EP1506304A2 - Nucleic acid-associated proteins - Google Patents

Abstract

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

Description

NUCLEIC ACID-ASSOCIATED PROTEINS

TECHNICAL FIELD

The invention relates to novel nucleic acids, nucleic acid-associated proteins encoded by these nucleic acids, and to the use of these nucleic acids and proteins in the diagnosis, treatment, and prevention of cell proliferative, neurological, developmental, and autoinimune/iiiflammatory disorders, and infections. The invention also relates to the assessment of the effects of exogenous compounds on the expression of nucleic acids and nucleic acid-associated proteins.

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. Transcription Factors

Transcriptional regulatory proteins are essential for the control of gene expression. Some of these proteins function as transcription factors that initiate, activate, repress, or terminate gene transcription. Transcription factors generally bind to the promoter, enhancer, and upstream regulatory regions of a gene in a sequence-specific manner, although some factors bind regulatory elements within or downstream of a gene coding region. Transcription factors may bind to a specific region of DNA singly or as a complex with other accessory factors. (Reviewed in Lewin, B. (1990) Genes IV, Oxford University Press, New York, NY, and Cell Press, Cambridge, MA, pp. 554-570.)

The double helix 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 β 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 α helices connected at a fixed angle by a short chain of amino acids. One of the helices binds to the major groove. Helix-turn-helix motifs are exemplified by the homeobox motif which is present in homeodomain proteins. These proteins are critical for specifying the anterior-posterior body axis during development and are conserved throughout the animal kingdom. The Antennapedia and Ultrabithorax proteins of Drosophila melanogaster are prototypical homeodomain proteins. (Pabo, CO. and R.T. Sauer (1992) Annu. Rev. Biochem. 61:1053-1095.)

The zinc finger motif, which binds zinc ions, generally contains tandem repeats of about 30 amino acids consisting of periodically spaced cysteine and histidine residues. Examples of this sequence pattern, designated C2H2 and C3HC4 ("RING" finger), have been described. (Lewin, supra.) Zinc finger proteins each contain an oc helix and an antiparallel β sheet whose proximity and conformation are maintained by the zinc ion. Contact with DNA is made by the arginine preceding the helix and by the second, third, and sixth residues of the helix. Variants of the zinc finger motif include poorly defined cysteine-rich motifs which bind zinc or other metal ions. These motifs may not contain histidine residues and are generally nonrepetitive. The zinc finger motif may be repeated in a tandem array within a protein, such that the α helix of each zinc finger in the protein makes contact with the major groove of the DNA double helix. This repeated contact between the protein and the DNA produces a strong and specific DNA-protein interaction. The strength and specificity of the interaction can be regulated by the number of zinc finger motifs within the protein. Though originally identified in DNA-binding proteins as regions that interact directly with DNA, zinc fingers occur in a variety of proteins that do not bind DNA (Lodish, H. et al. (1995) Molecular Cell Biology. Scientific American Books, New York, NY, pp. 447-451). For example, Galcheva-Gargova, Z. et al. (1996) Science 272:1797-1802) have identified zinc finger proteins that interact with various cytokine receptors.

The C2H2-type zinc finger signature motif contains a 28 amino acid sequence, including 2 conserved Cys and 2 conserved His residues in a C-2-C-12-H-3-H type motif. The motif generally occurs in multiple tandem repeats. A cysteine-rich domain including the motif Asp-His-His-Cys (DHHC-CRD) has been identified as a distinct subgroup of zinc finger proteins. The DHHC-CRD region has been implicated in growth and development. One DHHC-CRD mutant shows defective function of Ras, a small membrane-associated GTP-binding protein that regulates cell growth and differentiation, while other DHHC-CRD proteins probably function in pathways not involving Ras (Bartels, D.J. et al. (1999) Mol. Cell Biol. 19:6775-6787).

Zinc-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 ammo-terminal end of zinc finger transcription factors. SCAN domains are most often linked to C2H2 zinc finger motifs through their carboxyl-terminal end. Biochemical binding studies have established the SCAN domain as a selective hetero- and homotypic oligomerization domain. SCAN domain-mediated protein complexes may function to modulate the biological function of transcription factors (Schumacher, C. et al. (2000) J. Biol. Chem. 275:17173-17179).

The KRAB (Kruppel-associated box) domain is a conserved amino acid sequence spanning approximately 75 amino acids and is found in almost one-third of the 300 to 700 genes encoding C2H2 zinc fingers. The KRAB domain is found N-terminally with respect to the finger repeats. The KRAB domain is generally encoded by two 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 KRAB-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 (Williams, A . et al. (1999) Mol. Cell Biol. 19:8526-8535). A subgroup of highly related human KRAB zinc finger proteins detectable in all human tissues is highly expressed in human T lymphoid cells (Bellefroid, E.J. et al. (1993) EMBO J. 12:1363-1374). The ZNF85 KRAB zinc finger gene, a member of the human ZNF91 family, is highly expressed in normal adult testis, in seminomas, and in the NT2/D1 teratocarcinoma cell line (Poncelet, D.A. et al. (1998) DNA Cell Biol.17:931-943).

The C4 motif is found in hormone-regulated proteins. The C4 motif generally includes only 2 repeats. A number of eukaryotic and viral proteins contain a conserved cysteine-rich domain of 40 to 60 residues (called C3HC4 zinc-finger or RING 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(l to 3)-H-x(2 to3)-C-x(2)-C-x(4 to 48)-C-x(2)-C. The PHD finger is a C4HC3 zinc-finger-like motif found in nuclear proteins thought to be involved in chromatin-mediated 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 β sheets and an helix, followed by a long loop to the C-terminal end of the finger (Ominchinski, J.G. (1993) Science 261:438-446). The helix and the loop connecting the two β-sheets contact the major groove of the DNA, while the C-terminal part, which determines the specificity of binding, wraps around into the minor groove.

The 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 finger motifs with the LIM consensus sequence. The C-terminal domain of actin- binding LIM protein shows sequence similarity to known actin-binding proteins such as dematin and villin. Actin-binding LEvI 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 murine 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 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 API involved in cell growth and the determination of cell lineage (Papavassiliou, A.G. (1995) N. Engl. J. Med. 332:45-47).

The helix-loop-helix motif (HLH) consists of a short α helix connected by a loop to a longer α helix. The loop is flexible and allows the two helices to fold back against each other and to bind to DNA. The transcription factor Myc contains a prototypical HLH motif. The NF-kappa-B Rel signature defines a family of eukaryotic transcription factors involved in oncogenesis, embryonic development, differentiation and immune response. Most transcription factors containing the Rel homology domain (RHD) bind as dimers to a consensus DNA sequence motif termed kappa-B. Members of the Rel family share a highly conserved 300 amino acid domain termed the Rel homology domain. The characteristic Rel C-terminal domain is involved in gene activation and cytoplasmic anchoring functions. Proteins known to contain the RHD domain include vertebrate nuclear factor NF-kappa-B, which is a heterodimer of a DNA-binding subunit and the transcription factor p65, mammalian transcription factor RelB, and vertebrate proto-oncogene c-rel, a protein associated with differentiation and lymphopoiesis (Kabrun, N. and Enrietto, P.J. (1994) Semin. Cancer Biol. 5:103-112).

A DNA binding motif termed ARID (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 MTA1 and protein ER1. The Caenorhabditis elegans gene egl-27 is required for embryonic patterning MTA1, a human gene with elevated expression in metastatic carcinomas, is a component of a protein complex with histone deacetylase and nucleosome remodelling activities (Solari, F. et al. (1999) Development 126:2483-2494). The ELM2 domain is usually found to the N terminus of a myb-like DNA binding domain. ELM2 is also found associated with an ARID DNA.

The Iroquois (Irx) family of genes are found in nematodes, insects and vertebrates. Irx genes usually occur in one or two genomic clusters of three genes each and encode transcriptional controllers that possess a characteristic homeodomain. The Irx genes function early in development to specify the identity of diverse territories of the body. Later in development in both Drosophila and vertebrates, the Irx genes function again to subdivide those territories into smaller domains. (For a review of Iroquois genes, see Cavodeassi, F. et al. (2001) Development 128:2847-2855.) For example, mouse and human Irx4 proteins are 83% conserved and their 63-aa homeodomain is more than 93% identical to that of the Drosophila Iroquois patterning genes. Irx4 transcripts are predominantly expressed in the cardiac ventricles. The homeobox gene Irx4 mediates ventricular differentiation during cardiac development (Bruneau, B.G. et al. (2000) Dev. Biol. 217:266-77). Histidine triad (HIT) proteins share residues in distinctive dimeric, 10-stranded half-barrel structures that form two identical purine nucleotide-binding sites. Hint (histidine triad nucleotide-binding protein)-related proteins, found in all forms of life, and fragile histidine triad (Fhit)-related proteins, found in animals and fungi, represent the two main branches of the HIT superfamily. Fhit homologs bind and cleave diadenosine polyphosphates. Fhit-Ap(n)A complexes appear to function in a proapoptotic tumor suppression pathway in epithelial tissues (Brenner C. et al. (1999) J. Cell Physiol.l81:179-187).

Most transcription factors contain characteristic DNA binding motifs, and variations on the above motifs and new motifs have been and are currently being characterized. (Faisst, S. and S. Meyer (1992) Nucleic Acids Res. 20:3-26.) 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, HI, 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. HI 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 WTl is a tumor-suppressor protein that is inactivated in children with Wil 's tumor. The oncogene bcl-6, which plays an important role in large-cell lymphoma, is also a zinc-finger protein (Papavassiliou, A.G. (1995) N. Engl. J. Med. 332:45-47). Chromosomal translocations may also produce chimeric loci that fuse the coding sequence of one gene with the regulatory regions of a second unrelated gene. Such an arrangement likely results in inappropriate gene transcription, 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. Harrison'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 (SALL1) (Engelkamp, D. and V. van : Heyningen (1996) Curr. Opin. Genet. Dev. 6:334-342; Kohlhase, J. et al. (1999) Am. J. Hum. Genet. 64:435-445).

Human acute leukemias involve reciprocal chromosome translocations that fuse the ALL-1 gene located at chromosome region llq23 to a series of partner genes positioned on a variety of human chromosomes. The fused genes encode chimeric proteins. The AF17 gene encodes a protein of 1093 amino acids, containing a leucine-zipper dimerization motif located 3' of the fusion point and a cysteine-rich domain at the N terminus that shows homology to a domain within the protein Brl40 (peregrin) (Prasad R. et al. (1994) Proc. Natl. Acad. Sci. U S A 91:8107-8111). SYNTHESIS OF NUCLEIC ACIDS Polymerases

DNA and RNA replication are critical processes for cell replication and function. DNA and RNA replication are mediated by the enzymes DNA and RNA polymerase, respectively, by a "templating" process in which the nucleptide sequence of a DNA or RNA strand is copied by complementary base-pairing into a complementary nucleic acid sequence of either DNA or RNA. However, there are fundamental differences between the two processes. DNA polymerase catalyzes the stepwise addition of a deoxyribonucleotide to the 3 -OH end of a polynucleotide strand (the primer strand) that is paired to a second (template) strand. The new DNA strand therefore grows in the 5' to 3 'direction (Alberts, B. et al. (1994) The Molecular Biology 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 polymerase. Because DNA exists as a double-stranded helix, each of the two strands may serve as a template for the formation of a new complementary strand. Each of the two daughter cells of a dividing cell therefore inherits a new DNA double helix containing one old and one new strand. Thus, DNA is said to be replicated "semiconservatively" by DNA polymerase. In addition to the synthesis of new DNA, DNA polymerase is also involved in the repair of damaged DNA as discussed below under "Ligases."

In contrast to DNA polymerase, RNA polymerase uses a DNA template strand to "transcribe" DNA into RNA using ribonucleotide triphosphates as substrates. Like DNA polymerization, RNA polymerization proceeds in a 5' to 3' direction by addition of a ribonucleoside monophosphate to the 3'-OH end of a growing RNA chain. DNA transcription generates messenger RNAs (mRNA) that carry information for protein synthesis, as well as the transfer, ribosomal, and other RNAs that have structural or catalytic functions. In eukaryotes, three discrete RNA polymerases synthesize the three different types of RNA (Alberts, supra, pp. 367-368). RNA polymerase I makes the large ribosomal RNAs, RNA polymerase II makes the mRNAs that will be translated into proteins, and RNA polymerase in makes a variety of small, stable RNAs, including 5S ribosomal RNA and the transfer RNAs (tRNA). In all cases, RNA synthesis is initiated by binding of the RNA polymerase to a promoter region on the DNA and synthesis begins at a start site within the promoter. Synthesis is completed at a stop (termination) signal in the DNA whereupon both the polymerase and the completed RNA chain are released. Ligases

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, 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 nucleotide(s) 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, 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 invaded by retroviruses, and RNase H is an important enzyme in the retroviral replication cycle. Pancreatic RNase secreted by the pancreas into the intestine hydrolyzes RNA present in ingested foods. RNase activity in serum and cell extracts is elevated in a variety of cancers and infectious diseases (Schein, CH. (1997) Nat. Biotechnol. 15:529-536). Regulation of RNase activity is being investigated as a means to control tumor angiogenesis, allergic reactions, viral infection and replication, and fungal infections. MODIFICATION OF NUCLEIC ACIDS 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, supra pp. 448-451). In RNA metabolism, "tRNA methylase" produces one of several nucleotide modifications in tRNA that affect the conformation and base-pairing of the molecule and facilitate the recognition of the appropriate mRNA codons by specific tRNAs. The primary methylation pattern is the dimethylation of guanine residues to form N,N-dimethyl guanine. Helicases and Single-stranded Binding Proteins

Helicases are enzymes that destabilize and unwind double helix structures in both DNA and RNA. Since DNA replication occurs more or less simultaneously on both strands, the two strands must first separate to generate a replication "fork" for DNA polymerase to act on. Two types of replication proteins contribute to this process, DNA helicases and single-stranded binding proteins. DNA helicases hydrolyze ATP and use the energy of hydrolysis to separate the DNA strands. Single-stranded binding proteins (SSBs) then bind to the exposed DNA strands, without covering the bases, thereby temporarily stabilizing them for templating by the DNA polymerase (Alberts, supra, pp. 255-256).

RNA helicases also alter and regulate RNA conformation and secondary structure. Like the DNA helicases, RNA helicases utilize energy derived from ATP hydrolysis to destabilize and unwind RNA duplexes. The most well-characterized and ubiquitous family of RNA helicases is the DEAD- box family, so named for the conserved B-type ATP-binding motif which is diagnostic of proteins in this family. Over 40 DEAD-box helicases have been identified in organisms as diverse as bacteria, insects, yeast, amphibians, mammals, and plants. DEAD-box helicases function in diverse processes such as translation initiation, splicing, ribosome assembly, and RNA editing, transport, and stability. Examples of these RNA helicases include yeast Drsl protein, which is involved in ribosomal RNA processing; yeast I'lFl and T1F2 and mammalian eIF-4A, which are essential to the initiation of RNA translation; and human p68 antigen, which regulates cell growth and division (Ripmaster, T.L. et al. (1992) Proc. Natl. Acad. Sci. USA 89:11131-11135; Chang, T.-H. et al. (1990) Proc. Natl. Acad. Sci. USA 87:1571-1575). These RNA helicases demonstrate strong sequence homology over a stretch of some 420 amino acids. Included among these conserved sequences are the consensus sequence for the A motif of an ATP binding protein; the "DEAD box" sequence, associated with ATPase activity; the sequence SAT, associated with the actual helicase unwinding region; and an octapeptide consensus sequence, required for RNA binding and ATP hydrolysis (Pause, A. et al. (1993) Mol. Cell Biol. 13:6789-6798). Differences outside of these conserved regions are believed to reflect differences in the functional roles of individual proteins (Chang, 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 role in the progression of neuroblastoma (Nb) and retinoblastoma (Rb) tumors (Godbout, R. et al. (1998) J. Biol. Chem. 273:21161-21168). These observations suggest that DDX1 may promote or enhance tumor progression by altering the normal secondary structure and expression levels of RNA in cancer cells. Other DEAD-box helicases have been implicated either directly or indirectly in tumorigenesis. (Discussed in Godbout, supra.) For example, murine p68 is mutated in ultraviolet light-induced tumors, and human DDX6 is located at a chromosomal breakpoint associated with B-cell lymphoma. Similarly, a chimeric protein comprised of DDX10 and NUP98, a nucleoporin protein, may be involved in the pathogenesis of certain myeloid malignancies. Topoisomerases

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 U 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, supra, pp.260-262). Type II topoisomerases are 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 multi-drug resistance (MDR) as it appears to aid in the repair of DNA damage inflicted by DNA binding agents such as doxorubicin and vincristine.

The topoisomerase I family includes topoisomerases I and III (topo I and topo HI). 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 JHs 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 EH has been suggested to play a role in meiotic recombination. A mouse topo _U 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 (Hoc and πβ) 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 Ha isoform appears to be responsible for unlinking DNA during chromosome segregation. Cell lines expressing Eoc but not Uβ suggest that πβ is dispensable in cellular processes; however, Hβ knockout mice died perinataUy due to a failure in neural development. That the major abnormalities occurred in predominantly late developmental events (neurogenesis) suggests that Hβ is needed not at mitosis, but rather during DNA repair (Yang, X. et al. (2000) Science 287:131-134).

Topoisomerases have been implicated in a number of disease states, and topoisomerase poisons have proven to be effective anti-tumor drugs for some human malignancies. Topo I is mislocalized in Fanconi's anemia, and may be involved in the chromosomal breakage seen in this disorder (Wunder, E. (1984) Hum. Genet. 68:276-281). Overexpression of a truncated topo HI in ataxia-telangiectasia (A-T) cells partially suppresses the A-T phenotype, probably through a dominant negative mechanism. This suggests that topo El is deregulated in A-T (Fritz, E. et al. (1997) Proc. Natl. Acad. Sci. USA 94:4538-4542). Topo in 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 II can break DNA in the region of the A-T gene (ATM), which controls all DNA damage-responsive cell cycle checkpoints (Kaufmann, W.K. (1998) Proc. Soc. Exp. Biol. Med. 217:327-334). The ability of topoisomerases to break DNA has been used as the basis of antitumor drugs. Topoisomerase poisons act by increasing the number of dead-end covalent DNA-enzyme complexes in the 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, 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 riot normally change the arrangement of genes in a chromosome. In site-specific recombination, the recombinase recognizes specific nucleotide sequences present in one or both of the recombining molecules. Base-pairing is not involved in this form of recombination and therefore it does not require DNA homology between the recombining molecules. Unlike general recombination, this form of recombination can alter the relative positions of nucleotide sequences in chromosomes. RNA METABOLISM

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, 28S, 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, LI, 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 Garber, 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). 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 (Dufiier and Thomas (1999) Εxp. Cell. Res. 253:100-109). Some of the ribosomal proteins, including LI, also function as translational repressors by binding to polycistronic mRNAs encoding ribosomal proteins (reviewed in Liljas, supra and Garber, 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 L13a has been shown to induce apoptosis by arresting cell growth in the G2/M phase of the cell cycle. Inhibition of expression of L13a 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 L13a, rp22 and rp23, results in severe growth retardation and death. A closely related ribosomal protein, L7, arrests cells in Gl 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 LI, 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 Biology. 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 UI, 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).

Several splicing regulatory proteins have been identified in Drosophila. Human (HsSWAP) and mouse (MmSWAP) homologs of the suppressor-of- white-apricot (su(wa)) gene have been cloned and characterized. HsSWAP and MmSWAP both have five highly homologous regions to su(wa), including an argiriine/serine-rich domain and two repeated modules that are homologous to regions in the constitutive splicing factor, SPP91/PRP21. Mammalian SWAP mRNAs are alternatively spliced at the same splice sites as in Drosophila. The splice junctions of the Drosophila and mammalian regulated introns are conserved. Thus, research suggests that the mammalian SWAP gene functions as a vertebrate alternative splicing regulator (Denhez, F. and Lafyatis, R. (1994) Biol. Chem. 269:16170-16179).

Serine- and arginine-rich pre-mRNA splicing factors (SR proteins) are phosphorylated before they regulate splicing events. SRrp86 (SR-related protein of 86 kDa) is a novel SR protein containing a single aniino-terminal RNA recognition motif and two carboxy-terminal domains rich in serine- arginine (SR) dipeptides. SRrp86 activates splicing in the presence of SRp20. However, it inhibits the in vitro and m vivo activation of specific splice sites by SR proteins, including ASF/SF2, SC35, and SRp55. Research suggests that pairwise combination of SRrp86 with specific SR proteins leads to altered splicing efficiency and differential splice site selection (Barnard, D.C. and Patton, J.G. (2000) Mol. Cell. Biol. 20:3049-3057).

Heterogeneous nuclear ribonucleoproteins (hriRNPs) have been identified mat 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 β-strands and two α-helices arranged in an /β 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 melanogaster 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 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 -untranslated (non-coding) region and (ii) the addition of a poly(A) tract to the 5 'mRNA fragment. The presence of s-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 (Rϋegsegger, 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 mitochondrial form, for each different amino acid. The 20 aaRS enzymes can be divided into two structural classes. Class I enzymes add amino acids to the 2' hydroxyl at the 3' end of tRNAs while Class II enzymes add amino acids to the 3' hydroxyl at the 3' end of tRNAs. Each class is characterized by a distinctive topology of the catalytic domain. Class I enzymes contain a catalytic domain based on the nucleotide-binding Rossman 'fold'. In particular, a consensus tetrapeptide motif is highly conserved (Prosite Document PDOC00161, Aminoacyl-transfer RNA synthetases class-I signature). Class I enzymes are specific for arginine, cysteine, glutamic acid, glutamine, isoleucine, leucine, methionine, tyrosine, tryptophan, and valine. Class II enzymes contain a central catalytic domain, which consists of a seven-stranded antiparallel β-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 π enzymes are specific for alanine, asparagine, aspartic acid, glycine, histidine, lysine, phenylalanine, proline, serine, and threonine. Certain aaRSs also have editing functions. IleRS, for example, can misactivate valine to form

Val-tRNA^πe, but this product is cleared by a hydrolytic activity that destroys the mischarged product. This editing activity is located within a second catalytic site found in the connective polypeptide 1 region (CP1), a long insertion sequence within the Rossman fold domain of Class I enzymes (Schimmel, P. et al. (1998) FASEB J. 12:1599-1609). AaRSs also play a role in tRNA processing. It has been shown that mature tRNAs are charged with their respective amino acids in the nucleus before export to the cytoplasm, and charging may serve as a quality control mechanism to insure the tRNAs are functional (Martinis, S.A. et al. (1999) EMBO J. 18:4591-4596).

Under optimal conditions, polypeptide synthesis proceeds at a rate of approximately 40 amino acid residues per second. The rate of misincorporation during translation in on the order of 10^ and is primarily the result of aminoacyl-t-RNAs being charged with the incorrect amino acid. Incorrectly charged tRNA are toxic to cells as they result in the incorporation of incorrect amino acid residues into an elongating polypeptide. The rate of translation is presumed to be a compromise between the optimal rate of elongation and the need for translational fidelity. Mathematical calculations predict that 10^ is indeed the maximum acceptable error rate for protein synthesis in a biological system (reviewed in Stryer, supra; and Watson, J. et al. (1987) The Benjamin/Cummings Publishing Co., Inc. Menlo Park, CA). A particularly error prone aminoacyl-tRNA charging event is the charging of tRNA^Gl with Gin. A mechanism exits for the correction of this mischarging event which likely has its origins in evolution. Gin 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-tRNA^Gln based on the transformation of Glu-tRNA^Gln (synthesized by Glu-tRNA synthetase, GluRS) using the enzyme Glu- tRNA^Gln amidotransferase (Glu-AdT). The reactions involved in the transamidation pathway are as follows (Curnow, A.W. et al. (1997) Nucleic Acids Symposium 36:2-4):

GluRS _tRNAoin _{+ Glu + Aτp →} Glu-tRNA^Gln + AMP + PP_i

Glu-AdT Glu-tRNA^Gln + Gin + ATP - Gln-tRNA^Gln + Glu + ADP + P A similar enzyme, Asp-tRNA^" amidotransferase, exists in Archaea, which transforms Asp- tRNA^" to Asn-tRNA^". Formylase, the enzyme that transforms Met-tRNA™⁶¹ to fMet-tRNA¹¹** in eubacteria, is likely to be a related enzyme. A hydrolytic activity has also been identified that destroys mischarged Val-tRNA^πe (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^Gln. In fact, deletion of the Glu-AdT operon in Gram positive bacteria is lethal (Curnow, A.W. et al. (1997) Proc. Natl. Acad. Sci. USA 94:11819-11826). The existence of GluRS activity in other organisms has been inferred by the high degree of conservation in translation machinery in nature; however, GluRS has not been identified in all organisms, including 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-oc, 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 5. 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 autoimmune diseases such as rheumatic arthritis, dermatomyositis and polymyositis, and correlate strongly with complicating interstitial lung disease (ILD) (Freist, W. et al. (1999) Biol. Chem. 380:623-646; Freist, W. et al.

(1996) Biol. Chem. Hoppe Seyler 377:343-356). These antibodies appear to be generated in response to viral infection, and coxsackie virus has been used to induce experimental viral myositis 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 Modifications The modified ribonucleoside, pseudouridine (ψ), is present ubiquitously in the anticodon regions of transfer RNAs (tRNAs), large and small ribosomal RNAs (rRNAs), and small nuclear RNAs (snRNAs). y is the most common of the modified nucleosides (i.e., other than G, A, U, and C) present in tRNAs. Only a few yeast tRNAs that are not involved in protein synthesis do not contain ψ (Cortese, R. et al. (1974) J. Biol. Chem. 249:1103-1108). The enzyme responsible for the conversion of uridine to ψ, pseudouridine synthase (pseudouridylate synthase), was first isolated from Salmonella typhimurium (Arena, F. et al. (1978) Nucleic Acids Res. 5:4523-4536). The enzyme has since been isolated from a number of mammals, including steer and mice (Green, CJ. 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 cation (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 (reviewed in Smith, CM. 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 ψ in the anticodon loop of tRNAs results in reduced growth in both bacteria (Singer, C.E. et al. (1972) Nature New Biol. 238:72-74) and yeast (Lecointe, F. (1998) J. Biol. Chem. 273:1316-1323), although the genetic defect is not lethal.

Another ribonucleoside modification that occurs primarily in eukaryotic cells is the conversion of guanosine to NSN^dimethylguanosine (m² ₂G) at position 26 or 10 at the base of the D-stem of cytosolic and mitochondrial tRNAs. This posttranscriptional modification is believed to stabilize tRNA structure by preventing the formation of alternative tRNA secondary and tertiary structures. Yeast tRNA^Asp 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 m² ₂G is a 63 kDa S-adenosylmethionine (SAM)-dependent tRNA N^N^-dimethyl-guanosine methyltransferase (also referred to as the TRM1 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 for base pairing at positions C11-G24 and G10-C25 immediately preceding the G26 to be modified, with other structural features of the tRNA also being required for the proper presentation of the G26 substrate (Edqvist. J. et al. (1992) Nucleic Acids Res. 20:6575-6581). Studies in yeast suggest that cells carrying a weak ochre tRNA suppressor (sup3-i) are unable to suppress translation termination in the absence of TRM activity, suggesting a role for TRM in modifying the frequency of suppression in eukaryotic cells (Niederberger, C. et al. (1999) FEBS Lett. 464:67-70), in addition to the more general function of ensuring the proper three-dimensional structures for tRNA. Translation Initiation

Initiation of translation can be divided into three stages. The first stage brings an initiator transfer RNA (Met-tRNA_f) together with the 40S ribosomal subunit to form the 43S preinitiation complex. The second stage binds the 43 S 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, elFIA and eEF3 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-tRNA_f, elFIA, eIF3, and 40S ribosomal subunit together make up the 43S preinitiation complex (Pain, supra).

Additional factors are required for binding of the 43 S 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 eIF4G. eIF4E recognizes and binds to the mRNA 5 -terminal m⁷GTP 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 eIF4E, the central third interacts with eIF4A, and the C-terminal third interacts with eIF3 bound to the 43 S 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 43 S preinitiation complex. The helicase activity of eIF4A is thought to function in removing this secondary structure to facilitate binding of the 43 S preinitiation complex (Pain, supra). Translation Elongation

Elongation is the process whereby additional amino acids are joined to the initiator methionine to form the complete polypeptide chain. The elongation factors EFl α, EFl β γ, and EF2 are involved in elongating the polypeptide chain following initiation. EFl α is a GTP-binding protein. In EFl α's GTP-bound form, it brings an aminoacyl-tRNA to the ribosome' s A site. The amino acid attached to the newly arrived aminoacyl-tRNA forms a peptide bond with the initiatior methionine. The GTP on EFl is hydrolyzed to GDP, and EFl α -GDP dissociates from the ribosome. EFl β γ binds EFl - GDP and induces the dissociation of GDP from EFl , allowing EFl α to bind GTP and a new cycle to begin.

As subsequent aminoacyl-tRNAs are brought to the ribosome, EF-G, another GTP-binding protein, catalyzes the translocation of tRNAs from the A site to the P site and finally to the E site of the ribosome. This allows the ribosome and the mRNA to remain attached during translation. Translation Termination

The release factor eRF carries out termination of translation. eRF recognizes stop codons in the mRNA, leading to the release of the polypeptide chain from the ribosome. Expression profiling

Microarrays are analytical tools used in bioanalysis. A microarray has a plurality of molecules spatially distributed over, and stably associated with, the surface of a solid support. Microarrays of polypeptides, polynucleotides, and/or antibodies have been developed and find use in a variety of applications, such as gene sequencing, monitoring gene expression, gene mapping, bacterial identification, drug discovery, and combinatorial chemistry.

One area in particular in which microarrays find use is in gene expression analysis. Array technology can provide a simple way to explore the expression of a single polymorphic gene or the expression profile of a large number of related or unrelated genes. When the expression of a single gene is examined, arrays are employed to detect the expression of a specific gene or its variants. When an expression profile is examined, arrays provide a platform for identifying genes that are tissue specific, are affected by a substance being tested in a toxicology assay, are part of a signaling cascade, carry out housekeeping functions, or are specifically related to a particular genetic predisposition, condition, disease, or disorder.

