JP2005512531A - Nucleic acid binding protein - Google Patents

Abstract

  The present invention provides human nucleic acid binding protein (NAAP) and polynucleotides that identify and encode NAAP. The present invention also provides expression vectors, host cells, antibodies, agonists and antagonists. The present invention also provides a method for diagnosing, treating or preventing a disease associated with abnormal expression of NAAP.

Description

  The present invention relates to nucleic acid sequences and amino acid sequences of nucleic acid binding proteins. The present invention also relates to diagnosis / treatment / prevention of cell proliferation abnormalities, neurological diseases, neurological diseases, developmental disorders, autoimmune / inflammatory diseases, and infectious diseases using these sequences. The invention further relates to a calculation of the effect of foreign compounds on the expression of nucleic acid and amino acid sequences of nucleic acid binding proteins.

  Multicellular organisms are composed of a variety of cell types that differ significantly in structure and function. Cell types are determined by the characteristics of gene expression patterns, and different cell types express a set of overlapping but distinct genes during development. Spatial and temporal regulation of gene expression is crucial for cell proliferation, cell differentiation, apoptosis, and other processes required for organism development. Furthermore, gene expression is regulated in response to extracellular signals. Extracellular signals mediate intercellular communication and coordinate the actions of various cell types. Proper gene regulation also allows cells to function efficiently. In order to ensure this, only genes that require that function are expressed at a given time.

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

  The double helix structure and repeat sequences of DNA produce topological and chemical features that can be recognized by transcription factors. These features include hydrogen bond donor and acceptor groups, hydrophobic patches, major and minor grooves, and regularly repeated stretches of sequences (inducing a clear group of bends in the helix). is there. Usually, a group of DNA sequence motifs specifically recognized by a transcription factor is about 20 nucleotides in length. Multiple adjacent transcription factor binding motifs may be required for gene regulation.

  Many transcription factors incorporate DNA-binding structural motifs. These motifs consist of α helices or β sheets that bind to the major groove of DNA. Four well-characterized motifs are the helix turn helix, Zn finger, leucine zipper, and helix loop helix. Proteins having these motifs act as monomers alone or interact with DNA by forming homodimers or heterodimers.

  The helix-turn-helix motif consists of two α-helices that are connected at a fixed angle by a short chain of amino acids. One helix joins the main groove. One example of a helix-turn-helix motif is a homeobox motif, which is in a homeodomain protein. Homeodomain proteins are important in determining the anterior-posterior body axis during development and are conserved throughout the animal kingdom. Drosophila Drosophila Antennapedia and Ultrabithorax proteins are prototypical homeodomain proteins (Pabo, C.O. and R.T. Sauer (1992) Annu. Rev. Biochem. 61: 1053-1095).

The Zn finger motif binds to zinc ions. This motif generally has a tandem repeat group of about 30 amino acids consisting of cysteine and histidine residues arranged periodically. Two examples of this sequence pattern, C2H2 and C3HC4 ("RING" fingers) have already been described (Lewin, supra). Each of the Zn finger proteins has one antiparallel β sheet and one α helix, and their proximity and conformation are maintained by zinc ions. Contact with DNA is made by the arginine residue in front of the α-helix and by the second, third, and sixth residues of the α-helix. Examples of variants of Zn finger motifs include the less well-understood cysteine-rich motifs, which bind to zinc or other metal ions. These mutant motifs may have no histidine residues and are generally non-repetitive. The Zn finger motif can be repeated in tandem within the protein so that the alpha helix of each Zn finger of the protein can contact the main groove of the DNA double helix. This repeated contact between protein and DNA results in a strong and specific DNA-protein interaction. The strength and specificity of this interaction can be regulated by the number of Zn finger motifs in the protein. Zn fingers were first identified in DNA-binding proteins as regions that interact directly with DNA, but they also appear in various proteins that do not bind to DNA (Lodish, H. et al. (1995) Molecular Cell Biology , Scientific American Books, New York NY, 447-451). For example, Galcheva-Gargova, Z. et al. ((1996) Science 272: 1797-1802) have identified a group of Zn finger proteins that interact with various cytokine receptors.

  The C2H2 type Zn finger signature motif has a sequence of 28 amino acids, which includes two conserved Cys residues and two conserved His residues in one C-2- Included within C-12-H-3-H type motifs. C2H2 type motifs generally appear in multiple tandem repeats. A cysteine-rich domain that contains the motif Asp-His-His-Cys (DHHC-CRD) has been identified as a unique subgroup of Zn finger proteins. The DHHC-CRD region is thought to be involved in growth and development. Certain DHHC-CRD mutants show a loss of Ras function. Ras is a small membrane-bound GTP-binding protein that regulates cell growth and differentiation. On the other hand, other DHHC-CRD proteins probably function in pathways unrelated to Ras (Bartels, D.J. et al. (1999) Mol. Cell Biol. 19: 6775-6787).

  Zn finger transcription factors are often accompanied by modular sequence motifs. Examples of this motif are the Kruppel-associated box (KRAB) and the SCAN domain. For example, the hypoalphalipoproteinemia susceptibility gene ZNF202 encodes a SCAN box and a KRAB domain (followed by 8 C2H2 Zn finger motifs) (Honer, C. et al. (2001) Biochim Biophys. Acta 1517: 441-448). The SCAN domain is a highly conserved leucine-rich motif, consisting of about 60 amino acids and found at the amino terminus of the Zn finger transcription factor. SCAN domains are most often linked to C2H2 Zn finger motifs, and this linkage is made through their carboxyl terminus. Biochemical binding studies have identified the SCAN domain as a selective hetero- and homotypic oligomerization domain. The protein complex mediated by the SCAN domain appears to function to regulate 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 about 75 amino acids. The KRAB domain is found in almost one-third of the 300-700 genes that encode C2H2 Zn fingers. The KRAB domain is found N-terminal to the finger repeat. A 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 to repress transcription. To achieve transcriptional repression, either KRAB-associated protein-1 (a type of transcriptional corepressor) or KRAB-A interacting protein is recruited. Proteins with a KRAB domain appear to play a regulatory role during development (Williams, A.J. et al. (1999) Mol. Cell Biol. 19: 8526-8535). A subgroup of highly related human KRAB Zn finger proteins that can be detected in all human tissues is highly expressed on human T lymphocytes (Bellefroid, EJ et al. (1993) EMBO J. 12: 13631374 ). The ZNF85 KRAB Zn finger gene is a member of the human ZNF91 family and is highly expressed in normal adult testis, seminoma, and NT2 / D1 teratocarcinoma cell lines (Poncelet, DA et al. (1998) DNA Cell Biol. 17: 931-943).

  The C4 motif is found in hormone-regulated proteins. C4 motifs generally have only two repeats. Many eukaryotic and viral proteins have certain conserved cysteine-rich domains, which consist of 40-60 residues and are called C3HC4 Zn fingers or RING fingers. This domain binds to two zinc atoms. It is also probably involved in mediating protein-protein interactions. The three-dimensional “cross-brace” structure of the zinc ligation system is unique to the RING domain. The spacing between cysteine groups in such domains is Cx (2) -Cx (9-39) -Cx (1-3) -Hx (2-3) -Cx (2) -Cx (4-48)- Cx (2) -C. PHD fingers are some C4HC3 Zn finger-like motifs found in nuclear proteins that appear to be involved in chromatin-mediated transcriptional regulation.

  GATA-type transcription factors contain one or two Zn finger domains and specifically bind to a region of DNA with GATA, a contiguous nucleotide sequence. NMR studies show that this Zn finger has two irregular antiparallel β-sheets and one α-helix, followed by a long loop to the C-terminus of the finger (Ominchinski, JG (1993 ) Science 261: 438-446). The helix and the loop connecting the two β-sheets contact the main groove of DNA, while the C-terminal part (site that determines the binding characteristics) wraps around into the minor groove. minor groove).

  The LIM motif has about 60 amino acid residues and contains 7 conserved cysteine residues and 1 histidine within a consensus sequence (Schmeichel, KL and Beckerle, MC (1994) Cell 79: 211 -219). The LIM family includes transcription factors and cytoskeletal proteins that can be involved in development, differentiation, and cell growth. An example actin-binding LIM protein may play a role in the regulation of cytoskeleton and cell morphogenesis (Roof, D.J. et al. (1997) J. Cell Biol. 138: 575-588). There are four double Zn finger motif groups with LIM consensus sequences in the N-terminal domain of the actin-binding LIM protein. The C-terminal domain of the actin-binding LIM protein shows sequence similarity to known actin-binding proteins (eg, dematin and villin). Actin-binding LIM protein binds to F-actin. This binding takes place through the dematin-like C-terminal domain. The LIM domain appears to mediate protein-protein interactions with other LIM binding proteins.

  Myeloid cell development is controlled by a group of tissue-specific transcription factors. Myeloid Zn finger proteins (MZF) include MZF-1 and MZF-2. MZF-1 functions in the regulation of neutrophilic granulocyte development. One mouse homologue MZF-2 is expressed on myeloid cell populations, particularly cells committed to the neutrophil lineage. MZF-2 is downregulated by G-CSF. It also appears to have a unique function in neutrophil development (Murai, K. et al. (1997) Genes Cells 2: 581-591).

  The leucine zipper motif has a stretch of amino acids rich in leucine, which can form an amphipathic α-helix. This structure provides the basis for dimerization of two leucine zipper proteins. The region adjacent to the leucine zipper is usually basic and is positioned so as to be optimal for binding to the major groove upon protein dimerization. Proteins having such motifs are collectively referred to as bZIP transcription factors. Leucine zipper motifs are found in the proto-oncogenes Fos and Jun. Fos and Jun constitute AP1, a heterodimeric transcription factor. AP1 is involved in cell growth and cell lineage determination (Papavassiliou, A.G. (1995) N. Engl. J. Med. 332: 45-47).

  A helix-loop-helix motif (HLH) consists of one short alpha helix and a long alpha helix connected by a loop. Since this loop is flexible, the two helices can bend relative to each other and bind to DNA. Myc, a transcription factor, has a prototypical HLH motif.

  The NF-κ-B / Rel signature defines a family of eukaryotic transcription factors. This transcription factor is involved in carcinogenesis, embryonic development, differentiation, and immune responses. Most transcription factors with a Rel homology domain (RHD) bind as a dimer to κ-B, a consensus DNA sequence motif. Rel family members share a highly conserved 300 amino acid domain, the Rel homology domain. The characteristic Rel C-terminal domain is involved in gene activation and cytoplasmic anchoring functions. Examples of proteins known to have RHD domains include vertebrate nuclear factor NF-κ-B (heterodimer of a DNA binding subunit and transcription factor p65), mammalian transcription factor RelB, and There is a vertebrate proto-oncogene c-rel, a protein involved in differentiation and lymphogenesis (Kabrun, N., and Enrietto, PJ (1994) Semin. Cancer Biol. 5: 103-112 ).

  DNA binding motifs called ARIDs (AT-rich interaction domains) characterize an evolutionarily conserved family of proteins. The approximately 100 residue ARID sequence is present in a series of proteins that are strongly implicated in the regulation of cell growth, differentiation, and tissue-specific gene expression. Examples of ARID proteins include Bright (a regulator of B cell-specific gene expression), dead ringer (involved in development), and MRF-2 (suppresses expression from cytomegalovirus enhancers) (Dallas, PB et al. (2000) Mol. Cell Biol. 20: 3137-3146).

The ELM2 (Egl-27 and MTA1 homology 2) domain is found in the tumor metastasis associated protein MTA1 and protein ER1. The nematode ( Caenorhabditis elegans ) gene egl 27 is required for embryonic pattern formation. MTA1 is a human gene whose expression is increased in metastatic cancers. This gene is a component of a protein complex with histone deacetylase activity and nucleosome remodeling activity (Solari, F. et al. ( 1999) Development 126: 24832494). The ELM2 domain is usually found at the N-terminus of the myb-like DNA binding domain. ELM2 is also seen to be associated with certain ARID DNA.

  Iroquois (Irx) family genes are found in nematodes, insects and vertebrates. The Irx gene cluster usually consists of one or two genome clusters consisting of three genes, and encodes a transcriptional regulator having a characteristic homeodomain. Irx genes can function early in development to identify the uniqueness of various regions of the body. The Irx gene cluster functions again later in development in both Drosophila and vertebrates, dividing these regions into smaller domains. (For a review of the Iroquois gene, see Cavodeassi, F. et al. (2001) Development 128: 28472855.) For example, mouse and human Irx4 proteins are 83% conservative and their 63-aa homeodomain is Drosophila Iroquois has over 93% identity with the same domain of patterned genes. Irx4 transcripts are mainly expressed in the ventricles. The homeobox gene Irx4 mediates ventricular differentiation during cardiac development (Bruneau, B.G. et al. (2000) Dev. Biol. 217: 26677).

  The histidine triplet (HIT) protein shares residues within different dimeric, 10-strand half-barrel structures that form two identical purine nucleotide binding sites . Proteins related to Hint (histidine triplet nucleotide binding protein) are present in all forms of life, and proteins related to fragile histidine triplet (Fhit) are present in animals and fungi. The group of proteins represents the two major branches of the HIT superfamily. Fhit homologues bind to and cleave diadenosine polyphosphate. The Fhit-Ap (n) A complex appears to function in the pro-apoptotic tumor suppressor pathway in epithelial tissues (Brenner C. et al. (1999) J. Cell Physiol. 181: 179187).

  Peroxisome proliferator-activated receptor gamma (PPARgamma) is a nuclear receptor that regulates the expression of numerous genes involved in adipocyte differentiation, lipid storage and insulin sensitization. PPARgamma binds to and activates fatty acid derivatives and prostaglandin J2. Thiazolidinedione is a synthetic ligand and agonist of this receptor (Rocchi, S. and Auwerx, J. (2000) Br. J. Nutr. 84: S223227). Thiazolidinedione or PPAR gamma agonists improve insulin sensitization, reduce blood glucose, and lower blood pressure in type II diabetic subjects ((Lebovitz, HE and Banerji, MA (2001) Recent Prog. Horm. Res. 56: 265294 ).

  Most transcription factors have characteristic DNA-binding motifs, and various variants of these motifs and novel motifs have been characterized to date and are still characterized (Faisst , S. and S. Meyer (1992) Nucl. Acids Res. 20: 3-26).

Regulation of chromatin-related protein gene expression depends on providing a transcriptional mechanism at the transcription start site of each gene. Proteins that bind to DNA may be involved in this regulation. Chromatin proteins can affect regulatory sequence and accessibility of the start site. On the other hand, transcription factors can increase or decrease the affinity of the RNA polymerase complex at the start site. Eukaryotic nuclear DNA is organized into chromatin, but its compact organization of chromatin limits the accessibility of DNA to transcription factors and contributes to the physical organization of DNA, It plays an important role in regulation (Lewin, supra, pages 409-410). Two types of chromatin are observed. Authentic chromatin (some of which is transcribed) and heterochromatin (heterochromatin) (closely packed, most of which are inaccessible for transcription). The compact structure of chromatin is determined and influenced by chromatin binding proteins. Such proteins include histones, high mobility group (HMG) proteins, and chromodomain proteins. There are five classes of histones, H1, H2A, H2B, H3 and H4, all of which are highly basic low molecular weight proteins. Nucleosomes, which are the basic units of chromatin, have 200 base pairs of DNA and two copies of H2A, H2B, H3, and H4. H1 links adjacent nucleosome groups. HMG protein is a low molecular weight non-histone protein that appears to play a role in unwinding DNA and stabilizing single-stranded DNA. Chromodomain proteins play an important role in the formation of highly compacted heterochromatin. Heterochromatin is transcriptionally silent (inactive). Heterochromatin-like protein complex formation is also involved in the regulation of gene expression and genome organization. Gene regulation also occurs through modifications such as histone acetylation and DNA methylation that affect chromatin structure.

  The chromodomain proteins are Polycomb (Pc) group, heterochromatin protein 1 (HP1) group, CHD group (chromodomain, helicase / ATPase and DNA binding group), SUV39 group, and retinoblastoma binding protein 1 Divided into several classes such as (RBP1) group. Pc chromodomain proteins consist of more than 300 amino acids in length and share a C-terminal region called the Pc box. For example, Pc protein functions to suppress the Hox gene, but the activity of the Hox gene is restricted to a precisely restricted pattern during normal development. Proteins that interact with Pc chromodomain proteins include Ring1A, Bmi-1, Rae-28 / Mph1, Mel18 and RYBP. HP1-like chromodomain proteins are generally less than 200 amino acids long and contain a stretch of negatively charged amino acids near the N-terminus separated by a “hinge” region from the C-terminal region, which is a repeat of the chromodomain. Sharing. Deletion of mammalian Pc1 results in severe lymphocyte proliferative defects. Mammalian HP1 proteins include HP1α, HP1β, and HP1γ. Proteins that interact with HP-1 chromodomain proteins include INCENP, TIF1α, BRG1 / SNF2β, H1 / H5-like proteins, hRAD54L, bLAP, KAP-1 / Tif1β, laminin B receptor (LBR), SP100, and CAF -1 is included. Proteins that interact with CHD chromodomain proteins include HDAC1 / 2, RbAp46 / 48, and mtal. Mammalian CHD proteins are approximately 200 kilodaltons, are highly modular, and have several sequence motifs that show consistent positions along the length of the protein. Some members contain PHD Zn fingers. RBP1 contains an ADR domain and a chromodomain (for a review of chromodomain proteins, see Jones D.O. et al. (2000) BioEssays 22: 124-137).

Diseases and disorders associated with gene regulation Many tumor diseases in humans can be caused by inappropriate gene expression. Malignant cell growth can occur by overexpression of tumor promoting genes or insufficient expression of tumor suppressor genes (Cleary, ML (1992) Cancer Surv. 15: 89-104). The zinc finger transcriptional regulator WT1 is a tumor suppressor protein that is inactivated in children with Wilms tumor. Oncogene bcl-6, which plays an important role in large cell lymphoma, is also a Zn finger protein (Papavassiliou, AG (1995) N. Engl. J. Med. 332: 45-47). Chromosomal translocations can also generate a chimeric loci that fuses the coding sequence of one gene with the regulatory region of another unrelated gene. Such sequences probably cause inappropriate gene transcription. It can also cause malignant tumors. For example, in Burkitt lymphoma, the transcription factor Myc is translocated to the immunoglobulin heavy chain locus, greatly enhancing Myc expression, resulting in rapid cell growth resulting in leukemia (Latchman, DS (1996) N. Engl. J. Med. 334: 28-33).

In addition, the immune system responds to infection and trauma by activating a cascade of events that regulate the gradual selection, amplification, and recruitment of cellular defense mechanisms. A complex and balanced program of gene activation and repression is involved in this process. However, excessive activity of the immune system caused by inappropriate or insufficient regulation of gene expression can cause considerable damage to tissues and organs. This injury is well documented in the literature on immune responses associated with arthritis, allergens, heart attacks, strokes, and infections (Isselbacher et al. Harrison's Principles of Internal Medicine , 13th edition, McGraw Hill, Inc. and Teton Data Systems Software, 1996). The causative gene for autoimmune multiglandular endocrine insufficiency candidiasis ectodermal dystrophy (APECED) has recently been isolated and found to encode a protein with two PHD-type Zn finger motifs (Bjorses , P. et al. (1998) Hum. Mol. Genet. 7: 1547-1553).

  Furthermore, the production of multicellular organisms is based on the induction and regulation of cell differentiation at the appropriate stage of development. Central to this process is differential gene expression that confers uniqueness to cells and tissues throughout the body. Failure to regulate gene expression during development can lead to developmental / developmental disorders. Human developmental disorders resulting from mutations in Zn finger transcriptional regulators include: Urogenital developmental abnormalities (related to WT1); Greig craniopolysyndactyly, Pallister-Hall syndrome, and postaxial polydactyly type A (related to GLI3); and anus Townes-Brocks syndrome (SALL1-related) characterized by abnormalities in the kidneys, limbs and ears (Engelkamp, D. and van Heyningen, V. (1996) Curr. Opin. Genet. Dev. 6: 334-342; Kohlhase, J. et al. (1999) Am. J. Hum. Genet. 64: 435-445).

  Pax genes, also called paired-box genes, are a family of developmentally regulated genes that encode nuclear transcription factors. These are characterized by a pair of domains that are conserved amino acid motifs with DNA binding activity. In vertebrates, the Pax gene is also involved in embryogenesis. Mutations in four of the nine characterized Pax gene families are congenital humans such as Waardenburg syndrome (PAX3), aniridia (PAX6), Peter's malformation (PAX6) and renal deficiency syndrome (PAX2). Associated with disease. Vertebrate pax genes regulate organogenesis in the kidney, eye, ear, nose, limb muscles, spine and brain. Vertebrate Pax genes are involved in pattern formation during embryogenesis (Dahl, E. et al. (1997) Bioessays 19: 755765).

  In human acute leukemia, chromosomal reciprocal translocations are involved, and such translocations fuse the ALL1 gene in chromosomal region 11q23 to a series of partner genes located on various human chromosomes. The fusion gene encodes a chimeric protein. The AF17 gene encodes a 1093 amino acid protein, which is a leucine zipper dimerization motif 3 'to the fusion point and an N-terminal cysteine-rich domain that shows homology to domains within the protein Br140 (peregrin) (Prasad R. et al. (1994) Proc. Natl. Acad. Sci. USA 91: 81078111). Translin is a DNA-binding protein that specifically binds to consensus sequences at breakpoint junctions of chromosomal translocations in many cases of lymphoid malignancies (Aoki, K. et al. (1997) FEBS Lett. 401: 109112).

Nucleic acid synthesis
Polymerase
DNA and RNA replication is a vital process for cell replication and function. The enzymes that mediate the replication of DNA and RNA are DNA polymerase and RNA polymerase, respectively, and this mediation is performed by a “templating” process. In this process, the nucleotide sequence of a DNA or RNA strand is copied to a complementary nucleic acid sequence of either DNA or RNA by complementary base pairing. However, there are fundamental differences between the two processes.

The reaction catalyzed by DNA polymerase is the sequential addition of deoxyribonucleotides to the 3′-OH end of a polynucleotide strand (primer strand), which is paired with a second (template) strand. New DNA strands therefore grow in the 5 'to 3' direction (Alberts, B. et al. (1994) The Molecular Biology of the Cell , Garland Publishing Inc., New York, NY, pages 251-254). The substrate for this polymerization reaction is the corresponding deoxynucleotide triphosphate, which needs to base pair with the correct nucleotide on the template strand in order to be recognized by the polymerase. Since DNA exists as a double helix, each of the duplexes can be a template for the formation of a new complementary strand. Thus, each of the two daughter cells of a dividing cell inherits a new DNA double helix, one old and one new strand. Thus, DNA is said to be “semi-conservatively” replicated by DNA polymerase. In addition to the synthesis of new DNA, DNA polymerase is also involved in repairing damaged DNA. This is described in “ Ligase ” below.

  In contrast to DNA polymerase, RNA polymerase "transcribes" DNA into RNA using a single DNA template strand, which uses ribonucleotide triphosphate as a substrate. Like DNA polymerization, RNA polymerization proceeds in the 5 'to 3' direction by the addition of ribonucleoside monophosphate to the 3'-OH end of the growing RNA strand. DNA transcription produces messenger RNA (mRNA), which carries information for protein synthesis. DNA transcription also produces transfer RNA, ribosomal RNA, and other RNAs with structural or catalytic functions. In eukaryotic cells, three separate RNA polymerases synthesize three types of RNA (Alberts, supra, pages 367-368). RNA polymerase I makes large ribosomal RNA, RNA polymerase II makes mRNA that is translated into protein, and RNA polymerase III makes various small stable RNAs (such as 5S ribosomal RNA and transfer RNA (tRNA)). In all cases, RNA synthesis is initiated by the binding of RNA polymerase to a promoter region on the DNA, which begins at the start site within the promoter. Completion of synthesis is accomplished by a stop (termination) signal in the DNA where the polymerase and the completed RNA strand are released.

Ligase
Through the process of DNA repair, accidental base changes (such as oxidative damage, hydrolytic attack, and random methylation of DNA) are corrected before DNA replication or transcription occurs. Due to the efficiency of the DNA repair process, only less than a thousandth of an accidental base change will mutate (Alberts, supra, pages 245-249). Three steps common to most types of DNA repair are: (1) excision of damaged or altered bases or nucleotides by DNA nucleases. (2) Inserting the correct nucleotide into the gap left by the excised nucleotide. This is done by DNA polymerase using the complementary strand as a template. (3) Sealing with DNA ligase of the break remaining between the inserted nucleotide (s) and the existing DNA strand. In the last reaction, DNA ligase uses energy from ATP hydrolysis to activate the 5 'end of the nicked phosphodiester bond and then form a new bond with the 3'-OH of the DNA strand. Bloom syndrome is an inherited human disease, but this patient shows an increased incidence of cancer due to a partial deficiency of this DNA ligation (Alberts, supra, page 247).

Nuclease The nuclease enzyme includes DNase that hydrolyzes DNA and RNase that hydrolyzes RNA. The two nucleases serve different purposes in nucleic acid metabolism. Nucleases hydrolyze phosphodiester bonds between adjacent nucleotides. Endonucleases hydrolyze at internal positions, and exonucleases hydrolyze at 3 'or 5' terminal nucleotide positions. For example, certain DNA exonuclease activities in DNA polymerases work to remove improperly paired nucleotides attached to the 3'-OH ends of DNA strands grown by polymerases I will provide a. As mentioned above, DNA endonuclease activity is involved in the excision step of the DNA repair process.

  RNases also provide various functions. For example, RNase P is a ribonucleoprotein enzyme that cleaves the 5 ′ end of tRNA precursors as part of their maturation process. RNase H digests RNA strands of certain RNA / DNA hybrids. Such hybrids occur in cells invaded by retroviruses, and RNase H is an important enzyme in the retroviral replication cycle. Pancreatic RNase is secreted into the intestine by the pancreas and hydrolyzes RNA in the ingested food. Increased RNase activity in serum and cell extracts is seen in various cancers and infectious diseases (Schein, C.H. (1997) Nat. Biotechnol. 15: 529-536). Modulation of RNase activity has been studied as a means of controlling tumor angiogenesis, allergic reactions, viral infection and replication, and fungal infection.

Nucleic acid modification
Methylase specific nucleotide methylation occurs in both DNA and RNA and provides separate functions in the two macromolecules. Methylation of cytosine residues in DNA forms 5-methylcytosine, which occurs specifically within CG sequences that are base-paired to each other within the DNA duplex. This pattern of methylation is passed between generations during DNA replication. Enzymes that do this are called "maintenance methylases" and act preferentially on CG sequences that are base paired with already methylated CG sequences. Such methylation distinguishes between active and inactive genes by preventing the binding of regulatory proteins that “turn on” the gene while allowing the binding of proteins that inactivate the gene. (Alberts et al., Pp. 448-451). In RNA metabolism, “tRNA methylase” causes one of several nucleotide modifications in tRNA. This modification affects the conformation and base pairing of this molecule and promotes recognition of the appropriate mRNA codons by specific tRNAs. The main methylation pattern is dimethylation of guanine residues, which form N, N-dimethylguanine.

Helicases and single-stranded binding protein helicase enzymes destabilize and unwind the double helix structure of both DNA and RNA. Since DNA replication occurs on two strands almost simultaneously, the two strands must first be separated to produce a replica “fork” so that the DNA polymerase can act. Two replication proteins contribute to this process. DNA helicase and single-stranded binding protein. DNA helicase hydrolyzes ATP and uses its energy to separate DNA strands. Single-stranded binding proteins (SSB) then bind to the exposed DNA strands, but do not cover the bases and temporarily stabilize them for template removal by DNA polymerase (Alberts, supra. -Page 256).

  RNA helicase transforms and regulates RNA conformation and secondary structure. Like DNA helicases, RNA helicases utilize energy from ATP hydrolysis to destabilize and unwind RNA duplexes. The best characterized and ubiquitous family of RNA helicases is the DEAD box family, whose name is derived from the conserved B-type ATP binding motif. This motif is characteristic for this family of proteins. Over 40 DEAD box helicases have been identified in diverse organisms ranging from bacteria, insects, yeasts, amphibians, mammals, and plants. DEAD box helicases function in a variety of processes. Such processes include, for example, translation initiation, splicing, ribosome assembly, and RNA editing, transport, and stability. Examples of these RNA helicases include yeast Drs1 protein (involved in ribosomal RNA processing), yeast TIF1 and TIF2 and mammalian eIF-4A (which are essential for initiation of RNA translation), and human p68 antigen (cell growth) (Ripmaster, TL 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 exhibit strong sequence homology over a stretch of about 420 amino acids. These conserved sequences include the A motif consensus sequence of a certain ATP binding protein, the “DEAD box” sequence (involved in ATPase activity), the sequence SAT (involved in the actual helicase unwinding region), and RNA There is an octapeptide consensus sequence required for binding and ATP hydrolysis (Pause, A. et al. (1993) Mol. Cell Biol. 13: 6789-6798). Differences outside these conserved regions appear to reflect differences in the functional role of individual proteins (Chang, T.H. et al. (1990) Proc. Natl. Acad. Sci. USA 87: 1571-1575).

  Several DEAD box helicases play a tissue-specific and stage-specific role in spermatogenesis and embryogenesis. Overexpression of DEAD box 1 protein (DDX1) appears to play a role in the progression of neuroblastoma (Nb) and retinoblastoma (Rb) (Godbout, R. et al. (1998) J. Biol. Chem. 273: 21161-21168). These observations suggest that DDX1 promotes or enhances tumor progression by altering the normal secondary structure and expression level of RNA in cancer cells. Another group of DEAD box helicases appears to be directly or indirectly involved in tumorigenesis (discussed above in Godbout). For example, murine p68 is mutated in UV-induced tumors and human DDX6 is located at a chromosomal breakpoint associated with B-cell lymphoma. Similarly, a chimeric protein consisting of DDX10 and NUP98 (one nuclear pore protein: nucleoporin) may be involved in the pathogenesis of certain myeloid malignancies.

  RuvA, RuvB and RuvC proteins are involved in the later stages of homologous recombination and recombination repair of damaged DNA. RuvA and RuvB form a complex that promotes ATP-dependent branch migration of the Holliday junction for the formation of heteroduplex DNA. RuvA acts as a specificity factor targeting RuvB, the motor that moves the junction to the junction. Two RuvA tetramers sandwich the junction in between and hold it in an unfolded square planar configuration. RuvB's hexameric rings are facing each other across a junction and use the free energy generated by ATP hydrolysis to “pump” DNA through the RuvAB complex, creating a novel dual helicase action. Facilitate. A third protein, RuvC endonuclease, separates the Holliday junction by nicking the two DNA strands. Genetic and biochemical studies have shown that the direct interaction between these three proteins, possibly through the formation of the RuvABC complex, is associated with crossover migration and degradation (West, SC ( 1997) Annu. Rev. Genet. 31: 213244).

Topoisomerase
In addition to separating the DNA strands prior to replication, the two strands must be “unwound” from each other prior to separation by the DNA helicase. Proteins that perform this function are known as DNA topoisomerases. DNA topoisomerase effectively acts as a reversible nuclease to hydrolyze phosphodiesterase bonds in the DNA strand, allowing the duplex to freely rotate with respect to each other, and then unwinding the helix, Recombine the original phosphodiester bond in between. Topoisomerase is an essential enzyme responsible for the topological rearrangement of DNA produced by transcription, replication, chromatin formation, recombination, and chromosome segregation. Introduction of higher-order helical coils into DNA can be done by passage of a processive enzyme (eg RNA polymerase) or by separation before replication of the DNA strand by helicase. During the process of DNA synthesis, storage and repair, knotting and concatenation can occur. All topoisomerase functions by breaking the phosphodiester bond in the ribose-phosphate backbone of DNA. Certain catalytic tyrosine residues in this enzyme produce reaction intermediates by nucleophilic attack on the fragile phosphodiester bond. In this intermediate, a covalent bond is formed between the enzyme and one end of the broken strand. Certain tyrosine DNA phosphodiesterases function in DNA repair by hydrolyzing this bond in the accidental dead endopoisomerase I-DNA intermediate (Pouliot, JJ et al. (1999) Science 286: 552-555).

  There are two types of DNA topoisomerases, type I and type II. Type I topoisomerase acts as a monomer and cuts into single strands of DNA. On the other hand, type II topoisomerase acts as a homodimer and cleaves both strands. DNA topoisomerase I creates a single-strand break in the DNA helix, allowing the duplex of the helix to rotate about the remaining phosphodiester bond in the opposite strand. DNA topoisomerase I makes temporary breaks in both strands of the DNA helix, where two double helices cross each other. This topoisomerase II can effectively separate two linked circular DNAs (Alberts, supra, pages 260-262). Type II topoisomerase is almost limited to proliferating cells (such as cancer cells) in eukaryotes. For this reason, topoisomerase II is a target for anticancer agents. Topoisomerase II is thought to be involved in multidrug resistance (MDR). The reason is that this enzyme may help repair DNA damage by DNA binding agents (such as doxorubicin or vincristine).

  The topoisomerase I family includes topoisomerase I (TopoI) and topoisomerase III (TopoIII). As the crystal structure of human topoisomerase I suggests, this enzyme partially controls the rotation about the intact DNA strand. In this “controlled rotation” model, protein-DNA interactions limit this rotation. This is driven by a torsional strain in the DNA (Stewart, L. et al. (1998) Science 379: 1534-1541). Structurally, TopoI can be recognized by a catalytic tyrosine residue in its active site region and a number of other conserved residues. TopoI appears to function during transcription. Two TopoIIIs are known in humans and are homologous to prokaryotic topoisomerases, with conserved tyrosines and active site signatures specific to this family. TopoIII has been suggested to play a role in meiotic recombination. Certain TopoIII in mice are highly expressed in testis tissue, and the increase in expression is accompanied by an increase in the number of cells in the pachytene (Seki, T. et al. (1998) J. Biol. Chem. 273 : 28553-28556).

  The topoisomerase II family includes two isoenzymes (IIα and IIβ), which are encoded by separate genes. TopoII preferentially cleaves double-stranded DNA in a reproducible, non-random manner in certain AT-rich regions, but the principle of cleavage site selectivity is unknown. Structurally, TopoII has four domains. The first two are structurally similar and probably have distant homology to the similar domain of eukaryotic TopoI. The second domain has catalytic tyrosine and a highly conserved pentapeptide. The IIα isoform appears to be involved in unlinking DNA during chromosome segregation. Cell lines that express IIα but not IIβ suggest that IIβ is non-essential for cellular processes, but IIβ knockout mice died in the perinatal period due to a failure in neurogenesis. The fact that the main abnormality occurred mainly at late developmental events (neurogenesis) suggests that IIβ is required for DNA repair rather than mitosis (Yang, X. et al. (2000) Science 287: 131- 134).

