr Lys Cys Arg Asp Cys Gly Lys Val Phe Ile Phe Pro Ser Ala Leu Arg Thr His Glu Arg Thr His Thr Gly Glu Lys Pro Tyr Glu Cys Lys Gln Cys Gly Lys Ala Phe Ser Cys Ser Ser Tyr Ile Arg Ile His Lys Arg Thr His Thr Gly Glu Lys Pro Tyr Glu Cys Lys Glu Cys Gly Lys Ala Phe Ile Tyr Pro Thr Ser Phe Gln Gly His Met Arg' Met His Thr Gly Glu Lys Pro Tyr Lys Cys Lys Glu Cys Gly Lys Ala Phe Ser Leu His Ser Ser Phe Gln Arg His Thr Arg Ile His Asn Tyr Glu Lys Pro Leu Glu Cys Lys Gln Cys Gly Lys Ala Phe Ser Val Ser Thr Ser Leu Lys Lys His Met Arg Met His Asn Arg <210> 4 <211> 426 <212> PRT
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte ID No: 3495982CD1 <400> 4 Met Arg Arg Asn Ser Ser Leu Ser Phe Gln Met Glu Arg Pro Leu Glu Glu Gln Val Gln Ser Lys Trp Ser Ser Ser Gln Gly Arg Thr Gly Thr Gly Gly Ser Asp Val Leu Gln Met Gln Asn Ser Glu His His Gly Gln Ser Ile Lys Thr Gln Thr Asp Ser Ile Ser Leu Glu Asp Val Ala Val Asn Phe Thr Leu Glu Glu Trp Ala Leu Leu Asp Pro Gly Gln Arg Asn Ile Tyr Arg Asp Val Met Arg Ala'Thr Phe Lys Asn Leu Ala Cys Ile Gly Glu Lys Trp Lys Asp Gln Asp Ile Glu Asp Glu His Lys Asn Gln Gly Arg Asn Leu Arg Ser Pro Met Val Glu Ala Leu Cys Glu Asn Lys Glu Asp Cys Pro Cys Gly Lys Ser Thr Ser Gln Ile Pro Asp Leu Asn Thr Asn Leu Glu Thr Pro Thr Gly Leu Lys Pro Cys Asp Cys Ser Val Cys Gly Glu Val Phe Met His Gln Val Ser Leu Asn Arg His Met Arg Ser His Thr Glu Gln Lys Pro Asn Glu Cys His Glu Tyr Gly Glu Lys Pro His Lys Cys Lys Glu Cys Gly Lys Thr Phe Thr Arg Ser Ser Ser Ile Arg Thr His Glu Arg Ile His Thr Gly Glu Lys Pro Tyr Glu Cys Lys Glu Cys Gly Lys Ala Phe Ala Phe Leu Phe Ser Phe Arg Asn His Ile Arg Ile His Thr Gly Glu Thr Pro Tyr Glu Cys Lys Glu Cys Gly Lys Ala Phe Arg Tyr Leu Thr Ala Leu Arg Arg His Glu Lys Asn His Thr Gly Glu Lys Pro Tyr Lys Cys Lys Gln Cys Gly Lys Ala Phe Ile Tyr Tyr Gln Pro Phe Leu Thr His Glu Arg Thr His Thr Gly Glu Lys Pro Tyr Glu Cys Lys Gln Cys Gly Lys Ala Phe Ser Cys Pro Thr Tyr Leu Arg Ser His Glu Lys Thr His Thr Gly Glu Lys Pro Phe Val Cys Arg Glu Cys Gly Arg Ala Phe Phe Ser His Ser Ser Leu Arg Lys His Val Ser His His Thr Arg Pro Pro Val Leu Phe Phe Phe Phe Glu Thr Glu Ser Leu Pro Arg Leu Glu Cys Ser Gly Ala Ile Ser Ala Tyr Cys Lys Leu Arg Leu Leu Gly Ser Arg His Ser Pro Ala Ser A1a Ser Arg Val Ala Gly Thr Thr Gly Ala Arg His His Ala Arg Leu Ile Phe Cys Ile Phe Ser Gly Asp Gly Val Ser Pro Cys <210> 5 <211> 786 <212> PRT
<213> Homo Sapiens <220>
<221> misc feature <223> Incyte ID No: 7477891CD1 <400> 5 Met Ala Asn Asn Tyr Lys Lys Ile Val Leu Leu Lys Gly Leu Glu Val Ile Asn Asp Tyr His Phe Arg Ile Val Lys Ser Leu Leu Ser Asn Asp Leu Lys Leu Asn Pro Lys Met Lys Glu Glu Tyr Asp Lys Ile Gln Ile Ala Asp Leu Met Glu Glu Lys Phe Pro Gly Asp Ala Gly Leu Gly Lys Leu Ile Glu Phe Phe Lys Glu Ile Pro Thr Leu Gly Asp Leu Ala Glu Thr Leu Lys Arg Glu Lys Leu Lys Val Lys Gly Ile Ile Pro Ser Lys Lys Thr Lys Gln Lys Glu Val Tyr Pro Ala Thr Pro Ala Cys Thr Pro Ser Asn Arg Leu Thr Ala Lys Gly Ala Glu Glu Thr Leu Gly Pro Gln Lys Arg Lys Lys Pro Ser Glu Glu Glu Thr Gly Thr Lys Arg Ser Lys Met Ser Lys Glu Gln Thr Arg Pro Ser Cys Ser Ala Gly Ala Ser Thr Ser Thr Ala Met Gly Arg Ser Pro Pro Pro Gln Thr Ser Ser Ser Ala Pro Pro Asn Thr Ser Ser Thr Glu Ser Leu Lys Pro Leu Ala Asn Arg His Ala Thr Ala Ser Lys Asn Ile Phe Arg Glu Asp Pro Ile Ile Ala Met Val Leu Asn Ala Thr Lys Val Phe Lys Tyr Glu Ser Ser Glu Asn Glu Gln Arg Arg Met Phe His Ala Thr Val Ala Thr Gln Thr Gln Phe Phe His Val Lys Val Leu Asn Ile Asn Leu Lys Arg Lys Phe Ile Lys Lys Arg Ile Ile Ile Ile Ser Asn Tyr Ser Lys Arg Asn Ser Leu Leu Glu Val Asn Glu Ala Ser Ser Val Ser Glu Ala Gly Pro Asp Gln Thr Phe Glu Val Pro Lys Asp Ile Ile Arg Arg Ala Lys Lys Ile Pro Lys Ile Asn Ile Leu His Lys Gln Thr Ser Gly Tyr Ile Val Tyr Gly Leu Phe Met Leu His Thr Lys Ile Val Asn Arg Lys Thr Thr Ile Tyr Glu Ile Gln Asp Lys Thr Gly Ser Met Ala Val Val Gly Lys Gly Glu Cys His Asn Ile Pro Cys Glu Lys Gly Asp Lys Leu Arg Leu Phe Cys Phe Arg Leu Arg Lys Arg Glu Asn Met Ser Lys Leu Met Ser Glu Met His Ser Phe Ile Gln Ile Gln Lys Asn Thr Asn Gln Arg Ser His Asp Ser Arg Ser Met Ala Leu Pro Gln Glu Gln Ser Gln His Pro Lys Pro Ser Glu Ala Ser Thr Thr Leu Pro Glu Ser His Leu Lys Thr Pro Gln Met Pro Pro Thr Thr Pro Ser Ser Ser Phe Phe Thr Lys Lys Ser Glu Asp Thr Ile Ser Lys Met Asn Asp Phe Met Arg Met Gln Ile Leu Lys Glu Gly Ser His Phe Pro Gly Pro Phe Met Thr Ser Ile Gly Pro Ala Glu Ser His Pro His Thr Pro Gln Met Pro Pro Ser Thr Pro Ser Ser Ser Phe Leu Thr Thr Lys Ser Glu Asp Thr Tle Ser Lys Met Asn Asp Phe Met Arg Met Gln Ile Leu Lys Glu Gly Ser His Phe Pro Gly Pro Phe Met Thr Ser Tle Gly Pro Ala Glu Ser His Pro His Thr Pro Gln Met Pro Pro Ser Thr Pro Ser Ser Ser Phe Leu Thr Thr Leu Lys Pro Arg Leu Lys Thr Glu Pro Glu Glu Val Ser Ile Glu Asp Ser Ala Gln Ser Asp Leu Lys Glu Val Met Val Leu Asn Ala Thr Glu Ser Phe Val Tyr Glu Pro Lys Glu Gln Lys Lys Met Phe His Ala Thr Val Ala Thr Glu Asn Glu Val Phe Arg Val Lys Val Phe Asn Ile Asp Leu Lys Glu Lys Phe Thr Pro Lys Lys Ile Ile Ala Ile Ala Asn Tyr Val Cys Arg Asn Gly Phe Leu Glu Val Tyr Pro Phe Thr Leu Val Ala Asp Val Asn Ala Asp Arg Asn Met Glu Ile Pro Lys Gly Leu Ile Arg Ser Ala Ser Val Thr Pro Lys Ile Asn Gln Leu Cys Ser Gln Thr Lys Gly Ser Phe Val Asn Gly Val Phe Glu Val His Lys Lys Asn Val Arg Gly Glu Phe Thr Tyr Tyr Glu Ile Gln Asp Asn Thr Gly Lys Met Glu Val Val Val His Gly Arg Leu Thr Thr Ile Asn Cys Glu Glu Gly Asp Lys Leu Lys Leu Thr Cys Phe Glu Leu Ala Pro Lys Ser Gly Asn Thr Gly Glu Leu Arg Ser Val Ile His Ser His Ile Lys Val Ile Lys Thr Arg Lys Asn Lys Lys Asp Ile Leu Asn Pro Asp Ser Ser Met Glu Thr Ser Pro Asp Phe Phe Phe <210> 6 <211> 617 <212> PRT
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte ID No: 72688352CD1 <400> 6 Met Ile Lys Ser Gln Glu Ser Leu Thr Leu Glu Asp Val Ala Val Glu Phe Thr Trp Glu Glu Trp Gln Leu Leu Gly Pro Ala Gln Lys Asp Leu Tyr Arg Asp Val Met Leu Glu Asn Tyr Ser Asn Leu Val Ser Val Gly Tyr Gln Ala Ser Lys Pro Asp Ala Leu Phe Lys Leu Glu Gln Gly Glu Pro Trp Thr Val Glu Asn Glu Ile His Ser Gln Ile Cys Pro Glu Ile Lys Lys Val Asp Asn His Leu Gln Met His Ser Gln Lys Gln Arg Cys Leu Lys Arg Val Glu Gln Cys His Lys His Asn Ala Phe Gly Asn Ile Ile His Gln Arg Lys Ser Asp Phe 110 115 . 120 Pro Leu Arg Gln Asn His Asp Thr Phe Asp Leu His Gly Lys Ile Leu Lys Ser Asn Leu Ser Leu Val Asn Gln Asn Lys Arg Tyr Glu Ile Lys Asn Ser Val Gly Val Asn Gly Asp Gly Lys Ser Phe Leu His Ala Lys His Glu Gln Phe His Asn Glu Met Asn Phe Pro Glu Gly Gly Asn Ser Val Asn Thr Asn Ser Gln~Phe Ile Lys His Gln Arg Thr Gln Asn Ile Asp Lys Pro His Val Cys Thr Glu Cys Gly Lys Ala Phe Leu Lys Lys Ser Arg Leu Ile Tyr His Gln Arg Val His Thr Gly Glu Lys Pro His Gly Cys Ser Ile Cys Gly Lys Ala Phe Ser Arg Lys Ser Gly Leu Thr Glu His Gln Arg Asn His Thr Gly Glu Lys Pro Tyr Glu Cys Thr Glu Cys Asp Lys Ala Phe Arg Trp Lys Ser Gln Leu Asn Ala His Gln Lys Ile His Thr Gly Glu Lys Ser Tyr Ile Cys Ser Asp Cys Gly Lys Gly Phe Ile Lys Lys Ser Arg Leu Ile Asn His Gln Arg Val His Thr Gly Glu Lys Pro His Gly Cys Ser Leu Cys Gly Lys Ala Phe Ser Lys Arg Ser Arg Leu Thr Glu His Gln Arg Thr His Thr Gly Glu Lys Pro Tyr Glu 335 340 ~ 345 Cys Thr Glu Cys Asp Lys Ala Phe Arg Trp Lys Ser Gln Leu Asn Ala His Gln Lys Ala His Thr Gly Glu Lys Ser Tyr Ile Cys Arg Asp Cys Gly Lys Gly Phe Ile Gln Lys Gly Asn Leu Ile Val His 380 385 ° 390 Gln Arg Ile His Thr Gly Glu Lys Pro Tyr Ile Cys Asn Glu Cys Gly Lys Gly Phe Ile Gln Lys Gly Asn Leu Leu Ile His Arg Arg Thr His Thr Gly Glu Lys Pro Tyr Val Cys Asn Glu Cys Gly Lys Gly Phe Ser Gln Lys Thr Cys Leu Ile Ser His Gln Arg Phe His Thr Gly Lys Thr Pro Phe Val Cys Thr Glu Cys Gly Lys Ser Cys Ser His Lys Ser Gly Leu Ile Asn His Gln Arg Ile His Thr Gly Glu Lys Pro Tyr Thr Cys Ser Asp Cys Gly Lys Ala Phe Arg Asp Lys Ser Cys Leu Asn Arg His Arg Arg Thr His Thr Gly Glu Arg Pro Tyr Gly Cys Ser Asp Cys Gly Lys Ala Phe Ser His Leu Ser Cys Leu Val Tyr His Lys Gly Met Leu His Ala Arg Glu Lys Cys Val Gly Ser Val Lys Leu Glu Asn Pro Cys Ser Glu Ser His Ser Leu Ser His Thr Arg Asp Leu Ile Gln Asp Lys Asp Ser Val Asn Met Val Thr Leu Gln Met Pro Ser Val Ala Ala Gln Thr Ser Leu Thr Asn Ser Ala Phe Gln Ala Glu Ser Lys Val Ala Ile Val Ser Gln Pro Val Ala Arg Ser Ser Val Ser Ala Asp Ser Arg Ile Cys &05 610 615 Thr Glu <210> 7 <211> 249 <212> PRT
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte ID No: 7490652CD1 <400> 7 Met Ala Val Gly Lys Asn Lys His Leu Met Lys Gly Gly Lys Lys Gly Ala Glu Asn Arg Val Val Asp Pro Phe Ser Lys Lys Asp Trp Cys Asp Val Lys Ala Leu Ala Met Phe Asn Ile Arg Asn Ile Gly Glu Thr Leu Val Thr Arg Thr Arg Gly Thr Lys Ile Ala Ser Asp Ser Leu Lys Arg Arg Val Phe Glu Val Ser Leu Ala Asp Leu Gln Asn Asp Glu Val Ala Phe Arg Lys Phe Lys Leu Ile Ala Glu Asp Val Gln Lys Lys Thr Asn Phe Gln Gly Met Asp Leu Pro Asp Glu Met Cys Ser Val Val Lys Lys Trp Gln Thr Met Ile Glu Pro His Ile Asp Val Lys Thr Thr Asp Gly Tyr Leu Phe His Leu Leu Cys Asp Phe Thr Lys Lys His Asn Leu Ile Gln Lys Ala Ser Tyr Ala Gln His Gln Gln Val Cys Glu Ile Gln Lys Lys Met Met Glu Ile Met Thr Lys Gly Ala Asn Asp Leu Lys Glu Val Val Asn Lys Leu Ile Pro Gly Ser Thr Gly Lys Glu Lys Leu Cys Leu Ser Ile Tyr Leu Leu His Asp Val Phe Val Arg Lys Val Lys Met Leu Lys Met Pro Lys Phe Asp Leu Gly Lys Phe Met Gly Asn Cys Ser Gly Lys Ala Thr Gly Asp Glu Thr Gly Ala Lys Val Glu Leu Ala Asp Gly Tyr Glu Ala Leu Val Gln Glu Ser Val <210> 8 <211> 384 <212> PRT
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte ID No: 7489744CD1 <400> 8 Met Glu Val Ile Val Glu Asn Leu His Leu Pro Thr Ser Pro Ile Pro Pro Val Ala Gly Ala Glu Ser Gly Pro Gln Arg Ala Leu Ser Ser Pro Thr Ala Ala Ala Gly Leu Val Thr Ile Thr Pro Arg Glu Glu Pro Gln Leu Pro Gln Pro Ala Pro Val Thr Ile Thr Ala Thr Met Ser Ser Glu Ala Glu Thr Gln Gln Pro Pro Ala Ala Pro Pro Ala Ala Pro Ala Leu Ser Ala Ala Asp Thr Lys Pro Gly Thr Thr Gly Ser Gly Ala Gly Ser Gly Gly Pro Gly Gly Leu Thr Ser Ala Ala Pro Ala Gly Gly Asp Lys Lys Val Ile Ala Thr Lys Val Leu Gly Thr Val Lys Trp Phe Asn Val Arg Asn Gly Tyr Gly Phe Ile Asn Arg Asn Asp Thr Lys G1u Asp Val Phe Val His Gln Thr Ala Ile Lys Lys Asn Asn Pro Arg Lys Tyr Leu Arg Ser Val Gly Asp Gly Glu Thr Val Glu Phe Asp Val Val Glu Gly Glu Lys Gly Ala Glu Ala Ala Asn Val Thr Gly Pro Gly Gly Val Pro Val Gln Gly Ser Lys Tyr Ala Ala Asp Arg Asn His Tyr Arg Arg Tyr Pro Arg Arg Arg Gly Pro Pro Arg Asn Tyr Gln Gln Asn Tyr Gln Asn Ser Glu Ser Gly Glu Lys Asn Glu Gly Ser Glu Ser Ala Pro Glu Gly Gln Ala Gln Gln Arg Arg Pro Tyr Arg Arg Arg Arg Phe Pro Pro Tyr Tyr Met Arg Arg Pro Tyr Gly Arg Arg Pro Gln Tyr Ser Asn Pro Pro Val Gln Gly Glu Val Met Glu Gly Ala Asp Asn Gln Gly Ala Gly Glu Gln Gly Arg Pro Val Arg Gln Asn Met Tyr Arg Gly Tyr Arg Pro Arg Phe Arg Arg Gly Pro Pro Arg Gln Arg Gln Pro Arg Glu Asp Gly Asn Glu Glu Asp Lys Glu Asn Gln Gly Asp Glu Thr Gln Gly Gln Gln Pro Pro Gln Arg Arg Tyr Arg Arg Asn Phe Asn Tyr Arg Arg Arg Arg Pro Glu Asn Pro Lys Pro Gln Asp Gly Gln Glu Thr Lys Ala Ala Asp Pro Pro A1a Glu Asn Ser Ser Ala Pro Glu Ala Glu Gln Gly Gly Ala Glu <210> 9 <211> 312 <212> PRT
<213> Homo sapiens <220>
<221> misc feature <223> Incyte ID No: 3363382CD1 <400> 9 Met Ala Asp Gly Asp Ser Gly Ser Glu Arg Gly Gly Gly Gly Gly Pro Cys Gly Phe Gln Pro Ala Ser Arg Gly Gly Gly Glu Gln Glu Thr Gln Glu Leu Ala Ser Lys Arg Leu Asp Ile Gln Asn Lys Arg Phe Tyr Leu Asp Val Lys Gln Asn Ala Lys Gly Arg Phe Leu Lys Ile Ala Glu Val Gly Ala Gly Gly Ser Lys Ser Arg Leu Thr Leu Ser Met Ala Val Ala Ala Glu Phe Arg Asp Ser Leu Gly Asp Phe Ile Glu His Tyr Ala Gln Leu Gly Pro Ser Ser Pro Glu Gln Leu Ala Ala Gly Ala Glu Glu Gly Gly Gly Pro Arg Arg Ala Leu Lys Ser Glu Phe Leu Val Arg Glu Asn Arg Lys Tyr Tyr Leu Asp Leu Lys Glu Asn Gln Arg Gly Arg Phe Leu Arg Ile Arg Gln Thr Val Asn Arg Gly Gly Gly Gly Phe Gly Ala Gly Pro Gly Pro Gly Gly Leu Gln Ser Gly Gln Thr Ile Ala Leu Pro Ala Gln Gly Leu Ile Glu Phe Arg Asp Ala Leu Ala Lys Leu Ile Asp Asp Tyr Gly Gly Glu Asp Asp Glu Leu Ala Gly Gly Pro Gly Gly Gly Ala Gly Gly Pro Gly Gly Gly Leu Tyr Gly Glu Leu Pro Glu Gly Thr Ser Ile Thr Val Asp Ser Lys Arg Phe Phe Phe Asp Val Gly Cys Asn Lys Tyr Gly Val Phe Leu Arg Val Ser Glu Val Lys Pro Ser Tyr Arg ' 245 250 255 Asn Ala Ile Thr Val Pro Phe Lys Ala Trp Gly Lys Phe Gly Gly Ala Phe Cys Arg Tyr Ala Asp Glu Met Lys Glu Ile Gln Glu Arg Gln Arg Asp Lys Leu Tyr Glu Arg Arg Gly Gly Gly Ser Gly Gly Gly Glu Glu Ser Glu Gly Glu Glu Val Asp Glu Asp <210> 10 <211> 441 <212> PRT
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte ID No: 7491148CD1 <400> 10 Met Lys Asp His Asp Ala Ile Lys Leu Phe Val Gly Gln Ile Pro Arg Gly Leu Asp Glu Gln Asp Leu Lys Pro Leu Phe Glu Glu Phe Gly Arg Ile Tyr Glu Leu Thr Val Leu Lys Asp Arg Leu Thr~Gly Leu His Lys Gly Cys Ala Phe~Leu Thr Tyr Cys Ala Arg Asp Ser Ala Leu Lys Ala Gln Ser Ala Leu His Glu Gln Lys Thr Leu Pro Gly Phe His Tle Leu Asn Asn Asn Asn Asn Asn Lys Asn Arg Pro Glu Asp Arg Lys Leu Phe Val Gly Met Leu Gly Lys Gln Gln Gly Glu Glu Asp Val Arg Arg Leu Phe Gln Pro Phe Gly His Ile Glu Glu Cys Thr Val Leu Arg Ser Pro Asp Gly Thr Ser Lys Gly Cys Ala Phe Val Lys Phe Gly Ser Gln Gly Glu Ala Gln Ala Ala Ile Arg Gly Leu His Gly Ser Arg Thr Met Ala Gly Ala Ser Ser Ser Leu Val Val Lys Leu Ala Asp Thr Asp Arg Glu Arg Ala Leu Arg Arg Met Gln Gln Met Ala Gly His Leu Gly Ala Phe His Pro Ala Pro Leu Pro Leu Gly Ala Cys Gly Ala Tyr Thr Thr Ala Ile Leu Gln His Gln Ala Ala Leu Leu Ala Ala Ala Gln Gly Pro Gly Leu G1y Pro Val Ala Ala Val Ala Ala Gln Met Gln His Val Ala Ala Phe Ser Leu Val Ala Ala Pro Leu Leu Pro Ala Ala Ala Ala Asn Ser Pro Pro Gly Ser Gly Pro Gly Thr Leu Pro Gly Leu Pro Ala Pro Ile Gly~Val Asn Gly Val Arg Pro Ser Asp Thr Pro Arg Ser Asn Gly Gln Pro Gly Ser Asp Thr Leu Tyr Asn Asn Gly Leu Ser Pro Tyr Pro Ala Gln Ser Pro Gly Val Ala Asp Pro Leu Gln Gln Ala Tyr Ala Gly Met His His Tyr Ala Ala Ala Tyr Pro Ser Ala Tyr Ala Pro Val Ser Thr Ala Phe Pro Gln Gln Pro Ser Ala Leu Pro Gln Gln Gln Arg Glu Gly Pro Glu Gly Cys Asn Leu Phe Ile Tyr His Leu Pro Gln Glu Phe Gly Asp Ala Glu Leu Ile Gln Thr Phe Leu Pro Phe Gly Ala Val Val Ser Ala Lys Val Phe Val Asp Arg Ala Thr Asn Gln Ser Lys Cys Phe Gly Phe Val Ser Phe Asp Asn Pro Thr Ser Ala Gln Thr Ala Ile Gln Ala Met Asn Gly Phe Gln Ile Gly Met Lys Arg Leu Lys Val Gln Leu Lys Arg Pro Lys Asp Ala Asn Arg Pro Tyr <210> 11 <211> 493 <212> PRT
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte ID No: 8126343CD1 <400> 11 Met Ala Thr Asp Leu Pro Ile Met Ala Arg Gly Pro Ala Arg Ser Ala Ala Pro Ala Gly Gly Ser Ser Ser Gly Cys Gly Ala Arg Gln Gly Arg Ala Gly Gly Gly Val Leu Ala Met Ala Gly Leu Ser Asp Leu Glu Leu Arg Arg Glu Leu Gln Ala Leu Gly Phe Gln Pro Gly Pro Ile Thr Asp Thr Thr Arg Asp Val Tyr Arg Asn Lys Leu Arg Arg Leu Arg Gly Glu Ala Arg Leu Arg Asp Glu Glu Arg Leu Arg Glu Glu Ala Arg Pro Arg Gly Glu Glu Arg Leu Arg Glu Glu Ala Arg Leu Arg Glu Asp Ala Pro Leu Arg Ala Arg Pro Ala Ala Ala Ser Pro Arg Ala Glu Pro Trp Leu Ser Gln Pro Ala Ser Gly Ser Ala Tyr Ala Thr Pro Gly Ala Tyr Gly Asp Ile Arg Pro Ser Ala Ala Ser Trp Val Gly Ser Arg Gly Leu Ala Tyr Pro Ala Arg Pro Ala Gln Leu Arg Arg Arg Ala Ser Val Arg Gly Ser Ser Glu Glu Asp Glu Asp Ala Arg Thr Pro Asp Arg Ala Thr Gln Gly Pro Gly Leu Ala Ala Arg Arg Trp Trp Ala Ala Ser Pro Ala Pro Ala Arg Leu Pro Ser Ser Leu Leu Gly Pro Asp Pro Arg Pro Gly Leu Arg Ala Thr Arg Ala Gly Pro Ala Gly Ala Ala Arg Ala Arg Pro Glu Val Gly Arg Arg Leu Glu Arg Trp Leu Ser Arg Leu Leu Leu Trp Ala Ser Leu Gly Leu Leu Leu Val Phe Leu Gly Ile Leu Trp Val Lys Met Gly Lys Pro Ser Ala Pro Gln Glu Ala Glu Asp Asn Met Lys Leu Leu Pro Val Asp Cys Glu Arg Lys Thr Asp Glu Phe Cys Gln Ala Lys Gln Lys Ala Ala Leu Leu Glu Leu Leu His Glu Leu Tyr Asn Phe Leu Ala Ile Gln Ala Gly'Asn Phe Glu Cys Gly Asn Pro Glu Asn Leu Lys Ser Lys Cys Ile Pro Val Met Glu Ala Gln Glu Tyr Ile Ala Asn Val Thr Ser Ser Ser Ser Ala Lys Phe Glu Ala Ala Leu Thr Trp Ile Leu Ser Ser Asn Lys Asp Val Gly Ile Trp Leu Lys Gly Glu Asp Gln Ser Glu Leu Val Thr Thr Val Asp Lys Val Val Cys Leu Glu Ser Ala His Pro Arg Met Gly Val Gly Cys Arg Leu Ser Arg Ala Leu Leu Thr Ala Val Thr Asn Val Leu Ile Phe Phe Trp Cys Leu Ala Phe Leu Trp Gly Leu Leu Tle Leu Leu Lys Tyr Arg Trp Arg Lys Leu Glu Glu Glu Glu Gln Ala Met Tyr Glu Met Val Lys Lys Ile Ile Asp Val Val Gln Asp His Tyr Val Asp Trp Glu Gln Asp Met Glu Arg Tyr Pro Tyr Val Gly Ile Leu His Val Arg Asp Ser Leu Ile Pro Pro Gln Ser Arg <210> 12 <211> 553 <212> PRT
<213> Homo sapiens <220>
<221> misc_feature <223> Incyte ID No: 7044055CD1 <400> I2 Met Ala Ala Val Ser Leu Arg Leu Gly Asp Leu Val Trp Gly Lys Leu Gly Arg Tyr Pro Pro Trp Pro Gly Lys Ile Val Asn Pro Pro Lys Asp Leu Lys Lys Pro Arg Gly Lys Lys Cys Phe Phe Val Lys Phe Phe Gly Thr Glu Asp His Ala Trp Ile Lys Val Glu Gln Leu Lys Pro Tyr His Ala His Lys Glu Glu Met Ile Lys Ile Asn Lys Gly Lys Arg Phe Gln Gln Ala Val Asp Ala Val Glu Glu Phe Leu Arg Arg Ala Lys Gly Lys Asp Gln Thr Ser Ser His Asn Ser Ser Asp Asp Lys Asn Arg Arg Asn Ser Ser Glu Glu Arg Ser Arg Pro Asn Ser Gly Asp Glu Lys Arg Lys Leu Ser Leu Ser Glu Gly Lys Val Lys Lys Asn Met Gly Glu Gly Lys Lys Arg Val Ser Ser Gly Ser Ser Glu Arg Gly Ser Lys Ser Pro Leu Lys Arg Ala Gln Glu Gln Ser Pro Arg Lys Arg Gly Arg Pro Pro Lys Asp Glu Lys Asp Leu Thr Ile Pro Glu Ser Ser Thr Val Lys Gly Met Met Ala Gly Pro Met Ala Ala Phe Lys Trp Gln Pro Thr Ala Ser Glu Pro Val Lys Asp Ala Asp Pro His Phe His His Phe Leu Leu Ser Gln Thr Glu Lys Pro Ala Val Cys Tyr Gln Ala Ile Thr Lys Lys Leu Lys Ile Cys Glu Glu Glu Thr Gly Ser Thr Ser Ile Gln Ala Ala Asp Ser Thr Ala Val Asn Gly Ser Ile Thr Pro Thr Asp Lys Lys Ile Gly Phe Leu Gly Leu Gly Leu Met Gly Ser Gly Ile Val Ser Asn Leu Leu Lys Met Gly His Thr Val Thr Val Trp Asn Arg Thr Ala Glu Lys Cys Asp Leu Phe Ile Gln Glu Gly Ala Arg Leu Gly Arg Thr Pro Ala Glu Val Val Ser Thr Cys Asp Ile Thr Phe Ala Cys Val Ser Asp Pro Lys Ala Ala Lys Asp Leu Val Leu Gly Pro Ser Gly Val Leu Gln Gly Ile Arg Pro Gly Lys Cys Tyr Val Asp Met Ser Thr Val Asp Ala Asp Thr Val Thr Glu Leu Ala Gln Val Ile Val Ser Arg Gly Gly Arg Phe Leu Glu Ala Pro Val Ser Gly Asn Gln Gln Leu Ser Asn Asp Gly Met Leu Val Ile Leu Ala Ala Gly Asp Arg Gly Leu Tyr Glu Asp Cys Ser Ser Cys Phe Gln Ala Met Gly Lys Thr Ser Phe Phe Leu Gly Glu Val Gly Asn Ala Ala Lys Met Met Leu Ile Val Asn Met Val Gln Gly Ser Phe Met Ala Thr 440 , 445 450 Ile Ala Glu Gly Leu Thr Leu Ala Gln Val Thr Gly Gln Ser Gln Gln Thr Leu Leu Asp Ile Leu Asn Gln Gly Gln Leu Ala Ser Ile Phe Leu Asp Gln Lys Cys Gln Asn Ile Leu Gln Gly Asn Phe Lys Pro Asp Phe Tyr Leu Lys Tyr Ile Gln Lys Asp Leu Arg Leu Ala Ile Ala Leu Gly Asp Ala Val Asn His Pro Thr Pro Met Ala Ala Ala Ala Asn Glu Val Tyr Lys Arg Ala Lys Ala Leu Asp Gln Ser Asp Asn Asp Met Ser Ala Val Tyr Arg Ala Tyr Ile His <210> 13 <211> 1726 <212> PRT