RNA Expression Array technology can provide a simple way to explore the expression of a single polymorphic gene or the expression profile of a large number of related or unrelated genes. When the expression of a single gene is examined, arrays are employed to detect the expression of a specific gene or its variants. When an expression profile is examined, arrays provide a platform for 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 measuring the toxic response to potential therapeutic compounds and of the metabolic response to therapeutic agents. Diseases treated with steroids and disorders caused by the metabolic response to treatment with steroids include adenomatosis, cholestasis, cirrhosis, hemangioma, Henoch-Schonlein purpura, hepatitis, hepatoceUular and metastatic carcinomas, idiopathic thrombocytopenic purpura, porphjria, sarcoidosis, and Wilson disease. Response may be measured by comparing both the levels and sequences expressed in tissues from subjects exposed to or treated with steroid compounds such as mifepristone, progesterone, beclomethasone, medroxyprogesterone, budesonide, prednisone, dexamethasone, betamethasone, or danazol with the levels and sequences expressed in normal untreated tissue.

Steroids are a class of lipid-soluble molecules, including cholesterol, bile acids, vitamin D, and hormones, that share a common four-ring structure based on cyclopentanoperhydrophenanthrene and that carrry out a wide variety of functions. Cholesterol, for example, is a component of ceU membranes that controls membrane fluidity. It is also a precursor for bile acids which solubilize lipids and facilitate absorption in the smaU intestine during digestion. Vitamin D regulates the absorption of calcium in the small intestine and controls the concentration of calcium in plasma. Steroid hormones, produced by the adrenal cortex, ovaries, and testes, include glucocorticoids, mineralocorticoids, androgens, and estrogens. They control various biological processes by binding to intraceUular receptors that regulate transcription of specific genes in the nucleus. Glucocorticoids, for example, increase blood glucose concentrations by regulation of gluconeogenesis in the liver, increase blood concentrations of fatty acids by promoting lipolysis in adipose tissues, modulate sensitivity to catcholamines in the central nervous system, and reduce inflammation. The principal mineralocorticoid, aldosterone, is produced by the adrenal cortex and acts on ceUs of the distal tubules of the kidney to enhance sodium ion reabsorption. Androgens, produced by the interstitial cells of Leydig in the testis, include the male sex hormone testosterone, which triggers changes at puberty, the production of sperm and maintenance of secondary sexual characteristics. Female sex hormones, estrogen and progesterone, are produced by the ovaries and also by the placenta and adrenal cortex of the fetus during pregnancy. Estrogen regulates female reproductive processes and secondary sexual characteristics. Progesterone regulates changes in the endometrium during the menstrual cycle and pregnancy.

Steroid hormones are widely used for fertility control and in anti-inflammatory treatments for physical injuries and diseases such as arthritis, asthma, and auto-immune disorders. Progesterone, a naturaUy occurring progestin, is primarily used to treat amenorrhea, abnormal uterine bleeding, or as a contraceptive. Endogenous progesterone is responsible for inducing secretory activity in the endometrium of the estrogen-primed uterus in preparation for the implantation of a fertilized egg and for the maintenance of pregnancy. It is secreted from the corpus luteum in response to luteinizing hormone (LH). The primary contraceptive effect of exogenous progestins involves the suppression of the midcycle surge of LH. At the ceUular level, progestins diffuse freely into target ceUs and bind to the progesterone receptor. Target ceUs include the female reproductive tract, the mammary gland, the hypothalamus, and the pituitary. Once bound to the receptor, progestins slow the frequency of release of gonadotropin releasing hormone from the hypothalamus and blunt the pre-ovulatory LH surge, thereby preventing foUicular maturation and ovulation. Progesterone has minimal estrogenic and androgenic activity. Progesterone is metabolized hepaticaUy to pregnanediol and conjugated with glucuronic acid.

Medroxyprogesterone (MAH), also known as 6α-methyl-17-hydroxyprogesterone, is a synthetic progestin with a pharmacological activity about 15 times greater than progesterone. MAH is used for the treatment of renal and endometrial carcinomas, amenorrhea, abnormal uterine bleeding, and endometriosis associated with hormonal imbalance. MAH has a stimulatory effect on respiratory centers and has been used in cases of low blood oxygenation caused by sleep apnea, chronic obstructive pulmonary disease, or hypercapnia.

Mifepristone, also known as RU-486, is an antiprogesterone drug that blocks receptors of progesterone. It counteracts the effects of progesterone, which is needed to sustain pregnancy. Mifepristone induces spontaneous abortion when administered in early pregnancy foUowed by treatment with the prostaglandin, misoprostol. Further, studies show that mifepristone at a substantiaUy lower dose can be highly effective as a postcoital contraceptive when administered within five days after unprotected intercourse, thus providing women with a "morning-after piU" in case of contraceptive failure or sexual assault. Mifepristone also has potential uses in the treatment of breast and ovarian cancers in cases in which tumors are progesterone-dependent. It interferes with steroid- dependent growth of brain meningiomas, and may be useful in treatment of endometriosis where it blocks the estrogen-dependent growth of endometrial tissues. It may also be useful in treatment of uterine fibroid tumors and Cushing's Syndrome. Mifepristone binds to glucocorticoid receptors and interferes with cortisol binding. Mifepristone also may act as an anti-glucocorticoid and be effective for treating conditions where cortisol levels are elevated such as AIDS, anorexia nervosa, ulcers, diabetes, Parkinson's disease, multiple sclerosis, and Alzheimer's disease.

Danazol is a synthetic steroid derived from ethinyl testosterone. Danazol indirectly reduces estrogen production by lowering pituitary synthesis of foUicle-stimulating hormone and LH. Danazol also binds to sex hormone receptors in target tissues, thereby exhibiting anabolic, antiestrognic, and weakly androgenic activity. Danazol does not possess any progestogenic activity, and does not suppress normal pituitary release of corticotropin or release of cortisol by the adrenal glands. Danazol is used in the treatment of endometriosis to relieve pain and inhibit endometrial ceU growth. It is also used to treat fibrocystic breast disease and hereditary angioedema.

Corticosteroids are used to relieve inflammation and to suppress the immune response. They inhibit eosinopbil, basophil, and airway epitheUal ceU function by regulation of cytokines that mediate the inflammatory response. They inhibit leukocyte infiltration at the site of inflammation, interfere in the function of mediators of the inflammatory response, and suppress the humoral immune response. Corticosteroids are used to treat aUergies, asthma, arthritis, and skin conditions. Beclomethasone is a synthetic glucocorticoid that is used to treat steroid-dependent asthma, to relieve symptoms associated with aUergic or nonaUergic (vasomotor) rhinitis, or to prevent recurrent nasal polyps foUowing surgical removal. The anti-inflammatory and vasoconstrictive effects of intranasal beclomethasone are 5000 times greater than those produced by hydrocortisone. Budesonide is a corticosteroid used to control symptoms associated with allergic rhinitis or asthma. Budesonide has high topical anti-inflammatory activity but low systemic activity. Dexamethasone is a synthetic glucocorticoid used in anti- inflammatory or immunosuppressive compositions. It is also used in inhalants to prevent symptoms of asthma. Due to its greater ability to reach the central nervous system, dexamethasone is usuaUy the treatment of choice to control cerebral edema. Dexamethasone is approximately 20-30 times more potent than hydrocortisone and 5-7 times more potent than prednisone. Prednisone is metabolized in the liver to its active form, prednisolone, a glucocorticoid with anti-inflammatory properties. Prednisone is approximately 4 times more potent than hydrocortisone and the duration of action of prednisone is intermediate between hydrocortisone and dexamethasone. Prednisone is used to treat aUograft rejection, asthma, systemic lupus erythematosus, arthritis, ulcerative colitis, and other inflammatory conditions. Betamethasone is a synthetic glucocorticoid with antiinflammatory and immunosuppressive activity and is used to treat psoriasis and fungal infections, such as athlete's foot and ringworm.

The anti-inflammatory actions of corticosteroids are thought to involve phospholipase A₂ inhibitory proteins, coUectively caUed lipocortins. Lipocortins, in turn, control the biosynthesis of potent mediators of inflammation such as prostaglandins and leukotrienes by inhibiting the release of the precursor molecule arachidonic acid. Proposed mechanisms of action include decreased IgE synthesis, increased number of β-adrenergic receptors on leukocytes, and decreased arachidonic acid metabolism. During an immediate aUergic reaction, such as in chronic bronchial asthma, aUergens bridge the IgE antibodies on the surface of mast ceUs, which triggers these ceUs to release chemotactic substances. Mast ceU influx and activation, therefore, is partiaUy responsible for the inflammation and hyperirritability of the oral mucosa in asthmatic patients. This inflammation can be retarded by administration of corticosteroids. CA3+ Steroids

Array technology can provide a simple way to explore the expression of a single polymorphic gene or the expression profile of a large number of related or unrelated genes. When the expression of a single gene is examined, arrays are employed to detect the expression of a specific gene or its variants. When an expression profile is examined, arrays provide a platform for examining which genes are tissue specific, carrying out housekeeping functions, parts of a signaling cascade, or specificaUy related to a particular genetic predisposition, condition, disease, or disorder. The potential application of gene expression profiling is particularly relevant to measuring the toxic response to potential therapeutic compounds and of the metabolic response to therapeutic agents. Diseases treated with steroids and disorders caused by the metaboUc response to treatment with steroids mclude adenomatosis, cholestasis, cirrhosis, hemangioma, Henoch-Schonlein purpura, hepatitis, hepatoceUular and metastatic carcinomas, idiopathic thrombocytopenic purpura, porphyria, sarcoidosis, and Wilson disease. Response may be measured by comparing both the levels and sequences expressed in tissues from subjects exposed to or treated with steroid compounds such as mifepristone, progesterone, beclomethasone, medroxyprogesterone, budesonide, prednisone, dexamethasone, betamethasone, or danazol with the levels and sequences expressed in normal untreated tissue.

The effects upon liver metabolism and hormone clearance mechanisms are important to understand the pharmacodynamics of a drug. The human C3 A ceU line is a clonal derivative of HepG2/C3 (hepatoma ceU hne, isolated from a 15-year-old male with liver tumor), which was selected for strong contact inhibition of growth. The use of a clonal population enhances the reproducibility of the ceUs. C3A ceUs have many characteristics of primary human hepatocytes in culture: i) expression of insulin receptor and insulin-like growth factor II receptor; ii) secretion of a high ratio of serum albumin compared with α-fetoprotein iii) conversion of ammonia to urea and glutamine; iv) metabolize aromatic amino acids; and v) prohferate in glucose-free and insulin-free medium. The C3 A ceU line is now weU established as an in vitro model of the mature human liver (Mickelson et al. (1995) Hepatology 22:866-875; Nagendra et al. (1997) Am I Physiol 272:G408-G416). Vascular endothekal activation

Activation of vascular endothekum is a central event in a wide range of physiological and disease processes such as vascular tone regulation, coagulation and thrombosis, atherosclerosis, inflammation and some infectious diseases. Blood vessel waUs are composed of two tissue layers: an endothelial ceU (EC) layer which comprises the lumenal surface of the vessel, and an underlying vascular smooth muscle ceU (VSMC) layer. Through dynamic interactions with each other and with surrounding tissues, the vascular endothelium and smooth muscle tissues maintain vascular tone, control selective permeabihty of the vascular waU, direct vessel remodeling and angiogenesis, and modulate inflammatory and immune responses.

The inflammatory response is a complex vascular reaction mediated by numerous cytokines, chemokines, growth factors, and other signaling molecules expressed by activated ECs, VSMCs and leukocytes. Inflammation protects the organism during trauma and infection, but can also lead to pathological conditions such as atherosclerosis.

Human coronary artery endotheUal ceUs (HCAECs) are primary ceUs derived from the endothekum of a human coronary artery. HCAECs are used as an experimental model for investigating the role of the endothekum in human vascular biology in vitro. Human umbikcal artery endotheUal ceUs (HUAECs) are primary ceUs derived from the endothekum of an umbikcal artery. Human uterine myometrium microvascular endothekal ceUs (UtMVECs) are primary ceUs derived from the uterine myometrium microvasculature. Human Hiac Artery Endothekal CeUs (EHAECs) are primary ceUs derived from the endothekum of an ikac artery. Human umbikcal vein endothekal ceUs (HUVECs) are a primary ceU line derived from the endothe-hum of the human umbikcal vein. ECV304 is a human endothekal line. Breast cancer

There are more than 180,000 new cases of breast cancer diagnosed each year, and the mortakty rate for breast cancer approaches 10% of aU deaths in females between the ages of 45-54 (Gish, K. (1999) AWIS Magazine 28:7-10). However the survival rate based on early diagnosis of locakzed 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, CM. 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 epithekal ceUs.

CeU lines derived from human mammary epithekal ceUs at various stages of breast cancer provide a useful model to study the process of makgnant transformation and tumor progression as it has been shown that these ceU lines retain many of the properties of their parental tumors for lengthy culture periods (Wistuba, I.I. et al. (1998) Clin. Cancer Res. 4:2931-2938). Such a model is particularly useful for comparing phenotypic and molecular characteristics of human mammary epithekal ceUs at various stages of makgnant transformation.

Estrogen stimulation plays a critical role in the development of normal mammary epithehum. Estradiol has a direct mitogenic effect on breast cancer ceUs, causing them to divide more rapidly by shortening their ceU cycle. Also, estradiol induces a large number of enzymes and other proteins involved in nucleic acid synthesis in isolated breast cancer ceU knes. Estradiol may increase the expression of the EGF receptor in response to TGF-α and EGF. In addition, estrogens may promote prokferation of tumor cells by inducing the synthesis of TGF-α and EGF, and may block growth factors that would normally inhibit tumor ceU growth. Estrogen receptor (ER) has been investigated extensively as a prognostic marker in breast cancer. Patients whose tumors display high levels of estrogen receptor have a significantly better prognosis than patients with receptor-negative tumors. W ien ER is lost or ceUs expressing the ER are selected against by therapeutic treatments, the tumor becomes more aggressive. Lung cancer

Lung cancer is the leading cause of cancer death for men and the second leading cause of cancer death for women in the U.S. Lung cancers are divided into four histopathologicaUy distinct groups. Three groups (squamous ceU carcinoma, adenocarcinoma, and large ceU carcinoma) are classified as non-smaU ceU lung cancers (NSCLCs). The fourth group of cancers is referred to as smaU ceU lung cancer (SCLC). Deletions on chromosome 3 are common in this disease and are thought to indicate the presence of a tumor suppressor gene in this region. Activating mutations in K- ras are commonly found in lung cancer and are the basis of one of the mouse models for the disease. Ovarian Cancer

Ovarian cancer is the leading cause of death from a gynecologic cancer. The majority of ovarian cancers are derived from epithekal ceUs, and 70% of patients with epithekal ovarian cancers present with late-stage disease. As a result, the long-term survival rates for this disease is very low. Identification of early-stage markers for ovarian cancer would significantly increase the survival rate. The molecular events that lead to ovarian cancer are poorly understood. Some of the known aberrations include mutation of p53 and microsatelkte instabikty. Since gene expression patterns are likely to vary when normal ovary is compared to ovarian tumors, examination of gene expression in these tissues to identify possible markers for ovarian cancer is particularly relevant to improving diagnosis, prognosis, and treatment of this disease. Colon Cancer

Colorectal cancer is the second leading cause of cancer deaths in the United States. Colon cancer is associated with aging, since 90% of the total cases occur in individuals over the age of 55. A widely accepted hypothesis is that several contributing genetic mutations must accumulate over time in an individual who develops the disease. To understand the nature of genetic alterations in colorectal cancer, a number of studies have focused on the inherited syndromes. The first known inherited syndrome, Famikal Adenomatous Polyposis (FAP), is caused by mutations in the Adenomatous Polyposis Cok gene (APC), resulting in truncated or inactive forms of the protein. This tumor suppressor gene has been mapped to chromosome 5q. The second known inherited syndrome is hereditary nonpolyposis colorectal cancer (HNPCC), which is caused by mutations in mismatch repair genes.

Although hereditary colon cancer syndromes occur in a smaU percentage of the population and most colorectal cancers are considered sporadic, knowledge from studies of the hereditary syndromes can be generaUy appked. For instance, somatic mutations in APC occur in at least 80% of ^discriminate colon tumors. APC mutations are thought to be the initiating event in the disease. Other mutations occur subsequently. Approximately 50% of colorectal cancers contain activating mutations in ras, while 85% contain inactivating mutations in p53. Changes in these genes lead to gene expression changes in colon cancer. Less is understood about downstream targets of these mutations and the role they may play in cancer development and progression. Iru^ammatorv/Immune Response

Blood vessel waUs are composed of two tissue layers: an endothekal cell (EC) layer which comprises the lumenal surface of the vessel, and an underlying vascular smooth muscle ceU (VSMC) layer. Through dynamic interactions with each other and with surrounding tissues, the vascular endothekum and smooth muscle tissues maintain vascular tone, control selective permeabikty of the vascular waU, direct vessel remodeling and angiogenesis, and modulate inflammatory and immune responses.

The inflammatory response is a complex vascular reaction mediated by numerous cytokines, chemokines, growth factors, and other signaling molecules expressed by activated ECs, VSMCs and leukocytes. Inflammation protects the organism during trauma and infection, but can also lead to pathological conditions such as atherosclerosis. The pro-inflammatory cytokines, interleukin (IL)-l and tumor necrosis factor (TNF), are secreted by a smaU number of activated macrophages or other ceUs and can set off a cascade of vascular changes, largely through their abikty to alter gene expression patterns in ECs and VSMCs. These vascular changes include vasodilation and increased permeabikty of micro vasculature, edema, and leukocyte extravasation and transmigration across the vessel waU. Ultimately, leukocytes, particularly neutrophils and monocytes/macrophages, accumulate in the extravascular space, where they remove injurious agents by phagocytosis and oxidative killing, a process accompanied by release of toxic factors, such as proteases and reactive oxygen species. Tumor necrosis factor-α (TNF-α ) is a pleiotropic factor that exerts a variety of effects, such as growth promotion, growth inhibition, angiogenesis, cytotoxicity, inflammation, and immunomodulation. This cytokine is synthesized mainly by macrophages in response to invasive stimuU as an active 26 kDa membrane-bound precursor that is cleaved proteolyticaUy to a mature 17 kDa form with the prosequence polypeptide remaining associated to the membrane. The peptide is as bioactive as a 51 kDa trimer, which can be recognized by TNF-α receptors. TNF-α receptors are present in the majority of ceU types. IL-1 and TNF induce pro-inflammatory, thrombotic, and anti- apoptotic changes in gene expression by signaling through receptors on the surface of ECs and VSMCs; these receptors activate transcription factors such as NFfcB as weU as AP-1, IRF-1, and NF-GMa, leading to alterations in gene expression. Genes known to be differentiaUy regulated in EC by Db-1 and TNF include E selectin, VCAM-1, ICAM-1, PAF, I&Bα, IAP-1, MCP-1, eotaxin, ENA- 78, G-CSF, A20, ICE, and complement C3 component. A key event in inflammation, adhesion and transmigration of blood leukocytes across the vascular endothekum, for example, is mediated by increased expression of E selectin, P selectin, ICAM-1, and VCAM-1 on activated endothekum.

Several investigators have examined changes in vascular ceU gene expression associated with various inflammatory diseases or model systems. Examining human umbikcal vein endothekal ceUs (HUVEC) activated by recombinant TNFα or conditioned medium from activated human primary monocytes, Horrevoets et al. (1999; Blood 93:3418-3431) identified 106 differentiaUy regulated genes. In a similar approach, deVries et aL (2000; JBC 275:23939-23947) identified 40 differentiaUy regulated genes in umbikcal cord artery-derived smooth muscle ceUs activated by conditioned media from cultured macrophages after stimulation with oxidized LDL particles. In both studies, many of the identified genes were already known to be involved in inflammation. Comparing expression profiles from inflammatory diseased tissues, cultured macrophages, chondrocyte ceU lines, primary chondrocytes, and synoviocytes, HeUer et al. (1997; Proc Natl Acad Sci USA 94:2150-2155) identifed candidate genes involved in inflammatory responses, including TNF, 1L-1 IL-6, IL-8 G-CSF, RANTES, and V-CAM. From this candidate gene set, tissue inhibitor of metaUoproteinase 1, ferritin hght chain, and manganese superoxide dismutase were found to be differentiaUy expressed in rheumatoid arthritis (RA) relative to inflammatory bowel disease (IBD). Further, IL-3, chemokine Groα, and metaUoproteinase matrix metaUo-elastase were expressed in both RA and IBD. Most recently, in an analysis of cultured aortic smooth muscle ceUs treated with TNFα, Haley et al. (2000; Circulation 102:2185-2189) found a 20-fold increase in eotaxin, an eosinophil chemotactic factor. The overexpression of eotaxin and its receptor CCR3 in atherosclerotic lesions was confirmed by northern analysis.

Array technology can provide a simple way to explore the expression of a single polymorphic gene or the expression profile of a large number of related or unrelated genes. When the expression of a single gene is examined, arrays are employed to detect the expression of a specific gene or its variants. When an expression profile is examined, arrays provide a platform for identifying genes that are tissue specific, carry out housekeeping functions, are part of a signaling cascade, or are specificaUy related to a particular genetic predisposition, condition, disease, or disorder. The potential appkcation 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 coronary artery disease may be compared with the levels and sequences expressed in normal vascular tissue.

DNA-based array technology is also useful as a method of analyzing ceU signaling pathways. For example, protein kinase C (PKC) is a family of serine/threonine kinases which play a critical role in many signal transduction pathways in the ceU (Kanashiro, CA. and Khaki, R.A. (1998) Clin. Exp. Pharmacol. Physiol. 25:974-985). Phorbol 12-myristate 13-acetate (PMA) is a broad activator of the protein kinase C-dependent pathways. PMA promotes tumors in ceUs by over activating PKC pathways. PKC is also affected by intraceUular calcium levels. Internal calcium flux has been shown have many effects on ceUs, particular in the areas of ceU activation and prokferation (Cole, K, and

Kohn, E. (1994) Cancer Metastasis Rev. 13:31-44). Ionomycin is a calcium ionophore that permits the entry of calcium into the ceU, hence increasing the cytosohc calcium concentration. Thus the combination of PMA and ionomycin activates two of the major signaling pathways used by mammakan ceUs to interact with their environment. In T ceUs, for example, the combination of PMA and ionomycin mimics the type of secondary signaling events ekcited during optimal T ceU activation, while in endothekal ceUs, PMA and ionomycin reproduce the effect of inflammatory mediatiors.

CeU lines are widely used in experimental biology to model human ceU behavior. lurkat, an acute T-ceU leukemia ceU line that grows actively in the absence of external stimuk, is used to study signaling in human T ceUs. ECV304, a ceU line derived from the endothekum of the human umbikcal vein, is used to study the functional biology of human endothekal ceUs. Neurological disorders

Characterization of region-specific gene expression in the human brain provides a context and background for molecular neurobiology on a variety of neurological disorders. For example, Alzheimer's disease (AD) is a progressive, neurodestructive process of the human neocortex, characterized by the deterioration of memory and higher cognitive function. A progressive and irreversible brain disorder, AD is characterized by three major pathogenic episodes involving (a) an aberrant processing and deposition of beta-amyloid precursor protein (betaAPP) to form neurotoxic beta-amyloid (betaA) peptides and an aggregated insoluble polymer of betaA that forms the senile plaque, (b) the estabkshment of intraneuronal neuritic tau pathology yielding widespread deposits of agyrophikc neurofibriUary tangles (NFT) and (c) the initiation and prokferation of a brain-specific inflammatory response. These three seemingly disperse attributes of AD etiopathogenesis are linked by the fact that proinflammatory microgka, reactive astrocytes and their associated cytokines and chemokines are associated with the biology of the microtubule associated protein tau, betaA speciation and aggregation. Missense mutations in the presenikn genes PS1 and PS2, impkcated in early onset famikal AD, cause abnormal betaAPP processing with resultant overproduction of betaA42 and related neurotoxic peptides. Specific betaA fragments such as betaA42 can further potentiate proinflammatory mechanisms. Expression of the inducible oxidoreductase cyclooxygenase-2 and cytosokc phosphokpase A2 (cPLA2) are strongly activated during cerebral ischemia and trauma, epilepsy and AD, indicating the induction of proinflammatory gene pathways as a response to brain injury. Neurotoxic metals such as aluminum and zinc, both impkcated in AD etiopathogenesis, and arachidonic acid, a major metabokte of brain cPLA2 activity, each polymerize hyperphosphorylated tau to form NFT-kke bundles. Studies have identified a reduced risk for AD in patients aged over 70 years who were previously treated with non-steroidal anti-inflammatory drugs for non-CNS afflictions that include arthritis. (For a review of the interrelationships between the mechanisms of PS 1 , PS2 and betaAPP gene expression, tau and betaA deposition and the induction, regulation and prokferation in AD of the neuroinflammatory response, see Lukiw W.J, and Bazan N.G.(2000) Neurochem. Res. 2000 25:1173-1184).

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 ceU prokferative, 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.

There is a need in the art for new compositions, including nucleic acids and proteins, for the diagnosis, prevention, and treatment of ceU prokferative, neurological, developmental, and autoimmune/inflammatory disorders, and infections.

SUMMARY OF THE INVENTION

Various embodiments of the invention provide purified polypeptides, nucleic acid-associated proteins, referred to coUectively as 'NAAP' and individuaUy 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,' 'NAAP-16,' 'NAAP-17,' 'NAAP-18,' 'NAAP- 19,' 'NAAP-20,' 'NAAP-21,' 'NAAP-22,' 'NAAP-23,' 'NAAP-24,' 'NAAP-25,' 'NAAP-26,'

'NAAP-27,' 'NAAP-28,' 'NAAP-29,' 'NAAP-30,' 'NAAP-31,' 'NAAP-32,' 'NAAP-33,' 'NAAP- 34,' 'NAAP-35,' 'NAAP-36,' 'NAAP-37,' 'NAAP-38,' 'NAAP-39,' 'NAAP-40,' 'NAAP-41,' 'NAAP-42,' 'NAAP-43,' 'NAAP-44,' 'NAAP-45,' 'NAAP-46,' 'NAAP-47,' 'NAAP-48,' 'NAAP- 49,' 'NAAP-50,' 'NAAP-51,' 'NAAP-52,' 'NAAP-53,' 'NAAP-54,' 'NAAP-55,' 'NAAP-56,' and 'NAAP-57' and methods for using these proteins and their encoding polynucleotides for the detection, diagnosis, and treatment of diseases and medical conditions. Embodiments also provide methods for utikzing the purified nucleic acid-associated proteins and/or their encoding polynucleotides for faciktating the drug discovery process, including determination of efficacy, dosage, toxicity, and pharmacology. Related embodiments provide methods for utikzing the purified nucleic acid-associated proteins and/or their encoding polynucleotides for investigating the pathogenesis of diseases and medical conditions. An embodiment provides an isolated polypeptide selected from the group consisting of a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ED NO:l- 57, b) a polypeptide comprising a naturaUy occurring amino acid sequence at least 90% identical or at least about 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NO:l-57, c) a biologicaUy active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:l-57, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:l-57. Another embodiment provides an isolated polypeptide comprising an amino acid sequence of SEQ ID NO: 1-57.

StiU another embodiment provides an isolated polynucleotide encoding a polypeptide selected from the group consisting of a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 1-57, b) a polypeptide comprising a naturally occurring amino acid sequence at least 90% identical or at least about 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NO:l-57, c) a biologicaUy active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1-57, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:l-57. In another embodiment, the polynucleotide encodes a polypeptide selected from the group consisting of SEQ ID NO: 1-57. In an alternative embodiment, the polynucleotide is selected from the group consisting of SEQ ID NO:58-l 14.

StiU another embodiment provides a recombinant polynucleotide comprising a promoter sequence operably linked to a polynucleotide encoding a polypeptide selected from the group consisting of a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ED NO: 1-57, b) a polypeptide comprising a naturaUy occurring amino acid sequence at least 90% identical or at least about 90% identical to an amino acid sequence selected from the group consisting of SEQ ED NO:l-57, c) a biologicaUy active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1-57, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1-57. Another embodiment provides a ceU transformed with the recombinant polynucleotide. Yet another embodiment provides a transgenic organism comprising the recombinant polynucleotide.

Another embodiment provides a method for producing a polypeptide selected from the group consisting of a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 1-57, b) a polypeptide comprising a naturaUy occurring amino acid sequence at least 90% identical or at least about 90% identical to an amino acid sequence selected from the group consisting of SEQ ED NO: 1-57, c) a biologicaUy active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1-57, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1-57. The method comprises a) culturing a ceU under conditions suitable for expression of the polypeptide, wherein said ceU is transformed with a recombinant polynucleotide comprising a promoter sequence operably linked to a polynucleotide encoding the polypeptide, and b) recovering the polypeptide so expressed.

Yet another embodiment provides an isolated antibody which specificaUy binds to a polypeptide selected from the group consisting of a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 1-57, b) a polypeptide comprising a naturaUy occurring amino acid sequence at least 90% identical or at least about 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NO:l-57, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:l-57, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:l-57. StiU yet another embodiment provides an isolated polynucleotide selected from the group consisting of a) a polynucleotide comprising a polynucleotide sequence selected from the group consisting of SEQ ID NO:58-114, b) a polynucleotide comprising a naturaUy occurring polynucleotide sequence at least 90% identical or at least about 90% identical to a polynucleotide sequence selected from the group consisting of SEQ ID NO:58-l 14, c) a polynucleotide complementary to the polynucleotide of a), d) a polynucleotide complementary to the polynucleotide of b), and e) an RNA equivalent of a)-d). In other embodiments, the polynucleotide can comprise at least about 20, 30, 40, 60, 80, or 100 contiguous nucleotides. Yet another embodiment provides a method for detecting a target polynucleotide in a sample, said target polynucleotide being selected from the group consisting of a) a polynucleotide comprising a polynucleotide sequence selected from the group consisting of SEQ ID NO:58-114, b) a polynucleotide comprising a naturaUy occurring polynucleotide sequence at least 90% identical or at least about 90% identical to a polynucleotide sequence selected from the group consisting of SEQ ID NO:58-l 14, 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 specificaUy hybridizes to said target polynucleotide, under conditions whereby a hybridization complex is formed between said probe and said target polynucleotide or fragments thereof, and b) detecting the presence or absence of said hybridization complex. In a related embodiment, the method can include detecting the amount of the hybridization complex. In stiU other embodiments, the probe can comprise at least about 20, 30, 40, 60, 80, or 100 contiguous nucleotides. StiU yet another embodiment provides a method for detecting a target polynucleotide in a sample, said target polynucleotide being selected from the group consisting of a) a polynucleotide comprising a polynucleotide sequence selected from the group consisting of SEQ ID NO:58-114, b) a polynucleotide comprising a naturaUy occurring polynucleotide sequence at least 90% identical or at least about 90% identical to a polynucleotide sequence selected from the group consisting of SEQ ID NO:58-l 14, 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) amphfying said target polynucleotide or fragment thereof using polymerase chain reaction ampkfication, and b) detecting the presence or absence of said ampkfied target polynucleotide or fragment thereof. In a related embodiment, the method can include detecting the amount of the ampkfied target polynucleotide or fragment thereof.

