AU3208999A - Identification of a cdna associated with ischemia in human heart tissue - Google Patents
Identification of a cdna associated with ischemia in human heart tissue Download PDFInfo
- Publication number
- AU3208999A AU3208999A AU32089/99A AU3208999A AU3208999A AU 3208999 A AU3208999 A AU 3208999A AU 32089/99 A AU32089/99 A AU 32089/99A AU 3208999 A AU3208999 A AU 3208999A AU 3208999 A AU3208999 A AU 3208999A
- Authority
- AU
- Australia
- Prior art keywords
- nucleic acid
- protein
- seq
- sequence
- acid molecule
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Abandoned
Links
- 210000005003 heart tissue Anatomy 0.000 title description 20
- 208000028867 ischemia Diseases 0.000 title description 3
- 108090000623 proteins and genes Proteins 0.000 claims description 200
- 102000004169 proteins and genes Human genes 0.000 claims description 168
- 150000007523 nucleic acids Chemical class 0.000 claims description 101
- 102000039446 nucleic acids Human genes 0.000 claims description 95
- 108020004707 nucleic acids Proteins 0.000 claims description 95
- 239000003795 chemical substances by application Substances 0.000 claims description 78
- 238000000034 method Methods 0.000 claims description 63
- 230000014509 gene expression Effects 0.000 claims description 61
- 108090000765 processed proteins & peptides Proteins 0.000 claims description 36
- 230000027455 binding Effects 0.000 claims description 30
- 230000000694 effects Effects 0.000 claims description 29
- 125000003275 alpha amino acid group Chemical group 0.000 claims description 26
- 102000004196 processed proteins & peptides Human genes 0.000 claims description 25
- 239000012634 fragment Substances 0.000 claims description 24
- 239000013598 vector Substances 0.000 claims description 19
- 229920001184 polypeptide Polymers 0.000 claims description 17
- 150000001413 amino acids Chemical class 0.000 claims description 16
- 238000004519 manufacturing process Methods 0.000 claims description 8
- 239000002773 nucleotide Substances 0.000 claims description 8
- 125000003729 nucleotide group Chemical group 0.000 claims description 8
- 238000006467 substitution reaction Methods 0.000 claims description 8
- 239000000758 substrate Substances 0.000 claims description 5
- 239000002253 acid Substances 0.000 claims description 3
- 238000012258 culturing Methods 0.000 claims description 3
- 235000018102 proteins Nutrition 0.000 description 147
- 210000004027 cell Anatomy 0.000 description 67
- 239000002299 complementary DNA Substances 0.000 description 31
- 238000003752 polymerase chain reaction Methods 0.000 description 26
- 239000000523 sample Substances 0.000 description 26
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 20
- 230000000302 ischemic effect Effects 0.000 description 18
- 239000013615 primer Substances 0.000 description 17
- 241001465754 Metazoa Species 0.000 description 16
- 235000001014 amino acid Nutrition 0.000 description 16
- 108020004414 DNA Proteins 0.000 description 14
- 239000000284 extract Substances 0.000 description 14
- 239000000203 mixture Substances 0.000 description 14
- 210000002216 heart Anatomy 0.000 description 13
- 230000009261 transgenic effect Effects 0.000 description 11
- 230000001413 cellular effect Effects 0.000 description 10
- 230000001105 regulatory effect Effects 0.000 description 10
- 108091034117 Oligonucleotide Proteins 0.000 description 9
- 150000001875 compounds Chemical class 0.000 description 9
- 239000000126 substance Substances 0.000 description 9
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 8
- 238000009396 hybridization Methods 0.000 description 8
- 239000001301 oxygen Substances 0.000 description 8
- 229910052760 oxygen Inorganic materials 0.000 description 8
- 210000001519 tissue Anatomy 0.000 description 8
- 206010048858 Ischaemic cardiomyopathy Diseases 0.000 description 7
- 241000124008 Mammalia Species 0.000 description 7
- 239000013604 expression vector Substances 0.000 description 7
- 239000003550 marker Substances 0.000 description 7
- 108020004999 messenger RNA Proteins 0.000 description 7
- 208000031225 myocardial ischemia Diseases 0.000 description 7
- 239000011541 reaction mixture Substances 0.000 description 7
- 241000894007 species Species 0.000 description 7
- 108091026890 Coding region Proteins 0.000 description 6
- ZHNUHDYFZUAESO-UHFFFAOYSA-N Formamide Chemical compound NC=O ZHNUHDYFZUAESO-UHFFFAOYSA-N 0.000 description 6
- 108091000080 Phosphotransferase Proteins 0.000 description 6
- 230000031018 biological processes and functions Effects 0.000 description 6
- 230000015572 biosynthetic process Effects 0.000 description 6
- 230000009054 pathological process Effects 0.000 description 6
- 102000020233 phosphotransferase Human genes 0.000 description 6
- 239000000725 suspension Substances 0.000 description 6
- 238000003786 synthesis reaction Methods 0.000 description 6
- 108020004635 Complementary DNA Proteins 0.000 description 5
- 238000000636 Northern blotting Methods 0.000 description 5
- 108091028043 Nucleic acid sequence Proteins 0.000 description 5
- 208000037273 Pathologic Processes Diseases 0.000 description 5
- 108010021757 Polynucleotide 5'-Hydroxyl-Kinase Proteins 0.000 description 5
- 102000008422 Polynucleotide 5'-hydroxyl-kinase Human genes 0.000 description 5
- 108700019146 Transgenes Proteins 0.000 description 5
- 230000004075 alteration Effects 0.000 description 5
- 238000003556 assay Methods 0.000 description 5
- 230000001684 chronic effect Effects 0.000 description 5
- 208000029078 coronary artery disease Diseases 0.000 description 5
- 210000003527 eukaryotic cell Anatomy 0.000 description 5
- 238000010195 expression analysis Methods 0.000 description 5
- 239000000499 gel Substances 0.000 description 5
- 230000002068 genetic effect Effects 0.000 description 5
- 238000003780 insertion Methods 0.000 description 5
- 230000037431 insertion Effects 0.000 description 5
- 239000013612 plasmid Substances 0.000 description 5
- 239000007787 solid Substances 0.000 description 5
- 238000010561 standard procedure Methods 0.000 description 5
- YBJHBAHKTGYVGT-ZKWXMUAHSA-N (+)-Biotin Chemical compound N1C(=O)N[C@@H]2[C@H](CCCCC(=O)O)SC[C@@H]21 YBJHBAHKTGYVGT-ZKWXMUAHSA-N 0.000 description 4
- 206010059866 Drug resistance Diseases 0.000 description 4
- 102000004190 Enzymes Human genes 0.000 description 4
- 108090000790 Enzymes Proteins 0.000 description 4
- MWUXSHHQAYIFBG-UHFFFAOYSA-N Nitric oxide Chemical compound O=[N] MWUXSHHQAYIFBG-UHFFFAOYSA-N 0.000 description 4
- 241000700159 Rattus Species 0.000 description 4
- 108010008281 Recombinant Fusion Proteins Proteins 0.000 description 4
- 102000007056 Recombinant Fusion Proteins Human genes 0.000 description 4
- 230000002411 adverse Effects 0.000 description 4
- 230000001580 bacterial effect Effects 0.000 description 4
- 239000000872 buffer Substances 0.000 description 4
- 108091092328 cellular RNA Proteins 0.000 description 4
- 230000000295 complement effect Effects 0.000 description 4
- 239000003814 drug Substances 0.000 description 4
- 229940088598 enzyme Drugs 0.000 description 4
- 230000004927 fusion Effects 0.000 description 4
- 230000003053 immunization Effects 0.000 description 4
- 230000001404 mediated effect Effects 0.000 description 4
- 238000010369 molecular cloning Methods 0.000 description 4
- 230000026731 phosphorylation Effects 0.000 description 4
- 238000006366 phosphorylation reaction Methods 0.000 description 4
- 150000003839 salts Chemical class 0.000 description 4
- -1 that which encodes Chemical class 0.000 description 4
- 241000283690 Bos taurus Species 0.000 description 3
- 102000000584 Calmodulin Human genes 0.000 description 3
- 108010041952 Calmodulin Proteins 0.000 description 3
- 206010007572 Cardiac hypertrophy Diseases 0.000 description 3
- 208000006029 Cardiomegaly Diseases 0.000 description 3
- 241000588724 Escherichia coli Species 0.000 description 3
- 102100034343 Integrase Human genes 0.000 description 3
- 241000699660 Mus musculus Species 0.000 description 3
- 241000699670 Mus sp. Species 0.000 description 3
- 108010074596 Myosin-Light-Chain Kinase Proteins 0.000 description 3
- 108700026244 Open Reading Frames Proteins 0.000 description 3
- 241000283973 Oryctolagus cuniculus Species 0.000 description 3
- 108010092799 RNA-directed DNA polymerase Proteins 0.000 description 3
- 108700008625 Reporter Genes Proteins 0.000 description 3
- 108091006629 SLC13A2 Proteins 0.000 description 3
- 241000251539 Vertebrata <Metazoa> Species 0.000 description 3
- JLCPHMBAVCMARE-UHFFFAOYSA-N [3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-[[3-[[3-[[3-[[3-[[3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-hydroxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methyl [5-(6-aminopurin-9-yl)-2-(hydroxymethyl)oxolan-3-yl] hydrogen phosphate Polymers Cc1cn(C2CC(OP(O)(=O)OCC3OC(CC3OP(O)(=O)OCC3OC(CC3O)n3cnc4c3nc(N)[nH]c4=O)n3cnc4c3nc(N)[nH]c4=O)C(COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3CO)n3cnc4c(N)ncnc34)n3ccc(N)nc3=O)n3cnc4c(N)ncnc34)n3ccc(N)nc3=O)n3ccc(N)nc3=O)n3ccc(N)nc3=O)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cc(C)c(=O)[nH]c3=O)n3cc(C)c(=O)[nH]c3=O)n3ccc(N)nc3=O)n3cc(C)c(=O)[nH]c3=O)n3cnc4c3nc(N)[nH]c4=O)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)O2)c(=O)[nH]c1=O JLCPHMBAVCMARE-UHFFFAOYSA-N 0.000 description 3
- 125000000539 amino acid group Chemical group 0.000 description 3
- 238000002820 assay format Methods 0.000 description 3
- 230000008827 biological function Effects 0.000 description 3
- 230000017531 blood circulation Effects 0.000 description 3
- 210000004899 c-terminal region Anatomy 0.000 description 3
- 239000000470 constituent Substances 0.000 description 3
- 230000009089 cytolysis Effects 0.000 description 3
- 238000012217 deletion Methods 0.000 description 3
- 230000037430 deletion Effects 0.000 description 3
- 230000001419 dependent effect Effects 0.000 description 3
- 238000004520 electroporation Methods 0.000 description 3
- 230000001747 exhibiting effect Effects 0.000 description 3
- 238000009472 formulation Methods 0.000 description 3
- 238000002649 immunization Methods 0.000 description 3
- 238000002347 injection Methods 0.000 description 3
- 239000007924 injection Substances 0.000 description 3
- 208000037906 ischaemic injury Diseases 0.000 description 3
- 230000004048 modification Effects 0.000 description 3
- 238000012986 modification Methods 0.000 description 3
- 210000000107 myocyte Anatomy 0.000 description 3
- 238000002360 preparation method Methods 0.000 description 3
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 description 3
- 239000000047 product Substances 0.000 description 3
- 108020003175 receptors Proteins 0.000 description 3
- 102000005962 receptors Human genes 0.000 description 3
- 239000000243 solution Substances 0.000 description 3
- 230000001225 therapeutic effect Effects 0.000 description 3
- 230000009466 transformation Effects 0.000 description 3
- 230000024883 vasodilation Effects 0.000 description 3
- 235000012431 wafers Nutrition 0.000 description 3
- 206010003445 Ascites Diseases 0.000 description 2
- 241000282472 Canis lupus familiaris Species 0.000 description 2
- 206010007559 Cardiac failure congestive Diseases 0.000 description 2
- 208000024172 Cardiovascular disease Diseases 0.000 description 2
- 241000282693 Cercopithecidae Species 0.000 description 2
- 241000699800 Cricetinae Species 0.000 description 2
- 102000053602 DNA Human genes 0.000 description 2
- 201000010046 Dilated cardiomyopathy Diseases 0.000 description 2
- 206010048554 Endothelial dysfunction Diseases 0.000 description 2
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 2
- 241000282326 Felis catus Species 0.000 description 2
- 241000282412 Homo Species 0.000 description 2
- 102000004289 Interferon regulatory factor 1 Human genes 0.000 description 2
- 108090000890 Interferon regulatory factor 1 Proteins 0.000 description 2
- 241001529936 Murinae Species 0.000 description 2
- 241000699666 Mus <mouse, genus> Species 0.000 description 2