  Topoisomerase is believed to be involved in a number of pathologies, and topoisomerase poisons have been shown to be effective antitumor agents for several human malignant cancers. TopoI is localized in the wrong place in Fanconi's anemia. It is also believed to be involved in chromosome breaks seen in this disease (Wunder, E. (1984) Hum. Genet. 68: 276281). Abbreviated TopoIII overexpression in telangiectasia ataxia (A-T) cells partially suppresses the A-T phenotype, probably due to a dominant negative mechanism. This suggests that TopoIII is deregulated in A-T (Fritz, E. et al. (1997) Proc. Natl. Acad. Sci. USA 94: 4538-4542). TopoIII also interacts with the Bloom's Syndrome gene product. It has also been suggested to have a role as a tumor suppressor (Wu, L. et al. (2000) J. Biol. Chem. 275: 9636-9644). Abnormal TopoII activity is often associated with cancer and increased cancer risk. Significantly reduced Topo II activity has been seen in several, if not all, A-T cell lines (Mohamed, R. et al. (1987) Biochem. Biophys. Res. Commun. 149: 233-238). On the other hand, TopoII can perform DNA fragmentation in the region of the A-T gene (ATM). ATM controls all DNA damage responsive cell cycle checkpoints (Kaufmann, W.K. (1998) Proc. Soc. Exp. Biol. Med. 217: 327-334). The ability of topoisomerase to disrupt DNA has been used as a basis for antitumor drugs. Topoisomerase poisons act by increasing the intracellular number of dead-end covalent DNA-enzyme complexes, ultimately triggering the cell death pathway (Fortune, JM and N. Osheroff (2000) Prog. Nucleic Acid Res. Mol. Biol. 64: 221-253; Guichard, SM and MK Danks (1999) Curr. Opin. Oncol. 11: 482-489). Antibodies against TopoI are found in the sera of patients with systemic sclerosis, and the levels of this antibody are likely to be used as lung markers involved in this disease (Diot, E. et al. (1999) Chest 116: 715-720). Finally, the human TopoI DNA binding domain has been used as a DNA delivery vehicle for gene therapy (Chen, T.Y. et al. (2000) Appl. Microbiol. Biotechnol. 53: 558-567).

Recombinase gene recombination is a reorganization process of DNA sequences in the genome of certain organisms provides a genetic mutation of the organism in response to changes in the environment. DNA recombination allows mutations in specific combinations of genes in an individual's genome, and can vary in the timing and level of expression of these genes (see Alberts, pages 263-273, supra). See). Two broad classes of genetic recombination (universal recombination and site-specific recombination) are widely recognized. Universal recombination involves gene exchange between any homologous pair of DNA sequences. The position of these sequences is usually on two copies of the same chromosome. Recombinase, an enzyme that aids in this process, allows you to “nick” one strand of the DNA duplex somewhat randomly and exchange it for a complementary strand in the other duplex To. This process usually does not change the organization of genes in the chromosome. In site-specific recombination, the recombinase recognizes a specific nucleotide sequence in one or both of the recombining molecules. Since base pairing is not involved in this form of recombination, DNA homology between recombining molecules is not necessary. Unlike universal recombination, site-specific recombination can alter the relative position of nucleotide sequences within a chromosome.

RNA metabolism Ribonucleic acid (RNA) is a linear single-stranded polymer (consisting of four nucleotides, ATP, CTP, UTP, and GTP). In most organisms, RNA is transcribed as a copy of deoxyribonucleic acid (DNA), the organism's genetic material. In retroviruses, RNA works as genetic material instead of DNA. RNA copies of genetic material either encode proteins or play various structural, catalytic, or regulatory roles within an organism. The classification of RNA is based on its intracellular localization and function. Messenger RNA (mRNA) encodes a polypeptide. Ribosomal RNA (rRNA) is combined with ribosomal proteins to form ribosomes. Ribosomes are cytoplasmic particles that translate mRNA into polypeptides. Transfer RNA (tRNA) is a cytosolic adapter molecule, whose function in mRNA translation is the recognition of both the mRNA codon and the amino acids that fit that codon. Heteronuclear RNA (hnRNA) includes mRNA precursors and other various sizes of nuclear RNA. Nuclear small RNA (snRNA) is part of the nuclear spliceosome complex. This complex removes non-coding sequences (introns) intervening in the mRNA precursor and recombines exons.

  Proteins associate with RNA during transcription of RNA from DNA, RNA processing, and translation of mRNA into protein. Proteins also associate with RNA when it is utilized for structural, catalytic, or regulatory purposes.

RNA processing ribosomal RNA (rRNA) combines with ribosomal proteins to form ribosomes. Ribosomes are cytoplasmic particles that translate messenger RNA (mRNA) into a polypeptide. Eukaryotic ribosomes are composed of 60S (large) and 40S (small) subunits, which together form an 80S ribosome. In addition to 18S, 28S, 5S, and 5.8S rRNA, ribosomes contain more than 50 to 80 separate ribosomal proteins, depending on the type of organism. The classification of ribosomal proteins depends on the subunit to which they belong (ie L if associated with a large 60S subunit, S if a small 40S subunit). The Escherichia coli ribosome has been studied the most in detail and has 50 proteins, many of which are conserved in all living organisms. Elucidation of the structure of nine ribosomal proteins has been made with a resolution of less than 3.0D (ie S5, S6, S17, L1, L6, L9, L12, L14, L30), and common motif groups (for example ba-b proteins) Fold group) and acidic and basic RNA-binding motif groups located between b-strands. Most ribosomal proteins appear to be in direct contact with rRNA (reviewed in Liljas, A. and Garber, M. (1995) Curr. Opin. Struct. Biol. 5: 721-727; also Woodson, SA and Leontis, NB (1998) Curr. Opin. Struct. Biol. 8: 294-300; see Ramakrishnan, V. and White, SW (1998) Trends Biochem. Sci. 23: 208-212).

  Ribosomal proteins can undergo post-translational modifications and interact with other ribosome-related proteins to regulate translation. For example, the highly homologous 40S ribosomal protein S6 kinases S6K1 and S6K2 play a major role in regulating cell growth, which controls the biosynthesis of the translation components that make up protein synthesizers (eg ribosomal proteins) To do. In the case of S6K1, at least 8 phosphorylation sites appear to mediate kinase activation in a hierarchical manner (Dufner and Thomas (1999) Exp. Cell. Res. 253: 100-109). Several ribosomal proteins (including L1) also function as translational repressors, which bind to the polycistronic mRNA that encodes the ribosomal protein (reviewed above in Liljas and Garber).

  Recent evidence suggests that many ribosomal proteins have secondary functions that are independent of their involvement in protein biosynthesis. These proteins function as regulators of cell growth and in some cases as inducers of cell death. For example, expression of the human ribosomal protein L13a has been shown to induce apoptosis. This is done by stopping cell growth in the G2 / M phase of the cell cycle. Suppression of L13a expression induces apoptosis in target cells, suggesting that this protein is required for cell survival at the proper dose. Similar results have been obtained with yeast. Here, inactivation of rp22 and rp23, which are yeast homologues of L13a, caused severe growth retardation and death. A very closely related ribosomal protein, L7, arrests cells in the G1 phase and also induces apoptosis. Thus, a subset of ribosomal proteins appear to function as cell cycle checkpoints and appear to constitute a new family of cell growth regulators.

  Individual ribosomal proteins were mapped to the surface of intact ribosomes. For this, a 3D immunocryoelectron microscopy was used to visualize the antibodies produced against specific ribosomal proteins. Although progress has been made towards mapping L1, L7, and L12, elucidation of the structure of intact ribosomes is only 20-25D resolution, and there is a discrepancy between the different crude structures. (Frank, J. (1997) Curr. Opin. Struct. Biol. 7: 266-272).

Three separate sites have been identified on the ribosome. The aminoacyl tRNA acceptor site (A site) receives a charged tRNA with the amino acid charged (the exception is the starting tRNA). The peptidyl tRNA site (P site) binds to the nascent polypeptide and amino acids from the A site are added to this extending chain. The deacylated tRNA group is released from the ribosome after binding in the escape site (E site). For an overview of ribosome structure, see Stryer, L. (1995) Biochemistry WH Freeman and Company, New York NY 888-908l; Lodish, H. et al. (1995) Molecular Cell Biology Scientific American Books, New York NY 119-138. And Lewin, B (1997) Genes VI Oxford University Press, Inc. New York, NY.

A variety of proteins are required for the processing of RNA transcribed in the nucleus. Pre-stages of mRNA processing include capping with methylguanosine to the 5 ′ end, polyadenylation of the 3 ′ end, and splicing to remove introns. The primary RNA transcript from DNA is a faithful copy of a gene with both exon and intron sequences, and the intron sequence must be excised from the RNA transcript to produce the mRNA that encodes the protein. . "Splicing" of mRNA is done in the nucleus with the aid of a large multicomponent ribonucleoprotein complex (known as the spliceosome). The spliceosome complex consists of five low molecular nuclear ribonucleoprotein particles (snRNP), U1, U2, U4, U5, and U6. Each snRNP contains one type of snRNA and about 10 types of proteins. Several snRNP RNA components recognize and base pair with intron consensus sequences. The protein component mediates spliceosome assembly and the splicing reaction. Autoantibodies to the snRNP protein are found in the blood of patients with systemic lupus erythematosus (Stryer, L. (1995) Biochemistry WH Freeman and Company, New York NY, page 863).

  The role of heterogeneous nuclear ribonucleoprotein (hnRNP) has been identified in splicing, export of mature RNA to the cytoplasm, and mRNA translation (Biamonti, G. et al. (1998) Clin. Exp. Rheumatol. 16: 317-326). Examples of hnRNP are the yeast proteins Hrp1p (involved in RNA cleavage and polyadenylation at the 3 ′ end), Cbp80p (involved in capping the 5 ′ end of RNA), and Npl3p (mRNA from the nucleus) Mammalian homologue of hnRNP A1 (Shen, EC et al. (1998) Genes Dev. 12: 679-691). hnRNP has been shown to be an important target of autoimmune responses in rheumatic diseases (supra Biamonti).

Many snRNP and hnRNP proteins are characterized by certain RNA recognition motifs (RRM) (reviewed in Birney, E. et al. (1993) Nucleic Acids Res. 21: 5803-5816). The RRM is approximately 80 amino acids long and forms four β strands and two α helices organized in an α / β sandwich. Within the RRM is the core RNP-1 octapeptide motif and surrounding conserved sequences. In addition to the snRNP protein, examples of RNA-binding proteins having the above motif group include heteronuclear ribonucleoprotein that stabilizes nascent RNA and factors that regulate alternative splicing. Alternative splicing factors include developmentally regulated proteins, specific examples of which have been identified in lower eukaryotes (eg, Drosophila and Caenorhabditis elegans ). Each of these proteins plays an important role in the developmental process (eg, pattern formation and sex determination) (see, eg, Hodgkin, J. et al. (1994) Development 120: 3681-3689).

  The 3 ′ end of most eukaryotic mRNAs is also subject to post-transcriptional modification by polyadenylation. Polyadenylation proceeds through two enzymatically distinct steps. (i) The nucleolytic cleavage of the nascent mRNA is made with a group of cis-acting polyadenylation signals within the 3 ′ untranslated (non-coding) region, and (ii) poly (A) tract Added to the 5 'mRNA fragment. The presence of cis-acting RNA sequences is necessary for both steps. These sequences include 5'-AAUAAA-3 '(10-30 nucleotides upstream of the cleavage site) and less conserved GU-rich or U-rich sequence elements (10-30 cleavage sites). Nucleotide downstream). Cleavage factor (CstF), cleavage factor I (CF I), and cleavage factor II (CF II) are involved in the cleavage reaction, while cleavage polyadenylation factor (CPSF) and poly (A) polymerase (PAP) are Required for both cleavage and polyadenylation. An additional enzyme, poly (A) -binding protein II (PAB II), promotes poly (A) tract elongation (Ruegsegger, U. et al. (1996) J. Biol. Chem. 271: 6107-6113; and its (References).

Translation The correct translation of the genetic code depends on each amino acid forming a linkage with the appropriate transfer RNA (tRNA). Aminoacyl-tRNA synthetase (aaRS) is an essential protein found in all living organisms. aaRS is responsible for the activation of amino acids and their correct attachment to their cognate tRNA as the first step in protein biosynthesis. Prokaryotes have at least 20 different types of aaRS and one aaRS for each amino acid, while eukaryotes usually have two aaRSs, cytosolic and mitochondrial, 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 the tRNA, and class II enzymes add amino acids to the 3 ′ hydroxyl at the 3 ′ end of the tRNA. Each class is characterized by a unique topology of the catalytic domain. Class I enzymes have a catalytic domain based on nucleotide-bound “Rosman folds”. In particular, certain consensus tetrapeptide motifs are highly conserved (Prosite Document PDOC00161, Aminoacyl-transfer RNA synthetases class-I signature). Class I enzymes are specific for arginine, cysteine, glutamic acid, glutamine, isoleucine, leucine, methionine, tyrosine, tryptophan and valine. Class II enzymes have a central catalytic domain (consisting of a seven-stranded antiparallel β-sheet domain) and an N-terminal and C-terminal regulatory domain. Class II enzymes are divided into two groups based on the heterodimeric or homodimeric structure of the enzyme, the latter being further divided by the structure of the N-terminal and C-terminal regulatory domains (Hartlein, M. and Cusack, S (1995) J. Mol. Evol. 40: 519-530). Class II enzymes are specific for alanine, asparagine, aspartic acid, glycine, histidine, lysine, phenylalanine, proline, serine and threonine.

Some types of aaRS also have an editing function. For example, IleRS may mistakenly activate valine to form Val-tRNA ^Ile , but this product is removed by some hydrolysis action that destroys the accidentally charged product. This editing activity is located in the second catalytic site found in the connective polypeptide 1 region (CP1), a long insert within the Rossman fold domain of class I enzymes (Schimmel, P. et al. (1998) FASEB J. 12: 1599-1609). AaRS also plays a role in tRNA processing. Mature tRNAs have been shown to be charged with their respective amino acids in the nucleus before being exported to the cytoplasm. In addition, the binding with this amino acid functions as a quality control mechanism, and it is possible that tRNA is maintained functionally (Martinis, SA et al. (1999) EMBO J. 18: 4591-4596).

The rate of progress of polypeptide synthesis under optimal conditions is about 40 amino acid residues / second. The rate of misincorporation during translation is in the order of 10 ^-4 , and the main cause is that the aminoacyl-t-RNA was charged with the wrong amino acid. Incorrectly charged tRNAs are cytotoxic. The reason is that they incorporate incorrect amino acid residues into the growing polypeptide. The speed of translation is presumed to be determined by a compromise between the optimal extension speed and the need for translation fidelity. In mathematical calculations, 10 ^-4 is the maximum allowable error rate for protein synthesis in biological systems (reviewed above by Stryer 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 binding of tRNA ^Gln to glutamine (Gln). There is a mechanism to correct this mischarging, which seems to have its origin in evolution. Gln was in the last group of 20 natural amino acids used in polypeptide synthesis and appearing in nature. Gram-positive eubacteria, cyanobacteria, Archaeae, and eukaryotic organelles have a noncanonical pathway for the synthesis of Gln-tRNA ^Gln , which is the Glu-tRNA ^Gln ( Glu-tRNA synthetase GluRS is synthesized) and the enzyme Glu-tRNA ^Gln amide transferase (Glu-AdT) is used. The reactions involved in the transamidation pathway are as follows (Curnow, AW et al. (1997) Nucleic Acids Symposium 36: 2-4).

GluRS
tRNA ^Gln + Glu + ATP ⇒ Glu-tRNA ^Gln + AMP + PP _i

Glu-AdT
Glu-tRNA ^Gln + Gln + ATP ⇒ Gln-tRNA ^Gln + Glu + ADP + P

A similar enzyme, Asp-tRNA ^Asn amide transferase, is present in primordial bacteria and converts Asp-tRNA ^Asn to Asn-tRNA ^Asn . The formylase enzyme converts Met-tRNA ^fMet to fMet-tRNA ^fMet in eubacteria, which appears to be a closely related enzyme. Certain hydrolytic activities have also been identified to destroy mischarged Val-tRNA ^Ile (Schimmel, P. et al. (1998) FASEB J. 12: 1599-1609). One possible scenario for the evolution of Glu-AdT in primitive biological forms was the absence of a specific glutaminyl-tRNA synthetase (GlnRS) required an alternative pathway for the synthesis of Gln-tRNA ^Gln That's it. In fact, the deletion of the Glu-AdT operon in Gram-positive bacteria is lethal (Curnow, AW et al. (1997) Proc. Natl. Acad. Sci. USA 94: 11819-11826). Although the presence of GluRS activity in other organisms has been speculated by a high degree of conservation in the natural translation machinery, GluRS has not been identified in all organisms including humans. Such enzymes appear to serve to ensure translation fidelity and reduce the synthesis of defective polypeptides.

In addition to functions in protein synthesis, certain aminoacyl-tRNA synthetase groups play a role in cell fidelity, RNA splicing, RNA transport, apoptosis, and regulation of transcription and translation. For example, human tyrosyl-tRNA synthetase can be proteolytically cleaved into two parts with distinct cytokine activities. The C-terminal domain is chemotactic for monocytes and leukocytes and promotes the generation of myeloperoxidase, tumor necrosis factor-α and tissue factor. The N-terminal domain binds to the interleukin-8 type A receptor and functions as an interleukin 8-like cytokine. Human tyrosyl-tRNA synthetase (TyrRS) is secreted from tumor cells in the apoptotic stage. It may also accelerate apoptosis (Wakasugi, K and Schimmel, P (1999) Science 284: 147-151). Mitochondrial type Neurospora crassa TyrRS and S. cerevisiae LeuRS are essential for several group I intron splicing activities, and human mitochondrial LeuRS as yeast LeuRS in null strains of yeast Can be substituted. Several bacterial aaRSs are involved in the regulation of their own transcription or translation (see Martinis, supra). Several aaRSs can synthesize diadenosine oligophosphates, a class of signaling molecules that have a role in cell proliferation, differentiation, and apoptosis (Kisselev, LL et al. (1998) FEBS Lett. 427: 157-163; Vartanian, A. et al. (1999) FEBS Lett. 456: 175-180).
Autoantibodies against aminoacyl-tRNA are produced by patients with autoimmune diseases such as rheumatoid arthritis, dermatomyositis, and polymyositis. It also correlates with complex interstitial lung disease (ILD) intensity (Freist, W. et al. (1999) Biol. Chem. 380: 623-646; Freist, W. et al. (1996) Biol. Chem. Hoppe Seyler 377: 343-356). These autoantibodies appear to be produced in response to viral infection, and Coxsackie virus has been used to induce experimental viral myositis in animals.

Comparison of the aaRS structure between the human body and pathogens has helped in the design of new antibiotics (Schimmel, supra). Site-specific incorporation of unnatural amino acid groups into proteins is possible in vivo using genetically engineered aaRS groups (Liu, DR et al. (1997) Proc. Natl. Acad. Sci. USA 94: 10092-10097).

is a modified modified ribonucleosides of tRNA pseudouridine ([psi) is unevenly distributed to the anticodon region of transition RNA (tRNA), large and small ribosomal RNA (rRNA), and nuclear low molecular RNA (snRNA). Ψ is the most common modified nucleoside present in tRNA (ie other than G, A, U, and C). Only a few yeast tRNAs unrelated to protein synthesis have no ψ (Cortese, R. et al. (1974) J. Biol. Chem. 249: 1103-1108). Pseudouridine synthase (Pseudouridylate Synthase), the enzyme responsible for the conversion of uridine to Ψ, was first isolated from Salmonella typhimurium (Arena, F. et al. (1978) Nucleic Acids Res. 5: 4523- 4536). This enzyme has since been isolated from a number of mammals (eg, steers and mice) (Green, CJ et al. (1982) J. Biol. Chem. 257: 3045-52 and Chen, J. and Patton, JR. (1999) RNA 5: 409-419). tRNA pseudouridine synthase is the most widely studied member of this family. Optimal activities of these enzymes require thiol donors (such as cysteine) and monovalent cations (such as ammonia and potassium). No additional cofactors or high energy molecules (ATP or GTP) are required (Green, supra). Another eukaryotic pseudouridine synthase group has been identified and appears to be specific for rRNA (reviewed by Smith, CM and Steitz, JA (1997) Cell 89: 669-672). Also, certain bispecific enzymes have been identified that utilize both tRNA and rRNA substrates (Wrzesinski, J. et al. (1995) RNA 1: 437-448). When Ψ is absent within the anticodon loop of tRNA, both bacteria (Singer, CE et al. (1972) Nature New Biol. 238: 72-74) and yeast (Lecointe, F. (1998) 273: 1316-1323) Growth is reduced in, but this gene defect is not lethal.

Another ribonucleoside modification occurs primarily in eukaryotic cells. This is a conversion from guanosine to N ² , N ² -dimethylguanosine (m ² ₂ G), occurring at base position 26 or 10 in the D-stem of cytosolic and mitochondrial tRNAs. This post-transcriptional modification appears to stabilize the tRNA structure by preventing the formation of alternative tRNA secondary and tertiary structures. Yeast tRNA ^Asp is unique in that it does not have this modification. This modification does not occur in eubacteria, probably because the tRNA structure in these cells and organelles is constrained by the sequence, and post-transcriptional modifications do not need to prevent the formation of another structure (Steinberg, S. and Cedergren, R. (1995) RNA 1: 886-891 and references). 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 -dimethylguanosine methyltransferase (also called the TRM1 gene product, this Referred to herein as TRM) (Edqvist, J. (1995) Biochimie 77: 54-61). This enzyme is localized in both the nucleus and mitochondria (Li, JM. Et al. (1989) J. Cell Biol. 109: 1411-1419). Based on studies with Xenopus TRM, base pairing at the C11-G24 and G10-C25 positions just before G26 to be modified may have certain requirements, and another structure of tRNA The characteristic features may also be necessary for proper presentation of the G26 substrate (Edqvist. J. et al. (1992) Nucleic Acids Res. 20: 6575-6581). Studies in yeast suggest that cells with certain weak ocher tRNA suppressors (sup3-i) cannot suppress translation termination without TRM activity, so TRM is the correct tertiary tRNA in eukaryotic cells. In addition to the more general function of securing the original structure, this suggests that it also has a role of adjusting the frequency of inhibition (Niederberger, C. et al. (1999) FEBS Lett. 464: 67-70).

Translation start The start of translation can be divided into three stages. The first stage, to form the 43S pre-initiation complex from one start transfer RNA and (Met-tRNA _f) and 40S ribosomal subunits. The second stage binds the 43S pre-initiation complex to mRNA and then moves the complex to the correct AUG initiation codon. The third stage directs the 60S ribosomal subunit to the 40S subunit, producing an 80S ribosome at the start codon. Translational regulation primarily involves the first and second stages of the initiation process (VM Pain (1996) Eur. J. Biochem. 236: 747-771).

Several initiation factors, many of which have multiple subunits, are involved in the complex of the initiation tRNA with the 40S ribosomal subunit. eIF2 is a guanine nucleotide binding protein that mobilizes the starting tRNA to the 40S ribosomal subunit. eIF2 associates with the initiating tRNA only when bound to GTP. eIF2B is a kind of guanine nucleotide exchange protein, and is responsible for the conversion of eIF2 from a GDP binding inactive form to a GTP binding active form. Two other factors, eIF1A and eIF3, bind to and stabilize the 40S subunit, which interacts with 18S ribosomal RNA and specific ribosomal structural proteins. eIF3 is also involved in the association of 40S ribosomal subunit with mRNA. Met-tRNA _f , eIF1A, eIF3 and 40S ribosomal subunit together form a 43S pre-initiation complex (Pain, supra).

Multiple additional factors are required for the 43S pre-initiation complex to bind to the mRNA molecule, and this process is regulated at several levels. The complex called eIF4F is a complex composed of three kinds of proteins, eIF4E, eIF4A, and eIF4G. eIF4E recognizes and binds to the 5′-terminal m ⁷ GTP cap of mRNA, eIF4A is a bidirectional RNA-dependent helicase, and eIF4G is a kind of scaffold polypeptide. eIF4G has three binding domains. The N-terminal third of eIF4G interacts with eIF4E, the central third interacts with eIF4A, and the C-terminal third interacts with eIF3 bound to the 43S pre-initiation complex. To do. Thus, eIF4G bridges the 40S ribosomal subunit and mRNA (MW Hentze (1997) Science 275: 500-501).

  The ability of eIF4F to initiate 43S pre-initiation complex is regulated by the 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 a secondary structure that prevents binding of the 43S pre-initiation complex. The helicase activity of eIF4A appears to function to remove this secondary structure and promote binding of the 43S pre-initiation complex (Pain, supra).

In the translation elongation process, an additional group of amino acids bind to the starting methionine to form a complete polypeptide chain. Elongation factors EF1α, EF1βγ, and EF2 are involved in the elongation of the polypeptide chain after initiation. EF1α is a GTP binding protein. In the GTP-bound form of EF1α, EF1α guides aminoacyl tRNA to the A site of the ribosome. The amino acid attached to the newly arrived aminoacyl-tRNA forms a peptide bond with the initiating methionine. GTP on EF1α is hydrolyzed to GDP, and EF1α-GDP dissociates from the ribosome. By binding EF1βγ to EF1α-GDP and dissociating GDP from EF1α, EF1α can bind to GTP and a new cycle starts.

  In turn, aminoacyl tRNAs are directed to the ribosome and EF-G (another GTP binding protein) catalyzes the transfer of tRNA from the A site to the P site and finally to the E site of the ribosome. This allows the ribosome and mRNA to remain bound during translation.

A translation termination releasing factor (release factor) eRF to terminate the translation. eRF recognizes a stop codon in the mRNA and releases the polypeptide chain from the ribosome.

Expression profiling array technology can provide an easy way to explore the expression of a single polymorphic gene or the expression profile of many related or unrelated genes. When testing the expression of a single gene, the array is used to detect the expression of a particular gene or variant thereof. When testing expression profiles, the array provides a platform to identify genes such as: That is, which genes are tissue specific, affected by the substance being tested in the toxicity assay, are part of a signaling cascade, perform housekeeping functions, or have a specific genetic predisposition or condition Identification of genes that are specifically associated with a disease or disorder.

  The discovery of novel nucleic acid binding proteins and the polynucleotides that encode them can provide a new group of compositions to meet the needs of the art. These novel compositions are useful in the diagnosis, treatment, and prevention of cell proliferation abnormalities, neurological diseases, developmental disorders, autoimmune / inflammatory diseases, and infectious diseases. It is also useful for calculating the effects of foreign compounds on expression.

  The present invention is generally referred to as “NAAP”, and individually “NAAP-1”, “NAAP-2”, “NAAP-3”, “NAAP-4”, “NAAP-5”, “NAAP-6”, respectively. , “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 and NAAP- A purified polypeptide, which is a nucleic acid binding protein, referred to as “23” is provided. In certain embodiments, the invention provides: (a) a polypeptide consisting of an amino acid sequence selected from the group consisting of SEQ ID NO: 1-23; (b) an amino acid sequence selected from the group consisting of SEQ ID NO: 1-23; A polypeptide comprising a natural amino acid sequence that is at least 90% identical, (c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1-23, and (d) SEQ An isolated polypeptide selected from the group consisting of immunogenic fragments of a polypeptide having an amino acid sequence selected from the group consisting of ID NO: 1-23 is provided. In one aspect, an isolated polypeptide consisting of the amino acid sequence of SEQ ID NO: 1-23 is provided.

  The present invention also relates to (a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 1-23, (b) at least 90% of the amino acid sequence selected from the group consisting of SEQ ID NO: 1-23 (C) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1-23, and (d) SEQ ID NO An isolated polynucleotide is provided that encodes a polypeptide selected from the group consisting of immunogenic fragments of a polypeptide having an amino acid sequence selected from the group consisting of: 1-23. In one embodiment, the polynucleotide encodes a polypeptide selected from the group consisting of SEQ ID NO: 1-23. In another embodiment, the polynucleotide is selected from the group having SEQ ID NO: 24-46.

  The invention further comprises (a) a polypeptide having a certain amino acid sequence selected from the group consisting of SEQ ID NO: 1-23, and (b) a certain amino acid sequence selected from the group consisting of SEQ ID NO: 1-23 (C) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1-23 And (d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1-23, and a polynucleotide encoding a polypeptide selected from the group consisting of: A recombinant polynucleotide having a promoter sequence linked to is provided. In one embodiment, the present invention provides a cell transformed with this recombinant polynucleotide. In another embodiment, the present invention provides a transgenic organism having this recombinant polynucleotide.

  The present invention further relates to (a) a polypeptide consisting of an amino acid sequence selected from the group consisting of SEQ ID NO: 1-23, and (b) an amino acid sequence selected from the group consisting of SEQ ID NO: 1-23; A polypeptide comprising a natural amino acid sequence having 90% or more identity, and (c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1-23 And (d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1-23, and a method for producing a polypeptide selected from the group consisting of: The production method comprises the steps of (a) culturing cells transformed with the recombinant polynucleotide under conditions suitable for polypeptide expression, and (b) recovering the polypeptide so expressed. The recombinant polynucleotide has a promoter sequence operably linked to the polynucleotide encoding the polypeptide.

  The invention further comprises (a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 1-23, (b) at least 90% of an amino acid sequence selected from the group consisting of SEQ ID NO: 1-23 A polypeptide comprising a native amino acid sequence that is identical, (c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1-23, and (d) SEQ ID NO: 1 An isolated antibody is provided that specifically binds to a polypeptide selected from the group consisting of immunogenic fragments of a polypeptide having an amino acid sequence selected from the group consisting of -23.

  The invention further comprises (a) a polynucleotide having a polynucleotide sequence selected from the group consisting of SEQ ID NO: 24-46, (b) a polynucleotide sequence selected from the group consisting of SEQ ID NO: 24-46 and at least A polynucleotide comprising a natural polynucleotide sequence having 90% identity, a polynucleotide sequence complementary to (c) (a), a polynucleotide sequence complementary to (d) (b), and (e) ( Provided is an isolated polynucleotide selected from the group consisting of the RNA equivalents of a) to (d). In one embodiment, the polynucleotide consists of at least 60 contiguous nucleotide groups.

  The invention further provides a method of detecting a target polynucleotide in a sample. Wherein the target polynucleotide is (a) a polynucleotide having a polynucleotide sequence selected from the group consisting of SEQ ID NO: 24-46, (b) a polynucleotide sequence selected from the group consisting of SEQ ID NO: 24-46 (C) a polynucleotide complementary to (a), a polynucleotide complementary to (d) (b), and (e) ( a) having a polynucleotide sequence selected from the group consisting of RNA equivalents of a) to (d). The detection method comprises: (a) hybridizing the sample with a probe consisting of at least 20 consecutive nucleotide groups consisting of a sequence complementary to the target polynucleotide in the sample; and (b) The process comprises detecting the presence or absence of the hybridization complex, and optionally detecting the amount of the complex if present. The probe specifically hybridizes to the target polynucleotide under conditions such that a hybridization complex is formed between the probe and the target polynucleotide or fragment thereof. In one embodiment, the probe consists of at least 60 contiguous nucleotide groups.

  The present invention also provides a method for detecting a target polynucleotide in a sample. Wherein the target polynucleotide is (a) a polynucleotide comprising a polynucleotide sequence selected from the group consisting of SEQ ID NO: 24-46, (b) a polynucleotide selected from the group consisting of SEQ ID NO: 24-46 A polynucleotide comprising a natural polynucleotide sequence at least 90% identical to the sequence, (c) a polynucleotide complementary to the polynucleotide of (a), (d) a polynucleotide complementary to the polynucleotide of (b), And (e) having the sequence of a polynucleotide selected from the group consisting of RNA equivalents of (a)-(d). The detection method comprises: (a) a process of amplifying a target polynucleotide or fragment thereof using polymerase chain reaction amplification; and (b) detecting the presence / absence of the amplified target polynucleotide or fragment thereof, Optionally including detecting the amount of the polynucleotide or fragment thereof, if present.

  The invention further provides a composition comprising an effective amount of a polypeptide and a pharmaceutically acceptable excipient. An effective amount of a polypeptide is (a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 1-23, (b) an amino acid sequence selected from the group consisting of SEQ ID NO: 1-23 and at least A polypeptide comprising a natural amino acid sequence that is 90% identical, (c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1-23, and (d) SEQ ID Selected from the group consisting of immunogenic fragments of polypeptides having an amino acid sequence selected from the group consisting of NO: 1-23. In one embodiment, the composition has an amino acid sequence selected from the group consisting of SEQ ID NO: 1-23. Furthermore, the present invention provides a method comprising administering the composition to a patient in need of treatment of a disease or condition associated with reduced expression of functional NAAP.

  The present invention also provides (a) a polypeptide having a certain amino acid sequence selected from the group consisting of SEQ ID NO: 1-23, (b) a certain amino acid sequence selected from the group consisting of SEQ ID NO: 1-23 (C) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1-23, and (d) In order to confirm the effectiveness as an agonist of a polypeptide selected from the group consisting of an immunogenic fragment of a polypeptide having a certain amino acid sequence selected from the group consisting of SEQ ID NO: 1-23 Methods for screening compounds are provided. The screening method comprises (a) a process of exposing a sample having the polypeptide to a certain compound, and (b) a process of detecting agonist activity in the sample. Alternatively, the present invention provides a composition having an agonist compound identified by this method and an acceptable pharmaceutical excipient. In yet another alternative, the present invention provides a method of administering this composition to a patient in need of treatment of a disease or condition that involves a decrease in the expression of functional NAAP.

  The invention further comprises (a) a polypeptide having a certain amino acid sequence selected from the group consisting of SEQ ID NO: 1-23, and (b) a certain amino acid sequence selected from the group consisting of SEQ ID NO: 1-23 (C) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1-23, and (d) ) SEQ ID NO: 1-23 an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of: a method of screening a compound for the effectiveness as a antagonist of a polypeptide selected from the group consisting of provide. The screening method consists of (a) exposing a sample containing the polypeptide to a certain compound, and (b) detecting antagonistic activity in the sample. In one embodiment, the present invention provides a composition comprising an antagonist compound identified by this method and a pharmaceutically acceptable excipient. In yet another alternative, the present invention provides a method comprising administration of this composition to a patient in need of treatment of a disease or condition associated with overexpression of functional NAAP.

  The invention further comprises (a) a polypeptide having a certain amino acid sequence selected from the group consisting of SEQ ID NO: 1-23, and (b) a certain amino acid sequence selected from the group consisting of SEQ ID NO: 1-23 A polypeptide having a certain natural amino acid sequence with at least 90% identity to, (c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1-23, or (D) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1-23, and a method for screening a certain compound that specifically binds to a certain polypeptide selected from the group consisting of provide. The screening method comprises: (a) mixing the polypeptide with at least one test compound under appropriate conditions; (b) detecting binding of the test compound to the polypeptide, thereby And a process of identifying a compound that specifically binds.

  The invention further comprises (a) a polypeptide comprising an amino acid sequence selected from the group having SEQ ID NO: 1-23, (b) at least 90% of an amino acid sequence selected from the group consisting of SEQ ID NO: 1-23 (C) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group having SEQ ID NO: 1-23, or (d) SEQ ID NO A method is provided for screening for compounds that modulate the activity of a polypeptide selected from the group consisting of immunogenic fragments of a polypeptide having an amino acid sequence selected from the group having: 1-23. The screening method includes: (a) a step of mixing a polypeptide with at least one test compound under conditions where the activity of the polypeptide is allowed; and (b) a step of calculating 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, the change in the activity of the polypeptide in the presence of the test compound. Means a compound that modulates the activity of a polypeptide.

  The present invention further provides a method of screening certain compounds for the effect of altering the expression of a target polynucleotide having a certain polynucleotide sequence selected from the group consisting of SEQ ID NOs: 24-46. The method comprises (a) exposing a sample having the target polynucleotide to a compound, (b) detecting alterations in expression of the target polynucleotide, and (c) a variable amount of the compound. The process consists of comparing the expression of the target polynucleotide in the presence to the expression in the absence of the compound.

