US20160282350A1 - Methods of diagnosing cancer - Google Patents
Methods of diagnosing cancer Download PDFInfo
- Publication number
- US20160282350A1 US20160282350A1 US15/178,595 US201615178595A US2016282350A1 US 20160282350 A1 US20160282350 A1 US 20160282350A1 US 201615178595 A US201615178595 A US 201615178595A US 2016282350 A1 US2016282350 A1 US 2016282350A1
- Authority
- US
- United States
- Prior art keywords
- antibody
- protein
- seq
- cdna
- cancer
- 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
- 238000000034 method Methods 0.000 title claims abstract description 99
- 206010028980 Neoplasm Diseases 0.000 title claims abstract description 22
- 201000011510 cancer Diseases 0.000 title claims abstract description 17
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 204
- 102000004169 proteins and genes Human genes 0.000 claims abstract description 176
- 230000014509 gene expression Effects 0.000 claims abstract description 76
- 108010076504 Protein Sorting Signals Proteins 0.000 claims abstract description 38
- 108090000765 processed proteins & peptides Proteins 0.000 claims abstract description 32
- 102000004196 processed proteins & peptides Human genes 0.000 claims abstract description 22
- 239000012472 biological sample Substances 0.000 claims abstract description 16
- 229920001184 polypeptide Polymers 0.000 claims abstract description 14
- 230000002596 correlated effect Effects 0.000 claims abstract description 3
- 125000003275 alpha amino acid group Chemical group 0.000 claims abstract 8
- 239000013068 control sample Substances 0.000 claims abstract 4
- 210000001519 tissue Anatomy 0.000 claims description 90
- 241000282414 Homo sapiens Species 0.000 claims description 31
- 239000012634 fragment Substances 0.000 claims description 28
- 206010006187 Breast cancer Diseases 0.000 claims description 14
- 102000008394 Immunoglobulin Fragments Human genes 0.000 claims description 12
- 108010021625 Immunoglobulin Fragments Proteins 0.000 claims description 12
- -1 tissues Substances 0.000 claims description 11
- 208000026310 Breast neoplasm Diseases 0.000 claims description 10
- 208000033133 Testicular seminomatous germ cell tumor Diseases 0.000 claims description 8
- 208000009956 adenocarcinoma Diseases 0.000 claims description 8
- 208000024662 testicular seminoma Diseases 0.000 claims description 8
- 208000006265 Renal cell carcinoma Diseases 0.000 claims description 7
- 201000008395 adenosquamous carcinoma Diseases 0.000 claims description 7
- 206010044412 transitional cell carcinoma Diseases 0.000 claims description 7
- 208000015347 renal cell adenocarcinoma Diseases 0.000 claims description 6
- 206010060862 Prostate cancer Diseases 0.000 claims description 5
- 208000000236 Prostatic Neoplasms Diseases 0.000 claims description 5
- 201000009439 mucinous ovarian cystadenoma Diseases 0.000 claims description 5
- 239000000284 extract Substances 0.000 claims description 3
- 102000001706 Immunoglobulin Fab Fragments Human genes 0.000 claims description 2
- 108010054477 Immunoglobulin Fab Fragments Proteins 0.000 claims description 2
- 210000001124 body fluid Anatomy 0.000 claims description 2
- 208000005718 Stomach Neoplasms Diseases 0.000 claims 1
- 239000010839 body fluid Substances 0.000 claims 1
- 206010017758 gastric cancer Diseases 0.000 claims 1
- 201000011549 stomach cancer Diseases 0.000 claims 1
- 235000018102 proteins Nutrition 0.000 description 166
- 239000002299 complementary DNA Substances 0.000 description 110
- 210000004027 cell Anatomy 0.000 description 86
- 108020004635 Complementary DNA Proteins 0.000 description 76
- 239000000523 sample Substances 0.000 description 69
- 238000009396 hybridization Methods 0.000 description 59
- 125000003729 nucleotide group Chemical group 0.000 description 50
- 150000001413 amino acids Chemical class 0.000 description 49
- 239000002773 nucleotide Substances 0.000 description 49
- 238000004458 analytical method Methods 0.000 description 48
- 150000007523 nucleic acids Chemical class 0.000 description 39
- 238000003556 assay Methods 0.000 description 38
- 102000039446 nucleic acids Human genes 0.000 description 37
- 108020004707 nucleic acids Proteins 0.000 description 37
- 239000013598 vector Substances 0.000 description 36
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 35
- 108020004414 DNA Proteins 0.000 description 31
- 241000700159 Rattus Species 0.000 description 28
- 150000001875 compounds Chemical class 0.000 description 24
- 230000000295 complement effect Effects 0.000 description 23
- 239000003446 ligand Substances 0.000 description 23
- 108020004999 messenger RNA Proteins 0.000 description 23
- 210000000481 breast Anatomy 0.000 description 21
- 241000699666 Mus <mouse, genus> Species 0.000 description 20
- 239000003795 chemical substances by application Substances 0.000 description 19
- 229940024606 amino acid Drugs 0.000 description 18
- 235000001014 amino acid Nutrition 0.000 description 18
- 230000027455 binding Effects 0.000 description 18
- 208000035475 disorder Diseases 0.000 description 18
- 238000003384 imaging method Methods 0.000 description 18
- 239000012528 membrane Substances 0.000 description 18
- 238000002864 sequence alignment Methods 0.000 description 18
- 201000010099 disease Diseases 0.000 description 17
- 230000001105 regulatory effect Effects 0.000 description 17
- 238000012163 sequencing technique Methods 0.000 description 17
- 241001465754 Metazoa Species 0.000 description 16
- 238000002493 microarray Methods 0.000 description 16
- 239000000203 mixture Substances 0.000 description 16
- 230000009870 specific binding Effects 0.000 description 16
- 239000000758 substrate Substances 0.000 description 16
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 15
- 230000000694 effects Effects 0.000 description 15
- 239000003814 drug Substances 0.000 description 14
- 238000005516 engineering process Methods 0.000 description 14
- 239000013615 primer Substances 0.000 description 14
- 238000011282 treatment Methods 0.000 description 14
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 12
- 238000002372 labelling Methods 0.000 description 12
- 239000013612 plasmid Substances 0.000 description 12
- 102000005962 receptors Human genes 0.000 description 12
- 108020003175 receptors Proteins 0.000 description 12
- 238000012360 testing method Methods 0.000 description 12
- 102000003688 G-Protein-Coupled Receptors Human genes 0.000 description 11
- 108090000045 G-Protein-Coupled Receptors Proteins 0.000 description 11
- 238000006243 chemical reaction Methods 0.000 description 11
- 239000003153 chemical reaction reagent Substances 0.000 description 11
- 238000012216 screening Methods 0.000 description 11
- 210000004291 uterus Anatomy 0.000 description 11
- DBMJMQXJHONAFJ-UHFFFAOYSA-M Sodium laurylsulphate Chemical compound [Na+].CCCCCCCCCCCCOS([O-])(=O)=O DBMJMQXJHONAFJ-UHFFFAOYSA-M 0.000 description 10
- 201000008274 breast adenocarcinoma Diseases 0.000 description 10
- 239000013604 expression vector Substances 0.000 description 10
- 238000000746 purification Methods 0.000 description 10
- 108091034117 Oligonucleotide Proteins 0.000 description 9
- 108091000080 Phosphotransferase Proteins 0.000 description 9
- 238000003491 array Methods 0.000 description 9
- 230000004663 cell proliferation Effects 0.000 description 9
- 230000002068 genetic effect Effects 0.000 description 9
- 102000020233 phosphotransferase Human genes 0.000 description 9
- 239000000243 solution Substances 0.000 description 9
- 238000013518 transcription Methods 0.000 description 9
- 230000035897 transcription Effects 0.000 description 9
- 238000013519 translation Methods 0.000 description 9
- 230000014616 translation Effects 0.000 description 9
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 9
- 108060003393 Granulin Proteins 0.000 description 8
- 230000009918 complex formation Effects 0.000 description 8
- 230000003993 interaction Effects 0.000 description 8
- 230000004048 modification Effects 0.000 description 8
- 238000012986 modification Methods 0.000 description 8
- 210000001672 ovary Anatomy 0.000 description 8
- 102000040430 polynucleotide Human genes 0.000 description 8
- 108091033319 polynucleotide Proteins 0.000 description 8
- 239000002157 polynucleotide Substances 0.000 description 8
- 238000011160 research Methods 0.000 description 8
- 210000001258 synovial membrane Anatomy 0.000 description 8
- 230000001225 therapeutic effect Effects 0.000 description 8
- 230000009261 transgenic effect Effects 0.000 description 8
- 241000699670 Mus sp. Species 0.000 description 7
- 241000283984 Rodentia Species 0.000 description 7
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 7
- 235000014680 Saccharomyces cerevisiae Nutrition 0.000 description 7
- 208000006673 asthma Diseases 0.000 description 7
- 230000001580 bacterial effect Effects 0.000 description 7
- 230000015572 biosynthetic process Effects 0.000 description 7
- 239000000872 buffer Substances 0.000 description 7
- 230000023549 cell-cell signaling Effects 0.000 description 7
- 229940079593 drug Drugs 0.000 description 7
- 230000012010 growth Effects 0.000 description 7
- 238000000338 in vitro Methods 0.000 description 7
- 210000002784 stomach Anatomy 0.000 description 7
- 239000000126 substance Substances 0.000 description 7
- KDCGOANMDULRCW-UHFFFAOYSA-N 7H-purine Chemical compound N1=CNC2=NC=NC2=C1 KDCGOANMDULRCW-UHFFFAOYSA-N 0.000 description 6
- 102000004190 Enzymes Human genes 0.000 description 6
- 108090000790 Enzymes Proteins 0.000 description 6
- 208000023105 Huntington disease Diseases 0.000 description 6
- 241000283973 Oryctolagus cuniculus Species 0.000 description 6
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 6
- 206010003246 arthritis Diseases 0.000 description 6
- 210000002459 blastocyst Anatomy 0.000 description 6
- 210000000845 cartilage Anatomy 0.000 description 6
- 238000010367 cloning Methods 0.000 description 6
- 238000010276 construction Methods 0.000 description 6
- 238000003745 diagnosis Methods 0.000 description 6
- 229940088598 enzyme Drugs 0.000 description 6
- 238000002474 experimental method Methods 0.000 description 6
- 230000006870 function Effects 0.000 description 6
- 239000003112 inhibitor Substances 0.000 description 6
- 230000003834 intracellular effect Effects 0.000 description 6
- 201000010260 leiomyoma Diseases 0.000 description 6
- 238000004519 manufacturing process Methods 0.000 description 6
- 239000003550 marker Substances 0.000 description 6
- 230000001817 pituitary effect Effects 0.000 description 6
- 230000008569 process Effects 0.000 description 6
- 239000011541 reaction mixture Substances 0.000 description 6
- 230000001850 reproductive effect Effects 0.000 description 6
- 239000011347 resin Substances 0.000 description 6
- 229920005989 resin Polymers 0.000 description 6
- 201000000980 schizophrenia Diseases 0.000 description 6
- 101000700937 Amsacta albistriga Sex-specific storage protein 1 Proteins 0.000 description 5
- 108091035707 Consensus sequence Proteins 0.000 description 5
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 5
- 102000010834 Extracellular Matrix Proteins Human genes 0.000 description 5
- 108010037362 Extracellular Matrix Proteins Proteins 0.000 description 5
- 108091006027 G proteins Proteins 0.000 description 5
- 102000030782 GTP binding Human genes 0.000 description 5
- 108091000058 GTP-Binding Proteins 0.000 description 5
- 102000007056 Recombinant Fusion Proteins Human genes 0.000 description 5
- 108010008281 Recombinant Fusion Proteins Proteins 0.000 description 5
- 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 5
- 108060000200 adenylate cyclase Proteins 0.000 description 5
- 102000030621 adenylate cyclase Human genes 0.000 description 5
- 239000005557 antagonist Substances 0.000 description 5
- 210000004556 brain Anatomy 0.000 description 5
- 230000008859 change Effects 0.000 description 5
- 238000001514 detection method Methods 0.000 description 5
- 230000004069 differentiation Effects 0.000 description 5
- 239000003623 enhancer Substances 0.000 description 5
- 238000011156 evaluation Methods 0.000 description 5
- 210000003499 exocrine gland Anatomy 0.000 description 5
- 239000000499 gel Substances 0.000 description 5
- 206010020718 hyperplasia Diseases 0.000 description 5
- 238000011534 incubation Methods 0.000 description 5
- 125000005647 linker group Chemical group 0.000 description 5
- 229920000642 polymer Polymers 0.000 description 5
- 238000002360 preparation method Methods 0.000 description 5
- 231100000419 toxicity Toxicity 0.000 description 5
- 230000001988 toxicity Effects 0.000 description 5
- 231100000041 toxicology testing Toxicity 0.000 description 5
- VQPIHIGAMRSAAN-WMUFLLRFSA-N (3S)-3-[[(2S)-6-amino-2-[[2-[[(2S)-2-[[(2S)-6-amino-2-[[2-[[(2S)-2-[[(2S)-2-[[(2S)-2-[[2-[[(2S)-5-amino-2-[[(2S)-2-[[(2S)-6-amino-2-[[(2S)-2-[[(2S)-2-[[(2S)-2-amino-5-carbamimidamidopentanoyl]amino]-4-methylpentanoyl]amino]-3-(4-hydroxyphenyl)propanoyl]amino]hexanoyl]amino]-3-phenylpropanoyl]amino]-5-oxopentanoyl]amino]acetyl]amino]propanoyl]amino]-4-methylpentanoyl]amino]-4-methylpentanoyl]amino]acetyl]amino]hexanoyl]amino]-3-phenylpropanoyl]amino]acetyl]amino]hexanoyl]amino]-4-[[(2S)-1-[[(2S)-4-amino-1-[[(2S)-1-[[(2S)-1-[[(2S)-6-amino-1-[[(2S)-6-amino-1-[[(2S)-1-[[(2S)-1-[[(2S)-1-[[(2S)-1-[[(2S)-1-[[(2S)-1-[[(2S)-1-[[2-[[(2S)-1-[[(2S)-1-[[(2S)-1-[[(2S)-1-[[2-[[2-[[(2S)-1-[[(2S)-1-[[(2S,3R)-1-[[(2S)-5-amino-1-[[(2S)-1-[[(2S,3R)-1-[[(1S)-1-carboxy-2-phenylethyl]amino]-3-hydroxy-1-oxobutan-2-yl]amino]-3-methyl-1-oxobutan-2-yl]amino]-1,5-dioxopentan-2-yl]amino]-3-hydroxy-1-oxobutan-2-yl]amino]-3-hydroxy-1-oxopropan-2-yl]amino]-1-oxopropan-2-yl]amino]-2-oxoethyl]amino]-2-oxoethyl]amino]-4-methyl-1-oxopentan-2-yl]amino]-3-carboxy-1-oxopropan-2-yl]amino]-4-methyl-1-oxopentan-2-yl]amino]-1-oxopropan-2-yl]amino]-2-oxoethyl]amino]-3-(4-hydroxyphenyl)-1-oxopropan-2-yl]amino]-4-methyl-1-oxopentan-2-yl]amino]-3-carboxy-1-oxopropan-2-yl]amino]-3-hydroxy-1-oxopropan-2-yl]amino]-3-methyl-1-oxobutan-2-yl]amino]-3-methyl-1-oxobutan-2-yl]amino]-3-methyl-1-oxobutan-2-yl]amino]-1-oxohexan-2-yl]amino]-1-oxohexan-2-yl]amino]-3-(4-hydroxyphenyl)-1-oxopropan-2-yl]amino]-3-methyl-1-oxobutan-2-yl]amino]-1,4-dioxobutan-2-yl]amino]-3-(4-hydroxyphenyl)-1-oxopropan-2-yl]amino]-4-oxobutanoic acid Chemical compound CC(C)C[C@H](NC(=O)[C@H](CC(C)C)NC(=O)[C@H](C)NC(=O)CNC(=O)[C@H](CCC(N)=O)NC(=O)[C@H](Cc1ccccc1)NC(=O)[C@H](CCCCN)NC(=O)[C@H](Cc1ccc(O)cc1)NC(=O)[C@H](CC(C)C)NC(=O)[C@@H](N)CCCNC(N)=N)C(=O)NCC(=O)N[C@@H](CCCCN)C(=O)N[C@@H](Cc1ccccc1)C(=O)NCC(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](Cc1ccc(O)cc1)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](Cc1ccc(O)cc1)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CO)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](Cc1ccc(O)cc1)C(=O)NCC(=O)N[C@@H](C)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC(C)C)C(=O)NCC(=O)NCC(=O)N[C@@H](C)C(=O)N[C@@H](CO)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](Cc1ccccc1)C(O)=O VQPIHIGAMRSAAN-WMUFLLRFSA-N 0.000 description 4
- OPIFSICVWOWJMJ-AEOCFKNESA-N 5-bromo-4-chloro-3-indolyl beta-D-galactoside Chemical compound O[C@@H]1[C@@H](O)[C@@H](O)[C@@H](CO)O[C@H]1OC1=CNC2=CC=C(Br)C(Cl)=C12 OPIFSICVWOWJMJ-AEOCFKNESA-N 0.000 description 4
- 102000007347 Apyrase Human genes 0.000 description 4
- 108010007730 Apyrase Proteins 0.000 description 4
- 241000894006 Bacteria Species 0.000 description 4
- BHPQYMZQTOCNFJ-UHFFFAOYSA-N Calcium cation Chemical compound [Ca+2] BHPQYMZQTOCNFJ-UHFFFAOYSA-N 0.000 description 4
- 102000053642 Catalytic RNA Human genes 0.000 description 4
- 108090000994 Catalytic RNA Proteins 0.000 description 4
- 241000282693 Cercopithecidae Species 0.000 description 4
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 description 4
- 108010014303 DNA-directed DNA polymerase Proteins 0.000 description 4
- 102000016928 DNA-directed DNA polymerase Human genes 0.000 description 4
- 208000006402 Ductal Carcinoma Diseases 0.000 description 4
- 241000588724 Escherichia coli Species 0.000 description 4
- 206010061968 Gastric neoplasm Diseases 0.000 description 4
- KFZMGEQAYNKOFK-UHFFFAOYSA-N Isopropanol Chemical compound CC(C)O KFZMGEQAYNKOFK-UHFFFAOYSA-N 0.000 description 4
- 241000124008 Mammalia Species 0.000 description 4
- 108091028043 Nucleic acid sequence Proteins 0.000 description 4
- 239000004677 Nylon Substances 0.000 description 4
- 108091093037 Peptide nucleic acid Proteins 0.000 description 4
- 102000040945 Transcription factor Human genes 0.000 description 4
- 108091023040 Transcription factor Proteins 0.000 description 4
- 239000000556 agonist Substances 0.000 description 4
- 230000003321 amplification Effects 0.000 description 4
- 238000010171 animal model Methods 0.000 description 4
- 210000000625 blastula Anatomy 0.000 description 4
- 210000004899 c-terminal region Anatomy 0.000 description 4
- 238000004113 cell culture Methods 0.000 description 4
- 210000000349 chromosome Anatomy 0.000 description 4
- 238000012217 deletion Methods 0.000 description 4
- 230000037430 deletion Effects 0.000 description 4
- 238000011161 development Methods 0.000 description 4
- 230000018109 developmental process Effects 0.000 description 4
- 239000012153 distilled water Substances 0.000 description 4
- 239000003937 drug carrier Substances 0.000 description 4
- 210000001671 embryonic stem cell Anatomy 0.000 description 4
- 230000001605 fetal effect Effects 0.000 description 4
- 102000037865 fusion proteins Human genes 0.000 description 4
- 108020001507 fusion proteins Proteins 0.000 description 4
- RWSXRVCMGQZWBV-WDSKDSINSA-N glutathione Chemical compound OC(=O)[C@@H](N)CCC(=O)N[C@@H](CS)C(=O)NCC(O)=O RWSXRVCMGQZWBV-WDSKDSINSA-N 0.000 description 4
- UYTPUPDQBNUYGX-UHFFFAOYSA-N guanine Chemical compound O=C1NC(N)=NC2=C1N=CN2 UYTPUPDQBNUYGX-UHFFFAOYSA-N 0.000 description 4
- 210000004408 hybridoma Anatomy 0.000 description 4
- 238000003018 immunoassay Methods 0.000 description 4
- 230000001965 increasing effect Effects 0.000 description 4
- 108010045069 keyhole-limpet hemocyanin Proteins 0.000 description 4
- 210000003734 kidney Anatomy 0.000 description 4
- 210000003127 knee Anatomy 0.000 description 4
- 239000003068 molecular probe Substances 0.000 description 4
- 238000003199 nucleic acid amplification method Methods 0.000 description 4
- 229920001778 nylon Polymers 0.000 description 4
- 239000008194 pharmaceutical composition Substances 0.000 description 4
- 230000026731 phosphorylation Effects 0.000 description 4
- 238000006366 phosphorylation reaction Methods 0.000 description 4
- 238000004393 prognosis Methods 0.000 description 4
- 230000010076 replication Effects 0.000 description 4
- 210000003705 ribosome Anatomy 0.000 description 4
- 108091092562 ribozyme Proteins 0.000 description 4
- 238000007423 screening assay Methods 0.000 description 4
- 241000894007 species Species 0.000 description 4
- 238000003860 storage Methods 0.000 description 4
- 238000006467 substitution reaction Methods 0.000 description 4
- 238000003786 synthesis reaction Methods 0.000 description 4
- 229940124597 therapeutic agent Drugs 0.000 description 4
- 238000002560 therapeutic procedure Methods 0.000 description 4
- 238000012546 transfer Methods 0.000 description 4
- 230000009466 transformation Effects 0.000 description 4
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 description 3
- 229920001817 Agar Polymers 0.000 description 3
- 208000024827 Alzheimer disease Diseases 0.000 description 3
- 241000283690 Bos taurus Species 0.000 description 3
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 description 3
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 3
- 241000238631 Hexapoda Species 0.000 description 3
- 241000282412 Homo Species 0.000 description 3
- 206010062767 Hypophysitis Diseases 0.000 description 3
- 108060003951 Immunoglobulin Proteins 0.000 description 3
- 206010061218 Inflammation Diseases 0.000 description 3
- ZMXDDKWLCZADIW-UHFFFAOYSA-N N,N-Dimethylformamide Chemical compound CN(C)C=O ZMXDDKWLCZADIW-UHFFFAOYSA-N 0.000 description 3
- 108010038807 Oligopeptides Proteins 0.000 description 3
- 102000015636 Oligopeptides Human genes 0.000 description 3
- 238000012408 PCR amplification Methods 0.000 description 3
- 108010002747 Pfu DNA polymerase Proteins 0.000 description 3
- 102000001253 Protein Kinase Human genes 0.000 description 3
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 3
- 108010006785 Taq Polymerase Proteins 0.000 description 3
- 239000007983 Tris buffer Substances 0.000 description 3
- 239000002253 acid Substances 0.000 description 3
- 230000004913 activation Effects 0.000 description 3
- 239000002671 adjuvant Substances 0.000 description 3
- 239000008272 agar Substances 0.000 description 3
- 239000011543 agarose gel Substances 0.000 description 3
- 150000001412 amines Chemical class 0.000 description 3
- 230000000890 antigenic effect Effects 0.000 description 3
- 108010068991 arginyl-threonyl-prolyl-prolyl-prolyl-seryl-glycine Proteins 0.000 description 3
- 230000004071 biological effect Effects 0.000 description 3
- 230000033228 biological regulation Effects 0.000 description 3
- FPPNZSSZRUTDAP-UWFZAAFLSA-N carbenicillin Chemical compound N([C@H]1[C@H]2SC([C@@H](N2C1=O)C(O)=O)(C)C)C(=O)C(C(O)=O)C1=CC=CC=C1 FPPNZSSZRUTDAP-UWFZAAFLSA-N 0.000 description 3
- 229960003669 carbenicillin Drugs 0.000 description 3
- 230000001413 cellular effect Effects 0.000 description 3
- 238000012875 competitive assay Methods 0.000 description 3
- 230000008878 coupling Effects 0.000 description 3
- 238000010168 coupling process Methods 0.000 description 3
- 238000005859 coupling reaction Methods 0.000 description 3
- 235000018417 cysteine Nutrition 0.000 description 3
- 230000001086 cytosolic effect Effects 0.000 description 3
- 238000007865 diluting Methods 0.000 description 3
- 238000009826 distribution Methods 0.000 description 3
- 210000003979 eosinophil Anatomy 0.000 description 3
- 230000005284 excitation Effects 0.000 description 3
- 230000013595 glycosylation Effects 0.000 description 3
- 238000006206 glycosylation reaction Methods 0.000 description 3
- 210000002216 heart Anatomy 0.000 description 3
- HNDVDQJCIGZPNO-UHFFFAOYSA-N histidine Natural products OC(=O)C(N)CC1=CN=CN1 HNDVDQJCIGZPNO-UHFFFAOYSA-N 0.000 description 3
- 229910052739 hydrogen Inorganic materials 0.000 description 3
- 239000001257 hydrogen Substances 0.000 description 3
- 230000003053 immunization Effects 0.000 description 3
- 102000018358 immunoglobulin Human genes 0.000 description 3
- 230000004054 inflammatory process Effects 0.000 description 3
- 108010044426 integrins Proteins 0.000 description 3
- 102000006495 integrins Human genes 0.000 description 3
- 238000011835 investigation Methods 0.000 description 3
- 210000004072 lung Anatomy 0.000 description 3
- 239000002609 medium Substances 0.000 description 3
- 210000000056 organ Anatomy 0.000 description 3
- 238000012545 processing Methods 0.000 description 3
- 230000002062 proliferating effect Effects 0.000 description 3
- 210000002307 prostate Anatomy 0.000 description 3
- 108060006633 protein kinase Proteins 0.000 description 3
- 230000002285 radioactive effect Effects 0.000 description 3
- 238000011084 recovery Methods 0.000 description 3
- 230000009467 reduction Effects 0.000 description 3
- 150000003839 salts Chemical class 0.000 description 3
- 229940016590 sarkosyl Drugs 0.000 description 3
- 108700004121 sarkosyl Proteins 0.000 description 3
- 239000011780 sodium chloride Substances 0.000 description 3
- KSAVQLQVUXSOCR-UHFFFAOYSA-M sodium lauroyl sarcosinate Chemical compound [Na+].CCCCCCCCCCCC(=O)N(C)CC([O-])=O KSAVQLQVUXSOCR-UHFFFAOYSA-M 0.000 description 3
- 239000007790 solid phase Substances 0.000 description 3
- 210000000130 stem cell Anatomy 0.000 description 3
- 238000001356 surgical procedure Methods 0.000 description 3
- 230000032258 transport Effects 0.000 description 3
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 description 3
- 238000009281 ultraviolet germicidal irradiation Methods 0.000 description 3
- MTCFGRXMJLQNBG-REOHCLBHSA-N (2S)-2-Amino-3-hydroxypropansäure Chemical compound OC[C@H](N)C(O)=O MTCFGRXMJLQNBG-REOHCLBHSA-N 0.000 description 2
- 125000003088 (fluoren-9-ylmethoxy)carbonyl group Chemical group 0.000 description 2
- 108091023043 Alu Element Proteins 0.000 description 2
- 108091023037 Aptamer Proteins 0.000 description 2
- 101001007348 Arachis hypogaea Galactose-binding lectin Proteins 0.000 description 2
- 201000001320 Atherosclerosis Diseases 0.000 description 2
- 101000651671 Bombyx mori Sex-specific storage-protein 2 Proteins 0.000 description 2
- 108050007957 Cadherin Proteins 0.000 description 2
- 102000000905 Cadherin Human genes 0.000 description 2
- OYPRJOBELJOOCE-UHFFFAOYSA-N Calcium Chemical compound [Ca] OYPRJOBELJOOCE-UHFFFAOYSA-N 0.000 description 2
- 241000288950 Callithrix jacchus Species 0.000 description 2
- 241000283707 Capra Species 0.000 description 2
- 239000004971 Cross linker Substances 0.000 description 2
- IVOMOUWHDPKRLL-KQYNXXCUSA-N Cyclic adenosine monophosphate Chemical compound C([C@H]1O2)OP(O)(=O)O[C@H]1[C@@H](O)[C@@H]2N1C(N=CN=C2N)=C2N=C1 IVOMOUWHDPKRLL-KQYNXXCUSA-N 0.000 description 2
- 102000053602 DNA Human genes 0.000 description 2
- 108010017826 DNA Polymerase I Proteins 0.000 description 2
- 102000004594 DNA Polymerase I Human genes 0.000 description 2
- 238000001712 DNA sequencing Methods 0.000 description 2
- 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 2
- 238000002965 ELISA Methods 0.000 description 2
- 241000196324 Embryophyta Species 0.000 description 2
- 102100031780 Endonuclease Human genes 0.000 description 2
- 108010042407 Endonucleases Proteins 0.000 description 2
- 108050001049 Extracellular proteins Proteins 0.000 description 2
- KRHYYFGTRYWZRS-UHFFFAOYSA-N Fluorane Chemical compound F KRHYYFGTRYWZRS-UHFFFAOYSA-N 0.000 description 2
- ZHNUHDYFZUAESO-UHFFFAOYSA-N Formamide Chemical compound NC=O ZHNUHDYFZUAESO-UHFFFAOYSA-N 0.000 description 2
- 108010024636 Glutathione Proteins 0.000 description 2
- 229920002683 Glycosaminoglycan Polymers 0.000 description 2
- NTYJJOPFIAHURM-UHFFFAOYSA-N Histamine Chemical compound NCCC1=CN=CN1 NTYJJOPFIAHURM-UHFFFAOYSA-N 0.000 description 2
- 101000664506 Homo sapiens Spermatogenesis-associated protein 1 Proteins 0.000 description 2
- 206010020772 Hypertension Diseases 0.000 description 2
- 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 2
- 108010002352 Interleukin-1 Proteins 0.000 description 2
- 108091092195 Intron Proteins 0.000 description 2
- 108090000862 Ion Channels Proteins 0.000 description 2
- 102000004310 Ion Channels Human genes 0.000 description 2
- XEEYBQQBJWHFJM-UHFFFAOYSA-N Iron Chemical compound [Fe] XEEYBQQBJWHFJM-UHFFFAOYSA-N 0.000 description 2
- XUJNEKJLAYXESH-REOHCLBHSA-N L-Cysteine Chemical compound SC[C@H](N)C(O)=O XUJNEKJLAYXESH-REOHCLBHSA-N 0.000 description 2
- ROHFNLRQFUQHCH-YFKPBYRVSA-N L-leucine Chemical compound CC(C)C[C@H](N)C(O)=O ROHFNLRQFUQHCH-YFKPBYRVSA-N 0.000 description 2
- ROHFNLRQFUQHCH-UHFFFAOYSA-N Leucine Natural products CC(C)CC(N)C(O)=O ROHFNLRQFUQHCH-UHFFFAOYSA-N 0.000 description 2
- 208000000265 Lobular Carcinoma Diseases 0.000 description 2
- 241000282567 Macaca fascicularis Species 0.000 description 2
- 241000282560 Macaca mulatta Species 0.000 description 2
- 230000004988 N-glycosylation Effects 0.000 description 2
- 108091005461 Nucleic proteins Proteins 0.000 description 2
- 206010061535 Ovarian neoplasm Diseases 0.000 description 2
- 229910019142 PO4 Inorganic materials 0.000 description 2
- 102000004861 Phosphoric Diester Hydrolases Human genes 0.000 description 2
- 108090001050 Phosphoric Diester Hydrolases Proteins 0.000 description 2
- ZYFVNVRFVHJEIU-UHFFFAOYSA-N PicoGreen Chemical compound CN(C)CCCN(CCCN(C)C)C1=CC(=CC2=[N+](C3=CC=CC=C3S2)C)C2=CC=CC=C2N1C1=CC=CC=C1 ZYFVNVRFVHJEIU-UHFFFAOYSA-N 0.000 description 2
- NQRYJNQNLNOLGT-UHFFFAOYSA-N Piperidine Chemical compound C1CCNCC1 NQRYJNQNLNOLGT-UHFFFAOYSA-N 0.000 description 2
- 239000002202 Polyethylene glycol Substances 0.000 description 2
- 101710182846 Polyhedrin Proteins 0.000 description 2
- ZLMJMSJWJFRBEC-UHFFFAOYSA-N Potassium Chemical compound [K] ZLMJMSJWJFRBEC-UHFFFAOYSA-N 0.000 description 2
- 108010029485 Protein Isoforms Proteins 0.000 description 2
- 102000001708 Protein Isoforms Human genes 0.000 description 2
- 108010009341 Protein Serine-Threonine Kinases Proteins 0.000 description 2
- 102000009516 Protein Serine-Threonine Kinases Human genes 0.000 description 2
- 102000003800 Selectins Human genes 0.000 description 2
- 108090000184 Selectins Proteins 0.000 description 2
- 229920002684 Sepharose Polymers 0.000 description 2
- MTCFGRXMJLQNBG-UHFFFAOYSA-N Serine Natural products OCC(N)C(O)=O MTCFGRXMJLQNBG-UHFFFAOYSA-N 0.000 description 2
- VMHLLURERBWHNL-UHFFFAOYSA-M Sodium acetate Chemical compound [Na+].CC([O-])=O VMHLLURERBWHNL-UHFFFAOYSA-M 0.000 description 2
- 102100038832 Spermatogenesis-associated protein 1 Human genes 0.000 description 2
- DTQVDTLACAAQTR-UHFFFAOYSA-N Trifluoroacetic acid Chemical compound OC(=O)C(F)(F)F DTQVDTLACAAQTR-UHFFFAOYSA-N 0.000 description 2
- 239000007984 Tris EDTA buffer Substances 0.000 description 2
- IVOMOUWHDPKRLL-UHFFFAOYSA-N UNPD107823 Natural products O1C2COP(O)(=O)OC2C(O)C1N1C(N=CN=C2N)=C2N=C1 IVOMOUWHDPKRLL-UHFFFAOYSA-N 0.000 description 2
- ISAKRJDGNUQOIC-UHFFFAOYSA-N Uracil Chemical compound O=C1C=CNC(=O)N1 ISAKRJDGNUQOIC-UHFFFAOYSA-N 0.000 description 2
- XSQUKJJJFZCRTK-UHFFFAOYSA-N Urea Chemical compound NC(N)=O XSQUKJJJFZCRTK-UHFFFAOYSA-N 0.000 description 2
- DRTQHJPVMGBUCF-XVFCMESISA-N Uridine Chemical compound O[C@@H]1[C@H](O)[C@@H](CO)O[C@H]1N1C(=O)NC(=O)C=C1 DRTQHJPVMGBUCF-XVFCMESISA-N 0.000 description 2
- 241000700605 Viruses Species 0.000 description 2
- 230000021736 acetylation Effects 0.000 description 2
- 238000006640 acetylation reaction Methods 0.000 description 2
- 125000000539 amino acid group Chemical group 0.000 description 2
- 210000004102 animal cell Anatomy 0.000 description 2
- 230000005875 antibody response Effects 0.000 description 2
- 210000000628 antibody-producing cell Anatomy 0.000 description 2
- 210000004436 artificial bacterial chromosome Anatomy 0.000 description 2
- 210000004507 artificial chromosome Anatomy 0.000 description 2
- 210000001106 artificial yeast chromosome Anatomy 0.000 description 2
- 239000011324 bead Substances 0.000 description 2
- 230000003115 biocidal effect Effects 0.000 description 2
- 210000000601 blood cell Anatomy 0.000 description 2
- 208000030270 breast disease Diseases 0.000 description 2
- 208000014581 breast ductal adenocarcinoma Diseases 0.000 description 2
- 201000003714 breast lobular carcinoma Diseases 0.000 description 2
- 239000011575 calcium Substances 0.000 description 2
- 229910052791 calcium Inorganic materials 0.000 description 2
- 230000003197 catalytic effect Effects 0.000 description 2
- 230000005754 cellular signaling Effects 0.000 description 2
- 238000012512 characterization method Methods 0.000 description 2
- 230000001684 chronic effect Effects 0.000 description 2
- 238000003776 cleavage reaction Methods 0.000 description 2
- 230000021615 conjugation Effects 0.000 description 2
- 230000000875 corresponding effect Effects 0.000 description 2
- IDLFZVILOHSSID-OVLDLUHVSA-N corticotropin Chemical compound C([C@@H](C(=O)N[C@@H](CO)C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC=1NC=NC=1)C(=O)N[C@@H](CC=1C=CC=CC=1)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)NCC(=O)N[C@@H](CCCCN)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H](C(C)C)C(=O)NCC(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CC=1C=CC(O)=CC=1)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H](CC(N)=O)C(=O)NCC(=O)N[C@@H](C)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CO)C(=O)N[C@@H](C)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](C)C(=O)N[C@@H](CC=1C=CC=CC=1)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC=1C=CC=CC=1)C(O)=O)NC(=O)[C@@H](N)CO)C1=CC=C(O)C=C1 IDLFZVILOHSSID-OVLDLUHVSA-N 0.000 description 2
- ATDGTVJJHBUTRL-UHFFFAOYSA-N cyanogen bromide Chemical group BrC#N ATDGTVJJHBUTRL-UHFFFAOYSA-N 0.000 description 2
- 229940095074 cyclic amp Drugs 0.000 description 2
- XUJNEKJLAYXESH-UHFFFAOYSA-N cysteine Natural products SCC(N)C(O)=O XUJNEKJLAYXESH-UHFFFAOYSA-N 0.000 description 2
- 230000003247 decreasing effect Effects 0.000 description 2
- 230000001419 dependent effect Effects 0.000 description 2
- 238000001212 derivatisation Methods 0.000 description 2
- 239000003599 detergent Substances 0.000 description 2
- 230000009274 differential gene expression Effects 0.000 description 2
- 230000029087 digestion Effects 0.000 description 2
- VYFYYTLLBUKUHU-UHFFFAOYSA-N dopamine Chemical compound NCCC1=CC=C(O)C(O)=C1 VYFYYTLLBUKUHU-UHFFFAOYSA-N 0.000 description 2
- 239000000975 dye Substances 0.000 description 2
- 238000000295 emission spectrum Methods 0.000 description 2
- 210000004696 endometrium Anatomy 0.000 description 2
- 230000002255 enzymatic effect Effects 0.000 description 2
- 210000003527 eukaryotic cell Anatomy 0.000 description 2
- 210000003754 fetus Anatomy 0.000 description 2
- MHMNJMPURVTYEJ-UHFFFAOYSA-N fluorescein-5-isothiocyanate Chemical compound O1C(=O)C2=CC(N=C=S)=CC=C2C21C1=CC=C(O)C=C1OC1=CC(O)=CC=C21 MHMNJMPURVTYEJ-UHFFFAOYSA-N 0.000 description 2
- 238000001943 fluorescence-activated cell sorting Methods 0.000 description 2
- 102000034356 gene-regulatory proteins Human genes 0.000 description 2
- 108091006104 gene-regulatory proteins Proteins 0.000 description 2
- 239000011521 glass Substances 0.000 description 2
- 229960003180 glutathione Drugs 0.000 description 2
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 description 2
- 230000036541 health Effects 0.000 description 2
- 238000002744 homologous recombination Methods 0.000 description 2
- 230000006801 homologous recombination Effects 0.000 description 2
- 229940088597 hormone Drugs 0.000 description 2
- 239000005556 hormone Substances 0.000 description 2
- 210000000688 human artificial chromosome Anatomy 0.000 description 2
- FDGQSTZJBFJUBT-UHFFFAOYSA-N hypoxanthine Chemical compound O=C1NC=NC2=C1NC=N2 FDGQSTZJBFJUBT-UHFFFAOYSA-N 0.000 description 2
- 230000028993 immune response Effects 0.000 description 2
- 229940072221 immunoglobulins Drugs 0.000 description 2
- 238000001727 in vivo Methods 0.000 description 2
- 238000011065 in-situ storage Methods 0.000 description 2
- 229910052500 inorganic mineral Inorganic materials 0.000 description 2
- 238000003780 insertion Methods 0.000 description 2
- 230000037431 insertion Effects 0.000 description 2
- 230000002452 interceptive effect Effects 0.000 description 2
- 230000031146 intracellular signal transduction Effects 0.000 description 2
- 206010073096 invasive lobular breast carcinoma Diseases 0.000 description 2
- 231100000225 lethality Toxicity 0.000 description 2
- 230000029226 lipidation Effects 0.000 description 2
- 150000002632 lipids Chemical class 0.000 description 2
- 239000007788 liquid Substances 0.000 description 2
- 230000007774 longterm Effects 0.000 description 2
- 239000006166 lysate Substances 0.000 description 2
- 210000004962 mammalian cell Anatomy 0.000 description 2
- 210000001161 mammalian embryo Anatomy 0.000 description 2
- 238000013507 mapping Methods 0.000 description 2
- 239000011159 matrix material Substances 0.000 description 2
- 230000007246 mechanism Effects 0.000 description 2
- 239000011707 mineral Substances 0.000 description 2
- 208000004707 mucinous cystadenoma Diseases 0.000 description 2
- 230000035772 mutation Effects 0.000 description 2
- 210000000754 myometrium Anatomy 0.000 description 2
- 230000001537 neural effect Effects 0.000 description 2
- 230000003472 neutralizing effect Effects 0.000 description 2
- 239000008188 pellet Substances 0.000 description 2
- 238000010647 peptide synthesis reaction Methods 0.000 description 2
- 210000005259 peripheral blood Anatomy 0.000 description 2
- 239000011886 peripheral blood Substances 0.000 description 2
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 description 2
- 239000010452 phosphate Substances 0.000 description 2
- 239000002953 phosphate buffered saline Substances 0.000 description 2
- 125000002467 phosphate group Chemical group [H]OP(=O)(O[H])O[*] 0.000 description 2
- 230000035479 physiological effects, processes and functions Effects 0.000 description 2
- 210000003635 pituitary gland Anatomy 0.000 description 2
- 229920001223 polyethylene glycol Polymers 0.000 description 2
- 239000011148 porous material Substances 0.000 description 2
- 230000001323 posttranslational effect Effects 0.000 description 2
- 239000011591 potassium Substances 0.000 description 2
- 229910052700 potassium Inorganic materials 0.000 description 2
- 239000002243 precursor Substances 0.000 description 2
- 239000000047 product Substances 0.000 description 2
- 230000000644 propagated effect Effects 0.000 description 2
- 201000005825 prostate adenocarcinoma Diseases 0.000 description 2
- 230000006337 proteolytic cleavage Effects 0.000 description 2
- 238000011002 quantification Methods 0.000 description 2
- 238000003127 radioimmunoassay Methods 0.000 description 2
- 238000010839 reverse transcription Methods 0.000 description 2
- 230000007017 scission Effects 0.000 description 2
- 239000006152 selective media Substances 0.000 description 2
- 230000035945 sensitivity Effects 0.000 description 2
- QZAYGJVTTNCVMB-UHFFFAOYSA-N serotonin Chemical compound C1=C(O)C=C2C(CCN)=CNC2=C1 QZAYGJVTTNCVMB-UHFFFAOYSA-N 0.000 description 2
- 230000011664 signaling Effects 0.000 description 2
- 239000011734 sodium Substances 0.000 description 2
- 229910052708 sodium Inorganic materials 0.000 description 2
- 239000001632 sodium acetate Substances 0.000 description 2
- 235000017281 sodium acetate Nutrition 0.000 description 2
- 210000001550 testis Anatomy 0.000 description 2
- 231100001274 therapeutic index Toxicity 0.000 description 2
- RWQNBRDOKXIBIV-UHFFFAOYSA-N thymine Chemical compound CC1=CNC(=O)NC1=O RWQNBRDOKXIBIV-UHFFFAOYSA-N 0.000 description 2
- 231100000027 toxicology Toxicity 0.000 description 2
- 230000002103 transcriptional effect Effects 0.000 description 2
- 230000001131 transforming effect Effects 0.000 description 2
- 238000011820 transgenic animal model Methods 0.000 description 2
- 238000010396 two-hybrid screening Methods 0.000 description 2
- 241000701447 unidentified baculovirus Species 0.000 description 2
- 238000011144 upstream manufacturing Methods 0.000 description 2
- 210000001635 urinary tract Anatomy 0.000 description 2
- 230000002792 vascular Effects 0.000 description 2
- 238000012795 verification Methods 0.000 description 2
- 230000003612 virological effect Effects 0.000 description 2
- 229940088594 vitamin Drugs 0.000 description 2
- 239000011782 vitamin Substances 0.000 description 2