Another embodiment provides a composition comprising an effective amount of a polypeptide selected from the group consisting of a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ED NO:l-57, b) a polypeptide comprising a naturaUy occurring amino acid sequence at least 90% identical or at least about 90% identical to an amino acid sequence selected from the group consisting of SEQ ED NO:l-57, c) a biologicaUy active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ED NO: 1-57, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1-57, and a pharmaceuticaUy acceptable excipient. In one embodiment, the composition can comprise an amino acid sequence selected from the group consisting of SEQ ED NO: 1-57. Other embodiments provide a method of treating a disease or condition associated with decreased or abnormal expression of functional NAAP, comprising administering to a patient in need of such treatment the composition.

Yet another embodiment provides a method for screening a compound for effectiveness as an agonist of a polypeptide selected from the group consisting of a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ED NO:l-57, b) a polypeptide comprising a naturaUy occurring amino acid sequence at least 90% identical or at least about 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NO:l-57, c) a biologicaUy active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:l-57, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ DZ) NO:l-57. The method comprises a) exposing a sample comprising the polypeptide to a compound, and b) detecting agonist activity in the sample. Another embodiment provides a composition comprising an agonist compound identified by the method and a pharmaceuticaUy acceptable excipient. Yet another embodiment provides a method of treating a disease or condition associated with decreased expression of functional NAAP, comprising administering to a patient in need of such treatment the composition.

StiU yet another embodiment provides a method for screening a compound for effectiveness as an antagonist of a polypeptide selected from the group consisting of a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 1-57, b) a polypeptide comprising a naturaUy occurring amino acid sequence at least 90% identical or at least about 90% identical to an amino acid sequence selected from the group consisting of SEQ ED NO: 1-57, c) a biologicaUy active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:l-57, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:l-57. The method comprises a) exposing a sample comprising the polypeptide to a compound, and b) detecting antagonist activity in the sample. Another embodiment provides a composition comprising an antagonist compound identified by the method and a pharmaceuticaUy acceptable excipient. Yet another embodiment provides a method of treating a disease or condition associated with overexpression of functional NAAP, comprising administering to a patient in need of such treatment the composition.

Another embodiment provides a method of screening for a compound that specificaUy 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 ED NO: 1-57, b) a polypeptide comprising a naturaUy occurring amino acid sequence at least 90% identical or at least about 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NO:l-57, c) a biologicaUy active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:l-57, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1-57. 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 specificaUy binds to the polypeptide.

Yet another embodiment provides a method of screening for a compound that modulates the activity of a polypeptide selected from the group consisting of a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:l-57, b) a polypeptide comprising a naturaUy occurring amino acid sequence at least 90% identical or at least about 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NO:l-57, c) a biologicaUy active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ED NO:l-57, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consistmg of SEQ ID NO:l-57. The method comprises a) combining the polypeptide with at least one test compound under conditions permissive for the activity of the polypeptide, b) assessing the activity of the polypeptide in the presence of the test compound, and c) comparing the activity of the polypeptide in the presence of the test compound with the activity of the polypeptide in the absence of the test compound, wherein a change in the activity of the polypeptide in the presence of the test compound is indicative of a compound that modulates the activity of the polypeptide. StiU yet another embodiment provides a method for screening a compound for effectiveness in altering expression of a target polynucleotide, wherein said target polynucleotide comprises a polynucleotide sequence selected from the group consisting of SEQ ID NO:58-114, the method comprising a) exposing a sample comprising the target polynucleotide to a compound, b) detecting altered expression of the target polynucleotide, and c) comparing the expression of the target polynucleotide in the presence of varying amounts of the compound and in the absence of the compound.

Another embodiment provides a method for assessing toxicity of a test compound, said method comprising a) treating a biological sample containing nucleic acids with the test compound; b) hybridizing the nucleic acids of the treated biological sample with a probe comprising at least 20 contiguous nucleotides of a polynucleotide selected from the group consisting of i) a polynucleotide comprising a polynucleotide sequence selected from the group consisting of SEQ DD NO:58-l 14, ii) a polynucleotide comprising a naturaUy occurring polynucleotide sequence at least 90% identical or at least about 90% identical to a polynucleotide sequence selected from the group consisting of SEQ DD NO:58-l 14, 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 NO:58-114, ii) a polynucleotide comprising a naturally occurring polynucleotide sequence at least 90% identical or at least about 90% identical to a polynucleotide sequence selected from the group consisting of SEQ DD NO:58-l 14, iii) a polynucleotide complementary to the polynucleotide of i), iv) a polynucleotide complementary to the polynucleotide of ii), and v) an RNA equivalent of i)-iv).

Alternatively, the target polynucleotide can comprise a fragment of a polynucleotide selected from the group consisting of i)-v) above; c) quantifying the amount of hybridization complex; and d) comparing the amount of hybridization complex in the treated biological sample with the amount of hybridization complex in an untreated biological sample, wherein a difference in the amount of hybridization complex in the treated biological sample is indicative of toxicity of the test compound.

BRIEF DESCRIPTION OF THE TABLES

Table 1 summarizes the nomenclature for full length polynucleotide and polypeptide embodiments of the invention. Table 2 shows the GenBank identification number and annotation of the nearest GenBank homolog, and the PROTEOME database identification numbers and annotations of PROTEOME database homologs, for polypeptide embodiments of the invention. The probabikty scores for the matches between each polypeptide and its homolog(s) are also shown.

Table 3 shows structural features of polypeptide embodiments, including predicted motifs and domains, along with the methods, algorithms, and searchable databases used for analysis of the polypeptides.

Table 4 ksts the cDNA and/or genomic DNA fragments which were used to assemble polynucleotide embodiments, along with selected fragments of the polynucleotides.

Table 5 shows representative cDNA kbraries for polynucleotide embodiments.

Table 6 provides an appendix which describes the tissues and vectors used for construction of the cDNA kbraries shown in Table 5. Table 7 shows the tools, programs, and algorithms used to analyze polynucleotides and polypeptides, along with appkcable descriptions, references, and threshold parameters.

Table 8 shows single nucleotide polymorphisms found in polynucleotide sequences of the invention, along with aUele frequencies in different human populations.

DESCRIPTION OF THE INVENTION

Before the present proteins, nucleic acids, and methods are described, it is understood that embodiments of the invention are not limited to the particular machines, instruments, materials, and methods described, as these may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the invention.

As used herein and in the appended claims, the singular forms "a," "an," and "the" include plural reference unless the context clearly dictates otherwise. Thus, for example, a reference to "a host ceU" includes a plurakty of such host cells, and a reference to "an antibody" is a reference to one or more antibodies and equivalents thereof known to those skiUed in the art, and so forth. Unless defined otherwise, aU technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skiU 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. AU pubkcations mentioned herein are cited for the purpose of describing and disclosing the cell lines, protocols, reagents and vectors which are reported in the pubkcations and which might be used in connection with various embodiments of the invention. Nothing herein is to be construed as an admission that the invention is not entitled to antedate such disclosure by virtue of prior invention. DEFINITIONS

"NAAP" refers to the amino acid sequences of substantiaUy purified NAAP obtained from any species, particularly a mammakan 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, smaU 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 "aUekc variant" is an alternative form of the gene encoding NAAP. AUekc 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 aUekc variants of its naturally occurring form. Common mutational changes which give rise to aUekc variants are generaUy 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 okgonucleotide probe of the polynucleotide encoding NAAP, and improper or unexpected hybridization to aUekc variants, with a locus other than the normal chromosomal locus for the polynucleotide encoding NAAP. The encoded protein may also be "altered," and may contain deletions, insertions, or substitutions of amino acid residues which produce a silent change and result in a functionaUy equivalent NAAP. Dekberate amino acid substitutions may be made on the basis of one or more similarities in polarity, charge, solubikty, hydrophobicity, hydrophikcity, 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 hydrophikcity values may include: asparagine and glutamine; and serine and threonine. Amino acids with uncharged side chains having similar hydrophikcity values may include: leucine, isoleucine, and vakne; glycine and alanine; and phenylalanine and tyrosine.

The terms "amino acid" and "amino acid sequence" can refer to an okgopeptide, a peptide, a polypeptide, or a protein sequence, or a fragment of any of these, and to naturaUy occurring or synthetic molecules. Where "amino acid sequence" is recited to refer to a sequence of a naturaUy occurring protein molecule, "amino acid sequence" and like terms are not meant to kmit the amino acid sequence to the complete native amino acid sequence associated with the recited protein molecule.

"AmpUfication" relates to the production of additional copies of a nucleic acid. Ampkfication may be carried out using polymerase chain reaction (PCR) technologies or other nucleic acid ampkfication technologies weU 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, anticalins, nucleic acids, carbohydrates, smaU 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 weU as to fragments thereof, such as Fab, F(ab')₂, and Fv fragments, which are capable of binding an epitopic determinant. Antibodies that bind NAAP polypeptides can be prepared using intact polypeptides or using fragments containing smaU peptides of interest as the immunizing antigen. The polypeptide or okgopeptide 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 chemicaUy coupled to peptides include bovine serum albumin, thyroglobukn, 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 ekcit the immune response) for binding to an antibody.

The term "aptamer" refers to a nucleic acid or okgonucleotide 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 kbraries. Aptamer compositions may be double-stranded or single-stranded, and may include deoxyribonucleotides, ribonucleotides, nucleotide derivatives, or other nucleotide-kke 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'-NH₂), which may improve a desired property, e.g., resistance to nucleases or longer kfetime 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 specificaUy cross-linked to their cognate kgands, e.g., by photo-activation of a cross-linker (Brody, E.N. and L. Gold (2000) J. Biotechnol. 74:5-13).

The term "intramer" refers to an aptamer which is expressed in vivo. For example, a vaccinia virus-based RNA expression system has been used to express specific RNA aptamers at high levels in the cytoplasm of leukocytes (Blind, M. et al. (1999) Proc. Natl. Acad. Sci. USA 96:3606-3610).

The term "spiegelmer" refers to an aptamer which includes L-DNA, L-RNA, or other left- handed nucleotide derivatives or nucleotide-kke molecules. Aptamers containing left-handed nucleotides are resistant to degradation by naturally occurring enzymes, which normally act on substrates containing right-handed nucleotides. The term "antisense" refers to any composition capable of base-pairing with the "sense"

(coding) strand of a polynucleotide having a specific nucleic acid sequence. Antisense compositions may include DNA; RNA; peptide nucleic acid (PNA); oUgonucleotides having modified backbone linkages such as phosphorothioates, methylphosphonates, or benzylphosphonates; okgonucleotides having modified sugar groups such as 2'-methoxyethyl sugars or 2'-methoxyethoxy sugars; or okgonucleotides 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 ceU, the complementary antisense molecule base-pairs with a naturaUy occurring nucleic acid sequence produced by the ceU 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 "biologicaUy active" refers to a protein having structural, regulatory, or biochemical functions of a naturaUy occurring molecule. Likewise, "immunologicaUy active" or "immunogenic" refers to the capabikty of the natoral, recombinant, or synthetic NAAP, or of any okgopeptide thereof, to induce a specific immune response in appropriate animals or ceUs and to bind with specific antibodies.

"Complementary" describes the relationship between two single-stranded nucleic acid sequences that anneal by base-pairing. For example, 5'-AGT-3' pairs with its complement, 3'-TCA-5'.

A "composition comprising a given polynucleotide" and a "composition comprising a given polypeptide" can refer to any composition containing the given polynucleotide or polypeptide. The composition may comprise a dry formulation or an aqueous solution. Compositions comprising polynucleotides encoding NAAP or fragments of NAAP maybe employed as hybridization probes. The probes may be stored in freeze-dried form and may be associated with a stabiUzing agent such as a carbohydrate. In hybridizations, the probe maybe deployed in an aqueous solution containing salts (e.g., NaCl), detergents (e.g., sodium dodecyl sulfate; SDS), and other components (e.g., Denhardt's solution, dry milk, salmon sperm DNA, etc.). "Consensus sequence" refers to a nucleic acid sequence which has been subjected to repeated DNA sequence analysis to resolve uncaUed bases, extended using the XL-PCR kit (Appked 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 (Accekys, Burlington MA) or Phrap (University of Washington, Seattle WA). Some sequences have been both extended and assembled to produce the consensus sequence.

"Conservative amino acid substitutions" are those substitutions that are predicted to least interfere with the properties of the original protein, i.e., the structure and especiaUy 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, Gin, His

Asp Asn, Glu

Cys Ala, Ser

Gin Asn, Glu, His

Glu Asp, Gin, His Gly Ala

His Asn, Arg, Gin, Glu

De Leu, Val

Leu De, Val

Lys Arg, Gin, Glu Met Leu, De

Phe His, Met, Leu, Trp, Tyr

Ser Cys, Thr

Thr Ser, Val

Trp Phe, Tyr Tyr His, Phe, Trp Val De, Leu, Thr

Conservative amino acid substitutions generaUy maintain (a) the structure of the polypeptide backbone in the area of the substitution, for example, as a beta sheet or alpha hekcal 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 chernicaUy 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 maybe assembled through the novel reassortment of stable substructures, thus aUowing acceleration of the evolution of new protein functions.

A "fragment" is a unique portion of NAAP or a polynucleotide encoding NAAP which can be identical in sequence to, but shorter in length than, the parent sequence. A fragment may comprise up to the entire length of the defined sequence, minus one nucleotide/amino acid residue. For example, a fragment may comprise from about 5 to about 1000 contiguous nucleotides or amino acid residues. A fragment used as a probe, primer, antigen, therapeutic molecule, or for other purposes, may be at least 5, 10, 15, 16, 20, 25, 30, 40, 50, 60, 75, 100, 150, 250 or at least 500 contiguous nucleotides or amino acid residues in length. Fragments may be preferentiaUy selected from certain regions of a molecule. For example, a polypeptide fragment may comprise a certain length of contiguous amino acids selected from the first 250 or 500 amino acids (or first 25% or 50%) of a polypeptide as shown in a certain defined sequence. Clearly these lengths are exemplary, and any length that is supported by the specification, including the Sequence Listing, tables, and figures, may be encompassed by the present embodiments. A fragment of SEQ ED NO:58-114 can comprise a region of unique polynucleotide sequence that specificaUy identifies SEQ ED NO:58-114, for example, as distinct from any other sequence in the genome from which the fragment was obtained. A fragment of SEQ ED NO:58-l 14 can be employed in one or more embodiments of methods of the invention, for example, in hybridization and ampkfication technologies and in analogous methods that distinguish SEQ DD NO:58-l 14 from related polynucleotides. The precise length of a fragment of SEQ ID NO:58-l 14 and the region of SEQ DD NO:58-l 14 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 DD NO:l-57 is encoded by a fragment of SEQ DD NO:58-114. A fragment of SEQ ID NO: 1-57 can comprise a region of unique amino acid sequence that specificaUy identifies SEQ DD NO:l-57. For example, a fragment of SEQ DD NO:l-57 can be used as an immunogenic peptide for the development of antibodies that specificaUy recognize SEQ DD NO:l-57. The precise length of a fragment of SEQ ID NO:l-57 and the region of SEQ DD NO:l-57 to which the fragment corresponds can be determined based on the intended purpose for the fragment using one or more analytical methods described herein or otherwise known in the art.

A "fuU length" polynucleotide is one containing at least a translation initiation codon (e.g., methionine) foUowed by an open reading frame and a translation termination codon. A "fuU length" polynucleotide sequence encodes a "fuH length" polypeptide sequence.

"Homology" refers to sequence similarity or, alternatively, sequence identity, between two or more polynucleotide sequences or two or more polypeptide sequences.

The terms "percent identity" and "% identity," as appked to polynucleotide sequences, refer to the percentage of identical residue matches between at least two polynucleotide sequences akgned 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 akgnment between two sequences, and therefore achieve a more meaningful comparison of the two sequences.

Percent identity between polynucleotide sequences may be determined using one or more computer algorithms or programs known in the art or described herein. For example, percent identity can be determined using the default parameters of the CLUSTAL V algorithm as incorporated into the MEG ALIGN version 3.12e sequence akgnment program. This program is part of the LASERGENE software package, a suite of molecular biological analysis programs (DNASTAR, Madison Wl). 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 akgnments of polynucleotide sequences, the default parameters are set as foUows: Ktuple=2, gap penalty=5, window=4, and "diagonals saved"=4. The "weighted" residue weight table is selected as the default.

Alternatively, a suite of commonly used and freely available sequence comparison algorithms which can be used is provided by the National Center for Biotechnology Information (NCBI) Basic Local Akgnment 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 akgn a known polynucleotide sequence with other polynucleotide sequences from a variety of databases. Also available is a tool caUed "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 2.0.12 (April-21-2000) set at default parameters. Such default parameters may be, for example:

Matrix: BLOSUM62

Reward for match: 1

Penalty for mismatch: -2

Open Gap: 5 and Extension Gap: 2 penalties Gap x drop-off: 50

Expect: 10

Word Size: 11

Filter: on

Percent identity may be measured over the length of an entire defined sequence, for example, as defined by a particular SEQ DD 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 aU encode substantiaUy the same protein.

The phrases "percent identity" and "% identity," as appked to polypeptide sequences, refer to the percentage of identical residue matches between at least two polypeptide sequences akgned using a standardized algorithm. Methods of polypeptide sequence akgnment are weU-known. Some akgnment methods take into account conservative amino acid substitutions. Such conservative substitutions, explained in more detail above, generaUy preserve the charge and hydrophobicity at the site of substitution, thus preserving the structure (and therefore function) of the polypeptide. The phrases "percent similarity" and "% similarity," as appked to polypeptide sequences, refer to the percentage of residue matches, including identical residue matches and conservative substitutions, between at least two polypeptide sequences akgned using a standardized algorithm. In contrast, conservative substitotions are not included in the calculation of percent identity between polypeptide sequences.

Percent identity between polypeptide sequences may be determined using the default parameters of the CLUSTAL V algorithm as incorporated into the MEGALIGN version 3.12e sequence akgnment program (described and referenced above). For pairwise akgnments of polypeptide sequences using CLUSTAL V, the default parameters are set as foUows: Ktaple=l, gap penalty=3, window=5, and "diagonals saved"=5. The PAM250 matrix is selected as the default residue weight table. Alternatively the NCBI BLAST software suite may be used. For example, for a pairwise comparison of two polypeptide sequences, one may use the "BLAST 2 Sequences" tool Version 2.0.12 (April-21-2000) withblastp set at default parameters. Such default parameters maybe, for example:

Matrix: BLOSUM62 Open Gap: 11 and Extension Gap: 1 penalties

Gap x drop-off: 50

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 DD 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 aU of the elements required for chromosome repkcation, 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 stiU retains its original binding abiUty.

"Hybridization" refers to the process by which a polynucleotide strand anneals with a complementary strand through base pairing under defined hybridization conditions. Specific hybridization is an indication that two nucleic acid sequences share a high degree of complementarity. Specific hybridization complexes form under permissive annealing conditions and remain hybridized after the "washing" step(s). The washing step(s) is particularly important in deteπnining the stringency of the hybridization process, with more stringent conditions aUowing 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 skiU in the art and may be consistent among hybridization experiments, whereas wash conditions may be varied among experiments to achieve the desired stringency, and therefore hybridization specificity. Permissive annealing conditions occur, for example, at 68°C in the presence of about 6 x SSC, about 1% (w/v) SDS, and about 100 μg/ml sheared, denatured salmon sperm DNA.

GeneraUy, stringency of hybridization is expressed, in part, with reference to the temperature under which the wash step is carried out. Such wash temperatures are typicaUy selected to be about 5°C to 20°C lower than the thermal melting point (T, for the specific sequence at a defined ionic strength and pH. The T_m 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 T_m and conditions for nucleic acid hybridization are weU known and can be found in Sambrook, J. and D.W. RusseU (2001; Molecular Cloning: A Laboratory Manual, 3rd ed., vol. 1-3, Cold Spring Harbor Press, Cold Spring Harbor NY, ch. 9).

High stringency conditions for hybridization between polynucleotides of the present invention include wash conditions of 68°C in the presence of about 0.2 x SSC and about 0.1% SDS, for 1 hour. Alternatively, temperatures of about 65°C, 60°C, 55°C, or 42°C may be used. SSC concentration may be varied from about 0.1 to 2 x SSC, with SDS being present at about 0.1%. TypicaUy, 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 μg/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 wiU be readily apparent to those of ordinary skiU in the art. Hybridization, particularly under high stringency conditions, may be suggestive of evolutionary similarity between the nucleotides. Such similarity is strongly indicative of a similar role for the nucleotides and their encoded polypeptides. The term "hybridization complex" refers to a complex formed between two nucleic acids by virtue of the formation of hydrogen bonds between complementary bases. A hybridization complex may be formed in solution (e.g., C₀t or Rot analysis) or formed between one nucleic acid present in solution and another nucleic acid immobikzed on a sokd support (e.g., paper, membranes, filters, chips, pins or glass skdes, or any other appropriate substrate to which ceUs or their nucleic acids have been fixed).

The words "insertion" and "addition" refer to changes in an amino acid or polynucleotide sequence resulting in the addition of one or more amino acid residues or nucleotides, respectively.

"Immune response" can refer to conditions associated with inflammation, trauma, immune disorders, or infectious or genetic disease, etc. These conditions can be characterized by expression of various factors, e.g., cytokines, chemokines, and other signaling molecules, which may affect ceUular and systemic defense systems.

An "immunogenic fragment" is a polypeptide or okgopeptide fragment of NAAP which is capable of ekciting an immune response when introduced into a kving organism, for example, a mammal. The term "immunogenic fragment" also includes any polypeptide or okgopeptide 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 plurakty of polynucleotides, polypeptides, antibodies, or other chemical compounds on a substrate.

The terms "element" and "array element" refer to a polynucleotide, polypeptide, antibody, or other chemical compound having a unique and defined position on a microarray.

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, okgonucleotide, 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-kke 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 knked 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 okgonucleotide of at least about 5 nucleotides in length linked to a peptide backbone of amino acid residues ending in lysine. The terminal lysine confers solubikty to the composition. PNAs preferentiaUy bind complementary single stranded DNA or RNA and stop transcript elongation, and may be pegylated to extend their kfespan in the ceU.

"Post-translational modification" of an NAAP may involve Upidation, glycosylation, phosphorylation, acetylation, racemization, proteolytic cleavage, and other modifications known in the art. These processes may occur syntheticaUy or biochemicaUy. Biochemical modifications wiU vary by ceU type depending on the enzymatic mikeu of NAAP. "Probe" refers to nucleic acids encoding NAAP, their complements, or fragments thereof, which are used to detect identical, aUekc or related nucleic acids. Probes are isolated okgonucleotides or polynucleotides attached to a detectable label or reporter molecule. Typical labels include radioactive isotopes, kgands, chemiluminescent agents, and enzymes. "Primers" are short nucleic acids, usuaUy DNA okgonucleotides, 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 ampkfication (and identification) of a nucleic acid, e.g., by the polymerase chain reaction (PCR).

Probes and primers as used in the present invention typicaUy comprise at least 15 contiguous nucleotides of a known sequence. In order to enhance specificity, longer probes and primers may also be employed, such as probes and primers that comprise at least 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, or at least 150 consecutive nucleotides of the disclosed nucleic acid sequences. Probes and primers may be considerably longer than these examples, and it is understood that any length supported by the specification, including the tables, figures, and Sequence Listing, may be used.

Methods for preparing and using probes and primers are described in, for example, Sambrook, J. and D.W. RusseU (2001; Molecular Cloning: A Laboratory Manual, 3rd ed., vol. 1-3, Cold Spring Harbor Press, Cold Spring Harbor NY), Ausubel, F.M. et al. (1999; Short Protocols in Molecular Biology, 4^th ed., John Wiley & Sons, New York NY), and Innis, M. et al. (1990; PCR Protocols, A Guide to Methods and Appkcations, Academic Press, San Diego CA). PCR primer pairs can be derived from a known sequence, for example, by using computer programs intended for that purpose such as Primer (Version 0.5, 1991, Whitehead Institute for Biomedical Research, Cambridge MA). Okgonucleotides 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 okgonucleotides 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 capabikties. For example, the PrimOU primer selection program (available to the pubkc from the Genome Center at University of Texas South West Medical Center, DaUas 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 pubkc from the Whitehead Institute/MIT Center for Genome Research, Cambridge MA) aUows the user to input a "mispriming kbrary," in which sequences to avoid as primer binding sites are user-specified. Primer3 is useful, in particular, for the selection of okgonucleotides 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 pubkc from the UK Human Genome Mapping Project Resource Centre, Cambridge UK) designs primers based on multiple sequence akgnments, thereby aUowing selection of primers that hybridize to either the most conserved or least conserved regions of akgned nucleic acid sequences. Hence, this program is useful for identification of both unique and conserved okgonucleotides and polynucleotide fragments. The okgonucleotides and polynucleotide fragments identified by any of the above selection methods are useful in hybridization technologies, for example, as PCR or sequencing primers, microanay elements, or specific probes to identify fully or partiaUy complementary polynucleotides in a sample of nucleic acids. Methods of okgonucleotide selection are not limited to those described above.

A "recombinant nucleic acid" is a nucleic acid that is not naturaUy 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 accompkshed by chemical synthesis or, more commonly, by the artificial manipulation of isolated segments of nucleic acids, e.g., by genetic engineering techniques such as those described in Sambrook and RusseU (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 ceU.

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 usuaUy derived from untranslated regions of a gene and includes enhancers, promoters, introns, and 5' and 3' untranslated regions (UTRs). Regulatory elements interact with host or viral proteins which control transcription, translation, or RNA stabikty. "Reporter molecules" are chemical or biochemical moieties used for labeling a nucleic acid, amino acid, or antibody. Reporter molecules include radionuckdes; enzymes; fluorescent, chemiluminescent, or chromogenic agents; substrates; cofactors; inhibitors; magnetic particles; and other moieties known in the art.

An "RNA equivalent," in reference to a DNA molecule, is composed of the same linear sequence of nucleotides as the reference DNA molecule with the exception that aU 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 ceU, chromosome, organelle, or membrane isolated from a ceU; a ceU; genomic DNA, RNA, or cDNA, in solution or bound to a substrate; a tissue; a tissue print; etc.

The terms "specific binding" and "specificaUy binding" refer to that interaction between a protein or peptide and an agonist, an antibody, an antagonist, a small molecule, or any natoral 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 wiU reduce the amount of labeled A that binds to the antibody.

The term "substantiaUy purified" refers to nucleic acid or amino acid sequences that are removed from their natural environment and are isolated or separated, and are at least about 60% free, preferably at least about 75% free, and most preferably at least about 90% free from other components with which they are naturaUy 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, skdes, wafers, fibers, magnetic or nonmagnetic beads, gels, tubing, plates, polymers, microparticles and capiUaries. The substrate can have a variety of surface forms, such as weUs, trenches, pins, channels and pores, to which polynucleotides or polypeptides are bound.

A "transcript image" or "expression profile" refers to the coUective pattern of gene expression by a particular ceU type or tissue under given conditions at a given time.

"Transformation" describes a process by which exogenous DNA is introduced into a recipient ceU. Transformation may occur under natural or artificial conditions according to various methods weU 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 ceU. The method for transformation is selected based on the type of host ceU being transformed and may include, but is not limited to, bacteriophage or viral infection, electroporation, heat shock, kpofection, and particle bombardment. The term "transformed cells" includes stably transformed ceUs in which the inserted DNA is capable of repkcation either as an autonomously repkcating plasmid or as part of the host chromosome, as weU as transiently transformed ceUs 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 ceUs of the organism contains heterologous nucleic acid introduced by way of human intervention, such as by transgenic techniques weU known in the art. The nucleic acid is introduced into the ceU, directly or indirectly by introduction into a precursor of the ceU, by way of dekberate genetic manipulation, such as by microinjection or by infection with a recombinant virus. In another embodiment, the nucleic acid can be introduced by infection with a recombinant viral vector, such as a lentiviral vector (Lois, C. et al. (2002) Science 295:868-872). The term genetic manipulation does not include classical cross-breeding, or in vitro fertikzation, but rather is directed to the introduction of a recombinant DNA molecule. The transgenic organisms contemplated in accordance with the present invention include bacteria, cyanobacteria, fungi, plants and animals. The isolated DNA of the present invention can be introduced into the host by methods known in the art, for example infection, transfection, transformation or transconjugation. Techniques for transferring the DNA of the present invention into such organisms are widely known and provided in references such as Sambrook and RusseU (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 2.0.9 (May-07- 1999) set at default parameters. Such a pair of nucleic acids may show, for example, at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% or greater sequence identity over a certain defined length. A variant may be described as, for example, an "aUekc" (as defined above), "spkce," "species," or "polymorphic" variant. A spkce variant may have significant identity to a reference molecule, but wiU generaUy have a greater or lesser number of polynucleotides due to alternate spkcing of exons during mRNA processing. The conesponding polypeptide may possess additional functional domains or lack domains that are present in the reference molecule. Species variants are polynucleotides that vary from one species to another. The resulting polypeptides wiU generaUy 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 or sequence similarity to the particular polypeptide sequence over a certain length of one of the polypeptide sequences using blastp with the "BLAST 2 Sequences" tool Version 2.0.9 (May-07-1999) set at default parameters. Such a pair of polypeptides may show, for example, at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% or greater sequence identity or sequence similarity over a certain defined length of one of the polypeptides.

THE INVENTION Various embodiments of the invention include new human nucleic acid-associated proteins (NAAP), the polynucleotides encoding NAAP, and the use of these compositions for the diagnosis, treatment, or prevention of ceU prokferative, neurological, developmental, and autoimmune/inflammatory disorders, and infections. Table 1 summarizes the nomenclature for the fuU length polynucleotide and polypeptide embodiments of the invention. Each polynucleotide and its corresponding polypeptide are correlated to a single Incyte project identification number (Incyte Project DD). Each polypeptide sequence is denoted by both a polypeptide sequence identification number (Polypeptide SEQ DD NO:) and an Incyte polypeptide sequence number (Incyte Polypeptide ED) as shown. Each polynucleotide sequence is denoted by both a polynucleotide sequence identification number (Polynucleotide SEQ DD NO:) and an Incyte polynucleotide consensus sequence number (Incyte Polynucleotide DD) as shown. Column 6 shows the Incyte DD numbers of physical, fuU length clones corresponding to the polypeptide and polynucleotide sequences of the invention. The fuU length clones encode polypeptides which have at least 95% sequence identity to the polypeptide sequences shown in column 3. Table 2 shows sequences with homology to polypeptide embodiments of the invention as identified by BLAST analysis against the GenBank protein (genpept) database and the PROTEOME database. Columns 1 and 2 show the polypeptide sequence identification number (Polypeptide SEQ DD NO:) and the corresponding Incyte polypeptide sequence number (Incyte Polypeptide DD) for polypeptides of the invention. Column 3 shows the GenBank identification number (GenBank DD NO:) of the nearest GenBank homolog and the PROTEOME database identification numbers

(PROTEOME DD NO:) of the nearest PROTEOME database homologs. Column 4 shows the probabikty scores for the matches between each polypeptide and its homolog(s). Column 5 shows the annotation of the GenBank and PROTEOME database homolog(s) along with relevant citations where appkcable, aU 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 DD NO:) and the corresponding Incyte polypeptide sequence number (Incyte Polypeptide DD) for each polypeptide of the invention. Column

3 shows the number of amino acid residues in each polypeptide. Column 4 shows potential phosphorylation sites, and column 5 shows potential glycosylation sites, as determined by the MOTIFS program of the GCG sequence analysis software package (Accekys, Burkngton MA). Column 6 shows amino acid residues comprising signature sequences, domains, and motifs. Column 7 shows analytical methods for protein structure/function analysis and in some cases, searchable databases to which the analytical methods were appked.

