CA2433027A1 - Nucleic acid-associated proteins - Google Patents

Abstract

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

Description

NUCLEIC ACID-ASSOCIATED PROTEINS
TECHNICAL FIELD
This invention relates to nucleic acid and amino acid sequences of nucleic acid-associated proteins and to the use of these sequences in the diagnosis, treatment, and prevention of cell proliferative, neurological, developmental, and autoimmune/inflammatory disorders, and infections, and in the assessment of the effects of exogenous compounds on the expression of nucleic acid and amino acid sequences of nucleic acid-associated proteins.
to 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 types 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.
Transcriution 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 Yorlc, NY, and Cell Press, Cambridge, MA, pp. 554-570.) The double helix structure 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 helices or 13 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 cc 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 LTltrabithorax proteins of Drosophila melanogaster are prototypical homeodomain proteins. (Patio, C.O. and R.T.
Sauer (1992) Ann. Rev.
Biochem. 61:1053-1095.) Mouse HES-6 is a member of the Hairy/Bnhancer-of split (HES) family of basic helix-loop-helix transcription factors. HES genes act as nuclear effectors of Notch signaling to regulate the transcriptional activity of several Notch target genes. HES-6 is expressed in all neuxogenic placodes and their derivatives and in the brain, where it is patterned along both the anteroposterior and dorsoventral axes. HES-6 is also expressed in embryonic tissues where Notch signaling contxols cell-fate decisions, such as the trunk, the dorsal root ganglia, myotomes, and thymus. In the limb buds HES-6 is expressed in skeletal muscle and presumptive tendons. It is also expressed in epithelial cells of the embryonic respiratory, urinary and digestive systems (Vasiliauskas, D. and Stern C.D. (2000) Mech. Dev. 98:133-137; Pissarra, L. et al. (2000) Mech Dev 95:275-278).
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" forger), have been described. (Lewin, su ra.) Zinc forger 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 forger 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 a 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 forgers occur in a variety of proteins that do not bind DNA (Lodish, H. et al.
(1995) Molecular Cell Biolosy, Scientific American Books, New York, NY, pp. 447-451). For example, Galcheva-Gargova, Z. et al. (1996) Science 272:1797-1802) have identified zinc forger 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-finger 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 (Honer, C. et al. (2001) Biochim.
Biophys. Acta 1517:441-448). 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 forger 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 forgers. The KRAB domain is found N-terminally with respect to the finger repeats. The KRAB domain is generally encoded by two exons; the KRAB-A region or box is encoded by one exon and the KRAB-B region or box is encoded by a second exon. The function of the KRAB
domain is the repression of transcription. Transcription repression is accomplished by recruitment of either the DRAB-associated protein-1, a transcriptional corepressor, or the KRAB-A interacting protein. Proteins containing the KRAB domain are likely to play a regulatory role during development (VVilliams, A.J. et al., (1999) Mol. Cell Biol. 19:8526-8535). A
subgroup of highly related human KRAB zinc forger 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 KRAB
zinc forger 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 Kruppel protein regulates Drosophila segmentation. There are approximately 300 genes which encode such proteins in the whole human genome. In fact, more than 100 different mRNAs encoding Kruppel multifmgered proteins, most of them novel, have been found in the human placenta. The sequences of the 106 finger repeats present in nine of these proteins are highly homologous. There are a few positions located in the alpha-helical structure which show variability.
Research implies that this variability impacts the DNA-binding specificity of the proteins (Bellefroid, E.J. et al. (1989) DNA 8:377-387).
ZNF143 is a human zinc finger Kruppel family protein of the GLI type. It is 84°lo identical to the Xenopus laevis selenocysteine tRNA gene transcription activating factor (Staf). Staf is implicated in the enhanced transcription of small nuclear RNA (snRNA) and snRNA-type genes by RNA
polymerases II (Pol II) and III (Pol III). Staf also possesses the capacity to stimulate expression from a Pol II mRNA promoter. ZNF143, along with the related ZNF138 and ZNF139, is localized to chromosome regions 7q11.2, 7q21.3-q22.1, and 11p15.3-p15.4. These regions are involved in deletions and/or translocations associated with Williams syndrome, split hand and foot disease (SHFD1), and Beckwith-Wiedemann syndrome, respectively, suggesting that ZNF143 gene is involved in developmental and malignant disorders. ZNF143 mRNAs are expressed in many normal adult tissues, including leukocytes, colon, small intestine, ovary, testis, prostate, thymus, and spleen tissues. Further, transcription of the mouse chaperone-encoding Ccta gene is regulated by ZNF143 and another Staf family zinc-finger transcription factor, ZNF76, implying that these RNA and chaperone genes are coregulated to facilitate synthesis of mature proteins during active cell growth (Tommerup,N. and Vissing,H. (1995) Genomics 27: 259-264 ;Myslinski, E. et al.
(1998) J. Biol.
Chem. 273:21998-2006; Kubota, H. et al. (2000) J. Biol. Chem. 275:28641-28648).
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 finger) 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 forger is a C4HC3 zinc-finger-like motif found in nuclear proteins thought to be involved in chromatin-mediated 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 cc helix, followed by a long loop to the C-termnnal end of the finger (Ominchinski, J.G.
(1993) Science 261:438-446). 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 xmnor groove.
The LIM 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 Beckerle, M.C.
(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 forger 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 LIM protein binds to F-actin through its dematin-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 and MZF-2. MZF-1 functions in regulation of the development of neutrophilic granulocytes. A marine homolog MZF-2 is expressed in myeloid cells, particularly in the cells committed 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 of 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 mouse kreisler (kr) mutation causes segmentation abnormalities in the caudal hindbrain and defective inner ear development. The kr cDNA encodes a basic domain-leucine zipper (bZIP) transcription factor in which a serine is substituted for an asparagine residue conserved in the DNA-binding domain of all known bZIP family members. The identity, expression, and mutant phenotype of kr indicate an early role in axial patterning and provide insights into the molecular and , embryologic mechanisms that govern hindbrain and otic development (Cordes, S.P. and Barsh, G.S.
(1994) Cel179:1025-1034).
The helix-loop-helix motif (HLH) consists of a short oc helix connected by a loop to a longer cc 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 ReIB, and vertebrate proto-oncogene c-rel, a protein associated with differentiation and lymphopoiesis (Kabrun, N., and Enrietto, P.J. (1994) Semin. Cancer Biol. 5:103-112).
A DNA binding motif termed ARm (AT-rich interactive domain) distinguishes an evolutionarily conserved family of proteins. The approximately 100-residue ARID sequence is present in a series of proteins strongly implicated in the regulation of cell growth, development, and tissue-specific gene expression. ARID 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 MTAl and protein ERl. The Caenorhabditis ele ans 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.
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) Nucl. Acids Res. 20:3-26.) 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 histones, Hl, 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 Many neoplastic disorders in humans can be attributed to inappropriate gene expression.
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 Wilm's tumor. The oncogene bcl-6, which plays an important role in large-cell lymphoma, is also a zinc-forger 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, 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).
In 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 et al. Harxison's Principles of Internal Medicine, 13/e, McGraw Hill, Inc.
and Teton Data Systems Software, 1996.) 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 WTl; Greig cephalopolysyndactyly, Pallister-Hall syndrome, and postaxial polydactyly type A (GLI3), and Townes-Brocks syndrome, characterized by anal, renal, limb, and ear abnormalities (SALLl) (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).
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 polymerise, 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, thexe are fundamental differences between the two processes.
DNA polymerise catalyzes the stepwise addition of a deoxyribonucleotide to the 3'-OH end of a polynucleotide strand (the primer strand) that is paixed to a second (template) strand. The new DNA strand therefore grows in the 5' to 3' direction (Alberts, B. et al.
(1994) The Molecular Bioloay of the Cell, Garland 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 polymerise.
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 polymerise. In addition to the synthesis of new DNA, DNA polymerise is also involved in the repair of damaged DNA as discussed below under "Ligases."
In contrast to DNA polymerise, RNA polymerise 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. W eukaryotes, three discrete RNA
polymerises synthesize the three different types of RNA (Alberts et al., supra pp. 367-368). RNA polymerise I
makes the large ribosomal RNAs, RNA polymerise II makes the mRNAs that will be translated into proteins, and RNA polymerise III makes a variety of small, stable RNAs, including SS
ribosomal RNA and the transfer RNAs (tRNA). In all cases, RNA synthesis is initiated by binding of the RNA polymerise 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 polymerise and the completed RNA chain are released.
Li ases 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 et al., supra pp. 245-249). The three steps common to most types of DNA repair are (1) excision of 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 et al., supra 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 invadedby 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.
MODTFICATION OF NUCLEIC ACIDS
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 an 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 et al. 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 et al. su ra 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 TIF2 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) Proe. 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. 13:67$9-6798). Differences outside of these conserved regions are believed to reflect differences in the functional roles of individual proteins (Chang, T.H. et al.
(1990) Proc. Natl. Acad.
Sci. USA 87:1571-1575).
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 xole 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 rnay 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.
(Discussed in Godbout, supra.) For example, marine p68 is mutated in ultraviolet light-induced tumors, and human DDX6 is located at a chromosomal breakpoint associated with B-cell lymphoma.
Similarly, a chimeric protein comprised of DDX10 and NUP98, a nucleoporin protein, may be involved in the pathogenesis of certain myeloid malignancies.
Tonoisomerases 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. All 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 II
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 1I 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 et al. su ra pp.260-262). Type II
topoisomerases axe largely confined to proliferating cells in eukaryotes, such as cancer cells. For this reason they are targets for anticancer drugs. Topoisomerase.II has been implicated in mufti-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 ~. 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 are 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 III
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 (IIcc and II(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 II 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 IIa isoform appears to be responsible for unlinking DNA during chromosome segregation. Cell lines expressing Ifot but not II~3 suggest that II~i 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 2S disorder (Wunder, 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 II activity is often associated with cancer or increased cancer risk. Greatly lowered topo II 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 lI can break DNA in the region of the A-T gene (ATM), which controls all DNA damage-responsive cell cycle checkpoints (Kaufmann, W.I~. (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 cell, 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. (See Alberts et al. 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, recombinases, 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 recpmbination and therefore it does not require DNA homology between the recombining molecules. Unlike general recombination, this form of recombination can altex the relative positions of nucleotide sequences in chromosomes.
RNA METABOLISM
Ribonucleic acid (RNA) is a linear single-stranded polymer of four nucleotides, ATP, CTP, UTP, 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 60S (large) subunit and a 40S (small) subunit, which together form the 80S ribosome. In addition to the 18S, 285, 5S, and 5.8S rRNAs, ribosomes 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 60S 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, S17, 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 Garbex, M. (1995) Curr. Opin. Struct. Biol. 5:721-727; see also Woodson, S.A. and Leontis, N.B. (1998) Curr. Opin. Struct. Biol. 8:294-300;
Ramakrishnan, V, and White, S.W. (1998) Trends Biochem. Sci. 23:208-212).
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 spliceosomal complex is comprised of five small nuclear ribonucleoprotein particles (snRNPs) designated Ul, 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, L. (1995) Biochemistry W.H. Freeman and Company, New York NY, p. 863).
Heterogeneous nuclear ribonucleoproteins (hnRNPs) 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, 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 Drosophila melano.a~ stet and Caenorhabditis elegans. These proteins play key roles in developmental processes such as pattern formation and sex determination, respectively. (See, for example, Hodgkin, J.
et al. (1994) Development 1203681-3689.) RNA Stability and Degradation RNA helicases alter and regulate RNA conformation and secondary structure by using 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. Some DEAD-box helicases play tissue- and stage-specific roles in spermatogenesis and embryogenesis. All DEAD-box helicases contain several conserved sequence motifs spread out over about 420 amino acids. These motifs include an A-type ATP binding motif, the DEAD-box/B-type ATP-binding motif, a serine/arginine/threonine tripeptide of unknown function, and a C-terminal glycine-rich motif with a possible role in substrate binding and unwinding. In addition, alignment of divergent DEAD-box helicase sequences has shown that 37 amino acid residues are identical among these sequences, suggesting that conservation of these residues is important for helicase function. (Reviewed in Linden P. et al.
{1989) Nature 337:121-122.) Overexpression of the DEAD-box 1 protein (DDX1) may play a role in the progression of neuroblastoma (Nb) and retinoblastoma (Rb) tumors. 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 ultraviolet Light-induced tumors, B-cell lymphoma, and myeloid malignancies.
(Reviewed in Godbout, R. et al. (1998) J. Biol. Chem. 273:21161-21168.) Ribonucleases (RNases) catalyze the hydrolysis of phosphodiester bonds in RNA
chains, thus cleaving the RNA. 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. RNase H domains are often found as a domain associated with reverse transcriptases. 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.
Ribosomal proteins may undergo post-translational modifications or interact with other ribosome-associated proteins to regulate translation. For example, the highly homologous 40S
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, A.
supra and Garber, M. supra).
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 L1, L7, and L12 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) Curr. 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. 888-9081; Lodish, H. et al. (1995) Molecular Cell Biolo~y Scientific American Books, New York NY 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 ribonucleoprotein complex known as a spliceosome. The spliceosomal complex is comprised of five small nuclear ribonucleoprotein particles (snRNPs) designated Ul, 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, L. (1995) Biochemistry W.H. Freeman and Company, New York NY, p. 863).
Heterogeneous nucleax ribonucleoproteins (hnRNPs) 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 Al, 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, 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 l(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 Drosophila melano a~ ster and Caenorhabditis ele ans. These proteins play key roles in developmental processes such as pattern formation and sex determination, respectively. (See, for example, Hodgkin, J.
et al. (1994) Development 120:3681-3689.) The 3' ends of most eukaryote mRNAs are also posttranscriptionally 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'-untxanslated (non-coding) region and (ii) the addition of a poly(A) tract to the 5' mRNA fragment.
The presence of cis-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.
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 mitochondria) 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 If enzymes contain a central catalytic domain, which consists of a seven-stranded antiparallel 13-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 Cusack, S. (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-tRNA"e, 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 10-20 4 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, L. supra and Watson, J. et al. (1987) The Benjamin/Cummings Publishing Co., Inc. Menlo Park, CA). A particularly error prone aminoacyl-tRNA charging event is ~ the charging of tRNAc'" 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 noncanonical pathway for the synthesis of Gln-tRNAG'"
based on the transformation of Glu-tRNA~'° (synthesized by Glu-tRNA
synthetase, GIuRS) using the enzyme Glu-tRNA~'" 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):
GIuRS
tRNAc'n + Glu + ATP ~ Glu-tRNAc'" + AMP + Pp;
Glu-AdT
Glu-tRNAc'° + Gln + ATP ~ Gln-tRNAG'° + Glu + ADP + P
A similar enzyme, Asp-tRNAAs° amidotransferase, exists in Archaea, which transforms Asp-tRNAAs" to Asn-tRNA"S°. Formylase, the enzyme that transforms Met-tRNA~e' to fMet-tRNAtr''e' in eubacteria, is likely to be a related enzyme. A hydrolytic activity has also been identified that destroys mischarged Val-tRNAi'e (Schimmel, P. et al. (1998) FASEB J. 12:1599-1609). One likely scenario for the evolution of Glu-AdT in primitive life forms is the absence of a specific glutaminyl- -tRNA synthetase (GlnRS), requiring an alternative pathway for the synthesis of Gln-tRNA~'°. In fact, deletion of the Glu-AdT operon in Gram positive bacteria is lethal (Curnow, A.W. et al. (1997) Proc.
Natl. Acad. Sci. U.S.A. 94:11819-11826). The existence of GIuRS activity in other organisms has been inferred by the high degree of conservation in translation machinery in nature; however, GIuRS
has not been identified in all organisms, including Homo 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 fidelity, 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-a, and tissue factor. The N-terminal domain binds to the interleukin-8 type A receptor and functions as an 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 regulating their own transcription or translation (Martinis, supra). 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 autoixnmune 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 a1. (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 in animals.
Comparison of aaRS structures between humans and pathogens has been useful in the design of novel antibiotics (Schimmel, supra). Genetically engineered aaRSs have been utilized to allow site-specific incorporation of unnatural amino acids into proteins in vivo (Liu, D.R. et al. (1997) Proc.
Natl. Acad. Sci. USA 94:10092-10097).
tRNA Charging Protein biosynthesis depends on each amino acid forming a linkage with the appropriate tRNA. The aminoacyl-tRNA synthetases are responsible for the activation and correct attachment of an amino acid with its cognate tRNA. The 20 aminoacyl-tRNA synthetase 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'. Class II enzymes contain a central catalytic domain, which consists of a seven-stranded antiparallel 13-sheet motif, 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 Cusack, S. (1995) J. Mol.
Evol. 40:519-530).
Autoantibodies against aminoacyl-tRNAs are generated by patients with dermatomyositis and polymyositis, and correlate strongly with complicating interstitial lung disease (ILD). These antibodies appear to be generated in response to viral infection, and coxsackie virus has been used to induce experimental viral myositis in animals.
tRNA Modifications The modified ribonucleoside, pseudouridine (tu), is present ubiquitously in the anticodon regions of transfer RNAs (tRNAs), large and small ribosomal RNAs (rRNAs), and small nuelear 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 ~r (Cortese, R. et al. (1974) J. Biol. Chem. 249:1103-1108). The enzyme responsible for the conversion of uridine to ~, pseudouridine synthase (pseudouridylate synthase), was first isolated from Salmonella txphimurium (Arena, F, et al. (1978) Nuc. 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 Patton, J.R. (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, supra). Other eukaryotic pseudouridine synthases have been identified that appear to be specific for rRNA (revieved in Smith, C.M. and Steitz, J.A. (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 yr 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) 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 (mZ2G) 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 tRNAAsP 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 posttranscriptional modification to prevent the formation of alternative structures (Steinberg, S. and Cedergren, R. (1995) RNA 1:886-891, and references within). The enzyme responsible for the conversion of guanosine to mz2G is a 63 kDa S-adenosylmethionine (SAM)-dependent tRNA NZ,NZ-dimethyl-guanosine methyltransferase (also referred to as the TRMl 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 Xenopus laevis, there appears to be a requirement fox 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. (I992) Nuc. Acids Res. 20:6575-81).
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.
Translation Initiation Initiation of translation can be divided into three stages. The first stage brings an initiator transfer RNA (Mat-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 (V.M.
Pain (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. eIF2, a guanine nucleotide binding protein, recruits the initiator tRNA to the 40S ribosomal subunit. Only when eIF2 is bound to GTP
does it associate with the initiator tRNA. eIF2B, a guanine nucleotide exchange protein, is responsible for converting eIF2 from the GDP-bound inactive form to the GTP-bound active form.
Two other factors, elFlA and eIF3 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, eIF3, and 40S ribosomal subunit together make up the 43S preinitiation complex (Pain, supra).
eIF2 plays a central role in the maintenance of a rate-limiting step in mRNA
translation. In this step, eIF2 binds GTP and Met-tRNAi and transfers Met-tRNAi to the 40S
ribosomal subunit. At the end of the initiation process, GTP bound to elF2 is hydrolyzed to GDP and the eIF2.GDP complex is released from the ribosome. The exchange of GDP bound to eIF2 for GTP is a prerequisite to binding Met-tRNAi and is mediated by a second initiation factor, eIF2B, a guanine nucleotide-exchange factor. Phosphorylation of eIF2 on its alpha- subunit converts eIF2 from a substrate of elF2B into a competitive inhibitor. Thus, phosphorylation of elF2 alpha effectively prevents formation of the eIF2.GTP.Met-tRNAi complex and inhibits global protein synthesis.
Phosphorylation of eIF2 alpha occurs under a variety of conditions including viral infection, apoptosis, nutrient deprivation, heme-deprivation, and certain stresses. The 5'-untranslated region of hepatitis C virus (HCV) functions as an internal ribosome entry site (IRES) to initiate translation of HCV proteins. eIF2Bgamma and eIF2gamma are cellular factors involved in HCV
IRES-mediated translation (Kimball, S.R. (1999) Int. J. Biochem. Cell Biol. 31:25-29; Webb, B.L. and Proud, C.G.
(1997) Int. J. Biochem. Cell Biol. 29:1127-1131; Kruger M. et al. (2000) Proc.
Natl. Acad. Sci. U S A
97:8566-8571).
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. elF4E 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 eIF4G 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 (M.W.
Hentze (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 eIF4A is thought to function in removing this secondary structure to facilitate binding of the 43S
preinitiation complex (Pain, su era).
Translation Elon ag tion Elongation is the process whereby additional amino acids are joined to the initiator methionine to form the complete polypeptide chain. The elongation factors EF1 a, EF1 (3'y, and EF2 are involved in elongating the polypeptide chain following initiation. EF1 a is a GTP-binding protein. In EF1 cc's GTP-bound form, it brings an aminoacyl-tRNA to the ribosome's A site. The amino acid attached to the newly axrived aminoacyl-tRNA forms a peptide bond with the initiation methionine. The GTP on EFl oc is hydrolyzed to GDP, and EFl a -GDP dissociates from the ribosome. EF1 [3'y binds EFl cc -GDP and induces the dissociation of GDP
fromEF1 ot, allowing EFl cc 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 processivity of translation.
Translation Termination The release factor eRF carries out termination of translation. eRF recognizes stop codons in the nnRIVA, leading to the release of the polypeptide chain from the ribosome.
Treacher Collins Syndrome (TCS) is the most common of the human mandibulofacial dysostosis disorders. It shows autosomal dominant inheritance and occurs in 1 of 50,000 live births, with approximately 60% arising from new mutations. TCS symptoms show wide variability. The disease is deduced to be a result of interference in the development of the first and second branchial arches. The TCS gene, TCOFl, is localized to chromosome 5q31-33.3. There are ten identified mutations imTCOFl consisting of nonsense mutations, insertions, deletions, or splicing mutations that apparently Iead to premature termination of translation. Moreover, all are unique to each human family. TCOFl encodes a low complexity protein of 1,411 amino acids, with repeated motifs that mirror the organization of its exons. These motifs are shared with nucleolar trafficking proteins in other species and are highly phosphorylated by casein kinase. The full-length TCOF1 protein sequence also contains nuclear and nucleolar localization signals and several polymorphisms. This data suggests that TCS results from defects in a nucleolar trafficking protein that is critically required during human craniofacial development (Wise, C.A. et al. (1997) Proc. Natl.
Acad. Sci. U.S.A.
94:3110-3115).
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% of all deaths in females between the ages of 45-54 (Gish, K. (I999) 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, supra).
However, this type of hereditary breast cancer accounts for only about 5% to 9% of breast cancers, while the vast majority of breast cancer is due to non-inherited mutations that occur in breast epithelial cells.
The relationship between expression of epidermal growth factor (EGF) and its receptor, EGFR, to human mammary carcinoma has been particularly well studied. (See Khazaie, K. et al.
(1993) Cancer and Metastasis Rev. 12:255-274, and references cited therein for a review of this area.) Overexpression of EGFR, particularly coupled with down-regulation of the estrogen receptor, is a marker of poor prognosis in breast cancer patients. In addition, EGFR
expression in breast tumor S metastases is frequently elevated relative to the primary tumor, suggesting that EGFR is involved in tumor progression and metastasis. This is supported by accumulating evidence that EGF has effects on cell functions related to metastatic potential, such as cell motility, chemotaxis, secretion and differentiation. Changes in expression of other members of the erbB receptor family, of which EGFR
is one, have also been implicated in breast cancer. The abundance of erbB
receptors, such as HER-2/neu, HER-3, and HER-4, and their ligands in breast cancer points to their functional importance in the pathogenesis of the disease, and may therefore provide targets for therapy of the disease (Bacus, S. S. et al. (1994) Am. J. Clin. Pathol. 102:513-S24). Other known markers of breast cancer include a human secreted frizzled protein mRNA that is downregulated in breast tumors;
the matrix G1a protein which is overexpressed 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;
Ulrix, W. et al (1999) FEBS Lett 455:23-26; Sager, R. et al. (1996) Curr. Top. Microbiol. Immunol.
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) Clin. 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.
Preadipocyte Cells The most important function of adipose tissue is its ability to store and release fat during periods of feeding and fasting. White adipose tissue is the major energy reserve in periods of excess energy use, and its primary purpose is mobilization during energy deprivation.
Understanding how the various molecules regulate adiposity and energy balance in physiological and pathophysiological situations may lead to the development of novel therapeutics for human obesity. Adipose tissue is also one of the important target tissues for insulin. Adipogenesis and insulin resistance in type II
diabetes are linked and present intriguing relations. Most patients with type II diabetes are obese and obesity in turn causes insulin resistance.

The majority of research in adipocyte biology to date has been done using transformed mouse preadipocyte cell lines. The culture condition, which stimulates mouse preadipocyte differentiation is different from that for inducing human primary preadipocyte differentiation.
In addition, primary cells are diploid and may therefore reflect the in vivo context better than aneuploid cell lines.
Understanding the gene expression profile during adipogenesis in human will lead to understanding the fundamental mechanism of adiposity regulation. Furthermore, through comparing the gene expression profiles of adipogenesis between donor with normal weight and donor with obesity, identification of crucial genes, potential drug targets for obesity and type II diabetes, will be possible.
Peroxisome Proliferator-activated Receptor Gamma A og nist Thiazolidinediones (TZDs) act as agonists for the peroxisome-proliferator-activated receptor gamma (PPARy), a member of the nuclear hormone receptor superfamily. TZDs reduce hyperglycemia, hyperinsulinemia, and hypertension, in part by promoting glucose metabolism and inhibiting gluconeogenesis. Roles for PPARy and its agonists have been demonstrated in a wide range of pathological conditions including diabetes, obesity, hypertension, atherosclerosis, polycystic ovarian syndrome, and cancers such as breast, prostate, liposarcoma, and colon cancer.
The mechanism by which TZDs and other PPARy agonists enhance insulin sensitivity is not fully understood, but may involve the ability of PPARy to promote adipogenesis. When ectopically expressed in cultured preadipocytes, PPARy is a potent inducer of adipocyte differentiation. TZDs, in combination with insulin and other factors, can also enhance differentiation of human preadipocytes in culture (Adams et a1. (1997) J. Clin. Invest. 100:3149-3153).
The relative potency of different TZDs in promoting adipogenesis in vitro is proportional to both their insulin sensitizing effects in vivo, and their ability to bind and activate PPARy in vitro.
Interestingly, adipocytes derived from omental adipose depots are refractory to the effects of TZDs. It has therefore been suggested that the insulin sensitizing effects of TZDs may result from their ability to promote adipogenesis in subcutaneous adipose depots (Adams et al., ibid). Further, dominant negative mutations in the PPARy gene have been identified in two non-obese subjects with severe insulin resistance, hypertension, and overt non-insulin dependent diabetes mellitus (NIDDM) (Barroso et aI. (1998) Nature 402:880-883).
NIDDM is the most common form of diabetes mellitus, a chronic metabolic disease that affects 143 million people worldwide. NIDDM is characterized by abnormal glucose and lipid metabolism that result from a combination of peripheral insulin resistance and defective insulin secretion. NTDDM has a complex, progressive etiology and a high degree of heritability. Numerous complications of diabetes including heart disease, stroke, renal failure, retinopathy, and peripheral neuropathy contribute to the high rate of morbidity and mortality.
At the molecular level, PPARy functions as a ligand activated transcription factor. In the presence of ligand, PPARy forms a heterodimer with the retinoid X receptor (RXR) which then activates transcription of target genes containing one or more copies of a PPARy response element (PPRE). Many genes important in lipid storage and metabolism contain PPREs and have been identified as PPARy targets, including PEPCK, aP2, LPL, ACS, and FAT-P
(Auwerx, J. (1999) Diabetologia 42:1033-1049). Multiple ligands for PPARy have been identified.
These include a variety of fatty acid metabolites; synthetic drugs belonging to the TZD class, such as Pioglitazone and Rosiglitazone (BRL49653); and certain non-glitazone tyrosine analogs such as GI262570 and GW 1929. The prostaglandin derivative 15-dPGJ2 is a potent endogenous ligand for PPARy.
Expression of PPARy is very high in adipose but barely detectable in skeletal muscle, the primary site for insulin stimulated glucose disposal in the body. PPARy is also moderately expressed in large intestine, kidney, liver, vascular smooth muscle, hematopoietic cells, and macrophages. The high expression of PPARy in adipose suggests that the insulin sensitizing effects of TZDs may result from alterations in the expression of one or more PPARy regulated genes in adipose tissue.
Identification of PPARy target genes will contribute to better drug design and the development of novel therapeutic strategies for diabetes, obesity, and other conditions.
Systematic attempts to identify PPARy target genes have been made in several rodent models of obesity and diabetes (Suzuki et al. (2000) Jpn. J. Pharmacol. 84:113-123;
Way et al. (2001) Endocrinology 142:1269-1277). However, a serious drawback of the rodent gene expression studies is that significant differences exist between human and rodent models of adipogenesis, diabetes, and obesity (Taylor (1999) Cell 97:9-12; Gregoire et al. (1998) Physiol. Reviews 78:783-809). Therefore, an unbiased approach to identifying TZD regulated genes in primary cultures of human tissues is necessary to fully elucidate the molecular basis for diseases associated with PPARy activity.
Array teclmology 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 examining which genes are tissue specific, carrying out housekeeping functions, parts of a signaling cascade, or specifically related to a particular genetic predisposition, condition, disease, or disorder. The potential application of gene expression profiling is particularly relevant to improving diagnosis, prognosis, and treatment of disease. For example, both the levels and sequences expressed in tissues from subjects with diabetes may be compared with the levels and sequences expressed in normal tissue.
The discovery of new nucleic acid-associated proteins, and the polynucleotides encoding them, satisfies a need in the art by providing new compositions which are useful in the diagnosis, prevention, and treatment of cell proliferative, neurological, developmental, and autoimmune/inflammatory disorders, and infections, and in the assessment of the effects of exogenous compounds on the expression of nucleic acid and amino acid sequences of nucleic acid-associated proteins.
SUMMARY OF THE INVENTION
The invention features purified polypeptides, nucleic acid-associated proteins, referred to collectively as "NAAP" and individually as "NAAP-1," "NAAP-2," "NAAP-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," and "NAAP-I6." In one aspect, the invention provides an isolated polypeptide selected from the group consisting of a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID N0:1-16, b) a polypeptide comprising a naturally occurring amino acid sequence at least 90% identical to an amino acid sequence selected from the group consisting of SEQ 1D N0:1-16, c) a biologically active fragment of a polypeptide having an annino acid sequence selected from the group consisting of SEQ ID
NO: I-16, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1-16. In one alternative, the invention provides an isolated polypeptide comprising the amino acid sequence of SEQ m N0:1-16.
The invention further provides an isolated polynucleotide encoding a polypeptide selected from the group consisting of a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ m N0:1-16, b) a polypeptide comprising a naturally occurring amino acid sequence at least 90% identical to an amino acid sequence selected from the group consisting of SEQ
m NO:l-16, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ m N0:1-I6, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ m NO: I-16.
In one alternative, the polynucleotide encodes a polypeptide selected from the group consisting of SEQ m NO:1-I6. In another alternative, the polynucleotide is selected from the group consisting of SEQ m N0:17-32.
Additionally, the invention provides a recombinant polynucleotide comprising a promoter sequence operably linked to a polynucleotide encoding a polypeptide selected from the group consisting of a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:1-16, b) a polypeptide comprising a naturally occurring amino acid sequence at least 90% identical to an amino acid sequence selected from the group consisting of SEQ m N0:1-16, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ lD NO: I-16, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ m NO:1-16. In one alternative, the invention provides a cell transformed with the recombinant polynucleotide. In another alternative, the invention provides a transgenic organism comprising the recombinant polynucleotide.
The invention also provides a method for producing a polypeptide selected from the group consisting of a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ m N0:1-16, 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:1-16, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ m NO:1-16, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ m N0:1-16. The method comprises a) culturing a cell under conditions suitable for expression of the polypeptide, wherein said cell is transformed with a recombinant polynucleotide comprising a promoter sequence operably linked to a polynucleotide encoding the polypeptide, and b) recovering the polypeptide so expressed.
Additionally, the invention provides an isolated antibody which specifically binds to a polypeptide selected from the group consisting of a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ m NO:1-16, b) a polypeptide comprising a naturally occurring amino acid sequence at least 90% identical to an amino acid sequence selected from the group consisting of SEQ m NO:1-16, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ m NO:1-16, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ m NO:1-16.
The invention further provides an isolated polynucleotide selected from the group consisting of a) a polynucleotide comprising a polynucleotide sequence selected from the group consisting of SEQ m N0:17-32, b) a polynucleotide comprising a naturally occurring polynucleotide sequence at least 90% identical to a polynucleotide sequence selected from the group consisting of SEQ m N0:17-32, 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).
Tn one alternative, the polynucleotide comprises at least 60 contiguous nucleotides.
Additionally, the invention provides a method for detecting a target polynucleotide in a sample, said target polynucleotide having a sequence of a polynucleotide selected from the group consisting of a) a polynucleotide comprising a polynucleotide sequence selected from the group consisting of SEQ m N0:17-32, b) a polynucleotide comprising a naturally occurring polynucleotide sequence at least 90% identical to a polynucleotide sequence selected from the group consisting of SEQ 1D N0:17-32, c) a polynucleotide complementary to the polynucleotide of a), d) a polynucleotide complementary to the polynucleotide of b), and e) an RNA
equivalent of a)-d). The method comprises a) hybridizing the sample with a probe comprising at least 20 contiguous nucleotides comprising a sequence complementary to said target polynucleotide in the sample, and which probe specifically hybridizes to said target polynucleotide, under conditions whereby a hybridization complex is formed between said probe and said target polynucleotide or fragments thereof, and b) detecting the presence or absence of said hybridization complex, and optionally, if present, the amount thereof. In one alternative, the probe comprises at Ieast 60 contiguous nucleotides.
The invention further provides a method for detecting a target polynucleotide in a sample, said target polynucleotide having a sequence of a polynucleotide selected from the group consisting of a) a polynucleotide comprising a polynucleotide sequence selected from the group consisting of SEQ ID N0:17-32, b) a polynucleotide comprising a naturally occurring polynucleotide sequence at least 90% identical to a polynucleotide sequence selected from the group consisting of SEQ m N0:17-32, c) a polynucleotide complementary to the polynucleotide of a), d) a polynucleotide complementary to the polynucleotide of b), and e) an RNA equivalent of a)-d).
The method comprises a) amplifying said target polynucleotide or fragment thereof using polymerase chain reaction amplification, and b) detecting the presence or absence of said amplified target polynucleotide or fragment thereof, and, optionally, if present, the amount thereof.
The invention further provides a composition comprising an effective amount of a polypeptide selected from the group consisting of a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ~ NO:1-16, 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:1-16, c) a biologically active fragment of a poTypeptide having an amino acid sequence selected from the group consisting of SEQ ID
NO:1-16, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ m NO:1-16, and a pharmaceutically acceptable excipient. In one embodiment, the composition comprises an amino acid sequence selected from the group consisting of SEQ m N0:1-16. The invention additionally 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.
The invention also provides a method for screening a compound for effectiveness as an agonist of a polypeptide selected from the group consisting of a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ >D NO: l-16, b) a polypeptide comprising a naturally occurring amino acid sequence at least 90% identical to an amino acid sequence selected from the group consisting of SEQ m NO:1-16, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ll~
NO:1-16, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ m NO:1-16. The method comprises a) exposing a sample comprising the polypeptide to a compound, and b) detecting agonist activity in the sample. In one alternative, the invention provides a composition comprising an agonist compound identified by the method and a pharmaceutically acceptable excipient. In another alternative, the invention provides a method of treating a disease or condition associated with decreased expression of functional NAAP, comprising administering to a patient in need of such treatment the composition.
Additionally, the invention provides a method fox 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 N0:1-16, b) a polypeptide comprising a naturally occurring amino acid sequence at least 90% identical to an amino acid sequence selected from the group consisting of SEQ m NO:1-16, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ m NO:1-16, and d) an ixnmunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ m NO:1-16. The method comprises a) exposing a sample comprising the polypeptide to a compound, and b) detecting antagonist activity in the sample.
In one alternative, the invention provides a composition comprising an antagonist compound identified by the method and a pharmaceutically acceptable excipient. In another alternative, the invention provides a method of treating a disease or condition associated with overexpression of functional NAAP, comprising administering to a patient in need of such treatment the composition.
The invention further provides a method of screening for a compound that specifically binds to a polypeptide selected from the group consisting of a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ m NO:1-16, 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 ll~ NO:1-16, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ m NO:1-16, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1-16. The method comprises a) combining the polypeptide with at least one test compound under suitable conditions, and b) detecting binding of the polypeptide to the test compound, thereby identifying a compound that specifically binds to the polypeptide.
The invention further provides a method of screening for a compound that modulates the activity of a polypeptide selected from the group consisting of a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ m NO:1-16, b) a polypeptide comprising a naturally occurring amino acid sequence at least 90% identical to an amino acid sequence selected from the group consisting of SEQ m N0:1-16, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ m NO:1-16, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ lD NO:1-16. 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 a polypeptide in the presence of the test compound with the activity of the polypeptide in the absence of the test compound, wherein a change in the activity of the polypeptide in the presence of the test compound is indicative of a compound that modulates the activity of the polypeptide.
The invention further provides a method for screening a compound for effectiveness in altering expression of a target polynucleotide, wherein said target polynucleotide comprises a polynucleotide sequence selected from the group consisting of SEQ ID N0:17-32, the method comprising a) exposing a sample comprising the target polynucleotide to a compound, b) detecting altered expression of the target polynucleotide, and c) comparing the expression of the target polynucleotide in the presence of varying amounts of the compound and in the absence of the compound.
The invention further provides a method for assessing toxicity of a test compound, said method comprising a) treating a biological sample containing nucleic acids with the test compound;
b) hybridizing the nucleic acids of the treated biological sample with a probe comprising at least 20 contiguous nucleotides of a polynucleotide selected. from the group consisting of i) a polynucleotide comprising a polynucleotide sequence selected from the group consisting of SEQ
ID NO:17-32, ii) a polynucleotide comprising a naturally occurring polynucleotide sequence at least 90% identical to a polynucleotide sequence selected from the group consisting of SEQ ID N0:17-32, iii) a polynucleotide having a sequence complementary to i), iv) a polynucleotide complementary to the polynucleotide of ii), and v) an RNA equivalent of i)-iv). Hybridization occurs under conditions whereby a specific hybridization complex is formed between said probe and a target polynucleotide in the biological sample, said target polynucleotide selected from the group consisting of i) a polynucleotide comprising a polynucleotide sequence selected from the group consisting of SEQ ID
N0:17-32, ii) a polynucleotide comprising a naturally occurring polynucleotide sequence at least 90% identical to a polynucleotide sequence selected from the group consisting of SEQ ID N0:17-32, iii) a polynucleotide complementary to the polynucleotide of i), iv) a polynucleotide complementary to the polynucleotide of ii), and v) an RNA equivalent of i)-iv).
Alternatively, the target polynucleotide comprises a fragment of a polynucleotide sequence selected from the group consisting of i)-v) above; c) quantifying the amount of hybridization complex; and d) comparing the amount of hybridization complex in the treated biological sample with the amount of hybridization complex in an untreated biological sample, wherein a difference in the amount of hybridization complex in the treated biological sample is indicative of toxicity of the test compound.