  The present invention further provides a method for calculating the toxicity of a test compound. This method includes the following processes. (A) A process of treating a biological sample having a nucleic acid group with a test compound. (B) A process of hybridizing the nucleic acid group of the treated biological sample. The following probe is used in this process. (I) a polynucleotide having a polynucleotide sequence selected from the group consisting of SEQ ID NO: 24-46, (ii) at least a polynucleotide sequence selected from the group consisting of SEQ ID NO: 24-46 A polynucleotide having a natural polynucleotide sequence with 90% identity, (iii) a polynucleotide having a sequence complementary to (i), (iv) a polynucleotide complementary to the polynucleotide of (ii), (v ) A probe consisting of at least 20 contiguous nucleotide groups of a polynucleotide selected from the group consisting of the RNA equivalents of (i) to (iv). Hybridization occurs under conditions such that a specific hybridization complex is formed between the probe and a target polynucleotide in the biological sample. The target polynucleotide is (i) a polynucleotide having a polynucleotide sequence selected from the group consisting of SEQ ID NO: 24-46, (ii) a certain selected from the group consisting of SEQ ID NO: 24-46 A polynucleotide having a natural polynucleotide sequence having at least 90% identity to the polynucleotide sequence, (iii) a polynucleotide complementary to the polynucleotide of (i), (iv) complementary to the polynucleotide of (ii) Selected from the group consisting of: (v) (i) to (iv) RNA equivalents. Alternatively, the target polynucleotide has a fragment of a polynucleotide sequence selected from the group consisting of (i) to (v) above. The toxicity calculation method further includes (c) the process of quantifying the amount of hybridization complex, and (d) the amount of hybridization complex of the treated biological sample, compared to the amount of hybridization of the untreated biological sample. A difference in the amount of hybridization complex in the treated biological sample, including the process of comparing to the amount of hybridization complex, indicates the toxicity of the test compound.

(Description of the present invention)
Although the proteins, nucleotide sequences and methods of the present invention will be described, it should be understood before that that the present invention is not limited to the specific apparatus, materials and methods described and may be modified. Also, the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of the invention which is limited only by the claims. Please also understand.

  As used in the claims and in the specification, the singular forms “a” and “its” may refer to the plural unless the context clearly dictates otherwise. Please note that. Thus, for example, when “a certain host cell” is described, there may be a plurality of such host cells, when “a certain antibody” is described, one or more antibodies, and It also refers to antibody equivalents known to those skilled in the art.

  All technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art unless otherwise defined. Although any devices, materials, and methods similar or equivalent to those described herein can be used to practice or test the present invention, suitable devices, materials, and methods are now described. All publications referred to in this invention are cited for purposes of describing and disclosing cells, protocols, reagents and vectors that have been reported in the publication and may be used in connection with the present invention. . No disclosure in this specification is an admission that the invention is not entitled to antedate such disclosure by virtue of prior art.

(Definition)
The term “NAAP” refers to substantially purified NAAP obtained from all species (especially mammals including cattle, sheep, pigs, mice, horses and humans), such as natural, synthetic, semi-synthetic or recombinant. The amino acid sequence of

  The term “agonist” refers to a molecule that enhances or mimics the biological activity of NAAP. Examples of agonists include proteins, nucleic acids, carbohydrates, small molecules, etc. that modulate NAAP activity by interacting directly with NAAP or by acting on a component of a biological pathway in which NAAP is involved. Any compound or composition may be included.

  The term “allelic variant” refers to another form of the gene encoding NAAP. Allelic variants can be made from at least one mutation in a nucleic acid sequence. Transformed mRNA or polypeptide can also be made. Its structure or function may or may not change. A gene may not have any naturally occurring allelic variants or may have more than one. The usual mutational changes that give rise to allelic variants are generally attributed to natural deletions, additions or substitutions of nucleotides. Each of these changes can occur once or several times within a given sequence, alone or together with other changes.

  The sequences included in the “transformed / modified” nucleic acid sequence encoding NAAP have various nucleotide deletions, insertions, or substitutions, so that the same polypeptide as NAAP or NAAP There are sequences that give rise to polypeptides with at least one of the functional characteristics. This definition includes improper or unexpected hybridization to a group of allelic variants at a position that is not a normal chromosomal locus for a polynucleotide sequence encoding NAAP, and also includes a polynucleotide encoding NAAP. Of polymorphisms that are readily detectable or difficult to detect using certain oligonucleotide probes. The encoded protein may also be “transformed / modified” and may have deletions, insertions or substitutions of amino acid residues that result in a silent change resulting in a functionally equivalent NAAP. . Intentional amino acid substitutions are in terms of the polarity, charge, solubility, hydrophobicity, hydrophilicity, and / or amphipathic nature of the residue group, as long as NAAP activity is retained biologically or immunologically. Can be made based on similarity. For example, negatively charged amino acids include aspartic acid and glutamic acid, and positively charged amino acids include lysine and arginine. Amino acids having uncharged polar side chains with similar hydrophilicity values can include asparagine and glutamine, and serine and threonine. Amino acids having uncharged side chains with similar hydrophilicity values may include leucine, isoleucine and valine, glycine and alanine, and phenylalanine and tyrosine.

  The term “amino acid” or “amino acid sequence” refers to an oligopeptide, peptide, polypeptide or protein sequence or fragment thereof and refers to a natural or synthetic molecule. As used herein, “amino acid sequence” refers to the amino acid sequence of a natural protein molecule, and “amino acid sequence” and similar terms shall limit the amino acid sequence to the complete native amino acid sequence associated with the listed protein molecule. It is not something to do.

  “Amplification” refers to the additional replication of a nucleic acid sequence. Amplification usually uses polymerase chain reaction (PCR) techniques known to those skilled in the art.

  The term “antagonist” refers to a molecule that inhibits or attenuates the biological activity of NAAP. Antagonists include proteins such as antibodies, nucleic acids, carbohydrates, small molecules, any molecule that interacts directly with NAAP or acts on components of biological pathways involving NAAP to regulate NAAP activity. Other compounds and compositions can be included.

The term “antibody” refers to intact immunoglobulin molecules and fragments thereof, such as Fab, F (ab ′) ₂ and Fv fragments, that are capable of binding antigenic determinants. For the production of antibodies that bind to the NAAP polypeptide group, an intact polypeptide group can be used as an immunizing antigen, or a fragment group having the small peptide group can be used. The polypeptide or oligopeptide used to immunize an animal (mouse, rat, rabbit, etc.) can be derived from a polypeptide or oligopeptide obtained by RNA translation or chemical synthesis, and can be used as a carrier protein if desired. Conjugation is also possible. Commonly used carriers that chemically bond with peptides include bovine serum albumin, thyroglobulin, and mussel hemocyanin (KLH). Binding The binding peptide is used to immunize an animal.

  The term “antigenic determinant” refers to a region of a molecule (ie, an epitope) that makes contact with a particular antibody. When immunizing a host animal with a protein or protein fragment, multiple regions of the protein can trigger the production of antibodies that specifically bind to an antigenic determinant (a specific region or three-dimensional structure of the protein). An antigenic determinant can compete with an intact antigen (ie, an immunogen used to elicit an immune response) in binding to the antibody.

The term “aptamer” refers to a nucleic acid or oligonucleotide that binds to a specific molecular target. Aptamers are derived from in vitro evolution processes (eg, SELEX (abbreviation of Systematic Evolution of Ligands by EXponential Enrichment), described in US Pat. No. 5,270,163). This is a process of selecting target-specific aptamer sequences from a large group of 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 component of an aptamer can have modified sugar groups (e.g., the 2′-OH group of a ribonucleotide is replaced with 2′-F or 2′-NH ₂ ), and these sugar groups May improve desired properties, for example, resistance to nucleases or longer life in blood. In order to slow down the rate at which aptamers are removed from the circulatory system, aptamers can be conjugated to molecules such as high molecular weight carriers. Aptamers can be specifically cross-linked with their homologous ligands, for example by photoactivation of certain cross-linking agents (see, for example, Brody, EN and L. Gold (2000) J. Biotechnol. 74: 5-13). ).

  The term “intramer” means an aptamer that is expressed in vivo. For example, certain RNA aptamers are expressed at high levels in the cytoplasm of leukocytes using an RNA expression system based on vaccinia virus (Blind, M. et al. (1999) Proc. Natl Acad. Sci. USA 96: 3606-3610).

  The term “spiegelmer” refers to aptamers comprising L-DNA, L-RNA or other left-handed nucleotide derivatives or nucleotide-like molecules. Aptamers containing levorotatory nucleotides are resistant to degradation by natural enzymes that normally act on substrates containing dextrorotatory nucleotides.

  The term “antisense” refers to any composition capable of base pairing with the “sense” (coding) strand of a particular nucleic acid sequence. Antisense compositions include DNA, RNA, peptide nucleic acids (PNA), modified backbone linkages such as phosphorothioic acid, methylphosphonic acid or benzylphosphonic acid, modified sugar groups such as 2 ' There are oligonucleotides with -methoxyethyl sugar or 2'-methoxyethoxy sugar, or oligonucleotides with modified bases such as 5-methylcytosine, 2-deoxyuracil or 7-deaza-2'-deoxyguanosine obtain. Antisense molecules can be produced by any method, such as chemical synthesis or transcription. When introduced into a cell, a complementary antisense molecule forms a base pair with the natural nucleic acid sequence produced by the cell and forms a duplex that prevents transcription or translation. The expression “negative” or “minus” may refer to the antisense strand of a reference DNA molecule, and the expression “positive” or “plus” may refer to the sense strand of a reference DNA molecule.

  The term “biologically active” refers to a protein having the structural, regulatory or biochemical functions of a natural molecule. Similarly, the term “immunologically active” or “immunogenic” means that natural or recombinant NAAP, synthetic NAAP, or any oligopeptide thereof is suitable for a specific immune response in an appropriate animal or cell. Refers to the ability to bind to a specific antibody.

  The term “complementary” or “complementarity” refers to the relationship between two single stranded nucleic acids that anneal by base pairing. For example, the sequence “5′-AGT-3 ′” is paired with the complementary sequence “3′-TCA-5 ′”.

  “Composition comprising (having) a polynucleotide sequence of” and “composition having an amino acid sequence of” refer to a wide range of optional components comprising a given polynucleotide or amino acid sequence. The composition can include a dry formulation or an aqueous solution. A composition having a polynucleotide sequence encoding NAAP or a fragment of NAAP can be used as a hybridization probe. These probes can be stored in lyophilized form and can be conjugated with stabilizers such as carbohydrates. In hybridization, the probe is dispersed in an aqueous solution containing a salt (eg, NaCl), a surfactant (eg, sodium dodecyl sulfate; SDS) and other components (eg, Denhart's solution, milk powder, salmon sperm DNA, etc.) be able to.

  The “consensus sequence” is obtained by repeatedly analyzing the DNA sequence in order to separate unnecessary bases, and extending in the 5 ′ and / or 3 ′ direction using an XL-PCR kit (Applied Biosystems, Foster City CA) Resequenced nucleic acid sequences or one or more duplicates using GELVIEW fragment assembly system (GCG, Madison, WI) or fragment assembly computer programs such as Phrap (University of Washington, Seattle WA) Refers to nucleic acid sequences assembled from cDNA, EST, or genomic DNA fragments. Some sequences perform both extension and assembly to create a consensus sequence.

A “conservative amino acid substitution” is a substitution that is predicted to cause little loss of the properties of the original protein when the substitution is made, that is, the structure and particularly the function of the protein are preserved, and such substitution greatly changes. Refers to no replacement. The table below shows the amino acids that can be substituted for the original amino acids in the protein and are recognized as conservative amino acid substitutions.

  Conservative amino acid substitutions typically include (a) the backbone structure of the polypeptide in the substitution region, eg, a β sheet or α helix structure, (b) the charge or hydrophobicity of the molecule at the substitution site, and / or (c) the majority of the side chain. Is retained.

  “Deletion” refers to a change in amino acid or nucleotide sequence that results in the loss of one or several amino acid residues or nucleotides.

  The term “derivative” refers to a chemical modification of a polypeptide or polynucleotide. For example, replacement of hydrogen with an alkyl group, acyl group, hydroxyl group or amino group can be included in chemical modification of the polynucleotide. A polynucleotide derivative encodes a polypeptide that retains at least one biological or immunological function of the natural molecule. Polypeptide derivatives have been modified by glycosylation, polyethylene glycolation, or any similar process that retains at least one biological or immunological function of the polypeptide of derived origin It is a polypeptide.

  “Detectable label” refers to a reporter molecule or enzyme capable of producing a measurable signal and covalently or non-covalently bound to a polynucleotide or polypeptide.

  “Differential expression” refers to an increase (upregulation) or decrease (downregulation), or a loss of gene expression or protein expression, as determined by comparing at least two different samples. Such a comparison can be made, for example, between a treated sample and an untreated sample, or a pathological sample and a healthy sample.

  “Exon shuffling” refers to the recombination of different coding regions (exons). Since an exon can represent one structural or functional domain of the encoded protein, new combinations of stable substructures can be assembled and new protein functions evolved Can be promoted.

  The term “fragment” refers to a unique portion of a NAAP or NAAP-encoding polynucleotide that is identical in sequence to its parent sequence but shorter in length than the parent sequence. Fragments can have a length that is shorter than the total length of the defined sequence minus one nucleotide / amino acid residue. For example, a fragment may have 5 to 1000 consecutive nucleotide or amino acid residues. Fragments used as probes, primers, antigens, therapeutic molecules or for other purposes are at least 5, 10, 15, 16, 20, 25, 30, 40, 50, 60, 75, 100, 150, It can be 250 or 500 contiguous nucleotides or amino acid residue lengths. Fragments can be preferentially selected from specific regions of a molecule. For example, a polypeptide fragment is a length selected from the first 250 or 500 amino acids (or the first 25% or 50%) of a polypeptide as found within a defined sequence. Can have a continuous amino acid. These lengths are clearly given by way of example, and in embodiments of the invention can be any length supported by this specification including the sequence listing, tables and drawings.

  The fragment of SEQ ID NO: 24-46 contains a region of unique polynucleotide sequence that unambiguously identifies SEQ ID NO: 24-46, for example, different from other sequences in the genome from which the fragment was obtained. Certain fragments of SEQ ID NO: 24-46 are useful, for example, in hybridization and amplification techniques, or similar methods that distinguish SEQ ID NO: 24-46 from related polynucleotide sequences. The exact length of the fragment of SEQ ID NO: 24-46 and the region of SEQ ID NO: 24-46 corresponding to the fragment can be routinely determined by one skilled in the art based on the intended purpose for the fragment .

  Certain fragments of SEQ ID NO: 1-23 are encoded by certain fragments of SEQ ID NO: 24-46. The fragment of SEQ ID NO: 1-23 contains a unique amino acid sequence region that specifically identifies SEQ ID NO: 1-23. For example, the fragment of SEQ ID NO: 1-23 is useful as an immunogenic peptide to produce an antibody that specifically recognizes SEQ ID NO: 1-23. The exact length of the fragment of SEQ ID NO: 1-23 and the region of SEQ ID NO: 1-23 that the fragment corresponds to can be routinely determined by those skilled in the art based on the intended purpose for the fragment. is there.

  A “full length” polynucleotide sequence is a sequence having at least one translation start codon (eg, methionine) followed by an open reading frame and a translation stop codon. A “full length” polynucleotide sequence encodes a “full length” polypeptide sequence.

  The term “homology” means sequence similarity, interchangeability, or sequence identity of two or more polynucleotide sequences or two or more polypeptide sequences.

  The term “percent match” or “% match” as applied to a polynucleotide sequence refers to the percentage of residues that match between at least two or more polynucleotide sequences that are aligned using a standardized algorithm. Since the standardization algorithm optimizes the alignment between the two sequences, a gap group can be inserted into the two sequences to be compared in a standardized and reproducible manner, so that the two sequences can be compared more significantly.

  The percent identity between polynucleotide sequences can be determined using the default parameters of the CLUSTAL V algorithm such as those incorporated into the MEGALIGN version 3.12e sequence alignment program. This program is part of the LASERGENE software package (a set of molecular biological analysis programs) (DNASTAR, Madison WI). This CLUSTAL V is described in Higgins, D.G. and P.M. Sharp (1989) CABIOS 5: 151-153, Higgins, D.G. et al. (1992) CABIOS 8: 189-191. The default parameters when aligning polynucleotide sequences in pairs are set as Ktuple = 2, gap penalty = 5, window = 4, and “diagonals saved” = 4. A “weighted” residue weight table is selected as the default. CLUSTAL V reports the percent identity as a “percent similarity” between aligned polynucleotide sequence pairs.

  Alternatively, the National Local Center for Biotechnology Information (NCBI) Basic Local Alignment Search Tool (BLAST) is commonly used and provides a set of free sequence comparison algorithms (Altschul, SF et al. (1990) ) J. Mol. Biol. 215: 403-410). This algorithm is available from several sources, and is also available from NCBI in Bethesda, Maryland and the Internet (http://www.ncbi.nlm.nih.gov/BLAST/). The BLAST software suite includes a variety of sequence analysis programs, one of which is “blastn” that aligns a known polynucleotide sequence with another polynucleotide sequence obtained from various databases. In addition, a tool called “BLAST 2 Sequences”, which is used to directly compare two nucleotide sequences in a pair-wise manner, can be used. “BLAST 2 Sequences” can be used interactively by accessing http://www.ncbi.nlm.nih.gov/gorf/bl2.html. The “BLAST 2 Sequences” tool can be used for both blastn and blastp (described below). BLAST programs are typically used with gaps and other parameters set to default settings. For example, to compare two nucleotide sequences, blastn may be performed using the “BLAST 2 Sequences” tool Version 2.0.12 (April 21, 2000) set to default parameters. An example of setting default parameters is shown below.

Matrix: BLOSUM62
Reward for match: 1
Penalty for mismatch: -2
Open Gap: 5 and Extension Gap: 2 penalties
Gap x drop-off: 50
Expect: 10
Word Size: 11
Filter: on

  The percent identity can be measured over the full length of a defined sequence (eg, a sequence defined with a particular SEQ ID number). Alternatively, the percentage match for shorter lengths, eg, lengths of defined fragments obtained from larger sequences (eg, fragments of at least 20, 30, 40, 50, 70, 100 or 200 consecutive nucleotides) May be measured. The lengths listed here are merely exemplary, and the percent identity can be measured using any fragment length supported by the sequences described herein, including tables, figures, and sequence listings. It should be understood that a certain length can be described.

  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 should be understood that this degeneracy can be used to cause changes in a nucleic acid sequence to produce a large number of nucleic acid sequences in which all nucleic acid sequences encode substantially the same protein.

  The term “percent match” or “% identity” as used for a polypeptide sequence refers to the percentage of matching residues between two or more polypeptide sequences that are aligned using a standardized algorithm. Methods for polypeptide sequence alignment are known. Some alignment methods take into account conservative amino acid substitutions. Such conservative substitutions as detailed above usually conserve the structure of the polypeptide (and therefore also the function) since it preserves the charge and hydrophobicity of the substitution site.

  The percent identity between polypeptide sequences can be determined using the default parameters of the CLUSTAL V algorithm, such as those incorporated into the MEGALIGN version 3.12e sequence alignment program (see above for explanation). Default parameters for pairwise alignment of polypeptide sequences using CLUSTAL V are set as Ktuple = 1, gap penalty = 3, window = 5, “diagonals saved” = 5. The PAM250 matrix is selected as the default residue weight table. As with polynucleotide alignment, the percent identity of aligned polypeptide sequence pairs is reported by CLUSTAL V as “percent similarity”.

  Alternatively, the NCBI BLAST software suite may be used. For example, when comparing two polypeptide sequences pairwise, one would use blastp with the “BLAST 2 Sequences” tool Version 2.0.12 (April 21, 2000) set with default parameters. . An example of setting default parameters is shown below.

Matrix: BLOSUM62
Open Gap: 11 and Extension Gap: 1 penalties
Gap x drop-off: 50
Expect: 10
Word Size: 3
Filter: on

  The percent identity can be measured for the full length of a defined polypeptide sequence (eg, a sequence defined by a particular SEQ ID number). Alternatively, a shorter length, eg, the length of a fragment derived from a defined larger polypeptide sequence (eg, a fragment of at least 15, 20, 30, 40, 50, 70, or 150 contiguous residues) The coincidence rate may be measured. The lengths listed here are merely exemplary, and the percent identity can be measured using any fragment length supported by the sequences described herein, including tables, figures, and sequence listings. It should be understood that a certain length can be described.

  “Human Artificial Chromosome (HAC)” is a linear microchromosome containing all the elements necessary for chromosomal replication, segregation and maintenance, which may contain a DNA sequence of about 6 kb (kilobase) to 10 Mb in size. .

  The term “humanized antibody” refers to an antibody molecule in which the amino acid sequence of the non-antigen binding region has been altered to resemble a human antibody while retaining its original binding ability.

  “Hybridization” is an annealing process in which a single-stranded polynucleotide forms a base pair with a complementary single-stranded polynucleotide under predetermined hybridization conditions. Specific hybridization is an indication that two nucleic acid sequences share high complementarity. Specific hybridization complexes are formed under permissive annealing conditions and remain hybridized after the “wash” step. The washing step is particularly important in determining the stringency of the hybridization process, and more stringent conditions reduce non-specific binding (ie, binding of pairs between nucleic acid strands that are not perfectly matched). The permissive conditions for nucleic acid sequence annealing can be routinely determined by those skilled in the art. The permissive conditions can be constant for any hybridization experiment, but the wash conditions can be varied from experiment to experiment to obtain the desired stringency and thus also hybridization specificity. Permissive annealing conditions are, for example, at a temperature of 68 ° C., in the presence of about 6 × SSC, about 1% (w / v) SDS, and about 100 μg / ml shear-denatured salmon sperm DNA. .

In general, the stringency of hybridization can be expressed to some extent relative to the temperature at which the wash step is performed. Such washing temperatures are usually selected to be about 5-20 ° C. below the melting point (Tm) of the specific sequence at a given ionic strength and pH. This Tm is the temperature at which 50% of the target sequence hybridizes to a perfectly matched probe under a given ionic strength and pH. The formula for calculating Tm and hybridization conditions for nucleic acids are well known and are described in Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual , 2nd edition, volumes 1-3, Cold Spring Harbor Press, Plainview NY. Refer to Chapter 9 of Volume 2 in particular.

  High stringency hybridization between polynucleotides of the present invention involves a 1 hour wash step at about 68 ° C. in the presence of about 0.2 × SSC and about 1% SDS. Alternatively, it may be performed at a temperature of about 65 ° C, 60 ° C, 55 ° C or 42 ° C. SSC concentrations can vary in the range of about 0.1-2 × SSC in the presence of about 0.1% SDS. Usually, a blocking agent is used to prevent nonspecific hybridization. Such blocking agents include, for example, about 100-200 μg / ml denatured salmon sperm DNA. For example, organic solvents such as formamide at a concentration of about 35-50% v / v may be used for hybridization of RNA and DNA under specific conditions. Useful variations of the wash conditions will be apparent to those skilled in the art. Hybridization can indicate evolutionary similarity between nucleotides, particularly under high stringency conditions. Evolutionary similarity strongly suggests a similar role for nucleotide groups and nucleotide-encoded polypeptides.

The term “hybridization complex” refers to a complex of two nucleic acid sequences formed by hydrogen bonding between complementary bases. Hybridization complexes can be formed in solution (such as C ₀ t or R ₀ t analysis). Alternatively, one nucleic acid sequence is present in a dissolved state and the other nucleic acid sequence is a solid support (eg, paper, membrane, filter, chip, pin or glass slide, or other suitable substrate that is a cell or nucleic acid thereof. Can be formed between two nucleic acid sequences that are immobilized on a substrate to which is immobilized.

  The term “insertion” or “addition” refers to a change in amino acid sequence or nucleic acid sequence to which one or more amino acid residues or nucleotides are added, respectively.

  "Immune response" can refer to symptoms associated with inflammation, trauma, immune disorders, infectious diseases or genetic diseases. These symptoms can be characterized by the expression of various factors that can affect the cellular and systemic defense systems, such as cytokines, chemokines, and other signaling molecules.

  The term “immunogenic fragment” refers to a polypeptide or oligopeptide fragment of NAAP that can elicit an immune response when introduced into an organism (eg, a mammal). The term “immunoantigenic fragment” also includes any polypeptide or oligopeptide fragment of NAAP useful in any antibody production method as disclosed herein or as known in the art. .

  The term “microarray” refers to the organization of a plurality of polynucleotides, polypeptides or other compounds on a substrate.

  The term “element” or “array element” refers to a polynucleotide, polypeptide or other compound having a specific designated position on a microarray.

  The term “modulate” or “modulate activity” refers to altering the activity of NAAP. For example, modulation can cause changes in NAAP protein activity, binding properties, or other biological, functional, or immunological properties.

  The terms “nucleic acid” and “nucleic acid sequence” refer to nucleotides, oligonucleotides, polynucleotides or fragments thereof. The terms “nucleic acid” and “nucleic acid sequence” also refer to DNA or RNA of genomic or synthetic origin that can be single-stranded or double-stranded, or can represent the sense or antisense strand It may also refer to peptide nucleic acid (PNA) or any DNA-like or RNA-like substance.

  “Functionally linked” refers to a state in which a first nucleic acid sequence and a second nucleic acid sequence are in a functional relationship. For example, a promoter is operably linked to a coding sequence if the promoter affects the transcription or expression of the coding sequence. Functionally linked DNA sequences can be very close or contiguous, and can be in the same reading frame if necessary to join two protein coding regions.

  “Peptide nucleic acid (PNA)” refers to an antisense molecule or anti-gene agent comprising an oligonucleotide of at least about 5 nucleotides in length, attached to the peptide backbone of amino acid residues ending in lysine. The terminal lysine imparts solubility to this composition. PNA binds preferentially to complementary single-stranded DNA or RNA to stop transcription elongation and can be polyethyleneglycolized to extend the life of PNA in cells.

  “Post-translational modifications” of NAAP may include lipidation, glycosylation, phosphorylation, acetylation, racemization, proteolytic cleavage, and other modifications known in the art. These processes can occur synthetically or biochemically. Biochemical modifications depend on the NAAP enzyme environment and will vary with cell type.

  A “probe” refers to a nucleic acid sequence that encodes NAAP, a complementary sequence thereof, or a fragment thereof, and is used to detect the same sequence, an allelic nucleic acid sequence, or a related nucleic acid sequence. A probe is an isolated oligonucleotide or polynucleotide that is a sequence attached to a detectable label or reporter molecule. Typical labels include radioactive isotopes, ligands, chemiluminescent reagents and enzymes. A “primer” is a short nucleic acid, usually a DNA oligonucleotide, that can be annealed to a target polynucleotide by forming complementary base pairs. The primer can then be extended along the target DNA strand by a DNA polymerase enzyme. Primer pairs can be used for amplification (and identification) of nucleic acid sequences, for example, by polymerase chain reaction (PCR).

  The probes and primers used in the present invention usually consist of at least 15 consecutive nucleotide groups of known sequence. To increase specificity, longer probes and primers, such as probes consisting of at least 20, 25, 30, 40, 50, 60, 70, 80, 90, 100 or at least 150 consecutive nucleotides of the disclosed nucleic acid sequence And primers may also be used. Some probes and primers are much longer than this. It should be understood that nucleotides of any length supported by this specification, including tables, drawings and sequence listings may be used.

For the preparation and use of probes and primers, see Sambrook, J. et al. (1989) Molecular Cloning: A Laboratory Manual , 2nd edition, 1-3, Cold Spring Harbor Press, Plainview NY, Ausubel, FM et al., (1987 ) Current Protocols in Molecular Biology , Greene Publ. Assoc. & Wiley-Intersciences, New York NY, Innis et al. (1990) PCR Protocols, A Guide to Methods and Applications Academic Press, San Diego CA, etc. To obtain a PCR primer pair from a known sequence, for example, a computer program therefor, such as Primer (Version 0.5, 1991, Whitehead Institute for Biomedical Research, Cambridge MA) can be used.

  The selection of oligonucleotides to be used as primers is performed using software well known in the art for such purposes. For example, OLIGO 4.06 software is useful for selecting PCR primer pairs up to 100 nucleotides each, derived from oligonucleotides and up to 5,000 larger polynucleotides up to 32 kilobases of input polynucleotide sequences. It is also useful for analyzing sequences. Similar primer selection programs incorporate additional functionality for extended capabilities. For example, the PrimOU primer selection program (available to the general public from the Genome Center of the University of Texas Southwestern Medical Center in Dallas, Texas) can select specific primers from the megabase sequence, and thus the entire genome range. Useful for designing primers. The Primer3 primer selection program (available from the Whitehead Institute / MIT Center for Genome Research, Cambridge, Mass.) Allows the user to enter a “mispriming library” that allows the user to specify sequences to be avoided as primer binding sites. Primer3 is particularly useful for selection of oligonucleotides for microarrays (the source code for the latter two primer selection programs can be obtained from the respective sources and modified to meet user-specific needs). The PrimerGen program (available from the UK Human Genome Mapping Project in Cambridge, UK-publicly available from the Resource Center) designs primers based on multiple sequence alignments, thereby maximally conserving or minimally conserving aligned nucleic acid sequences Allows selection of primers that hybridize to any of the regions. This program is therefore useful for the identification of unique and conserved oligonucleotide and polynucleotide fragments. Oligonucleotides and polynucleotide fragments identified by any of the above selection methods identify complete or partially complementary polynucleotides in hybridization techniques, eg, as PCR or sequencing primers, as microarray elements, or in nucleic acid samples Useful as a specific probe. The method for selecting the oligonucleotide is not limited to the above method.

  As used herein, “recombinant nucleic acid” is not a natural sequence, but a sequence that is an artificial combination of separated segments of two or more sequences. This artificial combination is often achieved by chemical synthesis, but more commonly by artificial manipulation of isolated segments of nucleic acids, for example by genetic engineering techniques such as those described in Sambrook (supra). To do. The term recombinant nucleic acid also includes nucleic acids in which a portion of the nucleic acid has been modified by simple addition, substitution or deletion. Often recombinant nucleic acids include nucleic acid sequences operably linked to promoter sequences. Such recombinant nucleic acids can be part of a vector that is used, for example, to transform a cell.

  Alternatively, such a recombinant nucleic acid can be part of a viral vector, which can be based on, for example, vaccinia virus. Such vectors can be used to inoculate a mammal and the recombinant nucleic acid is expressed to induce a protective immune response in the mammal.

  “Regulators” are nucleic acid sequences that are usually derived from untranslated regions of a gene, and include enhancers, promoters, introns, and 5 ′ and 3 ′ untranslated regions (UTRs). Regulatory elements interact with host or viral proteins that control transcription, translation or RNA stability.

  A “reporter molecule” is a chemical or biochemical moiety used to label a nucleic acid, amino acid or antibody. Reporter molecules include radionuclides, enzymes, fluorescent agents, chemiluminescent agents, color formers, substrates, cofactors, inhibitors, magnetic particles and other components known in the art.

  In the present specification, the “RNA equivalent” for a DNA sequence is composed of the same linear nucleic acid sequence as the reference DNA sequence, but the thymine of the nitrogenous base is replaced with uracil, and the backbone of the sugar chain is It consists of ribose instead of deoxyribose.

  The term “sample” is used in its broadest sense. Samples that are presumed to contain NAAP, NAAP-encoding nucleic acid groups, or fragments thereof include body fluids, cells and cell-isolated chromosomes, organelles, and membrane extracts. There can be cells, genomic DNA, RNA, cDNA, tissue, tissue prints, etc. present in solution or fixed to a substrate.

  The terms “specific binding” and “specifically bind” refer to the interaction between a protein or peptide and an agonist, antibody, antagonist, small molecule, or any natural or synthetic binding composition. This interaction depends on whether there is a specific structure of the protein (eg, an antigenic determinant or epitope) that the binding molecule recognizes. For example, if the antibody is specific for epitope “A”, the presence of a polypeptide comprising epitope A (ie, free, unlabeled A) in a reaction involving free label A and the antibody is: Reduce the amount of labeled A bound to the antibody.

  The term “substantially purified” refers to a nucleic acid or amino acid sequence that has been removed from the natural environment, isolated or separated, and is at least about 60%, preferably at least about, about other naturally associated components. 75%, most preferred refers to those without at least about 90%.

  “Substitution” is the replacement of one or more amino acids or nucleotides with another amino acid or nucleotide, respectively.

  The term “substrate” refers to any suitable solid or semi-solid support, including membranes and filters, chips, slides, wafers, fibers, magnetic or non-magnetic beads, gels, tubes, plates, polymers, microparticles, capillaries. Is included. The substrate can have various surface forms such as wells, grooves, pins, channels, holes, and the like, and polynucleotides and polypeptides are bound to the substrate surface.

  A “transcript image” or “expression profile” refers to the pattern of collective gene expression by a particular cell type or tissue at a given time under given conditions.

  “Transformation” is the process by which foreign DNA is introduced into a recipient cell. Transformation can occur under natural or artificial conditions according to various methods known in the art, and is any known method that inserts an exogenous nucleic acid sequence into a prokaryotic or eukaryotic host cell. Based on this method. The method of transformation is selected according to the type of host cell to be transformed. Non-limiting transformation methods include bacteriophage or viral infection, electroporation, heat shock, lipofection, and particle bombardment. A “transformed cell” includes a stably transformed cell that can replicate as a plasmid in which the introduced DNA replicates autonomously or as part of a host chromosome. Furthermore, cells that transiently express introduced DNA or RNA in a limited time are also included.

As used herein, a “transgenic organism” is any organism, including but not limited to animals and plants, in which one or more cells of the organism are, for example, used in the art by human involvement. Have heterologous nucleic acid introduced by well-known transgenic technology. Nucleic acids are introduced into cells either directly or indirectly by introduction into cell precursors, by planned genetic manipulation, for example, by microinjection or by infection with recombinant viruses. In some embodiments, 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 engineering does not refer to classical crossbreeding or in vitro fertilization but to the introduction of recombinant DNA molecules. Genetic transformants contemplated according to the present invention include bacteria, cyanobacteria, fungi and animals and plants. The isolated DNA of the present invention can be introduced into a host by methods known in the art, such as infection, transfection, transformation or transconjugation. Techniques for transferring the DNA of the present invention into such organisms are well known and are described in references such as Sambrook et al. (1989), supra.

  A “variant / mutant sequence” of a particular nucleic acid sequence is defined as a nucleic acid sequence having at least 40% sequence identity with the particular nucleic acid sequence over the length of the entire nucleic acid sequence. For definition, blastn is executed using the “BLAST 2 Sequences” tool Version 2.0.9 (May 7, 1999) set to the default parameters. Such nucleic acid pairs are, for example, at least 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95% for a given length, It may show 96%, 97%, 98%, 99% or more homology. Certain variants may be described, for example, as “allelic” variants (described above), “splice” variants, “species” variants or “polymorphic” variants. A splice variant may have considerable homology with a reference molecule, but will usually have more or fewer nucleotides due to alternative splicing of exons during mRNA processing. Corresponding polypeptides may have additional functional domain groups or may lack domain groups present in the reference molecule. Species variants are polynucleotide sequences that vary from species to species. The resulting polypeptides usually have significant amino acid homology with each other. Polymorphic variants differ in the polynucleotide sequence of a particular gene between individuals of a given species. Polymorphic variant sequences can also include “single nucleotide polymorphisms” (SNPs) that differ in one nucleotide of the polynucleotide sequence. The presence of SNPs can indicate, for example, a particular population, condition or disease propensity.

A “variant” of a particular polypeptide sequence is defined as a polypeptide sequence that has at least 40% sequence identity to the particular polypeptide sequence throughout the length of one of the polypeptide sequences. For definition, blastp is executed by using “BLAST 2 Sequences” tool Version 2.0.9 (May 7, 1999) set as default parameters. Such polypeptide pairs are, for example, at least 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94% for one predetermined length of the polypeptide. 95%, 96%, 97%, 98%, 99% or more sequence identity.
(invention)
The present invention is based on the discovery of a novel group of human nucleic acid binding proteins (NAAP) and polynucleotides encoding NAAP. It is also based on the development of diagnosis, treatment, and prevention of cell proliferation abnormalities, neurological diseases, developmental disorders, autoimmune / inflammatory disorders, and infectious diseases utilizing these compositions.