- 102000003505 Myosin Human genes 0.000 description 2
- 108060008487 Myosin Proteins 0.000 description 2
- 102100035044 Myosin light chain kinase, smooth muscle Human genes 0.000 description 2
- 238000012408 PCR amplification Methods 0.000 description 2
- 241001494479 Pecora Species 0.000 description 2
- 108020004511 Recombinant DNA Proteins 0.000 description 2
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 2
- 241000282887 Suidae Species 0.000 description 2
- 230000009471 action Effects 0.000 description 2
- 238000000137 annealing Methods 0.000 description 2
- 239000005557 antagonist Substances 0.000 description 2
- 239000003146 anticoagulant agent Substances 0.000 description 2
- 239000000427 antigen Substances 0.000 description 2
- 108091007433 antigens Proteins 0.000 description 2
- 102000036639 antigens Human genes 0.000 description 2
- 229960004676 antithrombotic agent Drugs 0.000 description 2
- 239000011324 bead Substances 0.000 description 2
- 230000004071 biological effect Effects 0.000 description 2
- 229960002685 biotin Drugs 0.000 description 2
- 235000020958 biotin Nutrition 0.000 description 2
- 239000011616 biotin Substances 0.000 description 2
- 210000004369 blood Anatomy 0.000 description 2
- 239000008280 blood Substances 0.000 description 2
- 239000000969 carrier Substances 0.000 description 2
- 238000004113 cell culture Methods 0.000 description 2
- 208000026106 cerebrovascular disease Diseases 0.000 description 2
- 238000006243 chemical reaction Methods 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- 238000010367 cloning Methods 0.000 description 2
- 238000010276 construction Methods 0.000 description 2
- 238000011161 development Methods 0.000 description 2
- 201000011304 dilated cardiomyopathy 1A Diseases 0.000 description 2
- 229940079593 drug Drugs 0.000 description 2
- 230000008694 endothelial dysfunction Effects 0.000 description 2
- 230000002255 enzymatic effect Effects 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 210000004602 germ cell Anatomy 0.000 description 2
- 239000011521 glass Substances 0.000 description 2
- 210000005260 human cell Anatomy 0.000 description 2
- 230000002209 hydrophobic effect Effects 0.000 description 2
- 230000002163 immunogen Effects 0.000 description 2
- 230000001939 inductive effect Effects 0.000 description 2
- 239000006166 lysate Substances 0.000 description 2
- 210000004962 mammalian cell Anatomy 0.000 description 2
- 238000002156 mixing Methods 0.000 description 2
- 238000012544 monitoring process Methods 0.000 description 2
- 230000002107 myocardial effect Effects 0.000 description 2
- 210000004165 myocardium Anatomy 0.000 description 2
- 238000007911 parenteral administration Methods 0.000 description 2
- 238000010647 peptide synthesis reaction Methods 0.000 description 2
- 239000008194 pharmaceutical composition Substances 0.000 description 2
- 230000008569 process Effects 0.000 description 2
- 238000000746 purification Methods 0.000 description 2
- 238000011084 recovery Methods 0.000 description 2
- 230000010076 replication Effects 0.000 description 2
- 108091008146 restriction endonucleases Proteins 0.000 description 2
- 230000003248 secreting effect Effects 0.000 description 2
- 238000012163 sequencing technique Methods 0.000 description 2
- 210000002027 skeletal muscle Anatomy 0.000 description 2
- 150000003384 small molecules Chemical class 0.000 description 2
- 239000001509 sodium citrate Substances 0.000 description 2
- NLJMYIDDQXHKNR-UHFFFAOYSA-K sodium citrate Chemical compound O.O.[Na+].[Na+].[Na+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O NLJMYIDDQXHKNR-UHFFFAOYSA-K 0.000 description 2
- 239000007790 solid phase Substances 0.000 description 2
- 238000007910 systemic administration Methods 0.000 description 2
- 210000001550 testis Anatomy 0.000 description 2
- 238000013518 transcription Methods 0.000 description 2
- 230000035897 transcription Effects 0.000 description 2
- 238000011830 transgenic mouse model Methods 0.000 description 2
- 238000013519 translation Methods 0.000 description 2
- 230000000304 vasodilatating effect Effects 0.000 description 2
- 238000001086 yeast two-hybrid system Methods 0.000 description 2
- ZOOGRGPOEVQQDX-UUOKFMHZSA-N 3',5'-cyclic GMP Chemical compound C([C@H]1O2)OP(O)(=O)O[C@H]1[C@@H](O)[C@@H]2N1C(N=C(NC2=O)N)=C2N=C1 ZOOGRGPOEVQQDX-UUOKFMHZSA-N 0.000 description 1
- DEXFNLNNUZKHNO-UHFFFAOYSA-N 6-[3-[4-[2-(2,3-dihydro-1H-inden-2-ylamino)pyrimidin-5-yl]piperidin-1-yl]-3-oxopropyl]-3H-1,3-benzoxazol-2-one Chemical compound C1C(CC2=CC=CC=C12)NC1=NC=C(C=N1)C1CCN(CC1)C(CCC1=CC2=C(NC(O2)=O)C=C1)=O DEXFNLNNUZKHNO-UHFFFAOYSA-N 0.000 description 1
- 108700028369 Alleles Proteins 0.000 description 1
- 208000024827 Alzheimer disease Diseases 0.000 description 1
- 102000008873 Angiotensin II receptor Human genes 0.000 description 1
- 108050000824 Angiotensin II receptor Proteins 0.000 description 1
- 206010060965 Arterial stenosis Diseases 0.000 description 1
- 206010003211 Arteriosclerosis coronary artery Diseases 0.000 description 1
- 206010003225 Arteriospasm coronary Diseases 0.000 description 1
- BSYNRYMUTXBXSQ-UHFFFAOYSA-N Aspirin Chemical compound CC(=O)OC1=CC=CC=C1C(O)=O BSYNRYMUTXBXSQ-UHFFFAOYSA-N 0.000 description 1
- 208000037260 Atherosclerotic Plaque Diseases 0.000 description 1
- 241000972773 Aulopiformes Species 0.000 description 1
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 1
- 241000283707 Capra Species 0.000 description 1
- 102000014914 Carrier Proteins Human genes 0.000 description 1
- 108010078791 Carrier Proteins Proteins 0.000 description 1
- 241000700198 Cavia Species 0.000 description 1
- 108010035563 Chloramphenicol O-acetyltransferase Proteins 0.000 description 1
- 208000003890 Coronary Vasospasm Diseases 0.000 description 1
- 241000699802 Cricetulus griseus Species 0.000 description 1
- FBPFZTCFMRRESA-FSIIMWSLSA-N D-Glucitol Natural products OC[C@H](O)[C@H](O)[C@@H](O)[C@H](O)CO FBPFZTCFMRRESA-FSIIMWSLSA-N 0.000 description 1
- FBPFZTCFMRRESA-JGWLITMVSA-N D-glucitol Chemical compound OC[C@H](O)[C@@H](O)[C@H](O)[C@H](O)CO FBPFZTCFMRRESA-JGWLITMVSA-N 0.000 description 1
- 102000012410 DNA Ligases Human genes 0.000 description 1
- 108010061982 DNA Ligases Proteins 0.000 description 1
- 239000003155 DNA primer Substances 0.000 description 1
- 238000001712 DNA sequencing Methods 0.000 description 1
- 102000004163 DNA-directed RNA polymerases Human genes 0.000 description 1
- 108090000626 DNA-directed RNA polymerases Proteins 0.000 description 1
- 229920002307 Dextran Polymers 0.000 description 1
- 101100125027 Dictyostelium discoideum mhsp70 gene Proteins 0.000 description 1
- 206010061818 Disease progression Diseases 0.000 description 1
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 1
- LVGKNOAMLMIIKO-UHFFFAOYSA-N Elaidinsaeure-aethylester Natural products CCCCCCCCC=CCCCCCCCC(=O)OCC LVGKNOAMLMIIKO-UHFFFAOYSA-N 0.000 description 1
- 241000283086 Equidae Species 0.000 description 1
- 241000283073 Equus caballus Species 0.000 description 1
- 229920001917 Ficoll Polymers 0.000 description 1
- 108090000331 Firefly luciferases Proteins 0.000 description 1
- 101150031823 HSP70 gene Proteins 0.000 description 1
- 206010019280 Heart failures Diseases 0.000 description 1
- HTTJABKRGRZYRN-UHFFFAOYSA-N Heparin Chemical compound OC1C(NC(=O)C)C(O)OC(COS(O)(=O)=O)C1OC1C(OS(O)(=O)=O)C(O)C(OC2C(C(OS(O)(=O)=O)C(OC3C(C(O)C(O)C(O3)C(O)=O)OS(O)(=O)=O)C(CO)O2)NS(O)(=O)=O)C(C(O)=O)O1 HTTJABKRGRZYRN-UHFFFAOYSA-N 0.000 description 1
- 241000238631 Hexapoda Species 0.000 description 1
- 101001050288 Homo sapiens Transcription factor Jun Proteins 0.000 description 1
- 206010020772 Hypertension Diseases 0.000 description 1
- DGAQECJNVWCQMB-PUAWFVPOSA-M Ilexoside XXIX Chemical compound C[C@@H]1CC[C@@]2(CC[C@@]3(C(=CC[C@H]4[C@]3(CC[C@@H]5[C@@]4(CC[C@@H](C5(C)C)OS(=O)(=O)[O-])C)C)[C@@H]2[C@]1(C)O)C)C(=O)O[C@H]6[C@@H]([C@H]([C@@H]([C@H](O6)CO)O)O)O.[Na+] DGAQECJNVWCQMB-PUAWFVPOSA-M 0.000 description 1
- 108700002232 Immediate-Early Genes Proteins 0.000 description 1
- 108060003951 Immunoglobulin Proteins 0.000 description 1
- 108091092195 Intron Proteins 0.000 description 1
- 108010025815 Kanamycin Kinase Proteins 0.000 description 1
- 125000002842 L-seryl group Chemical group O=C([*])[C@](N([H])[H])([H])C([H])([H])O[H] 0.000 description 1
- 229930193140 Neomycin Natural products 0.000 description 1
- 239000000020 Nitrocellulose Substances 0.000 description 1
- 108020004711 Nucleic Acid Probes Proteins 0.000 description 1
- XDMCWZFLLGVIID-SXPRBRBTSA-N O-(3-O-D-galactosyl-N-acetyl-beta-D-galactosaminyl)-L-serine Chemical compound CC(=O)N[C@H]1[C@H](OC[C@H]([NH3+])C([O-])=O)O[C@H](CO)[C@H](O)[C@@H]1OC1[C@H](O)[C@@H](O)[C@@H](O)[C@@H](CO)O1 XDMCWZFLLGVIID-SXPRBRBTSA-N 0.000 description 1
- 108700020796 Oncogene Proteins 0.000 description 1
- 241000282579 Pan Species 0.000 description 1
- 102000007079 Peptide Fragments Human genes 0.000 description 1
- 108010033276 Peptide Fragments Proteins 0.000 description 1
- 108010001014 Plasminogen Activators Proteins 0.000 description 1
- 102000001938 Plasminogen Activators Human genes 0.000 description 1
- 241000276498 Pollachius virens Species 0.000 description 1
- 108010071563 Proto-Oncogene Proteins c-fos Proteins 0.000 description 1
- 102000007568 Proto-Oncogene Proteins c-fos Human genes 0.000 description 1
- 239000013614 RNA sample Substances 0.000 description 1
- 230000004570 RNA-binding Effects 0.000 description 1
- 206010038997 Retroviral infections Diseases 0.000 description 1
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 1
- 238000002105 Southern blotting Methods 0.000 description 1
- 108010090804 Streptavidin Proteins 0.000 description 1
- 108010023197 Streptokinase Proteins 0.000 description 1
- 206010049418 Sudden Cardiac Death Diseases 0.000 description 1
- 239000004098 Tetracycline Substances 0.000 description 1
- 241000906446 Theraps Species 0.000 description 1
- 102100023132 Transcription factor Jun Human genes 0.000 description 1
- 239000007983 Tris buffer Substances 0.000 description 1
- 108090000435 Urokinase-type plasminogen activator Proteins 0.000 description 1
- 102000003990 Urokinase-type plasminogen activator Human genes 0.000 description 1
- 208000036142 Viral infection Diseases 0.000 description 1
- DPXJVFZANSGRMM-UHFFFAOYSA-N acetic acid;2,3,4,5,6-pentahydroxyhexanal;sodium Chemical compound [Na].CC(O)=O.OCC(O)C(O)C(O)C(O)C=O DPXJVFZANSGRMM-UHFFFAOYSA-N 0.000 description 1
- 229960001138 acetylsalicylic acid Drugs 0.000 description 1
- NIXOWILDQLNWCW-UHFFFAOYSA-N acrylic acid group Chemical group C(C=C)(=O)O NIXOWILDQLNWCW-UHFFFAOYSA-N 0.000 description 1
- 239000004480 active ingredient Substances 0.000 description 1
- 239000013543 active substance Substances 0.000 description 1
- 230000001154 acute effect Effects 0.000 description 1
- 102000030621 adenylate cyclase Human genes 0.000 description 1
- 108060000200 adenylate cyclase Proteins 0.000 description 1
- 239000002671 adjuvant Substances 0.000 description 1
- 239000000556 agonist Substances 0.000 description 1
- 229960000723 ampicillin Drugs 0.000 description 1
- AVKUERGKIZMTKX-NJBDSQKTSA-N ampicillin Chemical compound C1([C@@H](N)C(=O)N[C@H]2[C@H]3SC([C@@H](N3C2=O)C(O)=O)(C)C)=CC=CC=C1 AVKUERGKIZMTKX-NJBDSQKTSA-N 0.000 description 1
- 230000003321 amplification Effects 0.000 description 1
- 230000000692 anti-sense effect Effects 0.000 description 1
- 230000002785 anti-thrombosis Effects 0.000 description 1
- 230000005875 antibody response Effects 0.000 description 1
- 229940127090 anticoagulant agent Drugs 0.000 description 1
- 230000000890 antigenic effect Effects 0.000 description 1
- 229940127218 antiplatelet drug Drugs 0.000 description 1
- 238000013459 approach Methods 0.000 description 1
- 239000007864 aqueous solution Substances 0.000 description 1
- 230000008321 arterial blood flow Effects 0.000 description 1
- 230000003143 atherosclerotic effect Effects 0.000 description 1
- 238000000211 autoradiogram Methods 0.000 description 1