BRIEF DESCRIPTION OF THE TABLES
Table 1 summarizes the nomenclature for the full length polynucleotide and polypeptide sequences of the present invention.
Table 2 shows the GenBank identification number and annotation of the nearest GenBank homolog, and the PROTEOME database identification numbers and annotations of PROTEOME
database homologs, for polypeptides of the invention. The probability scores for the matches between each polypeptide and its homolog(s) are also shown.
Table 3 shows structural features of polypeptide sequences of the invention, including predicted motifs and domains, along with the methods, algorithms, and searchable databases used for analysis of the polypeptides.
Table 4 lists the cDNA andlor genomic DNA fragments which were used to assemble polynucleotide sequences of the invention, along with selected fragments of the polynucleotide sequences.
Table 5 shows the representative cDNA library for polynucleotides of the invention.
Table 6 provides an appendix which describes the tissues and vectors used for construction of the cDNA Libraries shown in Table 5.
Table 7 shows the tools, programs, and algorithms used to analyze the polynucleotides and polypeptides of the invention, along with applicable descriptions, references, and threshold parameters.
DESCRIPTION OF THE INVENTION
Before the present proteins, nucleotide sequences, and methods are described, it is understood that this invention is not limited to the particular machines, materials and methods described, as these may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to Limit the scope of the present invention which will be limited only by the appended claims.
Tt must be noted that as used herein and in the appended claims, the singular forms "a," "an,"
and "the" include plural reference unless the context clearly dictates otherwise. Thus, for example, a ' reference to "a host cell" includes a plurality of such host cells, and a reference to "an antibody" is a reference to one or more antibodies and equivalents thereof known to those skilled in the art, and so forth.
Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art to which this invention belongs.
Although any machines, materials, and methods similar or equivalent to those described herein can be used to practice or test the present invention, the preferred machines, materials and methods are now described. All publications mentioned herein are cited for the purpose of describing and disclosing the cell lines, protocols, reagents and vectors which are reported in the publications and which might be used in connection with the invention. Nothing herein is to be construed as an admission that the invention is not entitled to antedate such disclosure by virtue of prior invention.
DEFINITIONS
"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 sequence 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 similarity 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 glutamic acid, and positively charged amino acids may include lysine and arginine. Amino acids with uncharged polar side chains having similar hydrophilicity values may include: asparagine and glutamine; and serine and threonine.
Amino acids with uncharged side chains having similar hydrophilicity values may include: leucine, isoleucine, and valine; glycine and alanine; and phenylalanine and tyrosine.
The terms "amino acid" and "amino acid sequence" refer to an oligopeptide, peptide, polypeptide, or protein sequence, or a fragment of any of these, and to naturally occurring or synthetic molecules. Where "amino acid sequence" is recited to refer to a sequence of a naturally occurring protein molecule, "amino acid sequence" and like terms are not meant to limit the amino acid sequence to the complete native amino acid sequence associated with the recited protein molecule.
"Amplification" relates to the production of additional copies of a nucleic acid sequence.
Amplification is generally carried out using polymerase chain reaction (PCR) technologies well known in the art.
The term "antagonist" refers to a molecule which inhibits or attenuates the biological activity of NAAP. Antagonists may include proteins such as antibodies, 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')z, 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 in vitro evolutionary process (e.g., SELEX
(Systematic Evolution of Ligands by EXponential Enrichment), described in U.S.
Patent No.
5,270,163), which selects for target-specific aptamer sequences from large combinatorial libraries.
Aptamer compositions may be double-stranded or single-stranded, and may include deoxyribonucleotides, ribonucleotides, nucleotide derivatives, or other nucleotide-like molecules.

The nucleotide components of an aptamer may have modified sugar groups (e.g., the 2'-OH group of a ribonucleotide may be replaced by 2'-F or 2'-NHZ), which may improve a desired property, e.g., resistance to nucleases or longer lifetime in blood. Aptamers may be conjugated to other molecules, e.g., a high molecular weight carrier to slow clearance of the aptamer from the circulatory system.
Aptamers may be specifically cross-linked to their cognate ligands, e.g., by photo-activation of a cross-linker. (See, e.g., Brody, E.N. and L. Gold (2000) J. Biotechnol. 74:5-13.) The term "intramer" refers to an aptamer which is expressed in vivo. For example, a vaccinia virus-based RNA expression system has been used to express specific RNA
aptamers at high levels in the cytoplasm of leukocytes (Blind, M. et al. (1999) Proc. Natl Acad. Sci. USA
96:3606-3610).
The term "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 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 sequence" and a "composition comprising a given amino acid sequence" refer broadly to any composition containing the given polynucleotide or amino acid sequence. The composition may comprise a dry formulation or an aqueous solution.

Compositions comprising polynucleotide sequences encoding 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., NaCI), detergents (e.g., sodium dodecyl sulfate; SDS), and other components (e.g., Denhardt's solution, dry milk, salmon sperm DNA, etc.).
"Consensus sequence" refers to a nucleic acid sequence which has been subjected to repeated DNA sequence analysis to resolve uncalled bases, extended using the XL-PCR kit (Applied Biosystems, Foster City CA) in the 5' and/or the 3' direction, and resequenced, or which has been assembled from one or more overlapping cDNA, EST, or genomic DNA fragments using a computer program for fragment assembly, such as the GELVIEW fragment assembly system (GCG, Madison WI) or Phrap (University of Washington, Seattle WA). Some sequences have been both extended and assembled to produce the consensus sequence.
"Conservative amino acid substitutions" are those substitutions that are predicted to Ieast 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, Ser Gln Asn, Glu, His Glu Asp, Gln, His Gly Ala His Asn, Arg, Gln, Glu Ile Leu, Val Leu lle, 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 the polynucleotide encoding NAAP
which is identical in sequence to but shorter in length than the parent sequence. A
fragment may comprise up to the entire length of the defined sequence, minus one nucleotide/amino acid residue. For example, a fragment may comprise from 5 to 1000 contiguous nucleotides or amino acid residues. A fragment used as a probe, primer, antigen, therapeutic molecule, or for other purposes, may be at least 5, 10, 15, 16, 20, 25, 30, 40, 50, 60, 75, 100, 150, 250 or at least 500 contiguous nucleotides or amino acid 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°l0) 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 1D N0:17-32 comprises a region of unique polynucleotide sequence that specifically identifies SEQ m N0:17-32, for example, as distinct from any other sequence in the genome from which the fragment was obtained. A fragment of SEQ m N0:17-32 is useful, for example, in hybridization and amplification technologies and in analogous methods that distinguish SEQ ID N0:17-32 from related polynucleotide sequences. The precise length of a fragment of SEQ

ID N0:17-32 and the xegion of SEQ ID N0:17-32 to which the fragment corresponds are routinely determinable by one of ordinary skill in the art based on the intended purpose for the fragment.
A fragment of SEQ ID NO:1-16 is encoded by a fragment of SEQ ID N0:17-32. A
fragment of SEQ ID N0:1-16 compxises a region of unique amino acid sequence that specifically identifies SEQ ID NO:1-16. For example, a fxagment of SEQ ID NO:1-16 is useful as an immunogenic peptide for the development of antibodies that specifically recognize SEQ ID NO:1-16.
The precise length of a fragment of SEQ ID N0:1-16 and the region of SEQ ID NO: l-16 to which the fragment corresponds are routinely determinable by one of ordinary skill in the art based on the intended puxpose for the fragment.
A "full length" polynucleotide sequence is one containing at least a translation initiation codon (e.g., methionine) followed by an open reading frame and a translation termination codon. A
"full length" polynucleotide sequence encodes a "full length" polypeptide sequence.
"Homology" refers to sequence similarity or, interchangeably, sequence identity, between two or more polynucleotide sequences or two or more polypeptide sequences.
The terms "percent identity" and "% identity," as applied to polynucleotide sequences, refer to the percentage of residue matches between at least two polynucleotide sequences aligned using a standardized algorithm. Such an algorithm may insert, in a standardized and reproducible way, gaps in the sequences being compared in order to optimize alignment between two sequences, and therefore achieve a more meaningful comparison of the two sequences.
Percent identity between polynucleotide sequences may be determined using the default parameters of the CLUSTAL V algorithm as incorporated into the MEGALIGN
version 3.12e sequence alignment program. This program is part of the LASERGENE software package, a suite of molecular biological analysis programs (DNASTAR, Madison WI). CLUSTAL V is described in Higgins, D.G. and P.M. Sharp (1989) CABIOS 5:151-153 and in Higgins, D.G. et al. (1992) CABIOS
8:189-191. For pairwise alignments of polynucleotide sequences, the default parameters are set as follows: Ktuple=2, gap penalty=5, window=4, and "diagonals saved"=4. The "weighted" residue weight table is selected as the default. Percent identity is reported by CLUSTAL V as the "percent similarity" between aligned polynucleotide sequences.
Alternatively, a suite of commonly used and freely available sequence comparison algorithms is provided by the National Center for Biotechnology Information (NCBI) Basic Local Alignment Search Tool (BLAST) (Altschul, S.F. et al. (1990) J. Mol. Biol. 215:403-410), which is available from several sources, including the NCBI, Bethesda, MD, and on the Internet at http://www.ncbi.nlm.nih.gov/BLAST/. The BLAST software suite includes various sequence analysis programs including "blastn," that is used to align a known polynucleotide sequence with other polynucleotide sequences from a variety of databases. Also available is a tool called "BLAST 2 Sequences" that is used for direct pairwise comparison of two nucleotide sequences. "BLAST 2 Sequences" can be accessed and used interactively at http://www.ncbi.nlm.nih.gov/gorf/bl2.html.
The "BLAST 2 Sequences" tool can be used for both blastn and blastp (discussed below). BLAST
programs are commonly used with gap and other parameters set to default settings. For example, to compare two nucleotide sequences, one may use blastn with the "BLAST 2 Sequences" tool Version 2Ø12 (April-21-2000) set at default parameters. Such default parameters may be, fox example:
Matrix: BLOSUM62 Reward for match: 1 Penalty for mismatch: -2 Open Gap: S and Extension Gap: 2 penalties Gap x drop-off.' S0 Expect: 10 Word Size: I1 Filter: on Percent identity may be measured over the length of an entire defined sequence, for example, as defined by a particular SEQ ll~ number, or may be measured over a shorter length, for example, over the length of a fragment taken from a larger, defined sequence, for instance, a fragment of at least 20, at least 30, at least 40, at least 50, at least 70, at least 100, or at least 200 contiguous nucleotides. Such lengths are exemplary only, and it is understood that any fragment length supported by the sequences shown herein, in the tables, figures, or Sequence Listing, may be used to describe a length over which percentage identity may be measured.
Nucleic acid sequences that do not show a high degree of identity may nevertheless encode similar amino acid sequences due to the degeneracy of the genetic code. It is understood that changes in a nucleic acid sequence can be made using this degeneracy to produce multiple nucleic acid sequences that all encode substantially the same protein.
The phrases "percent identity" and "% identity," as applied to polypeptide sequences, refer to the percentage of residue matches between at least two polypeptide sequences aligned using a standardized algorithm. Methods of polypeptide sequence alignment are well-known. Some alignment methods take into account conservative amino acid substitutions.
Such conservative substitutions, explained in more detail above, generally preserve the charge and hydrophobicity at the site of substitution, thus preserving the structure (and therefore function) of the polypeptide.
Percent identity between polypeptide sequences may be determined using the default parameters of the CLUSTAL V algorithm as incorporated into the MEGALIGN
version 3.12e sequence alignment program (described and referenced above). For pairwise alignments of polypeptide sequences using CLUSTAL V, the default parameters are set as follows: Ktuple=1, gap penalty=3, window=5, and "diagonals saved"=5. The PAM250 matrix is selected as the default residue weight table. As with polynucleotide alignments, the percent identity is reported by CLUSTAL V as the "percent similarity" between aligned polypeptide sequence pairs.
Alternatively the NCBI BLAST software suite may be used. For example, for a pairwise comparison of two polypeptide sequences, one may use the "BLAST 2 Sequences"
tool Version 2Ø12 (April-21-2000) with blastp set at default parameters. Such default parameters may be, for example:
Matrix: BLOSUM62 Open Gap: 1l and Extension Gap: 1 penalties Gap x drop-off.' S0 Expect: 10 Word Size: 3 Filter: on Percent identity may be measured over the length of an entire defined polypeptide sequence, for example, as defined by a particular SEQ ID number, or may be measured over a shorter length, for example, over the length of a fragment taken from a larger, defined polypeptide sequence, for instance, a fragment of at least 15, at least 20, at least 30, at least 40, at least 50, at least 70 or at least 150 contiguous residues. Such lengths are exemplary only, and it is understood that any fragment length supported by the sequences shown herein, in the tables, figures or Sequence Listing, may be used to describe a length over which percentage identity may be measured.
"Human artificial chromosomes" (HACs) are linear microchromosomes which may contain DNA sequences of about 6 kb to 10 Mb in size and which contain all of the elements required for chromosome replication, segregation and maintenance.
The term "humanized antibody" refers to an antibody molecule in which the amino acid sequence in the non-antigen binding regions has been altered so that the antibody more closely resembles a human antibody, and still retains its original binding ability.
"Hybridization" refers to the process by which a polynucleotide strand anneals with a complementary strand through base pairing under defined hybridization conditions. Specific hybridization is an indication that two nucleic acid sequences share a high degree of complementarzty.
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 I00 ,ug/mI 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 (Tm) for the specific sequence at a defined ionic strength and pH. The Tm is the temperature (under defined ionic strength and pH) at which 50% of the target sequence hybridizes to a perfectly matched probe. An equation for calculating Tm and conditions for nucleic acid hybridization are well known and can be found in Sambrook, J. et al.
(1989) Molecular Cloning: A Laboratory Manual, 2nd 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 ~g/ml. Organic solvent, such as formamide at a concentration of about 35-50% v/v, may also be used under particular circumstances, such as for RNA:DNA hybridizations. Useful variations on these wash conditions will be readily apparent to those of ordinary skill in the art. Hybridization, particularly under high stringency conditions, may be suggestive of evolutionary similarity between the nucleotides. Such simitlarity is strongly indicative of a similar role for the nucleotides and their encoded polypeptides.
The term "hybridization complex" refers to a complex formed between two nucleic acid sequences by virtue of the formation of hydrogen bonds between complementary bases. A
hybridization complex may be formed in solution (e.g., Cot or Rot analysis) or formed between one nucleic acid sequence present in solution and another nucleic acid sequence immobilized on a solid support (e.g., paper, membranes, filters, chips, pins or glass slides, or any other appropriate substrate to which cells or their nucleic acids have been fixed).
The words "insertion" and "addition" refer to changes in an amino acid or nucleotide sequence resulting in the addition of one or more amino acid residues or nucleotides, respectively.
"Immune response" can refer to conditions associated with inflammation, trauma, immune disorders, or infectious or genetic disease, etc. These conditions can be characterized by expression of various factors, e.g., cytokines, chemokines, and other signaling molecules, which may affect cellular and systemic defense systems.
An "immunogenic fragment" is a polypeptide or oligopeptide fragment of NAAP
which is capable of eliciting an immune response when introduced into a living organism, for example, a mammal. The term "imnnunogenic 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 art.
The term "microarray" refers to an arrangement of a plurality of polynucleotides, polypeptides, or other chemical compounds on a substrate.
The terms "element" and "array element" refer to a polynucleotide, polypeptide, or other chennical compound having a unique and defined position on a microarray.
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, oligonucleotide, polynucleotide, or any fragment thereof. These phrases also refer to DNA or RNA of genomic or synthetic origin which may be single-stranded or double-stranded and may represent the sense or the antisense strand, to peptide nucleic acid (PNA), or to any DNA-like or RNA-like material.
"Operably linked" refers to the situation in which a first nucleic acid sequence is placed in a functional relationship with a second nucleic acid sequence. For instance, a promoter is operably linked to a coding sequence if the promoter affects the transcription or expression of the coding sequence. Operably linked DNA sequences may be in close proximity or contiguous and, where necessary to join two protein coding regions, in the same reading frame.
"Peptide nucleic acid" (PNA) refers to an antisense molecule or anti-gene agent which comprises an oligonucleotide of at least about 5 nucleotides in length linked to a peptide backbone of amino acid residues ending in lysine. The terminal lysine confers solubility to the composition.
PNAs preferentially bind complementary single stranded DNA or RNA and stop transcript elongation, and may be pegylated to extend their lifespan in the cell.
"Post-translational modification" of an 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 acid sequences encoding NAAP, their complements, or fragments thereof, which are used to detect identical, allelic or related nucleic acid sequences. Probes are isolated oligonucleotides or polynucleotides attached to a detectable label or reporter molecule.
Typical labels include radioactive isotopes, ligands, chemiluminescent agents, and enzymes.
"Primers" are short nucleic acids, usually DNA oligonucleotides, which may be annealed to a target polynucleotide by complementary base-pairing. The primer may then be extended along the target DNA strand by a DNA polymerase enzyme. Primer pairs can be used for amplification (and identification) of a nucleic acid sequence, e.g., by the polymerase chain reaction (PCR).
Probes and primers as used in the present invention typically comprise at least 15 contiguous nucleotides of a known sequence. In order to enhance specificity, longer probes and primers may also be employed, such as probes and primers that comprise at least 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, or at least 150 consecutive nucleotides of the disclosed nucleic acid sequences. Probes and primers may be considerably longer than these examples, and it is understood that any length supported by the specification, including the tables, figures, and Sequence Listing, may be used.
Methods for preparing and using probes and primers are described in the references, for example Sambrook, J. et al. (1989) Molecular Cloning: A Laboratory Manual, 2°d ed., vol. 1-3, Cold Spring Harbor Press, Plainview NY; Ausubel, F.M. et al. (1987) Current Protocols in Molecular Biolo~y, Greene Publ. Assoc. & Wiley-Intersciences, New York NY; Innis, M. et al. (1990) PCR
Protocols, A Guide to Methods and Applications, Acadennic Press, San Diego CA.
PCR primer pairs can be derived from a known sequence, for example, by using computer programs intended fox that purpose such as Primer (Version 0.5, 1991, Whitehead Institute for Biomedical Research, Cambridge MA).
Oligonucleotides for use as primers are selected using software known in the art for such purpose. For example, OLIGO 4.06 software is useful for the selection of PCR
primer pairs of up to 100 nucleotides each, and for the analysis of oligonucleotides and larger polynucleotides of up to 5,000 nucleotides from an input polynucleotide sequence of up to 32 kilobases.
Similar primer selection programs have incorporated additional features for expanded capabilities. For example, the PrimOU primer selection program (available to the public from the Genome Center at University of Texas South West Medical Center, Dallas TX) is capable of choosing specific primers from megabase sequences and is thus useful for designing primers on a genome-wide scope. The Primer3 primer selection program (available to the public from the Whitehead Institute/MIT Center for Genome Research, Cambridge MA) allows the user to input a "mispriming library," in which sequences to avoid as primer binding sites are user-specified. Primer3 is useful, in particular, for the selection of oligonucleotides for microarrays. (The source code for the latter two primer selection programs may also be obtained from their respective sources and modified to meet the user's specific needs.) The PrimeGen program (available to the public from the UK Human Genome Mapping Project Resource Centre, Cambridge UK) designs primers based on multiple sequence alignments, thereby allowing selection of primers that hybridize to either the most conserved or least conserved regions of aligned nucleic acid sequences. Hence, this program is useful for identification of 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 sequence that is not naturally occurring or has a sequence that is made by an artificial combination of two or more otherwise separated segments of sequence.
This artificial combination is often accomplished by chemical synthesis or, more commonly, by the artificial manipulation of isolated segments of nucleic acids, e.g., by genetic engineering techniques such as those described in Sambrook, supra. The term recombinant includes nucleic acids that have been altered solely by addition, substitution, or deletion of a portion of the nucleic acid. Frequently, a recombinant nucleic acid may include a nucleic acid sequence operably linked to a promoter sequence. Such a recombinant nucleic acid may be part of a vector that is used, for example, to transform a cell.
Alternatively, such recombinant nucleic acids may be part of a viral vector, e.g., based on a vaccinia virus, that could be use to vaccinate a mammal wherein the recombinant nucleic acid is expressed, inducing a protective immunological response in the mammal.
A "regulatory element" refers to a nucleic acid sequence usually derived from untxanslated regions of a gene and includes enhancexs, promoters, introns, and 5' and 3' untranslated regions (ITTRs). Regulatory elements interact with host or viral proteins which control transcription, translation, or RNA stability.
"Reporter molecules" are chemical or biochemical moieties used for labeling a nucleic acid, amino acid, or antibody. Reporter molecules include radionuclides; enzymes;
fluorescent, chemiluminescent, or chromogenic agents; substrates; cofactors; inhibitors;
magnetic particles; and other moieties known in the art.
An "RNA equivalent," in reference to a DNA sequence, is composed of the same linear sequence of nucleotides as the reference DNA sequence with the exception that all occurrences of the nitrogenous base thymine are replaced with uracil, and the sugar backbone is composed of ribose instead of deoxyribose.
The term "sample" is used in its broadest sense. A sample suspected of containing 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" refex to that interaction between a protein or peptide and an agonist, an antibody, an antagonist, a small molecule, or any natural or synthetic binding composition. The interaction is dependent upon the presence of a particular structure of the protein, e.g., the antigenic determinant or epitope, recognized by the binding molecule. For example, if an antibody is specific for epitope "A," the presence of a polypeptide comprising the epitope A, or the presence of free unlabeled A, in a reaction containing free labeled A
and the antibody will reduce the amount of labeled A that binds to the antibody.
The term "substantially purified" refers to nucleic acid or amino acid sequences that are removed from their natural environment and are isolated or separated, and are at least 60% free, preferably at least 75% free, and most preferably at Ieast 90% free from other components with which they are naturally associated.
A "substitution" refers to the replacement of one or more amino acid residues or nucleotides by different amino acid residues or nucleotides, respectively.
"Substrate" refers to any suitable rigid or semi-rigid support including membranes, filters, chips, slides, wafers, fibers, magnetic or nonmagnetic beads, gels, tubing, plates, polymers, microparticles and capillaries. The substrate can have a variety of surface forms, such as wells, trenches, pins, channels and pores, to which polynucleotides or polypeptides are bound.
A "transcript image" or "expression profile" refers to the collective pattern of gene expression by a particular cell type or tissue under given conditions at a given time.
"Transformation" describes a process by which exogenous DNA is introduced into a recipient cell. Transformation may occur under natural or artificial conditions according to various methods well known in the art, and may rely on any known method for the insertion of foreign nucleic acid sequences into a prokaryotic or eukaryotic host cell. The method for transformation is selected based on the type of host cell being transformed and may include, but is not limited to, bacteriophage or viral infection, electroporation, heat shock, Iipofection, and particle bombardment. The term "transformed cells" includes stably transformed cells in which the inserted DNA is capable of replication either as an autonomously replicating plasmid or as part of the host chromosome, as well as transiently transformed cells which express the inserted DNA or RNA for limited periods of time.
A "transgenic organism," as used herein, is any organism, including but not limited to animals and plants, in which one or more of the cells of the organism contains heterologous nucleic acid introduced by way of human intervention, such as by transgenic techniques well known in the art. The nucleic acid is introduced into the cell, directly or indirectly by introduction into a precursor of the cell, by way of deliberate genetic manipulation, such as by microinjection or by infection with a recombinant virus. The term genetic manipulation does not include classical cross-breeding, or in vitro fertilization, but rather is directed to the introduction of a recombinant DNA molecule. The transgenic organisms contemplated in accordance with the present invention include bacteria, cyanobacteria, fungi, plants and animals. The isolated DNA of the present invention can be introduced into the host by methods known in the art, for example infection, transfection, transformation or transconjugation. Techniques for transferring the DNA of the present invention into such organisms are widely known and provided in references such as Sambrook et al. (1989), 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-I999) 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 polynucleotide sequences that vary from one species to another. The resulting polypeptides will generally have significant amino acid identity relative to each other. A polymorphic variant is a variation in the polynucleotide sequence of a particular gene between individuals of a given species. Polymorphic variants also may encompass "single-nucleotide polymorphisms" (SNPs) in which the polynucleotide sequence varies by one nucleotide base. The 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 2S least 60%, at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at Least 98%, or at least 99%
or greater sequence identity over a certain defined length of one of the polypeptides.
THE INVENTION
The invention is based on the discovery of new human 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, developmental, and autoimmune/inflammatory disorders, and infections.
Table 1 summarizes the nomenclature for the full length polynucleotide and polypeptide sequences of the invention. Each polynucleotide and its corresponding polypeptide are correlated to a single Incyte project identification numbex (Incyte Project ID). Each polypeptide sequence is denoted by both a polypeptide sequence identification number (Polypeptide SEQ lD NO:) and an Incyte polypeptide sequence number (Incyte Polypeptide ID) as shown. Each polynucleotide sequence is denoted by both a polynucleotide sequence identification number (Polynucleotide SEQ ID NO:) and an Incyte polynucleotide consensus sequence number (Incyte Polynucleotide ID) as shown.
Table 2 shows sequences with homology to the polypeptides of 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 ll~ NO:) of the nearest GenBank homolog. Column 4 shows the probability scores for the matches between each polypeptide and its homolog(s). Column 5 shows the annotation of the GenBank homolog(s) along with relevant citations where applicable, all of which are expressly incorporated by reference herein.
Table 3 shows various structural features of the polypeptides of the invention. Columns 1 and 2 show the polypeptide sequence identification number (SEQ ID NO:) and the corresponding Incyte polypeptide sequence number (Incyte Polypeptide ID) for each polypeptide of the invention.
Column 3 shows the number of amino acid residues in each polypeptide. Column 4 shows potential phosphorylation sites, and column 5 shows potential glycosylation sites, as determined by the MOT1FS 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 ID N0:2 is 38% identical from amino acid residues 210 to 768 to Haemophilus influenzae (Rd) transcription accessory protein (tex) (GenBank ID
g1573555) as determined by the Basic Local Alignment Search Tool (BLAST). (See Table 2.) The BLAST
probability score is 7.9e-105, which indicates the probability of obtaining the observed polypeptide sequence alignment by chance. SEQ ID N0:2 also contains an S 1 RNA binding domain as determined by searching for statistically significant matches in the hidden Markov model (HMM)-based PFAM database of conserved protein family domains. (See Table 3.) Data from BLIMPS and additional BLAST analyses provide further corroborative evidence that SEQ 1D
N0:2 is a transcriptional regulator.
In an alternative example, SEQ ID N0:3 is 92% identical to zebxafish emx2 hemeoprotein (GenBank ID g1089816) as determined by the Basic Local Alignment Search Tool (BLAST). (See Table 2.) The BLAST probability score is 3.2e-126, which indicates the probability of obtaining the observed polypeptide sequence alignment by chance. SEQ ID N0:3 also contains a homeobox domain as determined by searching for statistically significant matches in the hidden Markov model (HMM)-based PFAM database of conserved protein family domains. (See Table 3.) Data from BLIMPS, MOTIFS, and PROFILESCAN analyses provide further corroborative evidence that SEQ
ID N0:3 is a homeobox protein.
In an alternative example, SEQ ID N0:4 is 38% identical to an Arabidopsis thaliana bromodomain-containing transcription factor (GenBank ID g6850321) as determined by the BLAST
analysis with a probability score of 4.8e-52. SEQ ID N0:4 also contains a bromodomain as determined by searching for statistically significant matches in the hidden Markov model (HMM)-based PFAM database of conserved protein family domains. Data from BLIMPS and PROFILESCAN analyses provide further corroborative evidence that SEQ ID N0:4 is a bromodomain protein.
In an alternative example, SEQ ID N0:5 is 58% identical to human RET finger protein-like 3 (GenBank m g3417319) as determined by the BLAST analysis with a probability score of 2.1e-83.
SEQ ID N0:5 also contains a RING finger domain, characteristic of transcription factors, as determined by searching for statistically significant matches in the hidden Markov model (HMM)-based PFAM database of conserved protein family domains.
In an alternative example, SEQ ID N0:6 is 34% identical to a human acid forger protein, with similarity to C3HC4-type zinc finger proteins (GenBaxik ID g563127), as determined by the BLAST
analysis, with a probability score of 1.3e-23. SEQ ID N0:6 also contains zinc forger domains as determined by searching for statistically significant matches in the hidden Markov model (HMM)-based PFAM database of conserved protein family domains. Data from PROFILESCAN
and MOTIFS analyses provide further corroborative evidence that SEQ ID N0:6 is a zinc finger protein.
In an alternative example, SEQ ID N0:7 is 52% identical to the human CAGH44 protein (GenBank ID g2565057) as determined by the BLAST analysis with a probability score of 2.7e-46.
SEQ ID N0:7 also contains a fork-head domain as determined by searching for statistically significant matches in the hidden Markov model (HMM)-based PFAM database of conserved protein family domains. Data from BLIMPS, PROF1I,ESCAN, and MOTIFS analyses provide further corroborative evidence that SEQ ID N0:7 contains fork-head and zinc finger domains, characteristic of DNA-binding proteins.
In an alternative example, SEQ ID N0:8 is 91 % identical to the murine OASIS
CREB/ATF-family transcription factor (GenBank ID g4519621), as determined by the Basic Local Alignment Search Tool (BLAST). (See Table 2.) The BLAST probability score is 9.8e-251, which indicates the probability of obtaining the observed polypeptide sequence alignment by chance. SEQ ID N0:8 also contains a bZIP transcription factor domain as determined by searching for statistically significant matches in the hidden Markov model (HMM)-based PFAM database of conserved protein family domains. (See Table 3.) Data from BLIMPS and MOTIFS analyses provide further corroborative evidence that SEQ m N0:8 is a transcription factor.
In an alternative example, SEQ ID NO:11 is 58% identical to a human Kruppel-type zinc finger protein (GenBank ID g4519270) as determined by the Basic Local Alignment Search Tool (BLAST). (See Table 2.) The BLAST probability score is 2.0e-213, which indicates the probability of obtaining the observed polypeptide sequence alignment by chance. SEQ ID N0:
I I also contains a KRAB box and a C2H2 type Zinc finger domain as determined by searching for statistically significant matches in the hidden Markov model (HMM)-based PFAM database of conserved protein family domains. (See Table 3.) Data from BLIMPS and MOTIFS analyses provide further corroborative evidence that SEQ ll~ NO: l I is a zinc finger protein.
In an alternative example, SEQ ID N0:14 is 64% identical to Drosophila melano a~ ster protease (GenBank ID g2791289) as determined by the Basic Local Alignment Search Tool (BLAST). (See Table 2.) The BLAST probability score is 0.0, which indicates the probability of obtaining the observed polypeptide sequence alignment by chance. SEQ ID N0:14 also contains a reverse transcriptase (RNA-dependent DNA polymerase) domain, a "phage"
integrase family signature sequence, and an integrase core domain as determined by searching for statistically significant matches in the hidden Markov model (HMM)-based PFAM database of conserved protein family domains. (See Table 3.) Data from BL1MPS and additional BLAST analyses provide further corroborative evidence that SEQ ID N0:14 is a retrovirus-related polyprotein.
SEQ ID NO:1, SEQ ID N0:9, SEQ ID N0:10, SEQ ID N0:12, SEQ ID N0:13, SEQ ID
N0:15 and SEQ ID N0:16 were analyzed and annotated in a similar manner. The algorithms and parameters for the analysis of SEQ ID NO:1-16 are described in Table 7.
As shown in Table 4, the full length polynucleotide sequences of the present invention were assembled using cDNA sequences or coding (exon) sequences derived from genomic DNA, or any combination of these two types of sequences. Column 1 lists the polynucleotide sequence identification number (Polynucleotide SEQ ID NO:), the corresponding Incyte polynucleotide consensus sequence number (Incyte ID) for each polynucleotide of the invention, and the length of each polynucleotide sequence in basepairs. Column 2 shows the nucleotide start (5') and stop (3') positions of the cDNA and/or genomic sequences used to assemble the full length polynucleotide sequences of the invention, and of fragments of the polynucleotide sequences which are useful, for example, in hybridization or amplification technologies that identify SEQ ID
N0:17-32 or that distinguish between SEQ ID N0:17-32 and related polynucleotide sequences.
The polynucleotide fragments described in Column 2 of Table 4 may refer specifically, for example, to Incyte cDNAs derived from tissue-specific cDNA libraries or from pooled cDNA
libraries. Alternatively, the polynucleotide fragments described in column 2 may refer to GenBank cDNAs or ESTs which contributed to the assembly of the full length polynucleotide sequences. In addition, the polynucleotide fragments described in column 2 may identify sequences derived from the ENSEMBL (The Sanger Centre, Cambridge, UK) database (i.e., those sequences including the designation "ENST"). Alternatively, the polynucleotide fragments described in column 2 may be derived from the NCBI RefSeq Nucleotide Sequence Records Database (i.e., those sequences including the designation "NM" or "NT") or the NCBI RefSeq Protein Sequence Records (i.e., those sequences including the designation "NP"). Alternatively, the polynucleotide fragments described in column 2 may refer to assemblages of both cDNA and Genscan-predicted exons brought together by an "exon stitching" algorithm. For example, a polynucleotide sequence identified as FL XXXXXX Nl Nz_YYYYY_N3 Nø represents a "stitched" sequence in which XXXXXX
is the identification number of the cluster of sequences to which the algorithm was applied, and YYYYY is the number of the prediction generated by the algorithm, and N~,a,3..,, if present, represent specific exons that may have been manually edited during analysis (See Example V).
Alternatively, the polynucleotide fragments in column 2 may refer to assemblages of exons brought together by an "exon-stretching" algorithm. For example, a polynucleotide sequence identified as FLXXXXXX_gAAAAA_gBBBBB_1 N is a "stretched" sequence, with XXXXXX being the Incyte project identification number, gAAAA.A 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 1V and Example V).
Prefix Type of analysis andlor examples of programs GNN, GFG,Exon prediction from genomic sequences using, ENST for example, GENSCAN (Stanford University, CA, USA) or FGENES
(Computer Genomics Group, The Sanger Centre, Cambridge, UI~).

GBI Hand-edited analysis of genomic sequences.

