RPSL(+) - A DOMINANT SELECTABLE MARKER FOR GENE REPLACEMENT IN MYCOBACTERIA
RPSL(+) - A DOMINANT SELECTABLE MARKER FOR GENE REPLACEMENT IN MYCOBACTERIA
复制标题
DOI：
10.1111/j.1365-2958.1995.tb02324.x
复制
发表时间：
1995-06-01
影响因子：
3.6
通讯作者：
BOTTGER, EC
中科院分区：
文献类型：
--
作者：
SANDER, P;MEIER, A;BOTTGER, EC
分枝杆菌的分子遗传操作将受益于通过同源重组有效选择双交换事件的程序。在这里，我们描述了一个载体宿主系统的基因替换分枝杆菌，其效用进行了研究，使用功能失活的耻垢分枝杆菌的pyrF基因作为模型。该系统基于编码核糖体蛋白S12的野生型rpsL基因在链霉素抗性宿主中的表达。由于缺乏分枝杆菌来源，质粒不能在分枝杆菌中自主复制。用于维持克隆序列的第一选择由卡那霉素抗性基因赋予。链霉素的第二次同时选择是针对含有编码rpsL基因的链霉素敏感等位基因的基因的克隆序列的维持。通过将用于正选择的基因和用于负选择的基因分别置于目标靶基因之内和之外，获得基因置换。一步双重选择程序提供了一种严格区分双交换基因替换与单交换事件同源重组的方法。该系统应该有相当大的潜力，属或种的单交叉事件，甚至非法重组是占主导地位的重组机制。它也应该被广泛用于构建没有可选择表型的突变体。
Molecular genetic manipulations in mycobacteria would benefit from procedures which efficiently select for double-crossover events by homologous recombination. Here we describe a vector-host system for gene replacement in mycobacteria, the utility of which was investigated using functional inactivation of the pyrF gene in Mycobacterium smegmatis as a model. This system is based on the expression of the wildtype rpsL gene coding for ribosomal protein S12 in a streptomycin-resistant host. Owing to the absence of a mycobacterial origin the plasmids are unable to replicate autonomously in mycobacteria. The first selection for maintenance of cloned sequences is conferred by the kanamycin-resistance gene. The second simultaneous selection by streptomycin is against maintenance of cloned sequences which contain the gene encoding the streptomycin-sensitive allele of the rpsL gene. By placing the gene for positive selection and that used for negative selection within and outside the target gene of interest, respectively, gene replacement is obtained. A one-step double selection procedure provides a means to distinguish strictly between gene replacement by double crossover versus homologous recombination by single crossover events. The system should have considerable potential for genera or species where single-crossover events or even illegitimate recombination are the predominant recombination mechanisms. It should also be of wide use for the construction of mutants without a selectable phenotype.