Together, Tables 2 and 3 summarize the properties of polypeptides of the invention, and these properties estabksh that the claimed polypeptides are nucleic acid-associated proteins. For example, SEQ DD NO:ll is 100% identical, from residue Ml to residue L174 and from residue Q175 to residue Q488, to a human protein similar to interferon regulatory factor 5 (GenBank DD gl3278720) as determined by the Basic Local Akgnment Search Tool (BLAST). (See Table 2.) The BLAST probabikty score is 5.8e-273, which indicates the probabikty of obtaining the observed polypeptide sequence akgnment by chance. SEQ ED NO: 11 also has homology to human and mouse interferon regulatory factor 5, a transcription factor that induces expression of members of the interferon A family in response to NDV viral infection, as determined by BLAST analysis using the PROTEOME database. SEQ DD NO: 11 also contains an interferon regulatory factor transcription factor domain as determined by searching for statisticaUy significant matches in the hidden Markov model (HMM)- based PFAM database of conserved protein family domains. (See Table 3.) Data from BLIMPS, MOT FS, and PROFILESCAN analyses provide further coπoborative evidence that SEQ ID NO: 11 is a member of the interferon regulatory factor (IRF) family of transcription factors. hi an alternative example, SEQ DD NO:24 is 96% identical, from residue Ml to residue R133, to human hMBFlalpha (GenBank DD g6526355) as determined by the Basic Local Akgnment Search Tool (BLAST). (See Table 2.) The BLAST probabikty score is 5.0e-63, which indicates the probabikty of obtaining the observed polypeptide sequence akgnment by chance. SEQ DD NO:24 also has homology to endothekal differentiation-related factor-1, a putative transcriptional coactivator that binds to calmodukn (CALM1), in a calcium-dependent manner, and to the TATA-binding protein (TBP), has transcription factor function, and are EDF1 transcription coactivators, as determined by BLAST analysis using the PROTEOME database. SEQ DD NO:24 also contains a hekx-torn-hekx domain as determined by searching for statisticaUy significant matches in the hidden Markov model (HMM)-based PFAM database of conserved protein family domains. (See Table 3.) Data from additional BLAST analyses provide further corroborative evidence that SEQ DD NO:24 is a transcription factor regulator.

In an alternative example, SEQ DD NO:35 is 84% identical, from residue M932 to residue G1638, to human ras-responsive element binding protein (GenBank DD gl654112) as determined by the Basic Local Akgnment Search Tool (BLAST). (See Table 2.) The BLAST probabikty score is 0.0, which indicates the probabikty of obtaining the observed polypeptide sequence akgnment by chance. SEQ DD NO:35 also has homology to nuclear transcription factors, as determined by BLAST analysis using the PROTEOME database. SEQ ED NO:35 also contains a zinc finger domain as determined by searching for statisticaUy 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 DD NO:35 is a transcription factor.

In an alternative example, SEQ DD NO:55 is 85% identical, from residue Ml to residue V508, to rat serine-arginine-rich spkcing regulatory protein, SRRP86 (GenBank DD g7158880) as determined by the Basic Local Akgnment Search Tool (BLAST). (See Table 2.) The BLAST probabikty score is 4.8e-225, which indicates the probabikty of obtaining the observed polypeptide sequence akgnment by chance. SEQ DD NO:55 also has homology to proteins that are locakzed to the nucleus, function as RNA-binding proteins, and are serme-arginine-rich proteins, as determined by BLAST analysis using the PROTEOME database. SEQ DD NO:55 also contains an RNA recognition motif as determined by searching for statisticaUy significant matches in the hidden Markov model (HMM)-based PFAM database of conserved protein family domains. (See Table 3.) Data from BLIMPS, and other BLAST analyses provide further corroborative evidence that SEQ DD NO:55 is a serine-arginine-rich spkcing regulatory protein.

SEQ DD NO:1-10, SEQ DD NO:12-23, SEQ DD NO:25-34, SEQ DD NO:36-54 and SEQ DD NO:56-57 were analyzed and annotated in a similar manner. The algorithms and parameters for the analysis of SEQ DD NO: 1-57 are described in Table 7. As shown in Table 4, the fuU length polynucleotide embodiments were assembled using cDNA sequences or coding (exon) sequences derived from genomic DNA, or any combination of these two types of sequences. Column 1 ksts the polynucleotide sequence identification number (Polynucleotide SEQ DD NO:), the corresponding Incyte polynucleotide consensus sequence number (Incyte DD) 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 fuU length polynucleotide embodiments, and of fragments of the polynucleotides which are useful, for example, in hybridization or ampkfication technologies that identify SEQ DD NO:58-114 or that distinguish between SEQ DD NO:58-114 and related polynucleotides. The polynucleotide fragments described in Column 2 of Table 4 may refer specificaUy, for example, to Incyte cDNAs derived from tissue-specific cDNA kbraries or from pooled cDNA kbraries. Alternatively, the polynucleotide fragments described in column 2 may refer to GenBank cDNAs or ESTs which contributed to the assembly of the fuU length polynucleotides. In addition, the polynucleotide fragments described in column 2 may identify sequences derived from the ENSEMBL (The Sanger Centre, Cambridge, UK) database (i.e., those sequences including the designation "ENST"). Alternatively, the polynucleotide fragments described in column 2 may be derived from the NCBI RefSeq Nucleotide Sequence Records Database (Le. , 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_N₁_N₂_YYYYY_N₃_N₄ represents a "stitched" sequence in which XXXXXX is the identification number of the cluster of sequences to which the algorithm was appked, and YYYYY is the number of the prediction generated by the algorithm, and N_h23 , if present, represent specific exons that may have been manuaUy 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_l_N is a "stretched" sequence, with XXXXXX being the Incyte project identification number, gAAAAA being the GenBank identification number of the human genomic sequence to which the "exon-stretching" algorithm was appked, 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 (Le. , 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 ksts examples of component sequence prefixes and conesponding sequence analysis methods associated with the prefixes (see Example IV and Example V).

ENCY FuU 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 kbraries for those fuU length polynucleotides which were assembled using Incyte cDNA sequences. The representative cDNA kbrary is the Incyte cDNA kbrary which is most frequently represented by the Incyte cDNA sequences which were used to assemble and confirm the above polynucleotides. The tissues and vectors which were used to construct the cDNA kbraries shown in Table 5 are described in Table 6.

Table 8 shows single nucleotide polymorphisms (SNPs) found in polynucleotide sequences of the invention, along with aUele frequencies in different human populations. Columns 1 and 2 show the polynucleotide sequence identification number (SEQ DD NO:) and the corresponding Incyte project identification number (PDD) for polynucleotides of the invention. Column 3 shows the Incyte identification number for the EST in which the SNP was detected (EST DD), and column 4 shows the identification number for the SNP (SNP DD). Column 5 shows the position within the EST sequence at which the SNP is located (EST SNP), and column 6 shows the position of the SNP within the fuU- length polynucleotide sequence (CB1 SNP). Column 7 shows the aUele found in the EST sequence. Columns 8 and 9 show the two aUeles found at the SNP site. Column 10 shows the amino acid encoded by the codon including the SNP site, based upon the allele found in the EST. Columns 11-14 show the frequency of allele 1 in four different human populations. An entry of n/d (not detected) indicates that the frequency of aUele 1 in the population was too low to be detected, while n a (not available) indicates that the allele frequency was not determined for the population.

The invention also encompasses NAAP variants. Various embodiments of NAAP variants can have at least about 80%, at least about 90%, or at least about 95% amino acid sequence identity to the NAAP amino acid sequence, and can contain at least one functional or structural characteristic of NAAP.

Various embodiments also encompass polynucleotides which encode NAAP. In a particular embodiment, the invention encompasses a polynucleotide sequence comprising a sequence selected from the group consisting of SEQ DD NO:58-114, which encodes NAAP. The polynucleotide sequences of SEQ DD NO:58-l 14, as presented in the Sequence Listing, embrace the equivalent RNA sequences, wherein occurrences of the nitrogenous base thymine are replaced with uracil, and the sugar backbone is composed of ribose instead of deoxyribose.

The invention also encompasses variants of a polynucleotide encoding NAAP. In particular, such a variant polynucleotide wiU have at least about 70%, or alternatively at least about 85%, or even at least about 95% polynucleotide sequence identity to a polynucleotide encoding NAAP. A particular aspect of the invention encompasses a variant of a polynucleotide comprising a sequence selected from the group consisting of SEQ ED NO:58-114 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 ED NO:58-l 14. Any one of the polynucleotide variants described above can encode a polypeptide which contains at least one functional or structural characteristic of NAAP.

In addition, or in the alternative, a polynucleotide variant of the invention is a spkce variant of a polynucleotide encoding NAAP. A spkce variant may have portions which have significant sequence identity to a polynucleotide encoding NAAP, but wiU generaUy have a greater or lesser number of polynucleotides due to additions or deletions of blocks of sequence arising from alternate spkcing of exons during mRNA processing. A spkce variant may have less than about 70%, or alternatively less than about 60%, or alternatively less than about 50% polynucleotide sequence identity to a polynucleotide encoding NAAP over its entire length; however, portions of the spkce variant wiU have at least about 70%, or alternatively at least about 85%, or alternatively at least about 95%, or alternatively 100% polynucleotide sequence identity to portions of the polynucleotide encoding NAAP. Any one of the spkce variants described above can encode a polypeptide which contains at least one functional or structural characteristic of NAAP.

It wiU be appreciated by those skiUed 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 natoraUy 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 appked to the polynucleotide sequence of natoraUy occurring NAAP, and aU such variations are to be considered as being specificaUy disclosed.

Although polynucleotides which encode NAAP and its variants are generaUy capable of hybridizing to polynucleotides encoding natoraUy occurring NAAP under appropriately selected conditions of stringency, it may be advantageous to produce polynucleotides encoding NAAP or its derivatives possessing a substantiaUy different codon usage, e.g., inclusion of non-naturaUy 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 utikzed by the host. Other reasons for substantiaUy 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-kfe, than transcripts produced from the naturaUy occurring sequence. The invention also encompasses production of polynucleotides which encode NAAP and

NAAP derivatives, or fragments thereof, entirely by synthetic chemistry. After production, the synthetic polynucleotide may be inserted into any of the many available expression vectors and ceU systems using reagents weU known in the art. Moreover, synthetic chemistry may be used to introduce mutations into a polynucleotide encoding NAAP or any fragment thereof. Embodiments of the invention can also include polynucleotides that are capable of hybridizing to the claimed polynucleotides, and, in particular, to those having the sequences shown in SEQ DD NO:58-114 and fragments thereof, under various conditions of stringency (Wahl, GM. 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 (Appked Biosystems), thermostable T7 polymerase (Amersham Biosciences, Piscataway NJ), or combinations of polymerases and proofreading exonucleases such as those found in the ELONGASE ampkfication system (Invitrogen, Carlsbad CA). Preferably, sequence preparation is automated with machines such as the MICROLAB 2200 kquid transfer system (Hamilton, Reno NV), PTC200 thermal cycler (MJ Research, Watertown MA) and ABI CATALYST 800 thermal cycler (Appked Biosystems). Sequencing is then carried out using either the ABI 373 or 377 DNA sequencing system (Appked Biosystems), the MEGABACE 1000 DNA sequencing system (Amersham Biosciences), or other systems known in the art. The resulting sequences are analyzed using a variety of algorithms which are weU known in the art (Ausubel et al., supra, ch. 7; Meyers, R.A. (1995) Molecular Biology and Biotechnology. Wiley VCH, New York NY, pp. 856-853). The nucleic acids encoding NAAP may be extended utikzing 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 ampkfy unknown sequence from genomic DNA within a cloning vector (Sarkar, G. (1993) PCR Methods Appkc. 2:318-322). Another method, inverse PCR, uses primers that extend in divergent directions to ampkfy unknown sequence from a circularized template. The template is derived from restriction fragments comprising a known genomic locus and sunounding sequences (Trigka, T. et al. (1988) Nucleic Acids Res. 16:8186). A third method, capture PCR, involves PCR ampkfication of DNA fragments adjacent to known sequences in human and yeast artificial chromosome DNA (Lagerstrom, M. et al. (1991) PCR Methods Appkc. 1:111-119). In this method, multiple restriction enzyme digestions and kgations may be used to insert an engineered double-stranded sequence into a region of unknown sequence before performing PCR. Other methods which may be used to retrieve unknown sequences are known in the art (Parker, J.D. et al. (1991) Nucleic Acids Res. 19:3055-3060). Additionally, one may use PCR, nested primers, and PROMOTERFLNDER kbraries (Clontech, Palo Alto CA) to walk genomic DNA. This procedure avoids the need to screen kbraries and is useful in finding intron/exon junctions. For aU PCR-based methods, primers maybe designed using commerciaUy available software, such as OLIGO 4.06 primer analysis software (National Biosciences, Plymouth MN) or another appropriate program, to be about 22 to 30 nucleotides in length, to have a GC content of about 50% or more, and to anneal to the template at temperatures of about 68°C to 72°C

When screening for full length cDNAs, it is preferable to use kbraries that have been size-selected to include larger cDNAs. In addition, random-primed kbraries, which often include sequences containing the 5' regions of genes, are preferable for situations in which an okgo d(T) kbrary does not yield a fuU-length cDNA. Genomic kbraries may be useful for extension of sequence into 5' non-transcribed regulatory regions.

CapiUary electrophoresis systems which are commerciaUy available may be used to analyze the size or confirm the nucleotide sequence of sequencing or PCR products. In particular, capiUary sequencing may employ flowable polymers for electrophoretic separation, four different nucleotide- specific, laser-stimulated fluorescent dyes, and a charge coupled device camera for detection of the emitted wavelengths. Output kght intensity may be converted to electrical signal using appropriate software (e.g., GENOTYPER and SEQUENCE NAVIGATOR, Appked Biosystems), and the entire process from loading of samples to computer analysis and electronic data display may be computer controlled. CapiUary electrophoresis is especiaUy preferable for sequencing smaU DNA fragments which may be present in limited amounts in a particular sample.

In another embodiment of the invention, polynucleotides or fragments thereof which encode NAAP may be cloned in recombinant DNA molecules that direct expression of NAAP, or fragments or functional equivalents thereof, in appropriate host ceUs. Due to the inherent degeneracy of the genetic code, other polynucleotides which encode substantially the same or a functionaUy equivalent polypeptides may be produced and used to express NAAP.

The polynucleotides of the invention can be engineered using methods generaUy 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 okgonucleotides may be used to engineer the nucleotide sequences. For example, okgonucleotide-mediated site-directed mutagenesis may be used to introduce mutations that create new restriction sites, alter glycosylation patterns, change codon preference, produce spkce 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, EC. 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 abikty to bind to other molecules or compounds. DNA shuffling is a process by which a kbrary of gene variants is produced using PCR-mediated recombination of gene fragments. The kbrary is then subjected to selection or screening procedures that identify those gene variants with the desired properties. These prefeπed 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 naturaUy occurring genes in a directed and controUable manner. In another embodiment, polynucleotides encoding NAAP may be synthesized, in whole or in part, using one or more chemical methods weU known in the art (Caruthers, M.H. et al. (1980) Nucleic Acids Symp. Ser. 7:215-223; Horn, T. et al. (1980) Nucleic Acids Symp. Ser. 7:225-232). Alternatively, NAAP itself or a fragment thereof may be synthesized using chemical methods known in the art. For example, peptide synthesis can be performed using various solution-phase or sokd-phase techniques (Creighton, T. (1984) Proteins, Structures and Molecular Properties. WH Freeman, New York NY, pp. 55-60; Roberge, J.Y. et al. (1995) Science 269:202-204). Automated synthesis may be achieved using the ABI 431 A peptide synthesizer (Appked 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 naturaUy occurring polypeptide. The peptide may be substantiaUy purified by preparative high performance Uquid chromatography (Chiez, R.M. and F.Z. Regnier (1990) Methods Enzymol. 182:392-421). The composition of the synthetic peptides may be confirmed by amino acid analysis or by sequencing (Creighton, supra, pp. 28-53).

In order to express a biologicaUy active NAAP, the polynucleotides encoding NAAP or derivatives thereof may be inserted into an appropriate expression vector, i.e., a vector which contains the necessary elements for transcriptional and translational control of the inserted coding sequence in a suitable host. These elements include regulatory sequences, such as enhancers, constitutive and inducible promoters, and 5' and 3 'untranslated regions in the vector and in polynucleotides encoding NAAP. Such elements may vary in their strength and specificity. Specific initiation signals may also be used to achieve more efficient translation of polynucleotides encoding NAAP. Such signals include the ATG initiation codon and adjacent sequences, e.g. the Kozak sequence. In cases where a polynucleotide sequence encoding NAAP and its initiation codon and upstream regulatory sequences are inserted into the appropriate expression vector, no additional transcriptional or translational control signals may be needed. However, in cases where only coding sequence, or a fragment thereof, is inserted, exogenous translational control signals including an in-frame ATG initiation codon should be provided by the vector. Exogenous translational elements and initiation codons may be of various origins, both natoral and synthetic. The efficiency of expression may be enhanced by the inclusion of enhancers appropriate for the particular host ceU system used (Scharf, D. et al. (1994) Results Probl. CeU Differ. 20:125-162).

Methods which are weU known to those skiUed in the art may be used to construct expression vectors containing polynucleotides encoding NAAP and appropriate transcriptional and translational control elements. These methods include in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination (Sambrook and RusseU, supra, ch. 1-4, and 8; Ausubel et al., supra, ch. 1, 3, and 15).

A variety of expression vector/host systems may be utikzed to contain and express polynucleotides encoding NAAP. These include, but are not limited to, microorganisms such as bacteria transformed with recombinant bacteriophage, plasmid, or cosmid DNA expression vectors; yeast transformed with yeast expression vectors; insect ceU systems infected with viral expression vectors (e.g., baculovirus); plant ceU systems transformed with viral expression vectors (e.g., caukflower mosaic virus, CaMV, or tobacco mosaic virus, TMV) or with bacterial expression vectors (e.g., Ti or pBR322 plasmids); or animal ceU systems (Sambrook and RusseU, supra; Ausubel et al., supra; Van Heeke, G. and S.M. Schuster (1989) J. Biol. Chem. 264:5503-5509; Engelhard, E.K. et al. (1994) Proc. Natl. Acad. Sci. USA 91:3224-3227; Sandig, V. et al. (1996) Hum. Gene Ther. 7:1937- 1945; Takamatsu, N. (1987) EMBO J. 6:307-311: The McGraw HiU Yearbook of Science and Technology (1992) McGraw HiU, New York NY, pp. 191-196; Logan, J. and T. Shenk (1984) Proc. Natl. Acad. Sci. USA 81:3655-3659; Harrington, J.J. et al. (1997) Nat. Genet. 15:345-355). Expression vectors derived from retroviruses, adenoviruses, or herpes or vaccinia viruses, or from various bacterial plasmids, may be used for dekvery of polynucleotides to the targeted organ, tissue, or ceU population (Di Nicola, M. et al. (1998) Cancer Gen. Ther. 5:350-356; Yu, M. et al. (1993) Proc. Natl. Acad. Sci. USA 90:6340-6344; BuUer, R.M. et al. (1985) Nature 317:813-815; McGregor, D.P. et al. (1994) Mol. Immunol. 31:219-226; Verma, I.M. and N. Somia (1997) Nature 389:239-242). The invention is not limited by the host ceU employed. In bacterial systems, a number of cloning and expression vectors may be selected depending upon the use intended for polynucleotides encoding NAAP. For example, routine cloning, subcloning, and propagation of polynucleotides encoding NAAP can be achieved using a multifunctional E. coli vector such as PBLUESCR (Stratagene, La JoUa CA) or PSPORT1 plasmid (Invitrogen). Ligation of polynucleotides encoding NAAP into the vector's multiple cloning site disrupts the lacZ gene, allowing a colorimetric screening procedure for identification of transformed bacteria containing recombinant molecules. In addition, these vectors may be useful for in vitro transcription, dideoxy sequencing, single strand rescue with helper phage, and creation of nested deletions in the cloned sequence (Van Heeke, G. and S.M. Schuster (1989) J. Biol. Chem. 264:5503-5509). When large quantities of NAAP are needed, e.g. for the production of antibodies, vectors which direct high level expression of NAAP may be used. For example, vectors containing the strong, inducible SP6 or T7 bacteriophage promoter may be used.

Yeast expression systems may be used for production of NAAP. A number of vectors containing constitutive or inducible promoters, such as alpha factor, alcohol oxidase, and PGH promoters, may be used in the yeast Saccharomyces cerevisiae or Pichia pastoris. In addition, such vectors direct either the secretion or intraceUular retention of expressed proteins and enable integration of foreign polynucleotide sequences into the host genome for stable propagation (Ausubel et al., supra; Bitter, G.A. et al. (1987) Methods Enzymol. 153:516-544; Scorer, CA. et al. (1994) Bio/Technology 12:181-184).

Plant systems may also be used for expression of NAAP. Transcription of polynucleotides encoding NAAP may be driven by viral promoters, e.g., the 35S and 19S promoters of CaMV used alone or in combination with the omega leader sequence from TMV (Takamatsu, N. (1987) EMBO J. 6:307-311). Alternatively, plant promoters such as the smaU subunit of RUBISCO or heat shock promoters maybe used (Coruzzi, G. et al. (1984) EMBO J. 3:1671-1680; Brogke, R. et al. (1984) Science 224:838-843; Winter, J. et al. (1991) Results Probl. CeU Differ. 17:85-105). These constructs can be introduced into plant ceUs by direct DNA transformation or pathogen-mediated transfection (The McGraw HiU Yearbook of Science and Technology (1992) McGraw Hill, New York NY, pp. 191-196).

In mammakan ceUs, a number of viral-based expression systems may be utikzed. In cases where an adenovirus is used as an expression vector, polynucleotides encoding NAAP may be kgated into an adenovirus transcription/translation complex consisting of the late promoter and tripartite leader sequence. Insertion in a non-essential El or E3 region of the viral genome may be used to obtain infective virus which expresses NAAP in host cells (Logan, J. and T. Shenk (1984) Proc. Natl. Acad. Sci. USA 81:3655-3659). In addition, transcription enhancers, such as the Rous sarcoma virus (RSV) enhancer, may be used to increase expression in mammakan host ceUs. SV40 or EBV-based vectors may also be used for high-level protein expression.

Human artificial chromosomes (HACs) may also be employed to dekver 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 dekvered via conventional dekvery methods (kposomes, polycationic amino polymers, or vesicles) for therapeutic purposes (Harrington, J.J. et al. (1997) Nat. Genet. 15:345-355).

For long term production of recombinant proteins in mammakan systems, stable expression of NAAP in ceU lines is preferred. For example, polynucleotides encoding NAAP can be transformed into ceU lines using expression vectors which may contain viral origins of repkcation and/or endogenous expression elements and a selectable marker gene on the same or on a separate vector. FoUowing the introduction of the vector, ceUs 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 aUows growth and recovery of ceUs which successfuUy express the introduced sequences. Resistant clones of stably transformed ceUs may be propagated using tissue culture techniques appropriate to the ceU type. Any number of selection systems may be used to recover transformed cell lines. These include, but are not Umited to, the herpes simplex virus thymidine kinase and adenine phosphoribosyltransferase genes, for use in tkr and apr ceUs, respectively (Wigler, M. et al. (1977) CeU 11:223-232; Lowy, I. et al. (1980) CeU 22:817-823). Also, antimetabokte, antibiotic, or herbicide resistance can be used as the basis for selection. For example, dhfr confers resistance to methotrexate; neo confers resistance to the aminoglycosides neomycin and G-418; and als and pat confer resistance to chlorsulfuron and phosphinotricin acetyltransferase, respectively (Wigler, M. et al. (1980) Proc. Natl. Acad. Sci. USA 77:3567-3570; Colbere-Garapin, F. et al. (1981) J. Mol. Biol. 150:1-14). Additional selectable genes have been described, e.g., trpB and hisD, which alter ceUular requirements for metaboktes (Hartman, S.C. and R.C Mulkgan (1988) Proc. Natl. Acad. Sci. USA 85:8047-8051). Visible markers, e.g., anthocyanins, green fluorescent proteins (GFP; Clontech), β- glucuronidase and its substrate β-glucuronide, or luciferase and its substrate luciferin may be used. These markers can be used not only to identify transformants, but also to quantify the amount of transient or stable protein expression attributable to a specific vector system (Rhodes, CA. (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 ceUs containing polynucleotides encoding NAAP can be identified by the absence of marker gene function. Alternatively, a marker gene can be placed in tandem with a sequence encoding NAAP under the control of a single promoter. Expression of the marker gene in response to induction or selection usually indicates expression of the tandem gene as weU.

In general, host ceUs that contain the polynucleotide encoding NAAP and that express NAAP may be identified by a variety of procedures known to those of skiU in the art. These procedures include, but are not limited to, DNA-DNA or DNA-RNA hybridizations, PCR ampkfication, 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 ceU sorting (FACS). A two-site, monoclonal-based immunoassay utikzing monoclonal antibodies reactive to two non-interfering epitopes on NAAP is prefened, but a competitive binding assay may be employed. These and other assays are weU known in the art

(Hampton, R. et al. (1990) Serological Methods, a Laboratory Manual, APS Press, St. Paul MN, Sect. IV; Cokgan, J.E. et al. (1997) Current Protocols in Immunology, Greene Pub. Associates and Wiley- Interscience, New York NY; Pound, J.D. (1998) Immunochemical Protocols, Humana Press, Totowa NJ). A wide variety of labels and conjugation techniques are known by those skiUed 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 okgolabekng, nick translation, end-labeling, or PCR ampkfication using a labeled nucleotide. Alternatively, polynucleotides encoding NAAP, or any fragments thereof, may be cloned into a vector for the production of an mRNA probe. Such vectors are known in the art, are commerciaUy 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 maybe conducted using a variety of commerciaUy available kits, such as those provided by Amersham Biosciences, Promega (Madison Wl), and US Biochemical. Suitable reporter molecules or labels which may be used for ease of detection include radionuckdes, enzymes, fluorescent, chemiluminescent, or chromogenic agents, as weU as substrates, cofactors, inhibitors, magnetic particles, and the like.

Host ceUs transformed with polynucleotides encoding NAAP may be cultured under conditions suitable for the expression and recovery of the protein from ceU culture. The protein produced by a transformed ceU may be secreted or retained intraceUularly depending on the sequence and/or the vector used. As wiU 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 ceU membrane.

In addition, a host ceU strain may be chosen for its abikty to modulate expression of the inserted polynucleotides or to process the expressed protein in the desired fashion. Such modifications of the polypeptide include, but are not kmited to, acetylation, carboxylation, glycosylation, phosphorylation, kpidation, 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 ceUs which have specific ceUular machinery and characteristic mechanisms for post-translational activities (e.g., CHO, HeLa, MDCK, HEK293, and WI38) are available from the American Type Culture CoUection (ATCC, Manassas VA) and may be chosen to ensure the correct modification and processing of the foreign protein. In another embodiment of the invention, natoral, modified, or recombinant polynucleotides encoding NAAP may be kgated 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 commerciaUy available antibody may facihtate the screening of peptide kbraries for inhibitors of NAAP activity. Heterologous protein and peptide moieties may also faciktate purification of fusion proteins using commerciaUy available affinity matrices. Such moieties include, but are not limited to, glutathione S-transferase (GST), maltose binding protein (MBP), thioredoxin (Trx), calmodukn binding peptide (CBP), 6-His, FLAG, c-myc, and hemagglutinin (HA). GST, MBP, Trx, CBP, and 6-His enable purification of their cognate fusion proteins on immobikzed glutathione, maltose, phenylarsine oxide, calmodukn, and metal-chelate resins, respectively. FLAG, c-myc, and hemagglutinin (HA) enable immunoaffinity purification of fusion proteins using commerciaUy available monoclonal and polyclonal antibodies that specificaUy 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 et al. (supra, ch. 10 and 16). A variety of commercially available kits may also be used to faciktate expression and purification of fusion proteins.

In another embodiment, synthesis of radiolabeled NAAP may be achieved in vitro using the TNT rabbit reticulocyte lysate or wheat 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, fragments of NAAP, or variants of NAAP maybe used to screen for compounds that specificaUy bind to NAAP. One or more test compounds may be screened for specific binding to NAAP. In various embodiments, 1, 2, 3, 4, 5, 10, 20, 50, 100, or 200 test compounds can be screened for specific binding to NAAP. Examples of test compounds can include antibodies, anticakns, okgonucleotides, proteins (e.g., kgands or receptors), or smaU molecules.

In related embodiments, variants of NAAP can be used to screen for binding of test compounds, such as antibodies, to NAAP, a variant of NAAP, or a combination of NAAP and/or one or more variants NAAP. In an embodiment, a variant of NAAP can be used to screen for compounds that bind to a variant of NAAP, but not to NAAP having the exact sequence of a sequence of SEQ DD NO:l-57. NAAP variants used to perform such screening can have a range of about 50% to about 99% sequence identity to NAAP, with various embodiments having 60%, 70%, 75%, 80%, 85%, 90%, and 95% sequence identity.

In an embodiment, a compound identified in a screen for specific binding to NAAP can be closely related to the natural kgand of NAAP, e.g., a kgand or fragment thereof, a natural substrate, a structural or functional mimetic, or a natural binding partner (Cokgan, J.E. et al. (1991) Current Protocols in Immunology l(2):Chapter 5). In another embodiment, the compound thus identified can be a natoral kgand of a receptor NAAP (Howard, A.D. et al. (2001) Trends Pharmacol. Sci.22:132- 140; Wise, A. et al. (2002) Drug Discovery Today 7:235-246).

