Constriction of a fused operon consisting of the recA and kan (kanamycin resistance) genes and regulation of its expression by the lexA gene.
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.
D003001:Cloning, Molecular; D004352:Drug Resistance, Microbial; D004926:Escherichia coli / Q000235:genetics*; D005786:Gene Expression Regulation; D005796:Genes; D007612:Kanamycin / Q000494:pharmacology*; D009876:Operon; D010641:Phenotype; D010957:Plasmids; D011995:Recombination, Genetic
D007612:Kanamycin