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DNA Replication and Copy-number Control of Bacillus plasmid

芽孢杆菌质粒的 DNA 复制和拷贝数控制

基本信息

批准号：
62560104
负责人：
SEKI Tatsuji
金额：
$ 1.15万
依托单位：
Osaka 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：
1987
资助国家：
日本
起止时间：
1987 至 1988
项目状态：
已结题

项目摘要

The structure of a 1.5-kb DNA sequence that is necessary and sufficient for the DNA replication of an 8.2-kb cryptic plasmid, pFTB14, isolated from a strain of Bacillus amyloliquefaciens has been characterized. The 1.5-kb DNA sequence contains an open reading frame, rep, stretching for 1017 bp, a promoter region for rep expression, and a possible replication origin. Genetic analysis by deletion and insertion mutations suggested that the rep product is trans-active and essential for plasmid automonous replication. The predicted rep protein is a basic protein, as are the RepC protein of pT181, RepB of pUB101 and protein A of pC194 which are found in staphylococci and used as Bacillus vectors. The redicted rep protein has highly similar amino acid sequences with protein A of pC194 and RepB of pUB101 through the protein molecule, but not with RepC of pT181 or protein RepH encoded by and initiating the replication of pC194.In order to obtain rep protein, it was overproduced in Escherichia coli by the promoter system of lambda phage, and purified partially through DEAE-Toyopearl chromatography. The molecular weight of the rep protein was estimated to be 39,600 dal as same as that expected from DNA sequences. By the result of gel-shift assay analysis the protein was considered as DNA binding protein because of the binding activity on the restricted DNA sequence of 1.5-kb region, which is similar to the DNA regions for the replication origin of pC194 and pUB101.The above results suggested the rep protein regulates DNA replication of PFTB14 through binding with the origin of DNA replication.

从一株解淀粉芽孢杆菌中分离的8.2kb隐蔽质粒pFTB14的DNA复制所必需的和充分的1.5kb DNA序列的结构已经被表征。1.5kb的DNA序列含有一个开放阅读框REP，延伸1017bp，REP表达的启动子区域，以及一个可能的复制起点。通过缺失和插入突变进行的遗传分析表明，rep产物是反式活性的，是质粒自动复制所必需的。预测的rep蛋白是一种碱性蛋白，pT181的RepC蛋白、pUB101的RepB蛋白和pC194的A蛋白也是碱性蛋白，它们被用来作为芽孢杆菌的载体。Rep蛋白的氨基酸序列与pC194的蛋白A和pUB101的RepB高度相似，但与pT181的RepC和由pC194编码并启动复制的Rep蛋白不同。为了获得Rep蛋白，利用lambda噬菌体的启动子系统在大肠杆菌中大量表达，并通过DEAE-Toyota opearl柱层析进行部分纯化。Rep蛋白的分子量估计为39,600dal，与从DNA序列中预期的相同。凝胶漂移分析结果表明，该蛋白与pC194和pUB101的DNA复制起始区相似，是DNA结合蛋白。以上结果表明，该蛋白通过与DNA复制起始点的结合来调节PFTB14的DNA复制。