EP1427841A2 - Proteines associees a des acides nucleiques - Google Patents
Proteines associees a des acides nucleiquesInfo
- Publication number
- EP1427841A2 EP1427841A2 EP02761035A EP02761035A EP1427841A2 EP 1427841 A2 EP1427841 A2 EP 1427841A2 EP 02761035 A EP02761035 A EP 02761035A EP 02761035 A EP02761035 A EP 02761035A EP 1427841 A2 EP1427841 A2 EP 1427841A2
- Authority
- EP
- European Patent Office
- Prior art keywords
- polynucleotide
- seq
- polypeptide
- amino acid
- sequence
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Ceased
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Definitions
- the invention relates to novel nucleic acids, nucleic acid-associated proteins encoded by these nucleic acids, and to the use of these nucleic acids and proteins i ⁇ the diagnosis, treatment, and prevention of cell proliferative, neurological, reproductive, developmental, autoimmune/inflammatory, and DNA repair disorders, and infections.
- the invention also relates to the assessment of the effects of exogenous compounds on the expression of nucleic acids and nucleic acid-associated proteins.
- Multicellular organisms are comprised of diverse cell types that differ dramatically both in structure and function.
- the identity of a cell is determined by its characteristic pattern of gene expression, and different cell typ s express overlapping but distinctive sets of genes throughout development. Spatial and temporal regulation of gene expression is critical for the control of cell proliferation, cell differentiation,! apoptosis, and other processes that contribute to organismal development.
- gene expression is regulated in response to extracellular signals that mediate cell-cell communication and coordinate the activities of different cell types. Appropriate gene regulation also ensures that cells function efficiently by expressing only those genes whose functions are required at a given time.
- the cell nucleus contains all of the genetic information of the cell in the form of DNA, and the components and machinery necessary for replication of DNA and for transcription of DNA into RNA.
- DNA is organized into compact structures in the nucleus by interactions with various DNA-binding proteins such as histones and non-histone chromosomal proteins.
- DNA-specific nucleases, DNAses partially degrade these compacted structures prior to DNA replication or transcription.
- DNA replication takes place with the aid of DNA helicases which unwind the double-stranded DNA helix, and DNA polymerases that duplicate the separated DNA strands.
- Transcriptional regulatory proteins are essential for the control of gene expression. Some of these proteins function as transcription factors that initiate, activate, repress, or terminate gene transcription. Transcription factors generally bind to the promoter, enhancer, and upstream regulatory regions of a gene in a sequence-specific manner, although some factors bind regulatory elements within or downstream of a gene coding region. Transcription factors may bind to a specific region of DNA singly or as a complex with other accessory factors (reviewed in Lewin, B. (1990) Genes IV. Oxford University Press, New York NY, and Cell Press, Cambridge MA, pp. 554-570).
- the double helix structure and repeated sequences of DNA create topological and chemical features which can be recognized by transcription factors. These features are hydrogen bond donor and acceptor groups, hydrophobic patches, major and minor grooves, and regular, repeated stretches of sequence which induce distinct bends in the helix.
- transcription factors recognize specific DNA sequence motifs of about 20 nucleotides in length. Multiple, adjacent transcription factor-binding motifs maybe required for gene regulation.
- DNA-binding structural motifs which comprise either a helices or ⁇ sheets that bind to the major groove of DNA.
- structural motifs are helix-turn-helix, zinc finger, leucine zipper, and helix-loop-helix. Proteins containing these motifs may act alone as monomers, or they may form homo- or heterodimers that interact with DNA.
- the helix-turn-helix motif consists of two ⁇ helices connected at a fixed angle by a short chain of amino acids. One of the helices binds to the major groove. Helix-turn-helix motifs are exemplified by the homeobox motif which is present in homeodomain proteins. These proteins are critical for specifying the anterior-posterior body axis during development and are conserved throughout the animal kingdom.
- the Antennapedia and Ultrabithorax proteins of Drosophila melanogaster are prototypical homeodomain proteins (Pabo, CO. and R.T. Sauer (1992) Annu. Rev. Biochem. 61:1053-1095).
- Homeobox genes are a family of highly conserved regulatory genes that encode transcription factors.
- the helix-loop-helix motif (HLH) consists of a short a helix connected by a loop to a longer a helix.
- the loop is flexible and allows the two helices to fold back against each other and to bind to DNA.
- the protooncogene Myc a transcription factor that activates genes required for cellular proliferation, contains a prototypical HLH motif.
- the zinc finger motif which binds zinc ions, generally contains tandem repeats of about 30 amino acids consisting of periodically spaced cysteine and histidhie residues. Examples of this sequence pattern, designated C2H2 and C3HC4 ("RING" finger), have been described (Lewin, supra).
- Zinc finger proteins each contain an ⁇ helix and an antiparallel ⁇ sheet whose proximity and ⁇ conformation are maintained by the zinc ion.
- Contact with DNA is made by the arginine preceding the ⁇ helix and by the second, third, and sixth residues of the helix.
- Variants of the zinc finger motif include poorly defined cysteine-rich motifs which bind zinc or other metal ions.
- the zinc finger motif may be repeated in a tandem array within a protein, such that the ⁇ helix of each zinc finger in the protein makes contact with the major groove of the DNA double helix. This repeated contact between the protein and the DNA produces a strong and specific DNA-protein interaction. The strength and specificity of the interaction can be regulated by the number of zinc finger motifs within the protein. Though originally identified in DNA-binding proteins as regions that interact directly with DNA, zinc fingers occur in a variety of proteins that do not bind DNA (Lodish, H. et al. (1995) Molecular Cell Biology, Scientific American Books, New York NY, pp. 447-451).
- the C2H2-type zinc finger signature motif contains a 28 amino acid sequence, including 2 conserved Cys and 2 conserved His residues in a C-2-C-12-H-3-H type motif. The motif generally occurs in multiple tandem repeats.
- a cysteine-rich domain including the motif Asp-His-His-Cys (DHHC-CRD) has been identified as a distinct subgroup of zinc finger proteins. The DHHC-CRD region has been implicated in growth and development.
- DHHC-CRD mutant shows defective function of Ras, a small membrane-associated GTP-binding protein that regulates cell growth and differentiation, while other DHHC-CRD proteins probably function in pathways not involving Ras (Bartels, D.J. et al. (1999) Mol. Cell Biol. 19:6775-6787).
- Zinc-finger transcription factors are often accompanied by modular sequence motifs such as the Kruppel-associated box (KRAB) and the SCAN domain.
- KRAB Kruppel-associated box
- the hypoalphalipoproteinemia susceptibility gene ZNF202 encodes a SCAN box and a KRAB domain followed by eight C2H2 zinc-finger motifs (Honer, C. et al. (2001) Biochim. Biophys. Acta 1517:441-448).
- the SCAN domain is a highly conserved, leucine-rich motif of approximately 60 amino acids found at the ammo-terminal end of zinc finger transcription factors. SCAN domains are most often linked to C2H2 zinc finger motifs through their carboxyl-terminal end.
- SCAN domain-mediated protein complexes may function to modulate the biological function of transcription factors (Schumacher, C. et al. (2000) J. Biol. Chem. 275:17173-17179).
- the KRAB (Kruppel-associated box) domain is a conserved amino acid sequence spanning approximately 75 amino acids and is found in almost one-third of the 300 to 700 genes encoding C2H2 zinc fingers.
- the KRAB domain is found N-terminally with respect to the finger repeats.
- the KRAB domain is generally encoded by two exons; the KRAB-A region or box is encoded by one exon and the KRAB-B region or box is encoded by a second exon.
- the function of the KRAB domain is the repression of transcription. Transcription repression is accomplished by recruitment of either the KRAB -associated protein-1, a transcriptional corepressor, or the KRAB-A interacting protein.
- Proteins containing the KRAB domain are likely to play a regulatory role during development (Williams, A.J. et al. (1999) Mol. Cell Biol. 19:8526-8535).
- a subgroup of highly related human KRAB zinc finger proteins detectable in all human tissues is highly expressed in human T lymphoid cells (BeUefroid, E.J. et al. (1993) EMBO J. 12:1363-1374).
- the ZNF85 KRAB zinc finger gene a member of the human ZNF91 family, is highly expressed in normal adult testis, in seminomas, and in the NT2/D1 teratocarcinoma cell line (Poncelet, D.A. et al. (1998) DNA Cell Biol.17:931-943).
- the C4 motif is found in hormone-regulated proteins.
- the C4 motif generally includes only 2 repeats.
- a number of eukaryotic and viral proteins contain a conserved cysteine-rich domain of 40 to 60 residues (called C3HC4 zinc-finger or RLNG finger) that binds two atoms of zinc, and is probably involved in mediating protein-protein interactions.
- the 3D "cross-brace" structure of the zinc ligation system is unique to the RING domain.
- the spacing of the cysteines in such a domain is C-x(2)-C-x(9 to 39)-C-x(l to 3)-H-x(2 to3)-C-x(2)-C-x(4 to 48)-C-x(2)-C.
- the PHD finger is a C4HC3 zinc-finger-like motif found in nuclear proteins thought to be involved in chromatin-mediated transcriptional regulation.
- GATA-type transcription factors contain one or two zinc finger domains which bind specifically to a region of DNA that contains the consecutive nucleotide sequence GATA.
- NMR studies indicate that the zinc finger comprises two irregular anti-parallel ⁇ sheets and an ⁇ helix, followed by a long loop to the C-terminal end of the finger (Om ⁇ ichinski, J.G. (1993) Science 261:438-446).
- the helix and the loop connecting the two ⁇ -sheets contact the major groove of the DNA, while the C-terminal part, which determines the specificity of binding, wraps around into the minor groove.
- the LIM motif consists of about 60 amino acid residues and contains seven conserved cysteine residues and a histidine within a consensus sequence (Schmeichel, K.L. and M.C. Beckerle (1994) Cell 79:211-219).
- the LIM family includes transcription factors and cytoskeletal proteins which may be involved in development, differentiation, and cell growth.
- actin-binding LHVl protein which may play roles in regulation of the cytoskeleton and cellular morphogenesis (Roof, D.J. et al. (1997) J. Cell Biol. 138:575-588).
- the N-terminal domain of actin-binding LEV! protein has four double zinc finger motifs with the LIM consensus sequence.
- actin-binding LIM protein shows sequence similarity to known actin-binding proteins such as dematin and villin.
- Actin-binding LIM protein binds to F-actin through its dematin-like C-terminal domain.
- the LIM domain may mediate protein-protein interactions with other LIM-binding proteins.
- Myeloid cell development is controlled by tissue-specific transcription factors.
- Myeloid zinc finger proteins (MZF) include MZF-1 and MZF-2.
- MZF-1 functions in regulation of the development of neutrophilic granulocytes.
- a murine homolog MZF-2 is expressed in myeloid cells, particularly in the cells committed to the neutrophilic lineage.
- MZF-2 is down-regulated by G-CSF and appears to have a unique function in neutrophil development (Murai, K. et al. (1997) Genes Cells 2:581-591).
- the leucine zipper motif comprises a stretch of amino acids rich in leucine which can form an amphipathic helix. This structure provides the basis for dimerization of two leucine zipper proteins. The region adjacent to the leucine zipper is usually basic, and upon protein dimerization, is optimally positioned for binding to the major groove. Proteins containing such motifs are generally refe ⁇ ed to as bZIP transcription factors.
- the leucine zipper motif is found in the proto-oncogenes Fos and Jun, which comprise the heterodimeric transcription factor API involved in cell growth and the determination of cell lineage (Papavassiliou, A.G. (1995) N. Engl. J. Med. 332:45-47).
- the helix-loop-helix motif (HLH) consists of a short ⁇ helix connected by a loop to a longer ⁇ helix. The loop is flexible and allows the two helices to fold back against each other and to bind to DNA.
- the transcription factor Myc contains a prototypical HLH motif.
- the NF-kappa-B/Rel signature defines a family of eukaryotic transcription factors involved in oncogenesis, embryonic development, differentiation and immune response. Most transcription factors containing the Rel homology domain (RHD) bind as dimers to a consensus DNA sequence motif termed kappa-B. Members of the Rel family share a highly conserved 300 amino acid domain termed the Rel homology domain.
- the characteristic Rel C-terminal domain is involved in gene activation and cytoplasmic anchoring functions.
- Proteins known to contain the RHD domain include vertebrate nuclear factor NF-kappa-B, which is a heterodimer of a DNA-binding subunit and the transcription factor p65, mammalian transcription factor RelB, and vertebrate proto-oncogene c-rel, a protein associated with differentiation and lymphopoiesis (Kabrun, N. and PJ. Enrietto (1994) Semin. Cancer Biol. 5:103-112).
- a DNA binding motif termed ARID AT-rich interactive domain distinguishes an evolutionarily conserved family of proteins.
- ARID sequence is present in a series of proteins strongly implicated in the regulation of cell growth, development, and tissue-specific gene expression.
- ARID proteins include Bright (a regulator of B -cell-specific gene expression), dead ringer (involved in development), and MRF-2 (which represses expression from the cytomegalovirus enhancer) (Dallas, P.B. et al. (2000) Mol. Cell. Biol. 20:3137-3146).
- the ELM2 (Egl-27 and MTAl homology 2) domain is found in metastasis-associated protein MTAl and protein ER1.
- the Caenorhabditis elegans gene egl-27 is required for embryonic patterning MTAl, a human gene with elevated expression in metastatic carcinomas, is a component of a protein complex with histone deacetylase and nucleosome remodelling activities (Solari, F. et al. (1999) Development 126:2483-2494).
- the ELM2 domain is usually found to the N terminus of a myb-like DNA binding domain. ELM2 is also found associated with an ARID DNA.
- the Iroquois (Irx) family of genes are found in nematodes, insects and vertebrates. Irx genes usually occur in one or two genomic clusters of three genes each and encode transcriptional controllers that possess a characteristic homeodomain. The Irx genes function early in development to specify the identity of diverse territories of the body. Later in development in both Drosophila and vertebrates, the Ix ⁇ genes function again to subdivide those territories into smaller domains (reviewed in Cavodeassi, F. et al. (2001) Development 128:2847-2855). For example, mouse and human Irx4 proteins are 83% conserved and their 63-aa homeodomain is more than 93% identical to that of the Drosophila Iroquois patterning genes. Irx4 transcripts are predominantly expressed in the cardiac ventricles. The homeobox gene Irx4 mediates ventricular differentiation during cardiac development (Bruneau, B.G. et al. (2000) Dev. Biol. 217:266-77).
- Histidine triad (HIT) proteins share residues in distinctive dimeric, 10-stranded half-barrel structures that form two identical purine nucleotide-binding sites.
- Hint histidine triad nucleotide-binding protein
- Fhit fragile histidine triad
- T-box genes such as Brachyw ⁇ (T) are essential for tissue specification in development.
- the fundamental unit of chromatin the nucleosome, consists of 200 base pairs of DNA associated with two copies each of H2A, H2B, H3, and H4.
- HI links adjacent nucleosomes.
- HMG proteins are low molecular weight, non-histone proteins that may play a role in unwinding DNA and stabilizing single-stranded DNA.
- Chromodomain proteins play a key role in the formation of highly compacted heterochromatin, which is transcriptionally silent.
- the zinc finger-type transcriptional regulator WT1 is a tumor-suppressor protein that is inactivated in children with Wil 's tumor. Deletions of the WTl gene, or point mutations which destroy the DNA-binding activity of the protein, are associated with development of the pediatric nephroblastoma, Wilms tumor, and Denys-Drash syndrome. (Rauscher, F.J. (1993) FASEB J.
- Oncogene bcl-6 which plays an important role in large-cell lymphoma, is also a zinc-finger protein (Papavassiliou, A.G. (1995) N. Engl. J. Med. 332:45-47). Chromosomal translocations may also produce chimeric loci that fuse the coding sequence of one gene with the regulatory regions of a second unrelated gene. Such an arrangement likely results in inappropriate gene transcription, potentially contributing to malignancy. In Burkitt's lymphoma, for example, the transcription factor Myc is translocated to the immunoglobulin heavy chain locus, greatly enhancing Myc expression and resulting in rapid cell growth leading to leukemia (Latchman, D.S. (1996) N.
- Staf50 for Stimulated trans-acting factor of 50 kDa
- Staf50 for Stimulated trans-acting factor of 50 kDa
- Staf50 appears to mediate the antiviral activity of interferon by down-regulating the viral transcription directed by the long terminal repeat promoter region of human immunodeficiency virus type-1 in transfected cells.
- APECED autoimmune polyendocrinopathy-candidiasis- ectodermal dystrophy
- Human acute leukemias involve reciprocal chromosome translocations that fuse the ALL-1 gene located at chromosome region 1 lq23 to a series of partner genes positioned on a variety of human chromosomes.
- the fused genes encode chimeric proteins.
- the AF17 gene encodes a protein of 1093 amino acids, containing a leucine-zipper dimerization motif located 3' of the fusion point and a cysteine-rich domain at the N terminus that shows homology to a domain within the protein Brl40 (peregrin) (Prasad R. et al. (1994) Proc. Natl. Acad. Sci. USA 91:8107-8111).
- Impaired transcriptional regulation may lead to Alzheimer's disease, a progressive neurodegenerative disorder that is characterized by the formation of senile plaques and neurofibrillary tangles containing amyloid beta peptide.
- These plaques are found in limbic and association cortices of the brain, including hippocampus, temporal cortices, cingulate cortex, amygdala, nucleus basalis and locus caeruleus.
- cingulate cortex amygdala
- nucleus basalis nucleus basalis and locus caeruleus.
- cerebral changes are visible in the cingulate cortex (Mmoshima, S. et al. (1997) Ann. Neurol. 42:85-94).
- accumulating plaques damage the neuronal architecture in limbic areas and eventually cripple the memory process.
- DNA and RNA replication are critical processes for cell replication and function.
- DNA and RNA replication are mediated by the enzymes DNA and RNA polymerase, respectively, by a "templating" process in which the nucleotide sequence of a DNA or RNA strand is copied by complementary base-pairing into a complementary nucleic acid sequence of either DNA or RNA.
- templating the process in which the nucleotide sequence of a DNA or RNA strand is copied by complementary base-pairing into a complementary nucleic acid sequence of either DNA or RNA.
- DNA polymerase catalyzes the stepwise addition of a deoxyribonucleoti.de to the 3'-OH end of a polynucleotide strand (the primer strand) that is paired to a second (template) strand.
- the new DNA strand therefore grows in the 5' to 3' direction (Alberts, B. et al. (1994) The Molecular Biology of the Cell, Garland PubHshing Inc., New York NY, pp 251-254).
- the substrates for the polymerization reaction are the corresponding deoxynucleotide triphosphates which must base-pair with the correct nucleotide on the template strand in order to be recognized by the polymerase.
- DNA polymerase In addition to the synthesis of new DNA, DNA polymerase is also involved in the repair of damaged DNA as discussed below under “Ligases.” In contrast to DNA polymerase, RNA polymerase uses a DNA template strand to "transcribe" DNA into RNA using ribonucleotide triphosphates as substrates.
- RNA polymerization proceeds in a 5' to 3' direction by addition of a ribonucleoside monophosphate to the 3'-OH end of a growing RNA chain.
- DNA transcription generates messenger RNAs (mRNA) that carry information for protein synthesis, as well as the transfer, ribosomal, and other RNAs that have structural or catalytic functions.
- mRNA messenger RNAs
- three discrete RNA polymerases synthesize the three different types of RNA (Alberts, supra, pp. 367-368).
- RNA polymerase I makes the large ribosomal RNAs
- RNA polymerase E makes the mRNAs that will be translated into proteins
- RNA polymerase IH makes a variety of small, stable RNAs, including 5S ribosomal RNA and the transfer RNAs (tRNA).
- RNA synthesis is initiated by binding of the RNA polymerase to a promoter region on the DNA and synthesis begins at a start site within the promoter. Synthesis is completed at a stop (termination) signal in the DNA whereupon both the polymerase and the completed RNA chain are released.
- DNA repair is the process by which accidental base changes, such as those produced by oxidative damage, hydrolytic attack, or uncontrolled methylation of DNA, are co ⁇ ected before replication or transcription of the DNA can occur. Because of the efficiency of the DNA repair process, fewer than one in a thousand accidental base changes causes a mutation (Alberts, supra, pp. 245-249).
- the three steps common to most types of DNA repair are (1) excision of the damaged or altered base or nucleotide by DNA nucleases, (2) insertion of the correct nucleotide in the gap left by the excised nucleotide by DNA polymerase using the complementary strand as the template and, (3) sealing the break left between the inserted nucleotide(s) and the existing DNA strand by DNA ligase.
- DNA ligase uses the energy from ATP hydrolysis to activate the 5' end of the broken phosphodiester bond before forming the new bond with the 3 -OH of the DNA strand.
- Bloom's syndrome an inlierited human disease, individuals are partially deficient in DNA ligation and consequently have an increased incidence of cancer (Alberts, supra, p. 247).
- Nucleases comprise enzymes that hydrolyze both DNA (DNase) and RNA (Rnase). They serve different purposes in nucleic acid metabolism. Nucleases hydrolyze the phosphodiester bonds between adjacent nucleotides either at internal positions (endonucleases) or at the terminal 3' or 5' nucleotide positions (exonucleases).
- a DNA exonuclease activity in DNA polymerase serves to remove improperly paired nucleotides attached to the 3'-OH end of the growing DNA strand by the polymerase and thereby serves a "proofreading" function.
- DNA endonuclease activity is involved in the excision step of the DNA repair process. RNases also serve a variety of functions.
- RNase P is a ribonucleoprotein enzyme which cleaves the 5' end of pre-tRNAs as part of their maturation process.
- RNase H digests the RNA strand of an RNA DNA hybrid. Such hybrids occur in cells invaded by retroviruses, and RNase H is an important enzyme in the retroviral replication cycle.
- Pancreatic RNase secreted by the pancreas into the intestine hydrolyzes RNA present in ingested foods.
- RNase activity in serum and cell extracts is elevated in a variety of cancers and infectious diseases (Schein, CH. (1997) Nat. Biotechnol. 15:529-536). Regulation of RNase activity is being investigated as a means to control tumor angiogenesis, allergic reactions, viral infection and replication, and fungal infections. MODIFICATION OF NUCLEIC ACIDS DNA Repair
- Damage to DNA consists of any change that modifies the structure of the molecule. Changes to DNA can be divided into two general classes, single base changes and structural distortions. Single base changes affect the sequence but not the overall structure of the DNA. Since single base changes do not affect transcription or replication, they exert their effect on future generations. Structural distortions affect the structure of the DNA. A single strand nick or removal of a base may prevent a strand from acting as a viable template for synthesis of DNA or RNA.
- Intrastrand or interstrand covalent linkage between bases may distort the structure of the double helix and interfere with transcription and replication. Any damage to DNA can produce a mutation, and the mutation may produce a disorder, such as cancer.
- Repair systems can be divided into three general types, direct repair, excision repair, and retrieval systems.
- cells become exceedingly sensitive to environmental mutagens, such as ultraviolet irradiation.
- disorders associated with a loss in DNA repair systems often exhibit a high sensitivity to environmental mutagens. Examples of such disorders include xeroderma pigmentosum, Bloom's syndrome, and Werner's syndrome. Xeroderma pigmentosum results in a hypersensitivity to sunlight, especially ultraviolet, and produces skin defects.
- Bloom's syndrome results in an increased frequency of chromosomal aberrations, including sister chromosome exchanges (Yamagata, K. et al. (1998) Proc. Natl. Acad. Sci. USA 95:8733-8738).
- Direct repair involves the reversal or simple removal of the damaged region of DNA. Mismatches involving normal bases are repaired based on certain biases within the repair system. For example, mismatched GT base pairs are frequently caused by deamination of 5-methyl-cytosine to form thymine. Therefore, repair systems convert mismatched GT pairs to GC, instead of AT. Repair also favors the non-methylated strand hemimethylated DNA, since this strand represents the newly synthesized daughter strand.
- C-5 cytosine-specific DNA methylases are enzymes that specifically methylate the C-5 carbon of cytosines in DNA (Kumar, S. et al. (1994) Nucleic Acids Res. 22:1-10). Excision repair is a system in which mispaired or damaged bases are removed from DNA and a new stretch of DNA is synthesized to replace them.
- the damaged structure is recognized by an endonuclease that cleaves the DNA strand on both sides of the damage.
- a 5 -3 ' exonuclease removes a stretch of the damaged DNA strand.
- the resulting single-stranded region serves as a template for a DNA polymerase to synthesize a replacement for the excised sequence.
- DNA ligase covalently links the 3 ' end of the new material to the old material.
- DNA polymerase beta serves as the DNA repair polymerase. Mutations in the human DNA polymerase beta gene are associated with several types of cancer (Bhattacharyya, N. et al. (1999) DNA Cell Biol. 18:549-554; Matsuzaki, J. et al. (1996) Mol. Carcinog. 15:38-43).
- Methylation of specific nucleotides occurs in both DNA and RNA, and serves different functions in the two macromolecules. Methylation of cytosine residues to form 5 -methyl cytosine in DNA occurs specifically in CG sequences which are base-paired with one another in the DNA double-helix. The pattern of methylation is passed from generation to generation during DNA replication by an enzyme called "maintenance methylase" that acts preferentially on those CG sequences that are base-paired with a CG sequence that is already methylated. Such methylation appears to distinguish active from inactive genes by preventing the binding of regulatory proteins that "turn on” the gene, but permiting the binding of proteins that inactivate the gene (Alberts, supra, pp. 448-451).
- tRNA methylase produces one of several nucleotide modifications in tRNA that affect the conformation and base-pairing of the molecule and facilitate the recognition of the appropriate mRNA codons by specific tRNAs.
- the primary methylation pattern is the dimethylation of guanine residues to form N,N-dimethyl guanine.
- Helicases are enzymes that destabilize and unwind double helix structures in both DNA and RNA. Since DNA replication occurs more or less simultaneously on both strands, the two strands must first separate to generate a replication "fork" for DNA polymerase to act on. Two types of replication proteins contribute to this process, DNA helicases and single-stranded binding proteins. DNA helicases hydrolyze ATP and use the energy of hydrolysis to separate the DNA strands. Single-stranded binding proteins (SSBs) then bind to the exposed DNA strands, without covering the bases, thereby temporarily stabilizing them for templating by the DNA polymerase (Alberts, supra, pp. 255-256).
- SSBs Single-stranded binding proteins
- RNA helicases also alter and regulate RNA conformation and secondary structure. Like the DNA helicases, RNA helicases utilize energy derived from ATP hydrolysis to destabilize and unwind RNA duplexes.
- the most well-characterized and ubiquitous family of RNA helicases is the DEAD- box family, so named for the conserved B-type ATP-binding motif which is diagnostic of proteins in this family.
- DEAD-box helicases Over 40 DEAD-box helicases have been identified in organisms as diverse as bacteria, insects, yeast, amphibians, mammals, and plants. DEAD-box helicases function in diverse processes such as translation initiation, splicing, ribosome assembly, and RNA editing, transport, and stability.
- RNA helicases examples include yeast Drsl protein, which is involved in ribosomal RNA processing; yeast TJJF1 and TTF2 and mammalian eIF-4A, which are essential to the initiation of RNA translation; and human p68 antigen, which regulates cell growth and division (Ripmaster, T.L. et al. (1992) Proc. Natl. Acad. Sci. USA 89:11131-11135; Chang, T.-H. et al. (1990) Proc. Natl. Acad. Sci. USA 87:1571-1575). These RNA helicases demonstrate strong sequence homology over a stretch of some 420 amino acids.
- conserved sequences include the consensus sequence for the A motif of an ATP binding protein; the "DEAD box” sequence, associated with ATPase activity; the sequence SAT, associated with the actual helicase unwinding region; and an octapeptide consensus sequence, required for RNA binding and ATP hydrolysis (Pause, A. et al. (1993) Mol. Cell Biol. 13 :6789-6798). Differences outside of these conserved regions are believed to reflect differences in the functional roles of individual proteins (Chang et al., supra).
- DEAD-box helicases play tissue- and stage-specific roles in spermatogenesis and embryogenesis.
- Overexpression of the DEAD-box 1 protein (DDXl) may play a role in the progression of neuroblastoma (Nb) and retinoblastoma (Rb) tumors (Godbout, R. et al. (1998) J. Biol. Chem. 273:21161-21168).
- Nb neuroblastoma
- Rb retinoblastoma
- DDXl may promote or enhance tamor progression by altering the normal secondary structure and expression levels of RNA in cancer cells.
- Other DEAD-box helicases have been implicated either directly or indirectly in tumorigenesis (Godbout et al., supra).
- murine p68 is mutated in ultraviolet light-induced tumors
- human DDX6 is located at a chromosomal breakpoint associated with B-cell lymphoma.
- a chimeric protein comprised of DDXl 0 and NUP98 , a nucleoporin protein, may be involved in the pathogenesis of certain myeloid malignancies. Topoisomerases
- DNA topoisomerase effectively acts as a reversible nuclease that hydrolyzes a phosphodiesterase bond in a DNA strand, permits the two strands to rotate freely about one another to remove the strain of the helix, and then rejoins the original phosphodiester bond between the two strands.
- Topoisomerases are essential enzymes responsible for the topological rea ⁇ angement of DNA brought about by transcription, replication, chromatin formation, recombination, and chromosome segregation.
- Superhelical coils are introduced into DNA by the passage of processive enzymes such as RNA polymerase, or by the separation of DNA strands by a helicase prior to replication. Knotting and concatenation can occur in the process of DNA synthesis, storage, and repair. All topoisomerases work by breaking a phosphodiester bond in the ribose- phosphate backbone of DNA. A catalytic tyrosine residue on the enzyme makes a nucleophilic attack on the scissile phosphodiester bond, resulting in a reaction intermediate in which a covalent bond is formed between the enzyme and one end of the broken strand.
- a tyrosine-DNA phosphodiesterase functions in DNA repair by hydrolyzing this bond in occasional dead-end topoisomerase I-DNA intermediates (Pouliot, JJ. et al. (1999) Science 286:552-555).
- Type I topoisomerases work as monomers, making a break in a single strand of DNA while type ⁇ topoisomerases, working as homodirners, cleave both strands.
- DNA Topoisomerase I causes a single-strand break in a DNA helix to allow the rotation of the two strands of the helix about the remaining phosphodiester bond in the opposite strand.
- DNA topoisomerase II causes a transient break in both strands of a DNA helix where two double helices cross over one another. This type of topoisomerase can efficiently separate two interlocked DNA circles (Alberts, supra, pp. 260-262).
- Topoisomerase II has been implicated in multi-drug resistance (MDR) as it appears to aid in the repair of DNA damage inflicted by DNA binding agents such as doxorubicin and vincristine.
- MDR multi-drug resistance
- topoisomerase I family includes topoisomerases I and HI (topo I and topo HI).
- the crystal structure of human topoisomerase I suggests that rotation about the intact DNA strand is partially controlled by the enzyme.
- protein-DNA interactions limit the rotation, which is driven by torsional strain in the DNA (Stewart, L. et al. (1998) Science
- topo I can be recognized by its catalytic tyrosine residue and a number of other conserved residues in the active site region. Topo I is thought to function during transcription. Two topo His are known in humans, and they are homologous to prokaryotic topoisomerase I, with a conserved tyrosine and active site signature specific to this family. Topo UI has been suggested to play a role in meiotic recombination. A mouse topo HI is highly expressed in testis tissue and its expression increases with the increase in the number of cells in pachytene (Seki, T. et al. (1998) J. Biol. Chem. 273:28553-28556). The topoisomerase H family includes two isozymes (H and H ⁇ ) encoded by different genes.
- Topo H cleaves double stranded DNA in a reproducible, nonrandom fashion, preferentially in an AT rich region, but the basis of cleavage site selectivity is not known.
- topo H is made up of four domains, the first two of which are structurally similar and probably distantly homologous to similar domains in eukaryotic topo I. The second domain bears the catalytic tyrosine, as well as a highly conserved pentapeptide.
- the H isoform appears to be responsible for unlinking DNA during chromosome segregation.
- Cell lines expressing H ⁇ but not H ⁇ suggest that H ⁇ is dispensable in cellular processes; however, H ⁇ knockout mice died perinatally due to a failure in neural development. That the major abnormalities occurred in predominantly late developmental events (neurogenesis) suggests that H ⁇ is needed not at mitosis, but rather during DNA repair (Yang, X. et al. (2000) Science 287:131-134).