<213> Homo sapiens <220>
<221> misc feature <223> Incyte ID No: 7493424CD1 <400> 13 Met Lys Ala Gln Lys Ser Gly Lys Glu Gln Gln Leu Asp Ile Met 1 5 ~ 10 15 Asn Lys Gln Tyr Gln Gln Leu Glu Ser Arg Leu Asp Glu Ile Leu Ser Arg Ile Ala Lys Glu Thr Glu Glu Ile Lys Asp Leu Glu Glu Gln Leu Thr Glu Gly Gln Ile Ala Ala Asn Glu Ala Leu Lys Lys Asp Leu Glu Gly Val Ile Ser Gly Leu Gln Glu Tyr Leu Gly Thr Ile Lys Gly Gln Ala Thr Gln Ala Gln Asn Glu Cys Arg Lys Leu Arg Asp Glu Lys Glu Thr Leu Leu Gln Arg Leu Thr Glu Val Glu Gln Glu Arg Asp Gln Leu Glu Ile VaI AIa Met Asp AIa Glu Asn Met Arg Lys Glu Leu Ala Glu Leu Glu Ser Ala Leu Gln Glu Gln His Glu Val Asn Ala Ser Leu Gln Gln Thr Gln Gly Asp Leu Ser Ala Tyr Glu Ala Glu Leu Glu Ala Arg Leu Asn Leu Arg Asp Ala Glu Ala Asn Gln Leu Lys Glu Glu Leu Glu Lys Val Thr Arg Leu Thr Gln Leu Glu Gln Ser Ala Leu Gln Ala Glu Leu Glu Lys Glu Arg Gln Ala Leu Lys Asn Ala Leu Gly Lys Ala Gln Phe Ser Glu Glu Lys Glu Gln Glu Asn Ser Glu Leu His Ala Lys Leu Lys His Leu Gln Asp Asp Asn Asn Leu Leu Lys Gln Gln Leu Lys Asp Phe Gln Asn His Leu Asn His Val Val Asp Gly Leu Val Arg Pro Glu Glu Val Ala Ala Arg Val Asp Glu Leu Arg Arg Lys Leu Lys Leu Gly Thr Gly Glu Met Asn Ile His Ser Pro Ser Asp Val Leu Gly Lys Ser Leu Ala Asp Leu Gln Lys Gln Phe Ser Glu Ile Leu Ala Arg Ser Lys Trp Glu Arg Asp Glu Ala Gln Val Arg Glu Arg Lys Leu Gln Glu Glu Met Ala Leu Gln Gln Glu Lys Leu Ala Thr Gly Gln G1u Glu Phe Arg Gln Ala Cys Glu Arg Ala Leu Glu Ala Arg Met Asn Phe Asp Lys Arg Gln His Glu Ala Arg Ile Gln Gln Met Glu Asn Glu Ile His Tyr Leu Gln Glu Asn Leu Lys Ser Met Glu Glu Ile Gln Gly Leu Thr Asp Leu Gln Leu Gln Glu Ala Asp Glu Glu Lys Glu Arg Ile Leu Ala Gln Leu Arg Glu Leu Glu Lys Lys Lys Lys Leu Glu Asp Ala Lys Ser Gln Glu Gln Val Phe Gly Leu Asp Lys Glu Leu Lys Lys Leu Lys Lys Ala Val Ala Thr Ser Asp Lys Leu Ala Thr Ala Glu Leu Thr Ile Ala Lys Asp Gln Leu Lys Ser Leu His Gly Thr Val Met Lys Ile Asn Gln Glu Arg Ala Glu Glu Leu Gln Glu Ala Glu Arg Phe Ser Arg Lys Ala Ala Gln Ala Ala Arg Asp Leu Thr Arg Ala Glu Ala Glu Ile Glu Leu Leu Gln Asn Leu Leu Arg Gln Lys Gly Glu Gln Phe Arg Leu Glu Met Glu Lys Thr Gly Val Gly Thr Gly Ala Asn Ser Gln Val Leu Glu Ile Glu Lys Leu Asn Glu Thr Met Glu Arg Gln Arg Thr Glu Ile Ala Arg Leu Gln Asn Val Leu Asp Leu Thr Gly Ser Asp Asn Lys Gly Gly Phe Glu Asn Val Leu Glu Glu Ile Ala Glu Leu Arg Arg Glu Val Ser Tyr Gln Asn Asp Tyr Ile Ser Ser Met Ala Asp Pro Phe Lys Arg Arg Gly Tyr Trp Tyr Phe Met Pro Pro Pro Pro Ser Ser Lys Val Ser Ser His Ser Ser Gln Ala Thr Lys Asp Ser Gly Val Gly Leu Lys Tyr Ser Ala Ser Thr Pro Val Arg Lys Pro Arg Pro Gly Gln Gln Asp Gly Lys Glu Gly Ser Gln Pro Pro Pro Ala Ser Gly Tyr Trp Val Tyr Ser Pro Ile Arg Ser Gly Leu His Lys Leu Phe Pro Ser Arg Asp Ala Asp Ser Gly Gly Asp Ser Gln Glu Glu Ser Glu Leu Asp Asp Gln Glu Glu Pro Pro Phe Val Pro Pro Pro Gly Tyr Met Met Tyr Thr Val Leu Pro Asp Gly Ser Pro Val Pro Gln Gly Met Ala Leu Tyr Ala Pro Pro Pro Pro Leu Pro Asn Asn Ser Arg Pro Leu Thr Pro Gly Thr Val Val Tyr Gly Pro Pro Pro Ala Gly Ala Pro Met Val Tyr Gly Pro Pro Pro Pro Asn Phe Ser Ile Pro Phe Ile Pro Met Gly Val Leu His Cys Asn Val Pro Glu His His Asn Leu Glu Asn Glu Val Ser Arg Leu Glu Asp Ile Met Gln His Leu Lys Ser Lys Lys Arg Glu Glu Arg Trp Met,Arg Ala Ser Lys Arg Gln Ser GIu Lys Glu Met Glu Glu Leu His His Asn Ile Asp Asp Leu Leu Gln Glu Lys Lys Ser Leu Glu Cys Glu Val Glu GIu Leu His Arg Thr Val Gln Lys Arg Gln G1n Gln Lys Asp Phe Ile Asp Gly Asn Val Glu Ser Leu Met Thr Glu Leu Glu Ile Glu Lys Ser Leu Lys His His Glu Asp Ile Val Asp Glu Ile Glu Cys Ile Glu Lys Thr Leu Leu Lys Arg Arg Ser Glu Leu Arg Glu Ala Asp Arg Leu Leu Ala Glu Ala Glu Ser Glu Leu Ser Cys Thr Lys Glu Lys Thr Lys Asn Ala Val Glu Lys Phe Thr Asp Ala Lys Arg Ser Leu Leu Gln Thr Glu Ser Asp Ala Glu Glu Leu Glu Arg Arg Ala Gln Glu Thr Ala Val Asn Leu Val Lys Ala Asp Gln Gln Leu Arg Ser Leu Gln Ala Asp Ala Lys Asp Leu Glu Gln His Lys Ile Lys Gln Glu Glu Ile Leu Lys Glu Ile Asn Lys Ile Val Ala Ala Lys Asp Ser Asp Phe Gln Cys Leu Ser Lys Lys Lys Glu Lys Leu Thr Glu Glu Leu Gln Lys Leu Gln Lys Asp Ile Glu Met Ala Glu Arg Asn Glu Asp His His Leu Gln Val Leu Lys Glu Ser Glu Val Leu Leu Gln Ala Lys Arg Ala Glu Leu Glu Lys Leu Lys Ser Gln Val Thr Ser Gln Gln Gln Glu Met Ala Val Leu Asp Arg Gln Leu Gly His Lys Lys Glu Glu Leu His Leu Leu Gln Gly Ser Met Val Gln Ala Lys Ala Asp Leu GIn GIu Ala Leu Arg Leu GIy Glu Thr Glu Val Thr Glu Lys Cys Asn His Ile Arg Glu Val Lys Ser Leu Leu Glu Glu Leu Ser Phe Gln Lys Gly Glu Leu Asn Val Gln Ile Ser Glu Arg Lys Thr Gln Leu Thr Leu Ile Lys Gln Glu Ile Glu Lys Glu Glu Glu Asn Leu Gln Val Val Leu Arg Gln Met Ser Lys His Lys Thr Glu Leu Lys Asn Ile Leu Asp Met Leu Gln Leu Glu Asn His Glu Leu Gln Gly Leu Lys Leu Gln His Asp Gln Arg Val Ser Glu Leu Glu Lys Thr Gln Val Ala Val Leu Glu Glu Lys Leu Glu Leu Glu Asn Leu Gln Gln Ile Ser Gln Gln Gln Lys Gly Glu Ile Glu Trp Gln Lys Gln Leu Leu Glu Arg Asp Lys Arg Glu Ile Glu Arg Met Thr Ala Glu Ser Arg Ala Leu Gln Ser Cys Val Glu Cys Leu Ser Lys Glu Lys Glu Asp Leu Gln Glu Lys Cys Asp Ile Trp Glu Lys Lys Leu Ala Gln Thr Lys Arg Val Leu Ala Ala Ala Glu Glu Asn Ser Lys Met Glu Gln Ser Asn Leu Glu Lys Leu Glu Leu Asn Val Arg Lys Leu Gln Gln Glu Leu Asp Gln Leu Asn Arg Asp Lys Leu Ser Leu His Asn Asp Ile Ser Ala Met Gln Gln Gln Leu Gln Glu Lys Arg Glu Ala Val Asn Ser Leu Gln Glu Glu Leu Ala Asn Val Gln Asp His Leu Asn Leu Ala Lys Gln Asp Leu Leu His Thr Thr Lys His Gln Asp Val Leu Leu Ser Glu Gln Thr Arg Leu Gln Lys Asp Ile Ser Glu Trp Ala Asn Arg Phe Glu Asp Cys GIn Lys Glu Glu GIu Thr Lys Gln Gln Gln Leu Gln Val Leu Gln Asn Glu Ile Glu Glu Asn Lys Leu Lys Leu Val Gln Gln Glu Met Met Phe Gln Arg Leu Gln Lys Glu Arg Glu Ser Glu Glu Ser Lys Leu Glu Thr Ser Lys Val Thr Leu Lys Glu Gln Gln His Gln Leu Glu Lys Glu Leu Thr Asp Gln Lys Ser Lys Leu Asp Gln Val Leu Ser Lys Val Leu Ala Ala Glu Glu Arg Val Arg Thr Leu Gln Glu Glu Glu Arg Trp Cys Glu Ser Leu Glu Lys Thr Leu Ser Gln Thr Lys Arg Gln Leu Ser Glu Arg Glu Gln Gln Leu Val-Glu Lys Ser Gly Glu Leu Leu Ala Leu Gln Lys Glu Ala Asp Ser Met Arg Ala Asp Phe Ser Leu Leu Arg Asn Gln Phe Leu Thr Glu Arg Lys Lys Ala Glu Lys Gln Val Ala Ser Leu Lys Glu Ala Leu Lys Ile Gln Arg Ser Gln Leu Glu Lys Asn Leu Leu Met Ala Asn Gln Lys Asp Leu Glu Arg Arg Gln Met Glu Ile Ser Asp Ala Met Arg Thr Leu Lys Ser Glu Val Lys Asp Glu Ile Arg Thr Ser Leu Lys Asn Leu Asn Gln Phe Leu Pro Glu Leu Pro Ala Asp Leu Glu Ala Ile Leu Glu Arg Asn Glu Asn Leu Glu Gly Glu Leu Glu Ser Leu Lys Glu Asn Leu Pro Phe Thr Met Asn Glu Gly Pro Phe Glu Glu Lys Leu Asn Phe Ser Gln Val His I1e Met Asp Glu His Trp Arg Gly Glu Ala Leu Arg Glu Lys Leu Arg His Arg Glu Asp Arg Leu Lys Ala Gln Leu Arg His Cys Met Ser Lys Gln Ala Glu Val Leu Ile Lys Gly Lys Arg Gln Thr Glu Gly Thr Leu His Ser Leu Arg Arg Gln Val Asp Ala Leu Gly Glu Leu Val Thr Ser Thr Ser Ala Asp Ser Ala Ser Ser Pro Ser Leu Ser Gln Leu Glu Ser Ser Leu Thr Glu Asp Ser Gln Leu Gly Gln Asn Gln Glu Lys Asn Ala Ser Ala Arg <210> 14 <211> 747 <212> PRT
<213> Homo sapiens <220>
<221> misc_feature <223> Incyte ID No: 1482140CD1 <400> 14 Met Ala Leu Gly Leu Gln Arg Ala Arg Pro Ala Leu Ser Cys Gly Val Ile Ser Pro Pro Cys Ala Pro Thr Arg Asn Ser His Pro Gly Pro Gly Cys Thr Ala Ser Pro Pro Ala Pro Pro Gly Trp Pro Phe Ser,Gln Arg Gly Pro Gly Arg Trp Ser Thr Thr Glu Leu Arg Lys Glu Lys Ser Arg Asp Ala Ala Arg Ser Arg Arg Ser Gln Glu Thr Glu Val Leu Tyr Gln Leu Ala His Thr Leu Pro Phe Ala Arg Gly Val Ser Ala His Leu Asp Lys Ala Ser Ile Met Arg Leu Thr Ile Ser Tyr Leu Arg Met His Arg Leu Cys Ala Ala Gly Glu Trp Asn Gln Val Gly Ala Gly Glu Asn His Trp Met Leu Leu Leu Lys Ala Leu Glu Gly Phe Val Met Val Leu Thr Ala Glu Gly Asp Met Ala Tyr Leu Ser Glu Asn Val Ser Lys His Leu Gly Leu Ser Gln Leu Glu Leu Ile Gly His Ser Ile Phe Asp Phe Ile His Pro Cys Asp Gln Glu Glu Leu Gln Asp Ala Leu Thr Pro Gln Gln Thr Leu Ser Arg Arg Lys Val Glu Ala Pro Thr Glu Arg Cys Phe Ser Leu Arg Met Lys Ser Thr Leu Thr Ser Arg Gly Arg Thr Leu Asn Leu Lys Ala Ala Thr Trp Lys Val Leu Asn Cys Ser Gly His Met Arg Ala Tyr Lys Pro Pro Ala Gln Thr Ser Pro Ala Gly Ser Pro Asp Ser Glu Pro Pro Leu Gln Cys Leu Val Leu Ile Cys Glu Ala Ile Pro His Pro Gly Ser Leu Glu Pro Pro Leu Gly Arg Gly Ala Phe Leu Ser Arg His Ser Leu Asp Met Lys Phe Thr Tyr Cys Asp Asp Arg Ile Ala Glu Val Ala Gly Tyr Ser Pro Asp Asp Leu Ile Gly Cys Ser Ala Tyr Glu Tyr Ile His Ala Leu Asp Ser Asp Ala Val Ser Lys Ser Ile His Thr Leu Leu Ser Lys Gly Gln Ala Val Thr Gly Gln Tyr Arg Phe Leu Ala Arg Ser Gly Gly Tyr Leu Trp Thr Gln Thr Gln Ala Thr Val Val Ser Gly Gly Arg Gly Pro Gln Ser Glu Ser Ile Val Cys Val His Phe Leu Ile Ser Gln Val Glu Glu Thr Gly Val Val Leu Ser Leu Glu Gln Thr Glu Gln His Ser Arg Arg Pro Ile Gln Arg Gly Ala Pro Ser Gln Lys Asp Thr Pro Asn Pro Gly Asp Ser Leu Asp Thr Pro Gly Pro Arg Ile Leu Ala Phe Leu 425 430 435 ..
His Pro Pro Ser Leu Ser Glu Ala Ala Leu Ala Ala Asp Pro Arg Arg Phe Cys Ser Pro Asp Leu Arg Arg Leu Leu Gly Pro Ile Leu Asp Gly Ala Ser Val Ala Ala Thr Pro Ser Thr Pro Leu Ala Thr Arg His Pro Gln Ser Pro Leu Ser Ala Asp Leu Pro Asp Glu Leu Pro Val Gly Thr Glu Asn Val His Arg Leu Phe Thr Ser Gly Lys Asp Thr Glu Ala Val Glu Thr Asp Leu Asp Ile Ala Gln Asp Ala Asp Ala Leu Asp Leu Glu Met Leu Ala Pro Tyr Ile Ser Met Asp Asp Asp Phe Gln Leu Asn Ala Ser Glu Gln Leu Pro Arg Ala Tyr His Arg Pro Leu Gly Ala Val Pro Arg Pro Arg Ala Arg Ser Phe His Gly Leu Ser Pro Pro Ala Leu Glu Pro Ser Leu Leu Pro Arg Trp Gly Ser Asp Pro Arg Leu Ser Cys Ser Ser Pro Ser Arg Gly Asp Pro Ser Ala Ser Ser Pro Met Ala Gly Ala Arg Lys Arg Thr Leu Ala Gln Ser Ser G1u Asp Glu Asp Glu Gly Val Glu Leu Leu Gly Val Arg Pro Pro Lys Arg Ser Pro Ser Pro Glu His Glu Asn Phe Leu Leu Phe Pro Leu Ser Leu Ser Phe Leu Leu Thr Gly Gly Pro Ala Pro Gly Ser Leu Gln Asp Pro Thr Glu Leu Thr Gln Phe Leu Leu Ser Val Leu Ser Phe Pro Ile Leu Asp Pro Tyr Pro Leu Gly Cys Ala Ala Pro Gly Leu His Ala Ser Pro Phe Ser Leu Pro Thr Ile Ser Val Pro Gln Asn Pro Leu His Ser Pro Pro Gln Pro Ser Arg His Ala Leu Thr Leu Thr Leu Pro His Met Phe Gly Ala Pro Gly Ala Pro Ser Pro Leu Gly Trp Phe Ala Ile <210> 15 <211> 2759 <212> PRT
<213> Homo Sapiens <220>
<221> misc feature <223> Incyte ID No: 394992CD1 <400> 15 Met Val Ser Glu Glu Glu Glu Glu Glu Asp Gly Asp Ala Glu Glu Thr Gln Asp Ser Glu Asp Asp Glu Glu Asp Glu Met Glu Glu Asp Asp Asp Asp Ser Asp Tyr Pro Glu Glu Met Glu Asp Asp Asp Asp 35 40 45 .
Asp Ala Ser Tyr Cys Thr Glu Ser Ser Phe Arg Ser His Ser Thr Tyr Ser Ser Thr Pro Gly Arg Arg Lys Pro Arg Val His Arg Pro Arg Ser Pro Ile Leu Glu Glu Lys Asp Ile Pro Pro Leu Glu Phe Pro Lys Ser Ser Glu Asp Leu Met Val Pro Asn Glu His Ile Met Asn Val Ile Ala Ile Tyr Glu Val Leu Arg Asn Phe Gly Thr Val Leu Arg Leu Ser Pro Phe Arg Phe Glu Asp Phe Cys Ala Ala Leu Val Ser Gln Glu Gln Cys Thr Leu Met Ala Glu Met His Val Val Leu Leu Lys Ala Val Leu Arg Glu Glu Asp Thr Ser Asn Thr Thr Phe Gly Pro Ala Asp Leu Lys Asp Ser Val Asn Ser Thr Leu Tyr Phe Ile Asp Gly Met Thr Trp Pro Glu Val Leu Arg Val Tyr Cys Glu Ser Asp Lys Glu Tyr His His Val Leu Pro Tyr Gln Glu Ala Glu Asp Tyr Pro Tyr Gly Pro Val Glu Asn Lys Ile Lys Val Leu Gln Phe Leu Val Asp Gln Phe Leu Thr Thr Asn Ile Ala Arg Glu Glu Leu Met Ser Glu Gly Val Ile Gln Tyr Asp Asp His Cys Arg Val Cys His Lys Leu Gly Asp Leu Leu Cys Cys Glu Thr Cys Ser Ala Val Tyr His Leu Glu Cys Val Lys Pro Pro Leu Glu Glu Val Pro Glu Asp Glu Trp Gln Cys Glu Val Cys Val Ala His Lys Val Pro Gly Val Thr Asp Cys Val Ala Glu Ile Gln Lys Asn Lys Pro Tyr Ile Arg His Glu Pro Ile Gly Tyr Asp Arg Ser Arg Arg Lys Tyr Trp Phe Leu Asn Arg Arg Leu Ile Ile Glu Glu Asp Thr Glu Asn Glu Asn Glu Lys Lys Ile Trp Tyr Tyr Ser Thr Lys Val Gln Leu Ala Glu Leu Ile Asp Cys Leu Asp Lys Asp Tyr Trp Glu Ala Glu Leu Cys Lys Ile Leu Glu Glu Met Arg Glu Glu Ile His Arg His Met Asp Ile Thr Glu Asp Leu Thr Asn Lys Ala Arg Gly Ser Asn Lys Ser Phe Leu Ala Ala Ala Asn Glu Glu Ile Leu Glu Ser Ile Arg Ala Lys Lys Gly Asp Ile Asp Asn Val Lys Ser Pro Glu Glu Thr Glu Lys Asp Lys Asn Glu Thr Glu Asn Asp Ser Lys Asp 440 445 ' 450 Ala Glu Lys Asn Arg Glu Glu Phe Glu Asp Gln Ser Leu Glu Lys Asp Ser Asp Asp Lys Thr Pro Asp Asp Asp Pro Glu Gln Gly Lys Ser Glu Glu Pro Thr Glu Val Gly Asp Lys Gly Asn Ser Val Ser Ala Asn Leu Gly Asp Asn Thr Thr Asn Ala Thr Ser Glu Glu Thr Ser Pro Ser Glu Gly Arg Ser Pro Val Gly Cys Leu Ser Glu Thr Pro Asp Ser Ser Asn Met Ala Glu Lys Lys Val Ala Ser Glu Leu Pro Gln Asp Val Pro Val Gly Asp Phe Lys Ser Glu Lys Ser Asn Gly Glu Leu Ser Glu Ser Pro Gly Ala Gly Lys Gly Ala Ser Gly Ser Thr Arg Ile Ile Thr Arg Leu Arg Asn Pro Asp Ser Lys Leu Ser Gln Leu Lys Ser Gln Gln Val Ala Ala Ala Ala His Glu Ala Asn Lys Leu Phe Lys Glu Gly Lys Glu Val Leu Val Val Asn Ser Gln Gly Glu Ile Ser Arg Leu Ser Thr Lys Lys Glu Val Ile Met Lys Gly Asn Ile Asn Asn Tyr Phe Lys Leu Gly Gln Glu Gly Lys Tyr Arg Val Tyr His Asn Gln Tyr Ser Thr Asn Ser Phe Ala Leu Asn Lys His Gln His Arg Glu Asp His Asp Lys Arg Arg His Leu Ala His Lys Phe Cys Leu Thr Pro Ala Gly Glu Phe Lys Trp Asn Gly Ser Val His Gly Ser Lys Val Leu Thr Ile Ser Thr Leu Arg Leu Thr Ile Thr Gln Leu Glu Asn Asn Ile Pro Ser Ser Phe Leu His Pro Asn Trp Ala Ser His Arg Ala Asn Trp Ile Lys Ala Val Gln Met Cys Ser Lys Pro Arg Glu Phe Ala Leu Ala Leu Ala Ile Leu Glu Cys Ala Val Lys Pro Val Val Met Leu Pro Ile Trp Arg Glu Ser Leu Gly His Thr Arg Leu His Arg Met Thr Ser Ile Glu Arg Glu Glu Lys Glu Lys Val Lys Lys Lys Glu Lys Lys Gln Glu Glu Glu Glu Thr Met Gln Gln Ala Thr Trp Val Lys Tyr Thr Phe Pro Val Lys His Gln Val Trp Lys Gln Lys Gly Glu Glu Tyr Arg Val Thr Gly Tyr Gly Gly Trp Ser Trp Ile Ser Lys Thr His Val Tyr Arg Phe Val Pro Lys Leu Pro Gly Asn Thr Asn Val Asn Tyr Arg Lys Ser Leu Glu Gly Thr Lys Asn Asn Met Asp Glu Asn Met Asp Glu Ser Asp Lys Arg Lys Cys Ser Arg Ser Pro Lys Lys Ile Lys Ile Glu Pro Asp Ser Glu Lys Asp Glu Val Lys Gly Ser Asp Ala Ala Lys Gly Ala Asp Gln Asn GIu Met Asp Ile Ser Lys Ile Thr Glu Lys Lys Asp Gln Asp Val Lys Glu Leu Leu Asp Ser Asp Ser Asp Lys Pro Cys Lys Glu Glu Pro Met Glu Val Asp Asp Asp Met Lys Thr Glu Ser His Val Asn Cys Gln Glu Ser Ser Gln Val Asp Val Val Asn Val Ser Glu Gly Phe His Leu Arg Thr Ser Tyr Lys Lys Lys Thr Lys Ser Ser Lys Leu Asp Gly Leu Leu Glu Arg Arg Ile Lys Gln Phe Thr Leu Glu Glu Lys Gln Arg Leu Glu Lys 995 ' 1000 1005 Ile Lys Leu Glu Gly Gly Ile Lys Gly Tle Gly Lys Thr Ser Thr Asn Ser Ser Lys Asn Leu Ser Glu Ser Pro Val Ile Thr Lys Ala Lys Glu Gly Cys Gln Ser Asp Ser Met Arg Gln Glu Gln Ser Pro Asn Ala Asn Asn Asp Gln Pro Glu Asp Leu Ile Gln Gly Cys Ser Glu Ser Asp Ser Ser Val Leu Arg Met Ser Asp Pro Ser His Thr Thr Asn Lys Leu Tyr Pro Lys Asp Arg Val Leu Asp Asp Val Ser Ile Arg Ser Pro Glu Thr Lys Cys Pro Lys Gln Asn Ser Ile Glu Asn Asp Ile Glu Glu Lys Val Ser Asp Leu Ala Ser Arg Gly Gln Glu Pro Ser Lys Ser Lys Thr Lys Gly Asn Asp Phe Phe Ile Asp Asp Ser Lys Leu Ala Ser Ala Asp Asp Ile Gly Thr Leu Ile Cys Lys Asn Lys Lys Pro Leu Ile Gln Glu Glu Ser Asp Thr Ile Val Ser Ser Ser Lys Ser Ala Leu His Ser Ser Val Pro Lys Ser Thr Asn Asp Arg Asp Ala Thr Pro Leu Ser Arg Ala Met Asp Phe Glu Gly Lys Leu Gly Cys Asp Ser Glu Ser Asn Ser Thr Leu Glu Asn Ser Ser Asp Thr Val Ser Ile Gln Asp Ser Ser Glu Glu Asp Met Ile Val Gln Asn Ser Asn Glu Ser Ile Ser Glu Gln Phe Arg Thr Arg Glu Gln Asp Val Glu Val Leu Glu Pro Leu Lys Cys Glu Leu Val Ser Gly Glu Ser Thr Gly Asn Cys Glu Asp Arg Leu Pro Va1 Lys Gly Thr Glu Ala Asn Gly Lys Lys Pro Ser Gln Gln Lys Lys Leu Glu Glu Arg Pro Val Asn Lys Cys Ser Asp Gln Ile Lys Leu Lys Asn Thr Thr Asp Lys Lys Asn Asn Glu Asn Arg Glu Ser Glu Lys Lys Gly Gln Arg Thr Ser Thr Phe Gln Tle Asn Gly Lys Asp Asn Lys Pro Lys Ile Tyr Leu Lys Gly Glu Cys Leu Lys Glu Ile Sex Glu Ser Arg Val Val Ser Gly Asn Val GIu Pro Lys Val Asn Asn Ile Asn Lys Ile Ile Pro Glu Asn Asp Ile Lys Ser Leu Thr Val Lys Glu Ser Ala Ile Arg Pro Phe Ile Asn Gly Asp Val Ile Met Glu Asp Phe Asn Glu Arg Asn Ser Ser Glu Thr Lys Ser His Leu Leu Ser Ser Ser Asp Ala Glu Gly Asn Tyr Arg Asp Ser Leu Glu Thr Leu Pro Ser Thr Lys Glu Ser Asp Ser Thr Gln Thr Thr Thr Pro Ser Ala Ser Cys Pro Glu Ser Asn Ser Val Asn Gln Val Glu Asp Met Glu Ile Glu Thr Ser Glu Val Lys Lys Val Thr Ser Ser Pro Ile Thr Ser Glu Glu Glu Ser Asn Leu Ser Asn Asp Phe Ile Asp Glu Asn Gly Leu Pro Ile Asn Lys Asn Glu Asn Val Asn Gly Glu Ser Lys Arg Lys Thr Val Ile Thr Glu VaI Thr Thr Met Thr Ser Thr Val Ala Thr Glu Ser Lys Thr Val Ile Lys Val Glu Lys Gly Asp Lys Gln Thr Val Val Ser Ser Thr Glu Asn Cys Ala Lys Ser Thr Val Thr Thr Thr Thr Thr Thr Val Thr Lys Leu Ser Thr Pro Ser Thr Gly Gly Ser Val Asp