通过在大肠杆菌中进行功能互补从脆弱拟杆菌中克隆亮氨酸合成酶B基因（leuB基因）

Molecular cloning of the leuB gene from Bacteroides fragilis by functional complementation in Escherichia coli.

作者信息

Sarker M R, Akimoto S, Ono T, Kinouchi T, Ohnishi Y

机构信息

Department of Bacteriology, School of Medicine, University of Tokushima, Japan.

出版信息

Microbiol Immunol. 1995;39(1):19-25. doi: 10.1111/j.1348-0421.1995.tb02163.x.

DOI:10.1111/j.1348-0421.1995.tb02163.x

PMID:7783674

Abstract

Clones containing the Bacteroides fragilis leuB-complementing gene were isolated by screening of a B. fragilis genomic library constructed in Escherichia coli. One recombinant clone, designated pOT865, with the smallest DNA insert (4.5 kb) could complement three independent leuB mutations in E. coli and the leuB-complementing determinant in pOT865 was localized to a region of 1.5-kb DNA. The results of Southern blot analysis suggested that a single copy of the cloned gene was present in the B. fragilis genome. The cloned fragment appeared to contain a sequence that could function as promoter in E. coli and direct the synthesis of a 42-kDa protein. These results suggest that the cloned segment contains the structural gene for beta-isopropylmalate dehydrogenase (leuB).

摘要

通过筛选在大肠杆菌中构建的脆弱拟杆菌基因组文库，分离出了含有脆弱拟杆菌亮氨酸B互补基因的克隆。一个重组克隆，命名为pOT865，其DNA插入片段最小（4.5 kb），能够互补大肠杆菌中的三个独立亮氨酸B突变，并且pOT865中的亮氨酸B互补决定簇定位于1.5 kb DNA区域。Southern印迹分析结果表明，克隆基因在脆弱拟杆菌基因组中以单拷贝形式存在。克隆片段似乎包含一个在大肠杆菌中可作为启动子并指导合成42 kDa蛋白质的序列。这些结果表明，克隆片段包含β-异丙基苹果酸脱氢酶（亮氨酸B）的结构基因。