WO2003023003A2 - Proteines associees a des acides nucleiques - Google Patents

Proteines associees a des acides nucleiques Download PDF

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WO2003023003A2
WO2003023003A2 PCT/US2002/028540 US0228540W WO03023003A2 WO 2003023003 A2 WO2003023003 A2 WO 2003023003A2 US 0228540 W US0228540 W US 0228540W WO 03023003 A2 WO03023003 A2 WO 03023003A2
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seq
polynucleotide
polypeptide
amino acid
sequence
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PCT/US2002/028540
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WO2003023003A3 (fr
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Y. Tom Tang
Jennifer L. Jackson
Jennifer A. Griffin
Vicki S. Elliott
Ian J. Forsythe
Shanya D. Becha
Thomas W. Richardson
Ernestine A. Lee
William W. Sprague
Brooke M. Emerling
Kavitha Thangavelu
Bridget A. Warren
Uyen K. Tran
Henry Yue
Yuming Xu
Huibin Yue
Joana X. Li
April J. A. Hafalia
Bharati Sanjanwala
Joseph P. Marquis
Ann E. Gorvad
Soo Yeun Lee
Craig H. Ison
Mariah R. Baughn
Narinder K. Chawla
Danniel B. Nguyen
Anita Swarnakar
Yeganeh Zebarjadian
Purvi Shah
Michael Thornton
Monique G. Yao
Farrah A. Khan
Ameena R. Gandhi
Junming Yang
Amy E. Kable
Neil Burford
Jayalaxmi Ramkumar
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Incyte Genomics, Inc.
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Priority to CA002459442A priority patent/CA2459442A1/fr
Publication of WO2003023003A2 publication Critical patent/WO2003023003A2/fr
Publication of WO2003023003A3 publication Critical patent/WO2003023003A3/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value

Definitions

  • 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 autoimmune/inflammatory 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.
  • 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.
  • 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.
  • 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)
  • 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.
  • 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.
  • DNA-binding structural motifs which comprise either a helices or ⁇ sheets that bind to the major groove of DNA.
  • 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 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.
  • 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 ⁇ helix and an antiparallel ⁇ 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.
  • the zinc finger motif may be repeated in a tandem array within a protein, such that the ⁇ 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, DJ. et al. (1999) Mol. Cell Biol.
  • Zinc-finger transcription factors are often accompanied by modular sequence motifs such as the Kruppel-associated box (KRAB) and the SCAN domain.
  • KRAB Kruppel-associated box
  • the hypoalphalipoproteinemia susceptibility gene ZNF202 encodes a SCAN box and a KRAB domain followed by eight C2H2 zinc-finger motifs (Honer, C. et al. (2001) Biochim. Biophys. Acta 1517:441-448).
  • the SCAN domain is a highly conserved, leucine-rich motif of approximately 60 amino acids found at the amino-terminal end of zinc finger transcription factors. SCAN domains are most often linked to C2H2 zinc finger motifs through their carboxyl-terminal end.
  • 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, AJ. 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 . 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 seminornas, and in the NT2/D1 teratocarcinoma cell line (Poncelet, D.A. et al.
  • 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.
  • C3HC4 zinc-finger or RING finger a conserved cysteine-rich domain of 40 to 60 residues 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
  • 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 ⁇ 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 ⁇ -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 LLM 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.
  • actin- binding LLM 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 LBVI protein has four double zinc finger motifs with the LIM consensus sequence.
  • the C- terminal domain of actin-binding LJ protein shows sequence similarity to known actin-binding proteins such as dematin and villin.
  • Actin-binding LLM 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 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 dete ⁇ ination of cell lineage (Papavassiliou, A.G. (1995) N. Engl. J. Med. 332:45-47).
  • the helix-loop-helix motif (HLH) consists of a short ⁇ helix connected by a loop to a longer 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).
  • 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
  • ARID AT-rich interactive domain
  • 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 (lrx) family of genes are found in nematodes, insects and vertebrates. Lrx genes usually occur in one or two genomic clusters of three genes each and encode transcriptional controllers that possess a characteristic homeodomain. The lrx genes function early in development to specify the identity of diverse territories of the body. Later in development in both Drosophila and vertebrates, the lrx genes function again to subdivide those territories into smaller domains. (For a review of Iroquois genes, see Cavodeassi, F. et al.
  • 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
  • Fhit fragile histidine triad
  • chromatin 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 chromodornain proteins.
  • HMG high mobility group
  • chromodornain 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. Chromodornain 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.
  • the zinc finger-type transcriptional regulator WT1 is a tumor-suppressor protein that is inactivated in children with Wilm' s tumor.
  • the oncogene bcl-6 which plays an important role in large-cell lymphoma, is also a zinc-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.
  • 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).
  • 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.
  • 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.
  • the causative gene for autoimmune polyendocrinopathy- candidiasis-ectodermal dystrophy was recently isolated and found to encode a protein with two PHD-type zinc finger motifs (Bjorses, P. et al. (1998) Hum. Mol. Genet. 7:1547-1553). Furthermore, the generation of multicellular organisms is based upon the induction and coordination of cell differentiation at the appropriate stages of development. Central to this process is differential gene expression, which confers the distinct identities of cells and tissues throughout the body. Failure to regulate gene expression during development could result in developmental disorders.
  • Human developmental disorders caused by mutations in zinc finger-type transcriptional regulators include: urogenital developmental abnormalities associated with WT1; Greig cephalopolysyndactyly, Pallister-Hall syndrome, and postaxial polydactyly type A (GLI3), and Townes-Brocks syndrome, characterized by anal, renal, limb, and ear abnormalities (SALLl) (Engelkamp, D. and V. van Heyningen (1996) Curr. Opin. Genet. Dev. 6:334-342; Kohlhase, J. et al. (1999) Am. J. Hum. Genet. 64:435-445).
  • Human acute leukemias involve reciprocal chromosome translocations that fuse the ALL-1 gene located at chromosome region 1 lq23 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 Polvmerases DNA and RNA replication are critical processes for cell replication and function. DNA and
  • RNA replication are mediated by the enzymes DNA and RNA polymerase, respectively, by a "templating" process in which the nucleotide sequence of a DNA or RNA strand is copied by complementary base-pairing into a complementary nucleic acid sequence of either DNA or RNA.
  • 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.
  • 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.
  • 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.
  • DNA is said to be replicated "semiconservatively" by DNA polymerase.
  • DNA polymerase is also involved in the repair of damaged DNA as discussed below under “Ligases.”
  • 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).
  • mRNA messenger RNAs
  • RNA polymerase I makes the large ribosomal RNAs
  • RNA polymerase LI makes the mRNAs that will be translated into proteins
  • RNA polymerase HI makes a variety of small, stable RNAs, including 5S ribosomal RNA and the transfer RNAs (tRNA).
  • 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.
  • 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.
  • 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.
  • 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 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 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.
  • DNA endonuclease activity is involved in the excision step of the DNA repair process. RNases also serve a variety of functions.
  • 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).
  • 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 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).
  • SSBs Single- stranded binding proteins
  • 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.
  • DEAD-box helicases 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.
  • RNA helicases examples include yeast Drsl protein, which is involved in ribosomal RNA processing; yeast TIFl and TIF2 and mammalian eIF-4A, which are essential to the initiation of RNA translation; and human p68 antigen, which regulates cell growth and division (Ripmaster, T.L. et al. (1992) Proc. Natl. Acad. Sci. USA 89:11131-11135; Chang, T.-H. et al. (1990) Proc. Natl. Acad. Sci. USA 87: 1571-1575). These RNA helicases demonstrate strong sequence homology over a stretch of some 420 amino acids.
  • conserved sequences include 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).
  • 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).
  • Nb neuroblastoma
  • Rb retinoblastoma
  • 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.
  • DDX6 is located at a chromosomal breakpoint associated with B-cell lymphoma.
  • a chimeric protein comprised of DDX10 and NUP98, a nucleoporin protein, may be involved in the pathogenesis of certain myeloid malignancies. 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, JJ. et al. (1999) Science 286:552-555).
  • Type I topoisomerases work as monomers, making a break in a single strand of DNA while type LI 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 LI 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).
  • Topoisomerase LI 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.
  • MDR multi-drug resistance
  • 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.
  • protein-DNA interactions limit the rotation, which is driven by torsional strain in the DNA (Stewart, L. et al. (1998) Science 379: 1534-1541).
  • 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.
  • topo LUs Two topo LUs 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 HI has been suggested to play a role in meiotic recombination. A mouse topo III is highly expressed in testis tissue and its expression increases with the increase in the number of cells in pachytene (Seki, T. et al. (1998) J. Biol. Chem. 273:28553-28556). The topoisomerase LI family includes two isozymes (U ⁇ and ⁇ ) encoded by different genes.
  • Topo LI 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.
  • 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 Ha but not U ⁇ suggest that U ⁇ is dispensable in cellular processes; however, ⁇ knockout mice died perinatally due to a failure in neural development. That the major abnormalities occurred in predominantly late developmental events (neurogenesis) suggests that Ll ⁇ 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 LH is deregulated in A-T (Fritz, E. et al. (1997) Proc. Natl. Acad. Sci.
  • A-T ataxia-telangiectasia
  • Topo HI 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 H activity is often associated with cancer or increased cancer risk. Greatly lowered topo H 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 H can break DNA in the region of the A-T gene (ATM), which controls all DNA damage-responsive cell cycle checkpoints (Kaufmann, W.K.
  • ATM A-T gene
  • 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.
  • RNA METABOLISM enzymes, recombinases, that "nick" one strand of a DNA duplex more or less randomly and permit exchange with a complementary strand on another duplex.
  • the process does not normally change the arrangement of genes in a chromosome.
  • the recombinase recognizes specific nucleotide sequences present in one or both of the recombfning 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 Ribonucleic acid
  • DNA deoxyribonucleic acid
  • 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 are assembled, along with ribosomal proteins, into ribosomes, which are cytoplasmic particles that translate mRNA into polypeptides.
  • Transfer RNAs tRNAs
  • tRNAs Transfer RNAs
  • hnRNAs Heterogeneous nuclear RNAs
  • hnRNAs include mRNA precursors and other nuclear RNAs of various sizes.
  • Small nuclear RNAs snRNAs
  • 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.
  • Ribosomal RNAs 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.
  • 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, Ll, 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.
  • 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).
  • S6K1 and S6K2 the highly homologous 40S ribosomal protein S6 kinases
  • S6K1 and S6K2 the highly homologous 40S ribosomal protein S6 kinases
  • S6K1 and S6K2 the highly homologous 40S ribosomal protein S6 kinases
  • S6K1 and S6K2 the highly homologous 40S ribosomal protein S6 kinases
  • at least eight phosphorylation sites are believed to mediate kinase activation in a hierarchical fashion (Dufher and Thomas (19
  • 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.
  • L13a 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.
  • ribosomal protein L7
  • ribosomal proteins may function as cell cycle checkpoints and compose a new family of cell proliferation regulators.
  • the aminoacyl-tRNA acceptor site receives charged tRNAs (with the exception of the mitiator-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.
  • RNA 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.
  • spliceosomal complex is comprised of five small nuclear ribonucleoprotein particles (snRNPs) designated Ul, U2, U4, U5, and U6.
  • snRNPs small nuclear ribonucleoprotein particles
  • Ul small nuclear ribonucleoprotein particles
  • U2, U4, U5 small nuclear ribonucleoprotein particles
  • U6 small nuclear ribonucleoprotein particles
  • 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).
  • hnRNPs Heterogeneous nuclear ribonucleoproteins
  • 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.G.
  • 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).
  • RRM RNA recognition motif
  • the RRM is about 80 amino acids in length and forms four ⁇ -strands and two -helices arranged in an / ⁇ sandwich.
  • the RRM contains a core RNP-1 octapeptide motif along with surrounding conserved sequences.
  • 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 protems 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 c ⁇ -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 cw-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 ⁇
  • CstF Cleavage stimulation factor
  • CF I cleavage factor I
  • CF H cleavage factor H
  • CPSF polyadenylation specificity factor
  • PAP poly(A) polymerase
  • aaRSs aminoacyl-tRNA synthetases
  • the 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 H 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 H enzymes contain a central catalytic domain, which consists of a seven-stranded antiparallel ⁇ -sheet domain, as well as N- and C- terminal regulatory domains.
  • Class H 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 H enzymes are specific for alanine, asparagine, aspartic acid, glycine, histidine, lysine, phenylalanine, proline, serine, and threonine.
  • aaRSs also have editing functions.
  • fleRS for example, can misactivate valine to form Val-tRNA ne , 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).
  • CP1 connective polypeptide 1 region
  • AaRSs also play a role in tRNA processing.
  • 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 " 4 and is primarily the result of aminoacyl-t-RNAs being charged with the incorrect amino acid. Incorrectly charged tRNA are toxic to cells as they result in the incorporation of incorrect amino acid residues into an elongating polypeptide.
  • the rate of translation is presumed to be a compromise between the optimal rate of elongation and the need for translational fidelity.
  • Mathematical calculations predict that IO "4 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 Gln with Gin.
  • 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):
  • Glu-AdT Glu-tRNA GIn + Gin + ATP ⁇ Gln-tRNA Gln + Glu + ADP + P
  • Asp-tRNA Asn amidotransferase exists in Archaea, which transforms Asp- tRNA Asn to Asn-tRNA Asn .
  • Formylase the enzyme that transforms Met-tRNATM 6 ' to fMet-tRNA ⁇ ' in eubacteria, is likely to be a related enzyme.
  • a hydrolytic activity has also been identified that destroys mischarged Val-tRNA Ile (Schimmel, P. et al. (1998) FASEB J. 12:1599-1609).
  • Glu-AdT 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 G,n .
  • GlnRS glutaminyl- tRNA synthetase
  • 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.
  • tyrosyl-tRNA synthetases 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.
  • 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- ⁇ , and tissue factor.
  • the N-terminal domain binds to the interleukin-8 type A receptor and functions as an interleukin-8-like cytokine.
  • Mitochondrial Neurospora crassa TyrRS and S. cerevisiae LeuRS are essential factors for certain group I intron splicing activities, and human mitochondrial LeuRS can substitute for the yeast LeuRS in a yeast null strain.
  • Certain bacterial aaRSs are involved in regulating their own transcription or translation (Martinis, supra).
  • 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).
  • aaRSs 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 ( ⁇ ), 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.
  • 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, C.J. et al. (1982) J. Biol. Chem. 257:3045-52; and Chen, J. and Patton, J.R. (1999) RNA 5:409-419).
  • tRNA pseudouridine synthases have been the most extensively studied members of the family. They require a thiol donor (e.g., cysteine) and a monovalent cation (e.g., ammonia or potassium) for optimal activity.
  • ribonucleoside modification that occurs primarily in eukaryotic cells is the conversion of guanosine to INP ⁇ -dimethylguanosine (m 2 2 G) 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.
  • the enzyme responsible for the conversion of guanosine to m 2 2 G is a 63 kDa S- adenosylmethionine (SAM)-dependent tRNA N 2 ,N 2 -dimethyl-guanosine methyltransferase (also referred to as the TRM1 gene product and herein referred to as TRM) (Edqvist, J. (1995) Biochimie 77:54-61).
  • SAM S- adenosylmethionine
  • TRM1 gene product also referred to as the TRM1 gene product and herein referred to as TRM
  • the enzyme localizes to both the nucleus and the mitochondria (Li, J-M. et al. (1989) J. Cell Biol. 109: 1411-1419).
  • 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 43S preinitiation complex to the mRNA, followed by migration of the complex to the correct AUG initiation codon.
  • the third stage brings the 60S ribosomal subunit to the 40S subunit to generate an 80S ribosome at the inititation codon.
  • Regulation of translation primarily involves the first and second stage in the initiation process (V.M. Pain (1996) Eur. J. Biochem. 236:747-771).
  • eIF2 a guanine nucleotide binding protein, recruits the initiator tRNA to the 40S ribosomal subunit. Only when eIF2 is hound 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.
  • elFIA and eIF3 bind and stabilize the 40S subunit by interacting with the 18S ribosomal RNA and specific ribosomal structural proteins.
  • eIF3 is also involved in association of the 40S ribosomal subunit with mRNA.
  • the Met-tRNA f , elFIA, eEF3, and 40S ribosomal subunit together make up the 43S preinitiation complex (Pain, supra). Additional factors are required for binding of the 43S preinitiation complex to an mRNA molecule, and the process is regulated at several levels.
  • eIF4F is a complex consisting of three proteins: eLF4E, eLF4A, and eIF4G.
  • eIF4E recognizes and binds to the mRNA 5 -terminal m 7 GTP cap, eIF4A is a bidirectional RNA-dependent helicase, and eIF4G is a scaffolding polypeptide. eIF4G has three binding domains. The N-terminal third of eIF4G interacts with eIF4E, the central third interacts with eLF4A, and the C-terminal third interacts with eIF3 bound to the 43S preinitiation complex. Thus, eIF4G acts as a bridge between the 40S ribosomal subunit and the mRNA (M.W. Hentze (1997) Science 275:500-501).
  • the ability of eIF4F to initiate binding of the 43S preinitiation complex is regulated by structural features of the mRNA.
  • the mRNA molecule has an untranslated region (UTR) between the 5' cap and the AUG start codon. In some mRNAs this region forms secondary structures that impede binding of the 43S preinitiation complex.
  • the helicase activity of eIF4A is thought to function in removing this secondary structure to facilitate binding of the 43S preinitiation complex (Pain, supra).
  • Translation Elongation Elongation is the process whereby additional amino acids are joined to the initiator methionine to form the complete polypeptide chain.
  • EF1 is a GTP-binding protein. In EF1 ⁇ '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 EF1 is hydrolyzed to GDP, and EF1 -GDP dissociates from the ribosome.
  • EF1 ⁇ ⁇ binds EF1 ⁇ -GDP and induces the dissociation of GDP from EF1 , allowing EF1 ⁇ to bind GTP and a new cycle to begin.
  • 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.
  • 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.
  • arrays are employed to detect the expression of a specific gene or its variants.
  • 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.
  • PBMCs Human peripheral blood mononuclear cells contain about 52% lymphocytes (12% B lymphocytes, 40% T lymphocytes ⁇ 25% CD4+ and 15% CD8+ ⁇ ), 20% NK cells, 25% monocytes, and 3% various cells that include dendritic cells and progenitor cells.
  • Granulocyte-monocyte colony stimulating factor can support the in vitro colony formation of granulocyte-macrophage progenitors.
  • GM-CSF is a growth factor for erythroid, megakaryocyte, and eosinophil progenitors.
  • Lymphocytes (T and B) monocytes, macrophages, mast cells, endothelial cells, and fibroblasts can produce GM-CSF upon activation.
  • GM-CSF exerts its biological effects by binding to specific cell surface receptors.
  • LIF Leukemia inhibitory factor
  • Human LIF cDNA encodes a 202 amino acid residue polypeptide with a 22 amino acid residue signal peptide that is cleaved to yield a 180 amino acid residue mature human LIF.
  • Human aortic endothelial cells are primary cells derived from the endothelium of a human aorta. HAECs have been used as an experimental model for investigating the role of the endothelium in human vascular biology in vitro. Activation of the vascular endothelium is a central event in a wide range of both physiological and pathophysiological processes including vascular tone regulation, coagulation and thrombosis, atherosclerosis, and inflammation.
  • TNF- ⁇ is a pleiotropic cytokine involved in the mediation of inflammatory responses through activation of multiple signal transduction pathways.
  • TNF- ⁇ is produced by activated lymphocytes, macrophages, and other white blood cells, and is known to activate endothelial cells. Monitoring the endothelial cells' response to TNF- ⁇ at the level of the mRNA expression can provide information necessary for better understanding of both TNF- ⁇ signaling pathways and endothelial cell biology.
  • RPMI 6666 is a B cell lymphoblast cell line derived from peripheral blood affected by
  • RPMI 6666 cells are immature lymphocyte-producing immunoglobulins, and present cell-associated Epstein-Barr virus (EBV) particules.
  • EBV Epstein-Barr virus
  • LPS Lipopolysaccharide
  • monocytes elicit a variety of inflammatory responses, and because it activates complement by the alternative (properdin) pathway, it is often part of the pathology of gram- negative bacterial infections.
  • LPS released into the bloodstream by lysing gram-negative bacteria is first bound by LPS-binding proteins.
  • the LPS-binding protein complex interacts with CD 14 receptors on monocytes, macrophages, B cells, and other types of receptors on endothelial cells.
  • Activation of human B cells with LPS results in mitogenesis as well as immunoglobulin synthesis.
  • Breast cancer develops through a multi-step process whereby pre-malignant mammary epithelial cells undergo a relatively defined sequence of events leading to tumor formation.
  • the H14 medium used to grow an HMT-3522 mammary tumor progression cell model, contains the basal medium DMEM-F12 (1:1, v/v) supplemented with growth factors and growth hormones.
  • MCF-10A is a breast mammary gland (luminal ductal characteristics) cell line isolated from a 36-year-old woman with fibrocystic breast disease. MCF-10A expresses cytoplasmic keratins, epithelial sialomucins, and milkfat globule antigens. MCF7 is a nonmalignant breast adenocarcinoma cell line isolated from pleural effusion. MCF7 has retained characteristics of the mammary epithelium such as the ability to process estradiol via cytoplasmic estrogen receptors and the capacity to form domes in culture.
  • T-47D is a breast carcinoma cell line isolated from a pleural effusion from a donor with an infiltrating ductal carcinoma of the breast.
  • Sk-BR-3 is a breast adenocarcinoma cell line isolated from a malignant pleural effusion, that forms poorly differentiated adenocarcinoma when injected into nude mice.
  • BT-20 is a breast carcinoma cell line derived in vitro from the cells emigrating out of thin slices of a tumor mass.
  • MDA-mb-231 is a breast tumor cell line isolated from pleural effusion. It forms poorly differentiated adenocarcinoma in nude mice and ALS treated BALB/c mice. It also expresses the Wnt3 oncogene, EGF, and TGF- ⁇ .
  • the C3A cell line is a clonal derivative of HepG2, which is a hepatoma cell line isolated from a donor with a liver tumor.
  • C3A cells express both the insulin receptor and the insulin-like growth factor H receptor.
  • C3A cells show strong contact inhibition of growth.
  • Insulin resistance is central to the pathophysiology of type H diabetes and a number of other disease states. Down-regulation and reduced kinase activity of the insulin receptor play a role in insulin resistance. Also important are defects in the intracellular responses to insulin involving insulin receptor substrate (IRS-1) tyrosine phosphorylation and IRS/PI3 kinase interaction.
  • IRS-1 insulin receptor substrate
  • IRS/PI3 kinase interaction The major target for insulin signaling to glucose transport is a complex membrane trafficking pathway.
  • PI3 kinase activation by insulin plays a role in glucose transport.
  • LY294002 is a PI3 kinase specific inhibitor that promotes cell cycle arrest of C3A cells. This factor also appears to enhance the metabolic activity of the cells, especially with respect to proteins such as P450s.
  • the C3a cell line may be used to investigate transcript profiling in response to Clofibrate. dissolved in carboxymethyl cellulose. Fibric acid agents can have widely differing effects on lipoprotein levels. Among all patients, hyperlipidemic subjects with ApoE2/ApoE2 homozygosity respond best to gem-fibrozil therapy. Elevated triglyceride and cholesterol levels may be lowered, and tuboeruptive and palmar xanthomas may regress. Improvement in angina and intermittent claudication also occurs.
  • Second generation agents such as fenofibrate, bezafibrate, and ciprofibrate, lower VLDL levels to a degree similar to that produced by gemfibrozil, but they also decrease LDL levels by 15-20%.