- Topoisomerases have been implicated in a number of disease states, and topoisomerase poisons have proven to be effective anti-tumor drugs for some human malignancies. Topo I is mislocalized in Fanconi's anemia, and may be involved in the chromosomal breakage seen in this disorder (Wunder, E. (1984) Hum. Genet. 68:276-281). Overexpression of a truncated topo HI in ataxia-telangiectasia (A-T) cells partially suppresses the A-T phenotype, probably through a dominant negative mechanism. This suggests that topo HE is deregulated in A-T (Fritz, E. et al. (1997) Proc. Natl. Acad. Sci.
- A-T ataxia-telangiectasia
- Topo JJJ also interacts with the Bloom's Syndrome gene product, and has been suggested to have a role as a tumor suppressor (Wu, L. et al. (2000) J. Biol. Chem. 275:9636-9644). Abe ⁇ ant topo H activity is often associated with cancer or increased cancer risk. Greatly lowered topo H activity has been found in some, but not all A-T cell hues (Mohamed, R. et al. (1987) Biochem. Biophys. Res. Commun. 149:233-238).
- topo H can break DNA in the region of the A-T gene (ATM), which controls all DNA damage-responsive cell cycle checkpoints (Kaufmann, W.K. (1998) Proc. Soc. Exp. Biol. Med. 217:327-334).
- ATM A-T gene
- Topoisomerase poisons act by increasing the number of dead-end covalent DNA-enzyme complexes in the cell, ultimately triggering cell death pathways (Fortune, J.M. and N. Osheroff (2000) Prog. Nucleic Acid Res. Mol. Biol. 64:221-253; Guichard, S.M. and M.K. Danks (1999) Cu ⁇ . Opin.
- DNA recombination allows variation in the particular combination of genes present in an individual's genome, as well as the timing and level of expression of these genes (Alberts, supra, pp. 263-273).
- Two broad classes of genetic recombination are commonly recognized, general recombination and site-specific recombination.
- General recombination involves genetic exchange between any homologous pair of DNA sequences usually located on two copies of the same chromosome. The process is aided by enzymes, recombinases, that "nick" one strand of a DNA duplex more or less randomly and permit exchange with a complementary strand on another duplex. The process does not normally change the arrangement of genes in a chromosome.
- RNA METABOLISM Much of the regulation of gene expression in eucaryotic cells occurs at the posttranscriptional level. Messenger RNAs (mRNA), which are produced in the cell nucleus from primary transcripts of protein-encoding genes, are processed and transported to the cytoplasm where the protein synthesis machinery is located.
- RNA-binding proteins are a group of proteins that participate in the processing, editing, transport, localization, and posttranscriptional regulation of mRNAs, and comprise the protein component of ribosomes as well.
- the RNA-binding activity of many of these proteins is mediated by a series of RNA-binding motifs identified within them. These domains include the RNP motif, the arginine-rich motif, the RGG box, and the KH motif. (Reviewed in Burd, CG. and Dreyfuss, G. (1994) Science 265:615-621.)
- the RNP motif is the most widely found and best characterized of these motifs.
- the RNP motif is composed of 90-100 amino acids which form an RNA-binding domain and is found in one or more copies in proteins that bind pre-mRNA, mRNA, pre-ribosomal RNA, and small nuclear RNAs.
- the RNP motif is composed of two short sequences (RNP-1 and RNP-2) and a number of other mostly hydrophobic, conserved amino acids interspersed throughout the motif.
- Ribonucleic acid (RNA) is a linear single-stranded polymer of four nucleotides, ATP, CTP, UTP, and GTP.
- RNA is transcribed as a copy of deoxyribonucleic acid (DNA), the genetic material of the organism.
- DNA deoxyribonucleic acid
- RNA copies of the genetic material encode proteins or serve various structural, catalytic, or regulatory roles in organisms.
- RNA is classified according to its cellular localization and function.
- Messenger RNAs (mRNAs) encode polypeptides.
- Ribosomal RNAs (rRNAs) are assembled, along with ribosomal proteins, into ribosomes, which are cytoplasmic particles that translate mRNA into polypeptides.
- Transfer RNAs are cytosolic adaptor molecules that function in mRNA translation by recognizing both an mRNA codon and the amino acid that matches that codon.
- Heterogeneous nuclear RNAs include mRNA precursors and other nuclear RNAs of various sizes.
- Small nuclear RNAs are a part of the nuclear spliceosome complex that removes intervening, non-coding sequences (introns) and rejoins exons in pre-mRNAs.
- RNA Processing proteins are associated with RNA during its transcription from DNA, RNA processing, and translation of mRNA into protein. Proteins are also associated with RNA as it is used for structural, catalytic, and regulatory purposes.
- Ribosomal RNAs are assembled, along with ribosomal proteins, into ribosomes, which are cytoplasmic particles that translate messenger RNA (mRNA) into polypeptides.
- the eukaryotic ribosome is composed of a 60S (large) subunit and a 40S (small) subunit, which together form the 80S ribosome.
- ribosomes contain from 50 to over 80 different ribosomal proteins, depending on the organism. Ribosomal proteins are classified according to which subunit they belong (i.e., L, if associated with the large 60S large subunit or S if associated with the small 40S subunit).
- E. coli ribosomes have been the most thoroughly studied and contain 50 proteins, many of which are conserved in all life forms.
- the structures of nine ribosomal proteins have been solved to less than 3.0D resolution (i.e., S5, S6, S17, LI, L6, L9, L12, L14, L30), revealing common motifs, such as b-a-b protein folds in addition to acidic and basic RNA-binding motifs positioned between b-strands.
- Most ribosomal proteins are believed to contact rRNA directly (reviewed in Liljas, A. and M. Garber (1995) Curr. Opin. Struct. Biol. 5:721-727; see also Woodson, S.A. and N.B. Leontis (1998) Curr. Opin. Struct. Biol. 8:294-300; Ramakrishnan, V. and S.W. White (1998) Trends Biochem. Sci. 23:208-212).
- Ribosomal proteins may undergo post-translational modifications or interact with other ribosome-associated proteins to regulate translation.
- the highly homologous 40S ribosomal protein S6 kinases (S6K1 and S6K2) play a key role in the regulation of cell growth by controlling the biosynthesis of translational components which make up the protein synthetic apparatus (including the ribosomal proteins).
- S6K1 and S6K2 the highly homologous 40S ribosomal protein S6 kinases
- S6K1 and S6K2 the highly homologous 40S ribosomal protein S6 kinases
- S6K1 and S6K2 the highly homologous 40S ribosomal protein S6 kinases
- at least eight phosphorylation sites are believed to mediate kinase activation in a hierarchical fashion (Dufner and Thomas (1999) Exp. Cell. Res. 253 :100-109).
- ribosomal proteins have secondary functions independent of their involvement in protein biosynthesis. These proteins function as regulators of cell proliferation and, in some instances, as inducers of cell death.
- L13a human ribosomal protein L13a has been shown to induce apoptosis by a ⁇ esting cell growth in the G2/M phase of the cell cycle. Inhibition of expression of L13a induces apoptosis in target cells, which suggests that this protein is necessary, in the appropriate amount, for cell survival.
- Similar results have been obtained in yeast where inactivation of yeast homologues of L13a, rp22 and rp23, results in severe growth retardation and death.
- ribosomal protein L7
- ribosomal proteins may function as cell cycle checkpoints and compose a new family of cell proliferation regulators.
- the aminoacyl-tRNA acceptor site receives charged tRNAs (with the exception of the initiator-tRNA).
- the peptidyl-tRNA site (P site) binds the nascent polypeptide as the amino acid from the A site is added to the elongating chain.
- Deacylated tRNAs bind in the exit site (E site) prior to their release from the ribosome.
- RNA processing steps include capping at the 5' end with methylguanosine, polyadenylating the 3' end, and splicing to remove introns.
- the primary RNA transript from DNA is a faithful copy of the gene containing both exon and intron sequences, and the latter sequences must be cut out of the RNA transcript to produce a mRNA that codes for a protein.
- This "splicing" of the mRNA sequence takes place in the nucleus with the aid of a large, multicomponent ribonucleoprotein complex known as a spliceosome.
- the spliceosomal complex is comprised of five small nuclear ribonucleoprotein particles (snRNPs) designated Ul, U2, U4, U5, and U6.
- snRNPs small nuclear ribonucleoprotein particles
- Ul small nuclear ribonucleoprotein particles
- U2, U4, U5, and U6 small nuclear ribonucleoprotein particles
- Each snRNP contains a single species of snRNA and about ten proteins.
- the RNA components of some snRNPs recognize and base-pair with intron consensus sequences.
- the protein components mediate spliceosome assembly and the splicing reaction.
- Autoantibodies to snRNP proteins are found in the blood of patients with systemic lupus erythematosus (Stryer, supra, p. 863).
- hnRNPs Heterogeneous nuclear ribonucleoproteins
- Some examples of hnRNPs include the yeast proteins Hrplp, involved in cleavage and polyadenylation at the 3 'end of the RNA; Cbp80p, involved in capping the 5' end of the RNA; and Npl3p, a homolog of mammalian hnRNP Al, involved in export of mRNA from the nucleus (Shen, E.G. et al. (1998) Genes Dev. 12:679-691). HnRNPs have been shown to be important targets of the autoimmune response in rheumatic diseases (Biamonti et al., supra).
- RNA recognition motif RRM
- the RRM is about 80 amino acids in length and forms four ⁇ -strands and two ⁇ -helices a ⁇ anged in an / ⁇ sandwich.
- the RRM contains a core RNP-1 octapeptide motif along with su ⁇ ounding conserved sequences.
- examples of RNA-binding proteins which contain the above motifs include heteronuclear ribonucleoproteins which stabilize nascent RNA and factors which regulate alternative splicing.
- Alternative splicing factors include developmentally regulated proteins, specific examples of which have been identified in lower eukaryotes such as Drosophila melanogaster and Caenorhabditis elegans. These proteins play key roles in developmental processes such as pattern formation and sex determination, respectively (Hodgkin, J. et al. (1994) Development 120:3681-3689). The 3' ends of most eukaryote mRNAs are also posttranscriptionally modified by polyadenylation.
- Polyadenylation proceeds tlirough two enzymatically distinct steps: (i) the endonucleolytic cleavage of nascent mRNAs at ds-acting polyadenylation signals in the 3 -untranslated (non-coding) region and (ii) the addition of a poly(A) tract to the 5 'mRNA fragment.
- the presence of cw-acting RNA sequences is necessary for both steps. These sequences include 5 - AAUAAA-3' located 10-30 nucleotides upstream of the cleavage site and a less well-conserved GU ⁇ or U-rich sequence element located 10-30 nucleotides downstream of the cleavage site.
- CstF Cleavage stimulation factor
- CF I cleavage factor I
- CF H cleavage factor H
- CPSF polyadenylation specificity factor
- PAP poly(A) polymerase
- aaRSs aminoacyl-tRNA synthetases
- the aaRSs are essential proteins found in all living organisms.
- the aaRSs are responsible for the activation and co ⁇ ect attachment of an amino acid with its cognate tRNA, as the first step in protein biosynthesis.
- Prokaryotic organisms have at least twenty different types of aaRSs, one for each different amino acid, while eukaryotes usually have two aaRSs, a cytosolic form and a mitochondrial form, for each different amino acid.
- the 20 aaRS enzymes can be divided into two structural classes.
- Class I enzymes add amino acids to the 2' hydroxyl at the 3' end of tRNAs while Class H enzymes add amino acids to the 3 ' hydroxyl at the 3 ' end of tRNAs.
- Each class is characterized by a distinctive topology of the catalytic domain.
- Class I enzymes contain a catalytic domain based on the nucleotide-binding Rossman 'fold'. In particular, a consensus tetrapeptide motif is highly conserved (Prosite Document PDOC00161, Aminoacyl-transfer RNA synthetases class-I signature).
- Class I enzymes are specific for arginine, cysteine, glutamic acid, glutamine, isoleucine, leucine, methionine, tyrosine, tryptophan, and valine.
- Class H enzymes contain a central catalytic domain, which consists of a seven-stranded antiparallel ⁇ -sheet domain, as well as N- and C- terminal regulatory domains.
- Class H enzymes are separated into two groups based on the heterodimeric or homodimeric structure of the enzyme; the latter group is further subdivided by the structure of the N- and C-terminal regulatory domains (Hartlein, M. and S. Cusack (1995) J. Mol. Evol. 40:519-530).
- Class H enzymes are specific for alanine, asparagine, aspartic acid, glycine, histidine, lysine, phenylalanine, proline, serine, and threonine. Certain aaRSs also have editing functions. HeRS, for example, can misactivate valine to form
- Val-tRNA 1116 is cleared by a hydrolytic activity that destroys the mischarged product.
- This editing activity is located within a second catalytic site found in the connective polypeptide 1 region (CP1), a long insertion sequence within the Rossman fold domain of Class I enzymes (Schimmel, P. et al. (1998) FASEB J. 12:1599-1609).
- AaRSs also play a role in tRNA processing. It has been shown that mature tRNAs are charged with their respective amino acids in the nucleus before export to the cytoplasm, and charging may serve as a quality control mechanism to insure the tRNAs are functional (Martinis, S.A. et al. (1999) EMBO J. 18:4591-4596).
- polypeptide synthesis proceeds at a rate of approximately 40 amino acid residues per second.
- the rate of misincorporation during translation in on the order of 10 " and is primarily the result of aminoacyl-t-RNAs being charged with the inco ⁇ ect amino acid.
- Inco ⁇ ectly charged tRNA are toxic to cells as they result in the incorporation of inco ⁇ ect amino acid residues into an elongating polypeptide.
- the rate of translation is presumed to be a compromise between the optimal rate of elongation and the need for translational fidelity.
- Mathematical calculations predict that 10 "4 is indeed the maximum acceptable e ⁇ or rate for protein synthesis in a biological system (reviewed in Stryer, supra; and Watson, J. et al.
- a particularly e ⁇ or prone aminoacyl-tRNA charging event is the charging of tRNA Gln with Gin.
- Gram positive eubacteria, cyanobacteria, Archeae, and eukaryotic organelles possess a noncanonical pathway for the synthesis of Gln-tRNA GIn based on the transformation of Gh ⁇ -tRNA Gln (synthesized by Glu-tRNA synthetase, GluRS) using the enzyme Glu- tRNA G]n amidotransferase (Glu-AdT).
- the reactions involved in the transamidation pathway are as follows (Curnow, A.W. et al. (1997) Nucleic Acids Symposium 36:2-4):
- Asp-tRNA ⁇ 11 amidotransferase exists in Archaea, which transforms Asp- tRNA ⁇ 11 to Asn-tRNA ⁇ 11 .
- Formylase the enzyme that transforms Met-tRNA Met to fMet-tRNA Met in eubacteria, is likely to be a related enzyme.
- a hydrolytic activity has also been identified that destroys mischarged Val-tRNA" 0 (Schimmel, P. et al. (1998) FASEB J. 12:1599-1609).
- Glu-AdT One likely scenario for the evolution of Glu-AdT in primitive life forms is the absence of a specific glutaminyl-tRNA synthetase (GlnRS), requiring an alternative pathway for the synthesis of Gln-tRNA Gl11 .
- GlnRS glutaminyl-tRNA synthetase
- deletion of the Glu-AdT operon in Gram positive bacteria is lethal (Curnow, A.W. et al. (1997) Proc. Natl. Acad. Sci. USA 94:11819-11826).
- the existence of GluRS activity in other organisms has been infe ⁇ ed by the high degree of conservation in translation machinery in nature; however, GluRS has not been identified in all organisms, including Homo sapiens. Such an enzyme would be responsible for ensuring translational fidelity and reducing the synthesis of defective polypeptides.
- tyrosyl-tRNA synthetases In addition to their function in protein synthesis, specific aminoacyl tRNA synthetases also play roles in cellular fidelity, RNA splicing, RNA trafficking, apoptosis, and transcriptional and translational regulation.
- human tyrosyl-tRNA synthetase can be proteolyticalry cleaved into two fragments with distinct cytokine activities.
- the carboxy-terminal domain exhibits monocyte and leukocyte chemotaxis activity as well as stimulating production of myeloperoxidase, tumor necrosis factor- ⁇ , and tissue factor.
- the N-terminal domain binds to the interleukin-8 type A receptor and functions as an interleukin-8-like cytokine.
- Mitochondrial Neurospora crassa TyrRS and S. cerevisiae LeuRS are essential factors for certain group I intron splicing activities, and human mitochondrial LeuRS can substitute for the yeast LeuRS in a yeast null strain.
- Certain bacterial aaRSs are involved in regulating their own transcription or translation (Martinis et al, supra).
- aaRSs are able to synthesize diadenosine oligophosphates, a class of signalling molecules with roles in cell proliferation, differentiation, and apoptosis (Kisselev, L.L et al. (1998) FEBS Lett. 427:157-163; Vartanian, A. et al. (1999) FEBS Lett. 456:175-180).
- Autoantibodies against aminoacyl-tRNAs are generated by patients with autoimmune diseases such as rheumatic arthritis, dermatomyositis and polymyositis, and correlate strongly with complicating interstitial lung disease (ILD) (Freist, W. et al. (1999) Biol. Chem.
- tRNA Modifications The modified ribonucleoside, pseudouridine ( ⁇ ), is present ubiquitously in the anticodon regions of transfer RNAs (tRNAs), large and small ribosomal RNAs (rRNAs), and small nuclear RNAs (snRNAs).
- tRNAs transfer RNAs
- rRNAs large and small ribosomal RNAs
- snRNAs small nuclear RNAs
- y is the most common of the modified nucleosides (i.e., other than G, A, U, and C) present in tRNAs. Only a few yeast tRNAs that are not involved in protein synthesis do not contain ⁇ (Cortese, R. et al. (1974) J. Biol. Chem. 249:1103-1108).
- tRNA pseudouridine synthases have been the most extensively studied members of the family.
- thiol donor e.g., cysteine
- monovalent cation e.g., ammonia or potassium
- Additional cofactors or high energy molecules e.g., ATP or GTP
- ATP or GTP e.g., ATP or GTP
- eukaryotic pseudouridine synthases have been identified that appear to be specific for rRNA (reviewed in Smith, CM. and J.A. Steitz (1997) Cell 89:669-672) and a dual-specificity enzyme has been identified that uses both tRNA and rRNA substrates (Wrzesinski, J. et al. (1995) RNA 1: 437-448).
- RNA 1:886-891 This posttranscriptional modification is believed to stabilize tRNA structure by preventing the formation of alternative tRNA secondary and tertiary structures.
- Yeast tRNA ⁇ is unusual in that it does not contain this modification. The modification does not occur in eubacteria, presumably because the structure of tRNAs in these cells and organelles is sequence constrained and does not require posttranscriptional modification to prevent the formation of alternative structures (Steinberg, S. and R. Cedergren (1995) RNA 1:886-891, and references within).
- the enzyme responsible for the conversion of guanosine to m 2 2 G is a 63 kDa 5-adenosylmethionine (SAM)-dependent tRNA N ⁇ N imethyl-guanosine methyltransferase (also refe ⁇ ed to as the TRM1 gene product and herein refe ⁇ ed to as TRM) (Edqvist, J. (1995) Biochimie 77:54-61).
- SAM 5-adenosylmethionine
- Initiation of translation can be divided into three stages.
- the first stage brings an initiator transfer RNA (Met-tRNA f ) together with the 40S ribosomal subunit to form the 43S preinitiation complex.
- the second stage binds the 43 S preinitiation complex to the mRNA, followed by migration of the complex to the co ⁇ ect AUG initiation codon.
- the third stage brings the 60S ribosomal subunit to the 40S subunit to generate an 80S ribosome at the inititation codon.
- Regulation of translation primarily involves the first and second stage in the initiation process (Pain, V.M. (1996) Eur. J. Biochem. 236:747-771).
- eTF2 a guanine nucleotide binding protein
- eIF2B a guanine nucleotide exchange protein
- eIF3 a guanine nucleotide exchange protein
- eIF3 is also involved in association of the 40S ribosomal subunit with mRNA.
- Met-tRNA f , elFIA, eIF3, and 40S ribosomal subunit together make up the 43 S preinitiation complex (Pain, supra).
- eIF4F is a complex consistmg of three proteins: eIF4E, eIF4A, and eIF4G.
- eIF4E recognizes and binds to the mRNA 5 -terminal m 7 GTP cap
- eIF4A is a bidirectional RNA-dependent helicase
- eIF4G is a scaffolding polypeptide.
- eIF4G has three binding domains.
- eIF4G acts as a bridge between the 40S ribosomal subunit and the mRNA (Hentze, M.W. (1997) Science 275:500-501).
- the ability of eIF4F to initiate binding of the 43 S preinitiation complex is regulated by structural features of the mRNA.
- the mRNA molecule has an untranslated region (UTR) between the 5' cap and the AUG start codon. In some mRNAs tins region forms secondary structures that impede binding of the 43 S preinitiation complex.
- the helicase activity of eJJF4A is thought to function in removing this secondary structure to facilitate binding of the 43 S preinitiation complex (Pain, supra).
- Elongation is the process whereby additional amino acids are joined to the initiator methionine to form the complete polypeptide chain.
- the elongation factors EFl ⁇ , EFl ⁇ , and EF2 are involved in elongating the polypeptide chain following initiation.
- EFl ⁇ is a GTP-binding protein. In EFl ⁇ 's GTP-bound form, it brings an aminoacyl-tRNA to the ribosome's A site. The amino acid attached to the newly arrived aminoacyl-tRNA forms a peptide bond with the initiatior methionine.
- the GTP on EFl ⁇ is hydrolyzed to GDP, and EFl ⁇ -GDP dissociates from the ribosome.
- EFl ⁇ binds EFl ⁇ -GDP and induces the dissociation of GDP from EFl ⁇ , allowing EFl ⁇ to bind GTP and a new cycle to begin.
- EF-G another GTP-binding protein, catalyzes the translocation of tRNAs from the A site to the P site and finally to the E site of the ribosome. This allows the ribosome and the mRNA to remain attached during translation.
- the release factor eRF carries out termination of translation. eRF recognizes stop codons in the mRNA, leading to the release of the polypeptide chain from the ribosome.
- Expression profiling Microarrays are analytical tools used in bioanalysis. A microa ⁇ ay has a plurality of molecules spatially distributed over, and stably associated with, the surface of a solid support. Microarrays of polypeptides, polynucleotides, and/or antibodies have been developed and find use in a variety of applications, such as gene sequencing, monitoring gene expression, gene mapping, bacterial identification, drug discovery, and combinatorial chemistry. One area in particular in which microa ⁇ ays find use is in gene expression analysis.
- a ⁇ ay technology can provide a simple way to explore the expression of a single polymorphic gene or the expression profile of a large number of related or unrelated genes.
- arrays are employed to detect the expression of a specific gene or its variants.
- a ⁇ ays provide a platform for identifying genes that are tissue specific, are affected by a substance being tested in a toxicology assay, are part of a signaling cascade, carry out housekeeping functions, or are specifically related to a particular genetic predisposition, condition, disease, or disorder.
- EGFR to human mammary carcinoma has been particularly well studied (see Khazaie, K. et al. (1993) Cancer and Metastasis Rev. 12:255-274, and references cited therein for a review of this area).
- EGFR expression in breast tumor metastases is frequently elevated relative to the primary tamor, suggesting that EGFR is involved in tumor progression and metastasis. This is supported by accumulating evidence that EGF has effects on cell functions related to metastatic potential, such as cell motility, chemotaxis, secretion and differentiation.
- erbB receptors such as HER- 2/neu, HER-3 , and HER-4, and their ligands in breast cancer points to their functional importance in the pathogenesis of the disease, and may therefore provide targets for therapy of the disease (Bacus, S. S. et al. (1994) Am. J. Clin. Pathol. 102:S13-S24).
- a human secreted frizzled protein mRNA that is downregulated in breast tumors includes a human secreted frizzled protein mRNA that is downregulated in breast tumors; the matrix Gla protein which is overexpressed is human breast carcinoma cells; Drgl or RTP, a gene whose expression is diminished in colon, breast, and prostate tumors; maspin, a tumor suppressor gene downregulated in invasive breast carcinomas; and CaN19, a member of the S100 protein family, all of which are down regulated in mammary carcinoma cells relative to normal mammary epithelial cells (Zhou, Z. et al. (1998) Int. J. Cancer 78:95-99; Chen, L. et al. (1990) Oncogene 5:1391-1395; Ulrix, W.
- Cell lines derived from human mammary epithelial cells at various stages of breast cancer provide a useful model to study the process of malignant transformation and tamor progression as it has been shown that these cell lines retain many of the properties of their parental tumors for lengthy culture periods (Wistaba, II. et al. (1998) Clin. Cancer Res. 4:2931-2938). Such a model is particularly useful for comparing phenotypic and molecular characteristics of human mammary epithelial cells at various stages of malignant transformation.
- the immune system responds to infection or trauma by activating a cascade of events that coordinate the progressive selection, amplification, and mobilization of cellular defense mechanisms.
- a complex and balanced program of gene activation and repression is involved in this process.
- hyperactivity of the immune system as a result of improper or insufficient regulation of gene expression may result in considerable tissue or organ damage. This damage is well documented in immunological responses associated with arthritis, allergens, heart attack, stroke, and infections (Ha ⁇ ison's Principles of Internal Medicine, 13/e, McGraw Hill, Inc. and Teton Data Systems
- Staf50 for Stimulated trans-acting factor of 50 kDa
- Staf50 appears to mediate the antiviral activity of interferon by down-regulating the viral transcription directed by the long terminal repeat promoter region of human immunodeficiency virus type-1 in transfected cells (Tissot, C (1995) J. Biol. Chem. 270:14891-14898).
- DC Dendritic cells
- APC antigen presenting cells
- DC differentiate into separate subsets of mature immune cells that sustain and regulate immune responses following initial contact with antigen.
- DC subsets include those that preferentially induce particular T helper 1 (Thl) or T helper 2 (Th2) responses and those that regulate B cell responses.
- DC are being used with increasing frequency to manipulate immune responses, either to downregulate abe ⁇ ant autoimmune response or to enhance vaccination or tumor-specific response.
- DC are functionally specialized in co ⁇ elation with their particular differentiation state.
- CD34+ myeloid cells found in the bone marrow mature in response to signals into CD 14+ CDI lc+ monocytes.
- An innate or antigen non-specific response takes place initially when monocytes circulate to nonlymphoid tissues and respond to lipopolysaccharide (LPS), a bacterialfy-derived mitogen, and viruses.
- LPS lipopolysaccharide
- Such direct encounters with antigen cause secretion of pro-inflammatory cytokines that attract and regulate natural killer cells, macrophages, and eosinophils in the first line of defense against invading pathogens.
- Monocytes then mature into DC, which efficiently capture antigen through endocytosis and antigen-receptor uptake.
- Antigen processing and presentation trigger activation and differentiation into mature DC that express MHC class H molecules on the cell surface and efficiently activate T-cells, initiating antigen-specific T-cell and B-cell responses.
- T-cells activate DC througli CD40 ligand - CD40 interactions, which stimulate expression of the costimulatory molecules CD80 and CD86, the latter most potent in amplifying T-cell responses.
- DC interaction via CD40 with T cells also stimulates the production of inflammatory cytokines such as TNF alpha and IL-1.
- RANK a member of the TNF receptor family by its ligand, TRANCE, which is expressed on activated T cells, enhances the survival of DC through inhibition of apoptosis, thereby enhancing T cell activation.
- TRANCE a member of the TNF receptor family by its ligand
- PBMCs Human peripheral blood mononuclear cells
- PBMCs can be classified into discrete cellular populations representing the major components of the immune system.
- PBMCs contain about 52% lymphocytes (12% B lymphocytes, 40% T lymphocytes ⁇ 25% CD4+ and 15% CD8+ ⁇ ), 20% NK cells, 25% monocytes, and 3% various cells that include dendritic cells and progenitor cells.
- the proportions, as well as the biology of these cellular components tend to vary slightly between healthy individuals, depending on factors such as age, past medical history, and genetic backgrounds.
- Steroid Hormones Steroid Hormones
- Steroids are a class of lipid-soluble molecules, including cholesterol, bile acids, vitamin D, and hormones, that share a common four-ring structure based on cyclopentanoperhydrophenanthrene and that carrry out a wide variety of functions.
- Cholesterol for example, is a component of cell membranes that controls membrane fluidity. It is also a precursor for bile acids which solubilize lipids and facilitate absorption in the small intestine during digestion. Vitamin D regulates the absorption of calcium in the small intestine and controls the concentration of calcium in plasma.
- Steroid hormones produced by the adrenal cortex, ovaries, and testes, include glucocorticoids, mineralocorticoids, androgens, and estrogens.
- Glucocorticoids for example, increase blood glucose concentrations by regulation of gluconeogenesis in the liver, increase blood concentrations of fatty acids by promoting lipolysis in adipose tissues, modulate sensitivity to catcholamines in the central nervous system, and reduce inflammation.
- the principal mineralocorticoid, aldosterone, is produced by the adrenal cortex and acts on cells of the distal tubules of the kidney to enhance sodium ion reabsorption.
- Androgens produced by the interstitial cells of Leydig in the testis, include the male sex hormone testosterone, which triggers changes at puberty, the production of sperm and maintenance of secondary sexual characteristics.
- Female sex hormones, estrogen and progesterone are produced by the ovaries and also by the placenta and adrenal cortex of the fetus during pregnancy.
- Estrogen regulates female reproductive processes and secondary sexual characteristics.
- Progesterone regulates changes in the endometrium during the menstrual cycle and pregnancy.
- Progesterone a naturally occurring progestin, is primarily used to treat amenorrhea, abnormal uterine bleeding, or as a contraceptive. Endogenous progesterone is responsible for inducing secretory activity in the endometrium of the estrogen-primed uterus in preparation for the implantation of a fertilized egg and for the maintenance of pregnancy. It is secreted from the corpus luteu in response to luteinizing hormone (LH). The primary contraceptive effect of exogenous progestins involves the suppression of the midcycle surge of LH.
- LH luteinizing hormone
- progestins diffuse freely into target cells and bind to the progesterone receptor.
- Target cells include the female reproductive tract, the mammary gland, the hypothalamus, and the pitaitary. Once bound to the receptor, progestins slow the frequency of release of gonadotropin releasing hormone from the hypothalamus and blunt the pre-ovulatory LH surge, thereby preventing follicular maturation and ovulation.
- Progesterone has minimal estrogenic and androgenic activity. Progesterone is metabolized hepatically to pregnanediol and conjugated with glucuronic acid.
- MAH Medroxyprogesterone
- 6 ⁇ -methyl-17-hydroxyprogesterone is a synthetic progestin with a pharmacological activity about 15 times greater than progesterone.
- MAH is used for the treatment of renal and endometrial carcinomas, amenorrhea, abnormal uterine bleeding, and endometriosis associated with hormonal imbalance.
- MAH has a stimulatory effect on respiratory centers and has been used in cases of low blood oxygenation caused by sleep apnea, chronic obstructive pulmonary disease, or hypercapnia.
- Mifepristone also known as RU-486, is an antiprogesterone drug that blocks receptors of progesterone. It counteracts the effects of progesterone, which is needed to sustain pregnancy. Mifepristone induces spontaneous abortion when administered in early pregnancy followed by treatment with the prostaglandin, misoprostol. Further, studies show that mifepristone at a substantially lower dose can be Mghly effective as a postcoital contraceptive when administered within five days after unprotected intercourse, thus providing women with a "morning-after pill" in case of contraceptive failure or sexual assault. Mifepristone also has potential uses in the treatment of breast and ovarian cancers in cases in which tumors are progesterone-dependent.
- Mifepristone binds to glucocorticoid receptors and interferes with cortisol binding. Mifepristone also may act as an anti-glucocorticoid and be effective for treating conditions where cortisol levels are elevated such as AIDS, anorexia nervosa, ulcers, diabetes, Parkinson's disease, multiple sclerosis, and Alzheimer's disease.
- Danazol is a synthetic steroid derived from ethinyl testosterone.
- Danazol indirectly reduces estrogen production by lowering pituitary synthesis of folHcle-stimulating hormone and LH. Danazol also binds to sex hormone receptors in target tissues, thereby exhibiting anabolic, antiestrognic, and weakly androgenic activity. Danazol does not possess any progestogenic activity, and does not suppress normal pituitary release of corticotropin or release of cortisol by the adrenal glands. Danazol is used in the treatment of endometriosis to relieve pain and inhibit endometrial cell growth. It is also used to treat fibrocystic breast disease and hereditary angioedema.