In other embodiments, a compound identified in a screen for specific binding to NAAP can be closely related to the natural receptor to which NAAP binds, at least a fragment of the receptor, or a fragment of the receptor including aU or a portion of the kgand binding site or binding pocket. For example, the compound may be a receptor for NAAP which is capable of propagating a signal, or a decoy receptor for NAAP which is not capable of propagating a signal (Ashkenazi, A. and V.M. Divit (1999) Curr. Opin. CeU Biol. 11:255-260; Mantovani, A. et al. (2001) Trends Immunol. 22:328-336). The compound can be rationaUy designed using known techniques. Examples of such techniques include those used to construct the compound etanercept (ENBREL; Amgen Inc., Thousand Oaks CA), which is efficacious for treating rheumatoid arthritis in humans. Etanercept is an engineered p75 tumor necrosis factor (TNF) receptor dimer linked to the Fc portion of human IgG_! (Taylor, P.C et al. (2001) GUT. Opin. Immunol. 13:611-616).

In one embodiment, two or more antibodies having similar or, alternatively, different specificities can be screened for specific binding to NAAP, fragments of NAAP, or variants of NAAP. The binding specificity of the antibodies thus screened can thereby be selected to identify particular fragments or variants of NAAP. In one embodiment, an antibody can be selected such that its binding specificity aUows for preferential identification of specific fragments or variants of NAAP. In another embodiment, an antibody can be selected such that its binding specificity aUows for preferential diagnosis of a specific disease or condition having increased, decreased, or otherwise abnormal production of NAAP.

In an embodiment, anticakns can be screened for specific binding to NAAP, fragments of NAAP, or variants of NAAP. Anticakns are kgand-binding proteins that have been constructed based on a kpocakn scaffold (Weiss, G.A. and H.B. Lowman (2000) Chem. Biol. 7:R177-R184; Skena, A. (2001) J. Biotechnol. 74:257-275). The protein architecture of kpocalins can include a beta-barrel having eight antiparaUel beta-strands, which supports four loops at its open end. These loops form the natoral kgand-binding site of the kpocalins, a site which can be re-engineered in vitro by amino acid substitutions to impart novel binding specificities. The amino acid substitutions can be made using methods known in the art or described herein, and can include conservative substitotions (e.g., substitotions that do not alter binding specificity) or substitutions that modestly, moderately, or significantly alter binding specificity. In one embodiment, screening for compounds which specificaUy bind to, stimulate, or inhibit

NAAP involves producing appropriate cells which express NAAP, either as a secreted protein or on the ceU membrane. Preferred ceUs can include ceUs from mammals, yeast, Drosophila, or E. coli. CeUs expressing NAAP or ceU 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 sokd 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. AdditionaUy, the assay may be carried out using ceU-free preparations, chemical kbraries, or natural product mixtures, and the test compound(s) may be free in solution or affixed to a sokd support.

An assay can be used to assess the abikty of a compound to bind to its natoral kgand and/or to inhibit the binding of its natoral kgand to its natural receptors. Examples of such assays include radio- labeling assays such as those described in U.S. Patent No. 5,914,236 and U.S. Patent No. 6,372,724. In a related embodiment, one or more amino acid substitutions can be introduced into a polypeptide compound (such as a receptor) to improve or alter its abikty to bind to its natural kgands (Matthews, D.J. and J.A. WeUs. (1994) Chem. Biol. 1:25-30). In another related embodiment, one or more amino acid substitotions can be introduced into a polypeptide compound (such as a kgand) to improve or alter its abikty to bind to its natural receptors (Cunningham, B.C. and J.A. WeUs (1991) Proc. Natl. Acad. Sci. USA 88:3407-3411; Lowman, HB. et al. (1991) J. Biol. Chem. 266:10982-10988).

NAAP, fragments of NAAP, or variants of NAAP may be used to screen for compounds that modulate the activity of NAAP. Such compounds may include agonists, antagonists, or partial or inverse agonists. In one embodiment, an assay is performed under conditions permissive for NAAP activity, wherein NAAP is combined with at least one test compound, and the activity of NAAP in the presence of a test compound is compared with the activity of NAAP in the absence of the test compound. A change in the activity of NAAP in the presence of the test compound is indicative of a compound that modulates the activity of NAAP. Alternatively, a test compound is combined with an in vitro or ceU-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 plurakty of test compounds may be screened.

In another embodiment, polynucleotides encoding NAAP or their mammakan homologs may be "knocked out" in an animal model system using homologous recombination in embryonic stem (ES) ceUs. Such techniques are weU 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 ceUs, such as the mouse 129/SvJ ceU line, are derived from the early mouse embryo and grown in culture. The ES ceUs 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 conesponding 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) Ckn. Invest. 97:1999-2002; Wagner, K.U. et al. (1997) Nucleic Acids Res. 25:4323-4330). Transformed ES ceUs are identified and microinjected into mouse ceU blastocysts such as those from the C57BL/6 mouse strain. The blastocysts are surgicaUy 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 ceUs have the potential to differentiate into at least eight separate ceU kneages including endoderm, mesoderm, and ectodermal ceU types. These ceU lineages differentiate into, for example, neural ceUs, hematopoietic kneages, 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 ceUs, and the injected sequence integrates into the animal ceU genome. Transformed ceUs are injected into blastulae, and the blasrulae are implanted as described above. Transgenic progeny or inbred Unes are studied and treated with potential pharmaceutical agents to obtain information on treatment of a human disease. Alternatively, a mammal inbred to overexpress NAAP, e.g., by secreting NAAP in its milk, may also serve as a convenient source of that protein (Janne, J. et al. (1998) Biotechnol. Annu. Rev. 4:55-74). THERAPEUTICS

Chemical and structural similarity, e.g., in the context of sequences and motifs, exists between regions of NAAP and nucleic acid-associated proteins. In addition, examples of tissues expressing NAAP can be found in Table 6 and can also be found in Example XI. Therefore, NAAP appears to play a role in ceU prokferative, 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 ceU prokferative 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, colon, gaU bladder, gangka, gastrointestinal tract, heart, kidney, kver, lung, muscle, ovary, pancreas, parathyroid, penis, prostate, sakvary 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 demyeknating diseases, bacterial and viral meningitis, brain abscess, subdural empyema, epidural abscess, suppurative intracranial thrombophlebitis, myektis and radicuktis, viral central nervous system disease, prion diseases including kuru, Creutzfeldt-Jakob disease, and Gerstmann-Straussler-Scheinker syndrome, fatal famikal insomnia, nutritional and metabokc diseases of the nervous system, neurofibromatosis, tuberous sclerosis, cerebeUoretinal 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, metabokc, 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 abnormakties, and mental retardation), Smith-Magenis syndrome, myelodysplastic syndrome, hereditary mucoepithekal 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 (AEDS), Addison' s disease, adult respiratory distress syndrome, aUergies, ankylosing spondyktis, 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 melktas, emphysema, episodic lymphopenia with lymphocytotoxins, erythroblastosis fetaks, erythema nodosum, atrophic gastritis, glomerulonephritis, Goodpasture's syndrome, gout, Graves' disease, Hashimoto's thyroiditis, hypereosinophika, 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 coktis, uveitis, Werner syndrome, compkcations of cancer, hemodialysis, and extracorporeal circulation, viral, bacterial, fungal, parasitic, protozoal, and helminthic infections, and trauma; and an infection, such as those caused by a viral agent classified as adenovirus, arenavirus, bunyavirus, cakcivirus, coronavirus, filovirus, hepadnavirus, herpesvirus, flavivirus, orthomyxovirus, parvovirus, papovavirus, paramyxovirus, picornavirus, poxvirus, reovirus, retrovirus, rhabdovirus, or togavirus; an infection caused by a bacterial agent classified as pneumococcus, staphylococcus, streptococcus, baciUus, corynebacterium, clostridium, meningococcus, gonococcus, ksteria, moraxeUa, kingeUa, haemophilus, legioneUa, bordeteUa, gram-negative enterobacterium including shigeUa, saknoneUa, or campylobacter, pseudomonas, vibrio, bruceUa, franciseUa, yersinia, bartoneUa, norcardium, actinomyces, mycobacterium, spirochaetale, rickettsia, chlamydia, or mycoplasma; an infection caused by a fungal agent classified as aspergiUus, blastomyces, dermatophytes, cryptococcus, coccidioides, malasezzia, histoplasma, or other mycosis-causing fungal agent; and an infection caused by a parasite classified as plasmodium or malaria-causing, parasitic entamoeba, leishmania, trypanosoma, toxoplasma, pneumocystis carinii, intestinal protozoa such as giardia, trichomonas, tissue nematode such as trichineUa, 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 substantiaUy 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 stiU 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 ksted 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 ceU prokferative, neurological, developmental, and autoimmune/inflammatory disorders, and infections described above. In one aspect, an antibody which specificaUy binds NAAP may be used directly as an antagonist or indirectly as a targeting or dekvery mechanism for bringing a pharmaceutical agent to ceUs 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 protein, agonist, antagonist, antibody, complementary sequence, or vector embodiments may be administered in combination with other appropriate therapeutic agents. Selection of the appropriate agents for use in combination therapy may be made by one of ordinary skiU in the art, according to conventional pharmaceutical principles. The combination of therapeutic agents may act synergisticaUy 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 generaUy known in the art. In particular, purified NAAP may be used to produce antibodies or to screen kbraries of pharmaceutical agents to identify those which specificaUy bind NAAP. Antibodies to NAAP may also be generated using methods that are weU 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 kbrary. In an embodiment, neutrakzing antibodies (i.e., those which inhibit dimer formation) can be used therapeuticaUy. Single chain antibodies (e.g., from camels or Uamas) may be potent enzyme inhibitors and may have appkcation in the design of peptide mimetics, and in the development of immuno-adsorbents and biosensors (Muyldermans, S. (2001) J. Biotechnol. 74:277-302).

For the production of antibodies, various hosts including goats, rabbits, rats, mice, camels, dromedaries, llamas, humans, and others may be immunized by injection with NAAP or with any fragment or okgopeptide thereof which has immunogenic properties. Depending on the host species, various adjuvants maybe 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 (baciUi Calmette-Guerin) and Corynebacterium parvum are especiaUy preferable.

It is prefened that the okgopeptides, peptides, or fragments used to induce antibodies to NAAP have an amino acid sequence consisting of at least about 5 amino acids, and generaUy wiU consist of at least about 10 amino acids. It is also preferable that these okgopeptides, peptides, or fragments are substantiaUy identical to a portion of the amino acid sequence of the natoral 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 ceU knes in cultore. These include, but are not limited to, the hybridoma technique, the human B-cell hybridoma technique, and the EBV-hybridoma technique (Kohler, G. et al. (1975) Nature 256:495-497; Kozbor, D. et al. (1985) J. Immunol. Methods

81:31-42; Cote, R.J. et al. (1983) Proc. Natl. Acad. Sci. USA 80:2026-2030; Cole, S.P. et al. (1984)

Mol. CeU Biol. 62:109-120).

In addition, techniques developed for the production of "chimeric antibodies," such as the spkcing of mouse antibody genes to human antibody genes to obtain a molecule with appropriate antigen specificity and biological activity, can be used (Morrison, S.L. et al. (1984) Proc. Natl. Acad.

Sci. USA 81:6851-6855; Neuberger, M.S. et al. (1984) Nature 312:604-608; Takeda, S. et al. (1985)

Nature 314:452-454). Alternatively, techniques described for the production of single chain antibodies may be adapted, using methods known in the art, to produce NAAP-specific single chain antibodies. Antibodies with related specificity, but of distinct idiotypic composition, may be generated by chain shuffling from random combinatorial immunoglobulin kbraries (Burton, D.R. (1991) Proc. Natl. Acad.

Sci. USA 88:10134-10137).

Antibodies may also be produced by inducing in vivo production in the lymphocyte population or by screening immunoglobulin kbraries or panels of highly specific binding reagents as disclosed in the kteratare (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 kmited 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 kbraries may be constructed to aUow rapid and easy identification of monoclonal Fab fragments with the desired specificity (Huse, W.D. et al. (1989)

Science 246:1275-1281).

Various immunoassays may be used for screening to identify antibodies having the desired specificity. Numerous protocols for competitive binding or immunoradiometric assays using either polyclonal or monoclonal antibodies with estabkshed specificities are weU known in the art. Such immunoassays typicaUy involve the measurement of complex formation between NAAP and its specific antibody. A two-site, monoclonal-based immunoassay utikzing monoclonal antibodies reactive to two non-interfering NAAP epitopes is generaUy used, but a competitive binding assay may also be employed (Pound, supra). Various methods such as Scatchard analysis in conjunction with radioimmunoassay techniques may be used to assess the affinity of antibodies for NAAP. Affinity is expressed as an association constant, K_a, 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_a deteπnined for a preparation of polyclonal antibodies, which are heterogeneous in their affinities for multiple NAAP epitopes, represents the average affinity, or avidity, of the antibodies for NAAP. The K_a 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 K_a ranging from about IO⁹ to IO¹² L/mole are preferred for use in immunoassays in which the NAAP- antibody complex must withstand rigorous manipulations. Low-affinity antibody preparations with K_a ranging from about IO⁶ to IO⁷ L/mole are prefened 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; LiddeU, 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 quakty and suitabikty of such preparations for certain downstream appkcations. For example, a polyclonal antibody preparation containing at least 1-2 mg specific antibody/ml, preferably 5-10 mg specific antibody/ml, is generaUy employed in procedures requiring precipitation of NAAP-antibody complexes. Procedures for evaluating antibody specificity, titer, and avidity, and guidelines for antibody quakty and usage in various appkcations, are generaUy available (Catty, supra; Cokgan et al., supra). In another embodiment of the invention, polynucleotides encoding NAAP, or any fragment or complement thereof, may be used for therapeutic purposes. In one aspect, modifications of gene expression can be achieved by designing complementary sequences or antisense molecules (DNA, RNA, PNA, or modified okgonucleotides) to the coding or regulatory regions of the gene encoding NAAP. Such technology is weU known in the art, and antisense okgonucleotides or larger fragments can be designed from various locations along the coding or control regions of sequences encoding NAAP (Agrawal, S., ed. (1996) Antisense Therapeutics, Humana Press, Totawa NJ).

In therapeutic use, any gene dekvery system suitable for introduction of the antisense sequences into appropriate target ceUs can be used. Antisense sequences can be dekvered intraceUularly in the form of an expression plasmid which, upon transcription, produces a sequence complementary to at least a portion of the ceUular sequence encoding the target protein (Slater, J.E. et al. (1998) J. AUergy Clin. Immunol. 102:469-475; Scanlon, K.J. et al. (1995) 9:1288-1296). Antisense sequences can also be introduced intraceUularly through the use of viral vectors, such as retrovirus and adeno-associated virus vectors (MiUer, A.D. (1990) Blood 76:271; Ausubel et al, supra; Uckert, W. and W. Walther (1994) Pharmacol. Ther. 63:323-347). Other gene dekvery mechanisms include kposome-derived systems, artificial viral envelopes, and other systems known in the art (Rossi, J.J. (1995) Br. Med. Bull. 51:217-225; Boado, R.J. et al. (1998) J. Pharm. Sci. 87:1308-1315; Morris, M.C. et al. (1997) Nucleic Acids Res. 25:2730-2736).

In another embodiment of the invention, polynucleotides encoding NAAP may be used for somatic or germkne gene therapy. Gene therapy may be performed to (i) correct a genetic deficiency (e.g., in the cases of severe combined immunodeficiency (SCED)-Xl 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) CeU 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, famikal hypercholesterolemia, and hemophika resulting from Factor VIE or Factor IX deficiencies (Crystal, R.G. (1995) Science 270:404-410; Verma, I.M. and N. Somia (1997) Natore 389:239-242)), (ii) express a conditionaUy lethal gene product (e.g., in the case of cancers which result from unregulated cell prokferation), or (in) express a protein which affords protection against intraceUular parasites (e.g., against human retroviruses, such as human immunodeficiency virus (IEV) (Baltimore, D. (1988) Natore 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 Trypanosoma 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 ceUs may aUeviate 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 mammakan expression vectors encoding NAAP and introducing these vectors by mechanical means into NAAP-deficient ceUs. Mechanical transfer technologies for use with ceUs in vivo or ex vitro include (i) direct DNA microinjection into individual ceUs, (ii) baUistic gold particle dekvery, (iii) kposome-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; Ivies, Z. (1997) CeU 91:501-510; Boulay, J.-L. and H Recipon (1998) Cuπ. 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-SCRE , PCMV-TAG, PEGSH/PERV (Stratagene, La JoUa CA), and PTET-OFF, PTET-ON, PTRE2, PTRE2-LUC, PTK-HYG (Clontech, Palo Alto CA). NAAP may be expressed using (i) a constitotively active promoter, (e.g., from cytomegalovirus (CMV), Rous sarcoma virus (RSV), SV40 virus, thymidine kinase (TK), or β-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), commerciaUy available in the T-REX plasmid (Invitrogen)); the ecdysone-inducible promoter (available in the plasmids PVGRXR and PESTD; Invitrogen); the FK506/rapamycin inducible promoter; or the RU486/mifepristone inducible promoter (Rossi, F.M.V. and H.M. Blau, supra)), or (in) a tissue-specific promoter or the native promoter of the endogenous gene encoding NAAP from a normal individual.

CommerciaUy available Uposome transformation kits (e.g., the PERFECT LD?DD TRANSFECTION KIT, available from Invitrogen) aUow one with ordinary skiU in the art to dekver polynucleotides to target ceUs in culture and require minimal effort to optimize experimental parameters. In the alternative, transformation is performed using the calcium phosphate method (Graham, F.L. and A.J. Eb (1973) Virology 52:456-467), or by electroporation (Neumann, E. et al. (1982) EMBO J. 1:841-845). The introduction of DNA to primary cells requires modification of these standardized mammakan 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 s-acting RNA sequences and coding sequences required for efficient vector propagation. Retrovirus vectors (e.g., PFB and PFBNEO) are commerciaUy available (Stratagene) and are based on pubkshed 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 ceUs 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. Mffler (1988) J. Virol. 62:3802-3806; DuU, 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 ceU lines producing high transducing efficiency retroviral supernatant") discloses a method for obtaining retrovirus packaging ceU lines and is hereby incorporated by reference. Propagation of retrovirus vectors, transduction of a population of ceUs (e.g., CD4⁺ T-ceUs), and the retorn of transduced ceUs to a patient are procedures weU known to persons skiUed in the art of gene therapy and have been weU documented (Ranga, U. et al. (1997) J. Virol. 71:7020-7029; Bauer, G. et al. (1997) Blood 89:2259-2267; Bonyhadi, M.L. (1997) J. Virol. 71:4707-4716; Ranga, U. et al. (1998) Proc. Natl. Acad. Sci. USA 95:1201-1206; Su, L. (1997) Blood 89:2283-2290).

In an embodiment, an adenovirus-based gene therapy dekvery system is used to dekver polynucleotides encoding NAAP to ceUs which have one or more genetic abnormakties with respect to the expression of NAAP. The construction and packaging of adenovirus-based vectors are weU known to those with ordinary skill in the art. Repkcation 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). PotentiaUy 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, I.M. and N. Somia (1997; Natore 18:389:239-242).

In another embodiment, a herpes-based, gene therapy dekvery system is used to dekver polynucleotides encoding NAAP to target ceUs which have one or more genetic abnormakties with respect to the expression of NAAP. The use of herpes simplex virus (HSV)-based vectors may be especiaUy valuable for introducing NAAP to ceUs of the central nervous system, for which HSV has a tropism. The construction and packaging of herpes-based vectors are weU known to those with ordinary skill in the art. A repkcation-competent herpes simplex virus (HSV) type 1 -based vector has been used to dekver 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 transfeπed to a ceU 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). 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 ceUs with herpesvirus are techniques weU known to those of ordinary skiU in the art.

In another embodiment, an alphavirus (positive, single-stranded RNA virus) vector is used to dekver polynucleotides encoding NAAP to target ceUs. The biology of the prototypic alphavirus, Sernkki 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 repkcation, a subgenomic RNA is generated that normaUy encodes the viral capsid proteins. This subgenomic RNA repkcates to higher levels than the fuU 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 ceUs. While alphavirus infection is typicaUy associated with cell lysis within a few days, the abikty to estabksh a persistent infection in hamster normal kidney ceUs (BHK-21) with a variant of Sindbis virus (SIN) indicates that the lytic repkcation of alphaviruses can be altered to suit the needs of the gene therapy appkcation (Dryga, S.A. et al. (1997) Virology 228:74-83). The wide host range of alphaviruses wiU allow the introduction of NAAP into a variety of ceU types. The specific transduction of a subset of ceUs in a population may require the sorting of ceUs prior to transduction. The methods of manipulating infectious cDNA clones of alphaviruses, performing alphavirus cDNA and RNA transfections, and performing alphavirus infections, are weU known to those with ordinary skiU in the art.

Okgonucleotides 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 hekx base-pairing methodology. Triple hekx pairing is useful because it causes inhibition of the abikty of the double hekx to open sufficiently for the binding of polymerases, transcription factors, or regulatory molecules. Recent therapeutic advances using triplex DNA have been described in the kterature (Gee, J.E. et al. (1994) in Huber, B.E. and B.I. Carr, Molecular and Immunologic Approaches, Futara Pubkshing, 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, foUowed by endonucleolytic cleavage. For example, engineered hammerhead motif ribozyme molecules may specificaUy and efficiently catalyze endonucleolytic cleavage of RNA molecules 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, conesponding to the region of ttie target gene containing the cleavage site, may be evaluated for secondary structural features which may render the okgonucleotide inoperable. The suitabikty of candidate targets may also be evaluated by testing accessibikty to hybridization with complementary okgonucleotides using ribonuclease protection assays.

Complementary ribonucleic acid molecules and ribozymes may be prepared by any method known in the art for the synthesis of nucleic acid molecules. These include techniques for chemicaUy synthesizing okgonucleotides such as sokd phase phosphoramidite chemical synthesis. Alternatively, RNA molecules may be generated by in vitro and in vivo transcription of DNA molecules encoding NAAP. Such DNA sequences may be incorporated into a wide variety of vectors with suitable RNA polymerase promoters such as T7 or SP6. Alternatively, these cDNA constructs that synthesize complementary RNA, constitotively or inducibly, can be introduced into ceU lines, ceUs, or tissues. RNA molecules may be modified to increase intracellular stabikty and half-Ufe. 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 aU of these molecules by the inclusion of nontraditional bases such as inosine, queosine, and wybutosine, as weU as acetyl-, methyl-, thio-, and similarly modified forms of adenine, cytidine, guanine, thymine, and uridine which are not as easily recognized by endogenous endonucleases.

In other embodiments of the invention, the expression of one or more selected polynucleotides of the present invention can be altered, inhibited, decreased, or silenced using RNA interference (RNAi) or post-transcriptional gene silencing (PTGS) methods known in the art. RNAi is a posttranscriptional mode of gene silencing in which double-stranded RNA (dsRNA) introduced into a targeted ceU specificaUy suppresses the expression of the homologous gene (i.e., the gene bearing the sequence complementary to the dsRNA). This effectively knocks out or substantiaUy reduces the expression of the targeted gene. PTGS can also be accompkshed by use of DNA or DNA fragments as weU. RNAi methods are described by Fire, A. et al. (1998; Nature 391:806-811) and Gura, T. (2000; Natore 404:804-808). PTGS can also be initiated by introduction of a complementary segment of DNA into the selected tissue using gene dekvery and/or viral vector dekvery methods described herein or known in the art. RNAi can be induced in mammakan ceUs by the use of small interfering RNA also known as siRNA. SiRNA are shorter segments of dsRNA (typicaUy about 21 to 23 nucleotides in length) that result in vivo from cleavage of introduced dsRNA by the action of an endogenous ribonuclease. SiRNA appear to be the mediators of the RNAi effect in mammals. The most effective siRNAs appear to be 21 nucleotide dsRNAs with 2 nucleotide 3' overhangs. The use of siRNA for inducing RNAi in mammakan cells is described by Elbashir, S.M. et al. (2001; Nature 411:494-498).

SiRNA can either be generated indirectly by introduction of dsRNA into the targeted ceU, or directly by mammakan transfection methods and agents described herein or known in the art (such as kposome-mediated transfection, viral vector methods, or other polynucleotide dekvery/introductory methods). Suitable SiRNAs can be selected by examining a transcript of the target polynucleotide (e.g., mRNA) for nucleotide sequences downstream from the AUG start codon and recording the occurrence of each nucleotide and the 3 ' adjacent 19 to 23 nucleotides as potential siRNA target sites, with sequences having a 21 nucleotide length being preferred. Regions to be avoided for target siRNA sites include the 5' and 3 'untranslated regions (UTRs) and regions near the start codon (within 75 bases), as these may be richer in regulatory protein binding sites. UTR-binding proteins and/or translation initiation complexes may interfere with binding of the siRNP endonuclease complex. The selected target sites for siRNA can then be compared to the appropriate genome database (e.g., human, etc.) using BLAST or other sequence comparison algorithms known in the art. Target sequences with significant homology to other coding sequences can be eliminated from consideration. The selected SiRNAs can be produced by chemical synthesis methods known in the art or by in vitro transcription using commerciaUy available methods and kits such as the SILENCER siRNA construction kit (Ambion, Austin TX).

In alternative embodiments, long-term gene silencing and/or RNAi effects can be induced in selected tissue using expression vectors that continuously express siRNA. This can be accompkshed using expression vectors that are engineered to express hairpin RNAs (shRNAs) using methods known in the art (see, e.g., Brummelkamp, T.R. et al. (2002) Science 296:550-553; and Paddison, P.J. et al. (2002) Genes Dev. 16:948-958). In these and related embodiments, shRNAs can be dekvered to target ceUs using expression vectors known in the art. An example of a suitable expression vector for dekvery of siRNA is the PSE.ENCER1.0-U6 (circular) plasmid (Ambion). Once dekvered to the target tissue, shRNAs are processed in vivo into siRNA-like molecules capable of carrying out gene- specific silencing.

In various embodiments, the expression levels of genes targeted by RNAi or PTGS methods can be determined by assays for mRNA and/or protein analysis. Expression levels of the mRNA of a targeted gene, can be determined by northern analysis methods using, for example, the NORTHERNMAX-GLY kit (Ambion); by microanay methods; by PCR methods; by real time PCR methods; and by other RNA/polynucleotide assays known in the art or described herein. Expression levels of the protein encoded by the targeted gene can be determined by Western analysis using standard techniques known in the art.

An additional embodiment of the invention encompasses a method for screening for a compound which is effective in altering expression of a polynucleotide encoding NAAP. Compounds which may be effective in altering expression of a specific polynucleotide may include, but are not limited to, okgonucleotides, antisense okgonucleotides, triple hekx-forming okgonucleotides, 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 specificaUy inhibits expression of the polynucleotide encoding NAAP may be herapeuticaUy useful, and in the treatment of disorders associated with decreased NAAP expression or activity, a compound which specificaUy promotes expression of the polynucleotide encoding NAAP may be therapeuticaUy useful.

In various embodiments, one or more test compounds maybe 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, commerciaUy-available or proprietary kbrary of nataraUy-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 kbrary of chemical compounds created combinatoriaUy or randomly. A sample comprising a polynucleotide encoding NAAP is exposed to at least one test compound thus obtained. The sample may comprise, for example, an intact or permeabikzed ceU, or an in vitro ceU-free or reconstituted biochemical system. Alterations in the expression of a polynucleotide encoding NAAP are assayed by any method commonly known in the art. TypicaUy, 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 ceU line such as HeLa ceU (Clarke, M.L. et al. (2000) Biochem. Biophys. Res. Commun. 268:8-13). A particular embodiment of the present invention involves screening a combinatorial kbrary of okgonucleotides (such as deoxyribonucleotides, ribonucleotides, peptide nucleic acids, and modified okgonucleotides) 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 ceUs or tissues are available and equaUy suitable for use in vivo, in vitro, and ex vivo. For ex vivo therapy, vectors may be introduced into stem ceUs taken from the patient and clonaUy propagated for autologous transplant back into that same patient. Dekvery by transfection, by kposome injections, or by polycationic amino polymers may be achieved using methods which are weU known in the art (Goldman, C.K. et al. (1997) Nat. Biotechnol. 15:462- 466).

Any of the therapeutic methods described above may be appked 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 pharmaceuticaUy acceptable excipient. Excipients may include, for example, sugars, starches, ceUuloses, gums, and proteins. Various formulations are commonly known and are thoroughly discussed in the latest edition of Remington's Pharmaceutical Sciences (Maack PubUshing, Easton PA). Such compositions may consist of NAAP, antibodies to NAAP, and mimetics, agonists, antagonists, or inhibitors of NAAP.

In various embodiments, the compositions described herein, such as pharmaceutical compositions, may be administered by any number of routes including, but not limited to, oral, intravenous, intramuscular, intra-arterial, intrameduUary, intrathecal, intraventricular, pulmonary, transdermal, subcutaneous, intraperitoneal, intranasal, enteral, topical, subkngual, or rectal means. Compositions for pulmonary administration may be prepared in kquid or dry powder form. These compositions are generaUy aerosokzed immediately prior to inhalation by the patient. In the case of smaU molecules (e.g. traditional low molecular weight organic drugs), aerosol dekvery of fast- acting formulations is weU-known in the art. In the case of macromolecules (e.g. larger peptides and proteins), recent developments in the field of pulmonary dekvery via the alveolar region of the lung have enabled the practical dekvery of drugs such as insulin to blood circulation (see, e.g., Patton, J.S. et al., U.S. Patent No. 5,997,848). Pulmonary dekvery aUows administration without needle injection, and obviates the need for potentiaUy 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 weU within the capabikty of those skiUed in the art.

Speciakzed forms of compositions may be prepared for direct intraceUular dekvery of macromolecules comprising NAAP or fragments thereof. For example, kposome preparations containing a ceU-impermeable macromolecule may promote ceU fusion and intraceUular dekvery of the macromolecule. Alternatively, NAAP or a fragment thereof may be joined to a short cationic N- terminal portion from the HIV Tat-1 protein. Fusion proteins thus generated have been found to transduce into the ceUs of aU 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 initiaUy either in ceU culture assays, e.g., of neoplastic ceUs, 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 therapeuticaUy 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 amekorates 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 calculatmg the ED₅₀ (the dose therapeuticaUy effective in 50% of the population) or LD₅₀ (the dose lethal to 50% of the population) statistics. The dose ratio of toxic to therapeutic effects is the therapeutic index, which can be expressed as the LD₅₀/ED₅₀ ratio. Compositions which exhibit large therapeutic indices are prefened. The data obtained from ceU 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 ED₅₀ with kttle 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 wiU be determined by the practitioner, in kght 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-Ufe and clearance rate of the particular formulation.