Ile Ile Ser Val Lys Glu Gln Ser Lys Thr Val Val Thr Thr Thr Val Thr Asp Ser Leu Thr Thr Thr Gly Gly Thr Leu Val Thr Ser Met Thr Val Ser Lys Glu Tyr Ser Thr Arg Asp Lys Val Lys Leu Met Lys Phe Ser Arg Pro Lys Lys Thr Arg Ser Gly Thr Ala Leu Pro Ser Tyr Arg Lys Phe Val Thr Lys Ser Ser Lys Lys Ser Ile Phe Val Leu Pro Asn Asp Asp Leu Lys Lys Leu Ala Arg Lys Gly Gly Ile Arg Glu Val Pro Tyr Phe Asn Tyr Asn Ala Lys Pro Ala Leu Asp Ile Trp Pro Tyr Pro Ser Pro Arg Pro Thr Phe GIy Ile Thr Trp Arg Tyr Arg Leu Gln Thr Val Lys Ser Leu Ala Gly Val Ser Leu Met Leu Arg Leu Leu Trp Ala Ser Leu Arg Trp Asp Asp Met Ala Ala Lys Ala Pro Pro Gly Gly Gly Thr Thr Arg Thr Glu Thr Ser Glu Thr Glu Ile Thr Thr Thr Glu Ile Ile Lys Arg Arg Asp Val Gly Pro Tyr Gly Ile Arg Ser Glu Tyr Cys Ile Arg Lys Ile Ile Cys Pro Ile Gly Val Pro Glu Thr Pro Lys Glu Thr Pro Thr Pro Gln Arg Lys Gly Leu Arg Ser Ser Ala Leu Arg Pro Lys Arg Pro Glu Thr Pro Lys Gln Thr Gly Pro Val Ile Ile Glu Thr Trp Val Ala Glu Glu Glu Leu Glu Leu Trp Glu Ile Arg Ala Phe Ala Glu Arg Val Glu Lys Glu Lys Ala Gln Ala Val Glu Gln Gln Ala Lys Lys Arg Leu Glu Gln Gln Lys Pro Thr Val Ile Ala Thr Ser Thr Thr Ser Pro Thr Ser Ser Thr Thr Ser'Thr Ile Ser Pro Ala Gln Lys Val Met Val Ala Pro Ile Ser Gly Ser Val Thr Thr Gly Thr Lys Met Val Leu Thr Thr Lys Val Gly Ser Pro Ala Thr Val Thr Phe Gln Gln Asn Lys Asn Phe His Gln Thr Phe Ala Thr Trp Val Lys Gln Gly Gln Ser Asn Ser Gly Val Val Gln Val Gln Gln Lys Val Leu Gly Ile Ile Pro Ser Ser Thr Gly Thr Ser Gln Gln Thr Phe Thr Ser Phe Gln Pro Arg Thr Ala Thr Val Thr Ile Arg Pro Asn Thr 5er Gly Ser Gly Gly Thr Thr Ser Asn Ser Gln Val Ile Thr Gly Pro Gln Ile Arg Pro Gly Met Thr Val Ile Arg Thr Pro Leu Gln Gln Ser Thr Leu Gly Lys Ala Ile Ile Arg Thr Pro Val Met Val Gln Pro Gly Ala Pro Gln Gln Val Met Thr Gln Ile Ile Arg Gly Gln Pro Val Ser Thr Ala Val Ser Ala Pro Asn Thr Val Ser Ser Thr Pro Gly Gln Lys Ser Leu Thr Ser Ala Thr Ser Thr Ser Asn Ile Gln Ser Ser Ala Ser Gln Pro Pro Arg Pro Gln Gln Gly Gln Val Lys Leu Thr Met Ala Gln Leu Thr Gln Leu Thr Gln Gly His Gly Gly Asn Gln Gly Leu Thr Val Val Tle Gln Gly Gln Gly Gln Thr Thr Gly Gln Leu Gln Leu Ile Pro Gln Gly Val Thr Val Leu Pro Gly Pro Gly Gln Gln Leu Met Gln Ala Ala Met Pro Asn Gly Thr Val Gln Arg Phe Leu Phe Thr Pro Leu Ala Thr Thr Ala Thr Thr Ala Ser Thr Thr Thr Thr Thr Val Ser Thr Thr Ala Ala Gly Thr Gly 2150 2155 2160 ~ ' Glu Gln Arg Gln Ser Lys Leu Ser Pro Gln Met Gln Val His Gln Asp Lys Thr Leu Pro Pro Ala Gln Ser Ser Ser Val Gly Pro Ala Glu Ala Gln Pro Gln Thr Ala Gln Pro Ser Ala Gln Pro Gln Pro Gln Thr Gln Pro Gln Ser Pro Ala Gln Pro Glu Val Gln Thr Gln Pro Glu Val Gln Thr Gln Thr Thr Val Ser Ser His Val Pro Ser Glu Ala Gln Pro Thr His Ala Gln Ser Ser Lys Pro Gln Val Ala Ala Gln Ser Gln Pro Gln Ser Asn Val Gln Gly Gln Ser Pro Val Arg Val Gln Ser Pro Ser Gln Thr Arg Ile Arg Pro Ser Thr Pro Ser Gln Leu Ser Pro Gly Gln Gln Ser Gln Val Gln Thr Thr Thr Ser Gln Pro Ile Pro Ile Gln Pro His Thr Ser Leu Gln Ile Pro Ser Gln Gly Gln Pro Gln Ser Gln Pro Gln Val Val Met Lys His Asn Ala Val Ile Glu His Leu Lys Gln Lys Lys Ser Met Thr Pro Ala Glu Arg Glu Glu Asn Gln Arg Met Ile Val Cys Asn Gln Val Met Lys Tyr Ile Leu Asp Lys Ile Asp Lys Glu Glu Lys Gln Ala Ala Lys Lys Arg Lys Arg Glu Glu Ser Val Glu Gln Lys Arg Ser Lys Gln Asn Ala Thr Lys Leu Ser Ala Leu Leu Phe Lys His Lys Glu Gln Leu Arg Ala Glu Ile Leu Lys Lys Arg Ala Leu Leu Asp Lys Asp Leu Gln Ile Glu Val Gln Glu Glu Leu Lys Arg Asp Leu Lys Ile Lys Lys Glu Lys Asp Leu Met Gln Leu Ala Gln Ala Thr Ala Val Ala Ala Pro Cys Pro Pro Val Thr Pro Ala Pro Pro Ala 2450 2455 ~ 2460 Pro Pro Ala Pro Pro Pro Ser Pro Pro Pro Pro Pro Ala Val Gln His Thr Gly Leu Leu Ser Thr Pro Thr Leu Pro Ala Ala Ser Gln Lys Arg Lys Arg Glu Glu Glu Lys Asp Ser Ser Ser Lys Ser Lys Lys Lys Lys Met Ile Ser Thr Thr Ser Lys Glu Thr Lys Lys Asp Thr Lys Leu Tyr Cys Ile Cys Lys Thr Pro Tyr Asp Glu Ser Lys Phe Tyr Ile Gly Cys Asp Leu Cys Thr Asn Trp Tyr His Gly Glu Cys Val Gly Ile Thr Glu Lys Glu Ala Lys Lys Met Asp Val Tyr Ile Cys Asn Asp Cys Lys Arg Ala Gln Glu Gly Ser Ser Glu Glu Leu Tyr Cys Tle Cys Arg Thr Pro Tyr Asp Glu Ser Gln Phe Tyr Ile Gly Cys Asp Arg Cys Gln Asn Trp Tyr His Gly Arg Cys Val Gly Ile Leu Gln Ser Glu Ala Glu Leu Ile Asp Glu Tyr Val Cys Pro Gln Cys Gln Ser Thr Glu Asp Ala Met Thr Val Leu Thr Pro Leu Thr Glu Lys Asp Tyr Glu Gly Leu Lys Arg Val Leu Arg Ser 2&45 2650 2655 Leu Gln Ala His Lys Met Ala Trp Pro Phe Leu Glu Pro Val Asp Pro Asn Asp Ala Pro Asp Tyr Tyr Gly Val Ile Lys Glu Pro Met Asp Leu Ala Thr Met Glu Glu Arg Val Gln Arg Arg Tyr Tyr Glu Lys Leu Thr Glu Phe Val Ala Asp Met Thr Lys Ile Phe Asp Asn Cys Arg Tyr Tyr Asn Pro Ser Asp Ser Pro Phe Tyr Gln Cys Ala Glu VaI Leu Glu Ser Phe Phe Val Gln Lys Leu Lys Gly Phe Lys Ala Ser Arg Ser His Asn Asn Lys Leu Gln Ser Thr Ala Ser <210> 16 <211> 613 <212> PRT
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte ID No: 5093550CD1 <400> 16 Met Asp Val Ala Ile Glu Phe Ser Val Glu Glu Trp Gln Cys Leu Asp Thr Ala Gln Gln Asn Leu Tyr Arg Asn Val Met Leu Glu Asn Tyr Arg Asn Leu Val Phe Leu Gly Ile Ala Val Ser Lys Pro Asp Leu Ile Thr Cys Leu Glu Gln Gly Lys Glu Pro Trp Asn Met Glu Arg His Glu Met Val Ala Lys Pro Pro Gly Met Cys Cys Tyr Phe Ala Gln Asp Leu Arg Pro Glu Gln Ser Ile Lys Ala Ser Leu Gln Arg Tle Ile Leu Arg Lys Tyr Glu Lys Cys Gly His His Asn Leu Gln Leu Lys Lys Gly Tyr Lys Ser Val Asp Glu Tyr Lys Val His Lys Gly Ser Tyr Asn Gly Phe Asn Gln Cys Leu Thr Thr Thr Gln Ser Lys Ile Phe Gln Cys Asp Lys Tyr Val Lys Asp Phe His Lys Phe Ser Asn Ser Asn Arg His Lys Thr Glu Lys Asn Pro Phe Lys Cys Lys Glu Cys Gly Lys Ser Phe Cys Val Leu Ser His Leu Thr Gln His Lys Arg Ile His Thr Thr Val Asn Ser Tyr Lys Leu Glu Glu Cys Gly Lys Ala Phe Asn Val Ser Ser Thr Leu Ser Gln His Lys Arg Ile His Thr Gly Gln Lys His Tyr Lys Cys Glu Glu Cys Gly Ile Ala Phe Asn Lys Ser Ser His Leu Asn Thr His Lys Ile Ile His Thr Gly Glu Lys Ser Tyr Lys Arg Glu Glu Cys Gly Lys Ala Phe Asn Ile Ser Ser His Leu Thr Thr His Lys Tle Ile His Thr Gly Glu Asn Ala Tyr Lys Cys Lys Glu Cys Gly Lys Ala Phe Asn Gln Ser Ser Thr Leu Thr Arg His Lys Ile Ile His Ala Gly Glu Lys Pro Tyr Ile Cys Glu His Cys Gly Arg Ala Phe Asn Gln Ser Ser Asn Leu Thr Lys His Lys Arg Ile His Thr Gly Asp Lys Pro Tyr Lys Cys Glu Glu Cys Gly Lys Ala Phe Asn Val Ser Ser Thr Leu Thr Gln His Lys Arg Ile His Thr Gly Glu Lys Pro Tyr Lys Cys Glu Glu Cys Gly Lys Ala Phe Asn Val Ser Ser Thr Leu Thr Gln His Lys Arg Ile His Thr Gly Glu Lys Pro Tyr Lys Cys Glu Glu Cys Gly Lys Ala Phe Asn Thr Ser Ser His Leu Thr Thr 395 400 ~ 405 His Lys Arg Ile His Thr Gly Glu Lys Pro Tyr Lys Cys Glu Glu Cys Gly Lys Ala Phe Asn Gln Phe Ser Gln Leu Thr Thr His Lys Ile Ile His Thr Gly Glu Lys Pro Tyr Lys Cys Lys Glu Cys Gly Lys Ala Phe Lys Arg Ser Ser Asn Leu Thr Glu His Arg Ile Ile His Thr Gly Glu Lys Pro Tyr Lys Cys Glu Glu Cys Gly Lys Ala Phe Asn Leu Ser Ser His Leu Thr Thr His Lys Lys Ile His Thr Gly Glu Lys Pro Tyr Lys Cys Lys Glu Cys Gly Lys Ala Phe Asn Gln Ser Ser Thr Leu Ala Arg His Lys Ile Ile His Ala Gly Glu Lys Pro Tyr Lys Cys Glu Glu Cys Gly Lys Ala Phe Tyr Gln Tyr Ser Asn Leu Thr Gln His Lys Ile Tle His Thr Gly Glu Lys Pro Tyr Lys Cys Glu Glu Cys Gly Lys Ala Phe Asn Trp Ser Ser Thr Leu Thr Lys His Lys Val Ile His Thr Gly Glu Lys Pro Tyr Lys Cys Lys Glu Cys Gly Lys Ala Phe Asn Gln Cys Ser Asn Leu Thr Thr His Lys Lys Ile His Ala Val Glu Lys Ser Asp Lys <210> 17 <211> 240 <212> PRT
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte ID No: 7487977CD1 <400> 17 Met Ser Lys Pro Val Asp His Val Lys Arg Pro Met Asn Ala Phe Met Val Trp Ser Arg Ala Gln Arg Arg Lys Met Ala Gln Glu Asn Pro Lys Met His Asn Ser Glu Ile Ser Lys Arg Leu Gly Ala Glu Trp Lys Leu Leu Ser Glu Ala Glu Lys Arg Pro Tyr Ile Asp Glu Ala Lys Arg Leu Arg Ala Gln His Met Lys Glu His Pro Asp Tyr Lys Tyr Arg Pro Arg Arg Lys Pro Lys Asn Leu Leu Lys Lys Asp Arg Tyr Val Phe Pro Leu Pro Tyr Leu Gly Asp Thr Asp Pro Leu Lys Ala Ala Gly Leu Pro Val Gly Ala Ser Asp Gly Leu Leu Ser Ala Pro Glu Lys Ala Arg AIa Phe Leu Pro Pro AIa Ser Ala Pro Tyr Ser Leu Leu Asp Pro Ala Gln Phe Ser Ser Ser Ala Ile Gln Lys Met Gly Glu Val Pro His Thr Leu Ala Thr Gly Ala Leu Pro Tyr Ala Ser Thr Leu Gly Tyr Gln Asn Gly Ala Phe Gly Ser Leu Ser Cys Pro Ser Gln His Thr His Thr His Pro Ser Pro Thr Asn Pro Gly Tyr Val Val Pro Cys Asn Cys Thr Ala Trp Ser Ala Ser Thr Leu Gln Pro Pro Val Ala Tyr Ile Leu Phe Pro Gly Met Thr Lys Thr Gly Ile Asp Pro Tyr Ser Ser Ala His Ala Thr Ala Met <210> 18 <211> 555 <212> PRT
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte ID No: 1706514CD1 <400> 18 Met Asp Ser Val Val Phe Glu Asp Val Ala Val Asp Phe Thr Leu Glu Glu Trp Ala Leu Leu Asp Ser Ala Gln Arg Asp Leu Tyr Arg Asp Val Met Leu Glu Thr Phe Arg Asn Leu Ala Ser Val Asp Asp Gly Thr Gln Phe Lys Ala Asn Gly Ser Val Ser Leu Gln Asp Met Tyr Gly Gln Glu Lys Ser Lys Glu Gln Thr Ile Pro Asn Phe Thr Gly Asn Asn Ser Cys Ala Tyr Thr Leu Glu Lys Asn Cys Glu Gly Tyr Gly Thr Glu Asp His His Lys Asn Leu Arg Asn His Met Val Asp Arg Phe Cys Thr His Asn Glu Gly Asn Gln Tyr Gly Glu Ala Ile His Gln Met Pro Asp Leu Thr Leu His Lys Lys Val Ser Ala Gly Glu Lys Pro Tyr Glu Cys Thr Lys Cys Arg Thr Val Phe Thr His Leu Ser Ser Leu Lys Arg His Val Lys Ser His Cys Gly Arg Lys Ala Pro Pro Gly Glu Glu Cys Lys Gln Ala Cys Ile Cys Pro Ser His Leu His Ser His Gly Arg Thr Asp Thr Glu Glu Lys Pro Tyr Lys Cys Gln Ala Cys Gly Gln Thr Phe Gln His Pro Arg Tyr Leu Ser His His Val Lys Thr His Thr Ala Glu Lys Thr Tyr Lys Cys Glu Gln Cys Arg Met Ala Phe Asn Gly Phe Ala Ser Phe Thr Arg His Val Arg Thr His Thr Lys Asp Arg Pro Tyr Lys Cys Gln Glu Cys Gly Arg Ala Phe Ile Tyr Pro Ser Thr Phe Gln Arg His Met Thr Thr His Thr Gly Glu Lys Pro Tyr Lys Cys Gln His Cys Gly Lys Ala Phe Thr Tyr Pro Gln Ala Phe Gln Arg His Glu Lys Thr His Thr Gly Glu Lys Pro Tyr Glu Cys Lys Gln Cys Gly Lys Thr Phe Ser Trp Ser Glu Thr Leu Arg Val His Met Arg Ile His Thr Gly Asp Lys Leu Tyr Lys Cys Glu His Cys Gly Lys Ala Phe 335 . 340 345 Thr Ser Ser Arg Ser Phe Gln Gly His Leu Arg Thr His Thr Gly Glu Lys Pro Tyr Glu Cys Lys Gln Cys Gly Lys Ala Phe Thr Trp Ser Sex Thr Phe Arg Glu His Val Arg Ile His Thr Gln Glu Gln Leu Tyr Lys Cys Glu Gln Cys Gly Lys Ala Phe Thr Ser Ser Arg Ser Phe Arg Gly His Leu Arg Thr His Thr Gly Glu Lys Pro Tyr Glu Cys Lys Gln Cys Gly Lys Thr Phe Thr Trp Ser Ser Thr Phe Arg Glu His Val Arg Ile His Thr Gln Glu Gln Leu His Lys Cys Glu His Cys Gly Lys Ala Phe Thr Ser Ser Arg Ala Phe Gln Gly His Leu Arg Met His Thr Gly Glu Lys Pro Tyr Glu Cys Lys Gln Cys Gly Lys Thr Phe Thr Trp Ser Ser Thr Leu His Asn His Val Arg Met His Thr Gly Glu Lys Pro His Lys Cys Lys Gln Cys Gly Met Ser Phe Lys Trp His Ser Ser Phe Arg Asn His Leu Arg Met His Thr Gly Gln Lys Ser His Glu Cys Gln Ser Tyr Ser Lys Ala Phe Ser Cys Gln Val Ile Leu Ser Lys Thr Ser Glu Ser Thr His <210> 19 <211> 184 <212> PRT
<213> Homo Sapiens <220>
<221> misc feature <223> Incyte ID No: 7488247CD1 <400> 19 Met Ala Asp His Leu Met Leu Ala Glu Gly Tyr Arg Leu Val Gln Arg Pro Pro Ser Ala Ala Ala Ala His Gly Pro His Ala Leu Arg Thr Leu Pro Pro Tyr Ala Gly Pro Gly Leu Asp Ser Gly Leu Arg Pro Arg Gly Ala Pro Leu Gly Pro Pro Pro Pro Arg Gln Pro Gly Ala Leu Ala Tyr Gly Ala Phe Gly Pro Pro Ser Ser Phe Gln Pro Phe Pro Ala Val Pro Pro Pro Ala Ala Gly Ile Ala His Leu Gln Pro Val Ala Thr Pro Tyr Pro Gly Arg Ala Ala Ala Pro Pro Asn Ala Pro Gly Gly Pro Pro Gly Pro Gln Pro Ala Pro Ser Ala Ala Ala Pro Pro Pro Pro Ala His Ala Leu Gly Gly Met Asp Ala Glu Leu Ile Asp Glu Glu Ala Leu Thr Ser Leu Glu Leu Glu Leu Gly Leu His Arg Val Arg Glu Leu Pro Glu Leu Phe Leu Gly Gln Ser Glu Phe Asp Cys Phe Ser Asp Leu Gly Ser Ala Pro Pro Ala Gly Ser Val Ser Cys <210> 20 <211> 553 <212> PRT
<213> Homo Sapiens <220>
<221> misc feature <223> Incyte ID No: 1427269CD1 <400> 20 Met Pro Gly Met Met Glu Lys Gly Pro Glu Leu Leu Gly Lys Asn Arg Ser Ala Asn Gly Ser Ala Lys Ser Pro Ala Gly Gly Gly Gly Ser Gly Ala Ser Ser Thr Asn Gly Gly Leu His Tyr Ser Glu Pro Glu Ser Gly Cys Ser Ser Asp Asp Glu His Asp Val Gly Met Arg Val Gly Ala Glu Tyr Gln Ala Arg Ile Pro Glu Phe Asp Pro Gly Ala Thr Lys Tyr Thr Asp Lys Asp Asn Gly Gly Met Leu Val Trp Ser Pro Tyr His Ser Ile Pro Asp Ala Lys Leu Asp Glu Tyr Ile 95 100 10.5 Ala Ile Ala Lys Glu Lys His Gly Tyr Asn Val Glu Gln Ala Leu Gly Met Leu Phe Trp His Lys His Asn Ile Glu Lys Ser Leu Ala Asp Leu Pro Asn Phe Thr Pro Phe Pro Asp Glu Trp Thr Val Glu Asp Lys Val Leu Phe Glu Gln Ala Phe Ser Phe His Gly Lys Ser Phe His Arg Ile Gln Gln Met Leu Pro Asp Lys Thr Ile Ala Ser Leu Val Lys Tyr Tyr Tyr Ser Trp Lys Lys Thr Arg Ser Arg Thr Ser Leu Met Asp Arg Gln Ala Arg Lys Leu Ala Asn Arg His Asn Gln Gly Asp Ser Asp Asp Asp Val Glu Glu Thr His Pro Met Asp Gly Asn Asp Ser Asp Tyr Asp Pro Lys Lys Glu Ala Lys Lys Glu Gly Asn Thr Glu Gln Pro Val Gln Thr Ser Lys Ile Gly Leu Gly Arg Arg Glu Tyr Gln Ser Leu Gln His Arg His His Ser Gln Arg Ser Lys Cys Arg Pro Pro Lys Gly Met Tyr Leu Thr Gln Glu Asp Val Val Ala Val Ser Cys Ser Pro Asn Ala Ala Asn Thr Ile Leu Arg Gln Leu Asp Met Glu Leu Ile Ser Leu Lys Arg Gln Val Gln Asn Ala Lys Gln Val Asn Ser Ala Leu Lys Gln Lys Met Glu Gly Gly Ile Glu Glu Phe Lys Pro Pro Glu Ser Asn Gln Lys Ile Asn Ala Arg Trp Thr Thr Glu Glu Gln Leu Leu Ala Val Gln Gly Val Arg Lys Tyr Gly Lys Asp Phe Gln Ala Ile Ala Asp Val Ile Gly 365 370 ' 375 Asn Lys Thr Val Gly Gln Val Lys Asn Phe Phe Val Asn Tyr Arg Arg Arg Phe Asn Leu Glu Glu Val Leu Gln Glu Trp Glu Ala Glu Gln Gly Thr Gln Ala Ser Asn Gly Asp Ala Ser Thr Leu Gly.Glu Glu Thr Lys Ser Ala Ser Asn Val Pro Ser Gly Lys Ser Thr Asp Glu Glu Glu Glu Ala Gln Thr Pro Gln Ala Pro Arg Thr Leu Gly Pro Ser Pro Pro Ala Pro Ser Ser Thr Pro Thr Pro Thr Ala Pro Ile Ala Thr Leu Asn Gln Pro Pro Pro Leu Leu Arg Pro Thr Leu Pro Ala Ala Pro Ala Leu His Arg Gln Pro Pro Pro Leu G1n Gln Gln Ala Arg Phe Ile Gln Pro Arg Pro Thr Leu Asn Gln Pro Pro Pro Pro Leu Ile Arg Pro Ala Asn Ser Met Pro Pro Arg Leu Asn Pro Arg Pro Val Leu Ser Thr Val Gly Gly Gln Gln Pro Pro Ser Leu Ile Gly Ile Gln Thr Asp Ser Gln Ser Ser Leu His <210> 21 <211> 371 <212> PRT
<213> Homo sapiens <220>
<221> misc_feature <223> Incyte ID No: 103135CD1 <400> 21 Met Asp Met Ala Gln Glu Pro Val Thr Phe Arg Asp Val Ala Ile Tyr Phe Ser Arg Glu Glu Trp Ala Cys Leu Glu Pro Ser Gln Arg Ala Leu Tyr Arg Asp Val Met Leu Asp Asn Phe Ser Ser Val Ala Ala Leu Gly Phe Cys Ser Pro Arg Pro Asp Leu Val Ser Arg Leu Glu Gln Trp Glu Glu Pro Trp Val Glu Asp Arg Glu Arg Pro Glu 65 70 ~ 75 Phe Gln Ala Val Gln Arg Gly Pro Arg Pro Gly Ala Arg Lys Ser Ala Asp Pro Lys Arg His Cys Asp His Pro Ala Trp Ala His Lys 95 100 . 105 Lys Thr His Val Arg Arg Glu Arg Ala Arg Glu Gly Ser Ser Phe Arg Lys Gly Phe Arg Leu Asp Thr Asp Asp Gly Gln Leu Pro Arg Ala Ala Pro Glu Arg Thr Asp Ala Lys Pro Thr Ala Phe Pro Cys Gln Val Leu Thr Gln Arg Cys Gly Arg Arg Pro Gly Arg Arg Glu 155 l60 165 Arg Arg Lys Gln Arg Ala Val Glu Leu Ser Phe Tle Cys Gly Thr Cys Gly Lys Ala Leu Ser Cys His Ser Arg Leu Leu Ala His Gln 185 l90 195 Thr Val His Thr Gly Thr Lys Ala Phe Glu Cys Pro Glu Cys Gly Gln Thr Phe Arg Trp Ala Ser Asn Leu Gln Arg His Gln Lys Asn His Thr Arg Glu Lys Pro Phe Cys Cys Glu Ala Cys Gly Gln Ala Phe Ser Leu Lys Asp Arg Leu Ala Gln His Arg Lys Val His Thr Glu His Arg Pro Tyr Ser Cys Gly Asp Cys Gly Lys Ala Phe Lys Gln Lys Ser Asn Leu Leu Arg His Gln Leu Val His Thr Gly Glu Arg Pro Phe Tyr Cys Ala Asp Cys Gly Lys Ala Phe Arg Thr Lys Glu Asn Leu Ser His His Gln Arg Val His Ser Gly Glu Lys Pro Tyr Thr Cys Ala Glu Cys Gly Lys Ser Phe Arg Trp Pro Lys Gly Phe Ser Ile His Arg Arg Leu His Leu Thr Lys Arg Phe Tyr Glu Cys Gly His Cys Gly Lys Gly Phe Arg His Leu Gly Phe Phe Thr Arg His Gln Arg Thr His Arg His Gly Glu Val <210> 22 <211> 837 <212> PRT
<213> Homo Sapiens <220>
<221> misc feature <223> Incyte ID No: 1907346CD1 <400> 22 Met Leu Pro Lys Glu Glu Val Trp Lys Lys Arg Lys Arg Lys Glu Lys Glu Ser Gly Met Ala Leu Thr Gln Val Arg Leu Thr Phe Arg Asp Val Ala Ile Glu Phe Ser Gln Glu Glu Trp Lys Cys Leu Asp Pro Ala Gln Arg Ile Leu Tyr Arg Asp Val Met Leu Glu Asn Tyr Trp Asn Leu Val Ser Leu Gly Leu Cys His Phe Asp Met Asn Ile Ile Ser Met Leu Glu Glu Gly Lys Glu Pro Trp Thr Val Lys Ser Cys Val Lys Ile Ala Arg Lys Pro Arg Thr Arg Glu Cys Val Lys Gly Val Val Thr Asp Ile Pro Pro Lys Cys Thr Ile Lys Asp Leu Leu Pro Lys Glu Lys Ser Ser Thr Glu Ala Val Phe His Thr Val Val Leu Glu Arg His Glu Ser Pro Asp Ile Glu Asp Phe Ser Phe Lys Glu Pro Gln Lys Asn Val His Asp Phe Glu Cys Gln Trp Arg Asp Asp Thr Gly Asn Tyr Lys Gly Val Leu Met Ala Gln Lys Glu Gly Lys Arg Asp Gln Arg Asp Arg Arg Asp Ile Glu Asn Lys Leu Met Asn Asn Gln Leu Gly Val Ser Phe His Ser His Leu Pro Glu Leu Gln Leu Phe Gln Gly Glu Gly Lys Met Tyr Glu Cys Asn Gln Val Glu Lys Ser Thr Asn Asn Gly Ser Ser Val Ser Pro Leu Gln Gln Tle Pro Ser Ser Val Gln Thr His Arg Ser Lys Lys Tyr His Glu Leu Asn His Phe Ser Leu Leu Thr Gln Arg Arg Lys Ala Asn Ser Cys Gly Lys Pro Tyr Lys Cys Asn Glu Cys Gly Lys Ala Phe Thr Gln Asn Ser Asn Leu Thr Ser His Arg Arg Ile His Ser Gly Glu Lys Pro Tyr Lys Cys Ser Glu Cys Gly Lys Thr Phe Thr Val Arg Ser Asn Leu Thr Ile His Gln Val Ile His Thr Gly Glu Lys Pro Tyr Lys Cys His Glu Cys Gly Lys Val Phe Arg His Asn Ser Tyr Leu Ala Thr His Arg Arg Ile His Thr Gly Glu Lys Pro Tyr Lys Cys Asn Glu Cys Gly Lys Ala Phe Arg Gly His Ser Asn Leu Thr Thr His Gln Leu Ile His Thr Gly Glu Lys Pro Phe Lys Cys Asn Glu Cys Gly Lys Leu Phe Thr Gln Asn Ser His Leu Ile Ser His Trp Arg Ile His Thr Gly Glu Lys Pro Tyr Lys Cys Asn Glu Cys Gly Lys Ala Phe Ser Val Arg Ser Ser Leu Ala Ile His Gln Thr 21e His Thr Gly Glu Lys Pro Tyr Lys Cys Asn Glu Cys Gly Lys Val Phe Arg Tyr Asn Ser Tyr Leu Gly Arg His Arg Arg VaI