- 235000013343 vitamin Nutrition 0.000 description 2
- 229930003231 vitamin Natural products 0.000 description 2
- 108010047303 von Willebrand Factor Proteins 0.000 description 2
- 102100036537 von Willebrand factor Human genes 0.000 description 2
- 229960001134 von willebrand factor Drugs 0.000 description 2
- 238000005406 washing Methods 0.000 description 2
- 210000005253 yeast cell Anatomy 0.000 description 2
- JWZZKOKVBUJMES-UHFFFAOYSA-N (+-)-Isoprenaline Chemical compound CC(C)NCC(O)C1=CC=C(O)C(O)=C1 JWZZKOKVBUJMES-UHFFFAOYSA-N 0.000 description 1
- ASWBNKHCZGQVJV-UHFFFAOYSA-N (3-hexadecanoyloxy-2-hydroxypropyl) 2-(trimethylazaniumyl)ethyl phosphate Chemical compound CCCCCCCCCCCCCCCC(=O)OCC(O)COP([O-])(=O)OCC[N+](C)(C)C ASWBNKHCZGQVJV-UHFFFAOYSA-N 0.000 description 1
- GMVPRGQOIOIIMI-UHFFFAOYSA-N (8R,11R,12R,13E,15S)-11,15-Dihydroxy-9-oxo-13-prostenoic acid Natural products CCCCCC(O)C=CC1C(O)CC(=O)C1CCCCCCC(O)=O GMVPRGQOIOIIMI-UHFFFAOYSA-N 0.000 description 1
- 229930182837 (R)-adrenaline Natural products 0.000 description 1
- UCTWMZQNUQWSLP-VIFPVBQESA-N (R)-adrenaline Chemical compound CNC[C@H](O)C1=CC=C(O)C(O)=C1 UCTWMZQNUQWSLP-VIFPVBQESA-N 0.000 description 1
- UHDGCWIWMRVCDJ-UHFFFAOYSA-N 1-beta-D-Xylofuranosyl-NH-Cytosine Natural products O=C1N=C(N)C=CN1C1C(O)C(O)C(CO)O1 UHDGCWIWMRVCDJ-UHFFFAOYSA-N 0.000 description 1
- UFBJCMHMOXMLKC-UHFFFAOYSA-N 2,4-dinitrophenol Chemical compound OC1=CC=C([N+]([O-])=O)C=C1[N+]([O-])=O UFBJCMHMOXMLKC-UHFFFAOYSA-N 0.000 description 1
- HVAUUPRFYPCOCA-AREMUKBSSA-N 2-O-acetyl-1-O-hexadecyl-sn-glycero-3-phosphocholine Chemical compound CCCCCCCCCCCCCCCCOC[C@@H](OC(C)=O)COP([O-])(=O)OCC[N+](C)(C)C HVAUUPRFYPCOCA-AREMUKBSSA-N 0.000 description 1
- ZPZDIFSPRVHGIF-UHFFFAOYSA-N 3-aminopropylsilicon Chemical compound NCCC[Si] ZPZDIFSPRVHGIF-UHFFFAOYSA-N 0.000 description 1
- FWMNVWWHGCHHJJ-SKKKGAJSSA-N 4-amino-1-[(2r)-6-amino-2-[[(2r)-2-[[(2r)-2-[[(2r)-2-amino-3-phenylpropanoyl]amino]-3-phenylpropanoyl]amino]-4-methylpentanoyl]amino]hexanoyl]piperidine-4-carboxylic acid Chemical compound C([C@H](C(=O)N[C@H](CC(C)C)C(=O)N[C@H](CCCCN)C(=O)N1CCC(N)(CC1)C(O)=O)NC(=O)[C@H](N)CC=1C=CC=CC=1)C1=CC=CC=C1 FWMNVWWHGCHHJJ-SKKKGAJSSA-N 0.000 description 1
- 108020003589 5' Untranslated Regions Proteins 0.000 description 1
- 229930024421 Adenine Natural products 0.000 description 1
- GFFGJBXGBJISGV-UHFFFAOYSA-N Adenine Chemical compound NC1=NC=NC2=C1N=CN2 GFFGJBXGBJISGV-UHFFFAOYSA-N 0.000 description 1
- 229920000936 Agarose Polymers 0.000 description 1
- 108010049777 Ankyrins Proteins 0.000 description 1
- 102000008102 Ankyrins Human genes 0.000 description 1
- 102100022716 Atypical chemokine receptor 3 Human genes 0.000 description 1
- 241000972773 Aulopiformes Species 0.000 description 1
- 241001203868 Autographa californica Species 0.000 description 1
- 206010004446 Benign prostatic hyperplasia Diseases 0.000 description 1
- 102000004506 Blood Proteins Human genes 0.000 description 1
- 108010017384 Blood Proteins Proteins 0.000 description 1
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 1
- 101150111062 C gene Proteins 0.000 description 1
- 102400000140 C5a anaphylatoxin Human genes 0.000 description 1
- 101800001654 C5a anaphylatoxin Proteins 0.000 description 1
- 102400000113 Calcitonin Human genes 0.000 description 1
- 108060001064 Calcitonin Proteins 0.000 description 1
- 208000005623 Carcinogenesis Diseases 0.000 description 1
- 201000009030 Carcinoma Diseases 0.000 description 1
- 208000031229 Cardiomyopathies Diseases 0.000 description 1
- 108010078791 Carrier Proteins Proteins 0.000 description 1
- 102000014914 Carrier Proteins Human genes 0.000 description 1
- 108091006146 Channels Proteins 0.000 description 1
- 102000019034 Chemokines Human genes 0.000 description 1
- 108010012236 Chemokines Proteins 0.000 description 1
- VEXZGXHMUGYJMC-UHFFFAOYSA-M Chloride anion Chemical compound [Cl-] VEXZGXHMUGYJMC-UHFFFAOYSA-M 0.000 description 1
- 206010008631 Cholera Diseases 0.000 description 1
- 206010008674 Cholinergic syndrome Diseases 0.000 description 1
- 208000031404 Chromosome Aberrations Diseases 0.000 description 1
- 108020004705 Codon Proteins 0.000 description 1
- 108700010070 Codon Usage Proteins 0.000 description 1
- 206010009944 Colon cancer Diseases 0.000 description 1
- 208000035473 Communicable disease Diseases 0.000 description 1
- 108020004394 Complementary RNA Proteins 0.000 description 1
- 206010010356 Congenital anomaly Diseases 0.000 description 1
- 101710095468 Cyclase Proteins 0.000 description 1
- 206010063057 Cystitis noninfective Diseases 0.000 description 1
- 206010011803 Cystocele Diseases 0.000 description 1
- UHDGCWIWMRVCDJ-PSQAKQOGSA-N Cytidine Natural products O=C1N=C(N)C=CN1[C@@H]1[C@@H](O)[C@@H](O)[C@H](CO)O1 UHDGCWIWMRVCDJ-PSQAKQOGSA-N 0.000 description 1
- 108090000695 Cytokines Proteins 0.000 description 1
- 102000004127 Cytokines Human genes 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
- 102000004163 DNA-directed RNA polymerases Human genes 0.000 description 1
- 108090000626 DNA-directed RNA polymerases Proteins 0.000 description 1
- 206010012289 Dementia Diseases 0.000 description 1
- 102000012545 EGF-like domains Human genes 0.000 description 1
- 108050002150 EGF-like domains Proteins 0.000 description 1
- 201000009051 Embryonal Carcinoma Diseases 0.000 description 1
- 201000009273 Endometriosis Diseases 0.000 description 1
- 102000004533 Endonucleases Human genes 0.000 description 1
- 241001635598 Enicostema Species 0.000 description 1
- 102400001368 Epidermal growth factor Human genes 0.000 description 1
- 101800003838 Epidermal growth factor Proteins 0.000 description 1
- 241000206602 Eukaryota Species 0.000 description 1
- 108700024394 Exon Proteins 0.000 description 1
- 108060002716 Exonuclease Proteins 0.000 description 1
- LLQPHQFNMLZJMP-UHFFFAOYSA-N Fentrazamide Chemical compound N1=NN(C=2C(=CC=CC=2)Cl)C(=O)N1C(=O)N(CC)C1CCCCC1 LLQPHQFNMLZJMP-UHFFFAOYSA-N 0.000 description 1
- 102000012673 Follicle Stimulating Hormone Human genes 0.000 description 1
- 108010079345 Follicle Stimulating Hormone Proteins 0.000 description 1
- 208000009774 Follicular Cyst Diseases 0.000 description 1
- 101150097368 GPR19 gene Proteins 0.000 description 1
- 241000287828 Gallus gallus Species 0.000 description 1
- 108700028146 Genetic Enhancer Elements Proteins 0.000 description 1
- 102000053187 Glucuronidase Human genes 0.000 description 1
- 108010060309 Glucuronidase Proteins 0.000 description 1
- 229920002527 Glycogen Polymers 0.000 description 1
- 239000000579 Gonadotropin-Releasing Hormone Substances 0.000 description 1
- 108010043121 Green Fluorescent Proteins Proteins 0.000 description 1
- 102000004144 Green Fluorescent Proteins Human genes 0.000 description 1
- 101000678890 Homo sapiens Atypical chemokine receptor 3 Proteins 0.000 description 1
- 101001059454 Homo sapiens Serine/threonine-protein kinase MARK2 Proteins 0.000 description 1
- 101000938391 Homo sapiens Transmembrane protein Proteins 0.000 description 1
- 101000666856 Homo sapiens Vasoactive intestinal polypeptide receptor 1 Proteins 0.000 description 1
- 108090000144 Human Proteins Proteins 0.000 description 1
- 102000003839 Human Proteins Human genes 0.000 description 1
- UFHFLCQGNIYNRP-UHFFFAOYSA-N Hydrogen Chemical compound [H][H] UFHFLCQGNIYNRP-UHFFFAOYSA-N 0.000 description 1
- UGQMRVRMYYASKQ-UHFFFAOYSA-N Hypoxanthine nucleoside Natural products OC1C(O)C(CO)OC1N1C(NC=NC2=O)=C2N=C1 UGQMRVRMYYASKQ-UHFFFAOYSA-N 0.000 description 1
- 102000013463 Immunoglobulin Light Chains Human genes 0.000 description 1
- 108010065825 Immunoglobulin Light Chains Proteins 0.000 description 1
- 108050006430 Immunoglobulin-like domains Proteins 0.000 description 1
- 102000016844 Immunoglobulin-like domains Human genes 0.000 description 1
- 229930010555 Inosine Natural products 0.000 description 1
- UGQMRVRMYYASKQ-KQYNXXCUSA-N Inosine Chemical compound O[C@@H]1[C@H](O)[C@@H](CO)O[C@H]1N1C2=NC=NC(O)=C2N=C1 UGQMRVRMYYASKQ-KQYNXXCUSA-N 0.000 description 1
- 108010025815 Kanamycin Kinase Proteins 0.000 description 1
- WHUUTDBJXJRKMK-VKHMYHEASA-N L-glutamic acid Chemical compound OC(=O)[C@@H](N)CCC(O)=O WHUUTDBJXJRKMK-VKHMYHEASA-N 0.000 description 1
- AGPKZVBTJJNPAG-WHFBIAKZSA-N L-isoleucine Chemical compound CC[C@H](C)[C@H](N)C(O)=O AGPKZVBTJJNPAG-WHFBIAKZSA-N 0.000 description 1
- AYFVYJQAPQTCCC-GBXIJSLDSA-N L-threonine Chemical compound C[C@@H](O)[C@H](N)C(O)=O AYFVYJQAPQTCCC-GBXIJSLDSA-N 0.000 description 1
- QIVBCDIJIAJPQS-VIFPVBQESA-N L-tryptophane Chemical compound C1=CC=C2C(C[C@H](N)C(O)=O)=CNC2=C1 QIVBCDIJIAJPQS-VIFPVBQESA-N 0.000 description 1
- KZSNJWFQEVHDMF-BYPYZUCNSA-N L-valine Chemical compound CC(C)[C@H](N)C(O)=O KZSNJWFQEVHDMF-BYPYZUCNSA-N 0.000 description 1
- 108090001090 Lectins Proteins 0.000 description 1
- 102000004856 Lectins Human genes 0.000 description 1
- 108050006654 Lipocalin Proteins 0.000 description 1
- 102000019298 Lipocalin Human genes 0.000 description 1
- 108060001084 Luciferase Proteins 0.000 description 1
- 239000005089 Luciferase Substances 0.000 description 1
- 208000019693 Lung disease Diseases 0.000 description 1
- 239000006142 Luria-Bertani Agar Substances 0.000 description 1
- JLVVSXFLKOJNIY-UHFFFAOYSA-N Magnesium ion Chemical compound [Mg+2] JLVVSXFLKOJNIY-UHFFFAOYSA-N 0.000 description 1
- 206010027514 Metrorrhagia Diseases 0.000 description 1
- 108010006519 Molecular Chaperones Proteins 0.000 description 1
- 108010085220 Multiprotein Complexes Proteins 0.000 description 1
- 102000007474 Multiprotein Complexes Human genes 0.000 description 1
- 241000187479 Mycobacterium tuberculosis Species 0.000 description 1
- 108090000189 Neuropeptides Proteins 0.000 description 1
- 238000000636 Northern blotting Methods 0.000 description 1
- 108700026244 Open Reading Frames Proteins 0.000 description 1
- 102000016978 Orphan receptors Human genes 0.000 description 1
- 108070000031 Orphan receptors Proteins 0.000 description 1
- 102000016610 Oxidized LDL Receptors Human genes 0.000 description 1
- 108010028191 Oxidized LDL Receptors Proteins 0.000 description 1
- 102400000050 Oxytocin Human genes 0.000 description 1
- 101800000989 Oxytocin Proteins 0.000 description 1
- XNOPRXBHLZRZKH-UHFFFAOYSA-N Oxytocin Natural products N1C(=O)C(N)CSSCC(C(=O)N2C(CCC2)C(=O)NC(CC(C)C)C(=O)NCC(N)=O)NC(=O)C(CC(N)=O)NC(=O)C(CCC(N)=O)NC(=O)C(C(C)CC)NC(=O)C1CC1=CC=C(O)C=C1 XNOPRXBHLZRZKH-UHFFFAOYSA-N 0.000 description 1
- 108091007261 P2U receptors Proteins 0.000 description 1
- 102100037601 P2X purinoceptor 4 Human genes 0.000 description 1
- 238000009004 PCR Kit Methods 0.000 description 1
- 102000057297 Pepsin A Human genes 0.000 description 1
- 108090000284 Pepsin A Proteins 0.000 description 1
- 108091005804 Peptidases Proteins 0.000 description 1
- 102000035195 Peptidases Human genes 0.000 description 1
- 102000015439 Phospholipases Human genes 0.000 description 1
- 108010064785 Phospholipases Proteins 0.000 description 1
- 102000045595 Phosphoprotein Phosphatases Human genes 0.000 description 1
- 108700019535 Phosphoprotein Phosphatases Proteins 0.000 description 1
- 108010003541 Platelet Activating Factor Proteins 0.000 description 1
- 208000004403 Prostatic Hyperplasia Diseases 0.000 description 1
- 239000004365 Protease Substances 0.000 description 1
- 102000004022 Protein-Tyrosine Kinases Human genes 0.000 description 1
- 108090000412 Protein-Tyrosine Kinases Proteins 0.000 description 1
- 102000001744 Purinergic P2Y2 Receptors Human genes 0.000 description 1
- 108010080192 Purinergic Receptors Proteins 0.000 description 1
- CZPWVGJYEJSRLH-UHFFFAOYSA-N Pyrimidine Chemical compound C1=CN=CN=C1 CZPWVGJYEJSRLH-UHFFFAOYSA-N 0.000 description 1
- 108010092799 RNA-directed DNA polymerase Proteins 0.000 description 1
- MUPFEKGTMRGPLJ-RMMQSMQOSA-N Raffinose Natural products O(C[C@H]1[C@@H](O)[C@H](O)[C@@H](O)[C@@H](O[C@@]2(CO)[C@H](O)[C@@H](O)[C@@H](CO)O2)O1)[C@@H]1[C@H](O)[C@@H](O)[C@@H](O)[C@@H](CO)O1 MUPFEKGTMRGPLJ-RMMQSMQOSA-N 0.000 description 1
- 108020004511 Recombinant DNA Proteins 0.000 description 1
- 108700008625 Reporter Genes Proteins 0.000 description 1
- 102000006382 Ribonucleases Human genes 0.000 description 1
- 108010083644 Ribonucleases Proteins 0.000 description 1
- 241000714474 Rous sarcoma virus Species 0.000 description 1
- 101100348089 Saccharomyces cerevisiae (strain ATCC 204508 / S288c) BUR6 gene Proteins 0.000 description 1
- 108010031271 Saccharomyces cerevisiae Proteins Proteins 0.000 description 1
- 229920005654 Sephadex Polymers 0.000 description 1
- 239000012507 Sephadex™ Substances 0.000 description 1
- 238000012300 Sequence Analysis Methods 0.000 description 1
- 102100028904 Serine/threonine-protein kinase MARK2 Human genes 0.000 description 1
- 108010071390 Serum Albumin Proteins 0.000 description 1
- 102000007562 Serum Albumin Human genes 0.000 description 1
- 241000256248 Spodoptera Species 0.000 description 1
- 101000857870 Squalus acanthias Gonadoliberin Proteins 0.000 description 1
- 108091081024 Start codon Proteins 0.000 description 1
- 241000282887 Suidae Species 0.000 description 1
- NINIDFKCEFEMDL-UHFFFAOYSA-N Sulfur Chemical compound [S] NINIDFKCEFEMDL-UHFFFAOYSA-N 0.000 description 1
- ZMZDMBWJUHKJPS-UHFFFAOYSA-M Thiocyanate anion Chemical compound [S-]C#N ZMZDMBWJUHKJPS-UHFFFAOYSA-M 0.000 description 1
- AYFVYJQAPQTCCC-UHFFFAOYSA-N Threonine Natural products CC(O)C(N)C(O)=O AYFVYJQAPQTCCC-UHFFFAOYSA-N 0.000 description 1
- 239000004473 Threonine Substances 0.000 description 1
- 108090000190 Thrombin Proteins 0.000 description 1
- 208000007536 Thrombosis Diseases 0.000 description 1
- 102100032316 Transcription factor Sp6 Human genes 0.000 description 1
- 108700019146 Transgenes Proteins 0.000 description 1
- QIVBCDIJIAJPQS-UHFFFAOYSA-N Tryptophan Natural products C1=CC=C2C(CC(N)C(O)=O)=CNC2=C1 QIVBCDIJIAJPQS-UHFFFAOYSA-N 0.000 description 1
- 102000014384 Type C Phospholipases Human genes 0.000 description 1
- 108010079194 Type C Phospholipases Proteins 0.000 description 1
- MUPFEKGTMRGPLJ-UHFFFAOYSA-N UNPD196149 Natural products OC1C(O)C(CO)OC1(CO)OC1C(O)C(O)C(O)C(COC2C(C(O)C(O)C(CO)O2)O)O1 MUPFEKGTMRGPLJ-UHFFFAOYSA-N 0.000 description 1
- 108090000848 Ubiquitin Proteins 0.000 description 1
- 102000044159 Ubiquitin Human genes 0.000 description 1
- 208000006374 Uterine Cervicitis Diseases 0.000 description 1
- 206010046798 Uterine leiomyoma Diseases 0.000 description 1
- KZSNJWFQEVHDMF-UHFFFAOYSA-N Valine Natural products CC(C)C(N)C(O)=O KZSNJWFQEVHDMF-UHFFFAOYSA-N 0.000 description 1
- 208000024248 Vascular System injury Diseases 0.000 description 1
- 208000012339 Vascular injury Diseases 0.000 description 1
- 241000251539 Vertebrata <Metazoa> Species 0.000 description 1
- MMWCIQZXVOZEGG-HOZKJCLWSA-N [(1S,2R,3S,4S,5R,6S)-2,3,5-trihydroxy-4,6-diphosphonooxycyclohexyl] dihydrogen phosphate Chemical compound O[C@H]1[C@@H](O)[C@H](OP(O)(O)=O)[C@@H](OP(O)(O)=O)[C@H](O)[C@H]1OP(O)(O)=O MMWCIQZXVOZEGG-HOZKJCLWSA-N 0.000 description 1
- 230000005856 abnormality Effects 0.000 description 1
- 238000002835 absorbance Methods 0.000 description 1
- 125000000218 acetic acid group Chemical group C(C)(=O)* 0.000 description 1
- OIPILFWXSMYKGL-UHFFFAOYSA-N acetylcholine Chemical compound CC(=O)OCC[N+](C)(C)C OIPILFWXSMYKGL-UHFFFAOYSA-N 0.000 description 1
- 229960004373 acetylcholine Drugs 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 239000012190 activator Substances 0.000 description 1
- 239000004480 active ingredient Substances 0.000 description 1
- 239000013543 active substance Substances 0.000 description 1
- 231100000215 acute (single dose) toxicity testing Toxicity 0.000 description 1
- 238000011047 acute toxicity test Methods 0.000 description 1
- 125000002252 acyl group Chemical group 0.000 description 1
- 230000010933 acylation Effects 0.000 description 1
- 238000005917 acylation reaction Methods 0.000 description 1
- 229960000643 adenine Drugs 0.000 description 1
- 238000001042 affinity chromatography Methods 0.000 description 1
- 125000000217 alkyl group Chemical group 0.000 description 1
- 229960000711 alprostadil Drugs 0.000 description 1
- 150000001408 amides Chemical class 0.000 description 1
- 150000003862 amino acid derivatives Chemical class 0.000 description 1
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 description 1
- 229910052921 ammonium sulfate Inorganic materials 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
- 229930002877 anthocyanin Natural products 0.000 description 1
- 235000010208 anthocyanin Nutrition 0.000 description 1
- 239000004410 anthocyanin Substances 0.000 description 1
- 150000004636 anthocyanins Chemical class 0.000 description 1
- 230000000340 anti-metabolite Effects 0.000 description 1
- 230000002788 anti-peptide Effects 0.000 description 1
- 230000000692 anti-sense effect Effects 0.000 description 1
- 239000002256 antimetabolite Substances 0.000 description 1
- 229940100197 antimetabolite Drugs 0.000 description 1
- 210000000709 aorta Anatomy 0.000 description 1
- 238000013459 approach Methods 0.000 description 1
- 230000003305 autocrine Effects 0.000 description 1
- 108010058966 bacteriophage T7 induced DNA polymerase Proteins 0.000 description 1
- 210000000227 basophil cell of anterior lobe of hypophysis Anatomy 0.000 description 1
- 238000003287 bathing Methods 0.000 description 1
- 230000006399 behavior Effects 0.000 description 1
- 230000003542 behavioural effect Effects 0.000 description 1
- 230000008901 benefit Effects 0.000 description 1
- 102000005936 beta-Galactosidase Human genes 0.000 description 1
- 108010005774 beta-Galactosidase Proteins 0.000 description 1
- DRTQHJPVMGBUCF-PSQAKQOGSA-N beta-L-uridine Natural products O[C@H]1[C@@H](O)[C@H](CO)O[C@@H]1N1C(=O)NC(=O)C=C1 DRTQHJPVMGBUCF-PSQAKQOGSA-N 0.000 description 1
- 108091008324 binding proteins Proteins 0.000 description 1
- 238000004166 bioassay Methods 0.000 description 1
- 230000000035 biogenic effect Effects 0.000 description 1
- 238000001574 biopsy Methods 0.000 description 1
- 229930189065 blasticidin Natural products 0.000 description 1
- 239000002981 blocking agent Substances 0.000 description 1
- 230000000903 blocking effect Effects 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 210000004204 blood vessel Anatomy 0.000 description 1
- 210000000988 bone and bone Anatomy 0.000 description 1
- 229940098773 bovine serum albumin Drugs 0.000 description 1
- 210000005013 brain tissue Anatomy 0.000 description 1
- 238000009395 breeding Methods 0.000 description 1
- 230000001488 breeding effect Effects 0.000 description 1
- 101150046240 bsd gene Proteins 0.000 description 1
- 244000309464 bull Species 0.000 description 1
- 239000006227 byproduct Substances 0.000 description 1
- 238000010804 cDNA synthesis Methods 0.000 description 1
- BBBFJLBPOGFECG-VJVYQDLKSA-N calcitonin Chemical compound N([C@H](C(=O)N[C@@H](CC(C)C)C(=O)NCC(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CO)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC=1NC=NC=1)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC=1C=CC(O)=CC=1)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H]([C@@H](C)O)C(=O)NCC(=O)N[C@@H](CO)C(=O)NCC(=O)N[C@@H]([C@@H](C)O)C(=O)N1[C@@H](CCC1)C(N)=O)C(C)C)C(=O)[C@@H]1CSSC[C@H](N)C(=O)N[C@@H](CO)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CO)C(=O)N[C@@H]([C@@H](C)O)C(=O)N1 BBBFJLBPOGFECG-VJVYQDLKSA-N 0.000 description 1
- 229960004015 calcitonin Drugs 0.000 description 1
- 229910001424 calcium ion Inorganic materials 0.000 description 1
- 230000036952 cancer formation Effects 0.000 description 1
- 230000000711 cancerogenic effect Effects 0.000 description 1
- 239000004202 carbamide Substances 0.000 description 1
- 235000014633 carbohydrates Nutrition 0.000 description 1
- 150000001720 carbohydrates Chemical class 0.000 description 1
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 description 1
- 230000021523 carboxylation Effects 0.000 description 1
- 238000006473 carboxylation reaction Methods 0.000 description 1
- 231100000504 carcinogenesis Toxicity 0.000 description 1
- 231100000315 carcinogenic Toxicity 0.000 description 1
- 210000004413 cardiac myocyte Anatomy 0.000 description 1
- 239000005018 casein Substances 0.000 description 1
- BECPQYXYKAMYBN-UHFFFAOYSA-N casein, tech. Chemical compound NCCCCC(C(O)=O)N=C(O)C(CC(O)=O)N=C(O)C(CCC(O)=N)N=C(O)C(CC(C)C)N=C(O)C(CCC(O)=O)N=C(O)C(CC(O)=O)N=C(O)C(CCC(O)=O)N=C(O)C(C(C)O)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=O)N=C(O)C(CCC(O)=O)N=C(O)C(COP(O)(O)=O)N=C(O)C(CCC(O)=N)N=C(O)C(N)CC1=CC=CC=C1 BECPQYXYKAMYBN-UHFFFAOYSA-N 0.000 description 1
- 235000021240 caseins Nutrition 0.000 description 1
- 230000010261 cell growth Effects 0.000 description 1
- 210000000170 cell membrane Anatomy 0.000 description 1
- 206010008323 cervicitis Diseases 0.000 description 1
- 210000003679 cervix uteri Anatomy 0.000 description 1
- 230000003196 chaotropic effect Effects 0.000 description 1
- 239000013522 chelant Substances 0.000 description 1
- 238000007385 chemical modification Methods 0.000 description 1
- 210000004978 chinese hamster ovary cell Anatomy 0.000 description 1
- 229960005091 chloramphenicol Drugs 0.000 description 1
- WIIZWVCIJKGZOK-RKDXNWHRSA-N chloramphenicol Chemical compound ClC(Cl)C(=O)N[C@H](CO)[C@H](O)C1=CC=C([N+]([O-])=O)C=C1 WIIZWVCIJKGZOK-RKDXNWHRSA-N 0.000 description 1
- YTRQFSDWAXHJCC-UHFFFAOYSA-N chloroform;phenol Chemical compound ClC(Cl)Cl.OC1=CC=CC=C1 YTRQFSDWAXHJCC-UHFFFAOYSA-N 0.000 description 1
- 210000001612 chondrocyte Anatomy 0.000 description 1
- 238000004587 chromatography analysis Methods 0.000 description 1
- 230000002759 chromosomal effect Effects 0.000 description 1
- 231100000005 chromosome aberration Toxicity 0.000 description 1
- 238000003200 chromosome mapping Methods 0.000 description 1
- 201000003139 chronic cystitis Diseases 0.000 description 1
- 231100000132 chronic toxicity testing Toxicity 0.000 description 1
- 238000004140 cleaning Methods 0.000 description 1
- 239000011248 coating agent Substances 0.000 description 1
- 238000000576 coating method Methods 0.000 description 1
- 230000001427 coherent effect Effects 0.000 description 1
- 210000001072 colon Anatomy 0.000 description 1
- 208000029742 colonic neoplasm Diseases 0.000 description 1
- 238000004440 column chromatography Methods 0.000 description 1
- 238000002648 combination therapy Methods 0.000 description 1
- 230000009137 competitive binding Effects 0.000 description 1
- 230000002860 competitive effect Effects 0.000 description 1
- 239000003184 complementary RNA Substances 0.000 description 1
- 238000004590 computer program Methods 0.000 description 1
- 108091036078 conserved sequence Proteins 0.000 description 1
- 238000011109 contamination Methods 0.000 description 1
- 238000005112 continuous flow technique Methods 0.000 description 1
- 210000004351 coronary vessel Anatomy 0.000 description 1
- 239000007822 coupling agent Substances 0.000 description 1
- 239000013078 crystal Substances 0.000 description 1
- 210000004748 cultured cell Anatomy 0.000 description 1
- 238000012258 culturing Methods 0.000 description 1
- 150000001945 cysteines Chemical class 0.000 description 1
- 201000003146 cystitis Diseases 0.000 description 1
- UHDGCWIWMRVCDJ-ZAKLUEHWSA-N cytidine Chemical compound O=C1N=C(N)C=CN1[C@H]1[C@H](O)[C@@H](O)[C@H](CO)O1 UHDGCWIWMRVCDJ-ZAKLUEHWSA-N 0.000 description 1
- RGWHQCVHVJXOKC-SHYZEUOFSA-J dCTP(4-) Chemical compound O=C1N=C(N)C=CN1[C@@H]1O[C@H](COP([O-])(=O)OP([O-])(=O)OP([O-])([O-])=O)[C@@H](O)C1 RGWHQCVHVJXOKC-SHYZEUOFSA-J 0.000 description 1
- 208000036654 deficiency anemia Diseases 0.000 description 1
- 238000004925 denaturation Methods 0.000 description 1
- 230000036425 denaturation Effects 0.000 description 1
- 238000010511 deprotection reaction Methods 0.000 description 1
- 210000002249 digestive system Anatomy 0.000 description 1
- 238000006471 dimerization reaction Methods 0.000 description 1
- LOKCTEFSRHRXRJ-UHFFFAOYSA-I dipotassium trisodium dihydrogen phosphate hydrogen phosphate dichloride Chemical compound P(=O)(O)(O)[O-].[K+].P(=O)(O)([O-])[O-].[Na+].[Na+].[Cl-].[K+].[Cl-].[Na+] LOKCTEFSRHRXRJ-UHFFFAOYSA-I 0.000 description 1
- BNIILDVGGAEEIG-UHFFFAOYSA-L disodium hydrogen phosphate Chemical compound [Na+].[Na+].OP([O-])([O-])=O BNIILDVGGAEEIG-UHFFFAOYSA-L 0.000 description 1
- 229910000397 disodium phosphate Inorganic materials 0.000 description 1
- 235000019800 disodium phosphate Nutrition 0.000 description 1
- 229960003638 dopamine Drugs 0.000 description 1
- 231100000673 dose–response relationship Toxicity 0.000 description 1
- 229940000406 drug candidate Drugs 0.000 description 1
- 206010013663 drug dependence Diseases 0.000 description 1
- 238000007876 drug discovery Methods 0.000 description 1
- 238000007878 drug screening assay Methods 0.000 description 1
- 238000002651 drug therapy Methods 0.000 description 1
- 230000004064 dysfunction Effects 0.000 description 1
- 230000002900 effect on cell Effects 0.000 description 1
- 239000012636 effector Substances 0.000 description 1
- 238000001962 electrophoresis Methods 0.000 description 1
- 210000002257 embryonic structure Anatomy 0.000 description 1
- 239000000839 emulsion Substances 0.000 description 1
- 210000003372 endocrine gland Anatomy 0.000 description 1
- 210000001900 endoderm Anatomy 0.000 description 1
- 201000003908 endometrial adenocarcinoma Diseases 0.000 description 1
- 230000002357 endometrial effect Effects 0.000 description 1
- 208000029382 endometrium adenocarcinoma Diseases 0.000 description 1
- 230000002616 endonucleolytic effect Effects 0.000 description 1
- 210000002889 endothelial cell Anatomy 0.000 description 1
- 229940116977 epidermal growth factor Drugs 0.000 description 1
- 230000001973 epigenetic effect Effects 0.000 description 1
- 229960005139 epinephrine Drugs 0.000 description 1
- 210000003238 esophagus Anatomy 0.000 description 1
- 238000005530 etching Methods 0.000 description 1
- 102000013165 exonuclease Human genes 0.000 description 1
- 239000003777 experimental drug Substances 0.000 description 1
- 210000002744 extracellular matrix Anatomy 0.000 description 1
- 239000003925 fat Substances 0.000 description 1
- 235000019197 fats Nutrition 0.000 description 1
- 230000008175 fetal development Effects 0.000 description 1
- 239000000835 fiber Substances 0.000 description 1
- 239000007850 fluorescent dye Substances 0.000 description 1
- 229940028334 follicle stimulating hormone Drugs 0.000 description 1
- 230000037433 frameshift Effects 0.000 description 1
- 210000001652 frontal lobe Anatomy 0.000 description 1
- 238000010230 functional analysis Methods 0.000 description 1
- 230000002538 fungal effect Effects 0.000 description 1
- 229930182830 galactose Natural products 0.000 description 1
- 239000007789 gas Substances 0.000 description 1
- 238000001502 gel electrophoresis Methods 0.000 description 1
- 238000003209 gene knockout Methods 0.000 description 1
- 231100000089 gene mutation induction Toxicity 0.000 description 1
- 238000001415 gene therapy Methods 0.000 description 1
- 231100000025 genetic toxicology Toxicity 0.000 description 1
- 231100000024 genotoxic Toxicity 0.000 description 1
- 230000001738 genotoxic effect Effects 0.000 description 1
- 210000004602 germ cell Anatomy 0.000 description 1
- 229930195712 glutamate Natural products 0.000 description 1
- 229940049906 glutamate Drugs 0.000 description 1
- 229940096919 glycogen Drugs 0.000 description 1
- XLXSAKCOAKORKW-AQJXLSMYSA-N gonadorelin Chemical compound C([C@@H](C(=O)NCC(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N1[C@@H](CCC1)C(=O)NCC(N)=O)NC(=O)[C@H](CO)NC(=O)[C@H](CC=1C2=CC=CC=C2NC=1)NC(=O)[C@H](CC=1N=CNC=1)NC(=O)[C@H]1NC(=O)CC1)C1=CC=C(O)C=C1 XLXSAKCOAKORKW-AQJXLSMYSA-N 0.000 description 1
- 229940035638 gonadotropin-releasing hormone Drugs 0.000 description 1
- 210000003714 granulocyte Anatomy 0.000 description 1
- 239000005090 green fluorescent protein Substances 0.000 description 1
- 230000003394 haemopoietic effect Effects 0.000 description 1
- 238000010438 heat treatment Methods 0.000 description 1
- 230000002363 herbicidal effect Effects 0.000 description 1
- 239000004009 herbicide Substances 0.000 description 1
- 238000013537 high throughput screening Methods 0.000 description 1
- 229960001340 histamine Drugs 0.000 description 1
- 230000013632 homeostatic process Effects 0.000 description 1
- 210000005260 human cell Anatomy 0.000 description 1
- 210000003917 human chromosome Anatomy 0.000 description 1
- BHEPBYXIRTUNPN-UHFFFAOYSA-N hydridophosphorus(.) (triplet) Chemical compound [PH] BHEPBYXIRTUNPN-UHFFFAOYSA-N 0.000 description 1
- 150000002431 hydrogen Chemical class 0.000 description 1
- 230000002209 hydrophobic effect Effects 0.000 description 1
- 125000002887 hydroxy group Chemical group [H]O* 0.000 description 1
- 238000009802 hysterectomy Methods 0.000 description 1
- 230000001900 immune effect Effects 0.000 description 1
- 238000002649 immunization Methods 0.000 description 1
- 230000000984 immunochemical effect Effects 0.000 description 1
- 230000002163 immunogen Effects 0.000 description 1
- 230000016784 immunoglobulin production Effects 0.000 description 1
- 238000010348 incorporation Methods 0.000 description 1
- 230000001939 inductive effect Effects 0.000 description 1
- 239000012678 infectious agent Substances 0.000 description 1
- 230000001524 infective effect Effects 0.000 description 1
- 230000028709 inflammatory response Effects 0.000 description 1
- 230000002401 inhibitory effect Effects 0.000 description 1
- 230000005764 inhibitory process Effects 0.000 description 1
- 230000000977 initiatory effect Effects 0.000 description 1
- 238000002347 injection Methods 0.000 description 1
- 239000007924 injection Substances 0.000 description 1
- 229960003786 inosine Drugs 0.000 description 1
- 230000010354 integration Effects 0.000 description 1
- 238000009830 intercalation Methods 0.000 description 1
- 230000002687 intercalation Effects 0.000 description 1
- 201000006334 interstitial nephritis Diseases 0.000 description 1
- PNDPGZBMCMUPRI-UHFFFAOYSA-N iodine Chemical compound II PNDPGZBMCMUPRI-UHFFFAOYSA-N 0.000 description 1
- 229910052742 iron Inorganic materials 0.000 description 1
- 238000002955 isolation Methods 0.000 description 1
- 229960000310 isoleucine Drugs 0.000 description 1
- AGPKZVBTJJNPAG-UHFFFAOYSA-N isoleucine Natural products CCC(C)C(N)C(O)=O AGPKZVBTJJNPAG-UHFFFAOYSA-N 0.000 description 1
- 229940039009 isoproterenol Drugs 0.000 description 1
- 229960000318 kanamycin Drugs 0.000 description 1
- SBUJHOSQTJFQJX-NOAMYHISSA-N kanamycin Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CN)O[C@@H]1O[C@H]1[C@H](O)[C@@H](O[C@@H]2[C@@H]([C@@H](N)[C@H](O)[C@@H](CO)O2)O)[C@H](N)C[C@@H]1N SBUJHOSQTJFQJX-NOAMYHISSA-N 0.000 description 1
- 229930027917 kanamycin Natural products 0.000 description 1
- 229930182823 kanamycin A Natural products 0.000 description 1
- 101150066555 lacZ gene Proteins 0.000 description 1
- 239000002523 lectin Substances 0.000 description 1
- 230000000503 lectinlike effect Effects 0.000 description 1
- 231100000518 lethal Toxicity 0.000 description 1
- 230000001665 lethal effect Effects 0.000 description 1
- 208000032839 leukemia Diseases 0.000 description 1
- 210000000265 leukocyte Anatomy 0.000 description 1
- 150000002617 leukotrienes Chemical class 0.000 description 1
- 238000009630 liquid culture Methods 0.000 description 1
- XIXADJRWDQXREU-UHFFFAOYSA-M lithium acetate Chemical compound [Li+].CC([O-])=O XIXADJRWDQXREU-UHFFFAOYSA-M 0.000 description 1
- 210000004185 liver Anatomy 0.000 description 1
- 239000012139 lysis buffer Substances 0.000 description 1
- 210000002540 macrophage Anatomy 0.000 description 1
- 238000012423 maintenance Methods 0.000 description 1
- 208000029565 malignant colon neoplasm Diseases 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 230000001404 mediated effect Effects 0.000 description 1
- 238000002483 medication Methods 0.000 description 1
- PSGAAPLEWMOORI-PEINSRQWSA-N medroxyprogesterone acetate Chemical compound C([C@@]12C)CC(=O)C=C1[C@@H](C)C[C@@H]1[C@@H]2CC[C@]2(C)[C@@](OC(C)=O)(C(C)=O)CC[C@H]21 PSGAAPLEWMOORI-PEINSRQWSA-N 0.000 description 1
- 210000003716 mesoderm Anatomy 0.000 description 1
- 229910052751 metal Inorganic materials 0.000 description 1
- 239000002184 metal Substances 0.000 description 1
- 229910021645 metal ion Inorganic materials 0.000 description 1
- WSFSSNUMVMOOMR-NJFSPNSNSA-N methanone Chemical compound O=[14CH2] WSFSSNUMVMOOMR-NJFSPNSNSA-N 0.000 description 1
- 229930182817 methionine Natural products 0.000 description 1
- 125000001360 methionine group Chemical group N[C@@H](CCSC)C(=O)* 0.000 description 1
- 238000010208 microarray analysis Methods 0.000 description 1
- 239000011859 microparticle Substances 0.000 description 1
- 238000000386 microscopy Methods 0.000 description 1
- 230000005012 migration Effects 0.000 description 1
- 238000013508 migration Methods 0.000 description 1
- 235000013336 milk Nutrition 0.000 description 1
- 239000008267 milk Substances 0.000 description 1
- 210000004080 milk Anatomy 0.000 description 1
- 210000003470 mitochondria Anatomy 0.000 description 1
- 238000001823 molecular biology technique Methods 0.000 description 1
- 238000010369 molecular cloning Methods 0.000 description 1
- 125000004573 morpholin-4-yl group Chemical group N1(CCOCC1)* 0.000 description 1
- 230000003551 muscarinic effect Effects 0.000 description 1
- 210000002346 musculoskeletal system Anatomy 0.000 description 1
- 210000005036 nerve Anatomy 0.000 description 1
- 210000000653 nervous system Anatomy 0.000 description 1
- 210000003061 neural cell Anatomy 0.000 description 1
- 210000002569 neuron Anatomy 0.000 description 1
- 238000006386 neutralization reaction Methods 0.000 description 1
- 230000009871 nonspecific binding Effects 0.000 description 1
- 238000007826 nucleic acid assay Methods 0.000 description 1
- 230000003287 optical effect Effects 0.000 description 1
- 210000003463 organelle Anatomy 0.000 description 1
- 201000008482 osteoarthritis Diseases 0.000 description 1
- 230000002018 overexpression Effects 0.000 description 1
- 210000003101 oviduct Anatomy 0.000 description 1
- 229940094443 oxytocics prostaglandins Drugs 0.000 description 1
- XNOPRXBHLZRZKH-DSZYJQQASA-N oxytocin Chemical compound C([C@H]1C(=O)N[C@H](C(N[C@@H](CCC(N)=O)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CSSC[C@H](N)C(=O)N1)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H](CC(C)C)C(=O)NCC(N)=O)=O)[C@@H](C)CC)C1=CC=C(O)C=C1 XNOPRXBHLZRZKH-DSZYJQQASA-N 0.000 description 1
- 229960001723 oxytocin Drugs 0.000 description 1
- 230000003076 paracrine Effects 0.000 description 1
- 210000003681 parotid gland Anatomy 0.000 description 1
- 230000007170 pathology Effects 0.000 description 1
- 230000037361 pathway Effects 0.000 description 1
- 239000013610 patient sample Substances 0.000 description 1
- 229940111202 pepsin Drugs 0.000 description 1
- 239000000813 peptide hormone Substances 0.000 description 1
- 239000008177 pharmaceutical agent Substances 0.000 description 1
- 230000009120 phenotypic response Effects 0.000 description 1
- 239000008363 phosphate buffer Substances 0.000 description 1
- 150000008300 phosphoramidites Chemical class 0.000 description 1
- 230000000704 physical effect Effects 0.000 description 1
- 239000004033 plastic Substances 0.000 description 1
- 229920003023 plastic Polymers 0.000 description 1
- 239000002985 plastic film Substances 0.000 description 1
- 229920006255 plastic film Polymers 0.000 description 1
- 230000010118 platelet activation Effects 0.000 description 1
- 229920001983 poloxamer Polymers 0.000 description 1
- 229920000352 poly(styrene-co-divinylbenzene) Polymers 0.000 description 1
- 238000002264 polyacrylamide gel electrophoresis Methods 0.000 description 1
- 229920000447 polyanionic polymer Polymers 0.000 description 1
- 229920002704 polyhistidine Polymers 0.000 description 1
- 239000002952 polymeric resin Substances 0.000 description 1
- 102000054765 polymorphisms of proteins Human genes 0.000 description 1
- 229920005862 polyol Polymers 0.000 description 1
- 150000003077 polyols Chemical class 0.000 description 1
- 230000004481 post-translational protein modification Effects 0.000 description 1
- 230000007542 postnatal development Effects 0.000 description 1
- 238000007781 pre-processing Methods 0.000 description 1
- 238000001556 precipitation Methods 0.000 description 1
- 230000035935 pregnancy Effects 0.000 description 1
- 238000002953 preparative HPLC Methods 0.000 description 1
- 238000011809 primate model Methods 0.000 description 1
- 239000002987 primer (paints) Substances 0.000 description 1
- 230000037452 priming Effects 0.000 description 1
- 210000001236 prokaryotic cell Anatomy 0.000 description 1
- 230000035755 proliferation Effects 0.000 description 1
- AAEVYOVXGOFMJO-UHFFFAOYSA-N prometryn Chemical compound CSC1=NC(NC(C)C)=NC(NC(C)C)=N1 AAEVYOVXGOFMJO-UHFFFAOYSA-N 0.000 description 1
- 230000001915 proofreading effect Effects 0.000 description 1
- GMVPRGQOIOIIMI-DWKJAMRDSA-N prostaglandin E1 Chemical compound CCCCC[C@H](O)\C=C\[C@H]1[C@H](O)CC(=O)[C@@H]1CCCCCCC(O)=O GMVPRGQOIOIIMI-DWKJAMRDSA-N 0.000 description 1
- XEYBRNLFEZDVAW-UHFFFAOYSA-N prostaglandin E2 Natural products CCCCCC(O)C=CC1C(O)CC(=O)C1CC=CCCCC(O)=O XEYBRNLFEZDVAW-UHFFFAOYSA-N 0.000 description 1
- 150000003180 prostaglandins Chemical class 0.000 description 1
- 125000006239 protecting group Chemical group 0.000 description 1
- 230000004952 protein activity Effects 0.000 description 1
- 238000002731 protein assay Methods 0.000 description 1
- 238000000159 protein binding assay Methods 0.000 description 1
- 230000012846 protein folding Effects 0.000 description 1
- 230000018883 protein targeting Effects 0.000 description 1
- 229940063222 provera Drugs 0.000 description 1
- 230000002685 pulmonary effect Effects 0.000 description 1
- 150000003212 purines Chemical class 0.000 description 1
- 150000003230 pyrimidines Chemical class 0.000 description 1
- 238000004445 quantitative analysis Methods 0.000 description 1
- 230000006340 racemization Effects 0.000 description 1
- 230000005855 radiation Effects 0.000 description 1
- MUPFEKGTMRGPLJ-ZQSKZDJDSA-N raffinose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO[C@@H]2[C@@H]([C@@H](O)[C@@H](O)[C@@H](CO)O2)O)O1 MUPFEKGTMRGPLJ-ZQSKZDJDSA-N 0.000 description 1
- 239000011535 reaction buffer Substances 0.000 description 1
- 238000003753 real-time PCR Methods 0.000 description 1
- 238000003259 recombinant expression Methods 0.000 description 1
- 238000010188 recombinant method Methods 0.000 description 1
- 230000006798 recombination Effects 0.000 description 1
- 230000002829 reductive effect Effects 0.000 description 1
- 238000007634 remodeling Methods 0.000 description 1
- 230000003252 repetitive effect Effects 0.000 description 1
- 230000003362 replicative effect Effects 0.000 description 1