  • the fibric acids lower the levels of triglyceride-rich lipoproteins, such as VLDL, modestly raise HDL levels, and have variable effects on LDL levels.
  • Fibric acid agents may also alter the composition of the VLDL (see Goodman and Gilman's The Pharmacological Basis of Therapeutics, Goodman, L.S. et al. (eds.) 9 th edition, McGraw-Hill, 1996).
  • DMSO is a vehicle routinely used for dissolving drug substances, particularly for compounds with low solubility in aqueous solution. DMSO concentrations of 1-2% can have some biological effects on cells in culture. There is a need in the art for new compositions, including nucleic acids and proteins, for the diagnosis, prevention, and treatment of cell proliferative, neurological, developmental, and autoimmune/inflammatory disorders, and infections.
  • Various embodiments of the invention provide purified polypeptides, nucleic acid-associated proteins, referred to collectively as 'NAAP' and individually as 'NAAP-1,' 'NAAP-2,' 'NAAP-3,' 'NAAP-4,' 'NAAP-5,' 'NAAP-6,' 'NAAP-7,' 'NAAP-8,' 'NAAP-9,' 'NAAP-10,' 'NAAP-11,' 'NAAP-12,' 'NAAP-13,' 'NAAP-14,' 'NAAP-15,' '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,'
  • Embodiments also provide methods for utilizing the purified nucleic acid-associated proteins and/or their encoding polynucleotides for facilitating the drug discovery process, including determination of efficacy, dosage, toxicity, and pharmacology.
  • Related embodiments provide methods for utilizing the purified nucleic acid-associated proteins and/or their encoding polynucleotides for investigating the pathogenesis of diseases and medical conditions.
  • An embodiment provides an isolated polypeptide selected from the group consisting of a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 1- 56, 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: 1-56, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1-56, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1-56.
  • Another embodiment provides an isolated polypeptide comprising an amino acid sequence of SEQ ID NO: 1-56.
  • Still another embodiment provides an isolated polynucleotide encoding a polypeptide selected from the group consisting of a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ED NO: 1-56, 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: 1-56, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1-56, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1-56.
  • polynucleotide encodes a polypeptide selected from the group consisting of SEQ ID NO: 1-56. In an alternative embodiment, the polynucleotide is selected from the group consisting of SEQ ID NO:57-l 12.
  • Still another embodiment provides a recombinant polynucleotide comprising a promoter sequence operably linked to a polynucleotide encoding a polypeptide selected from the group consisting of a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 1-56, 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: 1-56, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1-56, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1-56.
  • Another embodiment provides a cell transformed with the recombinant polynucleotide.
  • Yet another embodiment provides a transgenic organism comprising the recombinant polynucleotide.
  • Another embodiment provides a method for producing a polypeptide selected from the group consisting of a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 1-56, 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 ED NO: 1-56, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1-56, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1-56.
  • the method comprises a) culturing a cell under conditions suitable for expression of the polypeptide, wherein said cell is transformed with a recombinant polynucleotide comprising a promoter sequence operably linked to a polynucleotide encoding the polypeptide, and b) recovering the polypeptide so expressed.
  • Yet another embodiment provides an isolated antibody which specifically binds to a polypeptide selected from the group consisting of a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ED NO: 1-56, 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: 1-56, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1-56, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ED NO: 1-56.
  • Still yet another embodiment provides an isolated polynucleotide selected from the group consisting of a) a polynucleotide comprising a polynucleotide sequence selected from the group consisting of SEQ D NO:57-l 12, b) a polynucleotide comprising a naturally occurring polynucleotide sequence at least 90% identical or at least about 90% identical to a polynucleotide sequence selected from the group consisting of SEQ ID NO:57-l 12, 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 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: 57-112, b) a polynucleotide comprising a naturally occurring polynucleotide sequence at least 90% identical or at least about 90% identical to a polynucleotide sequence selected from the group consisting of SEQ ID NO:57-l 12, c) a polynucleotide complementary to the polynucleotide of a), d) a polynucleotide complementary to the polynucleotide of b), and e) an RNA equivalent of a)-d).
  • the method comprises a) hybridizing the sample with a probe comprising at least 20 contiguous nucleotides comprising a sequence complementary to said target polynucleotide in the sample, and which probe specifically hybridizes to said target polynucleotide, under conditions whereby a hybridization complex is formed between said probe and said target polynucleotide or fragments thereof, and b) detecting the presence or absence of said hybridization complex.
  • the method can include detecting the amount of the hybridization complex.
  • the probe can comprise at least about 20, 30, 40, 60, 80, or 100 contiguous nucleotides.
  • Still yet another embodiment provides a method for detecting a target polynucleotide in a sample, said target polynucleotide being selected from the group consisting of a) a polynucleotide comprising a polynucleotide sequence selected from the group consisting of SEQ ID NO:57-l 12, b) a polynucleotide comprising a naturally occurring polynucleotide sequence at least 90% identical or at least about 90% identical to a polynucleotide sequence selected from the group consisting of SEQ ID NO:57-112, 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).
  • a target polynucleotide being selected from the group consisting of a) a polynucleotide comprising a polynucleot
  • the method comprises a) amplifying said target polynucleotide or fragment thereof using polymerase chain reaction amplification, and b) detecting the presence or absence of said amplified target polynucleotide or fragment thereof.
  • the method can include detecting the amount of the amplified target polynucleotide or fragment thereof.
  • compositions 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 ID NO: 1-56, 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 ED NO: 1-56, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1-56, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ED NO: 1-56, and a pharmaceutically acceptable excipient.
  • the composition can comprise an amino acid sequence selected from the group consisting of SEQ ID NO: 1-56.
  • Other embodiments provide a method of treating a disease or condition associated with decreased or abnormal expression of functional NAAP, comprising administering to a patient in need of such treatment the composition.
  • Yet another embodiment provides a method for screening a compound for effectiveness as an agonist of a polypeptide selected from the group consisting of a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 1-56, 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 ED NO: 1-56, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1-56, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1-56.
  • the method comprises a) exposing a sample comprising the polypeptide to a compound, and b) detecting agonist activity in the sample.
  • Another embodiment provides a composition comprising an agonist compound identified by the method and a pharmaceutically acceptable excipient.
  • Yet another embodiment provides a method of treating a disease or condition associated with decreased expression of functional NAAP, comprising administering to a patient in need of such treatment the composition.
  • Still yet another embodiment provides a method for screening a compound for effectiveness as an antagonist of a polypeptide selected from the group consisting of a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ TD NO: 1-56, 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: 1-56, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1-56, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1-56.
  • the method comprises a) exposing a sample comprising the polypeptide to a compound, and b) detecting antagonist activity in the sample.
  • Another embodiment provides a composition comprising an antagonist compound identified by the method and a pharmaceutically acceptable excipient.
  • Yet another embodiment provides a method of treating a disease or condition associated with overexpression of functional NAAP, comprising administering to a patient in need of such treatment the composition.
  • Another embodiment provides a method of screening for a compound that specifically binds to a polypeptide selected from the group consisting of a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 1-56, 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: 1-56, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ED NO: 1-56, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ED NO: 1-56.
  • the method comprises a) combining the polypeptide with at least one test compound under suitable conditions, and b) detecting binding of the polypeptide to the test compound, thereby identifying a compound that specifically binds to the polypeptide.
  • 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: 1-56, 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: 1-56, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1-56, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ED NO: 1-56.
  • the method comprises a) combining the polypeptide with at least one test compound under conditions permissive for the activity of the polypeptide, b) assessing the activity of the polypeptide in the presence of the test compound, and c) comparing the activity of the polypeptide in the presence of the test compound with the activity of the polypeptide in the absence of the test compound, wherein a change in the activity of the polypeptide in the presence of the test compound is indicative of a compound that modulates the activity of the polypeptide.
  • Still yet another embodiment provides a method for screening a compound for effectiveness in altering expression of a target polynucleotide, wherein said target polynucleotide comprises a polynucleotide sequence selected from the group consisting of SEQ ED NO:57-l 12, 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 ED NO:57-112, 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 ED NO:57-l 12, iii) a polynucleotide having a sequence complementary to i), iv) a polynucleotide complementary to the polynucleotide of ii), and v) an RNA equivalent of
  • 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 ED NO:57-l 12, 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 ED NO:57-l 12, 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).
  • 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.
  • 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 probability scores for the matches between each polypeptide and its homolog(s) are also shown.
  • Table 3 shows structural features of polypeptide embodiments, including predicted motifs and domains, along with the methods, algorithms, and searchable databases used for analysis of the polypeptides.
  • Table 4 lists the cDNA and/or genomic DNA fragments which were used to assemble polynucleotide embodiments, along with selected fragments of the polynucleotides.
  • Table 5 shows representative cDNA libraries for polynucleotide embodiments.
  • Table 6 provides an appendix which describes the tissues and vectors used for construction of the cDNA libraries shown in Table 5.
  • Table 7 shows the tools, programs, and algorithms used to analyze polynucleotides and polypeptides, along with applicable descriptions, references, and threshold parameters.
  • NAAP refers to the amino acid sequences of substantially purified NAAP obtained from any species, particularly a mammalian species, including bovine, ovine, porcine, murine, equine, and human, and from any source, whether natural, synthetic, semi-synthetic, or recombinant.
  • agonist refers to a molecule which intensifies or mimics the biological activity of NAAP.
  • Agonists may include proteins, nucleic acids, carbohydrates, small molecules, or any other compound or composition which modulates the activity of NAAP either by directly interacting with NAAP or by acting on components of the biological pathway in which NAAP participates.
  • allelic variant is an alternative form of the gene encoding NAAP. Allelic variants may result from at least one mutation in the nucleic acid sequence and may result in altered mRNAs or in polypeptides whose structure or function may or may not be altered. A gene may have none, one, or many allelic variants of its naturally occurring form. Common mutational changes which give rise to allelic variants are generally ascribed to natural deletions, additions, or substitutions of nucleotides. Each of these types of changes may occur alone, or in combination with the others, one or more times in a given sequence.
  • altered nucleic acid sequences encoding NAAP include those sequences with deletions, insertions, or substitutions of different nucleotides, resulting in a polypeptide the same as NAAP or a polypeptide with at least one functional characteristic of NAAP. Included within this definition are polymorphisms which may or may not be readily detectable using a particular oligonucleotide probe of the polynucleotide encoding NAAP, and improper or unexpected hybridization to allelic variants, with a locus other than the normal chromosomal locus for the polynucleotide encoding NAAP.
  • the encoded protein may also be "altered,” and may contain deletions, insertions, or substitutions of amino acid residues which produce a silent change and result in a functionally equivalent NAAP.
  • Deliberate amino acid substitutions may be made on the basis of one or more similarities in polarity, charge, solubility, hydrophobicity, hydrophilicity, and/or the amphipathic nature of the residues, as long as the biological or immunological activity of NAAP is retained.
  • negatively charged amino acids may include aspartic acid and glutamic acid
  • positively charged amino acids may include lysine and arginine.
  • Amino acids with uncharged polar side chains having similar hydrophilicity values may include: asparagine and glutamine; and serine and threonine.
  • Amino acids with uncharged side chains having similar hydrophilicity values may include: leucine, isoleucine, and valine; glycine and alanine; and phenylalanine and tyrosine.
  • amino acid and amino acid sequence can refer to an oligopeptide, a peptide, a polypeptide, or a protein sequence, or a fragment of any of these, and to naturally occurring or synthetic molecules. Where "amino acid sequence” is recited to refer to a sequence of a naturally occurring protein molecule, “amino acid sequence” and like terms are not meant to limit the amino acid sequence to the complete native amino acid sequence associated with the recited protein molecule.
  • Amplification relates to the production of additional copies of a nucleic acid. Amplification may be carried out using polymerase chain reaction (PCR) technologies or other nucleic acid amplification technologies well known in the art.
  • PCR polymerase chain reaction
  • antagonists refers to a molecule which inhibits or attenuates the biological activity of NAAP. Antagonists may include proteins such as antibodies, anticalins, nucleic acids, carbohydrates, small molecules, or any other compound or composition which modulates the activity of NAAP either by directly interacting with NAAP or by acting on components of the biological pathway in which NAAP participates.
  • antibody refers to intact immunoglobulin molecules as well as to fragments thereof, such as Fab, F(ab') 2 , and Fv fragments, which are capable of binding an epitopic determinant.
  • Antibodies that bind NAAP polypeptides can be prepared using intact polypeptides or using fragments containing small peptides of interest as the immunizing antigen.
  • the polypeptide or oligopeptide used to immunize an animal e.g., a mouse, a rat, or a rabbit
  • an animal e.g., a mouse, a rat, or a rabbit
  • RNA e.g., a mouse, a rat, or a rabbit
  • antigenic determinant refers to that region of a molecule (i.e., an epitope) that makes contact with a particular antibody.
  • an antigenic determinant may compete with the intact antigen (i.e., the immunogen used to elicit the immune response) for binding to an antibody.
  • aptamer refers to a nucleic acid or oligonucleotide molecule that binds to a specific molecular target.
  • Aptamers are derived from an in vitro evolutionary process (e.g., SELEX (Systematic Evolution of Ligands by Exponential Enrichment), described in U.S. Patent No. 5,270,163), which selects for target-specific aptamer sequences from large combinatorial libraries.
  • Aptamer compositions may be double-stranded or single-stranded, and may include deoxyribonucleotides, ribonucleotides, nucleotide derivatives, or other nucleotide-like molecules.
  • the nucleotide components of an aptamer may have modified sugar groups (e.g., the 2'-OH group of a ribonucleotide may be replaced by 2'-F or 2'-NH 2 ), which may improve a desired property, e.g., resistance to nucleases or longer lifetime in blood.
  • Aptamers may be conjugated to other molecules, e.g., a high molecular weight carrier to slow clearance of the aptamer from the circulatory system.
  • Aptamers may be specifically cross-linked to their cognate ligands, e.g., by photo-activation of a cross-linker (Brody, E.N. andL. Gold (2000) J. Biotechnol. 74:5-13).
  • introduction refers to an aptamer which is expressed in vivo.
  • 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).
  • spiegelmer refers to an aptamer which includes L-DNA, L-RNA, or other left- handed nucleotide derivatives or nucleotide-like molecules. Aptamers containing left-handed nucleotides are resistant to degradation by naturally occurring enzymes, which normally act on substrates containing right-handed nucleotides.
  • antisense refers to any composition capable of base-pairing with the "sense" (coding) strand of a polynucleotide having a specific nucleic acid sequence.
  • Antisense compositions may include DNA; RNA; peptide nucleic acid (PNA); oligonucleotides having modified backbone linkages such as phosphorothioates, methylphosphonates, or benzylphosphonates; oligonucleotides having modified sugar groups such as 2'-methoxyethyl sugars or 2'-methoxyethoxy sugars; or oligonucleotides having modified bases such as 5-methyl cytosine, 2'-deoxyuracil, or 7-deaza-2'- deoxyguanosine.
  • Antisense molecules may be produced by any method including chemical synthesis or transcription.
  • the complementary antisense molecule base-pairs with a naturally occurring nucleic acid sequence produced by the cell to form duplexes which block either transcription or translation.
  • the designation "negative” or “minus” can refer to the antisense strand, and the designation “positive” or “plus” can refer to the sense strand of a reference DNA molecule.
  • biologically active refers to a protein having structural, regulatory, or biochemical functions of a naturally occurring molecule.
  • immunologically active or “immunogenic” refers to the capability of the natural, recombinant, or synthetic NAAP, or of any oligopeptide thereof, to induce a specific immune response in appropriate animals or cells and to bind with specific antibodies.
  • compositions comprising a given polynucleotide and a “composition comprising a given polypeptide” can refer to any composition containing the given polynucleotide or polypeptide.
  • the composition may comprise a dry formulation or an aqueous solution.
  • Compositions comprising polynucleotides encoding NAAP or fragments of NAAP may be employed as hybridization probes. The probes may be stored in freeze-dried form and may be associated with a stabilizing agent such as a carbohydrate.
  • the probe may be deployed in an aqueous solution containing salts (e.g., NaCl), detergents (e.g., sodium dodecyl sulfate; SDS), and other components (e.g., Denhardt's solution, dry milk, salmon sperm DNA, etc.).
  • salts e.g., NaCl
  • detergents e.g., sodium dodecyl sulfate; SDS
  • other components e.g., Denhardt's solution, dry milk, salmon sperm DNA, etc.
  • Consensus sequence refers to a nucleic acid sequence which has been subjected to repeated DNA sequence analysis to resolve uncalled bases, extended using the XL-PCR kit (Applied Biosystems, Foster City CA) in the 5' and or the 3' direction, and resequenced, or which has been assembled from one or more overlapping cDNA, EST, or genomic DNA fragments using a computer program for fragment assembly, such as the GELVEEW fragment assembly system (Accelrys, Burlington MA) or Phrap (University of Washington, Seattle WA). Some sequences have been both extended and assembled to produce the consensus sequence.
  • Constant amino acid substitutions are those substitutions that are predicted to least interfere with the properties of the original protein, i.e., the structure and especially the function of the protein is conserved and not significantly changed by such substitutions.
  • the table below shows amino acids which may be substituted for an original amino acid in a protein and which are regarded as conservative amino acid substitutions.
  • Conservative amino acid substitutions generally maintain (a) the structure of the polypeptide backbone in the area of the substitution, for example, as a beta sheet or alpha helical conformation, (b) the charge or hydrophobicity of the molecule at the site of the substitution, and/or (c) the bulk of the side chain.
  • a “deletion” refers to a change in the amino acid or nucleotide sequence that results in the absence of one or more amino acid residues or nucleotides.
  • derivative refers to a chemically modified polynucleotide or polypeptide. Chemical modifications of a polynucleotide can include, for example, replacement of hydrogen by an alkyl, acyl, hydroxyl, or amino group.
  • a derivative polynucleotide encodes a polypeptide which retains at least one biological or immunological function of the natural molecule.
  • a derivative polypeptide is one modified by glycosylation, pegylation, or any similar process that retains at least one biological or immunological function of the polypeptide from which it was derived.
  • a “detectable label” refers to a reporter molecule or enzyme that is capable of generating a measurable signal and is covalently or noncovalently joined to a polynucleotide or polypeptide.
  • “Differential expression” refers to increased or upregulated; or decreased, downregulated, or absent gene or protein expression, determined by comparing at least two different samples. Such comparisons may be carried out between, for example, a treated and an untreated sample, or a diseased and a normal sample.
  • Exon shuffling refers to the recombination of different coding regions (exons). Since an exon may represent a structural or functional domain of the encoded protein, new proteins may be assembled through the novel reassortment of stable substructures, thus allowing acceleration of the evolution of new protein functions.
  • a “fragment” is a unique portion of NAAP or a polynucleotide encoding NAAP which can be identical in sequence to, but shorter in length than, the parent sequence.
  • a fragment may comprise up to the entire length of the defined sequence, minus one nucleotide/amino acid residue.
  • a fragment may comprise from about 5 to about 1000 contiguous nucleotides or amino acid residues.
  • a fragment used as a probe, primer, antigen, therapeutic molecule, or for other purposes may be at least 5, 10, 15, 16, 20, 25, 30, 40, 50, 60, 75, 100, 150, 250 or at least 500 contiguous nucleotides or amino acid residues in length. Fragments may be preferentially selected from certain regions of a molecule.
  • 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.
  • a fragment of SEQ ID NO:57-l 12 can comprise a region of unique polynucleotide sequence that specifically identifies SEQ ID NO:57-112, for example, as distinct from any other sequence in the genome from which the fragment was obtained.
  • a fragment of SEQ ID NO:57-l 12 can be employed in one or more embodiments of methods of the invention, for example, in hybridization and amplification technologies and in analogous methods that distinguish SEQ ED NO:57-112 from related polynucleotides.
  • the precise length of a fragment of SEQ ED NO:57-l 12 and the region of SEQ ID NO:57-112 to which the fragment corresponds are routinely determinable by one of ordinary skill in the art based on the intended purpose for the fragment.
  • a fragment of SEQ ID NO: 1-56 is encoded by a fragment of SEQ ED NO:57-l 12.
  • a fragment of SEQ ID NO: 1-56 can comprise a region of unique amino acid sequence that specifically identifies SEQ ED NO: 1-56.
  • a fragment of SEQ ID NO: 1-56 can be used as an immunogenic peptide for the development of antibodies that specifically recognize SEQ ID NO: 1-56.
  • the precise length of a fragment of SEQ ID NO: 1-56 and the region of SEQ ID NO: 1-56 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 “full length” polynucleotide is one containing at least a translation initiation codon (e.g., methionine) followed by an open reading frame and a translation termination codon.
  • a “full length” polynucleotide sequence encodes a "full length” polypeptide sequence.
  • Homology refers to sequence similarity or, alternatively, sequence identity, between two or more polynucleotide sequences or two or more polypeptide sequences.
  • percent identity and % identity refer to the percentage of identical residue matches between at least two polynucleotide sequences aligned using a standardized algorithm. Such an algorithm may insert, in a standardized and reproducible way, gaps in the sequences being compared in order to optimize alignment between two sequences, and therefore achieve a more meaningful comparison of the two sequences.
  • Percent identity between polynucleotide sequences may be determined using one or more computer algorithms or programs known in the art or described herein. For example, percent identity can be determined using the default parameters of the CLUSTAL V algorithm as incorporated into the MEGALIGN version 3.12e sequence alignment program. This program is part of the LASERGENE software package, a suite of molecular biological analysis programs (DNASTAR, Madison 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).
  • the BLAST software suite includes various sequence analysis programs including "blastn,” that is used to align a known polynucleotide sequence with other polynucleotide sequences from a variety of databases. Also available is a tool called “BLAST 2 Sequences” that is used for direct pairwise comparison of two nucleotide sequences. "BLAST 2 Sequences” can be accessed and used interactively at http://www.ncbi.nlm.nih.gov/gorf/bl2.html. The "BLAST 2 Sequences” tool can be used for both blastn and blastp (discussed below). BLAST programs are commonly used with gap and other parameters set to default settings.
  • BLAST 2 Sequences 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
  • Percent identity may be measured over the length of an entire defined sequence, for example, as defined by a particular SEQ ID number, or may be measured over a shorter length, for example, over the length of a fragment taken from a larger, defined sequence, for instance, a fragment of at least 20, at least 30, at least 40, at least 50, at least 70, at least 100, or at least 200 contiguous nucleotides.
  • Such lengths are exemplary only, and it is understood that any fragment length supported by the sequences shown herein, in the tables, figures, or Sequence Listing, may be used to describe a length over which percentage identity may be measured.
  • nucleic acid sequences that do not show a high degree of identity may nevertheless encode similar amino acid sequences due to the degeneracy of the genetic code. It is understood that changes in a nucleic acid sequence can be made using this degeneracy to produce multiple nucleic acid sequences that all encode substantially the same protein.
  • percent identity and % identity refer to the percentage of identical residue matches between at least two polypeptide sequences aligned using a standardized algorithm.
  • Methods of polypeptide sequence alignment are well-known. Some alignment methods take into account conservative amino acid substitutions. Such conservative substitutions, explained in more detail above, generally preserve the charge and hydrophobicity at the site of substitution, thus preserving the structure (and therefore function) of the polypeptide.
  • percent similarity and % similarity refer to the percentage of residue matches, including identical residue matches and conservative substitutions, between at least two polypeptide sequences aligned using a standardized algorithm. In contrast, conservative substitutions are not included in the calculation of percent identity between polypeptide sequences.
  • the PAM250 matrix is selected as the default residue weight table.
  • 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) with blastp set at default parameters.