His Thr Gly Glu Lys Pro Tyr Lys Cys Asn Glu Cys Gly Lys Ala Phe Ser Met His Ser Asn Leu Ala Thr His Gln Val Ile His Thr Gly Thr Lys Pro Phe Lys Cys Asn Glu Cys Ser Lys Val Phe Thr Gln Asn Ser Gln Leu Ala Asn His Arg Arg Met His Thr Gly Glu Lys Thr Tyr Lys Cys Asn Glu Cys Gly Lys Ala Phe Ser Val Arg Ser Ser Leu Thr Thr His Gln Ala Ile His Ser Gly Glu Lys Pro Tyr Lys Cys Ile Glu Cys Gly Lys Ser Phe Thr Gln Lys Ser His Leu Arg Ser His Arg Gly Ile His Ser Gly Glu Lys Pro Tyr Lys Cys Asn Glu Cys Gly Lys Val Phe Ala Gln Thr Ser Gln Leu Ala Arg His Trp Arg Val His Thr Gly Glu Lys Pro Tyr Lys Cys Asn Asp Cys Gly Arg Ala Phe Ser Asp Arg Ser Ser Leu Thr Phe His Gln AIa IIe His Thr Gly Glu Lys Pro Tyr Lys Cys His GIu Cys Gly Lys Val Phe Arg His Asn Ser Tyr Leu Ala Thr His Arg Arg Ile His Thr Gly Glu Lys Pro Tyr Lys Cys Asn Glu Cys Gly Lys Ala Phe Ser Met His Ser Asn Leu Thr Thr His Lys Val IIe His Thr Gly Glu Lys Pro Tyr Lys Cys Asn Gln Cys Gly Lys Val Phe Thr Gln Asn Ser His Leu Ala Asn His Gln Arg Thr His Thr Gly Glu Lys Pro Tyr Arg Cys Asn Glu Cys Gly Lys Ala Phe Ser Val Arg Ser Ser Leu Thr Thr His Gln Ala Ile His Thr Gly Lys Lys Pro Tyr Lys Cys Asn Glu Cys Gly Lys Val Phe Thr Gln Asn Ala His Leu Ala Asn His Arg Arg Ile His Thr Gly Glu Lys Pro Tyr Arg Cys Thr Glu Cys Gly Lys Ala Phe Arg Val Arg Ser Ser Leu Thr Thr His Met Ala Ile His Thr Gly Glu Lys Arg Tyr Lys Cys Asn Glu Cys Gly Lys Val Phe Arg Gln Ser Ser Asn Leu Ala Ser His His Arg Met His Thr Gly Glu Lys Pro Tyr Lys <210> 23 <211> 549 <212> PRT
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte ID No: 3041036CD1 <400> 23 Met Ala Ala Gln Leu Leu Thr Asp Glu Ala Leu Glu Ser Val Thr Phe Arg Asp Val Thr Val Asp Phe Thr Gln Glu Glu Trp Gln Gln Leu Glu Pro Ala Gln Lys Asp Leu Tyr Arg Asp Val Met Leu Glu Asn Tyr Arg Asn Leu Val Ser Leu Asp Trp Glu Thr Arg Pro Glu Met Lys Glu Leu Asp Pro Lys Asn Asp Ile Ser Glu Asp Lys Leu Ser Val Val Gly Glu Ala Thr Gly Gly Pro Thr Arg Asn Gly Ala Arg Gly Pro Gly Ser Glu Gly Val Trp Glu Pro Gly Ser Trp Pro Glu Arg Pro Arg Gly Asp Ala Gly Ala Glu Trp Glu Pro Leu Gly Ile Pro Gln Gly Asn Lys Leu Leu Gly Gly Ser Val Pro Ala Cys His Glu Leu Lys Ala Phe Ala Asn Gln Gly Cys Val Leu Val Pro Pro Arg Leu Asp Asp Pro Thr Glu Lys Gly Ala Cys Pro Pro Val Arg Arg Gly Lys Asn Phe Ser Ser Thr Ser Asp Leu Ser Lys Pro Pro Met Pro Cys Glu Glu Lys Lys Thr Tyr Asp Cys Ser Glu Cys Gly Lys Ala Phe Ser Arg Ser Ser Ser Leu Ile Lys His Gln Arg Ile His Thr Gly Glu Lys Pro Phe Glu Cys Asp Thr Cys Gly Lys His Phe Ile Glu Arg Ser Ser Leu Thr Ile His Gln Arg Val His Thr Gly Glu Lys Pro Tyr Ala Cys Gly Asp Cys Gly Lys Ala Phe Ser Gln Arg Met Asn Leu Thr Val His Gln Arg Thr His Thr Gly Glu Lys Pro Tyr Val Cys Asp Val Cys Gly Lys Ala Phe Arg Lys Thr Ser Ser Leu Thr Gln His Glu Arg Ile His Thr Gly Glu Lys Pro Tyr Ala Cys Gly Asp Cys Gly Lys Ala Phe Ser Gln Asn Met His Leu Ile Val His Gln Arg Thr His Thr Gly Glu Lys Pro Tyr Val Cys Pro Glu Cys Gly Arg Ala Phe Ser Gln Asn Met His Leu Thr Glu His Gln Arg Thr His Thr Gly Glu Lys Pro Tyr Ala Cys Lys Glu Cys Gly Lys Ala Phe Asn Lys Ser Ser Ser Leu Thr Leu His Gln Arg Asn His Thr Gly Glu Lys Pro Tyr Val Cys Gly Glu Cys Gly Lys Ala Phe Ser Gln Ser Ser Tyr Leu Ile Gln His Gln Arg Phe His Ile Gly Val Lys Pro Phe Glu Cys Ser Glu Cys Gly Lys Ala Phe Ser Lys Asn Ser Ser Leu Thr Gln His Gln Arg Ile His Thr Gly Glu Lys Pro Tyr Glu Cys Tyr Ile Cys Lys Lys His Phe Thr Gly Arg Ser Ser Leu Ile Val His Gln Ile Val His Thr Gly Glu Lys Pro Tyr Val Cys Gly Glu Cys Gly Lys Ala Phe Ser Gln Ser Ala Tyr Leu Ile Glu His Gln Arg Ile His Thr Gly Glu Lys Pro Tyr Arg Cys Gly Gln Cys Gly Lys Ser Phe Ile Lys Asn Ser Ser Leu Thr Val His Gln Arg Ile His Thr Gly Glu Lys Pro Tyr Arg Cys Gly Glu Cys Gly Lys Thr Phe Ser Arg Asn Thr Asn Leu Thr Arg His Leu Arg Ile His Thr <210> 24 <211> 555 <212> PRT
<213> Homo Sapiens <220>
<221> misc_feature <223> Tncyte ID No: 3856879CD1 <400> 24 Met Ala Ala Ala Arg Leu Leu Pro Val Pro Ala Gly Pro Gln Ala 1 5 10 ~ 15 Lys Leu Thr Phe Glu Asp Val Ala Val Leu Leu Ser Gln Asp Glu Trp Asp Arg Leu Cys Pro Ala Gln Arg Gly Leu Tyr Arg Asn Val Met Met Glu Thr Tyr Gly Asn Val Val Ser Leu Gly Leu Pro Gly Ser Lys Pro Asp Ile Ile Ser Gln Leu Glu Arg Gly Glu Asp Pro Trp Val Leu Asp Arg Lys Gly Ala Lys~Lys Ser Gln Gly Leu Trp Ser Asp Tyr Ser Asp Asn Leu Lys Tyr Asp His Thr Thr Ala Cys Thr Gln Gln Asp Ser Leu Ser Cys Pro Trp Glu Cys Glu Thr Lys Gly Glu Ser Gln Asn Thr Asp Leu Ser Pro Lys Pro Leu Ile Ser Glu Gln Thr Val Ile Leu Gly Lys Thr Pro Leu Gly Arg Ile Asp Gln Glu Asn Asn Glu Thr Lys Gln Ser Phe Cys Leu Ser Pro Asn Ser Val Asp His Arg Glu Val Gln Val Leu Ser Gln Ser Met Pro Leu Thr Pro His Gln Ala Val Pro Ser Gly Glu Arg Pro Tyr Met Cys Val Glu Cys Gly Lys Cys Phe Gly Arg Ser Ser His Leu Leu Gln His Gln Arg Ile His Thr Gly Glu Lys Pro Tyr Val Cys Ser Val Cys Gly Lys Ala Phe Ser Gln Ser Ser Val Leu Ser Lys His Arg Arg Ile His Thr Gly Glu Lys Pro Tyr Glu Cys Asn Glu Cys Gly Lys Ala Phe Arg Val Ser Ser Asp Leu Ala Gln His His Lys Ile His Thr Gly Glu Lys Pro His Glu Cys Leu Glu Cys Arg Lys Ala Phe Thr Gln Leu Ser His Leu Ile Gln His Gln Arg Ile His Thr Gly Glu Arg Pro Tyr Val Cys Pro Leu Cys Gly Lys Ala Phe Asn His Ser Thr Val Leu Arg Ser His Gln Arg Val His Thr Gly Glu Lys Pro His Arg Cys Asn Glu Cys Gly Lys Thr Phe Ser Val Lys Arg Thr Leu Leu Gln His Gln Arg Ile His Thr Gly Glu Lys Pro Tyr Thr Cys Ser Glu Cys Gly Lys Ala Phe Ser Asp Arg Ser Val Leu Ile Gln His His Asn Val His Thr Gly Glu Lys Pro Tyr Glu Cys Ser Glu Cys Gly Lys Thr Phe Ser His Arg Ser Thr Leu Met Asn His Glu Arg Ile His Thr Glu Glu Lys Pro Tyr Ala Cys Tyr Glu Cys Gly Lys Ala Phe Val Gln His Ser His Leu Ile Gln His Gln Arg Val His Thr Gly Glu Lys Pro Tyr Val Cys Gly Glu Cys Gly His Ala Phe Ser Ala Arg Arg Ser Leu Ile Gln His Glu Arg Ile His Thr Gly Glu Lys Pro Phe Gln Cys Thr Glu Cys Gly Lys Ala Phe Ser Leu Lys Ala Thr Leu Ile Val His Leu Arg Thr His Thr Gly Glu Lys Pro Tyr Glu Cys Asn Ser Cys Gly Lys Ala Phe Ser Gln Tyr Ser Val Leu Ile Gln His Gln Arg Ile His Thr Gly Glu Lys Pro Tyr Glu Cys Gly Glu Cys Gly Arg Ala Phe Asn Gln His Gly His Leu Ile Gln His Gln Lys Val His Arg Lys Leu <210> 25 <211> 601 <212> PRT
<213> Homo Sapiens <220>
<221> misc feature <223> Incyte ID No: 4178665CD1 <400> 25 Met Leu Cys Trp Leu Gln Glu Asn Asn Phe Cys Leu Leu Leu Cys Phe Leu Ser Gly Leu Leu Ser Arg His Lys Thr Lys Lys Leu Ser Ser Glu Lys Asp Ile His Glu Ile Ser Leu Ser Lys Glu Ser Ile Ile Glu Lys Ser Lys Thr Leu Arg Leu Lys Gly Ser Ile Phe Arg Asn Glu Trp Gln Asn Lys Ser Glu Phe Glu Gly Gln Gln Gly Leu Lys Glu Arg Ser Ile Ser Gln Lys Lys Ile Val Ser Lys Lys Met Ser Thr Asp Arg Lys Arg Pro Ser Phe Thr Leu Asn Gln Arg Ile His Asn Ser Glu Lys Ser Cys Asp Ser His Leu Val Gln His Gly Lys Ile Asp Ser Asp Val Lys His Asp Cys Lys Glu Cys Gly Ser Thr Phe Asn Asn Val Tyr Gln Leu Thr Leu His Gln Lys Ile His Thr Gly Glu Lys Ser Cys Lys Cys Glu Lys Cys Gly Lys Val Phe Ser His Ser Tyr Gln Leu Thr Leu His Gln Arg Phe His Thr Ghy Glu Lys Pro Tyr Glu Cys Gln Glu Cys Gly Lys Thr Phe Thr Leu Tyr Pro Gln Leu Asn Arg His Gln Lys Ile His Thr Gly Lys Lys Pro Tyr Met Cys Lys Lys Cys Asp Lys Gly Phe Phe Ser Arg Leu Glu Leu Thr Gln His Lys Arg Ile His Thr Gly Lys Lys Ser Tyr Glu Cys Lys Glu Cys Gly Lys Val Phe Gln Leu Ile Phe Tyr Phe Lys Glu His Glu Arg Ile His Thr Gly Lys Lys Pro Tyr Glu Cys Lys Glu Cys Gly Lys Ala Phe Ser Val Cys Gly Gln Leu Thr Arg His Gln Lys Ile His Thr Gly Val Lys Pro Tyr Glu Cys Lys Glu Cys Gly Lys Thr Phe Arg Leu Ser Phe Tyr Leu Thr Glu His Arg Arg Thr His Ala Gly Lys Lys Pro Tyr Glu Cys Lys Glu Cys Gly Lys Ser Phe Asn Val Arg Gly Gln Leu Asn Arg His Lys Thr Ile His Thr Gly Ile Lys Pro'Phe Ala Cys Lys Val Cys Glu Lys Ala Phe Ser Tyr Ser Gly Asp Leu Arg Val His Ser Arg Ile His Thr Gly Glu Lys Pro Tyr Glu Cys Lys Glu Cys Gly Lys Ala Phe Met Leu Arg Ser Val Leu Thr Glu His Gln Arg Leu His Thr Gly Val Lys Pro Tyr Glu Cys Lys Glu Cys Gly Lys Thr Phe Arg Val Arg Ser Gln Ile Ser Leu His Lys Lys Ile His Thr Asp Val Lys Pro Tyr Lys Cys Val Arg Cys Gly Lys Thr Phe Arg Phe Gly Phe Tyr Leu Thr Glu His Gln Arg Ile His Thr Gly Glu Lys Pro Tyr Lys Cys Lys GIu Cys Gly Lys Ala Phe Ile Arg Arg Gly Asn Leu Lys Glu His Leu Lys Ile His Ser Gly Leu Lys Pro Tyr Asp Cys Lys Glu Cys Gly Lys Ser Phe Ser Arg Arg G1y Gln Phe Thr Glu His Gln Lys Ile His Thr Gly Val Lys Pro Tyr Lys Cys Lys Glu Cys Gly Lys Ala Phe Ser Arg Ser Val Asp Leu Arg Ile His Gln Arg Ile His Thr Gly Glu Lys Pro Tyr Glu Cys Lys Gln Cys Gly Lys Ala Phe Arg Leu Asn Ser His Leu Thr Glu His Gln Arg Ile His Thr Gly Glu Lys Pro Tyr Glu Cys Lys Val Cys Arg Lys Ala Phe Arg Gln Tyr Ser His Leu Tyr Gln His Gln Lys Thr His Asn Val Ile <210> 26 <211> 743 <212> PRT
<213> Homo sapiens <220>
<221> misc feature <223> Incyte ID No: 7493326CD1 <400> 26 Met Met Gln Ala Gln Glu Ser Leu Thr Leu Glu Asp Val Ala Val Asp Phe Thr Trp Glu Glu Trp Gln Phe Leu Ser Pro Ala Gln Lys Asp Leu Tyr Arg Asp Val Met Leu Glu Asn Tyr Ser Asn Leu Val Ala Val Gly Tyr Gln Ala Ser Lys Pro Asp Ala Leu Ser Lys Leu Glu Arg Gly Glu Glu Thr Cys Thr.Thr Glu Asp Glu Ile Tyr Ser Arg Ile Cys Ser Asp Sex Gly Gly Ala Ser Gly Gly Ala Tyr Ala Glu Ile Arg Lys Ile Asp Asp Pro Leu Gln His His Leu Gln Asn Gln Ser Ile Gln Lys Sex Val Lys Gln Cys His Glu Gln Asn Met Phe Gly Asn Ile Val Asn Gln Asn Lys Gly His Phe Leu Leu Lys Gln Asp Cys Asp Thr Phe Asp Leu His Glu Lys Pro Leu Lys Ser Asn Leu Ser Phe Glu Asn Gln Lys Arg Ser Ser Gly Leu Lys Asn Ser Ala Glu Phe Asn Arg Asp Gly Lys Ser Leu Phe His Ala Asn His Lys Gln Phe Tyr Thr Glu Met Lys Phe Pro Ala Ile Ala Lys Pro Ile Asn Lys Ser Gln Phe Ile Lys Gln Gln Arg Thr His Asn Ile Glu Asn Ala His Val Cys Ser Glu Cys Gly Lys Ala Phe Leu Lys Leu Ser Gln Phe Ile Asp His Gln Arg Val His Thr Gly Glu Lys Pro His Val Cys Ser Met Cys Gly Lys Ala Phe Ser Arg Lys Ser Arg Leu Met Asp His Gln Arg Thr His Thr Glu Leu Lys His Tyr Glu Cys Thr Glu Cys Asp Lys Thr Phe Leu Lys Lys Ser Gln Leu Asn Ile His Gln Lys Thr His Met Gly Gly Lys Pro Tyr Thr Cys Ser Gln Cys Gly Lys Ala Phe Ile Lys Lys Cys Arg Leu Ile Tyr His Gln Arg Thr His Thr Gly Glu Lys Pro His Gly Cys Ser Val Cys Gly Lys Ala Phe Ser Thr Lys Phe Ser Leu Thr Thr His Gln Lys Thr His Thr Gly Glu Lys Pro Tyr Ile Cys Ser Glu Cys Gly Lys Gly Phe Ile Glu Lys Arg Arg Leu Thr Ala His His Arg Thr His Thr Gly Glu Lys Pro Phe Ile Cys Asn Lys Cys Gly Lys Gly Phe Thr Leu Lys Asn Ser Leu Ile Thr His Gln Gln Thr His Thr Gly Glu Lys Leu Tyr Thr Cys Ser Glu Cys Gly Lys Gly Phe Ser Met Lys His Cys Leu Met Val His Gln Arg Thr His Thr Gly Glu Lys Pro Tyr Lys Cys Asn Glu Cys Gly Lys Gly Phe Ala Leu Lys Ser Pro Leu Ile Arg His Gln Arg Thr His Thr Gly Glu Lys Pro Tyr Val Cys Thr Glu Cys Arg Lys Gly Phe Thr Met Lys Ser Asp Leu Ile Val His Gln Arg Thr His Thr Ala Glu Lys Pro Tyr Ile Cys Asn Asp Cys Gly Lys Gly Phe Thr Val Lys Ser Arg Leu Ile Val His Gln Arg Thr His Thr Gly Glu Lys Pro Tyr Val Cys Gly Glu Cys Gly Lys Gly Phe Pro Ala Lys Ile Arg Leu Met Gly His Gln Arg Thr His Thr Gly Glu Lys Pro Tyr Ile Cys Asn Glu Cys GIy Lys Gly Phe Thr Glu Lys Ser His Leu Asn Val His Arg Arg Thr His Thr GIy Glu Lys Pro Tyr Val Cys Ser Glu Cys Gly Lys Gly Leu Leu Gly Arg Ala Cys Ser Leu His His Gln Ala Asn Ser Tyr Trp Gly Glu Lys Pro Tyr Ile Cys Asn Glu Cys Gly Lys Gly Phe Ser Met Lys Ser Thr Leu Ser Ile His Gln Gln Thr His Thr Gly Glu Lys Pro Tyr Lys Cys Asn Glu Cys Asp Lys Thr Phe Arg Lys Lys Thr Cys Leu Ile Gln His Gln Arg Phe His Thr Gly Lys Thr Ser Phe Ala Cys Thr Glu Cys Gly Lys Phe Ser Leu Arg Lys Asn Asp Leu Ile Thr His Gln Arg Ile His Thr Gly Glu Lys Pro Tyr Lys Cys Ser Asp Cys Gly Lys Ala Phe Thr Thr Lys Ser Gly Leu Asn Val His Gln Arg Lys His Thr Gly Glu Arg Pro Tyr Gly Cys Ser Asp Cys Gly Lys Ala Phe Ala His Leu Ser Ile Leu Val Lys His Lys Arg Ile His Arg <210> 27 <211> 490 <212> PRT
<213> Homo Sapiens <220>
<221> misc feature <223> Incyte ID No: 1553836CD1 <400> 27 Met Lys Met Arg Arg Ile Lys Pro Ala Ala Thr Ser His Val Glu Gly Ser Gly Gly Val Ser Ala Lys Gly Lys Arg Lys Pro Arg Gln Glu Glu Asp Glu Asp Tyr Arg Glu Phe Pro Gln Lys Lys His Lys Leu Tyr Gly Arg Lys Gln Arg Pro Lys Thr Gln Pro Asn Pro Lys Ser Gln Ala Arg Arg Ile Arg Lys Glu Pro Pro Val Tyr Ala Ala Gly Ser Leu Glu Glu Gln Trp Tyr Leu Glu Ile Val Asp Lys Gly Ser Val Ser Cys Pro Thr Cys Gln Ala Val Gly Arg Lys Thr Ile Glu Gly Leu Lys Lys His Met Glu Asn Cys Lys Gln Glu Met Phe Thr Cys His His Cys Gly Lys Gln Leu Arg Ser Leu Ala Gly Met Lys Tyr His Val Met Ala Asn His Asn Ser Leu Pro Ile Leu Lys Ala Gly Asp Glu Ile Asp Glu Pro Ser Glu Arg Glu Arg Leu Arg Thr Val Leu Lys Arg Leu Gly Lys Leu Arg Cys Met Arg Glu Ser Cys Ser Ser Ser Phe Thr Ser Ile Met Gly Tyr Leu Tyr His Val Arg Lys Cys Gly Lys Gly Ala Ala Glu Leu Glu Lys Met Thr Leu Lys Cys His His Cys Gly Lys Pro Tyr Arg Ser Lys Ala Gly Leu Ala Tyr His Leu Arg Ser Glu His Gly Pro Ile Ser Phe Phe Pro Glu Ser Gly Gln Pro Glu Cys Leu Lys Glu Met Asn Leu Glu Ser Lys Ser Gly Gly Arg Val Gln Arg Arg Ser Ala Lys Ile Ala Val Tyr His Leu Gln Glu Leu Ala Ser Ala Glu Leu Ala Lys Glu Trp Pro Lys Arg Lys Val Leu Gln Asp Leu Val Pro Asp Asp Arg Lys Leu Lys Tyr Thr Arg Pro Gly Leu Pro Thr Phe Ser Gln Glu Val Leu His Lys Trp Lys Thr Asp Ile Lys Lys Tyr His Arg Tle Gln Cys Pro Asn Gln Gly Cys Glu Ala Val Tyr Ser Ser Val Ser Gly 335 ~ 340 345 Leu Lys Ala His Leu Gly Ser Cys Thr Leu Gly Asn Phe Val Ala Gly Lys Tyr Lys Cys Leu Leu Cys Gln Lys Glu Phe Val Ser Glu Ser Gly Val Lys Tyr His Ile Asn Ser Val His Ala Glu Asp Trp Phe Val Val Asn Pro Thr Thr Thr Lys Ser Phe Glu Lys Leu Met Lys Ile Lys Gln Arg Gln Gln Glu Glu Glu Lys Arg Arg Gln Gln His Arg Ser Arg Arg Sex Leu Arg Arg Arg Gln Gln Pro Gly Ile Glu Leu Pro Glu Thr Glu Leu Ser Leu Arg Val Gly Lys Asp Gln Arg Arg Asn Asn Glu Glu Leu Val Val Ser Ala Ser Cys Lys Glu Pro Glu Gln Glu Pro Val Pro Ala Gln Phe Gln Lys Val Lys Pro Pro Lys Thr Asn His Lys Arg Gly Arg Lys <210> 28 .
<211> 665 <212> PRT
<213> Homo sapiens <220>
<221> misc_feature <223> Incyte ID No: 1908201CD1 <400> 28 Met Pro Leu Arg Asp Lys Tyr Cys Gln Thr Asp His His His His Gly Cys Cys Glu Pro Val Tyr Ile Leu Glu Pro Gly Asp Pro Pro Leu Leu Gln Gln Pro Leu Gln Thr Ser Lys Ser Gly Ile Gln Gln Ile Ile Glu Cys Phe Arg Ser Gly Thr Lys Gln Leu Lys His Ile Leu Leu Lys Asp VaI Asp Thr Ile Phe Glu Cys Lys Leu Cys Arg Ser Leu Phe Arg Gly Leu Pro Asn Leu Ile Thr His Lys Lys Phe Tyr Cys Pro Pro Ser Leu Gln Met Asp Asp Asn Leu Pro Asp Val Asn Asp Lys Gln Ser Gln Ala Ile Asn Asp Leu Leu Glu Ala Ile Tyr Pro Ser Val Asp Lys Arg Glu Tyr Ile Ile Lys Leu Glu Pro Ile Glu Thr Asn Gln Asn Ala Val Phe Gln Tyr Ile Ser Arg Thr Asp Asn Pro Ile Glu Val Thr Glu Ser Ser Ser Thr Pro Glu Gln Thr Glu Val Gln Ile Gln Glu Thr Ser Thr Glu Gln Ser Lys Thr Val Pro Val Thr Asp Thr Glu Val Glu Thr Val Glu Pro Pro Pro Val Glu Ile Val Thf Asp Glu Val Ala Pro Thr Ser Asp Glu Gln Pro Gln Glu Ser Gln Ala Asp Leu Glu Thr Ser Asp Asn Ser Asp Phe Gly His Gln Leu Ile Cys Cys Leu Cys Arg Lys Glu Phe Asn Ser Arg Arg Gly Val Arg Arg His Ile Arg Lys Val His Lys Lys Lys Met Glu Glu Leu Lys Lys Tyr Ile Glu Thr Arg Lys Asn Pro 2&0 265 270 Asn Gln Ser Ser Lys Gly Arg Ser Lys Asn Val Leu Val Pro Leu Ser Arg Ser Cys Pro Val Cys Cys Lys Ser Phe Ala Thr Lys Ala Asn Val Arg Arg His Phe Asp Glu Val His Arg Gly Leu Arg Arg Asp Ser Ile Thr Pro Asp Ile Ala Thr Lys Pro Gly Gln Pro Leu Phe Leu Asp Ser Ile Ser Pro Lys Lys Ser Phe Lys Thr Arg Lys Gln Lys Ser Ser Ser Lys Ala Glu Tyr Asn Leu Thr Ala Cys Lys Cys Leu Leu Cys Lys Arg Lys Tyr Ser Ser Gln Ile Met Leu Lys Arg His Met Gln Ile Val His Lys Tle Thr Leu Ser Gly Thr Asn Ser Lys Arg GIu Lys Gly Pro Asn Asn Thr Ala Asn Ser Ser Glu Ile Lys Val Lys Val Glu Pro Ala Asp Ser Val Glu Ser Ser Pro Pro Ser Ile Thr His Ser Pro Gln Asn Glu Leu Lys Gly Thr Asn His Ser Asn Glu Lys Lys Asn Thr Pro Ala Ala Gln Lys Asn Lys Val Lys Gln Asp Ser Glu Ser Pro Lys Ser Thr Ser Pro Ser Ala Ala Gly Gly Gln Gln Lys Thr Arg Lys Pro Lys Leu Ser Ala Gly Phe Asp Phe Lys Gln Leu Tyr Cys Lys Leu Cys Lys Arg Gln Phe Thr Ser Lys Gln Asn Leu Thr Lys His Ile Glu Leu His Thr Asp Gly Asn Asn Ile Tyr Val Lys Phe Tyr Lys Cys Pro Leu Cys Thr Tyr Glu Thr Arg Arg Lys Arg Asp Val Ile Arg His Ile Thr Val Val His Lys Lys Ser Ser Arg Tyr Leu Gly Lys Ile Thr Ala Ser Leu Glu Ile Arg Ala Ile Lys Lys Pro Ile Asp Phe Val Leu Asn Lys Val Ala Lys Arg Gly Pro Ser Arg Asp Glu Ala Lys His Ser Asp Ser Lys His Asp Gly Thr Ser Asn Ser Pro Ser Lys Lys Tyr Glu Val Ala Asp Val Gly Ile Glu Val Lys Val Thr Lys Asn Phe Ser Leu His Arg Cys Asn Lys Cys Gly Lys Ala Phe Ala Lys Lys Thr Tyr Leu Glu His His Lys Lys Thr His Lys Ala Asn Ala Ser Asn Ser Pro Glu Gly Asn Lys Thr Lys Gly Arg Ser Thr Arg Ser Lys Ala Leu Val Trp <210> 29 <211> 570 <212> PRT