  Table 1 summarizes the nomenclature for the full-length polynucleotide and polypeptide sequences of the present invention. Each polynucleotide and its corresponding polypeptide correlates to one Incyte project identification number (Incyte project ID). Each polypeptide sequence was represented by a polypeptide sequence identification number (polypeptide SEQ ID NO :) and an Incyte polypeptide sequence number (Incyte polypeptide ID). Each polynucleotide sequence was represented by a polynucleotide sequence identification number (polynucleotide SEQ ID NO :) and an Incyte polynucleotide consensus sequence number (Incyte polynucleotide ID).

  Table 2 shows the sequences with homology to the polypeptides of the present invention as identified by BLAST analysis against the GenBank protein (genpept) 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 each polypeptide of the present invention. Column 3 shows the GenBank identification number (Genbank ID NO :) of the closest homolog of GenBank. Column 4 shows the probability score for a match between each polypeptide and one or more of its homologues. Column 5 indicates appropriate citations where applicable and one or more GenBank homologue annotations, all of which are hereby incorporated by reference.

  Table 3 shows various structural features of the polypeptides of the invention. Columns 1 and 2 each 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 of each polypeptide. Columns 4 and 5 show potential phosphorylation and glycosylation sites, respectively, as determined by the GCG sequence analysis software package MOTIFS program (Genetics Computer Group, Madison WI). Column 6 shows the amino acid residues that include the signature sequence, domain, and motif. Column 7 shows the analysis method for the analysis of the structure / function of the protein, and the applicable part further shows a searchable database used for the analysis method.

  Tables 2 and 3 together summarize the properties of each polypeptide of the present invention, which establishes that the claimed polypeptide is a nucleic acid binding protein. For example, SEQ ID NO: 2 has been shown by the Basic Local Alignment Search Tool (BLAST) to have 42% identity from residue Q784 to F1175 with Arabidopsis putative ATP-dependent RNA helicase A (GenBank ID g4510377) ( (See Table 2). The BLAST probability score is 1.9e-168, indicating the probability that an observed polypeptide sequence alignment will be obtained by chance. SEQ ID NO: 2 also has a DEAD / DEAH box helicase domain, which searches for statistically significant matches in the conserved protein family domain PFAM database based on the Hidden Markov Model (HMM) (See Table 3). Data from BLIMPS, MOTIFS, and PROFILESCAN analyzes provide further empirical evidence that SEQ ID NO: 2 is a DEAD / DEAH box helicase.

  In another example, SEQ ID NO: 4 represents 37% from L128 to P513 residues, 58% from F624 to L707 residues and 58% from K9 residues to Y49 from mouse ERG binding protein ESET (GenBank ID g3644042). It was confirmed by the Basic Local Alignment Search Tool (BLAST) that it has 26% identity to the residue (see Table 2). The BLAST probability score is 1.5e-88, indicating the probability that an observed polypeptide sequence alignment will be obtained by chance. SEQ ID NO: 4 also has a SET domain, which is determined by searching for statistically significant matches in the conserved protein family domain PFAM database based on the Hidden Markov Model (HMM). (See Table 3). Data from BLIMPS and BLAST analysis of the PRODOM and DOMO databases provide further confirmatory evidence that SEQ ID NO: 4 contains a SET domain and is a transcription factor.

  As another example, SEQ ID NO: 8 has been shown by the Basic Local Alignment Search Tool (BLAST) to be 100% identical to human putative helicase RUVBL (GenBank ID g8886769) from residue M22 to residue C665. (See Table 2). The BLAST probability score is 8e-239, indicating the probability that an observed polypeptide sequence alignment will be obtained by chance. SEQ ID NO: 8 also has an ATP-degrading enzyme domain, which searches for a statistically significant match in the PFAM database of conserved protein family domains based on the Hidden Markov Model (HMM). Determined (see Table 3). Data from BLIMPS and MOTIFS analyzes provide evidence to further confirm that SEQ ID NO: 8 is a helicase.

  In another example, SEQ ID NO: 14 has 98% identity to the human paired box (PAX) protein (GenBank ID g409139) from F13 to K312 residues Basic Local Alignment Search Tool (BLAST) (See Table 2). The BLAST probability score is 1.4e-156, indicating the probability that an observed polypeptide sequence alignment will be obtained by chance. SEQ ID NO: 14 also has a paired box domain, which searches for statistically significant matches in the conserved protein family domain PFAM database based on the Hidden Markov Model (HMM). Determined (see Table 3). Data from BLIMPS, MOTIFS, and PROFILESCAN analyzes provide further empirical evidence that SEQ ID NO: 14 is a PAX protein. SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5-7, SEQ ID NO: 9-13 and SEQ ID NO: 15-23 were similarly analyzed and annotated. The algorithm and parameters for analysis of SEQ ID NO: 1-23 are listed in Table 7.

  As shown in Table 4, the full-length polynucleotide sequences of the present invention were assembled using cDNA sequences or coding (exon) sequences derived from genomic DNA, or any combination of these two sequences. Column 1 shows the polynucleotide sequence identification number (polynucleotide SEQ ID NO) and the corresponding Incyte polynucleotide consensus sequence number (Incyte ID) for each polynucleotide of the invention, and the length of each polynucleotide sequence in base pairs. Yes. Column 2 shows the nucleotide start position (5 ′) and stop position (3 ′) of the cDNA sequence and / or genomic sequence used in the assembly of the full-length polynucleotide sequence of the present invention, and SEQ ID NO: 24-46. Nucleotide start position (5 ′) of a fragment of a polynucleotide sequence that is useful for identifying or distinguishing SEQ ID NO: 24-46 from a related polynucleotide sequence (e.g., hybridization or amplification techniques) And the end position (3 ').

The polynucleotide fragment described in column 2 of Table 4 may particularly refer to, for example, Incyte cDNA derived from a tissue-specific cDNA library or a pooled cDNA library. Alternatively, the polynucleotide fragment listed in column 2 may refer to a GenBank cDNA or EST that contributed to the assembly of the full length polynucleotide sequence. In addition, the polynucleotide fragment in row 2 can identify sequences derived from the ENSEMBL (The Sanger Centre, Cambridge, UK) database (ie, sequences that include the “ENST” designation). Alternatively, the polynucleotide fragment described in column 2 may be derived from the NCBI RefSeq Nucleotide Sequence Records database (ie, sequences containing the designation “NM” or “NT”) and from the NCBI RefSeq Protein Sequence Records. In some cases (ie, a sequence containing the designation “NP”). Alternatively, the polynucleotide fragments in column 2 may refer to both cDNA and Genscan predicted exon assemblies combined by an “exon-stitching” algorithm. For example, a polynucleotide identified as FL_XXXXXX_N ₁ _N ₂ _YYYYY_N ₃ _N ₄ has a cluster identification number of the sequence to which the algorithm is applied is XXXXXX, and a prediction number generated by the algorithm is YYYYY (if present N) _{1,2,3 ..} is a “stitched” sequence that is a particular exon that may have been manually edited during the analysis (see Example 5 ). Alternatively, the polynucleotide fragments in row 2 may refer to an assembly of exons joined together by an “exon-stretching” algorithm. For example, the polynucleotide sequence identified as FLXXXXXX_gAAAAA_gBBBBB_1_N is a “stretched” sequence. XXXXXX is the Incyte project identification number, gAAAAA is the GenBank identification number of the human genome sequence to which the `` exon stretching '' algorithm is applied, gBBBBB is the GenBank identification number or NCBI RefSeq identification number of the closest GenBank protein homologue, and N is a specific number Refers to an exon (see Example 5 ). When a RefSeq sequence is used as a protein homolog for the “exon stretching” algorithm, the RefSeq identification number (represented by “NM”, “NP”, or “NT”) is the GenBank identifier (ie, , GBBBBB) may be used instead.

Alternatively, prefix codes identify constituent sequences that have been manually edited, predicted from genomic DNA sequences, or derived from a combination of sequence analysis methods. The following table lists the constituent sequence prefix codes and examples of how to analyze the same sequence corresponding to the prefix code (see Examples 4 and 5 ).

  In some cases, in order to confirm the final consensus polynucleotide sequence, an Incyte cDNA coverage that overlapped with the sequence coverage shown in Table 4 was obtained, but no corresponding Incyte cDNA identification number was given.

  Table 5 shows a representative cDNA library for full-length polynucleotide sequences assembled using Incyte cDNA sequences. A representative cDNA library is an Incyte cDNA library, which is most often represented by a group of Incyte cDNA sequences, which were used to assemble and confirm the above polynucleotide sequences. The tissues and vectors used to create the cDNA library are shown in Table 5 and described in Table 6.

  The present invention also includes NAAP variants. Preferred NAAP variants have at least one functional or structural characteristic of NAAP and are at least about 80% amino acid sequence identity, or at least about 90% amino acid sequence identity to the NAAP amino acid sequence. Or a sequence having at least about 95% amino acid sequence identity.

  The present invention also provides a polynucleotide encoding NAAP. In certain embodiments, the present invention provides a polynucleotide sequence having a sequence selected from the group consisting of SEQ ID NOs: 24-46, which encodes NAAP. The polynucleotide sequence of SEQ ID NO: 24-46 also includes an equivalent RNA sequence as shown in the sequence listing, where the occurrence of the nitrogen base thymine is replaced by uracil and the sugar backbone is not deoxyribose. Consists of ribose.

  The invention also includes variants of polynucleotide sequences encoding NAAP. In particular, such a variant polynucleotide sequence has at least about 70% polynucleotide sequence identity, or at least about 85% polynucleotide sequence identity with a polynucleotide sequence encoding NAAP, and at least There will be as much as about 95% polynucleotide sequence identity. In certain embodiments of the invention, the polynucleotide sequence identity is at least about 70%, or at least about 85%, or at least about 95% with a nucleic acid sequence selected from the group consisting of SEQ ID NO: 24-46 Such as variants of polynucleotide sequences having a sequence selected from the group consisting of SEQ ID NOs: 24-46. Any of the polynucleotide variants described above can encode an amino acid sequence having at least one functional or structural characteristic of NAAP.

  Additionally or alternatively, certain polynucleotide variants of the invention are splice variants of a polynucleotide sequence encoding NAAP. Some splice variants may have multiple portions with significant sequence identity to the polynucleotide sequence encoding NAAP, but the addition of several blocks of sequences or the addition of several blocks of sequences resulting from alternative splicing of exons during mRNA processing. Deletions will usually have more or fewer polynucleotides. In some splice variants, less than about 70%, or less than about 60%, or less than about 50% of polynucleotide sequence identity is found over the entire length with the polynucleotide sequence encoding NAAP. , Some portions of the splice variant include at least about 70%, or at least about 85%, or at least about 95%, or even 100% of the polynucleotide with each portion of the polynucleotide sequence encoding NAAP. It will have sequence identity. Any of the splice variant sequences described above can encode an amino acid sequence having at least one functional or structural feature of NAAP.

  The degeneracy of the genetic code creates a variety of polynucleotide sequences that encode NAAP, some of which have minimal similarity to any known native gene polynucleotide sequences. Will be understood. Thus, the present invention may cover all possible polynucleotide sequence variations that can be produced by selection of combinations based on possible codon selection. These combinations are based on the standard triplet genetic code applied to the native NAAP polynucleotide sequences and should be considered that all mutations are clearly disclosed.

  Nucleotide sequences encoding NAAP and its variants are generally capable of hybridizing to the nucleotide sequence of native NAAP under suitably selected stringency conditions, but include substantially different codons, such as including non-natural codon groups. It may be beneficial to create a nucleotide sequence that encodes NAAP or derivatives thereof having use. Codons can be selected to increase the expression rate of peptides generated in a particular eukaryotic or prokaryotic host based on the frequency with which the host utilizes the particular codon. Another reason for substantially altering the nucleotide sequence encoding NAAP and its derivatives without altering the encoded amino acid sequence is to provide RNA with favorable properties such as longer half-life than transcripts made from the native sequence Sometimes a transcript is made.

  The present invention also includes a process in which DNA sequences or fragments thereof encoding NAAP and its derivatives are completely generated by synthetic chemistry. After production, this synthetic sequence can be inserted into any of a number of available expression vectors and cell lines using reagents known in the art. In addition, synthetic chemistry can be used to mutagenize sequences encoding NAAP or any fragment thereof.

  The invention further includes polynucleotide sequences that are capable of hybridizing under the various stringency conditions to the claimed polynucleotide sequences, particularly SEQ ID NO: 24-46 and fragments thereof (e.g. Wahl, GM and SL Berger (1987) Methods Enzymol. 152: 399-407, Kimmel, AR (1987) Methods Enzymol. 152: 507-511, etc.). Hybridization conditions, including annealing and washing conditions, are described in “Definitions”.

Methods for DNA sequencing are known in the art, and any embodiment of the present invention can be performed using DNA sequencing methods. Enzymes can be used for DNA sequencing methods, such as Klenow fragment of DNA polymerase I, SEQUENASE (US Biochemical, Cleveland OH), Taq polymerase (Applied Biosystems), thermostable T7 polymerase (Amersham, Pharmacia Biotech, Piscataway NJ). ) Can be used. Alternatively, a polymerase and proofreading exonuclease can be used in combination, as seen, for example, in the ELONGASE amplification system (Life Technologies, Gaithersburg MD). Preferably, sequence preparation is automated using equipment such as the MICROLAB 2200 liquid transfer system (Hamilton, Reno, NV), the PTC200 thermal cycler (MJ Research, Watertown MA) and the ABI CATALYST 800 thermal cycler (Applied Biosystems). The sequencing is then performed using ABI 373 or 377 DNA sequencing system (Applied Biosystems), MEGABACE 1000 DNA sequencing system (Molecular Dynamics, Sunnyvale CA) or other methods well known in the art. The resulting sequence is analyzed using various algorithms well known in the art (Ausubel, FM (1997) Short Protocols in Molecular Biology , John Wiley & Sons, New York NY, unit 7.7, Meyers, RA (1995) Molecular Biology and Biotechnology , Wiley VCH, New York NY, pages 856-853).

  Using various PCR-based methods well-known in the art and partial nucleotide sequences, NAAP-encoding nucleic acid sequences can be extended to detect upstream sequences such as promoters and regulatory elements. obtain. For example, restriction site PCR, one of the methods that can be used, is a method of amplifying an unknown sequence from genomic DNA in a cloning vector using a universal primer and a nested primer (for example, Sarkar, G. ( 1993) PCR Methods Applic. 2: 318--322). Another method is reverse PCR, which amplifies an unknown sequence from a circularized template using primers extended in a wide range of directions. The template is obtained from a group of restriction enzymes consisting of a certain known genomic locus and its surrounding sequences (see, eg, Triglia, T. et al. (1988) Nucleic Acids Res. 16: 8186). A third method is a capture PCR method, which is involved in a method for PCR amplification of DNA fragments adjacent to known sequences of human and yeast artificial chromosome DNA. (See Lagerstrom, M. et al. (1991) PCR Methods Applic 1: 111-119). In this method, it is possible to insert a recombinant double-stranded sequence into an unknown sequence region using a plurality of restriction enzyme digestion and ligation reactions before PCR. Several other methods that can be used to search for unknown sequences are also known in the art (see, eg, Parker, J.D. et al. (1991) Nucleic Acids Res. 19: 30553060). In addition, genomic DNA can be walked using PCR, nested primers, and PROMOTERFINDER libraries (Clontech, Palo Alto CA). This procedure is useful for discovery of intron / exon junctions without the need to screen libraries. All PCR-based methods use commercially available software such as OLIGO 4.06 primer analysis software (National Biosciences, Plymouth MN) or another suitable program, about 22-30 nucleotides in length and about 50% GC content. Thus, the primer group can be designed to anneal to the template at a temperature of about 68 ° C. to 72 ° C.

  When screening for full-length cDNA groups, it is preferable to use library groups that are size-selected to include larger cDNA groups. In addition, random primer libraries often contain sequences having the 5 ′ region of the gene and are suitable for situations in which an oligo d (T) library cannot produce a full-length cDNA. Genomic libraries may be useful for extending sequences into the 5 ′ non-transcribed regulatory region.

  Commercially available capillary electrophoresis systems can be used to analyze the size of the sequencing or PCR product or to confirm its nucleotide sequence. Specifically, capillary sequencing is emitted with a flowable polymer for electrophoretic separation and a laser activated fluorophore (fluorescent dye) that is specific for four different nucleotides. And a CCD camera used for detection of the different wavelengths. The output / light intensity can be converted to an electrical signal using suitable software (Applied Biosystems GENOTYPER, SEQUENCE NAVIGATOR, etc.). The entire process from sample loading to computer analysis and electronic data display is computer controllable. Capillary electrophoresis is particularly suitable for sequencing small DNA fragments that are present in small amounts in a particular sample.

  In another embodiment of the invention, a polynucleotide sequence encoding NAAP or a fragment thereof may be cloned into a recombinant DNA molecule to express NAAP, a fragment thereof or a functional equivalent in an appropriate host cell. Is possible. Due to the degeneracy inherent in the genetic code, another group of DNA sequences encoding substantially the same or functionally equivalent amino acid sequences can be produced and these sequences can be used for NAAP expression.

  For various purposes, the nucleotide sequences of the present invention can be recombined using a number of methods generally known in the art to modify the sequences encoding NAAP. The various purposes of this recombination include, but are not limited to, cloning of the gene product or modification of processing and / or expression. It is possible to recombine nucleotide sequences using random fragmentation of gene fragments and synthetic oligonucleotides and DNA shuffling by PCR reassembly. For example, site-directed mutagenesis via oligonucleotides can be used to introduce mutations that create new restriction sites, change glycosylation patterns, change codon preference, generate splice variants, and the like.

  Nucleotides of the present invention may be synthesized using MOLECULARBREEDING (Maxygen Inc., Santa Clara CA; U.S. Pat.No. 5,837,458; Chang, C.-C. et al. (1999) Nat. Biotechnol. 17: 793-797; Christians, FC et al. Nat. Biotechnol. 17: 259-264; Crameri, A. et al. (1996) Nat. Biotechnol. 14: 315-319). Alternatively, NAAP biological properties, such as the ability to bind to other molecules and compounds, can be altered or improved. DNA shuffling is a process of creating a library of gene variants using PCR-mediated recombination of gene fragments. The library is then subjected to a selection or screening procedure that identifies a group of genetic variants with the desired characteristics. These suitable variants can then be pooled and further repeated for DNA shuffling and selection / screening. Thus, genetic diversity is created by “artificial” breeding and rapid molecular evolution. For example, single gene fragments with random point mutations can be recombined, screened, and then shuffled until the desired properties are optimized. Alternatively, recombine a given gene with homologous genes from the same gene family, either from the same species or from different species, thereby maximizing the genetic diversity of multiple naturally occurring genes in a directed and controllable manner It can be made.

According to another embodiment, the NAAP-encoding sequence can be synthesized in whole or in part using chemical methods well known in the art (eg, Caruthers. MH et al. (1980) Nucl. Acids Res Symp. Ser 7: 215-223; and Horn, T. et al. (1980) Nucl. Acids Res. Symp. Ser. 225-232). Alternatively, NAAP itself or a fragment thereof can be synthesized using chemical methods. For example, peptide synthesis can be performed using a variety of liquid or solid phase techniques (eg, Creighton, T. (1984) Proteins, Structures and Molecular Properties , WH Freeman, New York NY, 55-60, Roberge, JY et al. ( 1995) Science 269: 202204). Automated synthesis can be achieved using an ABI 431A peptide synthesizer (Applied Biosystems). Further, certain natural polypeptides may be modified by directly altering the amino acid sequence of NAAP or any part thereof during direct synthesis and / or in combination with sequences from other proteins or any part thereof. Polypeptides or mutant polypeptides having the sequence can be made.

  This peptide can be substantially purified using preparative high performance liquid chromatography (see, for example, Chiez, R.M. and F.Z. Regnier (1990) Methods Enzymol. 182: 392-421). The composition of this synthetic peptide can be confirmed by amino acid analysis or sequencing (see Creighton, pages 28-53, etc.).

  In order to express biologically active NAAP, a nucleotide sequence encoding NAAP or a derivative thereof may be inserted into a suitable expression vector. A suitable expression vector is a vector having elements necessary for controlling transcription and translation of a coding sequence inserted into a suitable host. Necessary elements include regulatory sequences in the vector and polynucleotide sequences encoding NAAP (enhancers, constitutive and expression-inducible promoters, 5 ′ and 3 ′ untranslated regions, etc.). Necessary elements vary in strength and specificity. Specific initiation signals can be used to achieve more effective translation of NAAP-encoding sequences. Examples of initiation signals include ATG initiation codons and nearby sequences such as Kozak sequences. If the NAAP-encoding sequence, its initiation codon, and upstream regulatory sequences are inserted into a suitable expression vector, no additional transcriptional or translational control signals may be required. However, when only the coding sequence or a fragment thereof is inserted, an exogenous translational control signal such as an in-frame ATG start codon should be included in the expression vector. Exogenous translation elements and initiation codons can originate from a variety of natural and synthetic products. Inclusion of an enhancer suitable for the particular host cell system used can enhance expression efficiency (see, eg, Scharf, D. et al. (1994) Results Probl. Cell Differ. 20: 125-162) .

Methods which are well known to those skilled in the art can be used to create expression vectors with sequences encoding NAAP and suitable transcriptional and translational control elements. These methods include in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination techniques (eg, Sambrook, J. et al. (1989) Molecular Cloning. A Laboratory Manual , Cold Spring Harbor Press, Plainview NY, Chapters 4, 8 and 16-17, Ausubel, FM et al. (1995) Current Protocols in Molecular Biology , John Wiley & Sons, New York NY, Chapters 9, 13 and 16).

  A variety of expression vector / host systems can be used to retain and express sequences encoding NAAP. Such expression vectors / host systems include, but are not limited to, microorganisms such as bacteria transformed with recombinant bacteriophage, plasmid or cosmid DNA expression vectors, and yeast transformed with yeast expression vectors. Or transformed with an insect cell line infected with a viral expression vector (eg baculovirus), a viral expression vector (eg cauliflower mosaic virus: CaMV or tobacco mosaic virus: TMV) or a bacterial expression vector (eg Ti plasmid or pBR322 plasmid) Plant cell lines and animal cell lines. (Sambrook, supra, Ausubel, Van Heeke, G. and SM Schuster (1989) J. Biol. Chem. 264: 5503-5509, Engelhard, EK et al. (1994) Proc. Natl. Acad. Sci. USA 91: 3224-3227, Sandig, V. et al. (1996) Hum. Gene Ther. 7: 1937-1945, Takamatsu, N. (1987) EMBOJ. 6: 307-311, The McGraw Hill Science and Technology Yearbook Yearbook of Science and Technology) (1992) McGraw Hill New York NY, 191-196, Logan, J. et al. T. Shenk (1984) Proc. Natl. Acad. Sci. USA 81: 3655-3659, Harrington, JJ et al. (1997) Nat. Genet. 15: 345-355 etc.) Target nucleotide sequences using expression vectors derived from retroviruses, adenoviruses, herpesviruses or vaccinia viruses, or from various bacterial plasmids Can be delivered to organs, tissues or cell populations (Di Nicola, M. et al. (1998) Cancer Gen. Ther. 5 (6): 350-356, Yu, M. et al. (1993) Proc. Natl. Acad. Sci. USA 90 (13): 6340-63 44, Buller, RM et al. (1985) Nature 317 (6040): 813-815, McGregor, DP et al. (1994) Mol.Immunol. 31 (3): 219-226, Verma, IM and N. Somia (1997) Nature 389: 239-242 etc.). The present invention is not limited by the host cell used.

In bacterial systems, a number of cloning and expression vectors may be selected depending upon the use intended for polynucleotide sequences encoding NAAP. For example, a multifunctional E. coli vector such as PBLUESCRIPT (Stratagene, La Jolla CA) or PSPORT1 plasmid (Life Technologies) can be used for routine cloning, subcloning and propagation of a polynucleotide sequence encoding NAAP. Ligation of the NAAP-encoding sequence into the multicloning site of the vector disrupts the lacZ gene, allowing a colorimetric screening method for the identification of transformed bacteria containing recombinant molecules. In addition, these vectors may be useful for in vitro transcription in cloned sequences, dideoxy sequencing, single-stranded rescue with helper phage, and generation of nested deletions (e.g., Van Heeke, G. And SM Schuster (1989) J. Biol. Chem. 264: 5503-5509). For example, when a large amount of NAAP is required for antibody production or the like, a vector that induces NAAP expression at a high level can be used. For example, vectors containing a strong inducible SP6 bacteriophage promoter or an inducible T7 bacteriophage promoter can be used.

A yeast expression system can also be used to produce NAAP. A large number of vectors containing constitutive or inducible promoters such as α-factor, alcohol oxidase, and PGH promoter can be used for Saccharomyces cerevisiae or Pichia pastoris . In addition, such vectors indicate whether the expressed protein is secreted or retained in the cell, allowing the incorporation of foreign sequences into the host genome for stable growth. (See, eg, Ausubel, 1995, supra, Bitter, GA et al. (1987) Methods Enzymol. 153: 516-544 and Scorer. CA et al. (1994) Bio / Technology 12: 181-184).

Plant systems can also be used for NAAP expression. Transcription of NAAP-encoding sequences can be achieved by viral promoters such as CaMV-derived 35S and 19S as used alone or in combination with omega leader sequences from TMV (Takamatsu, N. (1987) EMBO J 6: 307-311). Promoted by a promoter. Alternatively, plant promoters such as the small subunit of RuBisCO, or heat shock promoters can be used (eg, Coruzzi, G. et al. (1984) EMBO J. 3: 1671-1680; Broglie, R. et al. (1984) Science 224: 838-843; and Winter, J. et al. (1991) Results Probl. Cell Differ. 17: 85-105). These constructs can be introduced into plant cells by direct DNA transformation or by pathogen-mediated transfection. (See The McGraw Hill Yearbook of Science and Technology (1992) McGraw Hill New York NY, pages 191-196).

  In mammalian cells, a number of virus-based expression systems can be utilized. When an adenovirus is used as an expression vector, a sequence group encoding NAAP can be ligated to an adenovirus transcription / translation complex consisting of a late promoter and a triple leader sequence. By inserting into the nonessential E1 or E3 region of the adenovirus genome, infectious viruses that express NAAP in host cells can be obtained (eg, Logan, J. and T. Shenk (1984) Proc. Natl. Acad Sci. USA 81: 3655-3659). In addition, transcription enhancers such as the Rous sarcoma virus (RSV) enhancer can be used to increase expression in mammalian host cells. Proteins can also be expressed at high levels using vectors based on SV40 or EBV.

  Human artificial chromosomes (HACs) can also be used to deliver fragments of DNA that are larger than those contained in and expressed from a plasmid. Approximately 6 kb to 10 Mb of HAC is made for treatment and is delivered by conventional delivery methods (liposomes, polycation amino polymers, or vesicles) (eg, Harrington, JJ et al. (1997) Nat. Genet. 15: 345). -355).

  For long-term production of recombinant proteins in mammalian systems, stable expression of NAAP in cell lines is desirable. For example, expression vectors and selectable marker genes on the same vector or on different vectors can be used to transform NAAP-encoding sequences into cell lines. The expression vector used may have a replication element of viral origin and / or an endogenous expression element. After the introduction of the vector, the cells can be grown in enhanced medium for about 1-2 days before being transferred to the selective medium. The purpose of the selectable marker is to confer resistance to the selective agent, and the presence of the selectable marker allows growth and recovery of cells that successfully express the introduced sequence. Resistant clones of stably transformed cells can be propagated using tissue culture techniques appropriate to the cell type.

Any number of selection systems can be used to recover transformed cell lines. Such a selection system includes, but is not limited to, the herpes simplex virus thymidine kinase gene used for tk ^- cells and the herpes simplex virus adenine phosphoribosyltransferase gene used for apr ^- cells. (See Wigler, M. et al. (1977) Cell 11: 223-232, Lowy, I. et al. (1980) Cell 22: 817-823). Moreover, tolerance to antimetabolites, antibiotics or herbicides can be used as a basis for selection. For example, dhfr confers resistance to methotrexate, neo confers resistance to aminoglycoside neomycin and G-418, als confers resistance to chlorsulfuron, and pat confers resistance to phosphinothricin acetyltransferase (Wigler, M. (1980) Proc. Natl. Acad. Sci. USA 77: 3567-3570, Colbere-Garapin, F. et al. (1981) J. Mol. Biol. 150: 1-14 etc.). Other selectable genes, such as trpB and hisD that alter cellular requirements for metabolism, are described in the literature (Hartman, SC and RC Mulligan (1988) Proc. Natl. Acad. Sci. USA. 85: 8047-8051 etc.). Visible markers such as anthocyanins, green fluorescent protein (GFP; Clontech), β-glucuronidase and its substrate β-glucuronide, or luciferase and its substrate luciferin may be used. These markers can be used not only to identify transformants, but also to quantify transient or stable protein expression due to specific vector systems (Rhodes, CA (1995) Methods Mol. Biol. 55: 121 (See -131 etc.)

  Even if the presence or absence of marker gene expression suggests the presence of the target gene, the presence and expression of the gene may need to be confirmed. For example, if a sequence encoding NAAP is inserted into a marker gene sequence, transformed cells having a sequence encoding NAAP can be identified by the lack of marker gene function. Alternatively, a marker gene can be placed in tandem with one sequence encoding NAAP under the control of a single promoter. Expression of a marker gene in response to induction or selection usually also indicates tandem gene expression.

  In general, host cells containing a nucleic acid sequence encoding NAAP and expressing NAAP can be identified using a variety of methods well known to those skilled in the art. Non-limiting methods well known to those skilled in the art include DNA-DNA or DNA-RNA hybridization, PCR amplification, and detection, quantification, or both of nucleic acid or protein sequences. There are also protein bioassays or immunoassays, including membrane-based, solution-based or chip-based techniques to do

Immunological methods for detecting and measuring NAAP expression using specific polyclonal or specific monoclonal antibodies are known in the art. Examples of such techniques include enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), fluorescence activated cell sorting (FACS) and the like. A two-site, monoclonal-based immunoassay using a group of monoclonal antibodies that react with two non-interfering epitopes on NAAP is preferred, but a competitive binding test can also be used. These and other assays are known in the art (Hampton. R. et al. (1990) Serological Methods, a Laboratory Manual. APS Press. St Paul. MN, Sect. IV, Coligan, JE et al. (1997). ) Current Protocols in Immunology , Greene Pub. Associates and Wiley-Interscience, New York NY, Pound, JD (1998) Immunochemical Protocols , Humans Press, Totowa NJ, etc.).

A wide variety of labeling and conjugation methods are known to those skilled in the art and can be used in various nucleic acid and amino acid assays. Methods for generating labeled hybridization or PCR probes for detecting sequences related to NAAP-encoding polynucleotides include oligo-labeling, nick translation, end-labeling, or labeling PCR amplification using the synthesized nucleotides is included. Alternatively, the NAAP-encoding sequence, or any fragment thereof, can be cloned into a vector for generating mRNA probes. Such vectors are known in the art and are also commercially available and can be used to synthesize RNA probes in vitro with the addition of a suitable RNA polymerase such as T7, T3 or SP6 and labeled nucleotides. Can do. Such methods can be performed using various kits commercially available from, for example, Amersham Pharmacia Biotech, Promega (Madison WI), US Biochemical, and the like. Suitable reporter molecules or labels that can be used to facilitate detection include substrates, cofactors, inhibitors, magnetic particles, as well as radionuclides, enzymes, fluorescent agents, chemiluminescent agents, color formers, and the like.

  Host cells transformed with a nucleotide sequence encoding NAAP are cultured under conditions suitable for expression and recovery of this protein in cell culture media. Whether a protein produced from a transformed cell is secreted or remains intracellular depends on the sequence used, the vector, or both. One skilled in the art will appreciate that expression vectors having NAAP-encoding polynucleotides can be designed to have signal sequences that direct NAAP secretion across prokaryotic or eukaryotic membranes.

  Furthermore, selection of the host cell line may be made by its ability to modulate the expression of the inserted sequences or to process the expressed protein in the desired form. Such polypeptide modifications include, but are not limited to, acetylation, carboxylation, glycosylation, phosphorylation, lipidation and acylation. Post-translational processing that cleaves the “prepro” or “pro” form of the protein can be used to identify induction, folding and / or activity of the protein to the target. Various host cells (eg CHO, HeLa, MDCK, HEK293, WI38, etc.) with specific cellular equipment and characteristic mechanisms for post-translational activity are available from the American Type Culture Collection (ATCC, Manassas, VA). And can be selected to ensure correct modification and processing of the foreign protein.

  In another embodiment of the present invention, in any host system described above, by ligating a natural, modified or recombinant nucleic acid sequence encoding NAAP to a heterologous sequence, Translation of certain fusion proteins can occur. For example, a chimeric NAAP protein that contains a heterologous moiety that can be recognized by a commercially available antibody can facilitate the screening of peptide libraries for inhibitors of NAAP activity. Also, heterologous protein portions and heterologous peptide portions can facilitate the 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, There is hemagglutinin (HA). GST on immobilized glutathione, MBP on maltose, Trx on phenylarsine oxide, CBP on calmodulin, and 6-His on metal chelate resins allow for purification of cognate fusion proteins. FLAG, c-myc and hemagglutinin (HA) allow immunoaffinity purification of fusion proteins using commercially available monoclonal and polyclonal antibodies that specifically recognize these epitope tags. Also, if the fusion protein is genetically engineered to have a proteolytic cleavage site between the sequence encoding NAAP and the heterologous protein sequence, NAAP can be cleaved from the heterologous portion after purification. Fusion protein expression and purification methods are described in Ausubel (1995), chapter 10, supra. Various commercially available kits can also be used to facilitate the expression and purification of the fusion protein.

In a further embodiment of the present invention, radiolabeled NAAP can be synthesized in vitro using a TNT rabbit reticulocyte lysate or wheat germ extract system (Promega). These systems couple the transcription and translation of protein coding sequences operably linked to a T7, T3 or SP6 promoter. Translation takes place in the presence of a radiolabeled amino acid precursor such as ³⁵ S methionine.

  The NAAP or fragment thereof of the present invention can be used to screen for compounds that specifically bind to NAAP. At least one or more test compounds can be used to screen for specific binding to NAAP. Test compounds include antibodies, oligonucleotides, proteins (eg receptors) or small molecules.

In certain embodiments, the compound thus identified is closely related to a natural ligand of NAAP, such as a ligand or fragment thereof, or a natural substrate, structural or functional mimetic, Alternatively, it is a natural binding partner (see Chapter 5 of Coligan, JE et al. (1991) Current Protocols in Immunology 1 (2)). Similarly, the compound may be closely related to the natural receptor to which NAAP binds, or at least some fragment of the receptor, such as the ligand binding site. In either case, the compound can be rationally designed using known techniques. In some embodiments, screening for these compounds includes the generation of suitable cell populations that express NAAP as secreted proteins or on the cell membrane. Suitable cells include cells from mammals, yeast, Drosophila, or E. coli. Cells expressing NAAP or cell membrane fractions containing NAAP are then contacted with a test compound and analyzed for binding, stimulation, or inhibition of activity of either NAAP or this compound.

  Some assays simply allow the test compound to be conjugated experimentally to the polypeptide and the binding detected by a fluorescent dye, radioisotope, enzyme conjugate or other detectable label. For example, the assay can include mixing at least one test compound with NAAP in solution or immobilized on a solid support and detecting binding of NAAP to the compound. Alternatively, detection and measurement of test compound binding in the presence of labeled competitor can be performed. In addition, this assay can be performed using cell-free reconstituted specimens, chemical libraries or natural product mixtures, where the test compound is released in solution or immobilized on a solid support.