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

INCY Full length transcript and exon prediction from mapping of EST
sequences to the genome. Genomic location and EST composition data are combined to predict the exons and resulting transcript.
In some cases, Incyte cDNA coverage redundant with the sequence coverage shown in Table 4 was obtained to confirm the final consensus polynucleotide sequence, but the relevant Incyte cDNA
identification numbers are not shown.
Table 5 shows the representative cDNA libraries for those full length polynucleotide sequences which were assembled using Incyte cDNA sequences. The representative cDNA library is the Incyte cDNA library which is most frequently represented by the Incyte cDNA sequences which were used to assemble and confirm the above polynucleotide sequences. The tissues and vectors which were used to construct the cDNA libraries shown in Table 5 are described in Table 6.
The invention also encompasses 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.
The invention also encompasses polynucleotides which encode NAAP. In a particular embodiment, the invention encompasses a polynucleotide sequence comprising a sequence selected from the group consisting of SEQ ll~ N0:17-32, which encodes NAAP. The polynucleotide sequences of SEQ m N0:17-32, as presented in the Sequence Listing, embrace the equivalent RNA
sequences, wherein occurrences of the nitrogenous base thymine are replaced with uracil, and the sugar backbone is composed of ribose instead of deoxyribose.
The invention also encompasses a variant of a polynucleotide sequence encoding NAAP. In particular, such a variant polynucleotide sequence will have at least about 70%, or alternatively at least about 85%, or even at least about 95% polynucleotide sequence identity to the polynucleotide sequence encoding NAAP. A particular aspect of the invention encompasses a variant of a polynucleotide sequence comprising a sequence selected from the group consisting of SEQ m N0:17-32 which has at least about 70%, or alternatively at least about 85%, or even at least about 95% polynucleotide sequence identity to a nucleic acid sequence selected from the group consisting of SEQ ID N0:17-32. Any one of the polynucleotide variants described above can encode an amino acid sequence which contains at least one functional or structural characteristic of NAAP.
In addition, or in the alternative, a polynucleotide variant of the invention is a splice variant of a polynucleotide sequence encoding NAAP. A splice variant may have portions which have significant sequence identity to the polynucleotide sequence 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 50%
polynucleotide sequence identity to the polynucleotide sequence encoding NAAP
over its entire length; however, portions of the splice variant will have at least about 70%, or alternatively at least about 85%, or alternatively at least about 95%, or alternatively 100%
polynucleotide sequence identity to portions of the polynucleotide sequence encoding NAAP. For example, a polynucleotide comprising a sequence of SEQ ID N0:29 is a splice variant of a polynucleotide comprising a sequence of SEQ ID NO:25. Any one of the splice variants described above can encode an amino acid sequence which contains at least one functional or structural characteristic of 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 nucleotide sequences which encode NAAP and its variants are generally capable of hybridizing to the nucleotide sequence of the naturally occurring NAAP under appropriately selected conditions of stringency, it may be advantageous to produce nucleotide sequences 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 which 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 DNA sequences which encode NAAP
and NAAP derivatives, or fragments thereof, entirely by synthetic chemistry. After production, the synthetic sequence may be inserted into any of the many available expression vectors and cell systems using reagents well known in the art. Moreover, synthetic chemistry may be used to introduce mutations into a sequence encoding NAAP or any fragment thereof.
Also encompassed by the invention are polynucleotide sequences that are capable of hybridizing to the claimed polynucleotide sequences, and, in particular, to those shown in SEQ ID
N0:17-32 and fragments thereof under various conditions of stringency. (See, e.g., Wahl, G.M. and S.L. Berger (1987) Methods Enzymol. 152:399-407; Kimmel, A.R. (1987) Methods Enzymol.
152:507-511.) Hybridization conditions, including annealing and wash conditions, are described in "Definitions."
Methods for DNA sequencing are well known in the art and may be used to practice any of the embodiments of the invention. The methods may employ such enzymes as the Klenow fragment of DNA polymerase I, SEQUENASE (US Biochemical, Cleveland OH), Taq polymerase (Applied Biosystems), thermostable T7 polymerase (Amersham Pharmacia Biotech, Piscataway NJ), or combinations of polymerases and proofreading exonucleases such as those found in the ELONGASE
amplification system (Life Technologies, Gaithersburg MD). Preferably, sequence preparation is automated with machines such as the MICROLAB 2200 liquid transfer system (Hamilton, Reno NV), PTC200 thermal cycler (MJ Research, Watertown MA) and ABI CATALYST 800 thermal cycler (Applied Biosystems). Sequencing is then carried out using either the ABI 373 or 377 DNA
sequencing system (Applied Biosystems), the MEGABACE 1000 DNA sequencing system (Molecular Dynamics, Sunnyvale CA), or other systems known in the art. The resulting sequences are analyzed using a variety of algorithms which are well known in the art.
(See, e.g., Ausubel, F.M.
(1997) Short Protocols in Molecular Biolo~y, John Wiley & Sons, New York NY, unit 7.7; Meyers, R.A. (1995) Molecular Biology and Biotechnolo~y, Wiley VCH, New York NY, pp.
856-853.) The nucleic acid sequences encoding 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. (See, e.g., Sarkar, G. (1993) PCR Methods Applic.
2:318-322.) Another method, inverse PCR, uses primers that extend in divergent directions to amplify unknown sequence from a circularized template. The template is derived from restriction fragments comprising a known genomic locus and surrounding sequences. (See, e.g., Triglia, T. et al. (1988) Nucleic Acids Res. 16:8186.) A third method, capture PCR, involves PCR amplification of DNA
fragments adjacent to known sequences in human and yeast artificial chromosome DNA.
(See, e.g., Lagerstrom, M. et al. (1991) PCR Methods Applic. 1:111-119.) In this method, multiple restriction enzyme digestions and ligations may be used to insert an engineered double-stranded sequence into a region of unknown sequence before performing PCR. Other methods which may be used to retrieve unknown sequences are known in the art. (See, e.g., Parker, J.D. et al. (1991) Nucleic Acids Res.
19:3055-3060). Additionally, one may use PCR, nested primers, and PROMOTERFINDER libraries (Clontech, Palo Alto CA) to walk genomic DNA. This procedure avoids the need to screen libraries and is useful in finding intron/exon junctions. For all PCR-based methods, primers may be designed using comnnercially available software, such as OLIGO 4.06 primer analysis software (National Biosciences, Plymouth MN) or another appropriate program, to be about 22 to 30 nucleotides in length, to have a GC content of about 50°7o 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 fox electrophoretic separation, four different nucleotide specific, laser-stimulated fluorescent dyes, and a charge coupled device camera for detection of the emitted wavelengths. Output/light intensity may be converted to electrical signal using appropriate software (e.g., GENOTYPER and SEQUENCE NAVIGATOR, Applied Biosystems), and the entire process from loading of samples to computer analysis and electronic data display may be computer controlled. Capillary electrophoresis is especially preferable for sequencing small DNA fragments which may be present in limited amounts in a particular sample.
In another embodiment of the invention, polynucleotide sequences or fragments thereof which encode 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 DNA sequences which encode substantially the same or a functionally equivalent amino acid sequence may be produced and used to express NAAP.
The nucleotide sequences of the present 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.
The nucleotides of the present invention may be subjected to DNA shuffling techniques such as MOLECULARBREEDING (Maxygen Inc., Santa Clara CA; described in U.S. Patent No.
5,837,458; Chang, C.-C. et al. (1999) Nat. Biotechnol. 17:793-797; Christians, F.C. et al. (1999) Nat.
Biotechnol. 17:259-264; and Crameri, A. et al. (1996) Nat. Biotechnol. 14:315-319) to alter or improve the biological properties of 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 evolution. For example, fragments of a single gene containing random point mutations may be recombined, screened, and then reshuffled until the desired properties are optimized. Alternatively, fragments of a given gene may be recombined with fragments of homologous genes in the same gene family, either from the same or different species, thereby maximizing the genetic diversity of multiple naturally occurring genes in a directed and controllable manner.
In another embodiment, sequences encoding NAAP may be synthesized, in whole or in part, using chemical methods well known in the art. (See, e.g., Caruthers, M.H. et al. (1980) Nucleic Acids Symp. Ser. 7:215-223; and Horn, T. et al. (1980) Nucleic Acids Symp. Ser.
7:225-232.) Alternatively, NAAP itself or a fragment thereof may be synthesized using chemical methods. For example, peptide synthesis can be performed using various solution-phase or solid-phase techniques.
(See, e.g., Creighton, T. (1984) Proteins. Structures and Molecular Pro ep roes, WH Freeman, New York NY, pp. 55-60; and Roberge, J.Y. et al. (1995) Science 269:202-204.) Automated synthesis may be achieved using the ABI 431A peptide synthesizer (Applied Biosystems).
Additionally, the amino acid sequence of 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. (See, e.g., Chiez, R.M. and F.Z. Regnier (1990) Methods Enzymol. 182:392-421.) The composition of the synthetic peptides may be confirmed by amino acid analysis or by sequencing. (See, e.g., Creighton, supra, pp. 28-53.) In order to express a biologically active NAAP, the nucleotide sequences 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 polynucleotide sequences encoding NAAP. Such elements may vary in their strength and specificity.
Specific initiation signals may also be used to achieve more efficient translation of sequences encoding NAAP. Such signals include the ATG initiation codon and adjacent sequences, e.g. the Kozak sequence. In cases where sequences 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. (See, e.g., Scharf, D. et al. (1994) Results Probl. Cell Differ. 20:125-162.) Methods which are well known to those skilled in the art may be used to construct expression vectors containing sequences encoding NAAP and appropriate transcriptional and translational control elements. These methods include in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination. (See, e.g., Sambrook, J. et al. (1989) Molecular Cloning, A
Laboratory Manual, Cold Spring Harbor Press, Plainview NY, ch. 4, 8, and 16-17; Ausubel, F.M. et al. (I995) Current Protocols in Molecular Biolo~y, John Wiley & Sons, New York NY, ch. 9, 13, and 16.) A variety of expression vector/host systems may be utilized to contain and express sequences encoding 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. (See, e.g., Sambrook, supra; Ausubel, supra; Van Heeke, G. and S.M. Schuster (1989) J. Biol. Chem. 264:5503-5509; Engelhard, E.I~. 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 Yeaxbook 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 Harnngton, J.J. et al. (1997) Nat. Genet. 15:345-355.) Expression vectors derived from retroviruses, adenoviruses, or herpes or vaccinia viruses, or from various bacterial plasmids, may be used for delivery of nucleotide sequences to the targeted organ, tissue, or cell population. (See, e.g., Di Nicola, M. et al. (1998) Cancer Gen. Ther. 5(6):350-356; Yu, M. et al. (1993) Proc. Natl. Acad. Sci.
USA 90(13):6340-6344; Buller, R.M. et al. (1985) Nature 317(6040):8I3-815;
McGregor, D.P. et al.
(1994) Mol. Immunol. 31(3):219-226; and Verma, LM. and N. Somia (1997) Nature 389:239-242.) The invention is not limited by the host cell employed.
In bacterial systems, a number of cloning and expression vectors may be selected depending upon the use intended for polynucleotide sequences encoding NAAP. For example, routine cloning, subcloning, and propagation of polynucleotide sequences encoding NAAP can be achieved using a multifunctional E. coli vector such as PBLUESCRIPT (Stratagene, La Jolla CA) or PSPORT1 plasmid (Life Technologies). Ligation of sequences encoding 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 in vitro transcription, dideoxy sequencing, single strand rescue with helper phage, and creation of nested deletions in the cloned sequence. (See, e.g., Van Heeke, G. and S.M.
Schuster (1989) J. Biol.
Chem. 264:5503-5509.) When large quantities of 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 cerevisiae or Pichia pastoris. In addition, such vectors direct either the secretion or intracellular retention of expressed proteins and enable integration of foreign sequences into the host genome for stable propagation.
(See, e.g., Ausubel, 1995, supra; Bitter, G.A. et al. (1987) Methods Enzymol. 153:516-544; and Scorer, C.A. et al. (1994) Bio/Technology 12:181-184.) Plant systems may also be used for expression of NAAP. Transcription of sequences 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 RUBISCO or heat shock promoters may be used. (See, e.g., Coruzzi, G. et al. (1984) EMBO J. 3:1671-1680; Broglie, R. et al.
(1984) Science 224:838-843; and Winter, J. et al. (1991) Results Probl. Cell Differ. 17:85-105.) These constructs can be introduced into plant cells by direct DNA
transformation or pathogen-mediated transfection. (See, e.g., The McGraw Hill Yearbook of Science and Technolo~y (1992) McGraw Hill, New York NY, pp. 191-196.) In mammalian cells, a number of viral-based expression systems may be utilized. In eases where an adenovirus is used as an expression vector, sequences encoding NAAP
may be ligated 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. (See, e.g., Logan, J. and T. Shenk (1984) Proc.
Natl. Acad. Sci. USA 81:3655-3659.) In addition, transcription enhancers, such as the Rous sarcoma virus (RSV) enhancer, may be used to increase expression in mammalian host cells. SV40 or EBV-based vectors may also be used for high-level protein expression.
Human artificial chromosomes (HACs) may also be employed to deliver larger fragments of DNA than can be contained in and expressed from a plasmid. HACs of about 6 kb to 10 Mb are constructed and delivered via conventional delivery methods (liposomes, polycationic amino polymers, or vesicles) for therapeutic purposes. (See, e.g., Haxrington, 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, sequences 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 include, but are not limited to, the herpes simplex virus thymidine kinase and adenine phosphoribosyltransferase genes, for use in tk- and apr cells, respectively.
(See, e.g., Wigler, M. et al. (1977) Cell 11:223-232; Lowy, I. et al. (1980) Cell 22:817-823.) Also, antimetabolite, antibiotic, or herbicide resistance can be used as the basis for selection. For example, dhfr confers resistance to methotrexate; zzeo confers resistance to the aminoglycosides neomycin and G-418; and als and pat confer resistance to chlorsulfuron and phosphinotricin acetyltransferase, respectively. (See, e.g., Wigler, M. et al. (1980) Proc. Natl. Acad. Sci. USA 77:3567-3570; Colbere-Garapin, F. et al. (1981) J. Mol. Biol. 150:1-14.) Additional selectable genes have been described, e.g., trpB and hisD, which alter cellular requirements for metabolites. (See, e.g., Hartman, S.C. and R.C. Mulligan (1988) Proc.
Natl. Acad. Sci. USA 85:8047-8051.) Visible markers, e.g., anthocyanins, green fluorescent proteins (GFP; Clontech), I3 glucuronidase and its substrate 13-glucuronide, or Iuciferase and its substrate luciferin may be used. These markers can be used not only to identify transformants, but also to quantify the amount of transient or stable protein expression attributable to a specific vector system.
(See, e.g., Rhodes, C.A. (1995) Methods Mol. Biol. 55:121-131.) Although the presence/absence of marker gene expression suggests that the gene of interest is also present, the presence and expression of the gene may need to be confirmed. For example, if the sequence encoding NAAP is inserted within a marker gene sequence, transformed cells containing sequences 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 nucleic acid sequence encoding NAAP
and that express NAAP may be identified by a variety of procedures known to those of skill in the art. These procedures include, but are not limited to, DNA-DNA or DNA-RNA hybridizations, PCR
amplification, and protein bioassay or immunoassay techniques which include membrane, solution, or chip based technologies for the detection and/or quantification of nucleic acid or protein sequences.
Immunological methods for detecting and measuring the expression of 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. (See, e.g., Hampton, R. et al. (1990) Serological Methods, a Laboratory Manual, APS
Press, St. Paul MN, Sect. IV; Coligan, J.E. et al. (1997) Current Protocols in Immunolo~y, Greene Pub. Associates and Wiley-Interscience, New York NY; and Pound, J.D. (1998) Immunochemical Protocols, Humana Press, Totowa NJ.) A wide variety of labels and conjugation techniques are known by those skilled in the art and may be used in various nucleic acid and amino acid assays. Means for producing labeled hybridization or PCR probes for detecting sequences related to polynucleotides encoding NAAP
include oligolabeling, nick translation, end-labeling, or PCR amplification using a labeled nucleotide.
Alternatively, the sequences 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 in vitro by addition of an appropriate RNA polymerase such as T7, T3, or SP6 and labeled nucleotides. These procedures may be conducted using a variety of commercially available kits, such as those provided by Amersham Pharmacia Biotech, Promega (Madison WI), and US Biochemical. Suitable reporter molecules or labels which may be used for ease of detection include radionuclides, enzymes, fluorescent, chemiluminescent, or chromogenic agents, as well as substrates, cofactors, inhibitors, magnetic particles, and the Like.
Host cells transformed with nucleotide sequences encoding 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 ifs ability to modulate expression of the inserted sequences or to process the expressed protein in the desired fashion.
Such modifications of the polypeptide include, but are not limited to, acetylation, carboxylation, glycosylation, phosphorylation, lipidation, and acylation. Post-translational processing which cleaves a "prepro" or "pro" form of the protein may also be used to specify protein targeting, folding, and/or activity.
Different host cells which have specific cellular machinery and characteristic mechanisms for post-translational activities (e.g., CHO, HeLa, MDCK, HEK293, and WI38) are available from the American Type Culture Collection (ATCC, Manassas VA) and may be chosen to ensure the correct modification and processing of the foreign protein.
In another embodiment of the invention, natural, modified, or recombinant nucleic acid sequences encoding 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 immunoaffmity 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 rnay also be used to facilitate expression and purification of fusion proteins.
In a further embodiment of the invention, synthesis of radiolabeled NAAP may be achieved in vitro using the TNT rabbit reticulocyte lysate or wheat germ extract system (Promega). These systems couple transcription and translation of protein-coding sequences operably associated with the T7, T3, or SP6 promoters. Translation takes place in the presence of a radiolabeled amino acid precursor, for example, 35S-methionine.
NAAP of the present invention or fragments thereof may be used to screen for compounds that specifically bind to NAAP. At least one and up to a plurality of test compounds may be screened for specific binding to NAAP. Examples of test compounds include antibodies, oligonucleotides, proteins (e.g., receptors), or small molecules.
In one embodiment, the compound thus identified is closely related to the natural ligand of NAAP, e.g., a ligand or fragment thereof, a natural substrate, a structural or functional mimetic, or a natural binding partner. (See, e.g., Coligan, J.E. et al. (1991) Current Protocols in Immunolo~y 1(2):

Chapter 5.) Similarly, the compound can be closely related to the natural receptor to which NAAP
binds, or to at least a fragment of the receptor, e.g., the ligand binding site. In either case, the compound can be rationally designed using known techniques. In one embodiment, screening for these compounds involves producing appropriate cells which express NAAP, either as a secreted protein or on the cell membrane. Preferred cells include cells from mammals, yeast, Drosophila, 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.
NAAP of the present invention or fragments thereof 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 in vitro 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. (See, e.g., U.S. Patent No. 5,175,383 and U.S. Patent No. 5,767,337.) For example, mouse ES cells, such as the mouse 1291SvJ cell line, are derived from the early mouse embryo and grown in culture. The ES cells are transformed with a vector containing the gene of interest disrupted by a marker gene, e.g., the neomycin phosphotransferase gene (neo; Capecchi, M.R.
(1989) Science 244:1288-1292). The vector integrates into the corresponding region of the host genome by homologous recombination. Alternatively, homologous recombination takes place using the Cre-loxP system to knockout a gene of interest in a tissue- or developmental stage-specific manner (Marth, J.D. (1996) Clin. Invest. 97:1999-2002; Wagner, K.U. et al.
(1997) Nucleic Acids Res. 25:4323-4330). Transformed ES cells are identified and microinjected into mouse cell blastocysts such as those from the C57BL/6 mouse strain. The blastocysts are surgically transferred to pseudopregnant dams, and the resulting chimeric progeny are genotyped and bred to produce heterozygous or homozygous strains. Transgenic animals thus generated may be tested with potential therapeutic or toxic agents.
Polynucleotides encoding 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 is closely associated with prostate and lung tumor tissues, umbilical cord blood and umbilical cord blood dendritic cells, and brain tissue, examples of which can be found in Table 6.
Therefore, NAAP appeaxs to play a role in cell proliferative, neurological, developmental, and autoimmune/inflammatory 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 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 developmental disorder such as renal tubular acidosis, anemia, Cushing's syndrome, achondroplastic dwarfism, Duchenne and Becker muscular dystrophy, epilepsy, gonadal dysgenesis, WAGR
syndrome (Wilms' tumor, aniridia, genitourinary abnormalities, and mental retardation), Smith-Magenis syndrome, myelodysplastic syndrome, hereditary mucoepithelial dysplasia, hereditary keratodermas, hereditary neuropathies such as Charcot-Marie-Tooth disease and neurofibromatosis, hypothyroidism, hydrocephalus, seizure disorders such as Syndenham's chorea and cerebral palsy, spina bifida, anencephaly, craniorachischisis, congenital glaucoma, cataract, and sensorineural hearing loss; 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-ectoderma,l dystrophy (APECED), bronchitis, cholecystitis, contact dermatitis, Crohn's disease, atopic dermatitis, dermatomyositis, diabetes mellitus, emphysema, episodic lymphopenia with lymphocytotoxins, erythroblastosis fetalis, erythema nodosum, atrophic gastritis, glomerulonephritis, Goodpasture's syndrome, gout, Graves' disease, Hashimoto's thyroiditis, hypereosinophilia, irritable bowel syndrome, multiple sclerosis, myasthenia gravis, myocardial or pericardial inflammation, osteoarthritis, osteoporosis, pancreatitis, polymyositis, psoriasis, Reiter's syndrome, rheumatoid arthritis, scleroderma, Sjogren's syndrome, systemic anaphylaxis, systemic lupus erythematosus, systemic sclerosis, thrombocytopenic purpura, ulcerative colitis, uveitis, Werner syndrome, complications of cancer, hemodialysis, and extracorporeal circulation, protozoal, and helminthic infections, and trauma;
and 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;
infections 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; infections caused by a fungal agent classified as aspergillus, blastomyces, dermatophytes, cryptococcus, coccidioides, malasezzia, histoplasma, or other mycosis-causing fungal agent; and infections 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.
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, developmental, and autoimmunelinflammatory 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.
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 of the proteins, antagonists, antibodies, agonists, complementary sequences, or vectors of the invention may be administered in combination with other appropriate therapeutic agents. Selection of the appropriate agents for use in combination therapy may be made by one of ordinary skill in the art, according to conventional pharmaceutical principles. The combination of therapeutic agents may act synergistically to effect the treatment or prevention of the various disorders described above. Using this approach, one may be able to achieve therapeutic efficacy with lower dosages of each agent, thus reducing the potential for adverse side effects.
An antagonist of 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.
For the production of antibodies, various hosts including goats, rabbits, rats, mice, 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 immunological response. Such adjuvants include, but are not limited to, Freund's, mineral gels such as aluminum hydroxide, and surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, KLH, and dinitrophenol. Among adjuvants used in humans, BCG (bacilli Calmette-Guerin) and Corynebacterium parvum are especially preferable.
It is preferred that the oligopeptides, peptides, or fragments used to induce antibodies to 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. (See, e.g., Kohler, G. et al. (1975) Nature 256:495-497; Kozbor, D.
et al. (1985) J.
Immunol. Methods 81:31-42; Cote, R.J. et al. (1983) Proc. Natl. Acad. Sci. USA
80:2026-2030; and Cole, S.P. et al. (1984) Mol. Cell Biol. 62:109-120.) In addition, techniques developed for the production of "chimeric antibodies,"
such as the splicing of mouse antibody genes to human antibody genes to obtain a molecule with appropriate antigen specificity and biological activity, can be used. (See, e.g., Morrison, S.L. et al. (1984) Proc.
Natl. Acad. Sci. USA 81:6851-6855; Neuberger, M.S. et al. (1984) Nature 312:604-608; and Takeda, S. et al. (1985) Nature 314:452-454.) Alternatively, techniques described for the production of single chain antibodies may be adapted, using methods known in the art, to produce 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. (See, e.g., Burton, D.R. (1991) Proc. Natl. Acad. Sci. USA 88:10134-10137.) Antibodies may also be produced by inducing in vivo production in the lymphocyte population or by screening irnmunoglobulin libraries or panels of highly specific binding reagents as disclosed in the literature. (See, e.g., Orlandi, R. et al. (1989) Proc. Natl.
Acad. Sci. USA
86:3833-3837; Winter, G. et al. (1991) Nature 349:293-299.) Antibody fragments which contain specific binding sites for 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.
(See, e.g., Huse, W.D.
et al. (1989) Science 246:1275-1281.) Various immunoassays may be used for screening to identify antibodies having the desired specificity. Numerous protocols for competitive binding or imrnunoradiometric assays using either 2S 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, su,~ra,).
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, Ka, 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 K~ 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 Ka 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 10'2 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' L/mole are preferred for use in immunopurification 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. (See, e.g., Catty, supra, and Coligan et al. supra.) In another embodiment of the invention, the 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. (See, e.g., Agrawal, S., ed. (1996) Antisense Therapeutics, Humana Press Inc., Totawa NJ.) In therapeutic use, any gene delivery system suitable for introduction of the antisense sequences into appropriate target cells can be used. Antisense sequences can be delivered intracellularly in the form of an expression plasmid which, upon transcription, produces a sequence complementary to at least a portion of the cellular sequence encoding the target protein. (See, e.g., Slater, J.E. et al. (1998) J. Allergy Clin. Immunol. 102(3):469-475; and Scanlon,'K.J. et al. (1995) 9(13):1288-1296.) Antisense sequences can also be introduced intracellularly through the use of viral vectors, such as retrovirus and adeno-associated virus vectors. (See, e.g., Miller, A.D. (1990) Blood 76:271; Ausubel, supra; Uckert, W. and W. Walther (1994) Pharmacol. Ther.
63(3):323-347.) Other gene delivery mechanisms include liposome-derived systems, artificial viral envelopes, and other systems known in the art. (See, e.g., Rossi, J.J. (1995) Br. Med. Bull.
51(1):217-225; Boado, R.J. et al. (1998) J. 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 (SCID)-X1 disease characterized by X-linked inheritance (Cavazzana-Calvo, M. et al. (2000) Science 288:669-672), severe combined immunodeficiency syndrome associated with an inherited adenosine deaminase (ADA) deficiency (Blaese, R.M, et al. (1995) Science 270:475-480; Bordignon, C. et al. (1995) Science 270:470-475), cystic fibrosis (Zabner, J. et al. (1993) Cell 75:207-216; Crystal, R.G. et al. (1995) Hum. Gene Therapy 6:643-666; Crystal, R.G. et al. (1995) Hum. Gene Therapy 6:667-703), thalassamias, familial hypercholesterolemia, and hemophilia resulting from Factor VIII or Factor IX
deficiencies (Crystal, R.G. (1995) Science 270:404-410; Verma, LM. and N. Somia (1997) Nature 389:239-242)), (ii) express a conditionally lethal gene product (e.g., in the case of cancers which result from unregulated cell proliferation), or (iii) express a protein which affords protection against intracellular parasites (e.g., against human retroviruses, such as human immunodeficiency virus (HIV) (Baltimore, D.
(1988) Nature 335:395-396; Poeschla, E. et al. (1996) Proc. Natl. Acad. Sci.
USA 93:11395-11399), hepatitis B or C virus (HBV, HCV); fungal parasites, such as Candida albicans and Paracoccidioides brasiliensis; and protozoan parasites such as Plasmodium falciparum and Try_panosoma cruzi). In the case where a genetic deficiency in NAAP expression or regulation causes disease, the expression of NAAP from an appropriate population of transduced cells may alleviate the clinical manifestations caused by the genetic deficiency.
In a further embodiment of the invention, diseases or disorders caused by deficiencies in 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 (Morgan, R.A. and W.F. Anderson (1993) Annu. Rev.
Biochem. 62:191-217; Ivics, Z. (1997) Cell 91:501-510; Boulay, J-L. and H.
Recipon (1998) Curr.
Opin. Biotechnol. 9:445-450).
Expression vectors that may be effective for the expression of NAAP include, but are not limited to, the PCDNA 3.1, EPITAG, PRCCMV2, PREP, PVAX, PCR2-TOPOTA vectors (Invitrogen, Carlsbad CA), PCMV-SCRIfT, 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, thymidine kinase (TK), or ~3-actin genes), (ii) an inducible promoter (e.g., the tetracycline-regulated promoter (Gossen, M. and H. Bujard (1992) Proc. Natl.

Acad. Sci. USA 89:5547-5551; Gossen, M. et al. (1995) Science 268:1766-1769;
Rossi, F.M.V. and H.M. Blau (1998) Curr. Opin. Biotechnol. 9:451-456), commercially available in the T-REX plasmid (Invitrogen)); the ecdysone-inducible promoter (available in the plasmids PVGRXR and PllVD;
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 KIT, available from Invitrogen) allow one with ordinary skill in the art to deliver polynucleotides to target cells in culture and require minimal effort to optimize experimental IO 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 axe 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 the alternative, 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, P.A. et al. ( 1999) Annu. Rev. Nutr. 19:511-544 and Verma, LM. and N. Somia (1997) Nature 18:389:239-242, both incorporated by reference herein.
In another alternative, a herpes-based, gene therapy delivery system is used to deliver polynucleotides encoding 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, W.F. et al. (1999) J. Virol. 73:519-532 and Xu, H. et al. (1994) Dev. Biol. 163:152-161, hereby incorporated by reference. The manipulation of cloned herpesvirus sequences, the generation of recombinant virus following the transfection of multiple plasmids containing different segments of the large herpesvirus genomes, the growth and propagation of herpesvirus, and the infection of cells with herpesvirus are techniques well known to those of ordinary skill in the art.
In another alternative, an alphavii-us (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 (BHK-21) with a variant of Sindbis virus (SIN) indicates that the lytic replication of alphaviruses can be altered to suit the needs of the gene therapy application (Dryga, S.A. et al. (1997) Virology 228:74-83). The wide host range of alphaviruses will allow the introduction of 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 polynierases, transcription factors, or regulatory molecules. Recent therapeutic advances using triplex DNA have been described in the literature. (See, e.g., Gee, J.E. et al. (1994) in Huber, B.E.
and B.I. Carr, Molecular and Immunolo ig~proaches, 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 sequences encoding NAAP.
Specific ribozyme cleavage sites within any potential RNA target are initially identified by scanning the target molecule for ribozyme cleavage sites, including the following sequences: GUA, GUU, and GUC. Once identified, short RNA sequences of between 15 and 20 ribonucleotides, corresponding to the region of the target gene containing the cleavage site, may be evaluated for secondary structural features which may render the oligonucleotide inoperable, The suitability of candidate targets may also be evaluated by testing accessibility to hybridization with complementary oligonucleotides using ribonuclease protection assays.
Complementary ribonucleic acid molecules and ribozymes of the invention may be prepared by any method known in the art for the synthesis of nucleic acid molecules.
These include techniques for chemically synthesizing oligonucleotides such as solid phase phosphoramidite chemical synthesis.
Alternatively, RNA molecules may be generated by in vitro and in vivo transcription of DNA
sequences encoding 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, thymine, and uridine which are not as easily recognized by endogenous endonucleases.
An additional embodiment of the invention encompasses a method for screening for a compound which is effective in altering expression of a polynucleotide encoding 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 altering polynucleotide expression; selection from an existing, commercially-available or proprietary library of naturally-occurring or non-natural chemical compounds; rational design of a compound based on chemical and/or structural properties of the target polynucleotide;
and selection from a library of chemical compounds created combinatorially or randomly. A sample comprising a polynucleotide encoding NAAP is exposed to at Ieast one test compound thus obtained. The sample may comprise, for example, an intact or permeabilized cell, or an in vitro cell-free or reconstituted biochemical system. Alterations in the expression of a polynucleotide encoding 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 Schizosaccharomyces pombe gene expression system (Atkins, D. et al. (1999) U.S. Patent No. 5,932,435; Arndt, G.M. et al.
(2000) Nucleic Acids Res. 28:E15) or a human cell line such as HeLa cell (Clarke, M.L. et al.
(2000) Biochem. Biophys.
Res. Commun. 268:8-13). A particular embodiment of the present invention involves screening a combinatorial library of oligonucleotides (such as deoxyribonucleotides, ribonucleotides, peptide nucleic acids, and modified oligonucleotides) for antisense activity against a specific polynucleotide sequence (Bruice, T.W. et al. (1997) U.S. Patent No. 5,686,242; Bruice, T.W.
et al. (2000) U.S.
Patent No. 6,022,691).
Many methods for introducing vectors into cells or tissues are available and equally suitable for use in yivo, in vitro, and ex vivo. For ex vivo therapy, vectors may be introduced into stem cells taken from the patient and clonally propagated for autologous transplant back into that same patient.
Delivery by transfection, by Iiposome injections, or by polycationic amino polymers may be achieved using methods which are well known in the art. (See, e.g., Goldman, C.K. et al. (1997) Nat.
Biotechnol. 15:462-466.) 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, intra-arterial, intramedullary, intrathecal, intraventricular, pulmonary, transdermal, subcutaneous, intraperitoneal, intranasal, enteral, topical, sublingual, or rectal means.

Compositions for pulmonary administration may be prepared in liquid or dry powder form.
These compositions are generally aerosolized immediately prior to inhalation by the patient. In the case of small molecules (e.g. traditional low molecular weight organic drugs), aerosol delivery of fast-acting formulations is well-known in the art. In the case of macromolecules (e.g. larger peptides and proteins), recent developments in the field of pulmonary delivery via the alveolar region of the lung have enabled the practical delivery of drugs such as insulin to blood circulation (see, e.g., Patton, J.S. et al., U.S. Patent No. 5,997,84.8). 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 this 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°Io of the population) or LDso (the dose lethal to 50°l0 of the population) statistics. The dose ratio of toxic to therapeutic effects is the therapeutic index, which can be expressed as the LDSO/EDSO ratio. Compositions which exhibit large therapeutic indices are preferred. The data obtained from cell culture assays and animal studies are used to formulate a range of dosage for human use. The dosage contained in such compositions is preferably within a range of circulating concentrations that includes the EDSO
with little or no toxicity.
The dosage varies within this range depending upon the dosage form employed, the sensitivity of the patient, and the route of administration.

The exact dosage will be determined by the practitioner, in light of factors related to the subject requiring treatment. Dosage and administration are adjusted to provide sufficient levels of the active moiety or to maintain the desired effect. Factors which may be taken into account include the severity of the disease state, the general health of the subject, the age, weight, and gender of the subject, time and frequency of administration, drug combination(s), reaction sensitivities, and response to therapy. Long-acting compositions may be administered every 3 to 4 days, every week, or biweekly depending on the half-life and clearance rate of the particular formulation.
Normal dosage amounts may vary from about 0.1 ,ug to 100,000 ~tg, 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 FAGS, 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, the polynucleotides encoding NAAP may be used for diagnostic purposes. The polynucleotides which may be used include oligonucleotide sequences, complementary RNA and DNA molecules, and PNAs. The polynucleotides may be used to detect and quantify gene expression in biopsied tissues in wluch expression of NAAP
may be correlated with disease. The diagnostic assay may be 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 polynucleotide sequences, 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 m N0:17-32 or from genomic sequences including promoters, enhancers, and introns of the NAAP
gene, Means for producing specific hybridization probes for DNAs encoding NAAP
include the cloning of polynucleotide sequences 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 in vitro by means of the addition of the appropriate RNA
polymerases and the appropriate labeled nucleotides. Hybridization probes may be labeled by a variety of reporter groups, for example, by radionuclides such as 32P or 35S, or by enzymatic labels, such as alkaline phosphatase coupled to the probe via avidin/biotin coupling systems, and the like.
Polynucleotide sequences 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 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 developmental disorder such as renal tubular acidosis, anemia, Cushing's syndrome, achondroplastic dwarfism, Duchenne and Becker muscular dystrophy, epilepsy, gonadal dysgenesis, WAGR syndrome (Wilms' tumor, aniridia, genitourinary abnormalities, and mental retardation), Smith-Magenis syndrome, myelodysplastic syndrome, hereditary mucoepithelial dysplasia, hereditary keratodermas, hereditary neuropathies such as Charcot-Marie-Tooth disease and neurofibromatosis, hypothyroidism, hydrocephalus, seizure disorders such as Syndenham's chorea and cerebral palsy, spina bifida, anencephaly, craniorachischisis, congenital glaucoma, cataract, and sensorineural hearing loss; 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, osteoarthritis, osteoporosis, pancreatitis, polymyositis, psoriasis, Reiter's syndrome, rheumatoid arthritis, scleroderma, Sjogren's syndrome, systemic anaphylaxis, systemic lupus erythematosus, systemic sclerosis, thrombocytopenic purpura, ulcerative colitis, uveitis, Werner syndrome, complications of cancer, hemodialysis, and extracorporeal circulation, protozoal, and helminthic infections, and trauma; and 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; infections 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; infections caused by a fungal agent classified as aspergillus, blastomyces, dermatophytes, cryptococcus, coccidioides, malasezzia, histoplasma, or other mycosis-causing fungal agent; and infections 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. The polynucleotide sequences 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, the nucleotide sequences encoding NAAP may be useful in assays that detect the presence of associated disorders, particularly those mentioned above. The nucleotide 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 quantified and compared with a standard value. If the amount of signal in the patient sample is significantly altered in comparison to a control sample then the presence of altered levels of nucleotide sequences encoding 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.
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 in vitro. Oligomers will preferably contain a fragment of a polynucleotide encoding NAAP, or a fragment of a polynucleotide complementary to the polynucleotide encoding 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 the polynucleotide sequences 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 the polynucleotide sequences 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).
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. (See, e.g., Melby, P.C. et al. (1993) J. hnmunol. 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 polynucleotide sequences described herein may be used as elements on a microarray. The microarray can be used in transcript imaging techniques which monitor the relative expression levels of large numbers of genes simultaneously as described below. The microarray may also be used to identify genetic variants, mutations, and polymorplusms. 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 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 in vitro model systems and preclinical evaluation of pharmaceuticals, as well as toxicological testing of industrial and naturally-occurring environmental compounds. All compounds induce characteristic gene expression patterns, frequently termed molecular fingerprints or toxicant signatures, which are indicative of mechanisms of action and toxicity (Nuwaysir, E.F. et al. (1999) Mol. Carcinog. 24:153-159; Steiner, S.
and N.L. Anderson (2000) Toxicol. Lett. 112-113:467-4.71, expressly incorporated by reference herein). If a test compound has a signature similar to that of a compound with known toxicity, it is likely to share those toxic properties. These fingerprints or signatures are most useful and refined when they contain expression information from a 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.govloc/news/toxchip.htm.) Therefore, it is important and desirable in toxicological screening using toxicant signatures to include all expressed gene sequences.
In one embodiment, the toxicity of a test compound is assessed by treating a biological sample containing nucleic acids with the test compound. Nucleic acids that are expressed in the treated biological sample are hybridized with one or more probes specific to the polynucleotides of the present invention, so that transcript levels corresponding to the polynucleotides of the present invention may be quantified. The transcript levels in the treated biological sample are compared with levels in an untreated biological sample. Differences in the transcript levels between the two samples are indicative of a toxic response caused by the test compound in the treated sample.
Another particular embodiment relates to the use of the polypeptide sequences of the present invention to analyze the proteome of a tissue or cell type. The term proteome refers to the global pattern of protein expression in a particular tissue or cell type. Each protein component of a proteome can be subjected individually to further analysis. Proteome expression patterns, or profiles, are analyzed by quantifying the number of expressed proteins and their relative abundance under given conditions and at a given time. A profile of a cell's proteome may thus be generated by separating and analyzing the polypeptides of a particular tissue or cell type.
In one embodiment, the separation is achieved using two-dimensional gel electrophoresis, in which proteins from a sample are separated by isoelectric focusing in the first dimension, and then according to molecular weight by sodium dodecyl sulfate slab gel electrophoresis in the second dimension (Steiner and Anderson, supra). The proteins are visualized in the gel as discrete and uniquely positioned spots, typically by staining the gel with an agent such as Coomassie Blue or silver or fluorescent stains. The optical density of each protein spot is generally proportional to the level of the protein in the sample. The optical densities of equivalently positioned protein spots from different samples, for example, from biological samples either treated or untreated with a test compound or therapeutic agent, are compared to identify any changes in protein spot density related to the treatment. The proteins in the spots are partially sequenced using, for example, standard methods employing chemical or enzymatic cleavage followed by mass spectrometry. The identity of the protein in a spot may be determined by comparing its partial sequence, preferably of at least 5 contiguous amino acid residues, to the polypeptide sequences of the present invention. In some cases, further sequence data may be obtained for definitive protein identification.
A proteomic profile may also be generated using antibodies specific for 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, r 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 profiling may be more reliable and informative in such cases.
In another embodiment, the toxicity of a test compound is assessed by treating a biological sample containing proteins with the test compound. Proteins that are expressed in the treated biological sample are separated so that the amount of each protein can be quantified. The amount of each protein is compared to the amount of the corresponding protein in an untreated biological sample. A difference in the amount of protein between the two samples is indicative of a toxic response to the test compound in the treated sample. Individual proteins are identified by sequencing the amino acid residues of the individual proteins and comparing these partial sequences to the polypeptides of the present invention.

In another embodiment, the toxicity of a test compound is assessed by treating a biological sample containing proteins with the test compound. Proteins from the biological sample are incubated with antibodies specific to the polypeptides of the present invention. The amount of protein recognized by the antibodies is quantified. The amount of protein in the treated biological sample is compared with the amount in an untreated biological sample. A
difference in the amount of protein between the two samples is indicative of a toxic response to the test compound in the treated sample.
Microarrays may be prepared, used, and analyzed using methods known in the art. (See, e.g., Brennan, T.M. et al. (1995) U.S. Patent No. 5,474,796; Schena, M. et al.
(1996) Proc. Natl. Acad. Sci.
USA 93:10614-10619; Baldeschweiler et al. (1995) PCT application W095/251116;
Shalom D. et al.
(1995) PCT application W095135505; 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, hereby expressly incorporated by reference.
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 mufti-gene family may potentially cause undesired cross hybridization during chromosomal mapping. The sequences may be mapped to a particular chromosome, to a specific region of a chromosome, or to artificial chromosome constructions, e.g., human artificial chromosomes (HACs), yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs), bacterial P1 constructions, or single chromosome cDNA libraries. (See, e.g., Harrington, J.J. et al. (1997) Nat.
Genet. 15:345-355; Price, C.M. (1993) Blood Rev. 7:127-134; and Trask, B.J.
(1991) Trends Genet.

7:149-154.) Once mapped, the nucleic acid sequences of the invention may be used to develop genetic linkage maps, for example, which correlate the inheritance of a disease state with the inheritance of a particular chromosome region or restriction fragment length polymorphism (RFLP).
(See, for example, Lander, E.S. and D. Botstein (1986) Proc. Natl. Acad. Sci.
USA 83:7353-7357.) Fluorescent in situ hybridization (FISH) may be correlated with other physical and genetic map data. (See, e.g., Heinz-Ulrich, et al. (1995) in Meyers, supra, pp. 965-968.) Examples of genetic map data can be found in various scientific journals or at the Online Mendelian Inheritance in Man (OM1M) World Wide Web site. Correlation between the location of the gene encoding 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.
In situ hybridization of chromosomal preparations and physical mapping techniques, such as linkage analysis using established chromosomal markers, may be used for extending genetic maps.
Often the placement of a gene on the chromosome of another mammalian species, such as mouse, may reveal associated markers even if the exact chromosomal locus is not known. This information is valuable to investigators searching for disease genes using positional cloning or other gene discovery techniques. Once the gene or genes responsible for a disease or syndrome have been crudely localized by genetic linkage to a particular genomic region, e.g., ataxia-telangiectasia to l 1q22-23, any sequences mapping to that area may represent associated or regulatory genes for further investigation. (See, e.g., Gatti, R.A. et al. (1988) Nature 336:577-580.) The nucleotide sequence of the instant invention may also be used to detect differences in the chromosomal location due to translocation, inversion, etc., among normal, carrier, or affected individuals.
In another embodiment of the invention, 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. (See, e.g., Geysen, et al. (1984) PCT
application WO84/03564.) 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 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 Iimitative of the remainder of the disclosure in any way whatsoever.