  • Such default parameters may be, for example: Matrix: BLOSUM62
  • Percent identity may be measured over the length of an entire defined polypeptide sequence, for example, as defined by a particular SEQ ID number, or may be measured over a shorter length, for example, over the length of a fragment taken from a larger, defined polypeptide sequence, for instance, a fragment of at least 15, at least 20, at least 30, at least 40, at least 50, at least 70 or at least 150 contiguous residues.
  • Such lengths are exemplary only, and it is understood that any fragment length supported by the sequences shown herein, in the tables, figures or Sequence Listing, may be used to describe a length over which percentage identity may be measured.
  • HACs Human artificial chromosomes
  • HACs are linear microchromosomes which may contain DNA sequences of about 6 kb to 10 Mb in size and which contain all of the elements required for chromosome replication, segregation and maintenance.
  • humanized antibody refers to an antibody molecule in which the amino acid sequence in the non-antigen binding regions has been altered so that the antibody more closely resembles a human antibody, and still retains its original binding ability.
  • Hybridization refers to the process by which a polynucleotide strand anneals with a complementary strand through base pairing under defined hybridization conditions. Specific hybridization is an indication that two nucleic acid sequences share a high degree of complementarity. Specific hybridization complexes form under permissive annealing conditions and remain hybridized after the "washing" step(s). The washing step(s) is particularly important in determining the stringency of the hybridization process, with more stringent conditions allowing less non-specific binding, i.e., binding between pairs of nucleic acid strands that are not perfectly matched.
  • Permissive conditions for annealing of nucleic acid sequences are routinely determinable by one of ordinary skill in the art and may be consistent among hybridization experiments, whereas wash conditions may be varied among experiments to achieve the desired stringency, and therefore hybridization specificity. Permissive annealing conditions occur, for example, at 68°C in the presence of about 6 x SSC, about 1% (w/v) SDS, and about 100 ⁇ g/ml sheared, denatured salmon sperm DNA.
  • wash temperatures are typically selected to be about 5°C to 20°C lower than the thermal melting point (T m ) for the specific sequence at a defined ionic strength and pH.
  • T m is the temperature (under defined ionic strength and pH) at which 50% of the target sequence hybridizes to a perfectly matched probe.
  • 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%.
  • blocking reagents are used to block non-specific hybridization. Such blocking reagents include, for instance, sheared and denatured salmon sperm DNA at about 100-200 ⁇ g/ml.
  • Organic solvent such as formamide at a concentration of about 35-50% v/v
  • RNA:DNA hybridizations Useful variations on these wash conditions will be readily apparent to those of ordinary skill in the art.
  • Hybridization particularly under high stringency conditions, may be suggestive of evolutionary similarity between the nucleotides. Such similarity is strongly indicative of a similar role for the nucleotides and their encoded polypeptides.
  • 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., C 0 t or Rgt analysis) or formed between one nucleic acid present in solution and another nucleic acid immobilized on a solid support (e.g., paper, membranes, filters, chips, pins or glass slides, or any other appropriate substrate to which cells or their nucleic acids have been fixed).
  • a solid support e.g., paper, membranes, filters, chips, pins or glass slides, or any other appropriate substrate to which cells or their nucleic acids have been fixed.
  • 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.
  • Immuno response can refer to conditions associated with inflammation, trauma, immune disorders, or infectious or genetic disease, etc. These conditions can be characterized by expression of various factors, e.g., cytokines, chemokines, and other signaling molecules, which may affect cellular and systemic defense systems.
  • an “immunogenic fragment” is a polypeptide or oligopeptide fragment of NAAP which is capable of eliciting an immune response when introduced into a living organism, for example, a mammal.
  • the term “immunogenic fragment” also includes any polypeptide or oligopeptide fragment of NAAP which is useful in any of the antibody production methods disclosed herein or known in the art.
  • microarray refers to an arrangement of a plurality of polynucleotides, polypeptides, antibodies, or other chemical compounds on a substrate.
  • array element refers to a polynucleotide, polypeptide, antibody, or other chemical compound having a unique and defined position on a microarray.
  • 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.
  • nucleic acid and nucleic acid sequence refer to a nucleotide, oligonucleotide, polynucleotide, or any fragment thereof. These phrases also refer to DNA or RNA of genomic or synthetic origin which may be single-stranded or double-stranded and may represent the sense or the antisense strand, to peptide nucleic acid (PNA), or to any DNA-like or RNA-like material.
  • PNA peptide nucleic acid
  • 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.
  • a promoter is operably linked to a coding sequence if the promoter affects the transcription or expression of the coding sequence.
  • Operably linked DNA sequences may be in close proximity or contiguous and, where necessary to join two protein coding regions, in the same reading frame.
  • PNA protein nucleic acid
  • PNA refers to an antisense molecule or anti-gene agent which comprises an oligonucleotide of at least about 5 nucleotides in length linked to a peptide backbone of amino acid residues ending in lysine. The terminal lysine confers solubility to the composition. PNAs preferentially bind complementary single stranded DNA or RNA and stop transcript elongation, and may be pegylated to extend their lifespan in the cell.
  • Probe refers to nucleic acids encoding NAAP, their complements, or fragments thereof, which are used to detect identical, allelic or related nucleic acids. Probes are isolated oligonucleotides or polynucleotides attached to a detectable label or reporter molecule. Typical labels include radioactive isotopes, ligands, chemiluminescent agents, and enzymes.
  • Primer pairs are short nucleic acids, usually DNA oligonucleotides, which may be annealed to a target polynucleotide by complementary base-pairing. The primer may then be extended along the target DNA strand by a DNA polymerase enzyme. Primer pairs can be used for amplification (and identification) of a nucleic acid, e.g., by the polymerase chain reaction (PCR).
  • PCR polymerase chain reaction
  • Probes and primers as used in the present invention typically comprise at least 15 contiguous nucleotides of a known sequence. In order to enhance specificity, longer probes and primers may also be employed, such as probes and primers that comprise at least 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, or at least 150 consecutive nucleotides of the disclosed nucleic acid sequences. Probes and primers may be considerably longer than these examples, and it is understood that any length supported by the specification, including the tables, figures, and Sequence Listing, may be used. Methods for preparing and using probes and primers are described in, for example,
  • 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).
  • Oligonucleotides for use as primers are selected using software known in the art for such purpose. For example, OLIGO 4.06 software is useful for the selection of PCR primer pairs of up to 100 nucleotides each, and for the analysis of oligonucleotides and larger polynucleotides of up to 5,000 nucleotides from an input polynucleotide sequence of up to 32 kilobases. Similar primer selection programs have incorporated additional features for expanded capabilities. For example, the PrimOU primer selection program (available to the public from the Genome Center at University of Texas South West Medical Center, Dallas TX) is capable of choosing specific primers from megabase sequences and is thus useful for designing primers on a genome-wide scope.
  • the Primer3 primer selection program (available to the public from the Whitehead Institute/MIT Center for Genome Research, Cambridge MA) allows the user to input a "mispriming library," in which sequences to avoid as primer binding sites are user-specified. Primer3 is useful, in particular, for the selection of oligonucleotides for microarrays. (The source code for the latter two primer selection programs may also be obtained from their respective sources and modified to meet the user's specific needs.)
  • the PrimeGen program (available to the public from the UK Human Genome Mapping Project Resource Centre, Cambridge UK) designs primers based on multiple sequence alignments, thereby allowing selection of primers that hybridize to either the most conserved or least conserved regions of aligned nucleic acid sequences.
  • this program is useful for identification of both unique and conserved oligonucleotides and polynucleotide fragments.
  • the oligonucleotides and polynucleotide fragments identified by any of the above selection methods are useful in hybridization technologies, for example, as PCR or sequencing primers, microarray elements, or specific probes to identify fully or partially complementary polynucleotides in a sample of nucleic acids. Methods of oligonucleotide selection are not limited to those described above.
  • a "recombinant nucleic acid” is a nucleic acid that is not naturally occurring or has a sequence that is made by an artificial combination of two or more otherwise separated segments of sequence. This artificial combination is often accomplished by chemical synthesis or, more commonly, by the artificial manipulation of isolated segments of nucleic acids, e.g., by genetic engineering techniques such as those described in Sambrook and Russell (supra).
  • the term recombinant includes nucleic acids that have been altered solely by addition, substitution, or deletion of a portion of the nucleic acid.
  • a recombinant nucleic acid may include a nucleic acid sequence operably linked to a promoter sequence.
  • Such a recombinant nucleic acid may be part of a vector that is used, for example, to transform a cell.
  • such recombinant nucleic acids may be part of a viral vector, e.g., based on a vaccinia virus, that could be use to vaccinate a mammal wherein the recombinant nucleic acid is expressed, inducing a protective immunological response in the mammal.
  • a “regulatory element” refers to a nucleic acid sequence usually derived from untranslated regions of a gene and includes enhancers, promoters, introns, and 5' and 3' untranslated regions (UTRs). Regulatory elements interact with host or viral proteins which control transcription, translation, or RNA stability.
  • Reporter molecules are chemical or biochemical moieties used for labeling a nucleic acid, amino acid, or antibody. Reporter molecules include radionuclides; enzymes; fluorescent, chemiluminescent, or chromogenic agents; substrates; cofactors; inhibitors; magnetic particles; and other moieties known in the art.
  • RNA equivalent in reference to a DNA molecule, is composed of the same linear sequence of nucleotides as the reference DNA molecule with the exception that all occurrences of the nitrogenous base thymine are replaced with uracil, and the sugar backbone is composed of ribose instead of deoxyribose.
  • sample is used in its broadest sense.
  • a sample suspected of containing NAAP, nucleic acids encoding NAAP, or fragments thereof may comprise a bodily fluid; an extract from a cell, chromosome, organelle, or membrane isolated from a cell; a cell; genomic DNA, RNA, or cDNA, in solution or bound to a substrate; a tissue; a tissue print; etc.
  • binding and “specifically binding” refer to that interaction between a protein or peptide and an agonist, an antibody, an antagonist, a small molecule, or any natural or synthetic binding composition. The interaction is dependent upon the presence of a particular structure of the protein, e.g., the antigenic determinant or epitope, recognized by the binding molecule. For example, if an antibody is specific for epitope "A,” the presence of a polypeptide comprising the epitope A, or the presence of free unlabeled A, in a reaction containing free labeled A and the antibody will reduce the amount of labeled A that binds to the antibody.
  • substantially purified refers to nucleic acid or amino acid sequences that are removed from their natural environment and are isolated or separated, and are at least about 60% free, preferably at least about 75% free, and most preferably at least about 90% free from other components with which they are naturally associated.
  • substitution refers to the replacement of one or more amino acid residues or nucleotides by different amino acid residues or nucleotides, respectively.
  • Substrate refers to any suitable rigid or semi-rigid support including membranes, filters, chips, slides, wafers, fibers, magnetic or nonmagnetic beads, gels, tubing, plates, polymers, microparticles and capillaries.
  • the substrate can have a variety of surface forms, such as wells, trenches, pins, channels and pores, to which polynucleotides or polypeptides are bound.
  • a “transcript image” or “expression profile” refers to the collective pattern of gene expression by a particular cell type or tissue under given conditions at a given time.
  • Transformation describes a process by which exogenous DNA is introduced into a recipient cell. Transformation may occur under natural or artificial conditions according to various methods well known in the art, and may rely on any known method for the insertion of foreign nucleic acid sequences into a prokaryotic or eukaryotic host cell. The method for transformation is selected based on the type of host cell being transformed and may include, but is not limited to, bacteriophage or viral infection, electroporation, heat shock, lipofection, and particle bombardment.
  • transformed cells includes stably transformed cells in which the inserted DNA is capable of replication either as an autonomously replicating plasmid or as part of the host chromosome, as well as transiently transformed cells which express the inserted DNA or RNA for limited periods of time.
  • a "transgenic organism,” as used herein, is any organism, including but not limited to animals and plants, in which one or more of the cells of the organism contains heterologous nucleic acid introduced by way of human intervention, such as by transgenic techniques well known in the art.
  • the nucleic acid is introduced into the cell, directly or indirectly by introduction into a precursor of the cell, by way of deliberate genetic manipulation, such as by microinjection or by infection with a recombinant virus.
  • the 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 fertilization, but rather is directed to the introduction of a recombinant DNA molecule.
  • the transgenic organisms contemplated in accordance with the present invention include bacteria, cyanobacteria, fungi, plants and animals.
  • the isolated DNA of the present invention can be introduced into the host by methods known in the art, for example infection, transfection, transformation or transconjugation. Techniques for transferring the DNA of the present invention into such organisms are widely known and provided in references such as Sambrook and Russell (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 "allelic” (as defined above), “splice,” “species,” or “polymorphic” variant.
  • a splice variant may have significant identity to a reference molecule, but will generally have a greater or lesser number of polynucleotides due to alternate splicing of exons during mRNA processing.
  • the corresponding polypeptide may possess additional functional domains or lack domains that are present in the reference molecule.
  • Species variants are polynucleotides that vary from one species to another. The resulting polypeptides will generally have significant amino acid identity relative to each other.
  • a polymorphic variant is a variation in the polynucleotide sequence of a particular gene between individuals of a given species.
  • Polymorphic variants also may encompass "single nucleotide polymorphisms" (SNPs) in which the polynucleotide sequence varies by one nucleotide base.
  • SNPs single nucleotide polymorphisms
  • 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.
  • NAAP nucleic acid-associated proteins
  • Table 1 summarizes the nomenclature for the full length polynucleotide and polypeptide embodiments of the invention. Each polynucleotide and its corresponding polypeptide are correlated to a single Incyte project identification number (Incyte Project ED). Each polypeptide sequence is denoted by both a polypeptide sequence identification number (Polypeptide SEQ ED 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 ID NO:) and an Incyte polynucleotide consensus sequence number (Incyte Polynucleotide JO) as shown.
  • Column 6 shows the Incyte ID numbers of physical, full length clones corresponding to the polypeptide and polynucleotide sequences of the invention. The full 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 the polypeptides of the invention as identified by BLAST analysis against the GenBank protein (genpept) database and the PROTEOME database.
  • Columns 1 and 2 show the polypeptide sequence identification number (Polypeptide SEQ ID NO:) and the corresponding Incyte polypeptide sequence number (Incyte Polypeptide ID) for polypeptides of the invention.
  • Column 3 shows the GenBank identification number (GenBank ID NO:) of the nearest GenBank homolog and the PROTEOME database identification numbers (PROTEOME ID NO:) of the nearest PROTEOME database homologs.
  • Column 4 shows the probability scores for the matches between each polypeptide and its homolog(s).
  • Column 5 shows the annotation of the GenBank and PROTEOME database homolog(s) along with relevant citations where applicable, all of which are expressly incorporated by reference herein.
  • Table 3 shows various structural features of the polypeptides of the invention.
  • Columns 1 and 2 show the polypeptide sequence identification number (SEQ ID NO:) and the corresponding Incyte polypeptide sequence number (Incyte Polypeptide ID) for each polypeptide of the invention.
  • Column 3 shows the number of amino acid residues in each polypeptide.
  • Column 4 shows potential phosphorylation sites, and column 5 shows potential glycosylation sites, as determined by the MOTIFS program of the GCG sequence analysis software package (Accelrys, Burlington 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 applied.
  • SEQ ED NO:6 is 58% identical, from residue L8 to residue H324, to human repressor transcription factor (GenBank ED gl017722) as determined by the Basic Local Alignment Search Tool (BLAST). (See Table 2.) The BLAST probability score is 3.4e-177, which indicates the probability of obtaining the observed polypeptide sequence alignment by chance.
  • SEQ ED NO: 6 also contains a neutral zinc maetllopeptidases, zinc-binding region signature and a zinc finger C2H2-type domain as determined by searching for statistically significant matches in the hidden Markov model (HMM)-based PFAM database of conserved protein family domains. (See Table 3.) Data from MOTIFS analyses and
  • SEQ ID NO: 17 is 70% identical, from residue Ml to residue G149, to Orvza sativa OsGRP2 (glycine-rich RNA-binding protein 2) (GenBank ID g2624328) as determined by the Basic Local Alignment Search Tool (BLAST). (See Table 2.)
  • the BLAST probability score is 3.6e-54, which indicates the probability of obtaining the observed polypeptide sequence alignment by chance.
  • SEQ ID NO: 17 also contains an RNA recognition motif domain as determined by searching for statistically significant matches in the hidden Markov model (HMM)-based PFAM database of conserved protein family domains.
  • HMM hidden Markov model
  • SEQ ID NO: 17 is an RNA-binding protein.
  • SEQ ED NO:21 is 44% identical, from residue Q5 to residue L246, to Kruppel-related protein ha0946 (GenBank ED g498152) as determined by the Basic Local Alignment Search Tool (BLAST). (See Table 2.) The BLAST probability score is 4.4e-49, which indicates the probability of obtaining the observed polypeptide sequence alignment by chance.
  • SEQ ID NO:21 also contains a KRAB box domain as determined by searching for statistically significant matches in the hidden Markov model (HMM)- based PFAM database of conserved protein family domains.
  • HMM hidden Markov model
  • SEQ ID NO:21 is a zinc finger protein.
  • SEQ ED NO:29 is 76% identical, from residue L287 to residue V422, and 71% identical, from residue Ml to residue K121, to human RP58 (GenBank ED g3005948) as determined by the Basic Local Alignment Search Tool (BLAST).
  • BLAST probability score is 1.3e-104, which indicates the probability of obtaining the observed polypeptide sequence alignment by chance.
  • SEQ ID NO:29 also contains a BTB/POZ domain and a C2H2-type zinc finger domain as determined by searching for statistically significant matches in the hidden Markov model (HMM)-based PFAM database of conserved protein family domains.
  • HMM hidden Markov model
  • SEQ ID NO:29 is a zinc finger protein, hi an alternative example, SEQ ED NO:37 is 84% identical, from residue A29 to residue L485, to human makorin 1 (GenBank ID g6601434) as determined by the Basic Local Alignment Search Tool (BLAST).
  • the BLAST probability score is 5.3E-213, which indicates the probability of obtaining the observed polypeptide sequence alignment by chance.
  • SEQ ED NO: 37 also contains a C3H4 type zinc finger domain as determined by searching for statistically significant matches in the hidden Markov model (HMM)-based PFAM database of conserved protein family domains. (See Table 3.) Data from BLIMPS, MOTH'S, and PROFELESCAN analyses provide further corroborative evidence that SEQ ED NO:37 is a zinc finger protein.
  • SEQ ID NO:51 is 97% identical, from residue Ml to residue 1383, to kruppel-like protein, HPF1 (GenBank ID gll917511) as determined by the Basic Local Alignment Search Tool (BLAST).
  • SEQ ID NO:51 is localized to the nucleus, binds DNA, and is a KRAB family zinc finger protein, as determined by BLAST analysis using the PROTEOME database. SEQ ID NO: 51 also contains a zinc finger, C2H2 type domain as determined by searching for statistically significant matches in the hidden Markov model (HMM)-based PFAM database of conserved protein family domains. (See Table 3.) Data from BLIMPS, MOTIFS, and other BLAST analyses provide further corroborative evidence that SEQ ID NO:51 is a KRAB family zinc finger protein.
  • SEQ ED NO: 1-5 SEQ ID NO:7-16, SEQ ID NO: 18- 20, SEQ ED NO:22-28, SEQ ID NO:30-36, SEQ ID NO:38-50, and SEQ H) NO:52-56 were analyzed and annotated in a similar manner.
  • the algorithms and parameters for the analysis of SEQ ID NO: 1-56 are described in Table 7.
  • Polynucleotide SEQ ID NO: the corresponding Incyte polynucleotide consensus sequence number (Incyte ID) for each polynucleotide of the invention, and the length of each polynucleotide sequence in basepairs.
  • Column 2 shows the nucleotide start (5') and stop (3') positions of the cDNA and/or genomic sequences used to assemble the full length polynucleotide embodiments, and of fragments of the polynucleotides which are useful, for example, in hybridization or amplification technologies that identify SEQ ID NO:57-l 12 or that distinguish between SEQ ID NO:57-l 12 and related polynucleotides.
  • the polynucleotide fragments described in Column 2 of Table 4 may refer specifically, for example, to Incyte cDNAs derived from tissue-specific cDNA libraries or from pooled cDNA libraries.
  • the polynucleotide fragments described in column 2 may refer to GenBank cDNAs or ESTs which contributed to the assembly of the full length polynucleotides.
  • 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").
  • the polynucleotide fragments described in column 2 may be derived from the NCBI RefSeq Nucleotide Sequence Records Database (i.e., those sequences including the designation "NM” or “NT”) or the NCBI RefSeq Protein Sequence Records (i.e., those sequences including the designation "NP”).
  • 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
  • ⁇ L___XXXXXX_N 1 _N 2 _YYYY_N 3 _1S 4 represents a "stitched" sequence in which XXXXX is the identification number of the cluster of sequences to which the algorithm was applied, and YYYYY is the number of the prediction generated by the algorithm, and N i 2 ⁇ 3 , if present, represent specific exons that may have been manually edited during analysis (See Example V).
  • the polynucleotide fragments in column 2 may refer to assemblages of exons brought together by an "exon-stretching" algorithm.
  • a polynucleotide sequence identified as FLXXXXX_gAAAAA_gBBBBB_l_N is a "stretched" sequence, with XXXXX being the Incyte project identification number, gAAAAA being the GenBank identification number of the human genomic sequence to which the "exon-stretching" algorithm was applied, gBBBBB being the GenBank identification number or ⁇ CBI RefSeq identification number of the nearest GenBank protein homolog, and N referring to specific exons (See Example V).
  • a RefSeq identifier (denoted by " ⁇ M,” “ ⁇ P,” or “NT”) may be used in place of the GenBank identifier (i.e., gBBBBB).
  • a prefix identifies component sequences that were hand-edited, predicted from genomic DNA sequences, or derived from a combination of sequence analysis methods. The following Table lists examples of component sequence prefixes and corresponding sequence analysis methods associated with the prefixes (see Example LV and Example V).
  • Incyte cDNA coverage redundant with the sequence coverage shown in Table 4 was obtained to confirm the final consensus polynucleotide sequence, but the relevant Incyte cDNA identification numbers are not shown.
  • Table 5 shows the representative cDNA libraries for those full length polynucleotides which were assembled using Incyte cDNA sequences.
  • the representative cDNA library is the Incyte cDNA library which is most frequently represented by the Incyte cDNA sequences which were used to assemble and confirm the above polynucleotides.
  • the tissues and vectors which were used to construct the cDNA libraries shown in Table 5 are described in Table 6.
  • 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.
  • polynucleotides which encode NAAP encompasses a polynucleotide sequence comprising a sequence selected from the group consisting of SEQ ID NO:57-l 12, which encodes NAAP.
  • the polynucleotide sequences of SEQ ED NO:57-l 12, 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.
  • a variant polynucleotide will 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 ID NO:57-l 12 which has at least about 70%, or alternatively at least about 85%, or even at least about 95% polynucleotide sequence identity to a nucleic acid sequence selected from the group consisting of SEQ ID NO:57-l 12.
  • Any one of the polynucleotide variants described above can encode a polypeptide which contains at least one functional or structural characteristic of NAAP.
  • a polynucleotide variant of the invention is a splice variant of a polynucleotide encoding NAAP.
  • a splice variant may have portions which have significant sequence identity to a polynucleotide encoding NAAP, but will generally have a greater or lesser number of polynucleotides due to additions or deletions of blocks of sequence arising from alternate splicing of exons during mRNA processing.
  • a splice variant may have less than about 70%, or alternatively less than about 60%, or alternatively less than about 50% polynucleotide sequence identity to a polynucleotide encoding NAAP over its entire length; however, portions of the splice variant will have at least about 70%, or alternatively at least about 85%, or alternatively at least about 95%, or alternatively 100% polynucleotide sequence identity to portions of the polynucleotide encoding NAAP.