The NAAP or fragment thereof of the present invention can be used to screen for compounds that modulate NAAP activity. Such compounds can include agonists, antagonists, partial agonists or inverse agonists and the like. In one embodiment, the assay is performed under conditions where NAAP activity is acceptable, wherein NAAP is mixed with at least one test compound, and the activity of NAAP in the presence of the test compound and in the absence of the test compound Compare with NAAP activity. A change in NAAP activity in the presence of the test compound suggests the presence of a compound that modulates NAAP activity. Alternatively, the assay is performed by mixing the test compound with an in vitro or cell-free system containing NAAP under conditions suitable for NAAP activity. In any of these assays, the test compound that modulates the activity of NAAP may be indirectly regulated, in which case no direct contact with the test compound is required. At least one or more test compounds can be screened.

  In another embodiment, homologous recombination in embryonic stem cells (ES cells) is used to “knock out” a polynucleotide encoding NAAP or a mammalian homolog thereof in an animal model system. Such techniques are well known in the art and are useful for creating animal models of human disease (see US Pat. Nos. 5,175,383 and 5,767,337, etc.). For example, mouse ES cells such as the 129 / SvJ cell line are derived from early mouse embryos and can be grown in culture. The ES cells are transformed with a vector having the target gene disrupted with a marker gene such as the neomycin phosphotransferase gene (neo: Capecchi, M.R. (1989) Science 244: 1288-1292). This vector is integrated into the corresponding region of the host genome by homologous recombination. Alternatively, homologous recombination is performed using the Cre-loxP system, and the target gene is knocked out in a tissue-specific or developmental stage-specific manner (Marth, JD (1996) Clin. Invest. 97: 1999-2002; Wagner, KU et al. (1997) Nucleic Acids Res. 25: 4323-4330). Transformed ES cells are identified and microinjected into mouse cell blastocysts collected from, for example, the C57BL / 6 mouse strain. These blastocysts are surgically introduced into pseudopregnant females, the genetic traits of the resulting chimeric progeny are identified, and they are crossed to create heterozygous or homozygous systems. Genetically modified animals produced in this way can be tested with potential therapeutic and toxic drugs.

Polynucleotides encoding NAAP can be manipulated in vitro in human blastocyst-derived ES cells. Human ES cells have the potential to differentiate into at least eight separate cell lineages, including endoderm, mesoderm and ectoderm cell types. These cell lines differentiate, for example, into neural cells, hematopoietic lineages and cardiomyocytes (Thomson, JA et al. (1998) Science 282: 1145-1147).

  Polynucleotides encoding NAAP can also be used to create “knock-in” humanized animals (pigs) or transgenic animals (mouse or rat) modeled on human disease. Using knock-in technology, a region of a polynucleotide encoding NAAP is injected into animal ES cells and the injected sequence is integrated into the animal cell genome. Transformed cells are injected into blastula and transplanted as described above. Test for genetically modified progeny or inbreds and treat with potential medicines to obtain information on the treatment of human disease. Alternatively, mammalian inbred lines that overexpress NAAP, for example, secreting NAAP into milk, can be a convenient protein source (Janne, J. et al. (1998) Biotechnol. Annu. Rev. 4:55). -74).

(Treatment)
There are chemical and structural similarities between the NAAP region and the nucleic acid binding protein region, for example, in sequence and motif content. See also Table 6 for examples of tissues that express NAAP. See also Example 11. Thus, NAAP is thought to play a role in cell proliferation abnormalities, neurological diseases, developmental disorders, and autoimmune / inflammatory diseases and infectious diseases. In the treatment of diseases associated with increased NAAP expression or activity, it is desirable to reduce NAAP expression or activity. It is also desirable to increase NAAP expression or activity in the treatment of diseases associated with decreased NAAP expression or activity.

  Thus, in one embodiment, NAAP or a fragment or derivative thereof can be administered to a subject for the treatment or prevention of a disease associated with decreased NAAP expression or activity. Among these diseases, but not limited to, cell proliferation abnormalities include actinic keratosis and atherosclerosis, bursitis, cirrhosis, hepatitis, mixed connective tissue disease (MCTD) , Myelofibrosis, paroxysmal nocturnal hemoglobinuria, polycythemia vera, psoriasis, primary thrombocythemia, and adenocarcinoma and leukemia, lymphoma, melanoma, myeloma, sarcoma, and teratocarcinoma, specifically Adrenal gland, bladder, bone, bone marrow, brain, breast, neck, gallbladder, ganglion, digestive tract, heart, kidney, liver, lung, muscle, ovary, pancreas, parathyroid gland, penis, prostate, salivary gland, skin, spleen, testis , Thymus, thyroid, uterine cancer, neurological diseases include sputum, ischemic cerebrovascular disease, stroke, cerebral neoplasm, Alzheimer's disease, Pick's disease, Huntington's disease, dementia, Parkinson's disease and other Extrapyramidal disorders, amyotrophic side hardening and other Motor neuron disorder, progressive neuromuscular atrophy, retinitis pigmentosa, hereditary ataxia, multiple sclerosis and other demyelinating diseases, bacterial and viral meningitis, brain abscess, subdural abscess, Epidural abscess, purulent intracranial thrombophlebitis, myelitis and radiculitis, viral central nervous system disease, prion disease (Kool, Creutzfeldt-Jakob disease, Gerstmann syndrome, and Gerstmann-Straussler-Scheinker syndrome ), Fatal familial insomnia, neurotrophic and metabolic diseases, neurofibromatosis, tuberous sclerosis, cerebelloretinal hemangioblastomatosis, trigeminal vascular syndrome, central nervous system Mental retardation and other developmental disorders, cerebral palsy, neuroskeletal disorders, autonomic nervous system disorders, cranial nerve disorders, spinal cord disorders, muscular dystrophy and other neuromuscular disorders, peripheral neuropathies, dermatomyositis and polymuscular muscles Hereditary, metabolic, endocrine, and addictive myopathy, myasthenia gravis, periodic limb paralysis, psychosis (moodness, anxiety disorder, schizophrenia), seasonal disorder (SAD), Inability to sit, amnesia, catatonia, diabetic neuropathy, extrapyramidal terminal defect syndrome, dystonia, schizophrenic mental disorder, postherpetic neuralgia, and Tourette's disease. It also includes developmental disorders, including tubular acidosis, anemia, Cushing syndrome, chondrogenic dystrophy, Duchenne-Becker muscular dystrophy, epilepsy, gonadal dysplasia, WAGR syndrome (Wilms tumor, aniridia) Genitourinary abnormalities, mental retardation), Smith- Magenis syndrome, myelodysplastic syndrome, hereditary mucosal epithelial dysplasia, hereditary keratoderma, Charcot-Marie-Tooth disease and neurofibromatosis Hereditary neuropathy, hypothyroidism, hydrocephalus, Syndenham's chorea and seizure disorders such as cerebral palsy, spinal cord fission, anencephalopathy, cranial spinal vertebrae, congenital glaucoma, cataract, sensory neuropathy Includes hearing loss, and autoimmune / inflammatory diseases include acquired immune deficiency syndrome (AIDS), Addison's disease, adult respiratory distress syndrome, allergy, ankylosing spondylitis Amyloidosis, anemia, asthma, atherosclerosis, autoimmune hemolytic anemia, autoimmune thyroiditis, autoimmune multiglandular endocrine ectodermal dystrophy (APECED), bronchitis, cholecystitis, contact Dermatitis, Crohn's disease, atopic dermatitis, dermatomyositis, diabetes mellitus, emphysema, lymphotoxin transient lymphopenia, fetal erythroblastosis, erythema nodosum, atrophic gastritis, glomerulonephritis, Goodpasture syndrome , Gout, Graves' disease, Hashimoto's thyroiditis, hypereosinophilia, irritable bowel syndrome, multiple sclerosis, myasthenia gravis, myocardial or pericardial inflammation, osteoarthritis, osteoporosis, pancreatitis, polymyositis , Psoriasis, Reiter syndrome, rheumatoid arthritis, scleroderma, Sjogren's syndrome, systemic anaphylaxis, systemic lupus erythematosus, systemic sclerosis, thrombocytopenia, ulcerative colitis Includes Werner syndrome, cancer complications, hemodialysis, extracorporeal circulation, viral infections, bacterial infections, fungal infections, parasitic infections, protozoal infections, helminth infections, trauma, also adenoviruses, arenaviruses , Bunyavirus, karchivirus, coronavirus, filovirus, hepadnavirus, herpes virus, flavivirus, orthomyxovirus, parvovirus, papovavirus, paramyxovirus, picornavirus, poxvirus, reovirus, retrovirus, Infection with viral mediators classified as rhabdovirus or togavirus, pneumococcal infection, staphylococci, streptococci, bacillus, corynebacterium, clostridium, meningococcus, gonococcus, listeria, molaxella, kingella , Hemophilus, les Onera, Bordetella, Gram-negative enterobacteria (including Shigella, Salmonella, Campylobacter), Pseudomonas, Vibrio, Brucella, Wild boar fungus, Yersinia, Bartonella, norcardium, Actinomycetes, Mycobacterium, Spirochete, Rickettsia, Infections caused by bacterial mediators classified as Chlamydia, Mycoplasma, Aspergillus, Blast myces, Dermatophytes, Cryptococcus, Coccidioides, malasezzia, histoplasma, or other fungal pathogenic fungi, and Plasmodim (parasite causing malaria), parasite amoeba, Leishmania spp., Trypanosoma spp., Toxoplasma spp., Pneumocystis carini spp., Giardia protozoa, Trichomonas spp. Extension insects, intestinal extension insects such as roundworm, lymphatic filariasis nematodes include infections by parasites that are classified as fluke and Jomushirui (tapeworms), such as schistosomes.

  In another embodiment, a vector capable of expressing NAAP or a fragment or derivative thereof for the treatment or prevention of diseases associated with decreased NAAP expression or activity, including but not limited to the diseases listed above. Can be administered to a subject.

  In yet another embodiment, a composition having substantially purified NAAP for the treatment or prevention of diseases associated with decreased NAAP expression or activity, including but not limited to the diseases listed above. The product can be administered to a subject together with a suitable pharmaceutical carrier.

  In yet another embodiment, an agonist that modulates NAAP activity is provided to a subject for the treatment or prevention of a disease associated with decreased NAAP expression or activity, including, but not limited to, the diseases listed above. Can be administered.

  In a further example, NAAP antagonists may be administered to a subject for the treatment or prevention of diseases associated with increased NAAP expression or activity. Examples of such diseases include, but are not limited to, cell proliferation abnormalities, neurological disorders, developmental disorders, autoimmune / inflammatory diseases, and infectious diseases as described above. In one aspect, an antibody that specifically binds to NAAP can be used directly as an antagonist or indirectly as a targeting or delivery mechanism that delivers the drug to cells or tissues that express NAAP.

  In another embodiment, a vector expressing a complementary sequence of a polynucleotide encoding NAAP is administered to a subject to treat a disease associated with increased NAAP expression or activity, including but not limited to the diseases described above. Can be treated or prevented.

  In another embodiment, any protein, antagonist, antibody, agonist, complementary sequence, or vector of the invention can be administered in combination with another suitable therapeutic agent. Suitable therapeutic agents for use in combination therapy can be selected by one skilled in the art according to conventional pharmaceutical principles. When combined with a therapeutic agent, it can provide a synergistic effect in the treatment or prevention of the various diseases described above. This method makes it possible to increase the pharmaceutical effect with a small amount of each drug, thereby reducing the possibility of side effects.

  An antagonist of NAAP can be produced using methods generally known in the art. Specifically, antibodies can be made using purified NAAP, or a library of therapeutic agents can be screened to identify those that specifically bind to NAAP. Antibodies to NAAP can also be produced using methods known in the art. Such antibodies can include, but are not limited to, polyclonal antibodies, monoclonal antibodies, chimeric antibodies, single chain antibodies, Fab fragments, and fragments produced by Fab expression libraries. Neutralizing antibodies (ie, antibodies that inhibit dimer formation) are usually suitable for therapy. Single chain antibodies (e.g. camels or llamas) are potent enzyme inhibitors and may have advantages in the design of peptidomimetics and in the development of immunosorbents and biosensors (Muyldermans, S. (2001) J. Biotechnol. 74: 277-302).

For the production of antibodies, various hosts including goats, rabbits, rats, mice, camels, dromedaries, llamas, humans, etc. are injected with NAAP, or any fragment or oligopeptide thereof with immunogenic properties. Can be immunized. Depending on the host species, various adjuvants can be used to enhance the immune response. Such adjuvants include, but are not limited to, Freund's adjuvant, mineral gel adjuvants such as aluminum hydroxide, lysolecithin, pluronic polyol, polyanion, peptide, oil emulsion, mussel hemocyanin (KLH), dinitrophenol, etc. There is a surfactant. Among adjuvants used in humans, BCG (Bacille Calmette-Guerin) and Corynebacterium parvum are particularly preferred.

  The oligopeptide, peptide or fragment used for inducing an antibody against NAAP preferably has an amino acid sequence consisting of at least about 5 amino acids, and generally consists of about 10 or more amino acids. These oligopeptides, peptides or fragments are also preferably identical to part of the amino acid sequence of the natural protein. Short stretches of NAAP amino acids can be fused with sequences of other proteins such as KLH to produce antibodies against this chimeric molecule.

  Monoclonal antibodies against NAAP can be made using any technique that produces antibody molecules by continuous cell lines in the culture medium. Such technologies include, but are not limited to, hybridoma technology, human B cell hybridoma technology, and EBV-hybridoma technology (Kohler, G. et al. (1975) Nature 256: 495-497, Kozbor, D. et al. (1985) .J. Immunol. Methods 81: 31-42, Cote, RJ et al. (1983) Proc. Natl. Acad. Sci. USA 80: 2026-2030, Cole, SP et al. (1984) Mol. Cell Biol. 62 : 109-120 etc.).

  Furthermore, techniques developed for making “chimeric antibodies” such as splicing mouse antibody genes into human antibody genes can be used to obtain molecules with suitable antigen specificity and biological activity (eg, Morrison, SL et al. (1984) Proc. Natl. Acad. Sci. 81: 68516855; Neuberger, MS et al. (1984) Nature 312: 604-608; Takeda, S. et al. (1985) Nature 314: 452-454 See). Alternatively, NAAP-specific single chain antibodies are generated using methods described in the art and applying the described techniques for the production of single chain antibodies. Antibodies with related specificity but different idiotype composition can also be generated by chain shuffling from random combinations of immunoglobulin libraries (Burton DR (1991) Proc. Natl. Acad. Sci. USA 88 : 10134-10137 etc.).

Antibody production can also be accomplished by induction of in vivo production in a lymphocyte population or by screening an immunoglobulin library or panel of highly specific binding reagents as disclosed in the literature. (See Orlando, R. et al. (1989) Proc. Natl. Acad. Sci. USA 86: 3833-3837, Winter, G. et al. (1991) Nature 349: 293-299).

Antibody fragments that contain specific binding sites for NAAP can also be produced. For example, but not by way of limitation, such fragments are produced by reducing the F (ab ′) ₂ fragment produced by pepsin digestion of the antibody molecule and the disulfide bridge of the F (ab ′) ₂ fragment. There is a Fab fragment to be. Alternatively, it is possible to quickly and easily identify a monoclonal Fab fragment having a desired specificity by preparing a Fab expression library (see Huse, WD et al. (1989) Science 246: 1275-1281). .

  Screening with various immunoassays (immunoassays) can identify antibodies with the desired specificity. Numerous protocols for immunoradiometric assays or competitive binding assays using either polyclonal or monoclonal antibodies with established specificity are known in the art. Such immunoassays usually involve the measurement of complex formation between NAAP and its specific antibody. Two-site monoclonal-based immunoassays are commonly used, using a group of monoclonal antibodies reactive against two non-interfering NAAP epitopes, but competitive binding studies are also available (Pound, supra).

Various methods such as Scatchard analysis in conjunction with radioimmunoassay techniques can be used to assess the affinity of an antibody for NAAP. The affinity is represented by a binding constant Ka, which is a value obtained by dividing the molar concentration of the NAAP antibody complex under equilibrium conditions by the molar concentration of free antibody and free antigen. Polyclonal antibodies have heterogeneous affinities for various NAAP epitopes, and Ka as determined for a given polyclonal antibody reagent represents the average affinity or binding activity of NAAP antibody groups. Monoclonal antibodies are monospecific for certain NAAP epitopes, and the Ka determined for certain reagents of the monoclonal antibody represents a true measure of affinity. High affinity antibody reagents with Ka values of 10 ⁹ to 10 ¹² L / mol are preferably used in immunoassays where the NAAP-antibody complex must withstand harsh manipulations. Low affinity antibody formulations with Ka values of 10 ⁶ to 10 ⁷ liter / mol are used for immunopurification and similar treatments where NAAP must eventually dissociate (preferably in an activated state) from the antibody. (Catty, D. (1988) Antibodies, Volume I: A Practical Approach , IRL Press, Washington, DC; Liddell, JE and Cryer, A. (1991) A Practical Guide to Monoclonal Antibodies , John Wiley & Sons, New York NY).

  The antibody titer and binding activity of the polyclonal antibody formulation can be further evaluated to determine the quality and suitability of such reagents for certain applications used later. For example, polyclonal antibody preparations containing at least 1-2 mg / ml specific antibody, preferably 5-10 mg / ml specific antibody, are generally used in processes where NAAP-antibody complexes must be precipitated. Antibody specificity, antibody titer, binding activity, and guidelines for antibody quality and use in various applications are generally available (see, for example, Catty literature, Coligan et al. Above).

In another embodiment of the invention, a polynucleotide encoding NAAP, or any fragment or complementary sequence thereof, can be used for therapeutic purposes. In some embodiments, gene expression can be modified by designing sequences or antisense molecules (DNA, RNA, PNA, or modified oligonucleotides) that are complementary to the coding and regulatory regions of the gene encoding NAAP. obtain. Such techniques are well known in the art, and antisense oligonucleotides or larger fragments can be designed from various positions along the control region of the sequence encoding NAAP, or along the coding region (e.g., Agrawal, (See S., ed. (1996) Antisense Therapeutics , Humana Press Inc., Totawa NJ.)

  For use in therapy, any gene delivery system suitable for introducing an antisense sequence into a suitable target cell can be used. Antisense sequences can be delivered into cells in the form of expression plasmids that produce sequences complementary to at least a portion of the cell sequence encoding the target protein during transcription (Slater, JE et al. (1998) J See Allergy Clin. Immunol. 102 (3): 469-475 and Scanlon, KJ et al. (1995) 9 (13): 1288-1296). Antisense sequences can also be introduced into cells using viral vectors such as retrovirus and adeno-associated virus vectors (Miller, AD (1990) Blood 76: 271, supra, Ausubel, Uckert, W. et al. And W. Walther (1994) Pharmacol. Ther. 63 (3): 323-347 etc.). Other gene delivery mechanisms include liposome systems, artificial viral envelopes and other systems known in the art (Rossi, JJ (1995) Br. Med. Bull. 51 (1): 217-225, Boado, RJ et al. (1998) J. Pharm. Sci. 87 (11): 1308-1315, Morris, MC et al. (1997) Nucleic Acids Res. 25 (14): 2730-2736.

In another embodiment of the invention, a polynucleotide encoding NAAP can be used for somatic or germ cell gene therapy. By carrying out gene therapy, (i) Severe combined immune deficiency (SCID) − characterized by gene deficiency (eg, X chromosome linkage inheritance (Cavazzana-Calvo, M. et al. (2000) Science 288: 669-672)) X1), severe combined immune deficiency associated with congenital adenosine deaminase (ADA) deficiency (Blaese, RM et al. (1995) Science 270: 475-480, Bordignon, C. et al. (1995) Science 270: 470- 475), cystic fibrosis (Zabner, J. et al. (1993) Cell 75: 207-216: Crystal, RG et al. (1995) Hum. Gene Therapy 6: 643-666, Crystal, RG et al. (1995) Hum. Gene Therapy 6: 667-703), thalassamia, familial hypercholesterolemia, hemophilia caused by factor VIII or factor IX deficiency (Crystal, RG (1995) Science 270: 404-410, Verma , IM and N. Somia. (1997) Nature 389: 239-242), (ii) expressing a conditional lethal gene product (eg, in the case of cancer resulting from uncontrolled cell growth), (iii) Intracellular parasites such as human retroviruses such as human immunodeficiency virus (HIV) (Baltimore, D. (1988) Nature 335: 395-396, Poeschla, E. et al. (1996) Proc. Natl. Acad. Sci. USA 93: 11395-11399), fungi parasites such as hepatitis B or C virus (HBV, HCV), Candida albicans and Paracoccidioides brasiliensis, and protozoan parasites such as Plasmodium falciparum and Trypanosoma cruzi ) Can be expressed. When a gene deficiency required for NAAP expression or regulation causes a disease, NAAP can be expressed from a suitable population of introduced cells to alleviate the expression of symptoms caused by the gene deficiency.

In a further embodiment of the invention, diseases and abnormalities caused by NAAP deficiency are treated by preparing mammalian expression vectors encoding NAAP and introducing these vectors into NAAP-deficient cells by mechanical means. Mechanical introduction techniques for in vivo or ex vitro cells include (i) direct DNA microinjection into individual cells, (ii) gene gun, (iii) transfection via liposomes, ( iv) Receptor-mediated gene transfer, and (v) Use of DNA transposons (Morgan, RA and WF Anderson (1993) Annu. Rev. Biochem. 62: 191-217, Ivics, Z. (1997) Cell 91 : 501-510; Boulay, JL. And H. Recipon (1998) Curr. Opin. Biotechnol. 9: 445-450).

  Expression vectors that can be effective for NAAP expression include, but are not limited to, PCDNA 3.1, EPITAG, PRCCMV2, PREP, PVAX, PCR2-TOPOTA vectors (Invitrogen, Carlsbad CA), PCMV-SCRIPT, PCMV-TAG, PEGSH / PERV (Stratagene, La Jolla CA), PTET-OFF, PTET-ON, PTRE2, PTRE2-LUC, PTK-HYG (Clontech, Palo Alto CA) are included. In order to express NAAP, (i) a constitutively active promoter (such as cytomegalovirus (CMV), rous sarcoma virus (RSV), SV40 virus, thymidine kinase (TK), or β-actin gene), (ii) Inducible promoters (eg, tetracycline regulatable promoters (Gossen, M. and H. Bujard (1992) Proc. Natl. Acad. Sci. USA 89: 5547-5551; Gossen, M. et al. (1995) Science 268: 1766-1769; Rossi, FMV and HM Blau (1998) Curr. Opin. Biotechnol. 9: 451-456)), ecdysone-inducible promoter ( Included in commercially available plasmids PVGRXR and PIND: Invitrogen), FK506 / rapamycin inducible promoter, or RU486 / mifepristone inducible promoter (Rossi, FMV and HM Blau, supra), or (iii) normal It is possible to use a natural promoter or a tissue-specific promoter of an endogenous gene encoding NAAP derived from an individual.

  Commercially available liposome transformation kits (eg, Invitrogen's PerFect Lipid Transfection Kit) allow those skilled in the art to deliver polynucleotides to target cells in culture. Also, the effort required to optimize experimental parameters is minimized. Alternatively, transformation using the calcium phosphate method (Graham. FL and AJ Eb (1973) Virology 52: 456-467) or electroporation (Neumann, B. et al. (1982) EMBO J. 1: 841-845) I do. In order to introduce DNA into primary culture cells, modifications to standardized mammalian transfection protocols are required.

In another embodiment of the present invention, a disease or disorder caused by a gene defect associated with NAAP expression can be identified as (i) a polyvirus encoding NAAP under the control of a retroviral terminal repeat (LTR) promoter or some independent promoter. Rev responsive element (RRE) with nucleotides, (ii) suitable RNA packaging signals, (iii) additional retroviral cis-acting RNA sequences and coding sequences necessary for efficient vector propagation A retroviral vector consisting of Retroviral vectors (eg PFB and PFBNEO) are commercially available from Stratagene and are based on published data (Riviere, I. et al. (1995) Proc. Natl. Acad. Sci. USA 92: 6733-6737). The above data is incorporated herein by reference. The vector is propagated in a suitable vector producing cell line (VPCL), which expresses an envelope gene with affinity for a receptor on the target cell or a pan-affinity envelope protein such as VSVg (Armentano, D. et al. (1987) J. Virol. 61: 1647-1650, Bender, MA et al. (1987) J. Virol. 61: 1639-1646, Adam, MA and AD Miller (1988) J. Virol. 62: 3802-3806, Dull , T. et al. (1998) J. Virol. 72: 8463-8471, Zufferey, R. et al. (1998) J. Virol. 72: 9873-9880). In US Pat. No. 5,910,434 to Rigg (“Method for obtaining retrovirus packaging cell lines producing high transducing efficiency retroviral supernatant”), a method for obtaining a retroviral packaging cell line is disclosed, with reference to It is a part of this specification. The propagation of retroviral vectors, transduction of cell populations (eg CD4 ⁺ T cells), and return of transduced cells to patients are methods well known to those skilled in the field of gene therapy and are described in numerous references. (Ranga, U. et al. (1997) J. Virol. 71: 7020-7029, Bauer, G. et al. (1997) Blood 89: 2259-2267, Bonyhadi, ML (1997) J. Virol. 71: 4707 -4716, Ranga, U. et al. (1998) Proc. Natl. Acad. Sci. USA 95: 1201-1206, Su, L. (1997) Blood 89: 2283-2290).

  Alternatively, a delivery system of adenoviral gene therapy is used to deliver a group of polynucleotides encoding NAAP to a group of cells having one or more genetic abnormalities associated with NAAP expression. The production and packaging of adenoviral vectors is well known to those skilled in the art. Replication-deficient adenovirus vectors have been shown to be able to be used in a variety of ways to introduce genes encoding various immunoregulatory proteins into intact islets (Csete, ME et al. (1995) Transplantation 27: 263268). Adenoviral vectors that may be used are described in US Pat. No. 5,707,618 (“Adenovirus vectors for gene therapy”) to Armentano, which is incorporated herein by reference. See also Antinozzi, P.A. et al. (1999) Annu. Rev. Nutr. 19: 511-544 and Verma, I.M. and N. Somia (1997) Nature 18: 389: 239-242 for adenoviral vectors. Both documents are hereby incorporated by reference.

  Alternatively, a herpes gene therapy delivery system is used to deliver a polynucleotide encoding NAAP to target cells having one or more genetic abnormalities associated with NAAP expression. Herpes simplex virus (HSV) vectors can be particularly beneficial when introducing NAAP into central neurons where HSV is tropic. The production and packaging of herpes vectors is known to those skilled in the art. Replication competent herpes simplex virus (HSV) type I vectors have been used to deliver reporter genes to primate eyes (Liu, X. et al. (1999) Exp. Eye Res. 169: 385- 395). The production of HSV-1 viral vectors is also disclosed in US Pat. No. 5,804,413 (“Herpes simplex virus swains for gene transfer”) granted to DeLuca, which is incorporated herein by reference. . US Pat. No. 5,804,413 describes a recombinant HSV d92 comprising a genome having at least one exogenous gene that is introduced into cells under the control of a promoter suitable for purposes including human gene therapy. The patent also discloses the generation and use of recombinant HSV lines lacking ICP4, ICP27 and ICP22. For HSV vectors, see also Goins, W.F. et al. (1999) J. Virol. 73: 519-532 and Xu, H. et al. (1994) Dev. Biol. 163: 152-161. Both documents are hereby incorporated by reference. Manipulation of cloned herpesvirus sequences, production of recombinant virus after transfection with a large number of plasmids containing different segments of the giant herpesvirus genome, herpesvirus growth and proliferation, and infection of herpesvirus cells Is a technique known to those skilled in the art.

  Alternatively, certain alphavirus (positive single stranded RNA virus) vectors are used to deliver a group of polynucleotides encoding NAAP to a group of target cells. Biological research on the prototype alphavirus Semliki Forest Virus (SFV) has been extensive and gene transfer vectors are based on the SFV genome (Garoff, H. and K.-J Li (1998) Cun. Opin. Biotech. 9: 464-469). During the replication of alpha viral RNA, a subgenomic RNA that normally encodes the viral capsid protein is created. This subgenomic RNA is replicated to a higher level than full-length genomic RNA, resulting in overproduction of capsid proteins compared to viral proteins with enzymatic activity (eg, proteases and polymerases). Similarly, by introducing a sequence encoding NAAP into a region encoding the capsid of the α virus genome, a large number of NAAP-encoding RNAs are produced in the vector-introduced cells, and NAAP is synthesized at a high level. Usually, infection with alpha virus is accompanied by cell lysis within a few days. On the other hand, the ability of a group of normal hamster kidney cells (BHK-21) having a certain variant of Sindbis virus (SIN) to establish a persistent infection can apply lytic replication of α viruses to gene therapy (Dryga, SA et al. (1997) Virology 228: 74-83). Since the α virus has a wide host range, NAAP can be introduced into various types of cells. Specific transduction of a subset of cells in a population may require cell sorting prior to transduction. Methods for treating alphavirus infectious cDNA clones, alphavirus cDNA and RNA transfection methods, and alphavirus infection methods are known to those skilled in the art.

  It is also possible to inhibit gene expression using an oligonucleotide derived from a transcription initiation site. This position is, for example, between about −10 and about +10 counting from the start site. Similarly, inhibition can be achieved using triple helix base pairing methods. Triple helix base pairing is useful because it inhibits the ability of the double helix to open sufficiently for the binding of polymerases, transcription factors or regulatory molecules. Recent therapeutic advances using triple helix DNA are described in the literature (Gee, JE et al. (1994) in: Huber, BE and BI Carr, Molecular and Immunologic Approaches, Futura Publishing Co., Mt. Kisco, NY, (See pages 163-177). Complementary sequences or antisense molecules can also be designed to block translation of mRNA by preventing transcripts from binding to ribosomes.

  Ribozymes are enzymatic RNA molecules, and ribozymes can also be used to catalyze the specific cleavage of RNA. The mechanism of ribozyme action involves sequence specific hybridization of ribozyme molecules to complementary target RNA and subsequent internal nucleotide strand breaks. For example, there is a possibility that a hammerhead ribozyme molecule produced by genetic engineering can specifically and effectively catalyze internal nucleotide strand breakage of a sequence encoding NAAP.

  Specific ribozyme cleavage sites within any RNA target are first identified by scanning the target molecule for ribozyme cleavage sites including GUA, GUU, GUC sequences. Once identified, assess whether a short RNA sequence of 15-20 ribonucleotides corresponding to the region of the target gene with the cleavage site has secondary structural features that render the oligonucleotide dysfunctional. Is possible. Assessment of the suitability of candidate targets can also be performed by testing accessibility to hybridization with complementary oligonucleotide groups using a ribonuclease protection assay.

The complementary ribonucleic acid molecules and ribozymes of the present invention can be made using any method well known in the art for nucleic acid molecule synthesis. As a production method, there is a method of chemically synthesizing oligonucleotides such as solid phase phosphoramidite chemical synthesis. Alternatively, RNA molecules can be produced by in vitro and in vivo transcription of DNA sequences encoding NAAP. Such DNA sequences can be incorporated into a variety of vectors using a suitable RNA polymerase promoter such as T7 or SP6. Alternatively, these cDNA products that synthesize complementary RNA constitutively or inducibly can be introduced into cell lines, cells or tissues.

  RNA molecules can be modified to increase intracellular stability and increase half-life. Possible modifications include, but are not limited to, the addition of flanking sequences at the 5 'end, 3' end, or both of the molecule, or phosphorothioate or 2 'O rather than phosphodiesterase linkages in the main chain of the molecule. -Use of methyl is included. This concept is originally in the production of PNA groups, but can be extended to all these molecules. For this purpose, acetyl-, methyl-, thio- and similar modifications of adenine, cytidine, guanine, thymine, and uridine that are not easily recognized by endogenous endonucleases, or unconventional bases such as inosine , Queosine, wybutosine.

  Further embodiments of the invention include methods of screening for compounds that are effective in altering the expression of a polynucleotide encoding NAAP. Non-limiting compounds that are effective in modifying the expression of a specific polynucleotide include oligonucleotides, antisense oligonucleotides, triple helix-forming oligonucleotides, transcription factors and other polypeptide transcriptional regulators, and specific polynucleotide sequences. There are non-polymeric chemical entities that can interact. Effective compounds can alter polynucleotide expression by acting as either inhibitors or enhancers of polynucleotide expression. Thus, in the treatment of diseases associated with increased NAAP expression or activity, compounds that specifically inhibit the expression of polynucleotides encoding NAAP are therapeutically useful and are associated with decreased NAAP expression or activity. In the treatment of disease, compounds that specifically promote the expression of a polynucleotide encoding NAAP may be therapeutically useful.

At least one or more test compounds can be screened for effectiveness in altering the expression of a particular polynucleotide. The test compound can be obtained by any method commonly known in the art. Such methods include chemical modification of compounds that are said to be effective in altering expression of the polynucleotide, selection of existing natural or non-natural compounds from existing commercial or private libraries, target poly There is a rational design of compounds based on chemical and / or structural properties of nucleotides, and selection from a library of combinatorially or randomly generated compounds. A sample having a polynucleotide encoding NAAP is exposed to at least one of the test compounds thus obtained. Samples can be, for example, intact cells, permeabilized cells, or in vitro cell-free or reconstituted biochemical systems. Alterations in the expression of a polynucleotide encoding NAAP are assayed by any method well known in the art. Usually, the expression of a specific nucleotide is detected by hybridization using a probe having a nucleotide sequence complementary to the sequence of a polynucleotide encoding NAAP. The amount of hybridization can be quantified, thereby forming the basis for comparison of expression of polynucleotides exposed and not exposed to one or more test compounds. Detection of a change in the expression of the polynucleotide exposed to the test compound indicates that the test compound is effective in altering the expression of the polynucleotide. For compounds effective in modifying the expression of specific polynucleotides, for example, Schizosaccharomyces pombe gene expression system (Atkins, D. et al. (1999) US Pat. No. 5,932,435, Arndt, GM et al. (2000) Nucleic Acids Res. 28: E15) or human cell lines such as HeLa cells (Clarke, ML et al. (2000) Biochem. Biophys. Res. Commun. 268: 8-13). Particular embodiments of the present invention are involved in screening combinatorial libraries of oligonucleotides (deoxyribonucleotides, ribonucleotides, peptide nucleic acids, modified oligonucleotides) for examining antisense activity against specific polynucleotide sequences. (Bruice, TW et al. (1997) US Pat. No. 5,686,242, Bruice, TW et al. (2000) US Pat. No. 6,022,691).

Numerous methods for introducing vectors into cells or tissues are available and are equally suitable for in vivo , in vitro and ex vivo use. For ex vivo treatment, the vector can be introduced into stem cells taken from the patient, cloned and expanded back to the patient by autologous transplantation. Delivery by transfection or by ribosome injection or polycation amino polymers can be performed using methods well known in the art (eg Goldman, CK et al. (1997) Nat. Biotechnol. 15: 462- 466).

  Any of the above treatment methods can be applied to all subjects in need of treatment including mammals such as humans, dogs, cats, cows, horses, rabbits, monkeys, and the like.

Certain further embodiments of the invention relate to the administration of certain compositions generally having certain active ingredients formulated with certain pharmaceutically acceptable excipients. Excipients include, for example, sugar, starch, cellulose, gum and protein. Various dosage forms are widely known, and details are described in the latest edition of Remington's Pharmaceutical Sciences (Maack Publishing, Easton PA). Such compositions consist of NAAP, NAAP antibodies, mimetics, agonists, antagonists, inhibitors, and the like.

