WO2002040656A2 - 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, and 84234, novel human guanine nucleotide dissociation stimulator, glycosyltransferase, dead type helicase, centaurin, dehydrogenase/reductase, and metal transporter family members and uses thereof - Google Patents

47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, and 84234, novel human guanine nucleotide dissociation stimulator, glycosyltransferase, dead type helicase, centaurin, dehydrogenase/reductase, and metal transporter family members and uses thereof Download PDF

Info

Publication number
WO2002040656A2
WO2002040656A2 PCT/US2001/045291 US0145291W WO0240656A2 WO 2002040656 A2 WO2002040656 A2 WO 2002040656A2 US 0145291 W US0145291 W US 0145291W WO 0240656 A2 WO0240656 A2 WO 0240656A2
Authority
WO
WIPO (PCT)
Prior art keywords
seq
amino acid
nucleic acid
domain
sequence
Prior art date
Application number
PCT/US2001/045291
Other languages
French (fr)
Other versions
WO2002040656A3 (en
Inventor
Rachel E. Meyers
Rory A.J. Curtis
Maria Alexandra Glucksmann
Original Assignee
Millennium Pharmaceuticals, Inc.
Priority date (The priority date 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 date listed.)
Filing date
Publication date
Application filed by Millennium Pharmaceuticals, Inc. filed Critical Millennium Pharmaceuticals, Inc.
Priority to AU2002239421A priority Critical patent/AU2002239421A1/en
Publication of WO2002040656A2 publication Critical patent/WO2002040656A2/en
Priority to US10/176,306 priority patent/US20030130485A1/en
Publication of WO2002040656A3 publication Critical patent/WO2002040656A3/en
Priority to US10/968,812 priority patent/US20050214893A1/en

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4702—Regulators; Modulating activity
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/1048—Glycosyltransferases (2.4)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/90—Isomerases (5.)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • DEAD Type Helicases A large number of biological processes require the unwinding from double-stranded or base-paired regions of DNA/DNA, RNA/RNA or RNA/DNA hybrids to single-stranded polynucleotides. These complex reactions are dependent on helicases, mechanochemical enzymes that couple the energy of nucleoside triphosphate hydrolysis to the dehybridization or unwinding of duplex nucleic acid molecules. Helicases comprise a large number of proteins that share high sequence similarity (Au Louis et al. (1999) Nucleic Acids Res 27(2):628-36).
  • helicases are involved in a number of different molecular mechanisms, including viral replication, RNA splicing, ribosome assembly, and initiation of translation including transcription regulation (e.g., SNF2, STH1, brahma, MOT1), maintenance of chromosome stability during mitosis (e.g., lodestar), and various aspects of processing DNA damage, including DNA excision repair (e.g., RAD16 and ERCC6), recombinational pathways (e.g., RAD54) and post-replication daughter strand gap repair (e.g., RAD5) (Eisen et al.
  • transcription regulation e.g., SNF2, STH1, brahma, MOT1
  • maintenance of chromosome stability during mitosis e.g., lodestar
  • processing DNA damage including DNA excision repair (e.g., RAD16 and ERCC6), recombinational pathways (e.g., RAD54) and post-replication daughter strand gap
  • Zinc transporter-3 encodes a putative transporter of zinc into synaptic vesicles.
  • ZnT-3 which is expressed in the brain and testis, is proposed to be a component of the complex that sequesters zinc in synaptic vesicles, thereby serving as a neuromodulator (Palmiter et al. (1996) Proc. Natl. Acad. Sci. USA 93:14934-14939).
  • ZnT-1, ZnT-2, and ZnT-3 share a common topology characterized by six membrane-spanning domains, a histidine-rich cytoplasmic loop between membrane spanning regions four and five, and a long C-terminal tail.
  • the invention features a nucleic acid molecule that encodes a 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 protein or polypeptide, e.g., a biologically active portion of the 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 protein.
  • vectors and host cells containing the 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 nucleic acid molecules of the invention e.g., vectors and host cells suitable for producing 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 nucleic acid molecules and polypeptides.
  • the invention provides nucleic acid fragments suitable as primers or hybridization probes for the detection of 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234-encoding nucleic acids.
  • the invention provides 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 polypeptides, e.g., a 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 polypeptide having the amino acid sequence shown in SEQ ID NO:2, SEQ ID NO:5, SEQ ID NO:8, SEQ ID NO:l 1, SEQ ID NO: 14, SEQ ID NO: 17, SEQ ID NO:20, SEQ ID NO:23, SEQ ID NO:26, OR SEQ ID NO:29 or the amino acid sequence encoded by the cDNA insert of the plasmid deposited with ATCC Accession Number as described herein; an amino acid sequence that is substantially identical to the amino acid sequence shown in SEQ ID NO:2, SEQ ID NO:5, SEQ ID NO:8, SEQ ID NO:l 1, SEQ ID NO: 14, SEQ ID NO: 17, S
  • Figure 1 depicts a hydropathy plot of human 47476. Relative hydrophobic residues are shown above the dashed horizontal line, and relative hydrophilic residues are below the dashed horizontal line. Numbers corresponding to positions in the amino acid sequence of human 47476 are indicated.
  • Figures 4A-4B depict alignments of the phorbol ester/diacylglycerol binding domain (Cl domain) of human 47476 with consensus amino acid sequences derived from a hidden Markov model (HMM) from PFAM and SMART.
  • the upper sequence of Figures 4A and 4B are the consensus amino acid sequences according to PFAM (SEQ ID NO: 36) and SMART (SEQ ID NO:37), respectively, while the lower amino acid sequences correspond to amino acids 541 to 590 of SEQ ID NO:2.
  • Figure 11 depicts an alignment ofthe PH domain of human 46842 and the corresponding consensus amino acid sequences derived from a hidden Markov model (HMM) from PFAM.
  • the upper sequence is the consensus amino acid sequence (SEQ ID NO:43), while the lower amino acid sequence corresponds to amino acids 269 to 363 of SEQ ID NO:l 1.
  • the upper sequence is the consensus amino acid sequence (SEQ ID NO: 53), while the lower amino acid sequence corresponds to the first cation efflux domain of human 64708 (amino acids 55 to 153 of SEQ ID NO:23).
  • the upper sequence is the consensus amino acid sequence (SEQ ID NO:54), while the lower amino acid sequence corresponds to the second cation efflux domain of human 64708 (amino acids 227 to 320 of SEQ ID NO:23).
  • Polypeptides ofthe invention include fragments which include: all or part of a hydrophobic sequence, i.e., a sequence above the dashed line, e.g., the sequence from about amino acid 35 to 50, from about 440 to 470, and from about 685 to 695 of SEQ ID NO:26; all or part of a hydrophilic sequence, i.e., a sequence below the dashed line, e.g., the sequence of from about amino acid 320 to 340, from about 555 to 575, and from about 750 to 765 of SEQ ID NO.26.
  • PFAM Accession Number SM0229 located at about amino acid residues 55 to 172 of SEQ ID NO:2; an EF-hand calcium-binding domain (PFAM Accession Number PF00036) located at about amino acid residues 470 to 498 of SEQ ID NO:2; a phorbol ester/diacylglycerol binding domain (Cl domain) (PFAM Accession Number PF00130) located at about amino acid residues 541 to 590 of SEQ ID NO:2; one predicted N-glycosylation site (PS00001) located at about amino acid residues 622 to 625 of SEQ ID NO:2; four predicted cAMP/cGMP-dependent protein kinase phosphorylation sites (PS00004) located at about amino acid rsidues 3 to 6, 7 to 10, 191 to 194 and 549 to 552 of SEQ ID NO:2; eight predicted Protein Kinase C phosphorylation sites (PS00005) located at about amino acid residues 6 to 8, 15 to 17, 35 to 37,
  • Human 46842 contains the following regions or other structural features: a PH domain (PFAM Accession Number PF00169) located at about amino acid residues 269 to 363 SEQ ID NO:l l; an ArfGAP domain (PFAM Accession Number PF01412) located at about amino acid residues 403 to 525 of SEQ ID NO: 11 ; two ankyrin repeat domains (PFAM Accession Number PF00023) located at about amino acid residues 702 to 734 and 735 to 767 of SEQ ID NO:l 1; an ArfGAP zinc ion coordinating motif located at about amino acid residues 421 to 440 of SEQ ID NO:l l; twelve Protein Kinase C phosphorylation sites (PS00005) located at about amino acid residues 12 to 14, 53 to 55, 102 to 104, 121 to 123, 160 to 162, 319 to 321, 347 to 349, 375 to 377, 492 to 494, 499 to 501, 544 to 546, and 549 to 5
  • Human 33201 The human 33201 sequence (see SEQ ID NO:13, as recited in Example 1), which is approximately 1718 nucleotides long including untranslated regions, contains a predicted methionine-initiated coding sequence of about 1056 nucleotides, including the termination codon.
  • the coding sequence encodes a 351 amino acid protein (see SEQ ID NO: 14, as recited in Example 1).
  • the human 84233 sequence (see SEQ ID NO:19, as recited in Example 1), which is approximately 2165 nucleotides long including untranslated regions, contains a predicted methionine-initiated coding sequence of about 963 nucleotides, including the termination codon.
  • the coding sequence encodes a 320 amino acid protein (see SEQ ID NO:20, as recited in Example 1).
  • SEQ ID NO:20 13-20 of SEQ ID NO:20; and four N-myristylation sites (PS00008) located at about amino acid residues 7-12, 42-47, 94-99, and 228-233 of SEQ ID NO:20.
  • a ras guanine nucleotide dissociation stimulator can include a "ras guanine nucleotide dissociation stimulator CDC25 family signature motif, defined by the sequence: [GAP]-[CT]-V-P-[FY]-X-X-X-X- [LINMFY]-X-[DN]-[LINM].
  • a ras guanine nucleotide dissociation stimulator CDC25 family signature motif can be involved in triggering the dissociation of a guanine nucleotide, e.g., GDP, from a ras or ras-like protein.
  • a 47476 polypeptide can further include a "phorbol ester/diacylglycerol binding domain (Cl domain)" or regions homologous with a “phorbol ester/diacylglycerol binding domain (Cl domain)".
  • the term "phorbol ester/diacylglycerol binding domain (Cl domain)” includes an amino acid sequence of about 30 to 100 amino acid residues in length and having a bit score for the alignment ofthe sequence to the phorbol ester/diacylglycerol binding domain (Cl domain) (HMM) of at least 30.
  • a phorbol ester/diacylglycerol binding domain includes at least about 35 to 75 amino acids, more preferably about 40 to 60 amino acid residues, or about 45 to 55 amino acids and has a bit score for the alignment ofthe sequence to the phorbol ester/diacylglycerol binding domain (Cl domain) (HMM) of at least 40, 50, 55, 59, or greater.
  • the phorbol ester/diacylglycerol binding domain (Cl domain) (HMM) has been assigned the PFAM Accession Number PF00130.
  • An alternative model (HMM) for the phorbol ester/diacylglycerol binding domain has been assigned the SMART Accession Number SM0109.
  • the glycosyl transferase family comprises a number of related enzymes that are capable of catalyzing the synthesis of glycoconjugates, including glycolipids, glycoproteins, and polysaccharides, by transferring an activated mono- or oligosaccharide residue to an existing acceptor molecule for the initiation or elongation ofthe carbohydrate chain.
  • the acceptor can be a lipid, a protein, a heterocyclic compound, or another carbohydrate residue.
  • Glycosyltransferases can be divided into numerous subfamilies based upon their specificity for sugar moieties and acceptor molecules.
  • the glycosyltransferase domain of human 67210 bears similarity to a subfamily designated "group 8" glycosyltransferases.
  • a 67210 polypeptide can include a "glycosyltransferase domain” or regions homologous with a "glycosyltransferase domain".
  • the term "glycosyl transferase domain” includes an amino acid sequence of about 100 to 450 amino acid residues in length, having a bit score for the alignment ofthe sequence to the glycosyltransferase domain (HMM) of at least 25.
  • a glycosyl transferase domain includes about 200 to 350 amino acid residues, or more preferably about 250 to 300 amino acids and has a bit score for the alignment ofthe sequence to the glycosyltransferase domain (HMM) of at least 30, 35, more 40, or more.
  • a 67210 polypeptide or protein has a "glycosyl transferase domain" or a region which includes at least about 100 to 450, more preferably about 200 to 350, or 250 to 300 amino acid residues and has at least about 60%, 70%, 80%, 90%, 95%, 98%, 99%, or more homology with a "glycosyltransferase,” e.g, the glycosyltransferase domain of human 67210 (e.g, residues 63 to 340 of SEQ ID NO:5).
  • a 67210 peptide can further include a signal sequence.
  • a "signal peptide” or “signal sequence” refers to a peptide of about 20 to 60, preferably about 25 to 55, or more preferably about 29 amino acid residues in length which occurs at the N-terminus of secretory and integral membrane proteins and which contains a majority of hydrophobic amino acid residues.
  • HMM DEAD type helicase domain
  • SEQ ID NO:39 amino acids 22 to 245 or 28 to 245 of SEQ ID NO:8
  • Figures 8A and 8B Alignments of the DEAD type helicase domain (amino acids 22 to 245 or 28 to 245 of SEQ ID NO:8) of human 49875 with a consensus amino acid sequence derived from a hidden Markov model according to PFAM (SEQ ID NO:39) or according to SMART (SEQ ID NO:40) are depicted in Figures 8A and 8B, respectively.
  • the term "conserved helicase C-terminal domain” is an amino acid sequence of at least 30 amino acid residues in length having a bit score for the alignment ofthe sequence to the C-terminal helicase domain (HMM) of at least 50.
  • a “conserved helicase C- terminal domain” preferably includes an amino acid sequence of about 20 to 140 amino acid residues in length, more preferably about 40 to 120 amino acid residues, or about 60 to 100 amino acids and having a bit score for the alignment ofthe sequence to the C-terminal helicase domain (HMM) of at least 50, preferably 80, and most preferably 90 or more.
  • the conserved helicase C-terminal domain (HMM) has been assigned the PFAM Accession Number PF00271).
  • a 49875 polypeptide or protein has at least one DEAD-box subfamily ATP-dependent helicase signature motif, or a region which includes at least 6, 7, 8, or even 9 amino acid residues and has at least 70%, 80%, 90%, or 100% homology with a "DEAD-box subfamily ATP-dependent helicase signature motif, e.g, DEAD-box subfamily ATP-dependent helicase signature motif of human 49875, e.g, about amino acid residues 169 to 177 of SEQ ID NO:8.
  • a 49875 family member can include at least one DEAD type helicase domain, and at least one conserved helicase C-terminal domain.
  • a 49875 activity can be an activity exerted by 49875 in a physiological milieu on, e.g, a 49875-responsive cell or on a 49875 substrate, e.g, ATP or a nucleic acid.
  • a 49875 activity can be determined in vivo or in vitro.
  • a 49875 activity is a direct activity, such as an association with a 49875 target molecule.
  • a 46842 polypeptide can include a "PH domain” or regions homologous with a "PH domain”.
  • dehydrogenase/reductase domain includes an amino acid sequence of about 200 to 400 amino acid residues in length and having a bit score for the alignment ofthe sequence to the dehydrogenase/reductase domain (HMM) of at least 40.
  • an dehydrogenase/reductase domain includes at least about 250-375 amino acids, more preferably about 275-350 amino acid residues, or about 310-340 amino acids and has a bit score for the alignment ofthe sequence to the dehydrogenase/reductase domain (HMM) of at least 50, 60, 70 or greater.
  • a 33201 family member can include at least one dehydrogenase/reductase domain. Furthermore, a 33201 family member can include: at least one, two, preferably three conserved glycine residues; at least one, two, three, preferably four-predicted N-glycosylation sites
  • a “target molecule” or “binding partner” is a molecule with which a 33201 protein binds or interacts in nature, e.g, an alcohol or a quinone.
  • a 33201 polypeptide or protein is an enzyme, e.g, an enzyme that catalyzes the oxidation and/or reduction of a 33201 substrate, e.g, a molecule that contains an alcohol group or a quinone.
  • a 33201 activity can also be an indirect activity, e.g, a cellular signaling activity mediated by a 33201 substrate, such that the activity ofthe 33201 upon the substrate alters its ability to signal, e.g, alters its concentration.
  • dehydrogenase activity means the ability to directly or indirectly remove a hydride from a substrate. Typically, after removal of a hydride from a substrate, electrons ofthe hydride are transferred to NAD+, NADP+, or other coenzyme (e.g, 3-acetylpyridine adenine dinucleotide phosphate) or hydride acceptor. For example, if the substrate has hydroxyl, dehydrogenation converts the hydroxyl to a keto group and produces NADH or NADPH and a proton. Hydride removal from substrate however does not require the presence of an acceptor. Free hydride can be detected optically by H+ binding to a dye molecule, for example
  • the 33201 molecules ofthe present invention are predicted to have one or more biological activities of dehydrogenase/reductase family members.
  • the 33201 proteins ofthe present invention can have one or more ofthe following activities: (1) the ability to metabolize an alcohol, e.g, to catalyze the reversible oxidation of ethanol to acetaldehyde; (2) the ability to metabolize or remove endogenous or non-endogenous (e.g, xenobiotic) substances, such as toxins; (3) the ability to catalyze the oxidation of retinoic acid; (4) the ability to catalyze the reduction of a substrate, e.g, quinone; (5) the ability to modulate dopamine metabolism; (6) the ability to reduce a bioreductive compound, e.g, a bioreductive antitumor quinone; (7) the ability to modulate cellular differentiation; (8) the ability to modulate cellular proliferation; or (9) the ability to
  • the 83378, 84233, 64708, 85041, and 84234 proteins contain a significant number of structural characteristics in common with members ofthe metal transporter family.
  • the term "family" when referring to the protein and nucleic acid molecules ofthe invention means two or more proteins or nucleic acid molecules having a common structural domain or motif and having sufficient amino acid or nucleotide sequence homology as defined herein.
  • family members can be naturally or non-naturally occurring and can be from either the same or different species.
  • a family can contain a first protein of human origin as well as other distinct proteins of human origin, or alternatively, can contain homologues of non-human origin, e.g, rat or mouse proteins.
  • Members of a family can also have common functional characteristics.
  • metal transporter proteins that increase cellular tolerance to divalent metal ions such as zinc, cadmium, and cobalt by mediating cation diffusion across membranes. Some metal transporter proteins are efflux pumps that remove divalent metal ions from cells. Other metal transporter proteins function to increase cellular tolerance to metal ions by mediating the sequestration of ions in subcellular compartments. Some metal transporter proteins are characterized by a topology comprising six membrane spanning domains, a histidine-rich loop between the fourth and fifth membrane spanning domains, and a long C-terminal tail. Examples of metal transporter proteins include ZnT-1, ZnT-2, and ZnT-3.
  • a cation efflux domain of a 83378 polypeptide preferably includes about 120-160 amino acids and has a bit score for the alignment ofthe sequence to the cation efflux domain (HMM) of at least 80, 130, or greater.
  • a cation efflux domain of a 84233 polypeptide preferably includes about 270 to 290 amino acids and has a bit score for the alignment ofthe sequence to the cation efflux domain (HMM) of at least 250 or greater.
  • a 83378, 84233, 64708, 85041, or 84234 polypeptide can include a "transmembrane domain” or regions homologous with a "transmembrane domain”.
  • Amino acid residues 34-51, 58-82, 101-119, 137-155, 202-219, and 232-249 ofthe 64708 protein are predicted to comprise transmembrane domains.
  • an N-terminal cytoplasmic domain is located at about amino acid residues 1-10 of SEQ ID NO: 17, 1-24 of SEQ ID NO:20, 1-33 of SEQ ID NO:23, 1-58 of SEQ ID NO:26, and 1-37 of SEQ ID NO:29.
  • a 83378, 84233, 64708, 85041, or 84234 polypeptide or protein has at least one cytoplasmic domain or a region which includes at least about 5, preferably about 10-300, and more preferably about 30-220 amino acid residues and has at least about 60%, 70%, 80%, 90%, 95%, 99%, or 100% homology with an "cytoplasmic domain," e.g, at least one cytoplasmic domain of human 83378, 84233, 64708, 85041, or 84234 (e.g, residues 1-10, 62-78, 135-240, and 300-485 of SEQ ID NO:17; residues 1-24, 75-91, 148-166, and 219-320 of SEQ ID NO:20; residues 1-33, 83-100, 156-201, and 250-461 of SEQ ID NO:23; residues 1-58, 120-128, 169-189, 259-266, 321-342, 438-485, 542-591, and 642-7
  • non-cytoplasmic loops include those domains ofthe protein that reside in the lumen ofthe organelle or the matrix or the intermembrane space.
  • a "non-cytoplasmic loop" can be found at about amino acids 32-43, 99-114, and 266-282 of SEQ ID NO:17; at about amino acids 50-57, 114-127, and 192-200 of SEQ ID NO:20; at about amino acids 52-57, 120-136, and 220-231 of SEQ ID NO:23; at about amino acids 78-98, 146-151, 215-238, 289-303, 363-418, 506-520, and 614-617 of SEQ ID NO:26; and at about amino acids 59-70, 124-140, and 257-262 of SEQ ID NO:29.
  • a "83378, 84233, 64708, 85041 , or 84234 activity” refers to an activity exerted by a 83378, 84233, 64708, 85041, or 84234 protein, polypeptide or nucleic acid molecule.
  • a 83378, 84233, 64708, 85041, or 84234 activity can be an activity exerted by 83378, 84233, 64708, 85041, or 84234 in a physiological milieu on, e.g, a 83378, 84233, 64708, 85041, or 84234-responsive cell or on a 83378, 84233, 64708, 85041, or 84234 substrate, e.g, a protein substrate.
  • a 83378, 84233, 64708, 85041, or 84234 activity can be determined in vivo or in vitro.
  • Examples of cellular proliferative and/or differentiative disorders include cancer, e.g, carcinoma, sarcoma, metastatic disorders or hematopoietic neoplastic disorders, e.g, leukemias.
  • a metastatic tumor can arise from a multitude of primary tumor types, including but not limited to those of prostate, colon, lung, breast and liver origin.
  • cancer hyperproliferative and neoplastic refer to cells having the capacity for autonomous growth. Examples of such cells include cells having an abnormal state or condition characterized by rapidly proliferating cell growth.
  • carcinoma is art recognized and refers to malignancies of epithelial or endocrine tissues including respiratory system carcinomas, gastrointestinal system carcinomas, genitourinary system carcinomas, testicular carcinomas, breast carcinomas, prostatic carcinomas, endocrine system carcinomas, and melanomas.
  • Exemplary carcinomas include those forming from tissue ofthe cervix, lung, prostate, breast, head and neck, colon and ovary.
  • carcinosarcomas e.g, which include malignant tumors composed of carcinomatous and sarcomatous tissues.
  • An "adenocarcinoma” refers to a carcinoma derived from glandular tissue or in which the tumor cells form recognizable glandular structures.
  • Immunological disorders or diseases include, but are not limited to, autoimmune diseases (including, for example, diabetes mellitus, arthritis (including rheumatoid arthritis, juvenile rheumatoid arthritis, osteoarthritis, psoriatic arthritis), multiple sclerosis, encephalomyelitis, myasthenia gravis, systemic lupus erythematosis, autoimmune thyroiditis, dermatitis (including atopic dermatitis and eczematous dermatitis), psoriasis, Sjogren's Syndrome, Crohn's disease, aphthous ulcer, ulceris, conjunctivitis, keratoconjunctivitis, ulcerative colitis, asthma, allergic asthma, cutaneous lupus eryfhematosus, scleroderma, vaginitis, proctitis, drug eruptions, leprosy reversal reactions, erythema nodosum leprosum, autoimmune u
  • disorders involving red blood cells include, but are not limited to, anemias, such as hemolytic anemias, including hereditary spherocytosis, hemolytic disease due to erythrocyte enzyme defects: glucose-6-phosphate dehydrogenase deficiency, sickle cell disease, thalassemia syndromes, paroxysmal nocturnal hemoglobinuria, immunohemolytic anemia, and hemolytic anemia resulting from trauma to red cells; and anemias of diminished erythropoiesis, including megaloblastic anemias, such as anemias of vitamin B 12 deficiency: pernicious anemia, and anemia of folate deficiency, iron deficiency anemia, anemia of chronic disease, aplastic anemia, pure red cell aplasia, and other forms of marrow failure.
  • anemias such as hemolytic anemias, including hereditary spherocytosis, hemolytic disease due to erythrocyte enzyme defects: glucose-6-phosphate dehydrogen
  • Neurological disorders or disorders involving the brain include, but are not limited to, disorders involving neurons, and disorders involving glia, such as astrocytes, oligodendrocytes, ependymal cells, and microglia; cerebral edema, raised intracranial pressure and herniation, and hydrocephalus; malformations and developmental diseases, such as neural tube defects, forebrain anomalies, posterior fossa anomalies, and syringomyelia and hydromyelia; perinatal brain injury; cerebrovascular diseases, such as those related to hypoxia, ischemia, and infarction, including hypotension, hypoperfusion, and low-flow states—global cerebral ischemia and focal cerebral ischemia—infarction from obstruction of local blood supply, intracranial hemorrhage, including intracerebral (intraparenchymal) hemorrhage, subarachnoid hemorrhage and ruptured berry aneurysms, and vascular malformations, hypertensive cerebrovascular disease
  • disorders involving the small intestine include the malabsorption syndromes such as, celiac sprue, tropical sprue (postinfectious sprue), whipple disease, disaccharidase (lactase) deficiency, abetalipoproteinemia, and tumors ofthe small intestine including adenomas and adenocarcinoma.
  • malabsorption syndromes such as, celiac sprue, tropical sprue (postinfectious sprue), whipple disease, disaccharidase (lactase) deficiency, abetalipoproteinemia, and tumors ofthe small intestine including adenomas and adenocarcinoma.
  • vascular diseases involving blood vessels include, but are not limited to, responses of vascular cell walls to injury, such as endothelial dysfunction and endothelial activation and intimal thickening; vascular diseases including, but not limited to, congenital anomalies, such as arteriovenous fistula, atherosclerosis, and hypertensive vascular disease, such as hypertension; inflammatory disease— the vasculitides, such as giant cell (temporal) arteritis, Takayasu arteritis, polyarteritis nodosa (classic), Kawasaki syndrome (mucocutaneous lymph node syndrome), microscopic polyanglitis (microscopic polyarteritis, hypersensitivity or leukocytoclastic anglitis), Wegener granulomatosis, thromboanglitis obliterans (Buerger disease), vasculitis associated with other disorders, and infectious arteritis; Raynaud disease; aneurysms and dissection, such as abdominal aortic aneurys
  • the encoded protein can be expressed recombinantly and the activity ofthe protein can be determined.
  • Biologically active portions of a 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 protein include peptides comprising amino acid sequences sufficiently homologous to or derived from the amino acid sequence ofthe 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 protein, e.g, the amino acid sequence shown in SEQ ID NO:2, SEQ ID NO:5, SEQ ID NO:8, SEQ ID NO:l 1, SEQ ID NO:14, SEQ ID NO:17, SEQ ID NO:20, SEQ ID NO:23, SEQ ID NO:26, or SEQ ID NO:29, which include less amino acids than the full length 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 proteins, and exhibit at least one activity of a 47476, 67210
  • Biologically active portions of a 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 protein can be used as targets for developing agents which modulate a 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 mediated activity, e.g, stimulation of guanine nucleoside dissociation from a GTPase protein, transfer of a sugar residue to another molecule, unwinding of a duplex nucleic acid molecule, stimulation ofthe hydrolysis of GTP molecule bound to a GTPase protein, catalyzsis ofthe oxidation of an alcohol group present on a molecule, or transport of metal ions across a lipid bilayer.
  • stimulation of guanine nucleoside dissociation from a GTPase protein transfer of a sugar residue to another molecule, unwinding of a duplex
  • the percent identity between two nucleotide sequences is determined using the GAP program in the GCG software package (available at http://www.gcg.com), using a NWSgapdna.CMP matrix and a gap weight of 40, 50, 60, 70, or 80 and a length weight of 1, 2, 3, 4, 5, or 6.
  • a particularly preferred set of parameters are a Blossum 62 scoring matrix with a gap penalty of 12, a gap extend penalty of 4, and a frameshift gap penalty of 5.
  • amino acid sequences that contain a common structural domain having at least about 60%, or 65% identity, likely 75% identity, more likely 85%, 90%.
  • “Misexpression or aberrant expression” refers to a non-wildtype pattern of gene expression at the RNA or protein level. It includes: expression at non- wild type levels, i.e, over- or under-expression; a pattern of expression that differs from wild type in terms ofthe time or stage at which the gene is expressed, e.g, increased or decreased expression (as compared with wild type) at a predetermined developmental period or stage; a pattern of expression that differs from wild type in terms of altered, e.g, increased or decreased, expression (as compared with wild type) in a predetermined cell type or tissue type; a pattern of expression that differs from wild type in terms ofthe splicing size, translated amino acid sequence, post-transitional modification, or biological activity ofthe expressed polypeptide; a pattern of expression that differs from wild type in terms ofthe effect of an environmental stimulus or extracellular stimulus on expression ofthe gene, e.g, a pattern of increased or decreased expression (as compared with wild type) in the presence
  • nucleic acid fragment suitable for use as a hybridization probe which can be used, e.g, to identify a nucleic acid molecule encoding a polypeptide ofthe invention, 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 mRNA, and fragments suitable for use as primers, e.g, PCR primers for the amplification or mutation of nucleic acid molecules.
  • the nucleic acid molecule can include only the coding region of SEQ ID NO:l, SEQ ID NO:4, SEQ ID NO:7, SEQ ID NO:10, SEQ ID NO:13, SEQ ID NO:16, SEQ ID NO: 19, SEQ ID NO:22, SEQ ID NO:25, and SEQ ID NO:28 (e.g, SEQ ID NO:3, SEQ ID NO:6, SEQ ID NO:9, SEQ ID NO: 12, SEQ ID NO: 15, SEQ ID NO: 18, SEQ ID NO:21, SEQ ID NO:24, SEQ ID NO:27, or SEQ ID NO:30) and, e.g, no flanking sequences which normally accompany the subject sequence.
  • an isolated nucleic acid molecule ofthe invention includes a nucleic acid molecule which is a complement (e.g, a full complement) ofthe nucleotide sequence shown in SEQ ID NO:l, SEQ ID NO:4, SEQ ID NO:7, SEQ ID NO:10, SEQ ID NO:13, SEQ ID NO:16, SEQ ID NO:19, SEQ ID NO:22, SEQ ID NO:25, or SEQ ID NO:28, or SEQ ID NO:3, SEQ ID NO:6, SEQ ID NO:9, SEQ ID NO:12, SEQ ID NO:15, SEQ ID NO:18, SEQ ID NO:21, SEQ ID NO:24, SEQ ID NO:27, or SEQ ID NO:30, or a portion of any of these nucleotide sequences.
  • the nucleic acid molecule ofthe invention is sufficiently complementary to the nucleotide sequence shown in SEQ ID NO:l, SEQ ID NO:4, SEQ ID NO:7, SEQ ID NO:10, SEQ ID NO:13, SEQ ID NO:16, SEQ ID NO:19, SEQ ID NO:22, SEQ ID NO:25, or SEQ ID NO:28, or SEQ ID NO:3, SEQ ID NO:6, SEQ ID NO:9, SEQ ID NO: 12, SEQ ID NO: 15, SEQ ID NO: 18, SEQ ID NO.21, SEQ ID NO:24, SEQ ID NO:27, or SEQ ID NO:30, such that it can hybridize (e.g, under a stringency condition described herein) to the nucleotide sequence shown in SEQ ID NO:l, SEQ ID NO:4, SEQ ID NO:7, SEQ ID NO:10, SEQ ID NO:13, SEQ ID NO:16, SEQ ID NO:19, SEQ ID NO:22, SEQ ID NO:
  • a nucleic acid molecule of the invention can include only a portion ofthe nucleic acid sequence of SEQ ID NO:l or SEQ ID NO:3.
  • such a nucleic acid molecule can include a fragment which can be used as a probe or primer or a fragment encoding a portion of a 47476 protein, e.g, an immunogenic or biologically active portion of a 47476 protein.
  • a fragment can comprise those nucleotides of SEQ ID NO:l, which encode a ras guanine nucleotide dissociation stimulator domain of human 47476.
  • nucleic acid includes a nucleotide sequence that includes part, or all, ofthe coding region and extends into either (or both) the 5' or 3' noncoding region.
  • Other embodiments include a fragment that includes a nucleotide sequence encoding an amino acid fragment described herein. Nucleic acid fragments can encode a specific domain or site described herein or fragments thereof, particularly fragments thereof which are at least 100 amino acids in length.
  • the nucleic acid fragments encode a specific domain or fragment thereof, wherein the domain or fragment is at least 25, 28, 45, 50, 150, 180 and 230 amino acids in length. Fragments also include nucleic acid sequences corresponding to specific amino acid sequences described above or fragments thereof. Nucleic acid fragments should not to be construed as encompassing those fragments that may have been disclosed prior to the invention.
  • a nucleic acid fragment can include a sequence corresponding to a domain, region, or functional site described herein.
  • a nucleic acid fragment can also include one or more domains, regions, or functional sites described herein.
  • a 47476 nucleic acid fragment can include a sequence corresponding to a ras guanine nucleotide dissociation stimulator domain, a guanine nucleotide dissociation stimulator domain N-terminal motif, an EF-hand calcium-binding domain, or a phorbol ester/diacylglycerolbinding domain (Cl domain).
  • the probe or primer is attached to a solid support, e.g, a solid support described herein.
  • the nucleic acid is a probe which is at least 10, 12, 15, 18, 20 and less than 200, more preferably less than 100, or less than 50, nucleotides in length. It should be identical, or differ by 1, or 2, or less than 5 or 10 nucleotides, from a sequence disclosed herein. If alignment is needed for this comparison the sequences should be aligned for maximum homology. "Looped" out sequences from deletions or insertions, or mismatches, are considered differences.
  • a nucleic acid fragment encoding a biologically active portion of 47476 includes a Ras guanine nucleotide dissociation stimulator domain, a guanine nucleotide dissociation stimulator domain N-terminal motif, an EF-handed calcium-binding domain, or a phorbol ester/diacylglycerol binding domain (Cl domain), e.g, about amino acid residues 195 to 381, 55 to 172, 470 to 498, and 541 to 590 of SEQ ID NO:2, respectively.
  • a nucleic acid fragment encoding a biologically active portion of a 47476 polypeptide may comprise a nucleotide sequence which is greater than 300 or more nucleotides in length
  • a nucleic acid in another embodiment, includes a nucleotide sequence that includes part, or all, ofthe coding region and extends into either (or both) the 5' or 3' noncoding region.
  • Other embodiments include a fragment which includes a nucleotide sequence encoding an amino acid fragment described herein.
  • Nucleic acid fragments can encode a specific domain or site described herein or fragments thereof, particularly fragments thereof which are at least 50 amino acids in length, preferably 75, 100, 150, 200, 250, 300, 325, 340 or more amino acids in length.
  • the nucleic acid fragments encode a specific domain or fragment thereof, wherein the domain or fragment is at least 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700 nucleic acids in length. Fragments also include nucleic acid sequences corresponding to specific amino acid sequences described above or fragments thereof. Nucleic acid fragments should not to be construed as encompassing those fragments that may have been disclosed prior to the invention.
  • a nucleic acid fragment encoding a "biologically active portion of a 67210 polypeptide” can be prepared by isolating a portion ofthe nucleotide sequence of SEQ ID NO:4 or 6, which encodes a polypeptide having a 67210 biological activity (e.g, the biological activities ofthe 67210 proteins are described herein), expressing the encoded portion ofthe 67210 protein (e.g, by recombinant expression in vitro) and assessing the activity ofthe encoded portion ofthe
  • a nucleic acid fragment encoding a biologically active portion of 67210 includes a glycosyltransferase domain, e.g, amino acid residues about 63 to 340 of SEQ ID NO:5.
  • a nucleic acid fragment encoding a biologically active portion of a 67210 polypeptide may comprise a nucleotide sequence which is greater than 200 or more nucleotides in length.
  • a nucleic acid includes a nucleotide sequence which is about
  • primers suitable for amplifying all or a portion of any ofthe following regions are provided: a DEAD-type helicase domain, e.g, about amino acid residues 22 to 245 of SEQ ID NO:8; a DEAD-box subfamily ATP-dependent helicase signature motif, e.g, about amino acid residuesl69 to 177 of SEQ ID NO:8; a conserved helicase C-terminal domain, e.g, about amino acid residues 281 to 363 of SEQ ID NO: 8; or an ATP/GTP-binding site motif A (P-loop), e.g, about amino acid residues 53 to 60 of SEQ ID NO:8.
  • a DEAD-type helicase domain e.g, about amino acid residues 22 to 245 of SEQ ID NO:8
  • a DEAD-box subfamily ATP-dependent helicase signature motif e.g, about amino acid residuesl69 to 177 of SEQ ID NO:8
  • a nucleic acid fragment differs by at least 1, 2, 3, 10, 20, or more nucleotides from a sequence disclosed in WO 01/55301, WO 01/57188 or WO 01/62927. Differences can include differing in length or sequence identity.
  • a nucleic acid in another embodiment, includes a nucleotide sequence that includes part, or all, of the coding region and extends into either (or both) the 5 ' or 3 ' noncoding region.
  • Other embodiments include a fragment which includes a nucleotide sequence encoding an amino acid fragment described herein.
  • Nucleic acid fragments can encode a specific domain or site described herein or fragments thereof, particularly fragments thereof which are at least 100, 200, 300, 400, or 500 amino acids in length. Fragments also include nucleic acid sequences corresponding to specific amino acid sequences described above or fragments thereof. Nucleic acid fragments should not to be construed as encompassing those fragments that may have been disclosed prior to the invention.
  • a probe/primer is an isolated or purified oligonucleotide.
  • the oligonucleotide typically includes a region of nucleotide sequence that hybridizes under a stringency condition described herein to at least about 7, 12 or 15, preferably about 20 or 25, more preferably about 30, 35, 40, 45, 50, 55, 60, 65, or 75 consecutive nucleotides of a sense or antisense sequence of SEQ ID NO: 10 or SEQ ID NO: 12, or of a naturally occurring allelic variant or mutant of SEQ ID NO: lOor SEQ ID NO: 12.
  • an oligonucleotide is less than about 200, 150, 120, or 100 nucleotides in length.
  • the probe or primer is attached to a solid support, e.g, a solid support described herein.
  • primers suitable for amplifying all or a portion of any ofthe following regions are provided: a PH domain, e.g, located at about amino acid residues 269 to 363 SEQ ID NO: 11 ; an ArfGAP domain, e.g, located at about amino acid residues 403 to 525 of SEQ ID NO:l 1; or an ankyrin repeat domain, e.g, located at about amino acid residues 702 to 734 or 735 to 767of SEQ ID NO:l l.
  • a nucleic acid in another embodiment, includes a nucleotide sequence that includes part, or all, ofthe coding region and extends into either (or both) the 5' or 3' noncoding region.
  • Other embodiments include a fragment which includes a nucleotide sequence encoding an amino acid fragment described herein.
  • Nucleic acid fragments can encode a specific domain or site described herein or fragments thereof, particularly fragments thereof which are at least 100 amino acids in length.
  • the nucleic acid fragments encode a specific domain or fragment thereof, wherein the domain or fragment is at least 105, or more preferably 110, 120, or even 130 amino acids in length.
  • Fragments also include nucleic acid sequences corresponding to specific amino acid sequences described above or fragments thereof. Nucleic acid fragments should not to be construed as encompassing those fragments that may have been disclosed prior to the invention.
  • the nucleic acid is a probe which is at least 10, 12, 15, 18, 20 and less than 200, more preferably less than 100, or less than 50, nucleotides in length. It should be identical, or differ by 1, or 2, or less than 5 or 10 nucleotides, from a sequence disclosed herein. If alignment is needed for this comparison the sequences should be aligned for maximum homology. "Looped" out sequences from deletions or insertions, or mismatches, are considered differences.
  • a nucleic acid fragment can encode an epitope bearing region of a polypeptide described herein.
  • a nucleic acid fragment encoding a "biologically active portion of a 33201 polypeptide” can be prepared by isolating a portion ofthe nucleotide sequence of SEQ ID NO: 13 or SEQ ID NO: 15, which encodes a polypeptide having a 33201 biological activity (e.g, the biological activities ofthe 33201 proteins are described herein), expressing the encoded portion ofthe 33201 protein (e.g, by recombinant expression in vitro) and assessing the activity ofthe encoded portion ofthe 33201 protein.
  • a nucleic acid fragment encoding a biologically active portion of 33201 can include a dehydrogenase/reductase domain, e.g, amino acid residues about 22 to 345 of SEQ ID NO: 14.
  • a nucleic acid fragment encoding a biologically active portion of a 33201 polypeptide may comprise a nucleotide sequence which is greater than 300 or more nucleotides in length.
  • a nucleic acid molecule ofthe invention can include only a portion ofthe nucleic acid sequence of SEQ ID NO:16, SEQ ID NO:19, SEQ ID NO:22, SEQ ID NO:25, or SEQ ID NO:16, SEQ ID NO:19, SEQ ID NO:22, SEQ ID NO:25, or SEQ ID NO:16, SEQ ID NO:19, SEQ ID NO:22, SEQ ID NO:25, or SEQ ID NO:16, SEQ ID NO:19, SEQ ID NO:22, SEQ ID NO:25, or SEQ ID
  • the nucleotide sequence determined from the cloning ofthe 83378, 84233, 64708, 85041, or 84234 gene allows for the generation of probes and primers designed for use in identifying and/or cloning other 83378, 84233, 64708, 85041, or 84234 family members, or fragments thereof, as well as 83378, 84233, 64708, 85041, or 84234 homologues, or fragments thereof, from other species.
  • a nucleic acid in another embodiment, includes a nucleotide sequence that includes part, or all, ofthe coding region and extends into either (or both) the 5' or 3' noncoding region.
  • Other embodiments include a fragment which includes a nucleotide sequence encoding an amino acid fragment described herein.
  • Nucleic acid fragments can encode a specific domain or site described herein or fragments thereof, particularly fragments thereof which are at least 50 amino acids in length.
  • the nucleic acid fragments encode a specific domain or fragment thereof, wherein the domain or fragment is at least 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, or 750 amino acids in length.
  • Fragments also include nucleic acid sequences corresponding to specific amino acid sequences described above or fragments thereof. Nucleic acid fragments should not to be construed as encompassing those fragments that may have been disclosed prior to the invention.
  • a nucleic acid fragment can include a sequence corresponding to a domain, region, or functional site described herein.
  • a nucleic acid fragment can also include one or more domain, region, or functional site described herein.
  • a 83378, 84233, 64708, 85041, or 84234 nucleic acid fragment can include a sequence corresponding to a cation efflux domain, a transmembrane domain, a cytoplasmic domain, or a non-cytoplasmic loop.
  • the probe or primer is attached to a solid support, e.g, a solid support described herein.
  • a kit of primers includes a forward primer that anneals to the coding strand and a reverse primer that anneals to the non-coding strand.
  • the forward primer can anneal to the start codon, e.g, the nucleic acid sequence encoding amino acid residue 1 of SEQ ID NO:17, SEQ ID NO:20, SEQ ID NO:23, SEQ ID NO:26, or SEQ ID NO:29.
  • the nucleic acid is a probe which is at least 10, 12, 15, 18, 20 and less than 200, more preferably less than 100, or less than 50, nucleotides in length. It should be identical, or differ by 1, or 2, or less than 5 or 10 nucleotides, from a sequence disclosed herein. If alignment is needed for this comparison the sequences should be aligned for maximum homology. "Looped" out sequences from deletions or insertions, or mismatches, are considered differences.
  • a probe or primer can be derived from the sense or anti-sense strand of a nucleic acid which encodes: a cation efflux domain (e.g, residues 11 to 133 or 231 to 389 of SEQ ID NO:17, residues 25 to 310 of SEQ ID NO:20, residues 55 to 153 or 227 to 320 of SEQ ID NO:23, residues 419 to 733 of SEQ ID NO:26, or residues 38 to 349 of SEQ ID NO:29); a transmembrane domain (e.g, residues 11-31, 44-61, 79-98, 115-134, 241-265, or 283-299 of SEQ ID NO: 17; residues 25-49, 58-74, 92-113, 128-147, 167-191, or 201-218 of SEQ ID NO:20; residues 34-51, 58-82, 101-119, 137-155, 202-219, or 232-249 of SEQ ID NO:23; residues 59-77
  • a set of primers is provided, e.g, primers suitable for use in a PCR, which can be used to amplify a selected region of a 83378, 84233, 64708, 85041, or 84234 sequence, e.g, a domain, region, site or other sequence described herein.
  • the primers should be at least 5, 10, or 50 base pairs in length and less than 100, or less than 200, base pairs in length.
  • the primers should be identical, or differs by one base from a sequence disclosed herein or from a naturally occurring variant.
  • a nucleic acid fragment can encode an epitope bearing region of a polypeptide described herein.
  • a nucleic acid fragment encoding a "biologically active portion of a 83378, 84233, 64708, 85041, or 84234 polypeptide" can be prepared by isolating a portion ofthe nucleotide sequence of SEQ ID NO: 16, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:28, or SEQ ID NO:30, which encodes a polypeptide having a 83378, 84233, 64708, 85041, or 84234 biological activity (e.g, the biological activities ofthe 83378, 84233, 64708, 85041, or 84234 proteins are described herein), expressing the encoded portion ofthe 83378, 84233, 64708, 85041, or 84234 protein (e.g, by recombinant expression in vitro) and assessing the activity ofthe encoded portion
  • a nucleic acid fragment encoding a biologically active portion of 83378, 84233, 64708, 85041, or 84234 includes a cation efflux domain, e.g, residues 11 to 133 or 231 to 389 of SEQ ID NO: 17, residues 25 to 310 of SEQ ID NO:20, residues 55 to 153 or 227 to 320 of SEQ ID NO:23, residues 419 to 733 of SEQ ID NO:26, or residues 38 to 349 of SEQ ID NO:29.
  • a cation efflux domain e.g, residues 11 to 133 or 231 to 389 of SEQ ID NO: 17, residues 25 to 310 of SEQ ID NO:20, residues 55 to 153 or 227 to 320 of SEQ ID NO:23, residues 419 to 733 of SEQ ID NO:26, or residues 38 to 349 of SEQ ID NO:29.
  • a nucleic acid includes a nucleotide sequence which is about 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, or more nucleotides in length and hybridizes under a stringency condition described herein to a nucleic acid molecule of SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:28, or SEQ ID NO:30.
  • a 83378 nucleic acid fragment differs by at least 1, 2, 3, 10, 20, or more nucleotides from the sequence of Genbank accession number AL359609 or a sequence disclosed in WO 01/62918, WO 01/55314, WO 01/55355. Differences can include differing in length or sequence identity.
  • a nucleic acid fragment can: include one or more nucleotides from SEQ ID NO: 16 or SEQ ID NO: 18 located outside the region of nucleotides 7-1044, 1085-1638, 1253-1638, 1299-1638, 723-1775 of SEQ ID NO:16; not include all ofthe nucleotides ofthe sequence of Genbank accession number AL359609 or a sequence disclosed in WO 01/62918, WO 01/55314, WO 01/55355, e.g, can be one or more nucleotides shorter (at one or both ends) than the sequence of Genbank accession number AL359609 or a sequence disclosed in WO 01/62918, WO 01/55314, WO 01/55355; or can differ by one or more nucleotides in the region of overlap.
  • a 64708 nucleic acid fragment differs by at least 1, 2, 3, 10, 20, or more nucleotides from the sequence of Genbank accession number AK000844 or a sequence disclosed in WO 01/57188, WO 01/57270, WO 01/57272, WO 01/57275, WO 01/57276, WO 01/57277, or WO 01/57278. Differences can include differing in length or sequence identity.
  • an isolated nucleic acid molecule ofthe invention has a nucleotide sequence encoding a protein having an amino acid sequence which differs, by at least 1, but less than 5, 10, 20, 50, or 100 amino acid residues that shown in SEQ ID NO:2, SEQ ID NO:5, SEQ ID NO:8, SEQ ID NO:l 1, SEQ ID NO: 14, SEQ ID NO: 17, SEQ ID NO:20, SEQ ID NO:23, SEQ ID NO:26, or SEQ ID NO:29. If alignment is needed for this comparison the sequences should be aligned for maximum homology.
  • the encoded protein can differ by no more than 5, 4, 3, 2, or 1 amino acid. "Looped" out sequences from deletions or insertions, or mismatches, are considered differences.
  • Nucleic acids ofthe inventor can be chosen for having codons, which are preferred, or non-preferred, for a particular expression system.
  • the nucleic acid can be one in which at least one codon, at preferably at least 10%, or 20% ofthe codons has been altered such that the sequence is optimized for expression in E. coli, yeast, human, insect, or CHO cells.
  • Nucleic acid variants can be naturally occurring, such as allelic variants (same locus), homologs (different locus), and orthologs (different organism) or can be non naturally occurring.
  • Non-naturally occurring variants can be made by mutagenesis techniques, including those applied to polynucleotides, cells, or organisms.
  • the variants can contain nucleotide substitutions, deletions, inversions and insertions. Variation can occur in either or both the coding and non- coding regions. The variations can produce both conservative and non-conservative amino acid substitutions (as compared in the encoded product).
  • Preferred 67210 variants include those that are correlated with glycosyltransferaseactivity.
  • Allelic variants of 67210 e.g, human 67210, include both functional and non-functional proteins.
  • Functional allelic variants are naturally occurring amino acid sequence variants ofthe 67210 protein within a population that maintain the ability to transfer an activated mono- or oligosaccharide residue to an existing acceptor molecule for the initiation or elongation of a carbohydrate chain.
  • Functional allelic variants will typically contain only conservative substitution of one or more amino acids of SEQ ID NO: 5, or substitution, deletion or insertion of non-critical residues in non-critical regions ofthe protein.
  • Preferred 46842 variants include those that are correlated with Arf GTPase stimulating activity.
  • the invention features, an isolated nucleic acid molecule which is antisense to 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234.
  • An "antisense" nucleic acid can include a nucleotide sequence which is complementary to a "sense" nucleic acid encoding a protein, e.g, complementary to the coding strand of a double-stranded cDNA molecule or complementary to an mRNA sequence.
  • the antisense nucleic acid molecule ofthe invention is an ⁇ - anomeric nucleic acid molecule.
  • An ⁇ -anomeric nucleic acid molecule forms specific double- stranded hybrids with complementary RNA in which, contrary to the usual ⁇ -units, the strands run parallel to each other (Gaultier et al. (1987) Nucleic Acids. Res. 15:6625-6641).
  • the antisense nucleic acid molecule can also comprise a 2'-o-methylribonucleotide (Inoue et al. (1987) Nucleic Acids Res. 15:6131-6148) or a chimeric RNA-DNA analogue (Inoue et al. (1987) FEBSLett. 215:327-330).
  • an antisense nucleic acid ofthe invention is a ribozyme.
  • a ribozyme having specificity for a 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234-encoding nucleic acid can include one or more sequences complementary to the nucleotide sequence of a 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 cDNA disclosed herein (i.e, SEQ ID NO: 1 , SEQ ID NO:4, SEQ ID NO:7, SEQ ID NO:10, SEQ ID NO:13, SEQ ID NO:16, SEQ ID NO:19, SEQ ID NO:22, SEQ ID NO:25, or SEQ ID NO:28, or SEQ ID NO:3, SEQ ID NO:6, SEQ ID NO:9, SEQ ID NO: 12, SEQ ID NO:15, SEQ ID NO:
  • RNA can be used to select a catalytic RNA having a specific ribonuclease activity from a pool of RNA molecules. See, e.g, Bartel, D. and Szostak, J.W. (1993) Science 261:1411-1418.
  • a 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 nucleic acid molecule can be modified at the base moiety, sugar moiety or phosphate backbone to improve, e.g, the stability, hybridization, or solubility ofthe molecule.
  • synthetic oligonucleotides with modifications see Toulme (2001) Nature Biotech. 19:17 and Faria et al. (2001) Nature Biotech. 19:40-44.
  • Such phosphoramidixe oligonucleotides can be effective antisense agents.
  • the deoxyribose phosphate backbone ofthe nucleic acid molecules can be modified to generate peptide nucleic acids (see Hyrup B. et al (1996) Bioorganic & Medicinal Chemistry 4: 5-23).
  • peptide nucleic acid or "PNA” refers to a nucleic acid mimic, e.g, a DNA mimic, in which the deoxyribose phosphate backbone is replaced by a pseudopeptide backbone and only the four natural nucleobases are retained.
  • the neutral backbone of a PNA can allow for specific hybridization to DNA and RNA under conditions of low ionic strength.
  • PNA oligomers can be synthesized using standard solid phase peptide synthesis protocols as described in Hyrup B. etal. (1996) supra and Perry-O'Keefe et al. Proc. Natl. Acad. Sci. 93: 14670-675.
  • PNAs of 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 nucleic acid molecules can also be used in the analysis of single base pair mutations in a gene, (e.g, by PNA-directed PCR clamping); as 'artificial restriction enzymes' when used in combination with other enzymes, (e.g, SI nucleases (Hyrup B. et al. (1996) supra)); or as probes or primers for DNA sequencing or hybridization (Hyrup B. et al. (1996) supra; Perry-O'Keefe supra).
  • the oligonucleotide may include other appended groups such as peptides (e.g, for targeting host cell receptors in vivo), or agents facilitating transport across the cell membrane (see, e.g, Letsinger et al. (1989) Proc. Natl. Acad. Sci USA 86:6553-6556; Lemaifre et al (1987) Proc. Natl. Acad. Sci. USA 84:648-652; PCT Publication No. W088/09810) or the blood-brain barrier (see, e.g, PCT Publication No. W089/10134).
  • peptides e.g, for targeting host cell receptors in vivo
  • agents facilitating transport across the cell membrane see, e.g, Letsinger et al. (1989) Proc. Natl. Acad. Sci USA 86:6553-6556; Lemaifre et al (1987) Proc. Natl. Acad. Sci. USA
  • oligonucleotides can be modified with hybridization-triggered cleavage agents (see, e.g, Krol et al. (1988) Bio-Techniques 6:958-976) or intercalating agents, (see, e.g, Zon (1988) Pharm. Res. 5:539-549).
  • the oligonucleotide may be conjugated to another molecule, (e.g, a peptide, hybridization triggered cross-linking agent, transport agent, or hybridization-triggered cleavage agent).
  • the invention also includes molecular beacon oligonucleotide primer and probe molecules having at least one region which is complementary to a 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 nucleic acid ofthe invention, two complementary regions one having a fluorophore and one a quencher such that the molecular beacon is useful for quantitating the presence ofthe 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 nucleic acid ofthe invention in a sample.
  • Molecular beacon nucleic acids are described, for example, in Lizardi et al, U.S. Patent No. 5,854,033; Nazarenko et al, U.S. Patent No. 5,866,336, and Livak et al, U.S. Patent 5,876,930.
  • the invention features, an isolated 47476 protein, or fragment, e.g, a biologically active portion, for use as immunogens or antigens to raise or test (or more generally to bind) anti-47476 antibodies.
  • 47476 protein can be isolated from cells or tissue sources using standard protein purification techniques.
  • 47476 protein or fragments thereof can be produced by recombinant DNA techniques or synthesized chemically.
  • Polypeptides ofthe invention include those that arise as a result ofthe existence of multiple genes, alternative transcription events, alternative RNA splicing events, and alternative translational and post-translational events.
  • the polypeptide can be expressed in systems, e.g, cultured cells, which result in substantially the same post-translational modifications present when expressed the polypeptide is expressed in a native cell, or in systems which result in the alteration or omission of post-translational modifications, e.g, glycosylation or cleavage, present when expressed in a native cell.
  • systems e.g, cultured cells, which result in substantially the same post-translational modifications present when expressed the polypeptide is expressed in a native cell, or in systems which result in the alteration or omission of post-translational modifications, e.g, glycosylation or cleavage, present when expressed in a native cell.
  • a 47476 polypeptide has one or more ofthe following characteristics
  • ras guanine nucleotide dissociation stimulator domain which is preferably about 70%, 80%), 90%, 95%, 98%, 99%, or more homologous to amino acid residues about 195 to 381 of SEQ ID NO:2;
  • (x) it has an EF-hand calcium-binding domain which is preferably about 70%, 80%, 90%, 95%, 98%, 99%, or more homologous to amino acid residues about 470 to 498 of SEQ ID NO:2; (xi) it has a phorbol ester/diacylglycerol binding domain (Cl domain) which is preferably about 70%, 80%, 90%>, 95%, 98%>, 99%), or more homologous to amino acid residues about 541 to 590 of SEQ ID NO:2;
  • guanine nucleotide dissociation stimulator domain N-terminal motif which is preferably about 70%, 80%, 90%, 95%, 98%, 99%, or more homologous to amino acid residues about 55 to 172 of SEQ ID NO:2;
  • differences are, preferably, differences or changes at a non-essential residue or a conservative substitution.
  • the differences are not in the ras guanine nucleotide dissociation stimulator domain, the EF-hand calcium-binding domain or the phorbol ester/diacylglycerol binding domain (Cl domain).
  • one or more differences are in the ras guanine nucleotide dissociation stimulator domain, the EF-hand calcium-binding domain, and/or the phorbol ester/diacylglycerol binding domain (Cl domain).
  • inventions include a protein that contain one or more changes in amino acid sequence, e.g, a change in an amino acid residue which is not essential for activity.
  • 47476 proteins differ in amino acid sequence from SEQ ID NO:2, yet retain biological activity.
  • the protein includes an amino acid sequence at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or more homologous to SEQ ID NO:2.
  • a 47476 protein or fragment is provided which varies from the sequence of SEQ ID NO.2 in regions defined by amino acids about 1 to 54, 173 to 194, 382 to 469, 499 to 540, and 591 to 672 by at least one but by less than 15, 10 or 5 amino acid residues in the protein or fragment, but which does not differ from SEQ ID NO.2 in regions defined by amino acids about 55 to 173, 195 to 381, 470 to 498 or 541 to 590 of SEQ ID NO:2. (If this comparison requires alignment the sequences should be aligned for maximum homology.
  • “Looped" out sequences from deletions or insertions, or mismatches, are considered differences.) In some embodiments the difference is at a non-essential residue or is a conservative substitution, while in others the difference is at an essential residue or is a non-conservative substitution.
  • a biologically active portion of a 47476 protein includes a ras guanine nucleotide dissociation stimulator domain.
  • other biologically active portions in which other regions ofthe protein are deleted, can be prepared by recombinant techniques and evaluated for one or more ofthe functional activities of a native 47476 protein.
  • the 47476 protein has an amino acid sequence shown in SEQ ID NO:2.
  • the 47476 protein is substantially identical to SEQ ID NO:2.
  • the 47476 protein is substantially identical to SEQ ID NO:2 and retains the functional activity ofthe protein of SEQ ID NO:2, as described in detail in the subsections above.
  • a 47476 polypeptide fragment differs by at least 1, 2, 3, 10, 20, or more amino acid residues of a previously disclosed sequence. Differences can include differing in length or sequence identity.
  • a fragment can: include one or more amino acid residues from SEQ ID NO: 5 outside the region of overlap with the previously disclosed amino acid sequence; not include all ofthe amino acid residues encoded by a previously disclosed polypeptide sequence, e.g, can be one or more amino acid residues shorter (at one or both ends) than a previously disclocsed polypeptide sequence, or can differ by one or more amino acid residues in the region of overlap.
  • the invention features, an isolated 67210 protein, or fragment, e.g, a biologically active portion, for use as immunogens or antigens to raise or test (or more generally to bind) anti-67210 antibodies.
  • 67210 protein can be isolated from cells or tissue sources using standard protein purification techniques.
  • 67210 protein or fragments thereof can be produced by recombinant DNA techniques or synthesized chemically.
  • Polypeptides ofthe invention include those which arise as a result ofthe existence of multiple genes, alternative transcription events, alternative RNA splicing events, and alternative translational and post-translational events.
  • a 67210 polypeptide has one or more ofthe following characteristics:
  • SMC coronary smooth muscle cells
  • the 67210 protein, or fragment thereof differs from the corresponding sequence in SEQ ID NO:2. In one embodiment it differs by at least one but by less than 15, 10 or 5 amino acid residues. In another it differs from the corresponding sequence in SEQ ID NO: 5 by at least one residue but less than 20%, 15%, 10% or 5% ofthe residues in it differ from the corresponding sequence in SEQ ID NO: 5. (If this comparison requires alignment the sequences should be aligned for maximum homology.
  • the 67210 protein has an amino acid sequence shown in SEQ ID NO:5. In other embodiments, the 67210 protein is substantially identical to SEQ ID NO:5. In yet another embodiment, the 67210 protein is substantially identical to SEQ ID NO: 5 and retains the functional activity ofthe protein of SEQ ID NO: 5, as described in detail in the subsections above.
  • a fragment can: include one or more amino acid residues from SEQ ID NO: 5 outside the region encoded by nucleotides 290 to 560, 675 to 1042, or 1152 to 1743 of SEQ ID NO:4; not include all ofthe amino acid residues encoded by a nucleotide sequence in Genbank accession number AC013776 or AC023550, e.g, can be one or more amino acid residues shorter (at one or both ends) than a sequence encoded by the nucleotide sequence in Genbank accession number AC013776 or AC023550, or can differ by one or more amino acid residues in the region of overlap.
  • the invention features, an isolated 49875 protein, or fragment, e.g, a biologically active portion, for use as immunogens or antigens to raise or test (or more generally to bind) anti-49875 antibodies.
  • 49875 protein can be isolated from cells or tissue sources using standard protein purification techniques.
  • 49875 protein or fragments thereof can be produced by recombinant DNA techniques or synthesized chemically.
  • Polypeptides ofthe invention include those that arise as a result ofthe existence of multiple genes, alternative transcription events, alternative RNA splicing events, and alternative translational and post-translational events.
  • the polypeptide can be expressed in systems, e.g, cultured cells, which result in substantially the same post-translational modifications present when expressed the polypeptide is expressed in a native cell, or in systems which result in the alteration or omission of post-translational modifications, e.g, glycosylation or cleavage, present when expressed in a native cell.
  • a 49875 polypeptide has one or more ofthe following characteristics:
  • a protein that contain one or more changes in amino acid sequence e.g, a change in an amino acid residue which is not essential for activity.
  • Such 49875 proteins differ in amino acid sequence from SEQ ID NO:8, yet retain biological activity.
  • the protein includes an amino acid sequence at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or more homologous to SEQ ID NO:8.
  • a 49875 protein or fragment which varies from the sequence of SEQ ID NO: 8 in regions defined by amino acids about amino acids 1-21 and 246-600 of SEQ ID NO: 8 by at least one but by less than 15, 10 or 5 amino acid residues in the protein or fragment but which does not differ from SEQ ID NO: 8 in regions defined by amino acids about 22-245. (If this comparison requires alignment the sequences should be aligned for maximum homology. "Looped" out sequences from deletions or insertions, or mismatches, are considered differences.) In some embodiments the difference is at a non-essential residue or is a conservative substitution, while in others the difference is at an essential residue or is a non-conservative substitution.
  • a biologically active portion of a 49875 protein includes a DEAD type helicase domain or a conserved C-terminal helicase domain.
  • other biologically active portions in which other regions ofthe protein are deleted, can be prepared by recombinant techniques and evaluated for one or more ofthe functional activities of a native 49875 protein.
  • the 49875 protein has an amino acid sequence shown in SEQ ID NO:8.
  • the 49875 protein is substantially identical to SEQ ID NO: 8.
  • the 49875 protein is substantially identical to SEQ ID NO: 8 and retains the functional activity ofthe protein of SEQ ID NO:8, as described in detail in the subsections above.
  • a fragment differs by at least 1, 2, 3, 10, 20, or more amino acid residues encoded by a sequence present in WO 01/55301, WO 01/57188 or WO 01/62927. Differences can include differing in length or sequence identity.
  • a fragment can: not include all ofthe amino acid residues encoded by a nucleotide sequence in WO 01/55301, WO 01/57188 or WO 01/62927, e.g, can be one or more amino acid residues shorter (at one or both ends) than a sequence encoded by a nucleotide sequence inWO 01/55301, WO 01/57188 or WO 01/62927.or can differ by one or more amino acid residues in the region of overlap.
  • the invention features, an isolated 46842 protein, or fragment, e.g, a biologically active portion, for use as immunogens or antigens to raise or test (or more generally to bind) anti-46842 antibodies.
  • 46842 protein can be isolated from cells or tissue sources using standard protein purification techniques.
  • 46842 protein or fragments thereof can be produced by recombinant DNA techniques or synthesized chemically.
  • Polypeptides ofthe invention include those which arise as a result ofthe existence of multiple genes, alternative transcription events, alternative RNA splicing events, and alternative translational and post-translational events.
  • the polypeptide can be expressed in systems, e.g, cultured cells, which result in substantially the same post-translational modifications present when expressed the polypeptide is expressed in a native cell, or in systems which result in the alteration or omission of post-translational modifications, e.g, glycosylation or cleavage, present when expressed in a native cell.
  • the 46842 protein, or fragment thereof differs from the corresponding sequence in SEQ ID NO: 11. In one embodiment it differs by at least one but by less than 15, 10 or 5 amino acid residues. In another it differs from the corresponding sequence in SEQ ID NO:l 1 by at least one residue but less than 20%, 15%, 10% or 5% ofthe residues in it differ from the corresponding sequence in SEQ ID NO:l 1. (If this comparison requires alignment the sequences should be aligned for maximum homology. "Looped" out sequences from deletions or insertions, or mismatches, are considered differences.) The differences are, preferably, differences or changes at a non essential residue or a conservative substitution.
  • the differences are not in the ArfGAP, PH or ankyrin repeat domains. In another preferred embodiment one or more differences are in the ArfGAP, PH or ankyrin repeat domains.
  • Other embodiments include a protein that contain one or more changes in amino acid sequence, e.g, a change in an amino acid residue which is not essential for activity.
  • Such 46842 proteins differ in amino acid sequence from SEQ ID NO:l 1, yet retain biological activity.
  • the protein includes an amino acid sequence at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or more homologous to SEQ ID NO:ll.
  • a 46842 protein or fragment is provided which varies from the sequence of SEQ ID NO:l 1 in regions defined by amino acids about 403 to 525 by at least one but by less than 15, 10 or 5 amino acid residues in the protein or fragment but which does not differ from SEQ ID NO:l 1 in regions defined by amino acids about 1 to 402, and/or 526 to 834. (If this comparison requires alignment the sequences should be aligned for maximum homology.
  • a biologically active portion of a 46842 protein includes an ArfGAP domain, a PH domain, or an ankyrin repeat domain.
  • other biologically active portions, in which other regions ofthe protein are deleted can be prepared by recombinant techniques and evaluated for one or more ofthe functional activities of a native 46842 protein.
  • the 46842 protein has an amino acid sequence shown in SEQ ID NO: 11. In other embodiments, the 46842 protein is substantially identical to SEQ ID NO: 11. In yet another embodiment, the 46842 protein is substantially identical to SEQ ID NO: 12.
  • a fragment differs by at least 1, 2, 3, 10, 20, or more amino acid residues encoded by a sequence present in KIAA1716 (see, e.g, Genbank accession number gi 12697977). Differences can include differing in length or sequence identity.
  • the invention features, an isolated 33201 protein, or fragment, e.g, a biologically active portion, for use as immunogens or antigens to raise or test (or more generally to bind) anti-33201 antibodies.
  • 33201 protein can be isolated from cells or tissue sources using standard protein purification techniques.
  • 33201 protein or fragments thereof can be produced by recombinant DNA techniques or synthesized chemically.
  • Polypeptides ofthe invention include those which arise as a result ofthe existence of multiple genes, alternative transcription events, alternative RNA splicing events, and alternative translational and post-translational events.
  • the polypeptide can be expressed in systems, e.g, cultured cells, which result in substantially the same post-translational modifications present when expressed the polypeptide is expressed in a native cell, or in systems which result in the alteration or omission of post-translational modifications, e.g, glycosylation or cleavage, present when expressed in a native cell.
  • a 33201 polypeptide has one or more ofthe following characteristics:
  • v it has a molecular weight, e.g, a deduced molecular weight, preferably ignoring any contribution of post-translational modifications, amino acid composition or other physical characteristic of a 33201 polypeptide, e.g, a polypeptide pf a SEQ ID NO:14;
  • dehydrogenase/reductase domain which is preferably about 70%, 80%, 90%), 95%, 98%, 99%, or more homolgous with amino acid residues about 22 to 345 of SEQ ID NO: 14;
  • “Looped” out sequences from deletions or insertions, or mismatches are considered differences.
  • the differences are, preferably, differences or changes at a non-essential residue or a conservative substitution.
  • the differences are not in the dehydrogenase/reductase domain (e.g, about amino acids 55-380 of SEQ ID NO: 14).
  • one or more differences are in the dehydrogenase/reductase domain, (e.g, about amino acids 55-380 of SEQ ID NO: 14).
  • inventions include a protein that contain one or more changes in amino acid sequence, e.g, a change in an amino acid residue which is not essential for activity.
  • Such 33201 proteins differ in amino acid sequence from SEQ ID NO: 14, yet retain biological activity.
  • the protein includes an amino acid sequence at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or more homologous to SEQ ID NO: 14.
  • a 33201 protein or fragment is provided which varies from the sequence of SEQ ID NO:14 in regions defined by amino acids about 1 to 21 and/or 346 to 351 of SEQ ID NO:14 by at least one but by less than 15, 10 or 5 amino acid residues in the protein or fragment, but which does not differ from SEQ ID NO: 14 in regions defined by amino acids about 22 to 345 of SEQ ID NO: 14. (If this comparison requires alignment the sequences should be aligned for maximum homology. "Looped" out sequences from deletions or insertions, or mismatches, are considered differences.) In some embodiments the difference is at a non-essential residue or is a conservative substitution, while in others the difference is at an essential residue or is a non- conservative substitution.
  • a biologically active portion of a 33201 protein includes a dehydrogenase/reductase domain.
  • other biologically active portions in which other regions ofthe protein are deleted, can be prepared by recombinant techniques and evaluated for one or more ofthe functional activities of a native 33201 protein.
  • the 33201 protein has an amino acid sequence shown in SEQ ID NO:14. In other embodiments, the 33201 protein is substantially identical to SEQ ID NO: 14. In yet another embodiment, the 33201 protein is substantially identical to SEQ ID NO:14 and retains the functional activity ofthe protein of SEQ ID NO:14, as described in detail in the subsections above.
  • the invention features, an isolated 83378, 84233, 64708, 85041, or 84234 protein, or fragment, e.g, a biologically active portion, for use as immunogens or antigens to raise or test (or more generally to bind) anti-83378, 84233, 64708, 85041, or 84234 antibodies.
  • 83378, 84233, 64708, 85041, or 84234 protein can be isolated from cells or tissue sources using standard protein purification techniques.
  • 83378, 84233, 64708, 85041, or 84234 protein or fragments thereof can be produced by recombinant DNA techniques or synthesized chemically.
  • a 83378, 84233, 64708, 85041, or 84234 polypeptide has one or more ofthe following characteristics:
  • (v) it has a molecular weight, e.g, a deduced molecular weight, preferably ignoring any contribution of post translational modifications, amino acid composition or other physical characteristic of SEQ ID NO: 17, SEQ ID NO:20, SEQ ID NO:23, SEQ ID NO:26, or SEQ ID NO:29;
  • a molecular weight e.g, a deduced molecular weight, preferably ignoring any contribution of post translational modifications, amino acid composition or other physical characteristic of SEQ ID NO: 17, SEQ ID NO:20, SEQ ID NO:23, SEQ ID NO:26, or SEQ ID NO:29;
  • cation efflux domain which a sequence similarity of preferably about 70%, 80%, 90% or 95% with about amino acid residues 11 to 133 or 231 to 389 of SEQ ID NO:17, residues 25 to 310 of SEQ ID NO:20, residues 55 to 153 or 227 to 320 of SEQ ID NO:23, residues 419 to 733 of SEQ ID NO:26, or residues 38 to 349 of SEQ ID NO:29; or
  • the 83378, 84233, 64708, 85041, or 84234 protein, or fragment thereof differs from the corresponding sequence in SEQ ID NO: 17, SEQ ID NO:20, SEQ ID NO:23, SEQ ID NO:26, or SEQ ID NO:29. In one embodiment it differs by at least one but by less than 15, 10 or 5 amino acid residues.
  • SEQ ID NO:17 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or more homologous to SEQ ID NO:17, SEQ ID NO:20, SEQ ID NO:23, SEQ ID NO:26, or SEQ ID NO:29.
  • a 83378 protein or fragment is provided which varies from the sequence of SEQ ID NO:17 in regions defined by amino acids about 1-10, 134-230, or 390-485 by at least one but by less than 15, 10 or 5 amino acid residues in the protein or fragment but which does not differ from SEQ ID NO: 17 in regions defmed by amino acids about 11-133 or 231-389.
  • a 84233 protein or fragment is provided which varies from the sequence of SEQ ID NO:20 in regions defined by amino acids about 1-24 or 311-320 by at least one but by less than 15, 10 or 5 amino acid residues in the protein or fragment but which does not differ from SEQ ID NO:20 in regions defined by amino acids about 25-310.
  • a 64708 protein or fragment which varies from the sequence of SEQ ID NO:23 in regions defined by amino acids about 1-54, 154- 226, or 321-461 by at least one but by less than 15, 10 or 5 amino acid residues in the protein or fragment but which does not differ from SEQ ID NO:23 in regions defined by amino acids about 55-153 or 227-320.
  • a 85041 protein or fragment is provided which varies from the sequence of SEQ ID NO:26 in regions defined by amino acids about 1-418 or 734-765 by at least one but by less than 15, 10 or 5 amino acid residues in the protein or fragment but which does not differ from SEQ ID NO:26 in regions defined by amino acids about 419-733.
  • a 84234 protein or fragment is provided which varies from the sequence of SEQ ID NO:29 in regions defined by amino acids about 1-37 or 350-376 by at least one but by less than 15, 10 or 5 amino acid residues in the protein or fragment but which does not differ from SEQ ID NO:29 in regions defined by amino acids about 38-349. (If this comparison requires alignment the sequences should be aligned for maximum homology. "Looped" out sequences from deletions or insertions, or mismatches, are considered differences.) In some embodiments the difference is at a non-essential residue or is a conservative substitution, while in others the difference is at an essential residue or is a non-conservative substitution. In one embodiment, a biologically active portion of a 83378, 84233, 64708, 85041, or
  • a 83378 fragment differs by at least 1, 2, 3, 10, 20, or more amino acid residues encoded by a sequence present in Genbank accession number AL359609 or a sequence disclosed in WO 01/62918, WO 01/55314, WO 01/55355. Differences can include differing in length or sequence identity.
  • a 84233 fragment differs by at least 1, 2, 3, 10, 20, or more amino acid residues encoded by a sequence present in Genbank accession number AX061210 or AX086187 or a sequence disclosed in WO 01/12659, WO 01/51628, or WO 00/78953. Differences can include differing in length or sequence identity.
  • a fragment can: include one or more amino acid residues from SEQ ID NO:20 outside the region encoded by nucleotides 420-2103, 998-1589, 1-429, 1146-1582, 996-1425, 998-1341, 996-1379, 996-1368 of SEQ ID NO: 19; not include all ofthe amino acid residues encoded by a nucleotide sequence in Genbank accession number AX061210 or AX086187 or a sequence disclosed in WO 01/12659, WO 01/51628, or WO 00/78953, e.g, can be one or more amino acid residues shorter (at one or both ends) than a sequence encoded by the nucleotide sequence in Genbank accession number AX061210 or AX086187 or a sequence disclosed in WO 01/12659, WO 01/51628, or WO 00/78953; or can differ by one or more amino acid residues in the region of overlap.
  • a 64708 fragment differs by at least 1, 2, 3, 10, 20, or more amino acid residues encoded by a sequence present in Genbank accession number AK000844 or a sequence disclosed in WO 01/57188, WO 01/57270, WO 01/57272, WO 01/57275, WO 01/57276, WO 01/57277, or WO 01/57278. Differences can include differing in length or sequence identity.
  • a fragment can: include one or more amino acid residues from SEQ ID NO:23 outside the region encoded by nucleotides 209-697, 918-1456, 918-1418, 1348- 1790, 20-442, 911-1280, or 911-1226 of SEQ ID NO:22; not include all ofthe amino acid residues encoded by a nucleotide sequence in Genbank accession number AK000844 or a sequence disclosed in WO 01/57188, WO 01/57270, WO 01/57272, WO 01/57275, WO
  • 01/57276, WO 01/57277, or WO 01/57278 can be one or more amino acid residues shorter (at one or both ends) than a sequence encoded by the nucleotide sequence in Genbank accession number AK000844 or a sequence disclosed in WO 01/57188, WO 01/57270, WO 01/57272, WO 01/57275, WO 01/57276, WO 01/57277, or WO 01/57278; or can differ by one or more amino acid residues in the region of overlap.
  • a 85041 fragment differs by at least 1, 2, 3, 10, 20, or more amino acid residues encoded by a sequence present in Genbank accession number AK022558, AK022818, or AF233321, or a sequence disclosed in WO 01/40466, WO 01/54472, WO 01/55318, or WO 01/53312. Differences can include differing in length or sequence identity.
  • a fragment can: include one or more amino acid residues from SEQ ID NO:26 outside the region encoded by nucleotides 55-2750, 1069-3010, 1347-3224, 1320-3010,1347- 3010, 602-3005, 483-2740, 1015-1942, 786-3259, 602-3005, 883-2051, 2139-3010, 94-809, or 1843-3010 of SEQ ID NO:25; not include all ofthe amino acid residues encoded by a nucleotide sequence in Genbank accession number AK022558, AK022818, or AF233321, or a sequence disclosed in WO 01/40466, WO 01/54472, WO 01/55318, or WO 01/53312, e.g, can be one or more amino acid residues shorter (at one or both ends) than a sequence encoded by the nucleotide sequence in Genbank accession number AK022558, AK022818, or AF233321, or a sequence disclosed
  • a 84234 fragment differs by at least 1, 2, 3, 10, 20, or more amino acid residues encoded by a sequence disclosed in WO 01/53312. Differences can include differing in length or sequence identity.
  • a fragment can: not include all ofthe amino acid residues encoded by a sequence disclosed in WO 01/53312, e.g, can be one or more amino acid residues shorter (at one or both ends) than a sequence disclosed in WO 01/53312; or can differ by one or more amino acid residues in the region of overlap.
  • non-47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 polypeptide refers to a polypeptide having an amino acid sequence corresponding to a protein which is not substantially homologous to the 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 protein, e.g, a protein which is different from the 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 protein and which is derived from the same or a different organism.
  • the 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 polypeptide ofthe fusion protein can correspond to all or a portion e.g, a fragment described herein of a 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 amino acid sequence.
  • a 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 fusion protein includes at least one (or two) biologically active portion of a 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 protein.
  • the fusion protein can be a 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 protein containing a heterologous signal sequence at its N-terminus.
  • expression and/or secretion of 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 can be increased through use of a heterologous signal sequence.
  • the 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 fusion proteins ofthe invention can be incorporated into pharmaceutical compositions and administered to a subject in vivo.
  • the 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 fusion proteins can be used to affect the bioavailability of a 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 substrate.
  • 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 fusion proteins may be useful therapeutically for the treatment of disorders caused by, for example, (i) aberrant modification or mutation of a gene encoding a 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 protein; (ii) mis-regulation ofthe 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 gene; and (iii) aberrant post-translational modification of a 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 protein.
  • Expression vectors are commercially available that already encode a fusion moiety (e.g, a GST polypeptide).
  • a 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234-encoding nucleic acid can be cloned into such an expression vector such that the fusion moiety is linked in-frame to the 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 protein.
  • the invention also features a variant of a 47476, 67210, 49875, 46842,
  • An agonist ofthe 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 proteins can retain substantially the same, or a subset, ofthe biological activities ofthe naturally occurring form of a 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 protein.
  • An antagonist of a 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 protein can inhibit one or more ofthe activities ofthe naturally occurring form ofthe 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 protein by, for example, competitively modulating a 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234- mediated activity of a 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 protein.
  • treatment of a subject with a variant having a subset ofthe biological activities ofthe naturally occurring form ofthe protein has fewer side effects in a subject relative to treatment with the naturally occurring form ofthe 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 protein.
  • Variants of a 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 protein can be identified by screening combinatorial libraries of mutants, e.g, truncation mutants, of a 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 protein for agonist or antagonist activity.
  • fragments e.g, N-terminal, C-terminal, or internal fragments of a 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 protein coding sequence can be used to generate a variegated population of fragments for screening and subsequent selection of variants of a 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 protein.
  • Variants in which a cysteine residues is added or deleted or in which a residue which is glycosylated is added or deleted are particularly preferred.
  • Recursive ensemble mutagenesis (REM), a new technique which enhances the frequency of functional mutants in the libraries, can be used in combination with the screening assays to identify 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 variants (Arkin and Yourvan (1992) Proc. Natl. Acad. Sci. USA 59:7811-7815; Delgrave et al. (1993) Protein Engineering 6:327-331).
  • REM Recursive ensemble mutagenesis
  • Cell based assays can be exploited to analyze a variegated 47476 library.
  • a library of expression vectors can be transfected into a cell line, e.g, a cell line, which ordinarily responds to 47476 in a substrate-dependent manner.
  • the transfected cells are then contacted with the substrate and the effect ofthe expression ofthe 47476 mutant on signaling by the substrate can be detected, e.g, by measuring GTP loading of a ras superfamily protein, measuring signal transduction that involves an activated ras superfamily protein, or by assaying for the changes induced by such signal transduction, e.g, cellular proliferation, differentiation, or migration.
  • Plasmid DNA can then be recovered from the cells which score for inhibition, or alternatively, potentiation of signaling by the 47476 substrate, and the individual clones further characterized.
  • Cell based assays can be exploited to analyze a variegated 67210 library.
  • a library of expression vectors can be transfected into a cell line, e.g, a cell line, which ordinarily responds to 67210 in a substrate-dependent manner.
  • the transfected cells are then contacted with the substrate and the effect ofthe expression ofthe 67210 mutant on signaling by the substrate can be detected, e.g, by measuring cellular properties influenced by 67210 activity, e.g, the appearance of particular glycosylated molecules on the cell surface, cellular adhesion, or signal transduction, e.g, as measured by cell proliferation or differentiation.
  • Plasmid DNA can then be recovered from the cells which score for inhibition, or alternatively, potentiation of signaling by the 67210 substrate, and the individual clones further characterized.
  • Cell based assays can be exploited to analyze a variegated 33201 library.
  • a library of expression vectors can be transfected into a cell line, e.g, a cell line, which ordinarily responds to 33201 in a substrate-dependent manner.
  • the transfected cells are then contacted with the substrate and the effect ofthe expression ofthe 33201 mutant on signaling by the substrate can be detected, e.g, by measuring a cellular property dependent upon the substrate, e.g, cellular proliferation or cellular differentiation.
  • Plasmid DNA can then be recovered from the cells which score for inhibition, or alternatively, potentiation of signaling by the substrate, and the individual clones further characterized.
  • Cell based assays can be exploited to analyze a variegated 83378, 84233, 64708, 85041, or 84234 library.
  • a library of expression vectors can be transfected into a cell line, e.g, a cell line, which ordinarily responds to 83378, 84233, 64708, 85041, or 84234 in a substrate-dependent manner.
  • the transfected cells are then contacted with the sustrate and the effect ofthe expression ofthe 83378, 84233, 64708, 85041, or 84234 mutant on signaling by the substrate can be detected, e.g, by measuring cation diffusion or a response ofthe cells to the substrate, e.g, a normal cellular response such as programmed cell death. Plasmid DNA can then be recovered from the cells which score for inhibition, or alternatively, potentiation of signaling by the substrate, and the individual clones further characterized.
  • polypeptide e.g, altering the sequence , e.g, by substitution or deletion of one or more residues of a non-conserved region, a domain or residue disclosed herein, and testing the altered polypeptide for the desired activity.
  • the invention features a method of making a fragment or analog of a 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 polypeptide a biological activity of a naturally occurring 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 polypeptide.
  • the method includes: altering the sequence, e.g, by substitution or deletion of one or more residues, of a 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 polypeptide, e.g, altering the sequence of a non- conserved region, or a domain or residue described herein, and testing the altered polypeptide for the desired activity.
  • VH and VL regions can be further subdivided into regions of hypervariability, termed “complementarity determining regions” ("CDR"), interspersed with regions that are more conserved, termed “framework regions” (FR).
  • CDR complementarity determining regions
  • FR framework regions
  • the extent ofthe framework region and CDR's has been precisely defined (see, Kabat, E.A, et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242, and Chothia, C. et al. (1987) J. Mol. Biol. 196:901-917, which are incorporated herein by reference).
  • Each VH and VL is composed of three CDR's and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.
  • the anti-47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 antibody can further include a heavy and light chain constant region, to thereby form a heavy and light immunoglobulin chain, respectively.
  • the antibody is a tetramer of two heavy immunoglobulin chains and two light immunoglobulin chains, wherein the heavy and light immunoglobulin chains are inter-connected by, e.g, disulfide bonds.
  • the heavy chain constant region is comprised of three domains, CHI, CH2 and CH3.
  • the light chain constant region is comprised of one domain, CL.
  • the variable region ofthe heavy and light chains contains a binding domain that interacts with an antigen.
  • the constant regions ofthe antibodies typically mediate the binding ofthe antibody to host tissues or factors, including various cells of the immune system (e.g, effector cells) and the first component (Clq) ofthe classical complement system.
  • Full-length immunoglobulin "heavy chains" (about 50 KDa or 446 amino acids), are similarly encoded by a variable region gene (about 116 amino acids) and one ofthe other aforementioned constant region genes, e.g, gamma (encoding about 330 amino acids).
  • antigen-binding fragments ofthe anti-47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 antibody include, but are not limited to: (i) a Fab fragment, a monovalent fragment consisting ofthe VL, VH, CL and CHI domains; (ii) a F(ab')2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fd fragment consisting ofthe VH and CHI domains; (iv) a Fv fragment consisting ofthe VL and VH domains of a single arm of an antibody, (v) a dAb fragment (Ward et al, (1989) Nature 341:544-546), which consists of a VH domain; and (vi) an isolated complementarity determining region (CDR).
  • CDR complementarity determining region
  • the two domains ofthe Fv fragment, VL and VH are coded for by separate genes, they can be joined, using recombinant methods, by a synthetic linker that enables them to be made as a single protein chain in which the VL and VH regions pair to form monovalent molecules (known as single chain Fv (scFv); see e.g., Bird et al. (1988) Science 242:423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879- 5883).
  • single chain Fv single chain Fv
  • Such single chain antibodies are also encompassed within the term "antigen-binding fragment" of an antibody.
  • the anti-47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 antibody can be a polyclonal or a monoclonal antibody.
  • the antibody can be recombinantly produced, e.g, produced by phage display or by combinatorial methods.
  • Phage display and combinatorial methods for generating anti-47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 antibodies are known in the art (as described in, e.g, Ladner et al. U.S. Patent No. 5,223,409; Kang et al. International Publication No.
  • 85041, or 84234 antibody is a fully human antibody (e.g, an antibody made in a mouse which has been genetically engineered to produce an antibody from a human immunoglobulin sequence), or a non-human antibody, e.g, a rodent (mouse or rat), goat, primate (e.g, monkey), camel antibody.
  • the non-human antibody is a rodent (mouse or rat antibody).
  • Method of producing rodent antibodies are known in the art.
  • Human monoclonal antibodies can be generated using transgenic mice carrying the human immunoglobulin genes rather than the mouse system.
  • Splenocytes from these transgenic mice immunized with the antigen of interest are used to produce hybridomas that secrete human mAbs with specific affinities for epitopes from a human protein (see, e.g. Wood et al. International Application WO 91/00906, Kucherlapati et al. PCT publication WO 91/10741; Lonberg et al. International Application WO 92/03918; Kay et al. International Application 92/03917; Lonberg, N. et al. 1994 Nature 368:856-859; Green, L.L. et al. 1994 Nature Genet. 7:13-21; Morrison, S.L. et al. 1994 Proc. Natl.
  • An anti-47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 antibody can be one in which the variable region, or a portion thereof, e.g, the CDR's, are generated in a non-human organism, e.g, a rat or mouse. Chimeric, CDR-grafted, and humanized antibodies are within the invention. Antibodies generated in a non-human organism, e.g, a rat or mouse, and then modified, e.g, in the variable framework or constant region, to decrease antigenicity in a human are within the invention.
  • Chimeric antibodies can be produced by recombinant DNA techniques known in the art. For example, a gene encoding the Fc constant region of a murine (or other species) monoclonal antibody molecule is digested with restriction enzymes to remove the region encoding the murine Fc, and the equivalent portion of a gene encoding a human Fc constant region is substituted (see Robinson et al. International Patent Publication PCT/US86/02269; Akira, et al, European Patent Application 184,187; Taniguchi, M, European Patent Application 171,496; Morrison et al, European Patent Application 173,494; Neuberger et al. International Application WO 86/01533; Cabilly et al. U.S.
  • a humanized or CDR-grafted antibody will have at least one or two but generally all three recipient CDR's (of heavy and or light immuoglobulin chains) replaced with a donor
  • the immunoglobulin providing the CDR's is called the "donor” and the immunoglobulin providing the framework is called the “acceptor.”
  • the donor immunoglobulin is a non-human (e.g, rodent).
  • the acceptor framework is a naturally-occurring (e.g, a human) framework or a consensus framework, or a sequence about 85% or higher, preferably 90%, 95%, 99% or higher identical thereto.
  • humanized antibodies in which specific amino acids have been substituted, deleted or added.
  • Preferred humanized antibodies have amino acid substitutions in the framework region, such as to improve binding to the antigen.
  • a humanized antibody will have framework residues identical to the donor framework residue or to another amino acid other than the recipient framework residue.
  • a selected, small number of acceptor framework residues ofthe humanized immunoglobulin chain can be replaced by the corresponding donor amino acids.
  • Prefened locations ofthe substitutions include amino acid residues adjacent to the CDR, or which are capable of interacting with a CDR (see e.g, US 5,585,089).
  • an antibody can be made by immunizing with purified 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 antigen, or a fragment thereof, e.g, a fragment described herein, membrane associated antigen, tissue, e.g, crude tissue preparations, whole cells, preferably living cells, lysed cells, or cell fractions, e.g, cytosolic fractions or membrane fractions.
  • tissue e.g, crude tissue preparations, whole cells, preferably living cells, lysed cells, or cell fractions, e.g, cytosolic fractions or membrane fractions.
  • a full-length 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 protein or, antigenic peptide fragment of 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 can be used as an immunogen or can be used to identify anti- 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 antibodies made with other immunogens, e.g, cells, membrane preparations, and the like.
  • the antigenic peptide of 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 should include at least 8 amino acid residues ofthe amino acid sequence shown in SEQ ID NO:2, SEQ ID NO:5, SEQ ID NO:8, SEQ ID NO:l 1, SEQ ID NO: 14, SEQ ID NO: 17, SEQ ID NO:20, SEQ ID NO:23, SEQ ID NO:26, or SEQ ID NO:29 and encompasses an epitope of 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234.
  • the antigenic peptide includes at least 10 amino acid residues, more preferably at least 15 amino acid residues, even more preferably at least 20 amino acid residues, and most preferably at least 30 amino acid residues.
  • fragments of 47476 which include, e.g, residues about 82 to 105, about 341 to 360, or about 521 to 540 of SEQ ID NO:2, can be used to make an antibody against a hydrophobic region ofthe 47476 protein; fragments of 47476, which include, e.g, residues about 55 to 173, about 195 to 381, about 470 to 498, or about 541 to 590 of SEQ ID NO:2, can be used to make an antibody against the a guanine nucleotide dissociation stimulator domain N-terminal motif, a ras guanine nucleotide dissociation stimulator domain, an EF-hand calcium-binding domain and a phorbol ester/diacylglycerol binding domain (Cl domain), respectively, ofthe 47476 protein.
  • Fragments of 49875 can be used as immunogens or used to characterize the specificity of an antibody.
  • fragments of 49875 which include, e.g, residues about 520 to 550 of SEQ ID NO:8, can be used to make antibodies against hydrophilic regions ofthe 49875 protein.
  • fragments of 49875 which include, e.g, residues about 285 to 295 of SEQ ID NO:8, can be used to make an antibody against a hydrophobic region ofthe 49875 protein; fragments of 49875 which include, e.g, residues about 22 to 245 of SEQ ID NO:8, can be used to make an antibody against the DEAD-type helicase region ofthe 49875 protein; fragments of 49875 which include, e.g, about amino acid residues 169 to 177 of SEQ ID NO:8, can be used to maka an antibody against a DEAD-box subfamily ATP-dependent helicase signature motif; and fragments of 49875 which include, e.g, residues about 281 to 363 of SEQ ID NO:8, can be used to make an antibody against the conserved helicase C-terminal region ofthe 49875 protein.
  • Fragments of 46842 can be used as immunogens or used to characterize the specificity of an antibody.
  • fragments of 46842 which include, e.g, residues about 567 to 580, 720 to 737, or 757 to 771 of SEQ ID NO:l 1, can be used to make antibodies against hydrophilic regions ofthe 46842 protein.
  • fragments of 46842 which include, e.g, residues about 431 to 439, from about 558 to 566, and from about 706 to 719 of SEQ ID NO:l 1, can be used to make an antibody against a hydrophobic region ofthe 46842 protein; fragments of 46842 which include, e.g, residues about 269 to 363 of SEQ ID NO:l 1, can be used to make an antibody against the PH domain region ofthe 46842 protein; fragments of 46842 which include, e.g, residues about 403 to 525 of SEQ ID NO: 11 , can be used to make an antibody against the ArfGAP domain region ofthe 46842 protein; and fragments of 46842 which include, e.g, residues about 702 to 734 or 735 to 767 of SEQ ID NO: 11 , can be used to make an antibody against the ankyrin repeat domains of 46842.
  • Fragments of 33201 can be used, e.g, to characterize the specificity of an antibody or to make immunogens.
  • fragments of 33201 which include, e.g, residues about 50 to 60, 82 to 90, or 205 to 210 of SEQ ID NO: 14, can be used to make antibodies against hydrophilic regions ofthe 33201 protein.
  • fragments of 33201 which include, e.g, residues about 70 to 80 or 158 to 178 of SEQ ID NO: 14, can be used to make an antibody against a hydrophobic region ofthe 33201 protein; fragments of 33201 which include residues from about 22 to 345 of SEQ ID NO: 14, or a fragment thereof, e.g, 22 to 50, 50 to 100, 150 to 200, 250 to 300, or 300 to 345 of SEQ ID NO:14, can be used to make an antibody against a dehydrogenase/reductase domain ofthe 33201 protein.
  • fragments of 83378, 84233, 64708, 85041 , or 84234 which include residues about amino acid residues 325 to 335, 340 to 350, or 415 to 430 of SEQ ID NO:17, about amino acid residues 220 to 230, 240 to 260, or 262 to 273 of SEQ ID NO:20, about amino acid residues 180 to 195, 290 to 300, or 340 to 350 of SEQ ID NO:23, about amino acid residues 35 to 50, 440 to 470, or 685 to 695 of SEQ ID NO:26, or about amino acid residues 59 to 70, 330 to 345, or 370 to 376 of SEQ ID NO:29 can be used to make an antibody against a hydrophobic region ofthe 83378, 84233, 64708, 85041, or 84234 protein.
  • Fragments of 83378, 84233, 64708, 85041, or 84234 which include about amino acid residues 32 to 43, 99 to 114, or 266 to 282 of SEQ ID NO: 17, about amino acid residues 50 to 57, 114 to 127, or 192 to 200 of SEQ ID NO:20, about amino acid residues 52 to 57, 120 to 136, or 220 to 231 of SEQ ID NO:23, about amino acid residues 78 to 98, 146 to 151, 215 to 238, 289 to 303, 363 to 418, 506 to 520, or 614 to 617 of SEQ ID NO:26, or about amino acid residues 59 to 70, 124 to 140, or 257 to 262 of SEQ ID NO:29 can be used to make an antibody against a non-cytoplasmic loop ofthe 83378, 84233, 64708, 85041, or 84234 protein.
  • Fragments of 83378, 84233, 64708, 85041, or 84234 which include about amino acid residues 1 to 10, 62 to 78, 135 to 240, or 300 to 485 of SEQ ID NO:17, about amino acid residues 1 to 24, 75 to 91, 148 to 166, or 219 to 320 of SEQ ID NO:20, about amino acid residues 1 to 33, 83 to 100, 156 to 201, or 250 to 461 of SEQ ID NO:23, about amino acid residues 1 to 58, 120 to 128, 169 to 189, 259 to 266, 321 to 342, 438 to 485, 542 to 591, or 642 to 765 of SEQ ID NO:26, or about amino acid residues 1 to 37, 88 to 104, 160 to 236, or 287 to 376 of SEQ ID NO:29 can be used to make an antibody against a cytoplasmic domain ofthe 83378, 84233, 64708, 85041, or 84234 protein.
  • Fragment of 83378, 84233, 64708, 85041, or 84234 which include about amino acid residues 11 to 133 or 231 to 389 of SEQ ID NO: 17, about amino acid residues 25 to 310 of SEQ ID NO:20, about amino acid residues 55 to 153 or 227 to 320 of SEQ ID NO:23, about amino acid residues 419 to 733 of SEQ ID NO:26, or about amino acid residues 38 to 349 of SEQ ID NO:29 can be used to make an antibody against the cation efflux region ofthe 83378, 84233, 64708, 85041, or 84234 protein.
  • Antibodies reactive with, or specific for, any of these regions, or other regions or domains described herein are provided. Antibodies which bind only native 47476, 67210, 49875, 46842, 33201, 83378, 84233,
  • 64708, 85041, or 84234 protein only denatured or otherwise non-native 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 protein, or which bind both, are with in the invention.
  • Antibodies with linear or conformational epitopes are within the invention. Conformational epitopes can sometimes be identified by identifying antibodies which bind to native but not denatured 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 protein.
  • Preferred epitopes encompassed by the antigenic peptide are regions of 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 are located on the surface ofthe protein, e.g, hydrophilic regions, as well as regions with high antigenicity.
  • an Emini surface probability analysis ofthe human 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 protein sequence can be used to indicate the regions that have a particularly high probability of being localized to the surface ofthe 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 protein.
  • the antibody can bind to the extracellular portion of a 67210, 83378, 84233, 64708, 85041, or 84234 protein, e.g, it can bind to a whole cell which expresses a 67210, 83378, 84233, 64708, 85041, or 84234 protein.
  • the antibody binds an intracellular portion of a 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 protein.
  • antibodies can bind one or more of purified antigen, membrane associated antigen, tissue, e.g, tissue sections, whole cells, preferably living cells, lysed cells, cell fractions, e.g, cytosolic fractions or membrane fractions.
  • the anti-47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 antibody can be a single chain antibody.
  • a single-chain antibody (scFV) may be engineered (see, for example, Colcher, D. et al. (1999) Ann N Y Acad Sci 880:263-80; and Reiter, Y. (1996) Clin Cancer Res 2:245-52).
  • the single chain antibody can be dimerized or multimerized to generate multivalent antibodies having specificities for different epitopes ofthe same target 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 protein.
  • the antibody has effector function and/or can fix complement. In other embodiments the antibody does not recruit effector cells; or fix complement.
  • the antibody has reduced or no ability to bind an Fc receptor.
  • it is a isotype or subtype, fragment or other mutant, which does not support binding to an Fc receptor, e.g, it has a mutagenized or deleted Fc receptor binding region.
  • an anti-47476 antibody alters (e.g, increases or decreases) that ability of 47476 to: (1) stimulate the exchange of guanine nucleotides (GTP for GDP) by a member ofthe ras superfamily of proteins; (2) bind calcium in a pentagonal bipyramidal configuration; (3) bind two zinc ions; (4) bind the second messenger diacylglycerol; (5) bind analogs of diacylglycerol, such as phorbol esters; or (6) activate one or more members ofthe ras superfamily of proteins.
  • GTP guanine nucleotides
  • an anti-67210 antibody alters (e.g, increases or decreases) the glycosyltransferase activity of a 67210 polypeptide.
  • the antibody can bind at or in proximity to the active site of a 67210 polypeptide or protein, e.g, to an epitope that is present within the glycosyl transferase domain, e.g, located at about amino acid residues 63 to 340 o SEQ ID NO:5.
  • an anti-49875 antibody alters (e.g, increases or decreases) the nucleic acid unwinding activity of a 49875 polypeptide.
  • an anti-46842 antibody alters (e.g, increases or decreases) the ArfGAP activity of a 46842 polypeptide.
  • the antibody can bind at or in proximity to a motif involved in ArfGAP catalytic activity, e.g, the sequence located at about residues 421 to 440 of SEQ ID NO:l 1.
  • an anti-33201 antibody alters (e.g, increases or decreases) the dehydrogenase/reductase activity of a 33201 polypeptide.
  • the antibody can bind at or in proximity to the active site, e.g, to an epitope that includes a residue located within a region from about 22 to 345 of SEQ ID NO:14, or a fragment thereof, e.g, 100 to 150, 150 to 200, 250 to 300, or 300 to 335 of SEQ ID NO:14.
  • an anti-83378, 84233, 64708, 85041, or 84234 antibody alters (e.g, increases or decreases) the cation diffusion activity of a 83378, 84233, 64708, 85041 , or 84234 polypeptide.
  • the antibody can be coupled to a toxin, e.g, a polypeptide toxin, e,g, ricin or diphtheria toxin or active fragment hereof, or a radioactive nucleus, or imaging agent, e.g. a radioactive, enzymatic, or other, e.g, imaging agent, e.g, a NMR contrast agent. Labels which produce detectable radioactive emissions or fluorescence are preferred.
  • An anti-47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 antibody (e.g, monoclonal antibody) can be used to isolate 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 by standard techniques, such as affinity chromatography or immunoprecipitation.
  • an anti-47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 antibody can be used to detect 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 protein (e.g, in a cellular lysate or cell supernatant) in order to evaluate the abundance and pattern of expression ofthe protein.
  • Anti-47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 antibodies can be used diagnostically to monitor protein levels in tissue as part of a clinical testing procedure, e.g, to determine the efficacy of a given treatment regimen. Detection can be facilitated by coupling (i.e, physically linking) the antibody to a detectable substance (i.e, antibody labelling). Examples of detectable substances include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, and radioactive materials.
  • suitable enzymes include horseradish peroxidase, alkaline phosphatase, ⁇ -galactosidase, or acetylcholinesterase;
  • suitable prosthetic group complexes include streptavidin/biotin and avidin/biotin;
  • suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin;
  • an example of a luminescent material includes luminol;
  • examples of bioluminescent materials include luciferase, luciferin, and aequorin, and examples of suitable radioactive material include I, I, S or H.
  • the invention also includes a nucleic acid which encodes an anti-47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 antibody, e.g, an anti-47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 antibody described herein. Also included are vectors which include the nucleic acid and cells transformed with the nucleic acid, particularly cells which are useful for producing an antibody, e.g, mammalian cells, e.g. CHO or lymphatic cells. The invention also includes cell lines, e.g, hybridomas, which make an anti-47476,
  • 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 antibody e.g, an antibody described herein, and method of using said cells to make a 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 antibody.
  • the invention includes, vectors, preferably expression vectors, containing a nucleic acid encoding a polypeptide described herein.
  • vector refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked and can include a plasmid, cosmid or viral vector.
  • the vector can be capable of autonomous replication or it can integrate into a host DNA.
  • Viral vectors include, e.g, replication defective retroviruses, adenoviruses and adeno-associated viruses.
  • a vector can include a 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 nucleic acid in a form suitable for expression ofthe nucleic acid in a host cell.
  • the recombinant expression vector includes one or more regulatory sequences operatively linked to the nucleic acid sequence to be expressed.
  • the term "regulatory sequence” includes promoters, enhancers and other expression control elements (e.g, poly adenylation signals). Regulatory sequences include those which direct constitutive expression of a nucleotide sequence, as well as tissue-specific regulatory and/or inducible sequences.
  • the design ofthe expression vector can depend on such factors as the choice ofthe host cell to be transformed, the level of expression of protein desired, and the like.
  • the expression vectors ofthe invention can be introduced into host cells to thereby produce proteins or polypeptides, including fusion proteins or polypeptides, encoded by nucleic acids as described herein (e.g, 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 proteins, mutant forms of 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 proteins, fusion proteins, and the like).
  • the recombinant expression vectors ofthe invention can be designed for expression of
  • polypeptides ofthe invention can be expressed in E. coli, insect cells (e.g, using baculoviras expression vectors), yeast cells or mammalian cells. Suitable host cells are discussed further in Goeddel, (1990) Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, CA.
  • the recombinant expression vector can be transcribed and translated in vitro, for example using T7 promoter regulatory sequences and T7 polymerase.
  • Fusion vectors add a number of amino acids to a protein encoded therein, usually to the amino terminus ofthe recombinant protein.
  • Such fusion vectors typically serve three purposes: 1) to increase expression of recombinant protein; 2) to increase the solubility of the recombinant protein; and 3) to aid in the purification ofthe recombinant protein by acting as a ligand in affinity purification.
  • a proteolytic cleavage site is introduced at the junction ofthe fusion moiety and the recombinant protein to enable separation ofthe recombinant protein from the fusion moiety subsequent to purification ofthe fusion protein.
  • Such enzymes, and their cognate recognition sequences include Factor Xa, thrombin and enterokinase.
  • Typical fusion expression vectors include pGEX (Pharmacia Biotech Inc; Smith, D.B. and Johnson, K.S.
  • GST glutathione S-transferase
  • Purified fusion proteins can be used in 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 activity assays, (e.g, direct assays or competitive assays described in detail below), or to generate antibodies specific for 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 proteins.
  • a fusion protein expressed in a retroviral expression vector ofthe present invention can be used to infect bone marrow cells which are subsequently transplanted into irradiated recipients. The pathology ofthe subject recipient is then examined after sufficient time has passed (e.g, six weeks).
  • nucleic acid sequence of the nucleic acid is altered into an expression vector so that the individual codons for each amino acid are those preferentially utilized in E. coli (Wada et al, (1992) Nucleic Acids Res. 20:2111-2118).
  • Such alteration of nucleic acid sequences ofthe invention can be carried out by standard DNA synthesis techniques.
  • the 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 expression vector can be a yeast expression vector, a vector for expression in insect cells, e.g, a baculoviras expression vector or a vector suitable for expression in mammalian cells.
  • the expression vector's control functions can be provided by viral regulatory elements.
  • commonly used promoters are derived from polyoma, Adenovirus 2, cytomegalovirus and Simian Virus 40.
  • the promoter is an inducible promoter, e.g, a promoter regulated by a steroid hormone, by a polypeptide hormone (e.g, by means of a signal transduction pathway), or by a heterologous polypeptide (e.g, the tetracycline-inducible systems, "Tet-On” and "Tet-Off ; see, e.g, Clontech Inc., CA, Gossen and Bujard (1992) Proc.
  • a promoter regulated by a steroid hormone e.g, by a polypeptide hormone (e.g, by means of a signal transduction pathway), or by a heterologous polypeptide (e.g, the tetracycline-inducible systems, "Tet-On" and "Tet-Off ; see, e.g, Clontech Inc., CA, Gossen and Bujard (1992) Proc.
  • a heterologous polypeptide e.g, the t
  • promoters are also encompassed, for example, the murine hox promoters (Kessel and Grass (1990) Science 249:374-379) and the ⁇ -fetoprotein promoter (Campes and Tilghman (1989) Genes Dev. 3:537- 546).
  • the invention further provides a recombinant expression vector comprising a DNA molecule ofthe invention cloned into the expression vector in an antisense orientation.
  • Regulatory sequences e.g, viral promoters and/or enhancers
  • operatively linked to a nucleic acid cloned in the antisense orientation can be chosen which direct the constitutive, tissue specific or cell type specific expression of antisense RNA in a variety of cell types.
  • the antisense expression vector can be in the form of a recombinant plasmid, phagemid or attenuated viras.
  • a host cell which includes a nucleic acid molecule described herein, e.g, a 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 nucleic acid molecule within a recombinant expression vector or a 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 nucleic acid molecule containing sequences which allow it to homologously recombine into a specific site ofthe host cell's genome.
  • the terms "host cell” and "recombinant host cell” are used interchangeably herein.
  • transformation and “transfection” are intended to refer to a variety of art-recognized techniques for introducing foreign nucleic acid (e.g, DNA) into a host cell, including calcium phosphate or calcium chloride co-precipitation, DEAE-dextran-mediated transfection, lipofection, or electroporation.
  • foreign nucleic acid e.g, DNA
  • a host cell including calcium phosphate or calcium chloride co-precipitation, DEAE-dextran-mediated transfection, lipofection, or electroporation.
  • a host cell ofthe invention can be used to produce (i.e, express) a 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 protein. Accordingly, the invention further provides methods for producing a 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 protein using the host cells ofthe invention.
  • the method includes culturing the host cell ofthe invention (into which a recombinant expression vector encoding a 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 protein has been introduced) in a suitable medium such that a 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 protein is produced.
  • the method further includes isolating a 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 protein from the medium or the host cell.
  • the invention features, a human cell, e.g, a hematopoietic, neural, muscle, or hepatic stem cell, transformed with nucleic acid which encodes a subject 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 polypeptide.
  • a human cell e.g, a hematopoietic, neural, muscle, or hepatic stem cell
  • nucleic acid which encodes a subject 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 polypeptide.
  • cells preferably human cells, e.g, human hematopoietic, neural, muscle, hepatic or fibroblast cells, in which an endogenous 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 is under the control of a regulatory sequence that does not normally control the expression ofthe endogenous 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 gene.
  • an endogenous 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 gene which is "transcriptionally silent," e.g, not normally expressed, or expressed only at very low levels, may be activated by inserting a regulatory element which is capable of promoting the expression of a normally expressed gene product in that cell.
  • Techniques such as targeted homologous recombinations, can be used to insert the heterologous DNA as described in, e.g, Chappel, US 5,272,071; WO 91/06667, published in May 16, 1991.
  • recombinant cells described herein can be used for replacement therapy in a subject.
  • a nucleic acid encoding a 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 polypeptide operably linked to an inducible promoter e.g, a steroid hormone receptor-regulated promoter
  • an inducible promoter e.g, a steroid hormone receptor-regulated promoter
  • the cell is cultivated and encapsulated in a biocompatible material, such as poly-lysine alginate, and subsequently implanted into the subject. See, e.g, Lanza (1996) Nat.
  • Production of 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 polypeptide can be regulated in the subject by admimstering an agent (e.g, a steroid hormone) to the subject.
  • an agent e.g, a steroid hormone
  • the implanted recombinant cells express and secrete an antibody specific for a 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 polypeptide.
  • the antibody can be any antibody or any antibody derivative described herein.
  • the invention provides non-human transgenic animals. Such animals are useful for studying the function and/or activity of a 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 protein and for identifying and/or evaluating modulators of 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 activity.
  • a "transgenic animal” is a non-human animal, preferably a mammal, more preferably a rodent such as a rat or mouse, in which one or more ofthe cells ofthe animal includes a transgene.
  • a transgenic animal can be one in which an endogenous 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 gene has been altered by, e.g, by homologous recombination between the endogenous gene and an exogenous DNA molecule introduced into a cell ofthe animal, e.g, an embryonic cell ofthe animal, prior to development ofthe animal.
  • Intronic sequences and polyadenylation signals can also be included in the transgene to increase the efficiency of expression ofthe transgene.
  • a tissue-specific regulatory sequence(s) can be operably linked to a transgene ofthe invention to direct expression of a 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 protein to particular cells.
  • a transgenic founder animal can be identified based upon the presence of a 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 transgene in its genome and/or expression of 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 mRNA in tissues or cells ofthe animals.
  • a transgenic founder animal can then be used to breed additional animals carrying the transgene.
  • proteins or polypeptides can be expressed in transgenic animals or plants, e.g, a nucleic acid encoding the protein or polypeptide can be introduced into the genome of an animal.
  • the nucleic acid is placed under the control of a tissue specific promoter, e.g, a milk or egg specific promoter, and recovered from the milk or eggs produced by the animal. Suitable animals are mice, pigs, cows, goats, and sheep.
  • the invention also includes a population of cells from a transgenic animal, as discussed, e.g, below.
  • the isolated nucleic acid molecules ofthe invention can be used, for example, to express a 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 protein (e.g, via a recombinant expression vector in a host cell in gene therapy applications), to detect a 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 mRNA (e.g, in a biological sample) or a genetic alteration in a 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 gene, and to modulate 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 activity, as described further below.
  • the 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 proteins can be used to treat disorders characterized by insufficient or excessive production of a 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 substrate or production of 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 inhibitors.
  • the 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 proteins can be used to screen for naturally occurring 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 substrates, to screen for drags or compounds which modulate 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 activity, as well as to treat disorders characterized by insufficient or excessive production of 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 protein or production of 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 protein forms which have decreased, aberrant or unwanted activity compared to 47476, 67210, 4
  • the anti-47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 antibodies of the invention can be used to detect and isolate 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 proteins, regulate the bioavailability of 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 proteins, and modulate 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 activity.
  • a method of evaluating a compound for the ability to interact with, e.g, bind, a subject 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 polypeptide is provided.
  • the method includes: contacting the compound with the subject 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 polypeptide; and evaluating ability ofthe compound to interact with, e.g, to bind or form a complex with the subject 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 polypeptide.
  • This method can be performed in vitro, e.g, in a cell free system, or in vivo, e.g, in a two-hybrid interaction trap assay.
  • This method can be used to identify naturally occurring molecules that interact with subject 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 polypeptide. It can also be used to find natural or synthetic inhibitors of subject 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 polypeptide. Screening methods are discussed in more detail below.
  • the invention provides methods (also referred to herein as "screening assays") for identifying modulators, i.e, candidate or test compounds or agents (e.g, proteins, peptides, peptidomimetics, peptoids, small molecules or other drags) which bind to 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 proteins, have a stimulatory or inhibitory effect on, for example, 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 expression or 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 activity, or have a stimulatory or inhibitory effect on, for example, the expression or activity of a 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708,
  • an activity of a 47476 protein can be assayed as follows: (a) introduce a 47476 necleic acid expression constract into a cell such that 47476 protein is produced; (b) activate a signal transduction pathway that utilizes a ras superfamily member that can be activated by a 47476 protein; and (c) evaluate the ability the 47476 protein or functional fragment thereof to modulate the activated signal transduction pathway, as compared to a control cell that lacks the 47476 expression constract. Assays for determining the activity level of a signal transduction pathway will depend on the particular signal transduction pathway.
  • an activity of a 49875 protein can be assayed as follows, (a) contact a nucleic acid, e.g, a nucleic acid duplex, with a 49875 protein or functional fragment thereof in the presence of an NTP, e.g, GTP or ATP, and (b) evaluate the ability ofthe 49875 protein or functional fragment thereof to cause the unwinding ofthe nucleic acid.
  • Assays for determining if a nucleic acid is wound or unwound are known in the art and include, e.g, gel electrophoresis.
  • an activity of a 46842 protein can be assayed as described in Kam et al. (2000), JBiol Chem 275:9653, or Dowler et al. (2000), Biochem J 351 :19, the contents of which are inco ⁇ orated herein by reference.
  • an activity of a 33201 protein can be assayed as follows: (a) contacting a cell that expresses a 33201 protein, or a fragment thereof, with a known substrate, e.g, an alcohol or quinone substrate; and (b) evaluate the ability the 33201 protein, or functional fragment thereof, to oxidize or reduce the substrate. Assays for determining whether a substrate has been oxidized or reduced are known in the art.
  • test compounds ofthe present invention can be obtained using any ofthe numerous approaches in combinatorial library methods known in the art, including: biological libraries; peptoid libraries (libraries of molecules having the functionalities of peptides, but with a novel, non-peptide backbone which are resistant to enzymatic degradation but which nevertheless remain bioactive; see, e.g, Zuckermann, R.N. et al. (1994) J Med. Chem. 37:2678-85); spatially addressable parallel solid phase or solution phase libraries; synthetic library methods requiring deconvolution; the 'one-bead one-compound' library method; and synthetic library methods using affinity chromatography selection.
  • the biological library and peptoid library approaches are limited to peptide libraries, while the other four approaches are applicable to peptide, non-peptide oligomer or small molecule libraries of compounds (Lam (1997) Anticancer Drug Des. 12:145).
  • an assay is a cell-based assay in which a cell which expresses a
  • 47476 protein or biologically active portion thereof is contacted with a test compound, and the ability ofthe test compound to modulate 47476 activity is determined. Determining the ability ofthe test compound to modulate 47476 activity can be accomplished, e.g, by monitoring the ability of 47476 polypeptide or proteins to activate one or more ras superfamily proteins, e.g, as measured by cellular characteristics at least partially controlled by ras superfamily members, e.g, cell shape, motility, growth, adhesion, or differentiation.
  • the cell for example, can be of mammalian origin, e.g, human.
  • an assay is a cell-based assay in which a cell which expresses a 49875 protein or biologically active portion thereof is contacted with a test compound, and the ability ofthe test compound to modulate 49875activity is determined. Determining the ability ofthe test compound to modulate 49875 activity can be accomplished, e.g, by monitoring the ability of 49875 molecules to unwind duplex nucleic acid molecules, e.g, as measured by changes in transcription, e.g, using a GFP construct and GFP expression as a readout, or changes in cellular behavior, e.g, changes in cell shape, motility, growth, adhesion, or differentiation.
  • the cell for example, can be of mammalian origin, e.g, human.
  • an assay is a cell-based assay in which a cell which expresses a 46842 protein or biologically active portion thereof is contacted with a test compound, and the ability ofthe test compound to modulate 46842 activity is determined. Determining the ability ofthe test compound to modulate 46842 activity can be accomplished, e.g, by monitoring the ability of 46842 molecules to stimulate the GTPase activity of an Arf or arf-like protein, e.g, as measured by changes in cell shape or protein trafficking, e.g, using an appropriate marker protein that is transported through the secretory pathway.
  • the cell for example, can be of mammalian origin, e.g, human.
  • an assay is a cell-based assay in which a cell which expresses a 83378, 84233, 64708, 85041, or 84234 protein or biologically active portion thereof is contacted with a test compound, and the ability ofthe test compound to modulate 83378, 84233, 64708, 85041, or 84234 activity is determined.
  • test compound to modulate 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 binding to a compound, e.g, a 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 substrate, or to bind to 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 can also be evaluated.
  • a compound e.g, a 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 substrate
  • a compound e.g, a 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 substrate
  • 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041 , or 84234 with or without the labeling of any of the interactants can be evaluated.
  • a microphysiometer can be used to detect the interaction of a compound with 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 without the labeling of either the compound or the 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234. McConnell, H. M. et al. (1992) Science 257:1906-1912.
  • a "microphysiometer” e.g, Cytosensor
  • LAPS light-addressable potentiometric sensor
  • Soluble and/or membrane-bound forms of isolated proteins can be used in the cell-free assays ofthe invention.
  • membrane-bound forms of the protein it may be desirable to utilize a solubilizing agent.
  • solubilizing agents include non-ionic detergents such as n-octylglucoside, n-dodecylglucoside, n-dodecylmaltoside, octanoyl-N-methylglucamide, decanoyl-N-methylglucamide, Triton® X-
  • Cell-free assays involve preparing a reaction mixture ofthe target gene protein and the test compound under conditions and for a time sufficient to allow the two components to interact and bind, thus forming a complex that can be removed and/or detected.
  • the interaction between two molecules can also be detected, e.g, using fluorescence energy transfer (FET) (see, for example, Lakowicz et al, U.S. Patent No. 5,631,169; Stavrianopoulos, et al, U.S. Patent No. 4,868,103).
  • FET fluorescence energy transfer
  • a fluorophore label on the first, 'donor' molecule is selected such that its emitted fluorescent energy will be absorbed by a fluorescent label on a second, 'acceptor' molecule, which in turn is able to fluoresce due to the absorbed energy.
  • the 'donor' protein molecule may simply utilize the natural fluorescent energy of tryptophan residues.
  • Labels are chosen that emit different wavelengths of light, such that the 'acceptor' molecule label may be differentiated from that ofthe 'donor'. Since the efficiency of energy transfer between the labels is related to the distance separating the molecules, the spatial relationship between the molecules can be assessed. In a situation in which binding occurs between the molecules, the fluorescent emission ofthe 'acceptor' molecule label in the assay should be maximal.
  • An FET binding event can be conveniently measured through standard fluorometric detection means well known in the art (e.g, using a fluorimeter). In another embodiment, determining the ability ofthe 47476, 67210, 49875, 46842,
  • 33201, 83378, 84233, 64708, 85041, or 84234 protein to bind to a target molecule can be accomplished using real-time Biomolecular Interaction Analysis (BIA) (see, e.g, Sjolander, S. and Urbaniczky, C. (1991) Anal. Chem. 63:2338-2345 and Szabo et al. (1995) Curr. Opin. Struct. Biol. 5:699-705).
  • Biomolecular Interaction Analysis see, e.g, Sjolander, S. and Urbaniczky, C. (1991) Anal. Chem. 63:2338-2345 and Szabo et al. (1995) Curr. Opin. Struct. Biol. 5:699-705.
  • "Surface plasmon resonance" or "BIA" detects biospecific interactions in real time, without labeling any ofthe interactants (e.g, BIAcore).
  • the target gene product or the test substance is anchored onto a solid phase.
  • the target gene product/test compound complexes anchored on the solid phase can be detected at the end ofthe reaction.
  • the target gene product can be anchored onto a solid surface, and the test compound, (which is not anchored), can be labeled, either directly or indirectly, with detectable labels discussed herein.
  • Binding of a test compound to a 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 protein, or interaction of a 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 protein with a target molecule in the presence and absence of a candidate compound can be accomplished in any vessel suitable for containing the reactants. Examples of such vessels include microtiter plates, test tubes, and micro-centrifuge tubes.
  • a fusion protein can be provided which adds a domain that allows one or both ofthe proteins to be bound to a matrix.
  • glutafhione-S- transferase/47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 fusion proteins or glutathione-S-transferase/target fusion proteins can be adsorbed onto glutathione sepharose beads (Sigma Chemical, St. Louis, MO) or glutathione derivatized microtiter plates, which are then combined with the test compound or the test compound and either the non- adsorbed target protein or 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 protein, and the mixture incubated under conditions conducive to complex formation
  • the beads or microtiter plate wells are washed to remove any unbound components, the matrix immobilized in the case of beads, complex determined either directly or indirectly, for example, as described above.
  • the complexes can be dissociated from the matrix, and the level of 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 binding or activity determined using standard techniques.
  • the non-immobilized component is added to the coated surface containing the anchored component. After the reaction is complete, unreacted components are removed (e.g, by washing) under conditions such that any complexes formed will remain immobilized on the solid surface.
  • the detection of complexes anchored on the solid surface can be accomplished in a number of ways. Where the previously non-immobilized component is pre-labeled, the detection of label immobilized on the surface indicates that complexes were formed.
  • an indirect label can be used to detect complexes anchored on the surface; e.g, using a labeled antibody specific for the immobilized component (the antibody, in turn, can be directly labeled or indirectly labeled with, e.g, a labeled anti-Ig antibody).
  • this assay is performed utilizing antibodies reactive with 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 protein or target molecules but which do not interfere with binding ofthe 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 protein to its target molecule.
  • Such antibodies can be derivatized to the wells ofthe plate, and unbound target or 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 protein trapped in the wells by antibody conjugation.
  • Methods for detecting such complexes include immunodetection of complexes using antibodies reactive with the 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 protein or target molecule, as well as enzyme-linked assays which rely on detecting an enzymatic activity associated with the 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 protein or target molecule.
  • cell free assays can be conducted in a liquid phase.
  • the reaction products are separated from unreacted components, by any of a number of standard techniques, including but not limited to: differential centrifugation (see, for example, Rivas, G, and Minton, A.P, (1993) Trends Biochem Sci 18:284-7); chromatography (gel filtration chromatography, ion-exchange chromatography); electrophoresis (see, e.g, Ausubel, F. et al, eds. Current Protocols in Molecular Biology 1999, J. Wiley: New York.); and immunoprecipitation (see, for example, Ausubel, F. et al, eds. (1999) Current Protocols in Molecular Biology, J. Wiley: New York).
  • differential centrifugation see, for example, Rivas, G, and Minton, A.P, (1993) Trends Biochem Sci 18:284-7
  • chromatography gel filtration chromatography, ion-exchange chromatography
  • electrophoresis see, e.g
  • the assay includes contacting the 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 protein or biologically active portion thereof with a known compound which binds 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 to form an assay mixture, contacting the assay mixture with a test compound, and determining the ability of the test compound to interact with a 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 protein, wherein determining the ability ofthe test compound to interact with a 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 protein includes determining the ability ofthe test compound to preferentially bind to 47476, 67210, 49875, 46842, 33
  • 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 protein through modulation ofthe activity of a downstream effector of a 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 target molecule.
  • the activity ofthe effector molecule on an appropriate target can be determined, or the binding ofthe effector to an appropriate target can be determined, as previously described.
  • a reaction mixture containing the target gene product and the binding partner is prepared, under conditions and for a time sufficient, to allow the two products to form complex.
  • the reaction mixture is provided in the presence and absence ofthe test compound.
  • the test compound can be initially included in the reaction mixture, or can be added at a time subsequent to the addition ofthe target gene and its cellular or extracellular binding partner.
  • Control reaction mixtures are incubated without the test compound or with a placebo.
  • the formation of any complexes between the target gene product and the cellular or extracellular binding partner is then detected.
  • the formation of a complex in the control reaction, but not in the reaction mixture containing the test compound indicates that the compound interferes with the interaction ofthe target gene product and the interactive binding partner.
  • complex formation within reaction mixtures containing the test compound and normal target gene product can also be compared to complex formation within reaction mixtures containing the test compound and mutant target gene product. This comparison can be important in those cases wherein it is desirable to identify compounds that disrapt interactions of mutant but not normal target gene products.
  • either the target gene product or the interactive cellular or extracellular binding partner is anchored onto a solid surface (e.g, a microtiter plate), while the non-anchored species is labeled, either directly or indirectly.
  • the anchored species can be immobilized by non-covalent or covalent attachments.
  • an immobilized antibody specific for the species to be anchored can be used to anchor the species to the solid surface.
  • the partner ofthe immobilized species is exposed to the coated surface with or without the test compound. After the reaction is complete, unreacted components are removed (e.g, by washing) and any complexes formed will remain immobilized on the solid surface.
  • a preformed complex ofthe target gene product and the interactive cellular or extracellular binding partner product is prepared in that either the target gene products or their binding partners are labeled, but the signal generated by the label is quenched due to complex formation (see, e.g, U.S. Patent No. 4,109,496 that utilizes this approach for immunoassays).
  • the addition of a test substance that competes with and displaces one ofthe species from the preformed complex will result in the generation of a signal above background. In this way, test substances that disrupt target gene product-binding partner interaction can be identified.
  • the 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 proteins can be used as "bait proteins" in a two-hybrid assay or three-hybrid assay (see, e.g, U.S. Patent No. 5,283,317; Zervos et al. (1993) Cell 72:223-232; Madura et al. (1993) J. Biol. Chem. 268:12046-12054; Bartel et al. (1993) Biotechniques 14:920-924; Iwabuchi et al.
  • Such 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234-bps can be activators or inhibitors of signals by the 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 proteins or 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 targets as, for example, downstream elements of a 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234-mediated signaling pathway.
  • the two-hybrid system is based on the modular nature of most transcription factors, which consist of separable DNA-binding and activation domains.
  • the assay utilizes two different DNA constructs.
  • the gene that codes for a 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 protein is fused to a gene encoding the DNA binding domain of a known transcription factor (e.g, GAL-4).
  • a DNA sequence, from a library of DNA sequences, that encodes an unidentified protein (“prey" or "sample" is fused to a gene that codes for the activation domain ofthe known transcription factor.
  • modulators of 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 expression are identified.
  • a cell or cell free mixture is contacted with a candidate compound and the expression of 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 mRNA or protein evaluated relative to the level of expression of 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 mRNA or protein in the absence ofthe candidate compound.
  • the candidate compound When expression of 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 mRNA or protein is greater in the presence ofthe candidate compound than in its absence, the candidate compound is identified as a stimulator of 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 mRNA or protein expression.
  • the level of 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 mRNA or protein expression can be determined by methods described herein for detecting 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 mRNA or protein.
  • This invention further pertains to novel agents identified by the above-described screening assays. Accordingly, it is within the scope of this invention to further use an agent identified as described herein (e.g, a 47476, 67210, 49875, 46842, 33201, 83378, 84233,
  • nucleic acid sequences identified herein can be used as polynucleotide reagents.
  • these sequences can be used to: (i) map their respective genes on a chromosome e.g, to locate gene regions associated with genetic disease or to associate 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 with a disease; (ii) identify an individual from a minute biological sample (tissue typing); and (iii) aid in forensic identification of a biological sample.
  • FISH Fluorescence in situ hybridization
  • clones larger than 1,000 bases have a higher likelihood of binding to a unique chromosomal location with sufficient signal intensity for simple detection.
  • 1,000 bases, and more preferably 2,000 bases will suffice to get good results at a reasonable amount of time.
  • Reagents for chromosome mapping can be used individually to mark a single chromosome or a single site on that chromosome, or panels of reagents can be used for marking multiple sites and/or multiple chromosomes. Reagents corresponding to noncoding regions of the genes actually are preferred for mapping purposes. Coding sequences are more likely to be conserved within gene families, thus increasing the chance of cross hybridizations during chromosomal mapping.
  • differences in the DNA sequences between individuals affected and unaffected with a disease associated with the 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 gene can be determined. If a mutation is observed in some or all ofthe affected individuals but not in any unaffected individuals, then the mutation is likely to be the causative agent ofthe particular disease. Comparison of affected and unaffected individuals generally involves first looking for structural alterations in the chromosomes, such as deletions or translocations that are visible from chromosome spreads or detectable using PCR based on that DNA sequence. Ultimately, complete sequencing of genes from several individuals can be performed to confirm the presence of a mutation and to distinguish mutations from polymorphisms.
  • RFLP restriction fragment length polymorphism
  • sequences ofthe present invention can also be used to determine the actual base-by-base DNA sequence of selected portions of an individual's genome.
  • the 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 nucleotide sequences described herein can be used to prepare two PCR primers from the 5' and 3' ends of the sequences. These primers can then be used to amplify an individual's DNA and subsequently sequence it. Panels of corresponding DNA sequences from individuals, prepared in this manner, can provide unique individual identifications, as each individual will have a unique set of such DNA sequences due to allelic differences.
  • allelic variation occurs to some degree in the coding regions of these sequences, and to a greater degree in the noncoding regions.
  • Each ofthe sequences described herein can, to some degree, be used as a standard against which DNA from an individual can be compared for identification purposes. Because greater numbers of polymorphisms occur in the noncoding regions, fewer sequences are necessary to differentiate individuals.
  • the noncoding sequences of SEQ ID NO:l, SEQ ID NO:4, SEQ ID NO:7, SEQ ID NO: 10, SEQ ID NO: 13, SEQ ID NO:16, SEQ ID NO: 19, SEQ ID NO:22, SEQ ID NO:25, or SEQ ID NO:28 can provide positive individual identification with a panel of perhaps 10 to 1,000 primers which each yield a noncoding amplified sequence of 100 bases.
  • a panel of reagents from 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 nucleotide sequences described herein is used to generate a unique identification database for an individual, those same reagents can later be used to identify tissue from that individual.
  • positive identification ofthe individual, living or dead can be made from extremely small tissue samples.
  • DNA-based identification techniques can also be used in forensic biology.
  • PCR technology can be used to amplify DNA sequences taken from very small biological samples such as tissues, e.g, hair or skin, or body fluids, e.g, blood, saliva, or semen found at a crime scene. The amplified sequence can then be compared to a standard, thereby allowing identification ofthe origin ofthe biological sample.
  • sequences ofthe present invention can be used to provide polynucleotide reagents, e.g, PCR primers, targeted to specific loci in the human genome, which can enhance the reliability of DNA-based forensic identifications by, for example, providing another "identification marker" (i.e. another DNA sequence that is unique to a particular individual).
  • another "identification marker” i.e. another DNA sequence that is unique to a particular individual.
  • actual base sequence information can be used for identification as an accurate alternative to patterns formed by restriction enzyme generated fragments.
  • Sequences targeted to noncoding regions of SEQ ID NO:l, SEQ ID NO:4, SEQ ID NO:7, SEQ ID NO:10, SEQ ID NO:13, SEQ ID NO: 16, SEQ ID NO:19, SEQ ID NO:22, SEQ ID NO:25, or SEQ ID NO:28 e.g, fragments derived from the noncoding regions of SEQ ID NO:l, SEQ ID NO:4, SEQ ID NO:7, SEQ ID NO: 10, SEQ ID NO:13, SEQ ID NO:16, SEQ ID NO:19, SEQ ID NO:22, SEQ ID NO:25, or SEQ ID NO:28 having a length of at least 20 bases, preferably at least 30 bases) are particularly appropriate for this use.
  • primers or probes can be used to screen tissue culture for contamination (i.e. screen for the presence of a mixture of different types of cells in a culture).
  • the present invention also pertains to the field of predictive medicine in which diagnostic assays, prognostic assays, and monitoring clinical trials are used for prognostic
  • the invention provides, a method of determining if a subject is at risk for a disorder related to a lesion in or the misexpression of a gene which encodes 47476, 67210,
  • disorders include, e.g, a disorder associated with the misexpression of 47476,
  • the method includes one or more ofthe following: detecting, in a tissue ofthe subject, the presence or absence of a mutation which affects the expression ofthe 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or
  • the method includes: ascertaining the existence of at least one of: a deletion of one or more nucleotides from the 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 gene; an insertion of one or more nucleotides into the gene, a point mutation, e.g, a substitution of one or more nucleotides ofthe gene, a gross chromosomal reanangement ofthe gene, e.g, a translocation, inversion, or deletion.
  • detecting the genetic lesion can include: (i) providing a probe/primer including an oligonucleotide containing a region of nucleotide sequence which hybridizes to a sense or antisense sequence from SEQ ID NO:l, SEQ ID NO:4, SEQ ID NO:7, SEQ ID NO: 10, SEQ ID NO:13, SEQ ID NO: 16, SEQ ID NO:19, SEQ ID NO:22, SEQ ID NO:25, or SEQ ID NO:28, or naturally occurring mutants thereof or 5' or 3' flanking sequences naturally associated with the 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 gene; (ii) exposing the probe/primer to nucleic acid ofthe tissue; and detecting, by hybridization, e.g, in situ hybridization, ofthe probe/primer to the nucleic acid, the presence or absence ofthe genetic lesion.
  • a probe/primer including an
  • detecting the misexpression includes ascertaining the existence of at least one of: an alteration in the level of a messenger RNA transcript of the
  • Methods ofthe invention can be used prenatally or to determine if a subject's offspring will be at risk for a disorder.
  • the method includes determining the structure of a 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 gene, an abnormal structure being indicative of risk for the disorder.
  • the method includes contacting a sample from the subject with an antibody to the 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 protein or a nucleic acid, which hybridizes specifically with the gene.
  • Diagnostic and prognostic assays ofthe invention include method for assessing the expression level of 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 molecules and for identifying variations and mutations in the sequence of 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 molecules.
  • the presence, level, or absence of 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 protein or nucleic acid in a biological sample can be evaluated by obtaining a biological sample from a test subject and contacting the biological sample with a compound or an agent capable of detecting 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 protein or nucleic acid (e.g, mRNA, genomic DNA) that encodes 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 protein such that the presence of 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 protein or nucleic acid is detected in the biological sample.
  • biological sample includes tissues, cells and biological fluids isolated from a subject, as well as tissues, cells and fluids present within a subject.
  • a preferred biological sample is serum.
  • the level of expression ofthe 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 gene can be measured in a number of ways, including, but not limited to: measuring the mRNA encoded by the 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 genes; measuring the amount of protein encoded by the 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 genes; or measuring the activity ofthe protein encoded by the 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 genes.
  • the isolated mRNA can be used in hybridization or amplification assays that include, but are not limited to, Southern or Northern analyses, polymerase chain reaction analyses and probe arrays.
  • One preferred diagnostic method for the detection of mRNA levels involves contacting the isolated mRNA with a nucleic acid molecule (probe) that can hybridize to the mRNA encoded by the gene being detected.
  • the nucleic acid probe can be, for example, a full- length 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 nucleic acid, such as the nucleic acid of SEQ ID NO:l, SEQ ID NO:4, SEQ ID NO:7, SEQ ID NO:10, SEQ ID NO:13, SEQ ID NO:16, SEQ ID NO:19, SEQ ID NO:22, SEQ ID NO:25, or SEQ ID NO:28, or a portion thereof, such as an oligonucleotide of at least 7, 15, 30, 50, 100, 250 or 500 nucleotides in length and sufficient to specifically hybridize under stringent conditions to 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 mRNA or genomic DNA.
  • the probe can be disposed on an address of an array, e.g, an array described below.
  • mRNA (or cDNA) is immobilized on a surface and contacted with the probes, for example by running the isolated mRNA on an agarose gel and transfening the mRNA from the gel to a membrane, such as nitrocellulose.
  • the probes are immobilized on a surface and the mRNA (or cDNA) is contacted with the probes, for example, in a two-dimensional gene chip array described below.
  • a skilled artisan can adapt known mRNA detection methods for use in detecting the level of mRNA encoded by the 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 genes.
  • the level of mRNA in a sample that is encoded by one of 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 can be evaluated with nucleic acid amplification, e.g, by rtPCR (Mullis (1987) U.S. Patent No. 4,683,202), ligase chain reaction (Barany (1991) Proc. Natl. Acad. Sci. USA 88:189-193), self sustained sequence replication (Guatelli et al, (1990) Proc. Natl. Acad. Sci. USA 87:1874-1878), transcriptional amplification system (Kwoh et al, (1989), Proc. Natl.
  • amplification primers are defined as being a pair of nucleic acid molecules that can anneal to 5' or 3' regions of a gene (plus and minus strands, respectively, or vice-versa) and contain a short region in between.
  • amplification primers are from about 10 to 30 nucleotides in length and flank a region from about 50 to 200 nucleotides in length. Under appropriate conditions and with appropriate reagents, such primers permit the amplification of a nucleic acid molecule comprising the nucleotide sequence flanked by the primers.
  • a cell or tissue sample can be prepared/processed and immobilized on a support, typically a glass slide, and then contacted with a probe that can hybridize to mRNA that encodes the 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 gene being analyzed.
  • the methods further contacting a control sample with a compound or agent capable of detecting 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 mRNA, or genomic DNA, and comparing the presence of 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 mRNA or genomic DNA in the control sample with the presence of 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 mRNA or genomic DNA in the test sample.
  • serial analysis of gene expression as described in U.S. Patent No. 5,695,937, is used to detect 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 transcript levels.
  • a variety of methods can be used to determine the level of protein encoded by 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234.
  • these methods include contacting an agent that selectively binds to the protein, such as an antibody with a sample, to evaluate the level of protein in the sample.
  • the antibody bears a detectable label.
  • Antibodies can be polyclonal, or more preferably, monoclonal. An intact antibody, or a fragment thereof (e.g. Fab or F(ab')2) can be used.
  • labeling with regard to the probe or antibody, is intended to encompass direct labeling ofthe probe or antibody by coupling (i.e, physically linking) a detectable substance to the probe or antibody, as well as indirect labeling ofthe probe or antibody by reactivity with a detectable substance. Examples of detectable substances are provided herein.
  • the detection methods can be used to detect 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 protein in a biological sample in vitro as well as in vivo.
  • In vivo techniques for detection of 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 protein include introducing into a subject a labeled anti-47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 antibody.
  • the antibody can be labeled with a radioactive marker whose presence and location in a subject can be detected by standard imaging techniques.