- 230000004044 response Effects 0.000 description 1
- 210000001995 reticulocyte Anatomy 0.000 description 1
- 230000002207 retinal effect Effects 0.000 description 1
- 206010039073 rheumatoid arthritis Diseases 0.000 description 1
- 239000003161 ribonuclease inhibitor Substances 0.000 description 1
- 238000011808 rodent model Methods 0.000 description 1
- 108010038196 saccharide-binding proteins Proteins 0.000 description 1
- 235000019515 salmon Nutrition 0.000 description 1
- 229920006298 saran Polymers 0.000 description 1
- 230000001953 sensory effect Effects 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 229940076279 serotonin Drugs 0.000 description 1
- 210000002966 serum Anatomy 0.000 description 1
- 238000012154 short term therapy Methods 0.000 description 1
- 238000002741 site-directed mutagenesis Methods 0.000 description 1
- 210000000329 smooth muscle myocyte Anatomy 0.000 description 1
- 230000003595 spectral effect Effects 0.000 description 1
- 230000002269 spontaneous effect Effects 0.000 description 1
- 238000010561 standard procedure Methods 0.000 description 1
- 239000012086 standard solution Substances 0.000 description 1
- 230000004936 stimulating effect Effects 0.000 description 1
- 230000007666 subchronic toxicity Effects 0.000 description 1
- 231100000195 subchronic toxicity Toxicity 0.000 description 1
- 208000011117 substance-related disease Diseases 0.000 description 1
- 235000000346 sugar Nutrition 0.000 description 1
- 239000011593 sulfur Substances 0.000 description 1
- 229910052717 sulfur Inorganic materials 0.000 description 1
- 230000004083 survival effect Effects 0.000 description 1
- 208000024891 symptom Diseases 0.000 description 1
- 229920003002 synthetic resin Polymers 0.000 description 1
- 125000004149 thio group Chemical group *S* 0.000 description 1
- 150000003573 thiols Chemical class 0.000 description 1
- RYYWUUFWQRZTIU-UHFFFAOYSA-K thiophosphate Chemical compound [O-]P([O-])([O-])=S RYYWUUFWQRZTIU-UHFFFAOYSA-K 0.000 description 1
- 125000000341 threoninyl group Chemical group [H]OC([H])(C([H])([H])[H])C([H])(N([H])[H])C(*)=O 0.000 description 1
- 229960004072 thrombin Drugs 0.000 description 1
- 229940113082 thymine Drugs 0.000 description 1
- 231100000331 toxic Toxicity 0.000 description 1
- 231100000167 toxic agent Toxicity 0.000 description 1
- 230000002588 toxic effect Effects 0.000 description 1
- 239000003440 toxic substance Substances 0.000 description 1
- 231100000440 toxicity profile Toxicity 0.000 description 1
- 239000003053 toxin Substances 0.000 description 1
- 231100000765 toxin Toxicity 0.000 description 1
- 108700012359 toxins Proteins 0.000 description 1
- 230000005758 transcription activity Effects 0.000 description 1
- 230000005026 transcription initiation Effects 0.000 description 1
- 230000001052 transient effect Effects 0.000 description 1
- 230000010474 transient expression Effects 0.000 description 1
- 102000035160 transmembrane proteins Human genes 0.000 description 1
- 108091005703 transmembrane proteins Proteins 0.000 description 1
- 238000011269 treatment regimen Methods 0.000 description 1
- GPRLSGONYQIRFK-MNYXATJNSA-N triton Chemical compound [3H+] GPRLSGONYQIRFK-MNYXATJNSA-N 0.000 description 1
- 238000001419 two-dimensional polyacrylamide gel electrophoresis Methods 0.000 description 1
- 108010087967 type I signal peptidase Proteins 0.000 description 1
- 125000001493 tyrosinyl group Chemical group [H]OC1=C([H])C([H])=C(C([H])=C1[H])C([H])([H])C([H])(N([H])[H])C(*)=O 0.000 description 1
- 238000002604 ultrasonography Methods 0.000 description 1
- 230000009452 underexpressoin Effects 0.000 description 1
- 241000701161 unidentified adenovirus Species 0.000 description 1
- 241000701366 unidentified nuclear polyhedrosis viruses Species 0.000 description 1
- 241001515965 unidentified phage Species 0.000 description 1
- 229940035893 uracil Drugs 0.000 description 1
- DRTQHJPVMGBUCF-UHFFFAOYSA-N uracil arabinoside Natural products OC1C(O)C(CO)OC1N1C(=O)NC(=O)C=C1 DRTQHJPVMGBUCF-UHFFFAOYSA-N 0.000 description 1
- 229940045145 uridine Drugs 0.000 description 1
- VBEQCZHXXJYVRD-GACYYNSASA-N uroanthelone Chemical compound C([C@@H](C(=O)N[C@H](C(=O)N[C@@H](CS)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CS)C(=O)N[C@H](C(=O)N[C@@H]([C@@H](C)CC)C(=O)NCC(=O)N[C@@H](CC=1C=CC(O)=CC=1)C(=O)N[C@@H](CO)C(=O)NCC(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CS)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCCNC(N)=N)C(O)=O)C(C)C)[C@@H](C)O)NC(=O)[C@H](CO)NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CO)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@@H](NC(=O)[C@H](CC=1NC=NC=1)NC(=O)[C@H](CCSC)NC(=O)[C@H](CS)NC(=O)[C@@H](NC(=O)CNC(=O)CNC(=O)[C@H](CC(N)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CS)NC(=O)[C@H](CC=1C=CC(O)=CC=1)NC(=O)CNC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CC=1C=CC(O)=CC=1)NC(=O)[C@H](CO)NC(=O)[C@H](CO)NC(=O)[C@H]1N(CCC1)C(=O)[C@H](CS)NC(=O)CNC(=O)[C@H]1N(CCC1)C(=O)[C@H](CC=1C=CC(O)=CC=1)NC(=O)[C@H](CO)NC(=O)[C@@H](N)CC(N)=O)C(C)C)[C@@H](C)CC)C1=CC=C(O)C=C1 VBEQCZHXXJYVRD-GACYYNSASA-N 0.000 description 1
- 208000024719 uterine cervix neoplasm Diseases 0.000 description 1
- 201000007954 uterine fibroid Diseases 0.000 description 1
- 229960005486 vaccine Drugs 0.000 description 1
- 239000004474 valine Substances 0.000 description 1
- 150000003722 vitamin derivatives Chemical class 0.000 description 1
- 235000012431 wafers Nutrition 0.000 description 1
- 210000000707 wrist Anatomy 0.000 description 1
- QAOHCFGKCWTBGC-QHOAOGIMSA-N wybutosine Chemical compound C1=NC=2C(=O)N3C(CC[C@H](NC(=O)OC)C(=O)OC)=C(C)N=C3N(C)C=2N1[C@@H]1O[C@H](CO)[C@@H](O)[C@H]1O QAOHCFGKCWTBGC-QHOAOGIMSA-N 0.000 description 1
- QAOHCFGKCWTBGC-UHFFFAOYSA-N wybutosine Natural products C1=NC=2C(=O)N3C(CCC(NC(=O)OC)C(=O)OC)=C(C)N=C3N(C)C=2N1C1OC(CO)C(O)C1O QAOHCFGKCWTBGC-UHFFFAOYSA-N 0.000 description 1
- 238000001086 yeast two-hybrid system Methods 0.000 description 1
- DGVVWUTYPXICAM-UHFFFAOYSA-N β‐Mercaptoethanol Chemical compound OCCS DGVVWUTYPXICAM-UHFFFAOYSA-N 0.000 description 1
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
- G01N33/57484—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
- C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
-
- 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
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
- C12N15/62—DNA sequences coding for fusion proteins
- C12N15/625—DNA sequences coding for fusion proteins containing a sequence coding for a signal sequence
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
- C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
- G01N33/57407—Specifically defined cancers
- G01N33/57415—Specifically defined cancers of breast
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
- C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
- C07K2319/02—Fusion polypeptide containing a localisation/targetting motif containing a signal sequence
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
- C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
- C07K2319/036—Fusion polypeptide containing a localisation/targetting motif targeting to the medium outside of the cell, e.g. type III secretion
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
- C07K2319/60—Fusion polypeptide containing spectroscopic/fluorescent detection, e.g. green fluorescent protein [GFP]
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
- C07K2319/61—Fusion polypeptide containing an enzyme fusion for detection (lacZ, luciferase)
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
- C07K2319/70—Fusion polypeptide containing domain for protein-protein interaction
- C07K2319/71—Fusion polypeptide containing domain for protein-protein interaction containing domain for transcriptional activaation, e.g. VP16
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
- C07K2319/80—Fusion polypeptide containing a DNA binding domain, e.g. Lacl or Tet-repressor
Definitions
- This invention relates to signal peptide-containing proteins, their encoding cDNAs, and antibodies which binds the proteins, which can be used in the diagnosis, prognosis, treatment and evaluation of therapies for disorders associated with cell proliferation and cell signaling.
- Protein transport is a quintessential process for both prokaryotic and eukaryotic cells. Transport of an individual protein usually occurs via an amino-terminal signal sequence which directs, or targets, the protein from its ribosomal assembly site to a particular cellular or extracellular location. Transport may involve any combination of several of the following steps: contact with a chaperone, unfolding, interaction with a receptor and/or a pore complex, addition of energy, and refolding. Moreover, an extracellular protein may be produced as an inactive precursor. Once the precursor has been exported, removal of the signal sequence by a signal peptidase activates the protein.
- HMMs hidden Markov models
- HMMs have a formal probabilistic basis and use position-specific scores for amino acids or nucleotides and for opening and extending an insertion or deletion.
- the algorithms are quite flexible in that they incorporate information from newly identified sequences to build even more successful patterns.
- To find signal sequences multiple unaligned sequences are compared to identify those which encode a peptide of 20 to 50 amino acids with an N-terminal methionine.
- receptors nuclear, 4 transmembrane, G protein coupled, and tyrosine kinase
- cytokines chemokines
- hormones growth and differentiation factors
- neuropeptides and vasomediators protein kinases, phosphatases, phospholipases, phosphodiesterases, nucleotide cyclases, matrix molecules (adhesion, cadherin, extracellular matrix molecules, integrin, and selectin), G proteins, ion channels (calcium, chloride, potassium, and sodium), proteases, transporter/pumps (amino acid, protein, sugar, metal and vitamin; calcium, phosphate, potassium, and sodium) and regulatory proteins. Receptors, kinases, and matrix proteins and diseases associated with their dysfunction are described below.
- GPCRs are a large group of receptors which transduce extracellular signals.
- GPCRs include receptors for biogenic amines such as dopamine, epinephrine, histamine, glutamate (metabotropic effect), acetylcholine (muscarinic effect), and serotonin; for lipid mediators of inflammation such as prostaglandins, platelet activating factor, and leukotrienes; for peptide hormones such as calcitonin, C5a anaphylatoxin, follicle stimulating hormone, gonadotropin releasing hormone, neurokinin, oxytocin, and thrombin; and for sensory signal mediators such as retinal photopigments and olfactory stimulatory molecules.
- biogenic amines such as dopamine, epinephrine, histamine, glutamate (metabotropic effect), acetylcholine (muscarinic effect), and serotonin
- lipid mediators of inflammation such as prostaglandin
- the structure of these highly-conserved receptors consists of seven hydrophobic transmembrane regions, an extracellular N-terminus, and a cytoplasmic C-terminus.
- the N-terminus interacts with ligands, and the C-terminus interacts with intracellular G proteins to activate second messengers such as cyclic AMP (cAMP), phospholipase C, inositol triphosphate, or ion channel proteins.
- cAMP cyclic AMP
- phospholipase C phospholipase C
- inositol triphosphate or ion channel proteins.
- Three extracellular loops alternate with three intracellular loops to link the seven transmembrane regions. The most conserved parts of these proteins are the transmembrane regions and the first two cytoplasmic loops.
- the kinases comprise the largest known group of proteins, a superfamily of enzymes with widely varied functions and specificities. Kinases regulate many different cell proliferation, differentiation, and signaling processes by adding phosphate groups to proteins. Receptor mediated extracellular events trigger the transfer of these high energy phosphate groups and activate intracellular signaling cascades. Activation is roughly analogous to the turning on a molecular switch, and in cases where signaling is uncontrolled may be associated with or produce inflammation and cancer.
- kinases are usually named after their substrate, their regulatory molecule, or after some aspect of a mutant phenotype. Almost all kinases contain a similar 250-300 amino acid catalytic domain.
- the N-terminal domain which contains subdomains I-IV, generally folds into a two-lobed structure which binds and orients the ATP (or GTP) donor molecule.
- the larger C terminal lobe which contains subdomains VIA-XI, binds the protein substrate and carries out the transfer of the gamma phosphate from ATP to the hydroxyl group of a serine, threonine, or tyrosine residue.
- Subdomain V spans the two lobes.
- the kinases may be categorized into families by the different amino acid sequences (between 5 and 100 residues) located on either side of, or inserted into loops of, the kinase domain. These amino acid sequences allow the regulation of each kinase as it recognizes and interacts with its target protein.
- the primary structure of the kinase domain is conserved and contains specific residues and identifiable motifs or patterns of amino acids.
- the serine threonine kinases represent one family which preferentially phosphorylates serine or threonine residues.
- MPs The matrix proteins
- the matrix proteins (MPs) provide structural support cell and tissue identity and autocrine, paracrine and juxtacrine properties for most eukaryotic cells (McGowan (1992) FASEB J 6:2895-2904).
- MPs include adhesion molecules, integrins and selectins, cadherins, lectins, lipocalins, and extracellular matrix proteins (ECMs).
- ECMs extracellular matrix proteins
- MPs possess many different domains which interact with soluble, extracellular molecules. These domains include collagen-like domains. EGF-like domains immunoglobulin-like domains, fibronectin-like domains, type A domain of von Willebrand factor (vWFA)-like modules. ankyrin repeat modules. RDG or RDG-like sequences. carbohydrate-binding domains, and calcium ion-binding domains.
- ECM proteins are frequently characterized by the presence of one or more domains which may contain a number of potential intracellular disulphide bridge motifs.
- domains which match the epidermal growth factor tandem repeat consensus are present within several known extracellular proteins that promote cell growth, development. and cell signaling.
- Other domains share internal homology and a regular distribution of single cysteines and cysteine doublets.
- cysteine arrangement generates the characteristic “double-loop” structure (Soltysik-Espanola et al.
- ECMPs are members of the vWFA-like module superfamily, a diverse group of proteins with a module sharing high sequence similarity.
- the vWFA-like module is found not only in plasma proteins, but also in plasma membrane and ECMPs (Colombatti and Bonaldo (1991) Blood 77:2305-2315). Crystal structure analysis of an integrin vWFA-like module shows a classic “Rossmann” fold and suggests a metal ion-dependent adhesion site for binding protein ligands (Lee et al. (1995) Cell 80:631-638).
- MPs function in the formation, growth, remodeling, and maintenance of bone, and in the mediation and regulation of inflammation.
- Biochemical changes that result from congenital, epigenetic, or infectious diseases affect the expression and balance of MPs. This balance, in turn, affects the activation, proliferation, differentiation, and migration of leukocytes and determines whether the immune response is appropriate or self-destructive (Roman (1996) Immunol. Res. 15:163-178).
- Adenylyl cyclases are a group of second messenger molecules which actively participate in cell signaling processes.
- the neural-specific type I AC is a Ca ++ -stimulated enzyme
- the human type VII is unresponsive to CA ++ and responds to prostaglandin E1 and isoproterenol. Characterization of these ACs, their tissue distribution, and the activators and inhibitors of the different types of ACs is the subject of various investigations (Nielsen et al. (1996) J. Biol. Chem. 271:33308-16; Hellevuo et al. (1995) J. Biol. Chem. 270:11581-9). AC interactions with kinases and G proteins in the intracellular signaling pathways of all tissues make them interesting candidate molecules for pharmaceutical research.
- ATP diphosphohydrolase is an enzyme expressed and secreted by quiescent endothelial cells and involved in vasomediation.
- the physiological role of ATPDase is to convert ATP and ADP to AMP. When this conversion occurs in the blood vessels during inflammatory response, it prevents extracellular ATP from causing vascular injury by inhibiting platelet activation and modulating vascular thrombosis (Robson et al. (1997) J. Exp. Med. 185:153-63).
- the present invention is based on the discovery of signal peptide-containing proteins, their encoding cDNAs and antibodies which specifically binds the proteins that are useful in the diagnosis, prognosis. treatment and evaluation of therapies for disorders associated with cell proliferation and cell signaling.
- the invention provides an isolated cDNA comprising a nucleic acid molecule selected from SEQ ID NOs: 1-15 and 17-78.
- SEQ ID NO: 17 encodes a protein having an amino acid sequence of SEQ ID NO: 16.
- the invention also provides isolated cDNAs comprising SEQ ID NOs: 18-78 which have from about 80% to about 100% sequence identity with NOs: 1-15 and 17.
- the invention additionally encompasses a complement of the cDNAs selected from SEQ ID NOs: 1-15 and 17-78.
- the cDNA of SEQ ID NO: 17 is a fragment or an oligonucleotide comprising a nucleic acid molecule selected from A 24 to G 44 , G 159 to C 182 , G 561 to A 596 , or A 1011 and T 1046 .
- the invention provides compositions comprising the cDNAs or their complements and a heterologous nucleotide sequence or a labeling moiety which may be used in methods of the invention, on a substrate, or in solution.
- the invention further provides a vector containing the cDNA. a host cell containing the vector, and a method for using the cDNA to make the human protein.
- the invention still further provides a transgenic cell line or organism comprising the vector containing a cDNA selected from SEQ ID NOs: 1-15 and 17-78.
- the invention provides a cDNA or the complement thereof which can be used in methods of detection. screening, and purification.
- the cDNA is a single-stranded RNA or DNA molecule, a peptide nucleic acid, a branched nucleic acid, and the like.
- the invention provides a method for using a cDNA to detect differential expression of a nucleic acid in a sample comprising hybridizing a eDNA to the nucleic acids, thereby forming hybridization complexes and comparing hybridization complex formation with at least one standard, wherein the comparison indicates differential expression of the cDNA in the sample.
- the method of detection further comprises amplifying the nucleic acids of the sample prior to hybridization.
- the method showing differential expression of the cDNA is used to diagnose a cancer.
- the invention additionally provides a method for using a composition of the invention to screen a plurality of molecules or compounds to identify or purify at least one ligand which specifically binds the cDNA, the method comprising combining the composition with the molecules or compounds under conditions allowing specific binding, and detecting specific binding to the composition, thereby identifying or purifying a ligand which binds the composition.
- the molecules or compounds are selected from aptamers, DNA molecules, RNA molecules, peptide nucleic acids, artificial chromosome constructions. peptides, transcription factors, repressors, and regulatory molecules.
- the invention provides a purified protein or a portion thereof selected from the group consisting of an amino acid sequence of SEQ ID NO: 16, a variant of SEQ ID NO: 16, an antigenic epitope of SEQ ID NO: 16, and a biologically active portion of SEQ ID NO: 16.
- the invention also provides a composition comprising the purified protein and a labeling moiety or a pharmaceutical carrier.
- the invention further provides a method of using the protein to treat a subject with cancer comprising administering to a patient in need of such treatment a composition containing the purified protein and a pharmaceutical carrier.
- the invention still further provides a method for using a protein to screen a library or a plurality of molecules or compounds to identify or purify at least one ligand, the method comprising combining the protein with the molecules or compounds under conditions to allow specific binding and detecting specific binding, thereby identifying or purifying a ligand which specifically binds the protein.
- the molecules or compounds are selected from DNA molecules, RNA molecules, peptide nucleic acids, peptides, proteins, mimetics, agonists, antagonists, antibodies, immunoglobulins. inhibitors, and drugs.
- the ligand is used to treat a subject with a cancer.
- the invention provides a method of using a protein having the amino acid sequence of SEQ ID NO: 16 to screen a plurality of antibodies to identify an antibody which specifically binds the protein comprising contacting isolated antibodies with the protein under conditions to form an antibody:protein complex, and dissociating the antibody from the protein, thereby obtaining antibody which specifically binds the protein.
- the invention also provides methods for using a protein having the amino acid sequence of SEQ ID NO: 16 to prepare and purify polyclonal and monoclonal antibodies which specifically bind the protein.
- the method for preparing a polyclonal antibody comprises immunizing a animal with protein under conditions to elicit an antibody response, isolating animal antibodies, attaching the protein to a substrate, contacting the substrate with isolated antibodies under conditions to allow specific binding to the protein, dissociating the antibodies from the protein, thereby obtaining purified polyclonal antibodies.
- the method for preparing and purifying monoclonal antibodies comprises immunizing a animal with a protein under conditions to elicit an antibody response, isolating antibody producing cells from the animal, fusing the antibody producing cells with immortalized cells in culture to form monoclonal antibody producing hybridoma cells, culturing the hybridoma cells, and isolating from culture monoclonal antibodies which specifically bind the protein.
- the invention provides purified polyclonal and monoclonal antibodies which bind specifically to a protein.
- the invention also provides a method for using an antibody to detect expression of a protein in a sample, the method comprising combining the antibody with a sample under conditions which allow the formation of antibody:protein complexes; and detecting complex formation, wherein complex formation indicates expression of the protein in the sample.
- the amount of complex formation when compared to standards is diagnostic of cancer.
- the invention provides a method for inserting a heterologous marker gene into the genomic DNA of a mammal to disrupt the expression of the endogenous polynucleotide.
- the invention also provides a method for using a cDNA to produce a model system, the method comprising constructing a vector containing a DNA selected from SEQ ID NOs: 1-15 and 17-78 transforming the vector into an embryonic stem cell, selecting a transformed embryonic stem cell, microinjecting the transformed embryonic stem cell into a blastocyst, thereby forming a chimeric blastocyst, transferring the chimeric blastocyst into a pseudopregnant dam, wherein the dam gives birth to a chimeric offspring containing the cDNA in its germ line, and breeding the chimeric mammal to produce a homozygous, model system.
- FIGS. 1A-1E show the amino acid sequence of SP16 (SEQ ID NO: 16) and nucleic acid sequence if its encoding cDNA (SEQ ID NO: 17). The alignment was produced using MacDNASIS PRO software (Hitachi Software Engineering, South San Francisco Calif.).
- FIG. 2 shows the amino acid sequence alignment between SP-16 (2547002; SEQ ID NO: 16) and bovine GPCR (GI 399711; SEQ ID NO: 79) produced using the MDGALIGN program of LASERGENE software (DNASTAR, Madison Wis.).
- Table I shows the sequence identification numbers, reference, Incyte Clone number, cDNA library, NCB1 sequence identifier and GenBank description for each of the signal peptide-containing proteins encoded by the cDNAs.
- Array refers to an ordered arrangement of at least two cDNAs or antibodies on a substrate. At least one of the cDNAs or antibodies represents a control or standard, and the other, a cDNA or antibody of diagnostic or therapeutic interest.
- the arrangement of two to about 40,000 cDNAs or of two to about 40,000 monoclonal or polyclonal antibodies on the substrate assures that the size and signal intensity of each labeled hybridization complex, formed between each cDNA and at least one nucleic acid, or antibody:protein complex, formed between each antibody and at least one protein to which the antibody specifically binds, is individually distinguishable.
- the “complement” of a cDNA of the Sequence Listing refers to a nucleic acid molecule which is completely complementary to the cDNA over its full length and which will hybridize to the cDNA or an mRNA under conditions of maximal stringency.
- cDNA refers to an isolated polynucleotide, nucleic acid molecule, or any fragment or complement thereof. It may have originated recombinantly or synthetically, may be double-stranded or single-stranded, represents coding and noncoding 3′ or 5′ sequence, and generally lacks introns.
- composition refers to the polynucleotide and a labeling moiety, a purified protein and a pharmaceutical carrier or a labeling moiety. an antibody and a labeling moiety, and the like.
- “Derivative” refers to a cDNA or a protein that has been subjected to a chemical modification. Derivatization of a cDNA can involve substitution of a nontraditional base such as queosine or of an analog such as hypoxanthine. Derivatization of a protein involves the replacement of a hydrogen by an acetyl, acyl, alkyl, amino, formyl, or morpholino group. Derivative molecules retain the biological activities of the naturally occurring molecules but may confer advantages such as longer lifespan or enhanced activity.
- “Differential expression” refers to an increased or up-regulated or a decreased or down-regulated expression as detected by presence. absence or at least two-fold change in the amount or abundance of a transcribed messenger RNA or translated protein in a sample.
- disorders associated with cell proliferation and cell signaling include cancers, genetic. and immune conditions. Each disorder is associated with expression of a signal peptide-containing protein or its specific encoding cDNA. These disorders include, but are not limited to, adenofibromatous hyperplasia as a prognostic of prostate cancer, asthma, arthritis, breast cancers such as ductal, lobular, and adenocarcinomas, Huntington's disease, mucinous cystadenoma of the ovary, renal cell cancer, schizophrenia stomach tumor, testicular seminoma, transitional cell carcinoma of the bladder, and uterine adenosquamous carcinoma.
- Fragments refers to a chain of consecutive nucleotides from about 50 to about 4000 base pairs in length. Fragments may be used in PCR or hybridization technologies to identify related nucleic acid molecules and in binding assays to screen for a ligand. Such ligands are useful as therapeutics to regulate replication, transcription or translation.
- a “hybridization complex” is formed between a cDNA and a nucleic acid of a sample when the purines of one molecule hydrogen bond with the pyrimidines of the complementary molecule, e.g., 5′-A-G-T-C-3′ base pairs with 3′-T-C-A-G-5′.
- Hybridization conditions, degree of complementarity and the use of nucleotide analogs affect the efficiency and stringency of hybridization reactions.
- Labeling moiety refers to any visible or radioactive label than can be attached to or incorporated into a cDNA or protein. Visible labels include but are not limited to anthocyanins, green fluorescent protein (OFP), 6 glucuronidase. luciferase, Cy3 and Cy5, and the like. Radioactive markers include radioactive forms of hydrogen, iodine, phosphorous, sulfur, and the like.
- Visible labels include but are not limited to anthocyanins, green fluorescent protein (OFP), 6 glucuronidase. luciferase, Cy3 and Cy5, and the like.
- Radioactive markers include radioactive forms of hydrogen, iodine, phosphorous, sulfur, and the like.
- Ligand refers to any agent, molecule, or compound which will bind specifically to a polynucleotide or to an epitope of a protein. Such ligands stabilize or modulate the activity of polynucleotides or proteins and may be composed of inorganic and/or organic substances including minerals, cofactors, nucleic acids, proteins, carbohydrates, fats. and lipids.
- Oligomer refers a single-stranded molecule from about 18 to about 60 nucleotides in length which may be used in hybridization or amplification technologies or in regulation of replication, transcription or translation. Equivalent terms are amplimer, primer, and oligomer.
- oligopeptide is an amino acid sequence from about five residues to about 15 residues that is used as part of a fusion protein to produce an antibody.
- “Portion” refers to any part of a protein used for any purpose; but especially, to an epitope for the screening of ligands or for the production of antibodies.
- Post-translational modification of a protein can involve lipidation, glycosylation, phosphorylation, acetylation, racemization, proteolytic cleavage, and the like. These processes may occur synthetically or biochemically. Biochemical modifications will vary by cellular location, cell type, pH, enzymatic milieu, and the like.
- Probe refers to a cDNA that hybridizes to at least one nucleic acid in a sample. Where targets are single-stranded, probes are complementary single strands. Probes can be labeled with reporter molecules for use in hybridization reactions including Southern, northern, in situ, dot blot, array, and like technologies or in screening assays.
- Protein refers to a polypeptide or any portion thereof.
- a “portion” of a protein refers to that length of amino acid sequence which would retain at least one biological activity, a domain identified by PFAM or PRINTS analysis or an antigenic epitope of the protein identified using Kyte-Doolittle algorithms of the PROTEAN program (DNASTAR).
- “Purified” refers to any molecule or compound that is separated from its natural environment and is from about 60% free to about 90% free from other components with which it is naturally associated.
- sample is used in its broadest sense as containing nucleic acids, proteins. antibodies. and the like.
- a sample may comprise a bodily fluid; the soluble fraction of a cell preparation, or an aliquot of media in which cells were grown; a chromosome, an organelle, or membrane isolated or extracted from a cell; genomic DNA, RNA, or cDNA in solution or bound to a substrate; a biopsy, a cell; a tissue; a tissue print; a fingerprint, buccal cells, skin, or hair; and the like.
- Similarity refers to the quantification (usually percentage) of nucleotide or residue matches between at least two sequences aligned using a standard algorithm such as Smith-Waterman alignment (Smith and Waterman (1981) J Mol Bioi 147:195-197) or BLAST2 (Altschul et al. (1997) Nucleic Acids Res 25:3389-3402).
- BLAST2 may be used in a reproducible way to insert gaps in one of the sequences in order to optimize alignment and to achieve a more meaningful comparison between them.
- similarity is greater than identity in that conservative substitutions (for example, valine for leucine or isoleucine) are counted in calculating the reported percentage. Substitutions which are considered to be conservative are well known in the art.
- Specific binding refers to a special and precise interaction between two molecules which is dependent upon their structure. particularly their molecular side groups. For example, the intercalation of a regulatory protein into the major groove of a DNA molecule or the binding between an epitope of a protein and an agonist, antagonist, or antibody.
- Substrate refers to any rigid or semi-rigid support to which cDNAs or proteins are bound and includes membranes, filters, chips, slides, wafers, fibers. magnetic or nonmagnetic beads, gels, capillaries or other tubing, plates, polymers, and microparticles with a variety of surface forms including wells, trenches, pins, channels and pores.
- a “transcript image” is a profile of gene transcription activity in a particular tissue at a particular time.
- “Variant” refers to molecules that are recognized variations of a cDNA or a protein encoded by the cDNA. Splice variants may be determined by BLAST score, wherein the score is at least 100, and most preferably at least 400. Allelic variants have a high percent identity to the cDNAs and may differ by about three bases per hundred bases. “Single nucleotide polymorphism” (SNP) refers to a change in a single base as a result of a substitution, insertion or deletion. The change may be conservative (purine for purine) or non-conservative (purine to pyrimidine) and may or may not result in a change in an encoded amino acid or its secondary, tertiary, or quaternary structure.
- SNP single nucleotide polymorphism
- the invention is based on the discovery of signal peptide-containing proteins, individually SP-1 through SP-16, and their encoding or regulating cDNAs, SEQ ID NOs: 1-15 and 17 which are characterized in TABLE I.
- U.S. Ser. No. 09/271,110, filed 17 Mar. 1999, is incorporated by reference herein in its entirety.
- the cDNAs and fragments thereof, the proteins and portions thereof, and an antibody which specifically binds each protein can be used directly or as compositions for the diagnosis, prognosis, treatment and evaluation of therapies for disorders associated with cell proliferation and cell signaling.
- SP-1 was identified in Incyte Clone 1221102 from the NEUTGMTOI cDNA library using a computer search for amino acid sequence alignments.
- a cDNA comprising the nucleic acid shown in SEQ ID NO: 1 and derived using Incyte Clone 1221102, which encompasses nucleotides 300-514 also found in Incyte clone 5269342F6 (SEQ ID NO:18) which was used on HumanGenomeGEM1 microarray, encodes a GPCR with homology to g1575512, the GPR19 gene.
- Electronic northern analysis showed the expression of this sequence in neuronal tissues and in stimulated granulocytes.
- the transcript image found in EXAMPLE VIII supported the northern analysis and showed four-fold, up-regulated expression of the cDNA encoding SP-1 in the brain from a subject diagnosed with Huntington's disease.
- SP-2 was identified in Incyte Clone 1457779 from the COLNFET02 cDNA library using a computer search for amino acid sequence alignments.
- a cDNA comprising the nucleic acid shown in SEQ ID NO: 2 and derived from Incyte Clone 1457779, which encompasses nucleotides 1-466 also found in Incyte clone 1457779F6 (SEQ ID NO: 22) which was used on LifeGEM1 microarray, encodes an ATP diphosphohydrolase with homology to g1842120.
- Electronic northern analysis showed the expression of this sequence in fetal colon, and transcript imaging showed that differential expression of SP-2 is diagnostic of stomach tumor.
- SP-3 was identified in Incyte Clone 1682433 from the PROSNOT15 cDNA library using a computer search for amino acid sequence alignments.
- a cDNA comprising the nucleic acid shown in SEQ ID NO: 3 and derived from Incyte Clone 1682433, which encompasses nucleotides 1-481 also found in Incyte clone 2444114F6 (SEQ ID NO: 26) which was used on LifeGEM1 microarray, encodes a signal peptide-containing protein with homology to g1010391, a transmembrane protein.
- Electronic northern analysis showed the expression of this sequence in fetal, cancerous or inflamed cells and tissues. Transcript imaging showed that differential expression of SP-3 is diagnostic of ductal carcinoma of the breast.
- SP-4 was identified in Incyte Clone 1899132 from the BLADTUTO6 cDNA library using a 35 (;omputer search for amino acid sequence alignments.
- Electronic northern analysis showed the expression of this sequence in cancerous and inflamed cells and tissues; transcript imaging showed that differential expression of SP-4 is diagnostic of uterine adenosquamous carcinoma.
- SP-5 was identified in Incyte Clone 1907344 from the CONNTUT01 cDNA library using a computer search for amino acid sequence alignments.
- Electronic northern analysis showed the expression of this sequence in cancerous and fetal or infant cells and tissues; transcript imaging showed that differential expression of SP-5 is diagnostic for adenocarcinoma of the breast.
- SP-6 was identified in Incyte Clone 1963651 from the BRSTNOT04 cDNA library using a computer search for amino acid sequence alignments.
- electronic northern analysis showed expression in ductal carcinoma; transcript imaging showed that differential expression of SP6 in ovary is diagnostic for mucinous cystadenoma.
- SP-7 was identified in Incyte Clone 1976095 from the PANCTUT02 cDNA library using a computer search for amino acid sequence alignments.
- Electronic northern analysis showed the expression of this sequence in cancerous and inflamed tissues; transcript imaging showed that differential expression of SP-7 in synovium or cartilage is diagnostic for arthritis.
- SP-8 was identified in Incyte Clone 2417676 from the HNT3AZT01 cDNA library using a computer search for amino acid sequence alignments.
- SP-9 was identified in Incyte Clone 1805538 from the SINTNOT13 cDNA library using a computer search for amino acid sequence alignments.
- Electronic northern analysis showed this sequence to be expressed in inflamed tissues; transcript imaging showed that differential expression of SP-9 is diagnostic of adenofibromatous hyperplasia and prognostic for prostate cancer.
- SP-10 was identified in Incyte Clone 1869688 from the SKINBIT01 cDNA library using a computer search for amino acid sequence alignments.
- SP-11 was identified in Incyte Clone 1880692 from the LEUKNOT03 cDNA library using a computer search for amino acid sequence alignments.
- SP-12 was identified in Incyte Clone 318060 from the EOSIHET02 cDNA library using a computer search for amino acid sequence alignments.
- electronic northern analysis showed this sequence to be expressed in nerve and blood cells; transcript imaging showed that differential expression of SP-12 is diagnostic for adenocarcinoma of the breast.