  The compositions used in the present invention can be administered by any number of routes including, but not limited to, oral, intravenous, intramuscular, intraarterial, intramedullary, intrathecal. , Intraventricular, lung, transdermal, subcutaneous, intraperitoneal, intranasal, intestinal, topical, sublingual or rectal.

  Compositions administered from the lungs can be prepared in liquid or dry powder form. Such compositions are usually aerosolized just before the patient inhales. In the case of small molecules (eg traditional low molecular weight organic drugs), aerosol delivery of fast acting formulations is known in the art. In the case of macromolecules (eg, larger peptides and proteins), recent improvements in pulmonary delivery through the alveolar region of the lung in the art can substantially transport drugs such as insulin into the blood circulation (See Patton, JS et al., US Pat. No. 5,997,848, etc.). Pulmonary delivery is superior in administration without needle injection and eliminates the need for penetration enhancers that may be toxic.

  Compositions suitable for use in the present invention include compositions containing as much active ingredient as necessary to achieve a given purpose. The determination of an effective dose is within the ability of those skilled in the art.

  A variety of specially shaped composition groups can be prepared to deliver macromolecules with NAAP or fragments thereof directly into cells. For example, a liposomal formulation comprising a cell-impermeable polymer can facilitate cell fusion and intracellular delivery of the polymer. Alternatively, NAAP or a fragment thereof can be attached to the short cationic N-terminus obtained from HIV Tat-1 protein. The fusion proteins thus generated have been shown to transduce cell groups of all tissues, including the brain, of a mouse model system (Schwarze, SR et al. (1999) Science 285: 1569- 1572).

  The therapeutically effective dose can be estimated initially for any compound in a cell culture assay, such as a cell culture assay for neoplastic cells, or in an animal model such as a mouse, rabbit, dog or pig. The animal model may also be used to determine the appropriate concentration range and route of administration. Such information can then be used to determine beneficial doses and routes for administration to humans.

The therapeutically effective dose refers to the amount of an active ingredient such as NAAP or a fragment thereof, an antibody of NAAP, an agonist or antagonist of NAAP, an inhibitor, etc., which recovers a symptom or condition. Therapeutic efficacy and toxicity can be measured by standard drug techniques in cell culture or animal experiments, eg, by measuring ED ₅₀ (50% therapeutically effective dose of a population) or LD ₅₀ (50% lethal dose of a population). Can be determined. The dose ratio of toxic to therapeutic effects is the therapeutic index and it can be expressed as the ratio LD ₅₀ / ED ₅₀ . Compositions that exhibit high therapeutic indices are desirable. Data obtained from cell culture assays and animal experiments is used to develop a range of dosage for human use. The dosage contained in such compositions is preferably in a range of blood concentrations that includes ED ₅₀ with little or no toxicity. Depending on the mode of administration used, patient sensitivity and route of administration, the dosage will vary within this range.

  The exact dosage will be determined by the practitioner in light of factors related to the subject that requires treatment. Dosage forms and dosages are adjusted to provide sufficient levels of the active ingredient or to maintain the desired effect. Factors related to the subject include the severity of the disease, the patient's general health, the subject's age, weight and sex (gender), time and frequency of administration, drug combination, response sensitivity and response to treatment. Long-acting compositions may be administered once every 3-4 days, once a week, or once every two weeks, depending on the half-life and clearance rate of the particular formulation.

  The usual dose depends on the route of administration, but is about 0.1-100.000 μg, up to a total of about 1 g. Guidance on specific doses and delivery methods is described in the literature and is usually available to practitioners. Those skilled in the art will utilize different formulations for nucleotides than for proteins or their inhibitors. Similarly, delivery of polynucleotides or polypeptides will be specific to particular cells, symptoms, sites, etc.

(Diagnosis)
In another example, an antibody that specifically binds to NAAP is used to diagnose a disorder characterized by NAAP expression or to monitor patients treated with NAAP or an agonist or antagonist or inhibitor of NAAP. Can be used in the assay. Antibodies useful for diagnostic purposes are made in the same manner as described above in the treatment section. NAAP diagnostic assays include methods of detecting NAAP from human body fluids or from cell or tissue extracts using antibodies and labels. The antibody can be modified or not, and can be labeled covalently or non-covalently with a reporter molecule. A wide variety of reporter molecules are known in the art and can be used, some of which have been described above.

  Various protocols for measuring NAAP, such as ELISA, RIA, FACS, etc., are well known in the art and provide a basis for diagnosing altered or abnormal levels of NAAP expression. Normal or standard NAAP expression values are obtained by mixing body fluids or cell extracts collected from normal mammals, such as human subjects, with antibodies to NAAP under conditions suitable for complex formation. To confirm. The amount of standard complex formation can be quantified by various methods such as photometry. The amount of NAAP expression in disease samples from subjects, controls, and biopsy tissues is compared to a standard value. The deviation between the standard value and the subject establishes the parameter for diagnosing the disease.

  According to another embodiment of the present invention, a polynucleotide encoding NAAP may be used for diagnosis. Polynucleotides that can be used include oligonucleotide sequences, complementary RNA and DNA molecules, and PNAs. These polynucleotides can be used to detect and quantify gene expression in biopsy tissues where NAAP expression can be correlated with disease. This diagnostic assay can be used to determine the presence of NAAP, as well as overexpression, and to monitor the regulation of NAAP levels during treatment.

  In certain embodiments, hybridization with PCR probes capable of detecting polynucleotide sequences, such as genomic sequences, encoding NAAP or closely related molecules can be used to identify nucleic acid sequences encoding NAAP. . Whether the probe is made from a highly specific region (eg, a 5 ′ regulatory region) or a slightly less specific region (eg, a conserved motif), The stringency of hybridization or amplification will determine whether the probe will identify only the native sequence encoding NAAP, or whether it will identify allelic variants or related sequences.

  The probe can also be used to detect related sequences and can have at least 50% sequence identity with any sequence encoding NAAP. The hybridization probe of the present invention can be DNA or RNA, and can be derived from the sequence of SEQ ID NO: 24-46, or a genomic sequence including a NAAP gene promoter, enhancer, and intron.

As a method for preparing a hybridization probe specific for DNA encoding NAAP, there is a method of cloning a polynucleotide sequence encoding NAAP or a NAAP derivative into a vector for preparing an mRNA probe. Vectors for making mRNA probes are known to those skilled in the art and are commercially available and used to synthesize RNA probes in vitro by adding a suitable RNA polymerase and a suitable labeled nucleotide. obtain. Hybridization probes can be labeled with various reporter populations. Examples of reporter populations include radionuclides such as ³² P or ³⁵ S, or enzyme labels such as alkaline phosphatase bound to a probe via an avidin / biotin binding system.

  A polynucleotide sequence encoding NAAP may be used in the diagnosis of diseases associated with NAAP expression. Among these diseases, but not limited to, cell proliferation abnormalities include actinic keratosis and atherosclerosis, bursitis, cirrhosis, hepatitis, mixed connective tissue disease (MCTD) , Myelofibrosis, paroxysmal nocturnal hemoglobinuria, polycythemia vera, psoriasis, primary thrombocythemia, and adenocarcinoma and leukemia, lymphoma, melanoma, myeloma, sarcoma, and teratocarcinoma, specifically Adrenal gland, bladder, bone, bone marrow, brain, breast, neck, gallbladder, ganglion, gastrointestinal tract, heart, kidney, liver, lung, muscle, ovary, pancreas, parathyroid gland, penis, prostate, salivary gland, skin, spleen, testis , Thymus, thyroid, uterine cancer, neurological diseases include sputum, ischemic cerebrovascular disorder, stroke, cerebral neoplasm, Alzheimer's disease, Pick's disease, Huntington's disease, dementia, Parkinson's disease and other Extrapyramidal disorders, amyotrophic side hardening and other Motor neuron disorder, progressive neuromuscular atrophy, retinitis pigmentosa, hereditary ataxia, multiple sclerosis and other demyelinating diseases, bacterial and viral meningitis, brain abscess, subdural abscess, Epidural abscess, purulent intracranial thrombophlebitis, myelitis and radiculitis, viral central nervous system disease, prion disease (Kool, Creutzfeldt-Jakob disease, Gerstmann syndrome, and Gerstmann-Straussler-Scheinker syndrome Fatal familial insomnia, neurotrophic and metabolic diseases, neurofibromatosis, tuberous sclerosis, cerebelloretinal hemangioblastomatosis, trigeminal neurovascular syndrome, central nervous system Mental retardation and other developmental disorders, cerebral palsy, neuroskeletal disorders, autonomic nervous system disorders, cranial nerve disorders, spinal cord disorders, muscular dystrophy and other neuromuscular disorders, peripheral neuropathies, dermatomyositis and polymuscular muscles Hereditary, metabolic, endocrine, and addictive myopathy, myasthenia gravis, periodic limb paralysis, psychosis (moodness, anxiety disorder, schizophrenia), seasonal disorder (SAD), Includes restlessness, amnesia, catatonia, diabetic neuropathy, extrapyramidal terminal defect syndrome, dystonia, schizophrenic mental disorder, postherpetic neuralgia, and Tourette's disease, including developmental disorders Among them, tubular acidosis, anemia, Cushing syndrome, chondrogenic dwarfism, Duchenne-Becker muscular dystrophy, sputum, gonadal malformation, WAGR syndrome (Wilms tumor, aniridia, genitourinary abnormalities, mental retardation) Smith- Magenis syndrome, myelodysplastic syndrome, hereditary mucosal epithelial dysplasia, hereditary keratoderma, Charcot-Marie-Tooth disease and neurofibromatosis Hereditary neuropathy, hypothyroidism, hydrocephalus, Syndenham's chorea, and seizure disorders such as cerebral palsy, spinal cord fission, anencephalopathy, cranial spinal vertebrae, congenital glaucoma, cataract, sensory nerve hearing Autoimmune / inflammatory diseases include acquired immune deficiency syndrome (AIDS), Addison's disease, adult respiratory distress syndrome, allergy, ankylosing spondylitis, amyloidosis, anemia, asthma, atherosclerosis , Autoimmune hemolytic anemia, autoimmune thyroiditis, autoimmune multiglandular endocrine candid ectoderm dystrophy (APECED), bronchitis, cholecystitis, contact dermatitis, Crohn's disease, atopic dermatitis, dermatomyositis , Diabetes mellitus, emphysema, lymphotoxic transient lymphopenia, fetal erythroblastosis, erythema nodosum, atrophic gastritis, glomerulonephritis, Goodpasture syndrome, gout, Graves' disease, Hashimoto Ko Thyroiditis, hypereosinophilia, irritable bowel syndrome, multiple sclerosis, myasthenia gravis, myocardial or pericardial inflammation, osteoarthritis, osteoporosis, pancreatitis, polymyositis, psoriasis, Reiter syndrome, Rheumatoid arthritis, scleroderma, Sjogren's syndrome, systemic anaphylaxis, systemic lupus erythematosus, systemic sclerosis, thrombocytopenia, ulcerative colitis, Werner syndrome, cancer complications, hemodialysis, extracorporeal circulation, virus Includes infections, bacterial infections, fungal infections, parasitic infections, protozoal infections, helminth infections, trauma, and also adenoviruses, arenaviruses, bunyaviruses, calciviruses, coronaviruses, filoviruses, hepa Donna virus, herpes virus, flavivirus, orthomyxovirus, parvovirus, papovavirus, paramyxovirus, picol Infections caused by viral mediators classified as viruses, poxviruses, reoviruses, retroviruses, rhabdoviruses, or togaviruses, pneumococcal infections, staphylococci, streptococci, bacillus, corynebacterium, clostridium, marrow Neisseria meningitidis, Neisseria gonorrhoeae, Listeria, Moraxella, Kingella, Hemophilus, Legionella, Bordetella, Gram-negative enterobacteria (including Shigella, Salmonella, Campylobacter), Pseudomonas, Vibrio, Brucella, Wild boar, Yersinia, Bartonella, norcardium, Infectious diseases caused by bacterial vectors classified as actinomycetes, mycobacteria, spirochaetale, rickettsia, chlamydia, mycoplasma, aspergillus, blast myces, dermatophytes, cryptococcus, coccidioides, m Infections caused by fungi classified as alasezzia, histoplasma, or other fungal pathogens, and plasmodim (parasites that cause malaria), parasitic amoeba, leishmania, trypanosoma, toxoplasma, pneumocystis carini, Intestinal protozoa such as Giardia, Intrinsic nematodes such as Trichomonas, Trichinella, Intestinal nematodes such as roundworm, Lymphatic filarial nematodes, Insects such as Schistosoma and tapeworms Included are infections caused by parasites classified as (Polypodidae). Polynucleotide sequences encoding NAAP can be detected using Southern or Northern methods, dot blotting, or other membrane-based techniques, PCR methods, which use body fluids or tissues collected from patients to detect altered NAAP expression. And can be used for dipstick, pin, and multi-format ELISA-based assays, and microarrays. Such qualitative or quantitative methods are known in the art.

  In certain embodiments, nucleotide sequences encoding NAAP may be useful in assays that detect related disorders, particularly those mentioned above. Nucleotide sequences encoding NAAP can be labeled by standard methods and added to body fluids or tissue samples taken from patients under conditions suitable for the formation of hybridization complexes. After a suitable incubation period, the sample is washed and the signal is quantified and compared to a standard value. If the amount of signal in the patient sample is significantly changed compared to the control sample, there is an associated disorder if the level of nucleotide sequences encoding NAAP is changed in the sample. Indicates. Such assays can also be used to evaluate specific therapeutic effects in animal experiments, clinical trials, or to monitor individual patient treatment.

In order to provide a basis for diagnosis of diseases associated with NAAP expression, a normal or standard profile of expression is established. This is achieved by mixing a body fluid or cell extract collected from a normal animal or human subject with a sequence encoding NAAP or a fragment thereof under conditions suitable for hybridization or amplification. Can be achieved. Standard hybridization can be quantified by comparing values obtained from experiments conducted with known amounts of substantially purified polynucleotides to values obtained from normal subjects. The standard value thus obtained can be compared with the value obtained from a sample obtained from a patient showing signs of disease. Deviation from standard values is used to determine the presence of the disease.
Once the presence of the disease is established and the treatment protocol is initiated, the hybridization assay can be repeated periodically to determine whether the patient's expression level has begun to approach that observed by normal subjects. The results obtained from continuous assays can be used to show the effect of treatment over a period of days to months.

  For cancer, the presence of an abnormal amount of transcripts (underexpressed or overexpressed) in living tissue from an individual indicates a predisposition to the disease or a method to detect the disease before clinical symptoms appear. Can be provided. This type of clearer diagnosis allows medical professionals to take advantage of preventive or aggressive treatment early on, thereby preventing the development or further progression of cancer.

Further diagnostic uses of oligonucleotides designed from NAAP-encoding sequences can include the use of PCR. These oligomers can be synthesized chemically, produced enzymatically, or produced in vitro . Oligomers preferably contain a fragment of a polynucleotide encoding NAAP, or a fragment of a polynucleotide complementary to a polynucleotide encoding NAAP, and are used to identify specific genes and conditions under optimized conditions. Is done. Oligomers can also be used for the detection, quantification, or both of closely related DNA or RNA sequences under moderately stringent conditions.

  In some embodiments, oligonucleotide primers derived from NAAP-encoding polynucleotide sequences can be used to detect single nucleotide polymorphisms (SNPs). SNPs are substitutions, insertions and deletions that often cause human congenital or acquired genetic diseases. Although not limited, SNP detection methods include single-stranded conformation polymorphism (SSCP) and fluorescent SSCP (fSSCP). In SSCP, DNA is amplified using oligonucleotide primers derived from a polynucleotide sequence group encoding NAAP and a polymerase chain reaction (PCR). This DNA can be derived from, for example, diseased or normal tissue, biopsy samples, body fluids, and the like. SNPs in DNA make a difference in the secondary and tertiary structure of single-stranded PCR products. Differences can be detected using gel electrophoresis in non-denaturing gels. In fSCCP, the oligonucleotide primer is fluorescently labeled. As a result, the amplimer can be detected by a high-throughput device such as a DNA sequencing machine. In addition, a sequence database analysis method called in silico SNP (in silico SNP, isSNP) identifies polymorphisms by comparing the sequences of individual overlapping DNA fragments as assembled into a common consensus sequence. Can do. These computer-based methods filter out sequence variations due to sequencing errors using the laboratory preparation of DNA and automated analysis of statistical models and DNA sequence chromatograms. In another embodiment, SNPs are detected and characterized, for example, by mass spectrometry using a high throughput MASSARRAY system (Sequenom, Inc., San Diego CA).

  SNPs can be used to study the genetic basis of human disease. For example, at least 16 common SNPs are associated with non-insulin dependent diabetes mellitus. SNPs are also useful for studying differences in outcomes of single gene diseases such as cystic fibrosis, sickle cell anemia, and chronic granulomatous disease. For example, a variant on mannose-binding lectin (MBL2) has been shown to correlate with adverse outcomes in cystic fibrosis lungs. SNPs also have utility in the pharmacogenomics of identifying genetic variants that affect patient responses to drugs such as life-threatening toxicities. For example, some mutations in N-acetyltransferase increase the incidence of peripheral neuropathy in response to the antituberculosis agent isoniazid, while mutations in the core promoter of the ALOX5 gene are anti-asthma drugs that target the 5-lipoxygenase pathway. Reduces clinical response to treatment. Analyzing the distribution of SNPs in different populations is useful for studying genetic drift, mutation, recombination and selection, as well as investigating the origin and migration of populations (Taylor, JG et al. (2001) Trends Mol. Med. 7: 507-512, Kwok, P.-Y and Z. Gu (1999) Mol. Med. Today 5: 538-543, Nowotny, P. et al. (2001) Curr. Opin. Neurobiol. 11 : 637-641).

  Methods that can be used to quantify NAAP expression include radiolabeling or biotin labeling of nucleotides, coamplification of control nucleic acids, and interpolation of results obtained from standard curves (eg, Melby, PC et al. ( 1993) J. Immunol. Methods 159: 235-244; Duplaa, C. et al. (1993) Anal. Biochem. 212: 229-236). Accelerate the quantification rate of multiple samples by performing high-throughput assays where the oligomer of interest is present in various dilutions and the quantification is rapid by spectrophotometric or colorimetric reactions .

  In yet another example, oligonucleotides or longer fragments derived from any of the polynucleotide sequences described herein can be used as elements in a microarray. Microarrays can be used in transcriptional imaging techniques that simultaneously monitor the relative expression levels of multiple genes. This is described below. Microarrays can also be used to identify genetic variants, mutations and polymorphisms. Use this information to determine gene function, understand the genetic basis of the disease, diagnose the disease, monitor disease progression / regression as a function of gene expression, and develop drug activity in disease treatment And can be monitored. In particular, this information can be used to develop a pharmacogenomic profile of the patient to select the most appropriate and effective treatment for the patient. For example, based on a patient's pharmacogenomic profile, a therapeutic agent that is highly effective against the patient and that exhibits few side effects can be selected.

  In another example, NAAP, a fragment of NAAP, or an antibody specific for NAAP can be used as an element on the microarray. Microarrays can be used to monitor or measure protein-protein interactions, drug-target interactions and gene expression profiles as described above.

  Certain embodiments relate to the use of the polynucleotides of the invention to produce a transcription image of a tissue or cell type. A transcript image represents a global pattern of gene expression by a particular tissue or cell type. Comprehensive gene expression patterns can be analyzed by quantifying the number and relative abundance of genes expressed at a given time under given conditions (Seilhamer et al. US Pat. No. 5,840,484 “Comparative Gene Transcript Analysis”). Which patent is hereby incorporated by reference). Thus, a transcript image can be generated by hybridizing a polynucleotide of the present invention or its complement to a whole tissue or cell type transcript or reverse transcript. In certain embodiments, hybridization is generated in a high throughput format such that a polynucleotide of the invention or its complement comprises a plurality of subsets of elements on a microarray. The resulting transcript image can provide a profile of gene activity.

Transcript images can be generated using transcripts isolated from tissues, cell lines, biopsies or biological samples thereof. The transcript image thus reflects gene expression in vivo for tissue or biopsy samples and in vitro for cell lines.

Transcript images that produce the expression profiles of polynucleotides of the invention can also be used in in vitro model systems and preclinical evaluation of drugs, or in connection with toxicity tests of industrial or natural environmental compounds. All compounds elicit characteristic gene expression patterns, often referred to as molecular fingerprints or toxicant signatures, that exhibit mechanisms of action and toxicity (Nuwaysir, EF et al. (1999) Mol. Carcinog. 24 : 15 3-159, Steiner, S. and NL Anderson (2000) Toxicol. Lett. 112-113: 467-471, which is hereby specifically incorporated by reference). If a test compound has a signature similar to that of a compound with known toxicity, it may share toxic properties. A fingerprint or signature is most useful and accurate when it contains expression information from multiple genes and gene families. Ideally, measurement of expression across the genome provides the highest quality signature. Even if there are genes whose expression is not altered by any tested compound, those genes are important because their expression levels can be used to normalize the remaining expression data. The normalize procedure is useful for comparing expression data after treatment with various compounds. Assigning gene function to elements of a toxic signature helps to interpret toxic mechanisms, but knowledge of gene function is not required for statistical matching of signature groups leading to toxicity prediction (eg, February 2000 29 Please refer to Press Release 00-02 published by the National Institute of Environmental Health Sciences on the day of this: http://www.niehs.nih.gov / oc / news / available at toxchip.htm). Therefore, it is important and desirable to include all expressed gene sequences in toxicological screening using toxicity signatures.

  In one embodiment, the toxicity of a test compound is calculated by treating a biological sample with nucleic acid with the test compound. Nucleic acid expressed in the treated biological sample can be hybridized with one or more probes specific for the polynucleotide of the invention, thereby quantifying the transcript level corresponding to the polynucleotide of the invention. The transcription level in the treated biological sample is compared to the level in the untreated biological sample. Differences in transcript levels between the two samples indicate a toxic response caused by the test compound in the treated sample.

  Another embodiment relates to analyzing the proteome of a tissue or cell type using the polypeptide sequences of the present invention. The term proteome refers to the global pattern of protein expression in a particular tissue or cell type. Each protein component of the proteome can be individually subject to further analysis. Proteome expression patterns or profiles can be analyzed by quantifying the number and relative abundance of proteins expressed at a given time under given conditions. Thus, a proteomic profile of a cell can be generated by isolating and analyzing polypeptides of a particular tissue or cell type. In some embodiments, the separation is achieved by two-dimensional gel electrophoresis in which the protein is separated from the sample by one-dimensional isoelectric focusing and separated according to molecular weight by two-dimensional sodium dodecyl sulfate slab gel electrophoresis. (Steiner and Anderson, above). Proteins are visualized in the gel as dispersed, unique points by staining the gel with a substance such as Coomassie Blue, or silver stain or fluorescent stain. The optical density of each protein spot is usually proportional to the protein level in the sample. The optical density of equally located protein spots from different samples, eg, biological samples either treated or untreated with the test compound or therapeutic agent, are compared to identify changes in protein spot density associated with the treatment. Proteins within the spot are partially sequenced using standard methods using, for example, chemical or enzymatic cleavage followed by mass spectrometry. The identity of a protein within a spot can be determined by comparing its subsequence, preferably at least 5 consecutive amino acid residues, to the polypeptide sequence of the invention. In some cases, additional sequence data is obtained for definitive protein identification.

  Proteomic profiles can also be generated by quantifying NAAP expression levels using antibodies specific for NAAP. In one embodiment, protein expression levels are quantified by using antibodies as elements on the microarray and exposing the microarray to the sample to detect the level of protein binding to each array element (Lueking, A. et al. (1999) Anal Biochern. 270: 103-111, Mendoze, LG et al. (1999) Biotechniques 27: 778-788). Detection can be performed in various ways known in the art, for example, a thiol-reactive or amino-reactive fluorescent compound can be reacted with a protein in the sample to detect the amount of fluorescent binding in each array element.

  Toxicity signatures at the proteome level are also useful for toxicological screening and should be analyzed in parallel with toxicity signatures at the transcriptional level. For some proteins in some tissues, there is a poor correlation between transcript abundance and protein abundance (Anderson, NL and J. Seilhamer (1997) Electrophoresis 18: 533-537). Proteome toxicity signatures may be useful in the analysis of compounds that do not affect so much but alter the proteome profile. Furthermore, since analysis of transcripts in body fluids is difficult due to rapid degradation of mRNA, proteome profiling can be more reliable and informative in such cases.

  In another example, the toxicity of a test compound is calculated by treating a biological sample containing the protein with the test compound. Proteins 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 the untreated biological sample. The difference in protein content of both samples indicates 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 subsequences to the polypeptides of the invention.

  In another example, the toxicity of a test compound is calculated by treating a biological sample containing the protein with the test compound. The protein obtained from the biological sample is incubated with an antibody specific for the polypeptide of the present invention. The amount of protein recognized by the antibody is quantified. The amount of protein in the treated biological sample is compared to the amount of protein in the untreated biological sample. The difference in protein content of both samples indicates a toxic response to the test compound in the treated sample.

Microarrays are prepared, used and analyzed using methods known in the art (Brennan, TM et al. (1995), US Pat. No. 5,474,796, Schena, M. et al. (1996) Proc. Natl. Acad. Sci. USA 93: 10614-10619, Baldeschweiler et al. (1995) PCT application WO95 / 251116, Shalon, D. et al. (1995) PCT application WO95 / 35505, Heller, RA et al. (1997) Proc. Natl. Acad. Sci. USA 94: 2150-2155, Heller, MJ et al. (1997) US Pat. No. 5,605,662 etc.). Various types of microarrays are known and described in detail in DNA Microarrays: A Practical Approach , M. Schena, edited (1999) Oxford University Press, London. This document is specifically incorporated herein by reference.

  In another embodiment of the invention, a group of nucleic acid sequences encoding NAAP may also be used to produce a group of hybridization probes useful for mapping native genomic sequences. Either a coding sequence or a non-coding sequence can be used, and in some cases a non-coding sequence is preferred over a coding sequence. For example, conservation of coding sequences within members of a multigene family can result in unwanted cross-hybridization during chromosomal mapping. The sequence may be on a particular chromosome, or on a particular region of a chromosome, or an artificially formed chromosome such as a human artificial chromosome (HAC), a yeast artificial chromosome (YAC), a bacterial artificial chromosome (BAC), Bacterial P1 product, or mapped to single chromosome cDNA libraries (eg Harrington, JJ et al. (1997) Nat. Genet. 15: 345-355, Price, CM (1993) Blood Rev. 7: 127-134 Trask, BJ (1991) Trends Genet. 7: 149-154). Once mapped, a genetic linkage map can be developed using the nucleic acid sequences of the present invention, eg, to correlate the inheritance of a disease state with the inheritance of a particular chromosomal region or with restriction enzyme fragment length polymorphism (RFLP) ( See, for example, Lander, ES and D. Botstein (1986) Proc. Natl. Acad. Sci. USA 83: 7353-7357).

Fluorescence in situ hybridization (FISH) can be correlated with other physical and genetic map data (see, for example, Heinz-Ulrich, et al. (1995) in Meyers, 965-968, supra). Examples of genetic map data can be found in various scientific journals or the Online Mendelian Inheritance in Man (OMIM) website. The correlation between the location of the gene encoding NAAP on a physical chromosomal map and a particular disease or a predisposition to a particular disease can help determine the region of DNA associated with this disease Can facilitate the cloning task of determining the position.

  The genetic map can be extended using physical mapping techniques such as linkage analysis using established chromosomal markers and chromosomal specimen in situ hybridization. By placing a gene on the chromosome of another mammal, such as a mouse, the associated markers can often be revealed even if the exact chromosomal locus is unknown. This information is valuable to researchers looking for disease genes using positional cloning or other gene discovery techniques. Once a gene (s) involved in a disease or syndrome is roughly positioned by genetic linkage to a specific genomic region, such as the 11q22-23 region of vasodilatory ataxia, it is mapped to that region Any sequence may represent a related or regulatory gene for further investigation (see, eg, Gatti, RA et al. (1988) Nature 336: 577580). The nucleotide sequence of the present invention can also be used for detecting a difference in chromosomal location among the healthy person, the owner, and the affected person due to translocation, inversion, and the like.

  In another embodiment of the invention, NAAP, catalytic fragments or immunogenic fragments or oligopeptides thereof may be used for screening a library of compounds in any of a variety of drug screening techniques. Fragments used for drug screening can be free in solution, fixed to a solid support, retained on the cell surface, or located intracellularly. Complex formation due to binding of NAAP to the drug being tested can also be measured.

  Another drug screening method is used to screen compounds with suitable binding affinity for the protein of interest with high throughput (see, eg, Geysen, et al. (1984) PCT application WO84 / 03564). In this method, a large number of different small molecule test compounds are synthesized on a solid substrate. The test compound is washed after reacting with NAAP or a fragment thereof. The bound NAAP is then detected by methods well known in the art. Purified NAAP can also be coated directly onto plates used in the drug screening techniques described above. Alternatively, non-neutralizing antibodies can be used to capture the peptide and immobilize the peptide on a solid support.

  In another example, a competitive drug screening assay can be used in which neutralizing antibodies capable of specific binding to NAAP compete with test compounds for binding to NAAP. In this way, antibodies can be used to detect the presence of any peptide that shares one or more antigenic determinants with NAAP.

  In another embodiment, the NAAP-encoding nucleotide sequence is a molecular biology technique that will be developed in the future, and features of the currently known nucleotide sequence (including but not limited to triplet genetic code, specific base pairing) It can be used for new technologies that depend on (including interactions, etc.).

  Without further details, those skilled in the art will be able to make full use of the present invention with the above description. Accordingly, the examples described below are for illustrative purposes only and do not limit the invention in any way.

  Reference to all patent applications, patents and publications mentioned above and below, especially U.S. Patent Application Nos. 60 / 288.598, 60 / 291.776, 60 / 292.172, and 60 / 293.564 This is a part of this specification.

1 Preparation of cDNA library
The Incyte cDNA group is derived from the cDNA library group described in the LIFESEQ GOLD database (Incyte Genomics, Palo Alto CA). Some tissues were homogenized and dissolved in guanidinium isothiocyanate solution, and other tissues were homogenized and dissolved in phenol or a suitable mixture of denaturants. TRIZOL (Life Technologies), which is an example of a mixed solution, is a single-phase solution of phenol and guanidine isothiocyanate. The resulting lysate was layered on a cesium chloride cushion solution and centrifuged or extracted with chloroform. RNA was precipitated from the lysate using either isopropanol, sodium acetate and ethanol, or another method.

  In order to increase the purity of RNA, extraction and precipitation of RNA with phenol was repeated as many times as necessary. In some cases, RNA was treated with DNase. In most libraries, poly (A +) RNA was isolated using oligo d (T) -linked paramagnetic particles (Promega), OLIGOTEX latex particles (QIAGEN, Chatsworth CA) or OLIGOTEX mRNA purification kit (QIAGEN). Alternatively, RNA was isolated directly from tissue lysates using another RNA isolation kit such as POLY (A) PURE mRNA purification kit (Ambion, Austin TX).

  In some cases, RNA was provided to Stratagene and Stratagene produced the corresponding cDNA library. If not, cDNA was synthesized using the UNIZAP vector system (Stratagene) or SUPERSCRIPT plasmid system (Life Technologies) using the recommended or similar methods known in the art to create a cDNA library (Ausubel, supra). , 1997, 5.1-6.6 units, etc.). Reverse transcription was initiated using oligo d (T) or random primers. A synthetic oligonucleotide adapter was ligated to double stranded cDNA and the cDNA was digested with a suitable restriction enzyme or group of enzymes. For most libraries, cDNA size selection (300-1000 bp) was performed using SEPHACRYL S1000, SEPHAROSE CL2B or SEPHAROSE CL4B column chromatography (Amersham Pharmacia Biotech) or preparative agarose gel electrophoresis. . The cDNA was ligated into compatible restriction enzyme sites of a suitable plasmid polylinker. Suitable plasmids include, for example, PBLUESCRIPT plasmid (Stratagene), PSPORT1 plasmid (Life Technologies), PCDNA2.1 plasmid (Invitrogen, Carlsbad CA), 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 containing Stratagene XL1-Blue, XL1-BIueMRF or SOLR, or Life Technologies DH5α, DH10B or ELECTROMAX DH10B.

2 Isolation of cDNA clones
The plasmid obtained as in Example 1 was recovered from the host cells by in vivo excision using the UNIZAP vector system (Stratagene) or by cell lysis. At least one of the following was used for purification of the plasmid. That is, Magic or WIZARD Minipreps DNA Purification System (Promega), AGTC Miniprep Purification Kit (Edge Biosystems, Gaithersburg MD), QIAWELL QIAWELL 8 Plasmid, QIAWELL 8 Plus Plasmid and QIAWELL 8 Ultra Plasmid Purification System, REAL Prep 96 Plasmid Purification Kit Either. The plasmid was precipitated and then resuspended in 0.1 ml distilled water and stored at 4 ° C. either lyophilized or not lyophilized.

  Alternatively, plasmid DNA was amplified from host cell lysates using direct binding PCR in a high throughput format (Rao, V.B. (1994) Anal. Biochem. 216: 1-14). Host cell lysis and thermal cycling processes were performed in a single reaction mixture. Samples are processed and stored in 384-well plates and the concentration of amplified plasmid DNA is determined fluorimetrically using PICOGREEN dye (Molecular Probes, Eugene OR) and FLUOROSKAN2 fluorescence scanner (Labsystems Oy, Helsinki, Finland) Quantified.

3 Sequencing and analysis
Incyte cDNA recovered from the plasmid as described in Example 2 was sequenced as shown below. Sequencing of cDNA can be performed using standard methods or high-throughput equipment such as ABI CATALYST 800 thermal cycler (Applied Biosystems) or PTC-200 thermal cycler (MJ Research) or HYDRA microdispenser (Robbins Scientific) or MICROLAB 2200 (Hamilton) liquid transfer. Processed in conjunction with the system. A cDNA sequencing reaction was prepared using a reagent provided by Amersham Pharmacia Biotech, or an ABI sequencing kit such as ABI PRISM BIGDYE Terminator cycle sequencing ready reaction kit (Applied Biosystems). For electrophoretic separation of cDNA sequencing reactions and detection of labeled polynucleotides, use the MEGABACE 1000 DNA sequencing system (Molecular Dynamics) or an ABI PRISM 373 or 377 sequence using standard ABI protocol and base calling software. Thing systems (Applied Biosystems) or other sequence analysis systems known in the art were used. Reading frames within the cDNA sequences were determined using standard methods (reviewed in Ausubel, 1997, 7.7 units supra). Several of the cDNA sequences were selected and the sequences were extended by the method described in Example 8 .