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • Zoology (AREA)
  • Genetics & Genomics (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Engineering & Computer Science (AREA)
  • Wood Science & Technology (AREA)
  • General Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Biochemistry (AREA)
  • Molecular Biology (AREA)
  • Biotechnology (AREA)
  • General Engineering & Computer Science (AREA)
  • Biomedical Technology (AREA)
  • Microbiology (AREA)
  • Toxicology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Biophysics (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Peptides Or Proteins (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)

Abstract

The invention provides isolated nucleic acids molecules, designated 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, and 84234 nucleic acid molecules, which encode novel Guanine Nucleotide Dissociation Stimulator, Glycosyltransferase, DEAD type helicase, Centaurin, Dehydrogenase/Reductase, and Metal Transporter family members. The invention also provides antisense nucleic acid molecules, recombinant expression vectors containing 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 nucleic acid molecules, host cells nt which the expression vectors have been introduced, and nonhuman transgenic animals in which a 474476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 gene has been introduced or disrupted. The invention still further provides isolated 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 proteins, fusion proteins, antigenic peptides and anti-47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 antibodies. Diagnostic methods utilizing compositions of the invention are also provided.

Description

47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, AND 84234, NOVEL HUMAN GUANLNE NUCLEOTIDE DISSOCIATION STIMULATOR, GLYCOSYLTRANSFERASE, DEAD TYPE HELICASE, CENTAURIN, DEHYDROGENASE/REDUCTASE, AND METAL TRANSPORTER
FAMILY MEMBERS AND USES THEREOF
Related Applications This application claims priority to U.S. provisional applications: 60/248,362, 60/248,331, and 60/248,365, all filed on November 14, 2000; and 60/250,077, 60/250,327, and 60/250,176, all filed on November 30, 2000. The contents of each of these provisional applications are incorporated herein by reference.
Background of the Invention
Guanine Nucleoside Dissociation Stimulators Ras and ras-like proteins are membrane-associated molecular switches that bind and slowly hydrolyze GTP to GDP. Cell-surface receptors that signal through tyrosine kinases activate ras and/or ras-like proteins by stimulating a guanine nucleotide exchange reaction. In the case of ras, genetic and biochemical studies have indicated that this exchange reaction is controlled by the ras guanine nucleotide exchange factor Son of sevenless (SOS). The balance between the GTP bound (active) and GDP bound (inactive) states of ras or a ras-like protein is regulated by the opposing action of proteins that activate the GTPase activity and proteins that promote the loss of bound GDP and the uptake of fresh GTP. The former proteins are GTPase-activating proteins, and the latter proteins are guanine nucleotide dissociation stimulators (GDSs) (or also as guanine nucleotide releasing (or exchange) factors (GRFs or GEFs)).
Proteins that act as GDSs can be classified into at least two families, CDC24 or CDC25, on the basis of sequence similarities. The size of the proteins in the CDC25 family range from 309 residues (LTE1) to 1596 residues (SOS). The sequence similarity shared by all GDS proteins is limited to a region of about 250 amino acids generally located in the C-terminal section (currently, the only exceptions are SOS and ralGDS, in which this domain makes up the central portion of the protein). This 250 amino acid domain has been shown to be essential for the activity of GDS proteins.
Glycosyltransferases Glycosyltransferases catalyze the synthesis of glycoconjugates, including glycolipids, glycoproteins, and polysaccharides, by transferring an activated mono- or oligosaccharide residue to an existing acceptor molecule for the initiation or elongation of the carbohydrate chain. A catalytic reaction is believed to involve the recognition of both the donor and acceptor by suitable domains, as well as the catalytic site of the enzyme (Amado et al. (1999) Biochim Biophys Ada 1473:35-53; Kapitonov et al. (1999) Glycobiology 9:961-78). Evidence indicates that formation of glycosidic linkages is covered by large homologous glycosyltransferase gene families, and that the existence of multiple enzyme isoforms provides a degree of redundancy as well as a higher level of regulation (Kapitonov et al. (1999), supra).
Glycosylation is the principal chemical modification to proteins as they pass through Golgi vesicles. Glycosyltransferases of the Golgi do not possess an obvious sequence homology which would suggest a common Golgi retention signal. However, they are all membrane proteins and share type II topology, consisting of an amino terminal cytoplasmic tail, a signal anchor transmembrane domain, a stem region, and a large luminal catalytic domain. The membrane-spanning domain and its flanking regions contain necessary and sufficient information for Golgi retention of these enzymes (Jaskiewicz (1997) Ada Biochim Pol 44:173- 9). ER localized glycosyltransferases can have either a type II topology, like the Golgi glycosyltransferases, or a type I topolgy, i.e., the N-terminus and catalytic domain reside in the lumen of the ER (Kapitonov et al. (1999), supra). Some glycosyltransferases are present on the cell surface and are thought to function as cell adhesion molecules by binding oligosaccharide substrates on adjacent cell surfaces or in the extracellular matrix. The best studied of these is beta 1,4-galactosyltransferase, which mediates sperm binding to the egg coat and selected cell interactions with the basal lamina (Shur (1993) Curr Opin Cell Biol 5:854-63).
DEAD Type Helicases A large number of biological processes require the unwinding from double-stranded or base-paired regions of DNA/DNA, RNA/RNA or RNA/DNA hybrids to single-stranded polynucleotides. These complex reactions are dependent on helicases, mechanochemical enzymes that couple the energy of nucleoside triphosphate hydrolysis to the dehybridization or unwinding of duplex nucleic acid molecules. Helicases comprise a large number of proteins that share high sequence similarity (Aubourg et al. (1999) Nucleic Acids Res 27(2):628-36). A sequence based classification has led to the definition of three superfamilies of helicases, namely SF1, SF2 and SF3 (Gorbalenya and Koonin (1993) Curr Opin Struct Biol 3:419-429). To date, SF2 is the best characterized superfamily, which includes the DEAD and DEAH box ("DEAD/H") helicases.
The DEAD box RNA helicase family has been defined by Linder et al. (1989), Nature 337:121-122, and named according to the highly conserved residues, Asp-Glu- Ala- Asp (D-E- A-D) motif. DEAD box RNA helicases differ mainly by the addition of N- and C-terminal sequences containing different targeting signals, RNA-binding motifs, and regions required for interactions with structural or regulatory proteins. Aubourg et al. (1999), supra.
Notwithstanding their shared nucleic acid unwinding activity, helicases are involved in a number of different molecular mechanisms, including viral replication, RNA splicing, ribosome assembly, and initiation of translation including transcription regulation (e.g., SNF2, STH1, brahma, MOT1), maintenance of chromosome stability during mitosis (e.g., lodestar), and various aspects of processing DNA damage, including DNA excision repair (e.g., RAD16 and ERCC6), recombinational pathways (e.g., RAD54) and post-replication daughter strand gap repair (e.g., RAD5) (Eisen et al. (1995) Nucleic Acid Res 23:2715-23; Schmid and Linder (1992) MolMicrobiol 6:283-292; and Stevenson et al. (1998) JPathol 184:351-359). Thus, helicases are important, inter alia, in replication, regulation of transcription, and cellular growth and differentiation.
Centaurins
Centaurin proteins are a family of regulatory proteins that control the small GTPase Arf. One subclass of centaurins, the centaurin-g proteins, typically has at least three types of canonical protein domains: a plekstrin homology (PH) domain, an Arf GAP domain, and ankyrin domains (for a review, see Jackson et al. (2000) Trends. Biochem. Set.25:489). Arf proteins are small guanine nucleotide binding proteins that regulate vesicular trafficking and the cytoskeleton. ArfGAP domains stimulate the intrinsic GTPase activity of Arf such that GTP bound to Arf is hydrolyzed to GDP. Centaurin proteins are implicated in the regulation of vesicle trafficking, and the actin cytoskeleton, such as in focal adhesions and membrane ruffling. As pivotal regulatory molecules in cell signalling networks, centaurins are potential modulators of cell motility, cell adhesion, and secretory events. Such physiological functions are commonly aberrant or perturbed in a number of diseases and pathologies.
Dehydrogenases/Reductases
The superfamily of dehydrogenases and reductases is comprised of numerous members, including dehydrogenase/reductases and quinone oxidoreductases. When members of the superfamily are aligned, only a few typically critical amino acid residues are conserved among the various proteins.
Humans possess nine variants of alcohol dehydrogenase/reductases (ADH). Dehydrogenase/reductases play fundamental roles in degradative, synthetic, and detoxification pathways. These enzymes catalyze the reversible oxidation of ethanol to acetaldehyde with the concomitant reduction of NAD. Some vertebrate ADHs metabolize alcohols other than ethanol, such as retinol (vitamin A). The ability of some ADHs to act as retinol dehydrogenases suggests that they may participate in the synthesis of retinoic acid, the active form of vitamin A involved in regulating cellular differentiation and embryonic development (Zgombic-Knoght et al. (1995) J. Biol. Chem. 270:10868-10877). Dehydrogenase/reductases have been implicated in a variety of developmental processes and pathophysiological disease states. For example, allelic variations of ADH2 and ADH3 appear to influence the susceptibility to alcoholism and alcoholic liver cirrhosis in Asians (Thomasson et al. (1991) Am. J. Hum Genet. 48:677-681, Chao et al. (1994) Hepatology 19:360-366, and Higuchi et al. (1995) Am. J. Psychiatry 152:1219-1221). Furthermore, several lines of evidence indicate that first-pass metabolism of alcohol in humans may occur in the liver via the activity of members of the mammalian ADH family. First-pass metabolism is the difference between the quantity of ethanol that reaches the systemic circulation by the intravenous route and the quantity that reaches the system circulation by the oral route (Yin et al. (1999) Enzymology and Molecular Biology of Carbonyl Metabolism 7, Plenum Publishers, New York). A role for dehydrogenase/reductases has also been proposed in the etiology of Parkinson's disease (Buervenich et al. (2000) Mov. Disord. 15:813-818). Quinone oxidoreductases catalyze the reduction of quinones. Members of this class of enzymes can contribute to the antitumor effects of certain bioreductive drugs by metabolizing anti-tumor quinones, thereby activating their cytotoxic effects (Fitzsimmons et al. (1996) J. Natl. Cancer Inst. 88:259-269).
Metal Transporters
The concentration of metallic ions such as cadmium, zinc, and cobalt is maintained within a narrow range in mammalian cells. A diverse family of metal ion transporter proteins, the "cation diffusion facilitator" family, contributes to the maintenance of cellular metallic ion homeostais (Paulsen et al. (1997) J. Membr. Biol 156:99-103).
Zinc is an essential component of metalloenzymes, transcription factors, and other proteins, but can be toxic to mammalian cells at high concentrations. When intracellular zinc exceeds a certain concentration, it is thought to trigger regulatory cellular processes. Various homeostatic mechanisms are thought to be used by cells to regulate intracellular zinc: regulation of zinc influx across the plasma membrane; regulation of zinc efflux across the plasma membrane; sequestration of zinc within subcellular compartments; and synthesis of molecules, e.g., metallothioneins, that bind tightly to zinc (Palmiter et al. (1996) EMBO J. 15:1784-1791; Palmiter et al. (1996) Proc. Natl. Acad. Sci. USA 93:14934-14939).
The genes encoding several zinc transporters have been cloned. Each of the proteins encoded by these genes appears to contribute to cellular resistance to zinc toxicity. Zinc transporter- 1 (ZnT-1) encodes a plasma membrane protein that stimulates zinc efflux. ZnT-1 appears to be activated by excess cellular zinc concentrations (Palmiter et al. (1995) EMBO J. 14:639-649). Zinc transporter-2 (ZnT-2) encodes a vesicular protein that promotes the vesicular sequestration of zinc. Thus, ZnT-2 appears to help protect cells from zinc toxicity by facilitating zinc transport into an endosomal/lysosomal compartment (Palmiter et al. (1996),
EMBOJ. 15:1784-1791). Zinc transporter-3 (ZnT-3) encodes a putative transporter of zinc into synaptic vesicles. ZnT-3, which is expressed in the brain and testis, is proposed to be a component of the complex that sequesters zinc in synaptic vesicles, thereby serving as a neuromodulator (Palmiter et al. (1996) Proc. Natl. Acad. Sci. USA 93:14934-14939). ZnT-1, ZnT-2, and ZnT-3 share a common topology characterized by six membrane-spanning domains, a histidine-rich cytoplasmic loop between membrane spanning regions four and five, and a long C-terminal tail.
Guanine nucleoside dissociation stimulators, glycosyltransferases, DEAD type helicases, centaurins, dehydrogenases/reductases, and metal transporters have all been implicated in human disease. Consequently, the isolation and characterization of additional guanine nucleoside dissociation stimulators, glycosyltransferases, DEAD type helicases, centaurins, dehydrogenases/reductases, and metal transporters will provide novel reagents for the treatment or prevention of disease, as well as new targets for the development of drugs that can be used to treat or prevent disease.
Summary of the Invention
The present invention is based, in part, on the discovery of novel Guanine Nucleotide Dissociation Stimulator, Glycosyltransferase, DEAD Type Helicase, Centaurin, Dehydrogenase/Reductase, and Metal Transporter family members, referred to herein as "47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, and 84234". The nucleotide sequences of cDNAs encoding 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, and 84234 are shown in SEQ ID NO:l, SEQ ID NO:4, SEQ ID NO:7, SEQ ID NO: 10, SEQ ID NO: 13, SEQ ID NO: 16, SEQ ID NO: 19, SEQ ID NO:22, SEQ ID NO:25, and SEQ ID NO:28,respectively, and the amino acid sequences of a 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, and 84234 polypeptide is shown in SEQ ID NO:2, SEQ ID NO:5, SEQ ID NO:8, SEQ ID NO:l 1, SEQ ID NO: 14, SEQ ID NO: 17, SEQ ID NO:20, SEQ ID NO:23, SEQ ID NO:26, and SEQ ID NO:29, respectively. In addition, the nucleotide sequences of the coding regions are depicted in SEQ ID NO:3, SEQ ID NO:6, SEQ ID NO:9, SEQ ID NO: 12, SEQ ID NO: 15, SEQ ID NO: 18, SEQ ID NO:21, SEQ ID NO:24, SEQ ID NO:27, and SEQ ID NO:30, respectively.
Accordingly, in one aspect, the invention features a nucleic acid molecule that encodes a 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 protein or polypeptide, e.g., a biologically active portion of the 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 protein. In a preferred embodiment the isolated nucleic acid molecule encodes a polypeptide having the amino acid sequence of SEQ ID NO:2, SEQ ID NO:5, SEQ ID NO:8, SEQ ID NO:l 1, SEQ ID NO: 14, SEQ ID NO: 17, SEQ ID NO:20, SEQ ID NO:23, SEQ ID NO:26, or SEQ ID NO:29. In other embodiments, the invention provides isolated 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 nucleic acid molecules having the nucleotide sequence shown in SEQ ID NO:l, SEQ ID NO:4, SEQ ID NO:7, SEQ ID NO: 10, SEQ ID NO-13, SEQ ID NO:16, SEQ ID NO:19, SEQ ID NO:22, SEQ ID NO:25, or SEQ ID NO:28, having the nucleotide sequence shown in SEQ ID NO:3, SEQ ID NO:6, SEQ ID NO:9, SEQ ID NO: 12, SEQ ID NO: 15, SEQ ID NO: 18, SEQ ID NO:21, SEQ ID NO:24, SEQ ID NO:27, or SEQ ID NO:30, or having the nucleotide sequence of the DNA insert of a plasmid deposited with an ATCC Accession Number as described herein. In still other embodiments, the invention provides nucleic acid molecules that are substantially identical (e.g., naturally occurring allelic variants) to the nucleotide sequence shown in SEQ ID NO:l, SEQ ID NO:4, SEQ ID NO:7, SEQ ID NO:10, SEQ ID NO:13, SEQ ID NO:16, SEQ ID NO: 19, SEQ ID NO:22, SEQ ID NO:25, and SEQ ID NO:28, the nucleotide sequence shown in SEQ ID NO:3, SEQ ID NO:6, SEQ ID NO:9, SEQ ID NO: 12, SEQ ID NO:15, SEQ ID NO: 18, SEQ ID NO:21, SEQ ID NO:24, SEQ ID NO:27, or SEQ ID NO:30, or the nucleotide sequence of the DNA insert of a plasmid deposited with an ATCC Accession Number as described herein. In other embodiments, the invention provides a nucleic acid molecule which hybridizes under a stringency condition described herein to a nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO:l, SEQ ID NO:4, SEQ ID NO:7, SEQ ID NO:10, SEQ ID NO:13, SEQ ID NO:16, SEQ ID NO:19, SEQ ID NO:22, SEQ ID NO:25, and SEQ ID NO:28,the nucleotide sequence of SEQ ID NO:3, SEQ ID NO:6, SEQ ID NO:9, SEQ ID NO:12, SEQ ID NO:15, SEQ ID NO:18, SEQ ID NO:21, SEQ ID NO:24, SEQ ID NO:27, or SEQ ID NO: 30, or the nucleotide sequence of the DNA insert of a plasmid deposited with an ATCC Accession Number as described herein, wherein the nucleic acid encodes a full length 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 protein or an active fragment thereof.
In a related aspect, the invention further provides nucleic acid constructs that include a 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 nucleic acid molecule described herein. In certain embodiments, the nucleic acid molecules of the invention are operatively linked to native or heterologous regulatory sequences. Also included, are vectors and host cells containing the 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 nucleic acid molecules of the invention e.g., vectors and host cells suitable for producing 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 nucleic acid molecules and polypeptides. In another related aspect, the invention provides nucleic acid fragments suitable as primers or hybridization probes for the detection of 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234-encoding nucleic acids.
In still another related aspect, isolated nucleic acid molecules that are antisense to a 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 encoding nucleic acid molecule are provided.
In another aspect, the invention features, 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 polypeptides, and biologically active or antigenic fragments thereof that are useful, e.g., as reagents or targets in assays applicable to treatment and diagnosis of 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234-mediated or - related disorders. In another embodiment, the invention provides 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 polypeptides having a 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 activity. Preferred polypeptides are 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 proteins including at least one Guanine Nucleotide Dissociation Stimulator, Glycosyltransferase, DEAD type helicase, Centaurin, Dehydrogenase/Reductase, or Metal Transporter domain, and, preferably, having a 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 activity, e.g., a 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 activity as described herein.
In other embodiments, the invention provides 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 polypeptides, e.g., a 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 polypeptide having the amino acid sequence shown in SEQ ID NO:2, SEQ ID NO:5, SEQ ID NO:8, SEQ ID NO:l 1, SEQ ID NO: 14, SEQ ID NO: 17, SEQ ID NO:20, SEQ ID NO:23, SEQ ID NO:26, OR SEQ ID NO:29 or the amino acid sequence encoded by the cDNA insert of the plasmid deposited with ATCC Accession Number as described herein; an amino acid sequence that is substantially identical to the amino acid sequence shown in SEQ ID NO:2, SEQ ID NO:5, SEQ ID NO:8, SEQ ID NO:l 1, SEQ ID NO: 14, SEQ ID NO: 17, SEQ ID NO:20, SEQ ID NO:23, SEQ ID NO:26, OR SEQ ID NO:29 or the amino acid sequence encoded by the cDNA insert of a plasmid deposited with an ATCC Accession Number as described herein; or an amino acid sequence encoded by a nucleic acid molecule having a nucleotide sequence which hybridizes under a stringency condition described herein to a nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO: 1 , SEQ ID NO:4, SEQ ID NO:7, SEQ ID NO:10, SEQ ID NO:13, SEQ ID NO:16, SEQ ID NO:19, SEQ ID NO:22, SEQ ID NO:25, and SEQ ID NO:28, the nucleotide sequence of SEQ ID NO:3, SEQ ID NO:6, SEQ ID NO:9, SEQ ID NO:12, SEQ ID NO:15, SEQ ID NO:18, SEQ ID NO:21, SEQ ID NO:24, SEQ ID NO:27, or SEQ ID NO:30, or the nucleotide sequence of the DNA insert of a plasmid deposited with an ATCC Accession Number as described herein, wherein the nucleic acid encodes a full length 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 protein or an active fragment thereof.
In a related aspect, the invention provides 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 polypeptides or fragments operatively linked to non-47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 polypeptides to form fusion proteins.
In another aspect, the invention features antibodies and antigen-binding fragments thereof, that react with, or more preferably specifically bind 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 polypeptides or fragments thereof, e.g., an extracellular domain or a catalytic domain of an 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 polypeptide.
In another aspect, the invention provides methods of screening for agents, e.g., compounds, that modulate the expression or activity of the 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 polypeptides or nucleic acids. In still another aspect, the invention provides a process for modulating 47476, 67210,
49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 polypeptide or nucleic acid expression or activity, e.g. using the screened compounds. In certain embodiments, the methods involve treatment of conditions related to aberrant activity or expression of the 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 polypeptides or nucleic acids, such as conditions involving aberrant or deficient cellular function resulting in a disorder, e.g., an immunological disorder, neurological disorder, metabolic disorder, cellular proliferation and/or differentiation disorder, disorder of metal ion imbalance, or a protein trafficing disorder.
In some embodiments, the agent, e.g., compound, is an inhibitor of a 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 polypeptide. Preferably, the inhibitor is chosen from a peptide, a phosphopeptide, a small organic molecule, a small inorganic molecule and an antibody (e.g., an antibody conjugated to a therapeutic moiety). In other embodiments, the compound is an inhibitor of a 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 nucleic acid, e.g., an antisense, a ribozyme, a double stranded RNA, or a triple helix molecule. In some embodiments, the agent, e.g., compound, is an activator of a 47476, 67210,
49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 polypeptide. Preferably, the activator is chosen from a peptide, a phosphopeptide, a small organic molecule, a small inorganic molecule and an antibody. In other embodiments, the compound activates (i.e., increases) the transcription or translation of a 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 nucleic acid, e.g., by directly or indirectly modulating the activity of a transcription or translation factor.
In some embodiments, the agent, e.g., compound is administered (e.g., to cells or to a subject) in combination with a second agent, e.g., a compound, e.g., a known therapeutic agent or compound. Such an agent, e.g., compound, could be used to treat or prevent immunological disorders, neurological disorders, metabolic disorders, cellular proliferation and/or differentiation disorders, disorders of metal ion imbalance, or a protein trafficing disorders.
In another aspect, the mvention features methods for treating or preventing a disorder characterized by aberrant cellular proliferation or differentiation of a 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234-expressing cell, in a subject. Preferably, the method includes administering to the subject (e.g., a mammal, e.g., a human) an effective amount of a compound (e.g., a compound identified using the methods described herein) that modulates the activity, or expression, of the 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 polypeptide or nucleic acid. In a preferred embodiment, the disorder is a cancerous or pre-cancerous condition. In a further aspect, the invention provides methods for evaluating the efficacy of a treatment of a disorder, e.g., an immunological disorder, neurological disorder, metabolic disorder, cellular proliferation and/or differentiation disorder, disorder of metal ion imbalance, or a protein trafficing disorder. The method includes: treating a subject, e.g., a patient or an animal, with a protocol under evaluation (e.g., treating a subject with one or more of: chemotherapy, radiation, and/or a compound identified using the methods described herein); and evaluating the expression of a 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 nucleic acid or polypeptide before and after treatment. A change, e.g., a decrease or increase, in the level of a 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 nucleic acid (e.g., mRNA) or polypeptide after treatment, relative to the level of expression before treatment, is indicative of the efficacy of the treatment of the disorder. The level of 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 nucleic acid or polypeptide expression can be detected by any method described herein.
In a preferred embodiment, the evaluating step includes obtaining a sample (e.g., a tissue sample, e.g., a biopsy, or a fluid sample) from the subject, before and after treatment and comparing the level of expressing of a 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 nucleic acid (e.g., mRNA) or polypeptide before and after treatment. In another aspect, the invention provides methods for evaluating the efficacy of a therapeutic or prophylactic agent (e.g., an anti-neoplastic agent). The method includes: contacting a sample with an agent (e.g., a compound identified using the methods described herein, a cytotoxic agent) and, evaluating the expression of 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 nucleic acid or polypeptide in the sample before and after the contacting step. A change, e.g., a decrease or increase, in the level of 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 nucleic acid (e.g., mRNA) or polypeptide in the sample obtained after the contacting step, relative to the level of expression in the sample before the contacting step, is indicative of the efficacy of the agent. The level of 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 nucleic acid or polypeptide expression can be detected by any method described herein. In a preferred embodiment, the sample includes cells obtained from the blood (e.g., hematopoietic cells, e.g., white blood cells or red blood cells), bone marrow, spleen, liver, kidney, stomach, a neural tissue, a cardiovascular tissue (e.g., heart, endothelial, or smooth muscle cells), or a cancerous tissue.a cancerous tissue, e.g., a cancerous lung, breast, or ovary tissue. The invention also provides assays for determining the activity of, or the presence or absence of, 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 polypeptides or nucleic acid molecules in a biological sample, including for disease diagnosis.
In further aspect, the invention provides assays for determining the presence or absence of a genetic alteration in a 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 polypeptide or nucleic acid molecule, including for disease diagnosis.
In another aspect, the invention features a two dimensional array having a plurality of addresses, each address of the plurality being positionally distinguishable from each other address of the plurality, and each address of the plurality having a unique capture probe, e.g., a nucleic acid or peptide sequence. At least one address of the plurality has a capture probe that recognizes a 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 molecule. In one embodiment, the capture probe is a nucleic acid, e.g., a probe complementary to a 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 nucleic acid sequence. In another embodiment, the capture probe is a polypeptide, e.g., an antibody specific for 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 polypeptides. Also featured is a method of analyzing a sample by contacting the sample to the aforementioned array and detecting binding of the sample to the array.
Other features and advantages of the invention will be apparent from the following detailed description, and from the claims.
Brief Description of the Drawings
Figure 1 depicts a hydropathy plot of human 47476. Relative hydrophobic residues are shown above the dashed horizontal line, and relative hydrophilic residues are below the dashed horizontal line. Numbers corresponding to positions in the amino acid sequence of human 47476 are indicated. Polypeptides of the invention include fragments which include: all or part of a hydrophobic sequence, i.e., a sequence above the dashed line, e.g., the sequence from about amino acid 82 to 105, from about 341 to 360, and from about 521 to 540 of SEQ ID NO:2; all or part of a hydrophilic sequence, i.e., a sequence below the dashed line, e.g., the sequence of from about amino acid 106 to 122, from about 325 to 340, and from about 500 to 520 of SEQ ID NO.2. Figures 2A-2C depict alignments of the ras guanine nucleotide dissociation stimulator domain of human 47476 with consensus amino acid sequences derived from a hidden Markov model (HMM) from PFAM and SMART. The upper sequence of Figure 2 A is the consensus amino acid sequence according to PFAM (SEQ ID NO:31), while the lower amino acid sequence corresponds to amino acids 195 to 381 of SEQ ID NO:2. Figure 2B shows an alignment of the ras guanine nucleotide dissociation stimulator domain consensus amino acid sequence according to SMART (SEQ ID NO:32) with amino acids 197 to 433 of SEQ ID NO:2, while Figure 2C shows an alignment of the guanine nucleotide dissociation stimulator N- terminal motif consensus sequence according to SMART (SEQ ID NO:33) with amino acids 55 to 172 of SEQ ID NO:2.
Figures 3A-3B depict alignments of the EF-hand calcium-binding domain of human 47476 with consensus amino acid sequences derived from a hidden Markov model (HMM) from PFAM and SMART, respectively. The upper sequence of Figure 3 A is the consensus amino acid sequence according to PFAM (SEQ ID NO: 34), while the lower amino acid sequence corresponds to amino acids 470 to 498 of SEQ ID NO:2. Figure 3B shows an alignment of the consensus amino acid sequence according to SMART (SEQ ID NO:35) with amino acids 470 to 498 of SEQ ID NO:2.
Figures 4A-4B depict alignments of the phorbol ester/diacylglycerol binding domain (Cl domain) of human 47476 with consensus amino acid sequences derived from a hidden Markov model (HMM) from PFAM and SMART. The upper sequence of Figures 4A and 4B are the consensus amino acid sequences according to PFAM (SEQ ID NO: 36) and SMART (SEQ ID NO:37), respectively, while the lower amino acid sequences correspond to amino acids 541 to 590 of SEQ ID NO:2.
Figure 5 depicts a hydropathy plot of human 67210. Relative hydrophobic residues are shown above the dashed horizontal line, and relative hydrophilic residues are below the dashed horizontal line. Numbers corresponding to positions in the amino acid sequence of human 67210 are indicated. Polypeptides of the invention include fragments which include: all or part of a hydrophobic sequence, i.e., a sequence above the dashed line, e.g., the sequence from about amino acid 10 to 25 of SEQ ID NO:5; all or part of a hydrophilic sequence, i.e., a sequence below the dashed line, e.g., the sequence of from about amino acid 240-260 of SEQ ID NO:5; a sequence which includes a Cys, or a glycosylation site Figure 6 depicts an aligmnent of the glycosyltransferase domain of human 67210 with a consensus amino acid sequence derived from a hidden Markov model (HMM) from PFAM. The upper sequence is the consensus amino acid sequence (SEQ ID NO: 38), while the lower amino acid sequence corresponds to amino acids 63 to 340 of SEQ ID NO: 5. Figure 7 depicts a hydropathy plot of human 49875. Relative hydrophobic residues are shown above the dashed horizontal line, and relative hydrophilic residues are below the dashed horizontal line. Numbers corresponding to positions in the amino acid sequence of human 49875 are indicated. Polypeptides of the invention include fragments which include: all or part of a hydrophobic sequence, i.e., a sequence above the dashed line, e.g., the sequence from about amino acid 285 to 295 of SEQ ID NO:8; all or part of a hydrophilic sequence, i.e., a sequence below the dashed line, e.g., the sequence of from about amino acid 520 to 550 of SEQ ID NO:8; a sequence which includes a Cys, or a glycosylation site.
Figures 8A-8B depict alignments of the DEAD-type helicase domain of human 49875 with consensus amino acid sequences derived from a hidden Markov model (HMM) from PFAM (PF00270) (Figure 8A) and from SMART (Figure 8B). The upper sequences are the consensus amino acid sequences according to PFAM (SEQ ID NO:39) and according to SMART (SEQ ID NO:40), while the lower amino acid sequences corresponds to amino acids 22 to 245 (Figure 8A) or amino acids 28 to 245 (Figure 8B) of SEQ ID NO:8.
Figures 9A-9B depict alignments of the conserved helicase C-terminal domain of human 49875 with consensus amino acid sequences derived from a hidden Markov model (HMM) from PFAM (PF00271) (Figure 9A) and from SMART (Figure 9B). The upper sequences are the consensus amino acid sequences according to PFAM (SEQ ID NO:41) and according to SMART (SEQ ID NO:42), while the lower amino acid sequences corresponds to amino acids 281 to 363 (Figure 9A) or amino acids 281 to 363 (Figure 9B) of SEQ ID NO:8. Figure 10 depicts a hydropathy plot of human 46842. Relative hydrophobic residues are shown above the dashed horizontal line, and relative hydrophilic residues are below the dashed horizontal line. The cysteine residues (cys) are indicated by short vertical lines just below the hydropathy trace. The numbers corresponding to the amino acid sequence of human 46842 are indicated. Polypeptides of the invention include fragments which include: all or part of a hydrophobic sequence, i.e., a sequence above the dashed line, e.g., the sequence from about amino acid 431 to 439, from about 558 to 566, and from about 706 to 719 of SEQ ID NO:l 1; all or part of a hydrophilic sequence, i.e., a sequence below the dashed line, e.g., the sequence of from about amino acid 567 to 580, from about 720 to 737, and from about 757 to 771 of SEQ ID NO:l 1; a sequence which includes a Cys, or a glycosylation site.
Figure 11 depicts an alignment ofthe PH domain of human 46842 and the corresponding consensus amino acid sequences derived from a hidden Markov model (HMM) from PFAM. The upper sequence is the consensus amino acid sequence (SEQ ID NO:43), while the lower amino acid sequence corresponds to amino acids 269 to 363 of SEQ ID NO:l 1.
Figure 12 depicts an alignment ofthe ArfGAP domain of human 46842 and the corresponding consensus amino acid sequences derived from a hidden Markov model (HMM) from PFAM. The upper sequence is the consensus amino acid sequence (SEQ ID NO:44), while the lower amino acid sequence corresponds to amino acids 403 to 525 of SEQ ID NO:l 1.
Figures 13A-13B depict alignments ofthe ankyrin domains of human 46842 and the corresponding consensus amino acid sequences derived from a hidden Markov model (HMM) from PFAM. The upper sequence is the consensus amino acid sequence (SEQ ID NO:45), while the lower amino acid sequence corresponds to amino acids 702 to 734 (Figure 13 A) and 735 to 767 (Figure 13B) of SEQ ID NO: 11.
Figure 14 depicts a hydropathy plot of human 33201. Relative hydrophobic residues are shown above the dashed horizontal line, and relative hydrophilic residues are below the dashed horizontal line. Numbers corresponding to positions in the amino acid sequence of human 33201 are indicated. Polypeptides ofthe invention include fragments which include: all or part of a hydrophobic sequence, i.e., a sequence above the dashed line, e.g., the sequence from about amino acid 70 to 80, and from about 158 to 178 of SEQ ID NO:2; all or part of a hydrophilic sequence, i.e., a sequence below the dashed line, e.g., the sequence of from about amino acid 50 to 60, from about 82 to 90, and from about 205 to 210 of SEQ ID NO: 14; a sequence which includes at least one Cys residue, or a glycosylation site.
Figures 15A-15B depicts an alignment ofthe dehydrogenase/reductase domain of human 33201 with a consensus amino acid sequence derived from a hidden Markov model (HMM) from PFAM. The upper sequence is the consensus amino acid sequence (SEQ ID NO:46), while the lower amino acid sequence corresponds to amino acids 22 to 345 of SEQ ID NO:14. Figure 16 depicts a hydropathy plot of human 83378. Relative hydrophobic residues are shown above the dashed horizontal line, and relative hydrophilic residues are below the dashed horizontal line. Numbers corresponding to positions in the amino acid sequence of human 83378 are indicated. Polypeptides ofthe invention include fragments which include: all or part of a hydrophobic sequence, i.e., a sequence above the dashed line, e.g., the sequence from about amino acid 325 to 335, from about 340 to 350, and from about 415 to 430 of SEQ ID NO: 17; all or part of a hydrophilic sequence, i.e., a sequence below the dashed line, e.g., the sequence of from about amino acid 150 to 160, from about 220 to 235, and from about 355 to 370 of SEQ ID NO: 17. Figures 17A-17B depict alignments ofthe first and second cation efflux domains of human 83378 with consensus amino acid sequences derived from hidden Markov models (HMM) from PFAM. In Figure 17A, the upper sequence is the consensus amino acid sequence (SEQ ID NO:47), while the lower amino acid sequence corresponds to the first cation efflux domain of human 83378 (amino acids 11 to 133 of SEQ ID NO: 17). Similarly, in Figure 17B, the upper sequence is the consensus amino acid sequence (SEQ ID NO:48), while the lower amino acid sequence corresponds to the second cation efflux domain of human 88378 (amino acids 231 to 389 of SEQ ID NO: 17).
Figures 18A-8B depicts an alignment ofthe amino acid sequence of human 83378 (upper sequence; SEQ ID NO: 17) with the amino acid sequence of rat ZnT-1 (lower sequence; GenBank3 Accession Number Q62720; SEQ ID NO:49).
Figures 18C-18D depicts an alignment ofthe amino acid sequence of human 83378 (lower sequence; SEQ ID NO: 17) with the amino acid sequence of human GenBank3 Accession Number AL359609 (upper sequence; SEQ ID NO:50).
Figure 19 depicts a hydropathy plot of human 84233. Relative hydrophobic residues are shown above the dashed horizontal line, and relative hydrophilic residues are below the dashed horizontal line. Numbers corresponding to positions in the amino acid sequence of human 84233 are indicated. Polypeptides ofthe invention include fragments which include: all or part of a hydrophobic sequence, i.e., a sequence above the dashed line, e.g., the sequence from about amino acid 220 to 230, from about 240 to 260, and from about 262 to 273 of SEQ ID NO:20; all or part of a hydrophilic sequence, i.e., a sequence below the dashed line, e.g., the sequence of from about amino acid 1 to 20, from about amino acid 80 to 90, and from about 150 to 160 of SEQ ID NO.20.
Figure 20 depicts an alignment ofthe cation efflux domain of human 84233 with a consensus amino acid sequence derived from a hidden Markov model (HMM) from PFAM. The upper sequence is the consensus amino acid sequence (SEQ ID NO:51), while the lower amino acid sequence corresponds to amino acids 25 to 310 of SEQ ID NO:20.
Figure 21 depicts an alignment ofthe amino acid sequence of human 84233 (lower sequence; SEQ ID NO:20) with the amino acid sequence of human ZnT-3 (upper sequence; GenBank3 Accession Number Q99726; SEQ ID NO:52). Figure 22 depicts a hydropathy plot of human 64708. Relative hydrophobic residues are shown above the dashed horizontal line, and relative hydrophilic residues are below the dashed horizontal line. Numbers corresponding to positions in the amino acid sequence of human 64708 are indicated. Polypeptides ofthe invention include fragments which include: all or part of a hydrophobic sequence, i.e., a sequence above the dashed line, e.g., the sequence from about amino acid 180 to 195, from about 290 to 300, and from about 340 to 350 of SEQ ID NO:23; all or part of a hydrophilic sequence, i.e., a sequence below the dashed line, e.g., the sequence of from about amino acid 123 to 133, from about 380 to 395, and from about 450 to 461 of SEQ ID NO:23.
Figures 23A-23B depict alignments ofthe first and second cation efflux domains of human 64708 with consensus amino acid sequences derived from hidden Markov models
(HMM) from PFAM. In Figure 23 A, the upper sequence is the consensus amino acid sequence (SEQ ID NO: 53), while the lower amino acid sequence corresponds to the first cation efflux domain of human 64708 (amino acids 55 to 153 of SEQ ID NO:23). Similarly, in Figure 23B, the upper sequence is the consensus amino acid sequence (SEQ ID NO:54), while the lower amino acid sequence corresponds to the second cation efflux domain of human 64708 (amino acids 227 to 320 of SEQ ID NO:23).
Figures 24A-24B depicts an alignment ofthe amino acid sequence of human 64708 (upper sequence; SEQ ID NO:23) with the amino acid sequence of murine ZnTll (lower sequence; GenBank3 Accession Number AF233321; SEQ ID NO:55). Figure 24C depicts an alignment ofthe amino acid sequence of human 64708 (lower sequence; SEQ ID NO:23) with the amino acid sequence of human GenBank3 Accession Number AK000844 (upper sequence; SEQ ID NO:56).
Figure 25 depicts a hydropathy plot of human 85041. Relative hydrophobic residues are shown above the dashed horizontal line, and relative hydrophilic residues are below the dashed horizontal line. Numbers corresponding to positions in the amino acid sequence of human 85041 are indicated. Polypeptides ofthe invention include fragments which include: all or part of a hydrophobic sequence, i.e., a sequence above the dashed line, e.g., the sequence from about amino acid 35 to 50, from about 440 to 470, and from about 685 to 695 of SEQ ID NO:26; all or part of a hydrophilic sequence, i.e., a sequence below the dashed line, e.g., the sequence of from about amino acid 320 to 340, from about 555 to 575, and from about 750 to 765 of SEQ ID NO.26.
Figures 26A-26B depicts an alignment ofthe cation efflux domain of human 85041 with a consensus amino acid sequence derived from a hidden Markov model (HMM) from PFAM. The upper sequence is the consensus amino acid sequence (SEQ ID NO: 57), while the lower amino acid sequence corresponds to amino acids 419 to 733 of SEQ ID NO:26.
Figures 27A-27B depicts an alignment ofthe amino acid sequence of human 85041 (upper sequence; SEQ ID NO:26) with the amino acid sequence of murine ZnTll (lower sequence; GenBank3 Accession Number AF233321; SEQ ID NO:55). Figure 28 depicts a hydropathy plot of human 84234. Relative hydrophobic residues are shown above the dashed horizontal line, and relative hydrophilic residues are below the dashed horizontal line. Numbers corresponding to positions in the amino acid sequence of human 84234 are indicated. Polypeptides ofthe invention include fragments which include: all or part of a hydrophobic sequence, i.e., a sequence above the dashed line, e.g., the sequence from about amino acid 59 to 70, from about 330 to 345, and from about 370 to 376 of SEQ ID NO:29; all or part of a hydrophilic sequence, i.e., a sequence below the dashed line, e.g., the sequence of from about amino acid 10 to 20, from about 165 to 175, and from about 190 to 230 of SEQ ID NO:29.
Figure 29 depicts an alignment ofthe cation efflux domain of human 84234 with a consensus amino acid sequence derived from a hidden Markov model (HMM) from PFAM. The upper sequence is the consensus amino acid sequence (SEQ ID NO: 58), while the lower amino acid sequence corresponds to amino acids 38 to 349 of SEQ ID NO:29.
Figure 30 depicts an alignment ofthe amino acid sequence of human 84234 (upper sequence; SEQ ID NO:29) with the amino acid sequence of murine ZnT12 (lower sequence; GenBank™ Accession Number AF233322; SEQ ID NO:59).
Detailed Description
Human 47476
The human 47476 sequence (see SEQ ID NO:l, as recited in Example 1), which is about 3134 nucleotides long, including untranslated regions, contains a predicted methionine-initiated coding sequence of about 2022 nucleotides, including the termination codon. The coding sequence encodes a 673 amino acid protein (see SEQ ID NO:2, as recited in Example 1).
Human 47476 contains the following regions or other structural features: a ras guanine nucleotide dissociation stimulator domain (PFAM Accession Number PF00617) located at about amino acid residues 195 to 381 of SEQ ID NO:2; a guanine nucleotide dissociation stimulator domain N-terminal motif (SMART
Accession Number SM0229) located at about amino acid residues 55 to 172 of SEQ ID NO:2; an EF-hand calcium-binding domain (PFAM Accession Number PF00036) located at about amino acid residues 470 to 498 of SEQ ID NO:2; a phorbol ester/diacylglycerol binding domain (Cl domain) (PFAM Accession Number PF00130) located at about amino acid residues 541 to 590 of SEQ ID NO:2; one predicted N-glycosylation site (PS00001) located at about amino acid residues 622 to 625 of SEQ ID NO:2; four predicted cAMP/cGMP-dependent protein kinase phosphorylation sites (PS00004) located at about amino acid rsidues 3 to 6, 7 to 10, 191 to 194 and 549 to 552 of SEQ ID NO:2; eight predicted Protein Kinase C phosphorylation sites (PS00005) located at about amino acid residues 6 to 8, 15 to 17, 35 to 37, 233 to 235, 316 to 318, 455 to 457, 547 to 549 and 668 to 670 of SEQ ID NO:2; fourteen predicted Casein Kinase II phosphorylation sites (PS00006) located at about amino acid residues 10 to 13, 35 to 38, 57 to 60, 210 to 213, 221 to 224, 360 to 363, 400 to 403, 418 to 421, 487 to 490, 517 to 520, 542 to 545, 552 to 555, 642 to 645 and 669 to 672 of SEQ ID NO.2; four predicted N-myristylation sites (PS00008) located at about amino acids 232 to 237, 245 to 250, 296 to 301 and 618 to 623 of SEQ ID NO:2; and one predicted Amidation site (PS00009) located at about amino acids 187 to 190 of SEQ
ID NO:2.
A plasmid containing the nucleotide sequence encoding human 47476 (clone "Fbh47476FL") was deposited with American Type Culture Collection (ATCC), 10801
University Boulevard, Manassas, VA 20110-2209, on and assigned Accession Number . This deposit will be maintained under the terms ofthe Budapest Treaty on the
International Recognition ofthe Deposit of Microorganisms for the Purposes of Patent Procedure. This deposit was made merely as a convenience for those of skill in the art and is not an admission that a deposit is required under 35 U.S.C. §112.
Human 67210
The human 67210 sequence (see SEQ ID NO:4, as recited in Example 1), which is approximately 1778 nucleotides long including untranslated regions, contains a predicted methionine-initiated coding sequence of about 1050 nucleotides, including the termination codon. The coding sequence encodes a 349 amino acid protein (see SEQ ID NO: 5, as recited in Example 1). The human 67210 protein of SEQ ID NO:5 and Figure 2 includes an amino- terminal hydrophobic amino acid sequence, consistent with a signal sequence, of about 29 amino acids (from amino acid 1 to about amino acid 29 of SEQ ID NO:5), which upon cleavage results in the production of a mature protein form.
Human 67210 contains the following regions or other structural features: a glycosyl transferase domain (PFAM Accession Number PF01501) located at about amino acid residues 63 to 340 of SEQ ID NO:5; a signal peptide located at about amino acids 1-29, which when cleaved gives a predicted mature protein of 319 amino acids, from about amino acid 30 to amino acid 349 of SEQ ID NO:5; one dileucine motif located at about amino acids 3 to 4 of SEQ ID NO:5; one predicted N-glycosylation site (PS00001) located at about amino acids 234 to 237 of SEQ ID NO.5; one Protein Kinase C phosphorylation site (PS00005) located at about amino acids 126 to 128 of SEQ ID NO:5; four Casein Kinase II phosphorylation sites (PS00006) located at about amino 43 to 46,
74 to 77, 127 to 130, and 189 to 192 of SEQ ID NO:5; one tyrosine kinase phosphorylation site (PS00007) located at about amino acid 253 to 260 of SEQ ID NO:5; and six N-myristoylation sites (PS00008) located at about amino acid 63 to 68, 86 to 91, 198 to 203, 218 to 223, 229 to 234, and 265 to 270 of SEQ ID NO:5.
A plasmid containing the nucleotide sequence encoding human 67210 (clone "Fbh67210FL") was deposited with American Type Culture Collection (ATCC), 10801
University Boulevard, Manassas, NA 20110-2209, on and assigned Accession Number . This deposit will be maintained under the terms ofthe Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent
Procedure. This deposit was made merely as a convenience for those of skill in the art and is not an admission that a deposit is required under 35 U.S.C. §112.
Human 49875 The human 49875 sequence (see SEQ ID NO:7, as recited in Example 1), which is approximately 2704 nucleotides long, including untranslated regions, contains a predicted methionine-initiated coding sequence of about 1803 nucleotides, including the termination codon. The coding sequence encodes a 600 amino acid protein (see SEQ ID NO: 8, as recited in
Example 1). Human 49875 contains the following regions or other structural features: a DEAD-type helicase domain (PFAM Accession Number PF00270) located at about amino acid residues 22 to 245 of SEQ ID NO:8; a DEAD-box subfamily ATP-dependent helicase signature motif (PS00039) located at about amino acid residues 169 to 177 of SEQ ID NO:8; a conserved helicase C-terminal domain (PFAM Accession Number PF00271 ) located at about amino acid residues 281 to 363 of SEQ ID NO:8; two N-glycosylation sites (PS00001) located at about amino acid residues 348 to 351, and 556 to 559 of SEQ ID NO:8; five Protein Kinase C phosphorylation sites (PS00005) located at about amino acid residues 57 to 59, 224 to 226, 359 to 361, 581 to 583, and 585 to 587 of SEQ ID NO:8; seven Casein Kinase II phosphorylation sites (PS00006) located at about amino acid residues 187 to 190, 224 to 227, 240 to 243, 507 to 510, 544 to 547, 570 to 573, and 594 to 597 of SEQ ID NO:8; seven N-myristoylation sites (PS00008) located at about amino acid residues 7 to 12, 56 to 61, 155 to 160, 199 to 204, 229 to 234, 320 to 325, and 577 to 582 of SEQ ID NO:8; two amidation sites (PS00009) located at about amino acid residues 510 to 513, and 581 to 584 of SEQ ID NO:8; and one ATP/GTP-binding site motif A (P-loop) (PS00017) located at about amino acid residues 53 to 60 of SEQ ID NO:8.
A plasmid containing the nucleotide sequence encoding human 49875 (clone "Fbh49875FL") was deposited with American Type Culture Collection (ATCC), 10801
University Boulevard, Manassas, NA 20110-2209, on and assigned Accession Number . This deposit will be maintained under the terms ofthe Budapest Treaty on the
International Recognition ofthe Deposit of Microorganisms for the Purposes of Patent Procedure. This deposit was made merely as a convenience for those of skill in the art and is not an admission that a deposit is required under 35 U.S.C. § 112.
Human 46842
The human 46842 sequence (see SEQ ID NO: 10, as recited in Example 1), which is approximately 2737 nucleotides long including untranslated regions, contains a predicted methionine-initiated coding sequence of about 2505 nucleotides, including the termination codon. The coding sequence encodes a 834 amino acid protein (see SEQ ID NO:l 1, as recited in Example 1).
Human 46842 contains the following regions or other structural features: a PH domain (PFAM Accession Number PF00169) located at about amino acid residues 269 to 363 SEQ ID NO:l l; an ArfGAP domain (PFAM Accession Number PF01412) located at about amino acid residues 403 to 525 of SEQ ID NO: 11 ; two ankyrin repeat domains (PFAM Accession Number PF00023) located at about amino acid residues 702 to 734 and 735 to 767 of SEQ ID NO:l 1; an ArfGAP zinc ion coordinating motif located at about amino acid residues 421 to 440 of SEQ ID NO:l l; twelve Protein Kinase C phosphorylation sites (PS00005) located at about amino acid residues 12 to 14, 53 to 55, 102 to 104, 121 to 123, 160 to 162, 319 to 321, 347 to 349, 375 to 377, 492 to 494, 499 to 501, 544 to 546, and 549 to 551 of SEQ ID NO:l 1; fourteen Casein Kinase II phosphorylation sites (PS00006) located at about amino acid residues 18 to 21, 84 to 87, 229 to 232, 253 to 256, 257 to 260, 387 to 390, 391 to 394, 500 to 503, 592 to 595, 629 to 632, 633 to 636, 645 to 648, 653 to 656, and 813 to 816 of SEQ ID NO:l l; three cAMP/cGMP-dependent protein kinase phosphorylation sites (PS00004) located at about amino acid residues 277 to 280, 493 to 496, and 577 to 580 of SEQ ID NO: 11 ; one tyrosine kinase phosphorylation site (PS00007) located at about amino acid residues 367 to 374 of SEQ ID NO:l l; one glycosaminoglycan attachment site (PS00002) located at about amino acid residues 610 to 613 of SEQ ID NO:l l; and ten N-myristylation sites (PS00008) located at about amino acids 42 to 47, 414 to 419,
434 to 439, 443 to 448, 449 to 454, 597 to 602, 611 to 616, 691 to 696, 726 to 731, and 808 to 813 of SEQ ID NO:ll.
A plasmid containing the nucleotide sequence encoding human 46842 was deposited with American Type Culture Collection (ATCC), 10801 University Boulevard, Manassas, NA 20110-2209, on and assigned Accession Number . This deposit will be maintained under the terms ofthe Budapest Treaty on the International Recognition ofthe Deposit of Microorganisms for the Purposes of Patent Procedure. This deposit was made merely as a convenience for those of skill in the art and is not an admission that a deposit is required under 35 U.S.C. §112.
Human 33201 The human 33201 sequence (see SEQ ID NO:13, as recited in Example 1), which is approximately 1718 nucleotides long including untranslated regions, contains a predicted methionine-initiated coding sequence of about 1056 nucleotides, including the termination codon. The coding sequence encodes a 351 amino acid protein (see SEQ ID NO: 14, as recited in Example 1).
Human 33201 contains the following regions or other structural features:
A dehydrogenase/reductase domain (PFAM Accession number PF00107) located at about amino acid residues 22 to 345 of SEQ ID NO: 14; three conserved glycine residues located at about amino acid residues 76, 93, and 224 of SEQ ID NO:14; one Protein Kinase C phosphorylation site (PS00005) located at about amino acid residues 154 to 156 of SEQ ID NO: 14; two Casein Kinase II phosphorylation sites (PS00006) located at about amino acid residues 60 to 63 and 91 to 94 of SEQ ID NO: 14; four N-glycosylation sites (PS00001) located at about amino acids 89 to 92, 155 to 158,
235 to 238 and 281 to 284 of SEQ ID NO:14: and eight N-myristylation sites (PS00008) located at about amino acids 16-21, 76-81, 153- 158, 162-167, 168-173, 185-190, 233-238, and 335-340 of SEQ ID NO:14.
A plasmid containing the nucleotide sequence encoding human 33201 (clone "Fbh67210FL") was deposited with American Type Culture Collection (ATCC), 10801
University Boulevard, Manassas, NA 20110-2209, on and assigned Accession Number This deposit will be maintained under the terms ofthe Budapest Treaty on the
International Recognition ofthe Deposit of Microorganisms for the Purposes of Patent Procedure. This deposit was made merely as a convenience for those of skill in the art and is not an admission that a deposit is required under 35 U.S.C. §112.
Human 83378
The human 83378 sequence (see SEQ ID NO: 16, as recited in Example 1), which is approximately 1827 nucleotides long including untranslated regions, contains a predicted methionine-initiated coding sequence of about 1458 nucleotides, including the termination codon. The coding sequence encodes a 485 amino acid protein (see SEQ ID NO: 17, as recited in Example 1).
Human 83378 contains the following regions or other structural features: a first and a second cation efflux domain (PFAM Accession Number PF01545) located at about amino acid residues 11 to 133 and 231 to 389 of SEQ ID NO: 17; six transmembrane domains, located at about 11 to 31, 44 to 61, 79 to 98, 115 to 134, 241 to 265, and 283 to 299 of SEQ ID NO: 17; four cytoplasmic domains located at about amino acids 1 to 10 (amino terminus), 62 to 78, 135 to 240, and 300 to 485 (carboxy terminus) of SEQ ID NO:17; three non-cytoplasmic loops located at about amino acids 32 to 43, 99 to 114, and 266 to
282 of SEQ ID NO:17; three N-glycosylation sites (PS00001) located at about amino acid residues 377-380, 471-474, and 481-484 of SEQ ID NO:17; one cAMP/cGMP-dependent protein kinase phosphorylation site (PS00004) located at about amino acid residues 62-65 of SEQ ID NO: 17; seven Protein Kinase C phosphorylation sites (PS00005) located at about amino acid residues 5-7, 8-10, 149-151, 192-194, 342-344, 363-365, and 446-448 of SEQ ID NO:17; five Casein Kinase II phosphorylation sites (PS00006) located at about amino acid residues 196-199, 219-222, 331-334, 446-449, and 473-476 of SEQ ID NO: 17; two tyrosine kinase phosphorylation sites (PS00007) located at about amino acid resdues
352-359 and 472-479 of SEQ ID NO: 17; and eight N-myristylation sites (PS00008) located at about amino acid residues 32-37, 54- 59, 81-86, 123-128, 161-166, 168-173, 186-191, and 467-472 of SEQ ID NO:17.
A plasmid containing the nucleotide sequence encoding human 83378 (clone "Fbh83378FL") was deposited with American Type Culture Collection (ATCC), 10801
University Boulevard, Manassas, NA 20110-2209, on and assigned Accession Number . This deposit will be maintained under the terms ofthe Budapest Treaty on the
International Recognition ofthe Deposit of Microorganisms for the Purposes of Patent Procedure. This deposit was made merely as a convenience for those of skill in the art and is not an admission that a deposit is required under 35 U.S.C. §112. Human 84233
The human 84233 sequence (see SEQ ID NO:19, as recited in Example 1), which is approximately 2165 nucleotides long including untranslated regions, contains a predicted methionine-initiated coding sequence of about 963 nucleotides, including the termination codon. The coding sequence encodes a 320 amino acid protein (see SEQ ID NO:20, as recited in Example 1).
Human 84233 contains the following regions or other structural features: a cation efflux domain (PFAM Accession Number PF01545) located at about amino acid residues 25 to 310 of SEQ ID NO:20; six transmembrane domains located at about amino acid residues 25 to 49, 58 to 74, 92 to 113, 128 to 147, 167 to 191, and 201 to 218 of SEQ ID NO:20; four cytoplasmic domains located at about amino acid residues 1 to 24 (amino terminus), 75 to 91, 148 to 166, and 219 to 320 (carboxy terminus) of SEQ ID NO:20; three non-cytoplasmic loops located at about amino acid residues 50 to 57, 114 to 127, and 192 to 200 of SEQ ID NO:20; two N-glycosylation sites (PS00001) located at about amino acid residues 162-165 and 234-237 of SEQ ID NO:20; one cAMP/cGMP-dependent protein kinase phosphorylation site (PS00004) located at about amino acid residues 81-84 of SEQ ID NO:20; four Protein Kinase C phosphorylation sites (PS00005) located at about amino acid residues 11-13, 75-77, 80-82, and 164-166 of SEQ ID NO:20; one Casein Kinase II phosphorylation site (PS00006) located at about amino acid residues 304-307 of SEQ ID NO:20; one tyrosine kinase phosphorylation site (PS00007) located at about amino acid residues
13-20 of SEQ ID NO:20; and four N-myristylation sites (PS00008) located at about amino acid residues 7-12, 42-47, 94-99, and 228-233 of SEQ ID NO:20.
A plasmid containing the nucleotide sequence encoding human 84233 (clone "Fbh84233FL") was deposited with American Type Culture Collection (ATCC), 10801
University Boulevard, Manassas, NA 20110-2209, on and assigned Accession Number This deposit will be maintained under the terms ofthe Budapest Treaty on the
International Recognition ofthe Deposit of Microorganisms for the Purposes of Patent Procedure. This deposit was made merely as a convenience for those of skill in the art and is not an admission that a deposit is required under 35 U.S.C. §112.
Human 64708
The human 64708 sequence (see SEQ ID NO:22, as recited in Example 1), which is approximately 2130 nucleotides long including untranslated regions, contains a predicted methionine-initiated coding sequence of about 1386 nucleotides, including the termination codon. The coding sequence encodes a 461 amino acid protein (see SEQ ID NO:23, as recited in Example 1).
Human 64708 contains the following regions or other structural features: a first and a second cation efflux domain (PFAM Accession Number PF01545) located at about amino acid residues 55 to 153 and 227 to 320 of SEQ ID NO:23; six transmembrane domains located at about amino acid residues 34 to 51, 58 to 82, 101 to 119, 137 to 155, 202 to 219, and 232 to 249 of SEQ ID NO:23; four cytoplasmic domains located at about amino acid residues 1 to 33 (amino terminus), 83 to 100, 156 to 201, and 250 to 461 (carboxy terminus) of SEQ ID NO:23; three non-cytoplasmic loops located at about amino acid residues 52 to 57, 120 to 136, and 220 to 231 of SEQ ID NO:23; one N-glycosylation site (PS00001) located at about amino acid residues 352-355 of SEQ ID NO.23; one cAMP/cGMP-dependent protein kinase phosphorylation site (PS00004) located at about amino acid residues 86-89 of SEQ ID NO:23; six Protein Kinase C phosphorylation sites (PS00005) located at about amino acid residues 31-33, 84-86, 134-136, 154-156, 250-252, and 317-319 of SEQ ID NO:23; two Casein Kinase II phosphorylation sites (PS00006) located at about amino acid residues 67-70 and 274-277 of SEQ ID NO:23; six N-myristylation sites (PS00008) located at about amino acid residues 140-145, 185- 190, 293-298, 412-417, 432-437, and 438-443 of SEQ ID NO:23; and one aminoacyl-transfer RNA synthetases class II site (PS00339) located at about amino acid residues 93-102 of SEQ ID NO:23.
A plasmid containing the nucleotide sequence encoding human 64708 (clone "Fbh64708FL") was deposited with American Type Culture Collection (ATCC), 10801
University Boulevard, Manassas, VA 20110-2209, on and assigned Accession Number . This deposit will be maintained under the terms ofthe Budapest Treaty on the
International Recognition ofthe Deposit of Microorganisms for the Purposes of Patent Procedure. This deposit was made merely as a convenience for those of skill in the art and is not an admission that a deposit is required under 35 U.S.C. §112.
Human 85041
The human 85041 sequence (see SEQ ID NO:25, as recited in Example 1), which is approximately 3304 nucleotides long including untranslated regions, contains a predicted methionine-initiated coding sequence of about 2298 nucleotides, including the termination codon. The coding sequence encodes a 765 amino acid protein (see SEQ ID NO:26, as recited in Example 1).
Human 85041 contains the following regions or other structural features: a cation efflux domain (PFAM Accession Number PF01545) located at about amino acid residues 419 to 733 of SEQ ID NO:26; 14 transmembrane domains located at about amino acid residues 59 to 77, 99 to 119, 129 to 145, 152 to 168, 190 to 214, 239 to 258, 267 to 288, 304 to 320, 343 to 362, 419 to 439, 486 to 505, 521 to 541, 592 to 613, and 618 to 641 of SEQ ID NO:26; eight cytoplasmic domains located at about amino acid residues 1 to 58 (amino terminus), 120 to 128, 169 to 189, 259 to 266, 321 to 342, 438 to 485, 542 to 591, and 642 to 765 (carboxy terminus) of SEQ ID NO:26; seven non-cytoplasmic loops located at about amino acid residues 78 to 98, 146 to 151, 215 to 238, 289 to 303, 363 to 418, 506 to 520, and 614 to 617 of SEQ ID NO:26; one N-glycosylation site (PS00001) located at about amino acid residues 721-724 of SEQ ID NO:26; one glycosaminoglycan attachment site (PS00002) located at about amino acid residues
143-146 of SEQ ID NO:26; one cAMP/cGMP-dependent protein kinase phosphorylation site (PS00004) located at about amino acid residues 225-228 of SEQ ID NO:26; eight Protein Kinase C phosphorylation sites (PS00005) located at about amino acid residues 26-28, 83-85, 224-226, 293-295, 366-368, 406-408, 675-677, and 754-756 of SEQ ID NO:26; three Casein Kinase II phosphorylation sites (PS00006) located at about amino acid residues 262-265, 430-433, and 675-678 of SEQ ID NO:26; one tyrosine kinase phosphorylation site (PS00007) located at about amino acid residues 750-757 of SEQ ID NO:26; and 15 N-myristylation sites (PS00008) located at about amino acid residues 14-19, 46-51,
102-107, 112-117, 144-149, 317-322, 347-352, 369-374, 437-442, 462-467, 529-534, 549-554, 579-584, 605-610, and 737-742 of SEQ ID NO:26.
A plasmid containing the nucleotide sequence encoding human 85041 (clone "Fbh85041FL") was deposited with American Type Culture Collection (ATCC), 10801 University Boulevard, Manassas, VA 20110-2209, on and assigned Accession Number . This deposit will be maintained under the terms ofthe Budapest Treaty on the
International Recognition ofthe Deposit of Microorganisms for the Purposes of Patent Procedure. This deposit was made merely as a convenience for those of skill in the art and is not an admission that a deposit is required under 35 U.S.C. §112.
Human 84234
The human 84234 sequence (see SEQ ID NO:28, as recited in Example 1), which is approximately 2637 nucleotides long including untranslated regions, contains a predicted methionine-initiated coding sequence of about 1131 nucleotides, including the termination codon. The coding sequence encodes a 376 amino acid protein (see SEQ ID NO:29, as recited in Example 1).
Human 84234 contains the following regions or other structural features: a cation efflux domain (PFAM Accession Number PF01545) located at about amino acid residues 38 to 349 of SEQ ID NO:29; six transmembrane domains located at about amino acid residues 38 to 58, 71 to 87, 105 to 123, 141 to 159, 237 to 256, and 263 to 286 of SEQ ID NO:29; four cytoplasmic domains located at about amino acid residues 1 to 37 (amino terminus), 88 to 104, 160 to 236, and 287 to 376 (carboxy terminus) of SEQ ID NO:29; three non-cytoplasmic loops located at about amino acid residues 59 to 70, 124 to 140, and 257 to 262 of SEQ ID NO:29; one N-glycosylation site (PS00001) located at about amino acid residues 45 to 48 of
SEQ ID NO:29; one glycosaminoglycan attachment site (PS00002) located at about amino acid residues 170-173 of SEQ ID NO:29; five Protein Kinase C phosphorylation sites (PS00005) located at about amino acid residues 5 to 7, 31 to 33, 34 to 36, 222 to 224, and 337 to 339 of SEQ ID NO:29; three Casein Kinase II phosphorylation sites (PS00006) located at about amino acid residues 5 to 8, 222 to 225, and 320 to 323 of SEQ ID NO:29; one tyrosine kinase phosphorylation site (PS00007) located at about amino acid residues 91 to 98 of SEQ ID NO:29; and seven N-myristylation sites (PS00008) located at about amino acid residues 56 to 61, 81 to 86, 126 to 131, 169 to 174, 201 to 206, 250 to 255, and 262 to 267 of SEQ ID NO:29.
A plasmid containing the nucleotide sequence encoding human 84234 (clone "Fbh84234FL") was deposited with American Type Culture Collection (ATCC), 10801
University Boulevard, Manassas, VA 20110-2209, on and assigned Accession Number . This deposit will be maintained under the terms ofthe Budapest Treaty on the
International Recognition ofthe Deposit of Microorganisms for the Purposes of Patent Procedure. This deposit was made merely as a convenience for those of skill in the art and is not an admission that a deposit is required under 35 U.S.C. §112.
Figure imgf000031_0001
Figure imgf000032_0001
47476 Polypeptide Characteristics
The 47476 protein contains a significant number of structural characteristics in common with members ofthe ras guanine nucleotide dissociation stimulator family of proteins, the EF- hand calcium-binding domain family of proteins, and the phorbol ester/diacylglycerol binding domain (Cl domain) family of proteins. The term "family" when referring to the protein and nucleic acid molecules ofthe invention means two or more proteins or nucleic acid molecules having a common structural domain or motif and having sufficient amino acid or nucleotide sequence homology as defined herein. Such family members can be naturally or non-naturally occurring and can be from either the same or different species. For example, a family can contain a first protein of human origin as well as other distinct proteins of human origin, or alternatively, can contain homologues of non-human origin, e.g., rat or mouse proteins. Members of a family can also have common functional characteristics.
A ras guanine nucleotide dissociation stimulator family of proteins is characterized by a common fold. Ras guanine nucleotide dissociation stimulator proteins interact with small guanine nucleotide binding proteins such as ras and/or ras family members, e.g., rac, rho, and CDC42, and via their interaction stimulate the small guanine nucleotide binding proteins to release the guanine nucleotide to which they are bound (usually GDP). After releasing the guanine numcleotide, the small guanine nucleotide binding proteins will bind to another guanine nucleotide. Cellular concentrations of GTP are about ten-fold greater than the concentrations of GDP, so after a small guanine nucleotide binding proteins releases the guanine nucleotide to which it is bound, it wll typically bind to a GTP molecule. Thus, ras guanine nucleotide dissociation stimulator proteins act to stimulate the exchange of GDP for GTP bound to small guanine nucleotide binding proteins, which results in the activation of small guanine nucleotide binding proteins. The interaction between ras and the guanine nucleotide dissociation stimulator SOS has been described in Boriack-Sjodin et al. (1998), Nature 394:337-343, the contents of which are incorporated herein by reference. A ras guanine nucleotide dissociation stimulator can include a "ras guanine nucleotide dissociation stimulator CDC25 family signature motif, defined by the sequence: [GAP]-[CT]-V-P-[FY]-X-X-X-X- [LINMFY]-X-[DN]-[LINM]. A ras guanine nucleotide dissociation stimulator CDC25 family signature motif, as defined, can be involved in triggering the dissociation of a guanine nucleotide, e.g., GDP, from a ras or ras-like protein.
A 47476 polypeptide can include a "ras guanine nucleotide dissociation stimulator domain" or regions which are homologous with a "ras guanine nucleotide dissociation stimulator domain". As used herein, the term "ras guanine nucleotide dissociation stimulator domain" includes an amino acid sequence of about 125 to 325 amino acid residues in length having a bit score for the alignment ofthe sequence to a ras guanine nucleotide dissociation stimulators domain (HMM) of at least 75. Preferably, a ras guanine nucleotide dissociation stimulator domain includes at least about 170 to 300 amino acids, more preferably about 180 to 250 amino acid residues, or about 185 to 240 amino acids and has a bit score for the alignment ofthe sequence to the ras guanine nucleotide dissociation stimulator domain (HMM) of at least 100, 150, 200, 225, 240, or greater. The ras guanine nucleotide dissociation stimulator domain (HMM) has been assigned the SMART Accession Number SM0147. An alternative model (HMM) for the ras guanine nucleotide dissociation stimulator domain has been assigned the PFAM Accession Number PF00617. Alignments ofthe ras guanine nucleotide dissociation stimulator domain (amino acids 195 to 381, or 197 to 433 of SEQ ID NO:2) of human 47476 with consensus amino acid sequences derived from a hidden Markov model according to PFAM (SEQ ID NO:31) and SMART (SEQ ID NO:32) are depicted in Figures 2A and 2B, respectively. In a preferred embodiment a 47476 polypeptide or protein includes a "ras guanine nucleotide dissociation stimulator domain" or a region which includes at least about 170 to 300 amino acids, more preferably about 180 to 250 amino acid residues, or about 185 to 240 amino acid residues and has at least about 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, or more homology with a "ras guanine nucleotide dissociation stimulator domain", e.g., the ras guanine nucleotide dissociation stimulator domain of human 47476 (e.g., residues 195 to 381 or 197 to 433 of SEQ ID NO:2). Some ras guanine nucleotide dissociation stimulator family members include a "ras guanine nucleotide dissociation stimulator N-terminal motif, which is a domain that is predominantly alpha helical, is located N-terminally to the ras guanine nucleotide dissociation stimulator domain, and is believed to play a structural role (i.e., non-catalytic role) within the context of the full-length protein.
A 47476 polypeptide can include a "ras guanine nucleotide dissociation stimulator N- terminal motif or or regions which are homologous with a "ras guanine nucleotide dissociation stimulator N-terminal motif.
As used herein, the term "ras guanine nucleotide dissociation stimulator N-terminal motif includes an amino acid sequence of about 75 to 250 amino acid residues in length having a bit score for the alignment ofthe sequence to the ras guanine nucleotide dissociation stimulator N-terminal motif domain profile (HMM) of at least 5. Preferably, a ras guanine nucleotide dissociation stimulator N-terminal motif includes at least about 100 to 200 amino acids, more preferably about 110 to 150 amino acid residues, or about 115 to 125 amino acids and has a bit score for the alignment ofthe sequence to the ras guanine nucleotide dissociation stimulator N-terminal motif domain profile (HMM) of at least 6, 7, 8, or greater. The ras guanine nucleotide dissociation stimulator N-terminal motif domain profile (HMM) has been assigned the SMART Accession Number SM0229. An alignment ofthe ras guanine nucleotide dissociation stimulator N-terminal motif (amino acids 55 to 172 of SEQ ID NO:2) of human 47476 with a consensus amino acid sequence (SEQ ID NO:33) derived from a hidden Markov model according to SMART is depicted in Figures 2C.
In a preferred embodiment a 47476 polypeptide or protein includes a "ras guanine nucleotide dissociation stimulator N-terminal motif or a region which includes at least about 100 to 200 amino acids, more preferably about 110 to 150 amino acid residues, or about 115 to 125 amino acid residues and has at least about 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, or more homology with a "ras guanine nucleotide dissociation stimulator N-terminal motif, e.g., the ras guanine nucleotide dissociation stimulator N-terminal motif of human 47476 (e.g., residues 55 to 172 of SEQ ID NO:2).
An EF-hand family of proteins is also characterized by a common fold. EF-hand domains typically consist of a twelve-residue loop flanked on both sides by twelve residue alpha-helical domains. In an EF-hand loop, a calcium ion can be coordinated in a pentagonal bipyramidal configuration. The six residues involved in the binding are in positions 1, 3, 5, 7, 9 and 12 ofthe loop. The invariant Glu or Asp at position 12 provides two oxygens for liganding Ca2+, a bidentate ligand. An EF-hand domain can include an "EF-hand calcium-binding motif, defined by the sequence: D-X-[DNS]-{ILVFYW}-[DENSTG]-[DNQGHRK]-{GP}-[LIVMC]- [DENQSTAGC]-X-X-[DE]-[LIVMFYW]. An EF-hand calcium-binding motif, as defined, can be involved in binding a calcium ion, e.g., a calcium ion present in the cytoplasm of a cell. A 47476 polypeptide can include an "EF-hand calcium-binding domain" or regions homologous with an "EF-hand calcium-binding domain".
As used herein, the term "EF-hand calcium-binding domain" includes an amino acid sequence of about 20 to 60 amino acid residues in length and having a bit score for the alignment ofthe sequence to the EF-hand calcium-binding domain (HMM) of at least 8. Preferably, an EF-hand calcium-binding domain includes at least about 20 to 50 amino acids, more preferably about 25 to 40 amino acid residues, or about 28 to 30 amino acids and has a bit score for the alignment ofthe sequence to the EF-hand calcium-binding domain (HMM) of at least 10, 11, 12, 13, 14, 15, or greater. The EF-hand calcium-binding domain (HMM) has been assigned the PFAM Accession Number PF00036. An alternative model (HMM) for the ras guanine nucleotide dissociation stimulator domain has been assigned the SMART Accession Number SM0054. Alignments ofthe EF-hand calcium-binding domain (amino acids 470 to 498 of SEQ ID NO:2) of human 47476 with consensus amino acid sequences derived from hidden Markov models according to PFAM (SEQ ID NO:34) and SMART (SEQ ID NO:35) are depicted in Figures 3A and 3B, respectively.
In a preferred embodiment a 47476 polypeptide or protein includes an "EF-hand calcium-binding domain" or a region which includes at least about 20 to 50, more preferably about 25 to 40, or about 28 to 30 amino acid residues and has at least about 50%, 60%, 70%, 80%, 90%), 95%), 98%, 99%, or more homology with an "EF-hand calcium-binding domain" e.g., the EF-hand calcium-binding domain of human 47476 (e.g., residues 470 to 498 of SEQ ID NO:2).
A phorbol ester/diacylglycerol binding family of proteins is characterized by a common fold. The following sequence pattern is representative of a phorbol ester/diacylglycerol binding domain (i.e, a Cl domain): H-X-[LIVMFYW]-X(8,11)-C-X(2)-C-X(3)-[LIVMFC]-X(5,10)-C- X(2)-C-X(4)-[HD]-X(2)-C-X(5,9)-C (SEQ ID NO:60). Phorbol esters can directly stimulate Protein Kinase C, and the N-terminal region of Protein Kinase C, known as Cl, has been shown to bind phorbol esters and diacylglycerols in a phospholipid and zinc-dependent fashion. The Cl region contains one or two copies of a cysteine-rich domain about 50 amino acids in length, and is essential for phorbol ester/diacylglycerol binding. The phorbol ester/diacylglycerol binding domain binds two zinc ions, the ligands of which are most likely the six cysteines and two histidines that are conserved within the phorbol ester/diacylglycerol binding domain.
A 47476 polypeptide can further include a "phorbol ester/diacylglycerol binding domain (Cl domain)" or regions homologous with a "phorbol ester/diacylglycerol binding domain (Cl domain)". As used herein, the term "phorbol ester/diacylglycerol binding domain (Cl domain)" includes an amino acid sequence of about 30 to 100 amino acid residues in length and having a bit score for the alignment ofthe sequence to the phorbol ester/diacylglycerol binding domain (Cl domain) (HMM) of at least 30. Preferably, a phorbol ester/diacylglycerol binding domain (Cl domain) includes at least about 35 to 75 amino acids, more preferably about 40 to 60 amino acid residues, or about 45 to 55 amino acids and has a bit score for the alignment ofthe sequence to the phorbol ester/diacylglycerol binding domain (Cl domain) (HMM) of at least 40, 50, 55, 59, or greater. The phorbol ester/diacylglycerol binding domain (Cl domain) (HMM) has been assigned the PFAM Accession Number PF00130. An alternative model (HMM) for the phorbol ester/diacylglycerol binding domain has been assigned the SMART Accession Number SM0109. Alignments ofthe phorbol ester/diacylglycerol binding domain (Cl domain) (amino acids 541 to 590 of SEQ ID NO:2) of human 47476 with consensus amino acid sequences derived from a hidden Markov model according to PFAM (SEQ ID NO: 36) and SMART (SEQ ID NO:37) are depicted in Figures 4A and 4B, respectively.
In a preferred embodiment a 47476 polypeptide or protein includes a "phorbol ester/diacylglycerol binding domain (Cl domain)" or a region which includes at least about 10 to 100, more preferably about 25 to 75, or about 40 to 60 amino acid residues and has at least about 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, or more homology with a "phorbol ester/diacylglycerol binding domain (Cl domain)" e.g, the phorbol ester/diacylglycerol binding domain (Cl domain) of human 47476 (e.g, residues 541 to 590 of SEQ ID NO:2). To identify the presence of a "Ras guanine nucleotide dissociation stimulator domain", a
"ras guanine nucleotide dissociation stimulator N-terminal motif, an "EF-hand calcium binding domain", or a "phorbol ester/diacylglycerol bining domain" in a 47476 protein sequence, and make the determination that a polypeptide or protein of interest has a particular profile, the amino acid sequence ofthe protein can be searched against the PFAM database of HMMs (e.g, the PFAM database, release 2.1) using the default parameters. For example, the hmmsf program, which is available as part ofthe HMMER package of search programs, is a family specific default program for MILPAT0063 and a score of 15 is the default threshold score for determining a hit. Alternatively, the threshold score for determining a hit can be lowered (e.g, to 8 bits). A description ofthe PFAM database can be found in Sonhammer et al. (1997) Proteins 28(3):405-420 and a detailed description of HMMs can be found, for example, in Gribskov et al. (1990) Meth. Enzymol. 183 : 146-159; Gribskov et al. (1987) Proc. Natl. Acad.
Sci. USA 84:4355-4358; Krogh et /.(1994) J Mol. Biol. 235:1501-1531; and Stultz et a/. (1993) Protein Sci. 2:305-314, the contents of which are incorporated herein by reference. A search was performed against the HMM database resulting in the identification of: a "ras guanine nucleotide dissociation stimulator domain" in the amino acid sequence of human 47476 located at about residues 195 to 381 of SEQ ID NO:2 (see Figure 2A); an "EF-hand calcium binding domain" in the amino acid sequence of human 47476 located at about residues 470 to 498 of SEQ ID NO:2 (see Figure 3A); and a "phorbol ester/diacylglycerol binding domain" in the amino acid sequence of human 47476 located at about residues 541 to 590 of SEQ ID NO:2 (see Figure 4A). Alternatively, to identify the presence of a "ras guanine nucleotide dissociation stimulator domain", a "ras guanine nucleotide dissociation stimulator N-terminal motif, an "EF-hand calcium binding domain", or a "phorbol ester/diacylglycerol binding domain" in a 47476 protein sequence, and make the determination that a polypeptide or protein of interest has a particular profile, the amino acid sequence ofthe protein can be searched against a SMART database (Simple Modular Architecture Research Tool) of HMMs as described in Schultz et al. (1998), Proc. Natl. Acad. Sci. USA 95:5857 and Schultz et al. (200) Nucl. Acids Res 28:231, the contents of which are incorporated herein by reference. The database contains domains identified by profiling with the hidden Markov models ofthe HMMer2 search program (R. Durbin et al. (1998) Biological sequence analysis: probabilistic models of proteins and nucleic acids. Cambridge University Press). The database also is extensively annotated and monitored by experts to enhance accuracy. A search was performed against the HMM database resulting in the identification of: a "ras guanine nucleotide dissociation stimulator domain" in the amino acid sequence of human 47476 at about residues 195 to 381 of SEQ ID NO:2 (see Figure 2B); a "ras guanine nucleotide dissociation stimulator N-terminal motif in the amino acid sequence of human 47476 at about residues 55 to 172 of SEQ ID NO:2 (see Figure 2C); an "EF-hand calcium binding domain" in the amino acid sequence of human 47476 at about residues 470 to 498 of SEQ ID NO:2 (see Figure 3B); and a "phorbol ester/diacylglycerol binding domain" in the amino acid sequence of human 47476 at about residues 541 to 590 of SEQ ID NO:2 (see Figure 4B).
A 47476 family member can include at least one ras guanine nucleotide dissociation stimulator domain, at least one ras guanine nucleotide dissociation sitmulator N-terminal motif, at least one EF-hand calcium-binding domain, and at least one phorbol ester/diacylglycerol binding domain (Cl domain). Furthermore, a 47476 family member can include: at least one predicted N-glycosylation sites (PS00001); at least one, two, three, and preferably four predicted cAMP- and cGMP-dependent protein kinase phosphorylation sites (PS00004); at least one, two, three, four, five, six, seven, and preferably eight predicted protein kinase C phosphorylation sites (PS00005); at least one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen and preferably fourteen predicted casein kinase II phosphorylation sites (PS00006); at least one, two, three, and preferably four predicted N-myristylation sites (PS00008); at least one predicted amidation site (PS00009); and at least one predicted phorbol ester/diacylglycerol binding domain (PS00479).
As the 47476 polypeptides ofthe invention may modulate 47476-mediated activities, they may be useful as of for developing novel diagnostic and therapeutic agents for 47476- mediated or related disorders, as described below.
As used herein, a "47476 activity", "biological activity of 47476" or "functional activity of 47476", refers to an activity exerted by a 47476 protein, polypeptide or nucleic acid molecule. For example, a 47476 activity can be an activity exerted by 47476 in a physiological milieu on, e.g, a 47476-responsive cell or on a 47476 substrate, e.g, a protein substrate. A 47476 activity can be determined in vivo or in vitro. In one embodiment, a 47476 activity is a direct activity, such as an association with a 47476 target molecule. A "target molecule" or "binding partner" is a molecule with which a 47476 protein binds or interacts in nature. In an exemplary embodiment, a 47476 polypeptide or protein has the ability to interact with, and thereby activate, ras and/or a ras-like protein.
A 47476 activity can also be an indirect activity, e.g, a cellular signaling activity mediated by another protein, e.g, ras or a ras-like protein, that the 47476 protein interacts with. Based on the above-described sequence similarities, the 47476 molecules ofthe present invention are predicted to have similar biological activities as ras guanine nucleotide dissociation stimulator (also known as "ras guanine nucleotide exchange factor") family members. For example, the 47476 proteins ofthe present invention can have one or more ofthe following activities: (1) the ability to stimulate the exchange of guanine nucleotides (e.g, GTP for GDP) by another protein, e.g, ras or a ras-like protein; (2) the ability to bind calcium ions; (3) the ability to bind zinc ions; (4) the ability to bind the second messenger, diacylglycerol; (5) the ability to bind analogs of diacylglycerol, such as phorbol esters; (6) the ability to activate members ofthe ras superfamily of proteins; (7) the ability to influence the acitivity of signaling pathways that include ras or ras-like proteins, e.g, growth factor signaling pathways or cellular adhesion signaling pathways; (8) the ability to influence cellular proliferation; (9) the ability to influence cellular differentiation; (10) the ability to influence cellular adhesion; (11) the ability to influence cell migration; or (12) has the ability to antagonize or inhibit, competitively or non- competitively, any of 1 - 11.
In addition, the 47476 molecules ofthe invention can be expected to function in tissues in which they are expressed. For example, human 47476 is expressed in blood cells (e.g, erythroid cells, megakaryocytes, neutrophils, periperal blood mononuclear cells), bone marrow mononuclear cells, spleen, and lung (see Example 2). Thus, the 47476 molecules can act as novel diagnostic targets and therapeutic agents for controlling aberrant or deficient signal transduction resulting in, e.g, hematopoeitic disorders, including, e.g, blood clotting disorders, autoimmune disorders, or disorders related to an inability to clear infections (e.g, viral viral or bacterial infections), as well as disorders related to abnormal cellular proliferation or differentiation, e.g, leukemias.
67210 The 67210 protein contains a significant number of structural characteristics in common with members ofthe glycosyl transferase family. The term "family" when referring to the protein and nucleic acid molecules ofthe invention means two or more proteins or nucleic acid molecules having a common structural domain or motif and having sufficient amino acid or nucleotide sequence homology as defined herein. Such family members can be naturally or non-naturally occurring and can be from either the same or different species. For example, a family can contain a first protein of human origin as well as other distinct proteins of human origin, or alternatively, can contain homologues of non-human origin, e.g, rat or mouse proteins. Members of a family can also have common functional characteristics. The glycosyl transferase family comprises a number of related enzymes that are capable of catalyzing the synthesis of glycoconjugates, including glycolipids, glycoproteins, and polysaccharides, by transferring an activated mono- or oligosaccharide residue to an existing acceptor molecule for the initiation or elongation ofthe carbohydrate chain. The acceptor can be a lipid, a protein, a heterocyclic compound, or another carbohydrate residue. Glycosyltransferases can be divided into numerous subfamilies based upon their specificity for sugar moieties and acceptor molecules. The glycosyltransferase domain of human 67210 bears similarity to a subfamily designated "group 8" glycosyltransferases. These enzymes comprise a subfamily whose members are involved in lipopolysaccharide biosynthesis and glycogen synthesis. Members of this family include lipopolysaccharide galactosyl-transferase, lipopolysaccharide glucosyltransferase 1, and glycogenin glucosyltransferase. Thus, this family includes enzymes critical for the proper function of many physiological systems, including carbohydrate and lipid metabolism, and cellular proliferation and differentiation.
A 67210 polypeptide can include a "glycosyltransferase domain" or regions homologous with a "glycosyltransferase domain". As used herein, the term "glycosyl transferase domain" includes an amino acid sequence of about 100 to 450 amino acid residues in length, having a bit score for the alignment ofthe sequence to the glycosyltransferase domain (HMM) of at least 25. Preferably, a glycosyl transferase domain includes about 200 to 350 amino acid residues, or more preferably about 250 to 300 amino acids and has a bit score for the alignment ofthe sequence to the glycosyltransferase domain (HMM) of at least 30, 35, more 40, or more. The glycosyl transferase domain (HMM) has been assigned the PFAM Accession Number PF01501. An alignment of glycosyl transferase domain (about amino acids 63 to 340 of SEQ ID NO:5) of human 67210 with a consensus amino acid sequence (SEQ ID NO:38) derived from a hidden Markov model is depicted in Figure 6.
In preferred embodiments, a 67210 polypeptide or protein has a "glycosyl transferase domain" or a region which includes at least about 100 to 450, more preferably about 200 to 350, or 250 to 300 amino acid residues and has at least about 60%, 70%, 80%, 90%, 95%, 98%, 99%, or more homology with a "glycosyltransferase," e.g, the glycosyltransferase domain of human 67210 (e.g, residues 63 to 340 of SEQ ID NO:5).
To identify the presence of a "glycosyltransferase" domain in a 67210 protein sequence, and make the determination that a polypeptide or protein of interest has a particular profile, the amino acid sequence ofthe protein can be searched against the PFAM database of HMMs (e.g, the Pfam database, release 2.1) using the default parameters. For example, the hmmsf program, which is available as part ofthe HMMER package of search programs, is a family specific default program for MILPAT0063 and a score of 15 is the default threshold score for determining a hit. Alternatively, the threshold score for determining a hit can be lowered (e.g, to 8 bits). A description ofthe PFAM database can be found in Sonhammer et al. (1997) Proteins 28(3):405-420 and a detailed description of HMMs can be found, for example, in Gribskov et α/.(1990) Meth. Enzymol. 183:146-159; Gribskov et α/.(1987) Proc. Natl. Acad. Sci. USA 84:4355-4358; Krogh et /.(1994) J Mol. Biol. 235:1501-1531; and Stultz et αt.(1993) Protein Sci. 2:305-314, the contents of which are incorporated herein by reference. A search was performed against the HMM database resulting in the identification of a "glycosyl transferase" domain in the amino acid sequence of human 67210 located at about residues 63 to 340 of SEQ ID NO:5 (see Figure 6).
A 67210 peptide can further include a signal sequence. As used herein, a "signal peptide" or "signal sequence" refers to a peptide of about 20 to 60, preferably about 25 to 55, or more preferably about 29 amino acid residues in length which occurs at the N-terminus of secretory and integral membrane proteins and which contains a majority of hydrophobic amino acid residues. For example, a signal sequence contains at least about 20 to 60, preferably about 25 to 55, or more preferably about 30 amino acid residues, and has at least about 40-70%, preferably about 50-65%, and more preferably about 55-60% hydrophobic amino acid residues (e.g, alanine, valine, leucine, isoleucine, phenylalanine, tyrosine, tryptophan, or proline). Such a "signal sequence", also referred to in the art as a "signal peptide", serves to direct a protein containing such a sequence to a lipid bilayer. For example, in one embodiment, a 67210 protein contains a signal sequence located at about amino acid residues 1 to 29 of SEQ ID NO:5. The "signal sequence" is cleaved during processing ofthe mature protein, and the mature 67210 protein corresponds to about amino acid residues 30 to 349 of SEQ ID NO:5.
A 67210 family member can include at least one glycosyl transferase domain and at least one signal peptide. A 67210 family member can further include: at least one dileucine motif; at least one predicted N-glycosylation site (PS00001); at least one predicted Protein Kinase C phosphorylation site (PS00005); at least one, two, three, preferably four predicted Casein Kinase II phosphorylation sites (PS00006); at least one predicted tyrosine kinase phosphorylation site (PS00007); and at least one, two, three, four, five, preferably six predicted N-myristoylation sites (PS00008).
As the 67210 polypeptides ofthe invention may modulate 67210-mediated activities, they may be useful as of for developing novel diagnostic and therapeutic agents for 67210- mediated or related disorders, as described below.
As used herein, a "67210 activity", "biological activity of 67210" or "functional activity of 67210", refers to an activity exerted by a 67210 protein, polypeptide or nucleic acid molecule. For example, a 67210 activity can be an activity exerted by 67210 in a physiological milieu on, e.g, a 67210-responsive cell or on a 67210 substrate, e.g, a protein substrate. A 67210 activity can be determined in vivo or in vitro. In one embodiment, a 67210 activity is a direct activity, such as an association with a 67210 target molecule. A "target molecule" or "binding partner" is a molecule with which a 67210 protein binds or interacts in nature. In an exemplary embodiment, 67210 is an enzyme that modifies (e.g, adds a sugar residue to) a lipid, protein, or carbohydrate substrate. A 67210 activity can also be an indirect activity, e.g, a cellular signaling activity mediated by interaction ofthe 67210 protein with a 67210 receptor. The features ofthe 67210 molecules ofthe present invention can provide similar biological activities as glycosyltransferase family members. For example, the 67210 proteins ofthe present invention can have one or more ofthe following activities: (1) catalyzes the transfer of an activated sugar residue to an acceptor molecule; (2) catalyzes the processing, folding, and secretion of glycoproteins; (3) catalyzes carbohydrate metabolism, e.g, lipopolysaccharide biosynthesis and glycogen synthesis; (4) catalyzes lipid metabolism; (5) modulates cell-cell interactions, e.g, between endothelial cells and blood cells; (6) modulates cell-matrix adhesive interactions; (7) modulates signal transduction, e.g, growth factor signaling; (8) modulates cell proliferation and/or differentiation; (9) modulates tumor cell growth, invasion and/or metastasis; (10) regulates myelin formation; (11) regulates viral and microbial adhesion; (12) modulates oligodendrocyte development; (13) controls sperm-egg binding; (14) regulates evasion of immune detection; (15) modulates xenograft rejection; or (16) has the ability to antagonize or inhibit, competitively or non-competitively, any of 1-15.
In addition, the 67210 molecules ofthe invention can be expected to function in tissues in which they are expressed. For example, human 67210 is expressed in cardiovascular tissues (e.g, arteries and smooth muscle cells), neural tissues (e.g, the brain cortex), and breast and ovary tissues (see Example 2). Thus, the 67210 molecules can act as novel diagnostic targets and therapeutic agents for controlling disorders of metabolic imbalance (e.g, disorders of lipopolysaccharide biosynthesis or glycogen synthesis), immunological disorders (e.g, autoimmune disorders or disorders associated with an inability to clear an infection, e.g, a viral or bacterial infection), cardiovascular disorders, nueurological disorders, or cellular proliferation and/or differentiation disorders, e.g, cancer.
49875 The 49875 protein contains a significant number of structural characteristics in common with members ofthe DEAD type helicase family. The term "family" when referring to the protein and nucleic acid molecules ofthe invention means two or more proteins or nucleic acid molecules having a common structural domain or motif and having sufficient amino acid or nucleotide sequence homology as defined herein. Such family members can be naturally or non-naturally occurring and can be from either the same or different species. For example, a family can contain a first protein of human origin as well as other distinct proteins of human origin, or alternatively, can contain homologues of non-human origin, e.g, rat or mouse proteins. Members of a family can also have common functional characteristics.
A DEAD type helicase family of proteins is characterized by a common DEAD-box subfamily ATP-dependent helicase signature motif. The DEAD-type helicase family comprises a number of related enzymes that share high structural homology and a common catalytic mechanism whereby the enzyme converts the energy from ATP hydrolysis into the mechanical energy required for unwinding of nucleic acid duplexes. For example, DEAD-type helicases catalyze the unwinding of ribonucleic acids during RNA splicing. Thus, this family includes enzymes critical for the proper function of many physiological systems, including replication, transcription, and cellular proliferation and differentiation.
