JPH0559710B2 - - Google Patents
Info
- Publication number
- JPH0559710B2 JPH0559710B2 JP60037168A JP3716885A JPH0559710B2 JP H0559710 B2 JPH0559710 B2 JP H0559710B2 JP 60037168 A JP60037168 A JP 60037168A JP 3716885 A JP3716885 A JP 3716885A JP H0559710 B2 JPH0559710 B2 JP H0559710B2
- Authority
- JP
- Japan
- Prior art keywords
- dna
- plasmid
- gene
- strain
- threonine
- 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.)
- Expired - Lifetime
Links
- 239000013612 plasmid Substances 0.000 claims description 61
- AYFVYJQAPQTCCC-GBXIJSLDSA-N L-threonine Chemical compound C[C@@H](O)[C@H](N)C(O)=O AYFVYJQAPQTCCC-GBXIJSLDSA-N 0.000 claims description 39
- 239000004473 Threonine Substances 0.000 claims description 34
- 241000186146 Brevibacterium Species 0.000 claims description 30
- AYFVYJQAPQTCCC-UHFFFAOYSA-N Threonine Natural products CC(O)C(N)C(O)=O AYFVYJQAPQTCCC-UHFFFAOYSA-N 0.000 claims description 29
- AGPKZVBTJJNPAG-WHFBIAKZSA-N L-isoleucine Chemical compound CC[C@H](C)[C@H](N)C(O)=O AGPKZVBTJJNPAG-WHFBIAKZSA-N 0.000 claims description 26
- 229960000310 isoleucine Drugs 0.000 claims description 26
- AGPKZVBTJJNPAG-UHFFFAOYSA-N isoleucine Natural products CCC(C)C(N)C(O)=O AGPKZVBTJJNPAG-UHFFFAOYSA-N 0.000 claims description 20
- 241000894006 Bacteria Species 0.000 claims description 16
- 108090000623 proteins and genes Proteins 0.000 claims description 16
- 241000186031 Corynebacteriaceae Species 0.000 claims description 14
- 238000012258 culturing Methods 0.000 claims description 11
- 238000004519 manufacturing process Methods 0.000 claims description 11
- 108010064711 Homoserine dehydrogenase Proteins 0.000 claims description 9
- 102100033451 Thyroid hormone receptor beta Human genes 0.000 claims description 9
- 108010071598 homoserine kinase Proteins 0.000 claims description 9
- 108020004414 DNA Proteins 0.000 description 55
- 229960002898 threonine Drugs 0.000 description 33
- 238000000034 method Methods 0.000 description 28
- 239000002609 medium Substances 0.000 description 26
- 101150055682 HK gene Proteins 0.000 description 23
- 210000004027 cell Anatomy 0.000 description 22
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 21
- 238000006243 chemical reaction Methods 0.000 description 16
- 239000013611 chromosomal DNA Substances 0.000 description 15
- 239000012634 fragment Substances 0.000 description 14
- ISWSIDIOOBJBQZ-UHFFFAOYSA-N Phenol Chemical compound OC1=CC=CC=C1 ISWSIDIOOBJBQZ-UHFFFAOYSA-N 0.000 description 12
- 210000001938 protoplast Anatomy 0.000 description 12
- IEDVJHCEMCRBQM-UHFFFAOYSA-N trimethoprim Chemical compound COC1=C(OC)C(OC)=CC(CC=2C(=NC(N)=NC=2)N)=C1 IEDVJHCEMCRBQM-UHFFFAOYSA-N 0.000 description 12
- 229960001082 trimethoprim Drugs 0.000 description 12
- 239000013598 vector Substances 0.000 description 12
- 101150113725 hd gene Proteins 0.000 description 11
- 239000000243 solution Substances 0.000 description 11
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 10
- 230000001580 bacterial effect Effects 0.000 description 10
- 108091008146 restriction endonucleases Proteins 0.000 description 10
- 241000186216 Corynebacterium Species 0.000 description 9
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 8
- UKAUYVFTDYCKQA-VKHMYHEASA-N L-homoserine Chemical compound OC(=O)[C@@H](N)CCO UKAUYVFTDYCKQA-VKHMYHEASA-N 0.000 description 8
- 238000000246 agarose gel electrophoresis Methods 0.000 description 8
- 229960005091 chloramphenicol Drugs 0.000 description 8
- 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 8
- 239000008103 glucose Substances 0.000 description 8
- 102000016943 Muramidase Human genes 0.000 description 7
- 108010014251 Muramidase Proteins 0.000 description 7
- 108010062010 N-Acetylmuramoyl-L-alanine Amidase Proteins 0.000 description 7
- 230000003698 anagen phase Effects 0.000 description 7
- 238000010438 heat treatment Methods 0.000 description 7
- 229960000274 lysozyme Drugs 0.000 description 7
- 239000004325 lysozyme Substances 0.000 description 7
- 235000010335 lysozyme Nutrition 0.000 description 7
- UKAUYVFTDYCKQA-UHFFFAOYSA-N -2-Amino-4-hydroxybutanoic acid Natural products OC(=O)C(N)CCO UKAUYVFTDYCKQA-UHFFFAOYSA-N 0.000 description 6
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 6
- 229930182844 L-isoleucine Natural products 0.000 description 6
- 201000004283 Shwachman-Diamond syndrome Diseases 0.000 description 6
- 239000011780 sodium chloride Substances 0.000 description 5
- 238000011426 transformation method Methods 0.000 description 5
- YBJHBAHKTGYVGT-ZKWXMUAHSA-N (+)-Biotin Chemical compound N1C(=O)N[C@@H]2[C@H](CCCCC(=O)O)SC[C@@H]21 YBJHBAHKTGYVGT-ZKWXMUAHSA-N 0.000 description 4
- 229920001817 Agar Polymers 0.000 description 4
- 241000186226 Corynebacterium glutamicum Species 0.000 description 4
- 102000012410 DNA Ligases Human genes 0.000 description 4
- 108010061982 DNA Ligases Proteins 0.000 description 4
- WHUUTDBJXJRKMK-VKHMYHEASA-N L-glutamic acid Chemical compound OC(=O)[C@@H](N)CCC(O)=O WHUUTDBJXJRKMK-VKHMYHEASA-N 0.000 description 4
- ROHFNLRQFUQHCH-YFKPBYRVSA-N L-leucine Chemical compound CC(C)C[C@H](N)C(O)=O ROHFNLRQFUQHCH-YFKPBYRVSA-N 0.000 description 4
- KDXKERNSBIXSRK-YFKPBYRVSA-N L-lysine Chemical compound NCCCC[C@H](N)C(O)=O KDXKERNSBIXSRK-YFKPBYRVSA-N 0.000 description 4
- ROHFNLRQFUQHCH-UHFFFAOYSA-N Leucine Natural products CC(C)CC(N)C(O)=O ROHFNLRQFUQHCH-UHFFFAOYSA-N 0.000 description 4
- 239000001888 Peptone Substances 0.000 description 4
- 108010080698 Peptones Proteins 0.000 description 4
- XSQUKJJJFZCRTK-UHFFFAOYSA-N Urea Chemical compound NC(N)=O XSQUKJJJFZCRTK-UHFFFAOYSA-N 0.000 description 4
- 239000008272 agar Substances 0.000 description 4
- 229940041514 candida albicans extract Drugs 0.000 description 4
- 238000010367 cloning Methods 0.000 description 4
- 238000005520 cutting process Methods 0.000 description 4
- -1 etc. Substances 0.000 description 4
- 230000012010 growth Effects 0.000 description 4
- 239000006166 lysate Substances 0.000 description 4
- 235000019319 peptone Nutrition 0.000 description 4
- 239000013600 plasmid vector Substances 0.000 description 4
- 238000002360 preparation method Methods 0.000 description 4
- 230000008929 regeneration Effects 0.000 description 4
- 238000011069 regeneration method Methods 0.000 description 4
- 239000006228 supernatant Substances 0.000 description 4
- 238000012546 transfer Methods 0.000 description 4
- 239000012138 yeast extract Substances 0.000 description 4
- 244000063299 Bacillus subtilis Species 0.000 description 3
- 235000014469 Bacillus subtilis Nutrition 0.000 description 3
- 102000053602 DNA Human genes 0.000 description 3
- 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 3
- 239000004472 Lysine Substances 0.000 description 3
- 239000002202 Polyethylene glycol Substances 0.000 description 3
- 229940024606 amino acid Drugs 0.000 description 3
- 235000001014 amino acid Nutrition 0.000 description 3
- 150000001413 amino acids Chemical class 0.000 description 3
- 230000002759 chromosomal effect Effects 0.000 description 3
- 238000003776 cleavage reaction Methods 0.000 description 3
- 230000002950 deficient Effects 0.000 description 3
- VHJLVAABSRFDPM-QWWZWVQMSA-N dithiothreitol Chemical compound SC[C@@H](O)[C@H](O)CS VHJLVAABSRFDPM-QWWZWVQMSA-N 0.000 description 3
- 229960002989 glutamic acid Drugs 0.000 description 3
- 238000004128 high performance liquid chromatography Methods 0.000 description 3
- 230000002934 lysing effect Effects 0.000 description 3
- 244000005700 microbiome Species 0.000 description 3
- 229910052757 nitrogen Inorganic materials 0.000 description 3
- 229920001223 polyethylene glycol Polymers 0.000 description 3
- 239000013587 production medium Substances 0.000 description 3
- 230000007017 scission Effects 0.000 description 3
- 238000005199 ultracentrifugation Methods 0.000 description 3
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 description 2
- LGVJIYCMHMKTPB-UHFFFAOYSA-N 3-hydroxynorvaline Chemical compound CCC(O)C(N)C(O)=O LGVJIYCMHMKTPB-UHFFFAOYSA-N 0.000 description 2
- QGZKDVFQNNGYKY-UHFFFAOYSA-N Ammonia Chemical compound N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 description 2
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 description 2
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 2
