Molecular cloning of the uvrD gene of Escherichia coli that controls ultraviolet sensitivity and spontaneous mutation frequency

Abstract

The uvrD gene of Escherichia coli that control UV sensitivity and spontaneous mutation frequency has been cloned with phage λ as vector. The increased sensitivity to ultraviolet light (UV) of uvrD3, uvrE502, recL152, and pdeB41 mutants, high mutability of uvrD3 and pdeB41 mutants, and conditional lethality of strain TS41 that carried pdeB41, polA1, and sup126 mutations were all suppressed by lysogenization of the mutant cells with λuvrD⁺. These results were consistent with the idea that the uvrD, uvrE, recL, and pdeB mutations are alleles of the uvrD gene. In addition to the uvD gene, λuvrD⁺ carried the corA gene that controls transport of Mg⁺⁺, Mn⁺⁺, and Co⁺⁺ through the cell membrane. Hybrid plasmids carrying both uvrD and corA genes were also constructed by using pKY2289 as a cloning vehicle. Orientational isomers that carried the same 12.0 kb fragment in the opposite direction were equally efficient in complementing the UvrD^- as well as CorA^- defects of the transformed host cells, suggesting that the DNA insert contains all the genetic signals needed to express the two gene products. Insertion of the γδ sequence into recombinant plasmids was performed to generate appropriate restriction endonuclease target sites in the cloned DNA fragments.