- Corticosteroids are used to relieve inflammation and to suppress the immune response. They inhibit eosinophil, basopbil, and airway epithelial cell function by regulation of cytokines that mediate the inflammatory response. They inhibit leukocyte infiltration at the site of inflammation, interfere in the function of mediators of the inflammatory response, and suppress the humoral immune response. Corticosteroids are used to treat allergies, asthma, arthritis, and skin conditions. Beclomethasone is a synthetic glucocorticoid that is used to treat steroid-dependent asthma, to relieve symptoms associated with allergic or nonallergic (vasomotor) rhinitis, or to prevent recurrent nasal polyps following surgical removal.
- intranasal beclomethasone is 5000 times greater than those produced by hydrocortisone.
- Budesonide is a corticosteroid used to control symptoms associated with allergic rhinitis or asthma.
- Budesonide has high topical anti-inflammatory activity but low systemic activity.
- Dexamethasone is a synthetic glucocorticoid used in anti- inflammatory or immunosuppressive compositions. It is also used in inhalants to prevent symptoms of asthma. Due to its greater ability to reach the central nervous system, dexamethasone is usually the treatment of choice to control cerebral edema.
- Dexamethasone is approximately 20-30 times more potent than hydrocortisone and 5-7 times more potent than prednisone.
- Prednisone is metabolized in the liver to its active form, prednisolone, a glucocorticoid with anti-inflammatory properties.
- Prednisone is approximately 4 times more potent than hydrocortisone and the duration of action of prednisone is intermediate between hydrocortisone and dexamethasone.
- Prednisone is used to treat allograft rejection, asthma, systemic lupus erythematosus, arthritis, ulcerative colitis, and other inflammatory conditions.
- Betamethasone is a synthetic glucocorticoid with antiinflammatory and immunosuppressive activity and is used to treat psoriasis and fungal infections, such as athlete's foot and ringworm.
- lipocortins phospholipase A 2 inhibitory proteins
- Lipocortins in tarn, control the biosynthesis of potent mediators of inflammation such as prostaglandins and leukotrienes by inhibiting the release of the precursor molecule arachidonic acid.
- Proposed mechanisms of action include decreased IgE synthesis, increased number of ⁇ -adrenergic receptors on leukocytes, and decreased arachidonic acid metabolism.
- allergens bridge the IgE antibodies on the surface of mast cells, which triggers these cells to release chemotactic substances.
- Mast cell influx and activation therefore, is partially responsible for the inflammation and hyperirritability of the oral mucosa in asthmatic patients. This inflammation can be retarded by administration of corticosteroids.
- compositions including nucleic acids and proteins, for the diagnosis, prevention, and treatment of cell proliferative, neurological, reproductive, developmental, autoimmune/inflammatory, and DNA repair disorders, and infections.
- NAAP purified polypeptides, nucleic acid-associated proteins, referred to collectively as “NAAP” and individually as “NAAP-1,” “NAAP-2,” “NAAP-3,” “NAAP-4,” “NAAP-5,” “NAAP-6,” “NAAP-7,” “NAAP-8,” “NAAP-9,” “NAAP-10,” “NAAP- 11,”.”NAAP-12,” “NAAP-13,” “NAAP-14,” “NAAP-15,” “NAAP-16,” “NAAP-17, “NAAP-18,” “NAAP-19,” “NAAP-20,” “NAAP-21,” “NAAP-22,” “NAAP-23,” “NAAP-24,” “NAAP-25,” “NAAP-26,” “NAAP-27,” “NAAP-28,” “NAAP-29,” “NAAP-30,” “NAAP-31,” “NAAP-32,”
- NAAP-33 “NAAP-34,” “NAAP-35,” and “NAAP-36,” and methods for using these proteins and their encoding polynucleotides for the detection, diagnosis, and treatment of diseases and medical conditions.
- Embodiments also provide methods for utilizing the purified nucleic acid-associated proteins and/or their encoding polynucleotides for facilitating the drug discovery process, including determination of efficacy, dosage, toxicity, and pharmacology.
- Related embodiments provide methods for utilizing the purified nucleic acid-associated proteins and/or their encoding polynucleotides for investigating the pathogenesis of diseases and medical conditions.
- An embodiment provides an isolated polypeptide selected from the group consisting of a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:l- 36, b) a polypeptide comprising a naturally occurring amino acid sequence at least 90% identical or at least about 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 1-36, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:l-36, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:l-36.
- Another embodiment provides an isolated polypeptide comprising an amino acid sequence of SEQ ID NO:l-36.
- Still another embodiment provides an isolated polynucleotide encoding a polypeptide selected from the group consisting of a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ JTD NO:l-36, b) a polypeptide comprising a naturally occu ⁇ ing amino acid sequence at least 90% identical or at least about 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 1-36, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1-36, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:l-36.
- polynucleotide encodes a polypeptide selected from the group consisting of SEQ JD NO: 1-36. In an alternative embodiment, the polynucleotide is selected from the group consisting of SEQ ID NO:37-72.
- Still another embodiment provides a recombinant polynucleotide comprising a promoter sequence operably linked to a polynucleotide encoding a polypeptide selected from the group consisting of a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 1-36, b) a polypeptide comprising a naturally occurring amino acid sequence at least 90% identical or at least about 90% identical to an amino acid sequence selected from the group consisting of SEQ JD NO:l-36, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1-36, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ JD NO: 1-36.
- Another embodiment provides a cell transformed with the recombinant polynucleotide.
- Yet another embodiment provides a transgenic organism comprising the recombinant polynucleotide.
- Another embodiment provides a method for producing a polypeptide selected from the group consisting of a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO.1-36, b) a polypeptide comprising a naturally occurring amino acid sequence at least 90% identical or at least about 90% identical to an amino acid sequence selected from the group consisting of SEQ JD NO:l-36, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:l-36, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:l-36.
- the method comprises a) culturing a cell under conditions suitable for expression of the polypeptide, wherein said cell is transformed with a recombinant polynucleotide comprising a promoter sequence operably linked to a polynucleotide encoding the polypeptide, and b) recovering the polypeptide so expressed.
- Yet another embodiment provides an isolated antibody which specifically binds to a polypeptide selected from the group consisting of a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:l-36, b) a polypeptide comprising a naturally occurring amino acid sequence at least 90% identical or at least about 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NO:l-36, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ JD NO: 1-36, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:l-36.
- Still yet another embodiment provides an isolated polynucleotide selected from the group consistmg of a) a polynucleotide comprising a polynucleotide sequence selected from the group consisting of SEQ ID NO:37-72, b) a polynucleotide comprising a naturally occurring polynucleotide sequence at least 90% identical or at least about 90% identical to a polynucleotide sequence selected from the group consisting of SEQ ID NO:37-72, c) a polynucleotide complementary to the polynucleotide of a), d) a polynucleotide complementary to the polynucleotide of b), and e) an RNA equivalent of a)-d).
- the polynucleotide can comprise at least about 20, 30, 40, 60, 80, or 100 contiguous nucleotides.
- Yet another embodiment provides a method for detecting a target polynucleotide in a sample, said target polynucleotide being selected from the group consisting of a) a polynucleotide comprising a polynucleotide sequence selected from the group consisting of SEQ ID NO:37-72, b) a polynucleotide comprising a naturally occurring polynucleotide sequence at least 90% identical or at least about 90% identical to a polynucleotide sequence selected from the group consisting of SEQ ID NO:37-72, c) a polynucleotide complementary to the polynucleotide of a), d) a polynucleotide complementary to the polynucleotide of b), and e) an RNA equivalent of a)-d).
- a target polynucleotide being selected from the group consisting of a) a polynucleotide comprising a polynucleot
- the method comprises a) hybridizing the sample with a probe comprising at least 20 contiguous nucleotides comprising a sequence complementary to said target polynucleotide in the sample, and which probe specifically hybridizes to said target polynucleotide, under conditions whereby a hybridization complex is formed between said probe and said target polynucleotide or fragments thereof, and b) detecting the presence or absence of said hybridization complex.
- the method can include detecting the amount of the hybridization complex.
- the probe can comprise at least about 20, 30, 40, 60, 80, or 100 contiguous nucleotides.
- Still yet another embodiment provides a method for detecting a target polynucleotide in a sample, said target polynucleotide being selected from the group consisting of a) a polynucleotide comprising a polynucleotide sequence selected from the group consisting of SEQ ID NO:37-72, b) a polynucleotide comprising a naturally occurring polynucleotide sequence at least 90% identical or at least about 90% identical to a polynucleotide sequence selected from the group consisting of SEQ ID NO:37-72, c) a polynucleotide complementary to the polynucleotide of a), d) a polynucleotide complementary to the polynucleotide of b), and e) an RNA equivalent of a)-d).
- a target polynucleotide being selected from the group consisting of a) a polynucleotide comprising a polynucleo
- the method comprises a) amplifying said target polynucleotide or fragment thereof using polymerase chain reaction amplification, and b) detecting the presence or absence of said amplified target polynucleotide or fragment thereof.
- the method can include detecting the amount of the amplified target polynucleotide or fragment thereof.
- compositions comprising an effective amount of a polypeptide selected from the group consisting of a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:l-36, b) a polypeptide comprising a naturally occurring amino acid sequence at least 90% identical or at least about 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NO:l-36, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1-36, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ JD NO:l-36, and a pharmaceutically acceptable excipient.
- the composition can comprise an amino acid sequence selected from the group consisting of SEQ JD NO:l-36.
- Other embodiments provide a method of treating a disease or condition associated with decreased or abnormal expression of functional NAAP, comprising administering to a patient in need of such treatment the composition.
- Yet another embodiment provides a method for screening a compound for effectiveness as an agonist of a polypeptide selected from the group consisting of a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 1-36, b) a polypeptide comprising a naturally occurring amino acid sequence at least 90% identical or at least about 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NO:l-36, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:l-36, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:l-36.
- the method comprises a) exposing a sample comprising the polypeptide to a compound, and b) detecting agonist activity in the sample.
- Another embodiment provides a composition comprising an agonist compound identified by the method and a pharmaceutically acceptable excipient.
- Yet another embodiment provides a method of treating a disease or condition associated with decreased expression of functional NAAP, comprising administering to a patient in need of such treatment the composition.
- Still yet another embodiment provides a method for screening a compound for effectiveness as an antagonist of a polypeptide selected from the group consisting of a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:l-36, b) a polypeptide comprising a naturally occurring amino acid sequence at least 90% identical or at least about 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 1-36, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:l-36, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ JD NO:l-36.
- the method comprises a) exposing a sample comprising the polypeptide to a compound, and b) detecting antagonist activity in the sample.
- Another embodiment provides a composition comprising an antagonist compound identified by the method and a pharmaceutically acceptable excipient.
- Yet another embodiment provides a method of treating a disease or condition associated with overexpression of functional NAAP, comprising administering to a patient in need of such treatment the composition.
- Another embodiment provides a method of screening for a compound that specifically binds to a polypeptide selected from the group consisting of a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 1-36, b) a polypeptide comprising a naturally occurring amino acid sequence at least 90% identical or at least about 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 1-36, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1-36, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ JD NO: 1-36.
- the method comprises a) combining the polypeptide with at least one test compound under suitable conditions, and b) detecting binding of the polypeptide to the test compound, thereby identifying a compound that specifically binds to the polypeptide.
- Yet another embodiment provides a method of screening for a compound that modulates the activity of a polypeptide selected from the group consisting of a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:l-36, b) a polypeptide comprising a naturally occurring amino acid sequence at least 90% identical or at least about 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 1-36, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ JD NO:l-36, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1-36.
- the method comprises a) combining the polypeptide with at least one test compound under conditions permissive for the activity of the polypeptide, b) assessing the activity of the polypeptide in the presence of the test compound, and c) comparing the activity of the polypeptide in the presence of the test compound with the activity of the polypeptide in the absence of the test compound, wherein a change in the activity of the polypeptide in the presence of the test compound is indicative of a compound that modulates the activity of the polypeptide.
- Still yet another embodiment provides a method for screening a compound for effectiveness in altering expression of a target polynucleotide, wherein said target polynucleotide comprises a polynucleotide sequence selected from the group consistmg of SEQ JD NO:37-72, the method comprising a) exposing a sample comprising the target polynucleotide to a compound, b) detecting altered expression of the target polynucleotide, and c) comparing the expression of the target polynucleotide in the presence of varying amounts of the compound and in the absence of the compound.
- Another embodiment provides a method for assessing toxicity of a test compound, said method comprising a) treating a biological sample containing nucleic acids with the test compound; b) hybridizing the nucleic acids of the treated biological sample with a probe comprising at least 20 contiguous nucleotides of a polynucleotide selected from the group consisting of i) a polynucleotide comprising a polynucleotide sequence selected from the group consisting of SEQ ID NO:37-72, ii) a polynucleotide comprising a naturally occurring polynucleotide sequence at least 90% identical or at least about 90% identical to a polynucleotide sequence selected from the group consisting of SEQ JD NO:37-72, iii) a polynucleotide having a sequence complementary to i), iv) a polynucleotide complementary to the polynucleotide of ii), and v) an RNA equivalent of
- Hybridization occurs under conditions whereby a specific hybridization complex is formed between said probe and a target polynucleotide in the biological sample, said target polynucleotide selected from the group consisting of i) a polynucleotide comprising a polynucleotide sequence selected from the group consisting of SEQ ID NO:37-72, ii) a polynucleotide comprising a naturally occurring polynucleotide sequence at least 90% identical or at least about 90% identical to a polynucleotide sequence selected from the group consisting of SEQ JD NO:37-72, iii) a polynucleotide complementary to the polynucleotide of i), iv) a polynucleotide complementary to the polynucleotide of ii), and v) an RNA equivalent of i)-iv).
- the target polynucleotide can comprise a fragment of a polynucleotide selected from the group consisting of i)-v) above; c) quantifying the amount of hybridization complex; and d) comparing the amount of hybridization complex in the treated biological sample with the amount of hybridization complex in an untreated biological sample, wherein a difference in the amount of hybridization complex in the treated biological sample is indicative of toxicity of the test compound.
- Table 1 summarizes the nomenclature for full length polynucleotide and polypeptide embodiments of the invention.
- Table 2 shows the GenBank identification number and annotation of the nearest GenBank homolog, and the PROTEOME database identification numbers and annotations of PROTEOME database homologs, for polypeptide embodiments of the invention. The probability scores for the matches between each polypeptide and its homolog(s) are also shown.
- Table 3 shows structural features of polypeptide embodiments, including predicted motifs and domains, along with the methods, algorithms, and searchable databases used for analysis of the polypeptides.
- Table 4 lists the cDNA and/or genomic DNA fragments which were used to assemble polynucleotide embodiments, along with selected fragments of the polynucleotides.
- Table 5 shows representative cDNA libraries for polynucleotide embodiments.
- Table 6 provides an appendix which describes the tissues and vectors used for construction of the cDNA libraries shown in Table 5.
- Table 7 shows the tools, programs, and algorithms used to analyze polynucleotides and polypeptides, along with applicable descriptions, references, and threshold parameters.
- Table 8 shows single nucleotide polymorphisms found in polynucleotide embodiments, along with allele frequencies in different human populations.
- a host cell includes a plurality of such host cells
- an antibody is a reference to one or more antibodies and equivalents thereof known to those skilled in the art, and so forth.
- NAAP refers to the amino acid sequences of substantially purified NAAP obtained from any species, particularly a mammalian species, including bovine, ovine, porcine, murine, equine, and human, and from any source, whether natural, synthetic, semi-synthetic, or recombinant.
- agonist refers to a molecule which intensifies or mimics the biological activity of NAAP.
- Agonists may include proteins, nucleic acids, carbohydrates, small molecules, or any other compound or composition which modulates the activity of NAAP either by directly interacting with NAAP or by acting on components of the biological pathway in which NAAP participates.
- An "allelic variant” is an alternative form of the gene encoding NAAP. Allelic variants may result from at least one mutation in the nucleic acid sequence and may result in altered mRNAs or in polypeptides whose structure or function may or may not be altered. A gene may have none, one, or many allelic variants of its naturally occurring form. Common mutational changes which give rise to allelic variants are generally ascribed to natural deletions, additions, or substitutions of nucleotides. Each of these types of changes may occur alone, or in combination with the others, one or more times in a given sequence.
- altered nucleic acid sequences encoding NAAP include those sequences with deletions, insertions, or substitutions of different nucleotides, resulting in a polypeptide the same as NAAP or a polypeptide with at least one functional characteristic of NAAP. Included within this definition are polymorphisms which may or may not be readily detectable using a particular oligonucleotide probe of the polynucleotide encoding NAAP, and improper or unexpected hybridization to allelic variants, with a locus other than the normal chromosomal locus for the polynucleotide encoding NAAP.
- the encoded protein may also be "altered,” and may contain deletions, insertions, or substitutions of amino acid residues which produce a silent change and result in a functionally equivalent NAAP.
- Deliberate amino acid substitutions may be made on the basis of one or more similarities in polarity, charge, solubility, hydrophobicity, hydrophilicity, and/or the amphipathic nature of the residues, as long as the biological or immunological activity of NAAP is retained.
- negatively charged amino acids may include aspartic acid and glutamic acid
- positively charged amino acids may include lysine and arginine.
- Ainino acids with uncharged polar side chains having similar hydrophilicity values may include: asparagine and glutamine; and serine and threonine.
- Amino acids with uncharged side chains having similar hydrophilicity values may include: leucine, isoleucine, and valine; glycine and alanine; and phenylalanine and tyrosine.
- PCR polymerase chain reaction
- Antagonist refers to a molecule which inhibits or attenuates the biological activity of NAAP.
- Antagonists may include proteins such as antibodies, anticalins, nucleic acids, carbohydrates, small molecules, or any other compound or composition which modulates the activity of NAAP either by directly interacting with NAAP or by acting on components of the biological pathway in which NAAP participates.
- antibody refers to intact immunoglobulin molecules as well as to fragments thereof, such as Fab, F(ab') 2 , and Fv fragments, which are capable of binding an epitopic determinant.
- Antibodies that bind NAAP polypeptides can be prepared using intact polypeptides or using fragments containing small peptides of interest as the immunizing antigen.
- the polypeptide or oligopeptide used to immunize an animal e.g., a mouse, a rat, or a rabbit
- an animal e.g., a mouse, a rat, or a rabbit
- ca ⁇ iers that are chemically coupled to peptides include bovine serum albumin, thyroglobulin, and keyhole limpet hemocyanin (KLH). The coupled peptide is then used to immunize the animal.
- antigenic determinant refers to that region of a molecule (i.e., an epitope) that makes contact with a particular antibody.
- an antigenic determinant may compete with the intact antigen (i.e., the immunogen used to elicit the immune response) for binding to an antibody.
- aptamer refers to a nucleic acid or oligonucleotide molecule that binds to a specific molecular target.
- Aptamers are derived from an in vitro evolutionary process (e.g., SELEX (Systematic Evolution of Ligands by Exponential Enrichment), described in U.S. Patent No. 5,270,163), which selects for target-specific aptamer sequences from large combinatorial libraries.
- Aptamer compositions maybe double-stranded or single-stranded, and may include deoxyribonucleotides, ribonucleotides, nucleotide derivatives, or other nucleotide-like molecules.
- the nucleotide components of an aptamer may have modified sugar groups (e.g., the 2'-OH group of a ribonucleotide may be replaced by 2 -F or 2 -NB j ), which may improve a desired property, e.g., resistance to nucleases or longer lifetime in blood.
- Aptamers may be conjugated to other molecules, e.g., a high molecular weight carrier to slow clearance of the aptamer from the circulatory system. Aptamers maybe specifically cross-linked to their cognate ligands, e.g., by photo-activation of a cross-linker (Brody, E.N. and L. Gold (2000) J. Biotechnol. 74:5-13).
- RNA aptamer refers to an aptamer which is expressed in vivo.
- a vaccinia virus-based RNA expression system has been used to express specific RNA aptamers at high levels in the cytoplasm of leukocytes (Blind, M. et al. (1999) Proc. Natl. Acad. Sci. USA 96:3606-3610).
- spiegelmer refers to an aptamer which includes L-DNA, L-RNA, or other left- handed nucleotide derivatives or nucleotide-like molecules. Aptamers containing left-handed nucleotides are resistant to degradation by naturally occurring enzymes, which normally act on substrates containing right-handed nucleotides.
- antisense refers to any composition capable of base-pairing with the "sense" (coding) strand of a polynucleotide having a specific nucleic acid sequence.
- Antisense compositions may include DNA; RNA; peptide nucleic acid (PNA); oligonucleotides having modified backbone linkages such as phosphorothioates, methylphosphonates, orbenzylphosphonates; oligonucleotides having modified sugar groups such as 2'-methoxyethyl sugars or 2'-methoxyethoxy sugars; or oligonucleotides having modified bases such as 5-methyl cytosine, 2'-deoxyuracil, or 7-deaza-2'- deoxyguanosine.
- Antisense molecules may be produced by any method including chemical synthesis or transcription. Once introduced into a cell, the complementary antisense molecule base-pairs with a naturally occu ⁇ ing nucleic acid sequence produced by the cell to form duplexes which block either transcription or translation.
- the designation "negative” or “minus” can refer to the antisense strand, and the designation “positive” or “plus” can refer to the sense strand of a reference DNA molecule.
- biologically active refers to a protein having structural, regulatory, or biochemical functions of a naturally occurring molecule.
- immunologically active or “immunogenic” refers to the capability of the nataral, recombinant, or synthetic NAAP, or of any oligopeptide thereof, to induce a specific immune response in appropriate animals or cells and to bind with specific antibodies.
- “Complementary” describes the relationship between two single-stranded nucleic acid sequences that anneal by base-pairing. For example, 5'-AGT-3' pairs with its complement, 3'-TCA-5'.
- composition comprising a given polynucleotide and a “composition comprising a given polypeptide” can refer to any composition containing the given polynucleotide or polypeptide.
- the composition may comprise a dry formulation or an aqueous solution.
- Compositions comprising polynucleotides encoding NAAP or fragments of NAAP maybe employed as hybridization probes.
- the probes maybe stored in freeze-dried form and maybe associated with a stabilizing agent such as a carbohydrate.
- the probe may be deployed in an aqueous solution containing salts (e.g., NaCl), detergents (e.g., sodium dodecyl sulfate; SDS), and other components (e.g., Denhardt's solution, dry milk, salmon sperm DNA, etc.).
- salts e.g., NaCl
- detergents e.g., sodium dodecyl sulfate; SDS
- other components e.g., Denhardt's solution, dry milk, salmon sperm DNA, etc.
- Consensus sequence refers to a nucleic acid sequence which has been subjected to repeated DNA sequence analysis to resolve uncalled bases, extended using the XL-PCR kit (Applied Biosystems, Foster City CA) in the 5' and/or the 3' direction, and resequenced, or which has been assembled from one or more overlapping cDNA, EST, or genomic DNA fragments using a computer program for fragment assembly, such as the GELVIEW fragment assembly system (GCG, Madison WI) or Phrap (University of Washington, Seattle WA). Some sequences have been both extended and assembled to produce the consensus sequence.
- Consative amino acid substitations are those substitations that are predicted to least interfere with the properties of the original protein, i.e., the structure and especially the function of the protein is conserved and not significantly changed by such substitations.
- the table below shows amino acids which may be substituted for an original amino acid in a protein and which are regarded as conservative amino acid substitations.
- Conservative amino acid substitations generally maintain (a) the structure of the polypeptide backbone in the area of the substitution, for example, as a beta sheet or alpha helical conformation, (b) the charge or hydrophobicity of the molecule at the site of the substitution, and/or (c) the bulk of the side chain.
- a “deletion” refers to a change in the amino acid or nucleotide sequence that results in the absence of one or more amino acid residues or nucleotides.
- derivative refers to a chemically modified polynucleotide or polypeptide. Chemical modifications of a polynucleotide can include, for example, replacement of hydrogen by an alkyl, acyl, hydroxyl, or amino group.
- a derivative polynucleotide encodes a polypeptide which retains at least one biological or immunological function of the nataral molecule.
- a derivative polypeptide is one modified by glycosylation, pegylation, or any similar process that retains at least one biological or immunological function of the polypeptide from which it was derived.
- a “detectable label” refers to a reporter molecule or enzyme that is capable of generating a measurable signal and is covalently or noncovalently joined to a polynucleotide or polypeptide.
- “Differential expression” refers to increased or upregulated; or decreased, downregulated, or absent gene or protein expression, determined by comparing at least two different samples. Such comparisons may be carried out between, for example, a treated and an untreated sample, or a diseased and a normal sample.
- Exon shuffling refers to the recombination of different coding regions (exons). Since an exon may represent a structural or functional domain of the encoded protein, new proteins may be assembled through the novel reassortment of stable substructures, thus allowing acceleration of the evolution of new protein functions.
- a “fragment” is a unique portion of NAAP or a polynucleotide encoding NAAP which can be identical in sequence to, but shorter in length than, the parent sequence.
- a fragment may comprise up to the entire length of the defined sequence, minus one nucleotide/amino acid residue.
- a fragment may comprise from about 5 to about 1000 contiguous nucleotides or amino acid residues.
- a fragment used as a probe, primer, antigen, therapeutic molecule, or for other purposes maybe at least 5, 10, 15, 16, 20, 25, 30, 40, 50, 60, 75, 100, 150, 250 or at least 500 contiguous nucleotides or amino acid residues in length. Fragments maybe preferentially selected from certain regions of a molecule.
- a polypeptide fragment may comprise a certain length of contiguous amino acids selected from the first 250 or 500 amino acids (or first 25% or 50%) of a polypeptide as shown in a certain defined sequence.
- these lengths are exemplary, and any length that is supported by the specification, including the Sequence Listing, tables, and figures, maybe encompassed by the present embodiments.
- a fragment of SEQ ID NO:37-72 can comprise a region of unique polynucleotide sequence that specifically identifies SEQ ID NO:37-72, for example, as distinct from any other sequence in the genome from which the fragment was obtained.
- a fragment of SEQ JD NO:37-72 can be employed in one or more embodiments of methods of the invention, for example, in hybridization and amplification technologies and in analogous methods that distinguish SEQ ID NO:37-72 from related polynucleotides.
- the precise length of a fragment of SEQ HO NO.37-72 and the region of SEQ ID NO:37-72 to which the fragment corresponds are routinely determinable by one of ordinary skill in the art based on the intended purpose for the fragment.
- a fragment of SEQ ID NO:l-36 is encoded by a fragment of SEQ JD NO:37-72.
- a fragment of SEQ JD NO:l-36 can comprise a region of unique amino acid sequence that specifically identifies SEQ ID NO:l-36.
- a fragment of SEQ ID NO:l-36 can be used as an immunogenic peptide for the development of antibodies that specifically recognize SEQ ID NO:l-36.
- the precise length of a fragment of SEQ JD NO:l-36 and the region of SEQ ID NO:l-36 to which the fragment corresponds can be determined based on the intended purpose for the fragment using one or more analytical methods described herein or otherwise known in the art.
- a “full length” polynucleotide is one containing at least a translation initiation codon (e.g., methionine) followed by an open reading frame and a translation termination codon.
- a “full length” polynucleotide sequence encodes a “full length” polypeptide sequence.
- “Homology” refers to sequence similarity or, interchangeably, sequence identity, between two or more polynucleotide sequences or two or more polypeptide sequences.
- percent identity and % identity refer to the percentage of residue matches between at least two polynucleotide sequences aligned using a standardized algorithm. Such an algorithm may insert, in a standardized and reproducible way, gaps in the sequences being compared in order to optimize alignment between two sequences, and therefore achieve a more meaningful comparison of the two sequences.
- Percent identity between polynucleotide sequences may be determined using one or more computer algorithms or programs known in the art or described herein. For example, percent identity can be determined using the default parameters of the CLUSTAL V algorithm as incorporated into the MEGALIGN version 3.12e sequence alignment program. This program is part of the
- LASERGENE software package a suite of molecular biological analysis programs (DNASTAR, Madison WI).
- CLUSTAL V is described in Higgins and Sharp (1989; CABIOS 5:151-153) and in Higgins et al. (1992; CABIOS 8:189-191).
- the "weighted” residue weight table is selected as the default. Percent identity is reported by CLUSTAL V as the "percent similarity" between aligned polynucleotide sequences.
- NCBI National Center for Biotechnology Information
- BLAST Local Alignment Search Tool
- BLAST 2 Sequences can be accessed and used interactively at http://www.ncbi.nlm.nih.gov/gorf/bl2.html.
- the "BLAST 2 Sequences” tool can be used for both blastn and blastp (discussed below).
- BLAST programs are commonly used with gap and other parameters set to default settings. For example, to compare two nucleotide sequences, one may use blastn with the "BLAST 2 Sequences" tool Version 2.0.12 (April-21-2000) set at default parameters. Such default parameters maybe, for example:
- Percent identity may be measured over the length of an entire defined sequence, for example, as defined by a particular SEQ ID number, or maybe measured over a shorter length, for example, over the length of a fragment taken from a larger, defined sequence, for instance, a fragment of at least 20, at least 30, at least 40, at least 50, at least 70, at least 100, or at least 200 contiguous nucleotides.
- Such lengths are exemplary only, and it is understood that any fragment length supported by the sequences shown herein, in the tables, figures, or Sequence Listing, may be used to describe a length over which percentage identity may be measured.
- nucleic acid sequences that do not show a high degree of identity may nevertheless encode similar amino acid sequences due to the degeneracy of the genetic code. It is understood that changes in a nucleic acid sequence can be made using this degeneracy to produce multiple nucleic acid sequences that all encode substantially the same protein.
- percent identity and % identity refer to the percentage of residue matches between at least two polypeptide sequences aligned using a standardized algorithm.
- Methods of polypeptide sequence alignment are well-known. Some alignment methods take into account conservative amino acid substitations. Such conservative substitations, explained in more detail above, generally preserve the charge andjhydrophobicity at the site of substitution, thus preserving the structure (and therefore function) of the polypeptide.
- NCBI BLAST software suite may be used.
- BLAST 2 Sequences Version 2.0.12 (April-21-2000) withblastp set at default parameters.
- Such default parameters maybe, for example: Matrix: BLOSUM62
- Percent identity may be measured over the length of an entire defined polypeptide sequence, for example, as defined by a particular SEQ ID number, or maybe measured over a shorter length, for example, over the length of a fragment taken from a larger, defined polypeptide sequence, for instance, a fragment of at least 15, at least 20, at least 30, at least 40, at least 50, at least 70 or at least 150 contiguous residues.
- Such lengths are exemplary only, and it is understood that any fragment length supported by the sequences shown herein, in the tables, figures or Sequence Listing, maybe used to describe a length over which percentage identity may be measured.
- HACs Human artificial chromosomes
- HACs are linear microchromosomes which may contain DNA sequences of about 6 kb to 10 Mb in size and which contain all of the elements required for chromosome replication, segregation and maintenance.
- humanized antibody refers to an antibody molecule in which the amino acid sequence in the non-antigen binding regions has been altered so that the antibody more closely resembles a human antibody, and still retains its original binding ability.
- Hybridization refers to the process by which a polynucleotide strand anneals with a complementary strand through base pairing under defined hybridization conditions. Specific hybridization is an indication that two nucleic acid sequences share a high degree of complementarity. Specific hybridization complexes form under permissive annealing conditions and remain hybridized after the "washing" step(s). The washing step(s) is particularly important in determining the stringency of the hybridization process, with more stringent conditions allowing less non-specific binding, i.e., binding between pairs of nucleic acid strands that are not perfectly matched.
- Permissive conditions for annealing of nucleic acid sequences are routinely determinable by one of ordinary skill in the art and maybe consistent among hybridization experiments, whereas wash conditions maybe varied among experiments to achieve the desired stringency, and therefore hybridization specificity. Permissive annealing conditions occur, for example, at 68°C in the presence of about 6 x SSC, about 1% (w/v) SDS, and about 100 ⁇ g/ml sheared, denatured salmon sperm DNA.
- wash temperatures are typically selected to be about 5°C to 20°C lower than the thermal melting point (T j consult) for the specific sequence at a defined ionic strength and pH.
- T j consult thermal melting point
- the T m is the temperature (under defined ionic strength and pH) at which 50% of the target sequence hybridizes to a perfectly matched probe.
- High stringency conditions for hybridization between polynucleotides of the present invention include wash conditions of 68°C in the presence of about 0.2 x SSC and about 0.1% SDS, for 1 hour. Alternatively, temperatares of about 65 °C, 60°C, 55°C, or 42°C maybe used. SSC concentration may be varied from about 0.1 to 2 x SSC, with SDS being present at about 0.1%.