- 102000004190 Enzymes Human genes 0.000 description 2
- 108090000790 Enzymes Proteins 0.000 description 2
- 241000588724 Escherichia coli Species 0.000 description 2
- WHUUTDBJXJRKMK-UHFFFAOYSA-N Glutamic acid Natural products OC(=O)C(N)CCC(O)=O WHUUTDBJXJRKMK-UHFFFAOYSA-N 0.000 description 2
- KDXKERNSBIXSRK-UHFFFAOYSA-N Lysine Natural products NCCCCC(N)C(O)=O KDXKERNSBIXSRK-UHFFFAOYSA-N 0.000 description 2
- WAEMQWOKJMHJLA-UHFFFAOYSA-N Manganese(2+) Chemical compound [Mn+2] WAEMQWOKJMHJLA-UHFFFAOYSA-N 0.000 description 2
- 108091034117 Oligonucleotide Proteins 0.000 description 2
- JGSARLDLIJGVTE-MBNYWOFBSA-N Penicillin G Chemical compound N([C@H]1[C@H]2SC([C@@H](N2C1=O)C(O)=O)(C)C)C(=O)CC1=CC=CC=C1 JGSARLDLIJGVTE-MBNYWOFBSA-N 0.000 description 2
- 239000004372 Polyvinyl alcohol Substances 0.000 description 2
- 108020004511 Recombinant DNA Proteins 0.000 description 2
- 239000002253 acid Substances 0.000 description 2
- DPKHZNPWBDQZCN-UHFFFAOYSA-N acridine orange free base Chemical compound C1=CC(N(C)C)=CC2=NC3=CC(N(C)C)=CC=C3C=C21 DPKHZNPWBDQZCN-UHFFFAOYSA-N 0.000 description 2
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 description 2
- 229910052921 ammonium sulfate Inorganic materials 0.000 description 2
- 235000011130 ammonium sulphate Nutrition 0.000 description 2
- 238000004458 analytical method Methods 0.000 description 2
- DZBUGLKDJFMEHC-UHFFFAOYSA-N benzoquinolinylidene Natural products C1=CC=CC2=CC3=CC=CC=C3N=C21 DZBUGLKDJFMEHC-UHFFFAOYSA-N 0.000 description 2
- 229960002685 biotin Drugs 0.000 description 2
- 235000020958 biotin Nutrition 0.000 description 2
- 239000011616 biotin Substances 0.000 description 2
- 239000000872 buffer Substances 0.000 description 2
- 239000001110 calcium chloride Substances 0.000 description 2
- 229910001628 calcium chloride Inorganic materials 0.000 description 2
- 235000011148 calcium chloride Nutrition 0.000 description 2
- 239000004202 carbamide Substances 0.000 description 2
- 229910052799 carbon Inorganic materials 0.000 description 2
- 238000005119 centrifugation Methods 0.000 description 2
- 238000007796 conventional method Methods 0.000 description 2
- 229940088598 enzyme Drugs 0.000 description 2
- 238000000855 fermentation Methods 0.000 description 2
- 230000004151 fermentation Effects 0.000 description 2
- 235000013922 glutamic acid Nutrition 0.000 description 2
- 239000004220 glutamic acid Substances 0.000 description 2
- 230000000977 initiatory effect Effects 0.000 description 2
- 238000003780 insertion Methods 0.000 description 2
- 230000037431 insertion Effects 0.000 description 2
- XEEYBQQBJWHFJM-UHFFFAOYSA-N iron Substances [Fe] XEEYBQQBJWHFJM-UHFFFAOYSA-N 0.000 description 2
- 229910052742 iron Inorganic materials 0.000 description 2
- 239000007788 liquid Substances 0.000 description 2
- 235000018977 lysine Nutrition 0.000 description 2
- WRUGWIBCXHJTDG-UHFFFAOYSA-L magnesium sulfate heptahydrate Chemical compound O.O.O.O.O.O.O.[Mg+2].[O-]S([O-])(=O)=O WRUGWIBCXHJTDG-UHFFFAOYSA-L 0.000 description 2
- 229940061634 magnesium sulfate heptahydrate Drugs 0.000 description 2
- 229910001437 manganese ion Inorganic materials 0.000 description 2
- 229910000402 monopotassium phosphate Inorganic materials 0.000 description 2
- 235000019796 monopotassium phosphate Nutrition 0.000 description 2
- PJNZPQUBCPKICU-UHFFFAOYSA-N phosphoric acid;potassium Chemical compound [K].OP(O)(O)=O PJNZPQUBCPKICU-UHFFFAOYSA-N 0.000 description 2
- 229920002451 polyvinyl alcohol Polymers 0.000 description 2
- 230000010076 replication Effects 0.000 description 2
- GHSJKUNUIHUPDF-UHFFFAOYSA-N s-(2-aminoethyl)-l-cysteine Chemical compound NCCSCC(N)C(O)=O GHSJKUNUIHUPDF-UHFFFAOYSA-N 0.000 description 2
- 229960000344 thiamine hydrochloride Drugs 0.000 description 2
- 235000019190 thiamine hydrochloride Nutrition 0.000 description 2
- 239000011747 thiamine hydrochloride Substances 0.000 description 2
- DPJRMOMPQZCRJU-UHFFFAOYSA-M thiamine hydrochloride Chemical compound Cl.[Cl-].CC1=C(CCO)SC=[N+]1CC1=CN=C(C)N=C1N DPJRMOMPQZCRJU-UHFFFAOYSA-M 0.000 description 2
- 230000009466 transformation Effects 0.000 description 2
- XDQDSNSFOMLZQF-PVQJCKRUSA-N (2r)-2-amino-3,3,3-trichloropropanoic acid Chemical compound OC(=O)[C@@H](N)C(Cl)(Cl)Cl XDQDSNSFOMLZQF-PVQJCKRUSA-N 0.000 description 1
- ZAYJDMWJYCTABM-CRCLSJGQSA-N (2s,3r)-2-azaniumyl-3-hydroxy-4-methylpentanoate Chemical compound CC(C)[C@@H](O)[C@H]([NH3+])C([O-])=O ZAYJDMWJYCTABM-CRCLSJGQSA-N 0.000 description 1
- GMKATDLSKRGLMZ-WHFBIAKZSA-N (2s,3s)-2-amino-n-hydroxy-3-methylpentanamide Chemical compound CC[C@H](C)[C@H](N)C(=O)NO GMKATDLSKRGLMZ-WHFBIAKZSA-N 0.000 description 1
- VZSRBBMJRBPUNF-UHFFFAOYSA-N 2-(2,3-dihydro-1H-inden-2-ylamino)-N-[3-oxo-3-(2,4,6,7-tetrahydrotriazolo[4,5-c]pyridin-5-yl)propyl]pyrimidine-5-carboxamide Chemical compound C1C(CC2=CC=CC=C12)NC1=NC=C(C=N1)C(=O)NCCC(N1CC2=C(CC1)NN=N2)=O VZSRBBMJRBPUNF-UHFFFAOYSA-N 0.000 description 1
- QJDRJTFSPVQEDV-UHFFFAOYSA-N 2-methylbutanethioic s-acid Chemical compound CCC(C)C(O)=S QJDRJTFSPVQEDV-UHFFFAOYSA-N 0.000 description 1
- 241000186361 Actinobacteria <class> Species 0.000 description 1
- 229920000936 Agarose Polymers 0.000 description 1
- GUBGYTABKSRVRQ-XLOQQCSPSA-N Alpha-Lactose Chemical compound O[C@@H]1[C@@H](O)[C@@H](O)[C@@H](CO)O[C@H]1O[C@@H]1[C@@H](CO)O[C@H](O)[C@H](O)[C@H]1O GUBGYTABKSRVRQ-XLOQQCSPSA-N 0.000 description 1
- VHUUQVKOLVNVRT-UHFFFAOYSA-N Ammonium hydroxide Chemical compound [NH4+].[OH-] VHUUQVKOLVNVRT-UHFFFAOYSA-N 0.000 description 1
- 239000004475 Arginine Substances 0.000 description 1
- 241000304886 Bacilli Species 0.000 description 1
- 229920002134 Carboxymethyl cellulose Polymers 0.000 description 1
- 241001485655 Corynebacterium glutamicum ATCC 13032 Species 0.000 description 1
- 230000007018 DNA scission Effects 0.000 description 1
- 229920002307 Dextran Polymers 0.000 description 1
- 241000588722 Escherichia Species 0.000 description 1
- 241001646716 Escherichia coli K-12 Species 0.000 description 1
- 229920001917 Ficoll Polymers 0.000 description 1
- 235000019766 L-Lysine Nutrition 0.000 description 1
- ODKSFYDXXFIFQN-BYPYZUCNSA-P L-argininium(2+) Chemical compound NC(=[NH2+])NCCC[C@H]([NH3+])C(O)=O ODKSFYDXXFIFQN-BYPYZUCNSA-P 0.000 description 1
- GUBGYTABKSRVRQ-QKKXKWKRSA-N Lactose Natural products OC[C@H]1O[C@@H](O[C@H]2[C@H](O)[C@@H](O)C(O)O[C@@H]2CO)[C@H](O)[C@@H](O)[C@H]1O GUBGYTABKSRVRQ-QKKXKWKRSA-N 0.000 description 1
- 102000003960 Ligases Human genes 0.000 description 1
- 108090000364 Ligases Proteins 0.000 description 1
- 241000144155 Microbacterium ammoniaphilum Species 0.000 description 1
- FYCWLJLGIAUCCL-DMTCNVIQSA-N O-methyl-L-threonine Chemical compound CO[C@H](C)[C@H](N)C(O)=O FYCWLJLGIAUCCL-DMTCNVIQSA-N 0.000 description 1
- FXDNYOANAXWZHG-VKHMYHEASA-N O-phospho-L-homoserine Chemical compound OC(=O)[C@@H](N)CCOP(O)(O)=O FXDNYOANAXWZHG-VKHMYHEASA-N 0.000 description 1
- FXDNYOANAXWZHG-UHFFFAOYSA-N O-phospho-L-homoserine Natural products OC(=O)C(N)CCOP(O)(O)=O FXDNYOANAXWZHG-UHFFFAOYSA-N 0.000 description 1
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 1
- 241000607720 Serratia Species 0.000 description 1
- 108010073771 Soybean Proteins Proteins 0.000 description 1
- 229920002472 Starch Polymers 0.000 description 1
- 229930006000 Sucrose Natural products 0.000 description 1
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose 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)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 description 1
- JZRWCGZRTZMZEH-UHFFFAOYSA-N Thiamine Natural products CC1=C(CCO)SC=[N+]1CC1=CN=C(C)N=C1N JZRWCGZRTZMZEH-UHFFFAOYSA-N 0.000 description 1
- GVBNSPFBYXGREE-CXWAGAITSA-N Visnadin Chemical compound C1=CC(=O)OC2=C1C=CC1=C2[C@@H](OC(C)=O)[C@@H](OC(=O)[C@H](C)CC)C(C)(C)O1 GVBNSPFBYXGREE-CXWAGAITSA-N 0.000 description 1
- 239000005862 Whey Substances 0.000 description 1
- 102000007544 Whey Proteins Human genes 0.000 description 1
- 108010046377 Whey Proteins Proteins 0.000 description 1
- 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 1
- 241000319304 [Brevibacterium] flavum Species 0.000 description 1
- 238000009825 accumulation Methods 0.000 description 1
- 150000003863 ammonium salts Chemical class 0.000 description 1
- ODKSFYDXXFIFQN-UHFFFAOYSA-N arginine Natural products OC(=O)C(N)CCCNC(N)=N ODKSFYDXXFIFQN-UHFFFAOYSA-N 0.000 description 1
