| Project/Area Number |
61571079
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| Research Category |
Grant-in-Aid for General Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Research Field |
Biological pharmacy
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| Research Institution | The Kitasato Insitute |
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Principal Investigator |
OMURA Satoshi The Kitasato Institute,Vice President., 副所長 (90050426)
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| Co-Investigator(Kenkyū-buntansha) |
IKEDA Haruo School of Pharmaceutical Sciences,Kitasato University,Research associate., 薬学部, 助手 (90159632)
田中 晴雄 北里大学, 薬学部, 教授 (40118823)
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| Project Period (FY) |
1986 – 1987
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| Project Status |
Completed (Fiscal Year 1987)
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| Budget Amount *help |
¥2,000,000 (Direct Cost: ¥2,000,000)
Fiscal Year 1987: ¥800,000 (Direct Cost: ¥800,000)
Fiscal Year 1986: ¥1,200,000 (Direct Cost: ¥1,200,000)
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| Keywords | Bifunctional cosmid / Cloning / 抗生物質 / シャトルコスミド |
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| Research Abstract |
1. Construction of Streptocyces-E. coli bifuctional cosmid.
Two kinds of bifunctionsl cosmid vectors have been constructed by using the cosmid pUR222::cos,which contains e. coli vector pURe222 and 0.47kb cohesive region of lambda phage genome. Bifunctional cosmid type I consists of Streptomyces multi-copy vector pIJ703 and pUR222::cos. Bifunctional cosmid type II consists of the replication region of a single-copy plasmid SCP of s. coelicolor A3 (2),pUR222::cos and thiostrepton resistance gene.
2. Studies of transduction condition and stability of recombinant plasmids.
Each bifunctional cosmid nector cut at a unique site was ligated with Sau3A-cut Streptomyces chromosomal DNA at the molar ratio from 1:3 to 3:1 and e. coli was tranxduced by the ligated DNA. The highest transduction was performed by the ligated DNA of 3:1 molar ratio of vector and chromosomal DNA. Both recombinant plasmids derived from bifunctional cosmid type I and II were stably propagated in e. coli. However,a part of region of recombinant plasmid derived from difunctionl cosmid type I was deleted.. 3. Cloning of polyketide-antibiotic biosynthetic genes.
Genomic libraries of kalafungin producer S. tanashiensis,nanaomycin methylester producer Streptomyces sp. OM-173 and avermectin producer S. avermitilis were constructed by using bifunctinal cosmid type II. Each clone was selected by colony hybridization method using a gene for the condensation step of actinorhodin biosyntheses,which was one of related compounds of kalajungin,as prode. Transformants of S. lividans introduced recombinant plasmids from clones of libraties of kalafungin and nanaomycin methylester procuders produced antimicrobial compounds.
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