- blocking reagents are used to block non-specific hybridization. Such blocking reagents include, for instance, sheared and denatured salmon sperm DNA at about 100-200 ⁇ g/ml.
- Organic solvent such as formamide at a concentration of about 35-50% v/v
- RNA:DNA hybridizations Useful variations on these wash conditions will be readily apparent to those of ordinary skill in the art.
- Hybridization particularly under high stringency conditions, may be suggestive of evolutionary similarity between the nucleotides. Such similarity is strongly indicative of a similar role for the nucleotides and their encoded polypeptides.
- hybridization complex refers to a complex formed between two nucleic acids by virtue of the formation of hydrogen bonds between complementary bases.
- a hybridization complex may be formed in solution (e.g., C 0 t or R 0 t analysis) or formed between one nucleic acid present in solution and another nucleic acid immobilized on a solid support (e.g., paper, membranes, filters, chips, pins or glass slides, or any other appropriate substrate to which cells or their nucleic acids have been fixed).
- insertion and “addition” refer to changes in an amino acid or polynucleotide sequence resulting in the addition of one or more amino acid residues or nucleotides, respectively.
- Immuno response can refer to conditions associated with inflammation, trauma, immune disorders, or infectious or genetic disease, etc. These conditions can be characterized by expression of various factors, e.g., cytokines, chemokines, and other signaling molecules, which may affect cellular and systemic defense systems.
- factors e.g., cytokines, chemokines, and other signaling molecules, which may affect cellular and systemic defense systems.
- an “immunogenic fragment” is a polypeptide or oligopeptide fragment of NAAP which is capable of eliciting an immune response when introduced into a living organism, for example, a mammal.
- the term “immunogenic fragment” also includes any polypeptide or oligopeptide fragment of NAAP which is useful in any of the antibody production methods disclosed herein or known in the art.
- microarray refers to an a ⁇ angement of a plurality of polynucleotides, polypeptides, antibodies, or other chemical compounds on a substrate.
- element and “array element” refer to a polynucleotide, polypeptide, antibody, or other chemical compound having a unique and defined position on a microa ⁇ ay.
- modulate refers to a change in the activity of NAAP. For example, modulation may cause an increase or a decrease in protein activity, binding characteristics, or any other biological, functional, or immunological properties of NAAP.
- nucleic acid and nucleic acid sequence refer to a nucleotide, oligonucleotide, polynucleotide, or any fragment thereof. These phrases also refer to DNA or RNA of genomic or synthetic origin which may be single-stranded or double-stranded and may represent the sense or the antisense strand, to peptide nucleic acid (PNA), or to any DNA-like or RNA-like material.
- PNA peptide nucleic acid
- operably linked refers to the situation in which a first nucleic acid sequencers placed in a functional relationship with a second nucleic acid sequence.
- a promoter is operably linked to a coding sequence if the promoter affects the transcription or expression of the coding sequence.
- Operably linked DNA sequences may be in close proximity or contiguous and, where necessary to join two protein coding regions, in the same reading frame.
- PNA protein nucleic acid
- PNA refers to an antisense molecule or anti-gene agent which comprises an oligonucleotide of at least about 5 nucleotides in length linked to a peptide backbone of amino acid residues ending in lysine. The terminal lysine confers solubility to the composition. PNAs preferentially bind complementary single stranded DNA or RNA and stop transcript elongation, and may be pegylated to extend their lifespan in the cell.
- Post-translational modification of an NAAP may involve lipidation, glycosylation, phosphorylation, acetylation, racemization, proteolytic cleavage, and other modifications known in the art. These processes may occur synthetically or biochemically. Biochemical modifications will vary by cell type depending on the enzymatic milieu of NAAP.
- Probe refers to nucleic acids encoding NAAP, their complements, or fragments thereof, which are used to detect identical, allelic or related nucleic acids.
- Probes are isolated oligonucleotides or polynucleotides attached to a detectable label or reporter molecule. Typical labels include radioactive isotopes, ligands, chemiluminescent agents, and enzymes.
- Primmers are short nucleic acids, usually DNA oligonucleotides, which may be annealed to a target polynucleotide by complementary base-pairing. The primer may then be extended along the target DNA strand by a DNA polymerase enzyme. Primer pairs can be used for amplification (and identification) of a nucleic acid, e.g., by the polymerase chain reaction (PCR).
- PCR polymerase chain reaction
- Probes and primers as used in the present invention typically comprise at least 15 contiguous nucleotides of a known sequence. In order to enhance specificity, longer probes and primers may also be employed, such as probes and primers that comprise at least 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, or at least 150 consecutive nucleotides of the disclosed nucleic acid sequences. Probes and primers may be considerably longer than these examples, and it is understood that any length supported by the specification, including the tables, figures, and Sequence Listing, maybe used.
- PCR primer pairs can be derived from a known sequence, for example, by using computer programs intended for that purpose such as Primer (Version 0.5, 1991, Whitehead Institute for Biomedical Research, Cambridge MA). Oligonucleotides for use as primers are selected using software known in the art for such purpose.
- OLIGO 4.06 software is useful for the selection of PCR primer pairs of up to 100 nucleotides each, and for the analysis of oligonucleotides and larger polynucleotides of up to 5,000 nucleotides from an input polynucleotide sequence of up to 32 kilobases.
- Similar primer selection programs have incorporated additional features for expanded capabilities.
- the PrimOU primer selection program (available to the public from the Genome Center at University of Texas South West Medical Center, Dallas TX) is capable of choosing specific primers from megabase sequences and is thus useful for designing primers on a genome- wide scope.
- the Primer3 primer selection program (available to the public from the Whitehead Institute/MIT Center for Genome Research, Cambridge MA) allows the user to input a "mispriming library," in which sequences to avoid as primer binding sites are user-specified. Primer3 is useful, in particular, for the selection of ohgonucleotides for microa ⁇ ays. (The source code for the latter two primer selection programs may also be obtained from their respective sources and modified to meet the user's specific needs.)
- the PrimeGen program (available to the public from the UK Human Genome Mapping Project Resource Centre, Cambridge UK) designs primers based on multiple sequence alignments, thereby allowing selection of primers that hybridize to either the most conserved or least conserved regions of aligned nucleic acid sequences.
- this program is useful for identification of both unique and conserved ohgonucleotides and polynucleotide fragments.
- the oligonucleotides and polynucleotide fragments identified by any of the above selection methods are useful in hybridization technologies, for example, as PCR or sequencing primers, microa ⁇ ay elements, or specific probes to identify fully or partially complementary polynucleotides in a sample of nucleic acids. Methods of oligonucleotide selection are not limited to those described above.
- a "recombinant nucleic acid” is a nucleic acid that is not naturally occurring or has a sequence that is made by an artificial combination of two or more otherwise separated segments of sequence. This artificial combination is often accomplished by chemical synthesis or, more commonly, by the artificial manipulation of isolated segments of nucleic acids, e.g., by genetic engineering techniques such as those described in Sambrook, supra.
- the term recombinant includes nucleic acids that have been altered solely by addition, substitution, or deletion of a portion of the nucleic acid.
- a recombinant nucleic acid may include a nucleic acid sequence operably linked to a promoter sequence. Such a recombinant nucleic acid may be part of a vector that is used, for example, to transform a cell.
- such recombinant nucleic acids may be part of a viral vector, e.g., based on a vaccinia virus, that could be use to vaccinate a mammal wherein the recombinant nucleic acid is expressed, inducing a protective immunological response in the mammal.
- a “regulatory element” refers to a nucleic acid sequence usually derived from untranslated regions of a gene and includes enhancers, promoters, introns, and 5' and 3' untranslated regions (UTRs). Regulatory elements interact with host or viral proteins which control transcription, translation, or RNA stability.
- Reporter molecules are chemical or biochemical moieties used for labeling a nucleic acid, amino acid, or antibody. Reporter molecules include radionuclides; enzymes; fluorescent, chemiluminescent, or chromogenic agents; substrates; cof actors; inhibitors; magnetic particles; and other moieties known in the art.
- An "RNA equivalent,” in reference to a DNA molecule, is composed of the same linear sequence of nucleotides as the reference DNA molecule with the exception that all occurrences of the nitrogenous base thymine are replaced with uracil, and the sugar backbone is composed of ribose instead of deoxyribose.
- sample is used in its broadest sense.
- a sample suspected of containing NAAP, nucleic acids encoding NAAP, or fragments thereof may comprise a bodily fluid; an extract from a cell, chromosome, organelle, or membrane isolated from a cell; a cell; genomic DNA, RNA, or cDNA, in solution or bound to a substrate; a tissue; a tissue print; etc.
- binding and “specifically binding” refer to that interaction between a protein or peptide and an agonist, an antibody, an antagonist, a small molecule, or any natural or synthetic binding composition. The interaction is dependent upon the presence of a particular structure of the protein, e.g., the antigenic determinant or epitope, recognized by the binding molecule. For example, if an antibody is specific for epitope "A,” the presence of a polypeptide comprising the epitope A, or the presence of free unlabeled A, in a reaction containing free labeled A and the antibody will reduce the amount of labeled A that binds to the antibody.
- substantially purified refers to nucleic acid or amino acid sequences that are removed from their natural environment and are isolated or separated, and are at least about 60% free, preferably at least about 75% free, and most preferably at least about 90% free from other components with which they are naturally associated.
- substitution refers to the replacement of one or more amino acid residues or nucleotides by different amino acid residues or nucleotides, respectively.
- Substrate refers to any suitable rigid or semi-rigid support including membranes, filters, chips, slides, wafers, fibers, magnetic or nonmagnetic beads, gels, tubing, plates, polymers, microparticles and capillaries.
- the substrate can have a variety of surface forms, such as wells, trenches, pins, channels and pores, to which polynucleotides or polypeptides are bound.
- a “transcript image” or “expression profile” refers to the collective pattern of gene expression by a particular cell type or tissue under given conditions at a given time.
- Transformation describes a process by which exogenous DNA is introduced into a recipient cell. Transformation may occur under natural or artificial conditions according to various methods well known in the art, and may rely on any known method for the insertion of foreign nucleic acid sequences into a prokaryotic or eukaryotic host cell. The method for transformation is selected based on the type of host cell being transformed and may include, but is not limited to, bacteriophage or viral infection, electroporation, heat shock, lipofection, and particle bombardment.
- transformed cells includes stably transformed cells in which the inserted DNA is capable of replication either as an autonomously replicating plasmid or as part of the host chromosome, as well as transiently transformed cells which express the inserted DNA or RNA for limited periods of time.
- a "transgenic organism,” as used herein, is any organism, including but not limited to animals and plants, in which one or more of the cells of the organism contains heterologous nucleic acid introduced by way of human intervention, such as by transgenic techniques well known in the art.
- the nucleic acid is introduced into the cell, directly or indirectly by introduction into a precursor of the cell, by way of deliberate genetic manipulation, such as by microinjection or by infection with a recombinant virus.
- the nucleic acid can be introduced by infection with a recombinant viral vector, such as a lentiviral vector (Lois, C et al. (2002) Science 295:868-872).
- the term genetic manipulation does not include classical cross-breeding, or in vitro fertilization, but rather is directed to the introduction of a recombinant DNA molecule.
- the transgenic organisms contemplated in accordance with the present invention include bacteria, cyanobacteria, fungi, plants and animals.
- the isolated DNA of the present invention can be introduced into the host by methods known in the art, for example infection, transfection, transformation or transconjugation. Techniques for transferring the DNA of the present invention into such organisms are widely known and provided in references such as Sambrook et al, supra.
- a "variant" of a particular nucleic acid sequence is defined as a nucleic acid sequence having at least 40% sequence identity to the particular nucleic acid sequence over a certain length of one of the nucleic acid sequences using blastn with the "BLAST 2 Sequences" tool Version 2.0.9 (May-07- 1999) set at default parameters.
- Such a pair of nucleic acids may show, for example, at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% or greater sequence identity over a certain defined length.
- a variant maybe described as, for example, an "allelic” (as defined above), "splice,” “species,” or “polymorphic” variant.
- a splice variant may have significant identity to a reference molecule, but will generally have a greater or lesser number of polynucleotides due to alternate splicing of exons during mRNA processing.
- the co ⁇ esponding polypeptide may possess additional functional domains or lack domains that are present in the reference molecule.
- Species variants are polynucleotides that vary from one species to another. The resulting polypeptides will generally have significant amino acid identity relative to each other.
- a polymorphic variant is a variation in the polynucleotide sequence of a particular gene between individuals of a given species.
- Polymorphic variants also may encompass "single nucleotide polymorphisms" (SNPs) in which the polynucleotide sequence varies by one nucleotide base.
- SNPs single nucleotide polymorphisms
- the presence of SNPs may be indicative of, for example, a certain population, a disease state, or a propensity for a disease state.
- a "variant" of a particular polypeptide sequence is defined as a polypeptide sequence having at least 40% sequence identity to the particular polypeptide sequence over a certain length of one of the polypeptide sequences using blastp with the "BLAST 2 Sequences" tool Version 2.0.9 (May-07- 1999) set at default parameters.
- Such a pair of polypeptides may show, for example, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% or greater sequence identity over a certain defined length of one of the polypeptides.
- NAAP nucleic acid-associated proteins
- Table 1 summarizes the nomenclature for the full length polynucleotide and polypeptide embodiments of the invention. Each polynucleotide and its corresponding polypeptide are correlated to a single Incyte project identification number (Incyte Project JD).
- Each polypeptide sequence is denoted by both a polypeptide sequence identification number (Polypeptide SEQ JD NO:) and an Incyte polypeptide sequence number (Incyte Polypeptide JD) as shown.
- Each polynucleotide sequence is denoted by both a polynucleotide sequence identification number (Polynucleotide SEQ ID NO:) and an Incyte polynucleotide consensus sequence number (Incyte Polynucleotide JD) as shown.
- Column 6 shows the Incyte JD numbers of physical, full length clones co ⁇ esponding to polypeptide and polynucleotide embodiments. The full length clones encode polypeptides which have at least 95% sequence identity to the polypeptides shown in column 3.
- Table 2 shows sequences with homology to the polypeptides of the invention as identified by BLAST analysis against the GenBank protein (genpept) database and the PROTEOME database.
- Columns 1 and 2 show the polypeptide sequence identification number (Polypeptide SEQ HO NO:) and the co ⁇ esponding Incyte polypeptide sequence number (Incyte Polypeptide ID) for polypeptides of the invention.
- Column 3 shows the GenBank identification number (GenBank JD NO:) of the nearest GenBank homolog and the PROTEOME database identification numbers (PROTEOME ID NO:) of the nearest PROTEOME database homologs.
- Column 4 shows the probability scores for the matches between each polypeptide and its homolog(s).
- Column 5 shows the annotation of the GenBank and PROTEOME database homolog(s) along with relevant citations where applicable, all of which are expressly incorporated by reference herein.
- Table 3 shows various structural features of the polypeptides of the invention.
- Columns 1 and 2 show the polypeptide sequence identification number (SEQ HO NO:) and the corresponding Incyte polypeptide sequence number (Incyte Polypeptide JD) for each polypeptide of the invention.
- Column 3 shows the number of amino acid residues in each polypeptide.
- Column 4 shows potential phosphorylation sites, and column 5 shows potential glycosylation sites, as determined by the MOTIF'S program of the GCG sequence analysis software package (Genetics Computer Group, Madison WI).
- Column 6 shows amino acid residues comprising signatare sequences, domains, and motifs.
- Column 7 shows analytical methods for protein structure/function analysis and in some cases, searchable databases to which the analytical methods were applied.
- SEQ ID NO:l is 88% identical, from residue Ml to residue L304, to mouse genomic screen homeobox protein 2 (GenBank JD gl042009) as determined by the Basic Local Ahgnment Search Tool (BLAST).
- the BLAST probability score is 2. le-146, which indicates the probability of obtaining the observed polypeptide sequence alignment by chance.
- SEQ HO NO: 1 also contains a homeobox domain as determined by searching for statistically significant matches in the hidden Markov model (HMM)-based PFAM database of conserved protein family domains. (See Table 3.) Data from BLIMPS, MOTIFS, PROFILESCAN, and additional BLAST analyses provide further corroborative evidence that SEQ HO NO: 1 is a homeobox protein.
- HMM hidden Markov model
- SEQ HO NO: 8 is 99% identical, from residue G24 to residue E384, to human DNA-binding protein B (GenBank HO gl81486) as determined by the Basic Local Alignment Search Tool (BLAST). (See Table 2.) The BLAST probability score is 5.8e-199, which indicates the probability of obtaining the observed polypeptide sequence ahgnment by chance. SEQ HO NO:8 also contains 'cold-shock' DNA-binding domain as determined by searching for statistically significant matches in the hidden Markov model (HMM)-based PFAM database of conserved protein family domains. (See Table 3.) Data from BLIMPS, MOTIFS, and PROFILESCAN analyses provide further co ⁇ oborative evidence that SEQ ID NO: 8 is a DNA-binding protein.
- HMM hidden Markov model
- SEQ JD NO:13 is 94% identical, from residue M780 to residue E1598, to human centriole associated protein CEP110 (GenBank HO g3435244) as determined by the Basic Local Ahgnment Search Tool (BLAST). (See Table 2.) The BLAST probability score is 0.0, which indicates the probability of obtaining the observed polypeptide sequence ahgnment by chance. Data from MOTIFS and further BLAST analyses provide co ⁇ oborative evidence that SEQ HO NO: 13 is a centriole associated protein.
- BLAST Basic Local Ahgnment Search Tool
- SEQ ID NO: 15 is 87% identical, from residue E435 to residue L2523, to human bromodomain PHD finger transcription factor (GenBank BO g6683492) as determined by the Basic Local Ahgnment Search Tool (BLAST).
- BLAST Basic Local Ahgnment Search Tool
- the BLAST probability score is 0.0, which indicates the probability of obtaining the observed polypeptide sequence alignment by chance.
- SEQ HO NO: 15 also contains a PHD finger domain and a bromodomain as determined by searching for statistically significant matches in the hidden Markov model (HMM)-based PFAM database of conserved protein family domains.
- HMM hidden Markov model
- PROFILESCAN analyses provide further co ⁇ oborative evidence that SEQ ID NO: 15 is a bromodomain PHD finger transcription factor.
- SEQ HO NO:21 is 41% identical, from residue T141 to residue E370, to human Kurppel-like zinc finger protein HZF2 (GenBank HO g8163824) as determined by the Basic Local Ahgnment Search Tool (BLAST).
- BLAST Basic Local Ahgnment Search Tool
- the BLAST probability score is 6.7e- 71, which indicates the probability of obtaining the observed polypeptide sequence alignment by chance.
- SEQ HO NO:21 also contains Zinc finger C2H2 type domains as determined by searching for statistically significant matches in the hidden Markov model (HMM)-based PFAM database of conserved protein family domains. (See Table 3.) Data from BLIMPS and MOTIFS analyses provide further co ⁇ oborative evidence that SEQ ID NO:21 is a C2H2 type zinc finger protein.
- SEQ HO NO:30 is 33% identical, from residue T556 to residue E1699, and 32% identical, from residue S10 to Y211, to Schizosaccharomyces pombe putative helicase
- SEQ ID NO:30 (GenBank ID g6901197) as determined by the Basic Local Ahgnment Search Tool (BLAST). (See Table 2.) The BLAST probability score is 2.9e-137, which indicates the probability of obtaining the observed polypeptide sequence alignment by chance. SEQ ID NO:30 also contains a DEAD DEAH box helicase domain as determined by searching for statistically significant matches in the hidden Markov model (HMM)-based PFAM database of conserved protein family domains. (See Table 3.) Data from BLAST analyses of the PRODOM and DOMO databases provide further corroborative evidence that SEQ HO NO:30 is a helicase.
- HMM hidden Markov model
- SEQ ID NO:33 is 97% identical, from residue Ml to residue V602, a murine T-box transcription factor (GenBank HO g3169261) as determined by the Basic Local Ahgnment Search Tool (BLAST).
- BLAST Basic Local Ahgnment Search Tool
- the BLAST probability score is 0.0, which indicates the probability of obtaining the observed polypeptide sequence ahgnment by chance.
- SEQ HO NO:33 also contains a T-box domain as determined by searching for statistically significant matches in the hidden Markov model (HMM)-based PFAM database of conserved protein family domains.
- HMM hidden Markov model
- Data from BLIMPS, PRODOM and DOMO BLAST, and MOTIFS analyses provide further co ⁇ oborative evidence that SEQ JD NO:33 is a transcription factor molecule.
- SEQ HO NO:l SEQ JD NO:8, SEQ BO NO:13, SEQ ID NO:15, SEQ HO NO:21, SEQ HO NO:30, and SEQ HO NO:33 are all molecules associated with nucleic acids.
- SEQ HO NO:2-7, SEQ ID NO:9-12, SEQ HO NO:14, SEQ HO NO:16- 20, SEQ HO NO:22-29, SEQ HO NO:31-32, and SEQ HO NO:34-36 were analyzed and annotated in a similar manner.
- the algorithms and parameters for the analysis of SEQ ID NO: 1-36 are described in Table 7.
- the full length polynucleotide embodiments were assembled using cDNA sequences or coding (exon) sequences derived from genomic DNA, or any combination of these two types of sequences.
- Column 1 hsts the polynucleotide sequence identification number (Polynucleotide SEQ ID NO:), the corresponding Incyte polynucleotide consensus sequence number (Incyte ID) for each polynucleotide of the invention, and the length of each polynucleotide sequence in basepairs.
- Column 2 shows the nucleotide start (5') and stop (3') positions of the cDNA and/or genomic sequences used to assemble the full length polynucleotide embodiments, and of fragments of the polynucleotides which are useful, for example, in hybridization or amplification technologies that identify SEQ HO NO:37-72 or that distinguish between SEQ HO NO:37-72 and related polynucleotides.
- the polynucleotide fragments described in Column 2 of Table 4 may refer specifically, for example, to Incyte cDNAs derived from tissue-specific cDNA libraries or from pooled cDNA libraries.
- the polynucleotide fragments described in column 2 may refer to GenBank cDNAs or ESTs which contributed to the assembly of the full length polynucleotides.
- the polynucleotide fragments described in column 2 may identify sequences derived from the ENSEMBL (The Sanger Centre, Cambridge, UK) database (Le., those sequences including the designation "ENST").
- the polynucleotide fragments described in column 2 may be derived from the NCBI RefSeq Nucleotide Sequence Records Database ⁇ ie., those sequences including the designation "NM” or “NT”) or the NCBI RefSeq Protein Sequence Records (i.e., those sequences including the designation "NP”).
- the polynucleotide fragments described in column 2 may refer to assemblages of both cDNA and Genscan-predicted exons brought together by an "exon stitching" algorithm.
- a polynucleotide sequence identified as FLJ ⁇ ⁇ Si_N 1 _N 2 _Y ⁇ YY ⁇ _N 3 _N 4 represents a "stitched" sequence in which XXXXX is the identification number of the cluster of sequences to which the algorithm was applied, and ITOTis the number of the prediction generated by the algorithm, and N 1A3 ..., if present, represent specific exons that may have been manually edited during analysis (See Example V).
- the polynucleotide fragments in column 2 may refer to assemblages of exons brought together by an "exon-stretching" algorithm.
- a polynucleotide sequence identified as ⁇ J XXXXX_gAAAAA_gBBBBB_l_N is a "stretched" sequence, with XXXXX being the Incyte project identification number, gAAAAA being the GenBank identification number of the human genomic sequence to which the "exon-stretching" algorithm was apphed, gBBBBB being the GenBank identification number or NCBI RefSeq identification number of the nearest GenBank protein homolog, and N referring to specific exons (See Example V).
- RefSeq identifier (denoted by " ⁇ M,” “ ⁇ P,” or “NT”) maybe used in place of the GenBank identifier ⁇ i.e., gBBBBB).
- a prefix identifies component sequences that were hand-edited, predicted from genomic DNA sequences, or derived from a combination of sequence analysis methods.
- the following Table lists examples of component sequence prefixes and co ⁇ esponding sequence analysis methods associated with the prefixes (see Example IV and Example V).
- Incyte cDNA coverage redundant with the sequence coverage shown in Table 4 was obtained to confirm the final consensus polynucleotide sequence, but the relevant Incyte cDNA identification numbers are not shown.
- Table 5 shows the representative cDNA libraries for those full length polynucleotides which were assembled using Incyte cDNA sequences.
- the representative cDNA library is the Incyte cDNA library which is most frequently represented by the Incyte cDNA sequences which were used to assemble and confirm the above polynucleotides.
- the tissues and vectors which were used to construct the cDNA libraries shown in Table 5 are described in Table 6.
- Table 8 shows single nucleotide polymorphisms (SNPs) found in polynucleotide embodiments, along with ahele frequencies in different human populations.
- Columns 1 and 2 show the polynucleotide sequence identification number (SEQ HO NO:) and the co ⁇ esponding Incyte project identification number (PflO) for polynucleotides of the invention.
- Column 3 shows the Incyte identification number for the EST in which the SNP was detected (EST ID), and column 4 shows the identification number for the SNP (SNP ID).
- EST SNP shows the position within the EST sequence at which the SNP is located (EST SNP), and column 6 shows the position of the SNP within the fuU-length polynucleotide sequence (CB1 SNP).
- CB1 SNP fuU-length polynucleotide sequence
- Column 7 shows the allele found in the EST sequence.
- Columns 8 and 9 show the two alleles found at the SNP site.
- Column 10 shows the amino acid encoded by the codon including the SNP site, based .upon the ahele found in the EST.
- Columns 11-14 show the frequency of ahele 1 in four different human populations. An entry of n/d (not detected) indicates that the frequency of ahele 1 in the population was too low to be detected, while n/a (not available) indicates that the ahele frequency was not determined for the population.
- NAAP variants are one which has at least about 80%, or alternatively at least about 90%, or even at least about 95% amino acid sequence identity to the NAAP amino acid sequence, and which contains at least one functional or structural characteristic of NAAP.
- polynucleotides which encode NAAP encompasses a polynucleotide sequence comprising a sequence selected from the group consisting of SEQ ID NO:37-72, which encodes NAAP.
- the polynucleotide sequences of SEQ HO NO:37-72, as presented in the Sequence Listing, embrace the equivalent RNA sequences, wherein occurrences of the nitrogenous base mymine are replaced with uracil, and the sugar backbone is composed of ribose instead of deoxyribose.
- the invention also encompasses variants of a polynucleotide encoding NAAP.
- a variant polynucleotide will have at least about 70%, or alternatively at least about 85%, or even at least about 95% polynucleotide sequence identity to a polynucleotide encoding NAAP.
- a particular aspect of the invention encompasses a variant of a polynucleotide comprising a sequence selected from the group consisting of SEQ ID NO:37-72 which has at least about 70%, or alternatively at least about 85%, or even at least about 95% polynucleotide sequence identity to a nucleic acid sequence selected from the group consisting of SEQ ID NO:37-72.
- Any one of the polynucleotide variants described above can encode a polypeptide which contains at least one functional or structural characteristic of NAAP.
- a polynucleotide variant of the invention is a sphce variant of a polynucleotide encoding NAAP.
- a sphce variant may have portions which have significant sequence identity to a polynucleotide encoding NAAP, but will generally have a greater or lesser number of polynucleotides due to additions or deletions of blocks of sequence arising from alternate splicing of exons during mRNA processing.
- a sphce variant may have less than about 70%, or alternatively less than about 60%, or alternatively less than about 50% polynucleotide sequence identity to a polynucleotide encoding NAAP over its entke length; however, portions of the splice variant wi have at least about 70%, or alternatively at least about 85%, or alternatively at least about 95%, or alternatively 100% polynucleotide sequence identity to portions of the polynucleotide encoding NAAP.
- a polynucleotide comprising a sequence of SEQ HO NO:72 is a sphce variant of a polynucleotide comprising a sequence of SEQ HO NO:50. Any one of the sphce variants described above can encode a polypeptide which contains at least one functional or structural characteristic of NAAP.
- polynucleotides which encode NAAP and its variants are generahy capable of hybridizing to polynucleotides encoding naturally occurring NAAP under appropriately selected conditions of stringency, it maybe advantageous to produce polynucleotides encoding NAAP or its derivatives possessing a substantially different codon usage, e.g., inclusion of non-naturally occurring codons. Codons may be selected to increase the rate at which expression of the peptide occurs in a particular prokaryotic or eukaryotic host in accordance with the frequency with which particular codons are utilized by the host.
- RNA transcripts having more desirable properties such as a greater half-hfe, than transcripts produced from the naturally occu ⁇ ing sequence.
- the invention also encompasses production of polynucleotides which encode NAAP and NAAP derivatives, or fragments thereof, entirely by synthetic chemistry.
- the synthetic polynucleotide maybe inserted into any of the many available expression vectors and cell systems using reagents weh known in the art.
- synthetic chemistry maybe used to introduce mutations into a polynucleotide encoding NAAP or any fragment thereof.
- Embodiments of the invention can also include polynucleotides that are capable of hybridizing to the claimed polynucleotides, and, in particular, to those having the sequences shown in SEQ HO NO:37-72 and fragments thereof, under various conditions of stringency (Wahl, G.M. and S.L. Berger (1987) Methods Enzymol. 152:399-407; Kimmel, A.R. (1987) Methods Enzymol. 152:507-511). Hybridization conditions, including annealing and wash conditions, are described in "Definitions.” Methods for DNA sequencing are weh known in the art and may be used to practice any of the embodiments of the invention.
- the methods may employ such enzymes as the Klenow fragment of DNA polymerase L SEQUENASE (US Biochemical, Cleveland OH), Taq polymerase (Applied Biosystems), thermostable T7 polymerase (Amersham Biosciences, Piscataway NJ), or combinations of polymerases and proofreading exonucleases such as those found in the ELONGASE amplification system (Invitrogen, Carlsbad CA).
- sequence preparation is automated with machines such as the MICROLAB 2200 liquid transfer system (Hamilton, Reno NV), PTC200 thermal cycler (MJ Research, Watertown MA) and ABI CATALYST 800 thermal cycler (Applied Biosystems).
- Sequencing is then carried out using either the ABI 373 or 377 DNA sequencing system (Applied Biosystems), the MEGABACE 1000 DNA sequencing system (Amersham Biosciences), or other systems known in the art.
- the resulting sequences are analyzed using a variety of algorithms which are weh known in the art (Ausubel, F.M. (1997) Short Protocols in Molecular Biology, John Wiley & Sons, New York NY, unit 7.7; Meyers, R.A. (1995) Molecular Biology and Biotechnology, Wiley VCH, New York NY, pp. 856-853).
- the nucleic acids encoding NAAP may be extended utilizing a partial nucleotide sequence and employing various PCR-based methods known in the art to detect upstream sequences, such as promoters and regulatory elements.
- various PCR-based methods known in the art to detect upstream sequences, such as promoters and regulatory elements.
- restriction-site PCR uses universal and nested primers to amplify unknown sequence from genomic DNA within a cloning vector (Sarkar, G. (1993) PCR Methods Apphc. 2:318-322).
- Another method, inverse PCR uses primers that extend in divergent directions to amplify unknown sequence from a circularized template.
- the template is derived from restriction fragments comprising a known genomic locus and su ⁇ ounding sequences (Trigha, T. et al.
- a third method involves PCR amplification of DNA fragments adjacent to known sequences in human and yeast artificial chromosome DNA (Lagerstrom, M. et al. (1991) PCR Methods Apphc. 1:111-119).
- multiple restriction enzyme digestions and hgations may be used to insert an engineered double-stranded sequence into a region of unknown sequence before performing PCR.
- Other methods which may be used to retrieve unknown sequences are known in the art (Parker, J.D. et al. (1991) Nucleic Acids Res. 19:3055-3060).
- primers may be designed using commercially available software, such as OLIGO 4.06 primer analysis software (National Biosciences, Plymouth MN) or another appropriate program, to be about 22 to 30 nucleotides in length, to have a GC content of about 50% or more, and to anneal to the template at temperatares of about 68°C to 72°C
- Genomic libraries may be useful for extension of sequence into 5' non-transcribed regulatory regions.
- Capillary electrophoresis systems which are commercially available may be used to analyze the size or confirm the nucleotide sequence of sequencing or PCR products.
- capillary sequencing may employ flowable polymers for electrophoretic separation, four different nucleotide- specific, laser-stimulated fluorescent dyes, and a charge coupled device camera for detection of the emitted wavelengths.
- Output/light intensity may be converted to electrical signal using appropriate software (e.g., GENOTYPER and SEQUENCE NAVIGATOR, Apphed Biosystems), and the entire process from loading of samples to computer analysis and electronic data display may be computer controlled.
