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Resistance Mechanisms to Bleomycin in a Producer Organism.

生产生物体对博莱霉素的耐药机制。

基本信息

批准号：
01550765
负责人：
SUGIYAMA Masanori
金额：
$ 1.28万
依托单位：
Hiroshima University
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (C)
财政年份：
1989
资助国家：
日本
起止时间：
1989 至 1990
项目状态：
已结题

项目摘要

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.

必须保护产生抗生素的生物体免受其自身抗生素的致命影响。从酶失活、靶位点抗性和膜通透性等方面研究了产生蛋白质合成抗生素抑制剂的链霉菌的抗性机制。博来霉素是一种糖肽类抗生素，被用作抗肿瘤剂;了解产生DNA合成抗生素抑制剂的链霉菌的耐药机制是否与蛋白质合成的不同是有意义的。本研究以产博来霉素的轮枝链霉菌ATCC 15003为研究对象，探讨其自身耐药机制，并克隆其耐药决定簇基因。初步实验表明，博莱霉素的抗菌活性消失后，与无细胞提取物和乙酰辅酶A孵育。当提取物在65 ℃孵育5分钟时，失活活性丧失，即使在乙酰辅酶A存在下也是如此。接下来，我试图克隆一个决定博来霉素生产者中博来霉素乙酰化酶的基因。用作宿主的微生物是S。lividans 66.我从S.轮枝菌ATCC 15003，命名为blm B。我发现了另一个基因（blmA），该基因赋予了S.含有blmB基因的7 kb片段中的lividans 66。分析作为700 bp片段获得的blmA基因的DNA序列。结果表明，blmA基因编码的蛋白质是由122个氨基酸组成的多肽，分子量为13197。在本研究中，我发现blumA和blmB基因在大肠杆菌中表达。初步实验表明，blmA基因编码的蛋白是博莱霉素的结合蛋白。我正在详细研究blm A蛋白的一些特性。