- 238000000376 autoradiography Methods 0.000 description 1
- 230000001042 autoregulative effect Effects 0.000 description 1
- 230000036772 blood pressure Effects 0.000 description 1
- 108010006025 bovine growth hormone Proteins 0.000 description 1
- 229940098773 bovine serum albumin Drugs 0.000 description 1
- 210000004556 brain Anatomy 0.000 description 1
- 238000010804 cDNA synthesis Methods 0.000 description 1
- 150000001718 carbodiimides Chemical class 0.000 description 1
- 235000014633 carbohydrates Nutrition 0.000 description 1
- 150000001720 carbohydrates Chemical class 0.000 description 1
- 239000001768 carboxy methyl cellulose Substances 0.000 description 1
- 230000003197 catalytic effect Effects 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 238000001311 chemical methods and process Methods 0.000 description 1
- 238000004587 chromatography analysis Methods 0.000 description 1
- 210000000349 chromosome Anatomy 0.000 description 1
- 238000012761 co-transfection Methods 0.000 description 1
- ZPUCINDJVBIVPJ-LJISPDSOSA-N cocaine Chemical compound O([C@H]1C[C@@H]2CC[C@@H](N2C)[C@H]1C(=O)OC)C(=O)C1=CC=CC=C1 ZPUCINDJVBIVPJ-LJISPDSOSA-N 0.000 description 1
- 208000014387 congenital coronary artery anomaly Diseases 0.000 description 1
- 230000021615 conjugation Effects 0.000 description 1
- 239000000356 contaminant Substances 0.000 description 1
- 238000013270 controlled release Methods 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 201000011639 coronary artery anomaly Diseases 0.000 description 1
- 201000011634 coronary artery vasospasm Diseases 0.000 description 1
- 208000026758 coronary atherosclerosis Diseases 0.000 description 1
- 239000012228 culture supernatant Substances 0.000 description 1
- 125000000151 cysteine group Chemical group N[C@@H](CS)C(=O)* 0.000 description 1
- 210000000805 cytoplasm Anatomy 0.000 description 1
- 230000002939 deleterious effect Effects 0.000 description 1
- 238000013461 design Methods 0.000 description 1
- 239000003599 detergent Substances 0.000 description 1
- 229960002086 dextran Drugs 0.000 description 1
- 229960000633 dextran sulfate Drugs 0.000 description 1
- 235000014113 dietary fatty acids Nutrition 0.000 description 1
- 230000029087 digestion Effects 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 230000005750 disease progression Effects 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 238000010494 dissociation reaction Methods 0.000 description 1
- 230000005593 dissociations Effects 0.000 description 1
- 238000009826 distribution Methods 0.000 description 1
- 101150052825 dnaK gene Proteins 0.000 description 1
- 230000003291 dopaminomimetic effect Effects 0.000 description 1
- 230000002222 downregulating effect Effects 0.000 description 1
- 239000003937 drug carrier Substances 0.000 description 1
- 230000004064 dysfunction Effects 0.000 description 1
- 210000001671 embryonic stem cell Anatomy 0.000 description 1
- 210000003038 endothelium Anatomy 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- LVGKNOAMLMIIKO-QXMHVHEDSA-N ethyl oleate Chemical compound CCCCCCCC\C=C/CCCCCCCC(=O)OCC LVGKNOAMLMIIKO-QXMHVHEDSA-N 0.000 description 1
- 229940093471 ethyl oleate Drugs 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 239000000194 fatty acid Substances 0.000 description 1
- 229930195729 fatty acid Natural products 0.000 description 1
- 239000010685 fatty oil Substances 0.000 description 1
- 239000003527 fibrinolytic agent Substances 0.000 description 1
- 239000012530 fluid Substances 0.000 description 1
- 230000006870 function Effects 0.000 description 1
- 108020001507 fusion proteins Proteins 0.000 description 1
- 102000037865 fusion proteins Human genes 0.000 description 1
- 239000007903 gelatin capsule Substances 0.000 description 1
- 238000010363 gene targeting Methods 0.000 description 1
- 230000004077 genetic alteration Effects 0.000 description 1
- 231100000118 genetic alteration Toxicity 0.000 description 1
- 210000002980 germ line cell Anatomy 0.000 description 1
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 description 1
- 239000010931 gold Substances 0.000 description 1
- 229910052737 gold Inorganic materials 0.000 description 1
- 230000012010 growth Effects 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 230000004217 heart function Effects 0.000 description 1
- 238000010438 heat treatment Methods 0.000 description 1
- 229960002897 heparin Drugs 0.000 description 1
- 229920000669 heparin Polymers 0.000 description 1
- 238000002744 homologous recombination Methods 0.000 description 1
- 230000006801 homologous recombination Effects 0.000 description 1
- 230000001900 immune effect Effects 0.000 description 1
- 238000003018 immunoassay Methods 0.000 description 1
- 230000005847 immunogenicity Effects 0.000 description 1
- 102000018358 immunoglobulin Human genes 0.000 description 1
- 239000012133 immunoprecipitate Substances 0.000 description 1
- 230000001771 impaired effect Effects 0.000 description 1
- 239000012535 impurity Substances 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 238000001727 in vivo Methods 0.000 description 1
- 238000011065 in-situ storage Methods 0.000 description 1
- 238000007918 intramuscular administration Methods 0.000 description 1
- 238000007912 intraperitoneal administration Methods 0.000 description 1
- 238000001990 intravenous administration Methods 0.000 description 1
- 238000002955 isolation Methods 0.000 description 1
- 210000003734 kidney Anatomy 0.000 description 1
- 210000003292 kidney cell Anatomy 0.000 description 1
- 238000000021 kinase assay Methods 0.000 description 1
- 210000000265 leukocyte Anatomy 0.000 description 1
- 239000002502 liposome Substances 0.000 description 1
- 210000004185 liver Anatomy 0.000 description 1
- 210000004072 lung Anatomy 0.000 description 1
- 210000004698 lymphocyte Anatomy 0.000 description 1
- 238000012423 maintenance Methods 0.000 description 1
- 210000001161 mammalian embryo Anatomy 0.000 description 1
- 239000011159 matrix material Substances 0.000 description 1
- 238000000520 microinjection Methods 0.000 description 1
- 239000002480 mineral oil Substances 0.000 description 1
- 235000010446 mineral oil Nutrition 0.000 description 1
- 239000003068 molecular probe Substances 0.000 description 1
- 230000004899 motility Effects 0.000 description 1
- 210000003205 muscle Anatomy 0.000 description 1
- 238000002703 mutagenesis Methods 0.000 description 1
- 231100000350 mutagenesis Toxicity 0.000 description 1
- 230000035772 mutation Effects 0.000 description 1
- 208000010125 myocardial infarction Diseases 0.000 description 1
- 229960004927 neomycin Drugs 0.000 description 1
- 229920001220 nitrocellulos Polymers 0.000 description 1
- 238000003199 nucleic acid amplification method Methods 0.000 description 1
- 239000002853 nucleic acid probe Substances 0.000 description 1
- 235000015097 nutrients Nutrition 0.000 description 1
- 238000002515 oligonucleotide synthesis Methods 0.000 description 1
- 210000000056 organ Anatomy 0.000 description 1
- 210000001672 ovary Anatomy 0.000 description 1
- 210000000496 pancreas Anatomy 0.000 description 1
- 239000000546 pharmaceutical excipient Substances 0.000 description 1
- 239000012071 phase Substances 0.000 description 1
- 239000006187 pill Substances 0.000 description 1
- 210000002826 placenta Anatomy 0.000 description 1
- 239000013600 plasmid vector Substances 0.000 description 1
- 229940127126 plasminogen activator Drugs 0.000 description 1
- 239000000106 platelet aggregation inhibitor Substances 0.000 description 1
- 229920002401 polyacrylamide Polymers 0.000 description 1
- 238000002264 polyacrylamide gel electrophoresis Methods 0.000 description 1
- 239000001267 polyvinylpyrrolidone Substances 0.000 description 1
- 229920000036 polyvinylpyrrolidone Polymers 0.000 description 1
- 235000013855 polyvinylpyrrolidone Nutrition 0.000 description 1
- 239000005373 porous glass Substances 0.000 description 1
- 230000037452 priming Effects 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 230000004853 protein function Effects 0.000 description 1
- 230000006798 recombination Effects 0.000 description 1
- 238000005215 recombination Methods 0.000 description 1
- 230000009467 reduction Effects 0.000 description 1
- 230000000630 rising effect Effects 0.000 description 1
- 235000019515 salmon Nutrition 0.000 description 1
- 108010073863 saruplase Proteins 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 230000028327 secretion Effects 0.000 description 1
- 239000013049 sediment Substances 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 239000008159 sesame oil Substances 0.000 description 1
- 235000011803 sesame oil Nutrition 0.000 description 1
- 238000010008 shearing Methods 0.000 description 1
- 230000035939 shock Effects 0.000 description 1
- 230000019491 signal transduction Effects 0.000 description 1
- 238000010583 slow cooling Methods 0.000 description 1
- 239000011734 sodium Substances 0.000 description 1
- 229910052708 sodium Inorganic materials 0.000 description 1
- 235000019812 sodium carboxymethyl cellulose Nutrition 0.000 description 1
- 229920001027 sodium carboxymethylcellulose Polymers 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- FQENQNTWSFEDLI-UHFFFAOYSA-J sodium diphosphate Chemical compound [Na+].[Na+].[Na+].[Na+].[O-]P([O-])(=O)OP([O-])([O-])=O FQENQNTWSFEDLI-UHFFFAOYSA-J 0.000 description 1
- 239000001488 sodium phosphate Substances 0.000 description 1
- 229910000162 sodium phosphate Inorganic materials 0.000 description 1
- 239000012064 sodium phosphate buffer Substances 0.000 description 1
- 229940048086 sodium pyrophosphate Drugs 0.000 description 1
- 239000002904 solvent Substances 0.000 description 1
- 238000000527 sonication Methods 0.000 description 1
- 239000000600 sorbitol Substances 0.000 description 1
- 210000004989 spleen cell Anatomy 0.000 description 1
- 239000003381 stabilizer Substances 0.000 description 1
- 229960005202 streptokinase Drugs 0.000 description 1
- 238000007920 subcutaneous administration Methods 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- 238000001308 synthesis method Methods 0.000 description 1
- 235000020357 syrup Nutrition 0.000 description 1
- 239000006188 syrup Substances 0.000 description 1
- 239000003826 tablet Substances 0.000 description 1
- 229960002180 tetracycline Drugs 0.000 description 1
- 229930101283 tetracycline Natural products 0.000 description 1
- 235000019364 tetracycline Nutrition 0.000 description 1
- 150000003522 tetracyclines Chemical class 0.000 description 1
- 235000019818 tetrasodium diphosphate Nutrition 0.000 description 1
- 239000001577 tetrasodium phosphonato phosphate Substances 0.000 description 1
- 229940124597 therapeutic agent Drugs 0.000 description 1
- 229960000103 thrombolytic agent Drugs 0.000 description 1
- 238000011200 topical administration Methods 0.000 description 1
- 238000001890 transfection Methods 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
- 238000011426 transformation method Methods 0.000 description 1
- 230000007704 transition Effects 0.000 description 1
- 150000003626 triacylglycerols Chemical class 0.000 description 1
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 description 1
- RYFMWSXOAZQYPI-UHFFFAOYSA-K trisodium phosphate Chemical compound [Na+].[Na+].[Na+].[O-]P([O-])([O-])=O RYFMWSXOAZQYPI-UHFFFAOYSA-K 0.000 description 1
- 241001515965 unidentified phage Species 0.000 description 1
- 230000003827 upregulation Effects 0.000 description 1
- 229960005356 urokinase Drugs 0.000 description 1
- 239000003981 vehicle Substances 0.000 description 1
- 230000003612 virological effect Effects 0.000 description 1
- 150000003722 vitamin derivatives Chemical class 0.000 description 1
- 229960005080 warfarin Drugs 0.000 description 1
- PJVWKTKQMONHTI-UHFFFAOYSA-N warfarin Chemical compound OC=1C2=CC=CC=C2OC(=O)C=1C(CC(=O)C)C1=CC=CC=C1 PJVWKTKQMONHTI-UHFFFAOYSA-N 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
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
- C12N9/10—Transferases (2.)