  • a polynucleotide comprising a sequence of SEQ ID NO: 110, a polynucleotide comprising a sequence of SEQ ID NO: 111, and a polynucleotide comprising a sequence of SEQ ID NO:62 are splice variants of each other; and a polynucleotide comprising a sequence of SEQ ID NO:67 and a polynucleotide comprising a sequence of SEQ TD NO:68 are splice variants of each other.
  • Any one of the splice variants described above can encode a polypeptide which contains at least one functional or structural characteristic of NAAP.
  • polynucleotides which encode NAAP and its variants are generally capable of hybridizing to polynucleotides encoding naturally occurring NAAP under appropriately selected conditions of stringency, it may be advantageous to produce polynucleotides encoding NAAP or its derivatives possessing a substantially different codon usage, e.g., inclusion of non-naturally occurring codons. Codons may be selected to increase the rate at which expression of the peptide occurs in a particular prokaryotic or eukaryotic host in accordance with the frequency with which particular codons are utilized by the host.
  • RNA transcripts having more desirable properties such as a greater half-life, than transcripts produced from the naturally occurring sequence.
  • the invention also encompasses production of polynucleotides which encode NAAP and NAAP derivatives, or fragments thereof, entirely by synthetic chemistry.
  • the synthetic polynucleotide may be inserted into any of the many available expression vectors and cell systems using reagents well known in the art.
  • synthetic chemistry may be used to introduce mutations into a polynucleotide encoding NAAP or any fragment thereof.
  • Embodiments of the invention can also include polynucleotides that are capable of hybridizing to the claimed polynucleotides, and, in particular, to those having the sequences shown in SEQ ID NO:57-l 12 and fragments thereof, under various conditions of stringency (Wahl, G.M. and S.L. Berger (1987) Methods Enzymol. 152:399-407; Kimmel, A.R. (1987) Methods Enzymol. 152:507-511). Hybridization conditions, including annealing and wash conditions, are described in "Definitions.”
  • Methods for DNA sequencing are well known in the art and may be used to practice any of the embodiments of the invention.
  • the methods may employ such enzymes as the Klenow fragment of DNA polymerase I, SEQUENASE (US Biochemical, Cleveland OH), Taq polymerase (Applied Biosystems), thermostable T7 polymerase (Amersham Biosciences, Piscataway NJ), or combinations of polymerases and proofreading exonucleases such as those found in the ELONGASE amplification system (Invitrogen, Carlsbad CA).
  • sequence preparation is automated with machines such as the MICROLAB 2200 liquid transfer system (Hamilton, Reno NV), PTC200 thermal cycler (MJ Research, Watertown MA) and ABI CATALYST 800 thermal cycler (Applied Biosystems). Sequencing is then carried out using either the ABI 373 or 377 DNA sequencing system (Applied Biosystems), the MEGABACE 1000 DNA sequencing system (Amersham Biosciences), or other systems known in the art. The resulting sequences are analyzed using a variety of algorithms which are well known in the art (Ausubel et al., supra, ch. 7; Meyers, R.A. (1995) Molecular Biologv and Biotechnology. Wiley VCH, New York NY, pp. 856-853).
  • machines such as the MICROLAB 2200 liquid transfer system (Hamilton, Reno NV), PTC200 thermal cycler (MJ Research, Watertown MA) and ABI CATALYST 800 thermal cycler (Applied Biosystems). Sequencing is then carried out using either the ABI 373
  • the nucleic acids encoding NAAP may be extended utilizing a partial nucleotide sequence and employing various PCR-based methods known in the art to detect upstream sequences, such as promoters and regulatory elements.
  • various PCR-based methods known in the art to detect upstream sequences, such as promoters and regulatory elements.
  • restriction-site PCR uses universal and nested primers to amplify unknown sequence from genomic DNA within a cloning vector (Sarkar, G. (1993) PCR Methods Applic. 2:318-322).
  • Another method, inverse PCR uses primers that extend in divergent directions to amplify unknown sequence from a circularized template.
  • the template is derived from restriction fragments comprising a known genomic locus and surrounding sequences (Triglia, T. et al.
  • a third method involves PCR amplification of DNA fragments adjacent to known sequences in human and yeast artificial chromosome DNA (Lagerstrom, M. et al. (1991) PCR Methods Applic. 1:111-119).
  • multiple restriction enzyme digestions and ligations may be used to insert an engineered double-stranded sequence into a region of unknown sequence before performing PCR.
  • Other methods which may be used to retrieve unknown sequences are known in the art (Parker, J.D. et al. (1991) Nucleic Acids Res. 19:3055-3060).
  • primers may be designed using commercially available software, such as OLIGO 4.06 primer analysis software (National Biosciences, Plymouth MN) or another appropriate program, to be about 22 to 30 nucleotides in length, to have a GC content of about 50% or more, and to anneal to the template at temperatures of about 68°C to 72°C.
  • Genomic libraries may be useful for extension of sequence into 5' non-transcribed regulatory regions.
  • Capillary electrophoresis systems which are commercially available may be used to analyze the size or confirm the nucleotide sequence of sequencing or PCR products, h particular, capillary sequencing may employ flowable polymers for electrophoretic separation, four different nucleotide- specific, laser-stimulated fluorescent dyes, and a charge coupled device camera for detection of the emitted wavelengths. Output/light intensity may be converted to electrical signal using appropriate software (e.g., GENOTYPER and SEQUENCE NAVIGATOR, Applied Biosystems), and the entire process from loading of samples to computer analysis and electronic data display may be computer controlled. Capillary electrophoresis is especially preferable for sequencing small DNA fragments which may be present in limited amounts in a particular sample.
  • polynucleotides or fragments thereof which encode NAAP may be cloned in recombinant DNA molecules that direct expression of NAAP, or fragments or functional equivalents thereof, in appropriate host cells. Due to the inherent degeneracy of the genetic code, other polynucleotides which encode substantially the same or a functionally equivalent polypeptides may be produced and used to express NAAP.
  • the polynucleotides of the invention can be engineered using methods generally known in the art in order to alter NAAP-encoding sequences for a variety of purposes including, but not limited to, modification of the cloning, processing, and/or expression of the gene product.
  • DNA shuffling by random fragmentation and PCR reassembly of gene fragments and synthetic oligonucleotides may be used to engineer the nucleotide sequences.
  • oligonucleotide-mediated site-directed mutagenesis may be used to introduce mutations that create new restriction sites, alter glycosylation patterns, change codon preference, produce splice variants, and so forth.
  • the nucleotides of the present invention may be subjected to DNA shuffling techniques such as MOLECULARBREEDLNG (Maxygen Inc., Santa Clara CA; described in U.S. Patent No. 5,837,458; Chang, C.-C. et al. (1999) Nat. Biotechnol. 17:793-797; Christians, F.C et al. (1999) Nat. Biotechnol. 17:259-264; and Crameri, A. et al. (1996) Nat. Biotechnol. 14:315-319) to alter or improve the biological properties of NAAP, such as its biological or enzymatic activity or its ability to bind to other molecules or compounds.
  • MOLECULARBREEDLNG Maxygen Inc., Santa Clara CA; described in U.S. Patent No. 5,837,458; Chang, C.-C. et al. (1999) Nat. Biotechnol. 17:793-797; Christians, F.C e
  • DNA shuffling is a process by which a library of gene variants is produced using PCR-mediated recombination of gene fragments. The library is then subjected to selection or screening procedures that identify those gene variants with the desired properties. These preferred variants may then be pooled and further subjected to recursive rounds of DNA shuffling and selection/screening.
  • genetic diversity is created through "artificial" breeding and rapid molecular evolution. For example, fragments of a single gene containing random point mutations may be recombined, screened, and then reshuffled until the desired properties are optimized. Alternatively, fragments of a given gene may be recombined with fragments of homologous genes in the same gene family, either from the same or different species, thereby maximizing the genetic diversity of multiple naturally occurring genes in a directed and controllable manner.
  • polynucleotides encoding NAAP may be synthesized, in whole or in part, using one or more chemical methods well known in the art (Caruthers, M.H. et al. (1980) Nucleic Acids Symp. Ser. 7:215-223; Horn, T. et al. (1980) Nucleic Acids Symp. Ser. 7:225-232).
  • NAAP itself or a fragment thereof may be synthesized using chemical methods known in the art.
  • peptide synthesis can be performed using various solution-phase or solid-phase techniques (Creighton, T. (1984) Proteins, Structures and Molecular Properties, WH Freeman, New York NY, pp. 55-60; Roberge, J.Y.
  • NAAP amino acid sequence of NAAP, or any part thereof, may be altered during direct synthesis and/or combined with sequences from other proteins, or any part thereof, to produce a variant polypeptide or a polypeptide having a sequence of a naturally occurring polypeptide.
  • the peptide may be substantially purified by preparative high performance liquid chromatography (Chiez, R.M. and F.Z. Regnier (1990) Methods Enzymol. 182:392-421).
  • the composition of the synthetic peptides may be confirmed by amino acid analysis or by sequencing (Creighton, supra, pp. 28-53).
  • 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.
  • a variety of expression vector/host systems may be utilized to contain and express polynucleotides encoding NAAP. These include, but are not limited to, microorganisms such as bacteria transformed with recombinant bacteriophage, plasmid, or cosmid DNA expression vectors; yeast transformed with yeast expression vectors; insect cell systems infected with viral expression vectors (e.g., baculovirus); plant cell systems transformed with viral expression vectors (e.g., cauliflower mosaic virus, CaMV, or tobacco mosaic virus, TMV) or with bacterial expression vectors (e.g., Ti or pBR322 plasmids); or animal cell systems (Sambrook and Russell, supra; Ausubel et al., supra; Van Heeke, G.
  • microorganisms such as bacteria transformed with recombinant bacteriophage, plasmid, or cosmid DNA expression vectors
  • yeast transformed with yeast expression vectors insect cell systems infected with viral expression vectors (e.g., bac
  • Expression vectors derived from retroviruses, adenoviruses, or herpes or vaccinia viruses, or from various bacterial plasmids may be used for delivery of polynucleotides to the targeted organ, tissue, or cell population (Di Nicola, M. et al. (1998) Cancer Gen. Ther. 5:350-356; Yu, M. et al. (1993) Proc. Natl. Acad. Sci. USA 90:6340-6344; Buller, 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 cell employed.
  • cloning and expression vectors may be selected depending upon the use intended for polynucleotides encoding NAAP.
  • routine cloning, subcloning, and propagation of polynucleotides encoding NAAP can be achieved using a multifunctional E. coli vector such as PBLUESCRIPT (Stratagene, La Jolla CA) or PSPORT1 plasmid (Invitrogen).
  • PBLUESCRIPT Stratagene, La Jolla CA
  • PSPORT1 plasmid Invitrogen.
  • 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).
  • vectors which direct high level expression of NAAP may be used.
  • 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.
  • such vectors direct either the secretion or intracellular retention of expressed proteins and enable integration of foreign polynucleotide sequences into the host genome for stable propagation (Ausubel 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. 3:17-311). Alternatively, plant promoters such as the small subunit of RUBISCO or heat shock promoters may be used (Coruzzi, G. et al. (1984) EMBO J. 3: 1671-1680; Broglie, R. et al. (1984) Science 224:838-843; Winter, J. et al. (1991) Results Probl. Cell Differ.
  • 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. 3:1311).
  • plant promoters such as the small sub
  • constructs can be introduced into plant cells by direct DNA transformation or pathogen-mediated transfection (The McGraw Hill Yearbook of Science and Technology (1992) McGraw Hill, New York NY, pp. 191-196).
  • mammalian cells a number of viral-based expression systems may be utilized.
  • polynucleotides encoding NAAP may be ligated into an adenovirus transcription/translation complex consisting of the late promoter and tripartite leader sequence. Insertion in a non-essential 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.
  • transcription enhancers such as the Rous sarcoma virus (RSV) enhancer, may be used to increase expression in mammalian host cells.
  • SV40 or EBV-based vectors may also be used for high-level protein expression.
  • HACs Human artificial chromosomes
  • HACs may also be employed to deliver larger fragments of DNA than can be contained in and expressed from a plasmid.
  • HACs of about 6 kb to 10 Mb are constructed and delivered via conventional delivery methods (liposomes, polycationic amino polymers, or vesicles) for therapeutic purposes (Harrington, J.J. et al. (1997) Nat. Genet. 15:345-355).
  • NAAP For long term production of recombinant proteins in mammalian systems, stable expression of NAAP in cell lines is preferred.
  • polynucleotides encoding NAAP can be transformed into cell lines using expression vectors which may contain viral origins of replication and/or endogenous expression elements and a selectable marker gene on the same or on a separate vector. Following the introduction of the vector, cells may be allowed to grow for about 1 to 2 days in enriched media before being switched to selective media.
  • the purpose of the selectable marker is to confer resistance to a selective agent, and its presence allows growth and recovery of cells which successfully express the introduced sequences.
  • Resistant clones of stably transformed cells may be propagated using tissue culture techniques appropriate to the cell type.
  • selection systems may be used to recover transformed cell lines. These include, but are not limited to, the herpes simplex virus thymidine kinase and adenine phosphoribosyltransferase genes, for use in tk ⁇ and apr cells, respectively (Wigler, M. et al. (1977) Cell 11:223-232; Lowy, I. et al. (1980) Cell 22:817-823). Also, antimetabolite, antibiotic, or herbicide resistance can be used as the basis for selection.
  • dhfr confers resistance to methotrexate
  • neo confers resistance to the aminoglycosides neomycin and G-418
  • als and pat confer resistance to chlorsulfuron and phosphinotricin acetyltransferase, respectively
  • trpB and hisD Additional selectable genes have been described, e.g., trpB and hisD, which alter cellular requirements for metabolites (Hartman, S.C.
  • Visible markers e.g., anthocyanins, green fluorescent proteins (GFP; Clontech), ⁇ - glucuronidase and its substrate ⁇ -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).
  • marker gene expression suggests that the gene of interest is also present, the presence and expression of the gene may need to be confirmed.
  • sequence encoding NAAP is inserted within a marker gene sequence
  • transformed cells containing polynucleotides encoding NAAP can be identified by the absence of marker gene function.
  • a marker gene can be placed in tandem with a sequence encoding NAAP under the control of a single promoter. Expression of the marker gene in response to induction or selection usually indicates expression of the tandem gene as well.
  • host cells that contain the polynucleotide encoding NAAP and that express NAAP may be identified by a variety of procedures known to those of skill in the art.
  • Means for producing labeled hybridization or PCR probes for detecting sequences related to polynucleotides encoding NAAP include oligolabeling, nick translation, end-labeling, or PCR amplification using a labeled nucleotide.
  • polynucleotides encoding NAAP, or any fragments thereof may be cloned into a vector for the production of an mRNA probe.
  • Such vectors are known in the art, are commercially available, and may be used to synthesize RNA probes in vitro by addition of an appropriate RNA polymerase such as T7, T3, or SP6 and labeled nucleotides.
  • reporter molecules or labels which may be used for ease of detection include radionuclides, enzymes, fluorescent, chemiluminescent, or chromogenic agents, as well as substrates, cofactors, inhibitors, magnetic particles, and the like.
  • Host cells transformed with polynucleotides encoding NAAP may be cultured under conditions suitable for the expression and recovery of the protein from cell culture.
  • the protein produced by a transformed cell may be secreted or retained intracellularly depending on the sequence and/or the vector used.
  • expression vectors containing polynucleotides which encode NAAP may be designed to contain signal sequences which direct secretion of NAAP through a prokaryotic or eukaryotic cell membrane.
  • a host cell strain may be chosen for its ability to modulate expression of the inserted polynucleotides or to process the expressed protein in the desired fashion.
  • modifications of the polypeptide include, but are not limited to, acetylation, carboxylation, glycosylation, phosphorylation, lipidation, and acylation.
  • Post-translational processing which cleaves a "prepro” or "pro” form of the protein may also be used to specify protein targeting, folding, and/or activity.
  • Different host cells which have specific cellular machinery and characteristic mechanisms for post-translational activities (e.g., CHO, HeLa, MDCK, HEK293, and WI38) are available from the American Type Culture Collection (ATCC, Manassas VA) and may be chosen to ensure the correct modification and processing of the foreign protein.
  • ATCC American Type Culture Collection
  • natural, modified, or recombinant polynucleotides encoding NAAP may be ligated to a heterologous sequence resulting in translation of a fusion protein in any of the aforementioned host systems.
  • a chimeric NAAP protein containing a heterologous moiety that can be recognized by a commercially available antibody may facilitate the screening of peptide libraries for inhibitors of NAAP activity.
  • Heterologous protein and peptide moieties may also facilitate purification of fusion proteins using commercially available affinity matrices.
  • Such moieties include, but are not limited to, glutathione S-transferase (GST), maltose binding protein (MBP), thioredoxin (Trx), calmodulin binding peptide (CBP), 6-His, FLAG, c-myc, and hemagglutinin (HA).
  • GST, MBP, Trx, CBP, and 6-His enable purification of their cognate fusion proteins on immobilized glutathione, maltose, phenylarsine oxide, calmodulin, and metal-chelate resins, respectively.
  • FLAG, c-myc, and hemagglutinin (HA) enable immunoaffinity purification of fusion proteins using commercially available monoclonal and polyclonal antibodies that specifically recognize these epitope tags.
  • a fusion protein may also be engineered to contain a proteolytic cleavage site located between the 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 facilitate expression and purification of fusion proteins.
  • 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, 35 S-methionine.
  • NAAP fragments of NAAP, or variants of NAAP may be used to screen for compounds that specifically bind to NAAP.
  • One or more test compounds may be screened for specific binding to NAAP.
  • 1, 2, 3, 4, 5, 10, 20, 50, 100, or 200 test compounds can be screened for specific binding to NAAP.
  • Examples of test compounds can include antibodies, anticalins, oligonucleotides, proteins (e.g., ligands or receptors), or small molecules.
  • 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.
  • 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 ID NO: 1-56.
  • 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.
  • a compound identified in a screen for specific binding to NAAP can be closely related to the natural ligand of NAAP, e.g., a ligand or fragment thereof, a natural substrate, a structural or functional mimetic, or a natural binding partner (Coligan, J.E. et al. (1991) Current Protocols in Immunology l(2):Chapter 5).
  • the compound thus identified can be a natural ligand of a receptor NAAP (Howard, A.D. et al. (2001) Trends Pharmacol. Sci.22: 132- 140; Wise, A. et al. (2002) Drug Discovery Today 7:235-246).
  • a compound identified in a screen for specific binding to NAAP can be closely related to the natural receptor to which NAAP binds, at least a fragment of the receptor, or a fragment of the receptor including all or a portion of the ligand binding site or binding pocket.
  • the compound may be a receptor for NAAP which is capable of propagating a signal, or a decoy receptor for NAAP which is not capable of propagating a signal (Ashkenazi, A. and V.M. Divit (1999) Curr. Opin. Cell Biol. 11:255-260; Mantovani, A. et al. (2001) Trends Immunol. 22:328-336).
  • the compound can be rationally designed using known techniques.
  • Etanercept is an engineered ⁇ 75 tumor necrosis factor (TNF) receptor dimer linked to the Fc portion of human IgGj (Taylor, P.C et al. (2001) Curr. Opin. Immunol. 13:611-616).
  • TNF tumor necrosis factor
  • 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.
  • an antibody can be selected such that its binding specificity allows for preferential identification of specific fragments or variants of NAAP.
  • an antibody can be selected such that its binding specificity allows for preferential diagnosis of a specific disease or condition having increased, decreased, or otherwise abnormal production of NAAP.
  • anticalins can be screened for specific binding to NAAP, fragments of
  • Anticalins are ligand-binding proteins that have been constructed based on a lipocalin scaffold (Weiss, G.A. and H.B. Lowman (2000) Chem. Biol. 7:R177-R184; Skerra, A. (2001) J. Biotechnol. 74:257-275).
  • the protein architecture of lipocalins can include a beta-barrel having eight antiparallel beta-strands, which supports four loops at its open end. These loops form the natural ligand-binding site of the lipocalins, a site which can be re-engineered in vitro by amino acid substitutions to impart novel binding specificities.
  • amino acid substitutions can be made using methods known in the art or described herein, and can include conservative substitutions (e.g., substitutions that do not alter binding specificity) or substitutions that modestly, moderately, or significantly alter binding specificity. In one embodiment, screening for compounds which specifically bind to, stimulate, or inhibit
  • NAAP involves producing appropriate cells which express NAAP, either as a secreted protein or on the cell membrane.
  • Preferred cells can include cells from mammals, yeast, Drosophila, or E. coli. Cells expressing NAAP or cell membrane fractions which contain NAAP are then contacted with a test compound and binding, stimulation, or inhibition of activity of either NAAP or the compound is analyzed.
  • An assay may simply test binding of a test compound to the polypeptide, wherein bindmg is detected by a fluorophore, radioisotope, enzyme conjugate, or other detectable label.
  • the assay may comprise the steps of combining at least one test compound with NAAP, either in solution or affixed to a solid support, and detecting the binding of NAAP to the compound.
  • the assay may detect or measure binding of a test compound in the presence of a labeled competitor.
  • the assay may be carried out using cell-free preparations, chemical libraries, or natural product mixtures, and the test compound(s) may be free in solution or affixed to a solid support.
  • An assay can be used to assess the ability of a compound to bind to its natural ligand and/or to inhibit the binding of its natural ligand to its natural receptors.
  • examples of such assays include radio-labeling assays such as those described in U.S. Patent No. 5,914,236 and U.S. Patent No. 6,372,724.
  • one or more amino acid substitutions can be introduced into a polypeptide compound (such as a receptor) to improve or alter its ability to bind to its natural ligands (Matthews, D.J. and J.A. Wells. (1994) Chem. Biol. 1:25-30).
  • one or more amino acid substitutions can be introduced into a polypeptide compound (such as a ligand) to improve or alter its ability to bind to its natural receptors (Cunningham, B.C. and J.A. Wells (1991) Proc. Natl. Acad. Sci. USA 88:3407-3411; Lowman, H.B. et al. (1991) J. Biol. Chem. 266:10982- 10988).
  • NAAP, fragments of NAAP, or variants of NAAP may be used to screen for compounds that modulate the activity of NAAP.
  • Such compounds may include agonists, antagonists, or partial or inverse agonists.
  • 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.
  • a test compound is combined with an in vitro or cell-free system comprising NAAP under conditions suitable for NAAP activity, and the assay is performed. In either of these assays, a test compound which modulates the activity of NAAP may do so indirectly and need not come in direct contact with the test compound. At least one and up to a plurality of test compounds may be screened.
  • polynucleotides encoding NAAP or their mammalian homologs may be "knocked out" in an animal model system using homologous recombination in embryonic stem (ES) cells.
  • ES embryonic stem
  • Such techniques are well known in the art and are useful for the generation of animal models of human disease (see, e.g., U.S. Patent No. 5,175,383 and U.S. Patent No. 5,767,337).
  • mouse ES cells such as the mouse 129/SvJ cell line, are derived from the early mouse embryo and grown in culture.
  • the ES cells are transformed with a vector containing the gene of interest disrupted by a marker gene, e.g., the neomycin phosphotransferase gene (neo; Capecchi, M.R. (1989) Science 244: 1288-1292).
  • a marker gene e.g., the neomycin phosphotransferase gene (neo; Capecchi, M.R. (1989) Science 244: 1288-1292).
  • the vector integrates into the corresponding region of the host genome by homologous recombination.
  • homologous recombination takes place using the Cre-loxP system to knockout a gene of interest in a tissue- or developmental stage-specific manner (Marth, J.D. (1996) Clin. Invest. 97:1999-2002; Wagner, K.U. et al. (1997) Nucleic Acids Res. 25:4323-4330).