- Capillary electrophoresis is especially preferable for sequencing small DNA fragments which may be present in limited amounts in a particular sample.
- polynucleotides or fragments thereof which encode NAAP maybe cloned in recombinant DNA molecules that direct expression of NAAP, or fragments or functional equivalents thereof, in appropriate host cells. Due to the inherent degeneracy of the genetic code, other polynucleotides which encode substantially the same or a functionahy equivalent polypeptides maybe produced and used to express NAAP.
- the polynucleotides of the invention can be engineered using methods generally known in the art in order to alter NAAP-encoding sequences for a variety of purposes including, but not limited to, modification of the cloning, processing, and/or expression of the gene product.
- DNA shuffling by random fragmentation and PCR reassembly of gene fragments and synthetic ohgonucleotides may be used to engineer the nucleotide sequences.
- ohgonucleotide-mediated site-directed mutagenesis maybe used to introduce mutations that create new restriction sites, alter glycosylation patterns, change codon preference, produce sphce variants, and so forth.
- the nucleotides of the present invention may be subjected to DNA shuffling techniques such as MOLECULARBREEDING (Maxygen Inc. ' , Santa Clara CA; described in U.S. Patent No. 5,837,458; Chang, C-C. et al. (1999) Nat. Biotechnol. 17:793-797; Christians, F.C et al. (1999) Nat. Biotechnol. 17:259-264; and Crameri, A. et al. (1996) Nat. Biotechnol. 14:315-319) to alter or improve the biological properties of NAAP, such as its biological or enzymatic activity or its ability to bind to other molecules or compounds.
- MOLECULARBREEDING Maxygen Inc. ' , Santa Clara CA; described in U.S. Patent No. 5,837,458; Chang, C-C. et al. (1999) Nat. Biotechnol. 17:793-797; Christians, F.C
- DNA shuffling is a process by which a library of gene variants is produced using PCR-mediated recombination of gene fragments. The library is then subjected to selection or screening procedures that identify those gene variants with the desired properties. These prefe ⁇ ed variants may then be pooled and further subjected to recursive rounds of DNA shuffling and selection/screening.
- genetic diversity is created through "artificial" breeding and rapid molecular evolution. For example, fragments of a single gene containing random point mutations may be recombined, screened, and then reshuffled until the desired properties are optimized.
- fragments of a given gene may be recombined with fragments of homologous genes in the same gene family, either from the same or different species, thereby maximizing the genetic diversity of multiple naturally occurring genes in a directed and controllable manner.
- polynucleotides encoding NAAP may be synthesized, in whole or in part, using one or more chemical methods weh known in the art (Carutliers, M.H. et al. (1980) Nucleic Acids Symp. Ser. 7:215-223; and Horn, T. et al. (1980) Nucleic Acids Symp. Ser. 7:225-232).
- NAAP itself or a fragment thereof may be synthesized using chemical methods known in the art. For example, peptide synthesis can be performed using various solution-phase or solid-phase techniques (Creighton, T. (1984) Proteins, Structares and Molecular Properties, WH
- NAAP amino acid sequence of NAAP, or any part thereof, may be altered during direct synthesis and/or combined with sequences from other proteins, or any part thereof, to produce a variant polypeptide or a polypeptide having a sequence of a naturally occurring polypeptide.
- the peptide may be substantially purified by preparative high performance liquid chromatography (Chiez, R.M. and F.Z. Regnier (1990) Methods Enzymol. 182:392-421).
- the composition of the synthetic peptides may be confirmed by amino acid analysis or by sequencing (Creighton, supra, pp. 28-53).
- the polynucleotides encoding NAAP or derivatives thereof may be inserted into an appropriate expression vector, i.e., a vector which contains the necessary elements for transcriptional and translational control of the inserted coding sequence in a suitable host.
- these elements include regulatory sequences, such as enhancers, constitutive and inducible promoters, and 5' and 3 'untranslated regions in the vector and in polynucleotides encoding NAAP. Such elements may vary in their strength and specificity.
- Specific initiation signals may also be used to achieve more efficient translation of polynucleotides encoding NAAP. Such signals include the ATG initiation codon and adjacent sequences, e.g. the Kozak sequence.
- Methods which are weh known to those skilled in the art may be used to construct expression vectors containing polynucleotides encoding NAAP and appropriate transcriptional and translational control elements. These methods include in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination (Sambrook et al, supra, ch. 4, 8, and 16-17; Ausubel, F.M. et al. (1995) Current Protocols in Molecular Biology, John Wiley & Sons, New York NY, ch. 9, 13, and 16).
- a variety of expression vector/host systems may be utilized to contain and express polynucleotides encoding NAAP. These include, but are not hrnited to, microorganisms such as bacteria transformed with recombinant bacteriophage, plasmid, or cosmid DNA expression vectors; yeast transformed with yeast expression vectors; insect ceh systems infected with viral expression vectors (e.g., baculovirus); plant ceh systems transformed with viral expression vectors (e.g., cauliflower mosaic virus, CaMV, or tobacco mosaic virus, TMV) or with bacterial expression vectors (e.g., Ti or pBR322 plasmids); or animal ceh systems (Sambrook et al, supra; Ausubel, supra; Van Heeke, G.
- microorganisms such as bacteria transformed with recombinant bacteriophage, plasmid, or cosmid DNA expression vectors; yeast transformed with yeast expression vectors; insect ceh systems infected with viral expression vectors (e.
- Expression vectors derived from retroviruses, adenoviruses, or herpes or vaccinia viruses, or from various bacterial plasmids may be used for dehvery of polynucleotides to the targeted organ, tissue, or ceh population (Di Nicola, M. et al. (1998) Cancer Gen. Ther. 5(6):350-356; Yu, M. et al. (1993) Proc. Natl. Acad. Sci. USA 90(13):6340-6344; Buller, R.M. et al. (1985) Nature 317(6040):813-815;
- a number of cloning and expression vectors may be selected depending upon the use intended for polynucleotides encoding NAAP. For example, routine cloning, subcloning, and propagation of polynucleotides encoding NAAP can be achieved using a multifunctional E. coli vector such as PBLUESCRIPT (Stratagene, La Jolla CA) or PSPORT1 plasmid (Invitrogen). Ligation of polynucleotides encoding NAAP into the vector's multiple cloning site disrupts the lacZ gene, allowing a colorimetric screening procedure for identification of transformed bacteria containing recombinant molecules.
- PBLUESCRIPT Stratagene, La Jolla CA
- PSPORT1 plasmid Invitrogen
- these vectors may be useful for in vitro transcription, dideoxy sequencing, single strand rescue with helper phage, and creation of nested deletions in the cloned sequence (Van Heeke, G. and S.M. Schuster (1989) J. Biol. Chem. 264:5503-5509).
- vectors which direct high level expression of NAAP may be used.
- vectors containing the strong, inducible SP6 or T7 bacteriophage promoter may be used.
- Yeast expression systems may be used for production of NAAP.
- a number of vectors containing constitutive or inducible promoters may be used in the yeast Saccharomyces cerevisiae or Pichia pastoris.
- constitutive or inducible promoters such as alpha factor, alcohol oxidase, and PGH promoters
- such vectors direct eitlier the secretion or mtiacehular retention of expressed proteins and enable integration of foreign polynucleotide sequences into the host genome for stable propagation (Ausubel, 1995, supra; Bitter, G.A. et al. (1987) Methods Enzymol. 153:516-544; and Scorer, CA. et al. (1994) Bio/Technology 12:181-184).
- Plant systems may also be used for expression of NAAP. Transcription of polynucleotides encoding NAAP may be driven by viral promoters, e.g., the 35S and 19S promoters of CaMV used alone or in combination with the omega leader sequence from TMV (Takamatsu, N. (1987) EMBO J. 3:1631). Alternatively, plant promoters such as the small subunit of RUBISCO or heat shock promoters maybe used (Coruzzi, G. et al. (1984) EMBO J. 3:1671-1680; Broghe, R. et al. (1984) Science 224:838-843; and Winter, J. et al. (1991) Results Probl. Ceh Differ. 17:85-105). These constructs can be introduced into plant cells by direct DNA transformation or pathogen-mediated transfection (The McGraw Hill Yearbook of Science and Technology (1992) McGraw Hill, New York NY, pp. 191-196).
- viral promoters e.g., the
- a number of viral-based expression systems may be utilized.
- polynucleotides encoding NAAP may be ligated into an adenovirus transcription/translation complex consisting of the late promoter and tripartite leader sequence. Insertion in a non-essential El or E3 region of the viral genome may be used to obtain infective virus which expresses NAAP in host cells (Logan, J. and T. Shenk (1984) Proc. Natl. Acad. Sci. USA 81:3655-3659).
- transcription enhancers such as the Rous sarcoma virus (RSV) enhancer, may be used to increase expression in mammalian host cells.
- SV40 or EBV-based vectors may also be used for high-level protein expression.
- HACs Human artificial chromosomes
- HACs Human artificial chromosomes
- hposomes polycationic amino polymers, or vesicles
- hposomes polycationic amino polymers, or vesicles
- NAAP in ceh lines is preferred.
- polynucleotides encoding NAAP can be transformed into ceh lines using expression vectors which may contain viral origins of rephcation and/or endogenous expression elements and a selectable marker gene on the same or on a separate vector. Following the introduction of the vector, cells may be ahowed to grow for about 1 to 2 days in enriched media before being switched to selective media.
- the purpose of the selectable marker is to confer resistance to a selective agent, and its presence allows growth and recovery of cells which successfully express the introduced sequences.
- Resistant clones of stably transformed cells maybe propagated using tissue culture techniques appropriate to the ceh type.
- any number of selection systems may be used to recover transformed ceh lines. These include, but are not limited to, the herpes simplex virus thymidine kinase and adenine phosphoribosyltransferase genes, for use in tk and apr cehs, respectively (Wigler, M. et al. (1977) CeU 11:223-232; Lowy, I. et al. (1980) Ceh 22:817-823). Also, antimetabohte, antibiotic, or herbicide resistance can be used as the basis for selection.
- dhfr confers resistance to methotrexate
- neo confers resistance to the aminoglycosides neomycin and G-418
- als and pat confer resistance to chlorsuhuron and phosphinotricin acetyltransferase, respectively
- trpB and hisD Additional selectable genes have been described, e.g., trpB and hisD, which alter cellular requirements for metabohtes (Hartman, S.C. and R.C.
- Visible markers e.g., anthocyanins, green fluorescent proteins (GFP; Clontech), ⁇ glucuronidase and its substrate ⁇ -glucuronide, or luciferase and its substrate luciferin maybe used. These markers can be used not only to identify transformants, but also to quantify the amount of transient or stable protein expression attributable to a specific vector system (Rhodes, CA. (1995) Methods Mol. Biol. 55:121-131).
- marker gene expression suggests that the gene of interest is also present, the presence and expression of the gene may need to be confirmed.
- sequence encoding NAAP is inserted within a marker gene sequence, transformed cehs containing polynucleotides encoding NAAP can be identified by the absence of marker gene function.
- a marker gene can be placed in tandem with a sequence encoding NAAP under the control of a single promoter. Expression of the marker gene in response to induction or selection usually indicates expression of the tandem gene as weh.
- host cehs that contain the polynucleotide encoding NAAP and that express NAAP may be identified by a variety of procedures known to those of skill in the art. These procedures include, but are not limited to, DNA-DNA or DNA-RNA hybridizations, PCR amplification, and protein bioassay or immunoassay techniques which include membrane, solution, or chip based technologies for the detection and/or quantification of nucleic acid or protein sequences.
- Immunological methods for detecting and measuring the expression of NAAP using either specific polyclonal or monoclonal antibodies are known in the art. Examples of such techniques include enzyme-linked immunosorbent assays (ELISAs), radioimmunoassays (RIAs), and fluorescence activated ceh sorting (FACS).
- ELISAs enzyme-linked immunosorbent assays
- RIAs radioimmunoassays
- FACS fluorescence activated ceh sorting
- Means for producing labeled hybridization or PCR probes for detecting sequences related to polynucleotides encoding NAAP include ohgolabeling, nick translation, end-labeling, or PCR amplification using a labeled nucleotide.
- polynucleotides encoding NAAP, or any fragments thereof maybe cloned into a vector for the production of an mRNA probe.
- RNA polymerase such as T7, T3, or SP6 and labeled nucleotides.
- T7, T3, or SP6 RNA polymerase
- Suitable reporter molecules or labels which may be used for ease of detection include radionuchdes, enzymes, fluorescent, chermluminescent, or chromogenic agents, as weh as substrates, cofactors, inhibitors, magnetic particles, and the like.
- Host cehs transformed with polynucleotides encoding NAAP may be cultured under conditions suitable for the expression and recovery of the protein from ceh culture.
- the protein produced by a transformed ceh may be secreted or retained intracehularly depending on the sequence and/or the vector used.
- expression vectors containing polynucleotides which encode NAAP maybe designed to contain signal sequences which direct secretion of NAAP througli a prokaryotic or eukaryotic ceh membrane.
- a host ceh strain may be chosen for its abihty to modulate expression of the inserted polynucleotides or to process the expressed protein in the desired fashion.
- modifications of the polypeptide include, but are not limited to, acetylation, carboxylation, glycosylation, phosphorylation, lipidation, and acylation.
- Post-translational processing which cleaves a "prepro” or "pro” form of the protein may also be used to specify protein targeting, folding, and/or activity.
- Different host cells which have specific cellular machinery and characteristic mechanisms for post-translational activities (e.g., CHO, HeLa, MDCK, HEK293, and WD 8) are available from the American Type Culture Collection (ATCC, Manassas VA) and may be chosen to ensure the correct modification and processing of the foreign protein.
- ATCC American Type Culture Collection
- nataral, modified, or recombinant polynucleotides encoding NAAP may be ligated to a heterologous sequence resulting in translation of a fusion protein in any of the aforementioned host systems.
- a chimeric NAAP protein containing a heterologous moiety that can be recognized by a commercially available antibody may facilitate the screening of peptide libraries for inhibitors of NAAP activity.
- Heterologous protein and peptide moieties may also facihtate purification of fusion proteins using commerciahy available affinity matrices.
- Such moieties include, but are not limited to, glutathione S-transferase (GST), maltose binding protein (MBP), thioredoxin (Trx), calmodulin binding peptide (CBP), 6-His, FLAG, c-myc, and hemagglutinin (HA).
- GST, MBP, Trx, CBP, and 6-His enable purification of then cognate fusion proteins on immobilized glutathione, maltose, phenylarsine oxide, calmodulin, and metal-chelate resins, respectively.
- FLAG, c-myc, and hemagglutinin (HA) enable immunoaffinity purification of fusion proteins using commercially available monoclonal and polyclonal antibodies that specifically recognize these epitope tags.
- a fusion protein may also be engineered to contain a proteolytic cleavage site located between the NAAP encoding sequence and the heterologous protein sequence, so that NAAP may be cleaved away from the heterologous moiety following purification. Methods for fusion protein expression and purification are discussed in Ausubel (1995, supra, ch. 10). A variety of commercially available kits may also be used to facilitate expression and purification of fusion proteins.
- synthesis of radiolabeled NAAP maybe achieved in vitro using the TNT rabbit reticulocyte lysate or wheat germ extract system (Promega). These systems couple transcription and translation of protein-coding sequences operably associated with the T7, T3, or SP6 promoters. Translation takes place in the presence of a radiolabeled amino acid precursor, for example, 35 S-me ⁇ taonine.
- NAAP fragments of NAAP, or variants of NAAP
- One or more test compounds may be screened for specific binding to NAAP.
- 1, 2, 3, 4, 5, 10, 20, 50, 100, or 200 test compounds can be screened for specific binding to NAAP.
- Examples of test compounds can include antibodies, anticalins, ohgonucleotides, proteins (e.g., ligands or receptors), or small molecules.
- variants of NAAP can be used to screen for binding of test compounds, such as antibodies, to NAAP, a variant of NAAP, or a combination of NAAP and/or one or more variants NAAP.
- a variant of NAAP can be used to screen for compounds that bind to a variant of NAAP, but not to NAAP having the exact sequence of a sequence of SEQ HO NO:l-36.
- NAAP variants used to perform such screening can have a range of about 50% to about 99% sequence identity to NAAP, with various embodiments having 60%, 70%, 75%, 80%, 85%, 90%, and 95% sequence identity.
- a compound identified in a screen for specific binding to NAAP can be closely related to the nataral ligand of NAAP, e.g., a ligand or fragment thereof, a nataral substrate, a structural or functional mimetic, or a nataral binding partner (Coligan, J.E. et al. (1991) Current
- the compound thus identified can be a nataral ligand of a receptor NAAP (Howard, A.D. et al. (2001) Trends Pharmacol. Sci.22:132- 140; Wise, A. et al. (2002) Drug Discovery Today 7:235-246).
- a compound identified in a screen for specific binding to NAAP can be closely related to the nataral receptor to which NAAP binds, at least a fragment of the receptor, or a fragment of the receptor including ah or a portion of the ligand binding site or binding pocket.
- the compound maybe a receptor for NAAP which is capable of propagating a signal, or a decoy receptor for NAAP which is not capable of propagating a signal (Ashkenazi, A. and V.M. Divit (1999) Cu ⁇ . Opin. Ceh Biol. 11:255-260; Mantovani, A. et al. (2001) Trends Immunol. 22:328-336).
- the compound can be rationally designed using known techniques. Examples of such techniques include those used to construct the compound etanercept (ENBREL; hnmunex Corp., Seattle WA), which is efficacious for treating rheumatoid arthritis in humans.
- Etanercept is an engineered p75 tamor necrosis factor (TNF) receptor dimer linked to the Fc portion of human IgG 2 (Taylor, P.C et al. (2001) Cu ⁇ . Opin. Immunol. 13:611-616).
- TNF tamor necrosis factor
- two or more antibodies having similar or, alternatively, different specificities can be screened for specific binding to NAAP, fragments of NAAP, or variants of NAAP.
- the binding specificity of the antibodies thus screened can thereby be selected to identify particular fragments or variants of NAAP.
- an antibody can be selected such that its binding specificity ahows for preferential identification of specific fragments or variants of NAAP.
- an antibody can be selected such that its binding specificity ahows for preferential diagnosis of a specific disease or condition having increased, decreased, or otherwise abnormal production of NAAP.
- anticalins can be screened for specific binding to NAAP, fragments of
- NAAP or variants of NAAP.
- Anticalins are hgand-binding proteins that have been constracted based on a lipocalin scaffold (Weiss, G.A. and H.B. Lowman (2000) Chem. Biol. 7:R177-R184; Ske ⁇ a, A. (2001) J. Biotechnol. 74:257-275).
- the protein architecture of hpocalins can include a beta-barrel having eight antiparahel beta-strands, which supports four loops at its open end. These loops form the nataral ligand-binding site of the Hpocalins, a site which can be re-engineered in vitro by amino acid substitutions to impart novel binding specificities.
- amino acid substitations can be made using methods known in the art or described herein, and can include conservative substitutions (e.g., substitations that do not alter binding specificity) or substitations that modestly, moderately, or significantly alter binding specificity. In one embodiment, screening for compounds which specifically bind to, stimulate, or inhibit
- NAAP involves producing appropriate cehs which express NAAP, either as a secreted protein or on the ceh membrane.
- Prefe ⁇ ed cells include cehs from mammals, yeast, Drosophila, or E. coli. Cehs expressing NAAP or ceh membrane fractions which contain NAAP are then contacted with a test compound and binding, stimulation, or inhibition of activity of either NAAP or the compound is analyzed.
- An assay may simply test binding of a test compound to the polypeptide, wherein binding is detected by a fluorophore, radioisotope, enzyme conjugate, or other detectable label.
- the assay may comprise the steps of combining at least one test compound with NAAP, either in solution or affixed to a solid support, and detecting the binding of NAAP to the compound.
- the assay may detect or measure binding of a test compound in the presence of a labeled competitor. Additionahy, the assay maybe carried out using cell-free preparations, chemical libraries, or nataral product mixtures, and the test compound(s) maybe free in solution or affixed to a solid support.
- An assay can be used to assess the abihty of a compound to bind to its natural hgand and/or to inhibit the binding of its nataral hgand to its nataral receptors.
- assays include radio- labeling assays such as those described in U.S. Patent No. 5,914,236 and U.S. Patent No. 6,372,724.
- one or more amino acid substitations can be introduced into a polypeptide compound (such as a receptor) to improve or alter its ability to bind to its nataral ligands (Matthews, D.J. and J.A. Wells. (1994) Chem. Biol. 1:25-30).
- one or more amino acid substitutions can be introduced into a polypeptide compound (such as a hgand) to improve or alter its ability to bind to its nataral receptors (Cunningham, B.C. and J.A. Wells (1991) Proc. Natl. Acad. Sci. USA 88:3407-3411; Lowman, H.B. et al. (1991) J. Biol. Chem. 266:10982-10988).
- a polypeptide compound such as a hgand
- NAAP, fragments of NAAP, or variants of NAAP maybe used to screen for compounds that modulate the activity of NAAP.
- Such compounds may include agonists, antagonists, or partial or inverse agonists.
- an assay is performed under conditions permissive for NAAP activity, wherein NAAP is combined with at least one test compound, and the activity of NAAP in the presence of a test compound is compared with the activity of NAAP in the absence of the test compound. A change in the activity of NAAP in the presence of the test compound is indicative of a compound that modulates the activity of NAAP.
- a test compound is combined with an in vitro or ceh-free system comprising NAAP under conditions suitable for NAAP activity, and the assay is performed.
- a test compound which modulates the activity of NAAP may do so indirectly and need not come in direct contact with the test compound. At least one and up to a plurahty of test compounds may be screened.
- polynucleotides encoding NAAP or then mammalian homologs may be "knocked out" in an animal model system using homologous recombination in embryonic stem (ES) cells.
- ES embryonic stem
- mouse ES cehs such as the mouse 129/SvJ ceh line
- the ES cells are transformed with a vector containing the gene of interest disrupted by a marker gene, e.g., the neomycin phosphotransferase gene ⁇ neo; Capecchi, M.R. (1989) Science 244: 1288-1292).
- the vector integrates into the corresponding region of the host genome by homologous recombination.
- homologous recombination takes place using the Cre-loxP ' system to knockout a gene of interest in a tissue- or developmental stage-specific manner (Marth, J.D. (1996) Clin. Invest.
- Transformed ES cehs are identified and microinjected into mouse ceh blastocysts such as those from the C57BL/6 mouse strain.
- the blastocysts are surgically transfe ⁇ ed to pseudopregnant dams, and the resulting chimeric progeny are genotyped and bred to produce heterozygous or homozygous strains.
- Transgenic animals thus generated maybe tested with potential therapeutic or toxic agents.
- Polynucleotides encoding NAAP may also be manipulated in vitro in ES cehs derived from human blastocysts.
- Human ES cehs have the potential to differentiate into at least eight separate ceh lineages including endoderm, mesoderm, and ectodermal cell types. These ceh lineages differentiate into, for example, neural cehs, hematopoietic lineages, and cardiomyocytes (Thomson, J.A. et al. (1998) Science 282:1145-1147).
- Polynucleotides encoding NAAP can also be used to create "knockin" humanized animals (pigs) or transgenic animals (mice or rats) to model human disease.
- knockin technology a region of a polynucleotide encoding NAAP is injected into animal ES cells, and the injected sequence integrates into the animal ceh genome.
- Transformed cehs are injected into blastulae, and the blastulae are implanted as described above.
- Transgenic progeny or inbred lines are studied and treated with potential pharmaceutical agents to obtain information on treatment of a human disease.
- a mammal inbred to overexpress NAAP e.g., by secreting NAAP in its milk, may also serve as a convenient source of that protein (Janne, J. et al. (1998) Biotechnol. Annu. Rev. 4:55-74).
- THERAPEUTICS may also serve as a convenient source of that protein (Janne, J. et al. (1998) Biotechnol. Annu. Rev. 4:55-74).
- NAAP appears to play a role in ceh prohferative, neurological, reproductive, developmental, autoimmune/inflammatory, and DNA repair disorders, and infections.
- it is desirable to decrease the expression or activity of NAAP In the treatment of disorders associated with decreased NAAP expression or activity, it is desirable to increase the expression or activity of NAAP.
- NAAP or a fragment or derivative thereof may be administered to a subject to treat or prevent a disorder associated with decreased expression or activity of NAAP.
- disorders include, but are not hmited to, a ceh proliferative disorder such as actinic keratosis, arteriosclerosis, atherosclerosis, bursitis, cirrhosis, hepatitis, mixed connective tissue disease (MCTD), myelofibrosis, paroxysmal nocturnal hemoglobinuria, polycyfhemia vera, psoriasis, primary thrombocythemia, and cancers including adenocarcinoma, leukemia, lymphoma, melanoma, myeloma, sarcoma, teratocarcinoma, and, in particular, a cancer of the adrenal gland, bladder, bone, bone marrow, brain, breast, cervix, gah bladder, ganglia, gastrointestinal tract, heart
- a vector capable of expressing NAAP or a fragment or derivative thereof may be administered to a subject to treat or prevent a disorder associated with decreased expression or activity of NAAP including, but not limited to, those described above.
- compositions comprising a substantially purified NAAP in conjunction with a suitable pharmaceutical carrier may be administered to a subject to treat or prevent a disorder associated with decreased expression or activity of NAAP including, but not limited to, those provided above.
- an agonist which modulates the activity of NAAP may be administered to a subject to treat or prevent a disorder associated with decreased expression or activity of NAAP including, but not hmited to, those hsted above.
- an antagonist of NAAP may be administered to a subject to treat or prevent a disorder associated with increased expression or activity of NAAP.
- disorders include, but are not limited to, those ceh proliferative, neurological, reproductive, developmental, autoimmune/inflammatory, and DNA repair disorders, and infections, described above.
- an antibody which specifically binds NAAP may be used directly as an antagonist or indirectly as a targeting or dehvery mechanism for bringing a pharmaceutical agent to cehs or tissues which express NAAP.
- a vector expressing the complement of the polynucleotide encoding NAAP may be administered to a subject to treat or prevent a disorder associated with increased expression or activity of NAAP including, but not limited to, those described above.
- any protein, agonist, antagonist, antibody, complementary sequence, or vector embodiments maybe administered in combination with other appropriate therapeutic agents. Selection of the appropriate agents for use in combination therapy may be made by one of ordinary skih in the art, according to conventional pharmaceutical principles.
- the combination of therapeutic agents may act synergisticahy to effect the treatment or prevention of the various disorders described above. Using this approach, one may be able to achieve therapeutic efficacy with lower dosages of each agent, thus reducing the potential for adverse side effects.
- An antagonist of NAAP may be produced using methods which are generahy known in the art. In particular, purified NAAP may be used to produce antibodies or to screen libraries of pharmaceutical agents to identify those which specifically bind NAAP.
- Antibodies to NAAP may also be generated using methods that are weh known in the art. Such antibodies may include, but are not hmited to, polyclonal, monoclonal, chimeric, and single chain antibodies, Fab fragments, and fragments produced by a Fab expression library. Neutralizing antibodies (i.e., those which inhibit dimer formation) are generahy prefe ⁇ ed for therapeutic use. Single chain antibodies (e.g., from camels or llamas) may be potent enzyme inhibitors and may have advantages in the design of peptide mimetics, and in the development of immuno-adsorbents and biosensors (Muyldermans, S. (2001) J. Biotechnol. 74:277-302).
- various hosts including goats, rabbits, rats, mice, camels, dromedaries, llamas, humans, and others may be immunized by injection with NAAP or with any fragment or oligopeptide thereof which has immunogenic properties.
- various adjuvants may be used to increase immunological response.
- adjuvants include, but are not limited to, Freund's, mineral gels such as aluminum hydroxide, and surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, KLH, and dinitrophenol.
- BCG Bacilli Calmette-Guerin
- Corynebacterium parvum are especiahy preferable. It is preferred that the oligopeptides, peptides, or fragments used to induce antibodies to
- NAAP have an amino acid sequence consisting of at least about 5 amino acids, and generahy wih consist of at least about 10 amino acids. It is also preferable that these ohgopeptides, peptides, or fragments are identical to a portion of the amino acid sequence of the nataral protein. Short stretches of NAAP amino acids maybe fused with those of another protein, such as KLH, and antibodies to the chimeric molecule may be produced.
- Monoclonal antibodies to NAAP may be prepared using any technique which provides for the production of antibody molecules by continuous ceh hnes in culture. These include, but are not hmited to, the hybridoma technique, the human B-ceh hybridoma technique, and the EBV-hybridoma technique (Kohler, G. et al. (1975) Natare 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; and Cole, S.P. et al.
- chimeric antibodies such as the splicing of mouse antibody genes to human antibody genes to obtain a molecule with appropriate antigen specificity and biological activity, can be used (Morrison, S.L. et al. (1984) Proc. Natl. Acad. Sci. USA 81:6851-6855; Neuberger, M.S. et al. (1984) Natare 312:604-608; and Takeda, S. et al.
- Antibodies may also be produced by inducing in vivo production in the lymphocyte population or by screening immunoglobulin libraries or panels of highly specific binding reagents as disclosed in the literature (Orlandi, R. et al. (1989) Proc. Natl. Acad. Sci. USA 86:3833-3837; Winter, G. et al. (1991) Nature 349:293-299).
- Antibody fragments which contain specific binding sites for NAAP may also be generated.
- fragments include, but are not limited to, F(ab') 2 fragments produced by pepsin digestion of the antibody molecule and Fab fragments generated by reducing the disulfide bridges of the F(ab')2 fragments.
- Fab expression libraries maybe constructed to ahow rapid and easy identification of monoclonal Fab fragments with the deshed specificity (Huse, W.D. et al. (1989) Science 246:1275-1281.)
- Various immunoassays may be used for screening to identify antibodies having the deshed specificity.
- Numerous protocols for competitive binding or immunoradiometric assays using either polyclonal or monoclonal antibodies with established specificities are weh known in the art.
- Such immunoassays typically involve the measurement of complex formation between NAAP and its specific antibody.
- a two-site, monoclonal-based immunoassay utilizing monoclonal antibodies reactive to two non-interfering NAAP epitopes is generahy used, but a competitive binding assay may also be employed (Pound, supra).
- Various methods such as Scatchard analysis in conjunction with radioimmunoassay techniques maybe used to assess the affinity of antibodies for NAAP.
- K a is defined as the molar concentration of NAAP-antibody complex divided by the molar concentrations of free antigen and free antibody under equihbrium conditions.
- the K a determined for a preparation of monoclonal antibodies, which are monospecific for a particular NAAP epitope, represents a true measure of affinity.
- Hgh-affinity antibody preparations with K a ranging from about 10 9 to 10 12 L/mole are prefe ⁇ ed for use in immunoassays in which the NAAP- antibody complex must withstand rigorous manipulations.
- Low-affinity antibody preparations with K a ranging from about 10 6 to 10 7 L/mole are preferred for use in immunopurification and similar procedures which ultimately require dissociation of NAAP, preferably in active form, from the antibody (Catty, D. (1988) Antibodies, Volume I: A Practical Approach, IRL Press, Washington DC; Liddeh, J.E. and A. Cryer (1991) A Practical Guide to Monoclonal Antibodies, John Wiley & Sons, New York NY).
- polyclonal antibody preparations may be further evaluated to determine the quahty and suitability of such preparations for certain downstream apphcations.
- a polyclonal antibody preparation containing at least 1-2 mg specific antibody/ml, preferably 5-10 mg specific antibody/ml is generahy employed in procedures requiring precipitation of NAAP-antibody complexes.
- Procedures for evaluating antibody specificity, titer, and avidity, and guidelines for antibody quahty and usage in various apphcations, are generahy available (Catty, supra, and Cohgan et al., supra).
- polynucleotides encoding NAAP may be used for therapeutic purposes.
- modifications of gene expression can be achieved by designing complementary sequences or antisense molecules (DNA, RNA, PNA, or modified oligonucleotides) to the coding or regulatory regions of the gene encoding NAAP.
- complementary sequences or antisense molecules DNA, RNA, PNA, or modified oligonucleotides
- antisense ohgonucleotides or larger fragments can be designed from various locations along the coding or control regions of sequences encoding NAAP (Agrawal, S., ed. (1996) Antisense Therapeutics, Humana Press Inc., Totawa NJ).