A 49875 polypeptide can include a "DEAD type helicase domain" or regions homologous with a "DEAD type helicase domain".
As used herein, the term "DEAD-type helicase domain" is an amino acid sequence of at least 100 amino acid residues in length and having a bit score for the alignment ofthe sequence to the DEAD type helicase domain (HMM) of at least 100. Preferably, a DEAD type helicase domain includes an amino acid sequence of about 100 to 350 amino acid residues in length, more preferably about 200 to 250 amino acid residues, or about 215 to 235 amino acids and having a bit score for the alignment ofthe sequence to the DEAD type helicase domain (HMM) of at least 100, preferably 150, and most preferably 180 or more. The DEAD type helicase domain (HMM) has been assigned the PFAM Accession Number PF00270
(http;//genome.wustl.edu/Pfam/.html). An alternative model (HMM) for the DEAD type helicase domain has been assigned the SMART Accession Number SM0487. Alignments of the DEAD type helicase domain (amino acids 22 to 245 or 28 to 245 of SEQ ID NO:8) of human 49875 with a consensus amino acid sequence derived from a hidden Markov model according to PFAM (SEQ ID NO:39) or according to SMART (SEQ ID NO:40) are depicted in Figures 8A and 8B, respectively.
In a preferred embodiment 49875 polypeptide or protein has a "DEAD-type helicase domain" or a region which includes at least about 100 to 350, more preferably about 200 to 250, or 215 to 235 amino acid residues and has at least about 60%, 70%, 80%, 90%, 95%, 98%, 99%, or more homology with a "DEAD-type helicase," e.g, the DEAD-type helicase domain of human 49875 (e.g, residues 22 to 245 of SEQ ID NO:8).
As used herein, the term "conserved helicase C-terminal domain" is an amino acid sequence of at least 30 amino acid residues in length having a bit score for the alignment ofthe sequence to the C-terminal helicase domain (HMM) of at least 50. A "conserved helicase C- terminal domain" preferably includes an amino acid sequence of about 20 to 140 amino acid residues in length, more preferably about 40 to 120 amino acid residues, or about 60 to 100 amino acids and having a bit score for the alignment ofthe sequence to the C-terminal helicase domain (HMM) of at least 50, preferably 80, and most preferably 90 or more. The conserved helicase C-terminal domain (HMM) has been assigned the PFAM Accession Number PF00271). An alternative model (HMM) for the conserved helicase C-terminal domain has been assigned the SMART Accession Number SM0490. Alignments ofthe conserved helicase C-terminal domain (amino acids 281 to 363 of SEQ ID NO:8) of human 49875 with consensus amino acid sequences derived from a hidden Markov model derived from PFAM (SEQ ID NO:41) and SMART (SEQ ID NO:42) are depicted in Figures 9A and 9B.
In a preferred embodiment 49875 polypeptide or protein has a "conserved helicase C- terminal domain" or a region which includes at least about 20 to 140, more preferably about 40 to 120, or 60 to 100 amino acid residues and has at least about 60%, 70% 80% 90% 95%, 99%, or 100% homology with a "conserved helicase C-terminal domain," e.g, the conserved helicase C-terminal domain of human 49875 (e.g, residues 281 to 363 of SEQ ID NO:8).
To identify the presence of a "DEAD type helicase domain" or a "conserved helicase C- terminal domain" in a 49875 protein sequence, and make the determination that a polypeptide or protein of interest has a particular profile, the amino acid sequence ofthe protein can be searched against the PFAM database of HMMs (e.g, the PFAM database, release 2.1) using the default parameters. For example, the hmmsf program, which is available as part ofthe HMMER package of search programs, is a family specific default program for MILPAT0063 and a score of 15 is the default threshold score for determining a hit. Alternatively, the threshold score for determining a hit can be lowered (e.g, to 8 bits). A description ofthe PFAM database can be found in Sonhammer et al. (1997) Proteins 28(3):405-420 and a detailed description of HMMs can be found, for example, in Gribskov et α/.(1990) Meth. Enzymol. 183:146-159; Gribskov et al(1987) Proc. Natl. Acad. Sci. USA 84:4355-4358; Krogh et a/. (1994) J Mol. Biol. 235:1501-1531; and Stultz et al.(1993) Protein Sci 2:305-314, the contents of which are incorporated herein by reference. A search was performed against the HMM database resulting in the identification of: a "DEAD type helicase domain" in the amino acid sequence of human 49875 at about residues 22 to 245 of SEQ ID NO:8 (see Figure 8A); and a "conserved helicase C-terminal domain" in the amino acid sequence of human 49875 at about residues 281 to 363 of SEQ ID NO:8 (see Figure 9A). Alternatively, to identify the presence of a "DEAD type helicase domain" or a "conserved helicase C-terminal domain" in a 49875 protein sequence, and make the determination that a polypeptide or protein of interest has a particular profile, the amino acid sequence ofthe protein can be searched against a SMART database (Simple Modular Architecture Research Tool) of HMMs as described in Schultz et al. (1998), Proc. Natl. Acad. Sci. USA 95:5857 and Schultz et al. (200) Nucl Acids Res 28:231, the contents of which are incoφorated herein by reference. The database contains domains identified by profiling with the hidden Markov models ofthe HMMer2 search program (R. Durbin et al. (1998) Biological sequence analysis: probabilistic models of proteins and nucleic acids. Cambridge University Press). The database also is extensively annotated and monitored by experts to enhance accuracy. A search was performed against the HMM database resulting in the identification of: a "DEAD type helicase domain" in the amino acid sequence of human 49875 at about residues 28 to 245 of SEQ ID NO:8 (see Figure 8B); and a "conserved helicase C-terminal domain" ("helicase_C") in the amino acid sequence of human 49875 at about residues 281 to 363 of SEQ ID NO:8 (see Figure 9B).
In some embodiments, a 49875 protein includes at least one DEAD-box subfamily ATP- dependent helicase signature motif. As used herein, a "DEAD-box subfamily ATP-dependent helicase signature motif includes a sequence of at least six amino acid residues defined by the sequence: [LIVMF]-[LIVMF]-D-E-A-D-[RKEN]-X-[LIVMFYGSTN] (SEQ ID NO:61). A DEAD-box subfamily ATP-dependent helicase signature motif, as defined, can be involved in unwinding a nucleic acid double helix, e.g, a DNA or RNA double helix. More preferably, a DEAD-box subfamily ATP-dependent helicase signature motif includes 7, 8, and most preferably 9 amino acid residues. The DEAD-box subfamily ATP-dependent helicase signature motif have been given the PROSITE Accession Number PS00039. In preferred embodiments, a 49875 polypeptide or protein has at least one DEAD-box subfamily ATP-dependent helicase signature motif, or a region which includes at least 6, 7, 8, or even 9 amino acid residues and has at least 70%, 80%, 90%, or 100% homology with a "DEAD-box subfamily ATP-dependent helicase signature motif, e.g, DEAD-box subfamily ATP-dependent helicase signature motif of human 49875, e.g, about amino acid residues 169 to 177 of SEQ ID NO:8. A 49875 family member can include at least one DEAD type helicase domain, and at least one conserved helicase C-terminal domain. Furthermore, a 49875 family member can include: at least one DEAD-box subfamily ATP-dependent helicase signature motif (PS00039); at least one, preferably two predicted N-glycosylation sites (PS00001); at least one, two, three, preferably four predicted protein kinase C phosphorylation sites (PS00005); at least one, two, three, four, five, six, preferably seven predicted casein kinase II phosphorylation sites (PS00006); at least one, two, three, four, five, six, preferably seven predicted N-myristylation sites (PS00008); at least one, preferably two predicted amidation sites (PS00009); and at least one ATP/GTP binding site (P-loop) (PS00017). As the 49875 polypeptides ofthe invention may modulate 49875-mediated activities, they may be useful as of for developing novel diagnostic and therapeutic agents for 49875- mediated or related disorders, as described below.
As used herein, a "49875 activity", "biological activity of 49875" or "functional activity of 49875", refers to an activity exerted by a 49875 protein, polypeptide or nucleic acid molecule. For example, a 49875 activity can be an activity exerted by 49875 in a physiological milieu on, e.g, a 49875-responsive cell or on a 49875 substrate, e.g, ATP or a nucleic acid. A 49875 activity can be determined in vivo or in vitro. In one embodiment, a 49875 activity is a direct activity, such as an association with a 49875 target molecule. A "target molecule" or "binding partner" is a molecule with which a 49875 protein binds or interacts in nature, e.g, ATP or a nucleic acid. In an exemplary embodiment, 49875 is an unwinding enzyme for a nucleic acid substrate, e.g, a double stranded nucleic acid substrate, e.g, double stranded RNA.
The features ofthe 49875 molecules ofthe present invention can provide similar biological activities as DEAD type helicase family members. For example, the 49875 proteins ofthe present invention can have one or more ofthe following activities: (1) binds nucleic acid molecules, e.g, RNA; (2) binds and hydrolyzes nucleoside 5 '-triphosphate (NTP), e.g, ATP or GTP; (3) catalyzes the separation of two complementary strands of a duplex nucleic acid molecules; (4) modulates replication; (5) modulates recombination; (6) modulates transcription; (7) modulates translation; (8) modulates RNA splicing; (9) modulates nucleic acid metabolism; (10) acts as a transcriptional regulator; or (11) has the ability to antagonize or inhibit, competitively or non-competitively, any of 1-10. As a result, the 49875 protein may have a critical function in one or more ofthe following physiological processes: (1) replication, e.g, eukaryotic or viral replication; (2) regulation of transcription; or (3) cell proliferation and differentiation.
In addition, the 49875 molecules ofthe invention can be expected to function in tissues in which they are expressed. For example, human 49875 is expressed in ovary and lung tissues, and the expression is upregulated in ovary and lung tumors(see Example 2). Thus, the 49875 molecules can act as novel diagnostic targets and therapeutic agents for controlling disorders of proliferation and/or differentiation, e.g, cancer, or immunological disorders.
46842 The 46842 protein contains a significant number of structural characteristics in common with members ofthe centaurin family. The 46842 protein has the organization of protein domains typical of centaurins, especially centaurin-γ family members. This domain structure is as follows: a PH domain, an ArfGAP domain, a first ankyrin domain, and a second ankyrin domain. The term "family" when referring to the protein and nucleic acid molecules ofthe invention means two or more proteins or nucleic acid molecules having a common structural domain or motif and having sufficient amino acid or nucleotide sequence homology as defined herein. Such family members can be naturally or non-naturally occurring and can be from either the same or different species. For example, a family can contain a first protein of human origin as well as other distinct proteins of human origin, or alternatively, can contain homologues of non-human origin, e.g, rat or mouse proteins. Members of a family can also have common functional characteristics.
PH domains are small domains found in a diverse class of proteins, including those involved in intracellular signaling and the cytoskeleton. The domain has been implicated in binding the βγ subunit of heterotrimeric G proteins, lipids (e.g, ascorbyl sterates, and phosphoinositols), and phosphoserine and phosphothreonine. The ligand specificity of PH domains can vary from domain to domain. The structures of multiple PH domains have been determined (for a review, see Riddihough (1994) Nat. Struct. Biol. 1:755-757). From analysis of such structures, it is evident that the PH domains represent a conserved fold consisting of two perpendicular β-sheets followed by an amphipathic α-helix despite the lack of absolutely conserved residues. However, the loop regions differ greatly in length and composition. A 46842 polypeptide can include a "PH domain" or regions homologous with a "PH domain".
As used herein, the term "PH domain" includes an amino acid sequence of about 80 to 120 amino acid residues in length and having a bit score for the alignment ofthe sequence to the PH domain (HMM) of at least 30. Preferably, a PH domain includes at least about 80 to 130 amino acids, more preferably about 85 to 120 amino acid residues, or about 90 to 100 amino acids and has a bit score for the alignment ofthe sequence to the PH domain (HMM) of at least 40, 50, 60 or greater. The PH domain (HMM) has been assigned the PFAM Accession Number PF00169. An alignment ofthe PH domain (amino acids 269 to 363 of SEQ ID NO:l 1) of human 46842 with a consensus amino acid sequence (SEQ ID NO:43) derived from a hidden Markov model is depicted in Figure 11.
In a preferred embodiment 46842 polypeptide or protein has a "PH domain" or a region which includes at least about 80 to 130 more preferably about 85 to 120 or 90 to 100 amino acid residues and has at least about 70%, 80%, 90%, 95%, 98%, 99%, or more homology with a "PH domain," e.g, the PH domain of human 46842 (e.g, residues 269 to 363 of SEQ ID NO:l 1). Preferably the PH domain ofthe 46842 polypeptide includes the tripeptide sequence "RWF", e.g, the sequence located at about amino acids 289 to 291 of SEQ ID NO:l 1. Typically, this tripeptide is part ofthe six characteristic residues ofthe PH domain motif, defined by the sequence: K-X-[GAST]-X(6,11)-[RK]-X-R-[ILVFWY]-[ILVFWY]. The PH domain motif has been described in Dowler et al. (2000), Biochem J 351 (Pt 1 ) : 19-31 , the contents of which are incorporated herein by reference.
A 46842 molecule can further include an ArfGAP domain. The ArfGAP domain differs structurally from other GAP domains, e.g, the SOS GAP domain. A signature feature ofthe ArfGAP structure is a bound zinc ion, and conserved cysteine residues for coordinating the zinc ion. An Arf GAP domain structure can be modeled based on the structure ofthe PAPβ protein (Mandiyan et al. (1999) EMBO J. 18:6890). This domain has a three-stranded β-sheet which is surrounded by five α-helices. The PAPβ structural model also contains two ankyrin repeats located on the carboxy terminal side ofthe ArfGAP. These two ankyrin repeats have an extensive surface which contacts the ArfGAP domain. The ArfGAP domain is highly evolved to bind to Arf small GTPases and to stimulated their ability to hydrolyze GTP to GDP. A 46842 polypeptide can include an "ArfGAP domain" or regions homologous with an "ArfGAP domain".
As used herein, the term "ArfGAP domain" includes an amino acid sequence of about 100 to 140 amino acid residues in length and having a bit score for the alignment ofthe sequence to the ArfGAP domain (HMM) of at least 195. Preferably, an ArfGAP domain includes the motif Cys-X-X-Cys-X( 16,17)-Cys-X-X-Cys (SEQ ID NO:62). Preferably, an ArfGAP domain includes at least about 100 to 140 amino acids, more preferably about 110 to 130 amino acid residues, or about 115 to 125 amino acids and has a bit score for the alignment ofthe sequence to the ArfGAP domain (HMM) of at least 100, 120, 140, 160, 180, preferably 190 or greater. The ArfGAP domain (HMM) has been assigned the PFAM Accession Number PF01412. An alignment ofthe ArfGAP domain (amino acids 403 to 525 of SEQ ID NO:l 1) of human 46842 with a consensus amino acid sequence (SEQ ID NO:44) derived from a hidden Markov model (PFAM) is depicted in Figure 12.
In a preferred embodiment, 46842 polypeptide or protein has a "ArfGAP domain" or a region which includes at least about 100 to 140 more preferably about 110 to 130 or 115 to 125 amino acid residues and has at least about 60%, 70% 80% 90% 95%, 99%, or 100% homology with a "ArfGAP domain," e.g, the ArfGAP domain of human 46842 (e.g, residues 403 to 525 of SEQ ID NO: 11). Preferably, the 46842 protein also includes the sequence matching the ArfGAP zinc ion coordinating motif, defined by the sequence: Cys-X-X-Cys-X(16,17)-Cys-X- X-Cys (SEQ ID NO:62). An ArfGAP zinc ion coordinating motif is present in the human 46842 sequence, located at about amino acid residues 421 to 440 of SEQ ID NO: 11.
A 46842 polypeptide can include at least one, preferably two "ankyrin repeats" or regions homologous with "ankyrin repeats".
Ankyrin repeats are short, approximately 30 to 40 amino acid elements form two α- helices, separated by a β-turn. The repeats are typically, consecutive, allowing the helices to pack against one another to form an accordion like structure. Key features of ankyrin repeats include highly conserved alanine, glycine, and dileucine residues.
As used herein, the term "ankyrin repeat" includes an amino acid sequence of about 30 to 50 amino acid residues in length and having a bit score for the alignment ofthe sequence to the ankyrin repeat (HMM) of at least 15. Preferably, a centaurin domain includes at least about 20 to 50 amino acids, more preferably about 25 to 45 amino acid residues, or about 30 to 40 amino acids and has a bit score for the alignment ofthe sequence to the centaurin domain (HMM) of at least 5, more preferably 10, 15, 16 or greater. The ankyrin repeat domain (HMM) has been assigned the PFAM Accession Number PF00023. An alignment ofthe ankyrin repeats (amino acids 702 to 734, and 735 to 767 of SEQ ID NO:l 1) of human 46842 with a consensus amino acid sequence (SEQ ID NO:45) derived from a PFAM hidden Markov model (HMM) is depicted in Figures 13A and 13B.
In a preferred embodiment, a 46842 polypeptide or protein has a "ankyrin repeat domain" or a region which includes at least about 20 to 50 more preferably about 25 to 45 or 30 to 40 amino acid residues and has at least about 60%, 70% 80% 90% 95%, 99%, or 100%) homology with a "ankyrin repeat domain", e.g, the ankyrin repeat domains of human 46842 (e.g, residues 702 to 734, and 735 to 767 of SEQ ID NO:l 1). Preferably, the ankyrin repeats include conserved residues, e.g, the conserved alanine located at about residue 710, glycine at about residue 714, dileucine at about residues 722 to 723, and glycine at about residues 726; the conserved alanine located at about residue 743, glycine at about residue 747, leucine at about residue 756, and glycine at about residues 759 of SEQ ID NO: 11.
To identify the presence of a "PH domain", an "ArfGAP domain", or an "ankyrin repeat domain" in a 46842 protein sequence, and make the determination that a polypeptide or protein of interest has a particular profile, the amino acid sequence ofthe protein can be searched against the Pfam database of HMMs (e.g, the Pfam database, release 2.1) using the default parameters. For example, the hmmsf program, which is available as part ofthe HMMER package of search programs, is a family specific default program for MILPAT0063 and a score of 15 is the default threshold score for determining a hit. Alternatively, the threshold score for determining a hit can be lowered (e.g, to 8 bits). A description ofthe Pfam database can be found in Sonhammer et al. (1997) Proteins 28(3):405-420 and a detailed description of HMMs can be found, for example, in Gribskov et /.(1990) Meth. Enzymol. 183:146-159; Gribskov et ah (1987) Proc. Natl Acad. Sci USA 84:4355-4358; Krogh et α/.(1994) J. Mol Biol. 235:1501- 1531; and Stultz et -./.(1993) Protein Sci. 2:305-314, the contents of which are incorporated herein by reference. A search was performed against the HMM database resulting in the identification of: a "PH domain" in the amino acid sequence of human 46842 at about residues 269 to 363 of SEQ ID NO: 11 (see Figure 11); an "ArfGAP domain" at about residues 403 to 525 of SEQ ID NO: 11 (see Figure 12); and two "ankyrin repeats" at about residues 702 to 734, and 735 to 767 of SEQ ID NO:l 1 (see Figures 13A and 13B).
Alternatively, to identify the presence of a PH domain, an ArfGAP domain, or an ankyrin repeat in a 46842 protein sequence, and make the determination that a polypeptide or protein of interest has a particular profile, the amino acid sequence ofthe protein can be also be searched against a SMART database (Simple Modular Architecture Research Tool), of HMMs as described in Schultz et al. (1998), Proc. Natl. Acad. Sci. USA 95:5857 and Schultz et al. (200) Nucl. Acids Res 28:231. The database contains domains identified by profiling with the hidden Markov models ofthe HMMer2 search program (R. Durbin et al. (1998) Biological sequence analysis: probabilistic models of proteins and nucleic acids. Cambridge University Press). The database also is extensively annotated and monitored by experts to enhance accuracy.
The human 46842 amino acid sequence can also be searched against a database of domains, e.g, the ProDom database (Corpet et al. (1999), Nucl. Acids Res. 27:263-267). For example, Figure 5 of USSN 60/250,327 shows a BLAST alignment ofthe amino terminal domain of human 46842 with a consensus amino acid sequence for oligophrenins derived from a ProDomainNo. 1568 (Release 1999.2; see also ProDom family PD023027 (ProDomain Release 2000.1). The ProDom protein domain database consists of an automatic compilation of homologous domains. Current versions of ProDom are built using recursive PSI-BLAST searches (Altschul SF et al. (1997) Nucleic Acids Res. 25:3389-3402; Gouzy et al. (1999) Computers and Chemistry 23:333-340.) ofthe SWISS-PROT 38 and TREMBL protein databases. The database automatically generates a consensus sequence for each domain. A BLAST search was performed against the ProDom database resulting in the identification of a consensus amino acid sequence for oligophrenins in the amino acid sequence of human 46842 at about residues 3 to 183 of SEQ ID NO: 11.
A 46842 family member can include at least one PH domain, at least one ArfGAP domain, and at least one, preferably two ankyrin repeats. Furthermore, a 46842 family member can include: at least one ArfGAP zinc ion coordinating motifs; at least one, two, three, four, five, six, seven, eight, nine, ten, eleven, preferably twelve predicted protein kinase C phosphorylation sites (PS00005); at least one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, preferably fourteen predicted casein kinase II phosphorylation sites (PS00006); at least one, two, preferably three predicted cAMP/cGMP-dependent protein kinase phosphorylation sites (PS00004); at least one predicted tyrosine kinase phosphorylation site (PS00007); at least one predicted glycosaminoglycan attachment site (PS00002); and at least one, two, three, four, five, six, seven, eight, nine, preferably ten predicted N-myristylation sites (PS00008).
As the 46842 polypeptides ofthe invention may modulate 46842-mediated activities, they may be useful as of for developing novel diagnostic and therapeutic agents for 46842- mediated or related disorders, as described below.
As used herein, a "46842 activity", "biological activity of 46842" or "functional activity of 46842", refers to an activity exerted by a 46842 protein, polypeptide or nucleic acid molecule on e.g, a 46842-responsive cell or on a 46842 substrate, e.g, a protein substrate, as determined in vivo or in vitro. In one embodiment, a 46842 activity is a direct activity, such as an association with a 46842 target molecule. A "target molecule" or "binding partner" is a molecule with which a 46842 protein binds or interacts in nature. In an exemplary embodiment, a 46842 polypeptide or protein is a GTP activating protein (GAP) for Arf proteins, e.g, a phosphoinositide-activated GAP protein.
A 46842 activity can also be an indirect activity, e.g, a cellular signaling activity mediated by another protein that interacts with a 46842 protein, e.g, an Arf or Arf-like protein. Based on the above-described sequence similarities, the 46842 molecules ofthe present invention are predicted to have similar biological activities as centaurin family members. For example, the 46842 proteins ofthe present invention can have one or more ofthe following activities: (1) Arf protein binding; (2) stimulation of Arf GTP hydrolysis; (3) phosphoinositide binding; (4) membrane binding; (5) binding and regulation of actin cytoskeletal structures, e.g, cortical actin, focal adhesions, and/or membrane ruffles; (6) modulation of vesicular transport, e.g, protein trafficing and secretion; or (7) the ability to antagonize or inhibit, competitively or non-competitively, any of 1-6.
In addition, the 46842 molecules ofthe invention can be expected to function in tissues in which they are expressed. Thus, the 46842 molecules can act as novel diagnostic targets and therapeutic agents for controlling cell motility and adhesion disorders (e.g, a metastatic disorder or an immunological disorder, e.g, a disorder related to an inability to clear an infection, e.g, a bacterial or viral infection), and secretory disorders (e.g, a neurological or viral disorder).
33201 The 33201 protein contains a significant number of structural characteristics in common with members ofthe dehydrogenase/reductase family. The term "family" when referring to the protein and nucleic acid molecules ofthe invention means two or more proteins or nucleic acid molecules having a common structural domain or motif and having sufficient amino acid or nucleotide sequence homology as defined herein. Such family members can be naturally or non-naturally occurring and can be from either the same or different species. For example, a family can contain a first protein of human origin as well as other distinct proteins of human origin, or alternatively, can contain homologues of non-human origin, e.g, rat or mouse proteins. Members of a family can also have common functional characteristics.
A dehydrogenase/reductase family member is an enzyme composed of two subunits. Members ofthe dehydrogenase/reductase family include alcohol dehydrogenase and quinone reductase. The , , and forms of these subunits can be mixed and matched among the different human forms ofthe dehydrogenases, creating mixed dimers that are still active. These dimers can exist as homo- or hetero-dimers. Dehydrogenase/reductases typically use two interactions to accomplish their reaction with ethanol. For example, the first is to use an associated zinc atom to position the alcoholic group on ethanol. The second is to use an NAD cofactor (from the vitamin niacin), which performs the reaction of oxidizing the alcohol.
Thus, a 33201 polypeptide can include a domain having dehydrogenase or a reductase activity. Furthermore, a 33201 polypeptide can have domain(s) that confer both dehydrogenase and reductase activity. The particular activity of such a polypeptide, i.e, whether it functions as a dehydrogenase or a reductase may depend upon the conditions, e.g, coenzyme availability, etc. Because ofthe reversibility ofthe reaction, the dehydrogenase and reductase domains of a 33201 polypeptide may be the same. Alternatively, the proteins may be bi-functional in that two separate domains confer dehydrogenase and reductase activity. The domains that confer these activities may therefore be located in the same or different regions ofthe polypeptide. Similarly, subsequences or fragments of 33201 can be capable of one of either ofthe activities, or can be capable of both dehydrogenase and reductase activity. A 33201 polypeptide can include a "dehydrogenase/reductase domain" or an "alcohol dehydrogenase domain" or regions homologous with a "dehydrogenase/reductase domain". Dehydrogenase/reductase domains typically participate in the reversible oxidation of alcohols to aldehydes. As used herein, the term "dehydrogenase/reductase domain" includes an amino acid sequence of about 200 to 400 amino acid residues in length and having a bit score for the alignment ofthe sequence to the dehydrogenase/reductase domain (HMM) of at least 40. Preferably, an dehydrogenase/reductase domain includes at least about 250-375 amino acids, more preferably about 275-350 amino acid residues, or about 310-340 amino acids and has a bit score for the alignment ofthe sequence to the dehydrogenase/reductase domain (HMM) of at least 50, 60, 70 or greater. The dehydrogenase/reductase domain (HMM) has been assigned the PFAM Accession Number PF00107. An alignment ofthe dehydrogenase/reductase domain (amino acids 22 to 345 of SEQ ID NO: 14) of human 33201 with a consensus amino acid sequence derived from a hidden Markov model is depicted in Figure 15. In one embodiment, a 33201 polypeptide is a zinc-containing dehydrogenase/reductase, e.g, is capable of binding one or two atoms of zinc.
When members ofthe superfamily are aligned, several amino acid residues are highly conserved among the various proteins. For example, a comparison of 106 proteins belonging to this superfamily reveals that only three residues are strictly conserved among all members (corresponding to Gly66, Gly86, and Gly201 of mammalian class I dehydrogenase/reductase) (Persson et al. (1994) Eur J.Biochem. 226:15-22). In preferred embodiments, a 33201 polypeptide or protein includes at least three conserved glycine residues, e.g, the three conserved glycine residues of human 33201 located at about amino acid residues 76, 93, and 224 of SEQ ID NO:14.
In a preferred embodiment 33201 polypeptide or protein has a "alcohol hydrogenase domain" or a region which includes at least about 250 to 375, more preferably about 275 to 350, or 310 to 340 amino acid residues and has at least about 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%), or more homology with a "dehydrogenase/reductase domain," e.g, the dehydrogenase/reductase domain of human 33201 (e.g, residues 22 to 345 of SEQ ID NO: 14). To identify the presence of an "dehydrogenase/reductase" domain in a 33201 protein sequence, and make the determination that a polypeptide or protein of interest has a particular profile, the amino acid sequence ofthe protein can be searched against the Pfam database of HMMs (e.g, the Pfam database, release 2.1) using the default parameters. For example, the hmmsf program, which is available as part ofthe HMMER package of search programs, is a family specific default program for MILPAT0063 and a score of 15 is the default threshold score for determining a hit. Alternatively, the threshold score for determining a hit can be lowered (e.g, to 8 bits). A description ofthe Pfam database can be found in Sonhammer et al. (1997) Proteins 28(3):405-420 and a detailed description of HMMs can be found, for example, in Gribskov et α/.(1990) Meth. Enzymol. 183:146-159; Gribskov et al.(1987) Proc Natl. Acad. Sci. USA 84:4355-4358; Krogh et /.(1994) J. Mol. Biol. 235:1501-1531; and Stultz et al(1993) Protein Sci. 2:305-314, the contents of which are incorporated herein by reference. A search was performed against the HMM database resulting in the identification of an
"dehydrogenase/reductase" domain in the amino acid sequence of human 33201 at about residues 22 to 345 of SEQ ID NO: 14 (see Figure 15).
A 33201 family member can include at least one dehydrogenase/reductase domain. Furthermore, a 33201 family member can include: at least one, two, preferably three conserved glycine residues; at least one, two, three, preferably four-predicted N-glycosylation sites
(PS00001); at least one predicted protein kinase C phosphorylation sites (PS00005); at least one, preferably two predicted casein kinase II phosphorylation sites (PS00006); and at least one, two, three, four, five, six, seven, preferably eight predicted N-myristylation sites (PS00008). As the 33201 polypeptides ofthe invention may modulate 33201 -mediated activities, they may be useful as of for developing novel diagnostic and therapeutic agents for 33201- mediated or related disorders, as described below.
As used herein, a "33201 activity", "biological activity of 33201" or "functional activity of 33201", refers to an activity exerted by a 33201 protein, polypeptide or nucleic acid molecule. For example, a 33201 activity can be an activity exerted by 33201 in a physiological milieu on, e.g, a 33201 -responsive cell or on a 33201 substrate, e.g, a protein substrate. A 33201 activity can be determined in vivo or in vitro. In one embodiment, a 33201 activity is a direct activity, such as an association with a 33201 target molecule. A "target molecule" or "binding partner" is a molecule with which a 33201 protein binds or interacts in nature, e.g, an alcohol or a quinone. In an exemplary embodiment, a 33201 polypeptide or protein is an enzyme, e.g, an enzyme that catalyzes the oxidation and/or reduction of a 33201 substrate, e.g, a molecule that contains an alcohol group or a quinone. A 33201 activity can also be an indirect activity, e.g, a cellular signaling activity mediated by a 33201 substrate, such that the activity ofthe 33201 upon the substrate alters its ability to signal, e.g, alters its concentration.
Depending on the conditions, such as cofactor and/or coenzyme availability, a 33201 polypeptide can have reductase or dehydrogenase activity. As used herein, the term "reductase activity" means the ability to add one or more hydrides to a substrate having, for example, a keto group. Typically, the hydride is provided by NADH, NADP, NADPH, or other coenzyme or hydride donor. For example, in the biological conversion of 4-androstenedione to testosterone, a hydrogen ion is transferred from NADPH to the substrate thereby forming NADP+ product. Coenzymes of 33201 polypeptide also include, but are not limited to NAD+ andNAD+ analogues (Plapp et al. (1986) Biochemistry 25:5396-5402 and Yamazaki et al. (1984) J. Biochem. 95:109-115), NADH, NADP+, and NADPH (LaRhee et al. (1984) Biochemistry 23:486-491 and Pollow et al. (1976) J. Steroid Biochem. 7:45-50). For example, a 33201 polypeptide can catalyze the reduction of quinone. A 33201 polypeptide can have a "dehydrogenase activity." As used herein, the term
"dehydrogenase activity" means the ability to directly or indirectly remove a hydride from a substrate. Typically, after removal of a hydride from a substrate, electrons ofthe hydride are transferred to NAD+, NADP+, or other coenzyme (e.g, 3-acetylpyridine adenine dinucleotide phosphate) or hydride acceptor. For example, if the substrate has hydroxyl, dehydrogenation converts the hydroxyl to a keto group and produces NADH or NADPH and a proton. Hydride removal from substrate however does not require the presence of an acceptor. Free hydride can be detected optically by H+ binding to a dye molecule, for example
Based on the above-described sequence similarities, the 33201 molecules ofthe present invention are predicted to have one or more biological activities of dehydrogenase/reductase family members. For example, the 33201 proteins ofthe present invention can have one or more ofthe following activities: (1) the ability to metabolize an alcohol, e.g, to catalyze the reversible oxidation of ethanol to acetaldehyde; (2) the ability to metabolize or remove endogenous or non-endogenous (e.g, xenobiotic) substances, such as toxins; (3) the ability to catalyze the oxidation of retinoic acid; (4) the ability to catalyze the reduction of a substrate, e.g, quinone; (5) the ability to modulate dopamine metabolism; (6) the ability to reduce a bioreductive compound, e.g, a bioreductive antitumor quinone; (7) the ability to modulate cellular differentiation; (8) the ability to modulate cellular proliferation; or (9) the ability to agonize or antagonize one or more ofthe activities 1-7.
Consequently, the 33201 molecules ofthe invention can be involved in pathological conditions, e.g, conditions involving cellular degeneration (e.g, neurodegeneration). Furthermore, the 33201 molecules ofthe invention can be allelic variants associated with a genetic disorder or predisposition due to decreased or increased activity/expression ofthe variant relative to wild-type activity, e.g, increased predisposition towards alcoholism or increased ethanol sensitivity due to a mutation in an dehydrogenase/reductase gene that correlates with predisposition/sensitivity or modulation of chemosensitivity, e.g, due to increased or decreased quinone reductase activity ofthe tissue relative to wild type activity.
In addition, the 33201 molecules ofthe invention can be expected to function in tissues in which they are expressed. Thus, the 33201 molecules can act as novel diagnostic targets and therapeutic agents for controlling one or more disorders, including metabolic disorders, liver disorders, kidney disorders, digestive disorders, and cellular proliferative and/or differentiative disorders.
83378, 84233, 64708, 85041, and 84234
The 83378, 84233, 64708, 85041, and 84234 proteins contain a significant number of structural characteristics in common with members ofthe metal transporter family. The term "family" when referring to the protein and nucleic acid molecules ofthe invention means two or more proteins or nucleic acid molecules having a common structural domain or motif and having sufficient amino acid or nucleotide sequence homology as defined herein. Such family members can be naturally or non-naturally occurring and can be from either the same or different species. For example, a family can contain a first protein of human origin as well as other distinct proteins of human origin, or alternatively, can contain homologues of non-human origin, e.g, rat or mouse proteins. Members of a family can also have common functional characteristics.
Members ofthe metal transporter family of proteins are membrane proteins that increase cellular tolerance to divalent metal ions such as zinc, cadmium, and cobalt by mediating cation diffusion across membranes. Some metal transporter proteins are efflux pumps that remove divalent metal ions from cells. Other metal transporter proteins function to increase cellular tolerance to metal ions by mediating the sequestration of ions in subcellular compartments. Some metal transporter proteins are characterized by a topology comprising six membrane spanning domains, a histidine-rich loop between the fourth and fifth membrane spanning domains, and a long C-terminal tail. Examples of metal transporter proteins include ZnT-1, ZnT-2, and ZnT-3. ZnT-1, a plasma membrane protein, functions as a zinc transporter, mediating the cellular efflux of zinc. ZnT-2 is located in vesicles within a cell and mediates the vesicular sequestration of zinc. ZnT-3 is thought to participate in the accumulation of zinc in synaptic vesicles. As the 83378, 84233, 64708, 85041, or 84234 proteins show homology to metal transporter proteins, they are likely to be involved in mediating tolerance to divalent metal ions, e.g, zinc, in the cells in which they are expressed. 83378, 84233, 64708, 85041, or 84234 proteins are predicted to be, like other members ofthe metal transporter family, transmembrane proteins, embedded either within the plasma membrane or the membrane of a subcellular organelle.
A 83378, 84233, 64708, 85041, or 84234 polypeptide can include a "cation efflux domain" or regions homologous with a "cation efflux domain."
As used herein, the term "cation efflux domain" includes an amino acid sequence of about 100 to 400 amino acid residues in length and having a bit score for the alignment ofthe sequence to the cation efflux domain (HMM) of at least 50. Preferably, a cation efflux domain includes at least about 150 to 350 amino acids, more preferably about 200 to 350 amino acid residues, or about 220 to 330 amino acids and has a bit score for the alignment ofthe sequence to the cation efflux domain (HMM) of at least 65 or greater. The cation efflux domain (HMM) has been assigned the PFAM Accession Number PF01545.
Alignments ofthe first and second cation efflux domains (amino 11 to 133 and 231 to 389 of SEQ ID NO: 17) of human 83378 with consensus amino acid sequences (SEQ ID NO:47 and SEQ ID NO:48) derived from hidden Markov models are depicted in Figures 17A and 17B. A cation efflux domain of a 83378 polypeptide preferably includes about 120-160 amino acids and has a bit score for the alignment ofthe sequence to the cation efflux domain (HMM) of at least 80, 130, or greater.
An alignment ofthe cation efflux domain (amino acids 25 to 310 of SEQ ID NO:20) of human 84233 with a consensus amino acid sequence (SEQ ID NO: 51) derived from a hidden Markov model is depicted in Figure 20. A cation efflux domain of a 84233 polypeptide preferably includes about 270 to 290 amino acids and has a bit score for the alignment ofthe sequence to the cation efflux domain (HMM) of at least 250 or greater.
Alignments ofthe first and second cation efflux domains (amino acids 55 to 153 and 227 to 320 of SEQ ID NO:23) of human 64708 with consensus amino acid sequences (SEQ ID NO:53 and SEQ ID NO:54) derived from hidden Markov models are depicted in Figures 23A and 23B. A cation efflux domain of a 64708 polypeptide preferably includes about 90 to 100 amino acids and has a bit score for the alignment ofthe sequence to the cation efflux domain (HMM) of at least 40 or greater.
An alignment ofthe cation efflux domain (amino acids 419 to 733 of SEQ ID NO:26) of human 85041 with a consensus amino acid sequence (SEQ ID NO:57) derived from a hidden Markov model is depicted in Figure 26. A cation efflux domain of a 85041 polypeptide preferably includes about 310 to 320 amino acids and has a bit score for the alignment ofthe sequence to the cation efflux domain (HMM) of at least 180 or greater.
An alignment ofthe cation efflux domain (amino acids 38 to 349 of SEQ ID NO:29) of human 84234 with a consensus amino acid sequence (SEQ ID NO:58) derived from a hidden Markov model is depicted in Figure 10. A cation efflux domain of a 84234 polypeptide preferably includes about 310 to 320 amino acids and has a bit score for the alignment ofthe sequence to the cation efflux domain (HMM) of at least 160 or greater.
In a preferred embodiment 83378, 84233, 64708, 85041, or 84234 polypeptide or protein has a "cation efflux domain" or a region which includes at least about 150 to 350 more preferably about 200 to 350 or 220 to 330 amino acid residues and has at least about 50%, 60%, 70% 80% 90% 95%, 99%, or 100% homology with a "cation efflux domain," e.g, the cation efflux domain of human 83378, 84233, 64708, 85041, or 84234 (e.g, residues 11 to 133 or 231 to 389 of SEQ ID NO: 17, residues 25 to 310 of SEQ ID NO:20, residues 55 to 153 or 227 to 320 of SEQ ID NO:23, residues 419 to 733 of SEQ ID NO:26, or residues 38 to 349 of SEQ ID NO:29).
To identify the presence of a "cation efflux" domain in a 83378, 84233, 64708, 85041, or 84234 protein sequence, and make the determination that a polypeptide or protein of interest has a particular profile, the amino acid sequence ofthe protein can be searched against the Pfam database of HMMs (e.g, the PFAM database, release 2.1) using the default parameters. For example, the hmmsf program, which is available as part ofthe HMMER package of search programs, is a family specific default program for MILPAT0063 and a score of 15 is the default threshold score for determining a hit. Alternatively, the threshold score for determining a hit can be lowered (e.g, to 8 bits). A description ofthe Pfam database can be found in Sonhammer et al. (1997) Proteins 28(3):405-420 and a detailed description of HMMs can be found, for example, in Gribskov et α/.(1990) Meth. Enzymol 183:146-159; Gribskov et α/.(1987) Proc. Natl. Acad. Sci. USA 84:4355-4358; Krogh et al.(l 994) J Mol. Biol. 235:1501-1531; and Srultz et α/.(1993) Protein Sci. 2:305-314, the contents of which are incorporated herein by reference. Searches were performed against the HMM database resulting in the identification of "cation efflux domains" in the amino acid sequence of human 83378, 84233, 64708, 85041, an 84234 at about: residues 11 to 133 and 231 to 389 of SEQ ID NO:17 (see Figures 17A and 17B); residues 25 to 310 of SEQ ID NO:20 (see Figure 20); residues 55 to 153 and 227 to 320 of SEQ ID NO:23 (see Figures 23A and 23B); residues 419 to 733 of SEQ ID NO:26 (see Figure 26), and residues 38 to 349 of SEQ ID NO:29 (see Figure 29).
A 83378, 84233, 64708, 85041, or 84234 polypeptide can include a "transmembrane domain" or regions homologous with a "transmembrane domain".
As used herein, the term "transmembrane domain" includes an amino acid sequence of about 15 amino acid residues in length which spans a phospholipid membrane. More preferably, a transmembrane domain includes about at least 20, 25, 30, 35, 40, or 45 amino acid residues and spans a phospholipid membrane. Transmembrane domains are rich in hydrophobic residues, and typically have an alpha-helical structure. In a preferred embodiment, at least 50%, 60%, 70%, 80%, 90%, 95% or more ofthe amino acids of a transmembrane domain are hydrophobic, e.g. , leucines, isoleucines, tyrosines, or tryptophans. Transmembrane domains are described in, for example, Zagotta W.N. et al, (1996) Annual Rev. Neurosci. 19: 235-263, the contents of which are incorporated herein by reference. Amino acid residues 11-31, 44-61, 79-98, 115-134, 241-265, and 283-299 ofthe 83378 protein (SEQ ID NO: 17) are predicted to comprise transmembrane domains. Accordingly, 83378 proteins having at least 50-60% homology, preferably about 60-70%, more preferably about 70-80%o, or about 80-90% homology with a transmembrane domain of human 83378 are within the scope ofthe invention. Amino acid residues 25-49, 58-74, 92-113, 128-147, 167-191, and 201-218 ofthe 84233 protein (SEQ ID NO:20) are predicted to comprise transmembrane domains. Accordingly, 84233 proteins having at least 50-60% homology, preferably about 60-70%, more preferably about 70-80%), or about 80-90% homology with a transmembrane domain of human 84233 are within the scope ofthe invention.
Amino acid residues 34-51, 58-82, 101-119, 137-155, 202-219, and 232-249 ofthe 64708 protein (SEQ ID NO:23) are predicted to comprise transmembrane domains.
Accordingly, 64708 proteins having at least 50-60% homology, preferably about 60-70%, more preferably about 70-80%, or about 80-90% homology with a transmembrane domain of human 64708 are within the scope ofthe invention.
Amino acid residues 59-77, 99-119, 129-145, 152-168, 190-214, 239-258, 267-288, 304- 320, 343-362, 419-439, 486-505, 521-541, 592-613, and 618-641 ofthe 85041 protein (SEQ ID NO:26) are predicted to comprise transmembrane domains. Accordingly, 85041 proteins having at least 50-60% homology, preferably about 60-70%, more preferably about 70-80%, or about 80-90% homology with a transmembrane domain of human 85041 are within the scope of the invention. Amino acid residues 38-58, 71-87, 105-123, 141-159, 237-256, and 263-286 ofthe
84234 protein (SEQ ID NO:29) are predicted to comprise transmembrane domains. Accordingly, 84234 proteins having at least 50-60%) homology, preferably about 60-70%, more preferably about 70-80%, or about 80-90%) homology with a transmembrane domain of human 84234 are within the scope ofthe invention. In one embodiment, a 83378, 84233, 64708, 85041, or 84234 protein includes at least one cytoplasmic domain. When located at the N-terminal domain the cytoplasmic domain is referred to herein as an "N-terminal cytoplasmic domain". As used herein, an "N-terminal cytoplasmic domain" includes an amino acid sequence having about 1-300, preferably about 1- 250, preferably about 1-200, more preferably about 1-150, more preferably about 1-100, more preferably about 1-80, or even more preferably about 1-60 amino acid residues in length and is located inside of a cell or intracellularly. The C-terminal amino acid residue of a "N-terminal cytoplasmic domain" is adjacent to an N-terminal amino acid residue of a transmembrane domain in a 83378, 84233, 64708, 85041, or 84234 protein. For example, an N-terminal cytoplasmic domain is located at about amino acid residues 1-10 of SEQ ID NO: 17, 1-24 of SEQ ID NO:20, 1-33 of SEQ ID NO:23, 1-58 of SEQ ID NO:26, and 1-37 of SEQ ID NO:29. In a preferred embodiment, a 83378, 84233, 64708, 85041, or 84234 polypeptide or protein has at least one cytoplasmic domain or a region which includes at least about 5, preferably about 10-300, and more preferably about 30-220 amino acid residues and has at least about 60%, 70%, 80%, 90%, 95%, 99%, or 100% homology with an "cytoplasmic domain," e.g, at least one cytoplasmic domain of human 83378, 84233, 64708, 85041, or 84234 (e.g, residues 1-10, 62-78, 135-240, and 300-485 of SEQ ID NO:17; residues 1-24, 75-91, 148-166, and 219-320 of SEQ ID NO:20; residues 1-33, 83-100, 156-201, and 250-461 of SEQ ID NO:23; residues 1-58, 120-128, 169-189, 259-266, 321-342, 438-485, 542-591, and 642-765 of SEQ ID NO:26; and residues 1-37, 88-104, 160-236, and 287-376 of SEQ ID NO:29). In another embodiment, a 52906, 33408, or 12189 protein includes at least one non- cytoplasmic loop. As used herein, the term "loop" includes an amino acid sequence that resides outside of a phospholipid membrane, having a length of at least about 4, preferably about 5-80, and more preferably about 5-60 amino acid residues, and has an amino acid sequence that connects two transmembrane domains within a protein or polypeptide. Non-cytoplasmic loops include extracellular domains (i.e, outside ofthe cell) and intracellular domains (i.e, within the cell). When referring to membrane-bound proteins found in intracellular organelles (e.g, mitochondria, endoplasmic reticulum, peroxisomes microsomes, vesicles, endosomes, and lysosomes), non-cytoplasmic loops include those domains ofthe protein that reside in the lumen ofthe organelle or the matrix or the intermembrane space. Accordingly, the N-terminal amino acid of a non-cytoplasmic loop is adjacent to a C-terminal amino acid of a transmembrane domain in a 83378, 84233, 64708, 85041, or 84234 molecule, and the C-terminal amino acid of a non-cytoplasmic loop is adjacent to an N-terminal amino acid of a transmembrane domain in a 83378, 84233, 64708, 85041, or 84234 molecule. As used herein, a "non-cytoplasmic loop" includes an amino acid sequence located outside of a cell or within an intracellular organelle. For example, a "non-cytoplasmic loop" can be found at about amino acids 32-43, 99-114, and 266-282 of SEQ ID NO:17; at about amino acids 50-57, 114-127, and 192-200 of SEQ ID NO:20; at about amino acids 52-57, 120-136, and 220-231 of SEQ ID NO:23; at about amino acids 78-98, 146-151, 215-238, 289-303, 363-418, 506-520, and 614-617 of SEQ ID NO:26; and at about amino acids 59-70, 124-140, and 257-262 of SEQ ID NO:29. In a preferred embodiment, a 83378, 84233, 64708, 85041 , or 84234 polypeptide or protein has at least one non-cytoplasmic loop or a region which includes at least about 4, preferably about 5-10, preferably about 10-20, and more preferably about 20-30 amino acid residues and has at least about 60%, 70%, 80%, 90%, 95%, 99%, or 100% homology with an "non-cytoplasmic loop," e.g, at least one non-cytoplasmic loop of human 83378, 84233, 64708, 85041, or 84234 (e.g, residues 32-43, 99-114, and 266-282 of SEQ ID NO:17; residues 50-57, 114-127, and 192-200 of SEQ ID NO:20; residues 52-57, 120-136, and 220-231 of SEQ ID NO:23; residues 78-98, 146-151, 215-238, 289-303, 363-418, 506-520, and 614-617 of SEQ ID NO:26; and residues 59-70, 124-140, and 257-262 of SEQ ID NO:29).
In another embodiment, a 83378, 84233, 64708, 85041, or 84234 protein includes a "C- terminal cytoplasmic domain", also referred to herein as a C-terminal cytoplasmic tail, in the sequence ofthe protein. As used herein, a "C-terminal cytoplasmic domain" includes an amino acid sequence having a length of at least about 30, preferably about 50-350, preferably about 60-250, more preferably about 80-220 amino acid residues and is located within a cell or within the cytoplasm of a cell. Accordingly, the N-terminal amino acid residue of a "C-terminal cytoplasmic domain" is adjacent to a C-terminal amino acid residue of a transmembrane domain in a 83378, 84233, 64708, 85041, or 84234 protein. For example, a C-terminal cytoplasmic domain is found at about amino acid residues 300-485 of SEQ ID NO: 17; at about amino acid residues 219-320 of SEQ ID NO:20; at about amino acid residues 250-461 of SEQ ID NO:23; at about amino acid residues 642-765 of SEQ ID NO:26; and at about amino acid residues 287- 376 of SEQ ID NO:29. In a preferred embodiment, a 83378, 84233, 64708, 85041, or 84234 polypeptide or protein has a C-terminal cytoplasmic domain or a region which includes at least about 50, preferably about 150-550, more preferably about 50-70 amino acid residues and has at least about 60%, 70% 80%) 90% 95%, 99%), or 100% homology with an "C-terminal cytoplasmic domain," e.g, the C-terminal cytoplasmic domain of human 83378, 84233, 64708, 85041, or 84234 (e.g, residues 300-485 of SEQ ID NO:17; residues 219-320 of SEQ ID NO:20; residues 250-461 of SEQ ID NO:23; residues 642-765 of SEQ ID NO:26; and residues 287-376 of SEQ ID NO.29).
Histidine residues in metal transporter proteins play important roles in binding to divalent metal ions such as zinc. Histidine residues located in the cytoplasmic domain between the fourth and fifth transmembrane domains as well as those located in the C-terminal cytoplasmic domain of metal trasnporter proteins are thought to be of particular importance. The 83378 protein has seven histidine residues in the C-terminal cytoplasmic domain (amino acid residues 300-485 of SEQ ID NO: 17). A preferred 83378 polypeptide has at least one, preferably two, three, four, five, six, or seven histidine residues in a C-terminal cytoplasmic domain. An alignment of 83378 with rat ZnT-1 (SEQ ID NO:49) is shown in Figure 20A. An alignment of 83378 with the amino acid sequence of human GenBank™ Accession Number AL359609 (SEQ ID NO:50) is shown in Figure 20B.
The 84233 protein has three histidine residues in the cytoplasmic domain between the fourth and fifth transmembrane domains (amino acid residues 148-166 of SEQ ID NO:20) and four histidine residues in the C-terminal cytoplasmic domain (amino acid residues 219-320 of SEQ ID NO:20). A preferred 84233 polypeptide has at least one, preferably two or three histidine residues in a cytoplasmic domain, e.g, a cytoplasmic domain located between the fourth and fifth transmembrane domains. A preferred 84233 polypeptide has at least one, preferably two, three, or four histidine residues in a C-terminal cytoplasmic domain. An alignment of 84233 with human ZnT-3 (SEQ ID NO:52) is shown in Figure 21. The 64708 protein has one histidine residue in the cytoplasmic domain between the fourth and fifth transmembrane domains (amino acid residues 156-201 of SEQ ID NO:23) and six histidine residues in the C-terminal cytoplasmic domain (amino acid residues 250-461 of SEQ ID NO:23). A preferred 64708 polypeptide has at least one histidine residue in a cytoplasmic domain, e.g, a cytoplasmic domain located between the fourth and fifth transmembrane domains. A preferred 64708 polypeptide has at least one, preferably two, three, four, five, or six histidine residues in a C-terminal cytoplasmic domain. An alignment of 64708 with murine ZnT-11 (SEQ ID NO:55) is shown in Figure 24A. An alignment of 64708 with the amino acid sequence of human GenBank™ Accession Number AK000844 (SEQ ID NO:56) is shown in Figure 24B. The 85041 protein has 15 histidine residue in the cytoplasmic domain between the twelfth and thirteenth transmembrane domains (amino acid residues 542-591 of SEQ ID NO:26) and six histidine residues in the C-terminal cytoplasmic domain (amino acid residues 642-765 of SEQ ID NO:26). A preferred 85041 polypeptide has at least one, preferably two, three, four, five, six, seven, eight, nine, 10, 11, 12, 13, 14, or 15 histidine residues in a cytoplasmic domain, e.g, a cytoplasmic domain located between the twelfth and thirteenth transmembrane domains. A preferred 85041 polypeptide has at least one, preferably two, three, four, five, or six histidine residues in a C-terminal cytoplasmic domain. An alignment of 85041 with murine ZnT-11 (SEQ ID NO: 55) is shown in Figure 27.
The 84234 protein has 21 histidine residue in the cytoplasmic domain between the fourth and fifth transmembrane domains (amino acid residues 160-236 of SEQ ID NO:29) and two histidine residues in the C-terminal cytoplasmic domain (amino acid residues 287-376 of SEQ ID NO:29). A preferred 84234 polypeptide has at least one, preferably two, three, four, five, six, seven, eight, nine, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21 histidine residues in a cytoplasmic domain, e.g, a cytoplasmic domainlocated between the fourth and fifth transmembrane domains. A preferred 84234 polypeptide has at least one, preferably two histidine residues in a C-terminal cytoplasmic domain. An alignment of 84234 with murine ZnT-12 (SEQ ID NO:59) is shown in Figure 30.
A 83378, 84233, 64708, 85041, or 84234 family member can include a cation efflux domain and at least one, preferably two, three, four, five, or six transmembrane domains. A 85041 family member can further include seven, eight, nine, 10, 11, 12, 13, or 14 transmembrane domains. Furthermore, a 83378, 84233, 64708, 85041, or 84234 family member can include at least one, two, three, or preferably four cytoplasmic domains. A 85041 family member can further include five six, seven, or eight cytoplasmic domains. Furthermore, a 83378, 84233, 64708, 85041, or 84234 family member can include at least one, two, or preferably three non-cytoplasmic loops. A 85041 family member can further include four, five six, or seven, non-cytoplasmic loops.
As the 83378, 84233, 64708, 85041, or 84234 polypeptides ofthe invention may modulate 83378, 84233, 64708, 85041, or 84234-mediated activities, they may be useful as of for developing novel diagnostic and therapeutic agents for 83378, 84233, 64708, 85041, or 84234-mediated or related disorders, as described below. As used herein, a "83378, 84233, 64708, 85041 , or 84234 activity", "biological activity of 83378, 84233, 64708, 85041, or 84234" or "functional activity of 83378, 84233, 64708, 85041, or 84234", refers to an activity exerted by a 83378, 84233, 64708, 85041, or 84234 protein, polypeptide or nucleic acid molecule. For example, a 83378, 84233, 64708, 85041, or 84234 activity can be an activity exerted by 83378, 84233, 64708, 85041, or 84234 in a physiological milieu on, e.g, a 83378, 84233, 64708, 85041, or 84234-responsive cell or on a 83378, 84233, 64708, 85041, or 84234 substrate, e.g, a protein substrate. A 83378, 84233, 64708, 85041, or 84234 activity can be determined in vivo or in vitro. In one embodiment, a 83378, 84233, 64708, 85041, or 84234 activity is a direct activity, such as an association with a 83378, 84233, 64708, 85041, or 84234 target molecule. A "target molecule" or "binding partner" is a molecule with which a 83378, 84233, 64708, 85041, or 84234 protein binds or interacts in nature, e.g, a divalent metal ion, e.g, zinc. In an exemplary embodiment, 83378, 84233, 64708, 85041, or 84234 is a transporter of divalent metal ions.
A 83378, 84233, 64708, 85041, or 84234 activity can also be an indirect activity, e.g, a cellular signaling activity mediated by interaction ofthe 83378, 84233, 64708, 85041, or 84234 protein with a 83378, 84233, 64708, 85041, or 84234 receptor. The features ofthe 83378, 84233, 64708, 85041, or 84234 molecules ofthe present invention can provide similar biological activities as metal transporter family members. For example, the 83378, 84233, 64708, 85041, or 84234 proteins ofthe present invention can have one or more ofthe following activities: (1) modulate cellular tolerance and/or resistance to a metal ion, e.g, zinc; (2) facilitate cation diffusion; (3) modulate cellular efflux of a metal ion, e.g, zinc; (4) modulate vesicular sequestration of a metal ion, e.g, zinc; (5) modulate sequestration of a metal ion, e.g, zinc, in synaptic vesicles; or (6) bind to a metal ion, e.g, zinc.
In addition, the 83378, 84233, 64708, 85041, or 84234 molecules ofthe invention can be expected to function in tissues in which they are expressed. Thus, the 83378, 84233, 64708, 85041, or 84234 molecules can act as novel diagnostic targets and therapeutic agents for controlling metal transport-related disorders, e.g, disorders associated with cellular toxicity resulting from aberrant or deficient cation diffusion. Furthermore, 83378, 84233, 64708, 85041, or 84234 molecules can be used for metal detoxification, e.g, to treat cells or individuals containing excessive or unwanted amounts of metal ions.
Accordingly, the 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 molecules can act as novel diagnostic targets and therapeutic agents for controlling one or more of cellular proliferative and/or differentiative disorders, immunological disorders (e.g, inflammatory disorders), red blood cell disorders, viral diseases, neurological disorders (e.g, brain disorders), pain or metabolic disorders, liver disorders, kidney disorders, disorders ofthe small intestine, disorder of metal ion imbalance, protein trafficking disorders, cardiovascular disorders, and disorders associated with bone metabolism. Examples of cellular proliferative and/or differentiative disorders include cancer, e.g, carcinoma, sarcoma, metastatic disorders or hematopoietic neoplastic disorders, e.g, leukemias. A metastatic tumor can arise from a multitude of primary tumor types, including but not limited to those of prostate, colon, lung, breast and liver origin. As used herein, the terms "cancer", "hyperproliferative" and "neoplastic" refer to cells having the capacity for autonomous growth. Examples of such cells include cells having an abnormal state or condition characterized by rapidly proliferating cell growth. Hyperproliferative and neoplastic disease states may be categorized as pathologic, i.e, characterizing or constituting a disease state, or may be categorized as non-pathologic, i.e, a deviation from normal but not associated with a disease state. The term is meant to include all types of cancerous growths or oncogenic processes, metastatic tissues or malignantly transformed cells, tissues, or organs, irrespective of histopathologic type or stage of invasiveness. "Pathologic hyperproliferative" cells occur in disease states characterized by malignant tumor growth. Examples of non-pathologic hyperproliferative cells include proliferation of cells associated with wound repair.
The terms "cancer" or "neoplasms" include malignancies ofthe various organ systems, such as affecting lung, breast, thyroid, lymphoid, gastrointestinal, and genito-urinary tract, as well as adenocarcinomas which include malignancies such as most colon cancers, renal-cell carcinoma, prostate cancer and/or testicular tumors, non-small cell carcinoma ofthe lung, cancer ofthe small intestine and cancer ofthe esophagus.
The term "carcinoma" is art recognized and refers to malignancies of epithelial or endocrine tissues including respiratory system carcinomas, gastrointestinal system carcinomas, genitourinary system carcinomas, testicular carcinomas, breast carcinomas, prostatic carcinomas, endocrine system carcinomas, and melanomas. Exemplary carcinomas include those forming from tissue ofthe cervix, lung, prostate, breast, head and neck, colon and ovary. The term also includes carcinosarcomas, e.g, which include malignant tumors composed of carcinomatous and sarcomatous tissues. An "adenocarcinoma" refers to a carcinoma derived from glandular tissue or in which the tumor cells form recognizable glandular structures.
The term "sarcoma" is art recognized and refers to malignant tumors of mesenchymal derivation. Examples of cellular proliferative and/or differentiative disorders ofthe colon include, but are not limited to, non-neoplastic polyps, adenomas, familial syndromes, colorectal carcinogenesis, colorectal carcinoma, and carcinoid tumors.
Examples of cellular proliferative and/or differentiative disorders ofthe liver include, but are not limited to, nodular hyperplasias, adenomas, and malignant tumors, including primary carcinoma ofthe liver and metastatic tumors.
Examples of cellular proliferative and/or differentiative disorders ofthe breast include, but are not limited to, proliferative breast disease including, e.g, epithelial hyperplasia, sclerosing adenosis, and small duct papillomas; tumors, e.g, stromal tumors such as fibroadenoma, phyllodes tumor, and sarcomas, and epithelial tumors such as large duct papilloma; carcinoma ofthe breast including in situ (noninvasive) carcinoma that includes ductal carcinoma in situ (including Paget's disease) and lobular carcinoma in situ, and invasive (infiltrating) carcinoma including, but not limited to, invasive ductal carcinoma, invasive lobular carcinoma, medullary carcinoma, colloid (mucinous) carcinoma, tubular carcinoma, and invasive papillary carcinoma, and miscellaneous malignant neoplasms. Disorders in the male breast include, but are not limited to, gynecomastia and carcinoma.
Examples of cellular proliferative and/or differentiative disorders ofthe lung include, but are not limited to, bronchogenic carcinoma, including paraneoplastic syndromes, bronchioloalveolar carcinoma, neuroendocrine tumors, such as bronchial carcinoid, miscellaneous tumors, and metastatic tumors; pathologies ofthe pleura, including inflammatory pleural effusions, noninflammatory pleural effusions, pneumothorax, and pleural tumors, including solitary fibrous tumors (pleural fibroma) and malignant mesothelioma.
Additional examples of proliferative disorders include hematopoietic neoplastic disorders. As used herein, the term "hematopoietic neoplastic disorders" includes diseases involving hyperplastic/neoplastic cells of hematopoietic origin. A hematopoietic neoplastic disorder can arise from myeloid, lymphoid or erythroid lineages, or precursor cells thereof. Preferably, the diseases arise from poorly differentiated acute leukemias, e.g, erythroblastic leukemia and acute megakaryoblastic leukemia. Additional exemplary myeloid disorders include, but are not limited to, acute promyeloid leukemia (APML), acute myelogenous leukemia (AML) and chronic myelogenous leukemia (CML) (reviewed in Vaickus, L. (1991) Crit Rev. in Oncol./Hemotol. 11 :267-97); lymphoid malignancies include, but are not limited to acute lymphoblastic leukemia (ALL) which includes B-lineage ALL and T-lineage ALL, chronic lymphocytic leukemia (CLL), prolymphocytic leukemia (PLL), hairy cell leukemia (HLL) and Waldenstrom's macroglobulinemia (WM). Additional forms of malignant lymphomas include, but are not limited to non-Hodgkin lymphoma and variants thereof, peripheral T cell lymphomas, adult T cell leukemia/lymphoma (ATL), cutaneous T-cell lymphoma (CTCL), large granular lymphocytic leukemia (LGF), Hodgkin's disease and Reed- Sternberg disease.
Immunological disorders or "inflammatory disorders" include disorders that involve hematopoieitic cells, e.g, a myeloid, lymphoid or erythroid cell, or a precursor cell thereof. Examples of such cells include myelocytic cells (polymorphonuclear cells), erythrocytic cells, lymphocytes, monocytes, reticular cells, plasma cells and megakaryocytes, as well as stem cells for the different lineages, and precursors for the committed progenitor cells, for example, precursors of blood cells (e.g, red blood cells, such as erythroblasts), macrophages (monoblasts), platelets (megakaryocytes), polymorphonuclear leucocytes (myeloblasts), and lymphocytes (lymphoblasts). Immunological disorders or diseases include, but are not limited to, autoimmune diseases (including, for example, diabetes mellitus, arthritis (including rheumatoid arthritis, juvenile rheumatoid arthritis, osteoarthritis, psoriatic arthritis), multiple sclerosis, encephalomyelitis, myasthenia gravis, systemic lupus erythematosis, autoimmune thyroiditis, dermatitis (including atopic dermatitis and eczematous dermatitis), psoriasis, Sjogren's Syndrome, Crohn's disease, aphthous ulcer, iritis, conjunctivitis, keratoconjunctivitis, ulcerative colitis, asthma, allergic asthma, cutaneous lupus eryfhematosus, scleroderma, vaginitis, proctitis, drug eruptions, leprosy reversal reactions, erythema nodosum leprosum, autoimmune uveitis, allergic encephalomyelitis, acute necrotizing hemorrhagic encephalopathy, idiopathic bilateral progressive sensorineural hearing loss, aplastic anemia, pure red cell anemia, idiopathic thrombocytopenia, polychondritis, Wegener's granulomatosis, chronic active hepatitis, Stevens-Johnson syndrome, idiopathic sprue, lichen planus, Graves' disease, sarcoidosis, primary biliary cirrhosis, uveitis posterior, and interstitial lung fibrosis), graft- versus-host disease, cases of transplantation, and allergy such as, atopic allergy.
Disorders involving red blood cells include, but are not limited to, anemias, such as hemolytic anemias, including hereditary spherocytosis, hemolytic disease due to erythrocyte enzyme defects: glucose-6-phosphate dehydrogenase deficiency, sickle cell disease, thalassemia syndromes, paroxysmal nocturnal hemoglobinuria, immunohemolytic anemia, and hemolytic anemia resulting from trauma to red cells; and anemias of diminished erythropoiesis, including megaloblastic anemias, such as anemias of vitamin B 12 deficiency: pernicious anemia, and anemia of folate deficiency, iron deficiency anemia, anemia of chronic disease, aplastic anemia, pure red cell aplasia, and other forms of marrow failure.
Disorders related to reduced platelet number, thrombocytopenia, include idiopathic thrombocytopenic purpura, including acute idiopathic thrombocytopenic purpura, drug-induced thrombocytopenia, HIV-associated thrombocytopenia, and thrombotic microangiopathies: thrombotic thrombocytopenic purpura and hemolytic-uremic syndrome. Bone marrow disorders include but are not limited to the following: diseases involving hematopoeitic stem cells; committed lymphoid progenitor cells; lymphoid cells including B and T-cells; committed myeloid progenitors, including monocytes, granulocytes, and megakaryocytes; and committed erythroid progenitors. These include but are not limited to the leukemias, including B-lymphoid leukemias, T-lymphoid leukemias, undifferentiated leukemias; erythroleukemia, megakaryoblastic leukemia, monocytic; [leukemias are encompassed with and without differentiation; chronic and acute lymphoblastic leukemia, chronic and acute lymphocytic leukemia, chronic and acute myelogenous leukemia, lymphoma, myelo dysplastic syndrome, chronic and acute myeloid leukemia, myelomonocytic leukemia; chronic and acute myeloblastic leukemia, chronic and acute myelogenous leukemia, chronic and acute promyelocytic leukemia, chronic and acute myelocytic leukemia, hematologic malignancies of monocyte-macrophage lineage, such as juvenile chronic myelogenous leukemia; secondary AML, antecedent hematological disorder; refractory anemia; aplastic anemia; reactive cutaneous angioendotheliomatosis; fibrosing disorders involving altered expression in dendritic cells, disorders including systemic sclerosis, E-M syndrome, epidemic toxic oil syndrome, eosinophilic fasciitis localized forms of scleroderma, keloid, and fibrosing colonopathy; angiomatoid malignant fibrous histiocytoma; carcinoma, including primary head and neck squamous cell carcinoma; sarcoma, including kaposi's sarcoma; fibroadanoma and phyllodes tumors, including mammary fibroadenoma; stromal tumors; phyllodes tumors, including histiocytoma; ery hroblastosis; neurofibromatosis; diseases ofthe vascular endothelium; demyelinating, particularly in old lesions; gliosis, vasogenic edema, vascular disease, Alzheimer's and Parkinson's disease; T-cell lymphomas; B-cell lymphomas. Disorders involving the spleen include, but are not limited to, splenomegaly, including nonspecific acute splenitis, congestive spenomegaly, and spenic infarcts; neoplasms, congenital anomalies, and rupture. Disorders associated with splenomegaly include infections, such as nonspecific splenitis, infectious mononucleosis, tuberculosis, typhoid fever, brucellosis, cytomegalovirus, syphilis, malaria, histoplasmosis, toxoplasmosis, kala-azar, trypanosomiasis, schistosomiasis, leishmaniasis, and echinococcosis; congestive states related to partial hypertension, such as cirrhosis ofthe liver, portal or splenic vein thrombosis, and cardiac failure; lymphohematogenous disorders, such as Hodgkin disease, non-Hodgkin lymphomas/leukemia, multiple myeloma, myeloproliferative disorders, hemolytic anemias, and thrombocytopenic purpura; immunologic-inflammatory conditions, such as rheumatoid arthritis and systemic lupus erythematosus; storage diseases such as Gaucher disease, Niemann-Pick disease, and mucopolysaccharidoses; and other conditions, such as amyloidosis, primary neoplasms and cysts, and secondary neoplasms.
Examples of viral diseases include, but are not limited to, Hepatitis B, Hepatitis C and Herpes Simplex Virus (HSV). Modulators of 47476, 67210, and 46842 activity, in particular, could be used to control viral diseases. The modulators can be used in the treatment and/or diagnosis of viral infected tissue or virus-associated tissue fibrosis, especially liver and liver fibrosis. Also, 47476, 67210, and 46842 modulators can be used in the treatment and/or diagnosis of virus-associated carcinoma, especially hepatocellular cancer. Neurological disorders or disorders involving the brain include, but are not limited to, disorders involving neurons, and disorders involving glia, such as astrocytes, oligodendrocytes, ependymal cells, and microglia; cerebral edema, raised intracranial pressure and herniation, and hydrocephalus; malformations and developmental diseases, such as neural tube defects, forebrain anomalies, posterior fossa anomalies, and syringomyelia and hydromyelia; perinatal brain injury; cerebrovascular diseases, such as those related to hypoxia, ischemia, and infarction, including hypotension, hypoperfusion, and low-flow states—global cerebral ischemia and focal cerebral ischemia—infarction from obstruction of local blood supply, intracranial hemorrhage, including intracerebral (intraparenchymal) hemorrhage, subarachnoid hemorrhage and ruptured berry aneurysms, and vascular malformations, hypertensive cerebrovascular disease, including lacunar infarcts, slit hemorrhages, and hypertensive encephalopathy; infections, such as acute meningitis, including acute pyogenic (bacterial) meningitis and acute aseptic (viral) meningitis, acute focal suppurative infections, including brain abscess, subdural empyema, and extradural abscess, chronic bacterial meningoencephalitis, including tuberculosis and mycobacterioses, neurosyphilis, and neuroborreliosis (Lyme disease), viral meningoencephalitis, including arthropod-borne (Arbo) viral encephalitis, Herpes simplex virus Type 1, Herpes simplex virus Type 2, Varicalla-zoster virus (Herpes zoster), cytomegalovirus, poliomyelitis, rabies, and human immunodeficiency virus 1, including HIV-1 meningoencephalitis (subacute encephalitis), vacuolar myelopathy, AIDS-associated myopathy, peripheral neuropathy, and AIDS in children, progressive multifocal leukoencephalopathy, subacute sclerosing panencephalitis, fungal meningoencephalitis, other infectious diseases of the nervous system; transmissible spongiform encephalopathies (prion diseases); demyelinating diseases, including multiple sclerosis, multiple sclerosis variants, acute disseminated encephalomyelitis and acute necrotizing hemorrhagic encephalomyelitis, and other diseases with demyelination; degenerative diseases, such as degenerative diseases affecting the cerebral cortex, including Alzheimer disease and Pick disease, degenerative diseases of basal ganglia and brain stem, including Parkinsonism, idiopathic Parkinson disease (paralysis agitans), progressive supranuclear palsy, corticobasal degenration, multiple system atrophy, including striatonigral degenration, Shy-Drager syndrome, and olivopontocerebellar atrophy, and Huntington disease; spinocerebellar degenerations, including spinocerebellar ataxias, including Friedreich ataxia, and ataxia-telanglectasia, degenerative diseases affecting motor neurons, including amyotrophic lateral sclerosis (motor neuron disease), bulbospinal atrophy (Kennedy syndrome), and spinal muscular atrophy; inborn errors of metabolism, such as leukodystrophies, including Krabbe disease, metachromatic leukodystrophy, adrenoleukodystrophy, Pelizaeus- Merzbacher disease, and Canavan disease, mitochondrial encephalomyopathies, including Leigh disease and other mitochondrial encephalomyopathies; toxic and acquired metabolic diseases, including vitamin deficiencies such as thiamine (vitamin Bl) deficiency and vitamin B12 deficiency, neurologic sequelae of metabolic disturbances, including hypoglycemia, hyperglycemia, and hepatic encephatopathy, toxic disorders, including carbon monoxide, methanol, ethanol, and radiation, including combined methotrexate and radiation-induced injury; tumors, such as gliomas, including astrocytoma, including fϊbrillary (diffuse) astrocytoma and glioblastoma multiforme, pilocytic astrocytoma, pleomorphic xanthoastrocytoma, and brain stem glioma, oligodendroglioma, and ependymoma and related paraventricular mass lesions, neuronal tumors, poorly differentiated neoplasms, including medulloblastoma, other parenchymal tumors, including primary brain lymphoma, germ cell tumors, and pineal parenchymal tumors, meningiomas, metastatic tumors, paraneoplastic syndromes, peripheral nerve sheath tumors, including schwannoma, neurofibroma, and malignant peripheral nerve sheath tumor (malignant schwannoma), and neurocutaneous syndromes (phakomatoses), including neurofibromotosis, including Type 1 neurofibromatosis (NF1) and TYPE 2 neurofibromatosis (NF2), tuberous sclerosis, and Von Hippel-Lindau disease.
Diseases of metabolic imbalance include, but are not limited to, obesity, anorexia nervosa, cachexia, lipid disorders, and diabetes. Examples of pain disorders include, but are not limited to, pain response elicited during various forms of tissue injury, e.g, inflammation, infection, and ischemia, usually referred to as hyperalgesia (described in, for example, Fields, H.L. (1987) Pain, New York:McGraw-Hill); pain associated with musculoskeletal disorders, e.g, joint pain; tooth pain; headaches; pain associated with surgery; pain related to irritable bowel syndrome; or chest pain.
Liver disorders can be associated with an accumulation in the liver of fibrous tissue, such as that resulting from an imbalance between production and degradation ofthe extracellular matrix accompanied by the collapse and condensation of preexisting fibers. In addition, liver disorders can involve hepatocellular necrosis or injury induced by a wide variety of agents including processes which disturb homeostasis., such as an inflammatory process, tissue damage resulting from toxic injury or altered hepatic blood flow, and infections (e.g, bacterial, viral and parasitic). Causes of liver fibrosis include, but are not limited to portal hypertension, inborn errors of metabolism, for example, fibrosis resulting from a storage disorder such as Gaucher's disease (lipid abnormalities) or a glycogen storage disease, Al- antitrypsin deficiency, a disorder mediating the accumulation (e.g, storage) of an exogenous substance, for example, hemochromatosis (iron-overload syndrome) and copper storage diseases (Wilson's disease), disorders resulting in the accumulation of a toxic metabolite (e.g, tyrosinemia, fructosemia and galactosemia) and peroxisomal disorders (e.g, Zellweger syndrome), or the administration of various chemicals or drugs, such as methotrexate, isonizaid, oxyphenisatin, methyldopa, chlorpromazine, tolbutamide or alcohol, or a vascular disorder such as obstruction of either the intrahepatic or extrahepatic bile flow or an alteration in hepatic circulation resulting, for example, from chronic heart failure, veno-occlusive disease, portal vein thrombosis or Budd-Chiari syndrome.
Disorders involving the kidney include, but are not limited to, congenital anomalies including, but not limited to, cystic diseases ofthe kidney, that include but are not limited to, cystic renal dysplasia, autosomal dominant (adult) polycystic kidney disease, autosomal recessive (childhood) polycystic kidney disease, and cystic diseases of renal medulla, which include, but are not limited to, medullary sponge kidney, and nephronophthisis-uremic medullary cystic disease complex, acquired (dialysis-associated) cystic disease, such as simple cysts; glomerular diseases including pathologies of glomerular injury that include, but are not limited to, in situ immune complex deposition, that includes, but is not limited to, anti-GBM nephritis, Heymann nephritis, and antibodies against planted antigens, circulating immune complex nephritis, antibodies to glomerular cells, cell-mediated immunity in glomerulonephritis, activation of alternative complement pathway, epithelial cell injury, and pathologies involving mediators of glomerular injury including cellular and soluble mediators, acute glomerulonephritis, such as acute proliferative (poststreptococcal, postinfectious) glomerulonephritis, including but not limited to, poststreptococcal glomerulonephritis and nonstreptococcal acute glomerulonephritis, rapidly progressive (crescentic) glomerulonephritis, nephrotic syndrome, membranous glomerulonephritis (membranous nephropathy), minimal change disease (lipoid nephrosis), focal segmental glomerulosclerosis, membranoproliferative glomerulonephritis, IgA nephropathy (Berger disease), focal proliferative and necrotizing glomerulonephritis (focal glomerulonephritis), hereditary nephritis, including but not limited to, Alport syndrome and thin membrane disease (benign familial hematuria), chronic glomerulonephritis, glomerular lesions associated with systemic disease, including but not limited to, systemic lupus erythematosus, Henoch-Schδnlein purpura, bacterial endocarditis, diabetic glomerulosclerosis, amyloidosis, fibrillary and immunotactoid glomerulonephritis, and other systemic disorders; diseases affecting tubules and interstitium, including acute tubular necrosis and tubulointerstitial nephritis, including but not limited to, pyelonephritis and urinary tract infection, acute pyelonephritis, chronic pyelonephritis and reflux nephropathy, and tubulointerstitial nephritis induced by drugs and toxins, including but not limited to, acute drug- induced interstitial nephritis, analgesic abuse nephropathy, nephropathy associated with nonsteroidal anti-inflammatory drugs, and other tubulointerstitial diseases including, but not limited to, urate nephropathy, hypercalcemia and nephrocalcinosis, and multiple myeloma; diseases of blood vessels including benign nephrosclerosis, malignant hypertension and accelerated nephrosclerosis, renal artery stenosis, and thrombotic microangiopathies including, but not limited to, classic (childhood) hemolytic-uremic syndrome, adult hemolytic-uremic syndrome/thrombotic thrombocytopenic purpura, idiopathic HUS/TTP, and other vascular disorders including, but not limited to, atherosclerotic ischemic renal disease, atheroembolic renal disease, sickle cell disease nephropathy, diffuse cortical necrosis, and renal infarcts; urinary tract obstruction (obstructive uropathy); urolithiasis (renal calculi, stones); and tumors ofthe kidney including, but not limited to, benign tumors, such as renal papillary adenoma, renal fibroma or hamartoma (renomedullary interstitial cell tumor), angiomyolipoma, and oncocytoma, and malignant tumors, including renal cell carcinoma (hypernephroma, adenocarcinoma of kidney), which includes urothelial carcinomas of renal pelvis.
Disorders involving the small intestine include the malabsorption syndromes such as, celiac sprue, tropical sprue (postinfectious sprue), whipple disease, disaccharidase (lactase) deficiency, abetalipoproteinemia, and tumors ofthe small intestine including adenomas and adenocarcinoma.
As used herein, disorders involving the heart, or "cardiovascular disease" or a "cardiovascular disorder" include diseases or disorders that affect the cardiovascular system, e.g, the heart, the blood vessels, and/or the blood. A cardiovascular disorder can be caused by an imbalance in arterial pressure, a malfunction ofthe heart, or an occlusion of a blood vessel, e.g, by a thrombus. A cardiovascular disorder includes, but is not limited to disorders such as arteriosclerosis, atherosclerosis, cardiac hypertrophy, ischemia reperfusion injury, restenosis, arterial inflammation, vascular wall remodeling, ventricular remodeling, rapid ventricular pacing, coronary microembolism, tachycardia, bradycardia, pressure overload, aortic bending, coronary artery ligation, vascular heart disease, valvular disease, including but not limited to, valvular degeneration caused by calcification, rheumatic heart disease, endocarditis, or complications of artificial valves; atrial fibrillation, long-QT syndrome, congestive heart failure, sinus node dysfunction, angina, heart failure, hypertension, atrial fibrillation, atrial flutter, pericardial disease, including but not limited to, pericardial effusion and pericarditis; cardiomyopathies, e.g, dilated cardiomyopathy or idiopathic cardiomyopathy, myocardial infarction, coronary artery disease, coronary artery spasm, ischemic disease, arrhythmia, sudden cardiac death, and cardiovascular developmental disorders (e.g, arteriovenous malformations, arteriovenous fistulae, raynaud's syndrome, neurogenic thoracic outlet syndrome, causalgia/reflex sympathetic dystrophy, hemangioma, aneurysm, cavernous angioma, aortic valve stenosis, atrial septal defects, atrioventricular canal, coarctation ofthe aorta, ebsteins anomaly, hypoplastic left heart syndrome, interruption ofthe aortic arch, mitral valve prolapse, ductus arteriosus, patent foramen ovale, partial anomalous pulmonary venous return, pulmonary atresia with ventricular septal defect, pulmonary atresia without ventricular septal defect, persistance ofthe fetal circulation, pulmonary valve stenosis, single ventricle, total anomalous pulmonary venous return, transposition ofthe great vessels, tricuspid atresia, truncus arteriosus, ventricular septal defects). A cardiovasular disease or disorder also can include an endothelial cell disorder and a hematological disorder.
A hematological disorder can include thrombosis. Thrombosis can result from platelet dysfunction, e.g, seen in myocardial infarction, angina, hypertension, lipid disorders, diabetes mellitus; myelodysplastic syndromes; myeloproliferative syndromes (including polycythemia vera and thombocythemia); thrombotic thrombocytopenic purpuras; HIV-induced platelet disorders (AIDS-Thrombocytopenia); heparin induced thrombocytopenia; mural cell alterations/interactions leading to platelet aggregation/degranulation, vascular endothelial cell activation injury, monocyte/macrophage extravasation and smooth muscle cell proliferation; autoimmune disorders such as, but not limited to vasculitis, antiphospholipid syndromes, systemic lupus erythromatosis; inflammatory diseases, such as, but not limited to immune activation; graft vs. host disease; radiation induced hypercoagulation; clotting factor dysregulation either hereditary (autosomal dominant or recessive) such as, but not limited to clotting factor pathways including protein C/S, Anti-thrombin III deficiency, and the Factor V Leiden mutation or acquired such as but not limited to autoimmune, cancer -associated and drug-induced dysregulation of clotting factors.
As used herein, an "endothelial cell disorder" includes a disorder characterized by aberrant, unregulated, or unwanted endothelial cell activity, e.g, proliferation, migration, angiogenesis, or vascularization; or aberrant expression of cell surface adhesion molecules or genes associated with angiogenesis, e.g, TIE-2, FLT and FLK. Endothelial cell disorders include tumorigenesis, tumor metastasis, psoriasis, diabetic retinopathy, endomefriosis, Grave's disease, ischemic disease (e.g, atherosclerosis), and chronic inflammatory diseases (e.g, rheumatoid arthritis).
Disorders involving blood vessels include, but are not limited to, responses of vascular cell walls to injury, such as endothelial dysfunction and endothelial activation and intimal thickening; vascular diseases including, but not limited to, congenital anomalies, such as arteriovenous fistula, atherosclerosis, and hypertensive vascular disease, such as hypertension; inflammatory disease— the vasculitides, such as giant cell (temporal) arteritis, Takayasu arteritis, polyarteritis nodosa (classic), Kawasaki syndrome (mucocutaneous lymph node syndrome), microscopic polyanglitis (microscopic polyarteritis, hypersensitivity or leukocytoclastic anglitis), Wegener granulomatosis, thromboanglitis obliterans (Buerger disease), vasculitis associated with other disorders, and infectious arteritis; Raynaud disease; aneurysms and dissection, such as abdominal aortic aneurysms, syphilitic (luetic) aneurysms, and aortic dissection (dissecting hematoma); disorders of veins and lymphatics, such as varicose veins, thrombophlebitis and phlebothrombosis, obstruction of superior vena cava (superior vena cava syndrome), obstruction of inferior vena cava (inferior vena cava syndrome), and lymphangitis and lymphedema; tumors, including benign tumors and tumor-like conditions, such as hemangioma, lymphangioma, glo us tumor (glomangioma), vascular ectasias, and bacillary angiomatosis, and intermediate-grade (borderline low-grade malignant) tumors, such as Kaposi sarcoma and hemangloendothelioma, and malignant tumors, such as angiosarcoma and hemangiopericytoma; and pathology of therapeutic interventions in vascular disease, such as balloon angioplasty and related techniques and vascular replacement, such as coronary artery bypass graft surgery.
"Bone metabolism" refers to direct or indirect effects in the formation or degeneration of bone structures, e.g, bone formation, bone resorption, etc, which may ultimately affect the concentrations in serum of calcium and phosphate. This term also includes activities mediated by proteins or small molecules that influence bone cells, e.g. osteoclasts and osteoblasts, resulting in bone formation and degeneration, such as the stimulation of differentiation of monocytes and mononuclear phagocytes into osteoclasts. Examples of bone metabolism disorders include, but are not limited to, osteoporosis, osteodystrophy, osteomalacia, rickets, osteitis fibrosa cystica, renal osteodystrophy, osteosclerosis, anti-convulsant treatment, osteopenia, fibrogenesis-imperfecta ossium, secondary hyperparathyrodism, hypoparathyroidism, hyperparathyroidism, cirrhosis, obstructive jaundice, drug induced metabolism, medullary carcinoma, chronic renal disease, rickets, sarcoidosis, glucocorticoid antagonism, malabsorption syndrome, steatorrhea, tropical sprue, idiopathic hypercalcemia and milk fever. The 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, and 84234 proteins, fragments thereof, and derivatives and other variants ofthe sequences in SEQ ID NO:2, SEQ ID NO:5, SEQ ID NO:8, SEQ ID NO:l 1, SEQ ID NO:14, SEQ ID NO: 17, SEQ ID NO:20, SEQ ID NO:23, SEQ ID NO:26, and SEQ ID NO:29 thereof are collectively referred to as "polypeptides or proteins ofthe invention" or "47476", "67210", "49875", "46842", "33201", "83378", "84233", "64708", "85041", or "84234" polypeptides or proteins. Nucleic acid molecules encoding such polypeptides or proteins are collectively referred to as "nucleic acids ofthe invention" or "47476", "67210", "49875", "46842", "33201", "83378", "84233", "64708", "85041", and "84234" nucleic acids. 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, and 84234 molecules refer to 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 nucleic acids, polypeptides, and antibodies.
As used herein, the term "nucleic acid molecule" includes DNA molecules (e.g, a cDNA or genomic DNA), RNA molecules (e.g, an mRNA) and analogs ofthe DNA or RNA. A DNA or RNA analog can be synthesized from nucleotide analogs. The nucleic acid molecule can be single-stranded or double-stranded, but preferably is double-stranded DNA. The term "isolated nucleic acid molecule" or "purified nucleic acid molecule" includes nucleic acid molecules that are separated from other nucleic acid molecules present in the natural source ofthe nucleic acid. For example, with regards to genomic DNA, the term "isolated" includes nucleic acid molecules which are separated from the chromosome with which the genomic DNA is naturally associated. Preferably, an "isolated" nucleic acid is free of sequences which naturally flank the nucleic acid (i.e, sequences located at the 5' and/or 3' ends ofthe nucleic acid) in the genomic DNA ofthe organism from which the nucleic acid is derived. For example, in various embodiments, the isolated nucleic acid molecule can contain less than about 5 kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5 kb or 0.1 kb of 5' and/or 3' nucleotide sequences which naturally flank the nucleic acid molecule in genomic DNA ofthe cell from which the nucleic acid is derived. Moreover, an "isolated" nucleic acid molecule, such as a cDNA molecule, can be substantially free of other cellular material, or culture medium when produced by recombinant techniques, or substantially free of chemical precursors or other chemicals when chemically synthesized.
As used herein, the term "hybridizes under low stringency, medium stringency, high stringency, or very high stringency conditions" describes conditions for hybridization and washing. Guidance for performing hybridization reactions can be found in Current Protocols in Molecular Biology, John Wiley & Sons, N.Y. (1989), 6.3.1-6.3.6, which is incorporated by reference. Aqueous and nonaqueous methods are described in that reference and either can be used. Specific hybridization conditions referred to herein are as follows: 1) low stringency hybridization conditions in 6X sodium chloride/sodium citrate (SSC) at about 45°C, followed by two washes in 0.2X SSC, 0.1 % SDS at least at 50°C (the temperature ofthe washes can be increased to 55°C for low stringency conditions); 2) medium stringency hybridization conditions in 6X SSC at about 45°C, followed by one or more washes in 0.2X SSC, 0.1% SDS at 60°C; 3) high stringency hybridization conditions in 6X SSC at about 45°C, followed by one or more washes in 0.2X SSC, 0.1 % SDS at 65°C; and preferably 4) very high stringency hybridization conditions are 0.5M sodium phosphate, 7% SDS at 65°C, followed by one or more washes at 0.2X SSC, 1% SDS at 65°C. Very high stringency conditions (4) are the preferred conditions and the ones that should be used unless otherwise specified.
Preferably, an isolated nucleic acid molecule ofthe invention that hybridizes under a stringency condition described herein to the sequence of SEQ ID NO:l, SEQ ID NO:4, SEQ ID NO:7, SEQ ID NO: 10, SEQ ID NO: 13, SEQ ID NO: 16, SEQ ID NO: 19, SEQ ID NO:22, SEQ ID NO:25, or SEQ ID NO:28, corresponds to a naturally-occurring nucleic acid molecule.
As used herein, a "naturally-occurring" nucleic acid molecule refers to an RNA or DNA molecule having a nucleotide sequence that occurs in nature. For example a naturally occurring nucleic acid molecule can encode a natural protein. As used herein, the terms "gene" and "recombinant gene" refer to nucleic acid molecules which include at least an open reading frame encoding a 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 protein. The gene can optionally further include non- coding sequences, e.g, regulatory sequences and introns. Preferably, a gene encodes a mammalian 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 protein or derivative thereof. An "isolated" or "purified" polypeptide or protein is substantially free of cellular material or other contaminating proteins from the cell or tissue source from which the protein is derived, or substantially free from chemical precursors or other chemicals when chemically synthesized. "Substantially free" means that a preparation of 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 protein is at least 10% pure. In a preferred embodiment, the preparation of 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 protein has less than about 30%, 20%, 10% and more preferably 5% (by dry weight), of non-47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 protein (also referred to herein as a "contaminating protein"), or of chemical precursors or non- 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 chemicals. When the 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 protein or biologically active portion thereof is recombinantly produced, it is also preferably substantially free of culture medium, i.e., culture medium represents less than about 20%, more preferably less than about 10%, and most preferably less than about 5% ofthe volume ofthe protein preparation. The invention includes isolated or purified preparations of at least 0.01, 0.1, 1.0, and 10 milligrams in dry weight.
A "non-essential" amino acid residue is a residue that can be altered from the wild-type sequence of 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 without abolishing or substantially altering a 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 activity. Preferably the alteration does not substantially alter the 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 activity, e.g, the activity is at least 20%, 40%, 60%, 70% or 80% of wild-type. An "essential" amino acid residue is a residue that, when altered from the wild-type sequence of 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234, results in abolishing a 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 activity such that less than 20% of the wild-type activity is present. For example, conserved amino acid residues in 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 are predicted to be particularly unamenable to alteration.
A "conservative amino acid substitution" is one in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g, lysine, arginine, histidine), acidic side chains (e.g, aspartic acid, glutamic acid), uncharged polar side chains (e.g, glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g, alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g, threonine, valine, isoleucine) and aromatic side chains (e.g, tyrosine, phenylalanine, tryptophan, histidine). Thus, a predicted nonessential amino acid residue in a 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 protein is preferably replaced with another amino acid residue from the same side chain family. Alternatively, in another embodiment, mutations can be introduced randomly along all or part of a 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 coding sequence, such as by saturation mutagenesis, and the resultant mutants can be screened for 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 biological activity to identify mutants that retain activity. Following mutagenesis of SEQ ID NO:l, SEQ ID NO:4, SEQ ID NO:7, SEQ ID NO:10, SEQ ID O:13, SEQ IDNO:16, SEQ ID NO:19, SEQ ID NO:22, SEQ ID NO.25, SEQ ID O.28, SEQ ID NO:3, SEQ IDNO:6, SEQ ID NO.9, SEQ ID NO: 12, SEQ ID NO: 15, SEQ ID NO: 18, SEQ ID NO:21, SEQ ID NO:24, SEQ ID NO:27, or SEQ ID NO:30, the encoded protein can be expressed recombinantly and the activity ofthe protein can be determined.