- C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
- C12N9/1205—Phosphotransferases with an alcohol group as acceptor (2.7.1), e.g. protein kinases
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K2217/00—Genetically modified animals
- A01K2217/05—Animals comprising random inserted nucleic acids (transgenic)
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Wood Science & Technology (AREA)
- Molecular Biology (AREA)
- Engineering & Computer Science (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Organic Chemistry (AREA)
- Zoology (AREA)
- Genetics & Genomics (AREA)
- Medicinal Chemistry (AREA)
- Microbiology (AREA)
- Biotechnology (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Biomedical Technology (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Peptides Or Proteins (AREA)
Description
WO 99/49062 PCT/US99/06662 IDENTIFICATION OF A cDNA ASSOCIATED WITH ISCHEMIA IN HUMAN HEART TISSUE 5 FIELD OF THE INVENTION The invention relates generally to the changes in gene expression in ischemic heart 10 tissue compared to normal human heart tissue. The invention relates specifically to a novel human gene which is up-regulated in ischemic human heart tissue. This application is related to U.S. Provisional Application Serial No. 60/079,377, filed March 26, 1998, which is incorporated herein by reference. 15 BACKGROUND OF THE INVENTION Cardiovascular disease is a general diagnostic category consisting of several separate diseases. Coronary heart disease and cerebrovascular disease are major components of cardiovascular disease with 478,530 dying of coronary heart disease and 144,070 dying of cerebrovascular disease in the U.S. in 1991. See Cecil Textbook of 20 Medicine, Bennet and Plum Eds., W.B. Saunders Co., 1996. Many of the acute forms of coronary heart disease are caused by coronary artery abnormalities such as coronary atherosclerosis. Among the more common causes and contributing factors in sudden cardiac death are chronic ischemic heart disease and ischemic cardiomyopathy. Chronic ischemic heart disease and ischemic cardiomyopathy are caused in part by 25 episodes of insufficient myocardial oxygen supply. Myocardial oxygen supply is governed by coronary blood flow and the ability of the myocardium to extract oxygen from the blood delivered to it. Unlike other organs, the heart always extracts oxygen with near maximal efficiency from the blood. Even under situations of minimal demand, there is little potential for enhanced oxygen extraction to counter increased oxygen demands. 30 Coronary blood flow, on the other hand, can increase several-fold in normal subjects as a result of coronary arterial vasodilation. Coronary arterial vasodilation is regulated by the coronary endothelium which releases vasodilatory substances, most importantly nitric oxide. In atherosclerotic coronary heart disease, endothelial dysfunction may diminish WO 99/49062 PCT/US99/06662 -2 production of vasodilatory substances, such as nitric oxide. Myocardial ischemia results when autoregulatory vasodilation is prevented, whether by flow-limiting coronary arterial stenosis or by endothelial dysfunction. In both cases, arterial blood flow can no longer increase proportional to rising oxygen demands. In other situations, myocardial ischemia 5 may occur when oxygen demands are constant but there is a primary decrease in coronary blood flow mediated via coronary artery spasm, rapid evolution of the underlying atherosclerotic plaque leading to a reduced coronary arterial lumen caliber, and/or intermittent microvascular plugging by platelet aggregates. At the molecular level, ischemia is characterized by the differential expression of 10 numerous genes compared to normal heart tissue. For instance, in human heart failure caused by ischemic cardiomyopathy, expression of the P,- and P 2 -adrenergic receptors of the adenyl cyclase signal transduction system is impaired by reductions in the expression of mRNA for each receptor (Ihl-Vahl et al., 1996, J Mol. Cell. Cardiol. 28:1-10). Ischemic injury is also known to lead to the differential expression of heat shock and 15 immediate early genes such as hsp70, c-fos, c-jun,jun-B as well the genes encoding angiotensin receptor subtypes (Plumier et al., 1996, J. Mol. Cell. Cardiol. 28:1251-1260; Wharton et al., 1998, J Pharmoc. Experiment. Therap. 284(1) 323-336; and Heads et al. 1995, J Mol. Cell. Cardiol. 27:2133-2148). The identification of new genes that are differentially expressed in ischemic heart 20 tissue will allow for the development of numerous diagnostic and therapeutic applications such as molecular probes and new agents which modulate the activity or expression of these genes. SUMMARY OF THE INVENTION 25 The present invention is based on our discovery of a new gene which is up regulated in ischemic heart tissue. The invention includes isolated nucleic acid molecules selected from the group consisting of an isolated nucleic acid molecule that encodes the amino acid sequence of SEQ ID No.2, an isolated nucleic acid molecule that encodes a fragment of at least 10 amino acids of SEQ ID No.2, an isolated nucleic acid molecule 30 which hybridizes to a nucleic acid molecule comprising SEQ ID No. 1 under conditions of sufficient stringency to produce a clear signal and an isolated nucleic acid molecule which hybridizes to a nucleic acid molecule that encodes the amino acid sequence of SEQ ID No.
WO 99/49062 PCT/US99/06662 -3 2 under conditions of sufficient stringency to produce a clear signal. The present invention further includes the nucleic acid molecules operably linked to one or more expression control elements, including a vector comprising the isolated nucleic acid molecules. The invention further includes host cells transformed to contain 5 the nucleic acid molecules of the invention and methods for producing a protein comprising the step of culturing a host cell transformed with the nucleic acid molecule of the invention under conditions in which the protein is expressed. The invention further provides an isolated polypeptide selected from the group consisting of an isolated polypeptide comprising the amino acid sequence of SEQ ID No.2, 10 an isolated polypeptide comprising a fragment of at least 10 amino acids of SEQ ID No.2, an isolated polypeptide comprising conservative amino acid substitutions of SEQ ID No.2 and naturally occurring amino acid sequence variants of SEQ ID No.2. The invention further provides an isolated antibody that binds to a polypeptide of the invention, including monoconal and polyclonal antibodies. 15 The invention further provides methods of identifying an agent which modulates the expression of a nucleic acid encoding the protein having the sequence of SEQ ID No.2 comprising the steps of exposing cells which express the nucleic acid to the agent and determining whether the agent modulates expression of said nucleic acid, thereby identifying an agent which modulates the expression of a nucleic acid encoding the protein 20 having the sequence of SEQ ID No.2. The invention further provides methods of identifying an agent which modulates at least one activity of a protein comprising the sequence of SEQ ID No.2 comprising the steps of exposing cells which express the protein to the agent and determining whether the agent modulates at least on activity of said protein, thereby identifying an agent which 25 modulates at least one activity of a protein comprising the sequence of SEQ ID No.2. The invention further provides methods of identifying binding partners for a protein comprising the sequence of SEQ ID No. 2, comprising the steps of exposing said protein to a potential binding partner and determining if the potential binding partner binds to said protein, thereby identifying binding partners for a protein comprising the sequence 30 of SEQID No. 2 The present invention further provides methods of modulating the expression of a nucleic acid encoding the protein having the sequence of SEQ ID No.2 comprising the step WO 99/49062 PCT/US99/06662 -4 of administering an effective amount of an agent which modulates the expression of a nucleic acid encoding the protein having the sequence of SEQ ID No.2. The invention also provides methods of modulating at least one activity of a protein comprising the sequence of SEQ ID No.2 comprising the step of administering an effective amount of an 5 agent which modulates at least one activity of a protein comprising the sequence of SEQ ID No.2. BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 Figure 1 is a section of an autoradiograph of the expression profile 10 generated from cDNAs made with RNA isolated from ischemic and control, non-ischemic heart tissue. Figure 2 Figure 2 shows the potential binding, phosphorylation and enzymatic sites of the protein of SEQ ID No.2. Figure 3 Figure 3 is a Northern blot of RNA isolated from various tissues. 15 Figure 4 Figure 4 is a PCR-expression analysis of RNA isolated from various tissues. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT I. General Description 20 The present invention is based in part on identifying a new gene that is differentially regulated or expressed in human ischemic heart tissue compared to normal human heart tissue. This gene, which may be distantly related to human myosin light chain kinases, encodes a protein predicted to consist of 819 amino acids. The protein can serve as a target for agents that can be used to modulate the 25 expression or activity of the protein. For example, agents may be identified which modulate biological processes associated with ischemic injury to the heart such as chronic ischemic heart disease and ischemic cardiomyopathy. Agents may also be identified which modulate the biological processes associated with recovery to ischemic injury to the heart. 30 The present invention is further based on the development of methods for isolating binding partners that bind to the protein. Probes based on the protein are used as capture probes to isolate potential binding partners, such as other proteins. Dominant negative WO 99/49062 PCT/US99/06662 -5 proteins, DNAs encoding these proteins, antibodies to these proteins, peptide fragments of these proteins or mimics of these proteins may be introduced into cells to affect function. Additionally, these proteins provide a novel target for screening of synthetic small molecules and combinatorial or naturally occurring compound libraries to discover novel 5 therapeutics to regulate heart function. II. Specific Embodiments A. The Protein Associated with Ischemic Heart Tissue The present invention provides isolated protein, allelic variants of the protein, and 10 conservative amino acid substitutions of the protein. As used herein, the protein or polypeptide refers to a protein that has the human amino acid sequence of that depicted in SEQ ID No.2. The invention includes naturally occurring allelic variants and proteins that have a slightly different amino acid sequence than that specifically recited above. Allelic variants, though possessing a slightly different amino acid sequence than those recited 15 above, will still have the same or similar biological functions associated with the 819 amino acid protein. As used herein, the family of proteins related to the 819 amino acid protein refer to proteins that have been isolated from organisms in addition to humans. The methods used to identify and isolate other members of the family of proteins related to the 819 amino 20 acid protein are described below. The proteins of the present invention are preferably in isolated form. As used herein, a protein is said to be isolated when physical, mechanical or chemical methods are employed to remove the protein from cellular constituents that are normally associated with the protein. A skilled artisan can readily employ standard purification methods to 25 obtain an isolated protein. The proteins of the present invention further include conservative variants of the proteins herein described. As used herein, a conservative variant refers to alterations in the amino acid sequence that do not adversely affect the biological functions of the protein. A substitution, insertion or deletion is said to adversely affect the protein when the altered 30 sequence prevents or disrupts a biological function associated with the protein. For example, the overall charge, structure or hydrophobic/hydrophilic properties of the protein can be altered without adversely affecting a biological activity. Accordingly, the amino WO 99/49062 PCT/US99/06662 -6 acid sequence can be altered, for example, to render the peptide more hydrophobic or more hydrophilic, without adversely affecting the biological activities of the protein. Ordinarily, the allelic variants, the conservative substitution variants, the members of the protein family, will have an amino acid sequence having at least 75% amino acid 5 sequence identity with the human sequence set forth in SEQ ID No.2, more preferably at least 80%, even more preferably at least 90%, and most preferably at least 95%. Identity or homology with respect to such sequences is defined herein as the percentage of amino acid residues in the candidate sequence that are identical with the known peptides, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent 10 homology, and not considering any conservative substitutions as part of the sequence identity. N-terminal, C-terminal or internal extensions, deletions, or insertions into the peptide sequence shall not be construed as affecting homology. Thus, the proteins of the present invention include molecules having the amino acid sequence disclosed in SEQ ID No.2; fragments thereof having a consecutive sequence 15 of at least about 3, 5, 10 or 15 amino acid residues of the 819 amino acid protein; amino acid sequence variants of such sequence wherein an amino acid residue has been inserted N- or C-terminal to, or within, the disclosed sequence; and amino acid sequence variants of the disclosed sequence, or their fragments as defined above, that have been substituted by another residue. Contemplated variants further include those containing predetermined 20 mutations by, e.g., homologous recombination, site-directed or PCR mutagenesis, and the corresponding proteins of other animal species, including but not limited to rabbit, rat, murine, porcine, bovine, ovine, equine and non-human primate species, and the alleles or other naturally occurring variants of the family of proteins; and derivatives wherein the protein has been covalently modified by substitution, chemical, enzymatic, or other 25 appropriate means with a moiety other than a naturally occurring amino acid (for example a detectable moiety such as an enzyme or radioisotope). As described below, members of the family of proteins can be used: 1) to identify agents which modulate at least one activity of the protein, including agents which may modulate phosphorylation mediated by the protein, 2) in methods of identifying binding 30 partners for the protein, 3) as an antigen to raise polyclonal or monoclonal antibodies, and 4) as a therapeutic agent.