- JGGLZQUGOKVDGS-VYTIMWRQSA-N aspartate semialdehyde Chemical compound O[C@@H]1[C@@H](NC(=O)C)CO[C@H](CO)[C@H]1O[C@@H]1[C@@H](NC(C)=O)[C@H](O)[C@H](O[C@@H]2[C@H]([C@@H](O[C@@H]3[C@@H]([C@H](O)[C@@H](O)[C@H](CO)O3)O[C@@H]3[C@@H]([C@H](O)[C@@H](O)[C@H](CO)O3)O[C@@H]3[C@H]([C@H](O)[C@@H](O)[C@H](CO)O3)O)[C@@H](O)[C@H](CO[C@@H]3[C@H]([C@H](O[C@@H]4[C@H]([C@H](O)[C@@H](O)[C@H](CO)O4)O)[C@@H](O)[C@H](CO)O3)O)O2)O)[C@H](CO)O1 JGGLZQUGOKVDGS-VYTIMWRQSA-N 0.000 description 1
- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 description 1
- ZAYJDMWJYCTABM-UHFFFAOYSA-N beta-hydroxy leucine Natural products CC(C)C(O)C(N)C(O)=O ZAYJDMWJYCTABM-UHFFFAOYSA-N 0.000 description 1
- 239000001768 carboxy methyl cellulose Substances 0.000 description 1
- 235000010948 carboxy methyl cellulose Nutrition 0.000 description 1
- 239000008112 carboxymethyl-cellulose Substances 0.000 description 1
- 230000000295 complement effect Effects 0.000 description 1
- 238000010276 construction Methods 0.000 description 1
- 241000186254 coryneform bacterium Species 0.000 description 1
- 238000012136 culture method Methods 0.000 description 1
- 239000012228 culture supernatant Substances 0.000 description 1
- 238000012217 deletion Methods 0.000 description 1
- 230000037430 deletion Effects 0.000 description 1
- 230000029087 digestion Effects 0.000 description 1
- 239000012153 distilled water Substances 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 238000001962 electrophoresis Methods 0.000 description 1
- 238000012869 ethanol precipitation Methods 0.000 description 1
- 239000013613 expression plasmid Substances 0.000 description 1
- 239000000284 extract Substances 0.000 description 1
- 239000001963 growth medium Substances 0.000 description 1
- 230000005764 inhibitory process Effects 0.000 description 1
- 229910001410 inorganic ion Inorganic materials 0.000 description 1
- 239000008101 lactose Substances 0.000 description 1
- SQQMAOCOWKFBNP-UHFFFAOYSA-L manganese(II) sulfate Chemical compound [Mn+2].[O-]S([O-])(=O)=O SQQMAOCOWKFBNP-UHFFFAOYSA-L 0.000 description 1
- 229910000357 manganese(II) sulfate Inorganic materials 0.000 description 1
- 229910021645 metal ion Inorganic materials 0.000 description 1
- 239000011785 micronutrient Substances 0.000 description 1
- 235000013369 micronutrients Nutrition 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 235000013379 molasses Nutrition 0.000 description 1
- 229940056360 penicillin g Drugs 0.000 description 1
- 230000035699 permeability Effects 0.000 description 1
- 239000002244 precipitate Substances 0.000 description 1
- 238000001556 precipitation Methods 0.000 description 1
- 230000035755 proliferation Effects 0.000 description 1
- 230000000644 propagated effect Effects 0.000 description 1
- 230000035484 reaction time Effects 0.000 description 1
- 230000001172 regenerating effect Effects 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 229940074404 sodium succinate Drugs 0.000 description 1
- ZDQYSKICYIVCPN-UHFFFAOYSA-L sodium succinate (anhydrous) Chemical compound [Na+].[Na+].[O-]C(=O)CCC([O-])=O ZDQYSKICYIVCPN-UHFFFAOYSA-L 0.000 description 1
- 229940001941 soy protein Drugs 0.000 description 1
- 239000008107 starch Substances 0.000 description 1
- 235000019698 starch Nutrition 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- WPLOVIFNBMNBPD-ATHMIXSHSA-N subtilin Chemical compound CC1SCC(NC2=O)C(=O)NC(CC(N)=O)C(=O)NC(C(=O)NC(CCCCN)C(=O)NC(C(C)CC)C(=O)NC(=C)C(=O)NC(CCCCN)C(O)=O)CSC(C)C2NC(=O)C(CC(C)C)NC(=O)C1NC(=O)C(CCC(N)=O)NC(=O)C(CC(C)C)NC(=O)C(NC(=O)C1NC(=O)C(=C/C)/NC(=O)C(CCC(N)=O)NC(=O)C(CC(C)C)NC(=O)C(C)NC(=O)CNC(=O)C(NC(=O)C(NC(=O)C2NC(=O)CNC(=O)C3CCCN3C(=O)C(NC(=O)C3NC(=O)C(CC(C)C)NC(=O)C(=C)NC(=O)C(CCC(O)=O)NC(=O)C(NC(=O)C(CCCCN)NC(=O)C(N)CC=4C5=CC=CC=C5NC=4)CSC3)C(C)SC2)C(C)C)C(C)SC1)CC1=CC=CC=C1 WPLOVIFNBMNBPD-ATHMIXSHSA-N 0.000 description 1
- 239000005720 sucrose Substances 0.000 description 1
- 235000019157 thiamine Nutrition 0.000 description 1
- 229960003495 thiamine Drugs 0.000 description 1
- 239000011721 thiamine Substances 0.000 description 1
- KYMBYSLLVAOCFI-UHFFFAOYSA-N thiamine Chemical compound CC1=C(CCO)SCN1CC1=CN=C(C)N=C1N KYMBYSLLVAOCFI-UHFFFAOYSA-N 0.000 description 1
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 description 1
- 235000013343 vitamin Nutrition 0.000 description 1
- 239000011782 vitamin Substances 0.000 description 1
- 229940088594 vitamin Drugs 0.000 description 1
- 229930003231 vitamin Natural products 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/0004—Oxidoreductases (1.)
- C12N9/0006—Oxidoreductases (1.) acting on CH-OH groups as donors (1.1)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/10—Transferases (2.)
- C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Organic Chemistry (AREA)
- Engineering & Computer Science (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Molecular Biology (AREA)
- Microbiology (AREA)
- Biotechnology (AREA)
- Biomedical Technology (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Medicinal Chemistry (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Description
ãçºæã®è©³çŽ°ãªèª¬æã
ïŒç£æ¥äžã®å©çšåéïŒ
æ¬çºæã¯ã¹ã¬ãªãã³åã¯ã€ãœãã€ã·ã³ã®è£œé æ³
ã«é¢ãã詳ããã¯çµæããã©ã¹ãããæããã³ãª
ããã«ã 现èãçšããçºé µæ³ã«ããã¹ã¬ãªãã³å
ã¯ã€ãœãã€ã·ã³ã®è£œé æ³ã«é¢ããã ïŒåŸæ¥ã®æè¡ïŒ åŸæ¥ãçºé µæ³ã«ããâã¹ã¬ãªãã³ãâã€ãœ
ãã€ã·ã³ã補é ããå Žåã埮çç©ãšããŠã¯èªç¶ç
ããåé¢ããèæ ªãŸãã¯è©²èæ ªã®äººå·¥å€ç°æ ªãçš
ããããŠãããâã¹ã¬ãªãã³ãçç£ãã人工å€
ç°æ ªã¯æ°å€ãç¥ãããŠããããã®å€ãã¯Î±âã¢ã
ãâβâããããã·åèé žïŒä»¥äžãAHVãšç¥èš
ãããïŒã«èæ§ãããããã¬ããã¯ããªãŠã å±ãŸ
ãã¯ã³ãªããã¯ããªãŠã å±ã«å±ããŠãããããã
ã®åŸ®çç©ã¯åç10ã20ïŒ ã§ïŒ¬âã¹ã¬ãªãã³ãçç£
ãããäŸãã°ãç±³åœç¹èš±ç¬¬3582471å·æ现æžãå
第3580810å·æ现æžåã³ç¹å ¬æ47â34956å·å ¬å ±ã«
ã¯AHVèæ§ã瀺ãããã¬ããã¯ããªãŠã å±ããš
ã·ãšãªãã¢å±åã³ã³ãªããã¯ããªãŠã å±ã«å±ãã
å€ç°æ ªãçšããã¹ã¬ãªãã³ã®è£œé æ³ãé瀺ãããŠ
ããããŸãããã¬ããã¯ããªãŠã å±åã³ã³ãªãã
ã¯ããªãŠã å±ã«å±ããå€ç°æ ªã«ããã¹ã¬ãªãã³ã®
çç£ã«ã€ããŠç¹éæ51â54984å·å ¬å ±ãå53â
101591å·å ¬å ±ãå54â32693å·å ¬å ±ãå54â35285
å·å ¬å ±ãå54â35286å·å ¬å ±ãå54â35288å·å ¬
å ±ãå54â37886å·å ¬å ±åã³å54â92692å·å ¬å ±ã«
ãé瀺ãããŠããã äžæ¹ãç±³åœç¹èš±ç¬¬4278765å·æ现æžãç¹éæ55
â131397å·å ¬å ±åã³å56â15696å·å ¬å ±ã«ã¯çµæ
ããã©ã¹ããDNAãçšããŠåœ¢è³ªè»¢æããããšã·
ãšãªãã¢ã»ã³ãªãçšããŠã¹ã¬ãªãã³ã補é ããæ¹
æ³ã瀺ãããŠããã ããã«ã欧å·ç¹èš±åºé¡å ¬é第0066129å·æ现æž
ã«ã¯AHVã«å¯Ÿããèæ§ãã³ã³ãããŒã«ããæè²
äœDNAæçãæ¿å ¥ãããŠãããã©ã¹ãããæã
ãã³ãªããã«ã 现èã䜿çšããã¹ã¬ãªãã³ã®çç£
ã«ã€ããŠèšèŒãããŠãããæŽã«ã欧å·ç¹èš±åºé¡å ¬
é第0131171å·ã«ã¯ããã¢ã»ãªã³ããããã²ããŒ
ãŒãã³ãŒãããéºäŒåãæ¿å ¥ãããŠãããã©ã¹ã
ããæããã³ãªããã«ã 现èãå¹é€ããŠãã€ãœã
ã€ã·ã³åã¯ã¹ã¬ãªãã³ãçç£ããããšãèšèŒãã
ãŠããã ãŸããã€ãœãã€ã·ã³ã«é¢ããç¶æ³ãã¹ã¬ãªãã³
ã®å Žåãšé ·äŒŒããŠãããã€ãœãã€ã·ã³ãçç£ãã
埮çç©ãšããŠã¯ã€ãœãã€ã·ã³ãããããµã¡ãŒãã«
察ããŠèæ§ãæããã»ã©ãã¢ã®å€ç°æ ªïŒç¹å ¬æ52
â30593å·å ¬å ±ïŒããã®çè²ã«ãã€ã·ã³ãèŠæ±ãã
ã³ãªããã¯ããªãŠã ã»ã°ã«ã¿ãã«ã ã®å€ç°æ ªïŒç¹
å ¬æ47â38995å·å ¬å ±ïŒãAHVã«èæ§ãæããã
ã¬ããã¯ããªãŠã å±åã³ã³ãªããã¯ããªãŠã å±ã®
å€ç°æ ªïŒç¹å ¬æ40â2880å·å ¬å ±ïŒãAHVã«èæ§
ãæãããã€ãã®çè²ã«ãªãžã³ãå¿ èŠãšãããã¬
ããã¯ããªãŠã å±ã®å€ç°æ ªïŒç¹å ¬æ51â6237å·å ¬
å ±ïŒãAHVåã³ïŒ¯âã¡ãã«ã¹ã¬ãªãã³ã«èæ§ã
æãããã¬ããã¯ããªãŠã å±ã®å€ç°æ ªïŒç¹å ¬æ51
â21077å·å ¬å ±ïŒãâïŒïŒâã¢ãããšãã«ïŒâã·ã¹
ãã€ã³ã«èæ§ãæããã³ãªããã¯ããªãŠã å±ã®å€
ç°æ ªïŒç¹å ¬æ52â4629å·å ¬å ±ïŒãïŒâã¢ããâïŒ
âã¡ãã«ããªé ªé žã«èæ§ãæãããšã·ãšãªãã¢å±
ã®å€ç°æ ªïŒç¹éæ53â69881å·å ¬å ±ïŒãAHVåã³
ããªã¯ããã¢ã©ãã³ã«èæ§ãæãããã¬ããã¯ã
ãªãŠã å±ã®å€ç°æ ªïŒç¹éæ54â35287å·å ¬å ±ïŒã
ããã ãã®ã»ãçµæããã©ã¹ããDNAãçšããŠåœ¢è³ª
転æããããšã·ãšãªãã¢ã»ã³ãªã¯ã¢ããé žã®çç£
èœãé«ãããšãé瀺ãããŠããïŒäŸãã°ç±³åœç¹èš±
第4278765å·æ现æžåç §ïŒãæŽã«æ¬§å·ç¹èš±åºé¡å ¬é
第0071023å·ã«ã¯ãAHVã«å¯Ÿããèæ§ãã³ã³ã
ããŒã«ããæè²äœDNAæçãæ¿å ¥ãããŠããã
ã©ã¹ãããæããã³ãªããã«ã 现èã䜿çšããã€
ãœãã€ã·ã³ã®çç£ã«ã€ããŠèšèŒãããŠããã ïŒçºæã解決ããããšããåé¡ç¹ïŒ ããããªããäžèšã®ã¹ã¬ãªãã³åã¯ã€ãœãã€ã·
ã³çç£èœãæãã现èã®ã¹ã¬ãªãã³åã¯ã€ãœãã€
ã·ã³çç£èœã¯ãæŽã«æ¹åããå¿ èŠãããã ïŒåé¡ç¹ã解決ããããã®ææ®µïŒ åäžã®ãããªç¶æ³äžã§æ¬çºæè ãã¯ããã¢ã»ãª
ã³ãããŒãŒïŒä»¥äžãHKããšèšãïŒãã³ãŒããã
éºäŒåïŒä»¥äžãHKãéºäŒåãšèšãïŒãçµèŸŒãŸã
ãŠãã第ïŒã®ãã©ã¹ããåã³ãã¢ã»ãªã³ãããã
ã²ããŒãŒïŒä»¥äžãHDããšèšãïŒãã³ãŒãããéº
äŒåïŒä»¥äžãHDéºäŒåããšèšãïŒãçµèŸŒãŸããŠ
ãã第ïŒã®ãã©ã¹ãããè²ããã³ãªããã«ã 现è
ããã¹ã¬ãªãã³åã³ã€ãœãã€ã·ã³ã®é«ãçç£æ§ã
æããããšãèŠãåºããã ã³ãªããã«ã 现èã¯ã奜æ°æ§ãã°ã©ã éœæ§æ¡¿è
ã§ãããéæé žæ§ã§ããŒããŒã¹ã»ãããŠã¢ã«ã»ãª
ãã»ãã¿ãŒããã€ãã€ããã¯ããªãªããžãŒç¬¬ïŒç
599é ïŒ1974ïŒã«èšèŒãããŠããŠããã®ãã¡ç¹ã«
以äžã«äŸç€ºãããããªïŒ¬âã°ã«ã¿ãã³é žã倧éã«
çç£ãããã®ãç¥ãããŠããã ãã¬ããã¯ããªãŠã ã»ãã€ããªã«ã¿ã