The disclosures of all patents, applications and publications, mentioned above and below, including U.S. Ser. No. 60/257,714, U.S. Ser. No. 60/260,081, U.S. Ser. No.
60/262,302, U.S. Ser.
No. 60/266, 088, U.S. Ser. No. [Attorney Docket No. PF-1249 P, filed October 29, 2001], and U.S.
Ser. No. 60/263,823, are expressly incorporated by reference herein.
EXAMPLES
I. Construction of cDNA Libraries Incyte cDNAs were derived from cDNA libraries described in the LIFESEQ GOLD
database (Incyte Genomics, Palo Alto CA). Some tissues were homogenized and lysed in guanidinium isothiocyanate, while others were homogenized and lysed in phenol or in a suitable mixture of denaturants, such as TRIZOL (Life Technologies), a monophasic solution of phenol and guanidine isothiocyanate. The resulting lysates were centrifuged over CsCI cushions or extracted with chloroform. RNA was precipitated from the lysates with either isopropanol or sodium acetate and ethanol, or by other routine methods.
Phenol extraction and precipitation of RNA were repeated as necessary to increase RNA
purity. In some cases, RNA was treated with DNase. For most libraries, poly(A)+ RNA was isolated using oligo d(T)-coupled paramagnetic particles (Promega), OLIGOTEX latex particles (QIAGEN, Chatsworth CA), or an OLIGOTEX mRNA purification kit (QIAGEN). Alternatively, RNA was isolated directly from tissue lysates using other RNA isolation kits, e.g., the POLY(A)PURE mRNA
purification kit (Ambion, Austin TX).
In some cases, Stratagene was provided with RNA and constructed the corresponding cDNA
libraries. Otherwise, cDNA was synthesized and cDNA libraries were constructed with the UNIZAP
vector system (Stratagene) or SUPERSCRIPT plasmid system (Life Technologies), using the recommended procedures or similar methods known in the art. (See, e.g., Ausubel, 1997, 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 SEPIiACRYL S 1000, SEPHAROSE CL2B, or SEPHAROSE CL4B column chromatography (Amersham Pharmacia Biotech) or preparative agarose gel electrophoresis. cDNAs were ligated into compatible restriction enzyme sites of the polylinker of a suitable plasmid, e.g., PBLUESCRIPT plasmid (Stratagene), PSPORT1 plasmid (Life Technologies), 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-BIueMRF, or SOLR from Stratagene or DHSa, DH10B, or ElectroMAX DH10B from Life Technologies.
II. Isolation of cDNA Clones Plasmids obtained as described in Example I were recovered from host cells by in vivo excision using the UNIZAP vector system (Stratagene) or by cell lysis.
Plasmids were purified using at least one of the following: a Magic or WIZARD Minipreps DNA purification system (Promega); an AGTC Miniprep purification kit (Edge Biosystems, Gaithersburg MD); and QIAWELL

8 Plasmid, QIAWELL 8 Plus Plasmid, QIAWELL 8 Ultra Plasmid purification systems or the R.E.A.L. PREP 96 plasmid purification kit from QIAGEN. Following precipitation, plasmids were resuspended in 0.1 ml of distilled water and stored, with or without lyophilization, at 4°C.
Alternatively, plasmid DNA was amplified from host cell lysates using direct link PCR in a high-throughput format (Rao, V.B. (1994) Anal. Biochem. 216:1-14). Host cell lysis and thermal cycling steps were carried out in a single reaction mixture. Samples were processed and stored in 384-well plates, and the concentration of amplified plasmid DNA was quantified fluorometrically using PICOGREEN dye (Molecular Probes, Eugene OR) and a FLUOROSKAN II
fluorescence scanner (Labsystems Oy, Helsinki, Finland).
III. Sequencing and Analysis Incyte cDNA recovered in plasmids as described in Example II were sequenced as follows.
Sequencing reactions were processed using standard methods or high-throughput instrumentation such as the ABI CATALYST 800 (Applied Biosystems) thermal cycler or the PTC-200 thermal cycler (MJ Research) in conjunction with the HYDRA microdispenser (Robbins Scientific) or the MICROLAB 2200 (Hamilton) liquid transfer system. cDNA sequencing reactions were prepared using reagents provided by Amersham Pharmacia Biotech or supplied in ABI
sequencing kits such as the ABI PRISM BIGDYE Terminator cycle sequencing ready reaction kit (Applied Biosystems).
Electrophoretic separation of cDNA sequencing reactions and detection of labeled polynucleotides were carried out using the MEGABACE 1000 DNA sequencing system (Molecular Dynamics); the ABI PRISM 373 or 377 sequencing system (Applied Biosystems) in conjunction with standard ABI
protocols and base calling software; or other sequence analysis systems known in the art. Reading frames within the cDNA sequences were identified using standard methods (reviewed in Ausubel, 1997, supra, unit 7.7). Some of the cDNA sequences were selected for extension using the techniques disclosed in Example VIII.
The polynucleotide sequences derived from Incyte cDNAs were validated by removing vector, linker, and poly(A) sequences and by masking ambiguous bases, using algorithms and programs based on BLAST, dynamic programming, and dinucleotide nearest neighbor analysis. The Incyte cDNA sequences or translations thereof were then queried against a selection of public databases such as the GenBank primate, rodent, mammalian, vertebrate, and eukaryote databases, and BLOCKS, PRINTS, DOMO, PRODOM; PROTEOME databases with sequences from Homo sa its ens, Rattus norve icus, Mus musculus, Caenorhabditis elegans, Saccharomyces cerevisiae, Schizosaccharomyces pombe, and Candida albicans (Incyte Genomics, Palo Alto CA); and hidden Markov model (HMM)-based protein family databases such as PFAM. (HMM is a probabilistic approach which analyzes consensus primary structures of gene families. See, for example, Eddy, S.R. (1996) Curr. Opin. Struct. Biol. 6:361-365.) The queries were performed using programs based on BLAST, FASTA, BLIMPS, and HMMER. The Incyte cDNA sequences were assembled to produce full length polynucleotide sequences. Alternatively, GenBank cDNAs, GenBank ESTs, stitched sequences, stretched sequences, or Genscan-predicted coding sequences (see Examples IV

10, and V) were used to extend Incyte cDNA assemblages to full length.
Assembly was performed using programs based on Phred, Phrap, and Consed, and cDNA assemblages were screened for open reading frames using programs based on GeneMark, BLAST, and FASTA. The full length polynucleotide sequences were translated to derive the corresponding full length polypeptide sequences. Alternatively, a polypeptide of the invention may begin at any of the methionine residues of the full length translated polypeptide. Full length polypeptide sequences were subsequently analyzed by querying against databases such as the GenBank protein databases (genpept), SwissProt, the PROTEOME databases, BLOCKS, PRINTS, DOMO, PRODOM, Prosite, and hidden Markov model (HMM)-based protein family databases such as PFAM. Full length polynucleotide sequences are also analyzed using MACDNASIS PRO software (Hitachi Software Engineering, South San Francisco CA) and LASERGENE software (DNASTAR). Polynucleotide and polypeptide sequence alignments are generated using default parameters specified by the CLUSTAL
algorithm as incorporated into the MEGALIGN multisequence alignment program (DNASTAR), which also calculates the percent identity between aligned sequences.
Table 7 summarizes the tools, programs, and algorithms used for the analysis and assembly of Incyte cDNA and full length sequences and provides applicable descriptions, references, and threshold parameters. The first column of Table 7 shows the tools, programs, and algorithms used, the second column provides brief descriptions thereof, the third column presents appropriate references, all of which are incorporated by reference herein in their entirety, and the fourth column presents, where applicable, the scores, probability values, and other parameters used to evaluate the strength of a match between two sequences (the higher the score or the lower the probability value, the greater the identity between two sequences).
The programs described above for the assembly and analysis of full length polynucleotide and polypeptide sequences were also used to identify polynucleotide sequence fragments from SEQ
ID N0:17-32. 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 (See Burge, C. and S. Marlin (1997) J. Mol. Biol.
268:78-94, and Burge, C. and S. Marlin (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 Incyte cDNA or public cDNA coverage of the Genscan-predicted sequences, thus providing evidence for transcription.
When Incyte cDNA
coverage was available, this information was used to correct or confirm the Genscan predicted sequence. Full length polynucleotide sequences were obtained by assembling Genscan-predicted coding sequences with Incyte cDNA sequences and/or public cDNA sequences using the assembly process described in Example III. Alternatively, full length polynucleotide sequences were derived entirely from edited or unedited Genscan-predicted coding sequences.
V. Assembly of Genomic Sequence Data with cDNA Sequence Data "Stitched" Sequences Partial cDNA sequences were extended with exons predicted by the Genscan gene identification program described in Example IV. Partial cDNAs assembled as described in Example Ilz were mapped to genomic DNA and parsed into clusters containing related cDNAs and Genscan exon predictions from one or more genomic sequences. Each cluster was analyzed using an algorithm based on graph theory and dynamic programming to integrate cDNA and genomic information, generating possible splice variants that were subsequently confirmed, edited, or extended to create a full length sequence. Sequence intervals in which the entire length of the interval was present on more than one sequence in the cluster were identified, and intervals thus identified were considered to be equivalent by transitivity. For example, if an interval was present on a cDNA and two genomic sequences, then all three intervals were considered to be equivalent. This process allows unrelated but consecutive genomic sequences to be brought together, bridged by cDNA
sequence. Intervals thus identified were then "stitched" together by the stitching algorithm in the order that they appear along their parent sequences to generate the longest possible sequence, as well as sequence variants.
Linkages between intervals which proceed along one type of parent sequence (cDNA to cDNA or genomic sequence to genomic sequence) were given preference over linkages which change parent type (cDNA to genomic sequence). The resultant stitched sequences were translated and compared by BLAST analysis to the genpept and gbpri public databases. Incorrect exons predicted by Genscan were corrected by comparison to the top BLAST hit from genpept. Sequences were further extended with additional cDNA sequences, or by inspection of genomic DNA, when necessary.
"Stretched" Sequences Partial DNA sequences were extended to full length with an algorithm based on BLAST
analysis. First, partial cDNAs assembled as described in Example III were queried against public databases such as the GenBank primate, rodent, mammalian, vertebrate, and eukaryote databases using the BLAST program. The nearest GenBank protein homolog was then compared by BLAST
analysis to either Incyte cDNA sequences or GenScan exon predicted sequences described in Example IV. A chimeric protein was generated by using the resultant high-scoring segment pairs (HSPs) to map the translated sequences onto the GenBank protein homolog.
Insertions or deletions may occur in the chimeric protein with respect to the original GenBank protein homolog. The GenBank protein homolog, the chimeric protein, or both were used as probes to search for homologous genomic sequences from the public human genome databases. Partial DNA sequences were therefore "stretched" or extended by the addition of homologous genomic sequences. The resultant stretched sequences were examined to determine whether it contained a complete gene.
VI. Chromosomal Mapping of NAAP Encoding Polynucleotides The sequences which were used to assemble SEQ ID N0:17-32 were compared with sequences from the Incyte LIFESEQ database and public domain databases using BLAST and other implementations of the Smith-Waterman algorithm. Sequences from these databases that matched SEQ ID N0:17-32 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 centiMoxgans, 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.gov/genemap/), can be employed to determine if previously identified disease genes map within or in proximity to the intervals indicated above. In this manner, SEQ ID
N0:20 was mapped to chromosome 8 within the interval from 125.80 to 140.60 centiMorgans, and SEQ ID N0:28 was mapped to chromosome 19 within the interval from 41.7 to 49.4 centiMorgans.
VII. Analysis of Polynucleotide Expression Northern analysis is a laboratory technique used to detect the presence of a transcript of a gene and involves the hybridization of a labeled nucleotide sequence to a membrane on which RNAs from a particular cell type or tissue have been bound. (See, e.g., Sambrook, sue, ch. 7; Ausubel (1995) 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 L1FESEQ (Incyte Genomics). This analysis is much faster than multiple membrane-based hybridizations. In addition, the sensitivity of the computer search can be modified to determine whether any particular match is categorized as exact or similar. The basis of the search is the product score, which is defined as:
BLAST Score x Percent Identity 5 x minimum {length(Seq. 1), length(Seq. 2)}
The product score takes into account both the degree of similarity between two sequences and the length of the sequence match. The product score is a normalized value between 0 and 100, and is calculated as follows: the BLAST score is multiplied by the percent nucleotide identity and the product is divided by (5 times the length of the shorter of the two sequences). The BLAST score is calculated by assigning a score of +5 for every base that matches in a high-scoring segment pair (HSP), and -4 for every mismatch. Two sequences may share more than one HSP
(separated by gaps). If there is more than one HSP, then the pair with the highest BLAST
score is used to calculate the product score. The product score represents a balance between fractional overlap and quality in a BLAST alignment. For example, a product score of 100 is produced only for 100%
identity over the entire length of the shorter of the two sequences being compared. A product score of 70 is produced either by 100% identity and 70% overlap at one end, or by 88% identity and 100% overlap at the other. A product score of 50 is produced either by 100% identity and 50%
overlap at one end, or 79%

identity and 100% overlap.
Alternatively, polynucleotide sequences encoding 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 TII]. Each cDNA
sequence is derived from a cDNA library constructed from a human tissue. Each human tissue is classified into one of the following organ/tissue categories: cardiovascular system; connective tissue;
digestive system; embryonic structures; endocrine system; exocrine glands;
genitalia, female;
genitalia, male; germ cells; heroic and immune system; liver; musculoskeletal system; nervous system; pancreas; respiratory system; sense organs; skin; stomatognathic system; unclassified/mixed;
or urinary tract. The number of libraries in each category is counted and divided by the total number of libraries across all categories. Similarly, each human tissue is classified into one of the following disease/condition categories: cancer, cell line, developmental, inflammation, neurological, trauma, cardiovascular, pooled, and other, and the number of libraries in each category is counted and divided by the total number of libraries across all categories. The resulting percentages reflect the tissue- and disease-specific expression of cDNA encoding NAAP. cDNA sequences and cDNA
library/tissue information are found in the LIFESEQ GOLD database (Incyte Genomics, Palo Alto CA).
VIII. Extension of NAAP Encoding Polynucleotides Full length polynucleotide sequences were also produced by extension of an appropriate fragment of the full length molecule using oligonucleotide primers designed from this fragment. One primer was synthesized to initiate 5' extension of the known fragment, and the other primer was synthesized to initiate 3' extension of the known fragment. The initial primers were designed using OLIGO 4.06 software (National Biosciences), or another appropriate program, to be about 22 to 30 nucleotides in length, to have a GC content of about 50% or more, and to anneal to the target sequence at temperatures of about 68°C to about 72°C. Any stretch of nucleotides which would result in hairpin structures and primer-primer dimerizations was avoided.
Selected human cDNA libraries were used to extend the sequence. If more than one extension was necessary or desired, additional or nested sets of primers were designed.
High fidelity amplification was obtained by PCR using methods well known in the art. PCR
was performed in 96-well plates using the PTC-200 thermal cycler (MJ Research, Inc.). The reaction mix contained DNA template, 200 nmol of each primer, reaction buffer containing Mg2+, (NH4)ZSO4, and 2-mercaptoethanol, Taq DNA polymerase (Amersham Pharmacia Biotech), ELONGASE enzyme (Life Technologies), and Pfu DNA polymerase (Stratagene), with the following parameters for primer pair PCI A and PCI B: Step 1: 94°C, 3 min; Step 2: 94°C, 15 sec;
Step 3: 60°C, 1 min; Step 4: 68°C, 2 min; Step 5: Steps 2, 3, and 4 repeated 20 times; Step 6: 68°C, 5 min; Step 7: storage at 4°C. In the alternative, the parameters for primer pair T7 and SK+ were as follows: Step 1: 94°C, 3 min; Step 2:

94°C, 15 sec; Step 3: 57°C, 1 min; Step 4: 68°C, 2 mun;
Step 5: Steps 2, 3, and 4 repeated 20 times;
Step 6: 68°C, 5 min; Step 7: storage at 4°C.
The concentration of DNA in each well was determined by dispensing 100 ~.l PICOGREEN
quantitation reagent (0.25% (v/v) PICOGREEN; Molecular Probes, Eugene OR) dissolved in 1X TE
and 0.5 p,1 of undiluted PCR product into each well of an opaque fluorimeter plate (Corning Costar, Acton MA), allowing the DNA to bind to the reagent. The plate was scanned in a Fluoroskan II
(Labsystems Oy, Helsinki, Finland) to measure the fluorescence of the sample and to quantify the concentration of DNA. A 5 ,u1 to 10 ,u1 aliquot of the reaction mixture was analyzed by electrophoresis on a 1 % agarose gel to determine which reactions were successful in extending the sequence.
The extended nucleotides were desalted and concentrated, transferred to 384-well plates, digested with CviJI cholera virus endonuclease (Molecular Biology Research, Madison WI), and sonicated or sheared prior to religation into pUC 18 vector (Amersham Pharmacia Biotech). For shotgun sequencing, the digested nucleotides were separated on low concentration (0.6 to 0.8%) agarose gels, fragments were excised, and agar digested with Agar ACE
(Promega). Extended clones were relegated using T4 ligase (New England Biolabs, Beverly MA) into pUC 18 vector (Amersham Pharmacia Biotech), treated with Pfu DNA polymerase (Stratagene) to fill-in restriction site overhangs, and transfected into competent E. coli cells. Transformed cells were selected on antibiotic-containing media, and individual colonies were picked and cultured overnight at 37°C in 384-well plates in LB/2x carb liquid media.
The cells were lysed, and DNA was amplified by PCR using Taq DNA polymerase (Amersham Pharmacia Biotech) and Pfu DNA polymerase (Stratagene) with the following parameters: Step 1: 94°C, 3 min; Step 2: 94°C, 15 sec; Step 3:
60°C, 1 min; Step 4: 72°C, 2 min;
Step 5: steps 2, 3, and 4 repeated 29 times; Step 6: 72°C, 5 min; Step 7: storage at 4°C. DNA was quantified by PICOGREEN reagent (Molecular Probes) as described above. Samples with low DNA
recoveries were reamplified using the same conditions as described above.
Samples were diluted with 20% dimethysulfoxide (1:2, v/v), and sequenced using DYENAMIC energy transfer sequencing primers and the DYENAMIC DIRECT kit (Amersham Pharmacia Biotech) or the ABI
PRISM
BIGDYE Terminator cycle sequencing ready reaction kit (Applied Biosystems).
In like manner, full length polynucleotide sequences are verified using the above procedure or are used to obtain 5' regulatory sequences using he above procedure along with oligonucleotides designed for such extension, and an appropriate genomic library.
IX. Labeling and Use of Individual Hybridization Probes Hybridization probes derived from SEQ ID N0:17-32 are employed to screen cDNAs, genomic DNAs, or mRNAs. Although the labeling of oligonucleotides, consisting of about 20 base pairs, is specifically described, essentially the same procedure is used with larger nucleotide fragments. Oligonucleotides are designed using state-of the-art software such as OLIGO 4.06 software (National Biosciences) and labeled by combining 50 pmol of each oligomer, 250 ,uCi of [y 32P] adenosine triphosphate (Amersham Pharmacia Biotech), and T4 polynucleotide kinase (DuPont NEN, Boston MA). The labeled oligonucleotides are substantially purified using a SEPHADEX G-25 superfine size exclusion dextran bead column (Amersham Pharmacia Biotech).
An aliquot containing 10' counts per minute of the labeled probe is used in a typical membrane-based hybridization analysis of human genomic DNA digested with one of the following endonucleases:
Ase I, Bgl II, Eco RI, Pst I, Xba I, or Pvu II (DuPont NEN).
The DNA from each digest is fractionated on a 0.7% agarose gel and transferred to nylon membranes (Nytran Plus, Schleicher & Schuell, Durham NH). Hybridization is carried out for 16 hours at 40°C. To remove nonspecific signals, blots are sequentially washed at room temperature under conditions of up to, for example, 0.1 x saline sodium citrate and 0.5%
sodium dodecyl sulfate.
Hybridization patterns are visualized using autoradiography or an alternative imaging means and compared.
X. Microarrays The linkage or synthesis of array elements upon a microarray can be achieved utilizing photolithography, piezoelectric printing (ink jet printing, See, e.g., Baldeschweiler, supra.), mechanical microspotting technologies, and derivatives thereof. The substrate in each of the aforementioned technologies should be uniform and solid with a non-porous surface (Schena (1999), supra). 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, LTV, chemical, or mechanical bonding procedures. A typical array may be produced using available methods and machines well known to those of ordinary skill in the art and may contain any appropriate number of elements. (See, e.g., Schena, M. et al. (1995) Science 270:467-470; Shalom 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 can be selected using software well known in the art such as LASERGENE software (DNASTAR). The array elements are hybridized with polynucleotides in a biological sample. The polynucleotides in the biological sample are conjugated to a fluorescent label or other molecular tag for ease of detection.
After hybridization, nonhybridized nucleotides from the biological sample are removed, and a fluorescence scanner is used to detect hybridization at each array element.
Alternatively, laser desorbtion and mass spectrometry may be used for detection of hybridization.
The degree of complementarity and the relative abundance of each polynucleotide which hybridizes to an element on the microarray may be assessed. In one embodiment, microarray preparation and usage is described in detail below.
Tissue or Cell Sample Preparation Total RNA is isolated from tissue samples using the guanidinium thiocyanate method and poly(A)* RNA is purified using the oligo-(dT) cellulose method. Each poly(A)~
RNA sample is reverse transcribed using MMLV reverse-transcriptase, 0.05 pg/~,l oligo-(dT) primer (2lmer), 1X
fixst strand buffer, 0.03 unitsl~,l RNase inhibitor, 500 ~,M dATP, 500 ~,M
dGTP, 500 ~,M dTTP, 40 ~,M dCTP, 40 ~.M dCTP-Cy3 (BDS) or dCTP-Cy5 (Amersham Pharmacia Biotech). The reverse transcription reaction is performed in a 25 ml volume containing 200 ng poly(A)+ RNA with GEMBRIGHT kits (Incyte). Specific control poly(A)+ RNAs are synthesized by in vitro transcription from non-coding yeast genomic DNA. After incubation at 37° C for 2 hr, each reaction sample (one with Cy3 and another with Cy5 labeling) is treated with 2.5 ml of 0.5M sodium hydroxide and incubated for 20 minutes at 85°C to the stop the reaction and degrade the RNA. Samples are purified using two successive CHROMA SPIN 30 gel filtration spin columns (CLONTECH
Laboratories, Inc.
(CLONTECH), Palo Alto CA) and after combining, both reaction samples are ethanol precipitated using 1 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 ~.15X SSC/0.2% SDS.
Microarray Preparation Sequences of the present invention are used to generate array elements. Each array element is amplified from bacterial cells containing vectors with cloned cDNA inserts.
PCR amplification uses primers complementary to the vector sequences flanking the cDNA insert.
Array elements are amplified in thirty cycles of PCR from an initial quantity of 1-2 ng to a final quantity greater than 5 ~.g. Amplified array elements are then purified using SEPHACRYL-400 (Amersham Pharmacia Biotech).
Purified array elements are immobilized on polymer-coated glass slides. Glass microscope slides (Corning) 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 p,1 of the array element DNA, at an average concentration of 100 ng/~,1, is loaded into the open capillary printing element by a high-speed robotic apparatus. The apparatus then deposits about 5 n1 of array element sample per slide.
Microarrays are UV-crosslinked using a STRATALINKER UV-crosslinker (Stratagene).
Microarrays are washed at room temperature once in 0.2% SDS and three times in distilled water.
Non-specific binding sites are blocked by incubation of microarrays in 0.2%
casein in phosphate buffered saline (PBS) (Tropix, Inc., Bedford MA) for 30 minutes at 60°
C followed by washes in 0.2% SDS and distilled water as before.
Hybridization Hybridization reactions contain 9 ~,l of sample mixture consisting of 0.2 ~,g each of Cy3 and Cy5 labeled cDNA synthesis products in 5X SSC, 0.2% SDS hybridization buffer.
The sample mixture is heated to 65°C for 5 minutes and is aliquoted onto the microarray surface and covered with an 1.8 cm2 coverslip. The arrays are transferred to a waterproof chamber having a cavity just slightly larger than a microscope slide. The chamber is kept at 100% humidity internally by the addition of 140 ~,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 and adding identical amounts of each to the hybridization mixture.
The output of the photomultiplier tube is digitized using a 12-bit RTI-835H
analog-to-digital (A/D) conversion board (Analog Devices, Inc., Norwood MA) installed in an IBM-compatible PC
computer. The digitized data are displayed as an image where the signal intensity is mapped using a linear 20-color transformation to a pseudocolor scale ranging from blue (low signal) to red (high signal). The data is also analyzed quantitatively. Where two different fluorophores are excited and measured simultaneously, the data are first corrected for optical crosstalk (due to overlapping emission spectra) between the fluorophores using each fluorophore's emission spectrum.
A grid is superimposed over the fluorescence signal image such that the signal from each spot is centered in each element of the grid. The fluorescence signal within each element is then integrated to obtain a numerical value corresponding to the average intensity of the signal. The software used for signal analysis is the GEMTOOLS gene expression analysis program (Incyte).
XI. 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.
XII. 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 T5 or T7 bacteriophage promoter in conjunction with the lac operator regulatory element. Recombinant vectors are transformed into suitable bacterial hosts, e.g., BL21(DE3).
Antibiotic resistant bacteria express NAAP upon induction with isopropyl beta-D-thiogalactopyranoside (7PTG). Expression of NAAP in eukaryotic cells is achieved by infecting insect or mammalian cell lines with recombinant Autog_raphica 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 polyhedrin promoter drives high levels of cDNA transcription. Recombinant baculovirus is used to infect Spodoptera 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., glutathione S-transferase (GST) or a peptide epitope tag, such as FLAG or 6-His, permitting rapid, single-step, affinity-based purification of recombinant fusion protein from crude cell lysates. GST, a 26 kilodalton enzyme from Schistosoma Lponicum, enables the purification of fusion proteins on immobilized glutathione under conditions that maintain protein activity and antigenicity (Amersham Pharmacia Biotech). Following purification, the GST moiety can be proteolytically cleaved from .15 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, supra, ch. 10 and 16). Purified NAAP obtained by these methods can be used directly in the assays shown in Examples XVI, XVII, XV QI, and XIX, where applicable.
XIII. 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 high levels of cDNA
expression. Vectors of choice include PCMV SPORT (Life Technologies) and PCR3.1 (Invitrogen, Carlsbad CA), both of which contain the cytomegalovirus promoter. 5-10 ,ug of recombinant vector are transiently transfected into a human cell line, for example, an endothelial or hematopoietic cell line, using either liposome formulations or electroporation. 1-2 ,ug 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, 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.
XIV. 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 rabbits 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. (See, e.g., Ausubel, 1995, supra, ch. 11.) Typically, oligopeptides of about 15 residues in length are synthesized using an ABI 431A
peptide synthesizer (Applied Biosystems) using FMOC chemistry and coupled to I~LH (Sigma-Aldrich, St. Louis MO) by reaction with N-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS) to increase immunogenicity. (See, e.g., Ausubel, 1995, su ra.) 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.
XV. Purification of Naturally Occurring NAAP Using Specific Antibodies Naturally occurring or recombinant NAAP is substantially purified by immunoaffinity 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 Pharmacia Biotech). After the coupling, the resin is blocked and washed according to the manufacturer's instructions.
Media containing NAAP are passed over the immunoaffmity 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.
XVI. Identification of Molecules Which Interact with NAAP
NAAP, or biologically active fragments thereof, are labeled with'z5I Bolton-Hunter reagent.
(See, e.g., Bolton, A.E. and W.M. Hunter (1973) Biochem. J. 133:529-539.) Candidate molecules previously arrayed in the wells of a mufti-well plate 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).
XVII. 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, LexAop LacZ, that consists of LexA DNA transcriptional control elements (LexAop) fused to sequences encoding the E. coli 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 LexAoP 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 solution (Aldrich, Milwaukee Wn in the presence of 100 ~.M dithiothreitol with 10% methanol added. The sample and zinc sulfate solutions are allowed to incubate for 20 min. The reaction solution is passed through a VYCAC column (Grace Vydac, Hesperia CA) with approximately 300 A
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, R. M. et al. ((2000) Biochemistry 39:8406-8417).
In the alternative, a method to determine nucleic acid binding activity of NAAP involves a polyacrylamide 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 5-10 min. 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 untransformed 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 ~.1 final volume) contain 15 mM HEPES, pH 7.9, 1.5 mM MgCl2, 10 mM
dithiothreitol, 3%
polyvinylalcohol, 1.5 ~iCi [methyl-3H]AdoMet (0.375 ,uM AdoMet) (DuPont-NEN), 0.6 ~,g NAAP, and acceptor substrate (e.g., 0.4 ~,g [35S]RNA, or 6-mercaptopurine (6-MP) to 1 mM final concentration). Reaction mixtures are incubated at 30°C for 30 min, then 65°C for 5 min.
Analysis of [methyl.-3H]RNA is as follows: (1) 50 ~.1 of 2 x loading buffer (20 mM Tris-HCI, pH 7.6, 1 M LiCI, 1 mM EDTA, 1 % sodium dodecyl sulphate (SDS)) and 50 ~,l oligo d(T)-cellulose (10 mglml in 1 x loading buffer) are added to the reaction mixture, and incubated at ambient temperature with shaking for 30 min. (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 p,1 of water into a 96-well collection plate, transferred to scintillation vials containing liquid scintillant, and radioactivity determined.
Analysis of [»zetlzyl-3H]6-MP is as follows: (1) 500 x,10.5 M borate buffer, pH 10.0, and then 2.5 ml of 20% (vlv) 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 min, 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%).
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, Mgz+, and varying amounts of NAAP in a Tris-HCl buffer, pH 7.5, at 37°C for 30 min. 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
expression. Vectors of choice include pCMV SPORT (Life Technologies) and pCR3.l (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 cytometiy 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 Biotech, 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 (W). A typical 500 ~.1 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 ~,Cilnmol tRNA). The reaction is initiated by the addition of <5 ~,l 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%
w/v). 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 HCI, followed by centrifugation. The supernatants from the washes axe 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 filtrate is proportional to the amount of peudouridine synthase activity in the sample (Ramamurthy, V. (I999) 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
MgCl2, 2 mM
dithiothreitol, 0.1 mM EDTA, and 1-2 fmol of [32P]-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 ~rMP and UMP
spots are evaluated after exposing the thin layer chromatography plates to film or a PHOSPHORIMAGER screen (Molecular Dynamics). Taking into account the relative number of uridylate residues in the substrate RNA, the relative amount t~rMP and UMP are determined and used to calculate the relative amount of yr per tRNA molecule (expressed in mol ~r /mol of tRNA or mol yr /mol of tRNA/min), which corresponds to the amount of pseudouridine synthase activity in the NAAP
sample (Lecointe, F. et aI.
(1998) J. Biol. Chem. 273:1316-1323).
Nz,Nz-dimethylguanosine transferase ((m22G)methyltransferase) activity of NAAP
is measured in a 160 ,u1 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]methionine (30-70 Ci/mM), 8 ~.g m22G-deficient tRNA or wild type tRNA from yeast, and approximately 100 ~.g 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 I ml in water containing 100 ~,g BSA. I 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'.,G-deficient, acid-insoluble tRNAs is proportional to the amount of NZ,Nz-dimethylguanosine transferase activity in the NAAP sample. Reactions comprising no substrate tRNAs, or wild-type tRNAs that have already been modified, serve as control reactions which should not yield acid-insoluble 3H-labeled products.
Polyadenylation activity of NAAP is measured using an in vitro polyadenylation reaction.
The reaction mixture is assembled on ice and comprises 10 p,1 of 5 mM
dithiothreitol, 0.025% (v/v) Nonidet P-40 detergent, 50 mM creatine phosphate, 6.5% (w/v) polyvinyl alcohol, 0.5 unit/~,l RNAGUARD (Amersham Pharmacia Biotech), 0.025 ~.g/p,l creatine kinase, 1.25 mM
cordycepin 5'-triphosphate, and 3.75 mM MgClz, in a total volume of 25 ~,1. 60 fmol of CstF, 50 fmol of CPSF, 240 fmol of PAP, 4 ~,1 of crude or partially purified CF II 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 mM 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 szP-labeled pre-mRNA template, along with 2.5 ~,g of unlabeled tRNA, in 1.5 ~,1 of water. Reactions are then incubated at 30 °C for 75-90 min and stopped by the addition of 75 ~.1 (approximately two-volumes) of proteinase K mix (0.2 M Tris-HCI, pH 7.9, 300 mM NaCI, 25 mM Na-EDTA, 2% (w/v) SDS), 1 ~.1 of 10 mg/ml proteinase K, 0.25 ~,1 of 20 mg/ml glycogen, and 23.75 ~.l 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, U. et al. (1996) J. Biol. Chem. 271:6107-6113; and references within).
tRNA synthetase activity is measured as the aminoacylation of a substrate tRNA
in the presence of ['4C]-labeled amino acid. NAAP is incubated with [14C]-labeled amino acid and the appropriate cognate tRNA (for example, ['4C]alanine and tRNA~'a) in a buffered solution. '4C-labeled product is separated from free [14C]amino acid by chromatography, and the incorporated'4C is quantified by scintillation counter. The amount of '4C-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 ['4C]-Glu-tRNAGIn (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 in water and spotted onto a cellulose TLC plate. The plate is developed in either isopropanol/formic acid/water or ammonia/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-tRNA~'° (adapted from Curnow, A.W. et al.
(1997) Proc. Natl. Acad. Sci.

U. S. A. 94:11819-26).
XVIII. Identification of NAAP Agonists and Antagonists Agonists or antagonists of NAAP activation or inhibition may be tested using the assays described in section XVII. Agonists cause an increase in NAAP activity and antagonists cause a decrease in NAAP activity.
XIX. NAAP Secretion Assay A high throughput assay may be used to identify polypeptides that are secreted in eukaryotic cells. In an example of such an assay, polypeptide expression libraries are constructed by fusing 5'-biased cDNAs to the 5'-end of a leaderless (3-lactamase gene. (3-lactamase is a convenient genetic reporter as it provides a high signal-to-noise ratio against low endogenous background activity and retains activity upon fusion to other proteins. A dual promoter system allows the expression of (3-lactamase fusion polypeptides in bacteria or eukaryotic cells, using the lae or CMV promoter, respectively.
Libraries are first transformed into bacteria, e.g., E. coli, to identify library members that encode fusion polypeptides capable of being secreted in a prokaryotic system.
Mammalian signal sequences direct the translocation of j3-lactamase fusion polypeptides into the periplasm of bacteria where it confers antibiotic resistance to carbenicillin. Carbenicillin-selected bacteria are isolated on solid media, individual clones are grown in liquid media, and the resulting cultures are used to isolate library member plasmid DNA.
Mammalian cells, e.g., 293 cells, are seeded into 96-well tissue culture plates at a density of about 40,000 cellslwell in 100 ~.l phenol red-free DME supplemented with 10%
fetal bovine serum (FBS) ( Life Technologies, Rockville, MD). The following day, purified plasmid DNAs isolated from carbenicillin-resistant bacteria are diluted with 15 ,u1 OPTI-MEM I
medium (Life Technologies) to a volume of 25 ~,1 for each well of cells to be transfected. In separate plates, 1 ~,1 LF2000 Reagent (Life Technologies) is diluted into 25 ~,1/well OPTI-MEM I. The 25 ~.1 diluted LF2000 Reagent is then combined with the 25 ~,1 diluted DNA, mixed briefly, and incubated for 20 minutes at room temperature. The resulting DNA-LF2000 reagent complexes are then added directly to each well of 293 cells. Cells are also transfected with appropriate control plasmids expressing either wild-type (3-lactamase, leaderless (3-lactamase, or, fox example, CD4-fused leaderless ~3-lactamase. 24 hrs following transfection, about 90 ,u1 of cell culture media are assayed at 37°C with 100 ~.M Nitrocefin (Calbiochem, San Diego CA) and 0.5 mM oleic acid (Sigma, St. Louis, MO) in 10 mM phosphate buffer (pH 7.0). Nitrocefm is a substrate for (3-lactamase that undergoes a noticeable color change from yellow to red upon hydrolysis. [3-lactamase activity is monitored over 20 min in a microtiter plate reader at 486 nm. Increased color absorption at 486 nm corresponds to secretion of a [3-lactamase fusion polypeptide in the transfected cell media, resulting from the presence of a eukaryotic signal sequence in the fusion polypeptide. Polynucleotide sequence analysis of the corresponding library member plasmid DNA is then used to identify the signal sequence-encoding cDNA. (Described in U.S. Patent application 09/803,317, filed March 9, 2001.) Various modifications and variations of the described methods and systems of the invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described in connection with certain embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention which are obvious to those skilled in molecular biology or related fields are intended to be within the scope of the following claims.