  • Transformed ES cells are identified and microinjected into mouse cell blastocysts such as those from the C57BL/6 mouse strain.
  • the blastocysts are surgically transferred to pseudopregnant dams, and the resulting chimeric progeny are genotyped and bred to produce heterozygous or homozygous strains.
  • Transgenic animals thus generated may be tested with potential therapeutic or toxic agents.
  • Polynucleotides encoding NAAP may also be manipulated in vitro in ES cells derived from human blastocysts.
  • Human ES cells have the potential to differentiate into at least eight separate cell lineages including endoderm, mesoderm, and ectodermal cell types. These cell lineages differentiate into, for example, neural cells, hematopoietic lineages, and cardiomyocytes (Thomson, J.A. et al. (1998) Science 282:1145-1147).
  • Polynucleotides encoding NAAP can also be used to create "knockin" humanized animals (pigs) or transgenic animals (mice or rats) to model human disease.
  • knockin technology a region of a polynucleotide encoding NAAP is injected into animal ES cells, and the injected sequence integrates into the animal cell genome.
  • Transformed cells are injected into blastulae, and the blastulae are implanted as described above.
  • Transgenic progeny or inbred lines are studied and treated with potential pharmaceutical agents to obtain information on treatment of a human disease.
  • 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 may also serve as a convenient source of that protein (Janne, J. et al. (1998) Biotechnol. Annu. Rev. 4:55-74).
  • NAAP appears to play a role in cell proliferative, neurological, developmental, and autoimmune/inflammatory disorders, and infections.
  • disorders associated with increased NAAP expression or activity it is desirable to decrease the expression or activity of NAAP.
  • 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.
  • disorders include, but are not limited to, a cell proliferative disorder such as actinic keratosis, arteriosclerosis, atherosclerosis, bursitis, cirrhosis, hepatitis, mixed connective tissue disease (MCTD), myelofibrosis, paroxysmal nocturnal hemoglobinuria, polycythemia vera, psoriasis, primary thrombocythemia, and cancers including adenocarcinoma, leukemia, lymphoma, melanoma, myeloma, sarcoma, teratocarcinoma, and, in particular, a cancer of the adrenal gland, bladder, bone, bone marrow, brain, breast, cervix, gall bladder, ganglia, gastrointestinal tract, heart, kidney, liver, lung, muscle, ovary, pancreas, parathyroid, penis, prostate, salivary glands, skin, spleen, testis, thymus
  • 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.
  • a composition comprising a substantially purified NAAP in conjunction with a suitable pharmaceutical carrier may be administered to a subject to treat or prevent a disorder associated with decreased expression or activity of NAAP including, but not limited to, those provided above.
  • an agonist which modulates the activity of NAAP may be administered to a subject to treat or prevent a disorder associated with decreased expression or activity of NAAP including, but not limited to, those listed above.
  • an antagonist of NAAP may be administered to a subject to treat or prevent a disorder associated with increased expression or activity of NAAP.
  • disorders include, but are not limited to, those cell proliferative, neurological, developmental, and autoimmune/inflammatory disorders, and infections described above.
  • an antibody which specifically binds NAAP may be used directly as an antagonist or indirectly as a targeting or delivery mechanism for bringing a pharmaceutical agent to cells or tissues which express NAAP.
  • 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.
  • any protein, agonist, antagonist, antibody, complementary sequence, or vector embodiments may be administered in combination with other appropriate therapeutic agents. Selection of the appropriate agents for use in combination therapy may be made by one of ordinary skill in the art, according to conventional pharmaceutical principles.
  • the combination of therapeutic agents may act synergistically to effect the treatment or prevention of the various disorders described above. Using this approach, one may be able to achieve therapeutic efficacy with lower dosages of each agent, thus reducing the potential for adverse side effects.
  • An antagonist of NAAP may be produced using methods which are generally known in the art.
  • purified NAAP may be used to produce antibodies or to screen libraries of pharmaceutical agents to identify those which specifically bind NAAP.
  • Antibodies to NAAP may also be generated using methods that are well known in the art. Such antibodies may include, but are not limited to, polyclonal, monoclonal, chimeric, and single chain antibodies, Fab fragments, and fragments produced by a Fab expression library.
  • neutralizing antibodies i.e., those which inhibit dimer formation
  • Single chain antibodies may be potent enzyme inhibitors and may have application in the design of peptide mimetics, and in the development of immuno-adsorbents and biosensors (Muyldermans, S. (2001) J. Biotechnol. 74:277-302).
  • 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 oligopeptide thereof which has immunogenic properties.
  • various adjuvants may be used to increase immunological response.
  • 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.
  • BCG Bacilli Calmette-Guerin
  • Corynebacterium parvum are especially preferable.
  • the oligopeptides, peptides, or fragments used to induce antibodies to NAAP have an amino acid sequence consisting of at least about 5 amino acids, and generally will consist of at least about 10 amino acids. It is also preferable that these oligopeptides, peptides, or fragments are substantially identical to a portion of the amino acid sequence of the natural protein. Short stretches of NAAP amino acids may be fused with those of another protein, such as KLH, and antibodies to the chimeric molecule may be produced.
  • Monoclonal antibodies to NAAP may be prepared using any technique which provides for the production of antibody molecules by continuous cell lines in culture. These include, but are not limited to, the hybridoma technique, the human B-cell hybridoma technique, and the EBV-hybridoma technique (Kohler, G. et al. (1975) Nature 256:495-497; Kozbor, D. et al. (1985) J. 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. Cell Biol. 62:109-120).
  • chimeric antibodies such as the splicing of mouse antibody genes to human antibody genes to obtain a molecule with appropriate antigen specificity and biological activity, can be used (Morrison, S.L. et al. (1984) Proc. Natl. Acad. Sci. USA 81:6851-6855; Neuberger, M.S. et al. (1984) Nature 312:604-608; Takeda, S. et al. (1985) Nature 314:452-454).
  • techniques described for the production of single chain antibodies may be adapted, using methods known in the art, to produce NAAP-specific single chain antibodies.
  • Antibodies with related specificity, but of distinct idiotypic composition may be generated by chain shuffling from random combinatorial immunoglobulin libraries (Burton, D.R. (1991) Proc. Natl. Acad. Sci. USA 88:10134-10137).
  • Antibodies may also be produced by inducing in vivo production in the lymphocyte population or by screening immunoglobulin libraries or panels of highly specific binding reagents as disclosed in the literature (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 he generated.
  • fragments include, but are not limited to, F(ab fragments produced by pepsin digestion of the antibody molecule and Fab fragments generated by reducing the disulfide bridges of the F(ab')2 fragments.
  • Fab expression libraries may be constructed to allow rapid and easy identification of monoclonal Fab fragments with the desired specificity (Huse, W.D. et al. (1989) Science 246:1275-1281).
  • immunoassays may be used for screening to identify antibodies having the desired specificity.
  • Numerous protocols for competitive binding or immunoradiometric assays using either polyclonal or monoclonal antibodies with established specificities are well known in the art.
  • Such immunoassays typically involve the measurement of comple formation between NAAP and its specific antibody.
  • a two-site, monoclonal-based immunoassay utilizing monoclonal antibodies reactive to two non-interfering NAAP epitopes is generally used, but a competitive binding assay may also be employed (Pound, supra).
  • K a is defined as the molar concentration of NAAP-antibody complex divided by the molar concentrations of free antigen and free antibody under equilibrium conditions.
  • K a association constant
  • 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 10 9 to IO 12 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 IO 6 to IO 7 L/mole are preferred for use in immunopurification and similar procedures which ultimately require dissociation of NAAP, preferably in active form, from the antibody (Catty, D. (1988) Antibodies, Volume I: A Practical Approach. ERL Press, Washington DC; Liddell, J.E. and A. Cryer (1991) A Practical Guide to Monoclonal Antibodies, John Wiley & Sons, New York NY).
  • polyclonal antibody preparations may be further evaluated to determine the quality and suitability of such preparations for certain downstream applications.
  • a polyclonal antibody preparation containing at least 1-2 mg specific antibody/ml, preferably 5-10 mg specific antibody/ml is generally employed in procedures requiring precipitation of NAAP-antibody complexes.
  • Procedures for evaluating antibody specificity, titer, and avidity, and guidelines for antibody quality and usage in various applications, are generally available (Catty, supra; Coligan et al., supra).
  • polynucleotides encoding NAAP may be used for therapeutic purposes.
  • modifications of gene expression can be achieved by designing complementary sequences or antisense molecules (DNA, RNA, PNA, or modified oligonucleotides) to the coding or regulatory regions of the gene encoding NAAP.
  • complementary sequences or antisense molecules DNA, RNA, PNA, or modified oligonucleotides
  • antisense oligonucleotides or larger fragments can be designed from various locations along the coding or control regions of sequences encoding NAAP (Agrawal, S., ed. (1996) Antisense Therapeutics. Humana Press, Totawa NJ).
  • Antisense sequences can be delivered intracellularly in the form of an expression plasmid which, upon transcription, produces a sequence complementary to at least a portion of the cellular sequence encoding the target protein (Slater, J.E. et al. (1998) J. Allergy Clin. Immunol. 102:469-475; Scanlon, K.J. et al. (1995) 9: 1288-1296).
  • Antisense sequences can also be introduced intracellularly through the use of viral vectors, such as retrovirus and adeno-associated virus vectors (Miller, A.D. (1990) Blood 76:271; Ausubel et al., supra; Uckert, W. and W. Walther (1994) Pharmacol. Ther. 63:323-347).
  • viral vectors such as retrovirus and adeno-associated virus vectors
  • Other gene delivery mechanisms include liposome-derived systems, artificial viral envelopes, and other systems known in the art (Rossi, J.J. (1995) Br. Med. Bull. 51:217-225; Boado, R.J. et al. (1998) J. Pharm. Sci. 87:1308-1315; Morris, M.C. et al. (1997) Nucleic Acids Res. 25:2730-2736).
  • polynucleotides encoding NAAP may be used for somatic or germline gene therapy.
  • Gene therapy may be performed to (i) correct a genetic deficiency (e.g., in the cases of severe combined immunodeficiency (SCID)-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.
  • SCID severe combined immunodeficiency
  • ADA adenosine deaminase
  • NAAP hepatitis B or C virus
  • fungal parasites such as Candida albicans and Paracoccidioides brasiliensis
  • protozoan parasites such as Plasmodiumfalciparum and Trypanosoma cruzi.
  • the expression of NAAP from an appropriate population of transduced cells may alleviate the clinical manifestations caused by the genetic deficiency.
  • diseases or disorders caused by deficiencies in NAAP are treated by constructing mammalian expression vectors encoding NAAP and introducing these vectors by mechanical means into NAAP-deficient cells.
  • Mechanical transfer technologies for use with cells in vivo or ex vitro include (i) direct DNA microinjection into individual cells, (ii) ballistic gold particle delivery, (iii) liposome-mediated transfection, (iv) receptor-mediated gene transfer, and (v) the use of DNA transposons (Morgan, R.A. and W.F. Anderson (1993) Annu. Rev. Biochem. 62:191-217; Ivies, Z. (1997) Cell 91:501-510; Boulay, J.-L. and H. Recipon (1998) Curr.
  • 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-SCRIPT, PCMV-TAG, PEGSH/PERV (Stratagene, La Jolla CA), and PTET-OFF, PTET-ON, PTRE2, PTRE2-LUC, PTK-HYG (Clontech, Palo Alto CA).
  • NAAP may be expressed using (i) a constitutively active promoter, (e.g., from cytomegalovirus (CMV), Rous sarcoma virus (RSV), SV40 virus, thymidine kinase (TK), or ⁇ -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.
  • a constitutively active promoter e.g., from cytomegalovirus (CMV), Rous sarcoma virus (RSV), SV40 virus, thymidine kinase (TK), or ⁇ -actin genes
  • liposome transformation kits e.g., the PERFECT LEPED TRANSFECTION KIT, available from Invitrogen
  • PERFECT LEPED TRANSFECTION KIT available from Invitrogen
  • transformation is performed using the calcium phosphate method (Graham, F.L. and A.J. Eb (1973) Virology 52:456-467), or by electroporation (Neumann, E. et al. (1982) EMBO J. 1:841-845).
  • the introduction of DNA to primary cells requires modification of these standardized mammalian transfection protocols.
  • 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 czs-acting RNA sequences and coding sequences required for efficient vector propagation.
  • Retrovirus vectors e.g., PFB and PFBNEO
  • Retrovirus vectors are commercially available (Stratagene) and are based on published data (Riviere, I. et al. (1995) Proc. Natl. Acad. Sci.
  • the vector is propagated in an appropriate vector producing cell line (VPCL) that expresses an envelope gene with a tropism for receptors on the target cells or a promiscuous envelope protein such as VSVg (Armentano, D. et al. (1987) J. Virol. 61:1647-1650; Bender, M.A. et al. (1987) J. Virol. 61:1639-1646; Adam, M.A. and A.D. Miller (1988) J. Virol. 62:3802-3806; Dull, T. et al. (1998) J. Virol. 72:8463-8471; Zufferey, R. et al. (1998) J.
  • VPCL vector producing cell line
  • U.S. Patent No. 5,910,434 to Rigg discloses a method for obtaining retrovirus packaging cell lines and is hereby incorporated by reference. Propagation of retrovirus vectors, transduction of a population of cells (e.g., CD4 + T- cells), and the return of transduced cells to a patient are procedures well known to persons skilled in the art of gene therapy and have been well documented (Ranga, U. et al. (1997) J. Virol. 71:7020- 7029; Bauer, G. et al.
  • an adenovirus-based gene therapy delivery system is used to deliver polynucleotides encoding NAAP to cells which have one or more genetic abnormalities with respect to the expression of NAAP.
  • the construction and packaging of adenovirus-based vectors are well known to those with ordinary skill in the art. Replication defective adenovirus vectors have proven to be versatile for importing genes encoding immunoregulatory proteins into intact islets in the pancreas (Csete, M.E. et al. (1995) Transplantation 27:263-268). Potentially useful adenoviral vectors are described in U.S. Patent No. 5,707,618 to Armentano ("Adenovirus vectors for gene therapy”), hereby incorporated by reference.
  • a herpes-based, gene therapy delivery system is used to deliver polynucleotides encoding NAAP to target cells which have one or more genetic abnormalities with respect to the expression of NAAP.
  • the use of herpes simplex virus (HSV)-based vectors may be especially valuable for introducing NAAP to cells of the central nervous system, for which HSV has a tropism.
  • the construction and packaging of herpes-based vectors are well known to those with ordinary skill in the art.
  • a replication-competent herpes simplex virus (HSV) type 1 -based vector has been used to deliver a reporter gene to the eyes of primates (Liu, X. et al. (1999) Exp. Eye Res. 169:385-395).
  • HSV-1 virus vector has also been disclosed in detail in U.S. Patent No. 5,804,413 to DeLuca ("Herpes simplex virus strains for gene transfer"), which is hereby incorporated by reference.
  • U.S. Patent No. 5,804,413 teaches the use of recombinant HSV d92 which consists of a genome containing at least one exogenous gene to be transferred to a cell under the control of the appropriate promoter for purposes including human gene therapy. Also taught by this patent are the construction and use of recombinant HSV strains deleted for ICP4, ICP27 and ICP22.
  • HSV vectors see also Goins, W.F. et al. (1999; J. Virol.
  • an alphavirus (positive, single-stranded RNA virus) vector is used to deliver polynucleotides encoding NAAP to target cells.
  • SFV Semliki Forest Virus
  • alphavirus infection is typically associated with cell lysis within a few days
  • BHK-21 hamster normal kidney cells
  • SEN Sindbis virus
  • the ability to establish a persistent infection in hamster normal kidney cells (BHK-21) with a variant of Sindbis virus (SEN) indicates that the lytic replication of alphaviruses can be altered to suit the needs of the gene therapy application (Dryga, S.A. et al. (1997) Virology 228:74-83).
  • the wide host range of alphaviruses will allow the introduction of NAAP into a variety of cell types.
  • the specific transduction of a subset of cells in a population may require the sorting of cells prior to transduction.
  • the methods of manipulating infectious cDNA clones of alphaviruses, performing alphavirus cDNA and RNA transfections, and performing alphavirus infections, are well known to those with ordinary skill in the art. Oligonucleotides derived from the transcription initiation site, e.g., between about positions
  • -10 and +10 from the start site may also be employed to inhibit gene expression.
  • inhibition can be achieved using triple helix base-pairing methodology.
  • Triple helix pairing is useful because it causes inhibition of the ability of the double helix to open sufficiently for the binding of polymerases, transcription factors, or regulatory molecules.
  • Recent therapeutic advances using triplex DNA have been described in the literature (Gee, J.E. et al. (1994) in Huber, B.E. and B.I. Carr, Molecular and Immunologic Approaches, Futura Publishing, Mt. Kisco NY, pp. 163-177).
  • a complementary sequence or antisense molecule may also be designed to block translation of mRNA by preventing the transcript from binding to ribosomes.
  • Ribozymes enzymatic RNA molecules
  • Ribozymes may also be used to catalyze the specific cleavage of RNA.
  • the mechanism of ribozyme action involves sequence-specific hybridization of the ribozyme molecule to complementary target RNA, followed by endonucleolytic cleavage.
  • engineered hammerhead motif ribozyme molecules may specifically and efficiently catalyze endonucleolytic cleavage of RNA molecules encoding NAAP.
  • RNA sequences of between 15 and 20 ribonucleotides, corresponding to the region of the target gene containing the cleavage site, may be evaluated for secondary structural features which may render the oligonucleotide inoperable.
  • the suitability of candidate targets may also be evaluated by testing accessibility to hybridization with complementary oligonucleotides using ribonuclease protection assays.
  • 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, constitutively or inducibly, can be introduced into cell lines, cells, or tissues. RNA molecules may be modified to increase intracellular stability and half-life.
  • flanking sequences at the 5' and/or 3' ends of the molecule Possible modifications include, but are not limited to, the addition of flanking sequences at the 5' and/or 3' ends of the molecule, or the use of phosphorothioate or 2' O-methyl rather than phosphodiesterase linkages within the backbone of the molecule.
  • This concept is inherent in the production of PNAs and can be extended in all of these molecules by the inclusion of nontraditional bases such as inosine, queosine, and wybutosine, as well as acetyl-, methyl-, thio-, and similarly modified forms of adenine, cytidine, guanine, thymine, and uridine which are not as easily recognized by endogenous endonucleases.
  • RNAi RNA interference
  • PTGS post-transcriptional gene silencing
  • RNAi is a post-transcriptional mode of gene silencing in which double-stranded RNA (dsRNA) introduced into a targeted cell specifically suppresses the expression of the homologous gene (i.e., the gene bearing the sequence complementary to the dsRNA). This effectively knocks out or substantially reduces the expression of the targeted gene.
  • dsRNA double-stranded RNA
  • PTGS can also be accomplished by use of DNA or DNA fragments as well. RNAi methods are described by Fire, A. et al.
  • PTGS can also be initiated by introduction of a complementary segment of DNA into the selected tissue using gene delivery and/or viral vector delivery methods described herein or known in the art.
  • RNAi can be induced in mammalian cells by the use of small interfering RNA also known as siRNA.
  • siRNA small interfering RNA also known as siRNA.
  • SiRNA are shorter segments of dsRNA (typically 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 mammalian 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 cell, or directly by mammalian transfection methods and agents described herein or known in the art (such as liposome-mediated transfection, viral vector methods, or other polynucleotide delivery/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.
  • mRNA target polynucleotide
  • 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 bmding 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 commercially available methods and kits such as the SILENCER siRNA construction kit (Ambion, Austin TX).
  • long-term gene silencing and/or RNAi effects can be induced in selected tissue using expression vectors that continuously express siRNA.
  • This can be accomplished 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).
  • shRNAs can be delivered to target cells using expression vectors known in the art.
  • An example of a suitable expression vector for delivery of siRNA is the PSILENCER1.0-U6 (circular) plasmid (Ambion).
  • 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 microarray 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, oligonucleotides, antisense oligonucleotides, triple helix-forming oligonucleotides, transcription factors and other polypeptide transcriptional regulators, and non- macromolecular chemical entities which are capable of interacting with specific polynucleotide sequences. Effective compounds may alter polynucleotide expression by acting as either inhibitors or promoters of polynucleotide expression.
  • a compound which specifically inhibits expression of the polynucleotide encoding NAAP may be therapeutically useful, and in the treatment of disorders associated with decreased NAAP expression or activity, a compound which specifically promotes expression of the polynucleotide encoding NAAP may be therapeutically useful.
  • one or more test compounds may be screened for effectiveness in altering expression of a specific polynucleotide.
  • a test compound may be obtained by any method commonly known in the art, including chemical modification of a compound known to be effective in altering polynucleotide expression; selection from an existing, commercially-available or proprietary library of naturally-occurring or non-natural chemical compounds; rational design of a compound based on chemical and/or structural properties of the target polynucleotide; and selection from a library of chemical compounds created combinatorially or randomly.
  • a sample comprising a polynucleotide encoding NAAP is exposed to at least one test compound thus obtained.
  • the sample may comprise, for example, an intact or permeabilized cell, or an in vitro cell-free or reconstituted biochemical system.
  • Alterations in the expression of a polynucleotide encoding NAAP are assayed by any method commonly known in the art.
  • 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 fo ⁇ ning the basis for a comparison of the expression of the polynucleotide both with and without exposure to one or more test compounds. Detection of a change in the expression of a polynucleotide exposed to a test compound indicates that the test compound is effective in altering the expression of the polynucleotide.
  • a screen for a compound effective in altering expression of a specific polynucleotide can be carried out, for example, using a Schizosaccharomyces pombe gene expression system (Atkins, D. et al. (1999) U.S. Patent No. 5,932,435; Arndt, G.M. et al. (2000) Nucleic Acids Res. 28:E15) or a human cell line such as HeLa cell (Clarke, M.L. et al. (2000) Biochem. Biophys. Res. Commun. 268:8-13).
  • a Schizosaccharomyces pombe gene expression system (Atkins, D. et al. (1999) U.S. Patent No. 5,932,435; Arndt, G.M. et al. (2000) Nucleic Acids Res. 28:E15) or a human cell line such as HeLa cell (Clarke, M.L. et al. (2000) Biochem. Bio
  • a particular embodiment of the present invention involves screening a combinatorial library of oligonucleotides (such as deoxyribonucleotides, ribonucleotides, peptide nucleic acids, and modified oligonucleotides) for antisense activity against a specific polynucleotide sequence (Bruice, T.W. et al. (1997) U.S. Patent No. 5,686,242; Bruice, T.W. et al. (2000) U.S. Patent No. 6,022,691). Many methods for introducing vectors into cells or tissues are available and equally suitable for use in vivo, in vitro, and ex vivo.
  • oligonucleotides such as deoxyribonucleotides, ribonucleotides, peptide nucleic acids, and modified oligonucleotides
  • vectors may be introduced into stem cells taken from the patient and clonally propagated for autologous transplant back into that same patient. Delivery by transfection, by liposome injections, or by polycationic amino polymers may be achieved using methods which are well known in the art (Goldman, C.K. et al. (1997) Nat. Biotechnol. 15:462- 466).
  • compositions which generally comprises an active ingredient formulated with a pharmaceutically acceptable excipient.
  • Excipients may include, for example, sugars, starches, celluloses, gums, and proteins.
  • formulations are commonly known and are thoroughly discussed in the latest edition of Remington's Pharmaceutical Sciences (Maack Publishing, Easton PA).
  • Such compositions may consist of NAAP, antibodies to NAAP, and mimetics, agonists, antagonists, or inhibitors of NAAP.