WO 99/49062 PCT/US99/06662 -7 B. Nucleic Acid Molecules The present invention further provides nucleic acid molecules that encode the protein having SEQ ID No.2 and the related proteins herein described, preferably in isolated form. As used herein, "nucleic acid" is defined as RNA or DNA that encodes a 5 peptide as defined above, or is complementary to nucleic acid sequence encoding such peptides, or hybridizes to such nucleic acid and remains stably bound to it under appropriate stringency conditions, or encodes a polypeptide sharing at least 75% sequence identity, preferably at least 80%, and more preferably at least 85%, with the peptide sequences. Specifically contemplated are genomic DNA, cDNA, mRNA and antisense 10 molecules, as well as nucleic acids based on alternative backbone or including alternative bases whether derived from natural sources or synthesized. Such hybridizing or complementary nucleic acids, however, are defined further as being novel and unobvious over any prior art nucleic acid including that which encodes, hybridizes under appropriate stringency conditions, or is complementary to nucleic acid encoding a protein according to 15 the present invention. "Stringent conditions" are those that (1) employ low ionic strength and high temperature for washing, for example, 0.015M NaCl/0.0015M sodium titrate/0.1% SDS at 50 0 C., or (2) employ during hybridization a denaturing agent such as formamide, for example, 50% (vol/vol) formamide with 0.1% bovine serum albumin/0.1% Ficoll/0.1% 20 polyvinylpyrrolidone/50 mM sodium phosphate buffer at pH 6.5 with 750 mM NaC1, 75 mM sodium citrate at 42 0 C. Another example is use of 50% formamide, 5 x SSC (0.75M NaC1, 0.075 M sodium citrate), 50 mM sodium phosphate (pH 6.8), 0.1% sodium pyrophosphate, 5 x Denhardt's solution, sonicated salmon sperm DNA (50 gg/ml), 0.1% SDS, and 10% dextran sulfate at 42 0 C., with washes at 42 0 C. in 0.2 x SSC and 0.1% SDS. 25 A skilled artisan can readily determine and vary the stringency conditions appropriately to obtain a clear and detectable hybridization signal. As used herein, a nucleic acid molecule is said to be "isolated" when the nucleic acid molecule is substantially separated from contaminant nucleic acid encoding other polypeptides from the source of nucleic acid. 30 The present invention further provides fragments of the encoding nucleic acid molecule. As used herein, a fragment of an encoding nucleic acid molecule refers to a small portion of the entire protein encoding sequence. The size of the fragment will be determined WO 99/49062 PCT/US99/06662 -8 by the intended use. For example, if the fragment is chosen so as to encode an active portion of the protein, the fragment will need to be large enough to encode the functional region(s) of the protein. If the fragment is to be used as a nucleic acid probe or PCR primer, then the fragment length is chosen so as to obtain a relatively small number of false positives during 5 probing/priming. Fragments of the encoding nucleic acid molecules of the present invention (i.e., synthetic oligonucleotides) that are used as probes or specific primers for the polymerase chain reaction (PCR), or to synthesize gene sequences encoding proteins of the invention can easily be synthesized by chemical techniques, for example, the phosphotriester method of Matteucci 10 et al., (J Am. Chem. Soc. 103:3185-3191, 1981) or using automated synthesis methods. In addition, larger DNA segments can readily be prepared by well known methods, such as synthesis of a group of oligonucleotides that define various modular segments of the gene, followed by ligation of oligonucleotides to build the complete modified gene. The encoding nucleic acid molecules of the present invention may further be 15 modified so as to contain a detectable label for diagnostic and probe purposes. A variety of such labels are known in the art and can readily be employed with the encoding molecules herein described. Suitable labels include, but are not limited to, biotin, radiolabeled nucleotides and the like. A skilled artisan can employ any of the art known labels to obtain a labeled encoding nucleic acid molecule. 20 Modifications to the primary structure itself by deletion, addition, or alteration of the amino acids incorporated into the protein sequence during translation can be made without destroying the activity of the protein. Such substitutions or other alterations result in proteins having an amino acid sequence encoded by a nucleic acid falling within the contemplated scope of the present invention. 25 C. Isolation of Other Related Nucleic Acid Molecules As described above, the identification of the human nucleic acid molecule having SEQ ID No.1 allows a skilled artisan to isolate nucleic acid molecules that encode other members of the protein family in addition to the human sequence herein described. Further, the 30 presently disclosed nucleic acid molecules allow a skilled artisan to isolate nucleic acid molecules that encode other members of the family of proteins in addition to the 819 amino acid protein having SEQ ID No.2.
WO 99/49062 PCT/US99/06662 -9 Essentially, a skilled artisan can readily use the amino acid sequence of SEQ ID No.2 to generate antibody probes to screen expression libraries prepared from appropriate cells. Typically, polyclonal antiserum from mammals such as rabbits immunized with the purified protein (as described below) or monoclonal antibodies can be used to probe a mammalian 5 cDNA or genomic expression library, such as lambda gtll library, to obtain the appropriate coding sequence for other members of the protein family. The cloned cDNA sequence can be expressed as a fusion protein, expressed directly using its own control sequences, or expressed by constructions using control sequences appropriate to the particular host used for expression of the enzyme. 10 Alternatively, a portion of the coding sequence herein described can be synthesized and used as a probe to retrieve DNA encoding a member of the protein family from any mammalian organism. Oligomers containing approximately 18-20 nucleotides (encoding about a 6-7 amino acid stretch) are prepared and used to screen genomic DNA or cDNA libraries to obtain hybridization under stringent conditions or conditions of sufficient stringency 15 to eliminate an undue level of false positives. Additionally, pairs of oligonucleotide primers can be prepared for use in a polymerase chain reaction (PCR) to selectively clone an encoding nucleic acid molecule. A PCR denature/anneal/extend cycle for using such PCR primers is well known in the art and can readily be adapted for use in isolating other encoding nucleic acid molecules. 20 D. rDNA molecules Containing a Nucleic Acid Molecule The present invention further provides recombinant DNA molecules (rDNAs) that contain a coding sequence. As used herein, a rDNA molecule is a DNA molecule that has been subjected to molecular manipulation in situ. Methods for generating rDNA molecules are 25 well known in the art. See for example, Sambrook et al., Molecular Cloning (1989). In the preferred rDNA molecules, a coding DNA sequence is operably linked to expression control sequences and/or vector sequences. The choice of vector and/or expression control sequences to which one of the protein family encoding sequences of the present invention is operably linked depends directly, as is 30 well known in the art, on the functional properties desired, e.g., protein expression, and the host cell to be transformed. A vector contemplated by the present invention is at least capable of directing the replication or insertion into the host chromosome, and preferably also expression, WO 99/49062 PCT/US99/06662 -10 of the structural gene included in the rDNA molecule. Expression control elements that are used for regulating the expression of an operably linked protein encoding sequence are known in the art and include, but are not limited to, inducible promoters, constitutive promoters, secretion signals, and other regulatory elements. 5 Preferably, the inducible promoter is readily controlled, such as being responsive to a nutrient in the host cell's medium. In one embodiment, the vector containing a coding nucleic acid molecule will include a prokaryotic replicon, i.e., a DNA sequence having the ability to direct autonomous replication and maintenance of the recombinant DNA molecule extrachromosomally in a prokaryotic host 10 cell, such as a bacterial host cell, transformed therewith. Such replicons are well known in the art. In addition, vectors that include a prokaryotic replicon may also include a gene whose expression confers a detectable marker such as a drug resistance. Typical bacterial drug resistance genes are those that confer resistance to ampicillin or tetracycline. Vectors that include a prokaryotic replicon can further include a prokaryotic or 15 bacteriophage promoter capable of directing the expression (transcription and translation) of the coding gene sequences in a bacterial host cell, such as E. coli. A promoter is an expression control element formed by a DNA sequence that permits binding of RNA polymerase and transcription to occur. Promoter sequences compatible with bacterial hosts are typically provided in plasmid vectors containing convenient restriction sites for insertion of a DNA 20 segment of the present invention. Typical of such vector plasmids are pUC8, pUC9, pBR322 and pBR329 available from Biorad Laboratories, (Richmond, CA), pPL and pKK223 available from Pharmacia, Piscataway, N.J. Expression vectors compatible with eukaryotic cells, preferably those compatible with vertebrate cells, can also be used to form a rDNA molecules the contains a coding sequence. 25 Eukaryotic cell expression vectors are well known in the art and are available from several commercial sources. Typically, such vectors are provided containing convenient restriction sites for insertion of the desired DNA segment. Typical of such vectors are pSVL and pKSV 10 (Pharmacia), pBPV-1/pML2d (International Biotechnologies, Inc.), pTDT1 (ATCC, #31255), the vector pCDM8 described herein, and the like eukaryotic expression vectors. 30 Eukaryotic cell expression vectors used to construct the rDNA molecules of the present invention may further include a selectable marker that is effective in an eukaryotic cell, preferably a drug resistance selection marker. A preferred drug resistance marker is the gene WO 99/49062 PCT/US99/06662 -11 whose expression results in neomycin resistance, i.e., the neomycin phosphotransferase (neo) gene. (Southern et al., J. Mol. Anal. Genet. 1:327-341, 1982.) Alternatively, the selectable marker can be present on a separate plasmid, and the two vectors are introduced by co transfection of the host cell, and selected by culturing in the appropriate drug for the selectable 5 marker. E. Host Cells Containing an Exogenously Supplied Coding Nucleic Acid Molecule The present invention further provides host cells transformed with a nucleic acid 10 molecule that encodes a protein of the present invention. The host cell can be either prokaryotic or eukaryotic. Eukaryotic cells useful for expression of a protein of the invention are not limited, so long as the cell line is compatible with cell culture methods and compatible with the propagation of the expression vector and expression of the gene product. Preferred eukaryotic host cells include, but are not limited to, yeast, insect and mammalian cells, 15 preferably vertebrate cells such as those from a mouse, rat, monkey or human cell line. Preferred eukaryotic host cells include Chinese hamster ovary (CHO) cells available from the ATCC as CCL61, NIH Swiss mouse embryo cells NIH/3T3 available from the ATCC as CRL 1658, baby hamster kidney cells (BHK), and the like eukaryotic tissue culture cell lines. Any prokaryotic host can be used to express a rDNA molecule encoding a protein of 20 the invention. The preferred prokaryotic host is E. coli. Transformation of appropriate cell hosts with a rDNA molecule of the present invention is accomplished by well known methods that typically depend on the type of vector used and host system employed. With regard to transformation of prokaryotic host cells, electroporation and salt treatment methods are typically employed; see, for example, Cohen et 25 al., Proc. Natl. Acad. Sci. USA 69:2110, 1972; and Maniatis et al., Molecular Cloning, A Laboratory Mammal, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1982). With regard to transformation of vertebrate cells with vectors containing rDNAs, electroporation, cationic lipid or salt treatment methods are typically employed; see, for example, Graham et al., Virol. 52:456, 1973; Wigler et al., Proc. Natl. Acad. Sci. USA 76:1373-76, 1979. 30 Successfully transformed cells, i.e., cells that contain a rDNA molecule of the present invention, can be identified by well known techniques including the selection for a selectable marker. For example, cells resulting from the introduction of an rDNA of the present invention WO 99/49062 PCT/US99/06662 -12 can be cloned to produce single colonies. Cells from those colonies can be harvested, lysed and their DNA content examined for the presence of the rDNA using a method such as that described by Southern, J. Mol. Biol. 98:503, 1975, or Berent et al., Biotech. 3:208, 1985 or the proteins produced from the cell assayed via an immunological method. 5 F. Production of Recombinant Proteins using a rDNA Molecule The present invention further provides methods for producing a protein of the invention using nucleic acid molecules herein described. In general terms, the production of a recombinant form of a protein typically involves the following steps: 10 First, a nucleic acid molecule is obtained that encodes a protein of the invention, such as the nucleic acid molecule depicted in SEQ ID No. 1, or nucleotides 136-2592 of SEQ ID No.1. If the encoding sequence is uninterrupted by introns, it is directly suitable for expression in any host. The nucleic acid molecule is then preferably placed in operable linkage with suitable 15 control sequences, as described above, to form an expression unit containing the protein open reading frame. The expression unit is used to transform a suitable host and the transformed host is cultured under conditions that allow the production of the recombinant protein. Optionally the recombinant protein is isolated from the medium or from the cells; recovery and purification of the protein may not be necessary in some instances where some impurities may 20 be tolerated. Each of the foregoing steps can be done in a variety of ways. For example, the desired coding sequences may be obtained from genomic fragments and used directly in appropriate hosts. The construction of expression vectors that are operable in a variety of hosts is accomplished using appropriate replicons and control sequences, as set forth above. The 25 control sequences, expression vectors, and transformation methods are dependent on the type of host cell used to express the gene and were discussed in detail earlier. Suitable restriction sites can, if not normally available, be added to the ends of the coding sequence so as to provide an excisable gene to insert into these vectors. A skilled artisan can readily adapt any host/expression system known in the art for use with the nucleic acid molecules of the 30 invention to produce recombinant protein.