<213> Homo Sapiens <220>
<221> misc-feature <223> Incyte ID No: 2827615CD1 <400> 29 Met Ser Lys Asp Leu Val Thr Phe Gly Asp Val Ala Val Asn Phe Ser Gln Glu Glu Trp Glu Trp Leu Asn Pro Ala Gln Arg Asn Leu Tyr Arg Lys Val Met Leu Glu Asn Tyr Arg Ser Leu Val Ser Leu Ala Gly Val Ser Val Ser Lys Pro Asp Val Ile Ser Leu Leu Glu Gln Gly Lys Glu Pro Trp Met Val Lys Lys Glu Gly Thr Arg Gly Pro Cys Pro Asp Trp Glu Tyr Val Phe Lys Asn Ser Glu Phe Ser Ser Lys Gln Glu Thr Tyr Glu Glu Ser Ser Lys Val Val Thr Val Gly Ala Arg His Leu Ser Tyr Ser Leu Asp Tyr Pro Ser Leu Arg Glu Asp Cys Gln Ser Glu Asp Trp Tyr Lys Asn Gln Leu Gly Ser Gln Glu Val His Leu Ser Gln Leu Ile Ile Thr His Lys Glu Ile Leu Pro Glu Val Gln Asn Lys Glu Tyr Asn Lys Ser Trp Gln Thr Phe His Gln Asp Thr Ile Phe Asp Ile Gln Gln Ser Phe Pro Thr Lys Glu Lys Ala His Lys His Glu Pro Gln Lys Lys Ser Tyr Arg Lys Lys Ser Val Glu Met Lys His Arg Lys Val Tyr Val Glu Lys Lys Leu Leu Lys Cys Asn Asp Cys Glu Lys Val Phe Asn Gln Ser Ser Ser Leu Thr Leu His Gln Arg Ile His Thr Gly Glu Lys Pro Tyr Ala Cys Val Glu Cys Gly Lys Thr Phe Ser Gln Ser Ala Asn Leu Ala Gln His Lys Arg Ile His Thr Gly Glu Lys Pro Tyr Glu Cys Lys Glu Cys Arg Lys Ala Phe Ser Gln Asn Ala His Leu Ala Gln His Gln Arg Val His Thr Gly Glu Lys Pro Tyr Gln Cys Lys Glu Cys Lys Lys Ala Phe Ser Gln Ile Ala His Leu Thr Gln His Gln Arg Val His Thr Gly Glu Arg Pro Phe Glu Cys Ile Glu Cys Gly Lys Ala Phe Ser Asn Gly Ser Phe Leu Ala Gln His Gln Arg Ile His Thr Gly Glu Lys Pro Tyr Val Cys Asn Val Cys Gly Lys Ala Phe Ser His Arg Gly Tyr Leu Ile Val His Gln Arg Ile His Thr Gly Glu Arg Pro Tyr Glu Cys Lys Glu Cys Arg Lys Ala Phe Ser Gln Tyr Ala His Leu Ala Gln His Gln Arg Val His Thr Gly Glu Lys Pro Tyr Glu Cys Lys Val Cys Arg Lys Ala Phe Ser Gln Ile Ala Tyr Leu Asp Gln His Gln Arg Val His Thr Gly Glu Lys Pro Tyr Glu Cys Ile Glu Cys Gly Lys Ala Phe Ser Asn Ser Ser Ser Leu Ala Gln His Gln Arg Ser His Thr Gly Glu Lys Pro Tyr Met Cys Lys Glu Cys Arg Lys Thr Phe Ser Gln Asn Ala Gly Leu Ala Gln His Gln Arg Ile His Thr Gly Glu Lys Pro Tyr Glu Cys Asn Val Cys Gly Lys Ala Phe Ser Tyr Ser Gly Ser Leu Thr Leu His Gln Arg Ile His Thr Gly Glu Arg Pro Tyr Glu Cys Lys Asp Cys Arg Lys Ser Phe Arg Gln Arg Ala His Leu Ala His His Glu Arg Ile His Thr Met Glu Ser Phe Leu Thr Leu Ser Ser Pro Ser Pro Ser Thr Ser Asn Gln Leu Pro Arg Pro Val G1y Phe Ile Ser <210> 30 <211> 1712 <212> PRT
<213> Homo Sapiens <220>
<22l> misc_feature <223> Incyte ID No: 4304550CD1 <400> 30 Met Glu Arg Asn Val Leu Thr Thr Phe Ser Gln Glu Met Ser Gln Leu Ile Leu Asn Glu Met Pro Lys Ala Glu Tyr Ser Ser Leu Phe Asn Asp Phe Val Glu Ser Glu Phe Phe Leu Ile Asp Gly Asp Ser Leu Leu Ile Thr Cys Ile Cys Glu Ile Ser Phe Lys Pro Gly Gln Asn Leu His Phe Phe Tyr Leu Val Glu Arg Tyr Leu Val Asp Leu Ile Ser Lys Gly Gly Gln Phe Thr Ile Val Phe Phe Lys Asp Ala Glu Tyr Ala Tyr Phe Asn Phe Pro Glu Leu Leu Ser Leu Arg Thr Ala Leu Ile Leu His Leu Gln Lys Asn Thr Thr Ile Asp Val Arg Thr Thr Phe Ser Arg Cys Leu Ser Lys Glu Trp Gly Ser Phe Leu 125 l30 135 Glu Glu Ser Tyr Pro Tyr Phe Leu Ile Val Ala Asp Glu Gly Leu Asn Asp Leu Gln Thr Gln Leu Phe Asn Phe Leu Ile Ile His Ser Trp Ala Arg Lys Val Asn Val Val Leu Ser Ser Gly Gln Glu Ser Asp Val Leu Cys Leu Tyr Ala Tyr Leu Leu Pro Ser Met Tyr Arg His Gln Ile Phe Ser Trp Lys Asn Lys Gln Asn Ile Lys Asp Ala 200 205 210 ., Tyr Thr Thr Leu Leu Asn Gln Leu Glu Arg Phe Lys Leu Ser Ala Leu Ala Pro Leu Phe Gly Ser Leu Lys Trp Asn Asn Ile Thr Glu Glu Ala His Lys Thr Val Ser Leu Leu Thr Gln Val Trp Pro Glu Gly Ser Asp Ile Arg Arg Val Phe Cys Val Thr Ser Cys Ser Leu Ser Leu Arg Met Tyr His Arg Phe Leu Gly Asn Arg Glu Pro Ser Ser Gly Gln Glu Thr Glu Ile Gln Gln Val Asn Ser Asn Cys Leu Thr Leu Gln Glu Met Glu Asp Leu Cys Lys Leu His Cys Leu Thr Val Val Phe Leu Leu His Leu Pro Leu Ser Gln Arg Ala Cys Ala Arg Val Ile Thr Ser His Trp Ala Glu Asp Met Lys Pro Leu Leu Gln Met Lys Lys Trp Cys Glu Tyr Phe Ile Leu Arg Asn Ile His Thr Phe Glu Phe Trp Asn Leu Asn Leu Ile His Leu Ser Asp Leu Asn Asp Glu Leu Leu Leu Lys Asn Ile Ala Phe Tyr Tyr Glu Asn Glu Asn Val Lys Gly Leu His Leu Asn Leu Gly Asp Thr Ile Met 395 400 . 405 Lys Asp Tyr Glu Tyr Leu Trp Asn Thr Ile Ser Lys Leu Val Arg Asp Phe Glu Val Gly Gln Pro Phe Pro Leu Arg Thr Thr Lys Val Cys Phe Leu Glu Lys Lys Pro Ser Pro Ile Lys Asp Ser Ser Asn Glu Met Val Pro Asn Leu Gly Phe Ile Pro Thr Ser Ser Phe Val Val Asp Lys Phe Ala Gly Asp Ile Leu Lys Asp Leu Pro Phe Leu Lys Ser Asp Asp Pro Ile Val Thr Ser Leu Val Lys Gln Lys Glu Phe Asp Glu Leu Val His Trp His Ser His Lys Pro Leu Ser Asp Asp Tyr Asp Arg Ser Arg Cys Gln Phe Asp Glu Lys Ser Arg Asp Pro Arg Val Leu Arg Ser Val Gln Lys Tyr His Val Phe Gln Arg Phe Tyr Gly Asn Ser Leu Glu Thr Val Ser Ser Lys Ile Ile Val Thr Gln Thr Ile Lys Ser Lys Lys Asp Phe Ser Gly Pro Lys Ser Lys Lys Ala His Glu Thr Lys Ala Glu Ile Ile Ala Arg Glu Asn Lys Lys Arg Leu Phe Ala Arg Glu Glu Gln Lys Glu Glu Gln Lys Trp Asn Ala Leu Ser Phe Ser Ile Glu Glu Gln Leu Lys Glu Asn Leu His Ser Gly Ile Lys Ser Leu Glu Asp Phe Leu Lys Ser Cys Lys Ser Ser Cys Val Lys Leu Gln Val Glu Met Val Gly Leu Thr Ala Cys Leu Lys Ala Trp Lys Glu His Cys Arg Ser Glu Glu Gly Lys Thr Thr Lys Asp Leu Ser Ile Ala Val Gln Val Met Lys Arg Ile His Ser Leu Met Glu Lys Tyr Ser Glu Leu Leu Gln Glu Asp Asp Arg Gln Leu Ile Ala Arg Cys Leu Lys Tyr Leu Gly Phe Asp Glu Leu Ala Ser Ser Leu His Pro Ala Gln Asp Ala Glu Asn Asp Val Lys Val Lys Lys Arg Asn Lys Tyr Ser Val Gly Ile Gly Pro Ala Arg Phe Gln Leu Gln Tyr Met Gly His Tyr Leu Ile Arg Asp G1u Arg Lys Asp Pro Asp Pro Arg Val Gln Asp Phe Ile Pro Asp Thr Trp Gln Arg Glu Leu Leu Asp Val Val Asp Lys Asn Glu Ser Ala Val Ile Val Ala Pro Thr Ser Ser Gly Lys Thr Tyr Ala Ser Tyr Tyr Cys Met Glu Lys Val Leu Lys Glu Ser Asp Asp Gly Val Val Val Tyr Val Ala Pro Thr Lys Ala Leu Val Asn Gln Val Ala Ala Thr Val Gln Asn Arg Phe Thr Lys Asn Leu Pro Ser Gly Glu Val Leu Cys Gly Val Phe Thr Arg Glu Tyr Arg His Asp Ala Leu Asn Cys Gln Val Leu Ile Thr Val Pro Ala Cys Phe Glu Ile Leu Leu Leu Ala Pro His Arg Gln Asn Trp Val Lys Lys Ile Arg Tyr Val Ile Phe Asp Glu Val His Cys Leu Gly Gly Glu Ile Gly Ala Glu Ile Trp Glu His Leu Leu Val Met Ile Arg Cys Pro Phe Leu Ala Leu Ser Ala Thr Ile Ser Asn Pro Glu His Leu Thr Glu Trp Leu Gln Ser Val Lys Trp Tyr Trp Lys Gln Glu Asp Lys Ile Ile Glu Asn Asn Thr Ala Ser Lys Arg His Val Gly Arg Gln Ala Gly Phe Pro Lys Asp Tyr Leu Gln Val Lys Gln Ser Tyr Lys Val Arg Leu Val Leu Tyr Gly Glu Arg Tyr Asn Asp Leu Glu Lys His Val Cys Ser Ile Lys His Gly Asp Ile His Phe Asp His Phe His Pro Cys Ala Ala Leu Thr Thr Asp His Ile Glu Arg Tyr Gly Phe Pro Pro Asp Leu Thr Leu Ser Pro Arg Glu Ser Ile Gln Leu Tyr Asp Ala Met Phe GIn Ile Trp Lys Ser Trp Pro Arg Ala Gln Glu Leu Cys Pro Glu Asn Phe Tle His Phe Asn Asn Lys Leu Val Ile Lys Lys Met Asp Ala Arg Lys Tyr Glu Glu Ser Leu Lys Ala Glu Leu Thr Ser Trp Ile Lys Asn Gly Asn Val Glu Gln Ala Arg Met Val Leu Gln Asn Leu Ser Pro Glu Ala Asp Leu Ser Pro Glu Asn Met Ile Thr Met Phe Pro Leu Leu Val Glu Lys Leu Arg Lys Met Glu Lys Leu Pro Ala Leu Phe Phe Leu Phe Lys Leu Gly Ala Val Glu Asn Ala A1a Glu Ser Val Ser Thr Phe Leu Lys Lys Lys Gln Glu Thr Lys Arg Pro Pro Lys Ala Asp Lys Glu Ala His Val Met Ala Asn Lys Leu Arg Lys Val Lys Lys Ser Ile Glu Lys Gln Lys Ile Ile Asp Glu Lys Ser Gln Lys Lys Thr Arg Asn Val Asp Gln Ser Leu Ile His Glu Ala Glu His Asp Asn Leu Val Lys Cys Leu Glu Lys Asn Leu Glu Ile Pro Gln Asp Cys Thr Tyr Ala Asp Gln Lys Ala Val Asp Thr Glu Thr Leu Gln Arg Val Phe Gly Arg Val Lys Phe Glu Arg Lys Gly Glu Glu Leu Lys Ala Leu Ala Glu Arg Gly Ile Gly Tyr His His Ser Ala Met Ser Phe Lys Glu Lys Gln Leu Val Glu Ile Leu Phe Arg Lys Gly Tyr Leu Arg Val Val Thr Ala Thr Gly Thr Leu Ala Leu Gly Val Asn Met Pro Cys Lys Ser Val Val Phe Ala Gln Asn Ser Val Tyr Leu Asp Ala Leu Asn Tyr Arg Gln Met Ser Gly Arg Ala Gly Arg Arg Gly Gln Asp Leu Met Gly Asp Val Tyr Phe Phe Asp Ile Pro Phe Pro Lys Ile Gly Lys Leu Ile Lys Ser Asn Val Pro Glu Leu Arg Gly His Phe Pro Leu Ser Ile Thr Leu VaI Leu Arg Leu Met Leu Leu Ala Ser Lys Gly Asp Asp Pro Glu Asp Ala Lys Ala Lys Val Leu Ser Val Leu Lys His Ser Leu Leu Ser Phe Lys Gln Pro Arg Val Met Asp Met Leu Lys Leu Tyr Phe Leu Phe Ser Leu Gln Phe Leu Val Lys Glu Gly Tyr Leu Asp Gln Glu Gly Asn Pro Met Gly Phe Ala Gly Leu Val Ser His Leu His Tyr His Glu Pro Ser Asn Leu Val Phe Val Ser Phe Leu Val Asn Gly Leu Phe His Asp Leu Cys Gln Pro Thr Arg Lys Gly Ser Lys His Phe Ser Gln Asp Val Met Glu Lys Leu Val Leu Val Leu Ala His Leu Phe Gly Arg Arg Tyr Phe Pro Pro Lys Phe Gln Asp Ala His Phe Glu Phe Tyr Gln Ser Lys Val Phe Leu Asp Asp Leu Pro Glu Asp Phe Ser Asp Ala Leu Asp Glu Tyr Asn Met Lys Ile Met Glu Asp Phe Thr Thr Phe Leu Arg Ile Val Ser Lys Leu Ala Asp Met Asn Gln Glu Tyr Gln Leu Pro Leu Ser Lys Ile Lys Phe Thr Gly Lys Glu Cys Glu Asp Ser Gln Leu Val Ser His Leu Met Ser Cys Lys Glu Gly Arg Val Ala Ile Ser Pro Phe Val Cys Leu Ser Gly Asn Phe Asp Asp Asp Leu Leu Arg Leu Glu Thr Pro Asn His Val Thr Leu Gly Thr Ile Gly Val Asn Arg Ser Gln Ala Pro Val Leu Leu Ser Gln Lys Phe Asp Asn Arg Gly Arg Lys Met Ser Leu Asn Ala Tyr Ala Leu Asp Phe Tyr Lys His Gly Ser Leu Ile Gly Leu Val Gln Asp Asn Arg Met Asn Glu Gly Asp Ala Tyr Tyr Leu Leu Lys Asp Phe Ala Leu Thr Ile Lys Ser Ile Ser Val Ser Leu Arg Glu Leu Cys Glu Asn Glu Asp Asp Asn Val Val Leu Ala Phe Glu Gln Leu Ser Thr Thr Phe Trp Glu Lys Leu Asn Lys Val <210> 31 <211> 780 <212> PRT
<213> Homo sapiens <220>
<221> mist feature <223> Incyte ID No: 7473738CD1 <400> 31 Met Ser Arg Phe Pro Ala Val Ala Gly Arg Ala Pro Arg Arg Gln Glu Glu Gly Glu Arg Pro Ile Glu Leu Gln Glu Glu Arg Pro Ser Ala Val Arg Ile Ala Asp Arg Glu Glu Lys Gly Cys Thr Ser Gln Glu Gly Gly Thr Thr Pro Thr Phe Pro Ile Gln Lys Gln Arg Lys Lys Leu Ile Gln Ala Val Arg Asp Asn Ser Phe Leu Ile Val Thr Gly Asn Thr Gly Ser Gly Lys Thr Thr Gln Leu Pro Lys Tyr Leu Tyr Glu Ala Gly Phe Ser Gln His Gly Met Ile Gly Val Thr Gln Pro Arg Lys Val Ala Ala Ile Ser Val Ala Gln Arg Val Ala Glu Glu Met Lys Cys Thr Leu Gly Ser Lys Val Gly Tyr Gln Val Arg Phe Asp Asp Cys Ser Ser Lys Glu Thr Ala Ile Lys Tyr Met Thr l40 145 150 Asp Gly Cys Leu Leu Lys His Ile Leu Gly Asp Pro Asn Leu Thr Lys Phe Ser Val Ile Ile Leu Asp Glu Ala His Glu Arg Thr Leu Thr Thr Asp Ile Leu Phe Gly Leu Leu Lys Lys Leu Phe Gln Glu Lys Ser Pro Asn Arg Lys Glu His Leu Thr Ser Gly Gly Thr Cys His Ala Thr Met Glu Leu Ala Lys Leu Ser Ala Phe Phe Gly Asn Cys Pro Ile Phe Asp Ile Pro Gly Arg Leu Tyr Pro Val Arg Glu Lys Phe Cys Asn Leu Ile Gly Pro Arg Asp Arg Glu Asn Thr Ala Tyr Ile Gln Ala Ile Val Lys Val Thr Met Asp Ile His Leu Asn Glu Met Ala Gly Asp Ile Leu Val Phe Leu Thr Gly Gln Phe Glu Ile G1u Lys Ser Cys Glu Leu Leu Phe Gln Met Ala Glu Ser Val Asp Tyr Asp Tyr Asp Val Gln Asp Thr Thr Leu Asp Gly Leu Leu 305 310 3l5 Ile Leu Pro Cys Tyr Gly Ser Met Thr Thr Asp Gln Gln Arg Arg Ile Phe Leu Pro Pro Pro Pro Gly Ile Arg Lys Cys Val Ile Ser Thr Asn Ile Ser Ala Thr Ser Leu Thr Ile Asp Gly Ile Arg Tyr Val Val Asp Gly Gly Phe Val Lys Gln Leu Asn His Asn Pro Arg Leu Gly Leu Asp Ile Leu Glu Val Val Pro Ile Ser Lys Ser Glu Ala Leu Gln Arg Ser Gly Arg Ala Gly Arg Thr Ser Ser Gly Lys Cys Phe Arg Ile Tyr Ser Lys Asp Phe Trp Asn Gln Cys Met Pro Asp His Val Ile Pro Glu Ile Lys Arg Thr Ser Leu Thr Ser Val Val Leu Thr Leu Lys Cys Leu Ala Ile His Asp Val Ile Arg Phe Pro Tyr Leu Asp Pro Pro Asn Glu Arg Leu Ile Leu Glu Ala Leu Lys Gln Leu Tyr Gln Cys Asp Ala Ile Asp Arg Ser Gly His Val Thr Arg Leu Gly Leu Ser Met Val Glu Phe Pro Leu Pro Pro His Leu Thr Cys Ala Val Ile Lys Ala Ala Ser Leu Asp Cys Glu Asp Leu Leu Leu Pro Ile Ala Ala Met Leu Ser Val Glu Asn Val Phe Ile Arg Pro Val Asp Pro Glu Tyr Gln Lys Glu Ala Glu Gln Arg His Arg Glu Leu Ala Ala Lys Ala Gly Gly Phe Asn Asp Phe Ala Thr Leu Ala Val Ile Phe Glu Gln Cys Lys Ser Ser Gly Ala Pro Ala Ser Trp Cys Gln Lys His Trp Ile His Trp Arg Cys Leu Phe Ser Ala Phe Arg Val Glu Ala Gln Leu Arg Glu Leu Ile Arg Lys Leu Lys Gln Gln Ser Asp Phe Pro Lys Glu Thr Phe Glu Gly Pro Lys His Glu Val Leu Arg Arg Cys Leu Cys Ala Gly Tyr Phe Lys Asn Val Ala Arg Arg Ser Val Gly Arg Thr Phe Cys Thr Met Asp Gly Arg Gly Ser Pro Val His Ile His~Pro Ser Ser Ala Leu His Glu Gln Glu Thr Lys Leu Glu Trp Ile Ile Phe His Glu Val Leu Val Thr Thr Lys Val Tyr Ala Arg Ile Val Cys Pro Ile Arg Tyr Glu Trp Val Arg Asp Leu Leu Pro Lys Leu His Glu Leu Asn Ala His Asp Leu Ser Ser Val Ala Arg Arg Glu Met Arg Glu Asp Ala Arg Arg Lys Trp Thr Asn Lys Glu Asn Val Lys Gln Leu Lys Asp Gly Ile Ser Lys Glu Val Leu Lys Lys Met Gln Arg Arg Asn Asp Asp Lys Ser Ile Ser Asp Ala Arg Ala Arg Phe Leu Glu Arg Lys Gln Gln Arg Ile Gln Asp His Ser Asp Thr Leu Lys Glu Thr Gly <210> 32 <211> 648 <212> PRT
<213> Homo sapiens <220>
<221> misc_feature <223> Incyte ID No: 4447743CD1 <400> 32 Met Glu Leu Thr Phe Arg Asp Val Ile Glu Phe Val Ala Ser Pro Glu Glu Trp Cys Leu Asp Pro Ala Gln Asn Leu Lys Gln Tyr Arg Asp Val Met Leu Glu Asn Tyr Arg Asn Leu Val Ser Leu Gly Phe Val Ile Ser Asn Pro Asp Leu Val Thr Cys Leu Glu Gln Ile Lys Glu Pro Cys Asn Leu Lys Ile His Glu Thr Ala Ala Lys Pro Pro Ala Ile Cys Ser Pro Phe Ser Gln Asp Leu Ser Pro Val Gln Gly Ile Glu Asp Ser Phe His Lys Leu Ile Leu Lys Arg Tyr Glu Lys Cys Gly His Glu Asn Leu Gln Leu Arg Lys Gly Cys Lys Arg Val l10 115 120 Asn Glu Cys Lys Val Gln Lys Gly Val Asn Asn Gly Val Tyr Gln Cys Leu Ser Thr Thr Gln Ser Lys Ile Phe Gln Cys Asn Thr Cys 140 l45 150 Val Lys Val Phe Ser Lys Phe Ser Asn Ser Asn Lys His Lys Ile Arg His Thr Gly Glu Lys Pro Phe Lys Cys Thr Glu Cys Gly Arg Ser Phe Tyr Met Ser His Leu Thr Gln His Thr Gly Ile His Ala Gly Glu Lys Pro Tyr Lys Cys Glu Lys Cys Gly Lys Ala Phe Asn Arg Ser Thr Ser Leu Ser Lys His Lys Arg Ile His Thr Gly Glu Lys Pro Tyr Thr Cys Glu Glu Cys Gly Lys Ala Phe Arg Arg Ser Thr Val Leu Asn Glu His Lys Lys Ile His Thr Gly Glu Lys Pro Tyr Lys Cys Glu Glu Cys Gly Lys Ala Phe Thr Arg Ser Thr Thr Leu Asn Glu His Lys Lys Ile His Thr Gly Glu Lys Pro Tyr Lys Cys Lys Glu Cys Gly Lys Ala Phe Arg Trp Ser Thr Ser Leu Asn Glu His Lys Asn Ile His Thr Gly Glu Lys Pro Tyr Lys Cys Lys Glu Cys Gly Lys Ala Phe Arg Gln Ser Arg Ser Leu Asn Glu His Lys Asn Ile His Thr Gly Glu Lys Pro Tyr Thr Cys Glu Lys Cys Gly Lys Ala Phe Asn Gln Ser Ser Ser Leu Ile Ile His Arg Ser Ile His Ser Glu Gln Lys Leu Tyr Lys Cys Glu Glu Cys Gly Lys Ala Phe Thr Trp Ser Ser Ser Leu Asn Lys His Lys Arg Ile His Thr GIy Glu Lys Pro Tyr Thr Cys GIu Glu Cys GIy Lys Ala Phe Tyr Arg Ser Ser His Leu Ala Lys His Lys Arg Tle His Thr Gly Glu Lys Pro Tyr Thr Cys Glu Glu Cys Gly Lys Ala Phe Asn Gln Ser Ser Thr Leu Ile Leu His Lys Arg Ile His Ser Gly Gln Lys Pro Tyr Lys Cys Glu Glu Cys Gly Lys Ala Phe Thr Arg Ser Thr Thr Leu Asn Glu His Lys Lys Ile His Thr Gly Glu Lys Pro Tyr Lys Cys Glu Glu Cys Gly Lys Ala Phe Ile Trp Ser Ala Ser Leu Asn Glu His Lys Asn Ile His Thr Gly Glu Lys Pro Tyr Lys Cys Lys Glu Cys Gly Lys Ala Phe Asn Gln Ser Ser Gly Leu Ile Ile His Arg Ser Ile His Ser Glu Gln Lys Leu Tyr Lys Cys Glu Glu Cys Gly Lys Ala Phe Thr Arg Ser Thr Ala Leu Asn Glu His Lys Lys Ile His Ser Gly Glu Lys Pro Tyr Lys Cys Lys Glu Cys Gly Lys Ala Tyr Asn Leu Ser Ser Thr Leu Thr Lys His Lys Arg Ile His Thr Gly Glu Lys Pro Phe Thr Cys Glu Glu Cys Gly Lys Ala Phe Asn Trp Ser Ser Ser Leu Thr Lys His Lys Ile Ile His Thr Gly Glu Lys Ser Tyr Lys Cys Glu Glu Cys Gly Lys Ala Phe Asn Arg Pro Ser Thr Leu Thr Val His Lys Arg Ile His Thr Gly Lys Glu His Ser <210> 33 <211> 602 <212> PRT