  • compositions described herein may be administered by any number of routes including, but not limited to, oral, intravenous, intramuscular, intra-arterial, intramedullary, intrathecal, intraventricular, pulmonary, transdermal, subcutaneous, intraperitoneal, intranasal, enteral, topical, sublingual, or rectal means.
  • routes including, but not limited to, oral, intravenous, intramuscular, intra-arterial, intramedullary, intrathecal, intraventricular, pulmonary, transdermal, subcutaneous, intraperitoneal, intranasal, enteral, topical, sublingual, or rectal means.
  • Compositions for pulmonary administration may be prepared in liquid or dry powder form.
  • compositions are generally aerosolized immediately prior to inhalation by the patient.
  • aerosol delivery of fast-acting formulations is well-known in the art.
  • macromolecules e.g. larger peptides and proteins
  • Pulmonary delivery allows administration without needle injection, and obviates the need for potentially toxic penetration enhancers.
  • compositions suitable for use in the invention include compositions wherein the active ingredients are contained in an effective amount to achieve the intended purpose.
  • the determination of an effective dose is well within the capability of those skilled in the art.
  • compositions may be prepared for direct intracellular delivery of macromolecules comprising NAAP or fragments thereof.
  • liposome preparations containing a cell-impermeable macromolecule may promote cell fusion and intracellular delivery of the macromolecule.
  • NAAP or a fragment thereof may be joined to a short cationic N- terminal portion from the HTV Tat-1 protein. Fusion proteins thus generated have been found to transduce into the cells of all tissues, including the brain, in a mouse model system (Schwarze, S.R. et al. (1999) Science 285:1569-1572).
  • the therapeutically effective dose can be estimated initially either in cell culture assays, e.g., of neoplastic cells, or in animal models such as mice, rats, rabbits, dogs, monkeys, or pigs.
  • An animal model may also be used to determine the appropriate concentration range and route of administration. Such information can then be used to determine useful doses and routes for administration in humans.
  • a therapeutically effective dose refers to that amount of active ingredient, for example NAAP or fragments thereof, antibodies of NAAP, and agonists, antagonists or inhibitors of NAAP, which ameliorates the symptoms or condition.
  • Therapeutic efficacy and toxicity may be determined by standard pharmaceutical procedures in cell cultures or with experimental animals, such as by calculating the ED 50 (the dose therapeutically effective in 50% of the population) or LD 50 (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 LD 50 /ED 50 ratio.
  • Compositions which exhibit large therapeutic indices are preferred.
  • the data obtained from cell culture assays and animal studies are used to formulate a range of dosage for human use.
  • the dosage contained in such compositions is preferably within a range of circulating concentrations that includes the ED 50 with little or no toxicity. The dosage varies within this range depending upon the dosage form employed, the sensitivity of the patient, and the route of administration.
  • Dosage and administration are adjusted to provide sufficient levels of the active moiety or to maintain the desired effect. Factors which may be taken into account include the severity of the disease state, the general health of the subject, the age, weight, and gender of the subject, time and frequency of administration, drug combination(s), reaction sensitivities, and response to therapy. Long-acting compositions may be administered every 3 to 4 days, every week, or biweekly depending on the half -life and clearance rate of the particular formulation.
  • Normal dosage amounts may vary from about 0.1 ⁇ g to 100,000 ⁇ g, up to a total dose of about 1 gram, depending upon the route of administration.
  • Guidance as to particular dosages and methods of delivery is provided in the literature and generally available to practitioners in the art. Those skilled in the art will employ different formulations for nucleotides than for proteins or their inhibitors. Similarly, delivery of polynucleotides or polypeptides will be specific to particular cells, conditions, locations, etc.
  • DIAGNOSTICS hi another embodiment, antibodies which specifically bind NAAP may be used for the diagnosis of disorders characterized by expression of NAAP, or in assays to monitor patients being treated with NAAP or agonists, antagonists, or inhibitors of NAAP.
  • Antibodies useful for diagnostic purposes may be prepared in the same manner as described above for therapeutics. Diagnostic assays for NAAP include methods which utilize the antibody and a label to detect NAAP in human body fluids or in extracts of cells or tissues.
  • the antibodies may be used with or without modification, and may be labeled by covalent or non-covalent attachment of a reporter molecule.
  • reporter molecules A wide variety of reporter molecules, several of which are described above, are known in the art and may be used.
  • NAAP a variety of protocols for measuring NAAP, including ELISAs, RIAs, and FACS, are known in the art and provide a basis for diagnosing altered or abnormal levels of NAAP expression.
  • Normal or standard values for NAAP expression are established by combining body fluids or cell extracts taken from normal mammalian subjects, for example, human subjects, with antibodies to NAAP under conditions suitable for complex formation. The amount of standard complex formation may be quantitated by various methods, such as photometric means. Quantities of NAAP expressed in subject, control, and disease samples from biopsied tissues are compared with the standard values. Deviation between standard and subject values establishes the parameters for diagnosing disease.
  • polynucleotides encoding NAAP may be used for diagnostic purposes.
  • the polynucleotides which may be used include oligonucleotides, complementary RNA and DNA molecules, and PNAs.
  • the polynucleotides may be used to detect and quantify gene expression in biopsied tissues in which expression of NAAP may be correlated with disease.
  • the diagnostic assay may be used to determine absence, presence, and excess expression of NAAP, and to monitor regulation of NAAP levels during therapeutic intervention.
  • hybridization with PCR probes which are capable of detecting polynucleotides, including genomic sequences, encoding NAAP or closely related molecules may be used to identify nucleic acid sequences which encode NAAP.
  • the specificity of the probe whether it is made from a highly specific region, e.g., the 5' regulatory region, or from a less specific region, e.g., a conserved motif, and the stringency of the hybridization or amplification will determine whether the probe identifies only naturally occurring sequences encoding NAAP, allelic variants, or related sequences. Probes may also be used for the detection of related sequences, and may have at least 50% sequence identity to any of the NAAP encoding sequences.
  • the hybridization probes of the subject invention may be DNA or RNA and may be derived from the sequence of SEQ ID NO:57-l 12 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.
  • vectors are known in the art, are commercially available, and may be used to synthesize RNA probes in vitro by means of the addition of the appropriate RNA polymerases and the appropriate labeled nucleotides.
  • Hybridization probes may be labeled by a variety of reporter groups, for example, by radionuclides such as 32 P or 35 S, or by enzymatic labels, such as alkaline phosphatase coupled to the probe via avidin/biotin coupling systems, and the like.
  • Polynucleotides encoding NAAP may be used for the diagnosis of disorders associated with expression of NAAP.
  • disorders include, but are not limited to, a cell proliferative disorder such as actinic keratosis, arteriosclerosis, atherosclerosis, bursitis, cirrhosis, hepatitis, mixed connective tissue disease (MCTD), myelofibrosis, paroxysmal nocturnal hemoglobinuria, polycythemia vera, psoriasis, primary thrombocythemia, and cancers including adenocarcinoma, leukemia, lymphoma, melanoma, myeloma, sarcoma, teratocarcinoma, and, in particular, a cancer of the adrenal gland, bladder, bone, bone marrow, brain, breast, cervix, gall bladder, ganglia, gastrointestinal tract, heart, kidney, liver, lung, muscle, ovary, pancreas, par
  • Polynucleotides encoding NAAP may be used in Southern or northern analysis, dot blot, or other membrane-based technologies; in PCR technologies; in dipstick, pin, and multiformat ELISA-like assays; and in microarrays utilizing fluids or tissues from patients to detect altered NAAP expression. Such qualitative or quantitative methods are well known in the art.
  • 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.
  • the sample is washed and the signal is quantified and compared with a standard value. H 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.
  • 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.
  • a normal or standard profile for expression is established. This may be accomplished by combining body fluids or cell extracts taken from normal subjects, either animal or human, with a sequence, or a fragment thereof, encoding NAAP, under conditions suitable for hybridization or amplification. Standard hybridization may be quantified by comparing the values obtained from normal subjects with values from an experiment in which a known amount of a substantially purified polynucleotide is used. Standard values obtained in this manner may be compared with values obtained from samples from patients who are symptomatic for a disorder. Deviation from standard values is used to establish the presence of a disorder.
  • 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.
  • the presence of an abnormal amount of transcript (either under- or overexpressed) in biopsied tissue from an individual may indicate a predisposition for the development of the disease, or may provide a means for detecting the disease prior to the appearance of actual clinical symptoms.
  • a more definitive diagnosis of this type may allow health professionals to employ preventative measures or aggressive treatment earlier, thereby preventing the development or further progression of the cancer.
  • oligonucleotides designed from the sequences encoding NAAP may involve the use of PCR. These oligomers may be chemically synthesized, generated enzymatically, or produced in vitro. Oligomers will preferably contain a fragment of a polynucleotide encoding NAAP, or a fragment of a polynucleotide complementary to the polynucleotide encoding NAAP, and will be employed under optimized conditions for identification of a specific gene or condition. Oligomers may also be employed under less stringent conditions for detection or quantification of closely related DNA or RNA sequences.
  • oligonucleotide primers derived from polynucleotides encoding NAAP may be used to detect single nucleotide polymorphisms (SNPs).
  • SNPs are substitutions, insertions and deletions that are a frequent cause of inherited or acquired genetic disease in humans.
  • Methods of SNP detection include, but are not limited to, single-stranded conformation polymorphism (SSCP) and fluorescent SSCP (fSSCP) methods.
  • SSCP single-stranded conformation polymorphism
  • fSSCP fluorescent SSCP
  • oligonucleotide primers derived from polynucleotides encoding NAAP are used to amplify DNA using the polymerase chain reaction (PCR).
  • the DNA may be derived, for example, from diseased or normal tissue, biopsy samples, bodily fluids, and the like.
  • SNPs in the DNA cause differences in the secondary and tertiary structures of PCR products in single-stranded form, and these differences are detectable using gel electrophoresis in non-denaturing gels.
  • the oligonucleotide primers are fluorescently labeled, which allows detection of the amplimers in high-throughput equipment such as DNA sequencing machines.
  • sequence database analysis methods termed in silico SNP (isSNP) are capable of identifying polymorphisms by comparing the sequence of individual overlapping DNA fragments which assemble into a common consensus sequence.
  • SNPs may be detected and characterized by mass spectrometry using, for example, the high throughput MASSARRAY system (Sequenom, Inc., San Diego CA).
  • SNPs may be used to study the genetic basis of human disease. For example, at least 16 common SNPs have been associated with non-insulin-dependent diabetes mellitus. SNPs are also useful for examining differences in disease outcomes in monogenic disorders, such as cystic fibrosis, sickle cell anemia, or chronic granulomatous disease. For example, variants in the mannose-binding lectin, MBL2, have been shown to be correlated with deleterious pulmonary outcomes in cystic fibrosis. SNPs also have utility in pharmacogenomics, the identification of genetic variants that influence a patient's response to a drug, such as life-threatening toxicity.
  • N-acetyl transferase is associated with a high incidence of peripheral neuropathy in response to the anti-tuberculosis drug isoniazid, while a variation in the core promoter of the ALOX5 gene results in diminished clinical response to treatment with an anti-asthma drug that targets the 5-lipoxygenase pathway.
  • Analysis of the distribution of SNPs in different populations is useful for investigating genetic drift, mutation, recombination, and selection, as well as for tracing the origins of populations and their migrations (Taylor, J.G. et al. (2001) Trends Mol. Med. 7:507-512; Kwok, P.-Y. and Z. Gu (1999) Mol. Med.
  • Methods which may also be used to quantify the expression of NAAP include radiolabeling or biotinylating nucleotides, coamplification of a control nucleic acid, and interpolating results from standard curves (Melby, P.C et al. (1993) J. 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 high-throughput format where the oligomer or polynucleotide of interest is presented in various dilutions and a spectrophotometric or colorimetric response gives rapid quantitation.
  • oligonucleotides or longer fragments derived from any of the polynucleotides described herein may be used as elements on a microarray.
  • the microarray can be used in transcript imaging techniques which monitor the relative expression levels of large numbers of genes simultaneously as described below.
  • the microarray may also be used to identify genetic variants, mutations, and polymorphisms. This information may be used to determine gene function, to understand the genetic basis of a disorder, to diagnose a disorder, to monitor progression/regression of disease as a function of gene expression, and to develop and monitor the activities of therapeutic agents in the treatment of disease. In particular, this information may be used to develop a pharmacogenomic profile of a patient in order to select the most appropriate and effective treatment regimen for that patient. For example, therapeutic agents which are highly effective and display the fewest side effects may be selected for a patient based on his/her pharmacogenomic profile.
  • NAAP fragments of NAAP, or antibodies specific for NAAP may be used as elements on a microarray.
  • the microarray may be used to monitor or measure protein-protein interactions, drug-target interactions, and gene expression profiles, as described above.
  • a particular embodiment relates to the use of the polynucleotides of the present invention to generate a transcript image of a tissue or cell type.
  • a transcript image represents the global pattern of gene expression by a particular tissue or cell type. Global gene expression patterns are analyzed by quantifying the number of expressed genes and their relative abundance under given conditions and at a given time (Seilhamer et al., "Comparative Gene Transcript Analysis," U.S. Patent No. 5,840,484; hereby expressly incorporated by reference herein).
  • a transcript image may be generated by hybridizing the polynucleotides of the present invention or their complements to the totality of transcripts or reverse transcripts of a particular tissue or cell type.
  • the hybridization takes place in high-throughput format, wherein the polynucleotides of the present invention or their complements comprise a subset of a plurality of elements on a microarray.
  • the resultant transcript image would provide a profile of gene activity.
  • Transcript images may be generated using transcripts isolated from tissues, cell lines, biopsies, or other biological samples. The transcript image may thus reflect gene expression in vivo, as in the case of a tissue or biopsy sample, or in vitro, as in the case of a cell line.
  • Transcript images which profile the expression of the polynucleotides of the present invention may also be used in conjunction with in vitro model systems and preclinical evaluation of pharmaceuticals, as well as toxicological testing of industrial and naturally-occurring environmental compounds. All compounds induce characteristic gene expression patterns, frequently termed molecular fingerprints or toxicant signatures, which are indicative of mechanisms of action and toxicity (Nuwaysir, E.F. et al. (1999) Mol. Carcinog. 24:153-159; Steiner, S. and N.L. Anderson (2000) Toxicol. Lett. 112-113:467-471). If a test compound has a signature similar to that of a compound with known toxicity, it is likely to share those toxic properties.
  • the toxicity of a test compound can be assessed by treating a biological sample containing nucleic acids with the test compound.
  • Nucleic acids that are expressed in the treated biological sample are hybridized with one or more probes specific to the polynucleotides of the present invention, so that transcript levels corresponding to the polynucleotides of the present invention may be quantified.
  • the transcript levels in the treated biological sample are compared with levels in an untreated biological sample. Differences in the transcript levels between the two samples are indicative of a toxic response caused by the test compound in the treated sample.
  • Another embodiment relates to the use of the polypeptides disclosed herein to analyze the proteome of a tissue or cell type.
  • proteome refers to the global pattern of protein expression in a particular tissue or cell type.
  • proteome expression patterns, or profiles are analyzed by quantifying the number of expressed proteins and their relative abundance under given conditions and at a given time.
  • a profile of a cell's proteome may thus be generated by separating and analyzing the polypeptides of a particular tissue or cell type.
  • the separation is achieved using two-dimensional gel electrophoresis, in which proteins from a sample are separated by isoelectric focusing in the first dimension, and then according to molecular weight by sodium dodecyl sulfate slab gel electrophoresis in the second dimension (Steiner and Anderson, supra).
  • the proteins are visualized in the gel as discrete and uniquely positioned spots, typically by staining the gel with an agent such as Coomassie Blue or silver or fluorescent stains.
  • the optical density of each protein spot is generally proportional to the level of the protein in the sample.
  • the optical densities of equivalently positioned protein spots from different samples, for example, from biological samples either treated or untreated with a test compound or therapeutic agent, are compared to identify any changes in protein spot density related to the treatment.
  • the proteins in the spots are partially sequenced using, for example, standard methods employing chemical or enzymatic cleavage followed by mass spectrometry.
  • the identity of the protein in a spot may be determined by comparing its partial sequence, preferably of at least 5 contiguous amino acid residues, to the polypeptide sequences of 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.
  • the antibodies are used as elements on a microarray, and protein expression levels are quantified by exposing the microarray to the sample and detecting the levels of protein bound to each array element (Lueking, A. et al. (1999) Anal. Biochem. 270: 103-111; Mendoze, L.G. et al. (1999) Biotechniques 27:778-788). Detection may be performed by a variety of methods known in the art, for example, by reacting the proteins in the sample with a thiol- or amino-reactive fluorescent compound and detecting the amount of fluorescence bound at each array element.
  • Toxicant signatures at the proteome level are also useful for toxicological screening, and should be analyzed in parallel with toxicant signatures at the transcript level.
  • There is a poor correlation between transcript and protein abundances for some proteins in some tissues (Anderson, N.L. and J. Seilhamer (1997) Electrophoresis 18:533-537), so proteome toxicant signatures may be useful in the analysis of compounds which do not significantly affect the transcript image, but which alter the proteomic profile.
  • the analysis of transcripts in body fluids is difficult, due to rapid degradation of mRNA, so proteomic profiling may be more reliable and informative in such cases.
  • the toxicity of a test compound is assessed by treating a biological sample containing proteins with the test compound.
  • Proteins that are expressed in the treated biological sample are separated so that the amount of each protein can be quantified.
  • the amount of each protein is compared to the amount of the corresponding protein in an untreated biological sample. A difference in the amount of protein between the two samples is indicative of a toxic response to the test compound in the treated sample.
  • Individual proteins are identified by sequencing the amino acid residues of the individual proteins and comparing these partial sequences to the polypeptides of the present invention.
  • 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 application W095/251116; Shalon, D. et al.
  • nucleic acid sequences encoding NAAP may be used to generate hybridization probes useful in mapping the naturally occurring genomic sequence. Either coding or noncoding sequences may be used, and in some instances, noncoding sequences may be preferable over coding sequences.
  • 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 libraries (Harrington, J.J. et al. (1997) Nat. Genet. 15:345-355; Price, CM. (1993) Blood Rev. 7:127-134; Trask, B.J.
  • HACs human artificial chromosomes
  • YACs yeast artificial chromosomes
  • BACs bacterial artificial chromosomes
  • nucleic acid sequences may be used to develop genetic linkage maps, for example, which correlate the inheritance of a disease state with the inheritance of a particular chromosome region or restriction fragment length polymorphism (RFLP) (Lander, E.S. and D. Botstein (1986) Proc. Natl. Acad. Sci. USA 83:7353-7357).
  • RFLP restriction fragment length polymorphism
  • Fluorescent in situ hybridization may be correlated with other physical and genetic map data (Heinz-Ulrich, et al. (1995) in Meyers, supra, pp. 965-968). Examples of genetic map data can be found in various scientific journals or at the Online Mendelian Inheritance in Man (OMEVI) World Wide Web site. Correlation between the location of the gene encoding NAAP on a physical map and a specific disorder, or a predisposition to a specific disorder, may help define the region of DNA associated with that disorder and thus may further positional cloning efforts. In situ hybridization of chromosomal preparations and physical mapping techniques, such as linkage analysis using established chromosomal markers, may be used for extending genetic maps.
  • a gene on the chromosome of another mammalian species 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.
  • the gene or genes responsible for a disease or syndrome have been crudely localized by genetic linkage to a particular genomic region, e.g., ataxia-telangiectasia to 1 lq22-23, 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.
  • NAAP its catalytic or immunogenic fragments, or oligopeptides thereof can be used for screening libraries of compounds in any of a variety of drug screening techniques.
  • the fragment employed in such screening may be free in solution, affixed to a solid support, borne on a cell surface, or located intracellularly. The formation of binding complexes between NAAP and the agent being tested may be measured.
  • Another technique for drug screening provides for high throughput screening of compounds having suitable binding affinity to the protein of interest (Geysen, et al. (1984) PCT application WO84/03564).
  • This method large numbers of different small test compounds are synthesized on a solid substrate. The test compounds are reacted with NAAP, or fragments thereof, and washed.
  • Bound NAAP is then detected by methods well known in the art. Purified NAAP can also be coated directly onto plates for use in the aforementioned drug screening techniques. Alternatively, non-neutralizing antibodies can be used to capture the peptide and immobilize it on a solid support.
  • 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.
  • Incyte cDNAs were derived from cDNA libraries described in the LEFESEQ 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.
  • TRIZOL Invitrogen
  • poly(A)+ RNA was isolated using oligo d(T)-coupled paramagnetic particles (Promega), OLIGOTEX latex particles (QIAGEN, Chatsworth CA), or an OLIGOTEX mRNA purification kit (QIAGEN).
  • Stratagene was provided with RNA and constructed the corresponding cDNA libraries.
  • cDNA was synthesized and cDNA libraries 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 oligo d(T) or random primers. Synthetic oligonucleotide adapters were ligated to double stranded cDNA, and the cDNA was digested with the appropriate restriction enzyme or enzymes.
  • the cDNA was size-selected (300-1000 bp) using SEPHACRYL S1000, SEPHAROSE CL2B, or SEPHAROSE CL4B column chromatography (Amersham Biosciences) or preparative agarose gel electrophoresis.
  • cDNAs were ligated into compatible restriction enzyme sites of the polylinker of a suitable plasmid, e.g., PBLUESCRIPT plasmid (Stratagene), PSPORT1 plasmid (Invitrogen, 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 cells including XLl-Blue, XL1- BlueMRF, or SOLR from Stratagene or DH5 ⁇ , DH10B, or ElectroMAX DH10B from Invitrogen.
  • Plasmids obtained as described in Example I were recovered from host cells by in vivo excision using the UNLZAP vector system (Stratagene) or by cell lysis. Plasmids were purified using at least one of the following: a Magic or WIZARD Minipreps DNA purification system (Promega); an AGTC Miniprep purification kit (Edge Biosystems, Gaithersburg MD); and QIAWELL 8 Plasmid, QIAWELL 8 Plus Plasmid, QIAWELL 8 Ultra Plasmid purification systems or the R.E. A.L. PREP 96 plasmid purification kit from QIAGEN.
  • a Magic or WIZARD Minipreps DNA purification system Promega
  • an AGTC Miniprep purification kit Edge Biosystems, Gaithersburg MD
  • plasmids were resuspended in 0.1 ml of distilled water and stored, with or without lyophilization, at 4°C Alternatively, plasmid DNA was amplified from host cell lysates using direct link PCR in a high-throughput format (Rao, V.B. (1994) Anal. Biochem. 216: 1-14). Host cell lysis and thermal cycling steps were carried out in a single reaction mixture. Samples were processed and stored in 384-well plates, and the concentration of amplified plasmid DNA was quantified fluorometrically using PICOGREEN dye (Molecular Probes, Eugene OR) and a FLUOROSKAN H fluorescence scanner (Labsystems Oy, Helsinki, Finland).
  • PICOGREEN dye Molecular Probes, Eugene OR
  • FLUOROSKAN H fluorescence scanner Labsystems Oy, Helsinki, Finland.
  • Incyte cDNA recovered in plasmids as described in Example H were sequenced as follows. Sequencing reactions were processed using standard methods or high-throughput instrumentation such as the ABI CATALYST 800 (Applied Biosystems) thermal cycler or the PTC-200 thermal cycler (MJ Research) in conjunction with the HYDRA microdispenser (Robbins Scientific) or the MICROLAB 2200 (Hamilton) liquid transfer system. cDNA sequencing reactions were prepared using reagents provided by Amersham Biosciences or supplied in ABI sequencing kits such as the ABI PRISM BIGDYE Terminator cycle sequencing ready reaction kit (Applied Biosystems).