- SP-13 was identified in Incyte Clone 396450 from the PITUNOT02 cDNA library using a computer search for amino acid sequence alignments.
- Electronic northern analysis showed this sequence to be expressed in hormone producing cells and tissues and inflamed cells and tissues; transcript imaging showed that differential expression of SP-13 is diagnostic for schizophrenia.
- SP-14 was identified in Incyte Clone 506333 from the TMLR3DT02 cDNA library using a computer search for amino acid sequence alignments.
- a nucleotide sequence SEQ ID NO: 14, derived from Incyte Clone 506333, which encompasses nucleotides 1-514 also found in Incyte clone 506333T6 (SEQ ID NO: 68) which was used on LifeGEM1 microarray, encodes a signal peptide-containing protein with homology to g2204110, adenylyl cyclase.
- Electronic northern analysis showed this sequence to be expressed in cancerous and inflamed cells and tissues; transcript imaging showed that differential expression of SP-14 is diagnostic of breast cancer, in particular lobular carcinoma of the breast.
- SP-15 was identified in Incyte Clone 764465 from the LUNGNOT04 cDNA library using a computer search for amino acid sequence alignments.
- Electronic northern analysis showed this sequence to be expressed in lung and in fetal liver; transcript imaging confirms the northern analysis and shows that differential expression of SP-15 when used with lung samples is diagnostic for asthma.
- SP-16 (SEQ ID NO: 16) was identified in Incyte Clone 2547002 from the UTRSNOT11 cDNA library using a computer search for amino acid sequence alignments.
- the invention encompasses a polypeptide comprising the amino acid sequence of SEQ ID NO: 16, as shown in FIGS. 1A-1E , SP-16 is 350 amino acids in length and has a G protein coupled receptor signature at S 125 GMQFLACISIDRYVAV (SEQ ID NO: 81); three potential N-glycosylation sites at N 6 , N 19 , and N 276 ; a potential glycosaminoglycan attachment site at S 48 ; and ten potential phosphorylation sites at S 25 , T 74 , T 177 , S 195 , T 223 , Y 269 , S 278 , S 309 , S 323 , and S 330 .
- SP-16 has 86% sequence identity with a bovine GPCR (g399711) and shares the GPCR signature, the N-glycosylation, the glycosaminoglycan attachment site, and the first nine of the phosphorylation sites with the bovine receptor ( FIG. 2 ). Fragments of the nucleic acid molecule useful for designing oligonucleotides or to be used directly as hybridization probes to distinguish between these homologous molecules include A 24 to G 44 , G 159 to C 182 , G 561 to A 596 , or A 1011 to T 1046 . mRNA encoding SP-16 was sparsely expressed in cDNA libraries. Electronic northern analysis (EXAMPLE VIII below) showed expression in breast adenocarcinoma; transcript imaging confirmed the northern analysis and showed that SP-16 is differentially expressed in breast adenocarcinoma and not in matched or normal breast tissues.
- EXAMPLE VIII Electronic northern analysis
- cDNA fragments encoding or regulating signal peptide-containing proteins were identified using BLAST2 with default parameters and the ZOOSEQ databases (Incyte Genomics, Palo Alto Calif.). These cDNAs have from about 80% to about 95% sequence identity to the human cDNA as shown in the table below.
- the first column shows the SEQ ID H for the human cDNA; the second column, the SEQ ID FR for fragment cDNAs; the third column, the sequence numbers for the fragments; the fourth column, the species; the fifth column, percent identity to the human cDNA; and the sixth column, the nucleotide, alignment (Nt H ) of the human and fragment cDNAs.
- the cDNAs of SEQ ID NOs: 1-15 and 17-78 may be used in hybridization, amplification, and screening technologies to identify and distinguish among the identical and related molecules in a sample.
- the cDNAs may also be used to produce transgenic cell lines or organisms which are model systems for cancers and upon which the toxicity and efficacy of potential therapeutic treatments may be tested. Toxicology studies, clinical trials, and subject/patient treatment profiles may be performed and monitored using the cDNAs, proteins, antibodies and molecules and compounds identified using the cDNAs and proteins of the present invention.
- mRNA is isolated from cells and tissues using methods which are well known to those skilled in the art and used to prepare the cDNA libraries.
- the Incyte cDNAs were isolated from cDNA libraries prepared as described in the EXAMPLES.
- the consensus sequences are chemically and/or electronically assembled from fragments including Incyte cDNAs and extension and/or shotgun sequences using computer programs such as PHRAP (P Green, University of Washington, Seattle Wash.), and the AUTOASSEMBLER application (ABI).
- PHRAP P Green, University of Washington, Seattle Wash.
- ABSEL application AUTOASSEMBLER application
- sequence preparation is automated with machines such as the MICROLAB 2200 system (Hamilton. Reno Nev.) and the DNA ENGINE thennal cycler (MJ Research, Watertown Mass.).
- Machines commonly used for sequencing include the ABI PRISM 3700, 377 or 373 DNA sequencing systems (ABI), the MEGABACE 1000 DNA sequencing system (APB), and the like.
- the sequences may be analyzed using a variety of algorithms well known in the art and described in Ausubel et al. (1997 ; Short Protocols in Molecular Biology , John Wiley & Sons, New York N.Y., unit 7.7) and in Meyers (1995 ; Molecular Biology and Biotechnology , Wiley VCH, New York N.Y., pp. 856-853).
- Shotgun sequencing may also be used to complete the sequence of a particular cloned insert of interest. Shotgun strategy involves randomly breaking the original insert into segments of various sizes and cloning these fragments into vectors. The fragments are sequenced and reassembled using overlapping ends until the entire sequence of the original insert is known. Shotgun sequencing methods are well known in the art and use thermostable DNA polymerases, heat-labile DNA polymerases, and primers chosen from representative regions flanking the cDNAs of interest. Incomplete assembled sequences are inspected for identity using various algorithms or programs such as CONSED (Gordon (1998) Genome Res 8; 195-202) which are well known in the art. Contaminating sequences, including vector or chimeric sequences, or deleted sequences can be removed or restored, respectively, organizing the incomplete assembled sequences into finished sequences.
- CONSED Gibco (1998) Genome Res 8; 195-202
- sequences of the invention may be extended using various PCR-based methods known in the art.
- the XL-PCR kit (ABI)
- nested primers and commercially available cDNA or genomic DNA libraries may be used to extend the . . . .
- primers may be designed using commercially available software to be about 22 to 30 nucleotides in length, to have a GC content of about 50% or more, and to anneal to a target molecule at temperatures from about 55C to about 68C.
- genomic, rather than cDNA libraries it is preferable to use genomic, rather than cDNA libraries.
- a probe may be designed or derived from unique regions such as the 5′ regulatory region or from a nonconserved region (i.e., 5′ or 3′ of the nucleotides encoding the conserved catalytic domain of the protein) and used in protocols to identify naturally occurring molecules encoding a signal peptide-containing protein, allelic variants, or related molecules.
- the probe may be DNA or RNA, may be single-stranded, and should have at least 50% sequence identity to a nucleic acid molecule selected from SEQ ID NOs: 1-15 and 17-78.
- Hybridization probes may be produced using oligolabeling, nick translation, end-labeling, or PCR amplification in the presence of a reporter molecule.
- a vector containing the cDNA or a fragment thereof may be used to produce an mRNA probe in vitro by addition of an RNA polymerase and labeled nucleotides. These procedures may be conducted using commercially available kits.
- the stringency of hybridization is determined by G+C content of the probe. salt concentration. and temperature. In particular, stringency can be increased by reducing the concentration of salt or raising the hybridization temperature.
- Hybridization can be performed at low stringency with buffers, such as 5 ⁇ SSC with 1% sodium dodecyl sulfate (SDS) at 60 C, which permits the formation of a hybridization complex between s that contain some mismatches. Subsequent washes are performed at higher stringency with buffers such as 0.2 ⁇ SSC with 0.1% SDS at either 45 C (medium stringency) or 68 C (high stringency). At high stringency, hybridization complexes will remain stable only where the nucleic acids are completely complementary.
- formamide can be added to the hybridization solution to reduce the temperature at which hybridization is performed, and background signals can be reduced by the use of detergents such as Sarkosyl or TRITON X-100 (Sigma-Aldrich, St Louis Mo.) and a blocking agent such as denatured salmon sperm DNA. Selection of components and conditions for hybridization are well known to those skilled in the art and are reviewed in Ausubel (supra) and Sambrook et al. (1989) Molecular Cloning, A Laboratory Manual , Cold Spring Harbor Press, Plainview N.Y.
- Arrays incorporating cDNAs or antibodies may be prepared and analyzed using methods well known in the art. Oligonucleotides or cDNAs may be used as hybridization probes or targets to monitor the expression level of large numbers of genes simultaneously or to identify genetic variants, mutations, and single nucleotide polymorphisms. Monoclonal or polyclonal antibodies may be used to detect or quantify expression of a protein in a sample. Such arrays may be used to determine gene function; to understand the genetic basis of a condition, disease, or disorder; to diagnose a condition, disease, or disorder; and to develop and monitor the activities of therapeutic agents. (See, e.g., Brennan et al. (1995) U.S. Pat. No.
- Hybridization probes are also useful in mapping the naturally occurring genomic sequence.
- the probes may be hybridized to a particular chromosome, a specific region of a chromosome, or an artificial chromosome construction.
- Such constructions include human artificial chromosomes (HAC), yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC), bacterial PI constructions, or the cDNAs of libraries made from single chromosomes.
- cDNAs encoding a signal peptide-containing protein may be cloned into a vector and used to express the protein, or portions thereof, in host cells.
- The can be engineered by such methods as DNA shuffling, as described in U.S. Pat. No. 5,830,721, and site-directed mutagenesis to create new restriction sites, alter glycosylation patterns, change codon preference to increase expression in a particular host, produce splice variants, extend half-life. and the like.
- the expression vector may contain transcriptional and translational control elements (promoters, enhancers. specific initiation signals. and polyadenylated 3′ sequence) from various sources which have been selected for their efficiency in a particular host.
- the vector, cDNA, and regulatory elements are combined using in vitro recombinant DNA techniques, synthetic techniques, and/or in vivo genetic recombination techniques well known in the art and described in Sambrook (supra, ch. 4, 8, 16 and 17).
- a variety of host systems may be transformed with an expression vector. These include, but are not limited to, bacteria transformed with recombinant bacteriophage, plasmid, or cosmid DNA expression vectors; yeast transformed with yeast expression vectors; insect cell systems transformed with baculovirus expression vectors; plant cell systems transformed with expression vectors containing viral and/or bacterial elements, or animal cell systems (Ausubel supra, unit 16).
- an adenovirus transcription/translation complex may be utilized in mammalian cells. After sequences are ligated into the E1 or E3 region of the viral genome, the infective virus is used to transform and express the protein in host cells.
- the Rous sarcoma virus enhancer or SV40 or EBV-based vectors may also be used for high-level protein expression.
- Routine cloning, subcloning, and propagation of s can be achieved using the multifunctional PBLUESCRIPT vector (Stratagene, La Jolla Calif.) or PSPORT1 plasmid (Life Technologies). Introduction of a into the multiple cloning site of these vectors disrupts the lacZ gene and allows colorimetric screening for transformed bacteria. In addition. these vectors may be useful for in vitro transcription, dideoxy sequencing, single strand rescue with helper phage, and creation of nested deletions in the cloned sequence.
- the vector can be stably transformed into cell lines along with a selectable or visible marker gene on the same or on a separate vector. After transformation, cells are allowed to grow for about 1 to 2 days in enriched media-and-then are-transferred to selective media. Selectable markers, antimetabolite, antibiotic, or herbicide resistance genes, confer resistance to the relevant selective agent and allow growth and recovery of cells which successfully express the introduced sequences. Resistant clones identified either by survival on selective media or by the expression of visible markers may be propagated using culture techniques. Visible markers are also used to estimate the amount of protein expressed by the introduced genes. Verification that the host cell contains the desired cDNA is based on DNA-DNA or DNA-RNA hybridizations or PCR amplification techniques.
- the host cell may be chosen for its ability to modify a recombinant protein in a desired fashion. Such modifications include acetylation, carboxylation, glycosylation, phosphorylation, lipidation, acylation and the like. Post-translational processing which cleaves a “prepro” form may also be used to specify protein targeting, folding, and/or activity. Different host cells available from the ATCC (Manassas Va.) which have specific cellular machinery and characteristic mechanisms for post-translational activities may be chosen to ensure the correct modification and processing of the recombinant protein.
- ATCC Manassas Va.
- Heterologous moieties engineered into a vector for ease of purification include glutathione Stransferase (GST), 6 ⁇ His, FLAG, MYC, and the like.
- GST and 6 ⁇ His are purified using commercially available affinity matrices such as immobilized glutathione and metal-chelate resins, respectively.
- FLAG and MYC are purified using commercially available monoclonal and polyclonal antibodies.
- a sequence encoding a proteolytic cleavage site may be part of the vector located between the protein and the heterologous moiety. Methods for recombinant protein expression and purification are discussed in Ausubel (supra, unit 16) and are commercially available.
- Proteins or portions thereof may be produced not only by recombinant methods, but also by using chemical methods well known in the art.
- Solid phase peptide synthesis may be carried out in a batchwise or continuous flow process which sequentially adds a-amino-and side chain-protected amino acid residues to an insoluble polymeric support via a linker group.
- a linker group such as methylarnine-derivatized polyethylene glycol is attached to poly(styrene-co-divinylbenzene) to form the support resin.
- the amino acid residues are N- ⁇ -protected by acid labile Boc (t-butyloxycarbonyl) or base-labile Fmoc (9-fluorenylmethoxycarbonyl).
- the carboxyl group of the protected amino acid is coupled to the amine of the linker group to anchor the residue to the solid phase support resin.
- Trifluoroacetic acid or piperidine are used to remove the protecting group in the case of Boc or Fmoc, respectively.
- Each additional amino acid is added to the anchored residue using a coupling agent or pre-activated amino acid derivative. and the resin is washed.
- the full length peptide is synthesized by sequential deprotection, coupling of derivitized amino acids, and washing with dichloromethane and/or N, N-dimethylformamide. The peptide is cleaved between the peptide carboxy terminus and the linker group to yield a peptide acid or amide.
- oligopeptide, peptide, or portion of protein used to induce antibodies should consist of at least about five amino acids, more preferably ten amino acids, which are identical to a portion of the natural protein. Oligopeptides may be fused with proteins such as KLH in order to produce antibodies to the chimeric molecule.
- Monoclonal antibodies may be prepared using any technique which provides for the production of antibodies by continuous cell lines in culture. These include, but are not limited to, the hybridoma technique, the human ⁇ -cell hybridoma technique, and the EBV-hybridoma technique. (See, e.g., Kohler et al. (1975) Nature 256:495-497; Kozbor et al. (1985) J. Immunol Methods 81:31-42; Cote et al. (1983) Proc Natl Acad Sci 80:2026-2030; and Cole et al (1984) Mol Cell Biol 62:109-120.)
- antibody fragments which contain specific binding sites for epitopes of the protein may also be generated.
- fragments include, but are not limited to, F(ab′)2 fragments produced by pepsin digestion of the antibody molecule and Fab fragments generated by reducing the disulfide bridges of the F(ab′)2 fragments.
- Fab expression libraries may be constructed to allow rapid and easy identification of monoclonal Flib fragments with the desired specificity. (See, e.g., Huse et al. (1989) Science 246:1275-1281.)
- a signal peptide-containing protein, or a portion thereof, may be used in screening assays of phagemid or ⁇ -lymphocyte immunoglobulin libraries to identify antibodies having the desired specificity.
- Numerous protocols for competitive binding or immunoassays using either polyclonal or monoclonal antibodies with established specificities are well known in the art. Such immunoassays typically involve the measurement of complex formation between the protein and its specific antibody.
- a two-site, monoclonal-based immunoassay utilizing monoclonal antibodies reactive to two non-interfering epitopes is preferred, but a competitive binding assay may also be employed (Pound (1998) Immunochemical Protocols, Humana Press, Totowa N.J.).
- reporter molecules and conjugation techniques are known by those skilled in the art and may be used in various nucleic acid, amino acid, and antibody assays. Synthesis of labeled molecules may be achieved using commercially available kits (Promega, Madison Wis.) for incorporation of a labeled nucleotide such as 32 P-dCTP (APB), Cy3-dCTP or Cy5-dCTP (Operon Technologies, Alameda Calif.), or amino acid such as 35 S-methionine (APB).
- APB 32 P-dCTP
- Cy3-dCTP Cy3-dCTP
- Cy5-dCTP Opon Technologies, Alameda Calif.
- amino acid such as 35 S-methionine (APB).
- Nucleotides and amino acids may be directly labeled with a variety of substances including fluorescent, chemiluminescent, or chromogenic agents, and the like, by chemical conjugation to amines, thiols and other groups present in the molecules using reagents such as BIODIPY or FITC (Molecular Probes, Eugene Oreg.).
- the cDNAs, fragments, oligonucleotides, complementary RNA and DNA molecules, and PNAs may be used to detect and quantify differential gene expression for diagnostic purposes.
- Disorders associated with expression of SP-1 through SP-16 include, but are not limited to, adenofibromatous hyperplasia as a prognostic of prostate cancer, asthma, arthritis, breast cancers such as ductal, lobular. and adeno-carcinomas, Huntington's disease, mucinous cystadenoma of the ovary, renal cell cancer, schizophrenia stomach tumor, testicular seminoma, transitional cell carcinoma of the bladder, and uterine adenosquamous carcinoma.
- the diagnostic assay may use hybridization or quantitative PCR to compare gene expression in a biological or biopsied subject sample to standard samples in order to detect differential gene expression. Qualitative and quantitative methods for this comparison are commercially available and well known in the art.
- the cDNA or probe may be labeled by standard methods and added to a biological sample from a subject under conditions for the formation of hybridization complexes. After an incubation period. the sample is washed and the amount of label (or signal) associated with hybridization complexes, is quantified and compared with a standard value. If complex formation in the subject sample is significantly altered (higher or lower) in comparison to either, a normal or disease standard, then differential expression indicates the presence of a disorder.
- normal and disease expression profiles are established. This is accomplished by combining a sample taken from normal subjects, either animal or human, with a cDNA under conditions for hybridization to occur. Standard hybridization complexes may be quantified by comparing the values obtained using normal subjects with values from an experiment in which a known amount of a purified sequence is used. Standard values obtained in this manner may be compared with values obtained from samples from patients who were diagnosed with a particular condition, disease, or disorder. Deviation from standard values toward those associated with a particular disorder is used to diagnose that disorder.
- Such assays may also be used to evaluate the efficacy of a particular therapeutic treatment regimen in animal studies or in clinical trials or to monitor the treatment of an individual patient. Once the presence of a condition is established and a treatment protocol is initiated, diagnostic assays may be repeated on a regular basis to determine if the level of expression in the patient begins to approximate that which is observed in a normal subject. The results obtained from successive assays may be used to show the efficacy of treatment over a period ranging from several days to years.
- Detection and quantification of a protein using either labeled amino acids or specific polyclonal or monoclonal antibodies which specifically bind the protein are known in the art. Examples of such techniques include two-dimensional polyacrylamide gel electrophoresis, enzyme-linked immunosorbent assays (ELISAs), radioimmunoassays (RIAs), and fluorescence activated cell sorting (FACS). These assays and their quantitation against purified. labeled standards are well known in the art (Ausubel, Supra, unit 10.1-10.6). A two-site, monoclonal-based immunoassay utilizing monoclonal antibodies reactive to two non-interfering epitopes is preferred, but a competitive binding assay may be employed. (See. e.g. Coligan et al. (1997) Current Protocols in Immunology , Wiley-Interscience, New York N.Y.; and Pound. supra.)
- each protein clearly plays a role in at least one of the described disorders (adenofibromatous hyperplasia as a prognostic of prostate cancer, asthma, arthritis, breast cancers such as ductal, lobular, and adena-carcinomas, Huntington's disease, mucinous cystadenoma of the ovary, renal cell cancer, schizophrenia stomach tumor, testicular seminoma, transitional cell carcinoma of the bladder, and uterine adenosquamous carcinoma) and SP-1 through SP-16 may be used either directly as a therapeutic or as a target for drug discovery.
- disorders adenofibromatous hyperplasia as a prognostic of prostate cancer, asthma, arthritis, breast cancers such as ductal, lobular, and adena-carcinomas, Huntington's disease, mucinous cystadenoma of the ovary, renal cell cancer, schizophrenia stomach tumor, testicular seminoma, transitional cell carcinoma of the bladder, and uterine adenosquamous
- increased expression of the protein may be treated by the delivery of an inhibitor, antagonist, antibody and the like or a pharmaceutical composition containing one or more of these molecules.
- Such delivery may be effected by methods well known in the art and may include delivery by an antibody specifically targeted to the diseased tissue.
- decreased expression of the protein late in the disease process may be treated by the delivery of the protein, an agonist, enhancer and the like or a pharmaceutical composition containing one or more of these molecules.
- delivery may be effected by methods well known in the art and may include delivery by an antibody specifically targeted to the diseased tissue.
- compositions may be administered in combination with other therapeutic agents.
- Selection of the agents for use in combination therapy may be made by one of ordinary skill in the art according to conventional pharmaceutical principles.
- a combination of therapeutic agents may act synergistically to affect treatment of a particular cancer at a lower dosage of each agent alone.
- Gene expression may be modified by designing complementary or antisense molecules (DNA, RNA, or PNA) to the control, 5′, 3′, or other regulatory regions of the gene encoding a signal peptidecontaining protein. Oligonucleotides designed to inhibit transcription initiation are preferred. Similarly, inhibition can be achieved using triple helix base-pairing which inhibits the binding of polymerases, transcription factors, or regulatory molecules (Gee et al. In: Huber and Carr (1994) Molecular and Immunologic Approaches , Futura Publishing, Mt. Kisco N.Y., pp. 163-177).
- a complementary molecule may also be designed to block translation by preventing binding between ribosomes and mRNA. In one alternative. a library or plurality of cDNAs may be screened to identify those which specifically bind a regulatory, nontranslated sequence.
- Ribozymes enzymatic RNA molecules, may also be used to catalyze the specific cleavage of RNA.
- the mechanism of ribozyme action involves sequence-specific hybridization of the ribozyme molecule to complementary target RNA followed by endonucleolytic cleavage at sites such as GUA, GUU, and GUC. Once such sites are identified, an oligonucleotide with the same sequence may be evaluated for secondary structural features which would render the oligonucleotide inoperable.
- the suitability of candidate wgets may also be evaluated by testing their hybridization with complementary oligonucleotides using ribonuclease protection assays.
- RNA molecules may be modified to increase intracellular stability and half-life by addition of flanking sequences at the 5′ and/or 3′ ends of the molecule or by the use of phosphorothioate or 2′ O-methyl rather than phosphodiesterase linkages within the backbone of the molecule. Modification is inherent in the production of PNAs and can be extended to other nucleic acid molecules.
- a cDNA encoding a signal peptide-containing protein may be used to screen a library or a plurality of molecules or compounds for specific binding affinity.
- the libraries may be aptamers, DNA molecules, RNA molecules, PNAs, peptides, proteins such as transcription factors, enhancers, or repressors, and other ligands which regulate the activity, replication, transcription, or translation of the endogenous gene.
- the assay involves combining a polynucleotide with a library or plurality of molecules or compounds under conditions allowing specific binding, and detecting specific binding to identify at least one molecule which specifically binds the single-stranded or double-stranded molecule.
- the cDNA of the invention may be incubated with a plurality of purified molecules or compounds and binding activity determined by methods well known in the art, e.g., a gelretardation assay (U.S. Pat. No. 6,010,849) or a commercially available reticulocyte lysate transcriptional assay.
- the cDNA may be incubated with nuclear extracts from biopsied and/or cultured cells and tissues. Specific binding between the cDNA and a molecule or compound in the nuclear extract is initially determined by gel shift assay and may be later confirmed by recovering and raising antibodies against that molecule or compound. When these antibodies are added into the assay, they cause a supershift in the gel-retardation assay.
- the cDNA may be used to purify a molecule or compound using affinity chromatography methods well known in the art.
- the cDNA is chemically reacted with cyanogen bromide groups on a polymeric resin or gel. Then a sample is passed over and reacts with or binds to the cDNA. The molecule or compound which is bound to the cDNA may be released from the cDNA by increasing the salt concentration of the flow-through medium and collected.
- the protein or a portion thereof may be used to purify a ligand from a sample.
- a method for using a protein or a portion thereof to purify a ligand would involve combining the protein or a portion thereof with a sample under conditions to allow specific binding, detecting specific binding between the protein and ligand, recovering the bound protein, and using a chaotropic agent to separate the protein from the purified ligand.
- a signal peptide-containing protein may be used to screen a plurality of molecules or compounds in any of a variety of screening assays.
- the portion of the protein employed in such screening may be free in solution, affixed to an abiotic or biotic substrate (e.g. borne on a cell surface), or located intracellularly.
- viable or fixed prokaryotic host cells that are stably transformed with recombinant nucleic acids that have expressed and positioned a peptide on their cell surface can be used in screening assays. The cells are screened against a plurality or libraries of ligands, and the specificity of binding or formation of complexes between the expressed protein and the ligand can be measured.
- the assay may be used to identify DNA molecules, RNA molecules, peptide nucleic acids, peptides, proteins, mimetics, agonists, antagonists, antibodies, immunoglobulins, inhibitors, and drugs or any other ligand, which specifically binds the protein.
- this invention comtemplates a method for high throughput screening using very small assay volumes and very small amounts of test compound as described in U.S. Pat. No. 5,876,946, incorporated herein by reference. This method is used to screen large numbers of molecules and compounds via specific binding.
- this invention also contemplates the use of competitive drug screening assays in which neutralizing antibodies capable of binding the protein specifically compete with a test compound capable of binding to the protein. Molecules or compounds identified by screening may be used in a model system to evaluate their toxicity, diagnostic, or therapeutic potential.
- compositions contain active ingredients in an effective amount to achieve a desired and intended purpose and a pharmaceutical carrier.
- the determination of an effective dose is well within the capability of those skilled in the art.
- the therapeutically effective dose may be estimated initially either in cell culture assays or in animal models. The animal model is also used to achieve a desirable concentration range and route of administration. Such information may then be used to determine useful doses and routes for administration in humans.
- a therapeutically effective dose refers to that amount of protein or inhibitor which ameliorates the symptoms or condition.
- Therapeutic efficacy and toxicity of such agents may be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., ED 50 (the dose therapeutically effective in 50% of the population) and LD 50 (the dose lethal to 50% of the population).
- the dose ratio between toxic and therapeutic effects is the therapeutic index, and it may be expressed as the ratio, LD 50 /ED 50 .
- Pharmaceutical compositions which exhibit large therapeutic indexes are preferred. The data obtained from cell culture assays and animal studies are used in formulating a range of dosage for human use.
- Animal models may be used as bioassays where they exhibit a phenotypic response similar to that of humans and where exposure conditions are relevant to human exposures. Mammals are the most common models. and most infectious agent, cancer, drug, and toxicity studies are performed on rodents such as rats or mice because of low cost, availability, lifespan, reproductive potential, and abundant reference literature. Inbred and outbred rodent strains provide a convenient model for investigation of the physiological consequences of under-or over-expression of genes of interest and for the development of methods for diagnosis and treatment of diseases. A mammal inbred to over-express a particular gene (for example, secreted in milk) may also serve as a convenient source of the protein expressed by that gene.
- Toxicology is the study of the effects of agents on living systems. The majority of toxicity studies are performed on rats or mice. Observation of qualitative and quantitative changes in physiology, behavior, homeostatic processes, and lethality in the rats or mice are used to generate a toxicity profile and to assess potential consequences on human health following exposure to the agent.
- Genotoxicology identifies and analyzes the effect of an agent on the rate of endogenous, spontaneous, and induced genetic mutations.
- Genotoxic agents usually have common chemical or physical properties that facilitate interaction with nucleic acids and are most harmful when chromosomal aberrations are transmitted to progeny.
- Toxicological studies may identify agents that increase the frequency of structural or functional abnormalities in the tissues of the progeny if administered to either parent before conception, to the mother during pregnancy. or to the developing organism. Mice and rats are most frequently used in these tests because their short reproductive cycle allows the production of the numbers of organisms needed to satisfy statistical requirements.
- Acute toxicity tests are based on a single administration of an agent to the subject to determine the symptomology or lethality of the agent. Three experiments are conducted: 1) an initial dose-range finding experiment, 2) an experiment to narrow the range of effective doses, and 3) a final experiment for establishing the dose-response curve.
- Subchronic toxicity tests are based on the repeated administration of an agent. Rat and dog are commonly used in these studies to provide data from species in different families. With the exception of carcinogenesis, there is considerable evidence that daily administration of an agent at high-dose concentrations for periods of three to four months will reveal most forms of toxicity in adult animals.
- Chronic toxicity tests with a duration of a year or more, are used to demonstrate either the absence of toxicity or the carcinogenic potential of an agent.
- studies are conducted on rats, a minimum of three test groups plus one control group are used, and animals are examined and monitored at the outset and at intervals throughout the experiment.
- Transgenic rodents that over-express or under-express a gene of interest may be inbred and used to model human diseases or to test therapeutic or toxic agents.
- the introduced gene may be activated at a specific time in a specific tissue type during fetal or postnatal development. Expression of the transgene is monitored by analysis of phenotype, of tissue-specific mRNA expression, or of serum and tissue protein levels in transgenic animals before, during, and after challenge with experimental drug therapies.
- Embryonic (ES) stem cells isolated from rodent embryos retain the potential to form embryonic tissues. When ES cells are placed inside a carrier embryo, they resume normal development and contribute to tissues of the live-born animal. ES cells are the preferred cells used in the creation of experimental knockout and knockin rodent strains.
- Mouse ES cells such as the mouse 129/SvJ cell line, are derived from the early mouse embryo and are grown under culture conditions well known in the art. Vectors used to produce a transgenic strain contain a disease gene candidate and a marker gene, the latter serves to identify the presence of the introduced disease gene.
- the vector is transformed into ES cells by methods well known in the art, and transformed ES cells are identified and microinjected into mouse cell blastocysts such as those from the C57BU6 mouse strain.
- the blastocysts are surgically transferred to pseudopregnant dams, and the resulting chimeric progeny are genotyped and bred to produce heterozygous or homozygous strains.
- ES cells derived from human blastocysts may be manipulated in vitro to differentiate into at least eight separate cell lineages. These lineages are used to study the differentiation of various cell types and tissues in vitro, and they include endoderm, mesoderm, and ectodermal cell types which differentiate into, for example, neural cells, hematopoietic lineages, and cardiomyocytes.
- a region of a gene is enzymatically modified to include a nonmammalian gene such as the neomycin phosphotransferase gene (neo; Capecchi (1989) Science 244:1288-1292).
- the modified gene is transformed into cultured ES cells and integrates into the endogenous genome by homologous recombination. The inserted sequence disrupts transcription and translation of the endogenous gene.
- Transformed cells are injected into rodent blastulae, and the blastulae are implanted into pseudopregnant dams.
- Transgenic progeny are crossbred to obtain homozygous inbred lines which lack a functional copy of the mammalian gene.
- the mammalian gene is a human gene.
- ES cells can be used to create knockin humanized animals (pigs) or transgenic animal models (mice or rats) of human diseases. With knockin technology. a region of a human gene is injected into animal ES cells. and the human sequence integrates into the animal cell genome. Transformed cells are injected into blastulae and the blastulae are implanted as described above. Transgenic progeny or inbred lines are studied and treated with potential pharmaceutical agents to obtain information on treatment of the analogous human condition. These methods have been used to model several human diseases.
- NHPs are the first choice test animal.
- NHPs and individual humans exhibit differential sensitivities to many drugs and toxins and can be classified as a range of phenotypes from “extensive metabolizers” to “poor metabolizers” of these agents.
- the cDNAs which encode the protein may be used in any molecular biology techniques that have yet to be developed. provided the new techniques rely on properties of cDNAs that are currently known. including, but not limited to, such properties as the triplet genetic code and specific base pair interactions.
- the UTRSNOT11 cDNA library was constructed from microscopically normal uterine tissue obtained from a 43-year-old female during a vaginal hysterectomy following diagnosis of uterine leiomyoma.
- Pathology indicated that the myometrium contained an intramural leiomyoma and a submucosal leiomyoma.
- the endometrium was proliferative, however, the cervix and fallopian tubes were unremarkable.
- the right and left ovaries contained corpus lutea.
- the patient presented with metrorrhagia and deficiency anemia Patient history included benign hypertension and atherosclerosis.
- Medications included PROVERA tablets (Pharmacia, Peapack N.J.), iron, and vitamins.
- Family history included benign hypertension, atherosclerosis, and malignant colon neoplasms.
- the frozen tissue was homogenized and lysed in TRIZOL reagent (1 gm tissue/10 ml reagent; Life Technologies) using a POLYTRON homogenizer (PT-3000; Brinkmann Instruments, Westbury N.Y.). After a brief incubation on ice, chloroform was added (1:5 v/v), and the lysate was centrifuged. The upper chloroform layer was removed to a fresh tube, and the RNA was extracted with isopropanol, resuspended in DEPC-treated water, and treated with DNAse for 25 min at 37 C.
- RNA was re-extracted three times with acid phenol-chloroform, pH 4.7, and precipitated with 0.3M sodium acetate and 2.5 volumes ethanol.
- the mRNA was isolated with the OLIGOTEX kit (Qiagen, Chatsworth Calif.) and used to construct the cDNA library.
- the mRNA was handled according to the recommended protocols in the SUPERSCRIPT plasmid system (Life Technologies).
- the cDNAs were fractionated on a SEPHAROSE CL4B column (APB), and those cDNAs exceeding 400 bp were ligated into pINCY1 plasmid.
- the plasmid was subsequently transformed into DH5a competent cells (Life Technologies).
- Plasmid DNA was released from the cells and purified using the REAL PREP 96 plasmid kit (Qiagen). This kit enabled the simultaneous purification of 96 samples in a 96-well block using multichannel reagent dispensers.
- the recommended protocol was employed except for the following changes: 1) the bacteria were cultured in 1 ml of sterile TERRIFIC BROTH (BD Biosciences, San Jose Calif.) with carbenicillin at 25 mg/L and glycerol at 0.4%; 2) after incubation for 19 hours, the cultures were lysed with 0.3 ml of lysis buffer; and 3) following isopropanol precipitation, the plasmid DNA pellet was resuspended in 0.1 ml of distilled water. After the last step in the protocol, samples were transferred to a 96-well block for storage at 4 C.
- the cDNAs were prepared for sequencing using the MICROLAB 2200 system (Hamilton) in combination with the DNA ENGINE thermal cyclers (MJ Research).
- the cDNAs were sequenced by the method of Sanger and Coulson (1975; J Mol Biol 94:441-448) using an ABI PRISM 373 or 377 sequencing system (ABI). Most of the isolates were sequenced according to standard ABI protocols and kits with solution volumes of 0.25 ⁇ -1.0 ⁇ concentrations or using standard solutions and dyes from APB.
- the cDNA sequence may be extended to full length using the Incyte clone, for example, SEQ ID NO:17, 2547002H1.
- a set of nested deletion sequencing templates was prepared from overnight liquid culture of clone 496071 using the ERASE-A-BASE system (Promega).
- Sequencing reactions were performed with the ABI PRISM Dye Terminator cycle sequencing kit with AMPLITAQ FS DNA polymerase (ABI). PCR was performed on a DNA ENGINE thermal cycler (MI Research). Reactions were analyzed on an ABI PRISM 310 genetic analyzer (ABI). Individual sequences were assembled and edited using ABI AutoAssembler software (ABI).
- extension is accomplished using oligonucleotide primers synthesized to initiate 5′ and 3′ extension of the known fragment.
- primers are designed using commercially available primer analysis software to be about 22 to 30 nucleotides in length, to have a GC content of about 50% or more, and to anneal to the target sequence at temperatures of about 68C to about 72 C. Any stretch of nucleotides that would result in hairpin structures and primer-primer dimerizations is avoided.
- Selected cDNA libraries are used as templates to extend the sequence. If more than one extension is necessary, additional or nested sets of primers are designed. Preferred libraries have been size-selected to include larger cDNAs and random primed to contain more sequences with 5′ or upstream regions of genes. Genomic libraries are used to obtain regulatory elements, especially extension into the 5′ promoter binding region.
- PCR is performed in 96-well plates using the DNA ENGINE thermal cycler (MJ Research).
- the reaction mix contains DNA template, 200 nmol of each primer, reaction buffer containing Mg 2+ , (NH 4 ) 2 SO 4 , and ⁇ -mercaptoethanol, Taq DNA polymerase (APB), ELONGASE enzyme (Life Technologies), and Pfu DNA polymerase (Stratagene), with the following parameters for primer pair PCI A and PCI B (Incyte Genomics): Step 1: 94C, three min; Step 2: 94C, 15 sec; Step 3: 60C, one min; Step 4: 68C, two min; Step 5: Steps 2, 3, and 4 repeated 20 times; Step 6: 68C, five min; Step 7: storage at 4 C.
- the parameters for primer pair T7 and SK+ are as follows: Step 1: 94C, three min; Step 2: 94C, 15 sec; Step 3: 57 C, one min; Step 4: 68C, two min; Step 5: Steps 2, 3, and 4 repeated 20 times; Step 6: 68C, five min; Step 7: storage at 4 C.
- the concentration of DNA in each well is determined by dispensing 100 ⁇ l PICOGREEN quantitation reagent (0.25% reagent in 1 ⁇ TE, v/v; Molecular Probes) and 0.5 ⁇ l of undiluted PCR product into each well of an opaque fluorimeter plate (Corning, Acton Mass.) and allowing the DNA to bind to the reagent.
- the plate is scanned in a Fluoroskan II (Labsystems Oy, Finland) to measure the fluorescence of the sample and to quantify the concentration of DNA.
- a 5 ⁇ A to 10 ⁇ l aliquot of the reaction mixture is analyzed by electrophoresis on a 1% agarose minigel to determine which reactions are successful in extending the sequence.
- the extended clones are desalted, concentrated, transferred to 384-well plates, digested with CviJI cholera virus endonuclease (Molecular Biology Research, Madison Wis.), and sonicated or sheared prior to religation into pUC18 vector (APB).
- CviJI cholera virus endonuclease Molecular Biology Research, Madison Wis.
- AGARACE enzyme Promega
- Extended clones are religated using T4 DNA ligase (New England Biolabs) into pUC18 vector (APB), treated with Pfu DNA polymerase (Stratagene) to fill-in restriction site overhangs, and transfected into E. coli competent cells. Transformed cells are selected on antibiotic-containing media, and individual colonies are picked and cultured overnight at 37 C in 384-well plates in LB/2 ⁇ carbenicillin liquid media.
- the cells are lysed, and DNA is amplified using primers, Taq DNA polymerase (APB) and Pfu DNA polymerase (Stratagene) with the following parameters: Step I: 94C, three min; Step 2: 94C, 15 sec; Step 3: 60C, one min; Step 4: 72 C, two min; Step 5: steps 2, 3, and 4 repeated 29 times; Step 6: 72 C, five min; Step 7: storage at 4 C.
- DNA is quantified using PICOGREEN quantitation reagent (Molecular Probes) as described above. Samples with low DNA recoveries are reamplified using the conditions described above.
- Samples are diluted with 20% dimethylsulfoxide (DMSO; 1:2, v/v), and sequenced using DYENAMIC energy transfer sequencing primers and the DYENAMIC DIRECT cycle sequencing kit (APB) or the PRISM BIGDYE terminator cycle sequencing kit (ABI).
- DMSO dimethylsulfoxide
- API DYENAMIC DIRECT cycle sequencing kit
- ABSI PRISM BIGDYE terminator cycle sequencing kit
- the cDNAs of the Sequence Listing or their deduced amino acid sequences were used to query databases such as GenBank, SwissProt, BLOCKS, and the like. These databases that contain previously identified and annotated sequences or domains were searched using BLAST or BLAST2 to produce alignments and to determine which sequences were exact matches or homologs. The alignments were to sequences of prokaryotic (bacterial) or eukaryotic (animal, fungal, or plant) origin. Alternatively, algorithms such as the one described in Smith and Smith (1992, Protein Engineering 5:35-51) could have been used to deal with primary sequence patterns and secondary structure gap penalties. All of the sequences disclosed in this application have lengths of at least 49 nucleotides, and no more than 12% uncalled bases (where N is recorded rather than A, C, G, or T).
- BLAST matches between a query sequence and a database sequence were evaluated statistically and only reported when they satisfied the threshold of 10 ⁇ 25 for nucleotides and 10 ⁇ 14 for peptides. Homology was also evaluated by product score calculated as follows: the % nucleotide or amino acid identity [between the query and reference sequences] in BLAST is multiplied by the % maximum possible BLAST score [based on the lengths of query and reference sequences] and then divided by 100. In comparison with hybridization procedures used in the laboratory, the stringency for an exact match was set from a lower limit of about 40 (with 1-2% error due to uncalled bases) to a 100% match of about 70.
- the BLAST software suite (available on the National Center for Biotechnology Information (“NCBI”) website, Bethesda Md., includes various sequence analysis programs including “blastn” that is used to align nucleotide sequences and BLAST2 that is used for direct pairwise comparison of either nucleotide or amino acid sequences.
- BLAST programs are commonly used with gap and other parameters set to default settings, e.g.: Matrix: BLOSUM62; Reward for match: 1; Penalty for mismatch: ⁇ 2; Open Gap: 5 and Extension Gap: 2 penalties; Gap x drop-off: 50; Expect: 10; Word Size: 11; and Filter: on. Identity is measured over the entire length of a sequence.
- cDNAs of this application were compared with assembled consensus sequences or templates found in the LIFESEQ GOLD database (Incyte Genomics).
- Component sequences from cDNA, extension, full length, and shotgun sequencing projects were subjected to PHRED analysis and assigned a quality score. All sequences with an acceptable quality score were subjected to various pre-processing and editing pathways to remove low quality 3′ ends, vector and linker sequences, polyA tails, Alu repeats, mitochondria) and ribosomal sequences, and bacterial contamination sequences.
- Edited sequences had to be at least 50 bp in length, and low-information sequences and repetitive elements such as dinucleotide repeats, Alu repeats, and the like, were replaced by “Ns” or masked.