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<110> INCYTE GENOMICS, INC.
BAUGHN, Mariah R.
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POLICKY,Jennifer L.
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YUE, Henry BATRA, Sajeev DING, Li LAL, Preeti G.
BOROWSKY, Mark L.
LU, Dyung Aina M.
GANDHI, Ameena R.
GRIFFIN, Jennifer A.
XU, Yuming AZIMZAI, Yalda GIETZEN, Kimberly J.
TANG, Y. Tom WARREN, Bridget A.
MASON, Patricia M.
BURFORD, Neil HAFALIA, April J.A.
LEE, Ernestine A.
YANG, Junming GORVAD, Ann E.
EMERLING, Brooke M.
MARQUIS, Joseph P.
LEE, Soo Yeun SWARNAKAR, Anita REDDY, Reddy <120> NUCLEIC ACID-ASSOCIATED PROTEINS
<130> PF-0869 PCT
<140> To Be Assigned <141> Herewith <150> 60/257,714; 60/260,081; 60/262,302; 60/263,823; 60/266,088; Unassigned <151> 2000-12-21; 2001-01-05; 2001-01-16; 2001-01-23; 2001-02-02; 2001-10-26 <160> 32 <170> PERL Program <210> 1 <211> 1096 <212> PRT
<213> Homo sapiens <220>
<221> misc_feature <223> Incyte ID No: 2530775CD1 <400> 1 Met Asp Cys Asn Ala Cys Met Ser Glu Glu Leu Trp GIy Met Phe Lys Thr Phe Pro Tyr Gln His Arg Tyr Arg Leu Tyr Gly Gln Trp Lys Asn Glu Thr Tyr Asn Ser His Pro Leu Leu Val Lys Val Lys Ala Gln Thr Ile Asp Arg Ala Lys Tyr Ile Met Lys Arg Leu Thr Lys Glu Asn Val Lys Pro Ser Gly Arg Gln Ile Gly Lys Leu Ser His Ser Asn Pro Thr Tle Leu Phe Asp Tyr Ile Leu Ser Gln Tle Gln Lys Tyr Asp Asn Leu Ile Thr Pro Val Val Asp Ser Leu Lys Tyr Leu Thr Ser Leu Asn Tyr Asp Val Leu Ala Tyr Cys Ile Ile Glu Ala Leu Ala Asn Pro Glu Lys Glu Arg Met Lys His Asp Asp 1.2 5 13 0 13 5 Thr Thr Ile Ser Ser Trp Leu Gln Ser Leu Ala Ser Phe Cys Gly Ala Val Phe Arg Lys Tyr Pro Ile Asp Leu Ala Gly Leu Leu Gln Tyr Val Ala Asn Gln Leu Lys Ala Gly Lys Ser Phe Asp Leu Leu Ile Leu Lys Glu Val Val Gln Lys Met Ala Gly Ile Glu Ile Thr Glu Glu Met Thr Met Glu Gln Leu Glu Ala Met Thr Gly Gly Glu Gln Leu Lys Ala Glu Gly Gly Tyr Phe Gly Gln Ile Arg Asn Thr Lys Lys Ser Ser Gln Arg Leu Lys Asp Ala Leu Leu Asp His Asp Leu Ala Leu Pro Leu Cys Leu Leu Met Ala Gln Gln Arg Asn Gly Val Ile Phe Gln Glu G1y Gly Glu Lys His Leu Lys Leu Val Gly Lys Leu Tyr Asp Gln Cys His Asp Thr Leu Val Gln Phe Gly Gly Phe Leu Ala Ser Asn Leu Ser Thr Glu Asp Tyr Ile Lys Arg Val Pro Ser Ile Asp Val Leu Cys Asn Glu Phe His Thr Pro His Asp Ala Ala Phe Phe Leu Ser Arg Pro Met Tyr Ala His His Ile Ser Ser Lys Tyr Asp Glu Leu Lys Lys Ser Glu Lys Gly Ser Lys Gln Gln His Lys Va1 His Lys Tyr Ile Thr Ser Cys Glu Met Val Met Ala Pro Val His Glu Ala Val Val Ser Leu His Va1 Ser Lys Val Trp Asp Asp I1e Ser Pro Gln Phe Tyr Ala Thr Phe Trp Ser Leu Thr Met Tyr Asp Leu A1a Val Pro His Thr Ser Tyr Glu Arg Glu Val Asn Lys Leu Lys Va1 Gln Met Lys Ala Ile Asp Asp Asn Gln G1u Met Pro Pro Asn Lys Lys Lys Lys G1u Lys Glu Arg Cys Thr A1a Leu Gln Asp Lys Leu Leu Glu Glu Glu Lys Lys Gln Met Glu His Val Gln Arg Va1 Leu Gln Arg Leu Lys Leu G1u Lys Asp Asn Trp Leu Leu Ala Lys Ser Thr Lys Asn Glu Thr Ile Thr Lys Phe Leu Gln Leu Cys Ile Phe Pro Arg Cys Ile Phe Ser Ala Ile Asp Ala Val Tyr Cys Ala Arg Phe Val Glu Leu Val His G1n Gln Lys ,,:
Thr Pro Asn Phe Ser Thr Leu Leu Cys Tyr Asp Arg Val Phe Ser Asp Ile Ile Tyr Thr Val Ala Ser Cys Thr Glu Asn Glu Ala Ser Arg Tyr Gly Arg Phe Leu Cys Cys Met Leu G1u Thr Val Thr Arg Trp His Ser Asp Arg Ala Thr Tyr Glu Lys Glu Cys Gly Asn Tyr Pro Gly Phe Leu Thr Ile Leu Arg Ala Thr Gly Phe Asp Gly Gly Asn Lys Ala Asp Gln Leu Asp Tyr Glu Asn Phe Arg His Val Val His Lys Trp His Tyr Lys Leu Thr Lys Ala Ser Val His Cys Leu Glu Thr Gly G1u Tyr Thr His I1e Arg Asn Ile Leu Ile Val Leu Thr Lys Ile Leu Pro Trp Tyr Pro Lys Val Leu Asn Leu Gly Gln Ala Leu Glu Arg Arg Val His Lys Ile Cys Gln Glu Glu Lys Glu Lys Arg Pro Asp Leu Tyr Ala Leu Ala Met Gly Tyr Ser Gly Gln Leu Lys Ser Arg Lys Ser Tyr Met Ile Pro Glu Asn Glu Phe His His Lys Asp Pro Pro Pro Arg Asn Ala Val Ala Ser Val Gln Asn Gly Pro Gly Gly Gly Pro Ser Ser Ser Ser Tle Gly Ser Ala Ser Lys Ser Asp G1u Ser Ser Thr G1u Glu Thr Asp Lys Ser Arg Glu Arg Ser Gln Cys Gly Val Lys Ala Val Asn Lys Ala Ser Ser Thr Thr Pro Lys Gly Asn Ser Ser Asn Gly Asn Ser Gly Ser Asn Ser Asn Lys Ala Va1 Lys Glu Asn Asp Lys Glu Lys Gly Lys Glu Lys Glu Lys Glu Lys Lys Glu Lys Thr Pro Ala Thr Thr Pro Glu Ala Arg Val Leu Gly Lys Asp Gly Lys Glu Lys Pro Lys Glu G1u Arg Pro Asn Lys Asp Glu Lys Ala Arg Glu Thr Lys Glu Arg Thr Pro Lys Ser Asp Lys Glu Lys Glu Lys Phe Lys Lys Glu Glu Lys Ala Lys Asp G1u Lys Phe Lys Thr Thr Val Pro Asn A1a Glu Ser Lys Ser Thr Gln Glu Arg Glu Arg Glu Lys Glu Pro Ser Arg Glu Arg Asp Ile Ala Lys Glu Met Lys Ser Lys Glu Asn Val Lys Gly Gly 875 880 ' 885 Glu Lys Thr Pro Val Ser Gly Ser Leu Lys Ser Pro Val Pro Arg Ser Asp Ile Pro Glu Pro Glu Arg G1u Gln Lys Arg Arg Lys Ile Asp Thr His Pro Ser Pro Ser His Ser Ser Thr Val Lys Asp Ser 920 925 . 930 Leu Ile Glu Leu Lys Glu Ser Ser Ala Lys Leu Tyr Ile Asn His Thr Pro Pro Pro Leu Ser Lys Ser Lys Glu Arg Glu Met Asp Lys Lys Asp Leu Asp Lys Ser Arg Glu Arg Ser Arg Glu Arg Glu Lys Lys Asp Glu Lys Asp Arg Lys Glu Arg Lys Arg Asp His Ser Asn Asn Asp Arg Glu Val Pro Pro Asp Leu Thr Lys Arg Arg Lys Glu Glu Asn Gly Thr Met Gly Val Ser Lys His Lys Ser Glu Ser Pro Cys Glu Ser Pro Tyr Pro Asn Glu Lys Asp Lys Glu Lys Asn Lys Ser Lys Ser Ser Gly Lys Glu Lys Gly Ser Asp Ser Phe Lys Ser Glu Lys Met Asp Lys Ile Ser Ser Gly GIy Lys Lys Glu Ser Arg His Asp Lys Glu Lys Ile Glu Lys Lys Glu Lys Arg Asp Ser Ser Gly Gly Lys Glu Glu Lys Lys His His Lys Ser Ser Asp Lys His Arg <210> 2 <211> 995 <212> PRT
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte ID No: 926296CD1 <400> 2 Met Ser Ser Leu Pro Arg Arg Ala Lys Val Gln Val Gln Asp Val Val Leu Lys Asp Glu Phe Ser Ser Phe Ser Glu Leu Ser Ser Ala Ser Glu Glu Asp Asp Lys Glu Asp Ser Ala Trp Glu Pro Gln Lys Lys Val Pro Arg Ser Arg Lys Gln Pro Pro Pro Lys Glu Ser Lys Pro Lys Arg Met Pro Arg Val Lys Lys Asn Ala Pro Gln Ile Ser Asp Gly Ser Glu Val Val Val Val Lys Glu Glu Leu Asn Ser Ser Val Ala Ile Ala Asp Thr Ala Leu Glu Asp Arg Lys Asn Lys Leu Asp Thr Val Gln Thr Leu Lys Thr A1a Lys Thr Lys Gln Lys Cys Ala Ala Gln Pro His Thr Val Arg Arg Thr Lys Lys Leu Lys Val Glu G1u Glu Thr Ser Lys Ala Ser Asn Leu Glu Gly Glu Ser Asn Ser Ser Glu Thr Pro Ser Thr Ser Thr Va1 Trp Gly Gly Thr Cys Lys Lys Glu Glu Asn Asp Asp Asp Phe Thr Phe Gly Gln Ser Ala Leu Lys Lys Ile Lys Thr Glu Thr Tyr Pro Gln G1y Gln Pro Val Lys Phe Pro Ala Asn Ala Asn Ser Thr Lys Glu Glu Val Glu Met Asn Trp Asp Met Val Gln Val Leu Ser Glu Arg Thr Asn Ile Glu Pro Trp Va1 Cys Ala Asn Tle I1e Arg Leu Phe Asn Asp Asp Asn Thr Ile Pro Phe Ile Ile Arg Tyr Arg Lys Glu Leu Ile Asn Asn Leu Asp Ala Asp Ser Leu Arg Glu Val Gln Gln Thr Leu Glu Glu Leu Arg Ala Val Ala Lys Lys Val His Ser Thr Ile Gln Lys Ile Lys Lys Glu Gly Lys Met Ser Glu Cys Leu Leu Lys Ala Met Leu Asn Cys Lys Thr Phe Glu Glu Leu Glu His Val Ser Ala Pro Tyr Lys Thr Gly Ser Lys Gly Thr Lys Ala Gln Arg Ala Arg Gln Leu Gly Leu Glu Gly Ala Ala Arg Ala Leu Leu Glu Lys Pro Gly Glu Leu Ser Leu Leu Ser Tyr Ile Arg Pro Asp Val Lys Gly Leu Ser Thr Leu Gln Asp Ile Glu Ile Gly Val Gln His Ile Leu Ala Asp Met Ile Ala Lys Asp Lys Asp Thr Leu Asp Phe Ile Arg Asn Leu Cys Gln Lys Arg His Val Cys Ile Gln Ser Ser Leu Ala Lys Val Ser Ser Lys Lys Val Asn Glu Lys Asp Val Asp Lys Phe Leu Leu Tyr Gln His Phe Ser Cys Asn Ile Arg Asn Ile His His His Gln Ile Leu Ala Ile Asn Arg Gly Glu Asn Leu Lys Val Leu Thr Val Lys Va1 Asn Ile Ser Asp Gly Val Lys Asp Glu Phe Cys Arg Trp Cys Ile Gln Asn Arg Trp Arg Pro Arg Ser Phe A1a Arg Pro Glu Leu Met Lys Ile Leu Tyr Asn Ser Leu Asn Asp Ser Phe Lys Arg Leu Tle Tyr Pro Leu Leu Cys Arg Glu Phe Arg Ala Lys Leu Thr Ser Asp Ala Glu Lys Glu Ser Val Met Met Phe Gly Arg Asn Leu Arg Gln Leu Leu Leu~Thr Ser Pro Val Pro Gly Arg Thr Leu Met Gly Val Asp Pro Gly Tyr Lys His G1y Cys Lys Leu Ala Ile Ile Ser Pro Thr Ser Gln Ile Leu His Thr Asp Val Val Tyr Leu His Cys Gly Gln Gly Phe Arg Glu Ala Glu Lys Ile Lys Thr Leu Leu Leu Asn Phe Asn Cys Ser Thr Val Val Ile Gly Asn Gly Thr Ala Cys Arg Glu Thr Glu Ala Tyr Phe Ala Asp Leu Ile Met Lys Asn Tyr Phe Ala Pro Leu Asp Val Val Tyr Cys Ile Val Ser Glu Ala Gly Ala Ser Ile Tyr Ser Val Ser Pro Glu Ala Asn Lys Glu Met Pro Gly Leu Asp Pro Asn Leu Arg Ser Ala Val Ser Ile Ala Arg Arg Val Gln Asp Pro Leu Ala Glu Leu Val Lys Ile Glu Pro Lys His Ile Gly Val G1y Met Tyr Gln His Asp Val Ser Gln Thr Leu Leu Lys Ala Thr Leu Asp Ser Val Val Glu Glu Cys Val Ser Phe Val Gly Val Asp Ile Asn Ile Cys Sex Glu Val Leu Leu Arg His Ile Ala Gly Leu Asn Ala Asn Arg Ala Lys Asn Ile Ile Glu Trp Arg Glu Lys Asn Gly Pro Phe Ile Asn Arg Glu Gln Leu Lys Lys Val Lys G1y Leu Gly Pro Lys Ser Phe Gln Gln Cys A1a Gly Phe Ile Arg Ile Asn Gln Asp Tyr Ile Arg Thr Phe Cys Ser Gln Gln Thr Glu Thr Ser Gly Gln Ile Gln Gly Val Ala Val Thr Ser Ser Ala Asp Va1 Glu Val Thr Asn Glu Lys Gln Gly Lys Lys Lys Ser 800 805 8l0 Lys Thr Ala Val Asn Val Leu Leu Lys Pro Asn Pro Leu Asp Gln Thr Cys Ile His Pro Glu Ser Tyr Asp Ile Ala Met Arg Phe Leu Ser Ser Ile Gly Gly Thr Leu Tyr Glu Val G1y Lys Pro Glu Met Gln Gln Lys Ile Asn Ser Phe Leu Glu Lys Glu Gly Met Glu Lys Ile Ala Glu Arg Leu Gln Thr Thr Val His Thr Leu Gln Val Ile Ile Asp Gly Leu Ser Gln Pro Glu Ser Phe Asp Phe Arg Thr Asp Phe Asp Lys Pro Asp Phe Lys Arg Ser Ile Val Cys Leu Glu Asp Leu Gln I1e Gly Thr Va1 Leu Thr Gly Lys Val Glu Asn Ala Thr Leu Phe Gly Ile Phe Va1 Asp Ile Gly Val Gly Lys Ser Gly Leu Ile Pro I1e Arg Asn Val Thr Glu Ala Lys Leu Ser Lys Thr Lys Lys Arg Arg Ser Leu Gly Leu Gly Pro Gly Glu Arg Val Glu Val Gln Val Leu Asn Ile Asp Ile Pro Arg Ser Arg Ile Thr Leu Asp Leu Ile Arg Val Leu <210> 3 <21l> 252 <212> PRT
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte ID No: 1322761CD1 <400> 3 Met Phe G1n Pro Ala Pro Lys Arg Cys Phe Thr Ile Glu Ser Leu 1 5. 10 15 Val A1a Lys Asp Ser Pro Leu Pro Ala Ser Arg Ser Glu Asp Pro Ile Arg Pro Ala Ala Leu Ser Tyr Ala Asn Ser Ser Pro Ile Asn Pro Phe Leu Asn Gly Phe His Ser Ala Ala Ala Ala Ala Ala Gly Arg Gly Val Tyr Ser Asn Pro Asp Leu Val Phe Ala Glu Ala Val Ser His Pro Pro Asn Pro Ala Val Pro Val His Pro Val Pro Pro Pro His Ala Leu Ala Ala His Pro Leu Pro Ser Ser His Ser Pro His Pro Leu Phe Ala Ser Gln Gln Arg Asp Pro Ser Thr Phe Tyr Pro Trp Leu Ile His Arg Tyr Arg Tyr Leu Gly His Arg Phe Gln Gly Asn Asp Thr Ser Pro G1u Ser Phe Leu Leu His Asn Ala Leu Ala Arg Lys Pro Lys Arg Ile Arg Thr Ala Phe Ser Pro Ser Gln Leu Leu Arg Leu Glu His Ala Phe Glu Lys Asn His Tyr Val Val Gly Ala Glu Arg Lys Gln Leu Ala His Ser Leu Ser Leu Thr Glu Thr Gln Val Lys Val Trp Phe Gln Asn Arg Arg Thr Lys Phe Lys Arg Gln Lys Leu Glu Glu Glu Gly Ser Asp Ser Gln Gln Lys Lys Lys Gly Thr His His Ile Asn Arg Trp Arg Ile Ala Thr Lys Gln Ala Ser Pro Glu G1u Ile Asp Val Thr Ser Asp Asp <210> 4 <211> 602 <212> PRT
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte ID No: 7472664CD1 <400> 4 Met Ser Phe Arg Pro Arg Ile Leu I1e Val Gly Glu Pro Gly Phe Gly Gln Gly Ser His Leu Ala Pro Ala Val Ile His Ala Leu Glu Lys Phe Thr Val Tyr Thr Leu Asp Ile Pro Val Leu Phe Gly Val Ser Thr Thr Ser Pro Glu Glu Thr Cys Ala Gln Val Ile Arg Glu Ala Lys Arg Thr Ala Pro Ser Ile Val Tyr Val Pro His Ile His Val Trp Trp Glu Ile Val Gly Pro Thr Leu Lys Ala Thr Phe Thr Thr Leu Leu Gln Asn Ile Pro Ser Phe Ala Pro Val Leu Leu Leu Ala Thr Ser Asp Lys Pro His Ser Ala Leu Pro Glu Glu Val Gln Glu Leu Phe Ile Arg Asp Tyr Gly Glu Ile Phe Asn Val Gln Leu Pro Asp Lys Glu Glu Arg Thr Lys Phe Phe Glu Asp Leu I1e Leu Lys Gln Ala A1a Lys Pro Pro Ile Ser Lys Lys Lys Ala Val Leu Gln Ala Leu Glu Val Leu Pro Val Ala Pro Pro Pro Glu Pro Arg Ser Leu Thr Ala Glu Glu Val Lys Arg Leu Glu Glu Gln Glu Glu Asp Thr Phe Arg Glu Leu Arg Ile Phe Leu Arg Asn Val Thr His Arg Leu Ala Ile Asp Lys Arg Phe Arg Val Phe Thr Lys Pro Val Asp Pro Asp Glu Val Pro Asp Tyr Val Thr Val Ile Lys Gln Pro Met Asp Leu Ser Ser Val Ile Ser Lys Ile Asp Leu His Lys Tyr Leu Thr Val Lys Asp Tyr Leu Arg Asp Ile Asp Leu Ile Cys Ser Asn Ala Leu Glu Tyr Asn Pro Asp Arg Asp Pro Gly Asp Arg Leu Ile Arg His Arg Ala Cys Ala Leu Arg Asp Thr Ala Tyr Ala Ile Ile Lys Glu Glu Leu Asp Glu Asp Phe Glu Gln Leu Cys Glu G1u Ile Gln Glu Ser Arg Lys Lys Arg Gly Cys Ser Ser Ser Lys Tyr Ala Pro Ser Tyr Tyr His Val Met Pro Lys Gln Asn Ser Thr Leu Val Gly Asp Lys Arg Ser Asp Pro Glu Gln Asn Glu Lys Leu Lys Thr Pro Ser Thr Pro Val Ala Cys Ser Thr Pro Ala Gln Leu Lys Arg Lys Ile Arg Lys Lys Ser Asn Trp Tyr Leu Gly Thr Ile Lys Lys Arg Arg Lys Ile Ser Gln Ala Lys Asp Asp Ser Gln Asn Ala Ile Asp His Lys Ile Glu Ser Asp Thr Glu G1u Thr Gln Asp Thr Ser Val Asp His Asn Glu Thr Gly Asn Thr Gly Glu Ser Ser Val Glu Glu Asn Glu Lys Gln Gln Asn Ala Ser Glu Ser Lys Leu Glu Leu Arg Asn Asn Ser Asn Thr Cys Asn Ile Glu Asn Glu Leu Glu Asp Ser Arg Lys Thr Thr Ala Cys Thr Glu Leu Arg Asp Lys Ile Ala Cys Asn Gly Asp Ala Ser Ser Ser Gln Ile Ile His Ile Ser Asp Glu Asn Glu Gly Lys Glu Met Cys Val Leu Arg Met Thr Arg Ala Arg Arg Ser Gln Val Glu Gln Gln Gln Leu Ile Thr Val Glu Lys Ala Leu Ala Ile Leu Ser G1n Pro Thr Pro Ser Leu Val Val Asp His Glu Arg Leu Lys Asn Leu Leu Lys Thr Val Val Lys Lys Ser Gln Asn Tyr Asn Ile Phe Gln Leu Glu Asn Leu Tyr Ala Val I1e Ser Gln Cys Ile Tyr Arg His Arg Lys Asp His Asp Lys Thr Ser Leu Ile Gln Lys Met Glu Gln Glu Val G1u Asn Phe Ser Cys Ser Arg <210> 5 <211> 280 <212> PRT
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte ID No: 7473124CD1 <400> 5 Met Pro Ile Tyr Ser Gln Thr Val Ala Met Ala Glu His Phe Lys Gln Ala Ser Ser Cys Pro Ile Cys Leu Asp Tyr Leu Glu Asn Pro Thr His Leu Lys Cys Gly Tyr Ile Cys Cys Leu Arg Cys Met Asn Ser Leu Arg Lys Gly Pro Asp G1y Lys Gly Val Leu Cys Pro Phe Cys Pro Val Val Ser Gln Lys Asn Asp Ile Arg Pro Ala Ala Gln Leu Gly Ala Leu Val Ser Lys Ile Lys Glu Leu Glu Pro Lys Val Arg Ala Val Leu Gln Met Asn Pro Arg Met Arg Lys Phe Gln Val Asp Met Thr Leu Asp Val Asp Thr Ala Asn Asn Asp Leu I1e Val Ser Glu Asp Leu Arg Arg Val Arg Cys Gly Asn Phe Arg Gln Asn Arg Lys Glu Gln Ala Glu Arg Phe Asp Thr Ala Leu Cys Val Leu Gly Thr Pro Arg Phe Thr Ser Gly Arg His Tyr Trp Glu Val Gly 155 160 l65 Val Gly Thr Ser Gln Val Trp Asp Val Gly Va1 Cys Lys Glu Ser Val Asn Arg Gln Gly Asn Val Val Leu Ser Ser Glu Leu Gly Phe Trp Thr Val Gly Leu Arg Gln Gly Gln Ile Tyr Phe Ala Ser Thr Lys Pro Val Thr Gly Leu Trp Val Ser Ser Gly Leu His Arg Va1 Gly Ile Tyr Leu Asp Ile Lys Thr Arg Ala Ile Ser Phe Tyr Asn Val Ser Asp Arg Ser His Ile Phe Thr Phe Thr Lys Ile Ser Ala Thr Glu Pro Leu Arg Pro Cys Phe Ala His Ala Asp Thr Ser Arg Asp Asp His Gly Tyr Leu Ser Val Cys Val <210> 6 <211> 221 <212> PRT
<213> Homo sapiens <220>
<221> misc_feature <223> Incyte ID No: 7473171CD1 <400> 6 Met Ala Ala Val Gly Pro Arg Thr Gly Pro Gly Thr Gly Ala Glu 1 5 l0 15 Ala Leu Ala Leu Ala Ala Glu Leu Gln Gly Glu Ala Thr Cys Ser 21e Cys Leu G1u Leu Phe Arg Glu Pro Val Ser Val Glu Cys Gly His Ser Phe Cys Arg Ala Cys Ile Gly Arg Cys Trp Glu Arg Pro Gly Ala Gly Ser Val Gly Ala Ala Thr Arg Ala Pro Pro Phe Pro Leu Pro Cys Pro Gln Cys Arg Glu Pro Ala Arg Pro Ser Gln Leu Arg Pro Asn Arg Gln Leu Ala A1a Val Ala Thr Leu Leu Arg Arg Phe Ser Leu Pro Ala Ala Ala Pro Gly Glu His G1y Ser Gln Ala 1l0 115 220 Ala Ala Ala Arg Ala Ala Ala Ala Arg Cys Gly Gln His Gly Glu Pro Phe Lys Leu Tyr Cys Gln Asp Asp Gly Arg Ala Ile Cys Val Val Cys Asp Arg Ala Arg Glu His Arg Glu His Ala Val Leu Pro Leu Asp Glu Ala Val Gln Glu Ala Lys Glu Leu Leu Glu Ser Arg Leu Arg Val Leu Lys Lys Glu Leu Glu Asp Cys Glu Val Phe Arg Ser Thr Glu Lys Lys Glu Ser Lys Glu Leu Leu Val Ser Gln Ala Pro Ala Gly Pro Pro Trp Asp Ile Thr Glu Ala <210> 7 <211> 668 <212> PRT
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte ID No: 7477026CD1 <400> 7 Met Met Val Glu Ser Ala Ser Glu Thr Ile Arg Ser Ala Pro Ser Gly Gln Asn Gly Val Gly Ser Leu Ser Gly Gln Ala Asp Gly Ser Ser Gly Gly Ala Thr Gly Thr Thr Ala Ser Gly Thr Gly Arg Glu Val Thr Thr Gly Ala Asp Ser Asn Gly G1u Met Ser Pro Ala Glu Leu Leu His Phe Gln G1n Gln Gln A1a Leu Gln Val Ala Arg Gln Phe Leu Leu Gln Gln Ala Ser Gly Leu Ser Ser Pro G1y Asn Asn Asp Ser Lys Gln Ser Ala Ser Ala Val Gln Val Pro Va1 Ser Val Ala Met Met Ser Pro Gln Met Leu Thr Pro Gln Gln Met Gln Gln Ile Leu Ser Pro Pro Gln Leu Gln Ala Leu Leu Gln G1n Gln Gln Ala Leu Met Leu Gln Gln Leu Gln Glu Tyr Tyr Lys Lys Gln Gln Glu G1n Leu His Leu G1n Leu Leu Thr Gln G1n Gln A1a Gly Lys Pro Gln Pro Lys Glu Ala Leu Gly Asn Lys G1n Leu Ala Phe Gln Gln Gln Leu Leu Gln Met Gln Gln Leu Gln Gln Gln His Leu Leu Asn Leu Gln Arg Gln Gly Leu Val Ser Leu Gln Pro Asn Gln Ala Ser Gly Pro Leu Gln Thr Leu Pro Gln Ala Ala Val Cys Pro Thr Asp Leu Pro G1n Leu Trp Lys Gly Glu Gly Ala Pro Gly Gln Pro Ala Glu Asp Ser Val Lys Gln Glu Gly Leu Asp~Leu Thr Gly Thr Ala Ala Thr Ala Thr Ser Phe Ala Ala Pro Pro Lys Val Ser Pro Pro Leu Ser His His Thr Leu Pro Asn Gly Gln Pro Thr Val Leu Thr Ser Arg Arg Asp Ser Ser Ser His Glu Glu Thr Pro Gly Ser His Pro Leu Tyr G1y His Gly Glu Cys Lys Trp Pro Gly Cys Glu Thr Leu Cys Glu Asp Leu G1y Gln Phe I1e Lys His Leu Asn Thr Glu His Ala Leu Asp Asp Arg Ser Thr A1a Gln Cys Arg Val GIn Met Gln Val Val Gln Gln Leu Glu Ile Gln Leu Ala Lys Glu Ser Glu Arg Leu Gln Ala Met Met Ala His Leu His Met Arg Pro Ser Glu Pro Lys Pro Phe Ser Gln Pro Val Thr Val Ser A1a Ala Asp Ser Phe Pro Asp Gly Leu Val His Pro Pro Thr Ser Ala Ala Ala Pro Val Thr Pro Leu Arg Pro Pro Gly Leu Gly Ser Ala Ser Leu His Gly Gly Gly Pro Ala Arg Arg Arg Ser Ser Asp Lys Phe Cys Ser Pro Ile Ser Ser G1u Leu Ala Gln Asn His Glu Phe Tyr Lys Asn Ala Asp Va1 Arg Pro Pro Phe Thr Tyr Ala Ser Leu Ile Arg Gln Ala Ile Leu Glu Thr Pro Asp Arg Gln Leu Thr Leu Asn Glu Tle Tyr Asn Trp Phe Thr Arg Met Phe Ala Tyr Phe Arg Arg Asn Thr Ala Thr Trp Lys Asn Ala Val Arg His Asn Leu Ser Leu His Lys Cys Phe Val Arg Val Glu Asn Val Lys Gly A1a Val Trp Thr Val Asp Glu Arg Glu Tyr Gln Lys Arg Arg Pro Pro Lys Met Thr Gly Ser Pro Thr Leu Val Lys Asn Met Ile Ser Gly Leu Ser Tyr Gly Ala Leu Asn Ala Ser Tyr Gln Ala Ala Leu Ala Glu Ser Ser Phe Pro Leu Leu Asn Ser Pro Gly Met Leu Asn Pro~Gly Ser Ala Ser Ser Leu Leu Pro Leu Ser His Asp Asp Val Gly AIa Pro Val Glu Pro Leu Pro Ser Asn G1y Ser Ser Ser Pro Pro Arg Leu Ser Pro Pro Gln Tyr Ser His G1n Val Gln Val Lys Glu Glu Pro Ala GIu Ala Glu GIu Asp Arg Gln Pro Gly Pro Pro Leu Gly Ala Pro Asn Pro Ser Ala Ser Gly Pro Pro Glu Asp Arg Asp Leu GIu Glu Glu Leu Pro Gly Glu Glu Leu Ser <210> 8 <211> 519 <212> PRT
<213> Homo sapiens <220>
<221> misc_feature <223> Incyte ID No: 6428773CD1 <400> 8 Met Asp Ala Val Leu GIu Pro Phe Pro AIa Asp Arg Leu Phe Pro 11!43 Gly Ser Ser Phe Leu Asp Leu Gly Asp Leu Asn Glu Ser Asp Phe Leu Asn Asn Ala His Phe Pro Glu His Leu Asp His Phe Thr Glu Asn Met G1u Asp Phe Ser Asn Asp Leu Phe Ser Ser Phe Phe Asp Asp Pro Val Leu Asp Glu Lys Ser Pro Leu Leu Asp Met G1u Leu Asp Ser Pro Thr Pro Gly Ile Gln Ala Glu His Ser Tyr Ser Leu Ser Gly Asp Ser Ala Pro Gln Ser Pro Leu Val Pro Ile Lys Met Glu Asp Thr Thr Gln Asp Ala Glu His Gly Ala Trp Ala Leu Gly His Lys Leu Cys Ser Ile Met Val Lys Gln Glu Gln Ser Pro Glu Leu Pro Val Asp Pro Leu Ala Ala Pro Ser Ala Met Ala Ala Ala Ala Ala Met A1a Thr Thr Pro Leu Leu Gly Leu Ser Pro Leu Ser Arg Leu Pro Ile Pro His Gln Ala Pro Gly Glu Met Thr G1n Leu Pro Val Ile Lys Ala Glu Pro Leu Glu Val Asn Gln Phe Leu Lys Val Thr Pro Glu Asp Leu Val Gln Met Pro Pro Thr Pro Pro Ser Ser His Gly Ser Asp Ser Asp G1y Ser Gln Ser Pro Arg Ser Leu Pro Pro Ser Ser Pro Val Arg Pro Met A1a Arg Ser Ser Thr Ala Ile Ser Thr Ser Pro Leu Leu Thr Ala Pro His Lys Leu Gln Gly Thr Ser Gly Pro Leu Leu Leu Thr Glu Glu Glu Lys Arg Thr Leu Ile Ala Glu Gly Tyr Pro Ile Pro Thr Lys Leu Pro Leu Thr Lys Ala Glu Glu Lys Ala Leu Lys Arg Val Arg Arg Lys Ile Lys Asn Lys Ile Ser Ala G1n Glu Ser Arg Arg Lys Lys Lys Glu Tyr Val Glu Cys Leu Glu Lys Lys Val Glu Thr Phe Thr Ser Glu Asn Asn Glu Leu Trp Lys Lys Val Glu Thr Leu Glu Asn Ala Asn Arg Thr Leu Leu Gln Gln Leu G1n Lys Leu Gln Thr Leu Val Thr Asn Lys Ile Ser Arg Pro Tyr Lys Met Ala Ala Thr Gln Thr Gly Thr Cys Leu Met Val Ala Ala Leu Cys Phe Val Leu Val Leu Gly Ser Leu Val Pro Cys Leu Pro Glu Phe Ser Ser Gly Ser Gln Thr Val Lys Glu Asp Pro Leu Ala Ala Asp Gly Val Tyr Thr Ala Ser Gln Met Pro Ser Arg Ser Leu Leu Phe Tyr Asp Asp Gly Ala Gly Leu Trp Glu Asp Gly Arg Ser Thr Leu Leu Pro Met Glu Pro Pro Asp Gly Trp Glu Ile Asn Pro Gly Gly Pro Ala G1u Gln Arg Pro Arg Asp His Leu Gln His Asp His Leu Asp Ser Thr His Glu Thr Thr Lys Tyr Leu Ser Glu Ala Trp Pro Lys Asp Gly Gly Asn Gly Thr Ser Pro Asp Phe Ser His Ser Lys Glu Trp Phe His Asp Arg Asp Leu Gly Pro Asn Thr Thr Ile Lys Leu Ser <210> 9 <211> 1256 <212> PRT
<213> Homo sapiens <220>
<221> misc_feature <223> Incyte ID No: 2749402CD1 <400> 9 Met Leu Phe Lys Leu Leu Gln Arg Gln Thr Tyr Thr Cys Leu Ser His Arg Tyr Gly Leu Tyr Val Cys Phe Leu Gly Val Val Val Thr Ile Val Ser Ala Phe Gln Phe Gly Glu Val Val Leu Glu Trp Ser Arg Asp Gln Tyr His Val Leu Phe Asp Ser Tyr Arg Asp Asn Ile Ala Gly Lys Ser Phe Gln Asn Arg Leu Cys Leu Pro Met Pro Ile Asp Val Val Tyr Thr Trp Val Asn Gly Thr Asp Leu Glu Leu Leu Lys Glu Leu Gln Gln Val Arg Glu Gln Met Glu Glu Glu Gln Lys Ala Met Arg Glu I1e Leu Gly Lys Asn Thr Thr Glu Pro Thr Lys Lys Ser Glu Lys Gln Leu Glu Cys Leu Leu Thr His Cys Ile Lys 125 ' 130 135 Va1 Pro Met Leu Val Leu Asp Pro Ala Leu Pro A1a Asn Ile Thr Leu Lys Asp Leu Pro Ser Leu Tyr Pro Ser Phe His Ser Ala Ser Asp Ile Phe Asn Val A1a Lys Pro Lys Asn Pro Ser Thr Asn Val Ser Val Val Val Phe Asp Ser Thr Lys Asp Val Glu Asp Ala His Ser Gly Leu Leu Lys Gly Asn Ser Arg Gln Thr Val Trp Arg Gly Tyr Leu Thr Thr Asp Lys Glu Val Pro Gly Leu Val Leu Met Gln Asp Leu Ala Phe Leu Ser Gly Phe Pro Pro Thr Phe Lys Glu Thr Asn Gln Leu Lys Thr Lys Leu Pro Glu Asn Leu Ser Ser Lys Val Lys Leu Leu Gln Leu Tyr Ser Glu Ala Ser Val Ala Leu Leu Lys Leu Asn Asn Pro Lys Asp Phe Gln Glu Leu Asn Lys Gln Thr Lys Lys Asn Met Thr Ile Asp Gly Lys Glu Leu Thr Ile Ser Pro Ala Tyr Leu Leu Trp Asp Leu Ser Ala Ile Ser Gln Ser Lys Gln Asp G1u Asp Ile Ser Ala Ser Arg Phe Glu Asp Asn Glu Glu Leu Arg Tyr Ser Leu Arg Ser Ile Glu Arg His Ala Pro Trp Val Arg Asn Ile Phe Ile Val Thr Asn Gly Gln Ile Pro Ser Trp Leu Asn Leu Asp Asn Pro Arg Val Thr Ile Val Thr His Gln Asp Val Phe Arg Asn Leu Ser His Leu Pro Thr Phe Ser Ser Pro Ala Ile Glu Ser His Ile His Arg Ile Glu Gly Leu Ser G1n Lys Phe Tle Tyr Leu Asn Asp Asp Val Met Phe Gly Lys Asp Val Trp Pro Asp Asp Phe Tyr Ser His Ser Lys Gly Gln Lys Val Tyr Leu Thr Trp Pro Val Pro Asn Cys Ala Glu Gly Cys Pro Gly Ser Trp Ile Lys Asp Gly Tyr Cys Asp Lys Ala Cys Asn Asn Sex A1a Cys Asp Trp Asp Gly Gly Asp Cys Ser Gly Asn Ser Gly Gly Ser Arg Tyr Ile Ala Gly Gly Gly Gly Thr Gly Ser Ile Gly Val Gly Gln Pro Trp Gln Phe Gly Gly Gly Ile Asn Ser Val Ser Tyr Cys Asn Gln G1y Cys AIa Asn Ser Trp Leu Ala Asp Lys Phe Cys Asp Gln Ala Cys Asn Val Leu Ser Cys Gly Phe Asp Ala G1y Asp Cys Gly Gln Asp His Phe His Glu Leu Tyr Lys Val Ile Leu Leu Pro Asn Gln Thr His Tyr Ile Ile Pro Lys Gly Glu Cys Leu Pro Tyr Phe Ser Phe Ala Glu Val Ala Lys Arg Gly Val Glu G1y Ala Tyr Ser Asp Asn Pro Ile Ile Arg His Ala Ser Ile Ala Asn Lys Trp Lys Thr Ile His Leu Ile Met His Ser Gly Met Asn Ala Thr Thr Ile His Phe Asn Leu Thr Phe Gln Asn Thr Asn Asp Glu Glu Phe Lys Met Gln IIe Thr Val Glu Val Asp Thr Arg Glu G1y Pro Lys Leu Asn Ser Thr Ala Gln Lys Gly Tyr Glu Asn Leu Val Ser Pro Ile Thr Leu Leu Pro Glu Ala G1u Tle Leu Phe Glu Asp Ile Pro Lys Glu Lys Arg Phe Pro Lys Phe Lys Arg His Asp Val Asn Ser Thr Arg Arg Ala Gln Glu Glu Val Lys Ile Pro Leu Val Asn Ile Ser Leu Leu Pro Lys Asp A1a Gln Leu Ser Leu Asn Thr Leu Asp Leu Gln Leu Glu His Gly Asp Ile Thr Leu Lys Gly Tyr Asn Leu Ser Lys Ser Ala Leu Leu Arg Ser Phe Leu Met Asn Ser Gln His Ala Lys I1e Lys Asn Gln Ala Ile Ile Thr Asp Glu Thr Asn Asp Ser Leu Val Ala Pro Gln Glu Lys Gln Val His Lys Ser Ile Leu Pro Asn Ser Leu Gly Val Ser Glu Arg Leu Gln Arg Leu Thr Phe Pro Ala Val Ser Val Lys Va1 Asn Gly His Asp Gln Gly Gln Asn Pro Pro Leu Asp Leu Glu Thr Thr Ala Arg Phe Arg Va1 Glu Thr His Thr Gln Lys Thr WO 02/50279 ~~. PCT/USO1/50256 Ile Gly Gly Asn Val Thr Lys Glu Lys Pro Pro Ser Leu Ile Val Pro Leu Glu Ser Gln Met Thr Lys Glu Lys Lys Ile Thr Gly Lys Glu Lys Glu Asn Ser Arg Met Glu Glu Asn Ala Glu Asn His Ile Gly Va1 Thr Glu Val Leu Leu Gly Arg Lys Leu Gln His Tyr Thr Asp Ser Tyr Leu Gly Phe Leu Pro Trp Glu Lys Lys Lys Tyr Phe Gln Asp Leu Leu Asp Glu Glu Glu Ser Leu Lys Thr Gln Leu Ala Tyr Phe Thr Asp Ser Lys Asn Thr Gly Arg Gln Leu Lys Asp Thr Phe Ala Asp Ser Leu Arg Tyr Val Asn Lys Ile Leu Asn Ser Lys Phe Gly Phe Thr Ser Arg Lys Val Pro Ala His Met Pro His Met Ile Asp Arg Ile VaI Met Gln GIu Leu Gln Asp Met Phe Pro Glu Glu Phe Asp Lys Thr Ser Phe His Lys Val Arg His Ser Glu Asp Met Gln Phe Ala Phe Ser Tyr Phe Tyr Tyr Leu Met Ser A1a Val Gln Pro Leu Asn Ile Ser Gln Val Phe Asp Glu Val Asp Thr Asp Gln Ser Gly Val Leu Ser Asp Arg Glu Ile Arg Thr Leu Ala Thr Arg Ile His Glu Leu Pro Leu Ser Leu Gln Asp Leu Thr Gly Leu Glu His Met Leu Ile Asn Cys Ser Lys Met Leu Pro Ala Asp Ile Thr Gln Leu Asn Asn Ile Pro Pro Thr Gln Glu Ser Tyr Tyr Asp Pro Asn Leu Pro Pro Val Thr Lys Ser Leu Val Thr Asn Cys Lys Pro Val Thr Asp Lys Ile His Lys Ala Tyr Lys Asp Lys Asn Lys Tyr Arg Phe Glu Ile Met Gly Glu Glu Glu Ile Ala Phe Lys Met Ile Arg Thr Asn Val Ser His Val Val Gly Gln Leu Asp Asp Ile Arg Lys Asn Pro Arg Lys Phe Val Cys Leu Asn Asp Asn 21e Asp His Asn His Lys Asp Ala Gln Thr Val Lys Ala Val Leu Arg Asp Phe Tyr Glu Ser Met Phe Pro Ile Pro Ser Gln Phe Glu Leu Pro Arg Glu Tyr Arg Asn Arg Phe Leu His Met His Glu Leu Gln Glu Trp Arg Ala Tyr Arg Asp Lys Leu Lys Phe Trp Thr His Cys Val Leu Ala Thr Leu Ile Met Phe Thr Ile Phe Ser Phe Phe Ala G1u Gln Leu Ile Ala Leu Lys Arg Lys Ile Phe Pro Arg Arg Arg Ile 1235 ~ 1240 1245 His Lys Glu Ala Ser Pro Asn Arg Ile Arg Val <210> 10 <212> 364 <212> PRT
<213> Homo sapiens 15!43 <220>
<221> misc_feature <223> Incyte ID No: 118539CD1 <400> 10 Met Glu His Asn Gly Ser Ala Ser Asn Ala Asp Lys Ile His Gln Asn Arg Leu Ser Ser Val Thr Glu Asp Glu Asp G1n Asp Ala Ala Leu Thr Ile Val Thr Va1 Leu Asp Lys Val Ala Ser Ile Val Asp Ser Va1 GIn Ala Ser Gln Lys Arg Ile Glu Glu Arg His Arg GIu Met Glu Asn Ala Ile Lys Ser Val Gln Ile Asp Leu Leu Lys Leu Ser Gln Ser His Ser Asn Thr Gly His Ile Ile Asn Lys Leu Phe Glu Lys Thr Arg Lys Va1 Ser Ala His Ile Lys Asp Val Lys Ala Arg Val Glu Lys Gln Gln Ile His Val Lys Lys Val Glu Val Lys Gln Glu Glu I1e Met Lys Lys Asn Lys Phe Arg Val Val Ile Phe Gln Glu Lys Phe Arg Cys Pro Thr Ser Leu Ser Val Val Lys Asp Arg Asn Leu Thr Glu Asn Gln Glu Glu Asp Asp Asp Asp Ile Phe Asp Pro Pro Val Asp Leu Ser Ser Asp Glu Glu Tyr Tyr Val Glu Glu Ser Arg Ser Ala Arg Leu Arg Lys Ser Gly Lys Glu His Ile Asp Asn Ile Lys Lys Ala Phe Ser Lys Glu Asn Met Gln Lys Thr Arg Gln Asn Leu Asp Lys Lys Val Asn Arg Ile Arg Thr Arg Ile 215 " 220 225 Val Thr Pro Glu Arg Arg Glu Arg Leu Arg Gln Ser Gly Glu Arg Leu Arg Gln Ser Gly Glu Arg Leu Arg Gln Ser Gly Glu Arg Phe Lys Lys Ser Ile Ser Asn Ala Ala Pro Ser Lys Glu Ala Phe Lys Met Arg Ser Leu Arg Lys Gly Lys Asp Arg Thr Val Ala Glu Gly Glu Glu Cys Ala Arg Glu Met Gly Val Asp I1e Ile Ala Arg Ser Glu Ser Leu Gly Pro Ile Ser Glu Leu Tyr Ser Asp Glu Leu Ser Gl.u Pro Glu His Glu Ala Ala Arg Pro Val Tyr Pro Pro His Glu Gly Arg Glu I1e Pro Thr Pro Glu Pro Leu Lys Val Thr Phe Lys Ser Gln Val Lys Val Glu Asp Asp Glu Ser Leu Leu Leu Asp Leu Lys His Ser Ser <210> 11 <211> 331 <212> PRT
<213> Homo Sapiens <220>
<221> misc feature <223> Incyte ID No: 4005918CD1 <400> 11 Met Thr Asp Pro Ser Leu Gly Leu Thr Val Pro Met Ala' Pro Pro Leu Ala Pro Leu Pro Pro Arg Asp Pro Asn Gly Ala Gly Ser Glu Trp Arg Lys Pro Gly Ala Va1 Ser Phe Ala Asp Val Ala Val Tyr Phe Ser Arg Glu Glu Trp Gly Cys Leu Arg Pro Ala Gln Arg Ala Leu Tyr Arg Asp Val Met Arg Glu Thr Tyr G1y His Leu Gly Ala Leu GIy Val GIy Gly Ser Lys Pro Ala Leu Ile Ser Trp Val Glu Glu Lys Ala Glu Leu Trp Asp Pro Ala Ala Gln Asp Pro Glu Val Ala Lys Cys Pro Thr Glu Ala Asp Pro AIa Asp Ser Arg Asn Lys Glu Glu Glu Arg Gln Arg Glu Gly Thr Gly Ala Leu Glu Lys Pro Asp Pro Val Ala Ala Gly Ser Pro Gly Leu Lys Ala Pro Gln Ala Pro Phe Ala Gly Leu Glu Gln Leu Ser Lys Ala Arg Arg Arg Ser Arg Pro Arg Phe Phe Ala His Pro Pro Val Pro Arg Ala Asp Gln Arg His Gly Cys Tyr Val Cys Gly Lys Ser Phe Ala Trp Arg Ser Thr Leu Val Glu His Ile Tyr Ser His Arg Gly Glu Lys Pro Phe His Cys Ala Asp Cys Gly Lys Gly Phe Gly His A1a Ser Ser Leu Ser Lys His Arg Ala Ile His Arg Gly Glu Arg Pro His Arg Cys Pro Glu Cys Gly Arg Ala Phe Met Arg Arg Thr Ala Leu Thr Ser His Leu Arg Val His Thr Gly Glu Lys Pro Tyr Arg Cys Pro Gln Cys Gly Arg Cys Phe Gly Leu Lys Thr Gly Met Ala Lys His Gln Trp Val His Arg Pro Gly Gly G1u Gly Arg Arg Gly Arg Arg Pro Gly G1y Leu Ser Val Thr Leu Thr Pro Val Arg Gly Asp Leu Asp Pro Pro Val Gly Phe Gln Leu Tyr Pro Glu Ile Phe Gln Glu Cys Gly <210> 12 <211> 670 <212> PRT
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte ID No: 5435937CD1 <400> 12 Met Asp Ser Val Ala Phe Glu Asp Val Ala Val Thr Phe Thr Gln Glu G1u Trp A1a Leu Leu Asp Pro Ser Gln Lys Asn Leu Cys Arg Asp Val Met Gln Glu Thr Phe Arg Asn Leu Ala Ser Ile Gly Lys Lys Trp Lys Pro Gln Asn Ile Tyr Val Glu Tyr Glu Asn Leu Arg Arg Asn Leu Arg Ile Va1 Gly Glu Arg Leu Phe Glu Ser Lys Glu Gly His Gln His Gly Glu Ile Leu Thr Gln Va1 Pro Asp Asp Met Leu Lys Lys Thr Thr Thr Gly Val Lys Ser Cys Glu Ser Ser Val Tyr Gly G1u Val Gly Ser Ala His Ser Ser Leu Asn Arg His Ile Arg Asp Asp Thr Gly His Lys Ala Tyr Glu Tyr Gln Glu Tyr Gly Gln Lys Pro Tyr Lys Cys Lys Tyr Cys Lys Lys Pro Phe Asn Cys Leu Ser Ser Val Gln Thr His Glu Arg Ala His Ser Gly Arg Lys Leu Tyr Val Cys Glu Glu Cys Gly Lys Thr Phe Ile Ser His Ser Asn Leu Gln Arg His Arg Ile Met His Arg Gly Asp Gly Pro Tyr Lys Cys Lys Phe Cys Gly Lys Ala Leu Met Phe Leu Ser Leu Tyr Leu Ile His Lys Arg Thr His Thr Gly Glu Lys Pro Tyr G1n Cys Lys Gln Cys Gly Lys Ala Phe Ser His Ser Ser Ser Leu Arg Ile His Glu Arg Thr His Thr Gly Glu Lys Pro Tyr Lys Cys Asn Glu Cys Gly Lys Ala Phe His Ser Ser Thr Cys Leu His Ala His Lys Arg Thr His Thr Gly Glu Lys Pro Tyr Glu Cys Lys Gln Cys Gly Lys Ala Phe Ser Ser Ser His Ser Phe Gln Ile His Glu Arg Thr His Thr Gly Glu Lys Pro Tyr G1u Cys Lys Glu Cys Gly Lys Ala Phe Lys Cys Pro Ser Ser Val Arg Arg His Glu Arg Thr His Ser Arg Lys Lys Pro Tyr Glu Cys Lys His Cys Gly Lys Val Leu Ser Tyr Leu Thr Ser Phe Gln Asn His Leu Gly Met His Thr Gly Glu Ile Ser His Lys Cys Lys I1e Cys Gly Lys Ala Phe Tyr Ser Pro Ser Ser Leu Gln Thr His Glu Lys Thr His Thr Gly G1u Lys Pro Tyr Lys Cys Asn Gln Cys Gly Lys Ala Phe Asn Ser Ser Ser Ser Phe Arg Tyr His Glu Arg Thr His Thr Gly Glu Lys Pro Tyr G1u Cys Lys Gln Cys Gly Lys Ala Phe Arg Ser Ala Ser Leu Leu Gln Thr His Gly Arg Thr His Thr Gly Glu Lys Pro Tyr Ala Cys Lys 440 445. 450 Glu Cys G1y Lys Pro Phe Ser Asn Phe Ser Phe Phe Gln Ile His Glu Arg Met His Arg Glu Glu Lys Pro Tyr Glu Cys Lys Gly Tyr Gly Lys Thr Phe Ser Leu Pro Ser Leu Phe His Arg His Glu Arg Thr His Thr Gly Gly Lys Thr Tyr Glu Cys Lys Gln Cys Gly Arg Ser Phe Asn Cys Ser Ser Ser Phe Arg Tyr His Gly Arg Thr His Thr Gly Glu Lys Pro Tyr Glu Cys Lys Gln Cys Gly Lys Ala Phe Arg Ser Ala Ser Gln Leu Gln Ile His G1y Arg Thr His Thr G1y Glu Lys Pro Tyr Glu Cys Lys Gln Cys Gly Lys Ala Phe Gly Ser Ala Ser His Leu Gln Met His Gly Arg Thr His Thr G1y Glu Lys Pro Tyr Glu Cys Lys Gln Cys Gly Lys Ser Phe Gly Cys Ala Ser Arg Leu Gln Met His Gly Arg Thr His Thr Gly Glu Lys Pro Tyr Lys Cys Lys Gln Cys Gly Lys Ala Phe G1y Cys Pro Ser Asn Leu Arg Arg His Gly Arg Thr His Thr Gly Glu Lys Pro Tyr Lys Cys Asn Gln Cys Gly Lys Val Phe Arg Cys Ser Ser Gln Leu G1n Val His Gly Arg Ala His Cys Ile Asp Thr Pro <210> 13 <211> 1196 <212> PRT
<213> Homo sapiens <220>
<221> misc_feature <223> Incyte ID No: 7503560CD1 <400> 13 Met Leu Phe Lys Leu Leu G1n Arg Gln Thr Tyr Thr Cys Leu Ser His Arg Tyr Gly Leu Tyr Val Cys Phe Leu Gly Val Val Val Thr Ile Val Ser Ala Phe Gln Phe Gly Glu Val Val Leu Glu Trp Ser Arg Asp Gln Tyr His Val Leu Phe Asp Ser Tyr Arg Asp Asn Ile Ala Gly Lys Ser Phe Gln Asn Arg Leu Cys Leu Pro Met Pro Ile Asp Val Va1 Tyr Thr Trp Val Asn Gly Thr Asp Leu Glu Leu Leu Lys Glu Leu Gln Gln Val Arg Glu Gln Met Glu G1u Glu Gln Lys Ala Met Arg Glu Ile Leu Gly Lys Asn Thr Thr Glu Pro Thr Lys Lys Ser Glu Lys Gln Leu Glu Cys Leu Leu Thr His Cys Ile Lys Val Pro Met Leu Val Leu Asp Pro Ala Leu Pro Ala Asn Ile Thr Leu Lys Asp Leu Pro Ser Leu Tyr Pro Ser Phe His Ser Ala Ser Asp Ile Phe Asn Val Ala Lys Pro Lys Asn Pro Ser Thr Asn Val Ser Val Val Val Phe Asp Ser Thr Lys Asp Val Glu Asp Ala His Ser Gly Leu Leu Lys Gly Asn Ser Arg Gln Thr Val Trp Arg Gly Tyr Leu Thr Thr Asp Lys Glu Val Pro Gly Leu Val Leu Met Gln Asp Leu Ala Phe Leu Ser Gly Phe Pro Pro Thr Phe Lys Glu Thr Asn Gln Leu Lys Thr Lys Leu Pro Glu Asn Leu Ser Ser Lys Val Lys Leu Leu Gln Leu Tyr Ser Glu Ala Ser Val Ala Leu Leu Lys Leu Asn Asn Pro Lys Asp Phe Gln Glu Leu Asn Lys Gln Thr Lys Lys Asn Met Thr Ile Asp Gly Lys Glu Leu Thr Ile Ser Pro Ala Tyr Leu Leu Trp Asp Leu Ser Ala Ile Ser Gln Ser Lys G1n Asp 305 310 . 315 Glu Asp Ile Ser Ala Ser Arg Phe Glu Asp Asn Glu Glu Leu Arg Tyr Ser Leu Arg Ser Ile Glu Arg His Ala Pro Trp Val Arg Asn Ile Phe Ile Val Thr Asn G1y Gln Ile Pro Ser Trp Leu Asn Leu Asp Asn Pro Arg Val Thr Ile Val Thr His Gln Asp Val Phe Arg Asn Leu Ser His Leu Pro Thr Phe Ser Ser Pro Ala Ile Glu Ser His Ile His Arg Ile Glu Gly Leu Ser Gln Lys Phe Ile Tyr Leu Asn Asp Asp Val Met Phe Gly Lys Asp Val Trp Pro Asp Asp Phe Tyr Ser His Ser Lys Gly Gln Lys Val Tyr Leu Thr Trp Pro Phe Gly Gly Gly Ile Asn Ser Val Ser Tyr Cys Asn Gln Gly Cys Ala Asn Ser Trp Leu Ala Asp Lys Phe Cys Asp Gln Ala Cys Asn Val Leu Ser Cys Gly Phe Asp Ala Gly Asp Cys Gly Gln Asp His Phe His Glu Leu Tyr Lys Val Ile Leu Leu Pro Asn Gln Thr His Tyr Ile Ile Pro Lys Gly Glu Cys Leu Pro Tyr Phe Ser Phe Ala Glu Val Ala Lys Arg Gly Val Glu Gly Ala Tyr Ser Asp Asn Pro Ile Ile Arg His Ala Ser Ile Ala Asn Lys Trp Lys Thr Ile His Leu Ile Met His Ser Gly Met Asn Ala Thr Thr Ile His Phe Asn Leu Thr Phe Gln Asn Thr Asn Asp Glu Glu Phe Lys Met Gln Ile Thr Val Glu Val Asp Thr Arg Glu Gly Pro Lys Leu Asn Ser Thr Ala Gln Lys Gly Tyr G1u Asn Leu Val Ser Pro Ile Thr Leu Leu Pro Glu Ala Glu Ile Leu Phe Glu Asp Ile Pro Lys Glu Lys Arg Phe Pro Lys Phe Lys Arg His Asp Val Asn Ser Thr Arg Arg Ala Gln Glu G1u Val Lys Ile Pro Leu Val Asn Ile Ser Leu Leu Pro Lys Asp Ala Gln Leu Ser Leu Asn Thr Leu Asp Leu Gln Leu Glu His Gly Asp Ile Thr Leu Lys Gly Tyr Asn Leu Ser Lys Ser Ala Leu Leu Arg Ser Phe Leu Met Asn Ser Gln His Ala Lys Ile Lys Asn Gln A1a Ile Ile Thr Asp Glu Thr Asn Asp Ser Leu Val Ala Pro Gln Glu Lys Gln Val His Lys Ser Ile Leu Pro Asn Ser Leu Gly Val Ser Glu Arg Leu Gln Arg Leu Thr Phe Pro Ala Val Ser Val Lys Val Asn Gly His Asp Gln GIy Gln Asn Pro Pro Leu Asp Leu Glu Thr Thr Ala Arg Phe Arg Val Glu Thr His Thr Gln Lys Thr Ile G1y Gly Asn Val Thr Lys Glu Lys Pro Pro Ser Leu Ile Val Pro Leu Glu Ser Gln Met Thr Lys Glu Lys Lys Ile Thr G1y Lys Glu Lys Glu Asn Ser Arg Met Glu Glu Asn Ala Glu Asn His Ile Gly Val Thr Glu Val Leu Leu Gly Arg Lys Leu Gln His Tyr Thr Asp Ser Tyr Leu Gly Phe Leu Pro Trp Glu Lys Lys Lys Tyr Phe G1n Asp Leu Leu Asp Glu Glu Glu Ser Leu Lys Thr Gln Leu Ala Tyr Phe Thr Asp Ser Lys Asn Thr G1y Arg Gln Leu Lys Asp Thr Phe Ala Asp Ser Leu Arg Tyr Val Asn Lys Ile Leu Asn Ser Lys Phe Gly Phe Thr Ser Arg Lys Val Pro Ala His Met Pro His Met Ile Asp Arg Ile Val Met Gln Glu Leu Gln Asp Met Phe Pro Glu 905 910 ' 915 Glu Phe Asp Lys Thr Ser Phe His Lys Val Arg His Ser Glu Asp Met Gln Phe Ala Phe Ser Tyr Phe Tyr Tyr Leu Met Ser Ala Val Gln Pro Leu Asn Ile Ser Gln Val Phe Asp Glu Val Asp Thr Asp Gln Ser Gly Val Leu Ser Asp Arg Glu Ile Arg Thr Leu Ala ,Thr Arg Ile His Glu Leu Pro Leu Ser Leu Gln Asp Leu Thr Gly Leu G1u His Met Leu Ile Asn Cys Ser Lys Met Leu Pro Ala Asp Ile Thr Gln Leu Asn Asn Ile Pro Pro Thr Gln Glu Ser Tyr Tyr Asp Pro Asn Leu Pro Pro Val Thr Lys Ser Leu Val Thr Asn Cys Lys Pro Val Thr Asp Lys Ile His Lys Ala Tyr Lys Asp Lys Asn Lys Tyr Arg Phe Glu Ile Met Gly Glu Glu Glu Ile Ala Phe Lys Met Ile Arg Thr Asn Val Ser His Val Val Gly G1n Leu Asp Asp Ile Arg Lys Asn Pro Arg Lys Phe Val Cys Leu Asn Asp Asn Ile Asp His Asn His Lys Asp Ala Gln Thr Val Lys Ala Val Leu Arg Asp Phe Tyr Glu Ser Met Phe Pro Ile Pro Ser Gln Phe Glu Leu Pro Arg Glu Tyr Arg Asn Arg Phe Leu His Met His Glu Leu Gln G1u Trp Arg Ala Tyr Arg Asp Lys Leu Lys Phe Trp Thr His Cys Val Leu Ala Thr Leu Ile Met Phe Thr Ile Phe Ser Phe Phe Ala Glu G1n Leu Ile Ala Leu Lys Arg Lys Ile Phe Pro Arg Arg Arg Ile His Lys Glu Ala Ser Pro Asn Arg Ile Arg Val <210> 14 <211> 2138 <212> PRT