Normal dosage amounts may vary from about 0.1 μg to 100,000 μg, up to a total dose of about 1 gram, depending upon the route of administration. Guidance as to particular dosages and methods of dekvery is provided in the kterature and generaUy available to practitioners in the art. Those skiUed in the art wiU employ different formulations for nucleotides than for proteins or their inhibitors. Similarly, dekvery of polynucleotides or polypeptides wiU be specific to particular ceUs, conditions, locations, etc. DIAGNOSTICS In another embodiment, antibodies which specificaUy 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 utiUze the antibody and a label to detect NAAP in human body fluids or in extracts of ceUs or tissues. The antibodies may be used with or without modification, and may be labeled by covalent or non-covalent attachment of a reporter molecule. A wide variety of reporter molecules, several of which are described above, are known in the art and may be used.

A variety of protocols for measuring NAAP, including ELISAs, RIAs, and FACS, are known in the art and provide a basis for diagnosing altered or abnormal levels of NAAP expression. Normal or standard values for NAAP expression are established by combining body fluids or ceU extracts taken from normal mammakan 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 estabhshes the parameters for diagnosing disease. In another embodiment of the invention, polynucleotides encoding NAAP may be used for diagnostic purposes. The polynucleotides which may be used include okgonucleotides, complementary RNA and DNA molecules, and PNAs. The polynucleotides may be used to detect and quantify gene expression in biopsied tissues in which expression of NAAP may be correlated with disease. The diagnostic assay 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 polynucleotides, including genomic sequences, encoding NAAP or closely related molecules may be used to identify nucleic acid sequences which encode NAAP. The specificity of the probe, whether it is made from a highly specific region, e.g., the 5'regulatory region, or from a less specific region, e.g., a conserved motif, and the stringency of the hybridization or ampkfication wiU determine whether the probe identifies only naturaUy occurring sequences encoding NAAP, aUekc 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 ED NO:58-l 14 or from genomic sequences including promoters, enhancers, and introns of the NAAP gene. Means for producing specific hybridization probes for polynucleotides encoding NAAP include the cloning of polynucleotides encoding NAAP or NAAP derivatives into vectors for the production of mRNA probes. Such vectors are known in the art, are commerciaUy 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 radionuckdes such as ³²P or ³⁵S, or by enzymatic labels, such as alkaline phosphatase coupled to the probe via avidin biotin coupling systems, and the kke.

Polynucleotides encoding NAAP may be used for the diagnosis of disorders associated with expression of NAAP. Examples of such disorders include, but are not limited to, a ceU prokferative 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, colon, gaU bladder, gangka, gastrointestinal tract, heart, kidney, kver, lung, muscle, ovary, pancreas, parathyroid, penis, prostate, sakvary glands, skin, spleen, testis, thymus, thyroid, and uterus; a neurological disorder such as epilepsy, ischemic cerebro vascular 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 demyeknating diseases, bacterial and viral meningitis, brain abscess, subdural empyema, epidural abscess, suppurative intracranial thrombophlebitis, myektis and radicuktis, viral central nervous system disease, prion diseases including kuru, Creutzfeldt-Jakob disease, and Gerstmann-Straussler-Scheinker syndrome, fatal famikal insomnia, nutritional and metabokc diseases of the nervous system, neurofibromatosis, tuberous sclerosis, cerebeUoretinal 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, metabokc, 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 tobular acidosis, anemia, Cushing's syndrome, achondroplastic dwarfism, Duchenne and Becker muscular dystrophy, epilepsy, gonadal dysgenesis, WAGR syndrome (Wilms' tamor, aniridia, genitourinary abnormakties, and mental retardation), Smith-Magenis syndrome, myelodysplastic syndrome, hereditary mucoepithekal 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 (ADDS), Addison's disease, adult respiratory distress syndrome, aUergies, ankylosing spondyktis, 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 meUitus, emphysema, episodic lymphopenia with lymphocytotoxins, erythroblastosis fetaks, erythema nodosum, atrophic gastritis, glomerulonephritis, Goodpastore's syndrome, gout, Graves' disease, Hashimoto's thyroiditis, hypereosinophika, irritable bowel syndrome, multiple sclerosis, myasthenia gravis, myocardial or pericardial inflammation, osteoarthritis, osteoporosis, pancreatitis, polymyositis, psoriasis, Reiter's syndrome, rheumatoid arthritis, scleroderma, Sjόgren's syndrome, systemic anaphylaxis, systemic lupus erythematosus, systemic sclerosis, thrombocytopenic purpura, ulcerative coktis, uveitis, Werner syndrome, compkcations of cancer, hemodialysis, and extracorporeal circulation, viral, bacterial, fungal, parasitic, protozoal, and helminthic infections, and trauma; and an infection, such as those caused by a viral agent classified as adenovirus, arenavirus, bunyavirus, cakcivirus, coronavirus, filovirus, hepadnavirus, herpesvirus, flavivirus, orthomyxovirus, parvoviru's, papovavirus, paramyxovirus, picornavirus, poxvirus, reovirus, retrovirus, rhabdovirus, or togavirus; an infection caused by a bacterial agent classified as pneumococcus, staphylococcus, streptococcus, baciUus, corynebacterium, clostridium, meningococcus, gonococcus, ksteria, moraxeUa, kingeUa, haemophilus, legioneUa, bordeteUa, gram-negative enterobacterium including shigeUa, saknoneUa, or campylobacter, pseudomonas, vibrio, bruceUa, franciseUa, yersinia, bartoneUa, norcardium, actinomyces, mycobacterium, spirochaetale, rickettsia, chlamydia, or mycoplasma; an infection caused by a fungal agent classified as aspergiUus, blastomyces, dermatophytes, cryptococcus, coccidioides, malasezzia, histoplasma, or other mycosis-causing fungal agent; and an infection caused by a parasite classified as plasmodium or malaria-causing, parasitic entamoeba, leishmania, trypanosoma, toxoplasma, pneumocystis carinii, intestinal protozoa such as giardia, trichomonas, tissue nematode such as trichineUa, intestinal nematode such as ascaris, lymphatic filarial nematode, trematode such as schistosoma, and cestode such as tapeworm. Polynucleotides encoding NAAP may be used in Southern or northern analysis, dot blot, or other membrane-based technologies; in PCR technologies; in dipstick, pin, and multiformat ELISA-kke assays; and in microarrays utikzing fluids or tissues from patients to detect altered NAAP expression. Such quaktative or quantitative methods are weU known in the art.

In a particular embodiment, polynucleotides encoding NAAP may be used in assays that detect the presence of associated disorders, particularly those mentioned above. Polynucleotides complementary to sequences encoding NAAP may be labeled by standard methods and added to a fluid or tissue sample from a patient under conditions suitable for the formation of hybridization complexes. After a suitable incubation period, the sample is washed and the signal is quantified and compared with a standard value. If the amount of signal in the patient sample is significantly altered in comparison to a control sample then the presence of altered levels of polynucleotides encoding 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 estabkshed. This may be accompkshed by combining body fluids or ceU extracts taken from normal subjects, either animal or human, with a sequence, or a fragment thereof, encoding NAAP, under conditions suitable for hybridization or ampkfication. 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 substantiaUy 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 estabksh the presence of a disorder.

Once the presence of a disorder is estabkshed 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 earker, thereby preventing the development or further progression of the cancer.

Additional diagnostic uses for okgonucleotides designed from the sequences encoding NAAP may involve the use of PCR. These okgomers may be chemicaUy synthesized, generated enzymaticaUy, or produced in vitro. Okgomers wiU preferably contain a fragment of a polynucleotide encoding NAAP, or a fragment of a polynucleotide complementary to the polynucleotide encoding NAAP, and wiU be employed under optimized conditions for identification of a specific gene or condition. Okgomers may also be employed under less stringent conditions for detection or quantification of closely related DNA or RNA sequences.

In a particular aspect, okgonucleotide primers derived from polynucleotides encoding NAAP may be used to detect single nucleotide polymorphisms (SNPs). SNPs are substitotions, 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, okgonucleotide primers derived from polynucleotides encoding NAAP are used to ampkfy 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 okgonucleotide primers are fluorescently labeled, which aUows detection of the ampkmers in high-throughput equipment such as DNA sequencing machines. AdditionaUy, sequence database analysis methods, termed in sikco 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 eπors using statistical models and automated analyses of DNA sequence chromatograms. In the alternative, SNPs may be detected and characterized by mass spectrometry using, for example, the high throughput MASSARRAY system (Sequenom, Inc., San Diego CA).

SNPs may be used to study the genetic basis of human disease. For example, at least 16 common SNPs have been associated with non-insulin-dependent diabetes meUitos. SNPs are also useful for examining differences in disease outcomes in monogenic disorders, such as cystic fibrosis, sickle ceU anemia, or chronic granulomatous disease. For example, variants in the mannose-binding lectin, MBL2, have been shown to be correlated with deleterious pulmonary outcomes in cystic fibrosis. SNPs also have utikty in pharmacogenomics, the identification of genetic variants that influence a patient's response to a drug, such as kfe-threatening toxicity. For example, a variation in N-acetyl transferase is associated with a high incidence of peripheral neuropathy in response to the anti-tuberculosis drug isoniazid, while a variation in the core promoter of the ALOX5 gene results in diminished cknical response to treatment with an anti-asthma drug that targets the 5-kpoxygenase pathway. Analysis of the distribution of SNPs in different populations is useful for investigating genetic drift, mutation, recombination, and selection, as weU as for tracing the origins of populations and their migrations (Taylor, J.G. et al. (2001) Trends Mol. Med. 7:507-512; Kwok, P.-Y. and Z. Gu (1999) Mol. Med. Today 5:538-543; Nowotny, P. et al. (2001) Our. Opin. Neurobiol. 11:637-641). Methods which may also be used to quantify the expression of NAAP include radiolabeling or biotinylating nucleotides, coampkfication of a control nucleic acid, and interpolating results from standard curves (Melby, P.C. et al. (1993) J. Immunol. Methods 159:235-244; Duplaa, C. et al. (1993) Anal. Biochem. 212:229-236). The speed of quantitation of multiple samples may be accelerated by running the assay in a Wgh-throughput format where the okgomer or polynucleotide of interest is presented in various dilutions and a spectrophotometric or colorimetric response gives rapid quantitation.

In further embodiments, okgonucleotides or longer fragments derived from any of the polynucleotides described herein may be used as elements on a microanay. The microaπay can be used in transcript imaging techniques which monitor the relative expression levels of large numbers of genes simultaneously as described below. The microarray may also be used to identify genetic variants, mutations, and polymorphisms. This information may be used to determine gene function, to understand the genetic basis of a disorder, to diagnose a disorder, to monitor progression/regression of disease as a function of gene expression, and to develop and monitor the activities of therapeutic agents in the treatment of disease. In particular, this information may be used to develop a pharmacogenomic profile of a patient in order to select the most appropriate and effective treatment regimen for that patient. For example, therapeutic agents which are highly effective and display the fewest side effects may be selected for a patient based on his her pharmacogenomic profile.

In another embodiment, NAAP, fragments of NAAP, or antibodies specific for NAAP may be used as elements on a microarray. The microarray may be used to monitor or measure protein- protein interactions, drug-target interactions, and gene expression profiles, as described above. A particular embodiment relates to the use of the polynucleotides of the present invention to generate a transcript image of a tissue or ceU type. A transcript image represents the global pattern of gene expression by a particular tissue or cell type. Global gene expression patterns are analyzed by quantifying the number of expressed genes and their relative abundance under given conditions and at a given time (Seilhamer et al., "Comparative Gene Transcript Analysis," U.S. Patent No. 5,840,484; hereby expressly incorporated by reference herein). Thus a transcript image may be generated by hybridizing the polynucleotides of the present invention or then complements to the totakty of transcripts or reverse transcripts of a particular tissue or ceU 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 plurakty of elements on a microanay. The resultant transcript image would provide a profile of gene activity.

Transcript images maybe generated using transcripts isolated from tissues, ceU 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 ceU 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 precknical evaluation of pharmaceuticals, as weU as toxicological testing of industrial and natoraUy-occurring environmental compounds. AU compounds induce characteristic gene expression patterns, frequently termed molecular fingerprints or toxicant signatures, which are indicative of mechanisms of action and toxicity (Nuwaysir, E.F. et al. (1999) Mol. Carcinog. 24:153-159; Steiner, S. and N.L. Anderson (2000) Toxicol. Lett. 112-113:467-471). If a test compound has a signature similar to that of a compound with known toxicity, it is Ukely 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 famikes. IdeaUy, a genome-wide measurement of expression provides the highest quakty signature. Even genes whose expression is not altered by any tested compounds are important as weU, as the levels of expression of these genes are used to normakze the rest of the expression data. The normakzation procedure is useful for comparison of expression data after treatment with different compounds. While the assignment of gene function to elements of a toxicant signature aids in interpretation of toxicity mechanisms, knowledge of gene function is not necessary for the statistical matching of signatures which leads to prediction of toxicity (see, for example, Press Release 00-02 from the National Institute of Environmental Health Sciences, released February 29, 2000, available at http://www.niehs.nih.gov/oc/news/toxchip.htm). Therefore, it is important and desirable in toxicological screening using toxicant signatures to include aU expressed gene sequences.

In an embodiment, the toxicity of a test compound can be assessed by treating a biological sample containing nucleic acids with the test compound. Nucleic acids that are expressed in the treated biological sample are hybridized with one or more probes specific to the polynucleotides of the present invention, so that transcript levels coπesponding to the polynucleotides of the present invention may be quantified. The transcript levels in the treated biological sample are compared with levels in an untreated biological sample. Differences in the transcript levels between the two samples are indicative of a toxic response caused by the test compound in the treated sample.

Another embodiment relates to the use of the polypeptides disclosed herein to analyze the proteome of a tissue or ceU type. The term proteome refers to the global pattern of protein expression in a particular tissue or ceU type. Each protein component of a proteome can be subjected individuaUy 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 ceU's proteome may thus be generated by separating and analyzing the polypeptides of a particular tissue or ceU 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 visuakzed in the gel as discrete and uniquely positioned spots, typicaUy by staining the gel with an agent such as Coomassie Blue or silver or fluorescent stains. The optical density of each protein spot is generaUy 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 partiaky sequenced using, for example, standard methods employing chemical or enzymatic cleavage foUowed by mass spectrometry. The identity of the protein in a spot may be determined by comparing its partial sequence, preferably of at least 5 contiguous amino acid residues, to the polypeptide sequences of interest. In some cases, further sequence data may be obtained for definitive protein identification.

A proteomic profile may also be generated using antibodies specific for NAAP to quantify the levels of NAAP expression. In one embodiment, the antibodies are used as elements on a microarray, and protein expression levels are quantified by exposing the microarray to the sample and detecting the levels of protein bound to each aπay element (Lueking, A. et al. (1999) Anal. Biochem. 270:103- 111; Mendoze, L.G. et al. (1999) Biotechniques 27:778-788). Detection maybe 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 paraUel with toxicant signatures at the transcript level. There is a poor correlation between transcript and protein abundances for some proteins in some tissues (Anderson, N.L. and J. Seilhamer (1997) Electrophoresis 18:533-537), so proteome toxicant signatures maybe useful in the analysis of compounds which do not significantly affect the transcript image, but which alter the proteomic profile. In addition, the analysis of transcripts in body fluids is difficult, due to rapid degradation of mRNA, so proteomic profikng may be more rekable 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 conesponding protein in an untreated biological sample. A difference in the amount of protein between the two samples is indicative of a toxic response to the test compound in the treated sample. Individual proteins are identified by sequencing the amino acid residues of the individual proteins and comparing these partial sequences to the polypeptides of the present invention.

In another embodiment, the toxicity of a test compound is assessed by treating a biological sample containing proteins with the test compound. Proteins from the biological sample are incubated with antibodies specific to the polypeptides of the present invention. The amount of protein recognized by the antibodies is quantified. The amount of protein in the treated biological sample is compared with the amount in an untreated biological sample. A difference in the amount of protein between the two samples is indicative of a toxic response to the test compound in the treated sample.

Microarrays may be prepared, used, and analyzed using methods known in the art (Brennan, T.M. et al. (1995) U.S. Patent No. 5,474,796; Schena, M. et al. (1996) Proc. Natl. Acad. Sci. USA 93:10614-10619; Baldeschweiler et al. (1995) PCT appkcation WO95/251116; Shalon, D. et al. (1995) PCT appkcation WO95/35505; HeUer, R.A. et al. (1997) Proc. Natl. Acad. Sci. USA 94:2150-2155; HeUer, M.J. et al. (1997) U.S. Patent No. 5,605,662). Various types of microanays are weU known and thoroughly described in Schena, M., ed. (1999; DNA Microarrays: A Practical Approach, Oxford University Press, London). In another embodiment of the invention, nucleic acid sequences encoding NAAP may be used to generate hybridization probes useful in mapping the naturaUy occurring genomic sequence. Either coding or noncoding sequences may be used, and in some instances, noncoding sequences may be preferable over coding sequences. For example, conservation of a coding sequence among members of a multi-gene family may potentiaUy 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 PI constructions, or single chromosome cDNA kbraries (Harrington, J.J. et al. (1997) Nat. Genet. 15:345- 355; Price, CM. (1993) Blood Rev. 7:127-134; Trask, BJ. (1991) Trends Genet. 7:149-154). Once mapped, the nucleic acid sequences may be used to develop genetic linkage maps, for example, which coπelate the inheritance of a disease state with the inheritance of a particular chromosome region or restriction fragment length polymorphism (RFLP) (Lander, E.S. and D. Botstein (1986) Proc. Natl. Acad. Sci. USA 83:7353-7357).

Fluorescent in situ hybridization (FISH) may be conelated with other physical and genetic map data (Heinz-Ulrich, et al. (1995) in Meyers, supra, pp. 965-968). Examples of genetic map data can be found in various scientific journals or at the Online Mendekan Inheritance in Man (OMEVI) World Wide Web site. Conelation 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 estabkshed chromosomal markers, maybe used for extending genetic maps. Often the placement of a gene on the chromosome of another mammakan 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 locakzed by genetic linkage to a particular genomic region, e.g., ataxia-telangiectasia to llq22-23, any sequences mapping to that area may represent associated or regulatory genes for further investigation (Gatti, R.A. et al. (1988) Nature 336:577-580). The nucleotide sequence of the instant invention may also be used to detect differences in the chromosomal location due to translocation, inversion, etc., among normal, carrier, or affected individuals.

In another embodiment of the invention, NAAP, its catalytic or immunogenic fragments, or okgopeptides thereof can be used for screening kbraries 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 sokd support, borne on a ceU surface, or located intraceUularly. The formation of binding complexes between NAAP and the agent being tested may be measured.

Another technique for drug screening provides for high throughput screening of compounds having suitable binding affinity to the protein of interest (Geysen, et al. (1984) PCT appkcation

WO84/03564). In this method, large numbers of different smaU test compounds are synthesized on a sokd substrate. The test compounds are reacted with NAAP, or fragments thereof, and washed. Bound NAAP is then detected by methods weU known in the art. Purified NAAP can also be coated directly onto plates for use in the aforementioned drug screening techniques. Alternatively, non-neutrakzing antibodies can be used to capture the peptide and immobikze it on a sokd support. In another embodiment, one may use competitive drug screening assays in which neutrakzing antibodies capable of binding NAAP specificaUy 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 bekeved that one skiUed in the art can, using the preceding description, utikze the present invention to its fuUest extent. The foUowing embodiments are, therefore, to be construed as merely iUustrative, and not limitative of the remainder of the disclosure in any way whatsoever.

The disclosures of aU patents, appkcations, and pubkcations mentioned above and below, including U.S. Ser. No. 60/343,004, U.S. Ser. No. 60/347,633, U.S. Ser. No. 60/359,498, and U.S. Ser. No. 60/351,749, are hereby expressly incorporated by reference.

EXAMPLES

I. Construction of cDNA Libraries

Incyte cDNAs were derived from cDNA kbraries described in the LIFESEQ GOLD database (Incyte Genomics, Palo Alto CA). Some tissues were homogenized and lysed in guanidinium isothiocyanate, while others were homogenized and lysed in phenol or in a suitable mixture of denaturants, such as TRIZOL (Invitrogen), a monophasic solution of phenol and guanidine isothiocyanate. The resulting lysates were centrifuged over CsCl cushions or extracted with chloroform. RNA was precipitated from the lysates with either isopropanol or sodium acetate and ethanol, or by other routine methods.

Phenol extraction and precipitation of RNA were repeated as necessary to increase RNA purity. In some cases, RNA was treated with DNase. For most kbraries, poly(A)+ RNA was isolated using okgo 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 kbraries. Otherwise, cDNA was synthesized and cDNA kbraries were constructed with the UNIZAP vector system (Stratagene) or SUPERSCRIPT plasmid system (Invitrogen), using the recommended procedures or similar methods known in the art (Ausubel et al., supra, ch. 5). Reverse transcription was initiated using okgo d(T) or random primers. Synthetic okgonucleotide adapters were kgated to double stranded cDNA, and the cDNA was digested with the appropriate restriction enzyme or enzymes. For most kbraries, the cDNA was size-selected (300-1000 bp) using SEPHACRYL S 1000, SEPHAROSE CL2B, or SEPHAROSE CL4B column chromatography (Amersham Biosciences) or preparative agarose gel electrophoresis. cDNAs were kgated into compatible restriction enzyme sites of the polyknker of a suitable plasmid, e.g., PBLUESCPJPT plasmid (Stratagene), PSPORT1 plasmid (Invitrogen, Carlsbad CA), PCDNA2.1 plasmid (Invitrogen), PBK- 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 ceUs including XLl-Blue, XLl-BlueMRF, or SOLR from Stratagene or DH5α, DH10B, or ElectroMAX DH10B from Invitrogen.

II. Isolation of cDNA Clones Plasmids obtained as described in Example I were recovered from host ceUs by in vivo excision using the UNIZAP vector system (Stratagene) or by ceU 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. FoUowing precipitation, plasmids were resuspended in 0.1 ml of distiUed water and stored, with or without lyophikzation, at 4°C

Alternatively, plasmid DNA was ampkfied from host ceU lysates using direct Unk PCR in a high-throughput format (Rao, V.B. (1994) Anal. Biochem. 216:1-14). Host ceU lysis and thermal cycling steps were carried out in a single reaction mixture. Samples were processed and stored in 384-weU plates, and the concentration of ampkfied plasmid DNA was quantified fluorometricaUy using PICOGREEN dye (Molecular Probes, Eugene OR) and a FLUOROSKAN E fluorescence scanner (Labsystems Oy, Helsinki, Finland).

III. Sequencing and Analysis

Incyte cDNA recovered in plasmids as described in Example E were sequenced as foUows. Sequencing reactions were processed using standard methods or high-throughput instrumentation such as the ABI CATALYST 800 (Appked Biosystems) thermal cycler or the PTC-200 thermal cycler (MJ Research) in conjunction with the HYDRA microdispenser (Robbins Scientific) or the MICROLAB 2200 (Hamilton) Uquid transfer system. cDNA sequencing reactions were prepared using reagents provided by Amersham Biosciences or suppked in ABI sequencing kits such as the ABI PRISM BIGDYE Terminator cycle sequencing ready reaction kit (Appked Biosystems). Electrophoretic separation of cDNA sequencing reactions and detection of labeled polynucleotides were carried out using the MEGABACE 1000 DNA sequencing system (Amersham Biosciences); the ABI PRISM 373 or 377 sequencing system (Appked Biosystems) in conjunction with standard ABI protocols and base calling software; or other sequence analysis systems known in the art. Reading frames within the cDNA sequences were identified using standard methods (Ausubel et al., supra, ch. 7). Some of the cDNA sequences were selected for extension using the techniques disclosed in Example VIE.

The polynucleotide sequences derived from Incyte cDNAs were vakdated 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 pubkc databases such as the GenBank primate, rodent, mammakan, vertebrate, and eukaryote databases, and BLOCKS, PRINTS, DOMO, PRODOM; PROTEOME databases with sequences from Homo sapiens, Rattus norvegicus, Mus musculus, Caenorhabditis elegans, Saccharomyces cerevisiae, Schizosaccharomyces pombe, and Candida albicans (Incyte Genomics, Palo Alto CA); hidden Markov model (HMM)-based protein family databases such as PFAM, ESfCY, and ΗGRFAM (Haft, D.H. et al. (2001) Nucleic Acids Res. 29:41-43); and HMM-based protein domain databases such as SMART (Schultz, J. et al. (1998) Proc. Natl. Acad. Sci. USA 95:5857-5864; Letonic, I. et al. (2002) Nucleic Acids Res. 30:242-244). (HMM is a probabikstic approach which analyzes consensus primary structures of gene famikes; see, for example, Eddy, S.R. (1996) Cun. 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 fuU length polynucleotide sequences. Alternatively, GenBank cDNAs, GenBank ESTs, stitched sequences, stretched sequences, or Genscan-predicted coding sequences (see Examples IV and V) were used to extend Incyte cDNA assemblages to fuU 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 fuU length polynucleotide sequences were translated to derive the conesponding fuU length polypeptide sequences. Alternatively, a polypeptide may begin at any of the methionine residues of the fuU length translated polypeptide. FuU length polypeptide sequences were subsequently analyzed by querying against databases such as the GenBank protein databases (genpept), SwissProt, the PROTEOME databases, BLOCKS, PRINTS, DOMO, PRODOM, Prosite, hidden Markov model (HMM)-based protein family databases such as PFAM, INCY, and ΗGRFAM; and HMM-based protein domain databases such as SMART. FuU length polynucleotide sequences are also analyzed using MACDNASIS PRO software (MiraiBio, Alameda CA) and LASERGENE software (DNASTAR). Polynucleotide and polypeptide sequence aUgnments are generated using default parameters specified by the CLUSTAL algorithm as incorporated into the MEGALIGN multisequence akgnment program (DNASTAR), which also calculates the percent identity between akgned sequences. Table 7 summarizes the tools, programs, and algorithms used for the analysis and assembly of

Incyte cDNA and fuU length sequences and provides appkcable 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, aU of which are incorporated by reference herein in their entirety, and the fourth column presents, where appkcable, the scores, probabikty values, and other parameters used to evaluate the strength of a match between two sequences (the higher the score or the lower the probabikty value, the greater the identity between two sequences).

The programs described above for the assembly and analysis of fuU length polynucleotide and polypeptide sequences were also used to identify polynucleotide sequence fragments from SEQ DD NO:58-l 14. Fragments from about 20 to about 4000 nucleotides which are useful in hybridization and ampkfication technologies are described in Table 4, column 2. IV. Identification and Editing of Coding Sequences from Genomic DNA

Putative nucleic acid-associated proteins were initiaUy identified by running the Genscan gene identification program against pubkc genomic sequence databases (e.g., gbpri and gbhtg). Genscan is a general-purpose gene identification program which analyzes genomic DNA sequences from a variety of organisms (Burge, C. and S. Karlin (1997) J. Mol. Biol. 268:78-94; Burge, C. and S. Karlin (1998) Curr. Opin. Struct. Biol. 8:346-354). The program concatenates predicted exons to form an assembled cDNA sequence extending from a methionine to a stop codon. The output of Genscan is a FASTA database of polynucleotide and polypeptide sequences. The maximum range of sequence for Genscan to analyze at once was set to 30 kb. To determine which of these Genscan predicted cDNA sequences encode nucleic acid-associated proteins, the encoded polypeptides were analyzed by querying against PFAM models for nucleic acid-associated proteins. Potential nucleic acid-associated proteins were also identified by homology to Incyte cDNA sequences that had been annotated as nucleic acid-associated proteins. These selected Genscan-predicted sequences were then compared by BLAST analysis to the genpept and gbpri pubkc 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 pubkc cDNA coverage of the Genscan-predicted sequences, thus providing evidence for transcription. When incyte cDNA coverage was available, this information was used to conect or confirm the Genscan predicted sequence. FuU length polynucleotide sequences were obtained by assembling Genscan-predicted coding sequences with Incyte cDNA sequences and/or pubkc cDNA sequences using the assembly process described in

Example IE. 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 IE 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 spkce variants that were subsequently confirmed, edited, or extended to create a fuU 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 aU three intervals were considered to be equivalent. This process aUows 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 pubkc databases. Incorrect exons predicted by Genscan were conected 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 fuU length with an algorithm based on BLAST analysis. First, partial cDNAs assembled as described in Example El were queried against pubkc databases such as the GenBank primate, rodent, mammakan, 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 pubkc 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 ED NO:58-114 were compared with sequences from the Incyte LIFESEQ database and pubkc domain databases using BLAST and other implementations of the Smith- Waterman algorithm. Sequences from these databases that matched SEQ DD NO:58-l 14 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 pubkc 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 aU sequences of that cluster, including its particular SEQ ED NO:, to that map location. Map locations are represented by ranges, or intervals, of human chromosomes. The map position of an interval, in centiMorgans, is measured relative to the terminus of the chromosome's p- arm. (The centiMorgan (cM) is a unit of measurement based on recombination frequencies between chromosomal markers. On average, 1 cM is roughly equivalent to 1 megabase (Mb) of DNA in humans, although this can vary widely due to hot and cold spots of recombination.) The cM distances are based on genetic markers mapped by Genethon which provide boundaries for radiation hybrid markers whose sequences were included in each of the clusters. Human genome maps and other resources available to the pubkc, 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. 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 ceU type or tissue have been bound (Sambrook and RusseU, supra, ch. 7; Ausubel et al., supra, ch. 4).

Analogous computer techniques applying BLAST were used to search for identical or related molecules in databases such as GenBank or LIFESEQ (Incyte Genomics). This analysis is much faster than multiple membrane-based hybridizations. In addition, the sensitivity of the computer search can be modified to determine whether any particular match is categorized as exact or similar. The basis of the search is the product score, which is defined as:

BLAST Score x Percent Identity 5 x minimum {length(Seq. 1), length(Seq. 2)}

The product score takes into account both the degree of similarity between two sequences and the length of the sequence match. The product score is a normakzed value between 0 and 100, and is calculated as foUows: the BLAST score is multipked 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). Jf 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 quakty in a BLAST akgnment. For example, a product score of 100 is produced only for 100% identity over the entire length of the shorter of the two sequences being compared. A product score of 70 is produced either by 100% identity and 70% overlap at one end, or by 88% identity and 100% overlap at the other. A product score of 50 is produced either by 100% identity and 50% overlap at one end, or 79% identity and 100% overlap. Alternatively, polynucleotides encoding NAAP are analyzed with respect to the tissue sources from which they were derived. For example, some full length sequences are assembled, at least in part, with overlapping Incyte cDNA sequences (see Example D3). Each cDNA sequence is derived from a cDNA kbrary constructed from a human tissue. Each human tissue is classified into one of the foUowing organ tissue categories: cardiovascular system; connective tissue; digestive system; embryonic structures; endocrine system; exocrine glands; genitaha, female; genitaka, male; germ cells; hemic and immune system; Uver; musculoskeletal system; nervous system; pancreas; respiratory system; sense organs; skin; stomatognathic system; unclassified/mixed; or urinary tract. The number of kbraries in each category is counted and divided by the total number of kbraries across aU categories. Similarly, each human tissue is classified into one of the foUowing disease/condition categories: cancer, ceU line, developmental, inflammation, neurological, trauma, cardiovascular, pooled, and other, and the number of kbraries in each category is counted and divided by the total number of kbraries across aU categories. The resulting percentages reflect the tissue- and disease-specific expression of cDNA encoding NAAP. cDNA sequences and cDNA kbrary/tissue information are found in the LIFESEQ GOLD database (Incyte Genomics, Palo Alto CA). VIII. Extension of NAAP Encoding Polynucleotides

FuU length polynucleotides are produced by extension of an appropriate fragment of the fuU length molecule using okgonucleotide 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 temperatares 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 kbraries were used to extend the sequence. If more than one extension was necessary or desired, additional or nested sets of primers were designed.

