1990 Fiscal Year Final Research Report Summary
Resistance Mechanisms to Bleomycin in a Producer Organism.
| Project/Area Number |
01550765
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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 |
発酵工学
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| Research Institution | Hiroshima University |
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Principal Investigator |
SUGIYAMA Masanori Faculty of Medicine, Associate Professor, 医学部, 助教授 (30106801)
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| Project Period (FY) |
1989 – 1990
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| Keywords | Anti-cancer drug / Antibiotics / Self-resistance / Bleomycin / Resistance gene |
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| Research Abstract |
Organisms which produce antibiotics must be protected from the lethal effect of their own antibiotics. The resistance mechanisms in streptomycetes which produce antibiotic inhibitors of protein synthesis have been studied with respect to enzymatic inactivation, resistance of target site and membrane permeability. Bleomycin is a glycopeptide antibiotic and is used as an antitumor agent ; it is of interest to know whether the resistance mechanisms in streptomycetes which produce antibiotic inhibitors of DNA synthesis are different from those of protein synthesis. In the present study, we attempted to investigate self-resistance mechanisms in a bleomycin-producing microorganism and to clone the gene (s) encoding the resistant determinant (s).
Streptomyces verticillus ATCC15003 was used as a bleomycin-producing microorganism. Initial experiments indicated that the antibacterial activity of bleomycin disappeared after incubation with the cell-free extract and acetyl coenzyme A. When the extract was incubated at 65 C for 5 min, the inactivating activity was lost, even in the presence of acetyl CoA. Next, I attempted to clone a gene (s) which determined a bleomycin-acetylating enzyme in the bleomycin producer. The organism used as a host was S. lividans 66. I obtained a gene coding for the enzyme as a 7 kb DNA fragment from S. verticillus ATCC15003 and named blm B. I found another gene (blmA) which conferred the bleomycin resistance to S. lividans 66 in the 7 kb fragment containing blmB gene. The blmA gene obtained as a 700 bp fragment was analyzed for DNA sequences. As a result, the protein encoded by blmA gene was a polypeptide consisting of 122 amino acids and the molecular weight was 13197. I found in the present study that blumA and blmB genes were expressed in Escherichia coli. The preliminary experiments suggested that the protein encoded by blmA gene was a binding protein to bleomycin. I am investigating some properties of the blm A protein in detail.
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[Publications] Misoka, F., Miyake, T., Miyoshi, K., Sugiyama, M., Sakamoto, S. and Fuwa, T.: "Overproduction of human insulin-like growth factor-II in Escherichia coli." Biotechnol. Lett.11. 839-844 (1989)
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