<213> Homo sapiens <220>
<221> misc_feature <223> Incyte ID No: 7472378CD1 <400> 14 Met Ser Lys Lys Phe Val Tyr Asn Leu Arg Lys Thr Thr Arg Ser Val Val Gly Val Pro Pro Asn Thr Asn Arg Pro Pro His Pro Val Arg Arg Pro Asp Ser Leu Leu Pro Ile Ser Glu Glu Pro Lys Ser 35 . 40 45 Ile Ser Ser Gln Thr Pro Asn Met Asp Ser Gly Asn Asp Ser Ala Arg Pro Thr Pro Ser Pro Leu Ala Pro Thr Val Ser Gly Ile Ser Ser Leu Tle Ser Thr Thr Phe Lys Pro Lys Asp Ile Met Ala Phe Val Glu His Leu Pro Thr Phe Asp Gly Thr Pro Arg Leu Leu Asp Arg Phe Ile Thr Ser Val Glu Glu Ile Leu Met Leu Ile Arg Gly Ala Asp Gln Thr Pro Tyr Gly Leu Leu Thr Leu Arg Thr Ile Arg Asn Lys Tle Ile Asp Arg Ala Asp Glu Ala Leu Glu Leu Ala Asn Thr Pro Leu Val Trp Asp G1u Ile Lys Ser Asn Leu Ile Arg Leu Tyr Ser Ser Lys Lys Ser Glu Ala Asn Leu Leu Ser Glu Leu Asn Thr Phe Ser Asp Asn Leu Thr Leu Gly Gln Leu Phe Phe Gly I1e Ser Lys Va1 Arg Ser Gln Leu Phe Ser Ile Leu Lys Asn Ser Glu His Asn Asn Thr Val Val Asp Ala Lys Lys Va1 Val Tyr Asn Glu Val Cys Leu Asn Ala Phe Met Thr Gly Leu Lys Glu Pro Leu Lys Thr Phe Val Arg Ile Lys Ser Pro Ser Thr Leu Glu Gln Ala Tyr Glu Gln Cys Gln Ile Glu Gln Thr Leu Tyr Arg Ala Gln Asn Lys Arg Thr Asn Arg Pro Glu Gln Gly Pro Asn Gly Ser Asp Asn Lys Thr Tyr Arg Asn Ser Tyr Asp Ser Asn Tyr Arg Ser Gly Arg Asn Asp Arg Asn Asp Arg Arg Gly Pro Tyr Ser Asn Ser Asn Ser Asn Ser Asn Ser G1y Gln Asn Arg Pro Phe Asn Ser His Asn Arg Thr Pro Gln Ser Gly Thr Lys Asp Asn Arg A1a Asn Thr Sex Asn Pro Phe Arg Ala Pro Ser His Ser Leu Asn Asn Ile Glu Glu Asn Pro Gln Pro Asp Ser Asn Phe Gln Gln Thr A1a Ser Gly Asn Gln Gln Gly Ser Thr His Ser Phe Ile Asp Pro Lys Tyr Val Asp Pro Arg Asn Cys Val Thr Leu Asp Thr Pro Ile Thr Leu Lys Thr Ala Leu Asn Ser Phe Lys Ile Tyr Gln Asn Val Ser Ile Pro Phe Pro Pro Glu Phe G1n Ile Thr Gly Lys Met Thr Leu Leu Pro Phe Lys Phe His Ser Tyr Phe Asp G1y Leu Ile Gly Met Asp Leu Leu Ser Tyr Leu Lys Thr Glu Ile Asp Leu Leu Asn Leu Asn Leu Lys Thr Pro Ser Thr I1e Ile Pro Leu Trp Thr His Ser Asn Ser Thr Ser Asn Val Phe Asn Ile Ser Gly His ~inr Lys Thr Ile Leu Pro Leu Pro Val Glu Thr Lys Gln Gly Asp Phe Tyr Ile Asp Ser Ile Thr Ile Asn Asp Asp Leu Ile Ile Ser Asp Gly Ile Tyr Asn Ala Gln Asn Asn Ile Ala Asn Phe Val Ile Thr Asn Tyr Ser Glu Arg Asp Gln Leu Leu Tyr Leu Glu Ser Pro Ile Lys Gly Met Pro Tyr Ser Thr Ala Asn Asn Val Glu Leu Phe Ser Ile Thr Ser Asp Thr Pro Gln Pro Gln Asn Ser A1a Ala Ser Leu G1n Ala Leu Gly Va1 Asp His Leu Ser Ser Glu Glu Lys Gln Ser Leu Leu Ser Leu Cys Lys Ser Tyr Leu Asp Ile Phe Tyr Asn Glu Asp Lys Ser Leu Thr Phe Thr Asn Lys Ile Thr His Thr Ile Lys Thr Thr Asp Asp Thr Pro Ile His Thr Lys Ser Tyr Arg Tyr Pro Tyr Ile His Lys Glu Glu Val Lys Lys Gln Ile Glu Ala Met Leu Asn Gln Asp Ile Ile Lys Ser Ser Tyr Ser Pro Trp Ser Ala Pro Val Trp Val Val Pro Lys Lys Ile Thr Pro Thr Gly Glu Gln Lys Trp Arg Leu Val Ile Asp Tyr Arg Lys Leu Asn Glu Lys Thr Ile Ser Asp Arg Tyr Pro Ile Pro Asn Ile Ala Asp Ile Leu Asp Arg Leu Gly Lys Ala Lys Tyr Phe Ser Thr Leu Asp Leu Ala Ser Gly Phe His Gln Ile Glu Met Asn Pro Asp Asp Thr Pro Lys Thr Ala Phe Thr Val Glu Gly Gly His Tyr Glu Phe Ile Arg Met Pro Phe Gly Leu Lys Asn Ala Pro Ala Thr Phe Gln Arg Val Met Asp Asn Ile Phe Gly Asp Leu Ile Gly Thr Ile Cys Leu Val Tyr Leu Asp Asp Ile Ile Ile Phe Ser Thr Ser Leu Gln Glu His Phe Ile His Leu Lys Thr Ile Phe Gly Arg Leu Arg Ser Ala Asn Phe Lys Val Gln Leu Thr Lys Ser Tyr Phe Leu Arg Arg Glu Thr Glu Phe Leu G1y His Ile Val Ser Gln Glu Gly Val Arg Pro Asn Pro Asn Lys Ile Glu Ala Ile Lys Asn Phe Pro Cys Pro His Ser Lys Lys Ser Ile Lys Ser Phe Leu Gly Leu Leu Gly Tyr Tyr Arg Lys Phe Ile Arg Asp Phe Ala Arg Leu Thr Gln Pro Met Thr G1n Lys Leu Arg Gly Asn Asn Lys Ser Ile Ile Ile Asp Asp Glu Phe Lys Lys A1a Phe Glu Tyr Cys Lys Thr Leu Leu Ser Asn Asp Pro Ile Leu Gln Tyr Pro Asp Phe Thr Lys Pro Phe Thr Leu Thr Thr Asp Ala Ser Asn Phe Ala Ile Gly A1a Val Leu Ser Gln Gly Pro Val His Ser Asp Arg Pro Val Cys Phe Ala Ser Arg Thr Leu Ser Ala Ala Glu Thr Asn Tyr Ser Thr Ile Glu Lys Glu Met Leu Ala Ile Ile Trp Ala Val Gln Tyr Phe Arg Pro Tyr Leu Phe Gly Arg Arg Phe Thr Ile Ile Thr Asp His Lys Pro Leu Thr Trp Leu Met Asn Phe Lys Gln Pro Asn Ser Lys Ile Val Arg Trp Arg Leu Gln Leu Gln Glu Tyr Asp Phe Glu Val Val Tyr Lys Lys Gly Ser Gln Asn Val Ile Ala Asp Ala Leu Ser Arg Pro Glu Ala Ser Val Asn His Asn Glu Ala Leu Ser Ile Pro Gln Asn Val Cys Pro Ile Ser Glu Lys Pro Leu Asn Asp Phe Asn Ile Gln Leu Leu Phe Lys Ile Thr Pro Asp Thr Asn Asn Ala Thr Leu Thr Pro Phe Lys His Lys Leu Arg Arg Glu Phe Cys Lys Pro Asn Phe Gln Tyr Asp Asp Va1 Val Cys Ile Leu Arg Gln Ser Leu Lys Pro Asn Lys Thr Cys Ala Val Phe Ala Pro Asp His Ile Phe Gln Met Val Glu Gln Ala Tyr Gln Thr Tyr Phe Ser Ala His Ser Gln Phe Lys Leu Ile Arg Cys Leu Ile Phe Leu Pro Glu Ile Thr Asp Ser Thr Glu Ile Glu Lys Ile Ile Thr Asp Tyr His Tyr Asn Ser Asn His Arg Gly Ile Asp Glu Thr Tyr Leu His Ile Lys Arg Gln Gln Phe Phe Pro His Met Lys G1u Arg Ile Thr Gln Leu Ile Arg Lys Cys Glu Thr Cys Leu Lys Leu Lys Tyr Asp Arg G1n Pro Gln Lys Ile Thr Tyr Gln Ile Ser Glu Leu Pro Ser Lys Pro Leu Asp Ile Leu His Ile Asp Ile Tyr Thr Ile Asn Lys Asn Tyr Asn Leu Thr Ile Ile Asp Lys Phe Ser Lys Phe Ala Ala Ala Tyr Pro Ile Thr Asn Arg Asn Cys Ile Asn Val Val Lys Ala Leu Lys His Phe Ile Ser Gln Phe Gly Ile Pro Lys Lys Leu Ile Tyr Asp Gln Gly Ala Glu Phe Ala Ser Asp Met Phe Asn Lys Phe Cys Thr Gln Phe Asn Ile Asp Leu His Val Thr Ser Phe Gln Gln Ser Ser Ser Asn Ser Pro Val Glu Arg Leu His Ser Thr Leu Thr Glu Ile Tyr Arg Ile Ile Leu Asp Val Arg Lys Gln Gln Lys Leu Ser Ser Glu His Asp Glu Ile Met Ser Glu Thr Leu Ile Thr Tyr Asn Asn Ala I1e His Ser Ala Thr Lys His Thr Pro Phe Glu Leu Phe Asn G1y Arg Thr His Ile Phe Asn Gln Thr Ile Gln Phe Asn Asn Glu His Asp Tyr Leu Thr Lys Leu Asn Glu Phe Arg G1u Lys Leu Tyr Pro Leu Ile Thr Asp Lys Leu Ser Asn Asp Val Val Arg Arg Thr Leu Lys Leu Asn Glu Thr Arg Thr Asp Pro Val Asp Leu Gln fro Asp Thr Leu Val Leu Arg Lys Glu Asn Arg Arg Asn Lys Ile Thr Pro Arg Phe Ser Ile His Lys Val Lys His Asp Lys Gly His Thr Leu Tle Thr Ala Arg Asn Gln Lys Leu His Lys Ser Lys Ile Arg Lys Thr Val Leu Lys Lys Asp Lys Ser Asn Asn Ala Ile His Val His Tyr Leu Asn Asp Asn Ala Pro Ile A1a Lys Ile Glu Leu G1y Lys Ala Leu Leu Ile Glu Arg Tyr Lys Ile Ile Ser His Val Ile Asn Leu Gln Asp Tyr Ser Arg Cys Met Glu Gln Phe His Leu Thr Ile Asn Lys Phe Asn Pro Asp Ser Thr Leu Thr Asp Ser Va1 Thr Ile Leu Lys Thr Lys Leu Thr Gln Ala Gln Val Lys Leu Lys Ala Leu Thr Pro Ser Tyr Arg Asn Lys Arg Gly Leu Tle Asn Gly Leu Gly Ser Leu Val Lys Val Val Thr Gly Asn Met Asp Ala Asn Asp Asn Lys Glu Ile His Glu Glu Leu Asp Asn Ile Lys Lys Asn Ser Glu Val Ser Asn Asp Asn Leu Gln Lys Gln Val Met Phe Asn Asn G1u Tle Leu Ile Arg Phe Glu Asn Ile Thr Asp His I1e Asn Asn Glu Gln I1e Leu Ile Ser Lys Phe Phe Asp Thr Ser Gln Asn Lys Ile Tyr Lys His Leu Asn Leu Gln Asp Thr Leu Leu Glu Glu Ile Gln Tyr Leu Asn Arg Ile Asn Tyr Asn Ile Glu Leu Phe Ile Asn His Leu Asn Asp Ile Thr Glu Ser Met Leu Leu Ala Lys Ile Asn Ile 21e Pro Lys Phe Ile Leu Asn Glu Gln Glu Met Asp Lys Ile Lys Thr Ile Leu Glu Lys Gln Asn Ile Thr Val Lys Asn Glu Gln Ser Ile Tyr Asn Phe Leu Gln Met Asn Thr Leu Asn Tyr Glu Gln Lys Ile Ile 25!43 Phe Asn Ile Lys Val Pro Ile Phe Lys G1n Pro Phe His Thr Leu Ala Arg Leu Val Pro Leu Pro Ile Asn Asn Thr Tyr Phe Val Ile Thr Pro Asn Tyr Leu Ala Tyr Asn Ile Asn Asn Lys Lys Phe His Met Thr Arg Lys Cys Pro Lys Leu Asp Asn Thr Phe Leu Cys Asp Glu Asn Phe Tyr Val Asp Thr Pro Gln Asn Asn Thr Cys Leu G1u His Leu Leu Asn Gly Glu Asn Ser Ser Cys Asp Val Arg Glu Thr Gly Pro Ile Thr Asp Val Phe Glu Ala Glu Arg Gly Tyr Ile Phe Ala Phe Asn Va1 Asn Lys Leu Lys Val Ser Leu Thr Asn Gly Ser Glu Leu Ser Ile Met Gly Ser Ala Ile Ile Arg Tyr Ile Asn Glu Thr Ile Gln Ile Asn Gly Ile Asp Tyr Asp Gly Thr Val Asp Thr Phe Pro Glu Gln Thr Asp Phe Asp Leu Pro Pro Met Arg Lys Val Thr Arg Asn Thr Thr Ile Thr Val Leu Ser Leu Glu Lys Leu His Leu Glu Ala Thr Gln Thr Met Asp Lys Ile Leu Ala Val His His Asn Thr Tle Gln His Thr Trp Thr Leu Tyr Thr Leu Leu Gly Leu Val Thr Phe Leu Ala Val Ile Leu Trp Leu His Arg Arg Thr Lys His Ile Val His Ile His Glu Asp His His Val Leu Thr Pro Ile Ala Lys Ser Lys Gly Lys Val Leu Ile Arg Asp Lys Leu Met Glu Arg Arg Asn Arg Arg Thr Glu Arg Thr Glu Lys Ala Arg Ile Trp Glu Val Thr Asp Arg Thr Val Arg Thr Trp Ile Gly Glu Ala Val Ala Ala Ala Ala A1a Asp Gly Val Thr Phe Ser Val Pro Val Thr Pro His Thr Phe Arg His Ser Tyr Ala Met His Met Leu Tyr Ala Gly Ile Pro Leu Lys Val Leu Gln Ser Leu Met Gly His Lys Ser Ile Ser Ser Thr Glu Val Tyr Thr Lys Val Phe Ala Leu Asp Val Ala Ala Arg His Arg Val Gln Phe Ala Met Pro Glu Ser Asp Ala Val Ala Met Leu Lys Gln Leu Ser <210> 15 <211> 257 <212> PRT
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte ID No: 1306049CD1 <400> 15 Met Glu Phe Pro Asp Leu Gly Ala His Cys Ser Glu Pro Ser Cys Gln Arg Leu Asp Phe Leu Pro Leu Lys Cys Asp Ala Cys Ser Gly Ile Phe Cys Ala Asp His Val Ala Tyr Ala Gln His His Cys Gly Ser Ala Tyr Gln Lys Asp Ile Gln Val Pro Val Cys Pro Leu Cys Asn Val Pro Val Pro Val Ala Arg G1y Glu Pro Pro Asp Arg Ala Val Gly G1u His Ile Asp Arg Asp Cys Arg Ser Asp Pro Ala Gln Gln Lys Arg Lys IIe Phe Thr Asn Lys Cys Glu Arg Ala Gly Cys Arg Gln Arg Glu Met Met Lys Leu Thr Cys Glu Arg Cys Ser Arg 210 l15 120 Asn Phe Cys Ile Lys His Arg His Pro Leu Asp His Asp Cys Ser Gly Glu Gly His Pro Thr Ser Arg Ala Gly Leu Ala Ala Ile Ser Arg Ala Gln Ala Val Ala Ser Thr Ser Thr Val Pro Ser Pro Ser Gln Thr Met Pro Ser Cys Thr Ser Pro Ser Arg Ala Thr Thr Arg Ser Pro Ser Trp Thr Ala Pro Pro Val I1e Ala Leu Gln Asn Gly Leu Ser Glu Asp Glu Ala Leu Gln Arg Ala Leu Glu Met Ser Leu Ala Glu Thr Lys Pro Gln Val Pro Ser Cys Gln Glu Glu Glu Asp Leu Ala Leu Ala Gln Ala Leu Ser Ala Ser Glu Ala Glu Tyr Gln Arg Gln Gln Ala Gln Ser Arg Ser Ser Lys Pro Ser Asn Cys Ser Leu Cys <210> 16 <212> 517 <222> PRT
<213> Homo sapiens <220>
<221> misc_feature <223> Incyte ID No: 3187174CD1 <400> 16 Met Ala Ala Pro Glu Gln Pro Leu A1a Ile Ser Arg Gly Cys Thr Ser Ser Ser Ser Leu Ser Pro Pro Arg Gly Asp Arg Thr Leu Leu Val Arg His Leu Pro Ala Glu Leu Thr Ala Glu Glu Lys Glu Asp Leu Leu Lys Tyr Phe Gly Ala Gln Ser Val Arg Val Leu Ser Asp Lys Gly Arg Leu Lys His Thr Ala Phe A1a Thr Phe Pro Asn Glu Lys Ala Ala Ile Lys Ala Leu Thr Arg Leu His Gln Leu Lys Leu Leu Gly His Thr Leu Val Val Glu Phe Ala Lys Glu Gln Asp Arg Val His Ser Pro Cys Pro Thr Ser Gly Ser Glu Lys Lys Lys Arg Ser Asp Asp Pro Val Glu Asp Asp Lys Glu Lys Lys Glu Leu Gly Tyr Leu Thr Val Glu Asn Gly Ile Ala Pro Asn His Gly Leu Thr Phe Pro Leu Asn Ser Cys Leu Lys Tyr Met Tyr Pro Pro Pro Ser Ser Thr Ile Leu Ala Asn Ile Val Asn Ala Leu Ala Ser Val Pro Lys Phe Tyr Val Gln Val Leu His Leu Met Asn Lys Met Asn Leu Pro Thr Pro Phe Gly Pro Ile Thr Ala Arg Pro Pro Met Tyr Glu Asp Tyr Met Pro Leu His Ala Pro Leu Pro Pro Thr Ser Pro Gln Pro Pro Glu Glu Pro Pro Leu Pro Asp Glu Asp Glu Glu Leu Ser Ser Glu Glu Ser Glu Tyr Glu Ser Thr Asp Asp Glu Asp Arg Gln Arg Met Asn Lys Leu Met Glu Leu Ala Asn Leu Gln Pro Lys Arg Pro Lys Thr Ile Lys Gln Arg His~Val Arg Lys Lys Lys Lys Lys Lys Asp Met Leu Asn Thr Pro Leu Cys Pro Ser His Ser Ser Leu His Pro Val Leu Leu Pro Ser Asp Val Phe Asp Gln Pro Gln Pro Va1 Gly Asn Lys Arg Ile Glu Phe His Ile Ser Thr Asp Met Pro Ala Ala Phe Lys Lys Asp Leu Glu Lys Glu Gln Asn Cys Glu Glu Lys Asn His Asp Leu Pro Ala Thr G1u Va1 Asp Ala Ser Asn Ile Gly Phe Gly Lys Ile Phe Pro Lys Ala Asn Leu Asp Ile Thr Glu 365 370 ' 375 Glu Ile Lys Glu Asp Ser Asp Glu Met Pro Ser Glu Cys Ile Ser Arg Arg Glu Leu Glu Lys Gly Arg Ile Ser Arg Glu Glu Met Glu Thr Leu Ser Va1 Phe Arg Ser Tyr Glu Pro Gly Glu Pro Asn Cys Arg Ile Tyr Val Lys Asn Leu Ala Lys His Va1 Gln Glu Lys Asp Leu Lys Tyr I1e Phe Gly Arg Tyr Val Asp Phe Ser Ser Glu Thr Gln Arg Ile Met Phe Asp Ile Arg Leu Met Lys Glu Gly Arg Met Lys Gly Gln Ala Phe Ile Gly Leu Pro Asn Glu Lys Ala Ala Ala Lys Ala Leu Lys Glu Ala Asn Gly Tyr Val Leu Phe Gly Lys Pro Met Val Val Gln Phe Ala Arg Ser Ala Arg Pro Lys Gln Asp Pro Lys Glu Gly Lys Arg Lys Cys <210> 17 <211> 4456 <212> DNA
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte ID No: 2530775CB2 <400> 17 caaaaacgcg atgtcgatct gacggatcac tcataggaca gctatgacgt cgcgtgctcg 60 cgtgcgttta ctcggtcttc ggtgcgagag aagataaaga aaaaacggaa gttatcctta 120 gctgtttgct tagcattact gaccaggtac tacttccatc tctttctttg atggactgca 180 atgcttgtat gtctgaggaa ctatggggaa tgtttaaaac atttccatat cagcatagat 240 atcgtctgta tggccagtgg aagaatgaaa cttataacag tcacccactt ttagtaaaag 300 ttaaagctca aacaatagac agagccaaat atatcatgaa gcgcctaacc aaggaaaatg 360 tgaagccttc tggaagacaa attgggaagt tgagccacag caatccaacc attttgtttg 420 attatatctt gtcacaaata cagaagtatg ataacttaat aacacctgta gtagattcat 480 tgaaatacct cacttcactg aattatgatg tcttggccta ttgtatcatt gaagctttag 540 ctaatccaga aaaggaaaga atgaaacatg atgacacaac catctcaagc tggcttcaga 600 gtctggctag tttctgtggt gcagtttttc gtaaatatcc aattgatctt gctggtcttc 660 ttcagtatgt tgccaatcag ctaaaggcgg gcaaaagttt tgacctgctt atattgaaag 720 aagtggtaca aaaaatggca ggaatagaaa ttacagagga aatgacaatg gagcaactag 780 aggctatgac tggtggagag cagctaaaag ctgagggtgg ttattttggt cagatcagaa 840 acactaaaaa atcctctcag agattaaagg atgctctatt ggaccatgat cttgcccttc 900 ctctctgtct gcttatggct cagcagagaa atggggtaat ctttcaggaa ggtggagaga 960 aacatttgaa acttgtggga aagctctatg accagtgtca tgataccctg gtgcagtttg 1020 gtgggttttt agcatctaat ctgagcacag aagattatat aaagcgagtg ccttcaattg 1080 atgtactctg taatgaattt catacacccc atgatgcagc atttttcctg tctaggccaa 1140 tgtatgccca tcatatttcg tcaaagtatg atgaacttaa aaaatcagaa aagggaagta 1200 aacagcaaca taaagttcat aagtacatta catcatgtga gatggtgatg gcgcctgtcc 1260 atgaagcagt ggtctcctta catgtttcca aagtctggga tgacatcagc cctcaattct 1320 atgctacatt ctggtcattg acaatgtatg accttgcagt tccacacacc agctatgaac 1380 gagaagtcaa taaacttaaa gtccagatga aagcaattga tgacaatcag gaaatgcccc 1440 caaataaaaa gaaaaaagag aaggagcgct gtactgccct tcaggacaag cttcttgaag 1500 aagaaaagaa acagatggaa catgtacaga gagttctaca gagattgaaa ctggaaaagg 1560 acaactggct tttagcaaaa tctaccaaaa atgagaccat cacaaaattt ctacagctgt 1620 gtatatttcc tcgatgtatt ttttcagcaa ttgatgctgt ttactgtgct cgttttgttg 1680 aattggtaca tcaacagaaa actccaaatt tttccacact tctttgctat gatcgagttt 1740 tctctgacat aatttacaca gttgcaagct gtactgaaaa tgaagccagt cgatacggaa 1800 ggtttctttg ctgcatgtta gagactgtga ccaggtggca tagtgataga gccacatatg 1860 aaaaggaatg tggaaactat ccaggattcc ttaccatatt acgggcaact ggatttgatg 1920 gtggaaataa ggctgatcaa ttagactatg aaaattttcg acatgttgta cataaatggc 1980 attacaaact aaccaaggca tcggtacatt gccttgaaac aggcgaatat actcacatca 2040 ggaatatctt gattgtgcta acaaaaatac ttccttggta cccaaaagtt ttgaatctgg 2100 gtcaagcttt ggaaagaaga gtacacaaaa tctgccaaga agaaaaagag aagaggccag 2160 atctatatgc attggctatg ggctactctg ggcagttgaa aagtagaaag tcatacatga 2220 tacctgaaaa tgagtttcat cacaaagacc cccctccgag gaatgcagtt gccagtgtgc 2280 aaaatgggcc tggtggtggg ccttcttcat catcaatagg aagtgcatct aaatcggatg 2340 aaagcagtac tgaggagact gataaatcaa gggagagatc tcagtgtggt gtgaaagctg 2400 ttaataaagc ttctagtacc acacctaaag ggaattcaag caatggaaat agtggctcta 2460 acagcaacaa agctgttaaa gaaaatgaca aagaaaaagg gaaagagaaa gaaaaagaga 2520 aaaaagaaaa gactccagct actactccag aggccagggt acttggtaaa gatggtaaag 2580 aaaaaccaaa ggaagagcgg ccaaataaag atgaaaaagc aagagagacc aaggaaagaa 2640 cgccgaagtc tgacaaagag aaagaaaaat tcaagaagga agaaaaagct aaagatgaga 2700 aatttaagac cactgtcccc°aacgcagaat caaaatcaac tcaagaaagg gaaagagaga 2760 aggagccatc cagagaaaga gatatagcaa aggaaatgaa atcaaaggaa aatgttaaag 2820 gaggagaaaa aacaccagtt tctgggtcct tgaaatcacc tgttcccaga tcagatattc 2880 cagagcctga aagggaacaa aaacgccgca aaattgatac tcacccttct ccatcacatt 2940 cctccacagt aaaggacagt ctcatcgaac tcaaggaatc ttcagcaaag ctctacatta 3000 atcatactcc tccaccactg tccaagagta aggagagaga aatggacaag aaagatttgg 3060 acaagtcaag ggaaagatcc agagaaagag agaaaaaaga tgaaaaggac aggaaagagc 3120 ggaaaaggga tcactcaaac aacgaccgtg aagtgccacc ggacttaacc aagagacgta 3180 aagaggagaa tggaacaatg ggggtttcaa aacataaaag tgaaagtcct tgtgaatctc 3240 cttatccaaa tgagaaagac aaggaaaaaa ataagtcaaa atcttcaggc aaagaaaaag 3300 gcagtgattc atttaaatct gagaagatgg ataaaatctc ctccggtggc aaaaaggagt 3360 ccaggcatga taaagaaaag atagaaaaga aagagaaacg ggacagttca ggaggaaagg 3420 aagagaagaa acatcataag tcctcggaca agcacagata atgaagactt tccatcaagg 3480 ctatggacag acccCCtaag ccgaaagtct cccagaggag gatgccaaag ctccgtgcgt 3540 cactctcaga atgatatctc ccataattaa gagcttctgg tgctgtccat ttaatgggaa 3600 tctgctttaa gtcagaagat gagtacacac caccatcctc ttgacgagac attccaaagt 3660 cactgatttt cagaacatta ttttcacctc caggcagttt cttacagcaa ggtccctgtg 3720 tatacatttt ttactcgaga tacaccatcc agaatcagca tctaatgaaa atttcactga 3780 gttttagttc attcttcttt ctctgaaagg agaggaaatc gcctccagga accagttcct 3840 taatgacgta gataggccgt ctttgtgtgt gaactcctat acttttgaca ttactgggat 3900 gattatattg cttgaggatt ttggcttcta gtaaaaattt taatttcagt tcctggggaa 3960 gacgttcttg acgtgtttta acagcaacag caattttatc ctttaatgtg accttaaatg 4020 tgtcaccaaa atttcccttg cctcttgtaa cgtggcacct ttatgattga gaacccattt 4080 cttattctcc taatggccat actgtgatac catgatgctc ttaattggaa cattgacttt 4140 tttttttttt tgcaatttaa acaattgaga taaaattcat ataacatcaa atttaccttt 4200 cacttttttt ttttcctttg ggggccaaag tcccgctgtt gtcccccggg tgggagtcaa 4260 ggggggagat ccgggcccac tgggaccccc gcccccgggg ttaaagaaat tccccttccc 4320 aagcaatttg gggggctggg gcccgaaaat tccttaaacc cggggggtgg gggttccctt 4380 ggcccaaaat ggggccaatg ccctttaaag ggggggaaaa aaaaccctgc tcaaaaaaag 4440 aattttccct ggtggc 4456 <210> 18 <211> 3662 <212> DNA
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte ID No: 926296CB1 <400> 18 acggaatctc gggtcttctg acgtgccggg cgggaagatg tcatcattgc caagaagagc 60 gaaagtacag gtccaggatg tggtactgaa agatgaattt tcttcattct ctgagttatc 120 atctgcctct gaagaagatg acaaggaaga tagtgcctgg gagccccaaa agaaagttcc 180 cagaagccgt aaacagcccc ctcccaagga atccaaacca aagaggatgc ctcgggtgaa 240 gaagaatgcc ccacagatca gtgatggctc agaagtcgtt gttgttaagg aggagctgaa 300 tagctctgtg gctattgctg atactgcttt agaagacaga aaaaataaat tggatactgt 360 acagactctg aaaacagcca agacaaaaca gaaatgtgca gcgcagccac atacagttcg 420 aaggactaaa aagctgaaag ttgaagaaga aaccagcaaa gccagcaact tagagggtga 480 gagtaatagt tcagagacac catccacaag cactgtgtgg ggaggtacat gcaagaagga 540 agagaatgac gatgacttta catttggtca gtccgcttta aagaaaatca agactgagac 600 atatcctcag gggcagcctg tcaagtttcc agcaaatgcc aatagtacta aagaggaggt 660 ggaaatgaat tgggacatgg tacaggtttt atctgagaga actaatattg aaccttgggt 720 ttgtgccaac atcattcgtc tctttaatga tgataacaca attcccttca ttatacgtta 780 tagaaaagag ctcattaata accttgatgc tgattccttg agagaagttc agcaaaccct 840 agaagagcta cgggctgttg caaagaaagt tcatagtaca atccagaaaa ttaagaagga 900 agggaagatg tctgagtgct tgttaaaagc catgctgaat tgtaaaactt ttgaagaact 960 agaacacgtg tctgctccat ataaaactgg aagcaaaggg actaaagccc agagagcaag 1020 acagttgggc ttagaaggag cagccagggc actgcttgag aaaccagggg agctcagtct 1080 gctatcgtac attaggcctg acgttaaagg gctttcaacg cttcaggata ttgaaatagg 1140 agtgcagcat attttagcag atatgattgc taaagacaaa gacacgcttg acttcattcg 1200 gaactt'gtgc cagaagagac atgtttgtat ccagtcatct ctggcaaaag tatcctcaaa 1260 aaaggtaaat gagaaagatg ttgataagtt tctgctctac cagcattttt cctgcaacat 1320 aagaaacatt caccatcatc agattctggc aattaaccgt ggagaaaatt tgaaggtact 1380 gacggttaag gtcaatattt ctgatggagt gaaggatgaa ttctgtaggt ggtgcatcca 1440 aaacaggtgg agaccacgta gctttgcaag gccagagtta atgaagatct tatataattc 1500 actgaatgat tcctttaaac gccttattta tcctcttctc tgtagagaat tcagagccaa 1560 actaacatca gatgcagaga aggaatcagt aatgatgttt ggacggaacc ttcgtcagct 1620 ccttttaaca agccctgttc cagggcgcac cttaatggga gtggatcctg gttataaaca 1680 tggttgcaaa ttagctataa tttctcctac tagtcagata cttcatactg atgtggttta 1740 cttgcattgt ggacaaggct tccgagaggc ggagaaaata aagacacttt tgctgaattt 1800 caactgcagc acagtagtga ttggaaatgg aactgcctgc agggaaacag aagcttactt 1860 tgctgacctg ataatgaaga attattttgc accactggat gttgtttact gtatcgtcag 1920 tgaagcagga gcatcaatct acagtgtcag ccctgaagct aacaaagaga tgccagggct 1980 ggaccctaat ttgagaagtg cagtttccat agcaaggcgt gtacaagatc cattagctga 2040 gctagtgaaa attgagccaa agcacattgg agttggaatg tatcagcatg acgtatccca 2100 gactttactc aaggcaacac tggacagtgt tgtagaagaa tgtgtcagct ttgtgggagt 2160 ggatattaac atctgttcag aagttttgtt aaggcatatt gcaggactca atgccaacag 2220 ggccaaaaat attattgaat ggcgagagaa aaatggaccc tttatcaacc gagaacagct 2280 gaagaaagtg aaagggctgg gcccaaaatc cttccaacag tgtgctggct tcatcagaat 2340 caaccaggat tatatccgaa cgttttgcag tcagcaaact gaaacttcag gccaaattca 2400 aggagttgct gtgacatctt cagcagacgt tgaggtcaca aatgagaagc agggcaaaaa 2460 gaagagcaaa actgcagtga atgttttact gaagccaaat cctttggacc aaacttgtat 2520 tcatccagaa tcatatgaca tagcaatgag gtttttgtca tccattggag ggacactgta 2580 tgaggttgga aagcctgaaa tgcaacaaaa aataaattca ttccttgaaa aggaaggaat 2640 ggagaaaatt gcagaaagat tgcaaacaac agtacacacc ttacaggtca tcatagatgg 2700 tctcagccag cctgaaagct ttgactttcg aacagatttt gataaacctg atttcaagag 2760 aagcatagta tgcctggaag atctgcagat tgggacagtt cttacaggca aagttgagaa 2820 tgccactctc tttggaattt ttgtggatat aggagtgggg aaatctgggc tgattcccat 2880 acgaaatgta acagaagcaa aactttcaaa aacaaagaag agaagaagcc ttggactggg 2940 ccccggagaa agagtggaag tccaagtact caacattgac atcccccgat ctaggattac 3000 tctggacctc attcgggtgt tatgagtatc ccacgaaggc cagacgctga ttttattttc 3060 tcatttccac agattgacaa ggataagtca gttgtttgta aactctaggt agcagatgag 3120 aaataattca cttaatatca gaaatatttt ccaaacactt tcctttattt tttcttctga 3180 ataaatagaa aaccaacagt ttgatttcct tttcccttaa aggaaacaac taatacacat 3240 tcttatatgg ctttatgtag taatagtttt ctgactaaaa ttttgttttt tattttttgt 3300 aatttatctt taactccttt tgcattttgt ataacagatt gcttaacttc tacttgccaa 3360 catctgcctt gctggacttg tatgggat.tg tcttcttgat ttgaattgta ccgtctttgt 3420 tgacacagta gggctgggca gtgtttaatc cttccatttt atagattttt ttttaatcag 3480 gccttttgga cttcattcat aattttgcaa taatctcttt tcccttgtca tgcaagccaa 3540 aaatatacca gtaaaacaga ttctgacgtg tttgtagtta tcaaatgaat ggctcgaaac 3600 acttctcaaa aggatatacg tattgacccc aacaataaat gtttgtggct agtgaaaaaa 3660 as 3662 <210> 19 <211> 2201 <212> DNA
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte ID No: 1322761CB1 <400> 19 ccccaaacaa acgagtcccc aattctcgtc cgtcctcgcc gcgggcagcg ggcggcggag 60 gcagcgtgcg gcggtcgcca ggagctggga gcccagggcg cccgctcctc ggcgcagcat 120 gttccagccg gcgcccaagc gctgcttcac catcgagtcg ctggtggcca aggacagtcc 180 CCtgCCCgCC tcgcgctccg aggaccccat ccgtcccgcg gcactcagct acgctaactc 240 cagccccata aatccgttcc tcaacggctt ccactcggcc gccgccgccg ccgccggtag 300 gggcgtctac tccaacccgg acttggtgtt cgccgaggcg gtctcgcacc cgcccaaccc 360 cgccgtgcca gtgcacccgg tgccgccgcc gCaCgCCCtg gCCg'CCCaCC CCCtaCCCtC 420 CtCgCaCtCg CCaCaCCCCC tattCgCCtC gcagcagcgg gatccgtcca CCttCtaCCC 480 ctggctcatc caccgctacc gatatctggg tcatcgcttc caagggaacg acactagccc 540 cgagagtttc cttttgcaca acgcgctggc ccgaaagccc aagcggatcc gaaccgcctt 600 CtCCCCgtCC CagCttCtaa ggctggaaca cgcctttgag aagaatcact acgtggtggg 660 cgccgaaagg aagcagctgg cacacagcct cagcctcacg gaaactcagg taaaagtatg 720 gtttcagaac cgaagaacaa agttcaaaag gcagaagctg gaggaagaag gctcagattc 780 gcaacaaaag aaaaaaggga cgcaccatat taaccggtgg agaatcgcca ccaagcaggc 840 gagtccggag gaaatagacg tgacctcaga tgattaaaaa cataaaccta accccacaga 900 aacggacaac atggagcaaa agagacaggg agaggtggag aaggaaaaaa ccctacaaaa 960 caaaaacaaa ccgcatacac gttcaccgag aaagggagag ggaatcggag ggagcagcgg 1020 aatgcggcga agactctgga cagcgagggc acagggtccc aaaccgaggc cgcgccaaga 1080 tggcagagga tggaggctcc ttcatcaaca agcgaccctc gtctaaagag gcagctgagt 1140 gagagacaca gagagaagga gaaagaggga gggagagaga gaaagagaga gaaagagaga 1200 gagagagaga gagaaagctg aacgtgcact ctgacaaggg gagctgtcaa tcaaacacca 1260 aaccggggag acaagatgat tggcaggtat tccgtttatc acagtccact taaaaaatga 1320 tgatgatgat aaaaaccacg acccaaccag gcacaggact tttttgtttt ttgcacttcg 1380 ctgtgtttcc cccccatctt taaaaataat tagtaataaa aaacaaaaat tccatatcta 1440 gccccatccc acacctgttt caaatccttg aaatgcatgt agcagttgtt gggcgaatgg 1500 tgtttaaaga ccgaaaatga attgtaattt tcttttcctt ttaaagacag gttctgtgtg 1560 ctttttattt tgattttttt tcccaagaaa tgtgcagtct gtaaacactt tttgatacct 1620 tctgatgtca aagtgattgt gcaagctaaa tgaagtaggc tcagcgatag tggtcctctt 1680 acagagaaac ggggagcagg acgacggggg ggctgggggt ggcgggggag ggtgcccaca 1740 aaaagaatca ggacttgtac tgggaaaaaa acccctaaat taattatatt tcttggacat 1800 tccctttcct aacatcctga ggcttaaaac cctgatgcaa acttctcctt tcagtggttg 1860 gagaaattgg ccgagttcaa ccattcactg caatgcctat tccaaacttt aaatctatct 1920 attgcaaaac ctgaaggact gtagttagcg gggatgatgt taagtgtggc caagcgcacg 1980 gcggcaagtt ttcaagcact gagtttctat tccaagatca tagacttact aaagagagtg 2040 acaaatgctt ccttaatgtc ttctatacca gaatgtaaat atttttgtgt tttgtgttaa 2100 tttgttagaa ttctaacaca ctatatactt ccaagaagta tgtcaatgtc aatattttgt 2160 caataaagat ttatcaatat gccctcaaaa aaaaaaaaaa a 2201 <210> 20 <211> 3188 <212> DNA
<213> Homo Sapiens <220>
<221> misc_feature <223> Tncyte ID No: 7472664CB1 <400> 20 gtgaatctag tctgtgtgat tttaaaactc ttgcttttga cctctgtgct atacttagtg 60 cttgtgtatt gcactcttaa aagttgattg attgttgatg gccattacta ccctataagt 120 ttactgggta aaagagattt tggtcttatt actcttaaat atttgctaac tatattccga 180 cgctgatttt taattttttt tttaaccatg gtcatgctaa tagagttttg tgtttctttg 240 tttggtcggc tggttctttt cttcccctgg ttgctttgga taggatactg tggagcagat 300 attaaatcaa tatgtgctga agctgcttta tgtgctttac gacgacgcta cccacagatc 360 tataccacta gtgagaaact gcagttggat ctctcttcaa ttaatatctc agctaaggat 420 ttcgaggtag ctatgcaaaa gatgatacca gcctcccaaa gagctgtgac atcacctggg 480 caggcactgt ccaccgttgt gaaaccactc ctgcaaaaca ctgttgacaa gattttagaa 540 gccctgcaga gagtatttcc acatgcagaa ttcagaacaa ataaaacatt agactcaggt 600 ataaaacttg attgaccatt taaagttagt ttttataatc agtaaagtga aatgtttcat 660 acataccaga aatatttatt tgtaaaagaa tctcttgaaa agaatttggt aatgctttac 720 ctgggatatt tgaaggaaat ttactccatg gattgccctt aatttaactt attgtaagtt 780 tggggtgcat ttttacattt acttattaat tcatatctac acaaatattt ttgattcttt 840 gaaaaaaatt taattgaagt atgctgactt ggtaactgaa gcaataaaga tttgctttgc 900 aatccaaaag ttactaatgt acaatttttt ggttttatag atatttcttg tcctctgcta 960 gaaagtgact tggcttacag tgatgatgat gttccatcag tttatgaaaa tggactttct 1020 cagaaatctt ctcataaggc aaaagacaat tttaattttc ttcatttgaa tagaaatgct 1080 tgttaccaac ctatgtcttt tcgaccaaga atattgatag taggagaacc aggatttggg 1140 caaggttctc acttggcacc agctgtcatt catgctttgg aaaagtttac tgtatataca 1200 ttagacattc ctgttctttt tggagttagt actacatccc ctgaagaaac atgtgcccag 1260 gtgattcgtg aagctaagag aacagcacca agtatagtgt atgttcctca tatccacgtg 1320 tggtgggaaa tagttggacc gacacttaaa gccacattta ccacattatt acagaatatt 1380 ccttcatttg ctccagtttt actacttgca acttctgaca aaccccattc cgctttgcca 1440 gaagaggtgc aagaattgtt tatccgtgat tatggagaga tttttaatgt ccagttaccg 1500 gataaagaag aacggacaaa attttttgaa gatttaattc taaaacaagc tgctaagcct 1560 cctatatcaa aaaagaaagc agttttgcag gctttggagg tactcccagt agcaccacca 1620 cctgagccaa gatcactgac agcagaagaa gtgaaacgac tagaagaaca agaagaagat 1680 acatttagag aactgaggat tttcttaaga aatgttacac ataggcttgc tattgacaag 1740 cgattccgag tgtttactaa gcctgttgac cctgatgagg ttcctgatta tgtcactgta 1800 ataaagcaac caatggacct ttcatctgta atcagtaaaa ttgatctaca caagtatctg 1860 actgtgaaag actatttgag agatattgat ctaatctgta gtaatgcctt agaatacaat 1920 ccagatagag atcctggaga tcgtcttatt aggcatagag cctgtgcttt aagagatact 1980 gcctatgcca taattaaaga agaacttgat gaagactttg agcagctctg tgaagaaatt 2040 caggaatcta gaaagaaaag aggttgtagc tcctccaaat atgccccgtc ttactaccat 2100 gtgatgccaa agcaaaattc cactcttgtt ggtgataaaa gatcagaccc agagcagaat 2160 gaaaagctaa agacaccgag tactcctgtg gcttgcagca ctcctgctca gttgaagagg 2220 aaaattcgca aaaagtcaaa ctggtactta ggcaccataa aaaagcgaag gaagatttca 2280 caggcaaagg atgatagcca gaatgccata gatcacaaaa ttgagagtga tacagaggaa 2340 actcaagaca caagtgtaga tcataatgag accggaaaca caggagagtc ttcggtggaa 2400 gaaaatgaaa aacagcaaaa tgcctctgaa agcaaactgg aattgagaaa taattcaaat 2460 acttgtaata tagagaatga gcttgaagac tctaggaaga ctacagcatg tacagaattg 2520 agagacaaga ttgcttgtaa tggagatgct tctagctctc agataataca tatttctgat 2580 gaaaatgaag gaaaagaaat gtgtgttctg cgaatgactc gagctagacg ttcccaggta 2640 gaacagcagc agctcatcac tgttgaaaag gctttggcaa ttctttctca gcctacaccc 2700 tcacttgttg tggatcatga gcgattaaaa aatcttttga agactgttgt taaaaaaagt 2760 caaaactaca acatatttca gttggaaaat ttgtatgcag taatcagcca atgtatttat 2820 cggcatcgca aggaccatga taaaacatca cttattcaga aaatggagca agaggtagaa 2880 aacttcagtt gttccagatg atgatgtcat ggtatcgagt attctttata ttcagttcct 2940 atttaagtca tttttgtcat gtccgcctaa ttgatgtagt atgaaaccct gcatctttaa 3000 ggaaaagatt aaaatagtaa aataaaagta tttaaacttt cctgatattt atgtacatat 3060 taagataaat gtcatgtgta agataactga taaatattgg aactttgcta gaacaagacc 3120 ctgtagtaat agtaataata gttgaagttt ggccaactct taataaagtt attttggtaa 3180 ctaaaaaa 3188 <210> 21 <211> 843 <212> DNA
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte ID No: 7473124CB1 <400> 21 atgcctattt actcccagac agtggccatg gctgaacact ttaaacaagc aagcagttgt 60 cctatctgcc tggattatct tgaaaacccc acgcacctga aatgtggata catctgttgc 120 ctccgatgca tgaactcact gcgaaagggg cccgatggga agggggtgct gtgccctttc 180 tgccctgtgg tctctcagaa aaatgacatc aggcccgctg cccagctggg ggcgctggtg 240 tccaagatca aggaactaga gcccaaggtg agagctgttc tgcagatgaa tccaaggatg 300 agaaagttcc aagtggatat gaccttggat gtggacacag ccaacaacga tctcatcgtt 360 tctgaagacc tgaggcgtgt ccgatgtggg aatttcagac agaataggaa ggagcaagct 420 gagaggttcg acactgccct gtgcgtcctg ggcacccctc gcttcacttc cggccgccat 480 tactgggagg tgggcgtggg caccagccaa gtgtgggatg tgggcgtgtg caaggaatct 540 gtgaaccgac aggggaacgt tgtactctct tcagaactcg gcttctggac tgtgggtttg 600 agacaaggac agatctactt tgccagcact aagcctgtga cgggtctctg ggtgagctca 660 ggtctacacc gagtggggat ttacctggat ataaaaacga gggccatttc cttctataat 720 gtcagtgata ggtcacatat cttcacattc acgaaaattt ctgctactga gccactgcgc 780 ccatgttttg ctcatgcaga tacaagtcgt gatgatcacg gatacttgag tgtgtgtgtg 840 taa 843 <210> 22 <211> 1102 <212> DNA
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte ID No: 7473171CB1 <400> 22 gctccgcgtt ctgtgaggtc cagtggccgc ccaggcgcga ccagatctgg gtgcgcggag 60 agcgcgcatg gcggctgtgg gaccgcggac cggccccgga accggcgccg aggctctagc 120 gctggcggca gagctgcagg gcgaggcgac gtgctccatc tgcctagagc tctttcgtga 180 gccggtgtcc gtcgagtgcg gccacagctt ctgccgcgcc tgcatagggc gctgctggga 240 gcgcccgggc gcggggtctg ttggggccgc cacccgcgcg CCCCCCttCC CaCtgCCCtg 3OO
tccgcagtgc cgcgagcccg cgcgccccag tcagctgcgg cccaaccggc agctggcggc 360 agtggccacg ctcctgcggc gcttcagcct gcccgcggct gccccgggag agcacgggtc 420 tcaggcggcc gcggcccggg cagcggctgc ccgctgcggg cagcatggcg aacccttcaa 480 gctctactgc caggacgacg gacgcgccat ctgcgtggtg tgcgaccgcg cccgcgagca 540 ccgcgagcac gccgtgctgc cgctggacga ggcggtgcag gaggccaagg agctcttgga 600 gtccaggctg agggtcttga agaaggaact ggaggactgt gaggtgttcc ggtccacgga 660 aaagaaggag agcaaggagc tgctggtgag ccaggcaccc gcaggccccc cgtgggacat 720 tacagaggcc tgagaactca gcaccagggc tcggtgtgtg tggtgttgga gtgtgtgcta 780 tggaaccgca gaatcgattt cagaaagata atagagtcca tattatatag ggtgtccaca 840 taattgttgt acaaaccaga gctttttaaa gtgaaaagca gtgctaaaat aattattgca 900 aaacaactgg cttaaactgg agctgtccca gcgaatcagg acgctcagtc actctgatat 960 tacgtaacat accagttagg gcctgcggaa gcatcttgta atggaacaca ttactatttc 1020 tgcagagaaa catggatatt caataagtgg gaatattaat acaataaaga gcctcatggc 1080 atgttttgtc aacaaaaaaa as 1102 <210> 23 <211> 2481 <212> DNA
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte ID No: 7477026CB1 <400> 23 gcagcccaca aagtgcgatg ccccgggctg cgaccggagc gagccccatg ccccgcgcgg 60 gctgacgggc cggagcccgc acggagcggc cgggcgggac aggtaccgct agagcgacat 120 gatggtggaa tctgcctcgg agacaatcag gtcggctcca tctggtcaga atggcgtggg 180 cagcctctct gggcaagccg atggcagcag cggcggggcc acagggacaa Ctgcaagtgg 240 cacgggcagg gaagtgacca cgggtgcaga cagcaatggt gagatgagtc ccgcagagct 300 gctgcacttc cagcagcaac aggctctcca agtggcccgg cagttcctgc tgcagcaggc 360 ctcaggcctg agctccccag ggaacaatga cagcaaacag tctgcctctg ctgtgcaggt 420 gcctgtgtcg gtggccatga tgtcgccgca gatgcttacc ccgcaacaga tgcagcagat 480 cctgtcgccc ccgcagctgc aggccttgct ccagcagcag caagccctca tgctccagca 540 gctacaggag tactacaaga agcagcagga gcagctccac ctgcagctcc tcacccagca 600 gcaggctggg aaaccgcagc ccaaagaggc actggggaac aagcagctgg ccttccagca 660 gcagctcctg caaatgcaac agttgcagca gcagcacctg ctcaacctgc agaggcaggg 720 gctggtcagc ctgcagccca accaagcctc ggggcccctc cagacccttc cgcaagcagc 780 tgtttgccca acagacctgc cccagctgtg gaagggcgag ggtgcccccg ggcagcctgc 840 cgaggacagc gtcaagcagg aggggctgga cctcactggc acggccgcca ccgctacctc 900 gtttgccgct CCCCCCaagg tCtCaCCCCC CCtCtCCCaC CataCCCtgC CCaaCggaCa 960 gcctactgtg ctcacatctc ggagagacag ctcttcccac gaggagaccc ccggctccca 1020 ccccctgtac ggacacggag agtgcaagtg gccaggctgt gagaccctgt gtgaagacct 1080 gggccagttt atcaaacacc tcaacacaga gcacgccctg gatgaccgga gtacagccca 1140 gtgccgggta cagatgcagg tggtgcagca gctggagatc cagctcgcca aggagagcga 1200 gcggctgcag gccatgatgg cccacctgca catgcggccc tcggagccca agcccttcag 2260 ccagccagtg accgtctctg cagcagactc attcccagat ggtctcgtgc accccccgac 1320 CtCggCCgCa gcccctgtca CCCCtCtaCg gCCCCCtggC ctgggctctg cctccctgca 1380 tggtgggggc ccagcccgtc ggagaagcag tgacaagttc tgctccccca tctcctcaga 1440 gctggcccag aatcatgagt tctacaagaa cgccgacgtc cggcccccct tcacctacgc 1500 CtCCCtCatC CgCCaggCCa tcctggaaac ccctgacagg cagctgaccc tgaatgagat 1560 ctataactgg ttcaccagga tgttcgccta tttccgcaga aacactgcca cctggaagaa 1620 cgccgtgcgc cacaacctca gcctgcacaa gtgcttcgtc cgcgtggaga acgtcaaggg 1680 tgccgtgtgg actgtggacg agcgggagta tcagaagcgg agaccgccaa agatgacagg 1740 gagccccacc ctggtgaaga acatgatctc tggcctcagc tatggagcac ttaatgccag 1800 ctaccaggcc gccctggccg agagcagctt ccccctcctc aacagccctg gcatgctgaa 1860 ccctggctcc gccagcagcc tgctgcccct cagccacgat gacgtgggtg cccccgtgga 1920 gccgctgccc agcaacggca gcagcagccc tcctcgcctc tccccgcccc agtacagcca 1980 ccaggtgcag gtgaaggagg agccagcaga ggcagaggaa gacaggcagc ccgggcctcc 2040 cctgggcgcc cctaacccca gcgcctcggg gcctccggaa gacagggacc tggaggagga 2100 gctgccggga gaagaactgt cctaagggcc tgtagtgacc ggcagggctg gggtgagacc 2160 cctcccttcc agaatccagg ccccatctcc cccaactcca cagcccctcc cgagcctcaa 2220 ggcaagtcca ggactcagac cggggaggcc cgggccagca gctcccagtg tgacctgaca 2280 aaaacacgta ggggcaggga cggtccccac ccccagggac acaacccctg gtcttggacc 2340 agtagaggac acggagggtt cagacccctc ctcagaccct ccccacatct gaaactgcct 2400 ccccccaacc accagcagca gcagggccct CCtCCCCC3C CagCtCtCCC CaCagggCCC 2460 ctcagcatca tggagacccg c 2481 <210> 24 <211> 2308 <212> DNA
<213> Homo Sapiens <220>