ATCC14020 ãã¬ããã¯ããªãŠã ã»ãµãã«ããªãã€ã¯ã
ATCC14066 ãã¬ããã¯ããªãŠã ã»ã€ã³ããªãªãã€ã«ã
ATCC14068 ãã¬ããã¯ããªãŠã ã»ã©ã¯ããã¢ãŒã¡ã³ã¿ã
ATCC13869 ãã¬ããã¯ããªãŠã ã»ããŒãŠã
ATCC13825 ãã¬ããã¯ããªãŠã ã»ãã©ãã
ATCC13826 ãã¬ããã¯ããªãŠã ã»ããªã²ãã¿ãªã¹
ATCC19240 ã³ãªããã¯ããªãŠã ã»ã¢ã»ãã¢ã·ããã€ã«ã
ATCC13870 ã³ãªããã¯ããªãŠã ã»ã¢ã»ãã°ã«ã¿ãã¯ã
ATCC15806 ã³ãªããã¯ããªãŠã ã»ã«ã«ããš
ATCC15991 ã³ãªããã¯ããªãŠã ã»ã°ã«ã¿ãã¯ã
ATCC13032ïŒ13060 ã³ãªããã¯ããªãŠã ã»ãªãªãŠã
ATCC15990 ã³ãªããã¯ããªãŠã ã»ã¡ã©ã»ã³ãŒã©
ATCC17965 ãã¯ããã¯ããªãŠã ã»ã¢ã³ã¢ãã¢ãã€ã©ã
ATCC15354 ã³ãªããã«ã 现èã«ã¯äžèšã®ãããªã°ã«ã¿ãã³
é žçç£æ§ãæãããã®ã®ã»ãã«ãã°ã«ã¿ãã³é žç
ç£æ§ã倱ã€ããã®åã³ãªãžã³ãã¢ã«ã®ãã³çã®ã¢
ããé žãçç£ããå€ç°æ ªãå«ãŸããã æ¬çºæã«ãŠäœ¿çšããããã©ã¹ãããã¯ã¿ãŒã¯ã
ã³ãªããã«ã 现è现èå ã«ãããŠå¢æ®ãåŸããã®
ã§ããã°ã©ã®ãããªãã®ã§ãè¯ããå ·äœçã«äŸç€º
ããã°ã以äžã®ãã®ãããã (1) pAM330 ç¹éæ58â67699åç § (2) pHM1519 ç¹éæ58â77895åç § (3) pAJ655 ç¹éæ58â192900åç § (4) pAJ611 ç¹éæ58â192900åç § (5) pAJ1844 ç¹éæ58â192900åç § ã³ãªããã«ã 现è现èå ã§å¢æ®å¯èœãªãã©ã¹ã
ãã®ãã®ä»ã®äŸãšããŠã¯pCG1ïŒç¹éæ57â
134500ïŒãpCG2ïŒç¹éæ58â35197ïŒãpCG4ïŒ
pCG11ïŒç¹éæ57â183799ïŒãããã æ¬çºæã«ãããŠã¯ããããã®ãã©ã¹ããã®ãã¡
ïŒçš®é¡ã®ãã©ã¹ãããçšãããããHKã¯ãâ
ã¹ã¬ãªãã³åã³ïŒ¬âã€ãœãã€ã·ã³çæã®äžéäœã§
ããâãã¢ã»ãªã³ãããã¹ããã¢ã»ãªã³ãçæ
ããåå¿ã觊åªããé µçŽ ã§ããã HKéºäŒåãåé¢ããæ¹æ³ã¯ãã³ãªããã«ã 现
èã®HKéºäŒåãæããŠããæ ªããããŸãæè²äœ
éºäŒåãæœåºãïŒäŸãã°H.Saito and K.Miura
Biochem.Biophys.Acta72ïŒ619ïŒïŒ1963ïŒã®æ¹æ³
ã䜿çšã§ããïŒããããé©åœãªå¶éé µçŽ ã§åæã
ããã€ãã§ãã³ãªããã«ã 现èã§å¢æ®ãåŸããã©
ã¹ãããã¯ã¿ãŒã«æ¥ç¶ããåŸãããçµæãDNA
ãçšããŠã³ãªããã«ã 现èã®HKæ¬ æå€ç°æ ªã圢
質転æããããHKçæ掻æ§ãä¿æããã«ããã€
ãèæ ªãåé¢ããããã«ããHKéºäŒåãåé¢ã§
ããã HDã¯ãã¢ã¹ãã«ããŒãã»ãã¢ã«ãããããã
ã¢ã»ãªã³ãçæããåå¿ã觊åªããé µçŽ ã§ããã HDéºäŒåãåé¢ããæ¹æ³ããHKéºäŒåãå
é¢ããæ¹æ³ãšåãããã«ããŠè¡ãããšãã§ããã
å³ã¡ãã³ãªããã«ã 现èã®HDæ¬ æå€ç°æ ªãçšã
ãããšã«ãããHDéºäŒåãåŸãããšãã§ããã HKéºäŒååã¯HDéºäŒåãåŸãããã®ã³ãªã
ãã«ã 现èã¯ãéæ§åã®ãã®ã§ãã€ãŠããããã
ã¹ã¬ãªãã³ã«ãããã€ãŒãããã¯é»å®³ã解é€ãã
ããããªHKåã¯HDãæããå€ç°æ ªãDNAäŸäž
æ ªãšããŠçšããæ¹ãããããçµæãåŸãããããš
ãå€ãããã®ãããªå€ç°æ ªã¯ãAHVã«èæ§ã®å€
ç°æ ªãšããŠåŸãããšãã§ããã HKéºäŒååã¯HDéºäŒåãåŸãããã«æè²äœ
éºäŒåãåæããéã«ãåæåå¿æéçã調ç¯ã
ãŠåæã®çšåºŠã調ç¯ããã°ãå·Ÿåºãçš®é¡ã®å¶éé µ
çŽ ã䜿çšã§ããã ãã¯ã¿ãŒDNAã¯ãæè²äœéºäŒåãåæããé
ã«çšããããå¶éé µçŽ ã«ããåæããããŸãã¯æ
è²äœDNAåæãã©ã°ã¡ã³ãåã³åæããããã¯
ã¿ãŒDNAã®ããããã®äž¡ç«¯ã«çžè£çãªå¡©åºé å
ãæãããªãªãŽãã¯ã¬ãªãããæ¥ç¶ããããŠãã€
ãã§ãã©ã¹ãããã¯ã¿ãŒãšæè²äœDNAãã©ã°ã¡
ã³ããšã®ã©ã€ã²ãŒã·ãšã³åå¿ã«ä»ãããã ãã®ããã«ããŠåŸããããæè²äœDNAãšãã¯
ã¿ãŒãã©ã¹ãããšã®çµæãDNAãã³ãªããã«ã
现èã«å±ããå容èãžå°å ¥ããã«ã¯ããšã·ãšãªã
ã¢ã»ã³ãªïŒ«â12ã«ã€ããŠå ±åãããŠããæ§ãª
ïŒMandelïŒM.and HigaïŒA.ïŒJ.Mol.ïŒBiol.ïŒ
53ïŒ159ïŒ1970ïŒå容è现èãå¡©åã«ã«ã·ãŠã ã§åŠ
çããŠDNAã®ééæ§ãå¢ãæ¹æ³ããŸãã¯ããã«
ã¹ã»ãºãããªã¹ã«ã€ããŠå ±åãããŠããæ§ã«
ïŒDuncanïŒC.H.ïŒWilsonïŒG.A.and YoungïŒF.
E.ïŒGeneïŒïŒïŒ153ïŒ1977ïŒïŒçŽ°èãDNAãåã
蟌ã¿åŸãæ§ã«ãªãå¢æ®æ®µéïŒããããã³ã³ããã³
ãã»ã«ïŒã«å°å ¥ããæ¹æ³ã«ããå¯èœã§ããããã
ãã¯ãããã«ã¹ã»ãºãããªã¹ãæŸç·èé¡ããã³é µ
æ¯ã«ã€ããŠç¥ãããŠããæ§ã«ïŒChangïŒS.and
ChoenïŒS.N.ïŒMolec.Gen.ïŒGenet.ïŒ168ïŒ111
ïŒ1979ïŒïŒBibbïŒM.J.ïŒWardïŒJ.M.and
HopwoodïŒO.A.ïŒNatureïŒ274ïŒ398ïŒ1978ïŒïŒ
HinnenïŒA.ïŒHicksïŒJ.B.and FinkïŒG.R.ïŒ
Proc.Natl.Acad.Sci.USAïŒ75 1929ïŒ1978ïŒïŒã
DNAå容èãããã©ã¹ããDNAã容æã«åã蟌
ãããããã©ã¹ããŸãã¯ã¹ããšããã©ã¹ãã«ããŠ
ãã©ã¹ãããDNAå容èã«å°å ¥ããããšãå¯èœ
ã§ããã ããããã©ã¹ãæ³ã§ã¯äžèšã®ããã«ã¹ã»ãºãã
ãªã¹ã«ãããŠäœ¿çšãããŠããæ¹æ³ã§ãå åé«ãé »
床ãåŸãããšãã§ããããç¹éæ57â183799ã«èš
èŒãããã³ãªããã¯ããªãŠã å±ãŸãã¯ãã¬ããã¯
ããªãŠã å±ã®ããããã©ã¹ãã«ããªãšãã¬ã³ã°ãª
ã³ãŒã«ãŸãã¯ããªããã«ã¢ã«ã³ãŒã«ãšäºäŸ¡éå±ã€
ãªã³ãšã®ååšäžã«DNAããšã蟌ãŸããæ¹æ³ãåœ
ç¶å©çšã§ãããããªãšãã¬ã³ã°ãªã³ãŒã«ãŸãã¯ã
ãªããã«ã¢ã«ã³ãŒã«ã®ãããã«ãã«ã«ããã·ã¡ã
ã«ã»ã«ããŒã¹ãããã¹ãã©ã³ããã€ã³ãŒã«ããã«
ãããã¯F68ïŒã»ã«ã瀟ïŒãªã©ã®æ·»å ã«ãã€ãŠ
DNAã®ãšã蟌ã¿ãä¿é²ãããæ¹æ³ã§ãåçã®çµ
æãåŸãããã ããããŠãHKéºäŒååã¯HDéºäŒåãçµèŸŒãŸ
ãããã©ã¹ãããåŸãããããã®äºã€ã®çµæãã
ã©ã¹ãããæããã³ãªããã«ã 现èã¯ãäºã€ã®ã
ã©ã¹ããã现èå ã«å ±åããããã®ã§ããã°ã€ãœ
ãã€ã·ã³åã¯ã¹ã¬ãªãã³ãçç£ãããå ±åãããª
ããã®ã§ããã°ãå ±åããããã©ã¹ãããã¯ã¿ãŒ
ã«å®¹æã«HKéºäŒååã³HDéºäŒåã移ãããã
ããšãã§ããã ã³ãªããã«ã 现è现èå ã§äºã€ã®ãã©ã¹ããã
å ±åãããããã«ã¯ãæ¬çºæè ãã®ç 究ã«ãã
ã°ããã©ã¹ããã®è€è£œéå§é åãç°ãªããã®ã§ã
ãã°å ±åãããããè€è£œéå§é åãåããã®ã§ã
ã€ãŠããä»ã®é åã倧ããç°ãªãã°å ±åãããã
ãšãããã äºã€ã®ãã©ã¹ãããä¿æããããã®ã³ãªããã«
ã 现è宿䞻ã¯ãã¹ã¬ãªãã³åã¯ã€ãœãã€ã·ã³èŠæ±
æ ªã§ãã€ãŠããããåéæ§æ ªã§ãã€ãŠããããã
ããAHVèæ§ãæããå€ç°æ ªãé垞奜ãŸããçµ
æãåŸãããç¹ã«ããé«ãã¹ã¬ãªãã³åã¯ã€ãœã
ã€ã·ã³çç£èœãæããèæ ªãæã奜ãŸããçµæã
åŸãããã åŸãããã¹ã¬ãªãã³åã¯ã€ãœãã€ã·ã³çç£èã
å¹é€ããæ¹æ³ã¯ãåŸæ¥ã®ã¹ã¬ãªãã³åã¯ã€ãœãã€
ã·ã³çç£èã®å¹é€æ¹æ³ãšç¹ã«å€ããªããå³ã¡ãå¹
å°ãšããŠã¯ãççŽ æºãçªçŽ æºãç¡æ©ã€ãªã³ãæŽã«
å¿ èŠã«å¿ããŠã¢ããé žããã¿ãã³çã®ææ©åŸ®éæ
é€çŽ ãå«æããéåžžã®ãã®ã§ãããççŽ æºãšããŠ
ã¯ãã°ã«ã³ãŒã¹ãã·ãŠã¯ããŒã¹ãã©ã¯ããŒã¹çå
ã³ãããã®å«æãã柱ç²å æ°Žå解液ãããšã€ãç³
èçãçšãããããçªçŽ æºãšããŠã¯ãã¢ã³ã¢ãã¢
ã¬ã¹ãã¢ã³ã¢ãã¢æ°Žãã¢ã³ã¢ããŠã å¡©ãã®ä»ã䜿
çšã§ããã å¹é€ã¯å¥œæ°çæ¡ä»¶äžã§å¹å°ã®PHåã³æž©åºŠãé©å®
調ç¯ãã€ã€ãå®è³ªçã«ã¹ã¬ãªãã³åã¯ã€ãœãã€ã·
ã³ã®çç£èç©ãåæ¢ãããŸã§è¡ãªãããã ããããŠå¹é€æ¶²äžã«ã¯èéã®ã¹ã¬ãªãã³åã¯ã€
ãœãã€ã·ã³ãçæèç©ããããå¹é€æ¶²ããã¹ã¬ãª
ãã³åã¯ã€ãœãã€ã·ã³ãæ¡åããã«ã¯ãéåžžã®æ¹
æ³ãé©çšã§ããã å®æœäŸ ïŒ ïŒ ãã¢ã»ãªã³ãããŒãŒïŒHKïŒéºäŒåã®ã¯ããŒ
ãã³ã° (1) HKéºäŒåãå«ãæè²äœDNAã®èª¿è£œ ãã¬ããã¯ããªãŠã ã»ã©ã¯ããã¢ãŒã¡ã³ã¿ã
ATCC13869ã®AHVèæ§çªç¶å€ç°æ ªAJ11188
ïŒFERMâP4190FERM BPâ577ïŒïŒç¹å ¬æ56â
3038ïŒãïŒã®CMGå¹å°ïŒãããã³ïŒïœïŒdlã
é µæ¯ãšãã¹ïŒïœïŒdlãã°ã«ã³ãŒã¹0.5ïœïŒdlãå
ã³NaCl0.5ïœïŒdlãå«ã¿ãPH7.2ã«èª¿æŽãããã®ïŒ
ã«æ€èãã30âã§çŽïŒæéæ¯çªå¹é€ãè¡ãªãã察
æ°å¢æ®æã®èäœãéããããã®èäœããªãŸããŒ
ã ã»SDSã§æº¶èãããã®ã¡ãéåžžã®ããšããŒã«åŠ
çæ³ã«ãããæè²äœDNAãæœåºç²Ÿè£œããæçµç
ã«3.5mgã®DNAãåŸãã (2) ãã¯ã¿ãŒDNAã®èª¿è£œ ãã¯ã¿ãŒãšããŠpAJ1844ïŒååé5.4ã¡ã¬ãã«ã
ã³ïŒãçšãããã®DNAã次ã®æ§ã«ããŠèª¿è£œããã ãŸãpAJ1844ããã©ã¹ãããšããŠä¿æãããã¬
ããã¯ããªãŠã ã»ã©ã¯ããã¢ãŒã¡ã³ã¿ã AJ12037
ïŒFERMâP7234ïŒã100mlã®CMGå¹å°ã«æ¥çš®ãã
30âã§å¯Ÿæ°å¢æ®æåŸæãŸã§å¹é€ããã®ã¡ããªãŸã
ãŒã SDSåŠçã«ãã溶èããã30000Ãïœ30åã®
è¶ é å¿ã«ããäžæž ãåŸããããšããŒã«åŠçã®ã®
ã¡ãïŒå®¹ã®ãšã¿ããŒã«ãå ããŠDNAãæ²æŸ±åå
ããããããå°éã®TENç·©è¡æ¶²ïŒ20mMããªã¹
å¡©é žå¡©ã20mM NaClã1mM EDTAïŒPH8.0ïŒã«
溶解åŸãã¢ã¬ããŒã¹ã²ã«é»æ°æ³³åã«ããåé¢åŸã
åãåºããŠpAJ1844ãã©ã¹ããDNAçŽ15ÎŒïœãåŸ
ãã (3) æè²äœDNAæçã®ãã¯ã¿ãŒãžã®æ¿å ¥ (1)ã§åŸãæè²äœDNA20ÎŒïœãš(2)ã§åŸããã©ã¹
ããDNA10ÎŒïœãšãå¶éãšã³ããã¯ã¬ã¢ãŒãŒPst
ã§ããããã37âãïŒæéåŠçããå®å šã«åæ
ããã65â10åã®ç±åŠçåŸãäž¡åå¿æ¶²ãæ··åãã
ATPåã³ãžããªã¹ã¬ã€ããŒã«ååšäžãT4ãã¢ãŒ
ãžç±æ¥ã®DNAãªã¬ãŒãŒã«ãã€ãŠ10âã24æé
DNAéã®é£çµåå¿ãè¡ã€ãã65âïŒåã®ç±åŠç
åŸãåå¿æ¶²ã«ïŒå容ã®ãšã¿ããŒã«ãå ããŠé£çµå
å¿çµäºåŸã®DNAãæ²æŸ±æ¡åããã (4) HKéºäŒåã®ã¯ããŒãã³ã° HKéºäŒåãæ¬ æãããã¬ããã¯ããªãŠã ã»ã©
ã¯ããã¢ãŒã¡ã³ã¿ã AJ12078ãå容èãšããŠçšã
ãã 圢質転æã®æ¹æ³ãšããŠã¯ãããããã©ã¹ããã©
ã³ã¹ããªãŒã¡ãŒã·ãšã³æ³ãçšããããŸããèæ ªã
ïŒmlã®CMG液äœå¹å°ã§å¯Ÿæ°å¢æ®æã®åæãŸã§å¹
é€ããããã·ãªã³ïŒ§ã0.6ãŠãããïŒmlæ·»å åŸã
ããã«1.5æéæ¯çªå¹é€ããé å¿åé¢ã«ããèæ ª
ãéããèäœã0.5Mã·ãŠãŒã¯ããŒã¹ã20mMã
ã¬ã€ã³é žã20mMå¡©åãã°ãã·ãŠã ã3.5ïŒ ãã
ãã»ã€ããã¹ïŒDifcoïŒãããªãSMMPå¹å°ïŒPH
6.5ïŒ0.5mlã§æŽæµããã次ãã§10mgïŒmlã®ãªãŸã
ãŒã ãå«ãSMMPå¹å°ã«æžæ¿ã30âã§20æéã
ãããã©ã¹ãåãå³ã€ãã6000Ãïœã10åéé å¿
åé¢åŸãããããã©ã¹ããSMMPã§æŽæµã0.5ml
ã®SMMPã«å床æžæ¿ããããã®æ§ã«ããŠåŸãã
ãããããã©ã¹ããš(3)ã§èª¿è£œããDNA10ÎŒïœã
5mM EDTAååšäžã§æ··åããããªãšãã¬ã³ã°ãª
ã³ãŒã«ãæçµæ¿åºŠã30ïŒ ã«ãªãæ§ã«æ·»å ããåŸã
DNAãããããã©ã¹ãã«åã蟌ãŸããçºã«å®€æž©
ã«ïŒåéæŸçœ®ããããã®ããããã©ã¹ãã
SMMPå¹å°ïŒmlã§æŽæµåŸãSMMPå¹å°ïŒmlã«å
æžæ¿ãã圢質çºçŸã®çºã30âã§ïŒæéå¹é€ããã
ãã®å¹é€æ¶²ãPH7.0ã®ããããã©ã¹ãåçå¹å°äž
ã«å¡åžãããããããã©ã¹ãåçå¹å°ã¯èžçæ°ŽïŒ
ãããããªã¹ïŒããããã·ã¡ãã«ïŒã¢ããã¡ã¿
ã³12ïœãECl0.5ïœãã°ã«ã³ãŒã¹10ïœãMgCl2ã»
6H2O8.1ïœãCaCl2ã»2H2O2.2ïœããããã³ïŒïœã
ç²æ«é µæ¯ãšãã¹ïŒïœãã«ã¶ããé žïŒDifco瀟ïŒïŒ
ïœãK2HPO40.2ïœãã³ãã¯é žãããªãŠã 135ïœã
å¯å€©ïŒïœåã³ã¯ãã©ã ããšãã³ãŒã«3ÎŒïœïŒãå«
ãã 30âã§ïŒé±éå¹é€åŸãçŽ10000åã®ã¯ãã©ã ã
ãšãã³ãŒã«èæ§ã³ãããŒãåºçŸããŠããã®ã§ãã
ãã¹ã¬ãªãã³ãå«ãŸãªãå¹å°ïŒThræ¬ å¹å°ïŒïŒïŒ
ïŒ ã°ã«ã³ãŒã¹ãïŒïŒ ç¡«é žã¢ã³ã¢ããŠã ã0.25ïŒ å°¿
çŽ ã0.1ïŒ ããé žäºæ°ŽçŽ ã«ãªãŠã ã0.04ïŒ ç¡«é žã
ã°ãã·ãŠã ïŒæ°Žå¡©ã2ppméã€ãªã³ã2ppmãã³ã¬
ã³ã€ãªã³ã200ÎŒïœïŒãµã€ã¢ãã³å¡©é žå¡©ã50ÎŒ
ïœïŒããªãã³ãPH7.0ãå¯å€©1.8ïŒ ïŒã«ã¬ããªã«
ããã¯ãã©ã ããšãã³ãŒã«èæ§ã§ãã€ãŠãã€ã¹ã¬
ãªãã³èŠæ±æ§ãæ¶å€±ããèæ ªïŒæ ªãåŸãã (5) 圢質転ææ ªã®ãã©ã¹ãã解æ ãããã®æ ªãã(2)ã§è¿°ã¹ãæ¹æ³ã«ããã溶è液
ã調補ããã¢ã¬ããŒã¹ã²ã«é»æ°æ³³åæ³ã«ãããã
ã©ã¹ããDNAãæ€åºãããšãããïŒæ ªã§ãã¯ã¿
ãŒã®pAJ1844ãããæããã«å€§ããªãã©ã¹ããã
æ€åºãããã ãããã®ãã¡ã®ä»£è¡šæ ªãAJ12079ïŒFERMâ
P7237ïŒFERM BPâ578ïŒãšåã¥ããã (6) åãã©ã³ã¹ããŒã¡ãŒã·ãšã³ AJ12079å ã®çµæããã©ã¹ããïŒpAJ211ïŒäž
ã«HKéºäŒåãååšããããšã確èªããããã«ã
ã®ãã©ã¹ããDNAãçšããã¬ããã¯ããªãŠã ã»
ã©ã¯ããã¢ãŒã¡ã³ã¿ã AJ12078ãå床圢質転æã
ãã çããã¯ãã©ã ããšãã³ãŒã«èæ§ã³ãããŒã®ã
ã¡ããããã10åãé£ãäžãã¹ã¬ãªãã³èŠæ±æ§ã
ãã¹ããããšããããããã®ããããèŠæ±æ§ãæ¶
倱ããŠãããäžèšã®çµæããã©ã¹ããäžã«HKéº
äŒåãååšããããšãæãããšãªã€ãã ïŒ HKéºäŒåãæãã2.9kb.PstDNAæçã®
pAJ224ãžã®ç§»ãå€ã (1) ããªã¡ãããªã èæ§ãæãããã¯ã¿ãŒãã©
ã¹ããpAJ228ã®é æ ãã¬ããã¯ããªãŠã ã»ã©ã¯ããã¢ãŒã¡ã³ã¿ã
AJ12036ããå€ç°èªå°ãããããªã¡ãããªã èæ§