- Antisense sequences can be delivered intracehularly in the form of an expression plasmid which, upon transcription, produces a sequence complementary to at least a portion of the cehular sequence encoding the target protein (Slater, J.E. et al. (1998) J. Ahergy Clin. Immunol. 102(3):469-475; and Scanlon, KJ. et al. (1995) 9(13):1288-1296).
- Antisense sequences can also be introduced intracehularly through the use of viral vectors, such as retrovirus and adeno-associated virus vectors (Miller, A.D.
- polynucleotides encoding NAAP may be used for somatic or germline gene therapy.
- Gene therapy may be performed to (i) correct a genetic deficiency (e.g., in the cases of severe combined immunodeficiency (SCHO)-Xl disease characterized by X- linked inheritance (Cavazzana-Calvo, M. et al. (2000) Science 288:669-672), severe combined immunodeficiency syndrome associated with an inherited adenosine deaminase (ADA) deficiency (Blaese, R.M. et al. (1995) Science 270:475-480; Bordignon, C et al.
- SCHO severe combined immunodeficiency
- ADA adenosine deaminase
- hepatitis B or C virus HBV, HCV
- fungal parasites such as Candida albicans and Paracoccidioides brasiliensis
- protozoan parasites such as Plasmodium falciparum and Trypanosoma cruzi.
- the expression of NAAP from an appropriate population of transduced cehs may alleviate the clinical manifestations caused by the genetic deficiency.
- diseases or disorders caused by deficiencies in NAAP are treated by constructing mammalian expression vectors encoding NAAP and introducing these vectors by mechanical means into NAAP-deficient cehs.
- Mechanical transfer technologies for use with cehs in vivo or ex vitro include (i) direct DNA microinjection into individual cells, (ii) ballistic gold particle dehvery, ( i) hposome-mediated transfection, (iv) receptor-mediated gene transfer, and (v) the use of DNA transposons (Morgan, R.A. and W.F. Anderson (1993) Annu. Rev. Biochem. 62:191-217; Ivies, Z. (1997) Ceh 91:501-510; Boulay, J-L and H. Recipon (1998) Cu ⁇ . Opin. Biotechnol. 9:445-450).
- Expression vectors that may be effective for the expression of NAAP include, but are not limited to, the PCDNA 3.1, EPITAG, PRCCMV2, PREP, PVAX, PCR2-TOPOTA vectors (Invitrogen, Carlsbad CA), PCMV-SCPJPT, PCMV-TAG, PEGSH/PERV (Stratagene, La Joha CA), and PTET-OFF, PTET-ON, PTRE2, PTRE2-LUC, PTK-HYG (Clontech, Palo Alto CA).
- NAAP maybe expressed using (i) a constitutively active promoter, (e.g., from cytomegalovirus (CMV), Rous sarcoma virus (RSV), SV40 virus, thymidine kinase (TK), or ⁇ -actin genes), (ii) an inducible promoter (e.g., the tetracychne-regulated promoter (Gossen, M. and H. Bujard (1992) Proc. Natl. Acad. Sci. USA 89:5547-5551; Gossen, M. et al. (1995) Science 268:1766-1769; Rossi, F.M.V. and HM. Blau (1998) Curr. Opin. Biotechnol.
- a constitutively active promoter e.g., from cytomegalovirus (CMV), Rous sarcoma virus (RSV), SV40 virus, thymidine kinase (TK), or ⁇ -actin genes
- FK506/rapamycin inducible promoter or the RU486/mifepristone inducible promoter (Rossi, F.M.V. and HM. Blau, supra)), or (hi) a tissue-specific promoter or the native promoter of the endogenous gene encoding NAAP from a normal individual.
- Commerciahy available hposome transformation kits e.g., the PERFECT LIPID TRANSFECTION KIT, available from Invitrogen
- transformation is performed using the calcium phosphate method (Graham, F.L. and A.J. Eb (1973) Virology 52:456-467), or by electroporation (Neumann, E. et al.
- diseases or disorders caused by genetic defects with respect to NAAP expression are treated by constructing a retrovirus vector consisting of (i) the polynucleotide encoding NAAP under the control of an independent promoter or the retrovirus long terminal repeat (LTR) promoter, (ii) appropriate RNA packaging signals, and ( i) a Rev-responsive element (RRE) along with additional retrovirus cw-acting RNA sequences and coding sequences required for efficient vector propagation.
- Retrovirus vectors e.g., PFB and PFBNEO
- Retrovirus vectors are commerciahy available (Stratagene) and are based on published data (Riviere, I. et al. (1995) Proc. Natl. Acad. Sci.
- the vector is propagated in an appropriate vector producing ceh line (VPCL) that expresses an envelope gene with a tropism for receptors on the target cells or a promiscuous envelope protein such as VSVg (Armentano, D. et al. (1987) J. Vhol. 61:1647-1650; Bender, M.A. et al. (1987) J. Vhol. 61:1639-1646; Adam, M.A. and A.D. Miher (1988) J. Vhol. 62:3802-3806; Duh, T. et al. (1998) J. Vhol. 72:8463-8471; Zufferey, R. et al.
- VSVg vector producing ceh line
- U.S. Patent No. 5,910,434 to Rigg discloses a method for obtaining retrovirus packaging ceh lines and is hereby incorporated by reference. Propagation of retrovirus vectors, transduction of a population of cehs (e.g., CD4 + T-cells), and the return of transduced cehs to a patient are procedures weh known to persons skilled in the art of gene therapy and have been weh documented (Ranga, U. et al. (1997) J. Vhol. 71:7020-7029; Bauer, G. et al.
- an adenovirus-based gene therapy dehvery system is used to dehver polynucleotides encoding NAAP to cehs which have one or more genetic abnormalities with respect to the expression of NAAP.
- the construction and packaging of adenovirus-based vectors are weh known to those with ordinary skih in the art.
- Replication defective adenovirus vectors have proven to be versatile for importing genes encoding immunoregulatory proteins into intact islets in the pancreas (Csete, M.E. et al. (1995) Transplantation 27:263-268). Potentiahy useful adenoviral vectors are described in U.S. Patent No.
- Adenovirus vectors for gene therapy hereby incorporated by reference.
- adenoviral vectors see also Antinozzi et al. (1999; Annu. Rev. Nutr. 19:511-544) and Verma and Somia (1997; Natare 18:389:239-242), both incorporated by reference herein.
- a herpes-based, gene therapy dehvery system is used to dehver polynucleotides encoding NAAP to target cehs which have one or more genetic abnormalities with respect to the expression of NAAP.
- herpes simplex virus (HSV)-based vectors may be especiahy valuable for introducing NAAP to cells of the central nervous system, for which HSV has a tropism.
- the construction and packaging of herpes-based vectors are weh known to those with ordinary skih in the art.
- a rephcation-competent herpes simplex virus (HSV) type 1-based vector has been used to dehver a reporter gene to the eyes of primates (Liu, X. et al. (1999) Exp. Eye Res. 169:385-395).
- the construction of a HSV-1 virus vector has also been disclosed in detail in U.S. Patent No.
- an alphavirus positive, single-stranded RNA virus
- RNA virus single-stranded RNA virus
- Semliki Forest Virus has been studied extensively and gene transfer vectors have been based on the SFV genome (Garoff, H. and K.-J. Li (1998) Cu ⁇ . Opin. Biotechnol. 9:464-469).
- alphavirus RNA replication a subgenomic RNA is generated that normahy encodes the viral capsid proteins. This subgenomic RNA replicates to higher levels than the full length genomic RNA, resulting in the overproduction of capsid proteins relative to the viral proteins with enzymatic activity (e.g., protease and polymerase).
- alphaviruses wih ahow the introduction of NAAP into a variety of ceh types.
- the specific transduction of a subset of cehs in a population may require the sorting of cells prior to transduction.
- the methods of manipulating infectious cDNA clones of alphaviruses, performing alphavirus cDNA and RNA transfections, and performing alphavirus infections, are weh known to those with ordinary skih in the art.
- Oligonucleotides derived from the transcription initiation site e.g., between about positions -10 and +10 from the start site, may also be employed to inhibit gene expression. Similarly, inhibition can be achieved using triple helix base-pairing methodology.
- Triple helix pairing is useful because it causes inhibition of the abihty of the double hehx to open sufficiently for the binding of polymerases, transcription factors, or regulatory molecules.
- Recent therapeutic advances using triplex DNA have been described in the literature (Gee, J.E. et al. (1994) in Huber, B.E. and B.I. Can, Molecular and Immunologic Approaches, Futara Publishing, Mt. Kisco NY, pp. 163-177).
- a complementary sequence or antisense molecule may also be designed to block translation of mRNA by preventing the transcript from binding to ribosomes.
- Ribozymes enzymatic RNA molecules, may also be used to catalyze the specific cleavage of RNA.
- the mechanism of ribozyme action involves sequence-specific hybridization of the ribozyme molecule to complementary target RNA, fohowed by endonucleolytic cleavage.
- engineered hammerhead motif ribozyme molecules may specifically and efficiently catalyze endonucleolytic cleavage of RNA molecules encoding NAAP.
- ribozyme cleavage sites within any potential RNA target are initially identified by scanning the target molecule for ribozyme cleavage sites, including the following sequences: GUA, GUU, and GUC
- short RNA sequences of between 15 and 20 ribonucleotides, co ⁇ esponding to the region of the target gene containing the cleavage site maybe evaluated for secondary stractural features which may render the oligonucleotide inoperable.
- the suitability of candidate targets may also be evaluated by testing accessibility to hybridization with complementary ohgonucleotides using ribonuclease protection assays.
- RNA molecules may be generated by in vitro and in vivo transcription of DNA molecules encoding NAAP. Such DNA sequences maybe incorporated into a wide variety of vectors with suitable RNA polymerase promoters such as T7 or SP6. Alternatively, these cDNA constructs that synthesize complementary RNA, constitutively or inducibly, can be introduced into ceh hnes, cehs, or tissues. RNA molecules may be modified to increase intracellular stability and half-life.
- flanking sequences at the 5' and/or 3' ends of the molecule Possible modifications include, but are not limited to, the addition of flanking sequences at the 5' and/or 3' ends of the molecule, or the use of phosphorothioate or 2' O-methyl rather than phosphodiesterase linkages within the backbone of the molecule.
- This concept is inherent in the production of PNAs and can be extended in ah of these molecules by the inclusion of nontraditional bases such as inosine, queosine, and wybutosine, as weh as acetyl-, methyl-, thio-, and similarly modified forms of adenine, cytidine, guanine, thymine, and uridine which are not as easily recognized by endogenous endonucleases.
- An additional embodiment of the invention encompasses a method for screening for a compound which is effective in altering expression of a polynucleotide encoding NAAP.
- Compounds which may be effective in altering expression of a specific polynucleotide may include, but are not limited to, ohgonucleotides, antisense ohgonucleotides, triple heUx-forming ohgonucleotides, transcription factors and other polypeptide transcriptional regulators, and non-macromolecular chemical entities which are capable of interacting with specific polynucleotide sequences. Effective compounds may alter polynucleotide expression by acting as either inhibitors or promoters of polynucleotide expression.
- a compound which specifically inhibits expression of the polynucleotide encoding NAAP maybe therapeutically useful
- a compound which specifically promotes expression of the polynucleotide encoding NAAP maybe therapeutically useful.
- At least one, and up to a plurality, of test compounds may be screened for effectiveness in altering expression of a specific polynucleotide.
- a test compound may be obtained by any method commonly known in the art, including chemical modification of a compound known to be effective in altering polynucleotide expression; selection from an existing, commerciahy-available or proprietary library of naturahy-occnrring or non-natural chemical compounds; rational design of a compound based on chemical and/or structural properties of the target polynucleotide; and selection from a library of chemical compounds created combinatoriahy or randomly.
- a sample comprising, a polynucleotide encoding NAAP is exposed to at least one test compound thus obtained.
- the sample may comprise, for example, an intact or permeabilized ceh, or an in vitro cell-free or reconstituted biochemical system.
- Alterations in the expression of a polynucleotide encoding NAAP are assayed by any method commonly known in the art.
- the expression of a specific nucleotide is detected by hybridization with a probe having a nucleotide sequence complementary to the sequence of the polynucleotide encoding NAAP.
- the amount of hybridization maybe quantified, thus forming the basis for a comparison of the expression of the polynucleotide both with and without exposure to one or more test compounds.
- a screen for a compound effective in altering expression of a specific polynucleotide can be carried out, for example, using a Schizosaccharomyces pombe gene expression system (Atkins, D. et al. (1999) U.S. Patent No. 5,932,435; Arndt, G.M. et al. (2000) Nucleic Acids Res. 28:E15) or a human ceh line such as HeLa ceh (Clarke, M.L. et al. (2000) Biochem. Biophys. Res.
- a particular embodiment of the present invention involves screening a combinatorial hbrary of ohgonucleotides (such as deoxyribonucleotides, ribonucleotides, peptide nucleic acids, and modified ohgonucleotides) for antisense activity against a specific polynucleotide sequence (Bruice, T.W. et al. (1997) U.S. Patent No. 5,686,242; Bruice, T.W. et al. (2000) U.S. Patent No. 6,022,691).
- ohgonucleotides such as deoxyribonucleotides, ribonucleotides, peptide nucleic acids, and modified ohgonucleotides
- vectors may be introduced into stem cehs taken from the patient and clonahy propagated for autologous transplant back into that same patient. Dehvery by transfection, by liposome injections, or by polycationic amino polymers may be achieved using methods which are weh known in the art (Goldman, C.K. et al. (1997) Nat. Biotechnol. 15:462- 466).
- compositions which generahy comprises an active ingredient formulated with a pharmaceutically acceptable excipient.
- Excipients may include, for example, sugars, starches, cehuloses, gums, and proteins.
- formulations are commonly known and are thoroughly discussed in the latest edition of Remington's Pharmaceutical Sciences (Maack PubHshing, Easton PA).
- Such compositions may consist of NAAP, antibodies to NAAP, and mimetics, agonists, antagonists, or inhibitors of NAAP.
- compositions utilized in this invention maybe administered by any number of routes including, but not limited to, oral, intravenous, intramuscular, intra-arterial, intrameduhary, intrathecal, intraventricular, pulmonary, transdermal, subcutaneous, intraperitoneal, intranasal, enteral, topical, sublingual, or rectal means.
- compositions for pulmonary administration maybe prepared in liquid or dry powder form. These compositions are generahy aerosolized immediately prior to inhalation by the patient.
- small molecules e.g. traditional low molecular weight organic drugs
- aerosol dehvery of fast- acting formulations is well-known in the art.
- macromolecules e.g. larger peptides and proteins
- recent developments in the field of pulmonary dehvery via the alveolar region of the lung have enabled the practical delivery of drags such as insulin to blood circulation (see, e.g., Patton, J.S. et al., U.S. Patent No. 5,997,848).
- compositions suitable for use in the invention include compositions wherein the active ingredients are contained in an effective amount to achieve the intended purpose. The determination of an effective dose is weh within the capabihty of those skilled in the art.
- Speciahzed forms of compositions may be prepared for direct intracehular dehvery of macromolecules comprising NAAP or fragments thereof.
- hposome preparations containing a ceh-impermeable macromolecule may promote ceh fusion and intracehular dehvery of the macromolecule.
- NAAP or a fragment thereof may be joined to a short cationic N- terminal portion from the HTV Tat-1 protein. Fusion proteins thus generated have been found to transduce into the cehs of ah tissues, including the brain, in a mouse model system (Schwarze, S.R. et al. (1999) Science 285:1569-1572).
- the therapeutically effective dose can be estimated initially either in ceh culture assays, e.g., of neoplastic cehs, or in animal models such as mice, rats, rabbits, dogs, monkeys, or pigs. An animal model may also be used to determine the appropriate concentration range and route of administration. Such information can then be used to determine useful doses and routes for administration in humans.
- a therapeuticahy effective dose refers to that amount of active ingredient, for example
- Therapeutic efficacy and toxicity maybe determined by standard pharmaceutical procedures in ceh cultures or with experimental animals, such as by calculating the ED 50 (the dose therapeuticahy effective in 50% of the population) or LD 50 (the dose lethal to 50% of the population) statistics.
- the dose ratio of toxic to therapeutic effects is the therapeutic index, which can be expressed as the LD 50 /ED 50 ratio.
- Compositions which exhibit large therapeutic indices are prefe ⁇ ed.
- the data obtained from ceh cultare assays and animal studies are used to formulate a range of dosage for human use.
- the dosage contained in such compositions is preferably within a range of chculating concentrations that includes the ED 50 with little or no toxicity. The dosage varies within this range depending upon the dosage form employed, the sensitivity of the patient, and the route of administration.
- the exact dosage wih be determined by the practitioner, in hght of factors related to the subject requiring treatment. Dosage and administration are adjusted to provide sufficient levels of the active moiety or to maintain the deshed effect. Factors which may be taken into account include the severity of the disease state, the general health of the subject, the age, weight, and gender of the subject, time and frequency of administration, drug combinations), reaction sensitivities, and response to therapy. Long-acting compositions maybe administered every 3 to 4 days, every week, or biweekly depending on the half-life and clearance rate of the particular formulation.
- Normal dosage amounts may vary from about 0.1 ⁇ g to 100,000 ⁇ g, up to a total dose of about 1 gram, depending upon the route of administration.
- Guidance as to particular dosages and methods of dehvery is provided in the literature and generahy available to practitioners in the art.
- Those skilled in the art wih employ different formulations for nucleotides than for proteins or their inhibitors.
- delivery of polynucleotides or polypeptides wih be specific to particular cells, conditions, locations, etc. DIAGNOSTICS
- antibodies which specifically bind NAAP maybe used for the diagnosis of disorders characterized by expression of NAAP, or in assays to monitor patients being treated with NAAP or agonists, antagonists, or inhibitors of NAAP.
- Antibodies useful for diagnostic purposes may be prepared in the same manner as described above for therapeutics. Diagnostic assays for NAAP include methods which utilize the antibody and a label to detect NAAP in human body fluids or in extracts of cehs or tissues.
- the antibodies may be used with or without modification, and may be labeled by covalent or non-covalent attachment of a reporter molecule.
- a wide variety of reporter molecules, several of which are described above, are known in the art and may be used.
- NAAP a variety of protocols for measuring NAAP, including ELISAs, RIAs, and FACS, are known in the art and provide a basis for diagnosing altered or abnormal levels of NAAP expression.
- Normal or standard values for NAAP expression are established by combining body fluids or ceh extracts taken from normal mammahan subjects, for example, human subjects, with antibodies to NAAP under conditions suitable for complex formation. The amount of standard complex formation may be quantitated by various methods, such as photometric means. Quantities of NAAP expressed in subject, control, and disease samples from biopsied tissues are compared with the standard values. Deviation between standard and subject values estabhshes the parameters for diagnosing disease.
- polynucleotides encoding NAAP may be used for diagnostic purposes.
- the polynucleotides which maybe used include ohgonucleotides, complementary RNA and DNA molecules, and PNAs.
- the polynucleotides may be used to detect and quantify gene expression in biopsied tissues in which expression of NAAP may be co ⁇ elated with disease.
- the diagnostic assay maybe used to dete ⁇ riine absence, presence, and excess expression of NAAP, and to monitor regulation of NAAP levels during therapeutic intervention.
- hybridization with PCR probes which are capable of detecting polynucleotides, including genomic sequences, encoding NAAP or closely related molecules may be used to identify nucleic acid sequences which encode NAAP.
- the specificity of the probe whether it is made from a highly specific region, e.g., the 5'regulatory region, or from a less specific region, e.g., a conserved motif, and the stringency of the hybridization or amplification wih determine whether the probe identifies only naturally occu ⁇ ing sequences encoding NAAP, ahelic variants, or related sequences.
- Probes may also be used for the detection of related sequences, and may have at least 50% sequence identity to any of the NAAP encoding sequences.
- the hybridization probes of the subject invention may be DNA or RNA and may be derived from the sequence of SEQ HO NO:37-72 or from genomic sequences including promoters, enhancers, and introns of the NAAP gene.
- Means for producing specific hybridization probes for polynucleotides encoding NAAP include the cloning of polynucleotides encoding NAAP or NAAP derivatives into vectors for the production of mRNA probes.
- vectors are known in the art, are commerciahy available, and may be used to synthesize RNA probes in vitro by means of the addition of the appropriate RNA polymerases and the appropriate labeled nucleotides.
- Hybridization probes may be labeled by a variety of reporter groups, for example, by radionuclides such as 32 P or 35 S, or by enzymatic labels, such as alkaline phosphatase coupled to the probe via avidin biotin coupling systems, and the like.
- Polynucleotides encoding NAAP may be used for the diagnosis of disorders associated with expression of NAAP.
- disorders include, but are not hmited to, a ceh proliferative disorder such as actinic keratosis, arteriosclerosis, atherosclerosis, bursitis, cirrhosis, hepatitis, mixed connective tissue disease (MCTD), myelofibrosis, paroxysmal noctarnal hemoglobinuria, polycythemia vera, psoriasis, primary thrombocythemia, and cancers including adenocarcinoma, leukemia, lymphoma, melanoma, myeloma, sarcoma, teratocarcinoma, and, in particular, a cancer of the adrenal gland, bladder, bone, bone marrow, brain, breast, cervix, gall bladder, ganglia, gastrointestinal tract, heart, kidney, liver, lung, muscle, ovary,
- Polynucleotides encoding NAAP maybe used in Southern or northern analysis, dot blot, or other membrane-based technologies; in PCR technologies; in dipstick, pin, and multiformat ELISA-like assays; and in microarrays utilizing fluids or tissues from patients to detect altered NAAP expression. Such qualitative or quantitative methods are weh known in the art.
- polynucleotides encoding NAAP may be used in assays that detect the presence of associated disorders, particularly those mentioned above.
- Polynucleotides complementary to sequences encoding NAAP maybe labeled by standard methods and added to a fluid or tissue sample from a patient under conditions suitable for the formation of hybridization complexes. After a suitable incubation period, the sample is washed and the signal is quantified and compared with a standard value. If the amount of signal in the patient sample is significantly altered in comparison to a control sample then the presence of altered levels of polynucleotides encoding NAAP in the sample indicates the presence of the associated disorder.
- Such assays may also be used to evaluate the efficacy of a particular therapeutic treatment regimen in animal studies, in clinical trials, or to monitor the treatment of an individual patient.
- a normal or standard profile for expression is established. This may be accomphshed by combining body fluids or ceh extracts taken from normal subjects, either animal or human, with a sequence, or a fragment thereof, encoding NAAP, under conditions suitable for hybridization or amphfication.
- Standard hybridization may be quantified by comparing the values obtained from normal subjects with values from an experiment in which a known amount of a substantially purified polynucleotide is used. Standard values obtained in this manner maybe compared with values obtained from samples from patients who are symptomatic for a disorder. Deviation from standard values is used to estabhsh the presence of a disorder.
- hybridization assays may be repeated on a regular basis to determine if the level of expression in the patient begins to approximate that which is observed in the normal subject.
- the results obtained from successive assays may be used to show the efficacy of treatment over a period ranging from several days to months.
- the presence of an abnormal amount of transcript (either under- or overexpressed) in biopsied tissue from an individual may indicate a predisposition for the development of the disease, or may provide a means for detecting the disease prior to the appearance of actual clinical symptoms.
- a more definitive diagnosis of this type may allow health professionals to employ preventative measures or aggressive treatment earlier, thereby preventing the development or further progression of the cancer.
- ohgonucleotides designed from the sequences encoding NAAP may involve the use of PCR. These ohgomers may be chemically synthesized, generated enzymaticahy, or produced in vitro.
- Ohgomers wih preferably contain a fragment of a polynucleotide encoding NAAP, or a fragment of a polynucleotide complementary to the polynucleotide encoding NAAP, and wih be employed under optimized conditions for identification of a specific gene or condition. Oligomers may also be employed under less stringent conditions for detection or quantification of closely related DNA or RNA sequences.
- ohgonucleotide primers derived from polynucleotides encoding NAAP maybe used to detect single nucleotide polymorphisms (SNPs).
- SNPs are substitutions, insertions and deletions that are a frequent cause of inherited or acquired genetic disease in humans.
- Methods of SNP detection include, but are not limited to, single-stranded conformation polymorphism (SSCP) and fluorescent SSCP (fSSCP) methods.
- SSCP single-stranded conformation polymorphism
- fSSCP fluorescent SSCP
- oligonucleotide primers derived from polynucleotides encoding NAAP are used to amplify DNA using the polymerase chain reaction (PCR).
- the DNA maybe derived, for example, from diseased or normal tissue, biopsy samples, bodily fluids, and the like.
- SNPs in the DNA cause differences in the secondary and tertiary structares of PCR products in single-stranded form, and these differences are detectable using gel electrophoresis in non-denaturing gels.
- the ohgonucleotide primers are fluorescently labeled, which ahows detection of the amplimers in Wgh-throughput equipment such as DNA sequencing machines.
- sequence database analysis methods termed in silico SNP (isSNP) are capable of identifying polymorphisms by comparing the sequence of individual overlapping DNA fragments which assemble into a common consensus sequence.
- SNPs may be detected and characterized by mass spectrometry using, for example, the high throughput MASSARRAY system (Sequenom, Inc., San Diego CA).
- SNPs maybe used to study the genetic basis of human disease. For example, at least 16 common SNPs have been associated with non-insulin-dependent diabetes me itas. SNPs are also useful for examining differences in disease outcomes in monogenic disorders, such as cystic fibrosis, sickle ceh anemia, or chronic granulomatous disease. For example, variants in the mannose-binding lectin, MBL2, have been shown to be co ⁇ elated with deleterious pulmonary outcomes in cystic fibrosis. SNPs also have utility in pharmacogenomics, the identification of genetic variants that influence a patient's response to a drag, such as life-tlireatening toxicity.
- N-acetyl transferase is associated with a high incidence of peripheral neuropathy in response to the anti-tuberculosis drug isoniazid, while a variation in the core promoter of the ALOX5 gene results in diminished clinical response to treatment with an anti-asthma drug that targets the 5-lipoxygenase pathway.
- Analysis of the distribution of SNPs in different populations is useful for investigating genetic drift, mutation, recombination, and selection, as weh as for tracing the origins of populations and their migrations (Taylor, J.G. et al. (2001) Trends Mol. Med. 7:507-512; Kwok, P.-Y. and Z. Gu (1999) Mol. Med. Today 5:538-543; Nowotny, P. et al. (2001) Curr. Opin. Neurobiol. 11:637-641).
- Methods which may also be used to quantify the expression of NAAP include radiolabeling or biotinylating nucleotides, coamplification of a control nucleic acid, and interpolating results from standard curves (Melby, P.C. et al. (1993) J. Immunol. Methods 159:235-244; Duplaa, C et al. (1993) Anal. Biochem. 212:229-236).
- the speed of quantitation of multiple samples maybe accelerated by running the assay in a Mgh-throughput format where the oligomer or polynucleotide of interest is presented in various dilutions and a spectrophotometric or colorimetric response gives rapid quantitation.
- ohgonucleotides or longer fragments derived from any of the polynucleotides described herein maybe used as elements on a microa ⁇ ay.
- the microarray can be used in transcript imaging techniques which monitor the relative expression levels of large numbers of genes simultaneously as described below.
- the microarray may also be used to identify genetic variants, mutations, and polymorphisms. This information may be used to determine gene function, to understand the genetic basis of a disorder, to diagnose a disorder, to monitor progression/regression of disease as a function of gene expression, and to develop and monitor the activities of therapeutic agents in the treatment of disease.
- this information may be used to develop a pharmacogenomic profile of a patient in order to select the most appropriate and effective treatment regimen for that patient.
- therapeutic agents which are highly effective and display the fewest side effects maybe selected for a patient based on his/her pharmacogenomic profile.
- NAAP fragments of NAAP, or antibodies specific for NAAP may be used as elements on a microa ⁇ ay.
- the microa ⁇ ay may be used to monitor or measure protein- protein interactions, drag-target interactions, and gene expression profiles, as described above.
- a particular embodiment relates to the use of the polynucleotides of the present invention to generate a transcript image of a tissue or ceh type.
- a transcript image represents the global pattern of gene expression by a particular tissue or ceh type. Global gene expression patterns are analyzed by quantifying the number of expressed genes and their relative abundance under given conditions and at a given time (See Seilhamer et al., "Comparative Gene Transcript Analysis," U.S. Patent No. 5,840,484, expressly incorporated by reference herein).
- a transcript image may be generated by hybridizing the polynucleotides of the present invention or their complements to the totality of transcripts or reverse transcripts of a particular tissue or ceh type.
- the hybridization takes place in high-throughput format, wherein the polynucleotides of the present invention or their complements comprise a subset of a plurality of elements on a microa ⁇ ay.
- the resultant transcript image would provide a profile of gene activity.
- Transcript images may be generated using transcripts isolated from tissues, ceh lines, biopsies, or other biological samples.
- the transcript image may thus reflect gene expression in vivo, as in the case of a tissue or biopsy sample, or in vitro, as in the case of a ceh line.
- Transcript images which profile the expression of the polynucleotides of the present invention may also be used in conjunction with in vitro model systems and prechnical evaluation of pharmaceuticals, as weh as toxicological testing of industrial and natarahy-occurring environmental compounds.
- Ah compounds induce characteristic gene expression patterns, frequently termed molecular fingerprints or toxicant signatures, which are indicative of mechanisms of action and toxicity (Nuwaysir, E.F. et al. (1999) Mol. Carcinog.
- test compound has a signature similar to that of a compound with known toxicity, it is likely to share those toxic properties. These fingerprints or signatures are most useful and refined when they contain expression information from a large number of genes and gene families. Ideahy, a genome-wide measurement of expression provides the highest quahty signatare. Even genes whose expression is not altered by any tested compounds are important as weh, as the levels of expression of these genes are used to normalize the rest of the expression data. The normalization procedure is useful for comparison of expression data after treatment with different compounds.
- the toxicity of a test compound can be assessed by treating a biological sample containing nucleic acids with the test compound.
- Nucleic acids that are expressed in the treated biological sample are hybridized with one or more probes specific to the polynucleotides of the present invention, so that transcript levels corresponding to the polynucleotides of the present invention may be quantified.
- the transcript levels in the treated biological sample are compared with levels in an untreated biological sample. Differences in the transcript levels between the two samples are indicative of a toxic response caused by the test compound in the treated sample.
- proteome refers to the global pattern of protein expression in a particular tissue or ceh type.
- proteome expression patterns, or profiles are analyzed by quantifying the number of expressed proteins and their relative abundance under given conditions and at a given time.
- a profile of a ceh's proteome may thus be generated by separating and analyzing the polypeptides of a particular tissue or ceh type.
- the separation is achieved using two-dimensional gel electrophoresis, in which proteins from a sample are separated by isoelectric focusing in the first dimension, and then according to molecular weight by sodium dodecyl sulfate slab gel electrophoresis in the second dimension (Steiner and Anderson, supra).
- the proteins are visualized in the gel as discrete and uniquely positioned spots, typically by staining the gel with an agent such as Coomassie Blue or silver or fluorescent stains.
- the optical density of each protein spot is generahy proportional to the level of the protein in the sample.
- the optical densities of equivalently positioned protein spots from different samples are compared to identify any changes in protein spot density related to the treatment.
- the proteins in the spots are partially sequenced using, for example, standard methods employing chemical or enzymatic cleavage fohowed by mass spectrometry.
- the identity of the protein in a spot maybe determined by comparing its partial sequence, preferably of at least 5 contiguous amino acid residues, to the polypeptide sequences of interest. In some cases, further sequence data maybe obtained for definitive protein identification.
- a proteomic profile may also be generated using antibodies specific for NAAP to quantify the levels of NAAP expression.
- the antibodies are used as elements on a microarray, and protein expression levels are quantified by exposing the microarray to the sample and detecting the levels of protein bound to each array element (Lueking, A. et al. (1999) Anal. Biochem. 270:103- 111; Mendoze, L.G. et al. (1999) Biotechniques 27:778-788).
- Detection maybe performed by a variety of methods known in the art, for example, by reacting the proteins in the sample with a thiol- or amino-reactive fluorescent compound and detecting the amount of fluorescence bound at each a ⁇ ay element.
- Toxicant signatures at the proteome level are also useful for toxicological screening, and should be analyzed in parallel with toxicant signatures at the transcript level.
- There is a poor co ⁇ elation between transcript and protein abundances for some proteins in some tissues (Anderson, N.L. and J. Seilhamer (1997) Electrophoresis 18:533-537), so proteome toxicant signatures maybe useful in the analysis of compounds which do not significantly affect the transcript image, but which alter the proteomic profile.