As used herein, a "biologically active portion" of a 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 protein includes a fragment of a 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 protein which participates in an interaction, e.g, an intramolecular or an inter-molecular interaction. An inter-molecular interaction can be a specific binding interaction or an enzymatic interaction (e.g, the interaction can be transient and a covalent bond is formed or broken). An inter-molecular interaction can be between a 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 molecule and a non-47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or
84234 molecule or between a first 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 molecule and a second 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 molecule (e.g, a dimerization interaction). Biologically active portions of a 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 protein include peptides comprising amino acid sequences sufficiently homologous to or derived from the amino acid sequence ofthe 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 protein, e.g, the amino acid sequence shown in SEQ ID NO:2, SEQ ID NO:5, SEQ ID NO:8, SEQ ID NO:l 1, SEQ ID NO:14, SEQ ID NO:17, SEQ ID NO:20, SEQ ID NO:23, SEQ ID NO:26, or SEQ ID NO:29, which include less amino acids than the full length 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 proteins, and exhibit at least one activity of a 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 protein. Typically, biologically active portions comprise a domain or motif with at least one activity ofthe 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 protein, e.g, stimulation of guanine nucleoside dissociation from a GTPase protein, transfer of a sugar residue to another molecule, unwinding of a duplex nucleic acid molecule, stimulation of the hydrolysis of GTP molecule bound to a GTPase protein, catalyzsis ofthe oxidation of an alcohol group present on a molecule, or transport of metal ions across a lipid bilayer. A biologically active portion of a 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 protein can be a polypeptide which is, for example, 10, 25, 50, 100, 200 or more amino acids in length. Biologically active portions of a 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 protein can be used as targets for developing agents which modulate a 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 mediated activity, e.g, stimulation of guanine nucleoside dissociation from a GTPase protein, transfer of a sugar residue to another molecule, unwinding of a duplex nucleic acid molecule, stimulation ofthe hydrolysis of GTP molecule bound to a GTPase protein, catalyzsis ofthe oxidation of an alcohol group present on a molecule, or transport of metal ions across a lipid bilayer.
Calculations of homology or sequence identity between sequences (the terms are used interchangeably herein) are performed as follows.
To determine the percent identity of two amino acid sequences, or of two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g, gaps can be introduced in one or both of a first and a second amino acid or nucleic acid sequence for optimal alignment and non-homologous sequences can be disregarded for comparison purposes). In a preferred embodiment, the length of a reference sequence aligned for comparison purposes is at least 30%, preferably at least 40%, more preferably at least 50%, 60%), and even more preferably at least 70%, 80%, 90%), 100%) ofthe length ofthe reference sequence. The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position (as used herein amino acid or nucleic acid "identity" is equivalent to amino acid or nucleic acid "homology"). The percent identity between the two sequences is a function ofthe number of identical positions shared by the sequences, taking into account the number of gaps, and the length of each gap, which need to be introduced for optimal alignment ofthe two sequences.
The comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm. In a preferred embodiment, the percent identity between two amino acid sequences is determined using the Needleman and Wunsch ((1970) J Mol. Biol. 48:444-453) algorithm which has been incorporated into the GAP program in the GCG software package (available at http://www.gcg.com), using either a Blossum 62 matrix or a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6. In yet another preferred embodiment, the percent identity between two nucleotide sequences is determined using the GAP program in the GCG software package (available at http://www.gcg.com), using a NWSgapdna.CMP matrix and a gap weight of 40, 50, 60, 70, or 80 and a length weight of 1, 2, 3, 4, 5, or 6. A particularly preferred set of parameters (and the one that should be used unless otherwise specified) are a Blossum 62 scoring matrix with a gap penalty of 12, a gap extend penalty of 4, and a frameshift gap penalty of 5.
The percent identity between two amino acid or nucleotide sequences can be determined using the algorithm of E. Meyers and W. Miller ((1989) CABIOS, 4:11-17) which has been incorporated into the ALIGN program (version 2.0), using a PAM120 weight residue table, a gap length penalty of 12 and a gap penalty of 4. The nucleic acid and protein sequences described herein can be used as a "query sequence" to perform a search against public databases to, for example, identify other family members or related sequences. Such searches can be performed using the NBLAST and XBLAST programs (version 2.0) of Altschul, et al. (1990) J Mol. Biol. 215:403-10. BLAST nucleotide searches can be performed with the NBLAST program, score = 100, wordlength = 12 to obtain nucleotide sequences homologous to 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 nucleic acid molecules ofthe invention. BLAST protein searches can be performed with the XBLAST program, score = 50, wordlength = 3 to obtain amino acid sequences homologous to 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 protein molecules of the invention. To obtain gapped alignments for comparison purposes, Gapped BLAST can be utilized as described in Altschul et al, (1997) Nucleic Acids Res. 25:3389-3402. When utilizing BLAST and Gapped BLAST programs, the default parameters ofthe respective programs (e.g, XBLAST and NBLAST) can be used. See http://www.ncbi.nlm.nih.gov.
Particularly preferred 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 polypeptides ofthe present invention have an amino acid sequence substantially identical to the amino acid sequence of SEQ ID NO:2, SEQ ID NO:5, SEQ ID NO:8, SEQ ID NO:l 1, SEQ ID NO:14, SEQ ID NO:17, SEQ ID NO:20, SEQ ID NO:23, SEQ ID NO:26, or SEQ ID NO:29. In the context of an amino acid sequence, the term "substantially identical" is used herein to refer to a first amino acid that contains a sufficient or minimum number of amino acid residues that are i) identical to, or ii) conservative substitutions of aligned amino acid residues in a second amino acid sequence such that the first and second amino acid sequences can have a common structural domain and/or common functional activity. For example, amino acid sequences that contain a common structural domain having at least about 60%, or 65% identity, likely 75% identity, more likely 85%, 90%. 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO:2, SEQ ID NO:5, SEQ ID NO:8, SEQ ID NO:l 1, SEQ ID NO: 14, SEQ ID NO: 17, SEQ ID NO:20, SEQ ID NO:23, SEQ ID NO:26, or SEQ ID NO:29 are termed substantially identical.
In the context of nucleotide sequence, the term "substantially identical" is used herein to refer to a first nucleic acid sequence that contains a sufficient or minimum number of nucleotides that are identical to aligned nucleotides in a second nucleic acid sequence such that the first and second nucleotide sequences encode a polypeptide having common functional activity, or encode a common structural polypeptide domain or a common functional polypeptide activity. For example, nucleotide sequences having at least about 60%>, or 65% identity, likely 75% identity, more likely 85%, 90%. 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO:l, SEQ ID NO:4, SEQ ID NO:7, SEQ ID NO: 10, SEQ ID NO: 13, SEQ ID NO: 16, SEQ ID NO: 19, SEQ ID NO:22, SEQ ID NO:25, or SEQ ID NO:28, or SEQ ID NO:3, SEQ ID NO:6, SEQ ID NO:9, SEQ ID NO:12, SEQ ID NO:15, SEQ ID NO: 18, SEQ ID NO:21, SEQ ID NO:24, SEQ ID NO:27, or SEQ ID NO:30 are termed substantially identical.
"Misexpression or aberrant expression", as used herein, refers to a non-wildtype pattern of gene expression at the RNA or protein level. It includes: expression at non- wild type levels, i.e, over- or under-expression; a pattern of expression that differs from wild type in terms ofthe time or stage at which the gene is expressed, e.g, increased or decreased expression (as compared with wild type) at a predetermined developmental period or stage; a pattern of expression that differs from wild type in terms of altered, e.g, increased or decreased, expression (as compared with wild type) in a predetermined cell type or tissue type; a pattern of expression that differs from wild type in terms ofthe splicing size, translated amino acid sequence, post-transitional modification, or biological activity ofthe expressed polypeptide; a pattern of expression that differs from wild type in terms ofthe effect of an environmental stimulus or extracellular stimulus on expression ofthe gene, e.g, a pattern of increased or decreased expression (as compared with wild type) in the presence of an increase or decrease in the strength ofthe stimulus.
"Subject," as used herein, refers to human and non-human animals. The term "non- human animals" ofthe invention includes all vertebrates, e.g, mammals, such as non-human primates (particularly higher primates), sheep, dog, rodent (e.g, mouse or rat), guinea pig, goat, pig, cat, rabbits, cow, and non-mammals, such as chickens, amphibians, reptiles, etc. In a preferred embodiment, the subject is a human. In another embodiment, the subject is an experimental animal or animal suitable as a disease model.
A "purified preparation of cells", as used herein, refers to an in vitro preparation of cells. In the case cells from multicellular organisms (e.g, plants and animals), a purified preparation of cells is a subset of cells obtained from the organism, not the entire intact organism. In the case of unicellular microorganisms (e.g, cultured cells and microbial cells), it consists of a preparation of at least 10% and more preferably 50% ofthe subject cells.
Various aspects ofthe invention are described in further detail below.
Isolated Nucleic Acid Molecules
In one aspect, the invention provides, an isolated or purified, nucleic acid molecule that encodes a 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 polypeptide described herein, e.g, a full-length 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 protein or a fragment thereof, e.g, a biologically active portion of 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 protein. Also included is a nucleic acid fragment suitable for use as a hybridization probe, which can be used, e.g, to identify a nucleic acid molecule encoding a polypeptide ofthe invention, 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 mRNA, and fragments suitable for use as primers, e.g, PCR primers for the amplification or mutation of nucleic acid molecules.
In one embodiment, an isolated nucleic acid molecule ofthe invention includes the nucleotide sequence shown in SEQ ID NO:l, SEQ ID NO:4, SEQ ID NO:7, SEQ ID NO:10, SEQ ID NO: 13, SEQ ID NO: 16, SEQ ID NO: 19, SEQ ID NO:22, SEQ ID NO:25, and SEQ ID NO:28, or a portion of any of these nucleotide sequences. In one embodiment, the nucleic acid molecule includes sequences encoding the human 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 protein (i.e., "the coding region" of SEQ ID NO:l, SEQ ID NO:4, SEQ ID NO:7, SEQ ID NO:10, SEQ ID NO:13, SEQ ID NO: 16, SEQ ID NO: 19, SEQ ID NO:22, SEQ ID NO:25, and SEQ ID NO:28, as shown in SEQ ID NO:3, SEQ ID NO:6, SEQ ID NO:9, SEQ ID NO: 12, SEQ ID NO: 15, SEQ ID NO: 18, SEQ ID NO:21, SEQ ID NO:24, SEQ ID NO:27, or SEQ ID NO:30), as well as 5' untranslated sequences. Alternatively, the nucleic acid molecule can include only the coding region of SEQ ID NO:l, SEQ ID NO:4, SEQ ID NO:7, SEQ ID NO:10, SEQ ID NO:13, SEQ ID NO:16, SEQ ID NO: 19, SEQ ID NO:22, SEQ ID NO:25, and SEQ ID NO:28 (e.g, SEQ ID NO:3, SEQ ID NO:6, SEQ ID NO:9, SEQ ID NO: 12, SEQ ID NO: 15, SEQ ID NO: 18, SEQ ID NO:21, SEQ ID NO:24, SEQ ID NO:27, or SEQ ID NO:30) and, e.g, no flanking sequences which normally accompany the subject sequence. In another embodiment, the nucleic acid molecule encodes a sequence corresponding to a mature fragment of a 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 protein.
In another embodiment, an isolated nucleic acid molecule ofthe invention includes a nucleic acid molecule which is a complement (e.g, a full complement) ofthe nucleotide sequence shown in SEQ ID NO:l, SEQ ID NO:4, SEQ ID NO:7, SEQ ID NO:10, SEQ ID NO:13, SEQ ID NO:16, SEQ ID NO:19, SEQ ID NO:22, SEQ ID NO:25, or SEQ ID NO:28, or SEQ ID NO:3, SEQ ID NO:6, SEQ ID NO:9, SEQ ID NO:12, SEQ ID NO:15, SEQ ID NO:18, SEQ ID NO:21, SEQ ID NO:24, SEQ ID NO:27, or SEQ ID NO:30, or a portion of any of these nucleotide sequences. In other embodiments, the nucleic acid molecule ofthe invention is sufficiently complementary to the nucleotide sequence shown in SEQ ID NO:l, SEQ ID NO:4, SEQ ID NO:7, SEQ ID NO:10, SEQ ID NO:13, SEQ ID NO:16, SEQ ID NO:19, SEQ ID NO:22, SEQ ID NO:25, or SEQ ID NO:28, or SEQ ID NO:3, SEQ ID NO:6, SEQ ID NO:9, SEQ ID NO: 12, SEQ ID NO: 15, SEQ ID NO: 18, SEQ ID NO.21, SEQ ID NO:24, SEQ ID NO:27, or SEQ ID NO:30, such that it can hybridize (e.g, under a stringency condition described herein) to the nucleotide sequence shown in SEQ ID NO:l, SEQ ID NO:4, SEQ ID NO:7, SEQ ID NO:10, SEQ ID NO:13, SEQ ID NO:16, SEQ ID NO:19, SEQ ID NO:22, SEQ ID NO:25, or SEQ ID NO:28, or SEQ ID NO:3, SEQ ID NO:6, SEQ ID NO:9, SEQ ID NO:12, SEQ ID O:15, SEQ ID NO:18, SEQ ID O:21, SEQ ID NO:24, SEQ ID NO:27, or SEQ ID NO:30, thereby forming a stable duplex.
In one embodiment, an isolated nucleic acid molecule ofthe present invention includes a nucleotide sequence which is at least about: 60%, 65%, 70%, 75%), 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more homologous to the entire length ofthe nucleotide sequence shown in SEQ ID NO:l, SEQ ID NO:4, SEQ ID NO:7, SEQ ID NO:10, SEQ ID NO:13, SEQ ID NO:16, SEQ ID NO:19, SEQ ID NO:22, SEQ ID NO:25, or SEQ ID NO:28, or SEQ ID NO:3, SEQ ID NO:6, SEQ ID NO:9, SEQ ID NO: 12, SEQ ID NO: 15, SEQ ID NO: 18, SEQ ID NO:21, SEQ ID NO:24, SEQ ID NO:27, or SEQ ID NO:30, or a portion, preferably ofthe same length, of any of these nucleotide sequences.
47476 Nucleic Acid Fragments
A nucleic acid molecule ofthe invention can include only a portion ofthe nucleic acid sequence of SEQ ID NO:l or SEQ ID NO:3. For example, such a nucleic acid molecule can include a fragment which can be used as a probe or primer or a fragment encoding a portion of a 47476 protein, e.g, an immunogenic or biologically active portion of a 47476 protein. A fragment can comprise those nucleotides of SEQ ID NO:l, which encode a ras guanine nucleotide dissociation stimulator domain of human 47476. The nucleotide sequence determined from the cloning ofthe 47476 gene allows for the generation of probes and primers designed for use in identifying and/or cloning other 47476 family members, or fragments thereof, as well as 47476 homologues, or fragments thereof, from other species. In another embodiment, a nucleic acid includes a nucleotide sequence that includes part, or all, ofthe coding region and extends into either (or both) the 5' or 3' noncoding region. Other embodiments include a fragment that includes a nucleotide sequence encoding an amino acid fragment described herein. Nucleic acid fragments can encode a specific domain or site described herein or fragments thereof, particularly fragments thereof which are at least 100 amino acids in length. Preferably, the nucleic acid fragments encode a specific domain or fragment thereof, wherein the domain or fragment is at least 25, 28, 45, 50, 150, 180 and 230 amino acids in length. Fragments also include nucleic acid sequences corresponding to specific amino acid sequences described above or fragments thereof. Nucleic acid fragments should not to be construed as encompassing those fragments that may have been disclosed prior to the invention.
A nucleic acid fragment can include a sequence corresponding to a domain, region, or functional site described herein. A nucleic acid fragment can also include one or more domains, regions, or functional sites described herein. Thus, for example, a 47476 nucleic acid fragment can include a sequence corresponding to a ras guanine nucleotide dissociation stimulator domain, a guanine nucleotide dissociation stimulator domain N-terminal motif, an EF-hand calcium-binding domain, or a phorbol ester/diacylglycerolbinding domain (Cl domain).
47476 probes and primers are provided. Typically a probe/primer is an isolated or purified oligonucleotide. The oligonucleotide typically includes a region of nucleotide sequence that hybridizes under a stringency condition described herein to at least about 7, 12 or 15, preferably about 20 or 25, more preferably about 30, 35, 40, 45, 50, 55, 60, 65, or 75 consecutive nucleotides of a sense or antisense sequence of SEQ ID NO:l or SEQ ID NO:3, or of a naturally occurring allelic variant or mutant of SEQ ID NO:l or SEQ ID NO:3. Preferably, an oligonucleotide is less than about 200, 150, 120, or 100 nucleotides in length.
In one embodiment, the probe or primer is attached to a solid support, e.g, a solid support described herein.
One exemplary kit of primers includes a forward primer that anneals to the coding strand and a reverse primer that anneals to the non-coding strand. The forward primer can anneal to the start codon, e.g, the nucleic acid sequence encoding amino acid residue 1 of SEQ ID NO:2. The reverse primer can anneal to the ultimate codon, e.g, the codon immediately before the stop codon, e.g, the codon encoding amino acid residue 673 of SEQ ID NO:2. In a preferred embodiment, the annealing temperatures ofthe forward and reverse primers differ by no more than 5, 4, 3, or 2°C.
In a preferred embodiment, the nucleic acid is a probe which is at least 10, 12, 15, 18, 20 and less than 200, more preferably less than 100, or less than 50, nucleotides in length. It should be identical, or differ by 1, or 2, or less than 5 or 10 nucleotides, from a sequence disclosed herein. If alignment is needed for this comparison the sequences should be aligned for maximum homology. "Looped" out sequences from deletions or insertions, or mismatches, are considered differences.
A probe or primer can be derived from the sense or anti-sense strand of a nucleic acid that encodes: a guanine nucleotide dissociation stimulator domain, e.g, located at about amino acid residues 195 to 381 of SEQ ID NO:2; a guanine nucleotide dissociation stimulator domain N-terminal motif, e.g, located at about amino acid residues 55 to 172 of SEQ ID NO:2; an EF- hand calcium-binding domain, e.g, located at about amino acid residues 470 to 498 of SEQ ID NO:2; or a phorbol ester/diacylglycerol binding domain (Cl domain), e.g, located at about amino acid residues 541 to 590 of SEQ ID NO:2.
In another embodiment a set of primers is provided, e.g, primers suitable for use in a PCR, which can be used to amplify a selected region of a 47476 sequence, e.g, a domain, region, site or other sequence described herein. The primers should be at least 5, 10, or 50 base pairs in length and less than 100, or less than 200, base pairs in length. The primers should be identical, or differs by one base from a sequence disclosed herein or from a naturally occurring variant. For example, primers suitable for amplifying all or a portion of any ofthe following regions are provided: a ras guanine nucleotide dissociation stimulator domain, e.g, located at about amino acid 195 to 381 of SEQ ID NO:2; a guanine nucleotide dissociation stimulator domain N-terminal motif , e.g, located at about amino acid residues 55 to 172 of SEQ ID NO:2; an EF-hand calcium-binding domain, e.g, located at about amino acid 470 to 498 of SEQ ID NO:2; or a phorbol ester/diacylglycerol binding domain (Cl domain), e.g, located at about amino acid 541 to 590 of SEQ ID NO:2.
A nucleic acid fragment can encode an epitope bearing region of a polypeptide described herein. A nucleic acid fragment encoding a "biologically active portion of a 47476 polypeptide" can be prepared by isolating a portion ofthe nucleotide sequence of SEQ ID NO:l or SEQ ID NO:3, which encodes a polypeptide having a 47476 biological activity (e.g, the biological activities ofthe 47476 proteins are described herein), expressing the encoded portion ofthe 47476 protein (e.g, by recombinant expression in vitro) and assessing the activity ofthe encoded portion ofthe 47476 protein. For example, a nucleic acid fragment encoding a biologically active portion of 47476 includes a Ras guanine nucleotide dissociation stimulator domain, a guanine nucleotide dissociation stimulator domain N-terminal motif, an EF-handed calcium-binding domain, or a phorbol ester/diacylglycerol binding domain (Cl domain), e.g, about amino acid residues 195 to 381, 55 to 172, 470 to 498, and 541 to 590 of SEQ ID NO:2, respectively. A nucleic acid fragment encoding a biologically active portion of a 47476 polypeptide, may comprise a nucleotide sequence which is greater than 300 or more nucleotides in length
In preferred embodiments, a nucleic acid fragment includes a nucleotide sequence which is about 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300 or more nucleotides in length and hybridizes under a stringency condition described herein to a nucleic acid molecule of SEQ ID NO:l, SEQ ID NO:3, or the nucleotide sequence ofthe DNA insert ofthe plasmid deposited with ATCC Accession Number as described herein.
In a preferred embodiment, a nucleic acid fragment differs by at least 1, 2, 3, 10, 20, or more nucleotides from a sequence previously disclosed. Differences can include differing in length or sequence identity. For example, a nucleic acid fragment can: include one or more nucleotides from SEQ ID NO:l or SEQ ID NO:3 located outside a region of overlap with a sequence previously disclosed; not include all ofthe nucleotides of a sequence previously disclosed, e.g, can be one or more nucleotides shorter (at one or both ends) than the previously disclosed sequence; or can differ by one or more nucleotides in the region of overlap. The nucleic acid fragment can include a sequence identical to the region of nucleotides 1 to 300, 300 to 800, 500 to 1000, 700 to 1300, 1000 to 1500, 1400 to 1900, 1800 to 2300, 2200 to 2700, 2600 to 3100 of SEQ ID NO:l.
67210 Nucleic Acid Fragments
A nucleic acid molecule ofthe invention can include only a portion ofthe nucleic acid sequence of SEQ ID NO:4 or SEQ ID NO:6. For example, such a nucleic acid molecule can include a fragment which can be used as a probe or primer or a fragment encoding a portion of a 67210 protein, e.g, an immunogenic or biologically active portion of a 67210 protein. A fragment can comprise those nucleotides of SEQ ID NO:4, which encode a glycosyltransferase domain of human 67210. The nucleotide sequence determined from the cloning ofthe 67210 gene allows for the generation of probes and primers designed for use in identifying and/or cloning other 67210 family members, or fragments thereof, as well as 67210 homologues, or fragments thereof, from other species.
In another embodiment, a nucleic acid includes a nucleotide sequence that includes part, or all, ofthe coding region and extends into either (or both) the 5' or 3' noncoding region. Other embodiments include a fragment which includes a nucleotide sequence encoding an amino acid fragment described herein. Nucleic acid fragments can encode a specific domain or site described herein or fragments thereof, particularly fragments thereof which are at least 50 amino acids in length, preferably 75, 100, 150, 200, 250, 300, 325, 340 or more amino acids in length. Preferably, the nucleic acid fragments encode a specific domain or fragment thereof, wherein the domain or fragment is at least 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700 nucleic acids in length. Fragments also include nucleic acid sequences corresponding to specific amino acid sequences described above or fragments thereof. Nucleic acid fragments should not to be construed as encompassing those fragments that may have been disclosed prior to the invention.
A nucleic acid fragment can include a sequence corresponding to a domain, region, or functional site described herein. A nucleic acid fragment can also include one or more domain, region, or functional site described herein. Thus, for example, a 67210 nucleic acid fragment can include a sequence corresponding to a glycosyltransferase domain.
67210 probes and primers are provided. Typically a probe/primer is an isolated or purified oligonucleotide. The oligonucleotide typically includes a region of nucleotide sequence that hybridizes under a stringency condition described herein to at least about 7, 12 or 15, preferably about 20 or 25, more preferably about 30, 35, 40, 45, 50, 55, 60, 65, or 75 consecutive nucleotides of a sense or antisense sequence of SEQ ID NO:4 or SEQ ID NO:6, or of a naturally occurring allelic variant or mutant of SEQ ID NO:4 or SEQ ID NO:6. Preferably, an oligonucleotide is less than about 200, 150, 120, or 100 nucleotides in length. In one embodiment, the probe or primer is attached to a solid support, e.g, a solid support described herein. One exemplary kit of primers includes a forward primer that anneals to the coding strand and a reverse primer that anneals to the non-coding strand. The forward primer can anneal to the start codon, e.g, the nucleic acid sequence encoding amino acid residue 1 of SEQ ID NO:5. The reverse primer can anneal to the ultimate codon, e.g, the codon immediately before the stop codon, e.g, the codon encoding amino acid residue 349 of SEQ ID NO:5. In a preferred embodiment, the annealing temperatures ofthe forward and reverse primers differ by no more than 5, 4, 3, or 2°C.
In a preferred embodiment the nucleic acid is a probe which is at least 10, 12, 15, 18, 20 and less than 200, more preferably less than 100, or less than 50, nucleotides in length. It should be identical, or differ by 1, or 2, or less than 5 or 10 nucleotides, from a sequence disclosed herein. If alignment is needed for this comparison the sequences should be aligned for maximum homology. "Looped" out sequences from deletions or insertions, or mismatches, are considered differences.
A probe or primer can be derived from the sense or anti-sense strand of a nucleic acid which encodes a glycosyltransferase domain (from amino acid 63 to 340 of SEQ ID NO:5). In another embodiment a set of primers is provided, e.g, primers suitable for use in a PCR, which can be used to amplify a selected region of a 67210 sequence, e.g, a domain, region, site or other sequence described herein. The primers should be at least 5, 10, or 50 base pairs in length and less than 100, or less than 200, base pairs in length. The primers should be identical, or differs by one base from a sequence disclosed herein or from a naturally occurring variant. For example, primers suitable for amplifying all or a portion of any ofthe following regions are provided: a glycosyltransferase domain, e.g, located at about amino acid 63 to 340 of SEQ ID NO:5.
A nucleic acid fragment can encode an epitope bearing region of a polypeptide described herein.
A nucleic acid fragment encoding a "biologically active portion of a 67210 polypeptide" can be prepared by isolating a portion ofthe nucleotide sequence of SEQ ID NO:4 or 6, which encodes a polypeptide having a 67210 biological activity (e.g, the biological activities ofthe 67210 proteins are described herein), expressing the encoded portion ofthe 67210 protein (e.g, by recombinant expression in vitro) and assessing the activity ofthe encoded portion ofthe
67210 protein. For example, a nucleic acid fragment encoding a biologically active portion of 67210 includes a glycosyltransferase domain, e.g, amino acid residues about 63 to 340 of SEQ ID NO:5. A nucleic acid fragment encoding a biologically active portion of a 67210 polypeptide, may comprise a nucleotide sequence which is greater than 200 or more nucleotides in length. In preferred embodiments, a nucleic acid includes a nucleotide sequence which is about
300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300 or more nucleotides in length and hybridizes under a stringency condition described herein to a nucleic acid molecule of SEQ ID NO:4, or SEQ ID NO:6.
In a preferred embodiment, a nucleic acid fragment differs by at least 1, 2, 3, 10, 20, or more nucleotides from the sequence of Genbank accession number AC013776 or AC023550. Differences can include differing in length or sequence identity. For example, a nucleic acid fragment can: include one or more nucleotides from SEQ ID NO:4 or SEQ ID NO:6 located outside the region of nucleotides 290 to 560, 675 to 1042, or 1152 to 1743 of SEQ ID NO:4; not include all ofthe nucleotides of Genbank accession number AC013776 or AC023550, e.g, can be one or more nucleotides shorter (at one or both ends) than the sequence of Genbank accession number AC013776 or AC023550; or can differ by one or more nucleotides in the region of overlap.
49875 Nucleic Acid Fragments
A nucleic acid molecule ofthe invention can include only a portion ofthe nucleic acid sequence of SEQ ID NO:7 or SEQ ID NO:9. For example, such a nucleic acid molecule can include a fragment which can be used as a probe or primer or a fragment encoding a portion of a 49875 protein, e.g, an immunogenic or biologically active portion of a 49875 protein. A fragment can comprise those nucleotides of SEQ ID NO:7, which encode a DEAD type helicase domain of human 49875. The nucleotide sequence determined from the cloning ofthe 49875 gene allows for the generation of probes and primers designed for use in identifying and/or cloning other 49875 family members, or fragments thereof, as well as 49875 homologues, or fragments thereof, from other species.
In another embodiment, a nucleic acid includes a nucleotide sequence that includes part, or all, ofthe coding region and extends into either (or both) the 5' or 3' noncoding region. Other embodiments include a fragment which includes a nucleotide sequence encoding an amino acid fragment described herein. Nucleic acid fragments can encode a specific domain or site described herein or fragments thereof, particularly fragments thereof which are at least 50 amino acids in length, preferably 100, 200, 250, 300, 350, 400, 450, 500, 550 or more amino acids in length. Fragments also include nucleic acid sequences corresponding to specific amino acid sequences described above or fragments thereof. Nucleic acid fragments should not to be construed as encompassing those fragments that may have been disclosed prior to the invention.
A nucleic acid fragment can include a sequence corresponding to a domain, region, or functional site described herein. A nucleic acid fragment can also include one or more domains, regions, or functional sites described herein. Thus, for example, a 49875 nucleic acid fragment can include a sequence corresponding to a DEAD-type helicase domain (amino acid residues 22 to 245 of SEQ ID NO: 8); a DEAD-box subfamily ATP-dependent helicase signature motif (amino acid 169 to 177 of SEQ ID NO:8); a conserved helicase C-terminal domain (amino acid residues 281 to 363 of SEQ ID NO:8); an ATP/GTP-binding site motif A (P-loop) (amino acid 53 to 60 of SEQ ID NO:8). 49875 probes and primers are provided. Typically a probe/primer is an isolated or purified oligonucleotide. The oligonucleotide typically includes a region of nucleotide sequence that hybridizes under a stringency condition described herein to at least about 7, 12 or 15, preferably about 20 or 25, more preferably about 30, 35, 40, 45, 50, 55, 60, 65, or 75 consecutive nucleotides of a sense or antisense sequence of SEQ ID NO: 7 or SEQ ID NO: 9, or of a naturally occurring allelic variant or mutant of SEQ ID NO:7 or SEQ ID NO:9. Preferably, an oligonucleotide is less than about 200, 150, 120, or 100 nucleotides in length.
In one embodiment, the probe or primer is attached to a solid support, e.g, a solid support described herein.
One exemplary kit of primers includes a forward primer that anneals to the coding strand and a reverse primer that anneals to the non-coding strand. The forward primer can anneal to the start codon, e.g, the nucleic acid sequence encoding amino acid residue 1 of SEQ ID NO:8. The reverse primer can anneal to the ultimate codon, e.g, the codon immediately before the stop codon, e.g, the codon encoding amino acid residue 600 of SEQ ID NO: 8. In a preferred embodiment, the annealing temperatures ofthe forward and reverse primers differ by no more than 5, 4, 3, or 2°C. In a preferred embodiment the nucleic acid is a probe which is at least 10, 12, 15, 18, 20 and less than 200, more preferably less than 100, or less than 50, nucleotides in length. It should be identical, or differ by 1, or 2, or less than 5 or 10 nucleotides, from a sequence disclosed herein. If alignment is needed for this comparison the sequences should be aligned for maximum homology. "Looped" out sequences from deletions or insertions, or mismatches, are considered differences.
A probe or primer can be derived from the sense or anti-sense strand of a nucleic acid which encodes: a DEAD-type helicase domain, e.g, about amino acid residues 22 to 245 of SEQ ID NO:8; a DEAD-box subfamily ATP-dependent helicase signature motif, e.g, about amino acid residuesl69 to 177 of SEQ ID NO:8; a conserved helicase C-terminal domain, e.g, about amino acid residues 281 to 363 of SEQ ID NO:8; or an ATP/GTP-binding site motif A (P-loop), e.g, about amino acid residues 53 to 60 of SEQ ID NO:8.
In another embodiment a set of primers is provided, e.g, primers suitable for use in a PCR, which can be used to amplify a selected region of a 49875 sequence, e.g, a domain, region, site or other sequence described herein. The primers should be at least 5, 10, or 50 base pairs in length and less than 100, or less than 200, base pairs in length. The primers should be identical, or differs by one base from a sequence disclosed herein or from a naturally occurring variant. For example, primers suitable for amplifying all or a portion of any ofthe following regions are provided: a DEAD-type helicase domain, e.g, about amino acid residues 22 to 245 of SEQ ID NO:8; a DEAD-box subfamily ATP-dependent helicase signature motif, e.g, about amino acid residuesl69 to 177 of SEQ ID NO:8; a conserved helicase C-terminal domain, e.g, about amino acid residues 281 to 363 of SEQ ID NO: 8; or an ATP/GTP-binding site motif A (P-loop), e.g, about amino acid residues 53 to 60 of SEQ ID NO:8.
A nucleic acid fragment can encode an epitope bearing region of a polypeptide described herein.
A nucleic acid fragment encoding a "biologically active portion of a 49875 polypeptide" can be prepared by isolating a portion ofthe nucleotide sequence of SEQ ID NO:7 or SEQ ID NO:9, which encodes a polypeptide having a 49875 biological activity (e.g, the biological activities ofthe 49875 proteins are described herein), expressing the encoded portion ofthe 49875 protein (e.g, by recombinant expression in vitro) and assessing the activity ofthe encoded portion ofthe 49875 protein. For example, a nucleic acid fragment encoding a biologically active portion of 49875 includes a DEAD type helicase domain, e.g, amino acid residues about 22 to 245 of SEQ ID NO:8. A nucleic acid fragment encoding a biologically active portion of a 49875 polypeptide, may comprise a nucleotide sequence which is greater than 150 or more nucleotides in length. In preferred embodiments, a nucleic acid includes a nucleotide sequence which is about
300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300 or more nucleotides in length and hybridizes under a stringency condition described herein to a nucleic acid molecule of SEQ ID NO:7, or SEQ ID NO:9.
In a preferred embodiment, a nucleic acid fragment differs by at least 1, 2, 3, 10, 20, or more nucleotides from a sequence disclosed in WO 01/55301, WO 01/57188 or WO 01/62927. Differences can include differing in length or sequence identity. For example, a nucleic acid fragment can: include one or more nucleotides from SEQ ID NO: 7 or SEQ ID NO: 9 located outside a region of overlap with a sequence disclosed in WO 01/55301, WO 01/57188 or WO 01/62927; not include all ofthe nucleotides of a sequence previously disclosed, e.g, can be one or more nucleotides shorter (at one or both ends) than a sequence disclosed in WO 01/55301, WO 01/57188 or WO 01/62927; or can differ by one or more nucleotides in the region of overlap.
46842 Nucleic Acid Fragments
A nucleic acid molecule ofthe invention can include only a portion ofthe nucleic acid sequence of SEQ ID NO: 10 or SEQ ID NO: 12, or the nucleotide sequence ofthe DNA insert of the plasmid deposited with ATCC Accession Number as described herein. For example, such a nucleic acid molecule can include a fragment which can be used as a probe or primer or a fragment encoding a portion of a 46842 protein, e.g, an immunogenic or biologically active portion of a 46842 protein. A fragment can comprise those nucleotides of SEQ ID NO:l which encode an ArfGAP domain of human 46842. The nucleotide sequence determined from the cloning ofthe 46842 gene allows for the generation of probes and primers designed for use in identifying and/or cloning other 46842 family members, or fragments thereof, as well as 46842 homologues, or fragments thereof, from other species.
In another embodiment, a nucleic acid includes a nucleotide sequence that includes part, or all, of the coding region and extends into either (or both) the 5 ' or 3 ' noncoding region. Other embodiments include a fragment which includes a nucleotide sequence encoding an amino acid fragment described herein. Nucleic acid fragments can encode a specific domain or site described herein or fragments thereof, particularly fragments thereof which are at least 100, 200, 300, 400, or 500 amino acids in length. Fragments also include nucleic acid sequences corresponding to specific amino acid sequences described above or fragments thereof. Nucleic acid fragments should not to be construed as encompassing those fragments that may have been disclosed prior to the invention.
A nucleic acid fragment can include a sequence corresponding to a domain, region, or functional site described herein. A nucleic acid fragment can also include one or more domain, region, or functional site described herein. Thus, for example, a 46842 nucleic acid fragment can include a sequence corresponding to an ArfGAP domain.
46842 probes and primers are provided. Typically a probe/primer is an isolated or purified oligonucleotide. The oligonucleotide typically includes a region of nucleotide sequence that hybridizes under a stringency condition described herein to at least about 7, 12 or 15, preferably about 20 or 25, more preferably about 30, 35, 40, 45, 50, 55, 60, 65, or 75 consecutive nucleotides of a sense or antisense sequence of SEQ ID NO: 10 or SEQ ID NO: 12, or of a naturally occurring allelic variant or mutant of SEQ ID NO: lOor SEQ ID NO: 12. Preferably, an oligonucleotide is less than about 200, 150, 120, or 100 nucleotides in length.
In one embodiment, the probe or primer is attached to a solid support, e.g, a solid support described herein.
One exemplary kit of primers includes a forward primer that anneals to the coding strand and a reverse primer that anneals to the non-coding strand. The forward primer can anneal to the start codon, e.g, the nucleic acid sequence encoding amino acid residue 1 of SEQ ID NO: 11. The reverse primer can anneal to the ultimate codon, e.g, the codon immediately before the stop codon, e.g, the codon encoding amino acid residue 834 of SEQ ID NO: 11. In a preferred embodiment, the annealing temperatures ofthe forward and reverse primers differ by no more than 5, 4, 3, or 2°C.
In a preferred embodiment the nucleic acid is a probe which is at least 10, 12, 15, 18, 20 and less than 200, more preferably less than 100, or less than 50, nucleotides in length. It should be identical, or differ by 1, or 2, or less than 5 or 10 nucleotides, from a sequence disclosed herein. If alignment is needed for this comparison the sequences should be aligned for maximum homology. "Looped" out sequences from deletions or insertions, or mismatches, are considered differences.
A probe or primer can be derived from the sense or anti-sense strand of a nucleic acid which encodes: a PH domain, e.g, located at about amino acid residues 269 to 363 SEQ ID NO: 11 ; an ArfGAP domain, e.g, located at about amino acid residues 403 to 525 of SEQ ID NO: 11 ; or an ankyrin repeat domain, e.g, located at about amino acid residues 702 to 734 or 735 to 767 of SEQ ID NO:l l.
In another embodiment a set of primers is provided, e.g, primers suitable for use in a PCR, which can be used to amplify a selected region of a 46842 sequence, e.g, a domain, region, site or other sequence described herein. The primers should be at least 5, 10, or 50 base pairs in length and less than 100, or less than 200, base pairs in length. The primers should be identical, or differs by one base from a sequence disclosed herein or from a naturally occurring variant. For example, primers suitable for amplifying all or a portion of any ofthe following regions are provided: a PH domain, e.g, located at about amino acid residues 269 to 363 SEQ ID NO: 11 ; an ArfGAP domain, e.g, located at about amino acid residues 403 to 525 of SEQ ID NO:l 1; or an ankyrin repeat domain, e.g, located at about amino acid residues 702 to 734 or 735 to 767of SEQ ID NO:l l.
A nucleic acid fragment can encode an epitope bearing region of a polypeptide described herein. A nucleic acid fragment encoding a "biologically active portion of a 46842 polypeptide" can be prepared by isolating a portion ofthe nucleotide sequence of SEQ ID NO: 10 or SEQ ID NO: 13, or the nucleotide sequence ofthe DNA insert ofthe plasmid deposited with ATCC Accession Number as disclosed herein, which encodes a polypeptide having a 46842 biological activity (e.g, the biological activities ofthe 46842 proteins are described herein), expressing the encoded portion ofthe 46842 protein (e.g, by recombinant expression in vitro) and assessing the activity ofthe encoded portion ofthe 46842 protein. For example, a nucleic acid fragment encoding a biologically active portion of 46842 includes an ArfGAP domain, e.g, about amino acid residues 403 to 525 of SEQ ID NO:l 1. A nucleic acid fragment encoding a biologically active portion of a 46842 polypeptide may comprise a nucleotide sequence which is greater than 300 or more nucleotides in length. In preferred embodiments, a nucleic acid includes a nucleotide sequence which is about 300, 400, 500, 600, 700, 800, 900, 1000, 1200, 1400, 1800 or more nucleotides in length and hybridizes under a stringency condition described herein to a nucleic acid molecule of SEQ ID NO:10 or SEQ ID NO:12. In a preferred embodiment, a nucleic acid fragment differs by at least 1, 2, 3, 10, 20, or more nucleotides from the sequence of GenBank accession number BAB21807 (KIAA1716). Differences can include differing in length or sequence identity. For example, a nucleic acid fragment can: include one or more nucleotides from SEQ ID NO: 10 or SEQ ID NO: 12 located outside the region of nucleotides 1 to 100, 50 to 109, 200 to 400, 600 to 800, 700 to 1200, 1200 to 1500, 1420 to 1950, or 2210 to 2425; not include all ofthe nucleotides of GenBank accession number BAB21807; or can differ by one or more nucleotides in the region of overlap.
33201 Nucleic Acid Fragments
A nucleic acid molecule ofthe invention can include only a portion ofthe nucleic acid sequence of SEQ ID NO : 13 or SEQ ID NO : 15. For example, such a nucleic acid molecule can include a fragment which can be used as a probe or primer or a fragment encoding a portion of a 33201 protein, e.g, an immunogenic or biologically active portion of a 33201 protein. A fragment can comprise those nucleotides of SEQ ID NO: 13, which encode a dehydrogenase/reductase domain of human 33201. The nucleotide sequence determined from the cloning ofthe 33201 gene allows for the generation of probes and primers designed for use in identifying and/or cloning other 33201 family members, or fragments thereof, as well as 33201 homologues, or fragments thereof, from other species.
In another embodiment, a nucleic acid includes a nucleotide sequence that includes part, or all, ofthe coding region and extends into either (or both) the 5' or 3' noncoding region. Other embodiments include a fragment which includes a nucleotide sequence encoding an amino acid fragment described herein. Nucleic acid fragments can encode a specific domain or site described herein or fragments thereof, particularly fragments thereof which are at least 100 amino acids in length. Preferably, the nucleic acid fragments encode a specific domain or fragment thereof, wherein the domain or fragment is at least 105, or more preferably 110, 120, or even 130 amino acids in length. Fragments also include nucleic acid sequences corresponding to specific amino acid sequences described above or fragments thereof. Nucleic acid fragments should not to be construed as encompassing those fragments that may have been disclosed prior to the invention.
A nucleic acid fragment can include a sequence corresponding to a domain, region, or functional site described herein. A nucleic acid fragment can also include one or more domains, regions, or functional sites described herein. Thus, for example, a 33201 nucleic acid fragment can include a sequence corresponding to a dehydrogenase/reductaseactivity.
33201 probes and primers are provided. Typically a probe/primer is an isolated or purified oligonucleotide. The oligonucleotide typically includes a region of nucleotide sequence that hybridizes under a stringency condition described herein to at least about 7, 12 or 15, preferably about 20 or 25, more preferably about 30, 35, 40, 45, 50, 55, 60, 65, or 75 consecutive nucleotides of a sense or antisense sequence of SEQ ID NO: 13 or SEQ ID NO: 15, or of a naturally occurring allelic variant or mutant of SEQ ID NO: 13 or SEQ ID NO: 15. Preferably, an oligonucleotide is less than about 200, 150, 120, or 100 nucleotides in length. In one embodiment, the probe or primer is attached to a solid support, e.g, a solid support described herein.
One exemplary kit of primers includes a forward primer that anneals to the coding strand and a reverse primer that anneals to the non-coding strand. The forward primer can anneal to the start codon, e.g, the nucleic acid sequence encoding amino acid residue 1 of SEQ ID NO: 14. The reverse primer can anneal to the ultimate codon, e.g, the codon immediately before the stop codon, e.g, the codon encoding amino acid residue 351 of SEQ ID NO:14. In a preferred embodiment, the annealing temperatures ofthe forward and reverse primers differ by no more than 5, 4, 3, or 2°C.
In a preferred embodiment the nucleic acid is a probe which is at least 10, 12, 15, 18, 20 and less than 200, more preferably less than 100, or less than 50, nucleotides in length. It should be identical, or differ by 1, or 2, or less than 5 or 10 nucleotides, from a sequence disclosed herein. If alignment is needed for this comparison the sequences should be aligned for maximum homology. "Looped" out sequences from deletions or insertions, or mismatches, are considered differences.
A probe or primer can be derived from the sense or anti-sense strand of a nucleic acid which encodes a dehydrogenase/reductase domain (e.g, about amino acid residues 22 to 345 of SEQ ID NO: 14). In another embodiment a set of primers is provided, e.g, primers suitable for use in a PCR, which can be used to amplify a selected region of a 33201 sequence, e.g, a domain, region, site or other sequence described herein. The primers should be at least 5, 10, or 50 base pairs in length and less than 100, or less than 200, base pairs in length. The primers should be identical, or differs by one base from a sequence disclosed herein or from a naturally occurring variant. For example, primers suitable for amplifying all or a portion of any ofthe following regions are provided: a dehydrogenase/reductase domain, e.g, from about amino acid residues 22 to 345 of SEQ ID NO:14.
A nucleic acid fragment can encode an epitope bearing region of a polypeptide described herein.
A nucleic acid fragment encoding a "biologically active portion of a 33201 polypeptide" can be prepared by isolating a portion ofthe nucleotide sequence of SEQ ID NO: 13 or SEQ ID NO: 15, which encodes a polypeptide having a 33201 biological activity (e.g, the biological activities ofthe 33201 proteins are described herein), expressing the encoded portion ofthe 33201 protein (e.g, by recombinant expression in vitro) and assessing the activity ofthe encoded portion ofthe 33201 protein. For example, a nucleic acid fragment encoding a biologically active portion of 33201 can include a dehydrogenase/reductase domain, e.g, amino acid residues about 22 to 345 of SEQ ID NO: 14. A nucleic acid fragment encoding a biologically active portion of a 33201 polypeptide, may comprise a nucleotide sequence which is greater than 300 or more nucleotides in length.
In preferred embodiments, a nucleic acid includes a nucleotide sequence which is about 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300 or more nucleotides in length and hybridizes under a stringency condition described herein to a nucleic acid molecule of SEQ ID NO:13, or SEQ ID NO:15. In a preferred embodiment, a nucleic acid fragment differs by at least 1, 2, 3, 10, 20, or more nucleotides from the sequence of Genbank accession number AC005520. Differences can include differing in length or sequence identity. For example, a nucleic acid fragment can: include one or more nucleotides from SEQ ID NO: 13 or SEQ ID NO: 15 located outside the region of nucleotides 1117 to 1423, 1425 to 1627, or 1629 to 1717; not include all ofthe nucleotides of Genbank accession AC005520, e.g, can be one or more nucleotides shorter (at one or both ends) than the sequence of Genbank accession number AC005520, or can differ by one or more nucleotides in the region of overlap.
83378, 84233, 64708, 85041, or 84234 Nucleic Acid Fragments
A nucleic acid molecule ofthe invention can include only a portion ofthe nucleic acid sequence of SEQ ID NO:16, SEQ ID NO:19, SEQ ID NO:22, SEQ ID NO:25, or SEQ ID
NO:28, or SEQ ID NO: 18, SEQ ID NO:21, SEQ ID NO:24, SEQ ID NO:27, or SEQ ID NO:30. For example, such a nucleic acid molecule can include a fragment which can be used as a probe or primer or a fragment encoding a portion of a 83378, 84233, 64708, 85041, or 84234 protein, e.g, an immunogenic or biologically active portion of a 83378, 84233, 64708, 85041, or 84234 protein. A fragment can comprise those nucleotides of SEQ ID NO:16, SEQ ID NO:19, SEQ ID NO:22, SEQ ID NO:25, or SEQ ID NO:28, which encode a cation efflux domain of human 83378, 84233, 64708, 85041, or 84234. The nucleotide sequence determined from the cloning ofthe 83378, 84233, 64708, 85041, or 84234 gene allows for the generation of probes and primers designed for use in identifying and/or cloning other 83378, 84233, 64708, 85041, or 84234 family members, or fragments thereof, as well as 83378, 84233, 64708, 85041, or 84234 homologues, or fragments thereof, from other species.
In another embodiment, a nucleic acid includes a nucleotide sequence that includes part, or all, ofthe coding region and extends into either (or both) the 5' or 3' noncoding region. Other embodiments include a fragment which includes a nucleotide sequence encoding an amino acid fragment described herein. Nucleic acid fragments can encode a specific domain or site described herein or fragments thereof, particularly fragments thereof which are at least 50 amino acids in length. Preferably, the nucleic acid fragments encode a specific domain or fragment thereof, wherein the domain or fragment is at least 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, or 750 amino acids in length. Fragments also include nucleic acid sequences corresponding to specific amino acid sequences described above or fragments thereof. Nucleic acid fragments should not to be construed as encompassing those fragments that may have been disclosed prior to the invention.
A nucleic acid fragment can include a sequence corresponding to a domain, region, or functional site described herein. A nucleic acid fragment can also include one or more domain, region, or functional site described herein. Thus, for example, a 83378, 84233, 64708, 85041, or 84234 nucleic acid fragment can include a sequence corresponding to a cation efflux domain, a transmembrane domain, a cytoplasmic domain, or a non-cytoplasmic loop.
83378, 84233, 64708, 85041, or 84234 probes and primers are provided. Typically a probe/primer is an isolated or purified oligonucleotide. The oligonucleotide typically includes a region of nucleotide sequence that hybridizes under a stringency condition described herein to at least about 7, 12 or 15, preferably about 20 or 25, more preferably about 30, 35, 40, 45, 50, 55, 60, 65, or 75 consecutive nucleotides of a sense or antisense sequence of SEQ ID NO: 16, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:28, or SEQ ID NO:30, or of a naturally occurring allelic variant or mutant of SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO:21 , SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:28, or SEQ ID NO:30. Preferably, an oligonucleotide is less than about 200, 150, 120, or 100 nucleotides in length.
In one embodiment, the probe or primer is attached to a solid support, e.g, a solid support described herein. One exemplary kit of primers includes a forward primer that anneals to the coding strand and a reverse primer that anneals to the non-coding strand. The forward primer can anneal to the start codon, e.g, the nucleic acid sequence encoding amino acid residue 1 of SEQ ID NO:17, SEQ ID NO:20, SEQ ID NO:23, SEQ ID NO:26, or SEQ ID NO:29. The reverse primer can anneal to the ultimate codon, e.g, the codon immediately before the stop codon, e.g, the codon encoding amino acid residue 485 of SEQ ID NO: 17, amino acid residue 320 of SEQ ID NO:20, amino acid residue 461 of SEQ ID NO:23, amino acid residue 765 of SEQ ID NO:26, or amino acid residue 376 of SEQ ID NO:29. In a preferred embodiment, the annealing temperatures ofthe forward and reverse primers differ by no more than 5, 4, 3, or 2°C.
In a preferred embodiment the nucleic acid is a probe which is at least 10, 12, 15, 18, 20 and less than 200, more preferably less than 100, or less than 50, nucleotides in length. It should be identical, or differ by 1, or 2, or less than 5 or 10 nucleotides, from a sequence disclosed herein. If alignment is needed for this comparison the sequences should be aligned for maximum homology. "Looped" out sequences from deletions or insertions, or mismatches, are considered differences. A probe or primer can be derived from the sense or anti-sense strand of a nucleic acid which encodes: a cation efflux domain (e.g, residues 11 to 133 or 231 to 389 of SEQ ID NO:17, residues 25 to 310 of SEQ ID NO:20, residues 55 to 153 or 227 to 320 of SEQ ID NO:23, residues 419 to 733 of SEQ ID NO:26, or residues 38 to 349 of SEQ ID NO:29); a transmembrane domain (e.g, residues 11-31, 44-61, 79-98, 115-134, 241-265, or 283-299 of SEQ ID NO: 17; residues 25-49, 58-74, 92-113, 128-147, 167-191, or 201-218 of SEQ ID NO:20; residues 34-51, 58-82, 101-119, 137-155, 202-219, or 232-249 of SEQ ID NO:23; residues 59-77, 99-119, 129-145, 152-168, 190-214, 239-258, 267-288, 304-320, 343-362, 419- 439, 486-505, 521-541, 592-613, or 618-641 of SEQ ID NO:26; or residues 38-58, 71-87, 105- 123, 141-159, 237-256, or 263-286 of SEQ ID NO:29); a cytoplasmic domain (e.g, residues 1- 10, 62-78, 135-240, or 300-485 of SEQ ID NO:17; residues 1-24, 75-91, 148-166, or 219-320 of SEQ ID NO:20; residues 1-33, 83-100, 156-201, or 250-461 of SEQ ID NO:23; residues 1- 58, 120-128, 169-189, 259-266, 321-342, 438-485, 542-591, or 642-765 of SEQ ID NO:26; and residues 1-37, 88-104, 160-236, or 287-376 of SEQ ID NO:29); or a non-cytoplasmic loop (residues 32-43, 99-114, or 266-282 of SEQ ID NO: 17; residues 50-57, 114-127, or 192-200 of SEQ ID NO:20; residues 52-57, 120-136, or 220-231 of SEQ ID NO:23; residues 78-98, 146- 151, 215-238, 289-303, 363-418, 506-520, or 614-617 of SEQ ID NO:26; or residues 59-70, 124-140, or 257-262 of SEQ ID NO:29).
In another embodiment a set of primers is provided, e.g, primers suitable for use in a PCR, which can be used to amplify a selected region of a 83378, 84233, 64708, 85041, or 84234 sequence, e.g, a domain, region, site or other sequence described herein. The primers should be at least 5, 10, or 50 base pairs in length and less than 100, or less than 200, base pairs in length. The primers should be identical, or differs by one base from a sequence disclosed herein or from a naturally occurring variant. For example, primers suitable for amplifying all or a portion of any ofthe following regions are provided a cation efflux domain (e.g, residues 11 to 133 or 231 to 389 of SEQ ID NO:17, residues 25 to 310 of SEQ ID NO:20, residues 55 to 153 or 227 to 320 of SEQ ID NO:23, residues 419 to 733 of SEQ ID NO:26, or residues 38 to 349 of SEQ ID NO:29); a transmembrane domain (e.g, residues 11-31, 44-61, 79-98, 115-134, 241-265, or 283-299 of SEQ ID NO:17; residues 25-49, 58-74, 92-113, 128-147, 167-191, or 201-218 of SEQ ID NO.20; residues 34-51, 58-82, 101-119, 137-155, 202-219, or 232-249 of SEQ ID NO:23; residues 59-77, 99-119, 129-145, 152-168, 190-214, 239-258, 267-288, 304- 320, 343-362, 419-439, 486-505, 521-541, 592-613, or 618-641 of SEQ ID NO:26; or residues 38-58, 71-87, 105-123, 141-159, 237-256, or 263-286 of SEQ ID NO:29); a cytoplasmic domain (e.g, residues 1-10, 62-78, 135-240, or 300-485 of SEQ ID NO:17; residues 1-24, 75- 91, 148-166, or 219-320 of SEQ ID NO:20; residues 1-33, 83-100, 156-201, or 250-461 of SEQ ID NO:23; residues 1-58, 120-128, 169-189, 259-266, 321-342, 438-485, 542-591, or 642-765 of SEQ ID NO:26; and residues 1-37, 88-104, 160-236, or 287-376 of SEQ ID NO:29); or a non-cytoplasmic loop (residues 32-43, 99-114, or 266-282 of SEQ ID NO:17; residues 50-57, 114-127, or 192-200 of SEQ ID NO :20; residues 52-57, 120-136, or 220-231 of SEQ ID NO:23; residues 78-98, 146-151, 215-238, 289-303, 363-418, 506-520, or 614-617 of SEQ ID NO:26; or residues 59-70, 124-140, or 257-262 of SEQ ID NO:29).
A nucleic acid fragment can encode an epitope bearing region of a polypeptide described herein.
A nucleic acid fragment encoding a "biologically active portion of a 83378, 84233, 64708, 85041, or 84234 polypeptide" can be prepared by isolating a portion ofthe nucleotide sequence of SEQ ID NO: 16, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:28, or SEQ ID NO:30, which encodes a polypeptide having a 83378, 84233, 64708, 85041, or 84234 biological activity (e.g, the biological activities ofthe 83378, 84233, 64708, 85041, or 84234 proteins are described herein), expressing the encoded portion ofthe 83378, 84233, 64708, 85041, or 84234 protein (e.g, by recombinant expression in vitro) and assessing the activity ofthe encoded portion of the 83378, 84233, 64708, 85041, or 84234 protein. For example, a nucleic acid fragment encoding a biologically active portion of 83378, 84233, 64708, 85041, or 84234 includes a cation efflux domain, e.g, residues 11 to 133 or 231 to 389 of SEQ ID NO: 17, residues 25 to 310 of SEQ ID NO:20, residues 55 to 153 or 227 to 320 of SEQ ID NO:23, residues 419 to 733 of SEQ ID NO:26, or residues 38 to 349 of SEQ ID NO:29. A nucleic acid fragment encoding a biologically active portion of a 83378, 84233, 64708, 85041, or 84234 polypeptide, may comprise a nucleotide sequence which is greater than 300 or more nucleotides in length.
In preferred embodiments, a nucleic acid includes a nucleotide sequence which is about 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, or more nucleotides in length and hybridizes under a stringency condition described herein to a nucleic acid molecule of SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:28, or SEQ ID NO:30. In a preferred embodiment, a 83378 nucleic acid fragment differs by at least 1, 2, 3, 10, 20, or more nucleotides from the sequence of Genbank accession number AL359609 or a sequence disclosed in WO 01/62918, WO 01/55314, WO 01/55355. Differences can include differing in length or sequence identity. For example, a nucleic acid fragment can: include one or more nucleotides from SEQ ID NO: 16 or SEQ ID NO: 18 located outside the region of nucleotides 7-1044, 1085-1638, 1253-1638, 1299-1638, 723-1775 of SEQ ID NO:16; not include all ofthe nucleotides ofthe sequence of Genbank accession number AL359609 or a sequence disclosed in WO 01/62918, WO 01/55314, WO 01/55355, e.g, can be one or more nucleotides shorter (at one or both ends) than the sequence of Genbank accession number AL359609 or a sequence disclosed in WO 01/62918, WO 01/55314, WO 01/55355; or can differ by one or more nucleotides in the region of overlap.
In a preferred embodiment, a 84233 nucleic acid fragment differs by at least 1, 2, 3, 10, 20, or more nucleotides from the sequence of Genbank accession number AX061210 or AX086187 or a sequence disclosed in WO 01/12659, WO 01/51628, or WO 00/78953. Differences can include differing in length or sequence identity. For example, a nucleic acid fragment can: include one or more nucleotides from SEQ ID NO: 19 or SEQ ID NO:21 located outside the region of nucleotides 420-2103, 998-1589, 1-429, 1146-1582, 996-1425, 998-1341, 996-1379, 996-1368 of SEQ ID NO:19; not include all ofthe nucleotides of Genbank accession number AX061210 or AX086187 or a sequence disclosed in WO 01/12659, WO 01/51628, or WO 00/78953, e.g, can be one or more nucleotides shorter (at one or both ends) than the sequence of Genbank accession number AX061210 or AX086187 or a sequence disclosed in WO 01/12659, WO 01/51628, or WO 00/78953; or can differ by one or more nucleotides in the region of overlap.
In a preferred embodiment, a 64708 nucleic acid fragment differs by at least 1, 2, 3, 10, 20, or more nucleotides from the sequence of Genbank accession number AK000844 or a sequence disclosed in WO 01/57188, WO 01/57270, WO 01/57272, WO 01/57275, WO 01/57276, WO 01/57277, or WO 01/57278. Differences can include differing in length or sequence identity. For example, a nucleic acid fragment can: include one or more nucleotides from SEQ ID NO:22 or SEQ ID NO:24 located outside the region of nucleotides 209-697, 918- 1456, 918-1418, 1348-1790, 20-442, 911-1280, or 911-1226 of SEQ ID NO:22; not include all ofthe nucleotides of Genbank accession number AK000844 or a sequence disclosed in WO 01/57188, WO 01/57270, WO 01/57272, WO 01/57275, WO 01/57276, WO 01/57277, or WO 01/57278, e.g, can be one or more nucleotides shorter (at one or both ends) than the sequence of Genbank accession number AK000844 or a sequence disclosed in WO 01/57188, WO 01/57270, WO 01/57272, WO 01/57275, WO 01/57276, WO 01/57277, or WO 01/57278; or can differ by one or more nucleotides in the region of overlap.
In a preferred embodiment, a 85041 nucleic acid fragment differs by at least 1, 2, 3, 10, 20, or more nucleotides from the sequence of Genbank accession number AK022558, AK022818, or AF233321, or a sequence disclosed in WO 01/40466, WO 01/54472, WO 01/55318, or WO 01/53312. Differences can include differing in length or sequence identity. For example, a nucleic acid fragment can: include one or more nucleotides from SEQ ID NO:25 or SEQ ID NO:27 located outside the region of nucleotides 55-2750, 1069-3010, 1347-3224, 1320-3010,1347-3010, 602-3005, 483-2740, 1015-1942, 786-3259, 602-3005, 883-2051, 2139- 3010, 94-809, or 1843-3010 of SEQ ID NO:25; not include all ofthe nucleotides of Genbank accession number AK022558, AK022818, or AF233321, or a sequence disclosed in WO 01/40466, WO 01/54472, WO 01/55318, or WO 01/53312, e.g, can be one or more nucleotides shorter (at one or both ends) than the sequence of Genbank accession number AK022558, AK022818, or AF233321, or a sequence disclosed in WO 01/40466, WO 01/54472, WO 01/55318, or WO 01/53312; or can differ by one or more nucleotides in the region of overlap. In a preferred embodiment, a 84234 nucleic acid fragment differs by at least 1, 2, 3, 10, 20, or more nucleotides from a sequence disclosed in WO 01/53312. Differences can include differing in length or sequence identity. For example, a nucleic acid fragment can: include one or more nucleotides from SEQ ID NO:28 or SEQ ID NO:30 located outside the region of nucleotides 129-1292 or 165-1292 of SEQ ID NO:28; not include all ofthe nucleotides of a sequence disclosed in WO 01/53312, e.g, can be one or more nucleotides shorter (at one or both ends) than the sequence of a sequence disclosed in WO 01/53312; or can differ by one or more nucleotides in the region of overlap.
47476, 67210, 49875. 46842, 33201, 83378, 84233, 64708, 85041, or 84234 Nucleic Acid Variants
The invention further encompasses nucleic acid molecules that differ from the nucleotide sequence shown in SEQ ID NO: 1 , SEQ ID NO:4, SEQ ID NO:7, SEQ ID NO: 10, SEQ ID NO:13, SEQ ID NO:16, SEQ ID NO: 19, SEQ ID NO:22, SEQ ID NO:25, or SEQ ID NO:28, or SEQ ID NO:3, SEQ ID NO:6, SEQ ID NO:9, SEQ ID NO: 12, SEQ ID NO: 15, SEQ ID NO:18, SEQ ID NO:21, SEQ ID NO:24, SEQ ID NO:27, or SEQ ID NO:30. Such differences can be due to degeneracy ofthe genetic code (and result in a nucleic acid which encodes the same 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 proteins as those encoded by the nucleotide sequence disclosed herein. In another embodiment, an isolated nucleic acid molecule ofthe invention has a nucleotide sequence encoding a protein having an amino acid sequence which differs, by at least 1, but less than 5, 10, 20, 50, or 100 amino acid residues that shown in SEQ ID NO:2, SEQ ID NO:5, SEQ ID NO:8, SEQ ID NO:l 1, SEQ ID NO: 14, SEQ ID NO: 17, SEQ ID NO:20, SEQ ID NO:23, SEQ ID NO:26, or SEQ ID NO:29. If alignment is needed for this comparison the sequences should be aligned for maximum homology. The encoded protein can differ by no more than 5, 4, 3, 2, or 1 amino acid. "Looped" out sequences from deletions or insertions, or mismatches, are considered differences. Nucleic acids ofthe inventor can be chosen for having codons, which are preferred, or non-preferred, for a particular expression system. E.g, the nucleic acid can be one in which at least one codon, at preferably at least 10%, or 20% ofthe codons has been altered such that the sequence is optimized for expression in E. coli, yeast, human, insect, or CHO cells.
Nucleic acid variants can be naturally occurring, such as allelic variants (same locus), homologs (different locus), and orthologs (different organism) or can be non naturally occurring. Non-naturally occurring variants can be made by mutagenesis techniques, including those applied to polynucleotides, cells, or organisms. The variants can contain nucleotide substitutions, deletions, inversions and insertions. Variation can occur in either or both the coding and non- coding regions. The variations can produce both conservative and non-conservative amino acid substitutions (as compared in the encoded product).
In a preferred embodiment, the nucleic acid differs from that of SEQ ID NO: 1 , SEQ ID NO:4, SEQ ID NO:7, SEQ ID NO:10, SEQ ID NO:13, SEQ ID NO:16, SEQ ID NO:19, SEQ ID NO:22, SEQ ID NO:25, or SEQ ID NO:28, or SEQ ID NO:3, SEQ ID NO:6, SEQ ID NO:9, SEQ ID NO:12, SEQ ID NO:15, SEQ ID NO:18, SEQ ID NO:21, SEQ ID NO:24, SEQ ID NO:27, or SEQ ID NO:30, e.g, as follows: by at least one but less than 10, 20, 30, or 40 nucleotides; at least one but less than 1 %, 5 %, 10% or 20% of the nucleotides in the subj ect nucleic acid. The nucleic acid can differ by no more than 5, 4, 3, 2, or 1 nucleotide. If necessary for this analysis the sequences should be aligned for maximum homology. "Looped" out sequences from deletions or insertions, or mismatches, are considered differences.
Orthologs, homologs, and allelic variants can be identified using methods known in the art. These variants comprise a nucleotide sequence encoding a polypeptide that is 50%, at least about 55%, typically at least about 70-75%, more typically at least about 80-85%, and most typically at least about 90-95% or more identical to the nucleotide sequence shown in SEQ ID NO:2, SEQ ID NO:5, SEQ ID NO:8, SEQ ID NO: 11, SEQ ID NO: 14, SEQ ID NO: 17, SEQ ID NO.20, SEQ ID NO:23, SEQ ID NO:26, or SEQ ID NO:29, or a fragment of this sequence. Such nucleic acid molecules can readily be identified as being able to hybridize under a stringency condition described herein, to the nucleotide sequence shown in SEQ ID NO:2, SEQ ID NO:5, SEQ ID NO:8, SEQ ID NO:l 1, SEQ ID NO:14, SEQ ID NO: 17, SEQ ID NO:20, SEQ ID NO:23, SEQ ID NO:26, or SEQ ID NO:29, or a fragment of the sequence. Nucleic acid molecules corresponding to orthologs, homologs, and allelic variants ofthe 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041 , or 84234 cDNAs ofthe invention can further be isolated by mapping to the same chromosome or locus as the 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 gene.
Preferred 47476 variants include those that are correlated with the ability to exchange guanine nucleotides (GTP for GDP); the ability to bind calcium; the ability to bind at least one and preferably two zinc ions; the ability to bind a second messenger, e.g, diacylglycerol; the ability to bind analogs of diacylglycerol, such as phorbol esters; or the ability to activate the ras superfamily of proteins.
Allelic variants of 47476, e.g, human 47476, include both functional and non-functional proteins. Functional allelic variants are naturally occurring amino acid sequence variants ofthe 47476 protein within a population that maintain the ability to exchange guanine nucleotides (GTP for GDP); to bind calcium; to bind at least one and preferably two zinc ions; to bind a second messenger, e.g, diacylglycerol; to bind analogs of diacylglycerol, such as phorbol esters; or to activate one or more members ofthe ras superfamily of proteins. Functional allelic variants will typically contain only conservative substitution of one or more amino acids of SEQ ID NO:2, or substitution, deletion or insertion of non-critical residues in non-critical regions ofthe protein. Non-functional allelic variants are naturally-occurring amino acid sequence variants ofthe 47476, e.g, human 47476, protein within a population that do not have the ability to: exchange guanine nucleotides (GTP for GDP); to bind calcium; bind at least one and preferably two zinc ions; bind a second messenger, e.g, diacylglycerol; bind analogs of diacylglycerol, such as phorbol esters; or to activate one or more members ofthe ras superfamily of proteins. Non-functional allelic variants will typically contain a non- conservative substitution, a deletion, or insertion, or premature truncation ofthe amino acid sequence of SEQ ID NO:2, or a substitution, insertion, or deletion in critical residues or critical regions ofthe protein.
Preferred 67210 variants include those that are correlated with glycosyltransferaseactivity. Allelic variants of 67210, e.g, human 67210, include both functional and non-functional proteins. Functional allelic variants are naturally occurring amino acid sequence variants ofthe 67210 protein within a population that maintain the ability to transfer an activated mono- or oligosaccharide residue to an existing acceptor molecule for the initiation or elongation of a carbohydrate chain. Functional allelic variants will typically contain only conservative substitution of one or more amino acids of SEQ ID NO: 5, or substitution, deletion or insertion of non-critical residues in non-critical regions ofthe protein. Non-functional allelic variants are naturally-occurring amino acid sequence variants ofthe 67210, e.g, human 67210, protein within a population that do not have the ability to transferr an activated mono- or oligosaccharide residue to an existing acceptor molecule for the initiation or elongation of a carbohydrate chain. Non-functional allelic variants will typically contain a non-conservative substitution, a deletion, or insertion, or premature truncation ofthe amino acid sequence of SEQ ID NO: 5, or a substitution, insertion, or deletion in critical residues or critical regions ofthe protein.
Preferred 49875 variants include those that are correlated with helicase activity, e.g, RNA or DNA helicase activity.
Allelic variants of 49875, e.g, human 49875, include both functional and non-functional proteins. Functional allelic variants are naturally occurring amino acid sequence variants ofthe 49875 protein within a population that maintain the ability to bind an NTP, e.g, ATP, and/or unwind a nucleic acid duplex. Functional allelic variants will typically contain only conservative substitution of one or more amino acids of SEQ ID NO:8, or substitution, deletion or insertion of non-critical residues in non-critical regions ofthe protein. Non-functional allelic variants are naturally-occurring amino acid sequence variants ofthe 49875, e.g, human 49875, protein within a population that do not have the ability to bind an NTP, e.g, ATP, and/or unwind a nucleic acid duplex. Non-functional allelic variants will typically contain a non- conservative substitution, a deletion, or insertion, or premature truncation ofthe amino acid sequence of SEQ ID NO: 8, or a substitution, insertion, or deletion in critical residues or critical regions ofthe protein.
Preferred 46842 variants include those that are correlated with Arf GTPase stimulating activity.
Allelic variants of 46842, e.g, human 46842, include both functional and non-functional proteins. Functional allelic variants are naturally occurring amino acid sequence variants ofthe 46842 protein within a population that maintain the ability to interact with, e.g, bind to, Arf proteins and phosphoinositides. Functional allelic variants will typically contain only conservative substitution of one or more amino acids of SEQ ID NO:l 1, or substitution, deletion or insertion of non-critical residues in non-critical regions ofthe protein. Non- functional allelic variants are naturally-occurring amino acid sequence variants ofthe 46842, e.g, human 46842, protein within a population that do not have the ability to interact with, e.g, bind to, Arf proteins and phosphoinositides. Non-functional allelic variants will typically contain a non-conservative substitution, a deletion, or insertion, or premature truncation ofthe amino acid sequence of SEQ ID NO: 11, or a substitution, insertion, or deletion in critical residues or critical regions ofthe protein.
Preferred 33201 variants include those that are correlated with the ability to metabolize alcohols and/or the ability to catalyze the reduction of quinines.
Allelic variants of 33201, e.g, human 33201, include both functional and non-functional proteins. Functional allelic variants are naturally occurring amino acid sequence variants ofthe 33201 protein within a population that maintain the ability to metabolize alcohols and/or the ability to catalyze the reduction of quinones. Functional allelic variants will typically contain only conservative substitution of one or more amino acids of SEQ ID NO: 14, or substitution, deletion or insertion of non-critical residues in non-critical regions ofthe protein. Nonfunctional allelic variants are naturally-occurring amino acid sequence variants ofthe 33201, e.g, human 33201, protein within a population that do not have the ability to metabolize alcohols and/or the ability to catalyze the reduction of quinones. Non-functional allelic variants will typically contain a non-conservative substitution, a deletion, or insertion, or premature truncation ofthe amino acid sequence of SEQ ID NO: 14, or a substitution, insertion, or deletion in critical residues or critical regions ofthe protein.
Preferred 83378, 84233, 64708, 85041, or 84234 variants include those that are correlated with the ability to facilitate cation diffusion.
Allelic variants of 83378, 84233, 64708, 85041, or 84234, e.g, human 83378, 84233, 64708, 85041, or 84234, include both functional and non-functional proteins. Functional allelic variants are naturally occurring amino acid sequence variants ofthe 83378, 84233, 64708, 85041, or 84234 protein within a population that maintain the ability to facilitate cation diffusion. Functional allelic variants will typically contain only conservative substitution of one or more amino acids of SEQ ID NO: 17, SEQ ID NO:20, SEQ ID NO:23, SEQ ID NO:26, or SEQ ID NO:29, or substitution, deletion or insertion of non-critical residues in non-critical regions ofthe protein. Non-functional allelic variants are naturally-occurring amino acid sequence variants ofthe 83378, 84233, 64708, 85041, or 84234, e.g, human 83378, 84233, 64708, 85041, or 84234, protein within a population that do not have the ability to facilitate cation diffusion. Non-functional allelic variants will typically contain a non-conservative substitution, a deletion, or insertion, or premature truncation ofthe amino acid sequence of SEQ ID NO: 17, SEQ ID NO:20, SEQ ID NO:23, SEQ ID NO:26, or SEQ ID NO:29, or a substitution, insertion, or deletion in critical residues or critical regions ofthe protein. Moreover, nucleic acid molecules encoding other 47476, 67210, 49875, 46842, 33201,
83378, 84233, 64708, 85041, or 84234 family members and, thus, which have a nucleotide sequence which differs from the 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 sequences of SEQ ID NO:l, SEQ ID NO:4, SEQ ID NO:7, SEQ ID NO:10, SEQ ID NO:13, SEQ ID NO: 16, SEQ ID NO:19, SEQ ID NO:22, SEQ ID NO:25, or SEQ ID NO:28, or SEQ ID NO:3, SEQ ID NO:6, SEQ ID NO:9, SEQ ID NO: 12, SEQ ID NO: 15, SEQ ID NO: 18, SEQ ID NO:21, SEQ ID NO:24, SEQ ID NO:27, or SEQ ID NO:30 are intended to be within the scope ofthe invention. Antisense Nucleic Acid Molecules, Ribozymes, and Modified 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 Nucleic Acid Molecules
In another aspect, the invention features, an isolated nucleic acid molecule which is antisense to 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234. An "antisense" nucleic acid can include a nucleotide sequence which is complementary to a "sense" nucleic acid encoding a protein, e.g, complementary to the coding strand of a double-stranded cDNA molecule or complementary to an mRNA sequence. The antisense nucleic acid can be complementary to an entire 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 coding strand, or to only a portion thereof (e.g, the coding region of human 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 corresponding to SEQ ID NO:l, SEQ ID NO:4, SEQ ID NO:7, SEQ ID NO:10, SEQ ID NO:13, SEQ ID NO:16, SEQ ID NO: 19, SEQ ID NO:22, SEQ ID NO:25, or SEQ ID NO:28, or SEQ ID NO:3, SEQ ID NO:6, SEQ ID NO:9, SEQ ID NO: 12, SEQ ID NO: 15, SEQ ID NO: 18, SEQ ID NO:21, SEQ ID NO:24, SEQ ID NO:27, or SEQ ID NO:30. In another embodiment, the antisense nucleic acid molecule is antisense to a "noncoding region" ofthe coding strand of a nucleotide sequence encoding 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 (e.g, the 5' and 3' untranslated regions).
An antisense nucleic acid can be designed such that it is complementary to the entire coding region of 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 mRNA, but more preferably is an oligonucleotide which is antisense to only a portion ofthe coding or noncoding region of 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 mRNA. For example, the antisense oligonucleotide can be complementary to the region surrounding the translation start site of 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 mRNA, e.g, between the -10 and +10 regions ofthe target gene nucleotide sequence of interest. An antisense oligonucleotide can be, for example, about 7, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, or more nucleotides in length.
An antisense nucleic acid ofthe invention can be constructed using chemical synthesis and enzymatic ligation reactions using procedures known in the art. For example, an antisense nucleic acid (e.g, an antisense oligonucleotide) can be chemically synthesized using naturally occurring nucleotides or variously modified nucleotides designed to increase the biological stability ofthe molecules or to increase the physical stability ofthe duplex formed between the antisense and sense nucleic acids, e.g, phosphorothioate derivatives and acridine substituted nucleotides can be used. The antisense nucleic acid also can be produced biologically using an expression vector into which a nucleic acid has been subcloned in an antisense orientation (i.e, RNA transcribed from the inserted nucleic acid will be of an antisense orientation to a target nucleic acid of interest, described further in the following subsection).
The antisense nucleic acid molecules ofthe invention are typically administered to a subject (e.g, by direct injection at a tissue site), or generated in situ such that they hybridize with or bind to cellular mRNA and/or genomic DNA encoding a 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 protein to thereby inhibit expression ofthe protein, e.g, by inhibiting transcription and/or translation. Alternatively, antisense nucleic acid molecules can be modified to target selected cells and then administered systemically. For systemic administration, antisense molecules can be modified such that they specifically bind to receptors or antigens expressed on a selected cell surface, e.g, by linking the antisense nucleic acid molecules to peptides or antibodies which bind to cell surface receptors or antigens. The antisense nucleic acid molecules can also be delivered to cells using the vectors described herein. To achieve sufficient intracellular concentrations ofthe antisense molecules, vector constructs in which the antisense nucleic acid molecule is placed under the control of a strong pol II or pol III promoter are preferred.
In yet another embodiment, the antisense nucleic acid molecule ofthe invention is an α- anomeric nucleic acid molecule. An α-anomeric nucleic acid molecule forms specific double- stranded hybrids with complementary RNA in which, contrary to the usual β-units, the strands run parallel to each other (Gaultier et al. (1987) Nucleic Acids. Res. 15:6625-6641). The antisense nucleic acid molecule can also comprise a 2'-o-methylribonucleotide (Inoue et al. (1987) Nucleic Acids Res. 15:6131-6148) or a chimeric RNA-DNA analogue (Inoue et al. (1987) FEBSLett. 215:327-330).
In still another embodiment, an antisense nucleic acid ofthe invention is a ribozyme. A ribozyme having specificity for a 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234-encoding nucleic acid can include one or more sequences complementary to the nucleotide sequence of a 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 cDNA disclosed herein (i.e, SEQ ID NO: 1 , SEQ ID NO:4, SEQ ID NO:7, SEQ ID NO:10, SEQ ID NO:13, SEQ ID NO:16, SEQ ID NO:19, SEQ ID NO:22, SEQ ID NO:25, or SEQ ID NO:28, or SEQ ID NO:3, SEQ ID NO:6, SEQ ID NO:9, SEQ ID NO: 12, SEQ ID NO:15, SEQ ID NO:18, SEQ ID NO:21, SEQ ID NO:24, SEQ ID NO:27, or SEQ ID NO:30), and a sequence having known catalytic sequence responsible for mRNA cleavage (see U.S. Pat. No. 5,093,246 or Haselhoff and Gerlach (1988) Nature 334:585-591). For example, a derivative of a Tetrahymena L-19 IVS RΝA can be constructed in which the nucleotide sequence ofthe active site is complementary to the nucleotide sequence to be cleaved in a 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234-encoding mRΝA. See, e.g, Cech et al U.S. Patent o. 4,987,071; and Cech et al. U.S. Patent No. 5,116,742. Alternatively, 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 mRNA can be used to select a catalytic RNA having a specific ribonuclease activity from a pool of RNA molecules. See, e.g, Bartel, D. and Szostak, J.W. (1993) Science 261:1411-1418. 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 gene expression can be inhibited by targeting nucleotide sequences complementary to the regulatory region ofthe 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 (e.g, the 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 promoter and/or enhancers) to form triple helical structures that prevent transcription ofthe 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 gene in target cells. See generally, Helene, C. (1991) Anticancer Drug Des. 6:569-84; Helene, C. i (1992) Ann. NY. Acad. Sci. 660:27-36; and Maher, L.J. (1992) Bioassays 14:807-15. The potential sequences that can be targeted for triple helix formation can be increased by creating a so-called
"switchback" nucleic acid molecule. Switchback molecules are synthesized in an alternating 5'- 3', 3'-5' manner, such that they base pair with first one strand of a duplex and then the other, eliminating the necessity for a sizeable stretch of either purines or pyrimidines to be present on one strand of a duplex. The invention also provides detectably labeled oligonucleotide primer and probe molecules. Typically, such labels are chemiluminescent, fluorescent, radioactive, or colorimetric.
A 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 nucleic acid molecule can be modified at the base moiety, sugar moiety or phosphate backbone to improve, e.g, the stability, hybridization, or solubility ofthe molecule. For non-limiting examples of synthetic oligonucleotides with modifications see Toulme (2001) Nature Biotech. 19:17 and Faria et al. (2001) Nature Biotech. 19:40-44. Such phosphoramidixe oligonucleotides can be effective antisense agents.
For example, the deoxyribose phosphate backbone ofthe nucleic acid molecules can be modified to generate peptide nucleic acids (see Hyrup B. et al (1996) Bioorganic & Medicinal Chemistry 4: 5-23). As used herein, the terms "peptide nucleic acid" or "PNA" refers to a nucleic acid mimic, e.g, a DNA mimic, in which the deoxyribose phosphate backbone is replaced by a pseudopeptide backbone and only the four natural nucleobases are retained. The neutral backbone of a PNA can allow for specific hybridization to DNA and RNA under conditions of low ionic strength. The synthesis of PNA oligomers can be performed using standard solid phase peptide synthesis protocols as described in Hyrup B. etal. (1996) supra and Perry-O'Keefe et al. Proc. Natl. Acad. Sci. 93: 14670-675.
PNAs of 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 nucleic acid molecules can be used in therapeutic and diagnostic applications. For example, PNAs can be used as antisense or antigene agents for sequence-specific modulation of gene expression by, for example, inducing transcription or translation arrest or inhibiting replication. PNAs of 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 nucleic acid molecules can also be used in the analysis of single base pair mutations in a gene, (e.g, by PNA-directed PCR clamping); as 'artificial restriction enzymes' when used in combination with other enzymes, (e.g, SI nucleases (Hyrup B. et al. (1996) supra)); or as probes or primers for DNA sequencing or hybridization (Hyrup B. et al. (1996) supra; Perry-O'Keefe supra).
In other embodiments, the oligonucleotide may include other appended groups such as peptides (e.g, for targeting host cell receptors in vivo), or agents facilitating transport across the cell membrane (see, e.g, Letsinger et al. (1989) Proc. Natl. Acad. Sci USA 86:6553-6556; Lemaifre et al (1987) Proc. Natl. Acad. Sci. USA 84:648-652; PCT Publication No. W088/09810) or the blood-brain barrier (see, e.g, PCT Publication No. W089/10134). In addition, oligonucleotides can be modified with hybridization-triggered cleavage agents (see, e.g, Krol et al. (1988) Bio-Techniques 6:958-976) or intercalating agents, (see, e.g, Zon (1988) Pharm. Res. 5:539-549). To this end, the oligonucleotide may be conjugated to another molecule, (e.g, a peptide, hybridization triggered cross-linking agent, transport agent, or hybridization-triggered cleavage agent). The invention also includes molecular beacon oligonucleotide primer and probe molecules having at least one region which is complementary to a 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 nucleic acid ofthe invention, two complementary regions one having a fluorophore and one a quencher such that the molecular beacon is useful for quantitating the presence ofthe 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 nucleic acid ofthe invention in a sample. Molecular beacon nucleic acids are described, for example, in Lizardi et al, U.S. Patent No. 5,854,033; Nazarenko et al, U.S. Patent No. 5,866,336, and Livak et al, U.S. Patent 5,876,930.
Isolated 47476 Polypeptides In another aspect, the invention features, an isolated 47476 protein, or fragment, e.g, a biologically active portion, for use as immunogens or antigens to raise or test (or more generally to bind) anti-47476 antibodies. 47476 protein can be isolated from cells or tissue sources using standard protein purification techniques. 47476 protein or fragments thereof can be produced by recombinant DNA techniques or synthesized chemically. Polypeptides ofthe invention include those that arise as a result ofthe existence of multiple genes, alternative transcription events, alternative RNA splicing events, and alternative translational and post-translational events. The polypeptide can be expressed in systems, e.g, cultured cells, which result in substantially the same post-translational modifications present when expressed the polypeptide is expressed in a native cell, or in systems which result in the alteration or omission of post-translational modifications, e.g, glycosylation or cleavage, present when expressed in a native cell.
In a preferred embodiment, a 47476 polypeptide has one or more ofthe following characteristics
(i) it has the ability to stimulate the exchange of guanine nucleotides (GTP for GDP) bound to small guanine nucleotide binding proteins, in particular, ras or ras-like proteins; (ii) it has the ability to bind calcium;
(iii) it has the ability to bind at least one and preferably two zinc ions; (iv) it has the ability to bind a second messenger, e.g, diacylglycerol or analogs thereof, such as phorbol esters; (v) it has the ability to activate a ras superfamily member; (vi) it has a molecular weight, e.g, a deduced molecular weight, preferably ignoring any contribution of post translational modifications, amino acid composition or other physical characteristic of a 47476 polypeptide, e.g, a polypeptide of SEQ ID NO:2;
(vii) it has an overall sequence similarity of at least 60%, more preferably at least 70%, 80%, 90%, 95%, 98%, 99%, or more with a polypeptide of SEQ ID NO:2;
(viii) it has a ras guanine nucleotide dissociation stimulator domain which is preferably about 70%, 80%), 90%, 95%, 98%, 99%, or more homologous to amino acid residues about 195 to 381 of SEQ ID NO:2;
(ix) it has a guanine nucleotide dissociation stimulator domain N-terminal motif which is preferably about 70%, 80%, 90%, 95%, 98%, 99%, or more homologous to amino acid residues about 55 to 172 of SEQ ID NO:2;
(x) it has an EF-hand calcium-binding domain which is preferably about 70%, 80%, 90%, 95%, 98%, 99%, or more homologous to amino acid residues about 470 to 498 of SEQ ID NO:2; (xi) it has a phorbol ester/diacylglycerol binding domain (Cl domain) which is preferably about 70%, 80%, 90%>, 95%, 98%>, 99%), or more homologous to amino acid residues about 541 to 590 of SEQ ID NO:2;
(xii) it has a guanine nucleotide dissociation stimulator domain N-terminal motif which is preferably about 70%, 80%, 90%, 95%, 98%, 99%, or more homologous to amino acid residues about 55 to 172 of SEQ ID NO:2;
(xiii) it has at least one predicted N-glycosylation site (PS00001);
(xiv) it has at least one, two, three, preferably four predicted cAMP/cGMP-dependent protein kinase phosphorylation sites (PS00004);
(xv) it has at least one, two, three, four, five, six, seven, preferably eight predicted Protein Kinase C phosphorylation sites (PS00005);
(xvi) it has at least one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, preferably fourteen predicted Casein Kinase II phosphorylation sites (PS00006);
(xvii) it has at least one, two, three, preferably four predicted N-myristylation sites (PS00008); and (xviii) it has at least one predicted Amidation site (PS00009). In a preferred embodiment the 47476 protein, or fragment thereof, differs from the corresponding sequence in SEQ ID NO:2. In one embodiment it differs by at least one but by less than 15, 10 or 5 amino acid residues. In another it differs from the corresponding sequence in SEQ ID NO:2 by at least one residue but less than 20%, 15%, 10% or 5% ofthe residues in it differ from the corresponding sequence in SEQ ID NO:2. (If this comparison requires alignment the sequences should be aligned for maximum homology. "Looped" out sequences from deletions or insertions, or mismatches, are considered differences.) The differences are, preferably, differences or changes at a non-essential residue or a conservative substitution. In a preferred embodiment, the differences are not in the ras guanine nucleotide dissociation stimulator domain, the EF-hand calcium-binding domain or the phorbol ester/diacylglycerol binding domain (Cl domain). In another preferred embodiment, one or more differences are in the ras guanine nucleotide dissociation stimulator domain, the EF-hand calcium-binding domain, and/or the phorbol ester/diacylglycerol binding domain (Cl domain).
Other embodiments include a protein that contain one or more changes in amino acid sequence, e.g, a change in an amino acid residue which is not essential for activity. Such
47476 proteins differ in amino acid sequence from SEQ ID NO:2, yet retain biological activity.
In one embodiment, the protein includes an amino acid sequence at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or more homologous to SEQ ID NO:2.
A 47476 protein or fragment is provided which varies from the sequence of SEQ ID NO.2 in regions defined by amino acids about 1 to 54, 173 to 194, 382 to 469, 499 to 540, and 591 to 672 by at least one but by less than 15, 10 or 5 amino acid residues in the protein or fragment, but which does not differ from SEQ ID NO.2 in regions defined by amino acids about 55 to 173, 195 to 381, 470 to 498 or 541 to 590 of SEQ ID NO:2. (If this comparison requires alignment the sequences should be aligned for maximum homology. "Looped" out sequences from deletions or insertions, or mismatches, are considered differences.) In some embodiments the difference is at a non-essential residue or is a conservative substitution, while in others the difference is at an essential residue or is a non-conservative substitution.
In one embodiment, a biologically active portion of a 47476 protein includes a ras guanine nucleotide dissociation stimulator domain. Moreover, other biologically active portions, in which other regions ofthe protein are deleted, can be prepared by recombinant techniques and evaluated for one or more ofthe functional activities of a native 47476 protein. In a preferred embodiment, the 47476 protein has an amino acid sequence shown in SEQ ID NO:2. In other embodiments, the 47476 protein is substantially identical to SEQ ID NO:2. In yet another embodiment, the 47476 protein is substantially identical to SEQ ID NO:2 and retains the functional activity ofthe protein of SEQ ID NO:2, as described in detail in the subsections above.
In a preferred embodiment, a 47476 polypeptide fragment differs by at least 1, 2, 3, 10, 20, or more amino acid residues of a previously disclosed sequence. Differences can include differing in length or sequence identity. For example, a fragment can: include one or more amino acid residues from SEQ ID NO: 5 outside the region of overlap with the previously disclosed amino acid sequence; not include all ofthe amino acid residues encoded by a previously disclosed polypeptide sequence, e.g, can be one or more amino acid residues shorter (at one or both ends) than a previously disclocsed polypeptide sequence, or can differ by one or more amino acid residues in the region of overlap.
Isolated 67210 Polypeptides In another aspect, the invention features, an isolated 67210 protein, or fragment, e.g, a biologically active portion, for use as immunogens or antigens to raise or test (or more generally to bind) anti-67210 antibodies. 67210 protein can be isolated from cells or tissue sources using standard protein purification techniques. 67210 protein or fragments thereof can be produced by recombinant DNA techniques or synthesized chemically. Polypeptides ofthe invention include those which arise as a result ofthe existence of multiple genes, alternative transcription events, alternative RNA splicing events, and alternative translational and post-translational events. The polypeptide can be expressed in systems, e.g, cultured cells, which result in substantially the same post-translational modifications present when expressed the polypeptide is expressed in a native cell, or in systems which result in the alteration or omission of post-translational modifications, e.g, glycosylation or cleavage, present when expressed in a native cell.
In a preferred embodiment, a 67210 polypeptide has one or more ofthe following characteristics:
(i) it has the ability to catalyze the transfer of an activated sugar residue to an acceptor molecule; (ii) it has the ability to catalyzes the processing, folding, and secretion of proteins;
(iii) it has a molecular weight, e.g, a deduced molecular weight, preferably ignoring any contribution of post translational modifications, amino acid composition or other physical characteristic of SEQ ID NO:5; (iv) it has an overall sequence similarity of at least 60%, more preferably at least 70, 80,
90, or 95%, with a polypeptide of SEQ ID NO:5;
(v) it has a glycosyl transferase domain which is preferably about 70%, 80%, 90% or 95%) homologous with amino acid residues about 63 to 340 of SEQ ID NO:5;
(vi) it can be found in any one or more of: coronary smooth muscle cells (SMC), normal artery, normal brain cortex, normal breast, and normal ovary;
(vii) it has at least one predicted signal peptide;
(viii) it has at least one dileucine motif;
(ix) it has at least one predicted N-glycosylation site (PS00001);
(x) it has at least one predicted Protein Kinase C phosphorylation site (PS00005); (xi) it has at least one, two, three, preferably four predicted Casein Kinase II phosphorylation sites (PS00006);
(xii) it has at least one predicted tyrosine kinase phosphorylation site (PS00007); and