å€ç°æ ªAJ12146ïŒFERMâP7672ïŒFERM BPâ
785ïŒãïŒã®CMGå¹å°ïŒãããã³ïŒïœïŒdlãé µ
æ¯ãšãã¹ïŒïœïŒdlãã°ã«ã³ãŒã¹0.5ïœïŒdlãåã³
NaCl0.5ïœïŒdlãå«ã¿ãPH7.2ã«èª¿æŽãããã®ïŒã«
æ€èãã30âã§çŽïŒæéæ¯çªå¹é€ãè¡ãªãã察æ°
å¢æ®æã®èäœãéããããã®èäœããªãŸããŒã ã»
SDSã§æº¶èãããã®ã¡ãéåžžã®ããšããŒã«åŠçæ³
ã«ãããæè²äœDNAãæœåºç²Ÿè£œããæçµçã«3.0
mgã®DNAãåŸãã (2) ãã¯ã¿ãŒãšããŠpAM330ãçšããã
pAM330ã¯æ¬¡ã®æ§ã«ããŠèª¿è£œããã ãŸãpAM330ããã©ã¹ãããšããŠä¿æãããã¬
ããã¯ããªãŠã ã»ã©ã¯ããã¢ãŒã¡ã³ã¿ã
ATCC13869ã100mlã®CMGå¹å°ã«æ¥çš®ãã30â
ã§å¯Ÿæ°å¢æ®æåŸæãŸã§å¹é€ããã®ã¡ããªãŸããŒã
SDSåŠçã«ãã溶èããã30000Ãïœ30åã®è¶ é
å¿ã«ããäžæž ãåŸããããšããŒã«åŠçã®ã®ã¡ãïŒ
容ã®ãšã¿ããŒã«ãå ããŠDNAãæ²æŸ±ç©ãšããŠå
åããããããå°éã®TENç·©è¡æ¶²ïŒ20mMããª
ã¹å¡©é žå¡©ã20mM NaClã1mM EDTAïŒPH8.0ïŒ
ã«æº¶è§£åŸãã¢ã¬ããŒã¹ã²ã«é»æ°æ³³åã«ããåé¢
åŸãåãåºããŠpAM330ãã©ã¹ããDNAçŽ15ÎŒïœ
ãåŸãã (3) (1)ã§åŸãæè²äœDNA20ÎŒïœãš(2)ã§åŸãã
ã©ã¹ããDNA10ÎŒïœãšãå¶éãšã³ããã¯ã¬
ã¢ãŒãŒMbo ã§ããããã37âã30åéåŠ
çããéšååæããã65âã10åã®ç±åŠç
åŸãäž¡åå¿æ¶²ãæ··åããATPåã³ãžããªã¹
ã¬ã€ããŒã«ååšäžãT4ãã¢ãŒãžç±æ¥ã®DNA
ãªã¬ãŒãŒã«ãã€ãŠ10âã24æéDNAéã®é£
çµåå¿ãè¡ã€ãã65âãïŒåã®ç±åŠçåŸãå
å¿æ¶²ã«ïŒå容ã®ãšã¿ããŒã«ãå ããŠé£çµåå¿
çµäºåŸã®DNAãæ²æŸ±æ¡åããã (4) ããªã¡ãããªã æåæ§ã®ãã¬ããã¯ããªãŠ
ã ã»ã©ã¯ããã¢ãŒã¡ã³ã¿ã AJ12036ãå容è
ãšããŠçšããã 圢質転æã®æ¹æ³ãšããŠã¯ãïŒâ(4)ã§è¿°ã¹ãæ¹æ³
ãçšãããããªã¡ãããªã ïŒSigma瀟ïŒ25ÎŒïœïŒ
ãå«ãåçå¹å°ã«ãŠã30âã§ïŒé±éå¹é€åŸãçŽ
100åã®ã³ãããŒãåºçŸããŠããã®ã§ããããã
ãªã¡ãããªã ãå«ãæå°å¹å°ïŒïŒïŒ ã°ã«ã³ãŒã¹ã
ïŒïŒ ç¡«é žã¢ã³ã¢ããŠã ã0.25ïŒ å°¿çŽ ã0.1ïŒ ãã
é žäºæ°ŽçŽ ã«ãªãŠã ã0.04ïŒ ç¡«é žãã°ãã·ãŠã ïŒæ°Ž
å¡©ã2ppméã€ãªã³ã2ppmãã³ã¬ã³ã€ãªã³ã
200ÎŒïœïŒãµã€ã¢ãã³å¡©é žå¡©ã50ÎŒïœïŒãããª
ã³ãPH7.0ãå¯å€©1.8ïŒ ãããªã¡ãããªã 50ÎŒïœïŒ
mlïŒã«ããªã«ããããªã¡ãããªã èæ§ïŒæ ªãåŸ
ãã (5) ãããã®æ ªããïŒâ(2)ã§è¿°ã¹ãæ¹æ³ã«ã
ãã溶è液ã調補ããã¢ã¬ããŒã¹ã²ã«é»æ°æ³³
åæ³ã«ããããã©ã¹ããDNAãæ€åºãããš
ããããã¯ã¿ãŒã®pAM330ãããæããã«å€§
ããªãã©ã¹ãããæ€åºãããããã®æ ªã
AJ12147ïŒFERMâP7673ïŒFERM BPâ
786ïŒãšåä»ããã (6) AJ12147ãæãããã©ã¹ããïŒpAJ228ïŒ
äžã«ããªã¡ãããªã èæ§éºäŒåãååšããã
ãšã確èªããããããã®ãã©ã¹ããDNAã
çšããã¬ããã¯ããªãŠã ã»ã©ã¯ããã¢ãŒã¡ã³
ã¿ã AJ12036ãå床ã圢質転æããã çããããªã¡ãããªã èæ§ãæããã³ãããŒã®
ãã¡ãããã10åãé£ãäžãã¢ã¬ããŒã¹é»æ°æ³³å
æ³ã«ãããã©ã¹ããNAãæ€åºãããšããããã
ãã®ãããã«ãpAJ228ãšåã倧ããã®ãã©ã¹ã
ããååšããŠãããäžèšçµæããã©ã¹ããäžã«ã
ãªã¡ãããªã èæ§ãè¡šçŸããéºäŒåãååšããã
ãšãæãããšãªã€ãã (7) ããªã¡ãããªã èæ§ãã©ã¹ããïŒpAJ228ïŒ
ãžã®HKéºäŒåïŒpAJ211ã®2.9kb PstDNA
æçïŒã®ç§»ããã pAJ228ã¯Pstã«ããåæéšäœãæããªããã
HKéºäŒåãå«ãpAJ211ç±æ¥ã®2.9kbã®Pstæ
çããã®ãŸãŸç§»ããããããšã¯äžå¯èœã§ãããã
ãã§ç¬¬ïŒå³ã«ç€ºããæ¹æ³ã§ç§»ããããè¡ã€ããå³
ã¡pAJ211ããå¶éé µçŽ Pstã«ããéšååæã§å
ãåºãã2.9kbã«DNAæçã®äž¡ç«¯ã«BamHïŒ
SalïŒPstã®åæéšäœããã€ã第ïŒå³ã«ç€ºãã
ãããªåæãªãªãŽãã¯ã¬ãªãããªã³ã«ãŒãT4ã
ã¢ãŒãžç±æ¥ã®DNAãªã¬ãŒãŒãçšããŠé£çµããã
åŸãå¶éé µçŽ BamHã§åæãããåŸããã
DNAæ··åç©ãã¢ã¬ããŒã¹ã»ã²ã«é»æ°æ³³åã«ããã
çŽ2.9kbã®DNAæçãåé¢æœåºãããšã¿ããŒã«æ²
柱ã«ããDNAæçãååãããããããŠåŸãã
ãDNAæçã¯2.9kbã®HKéºäŒåãå«ãDNAã®
䞡端ã«PstïŒSalïŒBamHã®åæéšäœã䞊
ãã æ§é ã«ãªã€ãŠããïŒç¬¬ïŒå³ïŒã äžæ¹ãpAJ228ã¯å¶éé µçŽ Mboã§éšååæã
ãåŸ65âïŒ10åã®ç±åŠçãããããã®åå¿æ¶²ã«å
è¿°ã®æ¹æ³ã§åŸãHKéºäŒåãå«ãDNAæçãå
ããT4DNAãªã¬ãŒãŒã§DNAéã®é£çµåå¿ãè¡
ã€ããåå¿åŸã65âãïŒåã®ç±åŠçãããïŒå容
ã®ãšã¿ããŒã«ã®æ·»å ã«ããæ²æŸ±æ¡åãããDNA
ããïŒâ(4)ãšåæ§ã®æ¹æ³ã«ããããªã¡ããã«ã æ
åæ§ã§ããã€ãHKéºäŒåãæ¬ æãããã¬ããã¯
ããªãŠã ã»ã©ã¯ããã¢ãŒã¡ã³ã¿ã AJ12078ãå容
èãšããŠããããã©ã¹ããã©ã³ã¹ããªãŒã¡ãŒã·ãš
ã³ãè¡ã€ãåŸãããªã¡ããã«ã 100ÎŒïœïŒmlãå«
ãããããã©ã¹ãåçå¹å°ã§å¹é€ããçµæçŽ200
åã®åçã³ãããŒãåºçŸããŠããã ãããããªã¡ããã«ã ã200ÎŒïœïŒmlãå«ã¿å
容èã®èŠæ±ç©è³ªã§ããã¹ã¬ãªãã³ãå«ãŸãªãæå°
å¹å°ã«ã¬ããªã«ããããªã¡ããã«ã èæ§ã§ããã€
ã¹ã¬ãªãã³èŠæ±æ§ãæ¶å€±ããèæ ªïŒæ ªãåŸããã
ããã®æ ªããïŒâ(2)ã«ç€ºããæ¹æ³ã«ãã溶è液ã
調補ããã¢ã¬ããŒã¹ã»ã²ã«é»æ°æ³³åã«ãããã©ã¹
ããDNAãæ€åºãããšãã14.9kbïŒ12.2kbïŒ
9.6kbïŒ6.5kbã®ãã©ã¹ãããæ€åºãããããã®ã
ã¡æãååéã®å°ãã6.5kbã®ãã©ã¹ããã
pAJ212ãšåä»ãããpAJ212ãä¿æããæ ªã1278
âHKæ ªãšåä»ããã pAJ212ã¯ãHKéºäŒåãé€ããŠãpAJ224ãšã
ãŠããã¬ããã¯ããªãŠã ã»ã©ã¯ããã¢ãŒã¡ã³ã¿ã
AJ12196ïŒFERMâP8015ïŒFERM BPâ787ïŒãš
ããŠå¯èšãããŠããã (9) åãã©ã³ã¹ããŒã¡ãŒã·ãšã³ AJ12078âHKå ã®çµæããã©ã¹ãã
ïŒpAJ212ïŒäžã«HKéºäŒåãååšããããšã確èª
ããããã«ãã®ãã©ã¹ããDNAãçšããã¬ãã
ã¯ããªãŠã ã»ã©ã¯ããã¢ãŒã¡ã³ã¿ã AJ12078ãå
床圢質転æããã çããããªã¡ããã«ã èæ§ã³ãããŒã®ãã¡ãã
ããã10åãé£ãäžãã¹ã¬ãªãã³èŠæ±æ§ããã¹ã
ãããšããããããã®ããããèŠæ±æ§ãæ¶å€±ããŠ
ãããäžèšã®çµæããã©ã¹ããäžã«HKéºäŒåã
ååšããããšãæãããšãªã€ãã ïŒ ãã¢ã»ãªã³ããããã²ããŒãŒïŒHDïŒéºäŒå
ã®ã¯ããŒãã³ã° (1) HDéºäŒåãå«ãæè²äœDNAã®èª¿è£œ ãã¬ããã¯ããªãŠã ã»ã©ã¯ããã¢ãŒã¡ã³ã¿ã
ATCC13869ã®Î±âã¢ããâβâãã€ãããã·å
èé žèæ§çªç¶å€ç°æ ªAJ11188ïŒFERMâP4190ïŒ
ïŒç¹å ¬æ56â3038ïŒãïŒã®CMGå¹å°ïŒãããã³
ïŒïœïŒdlãé µæ¯ãšãã¹ïŒïœïŒdlãã°ã«ã³ãŒã¹0.5
ïœïŒdlãåã³NaCl0.5ïœïŒdlãå«ã¿ãPH7.2ã«èª¿æŽ
ãããã®ïŒã«æ€èãã30âã§çŽïŒæéæ¯çªå¹é€ã
è¡ãªãã察æ°å¢æ®æã®èäœãéããããã®èäœã
ãªãŸããŒã ã»SDSã§æº¶èãããã®ã¡ãéåžžã®ããš
ããŒã«åŠçæ³ã«ãããæè²äœDNAãæœåºç²Ÿè£œãã
æçµçã«3.6mgã®DNAãåŸãã (2) ãã¯ã¿ãŒDNAã®èª¿è£œ ãã¯ã¿ãŒãšããŠpAJ1844ïŒç¹éæ58â192900å
ç §ïŒïŒååé5.4ã¡ã¬ãã«ãã³ïŒãçšãããã®
DNAã次ã®æ§ã«ããŠèª¿è£œããã pAJ1844ã¯ãã³ãªããã¯ããªãŠã ã»ã°ã«ã¿ããŠ
ã ATCC13058ãæããŠãããã©ã¹ããpHM1519
ããèªå°ããããã®ã§ããã ãŸãpAJ1844ããã©ã¹ãããšããŠä¿æãããã¬
ããã¯ããªãŠã ã»ã©ã¯ããã¢ãŒã¡ã³ã¿ã AJ12037
ïŒFERMâP7234ïŒïŒFERMâBP577ïŒã100mlã®
CMGå¹å°ã«æ¥çš®ãã30âã§å¯Ÿæ°å¢æ®æåŸæãŸã§
å¹é€ããã®ã¡ããªãŸããŒã SDSåŠçã«ãã溶èã
ãã30000Ãïœ30åã®è¶ é å¿ã«ããäžæž ãåŸãã
ããšããŒã«åŠçã®ã®ã¡ãïŒå®¹ã®ãšã¿ããŒã«ãå ã
ãŠDNAãæ²æŸ±ååããpAJ1844ãã©ã¹ããDNA
çŽ80ÎŒïœãåŸãã (3) æè²äœDNAæçã®ãã¯ã¿ãŒãžã®æ¿å ¥ (1)ã§åŸãæè²äœDNA40ÎŒïœããå¶éé µçŽ Pst
ã0.124ãŠãããã§30âã10åééšåæ¶åããã (2)ã§åŸãã©ã¹ããDNA10ÎŒïœã¯å¶éãšã³ãã
ã¯ã¬ã¢ãŒãŒPstã§37âãïŒæéåŠçããå®å šã«
åæããã65â10åã®ç±åŠçåŸãäž¡åå¿æ¶²ãæ··å
ããATPåã³ãžããªã¹ã¬ã€ããŒã«ååšäžãT4ã
ã¢ãŒãžç±æ¥ã®DNAãªã¬ãŒãŒã«ãã€ãŠ22âã15æ
éDNAéã®é£çµåå¿ãè¡ã€ãã65âïŒåã®ç±åŠ
çåŸãåå¿æ¶²ã«ïŒå容ã®ãšã¿ããŒã«ãå ããŠé£çµ
åå¿çµäºåŸã®DNAãæ²æŸ±æ¡åããã (4) HDéºäŒåã®ã¯ããŒãã³ã° HDéºäŒåãæ¬ æãããã¬ããã¯ããªãŠã ã»ã©
ã¯ããã¢ãŒã¡ã³ã¿ã AJ12019NRRLBâ15346ïŒ
ãå容èãšããŠçšãããïŒâ(4)ã§è¿°ã¹ãæ¹æ³ã§åœ¢
質転æããããªã€ãã 30âã§10æ¥éå¹é€åŸãã¯ãã©ã ããšãã³ãŒã«è
æ§ã§ãã€ãŠãã€ãã¢ã»ãªã³èŠæ±æ§ãæ¶å€±ãã
AJ12020ïŒFERMâBP269ïŒãåŸãã (5) 圢質転ææ ªã®ãã©ã¹ãã解æ ãããã®æ ªãã(2)ã§è¿°ã¹ãæ¹æ³ã«ããã溶è液
ã調補ããã¢ã¬ããŒã¹ã²ã«é»æ°æ³³åæ³ã«ãããã©
ã¹ããDNAãæ€åºãããšããã7.64Mdã®ãã©ã¹
ãããæ€åºããpAJ210ãšåã¥ããã (6) åãã©ã³ã¹ããŒã¡ãŒã·ãšã³ AJ12020å ã®çµæããã©ã¹ããïŒpAJ210ïŒäž
ã«HDéºäŒåãååšããããšã確èªããããã«ã
ã®ãã©ã¹ããDNAãçšããã¬ããã¯ããªãŠã ã»
ã©ã¯ããã¢ãŒã¡ã³ã¿ã AJ12019ãå床圢質転æã
ãã çããã¯ãã©ã ããšãã³ãŒã«èæ§ã³ãããŒã®ã
ã¡ããããã10åãé£ãäžããã¢ã»ãªã³èŠæ±æ§ã
ãã¹ãããšããããããã®ããããèŠæ±æ§ãæ¶å€±
ããŠãããäžèšã®çµæããã©ã¹ããäžã«HDéºäŒ
åãååšããããšãæãããšãªã€ãã ïŒ pAJ210ãšpAJ212ã®å ±åã«ããâã¹ã¬ãªã
ã³ã®çç£ pAJ210ãïŒâ(4)ã§çšãã圢質転ææ³ã«ããã
ã¹ã¬ãªãã³çç£èãAHVèæ§çªç¶å€ç°æ ªãã¬ã
ãã¯ããªãŠã ã»ã©ã¯ããã¢ãŒã¡ã³ã¿ã AJ11188
ïŒFERMâP4190ïŒäžã«å°å ¥ããŠã圢質転ææ ªã
ã¯ãã ããšãã³ãŒã«èæ§ã§éžæããèæ ªAJ12021
ïŒFERMâBP270ïŒæ ªãåŸãã åæ§ã«pAJ212ã圢質転ææ³ã«ããAJ11188æ ª
ã«å°å ¥ãã圢質転ææ ªãããªã¡ããã«ã èæ§ã§éž
æããèæ ª11188âHKæ ªãåŸãã ããã«pAJ212ãAJ12021æ ªã«å°å ¥ããã¯ãã©
ã ããšãã³ãŒã«èæ§ãã€ããªã¡ããã«ã èæ§ã§éž
æããäºçš®ã®ãã©ã¹ããpAJ210ãšpAJ212ãå«ã
AJ12209ïŒFERMâP8106ïŒFERM BPâ877ïŒæ ª
ãåŸãã A11188æ ªãAJ12021æ ªã11188âHKæ ªåã³
AJ12209æ ªãã¹ã¬ãªãã³çç£å¹å°ïŒã°ã«ã³ãŒã¹10
ïœïŒdlïŒïŒNH4ïŒ2SO43ïœïŒdlïŒKH2PO40.1ïœïŒ
dlïŒMgSO4ã»7H2O0.04ïœïŒdlïŒFeSO4ã»
7H2O10mgïŒMnSO4ã»5H2O10mgïŒãã¢ãã³ã»
HCl300ÎŒïœïŒãããªãã³100ÎŒïœïŒã倧è±èçœ
é žå æ°Žå解液ïŒãå³æ¶²ãïŒ45ïœïŒdlïŒå šçªçŽ ãšã
ãŠïŒãã€ãœãã€ã·ã³25mgïŒdlïŒãã€ã·ã³30ïœïŒdlïŒ
PH7.0ïŒCaCO35ïœïŒdlïŒå¥ã«æ®ºèããïŒïŒ30âã«
ãŠ72æéæ¯çªå¹é€ãããå¹é€åŸãé å¿äžæž äžã®ïŒ¬
âã¹ã¬ãªãã³ãâãªãžã³åã³ïŒ¬âãã¢ã»ãªã³ã
ãã€ã¯ããã€ãªã¢ãã»ã€åã¯é«é液äœã¯ãããã°
ã©ãã€ãŒã§å®éããããã®çµæã第ïŒè¡šã«ç€ºã
ãã å°ãpAJ210ïŒpAJ212ã¯å¯èšãããAJ12209æ ª
ããéåžžã®æ¹æ³ãçšããŠåé¢ãããã åãAJ12019ã¯AJ12020ãããAJ12078ã¯
AJ12079åã¯12078âHKãããAJ11188ã¯
AJ12021ïŒAJ12080åã¯AJ12209ãã宿䞻现èã
æãªãããšãªã宿䞻现èäžã®è€åãã©ã¹ãããé€
å»ããããšã«ãã容æã«åŸãããããã©ã¹ããã¯
宿䞻ããèªç¶ã«å€±ãªãããããšããããããé€å»ã
æäœã«ãã€ãŠé€ãããšãã§ããïŒBact.Rev.ïŒ
36ïŒp361â405ïŒ1972ïŒïŒã é€å»æäœã®äžäŸã¯ä»¥äžã®éãã§ããïŒå®¿äž»ã®ç
è²ãäžå®å šã«é»å®³ããæ¿åºŠïŒïŒâ50ïœïŒdlïŒã®ã¢
ã¯ãªãžã³ãªã¬ã³ãžãå«ãå¹å°ã«ãïŒmlåœãçŽ104
现èçšåºŠã«ãªãæ§ã«å°éã®èæ ªãæ¥çš®ã宿䞻èã®
çè²ãäžå®å šã«é»å®³ããŠãã27â37âã§äžå€å¹é€
ããïŒJ.Bacteriol.ïŒ88ïŒ261ïŒ1964ïŒãå¹é€æ¶²ãå¯
倩å¹å°ã«å¡åžãã27â37âã§äžå€å¹é€ãããå¹å°
äžã«åºçŸããã³ãããŒã®ãã¡ãã¯ãã©ã ããšãã³
ãŒã«ïŒ10ÎŒïœïŒmlïŒåã¯ããªã¡ããã«ã ïŒ50ÎŒ
ïœïŒmlïŒã«æåæ§ã瀺ãæ ªããã©ã¹ãããé€å»ã
ããŠããæ ªã§å³ã¡ãAJ12019ïŒAJ12078åã³
AJ11188ã§ããã ãè¡šã å®æœäŸ ïŒ ïŒâ(5)ïŒïŒâ(8)åã³ïŒâ(4)ã§åŸã圢質転ææ ª
AJ12079æ ªïŒAJ12078ïŒpAJ211ïŒ12078âHKæ ª
ïŒAJ12078ïŒpAJ212ïŒåã³AJ12020æ ª
ïŒAJ12019ïŒpAJ210ïŒã¯ïŒ¬âã€ãœãã€ã·ã³ãçç£
ããã第ïŒè¡šã¯ãåèæ ªã®å¹é€äžæž äžã®ïŒ¬âã€ãœ
ãã€ã·ã³ãâã¹ã¬ãªãã³ãâãªãžã³ãåã³ïŒ¬
âãã¢ã»ãªã³ããã€ã¯ããã€ãªã¢ãã»ã€åã¯é«é
液äœã¯ãããã°ã©ãã€ãŒã§å®éããçµæã§ããã ãè¡šã ãªããå¹é€ã¯ãïŒã§è¿°ã¹ãã¹ã¬ãªãã³çç£å¹å°
ããã€ãœãã€ã·ã³åã³ãã€ã·ã³ãé€å»ããå¹å°ã
çšãã30âã72æéæ¯çªå¹é€ããã ãã¬ããã¯ããªãŠã ã»ã©ã¯ããã¢ãŒã¡ã³ã¿ã
AJ12028ïŒFERM BPâ272ïŒã¯ãâã€ãœãã€ã·