- Edited sequences were subjected to assembly procedures in which the sequences were assigned to gene bins. Each sequence could only belong to one bin, and sequences in each bin were assembled to produce a template. Newly sequenced components were added to existing bins using BLAST and CROSSMATCH. To be added to a bin, the component sequences had to have a BLAST quality score greater than or equal to 150 and an alignment of at least 82% local identity. The sequences in each bin were assembled using PHRAP. Bins with several overlapping component sequences were assembled using DEEP PHRAP. The orientation of each template was determined based on the number and orientation of its component sequences.
- Bins were compared to one another, and those having local similarity of at least 82% were combined and reassembled. Bins having templates with less than 95% local identity were split. Templates were subjected to analysis by STITCHER/EXON MAPPER algorithms that determine the probabilities of the presence of splice variants, alternatively spliced exons, splice junctions, differential expression of alternative spliced genes across tissue types or disease states, and the like. Assembly procedures were repeated periodically, and templates were annotated using BLAST against GenBank databases such as GBpri.
- templates were subjected to BLAST, motif, and other functional analyses and categorized in protein hierarchies using methods described in U.S. Ser. No. 08/812,290 and U.S. Ser. No. 08/811,758, both filed Mar. 6, 1997; in U.S. Ser. No. 08/947,845, filed Oct. 9, 1997; and in U.S. Ser. No. 09/034,807, filed Mar. 4, 1998.
- templates were analyzed by translating each template in all three forward reading frames and searching each translation against the PFAM database of hidden Markov model-based protein families and domains using the HMMER software package (Washington University School of Medicine, St. Louis Mo.
- the cDNA was further analyzed using MACDNASIS PRO software (Hitachi Software Engineering), and LASERGENE software (DNASTAR) and queried against public databases such as the GenBank rodent, mammalian, vertebrate, prokaryote, and eukaryote databases, SwissProt, BLOCKS, PRINTS, PFAM, and Prosite.
- Radiation hybrid and genetic mapping data available from public resources such as the Stanford Human Genome Center (SHGC), Whitehead Institute for Genome Research (WIGR), and Généthon are used to determine if any of the cDNAs presented in the Sequence Listing have been mapped. Any fragment of a cDNA encoding a signal peptide-containing protein that has been mapped result in the assignment of all related fragments and regulatory sequences to the same location.
- the genetic map locations are described as ranges, or intervals, of human chromosomes. The map position of an interval, in cM (which is roughly equivalent to 1 megabase of human DNA), is measured relative to the terminus of the chromosomal p-arm.
- the cDNAs are applied to a substrate by one of the following methods.
- a mixture of cDNAs is fractionated by gel electrophoresis and transferred to a nylon membrane by capillary transfer.
- the cDNAs are individually ligated to a vector and inserted into bacterial host cells to form a library.
- the cDNAs are then arranged on a substrate by one of the following methods.
- bacterial cells containing individual clones are robotically picked and arranged on a nylon membrane.
- the membrane is placed on LB agar containing selective agent (carbenicillin, kanamycin, ampicillin, or chloramphenicol depending on the vector used) and incubated at 37 C for 16 hr.
- the membrane is removed from the agar and consecutively placed colony side up in 10% SDS, denaturing solution (1.5 M NaCl, 0.5 M NaOH), neutralizing solution (1.5 M NaCl, 1 M Tris, pH 8.0), and twice in 2 ⁇ SSC for 10 min each.
- the membrane is then UV irradiated in a STRATALINKER UV-crosslinker (Stratagene).
- cDNAs are amplified from bacterial vectors by thirty cycles of PCR using primers complementary to vector sequences flanking the insert. PCR amplification increases a starting concentration of 1-2 ng nucleic acid to a final quantity greater than 5 ⁇ g.
- Amplified nucleic acids from about 400 bp to about 5000 bp in length are purified using SEPHACRYL-400 beads (APB). Purified nucleic acids are arranged on a nylon membrane manually or using a dot/slot blotting manifold and suction device and are immobilized by denaturation, neutralization, and UV irradiation as described above.
- Purified nucleic acids are robotically arranged and immobilized on polymer-coated glass slides using the procedure described in U.S. Pat. No. 5,807,522.
- Polymer-coated slides are prepared by cleaning glass microscope slides (Corning, Acton Mass.) by ultrasound in 0.1% SDS and acetone, etching in 4% hydrofluoric acid (VWR Scientific Products, West Chester Pa.), coating with 0.05% aminopropyl silane (Sigma Aldrich) in 95% ethanol, and curing in a 110 C oven. The slides are washed extensively with distilled water between and after treatments.
- the nucleic acids are arranged on the slide and then immobilized by exposing the array to UV irradiation using a STRATALINKER UV-crosslinker (Stratagene). Arrays are then washed at room temperature in 0.2% SDS and rinsed three times in distilled water. Non-specific binding sites are blocked by incubation of arrays in 0.2% casein in phosphate buffered saline (PBS; Tropix, Bedford Mass.) for 30 min at 60 C; then the arrays are washed in 0.2% SDS and rinsed in distilled water as before.
- PBS phosphate buffered saline
- Hybridization probes derived from the cDNAs of the Sequence Listing are employed for screening cDNAs, mRNAs, or genomic DNA in membrane-based hybridizations. Probes are prepared by diluting the cDNAs to a concentration of 40-50 ng in 45 ⁇ l TE buffer, denaturing by heating to 100 C for five min, and briefly centrifuging. The denatured cDNA is then added to a REDIPRIME tube (APB), gently mixed until blue color is evenly distributed, and briefly centrifuged. Five ⁇ l of [ 32 P]dCTP is added to the tube, and the contents are incubated at 37 C for 10 min.
- APB REDIPRIME tube
- the labeling reaction is stopped by adding 5 ⁇ l of 0.2M EDTA, and probe is purified from unincorporated nucleotides using a PROBEQUANT G-50 microcolumn (APB).
- the purified probe is heated to 100 C for five min, snap cooled for two min on ice, and used in membrane-based hybridizations as described below.
- Hybridization probes derived from mRNA isolated from samples are employed for screening cDNAs of the Sequence Listing in array-based hybridizations.
- Probe is prepared using the GEMbright kit (Incyte Genomics) by diluting mRNA to a concentration of 200 ng in 9 ⁇ l TE buffer and adding 5 ⁇ l 5 ⁇ buffer, 1 ⁇ l 0.1 M DTT, 3 ⁇ l Cy3 or Cy5 labeling mix, 1 ⁇ l RNase inhibitor, 1 ⁇ l reverse transcriptase, and 5 ⁇ l 1 ⁇ yeast control mRNAs.
- Yeast control mRNAs are synthesized by in vitro transcription from noncoding yeast genomic DNA (W. Lei, unpublished).
- one set of control mRNAs at 0.002 ng, 0.02 ng. 0.2 ng, and 2 ng are diluted into reverse transcription reaction mixture at ratios of 1:100,000, 1:10,000, 1:1000, and 1:100 (w/w) to sample mRNA respectively.
- a second set of control mRNAs are diluted into reverse transcription reaction mixture at ratios of 1:3, 3:1, 1:10, 10:1, 1:25, and 25:1 (w/w).
- the reaction mixture is mixed and incubated at 37 C for two hr.
- the reaction mixture is then incubated for 20 min at 85 C, and probes are purified using two successive CHROMA SPIN+TE 30 columns (Clontech, Palo Alto Calif.).
- Purified probe is ethanol precipitated by diluting probe to 90 pl in DEPC-treated water, adding 2 ⁇ l 1 mg/ml glycogen, 60 ⁇ l 1.5 M sodium acetate, and 300 ⁇ l 100% ethanol.
- the probe is centrifuged for 20 min at 20,800 ⁇ g, and the pellet is resuspended in 12 ⁇ l resuspension buffer, heated to 65 C for five min, and mixed thoroughly. The probe is heated and mixed as before and then stored on ice. Probe is used in high density array-based hybridizations as described below.
- Membranes are pre-hybridized in hybridization solution containing 1% Sarkosyl and 1 ⁇ high phosphate buffer (0.5 M NaCl, 0.1 M Na2HPO4, 5 mM EDTA, pH 7) at 55 C for two hr.
- the probe diluted in 15 ml fresh hybridization solution, is then added to the membrane.
- the membrane is hybridized with the probe at 55 C for 16 hr.
- the membrane is washed for 15 min at 25 C in 1 mM Tris (pH 80), 1% Sarkosyl, and four times for 15 min each at 25 C in 1 mM Tris (pH 8.0).
- XOMAT-AR film Eastman Kodak, Rochester N.Y.
- XOMAT-AR film Eastman Kodak, Rochester N.Y.
- Probe is heated to 65 C for five min, centrifuged five min at 9400 rpm in a 5415 C microcentrifuge (Eppendorf Scientific, Westbury N.Y.), and then 18 ⁇ l is aliquoted onto the array surface and covered with a coverslip.
- the arrays are transferred to a waterproof chamber having a cavity just slightly larger than a microscope slide.
- the chamber is kept at 100% humidity internally by the addition of 140 pl of 5 ⁇ SSC in a corner of the chamber.
- the chamber containing the arrays is incubated for about 6.5 hr at 60 C.
- the arrays are washed for 10 min at 45 C in 1 ⁇ SSC, 0.1% SDS, and three times for 10 min each at 45 C in 0.1 ⁇ SSC, and dried.
- Hybridization reactions are performed in absolute or differential hybridization formats.
- absolute hybridization format probe from one sample is hybridized to array elements, and signals are detected after hybridization complexes form. Signal strength correlates with probe mRNA levels in the sample.
- differential hybridization format differential expression of a set of genes in two biological samples is analyzed. Probes from the two samples are prepared and labeled with different labeling moieties. A mixture of the two labeled probes is hybridized to the array elements, and signals are examined under conditions in which the emissions from the two different labels are individually detectable. Elements on the array that are hybridized to equal numbers of probes derived from both biological samples give a distinct combined fluorescence (Shalon WO95/35505).
- Hybridization complexes are detected with a microscope equipped with an Innova 70 mixed gas 10 W laser (Coherent, Santa Clara Calif.) capable of generating spectral lines at 488 nm for excitation of Cy3 and at 632 nm for excitation of Cy5.
- the excitation laser light is focused on the array using a 20 ⁇ microscope objective (Nikon, Melville N.Y.).
- the slide containing the array is placed on a computer-controlled X-Y stage on the microscope and raster-scanned past the objective with a resolution of 20 micrometers.
- the two fluorophores are sequentially excited by the laser.
- Emitted light is split, based on wavelength, into two photomultiplier tube detectors (PMT R1477, Hamamatsu Photonics Systems, Bridgewater N.J.) corresponding to the two fluorophores. Filters positioned between the array and the photomultiplier tubes are used to separate the signals. The emission maxima of the fluorophores used are 565 nm for Cy3 and 650 nm for Cy5. The sensitivity of the scans is calibrated using the signal intensity generated by the yeast control mRNAs added to the probe mix. A specific location on the array contains a complementary DNA sequence, allowing the intensity of the signal at that location to be correlated with a weight ratio of hybridizing species of 1:100,000.
- the output of the photomultiplier tube is digitized using a 12-bit RTI-835H analog-to-digital (A/D) conversion board (Analog Devices, Norwood Mass.) installed in an IBM-compatible PC computer.
- the digitized data are displayed as an image where the signal intensity is mapped using a linear 20-color transformation to a pseudocolor scale ranging from blue (low signal) to red (high signal).
- the data is also analyzed quantitatively. Where two different fluorophores are excited and measured simultaneously, the data are first corrected for optical crosstalk (due to overlapping emission spectra) between the fluorophores using the emission spectrum for each fluorophore.
- a grid is superimposed over the fluorescence signal image such that the signal from each spot is centered in each element of the grid.
- the fluorescence signal within each element is then integrated to obtain a numerical value corresponding to the average intensity of the signal.
- the software used for signal analysis is the GEMTOOLS program (Incyte Genomics).
- a transcript image was performed using the LIFESEQ GOLD database (Jun01 release, Incyte Genomics). This process allowed assessment of the relative abundance of the expressed polynucleotides in all of the cDNA libraries and reconfirmed the data submitted in U.S. Ser. No. 08/966,316, filed 7 Nov. 1997. Criteria for transcript imaging can be selected from category, number of cDNAs per library, library description, disease indication, clinical relevance of sample, and the like.
- All sequences and cDNA libraries in the LIFESEQ database have been categorized by system, organ/tissue and cell type. For each category, the number of libraries in which the sequence was expressed were counted and shown over the total number of libraries in that category.
- all normalized or pooled libraries which have high copy number sequences removed prior to processing, and all mixed or pooled tissues, which are considered non-specific in that they contain more than one tissue type or more than one subject's tissue, can be excluded from the analysis. Treated and untreated cell lines and/or fetal tissue data can also be disregarded or removed where clinical relevance is emphasized.
- fetal tissue may be emphasized wherever elucidation of inherited disorders or differentiation of particular cells or organs from stem cells (such as nerves, heart or kidney) would be furthered by removing clinical samples from the analysis.
- Transcript imaging can also be used to support data from other methodologies such as microarray analysis.
- the transcript images for SEQ ID NOs:1-15 and 17 are shown below.
- the first column shows library name; the second column, the number of cDNAs sequenced in that library; the third column, the description of the library; the fourth column, absolute abundance of the transcript in the library; and the fifth column, percentage abundance of the transcript in the library.
- Nervous System (Brain) Library cDNAs Description of Tissue Abundance % Abundance HNT2AGt01 5225 teratoCA line, hNT2, t/RA + MI 1 0.0191 BRADFDIT02 5908 frontal lobe, Huntington's, 57M 1 0.0169 BRAINOM01 24452 brain, infant, 10wF, NORM, WM 1 0.0041
- SEQ ID NO:1 is expressed four-fold higher in Huntington's disease with its associated dementia than in normal brain. Even though this GPCR is very sparsely expressed in human tissues; when SEQ ID NO:1 is used in a brain tissue-specific assay, it is diagnostic for Huntington's disease.
- SEQ ID NO:2 was greater than two-fold differentially expressed in biopsied sample from the stomach of a subject diagnosed with adenocarcinoma over cytologically normal tissue from the same subject. Expression was not found in any other cytologically normal stomach tissue which included STOMNOT01, STOMNOT08, and STOMTMR02. SEQ ID NO:2, when used in a stomach-specific assay, is diagnostic for adenocarcinoma.
- Exocrine Glands (Breast) Library cDNAs Description of Tissue Abundance % Abundance BRSTTUT16 3724 ductal carcinoma, 43F, m/BRSTTMT01 2 0.0537 BRSTNOR01 3107 breast, mw/BRSTTUT22, lobular CA, 59F 1 0.0322 BRSTTMT02 3240 PF changes, mw/BRSTTUT16, 46F 1 0.0309 BRSTNOTO9 3920 PF changes, mw/BRSTTUT08 adenoCA, 45F 1 0.0255 *Libraries made from normalized and pooled tissues were removed from this analysis
- SEQ ID NO:3 is differentially expressed in ductal carcinoma of the breast as compared with its matched cytologically normal BRSTTMTOI.
- SEQ ID NO:3 was not expressed in BRSTNOT25 and BRSTNOT35, normal breast tissues removed during breast reduction surgeries, and was not as highly expressed in tissues diagnosed with any other disease states or their cytologically normal matched tissues.
- SEQ ID NO:3, when used in a breast-specific assay including, but not limited to, ductal lavage, is diagnostic for ductal carcinoma.
- SEQ ID NO:4 is more than five-fold differentially expressed in adenosquamous carcinoma of the uterus. It was not differentially expressed in tissues from subjects diagnosed with cervicitis (UTRCNOP01, UTRCDIE01), endometriosis (UTREDIT07, UTREDIT14), cervical tumor (UTRCTUP01), endometrial adenocarcinoma (UTRSTUP03, UTRSTUPO4, UTRSTUP07), or leiomyoma (UTRSTUE01, UTRSTUT04, UTRSTUT05, UTRSTUT07) or in cytologically normal tissues (UTRCNOP01, UTREDME05, UTREDME06, UTREDMF01, UTREDMF02, UTREDMT07, UTRENONO3, UTRENOT10, UTRETMC01, UTRETUP01, UTRMTMR02, UTRMTMT01, UTRPNOM01, UTRSNONO3, UTRSNOP01, UTRSNOR01,
- Exocrine Glands (Breast) Library cDNAs Description of Tissue Abundance % Abundance BRSTTUT13 7631 breast adenoCA, 46F, m/BRSTNOT33 58 0.7601 BRSTNOT31 3102 breast, mw/ductal adenoCA, 57F 11 0.3546 BRSTNOT32 3766 nonfibrocyctic breast disease, 46F 13 0.345 Libraries made from normalized or pooled tissues and those containing less than 3000 cDNAs were removed from this analysis. indicates data missing or illegible when filed
- SEQ ID NO:5 is differentially expressed more than two-fold in adenocarcinoma of the breast when compared to expression in cytologically normal BRSTNOT31, BRSTNOT32 and matched BRSTNOT33.
- SEQ ID NO:5 was not differentially expressed in BRSTNOT25 and BRSTNOT35, normal breast tissues removed during breast reduction surgeries, and was not as highly expressed in tissues diagnosed with any other disease states or their cytologically normal matched tissues.
- SEQ ID when used in a breast-specific assay including, but not limited to, ductal lavage, is diagnostic for adenocarcinoma.
- SEQ ID NO:6 is differentially expressed more than two-fold in mucinous cystadenoma of the ovary when compared to expression in cytologically normal OVARNOT07, OVARNOT13, and OVARNOTO2 and in ovary tissue from a subject diagnosed with adenocarcinoma.
- SEQ ID NO:6 when used in a ovary-specific assay, is diagnostic for mucinous cystadenoma.
- Musculoskeletal System (Cartilage, Synovium) Library cDNAs Description of Tissue Abundance % Abundance CARCTXT02 3594 knee chondrocytes, M/F, t/IL-1 4 0.01113 SYNOOAT01 5674 synovium, knee, OA, 82F 5 0.0881 SYNONOT01 4046 synovium, 75M* 3 0.0741 SYNORAT03 5785 synovium, writs, rheuA, 56F 4 0.0691 SYNORAT05 3466 synovium, knee, rheuA, 62F 2 0.0577 SYNORAT04 5636 synovium, wrist, rheuA, 62F 3 0.0532 CARGDIT02 3440 cartilage, OA, M/F 1 0.0291 CARGDIT01 7229 cartilage, OA 2 0.0277 SYNORAB01 5053 synovium, hip, rheuA, 68F 1 0.0198 *insufficient clinical data to
- SEQ ID NO:7 is preferentially expressed in IL-1 treated chrondrocytes cultured from knee cartilage, in cartilage and synovia from subjects with rheumatoid and osteoarthritis. It was not expressed in normal control CARGNOT01. SEQ ID NO:7, when used in a tissue-specific assay, is diagnostic for arthritis.
- SEQ ID NO:8 was significantly expressed in testicular seminoma; it was not expressed in normal tissue from TESTN0001, TESTNOF01, TESTNOM01, TESTNON04, TESTNOP01, TESTNOT01, TESTNOT03, TESTNOT04, TESTNOT07, TESTNOT10, and TESTNOT11, or in embryonal carcinomas from TESTTUE02 and TESTTUT02.
- SEQ ID NO:7 when used in a clinically relevant, testicle-specific assay, is a diagnostic for testicular seminoma.
- SEQ ID NO:9 was specifically expressed in prostate tissue cytologically showing adenofibromatous hyperplasia and matched with adenocarcinoma of the prostate (see PROSTUT matches above). It was not expressed in tissues from subjects diagnosed with benign prostatic hyperplasia (PROSBPS05, PROSBPT03, PROSDIP01, PROSDIP02, and PROSDIP03), or prostatic IN (PROETMP06, PROETMP07). SEQ ID NO:9, when used in a prostate-specific assay, is diagnostic for AH and may serve as an early diagnostic marker for prostatic adenocarcinoma.
- Urinary Tract (Bladder) Description % Library cDNAs of Tissue Abundance Abundance BLADNOT05 3774 bladder mw/ 4 0.1060 BLADTUT04 TC CA in situ, 60M BLADDIT01 3775 bladder, chronic 1 0.0265 cystitis, 73M *Libraries made from normalized tissues were removed from this analysis.
- SEQ ID NO:10 showed five-fold differential expression in a cytologically normal bladder library which was matched with transitional cell carcinoma of the bladder. Expression of SEQ ID NO:10 was clearly distinct from that seen in tissue affected by chronic cystitis and was not seen in normal tissues, BLADNOR01, BLADNOT01, BLADNOT03, BLADNOT04, BLADNOT06, and BLADNOTO8 or in the tumor libraries, BLADTUE01, BLADTUT02, BLADTUT03, BLADTUT04, BLADTUT05, BLADTUT06, BLADTUTO7 and BLADTUT08, SEQ ID NO:10, when used in a bladder-specific assay, serves as an early diagnostic marker for transitional cell carcinoma of the bladder.
- Urinary Tract (Kidney) Library cDNAs Description of Tissue Abundance % Abundance KIDNTUT13 3771 renal cell CA, 51F 2 0.0530 KIDNTUT15 3941 renal cell CA, 65M m/KIDNNOT19 2 0.0507 KIDNNOT19 6952 mw/KIDNTUT15 renal cell CA, 65M 2 0.0288 KIDNTUT14 3861 renal cell CA, 43M, m/KIDNNOT20 1 0.0259 *Libraries made from normalized, subtracted, and pooled tissues were removed from this analysis.
- SEQ ID NO:11 is expressed in renal cell cancers and not expressed in cytologically normal kidney libraries (KIDNNOT01, KIDNNOT02, KIDNNOT20, KIDNNOT25, KIDNNOT26, KIDNNOT31, KIDNNOT32) or in KIDPTDE01 from a subject diagnosed with interstitial nephritis.
- SEQ ID NO:10 when used in a kidney-specific assay, serves as a diagnostic for renal cell cancer.
- SEQ ID NO:12 is expressed in adenocarcinoma of the breast and not expressed in cytologically normal matched tissue.
- SEQ ID NO:12 when used in a breast-specific assay including, but not limited to, ductal lavage, serves as a diagnostic for adenocarcinoma of the breast.
- SEQ ID NO:13 is highly overexpressed in the pituitary gland removed from a schizophenic subject with chronic pulmonary pulmonary disease. Such high expression levels were not seen in pooled normal tissue or in the pituitaries of subjects with cancers and Alzheimer's disease (AD). SEQ ID NO:13, when used in a tissue-specific assay, serves as a diagnostic for schizophrenia.
- SEQ ID NO:14 is differentially expressed in breast cancer, in particular, in lobular carcinoma. When used in a breast-specific assay including, but not limited to, ductal lavage, SEQ ID NO:14 serves as a diagnostic for breast cancer.
- SEQ ID NO:15 is 4-fold differentially expressed in peripheral blood, particularly eosinophils of asthmatics. When used in an assay of a lung sample, SEQ ID NO:15 is a diagnostic for asthma.
- SEQ ID NO:17 is expressed in adenocarcinoma of the breast and not expressed in cytologically normal matched tissue, BRSTNOT14. Expression was absent from BRSTNOT25 and BRSTNOT35, normal breast tissues removed during breast reduction surgeries. When used in a breastspecific assay, including, but not limited to, ductal lavage, and compared with cancerous and normal standards, expression of SEQ ID NO:17 is diagnostic for breast adenocarcinoma.
- the cDNA, an mRNA, or an antibody specifically binding the protein can serve a clinically relevant diagnostic marker for disorders associated with cell proliferation and cell signaling.
- SEQ ID NOs:1-15 and 17 were compared with all the other sequences in the LIFESEQ database (Incyte Genomics, Palo Alto Calif.) using BLAST analysis (Altschul (1993) supra); Altschul(1990) Supra). The results of the BLAST analyses were reported in THE INVENTION section above.
- Each of the Incyte clones is also used to screen northern blots.
- a probe is generated by EcoRl digestion of the plasmid containing the cDNA. The restriction digest is fractionated on a 1% agarose gel, a restriction fragment from about 400 to about 1400 nt in length is excised from the gel and purified on a QIAQUICK column (Qiagen). The fragment is comprised of the 5′ most region of the insert.
- the probe is prepared by random priming using the REDIPRIME labeling kit (APB) with REDIVUE [ ⁇ 32 P]d-CTP (3000 Ci/mmol; APB). Unincorporated radioactivity is removed by column chromatography using a SEPHADEX G-50 NICK column (APB).
- Blots are pre-hybridized in RAPID-HYB hybridization buffer (APB) for 1 hour at 65 C. Hybridizations are performed at 65 C using 0.5 ⁇ 10 cpm/ml probe for 1 hour. Blots are washed for 2 ⁇ 10 minutes in 1 ⁇ SSC, 0.1% SDS at room temperature followed by 2 stringent washes at 65 C in 0.2 ⁇ SSC, 0.1% SDS for 10 minutes each. Blots are wrapped in SARAN WRAP plastic film (Dow Chemical, Midland Mich.) and autoradiographed at ⁇ 70 C using 2 intensifying screens and HYPERFILM-MP (APB).
- APB RAPID-HYB hybridization buffer
- HEARNOT06 heart 44M HEAPNOT01 heart, coronary artery, plaque, pool SMCANOT01 smooth muscle cell line, aorta, M BRSTTUT14 breast tumor, adenocarcinoma, 62 F, mw/BRSTNOT14 UTRSNOT16 uterus, endometrium, 48F UTRSNOT11 uterus, myometrium, 43F UTRSNOT02 uterus, 34F LPARNOT02 parotid gland, 70M
- SEQ ID NO:17 is diagnostic for breast adenocarcinoma (bold above)
- Molecules complementary to the cDNA from about 5 (PNA) to about 5000 bp (complement of a cDNA insert), are used to detect or inhibit gene expression. Detection is described in Example VII.
- the complementary molecule is designed to bind to the most unique 5′ sequence and includes nucleotides of the 5′ UTR upstream of the initiation codon of the open reading frame.
- Complementary molecules include genomic sequences (such as enhancers or introns) and are used in “triple helix” base pairing to compromise the ability of the double helix to open sufficiently for the binding of polymerases, transcription factors, or regulatory molecules.
- a complementary molecule is designed to prevent ribosomal binding to the mRNA encoding the protein.
- Complementary molecules are placed in expression vectors and used to transform a cell line to test efficacy; into an organ, tumor, synovial cavity, or the vascular system for transient or short term therapy; or into a stem cell, zygote, or other reproducing lineage for long term or stable gene therapy.
- Transient expression lasts for a month or more with a non-replicating vector and for three months or more if elements for inducing vector replication are used in the transformation/expression system.
- the pUB61V5-His vector system (Invitrogen, Carlsbad Calif.) is used to express signal peptide-containing proteins in CHO cells.
- the vector contains the selectable bsd gene, multiple cloning sites, the promoter/enhancer sequence from the human ubiquitin C gene, a C-terminal V5 epitope for antibody detection with anti-V5 antibodies, and a C-terminal polyhistidine (6 ⁇ His) sequence for rapid purification on PROBOND resin (Invitrogen). Transformed cells are selected on media containing blasticidin.
- Spodoptera frueiperda (Sf9) insect cells are infected with recombinant Autographica californica nuclear polyhedrosis virus (baculovirus).
- the polyhedrin gene is replaced with the cDNA by homologous recombination and the polyhedrin promoter drives cDNA transcription.
- the protein is synthesized as a fusion protein with 6 ⁇ his which enables purification as described above. Purified protein is used in the following activity and to make antibodies.
- a signal peptide-containing protein is purified using polyacrylamide gel electrophoresis and used to immunize mice or rabbits. Antibodies are produced using the protocols well known in the art and summarized below. Alternatively, the amino acid sequence of signal peptide-containing proteins is analyzed using LASERGENE software (DNASTAR) to determine regions of high antigenicity. An antigenic epitope, usually found near the C-terminus or in a hydrophilic region is selected, synthesized, and used to raise antibodies.
- epitopes of about 15 residues in length are produced using an 431A peptide synthesizer (ABI) using Fmoc-chemistry and coupled to KLH (Sigma-Aldrich) by reaction with N-maleimidobenzoyl-N-hydroxysuccinimide ester to increase antigenicity.
- ABSI 431A peptide synthesizer
- KLH Sigma-Aldrich
- Rabbits are immunized with the epitope-KLH complex in complete Freund's adjuvant. Immunizations are repeated at intervals thereafter in incomplete Freund's adjuvant. After a minimum of seven weeks for mouse or twelve weeks for rabbit, antisera are drawn and tested for antipeptide activity.
- Testing involves binding the peptide to plastic, blocking with 1% bovine serum albumin, reacting with rabbit antisera, washing, and reacting with radio-iodinated goat anti-rabbit IgG. Methods well known in the art are used to determine antibody titer and the amount of complex formation.
- Naturally occurring or recombinant protein is purified by immunoaffinity chromatography using antibodies which specifically bind the protein.
- An immunoaffinity column is constructed by covalently coupling the antibody to CNBr-activated SEPHAROSE resin (APB). Media containing the protein is passed over the immunoaffinity column, and the column is washed using high ionic strength buffers in the presence of detergent to allow preferential absorbance of the protein. After coupling, the protein is eluted from the column using a buffer of pH 2-3 or a high concentration of urea or thiocyanate ion to disrupt antibody/protein binding, and the protein is collected.
- APB CNBr-activated SEPHAROSE resin
- the cDNA, or fragments thereof, or the protein, or portions thereof, are labeled with 3 P-dCTP, Cy3-dCTP, or Cy5-dCTP (APB), or with BIODIPY or FITC (Molecular Probes, Eugene Oreg.), respectively.
- Libraries of candidate molecules or compounds previously arranged on a substrate are incubated in the presence of labeled cDNA or protein. After incubation under conditions for either a nucleic acid or amino acid sequence, the substrate is washed, and any position on the substrate retaining label, which indicates specific binding or complex formation, is assayed, and the ligand is identified. Data obtained using different concentrations of the nucleic acid or protein are used to calculate affinity between the labeled nucleic acid or protein and the bound molecule.
- a yeast two-hybrid system MATCHMAKER LexA Two-Hybrid system (Clontech Laboratories, Palo Alto Calif.), is used to screen for peptides that bind the protein of the invention.
- a cDNA encoding the protein is inserted into the multiple cloning site of a pLexA vector, ligated, and transformed into E. coli cDNA, prepared from mRNA, is inserted into the multiple cloning site of a pB42AD vector, ligated, and transformed into E. coli construct a cDNA library.
- the pLexA plasmid and pB42AD-cDNA library constructs are isolated from E.
- Transformed yeast cells are plated on synthetic dropout (SD) media lacking histidine (-His), tryptophan (-Trp), and uracil (-Ura), and incubated at 30 C until the colonies have grown up and are counted.
- SD synthetic dropout
- the colonies are pooled in a minimal volume of 1 ⁇ TE (pH 7.5), replaced on SD/-His/-Leu/-Trp/-Ura media supplemented with 2% galactose (Gal), 1% raffinose (Raf), and 80 mg/ml 5-bromo-4-chloro-3-indolyl ⁇ -d-galactopyranoside (X-Gal), and subsequently examined for growth of blue colonies.
- Interaction between expressed protein and cDNA fusion proteins activates expression of a LEU2 reporter gene in EGY48 and produces colony growth on media lacking leucine (-Leu).
- Interaction also activates expression of ( ⁇ -galactosidase from the p8op-lacZ reporter construct that produces blue color in colonies grown on X-Gal.
- SP can be expressed in a mammalian cell line such as DLD-1 or HCT116 (ATCC; Manassas Va.) by transforming the cells with a eukaryotic expression vector encoding SF.
- a mammalian cell line such as DLD-1 or HCT116 (ATCC; Manassas Va.
- Other eukaryotic expression vectors such as those mentioned in EXAMPLE XI above, are commercially available, and the techniques to introduce them into cells are well known to those skilled in the art.
- the effect of SP on cell morphology can be visualized by microscopy; the effect on cell growth can be determined by measuring cell doubling-time; and the effect on tumorigenicity can be assessed by the ability of transformed cells to grow in a soft agar growth assay (Groden et al. (1995) Cancer Res. 55:1531-1539).
- Receptor SPs such as those encoded by SEQ ID NOs: 17, 15, 12, 6, and I can be expressed in heterologous expression systems and their biological activity tested utilizing the purinergic receptor system (P 2u ) as published by Erb et al. (1993; Proc Natl Acad Sci 90:10449-53). Because cultured K562 human leukemia cells lack P2U receptors, they can be transfected with expression vectors containing either normal or chimeric P 2u and loaded with fura-a, fluorescent probe for Ca ++ .
- P 2u purinergic receptor system
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Organic Chemistry (AREA)
- Genetics & Genomics (AREA)
- Molecular Biology (AREA)
- General Health & Medical Sciences (AREA)
- Immunology (AREA)
- Biomedical Technology (AREA)
- Biochemistry (AREA)
- Zoology (AREA)
- Medicinal Chemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Biotechnology (AREA)
- Biophysics (AREA)
- Hematology (AREA)
- Urology & Nephrology (AREA)
- Physics & Mathematics (AREA)
- Microbiology (AREA)
- Wood Science & Technology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Pathology (AREA)
- Analytical Chemistry (AREA)
- General Engineering & Computer Science (AREA)
- Gastroenterology & Hepatology (AREA)
- Oncology (AREA)
- Hospice & Palliative Care (AREA)
- Cell Biology (AREA)
- General Physics & Mathematics (AREA)
- Food Science & Technology (AREA)
- Toxicology (AREA)
- Plant Pathology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Pharmacology & Pharmacy (AREA)
- Animal Behavior & Ethology (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
Abstract
The invention provides a method of diagnosing the presence of cancer in a subject by detecting the expression of a signal peptide-containing protein, wherein the method comprises: providing a biological sample from the subject; contacting the biological sample with a polypeptide which specifically binds to antibodies present in the biological sample, wherein the polypeptide is selected from the group consisting of a polypeptide comprising the amino acid sequence of SEQ ID NO:16 and a polypeptide having an amino acid sequence at least about 90% identical to the amino acid sequence of SEQ ID NO:16; detecting the presence of bound polypeptide in the biological sample; comparing the presence of bound polypeptide in the biological sample with the presence of bound polypeptide in a control sample; wherein a higher presence in the biological sample as compared to the control sample is correlated with the presence of cancer.
Description
- This application is a Divisional of U.S. patent application Ser. No. 14/157,288, filed Jan. 16, 2014, which is a Divisional of U.S. patent application Ser. No. 13/763,102, filed Feb. 8, 2013, now abandoned, which is a continuation of U.S. patent application Ser. No. 11/979,577, filed Nov. 6, 2007, now abandoned, which is a Divisional of U.S. patent application Ser. No. 09/968,433, filed Oct. 1, 2001, now U.S. Pat. No. 7,321,023, which is a Continuation-in-Part of U.S. patent application Ser. No. 09/271,110, filed Mar. 17, 1999, now abandoned, which is a Continuation-in-Part of U.S. patent application Ser. No. 08/966,316, filed Nov. 7, 1997, now U.S. Pat. No. 5,932,445. The entire contents of these applications are incorporated herein by reference in their entirety.
- This invention relates to signal peptide-containing proteins, their encoding cDNAs, and antibodies which binds the proteins, which can be used in the diagnosis, prognosis, treatment and evaluation of therapies for disorders associated with cell proliferation and cell signaling.
- Protein transport is a quintessential process for both prokaryotic and eukaryotic cells. Transport of an individual protein usually occurs via an amino-terminal signal sequence which directs, or targets, the protein from its ribosomal assembly site to a particular cellular or extracellular location. Transport may involve any combination of several of the following steps: contact with a chaperone, unfolding, interaction with a receptor and/or a pore complex, addition of energy, and refolding. Moreover, an extracellular protein may be produced as an inactive precursor. Once the precursor has been exported, removal of the signal sequence by a signal peptidase activates the protein.
- Although amino-terminal signal sequences vary substantially, many patterns and overall properties are shared. Recently, hidden Markov models (HMMs), statistical alternatives to FASTA and Smith Waterman algorithms, have been used to find shared patterns, specifically consensus sequences (Pearson and Lipman (1988) Proc. Natl. Acad. Sci. 85:2444-2448; Smith and Waterman (1981) J. Mol. Biol. 147:195-197). Although they were initially developed to examine speech recognition patterns, HMMs have been used in biology to analyze protein and DNA sequences and to model protein structure (Krogh et al. (1994) J. Mol. Biol. 235:1501-1531; Collin et al. (1993) Protein Sci. 2:305-314). HMMs have a formal probabilistic basis and use position-specific scores for amino acids or nucleotides and for opening and extending an insertion or deletion. The algorithms are quite flexible in that they incorporate information from newly identified sequences to build even more successful patterns. To find signal sequences, multiple unaligned sequences are compared to identify those which encode a peptide of 20 to 50 amino acids with an N-terminal methionine.
- Some examples of the protein families which are known to have signal sequences are receptors (nuclear, 4 transmembrane, G protein coupled, and tyrosine kinase), cytokines (chemokines), hormones (growth and differentiation factors), neuropeptides and vasomediators, protein kinases, phosphatases, phospholipases, phosphodiesterases, nucleotide cyclases, matrix molecules (adhesion, cadherin, extracellular matrix molecules, integrin, and selectin), G proteins, ion channels (calcium, chloride, potassium, and sodium), proteases, transporter/pumps (amino acid, protein, sugar, metal and vitamin; calcium, phosphate, potassium, and sodium) and regulatory proteins. Receptors, kinases, and matrix proteins and diseases associated with their dysfunction are described below.
- G-protein coupled receptors, GPCRs are a large group of receptors which transduce extracellular signals. GPCRs include receptors for biogenic amines such as dopamine, epinephrine, histamine, glutamate (metabotropic effect), acetylcholine (muscarinic effect), and serotonin; for lipid mediators of inflammation such as prostaglandins, platelet activating factor, and leukotrienes; for peptide hormones such as calcitonin, C5a anaphylatoxin, follicle stimulating hormone, gonadotropin releasing hormone, neurokinin, oxytocin, and thrombin; and for sensory signal mediators such as retinal photopigments and olfactory stimulatory molecules. The structure of these highly-conserved receptors consists of seven hydrophobic transmembrane regions, an extracellular N-terminus, and a cytoplasmic C-terminus. The N-terminus interacts with ligands, and the C-terminus interacts with intracellular G proteins to activate second messengers such as cyclic AMP (cAMP), phospholipase C, inositol triphosphate, or ion channel proteins. Three extracellular loops alternate with three intracellular loops to link the seven transmembrane regions. The most conserved parts of these proteins are the transmembrane regions and the first two cytoplasmic loops. A conserved, acidic-Arg-aromatic triplet present in the second cytoplasmic loop may interact with the G proteins. The consensus pattern, [GSTALIVMYWC]-[GSTANCPDE]-{EDPKRH}-x(2)-[LIVMNQGA]-x(2)-[LIVMFT]-[GSTANC]-[LIVMFYWSTAC]-[DENH]-R-[FYVVCSH]-x(2)-[LIVM] (SEQ ID NO: 80) is characteristic of most proteins belonging to this group (Bolander (1994) Molecular Endocrinology, Academic Press. San Diego Calif.; Strosberg (1991) Eur J Biochem 196: 1-10).
- The kinases comprise the largest known group of proteins, a superfamily of enzymes with widely varied functions and specificities. Kinases regulate many different cell proliferation, differentiation, and signaling processes by adding phosphate groups to proteins. Receptor mediated extracellular events trigger the transfer of these high energy phosphate groups and activate intracellular signaling cascades. Activation is roughly analogous to the turning on a molecular switch, and in cases where signaling is uncontrolled may be associated with or produce inflammation and cancer.
- Kinases are usually named after their substrate, their regulatory molecule, or after some aspect of a mutant phenotype. Almost all kinases contain a similar 250-300 amino acid catalytic domain. The N-terminal domain, which contains subdomains I-IV, generally folds into a two-lobed structure which binds and orients the ATP (or GTP) donor molecule. The larger C terminal lobe, which contains subdomains VIA-XI, binds the protein substrate and carries out the transfer of the gamma phosphate from ATP to the hydroxyl group of a serine, threonine, or tyrosine residue. Subdomain V spans the two lobes.
- The kinases may be categorized into families by the different amino acid sequences (between 5 and 100 residues) located on either side of, or inserted into loops of, the kinase domain. These amino acid sequences allow the regulation of each kinase as it recognizes and interacts with its target protein. The primary structure of the kinase domain is conserved and contains specific residues and identifiable motifs or patterns of amino acids. The serine threonine kinases represent one family which preferentially phosphorylates serine or threonine residues. Many serine threonine kinases, including those from human, rabbit, rat, mouse, and chicken cells and tissues, have been described (Hardie and Hanks (1995) The Protein Kinase Facts Books, Vol 1:7-20 Academic Press, San Diego, Calif.).
- The matrix proteins (MPs) provide structural support cell and tissue identity and autocrine, paracrine and juxtacrine properties for most eukaryotic cells (McGowan (1992) FASEB J 6:2895-2904). MPs include adhesion molecules, integrins and selectins, cadherins, lectins, lipocalins, and extracellular matrix proteins (ECMs). MPs possess many different domains which interact with soluble, extracellular molecules. These domains include collagen-like domains. EGF-like domains immunoglobulin-like domains, fibronectin-like domains, type A domain of von Willebrand factor (vWFA)-like modules. ankyrin repeat modules. RDG or RDG-like sequences. carbohydrate-binding domains, and calcium ion-binding domains.
- For example. multidomain or mosaic proteins play an important role in the diverse functions of the ECMs (Engel et al. (1994) Development S35-42). ECM proteins (ECMPs) are frequently characterized by the presence of one or more domains which may contain a number of potential intracellular disulphide bridge motifs. For example, domains which match the epidermal growth factor tandem repeat consensus are present within several known extracellular proteins that promote cell growth, development. and cell signaling. Other domains share internal homology and a regular distribution of single cysteines and cysteine doublets. In the serum albumin family, cysteine arrangement generates the characteristic “double-loop” structure (Soltysik-Espanola et al. (1994) Dev Biol 165:73-85) important for ligand-binding (Kragh-Hansen (1990) Danish Med Bull 37:57-84). Other ECMPs are members of the vWFA-like module superfamily, a diverse group of proteins with a module sharing high sequence similarity. The vWFA-like module is found not only in plasma proteins, but also in plasma membrane and ECMPs (Colombatti and Bonaldo (1991) Blood 77:2305-2315). Crystal structure analysis of an integrin vWFA-like module shows a classic “Rossmann” fold and suggests a metal ion-dependent adhesion site for binding protein ligands (Lee et al. (1995) Cell 80:631-638).