- the analysis of transcripts in body fluids is difficult, due to rapid degradation of mRNA, so proteomic profiling may be more reliable and informative in such cases.
- the toxicity of a test compound is assessed by treating a biological sample containing proteins with the test compound.
- Proteins that are expressed in the treated biological sample are separated so that the amount of each protein can be quantified.
- the amount of each protein is compared to the amount of the co ⁇ esponding protein in an untreated biological sample. A difference in the amount of protein between the two samples is indicative of a toxic response to the test compound in the treated sample.
- Individual proteins are identified by sequencing the amino acid residues of the individual proteins and comparing these partial sequences to the polypeptides of the present invention.
- the toxicity of a test compound is assessed by treating a biological sample containing proteins with the test compound. Proteins from the biological sample are incubated with antibodies specific to the polypeptides of the present invention. The amount of protein recognized by the antibodies is quantified. The amount of protein in the treated biological sample is compared with the amount in an untreated biological sample. A difference in the amount of protein between the two samples is indicative of a toxic response to the test compound in the treated sample.
- Microarrays may be prepared, used, and analyzed using methods known in the art (Brennan,
- nucleic acid sequences encoding NAAP may be used to generate hybridization probes useful in mapping the naturally occu ⁇ ing genomic sequence. Either coding or noncoding sequences may be used, and in some instances, noncoding sequences may be preferable over coding sequences. For example, conservation of a coding sequence among members of a multi-gene family may potentially cause undesired cross hybridization during chromosomal mapping.
- sequences may be mapped to a particular chromosome, to a specific region of a chromosome, or to artificial chromosome constructions, e.g., human artificial chromosomes (HACs), yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs), bacterial PI constructions, or single chromosome cDNA libraries (Harrington, JJ. et al. (1997) Nat. Genet. 15:345- 355; Price, CM. (1993) Blood Rev. 7:127-134; and Trask, B J. (1991) Trends Genet. 7:149-154).
- HACs human artificial chromosomes
- YACs yeast artificial chromosomes
- BACs bacterial artificial chromosomes
- PI constructions or single chromosome cDNA libraries
- nucleic acid sequences may be used to develop genetic linkage maps, for example, which correlate the inheritance of a disease state with the inheritance of a particular chromosome region or restriction fragment length polymorphism (RFLP) (Lander, E.S. and D. Botstein (1986) Proc. Natl. Acad. Sci. USA 83:7353-7357).
- RFLP restriction fragment length polymorphism
- Fluorescent in situ hybridization maybe correlated with other physical and genetic map data (Heinz-Ulrich, et al. (1995) in Meyers, supra, pp. 965-968). Examples of genetic map data can be found in various scientific journals or at the Online Mendelian Inheritance in Man (OMDVI) World Wide Web site. Co ⁇ elation between the location of the gene encoding NAAP on a physical map and a specific disorder, or a predisposition to a specific disorder, may help define the region of DNA associated with that disorder and thus may further positional cloning efforts.
- In situ hybridization of chromosomal preparations and physical mapping techniques may be used for extending genetic maps. Often the placement of a gene on the chromosome of another mammahan species, such as mouse, may reveal associated markers even if the exact chromosomal locus is not known. This information is valuable to investigators searching for disease genes using positional cloning or other gene discovery techniques. Once the gene or genes responsible for a disease or syndrome have been crudely localized by genetic linkage to a particular genomic region, e.g., ataxia-telangiectasia to llq22-23, any sequences mapping to that area may represent associated or regulatory genes for further investigation (Gatti, R.A.
- NAAP nucleotide sequence of the instant invention may also be used to detect differences in the chromosomal location due to translocation, inversion, etc., among normal, carrier, or affected individuals.
- NAAP its catalytic or immunogenic fragments, or oligopeptides thereof can be used for screening libraries of compounds in any of a variety of drag screening techniques.
- the fragment employed in such screening may be free in solution, affixed to a solid support, borne on a ceh surface, or located intracehularly. The formation of binding complexes between NAAP and the agent being tested may be measured.
- Another technique for drug screening provides for high throughput screening of compounds having suitable binding affinity to the protein of interest (Geysen, et al. (1984) PCT apphcation WO84/03564).
- This method large numbers of different small test compounds are synthesized on a solid substrate. The test compounds are reacted with NAAP, or fragments thereof, and washed. Bound NAAP is then detected by methods weh known in the art. Purified NAAP can also be coated directly onto plates for use in the aforementioned drag screening techniques. Alternatively, non-neutralizing antibodies can be used to captare the peptide and immobilize it on a sohd support.
- nucleotide sequences which encode NAAP maybe used in any molecular biology techniques that have yet to be developed, provided the new techniques rely on properties of nucleotide sequences that are cu ⁇ ently known, including, but not limited to, such properties as the triplet genetic code and specific base pah interactions.
- Incyte cDNAs were derived from cDNA libraries described in the LTFESEQ GOLD database (Incyte Genomics, Palo Alto CA). Some tissues were homogenized and lysed in guanidinium isothiocyanate, while others were homogenized and lysed in phenol or in a suitable mixture of denaturants, such as TRIZOL (Invitrogen), a monophasic solution of phenol and guanidine isothiocyanate. The resulting lysates were centrifuged over CsCl cushions or extracted with chloroform. RNA was precipitated from the lysates with either isopropanol or sodium acetate and ethanol, or by other routine methods.
- TRIZOL Invitrogen
- poly(A)+ RNA was isolated using oligo d(T)-coupled paramagnetic particles (Promega), OLIGOTEX latex particles (QIAGEN, Chatsworth CA), or an OLIGOTEX mRNA purification kit (QIAGEN).
- Stratagene was provided with RNA and constracted the co ⁇ esponding cDNA libraries. Otherwise, cDNA was synthesized and cDNA libraries were constructed with the UNIZAP vector system (Stratagene) or SUPERSCRIPT plasmid system (Invitrogen), using the recommended procedures or similar methods known in the art (Ausubel, 1997, supra, units 5.1-6.6). Reverse transcription was initiated using oligo d(T) or random primers. Synthetic ohgonucleotide adapters were ligated to double stranded cDNA, and the cDNA was digested with the appropriate restriction enzyme or enzymes.
- the cDNA was size-selected (300-1000 bp) using SEPHACRYL S1000, SEPHAROSE CL2B, or SEPHAROSE CL4B column chromatography (Amersham Biosciences) or preparative agarose gel electrophoresis.
- cDNAs were hgated into compatible restriction enzyme sites of the polylinker of a suitable plasmid, e.g., PBLUESCRIPT plasmid (Stratagene), PSPORT1 plasmid (mvitrogen), 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 pL CY
- Recombinant plasmids were transformed into competent E. coli cehs including XLl-Blue, XLl-BlueMRF, or SOLR from Stratagene or DH5 ⁇ , DH10B, or ElectroMAX DH10B from Invitrogen.
- Plasmids obtained as described in Example I were recovered from host cehs by in vivo excision using the UNIZAP vector system (Stratagene) or by ceh lysis. Plasmids were purified using at least one of the following: a Magic or WIZARD Minipreps DNA purification system (Promega); an AGTC Miniprep purification kit (Edge Biosystems, Gaithersburg MD); and QIAWELL 8 Plasmid, QIAWELL 8 Plus Plasmid, QIAWELL 8 Ultra Plasmid purification systems or the R.E.A.L. PREP 96 plasmid purification kit from QIAGEN. Following precipitation, plasmids were resuspended in 0.1 ml of distilled water and stored, with or without lyophilization, at 4°C
- plasmid DNA was amplified from host ceh lysates using direct link PCR in a high-throughput format (Rao, V.B. (1994) Anal. Biochem. 216:1-14). Host ceh lysis and thermal cycling steps were ca ⁇ ied out in a single reaction mixture. Samples were processed and stored in 384-weh plates, and the concentration of amplified plasmid DNA was quantified fluorometricahy using PICOGREEN dye (Molecular Probes, Eugene OR) and a FLUOROSKAN H fluorescence scanner (Labsystems Oy, Helsinki, Finland).
- Incyte cDNA recovered in plasmids as described in Example H were sequenced as fohows. Sequencing reactions were processed using standard methods or Mgh-throughput instrumentation such as the ABI CATALYST 800 (Applied Biosystems) thermal cycler or the PTC-200 thermal cycler (MJ Research) in conjunction with the HYDRA microdispenser (Robbins Scientific) or the MICROLAB 2200 (Hamilton) liquid transfer system. cDNA sequencing reactions were prepared using reagents provided by Amersham Biosciences or supplied in ABI sequencing kits such as the ABI PRISM BIGDYE Terminator cycle sequencing ready reaction kit (Apphed Biosystems).
- Electrophoretic separation of cDNA sequencing reactions and detection of labeled polynucleotides were carried out using the MEGABACE 1000 DNA sequencing system (Amersham Biosciences); the ABI PRISM 373 or 377 sequencing system (Apphed Biosystems) in conjunction with standard ABI protocols and base calling software; or other sequence analysis systems known in the art. Reading frames within the cDNA sequences were identified using standard methods (reviewed in Ausubel, 1997, supra, unit 7.7). Some of the cDNA sequences were selected for extension using the techniques disclosed in Example VTA.
- the polynucleotide sequences derived from Incyte cDNAs were validated by removing vector, linker, and poly(A) sequences and by masking ambiguous bases, using algorithms and programs based on BLAST, dynamic programming, and dinucleotide nearest neighbor analysis.
- the Incyte cDNA sequences or translations thereof were then queried against a selection of pubhc databases such as the GenBank primate, rodent, mammalian, vertebrate, and eukaryote databases, and BLOCKS , PRINTS , DOMO, PRODOM; PROTEOME databases with sequences from Homo sapiens, Rattus norvegicus, Mus musculus, Caenorhabditis elegans, Saccharomyces cerevisiae, Schizosaccharomyces pombe, and Candida albicans (Incyte Genomics, Palo Alto CA); hidden Markov model (HMM)-based protein family databases such as PFAM, INCY, and TIGRFAM (Haft, D.H.
- HMM hidden Markov model
- HMM-based protein domain databases such as SMART (Schultz et al. (1998) Proc. Natl. Acad. Sci. USA 95:5857-5864; Letanic, I. et al. (2002) Nucleic Acids Res. 30:242-244).
- HMM is a probabilistic approach which analyzes consensus primary structures of gene families. See, for example, Eddy, S.R. (1996) Cu ⁇ . Opin. Struct. Biol. 6:361-365). The queries were performed using programs based on BLAST, FASTA, BLIMPS, and HMMER.
- the Incyte cDNA sequences were assembled to produce fuh length polynucleotide sequences.
- GenBank cDNAs, GenBank ESTs, stitched sequences, stretched sequences, or Genscan-predicted coding sequences were used to extend Incyte cDNA assemblages to fuh length. Assembly was performed using programs based on Phred, Phrap, and Consed, and cDNA assemblages were screened for open reading frames using programs based on GeneMark, BLAST, and FASTA.
- the fuh length polynucleotide sequences were translated to derive the corresponding fuh length polypeptide sequences.
- a polypeptide may begin at any of the methionine residues of the fuh length translated polypeptide.
- Fuh length polypeptide sequences were subsequently analyzed by querying against databases such as the GenBank protein databases (genpept), SwissProt, the PROTEOME databases, BLOCKS, PRINTS, DOMO, PRODOM, Prosite, hidden Markov model (HMM)-based protein family databases such as PFAM, INCY, and TIGRFAM; and HMM-based protein domain databases such as SMART.
- GenBank protein databases Genpept
- PROTEOME databases
- BLOCKS BLOCKS
- PRINTS DOMO
- PRODOM hidden Markov model
- Prosite Prosite
- HMM-based protein family databases such as PFAM, INCY, and TIGRFAM
- HMM-based protein domain databases such as SMART.
- Fuh length polynucleotide sequences are also analyzed using MACDNASIS PRO software (Hitachi Software Engineering, South San Francisco CA) and LA
- Polynucleotide and polypeptide sequence alignments are generated using default parameters specified by the CLUSTAL algorithm as incorporated into the MEGALIGN multisequence ahgnment program (DNASTAR), which also calculates the percent identity between aligned sequences.
- Table 7 summarizes the tools, programs, and algorithms used for the analysis and assembly of Incyte cDNA and fuh length sequences and provides apphcable descriptions, references, and threshold parameters.
- the first column of Table 7 shows the tools, programs, and algorithms used, the second column provides brief descriptions thereof, the third column presents appropriate references, ah of which are incorporated by reference herein in their entirety, and the fourth column presents, where apphcable, the scores, probability values, and other parameters used to evaluate the strength of a match between two sequences (the higher the score or the lower the probability value, the greater the identity between two sequences).
- Genscan is a general-purpose gene identification program which analyzes genomic DNA sequences from a variety of organisms (Burge, C and S. Karlin (1997) J. Mol. Biol. 268:78-94; Burge, C and S. Karhn (1998) Cu ⁇ . Opin. Struct. Biol. 8:346-354). The program concatenates predicted exons to form an assembled cDNA sequence extending from a methionine to a stop codon.
- Genscan is a FASTA database of polynucleotide and polypeptide sequences.
- the maximum range of sequence for Genscan to analyze at once was set to 30 kb.
- the encoded polypeptides were analyzed by querying against PFAM models for nucleic acid-associated proteins. Potential nucleic acid-associated proteins were also identified by homology to Incyte cDNA sequences that had been annotated as nucleic acid-associated proteins. These selected Genscan-predicted sequences were then compared by BLAST analysis to the genpept and gbpri pubhc databases.
- Genscan- predicted sequences were then edited by comparison to the top BLAST hit from genpept to co ⁇ ect e ⁇ ors in the sequence predicted by Genscan, such as extra or omitted exons.
- BLAST analysis was also used to find any Incyte cDNA or public cDNA coverage of the Genscan-predicted sequences, thus providing evidence for transcription.
- Incyte cDNA coverage was available, this information was used to co ⁇ ect or confirm the Genscan predicted sequence.
- Fuh length polynucleotide sequences were obtained by assembling Genscan-predicted coding sequences with Incyte cDNA sequences and/or pubhc cDNA sequences using the assembly process described in Example JR. Alternatively, fuh length polynucleotide sequences were derived entirely from edited or unedited Genscan-predicted coding sequences.
- Partial cDNA sequences were extended with exons predicted by the Genscan gene identification program described in Example IV. Partial cDNAs assembled as described in Example HI were mapped to genomic DNA and parsed into clusters containing related cDNAs and Genscan exon predictions from one or more genomic sequences. Each cluster was analyzed using an algorithm based on graph theory and dynamic programming to integrate cDNA and genomic information, generating possible splice variants that were subsequently confirmed, edited, or extended to create a fuh length sequence. Sequence intervals in which the entire length of the interval was present on more than one sequence in the cluster were identified, and intervals thus identified were considered to be equivalent by transitivity.
- Partial DNA sequences were extended to fuh length with an algorithm based on BLAST analysis.
- the nearest GenBank protein homolog was then compared by BLAST analysis to either Incyte cDNA sequences or GenScan exon predicted sequences described in Example IV.
- a chimeric protein was generated by using the resultant high-scoring segment pahs (HSPs) to map the translated sequences onto the GenBank protein homolog. Insertions or deletions may occur in the chimeric protein with respect to the original GenBank protein homolog.
- HSPs high-scoring segment pahs
- GenBank protein homolog The GenBank protein homolog, the chimeric protein, or both were used as probes to search for homologous genomic sequences from the pubhc human genome databases. Partial DNA sequences were therefore "stretched” or extended by the addition of homologous genomic sequences. The resultant stretched sequences were examined to determine whether it contained a complete gene.
- sequences which were used to assemble SEQ HO NO:37-72 were compared with sequences from the Incyte LIFESEQ database and pubhc domain databases using BLAST and other implementations of the Smith- Waterman algorithm. Sequences from these databases that matched SEQ HO NO:37-72 were assembled into clusters of contiguous and overlapping sequences using assembly algorithms such as Phrap (Table 7). Radiation hybrid and genetic mapping data available from pubhc resources such as the Stanford Human Genome Center (SHGC), Whitehead Institute for Genome Research (WIGR), and Genethon were used to determine if any of the clustered sequences had been previously mapped. Inclusion of a mapped sequence in a cluster resulted in the assignment of ah sequences of that cluster, including its particular SEQ ID NO:, to that map location.
- pubhc resources such as the Stanford Human Genome Center (SHGC), Whitehead Institute for Genome Research (WIGR), and Genethon were used to determine if any of the clustered sequences had been previously mapped.
- Map locations are represented by ranges, or intervals, of human chromosomes.
- the map position of an interval, in centiMorgans, is measured relative to the terminus of the chromosome's p- arm.
- centiMorgan cM
- centiMorgan is a unit of measurement based on recombination frequencies between chromosomal markers. On average, 1 cM is roughly equivalent to 1 megabase (Mb) of DNA in humans, although this can vary widely due to hot and cold spots of recombination.
- the cM distances are based on genetic markers mapped by Genethon which provide boundaries for radiation hybrid markers whose sequences were included in each of the clusters.
- Northern analysis is a laboratory technique used to detect the presence of a transcript of a gene and involves the hybridization of a labeled nucleotide sequence to a membrane on which RNAs from a particular ceh type or tissue have been bound (Sambrook et al., supra, ch. 7; Ausubel (1995) supra, ch. 4 and 16).
- the product score takes into account both the degree of similarity between two sequences and the length of the sequence match.
- the product score is a normalized value between 0 and 100, and is calculated as fohows: the BLAST score is multiplied by the percent nucleotide identity and the product is divided by (5 times the length of the shorter of the two sequences).
- the BLAST score is calculated by assigning a score of +5 for every base that matches in a high-scoring segment pair (HSP), and -4 for every mismatch. Two sequences may share more than one HSP (separated by gaps). H there is more than one HSP, then the pah with the highest BLAST score is used to calculate the product score.
- the product score represents a balance between fractional overlap and quahty in a BLAST alignment. For example, a product score of 100 is produced only for 100% identity over the entire length of the shorter of the two sequences being compared. A product score of 70 is produced either by 100% identity and 70% overlap at one end, or by 88% identity and 100% overlap at the other. A product score of 50 is produced either by 100% identity and 50% overlap at one end, or 79% identity and 100% overlap.
- polynucleotides encoding NAAP are analyzed with respect to the tissue sources from which they were derived. For example, some fuh length sequences are assembled, at least in part, with overlapping Incyte cDNA sequences (see Example HI).
- Each cDNA sequence is derived from a cDNA hbrary constructed from a human tissue.
- Each human tissue is classified into one of the following organ/tissue categories: cardiovascular system; connective tissue; digestive system; embryonic structares; endocrine system; exocrine glands; genitalia, female; genitalia, male; germ cehs; hemic and immune system; hver; musculoskeletal system; nervous system; pancreas; respiratory system; sense organs; skin; stomatognathic system; unclassified/mixed; or urinary tract.
- the number of libraries in each category is counted and divided by the total number of libraries across ah categories.
- each human tissue is classified into one of the following disease/condition categories: cancer, ceh line, developmental, inflammation, neurological, trauma, cardiovascular, pooled, and other, and the number of libraries in each category is counted and divided by the total number of libraries across ah categories. The resulting percentages reflect the tissue- and disease-specific expression of cDNA encoding NAAP.
- Fuh length polynucleotides are produced by extension of an appropriate fragment of the fuh length molecule using ohgonucleotide primers designed from this fragment.
- One primer was synthesized to initiate 5' extension of the known fragment, and the other primer was synthesized to initiate 3' extension of the known fragment.
- the initial primers were designed using OLIGO 4.06 software (National Biosciences), or another appropriate program, to be about 22 to 30 nucleotides in length, to have a GC content of about 50% or more, and to anneal to the target sequence at temperatares of about 68 °C to about 72 °C. Any stretch of nucleotides which would result in hairpin structares and primer-primer dimerizations was avoided. Selected human cDNA libraries were used to extend the sequence. If more than one extension was necessary or deshed, additional or nested sets of primers were designed.
- PCR was performed in 96-weh plates using the PTC-200 thermal cycler (MJ Research, Inc.).
- the reaction mix contained DNA template, 200 nmol of each primer, reaction buffer containing Mg 2+ , (NH ⁇ SO ⁇ and 2-mercaptoethanol, Taq DNA polymerase (Amersham Biosciences), ELONGASE enzyme
- Step 1 94°C, 3 min
- Step 2 94°C, 15 sec
- Step 3 60°C, 1 min
- Step 4 68°C, 2 min
- Step 5 Steps 2, 3, and 4 repeated 20 times
- Step 6 68°C, 5 min
- Step 7 storage at 4°C
- the parameters for primer pah T7 and SK+ were as follows: Step 1: 94 °C, 3 min; Step 2: 94°C 15 sec; Step 3: 57°C, 1 min; Step 4: 68°C, 2 min; Step 5: Steps 2, 3, and 4 repeated 20 times; Step 6: 68°C, 5 min; Step 7: storage at 4°C
- the concentration of DNA in each weh was determined by dispensing 100 ⁇ l PICOGREEN quantitation reagent (0.25% (v/v) PICOGREEN; Molecular Probes, Eugene OR) dissolved in IX TE and 0.5 jul of undiluted PCR product into each weh of an opaque fluorimeter plate (Corning Costar, Acton MA), allowing the DNA to bind to the reagent.
- the plate was scanned in a Fluoroskan H (Labsystems Oy, Helsinki, Finland) to measure the fluorescence of the sample and to quantify the concentration of DNA.
- a 5 ⁇ l to 10 ⁇ l aliquot of the reaction mixture was analyzed by electrophoresis on a 1 % agarose gel to determine which reactions were successful in extending the sequence.
- the extended nucleotides were desalted and concentrated, transfe ⁇ ed to 384-weh plates, digested with CviJI cholera viras endonuclease (Molecular Biology Research, Madison WI), and sonicated or sheared prior to rehgation into pUC 18 vector (Amersham Biosciences).
- the digested nucleotides were separated on low concentration (0.6 to 0.8%) agarose gels, fragments were excised, and agar digested with Agar ACE (Promega).
- Extended clones were rehgated using T4 ligase (New England Biolabs, Beverly MA) into pUC 18 vector (Amersham Biosciences), treated with Pfu DNA polymerase (Stratagene) to fill-in restriction site overhangs, and transfected into competent E. coli cehs. Transformed cehs were selected on antibiotic-containing media, and individual colonies were picked and cultured overnight at 37 °C in 384- weh plates in LB/2x carb liquid media.
- fuh length polynucleotides are verified using the above procedure or are used to obtain 5 'regulatory sequences using the above procedure along with oligonucleotides designed for such extension, and an appropriate genomic hbrary.
- SNPs single nucleotide polymorphisms
- LIFESEQ database Incyte Genomics
- Sequences from the same gene were clustered together and assembled as described in Example HI, allowing the identification of ah sequence variants in the gene.
- An algorithm consisting of a series of filters was used to distinguish SNPs from other sequence variants. Preliminary filters removed the majority of basecah e ⁇ ors by requiring a minimum Phred quahty score of 15, and removed sequence ahgnment errors and errors resulting from improper trimming of vector sequences, chimeras, and sphce variants.
- Certain SNPs were selected for further characterization by mass spectrometry using the high throughput MASSARRAY system (Sequenom, Inc.) to analyze ahele frequencies at the SNP sites in four different human populations.
- the Caucasian population comprised 92 individuals (46 male, 46 female), including 83 from Utah, four French, three deciualan, and two Amish individuals.
- the African population comprised 194 individuals (97 male, 97 female), ah African Americans.
- the Hispanic population comprised 324 individuals (162 male, 162 female), all Mexican Hispanic.
- the Asian population comprised 126 individuals (64 male, 62 female) with a reported parental breakdown of 43% Chinese, 31% Japanese, 13% Korean, 5% Vietnamese, and 8% other Asian. Ahele frequencies were first analyzed in the Caucasian population; in some cases those SNPs which showed no allelic variance in this population were not further tested in the other three populations.
- Hybridization probes derived from SEQ HO NO:37-72 are employed to screen cDNAs, genomic DNAs, or mRNAs. Although the labeling of ohgonucleotides, consisting of about 20 base pahs, is specifically described, essentially the same procedure is used with larger nucleotide fragments.
- Ohgonucleotides are designed using state-of-the-art software such as OLIGO 4.06 software (National Biosciences) and labeled by combining 50 pmol of each oligomer, 250 ⁇ Ci of [ ⁇ - 32 P] adenosine triphosphate (Amersham Biosciences), and T4 polynucleotide kinase (DuPont NEN, Boston MA).
- the labeled ohgonucleotides are substantially purified using a SEPHADEX G-25 superfine size exclusion dextran bead column (Amersham Biosciences). An aliquot containing 10 7 counts per minute of the labeled probe is used in a typical membrane-based hybridization analysis of human genomic DNA digested with one of the following endonucleases: Ase I, Bgl H, Eco Rl, Pst I, Xba I, or Pvu H (DuPont NEN).
- the DNA from each digest is fractionated on a 0.7% agarose gel and transferred to nylon membranes (Nytran Plus, Schleicher & Schuell, Durham NH). Hybridization is carried out for 16 hours at 40 X. To remove nonspecific signals, blots are sequentially washed at room temperature under conditions of up to, for example, 0.1 x saline sodium citrate and 0.5% sodium dodecyl sulfate. Hybridization patterns are visualized using autoradiography or an alternative imaging means and compared. XL Microarrays
- the linkage or synthesis of array elements upon a microa ⁇ ay can be achieved utilizing photohthography, piezoelectric printing (ink-jet printing; see, e.g., Baldeschweiler, supra), mechanical microspotting technologies, and derivatives thereof.
- the substrate in each of the aforementioned technologies should be uniform and sohd with a non-porous surface (Schena (1999), supra).
- Suggested substrates include sihcon, sihca, glass shdes, glass chips, and sihcon wafers.
- a procedure analogous to a dot or slot blot may also be used to a ⁇ ange and link elements to the surface of a substrate using thermal, UV, chemical, or mechanical bonding procedures.
- a typical a ⁇ ay may be produced using available methods and machines weh known to those of ordinary skih in the art and may contain any appropriate number of elements (Schena, M. et al. (1995) Science 270:467-470; Shalon, D. et al. (1996) Genome Res. 6:639-645; Marshah, A. and J. Hodgson (1998) Nat. Biotechnol. 16:27-31).
- Fuh length cDNAs, Expressed Sequence Tags (ESTs), or fragments or oligomers thereof may comprise the elements of the microa ⁇ ay. Fragments or ohgomers suitable for hybridization can be selected using software weh known in the art such as LASERGENE software (DNASTAR).
- the a ⁇ ay elements are hybridized with polynucleotides in a biological sample.
- the polynucleotides in the biological sample are conjugated to a fluorescent label or other molecular tag for ease of detection.
- a fluorescence scanner is used to detect hybridization at each a ⁇ ay element.
- microa ⁇ ay preparation and usage is described in detail below.
- Total RNA is isolated from tissue samples using the guanidinium thiocyanate method and poly(A) + RNA is purified using the oligo-(dT) cehulose method.
- Each poly(A) + RNA sample is reverse transcribed using MMLV reverse-transcriptase, 0.05 pg/ ⁇ l oligo-(dT) primer (21mer), IX first strand buffer, 0.03 units/ ⁇ l RNase inhibitor, 500 ⁇ M dATP, 500 ⁇ M dGTP, 500 ⁇ M dTTP, 40 ⁇ M dCTP, 40 ⁇ M dCTP-Cy3 (BDS) or dCTP-Cy5 (Amersham Biosciences).
- the reverse transcription reaction is performed in a 25 ml volume containing 200 ng poly(A) + RNA with GEMBRIGHT kits (Incyte).
- Specific control poly(A) + RNAs are synthesized by in vitro transcription from non-coding yeast genomic DNA. After incubation at 37° C for 2 hr, each reaction sample (one with Cy3 and another with Cy5 labeling) is treated with 2.5 ml of 0.5M sodium hydroxide and incubated for 20 minutes at 85° C to the stop the reaction and degrade the RNA. Samples are purified using two successive CHROMA SPIN 30 gel filtration spin columns (CLONTECH Laboratories, Inc.
- Sequences of the present invention are used to generate a ⁇ ay elements.
- Each a ⁇ ay element is amplified from bacterial cells containing vectors with cloned cDNA inserts.
- PCR amplification uses primers complementary to the vector sequences flanking the cDNA insert.
- Array elements are amplified in thirty cycles of PCR from an initial quantity of 1-2 ng to a final quantity greater than 5 ⁇ g. Amplified a ⁇ ay elements are then purified using SEPHACRYL-400 (Amersham Biosciences).
- Purified array elements are immobilized on polymer-coated glass shdes.
- Glass microscope slides (Corning) are cleaned by ultrasound in 0.1% SDS and acetone, with extensive distilled water washes between and after treatments.
- Glass shdes are etched in 4% hydrofluoric acid (VWR Scientific Products Corporation (VWR), West Chester PA), washed extensively in distihed water, and coated with 0.05% aminopropyl silane (Sigma) in 95% ethanol. Coated shdes are cured in a 110°C oven.
- Array elements are apphed to the coated glass substrate using a procedure described in U.S.
- Patent No. 5,807,522 incorporated herein by reference.
- 1 ⁇ l of the a ⁇ ay element DNA, at an average concentration of 100 ng/ ⁇ l, is loaded into the open capillary printing element by a high-speed robotic apparatus.
- the apparatus then deposits about 5 nl of a ⁇ ay element sample per shde.
- Microa ⁇ ays are UV-crosslinked using a STRATALINKER UV-crosshnker (Stratagene). Microarrays are washed at room temperature once in 0.2% SDS and three times in distihed water. Non-specific binding sites are blocked by incubation of microarrays in 0.2% casein in phosphate buffered saline (PBS) (Tropix, Inc., Bedford MA) for 30 minutes at 60° C fohowed by washes in 0.2% SDS and distihed water as before.
- PBS phosphate buffered saline
- Hybridization Hybridization reactions contain 9 ⁇ l of sample mixture consisting of 0.2 ⁇ g each of Cy3 and
- Cy5 labeled cDNA synthesis products in 5X SSC, 0.2% SDS hybridization buffer The sample mixture is heated to 65° C for 5 minutes and is aliquoted onto the microa ⁇ ay surface and covered with an 1.8 cm 2 covershp.
- the a ⁇ ays are transfe ⁇ ed to a waterproof chamber having a cavity just slightly larger than a microscope shde.
- the chamber is kept at 100% humidity internally by the addition of 140 ⁇ l of 5X SSC in a corner of the chamber.
- the chamber containing the a ⁇ ays is incubated for about 6.5 hours at 60X.
- the arrays are washed for 10 min at 45° C in a first wash buffer (IX SSC, 0.1% SDS), three times for 10 minutes each at 45° C in a second wash buffer (0.1X SSC), and dried.
- Detection Reporter-labeled hybridization complexes are detected with a microscope equipped with an Innova 70 mixed gas 10 W laser (Coherent, Inc., Santa Clara CA) capable of generating spectral lines at 488 nm for excitation of Cy3 and at 632 nm for excitation of Cy5.
- the excitation laser hght is focused on the a ⁇ ay using a 20X microscope objective (Nikon, Inc., Melville NY).
- the shde containing the array is placed on a computer-controlled X-Y stage on the microscope and raster- scanned past the objective.
- the 1.8 cm x 1.8 cm a ⁇ ay used in the present example is scanned with a resolution of 20 micrometers.
- a mixed gas multiline laser excites the two fluorophores sequentiahy. Emitted hght is split, based on wavelength, into two photomultiplier tube detectors (PMT R1477, Hamamatsu Photonics Systems, Bridgewater NJ) co ⁇ esponding to the two fluorophores. Appropriate filters positioned between the a ⁇ ay and the photomultiplier tabes are used to filter the signals.
- the emission maxima of the fluorophores used are 565 nm for Cy3 and 650 nm for Cy5.
- Each a ⁇ ay is typically scanned twice, one scan per fluorophore using the appropriate filters at the laser source, although the apparatus is capable of recording the spectra from both fluorophores simultaneously.
- the sensitivity of the scans is typically cahbrated using the signal intensity generated by a cDNA control species added to the sample mixture at a known concentration.
- a specific location on the a ⁇ ay contains a complementary DNA sequence, allowing the intensity of the signal at that location to be correlated with a weight ratio of hybridizing species of 1:100,000.
- the cahbration is done by labeling samples of the calibrating cDNA with the two fluorophores and adding identical amounts of each to the hybridization mixture.