ã³çç£èã§ãããâïŒïŒâã¢ãããšãã«âã·ã¹
ãã€ã³ãAHVãβâããããã·ãã€ã·ã³èæ§
ã§ããã€ã·ã³æ é€èŠæ±æ ªãšããŠè²çš®ããããã®ã§
ããããã®èæ ªãïŒâ(8)åã³ïŒâ(5)ã§åŸããã©ã¹
ããpAJ212åã³pAJ210DNAã§åœ¢è³ªè»¢æããïŒ
â(4)ãšåæ§ã®æ¹æ³ã§ã¯ãã ããšãã³ãŒã«åã³ããª
ã¡ãããªã èæ§åœ¢è³ªè»¢ææ ª12028DKæ ªãåŸãã ãŸãã¢ã¬ããŒã¹ã²ã«é»æ°æ³³åã«ãã圢質転ææ ª
ãpAJ210åã³pAJ212ãæããŠããããšã確èªã
ãã ïŒã§è¿°ã¹ãã¹ã¬ãªãã³çç£å¹å°ããã€ãœãã€ã·
ã³ãé€å»ããå¹å°ã§30âã72æé圢質転ææ ªãå¹
é€ããçç£ãããâã€ãœãã€ã·ã³ãâã¹ã¬ãª
ãã³ãâãªãžã³ãåã³ïŒ¬âãã¢ã»ãªã³éããã€
ã¯ããã€ãªã¢ãã»ã€åã¯é«é液äœã¯ãããã°ã©ã
ã€ãŒã§å®éãããçµæã第ïŒè¡šã«ç€ºããã ãè¡šã
ã«é¢ãã詳ããã¯çµæããã©ã¹ãããæããã³ãª
ããã«ã 现èãçšããçºé µæ³ã«ããã¹ã¬ãªãã³å
ã¯ã€ãœãã€ã·ã³ã®è£œé æ³ã«é¢ããã ïŒåŸæ¥ã®æè¡ïŒ åŸæ¥ãçºé µæ³ã«ããâã¹ã¬ãªãã³ãâã€ãœ
ãã€ã·ã³ã補é ããå Žåã埮çç©ãšããŠã¯èªç¶ç
ããåé¢ããèæ ªãŸãã¯è©²èæ ªã®äººå·¥å€ç°æ ªãçš
ããããŠãããâã¹ã¬ãªãã³ãçç£ãã人工å€
ç°æ ªã¯æ°å€ãç¥ãããŠããããã®å€ãã¯Î±âã¢ã
ãâβâããããã·åèé žïŒä»¥äžãAHVãšç¥èš
ãããïŒã«èæ§ãããããã¬ããã¯ããªãŠã å±ãŸ
ãã¯ã³ãªããã¯ããªãŠã å±ã«å±ããŠãããããã
ã®åŸ®çç©ã¯åç10ã20ïŒ ã§ïŒ¬âã¹ã¬ãªãã³ãçç£
ãããäŸãã°ãç±³åœç¹èš±ç¬¬3582471å·æ现æžãå
第3580810å·æ现æžåã³ç¹å ¬æ47â34956å·å ¬å ±ã«
ã¯AHVèæ§ã瀺ãããã¬ããã¯ããªãŠã å±ããš
ã·ãšãªãã¢å±åã³ã³ãªããã¯ããªãŠã å±ã«å±ãã
å€ç°æ ªãçšããã¹ã¬ãªãã³ã®è£œé æ³ãé瀺ãããŠ
ããããŸãããã¬ããã¯ããªãŠã å±åã³ã³ãªãã
ã¯ããªãŠã å±ã«å±ããå€ç°æ ªã«ããã¹ã¬ãªãã³ã®
çç£ã«ã€ããŠç¹éæ51â54984å·å ¬å ±ãå53â
101591å·å ¬å ±ãå54â32693å·å ¬å ±ãå54â35285
å·å ¬å ±ãå54â35286å·å ¬å ±ãå54â35288å·å ¬
å ±ãå54â37886å·å ¬å ±åã³å54â92692å·å ¬å ±ã«
ãé瀺ãããŠããã äžæ¹ãç±³åœç¹èš±ç¬¬4278765å·æ现æžãç¹éæ55
â131397å·å ¬å ±åã³å56â15696å·å ¬å ±ã«ã¯çµæ
ããã©ã¹ããDNAãçšããŠåœ¢è³ªè»¢æããããšã·
ãšãªãã¢ã»ã³ãªãçšããŠã¹ã¬ãªãã³ã補é ããæ¹
æ³ã瀺ãããŠããã ããã«ã欧å·ç¹èš±åºé¡å ¬é第0066129å·æ现æž
ã«ã¯AHVã«å¯Ÿããèæ§ãã³ã³ãããŒã«ããæè²
äœDNAæçãæ¿å ¥ãããŠãããã©ã¹ãããæã
ãã³ãªããã«ã 现èã䜿çšããã¹ã¬ãªãã³ã®çç£
ã«ã€ããŠèšèŒãããŠãããæŽã«ã欧å·ç¹èš±åºé¡å ¬
é第0131171å·ã«ã¯ããã¢ã»ãªã³ããããã²ããŒ
ãŒãã³ãŒãããéºäŒåãæ¿å ¥ãããŠãããã©ã¹ã
ããæããã³ãªããã«ã 现èãå¹é€ããŠãã€ãœã
ã€ã·ã³åã¯ã¹ã¬ãªãã³ãçç£ããããšãèšèŒãã
ãŠããã ãŸããã€ãœãã€ã·ã³ã«é¢ããç¶æ³ãã¹ã¬ãªãã³
ã®å Žåãšé ·äŒŒããŠãããã€ãœãã€ã·ã³ãçç£ãã
埮çç©ãšããŠã¯ã€ãœãã€ã·ã³ãããããµã¡ãŒãã«
察ããŠèæ§ãæããã»ã©ãã¢ã®å€ç°æ ªïŒç¹å ¬æ52
â30593å·å ¬å ±ïŒããã®çè²ã«ãã€ã·ã³ãèŠæ±ãã
ã³ãªããã¯ããªãŠã ã»ã°ã«ã¿ãã«ã ã®å€ç°æ ªïŒç¹
å ¬æ47â38995å·å ¬å ±ïŒãAHVã«èæ§ãæããã
ã¬ããã¯ããªãŠã å±åã³ã³ãªããã¯ããªãŠã å±ã®
å€ç°æ ªïŒç¹å ¬æ40â2880å·å ¬å ±ïŒãAHVã«èæ§
ãæãããã€ãã®çè²ã«ãªãžã³ãå¿ èŠãšãããã¬
ããã¯ããªãŠã å±ã®å€ç°æ ªïŒç¹å ¬æ51â6237å·å ¬
å ±ïŒãAHVåã³ïŒ¯âã¡ãã«ã¹ã¬ãªãã³ã«èæ§ã
æãããã¬ããã¯ããªãŠã å±ã®å€ç°æ ªïŒç¹å ¬æ51
â21077å·å ¬å ±ïŒãâïŒïŒâã¢ãããšãã«ïŒâã·ã¹
ãã€ã³ã«èæ§ãæããã³ãªããã¯ããªãŠã å±ã®å€
ç°æ ªïŒç¹å ¬æ52â4629å·å ¬å ±ïŒãïŒâã¢ããâïŒ
âã¡ãã«ããªé ªé žã«èæ§ãæãããšã·ãšãªãã¢å±
ã®å€ç°æ ªïŒç¹éæ53â69881å·å ¬å ±ïŒãAHVåã³
ããªã¯ããã¢ã©ãã³ã«èæ§ãæãããã¬ããã¯ã
ãªãŠã å±ã®å€ç°æ ªïŒç¹éæ54â35287å·å ¬å ±ïŒã
ããã ãã®ã»ãçµæããã©ã¹ããDNAãçšããŠåœ¢è³ª
転æããããšã·ãšãªãã¢ã»ã³ãªã¯ã¢ããé žã®çç£
èœãé«ãããšãé瀺ãããŠããïŒäŸãã°ç±³åœç¹èš±
第4278765å·æ现æžåç §ïŒãæŽã«æ¬§å·ç¹èš±åºé¡å ¬é
第0071023å·ã«ã¯ãAHVã«å¯Ÿããèæ§ãã³ã³ã
ããŒã«ããæè²äœDNAæçãæ¿å ¥ãããŠããã
ã©ã¹ãããæããã³ãªããã«ã 现èã䜿çšããã€
ãœãã€ã·ã³ã®çç£ã«ã€ããŠèšèŒãããŠããã ïŒçºæã解決ããããšããåé¡ç¹ïŒ ããããªããäžèšã®ã¹ã¬ãªãã³åã¯ã€ãœãã€ã·
ã³çç£èœãæãã现èã®ã¹ã¬ãªãã³åã¯ã€ãœãã€
ã·ã³çç£èœã¯ãæŽã«æ¹åããå¿ èŠãããã ïŒåé¡ç¹ã解決ããããã®ææ®µïŒ åäžã®ãããªç¶æ³äžã§æ¬çºæè ãã¯ããã¢ã»ãª
ã³ãããŒãŒïŒä»¥äžãHKããšèšãïŒãã³ãŒããã
éºäŒåïŒä»¥äžãHKãéºäŒåãšèšãïŒãçµèŸŒãŸã
ãŠãã第ïŒã®ãã©ã¹ããåã³ãã¢ã»ãªã³ãããã
ã²ããŒãŒïŒä»¥äžãHDããšèšãïŒãã³ãŒãããéº
äŒåïŒä»¥äžãHDéºäŒåããšèšãïŒãçµèŸŒãŸããŠ
ãã第ïŒã®ãã©ã¹ãããè²ããã³ãªããã«ã 现è
ããã¹ã¬ãªãã³åã³ã€ãœãã€ã·ã³ã®é«ãçç£æ§ã
æããããšãèŠãåºããã ã³ãªããã«ã 现èã¯ã奜æ°æ§ãã°ã©ã éœæ§æ¡¿è
ã§ãããéæé žæ§ã§ããŒããŒã¹ã»ãããŠã¢ã«ã»ãª
ãã»ãã¿ãŒããã€ãã€ããã¯ããªãªããžãŒç¬¬ïŒç
599é ïŒ1974ïŒã«èšèŒãããŠããŠããã®ãã¡ç¹ã«
以äžã«äŸç€ºãããããªïŒ¬âã°ã«ã¿ãã³é žã倧éã«
çç£ãããã®ãç¥ãããŠããã ãã¬ããã¯ããªãŠã ã»ãã€ããªã«ã¿ã
ATCC14020 ãã¬ããã¯ããªãŠã ã»ãµãã«ããªãã€ã¯ã
ATCC14066 ãã¬ããã¯ããªãŠã ã»ã€ã³ããªãªãã€ã«ã
ATCC14068 ãã¬ããã¯ããªãŠã ã»ã©ã¯ããã¢ãŒã¡ã³ã¿ã
ATCC13869 ãã¬ããã¯ããªãŠã ã»ããŒãŠã
ATCC13825 ãã¬ããã¯ããªãŠã ã»ãã©ãã
ATCC13826 ãã¬ããã¯ããªãŠã ã»ããªã²ãã¿ãªã¹
ATCC19240 ã³ãªããã¯ããªãŠã ã»ã¢ã»ãã¢ã·ããã€ã«ã
ATCC13870 ã³ãªããã¯ããªãŠã ã»ã¢ã»ãã°ã«ã¿ãã¯ã
ATCC15806 ã³ãªããã¯ããªãŠã ã»ã«ã«ããš
ATCC15991 ã³ãªããã¯ããªãŠã ã»ã°ã«ã¿ãã¯ã
ATCC13032ïŒ13060 ã³ãªããã¯ããªãŠã ã»ãªãªãŠã
ATCC15990 ã³ãªããã¯ããªãŠã ã»ã¡ã©ã»ã³ãŒã©
ATCC17965 ãã¯ããã¯ããªãŠã ã»ã¢ã³ã¢ãã¢ãã€ã©ã
ATCC15354 ã³ãªããã«ã 现èã«ã¯äžèšã®ãããªã°ã«ã¿ãã³
é žçç£æ§ãæãããã®ã®ã»ãã«ãã°ã«ã¿ãã³é žç
ç£æ§ã倱ã€ããã®åã³ãªãžã³ãã¢ã«ã®ãã³çã®ã¢
ããé žãçç£ããå€ç°æ ªãå«ãŸããã æ¬çºæã«ãŠäœ¿çšããããã©ã¹ãããã¯ã¿ãŒã¯ã
ã³ãªããã«ã 现è现èå ã«ãããŠå¢æ®ãåŸããã®
ã§ããã°ã©ã®ãããªãã®ã§ãè¯ããå ·äœçã«äŸç€º
ããã°ã以äžã®ãã®ãããã (1) pAM330 ç¹éæ58â67699åç § (2) pHM1519 ç¹éæ58â77895åç § (3) pAJ655 ç¹éæ58â192900åç § (4) pAJ611 ç¹éæ58â192900åç § (5) pAJ1844 ç¹éæ58â192900åç § ã³ãªããã«ã 现è现èå ã§å¢æ®å¯èœãªãã©ã¹ã
ãã®ãã®ä»ã®äŸãšããŠã¯pCG1ïŒç¹éæ57â
134500ïŒãpCG2ïŒç¹éæ58â35197ïŒãpCG4ïŒ
pCG11ïŒç¹éæ57â183799ïŒãããã æ¬çºæã«ãããŠã¯ããããã®ãã©ã¹ããã®ãã¡
ïŒçš®é¡ã®ãã©ã¹ãããçšãããããHKã¯ãâ
ã¹ã¬ãªãã³åã³ïŒ¬âã€ãœãã€ã·ã³çæã®äžéäœã§
ããâãã¢ã»ãªã³ãããã¹ããã¢ã»ãªã³ãçæ
ããåå¿ã觊åªããé µçŽ ã§ããã HKéºäŒåãåé¢ããæ¹æ³ã¯ãã³ãªããã«ã 现
èã®HKéºäŒåãæããŠããæ ªããããŸãæè²äœ
éºäŒåãæœåºãïŒäŸãã°H.Saito and K.Miura
Biochem.Biophys.Acta72ïŒ619ïŒïŒ1963ïŒã®æ¹æ³
ã䜿çšã§ããïŒããããé©åœãªå¶éé µçŽ ã§åæã
ããã€ãã§ãã³ãªããã«ã 现èã§å¢æ®ãåŸããã©
ã¹ãããã¯ã¿ãŒã«æ¥ç¶ããåŸãããçµæãDNA
ãçšããŠã³ãªããã«ã 现èã®HKæ¬ æå€ç°æ ªã圢
質転æããããHKçæ掻æ§ãä¿æããã«ããã€
ãèæ ªãåé¢ããããã«ããHKéºäŒåãåé¢ã§
ããã HDã¯ãã¢ã¹ãã«ããŒãã»ãã¢ã«ãããããã
ã¢ã»ãªã³ãçæããåå¿ã觊åªããé µçŽ ã§ããã HDéºäŒåãåé¢ããæ¹æ³ããHKéºäŒåãå
é¢ããæ¹æ³ãšåãããã«ããŠè¡ãããšãã§ããã
å³ã¡ãã³ãªããã«ã 现èã®HDæ¬ æå€ç°æ ªãçšã
ãããšã«ãããHDéºäŒåãåŸãããšãã§ããã HKéºäŒååã¯HDéºäŒåãåŸãããã®ã³ãªã
ãã«ã 现èã¯ãéæ§åã®ãã®ã§ãã€ãŠããããã
ã¹ã¬ãªãã³ã«ãããã€ãŒãããã¯é»å®³ã解é€ãã
ããããªHKåã¯HDãæããå€ç°æ ªãDNAäŸäž
æ ªãšããŠçšããæ¹ãããããçµæãåŸãããããš
ãå€ãããã®ãããªå€ç°æ ªã¯ãAHVã«èæ§ã®å€
ç°æ ªãšããŠåŸãããšãã§ããã HKéºäŒååã¯HDéºäŒåãåŸãããã«æè²äœ
éºäŒåãåæããéã«ãåæåå¿æéçã調ç¯ã
ãŠåæã®çšåºŠã調ç¯ããã°ãå·Ÿåºãçš®é¡ã®å¶éé µ
çŽ ã䜿çšã§ããã ãã¯ã¿ãŒDNAã¯ãæè²äœéºäŒåãåæããé
ã«çšããããå¶éé µçŽ ã«ããåæããããŸãã¯æ
è²äœDNAåæãã©ã°ã¡ã³ãåã³åæããããã¯
ã¿ãŒDNAã®ããããã®äž¡ç«¯ã«çžè£çãªå¡©åºé å
ãæãããªãªãŽãã¯ã¬ãªãããæ¥ç¶ããããŠãã€
ãã§ãã©ã¹ãããã¯ã¿ãŒãšæè²äœDNAãã©ã°ã¡
ã³ããšã®ã©ã€ã²ãŒã·ãšã³åå¿ã«ä»ãããã ãã®ããã«ããŠåŸããããæè²äœDNAãšãã¯
ã¿ãŒãã©ã¹ãããšã®çµæãDNAãã³ãªããã«ã
现èã«å±ããå容èãžå°å ¥ããã«ã¯ããšã·ãšãªã
ã¢ã»ã³ãªïŒ«â12ã«ã€ããŠå ±åãããŠããæ§ãª
ïŒMandelïŒM.and HigaïŒA.ïŒJ.Mol.ïŒBiol.ïŒ
53ïŒ159ïŒ1970ïŒå容è现èãå¡©åã«ã«ã·ãŠã ã§åŠ
çããŠDNAã®ééæ§ãå¢ãæ¹æ³ããŸãã¯ããã«
ã¹ã»ãºãããªã¹ã«ã€ããŠå ±åãããŠããæ§ã«
ïŒDuncanïŒC.H.ïŒWilsonïŒG.A.and YoungïŒF.
E.ïŒGeneïŒïŒïŒ153ïŒ1977ïŒïŒçŽ°èãDNAãåã
蟌ã¿åŸãæ§ã«ãªãå¢æ®æ®µéïŒããããã³ã³ããã³
ãã»ã«ïŒã«å°å ¥ããæ¹æ³ã«ããå¯èœã§ããããã
ãã¯ãããã«ã¹ã»ãºãããªã¹ãæŸç·èé¡ããã³é µ
æ¯ã«ã€ããŠç¥ãããŠããæ§ã«ïŒChangïŒS.and
ChoenïŒS.N.ïŒMolec.Gen.ïŒGenet.ïŒ168ïŒ111
ïŒ1979ïŒïŒBibbïŒM.J.ïŒWardïŒJ.M.and
HopwoodïŒO.A.ïŒNatureïŒ274ïŒ398ïŒ1978ïŒïŒ
HinnenïŒA.ïŒHicksïŒJ.B.and FinkïŒG.R.ïŒ
Proc.Natl.Acad.Sci.USAïŒ75 1929ïŒ1978ïŒïŒã
DNAå容èãããã©ã¹ããDNAã容æã«åã蟌
ãããããã©ã¹ããŸãã¯ã¹ããšããã©ã¹ãã«ããŠ
ãã©ã¹ãããDNAå容èã«å°å ¥ããããšãå¯èœ
ã§ããã ããããã©ã¹ãæ³ã§ã¯äžèšã®ããã«ã¹ã»ãºãã
ãªã¹ã«ãããŠäœ¿çšãããŠããæ¹æ³ã§ãå åé«ãé »
床ãåŸãããšãã§ããããç¹éæ57â183799ã«èš
èŒãããã³ãªããã¯ããªãŠã å±ãŸãã¯ãã¬ããã¯
ããªãŠã å±ã®ããããã©ã¹ãã«ããªãšãã¬ã³ã°ãª
ã³ãŒã«ãŸãã¯ããªããã«ã¢ã«ã³ãŒã«ãšäºäŸ¡éå±ã€
ãªã³ãšã®ååšäžã«DNAããšã蟌ãŸããæ¹æ³ãåœ
ç¶å©çšã§ãããããªãšãã¬ã³ã°ãªã³ãŒã«ãŸãã¯ã
ãªããã«ã¢ã«ã³ãŒã«ã®ãããã«ãã«ã«ããã·ã¡ã
ã«ã»ã«ããŒã¹ãããã¹ãã©ã³ããã€ã³ãŒã«ããã«
ãããã¯F68ïŒã»ã«ã瀟ïŒãªã©ã®æ·»å ã«ãã€ãŠ
DNAã®ãšã蟌ã¿ãä¿é²ãããæ¹æ³ã§ãåçã®çµ
æãåŸãããã ããããŠãHKéºäŒååã¯HDéºäŒåãçµèŸŒãŸ
ãããã©ã¹ãããåŸãããããã®äºã€ã®çµæãã
ã©ã¹ãããæããã³ãªããã«ã 现èã¯ãäºã€ã®ã
ã©ã¹ããã现èå ã«å ±åããããã®ã§ããã°ã€ãœ
ãã€ã·ã³åã¯ã¹ã¬ãªãã³ãçç£ãããå ±åãããª
ããã®ã§ããã°ãå ±åããããã©ã¹ãããã¯ã¿ãŒ
ã«å®¹æã«HKéºäŒååã³HDéºäŒåã移ãããã
ããšãã§ããã ã³ãªããã«ã 现è现èå ã§äºã€ã®ãã©ã¹ããã
å ±åãããããã«ã¯ãæ¬çºæè ãã®ç 究ã«ãã
ã°ããã©ã¹ããã®è€è£œéå§é åãç°ãªããã®ã§ã
ãã°å ±åãããããè€è£œéå§é åãåããã®ã§ã
ã€ãŠããä»ã®é åã倧ããç°ãªãã°å ±åãããã
ãšãããã äºã€ã®ãã©ã¹ãããä¿æããããã®ã³ãªããã«
ã 现è宿䞻ã¯ãã¹ã¬ãªãã³åã¯ã€ãœãã€ã·ã³èŠæ±
æ ªã§ãã€ãŠããããåéæ§æ ªã§ãã€ãŠããããã
ããAHVèæ§ãæããå€ç°æ ªãé垞奜ãŸããçµ
æãåŸãããç¹ã«ããé«ãã¹ã¬ãªãã³åã¯ã€ãœã
ã€ã·ã³çç£èœãæããèæ ªãæã奜ãŸããçµæã
åŸãããã åŸãããã¹ã¬ãªãã³åã¯ã€ãœãã€ã·ã³çç£èã
å¹é€ããæ¹æ³ã¯ãåŸæ¥ã®ã¹ã¬ãªãã³åã¯ã€ãœãã€
ã·ã³çç£èã®å¹é€æ¹æ³ãšç¹ã«å€ããªããå³ã¡ãå¹
å°ãšããŠã¯ãççŽ æºãçªçŽ æºãç¡æ©ã€ãªã³ãæŽã«
å¿ èŠã«å¿ããŠã¢ããé žããã¿ãã³çã®ææ©åŸ®éæ
é€çŽ ãå«æããéåžžã®ãã®ã§ãããççŽ æºãšããŠ
ã¯ãã°ã«ã³ãŒã¹ãã·ãŠã¯ããŒã¹ãã©ã¯ããŒã¹çå
ã³ãããã®å«æãã柱ç²å æ°Žå解液ãããšã€ãç³
èçãçšãããããçªçŽ æºãšããŠã¯ãã¢ã³ã¢ãã¢
ã¬ã¹ãã¢ã³ã¢ãã¢æ°Žãã¢ã³ã¢ããŠã å¡©ãã®ä»ã䜿
çšã§ããã å¹é€ã¯å¥œæ°çæ¡ä»¶äžã§å¹å°ã®PHåã³æž©åºŠãé©å®
調ç¯ãã€ã€ãå®è³ªçã«ã¹ã¬ãªãã³åã¯ã€ãœãã€ã·