(xiii) it has at least one, two, three, four, five, preferably six predicted N-myristoylation sites (PS00008). In a preferred embodiment the 67210 protein, or fragment thereof, differs from the corresponding sequence in SEQ ID NO:2. In one embodiment it differs by at least one but by less than 15, 10 or 5 amino acid residues. In another it differs from the corresponding sequence in SEQ ID NO: 5 by at least one residue but less than 20%, 15%, 10% or 5% ofthe residues in it differ from the corresponding sequence in SEQ ID NO: 5. (If this comparison requires alignment the sequences should be aligned for maximum homology. "Looped" out sequences from deletions or insertions, or mismatches, are considered differences.) The differences are, preferably, differences or changes at a non-essential residue or a conservative substitution. In a preferred embodiment the differences are not in the glycosyltransferase domain (amino acid 63 to 340 of SEQ ID NO:5). In another preferred embodiment one or more differences are in the glycosyltransferase domain (amino acid 63 to 340 of SEQ ID NO:5). Other embodiments include a protein that contain one or more changes in amino acid sequence, e.g, a change in an amino acid residue which is not essential for activity. Such 67210 proteins differ in amino acid sequence from SEQ ID NO: 5, yet retain biological activity. In one embodiment, the protein includes an amino acid sequence at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or more homologous to SEQ ID NO:5.
A 67210 protein or fragment is provided which varies from the sequence of SEQ ID NO:5 in regions defined by amino acids about 1 to 62 and 341 to 349 by at least one but by less than 15, 10 or 5 amino acid residues in the protein or fragment but which does not differ from SEQ ID NO: 5 in regions defined by amino acids about 63 to 340. (If this comparison requires alignment the sequences should be aligned for maximum homology. "Looped" out sequences from deletions or insertions, or mismatches, are considered differences.) In some embodiments the difference is at a non-essential residue or is a conservative substitution, while in others the difference is at an essential residue or is a non-conservative substitution. In one embodiment, a biologically active portion of a 67210 protein includes a glycosyltransferase domain. Moreover, other biologically active portions, in which other regions ofthe protein are deleted, can be prepared by recombinant techniques and evaluated for one or more ofthe functional activities of a native 67210 protein.
In a preferred embodiment, the 67210 protein has an amino acid sequence shown in SEQ ID NO:5. In other embodiments, the 67210 protein is substantially identical to SEQ ID NO:5. In yet another embodiment, the 67210 protein is substantially identical to SEQ ID NO: 5 and retains the functional activity ofthe protein of SEQ ID NO: 5, as described in detail in the subsections above.
In a preferred embodiment, a fragment differs by at least 1, 2, 3, 10, 20, or more amino acid residues encoded by a sequence present in Genbank accession number AC013776 or
AC023550. Differences can include differing in length or sequence identity. For example, a fragment can: include one or more amino acid residues from SEQ ID NO: 5 outside the region encoded by nucleotides 290 to 560, 675 to 1042, or 1152 to 1743 of SEQ ID NO:4; not include all ofthe amino acid residues encoded by a nucleotide sequence in Genbank accession number AC013776 or AC023550, e.g, can be one or more amino acid residues shorter (at one or both ends) than a sequence encoded by the nucleotide sequence in Genbank accession number AC013776 or AC023550, or can differ by one or more amino acid residues in the region of overlap.
Isolated 49875 Polypeptides
In another aspect, the invention features, an isolated 49875 protein, or fragment, e.g, a biologically active portion, for use as immunogens or antigens to raise or test (or more generally to bind) anti-49875 antibodies. 49875 protein can be isolated from cells or tissue sources using standard protein purification techniques. 49875 protein or fragments thereof can be produced by recombinant DNA techniques or synthesized chemically.
Polypeptides ofthe invention include those that arise as a result ofthe existence of multiple genes, alternative transcription events, alternative RNA splicing events, and alternative translational and post-translational events. The polypeptide can be expressed in systems, e.g, cultured cells, which result in substantially the same post-translational modifications present when expressed the polypeptide is expressed in a native cell, or in systems which result in the alteration or omission of post-translational modifications, e.g, glycosylation or cleavage, present when expressed in a native cell.
In a preferred embodiment, a 49875 polypeptide has one or more ofthe following characteristics:
(i) it has the ability to bind and hydrolyze ATG or GTP;
(ii) it has the ability to break the hydrogen bonds between the two strands of a nucleic acid duplex and unwind the duplex;
(iii) it has the ability to modulate replication; (iv) it has the ability to modulate transcription or translation;
(v) it has a molecular weight, e.g, a deduced molecular weight, preferably ignoring any contribution of post translational modifications, amino acid composition or other physical characteristic of SEQ ID NO:8;
(vi) it has an overall sequence similarity of at least 60%, more preferably at least 70%, 80%, 90%, 95%, 98%, 99%, or more with a polypeptide of SEQ ID NO:8;
(vii) it has a DEAD-type helicase domain which is preferably about 70%, 80%, 90%, 95%, 98%, 99%, or more homologous with amino acid residues about 22 to 245 of SEQ ID NO:8; or (vii) it has a conserved helicase C-terminal domain which is preferably about 70%, 80%, 90%, 95%, 98%, 99%o, or more homologous with amino acid residues about 281 to 363 of SEQ ID NO:8.
(viii) it has a conserved DEAD-box subfamily ATP-dependent helicase signature motif (PS00039);
(ix) it can be found in, e.g, breast, colon, lung and/or ovary normal and tumor tissue and placenta.
(x) it has at least one, preferably two predicted N-glycosylation sites;
(xi) it has at least one, two, three, four, preferably five predicted Protein Kinase C phosphorylation sites (PS00005);
(xii) it has at least one, two, three, four, five, six, preferably seven predicted Casein Kinase II phosphorylation sites (PS00006);
(xiii) it has at least one, two, three, four, five, six, preferably seven predicted N- myristoylation sites (PS00008); (xiv) it has at least one, preferably two predicted amidation sites (PS00009); or
(xv) it has at least one predicted ATP/GTP-binding site motif A (P-loop) (PS00017).
In a preferred embodiment the 49875 protein, or fragment thereof, differs from the corresponding sequence in SEQ ID: 8. In one embodiment it differs by at least one but by less than 15, 10 or 5 amino acid residues. In another it differs from the corresponding sequence in SEQ ID NO : 8 by at least one residue but less than 20%, 15%, 10% or 5% of the residues in it differ from the corresponding sequence in SEQ ID NO:8. (If this comparison requires alignment the sequences should be aligned for maximum homology. "Looped" out sequences from deletions or insertions, or mismatches, are considered differences.) The differences are, preferably, differences or changes at a non essential residue or a conservative substitution. In a preferred embodiment the differences are not in the DEAD type helicase domain (amino acids 22-245 of SEQ ID NO: 8). In another preferred embodiment one or more differences are in the DEAD type helicase domain (amino acids 22-245 of SEQ ID NO:8).
Other embodiments include a protein that contain one or more changes in amino acid sequence, e.g, a change in an amino acid residue which is not essential for activity. Such 49875 proteins differ in amino acid sequence from SEQ ID NO:8, yet retain biological activity. In one embodiment, the protein includes an amino acid sequence at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or more homologous to SEQ ID NO:8.
A 49875 protein or fragment is provided which varies from the sequence of SEQ ID NO: 8 in regions defined by amino acids about amino acids 1-21 and 246-600 of SEQ ID NO: 8 by at least one but by less than 15, 10 or 5 amino acid residues in the protein or fragment but which does not differ from SEQ ID NO: 8 in regions defined by amino acids about 22-245. (If this comparison requires alignment the sequences should be aligned for maximum homology. "Looped" out sequences from deletions or insertions, or mismatches, are considered differences.) In some embodiments the difference is at a non-essential residue or is a conservative substitution, while in others the difference is at an essential residue or is a non-conservative substitution.
In one embodiment, a biologically active portion of a 49875 protein includes a DEAD type helicase domain or a conserved C-terminal helicase domain. Moreover, other biologically active portions, in which other regions ofthe protein are deleted, can be prepared by recombinant techniques and evaluated for one or more ofthe functional activities of a native 49875 protein.
In a preferred embodiment, the 49875 protein has an amino acid sequence shown in SEQ ID NO:8. In other embodiments, the 49875 protein is substantially identical to SEQ ID NO: 8. In yet another embodiment, the 49875 protein is substantially identical to SEQ ID NO: 8 and retains the functional activity ofthe protein of SEQ ID NO:8, as described in detail in the subsections above.
In a preferred embodiment, a fragment differs by at least 1, 2, 3, 10, 20, or more amino acid residues encoded by a sequence present in WO 01/55301, WO 01/57188 or WO 01/62927. Differences can include differing in length or sequence identity. For example, a fragment can: not include all ofthe amino acid residues encoded by a nucleotide sequence in WO 01/55301, WO 01/57188 or WO 01/62927, e.g, can be one or more amino acid residues shorter (at one or both ends) than a sequence encoded by a nucleotide sequence inWO 01/55301, WO 01/57188 or WO 01/62927.or can differ by one or more amino acid residues in the region of overlap. Isolated 46842 Polypeptides
In another aspect, the invention features, an isolated 46842 protein, or fragment, e.g, a biologically active portion, for use as immunogens or antigens to raise or test (or more generally to bind) anti-46842 antibodies. 46842 protein can be isolated from cells or tissue sources using standard protein purification techniques. 46842 protein or fragments thereof can be produced by recombinant DNA techniques or synthesized chemically.
Polypeptides ofthe invention include those which arise as a result ofthe existence of multiple genes, alternative transcription events, alternative RNA splicing events, and alternative translational and post-translational events. The polypeptide can be expressed in systems, e.g, cultured cells, which result in substantially the same post-translational modifications present when expressed the polypeptide is expressed in a native cell, or in systems which result in the alteration or omission of post-translational modifications, e.g, glycosylation or cleavage, present when expressed in a native cell.
In a preferred embodiment, A 46842 polypeptide has one or more ofthe following characteristics:
(i) it has the ability to stimulate GTP hydrolysis of GTP bound by an Arf or Arf-like protein;
(ii) it has the ability to respond to phosphoinositide second messengers
(iii) it has a molecular weight, e.g, a deduced molecular weight, preferably ignoring any contribution of post translational modifications, amino acid composition or other physical characteristic of SEQ ID NO: 11 ;
(iv) it has an overall sequence similarity of at least 60%, more preferably at least 70%, 80%, 90%, 95%, 98%, 99%, or more with a polypeptide of SEQ ID NO:l 1;
(v) it has a PH domain which is preferably about 70%, 80%, 90%, 95%, 98%, 99%, or more homologous with amino acid residues about 269 to 363 of SEQ ID NO: 11 ;
(vi) it has an ArfGAP domain which is preferably about 70%, 80%, 90%, 95%, 98%, 99%, or more homologous with amino acid residues about 403 to 525 of SEQ ID NO: 11 ;
(vii) it has at least one, preferably two ankyrin domains which are preferably about 70%, 80%, 90%, 95%, 98%, 99%, or more homologous with amino acid residues about 702 to 734 or 735 to 767 of SEQ ID NO:ll;
(viii) it can colocalize with Arf proteins; (ix) it can localize to a vesicle membrane and/or the plasma membrane;
(x) it has the conserved cysteines for coordinating a zinc ion at about residues 421, 418, 438, AND 440 OF SEQ ID NO:l 1;
(xi) it has at least one, two, three, four, five, six, seven, eight, nine, ten, eleven, preferably twelve predicted Protein Kinase C phosphorylation sites (PS00005);
(xii) it has at least one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, preferably fourteen predicted Casein Kinase II phosphorylation sites (PS00006);
(xiii) it has at least one, two, preferably three predicted cAMP/cGMP-dependent protein kinase phosphorylation sites (PS00004); (xiv) it has at least one predicted tyrosine kinase phosphorylation site (PS00007);
(xv) it has at least one predicted glycosaminoglycan attachment site (PS00002); and
(xvi) it has at least one, two, three, four, five, six, seven, eight, nine, preferably ten predicted N-myristylation sites (PS00008).
In a preferred embodiment the 46842 protein, or fragment thereof, differs from the corresponding sequence in SEQ ID NO: 11. In one embodiment it differs by at least one but by less than 15, 10 or 5 amino acid residues. In another it differs from the corresponding sequence in SEQ ID NO:l 1 by at least one residue but less than 20%, 15%, 10% or 5% ofthe residues in it differ from the corresponding sequence in SEQ ID NO:l 1. (If this comparison requires alignment the sequences should be aligned for maximum homology. "Looped" out sequences from deletions or insertions, or mismatches, are considered differences.) The differences are, preferably, differences or changes at a non essential residue or a conservative substitution. In a preferred embodiment the differences are not in the ArfGAP, PH or ankyrin repeat domains. In another preferred embodiment one or more differences are in the ArfGAP, PH or ankyrin repeat domains. Other embodiments include a protein that contain one or more changes in amino acid sequence, e.g, a change in an amino acid residue which is not essential for activity. Such 46842 proteins differ in amino acid sequence from SEQ ID NO:l 1, yet retain biological activity.
In one embodiment, the protein includes an amino acid sequence at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or more homologous to SEQ ID NO:ll. A 46842 protein or fragment is provided which varies from the sequence of SEQ ID NO:l 1 in regions defined by amino acids about 403 to 525 by at least one but by less than 15, 10 or 5 amino acid residues in the protein or fragment but which does not differ from SEQ ID NO:l 1 in regions defined by amino acids about 1 to 402, and/or 526 to 834. (If this comparison requires alignment the sequences should be aligned for maximum homology. "Looped" out sequences from deletions or insertions, or mismatches, are considered differences.) In some embodiments the difference is at a non-essential residue or is a conservative substitution, while in others the difference is at an essential residue or is a non-conservative substitution.
In one embodiment, a biologically active portion of a 46842 protein includes an ArfGAP domain, a PH domain, or an ankyrin repeat domain. Moreover, other biologically active portions, in which other regions ofthe protein are deleted, can be prepared by recombinant techniques and evaluated for one or more ofthe functional activities of a native 46842 protein.
In a preferred embodiment, the 46842 protein has an amino acid sequence shown in SEQ ID NO: 11. In other embodiments, the 46842 protein is substantially identical to SEQ ID NO: 11. In yet another embodiment, the 46842 protein is substantially identical to SEQ ID
NO : 11 and retains the functional activity of the protein of SEQ ID NO : 11 , as described in detail in the subsections above. In still another embodiment, the protein includes the motif SLSSDSGLG at about amino acids 606 to 614 of SEQ ID NO: 11.
In a preferred embodiment, a fragment differs by at least 1, 2, 3, 10, 20, or more amino acid residues encoded by a sequence present in KIAA1716 (see, e.g, Genbank accession number gi 12697977). Differences can include differing in length or sequence identity. For example, a fragment can: include one or more amino acid residues from SEQ ID NO:l 1 outside the region of about amino acid residue 200 to 500, 300 to 600, or 400 to 834; not include all of the amino acid residues ofthe sequence in Genbank accession number gil2697977, e.g, can be one or more amino acid residues shorter (at one or both ends) than a sequence encoded by the nucleotide sequence in Genbank accession number gi 12697977, or can differ by one or more amino acid residues in the region of overlap.
Isolated 33201 Polypeptides
In another aspect, the invention features, an isolated 33201 protein, or fragment, e.g, a biologically active portion, for use as immunogens or antigens to raise or test (or more generally to bind) anti-33201 antibodies. 33201 protein can be isolated from cells or tissue sources using standard protein purification techniques. 33201 protein or fragments thereof can be produced by recombinant DNA techniques or synthesized chemically.
Polypeptides ofthe invention include those which arise as a result ofthe existence of multiple genes, alternative transcription events, alternative RNA splicing events, and alternative translational and post-translational events. The polypeptide can be expressed in systems, e.g, cultured cells, which result in substantially the same post-translational modifications present when expressed the polypeptide is expressed in a native cell, or in systems which result in the alteration or omission of post-translational modifications, e.g, glycosylation or cleavage, present when expressed in a native cell.
In a preferred embodiment, a 33201 polypeptide has one or more ofthe following characteristics:
(i) it has the ability to metabolize one or more alcohols;
(ii) it has the ability to catalyze the reduction of quinones; (iii) it has the ability to reduce a bioreductive compound, e.g, a bioreductive antitumor quinone;
(iv) it has the abilitity to metabolize and/or degrade toxins;
(v) it has a molecular weight, e.g, a deduced molecular weight, preferably ignoring any contribution of post-translational modifications, amino acid composition or other physical characteristic of a 33201 polypeptide, e.g, a polypeptide pf a SEQ ID NO:14;
(vi) it has an overall sequence similarity of at least 60%, more preferably at least 70%, 80%, 90%, 95%, 98%, 99%, or more with a polypeptide of SEQ ID NO: 14;
(vii) it has a dehydrogenase/reductase domain which is preferably about 70%, 80%, 90%), 95%, 98%, 99%, or more homolgous with amino acid residues about 22 to 345 of SEQ ID NO: 14;
(viii) it has at least two, preferably four, five, six and most preferably seven ofthe cysteines found in the amino acid sequence ofthe native protein;
(ix) it has at least one, two, preferably three conserved glycine residues;
(x) it has at least one predicted Protein Kinase C phosphorylation site (PS00005); (xi) it has at least one, preferably two predicted Casein Kinase II phosphorylation sites
(PS00006); (xii) it has at least one, two, three, preferably four predicted N-glycosylation sites (PS00001); and
(xiii) it has at least one, two, three, four, five, six, seven, preferably eight predicted N- myristylation sites (PS00008). In a preferred embodiment the 33201 protein, or fragment thereof, differs from the corresponding sequence in SEQ ID NO: 14. In one embodiment it differs by at least one but by less than 15, 10 or 5 amino acid residues. In another it differs from the corresponding sequence in SEQ ID NO: 14 by at least one residue but less than 20%, 15%, 10%) or 5% ofthe residues in it differ from the corresponding sequence in SEQ ID NO: 14. (If this comparison requires alignment the sequences should be aligned for maximum homology. "Looped" out sequences from deletions or insertions, or mismatches, are considered differences.) The differences are, preferably, differences or changes at a non-essential residue or a conservative substitution. In a preferred embodiment the differences are not in the dehydrogenase/reductase domain (e.g, about amino acids 55-380 of SEQ ID NO: 14). In another preferred embodiment one or more differences are in the dehydrogenase/reductase domain, (e.g, about amino acids 55-380 of SEQ ID NO: 14).
Other embodiments include a protein that contain one or more changes in amino acid sequence, e.g, a change in an amino acid residue which is not essential for activity. Such 33201 proteins differ in amino acid sequence from SEQ ID NO: 14, yet retain biological activity.
In one embodiment, the protein includes an amino acid sequence at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or more homologous to SEQ ID NO: 14.
A 33201 protein or fragment is provided which varies from the sequence of SEQ ID NO:14 in regions defined by amino acids about 1 to 21 and/or 346 to 351 of SEQ ID NO:14 by at least one but by less than 15, 10 or 5 amino acid residues in the protein or fragment, but which does not differ from SEQ ID NO: 14 in regions defined by amino acids about 22 to 345 of SEQ ID NO: 14. (If this comparison requires alignment the sequences should be aligned for maximum homology. "Looped" out sequences from deletions or insertions, or mismatches, are considered differences.) In some embodiments the difference is at a non-essential residue or is a conservative substitution, while in others the difference is at an essential residue or is a non- conservative substitution.
In one embodiment, a biologically active portion of a 33201 protein includes a dehydrogenase/reductase domain. Moreover, other biologically active portions, in which other regions ofthe protein are deleted, can be prepared by recombinant techniques and evaluated for one or more ofthe functional activities of a native 33201 protein.
In a preferred embodiment, the 33201 protein has an amino acid sequence shown in SEQ ID NO:14. In other embodiments, the 33201 protein is substantially identical to SEQ ID NO: 14. In yet another embodiment, the 33201 protein is substantially identical to SEQ ID NO:14 and retains the functional activity ofthe protein of SEQ ID NO:14, as described in detail in the subsections above.
In a preferred embodiment, a fragment differs by at least 1, 2, 3, 10, 20, or more amino acid residues encoded by a sequence present in Genbank accession number AC005520. Differences can include differing in length or sequence identity. For example, a fragment can include one or more amino acid residues from SEQ ID NO: 14 outside the region encoded by nucleotides 1117 to 1423, 1425 to 1627, or 1629 to 1717; not include all ofthe amino acid residues encoded by a nucleotide sequence in Genbank accession number AC005520, e.g, can be one or more amino acid residues shorter (at one or both ends) than a sequence encoded by the nucleotide sequence in Genbank accession number AC005520; or can differ by one or more amino acid residues in the region of overlap.
Isolated 83378, 84233, 64708, 85041, or 84234 Polypeptides
In another aspect, the invention features, an isolated 83378, 84233, 64708, 85041, or 84234 protein, or fragment, e.g, a biologically active portion, for use as immunogens or antigens to raise or test (or more generally to bind) anti-83378, 84233, 64708, 85041, or 84234 antibodies. 83378, 84233, 64708, 85041, or 84234 protein can be isolated from cells or tissue sources using standard protein purification techniques. 83378, 84233, 64708, 85041, or 84234 protein or fragments thereof can be produced by recombinant DNA techniques or synthesized chemically.
Polypeptides ofthe invention include those which arise as a result ofthe existence of multiple genes, alternative transcription events, alternative RNA splicing events, and alternative translational and post-translational events. The polypeptide can be expressed in systems, e.g, cultured cells, which result in substantially the same post-translational modifications present when expressed the polypeptide is expressed in a native cell, or in systems which result in the alteration or omission of post-translational modifications, e.g, glycosylation or cleavage, present when expressed in a native cell.
In a preferred embodiment, a 83378, 84233, 64708, 85041, or 84234 polypeptide has one or more ofthe following characteristics:
(i) it has the ability to modulate cellular tolerance and/or resistance to a metal ion, e.g, zinc; (ii) it has the ability to facilitate cation diffusion;
(iii) it has the ability to modulate cellular efflux of a metal ion, e.g, zinc;
(iv) it has the ability to modulate vesicular sequestration of a metal ion, e.g, zinc;
(v) it has a molecular weight, e.g, a deduced molecular weight, preferably ignoring any contribution of post translational modifications, amino acid composition or other physical characteristic of SEQ ID NO: 17, SEQ ID NO:20, SEQ ID NO:23, SEQ ID NO:26, or SEQ ID NO:29;
(vi) it has an overall sequence similarity of at least 60%, more preferably at least 70%, 80%, 90%, 95%, 98%, 99%, or more with a polypeptide of SEQ ID NO: 17, SEQ ID NO:20, SEQ ID NO:23, SEQ ID NO:26, or SEQ ID NO:29; (vii) it can be found in a membrane of a cell, e.g, a plasma membrane or a vesicular membrane;
(viii) it has a cation efflux domain which a sequence similarity of preferably about 70%, 80%, 90% or 95% with about amino acid residues 11 to 133 or 231 to 389 of SEQ ID NO:17, residues 25 to 310 of SEQ ID NO:20, residues 55 to 153 or 227 to 320 of SEQ ID NO:23, residues 419 to 733 of SEQ ID NO:26, or residues 38 to 349 of SEQ ID NO:29; or
(ix) it has at least one, two, three, four, five, six, preferably seven, or more histidine residues located on polypeptide regions present in the cytoplasm.
In a preferred embodiment the 83378, 84233, 64708, 85041, or 84234 protein, or fragment thereof, differs from the corresponding sequence in SEQ ID NO: 17, SEQ ID NO:20, SEQ ID NO:23, SEQ ID NO:26, or SEQ ID NO:29. In one embodiment it differs by at least one but by less than 15, 10 or 5 amino acid residues. In another it differs from the corresponding sequence in SEQ ID NO: 17, SEQ ID NO:20, SEQ ID NO:23, SEQ ID NO:26, or SEQ ID NO:29 by at least one residue but less than 20%, 15%, 10% or 5% ofthe residues in it differ from the corresponding sequence in SEQ ID NO: 17, SEQ ID NO:20, SEQ ID NO:23, SEQ ID NO:26, or SEQ ID NO:29. (If this comparison requires alignment the sequences should be aligned for maximum homology. "Looped" out sequences from deletions or insertions, or mismatches, are considered differences.) The differences are, preferably, differences or changes at a non essential residue or a conservative substitution. In a preferred embodiment the differences are not in the cation efflux domain. In another preferred embodiment one or more differences are in the cation efflux domain. Other embodiments include a protein that contain one or more changes in amino acid sequence, e.g, a change in an amino acid residue which is not essential for activity. Such 83378, 84233, 64708, 85041, or 84234 proteins differ in amino acid sequence from SEQ ID NO: 17, SEQ ID NO:20, SEQ ID NO:23, SEQ ID NO:26, or SEQ ID NO:29, yet retain biological activity. In one embodiment, the protein includes an amino acid sequence at least about 50%,
55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or more homologous to SEQ ID NO:17, SEQ ID NO:20, SEQ ID NO:23, SEQ ID NO:26, or SEQ ID NO:29.
A 83378 protein or fragment is provided which varies from the sequence of SEQ ID NO:17 in regions defined by amino acids about 1-10, 134-230, or 390-485 by at least one but by less than 15, 10 or 5 amino acid residues in the protein or fragment but which does not differ from SEQ ID NO: 17 in regions defmed by amino acids about 11-133 or 231-389. A 84233 protein or fragment is provided which varies from the sequence of SEQ ID NO:20 in regions defined by amino acids about 1-24 or 311-320 by at least one but by less than 15, 10 or 5 amino acid residues in the protein or fragment but which does not differ from SEQ ID NO:20 in regions defined by amino acids about 25-310. A 64708 protein or fragment is provided which varies from the sequence of SEQ ID NO:23 in regions defined by amino acids about 1-54, 154- 226, or 321-461 by at least one but by less than 15, 10 or 5 amino acid residues in the protein or fragment but which does not differ from SEQ ID NO:23 in regions defined by amino acids about 55-153 or 227-320. A 85041 protein or fragment is provided which varies from the sequence of SEQ ID NO:26 in regions defined by amino acids about 1-418 or 734-765 by at least one but by less than 15, 10 or 5 amino acid residues in the protein or fragment but which does not differ from SEQ ID NO:26 in regions defined by amino acids about 419-733. A 84234 protein or fragment is provided which varies from the sequence of SEQ ID NO:29 in regions defined by amino acids about 1-37 or 350-376 by at least one but by less than 15, 10 or 5 amino acid residues in the protein or fragment but which does not differ from SEQ ID NO:29 in regions defined by amino acids about 38-349. (If this comparison requires alignment the sequences should be aligned for maximum homology. "Looped" out sequences from deletions or insertions, or mismatches, are considered differences.) In some embodiments the difference is at a non-essential residue or is a conservative substitution, while in others the difference is at an essential residue or is a non-conservative substitution. In one embodiment, a biologically active portion of a 83378, 84233, 64708, 85041, or
84234 protein includes a cation efflux domain. Moreover, other biologically active portions, in which other regions ofthe protein are deleted, can be prepared by recombinant techniques and evaluated for one or more ofthe functional activities of anative 83378, 84233, 64708, 85041, or 84234 protein. In a prefened embodiment, the 83378, 84233, 64708, 85041, or 84234 protein has an amino acid sequence shown in SEQ ID NO: 17, SEQ ID NO:20, SEQ ID NO:23, SEQ ID NO:26, or SEQ ID NO:29. In other embodiments, the 83378, 84233, 64708, 85041, or 84234 protein is substantially identical to SEQ ID NO: 17, SEQ ID NO:20, SEQ ID NO:23, SEQ ID NO:26, or SEQ ID NO:29. In yet another embodiment, the 83378, 84233, 64708, 85041, or 84234 protein is substantially identical to SEQ ID NO: 17, SEQ ID NO:20, SEQ ID NO:23, SEQ ID NO:26, or SEQ ID NO:29 and retains the functional activity ofthe protein of SEQ ID NO:17, SEQ ID NO:20, SEQ ID NO:23, SEQ ID NO:26, or SEQ ID NO:29, as described in detail in the subsections above.
In a preferred embodiment, a 83378 fragment differs by at least 1, 2, 3, 10, 20, or more amino acid residues encoded by a sequence present in Genbank accession number AL359609 or a sequence disclosed in WO 01/62918, WO 01/55314, WO 01/55355. Differences can include differing in length or sequence identity. For example, a fragment can: include one or more amino acid residues from SEQ ID NO: 17 outside the region encoded by nucleotides 7-1044, 1085-1638, 1253-1638, 1299-1638, 723-1775 of SEQ ID NO:16; not include all ofthe amino acid residues encoded by a nucleotide sequence in Genbank accession number AL359609 or a sequence disclosed in WO 01/62918, WO 01/55314, WO 01/55355, e.g, can be one or more amino acid residues shorter (at one or both ends) than a sequence encoded by the nucleotide sequence in Genbank accession number AL359609 or a sequence disclosed in WO 01/62918, WO 01/55314, WO 01/55355; or can differ by one or more amino acid residues in the region of overlap. In a preferred embodiment, a 84233 fragment differs by at least 1, 2, 3, 10, 20, or more amino acid residues encoded by a sequence present in Genbank accession number AX061210 or AX086187 or a sequence disclosed in WO 01/12659, WO 01/51628, or WO 00/78953. Differences can include differing in length or sequence identity. For example, a fragment can: include one or more amino acid residues from SEQ ID NO:20 outside the region encoded by nucleotides 420-2103, 998-1589, 1-429, 1146-1582, 996-1425, 998-1341, 996-1379, 996-1368 of SEQ ID NO: 19; not include all ofthe amino acid residues encoded by a nucleotide sequence in Genbank accession number AX061210 or AX086187 or a sequence disclosed in WO 01/12659, WO 01/51628, or WO 00/78953, e.g, can be one or more amino acid residues shorter (at one or both ends) than a sequence encoded by the nucleotide sequence in Genbank accession number AX061210 or AX086187 or a sequence disclosed in WO 01/12659, WO 01/51628, or WO 00/78953; or can differ by one or more amino acid residues in the region of overlap.
In a preferred embodiment, a 64708 fragment differs by at least 1, 2, 3, 10, 20, or more amino acid residues encoded by a sequence present in Genbank accession number AK000844 or a sequence disclosed in WO 01/57188, WO 01/57270, WO 01/57272, WO 01/57275, WO 01/57276, WO 01/57277, or WO 01/57278. Differences can include differing in length or sequence identity. For example, a fragment can: include one or more amino acid residues from SEQ ID NO:23 outside the region encoded by nucleotides 209-697, 918-1456, 918-1418, 1348- 1790, 20-442, 911-1280, or 911-1226 of SEQ ID NO:22; not include all ofthe amino acid residues encoded by a nucleotide sequence in Genbank accession number AK000844 or a sequence disclosed in WO 01/57188, WO 01/57270, WO 01/57272, WO 01/57275, WO
01/57276, WO 01/57277, or WO 01/57278, e.g, can be one or more amino acid residues shorter (at one or both ends) than a sequence encoded by the nucleotide sequence in Genbank accession number AK000844 or a sequence disclosed in WO 01/57188, WO 01/57270, WO 01/57272, WO 01/57275, WO 01/57276, WO 01/57277, or WO 01/57278; or can differ by one or more amino acid residues in the region of overlap. In a preferred embodiment, a 85041 fragment differs by at least 1, 2, 3, 10, 20, or more amino acid residues encoded by a sequence present in Genbank accession number AK022558, AK022818, or AF233321, or a sequence disclosed in WO 01/40466, WO 01/54472, WO 01/55318, or WO 01/53312. Differences can include differing in length or sequence identity. For example, a fragment can: include one or more amino acid residues from SEQ ID NO:26 outside the region encoded by nucleotides 55-2750, 1069-3010, 1347-3224, 1320-3010,1347- 3010, 602-3005, 483-2740, 1015-1942, 786-3259, 602-3005, 883-2051, 2139-3010, 94-809, or 1843-3010 of SEQ ID NO:25; not include all ofthe amino acid residues encoded by a nucleotide sequence in Genbank accession number AK022558, AK022818, or AF233321, or a sequence disclosed in WO 01/40466, WO 01/54472, WO 01/55318, or WO 01/53312, e.g, can be one or more amino acid residues shorter (at one or both ends) than a sequence encoded by the nucleotide sequence in Genbank accession number AK022558, AK022818, or AF233321, or a sequence disclosed in WO 01/40466, WO 01/54472, WO 01/55318, or WO 01/53312; or can differ by one or more amino acid residues in the region of overlap. In a preferred embodiment, a 84234 fragment differs by at least 1, 2, 3, 10, 20, or more amino acid residues encoded by a sequence disclosed in WO 01/53312. Differences can include differing in length or sequence identity. For example, a fragment can: not include all ofthe amino acid residues encoded by a sequence disclosed in WO 01/53312, e.g, can be one or more amino acid residues shorter (at one or both ends) than a sequence disclosed in WO 01/53312; or can differ by one or more amino acid residues in the region of overlap.
47476, 67210, 49875. 46842, 33201, 83378, 84233, 64708, 85041, or 84234 Chimeric or Fusion Proteins
In another aspect, the invention provides 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 chimeric or fusion proteins. As used herein, a 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 "chimeric protein" or "fusion protein" includes a 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 polypeptide linked to a non-47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 polypeptide. A "non-47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 polypeptide" refers to a polypeptide having an amino acid sequence corresponding to a protein which is not substantially homologous to the 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 protein, e.g, a protein which is different from the 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 protein and which is derived from the same or a different organism. The 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 polypeptide ofthe fusion protein can correspond to all or a portion e.g, a fragment described herein of a 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 amino acid sequence. In a preferred embodiment, a 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 fusion protein includes at least one (or two) biologically active portion of a 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 protein. The non-47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 polypeptide can be fused to the N-terminus or C- terminus ofthe 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 polypeptide.
The fusion protein can include a moiety which has a high affinity for a ligand. For example, the fusion protein can be a GST-47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 fusion protein in which the 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 sequences are fused to the C-terminus ofthe GST sequences. Such fusion proteins can facilitate the purification of recombinant 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234. Alternatively, the fusion protein can be a 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 protein containing a heterologous signal sequence at its N-terminus. In certain host cells (e.g, mammalian host cells), expression and/or secretion of 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 can be increased through use of a heterologous signal sequence.
Fusion proteins can include all or a part of a serum protein, e.g, an IgG constant region, or human serum albumin.
The 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 fusion proteins ofthe invention can be incorporated into pharmaceutical compositions and administered to a subject in vivo. The 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 fusion proteins can be used to affect the bioavailability of a 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 substrate. 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 fusion proteins may be useful therapeutically for the treatment of disorders caused by, for example, (i) aberrant modification or mutation of a gene encoding a 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 protein; (ii) mis-regulation ofthe 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 gene; and (iii) aberrant post-translational modification of a 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 protein.
Moreover, the 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234-fusion proteins ofthe invention can be used as immunogens to produce anti-47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 antibodies in a subject, to purify 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 Hgands and in screening assays to identify molecules which inhibit the interaction of 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 with a 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 substrate.
Expression vectors are commercially available that already encode a fusion moiety (e.g, a GST polypeptide). A 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234-encoding nucleic acid can be cloned into such an expression vector such that the fusion moiety is linked in-frame to the 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 protein.
Variants of 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 Proteins In another aspect, the invention also features a variant of a 47476, 67210, 49875, 46842,
33201, 83378, 84233, 64708, 85041, or 84234 polypeptide, e.g, which functions as an agonist (mimetics) or as an antagonist. Variants ofthe 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 proteins can be generated by mutagenesis, e.g, discrete point mutation, the insertion or deletion of sequences or the truncation of a 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 protein. An agonist ofthe 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 proteins can retain substantially the same, or a subset, ofthe biological activities ofthe naturally occurring form of a 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 protein. An antagonist of a 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 protein can inhibit one or more ofthe activities ofthe naturally occurring form ofthe 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 protein by, for example, competitively modulating a 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234- mediated activity of a 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 protein. Thus, specific biological effects can be elicited by treatment with a variant of limited function. Preferably, treatment of a subject with a variant having a subset ofthe biological activities ofthe naturally occurring form ofthe protein has fewer side effects in a subject relative to treatment with the naturally occurring form ofthe 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 protein.
Variants of a 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 protein can be identified by screening combinatorial libraries of mutants, e.g, truncation mutants, of a 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 protein for agonist or antagonist activity.
Libraries of fragments e.g, N-terminal, C-terminal, or internal fragments of a 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 protein coding sequence can be used to generate a variegated population of fragments for screening and subsequent selection of variants of a 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 protein. Variants in which a cysteine residues is added or deleted or in which a residue which is glycosylated is added or deleted are particularly preferred.
Methods for screening gene products of combinatorial libraries made by point mutations or truncation, and for screening cDNA libraries for gene products having a selected property are known in the art. Such methods are adaptable for rapid screening ofthe gene libraries generated by combinatorial mutagenesis of 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 proteins. Recursive ensemble mutagenesis (REM), a new technique which enhances the frequency of functional mutants in the libraries, can be used in combination with the screening assays to identify 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 variants (Arkin and Yourvan (1992) Proc. Natl. Acad. Sci. USA 59:7811-7815; Delgrave et al. (1993) Protein Engineering 6:327-331).
Cell based assays can be exploited to analyze a variegated 47476 library. For example, a library of expression vectors can be transfected into a cell line, e.g, a cell line, which ordinarily responds to 47476 in a substrate-dependent manner. The transfected cells are then contacted with the substrate and the effect ofthe expression ofthe 47476 mutant on signaling by the substrate can be detected, e.g, by measuring GTP loading of a ras superfamily protein, measuring signal transduction that involves an activated ras superfamily protein, or by assaying for the changes induced by such signal transduction, e.g, cellular proliferation, differentiation, or migration. Plasmid DNA can then be recovered from the cells which score for inhibition, or alternatively, potentiation of signaling by the 47476 substrate, and the individual clones further characterized.
Cell based assays can be exploited to analyze a variegated 67210 library. For example, a library of expression vectors can be transfected into a cell line, e.g, a cell line, which ordinarily responds to 67210 in a substrate-dependent manner. The transfected cells are then contacted with the substrate and the effect ofthe expression ofthe 67210 mutant on signaling by the substrate can be detected, e.g, by measuring cellular properties influenced by 67210 activity, e.g, the appearance of particular glycosylated molecules on the cell surface, cellular adhesion, or signal transduction, e.g, as measured by cell proliferation or differentiation. Plasmid DNA can then be recovered from the cells which score for inhibition, or alternatively, potentiation of signaling by the 67210 substrate, and the individual clones further characterized.
Cell based assays can be exploited to analyze a variegated 49875 library. For example, a library of expression vectors can be transfected into a cell line, e.g, a cell line, which ordinarily responds to 49875 in a substrate-dependent manner. The transfected cells are then contacted with the substrate and the effect ofthe expression ofthe 49875 mutant on signaling by the substrate can be detected, e.g, by measuring a cellular process responsive to 49875 helicase activity, e.g, cellular proliferation or differentiation. Plasmid DNA can then be recovered from the cells which score for inhibition, or alternatively, potentiation of signaling by the 49875 substrate, and the individual clones further characterized.
Cell based assays can be exploited to analyze a variegated 46842 library. For example, a library of expression vectors can be transfected into a cell line, e.g, a cell line, which ordinarily responds to 46842 in a substrate-dependent manner. The transfected cells are then contacted with the substrate and the effect ofthe expression ofthe 46842 mutant on signaling by the substrate can be detected, e.g, by measuring GTP hydrolysis (e.g, ARF GTP hydrolysis), or cellular changes that reflect the presence or absence of Arf activity, e.g, changes in the transport of an appropriate marker protein, e.g, a cell surface or secreted marker protein. Plasmid DNA can then be recovered from the cells which score for inhibition, or alternatively, potentiation of signaling by the substrate, and the individual clones further characterized.
Cell based assays can be exploited to analyze a variegated 33201 library. For example, a library of expression vectors can be transfected into a cell line, e.g, a cell line, which ordinarily responds to 33201 in a substrate-dependent manner. The transfected cells are then contacted with the substrate and the effect ofthe expression ofthe 33201 mutant on signaling by the substrate can be detected, e.g, by measuring a cellular property dependent upon the substrate, e.g, cellular proliferation or cellular differentiation. Plasmid DNA can then be recovered from the cells which score for inhibition, or alternatively, potentiation of signaling by the substrate, and the individual clones further characterized.
Cell based assays can be exploited to analyze a variegated 83378, 84233, 64708, 85041, or 84234 library. For example, a library of expression vectors can be transfected into a cell line, e.g, a cell line, which ordinarily responds to 83378, 84233, 64708, 85041, or 84234 in a substrate-dependent manner. The transfected cells are then contacted with the sustrate and the effect ofthe expression ofthe 83378, 84233, 64708, 85041, or 84234 mutant on signaling by the substrate can be detected, e.g, by measuring cation diffusion or a response ofthe cells to the substrate, e.g, a normal cellular response such as programmed cell death. Plasmid DNA can then be recovered from the cells which score for inhibition, or alternatively, potentiation of signaling by the substrate, and the individual clones further characterized. In some embodiments, the cell based assays decribed above do not require the addition of a substrate, e.g, the cell may respond to simply the expression ofthe 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 polypeptide or protein. In such cases, the assay can be modified such that the 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 polypeptides or proteins are expressed by an inducible promoter. In another aspect, the invention features a method of making a 47476, 67210, 49875,
46842, 33201, 83378, 84233, 64708, 85041, or 84234 polypeptide, e.g, apeptide having a non- wild type activity, e.g, an antagonist, agonist, or super agonist of a naturally occurring 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 polypeptide, e.g, a naturally occurring 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 polypeptide. The method includes: altering the sequence of a 47476, 67210, 49875, 46842,
33201, 83378, 84233, 64708, 85041, or 84234 polypeptide, e.g, altering the sequence , e.g, by substitution or deletion of one or more residues of a non-conserved region, a domain or residue disclosed herein, and testing the altered polypeptide for the desired activity.
In another aspect, the invention features a method of making a fragment or analog of a 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 polypeptide a biological activity of a naturally occurring 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 polypeptide. The method includes: altering the sequence, e.g, by substitution or deletion of one or more residues, of a 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 polypeptide, e.g, altering the sequence of a non- conserved region, or a domain or residue described herein, and testing the altered polypeptide for the desired activity.
Anti-47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 Antibodies
In another aspect, the invention provides an anti-47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 antibody, or a fragment thereof (e.g, an antigen-binding fragment thereof). The term "antibody" as used herein refers to an immunoglobulin molecule or immunologically active portion thereof, i.e, an antigen-binding portion. As used herein, the term "antibody" refers to a protein comprising at least one, and preferably two, heavy (H) chain variable regions (abbreviated herein as VH), and at least one and preferably two light (L) chain variable regions (abbreviated herein as VL). The VH and VL regions can be further subdivided into regions of hypervariability, termed "complementarity determining regions" ("CDR"), interspersed with regions that are more conserved, termed "framework regions" (FR). The extent ofthe framework region and CDR's has been precisely defined (see, Kabat, E.A, et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242, and Chothia, C. et al. (1987) J. Mol. Biol. 196:901-917, which are incorporated herein by reference). Each VH and VL is composed of three CDR's and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.
The anti-47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 antibody can further include a heavy and light chain constant region, to thereby form a heavy and light immunoglobulin chain, respectively. In one embodiment, the antibody is a tetramer of two heavy immunoglobulin chains and two light immunoglobulin chains, wherein the heavy and light immunoglobulin chains are inter-connected by, e.g, disulfide bonds. The heavy chain constant region is comprised of three domains, CHI, CH2 and CH3. The light chain constant region is comprised of one domain, CL. The variable region ofthe heavy and light chains contains a binding domain that interacts with an antigen. The constant regions ofthe antibodies typically mediate the binding ofthe antibody to host tissues or factors, including various cells of the immune system (e.g, effector cells) and the first component (Clq) ofthe classical complement system.
As used herein, the term "immunoglobulin" refers to a protein consisting of one or more polypeptides substantially encoded by immunoglobulin genes. The recognized human immunoglobulin genes include the kappa, lambda, alpha (IgAl and IgA2), gamma (IgGl, IgG2, IgG3, IgG4), delta, epsilon and mu constant region genes, as well as the myriad immunoglobulin variable region genes. Full-length immunoglobulin "light chains" (about 25 KDa or 214 amino acids) are encoded by a variable region gene at the NH2-terminus (about 110 amino acids) and a kappa or lambda constant region gene at the COOH— terminus. Full-length immunoglobulin "heavy chains" (about 50 KDa or 446 amino acids), are similarly encoded by a variable region gene (about 116 amino acids) and one ofthe other aforementioned constant region genes, e.g, gamma (encoding about 330 amino acids).
The term "antigen-binding fragment" of an antibody (or simply "antibody portion," or "fragment"), as used herein, refers to one or more fragments of a full-length antibody that retain the ability to specifically bind to the antigen, e.g, 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 polypeptide or fragment thereof. Examples of antigen-binding fragments ofthe anti-47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 antibody include, but are not limited to: (i) a Fab fragment, a monovalent fragment consisting ofthe VL, VH, CL and CHI domains; (ii) a F(ab')2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fd fragment consisting ofthe VH and CHI domains; (iv) a Fv fragment consisting ofthe VL and VH domains of a single arm of an antibody, (v) a dAb fragment (Ward et al, (1989) Nature 341:544-546), which consists of a VH domain; and (vi) an isolated complementarity determining region (CDR). Furthermore, although the two domains ofthe Fv fragment, VL and VH, are coded for by separate genes, they can be joined, using recombinant methods, by a synthetic linker that enables them to be made as a single protein chain in which the VL and VH regions pair to form monovalent molecules (known as single chain Fv (scFv); see e.g., Bird et al. (1988) Science 242:423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879- 5883). Such single chain antibodies are also encompassed within the term "antigen-binding fragment" of an antibody. These antibody fragments are obtained using conventional techniques known to those with skill in the art, and the fragments are screened for utility in the same manner as are intact antibodies.
The anti-47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 antibody can be a polyclonal or a monoclonal antibody. In other embodiments, the antibody can be recombinantly produced, e.g, produced by phage display or by combinatorial methods. Phage display and combinatorial methods for generating anti-47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 antibodies are known in the art (as described in, e.g, Ladner et al. U.S. Patent No. 5,223,409; Kang et al. International Publication No. WO 92/18619; Dower et al. International Publication No. WO 91/17271; Winter et al. International Publication WO 92/20791 ; Markland et al. International Publication No. WO 92/15679; Breitling et al. International Publication WO 93/01288; McCafferty et al. International Publication No. WO 92/01047; Garrard et al. International Publication No. WO 92/09690; Ladner et al. International Publication No. WO 90/02809; Fuchs et al. (1991) Bio/Technology 9:1370-1372; Hay et al. (1992) Hum Antϊbod Hybridomas 3:81-85; Huse et al. (1989) Science 246:1275-1281; Griffths et al. (1993) EMBO J 12:725-734; Hawkins et al.
(1992) J Mol Biol 226:889-896; Clackson et al. (1991) Nature 352:624-628; Gram et al. (1992) EN4S 89:3576-3580; Garrad et al. (1991) Bio/Technology 9:1373-1377; Hoogenboom et al. (1991) Nuc Acid Res 19:4133-4137; and Barbas et al. (1991) PNAS 88:7978-7982, the contents of all of which are incorporated by reference herein). In one embodiment, the anti-47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708,
85041, or 84234 antibody is a fully human antibody (e.g, an antibody made in a mouse which has been genetically engineered to produce an antibody from a human immunoglobulin sequence), or a non-human antibody, e.g, a rodent (mouse or rat), goat, primate (e.g, monkey), camel antibody. Preferably, the non-human antibody is a rodent (mouse or rat antibody). Method of producing rodent antibodies are known in the art. Human monoclonal antibodies can be generated using transgenic mice carrying the human immunoglobulin genes rather than the mouse system. Splenocytes from these transgenic mice immunized with the antigen of interest are used to produce hybridomas that secrete human mAbs with specific affinities for epitopes from a human protein (see, e.g. Wood et al. International Application WO 91/00906, Kucherlapati et al. PCT publication WO 91/10741; Lonberg et al. International Application WO 92/03918; Kay et al. International Application 92/03917; Lonberg, N. et al. 1994 Nature 368:856-859; Green, L.L. et al. 1994 Nature Genet. 7:13-21; Morrison, S.L. et al. 1994 Proc. Natl. Acad. Sci. USA 81:6851-6855; Bruggeman et al. 1993 Year Immunol 7:33-40; Tuaillon et al. 1993 PNAS 90:3720-3724; Bruggeman et al. 1991 Eur J Immunol 21:1323-1326).
An anti-47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 antibody can be one in which the variable region, or a portion thereof, e.g, the CDR's, are generated in a non-human organism, e.g, a rat or mouse. Chimeric, CDR-grafted, and humanized antibodies are within the invention. Antibodies generated in a non-human organism, e.g, a rat or mouse, and then modified, e.g, in the variable framework or constant region, to decrease antigenicity in a human are within the invention.
Chimeric antibodies can be produced by recombinant DNA techniques known in the art. For example, a gene encoding the Fc constant region of a murine (or other species) monoclonal antibody molecule is digested with restriction enzymes to remove the region encoding the murine Fc, and the equivalent portion of a gene encoding a human Fc constant region is substituted (see Robinson et al. International Patent Publication PCT/US86/02269; Akira, et al, European Patent Application 184,187; Taniguchi, M, European Patent Application 171,496; Morrison et al, European Patent Application 173,494; Neuberger et al. International Application WO 86/01533; Cabilly et al. U.S. Patent No. 4,816,567; Cabilly et al, European Patent Application 125,023; Better et al. (1988 Science 240:1041-1043); Liu et al. (1987) PNAS 84:3439-3443; Liu et al, 1987, J Immunol. 139:3521-3526; Sun et al. (1987) PNAS 84:214- 218; Nishimura et al, 1987, Cane. Res. 47:999-1005; Wood et al. (1985) Nature 314:446-449; and Shaw et al, 1988, J Natl Cancer Inst. 80:1553-1559).
A humanized or CDR-grafted antibody will have at least one or two but generally all three recipient CDR's (of heavy and or light immuoglobulin chains) replaced with a donor
CDR. The antibody may be replaced with at least a portion of a non-human CDR or only some ofthe CDR's may be replaced with non-human CDR's. It is only necessary to replace the number of CDR's required for binding ofthe humanized antibody to a 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 or a fragment thereof. Preferably, the donor will be a rodent antibody, e.g, a rat or mouse antibody, and the recipient will be a human framework or a human consensus framework. Typically, the immunoglobulin providing the CDR's is called the "donor" and the immunoglobulin providing the framework is called the "acceptor." In one embodiment, the donor immunoglobulin is a non-human (e.g, rodent). The acceptor framework is a naturally-occurring (e.g, a human) framework or a consensus framework, or a sequence about 85% or higher, preferably 90%, 95%, 99% or higher identical thereto.
As used herein, the term "consensus sequence" refers to the sequence formed from the most frequently occurring amino acids (or nucleotides) in a family of related sequences (See e.g, Winnaker, From Genes to Clones (Nerlagsgesellschaft, Weinheim, Germany 1987). In a family of proteins, each position in the consensus sequence is occupied by the amino acid occurring most frequently at that position in the family. If two amino acids occur equally frequently, either can be included in the consensus sequence. A "consensus framework" refers to the framework region in the consensus immunoglobulin sequence.
An antibody can be humanized by methods known in the art. Humanized antibodies can be generated by replacing sequences ofthe Fv variable region which are not directly involved in antigen binding with equivalent sequences from human Fv variable regions. General methods for generating humanized antibodies are provided by Morrison, S. L, 1985, Science 229:1202- 1207, by Oi et al, 1986, BioTechniques 4:214, and by Queen et al. US 5,585,089, US 5,693,761 and US 5,693,762, the contents of all of which are hereby incorporated by reference. Those methods include isolating, manipulating, and expressing the nucleic acid sequences that encode all or part of immunoglobulin Fv variable regions from at least one of a heavy or light chain. Sources of such nucleic acid are well known to those skilled in the art and, for example, may be obtained from a hybridoma producing an antibody against a 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 polypeptide or fragment thereof. The recombinant DΝA encoding the humanized antibody, or fragment thereof, can then be cloned into an appropriate expression vector. Humanized or CDR-grafted antibodies can be produced by CDR-grafting or CDR substitution, wherein one, two, or all CDR's of an immunoglobulin chain can be replaced. See e.g, U.S. Patent 5,225,539; Jones et al. 1986 Nαt-.re 321:552-525; Verhoeyan et al. 1988 Science 239:1534; Beidler et al. 1988 J. Immunol. 141:4053-4060; Winter US 5,225,539, the contents of all of which are hereby expressly incorporated by reference. Winter describes a CDR-grafting method which may be used to prepare the humanized antibodies ofthe present invention (UK Patent Application GB 2188638A, filed on March 26, 1987; Winter US 5,225,539), the contents of which is expressly incorporated by reference.
Also within the scope ofthe invention are humanized antibodies in which specific amino acids have been substituted, deleted or added. Preferred humanized antibodies have amino acid substitutions in the framework region, such as to improve binding to the antigen. For example, a humanized antibody will have framework residues identical to the donor framework residue or to another amino acid other than the recipient framework residue. To generate such antibodies, a selected, small number of acceptor framework residues ofthe humanized immunoglobulin chain can be replaced by the corresponding donor amino acids. Prefened locations ofthe substitutions include amino acid residues adjacent to the CDR, or which are capable of interacting with a CDR (see e.g, US 5,585,089). Criteria for selecting amino acids from the donor are described in US 5,585,089, e.g, columns 12-16 of US 5,585,089, the e.g, columns 12-16 of US 5,585,089, the contents of which are hereby incorporated by reference. Other techniques for humanizing antibodies are described in Padlan et al. EP 519596 Al , published on December 23, 1992.
In preferred embodiments an antibody can be made by immunizing with purified 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 antigen, or a fragment thereof, e.g, a fragment described herein, membrane associated antigen, tissue, e.g, crude tissue preparations, whole cells, preferably living cells, lysed cells, or cell fractions, e.g, cytosolic fractions or membrane fractions.
A full-length 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 protein or, antigenic peptide fragment of 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 can be used as an immunogen or can be used to identify anti- 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 antibodies made with other immunogens, e.g, cells, membrane preparations, and the like. The antigenic peptide of 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 should include at least 8 amino acid residues ofthe amino acid sequence shown in SEQ ID NO:2, SEQ ID NO:5, SEQ ID NO:8, SEQ ID NO:l 1, SEQ ID NO: 14, SEQ ID NO: 17, SEQ ID NO:20, SEQ ID NO:23, SEQ ID NO:26, or SEQ ID NO:29 and encompasses an epitope of 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234. Preferably, the antigenic peptide includes at least 10 amino acid residues, more preferably at least 15 amino acid residues, even more preferably at least 20 amino acid residues, and most preferably at least 30 amino acid residues.
Fragments of 47476 can be used as immunogens or used to characterize the specificity of an antibody. For example, fragments of 47476 that include, e.g, residues about 106 to 122, about 325 to 340, or about 500 to 520 of SEQ ID NO:2, can be used to makeantibodies against hydrophilic regions ofthe 47476 protein. Similarly, fragments of 47476 which include, e.g, residues about 82 to 105, about 341 to 360, or about 521 to 540 of SEQ ID NO:2, can be used to make an antibody against a hydrophobic region ofthe 47476 protein; fragments of 47476, which include, e.g, residues about 55 to 173, about 195 to 381, about 470 to 498, or about 541 to 590 of SEQ ID NO:2, can be used to make an antibody against the a guanine nucleotide dissociation stimulator domain N-terminal motif, a ras guanine nucleotide dissociation stimulator domain, an EF-hand calcium-binding domain and a phorbol ester/diacylglycerol binding domain (Cl domain), respectively, ofthe 47476 protein. Fragments of 67210 can be used as immunogens or used to characterize the specificity of an antibody . For example, fragments of 67210 which include, e.g, residues about 240-260, can be used to make antibodies against hydrophilic regions ofthe 67210 protein. Similarly, fragments of 67210 which include, e.g, residues about 10-25 of SEQ ID NO:5, can be used to make an antibody against a hydrophobic region ofthe 67210 protein; and fragmentsof 67210 which include, e.g, residues about 63 to 340 of SEQ ID NO:5, can be used to make an antibody against the glycosyltransferase region ofthe 67210 protein.
Fragments of 49875 can be used as immunogens or used to characterize the specificity of an antibody. For example, fragments of 49875 which include, e.g, residues about 520 to 550 of SEQ ID NO:8, can be used to make antibodies against hydrophilic regions ofthe 49875 protein. Similarly, fragments of 49875 which include, e.g, residues about 285 to 295 of SEQ ID NO:8, can be used to make an antibody against a hydrophobic region ofthe 49875 protein; fragments of 49875 which include, e.g, residues about 22 to 245 of SEQ ID NO:8, can be used to make an antibody against the DEAD-type helicase region ofthe 49875 protein; fragments of 49875 which include, e.g, about amino acid residues 169 to 177 of SEQ ID NO:8, can be used to maka an antibody against a DEAD-box subfamily ATP-dependent helicase signature motif; and fragments of 49875 which include, e.g, residues about 281 to 363 of SEQ ID NO:8, can be used to make an antibody against the conserved helicase C-terminal region ofthe 49875 protein.
Fragments of 46842 can be used as immunogens or used to characterize the specificity of an antibody. For example, fragments of 46842 which include, e.g, residues about 567 to 580, 720 to 737, or 757 to 771 of SEQ ID NO:l 1, can be used to make antibodies against hydrophilic regions ofthe 46842 protein. Similarly, fragments of 46842 which include, e.g, residues about 431 to 439, from about 558 to 566, and from about 706 to 719 of SEQ ID NO:l 1, can be used to make an antibody against a hydrophobic region ofthe 46842 protein; fragments of 46842 which include, e.g, residues about 269 to 363 of SEQ ID NO:l 1, can be used to make an antibody against the PH domain region ofthe 46842 protein; fragments of 46842 which include, e.g, residues about 403 to 525 of SEQ ID NO: 11 , can be used to make an antibody against the ArfGAP domain region ofthe 46842 protein; and fragments of 46842 which include, e.g, residues about 702 to 734 or 735 to 767 of SEQ ID NO: 11 , can be used to make an antibody against the ankyrin repeat domains of 46842.
Fragments of 33201 can be used, e.g, to characterize the specificity of an antibody or to make immunogens. For example, fragments of 33201 which include, e.g, residues about 50 to 60, 82 to 90, or 205 to 210 of SEQ ID NO: 14, can be used to make antibodies against hydrophilic regions ofthe 33201 protein. Similarly, fragments of 33201 which include, e.g, residues about 70 to 80 or 158 to 178 of SEQ ID NO: 14, can be used to make an antibody against a hydrophobic region ofthe 33201 protein; fragments of 33201 which include residues from about 22 to 345 of SEQ ID NO: 14, or a fragment thereof, e.g, 22 to 50, 50 to 100, 150 to 200, 250 to 300, or 300 to 345 of SEQ ID NO:14, can be used to make an antibody against a dehydrogenase/reductase domain ofthe 33201 protein.
Fragments of 83378, 84233, 64708, 85041, or 84234 can be used, e.g, as immunogens or to characterize the specificity of an antibody. For example, fragments of 83378, 84233, 64708, 85041, or 84234 which include about amino acid residues 150 to 160, 220 to 235, or 355 to 370 of SEQ ID NO: 17, about amino acid residues 1 to 20, 80 to 90, or 150 to 160 of SEQ ID NO:20, about amino acid residues 123 to 133, 380 to 395, or 450 to 461 of SEQ ID NO:23, about amino acid residues 320 to 340, 555 to 575, or 750 to 765 of SEQ ID NO:26, or about amino acid residues 10 to 20, 165 to 175, or 190 to 230 of SEQ ID NO:29, can be used to make antibodies against hydrophilic regions ofthe 83378, 84233, 64708, 85041, or 84234 protein. Similarly fragments of 83378, 84233, 64708, 85041 , or 84234 which include residues about amino acid residues 325 to 335, 340 to 350, or 415 to 430 of SEQ ID NO:17, about amino acid residues 220 to 230, 240 to 260, or 262 to 273 of SEQ ID NO:20, about amino acid residues 180 to 195, 290 to 300, or 340 to 350 of SEQ ID NO:23, about amino acid residues 35 to 50, 440 to 470, or 685 to 695 of SEQ ID NO:26, or about amino acid residues 59 to 70, 330 to 345, or 370 to 376 of SEQ ID NO:29 can be used to make an antibody against a hydrophobic region ofthe 83378, 84233, 64708, 85041, or 84234 protein.
Fragments of 83378, 84233, 64708, 85041, or 84234 which include about amino acid residues 32 to 43, 99 to 114, or 266 to 282 of SEQ ID NO: 17, about amino acid residues 50 to 57, 114 to 127, or 192 to 200 of SEQ ID NO:20, about amino acid residues 52 to 57, 120 to 136, or 220 to 231 of SEQ ID NO:23, about amino acid residues 78 to 98, 146 to 151, 215 to 238, 289 to 303, 363 to 418, 506 to 520, or 614 to 617 of SEQ ID NO:26, or about amino acid residues 59 to 70, 124 to 140, or 257 to 262 of SEQ ID NO:29 can be used to make an antibody against a non-cytoplasmic loop ofthe 83378, 84233, 64708, 85041, or 84234 protein.
Fragments of 83378, 84233, 64708, 85041, or 84234 which include about amino acid residues 1 to 10, 62 to 78, 135 to 240, or 300 to 485 of SEQ ID NO:17, about amino acid residues 1 to 24, 75 to 91, 148 to 166, or 219 to 320 of SEQ ID NO:20, about amino acid residues 1 to 33, 83 to 100, 156 to 201, or 250 to 461 of SEQ ID NO:23, about amino acid residues 1 to 58, 120 to 128, 169 to 189, 259 to 266, 321 to 342, 438 to 485, 542 to 591, or 642 to 765 of SEQ ID NO:26, or about amino acid residues 1 to 37, 88 to 104, 160 to 236, or 287 to 376 of SEQ ID NO:29 can be used to make an antibody against a cytoplasmic domain ofthe 83378, 84233, 64708, 85041, or 84234 protein.
Fragment of 83378, 84233, 64708, 85041, or 84234 which include about amino acid residues 11 to 133 or 231 to 389 of SEQ ID NO: 17, about amino acid residues 25 to 310 of SEQ ID NO:20, about amino acid residues 55 to 153 or 227 to 320 of SEQ ID NO:23, about amino acid residues 419 to 733 of SEQ ID NO:26, or about amino acid residues 38 to 349 of SEQ ID NO:29 can be used to make an antibody against the cation efflux region ofthe 83378, 84233, 64708, 85041, or 84234 protein.
Antibodies reactive with, or specific for, any of these regions, or other regions or domains described herein are provided. Antibodies which bind only native 47476, 67210, 49875, 46842, 33201, 83378, 84233,
64708, 85041, or 84234 protein, only denatured or otherwise non-native 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 protein, or which bind both, are with in the invention. Antibodies with linear or conformational epitopes are within the invention. Conformational epitopes can sometimes be identified by identifying antibodies which bind to native but not denatured 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 protein.
Preferred epitopes encompassed by the antigenic peptide are regions of 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 are located on the surface ofthe protein, e.g, hydrophilic regions, as well as regions with high antigenicity. For example, an Emini surface probability analysis ofthe human 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 protein sequence can be used to indicate the regions that have a particularly high probability of being localized to the surface ofthe 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 protein.
In a preferred embodiment the antibody can bind to the extracellular portion of a 67210, 83378, 84233, 64708, 85041, or 84234 protein, e.g, it can bind to a whole cell which expresses a 67210, 83378, 84233, 64708, 85041, or 84234 protein. In another embodiment, the antibody binds an intracellular portion of a 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 protein. In preferred embodiments antibodies can bind one or more of purified antigen, membrane associated antigen, tissue, e.g, tissue sections, whole cells, preferably living cells, lysed cells, cell fractions, e.g, cytosolic fractions or membrane fractions.
The anti-47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 antibody can be a single chain antibody. A single-chain antibody (scFV) may be engineered (see, for example, Colcher, D. et al. (1999) Ann N Y Acad Sci 880:263-80; and Reiter, Y. (1996) Clin Cancer Res 2:245-52). The single chain antibody can be dimerized or multimerized to generate multivalent antibodies having specificities for different epitopes ofthe same target 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 protein. In a preferred embodiment the antibody has effector function and/or can fix complement. In other embodiments the antibody does not recruit effector cells; or fix complement.
In a preferred embodiment, the antibody has reduced or no ability to bind an Fc receptor. For example, it is a isotype or subtype, fragment or other mutant, which does not support binding to an Fc receptor, e.g, it has a mutagenized or deleted Fc receptor binding region.
In a preferred embodiment, an anti-47476 antibody alters (e.g, increases or decreases) that ability of 47476 to: (1) stimulate the exchange of guanine nucleotides (GTP for GDP) by a member ofthe ras superfamily of proteins; (2) bind calcium in a pentagonal bipyramidal configuration; (3) bind two zinc ions; (4) bind the second messenger diacylglycerol; (5) bind analogs of diacylglycerol, such as phorbol esters; or (6) activate one or more members ofthe ras superfamily of proteins. For example, the antibody can bind at or in proximity to the active site of a 47476 polypeptide or protein, e.g, to an epitope that is present within: a guanine nucleotide dissociation stimulator domain N-terminal motif, e.g, located at about amino acid residues 55 to 172 of SEQ ID NO:2; a guanine nucleotide dissociation stimulator domain, e.g, located at about amino acid residues 195 to 381 of SEQ ID NO:2; an EF-hand calcium-binding domain, e.g, located at about amino acid residues 470 to 498 of SEQ ID NO:2; or a phorbol ester/diacylglycerol binding domain (Cl domain), e.g, located at about amino acid residues 541 to 590 of SEQ ID NO:2. In a preferred embodiment, an anti-67210 antibody alters (e.g, increases or decreases) the glycosyltransferase activity of a 67210 polypeptide. For example, the antibody can bind at or in proximity to the active site of a 67210 polypeptide or protein, e.g, to an epitope that is present within the glycosyl transferase domain, e.g, located at about amino acid residues 63 to 340 o SEQ ID NO:5. In a preferred embodiment, an anti-49875 antibody alters (e.g, increases or decreases) the nucleic acid unwinding activity of a 49875 polypeptide. For example, the antibody can bind at or in proximity to the active site, e.g, to an epitope that includes a DEAD type helicase ATP binding motif, e.g, about amino acid residues 169 to 177 of SEQ ID NO:8.
In a preferred embodiment, an anti-46842 antibody alters (e.g, increases or decreases) the ArfGAP activity of a 46842 polypeptide. For example, the antibody can bind at or in proximity to a motif involved in ArfGAP catalytic activity, e.g, the sequence located at about residues 421 to 440 of SEQ ID NO:l 1.
In a preferred embodiment, an anti-33201 antibody alters (e.g, increases or decreases) the dehydrogenase/reductase activity of a 33201 polypeptide. For example, the antibody can bind at or in proximity to the active site, e.g, to an epitope that includes a residue located within a region from about 22 to 345 of SEQ ID NO:14, or a fragment thereof, e.g, 100 to 150, 150 to 200, 250 to 300, or 300 to 335 of SEQ ID NO:14.
In a preferred embodiment, an anti-83378, 84233, 64708, 85041, or 84234 antibody alters (e.g, increases or decreases) the cation diffusion activity of a 83378, 84233, 64708, 85041 , or 84234 polypeptide.
The antibody can be coupled to a toxin, e.g, a polypeptide toxin, e,g, ricin or diphtheria toxin or active fragment hereof, or a radioactive nucleus, or imaging agent, e.g. a radioactive, enzymatic, or other, e.g, imaging agent, e.g, a NMR contrast agent. Labels which produce detectable radioactive emissions or fluorescence are preferred. An anti-47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 antibody (e.g, monoclonal antibody) can be used to isolate 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 by standard techniques, such as affinity chromatography or immunoprecipitation. Moreover, an anti-47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 antibody can be used to detect 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 protein (e.g, in a cellular lysate or cell supernatant) in order to evaluate the abundance and pattern of expression ofthe protein. Anti-47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 antibodies can be used diagnostically to monitor protein levels in tissue as part of a clinical testing procedure, e.g, to determine the efficacy of a given treatment regimen. Detection can be facilitated by coupling (i.e, physically linking) the antibody to a detectable substance (i.e, antibody labelling). Examples of detectable substances include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, and radioactive materials. Examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, β-galactosidase, or acetylcholinesterase; examples of suitable prosthetic group complexes include streptavidin/biotin and avidin/biotin; examples of suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; an example of a luminescent material includes luminol; examples of bioluminescent materials include luciferase, luciferin, and aequorin, and examples of suitable radioactive material include I, I, S or H.
The invention also includes a nucleic acid which encodes an anti-47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 antibody, e.g, an anti-47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 antibody described herein. Also included are vectors which include the nucleic acid and cells transformed with the nucleic acid, particularly cells which are useful for producing an antibody, e.g, mammalian cells, e.g. CHO or lymphatic cells. The invention also includes cell lines, e.g, hybridomas, which make an anti-47476,
67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 antibody, e.g, an antibody described herein, and method of using said cells to make a 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 antibody.
Recombinant Expression Vectors, Host Cells and Genetically Engineered Cells In another aspect, the invention includes, vectors, preferably expression vectors, containing a nucleic acid encoding a polypeptide described herein. As used herein, the term "vector" refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked and can include a plasmid, cosmid or viral vector. The vector can be capable of autonomous replication or it can integrate into a host DNA. Viral vectors include, e.g, replication defective retroviruses, adenoviruses and adeno-associated viruses.
A vector can include a 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 nucleic acid in a form suitable for expression ofthe nucleic acid in a host cell. Preferably the recombinant expression vector includes one or more regulatory sequences operatively linked to the nucleic acid sequence to be expressed. The term "regulatory sequence" includes promoters, enhancers and other expression control elements (e.g, poly adenylation signals). Regulatory sequences include those which direct constitutive expression of a nucleotide sequence, as well as tissue-specific regulatory and/or inducible sequences. The design ofthe expression vector can depend on such factors as the choice ofthe host cell to be transformed, the level of expression of protein desired, and the like. The expression vectors ofthe invention can be introduced into host cells to thereby produce proteins or polypeptides, including fusion proteins or polypeptides, encoded by nucleic acids as described herein (e.g, 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 proteins, mutant forms of 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 proteins, fusion proteins, and the like). The recombinant expression vectors ofthe invention can be designed for expression of
47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 proteins in prokaryotic or eukaryotic cells. For example, polypeptides ofthe invention can be expressed in E. coli, insect cells (e.g, using baculoviras expression vectors), yeast cells or mammalian cells. Suitable host cells are discussed further in Goeddel, (1990) Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, CA. Alternatively, the recombinant expression vector can be transcribed and translated in vitro, for example using T7 promoter regulatory sequences and T7 polymerase.
Expression of proteins in prokaryotes is most often carried out in E. coli with vectors containing constitutive or inducible promoters directing the expression of either fusion or non- fusion proteins. Fusion vectors add a number of amino acids to a protein encoded therein, usually to the amino terminus ofthe recombinant protein. Such fusion vectors typically serve three purposes: 1) to increase expression of recombinant protein; 2) to increase the solubility of the recombinant protein; and 3) to aid in the purification ofthe recombinant protein by acting as a ligand in affinity purification. Often, a proteolytic cleavage site is introduced at the junction ofthe fusion moiety and the recombinant protein to enable separation ofthe recombinant protein from the fusion moiety subsequent to purification ofthe fusion protein. Such enzymes, and their cognate recognition sequences, include Factor Xa, thrombin and enterokinase. Typical fusion expression vectors include pGEX (Pharmacia Biotech Inc; Smith, D.B. and Johnson, K.S. (1988) Gene 67:31-40), pMAL (New England Biolabs, Beverly, MA) and pRIT5 (Pharmacia, Piscataway, NJ) which fuse glutathione S-transferase (GST), maltose E binding protein, or protein A, respectively, to the target recombinant protein.
Purified fusion proteins can be used in 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 activity assays, (e.g, direct assays or competitive assays described in detail below), or to generate antibodies specific for 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 proteins. In a preferred embodiment, a fusion protein expressed in a retroviral expression vector ofthe present invention can be used to infect bone marrow cells which are subsequently transplanted into irradiated recipients. The pathology ofthe subject recipient is then examined after sufficient time has passed (e.g, six weeks).
To maximize recombinant protein expression in E. coli is to express the protein in a host bacteria with an impaired capacity to proteolytically cleave the recombinant protein
(Gottesman, S, (1990) Gene Expression Technology: Methods in Enzymology 185, Academic
Press, San Diego, California 119-128). Another strategy is to alter the nucleic acid sequence of the nucleic acid to be inserted into an expression vector so that the individual codons for each amino acid are those preferentially utilized in E. coli (Wada et al, (1992) Nucleic Acids Res. 20:2111-2118). Such alteration of nucleic acid sequences ofthe invention can be carried out by standard DNA synthesis techniques.
The 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 expression vector can be a yeast expression vector, a vector for expression in insect cells, e.g, a baculoviras expression vector or a vector suitable for expression in mammalian cells. When used in mammalian cells, the expression vector's control functions can be provided by viral regulatory elements. For example, commonly used promoters are derived from polyoma, Adenovirus 2, cytomegalovirus and Simian Virus 40.
In another embodiment, the promoter is an inducible promoter, e.g, a promoter regulated by a steroid hormone, by a polypeptide hormone (e.g, by means of a signal transduction pathway), or by a heterologous polypeptide (e.g, the tetracycline-inducible systems, "Tet-On" and "Tet-Off ; see, e.g, Clontech Inc., CA, Gossen and Bujard (1992) Proc.
Natl. Acad. Sci. USA 89:5547, and Paillard (1989) Human Gene Therapy 9:983).
In another embodiment, the recombinant mammalian expression vector is capable of directing expression ofthe nucleic acid preferentially in a particular cell type (e.g, tissue- specific regulatory elements are used to express the nucleic acid). Non-limiting examples of suitable tissue-specific promoters include the albumin promoter (liver-specific; Pinkert et al.
(1987) Genes Dev. 1 :268-277), lymphoid-specific promoters (Calame and Eaton (1988) Adv.
Immunol 43:235-275), in particular promoters of T cell receptors (Winoto and Baltimore
(1989) EMBO J. 8:729-733) and immunoglobulins (Banerji et al (1983) Cell 33:729-740; Queen and Baltimore (1983) Cell 33:741-748), neuron-specific promoters (e.g, the neurofilament promoter; Byme and Ruddle (1989) Proc. Natl Acad. Sci. USA 86:5473-5477), pancreas-specific promoters (Edlund et al. (1985) Science 230:912-916), and mammary gland- specific promoters (e.g, milk whey promoter; U.S. Patent No. 4,873,316 and European Application Publication No. 264,166). Developmentally-regulated promoters are also encompassed, for example, the murine hox promoters (Kessel and Grass (1990) Science 249:374-379) and the α-fetoprotein promoter (Campes and Tilghman (1989) Genes Dev. 3:537- 546).
The invention further provides a recombinant expression vector comprising a DNA molecule ofthe invention cloned into the expression vector in an antisense orientation. Regulatory sequences (e.g, viral promoters and/or enhancers) operatively linked to a nucleic acid cloned in the antisense orientation can be chosen which direct the constitutive, tissue specific or cell type specific expression of antisense RNA in a variety of cell types. The antisense expression vector can be in the form of a recombinant plasmid, phagemid or attenuated viras.
Another aspect the invention provides a host cell which includes a nucleic acid molecule described herein, e.g, a 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 nucleic acid molecule within a recombinant expression vector or a 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 nucleic acid molecule containing sequences which allow it to homologously recombine into a specific site ofthe host cell's genome. The terms "host cell" and "recombinant host cell" are used interchangeably herein. Such terms refer not only to the particular subject cell but to the progeny or potential progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope ofthe term as used herein.
A host cell can be any prokaryotic or eukaryotic cell. For example, a 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 protein can be expressed in bacterial cells (such as E. coli), insect cells, yeast or mammalian cells (such as Chinese hamster ovary cells (CHO) or COS cells (African green monkey kidney cells CV-1 origin SV40 cells; Gluzman (1981) Ce/Z223: 175-182)). Other suitable host cells are known to those skilled in the art. Vector DNA can be introduced into host cells via conventional transformation or transfection techniques. As used herein, the terms "transformation" and "transfection" are intended to refer to a variety of art-recognized techniques for introducing foreign nucleic acid (e.g, DNA) into a host cell, including calcium phosphate or calcium chloride co-precipitation, DEAE-dextran-mediated transfection, lipofection, or electroporation.
A host cell ofthe invention can be used to produce (i.e, express) a 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 protein. Accordingly, the invention further provides methods for producing a 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 protein using the host cells ofthe invention. In one embodiment, the method includes culturing the host cell ofthe invention (into which a recombinant expression vector encoding a 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 protein has been introduced) in a suitable medium such that a 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 protein is produced. In another embodiment, the method further includes isolating a 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 protein from the medium or the host cell.
In another aspect, the invention features, a cell or purified preparation of cells which include a 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 transgene, or which otherwise misexpress 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234. The cell preparation can consist of human or non-human cells, e.g, rodent cells, e.g, mouse or rat cells, rabbit cells, or pig cells. In prefened embodiments, the cell or cells include a 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 transgene, e.g, a heterologous form of a 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234, e.g, a gene derived from humans (in the case of a non-human cell). The 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 transgene can be misexpressed, e.g, overexpressed or underexpressed. In other preferred embodiments, the cell or cells include a gene that mis-expresses an endogenous 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234, e.g, a gene the expression of which is disrapted, e.g, a knockout. Such cells can serve as a model for studying disorders that are related to mutated or mis-expressed 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 alleles or for use in drug screening.
In another aspect, the invention features, a human cell, e.g, a hematopoietic, neural, muscle, or hepatic stem cell, transformed with nucleic acid which encodes a subject 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 polypeptide. Also provided are cells, preferably human cells, e.g, human hematopoietic, neural, muscle, hepatic or fibroblast cells, in which an endogenous 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 is under the control of a regulatory sequence that does not normally control the expression ofthe endogenous 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 gene. The expression characteristics of an endogenous gene within a cell, e.g, a cell line or microorganism, can be modified by inserting a heterologous DNA regulatory element into the genome ofthe cell such that the inserted regulatory element is operably linked to the endogenous 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 gene. For example, an endogenous 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 gene which is "transcriptionally silent," e.g, not normally expressed, or expressed only at very low levels, may be activated by inserting a regulatory element which is capable of promoting the expression of a normally expressed gene product in that cell. Techniques such as targeted homologous recombinations, can be used to insert the heterologous DNA as described in, e.g, Chappel, US 5,272,071; WO 91/06667, published in May 16, 1991.
In a preferred embodiment, recombinant cells described herein can be used for replacement therapy in a subject. For example, a nucleic acid encoding a 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 polypeptide operably linked to an inducible promoter (e.g, a steroid hormone receptor-regulated promoter) is introduced into a human or nonhuman, e.g, mammalian, e.g, porcine recombinant cell. The cell is cultivated and encapsulated in a biocompatible material, such as poly-lysine alginate, and subsequently implanted into the subject. See, e.g, Lanza (1996) Nat. Biotechnol. 14:1107; Joki et al. (2001) Nat. Biotechnol. 19:35; and U.S. Patent No. 5,876,742. Production of 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 polypeptide can be regulated in the subject by admimstering an agent (e.g, a steroid hormone) to the subject. In another preferred embodiment, the implanted recombinant cells express and secrete an antibody specific for a 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 polypeptide. The antibody can be any antibody or any antibody derivative described herein. Transgenic Animals
The invention provides non-human transgenic animals. Such animals are useful for studying the function and/or activity of a 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 protein and for identifying and/or evaluating modulators of 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 activity. As used herein, a "transgenic animal" is a non-human animal, preferably a mammal, more preferably a rodent such as a rat or mouse, in which one or more ofthe cells ofthe animal includes a transgene. Other examples of transgenic animals include non-human primates, sheep, dogs, cows, goats, chickens, amphibians, and the like. A transgene is exogenous DNA or a rearrangement, e.g, a deletion of endogenous chromosomal DNA, which preferably is integrated into or occurs in the genome ofthe cells of a transgenic animal. A transgene can direct the expression of an encoded gene product in one or more cell types or tissues ofthe transgenic animal, other transgenes, e.g, a knockout, reduce expression. Thus, a transgenic animal can be one in which an endogenous 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 gene has been altered by, e.g, by homologous recombination between the endogenous gene and an exogenous DNA molecule introduced into a cell ofthe animal, e.g, an embryonic cell ofthe animal, prior to development ofthe animal.
Intronic sequences and polyadenylation signals can also be included in the transgene to increase the efficiency of expression ofthe transgene. A tissue-specific regulatory sequence(s) can be operably linked to a transgene ofthe invention to direct expression of a 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 protein to particular cells. A transgenic founder animal can be identified based upon the presence of a 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 transgene in its genome and/or expression of 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 mRNA in tissues or cells ofthe animals. A transgenic founder animal can then be used to breed additional animals carrying the transgene. Moreover, transgenic animals carrying a transgene encoding a 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 protein can further be bred to other transgenic animals carrying other transgenes.
47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 proteins or polypeptides can be expressed in transgenic animals or plants, e.g, a nucleic acid encoding the protein or polypeptide can be introduced into the genome of an animal. In preferred embodiments the nucleic acid is placed under the control of a tissue specific promoter, e.g, a milk or egg specific promoter, and recovered from the milk or eggs produced by the animal. Suitable animals are mice, pigs, cows, goats, and sheep.
The invention also includes a population of cells from a transgenic animal, as discussed, e.g, below.
Uses
The nucleic acid molecules, proteins, protein homologues, and antibodies described herein can be used in one or more ofthe following methods: a) screening assays; b) predictive medicine (e.g, diagnostic assays, prognostic assays, monitoring clinical trials, and pharmacogenetics); and c) methods of treatment (e.g, therapeutic and prophylactic).
The isolated nucleic acid molecules ofthe invention can be used, for example, to express a 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 protein (e.g, via a recombinant expression vector in a host cell in gene therapy applications), to detect a 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 mRNA (e.g, in a biological sample) or a genetic alteration in a 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 gene, and to modulate 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 activity, as described further below. The 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 proteins can be used to treat disorders characterized by insufficient or excessive production of a 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 substrate or production of 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 inhibitors. In addition, the 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 proteins can be used to screen for naturally occurring 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 substrates, to screen for drags or compounds which modulate 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 activity, as well as to treat disorders characterized by insufficient or excessive production of 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 protein or production of 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 protein forms which have decreased, aberrant or unwanted activity compared to 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 wild type protein (e.g, immunological disorders, neurological disorders, metabolic disorders, cellular proliferation and/or differentiation disorders, disorders of metal ion imbalance, protein trafficing disorders, or cardiovascular disorders). Moreover, the anti-47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 antibodies of the invention can be used to detect and isolate 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 proteins, regulate the bioavailability of 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 proteins, and modulate 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 activity.
A method of evaluating a compound for the ability to interact with, e.g, bind, a subject 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 polypeptide is provided. The method includes: contacting the compound with the subject 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 polypeptide; and evaluating ability ofthe compound to interact with, e.g, to bind or form a complex with the subject 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 polypeptide. This method can be performed in vitro, e.g, in a cell free system, or in vivo, e.g, in a two-hybrid interaction trap assay. This method can be used to identify naturally occurring molecules that interact with subject 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 polypeptide. It can also be used to find natural or synthetic inhibitors of subject 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 polypeptide. Screening methods are discussed in more detail below.