WO 99/49062 PCT/US99/06662 - 13 G. Methods to Identify Binding Partners Another embodiment of the present invention provides methods for use in isolating and identifying binding partners of proteins of the invention. In detail, a protein of the invention is mixed with a potential binding partner or an extract or fraction of a cell under 5 conditions that allow the association of potential binding partners with the protein of the invention. After mixing, peptides, polypeptides, proteins or other molecules that have become associated with a protein of the invention are separated from the mixture. The binding partner that bound to the protein of the invention can then be removed and further analyzed. To identify and isolate a binding partner, the entire protein, for instance the 10 entire 819 amino acid protein of SEQ ID No.2 can be used. Alternatively, a fragment of the protein can be used. As used herein, a cellular extract refers to a preparation or fraction which is made from a lysed or disrupted cell. The preferred source of cellular extracts will be cells derived from human heart tissue, for instance, ischemic human heart tissue. Alternatively, 15 cellular extracts may be prepared from normal human heart tissue or available cell lines, particularly heart or muscle derived cell lines. A variety of methods can be used to obtain an extract of a cell. Cells can be disrupted using either physical or chemical disruption methods. Examples of physical disruption methods include, but are not limited to, sonication and mechanical shearing. 20 Examples of chemical lysis methods include, but are not limited to, detergent lysis and enzyme lysis. A skilled artisan can readily adapt methods for preparing cellular extracts in order to obtain extracts for use in the present methods. Once an extract of a cell is prepared, the extract is mixed with the protein of the invention under conditions in which association of the protein with the binding partner can 25 occur. A variety of conditions can be used, the most preferred being conditions that closely resemble conditions found in the cytoplasm of a human cell. Features such as osmolarity, pH, temperature, and the concentration of cellular extract used, can be varied to optimize the association of the protein with the binding partner. After mixing under appropriate conditions, the bound complex is separated from 30 the mixture. A variety of techniques can be utilized to separate the mixture. For example, antibodies specific to a protein of the invention can be used to immunoprecipitate the binding partner complex. Alternatively, standard chemical separation techniques such as WO 99/49062 PCT/US99/06662 -14 chromatography and density/sediment centrifugation can be used. After removal of nonassociated cellular constituents found in the extract, the binding partner can be dissociated from the complex using conventional methods. For example, dissociation can be accomplished by altering the salt concentration or pH of the 5 mixture. To aid in separating associated binding partner pairs from the mixed extract, the protein of the invention can be immobilized on a solid support. For example, the protein can be attached to a nitrocellulose matrix or acrylic beads. Attachment of the protein to a solid support aids in separating peptide/binding partner pairs from other constituents found 10 in the extract. The identified binding partners can be either a single protein or a complex made up of two or more proteins. Alternatively, the nucleic acid molecules of the invention can be used in a yeast two-hybrid system. The yeast two-hybrid system has been used to identify other protein partner pairs and can readily be adapted to employ the nucleic acid molecules herein 15 described. H. Methods to Identify Agents that Modulate the Expression a Nucleic Acid Encoding the Ischemic Heart Associated Protein Another embodiment of the present invention provides methods for identifying 20 agents that modulate the expression of a nucleic acid encoding a protein of the invention such as a protein having the amino acid sequence of SEQ ID No. 2. Such assays may utilize any available means of monitoring for changes in the expression level of the nucleic acids of the invention. As used herein, an agent is said to modulate the expression of a nucleic acid of the invention, for instance a nucleic acid encoding the protein having the 25 sequence of SEQ ID No.2, if it is capable of up- or down-regulating expression of the nucleic acid in a cell. In one assay format, cell lines that contain reporter gene fusions between the open reading frame defined by nucleotides 136-2592 of SEQ ID No.1 and any assayable fusion partner may be prepared. Numerous assayable fusion partners are known and readily 30 available including the firefly luciferase gene and the gene encoding chloramphenicol acetyltransferase (Alam et al. (1990) Anal. Biochem. 188:245-254). Cell lines containing the reporter gene fusions are then exposed to the agent to be tested under appropriate WO 99/49062 PCT/US99/06662 - 15 conditions and time. Differential expression of the reporter gene between samples exposed to the agent and control samples identifies agents which modulate the expression of a nucleic acid encoding the protein having the sequence of SEQ ID No.2. Additional assay formats may be used to monitor the ability of the agent to 5 modulate the expression of a nucleic acid encoding a protein of the invention such as the protein having SEQ ID No.2. For instance, mRNA expression may be monitored directly by hybridization to the nucleic acids of the invention. Cell lines are exposed to the agent to be tested under appropriate conditions and time and total RNA or mRNA is isolated by standard procedures such those disclosed in Sambrook et al. (Molecular Cloning: A 10 Laboratory Manual, 2nd Ed. Clod Spring Harbor Laboratory Press, 1989). Probes to detect differences in RNA expression levels between cells exposed to the agent and control cells may be prepared from the nucleic acids of the invention. It is preferable, but not necessary, to design probes which hybridize only with target nucleic acids under conditions of high stringency. Only highly complementary nucleic acid 15 hybrids form under conditions of high stringency. Accordingly, the stringency of the assay conditions determines the amount of complementarity which should exist between two nucleic acid strands in order to form a hybrid. Stringency should be chosen to maximize the difference in stability between the probe:target hybrid and potential probe:non-target hybrids. 20 Probes may be designed from the nucleic acids of the invention through methods known in the art. For instance, the G+C content of the probe and the probe length can affect probe binding to its target sequence. Methods to optimize probe specificity are commonly available in Sambrook et al. (Molecular Cloning: A Laboratory Approach, Cold Spring Harbor Press, NY, 1989) or Ausubel et al. (Current Protocols in Molecular 25 Biology, Greene Publishing Co., NY, 1995). Hybridization conditions are modified using known methods, such as those described by Sambrook et al. and Ausubel et al. as required for each probe. Hybridization of total cellular RNA or RNA enriched for polyA RNA can be accomplished in any available format. For instance, total cellular RNA or RNA enriched for polyA RNA can 30 be affixed to a solid support and the solid support exposed to at least one probe comprising at least one, or part of one of the sequences of the invention under conditions in which the probe will specifically hybridize. Alternatively, nucleic acid fragments comprising at least WO 99/49062 PCT/US99/06662 -16 one, or part of one of the sequences of the invention can be affixed to a solid support, such as a porous glass wafer. The glass wafer can then be exposed to total cellular RNA or polyA RNA from a sample under conditions in which the affixed sequences will specifically hybridize. Such glass wafers and hybridization methods, such as those 5 disclosed by Beattie (WO 95/11755), are widely available. By examining for the ability of a given probe to specifically hybridize to an RNA sample from an untreated cell population and from a cell population exposed to the agent, agents which up or down regulate the expression of a nucleic acid encoding the protein having the sequence of SEQ ID No.2 are identified. 10 I. Methods to Identify Agents that Modulate at Least One Activity of the Ischemic Heart Associated Protein Another embodiment of the present invention provides methods for identifying agents that modulate at least one activity of a protein of the invention such as the protein 15 having the amino acid sequence of SEQ ID No.2. Such methods or assays may utilize any means of monitoring or detecting the desired activity. In one format, the relative amounts of a protein of the invention between a cell population that has been exposed to the agent to be tested compared to an un-exposed control cell population may be assayed. In this format, probes such as specific antibodies 20 are used to monitor the differential expression of the protein in the different cell populations. Cell lines or populations are exposed to the agent to be tested under appropriate conditions and time. Cellular lysates may be prepared from the exposed cell line or population and a control, unexposed cell line or population. The cellular lysates are then analyzed with the probe. 25 Antibody probes are prepared by immunizing suitable mammalian hosts in appropriate immunization protocols using the peptides, polypeptides or proteins of the invention if they are of sufficient length, or, if desired, or if required to enhance immunogenicity, conjugated to suitable carriers. Methods for preparing immunogenic conjugates with carriers such as BSA, KLH, or other carrier proteins are well known in the 30 art. In some circumstances, direct conjugation using, for example, carbodiimide reagents may be effective; in other instances linking reagents such as those supplied by Pierce Chemical Co., Rockford, IL, may be desirable to provide accessibility to the hapten. The WO 99/49062 PCT/US99/06662 -17 hapten peptides can be extended at either the amino or carboxy terminus with a Cys residue or interspersed with cysteine residues, for example, to facilitate linking to a carrier. Administration of the immunogens is conducted generally by injection over a suitable time period and with use of suitable adjuvants, as is generally understood in the art. During the 5 immunization schedule, titers of antibodies are taken to determine adequacy of antibody formation. While the polyclonal antisera produced in this way may be satisfactory for some applications, for pharmaceutical compositions, use of monoclonal preparations is preferred. Immortalized cell lines which secrete the desired monoclonal antibodies may be 10 prepared using the standard method of Kohler and Milstein or modifications which effect immortalization of lymphocytes or spleen cells, as is generally known. The immortalized cell lines secreting the desired antibodies are screened by immunoassay in which the antigen is the peptide hapten, polypeptide or protein. When the appropriate immortalized cell culture secreting the desired antibody is identified, the cells can be cultured either in 15 vitro or by production in ascites fluid. The desired monoclonal antibodies are then recovered from the culture supernatant or from the ascites supernatant. Fragments of the monoclonals or the polyclonal antisera which contain the immunologically significant portion can be used as antagonists, as well as the intact antibodies. Use of immunologically reactive fragments, such as the Fab, Fab', 20 of F(ab') 2 fragments is often preferable, especially in a therapeutic context, as these fragments are generally less immunogenic than the whole immunoglobulin. The antibodies or fragments may also be produced, using current technology, by recombinant means. Regions that bind specifically to the desired regions of receptor can also be produced in the context of chimeras with multiple species origin. 25 In an alternative format, a specific activity of a protein of the invention may be assayed, such as the ability of the protein to phosphorylate a substrate such as myosin. Cell lines or populations are exposed under appropriate conditions to the agent to be tested. Agents which modulate the kinase activity of the protein of the invention are identified by assaying the kinase activity of the protein from the exposed cell line or 30 population and a control, unexposed cell line or population, thereby identifying agents which modulate the kinase activity of the protein. Kinase assays to measure the ability of the agent to modulate the kinase activity of WO 99/49062 PCT/US99/06662 -18 a protein of the invention are widely available such as the assays disclosed by Mishima et al. (1996) J Biochem. 119:906-913) and Michnoff et al. (1986) J1 Biol. Chem. 261:8320 8326. Alternative assay formats include actin-myosin motility assays such as those disclosed by Kohama et al. (1996) TIPS 17:284-287 or Warrick et al. (1987) Ann. Rev. 5 Cell. Biol. 3:379-421. Agents that are assayed in the above method can be randomly selected or rationally selected or designed. As used herein, an agent is said to be randomly selected when the agent is chosen randomly without considering the specific sequences involved in the association of the a protein of the invention alone or with its associated substrates, binding 10 partners, etc. An example of randomly selected agents is the use a chemical library or a peptide combinatorial library, or a growth broth of an organism. As used herein, an agent is said to be rationally selected or designed when the agent is chosen on a nonrandom basis which takes into account the sequence of the target site and/or its conformation in connection with the agent's action. As described in the 15 Examples, there are proposed binding sites for ATP/GTP and calmodulin as well as cAMP/cGMP kinase sites, TyrP sites and Ser/Thr kinase (catalytic) sites in the protein having SEQ ID No.2. Agents can be rationally selected or rationally designed by utilizing the peptide sequences that make up these sites. For example, a rationally selected peptide agent can be a peptide whose amino acid sequence is identical to the ATP or calmodulin 20 binding sites or domains. The agents of the present invention can be, as examples, peptides, small molecules, vitamin derivatives, as well as carbohydrates. A skilled artisan can readily recognize that there is no limit as to the structural nature of the agents of the present invention. The peptide agents of the invention can be prepared using standard solid phase (or 25 solution phase) peptide synthesis methods, as is known in the art. In addition, the DNA encoding these peptides may be synthesized using commercially available oligonucleotide synthesis instrumentation and produced recombinantly using standard recombinant production systems. The production using solid phase peptide synthesis is necessitated if non-gene-encoded amino acids are to be included. 30 Another class of agents of the present invention are antibodies immunoreactive with critical positions of proteins of the invention. Antibody agents are obtained by immunization of suitable mammalian subjects with peptides that contain as antigenic WO 99/49062 PCT/US99/06662 - 19 regions those portions of the protein intended to be targeted by the antibodies. J. Uses for Agents that Modulate at Least One Activity of the Ischemic Heart Associated Protein 5 As provided in the Examples, the proteins and nucleic acids of the invention, such as the protein having the amino acid sequence of SEQ ID No.1, are up-regulated in ischemic heart tissue. Agents that modulate or down-regulate the expression of the protein or agents such as agonists or antagonists of at least one activity of the protein may be used to modulate biological and pathologic processes associated with the protein's function and 10 activity. As used herein, a subject can be any mammal, so long as the mammal is in need of modulation of a pathological or biological process mediated by a protein of the invention. The term "mammal" is meant an individual belonging to the class Mammalia. The invention is particularly useful in the treatment of human subjects. 15 As used herein, a biological or pathological process mediated by a protein of the invention may include binding of substrates such as ATP, GTP or calmodulin or phosphorylation of a substrate such as skeletal myosin. Pathological processes refer to a category of biological processes which produce a deleterious effect. For example, expression or up-regulation of expression of a protein of 20 the invention is associated with chronic ischemic heart disease and ischemic cardiomyopathy. As used herein, an agent is said to modulate a pathological process when the agent reduces the degree or severity of the process. For instance, chronic ischemic heart disease or ischemic cardiomyopathy may be prevented or disease progression modulated after an ischemic event by the administration of agents which reduce or 25 modulate in some way the expression or at least one activity of a protein of the invention. The agents of the present invention can be provided alone, or in combination with other agents that modulate a particular pathological process. For example, an agent of the present invention can be administered in combination with anti-thrombotic agents. As used herein, two agents are said to be administered in combination when the two agents 30 are administered simultaneously or are administered independently in a fashion such that the agents will act at the same time. The agents of the present invention can be administered via parenteral, WO 99/49062 PCT/US99/06662 -20 subcutaneous, intravenous, intramuscular, intraperitoneal, transdermal, or buccal routes. Alternatively, or concurrently, administration may be by the oral route. The dosage administered will be dependent upon the age, health, and weight of the recipient, kind of concurrent treatment, if any, frequency of treatment, and the nature of the effect desired. 