ã³ã®çç£èç©ãåæ¢ãããŸã§è¡ãªãããã ããããŠå¹é€æ¶²äžã«ã¯èéã®ã¹ã¬ãªãã³åã¯ã€
ãœãã€ã·ã³ãçæèç©ããããå¹é€æ¶²ããã¹ã¬ãª
ãã³åã¯ã€ãœãã€ã·ã³ãæ¡åããã«ã¯ãéåžžã®æ¹
æ³ãé©çšã§ããã å®æœäŸ ïŒ ïŒ ãã¢ã»ãªã³ãããŒãŒïŒHKïŒéºäŒåã®ã¯ããŒ
ãã³ã° (1) HKéºäŒåãå«ãæè²äœDNAã®èª¿è£œ ãã¬ããã¯ããªãŠã ã»ã©ã¯ããã¢ãŒã¡ã³ã¿ã
ATCC13869ã®AHVèæ§çªç¶å€ç°æ ªAJ11188
ïŒFERMâP4190FERM BPâ577ïŒïŒç¹å ¬æ56â
3038ïŒãïŒã®CMGå¹å°ïŒãããã³ïŒïœïŒdlã
é µæ¯ãšãã¹ïŒïœïŒdlãã°ã«ã³ãŒã¹0.5ïœïŒdlãå
ã³NaCl0.5ïœïŒdlãå«ã¿ãPH7.2ã«èª¿æŽãããã®ïŒ
ã«æ€èãã30âã§çŽïŒæéæ¯çªå¹é€ãè¡ãªãã察
æ°å¢æ®æã®èäœãéããããã®èäœããªãŸããŒ
ã ã»SDSã§æº¶èãããã®ã¡ãéåžžã®ããšããŒã«åŠ
çæ³ã«ãããæè²äœDNAãæœåºç²Ÿè£œããæçµç
ã«3.5mgã®DNAãåŸãã (2) ãã¯ã¿ãŒDNAã®èª¿è£œ ãã¯ã¿ãŒãšããŠpAJ1844ïŒååé5.4ã¡ã¬ãã«ã
ã³ïŒãçšãããã®DNAã次ã®æ§ã«ããŠèª¿è£œããã ãŸãpAJ1844ããã©ã¹ãããšããŠä¿æãããã¬
ããã¯ããªãŠã ã»ã©ã¯ããã¢ãŒã¡ã³ã¿ã AJ12037
ïŒFERMâP7234ïŒã100mlã®CMGå¹å°ã«æ¥çš®ãã
30âã§å¯Ÿæ°å¢æ®æåŸæãŸã§å¹é€ããã®ã¡ããªãŸã
ãŒã SDSåŠçã«ãã溶èããã30000Ãïœ30åã®
è¶ é å¿ã«ããäžæž ãåŸããããšããŒã«åŠçã®ã®
ã¡ãïŒå®¹ã®ãšã¿ããŒã«ãå ããŠDNAãæ²æŸ±åå
ããããããå°éã®TENç·©è¡æ¶²ïŒ20mMããªã¹
å¡©é žå¡©ã20mM NaClã1mM EDTAïŒPH8.0ïŒã«
溶解åŸãã¢ã¬ããŒã¹ã²ã«é»æ°æ³³åã«ããåé¢åŸã
åãåºããŠpAJ1844ãã©ã¹ããDNAçŽ15ÎŒïœãåŸ
ãã (3) æè²äœDNAæçã®ãã¯ã¿ãŒãžã®æ¿å ¥ (1)ã§åŸãæè²äœDNA20ÎŒïœãš(2)ã§åŸããã©ã¹
ããDNA10ÎŒïœãšãå¶éãšã³ããã¯ã¬ã¢ãŒãŒPst
ã§ããããã37âãïŒæéåŠçããå®å šã«åæ
ããã65â10åã®ç±åŠçåŸãäž¡åå¿æ¶²ãæ··åãã
ATPåã³ãžããªã¹ã¬ã€ããŒã«ååšäžãT4ãã¢ãŒ
ãžç±æ¥ã®DNAãªã¬ãŒãŒã«ãã€ãŠ10âã24æé
DNAéã®é£çµåå¿ãè¡ã€ãã65âïŒåã®ç±åŠç
åŸãåå¿æ¶²ã«ïŒå容ã®ãšã¿ããŒã«ãå ããŠé£çµå
å¿çµäºåŸã®DNAãæ²æŸ±æ¡åããã (4) HKéºäŒåã®ã¯ããŒãã³ã° HKéºäŒåãæ¬ æãããã¬ããã¯ããªãŠã ã»ã©
ã¯ããã¢ãŒã¡ã³ã¿ã AJ12078ãå容èãšããŠçšã
ãã 圢質転æã®æ¹æ³ãšããŠã¯ãããããã©ã¹ããã©
ã³ã¹ããªãŒã¡ãŒã·ãšã³æ³ãçšããããŸããèæ ªã
ïŒmlã®CMG液äœå¹å°ã§å¯Ÿæ°å¢æ®æã®åæãŸã§å¹
é€ããããã·ãªã³ïŒ§ã0.6ãŠãããïŒmlæ·»å åŸã
ããã«1.5æéæ¯çªå¹é€ããé å¿åé¢ã«ããèæ ª
ãéããèäœã0.5Mã·ãŠãŒã¯ããŒã¹ã20mMã
ã¬ã€ã³é žã20mMå¡©åãã°ãã·ãŠã ã3.5ïŒ ãã
ãã»ã€ããã¹ïŒDifcoïŒãããªãSMMPå¹å°ïŒPH
6.5ïŒ0.5mlã§æŽæµããã次ãã§10mgïŒmlã®ãªãŸã
ãŒã ãå«ãSMMPå¹å°ã«æžæ¿ã30âã§20æéã
ãããã©ã¹ãåãå³ã€ãã6000Ãïœã10åéé å¿
åé¢åŸãããããã©ã¹ããSMMPã§æŽæµã0.5ml
ã®SMMPã«å床æžæ¿ããããã®æ§ã«ããŠåŸãã
ãããããã©ã¹ããš(3)ã§èª¿è£œããDNA10ÎŒïœã
5mM EDTAååšäžã§æ··åããããªãšãã¬ã³ã°ãª
ã³ãŒã«ãæçµæ¿åºŠã30ïŒ ã«ãªãæ§ã«æ·»å ããåŸã
DNAãããããã©ã¹ãã«åã蟌ãŸããçºã«å®€æž©
ã«ïŒåéæŸçœ®ããããã®ããããã©ã¹ãã
SMMPå¹å°ïŒmlã§æŽæµåŸãSMMPå¹å°ïŒmlã«å
æžæ¿ãã圢質çºçŸã®çºã30âã§ïŒæéå¹é€ããã
ãã®å¹é€æ¶²ãPH7.0ã®ããããã©ã¹ãåçå¹å°äž
ã«å¡åžãããããããã©ã¹ãåçå¹å°ã¯èžçæ°ŽïŒ
ãããããªã¹ïŒããããã·ã¡ãã«ïŒã¢ããã¡ã¿
ã³12ïœãECl0.5ïœãã°ã«ã³ãŒã¹10ïœãMgCl2ã»
6H2O8.1ïœãCaCl2ã»2H2O2.2ïœããããã³ïŒïœã
ç²æ«é µæ¯ãšãã¹ïŒïœãã«ã¶ããé žïŒDifco瀟ïŒïŒ
ïœãK2HPO40.2ïœãã³ãã¯é žãããªãŠã 135ïœã
å¯å€©ïŒïœåã³ã¯ãã©ã ããšãã³ãŒã«3ÎŒïœïŒãå«
ãã 30âã§ïŒé±éå¹é€åŸãçŽ10000åã®ã¯ãã©ã ã
ãšãã³ãŒã«èæ§ã³ãããŒãåºçŸããŠããã®ã§ãã
ãã¹ã¬ãªãã³ãå«ãŸãªãå¹å°ïŒThræ¬ å¹å°ïŒïŒïŒ
ïŒ ã°ã«ã³ãŒã¹ãïŒïŒ ç¡«é žã¢ã³ã¢ããŠã ã0.25ïŒ å°¿
çŽ ã0.1ïŒ ããé žäºæ°ŽçŽ ã«ãªãŠã ã0.04ïŒ ç¡«é žã
ã°ãã·ãŠã ïŒæ°Žå¡©ã2ppméã€ãªã³ã2ppmãã³ã¬
ã³ã€ãªã³ã200ÎŒïœïŒãµã€ã¢ãã³å¡©é žå¡©ã50ÎŒ
ïœïŒããªãã³ãPH7.0ãå¯å€©1.8ïŒ ïŒã«ã¬ããªã«
ããã¯ãã©ã ããšãã³ãŒã«èæ§ã§ãã€ãŠãã€ã¹ã¬
ãªãã³èŠæ±æ§ãæ¶å€±ããèæ ªïŒæ ªãåŸãã (5) 圢質転ææ ªã®ãã©ã¹ãã解æ ãããã®æ ªãã(2)ã§è¿°ã¹ãæ¹æ³ã«ããã溶è液
ã調補ããã¢ã¬ããŒã¹ã²ã«é»æ°æ³³åæ³ã«ãããã
ã©ã¹ããDNAãæ€åºãããšãããïŒæ ªã§ãã¯ã¿
ãŒã®pAJ1844ãããæããã«å€§ããªãã©ã¹ããã
æ€åºãããã ãããã®ãã¡ã®ä»£è¡šæ ªãAJ12079ïŒFERMâ
P7237ïŒFERM BPâ578ïŒãšåã¥ããã (6) åãã©ã³ã¹ããŒã¡ãŒã·ãšã³ AJ12079å ã®çµæããã©ã¹ããïŒpAJ211ïŒäž
ã«HKéºäŒåãååšããããšã確èªããããã«ã
ã®ãã©ã¹ããDNAãçšããã¬ããã¯ããªãŠã ã»
ã©ã¯ããã¢ãŒã¡ã³ã¿ã AJ12078ãå床圢質転æã
ãã çããã¯ãã©ã ããšãã³ãŒã«èæ§ã³ãããŒã®ã
ã¡ããããã10åãé£ãäžãã¹ã¬ãªãã³èŠæ±æ§ã
ãã¹ããããšããããããã®ããããèŠæ±æ§ãæ¶
倱ããŠãããäžèšã®çµæããã©ã¹ããäžã«HKéº
äŒåãååšããããšãæãããšãªã€ãã ïŒ HKéºäŒåãæãã2.9kb.PstDNAæçã®
pAJ224ãžã®ç§»ãå€ã (1) ããªã¡ãããªã èæ§ãæãããã¯ã¿ãŒãã©
ã¹ããpAJ228ã®é æ ãã¬ããã¯ããªãŠã ã»ã©ã¯ããã¢ãŒã¡ã³ã¿ã
AJ12036ããå€ç°èªå°ãããããªã¡ãããªã èæ§
å€ç°æ ªAJ12146ïŒFERMâP7672ïŒFERM BPâ
785ïŒãïŒã®CMGå¹å°ïŒãããã³ïŒïœïŒdlãé µ
æ¯ãšãã¹ïŒïœïŒdlãã°ã«ã³ãŒã¹0.5ïœïŒdlãåã³
NaCl0.5ïœïŒdlãå«ã¿ãPH7.2ã«èª¿æŽãããã®ïŒã«
æ€èãã30âã§çŽïŒæéæ¯çªå¹é€ãè¡ãªãã察æ°
å¢æ®æã®èäœãéããããã®èäœããªãŸããŒã ã»
SDSã§æº¶èãããã®ã¡ãéåžžã®ããšããŒã«åŠçæ³
ã«ãããæè²äœDNAãæœåºç²Ÿè£œããæçµçã«3.0
mgã®DNAãåŸãã (2) ãã¯ã¿ãŒãšããŠpAM330ãçšããã
pAM330ã¯æ¬¡ã®æ§ã«ããŠèª¿è£œããã ãŸãpAM330ããã©ã¹ãããšããŠä¿æãããã¬
ããã¯ããªãŠã ã»ã©ã¯ããã¢ãŒã¡ã³ã¿ã
ATCC13869ã100mlã®CMGå¹å°ã«æ¥çš®ãã30â
ã§å¯Ÿæ°å¢æ®æåŸæãŸã§å¹é€ããã®ã¡ããªãŸããŒã
SDSåŠçã«ãã溶èããã30000Ãïœ30åã®è¶ é
å¿ã«ããäžæž ãåŸããããšããŒã«åŠçã®ã®ã¡ãïŒ
容ã®ãšã¿ããŒã«ãå ããŠDNAãæ²æŸ±ç©ãšããŠå
åããããããå°éã®TENç·©è¡æ¶²ïŒ20mMããª
ã¹å¡©é žå¡©ã20mM NaClã1mM EDTAïŒPH8.0ïŒ
ã«æº¶è§£åŸãã¢ã¬ããŒã¹ã²ã«é»æ°æ³³åã«ããåé¢
åŸãåãåºããŠpAM330ãã©ã¹ããDNAçŽ15ÎŒïœ
ãåŸãã (3) (1)ã§åŸãæè²äœDNA20ÎŒïœãš(2)ã§åŸãã
ã©ã¹ããDNA10ÎŒïœãšãå¶éãšã³ããã¯ã¬
ã¢ãŒãŒMbo ã§ããããã37âã30åéåŠ
çããéšååæããã65âã10åã®ç±åŠç
åŸãäž¡åå¿æ¶²ãæ··åããATPåã³ãžããªã¹
ã¬ã€ããŒã«ååšäžãT4ãã¢ãŒãžç±æ¥ã®DNA
ãªã¬ãŒãŒã«ãã€ãŠ10âã24æéDNAéã®é£
çµåå¿ãè¡ã€ãã65âãïŒåã®ç±åŠçåŸãå
å¿æ¶²ã«ïŒå容ã®ãšã¿ããŒã«ãå ããŠé£çµåå¿
çµäºåŸã®DNAãæ²æŸ±æ¡åããã (4) ããªã¡ãããªã æåæ§ã®ãã¬ããã¯ããªãŠ
ã ã»ã©ã¯ããã¢ãŒã¡ã³ã¿ã AJ12036ãå容è
ãšããŠçšããã 圢質転æã®æ¹æ³ãšããŠã¯ãïŒâ(4)ã§è¿°ã¹ãæ¹æ³
ãçšãããããªã¡ãããªã ïŒSigma瀟ïŒ25ÎŒïœïŒ
ãå«ãåçå¹å°ã«ãŠã30âã§ïŒé±éå¹é€åŸãçŽ
100åã®ã³ãããŒãåºçŸããŠããã®ã§ããããã
ãªã¡ãããªã ãå«ãæå°å¹å°ïŒïŒïŒ ã°ã«ã³ãŒã¹ã
ïŒïŒ ç¡«é žã¢ã³ã¢ããŠã ã0.25ïŒ å°¿çŽ ã0.1ïŒ ãã
é žäºæ°ŽçŽ ã«ãªãŠã ã0.04ïŒ ç¡«é žãã°ãã·ãŠã ïŒæ°Ž
å¡©ã2ppméã€ãªã³ã2ppmãã³ã¬ã³ã€ãªã³ã
200ÎŒïœïŒãµã€ã¢ãã³å¡©é žå¡©ã50ÎŒïœïŒãããª
ã³ãPH7.0ãå¯å€©1.8ïŒ ãããªã¡ãããªã 50ÎŒïœïŒ
mlïŒã«ããªã«ããããªã¡ãããªã èæ§ïŒæ ªãåŸ
ãã (5) ãããã®æ ªããïŒâ(2)ã§è¿°ã¹ãæ¹æ³ã«ã
ãã溶è液ã調補ããã¢ã¬ããŒã¹ã²ã«é»æ°æ³³
åæ³ã«ããããã©ã¹ããDNAãæ€åºãããš
ããããã¯ã¿ãŒã®pAM330ãããæããã«å€§
ããªãã©ã¹ãããæ€åºãããããã®æ ªã
AJ12147ïŒFERMâP7673ïŒFERM BPâ
786ïŒãšåä»ããã (6) AJ12147ãæãããã©ã¹ããïŒpAJ228ïŒ
äžã«ããªã¡ãããªã èæ§éºäŒåãååšããã
ãšã確èªããããããã®ãã©ã¹ããDNAã
çšããã¬ããã¯ããªãŠã ã»ã©ã¯ããã¢ãŒã¡ã³
ã¿ã AJ12036ãå床ã圢質転æããã çããããªã¡ãããªã èæ§ãæããã³ãããŒã®
ãã¡ãããã10åãé£ãäžãã¢ã¬ããŒã¹é»æ°æ³³å
æ³ã«ãããã©ã¹ããNAãæ€åºãããšããããã
ãã®ãããã«ãpAJ228ãšåã倧ããã®ãã©ã¹ã
ããååšããŠãããäžèšçµæããã©ã¹ããäžã«ã
ãªã¡ãããªã èæ§ãè¡šçŸããéºäŒåãååšããã
ãšãæãããšãªã€ãã (7) ããªã¡ãããªã èæ§ãã©ã¹ããïŒpAJ228ïŒ
ãžã®HKéºäŒåïŒpAJ211ã®2.9kb PstDNA
æçïŒã®ç§»ããã pAJ228ã¯Pstã«ããåæéšäœãæããªããã
HKéºäŒåãå«ãpAJ211ç±æ¥ã®2.9kbã®Pstæ
çããã®ãŸãŸç§»ããããããšã¯äžå¯èœã§ãããã
ãã§ç¬¬ïŒå³ã«ç€ºããæ¹æ³ã§ç§»ããããè¡ã€ããå³
ã¡pAJ211ããå¶éé µçŽ Pstã«ããéšååæã§å
ãåºãã2.9kbã«DNAæçã®äž¡ç«¯ã«BamHïŒ
SalïŒPstã®åæéšäœããã€ã第ïŒå³ã«ç€ºãã
ãããªåæãªãªãŽãã¯ã¬ãªãããªã³ã«ãŒãT4ã
ã¢ãŒãžç±æ¥ã®DNAãªã¬ãŒãŒãçšããŠé£çµããã
åŸãå¶éé µçŽ BamHã§åæãããåŸããã
DNAæ··åç©ãã¢ã¬ããŒã¹ã»ã²ã«é»æ°æ³³åã«ããã
çŽ2.9kbã®DNAæçãåé¢æœåºãããšã¿ããŒã«æ²
柱ã«ããDNAæçãååãããããããŠåŸãã
ãDNAæçã¯2.9kbã®HKéºäŒåãå«ãDNAã®
䞡端ã«PstïŒSalïŒBamHã®åæéšäœã䞊
ãã æ§é ã«ãªã€ãŠããïŒç¬¬ïŒå³ïŒã äžæ¹ãpAJ228ã¯å¶éé µçŽ Mboã§éšååæã
ãåŸ65âïŒ10åã®ç±åŠçãããããã®åå¿æ¶²ã«å
è¿°ã®æ¹æ³ã§åŸãHKéºäŒåãå«ãDNAæçãå
ããT4DNAãªã¬ãŒãŒã§DNAéã®é£çµåå¿ãè¡
ã€ããåå¿åŸã65âãïŒåã®ç±åŠçãããïŒå容
ã®ãšã¿ããŒã«ã®æ·»å ã«ããæ²æŸ±æ¡åãããDNA
ããïŒâ(4)ãšåæ§ã®æ¹æ³ã«ããããªã¡ããã«ã æ
åæ§ã§ããã€ãHKéºäŒåãæ¬ æãããã¬ããã¯
ããªãŠã ã»ã©ã¯ããã¢ãŒã¡ã³ã¿ã AJ12078ãå容
èãšããŠããããã©ã¹ããã©ã³ã¹ããªãŒã¡ãŒã·ãš
ã³ãè¡ã€ãåŸãããªã¡ããã«ã 100ÎŒïœïŒmlãå«
ãããããã©ã¹ãåçå¹å°ã§å¹é€ããçµæçŽ200
åã®åçã³ãããŒãåºçŸããŠããã ãããããªã¡ããã«ã ã200ÎŒïœïŒmlãå«ã¿å
容èã®èŠæ±ç©è³ªã§ããã¹ã¬ãªãã³ãå«ãŸãªãæå°
å¹å°ã«ã¬ããªã«ããããªã¡ããã«ã èæ§ã§ããã€
ã¹ã¬ãªãã³èŠæ±æ§ãæ¶å€±ããèæ ªïŒæ ªãåŸããã
ããã®æ ªããïŒâ(2)ã«ç€ºããæ¹æ³ã«ãã溶è液ã
調補ããã¢ã¬ããŒã¹ã»ã²ã«é»æ°æ³³åã«ãããã©ã¹
ããDNAãæ€åºãããšãã14.9kbïŒ12.2kbïŒ
9.6kbïŒ6.5kbã®ãã©ã¹ãããæ€åºãããããã®ã
ã¡æãååéã®å°ãã6.5kbã®ãã©ã¹ããã
pAJ212ãšåä»ãããpAJ212ãä¿æããæ ªã1278
âHKæ ªãšåä»ããã pAJ212ã¯ãHKéºäŒåãé€ããŠãpAJ224ãšã
ãŠããã¬ããã¯ããªãŠã ã»ã©ã¯ããã¢ãŒã¡ã³ã¿ã
AJ12196ïŒFERMâP8015ïŒFERM BPâ787ïŒãš
ããŠå¯èšãããŠããã (9) åãã©ã³ã¹ããŒã¡ãŒã·ãšã³ AJ12078âHKå ã®çµæããã©ã¹ãã
ïŒpAJ212ïŒäžã«HKéºäŒåãååšããããšã確èª
ããããã«ãã®ãã©ã¹ããDNAãçšããã¬ãã
ã¯ããªãŠã ã»ã©ã¯ããã¢ãŒã¡ã³ã¿ã AJ12078ãå
床圢質転æããã çããããªã¡ããã«ã èæ§ã³ãããŒã®ãã¡ãã
ããã10åãé£ãäžãã¹ã¬ãªãã³èŠæ±æ§ããã¹ã
ãããšããããããã®ããããèŠæ±æ§ãæ¶å€±ããŠ
ãããäžèšã®çµæããã©ã¹ããäžã«HKéºäŒåã
ååšããããšãæãããšãªã€ãã ïŒ ãã¢ã»ãªã³ããããã²ããŒãŒïŒHDïŒéºäŒå
ã®ã¯ããŒãã³ã° (1) HDéºäŒåãå«ãæè²äœDNAã®èª¿è£œ ãã¬ããã¯ããªãŠã ã»ã©ã¯ããã¢ãŒã¡ã³ã¿ã
ATCC13869ã®Î±âã¢ããâβâãã€ãããã·å
èé žèæ§çªç¶å€ç°æ ªAJ11188ïŒFERMâP4190ïŒ
ïŒç¹å ¬æ56â3038ïŒãïŒã®CMGå¹å°ïŒãããã³
ïŒïœïŒdlãé µæ¯ãšãã¹ïŒïœïŒdlãã°ã«ã³ãŒã¹0.5
ïœïŒdlãåã³NaCl0.5ïœïŒdlãå«ã¿ãPH7.2ã«èª¿æŽ
ãããã®ïŒã«æ€èãã30âã§çŽïŒæéæ¯çªå¹é€ã
è¡ãªãã察æ°å¢æ®æã®èäœãéããããã®èäœã
ãªãŸããŒã ã»SDSã§æº¶èãããã®ã¡ãéåžžã®ããš
ããŒã«åŠçæ³ã«ãããæè²äœDNAãæœåºç²Ÿè£œãã
æçµçã«3.6mgã®DNAãåŸãã (2) ãã¯ã¿ãŒDNAã®èª¿è£œ ãã¯ã¿ãŒãšããŠpAJ1844ïŒç¹éæ58â192900å
ç §ïŒïŒååé5.4ã¡ã¬ãã«ãã³ïŒãçšãããã®
DNAã次ã®æ§ã«ããŠèª¿è£œããã pAJ1844ã¯ãã³ãªããã¯ããªãŠã ã»ã°ã«ã¿ããŠ
ã ATCC13058ãæããŠãããã©ã¹ããpHM1519
