Miki T, Kumahara H, Nakazawa A
Mol Gen Genet. 1981;183(1):25-31. doi: 10.1007/BF00270133.
The kanamycin resistance gene (kan) of transposon Tn5 was cloned into a derivative of plasmid pBR322. A DNA fragment containing the promoter-operator region of the recA gene was inserted into the promoter region of the cloned kan gene to produce a fused operon, recA-kan. Plasmid pMCR685 carrying recA-kan expressed a low level of activity of the kan gene product (kanamycin phosphotransferase; KPT) in the wild-type cells of Escherichia coli, while the plasmid showed an increased level of the activity in the SPr- mutant cells which produce the inactive lexA protein. The KPT activity in the wild-type cells harboring the plasmid increased 6- to 11-fold upon treatment of the cells with mitomycin C or nalidixic acid, both of which are known to induce synthesis of recA protein. Expression of the recA-kan operon fusion was remarkably repressed by the lexA gene cloned into a plasmid carrying the operon fusion. Higher concentrations of mitomycin C were required for maximal induction of KPT activity in the cells harboring the resulting plasmid pMCR687. These results strongly suggest that the lexA gene product can be itself repress the recA gene, and that pMCR687 is a useful vector to clone genes whose expression is harmful to the host cell growth.
转座子Tn5的卡那霉素抗性基因（kan）被克隆到质粒pBR322的一个衍生物中。将一个包含recA基因启动子-操纵子区域的DNA片段插入到克隆的kan基因的启动子区域，以产生一个融合操纵子recA-kan。携带recA-kan的质粒pMCR685在大肠杆菌野生型细胞中表达低水平的kan基因产物（卡那霉素磷酸转移酶；KPT）活性，而该质粒在产生无活性lexA蛋白的SPr-突变细胞中显示出较高水平的活性。在用丝裂霉素C或萘啶酸处理细胞后，携带该质粒的野生型细胞中的KPT活性增加了6至11倍，这两种物质都已知能诱导recA蛋白的合成。recA-kan操纵子融合的表达被克隆到携带操纵子融合的质粒中的lexA基因显著抑制。对于携带所得质粒pMCR687的细胞，需要更高浓度的丝裂霉素C才能最大程度地诱导KPT活性。这些结果有力地表明，lexA基因产物本身可以抑制recA基因，并且pMCR687是克隆其表达对宿主细胞生长有害的基因的有用载体。