5 The present invention further provides compositions containing one or more agents which modulate expression or at least one activity of a protein of the invention. While individual needs vary, determination of optimal ranges of effective amounts of each component is within the skill of the art. Typical dosages comprise 0.1 to 100 jg/kg body wt. The preferred dosages comprise 0.1 to 10 gg/kg body wt. The most preferred dosages 10 comprise 0.1 to 1 jg/kg body wt. In addition to the pharmacologically active agent, the compositions of the present invention may contain suitable pharmaceutically acceptable carriers comprising excipients and auxiliaries which facilitate processing of the active compounds into preparations which can be used pharmaceutically for delivery to the site of action. Suitable 15 formulations for parenteral administration include aqueous solutions of the active compounds in water-soluble form, for example, water-soluble salts. In addition, suspensions of the active compounds as appropriate oily injection suspensions may be administered. Suitable lipophilic solvents or vehicles include fatty oils, for example, sesame oil, or synthetic fatty acid esters, for example, ethyl oleate or triglycerides. 20 Aqueous injection suspensions may contain substances which increase the viscosity of the suspension include, for example, sodium carboxymethyl cellulose, sorbitol, and/or dextran. Optionally, the suspension may also contain stabilizers. Liposomes can also be used to encapsulate the agent for delivery into the cell. The pharmaceutical formulation for systemic administration according to the 25 invention may be formulated for enteral, parenteral or topical administration. Indeed, all three types of formulations may be used simultaneously to achieve systemic administration of the active ingredient. Suitable formulations for oral administration include hard or soft gelatin capsules, pills, tablets, including coated tablets, elixirs, suspensions, syrups or inhalations and 30 controlled release forms thereof. In practicing the methods of this invention, the compounds of this invention may be used alone or in combination, or in combination with other therapeutic or diagnostic WO 99/49062 PCT/US99/06662 -21 agents. In certain preferred embodiments, the compounds of this invention may be coadministered along with other compounds typically prescribed for these conditions according to generally accepted medical practice, such as anticoagulant agents, thrombolytic agents, or other antithrombotics, including platelet aggregation inhibitors, 5 tissue plasminogen activators, urokinase, prourokinase, streptokinase, heparin, aspirin, or warfarin. The compounds of this invention can be utilized in vivo, ordinarily in mammals, such as humans, sheep, horses, cattle, pigs, dogs, cats, rats and mice, or in vitro. K. Transgenic Animals 10 Transgenic animals containing a mutant, knock-out or modified gene corresponding to the cDNA sequence of SEQ ID No.1 or a construct to modify the expression level of the gene, for instance for up-regulating the expression, are also included in the invention. Transgenic animals are genetically modified animals into which recombinant, exogenous or cloned genetic material has been experimentally transferred. 15 Such genetic material is often referred to as a "transgene". The nucleic acid sequence of the transgene, in this case a form of SEQ ID No.1, may be integrated either at a locus of a genome where that particular nucleic acid sequence is not otherwise normally found or at the normal locus for the transgene. The transgene may consist of nucleic acid sequences derived from the genome of the same species or of a different species than the species of 20 the target animal. The term "germ cell line transgenic animal" refers to a transgenic animal in which the genetic alteration or genetic information was introduced into a germ line cell, thereby conferring the ability of the transgenic animal to transfer the genetic information to offspring. If such offspring in fact possess some or all of that alteration or genetic 25 information, then they too are transgenic animals. The alteration or genetic information may be foreign to the species of animal to which the recipient belongs, foreign only to the particular individual recipient, or may be genetic information already possessed by the recipient. In the last case, the altered or introduced gene may be expressed differently than the native gene. 30 Transgenic animals can be produced by a variety of different methods including transfection, electroporation, microinjection, gene targeting in embryonic stem cells and recombinant viral and retroviral infection (see, e.g., U.S. Patent No. 4,736,866; U.S. Patent WO 99/49062 PCT/US99/06662 -22 No. 5,602,307; Mullins et al. (1993) Hypertension 22(4):630-633; Brenin et al. (1997) Surg. Oncol. 6(2)99-110; Tuan (ed.), Recombinant Gene Expression Protocols, Methods in Molecular Biology No. 62, Humana Press (1997)). A number of recombinant or transgenic mice have been produced, including those 5 which express an activated oncogene sequence (U.S. Patent No. 4,736,866); express simian SV 40 T-antigen (U.S. Patent No. 5,728,915); lack the expression of interferon regulatory factor 1 (IRF-1) (U.S. Patent No. 5,731,490); exhibit dopaminergic dysfunction (U.S. Patent No. 5,723,719); express at least one human gene which participates in blood pressure control (U.S. Patent No. 5,731,489); display greater similarity to the conditions 10 existing in naturally occurring Alzheimer's disease (U.S. Patent No. 5,720,936); have a reduced capacity to mediate cellular adhesion (U.S. Patent No. 5,602,307); possess a bovine growth hormone gene (Clutter et al. (1996) Genetics 143(4):1753-1760); or, are capable of generating a fully human antibody response (McCarthy (1997) The Lancet 349(9049):405). 15 While mice and rats remain the animals of choice for most transgenic experimentation, in some instances it is preferable or even necessary to use alternative animal species. Transgenic procedures have been successfully utilized in a variety of non murine animals, including sheep, goats, pigs, dogs, cats, monkeys, chimpanzees, hamsters, rabbits, cows and guinea pigs (see, e.g., Kim et al. (1997) Mol. Reprod. Dev. 46(4):515 20 526; Houdebine (1995) Reprod. Nutr. Dev. 35(6):609-617; Petters (1994) Reprod. Fertil. Dev. 6(5):643-645; Schnieke et al. (1997) Science 278(5346):2130-2133; and Amoah (1997) J. Animal Science 75(2):578-585). The method of introduction of nucleic acid fragments into recombination competent mammalian cells can be by any method which favors co-transformation of 25 multiple nucleic acid molecules. Detailed procedures for producing transgenic animals are readily available to one skilled in the art, including the disclosures in U.S. Patent No. 5,489,743 and U.S. Patent No. 5,602,307. Without further description, it is believed that one of ordinary skill in the art can, using the preceding description and the following illustrative examples, make and utilize 30 the compounds of the present invention and practice the claimed methods. The following working examples therefore, specifically point out preferred embodiments of the present invention, and are not to be construed as limiting in any way the remainder of the WO 99/49062 PCT/US99/06662 - 23 disclosure. EXAMPLES Example 1 5 Identification of Differentially Expressed Ischemic Heart mRNA Heart tissue was obtained from five male patients with inotrope-dependent post ischemic cardiomyopathy exhibiting severe myocyte and or cardiac hypertrophy with at least three years since their first myocardial infarction. Heart tissue was also obtained from 5 female patients with idiopathic dilated cardiomyopathy exhibiting severe myocyte 10 and/or cardiac hypertrophy and CHF duration of at least 2 years. Total cellular RNA was prepared from the heart tissue described above as well as from control, non-ischemic heart tissue using the procedure of Newburger et al. (1981) J. Biol. Chem. 266(24): 16171-7 and Newburger et al. (1988) Proc. Natl. Acad. Sci. USA 85:5215-5219. 15 Synthesis of cDNA was performed as previously described by Prashar et al. in WO 97/05286 and in Prashar et al. (1996) Proc. Natl. Acad. Sci. USA 93:659-663. Briefly, cDNA was synthesized according to the protocol described in the GIBCO/BRL kit for cDNA synthesis. The reaction mixture for first-strand synthesis included 6 gg of total RNA, and 200 ng of a mixture of 1-base anchored oligo(dT) primers with all three 20 possible anchored bases (ACGTAATACGACTCACTATAGGGCGAATTGGGTCGACTTTTTTTTTTTTTTTTT n1 wherein nl=A/C or G) (SEQ ID No.3) along with other components for first-strand synthesis reaction except reverse transcriptase. This mixture was incubated at 65 0 C for 5m, chilled on ice and the process repeated. Alternatively, the reaction mixture may 25 include 10pg of total RNA, and 2 pmol of 1 of the 2-base anchored oligo(dT) primers a heel such as RP5.0 (CTCTCAAGGATCTTACCGCTTgAT) (SEQ ID No.4), or RP6.0
(TAATACCGCGCCACATAGCAT
1 8 CG) (SEQ ID No.5), or RP9.2
(CAGGGTAGACGACGCTACGCT
8 GA) (SEQ ID No.6) along with other components for first-strand synthesis reaction except reverse transcriptase. This mixture was then 30 layered with mineral oil and incubated at 65 0 C for 7 min followed by 50 0 C for another 7 min. At this stage, 2 .l of Superscript reverse transcriptase (200 units/Ml; GIBCO/BRL) was added quickly and mixed, and the reaction continued for 1 hr at 45-50oC. Second- WO 99/49062 PCT/US99/06662 - 24 strand synthesis was performed at 16oC for 2 hr. At the end of the reaction, the cDNAs were precipitated with ethanol and the yield of cDNA was calculated. In our experiments, =200 ng of cDNA was obtained from 10gg of total RNA. The adapter oligonucleotide sequences were 5 Al (TAGCGTCCGGCGCAGCGACGGCCAG) (SEQ ID No.7) and A2 (GATCCTGGCCGTCGGCTGTCTGTCGGCGC) (SEQ ID No.8). One microgram of oligonucleotide A2 was first phosphorylated at the 5' end using T4 polynucleotide kinase (PNK). After phosphorylation, PNK was heated denatured, and lyg of the oligonucleotide Al was added along with 10x annealing buffer (1 M NaC1/100 10 mM Tris-HC1, pH8.0/10 mM EDTA, pH8.0) in a final vol of 20 Al. This mixture was then heated at 65 0 C for 10 min followed by slow cooling to room temperature for 30 min, resulting in formation of the Y adapter at a final concentration of 100 ng/ul. About 20 ng of the cDNA was digested with 4 units ofBgl II in a final vol of 10 Al for 30 min at 37oC. Two microliters (=4 ng of digested cDNA) of this reaction mixture was then used for 15 ligation to 100 ng (= 50-fold) of the Y-shaped adapter in a final vol of 5 l for 16 hr at 15oC. After ligation, the reaction mixture was diluted with water to a final vol of 80 kl (adapter ligated cDNA concentration, = 50 pg/M) and heated at 65 C for 10 min to denature T4 DNA ligase, and 2-Al aliquots (with = 100 pg of cDNA) were used for PCR. The following sets of primers were used for PCR amplification of the adapter 20 ligated 3' -end cDNAs:
TGAAGCCGAGACGTCGGTCG(T)
8 nl, n2 (wherein nl, n2 = AA, AC, AG AT CA CC CG CT GA GC GG and GT) (SEQ ID No.9) as the 3' primer with Al as the 5' primer or alternatively RP 5.0, RP 6.0, or RP 9.2 used as 3' primers with primer A1.1 serving as the 5' primer. To detect the PCR products on the display gel, 24 pmol of 25 oligonucleotide Al or A1.1 was 5' -end-labeled using 15 Al of [y- 32 P]ATP (Amersham; 3000 Ci/mmol) and PNK in a final volume of 20 Al for 30 min at 370C. After heat denaturing PNK at 65 C for 20 min, the labeled oligonucleotide was diluted to a final concentration of 2 AM in 80 gl with unlabeled oligonucleotide A1.1. The PCR mixture (20/1) consisted of 2 Ml (= 100 pg) of the template, 2l of 10Ox PCR buffer (100 mM 30 Tris.HC1, pH 8.3/500 mM KC1), 2 Al of 15 mM MgCl 2 to yield 1.5 mM final Mg 2 + concentration optimum in the reaction mixture, 200 AM dNTPs, 200 nM each 5' and 3' PCR primers, and 1 unit of Amplitaq Gold. Primers and dNTPs were added after WO 99/49062 PCT/US99/06662 -25 preheating the reaction mixture containing the rest of the components at 85 0 C. This "hot start" PCR was done to avoid artefactual amplification arising out of arbitrary annealing of PCR primers at lower temperature during transition from room temperature to 94 0 C in the first PCR cycle. PCR consisted of 5 cycles of 94oC for 30 sec, 55oC for 2 min, and 72'C 5 for 60 sec followed by 25 cycles of 94oC for 30 sec, 60'C for 2 min, and 72oC for 60 sec. A higher number of cycles resulted in smeary gel patterns. PCR products ( 2 .5pl) were analyzed on 6% polyacrylamide sequencing gel. For double or multiple digestion following adapter ligation, 13.2 4l of the ligated cDNA sample was digested with a secondary restriction enzyme(s) in a final vol of 20 Ml. From this solution, 34 1 was used as 10 template for PCR. This template vol of 3 Ml carried = 100 pg of the cDNA and 10 mM MgCl 2 (from the 10x enzyme buffer), which diluted to the optimum of 1.5 mM in the final PCR vol of 20 1l. Since Mg 2 + comes from the restriction enzyme buffer, it was not included in the reaction mixture when amplifying secondarily cut cDNA. Individual cDNA fragments corresponding to mRNA species were separated by denaturing 15 polyacrylamide gel electrophoresis and visualized by autoradiography. Bands were extracted from the display gels as described by Liang et al. (1995 Curr. Opin. Immunol. 7:274-280), reamplified using the 5' and 3' primers, and subcloned into pCR-Script with high efficiency using the PCR-Script cloning kit from Stratagene. Plasmids were sequenced by cycle sequencing on an ABI automated sequencer. Alternatively, bands 20 were extracted (cored) from the display gels, PCR amplified and sequenced directly without subcloning. Figure 1 presents a section of an autoradiogram of the expression profile generated from cDNAs made from RNA isolated from control and ischemic heart tissue. Ci-25 is a band that corresponds to a cDNA derived from a mRNA species that is up 25 regulated in ischemic heart tissue in a female patient with idiopathic dilated cardiomyopathy exhibiting severe myocyte and/or cardiac hypertrophy and CHF duration of at least 2 years. The band corresponding to Ci-25 was sequenced. The sequence of CI 25 is: GGCTCACATCTGTAATCCCAGCACTTTGGGAGGCCAAGGTGGGCAGATTGCTG 30 GCCAACATGGTAAAACCCCATCTCTAAAGATATAAAAATTAGCTGGGCGTGGT GGCGCATACCTGTAATCCCAGCTACTTGGGAGGCTAAGGCACAAGAATCACTT
AAACAGGAGGCGGGGGTTGCAGTGAGCTGAGATCACACCACTGCACTCCAGC
WO 99/49062 PCT/US99/06662 - 26 CTGGGTGGCAGAGCAAAACTTTGTCCCCACCCCTGACAAAAAACAAACAAAC AAACAAAACAAAAAAAAAACCTGTCAATTCA (SEQ ID No.10). Example 2 5 Cloning of a Full Length cDNA Corresponding to Ci-25 The full length cDNA corresponding to Ci-25 band was obtained by the oligo-pulling method. Briefly, a gene-specific oligo was designed based on cDNA fragment Ci-25. The oligo was labeled with biotin and used to hybridize with 2 ug of single strand plasmid DNA (cDNA recombinants) from a human heart cDNA library 10 following the procedures of Sambrook et al.. The hybridized cDNAs were separated by streptavidin-conjugated beads and eluted by heating. The eluted cDNA was converted to double strand plasmid DNA and used to transform E. coli cells (DH10B) and the longest cDNA was screened. After confirmed by PCR using gene-specific primers, the cDNA clone was subjected to DNA sequencing. 15 The nucleotide sequence of the full-length cDNA corresponding to the differentially regulated Ci-25 band is set forth in SEQ ID No.1. The cDNA comprises 5532 base pairs with an open reading frame encoding a protein predicted to contain 819 amino acids. The predicted amino acid sequence is presented in SEQ ID Nos. 1 and 2. Comparison of the open reading to the sequences of known proteins and genes indicates 20 that the gene may be distantly related to a known myosin light chain kinase gene as it exhibits 61% identity to a myosin light chain kinase at the nucleotide sequence level. Example 3 Northern Blot and PCR Expression Analysis 25 The tissue distribution of RNA encoding the differentially regulated gene encoding the protein of SEQ ID NO.2 was analyzed by Northern Blot as well as PCR expression analysis of RNA isolated from various tissues. RNA was isolated from human heart, brain, placenta, lung, liver, skeletal muscle, kidney, leukocytes, testis and pancreas using standard procedures. Northern blots were prepared using a probe derived from SEQ ID 30 No.1 with hybridization conditions as described by Sambrook et al. PCR expression analysis was also performed using primers derived from SEQ ID No.1 using AmpliTaq Gold PCR amplification kits (Perkin Elmer). Figure 3 is a Northern blot demonstrating the WO 99/49062 PCT/US99/06662 - 27 presence of specific RNA in heart and skeletal muscle. Figure 4 is a PCR expression analysis demonstrating the presence of specific RNA in heart and testis. Example 4 5 Generation of Transgenic Mice Transgenic mice were generated using a gene corresponding to the cDNA sequence of SEQ ID No.1. Briefly, a cDNA fragment encoding the protein of SEQ ID No.2 was cloned into an alpha-MHC vector using standard techniques. The vector was then used to produce transgenic mice in accordance with standard techniques. Of the 22 resulting mice, 10 six were confirmed by Southern blot to be transgene positive. Although the present invention has been described in detail with reference to the examples above, it is understood that various modifications can be made without departing from the spirit of the invention. Accordingly, the invention is limited only by the 15 following claims. All cited patents and publications referred to in this application are herein incorporated by reference in their entirety. 20
Claims (22)
1. An isolated nucleic acid molecule selected from the group consisting of an isolated nucleic acid molecule that encodes the amino acid sequence of SEQ ID No.2, an 5 isolated nucleic acid molecule that encodes a fragment of at last 10 amino acids of SEQ ID No.2, an isolated nucleic acid molecule which hybridizes to a nucleic acid molecule comprising SEQ ID No. 1 under conditions of sufficient stringency to produce a clear signal and an isolated nucleic acid molecule which hybridizes to a nucleic acid molecule that encodes the amino acid sequence of SEQ ID No. 2 under conditions of sufficient 10 stringency to produce a clear signal.