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte ID No: 7497554CD1 <400> 33 Met Ser Glu Arg Arg Arg Ser Ala Val Ala Leu Ser Ser Arg Ala His Ala Phe Ser Val Glu Ala Leu Ile Gly Ser Asn Lys Lys Arg Lys Leu Arg Asp Trp Glu Glu Lys Gly Leu Asp Leu Ser Met Glu Ala Leu Ser Pro Ala Gly Pro Leu Gly Asp Thr Glu Asp Ala Ala Ala His Gly Leu Glu Pro His Pro Asp Ser Glu Gln Ser Thr Gly Ser Asp Ser Glu Val Leu Thr Glu Arg Thr Ser Cys Ser Phe Ser Thr His Thr Asp Leu Ala Ser Gly Ala Ala Gly Pro Val Pro Ala Ala Met Ser Ser Met Glu Glu Ile Gln Val Glu Leu Gln Cys Ala Asp Leu Trp Lys Arg Phe His Asp Ile Gly Thr Glu Met Ile Ile Thr Lys Ala Gly Arg Arg Met Phe Pro Ala Met Arg Val Lys Ile Thr Gly Leu Asp Pro Asn Gln Gln Tyr Tyr Ile Ala Met Asp Ile Val Pro Val Asp Asn Lys Arg Tyr Arg Tyr Val Tyr His Ser Ser Lys Trpe Met Val Ala Gly Asn Ala Asp Ser Pro Val Pro Pro Arg Val Tyr Ile His Pro Asp Ser Leu Ala Ser Gly Asp Thr Trp Met Arg Gln Val Val Ser Phe Asp Lys Leu Lys Leu Thr Asn Asn Glu Leu Asp Asp Gln GIy His Ile Ile Leu His Ser Met His Lys Tyr Gln Pro Arg Val His Val Ile Arg Lys Asp Phe Ser Ser Asp Leu Ser Pro Thr Lys Pro Val Pro Val Gly Asp Gly Val Lys Thr Phe Asn Phe Pro Glu Thr Val Phe Thr Thr Val Thr Ala Tyr Gln Asn Gln Gln Ile Thr Arg Leu Lys Ile Asp Arg Asn Pro Phe Ala Lys Gly Phe Arg Asp Ser Gly Arg Asn Arg Thr Gly Leu Glu Ala Ile Met Glu Thr Tyr Ala Phe Trp Arg Pro Pro Val Arg Thr Leu Thr Phe Glu Asp Phe Thr Thr Met Gln Lys Gln Gln Gly Gly Ser Thr Gly Thr Ser Pro Thr Thr Ser Ser Thr Gly Thr Pro Ser Pro Ser Ala Ser Ser His Leu Leu Ser Pro Ser Cys Ser Pro Pro Thr Phe His Leu Ala Pro Asn Thr Phe Asn Val Gly Cys Arg Glu Ser Gln Leu Cys Asn Leu Asn Leu Ser Asp Tyr Pro Pro Cys Ala Arg Ser Asn Met Ala Ala Leu Gln Ser Tyr Pro Gly Leu Ser Asp Ser Gly Tyr Asn Arg Leu Gln Ser Gly Thr Thr Ser Ala Thr Gln Pro Ser Glu Thr Phe Met Pro Gln Arg Thr Pro Ser Leu Ile Ser Gly Ile Pro Thr Pro Pro Sex Leu Pro Gly Asn Ser Lys Met Glu Ala Tyr 455 460 4&5 Gly Gly Gln Leu Gly Ser Phe Pro Thr Ser Gln Phe Gln Tyr Val 470 475 ~ 480 Met Gln Ala Gly Asn Ala Ala Ser Ser Ser Ser Ser Pro His Met Phe Gly Gly Ser His Met Gln Gln Ser Ser Tyr Asn Ala Phe Ser Leu His Asn Pro Tyr Asn Leu Tyr Gly Tyr Asn Phe Pro Thr Ser Pro Arg Leu Ala Ala Ser Pro Glu Lys Leu Ser Ala Ser Gln Ser Thr Leu Leu Cys Ser Ser Pro Ser Asn Gly Ala Phe Gly Glu Arg Gln Tyr Leu Pro Ser Gly Met Glu His Ser Met His Met Ile Ser Pro Ser Pro Asn Asn Gln Gln Ala Thr Asn Thr Cys Asp Gly Arg Gln Tyr Gly Ala Val Pro Gly Ser Ser Ser Gln Met Ser Val His Met Val <210> 34 <211> 388 <212> PRT
<213> Homo Sapiens <220>
<221> misc_featuxe <223> Incyte ID No: 7475843CD1 <400> 34 Met Leu Glu Asn Tyr Arg Asn Leu Val Ser Leu Gly Ile Ala Val Ser Lys Pro Asp Leu Ile Thr Cys Leu Glu Gln Arg Asn Glu Pro Trp Asn Val Lys Lys His Glu Thr Val Ala Arg His Pro Ala Val Ser Ser His Phe Thr Gln Asp Leu Leu Pro Glu His Gly Tle Lys Asp Ser Phe Gln Lys Val Ile Leu Arg Arg Tyr Gly Ser Tyr Gly Ile Glu Asn Leu Gln Leu Lys Lys Asp Trp Glu Ser Val Gly Glu Ser Lys Val Gln Lys Glu Cys Cys Asn Gly Leu Asn Gln Ser Leu Ser Thr Thr His Thr Lys Ile Phe Gln Phe Asn Lys Cys Val Lys Val Phe Ser Lys Ser Ser Asn Leu Asn Arg His Lys Ile Arg His Thr Gly Glu Ile Ser Ser Asn Cys Lys Glu Cys Asp Asn Ser Phe Tyr Ile Ser Ser Val Leu Thr Pro Leu Gln Arg IIe His Thr Ala Glu Lys Ser Tyr Lys Cys Lys Gln Cys Gly Lys Ala Phe Arg His Cys Ser Cys Phe Leu Glu His Glu Thr Ile His Asn Glu Glu Lys His Tyr Lys Cys Lys Glu Cys Gly Lys Val Phe Lys Ser Phe Thr Ser Leu Ser Asn His Ile Ile Ile His Thr Gly Lys Lys Leu Tyr Lys Cys Glu Glu Cys Gly Lys Ala Phe Asn His Ser Ser Asn His Ala Lys His Lys Lys Ile His Thr Gly Gln Lys Pro His Lys Cys Glu Glu Cys Gly Lys Ala Phe Asn Trp Phe Ser Tyr Leu Thr Leu His Lys Arg Ile His Thr Gly Glu Lys Pro Tyr Lys Cys Asp Glu Cys Gly Lys Ala Phe Asn Gln Cys Ser Asn Leu Thr Lys His Lys Arg Ile His Thr Gly Glu Lys Pro Tyr Lys Cys Glu Glu Cys Gly Lys Ala Phe Asn Arg Cys Ser His Leu Thr Glu His Lys Arg Ile His Thr Gly Glu Lys Pro Tyr Lys Cys Glu Glu Cys Gly Lys Val Phe Tle Ser Cys Ser Ser Leu Ser Asn His Lys Arg Ile His Thr Arg Glu Lys Cys Tyr Lys Ser Glu Glu Cys Gly Lys Thr Phe Asn His Cys Ser Asp Leu Asn Val Pro Glu Lys Ile His Thr <210> 35 <211> 480 <212> PRT
<213> Homo Sapiens <220>
<221> misc_~eature <223> Incyte ID No: 6319550CD1 <400> 35 Met Gly Ser Pro Ala Ala Pro G1u Gly Ala Leu Gly Tyr Val Arg 1 5 10 l5 Glu Phe Thr Arg His Ser Ser Asp Val Leu Gly Asn Leu Asn Glu Leu Arg Leu Arg Gly Ile Leu Thr Asp Val Thr Leu Leu Val Gly Gly Gln Pro Leu Arg Ala His Lys Ala Val Leu Ile Ala Cys Ser Gly Phe Phe Tyr Ser Ile Phe Arg Gly Arg Ala Gly Val Gly Val Asp Val Leu Ser Leu Pro Gly Gly Pro Glu Ala Arg Gly Phe Ala Pro Leu Leu Asp Phe Met Tyr Thr Ser Arg Leu Arg Leu Ser Pro Ala Thr Ala Pro Ala Val Leu Ala Ala Ala Thr Tyr Leu Gln Met Glu His Val Val Gln Ala Cys His Arg Phe Ile Gln Ala Ser Tyr Glu Pro Leu Gly Ile Ser Leu Arg Pro Leu Glu Ala Glu Pro Pro Thr Pro Pro Thr Ala Pro Pro Pro Gly Ser Pro Arg Arg Ser Glu Gly His Pro Asp Pro Pro Thr Glu Ser Arg Ser Cys Ser Gln Gly Pro Pro Ser Pro Ala Ser Pro Asp Pro Lys Ala Cys Asn Trp Lys Lys Tyr Lys Tyr Ile Val Leu Asn Ser Gln Ala Ser Gln Ala Gly Ser Leu Val Gly Glu Arg Ser Ser Gly Gln Pro Cys Pro Gln Ala WO 03/000864 ., ..._. .. . PCT/US02/21179 Arg Leu Pro Ser Gly Asp Glu Ala Ser Ser Ser Ser Ser Sex Ser Ser Ser Ser Ser Ser Glu Glu Gly Pro Ile Pro Gly Pro Gln Ser Arg Leu Ser Pro Thr Ala Ala Thr Val Gln Phe Lys Cys Gly Ala Pro Ala Ser Thr Pro Tyr Leu Leu Thr Ser Gln Ala Gln Asp Thr Ser Gly Ser Pro Ser Glu Arg Ala Arg Pro Leu Pro Gly Ser Glu 2g0 295 300 Phe Phe Ser Cys Gln Asn Cys Glu Ala Val Ala Gly Cys Ser Ser Gly Leu Asp Ser Leu Val Pro Gly Asp G1u Asp Lys Pro Tyr Lys Cys Gln Leu Cys Arg Ser Ser Phe Arg Tyr Lys Gly Asn Leu Ala Ser His Arg Thr Val His Thr Gly Glu Lys Pro Tyr His Cys Ser Ile Cys G1y Ala Arg Phe Asn Arg Pro A1a Asn Leu Lys Thr His Ser Arg Ile His Ser Gly Glu Lys Pro Tyr Lys Cys Glu Thr Cys Gly Ser Arg Phe Val Gln Val Ala His Leu Arg Ala His Val Leu Ile His Thr Gly Glu Lys Pro Tyr Pro Cys Pro Thr Cys Gly Thr Arg Phe Arg His Leu Gln Thr Leu Lys Ser His Val Arg Ile His Thr Gly Glu Lys Pro Tyr His Cys Asp Pro Cys Gly Leu His Phe Arg His Lys Ser Gln Leu Arg Leu His Leu Arg Gln Lys His Gly Ala Ala Thr Asn Thr Lys Val His Tyr His Ile Leu Gly Gly Pro <210> 36 <211> 790 <212> PRT
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte ID No: 7510064CD1 <400> 36 Met Ala Leu G1y Leu Gln Arg Ala Arg Pro Ala Leu Ser Cys Gly Val Ile Ser Pro Pro Cys Ala Pro Thr Arg Asn Ser His Pro Gly Pro Gly Cys Thr Ala Ser Pro Pro Ala Pro Pro Gly Trp Pro Phe Sex Gln Arg Gly Pro Gly Arg Trp Ser Thr Thr Glu Leu Arg Lys Glu Lys Ser Arg Asp Ala Ala Arg Sex Arg Arg Ser Gln Glu Thr Glu Val Leu Tyr Gln Leu Ala His Thr Leu Pro Phe Ala Axg Gly Val Ser Ala His Leu Asp Lys Ala Ser Ile Met Arg Leu Thr Ile Ser Tyr Leu Arg Met His Arg Leu Cys Ala Ala Gly Glu Trp Asn Gln Val Gly Ala Gly Gly Glu Pro Leu Asp Ala Cys Tyr Leu Lys Ala Leu Glu Gly Phe Val Met Val Leu Thr Ala Glu Gly Asp Met Ala Tyr Leu Ser Glu Asn Val Ser Lys His Leu Gly Leu Ser Gln Leu Glu Leu Ile Gly His Ser Ile Phe Asp Phe Ile His Pro Cys Asp Gln Glu Glu Leu Gln Asp Ala Leu Thr Pro Gln Gln Thr Leu Ser Arg Arg Lys Val Glu Ala Pro Thr Glu Arg Cys Phe Ser Leu Arg Met Lys Ser Thr Leu Thr Ser Arg Gly Arg Thr Leu Asn Leu Lys Ala Ala Thr Trp Lys Val Leu Asn Cys Ser Gly His Met Arg Ala Tyr Lys Pro Pro Ala Gln Thr Ser Pro Ala Gly Ser Pro Asp Ser Glu Pro Pro Leu Gln Cys Leu Val Leu Ile Cys Glu Ala Ile Pro His Pro Gly Ser Leu Glu Pro Pro Leu Gly Arg Gly Ala Phe Leu Ser Arg His Ser Leu Asp Met Lys Phe Thr Tyr Cys Asp Asp Arg Ile Ala Glu Val Ala Gly Tyr Ser Pro Asp Asp Leu Ile Gly Cys Ser Ala Tyr Glu Tyr Ile His Ala Leu Asp Ser Asp Ala Val Ser Lys Ser Ile His Thr Leu Leu Ser Lys Gly Gln Ala Val Thr Gly Gln Tyr Arg Phe Leu Ala Arg Ser Gly Gly Tyr Leu Trp Thr Gln Thr Gln Ala Thr Val Val Ser Gly Gly Arg Gly Pro Gln Ser Glu Ser Ile Val Cys Val His Phe Leu IIe Ser Gln Val Glu Glu Thr Gly Val Val Leu Ser Leu Glu Gln Thr Glu Gln His Ser Arg Arg Pro Ile Gln Arg Gly Ala Pro Ser Gln Lys Asp Thr Pro Asn Pro Gly Asp Ser Leu Asp Thr Pro Gly Pro Arg Ile Leu Ala Phe Leu His Pro Pro Ser Leu Ser Glu Ala Ala Leu Ala Ala Asp Pro Arg Arg Phe Cys Ser Pro Asp Leu Arg Arg Leu Leu Gly Pro Ile Leu Asp Gly Ala Ser Val Ala Ala Thr Pro Ser Thr Pro Leu Ala Thr Arg His Pro Gln Ser Pro Leu Ser Ala Asp Leu Pro Asp Glu Leu Pro Val Gly Thr Glu Asn Val His Arg Leu Phe Thr Ser Gly Lys Asp Thr Glu Ala Val Glu Thr Asp Leu Asp Ile Ala Gln Met Arg Lys Leu Lys Leu Arg Leu Leu Thr Thr Gly Thr Glu Leu Arg Ser Asp Gly Ala Gly Thr Ser Ala Lys Val His Pro Ser Pro Arg Leu Ile Leu Leu Pro Pro Ser Cys Pro Pro Gln Asp Ala Asp Ala Leu Asp Leu Glu Met Leu Ala Pro Tyr Ile Ser Met Asp Asp Asp Phe Gln Leu Asn Ala Ser Glu Gln Leu Pro Arg Ala Tyr His Arg Pro Leu Gly Ala Val Pro Arg Pro Arg Ala Arg Ser Phe His Gly Leu Ser Pro Pro Ala Leu Glu Pro Ser Leu Leu Pro Arg Trp Gly Ser Asp Pro Arg Leu Ser Cys Ser Ser Pro Ser Arg Gly Asp Pro Ser Ala Ser Ser Pro Met Ala Gly Ala Arg Lys Arg Thr Leu Ala Gln Ser Ser Glu Asp Glu Asp Glu Gly Val Glu Leu Leu Gly Val Arg Pro Pro Lys Arg Ser Pro Ser Pro Glu His Glu Asn Phe Leu Leu Phe Pro Leu Ser Leu Ser Phe Leu Leu Thr Gly Gly Pro Ala Pro Gly Ser Leu Gln Asp Pro Thr Glu Leu Thr Gln Phe Leu Leu Ser Val Leu Ser Phe Pro Ile Leu Asp Pro Tyr Pro Leu Gly Cys Ala Ala Pro Gly Leu His Ala Ser Pro Phe Ser Leu Pro Thr Ile Ser Val Pro Gln Asn Pro Leu His Phe Pro Pro Gln Pro Ser Arg His Ala Leu Thr Leu Thr Leu Pro His Met Phe Gly Ala Pro Gly Ala Pro Ser Pro Leu Gly Trp Phe Ala Ile <210> 37 <211> 1154 <212> DNA
<213> Homo Sapiens <220>
<221> misc feature <223> Incyte ID No: 7490148CB1 <400> 37 agcatcacgt gccagggtgg ggggctataa aatacccgag ccgggcgccg gcgggggacg 60 tgaggacagc cctctccggg gacccctttg ttcccagccc agacgccaac acctctgcgt 120 ccccaagggc ttgactgccc gtgtctgcgc ggctcccagg gcagagctta gaacactaga 180 ggagaggggt cgccgcgaac tgccggggct tccagccacc cacccctctc gacatgtcgc 240 gctccttcta tgtcgactcg ctcatcatca aggacacctc acggcctgcg ccctcgctgc 300 ctgaaccgca ccccgggccg gatttcttca tcccgcttgg catgccgccc ccattggtga 360 tgtccgtgtc cggccccggc tgcccgtccc gcaagagcgg cgcgttctgc gtgtgccctc 420 tctgcgtcac ttcgcacctg cactcctctc gggggtctgt gggccccgcc agcgggggcg 480 cagggccggg gtttcccggg cccggagaca gtggggtggc agggcccgca ggggcactgc 540 ctctgcttaa gggccagttc tcttcggctc ctggggacgc gcagttttgc ccgcgggtga 600 accatgcgca tcatcaccac cacccgccgc agcaccacca tcaccatcat cagccccagc 660 agcctggctc ggccgcggcg gcggcagcag cagcagcggc ggcggcggcc gcggcggcct 720 tggggcaccc gcagcaccac gcacctgtct gcaccgccac cacctacaac gtggcggacc 780 cgcggagatt ccactgcctc accatgggag gctctgacgc cagccaggta cccaatggca 840 agaggatgag gacggcgttc actagcacgc aactcctgga gctggagaga gaattctctt 900 ccaacatgta cctgtctcga ctccggagga ttgaaatcgc cacttacctg aacctgtcgg 960 agaagcaggt gaaaatctgg tttcagaacc gccgagtgaa gcacaagaag gaggggaagg 1020 gcacgcagag gaacagtcac gcgggctgca agtgcgtcgg gagccaggtg cactacgcgc 1080 gctccgagga tgaggactcc ctgtcgccgg cctcagccaa cgatgacaag gagatttccc 1140 ccttatgagg gagg 1154 <210> 38 <211> 754 <212> DNA
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte ID No: 7490301CB1 <400> 38 agccaaaatc tttgaaaggt tctcattgca gatccataat caaaccaccc cagccagaac 60 aatttctgcc tctacagcgc tgtcatggag cagacctgaa tctcacccat ggagacaggc 120 aggcaagcag gtgtgtctgc tgagatgttc gccatgcccc gagatctgaa gggcagcaac 180 aaggatggga tccctgagga cctagatggg aacttggaag aacccaggga tcaggaaggt 240 gagctcagga gtgaggatgt catggacctc acagaaggtg acaatgaggc etcagcctca 300 gctcctcctg cagccaaaag acggaaaaca gataccaaag gaaagaagga gaggaagccc 360 actgtggatg cagaggaggc tcagaggatg acaaccctgc tgtctgccat gtctgaggag 420 cagctgtccc gctacgaagt gtgtcgccgg tcagctttcc caaaagcatg cattgcgggt 480 ctgatgcggt ctatcactgg cagatcggtg tctgagaacg tggcgattgc catggctgga 540 atagccaagg tctttgttgg agaggtggtg gaagaggccc tggacgtgtg tgagatgtgg 600 ggagaaatgc ccccactgca gcccaagcat ttaagggagg ctgttcgcag gttaaagccc 660 aagggcctct tccccaacag caactacaaa aaaatcatgt tctaggccca aggccagacg 720 gaggggtctg tttgtgcagg aataagtacc gcat 754 <210> 39 <211> 2483 <212> DNA
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte ID No: 2383223CB1 <400> 39 gtgcgatcgg gttgtgctta gcttggggtc tcctggcccc ttgacgcgtc aggttgctgt 60 acccctgcat cggatgcgct gtaccctgcg ctggctccgt gaaccttagg gacaacaccg 120 ggacacccgc gaggccggaa aatggactca gtggcttttg aggatgtgtc tgtgagcttc 180 agccaggagg agtgggctct gctggctcct tcacagaaga aactctacag agatgtgatg 240 caggaaacat tcaagaacct ggcatctata ggggaaaaat gggaagaccc gaatgttgaa 300 gatcaacaca aaaaccaagg acgaaatcta agaagccata cgggagagag actctgtgaa 360 ggtaaagaag gtagtcaatg tgcagaaaac ttcagtccca atctcagtgt gacgaagaag 420 actgccggag taaaaccata tgagtgtact atctgtggaa aagccttcat gcgtctctca 480 tcccttacta gacacatgag gtctcacact ggatacgagc tatttgagaa gccatataaa 540 tgtaaggagt gtgagaaagc ctttagttat ctcaaatcct ttcaaagaca tgaaaggagt 600 cacactggag aaaaacccta taaatgtaaa caatgtggaa aaaccttcat atatcaccag 660 ccctttcaaa gacatgagcg gactcacatt ggagaaaaac cctatgaatg taagcaatgt 720 ggaaaagctc ttagttgttc cagttcgctt cgagttcatg aaaggattca cactggagaa 780 aagccctatg aatgtaaaca atgtgggaaa gccttcagtt gttccagttc tattcgagta 840 cacgaaagaa ctcacactgg agagaaaccc tatgcatgta aggaatgtgg gaaagccttc 900 atttcccaca caagtgttct aacacacatg ataacacaca acggagatag accttataaa 960 tgcaaagaat gtggaaaggc attcattttt cccagttttt tacgagtaca tgaaagaatt 1020 cacactggag agaaacccta taaatgtaaa caatgtggta aagccttcag atgttccacc 1080 tccattcaaa ttcatgaaag aattcatact ggagagaagc cctataaatg taaagaatgt 1140 gggaaatctt tcagtgcacg cccagccttt cgagtacacg tgagagtgca tactggagag 1200 aaaccctata agtgtaaaga atgtgggaaa gcctttagta gaatcagtta ctttcgaata 1260 catgaaagga ctcacactgg agagaaaccc tacgaatgta aaaaatgtgg gaaaactttc 1320 aattatcctc tagatttgaa aatccacaag agaaatcaca ctggagaaaa accctatgag 1380 tgtaaggaat gtgcaaaaac cttcatttct cttgagaact ttcgaagaca catgatcacc 1440 cacactggag acggacctta taaatgtagg gactgtggga aggtgttcat ttttcctagt 1500 gcgttacgaa cacatgaaag aactcacact ggagagaaac cctatgaatg taaacaatgt 1560 ggaaaagcct ttagttgttc tagttacatt cggatacata aaagaactca cactggggag 1620 aaaccttatg aatgtaagga atgcgggaag gcctttattt atcccacaag ctttcaagga 1680 cacatgagaa tgcatactgg agagaaaccc tataaatgta aagaatgtgg gaaggccttt 1740 agtcttcaca gttcctttca aagacataca agaattcaca attatgagaa acctcttgaa 1800 tgtaagcaat gtggaaaagc cttcagtgtg tccacatcct taaaaaaaca tatgagaatg 1860 cacaatcgat agaaactcta taaatgtgag aaataggaga aagttttcaa ttctaacaga 1920 tgctttcaaa gttgtgaaaa ttcccactga agagagaaat cctgtcaatg taagtaatat 1980 agaaagcgag atacaagatg attcatgtat agtcaggtac cacataatca tgtttcagca 2040 gcaatggacc atataggttg gtagccccat aagattatat aacacctaaa acatttctat 2100 caaccgcaat ttagtagcag tagtaacacc atagtgcagc acattattta agtgtttatg 2160 gtgctggtgt aaacgtgctg cactttcagt tgtataaaag tacaagacag cggccgggga 2220 cggtggctca cgcctgtaat cccagcactt tgggaggcca aggcgggcgg atcacgaggc 2280 caggagatca agaccatcct ggctaacacg gtaaaacccc atctctacta aaaatacaaa 2340 aaattagccg ggcgtggtgg tgggcgcctg tagtcccagc tacttgggag gctgaggcag 2400 gagaatggca taaacccagg aggccgagct gacagtgagc tgagatccgg ccaacagagc 2460 gagactctgt ctcaaaaaaa aaa 2483 <210> 40 <211> 2535 <212> DNA
<213> Homo sapiens <220>
<221> misc feature <223> Incyte ID No: 3495982CB1 <400> 40 agggtccaag cgggccgcgg ccgctgggac ggaactggtg ccctccgtgg acaattgtgt 60 tgaagcagaa attgttccgg atctcgggtc ggacacggaa gtcttcctgc agtgtttctg 120 gatgcgggga cagggatgcg caggaattcc agtctcagtt tccagatgga gcgacccctc 180 gaggagcaag tccagagcaa gtggtcgtct agtcaaggcc gcacaggaac aggagggtct 240 gatgtcctcc agatgcagaa cagtgaacac catggacaaa gcatcaagac tcaaactgac 300 tccatctccc ttgaggatgt ggctgtgaac ttcaccctgg aggagtgggc tttgctggat 360 cctggccaga ggaatatcta cagagatgtg atgcgggcaa ccttcaagaa cctggcctgt 420 ataggggaaa aatggaaaga ccaggatatt gaagatgaac acaaaaacca gggaagaaat 480 DEMANDE OU BREVET VOLUMINEUX
LA PRESENTE PARTIE DE CETTE DEMANDE OU CE BREVET COMPREND
PLUS D'UN TOME.