- The diversity, distribution and biochemistry of MPs is indicative of their many, overlapping roles in cell proliferation and cell signaling. MPs function in the formation, growth, remodeling, and maintenance of bone, and in the mediation and regulation of inflammation. Biochemical changes that result from congenital, epigenetic, or infectious diseases affect the expression and balance of MPs. This balance, in turn, affects the activation, proliferation, differentiation, and migration of leukocytes and determines whether the immune response is appropriate or self-destructive (Roman (1996) Immunol. Res. 15:163-178).
- Adenylyl cyclases (AC) are a group of second messenger molecules which actively participate in cell signaling processes. There are at least eight types of mammalian ACs which show regions of conserved sequence and are responsive to different stimuli. For example, the neural-specific type I AC is a Ca++-stimulated enzyme whereas the human type VII is unresponsive to CA++ and responds to prostaglandin E1 and isoproterenol. Characterization of these ACs, their tissue distribution, and the activators and inhibitors of the different types of ACs is the subject of various investigations (Nielsen et al. (1996) J. Biol. Chem. 271:33308-16; Hellevuo et al. (1995) J. Biol. Chem. 270:11581-9). AC interactions with kinases and G proteins in the intracellular signaling pathways of all tissues make them interesting candidate molecules for pharmaceutical research.
- ATP diphosphohydrolase (ATPDase) is an enzyme expressed and secreted by quiescent endothelial cells and involved in vasomediation. The physiological role of ATPDase is to convert ATP and ADP to AMP. When this conversion occurs in the blood vessels during inflammatory response, it prevents extracellular ATP from causing vascular injury by inhibiting platelet activation and modulating vascular thrombosis (Robson et al. (1997) J. Exp. Med. 185:153-63).
- The discovery of new signal peptide-containing proteins, their encoding cDNAs, and antibodies which bind the proteins satisfies a long standing need in the art by providing molecules and compositions which can be used in the diagnosis, prognosis, treatment and evaluation of therapies for disorders associated with cell proliferation and cell signaling.
- The present invention is based on the discovery of signal peptide-containing proteins, their encoding cDNAs and antibodies which specifically binds the proteins that are useful in the diagnosis, prognosis. treatment and evaluation of therapies for disorders associated with cell proliferation and cell signaling.
- The invention provides an isolated cDNA comprising a nucleic acid molecule selected from SEQ ID NOs: 1-15 and 17-78. SEQ ID NO: 17 encodes a protein having an amino acid sequence of SEQ ID NO: 16. The invention also provides isolated cDNAs comprising SEQ ID NOs: 18-78 which have from about 80% to about 100% sequence identity with NOs: 1-15 and 17. The invention additionally encompasses a complement of the cDNAs selected from SEQ ID NOs: 1-15 and 17-78. In one aspect, the cDNA of SEQ ID NO: 17 is a fragment or an oligonucleotide comprising a nucleic acid molecule selected from A24 to G44, G159 to C182, G561 to A596, or A1011 and T1046.
- The invention provides compositions comprising the cDNAs or their complements and a heterologous nucleotide sequence or a labeling moiety which may be used in methods of the invention, on a substrate, or in solution. The invention further provides a vector containing the cDNA. a host cell containing the vector, and a method for using the cDNA to make the human protein. The invention still further provides a transgenic cell line or organism comprising the vector containing a cDNA selected from SEQ ID NOs: 1-15 and 17-78. In a second aspect. the invention provides a cDNA or the complement thereof which can be used in methods of detection. screening, and purification. In a further aspect, the cDNA is a single-stranded RNA or DNA molecule, a peptide nucleic acid, a branched nucleic acid, and the like.
- The invention provides a method for using a cDNA to detect differential expression of a nucleic acid in a sample comprising hybridizing a eDNA to the nucleic acids, thereby forming hybridization complexes and comparing hybridization complex formation with at least one standard, wherein the comparison indicates differential expression of the cDNA in the sample. In one aspect, the method of detection further comprises amplifying the nucleic acids of the sample prior to hybridization. In another aspect, the method showing differential expression of the cDNA is used to diagnose a cancer.
- The invention additionally provides a method for using a composition of the invention to screen a plurality of molecules or compounds to identify or purify at least one ligand which specifically binds the cDNA, the method comprising combining the composition with the molecules or compounds under conditions allowing specific binding, and detecting specific binding to the composition, thereby identifying or purifying a ligand which binds the composition. In one aspect. the molecules or compounds are selected from aptamers, DNA molecules, RNA molecules, peptide nucleic acids, artificial chromosome constructions. peptides, transcription factors, repressors, and regulatory molecules.
- The invention provides a purified protein or a portion thereof selected from the group consisting of an amino acid sequence of SEQ ID NO: 16, a variant of SEQ ID NO: 16, an antigenic epitope of SEQ ID NO: 16, and a biologically active portion of SEQ ID NO: 16. The invention also provides a composition comprising the purified protein and a labeling moiety or a pharmaceutical carrier. The invention further provides a method of using the protein to treat a subject with cancer comprising administering to a patient in need of such treatment a composition containing the purified protein and a pharmaceutical carrier. The invention still further provides a method for using a protein to screen a library or a plurality of molecules or compounds to identify or purify at least one ligand, the method comprising combining the protein with the molecules or compounds under conditions to allow specific binding and detecting specific binding, thereby identifying or purifying a ligand which specifically binds the protein. In one aspect. the molecules or compounds are selected from DNA molecules, RNA molecules, peptide nucleic acids, peptides, proteins, mimetics, agonists, antagonists, antibodies, immunoglobulins. inhibitors, and drugs. In another aspect, the ligand is used to treat a subject with a cancer.
- The invention provides a method of using a protein having the amino acid sequence of SEQ ID NO: 16 to screen a plurality of antibodies to identify an antibody which specifically binds the protein comprising contacting isolated antibodies with the protein under conditions to form an antibody:protein complex, and dissociating the antibody from the protein, thereby obtaining antibody which specifically binds the protein.
- The invention also provides methods for using a protein having the amino acid sequence of SEQ ID NO: 16 to prepare and purify polyclonal and monoclonal antibodies which specifically bind the protein. The method for preparing a polyclonal antibody comprises immunizing a animal with protein under conditions to elicit an antibody response, isolating animal antibodies, attaching the protein to a substrate, contacting the substrate with isolated antibodies under conditions to allow specific binding to the protein, dissociating the antibodies from the protein, thereby obtaining purified polyclonal antibodies. The method for preparing and purifying monoclonal antibodies comprises immunizing a animal with a protein under conditions to elicit an antibody response, isolating antibody producing cells from the animal, fusing the antibody producing cells with immortalized cells in culture to form monoclonal antibody producing hybridoma cells, culturing the hybridoma cells, and isolating from culture monoclonal antibodies which specifically bind the protein.
- The invention provides purified polyclonal and monoclonal antibodies which bind specifically to a protein. The invention also provides a method for using an antibody to detect expression of a protein in a sample, the method comprising combining the antibody with a sample under conditions which allow the formation of antibody:protein complexes; and detecting complex formation, wherein complex formation indicates expression of the protein in the sample. In one aspect, the amount of complex formation when compared to standards is diagnostic of cancer.
- The invention provides a method for inserting a heterologous marker gene into the genomic DNA of a mammal to disrupt the expression of the endogenous polynucleotide. The invention also provides a method for using a cDNA to produce a model system, the method comprising constructing a vector containing a DNA selected from SEQ ID NOs: 1-15 and 17-78 transforming the vector into an embryonic stem cell, selecting a transformed embryonic stem cell, microinjecting the transformed embryonic stem cell into a blastocyst, thereby forming a chimeric blastocyst, transferring the chimeric blastocyst into a pseudopregnant dam, wherein the dam gives birth to a chimeric offspring containing the cDNA in its germ line, and breeding the chimeric mammal to produce a homozygous, model system.
-
FIGS. 1A-1E show the amino acid sequence of SP16 (SEQ ID NO: 16) and nucleic acid sequence if its encoding cDNA (SEQ ID NO: 17). The alignment was produced using MacDNASIS PRO software (Hitachi Software Engineering, South San Francisco Calif.). -
FIG. 2 shows the amino acid sequence alignment between SP-16 (2547002; SEQ ID NO: 16) and bovine GPCR (GI 399711; SEQ ID NO: 79) produced using the MDGALIGN program of LASERGENE software (DNASTAR, Madison Wis.). - Table I shows the sequence identification numbers, reference, Incyte Clone number, cDNA library, NCB1 sequence identifier and GenBank description for each of the signal peptide-containing proteins encoded by the cDNAs.
- It is understood that this invention is not limited to the particular machines, materials and methods described. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments and is not intended to limit the scope of the present invention which will be limited only by the appended claims. As used herein, the singular forms “a,” “an,” and “the” include plural reference unless the context clearly dictates otherwise. For example, a reference to “a host cell” includes a plurality of such host cells known to those skilled in the art.
- Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art to which this invention belongs. All publications mentioned herein are cited for the purpose of describing and disclosing the cell lines, protocols, reagents and vectors which are reported in the publications and which might be used in connection with the invention. Nothing herein is to be construed as an admission that the invention is not entitled to antedate such disclosure by virtue of prior invention.
- “Array” refers to an ordered arrangement of at least two cDNAs or antibodies on a substrate. At least one of the cDNAs or antibodies represents a control or standard, and the other, a cDNA or antibody of diagnostic or therapeutic interest. The arrangement of two to about 40,000 cDNAs or of two to about 40,000 monoclonal or polyclonal antibodies on the substrate assures that the size and signal intensity of each labeled hybridization complex, formed between each cDNA and at least one nucleic acid, or antibody:protein complex, formed between each antibody and at least one protein to which the antibody specifically binds, is individually distinguishable.
- The “complement” of a cDNA of the Sequence Listing refers to a nucleic acid molecule which is completely complementary to the cDNA over its full length and which will hybridize to the cDNA or an mRNA under conditions of maximal stringency.
- “cDNA” refers to an isolated polynucleotide, nucleic acid molecule, or any fragment or complement thereof. It may have originated recombinantly or synthetically, may be double-stranded or single-stranded, represents coding and noncoding 3′ or 5′ sequence, and generally lacks introns.
- A “composition” refers to the polynucleotide and a labeling moiety, a purified protein and a pharmaceutical carrier or a labeling moiety. an antibody and a labeling moiety, and the like.
- “Derivative” refers to a cDNA or a protein that has been subjected to a chemical modification. Derivatization of a cDNA can involve substitution of a nontraditional base such as queosine or of an analog such as hypoxanthine. Derivatization of a protein involves the replacement of a hydrogen by an acetyl, acyl, alkyl, amino, formyl, or morpholino group. Derivative molecules retain the biological activities of the naturally occurring molecules but may confer advantages such as longer lifespan or enhanced activity.
- “Differential expression” refers to an increased or up-regulated or a decreased or down-regulated expression as detected by presence. absence or at least two-fold change in the amount or abundance of a transcribed messenger RNA or translated protein in a sample.
- Disorders associated with cell proliferation and cell signaling include cancers, genetic. and immune conditions. Each disorder is associated with expression of a signal peptide-containing protein or its specific encoding cDNA. These disorders include, but are not limited to, adenofibromatous hyperplasia as a prognostic of prostate cancer, asthma, arthritis, breast cancers such as ductal, lobular, and adenocarcinomas, Huntington's disease, mucinous cystadenoma of the ovary, renal cell cancer, schizophrenia stomach tumor, testicular seminoma, transitional cell carcinoma of the bladder, and uterine adenosquamous carcinoma.
- “Fragment” refers to a chain of consecutive nucleotides from about 50 to about 4000 base pairs in length. Fragments may be used in PCR or hybridization technologies to identify related nucleic acid molecules and in binding assays to screen for a ligand. Such ligands are useful as therapeutics to regulate replication, transcription or translation.
- A “hybridization complex” is formed between a cDNA and a nucleic acid of a sample when the purines of one molecule hydrogen bond with the pyrimidines of the complementary molecule, e.g., 5′-A-G-T-C-3′ base pairs with 3′-T-C-A-G-5′. Hybridization conditions, degree of complementarity and the use of nucleotide analogs affect the efficiency and stringency of hybridization reactions.
- “Labeling moiety” refers to any visible or radioactive label than can be attached to or incorporated into a cDNA or protein. Visible labels include but are not limited to anthocyanins, green fluorescent protein (OFP), 6 glucuronidase. luciferase, Cy3 and Cy5, and the like. Radioactive markers include radioactive forms of hydrogen, iodine, phosphorous, sulfur, and the like.
- “Ligand” refers to any agent, molecule, or compound which will bind specifically to a polynucleotide or to an epitope of a protein. Such ligands stabilize or modulate the activity of polynucleotides or proteins and may be composed of inorganic and/or organic substances including minerals, cofactors, nucleic acids, proteins, carbohydrates, fats. and lipids.
- “Oligonucleotide” refers a single-stranded molecule from about 18 to about 60 nucleotides in length which may be used in hybridization or amplification technologies or in regulation of replication, transcription or translation. Equivalent terms are amplimer, primer, and oligomer.
- An “oligopeptide” is an amino acid sequence from about five residues to about 15 residues that is used as part of a fusion protein to produce an antibody.
- “Portion” refers to any part of a protein used for any purpose; but especially, to an epitope for the screening of ligands or for the production of antibodies.
- “Post-translational modification” of a protein can involve lipidation, glycosylation, phosphorylation, acetylation, racemization, proteolytic cleavage, and the like. These processes may occur synthetically or biochemically. Biochemical modifications will vary by cellular location, cell type, pH, enzymatic milieu, and the like.
- “Probe” refers to a cDNA that hybridizes to at least one nucleic acid in a sample. Where targets are single-stranded, probes are complementary single strands. Probes can be labeled with reporter molecules for use in hybridization reactions including Southern, northern, in situ, dot blot, array, and like technologies or in screening assays.
- “Protein” refers to a polypeptide or any portion thereof. A “portion” of a protein refers to that length of amino acid sequence which would retain at least one biological activity, a domain identified by PFAM or PRINTS analysis or an antigenic epitope of the protein identified using Kyte-Doolittle algorithms of the PROTEAN program (DNASTAR).
- “Purified” refers to any molecule or compound that is separated from its natural environment and is from about 60% free to about 90% free from other components with which it is naturally associated.
- “Sample” is used in its broadest sense as containing nucleic acids, proteins. antibodies. and the like. A sample may comprise a bodily fluid; the soluble fraction of a cell preparation, or an aliquot of media in which cells were grown; a chromosome, an organelle, or membrane isolated or extracted from a cell; genomic DNA, RNA, or cDNA in solution or bound to a substrate; a biopsy, a cell; a tissue; a tissue print; a fingerprint, buccal cells, skin, or hair; and the like.
- “Similarity” refers to the quantification (usually percentage) of nucleotide or residue matches between at least two sequences aligned using a standard algorithm such as Smith-Waterman alignment (Smith and Waterman (1981) J Mol Bioi 147:195-197) or BLAST2 (Altschul et al. (1997) Nucleic Acids Res 25:3389-3402). BLAST2 may be used in a reproducible way to insert gaps in one of the sequences in order to optimize alignment and to achieve a more meaningful comparison between them. Particularly in proteins, similarity is greater than identity in that conservative substitutions (for example, valine for leucine or isoleucine) are counted in calculating the reported percentage. Substitutions which are considered to be conservative are well known in the art.
- “Specific binding” refers to a special and precise interaction between two molecules which is dependent upon their structure. particularly their molecular side groups. For example, the intercalation of a regulatory protein into the major groove of a DNA molecule or the binding between an epitope of a protein and an agonist, antagonist, or antibody.
- “Substrate” refers to any rigid or semi-rigid support to which cDNAs or proteins are bound and includes membranes, filters, chips, slides, wafers, fibers. magnetic or nonmagnetic beads, gels, capillaries or other tubing, plates, polymers, and microparticles with a variety of surface forms including wells, trenches, pins, channels and pores.
- A “transcript image” is a profile of gene transcription activity in a particular tissue at a particular time.
- “Variant” refers to molecules that are recognized variations of a cDNA or a protein encoded by the cDNA. Splice variants may be determined by BLAST score, wherein the score is at least 100, and most preferably at least 400. Allelic variants have a high percent identity to the cDNAs and may differ by about three bases per hundred bases. “Single nucleotide polymorphism” (SNP) refers to a change in a single base as a result of a substitution, insertion or deletion. The change may be conservative (purine for purine) or non-conservative (purine to pyrimidine) and may or may not result in a change in an encoded amino acid or its secondary, tertiary, or quaternary structure.
- The invention is based on the discovery of signal peptide-containing proteins, individually SP-1 through SP-16, and their encoding or regulating cDNAs, SEQ ID NOs: 1-15 and 17 which are characterized in TABLE I. U.S. Ser. No. 09/271,110, filed 17 Mar. 1999, is incorporated by reference herein in its entirety. The cDNAs and fragments thereof, the proteins and portions thereof, and an antibody which specifically binds each protein can be used directly or as compositions for the diagnosis, prognosis, treatment and evaluation of therapies for disorders associated with cell proliferation and cell signaling.
- SP-1 was identified in Incyte Clone 1221102 from the NEUTGMTOI cDNA library using a computer search for amino acid sequence alignments. A cDNA comprising the nucleic acid shown in SEQ ID NO: 1 and derived using Incyte Clone 1221102, which encompasses nucleotides 300-514 also found in Incyte clone 5269342F6 (SEQ ID NO:18) which was used on HumanGenomeGEM1 microarray, encodes a GPCR with homology to g1575512, the GPR19 gene. Electronic northern analysis showed the expression of this sequence in neuronal tissues and in stimulated granulocytes. The transcript image found in EXAMPLE VIII supported the northern analysis and showed four-fold, up-regulated expression of the cDNA encoding SP-1 in the brain from a subject diagnosed with Huntington's disease.
- SP-2 was identified in Incyte Clone 1457779 from the COLNFET02 cDNA library using a computer search for amino acid sequence alignments. A cDNA comprising the nucleic acid shown in SEQ ID NO: 2 and derived from Incyte Clone 1457779, which encompasses nucleotides 1-466 also found in Incyte clone 1457779F6 (SEQ ID NO: 22) which was used on LifeGEM1 microarray, encodes an ATP diphosphohydrolase with homology to g1842120. Electronic northern analysis showed the expression of this sequence in fetal colon, and transcript imaging showed that differential expression of SP-2 is diagnostic of stomach tumor.
- SP-3 was identified in Incyte Clone 1682433 from the PROSNOT15 cDNA library using a computer search for amino acid sequence alignments. A cDNA comprising the nucleic acid shown in SEQ ID NO: 3 and derived from Incyte Clone 1682433, which encompasses nucleotides 1-481 also found in Incyte clone 2444114F6 (SEQ ID NO: 26) which was used on LifeGEM1 microarray, encodes a signal peptide-containing protein with homology to g1010391, a transmembrane protein. Electronic northern analysis showed the expression of this sequence in fetal, cancerous or inflamed cells and tissues. Transcript imaging showed that differential expression of SP-3 is diagnostic of ductal carcinoma of the breast.
- SP-4 was identified in Incyte Clone 1899132 from the BLADTUTO6 cDNA library using a 35 (;omputer search for amino acid sequence alignments. A nucleotide sequence, SEQ ID NO: 4. derived from Incyte Clone 1899132, which encompasses nucleotides 272-625 also found in Incyte clone 1899132F6 (SEQ ID NO: 31) which was used on LifeGEM1 microarray encodes a signal peptide containing protein with homology to g887602, a Saccharomyces cerevisiae protein. Electronic northern analysis showed the expression of this sequence in cancerous and inflamed cells and tissues; transcript imaging showed that differential expression of SP-4 is diagnostic of uterine adenosquamous carcinoma.
- SP-5 was identified in Incyte Clone 1907344 from the CONNTUT01 cDNA library using a computer search for amino acid sequence alignments. A nucleotide sequence, SEQ ID NO: 5, derived from Incyte Clone 1907344, which encompasses nucleotides 17-450 also found in Incyte clone 2487075F6 (SEQ ID NO: 35) which was used on HumanGenomeGEM1 microarray, encodes a signal peptide containing protein with homology to g33715, immunoglobulin light chain. Electronic northern analysis showed the expression of this sequence in cancerous and fetal or infant cells and tissues; transcript imaging showed that differential expression of SP-5 is diagnostic for adenocarcinoma of the breast.
- SP-6 was identified in Incyte Clone 1963651 from the BRSTNOT04 cDNA library using a computer search for amino acid sequence alignments. A nucleotide sequence, SEQ ID NO: 6, derived from Incyte Clone 1963651, which encompasses nucleotides 651-1090 also found in Incyte clone 1414964F6 (SEQ ID NO: 41) which was used on LifeGEM1 microarray, encodes a GPCR with homology to g1657623, orphan receptor RDC1. Although electronic northern analysis showed expression in ductal carcinoma; transcript imaging showed that differential expression of SP6 in ovary is diagnostic for mucinous cystadenoma.
- SP-7 was identified in Incyte Clone 1976095 from the PANCTUT02 cDNA library using a computer search for amino acid sequence alignments. A nucleotide sequence, SEQ ID NO: 7, derived from Incyte Clone 1976095, which encompasses nucleotides 74-525 also found in Incyte clone 1976095F6 (SEQ ID NO: 44) which was used on LifeGEM1 microarray, encodes a signal peptide-containing protein with homology to g2117185, a Mycobacterium tuberculosis protein. Electronic northern analysis showed the expression of this sequence in cancerous and inflamed tissues; transcript imaging showed that differential expression of SP-7 in synovium or cartilage is diagnostic for arthritis.
- SP-8 was identified in Incyte Clone 2417676 from the HNT3AZT01 cDNA library using a computer search for amino acid sequence alignments. A nucleotide sequence, SEQ ID NO: 8, derived from Incyte Clone 2417676, which encompasses nucleotides 2-363 also found in Incyte clone 2890678F6 (SEQ ID NO: 49) which was used on HumanGenomeGEM1 microarray, encodes a signal peptide-containing protein with homology to g2150012, a human transmembrane protein. Electronic northern analysis showed this sequence to be expressed in proliferating. cancerous or inflamed tissues; transcript imaging shows that differential expression of SP-8 is diagnostic for testicular seminoma.
- SP-9 was identified in Incyte Clone 1805538 from the SINTNOT13 cDNA library using a computer search for amino acid sequence alignments. A nucleotide sequence, SEQ ID NO: 9, derived from Incyte Clone 1805538, which encompasses nucleotides 15-419 also found in Incyte clone 2183094F6 (SEQ ID NO: 53) which was used on LifeGEM1 microarray, encodes a signal peptide-containing protein with homology to g294502, an extracellular matrix protein. Electronic northern analysis showed this sequence to be expressed in inflamed tissues; transcript imaging showed that differential expression of SP-9 is diagnostic of adenofibromatous hyperplasia and prognostic for prostate cancer.
- SP-10 was identified in Incyte Clone 1869688 from the SKINBIT01 cDNA library using a computer search for amino acid sequence alignments. A nucleotide sequence, SEQ ID NO: 10, derived from Incyte Clone 1869688, which encompasses nucleotides 1124-1380 also found in Incyte clone 2182042F6 (SEQ ID NO: 57) which was used on HumanGenomeGEM1 microarray, encodes a signal peptide-containing protein with homology to g1562, a G3 serine/threonine kinase. Electronic northern analysis showed this sequence to be expressed in proliferating tissues; transcript imaging showed that differential expression of SP-10 is diagnostic of transitional cell carcinoma of the bladder.
- SP-11 was identified in Incyte Clone 1880692 from the LEUKNOT03 cDNA library using a computer search for amino acid sequence alignments. A nucleotide sequence, SEQ ID NO: 11, derived from Incyte Clone 1880692, which encompasses nucleotides 12-309 also found in Incyte clone 1880692F6 (SEQ ID NO: 60) which was used on LifeGEMI microarray, encodes a signal peptide-containing protein with homology to g1487910, a C. elegans protein. Electronic northern analysis showed this sequence to be expressed in cancer and blood cells; transcript imaging showed that differential expression of SP-11 is diagnostic for renal cell cancer.
- SP-12 was identified in Incyte Clone 318060 from the EOSIHET02 cDNA library using a computer search for amino acid sequence alignments. A nucleotide sequence, SEQ ID NO: 12, derived from Incyte Clone 318060, which encompasses nucleotides 193-1244 also found in Incyte clone 1266985F6 (SEQ ID NO: 64) which was used on HumanGenomeGEM1 microarray, encodes a receptor with homology to g606788, an opioid GPCR. Although electronic northern analysis showed this sequence to be expressed in nerve and blood cells; transcript imaging showed that differential expression of SP-12 is diagnostic for adenocarcinoma of the breast.
- SP-13 was identified in Incyte Clone 396450 from the PITUNOT02 cDNA library using a computer search for amino acid sequence alignments. A nucleotide sequence, SEQ ID NO: 13, derived from Incyte Clone 396450, which encompasses nucleotides 1-277 also found in Incyte clone 396450R6 (SEQ ID NO: 65) which was used on LifeGEM1 microarray, encodes a signal peptide-containing protein with homology to g342279, opiomelanocortin. Electronic northern analysis showed this sequence to be expressed in hormone producing cells and tissues and inflamed cells and tissues; transcript imaging showed that differential expression of SP-13 is diagnostic for schizophrenia.
- SP-14 was identified in Incyte Clone 506333 from the TMLR3DT02 cDNA library using a computer search for amino acid sequence alignments. A nucleotide sequence. SEQ ID NO: 14, derived from Incyte Clone 506333, which encompasses nucleotides 1-514 also found in Incyte clone 506333T6 (SEQ ID NO: 68) which was used on LifeGEM1 microarray, encodes a signal peptide-containing protein with homology to g2204110, adenylyl cyclase. Electronic northern analysis showed this sequence to be expressed in cancerous and inflamed cells and tissues; transcript imaging showed that differential expression of SP-14 is diagnostic of breast cancer, in particular lobular carcinoma of the breast.
- SP-15 was identified in Incyte Clone 764465 from the LUNGNOT04 cDNA library using a computer search for amino acid sequence alignments. A nucleotide sequence, SEQ ID NO: 15, derived from Incyte Clone 764465, which encompasses nucleotides 49-528 also found in Incyte clone 764465R6 (SEQ ID NO: 69) which was used on LifeGEM1 microarray, encodes a receptor with homology to GI 1902984, lectin-like oxidized LDL receptor. Electronic northern analysis showed this sequence to be expressed in lung and in fetal liver; transcript imaging confirms the northern analysis and shows that differential expression of SP-15 when used with lung samples is diagnostic for asthma.
- SP-16 (SEQ ID NO: 16) was identified in
Incyte Clone 2547002 from the UTRSNOT11 cDNA library using a computer search for amino acid sequence alignments. A consensus sequence, SEQ ID NO: 17, was derived from the extension and assembly of the overlapping nucleic acid sequences of Incyte Clones 2741185T6, 2741185T6F6.comp, and 2741185H1 (BRSTIUT14) and 2547002F6 and 2547002H1 (UTRSNOT11), SEQ ID NOs: 72-76, respectively. - In one embodiment, the invention encompasses a polypeptide comprising the amino acid sequence of SEQ ID NO: 16, as shown in
FIGS. 1A-1E , SP-16 is 350 amino acids in length and has a G protein coupled receptor signature at S125GMQFLACISIDRYVAV (SEQ ID NO: 81); three potential N-glycosylation sites at N6, N19, and N276; a potential glycosaminoglycan attachment site at S48; and ten potential phosphorylation sites at S25, T74, T177, S195, T223, Y269, S278, S309, S323, and S330. SP-16 has 86% sequence identity with a bovine GPCR (g399711) and shares the GPCR signature, the N-glycosylation, the glycosaminoglycan attachment site, and the first nine of the phosphorylation sites with the bovine receptor (FIG. 2 ). Fragments of the nucleic acid molecule useful for designing oligonucleotides or to be used directly as hybridization probes to distinguish between these homologous molecules include A24 to G44, G159 to C182, G561 to A596, or A1011 to T1046. mRNA encoding SP-16 was sparsely expressed in cDNA libraries. Electronic northern analysis (EXAMPLE VIII below) showed expression in breast adenocarcinoma; transcript imaging confirmed the northern analysis and showed that SP-16 is differentially expressed in breast adenocarcinoma and not in matched or normal breast tissues. - cDNA fragments encoding or regulating signal peptide-containing proteins were identified using BLAST2 with default parameters and the ZOOSEQ databases (Incyte Genomics, Palo Alto Calif.). These cDNAs have from about 80% to about 95% sequence identity to the human cDNA as shown in the table below. The first column shows the SEQ IDH for the human cDNA; the second column, the SEQ IDFR for fragment cDNAs; the third column, the sequence numbers for the fragments; the fourth column, the species; the fifth column, percent identity to the human cDNA; and the sixth column, the nucleotide, alignment (NtH) of the human and fragment cDNAs.
-
SEQ IDH SEQ IDFR Clone No. Species Identity NtH Alignment 1 19 051293_Mm.1 Mouse 80% 1-518 1 20 703901370J1 Rat 84% 1-518 1 21 296771_Rn.1 Rat 81% 1-518 2 23 023793_Mm.1 Mouse 83% 307-606 2 24 701923941H1 Rat 84% 402-606 2 25 317489_Rn.1 Rat 84% 402-606 3 27 703711491J1 Dog 89% 817-1075 3 28 060931_Mm.3 Mouse 85% 95-1099 3 29 701926832H1 Rat 88% 801-1033 3 30 317017_Rn.1 Rat 88% 801-1033 4 32 026438_Mm.1 Mouse 84% 311-861 4 33 70298994H1 Rat 86% 489-731 4 34 286037_Rn.1 Rat 86% 341-731 5 36 703200737J1 Monkey 90% 280-450 5 37 071816_Mf.2 Monkey 86% 280-450 5 38 008837_Cf.1 Dog 89% 38-361 5 39 700298833H1 Rat 92% 263-450 5 40 274060_Rn.1 Rat 92% 263-450 6 42 031166_Mm.1 Mouse 87% 201-1803 6 43 203462_Rn.3 Rat 87% 776-1261 7 45 005653_Mf.1 Monkey 90% 519-700 7 46 007876_Cf.1 Dog 89% 134-700 7 47 003508_Mm.1 Mouse 83% 98-668 7 48 205363_Rn.4 Rat 84% 74-700 8 50 008780_Cf.1 Dog 93% 186-296 8 51 013606_Mm.1 Mouse 86% 37-357 8 52 248462_Rn.1 Rat 89% 110-313 9 54 001680_Cf.1 Dog 85% 148-201 9 55 021581_Mm.1 Mouse 82% 232-532 9 56 283960_Rn.1 Rat 86% 232-307 10 58 037196_Mm.1 Mouse 90% 192-1040 10 59 215631_Rn.1 Rat 88% 170-651 11 61 023463_Cf.1 Dog 90% 93-363 11 62 017863_Mm.1 Mouse 85% 179-619 11 63 300968_Rn.1 Rat 82% 179-647 13 66 019409_Mm.2 Mouse 83% 136-272 13 67 216194_Rn.7 Rat 84% 134-272 15 70 028681_Mm.2 Mouse 80% 54-215 15 71 211274_Rn.1 Rat 88% 56-114 17 77 000569_Mm.1 Mouse 87% 789-1091 17 78 251020_Rn.1 Rat 83% 180-820 - It will be appreciated by those skilled in the art that as a result of the degeneracy of the genetic code, a multitude of cDNAs encoding signal peptide-containing proteins, some bearing minimal similarity to the cDNAs of any known and naturally occurring gene, may be produced. Thus, the invention contemplates each and every possible variation of cDNA that could be made by selecting combinations based on possible codon choices. These combinations are made in accordance with the standard triplet genetic code as applied to the polynucleotide encoding naturally occurring signal peptide-containing proteins, and all such variations are to be considered as being specifically disclosed.
- The cDNAs of SEQ ID NOs: 1-15 and 17-78 may be used in hybridization, amplification, and screening technologies to identify and distinguish among the identical and related molecules in a sample. The cDNAs may also be used to produce transgenic cell lines or organisms which are model systems for cancers and upon which the toxicity and efficacy of potential therapeutic treatments may be tested. Toxicology studies, clinical trials, and subject/patient treatment profiles may be performed and monitored using the cDNAs, proteins, antibodies and molecules and compounds identified using the cDNAs and proteins of the present invention.
- cDNA Libraries
- In a particular embodiment disclosed herein, mRNA is isolated from cells and tissues using methods which are well known to those skilled in the art and used to prepare the cDNA libraries. The Incyte cDNAs were isolated from cDNA libraries prepared as described in the EXAMPLES. The consensus sequences are chemically and/or electronically assembled from fragments including Incyte cDNAs and extension and/or shotgun sequences using computer programs such as PHRAP (P Green, University of Washington, Seattle Wash.), and the AUTOASSEMBLER application (ABI). After verification of the 5′ and 3′ sequence, at least one of the representative cDNAs which encode a signal peptide-containing protein is designated a reagent. These reagent cDNAs are also used in the construction of human microarrays and are represented among the sequences on the Human Genome Gem Arrays (Incyte Genomics).
- Methods for sequencing nucleic acids are well known in the art and may be used to practice any of the embodiments of the invention. These methods employ enzymes such as the Klenow fragment of DNA polymerase I, SEQUENASE, Taq DNA polymerase and thermostable T7 DNA polymerase (Amersham Phannacia Biotech (APB), Piscataway N.J.), or combinations of polymerases and proofreading exonucleases such as those found in the ELONGASE amplification system (Life Technologies, Gaithersburg Md.). Preferably, sequence preparation is automated with machines such as the MICROLAB 2200 system (Hamilton. Reno Nev.) and the DNA ENGINE thennal cycler (MJ Research, Watertown Mass.). Machines commonly used for sequencing include the ABI PRISM 3700, 377 or 373 DNA sequencing systems (ABI), the MEGABACE 1000 DNA sequencing system (APB), and the like. The sequences may be analyzed using a variety of algorithms well known in the art and described in Ausubel et al. (1997; Short Protocols in Molecular Biology, John Wiley & Sons, New York N.Y., unit 7.7) and in Meyers (1995; Molecular Biology and Biotechnology, Wiley VCH, New York N.Y., pp. 856-853).
- Shotgun sequencing may also be used to complete the sequence of a particular cloned insert of interest. Shotgun strategy involves randomly breaking the original insert into segments of various sizes and cloning these fragments into vectors. The fragments are sequenced and reassembled using overlapping ends until the entire sequence of the original insert is known. Shotgun sequencing methods are well known in the art and use thermostable DNA polymerases, heat-labile DNA polymerases, and primers chosen from representative regions flanking the cDNAs of interest. Incomplete assembled sequences are inspected for identity using various algorithms or programs such as CONSED (Gordon (1998) Genome Res 8; 195-202) which are well known in the art. Contaminating sequences, including vector or chimeric sequences, or deleted sequences can be removed or restored, respectively, organizing the incomplete assembled sequences into finished sequences.
- The sequences of the invention may be extended using various PCR-based methods known in the art. For example, the XL-PCR kit (ABI), nested primers, and commercially available cDNA or genomic DNA libraries may be used to extend the . . . . For all PCR-based methods, primers may be designed using commercially available software to be about 22 to 30 nucleotides in length, to have a GC content of about 50% or more, and to anneal to a target molecule at temperatures from about 55C to about 68C. When extending a sequence to recover regulatory elements, it is preferable to use genomic, rather than cDNA libraries.
- The cDNA and fragments thereof can be used in hybridization technologies for various purposes. A probe may be designed or derived from unique regions such as the 5′ regulatory region or from a nonconserved region (i.e., 5′ or 3′ of the nucleotides encoding the conserved catalytic domain of the protein) and used in protocols to identify naturally occurring molecules encoding a signal peptide-containing protein, allelic variants, or related molecules. The probe may be DNA or RNA, may be single-stranded, and should have at least 50% sequence identity to a nucleic acid molecule selected from SEQ ID NOs: 1-15 and 17-78. Hybridization probes may be produced using oligolabeling, nick translation, end-labeling, or PCR amplification in the presence of a reporter molecule. A vector containing the cDNA or a fragment thereof may be used to produce an mRNA probe in vitro by addition of an RNA polymerase and labeled nucleotides. These procedures may be conducted using commercially available kits.
- The stringency of hybridization is determined by G+C content of the probe. salt concentration. and temperature. In particular, stringency can be increased by reducing the concentration of salt or raising the hybridization temperature. Hybridization can be performed at low stringency with buffers, such as 5×SSC with 1% sodium dodecyl sulfate (SDS) at 60 C, which permits the formation of a hybridization complex between s that contain some mismatches. Subsequent washes are performed at higher stringency with buffers such as 0.2×SSC with 0.1% SDS at either 45 C (medium stringency) or 68 C (high stringency). At high stringency, hybridization complexes will remain stable only where the nucleic acids are completely complementary. In some membrane-based hybridizations, preferably 35% or most preferably 50%, formamide can be added to the hybridization solution to reduce the temperature at which hybridization is performed, and background signals can be reduced by the use of detergents such as Sarkosyl or TRITON X-100 (Sigma-Aldrich, St Louis Mo.) and a blocking agent such as denatured salmon sperm DNA. Selection of components and conditions for hybridization are well known to those skilled in the art and are reviewed in Ausubel (supra) and Sambrook et al. (1989) Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Press, Plainview N.Y.
- Arrays incorporating cDNAs or antibodies may be prepared and analyzed using methods well known in the art. Oligonucleotides or cDNAs may be used as hybridization probes or targets to monitor the expression level of large numbers of genes simultaneously or to identify genetic variants, mutations, and single nucleotide polymorphisms. Monoclonal or polyclonal antibodies may be used to detect or quantify expression of a protein in a sample. Such arrays may be used to determine gene function; to understand the genetic basis of a condition, disease, or disorder; to diagnose a condition, disease, or disorder; and to develop and monitor the activities of therapeutic agents. (See, e.g., Brennan et al. (1995) U.S. Pat. No. 5,474,796; Schena et al. (1996) Proc Natl Acad Sci 93:10614-10619; Heller et al. (1997) Proc Natl Acad Sci 94:2150-2155; Heller et al. (1997) U.S. Pat. No. 5,605,662; and deWildt et al. (2000) Nature Biotechnol 18:989-994.)
- Hybridization probes are also useful in mapping the naturally occurring genomic sequence. The probes may be hybridized to a particular chromosome, a specific region of a chromosome, or an artificial chromosome construction. Such constructions include human artificial chromosomes (HAC), yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC), bacterial PI constructions, or the cDNAs of libraries made from single chromosomes.
- Anyone of a multitude of cDNAs encoding a signal peptide-containing protein may be cloned into a vector and used to express the protein, or portions thereof, in host cells. The can be engineered by such methods as DNA shuffling, as described in U.S. Pat. No. 5,830,721, and site-directed mutagenesis to create new restriction sites, alter glycosylation patterns, change codon preference to increase expression in a particular host, produce splice variants, extend half-life. and the like. The expression vector may contain transcriptional and translational control elements (promoters, enhancers. specific initiation signals. and polyadenylated 3′ sequence) from various sources which have been selected for their efficiency in a particular host. The vector, cDNA, and regulatory elements are combined using in vitro recombinant DNA techniques, synthetic techniques, and/or in vivo genetic recombination techniques well known in the art and described in Sambrook (supra, ch. 4, 8, 16 and 17).
- A variety of host systems may be transformed with an expression vector. These include, but are not limited to, bacteria transformed with recombinant bacteriophage, plasmid, or cosmid DNA expression vectors; yeast transformed with yeast expression vectors; insect cell systems transformed with baculovirus expression vectors; plant cell systems transformed with expression vectors containing viral and/or bacterial elements, or animal cell systems (Ausubel supra, unit 16). For example. an adenovirus transcription/translation complex may be utilized in mammalian cells. After sequences are ligated into the E1 or E3 region of the viral genome, the infective virus is used to transform and express the protein in host cells. The Rous sarcoma virus enhancer or SV40 or EBV-based vectors may also be used for high-level protein expression.
- Routine cloning, subcloning, and propagation of s can be achieved using the multifunctional PBLUESCRIPT vector (Stratagene, La Jolla Calif.) or PSPORT1 plasmid (Life Technologies). Introduction of a into the multiple cloning site of these vectors disrupts the lacZ gene and allows colorimetric screening for transformed bacteria. In addition. these vectors may be useful for in vitro transcription, dideoxy sequencing, single strand rescue with helper phage, and creation of nested deletions in the cloned sequence.
- For long term production of recombinant proteins. the vector can be stably transformed into cell lines along with a selectable or visible marker gene on the same or on a separate vector. After transformation, cells are allowed to grow for about 1 to 2 days in enriched media-and-then are-transferred to selective media. Selectable markers, antimetabolite, antibiotic, or herbicide resistance genes, confer resistance to the relevant selective agent and allow growth and recovery of cells which successfully express the introduced sequences. Resistant clones identified either by survival on selective media or by the expression of visible markers may be propagated using culture techniques. Visible markers are also used to estimate the amount of protein expressed by the introduced genes. Verification that the host cell contains the desired cDNA is based on DNA-DNA or DNA-RNA hybridizations or PCR amplification techniques.
- The host cell may be chosen for its ability to modify a recombinant protein in a desired fashion. Such modifications include acetylation, carboxylation, glycosylation, phosphorylation, lipidation, acylation and the like. Post-translational processing which cleaves a “prepro” form may also be used to specify protein targeting, folding, and/or activity. Different host cells available from the ATCC (Manassas Va.) which have specific cellular machinery and characteristic mechanisms for post-translational activities may be chosen to ensure the correct modification and processing of the recombinant protein.
- Recovery of Proteins from Cell Culture
- Heterologous moieties engineered into a vector for ease of purification include glutathione Stransferase (GST), 6×His, FLAG, MYC, and the like. GST and 6×His are purified using commercially available affinity matrices such as immobilized glutathione and metal-chelate resins, respectively. FLAG and MYC are purified using commercially available monoclonal and polyclonal antibodies. For ease of separation following purification, a sequence encoding a proteolytic cleavage site may be part of the vector located between the protein and the heterologous moiety. Methods for recombinant protein expression and purification are discussed in Ausubel (supra, unit 16) and are commercially available.