2. The isolated nucleic acid molecule of claim 1, wherein the nucleic acid molecule comprises the sequence of SEQ ID No.1. 15
3. The isolated nucleic acid molecule of claim 2, wherein the nucleic acid molecule consists of the sequence of SEQ ID No.1.
4. The isolated nucleic acid molecule of claim 1, wherein the nucleic acid molecule comprises nucleotides 136 to 2592 of SEQ ID No.1. 20
5. The isolated nucleic acid molecule of claim 2, wherein the nucleic acid molecule consists of nucleotides 136 to 2592 of SEQ ID No.1.
6. The isolated nucleic acid molecule of any one of claims 1-5, wherein said 25 nucleic acid molecule is operably linked to one or more expression control elements.
7. A vector comprising an isolated nucleic acid molecule of any one of claims 1-5.
8. A host cell transformed to contain the nucleic acid molecule of any one claims 30 1-5.
9. A host cell comprising a vector of claim 7. WO 99/49062 PCT/US99/06662 -29
10. A host cell of claim 9, wherein said host is selected from the group consisting of prokaryotic hosts and eukaryotic hosts.
11. A method for producing a protein comprising the step of culturing a host cell 5 transformed with the nucleic acid molecule of any one of claims 1-5 under conditions in which the protein is expressed.
12. The method of claim 11, wherein said host cell is selected from the group consisting of prokaryotic hosts and eukaryotic hosts. 10
13. An isolated polypeptide produced by the method of claim 11.
14. An isolated polypeptide selected from the group consisting of an isolated polypeptide comprising the amino acid sequence of SEQ ID No.2, an isolated polypeptide 15 comprising a fragment of at least 10 amino acids of SEQ ID No.2, an isolated polypeptide comprising conservative amino acid substitutions of SEQ ID No.2 and naturally occurring amino acid sequence variants of SEQ ID No.2.
15. An isolated antibody that binds to a polypeptide of either claim 13 or 14. 20
16. The antibody of claim 14 wherein said antibody is a monoclonal or polyclonal antibody.
17. A method of identifying an agent which modulates the expression of a nucleic 25 acid encoding the protein having the sequence of SEQ ID No.2 comprising the steps of: exposing cells which express the nucleic acid to the agent; and determining whether the agent modulates expression of said nucleic acid, thereby identifying an agent which modulates the expression of a nucleic acid encoding the protein having the sequence of SEQ ID No.2. 30
18. A method of identifying an agent which modulates at least one activity of a protein comprising the sequence of SEQ ID No.2 comprising the steps of: WO 99/49062 PCT/US99/06662 -30 exposing cells which express the protein to the agent; determining whether the agent modulates at least on activity of said protein, thereby identifying an agent which modulates at least one activity of a protein comprising the sequence of SEQ ID No.2. 5
19. The method of claim 19, wherein the agent modulates the ability of the protein to phosphorylate a substrate.
20. A method of identifying binding partners for a protein comprising the sequence 10 of SEQ ID No. 2, comprising the steps of: exposing said protein to a potential binding partner; and determining if the potential binding partner binds to said protein, thereby identifying binding partners for a protein comprising the sequence of SEQ ID No. 2. 15
21. A method of modulating the expression of a nucleic acid encoding the protein having the sequence of SEQ ID No.2 comprising the step of: administering an effective amount of an agent which modulates the expression of a nucleic acid encoding the protein having the sequence of SEQ ID No.2. 20
22. A method of modulating at least one activity of a protein comprising the sequence of SEQ ID No.2 comprising the step of: administering an effective amount of an agent which modulates at least one activity of a protein comprising the sequence of SEQ ID No.2. 25
Applications Claiming Priority (3)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US7937798P | 1998-03-26 | 1998-03-26 | |
US60079377 | 1998-03-26 | ||
PCT/US1999/006662 WO1999049062A1 (en) | 1998-03-26 | 1999-03-26 | IDENTIFICATION OF A cDNA ASSOCIATED WITH ISCHEMIA IN HUMAN HEART TISSUE |
Publications (1)
Publication Number | Publication Date |
---|---|
AU3208999A true AU3208999A (en) | 1999-10-18 |
Family
ID=22150166
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
AU32089/99A Abandoned AU3208999A (en) | 1998-03-26 | 1999-03-26 | Identification of a cdna associated with ischemia in human heart tissue |
Country Status (4)
Country | Link |
---|---|
EP (1) | EP1073756A4 (en) |
AU (1) | AU3208999A (en) |
CA (1) | CA2323574A1 (en) |
WO (1) | WO1999049062A1 (en) |
Families Citing this family (6)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CA2383244A1 (en) * | 1999-05-28 | 2000-12-07 | Sugen, Inc. | Protein kinases |
CA2398430A1 (en) * | 2000-01-25 | 2001-08-02 | Sugen, Inc. | Human protein kinases and protein kinase-like enzymes |
US20040072170A1 (en) * | 2000-05-30 | 2004-04-15 | Bunk Daniela Beck Nee | Novel target genes for diseases of the heart |
US9580515B2 (en) * | 2006-08-21 | 2017-02-28 | Zensun (Shanghai) Science & Technology, Co., Ltd. | Neukinase, a downstream protein of neuregulin |
JP2009242388A (en) * | 2008-03-14 | 2009-10-22 | National Cardiovascular Center | Application of heart-specific kinase for diagnosis and treatment of cardiac insufficiency |
WO2013053076A1 (en) | 2011-10-10 | 2013-04-18 | Zensun (Shanghai)Science & Technology Limited | Compositions and methods for treating heart failure |
Family Cites Families (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US6174993B1 (en) * | 1997-05-21 | 2001-01-16 | The Children's Medical Center Corp. | Short peptides which selectively modulate the activity of serine/threonine kinases |
WO2002024889A2 (en) * | 2000-09-12 | 2002-03-28 | The Government Of The United States Of America, As Represented By The Secretary Of The Department Of Health And Human Services | Optimized cardiac contraction through differential phosphorylation of myosin |
-
1999
- 1999-03-26 WO PCT/US1999/006662 patent/WO1999049062A1/en not_active Application Discontinuation
- 1999-03-26 EP EP99914188A patent/EP1073756A4/en not_active Withdrawn
- 1999-03-26 AU AU32089/99A patent/AU3208999A/en not_active Abandoned
- 1999-03-26 CA CA002323574A patent/CA2323574A1/en not_active Abandoned
Also Published As
Publication number | Publication date |
---|---|
CA2323574A1 (en) | 1999-09-30 |
EP1073756A4 (en) | 2003-01-22 |
EP1073756A1 (en) | 2001-02-07 |
WO1999049062A1 (en) | 1999-09-30 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
US6486299B1 (en) | Genes and proteins predictive and therapeutic for stroke, hypertension, diabetes and obesity | |
CA2301862A1 (en) | Atrial natriuretic factor mutants and ischemic stroke | |
AU3208999A (en) | Identification of a cdna associated with ischemia in human heart tissue | |
US6649377B1 (en) | Human aggrecanase and nucleic acid compositions encoding the same | |
US20050037454A1 (en) | Gene associated with bone disorders | |
WO2000011942A9 (en) | IDENTIFICATION OF A cDNA ASSOCIATED WITH ISCHEMIA IN HUMAN HEART TISSUE | |
US6399761B1 (en) | Nucleic acid encoding human potassium channel K+ nov1 protein | |
WO2001062767A1 (en) | IDENTIFICATION OF A cDNA ASSOCIATED WITH VENTRICULAR FUNCTION IN HUMAN MYOCARDIAL TISSUE | |
WO2000060122A1 (en) | IDENTIFICATION OF A cDNA ASSOCIATED WITH ISCHEMIA IN HUMAN HEART TISSUE | |
AU1908599A (en) | Mammalian alpha helical protein-1 | |
WO2003025152A2 (en) | IDENTIFICATION OF A cDNA ASSOCIATED WITH ISCHEMIA IN HUMAN HEART TISSUE | |
US6303770B1 (en) | Nucleic acids encoding mammalian alpha helical protein-1 | |
WO2001027287A2 (en) | Telomerase reverse transcriptase (tert) genes | |
US6803184B1 (en) | MPR-related ABC transporter encoding nucleic acids and methods of use thereof | |
WO2001005803A9 (en) | NOVEL cDNAs ASSOCIATED WITH RENAL DISEASE | |
WO2002012262A1 (en) | IDENTIFICATION OF cDNAs ASSOCIATED WITH BENIGN PROSTATIC HYPERPLASIA | |
WO2000052026A1 (en) | IDENTIFICATION OF A cDNA ASSOCIATED WITH COMPENSATORY HYPERTROPHY IN RENAL TISSUE | |
US20030079239A1 (en) | Gene Associated with bone disorders | |
US20040242468A1 (en) | Gene involved in mineral deposition and uses thereof | |
US20030099992A1 (en) | Genes associated with mast cell activation | |
WO2001005804A1 (en) | Novel genes associated with renal disease | |
JP4117359B2 (en) | Novel extracellular matrix protein specifically expressed in hair follicle placode and gene thereof | |
WO2000078788A1 (en) | NOVEL cDNA ASSOCIATED WITH RENAL DISEASE | |
US20030166881A1 (en) | Novel genes associated with allergic hypersensitivity and mast cell activation | |
US20030054385A1 (en) | Human ubiquitin-conjugating enzymes |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
MK5 | Application lapsed section 142(2)(e) - patent request and compl. specification not accepted |