New mutations in cloned Escherichia coli umuDC genes: novel phenotypes of strains carrying a umuC125 plasmid.
The umuDC locus of Escherichia coli is required for most mutagenesis by UV and many chemicals. Mutations in E. coli umuDC genes cloned on pBR322-derived plasmids were isolated by two methods. First, spontaneously-arising mutant umuDC plasmids that failed to confer cold-sensitive growth on a lexA51(Def) strain were isolated by selection. Second, mutant umuDC plasmids that affected apparent mutant yield after UV-irradiation in a strain carrying umuD+C+ in the chromosome were isolated by screening hydroxylamine-mutagenized umuD+C+ plasmids. pBR322-derived umuD+C+ plasmids inhibited the induction of the SOS response of lexA+ strains as measured by expression of din::Mu dl(lac Ap) fusions but most mutant plasmids did not. Mutant plasmids defective in complementation of chromosomal umuD44, umuC36, or both were found among those selected for failure to confer cold-sensitivity, whereas those identified by the screening procedure yielded mostly mutant plasmids with more complex phenotypes. We studied in greater detail a plasmid, pLM109, carrying the umuC125 mutation. This plasmid increased the sensitivity of lexA+ strains to killing by UV-irradiation but was able to complement the deficiencies of umuC mutants in UV mutagenesis. pLM109 failed to confer cold-sensitive growth on lexA(Def) strains but inhibited SOS induction in lexA+ strains. The effect of pLM109 on the UV sensitivity of lexA(Def) strains was similar to that of the parental umuD+C+ plasmid. The mutation responsible for the phenotypes of pLM109 was localized to a 615-bp fragment. DNA sequencing revealed that the umuC125 mutation was a G:C----A:T transition that changed codon 39 of umuC from GCC----GTC thus changing Ala39 to Val39. The implications of the umuC125 mutation for umuDC-dependent effects on UV-mutagenesis and cell survival after UV damage are discussed.
Marsh
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L
Department of Cell Biology, Albert Einstein College of Medicine, Yeshiva University, Bronx, NY 10461.
Nohmi
T
T
Hinton
S
S
Walker
G C
GC
D000483:Alleles; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics* / Q000254:growth & development / Q000528:radiation effects; D005798:Genes, Bacterial; D007700:Kinetics; D016685:Mitomycin / Q000494:pharmacology; D016296:Mutagenesis; D009154:Mutation; D010641:Phenotype; D010957:Plasmids; D013014:SOS Response, Genetics; D013696:Temperature; D014466:Ultraviolet Rays
D016685:Mitomycin