Polynucleotide sequences derived from Incyte cDNA sequences were validated by removing vector, linker and poly (A) sequences and masking ambiguous base pairs. In doing so, algorithms and programs based on BLAST, dynamic programming and adjacent dinucleotide frequency analysis were used. Secondly, Incyte cDNA sequences, or translations thereof, into public databases (eg GenBank primates and rodents, mammals, vertebrates, eukaryotes and BLOCKS, PRINTS, DOMO, PRODOM) and humans PROTEOME database (Incyte Genomics, Palo Alto CA) containing sequences from rat, mouse, nematode, budding yeast (Saccharomyces cerevisiae), fission yeast (Schizosaccharomyces pombe), and Candida albicans, and hidden markovs such as PFAM Protein family database based on model (HMM) and SMART (Schultz et al. (1998) Proc. Natl. Acad. Sci. USA 95: 5857-5864, Letunic, I. et al. (2002) Nucleic Acids Res. 30: 242- We queried against a database selected from HMM-based protein domain databases such as 244). (HMM is a probabilistic approach to analyzing the consensus primary structure of gene families. See, eg, Eddy, SR (1996) Cuff. Opin. Struct. Biol. 6: 361-365). Queries were made using programs based on BLAST, FASTA, BLIMPS, and HMMER. Incyte cDNA sequences were assembled to yield a full length polynucleotide sequence. Alternatively, the Incyte cDNA assembly can be extended to full length using GenBank cDNAs, GenBank ESTs, stitched sequences, stretched sequences, or Genscan predicted coding sequences (see Examples 4 and 5 ). I let you. Assembly was performed using a program based on Phred, Phrap and Consed, and a cDNA assembly was screened for open reading frames using a program based on GenMark, BLAST and FASTA. The full length polynucleotide sequence was translated to derive the corresponding full length polypeptide sequence. Alternatively, the polypeptides of the present invention can begin at any methionine residue of the full-length translated polypeptide. Subsequent queries for analysis of full-length polypeptide sequences include databases such as GenBank protein databases (genpept), SwissProt, PROTEOME databases, BLOCKS, PRINTS, DOMO, PRODOM and Prosite, PFAM, INCY, and TIGRFAM Hidden Markov Model (HMM) based protein family database group, and HMM based protein domain database group such as SMART. Full-length polynucleotide sequences are also analyzed using MACDNASIS PRO software (Hitachi Software Engineering, South San Francisco CA) and LASERGENE software (DNASTAR). Polynucleotide and polypeptide sequence alignments are generated using default parameters specified by the CLUSTAL algorithm, such as that incorporated into the MEGALIGN multi-sequence alignment program (DNASTAR), which also calculates the percent identity between aligned sequences.

  Table 7 gives an overview of the tools, programs and algorithms used for analysis and assembly of Incyte cDNA and full-length sequences, as well as applicable descriptions, references and threshold parameters. The tools, programs and algorithms used are shown in column 1 of Table 7 and a brief description thereof in column 2. Column 3 is a preferred reference, all of which are incorporated herein by reference in their entirety. Where applicable, column 4 indicates the score, probability value, and other parameters used to evaluate the strength with which the two sequences match (the higher the score or the lower the probability value, the distance between the two sequences). The homology is increased).

  The above programs used for the assembly and analysis of full-length polynucleotide sequences and polypeptide sequences were also used to identify the polynucleotide sequence fragments of SEQ ID NO: 24-46. A group of fragments of about 20 to about 4000 nucleotides useful for hybridization and amplification techniques is shown in column 2 of Table 4.

4. Identification and Editing of Coding Sequence from Genomic DNA Nucleic acid binding proteins for putative estimation were first identified by executing the Genscan gene identification program against public genome sequence databases (eg gbpri and gbhtg). Genscan is a universal gene identification program that analyzes genomic DNA sequences from various organisms (Burge, C and S. Karlin (1997) J. Mol. Biol. 268: 78-94, Burge, C and S. Karlin ( 1998) Cuff. Opin. Struct. Biol. 8: 346-354). The program links predicted exons to form an assembled cDNA sequence that spans from methionine to the stop codon. The output of Genscan is a FASTA database of polynucleotide and polypeptide sequences. The maximum range of sequences that Genscan analyzes at one time was set to 30 kb. To determine which of these Genscan predicted cDNA sequences encode nucleic acid binding proteins, the encoded polypeptides were analyzed by querying the PFAM model group for nucleic acid binding molecules. Potential nucleic acid binding proteins were also identified based on homology to Incyte cDNA sequences that were already annotated as nucleic acid binding proteins. The Genscan predicted sequences thus selected were then compared to the public databases genpept and gbpri by BLAST analysis. If necessary, Genscan predicted sequences were edited by comparison with the top BLAST hits 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 sequence, thus providing evidence of 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 construction process described in Example 3 . Alternatively, the full-length polynucleotide sequence is derived entirely from edited or unedited Genscan predicted coding sequences.

5 Assembly of genomic sequence data with cDNA sequence data
Stitched sequence
The partial cDNA sequence was extended using exons predicted by the Genscan gene identification program described in Example 4 . Partial cDNAs assembled as described in Example 3 were mapped to genomic DNA and resolved into clusters containing Genscan exons predicted from related cDNAs and one or more genomic sequences. Analyze each cluster using graph theory and dynamic programming-based algorithms to integrate cDNA and genomic information, and then generate potential splice variants that can be verified, edited or extended to yield full-length sequences did. A sequence segment group in which the total length of the segment is in two or more sequences was identified in the cluster, and the segment groups identified as such were considered to be equal due to transitivity. For example, if there is a section on one cDNA and two genomic sequences, all three sections were considered equal. This process can link unrelated but contiguous genomic sequences together by cDNA sequences. The sections thus identified are stitched together with a stitch algorithm in the order they appear along the parent sequence to create the longest possible sequence and variant sequence. Linkage between sections (cDNA-cDNA or genomic sequence-genomic sequence) generated along one type of parent sequence prevailed over linkage (cDNA-genomic sequence) that changes the type of parent. The resulting stitch sequences were translated and compared with public databases genpept and gbpri by BLAST analysis. The incorrect exon group predicted by Genscan was corrected by comparison with the top BLAST hits from genpept. If necessary, the sequences were further extended using additional cDNA sequences or by examination of genomic DNA.

Stretched sequence
The partial DNA sequence was extended to full length by an algorithm based on BLAST analysis. First, a BLAST program is used to query GenBank primates, rodents, mammals, vertebrates and eukaryotes databases such as the partial cDNA assembled as described in Example 3. It was. The closest GenBank protein homologue was then compared to either the Incyte cDNA sequence or the GenScan exon predicted sequence described in Example 4 by BLAST analysis. + The resulting high scoring segment pair (HSP) was used to produce a chimeric protein and the translated sequence was mapped onto the GenBank protein homologue. Insertions or deletions can occur in the chimeric protein relative to the original GenBank protein homologue. Homologous genomic sequences were searched from public human genome databases using GenBank protein homologues, chimeric proteins or both as probes. In this way, the partial DNA sequence was stretched or extended by the addition of homologous genomic sequences. The resulting stretch sequence was examined to determine whether it contained the complete gene.

6 Chromosome mapping of polynucleotides encoding NAAP
The sequences used to assemble SEQ ID NO: 24-46 were compared to sequences in the Incyte LIFESEQ database and the public domain database using BLAST and Smith-Waterman algorithms. These database sequences that matched SEQ ID NO: 24-46 were assembled into clusters of contiguous and overlapping sequences (Table 7) using an assembly algorithm such as Phrap (Table 7). Using the radiation hybrid and genetic map data available from public sources such as the Stanford Human Genome Center (SHGC), Whitehead Genome Research Institute (WIGR), Genethon, etc., any clustered sequences have already been Judged whether it was mapped. If the mapped sequence was contained in a cluster, the entire sequence of that cluster was assigned to a map location along with the individual SEQ ID numbers.

The location on the map is expressed as a range or section of the human chromosome. The location of a section of the map in centimorgan units is measured relative to the end of the p-arm of the chromosome (centiorgan (cM) is a unit of measure based on the frequency of recombination between chromosomal markers. 1 cM is roughly equivalent to 1 megabase (Mb) of DNA in humans, although this value varies widely with recombination hot and cold spots). The cM distance is based on a set of genetic markers mapped by Genethon that provide boundaries for radiation hybrid markers whose sequences are contained within each cluster. NCBI “GeneMap'99” (http://www.ncbi.nlm.nih.gov/genemap/) and other resources that have already been identified using resources such as human genome maps available to general individuals It can be determined whether it is mapped in or near the section.
7 Analysis of Polynucleotide Expression Northern analysis is an experimental technique used to detect the presence of transcribed genetic information, in which the labeled nucleotide sequence to the membrane to which RNA from a particular cell type or tissue is bound. Involved in hybridization. + (See, for example, Sambrook, Chapter 7 and Ausubel (1995) chapters 4 and 16 above).

  Using similar computer techniques applying BLAST, we searched for identical or related molecules in cDNA databases such as GenBank and LifeSeq (Incyte Genomics). Northern analysis is much faster than many membrane-based hybridizations. In addition, the sensitivity of computer searches can be modified to determine whether any particular match is classified as exact or homologous. The search criterion is a product score, which is defined by the following equation.

  The product score takes into account both the similarity between two sequences and the length with which the sequences match. The product score is a normalized value of 0 to 100, and is obtained as follows. Multiply the BLAST score by the nucleotide sequence identity and divide the product by 5 times the shorter length of the two sequences. To calculate the BLAST score, a score of +5 is assigned to each matching base in a high scoring segment pair (HSP) and -4 is assigned to each mismatched base. Two sequences can share more than one HSP (separated by gaps). If there are two or more HSPs, the product score is calculated using the segment pair with the highest BLAST score. The product score represents the balance between the fractional overlap and the quality of the BLAST alignment. For example, a product score of 100 is obtained only if there is a 100% match over the shorter length of the two sequences compared. The product score 70 is obtained when either one end is 100% coincident and 70% overlap, or the other end is 88% coincident and 100% overlap. A product score of 50 is obtained if either end is 100% coincident and 50% overlap, or 79% coincidence and 100% overlap.

Alternatively, the polynucleotide sequence encoding NAAP is analyzed against the tissue from which it was derived. For example, some full length sequences are assembled at least in part using overlapping Incyte cDNA sequences (see Example 3 ). Each cDNA sequence is derived from a cDNA library made from human tissue. Each human tissue is classified into one of the following organ / tissue categories. Cardiovascular system, connective tissue, digestive system, embryonic structure, endocrine system, exocrine gland, female reproductive organs, male reproductive organs, germ cells, blood and immune system, liver, musculoskeletal system, nervous system, pancreas, respiratory system, Sensory organs, skin, stomatognathic system, non-classified / mixed or urinary tract. Count the number of libraries in each category and divide by the total number of libraries in all categories. Similarly, each human tissue is classified into one of the following disease / condition categories: cancer, cell line, development, inflammation, nervousness, trauma, cardiovascular, pool, etc. Count the number of libraries in each category and divide by the total number of libraries in all categories. The resulting percentage reflects tissue-specific and disease-specific expression of the cDNA encoding NAAP. cDNA sequence and cDNA library / tissue information can be obtained from the LIFESEQ GOLD database (Incyte Genomics, Palo Alto CA).

8 Extended full-length polynucleotide sequences of polynucleotides encoding NAAP were also generated by extending appropriate fragments of full-length molecules using oligonucleotide primers designed from the fragments. One primer was synthesized to initiate a 5 ′ extension of a known fragment and another primer was synthesized to initiate a 3 ′ extension of a known fragment. The starting primer is about 22-30 nucleotides in length, has a GC content greater than about 50%, and is annealed to the target sequence at a temperature of about 68-72 ° C., OLIGO 4.06 software (National Biosciences) or another suitable Was designed from cDNA using a simple program. All nucleotide extensions that resulted in hairpin structures and primer-primer dimers were avoided.

  The sequence was extended using selected human cDNA libraries. Where more than one extension was necessary or desirable, additional primers or nested sets of primers were designed.

High fidelity amplification was obtained by PCR using methods well known to those skilled in the art. PCR was performed in 96 well plates using a PTC-200 thermal cycler (MJ Research, Inc.). The reaction mixture has template DNA and 200 nmol of each primer. In addition, a buffer containing Mg ²⁺ , (NH ₄ ) ₂ SO ₄ and 2-mercaptoethanol, Taq DNA polymerase (Amersham Pharmacia Biotech), ELONGASE enzyme (Life Technologies), Pfu DNA polymerase (Stratagene) is included. Amplification was performed on the primer set, PCI A and PCI B, with the following parameters. Step 1: 94 ° C, 3 minutes, Step 2: 94 ° C, 15 seconds, Step 3: 60 ° C, 1 minute, Step 4: 68 ° C, 2 minutes, Step 5: Repeat steps 2, 3, and 4 20 times . Step 6: Store at 68 ℃, 5 minutes, Step 7: Store at 4 ℃. Alternatively, amplification was performed on the primer set, T7 and SK +, with the following parameters: Step 1: 94 ° C, 3 minutes, Step 2: 94 ° C, 15 seconds, Step 3: 57 ° C, 1 minute, Step 4: 68 ° C, 2 minutes, Step 5: Repeat steps 2, 3, and 4 20 times . Step 6: Store at 68 ℃, 5 minutes, Step 7: Store at 4 ℃.

  The DNA concentration in each well is 1 x TE and 100 µl of PICOGREEN quantification reagent (0.25 (v / v) PICOGREEN; Molecular Probes, Eugene OR) dissolved in 0.5 µl of undiluted PCR product. It was distributed to each well of (Corning Costar, Acton MA) and measured so that DNA could bind to the reagent. Plates were scanned with Fluoroskan II (Labsystems Oy, Helsinki, Finland) to measure sample fluorescence and quantify DNA concentration. An aliquot of 5-10 μl of the reaction mixture was analyzed by electrophoresis on a 1% agarose gel to determine which reaction was successful in extending the sequence.

  Extended nucleotides are desalted and concentrated, transferred to a 384-well plate, digested with CviJI cholera virus endonuclease (Molecular Biology Research, Madison WI), sonicated or sheared, and pUC 18 vector (Amersham Pharmacia Biotech) Re-linked to. For shotgun sequencing, the digested nucleotides were separated on a low concentration (0.6-0.8%) agarose gel, the fragments were excised, and the agar was digested with Agar ACE (Promega). Elongated clones were religated to pUC 18 vector (Amersham Pharmacia Biotech) using T4 ligase (New England Biolabs, Beverly MA) and treated with Pfu DNA polymerase (Stratagene) to fill restriction site overhangs E. coli cells were transfected. Transfected cells were selected and transferred to a medium containing antibiotics, and each colony was excised and cultured overnight at 37 ° C. in a 384-well plate of LB / 2x carbenicillin culture.

  Cells were lysed and DNA was PCR amplified using Taq DNA polymerase (Amersham Pharmacia Biotech) and Pfu DNA polymerase (Stratagene) as follows. Step 1: 94 ° C, 3 minutes, Step 2: 94 ° C, 15 seconds, Step 3: 60 ° C, 1 minute, Step 4: 72 ° C, 2 minutes, Step 5: Repeat steps 2, 3, and 4 29 times . Step 6: Store at 72 ° C for 5 minutes, Step 7: Store at 4 ° C. DNA was quantified with PICOGREEN reagent (Molecular Probes) as described above. Samples with low DNA recovery were reamplified using the same conditions as above. Samples are diluted with 20% dimethyl sulfoxide (1: 2, v / v), and the DYENAMIC energy transfer sequencing primer and DYENAMIC DIRECT kit (Amersham Pharmacia Biotech) or ABI PRISM BIGDYE terminator cycle sequencing reaction kit (Terminator cycle sequencing ready) reaction kit) (Applied Biosystems).

  Similarly, full-length polynucleotide sequences were verified using the above procedure. Alternatively, 5 ′ regulatory sequences were obtained using full-length polynucleotides using the oligonucleotides designed for such extension and some appropriate genomic library in the above procedure.

9 Identification of Single Nucleotide Polymorphisms in Polynucleotides Encoding NAAP A common DNA sequence variant known as single nucleotide polymorphism (SNP) is SEQ ID NO: 24-46 using the LIFESEQ database (Incyte Genomics). Identified. As described in Example 3 , sequences from the same gene were clustered together to identify all the sequence variants within the gene. An algorithm consisting of a series of filters was used to distinguish SNPs from other sequence variants. The pre-filter eliminates the majority of base call errors by requiring a minimum Phred quality score of 15 and also eliminates errors caused by sequence alignment errors and improper trimming of vector sequences, chimeras and splice variants. Removed. The original chromatogram file in the vicinity of the putative SNP was analyzed by an automated procedure for advanced chromosome analysis. Clone error filters used statistically generated algorithms to identify errors introduced during the experimental process, such as those caused by reverse transcriptase, polymerase, or somatic mutation. The cluster error filter used statistically generated algorithms to identify errors due to clustering of closely related homologues or pseudogenes, or errors caused by contamination with non-human sequences. The last group of filters removed duplicates and SNPs present in immunoglobulins or T cell receptors.

  Several SNPs were selected for further characterization by mass spectrometry using a high-throughput MASSARRAY system (Sequenom, Inc.) to analyze allele frequencies at SNP sites in four different human populations. The white population consisted of 92 people (46 men and 46 women), including 83 Utah, 4 French, 3 Venezuela and 2 Amish. The African population consists of 194 African-Americans (97 men and 97 women). The Hispanic population is comprised of 324 Mexican Hispanics (162 men and 162 women). The Asian population consists of 126 people (64 men and 62 women), with a parent breakdown of 43% Chinese, 31% Japanese, 13% Korean, 5% Vietnamese, and 8% other Asians. Has been. The frequency of alleles was first analyzed in the white population, and in some cases, SNPs that did not show allelic variance in this population were not further examined in the other three populations.

10 Labeling and use of individual hybridization probes
A cDNA, mRNA, or genomic DNA is screened using a hybridization probe derived from SEQ ID NO: 24-46. Although specifically described for labeling oligonucleotides of about 20 base pairs, essentially the same procedure is used for larger nucleotide fragments. Oligonucleotides were designed using the latest software such as OLIGO 4.06 software (National Biosciences), each oligomer 50 pmol, [γ- ³² P] adenosine triphosphate (Amersham Pharmacia Biotech) 250 μCi, T4 polynucleotide kinase (DuPont NEN, Boston MA). The labeled oligonucleotide is substantially purified using a SEPHADEX G-25 ultrafine molecular size exclusion dextran bead column (Amersham Pharmacia Biotech). 10 ⁷ counts per minute in a typical membrane-based hybridization analysis of human genomic DNA digested with any one of Ase I, Bgl II, Eco RI, Pst I, Xba1 or Pvu II (DuPont NEN) An aliquot containing a labeled probe is used.

  DNA from each digest is fractionated on a 0.7% agarose gel and transferred to a nylon membrane (Nytran Plus, Schleicher & Schuell, Durham NH). Hybridization is performed at 40 ° C. for 16 hours. To remove non-specific signals, the blots are washed sequentially at room temperature, for example under conditions consistent with 0.1 × sodium citrate saline and 0.5% sodium dodecyl sulfate. Visualize and compare hybridization patterns using autoradiography or alternative imaging means.

11 Microarrays The concatenation or synthesis of array elements on a microarray is performed using photolithography, piezo printing (see inkjet printing, Baldeschweiler, etc.), mechanical microspotting techniques, and techniques derived therefrom. Can be achieved. In each of the above technologies, the substrate should be a solid with a uniform, non-porous surface (Schena (1999) supra). Recommended substrates include silicon, silica, glass slides, glass chips and silicon wafers. Alternatively, a procedure similar to dot blot or slot blot may be utilized to place and bind elements to the surface of the substrate using thermal, ultraviolet, chemical or mechanical bonding procedures. Conventional arrays can be made using available methods and machinery known to those skilled in the art and can have any suitable number of elements (eg, 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 etc.).

  Full-length cDNAs, expressed sequence tags (ESTs), or fragments or oligomers thereof can be elements of a microarray. Fragments or oligomers suitable for hybridization can be selected using software known in the art such as LASERGENE software (DNASTAR). The array element group is hybridized with the polynucleotide group in the biological sample. The polynucleotide in the biological sample is conjugated to a molecular tag such as a fluorescent label to facilitate detection. After hybridization, unhybridized nucleotides from the biological sample are removed and hybridization at each array element is detected using a fluorescent scanner. Alternatively, hybridization can also be detected using laser desorption and mass spectrometry. The degree of complementarity and relative abundance of each polynucleotide that hybridizes to an element on the microarray can be calculated. The preparation and use of the microarray in one embodiment is detailed below.

Tissue or Cell Sample Preparation Total RNA is isolated from tissue samples using the guanidinium thiocyanate method and poly (A) ⁺ RNA is purified using the oligo (dT) cellulose method. Each poly (A) ⁺ RNA sample was treated with MMLV reverse transcriptase, 0.05 pg / μl oligo (dT) primer (21mer), 1 × first strand synthesis buffer, 0.03 unit / μl RNase inhibitor, 500 μM dATP Reverse transcription using 500 μM dGTP, 500 μM dTTP, 40 μM dCTP, 40 μM dCTP-Cy3 (BDS) or dCTP-Cy5 (Amersham Pharmacia Biotech). The reverse transcription reaction is performed using a GEMBRIGHT kit (Incyte) in a volume of 25 ml containing 200 ng poly (A) ⁺ RNA. Specific control poly (A) ⁺ RNA is synthesized from non-coding yeast genomic DNA by in vitro transcription. After incubation at 37 ° C for 2 hours, each reaction sample (one Cy3 and the other Cy5 labeled) was treated with 2.5 ml of 0.5M sodium hydroxide and incubated at 85 ° C for 20 minutes to react. To break down RNA. Samples are purified using two consecutive CHROMA SPIN 30 gel filtration spin columns (CLONTECH Laboratories, Inc. (CLONTECH), Palo Alto CA). After mixing, the two reaction samples are ethanol precipitated with 1 ml glycogen (1 mg / ml), 60 ml sodium acetate and 300 ml 100% ethanol. The sample is then completely dried using SpeedVAC (Savant Instruments Inc., Holbrook NY) and resuspended in 14 μl of 5 × SSC / 0.2% SDS.

Preparation of microarrays Array elements are made using the sequences of the present invention. Each array element is amplified from a vector-containing bacterial cell with a cloned cDNA insert. PCR amplification uses primers complementary to the vector sequence located on the side of the cDNA insert. Array elements are amplified from an initial amount of 1-2 ng to a final amount greater than 5 μg in 30 cycles of PCR. The amplified array element is purified using SEPHACRYL-400 (Amersham Pharmacia Biotech).

  The purified array element is fixed on a polymer-coated slide glass. Microscope slides (Corning) are ultrasonically washed in 0.1% SDS and acetone and thoroughly washed with distilled water during and after treatment. The slides were etched in 4% hydrofluoric acid (VWR Scientific Products Corporation (VWR), West Chester PA), washed thoroughly in distilled water, and 0.05% aminopropylsilane (Sigma) in 95% ethanol. ) To coat. Coated slides are cured in an oven at 110 ° C.

  Array elements are added to the coated glass substrate using the method described in US Pat. No. 5,807,522. This patent is hereby incorporated by reference. 1 μl of array element DNA having an average concentration of 100 ng / μl is filled into an open capillary printing element by a high-speed machine. The instrument now adds approximately 5 nl of array element samples per slide.

  The microarray is UV crosslinked using a STRATALINKER UV crosslinking agent (Stratagene). The microarray is washed once with 0.2% SDS at room temperature and three times with distilled water. After incubating the microarray for 30 minutes at 60 ° C. in 0.2% casein in phosphate buffered saline (PBS) (Tropix, Inc., Bedford MA), 0.2% SDS and Nonspecific binding sites are blocked by washing with distilled water.

The hybridization hybridization reaction solution contains 9 μl of a sample mixture containing 0.2 μg of each of the Cy3 and Cy5 labeled cDNA synthesis products in 5 × SSC, 0.2% SDS hybridization buffer. The sample mixture is heated to 65 ° C. for 5 minutes, aliquoted on the microarray surface and covered with a 1.8 cm ² cover glass. The array is transferred to a waterproof chamber with a cavity slightly larger than the microscope slide. To keep the chamber at 100 humidity, add 140 μl of 5 × SSC to one corner of the chamber. The chamber with the array is incubated at 60 ° C. for about 6.5 hours. The array is washed in the first wash buffer (1 × SSC, 0.1% SDS) for 10 minutes at 45 ° C. and in the second wash buffer (0.1 × SSC) for 10 minutes at 45 ° C. 3 Wash once and dry.

To detect the detection reporter labeled hybridization complex, an Innova 70 mixed gas 10 W laser (Coherent, Inc., Santa Clara CA) capable of generating spectral lines at 488 nm for Cy3 excitation and 632 nm for Cy5 excitation. ) Is used. A 20 × microscope objective (Nikon, Inc., Melville NY) is used to focus the excitation laser light on the array. The slide containing the array is placed on a microscope, computer-controlled XY stage and raster scanned through the objective lens. The 1.8 cm × 1.8 cm array used in this example scans with a resolution of 20 μm.

  In two different scans, the mixed gas multiline laser sequentially excites the two fluorophores. The emitted light is separated based on wavelength and sent to two photomultiplier detectors (PMT R1477, Hamamatsu Photonics Systems, Bridgewater NJ) corresponding to the two fluorophores. A suitable filter group is placed between the array and the photomultiplier tube to filter the signal. The maximum emission wavelength of the fluorophore used is 565 nm for Cy3 and 650 nm for Cy5. The instrument can record spectra from both fluorophores simultaneously, but with a suitable filter in the laser source, it scans once for each fluorophore and typically scans each array twice.

  The sensitivity of the scan is usually calibrated using the signal intensity generated by the cDNA control species added to a known concentration of the sample mixture. A specific location on the array contains a complementary DNA sequence, and the intensity of the signal at that location is correlated to a weight ratio of hybridizing species of 1: 100,000. When two samples from different sources (such as cells to be tested and control cells) are each labeled with a different fluorescent dye and hybridized to a single array to identify genes that are differentially expressed The calibration is performed by labeling the sample of cDNA to be calibrated with two fluorescent dyes and adding equal amounts each to the hybridization mixture.

  The photomultiplier tube output is digitized using a 12-bit RTI-835H analog-to-digital (A / D) conversion board (Analog Devices, Inc., Norwood MA) installed on an IBM compatible PC computer. The digitized data is displayed as an image in which the signal intensity is mapped using a linear 20 color conversion from a blue (low signal) to red (high signal) pseudo color scale. Data is also analyzed quantitatively. When exciting and measuring two different fluorophores simultaneously, using the emission spectra of each fluorophore, the data is first corrected for optical crosstalk between the fluorophores (due to overlapping emission spectra).

  A grid is overlaid on the fluorescent signal image, whereby the signal from each spot is collected on each element of the grid. The fluorescence signals within each element are integrated to give a numerical value depending on the average intensity of the signal. The software used for signal analysis is the GEMTOOLS gene expression analysis program (Incyte).

Expression For example, steroid-treated liver cell lines were found to down-regulate the expression of SEQ ID NO: 35 relative to untreated liver cell line controls. Hepatoblastoma cells were obtained from a liver cancer patient (15 years old, male) and used to obtain a cell line expressing insulin receptor and insulin-like growth factor II receptor. Samples were treated with steroids such as progesterone, betamethasone, dexamethasone, prednisone, budesonide, medroxyprogesterone, and beclomethasone. Thus, SEQ ID NO: 35 may be useful for diagnosis and treatment of autoimmune / inflammatory diseases.

  In a further example, SEQ ID NO: 41 showed differential expression in preadipocyte tissue treated with PPAR-gamma agonist and differentiation medium relative to untreated tissue, which was confirmed by microarray analysis. . Accordingly, SEQ ID NO: 41 is useful in the treatment of metabolic diseases such as diabetes. Primary subcutaneous preadipocytes were isolated from adipose tissue of a woman (40 years old) with a body mass index (BMI) of 32.47. Preadipocytes were cultured in a differentiation medium containing active ingredients PPAR-gamma and human insulin to induce differentiation into adipocytes, and then switched to a culture medium containing only insulin. Differentiated adipocytes were compared to untreated preadipocytes in culture medium without inducer.

12 Complementary polynucleotides
A sequence complementary to a sequence encoding NAAP or any portion thereof is used to detect, reduce or inhibit the expression of native NAAP. Although the use of oligonucleotides containing about 15-30 base pairs is described, essentially the same procedure is used for smaller or larger sequence fragments. Oligo 4.06 software (National Biosciences) and NAAP coding sequences are used to design appropriate oligonucleotide groups. To inhibit transcription, a complementary oligonucleotide is designed from the most unique 5 'sequence and used to prevent the promoter from binding to the coding sequence. To inhibit translation, a complementary oligonucleotide is designed to prevent the ribosome from binding to transcripts encoding NAAP.

13 NAAP expression
NAAP expression and purification can be performed using bacterial or viral based expression systems. To express ATRS in bacteria, the cDNA is subcloned into a suitable vector having an antibiotic resistance gene and an inducible promoter that increases the level of cDNA transcription. Such promoters include, but are not limited to, the T5 or T7 bacteriophage promoter in combination with the lac operator regulatory element and the trp-lac (tac) hybrid promoter. The recombinant vector is transformed into a suitable bacterial host such as BL21 (DE3). To express NAAP in antibiotic resistant bacteria, induce with isopropyl β-D thiogalactopyranoside (IPTG). NAAP expression in eukaryotic cells is carried out by infecting insect cell lines or mammalian cell lines with a recombinant form of Autographica californica nuclear polyhedrosis virus (AcMNPV), commonly known as baculovirus. To replace the baculovirus nonessential polyhedrin gene with cDNA encoding NAAP, either homologous recombination or bacterial-mediated gene transfer with transfer plasmid mediation is performed. The infectivity of the virus is maintained and a high level of cDNA transcription is performed by a strong polyhedrin promoter. Recombinant baculoviruses are often used to infect Spodoptera frugiperda (Sf9) insect cells, but may also be used to infect human hepatocytes. In the latter case, further genetic modification of the baculovirus is required. (See Engelhard. EK et al. (1994) Proc. Natl. Acad. Sci. USA 91: 3224-3227, Sandig, V. et al. (1996) Hum. Gene Ther. 7: 1937-1945.).

In most expression systems, NAAP becomes a fusion protein synthesized with, for example, glutathione S-transferase (GST) or a peptide epitope tag such as FLAG or 6-His, so recombinant fusion from unpurified cell lysates Protein affinity-based purification can be performed rapidly in one step. GST is a 26 kDa enzyme from Schistosoma japonicum that allows purification of the fusion protein on immobilized glutathione while maintaining protein activity and antigenicity (Amersham Pharmacia Biotech). After purification, the GST moiety can be proteolytically cleaved from NAAP at a specifically engineered site. FLAG is an 8-amino acid peptide that allows immunoaffinity purification using commercially available monoclonal and polyclonal anti-FLAG antibodies (Eastman Kodak). 6-His, in which 6 histidine residues are continuously extended, allows purification on metal chelate resins (QIAGEN). Methods for protein expression and purification are described in Ausubel (1995) chapters 10 and 16 above. NAAP purified by these methods can be used directly to perform the applicable assays of Examples 17, 18, and 19 below.

14 Functional assays
The function of NAAP is assessed by the expression of sequences encoding NAAP at physiologically elevated levels in mammalian cell culture systems. Subclone the cDNA into a mammalian expression vector with a strong promoter that expresses cDNA at high levels. Vectors selected include the pCMV SPORT plasmid (Life Technologies) and the pCR 3.1 plasmid (Invitrogen, Carlsbad CA), both having a cytomegalovirus promoter. Using a liposomal formulation or electroporation, 5-10 μg of the recombinant vector is transiently transfected into a human cell line, such as an endothelial or hematopoietic cell line. In addition, 1-2 μg of plasmid containing the sequence encoding the labeled protein is simultaneously transfected. The expression of the labeled protein provides a means to distinguish between transfected and non-transfected cells. Moreover, expression of the cDNA from the recombinant vector can be accurately predicted by the expression of the labeled protein. The labeled protein can be selected from, for example, green fluorescent protein (GFP; Clontech), CD64 or CD64-GFP fusion protein. Identify transfected cells that express GFP or CD64-GFP using flow cytometry (FCM), an automated, laser-optical technology, and evaluate the apoptotic status and other cellular properties of the cells To do. FCM detects and measures the uptake of fluorescent molecules that diagnose phenomena that precede or occur simultaneously with cell death. These phenomena include changes in nuclear DNA content as measured by DNA staining with propidium iodide, changes in cell size and particle size as measured by forward and 90 ° side scatter, bromodeoxyuridine. Down-regulation of DNA synthesis measured by a decrease in the uptake of protein, alteration of protein expression in the cell surface and in cells measured by reactivity with specific antibodies, and fluorescein-conjugated annexin V protein to the cell surface There is a change in the plasma membrane composition measured by binding. For flow cytometry, see Ormerod, MG (1994) Flow Cytometry , There is a description in Oxford, New York NY.

  The effect of NAAP on gene expression can be assessed using highly purified cell populations transfected with sequences encoding NAAP and either CD64 or CD64-GFP. CD64 or CD64-GFP is expressed on the transfected cell surface and binds to a conserved region of human immunoglobulin G (IgG). Transfected and non-transformed cells can be efficiently separated using magnetic beads coated with either human IgG or antibodies to CD64 (DYNAL, Lake Success NY). mRNA can be purified from cells by methods well known to those skilled in the art. Expression of mRNA encoding NAAP and other genes of interest can be analyzed by Northern analysis or microarray technology.

15 Production of NAAP-specific antibodies Using polyacrylamide gel electrophoresis (PAGE; see, for example, Harrington, MG (1990) Methods Enzymol. 182: 488-495) or other purified NAAP. And immunize animals (rabbits, mice, etc.) using standard protocols to produce antibodies.

  Alternatively, the NAAP amino acid sequence can be analyzed using LASERGENE software (DNASTAR) to determine highly immunogenic regions and the corresponding oligopeptides synthesized and used to generate antibodies in a manner well known to those skilled in the art. Produce. The selection of suitable epitopes, such as those near the C-terminus or in adjacent hydrophilic regions, is well known in the art (see, eg, Ausubel, 1995, chapter 11 above).

  Usually, an oligopeptide of about 15 residues in length is synthesized using an ABI 431A peptide synthesizer (Applied Biosystems) using Fmoc chemistry, and by a reaction using N-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS). Bind to KLH (Sigma-Aldrich, St. Louis MO) to increase immunogenicity (see Ausubel, 1995 et al. Above). Rabbits are immunized with oligopeptide-KLH conjugate in complete Freund's adjuvant. In order to test the anti-peptide activity and anti-NAAP activity of the obtained antiserum, the peptide or NAAP is bound to a substrate, blocked with 1% BSA, washed with a rabbit antiserum, washed, and further radioactive. React with iodine-labeled goat anti-rabbit IgG.

16 Purification of natural NAAP using specific antibodies In order to substantially purify natural NAAP or recombinant NAAP, immunoaffinity chromatography using a group of antibodies specific for NAAP is performed. The immunoaffinity column is formed by covalently binding an activation chromatography resin such as CNBr-activated SEPHAROSE (Amersham Pharmacia Biotech) and an anti-NAAP antibody. After binding, the resin is blocked and washed according to the manufacturer's instructions.

  The culture solution containing NAAP is passed through an immunoaffinity column, and the column is washed under conditions that allow preferential adsorption of NAAP (for example, with a buffer having a high ionic strength in the presence of a surfactant). The column is eluted under conditions that break the binding of the antibody to NAAP (eg, with some pH 2-3 buffer, or a high concentration of a chaotrope such as urea or thiocyanate ions), and the NAAP is collected. .

17 Identification of molecules that interact with NAAP
NAAP, or a biologically active fragment thereof, is labeled with ¹²⁵ I Bolton Hunter reagent (see, eg, Bolton AE and WM Hunter (1973) Biochem. J. 133: 529-539). Candidate molecules pre-arranged in each well of the multi-well plate are incubated with labeled NAAP, washed, and all wells with labeled NAAP complex are assayed. Data obtained at various NAAP concentrations are used to calculate values for the quantity, affinity, and association of NAAP bound to the candidate molecule.

  Alternatively, the molecule that interacts with NAAP may be selected from the yeast two-hybrid system described in Fields, S. and O. Song (1989, Nature 340: 245-246), or MATCHMAKER. Analyze using a commercial kit based on a 2-hybrid system such as the system (Clontech).

  NAAP is also used in the PATHCALLING process (CuraGen Corp., New Haven CT) using a high-throughput yeast two-hybrid system to determine all interactions between proteins encoded by two large libraries of genes. (Nandabalan, K. et al. (2000) US Pat. No. 6,057,101).