  • Electrophoretic separation of cDNA sequencing reactions and detection of labeled polynucleotides were carried out using the MEGABACE 1000 DNA sequencing system (Amersham Biosciences); the ABI PRISM 373 or 377 sequencing system (Applied Biosystems) in conjunction with standard ABI protocols and base calling software; or other sequence analysis systems known in the art. Reading frames within the cDNA sequences were 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 VHI.
  • the polynucleotide sequences derived from Incyte cDNAs were validated by removing vector, linker, and poly (A) sequences and by masking ambiguous bases, using algorithms and programs based on BLAST, dynamic programming, and dinucleotide nearest neighbor analysis.
  • the Incyte cDNA sequences or translations thereof were then queried against a selection of public databases such as the GenBank primate, rodent, mammalian, vertebrate, and eukaryote databases, and BLOCKS, PRINTS, DOMO, PRODOM; PROTEOME databases with sequences from Homo sapiens, Rattus norvegicus, Mus musculus, Caenorhabditis elegans, Saccharomyces cerevisiae, Schizosaccharomyces pombe, and Candida albicans (Incyte Genomics, Palo Alto CA); hidden Markov model (HMM)-based protein family databases such as PFAM, INCY, and TIGRFAM (Haft, D.H.
  • HMM hidden Markov model
  • HMM-based protein domain databases such as SMART (Schultz, J. et al. (1998) Proc. Natl. Acad. Sci. USA 95:5857-5864; Letunic, I. et al. (2002) Nucleic Acids Res. 30:242-244).
  • HMM is a probabilistic approach which analyzes consensus primary structures of gene families; see, for example, Eddy, S.R. (1996) Curr. Opin. Struct. Biol. 6:361-365.
  • the queries were performed using programs based on BLAST, FASTA, BLIMPS, and HMMER.
  • Incyte cDNA sequences were assembled to produce full length polynucleotide sequences.
  • GenBank cDNAs, GenBank ESTs, stitched sequences, stretched sequences, or Genscan-predicted coding sequences were used to extend Incyte cDNA assemblages to full length. Assembly was performed using programs based on Phred, Phrap, and Consed, and cDNA assemblages were screened for open reading frames using programs based on
  • Table 7 summarizes the tools, programs, and algorithms used for the analysis and assembly of Incyte cDNA and full length sequences and provides applicable descriptions, references, and threshold parameters.
  • the first column of Table 7 shows the tools, programs, and algorithms used, the second column provides brief descriptions thereof, the third column presents appropriate references, all of which are incorporated by reference herein in their entirety, and the fourth column presents, where applicable, the scores, probability values, and other parameters used to evaluate the strength of a match between two sequences (the higher the score or the lower the probability value, the greater the identity between two sequences).
  • 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.
  • Genscan 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 protems were also identified by homology to Incyte cDNA sequences that had been annotated as nucleic acid-associated proteins. These selected Genscan-predicted sequences were then compared by BLAST analysis to the genpept and gbpri public databases.
  • Genscan-predicted sequences were then edited by comparison to the top BLAST hit from genpept to correct errors in the sequence predicted by Genscan, such as extra or omitted exons.
  • BLAST analysis was also used to find any Incyte cDNA or public cDNA coverage of the Genscan-predicted sequences, thus providing evidence for transcription. When Incyte cDNA coverage was available, this information was used to correct or confirm the Genscan predicted sequence.
  • Full length polynucleotide sequences were obtained by assembling Genscan-predicted coding sequences with Incyte cDNA sequences and/or public cDNA sequences using the assembly process described in Example HI. Alternatively, full length polynucleotide sequences were derived entirely from edited or unedited Genscan-predicted coding 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 EH were mapped to genomic DNA and parsed into clusters containing related cDNAs and Genscan exon predictions from one or more genomic sequences. Each cluster was analyzed using an algorithm based on graph theory and dynamic programming to integrate cDNA and genomic information, generating possible splice variants that were subsequently confirmed, edited, or extended to create a full length sequence. Sequence intervals in which the entire length of the interval was present on more than one sequence in the cluster were identified, and intervals thus identified were considered to be equivalent by transitivity.
  • Partial DNA sequences were extended to full length with an algorithm based on BLAST analysis.
  • GenBank primate a GenBank primate
  • rodent a rodent
  • mammalian a mammalian
  • vertebrate eukaryote databases
  • eukaryote databases using the BLAST program.
  • 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.
  • HSPs high-scoring segment
  • GenBank protein homolog The GenBank protein homolog, the chimeric protein, or both were used as probes to search for homologous genomic sequences from the public human genome databases. Partial DNA sequences were therefore "stretched” or extended by the addition of homologous genomic sequences. The resultant stretched sequences were examined to determine whether it contained a complete gene. VI. Chromosomal Mapping of NAAP Encoding Polynucleotides The sequences which were used to assemble SEQ ID NO: 57-112 were compared with sequences from the Incyte LEFESEQ database and public domain databases using BLAST and other implementations of the Smith-Waterman algorithm.
  • centiMorgans The map position of an interval, in centiMorgans, is measured relative to the terminus of the chromosome's p- arm.
  • centiMorgan 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.
  • Mb megabase
  • 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.
  • Northern analysis is a laboratory technique used to detect the presence of a transcript of a gene and involves the hybridization of a labeled nucleotide sequence to a membrane on which RNAs from a particular cell type or tissue have been bound (Sambrook and Russell, 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.
  • 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:
  • the product score takes into account both the degree of similarity between two sequences and the length of the sequence match.
  • the product score is a normalized value between 0 and 100, and is calculated as follows: the BLAST score is multiplied by the percent nucleotide identity and the product is divided by (5 times the length of the shorter of the two sequences).
  • the BLAST score is calculated by assigning a score of +5 for every base that matches in a high-scoring segment pair (HSP), and -4 for every mismatch. Two sequences may share more than one HSP (separated by gaps). H there is more than one HSP, then the pair with the highest BLAST score is used to calculate the product score.
  • the product score represents a balance between fractional overlap and quality in a BLAST alignment.
  • 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.
  • 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 IH). Each cDNA sequence is derived from a cDNA library constructed from a human tissue.
  • Each human tissue is classified into one of the following organ/tissue categories: cardiovascular system; connective tissue; digestive system; embryonic structures; endocrine system; exocrine glands; genitalia, female; genitalia, male; germ cells; hemic and immune system; liver; musculoskeletal system; nervous system; pancreas; respiratory system; sense organs; skin; stomatognathic system; unclassified/mixed; or urinary tract.
  • the number of libraries in each category is counted and divided by the total number of libraries across all categories.
  • each human tissue is classified into one of the following disease/condition categories: cancer, cell line, developmental, inflammation, neurological, trauma, cardiovascular, pooled, and other, and the number of libraries in each category is counted and divided by the total number of libraries across all categories. The resulting percentages reflect the tissue- and disease-specific expression of cDNA encoding NAAP.
  • cDNA sequences and cDNA library/tissue information are found in the LLFESEQ GOLD database (Incyte Genomics, Palo Alto CA). VIII. Extension of NAAP Encoding Polynucleotides
  • Full length polynucleotides are produced by extension of an appropriate fragment of the full length molecule using oligonucleotide primers designed from this fragment.
  • One primer was synthesized to initiate 5' extension of the known fragment, and the other primer was synthesized to initiate 3' extension of the known fragment.
  • the initial primers were designed using OLIGO 4.06 software (National Biosciences), or another appropriate program, to be about 22 to 30 nucleotides in length, to have a GC content of about 50% or more, and to anneal to the target sequence at temperatures of about 68 °C to about 72°C. Any stretch of nucleotides which would result in hairpin structures and primer-primer dimerizations was avoided.
  • Selected human cDNA libraries were used to extend the sequence. If more than one extension was necessary or desired, additional or nested sets of primers were designed. High fidelity amplification was obtained by PCR using methods well known in the art. PCR was performed in 96-well plates using the PTC-200 thermal cycler (MJ Research, Inc.).
  • the reaction mix contained DNA template, 200 nmol of each primer, reaction buffer containing Mg 2+ , (NH 4 ) 2 S0 4 , and 2-mercaptoethanol, Taq DNA polymerase (Amersham Biosciences), ELONGASE enzyme (Invitrogen), and Pfu DNA polymerase (Stratagene), with the following parameters for primer pair PCI A and 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
  • the parameters for primer pair T7 and SK+ were as follows: Step 1: 94°C, 3 min; Step 2: 94°C, 15 sec; Step 3: 57°C, 1 min; Step 4: 68°C, 2 min; Step 5: Steps 2, 3, and 4 repeated 20 times; Step 6: 68°C
  • the plate was scanned in a Fluoroskan H (Labsystems Oy, Helsinki, Finland) to measure the fluorescence of the sample and to quantify the concentration of DNA.
  • a 5 ⁇ l to 10 ⁇ l aliquot of the reaction mixture was analyzed by electrophoresis on a 1 % agarose gel to determine which reactions were successful in extending the sequence.
  • the extended nucleotides were desalted and concentrated, transferred to 384-well plates, digested with CviJI cholera virus endonuclease (Molecular Biology Research, Madison Wl), and sonicated or sheared prior to religation into pUC 18 vector (Amersham Biosciences).
  • CviJI cholera virus endonuclease Molecular Biology Research, Madison Wl
  • sonicated or sheared prior to religation into pUC 18 vector
  • the digested nucleotides were separated on low concentration (0.6 to 0.8%) agarose gels, fragments were excised, and agar digested with Agar ACE (Promega).
  • Extended clones were religated using T4 ligase (New England Biolabs, Beverly MA) into pUC 18 vector (Amersham Biosciences), treated with Pfu DNA polymerase (Stratagene) to fill-in restriction site overhangs, and transfected into competent E. coli cells. Transformed cells were selected on antibiotic-containing media, and individual colonies were picked and cultured overnight at 37° C in 384-well plates in LB/2x carb liquid media.
  • the cells were lysed, and DNA was amplified by PCR using Taq DNA polymerase (Amersham Biosciences) and Pfu DNA polymerase (Stratagene) with the following parameters: Step 1: 94°C, 3 min; Step 2: 94°C, 15 sec; Step 3: 60°C, 1 min; Step 4: 72°C, 2 min; Step 5: steps 2, 3, and 4 repeated 29 times; Step 6: 72 C C, 5 min; Step 7: storage at 4°C DNA was quantified by PICOGREEN reagent (Molecular Probes) as described above. Samples with low DNA recoveries were reamplified using the same conditions as described above.
  • SNPs single nucleotide polymorphisms
  • LEFESEQ database Incyte Genomics
  • Sequences from the same gene were clustered together and assembled as described in Example HI, allowing the identification of all sequence variants in the gene.
  • An algorithm consisting of a series of filters was used to distinguish SNPs from other sequence variants. Preliminary filters removed the majority of basecall errors by requiring a minimum Phred quality score of 15, and removed sequence alignment errors and errors resulting from improper trimming of vector sequences, chimeras, and splice variants.
  • An automated procedure of advanced chromosome analysis analysed the original chromatogram files in the vicinity of the putative SNP.
  • Clone error filters used statistically generated algorithms to identify errors introduced during laboratory processing, such as those caused by reverse transcriptase, polymerase, or somatic mutation.
  • Clustering error filters used statistically generated algorithms to identify errors resulting from clustering of close homologs or pseudogenes, or due to contamination by non-human sequences. A final set of filters removed duplicates and SNPs found in immunoglobulins or T-cell receptors.
  • Certain SNPs were selected for further characterization by mass spectrometry using the high throughput MASSARRAY system (Sequenom, Inc.) to analyze allele frequencies at the SNP sites in four different human populations.
  • the Caucasian population comprised 92 individuals (46 male, 46 female), including 83 from Utah, four French, three deciualan, and two Amish individuals.
  • the African population comprised 194 individuals (97 male, 97 female), all African Americans.
  • the Hispanic population comprised 324 individuals (162 male, 162 female), all Mexican Hispanic.
  • the Asian population comprised 126 individuals (64 male, 62 female) with a reported parental breakdown of 43% Chinese, 31% Japanese, 13% Korean, 5% Vietnamese, and 8% other Asian. Allele frequencies were first analyzed in the Caucasian population; in some cases those SNPs which showed no allelic variance in this population were not further tested in the other three populations.
  • Hybridization probes derived from SEQ ID NO:57-l 12 are employed to screen cDNAs, genomic DNAs, or mRNAs. Although the labeling of oligonucleotides, consisting of about 20 base pairs, is specifically described, essentially the same procedure is used with larger nucleotide fragments. Oligonucleotides are designed using state-of-the-art software such as OLIGO 4.06 software (National Biosciences) and labeled by combining 50 pmol of each oligomer, 250 ⁇ Ci of [ ⁇ - 32 P] adenosine triphosphate (Amersham Biosciences), and T4 polynucleotide kinase (DuPont NEN, Boston MA).
  • the labeled oligonucleotides are substantially purified using a SEPHADEX G-25 superfine size exclusion dextran bead column (Amersham Biosciences). An aliquot containing IO 7 counts per minute of the labeled probe is used in a typical membrane-based hybridization analysis of human genomic DNA digested with one of the following endonucleases: Ase I, Bgl H, Eco Rl, Pst I, Xba I, or Pvu H (DuPont NEN).
  • the DNA from each digest is fractionated on a 0.7% agarose gel and transferred to nylon membranes (Nytran Plus, Schleicher & Schuell, Durham NH). Hybridization is carried out for 16 hours at 40°C. To remove nonspecific signals, blots are sequentially washed at room temperature under conditions of up to, for example, 0.1 x saline sodium citrate and 0.5% sodium dodecyl sulfate. Hybridization patterns are visualized using autoradiography or an alternative imaging means and compared. XI. Microarrays
  • the linkage or synthesis of array elements upon a microarray can be achieved utilizing photolithography, piezoelectric printing (ink-jet printing; see, e.g., Baldeschweiler et al., supra), mechanical microspotting technologies, and derivatives thereof.
  • the substrate in each of the aforementioned technologies should be uniform and solid with a non-porous surface (Schena, M., ed. (1999) DNA Microarrays: A Practical Approach. Oxford University Press, London). Suggested substrates include silicon, silica, glass slides, glass chips, and silicon wafers.
  • a procedure analogous to a dot or slot blot may also be used to arrange and link elements to the surface of a substrate using thermal, UV, chemical, or mechanical bonding procedures.
  • a typical array may be produced using available methods and machines well known to those of ordinary skill in the art and may contain any appropriate number of elements (Schena, M. et al. (1995) Science 270:467-470; Shalon, D. et al. (1996) Genome Res. 6:639-645; Marshall, A. and J. Hodgson (1998) Nat. Biotechnol. 16:27-31).
  • Full length cDNAs, Expressed Sequence Tags (ESTs), or fragments or oligomers thereof may comprise the elements of the microarray. Fragments or oligomers suitable for hybridization can be selected using software well known in the art such as LASERGENE software (DNASTAR).
  • the array elements are hybridized with polynucleotides in a biological sample.
  • the polynucleotides in the biological sample are conjugated to a fluorescent label or other molecular tag for ease of detection.
  • a fluorescence scanner is used to detect hybridization at each array element.
  • laser desorbtion and mass spectrometry may be used for detection of hybridization.
  • the degree of complementarity and the relative abundance of each polynucleotide which hybridizes to an element on the microarray may be assessed.
  • microarray preparation and usage is described in detail below.
  • Total RNA is isolated from tissue samples using the guanidinium thiocyanate method and poly(A) + RNA is purified using the oligo-(dT) cellulose method.
  • Each poly (A) + RNA sample is reverse transcribed using MMLV reverse-transcriptase, 0.05 pg/ ⁇ l oligo-(dT) primer (21mer), IX first strand buffer, 0.03 units/ ⁇ l RNase inhibitor, 500 ⁇ M dATP, 500 ⁇ M dGTP, 500 ⁇ M dTTP, 40 ⁇ M dCTP, 40 ⁇ M 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 ⁇ l 5X SSC/0.2% SDS.
  • SpeedVAC SpeedVAC
  • Sequences of the present invention are used to generate array elements.
  • Each array element is amplified from bacterial cells containing vectors with cloned cDNA inserts.
  • PCR amplification uses primers complementary to the vector sequences flanking the cDNA insert.
  • Array elements are amplified in thirty cycles of PCR from an initial quantity of 1-2 ng to a final quantity greater than 5 ⁇ g. Amplified array elements are then purified using SEPHACRYL-400 (Amersham Biosciences).
  • Purified array elements are immobilized on polymer-coated glass slides.
  • Glass microscope slides (Corning) are cleaned by ultrasound in 0.1% SDS and acetone, with extensive distilled water washes between and after treatments.
  • Glass slides are etched in 4% hydrofluoric acid (VWR Scientific Products Corporation (VWR), West Chester PA), washed extensively in distilled water, and coated with 0.05% aminopropyl silane (Sigma) in 95% ethanol. Coated slides are cured in a 110°C oven.
  • Array elements are applied to the coated glass substrate using a procedure described in U.S. Patent No. 5,807,522, incorporated herein by reference.
  • 1 ⁇ l of the array element DNA, at an average concentration of 100 ng/ ⁇ l, is loaded into the open capillary printing element by a high-speed robotic apparatus. The apparatus then deposits about 5 nl of array element sample per slide.
  • Microarrays are UV-crosslinked using a STRATALINKER UV-crosslinker (Stratagene). Microarrays are washed at room temperature once in 0.2% SDS and three times in distilled water. Non-specific binding sites are blocked by incubation of microarrays in 0.2% casein in phosphate buffered saline (PBS) (Tropix, Inc., Bedford MA) for 30 minutes at 60° C followed by washes in 0.2% SDS and distilled water as before. Hybridization
  • Hybridization reactions contain 9 ⁇ l of sample mixture consisting of 0.2 ⁇ g each of Cy3 and Cy5 labeled cDNA synthesis products in 5X SSC, 0.2% SDS hybridization buffer.
  • the sample mixture is heated to 65° C for 5 minutes and is aliquoted onto the microarray surface and covered with an 1.8 cm 2 coverslip.
  • the arrays are transferred to a waterproof chamber having a cavity just slightly larger than a microscope slide.
  • the chamber is kept at 100% humidity internally by the addition of 140 ⁇ l of 5X SSC in a corner of the chamber.
  • the chamber containing the arrays is incubated for about 6.5 hours at 60° C.
  • the arrays are washed for 10 min at 45° C in a first wash buffer (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 light is focused on the array using a 20X microscope objective (Nikon, Inc., Melville NY).
  • the slide containing the array is placed on a computer-controlled X-Y stage on the microscope and raster- scanned past the objective.
  • the 1.8 cm x 1.8 cm array used in the present example is scanned with a resolution of 20 micrometers.
  • a mixed gas multiline laser excites the two fluorophores sequentially. Emitted light is split, based on wavelength, into two photomultiplier tube detectors (PMT R1477, Hamamatsu Photonics Systems, Bridgewater NJ) corresponding to the two fluorophores. Appropriate filters positioned between the array and the photomultiplier tubes are used to filter the signals.
  • the emission maxima of the fluorophores used are 565 nm for Cy3 and 650 nm for Cy5.
  • Each array is typically scanned twice, one scan per fluorophore using the appropriate filters at the laser source, although the apparatus is capable of recording the spectra from both fluorophores simultaneously.
  • the sensitivity of the scans is typically calibrated using the signal intensity generated by a cDNA control species added to the sample mixture at a known concentration.
  • a specific location on the array contains a complementary DNA sequence, allowing the intensity of the signal at that location to be correlated with a weight ratio of hybridizing species of 1 : 100,000.
  • the calibration is done by labeling samples of the calibrating cDNA with the two fluorophores and adding identical amounts of each to the hybridization mixture.
  • the output of the photomultiplier tube is digitized using a 12-bit RTI-835H analog-to-digital (AID) conversion board (Analog Devices, Inc., Norwood MA) installed in an IBM-compatible PC computer.
  • the digitized data are displayed as an image where the signal intensity is mapped using a linear 20-color transformation to a pseudocolor scale ranging from blue (low signal) to red (high signal).
  • the data is also analyzed quantitatively. Where two different fluorophores are excited and measured simultaneously, the data are first corrected for optical crosstalk (due to overlapping emission spectra) between the fluorophores using each fluorophore' s emission spectrum.
  • a grid is superimposed over the fluorescence signal image such that the signal from each spot is centered in each element of the grid.
  • the fluorescence signal within each element is then integrated to obtain a numerical value corresponding to the average intensity of the signal.
  • the software used for signal analysis is the GEMTOOLS gene expression analysis program (Incyte 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 differentially expressed.
  • peripheral blood mononuclear cells For example, expression of SEQ ED NO:64 was decreased in peripheral blood mononuclear cells by a factor of 2 or more after treatment with granulocyte-monocyte colony stimulating factor (GM-CSF), and was also decreased in peripheral blood mononuclear cells by a factor of 2 or more after treatment with leukemia inhibitory factor (LIF) as determined by microarray analysis.
  • GM-CSF granulocyte-monocyte colony stimulating factor
  • LIF leukemia inhibitory factor
  • peripheral blood mononuclear cells are isolated from freshly obtained peripheral blood of six healthy donors. PBMCs from each donor were placed in culture for 2 hours in the presence or absence of recombinant GM-CSF. GM-CSF treated PBMCs and untreated control PBMCs from the different donors are pooled according to their respective treatments.
  • peripheral blood mononuclear cells are isolated from freshly obtained peripheral blood of six healthy donors. PBMCs from each donor are placed in culture for 2 hours in the presence or absence of recombinant LIF. LTF-treated PBMCs and untreated control PBMCs from the different donors are pooled according to their respective treatments. Therefore, SEQ ID NO:64 is useful in diagnostic assays for cell proliferative disorders and autoimmune/inflammation disorders.
  • SEQ ED NO:76 showed increased expression in HAEC cells treated with TNF- ⁇ versus untreated cells as determined by microarray analysis. HAECs are grown to 85% confluency and then treated with TNF- ⁇ for 1, 2, 4, 6, 8, 10, 24, and 48 hours. These TNF- ⁇ treated cells are compared to untreated HAECs collected at 85% confluency (0 hour). Therefore, SEQ ED NO: 176 is useful in immune/inflammation disorders.
  • SEQ ID NO:76 showed decreased expression in dentate nucleus tissue versus pooled brain tissue as determined by microarray analysis. Samples from specific brain regions are obtained from 4 individual donors and compared to a pooled-brain control (Dn 4096) reconstituted from the purified mRNA isolated from the major regions of two male brains (Dn 3641 and Dn 3642). Therefore, SEQ ED NO:76 is useful in neurological disorders.
  • Dn 4096 pooled-brain control
  • SEQ TD NO: 99 showed decreased expression in RPMI 6666 cells treated with LPS versus untreated cells as determined by microarray analysis.
  • RPMI 6666 cells are stimulated in vitro with E. coli LPS for 0.5, 1, 2, 4, and 8 hours. These treated cells are compared to untreated RPMI 6666 cells kept in culture in the absence of stimuli.
  • SEQ ID NO:99 showed decreased expression in T-47D breast carcinoma cells and BT-20 breast carcinoma cells versus MCF-10A fibrocystic non-tumorigenic cells as determined by microarray analysis. The cells are grown in defined serum-free H14 medium to 70- 80% confluence prior to RNA harvest.
  • SEQ ID NO:99 showed decreased expression in C3A cells treated with
  • LY294002 for 6 hours versus untreated cells, as determined by microarray analysis.
  • Early confluent C3A cells are treated with LY294002 at 2, 0.5, 15, and 25 ⁇ g/ml for 1, 3, and 6 hours.