- Proteins or portions thereof may be produced not only by recombinant methods, but also by using chemical methods well known in the art. Solid phase peptide synthesis may be carried out in a batchwise or continuous flow process which sequentially adds a-amino-and side chain-protected amino acid residues to an insoluble polymeric support via a linker group. A linker group such as methylarnine-derivatized polyethylene glycol is attached to poly(styrene-co-divinylbenzene) to form the support resin. The amino acid residues are N-α-protected by acid labile Boc (t-butyloxycarbonyl) or base-labile Fmoc (9-fluorenylmethoxycarbonyl). The carboxyl group of the protected amino acid is coupled to the amine of the linker group to anchor the residue to the solid phase support resin. Trifluoroacetic acid or piperidine are used to remove the protecting group in the case of Boc or Fmoc, respectively. Each additional amino acid is added to the anchored residue using a coupling agent or pre-activated amino acid derivative. and the resin is washed. The full length peptide is synthesized by sequential deprotection, coupling of derivitized amino acids, and washing with dichloromethane and/or N, N-dimethylformamide. The peptide is cleaved between the peptide carboxy terminus and the linker group to yield a peptide acid or amide. These processes are described in the Novabiochem 1997/98 Catalog and Peptide Synthesis Handbook (San Diego Calif., pp. S1-S20). Automated synthesis may also be carried out on machines such as the ABI 431 A peptide synthesizer (ABI). A protein or portion thereof may be purified by preparative high performance liquid chromatography and its composition confirmed by amino acid analysis or by sequencing (Creighton (1984) Proteins, Structures and Molecular Properties, WH Freeman, New York N.Y.).
- Various hosts including, but not limited to, goats, rabbits, rats, mice, and human cell lines may be immunized by injection with a signal peptide-containing protein or any immunogenic portion thereof. Adjuvants such as Freund's, mineral gels, and surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemacyanin (KLH), and dinitrophenol may be used to increase immunological response. The oligopeptide, peptide, or portion of protein used to induce antibodies should consist of at least about five amino acids, more preferably ten amino acids, which are identical to a portion of the natural protein. Oligopeptides may be fused with proteins such as KLH in order to produce antibodies to the chimeric molecule.
- Monoclonal antibodies may be prepared using any technique which provides for the production of antibodies by continuous cell lines in culture. These include, but are not limited to, the hybridoma technique, the human β-cell hybridoma technique, and the EBV-hybridoma technique. (See, e.g., Kohler et al. (1975) Nature 256:495-497; Kozbor et al. (1985) J. Immunol Methods 81:31-42; Cote et al. (1983) Proc Natl Acad Sci 80:2026-2030; and Cole et al (1984) Mol Cell Biol 62:109-120.)
- Alternatively, techniques described for antibody production may be adapted, using methods known in the art, to produce epitope-specific, single chain antibodies. Antibody fragments which contain specific binding sites for epitopes of the protein may also be generated. For example, such fragments include, but are not limited to, F(ab′)2 fragments produced by pepsin digestion of the antibody molecule and Fab fragments generated by reducing the disulfide bridges of the F(ab′)2 fragments. Alternatively, Fab expression libraries may be constructed to allow rapid and easy identification of monoclonal Flib fragments with the desired specificity. (See, e.g., Huse et al. (1989) Science 246:1275-1281.)
- A signal peptide-containing protein, or a portion thereof, may be used in screening assays of phagemid or β-lymphocyte immunoglobulin libraries to identify antibodies having the desired specificity. Numerous protocols for competitive binding or immunoassays using either polyclonal or monoclonal antibodies with established specificities are well known in the art. Such immunoassays typically involve the measurement of complex formation between the protein and its specific antibody. A two-site, monoclonal-based immunoassay utilizing monoclonal antibodies reactive to two non-interfering epitopes is preferred, but a competitive binding assay may also be employed (Pound (1998) Immunochemical Protocols, Humana Press, Totowa N.J.).
- A wide variety of reporter molecules and conjugation techniques are known by those skilled in the art and may be used in various nucleic acid, amino acid, and antibody assays. Synthesis of labeled molecules may be achieved using commercially available kits (Promega, Madison Wis.) for incorporation of a labeled nucleotide such as 32P-dCTP (APB), Cy3-dCTP or Cy5-dCTP (Operon Technologies, Alameda Calif.), or amino acid such as 35S-methionine (APB). Nucleotides and amino acids may be directly labeled with a variety of substances including fluorescent, chemiluminescent, or chromogenic agents, and the like, by chemical conjugation to amines, thiols and other groups present in the molecules using reagents such as BIODIPY or FITC (Molecular Probes, Eugene Oreg.).
- The cDNAs, fragments, oligonucleotides, complementary RNA and DNA molecules, and PNAs may be used to detect and quantify differential gene expression for diagnostic purposes. Disorders associated with expression of SP-1 through SP-16 include, but are not limited to, adenofibromatous hyperplasia as a prognostic of prostate cancer, asthma, arthritis, breast cancers such as ductal, lobular. and adeno-carcinomas, Huntington's disease, mucinous cystadenoma of the ovary, renal cell cancer, schizophrenia stomach tumor, testicular seminoma, transitional cell carcinoma of the bladder, and uterine adenosquamous carcinoma. The diagnostic assay may use hybridization or quantitative PCR to compare gene expression in a biological or biopsied subject sample to standard samples in order to detect differential gene expression. Qualitative and quantitative methods for this comparison are commercially available and well known in the art.
- For example, the cDNA or probe may be labeled by standard methods and added to a biological sample from a subject under conditions for the formation of hybridization complexes. After an incubation period. the sample is washed and the amount of label (or signal) associated with hybridization complexes, is quantified and compared with a standard value. If complex formation in the subject sample is significantly altered (higher or lower) in comparison to either, a normal or disease standard, then differential expression indicates the presence of a disorder.
- In order to provide standards for establishing differential expression, normal and disease expression profiles are established. This is accomplished by combining a sample taken from normal subjects, either animal or human, with a cDNA under conditions for hybridization to occur. Standard hybridization complexes may be quantified by comparing the values obtained using normal subjects with values from an experiment in which a known amount of a purified sequence is used. Standard values obtained in this manner may be compared with values obtained from samples from patients who were diagnosed with a particular condition, disease, or disorder. Deviation from standard values toward those associated with a particular disorder is used to diagnose that disorder.
- Such assays may also be used to evaluate the efficacy of a particular therapeutic treatment regimen in animal studies or in clinical trials or to monitor the treatment of an individual patient. Once the presence of a condition is established and a treatment protocol is initiated, diagnostic assays may be repeated on a regular basis to determine if the level of expression in the patient begins to approximate that which is observed in a normal subject. The results obtained from successive assays may be used to show the efficacy of treatment over a period ranging from several days to years.
- Detection and quantification of a protein using either labeled amino acids or specific polyclonal or monoclonal antibodies which specifically bind the protein are known in the art. Examples of such techniques include two-dimensional polyacrylamide gel electrophoresis, enzyme-linked immunosorbent assays (ELISAs), radioimmunoassays (RIAs), and fluorescence activated cell sorting (FACS). These assays and their quantitation against purified. labeled standards are well known in the art (Ausubel, Supra, unit 10.1-10.6). A two-site, monoclonal-based immunoassay utilizing monoclonal antibodies reactive to two non-interfering epitopes is preferred, but a competitive binding assay may be employed. (See. e.g. Coligan et al. (1997) Current Protocols in Immunology, Wiley-Interscience, New York N.Y.; and Pound. supra.)
- Chemical and structural similarity, in particular annotation and motifs that suggest function. are described for SEQ ID NO:16 in THE INVENTION section and transcript images that suggest function for the proteins encoded or regulated by SEQ ID NO:1-15 are described in EXAMPLE VIII and EXAMPLE IX. In addition. the differential expression of each of the cDNAs was shown to be tissue-specific and associated with a particular disorder in EXAMPLE VIII. Thus, each protein clearly plays a role in at least one of the described disorders (adenofibromatous hyperplasia as a prognostic of prostate cancer, asthma, arthritis, breast cancers such as ductal, lobular, and adena-carcinomas, Huntington's disease, mucinous cystadenoma of the ovary, renal cell cancer, schizophrenia stomach tumor, testicular seminoma, transitional cell carcinoma of the bladder, and uterine adenosquamous carcinoma) and SP-1 through SP-16 may be used either directly as a therapeutic or as a target for drug discovery.
- In one embodiment, increased expression of the protein may be treated by the delivery of an inhibitor, antagonist, antibody and the like or a pharmaceutical composition containing one or more of these molecules. Such delivery may be effected by methods well known in the art and may include delivery by an antibody specifically targeted to the diseased tissue.
- In another embodiment, decreased expression of the protein late in the disease process may be treated by the delivery of the protein, an agonist, enhancer and the like or a pharmaceutical composition containing one or more of these molecules. Such delivery may be effected by methods well known in the art and may include delivery by an antibody specifically targeted to the diseased tissue.
- Any of these compositions may be administered in combination with other therapeutic agents. Selection of the agents for use in combination therapy may be made by one of ordinary skill in the art according to conventional pharmaceutical principles. A combination of therapeutic agents may act synergistically to affect treatment of a particular cancer at a lower dosage of each agent alone.
- Gene expression may be modified by designing complementary or antisense molecules (DNA, RNA, or PNA) to the control, 5′, 3′, or other regulatory regions of the gene encoding a signal peptidecontaining protein. Oligonucleotides designed to inhibit transcription initiation are preferred. Similarly, inhibition can be achieved using triple helix base-pairing which inhibits the binding of polymerases, transcription factors, or regulatory molecules (Gee et al. In: Huber and Carr (1994) Molecular and Immunologic Approaches, Futura Publishing, Mt. Kisco N.Y., pp. 163-177). A complementary molecule may also be designed to block translation by preventing binding between ribosomes and mRNA. In one alternative. a library or plurality of cDNAs may be screened to identify those which specifically bind a regulatory, nontranslated sequence.
- Ribozymes, enzymatic RNA molecules, may also be used to catalyze the specific cleavage of RNA. The mechanism of ribozyme action involves sequence-specific hybridization of the ribozyme molecule to complementary target RNA followed by endonucleolytic cleavage at sites such as GUA, GUU, and GUC. Once such sites are identified, an oligonucleotide with the same sequence may be evaluated for secondary structural features which would render the oligonucleotide inoperable. The suitability of candidate wgets may also be evaluated by testing their hybridization with complementary oligonucleotides using ribonuclease protection assays.
- Complementary nucleic acids and ribozymes of the invention may be prepared via recombinant expression, in vitro or in vivo, or using solid phase phosphoramidite chemical synthesis. In addition, RNA molecules may be modified to increase intracellular stability and half-life by addition of flanking sequences at the 5′ and/or 3′ ends of the molecule or by the use of phosphorothioate or 2′ O-methyl rather than phosphodiesterase linkages within the backbone of the molecule. Modification is inherent in the production of PNAs and can be extended to other nucleic acid molecules. Either the inclusion of nontraditional bases such as inosine, queosine, and wybutosine, or the modification of adenine, cytidine, guanine, thymine, and uridine with acetyl-, methyl-, thio-groups renders the molecule less available to endogenous endonucleases.
- A cDNA encoding a signal peptide-containing protein may be used to screen a library or a plurality of molecules or compounds for specific binding affinity. The libraries may be aptamers, DNA molecules, RNA molecules, PNAs, peptides, proteins such as transcription factors, enhancers, or repressors, and other ligands which regulate the activity, replication, transcription, or translation of the endogenous gene. The assay involves combining a polynucleotide with a library or plurality of molecules or compounds under conditions allowing specific binding, and detecting specific binding to identify at least one molecule which specifically binds the single-stranded or double-stranded molecule.
- In one embodiment, the cDNA of the invention may be incubated with a plurality of purified molecules or compounds and binding activity determined by methods well known in the art, e.g., a gelretardation assay (U.S. Pat. No. 6,010,849) or a commercially available reticulocyte lysate transcriptional assay. In another embodiment, the cDNA may be incubated with nuclear extracts from biopsied and/or cultured cells and tissues. Specific binding between the cDNA and a molecule or compound in the nuclear extract is initially determined by gel shift assay and may be later confirmed by recovering and raising antibodies against that molecule or compound. When these antibodies are added into the assay, they cause a supershift in the gel-retardation assay.
- In another embodiment, the cDNA may be used to purify a molecule or compound using affinity chromatography methods well known in the art. In one embodiment, the cDNA is chemically reacted with cyanogen bromide groups on a polymeric resin or gel. Then a sample is passed over and reacts with or binds to the cDNA. The molecule or compound which is bound to the cDNA may be released from the cDNA by increasing the salt concentration of the flow-through medium and collected.
- In a further embodiment, the protein or a portion thereof may be used to purify a ligand from a sample. A method for using a protein or a portion thereof to purify a ligand would involve combining the protein or a portion thereof with a sample under conditions to allow specific binding, detecting specific binding between the protein and ligand, recovering the bound protein, and using a chaotropic agent to separate the protein from the purified ligand.
- In a preferred embodiment, a signal peptide-containing protein may be used to screen a plurality of molecules or compounds in any of a variety of screening assays. The portion of the protein employed in such screening may be free in solution, affixed to an abiotic or biotic substrate (e.g. borne on a cell surface), or located intracellularly. For example, in one method, viable or fixed prokaryotic host cells that are stably transformed with recombinant nucleic acids that have expressed and positioned a peptide on their cell surface can be used in screening assays. The cells are screened against a plurality or libraries of ligands, and the specificity of binding or formation of complexes between the expressed protein and the ligand can be measured. Depending on the particular kind of molecules or compounds being screened, the assay may be used to identify DNA molecules, RNA molecules, peptide nucleic acids, peptides, proteins, mimetics, agonists, antagonists, antibodies, immunoglobulins, inhibitors, and drugs or any other ligand, which specifically binds the protein.
- In one aspect, this invention comtemplates a method for high throughput screening using very small assay volumes and very small amounts of test compound as described in U.S. Pat. No. 5,876,946, incorporated herein by reference. This method is used to screen large numbers of molecules and compounds via specific binding. In another aspect, this invention also contemplates the use of competitive drug screening assays in which neutralizing antibodies capable of binding the protein specifically compete with a test compound capable of binding to the protein. Molecules or compounds identified by screening may be used in a model system to evaluate their toxicity, diagnostic, or therapeutic potential.
- Pharmaceutical compositions contain active ingredients in an effective amount to achieve a desired and intended purpose and a pharmaceutical carrier. The determination of an effective dose is well within the capability of those skilled in the art. For any compound. the therapeutically effective dose may be estimated initially either in cell culture assays or in animal models. The animal model is also used to achieve a desirable concentration range and route of administration. Such information may then be used to determine useful doses and routes for administration in humans.
- A therapeutically effective dose refers to that amount of protein or inhibitor which ameliorates the symptoms or condition. Therapeutic efficacy and toxicity of such agents may be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., ED50 (the dose therapeutically effective in 50% of the population) and LD50 (the dose lethal to 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index, and it may be expressed as the ratio, LD50/ED50. Pharmaceutical compositions which exhibit large therapeutic indexes are preferred. The data obtained from cell culture assays and animal studies are used in formulating a range of dosage for human use.
- Animal models may be used as bioassays where they exhibit a phenotypic response similar to that of humans and where exposure conditions are relevant to human exposures. Mammals are the most common models. and most infectious agent, cancer, drug, and toxicity studies are performed on rodents such as rats or mice because of low cost, availability, lifespan, reproductive potential, and abundant reference literature. Inbred and outbred rodent strains provide a convenient model for investigation of the physiological consequences of under-or over-expression of genes of interest and for the development of methods for diagnosis and treatment of diseases. A mammal inbred to over-express a particular gene (for example, secreted in milk) may also serve as a convenient source of the protein expressed by that gene.
- Toxicology is the study of the effects of agents on living systems. The majority of toxicity studies are performed on rats or mice. Observation of qualitative and quantitative changes in physiology, behavior, homeostatic processes, and lethality in the rats or mice are used to generate a toxicity profile and to assess potential consequences on human health following exposure to the agent.
- Genetic toxicology identifies and analyzes the effect of an agent on the rate of endogenous, spontaneous, and induced genetic mutations. Genotoxic agents usually have common chemical or physical properties that facilitate interaction with nucleic acids and are most harmful when chromosomal aberrations are transmitted to progeny. Toxicological studies may identify agents that increase the frequency of structural or functional abnormalities in the tissues of the progeny if administered to either parent before conception, to the mother during pregnancy. or to the developing organism. Mice and rats are most frequently used in these tests because their short reproductive cycle allows the production of the numbers of organisms needed to satisfy statistical requirements.
- Acute toxicity tests are based on a single administration of an agent to the subject to determine the symptomology or lethality of the agent. Three experiments are conducted: 1) an initial dose-range finding experiment, 2) an experiment to narrow the range of effective doses, and 3) a final experiment for establishing the dose-response curve.
- Subchronic toxicity tests are based on the repeated administration of an agent. Rat and dog are commonly used in these studies to provide data from species in different families. With the exception of carcinogenesis, there is considerable evidence that daily administration of an agent at high-dose concentrations for periods of three to four months will reveal most forms of toxicity in adult animals.
- Chronic toxicity tests, with a duration of a year or more, are used to demonstrate either the absence of toxicity or the carcinogenic potential of an agent. When studies are conducted on rats, a minimum of three test groups plus one control group are used, and animals are examined and monitored at the outset and at intervals throughout the experiment.
- Transgenic rodents that over-express or under-express a gene of interest may be inbred and used to model human diseases or to test therapeutic or toxic agents. (See, e.g., U.S. Pat. No. 5,175,383 and U.S. Pat. No. 5,767,337.) In some cases, the introduced gene may be activated at a specific time in a specific tissue type during fetal or postnatal development. Expression of the transgene is monitored by analysis of phenotype, of tissue-specific mRNA expression, or of serum and tissue protein levels in transgenic animals before, during, and after challenge with experimental drug therapies.
- Embryonic (ES) stem cells isolated from rodent embryos retain the potential to form embryonic tissues. When ES cells are placed inside a carrier embryo, they resume normal development and contribute to tissues of the live-born animal. ES cells are the preferred cells used in the creation of experimental knockout and knockin rodent strains. Mouse ES cells, such as the mouse 129/SvJ cell line, are derived from the early mouse embryo and are grown under culture conditions well known in the art. Vectors used to produce a transgenic strain contain a disease gene candidate and a marker gene, the latter serves to identify the presence of the introduced disease gene. The vector is transformed into ES cells by methods well known in the art, and transformed ES cells are identified and microinjected into mouse cell blastocysts such as those from the C57BU6 mouse strain. The blastocysts are surgically transferred to pseudopregnant dams, and the resulting chimeric progeny are genotyped and bred to produce heterozygous or homozygous strains.
- ES cells derived from human blastocysts may be manipulated in vitro to differentiate into at least eight separate cell lineages. These lineages are used to study the differentiation of various cell types and tissues in vitro, and they include endoderm, mesoderm, and ectodermal cell types which differentiate into, for example, neural cells, hematopoietic lineages, and cardiomyocytes.
- In gene knockout analysis, a region of a gene is enzymatically modified to include a nonmammalian gene such as the neomycin phosphotransferase gene (neo; Capecchi (1989) Science 244:1288-1292). The modified gene is transformed into cultured ES cells and integrates into the endogenous genome by homologous recombination. The inserted sequence disrupts transcription and translation of the endogenous gene. Transformed cells are injected into rodent blastulae, and the blastulae are implanted into pseudopregnant dams. Transgenic progeny are crossbred to obtain homozygous inbred lines which lack a functional copy of the mammalian gene. In one example, the mammalian gene is a human gene.
- ES cells can be used to create knockin humanized animals (pigs) or transgenic animal models (mice or rats) of human diseases. With knockin technology. a region of a human gene is injected into animal ES cells. and the human sequence integrates into the animal cell genome. Transformed cells are injected into blastulae and the blastulae are implanted as described above. Transgenic progeny or inbred lines are studied and treated with potential pharmaceutical agents to obtain information on treatment of the analogous human condition. These methods have been used to model several human diseases.
- The field of animal testing deals with data and methodology from basic sciences such as physiology, genetics, chemistry, pharmacology and statistics. These data are paramount in evaluating the effects of therapeutic agents on non-human primates as they can be related to human health. Monkeys are used as human surrogates in vaccine and drug evaluations, and their responses are relevant to human exposures under similar conditions. Cynomolgus and Rhesus monkeys (Macaca fascicularis and Macaca mulatta, respectively) and Common Marmosets (Callithrix jacchus) are the most common non-human primates (NHPs) used in these investigations. Since great cost is associated with developing and maintaining a colony of NHPs. early research and toxicological studies are usually carried out in rodent models. In studies using behavioral measures such as drug addiction, NHPs are the first choice test animal. In addition, NHPs and individual humans exhibit differential sensitivities to many drugs and toxins and can be classified as a range of phenotypes from “extensive metabolizers” to “poor metabolizers” of these agents.
- In additional embodiments, the cDNAs which encode the protein may be used in any molecular biology techniques that have yet to be developed. provided the new techniques rely on properties of cDNAs that are currently known. including, but not limited to, such properties as the triplet genetic code and specific base pair interactions.
- The UTRSNOT11 cDNA library was constructed from microscopically normal uterine tissue obtained from a 43-year-old female during a vaginal hysterectomy following diagnosis of uterine leiomyoma. Pathology indicated that the myometrium contained an intramural leiomyoma and a submucosal leiomyoma. The endometrium was proliferative, however, the cervix and fallopian tubes were unremarkable. The right and left ovaries contained corpus lutea. The patient presented with metrorrhagia and deficiency anemia. Patient history included benign hypertension and atherosclerosis. Medications included PROVERA tablets (Pharmacia, Peapack N.J.), iron, and vitamins. Family history included benign hypertension, atherosclerosis, and malignant colon neoplasms.
- The frozen tissue was homogenized and lysed in TRIZOL reagent (1 gm tissue/10 ml reagent; Life Technologies) using a POLYTRON homogenizer (PT-3000; Brinkmann Instruments, Westbury N.Y.). After a brief incubation on ice, chloroform was added (1:5 v/v), and the lysate was centrifuged. The upper chloroform layer was removed to a fresh tube, and the RNA was extracted with isopropanol, resuspended in DEPC-treated water, and treated with DNAse for 25 min at 37 C. The RNA was re-extracted three times with acid phenol-chloroform, pH 4.7, and precipitated with 0.3M sodium acetate and 2.5 volumes ethanol. The mRNA was isolated with the OLIGOTEX kit (Qiagen, Chatsworth Calif.) and used to construct the cDNA library.
- The mRNA was handled according to the recommended protocols in the SUPERSCRIPT plasmid system (Life Technologies). The cDNAs were fractionated on a SEPHAROSE CL4B column (APB), and those cDNAs exceeding 400 bp were ligated into pINCY1 plasmid. The plasmid was subsequently transformed into DH5a competent cells (Life Technologies).
- Plasmid DNA was released from the cells and purified using the REAL PREP 96 plasmid kit (Qiagen). This kit enabled the simultaneous purification of 96 samples in a 96-well block using multichannel reagent dispensers. The recommended protocol was employed except for the following changes: 1) the bacteria were cultured in 1 ml of sterile TERRIFIC BROTH (BD Biosciences, San Jose Calif.) with carbenicillin at 25 mg/L and glycerol at 0.4%; 2) after incubation for 19 hours, the cultures were lysed with 0.3 ml of lysis buffer; and 3) following isopropanol precipitation, the plasmid DNA pellet was resuspended in 0.1 ml of distilled water. After the last step in the protocol, samples were transferred to a 96-well block for storage at 4 C.
- The cDNAs were prepared for sequencing using the MICROLAB 2200 system (Hamilton) in combination with the DNA ENGINE thermal cyclers (MJ Research). The cDNAs were sequenced by the method of Sanger and Coulson (1975; J Mol Biol 94:441-448) using an ABI PRISM 373 or 377 sequencing system (ABI). Most of the isolates were sequenced according to standard ABI protocols and kits with solution volumes of 0.25×-1.0× concentrations or using standard solutions and dyes from APB.
- The cDNA sequence may be extended to full length using the Incyte clone, for example, SEQ ID NO:17, 2547002H1. A set of nested deletion sequencing templates was prepared from overnight liquid culture of clone 496071 using the ERASE-A-BASE system (Promega).
- Sequencing reactions were performed with the ABI PRISM Dye Terminator cycle sequencing kit with AMPLITAQ FS DNA polymerase (ABI). PCR was performed on a DNA ENGINE thermal cycler (MI Research). Reactions were analyzed on an ABI PRISM 310 genetic analyzer (ABI). Individual sequences were assembled and edited using ABI AutoAssembler software (ABI).
- In the alternative, extension is accomplished using oligonucleotide primers synthesized to initiate 5′ and 3′ extension of the known fragment. These primers are designed using commercially available primer analysis software to be about 22 to 30 nucleotides in length, to have a GC content of about 50% or more, and to anneal to the target sequence at temperatures of about 68C to about 72 C. Any stretch of nucleotides that would result in hairpin structures and primer-primer dimerizations is avoided.
- Selected cDNA libraries are used as templates to extend the sequence. If more than one extension is necessary, additional or nested sets of primers are designed. Preferred libraries have been size-selected to include larger cDNAs and random primed to contain more sequences with 5′ or upstream regions of genes. Genomic libraries are used to obtain regulatory elements, especially extension into the 5′ promoter binding region.
- High fidelity amplification is obtained by PCR using methods such as that taught in U.S. Pat. No. 5,932,451. PCR is performed in 96-well plates using the DNA ENGINE thermal cycler (MJ Research). The reaction mix contains DNA template, 200 nmol of each primer, reaction buffer containing Mg2+, (NH4)2SO4, and β-mercaptoethanol, Taq DNA polymerase (APB), ELONGASE enzyme (Life Technologies), and Pfu DNA polymerase (Stratagene), with the following parameters for primer pair PCI A and PCI B (Incyte Genomics): Step 1: 94C, three min; Step 2: 94C, 15 sec; Step 3: 60C, one min; Step 4: 68C, two min; Step 5:
Steps 2, 3, and 4 repeated 20 times; Step 6: 68C, five min; Step 7: storage at 4 C. In the alternative, the parameters for primer pair T7 and SK+ (Stratagene) are as follows: Step 1: 94C, three min; Step 2: 94C, 15 sec; Step 3: 57 C, one min; Step 4: 68C, two min; Step 5:Steps 2, 3, and 4 repeated 20 times; Step 6: 68C, five min; Step 7: storage at 4 C. - The concentration of DNA in each well is determined by dispensing 100 μl PICOGREEN quantitation reagent (0.25% reagent in 1×TE, v/v; Molecular Probes) and 0.5 μl of undiluted PCR product into each well of an opaque fluorimeter plate (Corning, Acton Mass.) and allowing the DNA to bind to the reagent. The plate is scanned in a Fluoroskan II (Labsystems Oy, Finland) to measure the fluorescence of the sample and to quantify the concentration of DNA. A 5 μA to 10 μl aliquot of the reaction mixture is analyzed by electrophoresis on a 1% agarose minigel to determine which reactions are successful in extending the sequence.
- The extended clones are desalted, concentrated, transferred to 384-well plates, digested with CviJI cholera virus endonuclease (Molecular Biology Research, Madison Wis.), and sonicated or sheared prior to religation into pUC18 vector (APB). For shotgun sequences, the digested nucleotide sequences are separated on low concentration (0.6 to 0.8%) agarose gels, fragments are excised, and the agar is digested with AGARACE enzyme (Promega). Extended clones are religated using T4 DNA ligase (New England Biolabs) into pUC18 vector (APB), treated with Pfu DNA polymerase (Stratagene) to fill-in restriction site overhangs, and transfected into E. coli competent cells. Transformed cells are selected on antibiotic-containing media, and individual colonies are picked and cultured overnight at 37 C in 384-well plates in LB/2× carbenicillin liquid media.
- The cells are lysed, and DNA is amplified using primers, Taq DNA polymerase (APB) and Pfu DNA polymerase (Stratagene) with the following parameters: Step I: 94C, three min; Step 2: 94C, 15 sec; Step 3: 60C, one min; Step 4: 72 C, two min; Step 5:
steps 2, 3, and 4 repeated 29 times; Step 6: 72 C, five min; Step 7: storage at 4 C. DNA is quantified using PICOGREEN quantitation reagent (Molecular Probes) as described above. Samples with low DNA recoveries are reamplified using the conditions described above. Samples are diluted with 20% dimethylsulfoxide (DMSO; 1:2, v/v), and sequenced using DYENAMIC energy transfer sequencing primers and the DYENAMIC DIRECT cycle sequencing kit (APB) or the PRISM BIGDYE terminator cycle sequencing kit (ABI). - The cDNAs of the Sequence Listing or their deduced amino acid sequences were used to query databases such as GenBank, SwissProt, BLOCKS, and the like. These databases that contain previously identified and annotated sequences or domains were searched using BLAST or BLAST2 to produce alignments and to determine which sequences were exact matches or homologs. The alignments were to sequences of prokaryotic (bacterial) or eukaryotic (animal, fungal, or plant) origin. Alternatively, algorithms such as the one described in Smith and Smith (1992, Protein Engineering 5:35-51) could have been used to deal with primary sequence patterns and secondary structure gap penalties. All of the sequences disclosed in this application have lengths of at least 49 nucleotides, and no more than 12% uncalled bases (where N is recorded rather than A, C, G, or T).
- As detailed in Karlin (supra), BLAST matches between a query sequence and a database sequence were evaluated statistically and only reported when they satisfied the threshold of 10−25 for nucleotides and 10−14 for peptides. Homology was also evaluated by product score calculated as follows: the % nucleotide or amino acid identity [between the query and reference sequences] in BLAST is multiplied by the % maximum possible BLAST score [based on the lengths of query and reference sequences] and then divided by 100. In comparison with hybridization procedures used in the laboratory, the stringency for an exact match was set from a lower limit of about 40 (with 1-2% error due to uncalled bases) to a 100% match of about 70.
- The BLAST software suite (available on the National Center for Biotechnology Information (“NCBI”) website, Bethesda Md., includes various sequence analysis programs including “blastn” that is used to align nucleotide sequences and BLAST2 that is used for direct pairwise comparison of either nucleotide or amino acid sequences. BLAST programs are commonly used with gap and other parameters set to default settings, e.g.: Matrix: BLOSUM62; Reward for match: 1; Penalty for mismatch: −2; Open Gap: 5 and Extension Gap: 2 penalties; Gap x drop-off: 50; Expect: 10; Word Size: 11; and Filter: on. Identity is measured over the entire length of a sequence. Brenner et al., (1998; Proc Natl Acad Sci 95:6073-6078, incorporated herein by reference) analyzed BLAST for its ability to identify structural homologs by sequence identity and found 30% identity is a reliable threshold for sequence alignments of at least 150 residues and 40%, for alignments of at least 70 residues.
- The cDNAs of this application were compared with assembled consensus sequences or templates found in the LIFESEQ GOLD database (Incyte Genomics). Component sequences from cDNA, extension, full length, and shotgun sequencing projects were subjected to PHRED analysis and assigned a quality score. All sequences with an acceptable quality score were subjected to various pre-processing and editing pathways to remove low quality 3′ ends, vector and linker sequences, polyA tails, Alu repeats, mitochondria) and ribosomal sequences, and bacterial contamination sequences. Edited sequences had to be at least 50 bp in length, and low-information sequences and repetitive elements such as dinucleotide repeats, Alu repeats, and the like, were replaced by “Ns” or masked.
- Edited sequences were subjected to assembly procedures in which the sequences were assigned to gene bins. Each sequence could only belong to one bin, and sequences in each bin were assembled to produce a template. Newly sequenced components were added to existing bins using BLAST and CROSSMATCH. To be added to a bin, the component sequences had to have a BLAST quality score greater than or equal to 150 and an alignment of at least 82% local identity. The sequences in each bin were assembled using PHRAP. Bins with several overlapping component sequences were assembled using DEEP PHRAP. The orientation of each template was determined based on the number and orientation of its component sequences.
- Bins were compared to one another, and those having local similarity of at least 82% were combined and reassembled. Bins having templates with less than 95% local identity were split. Templates were subjected to analysis by STITCHER/EXON MAPPER algorithms that determine the probabilities of the presence of splice variants, alternatively spliced exons, splice junctions, differential expression of alternative spliced genes across tissue types or disease states, and the like. Assembly procedures were repeated periodically, and templates were annotated using BLAST against GenBank databases such as GBpri. An exact match was defined as having from 95% local identity over 200 base pairs through 100% local identity over 100 base pairs and a homolog match as having an E-value (or probability score) of ≦1×10−8. The templates were also subjected to frameshift FASTx against GENPEPT, and homolog match was defined as having an E-value of ≦1×10−8. Template analysis and assembly was described in U.S. Ser. No. 09/276,534, filed Mar. 25, 1999.
- Following assembly, templates were subjected to BLAST, motif, and other functional analyses and categorized in protein hierarchies using methods described in U.S. Ser. No. 08/812,290 and U.S. Ser. No. 08/811,758, both filed Mar. 6, 1997; in U.S. Ser. No. 08/947,845, filed Oct. 9, 1997; and in U.S. Ser. No. 09/034,807, filed Mar. 4, 1998. Then templates were analyzed by translating each template in all three forward reading frames and searching each translation against the PFAM database of hidden Markov model-based protein families and domains using the HMMER software package (Washington University School of Medicine, St. Louis Mo. The cDNA was further analyzed using MACDNASIS PRO software (Hitachi Software Engineering), and LASERGENE software (DNASTAR) and queried against public databases such as the GenBank rodent, mammalian, vertebrate, prokaryote, and eukaryote databases, SwissProt, BLOCKS, PRINTS, PFAM, and Prosite.
- Radiation hybrid and genetic mapping data available from public resources such as the Stanford Human Genome Center (SHGC), Whitehead Institute for Genome Research (WIGR), and Généthon are used to determine if any of the cDNAs presented in the Sequence Listing have been mapped. Any fragment of a cDNA encoding a signal peptide-containing protein that has been mapped result in the assignment of all related fragments and regulatory sequences to the same location. The genetic map locations are described as ranges, or intervals, of human chromosomes. The map position of an interval, in cM (which is roughly equivalent to 1 megabase of human DNA), is measured relative to the terminus of the chromosomal p-arm.
- The cDNAs are applied to a substrate by one of the following methods. A mixture of cDNAs is fractionated by gel electrophoresis and transferred to a nylon membrane by capillary transfer. Alternatively, the cDNAs are individually ligated to a vector and inserted into bacterial host cells to form a library. The cDNAs are then arranged on a substrate by one of the following methods. In the first method, bacterial cells containing individual clones are robotically picked and arranged on a nylon membrane. The membrane is placed on LB agar containing selective agent (carbenicillin, kanamycin, ampicillin, or chloramphenicol depending on the vector used) and incubated at 37 C for 16 hr. The membrane is removed from the agar and consecutively placed colony side up in 10% SDS, denaturing solution (1.5 M NaCl, 0.5 M NaOH), neutralizing solution (1.5 M NaCl, 1 M Tris, pH 8.0), and twice in 2×SSC for 10 min each. The membrane is then UV irradiated in a STRATALINKER UV-crosslinker (Stratagene).
- In the second method, cDNAs are amplified from bacterial vectors by thirty cycles of PCR using primers complementary to vector sequences flanking the insert. PCR amplification increases a starting concentration of 1-2 ng nucleic acid to a final quantity greater than 5 μg. Amplified nucleic acids from about 400 bp to about 5000 bp in length are purified using SEPHACRYL-400 beads (APB). Purified nucleic acids are arranged on a nylon membrane manually or using a dot/slot blotting manifold and suction device and are immobilized by denaturation, neutralization, and UV irradiation as described above. Purified nucleic acids are robotically arranged and immobilized on polymer-coated glass slides using the procedure described in U.S. Pat. No. 5,807,522. Polymer-coated slides are prepared by cleaning glass microscope slides (Corning, Acton Mass.) by ultrasound in 0.1% SDS and acetone, etching in 4% hydrofluoric acid (VWR Scientific Products, West Chester Pa.), coating with 0.05% aminopropyl silane (Sigma Aldrich) in 95% ethanol, and curing in a 110 C oven. The slides are washed extensively with distilled water between and after treatments. The nucleic acids are arranged on the slide and then immobilized by exposing the array to UV irradiation using a STRATALINKER UV-crosslinker (Stratagene). Arrays are then washed at room temperature in 0.2% SDS and rinsed three times in distilled water. Non-specific binding sites are blocked by incubation of arrays in 0.2% casein in phosphate buffered saline (PBS; Tropix, Bedford Mass.) for 30 min at 60 C; then the arrays are washed in 0.2% SDS and rinsed in distilled water as before.
- Hybridization probes derived from the cDNAs of the Sequence Listing are employed for screening cDNAs, mRNAs, or genomic DNA in membrane-based hybridizations. Probes are prepared by diluting the cDNAs to a concentration of 40-50 ng in 45 μl TE buffer, denaturing by heating to 100 C for five min, and briefly centrifuging. The denatured cDNA is then added to a REDIPRIME tube (APB), gently mixed until blue color is evenly distributed, and briefly centrifuged. Five μl of [32P]dCTP is added to the tube, and the contents are incubated at 37 C for 10 min. The labeling reaction is stopped by adding 5 μl of 0.2M EDTA, and probe is purified from unincorporated nucleotides using a PROBEQUANT G-50 microcolumn (APB). The purified probe is heated to 100 C for five min, snap cooled for two min on ice, and used in membrane-based hybridizations as described below.
- Hybridization probes derived from mRNA isolated from samples are employed for screening cDNAs of the Sequence Listing in array-based hybridizations. Probe is prepared using the GEMbright kit (Incyte Genomics) by diluting mRNA to a concentration of 200 ng in 9 μl TE buffer and adding 5
μl 5× buffer, 1 μl 0.1 M DTT, 3 μl Cy3 or Cy5 labeling mix, 1 μl RNase inhibitor, 1 μl reverse transcriptase, and 5μl 1× yeast control mRNAs. Yeast control mRNAs are synthesized by in vitro transcription from noncoding yeast genomic DNA (W. Lei, unpublished). As quantitative controls, one set of control mRNAs at 0.002 ng, 0.02 ng. 0.2 ng, and 2 ng are diluted into reverse transcription reaction mixture at ratios of 1:100,000, 1:10,000, 1:1000, and 1:100 (w/w) to sample mRNA respectively. To examine mRNA differential expression patterns, a second set of control mRNAs are diluted into reverse transcription reaction mixture at ratios of 1:3, 3:1, 1:10, 10:1, 1:25, and 25:1 (w/w). The reaction mixture is mixed and incubated at 37 C for two hr. The reaction mixture is then incubated for 20 min at 85 C, and probes are purified using two successive CHROMA SPIN+TE 30 columns (Clontech, Palo Alto Calif.). Purified probe is ethanol precipitated by diluting probe to 90 pl in DEPC-treated water, adding 2μl 1 mg/ml glycogen, 60 μl 1.5 M sodium acetate, and 300 μl 100% ethanol. The probe is centrifuged for 20 min at 20,800×g, and the pellet is resuspended in 12 μl resuspension buffer, heated to 65 C for five min, and mixed thoroughly. The probe is heated and mixed as before and then stored on ice. Probe is used in high density array-based hybridizations as described below. - Membranes are pre-hybridized in hybridization solution containing 1% Sarkosyl and 1× high phosphate buffer (0.5 M NaCl, 0.1 M Na2HPO4, 5 mM EDTA, pH 7) at 55 C for two hr. The probe, diluted in 15 ml fresh hybridization solution, is then added to the membrane. The membrane is hybridized with the probe at 55 C for 16 hr. Following hybridization, the membrane is washed for 15 min at 25 C in 1 mM Tris (pH 80), 1% Sarkosyl, and four times for 15 min each at 25 C in 1 mM Tris (pH 8.0). To detect hybridization complexes, XOMAT-AR film (Eastman Kodak, Rochester N.Y.) is exposed to the membrane overnight at −70 C, developed, and examined visually.
- Probe is heated to 65 C for five min, centrifuged five min at 9400 rpm in a 5415 C microcentrifuge (Eppendorf Scientific, Westbury N.Y.), and then 18 μl is aliquoted onto the array surface and covered with a coverslip. The arrays are transferred to a waterproof chamber having a cavity just slightly larger than a microscope slide. The chamber is kept at 100% humidity internally by the addition of 140 pl of 5×SSC in a corner of the chamber. The chamber containing the arrays is incubated for about 6.5 hr at 60 C. The arrays are washed for 10 min at 45 C in 1×SSC, 0.1% SDS, and three times for 10 min each at 45 C in 0.1×SSC, and dried.
- Hybridization reactions are performed in absolute or differential hybridization formats. In the absolute hybridization format, probe from one sample is hybridized to array elements, and signals are detected after hybridization complexes form. Signal strength correlates with probe mRNA levels in the sample. In the differential hybridization format, differential expression of a set of genes in two biological samples is analyzed. Probes from the two samples are prepared and labeled with different labeling moieties. A mixture of the two labeled probes is hybridized to the array elements, and signals are examined under conditions in which the emissions from the two different labels are individually detectable. Elements on the array that are hybridized to equal numbers of probes derived from both biological samples give a distinct combined fluorescence (Shalon WO95/35505).
- Hybridization complexes are detected with a microscope equipped with an Innova 70 mixed gas 10 W laser (Coherent, Santa Clara Calif.) capable of generating spectral lines at 488 nm for excitation of Cy3 and at 632 nm for excitation of Cy5. The excitation laser light is focused on the array using a 20× microscope objective (Nikon, Melville N.Y.). The slide containing the array is placed on a computer-controlled X-Y stage on the microscope and raster-scanned past the objective with a resolution of 20 micrometers. In the differential hybridization format, the two fluorophores are sequentially excited by the laser. Emitted light is split, based on wavelength, into two photomultiplier tube detectors (PMT R1477, Hamamatsu Photonics Systems, Bridgewater N.J.) corresponding to the two fluorophores. Filters positioned between the array and the photomultiplier tubes are used to separate the signals. The emission maxima of the fluorophores used are 565 nm for Cy3 and 650 nm for Cy5. The sensitivity of the scans is calibrated using the signal intensity generated by the yeast control mRNAs added to the probe mix. A specific location on the array contains a complementary DNA sequence, allowing the intensity of the signal at that location to be correlated with a weight ratio of hybridizing species of 1:100,000.