Screening Assays
The invention provides methods (also referred to herein as "screening assays") for identifying modulators, i.e, candidate or test compounds or agents (e.g, proteins, peptides, peptidomimetics, peptoids, small molecules or other drags) which bind to 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 proteins, have a stimulatory or inhibitory effect on, for example, 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 expression or 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 activity, or have a stimulatory or inhibitory effect on, for example, the expression or activity of a 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234- interacting molecule, e.g, substrate molecule. Compounds thus identified can be used to modulate the activity of target gene products (e.g, 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 genes) in a therapeutic protocol, to elaborate the biological function ofthe target gene product, or to identify compounds that disrupt normal target gene interactions.
In one embodiment, the invention provides assays for screening candidate or test compounds which are substrates of a 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 protein or polypeptide or a biologically active portion thereof. In another embodiment, the invention provides assays for screening candidate or test compounds that bind to or modulate an activity of a 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 protein or polypeptide or a biologically active portion thereof. In one embodiment, an activity of a 47476 protein can be assayed as follows: (a) introduce a 47476 necleic acid expression constract into a cell such that 47476 protein is produced; (b) activate a signal transduction pathway that utilizes a ras superfamily member that can be activated by a 47476 protein; and (c) evaluate the ability the 47476 protein or functional fragment thereof to modulate the activated signal transduction pathway, as compared to a control cell that lacks the 47476 expression constract. Assays for determining the activity level of a signal transduction pathway will depend on the particular signal transduction pathway. Nevertheless, such assays are known in the art and include, e.g, detection of GTP loading of ras or ras-like proteins, gel electrophoresis of downstream targets, e.g, MAP kinase, or detection of the level of phosphorylation of various components ofthe signaling pathway. In one embodiment, an activity of a 67210 protein can be assayed as follows: (a) contacting an acceptor molecule (e.g, a lipid, protein, or carbohydrate) with a 67210 protein or functional fragment thereof in the presence of an activated mono- or oligosaccharide residue; and (b) evaluating the ability the 67210 protein or functional fragment thereof to initiate or elongate the carbohydrate chain. Assays for determining whether a carbohydrate modification has been made are known in the art and include, e.g, gel electrophoresis or antibody affinity.
In one embodiment, an activity of a 49875 protein can be assayed as follows, (a) contact a nucleic acid, e.g, a nucleic acid duplex, with a 49875 protein or functional fragment thereof in the presence of an NTP, e.g, GTP or ATP, and (b) evaluate the ability ofthe 49875 protein or functional fragment thereof to cause the unwinding ofthe nucleic acid. Assays for determining if a nucleic acid is wound or unwound are known in the art and include, e.g, gel electrophoresis. In one embodiment, an activity of a 46842 protein can be assayed as described in Kam et al. (2000), JBiol Chem 275:9653, or Dowler et al. (2000), Biochem J 351 :19, the contents of which are incoφorated herein by reference.
In one embodiment, an activity of a 33201 protein can be assayed as follows: (a) contacting a cell that expresses a 33201 protein, or a fragment thereof, with a known substrate, e.g, an alcohol or quinone substrate; and (b) evaluate the ability the 33201 protein, or functional fragment thereof, to oxidize or reduce the substrate. Assays for determining whether a substrate has been oxidized or reduced are known in the art.
In one embodiment, an activity of a 83378, 84233, 64708, 85041, or 84234 protein can be assayed by measuring ion transport or by visualizing ions contained within a cell or a cellular compartment, e.g, in the presence and/or absence of a 83378, 84233, 64708, 85041, or 84234 protein. For example methods of detecting zinc transport or visualizing zinc within cellular compartments, e.g, using a zinc-specific fluorescent probe, are described in detail in, e.g, Palmiter et al. (1995) EMBOJ. 14:639-649, Palmiter et al. (1996) EMBOJ. 15:1784-1791, and Palmiter et al. (1996) Proc. Natl. Acad. Sci. USA 93:14934-14939, the contents of which are incorporated herein by reference.
The test compounds ofthe present invention can be obtained using any ofthe numerous approaches in combinatorial library methods known in the art, including: biological libraries; peptoid libraries (libraries of molecules having the functionalities of peptides, but with a novel, non-peptide backbone which are resistant to enzymatic degradation but which nevertheless remain bioactive; see, e.g, Zuckermann, R.N. et al. (1994) J Med. Chem. 37:2678-85); spatially addressable parallel solid phase or solution phase libraries; synthetic library methods requiring deconvolution; the 'one-bead one-compound' library method; and synthetic library methods using affinity chromatography selection. The biological library and peptoid library approaches are limited to peptide libraries, while the other four approaches are applicable to peptide, non-peptide oligomer or small molecule libraries of compounds (Lam (1997) Anticancer Drug Des. 12:145).
Examples of methods for the synthesis of molecular libraries can be found in the art, for example in: DeWitt et α/. (1993) Proc. Natl Acad. Sci. U.S.A. 90:6909; Erb et al. (1994) Proc. Natl. Acad. Sci. USA 91 :11422; Zuckermann et al. (1994). J. Med. Chem. 37:2678; Cho et al. (1993) Science 261 :1303; Carrell et al. (1994) Angew. Chem. Int. Ed. Engl. 33:2059; Carell et al. (1994) Angew. Chem. Int. Ed. Engl. 33:2061; and Gallop et al. (1994) J Med. Chem. 37:1233.
Libraries of compounds may be presented in solution (e.g, Houghten (1992) Biotechniques 13:412-421), or on beads (Lam (1991) Nature 354:82-84), chips (Fodor (1993) Nature 364:555-556), bacteria (Ladner, U.S. Patent No. 5,223,409), spores (Ladner U.S. Patent No. 5,223,409), plasmids (Cull et al. (1992) Proc Natl Acad Sci USA 89:1865-1869) or on phage (Scott and Smith (1990) Science 249:386-390; Devlin (1990) Science 249:404-406; Cwirla et al. (1990) Proc. Natl. Acad. Sci 87:6378-6382; Felici (1991) J. Mol. Biol. 222:301- 310; Ladner supra.). In one embodiment, an assay is a cell-based assay in which a cell which expresses a
47476 protein or biologically active portion thereof is contacted with a test compound, and the ability ofthe test compound to modulate 47476 activity is determined. Determining the ability ofthe test compound to modulate 47476 activity can be accomplished, e.g, by monitoring the ability of 47476 polypeptide or proteins to activate one or more ras superfamily proteins, e.g, as measured by cellular characteristics at least partially controlled by ras superfamily members, e.g, cell shape, motility, growth, adhesion, or differentiation. The cell, for example, can be of mammalian origin, e.g, human.
In another embodiment, an assay is a cell-based assay in which a cell which expresses a 67210 protein or biologically active portion thereof is contacted with a test compound, and the ability ofthe test compound to modulate 67210 activity is determined. Determining the ability ofthe test compound to modulate 67210 activity can be accomplished, e.g, by monitoring the ability of 67210 add sugar residues to an appropriate substrate, e.g, as measured by the appearance ofthe modified substrate molecule ofthe cell surface or by changes in signal transduction with in the cell that result in changes in cellular behavior, e.g, changes in cell shape, motility, growth, adhesion, or differentiation. The cell, for example, can be of mammalian origin, e.g, human.
In another embodiment, an assay is a cell-based assay in which a cell which expresses a 49875 protein or biologically active portion thereof is contacted with a test compound, and the ability ofthe test compound to modulate 49875activity is determined. Determining the ability ofthe test compound to modulate 49875 activity can be accomplished, e.g, by monitoring the ability of 49875 molecules to unwind duplex nucleic acid molecules, e.g, as measured by changes in transcription, e.g, using a GFP construct and GFP expression as a readout, or changes in cellular behavior, e.g, changes in cell shape, motility, growth, adhesion, or differentiation. The cell, for example, can be of mammalian origin, e.g, human.
In another embodiment, an assay is a cell-based assay in which a cell which expresses a 46842 protein or biologically active portion thereof is contacted with a test compound, and the ability ofthe test compound to modulate 46842 activity is determined. Determining the ability ofthe test compound to modulate 46842 activity can be accomplished, e.g, by monitoring the ability of 46842 molecules to stimulate the GTPase activity of an Arf or arf-like protein, e.g, as measured by changes in cell shape or protein trafficking, e.g, using an appropriate marker protein that is transported through the secretory pathway. The cell, for example, can be of mammalian origin, e.g, human.
In another embodiment, an assay is a cell-based assay in which a cell which expresses a 83378, 84233, 64708, 85041, or 84234 protein or biologically active portion thereof is contacted with a test compound, and the ability ofthe test compound to modulate 83378, 84233, 64708, 85041, or 84234 activity is determined. Determining the ability ofthe test compound to modulate 46842 activity can be accomplished, e.g, by monitoring the ability of 83378, 84233, 64708, 85041, or 84234 molecules to transport metal ions, e.g, as measured by changes in the subcellular location of metal ions, e.g, zinc ions, or in the cellular response to the presence of metal ions, e.g, programmed cell death. The cell, for example, can be of mammalian origin, e.g, human.
The ability ofthe test compound to modulate 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 binding to a compound, e.g, a 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 substrate, or to bind to 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 can also be evaluated. This can be accomplished, for example, by coupling the compound, e.g, the substrate, with a radioisotope or enzymatic label such that binding ofthe compound, e.g, the substrate, to 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 can be determined by detecting the labeled compound, e.g, substrate, in a complex. Alternatively, 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 could be coupled with a radioisotope or enzymatic label to monitor the ability of a test compound to modulate 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 binding to a 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 substrate in a complex. For example, compounds (e.g, 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708,
85041, or 84234 substrates) can be labeled with 125I, 35S, 14C, or 3H, either directly or indirectly, and the radioisotope detected by direct counting of radioemmission or by scintillation counting. Alternatively, compounds can be enzymatically labeled with, for example, horseradish peroxidase, alkaline phosphatase, or luciferase, and the enzymatic label detected by determination of conversion of an appropriate substrate to product.
The ability of a compound (e.g, a 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 substrate) to interact with 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041 , or 84234 with or without the labeling of any of the interactants can be evaluated. For example, a microphysiometer can be used to detect the interaction of a compound with 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 without the labeling of either the compound or the 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234. McConnell, H. M. et al. (1992) Science 257:1906-1912. As used herein, a "microphysiometer" (e.g, Cytosensor) is an analytical instrument that measures the rate at which a cell acidifies its environment using a light-addressable potentiometric sensor (LAPS). Changes in this acidification rate can be used as an indicator ofthe interaction between a compound and 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234. In yet another embodiment, a cell-free assay is provided in which a 47476, 67210,
49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 protein or biologically active portion thereof is contacted with a test compound and the ability ofthe test compound to bind to the 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 protein or biologically active portion thereof is evaluated. Preferred biologically active portions ofthe 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 proteins to be used in assays ofthe present invention include fragments which participate in interactions with non- 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 molecules, e.g, fragments with high surface probability scores.
Soluble and/or membrane-bound forms of isolated proteins (e.g, 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 proteins or biologically active portions thereof) can be used in the cell-free assays ofthe invention. When membrane-bound forms of the protein are used, it may be desirable to utilize a solubilizing agent. Examples of such solubilizing agents include non-ionic detergents such as n-octylglucoside, n-dodecylglucoside, n-dodecylmaltoside, octanoyl-N-methylglucamide, decanoyl-N-methylglucamide, Triton® X-
100, Triton® X-l 14, Thesit®, Isotridecypoly(ethylene glycol ether)n, 3-[(3- cholamidopropyl)dimethylamminio]-l -propane sulfonate (CHAPS), 3-[(3- cholamidopropyl)dimethylamminio]-2-hydroxy-l -propane sulfonate (CHAPSO), orN- dodecyl=N,N-dimemyl-3-ammonio- 1 -propane sulfonate.
Cell-free assays involve preparing a reaction mixture ofthe target gene protein and the test compound under conditions and for a time sufficient to allow the two components to interact and bind, thus forming a complex that can be removed and/or detected.
The interaction between two molecules can also be detected, e.g, using fluorescence energy transfer (FET) (see, for example, Lakowicz et al, U.S. Patent No. 5,631,169; Stavrianopoulos, et al, U.S. Patent No. 4,868,103). A fluorophore label on the first, 'donor' molecule is selected such that its emitted fluorescent energy will be absorbed by a fluorescent label on a second, 'acceptor' molecule, which in turn is able to fluoresce due to the absorbed energy. Alternately, the 'donor' protein molecule may simply utilize the natural fluorescent energy of tryptophan residues. Labels are chosen that emit different wavelengths of light, such that the 'acceptor' molecule label may be differentiated from that ofthe 'donor'. Since the efficiency of energy transfer between the labels is related to the distance separating the molecules, the spatial relationship between the molecules can be assessed. In a situation in which binding occurs between the molecules, the fluorescent emission ofthe 'acceptor' molecule label in the assay should be maximal. An FET binding event can be conveniently measured through standard fluorometric detection means well known in the art (e.g, using a fluorimeter). In another embodiment, determining the ability ofthe 47476, 67210, 49875, 46842,
33201, 83378, 84233, 64708, 85041, or 84234 protein to bind to a target molecule can be accomplished using real-time Biomolecular Interaction Analysis (BIA) (see, e.g, Sjolander, S. and Urbaniczky, C. (1991) Anal. Chem. 63:2338-2345 and Szabo et al. (1995) Curr. Opin. Struct. Biol. 5:699-705). "Surface plasmon resonance" or "BIA" detects biospecific interactions in real time, without labeling any ofthe interactants (e.g, BIAcore). Changes in the mass at the binding surface (indicative of a binding event) result in alterations ofthe refractive index of light near the surface (the optical phenomenon of surface plasmon resonance (SPR)), resulting in a detectable signal which can be used as an indication of real-time reactions between biological molecules.
In one embodiment, the target gene product or the test substance is anchored onto a solid phase. The target gene product/test compound complexes anchored on the solid phase can be detected at the end ofthe reaction. Preferably, the target gene product can be anchored onto a solid surface, and the test compound, (which is not anchored), can be labeled, either directly or indirectly, with detectable labels discussed herein.
It may be desirable to immobilize either 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234, an anti-47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 antibody or its target molecule to facilitate separation of complexed from uncomplexed forms of one or both ofthe proteins, as well as to accommodate automation ofthe assay. Binding of a test compound to a 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 protein, or interaction of a 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 protein with a target molecule in the presence and absence of a candidate compound, can be accomplished in any vessel suitable for containing the reactants. Examples of such vessels include microtiter plates, test tubes, and micro-centrifuge tubes. In one embodiment, a fusion protein can be provided which adds a domain that allows one or both ofthe proteins to be bound to a matrix. For example, glutafhione-S- transferase/47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 fusion proteins or glutathione-S-transferase/target fusion proteins can be adsorbed onto glutathione sepharose beads (Sigma Chemical, St. Louis, MO) or glutathione derivatized microtiter plates, which are then combined with the test compound or the test compound and either the non- adsorbed target protein or 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 protein, and the mixture incubated under conditions conducive to complex formation
(e.g, at physiological conditions for salt and pH). Following incubation, the beads or microtiter plate wells are washed to remove any unbound components, the matrix immobilized in the case of beads, complex determined either directly or indirectly, for example, as described above. Alternatively, the complexes can be dissociated from the matrix, and the level of 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 binding or activity determined using standard techniques. Other techniques for immobilizing either a 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 protein or a target molecule on matrices include using conjugation of biotin and streptavidin. Biotinylated 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 protein or target molecules can be prepared from biotin- NHS (N-hydroxy-succinimide) using techniques known in the art (e.g, biotinylation kit, Pierce Chemicals, Rockford, IL), and immobilized in the wells of streptavidin-coated 96 well plates (Pierce Chemical).
In order to conduct the assay, the non-immobilized component is added to the coated surface containing the anchored component. After the reaction is complete, unreacted components are removed (e.g, by washing) under conditions such that any complexes formed will remain immobilized on the solid surface. The detection of complexes anchored on the solid surface can be accomplished in a number of ways. Where the previously non-immobilized component is pre-labeled, the detection of label immobilized on the surface indicates that complexes were formed. Where the previously non-immobilized component is not pre-labeled, an indirect label can be used to detect complexes anchored on the surface; e.g, using a labeled antibody specific for the immobilized component (the antibody, in turn, can be directly labeled or indirectly labeled with, e.g, a labeled anti-Ig antibody).
In one embodiment, this assay is performed utilizing antibodies reactive with 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 protein or target molecules but which do not interfere with binding ofthe 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 protein to its target molecule. Such antibodies can be derivatized to the wells ofthe plate, and unbound target or 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 protein trapped in the wells by antibody conjugation. Methods for detecting such complexes, in addition to those described above for the GST- immobilized complexes, include immunodetection of complexes using antibodies reactive with the 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 protein or target molecule, as well as enzyme-linked assays which rely on detecting an enzymatic activity associated with the 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 protein or target molecule. Alternatively, cell free assays can be conducted in a liquid phase. In such an assay, the reaction products are separated from unreacted components, by any of a number of standard techniques, including but not limited to: differential centrifugation (see, for example, Rivas, G, and Minton, A.P, (1993) Trends Biochem Sci 18:284-7); chromatography (gel filtration chromatography, ion-exchange chromatography); electrophoresis (see, e.g, Ausubel, F. et al, eds. Current Protocols in Molecular Biology 1999, J. Wiley: New York.); and immunoprecipitation (see, for example, Ausubel, F. et al, eds. (1999) Current Protocols in Molecular Biology, J. Wiley: New York). Such resins and chromatographic techniques are known to one skilled in the art (see, e.g, Heegaard, N.H, (1998) JMol Recognit 11 :141-8; Hage, D.S., and Tweed, S.A. (1997) J Chromatogr B Biomed Sci Appl. 699:499-525). Further, fluorescence energy transfer may also be conveniently utilized, as described herein, to detect binding without further purification of the complex from solution.
In a preferred embodiment, the assay includes contacting the 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 protein or biologically active portion thereof with a known compound which binds 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 to form an assay mixture, contacting the assay mixture with a test compound, and determining the ability of the test compound to interact with a 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 protein, wherein determining the ability ofthe test compound to interact with a 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 protein includes determining the ability ofthe test compound to preferentially bind to 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 or biologically active portion thereof, or to modulate the activity of a target molecule, as compared to the known compound.
The target gene products ofthe invention can, in vivo, interact with one or more cellular or extracellular macromolecules, such as proteins. For the purposes of this discussion, such cellular and extracellular macromolecules are referred to herein as "binding partners." Compounds that disrupt such interactions can be useful in regulating the activity ofthe target gene product. Such compounds can include, but are not limited to molecules such as antibodies, peptides, and small molecules. The preferred target genes/products for use in this embodiment are the 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 genes herein identified. In an alternative embodiment, the invention provides methods for determining the ability ofthe test compound to modulate the activity of a 47476, 67210,
49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 protein through modulation ofthe activity of a downstream effector of a 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 target molecule. For example, the activity ofthe effector molecule on an appropriate target can be determined, or the binding ofthe effector to an appropriate target can be determined, as previously described. To identify compounds that interfere with the interaction between the target gene product and its cellular or extracellular binding partner(s), a reaction mixture containing the target gene product and the binding partner is prepared, under conditions and for a time sufficient, to allow the two products to form complex. In order to test an inhibitory agent, the reaction mixture is provided in the presence and absence ofthe test compound. The test compound can be initially included in the reaction mixture, or can be added at a time subsequent to the addition ofthe target gene and its cellular or extracellular binding partner. Control reaction mixtures are incubated without the test compound or with a placebo. The formation of any complexes between the target gene product and the cellular or extracellular binding partner is then detected. The formation of a complex in the control reaction, but not in the reaction mixture containing the test compound, indicates that the compound interferes with the interaction ofthe target gene product and the interactive binding partner. Additionally, complex formation within reaction mixtures containing the test compound and normal target gene product can also be compared to complex formation within reaction mixtures containing the test compound and mutant target gene product. This comparison can be important in those cases wherein it is desirable to identify compounds that disrapt interactions of mutant but not normal target gene products.
These assays can be conducted in a heterogeneous or homogeneous format. Heterogeneous assays involve anchoring either the target gene product or the binding partner onto a solid phase, and detecting complexes anchored on the solid phase at the end ofthe reaction. In homogeneous assays, the entire reaction is carried out in a liquid phase. In either approach, the order of addition of reactants can be varied to obtain different information about the compounds being tested. For example, test compounds that interfere with the interaction between the target gene products and the binding partners, e.g, by competition, can be identified by conducting the reaction in the presence ofthe test substance. Alternatively, test compounds that disrapt preformed complexes, e.g, compounds with higher binding constants that displace one ofthe components from the complex, can be tested by adding the test compound to the reaction mixture after complexes have been formed. The various formats are briefly described below.
In a heterogeneous assay system, either the target gene product or the interactive cellular or extracellular binding partner, is anchored onto a solid surface (e.g, a microtiter plate), while the non-anchored species is labeled, either directly or indirectly. The anchored species can be immobilized by non-covalent or covalent attachments. Alternatively, an immobilized antibody specific for the species to be anchored can be used to anchor the species to the solid surface. In order to conduct the assay, the partner ofthe immobilized species is exposed to the coated surface with or without the test compound. After the reaction is complete, unreacted components are removed (e.g, by washing) and any complexes formed will remain immobilized on the solid surface. Where the non-immobilized species is pre-labeled, the detection of label immobilized on the surface indicates that complexes were formed. Where the non-immobilized species is not pre-labeled, an indirect label can be used to detect complexes anchored on the surface; e.g, using a labeled antibody specific for the initially non-immobilized species (the antibody, in turn, can be directly labeled or indirectly labeled with, e.g, a labeled anti-Ig antibody). Depending upon the order of addition of reaction components, test compounds that inhibit complex formation or that disrupt preformed complexes can be detected. Alternatively, the reaction can be conducted in a liquid phase in the presence or absence ofthe test compound, the reaction products separated from unreacted components, and complexes detected; e.g, using an immobilized antibody specific for one ofthe binding components to anchor any complexes formed in solution, and a labeled antibody specific for the other partner to detect anchored complexes. Again, depending upon the order of addition of reactants to the liquid phase, test compounds that inhibit complex or that disrapt preformed complexes can be identified. In an alternate embodiment ofthe invention, a homogeneous assay can be used. For example, a preformed complex ofthe target gene product and the interactive cellular or extracellular binding partner product is prepared in that either the target gene products or their binding partners are labeled, but the signal generated by the label is quenched due to complex formation (see, e.g, U.S. Patent No. 4,109,496 that utilizes this approach for immunoassays). The addition of a test substance that competes with and displaces one ofthe species from the preformed complex will result in the generation of a signal above background. In this way, test substances that disrupt target gene product-binding partner interaction can be identified.
In yet another aspect, the 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 proteins can be used as "bait proteins" in a two-hybrid assay or three-hybrid assay (see, e.g, U.S. Patent No. 5,283,317; Zervos et al. (1993) Cell 72:223-232; Madura et al. (1993) J. Biol. Chem. 268:12046-12054; Bartel et al. (1993) Biotechniques 14:920-924; Iwabuchi et al. (1993) Oncogene 8:1693-1696; and Brent WO94/10300), to identify other proteins, which bind to or interact with 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 ("47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234-binding proteins" or "47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234-bp") and are involved in 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 activity. Such 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234-bps can be activators or inhibitors of signals by the 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 proteins or 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 targets as, for example, downstream elements of a 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234-mediated signaling pathway.
The two-hybrid system is based on the modular nature of most transcription factors, which consist of separable DNA-binding and activation domains. Briefly, the assay utilizes two different DNA constructs. In one constract, the gene that codes for a 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 protein is fused to a gene encoding the DNA binding domain of a known transcription factor (e.g, GAL-4). In the other construct, a DNA sequence, from a library of DNA sequences, that encodes an unidentified protein ("prey" or "sample") is fused to a gene that codes for the activation domain ofthe known transcription factor. (Alternatively the: 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 protein can be the fused to the activator domain.) If the "bait" and the "prey" proteins are able to interact, in vivo, forming a 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234-dependent complex, the DNA-binding and activation domains ofthe transcription factor are brought into close proximity. This proximity allows transcription of a reporter gene (e.g, lacZ) which is operably linked to a transcriptional regulatory site responsive to the transcription factor. Expression ofthe reporter gene can be detected and cell colonies containing the functional transcription factor can be isolated and used to obtain the cloned gene which encodes the protein which interacts with the 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 protein.
In another embodiment, modulators of 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 expression are identified. For example, a cell or cell free mixture is contacted with a candidate compound and the expression of 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 mRNA or protein evaluated relative to the level of expression of 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 mRNA or protein in the absence ofthe candidate compound. When expression of 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 mRNA or protein is greater in the presence ofthe candidate compound than in its absence, the candidate compound is identified as a stimulator of 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 mRNA or protein expression. Alternatively, when expression of 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 mRNA or protein is less (statistically significantly less) in the presence ofthe candidate compound than in its absence, the candidate compound is identified as an inhibitor of 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 mRNA or protein expression. The level of 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 mRNA or protein expression can be determined by methods described herein for detecting 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 mRNA or protein.
In another aspect, the invention pertains to a combination of two or more ofthe assays described herein. For example, a modulating agent can be identified using a cell-based or a cell free assay, and the ability ofthe agent to modulate the activity of a 47476.) 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 protein can be confirmed in vivo, e.g, in an animal such as an animal model for immunological disorders, neurological disorders, metabolic disorders, cellular proliferation and/or differentiation disorders, disorders of metal ion imbalance, protein trafficing disorders, or cardiovascular disorders.
This invention further pertains to novel agents identified by the above-described screening assays. Accordingly, it is within the scope of this invention to further use an agent identified as described herein (e.g, a 47476, 67210, 49875, 46842, 33201, 83378, 84233,
64708, 85041, or 84234 modulating agent, an antisense 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 nucleic acid molecule, a 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234-specific antibody, or a 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234-binding partner) in an appropriate animal model to determine the efficacy, toxicity, side effects, or mechanism of action, of treatment with such an agent. Furthermore, novel agents identified by the above-described screening assays can be used for treatments as described herein.
Detection Assays
Portions or fragments ofthe nucleic acid sequences identified herein can be used as polynucleotide reagents. For example, these sequences can be used to: (i) map their respective genes on a chromosome e.g, to locate gene regions associated with genetic disease or to associate 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 with a disease; (ii) identify an individual from a minute biological sample (tissue typing); and (iii) aid in forensic identification of a biological sample. These applications are described in the subsections below.
Chromosome Mapping
The 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 nucleotide sequences or portions thereof can be used to map the location ofthe 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 genes on a chromosome. This process is called chromosome mapping. Chromosome mapping is useful in correlating the 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 sequences with genes associated with disease.
Briefly, 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 genes can be mapped to chromosomes by preparing PCR primers (preferably 15-25 bp in length) from the 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 nucleotide sequences. These primers can then be used for PCR screening of somatic cell hybrids containing individual human chromosomes. Only those hybrids containing the human gene corresponding to the 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 sequences will yield an amplified fragment. A panel of somatic cell hybrids in which each cell line contains either a single human chromosome or a small number of human chromosomes, and a full set of mouse chromosomes, can allow easy mapping of individual genes to specific human chromosomes. (D'Eustachio P. et al. (1983) Science 220:919-924). Other mapping strategies e.g, in situ hybridization (described in Fan, Y. et al. (1990)
Proc. Natl. Acad. Sci. USA, 87:6223-27), pre-screening with labeled flow-sorted chromosomes, and pre-selection by hybridization to chromosome specific cDNA libraries can be used to map 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 to a chromosomal location. Fluorescence in situ hybridization (FISH) of a DNA sequence to a metaphase chromosomal spread can further be used to provide a precise chromosomal location in one step. The FISH technique can be used with a DNA sequence as short as 500 or 600 bases. However, clones larger than 1,000 bases have a higher likelihood of binding to a unique chromosomal location with sufficient signal intensity for simple detection. Preferably 1,000 bases, and more preferably 2,000 bases will suffice to get good results at a reasonable amount of time. For a review of this technique, see Verma et al, Human Chromosomes: A Manual of Basic Techniques ((1988) Pergamon Press, New York).
Reagents for chromosome mapping can be used individually to mark a single chromosome or a single site on that chromosome, or panels of reagents can be used for marking multiple sites and/or multiple chromosomes. Reagents corresponding to noncoding regions of the genes actually are preferred for mapping purposes. Coding sequences are more likely to be conserved within gene families, thus increasing the chance of cross hybridizations during chromosomal mapping.
Once a sequence has been mapped to a precise chromosomal location, the physical position o the sequence on the chromosome can be correlated with genetic map data. (Such data are found, for example, in V. McKusick, Mendelian Inheritance in Man, available on-line through Johns Hopkins University Welch Medical Library). The relationship between a gene and a disease, mapped to the same chromosomal region, can then be identified through linkage analysis (co-inheritance of physically adjacent genes), described in, for example, Egeland, J. et al. (1987) Nature, 325:783-787. Moreover, differences in the DNA sequences between individuals affected and unaffected with a disease associated with the 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 gene, can be determined. If a mutation is observed in some or all ofthe affected individuals but not in any unaffected individuals, then the mutation is likely to be the causative agent ofthe particular disease. Comparison of affected and unaffected individuals generally involves first looking for structural alterations in the chromosomes, such as deletions or translocations that are visible from chromosome spreads or detectable using PCR based on that DNA sequence. Ultimately, complete sequencing of genes from several individuals can be performed to confirm the presence of a mutation and to distinguish mutations from polymorphisms.
Tissue Typing
47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 sequences can be used to identify individuals from biological samples using, e.g, restriction fragment length polymorphism (RFLP). In this technique, an individual's genomic DNA is digested with one or more restriction enzymes, the fragments separated, e.g, in a Southern blot, and probed to yield bands for identification. The sequences ofthe present invention are useful as additional DNA markers for RFLP (described in U.S. Patent 5,272,057).
Furthermore, the sequences ofthe present invention can also be used to determine the actual base-by-base DNA sequence of selected portions of an individual's genome. Thus, the 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 nucleotide sequences described herein can be used to prepare two PCR primers from the 5' and 3' ends of the sequences. These primers can then be used to amplify an individual's DNA and subsequently sequence it. Panels of corresponding DNA sequences from individuals, prepared in this manner, can provide unique individual identifications, as each individual will have a unique set of such DNA sequences due to allelic differences.
Allelic variation occurs to some degree in the coding regions of these sequences, and to a greater degree in the noncoding regions. Each ofthe sequences described herein can, to some degree, be used as a standard against which DNA from an individual can be compared for identification purposes. Because greater numbers of polymorphisms occur in the noncoding regions, fewer sequences are necessary to differentiate individuals. The noncoding sequences of SEQ ID NO:l, SEQ ID NO:4, SEQ ID NO:7, SEQ ID NO: 10, SEQ ID NO: 13, SEQ ID NO:16, SEQ ID NO: 19, SEQ ID NO:22, SEQ ID NO:25, or SEQ ID NO:28 can provide positive individual identification with a panel of perhaps 10 to 1,000 primers which each yield a noncoding amplified sequence of 100 bases. If predicted coding sequences, such as those in SEQ ID NO:3, SEQ ID NO:6, SEQ ID NO:9, SEQ ID NO: 12, SEQ ID NO: 15, SEQ ID NO: 18, SEQ ID NO:21, SEQ ID NO:24, SEQ ID NO:27, or SEQ ID NO:30 are used, a more appropriate number of primers for positive individual identification would be 500-2,000.
If a panel of reagents from 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 nucleotide sequences described herein is used to generate a unique identification database for an individual, those same reagents can later be used to identify tissue from that individual. Using the unique identification database, positive identification ofthe individual, living or dead, can be made from extremely small tissue samples.
Use of Partial 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 Sequences in Forensic Biology DNA-based identification techniques can also be used in forensic biology. To make such an identification, PCR technology can be used to amplify DNA sequences taken from very small biological samples such as tissues, e.g, hair or skin, or body fluids, e.g, blood, saliva, or semen found at a crime scene. The amplified sequence can then be compared to a standard, thereby allowing identification ofthe origin ofthe biological sample. The sequences ofthe present invention can be used to provide polynucleotide reagents, e.g, PCR primers, targeted to specific loci in the human genome, which can enhance the reliability of DNA-based forensic identifications by, for example, providing another "identification marker" (i.e. another DNA sequence that is unique to a particular individual). As mentioned above, actual base sequence information can be used for identification as an accurate alternative to patterns formed by restriction enzyme generated fragments. Sequences targeted to noncoding regions of SEQ ID NO:l, SEQ ID NO:4, SEQ ID NO:7, SEQ ID NO:10, SEQ ID NO:13, SEQ ID NO: 16, SEQ ID NO:19, SEQ ID NO:22, SEQ ID NO:25, or SEQ ID NO:28 (e.g, fragments derived from the noncoding regions of SEQ ID NO:l, SEQ ID NO:4, SEQ ID NO:7, SEQ ID NO: 10, SEQ ID NO:13, SEQ ID NO:16, SEQ ID NO:19, SEQ ID NO:22, SEQ ID NO:25, or SEQ ID NO:28 having a length of at least 20 bases, preferably at least 30 bases) are particularly appropriate for this use.
The 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 nucleotide sequences described herein can further be used to provide polynucleotide reagents, e.g, labeled or labelable probes which can be used in, for example, an in situ hybridization technique, to identify a specific tissue. This can be very useful in cases where a forensic pathologist is presented with a tissue of unknown origin. Panels of such 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 probes can be used to identify tissue by species and/or by organ type. In a similar fashion, these reagents, e.g, 47476, 67210, 49875, 46842, 33201, 83378,
84233, 64708, 85041, or 84234 primers or probes can be used to screen tissue culture for contamination (i.e. screen for the presence of a mixture of different types of cells in a culture).
Predictive Medicine
The present invention also pertains to the field of predictive medicine in which diagnostic assays, prognostic assays, and monitoring clinical trials are used for prognostic
(predictive) purposes to thereby treat an individual.
Generally, the invention provides, a method of determining if a subject is at risk for a disorder related to a lesion in or the misexpression of a gene which encodes 47476, 67210,
49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234. Such disorders include, e.g, a disorder associated with the misexpression of 47476,
67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 gene, e.g, an immunological disorder, neurological disorder, metabolic disorder, cellular proliferation and/or differentiation disorder, disorder of metal ion imbalance, a protein trafficing disorder, or a cardiovascular disorder. The method includes one or more ofthe following: detecting, in a tissue ofthe subject, the presence or absence of a mutation which affects the expression ofthe 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or
84234 gene, or detecting the presence or absence of a mutation in a region which controls the expression ofthe gene, e.g, a mutation in the 5' control region; detecting, in a tissue ofthe subject, the presence or absence of a mutation which alters the structure ofthe 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 gene; detecting, in a tissue ofthe subject, the misexpression ofthe 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 gene, at the mRNA level, e.g, detecting a non- wild type level of a mRNA ; detecting, in a tissue ofthe subject, the misexpression ofthe gene, at the protein level, e.g, detecting a non-wild type level of a 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 polypeptide. In preferred embodiments the method includes: ascertaining the existence of at least one of: a deletion of one or more nucleotides from the 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 gene; an insertion of one or more nucleotides into the gene, a point mutation, e.g, a substitution of one or more nucleotides ofthe gene, a gross chromosomal reanangement ofthe gene, e.g, a translocation, inversion, or deletion. For example, detecting the genetic lesion can include: (i) providing a probe/primer including an oligonucleotide containing a region of nucleotide sequence which hybridizes to a sense or antisense sequence from SEQ ID NO:l, SEQ ID NO:4, SEQ ID NO:7, SEQ ID NO: 10, SEQ ID NO:13, SEQ ID NO: 16, SEQ ID NO:19, SEQ ID NO:22, SEQ ID NO:25, or SEQ ID NO:28, or naturally occurring mutants thereof or 5' or 3' flanking sequences naturally associated with the 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 gene; (ii) exposing the probe/primer to nucleic acid ofthe tissue; and detecting, by hybridization, e.g, in situ hybridization, ofthe probe/primer to the nucleic acid, the presence or absence ofthe genetic lesion.
In preferred embodiments detecting the misexpression includes ascertaining the existence of at least one of: an alteration in the level of a messenger RNA transcript of the
47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 gene; the presence of a non- ild type splicing pattern of a messenger RNA transcript ofthe gene; or a non- wild type level of 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234.
Methods ofthe invention can be used prenatally or to determine if a subject's offspring will be at risk for a disorder. In preferred embodiments the method includes determining the structure of a 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 gene, an abnormal structure being indicative of risk for the disorder.
In preferred embodiments the method includes contacting a sample from the subject with an antibody to the 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 protein or a nucleic acid, which hybridizes specifically with the gene. These and other embodiments are discussed below.
Diagnostic and Prognostic Assays
Diagnostic and prognostic assays ofthe invention include method for assessing the expression level of 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 molecules and for identifying variations and mutations in the sequence of 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 molecules.
Expression Monitoring and Profiling. The presence, level, or absence of 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 protein or nucleic acid in a biological sample can be evaluated by obtaining a biological sample from a test subject and contacting the biological sample with a compound or an agent capable of detecting 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 protein or nucleic acid (e.g, mRNA, genomic DNA) that encodes 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 protein such that the presence of 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 protein or nucleic acid is detected in the biological sample. The term "biological sample" includes tissues, cells and biological fluids isolated from a subject, as well as tissues, cells and fluids present within a subject. A preferred biological sample is serum. The level of expression ofthe 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 gene can be measured in a number of ways, including, but not limited to: measuring the mRNA encoded by the 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 genes; measuring the amount of protein encoded by the 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 genes; or measuring the activity ofthe protein encoded by the 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 genes. The level of mRNA corresponding to the 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 gene in a cell can be determined both by in situ and by in vitro formats.
The isolated mRNA can be used in hybridization or amplification assays that include, but are not limited to, Southern or Northern analyses, polymerase chain reaction analyses and probe arrays. One preferred diagnostic method for the detection of mRNA levels involves contacting the isolated mRNA with a nucleic acid molecule (probe) that can hybridize to the mRNA encoded by the gene being detected. The nucleic acid probe can be, for example, a full- length 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 nucleic acid, such as the nucleic acid of SEQ ID NO:l, SEQ ID NO:4, SEQ ID NO:7, SEQ ID NO:10, SEQ ID NO:13, SEQ ID NO:16, SEQ ID NO:19, SEQ ID NO:22, SEQ ID NO:25, or SEQ ID NO:28, or a portion thereof, such as an oligonucleotide of at least 7, 15, 30, 50, 100, 250 or 500 nucleotides in length and sufficient to specifically hybridize under stringent conditions to 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 mRNA or genomic DNA. The probe can be disposed on an address of an array, e.g, an array described below. Other suitable probes for use in the diagnostic assays are described herein.
In one format, mRNA (or cDNA) is immobilized on a surface and contacted with the probes, for example by running the isolated mRNA on an agarose gel and transfening the mRNA from the gel to a membrane, such as nitrocellulose. In an alternative format, the probes are immobilized on a surface and the mRNA (or cDNA) is contacted with the probes, for example, in a two-dimensional gene chip array described below. A skilled artisan can adapt known mRNA detection methods for use in detecting the level of mRNA encoded by the 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 genes.
The level of mRNA in a sample that is encoded by one of 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 can be evaluated with nucleic acid amplification, e.g, by rtPCR (Mullis (1987) U.S. Patent No. 4,683,202), ligase chain reaction (Barany (1991) Proc. Natl. Acad. Sci. USA 88:189-193), self sustained sequence replication (Guatelli et al, (1990) Proc. Natl. Acad. Sci. USA 87:1874-1878), transcriptional amplification system (Kwoh et al, (1989), Proc. Natl. Acad. Sci. USA 86:1173-1177), Q-Beta Replicase (Lizardi et al, (1988) Bio/Technology 6:1197), rolling circle replication (Lizardi et al, U.S. Patent No. 5,854,033) or any other nucleic acid amplification method, followed by the detection ofthe amplified molecules using techniques known in the art. As used herein, amplification primers are defined as being a pair of nucleic acid molecules that can anneal to 5' or 3' regions of a gene (plus and minus strands, respectively, or vice-versa) and contain a short region in between. In general, amplification primers are from about 10 to 30 nucleotides in length and flank a region from about 50 to 200 nucleotides in length. Under appropriate conditions and with appropriate reagents, such primers permit the amplification of a nucleic acid molecule comprising the nucleotide sequence flanked by the primers.
For in situ methods, a cell or tissue sample can be prepared/processed and immobilized on a support, typically a glass slide, and then contacted with a probe that can hybridize to mRNA that encodes the 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 gene being analyzed.
In another embodiment, the methods further contacting a control sample with a compound or agent capable of detecting 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 mRNA, or genomic DNA, and comparing the presence of 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 mRNA or genomic DNA in the control sample with the presence of 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 mRNA or genomic DNA in the test sample. In still another embodiment, serial analysis of gene expression, as described in U.S. Patent No. 5,695,937, is used to detect 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 transcript levels.
A variety of methods can be used to determine the level of protein encoded by 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234. In general, these methods include contacting an agent that selectively binds to the protein, such as an antibody with a sample, to evaluate the level of protein in the sample. In a preferred embodiment, the antibody bears a detectable label. Antibodies can be polyclonal, or more preferably, monoclonal. An intact antibody, or a fragment thereof (e.g. Fab or F(ab')2) can be used. The term "labeled", with regard to the probe or antibody, is intended to encompass direct labeling ofthe probe or antibody by coupling (i.e, physically linking) a detectable substance to the probe or antibody, as well as indirect labeling ofthe probe or antibody by reactivity with a detectable substance. Examples of detectable substances are provided herein. The detection methods can be used to detect 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 protein in a biological sample in vitro as well as in vivo. In vitro techniques for detection of 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 protein include enzyme linked immunosorbent assays (ELISAs), immunoprecipitations, immunofluorescence, enzyme immunoassay (EIA), radioimmunoassay (RIA), and Western blot analysis. In vivo techniques for detection of 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 protein include introducing into a subject a labeled anti-47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 antibody. For example, the antibody can be labeled with a radioactive marker whose presence and location in a subject can be detected by standard imaging techniques. In another embodiment, the sample is labeled, e.g, biotinylated and then contacted to the antibody, e.g, an anti-47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 antibody positioned on an antibody array (as described below). The sample can be detected, e.g, with avidin coupled to a fluorescent label. In another embodiment, the methods further include contacting the control sample with a compound or agent capable of detecting 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 protein, and comparing the presence of 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 protein in the control sample with the presence of 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 protein in the test sample.
The invention also includes kits for detecting the presence of 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 in a biological sample. For example, the kit can include a compound or agent capable of detecting 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 protein or mRNA in a biological sample; and a standard. The compound or agent can be packaged in a suitable container. The kit can further comprise instractions for using the kit to detect 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 protein or nucleic acid.
For antibody-based kits, the kit can include: (1) a first antibody (e.g, attached to a solid support) which binds to a polypeptide corresponding to a marker ofthe invention; and, optionally, (2) a second, different antibody which binds to either the polypeptide or the first antibody and is conjugated to a detectable agent. For oligonucleotide-based kits, the kit can include: (1) an oligonucleotide, e.g, a detectably labeled oligonucleotide, which hybridizes to a nucleic acid sequence encoding a polypeptide corresponding to a marker ofthe invention or (2) a pair of primers useful for amplifying a nucleic acid molecule corresponding to a marker ofthe invention. The kit can also includes a buffering agent, a preservative, or a protein stabilizing agent. The kit can also includes components necessary for detecting the detectable agent (e.g, an enzyme or a substrate). The kit can also contain a control sample or a series of control samples which can be assayed and compared to the test sample contained. Each component ofthe kit can be enclosed within an individual container and all ofthe various containers can be within a single package, along with instractions for interpreting the results ofthe assays performed using the kit.
The diagnostic methods described herein can identify subjects having, or at risk of developing, a disease or disorder associated with misexpressed or aberrant or unwanted 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 expression or activity. As used herein, the term "unwanted" includes an unwanted phenomenon involved in a biological response such as an immunological disorder, neurological disorder, metabolic disorder, cellular proliferation and/or differentiation disorder, disorder of metal ion imbalance, a protein trafficing disorder, or a cardiovascular disorder.
In one embodiment, a disease or disorder associated with aberrant or unwanted 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 expression or activity is identified. A test sample is obtained from a subject and 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 protein or nucleic acid (e.g, mRNA or genomic DNA) is evaluated, wherein the level, e.g, the presence or absence, of 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 protein or nucleic acid is diagnostic for a subject having or at risk of developing a disease or disorder associated with aberrant or unwanted 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 expression or activity. As used herein, a "test sample" refers to a biological sample obtained from a subject of interest, including a biological fluid (e.g, serum), cell sample, or tissue.
The prognostic assays described herein can be used to determine whether a subject can be administered an agent (e.g, an agonist, antagonist, peptidomimetic, protein, peptide, nucleic acid, small molecule, or other drug candidate) to treat a disease or disorder associated with aberrant or unwanted 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 expression or activity. For example, such methods can be used to determine whether a subject can be effectively treated with an agent for an immunological disorder, neurological disorder, metabolic disorder, cellular proliferation and/or differentiation disorder, disorder of metal ion imbalance, a protein trafficing disorder disorder, or a cardiovascular disorder. In another aspect, the invention features a computer medium having a plurality of digitally encoded data records. Each data record includes a value representing the level of expression of 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 in a sample, and a descriptor ofthe sample. The descriptor ofthe sample can be an identifier ofthe sample, a subject from which the sample was derived (e.g, a patient), a diagnosis, or a treatment (e.g, a preferred treatment). In a prefened embodiment, the data record further includes values representing the level of expression of genes other than 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 (e.g, other genes associated with a 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234-disorder, or other genes on an array). The data record can be structured as a table, e.g, a table that is part of a database such as a relational database (e.g, a SQL database ofthe Oracle or Sybase database environments).
Also featured is a method of evaluating a sample. The method includes providing a sample, e.g, from the subject, and determining a gene expression profile ofthe sample, wherein the profile includes a value representing the level of 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 expression. The method can further include comparing the value or the profile (i.e, multiple values) to a reference value or reference profile. The gene expression profile ofthe sample can be obtained by any ofthe methods described herein (e.g, by providing a nucleic acid from the sample and contacting the nucleic acid to an array). The method can be used to diagnose an immunological disorder, neurological disorder, metabolic disorder, cellular proliferation and/or differentiation disorder, disorder of metal ion imbalance, a protein trafficing disorder disorder, or a cardiovascular disorder in a subject wherein an increase or decrease in 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 expression is an indication that the subject has or is disposed to having an immunological disorder, neurological disorder, metabolic disorder, cellular proliferation and/or differentiation disorder, disorder of metal ion imbalance, a protein trafficing disorder, or a cardiovascular disorder. The method can be used to monitor a treatment for an immunological disorder, neurological disorder, metabolic disorder, cellular proliferation and/or differentiation disorder, disorder of metal ion imbalance, a protein trafficing disorder, or a cardiovascular disorder in a subject. For example, the gene expression profile can be determined for a sample from a subject undergoing treatment. The profile can be compared to a reference profile or to a profile obtained from the subject prior to treatment or prior to onset ofthe disorder (see, e.g, Golub et al. (1999) Science 286:531).
In yet another aspect, the invention features a method of evaluating a test compound (see also, "Screening Assays", above). The method includes providing a cell and a test compound; contacting the test compound to the cell; obtaining a subject expression profile for the contacted cell; and comparing the subject expression profile to one or more reference profiles. The profiles include a value representing the level of 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 expression. In a preferred embodiment, the subject expression profile is compared to a target profile, e.g, a profile for a normal cell or for desired condition of a cell. The test compound is evaluated favorably if the subject expression profile is more similar to the target profile than an expression profile obtained from an uncontacted cell.
In another aspect, the invention features, a method of evaluating a subject. The method includes: a) obtaining a sample from a subject, e.g, from a caregiver, e.g, a caregiver who obtains the sample from the subject; b) determining a subject expression profile for the sample. Optionally, the method further includes either or both of steps: c) comparing the subject expression profile to one or more reference expression profiles; and d) selecting the reference profile most similar to the subject reference profile. The subject expression profile and the reference profiles include a value representing the level of 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 expression. A variety of routine statistical measures can be used to compare two reference profiles. One possible metric is the length ofthe distance vector that is the difference between the two profiles. Each ofthe subject and reference profile is represented as a multi-dimensional vector, wherein each dimension is a value in the profile. The method can further include transmitting a result to a caregiver. The result can be the subject expression profile, a result of a comparison ofthe subject expression profile with another profile, a most similar reference profile, or a descriptor of any ofthe aforementioned. The result can be transmitted across a computer network, e.g, the result can be in the form of a computer transmission, e.g, a computer data signal embedded in a carrier wave. Also featured is a computer medium having executable code for effecting the following steps: receive a subject expression profile; access a database of reference expression profiles; and either i) select a matching reference profile most similar to the subject expression profile or ii) determine at least one comparison score for the similarity ofthe subject expression profile to at least one reference profile. The subject expression profile, and the reference expression profiles each include a value representing the level of 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 expression.
Arrays and Uses Thereof
In another aspect, the invention features an array that includes a substrate having a plurality of addresses. At least one address ofthe plurality includes a capture probe that binds specifically to a 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 molecule (e.g, a 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 nucleic acid or a 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 polypeptide). The array can have a density of at least than 10, 50, 100, 200, 500, 1,000, 2,000, or 10,000 or more addresses/cm , and ranges between. In a preferred embodiment, the plurality of addresses includes at least 10, 100, 500, 1,000, 5,000, 10,000, 50,000 addresses. In a preferred embodiment, the plurality of addresses includes equal to or less than 10, 100, 500, 1,000, 5,000, 10,000, or 50,000 addresses. The substrate can be a two-dimensional substrate such as a glass slide, a wafer (e.g, silica or plastic), a mass spectroscopy plate, or a three- dimensional substrate such as a gel pad. Addresses in addition to address ofthe plurality can be disposed on the array.
In a preferred embodiment, at least one address ofthe plurality includes a nucleic acid capture probe that hybridizes specifically to a 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 nucleic acid, e.g, the sense or anti-sense strand. In one preferred embodiment, a subset of addresses ofthe plurality of addresses has a nucleic acid capture probe for 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234. Each address ofthe subset can include a capture probe that hybridizes to a different region of a 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 nucleic acid. In another preferred embodiment, addresses ofthe subset include a capture probe for a 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 nucleic acid. Each address ofthe subset is unique, overlapping, and complementary to a different variant of 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 (e.g, an allelic variant, or all possible hypothetical variants). The array can be used to sequence 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 by hybridization (see, e.g, U.S. Patent No. 5,695,940).
An array can be generated by various methods, e.g, by photolithographic methods (see, e.g., U.S. Patent Nos. 5,143,854; 5,510,270; and 5,527,681), mechanical methods (e.g, ' directed-flow methods as described in U.S. Patent No. 5,384,261), pin-based methods (e.g, as described in U.S. Pat. No. 5,288,514), and bead-based techniques (e.g, as described in PCT US/93/04145).
In another preferred embodiment, at least one address ofthe plurality includes a polypeptide capture probe that binds specifically to a 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 polypeptide or fragment thereof. The polypeptide can be a naturally-occurring interaction partner of 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 polypeptide. Preferably, the polypeptide is an antibody, e.g, an antibody described herein (see "Anti-47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 Antibodies," above), such as a monoclonal antibody or a single-chain antibody.
In another aspect, the invention features a method of analyzing the expression of 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234. The method includes providing an array as described above; contacting the anay with a sample and detecting binding of a 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234-molecule (e.g, nucleic acid or polypeptide) to the anay. In a prefened embodiment, the anay is a nucleic acid anay. Optionally the method further includes amplifying nucleic acid from the sample prior or during contact with the anay.
In another embodiment, the anay can be used to assay gene expression in a tissue to ascertain tissue specificity of genes in the anay, particularly the expression of 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234. If a sufficient number of diverse samples is analyzed, clustering (e.g, hierarchical clustering, k-means clustering, Bayesian clustering and the like) can be used to identify other genes which are co-regulated with 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234. For example, the anay can be used for the quantitation ofthe expression of multiple genes. Thus, not only tissue specificity, but also the level of expression of a battery of genes in the tissue is ascertained. Quantitative data can be used to group (e.g, cluster) genes on the basis of their tissue expression per se and level of expression in that tissue. For example, anay analysis of gene expression can be used to assess the effect of cell- cell interactions on 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 expression. A first tissue can be perturbed and nucleic acid from a second tissue that interacts with the first tissue can be analyzed. In this context, the effect of one cell type on another cell type in response to a biological stimulus can be determined, e.g, to monitor the effect of cell- cell interaction at the level of gene expression.
In another embodiment, cells are contacted with a therapeutic agent. The expression profile ofthe cells is determined using the anay, and the expression profile is compared to the profile of like cells not contacted with the agent. For example, the assay can be used to determine or analyze the molecular basis of an undesirable effect ofthe therapeutic agent. If an agent is administered therapeutically to treat one cell type but has an undesirable effect on another cell type, the invention provides an assay to determine the molecular basis ofthe undesirable effect and thus provides the opportunity to co-administer a counteracting agent or otherwise treat the undesired effect. Similarly, even within a single cell type, undesirable biological effects can be determined at the molecular level. Thus, the effects of an agent on expression of other than the target gene can be ascertained and counteracted.
In another embodiment, the anay can be used to monitor expression of one or more genes in the array with respect to time. For example, samples obtained from different time points can be probed with the anay. Such analysis can identify and/or characterize the development of a 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234- associated disease or disorder; and processes, such as a cellular transformation associated with a 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234-associated disease or disorder. The method can also evaluate the treatment and/or progression of a 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234-associated disease or disorder The anay is also useful for ascertaining differential expression patterns of one or more genes in normal and abnormal cells. This provides a battery of genes (e.g. , including 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234) that could serve as a molecular target for diagnosis or therapeutic intervention.
In another aspect, the invention features an anay having a plurality of addresses. Each address ofthe plurality includes a unique polypeptide. At least one address ofthe plurality has disposed thereon a 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 polypeptide or fragment thereof. Methods of producing polypeptide anays are described in the art, e.g, in De Wildt et al. (2000). Nature Biotech. 18, 989-994; Lueking et al. (1999). Anal. Biochem. 270, 103-111; Ge, H. (2000). Nucleic Acids Res. 28, e3, 1-VII; MacBeath, G, and Schreiber, S.L. (2000). Science 289, 1760-1763; and WO 99/51773A1. In a prefened embodiment, each addresses ofthe plurality has disposed thereon a polypeptide at least 60, 70, 80,85, 90, 95 or 99 % identical to a 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 polypeptide or fragment thereof. For example, multiple variants of a 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 polypeptide (e.g, encoded by allelic variants, site-directed mutants, random mutants, or combinatorial mutants) can be disposed at individual addresses ofthe plurality. Addresses in addition to the address ofthe plurality can be disposed on the anay.
The polypeptide anay can be used to detect a 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 binding compound, e.g, an antibody in a sample from a subject with specificity for a 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 polypeptide or the presence of a 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234-binding protein or ligand.
The anay is also useful for ascertaining the effect ofthe expression of a gene on the expression of other genes in the same cell or in different cells (e.g., ascertaining the effect of 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 expression on the expression of other genes). This provides, for example, for a selection of alternate molecular targets for therapeutic intervention if the ultimate or downstream target cannot be regulated. In another aspect, the invention features a method of analyzing a plurality of probes. The method is useful, e.g, for analyzing gene expression. The method includes: providing a two dimensional anay having a plurality of addresses, each address ofthe plurality being positionally distinguishable from each other address ofthe plurality having a unique capture probe, e.g, wherein the capture probes are from a cell or subject which express 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 or from a cell or subject in which a 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 mediated response has been elicited, e.g, by contact ofthe cell with 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 nucleic acid or protein, or administration to the cell or subject 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 nucleic acid or protein; providing a two dimensional anay having a plurality of addresses, each address ofthe plurality being positionally distinguishable from each other address ofthe plurality, and each address ofthe plurality having a unique capture probe, e.g, wherein the capture probes are from a cell or subject which does not express 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 (or does not express as highly as in the case ofthe 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 positive plurality of capture probes) or from a cell or subject which in which a 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 mediated response has not been elicited (or has been elicited to a lesser extent than in the first sample); contacting the anay with one or more inquiry probes (which is preferably other than a 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 nucleic acid, polypeptide, or antibody), and thereby evaluating the plurality of capture probes. Binding, e.g, in the case of a nucleic acid, hybridization with a capture probe at an address ofthe plurality, is detected, e.g, by signal generated from a label attached to the nucleic acid, polypeptide, or antibody. In another aspect, the invention features a method of analyzing a plurality of probes or a sample. The method is useful, e.g, for analyzing gene expression. The method includes: providing a two dimensional anay having a plurality of addresses, each address ofthe plurality being positionally distinguishable from each other address ofthe plurality having a unique capture probe, contacting the anay with a first sample from a cell or subject which express or mis-express 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 or from a cell or subject in which a 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234-mediated response has been elicited, e.g, by contact ofthe cell with 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 nucleic acid or protein, or administration to the cell or subject 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 nucleic acid or protein; providing a two dimensional anay having a plurality of addresses, each address ofthe plurality being positionally distinguishable from each other address ofthe plurality, and each address ofthe plurality having a unique capture probe, and contacting the anay with a second sample from a cell or subject which does not express 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 (or does not express as highly as in the case ofthe 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 positive plurality of capture probes) or from a cell or subject which in which a 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 mediated response has not been elicited (or has been elicited to a lesser extent than in the first sample); and comparing the binding ofthe first sample with the binding ofthe second sample. Binding, e.g, in the case of a nucleic acid, hybridization with a capture probe at an address ofthe plurality, is detected, e.g, by signal generated from a label attached to the nucleic acid, polypeptide, or antibody. The same anay can be used for both samples or different anays can be used. If different anays are used the plurality of addresses with capture probes should be present on both anays.
In another aspect, the invention features a method of analyzing 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234, e.g, analyzing structure, function, or relatedness to other nucleic acid or amino acid sequences. The method includes: providing a 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 nucleic acid or amino acid sequence; comparing the 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 sequence with one or more preferably a plurality of sequences from a collection of sequences, e.g, a nucleic acid or protein sequence database; to thereby analyze 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234.
Detection of Variations or Mutations
The methods ofthe invention can also be used to detect genetic alterations in a 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 gene, thereby determining if a subject with the altered gene is at risk for a disorder characterized by misregulation in 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 protein activity or nucleic acid expression, such as an immunological disorder, neurological disorder, metabolic disorder, cellular proliferation and/or differentiation disorder, disorder of metal ion imbalance, a protein trafficking disorder, or a cardiovascular disorder. In prefened embodiments, the methods include detecting, in a sample from the subject, the presence or absence of a genetic alteration characterized by at least one of an alteration affecting the integrity of a gene encoding a 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234-protein, or the mis-expression ofthe 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 gene. For example, such genetic alterations can be detected by ascertaining the existence of at least one of 1) a deletion of one or more nucleotides from a 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 gene; 2) an addition of one or more nucleotides to a 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 gene; 3) a substitution of one or more nucleotides of a 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 gene, 4) a chromosomal reanangement of a 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 gene; 5) an alteration in the level of a messenger RNA transcript of a 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 gene, 6) abenant modification of a 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 gene, such as ofthe methylation pattern ofthe genomic DNA, 7) the presence of a non- wild type splicing pattern of a messenger RNA transcript of a 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 gene, 8) a non- wild type level of a 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or
84234-protein, 9) allelic loss of a 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 gene, and 10) inappropriate post-translational modification of a 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234-protein.
An alteration can be detected without a probe/primer in a polymerase chain reaction, such as anchor PCR or RACE PCR, or, alternatively, in a ligation chain reaction (LCR), the latter of which can be particularly useful for detecting point mutations in the 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234-gene. This method can include the steps of collecting a sample of cells from a subject, isolating nucleic acid (e.g, genomic, mRNA or both) from the sample, contacting the nucleic acid sample with one or more primers which specifically hybridize to a 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 gene under conditions such that hybridization and amplification ofthe 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234-gene (if present) occurs, and detecting the presence or absence of an amplification product, or detecting the size ofthe amplification product and comparing the length to a control sample. It is anticipated that PCR and/or LCR may be desirable to use as a preliminary amplification step in conjunction with any ofthe techniques used for detecting mutations described herein. Alternatively, other amplification methods described herein or known in the art can be used.
In another embodiment, mutations in a 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 gene from a sample cell can be identified by detecting alterations in restriction enzyme cleavage patterns. For example, sample and control DNA is isolated, amplified (optionally), digested with one or more restriction endonucleases, and fragment length sizes are determined, e.g, by gel electrophoresis and compared. Differences in fragment length sizes between sample and control DNA indicates mutations in the sample DNA. Moreover, the use of sequence specific ribozymes (see, for example, U.S. Patent No. 5,498,531) can be used to score for the presence of specific mutations by development or loss of a ribozyme cleavage site.
In other embodiments, genetic mutations in 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 can be identified by hybridizing a sample and control nucleic acids, e.g, DNA or RNA, two-dimensional anays, e.g, chip based anays. Such anays include a plurality of addresses, each of which is positionally distinguishable from the other. A different probe is located at each address ofthe plurality. A probe can be complementary to a region of a 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 nucleic acid or a putative variant (e.g, allelic variant) thereof. A probe can have one or more mismatches to a region of a 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 nucleic acid (e.g, a destabilizing mismatch). The anays can have a high density of addresses, e.g, can contain hundreds or thousands of oligonucleotides probes (Cronin, M.T. et al. (1996) Human Mutation 7: 244-255; Kozal, M.J. et al. (1996) Nature Medicine 2: 753-759). For example, genetic mutations in 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 can be identified in two-dimensional anays containing light-generated DNA probes as described in Cronin, M.T. et al. supra. Briefly, a first hybridization anay of probes can be used to scan through long stretches of DNA in a sample and control to identify base changes between the sequences by making linear anays of sequential overlapping probes. This step allows the identification of point mutations. This step is followed by a second hybridization anay that allows the characterization of specific mutations by using smaller, specialized probe anays complementary to all variants or mutations detected. Each mutation anay is composed of parallel probe sets, one complementary to the wild-type gene and the other complementary to the mutant gene.
In yet another embodiment, any of a variety of sequencing reactions known in the art can be used to directly sequence the 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 gene and detect mutations by comparing the sequence ofthe sample 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 with the conesponding wild-type (control) sequence. Automated sequencing procedures can be utilized when performing the diagnostic assays ((1995) Biotechniques 19:448), including sequencing by mass spectrometry. Other methods for detecting mutations in the 47476, 67210, 49875, 46842, 33201 ,
83378, 84233, 64708, 85041, or 84234 gene include methods in which protection from cleavage agents is used to detect mismatched bases in RNA/RNA or RNA/DNA heteroduplexes (Myers et al. (1985) Science 230:1242; Cotton et al. (1988) Proc. Natl Acad Sci USA 85:4397; Saleeba et al. (1992) Methods Enzymol. 217:286-295). In still another embodiment, the mismatch cleavage reaction employs one or more proteins that recognize mismatched base pairs in double-stranded DNA (so called "DNA mismatch repair" enzymes) in defined systems for detecting and mapping point mutations in 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 cDNAs obtained from samples of cells. For example, the mutY enzyme of E. coli cleaves A at G/A mismatches and the thymidine DNA glycosylase from HeLa cells cleaves T at G/T mismatches (Hsu et al. (1994) Carcinogenesis 15:1657-1662; U.S. Patent No. 5,459,039).
In other embodiments, alterations in electrophoretic mobility will be used to identify mutations in 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 genes. For example, single strand conformation polymorphism (SSCP) may be used to detect differences in electrophoretic mobility between mutant and wild type nucleic acids (Orita et al. (1989) Proc Natl Acad. Sci USA: 86:2766, see also Cotton (1993) Mutat. Res. 285:125-144; and Hayashi (1992) Genet. Anal. Tech. Appl. 9:73-79). Single-stranded DNA fragments of sample and control 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 nucleic acids will be denatured and allowed to renature. The secondary structure of single- stranded nucleic acids varies according to sequence, the resulting alteration in electrophoretic mobility enables the detection of even a single base change. The DNA fragments may be labeled or detected with labeled probes. The sensitivity ofthe assay may be enhanced by using RNA (rather than DNA), in which the secondary structure is more sensitive to a change in sequence. In a prefened embodiment, the subject method utilizes heteroduplex analysis to separate double stranded heteroduplex molecules on the basis of changes in electrophoretic mobility (Keen et al (1991 ) Trends Genet 7:5).
In yet another embodiment, the movement of mutant or wild-type fragments in polyacrylamide gels containing a gradient of denaturant is assayed using denaturing gradient gel electrophoresis (DGGE) (Myers et al. (1985) Nature 313:495). When DGGE is used as the method of analysis, DNA will be modified to insure that it does not completely denature, for example by adding a GC clamp of approximately 40 bp of high-melting GC-rich DNA by PCR. In a further embodiment, a temperature gradient is used in place of a denaturing gradient to identify differences in the mobility of control and sample DNA (Rosenbaum and Reissner (1987) Biophys Chem 265:12753).
Examples of other techniques for detecting point mutations include, but are not limited to, selective oligonucleotide hybridization, selective amplification, or selective primer extension (Saiki et al. (1986) Nature 324:163); Saiki et al. (1989) Proc. Natl Acad. Sci USA 86:6230). A further method of detecting point mutations is the chemical ligation of oligonucleotides as described in Xu et al. ((2001) Nature Biotechnol. 19:148). Adjacent oligonucleotides, one of which selectively anneals to the query site, are ligated together if the nucleotide at the query site ofthe sample nucleic acid is complementary to the query oligonucleotide; ligation can be monitored, e.g, by fluorescent dyes coupled to the oligonucleotides.
Alternatively, allele specific amplification technology that depends on selective PCR amplification may be used in conjunction with the instant invention. Oligonucleotides used as primers for specific amplification may carry the mutation of interest in the center ofthe molecule (so that amplification depends on differential hybridization) (Gibbs et al. (1989) Nucleic Acids Res. 17:2437-2448) or at the extreme 3' end of one primer where, under appropriate conditions, mismatch can prevent, or reduce polymerase extension (Prossner (1993) Tibtech 11 :238). In addition it may be desirable to introduce a novel restriction site in the region ofthe mutation to create cleavage-based detection (Gasparini et al (1992) Mol Cell Probes 6:1). It is anticipated that in certain embodiments amplification may also be performed using Taq ligase for amplification (Barany (1991) Proc. Natl. Acad. Sci USA 88:189). In such cases, ligation will occur only if there is a perfect match at the 3' end ofthe 5' sequence making it possible to detect the presence of a known mutation at a specific site by looking for the presence or absence of amplification.
In another aspect, the invention features a set of oligonucleotides. The set includes a plurality of oligonucleotides, each of which is at least partially complementary (e.g, at least 50%, 60%, 70%, 80%, 90%, 92%, 95%, 97%, 98%, or 99% complementary) to a 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 nucleic acid.
In a prefened embodiment the set includes a first and a second oligonucleotide. The first and second oligonucleotide can hybridize to the same or to different locations of SEQ ID NO:l, SEQ ID NO:4, SEQ ID NO:7, SEQ ID NO:10, SEQ ID NO:13, SEQ ID NO:16, SEQ ID NO: 19, SEQ ID NO:22, SEQ ID NO:25, or SEQ ID NO:28. Different locations can be different but overlapping, or non-overlapping on the same strand. The first and second oligonucleotide can hybridize to sites on the same or on different strands.
The set can be useful, e.g, for identifying SNP's, or identifying specific alleles of 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234. In a prefened embodiment, each oligonucleotide ofthe set has a different nucleotide at an intenogation position. In one embodiment, the set includes two oligonucleotides, each complementary to a different allele at a locus, e.g, a biallelic or polymorphic locus.
In another embodiment, the set includes four oligonucleotides, each having a different nucleotide (e.g, adenine, guanine, cytosine, or thymidine) at the intenogation position. The intenogation position can be a SNP or the site of a mutation. In another prefened embodiment, the oligonucleotides ofthe plurality are identical in sequence to one another (except for differences in length). The oligonucleotides can be provided with differential labels, such that an oligonucleotide that hybridizes to one allele provides a signal that is distinguishable from an oligonucleotide that hybridizes to a second allele. In still another embodiment, at least one of the oligonucleotides ofthe set has a nucleotide change at a position in addition to a query position, e.g, a destabilizing mutation to decrease the Tm ofthe oligonucleotide. In another embodiment, at least one oligonucleotide ofthe set has a non-natural nucleotide, e.g, inosine. In a prefened embodiment, the oligonucleotides are attached to a solid support, e.g, to different addresses of an anay or to different beads or nanoparticles. In a prefened embodiment the set of oligo nucleotides can be used to specifically amplify, e.g, by PCR, or detect, a 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 nucleic acid.
The methods described herein may be performed, for example, by utilizing pre- packaged diagnostic kits comprising at least one probe nucleic acid or antibody reagent described herein, which may be conveniently used, e.g., in clinical settings to diagnose patients exhibiting symptoms or family history of a disease or illness involving a 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 gene.
Use of 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 Molecules as Sunogate Markers
The 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 molecules ofthe invention are also useful as markers of disorders or disease states, as markers for precursors of disease states, as markers for predisposition of disease states, as markers of drug activity, or as markers ofthe pharmacogenomic profile of a subject. Using the methods described herein, the presence, absence and/or quantity ofthe 47476, 67210, 49875, 46842,
33201, 83378, 84233, 64708, 85041, or 84234 molecules ofthe invention may be detected, and may be conelated with one or more biological states in vivo. For example, the 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 molecules ofthe invention may serve as sunogate markers for one or more disorders or disease states or for conditions leading up to disease states. As used herein, a "surrogate marker" is an objective biochemical marker which conelates with the absence or presence of a disease or disorder, or with the progression of a disease or disorder (e.g, with the presence or absence of a tumor). The presence or quantity of such markers is independent ofthe disease. Therefore, these markers may serve to indicate whether a particular course of treatment is effective in lessening a disease state or disorder. Surrogate markers are of particular use when the presence or extent of a disease state or disorder is difficult to assess through standard methodologies (e.g, early stage tumors), or when an assessment of disease progression is desired before a potentially dangerous clinical endpoint is reached (e.g, an assessment of cardiovascular disease may be made using cholesterol levels as a sunogate marker, and an analysis of HIV infection may be made using HIV RNA levels as a sunogate marker, well in advance ofthe undesirable clinical outcomes of myocardial infarction or fully-developed AIDS). Examples ofthe use of sunogate markers in the art include: Koomen et al. (2000) J Mass. Spectrom. 35: 258-264; and James (1994) AIDS Treatment News Archive 209.
The 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 molecules ofthe invention are also useful as pharmacodynamic markers. As used herein, a "pharmacodynamic marker" is an objective biochemical marker which conelates specifically with drag effects. The presence or quantity of a pharmacodynamic marker is not related to the disease state or disorder for which the drug is being administered; therefore, the presence or quantity ofthe marker is indicative ofthe presence or activity ofthe drug in a subject. For example, a pharmacodynamic marker may be indicative ofthe concentration ofthe drug in a biological tissue, in that the marker is either expressed or transcribed or not expressed or transcribed in that tissue in relationship to the level ofthe drug. In this fashion, the distribution or uptake ofthe drag may be monitored by the pharmacodynamic marker. Similarly, the presence or quantity ofthe pharmacodynamic marker may be related to the presence or quantity ofthe metabolic product of a drug, such that the presence or quantity ofthe marker is indicative ofthe relative breakdown rate ofthe drug in vivo. Pharmacodynamic markers are of particular use in increasing the sensitivity of detection of drag effects, particularly when the drag is administered in low doses. Since even a small amount of a drug may be sufficient to activate multiple rounds of marker (e.g, a 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 marker) transcription or expression, the amplified marker may be in a quantity which is more readily detectable than the drug itself. Also, the marker may be more easily detected due to the nature ofthe marker itself; for example, using the methods described herein, anti-47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 antibodies may be employed in an immune-based detection system for a 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 protein marker, or 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234-sρecific radiolabeled probes may be used to detect a 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 mRNA marker. Furthermore, the use of a pharmacodynamic marker may offer mechanism-based prediction of risk due to drag treatment beyond the range of possible direct observations. Examples ofthe use of pharmacodynamic markers in the art include: Matsuda et al US 6,033,862; Hattis et al. (1991) Env. Health Persped. 90: 229-238; Schentag (1999) Am. J. Health-Syst. Pharm. 56 Suppl. 3: S21-S24; and Nicolau (1999) Am, J. Health-Syst. Pharm. 56 Suppl. 3: S16-S20.
The 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 molecules ofthe invention are also useful as pharmacogenomic markers. As used herein, a "pharmacogenomic marker" is an objective biochemical marker which conelates with a specific clinical drag response or susceptibility in a subject (see, e.g, McLeod et al. (1999) Eur. J. Cancer 35:1650-1652). The presence or quantity ofthe pharmacogenomic marker is related to the predicted response ofthe subject to a specific drug or class of drags prior to administration ofthe drag. By assessing the presence or quantity of one or more pharmacogenomic markers in a subject, a drag therapy which is most appropriate for the subject, or which is predicted to have a greater degree of success, may be selected. For example, based on the presence or quantity of RNA, or protein (e.g, 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 protein or RNA) for specific tumor markers in a subject, a drag or course of treatment may be selected that is optimized for the treatment ofthe specific tumor likely to be present in the subject. Similarly, the presence or absence of a specific sequence mutation in 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 DNA may conelate 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 drag response. The use of pharmacogenomic markers therefore permits the application ofthe most appropriate treatment for each subject without having to administer the therapy.
Pharmaceutical Compositions