18 Demonstration of NAAP activity
NAAP activity is measured by its ability to stimulate transcription of reporter genes (Liu, HY et al. (1997) EMBO J. 16: 5289-5298). This assay uses LexA _op -LacZ, a well-characterized reporter gene construct. This LexA _op -LacZ consists of a LexA DNA transcriptional regulatory element (LexA _op ) fused to a sequence encoding the E. coli LacZ enzyme. Methods for producing and expressing fusion genes, introducing fusion genes into cells, and measuring LacZ enzyme activity are well known to those skilled in the art. The sequence encoding NAAP is cloned into a plasmid that directs the synthesis of LexA-NAAP, a fusion protein consisting of a DNA binding domain derived from a LexA transcription factor and NAAP. The resulting plasmid encoding the LexA-NAAP fusion protein is introduced into yeast cells along with a plasmid having a LexA _op -LacZ reporter gene. The amount of LacZ enzyme activity associated with LexA-NAAP transfected cells compared to control cells is proportional to the amount of transcription stimulated by NAAP.

  Alternatively, NAAP activity is measured by its ability to bind to zinc. 5-10 μM sample solution (in 2.5 mM ammonium acetate solution (pH 7.4)) in the presence of 0.05 M zinc sulfate solution (Aldrich, Milwaukee WI) and 100 μM dithiothreitol (with 10% methanol) Mix under. Incubate the sample with the zinc sulfate solution for 20 minutes. The reaction solution is passed through a VYDAC column (approximately 300 angstrom bore size, 5 μM particle size) (Grace Vydac, Hesperia CA). Thereby, the zinc-sample complex is isolated from the solution and then subjected to a mass spectrometer (PE Sciex, Ontario, Canada). A functional atomic weight of 63.5 Da is used to quantify the zinc bound to the sample (this atomic weight was observed by Whittal, R. M. et al. (2000) Biochemistry 39: 8406-8417).

Alternatively, one method of determining NAAP nucleic acid binding activity involves a polyacrylamide gel migration mobility shift assay. In preparing this assay, NAAP is expressed by transforming a mammalian cell line, such as COS7, HeLa, or CHO, using a eukaryotic expression vector carrying NAAP cDNA. Following transformation, the cells are incubated for 48-72 hours under conditions suitable for the cell line to express and accumulate NAAP. Preparation of an extract with solubilized protein can be performed from cells expressing NAAP. This method is well known in the art. Each portion of the extract with NAAP is added to [ ³² P] -labeled RNA or DNA. It is possible to synthesize radioactive nucleic acids in vitro by techniques known in the art. The mixture is incubated at 25 ° C. in the presence of RNase and DNA-degrading enzyme inhibitors under buffered conditions for 5-10 minutes. After incubation, the sample is analyzed by polyacrylamide gel electrophoresis. Then, it is analyzed by autoradiograph method. The presence of a band on the autoradiogram indicates the formation of a complex between NAAP and the radioactive transcript. Similar mobility bands will not be seen in samples prepared with control extracts prepared from non-transformed cells.

Alternatively, one method of determining methylase activity of NAAP measures the transfer of radiolabeled methyl groups between the donor and acceptor substrates. The reaction mixture (final volume of 50 [mu] l), 15 mM of HEPES (pH 7.9), 1.5 mM of MgCl _2, 10 mM dithiothreitol, 3% polyvinyl alcohol, 1.5MyuCi [methyl - ³ H] AdoMet (0.375 μM AdoMet) (DuPont-NEN), 0.6 μg NAAP, and receptor substrate (eg, 0.4 μg [ ³⁵ S] RNA or 6-mercaptopurine (6-MP), final concentration up to 1 mM). The reaction mixture is incubated at 30 ° C for 30 minutes and then at 65 ° C for 5 minutes.

[Methyl - ³ H] Analysis of RNA are as follows. (1) 50 μl 2 × loading buffer (20 mM Tris-HCl (pH 7.6), 1 M LiCl, 1 mM EDTA, 1% sodium dodecyl sulfate (SDS)) and 50 μl oligo d (T) − Cellulose (10 mg / ml in 1 × loading buffer) is added to the reaction mixture and incubation is performed for 30 minutes with shaking at room temperature. (2) Transfer the reaction mixture to a 96-well filtration plate (connected to a vacuum device). (3) Wash each sample sequentially. This uses three 2.4 ml aliquots of 1 × oligo d (T) loading buffer (with 0.5% SDS or 0.1% SDS and without SDS). (4) Elution of RNA is performed in a 96-well collection plate with 300 μl of water, transferred to a scintillation vial (containing liquid scintillant), and radioactivity is determined.

[Methyl - ³ H] of 6-MP analysis is as follows. (1) Add 500 μl of 0.5 M borate buffer (pH 10.0) and then 2.5 ml of isoamyl alcohol diluted in toluene with 20% (v / v) to the reaction mixture. (2) Mix the sample well by vortexing for 10 seconds. (3) After centrifugation for 10 minutes at 700 g, transfer 1.5 ml of the organic phase to a scintillation vial (containing 0.5 ml absolute ethanol and liquid scintillant) and determine the radioactivity. (4) Correct the results for extraction of 6-MP into the organic phase (about 41%).

Alternatively, NAAP type I topoisomerase activity assays are based on the relaxation of certain supercoiled DNA substrates. Incubation of NAAP with its substrate is performed in a buffer lacking Mg ²⁺ and ATP to terminate the reaction and load the product onto an agarose gel. Discrimination of the transformed topoisomer from the supercoil substrate can be done by electrophoresis. This assay is specific for type I topoisomerase activity. The reason is that Mg ²⁺ and ATP are cofactors required for type II topoisomerase.

  An assay for NAAP type II topoisomerase activity can be performed based on the decatenation of certain kinetoplast DNA (KDNA) substrates. Incubation of NAAP is performed with KDNA to terminate the reaction and load the product onto an agarose gel. Discrimination of monomer circular KDNA from linked KDNA can be performed by electrophoresis. A commercial kit for measuring type I topoisomerase activity and type II topoisomerase activity is obtained from Topogen (Columbus OH).

NAAP's ATP-dependent RNA helicase unwinding activity can be measured by the method described in Zhang and Grosse (1994; Biochemistry 33: 3906-3912). The substrate for RNA unwinding contains ³² P-labeled RNA, which consists of two RNA strands (194 and 130 nucleotides long) and has a 17 base pair duplex region. The RNA substrate is incubated with variable amounts of NAAP in ATP, Mg ²⁺ , Tris-HCl buffer (pH 7.5) for 30 minutes at 37 ° C. Next, the single-stranded RNA product is separated from the double-stranded RNA substrate by electrophoresis through a 10% SDS-polyacrylamide gel and quantified by autoradiographic method. The amount of single stranded RNA recovered is proportional to the amount of NAAP in the preparation.

  Alternatively, an assay for NAAP function is assessed by expression of a sequence encoding NAAP at a physiologically elevated level in a mammalian cell culture system. Subclone the cDNA into a mammalian expression vector with a strong promoter that expresses cDNA at high levels. Vectors selected include pCMV SPORT (Life Technologies) and pCR3.1 (Invitrogen Corporation, Carlsbad CA), both of which have a cytomegalovirus promoter. Using a liposomal formulation or electroporation, 5-10 μg of the recombinant vector is transiently transfected into a human cell line, preferably an endothelial or hematopoietic cell line. In addition, 1-2 μg of plasmid containing the sequence encoding the labeled protein is simultaneously transfected.

  The expression of the labeled protein provides a means to distinguish between transfected and non-transfected cells. Moreover, expression of the cDNA from the recombinant vector can be accurately predicted by the expression of the labeled protein. The labeled protein can be selected from, for example, green fluorescent protein (GFP; CLONTECH), CD64 or CD64-GFP fusion protein. Identify transfected cells expressing GFP or CD64-GFP using flow cytometry (FCM), an automated, laser-optical technology, and determine the apoptotic status and other cellular properties of those cells evaluate.

FCM detects and measures the uptake of fluorescent molecules that diagnose phenomena that precede or occur simultaneously with cell death. These phenomena include changes in nuclear DNA content as measured by DNA staining with propidium iodide, changes in cell size and particle size as measured by forward and 90 ° side scatter, bromodeoxyuridine. Down-regulation of DNA synthesis measured by a decrease in the uptake of protein, changes in cell surface and protein expression measured by reactivity with specific antibodies, and fluorescein-conjugated annexin V protein on the cell surface There is a change in the plasma membrane composition measured by binding. The flow cytometry method is described in Ormerod, MG (1994) Flow Cytometry , Oxford, New York NY.

  The effect of NAAP on gene expression can be assessed using highly purified cell populations transfected with sequences encoding NAAP and either CD64 or CD64-GFP. CD64 or CD64-GFP is expressed on the transfected cell surface and binds to a conserved region of human immunoglobulin G (IgG). Transfected and non-transfected cells can be efficiently separated using magnetic beads coated with either human IgG or antibodies to CD64 (DYNAL, Inc., Lake Success NY). mRNA can be purified from cells by methods well known to those skilled in the art. Expression of mRNA encoding NAAP and other genes of interest can be analyzed by Northern analysis or microarray technology.

NAAP pseudouridine synthase activity was assayed using a tritium ( ³ H) release assay adapted from Nurse et al. ((1995) RNA 1: 102112) to isomerize ³ H-radiolabeled U in RNA. The release of ³ H from the C ₅ position of the pyrimidine component of uridylic acid (U) is measured when it becomes pseudouridine (Ψ). A typical 500 μl assay mix consists of 50 mM HEPES buffer (pH 7.5), 100 mM ammonium acetate, 5 mM dithiothreitol, 1 mM EDTA, 30 units RNase inhibitor, and 0.1-4.2 μM. [5 ³ H] tRNA (about 1 μCi / nmol of tRNA). To initiate the reaction, add less than 5 μl of a concentrated solution of NAAP (or a sample containing NAAP) and incubate for 5 minutes at 37 ° C. After adding NAAP, a portion of the reaction mixture is collected at various elapsed times (up to 30 minutes) and diluted in 1 ml 0.1 M HCl containing NoritSA3 (12% w / v) to react. Stop. The stopped reaction mixture is centrifuged for 5 minutes in a microcentrifuge at maximum speed and the supernatant is filtered through a glass wool stuffing. The pellet is washed by resuspension twice in 1 ml 0.1 M HCl and then centrifuged. Each of the washed supernatants is passed separately through a glass wool stuffing and mixed with the original filtrate. A part of the mixed filtrate is mixed with scintillation liquid (maximum 10 ml) and counted with a scintillation counter. .. The amount of ³ H released from RNA, also the amount of ³ H present in the soluble filtrate is proportional to the amount of pseudouridine synthase activity in a sample (Ramamurthy, V. (1999) J. Biol Chem 274: 22225-22230).

Alternatively, the NAAP pseudouridine synthase activity assay is performed at 30 to 37 ° C. in the following mixture. That is, 100 mM TrisHCl (pH 8.0), 100 mM ammonium acetate, 5 mM MgCl ₂ , 2 mM dithiothreitol, 0.1 mM EDTA, and 1-2 fmol [ ³² P] radiolabeled runoff transcript (Production is done in vitro with an appropriate RNA polymerase, ie T7 or SP6) as a substrate. Start the reaction by adding NAAP, or do not add to the reaction solution and use as a control sample. After incubation, RNA is extracted with phenol-chloroform and precipitated in ethanol. In addition, RNase T ₂ is used for complete hydrolysis to 3-nucleotide monophosphate. Two-dimensional thin layer chromatography is used for hydrolyzate analysis, and the amount of ³² P radiolabel (amount present in the ΨMP and UMP spots) is evaluated after exposing the thin layer chromatography plate to film or a PhosphorImager screen. Determine the relative amount of yMP and UMP taking into account the relative number of uridylate residues in the substrate RNA, and use this amount to calculate the relative amount of Ψ relative to the amount of tRNA molecules (mol Ψ / tRNA mole, Or expressed as mol Ψ / tRNA mol / min). This amount corresponds to the amount of pseudouridine synthase activity in the NAAP sample (Lecointe, F. et al. (1998) J. Biol. Chem. 273: 1316-1323).

Measurement of NAAP N ² , N ² dimethylguanosine transferase ((m ² ₂ G) methyltransferase) activity is carried out in a 160 μl reaction mixture containing: That 100 mM of Tris-HCl (pH 7.5), 0.1 mM of EDTA, 10 mM of MgCl _2, 20 mM NH ₄ of Cl, 1 mM dithiothreitol, 6.2 [mu] M of S- adenosyl-L-[methyl - ³ H] Contains methionine (30-70 Ci / mM), 8 μg m ² ₂ G-deficient tRNA or wild type tRNA (from yeast) and about 100 μg purified NAAP or NAAP containing sample. Incubate the reaction at 30 ° C. for 90 minutes and cool on ice. Dilute a portion of each reaction in 1 ml of water containing 100 μg BSA. 1 ml of 2 M HCl is added to each sample and the acidic insoluble product is allowed to settle on ice for 20 minutes before filtration for collection through a glass fiber filter. The collected material is washed several times with hydrochloric acid and quantified using a liquid scintillation counter. ^{The amount of 3} H incorporated into acidic insoluble tRNA lacking m ² ₂ G is proportional to the amount of N ² , N ² -dimethylguanosine transferase activity in the NAAP sample. Reactions that do not contain substrate tRNA, or reactions that contain already modified wild-type tRNA, served as control reactions, and this reaction should not yield acidic insoluble ³ H-labeled products.

In vitro polyadenylation reaction is used to measure NAAP polyadenylation activity. The reaction mixture is made on ice and includes: 10 μl 5 mM dithiothreitol, 0.025% (v / v) NONIDET P-40, 50 mM creatine phosphate, 6.5% (w / v) polyvinyl alcohol, 0.5 unit / μl RNAGUARD (Pharmacia), 0.025 μg / μl Bring creatine kinase, 1.25 mM cordycepin 5′-triphosphate, and 3.75 mM MgCl ₂ to a total volume of 25 μl. 60 fmol CstF, 50 fmol CPSF, 240 fmol PAP, 4 μl unpurified CF II, or partially purified CF II and a variable amount of CF I are then added to the reaction mixture. To adjust the volume to 23.5 μl, add a buffer with: 50 mM TrisHCl (pH 7.9), 10% (v / v) glycerol, and 0.1 mM NaEDTA. The final ammonium sulfate concentration should be less than 20 mM. To initiate the reaction (on ice), add 15 μmol of ³² P-labeled mRNA precursor template and 1.5 μl of water containing 2.5 μg of unlabeled tRNA. The reaction is then incubated. This is done at 30 ° C. for 75-90 minutes, and 75 μl (approximately 2 volumes) of proteinase K mix (0.2 M Tris-HCl (pH 7.9), 300 mM NaCl, 25 mM NaEDTA, 2 % (w / v SDS), 1 μl of 10 mg / ml proteinase K, 0.25 μl of 20 mg / ml glycogen and 23.75 μl of water. Following incubation, RNA is precipitated with ethanol and analysis is performed on a 6% (w / v) polyacrylamide, 8.3 M urea sequencing gel. The dried gel is sensitized by autoradiography or by a phosphoimager. To determine cleavage activity, the amount of cleavage product is compared to the amount of mRNA precursor template. Removing either polypeptide component from the reaction and substituting NAAP is useful for identifying specific biological functions of NAAP in mRNA precursor polyadenylation (Ruegsegger, U. et al. (1996) J. Biol. Chem. 271: 6107-6113 and references therein).

tRNA synthetase activity is measured as aminoacylation of the substrate tRNA in the presence of [ ¹⁴ C] -labeled amino acid. NAAP is incubated in buffer with [ ¹⁴ C] -labeled amino acid and a suitable cognate tRNA (eg, [ ¹⁴ C] alanine and tRNA ^ala ). ^{The 14} C-labeled product is separated from free [ ¹⁴ C] amino acid by chromatography, and the incorporated [ ¹⁴ C] is measured with a scintillation counter. In this assay, the amount of ¹⁴ C-labeled product is proportional to the activity of NAAP.

Alternatively, to measure NAAP activity, the NAAP-containing sample is incubated with a solution containing: [ ¹⁴ C] -GlutRNAGln (eg, 1 μM) that is 1 mM ATP, 5 mM Hepes-KOH (pH 7.0), 2.5 mM KCl, 1.5 mM magnesium chloride, and 0.5 mM DTT, and misacylated ) And similar concentrations of unlabeled L-glutamine. To stop the reaction, 3 M sodium acetate (pH 5.0) was added, and the mixture was extracted with the same amount of water-saturated phenol, and centrifuged to separate the aqueous and organic phases at 15,000 × g at room temperature. Do for 1 minute. Precipitate the aqueous phase with 3 volumes of ethanol at -70 ° C for 15 minutes. Centrifuge to collect the precipitated aminoacyl-tRNA at 15,000 xg for 15 minutes at 4 ° C. The pellet is resuspended with 25 mM KOH, deacylated for 10 minutes at 65 ° C., neutralized with 0.1 M HCl (final pH 6-7) and vacuum dried. Resuspend the dried pellet in water and spot on a cellulose TLC plate. Plate development is performed in isopropanol / formic acid / water or ammonia / water / chloroform / methanol. The image is subjected to densitometer analysis, and the relative amount of Glu and Gln is calculated based on the Rf value and relative intensity of the spot. Calculation of NAAP activity is based on the amount of Gln resulting from acylation and conversion of Glu as Glu-tRNA ^Gln (Curnow, AW et al. (1997) Proc. Natl. Acad. Sci. USA 94: 11819-26).

  Alternatively, NAAP activity is demonstrated by increased chromatin activity. Chromatin activity is proportional to susceptibility to RNase I (Dawson, B.A. et al. (1989) J. Biol. Chem. 264: 12830-12837). A NAAP-containing sample (NAAP +) and a control sample (NAAP-) were treated with DNA-degrading enzyme I, followed by 5-[(N-biotinamido) hexanoamido-ethyl-1 , 3-thiopropionyl-3-aminoallyl] -2'-deoxyuridine 5'-triphosphate (5-[(N-biotinamido) hexanoamido-ethyl-1,3-thiopropio-nyl-3-aminoallyl] -2 ' -deoxyuridine 5'-triphosphate) was inserted using nick repair techniques well known in the art. After purification and digestion with EcoRI restriction endonuclease, affinity isolation is performed by sequentially binding the biotinylated sequence to streptavidin and biotincellulose. The difference in biotinylation between the presence and absence of NAAP is proportional to NAAP activity.

19 Identification of NAAP agonists and antagonists
Agonists or antagonists of NAAP activation or inhibition can be tested using the assay described in Example 18 . Agonists increase NAAP activity and antagonists decrease NAAP activity.

It will be apparent to those skilled in the art that various modifications and variations can be made to the described method and system of the present invention without departing from the spirit or spirit of the invention. While the invention has been described with reference to several embodiments, it should be understood that the scope of the invention should not be unduly limited to such specific embodiments. . Indeed, various modifications of the described modes for carrying out the invention which are obvious to those skilled in molecular biology or related fields are intended to be within the scope of the following claims.
(Short description of the table)
Table 1 outlines the nomenclature for the full-length polynucleotide and polypeptide sequences of the present invention.

  Table 2 shows the GenBank identification number and the GenBank homologue annotation closest to the polypeptide of the invention. The probability score for each polypeptide and its GenBank homologue is also shown.

  Table 3 shows the structural features of the polynucleotide sequences of the present invention, including predicted motifs and domains, along with methods, algorithms and searchable databases for use in polypeptide analysis.

  Table 4 shows the cDNA and genomic DNA fragments used to assemble the polynucleotide sequences of the present invention, along with selected fragments of the polynucleotide sequences.

  Table 5 shows a representative cDNA library of the polynucleotides of the present invention.

  Table 6 is an appendix explaining the tissues and vectors used for the preparation of the cDNA library shown in Table 5.

  Table 7 shows the tools, programs, and algorithms used to analyze the polynucleotides and polypeptides of the present invention, along with applicable descriptions, references and threshold parameters.

Claims (101)

An isolated polypeptide selected from the group consisting of the following (a) to (f):
(A) a polypeptide having a certain amino acid sequence selected from the group consisting of SEQ ID NO: 1-23 (SEQ ID NO: 1 to 23) (b) SEQ ID NO: 1-12, SEQ ID NO: 14-18, And a polypeptide having a natural amino acid sequence which is at least 90% identical to an amino acid sequence selected from the group having SEQ ID NO: 23.
(C) a polypeptide having a natural amino acid sequence which is at least 99% identical to the amino acid sequence of SEQ ID NO: 21 (d) at least 98% identical to the amino acid sequence of SEQ ID NO: 22 (E) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-23 (f) a group having SEQ ID NOs: 1-23 An immunogenic fragment of a polypeptide having an amino acid sequence selected from 2. The isolated polypeptide of claim 1 having an amino acid sequence selected from the group consisting of SEQ ID NO: 1-23. 2. An isolated polynucleotide encoding the polypeptide of claim 1. An isolated polynucleotide encoding the polypeptide of claim 2. 5. The isolated polynucleotide of claim 4 having a polynucleotide sequence selected from the group consisting of SEQ ID NO: 24-46. A recombinant polynucleotide comprising a promoter sequence operably linked to the polynucleotide of claim 3. A cell transformed with the recombinant polynucleotide according to claim 6. A genetic transformant comprising the recombinant polynucleotide according to claim 6. A method for producing the polypeptide of claim 1, comprising:
(A) culturing cells transformed with a recombinant polynucleotide comprising a promoter sequence operably linked to a polynucleotide encoding the polypeptide of claim 1 under conditions suitable for expression of said polypeptide. Process,
(B) recovering the polypeptide so expressed. The method according to claim 9, characterized in that said polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 1-23. An isolated antibody that specifically binds to the polypeptide of claim 1. An isolated polynucleotide selected from the group consisting of the following (a) to (g).
(A) a polynucleotide comprising a polynucleotide sequence selected from the group having SEQ ID NO: 24-46 (b) from SEQ ID NO: 24-35, SEQ ID NO: 37-41 and SEQ ID NO: 43-46 A polynucleotide having a natural polynucleotide sequence such that at least 90% is identical to a polynucleotide sequence selected from the group (c) at least 96% identical to the polynucleotide sequence of SEQ ID NO: 42 A polynucleotide having a natural polynucleotide sequence such as: (d) a polynucleotide complementary to the polynucleotide of (a) (e) a polynucleotide complementary to the polynucleotide of (b) (f) a polynucleotide of (c) Polynucleotide complementary to nucleotide (g) RNA equivalent of (a)-(f) 13. An isolated polynucleotide comprising at least 60 contiguous nucleotides of the polynucleotide of claim 12. A method for detecting a target polynucleotide having the sequence of the polynucleotide of claim 12 in a sample comprising:
(A) hybridizing the sample with a probe having a group of at least 20 consecutive nucleotides having a sequence complementary to the target polynucleotide in the sample, the probe comprising a hybridization complex Specifically hybridizing to the target polynucleotide under conditions where a body is formed between the probe and the target polynucleotide or fragments thereof;
(B) detecting the presence / absence of the hybridization complex, and optionally detecting the amount of the complex, if there is a high level between the probe and the target polynucleotide or fragment. A method wherein the probe specifically hybridizes to the target polynucleotide under conditions such that a hybridization complex is formed. 15. The method of claim 14, wherein the probe comprises at least 60 consecutive nucleotides. A method for detecting a target polynucleotide having the sequence of the polynucleotide of claim 12 in a sample comprising:
(A) amplifying the target polynucleotide or fragment thereof using polymerase chain reaction amplification;
(B) detecting the presence or absence of the amplified target polynucleotide or a fragment thereof, and optionally detecting the amount of the target polynucleotide or a fragment thereof if present. A composition comprising the polypeptide of claim 1 and a pharmaceutically acceptable excipient. 18. The composition of claim 17, wherein the polypeptide has an amino acid sequence selected from the group consisting of SEQ ID NO: 1-23. 18. A method of treating a disease or condition associated with reduced expression of functional NAAP, comprising administering the composition of claim 17 to a patient in need of such treatment. A method of screening a compound to confirm the effectiveness of the polypeptide of claim 1 as an agonist, comprising:
(A) exposing a sample having the polypeptide of claim 1 to a compound;
(B) A screening method comprising the step of detecting agonist activity in the sample. 21. A composition comprising an agonist compound identified by the method of claim 20 and a pharmaceutically acceptable excipient. 22. A method of treating a disease or condition associated with reduced expression of functional NAAP, comprising administering the composition of claim 21 to a patient in need of such treatment. A method of screening a compound to confirm its effectiveness as an antagonist of the polypeptide of claim 1, comprising: (a) exposing a sample having the polypeptide of claim 1 to a compound;
(B) a screening method comprising the step of detecting antagonist activity in the sample. 24. A composition comprising an antagonist compound identified by the method of claim 23 and a pharmaceutically acceptable excipient. 25. A method of treating a disease or condition associated with overexpression of functional NAAP, comprising administering the composition of claim 24 to a patient in need of such treatment. A method for screening for a compound that specifically binds to the polypeptide of claim 1, comprising:
(A) mixing the polypeptide of claim 1 with at least one test compound under suitable conditions;
(B) detecting the binding of the polypeptide of claim 1 to a test compound, thereby identifying the compound that specifically binds to the polypeptide of claim 1. A method of screening for compounds that modulate (modulate) the activity of the polypeptide of claim 1 comprising:
(A) mixing the polypeptide of claim 1 with at least one test compound under conditions that permit the activity of the polypeptide of claim 1;
(B) calculating the activity of the polypeptide of claim 1 in the presence of a test compound;
(C) comparing the activity of the polypeptide of claim 1 in the presence of the test compound with the activity of the polypeptide of claim 1 in the absence of the test compound, 2. A method wherein the change in activity of the polypeptide of claim 1 in the presence of is indicative of a compound that modulates the activity of the polypeptide of claim 1. A method of screening for compounds effective to alter the expression of a target polynucleotide having the sequence of claim 5 comprising:
(A) exposing a sample containing the target polynucleotide to a compound under conditions suitable for expression of the target polynucleotide;
(B) detecting altered expression of the target polynucleotide;
(C) comparing the expression of the target polynucleotide in the presence of a variable amount of the compound and in the absence of the compound. A method for calculating the toxicity of a test compound comprising:
(A) treating a biological sample having a nucleic acid group with the test compound;
(B) hybridizing a nucleic acid group of the treated biological sample with a probe having at least 20 consecutive nucleotide groups of the polynucleotide of claim 12, wherein the hybridization comprises the probe and the probe 13. A polynucleotide sequence of the polynucleotide of claim 12 or a sequence thereof, wherein the target polynucleotide is formed under conditions that form a specific hybridization complex with a target polynucleotide of a biological sample. A polynucleotide comprising a fragment, the above process;
(C) quantifying the yield of the hybridization complex;
(D) comparing the amount of the hybridization complex in the treated biological sample with the amount of the hybridization complex in an untreated biological sample, A difference in the amount of the hybridization complex in a biological sample is indicative of the toxicity of the test compound. A diagnostic test for a condition or disease associated with the expression of NAAP in a biological sample, comprising:
(A) mixing the antibody with the polypeptide and binding the biological sample with the antibody of claim 11 under conditions suitable to form a complex of the antibody and the polypeptide. When,
(B) detecting the complex, wherein the presence of the complex correlates with the presence of the polypeptide in the biological sample. The antibody of claim 11, comprising
(A) Chimeric antibody (b) Single chain antibody (c) Fab fragment (d) F (ab ′) ₂ fragment (e) Humanized antibody 12. A composition comprising the antibody of claim 11 and an acceptable excipient. 35. A method for diagnosing a symptom or disease associated with NAAP expression in a subject, comprising the step of administering to the subject an effective amount of the composition of claim 32. 33. The composition of claim 32, wherein the antibody is labeled. 35. A method of diagnosing a symptom or disease associated with NAAP expression in a subject, comprising the step of administering to the subject an effective amount of the composition of claim 34. A method for preparing a polyclonal antibody having the specificity of the antibody of claim 11, comprising:
(A) immunizing an animal with a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1-23 or an immunogenic fragment thereof under conditions that elicit an antibody response The process of becoming
(B) isolating the antibody from the animal;
(C) a polyclonal that screens the isolated antibody with the polypeptide, thereby specifically binding to a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1-23 Identifying the antibody. 37. A polyclonal antibody produced by the method of claim 36. 38. A composition comprising the polyclonal antibody of claim 37 and a suitable carrier. A method for producing a monoclonal antibody having the specificity of the antibody according to claim 11, comprising:
(A) immunizing an animal with a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1-23 or an immunogenic fragment thereof under conditions that elicit an antibody response The process of becoming
(B) isolating cells producing antibodies from said animal;
(C) fusing the immortalized cell with the antibody-producing cell to form a hybridoma cell that produces a monoclonal antibody;
(D) culturing the hybridoma cells;
(E) isolating from the culture a monoclonal antibody that specifically binds to a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1-23. 40. A monoclonal antibody produced by the method of claim 39. 41. A composition comprising the monoclonal antibody of claim 40 and a suitable carrier. The antibody according to claim 11, which is produced by screening a Fab expression library. 12. The antibody according to claim 11, wherein the antibody is produced by screening a recombinant immunoglobulin library. A method of detecting a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 1-23,
(A) incubating the antibody of claim 11 with a sample under conditions that allow specific binding of the antibody to the polypeptide;
(B) including the step of detecting specific binding, wherein the specific binding suggests that a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 1-23 is present in the sample And how to. A method for purifying a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1-23, comprising:
(A) incubating the antibody of claim 11 with a sample under conditions that allow specific binding of the antibody to the polypeptide;
(B) separating the antibody from the sample to obtain a purified polypeptide having a certain amino acid sequence selected from the group consisting of SEQ ID NO: 1-23. A microarray, wherein at least one element of the microarray is a polynucleotide according to claim 13. A method for generating an expression profile of a sample comprising a polynucleotide comprising:
(A) labeling a sample polynucleotide;
(B) contacting the elements of the microarray of claim 46 with the labeled polynucleotides of the sample under conditions suitable for formation of a hybridization complex;
(C) quantifying the expression of the polynucleotide in the sample and producing a transcription image of the sample. An array having various nucleotide molecules attached to unique physical locations on a solid substrate, wherein at least one said nucleotide molecule is specific to at least 30 consecutive nucleotide groups of a target polynucleotide 13. An array having an initial oligonucleotide or polynucleotide sequence capable of hybridizing to said polynucleotide, wherein said target polynucleotide is the polynucleotide of claim 12. 49. The array of claim 48, wherein the initial sequence of the oligonucleotide or polynucleotide is completely complementary to at least 30 contiguous nucleotide groups of the target polynucleotide. 49. The array of claim 48, wherein the initial sequence of the oligonucleotide or polynucleotide is completely complementary to at least 60 contiguous nucleotide groups of the target polynucleotide. 49. The array of claim 48, wherein the initial sequence of the oligonucleotide or polynucleotide is completely complementary to the target polynucleotide. 49. The array of claim 48, wherein the array is a microarray. 49. The array of claim 48, further comprising the target polynucleotide hybridized to a nucleotide molecule having an initial sequence of the oligonucleotide or polynucleotide. 49. The array of claim 48, wherein a linker connects at least one of the nucleotide molecules and the solid substrate. 49. The array of claim 48, wherein each unique physical location on the substrate comprises a plurality of nucleotide molecules, wherein the plurality of nucleotide molecules at any single unique physical location comprises the same sequence. Each of the unique physical locations on the substrate includes a group of nucleotide molecules having a sequence that is different from the sequence of the nucleotide molecules at another unique physical location on the substrate. An array. 2. The polypeptide of claim 1 having the amino acid sequence of SEQ ID NO: 1. The polypeptide of claim 1 having the amino acid sequence of SEQ ID NO: 2. The polypeptide of claim 1 having the amino acid sequence of SEQ ID NO: 3. The polypeptide of claim 1 having the amino acid sequence of SEQ ID NO: 4. The polypeptide of claim 1 having the amino acid sequence of SEQ ID NO: 5. The polypeptide of claim 1 having the amino acid sequence of SEQ ID NO: 6. The polypeptide of claim 1 having the amino acid sequence of SEQ ID NO: 7. The polypeptide of claim 1 having the amino acid sequence of SEQ ID NO: 8. The polypeptide of claim 1 having the amino acid sequence of SEQ ID NO: 9. The polypeptide of claim 1 having the amino acid sequence of SEQ ID NO: 10. The polypeptide of claim 1 having the amino acid sequence of SEQ ID NO: 11. The polypeptide of claim 1 having the amino acid sequence of SEQ ID NO: 12. The polypeptide of claim 1 having the amino acid sequence of SEQ ID NO: 13. The polypeptide of claim 1 having the amino acid sequence of SEQ ID NO: 14. The polypeptide of claim 1 having the amino acid sequence of SEQ ID NO: 15. The polypeptide of claim 1 having the amino acid sequence of SEQ ID NO: 16. 2. The polypeptide of claim 1 having the amino acid sequence of SEQ ID NO: 17. The polypeptide of claim 1 having the amino acid sequence of SEQ ID NO: 18. The polypeptide of claim 1 having the amino acid sequence of SEQ ID NO: 19. The polypeptide of claim 1 having the amino acid sequence of SEQ ID NO: 20. The polypeptide of claim 1 having the amino acid sequence of SEQ ID NO: 21. The polypeptide of claim 1 having the amino acid sequence of SEQ ID NO: 22. The polypeptide of claim 1 having the amino acid sequence of SEQ ID NO: 23. The polynucleotide of Claim 12 having the polynucleotide sequence of SEQ ID NO: 24. The polynucleotide of Claim 12 having the polynucleotide sequence of SEQ ID NO: 25. The polynucleotide of Claim 12 having the polynucleotide sequence of SEQ ID NO: 26. The polynucleotide of Claim 12 having the polynucleotide sequence of SEQ ID NO: 27. The polynucleotide of Claim 12 having the polynucleotide sequence of SEQ ID NO: 28. The polynucleotide of claim 12 having the polynucleotide sequence of SEQ ID NO: 29. The polynucleotide of claim 12 having the polynucleotide sequence of SEQ ID NO: 30. The polynucleotide of Claim 12 having the polynucleotide sequence of SEQ ID NO: 31. The polynucleotide of Claim 12 having the polynucleotide sequence of SEQ ID NO: 32. The polynucleotide of Claim 12 having the polynucleotide sequence of SEQ ID NO: 33. The polynucleotide of claim 12 having the polynucleotide sequence of SEQ ID NO: 34. The polynucleotide of Claim 12 having the polynucleotide sequence of SEQ ID NO: 35. The polynucleotide of Claim 12 having the polynucleotide sequence of SEQ ID NO: 36. The polynucleotide of Claim 12 having the polynucleotide sequence of SEQ ID NO: 37. The polynucleotide of Claim 12 having the polynucleotide sequence of SEQ ID NO: 38. The polynucleotide of claim 12 having the polynucleotide sequence of SEQ ID NO: 39. The polynucleotide of Claim 12 having the polynucleotide sequence of SEQ ID NO: 40. The polynucleotide of Claim 12 having the polynucleotide sequence of SEQ ID NO: 41. The polynucleotide of Claim 12 having the polynucleotide sequence of SEQ ID NO: 42. The polynucleotide of Claim 12 having the polynucleotide sequence of SEQ ID NO: 43. The polynucleotide of claim 12 having the polynucleotide sequence of SEQ ID NO: 44. The polynucleotide of claim 12 having the polynucleotide sequence of SEQ ID NO: 45. The polynucleotide of claim 12 having the polynucleotide sequence of SEQ ID NO: 46.