<221> misc_feature <223> Incyte ID No: 6428773CB1 <400> 24 cgtttgccag cgctcaggca ggagctctgg actgggcgcg ccgccgccct ggagtgaggg 60 aagcccagtg gaagggggtc ccgggagccg gctgcgatgg acgccgtctt ggaacccttc 120 ccggccgaca ggctgttccc cggatccagc ttcctggact tgggggatct gaacgagtcg 180 gacttcctca acaatgcgca ctttcctgag cacctggacc actttacgga gaacatggag 240 gacttctcca atgacctgtt cagcagcttc tttgatgacc ctgtgctgga tgagaagagc 300 cctctattgg acatggaact ggactcccct acgccaggca tccaggcgga gcacagctac 360 tccctgagcg gcgactcagc gccccagagc ccccttgtgc ccatcaagat ggaggacacc 420 acccaagatg cagagcatgg agcatgggcg ctgggacaca aactgtgctc catcatggtg 480 aagcaggagc agagcccgga gctgcccgtg gaccctctgg ctgccccctc ggccatggct 540 gccgcggccg ccatggccac caccccgctg ctgggcctca gccccttgtc caggctgccc 600 atCCCCCaCC aggCCCCggg agagatgact cagctgccag tgatcaaagc agagcctctg 660 gaggtgaacc agttcctcaa agtgacaccg gaggacctgg tgcagatgcc tccgacgccc 720 cccagcagcc atggcagtga cagcgacggc tCCCagagtC CCCgCtCtCt gCCCCCCtCC 780 agccctgtca ggcccatggc gcgctcctcc acggccatct CCaCCtCCCC aCtCCtCaCt 840 gcccctcaca aattacaggg gacatcaggg ccactgctcc tgacagagga ggagaagcgg 900 accctgattg ctgagggcta ccccatcccc acaaaactcc ccctcaccaa agccgaggag 960 aaggccttga agagagtccg gaggaaaatc aagaacaaga tctcagccca ggagagccgt 1020 cgtaagaaga aggagtatgt ggagtgtcta gaaaagaagg tggagacatt tacatctgag 1080 aacaatgaac tgtggaagaa ggtggagacc ctggagaatg ccaacaggac cctgctccag 1140 cagctgcaga aactccagac tctggtcacc aacaagatct ccagacctta caagatggcc 1200 gccacccaga ctgggacctg cctcatggtg gcagccttgt gctttgttct ggtgctgggc 1260 tCCCtCgtgC CCtgCCttCC CgagttCtCC tccggctccc agactgtgaa ggaagacccc 1320 ctggccgcag acggcgtcta cacggccagc cagatgccct cccgaagcct cctattctac 1380 gatgacgggg caggcttatg ggaagatggc cgcagcaccc tgctgcccat ggagccccca 1440 gatggctggg aaatcaaccc cggggggccg gcagagcagc ggccccggga ccacctgcag 1500 catgatcacc tggacagcac ccacgagacc accaagtacc tgagtgaggc ctggcctaaa 2560 gacggtggaa acggcaccag ccccgacttc tcccactcca aggagtggtt ccacgacagg 1620 gatctgggcc ccaacaccac catcaaactc tcctaggcca tgccaagacc caggacatag 1680 gacggacccc tggtacccag aagaggagtt cttgctcact aacccggatc cgcctcgtgc 1740 ccctgcctcc tggagcttcc cattccagga gaaaaggctc cacttcccag cccttccttg 1800 cccctgacat ttggactctt cccttgggcc gaccactctg ttctcattct ccttcccacc 1860 aacatccatc cgtccttctc agacaaacca ctcactgggt accccacctc ctctctcata 1920 tgcccaacac gaCCaCtgCC tCCCtgCCCC CaCaCCtgCa CCCaaaCaga CaCatCaaCg 1980 CaCCCCaCtC aCagaCaCCC CttaCCCCaC CCCCa.Ctgta cagagaccaa gaacagaaat 2040 tgtttgtaaa taatgaacct tattttttat tattgccaat cccctaagat attgtatttt 2100 acaaatctcc ctcttccctt cgcccctccc ttgttttata ttttatgaag ttagtgcggg 2160 ctttgctgct ccctggccca ggaaagaggg actacctgac cctcacctgg cacccccctg 2220 ctgctgccca agccgctggg cctttttaat tgccaaactg ctctcttcat cagctcagca 2280 catgctttaa gaaagcaaaa ccaaaaaa 2308 <210> 25 <211> 4369 <212> DNA
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte ID No: 2749402CB1 <400> 25 gcaatgagcg gcgcccggag gctgtgacct gcgcgcggcg gcccgaccgg ggcccctgaa 60 tggcggctcg ctgaggcggc ggcggcggcg gcggctcagg ctcctcgggg cgtggcgtgg 120 cggtgaaggg gtgatgctgt tcaagctcct gcagagacag acctatacct gcctgtccca 180 caggtatggg ctctacgtgt gcttcttggg cgtcgttgtc accatcgtct ccgccttcca 240 gttcggagag gtggttctgg aatggagccg agatcaatac catgttttgt ttgattccta 300 tagagacaat attgctggaa agtcctttca gaatcggctt tgtctgccca tgccgattga 360 cgttgtttac acctgggtga atggcacaga tcttgaacta ctgaaggaac tacagcaggt 420 cagagaacag atggaggagg agcagaaagc aatgagagaa atccttggga aaaacacaac 480 ggaacctact aagaagagtg agaagcagtt agagtgtttg ctaacacact gcattaaggt 540 gccaatgctt gtcctggacc cagccctgcc agccaacatc accctgaagg acctgccatc 600 tctttatcct tcttttcatt ctgccagtga cattttcaat gttgcaaaac caaaaaaccc 660 ttctaccaat gtctcagttg ttgtttttga cagtactaag gatgttgaag atgcccactc 720 tggactgctt aaaggaaata gcagacagac agtatggagg ggctacttga caacagataa 780 agaagtccct ggattagtgc taatgcaaga tttggctttc ctgagtggat ttccaccaac 840 attcaaggaa acaaatcaac taaaaacaaa attgccagaa aatctttcct ctaaagtcaa 900 actgttgcag ttgtattcag aggccagtgt agcgcttcta aaactgaata accccaagga 960 ttttcaagaa ttgaataagc aaactaagaa gaacatgacc attgatggaa aagaactgac 1020 cataagtcct gcatatttat tatgggatct gagcgccatc agccagtcta agcaggatga 1080 agacatctct gccagtcgtt ttgaagataa cgaagaactg aggtactcat tgcgatctat 1140 cgagaggcat gcaccatggg ttcggaatat tttcattgtc accaacgggc agattccatc 1200 ctggctgaac cttgacaatc ctcgagtgac aatagtaaca caccaggatg tttttcgaaa 1260 tttgagccac ttgcctacct ttagttcacc tgctattgaa agtcacattc atcgcatcga 1320 agggctgtcc cagaagttta tttacctaaa tgatgatgtc atgtttggga aggatgtctg 1380 gccagatgat ttttacagtc actccaaagg ccagaaggtt tatttgacat ggcctgtgcc 1440 aaactgtgcc gagggctgcc caggttcctg gattaaggat ggctattgtg acaaggcttg 1500 taataattca gcctgcgatt gggatggtgg ggattgctct ggaaacagtg gagggagtcg 1560 ctatattgca ggaggtggag gtactgggag tattggagtt ggacagccct ggcagtttgg 1620 tggaggaata aacagtgtct cttactgtaa tcagggatgt gcgaattcct ggctcgctga 1680 taagttctgt gaccaagcat gcaatgtctt gtcctgtggg tttgatgctg gcgactgtgg 1740 gcaagatcat tttcatgaat tgtataaagt gatccttctc ccaaaccaga ctcactatat 1800 tattccaaaa ggtgaatgcc tgccttattt cagctttgca gaagtagcca aaagaggagt 1860 tgaaggtgcc tatagtgaca atccaataat tcgacatgct tctattgcca acaagtggaa 1920 aaccatccac ctcataatgc acagtggaat gaatgccacc acaatacatt ttaatctcac 1980 gtttcaaaat acaaacgatg aagagttcaa aatgcagata acagtggagg tggacacaag 2040 ggagggacca aaactgaatt ctacggccca gaagggttac gaaaatttag ttagtcccat 2100 aacacttctt ccagaggcgg aaatcctttt tgaggatatt cccaaagaaa aacgcttccc 2160 gaagtttaag agacatgatg ttaactcaac aaggagagcc caggaagagg tgaaaattcc 2220 cctggtaaat atttcactcc ttccaaaaga cgcccagttg agtctcaata ccttggattt 2280 gcaactggaa catggagaca tcactttgaa aggatacaat ttgtccaagt cagccttgct 2340 gagatcattt ctgatgaact cacagcatgc taaaataaaa aatcaagcta taataacaga 2400 tgaaacaaat gacagtttgg tggctccaca ggaaaaacag gttcataaaa gcatcttgcc 2460 aaacagctta ggagtgtctg aaagattgca gaggttgact tttcctgcag tgagtgtaaa 2520 agtgaatggt catgaccagg gtcagaatcc acccctggac ttggagacca cagcaagatt 2580 tagagtggaa actcacaccc aaaaaaccat aggcggaaat gtgacaaaag aaaagccccc 2640 atctctgatt gttccactgg aaagccagat gacaaaagaa aagaaaatca cagggaaaga 2700 aaaagagaac agtagaatgg aggaaaatgc tgaaaatcac ataggcgtta ctgaagtgtt 2760 acttggaaga aagctgcagc attacacaga tagttacttg ggctttttgc catgggagaa 2820 aaaaaagtat ttccaagatc ttctcgacga agaagagtca ttgaagacac aattggcata 2880 cttcactgat agcaaaaata ctgggaggca actaaaagat acatttgcag attccctcag 2940 atatgtaaat aaaattctaa atagcaagtt tggattcaca tcgcggaaag tccctgctca 3000 catgcctcac atgattgacc ggattgttat gcaagaactg caagatatgt tccctgaaga 3060 atttgacaag acgtcatttc acaaagtgcg ccattctgag gatatgcagt ttgccttctc 3120 ttatttttat tatctcatga gtgcagtgca gccactgaat atatctcaag tctttgatga 3180 agttgataca gatcaatctg gtgtcttgtc tgacagagaa atccgaacac tggctaccag 3240 aattcacgaa ctgccgttaa gtttgcagga tttgacaggt ctggaacaca tgctaataaa 3300 ttgctcaaaa atgcttcctg ctgatatcac gcagctaaat aatattccac caactcagga 3360 atcctactat gatcccaacc tgccaccggt cactaaaagt ctagtaacaa actgtaaacc 3420 agtaactgac aaaatccaca aagcatataa ggacaaaaac aaatataggt ttgaaatcat 3480 gggagaagaa gaaatcgctt ttaaaatgat tcgtaccaac gtttctcatg tggttggcca 3540 gttggatgac ataagaaaaa accctaggaa gtttgtttgc ctgaatgaca acattgacca 3600 caatcataaa gatgctcaga cagtgaaggc tgttctcagg gacttctatg aatccatgtt 3660 ccccatacct tcccaatttg aactgccaag agagtatcga aaccgtttcc ttcatatgca 3720 tgagctgcag gaatggaggg cttatcgaga caaattgaag ttttggaccc attgtgtact 3780 agcaacattg attatgttta ctatattctc attttttgct gagcagttaa ttgcacttaa 3840 gcggaagata tttcccagaa ggaggataca caaagaagct agtcccaatc gaatcagagt 3900 atagaagatc ttcatttgaa aaccatctac ctcagcattt actgagcatt ttaaaactca 3960 gcttcacaga gatgtctttg tgatgtgatg cttagcagtt tggcccgaag aaggaaaata 4020 tccagtacca tgctgttttg tggcatgaat atagcccact gaccaggaat tatttaacca 4080 acccactgaa aacttgtgtg ttgagcagct ctgaactgat tttactttta aagaatttgc 4140 tcatggacct gtcatccttt ttataaaaag gctcactgac aagagacagc tgttaatttc 4200 ccacagcaat cattgcagac taactttatt aggagaagcc tatgccagct gggagtgatt 4260 gctaagaggc tccagtcttt gcattccaaa gccttttgct aaagttttgc actttttcct 4320 ttttcatttc ccattttcaa gtagttacct aagttaacta gttacttcg 4369 <210> 26 <211> 1445 <212> DNA
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte ID No: 118539CB1 <400> 26 aacagtcccc ggtgctttgc aagggttcag attgcagaac agtcacatat gggtatttgt 60 agttataaat acttgaccgt tctctagggg tggggtttcc aactctttaa acaaccctgt 120 tgcctgttat caagctgact ttttgctcca agtgccagaa ttgattgtgg ttaggacatc 180 ttacgctgag aaataaataa aatggaacat aatgggtctg cttcaaatgc tgataaaatc 240 caccagaatc gcctgtcgag tgttacagaa gatgaagacc aagacgctgc tcttaccatt 300 gtgactgtgc tggacaaagt agcctccatc gtggacagtg tgcaggcaag ccagaagaga 360 atagaagaga gacacaggga aatggaaaat gccataaaat ccgtccagat tgacctgttg 420 aagctttcac agtcgcatag caatacaggg catatcatta acaaattgtt tgagaaaacc 480 cgaaaagtta gtgctcacat taaagatgtg aaagcccggg tggagaagca acaaattcat 540 gttaaaaaag ttgaagtcaa gcaagaggaa ataatgaaga aaaacaaatt ccgcgtggta 600 atattccagg agaagtttcg gtgtccgaca tccctgtctg ttgttaaaga cagaaaccta 660 actgagaacc aagaagagga tgatgatgat atctttgatc ccccagtaga tctgtcttcg 720 gatgaagaat attatgttga agaaagcaga tctgccaggc ttaggaagtc aggcaaggag 780 cacattgata atatcaagaa ggcattttcc aaagaaaaca tgcagaagac acggcagaat 840 cttgacaaga aagtgaacag aattagaact agaatagtga ccccggagag gagagagagg 900 ctaaggcagt caggagagag gctgagacag tcaggggaga ggctgagaca gtcaggggag 960 aggtttaaga aatctatttc taatgcagct ccctcaaagg aagcttttaa gatgcgcagc 1020 ctcaggaaag gtaaggaccg aacagtggct gaaggtgagg aatgtgccag ggagatgggt 1080 gtggacatca ttgccaggag cgagtctctg ggccccatca gtgagctcta ctctgatgag 1140 ctcagtgaac cagaacacga ggcagccagg ccggtgtatc ctccccatga aggaagggaa 1200 atccccaccc ccgagccttt aaaagttact tttaaatctc aggtgaaagt agaggatgat 1260 gaatctcttt tgttagattt aaagcactca tcgtaaagag gaattaagta tatcctaaat 1320 atgaatctcc taatcatgca gttttagttt gaatagtgta gtcgtctaca tttctgtgcc 1380 atgtaggaaa acataaatgt attttttttc ttatatttaa aatcttgaag ataatataaa 1440 taut 1445 <210> 27 <211> 1608 <212> DNA
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte ID No: 4005918CB1 <400> 27 ggcagcgggg gcgcggggag gggggcgtcg tcgtgggtac aattgcgcag gggcaaaggt 60 cgagaggtcg cgctgccgcc gttttatttg aagacatcgt ccagttctga ccatggactc 120 gcagccatcg gcccttagtt tccatcccct ctagtgggcc ttcgggggct ctactgacgt 180 ccctccttcc cttggtaccg ggccggggaa gtgttctcgg gcgcgggagg ttccgcatgc 240 ccaggcctgg ccaggggaga tgaccgatcc gtcgctgggg ctgacagtcc ccatggcgcc 300 gcctctggcc ccgctccctc cccgggaccc aaacggggcg ggatccgagt ggagaaagcc 360 cggggccgtg agcttcgccg acgtggccgt gtacttctcc cgggaggagt ggggctgcct 420 gcggcccgcg cagagggccc tgtaccggga cgtgatgcgg gagacctacg gccacctggg 480 cgcgctcgga gtcggaggca gcaagccggc gctcatctcc tgggtggagg agaaggccga 540 actgtgggat ccggctgccc aggatccgga ggtggcgaag tgtccgacag aagcggaccc 600 agcagattcc agaaacaagg aagaggaaag acaaagggaa gggacgggag ccctggagaa 660 gcccgaccct gtggccgccg ggtctcctgg gctgaaggct ccccaagccc cctttgccgg 720 gttggagcag ctgtccaagg cccggcgccg gagtcgcccc cgcttttttg cccacccccc 780 tgtcccccga gctgaccagc gtcacggctg ctacgtgtgc gggaagagct tcgcctggcg 840 ctccacactg gtggagcaca tttacagcca caggggcgag aagcccttcc actgcgcaga 900 ctgcggcaag ggcttcggcc acgcttcctc cctgagcaaa caccgggcca tccatcgtgg 960 ggagcggccc caccgctgtc ccgagtgtgg tcgggccttc atgcgccgca cggcgctgac 1020 ttctcacctg cgcgttcaca ctggcgagaa gccctaccgc tgcccgcagt gtggccgctg 1080 cttcggcctg aagaccggca tggccaagca ccaatgggtc catcggcccg ggggcgaggg 1140 gcgtaggggc cggcgccctg gggggctgtc tgtgaccctg actcctgtcc gcggggacct 1200 ggacccgcct gtgggcttcc agctgtatcc agagatattc caggaatgtg ggtgacggcc 1260 taaaaagtga ccatctagac attgtgggcg gcccgagatg ggctcagggg cccgaacctc 1320 tgcagcggcc tgcagggagg tcccagaatc caccgcaaga gctggcctgg ggtgcggaca 1380 gtctgatctt gggctctcag cagcctcttc tgccagcacc ttgctccccg ctgccctggg 1440 ctctccaagg ccccctttgc tgaggcaggg ctgaggtgag aaccccccag acctccatac 1500 agggaagcaa aagctgtttc tcctcccaga gatgctaaga ggattgaggt agagaagaac 1560 cttgttttct ctgttgtctt tttcttttta cttttttaat tttttgag 1608 <210> 28 <211> 2275 <212> DNA
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte ID No: 5435937CB1 <400> 28 ggctgagggg gcggggcccg gcgcttcgtc caatcagggg cgctgggcgg ggcctgccga 60 gtgtccatcg gggagggcga aggccctgct ctcctcggtt cccggctcca ggcggcgagc 120 tgaggttggg agcctggctt tcccctccga gagggttcag gtgcctctgc catagcttct 180 gtcgcctgtg ctgtgacccg cactggtcgt gggagtcacc tgaaaggcaa gaaatggatt 240 cagtggcctt tgaggatgtg gctgtgacct tcacccaaga ggagtgggct ttgctggatc 300 cttcccagaa aaatctctgt agagatgtga tgcaagaaac cttcaggaac ctggcctcta 360 tagggaaaaa atggaaacec cagaacatat atgtagagta cgaaaatcta aggagaaacc 420 taagaattgt gggagagaga ctctttgaaa gtaaagaagg tcatcagcat ggagaaattt 480 tgacccaggt tccagatgac atgctgaaga aaacaactac tggagtaaaa tcatgcgaaa 540 gcagtgtgta tggagaagta ggcagtgctc attcatctct taataggcac atcagagatg 600 acactggaca caaggcatat gagtatcaag aatatggaca gaaaccatat aaatgtaaat 660 actgtaaaaa acctttcaac tgtctctcct ctgttcagac acatgaaagg gctcatagtg 720 gaaggaaact ctatgtttgt gaggaatgcg gaaaaacatt tatttcccat tcaaaccttc 780 aaagacacag gataatgcac cgtggagatg gaccttataa gtgtaaattt tgtgggaaag 840 ccttgatgtt tctcagtttg tatcttatcc acaaacgaac tcacactgga gagaaaccat 900 atcaatgtaa acagtgtggt aaagccttta gtcattctag tagccttcga atacatgaaa 960 gaactcacac tggggagaag ccttataaat gtaatgaatg tgggaaagca ttccatagtt 1020 ccacatgcct tcatgctcat aaaagaactc acactgggga gaagccatat gaatgtaaac 1080 agtgtgggaa agccttcagc tcttcccatt cctttcaaat acatgaaaga actcacacgg 1140 gggagaagcc atatgaatgt aaggaatgtg gaaaagcatt caagtgtccc agttctgttc 2200 gcagacatga aagaacccac tctaggaaaa aaccctatga atgtaaacat tgtgggaaag 1260 tattatctta tcttaccagc tttcaaaacc acttgggaat gcacactgga gagatatctc 1320 ataaatgtaa gatatgtggg aaagcctttt attctcccag ttcacttcaa acacatgaaa 1380 aaactcacac tggagagaaa ccctataaat gcaaccaatg tggtaaagcc tttaattctt 1440 ccagttcctt ccgatatcat gaaagaactc acactggaga gaaaccttac gagtgtaagc 1500 aatgtgggaa agccttcaga tctgcctcac tccttcaaac acatggtagg actcacacgg 1560 gagagaaacc ctatgcatgt aaggaatgtg gaaaaccatt tagtaatttc tctttctttc 1620 aaatacatga aaggatgcac agagaagaga agccgtatga atgtaagggt tatgggaaaa 2680 cattcagttt gcccagttta tttcatagac atgaaaggac tcacactgga ggaaaaacct 1740 atgaatgcaa gcagtgtggc agatccttca actgttcgag ctcctttcga tatcatggaa 1800 ggactcacac tggagagaaa ccctatgaat gcaagcaatg tggaaaagcc ttcagatctg 1860 cctcacagct tcaaattcat ggaaggactc acactggaga gaaaccttat gaatgtaagc 1920 agtgtgggaa agcctttgga tctgcctcac accttcaaat gcatggaagg actcacactg 1980 gagagaaacc ctatgaatgt aagcagtgtg ggaagtcttt tggatgtgcc tcgcgacttc 2040 aaatgcatgg aaggactcac actggagaga aaccgtataa atgtaagcaa tgtgggaaag 2100 cttttggatg tccctcaaac cttcgaaggc atggaaggac tcacactgga gagaaaccct 2160 ataaatgtaa ccaatgtggt aaagtcttta gatgttcttc acaacttcaa gtgcatggaa 2220 gggctcactg catagacacc ccataacccc aggctttagg aggctgaggt ggggg 2275 <210> 29 <211> 4277 <212> DNA
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte ID No: 7503560CB1 <220>
<221> unsure <222> 4266 <223> a, t, c, g, or other <400> 29 gggagctgca atgagcggcg cccggaggct gtgacctgcg cgcggcggcc cgaccggggc 60 ccctgaatgg cggctcgctg aggcggcggc ggcggcggcg gctcaggctc ctcggggcgt 120 ggcgtggcgg tgaaggggtg atgctgttca agctcctgca gagacagacc tatacctgcc 180 tgtcccacag gtatgggctc tacgtgtgct tcttgggcgt cgttgtcacc atcgtctccg 240 ccttccagtt cggagaggtg gttctggaat ggagccgaga tcaataccat gttttgtttg 300 attcctatag agacaatatt gctggaaagt cctttcagaa tcggctttgt ctgcccatgc 360 cgattgacgt tgtttacacc tgggtgaatg gcacagatct tgaactactg aaggaactac 420 agcaggtcag agaacagatg gaggaggagc agaaagcaat gagagaaatc cttgggaaaa 480 acacaacgga acctactaag aagagtgaga agcagttaga gtgtttgcta acacactgca 540 ttaaggtgcc aatgcttgtc ctggacccag ccctgccagc caacatcacc ctgaaggacc 600 tgccatctct ttatccttct tttcattctg ccagtgacat tttcaatgtt gcaaaaccaa 660 aaaacccttc taccaatgtc tcagttgttg tttttgacag tactaaggat gttgaagatg 720 cccactctgg actgcttaaa ggaaatagca gacagacagt atggaggggc tacttgacaa 780 cagataaaga agtccctgga ttagtgctaa tgcaagattt ggctttcctg agtggatttc 840 caccaacatt caaggaaaca aatcaactaa aaacaaaatt gccagaaaat ctttcctcta 900 aagtcaaact gttgcagttg tattcagagg ccagtgtagc gcttctaaaa ctgaataacc 960 ccaaggattt tcaagaattg aataagcaaa ctaagaagaa catgaccatt gatggaaaag 1020 aactgaccat aagtcctgca tatttattat gggatctgag cgccatcagc cagtctaagc 1080 aggatgaaga catctctgcc agtcgttttg aagataacga agaactgagg tactcattgc 1140 gatctatcga gaggcatgca ccatgggttc ggaatatttt cattgtcacc aacgggcaga 1200 ttccatcctg gctgaacctt gacaatcctc gagtgacaat agtaacacac caggatgttt 1260 ttcgaaattt gagccacttg cctaccttta gttcacctgc tattgaaagt cacattcatc 1320 gcatcgaagg gctgtcccag aagtttattt acctaaatga tgatgtcatg tttgggaagg 1380 atgtctggcc agatgatttt tacagtcact ccaaaggcca gaaggtttat ttgacatggc 1440 cttttggtgg aggaataaac agtgtctctt actgtaatca gggatgtgcg aattcctggc 1500 tcgctgataa gttctgtgac caagcatgca atgtcttgtc ctgtgggttt gatgctggcg 1560 actgtgggca agatcatttt catgaattgt ataaagtgat ccttctccca aaccagactc 1620 actatattat tccaaaaggt gaatgcctgc cttatttcag ctttgcagaa gtagccaaaa 1680 gaggagttga aggtgcctat agtgacaatc caataattcg acatgcttct attgccaaca 1740 agtggaaaac catccacctc ataatgcaca gtggaatgaa tgccaccaca atacatttta 1800 atctcacgtt tcaaaataca aacgatgaag agttcaaaat gcagataaca gtggaggtgg 1860 acacaaggga gggaccaaaa ctgaattcta cagcccagaa gggttacgaa aatttagtta 1920 gtcccataac acttcttcca gaggcggaaa tcctttttga ggatattccc aaagaaaaac 1980 gcttcccgaa gtttaagaga catgatgtta actcaacaag gagagcccag gaagaggtga 2040 aaattcccct ggtaaatatt tcactccttc caaaagacgc ccagttgagt ctcaatacct 2100 tggatttgca actggaacat ggagacatca ctttgaaagg atacaatttg tccaagtcag 2160 ccttgctgag atcatttctg atgaactcac agcatgctaa aataaaaaat caagctataa 2220 taacagatga aacaaatgac agtttggtgg ctccacagga aaaacaggtt cataaaagca 2280 tcttgccaaa cagcttagga gtgtctgaaa gattgcagag gttgactttt cctgcagtga 2340 gtgtaaaagt gaatggtcat gaccagggtc agaatccacc cctggacttg gagaccacag 2400 caagatttag agtggaaact cacacccaaa aaaccatagg cggaaatgtg acaaaagaaa 2460 agcccccatc tctgattgtt ccactggaaa gccagatgac aaaagaaaag aaaatcacag 2520 ggaaagaaaa agagaacagt agaatggagg aaaatgctga aaatcacata ggcgttactg 2580 aagtgttact tggaagaaag ctgcagcatt acacagatag ttacttgggc tttttgccat 2640 gggagaaaaa aaagtatttc caagatcttc tcgacgaaga agagtcattg aagacacaat 2700 tggcatactt cactgatagc aaaaatactg ggaggcaact aaaagataca tttgcagatt 2760 ccctcagata tgtaaataaa attctaaata gcaagtttgg attcacatcg cggaaagtcc 2820 ctgctcacat gcctcacatg attgaccgga ttgttatgca agaactgcaa gatatgttcc 2880 ctgaagaatt tgacaagacg tcatttcaca aagtgcgcca ttctgaggat atgcagtttg 2940 ccttctctta tttttattat ctcatgagtg cagtgcagcc actgaatata tctcaagtct 3000 ttgatgaagt tgatacagat caatctggtg tcttgtctga cagagaaatc cgaacactgg 3060 ctaccagaat tcacgaactg ccgttaagtt tgcaggattt gacaggtctg gaacacatgc 3120 taataaattg ctcaaaaatg cttcctgctg atatcacgca gctaaataat attccaccaa 3180 ctcaggaatc ctactatgat cccaacctgc caccggtcac taaaagtcta gtaacaaact 3240 gtaaaccagt aactgacaaa atccacaaag catataagga caaaaacaaa tataggtttg 3300 aaatcatggg agaagaagaa atcgctttta aaatgattcg taccaacgtt tctcatgtgg 3360 ttggccagtt ggatgacata agaaaaaacc ctaggaagtt tgtttgcctg aatgacaaca 3420 ttgaccacaa tcataaagat gctcagacag tgaaggctgt tctcagggac ttctatgaat 3480 ccatgttccc cataccttcc caatttgaac tgccaagaga gtatcgaaac cgtttccttc 3540 atatgcatga gctgcaggaa tggagggctt atcgagacaa attgaagttt tggacccatt 3600 gtgtactagc aacattgatt atgtttacta tattctcatt ttttgctgag cagttaattg 3660 cacttaagcg gaagatattt cccagaagga ggatacacaa agaagctagt cccaatcgaa 3720 tcagagtata gaagatcttc atttgaaaac catctacctc agcatttact gagcatttta 3780 aaactcagct tcacagagat gtctttgtga tgtgatgctt agcagtttgg cccgaagaag 3840 gaaaatatcc agtaccatgc tgttttgtgg catgaatata gcccactgac caggaattat 3900 ttaaccaacc cactgaaaac ttgtgtgttg agcagctctg aactgatttt acttttaaag 3960 aatttgctca tggacctgtc atccttttta taaaaaggct cactgacaag agacagctgt 4020 taatttccca cagcaatcat tgcagactaa ctttattagg agaagcctat gccagctggg 4080 agtgattgct aagaggctcc agtctttgca ttccaaagcc ttttgctaaa gttttgcact 4140 tttttttttt catttcccat ttttaagtag ttactaagtt aactagttat tcttgcttct 4200 gagtataacg aattgggatg tctaaaccct atttttatag atgttattta aataatgcag 4260 catttncacc tcttttt 4277 <210> 30 <211> 6417 <212> DNA
<213> Homo sapiens <220>
<221> misc_feature <223> Incyte ID No: 7472378CB1 <400> 30 atgtcaaaaa agttcgttta caaccttagg aaaactacac gttcagttgt~tggagttcca 60 ccaaacacta ataggccccc acatcccgtt agacgtcctg actcccttct cccgatttcg 120 gaagaaccca aatcaatatc ttcccaaacc cccaatatgg actcgggaaa cgattctgcc 180 cgccccactc catcccctct ggcgcccact gtcagtggta ttagctcctt aatttcaact 240 acgttcaagc ctaaagatat catggcattt gttgagcatt tgccaacctt tgatggtaca 300 cctcgtctat tggacaggtt tatcactagc gtagaagaaa tcctgatgct catcagggga 360 gctgaccaaa caccgtatgg cctgcttact ctgaggacca tcaggaacaa aatcattgat 420 agggccgacg aagccttgga actggcaaat acccccttgg tttgggatga gattaaaagc 480 aatctcatcc gcctctactc gagcaagaaa agcgaggcca acttgttaag cgagcttaac 540 acattttcgg acaacctgac cttgggccaa ctgttctttg gtatatcaaa ggtgagaagc 600 caactcttct ccatactcaa aaacagcgaa cacaacaaca ctgttgtaga tgcaaaaaag 660 gttgtctaca acgaggtttg tctcaatgct tttatgactg gtttgaagga acctctcaag 720 actttcgtca ggataaagtc cccttctaca cttgaacagg cgtacgagca atgccaaata 780 gagcagacct tatatagggc acaaaacaag cgaaccaaca gaccagagca gggacccaat 840 ggatcagaca ataaaaccta ccgaaatagc tacgacagca attaccgcag cggacgtaac 900 gaccgaaatg accgtagggg accctactct aactctaact ctaactctaa ctCtggccaa 960 aatagaccat ttaattcaca caatcgcaca ccccaatccg gcaccaagga caaccgggcc 1020 aatacatcaa acccctttcg agcaccttca catagtttga ataatataga ggagaaccct 1080 caacctgatt cgaattttca gcaaacggcc tcgggaaacc aacagggctc tacacactcc 1140 ttcatcgacc caaaatatgt cgaccctagg aactgtgtga ccttagatac gcccataaca 1200 ctcaaaacag ccctgaacag ttttaaaata tatcaaaacg tctctatacc atttccaccg 1260 gaattccaaa tcacgggcaa aatgaccctt ctacctttca agttccactc ttattttgac 1320 ggattgatag gaatggactt attatcttac ctaaaaacag aaatagattt acttaaccta 1380 aatctaaaaa ccccaagtac cattataccc ttatggaccc acagtaactc aacttcaaac 1440 gtatttaata tctctggaca tacgaaaact attttgccac taccagtgga aaccaaacag 1500 ggcgacttct acatcgattc aattacaatc aatgatgact taataatatc agacgggatt 1560 tataatgccc aaaacaatat tgctaatttc gttatcacaa actatagcga gagggatcag 1620 ttattgtacc tcgagagccc gataaaaggc atgccatact ccacggccaa caatgttgaa 1680 cttttcagta tcacttcaga caccccacag ccccaaaact ccgcagcgtc gttacaagcc 1740 cttggcgtcg atcacctctc ctctgaagag aaacaaagcc tactttcact ttgcaaaagt 1800 tatctagata tcttctacaa tgaagacaaa tcattgacct tcaccaacaa gattacacac 1860 acgattaaaa ccacggacga cacccccatt catacaaaat cttatagata tccttacatt 1920 cataaagagg aggtcaaaaa acaaatagag gcaatgttaa atcaggacat tatcaaatcc 1980 agttattccc cgtggagcgc ccccgtctgg gtcgtcccaa agaaaatcac tcctacggga 2040 gagcaaaaat ggcgtctagt tatcgattat agaaaactca acgagaagac tatatccgat 2100 agatatccaa tacctaacat cgcggatatc ttagacagat tgggcaaagc caaatatttc 2160 tccacacttg atctggcaag tggattccat cagatagaaa tgaatcccga cgacacaccc 2220 aaaactgcat ttacagtaga ggggggccac tacgagttca ttagaatgcc gtttggcctc 2280 aaaaatgccc cagccacatt ccaaagggtg atggacaata tttttggaga ccttatcgga 2340 actatctgcc tagtttacct agatgatata ataattttct caacctcctt acaagaacac 2400 ttcatacact tgaaaactat ttttggaaga ctcagatctg ccaactttaa agtccaactc 2460 acaaaatcct acttcctcag gcgggagaca gaattccttg gccacatcgt ttcacaagaa 2520 ggtgttaggc caaatcccaa taagatcgaa gctataaaaa actttccatg tccccacagt 2580 aaaaagtcaa ttaagtcttt cctaggcttg ttgggatatt acagaaaatt tatcagagat 2640 tttgcgagac ttacccaacc catgacacaa aaattaaggg gaaacaataa atcgatcata 2700 atagatgatg aattcaaaaa ggcctttgaa tattgcaaaa ccttactgtc taacgaccca 2760 atcctccaat acccggactt tacaaaacct ttcacactaa ccacggacgc aagtaatttc 2820 gcaataggag ctgtcctatc ccaaggtccg gtgcatagtg ataggcccgt atgttttgct 2880 agtagaacct tgtcggctgc ggaaacaaat tattccacaa ttgagaagga aatgctggcc 2940 attatatggg cggtccaata cttcagaccc tacctctttg gcaggagatt cactataatc 3000 accgatcaca aaccactaac ttggttaatg aatttcaaac aaccaaattc taaaatagtt 3060 aggtggagac tccagcttca ggagtacgat ttcgaagtcg tctacaagaa aggctctcaa 3120 aatgtaattg ctgatgctct cagtagacca gaggcctctg tcaaccataa cgaagcccta 3180 tcaattcctc aaaatgtttg ccccatctca gagaaacccc ttaatgattt taatattcag 3240 ctcctgttca aaataacccc agatacaaat aacgccacac tgaccccgtt taaacacaaa 3300 cttaggaggg aattctgtaa acccaatttt cagtatgacg acgtagtttg cattcttagg 3360 cagtcgttaa aaccaaacaa gacatgcgcg gtatttgccc ccgaccacat ttttcaaatg 3420 gtggaacaag cctaccaaac ctacttctca gcccacagtc aatttaaact cattagatgt 3480 ttgatcttcc tccccgaaat tactgatagt acggagatcg aaaaaattat aaccgactat 3540 cactataata gtaaccatcg agggatcgat gaaacatatt tacacataaa acgacaacag 3600 ttcttcccac atatgaagga gagaataact cagttaattc gaaaatgtga aacatgttta 3660 aaattaaaat acgacagaca acctcaaaag atcacttacc aaatatccga actaccttca 3720 aaaccgttgg acatcttaca tatagacatt tatactatta acaaaaatta taaccttact 3780 attatcgata aattttctaa atttgcggct gcctacccta taactaatag gaattgcatt 3840 aacgtagtta aagccttaaa acatttcatt tcccaatttg gtattcccaa aaagctgatc 3900 tatgatcagg gagcagaatt cgctagcgat atgttcaata agttctgcac tcaatttaac 3960 attgacctac acgttacgtc ctttcaacaa tcctctagta actctcccgt tgaacggctt 4020 cactcgacac taactgagat ttacagaata atacttgacg tcaggaaaca acagaaaCtc 4080 agtagcgagc atgacgagat aatgtccgaa accctaatca catataataa cgctattcat 4140 tctgcaacta aacatacccc ctttgaacta tttaacggac gtactcatat attcaaccaa 4200 acaatccagt tcaataacga acacgactac ttaacgaaat taaatgaatt tcgcgagaag 4260 ttgtaccccc tcatcacgga caaactttca aatgacgtag ttaggagaac cctaaaatta 4320 aatgaaaccc gaacagaccc cgtagaccta caaccagaca ctttagtcct taggaaggaa 4380 aacagacgta ataagattac acccaggttt tcgattcaca aagtcaaaca cgacaaaggt 4440 catacattga taactgctag gaatcaaaaa ctacacaaat caaaaattcg aaaaacagtt 4500 ttgaaaaaag acaaaagcaa caacgctatc catgtccatt atttaaatga taacgcccct 4560 atagccaaga tagaactagg gaaagcctta ctaattgaga ggtacaaaat aattagtcat 4620 gtaatcaacc tacaagacta cagcagatgt atggaacaat tccatctgac cattaataaa 4680 tttaaccccg attccacgtt gacggactcc gtcacaattt taaaaaccaa attaacccaa 4740 gcccaagtaa agctcaaagc ccttacacct tcatatagaa acaaacgggg tttgattaac 4800 ggattgggga gtctagtaaa ggtggttacc ggcaacatgg atgccaacga caataaagaa 4860 atacatgaag aacttgacaa tataaagaaa aattccgaag tcagtaacga caatctccaa 4920 aaacaagtaa tgtttaacaa cgaaatactt atccggttcg aaaatatcac ggaccatata 4980 aataatgaac aaattttgat aagtaaattc tttgatacct cacaaaacaa aatatacaaa 5040 cacttaaact tacaagatac ccttctggaa gaaatacaat atttaaatag gattaattat 5100 aacatagaat tattcattaa ccacctaaac gacataacag aaagtatgct attggcgaaa 5160 ataaatataa ttcccaagtt catcctaaat gaacaagaaa tggataaaat aaaaacaata 5220 ctggaaaaac aaaatatcac agtcaaaaat gaacaaagta tatacaattt cctacaaatg 5280 aatacactaa attacgaaca aaagattatt tttaatatca aagtcccaat ttttaaacaa 5340 ccttttcata ccctcgccag actagttcca ttaccaataa ataacacata ttttgtaata 5400 accccaaatt acctagctta taatattaat aataagaaat ttcatatgac ccgtaaatgc 5460 cccaaactgg ataatacatt cttgtgcgac gagaacttct acgttgatac accacagaac 5520 aacacatgcc tggaacacct tttgaacgga gaaaacagtt cctgcgatgt acgggaaacc 5580 ggccccatca ccgacgtgtt cgaggcagag agaggttaca tcttcgcatt caacgtgaac 5640 aaactgaagg tatccctaac aaacggctcc gagctctcaa taatggggtc agccatcatc 5700 agatacatta acgaaacaat acagattaac ggtatcgatt acgacggcac ggttgacacg 5760 ttccctgaac agacggattt tgatcttccc cccatgcgaa aagtaactag gaataccact 5820 attacggtac taagcctaga aaaactgcac ctcgaagcca cccaaacaat ggataaaatc 5880 ctggccgtcc atcacaatac tatacagcac acctggacac tctacactct gctcggattg 5940 gtaacgttcc tagcagtcat cttatggctg caccgacgaa cgaaacacat cgtccacatc 6000 cacgaggatc atcacgtttt gaccccaatt gcaaaaagta aaggtaaggt attaattcgc 6060 gataagctca tggagcggcg taaccgtcgc acagaaagga cagagaaagc gcggatctgg 6120 gaagtgacgg acagaacggt caggacctgg attggggagg cggttgccgc cgctgctgct 6180 gacggtgtga cgttctctgt tccggtcaca ccacatacgt tccgccattc ctatgcgatg 6240 cacatgctgt atgccggtat accgctgaaa gttctgcaaa gcctgatggg acataagtcc 6300 atcagttcaa cggaagtcta cacgaaggtt tttgcgctgg atgtggctgc ccggcaccgg 6360 gtgcagtttg cgatgccgga gtctgatgcg gttgcgatgc tgaaacaatt atcctga 6417 <210> 31 <211> 1194 <212> DNA
<213> Homo sapiens <220>
<221> misC_feature <223> Incyte ID No: 1306049CB1 <400> 31 gccgcgcgcg ccgagggagg agcgggcgcc gggggccggc tggcgcgggg gctccgaccc 60 tgcccggctt ggcgatggag tttccggacc tcggcgctca ctgttcggag ccgagctgtc 120 agcgcttgga ttttctgccg cttaagtgtg atgcctgctc aggcatcttc tgcgcagacc 280 atgtggccta cgcccagcat cactgtggat ctgcttacca aaaggatatc caggtacctg 240 tgtgccctct ctgtaatgtg cctgtgcctg tggccagagg ggagccccct gaccgtgctg 300 tgggagagca cattgacaga gactgtcgct atgatccagc acagcaaaaa cgtaagatct 360 tcaccaataa gtgtgaacgc gctggctgcc ggcagcgaga aatgatgaaa ctgacctgtg 420 aacgctgtag ccgaaacttc tgcatcaagc accggcatcc actggaccat gattgctctg 480 gggaggggca cccaaccagc cgggcaggac ttgctgccat ctccagagca caagctgtgg 540 cttctacaag cactgtcccc agcccaagtc aaaccatgcc ttcctgtacc tctcccagca 600 gagccacaac ccgatctccg tcctggacag cccctccagt gattgctttg cagaatggcc 660 tgagtgagga tgaagctctg cagcgggccc tggaaatgtc cctggcagaa accaaacccc 720 aggttccaag ttgtcaggag gaagaagacc tagctttagc acaagcactg tcagccagtg 780 aggcagaata ccagcggcag caggcccaga gccgcagctc gaagccgtcc aactgcagcc 840 tgtgctaggg ccctgggctt ggggagggag gttcacctga ggaggactgt ggccctcaca 900 cctctagggt acacagggag aggaggcccg gagcaccctg gagggcagag acaagcggga 960 gtgatgtgga ggtcgccctg ggagcctctg gaaggccttg ctagtgctcc agctgcatgg 1020 aagagagcgg ctagcaactg ttccctggtt gggccctcag tggatgctgg ccaggcccta 1080 ctcttagccc cttcatcatg tcatctccct tatgctggag ctgccccgat gtggagtggg 1140 caggaagggg cctggaaaaa ataaaggatc ttggcagttg ataaaaaaaa aaaa 1194 <210> 32 <211> 2801 <212> DNA
<213> Homo sapiens <220>
<221> misc_feature <223> Incyte ID No: 3187174CB1 <400> 32 accttctttc cctgaagtgg ctggggttcc tgtttccttc tttgattgac aacttgtgtt 60 aaccctcgca catctctggg ccaatttttg cttgaaaatg gcagctcccg agcagccgct 120 tgcgatatca aggggatgca cgagctcctc ctcgctttcc ccgcctcggg gcgaccgaac 180 ccttctggtc aggcacctgc cggctgagct tactgctgag gagaaagagg acttgctgaa 240 gtacttcggg gctcagtctg tgcgggtcct gtcagataag gggcgactga aacatacagc 300 ttttgccaca ttccctaatg aaaaagcagc tataaaggca ttgacaagac tccatcaact 360 gaaactttta ggtcatactt tagtcgttga atttgcaaaa gagcaagatc gagttcactc 420 cccatgtccc acttcaggtt ctgaaaaaaa aaaaaggtct gatgaccctg tcgaagatga 480 taaagaaaaa aaagaacttg gttatttaac agtagaaaat ggaattgcac caaaccatgg 540 gctgactttt cctttaaatt catgcctcaa gtatatgtac ccaccacctt ccagcacaat 600 cctagcaaac attgtaaatg ccttggcaag cgtgcctaag ttctatgtac aggtccttca 660 tcttatgaat aaaatgaatt tgcccacacc ttttggacca attactgcgc gacctcccat 720 gtatgaagac tatatgccat tgcatgcacc tcttccaccc acatctcctc agccacctga 780 ggaacctcct ttgccagacg aggatgagga attatctagt gaagaatcag aatatgaaag 840 cactgatgat gaggaccgac agagaatgaa caaattaatg gaactagcaa atcttcagcc 900 caaaagacct aaaacaataa agcagcgcca tgtgagaaaa aaaaaaaaaa aaaaggatat 960 gttgaataca cctttgtgtc cttcacacag cagtttacat ccagtgctgt taccttcaga 1020 tgtatttgac caaccacaac ctgtaggtaa caaaagaatt gaattccata tatctaccga 1080 catgccagct gcatttaaga aagatttaga aaaggaacaa aattgtgagg aaaaaaatca 1140 tgatttacct gctactgaag ttgatgcatc caatatagga tttggaaaaa tcttccccaa 1200 agctaatttg gacatcacag aggagattaa agaagactct gatgaaatgc cttcagaatg 1260 tatttctaga agggaattgg aaaagggcag aatttctaga gaagaaatgg aaacactttc 1320 agttttcaga agttatgaac cgggtgaacc aaactgtaga atttatgtaa agaatttagc 1380 taaacatgtt caagaaaagg accttaaata tatttttgga agatatgttg acttttcatc 1440 agaaacacag cggatcatgt ttgatatacg tttgatgaaa gaaggtcgta tgaaaggaca 1500 agctttcatt ggacttccta atgaaaaagc agcagcaaaa gccttaaagg aagctaatgg 1560 atatgtgctt tttggaaaac ccatggtggt tcagtttgct cgatctgcta gaccaaaaca 1620 agatcctaag gaaggaaaaa gaaagtgtta aaaattaata aagaagcatt cctgcgtaaa 1680 tactgctgta atactgtcat gcaaagtgta tcctttcttg tcgtatcctt tttggggcag 1740 tgtttttttg tttttttcct agaaatgttt gtccttcccc cacctgttga tccaggttaa 1800 ggaatacttt tttacacttt attcaaatga aatatttcta aaattatttg tatagactga 1860 acagatcttt tatgtgtttt tagatttgtt gttgaatttt ctgtgctgtc ctttatataa 1920 ttttttgagg gaaagttagt gaatcaggtc aacttactta gagaatgtgt tcatttactt 1980 taacccagaa tacagtcttg tttcttctat ttgtatgttt cctaaaccta attcaataac 2040 atatgctttc tgttgtgtaa tatatctggt ttaggtattt ataatgtgtt taaaatttgg 2100 gcaaaggaaa tgtttttctt ttaaaaagta cttacattga aaattaagat gtctggatta 2160 ctatgtaaat tctagagagt agcagacctc tcatctgaag tcttagtgaa tctcttttga 2220 catagatagc aatagaagta tctttcttct ttcccctttc tttttctaaa caagagaaga 2280 aaagcgtaat agaggggaga acacataatg cccactaagg gtagtgcatt aaggaaaaac 2340 agtcttggca ggtatatagg aatagtggtt tccagactgg ttgatgaccg taatcaccaa 2400 gaacagtggt tctcagtctt ggctgcacat tgcagtgatc tggaacttaa atactaattc 2460 taaaagggtg cagtggctca tacctgtaat cccagcactt tgcaagtccg agatgggaga 2520 atcacttgag cccaggagtt tgagaccagc ctgggcaatg tagggagacc ctgtccctac 2580 aaaaaataca aaaattagcc aagtgtggtg gcttgcacct ctggtctcag ctacttggga 2640 tgctaaggca ggaggattac ttgagcccca gaggttgagg ttgcagtgaa ccatgatcac 2700 accactgcat tctagcctgg gtgacagagt gagaccctct ccctcctaaa aaaatcctta 2760 agaaatatat tgatgcttgg ttcctttggt cagaattttg a 2801