ããèªå°ããããã®ã§ããã ãŸãpAJ1844ããã©ã¹ãããšããŠä¿æãããã¬
ããã¯ããªãŠã ã»ã©ã¯ããã¢ãŒã¡ã³ã¿ã AJ12037
ïŒFERMâP7234ïŒïŒFERMâBP577ïŒã100mlã®
CMGå¹å°ã«æ¥çš®ãã30âã§å¯Ÿæ°å¢æ®æåŸæãŸã§
å¹é€ããã®ã¡ããªãŸããŒã SDSåŠçã«ãã溶èã
ãã30000Ãïœ30åã®è¶ é å¿ã«ããäžæž ãåŸãã
ããšããŒã«åŠçã®ã®ã¡ãïŒå®¹ã®ãšã¿ããŒã«ãå ã
ãŠDNAãæ²æŸ±ååããpAJ1844ãã©ã¹ããDNA
çŽ80ÎŒïœãåŸãã (3) æè²äœDNAæçã®ãã¯ã¿ãŒãžã®æ¿å ¥ (1)ã§åŸãæè²äœDNA40ÎŒïœããå¶éé µçŽ Pst
ã0.124ãŠãããã§30âã10åééšåæ¶åããã (2)ã§åŸãã©ã¹ããDNA10ÎŒïœã¯å¶éãšã³ãã
ã¯ã¬ã¢ãŒãŒPstã§37âãïŒæéåŠçããå®å šã«
åæããã65â10åã®ç±åŠçåŸãäž¡åå¿æ¶²ãæ··å
ããATPåã³ãžããªã¹ã¬ã€ããŒã«ååšäžãT4ã
ã¢ãŒãžç±æ¥ã®DNAãªã¬ãŒãŒã«ãã€ãŠ22âã15æ
éDNAéã®é£çµåå¿ãè¡ã€ãã65âïŒåã®ç±åŠ
çåŸãåå¿æ¶²ã«ïŒå容ã®ãšã¿ããŒã«ãå ããŠé£çµ
åå¿çµäºåŸã®DNAãæ²æŸ±æ¡åããã (4) HDéºäŒåã®ã¯ããŒãã³ã° HDéºäŒåãæ¬ æãããã¬ããã¯ããªãŠã ã»ã©
ã¯ããã¢ãŒã¡ã³ã¿ã AJ12019NRRLBâ15346ïŒ
ãå容èãšããŠçšãããïŒâ(4)ã§è¿°ã¹ãæ¹æ³ã§åœ¢
質転æããããªã€ãã 30âã§10æ¥éå¹é€åŸãã¯ãã©ã ããšãã³ãŒã«è
æ§ã§ãã€ãŠãã€ãã¢ã»ãªã³èŠæ±æ§ãæ¶å€±ãã
AJ12020ïŒFERMâBP269ïŒãåŸãã (5) 圢質転ææ ªã®ãã©ã¹ãã解æ ãããã®æ ªãã(2)ã§è¿°ã¹ãæ¹æ³ã«ããã溶è液
ã調補ããã¢ã¬ããŒã¹ã²ã«é»æ°æ³³åæ³ã«ãããã©
ã¹ããDNAãæ€åºãããšããã7.64Mdã®ãã©ã¹
ãããæ€åºããpAJ210ãšåã¥ããã (6) åãã©ã³ã¹ããŒã¡ãŒã·ãšã³ AJ12020å ã®çµæããã©ã¹ããïŒpAJ210ïŒäž
ã«HDéºäŒåãååšããããšã確èªããããã«ã
ã®ãã©ã¹ããDNAãçšããã¬ããã¯ããªãŠã ã»
ã©ã¯ããã¢ãŒã¡ã³ã¿ã AJ12019ãå床圢質転æã
ãã çããã¯ãã©ã ããšãã³ãŒã«èæ§ã³ãããŒã®ã
ã¡ããããã10åãé£ãäžããã¢ã»ãªã³èŠæ±æ§ã
ãã¹ãããšããããããã®ããããèŠæ±æ§ãæ¶å€±
ããŠãããäžèšã®çµæããã©ã¹ããäžã«HDéºäŒ
åãååšããããšãæãããšãªã€ãã ïŒ pAJ210ãšpAJ212ã®å ±åã«ããâã¹ã¬ãªã
ã³ã®çç£ pAJ210ãïŒâ(4)ã§çšãã圢質転ææ³ã«ããã
ã¹ã¬ãªãã³çç£èãAHVèæ§çªç¶å€ç°æ ªãã¬ã
ãã¯ããªãŠã ã»ã©ã¯ããã¢ãŒã¡ã³ã¿ã AJ11188
ïŒFERMâP4190ïŒäžã«å°å ¥ããŠã圢質転ææ ªã
ã¯ãã ããšãã³ãŒã«èæ§ã§éžæããèæ ªAJ12021
ïŒFERMâBP270ïŒæ ªãåŸãã åæ§ã«pAJ212ã圢質転ææ³ã«ããAJ11188æ ª
ã«å°å ¥ãã圢質転ææ ªãããªã¡ããã«ã èæ§ã§éž
æããèæ ª11188âHKæ ªãåŸãã ããã«pAJ212ãAJ12021æ ªã«å°å ¥ããã¯ãã©
ã ããšãã³ãŒã«èæ§ãã€ããªã¡ããã«ã èæ§ã§éž
æããäºçš®ã®ãã©ã¹ããpAJ210ãšpAJ212ãå«ã
AJ12209ïŒFERMâP8106ïŒFERM BPâ877ïŒæ ª
ãåŸãã A11188æ ªãAJ12021æ ªã11188âHKæ ªåã³
AJ12209æ ªãã¹ã¬ãªãã³çç£å¹å°ïŒã°ã«ã³ãŒã¹10
ïœïŒdlïŒïŒNH4ïŒ2SO43ïœïŒdlïŒKH2PO40.1ïœïŒ
dlïŒMgSO4ã»7H2O0.04ïœïŒdlïŒFeSO4ã»
7H2O10mgïŒMnSO4ã»5H2O10mgïŒãã¢ãã³ã»
HCl300ÎŒïœïŒãããªãã³100ÎŒïœïŒã倧è±èçœ
é žå æ°Žå解液ïŒãå³æ¶²ãïŒ45ïœïŒdlïŒå šçªçŽ ãšã
ãŠïŒãã€ãœãã€ã·ã³25mgïŒdlïŒãã€ã·ã³30ïœïŒdlïŒ
PH7.0ïŒCaCO35ïœïŒdlïŒå¥ã«æ®ºèããïŒïŒ30âã«
ãŠ72æéæ¯çªå¹é€ãããå¹é€åŸãé å¿äžæž äžã®ïŒ¬
âã¹ã¬ãªãã³ãâãªãžã³åã³ïŒ¬âãã¢ã»ãªã³ã
ãã€ã¯ããã€ãªã¢ãã»ã€åã¯é«é液äœã¯ãããã°
ã©ãã€ãŒã§å®éããããã®çµæã第ïŒè¡šã«ç€ºã
ãã å°ãpAJ210ïŒpAJ212ã¯å¯èšãããAJ12209æ ª
ããéåžžã®æ¹æ³ãçšããŠåé¢ãããã åãAJ12019ã¯AJ12020ãããAJ12078ã¯
AJ12079åã¯12078âHKãããAJ11188ã¯
AJ12021ïŒAJ12080åã¯AJ12209ãã宿䞻现èã
æãªãããšãªã宿䞻现èäžã®è€åãã©ã¹ãããé€
å»ããããšã«ãã容æã«åŸãããããã©ã¹ããã¯
宿䞻ããèªç¶ã«å€±ãªãããããšããããããé€å»ã
æäœã«ãã€ãŠé€ãããšãã§ããïŒBact.Rev.ïŒ
36ïŒp361â405ïŒ1972ïŒïŒã é€å»æäœã®äžäŸã¯ä»¥äžã®éãã§ããïŒå®¿äž»ã®ç
è²ãäžå®å šã«é»å®³ããæ¿åºŠïŒïŒâ50ïœïŒdlïŒã®ã¢
ã¯ãªãžã³ãªã¬ã³ãžãå«ãå¹å°ã«ãïŒmlåœãçŽ104
现èçšåºŠã«ãªãæ§ã«å°éã®èæ ªãæ¥çš®ã宿䞻èã®
çè²ãäžå®å šã«é»å®³ããŠãã27â37âã§äžå€å¹é€
ããïŒJ.Bacteriol.ïŒ88ïŒ261ïŒ1964ïŒãå¹é€æ¶²ãå¯
倩å¹å°ã«å¡åžãã27â37âã§äžå€å¹é€ãããå¹å°
äžã«åºçŸããã³ãããŒã®ãã¡ãã¯ãã©ã ããšãã³
ãŒã«ïŒ10ÎŒïœïŒmlïŒåã¯ããªã¡ããã«ã ïŒ50ÎŒ
ïœïŒmlïŒã«æåæ§ã瀺ãæ ªããã©ã¹ãããé€å»ã
ããŠããæ ªã§å³ã¡ãAJ12019ïŒAJ12078åã³
AJ11188ã§ããã ãè¡šã å®æœäŸ ïŒ ïŒâ(5)ïŒïŒâ(8)åã³ïŒâ(4)ã§åŸã圢質転ææ ª
AJ12079æ ªïŒAJ12078ïŒpAJ211ïŒ12078âHKæ ª
ïŒAJ12078ïŒpAJ212ïŒåã³AJ12020æ ª
ïŒAJ12019ïŒpAJ210ïŒã¯ïŒ¬âã€ãœãã€ã·ã³ãçç£
ããã第ïŒè¡šã¯ãåèæ ªã®å¹é€äžæž äžã®ïŒ¬âã€ãœ
ãã€ã·ã³ãâã¹ã¬ãªãã³ãâãªãžã³ãåã³ïŒ¬
âãã¢ã»ãªã³ããã€ã¯ããã€ãªã¢ãã»ã€åã¯é«é
液äœã¯ãããã°ã©ãã€ãŒã§å®éããçµæã§ããã ãè¡šã ãªããå¹é€ã¯ãïŒã§è¿°ã¹ãã¹ã¬ãªãã³çç£å¹å°
ããã€ãœãã€ã·ã³åã³ãã€ã·ã³ãé€å»ããå¹å°ã
çšãã30âã72æéæ¯çªå¹é€ããã ãã¬ããã¯ããªãŠã ã»ã©ã¯ããã¢ãŒã¡ã³ã¿ã
AJ12028ïŒFERM BPâ272ïŒã¯ãâã€ãœãã€ã·
ã³çç£èã§ãããâïŒïŒâã¢ãããšãã«âã·ã¹
ãã€ã³ãAHVãβâããããã·ãã€ã·ã³èæ§
ã§ããã€ã·ã³æ é€èŠæ±æ ªãšããŠè²çš®ããããã®ã§
ããããã®èæ ªãïŒâ(8)åã³ïŒâ(5)ã§åŸããã©ã¹
ããpAJ212åã³pAJ210DNAã§åœ¢è³ªè»¢æããïŒ
â(4)ãšåæ§ã®æ¹æ³ã§ã¯ãã ããšãã³ãŒã«åã³ããª
ã¡ãããªã èæ§åœ¢è³ªè»¢ææ ª12028DKæ ªãåŸãã ãŸãã¢ã¬ããŒã¹ã²ã«é»æ°æ³³åã«ãã圢質転ææ ª
ãpAJ210åã³pAJ212ãæããŠããããšã確èªã
ãã ïŒã§è¿°ã¹ãã¹ã¬ãªãã³çç£å¹å°ããã€ãœãã€ã·
ã³ãé€å»ããå¹å°ã§30âã72æé圢質転ææ ªãå¹
é€ããçç£ãããâã€ãœãã€ã·ã³ãâã¹ã¬ãª
ãã³ãâãªãžã³ãåã³ïŒ¬âãã¢ã»ãªã³éããã€
ã¯ããã€ãªã¢ãã»ã€åã¯é«é液äœã¯ãããã°ã©ã
ã€ãŒã§å®éãããçµæã第ïŒè¡šã«ç€ºããã ãè¡šã
第ïŒå³ã¯HKéºäŒåãçµã¿èŸŒãã pAJ212åã³
çºçŸçšãã©ã¹ããpAJ224ãé æããæ¹æ³ã§ããã
çºçŸçšãã©ã¹ããpAJ224ãé æããæ¹æ³ã§ããã
Claims (1)
- ïŒ ãã¬ããã¯ããªãŠã å±çŽ°èç±æ¥ã®ãã¢ã»ãªã³
ãããŒãŒãã³ãŒãããéºäŒåãçµèŸŒãŸããŠãã第
ïŒã®ãã©ã¹ããåã³ãã¬ããã¯ããªãŠã å±çŽ°èç±
æ¥ã®ãã¢ã»ãªã³ããããã²ããŒãŒãã³ãŒãããéº
äŒåãçµèŸŒãŸããŠãã第ïŒã®ãã©ã¹ãããæãã
ã³ãªããã«ã 现èãå¹é€ããããšãç¹åŸŽãšããã¹
ã¬ãªãã³åã¯ã€ãœãã€ã·ã³ã®è£œé æ³ã
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
JP3716885A JPS61195695A (ja) | 1985-02-26 | 1985-02-26 | ã¹ã¬ãªãã³åã¯ã€ãœãã€ã·ã³ã®è£œé æ³ |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
JP3716885A JPS61195695A (ja) | 1985-02-26 | 1985-02-26 | ã¹ã¬ãªãã³åã¯ã€ãœãã€ã·ã³ã®è£œé æ³ |
Publications (2)
Publication Number | Publication Date |
---|---|
JPS61195695A JPS61195695A (ja) | 1986-08-29 |
JPH0559710B2 true JPH0559710B2 (ja) | 1993-08-31 |
Family
ID=12490068
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
JP3716885A Granted JPS61195695A (ja) | 1985-02-26 | 1985-02-26 | ã¹ã¬ãªãã³åã¯ã€ãœãã€ã·ã³ã®è£œé æ³ |
Country Status (1)
Country | Link |
---|---|
JP (1) | JPS61195695A (ja) |
Families Citing this family (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JP3966583B2 (ja) * | 1997-06-23 | 2007-08-29 | ååéé µå·¥æ¥æ ªåŒäŒç€Ÿ | çºé µæ³ã«ããïœâã¢ããé žã®è£œé æ³ |
CA2319283A1 (en) | 1999-09-20 | 2001-03-20 | Kuniki Kino | Method for producing l-amino acids by fermentation |
JP4245746B2 (ja) | 1999-09-20 | 2009-04-02 | ååçºé µãã€ãªæ ªåŒäŒç€Ÿ | çºé µæ³ã«ããã¢ããé žã®è£œé æ³ |
JP2009153382A (ja) | 2006-03-30 | 2009-07-16 | Ajinomoto Co Inc | ã¡ã¿ããŒã«è³åæ§çŽ°èãçšããã«ã«ãã³é žã®è£œé æ³ |
Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JPS57186496A (en) * | 1981-05-11 | 1982-11-16 | Ajinomoto Co Inc | Preparation of l-threonine by fermentation |
JPS6030693A (ja) * | 1983-07-29 | 1985-02-16 | Kyowa Hakko Kogyo Co Ltd | âã€ãœãã€ã·ã³ã®è£œé æ³ |
-
1985
- 1985-02-26 JP JP3716885A patent/JPS61195695A/ja active Granted
Patent Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JPS57186496A (en) * | 1981-05-11 | 1982-11-16 | Ajinomoto Co Inc | Preparation of l-threonine by fermentation |
JPS6030693A (ja) * | 1983-07-29 | 1985-02-16 | Kyowa Hakko Kogyo Co Ltd | âã€ãœãã€ã·ã³ã®è£œé æ³ |
Also Published As
Publication number | Publication date |
---|---|
JPS61195695A (ja) | 1986-08-29 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
EP0143195B1 (en) | Recombinant dna having a phosphoenol pyruvate carboxylase gene inserted therein, bacteria carrying said recombinant dna and a process for producing amino acids using said bacteria | |
US4980285A (en) | Method for producing L-amino acids | |
CA1202257A (en) | Method for expressing a gene | |
JPH0547196B2 (ja) | ||
JPS6279788A (ja) | ã¢ããé žã®è£œé æ³ | |
JPS6411280B2 (ja) | ||
JPH06102024B2 (ja) | æ°èŠããã¢ãŒã¿ãŒåã³è©²ããã¢ãŒã¿ãŒãçšããéºäŒåçºçŸæ¹æ³ | |
JPH0353913B2 (ja) | ||
US4861722A (en) | Coryneform bacteria carrying recombinant plasmids and their use in the fermentative production of L-lysine | |
EP0137348B1 (en) | Recombinant dna, bacteria carrying said recombinant dna and a process for producing l-threonine or l-isoleucine using said bacteria | |
JPH0728749B2 (ja) | âã¢ã«ã®ãã³ã®è£œé æ³ | |
US4968609A (en) | Coryneform bacteria carrying recombinant DNA and a process for producing aromatic amino acids using said bacteria | |
JPH0559710B2 (ja) | ||
CA1283070C (en) | Process for production of l-phenylalanine by fermentation | |
US5017481A (en) | Coryneform bacteria carrying recombinant DNA and process for producing an aromatic amino acid by using the same | |
JPH06125779A (ja) | çµæãïœïœïœãæããã³ãªãå现èãçšããè³éŠæã¢ããé žã®è£œé æ³ | |
US4885245A (en) | Process for producing L-tryptophan | |
JPS6070093A (ja) | çºé µæ³ã«ããïœâããã·ã³ã®è£œé æ³ | |
JPH01160487A (ja) | ã·ã€ãã«ãã¯ã¿ãŒïœïœïŒãçµæããã©ãã¹ãã埮çç©ã®åœ¢è³ªè»¢ææ³ããªã·ã³ã®è£œæ³åã³ãªã·ã³çç£æ§ã«åœ¢è³ªè»¢æãã埮çç© | |
JPH055479B2 (ja) | ||
JPH0476678B2 (ja) | ||
JPS63237793A (ja) | âã¹ã¬ãªãã³ã®è£œé æ³ | |
JPH055480B2 (ja) | ||
JPH06102031B2 (ja) | çºé µæ³ã«ããïœâããšãã«ã¢ã©ãã³ã®è£œé æ³ | |
JPH0526467B2 (ja) |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
EXPY | Cancellation because of completion of term |