NOTE : Pour les tomes additionels, veuillez contacter 1e Bureau canadien des brevets JUMBO APPLICATIONS/PATENTS
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Claims (127)

What is claimed is:

1. An isolated polypeptide selected from the group consisting of:
a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:1-36, b) a polypeptide comprising a naturally occurring amino acid sequence at least 90%
identical to an amino acid sequence selected from the group consisting of SEQ
ID
NO:1, SEQ ID NO:3-7, SEQ ID NO:10-21, SEQ ID NO:24-30, SEQ ID NO:32, SEQ
ID NO:34, and SEQ ID NO:36, c) a polypeptide comprising a naturally occurring amino acid sequence at least 91%
identical to the amino acid sequence of SEQ ID NO:35, d) a polypeptide comprising a naturally occurring amino acid sequence at least 95%
identical to the amino acid sequence of SEQ ID NO:9, e) a polypeptide comprising a naturally occurring amino acid sequence at least 97%
identical to the amino acid sequence of SEQ ID NO:22, f) a polypeptide comprising a naturally occurring amino acid sequence at least 98%
identical to an amino acid sequence selected from the group consisting of SEQ
ID
NO:2 and SEQ ID NO:33, g) a polypeptide comprising a naturally occurring amino acid sequence at least 99%
identical to an amino acid sequence selected from the group consisting of SEQ
ID
NO:23 and SEQ ID NO:31, h) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1-36, and i) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1-36.