High fidekty ampkfication was obtained by PCR using methods weU known in the art. PCR was performed in 96-weU 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⁺, (NEL^SQ,, and 2-mercaptoethanol, Taq DNA polymerase (Amersham Biosciences), ELONGASE enzyme (Invitrogen), and Pfu DNA polymerase (Stratagene), with the foUowing 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 foUows: Step 1: 94°C, 3 min; Step 2: 94°C, 15 sec; Step 3: 57°C, 1 min; Step 4: 68°C, 2 min; Step 5: Steps 2, 3, and 4 repeated 20 times; Step 6: 68°C, 5 min; Step 7: storage at 4°C

The concentration of DNA in each weU was determined by dispensing 100 μl PICOGREEN quantitation reagent (0.25% (v/v) PICOGREEN; Molecular Probes, Eugene OR) dissolved in IX TE and 0.5 μl of undiluted PCR product into each weU of an opaque fluorimeter plate (Corning Costar, Acton MA), aUowing the DNA to bind to the reagent. The plate was scanned in a Fluoroskan E (Labsystems Oy, Helsinki, Finland) to measure the fluorescence of the sample and to quantify the concentration of DNA. A 5 l to 10 μl akquot 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-weU plates, digested with CviJI cholera virus endonuclease (Molecular Biology Research, Madison Wl), and sonicated or sheared prior to rekgation into pUC 18 vector (Amersham Biosciences). For shotgun sequencing, the digested nucleotides were separated on low concentration (0.6 to 0.8%) agarose gels, fragments were excised, and agar digested with Agar ACE (Promega). Extended clones were rekgated using T4 kgase (New England Biolabs, Beverly MA) into pUC 18 vector (Amersham

Biosciences), treated with Pfu DNA polymerase (Stratagene) to fiU-in restriction site overhangs, and transfected into competent E. coli ceUs. Transformed cells were selected on antibiotic-containing media, and individual colonies were picked and cultared overnight at 37 °C in 384-weU plates in LB/2x carb kquid media. The ceUs were lysed, and DNA was ampkfied by PCR using Taq DNA polymerase

(Amersham Biosciences) and Pfu DNA polymerase (Stratagene) with the foUowing 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 reampUfied 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 Biosciences) or the ABI PRISM BIGDYE Terminator cycle sequencing ready reaction kit (Appked Biosystems).

In like manner, fuU length polynucleotides are verified using the above procedure or are used to obtain 5' regulatory sequences using the above procedure along with okgonucleotides designed for such extension, and an appropriate genomic kbrary. IX. Identification of Single Nucleotide Polymorphisms in NAAP Encoding Polynucleotides

Common DNA sequence variants known as single nucleotide polymorphisms (SNPs) were identified in SEQ ED NO:58-l 14 using the LtFESEQ database (Incyte Genomics). Sequences from the same gene were clustered together and assembled as described in Example IE, allowing the identification of aU sequence variants in the gene. An algorithm consisting of a series of filters was used to distinguish SNPs from other sequence variants. Preliminary filters removed the majority of basecaU enors by requiring a niinimum Phred quakty score of 15, and removed sequence akgnment enors and errors resulting from improper trimming of vector sequences, chimeras, and spkce variants. An automated procedure of advanced chromosome analysis analysed the original chromatogram files in the vicinity of the putative SNP. Clone eπor filters used statisticaUy generated algorithms to identify enors introduced during laboratory processing, such as those caused by reverse transcriptase, polymerase, or somatic mutation. Clustering enor filters used statisticaUy generated algorithms to identify enors resulting from clustering of close homologs or pseudogenes, or due to contamination by non-human sequences. A final set of filters removed dupkcates and SNPs found in immunoglobulins or T-ceU receptors.

Certain SNPs were selected for further characterization by mass spectrometry using the high throughput MASSARRAY system (Sequenom, Inc.) to analyze aUele frequencies at the SNP sites in four different human populations. The Caucasian population comprised 92 individuals (46 male, 46 female), including 83 from Utah, four French, three Venezualan, and two Amish individuals. The African population comprised 194 individuals (97 male, 97 female), aU African Americans. The Hispanic population comprised 324 individuals (162 male, 162 female), aU Mexican Hispanic. The Asian population comprised 126 individuals (64 male, 62 female) with a reported parental breakdown of 43% Chinese, 31% Japanese, 13% Korean, 5% Vietnamese, and 8% other Asian. AUele frequencies were first analyzed in the Caucasian population; in some cases those SNPs which showed no aUekc variance in this population were not further tested in the other three populations. X. Labeling and Use of Individual Hybridization Probes

Hybridization probes derived from SEQ DD NO:58-114 are employed to screen cDNAs, genomic DNAs, or mRNAs. Although the labeling of okgonucleotides, consisting of about 20 base pairs, is specificaUy described, essentiaUy the same procedure is used with larger nucleotide fragments. Okgonucleotides are designed using state-of-the-art software such as OLIGO 4.06 software (National Biosciences) and labeled by combining 50 pmol of each okgomer, 250 μCi of [γ-³²P] adenosine triphosphate (Amersham Biosciences), and T4 polynucleotide kinase (DuPont NEN, Boston MA). The labeled okgonucleotides are substantiaUy purified using a SEPHADEX G-25 superfine size exclusion dextran bead column (Amersham Biosciences). An akquot containing IO⁷ 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 foUowing endonucleases: Ase I, Bgl D, Eco Rl, Pst I, Xba I, or Pvu E (DuPont NEN).

The DNA from each digest is fractionated on a 0.7% agarose gel and transfened to nylon membranes (Nytran Plus, Schleicher & SchueU, Durham NH). Hybridization is carried out for 16 hours at 40 °C. To remove nonspecific signals, blots are sequentiaUy 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 visuakzed using autoradiography or an alternative imaging means and compared. XI. Microarrays

The linkage or synthesis of array elements upon a microarray can be achieved utikzing photokthography, piezoelectric printing (ink-jet printing; see, e.g., Baldeschweiler et al., supra), mechanical microspotting technologies, and derivatives thereof. The substrate in each of the aforementioned technologies should be uniform and sokd with a non-porous surface (Schena, M., ed. (1999) DNA Microarrays: A Practical Approach, Oxford University Press, London). Suggested substrates include sikcon, sikca, glass skdes, glass chips, and sikcon wafers. Alternatively, a procedure analogous to a dot or slot blot may also be used to arrange and link elements to the surface of a substrate using thermal, UV, chemical, or mechanical bonding procedures. A typical array may be produced using available methods and machines weU known to those of ordinary skill in the art and may contain any appropriate number of elements (Schena, M. et al. (1995) Science 270:467-470; Shalon, D. et al. (1996) Genome Res. 6:639-645; MarshaU, A. and J. Hodgson (1998) Nat. Biotechnol. 16:27-31). FuU length cDNAs, Expressed Sequence Tags (ESTs), or fragments or okgomers thereof may comprise the elements of the microanay. Fragments or okgomers suitable for hybridization can be selected using software weU known in the art such as LASERGENE software (DNASTAR). The aπay 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 aπay 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 microanay 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 okgo-(dT) ceUulose method. Each poly(A)⁺ RNA sample is reverse transcribed using MMLV reverse-transcriptase, 0.05 pg/μl okgo-(dT) primer (21mer), IX first strand buffer, 0.03 units/μl RNase inhibitor, 500 μM dATP, 500 μM dGTP, 500 μM dTTP, 40 μM dCTP, 40 μM dCTP-Cy3 (BDS) or dCTP-Cy5 (Amersham Biosciences). The reverse transcription reaction is performed in a 25 ml volume containing 200 ng poly(A)⁺ RNA with GEMBRIGHT kits (Incyte Genomics). Specific control poly(A)⁺ RNAs are synthesized by in 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, 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 μl 5X SSC/0.2% SDS. Microarray Preparation Sequences of the present invention are used to generate array elements. Each array element is ampkfied from bacterial ceUs containing vectors with cloned cDNA inserts. PCR ampkfication uses primers complementary to the vector sequences flanking the cDNA insert. Array elements are ampkfied in thirty cycles of PCR from an initial quantity of 1-2 ng to a final quantity greater than 5 μg. Ampkfied array elements are then purified using SEPHACRYL-400 (Amersham Biosciences). Purified array elements are immobikzed on polymer-coated glass skdes. Glass microscope skdes (Corning) are cleaned by ultrasound in 0.1% SDS and acetone, with extensive distiUed water washes between and after treatments. Glass skdes are etched in 4% hydrofluoric acid (VWR Scientific Products Corporation (VWR), West Chester PA), washed extensively in distiUed water, and coated with 0.05% aminopropyl silane (Sigma) in 95% ethanol. Coated skdes are cured in a 110°C oven.

Aπay elements are appked to the coated glass substrate using a procedure described in U.S. Patent No. 5,807,522, incorporated herein by reference. 1 μl of the aπay element DNA, at an average

ill concentration of 100 ng/μl, is loaded into the open capiUary printing element by a high-speed robotic apparatus. The apparatus then deposits about 5 nl of aπay element sample per skde.

Microarrays are UV-crossknked using a STRATALINKER UV-crosslinker (Stratagene). Microaπays are washed at room temperature once in 0.2% SDS and three times in distiUed water. Non-specific binding sites are blocked by incubation of microaπays in 0.2% casein in phosphate buffered saline (PBS) (Tropix, Inc., Bedford MA) for 30 minutes at 60° C foUowed by washes in 0.2%

SDS and distiUed water as before.

Hybridization

Hybridization reactions contain 9 μl of sample mixture consisting of 0.2 μg 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 akquoted onto the microarray surface and covered with an 1.8 cm² coverskp. The aπays are transfeπed to a waterproof chamber having a cavity just skghtly larger than a microscope skde. The chamber is kept at 100% humidity internaUy by the addition of 140 μl of 5X SSC in a comer 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 (IX 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 Cy5. The excitation laser kght is focused on the aπay using a 20X microscope objective (Nikon, Inc., MelviUe NY). The skde containing the aπay is placed on a computer-controUed 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 sequentiaUy.

Emitted kght is spkt, based on wavelength, into two photomultipker tube detectors (PMT R1477, Hamamatsu Photonics Systems, Bridgewater NJ) coπesponding to the two fluorophores. Appropriate filters positioned between the array and the photomultipker tabes are used to filter the signals. The emission maxima of the fluorophores used are 565 nm for Cy3 and 650 nm for Cy5. Each aπay is typicaUy 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 cakbrated using the signal intensity generated by a cDNA control species added to the sample mixture at a known concentration. A specific location on the aπay contains a complementary DNA sequence, aUowing 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 ceUs), each labeled with a different fluorophore, are hybridized to a single aπay for the purpose of identifying genes that are differentiaUy expressed, the cakbration is done by labeling samples of the cakbrating cDNA with the two fluorophores and adding identical amounts of each to the hybridization mixture.

The output of the photomultipker tube is digitized using a 12-bit RTI-835H analog-to-digital (A/D) conversion board (Analog Devices, Inc., Norwood MA) instaUed in an IBM-compatible PC computer. The digitized data are displayed as an image where the signal intensity is mapped using a linear 20-color transformation to a pseudocolor scale ranging from blue (low signal) to red (high signal). The data is also analyzed quantitatively. Where two different fluorophores are excited and measured simultaneously, the data are first corrected for optical crosstalk (due to overlapping emission spectra) between the fluorophores using each fluorophore 's emission spectrum. A grid is superimposed over the fluorescence signal image such that the signal from each spot is centered in each element of the grid. The fluorescence signal within each element is then integrated to obtain a numerical value corresponding to the average intensity of the signal. The software used for signal analysis is the GEMTOOLS gene expression analysis program (Incyte Genomics). Array elements that exhibit at least about a two-fold change in expression, a signal-to-background ratio of at least about 2.5, and an element spot size of at least about 40%, are considered to be differentiaUy expressed. Expression

For example, both SEQ ED NO:62 and SEQ ED NO:71 showed differential expression in cancer ceU lines or tomorous tissue versus non-cancerous ceU lines or tissues, as determined by microarray analysis. The expression of SEQ ED NO:62 was decreased by at least two-fold in ovarian tumor tissue from a 79 year-old female donor as compared to matched normal ovarian tissue from the same donor. Matched normal and tumorigenic ovarian tissue samples were provided by the Huntsman Cancer Institute, (Salt Lake City, UT). Therefore, SEQ DD NO:62 is useful in diagnostic assays for ovarian cancer. The expression of SEQ ED NO:71 was increased by at least two-fold in lung squamous ceU carcinoma tissue as compared to matched normal lung tissue from the same donor. Matched normal and tumorigenic lung tissue samples were provided by the Roy Castle International Centre for Lung Cancer Research (Liverpool, UK). Therefore, SEQ ED NO:71 is useful in diagnostic assays for lung cancer.

In addition, the expression of SEQ ED NO:71 was increased at least two-fold in Tangier disease-derived fibroblasts compared to normal fibroblasts. Both types of ceUs were also cultured in the presence of cholesterol and compared with the same ceU type cultured in the absence of cholesterol. Human fibroblasts were obtained from skin explants from both normal subjects and two patients with homozygous Tangier disease. CeU lines were immortakzed by transfection with human papiUomavirus 16 genes E6 and E7 and a neomycin resistance selectable marker. TD derived cells are deficient in an assay of apoA-I mediated tritiated cholesterol efflux. Therefore, SEQ ED NO:71 is also useful in diagnostic assays for Tangier disease. In an alternative example, SEQ ED NO:79 and SEQ ED NO:81 were differentiaUy expressed in human breast tumor ceUs. Histological and molecular evaluation of breast tumors reveals that the development of breast cancer evolves through a multi-step process whereby pre-mahgnant mammary epithekal ceUs undergo a relatively defined sequence of events leading to tumor formation. An early event in tumor development is ductal hyperplasia. CeUs undergoing rapid neoplastic growth graduaUy progress to invasive carcinoma and become metastatic to the lung, bone, and potentiaUy other organs. Several variables that may influence the process of tumor progression and makgnant transformation include genetic factors, environmental factors, growth factors, and hormones. Based on the complexity of this process, it is critical to study a population of human mammary epithekal ceUs undergoing the process of makgnant transformation, and to associate specific stages of progression with phenotypic and molecular characteristics. Primary breast epithekal ceUs (HMECs) were compared to various breast carcinoma Unes at various stages of tumor progression.

Gene expression profiles of nonmakgnant mammary epithekal cells were compared to gene expression profiles of various breast carcinoma lines at different stages of tamor progression. The ceUs were grown in defined serum-free H14 medium to 70-80% confluence prior to RNA harvest. CeU lines compared included: a) HMEC, a primary breast epithekal ceU line isolated from a normal donor, b)MCF-10A, a breast mammary gland cell line isolated from a 36-year-old woman with fibrocystic breast disease, c)MCF7, a nonmakgnant breast adenocarcinoma ceU line isolated from the pleural effusion of a 69- year-old female, d)T-47D, a breast carcinoma ceU line isolated from a pleural effusion obtained from a 54-year-old female with an infiltrating ductal carcinoma of the breast, e)Sk- BR-3, a breast adenocarcinoma ceU line isolated from a makgnant pleural effusion of a 43 -year-old female, f)BT-20, a breast carcinoma cell line derived in vitro from ceUs emigrating out of thin skces of the tamor mass isolated from a 74-year-old female, g)MDA-mb-231, a breast tamor ceU line isolated from the pleural effusion of a 51-year-old female, and h)MDA-mb-435S, a spindle-shaped strain that evolved from the parent line (435) isolated by R. CaiUeau from pleural effusion of a 31 -year-old female with metastatic, ductal adenocarcinoma of the breast.

In various microanay experiments, both SEQ DD NO:79 and SEQ DD NO:81 were underexpressed by at least two-fold in the foUowing tamor ceU lines: MCF7 (nonmakgnant breast adenocarcinoma), T47D (breast carcinoma), BT-20 (breast carcinoma), MDA-mb-231 (breast tamor ceU line which also expresses the Wnt3 oncogene, EGF, and TGF-alpha), and MDA-mb-435S (spindle-shaped strain of breast tamor ceU evolved from a metastatic, ductal adenocarcinoma ceU of the breast). These experiments indicate that SEQ ED NO:79 and SEQ ED NO:81 exhibited significant differential expression patterns using microanay techniques, and further estabksh their utikty as diagnostic markers, disease staging or therapeutic agents which may be useful in a variety of conditions and diseases involving nucleic acid-associated proteins, including breast cancer.

In an alternative example, SEQ ED NO:90, SEQ ED NO:91, and SEQ ED NO:93 showed differential expression in tumorous or diseased tissue versus non-tumorous or healthy tissues, as determined by microanay analysis. Aπay elements that exhibited about at least a two-fold change in expression and a signal intensity over 250 units, a signal-to-background ratio of a least 2.5, and an element spot size of at least 40% were identified as differentiaUy expressed using the GEMTOOLS program (Incyte Genomics). For example, SEQ ED NO:90 showed increased expression in lung carcinoma tissue versus normal lung tissue as determined by microanay analysis. In one experiment, grossly uninvolved lung tissue from a 66 year-old male was compared to lung squamous ceU carcinoma tissue from the same donor (Roy Castle International Centre for Lung Cancer Research, Liverpool, UK). In a second experiment, grossly uninvolved lung tissue from a 66 year-old female was compared to lung adenocarcinoma tissue from the same donor (Roy Castle International Centre for Lung Cancer Research, Liverpool, UK). Therefore, SEQ ED NO:90 is useful in monitoring treatment of, and diagnostic assays for, lung cancer and other ceU prokferative disorders.

In another example, SEQ ED NO:90 showed differential expression in amygdala enterorhinal cortex tissue versus pooled brain tissue as determined by microarray analysis. Specific brain regions from 4 individual male donors (47, 48, 59, and 60 years old) were compared to a pooled brain control. The pooled brain control was reconstitated from the purified mRNA isolated from the major regions of the brain from two male brains (the 47 -year-old male and the 48-year-old male). Therefore, SEQ ED NO:90 is useful in monitoring treatment of, and diagnostic assays for, Alzheimer's disease and other neurological disorders.

In another example, SEQ DD NO:91 showed decreased expression in breast carcinoma ceU lines versus a primary breast epithekal ceU line isolated from a normal donor, as determined by microarray analysis. The gene expression profile of a nonmakgnant mammary epithekal ceU line was compared to the gene expression profiles of breast carcinoma knes at different stages of tamor progression. CeU lines compared included: a) BT-20, a breast carcinoma ceU line derived in vitro from the ceUs emigrating out of thin skces of tamor mass isolated from a 74-year-old female, b) BT- 474, a breast ductal carcinoma ceU line that was isolated from a sokd, invasive ductal carcinoma of the breast obtained from a 60-year-old woman, c) BT-483, a breast ductal carcinoma cell line that was isolated from a papiUary invasive ductal tamor obtained from a 23-year-old normal, menstruating, parous female with a family history of breast cancer, d) Hs 578T, a breast ductal carcinoma ceU line isolated from a 74-year-old female with breast carcinoma, e) MCF7, a nonmakgnant breast adenocarcinoma ceU line isolated from the pleural effusion of a 69-year-old female, f) MCF-10A, a breast mammary gland (luminal ductal characteristics) ceU line isolated, from a 36-year-old woman with fϊbrocystic breast disease, g) MDA-MB-468, a breast adenocarcinoma ceU line isolated from the pleural effusion of a 51 -year-old female with metastatic adenocarcinoma of the breast, and h) HMEC, a primary breast epithekal ceU line isolated from a normal donor. SEQ DD NO:91 showed decreased expression in lines a) through e) above. Therefore, SEQ DD NO:91 is useful in monitoring treatment of, and diagnostic assays for, breast cancer and other ceU prokferative disorders.

In another example, SEQ ED NO:93 showed decreased expression in breast carcinoma ceUs treated with estradiol versus untreated breast carcinoma ceUs as determined by microanay analysis. BT-20 is a breast carcinoma ceU Une derived in vitro from the ceUs emigrating out thin skces of the tamor mass isolated from a 74-year-old female. BT-20 ceUs were treated with α-estradiol for 4, 8, 14, 24, 36, and 48 hours. These treated ceUs were compared to untreated BT-20 ceUs kept in cultare for the same amount of time. Therefore, SEQ ED NO:93 is useful in monitoring treatment of, and diagnostic assays for, breast cancer and other ceU prokferative disorders.

In another example, SEQ ED NO:93 showed increased expression in coronary artery epithekal ceUs versus pooled vascular endothekal ceUs as determined by microanay analysis. Human vascular and microvascular endothekal ceUs were isolated from coronary artery, umbikcal artery and vein, uterine microvasculatare, ikac artery, dermal microvasculatare, pulmonary artery, aorta. AU ceU types in this experiment are used as experimental models for investigating the role of endothekum in human vascular biology. Therefore, SEQ ED NO:93 is useful in monitoring treatment of, and diagnostic assays for, cardiovascular and other autoimmune/inflammation disorders.

In an alternative example, SEQ ED NO:101, SEQ ED NO:103, SEQ ED NO:106, SEQ ED NO:112, and SEQ ED NO:113 showed differential expression in tamorous tissues versus non-tamorous tissues or in treated versus untreated ceU lines, as determined by microarray analysis. Array elements that exhibited about at least a two-fold change in expression and a signal intensity over 250 units, a signal-to-background ratio of a least 2.0, and an element spot size of at least 40% were identified as differentiaUy expressed using the GEMTOOLS program (Incyte Genomics).

For example, the expression of SEQ DD NO:101 was decreased at least two-fold in an ovarian adenocarcinoma when matched with normal tissue from the same donor. The tamorous ovary tissue was obtained from ovarian adenocarcinoma from a 79-year-old female. Normal ovary tissue was obtained from ovary from the same donor. Therefore, SEQ DD NO:101 is useful in diagnostic assays for ovarian adenocarcinoma. Matched normal and tumorigenic ovary tissue samples were provided by the Huntsman Cancer Institute, (Salt Lake City, UT). The gene expression profile of a nonmakgnant mammary epithekal ceU line (MCF-IOA) or a nonmakgnant mammary epithekal ceU line (HMEC) was compared to the gene expression profiles of breast carcinoma lines at different stages of tamor progression. CeU lines compared included: a) HMEC, a primary breast epithekal ceU line isolated from a normal donor, b) MCF-IOA, a breast mammary gland ceU line isolated from a 36-year-old woman with fibrocystic breast disease; c)MCF7, a nonmakgnant breast adenocarcinoma cek line isolated from the pleural effusion of a 69- year-old female; d)T-47D, a breast carcinoma ceU line isolated from a pleural effusion obtained from a 54- year-old female with an infiltrating ductal carcinoma of the breast; e)Sk-BR-3, a breast adenocarcinoma ceU line isolated from a makgnant pleural effusion of a 43-year-old female; f)BT-20, a breast carcinoma ceU line derived in vitro from tamor mass isolated from a 74-year-old female; g)MDA-mb-231, a breast tamor ceU line isolated from the pleural effusion of a 51 -year old female; and h) MDA-mb-435S, a spindle shaped strain that evolved from the parent line (435) isolated from the pleural effusion of a 31-year-old female with metastatic, ductal adenocarcinoma of the breast. AU ceU lines were grown in the suppker's recommended medium to 70-80% confluence prior to comparison. The expression of SEQ ED NO:101 was decreased at least two-fold in three (MCF7, T- 47D, and MDA-mb-231) of the seven ceU lines when compared with HMEC or MCF-IOA ceU lines. These experiments indicate that SEQ ED NO: 101 was significantly under-expressed in the breast tamor ceU lines tested, estabkshing the utikty of SEQ ED NO: 101 as a diagnostic marker for disease staging or as a potential therapeutic target for breast cancer.

In another example, SEQ ED NO: 103, exhibited differential expression by microarray analysis. Early confluent C3A ceUs were treated with mifepristone, progesterone, beclomethasone, medroxyprogesterone, budesonide, prednisone, dexamethasone, betamethasone, or danazol at concentrations of 1 μM, 10 μM, and 100 μM for 1, 3, and 6 hours. In aU cases mRNA from untreated early confluent C3A ceUs were prepared in paraUel as described below. The expression of SEQ ED NO: 103 was decreased at least two-fold in HCA3 ceUs when treated with Progesterone, Beclomethasone, Medroxyprogesterone, Bedusonide, Prednisone, Dexamethasone, and Betamethasone at doses between lμM and 100 μM for between 1 and 6 hours, and with Catechol plus water at lOOμM for 1, 3, and 6 hours. Therefore, SEQ ED NO:103 is useful as a diagnostic marker for disease staging or as a potential therapeutic target for kver disorders such as adenomatosis, cholestasis, cirrhosis, hemangioma, Henoch-Schonlein purpura, hepatitis, hepatoceUular and metastatic carcinomas, idiopathic thrombocytopenic purpura, porphyria, sarcoidosis, and Wilson disease.

In another example, the expression of SEQ ED NO: 106 was increased at least four-fold in lung squamous ceU carcinoma when matched with normal tissue from the same donor. The tamorous tissue was obtained from lung squamous ceU carcinoma from a 68-year-old female. Normal lung tissue was obtained from the same donor. Therefore, SEQ ED NO: 106 is useful in diagnostic assays for lung squamous cell carcinoma, in disease staging and as a potential therapeutic target. Matched normal and tumorigenic lung tissue samples are provided by the Roy Castle International Centre for Lung Cancer Research (Liverpool UK). Further, the expression of SEQ ED NO: 106 was decreased at least three-fold in colon tamor when matched with normal tissue from the same donor. Tumorous colon tissue was obtained from a 67 -year-old donor with moderately differentiated colon adenocarcinoma. Normal tissue was obtained from the colon of the same donor. Therefore, SEQ ED NO: 106 is useful as a diagnostic marker, for disease staging, or as a potential therapeutic target for colon adenocarcinoma. Matched normal and tumorigenic colon tissue samples were provided by the Huntsman Cancer Institute (Salt Lake City, UT).

In another example, the expression of SEQ ED NO: 112 was decreased at least two-fold in two colon adenocarcinomas when matched with normal tissue from the same donor. The tamorous tissue was obtained from a 64-year-old female with moderately differentiated colon adenocarcinoma and from an 83 -year old female with colon adenocarcinoma. Normal tissues were obtained from the colons of the same donors. Therefore, SEQ DD NO: 112 is useful as a diagnostic marker, for disease staging, or as a potential therapeutic target for colon adenocarcinoma. Matched normal and tumorigenic colon tissue samples are provided by the Huntsman Cancer Institute (Salt Lake City, UT).

In yet another example, SEQ ED NO: 113 showed differential expression in inflammatory responses as determined by microarray analysis. The expression of SEQ ED NO:l 13 was increased by at least two-fold in three ceU lines, Daudi (a B lymphoblast ceU line [Burkitt's lymphoma]), Jurkat (an acute T ceU leukemia ceU Une that grows in the absence of external stimuk), and THP-1 (a promonocyte ceU line isolated from the peripheral blood of a 1 -year-old male with acute monocytic leukemia). These three ceU lines are used extensively for the study of signaling in human T ceUs and B ceUs. These ceU lines were treated with various concentrations of PMA (a broad activator of protein kinase C-dependent (PKC) pathways), with or without Ionomycin (a calcium ionophore that permits the entry of calcium in the ceU), with or without Brefeldin A (an antiviral antibiotic factor produced by PeniciUium brefeldianum), with or without antibodies to CD3 or CD8, or with or without LPS (kpopolysaccharide). The expression of SEQ ED NO: 113 was increased by at least two-fold in three tamorous ceU lines from breast (MDA-mb-231, T-47D, and MCF7) when treated with 1 or 10 ng/ml of TNFα. See descriptions supra. The expression of SEQ ED NO:l 13 was also increased by at least 2.9-fold in DU 145, a prostate carcinoma ceU line isolated from a metastatic site in the brain of a 69-year-old male with widespread metastatic prostate carcinoma, when compared to Prec, a primary prostate epithekal ceU line isolated from a normal donor; and increased by at least two-fold in the foUowing endothekal ceU lines: ECV304 (a ceU line derived from the endothekum of the human umbikcal vein) treated with PMA and Ionomycin or TNFα; EUAEC (primary ceUs derived from the endothekum of an ikac artery); HMVEC (primary ceUs derived from microvascular of human skin endothekal ceUs); HUAEC (primary ceUs derived from the endothekum of an umbikcal artery); and UtMVECMyo (primary cells derived from the uterine myometrium microvasculatare) aU treated with 10 ng/ml TNF-α when compared to untreated ceUs of the same ceU line; and HUVEC (primary ceUs derived from the endothekum of the human umbikcal vein) treated with TNFα and cycloheximide when compared to untreated HUVEC ceUs treated with cycloheximide.