- the output of the photomultipher tube is digitized using a 12-bit RTT-835H analog-to-digital (A/D) conversion board (Analog Devices, Inc., Norwood MA) installed in an IBM-compatible PC computer.
- the digitized data are displayed as an image where the signal intensity is mapped using a linear 20-color transformation to a pseudocolor scale ranging from blue (low signal) to red (high signal).
- the data is also analyzed quantitatively. Where two different fluorophores are excited and measured simultaneously, the data are first co ⁇ ected for optical crosstalk (due to overlapping emission spectra) between the fluorophores using each fluorophore's emission spectrum.
- a grid is superimposed over the fluorescence signal image such that the signal from each spot is centered in each element of the grid.
- the fluorescence signal within each element is then integrated to obtain a numerical value co ⁇ esponding to the average intensity of the signal.
- the software used for signal analysis is the GEMTOOLS gene expression analysis program (Incyte).
- a ⁇ ay elements that exhibited at least about a two-fold change in expression, a signal-to-background ratio of at least 2.5, and an element spot size of at least 40% were identified as differentially expressed using the GEMTOOLS program (Incyte Genomics).
- SEQ ID NO:41 showed differential expression in several human breast cancer ceh lines, as determined by microa ⁇ ay analysis.
- HMEC is a human primary mammary epithelial ceh strain derived from normal mammary tissue (Clonetics, San Diego, CA). The following ceh lines were tested on microa ⁇ ays: MCF-10A is a human breast mammary gland ceh line isolated from a 36-year-old female with fibrocystic breast disease; SkBR3 is a breast adenocarcinoma ceh line isolated from a malignant pleural effusion of a 43 -year-old female; MCF7 is a breast adenocarcinoma ceh line derived from the pleural effusion of a 69-year-old female; T47D is a breast carcinoma ceh line derived from a pleural effusion from a 54-year-old female with an infiltrating ductal carcinoma of the breast; BT20 is a breast carcinoma ceh line derived in vitro from cells emigrating out of thin slices of a tumor mass isolated from a 74-year-old female;
- cDNAs from the five tamor ceh hnes representing various stages of breast tamor progression were compared with that of the non-mahgnant mammary epithelial ceh lines, HMEC or MCF-10A.
- SEQ HO NO:41 showed at least two-fold differential expression when comparing HMEC cehs versus Sk-BR-3 and T-47D cehs. Additionahy, SEQ HO NO:41 expression was decreased at least two-fold when comparing breast cehs from fibrocystic breast tissue versus BT-20, MCF-7, MDA-mb- 231, Sk-BR-3, and T-47D cancerous ceh hnes.
- SEQ ID NO:44 is upregulated 3.9 fold in DC as compared to monocytes, suggesting that SEQ ID NO:44, encoding SEQ JD NO:8, could be used for example, to understand the process by which monocytes differentiate into immature dendritic cehs and eventually allow manipulation of the immune system leading to potential immunotherapies for diseases such as cancer, AIDS, and infectious diseases; and enhancing vaccine efficacy.
- SEQ HO NO:48 is upregulated in six out of seven PBMC populations (each of which was obtained from a different donor) treated with Staphylococcal exotoxins (SEB).
- SEB Staphylococcal exotoxins
- the PBMCs were stimulated in vitro with SEB for 24 and 72 hours.
- the expression of SEQ HO NO:48 was higher after 24 hours and dropped after 72 hours. Therefore, SEQ HO NO:48 is useful in diagnostic assays for immune responses.
- SEQ HO NO:64 showed differential expression in the C3A ceh line, a well-established in vitro model of the mature human hver (Mickelson, J.K. et al. (1995) Hepatology 22:866-875; Nagendra, A.R. et al. (1997) Am. J. Physiol. 272:G408-G416), as determined by microa ⁇ ay analysis.
- the effects upon hver metabohsm and hormone clearance mechanisms are important to understand the pharmacodynamics of a drag.
- the human C3A ceh line is a clonal derivative of HepG2/C3 (hepatoma ceh line, isolated from a 15-year-old male with hver tamor), which was selected for strong contact inhibition of growth.
- HepG2/C3 hepatoma ceh line, isolated from a 15-year-old male with hver tamor
- the use of a clonal population enhances the reproducibihty of the cells.
- C3 A cehs have many characteristics of primary human hepatocytes in culture: i) expression of insulin receptor and insulin-like growth factor H receptor; h) secretion of a high ratio of serum albumin compared with ⁇ -fetoprotein; iii) conversion of ammonia to urea and glutamine; iv) abihtiy to metabolize aromatic amino acids; and v) proliferation in glucose-free and insuhn-free medium.
- SEQ ID NO:64 showed differential expression in C3A cehs treated with a variety of steroids including beclomethasone, medroxyprogesterone, budesonide, prednisone, dexamethasone, and progesterone, versus untreated C3A cells, as determined by microarray analysis. Therefore, SEQ ID NO:64 is useful for the diagnosis and monitoring of hver, endocrine, and reproductive diseases and in the diagnosis of and as a therapeutic target for inflammatory diseases and humoral immune response.
- Sequences complementary to the NAAP-encoding sequences, or any parts thereof, are used to detect, decrease, or inhibit expression of naturally occurring NAAP.
- ohgonucleotides comprising from about 15 to 30 base pahs is described, essentially the same procedure is used with smaller or with larger sequence fragments.
- Appropriate oligonucleotides are designed using OLIGO 4.06 software (National Biosciences) and the coding sequence of NAAP.
- a complementary ohgonucleotide is designed from the most unique 5' sequence and used to prevent promoter binding to the coding sequence.
- a complementary oligonucleotide is designed to prevent ribosomal binding to the NAAP-encoding transcript.
- NAAP is achieved using bacterial or virus-based expression systems.
- cDNA is subcloned into an appropriate vector containing an antibiotic resistance gene and an inducible promoter that directs high levels of cDNA transcription.
- promoters include, but are not hmited to, the tip-lac ⁇ tac) hybrid promoter and the T5 or T7 bacteriophage promoter in conjunction with the lac operator regulatory element.
- Recombinant vectors are transformed into suitable bacterial hosts, e.g., BL21(DE3).
- Antibiotic resistant bacteria express NAAP upon induction with isopropyl beta-D- thiogalactopyranoside (HPTG).
- NAAP in eukaryotic cehs is achieved by infecting insect or mammahan ceh lines with recombinant Autographica californica nuclear polyhedrosis virus (AcMNPV), commonly known as baculoviras.
- AcMNPV Autographica californica nuclear polyhedrosis virus
- the nonessential polyhedrin gene of baculoviras is replaced with cDNA encoding NAAP by either homologous recombination or bacterial-mediated transposition involving transfer plasmid intermediates. Viral infectivity is maintained and the strong polyhedrin promoter drives high levels of cDNA transcription.
- baculoviras Recombinant baculoviras is used to infect Spodoptera frugiperda (Sf9) insect cehs in most cases, or human hepatocytes, in some cases. Infection of the latter requires additional genetic modifications to baculoviras (See Engelhard, E.K. et al. (1994) Proc. Natl. Acad. Sci. USA 91:3224-3227; Sandig, Y. et al. (1996) Hum. Gene Ther.
- NAAP is synthesized as a fusion protein with, e.g., glutathione S- transferase (GST) or a peptide epitope tag, such as FLAG or 6-His, permitting rapid, single-step, affinity-based purification of recombinant fusion protein from crade ceh lysates.
- GST a 26-kilodalton enzyme from Schistosoma japonicum, enables the purification of fusion proteins on immobilized glutathione under conditions that maintain protein activity and antigenicity (Amersham Biosciences). Following purification, the GST moiety can be proteolyticahy cleaved from NAAP at specifically engineered sites.
- FLAG an 8-amino acid peptide
- 6-His a stretch of six consecutive histidine residues, enables purification on metal-chelate resins (QIAGEN). Methods for protein expression and purification are discussed in Ausubel (1995, supra, ch. 10 and 16). Purified NAAP obtained by these methods can be used directly in the assays shown in Examples XVH, XVm, and XIX, where apphcable. XIV. Functional Assays
- NAAP function is assessed by expressing the sequences encoding NAAP at physiologically elevated levels in mammahan ceh cultare systems.
- cDNA is subcloned into a mammahan expression vector containing a strong promoter that drives high levels of cDNA expression.
- Vectors of choice include PCMV SPORT plasmid (Invitrogen, Carlsbad CA) and PCR3.1 plasmid (Invitrogen), both of which contain the cytomegalovirus promoter. 5-10 ⁇ g of recombinant vector are transiently transfected into a human ceh line, for example, an endothelial or hematopoietic ceh line, using either liposome formulations or electroporation.
- 1-2 ⁇ g of an additional plasmid containing sequences encoding a marker protein are co-transfected.
- Expression of a marker protein provides a means to distinguish transfected cehs from nontransfected cehs and is a rehable predictor of cDNA expression from the recombinant vector.
- Marker proteins of choice include, e.g., Green Fluorescent Protein (GFP; Clontech), CD64, or a CD64-GFP fusion protein.
- FCM Flow cytometry
- FCM Flow cytometry
- FCM detects and quantifies the uptake of fluorescent molecules that diagnose events preceding or coincident with ceh death. These events include changes in nuclear DNA content as measured by staining of DNA with propidium iodide; changes in ceh size and granularity as measured by forward hght scatter and 90 degree side light scatter; down-regulation of DNA synthesis as measured by decrease in bromodeoxyuridrne uptake; alterations in expression of ceh surface and intracehular proteins as measured by reactivity with specific antibodies; and alterations in plasma membrane composition as measured by the binding of fluorescein-conjugated Annexin V protein to the ceh surface. Methods in flow cytometry are discussed in Ormerod, M.G. (1994) Flow Cytometry, Oxford, New York NY.
- NAAP The influence of NAAP on gene expression can be assessed using highly purified populations of cehs transfected with sequences encoding NAAP and either CD64 or CD64-GFP.
- CD64 and CD64-GFP are expressed on the surface of transfected cells and bind to conserved regions of human immunoglobulin G (IgG).
- Transfected cells are efficiently separated from nontransfected cehs using magnetic beads coated with either human IgG or antibody against CD64 (DYNAL, Lake Success NY).
- mRNA can be purified from the cells using methods weh known by those of skih in the art. Expression of mRNA encoding NAAP and other genes of interest can be analyzed by northern analysis or microa ⁇ ay techniques.
- XV Production of NAAP Specific Antibodies NAAP substantially purified using polyacrylamide gel electrophoresis (PAGE; see, e.g.,
- oligopeptides of about 15 residues in length are synthesized using an ABI 43 IA peptide synthesizer (Apphed Biosystems) using FMOC chemistry and coupled to KLH (Sigma- Aldrich, St. Louis MO) by reaction with N-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS) to increase immunogenicity (Ausubel, 1995, supra). Rabbits are immunized with the ohgopeptide-KLH complex in complete Freund's adjuvant.
- ABI 43 IA peptide synthesizer Apphed Biosystems
- KLH Sigma- Aldrich, St. Louis MO
- MBS N-maleimidobenzoyl-N-hydroxysuccinimide ester
- Resulting antisera are tested for antipeptide and anti-NAAP activity by, for example, binding the peptide or NAAP to a substrate, blocking with 1 % BSA, reacting with rabbit antisera, washing, and reacting with radio-iodinated goat anti-rabbit IgG.
- Naturally occurring or recombinant NAAP is substantially purified by immunoaffinity chromatography using antibodies specific for NAAP.
- An in ⁇ unoaffinity column is constracted by covalently coupling anti-NAAP antibody to an activated chromatographic resin, such as
- NAAP Media containing NAAP are passed over the immunoaffinity column, and the column is washed under conditions that ahow the preferential absorbance of NAAP (e.g., high ionic strength buffers in the presence of detergent).
- the column is eluted under conditions that disrupt antibody/NAAP binding (e.g., a buffer of pH 2 to pH 3 » or a high concentration of a chaotrope, such as urea or thiocyanate ion), and NAAP is cohected.
- NAAP or biologically active fragments thereof, are labeled with 125 I Bolton-Hunter reagent (Bolton, A.E. and W.M. Hunter (1973) Biochem. J. 133:529-539).
- Candidate molecules previously a ⁇ ayed in the wells of a multi-weh plate are incubated with the labeled NAAP, washed, and any wehs with labeled NAAP complex are assayed. Data obtained using different concentrations of NAAP are used to calculate values for the number, affinity, and association of NAAP with the candidate molecules.
- molecules interacting with NAAP are analyzed using the yeast two-hybrid system as described in Fields, S. and O. Song (1989) Natare 340:245-246, or using commerciahy available kits based on the two-hybrid system, such as the MATCHMAKER system (Clontech).
- NAAP may also be used in the PATHCALLING process (CuraGen Corp., New Haven CT) which employs the yeast two-hybrid system in a high-throughput manner to determine ah interactions between the proteins encoded by two large libraries of genes (Nandabalan, K. et al. (2000) U.S. Patent No. 6,057,101).
- NAAP activity is measured by its abihty to stimulate transcription of a reporter gene (Liu, O 03/000864
- the assay entails the use of a weh characterized reporter gene construct, LexA op -LacZ, that consists of LexA DNA transcriptional control elements (LexA op ) fused to sequences encoding the E. coli LacZ enzyme.
- LexA op -LacZ a weh characterized reporter gene construct
- the methods for constructing and expressing fusion genes, introducing them into cehs, and measuring LacZ enzyme activity, are weh known to those skilled in the art.
- Sequences encoding NAAP are cloned into a plasmid that directs the synthesis of a fusion protein, LexA-NAAP, consisting of NAAP and a DNA binding domain derived from the LexA transcription factor.
- the resulting plasmid encoding a LexA-NAAP fusion protein, is introduced into yeast cells along with a plasmid containing the LexA 0
- the amount of LacZ enzyme activity associated with LexA-NAAP transfected cells, relative to control cells, is proportional to the amount of transcription stimulated by the NAAP.
- NAAP activity is measured by its abihty to bind zinc.
- a 5-10 ⁇ M sample solution in 2.5 mM ammonium acetate solution at pH 7.4 is combined with 0.05 M zinc sulfate solutio (Aldrich, Milwaukee WI) in the presence of 100 ⁇ M dithiothreitol with 10% methanol added.
- the sample and zinc sulfate solutions are allowed to incubate for 20 minutes.
- the reaction solution is passed through a VYDAC column (Grace Vydac, Hesperia, CA) with approximately 300 Angstrom bore size and 5 ⁇ M particle size to isolate zinc-sample complex from the solution, and into a mass spectrometer (PE Sciex, Ontario, Canada).
- Zinc bound to sample is quantified using the functional atomic mass of 63.5 Da observed by Whittal et al. (2000; Biochemistry 39:8406-8417).
- a method to determine nucleic acid binding activity of NAAP involves a polyacrylamide gel mobility-shift assay.
- NAAP is expressed by transforming a mammahan cell line such as COS7, HeLa or CHO with a eukaryotic expression vector containing NAAP cDNA. The cells are incubated for 48-72 hours after transformation under conditions appropriate for the ceh line to allow expression and accumulation of NAAP.
- Extracts containing solubilized proteins can be prepared from cehs expressing NAAP by methods weh known in the art. Portions of the extract containing NAAP are added to [ 32 P]-labeled RNA or DNA. Radioactive nucleic acid can be synthesized in vitro by techniques weh known in the art.
- the mixtures are incubated at 25°C in the presence of RNase- and DNase-inhibitors under buffered conditions for 5-10 minutes. After incubation, the samples are analyzed by polyacrylamide gel electrophoresis followed by autoradiography. The presence of a band on the autoradiogram indicates the formation of a complex between NAAP and the radioactive transcript. A band of similar mobility wih not be present in samples prepared using control extracts prepared from untransformed cells.
- a method to dete ⁇ riine methylase activity of NAAP measures transfer of radiolabeled methyl groups between a donor substrate and an acceptor substrate.
- Reaction mixtures (50 ⁇ l final volume) contain 15 mM HEPES, pH 7.9, 1.5 mM MgCL,, 10 mM dithiothrehol, 3% polyvinylalcohol, 1.5 ⁇ Ci [met/ry/- 3 H]AdoMet (0.375 ⁇ M AdoMet) (DuPont-NEN), 0.6 ⁇ g NAAP, and acceptor substrate (e.g., 0.4 ⁇ g [ 35 S]RNA, or 6-mercaptopurine (6-MP) to 1 mM final concentration).
- acceptor substrate e.g., 0.4 ⁇ g [ 35 S]RNA, or 6-mercaptopurine (6-MP) to 1 mM final concentration.
- Reaction mixtures are incubated at 30X for 30 minutes, then 65°C for 5 minutes.
- Analysis of [me /- 3 H]RNA is as follows: (1) 50 ⁇ l of 2 x loading buffer (20 mM Tris-HCI, pH 7.6, 1 M LiCl, 1 mM EDTA, 1% sodium dodecyl sulphate (SDS)) and 50 ⁇ l ohgo d(T)-cehulose (10 mg/ml in 1 x loading buffer) are added to the reaction mixture, and incubated at ambient temperature with shaking for 30 minutes. (2) Reaction mixtures are transfe ⁇ ed to a 96-weh filtration plate attached to a vacuum apparatus.
- NAAP activity is proportional to the measured radioactivity.
- DNA repair activity of DNAME is measured as incorporation of [ 32 P]dATP into a plasmid treated with a DNA damaging agent, such as cisplatin or ultraviolet irradiation, relative to a control, untreated plasmid DNA (Coudore, F. et al. (1997) FEBS Lett. 414:581-584).
- Ceh extracts are purified from mammahan ceh lines, E. coli, or S. cerevisiae having compromised endogenous repair activities due to mutations in repair enzymes.
- Ceh extracts are prepared by hypotonic lysis of cehs fohowed by centrifugation at 300,000 x g. Extracts are treated with 63% ammonium sulfate to minimize non-specific nuclease activity.
- the repair synthesis assay is performed in a 50 ⁇ l reaction volume containing 200 ⁇ g protein in ceh extract, 300 ng damaged plasmid, 300 ng control plasmid, 4 ⁇ M dATP, 20 ⁇ M each dCTP, dTTP, and dGTP, 0.2 ⁇ M [ 3 P]dATP, 20 mM HEPES-KOH (pH 7.8), 2.5 ⁇ g creatine phosphokinase, 7 mM MgCl;, and 2 mM EGTA.
- reaction mixtures are treated with 200 ⁇ g/ml proteinase K and 0.5% SDS.
- Plasmid DNA is purified from reaction mixtures by phenol-chloroform extraction and ethanol precipitation. Data is quantified by gel electrophoresis of linearized plasmid fohowed by autoradiography, scintillation counting of excised DNA bands, and densitometry of the photographic negative of the gel to normalize for plasmid DNA recovery.
- type I topoisomerase activity of NAAP can be assayed based on the relaxation of a supercoiled DNA substrate.
- NAAP is incubated with its substrate in a buffer lacking Mg 2+ and ATP, the reaction is terminated, and the products are loaded on an agarose gel.
- Altered topoisomers can be distinguished from supercoiled substrate electrophoreticahy. This assay is specific for type I topoisomerase activity because Mg 2+ and ATP are necessary cofactors for type H topoisomerases.
- Type H topoisomerase activity of NAAP can be assayed based on the decatenation of a kinetoplast DNA (KDNA) substrate.
- KDNA kinetoplast DNA
- NAAP is incubated with KDNA, the reaction is terminated, and the products are loaded on an agarose gel.
- Monomeric circular KDNA can be distmguished from catenated KDNA electrophoreticahy. Kits for measuring type I and type H topoisomerase activities are available commerciahy from Topogen (Columbus OH).
- ATP-dependent RNA hehcase unwinding activity of NAAP can be measured by the method described by Zhang and Grosse (1994; Biochemistry 33 :3906-3912).
- the substrate for RNA unwinding consists of 32 P-labeled RNA composed of two RNA strands of 194 and 130 nucleotides in length containing a duplex region of 17 base-pahs.
- the RNA substrate is incubated together with ATP, Mg 2+ , and varying amounts of NAAP in a Tris-HCI buffer, pH 7.5, at 37°C for 30 minutes.
- the single-stranded RNA product is then separated from the double-stranded RNA substrate by electrophoresis through a 10% SDS-polyacrylamide gel, and quantitated by autoradiography.
- the amount of single-stranded RNA recovered is proportional to the amount of NAAP in the preparation.
- NAAP function is assessed by expressing the sequences encoding NAAP at physiologically elevated levels in mammahan ceh cultare systems.
- cDNA is subcloned into a mammahan expression vector containing a strong promoter that drives high levels of cDNA expression.
- Vectors of choice include pCMV SPORT (Life Technologies) and pCR3.1 (invitrogen Corporation, Carlsbad CA), both of which contain the cytomegalovirus promoter. 5-10 ⁇ g of recombinant vector are transiently transfected into a human ceh line, preferably of endothelial or hematopoietic origin, using either hposome formulations or electroporation.
- 1-2 ⁇ g of an additional plasmid containing sequences encoding a marker protein are co-transfected.
- Expression of a marker protein provides a means to distinguish transfected cehs from nontransfected cells and is a reliable predictor of cDNA expression from the recombinant vector.
- Marker proteins of choice include, e.g., Green Fluorescent Protein (GFP; CLONTECH), CD64, or a CD64-GFP fusion protein.
- FCM Flow cytometry
- FCM Flow cytometry
- FCM detects and quantifies the uptake of fluorescent molecules that diagnose events preceding or coincident with ceh death. These events include changes in nuclear DNA content as measured by staining of DNA with propidium iodide; changes in ceh size and granularity as measured by forward hght scatter and 90 degree side light scatter; down-regulation of DNA synthesis as measured by decrease in bromodeoxyuridine uptake; alterations in expression of ceh surface and intracehular proteins as measured by reactivity with specific antibodies; and alterations in plasma membrane composition as measured by the binding of fluorescein-conjugated Annexin V protein to the ceh surface. Methods in flow cytometry are discussed in Ormerod, M. G. (1994) Flow Cytometry, Oxford, New York NY.
- NAAP The influence of NAAP on gene expression can be assessed using highly purified populations of cehs transfected with sequences encoding NAAP and either CD64 or CD64-GFP.
- CD64 and CD64-GFP are expressed on the surface of transfected cells and bind to conserved regions of human immunoglobulin G (IgG).
- Transfected cells are efficiently separated from nontransfected cehs using magnetic beads coated with either human IgG or antibody against CD64 (DYNAL, Inc., Lake Success NY).
- mRNA can be purified from the cehs using methods weh known by those of skih in the art. Expression of mRNA encoding NAAP and other genes of interest can be analyzed by northern analysis or microa ⁇ ay techniques.
- Pseudouridine synthase activity of NAAP is assayed using a tritium ( 3 H) release assay modified from Nurse et al. (1995; RNA 1:102-112), which measures the release of 3 H from the C 5 position of the pyrimidine component of uridylate (TJ) when 3 H-radiolabeled U in RNA is isomerized to pseudouridine ( ⁇ ).
- 3 H tritium
- a typical 500 ⁇ l assay mixture contains 50 mM HEPES buffer (pH 7.5), 100 mM ammonium acetate, 5 mM dithiothreitol, 1 mM EDTA, 30 units RNase inhibitor, and 0.1-4.2 ⁇ M [5- 3 HJtRNA (approximately 1 ⁇ Ci/nmol tRNA).
- the reaction is initiated by the addition of ⁇ 5 ⁇ l of a concentrated solution of NAAP (or sample containing NAAP) and incubated for 5 min at 37 °C Portions of the reaction mixture are removed at various times (up to 30 min) f oho wing the addition of NAAP and quenched by dilution into 1 ml 0.1 M HCI containing Norit-SA3 (12% w/v). The quenched reaction mixtures are centrifuged for 5 min at maximum speed in a microcentrifuge, and the supernatants are filtered through a plug of glass wool. The pehet is washed twice by resuspension in 1 ml 0.1 M HCI, fohowed by centrifugation.
- the supernatants from the washes are separately passed through the glass wool plug and combined with the original filtrate.
- a portion of the combined filtrate is mixed with scmtihation fluid (up to 10 ml) and counted using a scmtihation counter.
- the amount of 3 H released from the RNA and present in the soluble filtrate is proportional to the amount of peudouridine synthase activity in the sample (Ramamurthy, V. (1999) J. Biol. Chem. 274:22225-22230).
- pseudouridine synthase activity of NAAP is assayed at 30 °C to 37 X in a mixture containing 100 mM Tris-HCI (pH 8.0), 100 mM ammonium acetate, 5 mM MgCL , , 2 mM dithiothreitol, 0.1 mM EDTA, and 1-2 fmol of [ 32 P]-radiolabeled runoff transcripts (generated in vitro by an appropriate RNA polymerase, i.e., T7 or SP6) as substrates.
- NAAP is added to initiate the reaction or omitted from the reaction in control samples.
- RNA is extracted with phenol-chloroform, precipitated in ethanol, and hydrolyzed completely to 3 -nucleotide monophosphates using RNase T 2 .
- the hydrolysates are analyzed by two-dimensional thin layer chromatography, and the amount of 32 P radiolabel present in the ⁇ MP and UMP spots are evaluated after exposing the thin layer chromatography plates to film or a Phosphorhnager screen.
- the relative amount ⁇ MP and UMP are determined and used to calculate the relative amount of ⁇ per tRNA molecule (expressed in mol ⁇ /mol of tRNA or mol ⁇ /mol of xRNA/minute), which corresponds to the amount of pseudouridine synthase activity in the NAAP sample (Lecointe, supra).
- JSP ⁇ -dimethylguanosine transferase ((m 2 2 G)methyltransferase) activity of NAAP is measured in a 160 ⁇ l reaction mixtare containing 100 mM Tris-HCI (pH 7.5), 0:1 mM EDTA, 10 mM MgCl 2 , 20 mM NHXl, ImM dithiothreitol, 6.2 ⁇ M S-adenosyl-L-[met ⁇ vZ- 3 H]methionine (30-70 Ci/mM), 8 ⁇ g m 2 2 G-deficient tRNA or wild type tRNA from yeast, and approximately 100 ⁇ g of purified NAAP or a sample comprising NAAP.
- the reactions are incubated at 30 X for 90 min and chilled on ice. A portion of each reaction is diluted to 1 ml in water containing 100 ⁇ g BSA. 1 ml of 2 M HCI is added to each sample and the acid insoluble products are ahowed to precipitate on ice for 20 min before being cohected by filtration through glass fiber filters. The collected material is washed several times with HCI and quantitated using a Hquid scintillation counter. The amount of 3 H incorporated into the m 2 2 G-deficient, acid-insoluble tRNAs is proportional to the amount of transferase activity in the NAAP sample.
- Reactions comprising no substrate tRNAs, or wild-type tRNAs that have already been modified, serve as control reactions which should not yield acid-insoluble 3 H-labeled products.
- Polyadenylation activity of NAAP is measured using an in vitro polyadenylation reaction.
- the reaction mixtare is assembled on ice and comprises 10 ⁇ l of 5 mM dithiothreitol, 0.025% (v/v) NONHOET P-40, 50 mM creatine phosphate, 6.5% (w/v) polyvinyl alcohol, 0.5 unit/ ⁇ l RNAGUARD (Pharmacia), 0.025 ⁇ g/ ⁇ l creatine kinase, 1.25 mM cordycepin 5'-triphosphate, and 3.75 mM MgCl 2 , in a total volume of 25 ⁇ l.
- Reactions are then incubated at 30 °C for 75-90 min and stopped by the addition of 75 ⁇ l (approximately two-volumes) of proteinase K mix (0.2 M Tris-HCI, pH 7.9, 300 mM NaCl, 25 mM Na-EDTA, 2% (w/v) SDS), 1 ⁇ l of 10 mg/ml proteinase K, 0.25 ⁇ l of 20 mg/m glycogen, and 23.75 ⁇ l of water). Following incubation, the RNA is precipitated with ethanol and analyzed on a 6% (w/v) polyacrylamide, 8.3 M urea sequencing gel. The dried gel is developed by autoradiography or using a phosphoimager.
- proteinase K mix 0.2 M Tris-HCI, pH 7.9, 300 mM NaCl, 25 mM Na-EDTA, 2% (w/v) SDS
- 1 ⁇ l of 10 mg/ml proteinase K 0.25 ⁇ l of 20
- Cleavage activity is determined by comparing the amount of cleavage product to the amount of pre-mRNA template.
- the omission of any of the polypeptide components of the reaction and substitution of NAAP is useful for identifying the specific biological function of NAAP in pre-mRNA polyadenylation (R ⁇ egsegger, supra; and references within).
- tRNA synthetase activity is measured as the aminoacylation of a substrate tRNA in the presence of [ 14 C]-labeled amino acid.
- NAAP is incubated with [ 14 C]-labeled amino acid and the appropriate cognate tRNA (for example, [ 14 C]alanine and tRNA aIa ) in a buffered solution.
- 14 C- labeled product is separated from free [ 14 Cjamino acid by chromatography, and the incorporated 1 C is quantified by scintiUation counter. The amount of 14 C-labeled product detected is proportional to the activity of NAAP in this assay.
- NAAP activity is measured by incubating a sample containing NAAP in a solution containing 1 mM ATP, 5 mM Hepes-KOH (pH 7.0), 2.5 mM KCI, 1.5 mM magnesium chloride, and 0.5 mM DTT along with misacylated [ 14 C]-Glu-tRNAGln (e.g., 1 ⁇ M) and a similar concentration of unlabeled L-glutamine.
- misacylated [ 14 C]-Glu-tRNAGln e.g., 1 ⁇ M
- the mixture is extracted with an equal volume of water-saturated phenol, and the aqueous and organic phases are separated by centrifugation at 15,000 x g at room temperature for 1 min.
- the aqueous phase is removed and precipitated with 3 volumes of ethanol at -70°C for 15 min.
- the precipitated aminoacyl-tRNAs are recovered by centrifugation at 15,000 x g at 4°C for 15 min.
- the pehet is resuspended in of 25 mM KOH, deacylated at 65°C for 10 rnin., neutralized with 0.1 M HCI (to final pH 6-7), and dried under vacuum.
- the dried pehet is resuspended in water and spotted onto a cellulose TLC plate.
- the plate is developed in either isopropanol/formic acid/water or ammonia/water/chloroform/ methanol.
- the image is subjected to densitomexric analysis and the relative amounts of Glu and Gin are calculated based on the Rf values and relative intensities of the spots.
- NAAP activity is calculated based on the amount of Gin resulting from the transformation of Glu while acylated as Glu-tRNA 0111 (adapted from Curnow, A.W. et al. (1997) Proc. Natl. Acad. Sci. USA 94:11819-26).
- Agonists or antagonists of NAAP activation or hihibition may be tested using the assays described in section XVfll. Agonists cause an increase in NAAP activity and antagonists cause a decrease in NAAP activity.
- ABI FACTURA A program that removes vector sequences and Applied Biosystems, Foster City, CA. masks ambiguous bases in nucleic acid sequences.
- ABI AutoAssembler A program that assembles nucleic acid sequences. Applied Biosystems, Foster City, CA.
- fastx score 100 or greater
- Phred A base-calling algorithm that examines automated Ewing, B. et al. (1998) Genome Res. sequencer traces with high sensitivity and probability. 8:175-185; Ewing, B. and P. Green (1998) Genome Res. 8:186-194.
- TMHMMER A program that uses a hidden Markov model (HMM) to Sonnhammer, E.L. et al. (1998) Proc. Sixth Intl. delineate transmembrane segments on protein sequences Conf. on Intelligent Systems for Mol. Biol., and determine orientation. Glasgow et al., eds., The Am. Assoc. for Artificial Intelligence Press, Menlo Park, CA, pp. 175-182.
- HMM hidden Markov model
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- 2002-06-21 EP EP02761035A patent/EP1427841A2/fr not_active Ceased
- 2002-06-21 CA CA002450969A patent/CA2450969A1/fr not_active Abandoned
- 2002-06-21 AU AU2002326332A patent/AU2002326332A1/en not_active Abandoned
- 2002-06-21 WO PCT/US2002/021179 patent/WO2003000864A2/fr not_active Application Discontinuation
Non-Patent Citations (1)
Title |
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See references of WO03000864A2 * |
Also Published As
Publication number | Publication date |
---|---|
AU2002326332A1 (en) | 2003-01-08 |
WO2003000864A3 (fr) | 2003-11-20 |
WO2003000864A8 (fr) | 2003-12-18 |
WO2003000864A2 (fr) | 2003-01-03 |
JP2005502329A (ja) | 2005-01-27 |
CA2450969A1 (fr) | 2003-01-03 |
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