Hosted T J, Baltz R H
Lilly Research Laboratories, A Division of Eli Lilly and Company, Indianapolis, Indiana 46285, USA.
J Bacteriol. 1997 Jan;179(1):180-6. doi: 10.1128/jb.179.1.180-186.1997.
We developed a gene replacement system using the rpsL gene of Streptomyces roseosporus and demonstrated its utility by constructing a deletion in the S. roseosporus glnA gene. A 1.3-kb BamHI fragment that hybridized to the Mycobacterium smegmatis rpsL gene was subcloned from an S. roseosporus cosmid library and sequenced. Plasmid pRHB514 containing the rpsL gene conferred streptomycin sensitivity (Sm(S)) to the Sm(r) S. roseosporus TH149. The temperature-sensitive plasmid pRHB543 containing rpsL and the S. roseosporus glnA gene disrupted with a hygromycin resistance (Hm(r)) gene was introduced into S. roseosporus TH149, and recombinants containing single and double crossovers were obtained after a temperature increase. Southern hybridization analysis revealed that single crossovers occurred in the glnA or rpsL genes and that double crossovers resulted in replacement of the chromosomal glnA gene with the disrupted glnA. Glutamine synthetase activity was undetectable in the recombinant containing the disrupted glnA gene.
我们利用玫瑰孢链霉菌的rpsL基因开发了一种基因置换系统，并通过构建玫瑰孢链霉菌谷氨酰胺合成酶基因（glnA）的缺失来证明其效用。从玫瑰孢链霉菌粘粒文库中克隆并测序了一个与耻垢分枝杆菌rpsL基因杂交的1.3 kb BamHI片段。含有rpsL基因的质粒pRHB514赋予了玫瑰孢链霉菌TH149对链霉素的敏感性（Sm(S)）。将含有rpsL基因和被潮霉素抗性（Hm(r)）基因破坏的玫瑰孢链霉菌glnA基因的温度敏感型质粒pRHB543导入玫瑰孢链霉菌TH149，升温后获得了含有单交换和双交换的重组体。Southern杂交分析表明，单交换发生在glnA或rpsL基因中，双交换导致染色体上的glnA基因被破坏的glnA所取代。在含有被破坏的glnA基因的重组体中未检测到谷氨酰胺合成酶活性。