Further, the expression of SEQ DD NO:l 13 was decreased by at least two-fold in HepG2 (a human hepatoma ceU line isolated from a 15-year-old male with a kver tamor) when treated with TNFα. SEQ ED NO:l 13 was decreased at least two-fold in five out of six breast tamor ceU lines (BT20, HMEC, MDA-mb-231, MDA-mb-435S, T-47D; see descriptions supra) when compared with MCF10A ceU line. The expression of SEQ ED NO:113 was decreased at least 2.5-fold in a colon cancer when matched with normal tissue from the same donor. The tamouous colon tissue was obtained from a 73-year-old female with colon cancer. Normal colon tissue was obtained from the colon of the same donor. Also, the expression of SEQ ED NO:l 13 was decreased at least two-fold in a donor with lung adenocarcinoma and decreased at least 2.5-fold in four donors with lung squamous ceU carcinoma. The adenocarcinoma tissue was obtained from a 66-year-old female with lung adenocarcinoma. The squamous ceU carcinoma tissue was obtained from a 73-year-old male, a 68- year-old female, a 66-year-old male, and a 73-year-old male. Normal lung tissues were obtained from the lungs of the same donors. AU matched normal and tamorous lung tissue samples were provided by the Roy Castle International Centre for Lung Cancer Research (Liverpool UK). Therefore, SEQ ED NO: 113 is useful in monitoring treatment of, and diagnostic assays for, autoimmune/inflammation disorders, breast cancer, colon cancer, and lung cancer. XII. Complementary Polynucleotides

Sequences complementary to the NAAP-encoding sequences, or any parts thereof, are used to detect, decrease, or inhibit expression of naturaUy occurring NAAP. Although use of okgonucleotides comprising from about 15 to 30 base pairs is described, essentiaUy the same procedure is used with smaUer or with larger sequence fragments. Appropriate okgonucleotides are designed using OLIGO 4.06 software (National Biosciences) and the coding sequence of NAAP. To inhibit transcription, a complementary okgonucleotide is designed from the most unique 5' sequence and used to prevent promoter binding to the coding sequence. To inhibit translation, a complementary okgonucleotide is designed to prevent ribosomal binding to the NAAP-encoding transcript. XIII. Expression of NAAP Expression and purification of NAAP is achieved using bacterial or virus-based expression systems. For expression of NAAP in bacteria, cDNA is subcloned into an appropriate vector containing an antibiotic resistance gene and an inducible promoter that directs high levels of cDNA transcription. Examples of such promoters include, but are not limited to, the trp-lac (tac) hybrid promoter and the 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 (IPTG). Expression of NAAP in eukaryotic ceUs is achieved by infecting insect or mammakan ceU lines with recombinant Autographica 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 ceUs in most cases, or human hepatocytes, in some cases. Infection of the latter requires additional genetic modifications to baculovirus (Engelhard, E.K. et al. (1994) Proc. Natl. Acad. Sci. USA 91:3224-3227; Sandig, V. et al. (1996) Hum. Gene Ther. 7:1937- 1945). In most expression systems, 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 ceU lysates. GST, a 26-kilodalton enzyme from Schistosoma japonicum, enables the purification of fusion proteins on immobikzed glutathione under conditions that maintain protein activity and antigenicity (Amersham Biosciences). FoUowing purification, the GST moiety can be proteolyticaUy cleaved from NAAP at specificaUy engineered sites. FLAG, an 8-amino acid peptide, enables immunoaffinity purification using commerciaUy available monoclonal and polyclonal anti-FLAG antibodies (Eastman Kodak). 6-His, a stretch of six consecutive histidine residues, enables purification on metal-chelate resins (QIAGEN). Methods for protein expression and purification are discussed in Ausubel et al. (supra, ch. 10 and 16). Purified NAAP obtained by these methods can be used directly in the assays shown in Examples XVE, XVEI, and XIX, where appkcable. XIV. Functional Assays

NAAP function is assessed by expressing the sequences encoding NAAP at physiologicaUy elevated levels in mammakan ceU culture systems. cDNA is subcloned into a mammakan expression vector containing a strong promoter that drives high levels of cDNA expression. Vectors of choice include PCMV SPORT plasmid (Invitrogen, Carlsbad CA) and PCR3.1 plasmid (Invitrogen), both of which contain the cytomegalovirus promoter. 5-10 μg of recombinant vector are transiently transfected into a human ceU line, for example, an endothekal or hematopoietic cell Une, using either kposome formulations or electroporation. 1-2 μg of an additional plasmid containing sequences encoding a marker protein are co-transfected. Expression of a marker protein provides a means to distinguish transfected ceUs from nontransfected cells and is a rehable 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 ceUs expressing GFP or CD64-GFP and to evaluate the apoptotic state of the ceUs and other cellular properties. FCM detects and quantifies the uptake of fluorescent molecules that diagnose events preceding or coincident with ceU death. These events include changes in nuclear DNA content as measured by staining of DNA with propidium iodide; changes in ceU size and granularity as measured by forward Ught scatter and 90 degree side kght scatter; down-regulation of DNA synthesis as measured by decrease in bromodeoxyuridine uptake; alterations in expression of cek surface and intraceUular 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 ceU 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 ceUs transfected with sequences encoding NAAP and either CD64 or CD64-GFP. CD64 and CD64-GFP are expressed on the surface of transfected ceUs and bind to conserved regions of human immunoglobulin G (IgG). Transfected ceUs are efficiently separated from nontransfected ceUs using magnetic beads coated with either human IgG or antibody against CD64 (DYNAL, Lake Success NY). mRNA can be purified from the ceUs using methods weU known by those of skiU in the art. Expression of mRNA encoding NAAP and other genes of interest can be analyzed by northern analysis or microanay techniques. XV. Production of NAAP Specific Antibodies

NAAP substantially purified using polyacrylamide gel electrophoresis (PAGE; see, e.g., Harrington, M.G. (1990) Methods Enzymol. 182:488-495), or other purification techniques, is used to immunize animals (e.g., rabbits, mice, etc.) and to produce antibodies using standard protocols. Alternatively, the NAAP amino acid sequence is analyzed using LASERGENE software (DNASTAR) to determine regions of high immunogenicity, and a corresponding okgopeptide is synthesized and used to raise antibodies by means known to those of skiU in the art. Methods for selection of appropriate epitopes, such as those near the C-terminus or in hydrophikc regions are weU described in the art (Ausubel et al., supra, ch. 11).

TypicaUy, okgopeptides of about 15 residues in length are synthesized using an ABI 431 A peptide synthesizer (Appked Biosystems) using FMOC chemistry and coupled to KLH (Sigma-

Aldrich, St. Louis MO) by reaction with N-maleinήdobenzoyl-N-hydroxysuccinimide ester (MBS) to increase immunogenicity (Ausubel et al., supra). Rabbits are immunized with the okgopeptide-KLH complex in complete Freund's adjuvant. Resulting antisera are tested for antipeptide and anti-NAAP activity by, for example, binding the peptide or NAAP to a substrate, blocking with 1% BSA, reacting with rabbit antisera, washing, and reacting with radio-iodinated goat anti-rabbit IgG. XVI. Purification of Naturally Occurring NAAP Using Specific Antibodies

NaturaUy occurring or recombinant NAAP is substantiaUy purified by inimunoafftnity chromatography using antibodies specific for NAAP. An immunoaffinity column is constructed by covalently coupling anti-NAAP antibody to an activated chromatographic resin, such as CNBr-activated SEPHAROSE (Amersham Biosciences). After the coupling, the resin is blocked and washed according to the manufacturer's instructions. Media containing NAAP are passed over the immunoaffinity column, and the column is washed under conditions that aUow 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 coUected. XVII. Identification of Molecules Which Interact with NAAP

NAAP, or biologicaUy active fragments thereof, are labeled with ¹²⁵I Bolton-Hunter reagent (Bolton, A.E. and W.M. Hunter (1973) Biochem. J. 133:529-539). Candidate molecules previously arrayed in the weUs of a multi-well plate are incubated with the labeled NAAP, washed, and any weUs 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 commerciaUy 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 aU interactions between the proteins encoded by two large kbraries of genes (Nandabalan, K. et al. (2000) U.S. Patent No. 6,057,101). XVIII. Demonstration of NAAP Activity NAAP activity is measured by its abikty 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 weU characterized reporter gene construct, LexA_op-LacZ, that consists of LexA DNA transcriptional control elements (LexA_op) fused to sequences encoding the E. cok LacZ enzyme. The methods for constructing and expressing fusion genes, introducing them into ceUs, and measuring LacZ enzyme activity, are weU known to those skilled in the art. Sequences encoding NAAP are cloned into a plasmid that directs the synthesis of a fusion protein, LexA-NAAP, consisting of NAAP and a DNA binding domain derived from the LexA transcription factor. The resulting plasmid, encoding a LexA-NAAP fusion protein, is introduced into yeast cells along with a plasmid containing the LexA_op-LacZ reporter gene. The amount of LacZ enzyme activity associated with LexA-NAAP transfected ceUs, relative to control ceUs, is proportional to the amount of transcription stimulated by the NAAP.

Alternatively, NAAP activity is measured by its abikty to bind zinc. A 5-10 μM sample solution in 2.5 mM ammonium acetate solution at pH 7.4 is combined with 0.05 M zinc sulfate solution (Aldrich, Milwaukee Wl) in the presence of 100 μM dithiothreitol with 10% methanol added. The sample and zinc sulfate solutions are aUowed to incubate for 20 minutes. The reaction solution is passed through a VYDAC column (Grace Vydac, Hesperia, CA) with approximately 300 Angstrom bore size and 5 μM 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 mobikty-shift assay. In preparation for this assay, NAAP is expressed by ttansforming a mammakan ceU line such as COS7, HeLa or CHO with a eukaryotic expression vector containing NAAP cDNA. The ceUs are incubated for 48-72 hours after transformation under conditions appropriate for the ceU line to aUow expression and accumulation of NAAP. Extracts containing solubikzed proteins can be prepared from ceUs expressing NAAP by methods weU 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 weU 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 minutes. After incubation, the samples are analyzed by polyacrylamide gel electrophoresis followed by autoradiography. The presence of a band on the autoradiogram indicates the formation of a complex between NAAP and the radioactive transcript. A band of similar mobikty wiU not be present in samples prepared using control extracts prepared from untransformed ceUs. 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 μl final volume) contain 15 mM HEPES, pH 7.9, 1.5 mM MgCl₂, 10 mM dithiothreitol, 3% polyvinylalcohol, 1.5 μCi [met/ry/-³H]AdoMet (0.375 μM AdoMet) (DuPont-NEN), 0.6 μg NAAP, and acceptor substrate (e.g., 0.4 μg [³⁵S]RNA, or 6-rnercaptopurine (6-MP) to 1 rnM final concentration). Reaction mixtures are incubated at 30°C for 30 minutes, then 65°C for 5 minutes.

Analysis of [met/ryZ-³H]RNA is as foUows: (1) 50 μl of 2 x loading buffer (20 mM Tris-HCl, pH 7.6, 1 M LiCl, 1 mM EDTA, 1% sodium dodecyl sulphate (SDS)) and 50 μl ohgo d(T)-ceUulose (10 mg/ml in 1 x loading buffer) are added to the reaction mixture, and incubated at ambient temperature with shaking for 30 minutes. (2) Reaction mixtures are transfeπed to a 96-weU filtration plate attached to a vacuum apparatus. (3) Each sample is washed sequentiaUy with three 2.4 ml akquots of 1 x okgo d(T) loading buffer containing 0.5% SDS, 0.1% SDS, or no SDS. (4) RNA is eluted with 300 μl of water into a 96-weU coUection plate, transfeπed to scintillation vials containing kquid scintiUant, and radioactivity determined.

Analysis of [met/ry/-³H]6-MP is as foUows: (1) 500 μl 0.5 M borate buffer, pH 10.0, and then 2.5 ml of 20% (v/v) isoamyl alcohol in toluene are added to the reaction mixtures. (2) The samples are mixed by vigorous vortexing for ten seconds. (3) After centrifugation at 700g for 10 minutes, 1.5 ml of the organic phase is transferred to scintillation vials containing 0.5 ml absolute ethanol and kquid scintiUant, 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 electrophoreticaUy. This assay is specific for type I topoisomerase activity because Mg²⁺ and ATP are necessary cofactors for type E topoisomerases.

Type E 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 electrophoreticaUy. Kits for measuring type I and type E topoisomerase activities are available commerciaUy from Topogen (Columbus OH).

ATP-dependent RNA hekcase unwinding activity of NAAP can be measured by the method described by Zhang and Grosse (1994; Biochemistry 33:3906-3912). The substrate for RNA unwinding consists of ³²P-labeled RNA composed of two RNA strands of 194 and 130 nucleotides in length containing a duplex region of 17 base-pairs. The RNA substrate is incubated together with ATP, Mg²⁺, and varying amounts of NAAP in a Tris-HCl buffer, pH 7.5, at 37°C for 30 minutes. The single-stranded RNA product is then separated from the double-stranded RNA substrate by electrophoresis through a 10% SDS-polyacrylamide gel, and quantitated by autoradiography. The amount of single-stranded RNA recovered is proportional to the amount of NAAP in the preparation. In the alternative, NAAP function is assessed by expressing the sequences encoding NAAP at physiologicaUy elevated levels in mammakan ceU cultare systems. cDNA is subcloned into a mammakan 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 Corporation, Carlsbad CA), both of which contain the cytomegalovirus promoter. 5-10 μg of recombinant vector are transiently transfected into a human ceU line, preferably of endothekal or hematopoietic origin, using either kposome formulations or electroporation. 1-2 μg of an additional plasmid containing sequences encoding a marker protein are co-transfected.

Expression of a marker protein provides a means to distinguish transfected ceUs from nontransfected ceUs and is a rehable 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 ceUs expressing GFP or CD64-GFP and to evaluate the apoptotic state of the ceUs and other ceUular properties.

FCM detects and quantifies the uptake of fluorescent molecules that diagnose events preceding or coincident with ceU death. These events include changes in nuclear DNA content as measured by staining of DNA with propidium iodide; changes in ceU size and granularity as measured by forward kght scatter and 90 degree side kght scatter; down-regulation of DNA synthesis as measured by decrease in bromodeoxyuridine uptake; alterations in expression of cell surface and intraceUular 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 ceU 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 ceUs transfected with sequences encoding NAAP and either CD64 or CD64-GFP. CD64 and CD64-GFP are expressed on the surface of transfected ceUs and bind to conserved regions of human immunoglobulin G (IgG). Transfected ceUs are efficiently separated from nontransfected cells using magnetic beads coated with either human IgG or antibody against CD64 (DYNAL, Inc., Lake Success NY). mRNA can be purified from the ceUs using methods well known by those of skiU in the art. Expression of mRNA encoding NAAP and other genes of interest can be analyzed by northern analysis or microanay 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 C₅ position of the pyrimidine component of uridylate (U) when ³H-radiolabeled U in RNA is isomerized to pseudouridine (ψ). A typical 500 μl 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 μM [5-³H]tRNA (approximately 1 μCi/nmol tRNA). The reaction is initiated by the addition of <5 μl 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 peUet is washed twice by resuspension in 1 ml 0.1 M HCl, foUowed by centrifugation. The supernatants from the washes are separately passed through the glass wool plug and combined with the original filtrate. A portion of the combined filtrate is mixed with scintiUation 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. (1999) J. Biol. Chem. 274:22225-22230).

In the alternative, pseudouridine synthase activity of NAAP is assayed at 30 °C to 37 °C in a mixture containing 100 mM Tris-HCl (pH 8.0), 100 mM ammonium acetate, 5 mM 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. FoUowing 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 ψMP and UMP spots are evaluated after exposing the thin layer chromatography plates to film or a Phosphorlmager screen. Taking into account the relative number of uridylate residues in the substrate RNA, the relative amount ψMP and UMP are determined and used to calculate the relative amount of ψ per tRNA molecule (expressed in mol ψ /mol of tRNA or mol ψ /mol of tRNAyminute), which corresponds to the amount of pseudouridine synthase activity in the NAAP sample (Lecointe, F. et al. (1998) J. Biol. Chem. 273:1316-1323). N^,>P-dimethylguanosine transferase ((m² ₂G)methyltransferase) activity of NAAP is measured in a 160 μl reaction mixture containing 100 mM Tris-HCl (pH 7.5), 0.1 mM EDTA, 10 mM MgCl₂, 20 mM NILCl, ImM dithiothreitol, 6.2 μM 5-adenosyl-L-[met/ιy/-³H]methionine (30-70 Ci/mM), 8 μg m² ₂G-deficient tRNA or wild type tRNA from yeast, and approximately 100 μg of purified NAAP or a sample comprising NAAP. The reactions are incubated at 30 °C for 90 min and chiUed on ice. A portion of each reaction is diluted to 1 ml in water containing 100 μg BSA. 1 ml of 2 M HCl is added to each sample and the acid insoluble products are aUowed to precipitate on ice for 20 min before being coUected by filtration through glass fiber filters. The coUected material is washed several times with HCl and quantitated using a Uquid scintiUation counter. The amount of ³H incorporated into the m² ₂G-deficient, acid-insoluble tRNAs is proportional to the amount of transferase activity in the NAAP sample. Reactions comprising no substrate tRNAs, or wild-type tRNAs that have akeady been modified, serve as control reactions which should not yield acid-insoluble ³H-labeled products.

Polyadenylation activity of NAAP is measured using an in vitro polyadenylation reaction. The reaction mixture is assembled on ice and comprises 10 μl of 5 mM dithiothreitol, 0.025% (v/v) NONEDET P-40, 50 mM creatine phosphate, 6.5% (w/v) polyvinyl alcohol, 0.5 unit μl RNAGUARD (Pharmacia), 0.025 μg/μl creatine kinase, 1.25 mM cordycepin 5'-triphosphate, and 3.75 mM MgClj, in a total volume of 25 μl. 60 fmol of CstF, 50 fmol of CPSF, 240 fmol of PAP, 4 μl of crude or partiaUy purified CF E and various amounts of amounts CF I are then added to the reaction mix. The volume is adjusted to 23.5 μl 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 ³²P-labeled pre-mRNA template, along with 2.5 μg of unlabeled tRNA, in 1.5 μl of water. Reactions are then incubated at 30 °C for 75-90 min and stopped by the addition of 75 μl (approximately two-volumes) of proteinase K mix (0.2 M Tris-HCl, pH 7.9, 300 mM NaCl, 25 mM Na-EDTA, 2% (w/v) SDS), 1 μl of 10 mg/ml proteinase K, 0.25 μl of 20 mg/ml glycogen, and 23.75 μl of water). FoUowing 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 (Ruegsegger, 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 [¹⁴C]-labeled amino acid. NAAP is incubated with [¹⁴C]-labeled amino acid and the appropriate cognate tRNA (for example, [¹⁴C]alanine and tRNA^ala) in a buffered solution. ¹⁴C- labeled product is separated from free [¹⁴C]amino acid by chromatography, and the incorporated ¹⁴C is quantified by scintiUation counter. The amount of ¹⁴C-labeled product detected is proportional to the activity of NAAP in this assay.

In the alternative, NAAP activity is measured by incubating a sample containing NAAP in a solution containing 1 mM ATP, 5 mM Hepes-KOH (pH 7.0), 2.5 mM KCl, 1.5 mM magnesium chloride, and 0.5 mM DTT along with misacylated [¹⁴C]-Glu-tRNAGln (e.g., 1 μM) and a similar concentration of unlabeled L-glutamine. FoUowing 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 peUet is resuspended in of 25 mM KOH, deacylated at 65°C for 10 min., neutrakzed with 0.1 M HCl (to final pH 6-7), and dried under vacuum. The dried peUet is resuspended in water and spotted onto a ceUulose 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 Gin are calculated based on the Rf values and relative intensities of the spots. NAAP activity is calculated based on the amount of Gin resulting from the transformation of Glu while acylated as Glu-tRNA^Gln (adapted from Curnow, A.W. et al. (1997) Proc. Natl. Acad. Sci. USA 94:11819-26). XIX. Identification of NAAP Agonists and Antagonists

Agonists or antagonists of NAAP activation or inhibition may be tested using the assays described in section XVTE. Agonists cause an increase in NAAP activity and antagonists cause a decrease in NAAP activity.

Various modifications and variations of the described compositions, methods, and systems of the invention wiU be apparent to those skilled in the art without departing from the scope and spirit of the invention. It wiU be appreciated that the invention provides novel and useful proteins, and their encoding polynucleotides, which can be used in the drug discovery process, as weU as methods for using these compositions for the detection, diagnosis, and treatment of diseases and conditions. Although the invention has been described in connection with certain embodiments, it should be understood that the invention as claimed should not be unduly kmited to such specific embodiments. Nor should the description of such embodiments be considered exhaustive or limit the invention to the precise forms disclosed. Furthermore, elements from one embodiment can be readily recombined with elements from one or more other embodiments. Such combinations can form a number of embodiments within the scope of the invention. It is intended that the scope of the invention be defined by the foUowing claims and their equivalents.

Table 1

Table 1

Table 2

Table 2

Table 2

Table 2

Table 2

Table 2

Table 2

Table 2

Table 2

Table 2

Table 2

Table 2

Table 2

Table 2

-. \

Table 2

Table 2

Table 2

Table 2

Table 2

Table 2

Table 2

Table 2

Table 2

Table 2

Table 3

Table 3

Table 3

Table 3

Table 3

Table 3

Table 3

Table 3

Table 3

Table 3

Table 3

Table 3

Table 3

Table 3

Table 3

Table 3

Table 3

Table 3

Table 3

Table 3

Table 3

Table 3

Table 3

Table 3

Table 3

Table 3

Table 3

Table 3

Table 3

Table 3

Table 3

Table 3

Table 3

Table 3

o so

Table 3

Table 3

Table 3

Table 3

Table 3

Table 3

Table 3

Table 4

Table 4

Table 4

t o o

Table 4

Table 4

t oo t

Table 4

Table 4

Table 4

Table 4

Table 4

to o -J

Table 4

Table 4

o t so

Table 4

Table 4

Table 4

Table 4

Table 4

Table 4

Table 4

Table 4

to -J

Table 4

Table 4

Table 4

Table 4

Table 4

Table 4

Table 4

t to

-p.

Table 4

Table 4

Table 4

Table 4

Table 5

t to so

Table 5

Table 6

Table 6

Table 6

Table 6

Table 6

Table 6

Table 6

Table 6

Table 6

Table 7

t *o σ.

Table 7

t

■p.

Table 7

Table 8

Table 8

Claims

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:l-57, b) a polypeptide comprising a naturally occurring amino acid sequence at least 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NO:l-2, SEQ ID NO:13, SEQ ID NO:16, SEQ ID NO:18-20, SEQ ID NO:22-23, SEQ ID NO:31-32, SEQ ID NO:36-39, SEQ ID NO:41, SEQ ID NO:43, SEQ ID NO:46, SEQ ID NO:51, and SEQ ID NO:55, c) a polypeptide comprising a naturally occurring amino acid sequence at least 91% identical to an amino acid sequence selected from the group consisting of SEQ ID NO:21, and SEQ ID NO:27, d) a polypeptide comprising a naturally occurring amino acid sequence at least 94% identical to an amino acid sequence selected from the group consisting of SEQ ID

NO: 17, SEQ ID NO:20, and SEQ ID NO:42, e) a polypeptide comprising a naturally occurring amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO:50, f) a polypeptide comprising a naturally occurring amino acid sequence at least 96% identical to the amino acid sequence of SEQ ID NO:19, g) a polypeptide comprising a naturally occurring amino acid sequence at least 98% identical to the amino acid sequence of SEQ ID NO:24, h) a polypeptide comprising a naturally occurring amino acid sequence at least 99% identical to an amino acid sequence selected from the group consisting of SEQ ID NO:9, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:52, and SEQ ID NO:57, i) a polypeptide consisting essentially of a naturally occurring amino acid sequence at least 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NO:3-8, SEQ ID NO:10-12, SEQ ID NO:14-15, SEQ ID NO:24-27, SEQ ID NO:29, SEQ ID NO:34, SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:44-45, SEQ ID NO:47-49, SEQ ID NO:53-54, and SEQ ID NO:56, j) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:l-57, and k) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:l-57.

2. An isolated polypeptide of claim 1 comprising an amino acid sequence selected from the group consisting of SEQ ID NO:l-57.

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:58-l 14.

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: 1-57.

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:58-l 14, 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:58-67, SEQ ID NO:69-86, SEQ ID NO:88, SEQ ID NO:93, SEQ ID NO:95- 96, SEQ ID NO:98, SEQ ID NO:100, SEQ ID NO:108, and SEQ ID NO:110-112, c) a polynucleotide comprising a naturally occurring polynucleotide sequence at least 91% identical to the polynucleotide sequence of SEQ ID NO:94, d) a polynucleotide comprising a naturally occurring polynucleotide sequence at least

93% identical to a polynucleotide sequence selected from the group consisting of SEQ ID NO: 106, and SEQ ID NO: 114, e) a polynucleotide comprising a naturally occurring polynucleotide sequence at least 94% identical to a polynucleotide sequence selected from the group consisting of SEQ ID NO:87, and SEQ ID NO: 103, f) a polynucleotide comprising a naturally occurring polynucleotide sequence at least 96% identical to a polynucleotide sequence selected from the group consisting of SEQ ID NO:89-90, SEQ ID NO:92, and SEQ ID NO:101, g) a polynucleotide comprising a naturally occurring polynucleotide sequence at least 97% identical to the polynucleotide sequence of SEQ ID NO:99, h) a polynucleotide comprising a naturally occurring polynucleotide sequence at least

98% identical to a polynucleotide sequence selected from the group consisting of SEQ ID NO:68, SEQ ID NO:91, and SEQ ID NO:109, i) a polynucleotide comprising a naturally occurring polynucleotide sequence at least 99% identical to a polynucleotide sequence selected from the group consisting of SEQ

ID NO: 102, and SEQ ID NO: 107, j) a polynucleotide consisting essentially of a naturally occurring polynucleotide sequence at least 90% identical to a polynucleotide sequence selected from the group consisting of SEQ ID NO:97, SEQ ID NO:104-105, and SEQ ID NO:113, k) a polynucleotide complementary to a polynucleotide of a),

1) a polynucleotide complementary to a polynucleotide of b), m) a polynucleotide complementary to a polynucleotide of c), n) a polynucleotide complementary to a polynucleotide of d), o) a polynucleotide complementary to a polynucleotide of e), p) a polynucleotide complementary to a polynucleotide of f), q) a polynucleotide complementary to a polynucleotide of g), r) a polynucleotide complementary to a polynucleotide of h), s) a polynucleotide complementary to a polynucleotide of i), t) a polynucleotide complementary to a polynucleotide of j), and u) an RNA equivalent of a)-t).

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:l-57.

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 method for 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, further comprising a label.

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: 1-57, or an immunogenic fragment thereof, under conditions to elicit an antibody response, b) isolating antibodies from the animal, and c) screening the isolated antibodies with the polypeptide, thereby identifying a polyclonal antibody which specifically binds to a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 1-57.

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:l-57, or an immunogenic fragment thereof, under conditions to elicit an antibody response, b) isolating antibody producing cells from the animal, c) fusing the antibody producing cells with immortalized cells to form monoclonal antibody-producing hybridoma cells, d) culturing the hybridoma cells, and e) isolating from the culture monoclonal antibody which specifically binds to a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:l-57.

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:l-57 in a sample, the method comprising: a) incubating the antibody of claim 11 with the sample under conditions to allow specific binding of the antibody and the polypeptide, and b) detecting specific binding, wherein specific binding indicates the presence of a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 1-57 in the sample.

45. A method of purifying a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:l-57 from a sample, the method comprising: a) incubating the antibody of claim 11 with the sample under conditions to allow specific binding of the antibody and the polypeptide, and b) separating the antibody from the sample and obtaining the purified polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID

NO:l-57.

46. A microanay 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 aπay 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 aπay 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 aπay of claim 48, wherein a linker joins at least one of said nucleotide molecules to said solid substrate.

55. An aπay 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:l.

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:l 1.

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

96. A polypeptide of claim 1, comprising the amino acid sequence of SEQ ID NO:41.

97. A polypeptide of claim 1 , comprising the amino acid sequence of SEQ D NO:42.

98. A polypeptide of claim 1, comprising the amino acid sequence of SEQ ID NO:43.

99. A polypeptide of claim 1, comprising the amino acid sequence of SEQ ID NO:44.

100. A polypeptide of claim 1, comprising the amino acid sequence of SEQ ID NO:45.

101. A polypeptide of claim 1, comprising the amino acid sequence of SEQ ID NO:46.

102. A polypeptide of claim 1, comprising the amino acid sequence of SEQ ID NO:47.

103. A polypeptide of claim 1, comprising the amino acid sequence of SEQ ID NO:48.

104. A polypeptide of claim 1, comprising the amino acid sequence of SEQ ID NO:49.

105. A polypeptide of claim 1, comprising the amino acid sequence of SEQ ID NO:50.

106. A polypeptide of claim 1, comprising the amino acid sequence of SEQ ID NO:51.

107. A polypeptide of claim 1, comprising the amino acid sequence of SEQ ID NO:52.

108. A polypeptide of claim 1, comprising the amino acid sequence of SEQ ID NO:53.

109. A polypeptide of claim 1, comprising the amino acid sequence of SEQ ID NO:54.

110. A polypeptide of claim 1, comprising the amino acid sequence of SEQ ID NO:55.

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

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

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

114. A polynucleotide of claim 12, comprising the polynucleotide sequence of SEQ ID

NO:59.

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

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

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

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

119. A polynucleotide of claim 12, comprising the polynucleotide sequence of SEQ ID

NO:64.

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

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

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

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

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

125. A polynucleotide of claim 12, comprising the polynucleotide sequence of SEQ ID

NO:70.

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

127. A polynucleotide of claim 12, comprising the polynucleotide sequence of SEQ ID

NO:72.

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

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

130. A polynucleotide of claim 12, comprising the polynucleotide sequence of SEQ ID

NO:75.

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

132. A polynucleotide of claim 12, comprising the polynucleotide sequence of SEQ ID

NO:77.

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

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

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

136. A polynucleotide of claim 12, comprising the polynucleotide sequence of SEQ ID

NO:81.

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

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

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

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

141. A polynucleotide of claim 12, comprising the polynucleotide sequence of SEQ ID

NO:86.

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

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

144. A polynucleotide of claim 12, comprising the polynucleotide sequence of SEQ ID NO:89.

145. A polynucleotide of claim 12, comprising the polynucleotide sequence of SEQ ID NO:90.

146. A polynucleotide of claim 12, comprising the polynucleotide sequence of SEQ ID NO:91.

147. A polynucleotide of claim 12, comprising the polynucleotide sequence of SEQ ID

NO:92.

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

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

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

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

152. A polynucleotide of claim 12, comprising the polynucleotide sequence of SEQ ID

NO:97.

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

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

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

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

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

158. A polynucleotide of claim 12, comprising the polynucleotide sequence of SEQ ID

NO:103.

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

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

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

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

163. A polynucleotide of claim 12, comprising the polynucleotide sequence of SEQ ID

NO:108.

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

165. A polynucleotide of claim 12, comprising the polynucleotide sequence of SEQ ED NO:110.

166. A polynucleotide of claim 12, comprising the polynucleotide sequence of SEQ ED NO:lll.

167. A polynucleotide of claim 12, comprising the polynucleotide sequence of SEQ ED NO:112.

168. A polynucleotide of claim 12, comprising the polynucleotide sequence of SEQ ED NO:113.

169. A polynucleotide of claim 12, comprising the polynucleotide sequence of SEQ ED

NO:114.