2. An isolated polypeptide of claim 1 comprising an amino acid sequence selected from the group consisting of SEQ ID NO:1-36.

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

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

5. An isolated polynucleotide of claim 4 comprising a polynucleotide sequence selected from the group consisting of SEQ ID NO:37-72.

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

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

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

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

10. A method of claim 9, wherein the polypeptide comprises. an amino acid sequence selected from the group consisting of SEQ ID NO:1-36.

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

12. An isolated polynucleotide selected from the group consisting of:
a) a polynucleotide comprising a polynucleotide sequence selected from the group consisting of SEQ ID NO:37-72, b) a polynucleotide comprising a naturally occurring polynucleotide sequence at least 90% identical to a polynucleotide sequence selected from the group consisting of SEQ
ID NO:37-43 and SEQ ID NO:46-71, c) a polynucleotide comprising a naturally occurring polynucleotide sequence at least 92% identical to the polynucleotide sequence of SEQ ID NO:72, d) a polynucleotide comprising a naturally occurring polynucleotide sequence at least 95% identical to the polynucleotide sequence of SEQ ID NO:45, e) a polynucleotide complementary to a polynucleotide of a), f) a polynucleotide complementary to a polynucleotide of b), g) a polynucleotide complementary to a polynucleotide of c), h) a polynucleotide complementary to a polynucleotide of d), and i) an RNA equivalent of a)-h).

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

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

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

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

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

18. A composition of claim 17, wherein the polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NO:1-36.

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

39. A method of making a monoclonal antibody with the specificity of the antibody of claim 11, the method comprising:
a) immunizing an animal with a polypeptide consisting of an amino acid sequence selected from the group consisting of SEQ ID NO:1-36, or an immunogenic fragment thereof, under conditions to elicit an antibody response, b) isolating antibody producing cells from the animal, c) fusing the antibody producing cells with immortalized cells to form monoclonal antibody-producing hybridoma cells, d) culturing the hybridoma cells, and e) isolating from the culture monoclonal antibody which specifically binds to a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:1-36.

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

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

42. The antibody of claim 21, wherein the antibody is produced by screening a Fab expression library.

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

44. A method of detecting a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:1-36 in a sample, the method comprising:
a) incubating the antibody of claim 11 with a sample under conditions to allow specific binding of the antibody and the polypeptide, and b) detecting specific binding, wherein specific binding indicates the presence of a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:1-36 in the sample.

45. A method of purifying a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:1-36 from a sample, the method comprising:
a) incubating the antibody of claim 11 with a sample under conditions to allow specific binding of the antibody and the polypeptide, and b) separating the antibody from the sample and obtaining the purified polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID
NO:1-36.

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

92. A polynucleotide of claim 12, comprising the polynucleotide sequence of SEQ ID NO:37.

93. A polynucleotide of claim 12, comprising the polynucleotide sequence of SEQ ID NO:38.

94. A polynucleotide of claim 12, comprising the polynucleotide sequence of SEQ ID NO:39.

95. A polynucleotide of claim 12, comprising the polynucleotide sequence of SEQ ID NO:40.

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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