- The output of the photomultiplier tube is digitized using a 12-bit RTI-835H analog-to-digital (A/D) conversion board (Analog Devices, Norwood Mass.) installed in an IBM-compatible PC computer. The digitized data are displayed as an image where the signal intensity is mapped using a linear 20-color transformation to a pseudocolor scale ranging from blue (low signal) to red (high signal). The data is also analyzed quantitatively. Where two different fluorophores are excited and measured simultaneously, the data are first corrected for optical crosstalk (due to overlapping emission spectra) between the fluorophores using the emission spectrum for each fluorophore. A grid is superimposed over the fluorescence signal image such that the signal from each spot is centered in each element of the grid. The fluorescence signal within each element is then integrated to obtain a numerical value corresponding to the average intensity of the signal. The software used for signal analysis is the GEMTOOLS program (Incyte Genomics).
- A transcript image was performed using the LIFESEQ GOLD database (Jun01 release, Incyte Genomics). This process allowed assessment of the relative abundance of the expressed polynucleotides in all of the cDNA libraries and reconfirmed the data submitted in U.S. Ser. No. 08/966,316, filed 7 Nov. 1997. Criteria for transcript imaging can be selected from category, number of cDNAs per library, library description, disease indication, clinical relevance of sample, and the like.
- All sequences and cDNA libraries in the LIFESEQ database have been categorized by system, organ/tissue and cell type. For each category, the number of libraries in which the sequence was expressed were counted and shown over the total number of libraries in that category. In some transcript images, all normalized or pooled libraries, which have high copy number sequences removed prior to processing, and all mixed or pooled tissues, which are considered non-specific in that they contain more than one tissue type or more than one subject's tissue, can be excluded from the analysis. Treated and untreated cell lines and/or fetal tissue data can also be disregarded or removed where clinical relevance is emphasized. Conversely, fetal tissue may be emphasized wherever elucidation of inherited disorders or differentiation of particular cells or organs from stem cells (such as nerves, heart or kidney) would be furthered by removing clinical samples from the analysis. Transcript imaging can also be used to support data from other methodologies such as microarray analysis.
- The transcript images for SEQ ID NOs:1-15 and 17 are shown below. The first column shows library name; the second column, the number of cDNAs sequenced in that library; the third column, the description of the library; the fourth column, absolute abundance of the transcript in the library; and the fifth column, percentage abundance of the transcript in the library.
-
-
Category: Nervous System (Brain) Library cDNAs Description of Tissue Abundance % Abundance HNT2AGt01 5225 teratoCA line, hNT2, t/RA + MI 1 0.0191 BRADFDIT02 5908 frontal lobe, Huntington's, 57M 1 0.0169 BRAINOM01 24452 brain, infant, 10wF, NORM, WM 1 0.0041 - In clinically-relevant brain samples, SEQ ID NO:1 is expressed four-fold higher in Huntington's disease with its associated dementia than in normal brain. Even though this GPCR is very sparsely expressed in human tissues; when SEQ ID NO:1 is used in a brain tissue-specific assay, it is diagnostic for Huntington's disease.
-
-
Category: Digestive System (Stomach) Library cDNAs Description of Tissue Abundance % Abundance STOMTUT01 2696 stomach adenoCA, 52M, m/STOMMOTO2 3 0.1113 STOMTDE01 3971 stomach, aw/esophagus adenoCA, 61M 2 0.0504 STOMNOTO2 3156 stomach, mw/adenoCA, 52M 1 0.0317 *Libraries made from normalized and pooled tissues were removed from this analysis - SEQ ID NO:2 was greater than two-fold differentially expressed in biopsied sample from the stomach of a subject diagnosed with adenocarcinoma over cytologically normal tissue from the same subject. Expression was not found in any other cytologically normal stomach tissue which included STOMNOT01, STOMNOT08, and STOMTMR02. SEQ ID NO:2, when used in a stomach-specific assay, is diagnostic for adenocarcinoma.
-
-
Category: Exocrine Glands (Breast) Library cDNAs Description of Tissue Abundance % Abundance BRSTTUT16 3724 ductal carcinoma, 43F, m/ BRSTTMT01 2 0.0537 BRSTNOR01 3107 breast, mw/BRSTTUT22, lobular CA, 59F 1 0.0322 BRSTTMT02 3240 PF changes, mw/BRSTTUT16, 46F 1 0.0309 BRSTNOTO9 3920 PF changes, mw/BRSTTUT08 adenoCA, 45F 1 0.0255 *Libraries made from normalized and pooled tissues were removed from this analysis - SEQ ID NO:3 is differentially expressed in ductal carcinoma of the breast as compared with its matched cytologically normal BRSTTMTOI. In addition, SEQ ID NO:3 was not expressed in BRSTNOT25 and BRSTNOT35, normal breast tissues removed during breast reduction surgeries, and was not as highly expressed in tissues diagnosed with any other disease states or their cytologically normal matched tissues. SEQ ID NO:3, when used in a breast-specific assay including, but not limited to, ductal lavage, is diagnostic for ductal carcinoma.
-
-
Category: Female Reproductive (Uterus) Library cDNAs Description of Tissue Abundance % Abundance UTRSTUC01 1175 uterus adenosquamousCA, F, pool 2 0.1702 UTRENOT09 2791 uterus, endometriun, aw/cystocele, 38F 1 0.0358 UTRSNOT05 6678 uterus, mw/leiomyoma, 45 F 1 0.0150 UTRSTUP05 16785 uterus serous papillary CA, F, pool 2 0.0119 UTRSTUP02 22349 uterus endometrial adenoCA, F, pool 2 0.0089 - SEQ ID NO:4 is more than five-fold differentially expressed in adenosquamous carcinoma of the uterus. It was not differentially expressed in tissues from subjects diagnosed with cervicitis (UTRCNOP01, UTRCDIE01), endometriosis (UTREDIT07, UTREDIT14), cervical tumor (UTRCTUP01), endometrial adenocarcinoma (UTRSTUP03, UTRSTUPO4, UTRSTUP07), or leiomyoma (UTRSTUE01, UTRSTUT04, UTRSTUT05, UTRSTUT07) or in cytologically normal tissues (UTRCNOP01, UTREDME05, UTREDME06, UTREDMF01, UTREDMF02, UTREDMT07, UTRENONO3, UTRENOT10, UTRETMC01, UTRETUP01, UTRMTMR02, UTRMTMT01, UTRPNOM01, UTRSNONO3, UTRSNOP01, UTRSNOR01, UTRSNOT01, UTRSNOT02, UTRSNOT06, UTRSNOT08, UTRSNOT10, UTRSNOT11; UTRSNOTI2, UTRSNOT16, UTRSNOT18, UTRSTDT01, UTRSTMC01, UTRSTME01, UTRSTMR01, and UTRSTMR02). SEQ ID NO:4, when used in a uterus-specific assay, is diagnostic for adenosquamous carcinoma.
-
-
Category: Exocrine Glands (Breast) Library cDNAs Description of Tissue Abundance % Abundance BRSTTUT13 7631 breast adenoCA, 46F, m/BRSTNOT33 58 0.7601 BRSTNOT31 3102 breast, mw/ductal adenoCA, 57F 11 0.3546 BRSTNOT32 3766 nonfibrocyctic breast disease, 46F 13 0.345 Libraries made from normalized or pooled tissues and those containing less than 3000 cDNAs were removed from this analysis. indicates data missing or illegible when filed - SEQ ID NO:5 is differentially expressed more than two-fold in adenocarcinoma of the breast when compared to expression in cytologically normal BRSTNOT31, BRSTNOT32 and matched BRSTNOT33. SEQ ID NO:5 was not differentially expressed in BRSTNOT25 and BRSTNOT35, normal breast tissues removed during breast reduction surgeries, and was not as highly expressed in tissues diagnosed with any other disease states or their cytologically normal matched tissues. SEQ ID when used in a breast-specific assay including, but not limited to, ductal lavage, is diagnostic for adenocarcinoma.
-
-
Category: Female Reproductive (Ovary) Library cDNAs Description of Tissue Abundance % Abundance OVARTUT02 3532 ovary tumor, mucinous cystademona, 51F 2 0.0566 OVARTUT07 3663 ovary, mw/follicular cysts, 28F 1 0.0273 OVARTUT13 3868 ovary, aw/leiomyoma, 47F 1 0.0259 OVARTUT07 4386 ovary tumor, adenoCA, 58F 1 0.0228 OVARNOT02 8870 ovary, aw/cardiomyopathy, 59F 1 0.0113 ‘Libraries made from normalized or pooled tissues were removed from this analysis. - SEQ ID NO:6 is differentially expressed more than two-fold in mucinous cystadenoma of the ovary when compared to expression in cytologically normal OVARNOT07, OVARNOT13, and OVARNOTO2 and in ovary tissue from a subject diagnosed with adenocarcinoma. SEQ ID NO:6 when used in a ovary-specific assay, is diagnostic for mucinous cystadenoma.
-
-
Category: Musculoskeletal System (Cartilage, Synovium) Library cDNAs Description of Tissue Abundance % Abundance CARCTXT02 3594 knee chondrocytes, M/F, t/IL-1 4 0.01113 SYNOOAT01 5674 synovium, knee, OA, 82F 5 0.0881 SYNONOT01 4046 synovium, 75M* 3 0.0741 SYNORAT03 5785 synovium, writs, rheuA, 56F 4 0.0691 SYNORAT05 3466 synovium, knee, rheuA, 62F 2 0.0577 SYNORAT04 5636 synovium, wrist, rheuA, 62F 3 0.0532 CARGDIT02 3440 cartilage, OA, M/ F 1 0.0291 CARGDIT01 7229 cartilage, OA 2 0.0277 SYNORAB01 5053 synovium, hip, rheuA, 68F 1 0.0198 *insufficient clinical data to rule out that this individual did not have some age-related arthritis. - SEQ ID NO:7 is preferentially expressed in IL-1 treated chrondrocytes cultured from knee cartilage, in cartilage and synovia from subjects with rheumatoid and osteoarthritis. It was not expressed in normal control CARGNOT01. SEQ ID NO:7, when used in a tissue-specific assay, is diagnostic for arthritis.
-
-
Category: Male Reproductive (Testes) Description % Library cDNAs of Tissue Abundance Abundance TESTTUT03 3812 testicular seminoma, 2 0.0525 45M - SEQ ID NO:8 was significantly expressed in testicular seminoma; it was not expressed in normal tissue from TESTN0001, TESTNOF01, TESTNOM01, TESTNON04, TESTNOP01, TESTNOT01, TESTNOT03, TESTNOT04, TESTNOT07, TESTNOT10, and TESTNOT11, or in embryonal carcinomas from TESTTUE02 and TESTTUT02. SEQ ID NO:7, when used in a clinically relevant, testicle-specific assay, is a diagnostic for testicular seminoma.
-
-
Category: Male Reproductive (Prostate) Library cDNAs Description of Tissue Abundance % Abundance PROSTMT05 3234 AH, mw/PROSTUT16 adenoCA, 55M 2 0.0618 PROSNOT19 3678 AH, mw/PROSTUT13 adenoCA, M 2 0.0544 PROSNOT07 3046 Ah, mw/PROSTUT05 adenoCA, 69M 1 0.0328 PROSTMT07 3104 AH, mw/ adenoCA 73M 1 0.0322 PROSDIN01 3421 AH, mw/PROSTUT10 adenoCA, 66, NORM 1 0.0292 PROSNOT28 3814 AH, mw/PROSTUT16 adenoCA, 55M 1 0.0262 PROSNOT15 4133 AH, mw/PROSTUT10 adenoCA, 66M 1 0.0242 PROSTMY01 6460 AH, mwPROSTUT16 adenoCA, 55M 1 0.0155 PROSBPT02 6583 AH, mw/adenoCA, 65M 1 0.0152 *Libraries made from subtracted or pooled tissues were removed from this analysis. - SEQ ID NO:9 was specifically expressed in prostate tissue cytologically showing adenofibromatous hyperplasia and matched with adenocarcinoma of the prostate (see PROSTUT matches above). It was not expressed in tissues from subjects diagnosed with benign prostatic hyperplasia (PROSBPS05, PROSBPT03, PROSDIP01, PROSDIP02, and PROSDIP03), or prostatic IN (PROETMP06, PROETMP07). SEQ ID NO:9, when used in a prostate-specific assay, is diagnostic for AH and may serve as an early diagnostic marker for prostatic adenocarcinoma.
-
-
Category: Urinary Tract (Bladder) Description % Library cDNAs of Tissue Abundance Abundance BLADNOT05 3774 bladder mw/ 4 0.1060 BLADTUT04 TC CA in situ, 60M BLADDIT01 3775 bladder, chronic 1 0.0265 cystitis, 73M *Libraries made from normalized tissues were removed from this analysis. - SEQ ID NO:10 showed five-fold differential expression in a cytologically normal bladder library which was matched with transitional cell carcinoma of the bladder. Expression of SEQ ID NO:10 was clearly distinct from that seen in tissue affected by chronic cystitis and was not seen in normal tissues, BLADNOR01, BLADNOT01, BLADNOT03, BLADNOT04, BLADNOT06, and BLADNOTO8 or in the tumor libraries, BLADTUE01, BLADTUT02, BLADTUT03, BLADTUT04, BLADTUT05, BLADTUT06, BLADTUTO7 and BLADTUT08, SEQ ID NO:10, when used in a bladder-specific assay, serves as an early diagnostic marker for transitional cell carcinoma of the bladder.
-
-
Category: Urinary Tract (Kidney) Library cDNAs Description of Tissue Abundance % Abundance KIDNTUT13 3771 renal cell CA, 51F 2 0.0530 KIDNTUT15 3941 renal cell CA, 65M m/ KIDNNOT19 2 0.0507 KIDNNOT19 6952 mw/KIDNTUT15 renal cell CA, 65M 2 0.0288 KIDNTUT14 3861 renal cell CA, 43M, m/ KIDNNOT20 1 0.0259 *Libraries made from normalized, subtracted, and pooled tissues were removed from this analysis. - SEQ ID NO:11 is expressed in renal cell cancers and not expressed in cytologically normal kidney libraries (KIDNNOT01, KIDNNOT02, KIDNNOT20, KIDNNOT25, KIDNNOT26, KIDNNOT31, KIDNNOT32) or in KIDPTDE01 from a subject diagnosed with interstitial nephritis. SEQ ID NO:10, when used in a kidney-specific assay, serves as a diagnostic for renal cell cancer.
-
-
Category: Exocrine Glands (Breast) Description % Library cDNAs of Tissue Abundance Abundance BRSTTUT15 6535 adenocaracinoma, 2 0.0306 46F, m/BRSTNOT17 - SEQ ID NO:12 is expressed in adenocarcinoma of the breast and not expressed in cytologically normal matched tissue. SEQ ID NO:12, when used in a breast-specific assay including, but not limited to, ductal lavage, serves as a diagnostic for adenocarcinoma of the breast.
-
-
Category: Endocrine Glands (Pituitary Gland) Library cDNAs Description of Tissue Abundance % Abundance PITUNOT06 6165 Pituitary aw/schizophrenia, COPD, 55M 808 13.1062 PITUNOT02 226 Pituitary, 15-75M/F, pool 4 1.7699 PITUNOT01 8390 Pituitary, 16-70M/F, pool 87 1.0369 PITUNOT03 2857 Pituitary aw/colon cancer, 46M 15 0.5250 PITUDIR01 5981 Pituitary aw/AD, mets adenoCA, 70F 14 0.2341 *Libraries made from normalized tissues were removed from this analysis, - SEQ ID NO:13 is highly overexpressed in the pituitary gland removed from a schizophenic subject with chronic pulmonary pulmonary disease. Such high expression levels were not seen in pooled normal tissue or in the pituitaries of subjects with cancers and Alzheimer's disease (AD). SEQ ID NO:13, when used in a tissue-specific assay, serves as a diagnostic for schizophrenia.
-
-
Category: Exocrine Glands (Breast) Library cDNAs Description of Tissue Abundance % Abundance BRSTTUT22 3774 Lobular CA/ BRSTNOT16 2 0.0530 BRSTNOT31 3102 mw/ductal adenoCA, 57F 1 0.0322 BRSTDIT01 3394 PF changes, mw/intraductal cancer, 48F 1 0.0295 BRSTNOT28 3734 PF changes, 40F 1 0.0268 BRSTNOT09 3920 PF changes, mw/BRSTTUT08 adenoCA, 45F 1 0.0255 BRSTNOT19 4019 mw/lobular CA, 67F 1 0.0249 BRSTNOT23 4056 NF breast disease, 35F 1 0.0247 BRSTNOT03 6777 PF changes, mw/BRSTTUT02 adenoCA, 54F 1 0.0148 BRSTNOT02 9077 PF changes, mw/BRSTTUT01 adenoCA, 55F 1 0.0110 BRSTNOT07 10055 PF changes, mw/intraductal adenoCA, 43F 1 0.0099 *Libraries made from normalized tissues were removed from this analysis. - SEQ ID NO:14 is differentially expressed in breast cancer, in particular, in lobular carcinoma. When used in a breast-specific assay including, but not limited to, ductal lavage, SEQ ID NO:14 serves as a diagnostic for breast cancer.
-
-
Category: Hemic Immune (Peripheral blood) Library cDNAs Description of Tissue Abundance % Abundance EOSINOT02 2356 eosinophils, asthma, M/ F 5 0.2122 MPHGNOT03 7791 macrophages, M/F 4 0.0513 EOSINOT01 2404 eosinophils, nonallergic, M/F1 0.0416 *Libraries made from treated cell lines were removed from this analysis. - SEQ ID NO:15 is 4-fold differentially expressed in peripheral blood, particularly eosinophils of asthmatics. When used in an assay of a lung sample, SEQ ID NO:15 is a diagnostic for asthma.
-
-
Category: Exocrine Gland (Breast) Description % Library* cDNAs of Tissue Abundance Abundance BRSTTUT14 3951 breast 1 0.0253 adenoCa, 62F, m/BRSTNOT14 - The transcript image confirms the information obtained in the original northern analysis (7 Nov. 1997). SEQ ID NO:17 is expressed in adenocarcinoma of the breast and not expressed in cytologically normal matched tissue, BRSTNOT14. Expression was absent from BRSTNOT25 and BRSTNOT35, normal breast tissues removed during breast reduction surgeries. When used in a breastspecific assay, including, but not limited to, ductal lavage, and compared with cancerous and normal standards, expression of SEQ ID NO:17 is diagnostic for breast adenocarcinoma.
- In assays using normal and cancerous standards and patient samples, the cDNA, an mRNA, or an antibody specifically binding the protein can serve a clinically relevant diagnostic marker for disorders associated with cell proliferation and cell signaling.
- SEQ ID NOs:1-15 and 17 were compared with all the other sequences in the LIFESEQ database (Incyte Genomics, Palo Alto Calif.) using BLAST analysis (Altschul (1993) supra); Altschul(1990) Supra). The results of the BLAST analyses were reported in THE INVENTION section above.
- Each of the Incyte clones is also used to screen northern blots. A probe is generated by EcoRl digestion of the plasmid containing the cDNA. The restriction digest is fractionated on a 1% agarose gel, a restriction fragment from about 400 to about 1400 nt in length is excised from the gel and purified on a QIAQUICK column (Qiagen). The fragment is comprised of the 5′ most region of the insert. The probe is prepared by random priming using the REDIPRIME labeling kit (APB) with REDIVUE [−32P]d-CTP (3000 Ci/mmol; APB). Unincorporated radioactivity is removed by column chromatography using a SEPHADEX G-50 NICK column (APB).
- Each commercial MTN blot (Clontech) contained approximately 2 ug of poly A+ per lane from various tissues. Otherwise, RNA was electrophoresed on a denaturing formaldehyde, 1.2% agarose gel, blotted on a nylon membrane, and fixed by UV irradiation.
- Blots are pre-hybridized in RAPID-HYB hybridization buffer (APB) for 1 hour at 65 C. Hybridizations are performed at 65 C using 0.5×10 cpm/ml probe for 1 hour. Blots are washed for 2×10 minutes in 1×SSC, 0.1% SDS at room temperature followed by 2 stringent washes at 65 C in 0.2×SSC, 0.1% SDS for 10 minutes each. Blots are wrapped in SARAN WRAP plastic film (Dow Chemical, Midland Mich.) and autoradiographed at −70 C using 2 intensifying screens and HYPERFILM-MP (APB).
- The northern analysis for SEQ ID NO:17,
Incyte clone 2547002, performed Wednesday, 5 Nov. 1997 showed expression in the following libraries of the LIFESEQ database (Incyte Genomics). -
Library Description HEARNOT06 heart, 44M HEAPNOT01 heart, coronary artery, plaque, pool SMCANOT01 smooth muscle cell line, aorta, M BRSTTUT14 breast tumor, adenocarcinoma, 62 F, mw/BRSTNOT14 UTRSNOT16 uterus, endometrium, 48F UTRSNOT11 uterus, myometrium, 43F UTRSNOT02 uterus, 34F LPARNOT02 parotid gland, 70M - When used in a breast sample specific assay and compared with cancerous and normal standards, SEQ ID NO:17 is diagnostic for breast adenocarcinoma (bold above)
- Molecules complementary to the cDNA, from about 5 (PNA) to about 5000 bp (complement of a cDNA insert), are used to detect or inhibit gene expression. Detection is described in Example VII. To inhibit transcription by preventing promoter binding, the complementary molecule is designed to bind to the most unique 5′ sequence and includes nucleotides of the 5′ UTR upstream of the initiation codon of the open reading frame. Complementary molecules include genomic sequences (such as enhancers or introns) and are used in “triple helix” base pairing to compromise the ability of the double helix to open sufficiently for the binding of polymerases, transcription factors, or regulatory molecules. To inhibit translation, a complementary molecule is designed to prevent ribosomal binding to the mRNA encoding the protein.
- Complementary molecules are placed in expression vectors and used to transform a cell line to test efficacy; into an organ, tumor, synovial cavity, or the vascular system for transient or short term therapy; or into a stem cell, zygote, or other reproducing lineage for long term or stable gene therapy. Transient expression lasts for a month or more with a non-replicating vector and for three months or more if elements for inducing vector replication are used in the transformation/expression system.
- Stable transformation of dividing cells with a vector encoding the complementary molecule produces a transgenic cell line, tissue, or organism (U.S. Pat. No. 4,736,866). Those cells that assimilate and replicate sufficient quantities of the vector to allow stable integration also produce enough complementary molecules to compromise or entirely eliminate activity of the cDNA encoding the protein.
- Expression and purification of the protein are achieved using either a mammalian or an insect cell expression system. The pUB61V5-His vector system (Invitrogen, Carlsbad Calif.) is used to express signal peptide-containing proteins in CHO cells. The vector contains the selectable bsd gene, multiple cloning sites, the promoter/enhancer sequence from the human ubiquitin C gene, a C-terminal V5 epitope for antibody detection with anti-V5 antibodies, and a C-terminal polyhistidine (6×His) sequence for rapid purification on PROBOND resin (Invitrogen). Transformed cells are selected on media containing blasticidin.
- Spodoptera frueiperda (Sf9) insect cells are infected with recombinant Autographica californica nuclear polyhedrosis virus (baculovirus). The polyhedrin gene is replaced with the cDNA by homologous recombination and the polyhedrin promoter drives cDNA transcription. The protein is synthesized as a fusion protein with 6×his which enables purification as described above. Purified protein is used in the following activity and to make antibodies.
- A signal peptide-containing protein is purified using polyacrylamide gel electrophoresis and used to immunize mice or rabbits. Antibodies are produced using the protocols well known in the art and summarized below. Alternatively, the amino acid sequence of signal peptide-containing proteins is analyzed using LASERGENE software (DNASTAR) to determine regions of high antigenicity. An antigenic epitope, usually found near the C-terminus or in a hydrophilic region is selected, synthesized, and used to raise antibodies. Typically, epitopes of about 15 residues in length are produced using an 431A peptide synthesizer (ABI) using Fmoc-chemistry and coupled to KLH (Sigma-Aldrich) by reaction with N-maleimidobenzoyl-N-hydroxysuccinimide ester to increase antigenicity.
- Rabbits are immunized with the epitope-KLH complex in complete Freund's adjuvant. Immunizations are repeated at intervals thereafter in incomplete Freund's adjuvant. After a minimum of seven weeks for mouse or twelve weeks for rabbit, antisera are drawn and tested for antipeptide activity.
- Testing involves binding the peptide to plastic, blocking with 1% bovine serum albumin, reacting with rabbit antisera, washing, and reacting with radio-iodinated goat anti-rabbit IgG. Methods well known in the art are used to determine antibody titer and the amount of complex formation.
- Naturally occurring or recombinant protein is purified by immunoaffinity chromatography using antibodies which specifically bind the protein. An immunoaffinity column is constructed by covalently coupling the antibody to CNBr-activated SEPHAROSE resin (APB). Media containing the protein is passed over the immunoaffinity column, and the column is washed using high ionic strength buffers in the presence of detergent to allow preferential absorbance of the protein. After coupling, the protein is eluted from the column using a buffer of pH 2-3 or a high concentration of urea or thiocyanate ion to disrupt antibody/protein binding, and the protein is collected.
- The cDNA, or fragments thereof, or the protein, or portions thereof, are labeled with 3 P-dCTP, Cy3-dCTP, or Cy5-dCTP (APB), or with BIODIPY or FITC (Molecular Probes, Eugene Oreg.), respectively. Libraries of candidate molecules or compounds previously arranged on a substrate are incubated in the presence of labeled cDNA or protein. After incubation under conditions for either a nucleic acid or amino acid sequence, the substrate is washed, and any position on the substrate retaining label, which indicates specific binding or complex formation, is assayed, and the ligand is identified. Data obtained using different concentrations of the nucleic acid or protein are used to calculate affinity between the labeled nucleic acid or protein and the bound molecule.
- A yeast two-hybrid system, MATCHMAKER LexA Two-Hybrid system (Clontech Laboratories, Palo Alto Calif.), is used to screen for peptides that bind the protein of the invention. A cDNA encoding the protein is inserted into the multiple cloning site of a pLexA vector, ligated, and transformed into E. coli cDNA, prepared from mRNA, is inserted into the multiple cloning site of a pB42AD vector, ligated, and transformed into E. coli construct a cDNA library. The pLexA plasmid and pB42AD-cDNA library constructs are isolated from E. coli and used in a 2:1 ratio to co-transform competent yeast EGY48[p8oplacZ] cells using a polyethylene glycol/lithium acetate protocol. Transformed yeast cells are plated on synthetic dropout (SD) media lacking histidine (-His), tryptophan (-Trp), and uracil (-Ura), and incubated at 30 C until the colonies have grown up and are counted. The colonies are pooled in a minimal volume of 1×TE (pH 7.5), replaced on SD/-His/-Leu/-Trp/-Ura media supplemented with 2% galactose (Gal), 1% raffinose (Raf), and 80 mg/ml 5-bromo-4-chloro-3-indolyl β-d-galactopyranoside (X-Gal), and subsequently examined for growth of blue colonies. Interaction between expressed protein and cDNA fusion proteins activates expression of a LEU2 reporter gene in EGY48 and produces colony growth on media lacking leucine (-Leu). Interaction also activates expression of (β-galactosidase from the p8op-lacZ reporter construct that produces blue color in colonies grown on X-Gal.
- Positive interactions between expressed protein and cDNA fusion proteins are verified by isolating individual positive colonies and growing them in SD/-Trpf-Ura liquid medium for 1 to 2 days at 30 C. A sample of the culture is plated on SD/-Trp/-Ura media and incubated at 30 C until colonies appear. The sample is replica-plated on SD/-Trp/-Ura and SD/-His/-Trp/-Ura plates. Colonies that grow on SD containing histidine but not on media lacking histidine have lost the pLexA plasmid. Histidinerequiring colonies are grown on SD/GallRaf/X-Gal/-Trp/-Ura, and white colonies are isolated and propagated. The pB42AD-cDNA plasmid, which contains a cDNA encoding a protein that physically interacts with the protein, is isolated from the yeast cells and characterized.
- SP can be expressed in a mammalian cell line such as DLD-1 or HCT116 (ATCC; Manassas Va.) by transforming the cells with a eukaryotic expression vector encoding SF. Other eukaryotic expression vectors, such as those mentioned in EXAMPLE XI above, are commercially available, and the techniques to introduce them into cells are well known to those skilled in the art. The effect of SP on cell morphology can be visualized by microscopy; the effect on cell growth can be determined by measuring cell doubling-time; and the effect on tumorigenicity can be assessed by the ability of transformed cells to grow in a soft agar growth assay (Groden et al. (1995) Cancer Res. 55:1531-1539).
- Receptor SPs such as those encoded by SEQ ID NOs: 17, 15, 12, 6, and I can be expressed in heterologous expression systems and their biological activity tested utilizing the purinergic receptor system (P2u) as published by Erb et al. (1993; Proc Natl Acad Sci 90:10449-53). Because cultured K562 human leukemia cells lack P2U receptors, they can be transfected with expression vectors containing either normal or chimeric P2u and loaded with fura-a, fluorescent probe for Ca++. Activation of properly assembled and functional extracellular SP-transmembrane/intracellular P2U receptors with extracellular UTP or ATP mobilizes intracellular Ca++ which reacts with fura-a and is measured spectrofluorometrically. Bathing the transfected K562 cells in microwells containing appropriate ligands will trigger binding and fluorescent activity identifying effectors of SP. The P2u system is also useful for identifying antagonists or inhibitors which block binding and prevent such fluorescent reactions.
- All patents and publications mentioned in the specification are incorporated by reference herein. Various modifications and variations of the described method and system of the invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described in connection with specific preferred embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention that are obvious to those skilled in the field of molecular biology or related fields are intended to be within the scope of the following claims.
Claims (16)
1.-20. (canceled)
21. A method of diagnosing the presence of cancer in a subject by detecting the expression of a signal peptide-containing protein, wherein the method comprises:
(a) providing a biological sample from the subject;
(b) contacting the biological sample with an antibody or antibody fragment which specifically binds to a signal peptide-containing protein present in the biological sample, wherein the antibody or antibody fragment specifically binds to a polypeptide selected from the group consisting of:
(i) a polypeptide comprising the amino acid sequence of SEQ ID NO:16, and
(ii) a polypeptide having an amino acid sequence at least about 90% identical to the amino acid sequence of SEQ ID NO:16,
(c) detecting the presence of bound antibody in the biological sample;
(d) comparing the presence of bound antibody in the biological sample with the presence of bound antibody in a control sample;
(e) wherein a higher presence in the biological sample as compared to the control sample is correlated with the presence of cancer.
22. The method of claim 21 , wherein the subject is a human.
23. The method of claim 21 , wherein the biological sample comprises body fluids, tissues, or extracts of such tissues.
24. The method of claim 21 , wherein the antibody or antibody fragment specifically binds to a polypeptide comprising the amino acid sequence of SEQ ID NO:16.
25. The method of claim 21 , wherein the antibody or antibody fragment specifically binds to a polypeptide consisting of the amino acid sequence of SEQ ID NO:16.
26. The method of claim 21 , wherein the antibody or antibody fragment is a monoclonal antibody.
27. The method of claim 21 , wherein the antibody or antibody fragment is a polyclonal antibody.
28. The method of claim 21 , wherein the antibody or antibody fragment is a Fab fragment.
29. The method of claim 21 , wherein the antibody or antibody fragment is a F(ab′)2 fragment.
30. The method of claim 21 , wherein the antibody or antibody fragment is a single-chain antibody.
31. The method of claim 21 , wherein the antibody or antibody fragment is labeled.
32. The method of claim 21 , wherein the antibody or antibody fragment is labeled to a reporter molecule.
33. The method of claim 21 , wherein the cancer is selected from the group consisting of adenocarcinomas, prostate cancer, breast cancer, renal cell cancer, stomach cancer, testicular seminoma, mucinous cystadenoma of the ovary, transitional cell carcinoma of the bladder, uterine adenosquamous carcinoma
34. The method of claim 33 , wherein the cancer is breast cancer.
35. The method of claim 34 , wherein the breast cancer is ductal or lobular.
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US15/178,595 US20160282350A1 (en) | 1997-11-07 | 2016-06-10 | Methods of diagnosing cancer |
Applications Claiming Priority (7)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US08/966,316 US5932445A (en) | 1997-11-07 | 1997-11-07 | Signal peptide-containing proteins |
US27111099A | 1999-03-17 | 1999-03-17 | |
US09/968,433 US7321023B2 (en) | 1997-11-07 | 2001-10-01 | SP16 protein |
US11/979,577 US20080181895A1 (en) | 1997-11-07 | 2007-11-06 | SP16 protein |
US13/763,102 US20130243781A1 (en) | 1997-11-07 | 2013-02-08 | Signal peptide-containing proteins |
US14/157,288 US20140155288A1 (en) | 1997-11-07 | 2014-01-16 | Methods for diagnosing cancer |
US15/178,595 US20160282350A1 (en) | 1997-11-07 | 2016-06-10 | Methods of diagnosing cancer |
Related Parent Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
US14/157,288 Division US20140155288A1 (en) | 1997-11-07 | 2014-01-16 | Methods for diagnosing cancer |
Publications (1)
Publication Number | Publication Date |
---|---|
US20160282350A1 true US20160282350A1 (en) | 2016-09-29 |
Family
ID=46280105
Family Applications (5)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
US09/968,433 Expired - Fee Related US7321023B2 (en) | 1997-11-07 | 2001-10-01 | SP16 protein |
US11/979,577 Abandoned US20080181895A1 (en) | 1997-11-07 | 2007-11-06 | SP16 protein |
US13/763,102 Abandoned US20130243781A1 (en) | 1997-11-07 | 2013-02-08 | Signal peptide-containing proteins |
US14/157,288 Abandoned US20140155288A1 (en) | 1997-11-07 | 2014-01-16 | Methods for diagnosing cancer |
US15/178,595 Abandoned US20160282350A1 (en) | 1997-11-07 | 2016-06-10 | Methods of diagnosing cancer |
Family Applications Before (4)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
US09/968,433 Expired - Fee Related US7321023B2 (en) | 1997-11-07 | 2001-10-01 | SP16 protein |
US11/979,577 Abandoned US20080181895A1 (en) | 1997-11-07 | 2007-11-06 | SP16 protein |
US13/763,102 Abandoned US20130243781A1 (en) | 1997-11-07 | 2013-02-08 | Signal peptide-containing proteins |
US14/157,288 Abandoned US20140155288A1 (en) | 1997-11-07 | 2014-01-16 | Methods for diagnosing cancer |
Country Status (1)
Country | Link |
---|---|
US (5) | US7321023B2 (en) |
Families Citing this family (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JP5087200B2 (en) * | 2000-07-07 | 2012-11-28 | モサイド・テクノロジーズ・インコーポレーテッド | Method and apparatus for synchronizing row and column access operations |
US20060194265A1 (en) * | 2001-10-23 | 2006-08-31 | Morris David W | Novel therapeutic targets in cancer |
US7885034B2 (en) * | 2006-09-13 | 2011-02-08 | International Business Machines Corporation | Contactless method for power and communications in a tape library |
BRPI1005159A2 (en) * | 2009-01-16 | 2018-04-03 | Teva Pharma | "Method of treating or preventing neutropenia in a human patient, method of treating or preventing leukopenia in a human patient, and method of reducing the incidence of infection" |
Citations (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US6838258B2 (en) * | 1999-10-05 | 2005-01-04 | Agensys, Inc. | G protein-coupled receptor up-regulated in prostate cancer and uses thereof |
Family Cites Families (25)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US4736866B1 (en) * | 1984-06-22 | 1988-04-12 | Transgenic non-human mammals | |
US5175383A (en) * | 1989-02-17 | 1992-12-29 | President And Fellows Of Harvard College | Animal model for benign prostatic disease |
US5194596A (en) * | 1989-07-27 | 1993-03-16 | California Biotechnology Inc. | Production of vascular endothelial cell growth factor |
US5350836A (en) * | 1989-10-12 | 1994-09-27 | Ohio University | Growth hormone antagonists |
US5578444A (en) * | 1991-06-27 | 1996-11-26 | Genelabs Technologies, Inc. | Sequence-directed DNA-binding molecules compositions and methods |
US5474796A (en) * | 1991-09-04 | 1995-12-12 | Protogene Laboratories, Inc. | Method and apparatus for conducting an array of chemical reactions on a support surface |
US5605662A (en) * | 1993-11-01 | 1997-02-25 | Nanogen, Inc. | Active programmable electronic devices for molecular biological analysis and diagnostics |
US5660827A (en) * | 1992-03-05 | 1997-08-26 | Board Of Regents, The University Of Texas System | Antibodies that bind to endoglin |
US5605793A (en) * | 1994-02-17 | 1997-02-25 | Affymax Technologies N.V. | Methods for in vitro recombination |
US5807522A (en) * | 1994-06-17 | 1998-09-15 | The Board Of Trustees Of The Leland Stanford Junior University | Methods for fabricating microarrays of biological samples |
US5767337A (en) * | 1995-07-31 | 1998-06-16 | Duke University | Creation of human apolipoprotein E isoform specific transgenic mice in apolipoprotein deficient "knockout" mice |
US6023659A (en) * | 1996-10-10 | 2000-02-08 | Incyte Pharmaceuticals, Inc. | Database system employing protein function hierarchies for viewing biomolecular sequence data |
US5953727A (en) * | 1996-10-10 | 1999-09-14 | Incyte Pharmaceuticals, Inc. | Project-based full-length biomolecular sequence database |
US6140060A (en) * | 1996-12-12 | 2000-10-31 | Chun; Jerold J. M. | Cloned lysophosphatidic acid receptors |
US6553317B1 (en) * | 1997-03-05 | 2003-04-22 | Incyte Pharmaceuticals, Inc. | Relational database and system for storing information relating to biomolecular sequences and reagents |
US5876946A (en) * | 1997-06-03 | 1999-03-02 | Pharmacopeia, Inc. | High-throughput assay |
US6737252B2 (en) * | 1997-07-25 | 2004-05-18 | Schering Corporation | 7 transmembrane receptor family member BLRX |
EP0899332A3 (en) * | 1997-08-15 | 2000-07-05 | Smithkline Beecham Corporation | The G-protein coupled receptor HFIAO41 |
US5994097A (en) * | 1997-08-28 | 1999-11-30 | Incyte Pharmaceuticals, Inc. | Polynucleotide encoding human G-protein coupled receptor |
US5932445A (en) * | 1997-11-07 | 1999-08-03 | Incyte Pharmaceuticals, Inc. | Signal peptide-containing proteins |
US5932451A (en) * | 1997-11-19 | 1999-08-03 | Incyte Pharmaceuticals, Inc. | Method for unbiased mRNA amplification |
US6057126A (en) * | 1997-12-24 | 2000-05-02 | Allelix Biopharmaceuticals, Inc. | Mammalian EDG-5 receptor homologs |
US6620615B1 (en) * | 1999-04-23 | 2003-09-16 | Curagen Corporation | G-protein coupled receptor—encoding nucleic acids |
US6835547B1 (en) * | 1999-10-12 | 2004-12-28 | Chemocentryx, Inc. | Methods for identifying modulators of CCX CKR activity |
US6998239B1 (en) * | 1999-10-12 | 2006-02-14 | Chemocentryx, Inc. | Method for identifying a modulator of the binding of CCX CKR polypeptide to a chemokine |
-
2001
- 2001-10-01 US US09/968,433 patent/US7321023B2/en not_active Expired - Fee Related
-
2007
- 2007-11-06 US US11/979,577 patent/US20080181895A1/en not_active Abandoned
-
2013
- 2013-02-08 US US13/763,102 patent/US20130243781A1/en not_active Abandoned
-
2014
- 2014-01-16 US US14/157,288 patent/US20140155288A1/en not_active Abandoned
-
2016
- 2016-06-10 US US15/178,595 patent/US20160282350A1/en not_active Abandoned
Patent Citations (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US6838258B2 (en) * | 1999-10-05 | 2005-01-04 | Agensys, Inc. | G protein-coupled receptor up-regulated in prostate cancer and uses thereof |
Also Published As
Publication number | Publication date |
---|---|
US20130243781A1 (en) | 2013-09-19 |
US20030073162A1 (en) | 2003-04-17 |
US7321023B2 (en) | 2008-01-22 |
US20140155288A1 (en) | 2014-06-05 |
US20080181895A1 (en) | 2008-07-31 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
US6524799B1 (en) | DNA encoding sparc-related proteins | |
US20020187472A1 (en) | Steap-related protein | |
US20020102569A1 (en) | Diagnostic marker for cancers | |
US20160282350A1 (en) | Methods of diagnosing cancer | |
US6566066B1 (en) | Aquaporin-8 variant | |
US6590089B1 (en) | RVP-1 variant differentially expressed in Crohn's disease | |
US20030166300A1 (en) | Growth-related inflammatory and immune response protein | |
US6444430B1 (en) | Ndr2-related proteins | |
US20030211515A1 (en) | Novel compounds | |
US6783955B2 (en) | Polynucleotides encoding human presenilin variant | |
US6632617B1 (en) | Tumor-associated antigen | |
US20020123054A1 (en) | Human angiopoietin | |
US20020025555A1 (en) | GPCR diagnostic for brain cancer | |
US20030165989A1 (en) | GPCR diagnostic for brain cancer | |
US20030175787A1 (en) | Vesicle membrane proteins | |
US20020055108A1 (en) | Human Sec6 vesicle transport protein | |
US20020127636A1 (en) | Ankyrin repeat domain 2 protein variant | |
US20020132238A1 (en) | Progesterone receptor complex p23-like protein | |
EP1319021A2 (en) | Atp-binding cassette protein | |
US20030166041A1 (en) | TIMM8b-related protein | |
US20030166501A1 (en) | Mucin-related tumor marker | |
US20020137038A1 (en) | Intestinal proteins | |
US20020146767A1 (en) | Human EMR1-like G protein-coupled receptor | |
US20030113317A1 (en) | Molecules associated with apoptosis | |
WO2002055706A2 (en) | Asip-related proteins |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
STCB | Information on status: application discontinuation |
Free format text: ABANDONED -- FAILURE TO RESPOND TO AN OFFICE ACTION |