Claims (87)

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:1-16, 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:1-2 and SEQ ID NO:4-16, c) a polypeptide comprising a naturally occurring amino acid sequence at least 95%
identical to an amino acid sequence of SEQ ID NO:3, d) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1-16, and e) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1-16.

2. An isolated polypeptide of claim 1 comprising an amino acid sequence selected from the group consisting of SEQ ID NO:1-16.

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:17-32.

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:1-16.

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:17-32, 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:17-18 and SEQ ID NO:20-32, c) a polynucleotide comprising a naturally occurring polynucleotide sequence at least 95% identical to the polynucleotide sequence of SEQ ID NO:19, d) a polynucleotide complementary to a polynucleotide of a), e) a polynucleotide complementary to a polynucleotide of b), f) a polynucleotide complementary to a polynucleotide of c), and g) an RNA equivalent of a)-f).

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

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

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

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

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

18. A composition of claim 17, wherein the polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NO:1-16.

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:1-16, or an immunogenic fragment thereof, under conditions to elicit an antibody response, b) isolating antibodies from said animal, and c) screening the isolated antibodies with the polypeptide, thereby identifying a polyclonal antibody which binds specifically to a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:1-16.

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:1-16, or an immunogenic fragment thereof, under conditions to elicit an antibody response, b) isolating antibody producing cells from the animal, c) fusing the antibody producing cells with immortalized cells to form monoclonal antibody-producing hybridoma cells, d) culturing the hybridoma cells, and e) isolating from the culture monoclonal antibody which binds specifically to a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:1-16.

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

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

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

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

44. A method of detecting a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:1-16 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:1-16 in the sample.

45. A method of purifying a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:1-16 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:1-16.

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 polynucleotide of claim 12, comprising the polynucleotide sequence of SEQ ID
NO:17.

73. A polynucleotide of claim 12, comprising the polynucleotide sequence of SEQ ID
NO:18.

74. A polynucleotide of claim 12, comprising the polynucleotide sequence of SEQ ID
NO:19.

75. A polynucleotide of claim 12, comprising the polynucleotide sequence of SEQ ID
NO:20.

76. A polynucleotide of claim 12, comprising the polynucleotide sequence of SEQ ID
NO:21.

77. A polynucleotide of claim 12, comprising the polynucleotide sequence of SEQ ID
NO:22.

78. A polynucleotide of claim 12, comprising the polynucleotide sequence of SEQ ID
NO:23.

79. A polynucleotide of claim 12, comprising the polynucleotide sequence of SEQ ID
NO:24.

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

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

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

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

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

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

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

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