  • the treated cells are compared to untreated early-confluent C3A cells.
  • SEQ ID NO: 99 showed decreased expression in C3A cells treated with 2000 ⁇ g/ml clofibrate versus untreated cells as determined by microarray analysis.
  • Early confluent C3A cells are treated with clofibrate in concentrations of 50, 200, 1600, and 2000 ⁇ g/ml dissolved, in 1% carboxymethyl cellulose, for 0, 1, 3, and 6 hours.
  • Parallel samples of C3A cells are treated with 1% carboxymethyl cellulose alone.
  • SEQ ED NO:99 showed decreased expression in C3A cells treated for 6 hours with .5 percent DMSO and in C3A cells treated for 6 hours with 2 percent DMSO, versus untreated cells, as determined by microarray analysis.
  • Early confluent C3A cells are treated with DMSO [0.1, 0.5, 1 and 2% (v/v) solutions in water] for 1, 3, and 6 hours.
  • Time matched control samples of untreated C3A cells are also run in parallel. Therefore, SEQ ID NO:99 is useful in diagnostic assays for cell proliferation disorders.
  • Sequences complementary to the NAAP-encoding sequences, or any parts thereof, are used to detect, decrease, or inhibit expression of naturally occurring NAAP.
  • oligonucleotides comprising from about 15 to 30 base pairs is described, essentially the same procedure is used with smaller or with larger sequence fragments.
  • Appropriate oligonucleotides are designed using OLIGO 4.06 software (National Biosciences) and the coding sequence of NAAP.
  • a complementary oligonucleotide is designed from the most unique 5' sequence and used to prevent promoter binding to the coding sequence.
  • To inhibit translation, a complementary oligonucleotide is designed to prevent ribosomal binding to the NAAP-encoding transcript.
  • NAAP is achieved using bacterial or virus-based expression systems.
  • cDNA is subcloned into an appropriate vector containing an antibiotic resistance gene and an inducible promoter that directs high levels of cDNA transcription.
  • 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 (EPTG).
  • NAAP in eukaryotic cells
  • baculovirus recombinant Autographica califomica nuclear polyhedrosis virus (AcMNPV), commonly known as baculovirus.
  • AcMNPV Autographica califomica nuclear polyhedrosis virus
  • 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 Spodopterafrugiperda (Sf9) insect cells in most cases, or human hepatocytes, in some cases.
  • NAAP is synthesized as a fusion protein with, e.g., glutathione S-transferase (GST) or a peptide epitope tag, such as FLAG or 6-His, permitting rapid, single-step, affinity-based purification of recombinant fusion protein from crude cell lysates.
  • GST glutathione S-transferase
  • a peptide epitope tag such as FLAG or 6-His
  • FLAG an 8-amino acid peptide
  • 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 XVH, XVHI, and XLX, where applicable. XIV. Functional Assays
  • NAAP function is assessed by expressing the sequences encoding NAAP at physiologically elevated levels in mammalian cell culture systems.
  • cDNA is subcloned into a mammalian expression vector containing a strong promoter that drives 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 ⁇ g of recombinant vector are transiently transfected into a human cell line, for example, an endothelial or hematopoietic cell line, using either liposome formulations or electroporation.
  • 1-2 ⁇ g of an additional plasmid containing sequences encoding a marker protein are co-transfected.
  • Expression of a marker protein provides a means to distinguish transfected cells from nontransfected cells and is a reliable predictor of cDNA expression from the recombinant vector.
  • Marker proteins of choice include, e.g., Green Fluorescent Protein (GFP; Clontech), CD64, or a CD64-GFP fusion protein.
  • FCM Flow cytometry
  • FCM detects and quantifies the uptake of fluorescent molecules that diagnose events preceding or coincident with cell death. These events include changes in nuclear DNA content as measured by staining of DNA with propidium iodide; changes in cell size and granularity as measured by forward light scatter and 90 degree side light scatter; down-regulation of DNA synthesis as measured by decrease in bromodeoxyuridine uptake; alterations in expression of cell surface and intracellular proteins as measured by reactivity with specific antibodies; and alterations in plasma membrane composition as measured by the binding of fluorescein-conjugated Annexin V protein to the cell surface. Methods in flow cytometry are discussed in Ormerod, M.G. (1994; Flow Cytometry, Oxford, New York NY).
  • NAAP The influence of NAAP on gene expression can be assessed using highly purified populations of cells transfected with sequences encoding NAAP and either CD64 or CD64-GFP.
  • CD64 and CD64-GFP are expressed on the surface of transfected cells and bind to conserved regions of human immunoglobulin G (IgG).
  • Transfected cells are efficiently separated from nontransfected cells using magnetic beads coated with either human IgG or antibody against CD64 (DYNAL, Lake Success NY).
  • mRNA can be purified from the cells using methods well known by those of skill in the art. Expression of mRNA encoding NAAP and other genes of interest can be analyzed by northern analysis or microarray techniques.
  • 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.
  • PAGE polyacrylamide gel electrophoresis
  • NAAP amino acid sequence is analyzed using LASERGENE software (DNASTAR) to determine regions of high immunogenicity, and a corresponding oligopeptide is synthesized and used to raise antibodies by means known to those of skill in the art.
  • LASERGENE software DNASTAR
  • Methods for selection of appropriate epitopes, such as those near the C-terminus or in hydrophilic regions are well described in the art (Ausubel et al., supra, ch. 11).
  • oligopeptides of about 15 residues in length are synthesized using an ABI 431 A peptide synthesizer (Applied Biosystems) using FMOC chemistry and coupled to KLH (Sigma- Aldrich, St. Louis MO) by reaction with N-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS) to increase immunogenicity (Ausubel et al., supra). Rabbits are immunized with the oligopeptide-KLH complex in complete Freund's adjuvant.
  • ABI 431 A peptide synthesizer Applied Biosystems
  • KLH Sigma- Aldrich, St. Louis MO
  • MBS N-maleimidobenzoyl-N-hydroxysuccinimide ester
  • 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.
  • Naturally occurring or recombinant NAAP is substantially purified by immunoaffinity chromatography using antibodies specific for NAAP.
  • An immunoaffinity column is constructed by covalently coupling anti-NAAP antibody to an activated chromatographic resin, such as CNBr-activated SEPHAROSE (Amersham Biosciences). After the coupling, the resin is blocked and washed according to the manufacturer's instructions. Media containing NAAP are passed over the immunoaffinity column, and the column is washed under conditions that allow the preferential absorbance of NAAP (e.g., high ionic strength buffers in the presence of detergent).
  • the column is eluted under conditions that disrupt antibody/NAAP binding (e.g., a buffer of pH 2 to pH 3, or a high concentration of a chaotrope, such as urea or thiocyanate ion), and NAAP is collected.
  • a buffer of pH 2 to pH 3, or a high concentration of a chaotrope, such as urea or thiocyanate ion e.g., a buffer of pH 2 to pH 3, or a high concentration of a chaotrope, such as urea or thiocyanate ion
  • NAAP or biologically active fragments thereof, are labeled with 125 I Bolton-Hunter reagent (Bolton, A.E. and W.M. Hunter (1973) Biochem. J. 133:529-539).
  • Bolton-Hunter reagent Bolton, A.E. and W.M. Hunter (1973) Biochem. J. 133:529-539.
  • Candidate molecules previously arrayed in the wells of a multi-well plate are incubated with the labeled NAAP, washed, and any wells with labeled NAAP complex are assayed. Data obtained using different concentrations of NAAP are used to calculate values for the number, affinity, and association of NAAP with the candidate molecules.
  • molecules interacting with NAAP are analyzed using the yeast two-hybrid system as described in Fields, S. and O. Song (1989; Nature 340:245-246), or using commercially available kits based on the two-hybrid system, such as the MATCHMAKER system (Clontech).
  • NAAP may also be used in the PATHCALLING process (CuraGen Corp., New Haven CT) which employs the yeast two-hybrid system in a high-throughput manner to determine all interactions between the proteins encoded by two large libraries of genes (Nandabalan, K. et al. (2000) U.S. Patent No. 6,057,101).
  • PATHCALLING process CuraGen Corp., New Haven CT
  • yeast two-hybrid system in a high-throughput manner to determine all interactions between the proteins encoded by two large libraries of genes
  • NAAP activity is measured by its ability to stimulate transcription of a reporter gene (Liu, H.Y. et al. (1997) EMBO J. 16:5289-5298).
  • the assay entails the use of a well characterized reporter gene construct, LexA op -LacZ, that consists of LexA DNA transcriptional control elements (LexA op ) fused to sequences encoding the E. coli LacZ enzyme.
  • LexA op -LacZ that consists of LexA DNA transcriptional control elements (LexA op ) fused to sequences encoding the E. coli LacZ enzyme.
  • the methods for constructing and expressing fusion genes, introducing them into cells, and measuring LacZ enzyme activity, are well known to those skilled in the art.
  • Sequences encoding NAAP are cloned into a plasmid that directs the synthesis of a fusion protein, LexA-NAAP, consisting of NAAP and a DNA binding domain derived from the LexA transcription factor.
  • LexA-NAAP a fusion protein
  • 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 cells, relative to control cells, is proportional to the amount of transcription stimulated by the NAAP.
  • NAAP activity is measured by its ability to bind zinc.
  • a 5-10 ⁇ M sample solution in 2.5 mM ammonium acetate solution at pH 7.4 is combined with 0.05 M zinc sulfate solution (Aldrich, Milwaukee WL) in the presence of 100 ⁇ M dithiothreitol with 10% methanol added.
  • the sample and zinc sulfate solutions are allowed to incubate for 20 minutes.
  • the reaction solution is passed through a VYDAC column (Grace Vydac, Hesperia, CA) with approximately 300 Angstrom bore size and 5 ⁇ M particle size to isolate zinc-sample complex from the solution, and into a mass spectrometer (PE Sciex, Ontario, Canada).
  • Zinc bound to sample is quantified using the functional atomic mass of 63.5 Da observed by Whittal, R. M. et al. ((2000) Biochemistry 39:8406- 8417).
  • a method to determine nucleic acid binding activity of NAAP involves a polyacrylamide gel mobility-shift assay, h preparation for this assay, NAAP is expressed by transforming a mammalian cell line such as COS7, HeLa or CHO with a eukaryotic expression vector containing NAAP cDNA. The cells are incubated for 48-72 hours after transformation under conditions appropriate for the cell line to allow expression and accumulation of NAAP. Extracts containing solubilized proteins can be prepared from cells expressing NAAP by methods well known in the art. Portions of the extract containing NAAP are added to [ 32 P] -labeled RNA or DNA. Radioactive nucleic acid can be synthesized in vitro by techniques well known in the art.
  • a method to determine methylase activity of NAAP measures transfer of radiolabeled methyl groups between a donor substrate and an acceptor substrate.
  • Reaction mixtures (50 ⁇ l final volume) contain 15 mM HEPES, pH 7.9, 1.5 mM MgCl 2 , 10 mM dithiothreitol, 3% polyvinylalcohol, 1.5 ⁇ Ci [met/r Z- 3 H]AdoMet (0.375 ⁇ M AdoMet) (DuPont-NEN), 0.6 ⁇ g NAAP, and acceptor substrate (e.g., 0.4 ⁇ g [ 35 S]RNA, or 6-mercaptopurine (6-MP) to 1 mM final concentration). Reaction mixtures are incubated at 30°C for 30 minutes, then 65°C for 5 minutes.
  • acceptor substrate e.g., 0.4 ⁇ g [ 35 S]RNA, or 6-mercaptopurine (6-MP)
  • 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 Mg 2+ and ATP, the reaction is terminated, and the products are loaded on an agarose gel. Altered topoisomers can be distinguished from supercoiled substrate electrophoretically.
  • This assay is specific for type I topoisomerase activity because Mg 2+ and ATP are necessary cofactors for type H topoisomerases.
  • Type H topoisomerase activity of NAAP can be assayed based on the decatenation of a kinetoplast DNA (KDNA) substrate.
  • KDNA kinetoplast DNA
  • NAAP is incubated with KDNA, the reaction is terminated, and the products are loaded on an agarose gel.
  • Monomeric circular KDNA can be distinguished from catenated KDNA electrophoretically. Kits for measuring type I and type H topoisomerase activities are available commercially from Topogen (Columbus OH).
  • ATP-dependent RNA helicase unwinding activity of NAAP can be measured by the method described by Zhang and Grosse (1994; Biochemistry 33:3906-3912).
  • the substrate for RNA unwinding consists of 32 P-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, Mg 2+ , and varying amounts of NAAP in a Tris-HCI 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.
  • NAAP function is assessed by expressing the sequences encoding NAAP at physiologically elevated levels in mammalian cell culture systems.
  • cDNA is subcloned into a mammalian expression vector containing a strong promoter that drives high levels of cDNA expression.
  • Vectors of choice include pCMV SPORT (Life Technologies) and pCR3.1 (Invitrogen Corporation, Carlsbad CA), both of which contain the cytomegalovirus promoter.
  • recombinant vector 5-10 ⁇ g of recombinant vector are transiently transfected into a human cell line, preferably of endothelial or hematopoietic origin, using either liposome formulations or electroporation. 1-2 ⁇ g of an additional plasmid containing sequences encoding a marker protein are co-transfected.
  • marker protein provides a means to distinguish transfected cells from nontransfected cells and is a reliable predictor of cDNA expression from the recombinant vector.
  • Marker proteins of choice include, e.g., Green Fluorescent Protein (GFP; CLONTECH), CD64, or a CD64-GFP fusion protein.
  • FCM Flow cytometry
  • FCM detects and quantifies the uptake of fluorescent molecules that diagnose events preceding or coincident with cell death. These events include changes in nuclear DNA content as measured by staining of DNA with propidium iodide; changes in cell size and granularity as measured by forward light scatter and 90 degree side light scatter; down-regulation of DNA synthesis as measured by decrease in bromodeoxyuridine uptake; alterations in expression of cell surface and intracellular proteins as measured by reactivity with specific antibodies; and alterations in plasma membrane composition as measured by the binding of fluorescein-conjugated Annexin V protein to the cell surface. Methods in flow cytometry are discussed in Ormerod, M. G. (1994) Flow Cytometry, Oxford, New York NY.
  • NAAP The influence of NAAP on gene expression can be assessed using highly purified populations of cells transfected with sequences encoding NAAP and either CD64 or CD64-GFP.
  • CD64-GFP are expressed on the surface of transfected cells and bind to conserved regions of human immunoglobulin G (IgG).
  • Transfected cells are efficiently separated from nontransfected cells using magnetic beads coated with either human IgG or antibody against CD64 (DYNAL, Inc., Lake Success NY).
  • mRNA can be purified from the cells using methods well known by those of skill in the art. Expression of mRNA encoding NAAP and other genes of interest can be analyzed by northern analysis or microarray techniques.
  • Pseudouridine synthase activity of NAAP is assayed using a tritium ( 3 H) release assay modified from Nurse et al. ((1995) RNA 1:102-112), which measures the release of 3 H from the C 5 position of the pyrimidine component of uridylate (U) when 3 H-radiolabeled U in RNA is isomerized to pseudouridine ( ⁇ ).
  • 3 H tritium
  • a typical 500 ⁇ l assay mixture contains 50 mM HEPES buffer (pH 7.5), 100 mM ammonium acetate, 5 mM dithiothreitol, 1 mM EDTA, 30 units RNase inhibitor, and 0.1-4.2 ⁇ M [5- 3 H]tRNA (approximately 1 ⁇ Ci/nmol tRNA).
  • the reaction is initiated by the addition of ⁇ 5 ⁇ l of a concentrated solution of NAAP (or sample containing NAAP) and incubated for 5 min at 37 °C.
  • Portions of the reaction mixture are removed at various times (up to 30 min) following the addition of NAAP and quenched by dilution into 1 ml 0.1 M HCI containing Norit-SA3 (12% w/v).
  • the quenched reaction mixtures are centrifuged for 5 min at maximum speed in a microcentrifuge, and the supematants are filtered through a plug of glass wool.
  • the pellet is washed twice by resuspension in 1 ml 0.1 M HCI, followed by centrifugation.
  • the supematants from the washes are separately passed through the glass wool plug and combined with the original filtrate.
  • a portion of the combined filtrate is mixed with scintillation fluid (up to 10 ml) and counted using a scintillation counter.
  • the amount of 3 H 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).
  • pseudouridine synthase activity of NAAP is assayed at 30 °C to 37 °C in a mixture containing 100 mM Tris-HCI (pH 8.0), 100 mM ammonium acetate, 5 mM MgCl 2 , 2 mM dithiothreitol, 0.1 mM EDTA, and 1-2 fmol of [ 32 P] -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.
  • RNA is extracted with phenol-chloroform, precipitated in ethanol, and hydrolyzed completely to 3-nucleotide monophosphates using RNase T 2 .
  • the hydrolysates are analyzed by two-dimensional thin layer chromatography, and the amount of 32 P radiolabel present in the ⁇ MP and UMP spots are evaluated after exposing the thin layer chromatography plates to film or a Phosphorlmager screen.
  • the relative amount ⁇ MP and UMP are determined and used to calculate the relative amount of ⁇ per tRNA molecule (expressed in mol ⁇ /mol of tRNA or mol ⁇ /mol of tRNA/minute), 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 2 ,N 2 -dimethylguanosine transferase ((m 2 2 G)methyltransferase) activity of NAAP is measured in a 160 ⁇ l reaction mixture containing 100 mM Tris-HCI (pH 7.5), 0.1 mM EDTA, 10 mM MgCl 2 , 20 mM NH 4 C1, lmM dithiothreitol, 6.2 ⁇ M S-adenosyl-L-[met/ ⁇ yZ- 3 H]methionine (30-70 Ci/mM), 8 ⁇ g m 2 2 G-deficient tRNA or wild type tRNA from yeast, and approximately 100 ⁇ g of purified NAAP or a sample comprising NAAP.
  • reactions are incubated at 30 °C for 90 min and chilled on ice. A portion of each reaction is diluted to 1 ml in water containing 100 ⁇ g BSA. 1 ml of 2 M HCI is added to each sample and the acid insoluble products are allowed to precipitate on ice for 20 min before being collected by filtration through glass fiber filters. The collected material is washed several times with HCI and quantitated using a liquid scintillation counter. The amount of 3 H incorporated into the m 2 2 G-deficient, acid-insoluble tRNAs is proportional to the amount of N 2 ,N 2 -dimethylguanosine transferase activity in the NAAP sample. Reactions comprising no substrate tRNAs, or wild-type tRNAs that have already been modified, serve as control reactions which should not yield acid-insoluble 3 H-labeled products.
  • Polyadenylation activity of NAAP is measured using an in vitro polyadenylation reaction.
  • the reaction mixture is assembled on ice and comprises 10 ⁇ l of 5 mM dithiothreitol, 0.025% (v/v) NONEDET P-40, 50 mM creatine phosphate, 6.5% (w/v) polyvinyl alcohol, 0.5 unit ⁇ l RNAGUARD (Pharmacia), 0.025 ⁇ g/ ⁇ l creatine kinase, 1.25 mM cordycepin 5'-triphosphate, and 3.75 mM MgCl 2 , in a total volume of 25 ⁇ l.
  • Reactions are then incubated at 30 °C for 75-90 min and stopped by the addition of 75 ⁇ l (approximately two-volumes) of proteinase K mix (0.2 M Tris- HCI, pH 7.9, 300 mM NaCl, 25 mM Na-EDTA, 2% (w/v) SDS), 1 ⁇ l of 10 mg/ml proteinase K, 0.25 ⁇ l of 20 mg/ml glycogen, and 23.75 ⁇ l of water). Following incubation, the RNA is precipitated with ethanol and analyzed on a 6% (w/v) polyacrylamide, 8.3 M urea sequencing gel. The dried gel is developed by autoradiography or using a phosphoimager.
  • proteinase K mix 0.2 M Tris- HCI, pH 7.9, 300 mM NaCl, 25 mM Na-EDTA, 2% (w/v) SDS
  • 1 ⁇ l of 10 mg/ml proteinase K 0.25 ⁇ l of
  • Cleavage activity is determined by comparing the amount of cleavage product to the amount of pre-mRNA template.
  • the omission of any of the polypeptide components of the reaction and substitution of NAAP is useful for identifying the specific biological function of NAAP in pre-mRNA polyadenylation (Riiegsegger, U. et al. (1996) J. Biol. Chem. 271:6107-6113; and references within).
  • tRNA synthetase activity is measured as the aminoacylation of a substrate tRNA in the presence of [ 14 C]-labeled amino acid.
  • NAAP is incubated with [ 14 C]-labeled amino acid and the appropriate cognate tRNA (for example, [ 14 C]alanine and tRNA ala ) in a buffered solution.
  • 1 C- labeled product is separated from free [ 14 C]amino acid by chromatography, and the incorporated 14 C is quantified by scintillation counter.
  • the amount of 14 C-labeled product detected is proportional to the activity of NAAP in this assay.
  • 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 KC1, 1.5 mM magnesium chloride, and 0.5 mM DTT along with misacylated [ 14 C] -Glu-tRNAGln (e.g., 1 ⁇ M) and a similar concentration of unlabeled L-glutamine.
  • misacylated [ 14 C] -Glu-tRNAGln e.g., 1 ⁇ M
  • the mixture is extracted with an equal volume of water-saturated phenol, and the aqueous and organic phases are separated by centrifugation at 15,000 x g at room temperature for 1 min.
  • the aqueous phase is removed and precipitated with 3 volumes of ethanol at -70°C for 15 min.
  • the precipitated aminoacyl-tRNAs are recovered by centrifugation at 15,000 x g at 4°C forl5 min.
  • the pellet is resuspended in of 25 mM KOH, deacylated at 65°C for 10 min., neutralized with 0.1 M HCI (to final pH 6-7), and dried under vacuum.
  • the dried pellet is resuspended in water and spotted onto a cellulose TLC plate.
  • the plate is developed in either isopropanol/formic acid/water or ammonia/water/chloroforrn/ methanol.
  • 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 XVHI. Agonists cause an increase in NAAP activity and antagonists cause a decrease in NAAP activity.

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Abstract

Plusieurs modes de réalisation de l'invention concernent des protéines associées à des acides nucléiques (NAAP) et des polynucléotides identifiant et codant les protéines NAAP. Des modes de réalisation de l'invention concernent également des vecteurs d'expression, des cellules hôtes, des anticorps, des agonistes et des antagonistes. D'autres modes de réalisation concernent enfin des procédés de diagnostic, de traitement ou de prévention de troubles associés à une expression aberrante des protéines NAAP.
PCT/US2002/028540 2001-09-07 2002-09-05 Proteines associees a des acides nucleiques WO2003023003A2 (fr)

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Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
1994, OXFORD UNIVERSITY PRESS, OXFORD article LEWIN: 'Genes V', pages 889 - 893, XP002964603 *
ABRINK ET AL.: 'Isolation of cDNA clones for 42 different kruppel-related zinc finger proteins expressed in the human monoblast cell line U-937' DNA AND CELL BIOLOGY vol. 14, no. 2, 1995, pages 125 - 136, XP002964607 *
BELLEFROID ET AL.: 'The human genome contains hundreds of genes coding for finger proteins of the kruppel type' DNA vol. 8, no. 6, 1989, pages 377 - 387, XP002964606 *
DENNY ET AL.: 'A zinc finger protein-encoding gene expressed in the post-meiotic phase of spermatogenesis' GENE vol. 106, 1991, pages 221 - 227, XP002964604 *
LICHTER ET AL.: 'Clustering of C2-H2 zinc finger motif sequences with telomeric and fragile site regions of human chromosomes' GENOMICS vol. 13, 1992, pages 999 - 1007, XP002964605 *

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