The nucleic acid and polypeptides, fragments thereof, as well as anti-47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 antibodies (also refened to herein as "active compounds") ofthe invention can be incorporated into pharmaceutical compositions. Such compositions typically include the nucleic acid molecule, protein, or antibody and a pharmaceutically acceptable canier. As used herein the language "pharmaceutically acceptable canier" includes solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration. Supplementary active compounds can also be incorporated into the compositions. A pharmaceutical composition is formulated to be compatible with its intended route of administration. Examples of routes of administration include parenteral, e.g, intravenous, intradermal, subcutaneous, oral (e.g, inhalation), transdermal (topical), transmucosal, and rectal administration. Solutions or suspensions used for parenteral, intradermal, or subcutaneous application can include the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose. pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide. The parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.
Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. For intravenous administration, suitable earners include physiological saline, bacteriostatic water, Cremophor EL™ (BASF, Parsippany, NJ) or phosphate buffered saline (PBS). In all cases, the composition must be sterile and should be fluid to the extent that easy syringability exists. It should be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. The canier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyetheylene glycol, and the like), and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance ofthe required particle size in the case of dispersion and by the use of surfactants. Prevention ofthe action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, tbimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as manitol, sorbitol, sodium chloride in the composition. Prolonged absorption ofthe injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate and gelatin. Sterile injectable solutions can be prepared by incorporating the active compound in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle which contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the prefened methods of preparation are vacuum drying and freeze-drying which yields a powder ofthe active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.
Oral compositions generally include an inert diluent or an edible canier. For the purpose of oral therapeutic administration, the active compound can be incoφorated with excipients and used in the form of tablets, troches, or capsules, e.g, gelatin capsules. Oral compositions can also be prepared using a fluid canier for use as a mouthwash. Pharmaceutically compatible binding agents, and/or adjuvant materials can be included as part ofthe composition. The tablets, pills, capsules, troches and the like can contain any ofthe following ingredients, or compounds of a similar nature: a binder such as microcrystalline cellulose, gum tragacanth or gelatin; an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel, or com starch; a lubricant such as magnesium stearate or Sterotes; a glidant such as colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; or a flavoring agent such as peppermint, methyl salicylate, or orange flavoring. For administration by inhalation, the compounds are delivered in the form of an aerosol spray from pressured container or dispenser which contains a suitable propellant, e.g, a gas such as carbon dioxide, or a nebulizer.
Systemic administration can also be by transmucosal or transdermal means. For transmucosal or transdermal administration, penetrants appropriate to the banier to be permeated are used in the formulation. Such penetrants are generally known in the art, and include, for example, for transmucosal administration, detergents, bile salts, and fusidic acid derivatives. Transmucosal administration can be accomplished through the use of nasal sprays or suppositories. For transdermal administration, the active compounds are formulated into ointments, salves, gels, or creams as generally known in the art. The compounds can also be prepared in the form of suppositories (e.g, with conventional suppository bases such as cocoa butter and other glycerides) or retention enemas for rectal delivery.
In one embodiment, the active compounds are prepared with earners that will protect the compound against rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Methods for preparation of such formulations will be apparent to those skilled in the art. The materials can also be obtained commercially from Alza Coφoration and Nova Pharmaceuticals, Inc. Liposomal suspensions (including liposomes targeted to infected cells with monoclonal antibodies to viral antigens) can also be used as pharmaceutically acceptable earners. These can be prepared according to methods known to those skilled in the art, for example, as described in U.S. Patent No. 4,522,811.
It is advantageous to formulate oral or parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the subject to be treated; each unit containing a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical canier.
Toxicity and therapeutic efficacy of such compounds can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD50 (the dose lethal to 50% ofthe population) and the ED50 (the dose therapeutically effective in 50%> ofthe population). The dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD50/ED50. Compounds which exhibit high therapeutic indices are prefened. While compounds that exhibit toxic side effects may be used, care should be taken to design a delivery system that targets such compounds to the site of affected tissue in order to minimize potential damage to uninfected cells and, thereby, reduce side effects.
The data obtained from the cell culture assays and animal studies can be used in formulating a range of dosage for use in humans. The dosage of such compounds lies preferably within a range of circulating concentrations that include the ED50 with little or no toxicity. The dosage may vary within this range depending upon the dosage form employed and the route of administration utilized. For any compound used in the method ofthe invention, the therapeutically effective dose can be estimated initially from cell culture assays. A dose may be formulated in animal models to achieve a circulating plasma concentration range that includes the IC50 (i.e., the concentration ofthe test compound which achieves a half-maximal inhibition of symptoms) as determined in cell culture. Such information can be used to more accurately determine useful doses in humans. Levels in plasma may be measured, for example, by high performance liquid chromatography.
As defined herein, a therapeutically effective amount of protein or polypeptide (i.e, an effective dosage) ranges from about 0.001 to 30 mg/kg body weight, preferably about 0.01 to 25 mg/kg body weight, more preferably about 0.1 to 20 mg/kg body weight, and even more preferably about 1 to 10 mg/kg, 2 to 9 mg/kg, 3 to 8 mg/kg, 4 to 7 mg/kg, or 5 to 6 mg/kg body weight. The protein or polypeptide can be administered one time per week for between about 1 to 10 weeks, preferably between 2 to 8 weeks, more preferably between about 3 to 7 weeks, and even more preferably for about 4, 5, or 6 weeks. The skilled artisan will appreciate that certain factors may influence the dosage and timing required to effectively treat a subject, including but not limited to the severity ofthe disease or disorder, previous treatments, the general health and/or age ofthe subject, and other diseases present. Moreover, treatment of a subject with a therapeutically effective amount of a protein, polypeptide, or antibody can include a single treatment or, preferably, can include a series of treatments. For antibodies, the prefened dosage is 0.1 mg/kg of body weight (generally 10 mg/kg to
20 mg/kg). If the antibody is to act in the brain, a dosage of 50 mg/kg to 100 mg/kg is usually appropriate. Generally, partially human antibodies and fully human antibodies have a longer half-life within the human body than other antibodies. Accordingly, lower dosages and less frequent administration is often possible. Modifications such as lipidation can be used to stabilize antibodies and to enhance uptake and tissue penetration (e.g, into the brain). A method for lipidation of antibodies is described by Cruikshank et al. ((1997) J. Acquired Immune Deficiency Syndromes and Human Retrovirology 14:193).
The present invention encompasses agents which modulate expression or activity. An agent may, for example, be a small molecule. For example, such small molecules include, but are not limited to, peptides, peptidomimetics (e.g, peptoids), amino acids, amino acid analogs, polynucleotides, polynucleotide analogs, nucleotides, nucleotide analogs, organic or inorganic compounds (i.e.,. including heteroorganic and organometallic compounds) having a molecular weight less than about 10,000 grams per mole, organic or inorganic compounds having a molecular weight less than about 5,000 grams per mole, organic or inorganic compounds having a molecular weight less than about 1 ,000 grams per mole, organic or inorganic compounds having a molecular weight less than about 500 grams per mole, and salts, esters, and other pharmaceutically acceptable forms of such compounds.
Exemplary doses include milligram or microgram amounts ofthe small molecule per kilogram of subject or sample weight (e.g, about 1 microgram per kilogram to about 500 milligrams per kilogram, about 100 micrograms per kilogram to about 5 milligrams per kilogram, or about 1 microgram per kilogram to about 50 micrograms per kilogram. It is furthermore understood that appropriate doses of a small molecule depend upon the potency of the small molecule with respect to the expression or activity to be modulated. When one or more of these small molecules is to be administered to an animal (e.g, a human) in order to modulate expression or activity of a polypeptide or nucleic acid ofthe invention, a physician, veterinarian, or researcher may, for example, prescribe a relatively low dose at first, subsequently increasing the dose until an appropriate response is obtained. In addition, it is understood that the specific dose level for any particular animal subject will depend upon a variety of factors including the activity ofthe specific compound employed, the age, body weight, general health, gender, and diet ofthe subject, the time of administration, the route of administration, the rate of excretion, any drug combination, and the degree of expression or activity to be modulated.
An antibody (or fragment thereof) may be conjugated to a therapeutic moiety such as a cytotoxin, a therapeutic agent or a radioactive ion. A cytotoxin or cytotoxic agent includes any agent that is detrimental to cells. Examples include taxol, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicin, doxorabicin, daunorabicin, dihydroxy anthracin dione, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, puromycin, maytansinoids, e.g., maytansinol (see US Patent No. 5,208,020), CC- 1065 (see US Patent Nos. 5,475,092, 5,585,499, 5,846,545) and analogs or homologs thereof. Therapeutic agents include, but are not limited to, antimetabolites (e.g., methotrexate, 6- mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil decarbazine), alkylating agents (e.g., mechlorethamine, thioepa chlorambucil, CC-1065, melphalan, carmustine (BSNU) and lomustine (CCNU), cyclothosphamide, busulfan, dibromomannitol, streptozotocin, mitomycin C, and cis-dichlorodiamine platinum (11) (DDP) cisplatin), anthracyclines (e.g., daunorubicin (formerly daunomycin) and doxorubicin), antibiotics (e.g., dactinomycin (formerly actinomycin), bleomycin, mithramycin, and anthramycin (AMC)), and anti-mitotic agents (e.g., vincristine, vinblastine, taxol and maytansinoids). Radioactive ions include, but are not limited to iodine, yttrium and praseodymium.
The conjugates ofthe invention can be used for modifying a given biological response, the drag moiety is not to be construed as limited to classical chemical therapeutic agents. For example, the drag moiety may be a protein or polypeptide possessing a desired biological activity. Such proteins may include, for example, a toxin such as abrin, ricin A, pseudomonas exotoxin, or diphtheria toxin; a protein such as tumor necrosis factor, α-interferon, β-interferon, nerve growth factor, platelet derived growth factor, tissue plasminogen activator; or, biological response modifiers such as, for example, lymphokines, interleukin-1 ("IL-1"), interleukin-2 ("1L-2"), interleukin-6 ("IL-6"), granulocyte macrophase colony stimulating factor ("GM- CSF"), granulocyte colony stimulating factor ("G-CSF"), or other growth factors. Alternatively, an antibody can be conjugated to a second antibody to form an antibody heteroconjugate as described by Segal in U.S. Patent No. 4,676,980.
The nucleic acid molecules ofthe invention can be inserted into vectors and used as gene therapy vectors. Gene therapy vectors can be delivered to a subject by, for example, intravenous injection, local administration (see U.S. Patent 5,328,470) or by stereotactic injection (see e.g, Chen et al. (1994) Proc. Natl Acad. Sci. USA 91:3054-3057). The pharmaceutical preparation ofthe gene therapy vector can include the gene therapy vector in an acceptable diluent, or can comprise a slow release matrix in which the gene delivery vehicle is imbedded. Alternatively, where the complete gene delivery vector can be produced intact from recombinant cells, e.g, retroviral vectors, the pharmaceutical preparation can include one or more cells which produce the gene delivery system.
The pharmaceutical compositions can be included in a container, pack, or dispenser together with instractions for administration. Methods of Treatment
The present invention provides for both prophylactic and therapeutic methods of treating a subject at risk of (or susceptible to) a disorder or having a disorder associated with abenant or unwanted 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 expression or activity. As used herein, the term "treatment" is defined as the application or administration of a therapeutic agent to a patient, or application or administration of a therapeutic agent to an isolated tissue or cell line from a patient, who has a disease, a symptom of disease or a predisposition toward a disease, with the puφose to cure, heal, alleviate, relieve, alter, remedy, ameliorate, improve or affect the disease, the symptoms of disease or the predisposition toward disease. A therapeutic agent includes, but is not limited to, small molecules, peptides, antibodies, ribozymes and antisense oligonucleotides.
With regards to both prophylactic and therapeutic methods of treatment, such treatments may be specifically tailored or modified, based on knowledge obtained from the field of pharmacogenomics. "Pharmacogenomics", as used herein, refers to the application of genomics technologies such as gene sequencing, statistical genetics, and gene expression analysis to drags in clinical development and on the market. More specifically, the term refers the study of how a patient's genes determine his or her response to a drag (e.g, a patient's "drug response phenotype", or "drag response genotype".) Thus, another aspect ofthe invention provides methods for tailoring an individual's prophylactic or therapeutic treatment with either the 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 molecules ofthe present invention or 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 modulators according to that individual's drag response genotype. Pharmacogenomics allows a clinician or physician to target prophylactic or therapeutic treatments to patients who will most benefit from the treatment and to avoid treatment of patients who will experience toxic drag-related side effects.
In one aspect, the invention provides a method for preventing in a subject, a disease or condition associated with an abenant or unwanted 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 expression or activity, by administering to the subject a 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 or an agent which modulates 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 expression or at least one 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 activity. Subjects at risk for a disease which is caused or contributed to by abenant or unwanted 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 expression or activity can be identified by, for example, any or a combination of diagnostic or prognostic assays as described herein. Administration of a prophylactic agent can occur prior to the manifestation of symptoms characteristic ofthe 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 abenance, such that a disease or disorder is prevented or, alternatively, delayed in its progression. Depending on the type of 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 abenance, for example, a 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234, 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 agonist or 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 antagonist agent can be used for treating the subject. The appropriate agent can be determined based on screening assays described herein. t is possible that some 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 disorders can be caused, at least in part, by an abnormal level of gene product, or by the presence of a gene product exhibiting abnormal activity. As such, the reduction in the level and/or activity of such gene products would bring about the amelioration of disorder symptoms.
The 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 molecules can act as novel diagnostic targets and therapeutic agents for controlling one or more of cellular proliferative and/or differentiative disorders, immunological disorders (e.g, inflammatory disorders), red blood cell disorders, viral diseases, neurological disorders (e.g, brain disorders), pain or metabolic disorders, liver disorders, kidney disorders, disorders ofthe small intestine, disorder of metal ion imbalance, protein trafficking disorders, cardiovascular disorders, and disorders associated with bone metabolism, as discussed above. Successful treatment of 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708,
85041, or 84234 disorders can be brought about by techniques that serve to inhibit the expression or activity of target gene products. For example, compounds, e.g, an agent identified using an assays described above, that proves to exhibit negative modulatory activity, can be used in accordance with the invention to prevent and/or ameliorate symptoms of 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 disorders. Such molecules can include, but are not limited to peptides, phosphopeptides, small organic or inorganic molecules, or antibodies (including, for example, polyclonal, monoclonal, humanized, anti- idiotypic, chimeric or single chain antibodies, and Fab, F(ab') and Fab expression library fragments, scFV molecules, and epitope-binding fragments thereof).
Further, antisense and ribozyme molecules that inhibit expression ofthe target gene can also be used in accordance with the invention to reduce the level of target gene expression, thus effectively reducing the level of target gene activity. Still further, triple helix molecules can be utilized in reducing the level of target gene activity. Antisense, ribozyme and triple helix molecules are discussed above.
It is possible that the use of antisense, ribozyme, and/or triple helix molecules to reduce or inhibit mutant gene expression can also reduce or inhibit the transcription (triple helix) and/or translation (antisense, ribozyme) of mRNA produced by normal target gene alleles, such that the concentration of normal target gene product present can be lower than is necessary for a normal phenotype. In such cases, nucleic acid molecules that encode and express target gene polypeptides exhibiting normal target gene activity can be introduced into cells via gene therapy method. Alternatively, in instances in that the target gene encodes an extracellular protein, it can be preferable to co-administer normal target gene protein into the cell or tissue in order to maintain the requisite level of cellular or tissue target gene activity.
Another method by which nucleic acid molecules may be utilized in treating or preventing a disease characterized by 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 expression is through the use of aptamer molecules specific for 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 protein. Aptamers are nucleic acid molecules having a tertiary structure which permits them to specifically bind to protein Hgands (see, e.g, Osborne, et al. (1997) Curr. Opin. Chem Biol. 1 : 5-9; and Patel, D.J. (1997) Curr Opin Chem Biol 1 :32-46). Since nucleic acid molecules may in many cases be more conveniently introduced into target cells than therapeutic protein molecules may be, aptamers offer a method by which 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 protein activity may be specifically decreased without the introduction of drugs or other molecules which may have pluripotent effects.
Antibodies can be generated that are both specific for target gene product and that reduce target gene product activity. Such antibodies may, therefore, by administered in instances whereby negative modulatory techniques are appropriate for the treatment of 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 disorders. For a description of antibodies, see the Antibody section above.
In circumstances wherein injection of an animal or a human subject with a 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 protein or epitope for stimulating antibody production is harmful to the subject, it is possible to generate an immune response against 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 through the use of anti-idiotypic antibodies (see, for example, Herlyn, D. (1999) Ann Med 31 :66-78; and Bhattacharya-Chatterjee, M, and Foon, K.A. (1998) Cancer Treat Res. 94:51- 68). If an anti-idiotypic antibody is introduced into a mammal or human subject, it should stimulate the production of anti-anti-idiotypic antibodies, which should be specific to the 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 protein. Vaccines directed to a disease characterized by 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 expression may also be generated in this fashion.
In instances where the target antigen is intracellular and whole antibodies are used, internalizing antibodies may be prefened. Lipofectin or liposomes can be used to deliver the antibody or a fragment ofthe Fab region that binds to the target antigen into cells. Where fragments ofthe antibody are used, the smallest inhibitory fragment that binds to the target antigen is prefened. For example, peptides having an amino acid sequence conesponding to the Fv region ofthe antibody can be used. Alternatively, single chain neutralizing antibodies that bind to intracellular target antigens can also be administered. Such single chain antibodies can be administered, for example, by expressing nucleotide sequences encoding single-chain antibodies within the target cell population (see e.g, Marasco et al. (1993) Proc. Natl. Acad. Sci. USA 90:7889-7893).
The identified compounds that inhibit target gene expression, synthesis and/or activity can be administered to a patient at therapeutically effective doses to prevent, treat or ameliorate 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 disorders. A therapeutically effective dose refers to that amount ofthe compound sufficient to result in amelioration of symptoms ofthe disorders. Toxicity and therapeutic efficacy of such compounds can be determined by standard pharmaceutical procedures as described above. The data obtained from the cell culture assays and animal studies can be used in formulating a range of dosage for use in humans. The dosage of such compounds lies preferably within a range of circulating concentrations that include the ED5o with little or no toxicity. The dosage can vary within this range depending upon the dosage form employed and the route of administration utilized. For any compound used in the method ofthe invention, the therapeutically effective dose can be estimated initially from cell culture assays. A dose can be formulated in animal models to achieve a circulating plasma concentration range that includes the IC5o (i.e., the concentration ofthe test compound that achieves a half-maximal inhibition of symptoms) as determined in cell culture. Such information can be used to more accurately determine useful doses in humans. Levels in plasma can be measured, for example, by high performance liquid chromatography. Another example of determination of effective dose for an individual is the ability to directly assay levels of "free" and "bound" compound in the serum ofthe test subject. Such assays may utilize antibody mimics and/or "biosensors" that have been created through molecular imprinting techniques. The compound which is able to modulate 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 activity is used as a template, or "imprinting molecule", to spatially organize polymerizable monomers prior to their polymerization with catalytic reagents. The subsequent removal ofthe imprinted molecule leaves a polymer matrix which contains a repeated "negative image" ofthe compound and is able to selectively rebind the molecule under biological assay conditions. A detailed review of this technique can be seen in Ansell, R. J. et al (1996) Current Opinion in Biotechnology 7:89-94 and in Shea, KJ. (1994) Trends in Polymer Science 2:166-173. Such "imprinted" affinity matrixes are amenable to ligand-binding assays, whereby the immobilized monoclonal antibody component is replaced by an appropriately imprinted matrix. An example ofthe use of such matrixes in this way can be seen in Vlatakis, G. et al (1993) Nature 361:645-647. Through the use of isotope-labeling, the "free" concentration of compound which modulates the expression or activity of 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 can be readily monitored and used in calculations of IC5o.
Such "imprinted" affinity matrixes can also be designed to include fluorescent groups whose photon-emitting properties measurably change upon local and selective binding of target compound. These changes can be readily assayed in real time using appropriate fiberoptic devices, in turn allowing the dose in a test subject to be quickly optimized based on its individual IC5o. An rudimentary example of such a "biosensor" is discussed in Kriz, D. et al (1995) Analytical Chemistry 67:2142-2144.
Another aspect ofthe invention pertains to methods of modulating 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 expression or activity for therapeutic puφoses. Accordingly, in an exemplary embodiment, the modulatory method ofthe invention involves contacting a cell with a 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 or agent that modulates one or more ofthe activities of 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 protein activity associated with the cell. An agent that modulates 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 protein activity can be an agent as described herein, such as a nucleic acid or a protein, a naturally-occuning target molecule of a 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 protein (e.g, a 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 substrate or receptor), a 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 antibody, a 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 agonist or antagonist, a peptidomimetic of a 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 agonist or antagonist, or other small molecule.
In one embodiment, the agent stimulates one or 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 activities. Examples of such stimulatory agents include active 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 protein and a nucleic acid molecule encoding 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234. In another embodiment, the agent inhibits one or more 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 activities. Examples of such inhibitory agents include antisense 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 nucleic acid molecules, anti-47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 antibodies, and 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 inhibitors. These modulatory methods can be performed in vitro (e.g, by culturing the cell with the agent) or, alternatively, in vivo (e.g, by administering the agent to a subject). As such, the present invention provides methods of treating an individual afflicted with a disease or disorder characterized by abenant or unwanted expression or activity of a
47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 protein or nucleic acid molecule. In one embodiment, the method involves administering an agent (e.g, an agent identified by a screening assay described herein), or combination of agents that modulates (e.g, up regulates or down regulates) 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 expression or activity. In another embodiment, the method involves administering a 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 protein or nucleic acid molecule as therapy to compensate for reduced, abenant, or unwanted 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 expression or activity.
Stimulation of 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 activity is desirable in situations in which 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 is abnormally downregulated and/or in which increased 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 activity is likely to have a beneficial effect. For example, stimulation of 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 activity is desirable in situations in which a 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 is dowmegulated and/or in which increased 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 activity is likely to have a beneficial effect. Likewise, inhibition of 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 activity is desirable in situations in which 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 is abnormally upregulated and/or in which decreased 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 activity is likely to have a beneficial effect.
Pharmacogenomics
The 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 molecules ofthe present invention, as well as agents, or modulators which have a stimulatory or inhibitory effect on 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 activity (e.g, 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 gene expression) as identified by a screening assay described herein can be administered to individuals to treat (prophylactically or therapeutically) 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 associated disorders (e.g, immunological disorders, neurological disorders, metabolic disorders, cellular proliferation and/or differentiation disorders, disorders of metal ion imbalance, protein trafficing disorders, or cardiovascular disorders) associated with abenant or unwanted 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 activity. In conjunction with such treatment, pharmacogenomics (i.e, the study ofthe relationship between an individual's genotype and that individual's response to a foreign compound or drug) may be considered. Differences in metabolism of therapeutics can lead to severe toxicity or therapeutic failure by altering the relation between dose and blood concentration ofthe pharmacologically active drag. Thus, a physician or clinician may consider applying knowledge obtained in relevant pharmacogenomics studies in determining whether to administer a 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 molecule or 47476, 67210, 49875, 46842,
33201, 83378, 84233, 64708, 85041, or 84234 modulator as well as tailoring the dosage and/or therapeutic regimen of treatment with a 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 molecule or 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 modulator. Pharmacogenomics deals with clinically significant hereditary variations in the response to drags due to altered drag disposition and abnormal action in affected persons. See, for example, Eichelbaum, M. et al. (1996) Clin. Exp. Pharmacol. Physiol. 23:983-985 and Linder, M.W. et al. (1997) Clin. Chem. 43:254-266. In general, two types of pharmacogenetic conditions can be differentiated. Genetic conditions transmitted as a single factor altering the way drags act on the body (altered drag action) or genetic conditions transmitted as single factors altering the way the body acts on drugs (altered drag metabolism). These pharmacogenetic conditions can occur either as rare genetic defects or as naturally-occuning polymoφhisms. For example, glucose-6-phosphate dehydrogenase deficiency (G6PD) is a common inherited enzymopathy in which the main clinical complication is haemolysis after ingestion of oxidant drags (anti-malarials, sulfonamides, analgesics, nitrofurans) and consumption of fava beans.
One pharmacogenomics approach to identifying genes that predict drug response, known as "a genome-wide association", relies primarily on a high-resolution map ofthe human genome consisting of already known gene-related markers (e.g, a "bi-allelic" gene marker map which consists of 60,000-100,000 polymoφhic or variable sites on the human genome, each of which has two variants.) Such a high-resolution genetic map can be compared to a map ofthe genome of each of a statistically significant number of patients taking part in a Phase II/III drag trial to identify markers associated with a particular observed drag response or side effect. Alternatively, such a high resolution map can be generated from a combination of some ten- million known single nucleotide polymoφhisms (SNPs) in the human genome. As used herein, a "SNP" is a common alteration that occurs in a single nucleotide base in a stretch of DNA. For example, a SNP may occur once per every 1000 bases of DNA. A SNP may be involved in a disease process, however, the vast majority may not be disease-associated. Given a genetic map based on the occunence of such SNPs, individuals can be grouped into genetic categories depending on a particular pattern of SNPs in their individual genome. In such a manner, treatment regimens can be tailored to groups of genetically similar individuals, taking into account traits that may be common among such genetically similar individuals.
Alternatively, a method termed the "candidate gene approach," can be utilized to identify genes that predict drag response. According to this method, if a gene that encodes a drag's target is known (e.g, a 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 protein ofthe present invention), all common variants of that gene can be fairly easily identified in the population and it can be determined if having one version ofthe gene versus another is associated with a particular drag response.
Alternatively, a method termed the "gene expression profiling," can be utilized to identify genes that predict drug response. For example, the gene expression of an animal dosed with a drug (e.g, a 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 molecule or 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 modulator ofthe present invention) can give an indication whether gene pathways related to toxicity have been turned on.
Information generated from more than one ofthe above pharmacogenomics approaches can be used to determine appropriate dosage and treatment regimens for prophylactic or therapeutic treatment of an individual. This knowledge, when applied to dosing or drag selection, can avoid adverse reactions or therapeutic failure and thus enhance therapeutic or prophylactic efficiency when treating a subject with a 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 molecule or 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 modulator, such as a modulator identified by one ofthe exemplary screening assays described herein. The present invention further provides methods for identifying new agents, or combinations, that are based on identifying agents that modulate the activity of one or more of the gene products encoded by one or more ofthe 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 genes ofthe present invention, wherein these products may be associated with resistance ofthe cells to a therapeutic agent. Specifically, the activity ofthe proteins encoded by the 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 genes ofthe present invention can be used as a basis for identifying agents for overcoming agent resistance. By blocking the activity of one or more ofthe resistance proteins, target cells, e.g, human cells, will become sensitive to treatment with an agent that the unmodified target cells were resistant to.
Monitoring the influence of agents (e.g, drugs) on the expression or activity of a 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 protein can be applied in clinical trials. For example, the effectiveness of an agent determined by a screening assay as described herein to increase 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 gene expression, protein levels, or upregulate 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 activity, can be monitored in clinical trials of subjects exhibiting decreased 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 gene expression, protein levels, or downregulated 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 activity. Alternatively, the effectiveness of an agent determined by a screening assay to decrease 47476, 67210, 49875, 46842, 33201, 83378,
84233, 64708, 85041, or 84234 gene expression, protein levels, or downregulate 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 activity, can be monitored in clinical trials of subjects exhibiting increased 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 gene expression, protein levels, or upregulated 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 activity. In such clinical trials, the expression or activity of a 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 gene, and preferably, other genes that have been implicated in, for example, a 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234-associated disorder can be used as a "read out" or markers ofthe phenotype of a particular cell. 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 Informatics
The sequence of a 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 molecule is provided in a variety of media to facilitate use thereof. A sequence can be provided as a manufacture, other than an isolated nucleic acid or amino acid molecule, which contains a 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234. Such a manufacture can provide a nucleotide or amino acid sequence, e.g, an open reading frame, in a form which allows examination ofthe manufacture using means not directly applicable to examining the nucleotide or amino acid sequences, or a subset thereof, as they exists in nature or in purified form. The sequence information can include, but is not limited to, 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 full-length nucleotide and/or amino acid sequences, partial nucleotide and/or amino acid sequences, polymoφhic sequences including single nucleotide polymoφhisms (SNPs), epitope sequence, and the like. In a prefened embodiment, the manufacture is a machine-readable medium, e.g, a magnetic, optical, chemical or mechanical information storage device. As used herein, "machine-readable media" refers to any medium that can be read and accessed directly by a machine, e.g, a digital computer or analogue computer. Non-limiting examples of a computer include a desktop PC, laptop, mainframe, server (e.g, a web server, network server, or server farm), handheld digital assistant, pager, mobile telephone, and the like. The computer can be stand-alone or connected to a communications network, e.g, a local area network (such as a VPN or intranet), a wide area network (e.g, an Extranet or the Internet), or a telephone network (e.g, a wireless, DSL, or ISDN network). Machine-readable media include, but are not limited to: magnetic storage media, such as floppy discs, hard disc storage medium, and magnetic tape; optical storage media such as CD-ROM; electrical storage media such as RAM, ROM, EPROM, EEPROM, flash memory, and the like; and hybrids of these categories such as magnetic/optical storage media.
A variety of data storage structures are available to a skilled artisan for creating a machine-readable medium having recorded thereon a nucleotide or amino acid sequence ofthe present invention. The choice ofthe data storage structure will generally be based on the means chosen to access the stored information. In addition, a variety of data processor programs and formats can be used to store the nucleotide sequence information ofthe present invention on computer readable medium. The sequence information can be represented in a word processing text file, formatted in commercially-available software such as WordPerfect and Microsoft Word, or represented in the form of an ASCII file, stored in a database application, such as DB2, Sybase, Oracle, or the like. The skilled artisan can readily adapt any number of data processor structuring formats (e.g., text file or database) in order to obtain computer readable medium having recorded thereon the nucleotide sequence information ofthe present invention. In a prefened embodiment, the sequence information is stored in a relational database (such as Sybase or Oracle). The database can have a first table for storing sequence (nucleic acid and/or amino acid sequence) information. The sequence information can be stored in one field (e.g, a first column) of a table row and an identifier for the sequence can be store in another field (e.g, a second column) ofthe table row. The database can have a second table, e.g, storing annotations. The second table can have a field for the sequence identifier, a field for a descriptor or annotation text (e.g, the descriptor can refer to a functionality ofthe sequence, a field for the initial position in the sequence to which the annotation refers, and a field for the ultimate position in the sequence to which the annotation refers. Non-limiting examples for annotation to nucleic acid sequences include polymoφhisms (e.g, SNP's) translational regulatory sites and splice junctions. Non-limiting examples for annotations to amino acid sequence include polypeptide domains, e.g, a domain described herein; active sites and other functional amino acids; and modification sites.
By providing the nucleotide or amino acid sequences ofthe invention in computer readable form, the skilled artisan can routinely access the sequence information for a variety of puφoses. For example, one skilled in the art can use the nucleotide or amino acid sequences of the invention in computer readable form to compare a target sequence or target structural motif with the sequence information stored within the data storage means. A search is used to identify fragments or regions ofthe sequences ofthe invention which match a particular target sequence or target motif. The search can be a BLAST search or other routine sequence comparison, e.g, a search described herein.
Thus, in one aspect, the invention features a method of analyzing 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234, e.g, analyzing structure, function, or relatedness to one or more other nucleic acid or amino acid sequences. The method includes: providing a 47476, 67210, 49875, 6842, 33201, 83378, 84233, 64708, 85041, or 84234 nucleic acid or amino acid sequence; comparing the 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 sequence with a second sequence, e.g, one or more preferably a plurality of sequences from a collection of sequences, e.g, a nucleic acid or protein sequence database to thereby analyze 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234. The method can be performed in a machine, e.g, a computer, or manually by a skilled artisan.
The method can include evaluating the sequence identity between a 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 sequence and a database sequence. The method can be performed by accessing the database at a second site, e.g, over the Internet. As used herein, a "target sequence" can be any DNA or amino acid sequence of six or more nucleotides or two or more amino acids. A skilled artisan can readily recognize that the longer a target sequence is, the less likely a target sequence will be present as a random occunence in the database. Typical sequence lengths of a target sequence are from about 10 to 100 amino acids or from about 30 to 300 nucleotide residues. However, it is well recognized that commercially important fragments, such as sequence fragments involved in gene expression and protein processing, may be of shorter length.
Computer software is publicly available which allows a skilled artisan to access sequence information provided in a computer readable medium for analysis and comparison to other sequences. A variety of known algorithms are disclosed publicly and a variety of commercially available software for conducting search means are and can be used in the computer-based systems ofthe present invention. Examples of such software include, but are not limited to, MacPattern (EMBL), BLASTN and BLASTX (NCBI).
Thus, the invention features a method of making a computer readable record of a sequence of a 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 sequence which includes recording the sequence on a computer readable matrix. In a prefened embodiment the record includes one or more ofthe following: identification of an ORF; identification of a domain, region, or site; identification ofthe start of transcription; identification ofthe transcription terminator; the full length amino acid sequence ofthe protein, or a mature form thereof; the 5' end ofthe translated region. In another aspect, the invention features, a method of analyzing a sequence. The method includes: providing a 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 sequence, or record, in machine-readable form; comparing a second sequence to the 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 sequence; thereby analyzing a sequence. Comparison can include comparing to sequences for sequence identity or determining if one sequence is included within the other, e.g, determining if the 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 sequence includes a sequence being compared. In a prefened embodiment the 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 or second sequence is stored on a first computer, e.g, at a first site and the comparison is performed, read, or recorded on a second computer, e.g, at a second site. E.g, the 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 or second sequence can be stored in a public or proprietary database in one computer, and the results ofthe comparison performed, read, or recorded on a second computer. In a prefened embodiment the record includes one or more ofthe following: identification of an ORF; identification of a domain, region, or site; identification ofthe start of transcription; identification ofthe transcription terminator; the full length amino acid sequence ofthe protein, or a mature form thereof; the 5' end ofthe translated region.
In another aspect, the invention provides a machine-readable medium for holding instructions for performing a method for determining whether a subject has a 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234-associated disease or disorder or a pre-disposition to a 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234-associated disease or disorder, wherein the method comprises the steps of determining 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 sequence information associated with the subject and based on the 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 sequence information, determining whether the subject has a 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234-associated disease or disorder or a pre-disposition to a 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234-associated disease or disorder and/or recommending a particular treatment for the disease, disorder or pre-disease condition.
The invention further provides in an electronic system and/or in a network, a method for determining whether a subject has a 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234-associated disease or disorder or a pre-disposition to a disease associated with a 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 wherein the method comprises the steps of determining 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 sequence information associated with the subject, and based on the 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 sequence information, determining whether the subject has a 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234-associated disease or disorder or a pre-disposition to a 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234-associated disease or disorder, and/or recommending a particular treatment for the disease, disorder or pre-disease condition. In a prefened embodiment, the method further includes the step of receiving information, e.g, phenotypic or genotypic information, associated with the subject and/or acquiring from a network phenotypic information associated with the subject. The information can be stored in a database, e.g, a relational database. In another embodiment, the method further includes accessing the database, e.g, for records relating to other subjects, comparing the 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 sequence ofthe subject to the 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 sequences in the database to thereby determine whether the subject as a 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234-associated disease or disorder, or a predisposition for such.
The present invention also provides in a network, a method for determining whether a subject has a 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 associated disease or disorder or a pre-disposition to a 47476, 67210, 49875, 46842, 33201 , 83378, 84233, 64708, 85041, or 84234-associated disease or disorder associated with 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234, said method comprising the steps of receiving 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 sequence information from the subject and/or information related thereto, receiving phenotypic information associated with the subject, acquiring information from the network conesponding to 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 and/or conesponding to a 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234-associated disease or disorder (e.g, an immunological disorder, neurological disorder, metabolic disorder, cellular proliferation and/or differentiation disorder, disorder of metal ion imbalance, a protein trafficing disorder, or a cardiovascular disorder), and based on one or more of the phenotypic information, the 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 information (e.g., sequence information and/or information related thereto), and the acquired information, determining whether the subject has a 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234-associated disease or disorder or a predisposition to a 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234- associated disease or disorder. The method may further comprise the step of recommending a particular treatment for the disease, disorder or pre-disease condition.
The present invention also provides a method for determining whether a subject has a 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 -associated disease or disorder or a pre-disposition to a 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234-associated disease or disorder, said method comprising the steps of receiving information related to 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 (e.g., sequence information and/or information related thereto), receiving phenotypic information associated with the subject, acquiring information from the network related to 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 and/or related to a 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234-associated disease or disorder, and based on one or more ofthe phenotypic information, the 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 information, and the acquired information, determining whether the subject has a 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234-associated disease or disorder or a pre-disposition to a 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234-associated disease or disorder. The method may further comprise the step of recommending a particular treatment for the disease, disorder or pre-disease condition.
This invention is further illustrated by the following examples that should not be construed as limiting. The contents of all references, patents and published patent applications cited throughout this application are incoφorated herein by reference.
EXAMPLES
Example 1: Identification and Characterization of Human 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, and 84234 cDNAs
The human 47476 nucleic acid sequence is recited as follows: CACCGAGGAGGCCCCAGCTCCCTAGGGGCTGAGAAGCTGGAGTCCTGGGCAAGGG GAGGAGCTGAGCCCTACTCTTGCAAGACCCCCGGCCTCCTCACCCCACGCGGGAAG CATGAACAGAAAAGACAGTAAGAGGAAGTCCCACCAGGAATGCACCGGAAAAAT AGGAGGGCGAGGCCGGCCCCGCCAAGTGCGCCGCCACAAGACATGCCCCAGCCCT CGGGAAATCAGCAAGGTCATGGCTTCCATGAACCTGGGCCTGCTGAGTGAGGGCG GCTGCAGCGAAGATGAGCTGCTGGAGAAATGCATCCAGTCCTTCGATTCAGCTGGC AGCCTGTGCCACGAGGACCACATGCTCAACATGGTGCTGGCCATGCACAGCTGGGT GCTGCCGTCCGCCGACCTGGCTGCCCGCCTGCTGACCTCATACCAGAAGGCCACAG GGGACACCCAGGAGCTGAGACGGCTGCAGATCTGTCACCTGGTCAGGTACTGGCTG ATGCGAC ACCCTGAGGTGATGCACCAGGATCCCC AGCTAGAAGAAGTCATAGGTC GTTTCTGGGCCACCGTGGCCCGGGAGGGCAACTCAGCCCAGAGAAGACTGGGAGA CTCTTCTGACCTCCTGAGCCCTGGTGGCCCTGGCCCCCCACTCCCAATGAGCAGCCC AGGCCTGGGCAAAAAGCGCAAAGTGTCCTTGCTTTTCGACCACTTGGAGACGGGGG AGCTGGCTCAGCACCTCACCTACCTGGAGTTCCGGTCCTTCCAGGCTATCACGCCCC AGGACCTGCGGAGCTACGTTTTGCAGGGCTCAGTACGAGGCTGCCCGGCCCTGGAG GGCTCCGTAGGTCTCAGCAACAGCGTGTCCCGCTGGGTGCAGGTGATGGTGCTGAG CCGTCCCGGGCCCCTACAGCGTGCACAGGTGCTGGACAAGTTCATTCACGTGGCAC AGAGGCTCCACCAGCTGCAGAATTTCAACACGCTGATGGCAGTCACAGGGGGCCTG TGTCACAGTGCCATCTCCAGACTCAAGGACTCCCATGCCCACCTGAGCCCTGACAG CACCAAGGCCCTCCTGGAGCTCACTGAGCTCCTTGCCTCCCACAACAACTACGCCC GCTACCGCCGCACCTGGGCTGGCTGCGCGGGTTTCCGGCTGCCTGTACTGGGCGTG CACCTCAAGGACCTGGTGTCCCTGCATGAGGCACAGCCCGACAGGTTGCCTGACGG CCGCCTGCACCTACCCAAGCTGAACAACCTCTACCTGCGGCTGCAGGAGCTGGTGG CCCTCCAAGGGCAGCATCCACCCTGCAGCGCCAATGAGGATCTGCTGCACCTGCTC ACGCTCTCCCTGGACCTCTTCTACACGGAAGACGAGATCTATGAGCTTTCTTATGCC CGGGAGCCGCGTTGTCCCAAGAGCCTGCCACCCTCCCCCTTCAATGCACCTCTGGT GGTGGAGTGGGCCCCTGGTGTGACACCCAAGCCGGACAGGGTCACACTGGGTCGG CATGTGGAGCAGCTGGTGGAGTCTGTGTTCAAGAATTATGACCCTGAAGGCCGAGG AACAATCTCTCAGGAGGACTTTGAGCGACTCTCGGGCAATTTTCCCTTCGCCTGCCA TGGGCTTCACCCACCCCCACGCCAGGGGAGAGGATCCTTCAGCAGAGAGGAGCTG ACAGGGTACCTGCTCCGGGCCAGCGCCATCTGCTCCAAGTTGGGCCTGGCCTTCCT GCACACCTTCCATGAGGTCACCTTCCGAAAGCCTACCTTCTGCGACAGCTGCAGTG GCTTCCTCTGGGGTGTCACCAAGCAAGGCTACCGCTGTCGGGAGTGCGGGCTGTGT TGCCACAAACACTGCAGAGACCAGGTGAAGGTAGAATGTAAGAAGAGGCCAGGGG CCAAGGGCGATGCAGGACCCCCCGGAGCTCCTGTCCCATCCACACCAGCTCCCCAT GCCAGCTGTGGCTCCGAGGAAAATCACTCCTACACGCTATCCCTGGAGCCTGAGAC TGGGTGCCAGCTTCGCCATGCCTGGACCCAGACTGAATCCCCACACCCTTCCTGGG AAACAGATACGGTCCCCTGCCCGGTGATGGACCCACCATCAACTGCATCCTCCAAG CTGGATTCCTAGACATCTCTTGGTCTCCTCTCTTCCTCACTCCCTTCCCCCAGTCAGT CCTGAGTCCTGCCGTCAGACTCTGGCAGGGCTCCCAGAGGTAGGCCTCCAGAGGCA TCCGCCTTCCCATCCACACTGCCTTTAGTGGTACGTCCGTCATCTTTTTCCCTGGAA GTGACTTTCCTCTTTTGCATCCTGGTGGATGCTAATCCTGCTCCCTTTCCCTGCACTT AGTATCTGTGCGGCTGTGTGTTGGATACATTTAGACTCAGGCCTCATTCTACCAACT CCCTACATCTCCATTTCTTTTTTTTTTTGAGACAGAGTCTTGCTCTGTCACCCAGGCT GGAGTGCAATGATGCAATCTCAGCTCACTGCAACTTCCGCCTCCTGGGTTCAAGTG ATTCTTCTGCCTCAGCCTCCCAAGTAGCTGGGATTACAGGCACGCGCCACCATGCC CGGCTAATTTTTGTATTTTTAGTAGAGACGGGGTTTCCCCATGTTGGCCAAGATGGT CTCAAACTCCTGACCTCATGATCCACCAGCCTCAGCCTCCCAAAGTGCTGGGAATA CAGGCGTGAGCCACCACGCCCAGCTCCTGTTTTTCTGCATAGAATGTGCTCCCTCTG ACAGCCTATACTTTTTCCCTCTTTATCTCGTTGAACGTCTGTCTTTCCTCAACAGGAG CATCAACTCCAGGAGAACAGGGATTTCTATCAATGTCATTCACTGCTGCATCCCCA GTGCCTAGAACAGGGCTGGCAGGTGGTAGGCGCCTGACAGATGCTTGTCTGATGCC TGAATGTCTCCTTACCCATGCCCACGGCACAGGATAGATGTGCTATAGGGCAAAGA ACTTTGAGGGTCGAGCAGCAGGGGCCCATTCAGTCCCAGGGAGCAGAGACCCCCC CAACCCCTTCACAAACCCCAACACCCCTGACTTGGCCCCCACAGAGAGAGGTCCTA CAGCTGTCATAAATTA-AATTTATTCTCTGGAAAAAAAAAAAAAAAAAAA (SEQ ID NO:l).
The human 47476 sequence (SEQ ID NO:l), which is approximately 3134 nucleotides long, includes an initiation codon (ATG) and a termination codon (TAA) which are underscored above. The region between and inclusive ofthe initiation codon and the termination codon is a methionine-initiated coding sequence of about 2022 nucleotides, including the termination codon (nucleotides indicated as "coding" of SEQ ID NO: 1 ; SEQ ID NO:3). The coding sequence encodes a 673 amino acid protein (SEQ ID NO:2), which is recited as follows: MNRKDSKRKSHQECTGKIGGRGRPRQVRRHKTCPSPREISKVMASMNLGLLSEGGCSE DELLEKCIQSFDSAGSLCHEDHMLNMVLAMHSWVLPSADLAARLLTSYQKATGDTQE LRRLQICHLVRYWLMRHPEVMHQDPQLEEVIGRFWATVAREGNSAQRRLGDSSDLLS PGGPGPPLPMSSPGLGKKRKVSLLFDHLETGELAQHLTYLEFRSFQAITPQDLRSYVLQ GSVRGCPALEGSVGLSNSVSRWVQVMVLSRPGPLQRAQVLDKFIHVAQRLHQLQNFN TLMAVTGGLCHSAISRLKDSHAHLSPDSTKALLELTELLASHNNYARYRRTWAGCAGF RLPVLGVHLKDLVSLHEAQPDRLPDGRLHLPKLNNLYLRLQELVALQGQHPPCSANED LLHLLTLSLDLFYTEDEIYELS YAREPRCPKSLPPSPFNAPLVVEWAPGVTPKPDRVTLG RHNEQLNESVFKNYDPEGRGTISQEDFERLSGNFPFACHGLHPPPRQGRGSFSREELTG YLLRASAICSKLGLAFLHTFHEVTFRKPTFCDSCSGFLWGNTKQGYRCRECGLCCHKH CRDQNKNECKKRPGAKGDAGPPGAPVPSTPAPHASCGSEEΝHSYTLSLEPETGCQLRH AWTQTESPHPSWETDTVPCPVMDPPSTASSKLDS (SEQ ID ΝO:2). The human 67210 nucleic acid sequence is recited as follows:
GAATTTGTAATACGACTCACTATAGGGAGTCGACCCACGCGTCCGGCGGGTCGGGG AGGAATATTCTTTTGGAAACGTAATATTGGCCTTGGGGCTCTCCAGCCCTTTGGGAC TTCCAATGGGATCTTAGAAGCAGCCGAAGCAGCGTGAGGGCGGCAGCCCAGGGCC AGCCACGATTTGAACGCTCTGCCTTGCAGCTCTTCTGGACCGAGGAGCCCAAAGCC CTACCCTCACCATTCACCAGGTTACAGTTCTTATCCGCGTGAATACACATGGCTCTG TTACGAAAAATTAATCAGGTGCTGCTGTTCCTTCTGATCGTGACCCTCTGTGTGATT CTGTATAAGAAAGTTCATAAGGGGACTGTGCCCAAGAATGACGCAGATGATGAAT CCGAGACTCCTGAAGAACTGGAAGAAGAGATTCCTGTGGTGATTTGTGCTGCAGCA GGGAGGATGGGTGCCACTATGGCTGCCATCAATAGCATCTACAGCAACACTGACGC CAACATCTTGTTCTATGTAGTGGGACTCCGGAATACTCTGACTCGAATACGAAAAT GGATTGAACATTCCAAACTGAGAGAAATAAACTTTAAAATCGTGGAATTCAACCCG ATGGTCCTCAAAGGGAAGATCAGACCAGACTCATCGAGGCCTGAATTGCTCCAGCC TCTGAACTTTGTTCGATTTTATCTCCCTCTACTTATCCACCAACACGAGAAAGTCAT CTATTTGGACGATGATGTAATTGTACAAGGTGATATCCAAGAACTGTATGACACCA CCTTGGCCCTGGGCCACGCGGCGGCTTTCTCAGATGACTGCGATTTGCCCTCTGCTC AGGACATAAACAGACTCGTGGGACTTCAGAACACATATATGGGCTATCTGGACTAC CGGAAGAAGGCCATCAAGGACCTTGGCATCAGCCCCAGCACCTGCTCTTTCAATCC TGGTGTGATTGTTGCCAACATGACAGAATGGAAGCACCAGCGCATCACCAAGCAAT TGGAGAAATGGATGCAAAAGAATGTGGAGGAAAACCTCTATAGCAGCTCCCTGGG AGGAGGGGTGGCCACCTCCCCAATGCTGATTGTGTTTCATGGGAAATATTCCACAA TTAACCCCCTGTGGCACATAAGGCACCTGGGCTGGAATCCAGATGCCAGATATTCG GAGCATTTTCTGCAGGAAGCTAAATTACTCCACTGGAATGGAAGACATAAACCTTG GGACTTCCCTAGTGTTCACAACGACTTATGGGAAAGCTGGTTTGTTCCTGACCCTGC AGGGATATTTAAACTCAATCACCATAGCTGATATAACTCTACCCTTAAAATATTCC CTGTATAGAAATGTGGAATTGTCCCTTTGTAGCCAACTATAACATTGTTCTTTATGA ATATTACCTTTGATACATATGATCCACAATATAAAAACCAAAAACTACTGTGTGCA AATTATACCTTGGACCATATAGGCATTGATTAACTTCTTTAAGTACATGTGATAACT ATGGAAATCAAGATTATGTGACTGAAAAACATAAAGGAAGAGACCCATCTAGATA ACAGCAATCAACCTGCTTAATTCTGAATGACAATTATATCCACAAATTTTTAAAACT TCTACATGTATTTTTCACATGAAGATCTCCTTAACAGGTTGCCAACCTTTTCTTTTAT AAAACTATTACATTTAAAATATGGACGTCTGAAAAATAAAATATTCATCATTTTTA AAAAAA-AA-AAAAAAΛMAAAAAANAAAAAAAAAAAA (SEQ ID NO:4).
The human 67210 sequence (SEQ ID NO:4), which is approximately 1778 nucleotides long. The nucleic acid sequence includes an initiation codon (ATG) and a termination codon (TGA) which are underscored above. The region between and inclusive ofthe initiation codon and the termination codon is a methionine-initiated coding sequence of about 1050 nucleotides, including the termination codon (nucleotides indicated as "coding" of SEQ ID NO:4; SEQ ID NO:6). The coding sequence encodes a 349 amino acid protein (SEQ ID NO:5), which is recited as follows:
MALLRKTNQNLLFLLINTLCVILYKKVHKGTVPKNDADDESETPEELEEEIPVVICAAAG RMGATMAAΓNSIYSNTDANILFYVVGLRNTLTRIRKWIEHSKLREΓNFKIVEFNPMVLKG KIRPDSSRPELLQPLNFVRFYLPLLIHQHEKVIYLDDDVIVQGDIQELYDTTLALGHAAA FSDDCDLPSAQDRNL^VGLQNTYMGYLDYRK-KAIKDLGISPSTCSFNPGVIVANMTEW KHQRITKQLEKWMQKNVEENLYSSSLGGGVATSPMLIVFHGKYSTINPLWHIRHLGWN PDARYSEHFLQEAKLLHWNGRHKPWDFPSVHNDLWESWFVPDPAGIFKLNHHS (SEQ ID NO:5). The human 49875 nucleic acid sequence is recited as follows: GGCGCGTTCGAGCAGCGGCGACCGACGCGGCGAAGGAGCGCGCCATGGAGCATGT GACAGAGGGCTCCTGGGAGTCGCTGCCTGTGCCGCTGCACCCGCAGGTGCTGGGCG CGCTGCGGGAGCTGGGCTTCCCGTACATGACGCCGGTGCAGTCCGCAACCATCCCT CTGTTCATGCGAAACAAAGATGTCGCTGCAGAAGCGGTCACAGGTAGTGGCAAAA CACTCGCTTTTGTCATCCCCATCCTGGAAATTCTTCTGAGAAGAGAAGAGAAGTTA AAAAAGAGTCAGGTTGGAGCCATAATCATCACCCCCACTCGAGAGCTGGCCATTCA AATAGACGAGGTCCTGTCGCATTTCACGAAGCACTTCCCCGAGTTCAGCCAGATTC TTTGGATCGGAGGCAGGAATCCTGGAGAAGATGTTGAGAGGTTTAAGCAACAAGG TGGGAACATCATTGTGGCCACTCCAGGCCGCTTGGAGGACATGTTCCGGAGGAAGG CCGAAGGCTTGGATCTGGCCAGCTGTGTGCGATCCCTGGATGTCCTGGTGTTGGAT GAGGCAGACAGACTTCTGGACATGGGGTTTGAGGCAAGCATCAACACCATTCTGGA GTTTTTGCCAAAGCAGAGGAGAACAGGCCTTTTCTCTGCCACTCAGACGCAGGAAG TGGAGAACCTGGTGAGAGCGGGCCTCCGGAACCCTGTCCGGGTCTCAGTGAAGGA GAAGGGCGTGGCAGCCAGCAGTGCCCAGAAGACCCCCTCCCGCCTGGAAAACTAC TACATGGTATGCAAGGCAGATGAGAAATTTAATCAGCTGGTCCATTTTCTTCGCAA TCATAAGCAGGAGAAACACCTGGTCTTCTTCAGCACCTGTGCCTGTGTGGAATACT ATGGGAAGGCTCTGGAAGTGCTGGTGAAGGGCGTGAAGATTATGTGCATTCACGG AAAGATGAAATATAAACGCAATAAGATCTTCATGGAGTTCCGCAAATTGCAAAGTG GGATTTTAGTGTGCACTGATGTGATGGCCCGGGGAATTGATATTCCTGAAGTCAAC TGGGTTTTGCAGTATGACCCTCCCAGCAATGCAAGTGCCTTCGTGCATCGCTGCGGT CGCACAGCTCGCATTGGCCACGGGGGCAGCGCTCTGGTGTTCCTCCTGCCCATGGA AGAGTCATACATCAATTTCCTTGCAATTAACCAAAAATGCCCCCTGCAGGAGATGA AGCCCCAGAGAAACACAGCGGACCTTCTGCCAAAACTCAAGTCCATGGCCCTGGCT GACAGAGCTGTGTTTGAAAAGGGCATGAAAGCTTTTGTGTCATATGTCCAAGCTTA TGCAAAGCATGAATGCAACCTGATTTTCAGATTAAAGGATCTTGATTTTGCCAGCCT TGCTCGAGGTTTTGCCCTGCTGAGGATGCCCAAGATGCCAGAATTGAGAGGAAAGC AGTTTCCAGATTTTGTGCCCGTGGACGTTAATACCGACACGATTCCATTTAAAGATA AAATCAGAGAAAAGCAGAGGCAGAAACTCCTGGAGCAACAAAGAAGAGAGAAAA CAGAA-AATGAAGGGAGAAGAAAATTCATAAAAAATAAAGCTTGGTCAAAGCAGAA GGCCA-AA-AAAGAAAAGAAGAAAAAAATGAATGAGAAAAGGAAAAGGGAAGAGG GTTCTGATATTGAAGATGAGGACATGGAAGAACTTCTTAATGACACAAGACTCTTG AAAAAΛCTTAAGAAAGGCAAAATAACTGAAGAAGAATTTGAGAAGGGCTTGTTGA CAACTGGCAAAAGAACAATCAAGACAGTGGATTTAGGGATCTCAGATTTGGAAGA TGACTGCTGATTCCAGTGCCACAGATGAACCCACAAGGACATAGCTGTTCCCTAAC TTGGTGGATGGCTCCAGTTTGCTTTTAACGAAAATCACAACTTCAGGAGACATCTG AAAAGAATGATGTCTCTGAAAGCTGTCCTTTCAGATGAGGGAGAAATGAAGGATTT CACACTTCAGAATATTTTACTAAAAACATTCCAGTCTTGGCCGGGTGCGGTGGCTCC TGCCTATAATCCCAGCACTTTGGGAGGCTGAGGCAGGAGGATCACTTGAGCCCAGG AGTTCAAGACCAGCCTGGGAAC ACAGCGAGACCCTCTC ATTAAAAACAACAAAAC AAAACAATTCCAGTCTTGGAGTAGTCTAACAGAAGAAAATGTAAAATTATTTGAGT GTAAATAATAGATGTCAGTATTTATCATGATGGGTCACATATAGACATATGTHCAT ATTATATATATATATATATATATATATATATATATATATATATATATATATATAAGC TCTTTTTTCTGAGGCTATTTTATAGTTATTTTTAAACATAAAGAYACASAAGTCTTCT TSACTTCTGATTTTCACAAACCATTCCTCAGTATCTTCAGGCATTTGWCCTCCTGAA TGTGCTTGGCCMTGGGCTTCAGTTATCCTTTGATGTCCTGCAGGGGTGGCTAATGTG CTGGGGTTTTTCTGTGTTAATAGTCMCAGTATTGTTTTATTGGTSAATAGCTGAAMA WCAGAGGGATTAAGTCATATTCCGGGAAAGAGAATTATAGTTTTTATGCCTCCTGT TGAATAAATGGTGTCMTGATTGCCTGGGTAAAAAAAAAAAAAAWAAAGGKCGKC CGYTAGACTATTTAGAGAAAAAA (SEQ ID NO:7).
The human 49875 sequence (SEQ ID NO:7), which is approximately 2704 nucleotides long. The nucleic acid sequence includes an initiation codon (ATG) and a termination codon (TGA) which are underscored above. The region between and inclusive ofthe initiation codon and the termination codon is a methionine-initiated coding sequence of about 1803 nucleotides, including the termination codon (nucleotides indicated as "coding" of SEQ ID NO:7; SEQ ID NO:9). The coding sequence encodes a 600 amino acid protein (SEQ ID NO:8), which is recited as follows:
MEHVTEGSWESLPVPLHPQVLGALRELGFPYMTPVQSATIPLFMRNKDVAAEAVTGSG KTLAFVIPILEILLRREEKLKKSQVGAIIITPTRELAIQIDEVLSHFTKHFPEFSQILWIGGR NPGEDVERFKQQGGNIIVATPGRLEDMFRRKAEGLDLASCVRSLDVLVLDEADRLLDM GFEASΓNΉLEFLPKQRRTGLFSATQTQEVENLVRAGLRNPVRVSVKEKGVAASSAQKT PSRLENYYMNCKADEKFNQLNHFLRNHKQEKHLNFFSTCACNEYYGKALENLNKGNK IMCIHGKMKYKRΝKIFMEFRKLQSGILVCTDVMARGIDIPEVΝWVLQYDPPSΝASAFV HRCGRTARIGHGGSALVFLLPMEESYΓΝFLAΓΝQKCPLQEMKPQRΝTADLLPKLKSMAL ADRAVFEKGMKAFVSYVQAYAKHECΝLIFRLKDLDFASLARGFALLRMPKMPELRGK QFPDFVPVDVΝTDTIPFKDKIREKQRQKLLEQQRREKTEΝEGRRKFIKΝKAWSKQKAK KEKKKKMNEKRKREEGSDIEDEDMEELLNDTRLLKKLKKGK1TEEEFEKGLLTTGKRTI KTVDLGISDLEDDC (SEQ ID NO:8).
The human 46842 nucleic acid sequence is recited as follows: CGGTCGGGGCGTTACGGCTGCCGCTCGGCGCCGCGGTCTCGTGCCAGTGAGCGCCG GGCGCCGCAGCCATGACCGTGGAGTTCGAGGAGTGCGTCAAGGACTCCCCGCGCTT CAGGGCGACCATTGACGAGGTGGAGACGGACGTGGTGGAGATTGAGGCCAAACTG GACAAGCTGGTGAAGCTGTGCAGTGGCATGGTGGAAGCCGGTAAGGCCTACGTCA GCACCAGCAGGCTTTTCGTGAGCGGCGTCCGCGACCTGTCCCAGCAGTGCCAGGGC GACACCGTCATCTCGGAATGTCTGCAGAGGTTCGCTGACAGCCTACAGGAGGTGGT GAACTACCACATGATCCTGTTTGACCAGGCCCAGAGGTCCGTGCGGCAGCAGCTCC AGAGCTTTGTCAAAGAGGATGTGCGGAAGTTCAAGGAGACAAAGAAGCAGTTTGA CAAGGTGCGGGAGGACCTGGAGCTGTCCCTGGTGAGGAACGCCCAGGCCCCGAGG CACCGGCCCCACGAGGTGGAGGAAGCCACCGGGGCCCTCACCCTCACCAGGAAGT GCTTCCGCCACCTGGCACTGGACTATGTGCTCCAGATCAATGTTCTGCAGGCCAAG AAGAAGTTTGAGATCCTGGACTCTATGCTGTCCTTCATGCACGCCCAGTCCAGCTTC TTCCAGCAGGGCTACAGCCTCCTGCACCAGCTGGACCCCTACATGAAGAAGCTGGC AGCCGAGCTGGACCAGCTGGTGATCGACTCTGCGGTGGAAAAGCGTGAGATGGAG CGAAAGCACGCCGCCATCCAGCAGCGGACGCTGCTGCAGGACTTCTCCTACGATGA GTCCAAAGTGGAGTTTGACGTGGACGCGCCCAGTGGGGTGGTGATGGAGGGCTAC CTCTTCAAGAGGGCCAGCAACGCTTTCAAGACATGGAACCGGCGCTGGTTCTCCAT TCAGAACAGCCAGCTGGTCTACCAGAAGAAGCTCAAGGATGCCCTCACCGTGGTGG TGGATGACCTCCGCCTGTGCTCTGTGAAGCCGTGTGAGGACATCGAGCGGAGGTTC TGCTTCGAGGTGCTGTCACCCACCAAGAGCTGCATGCTGCAGGCTGACTCCGAGAA GCTGCGGCAAGCCTGGGTCCAGGCTGTGCAGGCCAGCATCGCCTCCGCCTACCGCG AGAGCCCTGACAGTTGCTATAGCGAGAGGCTGGACCGCACAGCATCCCCGTCCACG AGCAGCATCGACTCCGCCACCGACACTCGGGAGCGTGGCGTGAAGGGCGAGAGTG TGCTGCAGCGTGTGCAGAGTGTGGCCGGCAACAGCCAGTGCGGCGACTGCGGCCA GCCGGACCCCCGCTGGGCCAGCATCAACCTGGGCGTGCTGCTCTGCATTGAGTGCT CCGGCATCCACAGGAGCCTGGGTGTCCACTGCTCCAAGGTGCGGTCCCTGACGCTG GACTCGTGGGAGCCTGAGCTGCTAAAGCTGATGTGTGAGCTTGGAAACAGCGCTGT GAATCAGATCTATGAGGCCCAGTGTGAGGGTGCAGGCAGCAGGAAACCCACAGCC AGCAGCTCCCGGCAGGACAAGGAGGCCTGGATCAAGGACAAATACGTGGAAAAGA AGTTTCTGCGGAAGGCGCCCATGGCACCAGCCCTGGAGGCCCCAAGACGCTGGAG GGTGCAGAAGTGCCTGCGGCCCCACAGCTCTCCCCGCGCTCCCACTGCCCGCCGCA AGGTCCGGCTTGAGCCCGTTCTGCCCTGTGTGGCCGCTCTGTCCTCAGTGGGCACCC TGGATCGTAAGTTCCGCCGAGACTCCCTCTTCTGTCCCGACGAGCTGGACTCGCTCT TCTCCTACTTCGACGCAGGGGCCGCAGGGGCTGGCCCTCGCAGTCTGAGTAGCGAC AGTGGCCTTGGGGGCAGCTCGGATGGCAGCTCGGACGTCCTGGCTTTCGGCTCGGG CTCTGTGGTGGACAGCGTCACTGAGGAGGAGGGTGCAGAGTCGGAGGAGTCCAGC GGTGAGGCAGACGGGGACACTGAGGCCGAGGCCTGGGGCCTGGCGGACGTGCGCG AGCTGCACCCGGGGCTCTTGGCGCACCGCGCAGCGCGTGCCCGCGACCTTCCTGCG CTGGCGGCGGCGCTGGCCCACGGGGCCGAGGTCAACTGGGCGGACGCGGAGGATG AGGGCAAGACGCCGCTGGTGCAGGCCGTGCTAGGGGGCTCCTTGATCGTCTGTGAG TTCCTGCTGCAAAACGGAGCGGACGTGAACCAAAGAGACAGCCGGGGCCGGGCGC CCCTGCACCACGCCACGCTGCTGGGCCGCACCGGCCAGGTTTGCCTGTTCCTGAAG CGGGGCGCGGACCAGCACGCCCTGGACCAAGAGCAGCGGGACCCGTTGGCCATCG CAGTGCAGGCGGCCAACGCTGACATCGTGACACTGCTCCGTCTGGCGCGCATGGCG GAGGAAATGCGCGAGGCCGAGGCTGCCCCTGGTCCCCCGGGCGCCCTGGCGGGCA GCCCCACGGAGCTCCAGTTCCGCAGGTGTATCCAGGAGTTCATCAGCCTCCACCTG GAAGAGAGCTAGGGCCGGGCGGGCCGGGCAGCTGCCACCCCGCCCGGCCCGACGC CCCGCATGCCCCGAAGTCCCTGGCGCCCACCCGGCCGCGGCCCTGCGTGTGACCCG CGGGTCGATACCTGGCAGCCCCAGTGCTGGGGCGCCGCGGCCCTGCTCGCCCAGGA GGAGAGCGA (SEQ ID NO: 10).
The human 46842 sequence (SEQ ID NO:10), which is approximately 2737 nucleotides long. The nucleic acid sequence includes an initiation codon (ATG) and a termination codon (TAG) which are underscored above. The region between and inclusive ofthe initiation codon and the termination codon is a methionine-initiated coding sequence of about 2505 nucleotides, including the termination codon (nucleotides indicated as "coding" of SEQ ID NO: 10; SEQ ID NO: 12). The coding sequence encodes a 834 amino acid protein (SEQ ID NO:l 1), which is recited as follows:
MTVEFEECVKDSPRFRATIDEVETDVVEIEAKLDKLVKLCSGMVEAGKAYVSTSRLFV SGVRDLSQQCQGDTVISECLQRFADSLQEVVNYHMILFDQAQRSVRQQLQSFVKEDVR KFKETKKQFDKVREDLELSLVRNAQAPRHRPHEVEEATGALTLTRKCFRHLALDYVLQ INVLQAKKKFEILDSMLSFMHAQSSFFQQGYSLLHQLDPYMKKLAAELDQLVIDSAVE OtEMEPvKHAAIQQRTLLQDFSYDESKVEFDVDAPSGVVMEGYLFKRASNAFKTWNRR WFSIQNSQLVYQKKLKDALTVVVDDLRLCSVKPCEDIERRFCFEVLSPTKSCMLQADSE KLRQAWVQAVQASIASAYRESPDSCYSERLDRTASPSTSSIDSATDTRERGVKGES VLQ RVQSVAGNSQCGDCGQPDPRWASLNLGVLLCIECSGIHRSLGVHCSKVRSLTLDSWEPE LLM-MCELGNSAVNQIYEAQCEGAGSl^KPTASSSRQDKEAWIKDKYVEK-KFLRKAPM APALEAPΪ-RWRVQKCLRPHSSPRAPTARRKVRLEPVLPCVAALSSVGTLDRKFRRDSLF CPDELDSLFSYFDAGAAGAGPRSLSSDSGLGGSSDGSSDVLAFGSGSVVDSVTEEEGAE SEESSGEADGDTEAEAWGLADVRELHPGLLAHRAA-RARDLPALAAALAHGAEVNWA DAEDEGKTPLVQAVLGGSLIVCEFLLQNGADVNQRDSRGRAPLHHATLLGRTGQVCLF LKRGADQHALDQEQRDPLAIAVQAANADIVTLLRLARMAEEMREAEAAPGPPGALAG SPTELQFRRCIQEFISLHLEES (SEQ ID NO: 11).
The human 33201 nucleic acid sequence is recited as follows: CACGCGTCCGGGCCTGGGGGCGAGCTGGGGTCGTGCAGTACAGCCTCTTTCCGGCA AATCACGCGAGATTTCGTTCACCCGGGCTCCACACGGAGTATTTTATACAGAAATC TTGTGAAACCACTGCCCAACCAGAGCAATGATTGTTCAAAGAGTGGTATTGAATTC TCGACCTGGAAAAAATGGTAATCCAGTGGCAGAGAATTTCCGAATGGAAGAAGTC TATTTACCAGATAATATTAATGAAGGACAAGTACAAGTTAGAACTCTTTATCTTTCT GTGGATCCTTACATGCGTTGTAGAATGAATGAAGACACTGGCACTGATTATATAAC ACCTTGGCAGCTATCTCAAGTCGTTGATGGTGGAGGTATTGGAATTATAGAAGAAA GCAAACACACAAATTTGACTAAAGGCGATTTTGTGACTTCTTTCTATTGGCCCTGGC AAACCAAGGTTATTCTGGATGGAAATAGCCTTGAAAAGGTAGACCCACAACTTGTG GATGGACACCTTTCATATTTTCTTGGAGCTATAGGTATGCCTGGTTTGACTTCCTTG ATTGGGATACAGGAAAAAGGTCATATAACTGCTGGATCTAATAAGACAATGGTTGT CAGTGGGGCCGCAGGTGCCTGTGGATCTGTGGCTGGGCAGATTGGCCATTTCTTAG GTTGTTCCAGAGTGGTGGGAATTTGTGGAACACATGAGAAATGCATCCTCTTGACC TCAGAACTGGGCTTTGATGCTGCAATTAATTATAAAAAAGACAATGTGGCAGAACA GCTCCGTGAATCATGCCCAGCTGGAGTGGATGTTTATTTTGACAATGTTGGTGGTAA CATCAGTGATACAGTGATAAGTCAGATGAATGAGAACAGCCACATCATCCTGTGTG GTCAAATTTCTCAGTACAACAAAGATGTGCCTTATCCTCCCCCGCTATCCCCTGCTA TAGAGGCAATCCAGAAAGAAAGAAACATCACAAGGGAAAGATTTCTGGTATTAAA TTATAAAGACAAATTTGAGCCTGGCATTCTACAGCTGAGTCAGTGGTTTAAAGAAG GAAAGCTAAAGATTAAAGAGACGGTAATAAATGGGTTGGAAAACATGGGAGCTGC ATTCCAGTCCATGATGACAGGAGGTAACATTGGAAAGCAGATAGTTTGCATTTCAG AAGAAATCTCTTTGTAATTGCTGTAAATGTCATCAAGGC AATCATAGATTTCTTTTC CATTTTGCATATTTTCAAAGATATGTTAAAAAATCCTTAGACTATACATAGCTCTTG ATTTAAATGTGATCATAGGTGTTATTTTTAGTTGCATAGGGTATTTGATACAATCAT TAATGGATCATACACAATAGGTTTTTAAAAATTAATAACTTTTAGTAATTACTTTTA TTAATTTAAAATAGAACACTTGAGAGGCACTTTGTAAAGATTTGTTAAACTGGAAA CGTTTTACATGATCTGATACAACCATTAATGAATCATACACAATAGGTTTTTTAAAA TTAATATTAATAACTTTTATTAATTTAAAATAGAATGCTTAAAATAAAATAGAATGC TTGAGAGGCACTGAGTAAAGATTTGTTGAACTGGAAATGTTTTACATGATTTTTAA ACTGAAACTTGGTGTAAAAATAGAATTGAGATGGCCTTTTTTTCACATTGTAGACTG AAAAGAGACTTAATGGTATGATGTGTACC (SEQ ID NO: 13). The human 33201 sequence (SEQ ID NO: 13), which is approximately 1718 nucleotides long. The nucleic acid sequence includes an initiation codon (ATG) and a termination codon (TAA) which are underscored above. The region between and inclusive ofthe initiation codon and the termination codon is a methionine-initiated coding sequence of about 1056 nucleotides,
DKFEPGILQLSQWFKEGKLKIKETN1NGLENMGAAFQSMMTGGNIGKQINCISEEISL (SEQ ID NO: 14).
The human 83378 nucleic acid sequence is recited as follows: GCCCTTATGGGCCGCTACTCTGGCAAGACGTGCCGGCTGCTCTTCATGCTGGTGCTC ACCGTCGCCTTCTTCGTGGCGGAGCTGGTCTCCGGCTACCTGGGCAACTCCATCGCG CTGCTCTCCGACTCCTTCAACATGCTCTCCGACCTGATCTCGCTGTGCGTGGGCCTG AGCGCCGGCTACATCGCCCGGCGCCCCACCCGGGGCTTCAGCGCCACCTACGGCTA CGCCCGCGCCGAGGTGGTGGGCGCGCTGAGCAACGCGGTCTTCCTCACCGCGCTCT GCTTCACCATCTTCGTGGAGGCCGTGCTGCGCCTGGCCCGGCCCGAGCGCATCGAT GACCCCGAGCTGGTGCTCATCGCCGGCGTCCTGGGGCTGTTGGTCAACGTGGTGGG GCTGCTCATCTTCCAGGACTGCGCCGCCTGGTTCGCGTGCTGCCTCCGGGGACGCA GTCGCCGCCTGCAGCAGCGGCAGCAGCTGGCGGAGGGCTGTGTCCCCGGCGCTTTC GGGGGGCCTCAGGGCGCGGAGGACCCGCGGCGCGCGGCGGACCCGACAGCCCCAG GCTCGGACTCGGCCGTAACCCTCCGGGGGACCTCGGTGGAAAGGAAGCGGGAGAA GGGGGCGACCGTGTTCGCAAACGTAGCAGGTGATTCCTTCAACACCCAGAATGAGC CAGAAGACATGATGAAAAAAGAGAAAAAGTCTGAAGCTCTGAATATCAGAGGTGT ACTTTTGCATGTGATGGGAGATGCCCTGGGGTCCGTGGTTGTGGTCATCACGGCCA TCATATTCTATGTGCTTCCCCTGAAGAGTGAGGACCCGTGTAACTGGCAGTGTTACA TTGACCCCAGCCTGACTGTCCTCATGGTCATCATCATTTTGTCATCTGCCTTCCCGCT TATCAAGGAGACCGCTGCCATTCTGCTACAGATGGTCCCAAAAGGAGTCAACATGG AAGAGCTGATGAGTAAACTCTCTGCTGTGCCTGGAATTAGCAGTGTACATGAAGTG CACATCTGGGAACTTGTAAGTGGAAAGATTATTGCCACCCTGCACATCAAGTATCC TAAGGACAGGGGATATCAAGATGCCAGCACAAAAATTCGAGAAATCTTCCACCAT GCGGGAATCCACAATGTGACCATCCAGTTTGAAAATGTGGACTTGAAGGAACCCCT GGAGCAGAAGGACTTACTGTTGCTCTGCAACTCACCCTGCATCTCCAAGGGCTGTG CTAAGCAGCTGTGTTGTCCCCCCGGGGCACTGCCTCTGGCTCACGTCAATGGCTGTG CTGAGCAAAATGGTGGGCCCTCTCTAGACACATACGGAAGTGATGGCCTCAGTAGA AGAGACGCAAGAGAAGTGGCTATTGAAGTGTCTTTGGATAGCTGTCTGAGTGACCA CGGACAAAGTCTTAACAAAACTCAGGAGGACCAATGTTATGTCAACAGAACGCATT TTTAATCTGGTACTCACATAATCAGACCATATAGACGAGGCACTTTGGAACCACAA GCTTGGCTCACAAAAAGAGCTTTCTGGGTTGTAGGCCCAGACTAGACTTGCAGCAT GCATGCTCTGTGTTCACTAGGGGTTGGCTGTTTGGGATTTTAGTTAAACGTGTCTGT GAATTTTTATTGTTAACTAACTCCTTTCCATTCCCCTGGGTGTCTCATGCTGCTCTTT GACTGTTTCAGCTTGAACATGCATTTTCTAAAGCAAACTGCACTAGTGTATATATCA GGGACATTAAAGTCTGGGACTGGGGCTCAATAAAAAAAAAAAAAAAAAAAAAAA AAAAAAAAAAA-AAAAAAAAAGGGCGGCCGC (SEQ ID NO: 16).
The human 83378 sequence (SEQ ID NO: 16) is approximately 1827 nucleotides long. The nucleic acid sequence includes an initiation codon (ATG) and a termination codon (TAA) which are underscored above. The region between and inclusive ofthe initiation codon and the termination codon is a methionine-initiated coding sequence of about 1458 nucleotides, including the termination codon (nucleotides indicated as "coding" of SEQ ID NO : 16; SEQ ID NO: 18). The coding sequence encodes a 485 amino acid protein (SEQ ID NO: 17), which is recited as follows:
MGRYSGKTCRLLFMLVLTVAFFVAELVSGYLGNSIALLSDSFNMLSDLISLCVGLSAGY IARRPTRGFSATYGYARAEVVGALSNAVFLTALCFTIFVEAVLRLARPERIDDPELVLIA GVLGLLVNVVGLL1FQDCAAWFACCLRGRSRRLQQRQQLAEGCVPGAFGGPQGAEDP i AADPTAPGSDSAVTLRGTSVERKREKGATVFANVAGDSFNTQNEPEDMMKKEKKS EALNIRGVLLHVMGDALGSVVVVITAIIFYVLPLKSEDPCNWQCYIDPSLTVLMVIIILSS AFPLIKETAAILLQMNPKGVNMEELMSKLSANPGISSNHEVHIWELVSGKIIATLHIKYP KDRGYQDASTKIREIFHHAGIHΝVTIQFEΝVDLKEPLEQKDLLLLCΝSPCISKGCAKQLC CPPGALPLAHVΝGCAEQΝGGPSLDTYGSDGLSRRDAREVAIEVSLDSCLSDHGQSLΝK TQEDQCYVΝRTHF (SEQ ID NO: 17).
The human 84233 nucleic acid sequence is recited as follows: CCGGGCAGGTACGCGGGGAAGCTCTTGAGCTCCTCTACCTCTTAGAAAGCACAATT GAATCAGATATCATATGAAAGACATACACACTTCATGTAATGCTACCTGCGAGTCT CCCTAGAAAAGCAGTTTTTGTAGGTGAAAACAATGAAGCCAGGTAATATTGCAAGG AGGCTGTAATTTTAGCAGACCTACCAACAACACTGATGTAGGAAGCTCATTATTTT AATTTCTGGAGCCTTTTAATTTTTTCTTTAGAAAGTGTATAAATAATTGCAGTGCTG CTTTGCTTCCAAAACTGGGCAGTGAGTTCAACAACAACGACAACAACAGCCGCAGC TCATCCTGGCCGTCATGGAGTTTCTTGAAAGAACGTATCTTGTGAATGATAAAGCT GCCAAGATGTATGCTTTCACACTAGAAAGGAGCTGCAAATGAACACTTCATAGCAA TGTGGAACTCCAACAGAAACCGGTGAATAAAGATCAGTGTCCCAGAGAGAGACCA GAGGAGCTGGAGTCAGGAGGCATGTACCACTGCCACAGTGGCTCCAAGCCCACAG AAAAGGGGGCGAATGAGTACGCCTATGCCAAGTGGAAACTCTGTTCTGCTTCAGCA ATATGCTTCATTTTCATGATTGCAGAGGTCGTGGGTGGGCACATTGCTGGGAGTCTT GCTGTTGTCACAGATGCTGCCCACCTCTTAATTGACCTGACCAGTTTCCTGCTCAGT CTCTTCTCCCTGTGGTTGTCATCGAAGCCTCCCTCTAAGCGGCTGACATTTGGATGG CACCGAGCAGAGATCCTTGGTGCCCTGCTCTCCATCCTGTGCATCTGGGTGGTGACT GGCGTGCTAGTGTACCTGGCATGTGAGCGCCTGCTGTATCCTGATTACCAGATCCA GGCGACTGTGATGATCATCGTTTCCAGCTGCGCAGTGGCGGCCAACATTGTACTAA CTGTGGTTTTGCACCAGAGATGCCTTGGCCACAATCACAAGGAAGTACAAGCCAAT GCCAGCGTCAGAGCTGCTTTTGTGCATGCCCTTGGAGATCTATTTCAGAGTATCAGT GTGCTAATTAGTGCACTTATTATCTACTTTAAGCCAGAGTATAAAATAGCCGACCC AATCTGCACATTCATCTTTTCCATCCTGGTCTTGGCCAGCACCATCACTATCTTAAA GGACTTCTCCATCTTACTCATGGAAGGTGTGCCAAAGAGCCTGAATTACAGTGGTG TGAAAGAGCTTATTTTAGCAGTCGACGGGGTGCTGTCTGTGCACAGCCTGCACATC TGGTCTCTAACAATGAATCAAGTAATTCTCTCAGCTCATGTTGCTACAGCAGCCAGC CGGGACAGCCAAGTGGTTCGGAGAGAAATTGCTAAAGCCCTTAGCAAAAGCTTTAC GATGCACTCACTCACCATTCAGATGGAATCTCCAGTTGACCAGGACCCCGACTGCC TTTTCTGTGAAGACCCCTGTGACTAGCTCAGTCACACCGTCAGTTTCCCAAATTTGA CAGGCCACCTTCAAACATGCTGCTATGCAGTTTCTGCATCATAGAAAATAAGGAAC CAAAGGAAGAAATTCATGTCATGGTGCAATGCATATTTTATCTATTTATTTAGTTGC ATTCACCATGAAGGAAGAGGCACTGAGATCCATCAATCAATTGGATTATATACTGA TCAGTAGCTGTGTTCAATTGCAGGAATGTGTATATAGATTATTCCTGAGTGGAGCC GAAGTAACAGCTGTTTGTAACTATCGGCAATACCAAATTCATCTCCCTTCCAATAAT GCATCTTGAGAACACATAGGTAAATTTGAACTCAGGAAAGTCTTACTAGAAATCAG TGGAAGGGACAAATAGTCACAAAATTTTACCAAAACATTAGAAACAAAAAATAAG GAGAGCCAAGTCAGGAATAAAAGTGACTCTGTATGCTAACGCCACATTAGAACTTG GTTCTCTCACCAAGCTGTAATGTGATTTTTTTTTCTACTCTGAATTGGAAATATGTAT GAATATACAGAGAAGTGCTTACAACTAATTTTTATTTACTTGTCACATTTTGGCAAT AAATCCCTCTTATTTCTAAAA-AAAAAAAAAAA-AAAAAAΛAAAAAAA-AAAAAAAAA AAAA-AAAAAAAAAAAAAAAAA-AAAAAA (SEQ ID NO:19). The human 84233 sequence (SEQ ID NO: 19) is approximately 2165 nucleotides long. The nucleic acid sequence includes an initiation codon (ATG) and a termination codon (TAG) which are underscored above. The region between and inclusive ofthe initiation codon and the termination codon is a methionine-initiated coding sequence of about 963 nucleotides, including the termination codon (nucleotides indicated as "coding" of SEQ ID NO:19; SEQ ID NO:21). The coding sequence encodes a 320 amino acid protein (SEQ ID NO:20), which is recited as follows:
MYHCHSGSKPTEKGANEYAYAKWKLCSASAICFIFMIAEVVGGHIAGSLAVVTDAAHL LIDLTSFLLSLFSLWLSSKPPSKRLTFGWHRAEILGALLSILCIWVVTGVLVYLACERLLY PDYQIQATNMIINSSCANAANINLTNVLHQRCLGHNHKEVQANASVRAAFVHALGDLF QSISVLISALIIYFKPEYKIADPICTFIFSILVLASTITILKDFSILLMEGVPKSLNYSGVKELI LAVDGVLSVHSLHIWSLTMNQVILSAHVATAASRDSQVVRREIAKALSKSFTMHSLTIQ MESPVDQDPDCLFCEDPCD (SEQ ID NO:20).
The human 64708 nucleic acid sequence is recited as follows: GCACGAGGGCGGGAGCTGTGCAGCTCCTTATCATGGGGACAATTCATCTCTTTCGA AAACCACAAAGATCCTTTTTTGGCAAGTTGTTACGGGAATTTAGACTTGTAGCAGC TGACCGAAGGTCCTGGAAGATACTGCTCTTTGGTGTAATAAACTTGATATGTACTG GCTTCCTGCTTATGTGGTGCAGTTCTACTAATAGTATAGCTTTAACTGCCTATACTT ACCTGACCATTTTTGATCTTTTTAGTTTAATGACATGTTTAATAAGTTACTGGGTAA CATTGAGGAAACCTAGCCCTGTCTATTCATTTGGGTTTGAAAGATTAGAAGTCCTG GCTGTATTTGCCTCCACAGTCTTGGCACAGTTGGGAGCTCTCTTTATATTAAAAGAA AGTGCAGAACGCTTTTTGGAACAGCCCGAGATACACACGGGAAGATTATTAGTTGG TACTTTTGTGGCTCTTTGTTTCAACCTGTTCACGATGCTTTCTATTCGGAATAAACCT TTTGCTTATGTCTCAGAAGCTGCTAGTACGAGCTGGCTTCAAGAGCATGTTGCAGAT CTTAGTCGAAGCTTGTGTGGAATTATTCCGGGACTTAGCAGTATCTTCCTTCCCCGA ATGAATCCATTTGTTTTGATTGATCTTGCTGGAGCATTTGCTCTTTGTATTACATATA TGCTCATTGAAATTAATAATTATTTTGCCGTAGACACTGCCTCTGCTATAGCTATTG CCTTGATGACATTTGGCACTATGTATCCCATGAGTGTGTACAGTGGGAAAGTCTTAC TCCAGACAACACCACCCCATGTTATTGGTCAGTTGGACAAACTCATCAGAGAGGTA TCTACCTTAGATGGAGTTTTAGAAGTCCGAAATGAACATTTTTGGACCCTAGGTTTT GGCTCATTGGCTGGATCAGTGCATGTAAGAATTCGACGAGATGCCAATGAACAAAT GGTTCTTGCTCATGTGACCAACAGGCTGTACACTCTAGTGTCTACTCTAACTGTTCA AATTTTCAAGGATGACTGGATTAGGCCTGCCTTATTGTCTGGGCCTGTTGCAGCCAA TGTCCTAAACTTTTCAGATCATCACGTAATCCCAATGCCTCTTTTAAAGGGTACTGA TGATTTGAACCCAGTTACATCAACTCCAGCTAAACCTAGTAGTCCACCTCCAGAATT TTCATTTAACACTCCTGGGAAAAATGTGAACCCAGTTATTCTTCTAAACACACAAA CAAGGCCTTATGGTTTTGGTCTCAATCATGGACACACACCTTACAGCAGCATGCTTA ATCAAGGACTTGGAGTTCCAGGAATTGGAGCAACTCAAGGATTGAGGACTGGTTTT ACAAATATACCAAGTAGATATGGAACTAATAATAGAATTGGACAACCAAGACCAT GATAGACTCTAACTTATTTTTATAAGGAATATTGACTCCTTGGCTTCCAATTTATTT AGTAATCCAACTTTGCATTGACTGTTTAATCATTTACTCTAAATGTTAGATAATAGT AGTCTTGTTCACATTTCATGAAACCTATGAAACTATATTTTTGTAAAATGTATTTGT GACAGTGAAATCCTCGTAAATGTTAAAGGCTTTAAATAGGCTTCCTTTAGAAAATG TGTTTCTTTAAATTTGGATTTTGGTATCTTTGGTTTTGTAGTTGACTGCAGTGTGATG TGACCTTACCTTTATAAGAGCCACTTGATGGAGTAGATCTGTCACATTACTAAGATA CGATATTTCTTTTTTTTTCCGAGACGGAGTCTTGCTCTGCCACTGTGCCCGGCCAAT ACATTATTATTAACTTAAGGCTGTACTTTATTAAGGCTTCCTTAGTTTTTGTTTTGTT TTGTTTTTTGAGATGGAGTCTCACTCTGTCGCCCAGGCTGGAATGCAGTGGCATGAT CTCAGCTCACTGCAACCTCTGCCTCCTGAGGCAGGAGAGTTGCTTGAACCTGGGGG GCGGAGGTTGCAGTGAGCCACTGCACTCCAGCCTGATGACAGAGCAAGACTCAGTC TCAAA-AATAAATA-AAAATAATAAAACCTCCATAAGTAATCCTGAAAAAAAAAAAA AAAAAAAAATCCGAGGGGGGGCGCCGGTACCCA (SEQ ID NO:22).
The human 64708 sequence (SEQ ID NO:22) is approximately 2130 nucleotides long. The nucleic acid sequence includes an initiation codon (ATG) and a termination codon (TGA) which are underscored above. The region between and inclusive ofthe initiation codon and the termination codon is a methionine-initiated coding sequence of about 1386 nucleotides, including the termination codon (nucleotides indicated as "coding" of SEQ ID NO:22; SEQ ID NO:24). The coding sequence encodes a 461 amino acid protein (SEQ ID NO:23), which is recited as follows: MGTIHLFRKPQRSFFGKLLREFRLNAADRRSWKILLFGN1NLICTGFLLMWCSSTNSIAL TAYTYLTIFDLFSLMTCLISYWVTLRKPSPVYSFGFERLEVLAVFASTVLAQLGALFILK ESAERFLEQPEIHTGRLLVGTFVALCFNLFTMLSIRNKPFAYVSEAASTSWLQEHVADLS RSLCGIIPGLSSIFLPRMNPFNLIDLAGAFALCITYMLIErNNYFANDTASAIAIALMTFGT MYPMSNYSGKNLLQTTPPHNIGQLDKLIRENSTLDGNLENRΝEHFWTLGFGSLAGSNH NRIRRDAΝEQMVLAHVTΝRLYTLVSTLTVQIFKDDWIRPALLSGPVAAΝVLΝFSDHHV IPMPLLKGTDDLΝPVTSTPAKPSSPPPEFSFΝTPGKΝVΝPVILLΝTQTRPYGFGLΝHGHT PYSSMLΝQGLGVPGIGATQGLRTGFTΝIPSRYGTΝΝRIGQPRP (SEQ ID ΝO:23). The human 85041 nucleic acid sequence is recited as follows: TTGCTGGGCCTGATGACGTGGCTTGGCAACGTCCCTACCGCCGCTGCTTCCCGGGA ACCTGGCGCCGCCGGAACTGATCGCGGCCTAGTCCCGACGCGTGTGTGCTAGTGAG CCGGAGCCGGCGACGGCGGCAGTGGCGGCCCGGCCTGCAGGAGCCCGACGGGGTC TCTGCCATGGGGGAGTGACGCGCCTGCACCCGCTGTTCCGCGGCAGCGGCGAGACA TGAGGAGACCCCGCGACAGGGGCAGCGGCGGCGGCTCGTGAGCCCCGGGATGGAG GAGAAATACGGCGGGGACGTGCTGGCCGGCCCCGGCGGCGGCGGCGGCCTTGGGC CGGTGGACGTACCCAGCGCTCGATTAACAAAATATATTGTGTTACTATGTTTCACTA AATTTTTGAAGGCTGTGGGACTTTTCGAATCATATGATCTCCTAAAAGCTGTTCACA TTGTTCAGTTCATTTTTATATTAAAACTTGGGACTGCATTTTTTATGGTTTTGTTTCA AAAGCCATTTTCTTCTGGGAAAACTATTACCAAACACCAGTGGATCAAAATATTTA AACATGCAGTTGCTGGGTGTATTATTTCACTCTTGTGGTTTTTTGGCCTCACTCTTTG TGGACCACTAAGGACTTTGCTGCTATTTGAGCACAGTGATATTGTTGTCATTTCACT ACTCAGTGTTTTGTTCACCAGTTCTGGAGGAGGACCAGCAAAGACAAGGGGAGCTG CTTTTTTCATTATTGCTGTGATCTGTTTATTGCTTTTTGACAATGATGATCTCATGGC TAAAATGGCTGAACACCCTGAAGGACATCATGACAGTGCTCTAACTCATATGCTTT ACACAGCCATTGCCTTCTTAGGTGTGGCAGATCACAAGGGTGGAGTATTATTGCTA GTACTGGCTTTGTGTTGTAAAGTTGGTTTTCATACAGCTTCCAGAAAGCTCTCTGTC GACGTTGGTGGAGCTAAACGTCTTCAAGCTTTATCTCATCTTGTTTCTGTGCTTCTCT TGTGCCCATGGGTCATTGTTCTTTCTGTGACAACTGAGAGTAAAGTGGAGTCTTGGT TTTCTCTCATTATGCCTTTTGCAACGGTTATCTTTTTTGTCATGATCCTGGATTTCTA CGTGGATTCCATTTGTTCAGTCAAAATGGAAGTTTCCAAATGTGCTCGTTATGGATC CTTTCCCATTTTTATTAGTGCTCTCCTTTTTGGAAATTTTTGGACACATCCAATAACA GACCAGCTTCGGGCTATGAACAAAGCAGCACACCAGGAGAGCACTGAACACGTCC TGTCTGGAGGAGTGGTAGTGAGTGCTATATTCTTCATTTTGTCTGCCAATATCTTAT CATCTCCCTCTAAGAGAGGACAAAAAGGTACCCTTATTGGATATTCTCCTGAAGGA ACACCTCTTTATAACTTCATGGGTGATGCTTTTCAGCATAGCTCTCAATCGATCCCT AGGTTTATTAAGGAATCACTAAAACAAATTCTTGAGGAGAGTGACTCTAGGCAGAT CTTTTACTTCTTGTGCTTGAATCTGCTTTTTACCTTTGTGGAATTATTCTATGGCGTG CTGACCAATAGTCTGGGCCTGATCTCGGATGGATTCCACATGCTTTTTGACTGCTCT GCTTTAGTCATGGGACTTTTTGCTGCCCTGATGAGTAGGTGGAAAGCCACTCGGATT TTCTCCTATGGGTACGGCCGAATAGAAATTCTGTCTGGATTTATTAATGGACTTTTT CTAATAGTAATAGCGTTTTTTGTGTTTATGGAGTCAGTGGCTARATTGATTGATCCT CCAGAATTAGACACTCACATGTTAACACCAGTYTCAGTTGGAGGGCTGATAGTAAA CCTTATTGGTATCTGTGCCTTTAGCCATGCCCATAGCCATGCCCATGGAGCTTCTCA AGGAAGCTGTCACTC ATCTGATC AC AGCCATTC ACAYCATATGC ATGGAC AC AGTG ACCATGGGCATGGTCACAGCCACGGATCTGCGGGTGGAGGCATGAATGCTAACAT GAGGGGTGTATTTCTACATGTTTTGGCAGATACACTTGGCAGCATTGGTGTGATCGT ATCCACAGTTCTTATAGAGCAGTTTGGATGGTTCATCGCTGACCCACTCTGTTCTCT TTTTATTGCTATATTAATATTTCTCAGTGTTGTTCCACTGATTAAAGATGCCTGCCAG GTTCTACTCCTGAGATTGCCACCAGAATATGAAAAAGAACTACATATTGCTTTAGA AAAGGTACAGGAAATTGAAGGATTAATATCATACCGAGACCCTCATTTTTGGCGTC ATTYTGCTAGTATTGTGGCAGGAACAATTCATATACAGGTGACATYTGATGTGCTA GAACAAAGAATAGTACRGCAGGTTACAGGAATACTTAAAGATGCTGGAGTAAACA ATTTAACAATTCAAGTGGAAAAGGAGGCATACTTTCAACATATGTCTGGCCTAAGT ACTGGATTTCATGATGTTCTGGCTATGACAAAACAAATGGAATCCATGAAATACTG CAAAGATGGTACTTACATCATGTGAGATAACTCAAGRATTACCCCTGGRGRATAAA CAATGAAGRTTAAATGACTCAGTATTTGTAATATTGCCAGAAGGATAAAAATTACA CATTAACTGTACAGAAACAGAGTTCCCTACTACTGGATCAAGGAATCTTTCTTGAA GGAAATTTAAATACAGAATGAAACATTAATGGTAAAAGTGGAGTAATTATTTAAAT TATGTGTATAAAAGGAATCAAATTTTGAGTAAACATGATGTATTACATCATCTTCA AAAATAGATATGATGGATTCTAGTGAAGACCAAAATTACTTCTGTTTACTTTCTATC AGGAAGCATCTCCATTGTAAATATGTATTTACATGTTTATTACAAAGACCCAAATG AAAAATTTTTAGTCCATTTTTTGCATAGCCTAAAGATAAAATAGGAATAAAAGTTC TATATTTATGGGATTTTCTGTATATAAAACTGGTTTCTAATTATAACTTAAGTCCATT AAGTAAAATCTGTATTGCCACTTTAAATGTAAACTAAATTATTTGGGAGAAACTTC AACCACTGATATGAGATAAGCAATGAGAATAGGGAAGTGTATAACATCACAGTTTT TGATGTATTACAAAAATCAACCACTTTATAAAATAAATTTTTTTTACTTTTGGTAAA AAAAAAAAAAAA-AAAAAAAAAAAAAAAAAAAAAAAAAAGCGGCCGCTGAATTCT AGNTAGAATTCAGCGGCCGCTGAATNCTA (SEQ ID NO:25).
The human 85041 sequence (SEQ ID NO:25) is approximately 3304 nucleotides long. The nucleic acid sequence includes an initiation codon (ATG) and a termination codon (TGA) which are underscored above. The region between and inclusive ofthe initiation codon and the termination codon is a methionine-initiated coding sequence of about 2298 nucleotides, including the termination codon (nucleotides indicated as "coding" of SEQ ID NO:25; SEQ ID NO:27). The coding sequence encodes a 765 amino acid protein (SEQ ID NO:26), which is recited as follows:
MEEKYGGDVLAGPGGGGGLGPNDNPSARLTKYINLLCFTKFLKAVGLFESYDLLKAN HINQFIFILKLGTAFFMNLFQKPFSSGKTITKHQWIKIFKHANAGCIISLLWFFGLTLCGPL RTLLLFEHSDINNISLLSNLFTSSGGGPAKTRGAAFFIIANICLLLFDΝDDLMAKMAEHPE GHHDSALTHMLYTAIAFLGVADHKGGVLLLVLALCCKVGFHTASRKLSVDVGGAKRL QALSHLVSVLLLCPWVIVLS VTTESKVES WFSLIMPFATVIFFVMILDF YVDSICSVKME VSKCARYGSFPIFISALLFGΝFWTHPITDQLRAMΝKAAHQESTEHVLSGGVVNSAIFFIL SAΝILSSPSKRGQKGTLIGYSPEGTPLYΝFMGDAFQHSSQSIPRFIKESLKQILEESDSRQI FYFLCLΝLLFTFVELFYGVLTΝSLGLISDGFHMLFDCSALVMGLFAALMSRWKATRIFS YGYGRIEILSGFIΝGLFLIVIAFFVFMESVARLIDPPELDTHMLTPVSVGGLIVΝLIGICAFS HAHSHAHGASQGSCHSSDHSHSHHMHGHSDHGHGHSHGSAGGGMΝAΝMRGVFLHN LADTLGSIGVIVSTVLIEQFGWFIADPLCSLFIAILIFLSVVPLIKDACQVLLLRLPPEYEKE LHIALEKIQEIEGLISYRDPHFWRHSASIVAGTIHIQVTSDVLEQRIVQQVTGILKDAGVΝ ΝLTIQVEKEAYFQHMSGLSTGFHDVLAMTKQMESMKYCKDGTYIM (SEQ ID ΝO:26). The human 84234 nucleic acid sequence is recited as follows: GTCGACCCACGCGTCCGGTCTGTGTCTGTCTGTGTCTCGCAGCGGCGCGCGGCCCC GGACAAGCGCTGGGGATTCCCGTTTGAGGCGTCACTACTGTCACTGCCATCACCCC ACGGAGCCACTTCTAGAGGGGAGTAGACCCGGCCCTTCGCCGGGCAGAGAAGATG TTGCCCCTGTCCATCAAAGACGATGAATACAAACCACCCAAGTTCAATTTGTTCGG CAAGATCTCGGGCTGGTTTAGGTCTATACTGTCCGACAAGACTTCCCGGAACCTGTT TTTCTTCCTGTGCCTGAACCTCTCTTTCGCTTTTGTGGAACTACTCTACGGCATCTGG AGCAACTGCTTAGGCTTGATTTCCGACTCTTTTCACATGTTTTTCGATAGCACTGCC ATTTTGGCTGGACTGGCAGCTTCTGTTATTTCAAAATGGAGAGATAATGATGCTTTC TCCTATGGGTATGTTAGAGCGGAAGTTCTGGCTGGCTTTGTCAATGGCCTATTTTTG ATCTTCACTGCTTTTTTTATTTTCTCAGAAGGAGTTGAGAGAGCATTAGCCCCTCCA GATGTCCACCATGAGAGACTGCTTCTTGTTTCCATTCTTGGGTTTGTGGTAAACCTA ATAGGAATATTTGTTTTCAAACATGGAGGTCATGGACATTCTCATGGCTCTGGCCAC GGACACAGTCATTCCCTCTTTAATGGTGCTCTAGATCAGGCACATGGCCATGTCGAT CATTGCCATAGCCATGAAGTGAAACATGGTGCTGCACATAGCCATGATCATGCTCA TGGACATGGACACTTTCATTCTCATGATGGCCCGTCCTTAAAAGAAACAACAGGAC CCAGCAGACAGATTTTACAAGGTGTATTTTTACATATCCTAGCAGATACACTTGGA AGTATTGGTGTAATTGCTTCTGCC ATC ATGATGCAAAATTTTGGTCTGATGATAGCA GATCCTATCTGTTCAATTCTTATAGCCATTCTTATAGTTGTAAGTGTTATTCCTCTTT TAAGAGAATCTGTTGGAATATTAATGCAGAGAACTCCTCCCCTATTAGAAAATAGT CTGCCTCAGTGCTATCAGAGGGTACAGCAGTTGCAAGGAGTTTACAGTTTACAGGA ACAGCACTTCTGGACTTTATGTTCTGACGTTTATGTTGGGACCTTGAAATTAATAGT AGCACCTGATGCTGATGCTAGGTGGATTTTAAGCCAAAC ACATAATATTTTTACTC A GGCTGGAGTGAGACAGCTCTACGTACAGATTGACTTTGCAGCCATGTAGTGAATGG AAAGAAATTATGCACCTTTTATGGACCAAATTTTTCTGCCAGTAAGAATTTCAGTTG TGGGCCTCCAGTCTTCTGGAATGTCTTCACTGCAGCTGCTGGAAATCACTGCTTTCA TTCCCACAAAACCAGTATTACTTTTTTTTAAAAAAAGAAAGAAATTGGAAATCTGT GCTATACGTAATGTCATAATTGTTGACGTTCTTCAGTATAATCATGTTTGTTAAATT
GTTTGTACCTTGCAAACTTATGAACCAAACCATATTGGGTTTTCAAAGTGCCTTTGT
ATCCAAAAATGTATTTGCCAGCATAGAAATGTACCATCAAGAGTCATGTATGTTTT ATTTTTGχτττTATTTTTT
GTGCAGTGGCATAATCTCAGCTCACTGCAACCTCTGCTTCCCGGGTTCAAGCTATTC TCCTGCCTCAGCCTTCTGAGTAGCTGCGACTATGGGCGTGTGCCACCATGCCCAGCT AATTTTTGCATTTTTAGTAGAGATGGGGTTTCACCATGTTGGCCAGGATGGTCTTGA TCTCTTGACCTCGTGATCCGTCCACCTCAGCCTCCCAAAGTGCTGGGATTACAGGTG TGAGCCACCACGCCCGGCTATGTTTTTTTTTTTTTTTTTTTTTTAACAATGGATATTC TGTAAAATATCCTGTACAAAACAAGACTATTGCCAGTAATGTATACAAATTGTCTTT TTGTGTACTTCCAGCATAGGTTTGGATATATGAACATTTTTCTTTTATTGTTTTATCT TCACAGAAAATAAAAGGTGTTAATTTGCTTTTTAAAACAAATTTAATTATCACATTT TAAATTACTTTGTGGACTGTGTTTTCAAAACTTTGCAAATTCATCTGTTACACAGAA AATTTTGACTTAAAACATCTGCTGAATTCCAAATTTCTGTAATAGCTACTGTATCTG TGATAAACTTTCCTATATCTTTCCTTGCCATTTCTATGGATTTTATAATGAAAGAAA AAGGCATTGGAGACTGAAGGCAGAAATGGTTGTGACAGTGCTGTTTGGCTTTTTCA TTCTTCAAATGCCAAGTCATCCACTTTGTTTTCCTGTTTAGGCTTTGCACAAATACA ATTGCTTTCAGGAATCCTAAAGCAGCATTTTATTGAGTTTGAATTATTAAAGGTACA GAGGAAATGTGGTGATGTAGAACTTTTCCTAACACAGGTATCTAGGAAGTAAGTGC TGAGTTGATTTTCTAGGTTCTTACGTATTTGAAAAATAAAATTGCAATTCGAGATAA AAAAAAAAAAAAAAAGGGCGGCCGC (SEQ ID NO:28). The human 84234 sequence (SEQ ID NO:28) is approximately 2637 nucleotides long.
The nucleic acid sequence includes an initiation codon (ATG) and a termination codon (TAG) which are underscored above. The region between and inclusive ofthe initiation codon and the termination codon is a methionine-initiated coding sequence of about 1131 nucleotides, including the termination codon (nucleotides indicated as "coding" of SEQ ID NO:28; SEQ ID NO:30). The coding sequence encodes a 376 amino acid protein (SEQ ID NO:29), which is recited as follows:
MLPLSIKDDEYKPPKFNLFGKISGWFRSILSDKTSRNLFFFLCLNLSFAFVELLYGIWSNC LGLISDSFHMFFDSTAILAGLAASVISKWRDNDAFSYGYVRAENLAGFVNGLFLIFTAFF IFSEGVERALAPPDVHHERLLLVSILGFVVNLIGIFVFKHGGHGHSHGSGHGHSHSLFNG ALDQAHGHVDHCHSHEVKHGAAHSHDHAHGHGHFHSHDGPSLKETTGPSRQILQGVF LHILADTLGSIGVIASAIMMQNFGLMIADPICSILIAILIVVSVIPLLRESVGILMQRTPPLL ENSLPQCYQRVQQLQGVYSLQEQHFWTLCSDVYVGTLKLIVAPDADARWILSQTHNIF TQAGVRQLYVQ1DFAAM (SEQ ID NO:29).
Example 2: Tissue Distribution of 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 mRNA by TaqMan Analysis
Endogenous human 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 gene expression was determined using the Perkin-Elmer/ABI 7700 Sequence Detection System which employs TaqMan technology. Briefly, TaqMan technology relies on standard RT-PCR with the addition of a third gene-specific oligonucleotide (refened to as a probe) which has a fluorescent dye coupled to its 5' end (typically 6-FAM) and a quenching dye at the 3' end (typically TAMRA). When the fluorescently tagged oligonucleotide is intact, the fluorescent signal from the 5' dye is quenched. As PCR proceeds, the 5' to 3' nucleolytic activity of Taq polymerase digests the labeled primer, producing a free nucleotide labeled with 6-FAM, which is now detected as a fluorescent signal. The PCR cycle where fluorescence is first released and detected is directly proportional to the starting amount ofthe gene of interest in the test sample, thus providing a quantitative measure ofthe initial template concentration. Samples can be internally controlled by the addition of a second set of primers/probe specific for a housekeeping gene such as GAPDH which has been labeled with a different fluorophore on the 5' end (typically VIC).
To determine the level of 47476 mRNA in various human tissues a primer/probe set was designed. Total RNA was prepared from a series of human tissues using an RNeasy kit from Qiagen. First strand cDNA was prepared from 1 μg total RNA using an oligo-dT primer and Superscript II reverse transcriptase (Gibco/BRL). cDNA obtained from approximately 50 ng total RNA was used per TaqMan reaction. Tissues tested include the human tissues and several cell lines shown in Table 1.
Table 1
Relative
Tissue Type Expression
Artery normal 0
Aorta diseased 0
Vein normal 0
Coronary SMC 0
HUVEC 0
Hemangioma 0
Heart normal 0
Heart CHF 0
Kidney 0
Skeletal Muscle 0
Adipose normal 0
Pancreas 0 primary osteoblasts 0
Osteoclasts (diff) 0
Skin normal 0
Spinal cord normal 0
Brain Cortex normal 0
Brain Hypothalamus normal 0
Nerve
Figure imgf000247_0001
As shown in Table 1, 47476 mRNA was detected in bone marrow mononuclear cells (BM-MNC), megakaryocytes, neutrophils, erythroid cells, lung (normal and diseased, i.e, COPD (chronic obstructive pulmonary disease) and spleen.
To determine the level of 67210 mRNA in various human tissues a primer/probe set was designed. Total RNA was prepared from a series of human tissues using an RNeasy kit from Qiagen. First strand cDNA was prepared from 1 μg total RNA using an oligo-dT primer and Superscript II reverse transcriptase (Gibco/BRL). cDNA obtained from approximately 50 ng total RNA was used per TaqMan reaction. Tissues tested include the human tissues and several cell lines shown in Table 2. Table 2
Figure imgf000247_0002
Figure imgf000248_0001
Megakaryocytes 0 Erythroid 0.412 positive control 44.8111
As shown in Table 2, 67210 mRNA was detected (average levels of expression greater than about 50) in coronary smooth muscle cells (SMC), normal artery, normal brain cortex, normal breast, and normal ovary. The highest level of 67210 expression was found in coronary SMC.
To determine the level of 49875 mRNA in various human tissues a primer/probe set was designed. Total RNA was prepared from a series of human tissues using an RNeasy kit from Qiagen. First strand cDNA was prepared from 1 μg total RNA using an oligo-dT primer and Superscript II reverse transcriptase (Gibco/BRL). cDNA obtained from approximately 50 ng total RNA was used per TaqMan reaction. Tissues tested include the human tissues shown in Table 3.
Table 3
Figure imgf000249_0001
Figure imgf000250_0001
* β2 Ct and target Ct values very close
* * Ct value less than 3
As shown in Table 3, 49875 mRNA was detected in normal (N) or tumor (T) breast, ovary, lung, and colon tissue samples. Significantly, 49875 mRNA was upregulated in a subset of ovary, lung, and colon tumors, as compared to the respective normal samples. This suggests that 49875 molecules play a role in tumor development, and that they can be used for diagnosis of ovary, lung, and colon tumors. 49875 mRNA was also detected in placenta tissue samples and in normal liver (N), as well as metastatic liver (Met), samples. Again, 49875 mRNA was upregulated in a subset ofthe metastatic liver samples, as compared to the normal liver tissue. Example 3: Tissue Distribution of 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 mRNA by Northern Analysis
Northern blot hybridizations with various RNA samples can be performed under standard conditions and washed under stringent conditions, i.e, 0.2xSSC at 65°C. A DNA probe conesponding to all or a portion ofthe 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 cDNA (SEQ ID NO:l, SEQ ID NO:4, SEQ ID NO:7, SEQ ID NO: 10, SEQ ID NO: 13, SEQ ID NO: 16, SEQ ID NO: 19, SEQ ID NO:22, SEQ ID NO:25, or
SEQ ID NO:28) can be used. The DNA was radioactively labeled with 32P-dCTP using the Prime-It Kit (Stratagene, La Jolla, CA) according to the instractions ofthe supplier. Filters containing mRNA from mouse hematopoietic and endocrine tissues, and cancer cell lines
(Clontech, Palo Alto, CA) can be probed in ExpressHyb hybridization solution (Clontech) and washed at high stringency according to manufacturer's recommendations.
Example 4: Recombinant Expression of 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 in Bacterial Cells In this example, 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or
84234 is expressed as a recombinant glutathione-S-transferase (GST) fusion polypeptide in E. coli and the fusion polypeptide is isolated and characterized. Specifically, 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 is fused to GST and this fusion polypeptide is expressed in E. coli, e.g, strain PEB199. Expression ofthe GST-47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 fusion protein in PEB199 is induced with IPTG. The recombinant fusion polypeptide is purified from crude bacterial lysates ofthe induced PEB199 strain by affinity chromatography on glutathione beads. Using polyacrylamide gel electrophoretic analysis ofthe polypeptide purified from the bacterial lysates, the molecular weight ofthe resultant fusion polypeptide is determined.
Example 5: Expression of Recombinant 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 Protein in COS Cells
To express the 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 gene in COS cells (e.g, COS-7 cells, CV-1 origin SV40 cells; Gluzman (1981) Ce/7E23:175-182), the pcDNA/Amp vector by Invitrogen Coφoration (San Diego, CA) is used. This vector contains an SV40 origin of replication, an ampicillin resistance gene, an E. coli replication origin, a CMV promoter followed by a polylinker region, and an S V40 intron and polyadenylation site. A DNA fragment encoding the entire 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 protein and an HA tag (Wilson et al. (1984) Cell 37:767) or a FLAG tag fused in-frame to its 3' end ofthe fragment is cloned into the polylinker region ofthe vector, thereby placing the expression ofthe recombinant protein under the control ofthe CMV promoter.
To constract the plasmid, the 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 DNA sequence is amplified by PCR using two primers. The 5' primer contains the restriction site of interest followed by approximately twenty nucleotides ofthe 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 coding sequence starting from the initiation codon; the 3' end sequence contains complementary sequences to the other restriction site of interest, a translation stop codon, the HA tag or FLAG tag and the last 20 nucleotides ofthe 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 coding sequence. The PCR amplified fragment and the pCDNA/Amp vector are digested with the appropriate restriction enzymes and the vector is dephosphorylated using the CIAP enzyme (New England Biolabs, Beverly, MA). Preferably the two restriction sites chosen are different so that the 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234_gene is inserted in the conect orientation. The ligation mixture is transformed into E. coli cells (strains HB101, DH5α, SURE, available from Stratagene Cloning Systems, La Jolla, CA, can be used), the transformed culture is plated on ampicillin media plates, and resistant colonies are selected. Plasmid DNA is isolated from transformants and examined by restriction analysis for the presence ofthe conect fragment. COS cells are subsequently transfected with the 47476, 67210, 49875, 46842, 33201,
83378, 84233, 64708, 85041, or 84234-ρcDNA/Amp plasmid DNA using the calcium phosphate or calcium chloride co-precipitation methods, DEAE-dextran-mediated transfection, lipofection, or electroporation. Other suitable methods for transfecting host cells can be found in Sambrook, J, Fritsh, E. F, and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual. 2nd, ed, Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. The expression ofthe 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 polypeptide is detected by radiolabelling (35S-methionine or 35S- cysteine available from NEN, Boston, MA, can be used) and immunoprecipitation (Harlow, E. and Lane, D. (1988) Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY) using an HA specific monoclonal antibody. Briefly, the cells are labeled for 8 hours with 35S-methionine (or 3^S-cysteine). The culture media are then collected and the cells are lysed using detergents (RIPA buffer, 150 mM NaCl, 1% NP-40, 0.1% SDS, 0.5% DOC, 50 mM Tris, pH 7.5). Both the cell lysate and the culture media are precipitated with an HA specific monoclonal antibody. Precipitated polypeptides are then analyzed by SDS-PAGE. Alternatively, DNA containing the 47476, 67210, 49875, 46842, 33201, 83378, 84233,
64708, 85041, or 84234 coding sequence is cloned directly into the polylinker ofthe pCDNA/Amp vector using the appropriate restriction sites. The resulting plasmid is transfected into COS cells in the manner described above, and the expression ofthe 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 polypeptide is detected by radiolabelling and immunoprecipitation using a 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234 specific monoclonal antibody.
Equivalents
Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments ofthe invention described herein. Such equivalents are intended to be encompassed by the following claims.

Claims

What is claimed is: 1. An isolated nucleic acid molecule selected from the group consisting of: a) a nucleic acid comprising the nucleotide sequence of SEQ ID NO: 1 , SEQ ID NO:4, SEQ ID NO:7, SEQ ID NO:10, SEQ ID NO:13, SEQ ID NO:16, SEQ ID NO:19, SEQ ID NO:22, SEQ ID NO:25, SEQ ID NO:28, SEQ ID NO:3, SEQ ID NO:6, SEQ ID NO:9, SEQ ID NO:12, SEQ ID NO:15, SEQ ID NO:18, SEQ ID NO:21, SEQ ID NO:24, SEQ ID NO:27, or SEQ ID NO: 30, or a full complement thereof; and b) a nucleic acid molecule which encodes a polypeptide comprising the amino acid sequence of SEQ ID NO:2, SEQ ID NO:5, SEQ ID NO:8, SEQ ID NO:l 1, SEQ ID NO: 14, SEQ ID NO: 17, SEQ ID NO:20, SEQ ID NO:23, SEQ ID NO:26, or SEQ ID NO:29.
2. The nucleic acid molecule of claim 1 , further comprising vector nucleic acid sequences.
3. The nucleic acid molecule of claim 1 , further comprising nucleic acid sequences encoding a heterologous polypeptide.
4. A host cell which contains the nucleic acid molecule of claim 1.
5. An isolated polypeptide comprising the amino acid sequence of SEQ ID NO:2, SEQ ID NO:5, SEQ ID NO:8, SEQ ID NO:l 1, SEQ ID NO:14, SEQ ID NO:17, SEQ ID NO:20, SEQ ID NO:23, SEQ ID NO:26, OR SEQ ID NO:29.
6. The polypeptide of claim 5 further comprising heterologous amino acid sequences.
7. An antibody or antigen-binding fragment thereof that selectively binds to a polypeptide of claim 5.
8. A method for producing a polypeptide comprising the amino acid sequence of SEQ ID NO:2, SEQ ID NO:5, SEQ ID NO:8, SEQ ID NO:l 1, SEQ ID NO:14, SEQ ID NO:17, SEQ ID NO:20, SEQ ID NO:23, SEQ ID NO:26, or SEQ ID NO:29, the method comprising culturing the host cell of claim 4 under conditions in which the nucleic acid molecule is expressed.
9. A method for detecting the presence of a polypeptide of claim 5 in a sample, comprising: a) contacting the sample with a compound which selectively binds to a polypeptide of claim 8; and b) determining whether the compound binds to the polypeptide in the sample.
10. The method of claim 9, wherein the compound which binds to the polypeptide is an antibody.
11. A kit comprising a compound which selectively binds to a polypeptide of claim 5 and instructions for use.
12. A method for detecting the presence of a nucleic acid molecule of claim 1 in a sample, comprising the steps of: a) contacting the sample with a nucleic acid probe or primer which selectively hybridizes to the nucleic acid molecule; and b) determining whether the nucleic acid probe or primer binds to a nucleic acid molecule in the sample.
13. The method of claim 12, wherein the sample comprises mRNA molecules and is contacted with a nucleic acid probe.
14. A kit comprising a compound which selectively hybridizes to a nucleic acid molecule of claim 1 and instractions for use.
15. A method for identifying a compound which binds to a polypeptide of claim 5 comprising the steps of: a) contacting a polypeptide, or a cell expressing a polypeptide of claim 5 with a test compound; and b) determining whether the polypeptide binds to the test compound.
16. A method for modulating the activity of a polypeptide of claim 5, comprising contacting a polypeptide or a cell expressing a polypeptide of claim 5 with a compound which binds to the polypeptide in a sufficient concentration to modulate the activity ofthe polypeptide.
17. A method of inhibiting abenant activity of a 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234-expressing cell, comprising contacting a 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234-expressing cell with a compound that modulates the activity or expression of a polypeptide of claim 5, in an amount which is effective to reduce or inhibit the abenant activity ofthe cell.
18. The method of claim 17, wherein the compound is selected from the group consisting of a peptide, a phosphopeptide, a small organic molecule, and an antibody.
19. The method of claim 17, wherein the cell is located in a cancerous or pre-cancerous tissue.
20. A method of treating or preventing a disorder characterized by abenant activity of a 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234-expressing cell, in a subject, comprising: administering to the subject an effective amount of a compound that modulates the activity or expression of a nucleic acid molecule of claim 1, such that the abenant activity ofthe 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, or 84234-expressing cell is reduced or inhibited.
PCT/US2001/045291 2000-11-14 2001-11-14 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, and 84234, novel human guanine nucleotide dissociation stimulator, glycosyltransferase, dead type helicase, centaurin, dehydrogenase/reductase, and metal transporter family members and uses thereof WO2002040656A2 (en)

Priority Applications (3)

Application Number Priority Date Filing Date Title
AU2002239421A AU2002239421A1 (en) 2000-11-14 2001-11-14 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, and 84234, novel human guanine nucleotide dissociation stimulator, glycosyltransferase, dead type helicase, centaurin, dehydrogenase/reductase, and metal transporter family members and uses thereof
US10/176,306 US20030130485A1 (en) 2000-11-14 2002-06-20 Novel human genes and methods of use thereof
US10/968,812 US20050214893A1 (en) 2000-11-14 2004-10-19 Novel human genes and methods of use thereof

Applications Claiming Priority (12)

Application Number Priority Date Filing Date Title
US24836200P 2000-11-14 2000-11-14
US24836500P 2000-11-14 2000-11-14
US24833100P 2000-11-14 2000-11-14
US60/248,331 2000-11-14
US60/248,365 2000-11-14
US60/248,362 2000-11-14
US25032700P 2000-11-30 2000-11-30
US25007700P 2000-11-30 2000-11-30
US25017600P 2000-11-30 2000-11-30
US60/250,327 2000-11-30
US60/250,176 2000-11-30
US60/250,077 2000-11-30

Related Child Applications (1)

Application Number Title Priority Date Filing Date
US10/176,306 Continuation-In-Part US20030130485A1 (en) 2000-11-14 2002-06-20 Novel human genes and methods of use thereof

Publications (2)

Publication Number Publication Date
WO2002040656A2 true WO2002040656A2 (en) 2002-05-23
WO2002040656A3 WO2002040656A3 (en) 2004-02-26

Family

ID=27559353

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2001/045291 WO2002040656A2 (en) 2000-11-14 2001-11-14 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, and 84234, novel human guanine nucleotide dissociation stimulator, glycosyltransferase, dead type helicase, centaurin, dehydrogenase/reductase, and metal transporter family members and uses thereof

Country Status (2)

Country Link
AU (1) AU2002239421A1 (en)
WO (1) WO2002040656A2 (en)

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2004078980A1 (en) * 2003-03-05 2004-09-16 National Institute Of Advanced Industrial Science And Technology Nucleic acid and method of examining canceration using the nucleic acid
EP1611887A2 (en) * 2004-06-10 2006-01-04 Abgenomics Corporation Modulation of peroxisome proliferator-activated receptors

Non-Patent Citations (6)

* Cited by examiner, † Cited by third party
Title
CLYDE-SMITH JODI ET AL: "Characterization of RasGRP2, a plasma membrane-targeted, dual specificity Ras/Rap exchange factor." JOURNAL OF BIOLOGICAL CHEMISTRY, vol. 275, no. 41, 13 October 2000 (2000-10-13), pages 32260-32267, XP002216973 ISSN: 0021-9258 *
EBINU J O ET AL: "RASGRP, A RAS GUANYL NUCLEOTIDE-RELEASING PROTEIN WITH CALCIUM- ANDDIACYLGLYCEROL-BINDING MOTIFS" SCIENCE, AMERICAN ASSOCIATION FOR THE ADVANCEMENT OF SCIENCE,, US, vol. 280, 15 May 1998 (1998-05-15), pages 1082-1086, XP000882708 ISSN: 0036-8075 *
LI L ET AL: "Cloning of Ras guanine nucleotide releasing protein 4, and evaluation of its expression in rat mast cells and thei bone marrow progenitors" MOLECULAR IMMUNOLOGY, - September 2002 (2002-09) pages 1283-1288, XP002216977 *
LORENZO PATRICIA S ET AL: "Phorbol esters modulate the Ras exchange factor RasGRP3." CANCER RESEARCH, vol. 61, no. 3, 1 February 2001 (2001-02-01), pages 943-949, XP002216974 ISSN: 0008-5472 *
REUTHERS G W ET AL: "RasGRP4 is a novel Ras activator isolated from acute myeloid leukemia" JOURNAL OF BIOLOGICAL CHEMISTRY, vol. 277, no. 34, - 23 August 2002 (2002-08-23) pages 30508-30514, XP002216976 *
YANG YI ET AL: "RasGRP4, a new mast cell-restricted Ras guanine nucleotide-releasing protein with calcium- and diacylglycerol-binding motifs. Identification of defective variants of this signaling protein in asthma, mastocytosis, and mast cell leukemia patients and demonstration of the importance of RasGRP4 in mast" JOURNAL OF BIOLOGICAL CHEMISTRY, vol. 277, no. 28, 12 July 2002 (2002-07-12), pages 25756-25774, XP002216975 ISSN: 0021-9258 *

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2004078980A1 (en) * 2003-03-05 2004-09-16 National Institute Of Advanced Industrial Science And Technology Nucleic acid and method of examining canceration using the nucleic acid
EP1611887A2 (en) * 2004-06-10 2006-01-04 Abgenomics Corporation Modulation of peroxisome proliferator-activated receptors
EP1611887A3 (en) * 2004-06-10 2006-02-08 Abgenomics Corporation Modulation of peroxisome proliferator-activated receptors

Also Published As

Publication number Publication date
AU2002239421A1 (en) 2002-05-27
WO2002040656A3 (en) 2004-02-26

Similar Documents

Publication Publication Date Title
US20020164769A1 (en) 32144, a novel human fatty acid amide hydrolase family member and uses thereof
EP1354049A2 (en) 16816 and 16839, human phospholipase c molecules and uses therefor
US20050214893A1 (en) Novel human genes and methods of use thereof
EP1335981A2 (en) 13237, 18480, 2245 or 16228 human protein kinase molecules and uses therefor
WO2002026803A2 (en) 22108 and 47916, novel human thioredoxin family members and uses thereof
EP1294899A2 (en) 16836, a human phospholipase c family member and uses thereof
WO2002040656A2 (en) 47476, 67210, 49875, 46842, 33201, 83378, 84233, 64708, 85041, and 84234, novel human guanine nucleotide dissociation stimulator, glycosyltransferase, dead type helicase, centaurin, dehydrogenase/reductase, and metal transporter family members and uses thereof
EP1270726A1 (en) 69624, a human transporter family member and uses therefor
EP1259620A2 (en) 2504, 15977, and 14760, novel protein kinase family members and uses therefor
US20030176330A1 (en) 55562 and 21617, novel human proteins and methods of use thereof
US20020016449A1 (en) 46743 and 27417, novel human acyltransferase family members and uses therefor
EP1309696A2 (en) 32140, human aldehyde dehydrogenase and uses therefor
US20020164320A1 (en) 56939, a novel human acyl-CoA thioesterase family member and uses thereof
US20020001807A1 (en) 21509 and 33770, novel human dehydrogenase family members and uses thereof
US20020009777A1 (en) 25552, a novel human methyltransferase family member and uses thereof
US20020155499A1 (en) 32624, a novel human UDP-glucuronosyl and glycosyl transferase family member and uses thereof
WO2002018558A2 (en) 27439, a human hydroxylase and uses therefor
WO2002010401A2 (en) 18431 and 32374, human protein kinase family members and uses therefor
WO2001068806A9 (en) 26934, a novel cytidine deaminase-like molecule and uses thereof
WO2002029040A2 (en) 25219, a novel human aminotransferase and uses therefor
WO2002038773A2 (en) A human phospholipase c and uses thereof
US20020123475A1 (en) 32626, a novel human UDP-glycosyltransferase and uses thereof
US20030119161A1 (en) 32132, a novel fucosyltransferase family member and uses therefor
WO2002033088A2 (en) Human carboxypeptidase, scramblase, and protocadherin family members and uses thereof
WO2001073049A2 (en) 33877 and 47179, human glycosyl transferase family members and uses thereof

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A2

Designated state(s): AE AG AL AM AT AU AZ BA BB BG BR BY BZ CA CH CN CO CR CU CZ DE DK DM DZ EC EE ES FI GB GD GE GH GM HR HU ID IL IN IS JP KE KG KP KR KZ LC LK LR LS LT LU LV MA MD MG MK MN MW MX MZ NO NZ PL PT RO RU SD SE SG SI SK SL TJ TM TR TT TZ UA UG US UZ VN YU ZA ZW

AL Designated countries for regional patents

Kind code of ref document: A2

Designated state(s): GH GM KE LS MW MZ SD SL SZ TZ UG ZW AM AZ BY KG KZ MD RU TJ TM AT BE CH CY DE DK ES FI FR GB GR IE IT LU MC NL PT SE TR BF BJ CF CG CI CM GA GN GQ GW ML MR NE SN TD TG

121 Ep: the epo has been informed by wipo that ep was designated in this application
DFPE Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101)
REG Reference to national code

Ref country code: DE

Ref legal event code: 8642

122 Ep: pct application non-entry in european phase
NENP Non-entry into the national phase

Ref country code: JP

WWW Wipo information: withdrawn in national office

Ref document number: JP

DPE2 Request for preliminary examination filed before expiration of 19th month from priority date (pct application filed from 20040101)