Isolation of insertion, deletion, and nonsense mutations of the uracil-DNA glycosylase (ung) gene of Escherichia coli K-12.
Two uracil-DNA glycosylase (ung) mutation selection procedures based upon the ability of uracil glycosylase to degrade the chromosomes of organisms containing uracil-DNA were devised to obtain a collection of well-defined ung alleles. In an enrichment procedure, lysogens were selected from Escherichia coli cultures infected with lambda pKanr phage containing uracil in their DNA. (These uracil-DNA phage were prepared by growth on host cells deficient in both dUTPase and uracil-DNA glycosylase.) The lysogenic Kanr population was enriched for uracil glycosylase-deficient mutants by a factor of 10(4). In a phage suicide selection procedure, lambda pung+ phage were unable to form plaques on dut ung cells containing uracil-DNA in their chromosomes, and all of the progeny were lambda pung-. Deletion, insertion (ung::Mu and ung::Tn10), nonsense, and missense mutants were isolated by using these procedures. Extracts of three insertion mutants contained no detectable enzyme activity. All of the other mutant isolates had less than 1% of the normal uracil glycosylase specific activity. The previously studied ung-1 allele, which was derived by N-methyl-N'-nitro-N-nitrosoguanidine mutagenesis, produced about 0.02% of the normal amount of uracil glycosylase activity. No significant phenotypic differences between ung-1 and ung::Tn10 alleles were observed. Variations of the lysogen selection procedure may be helpful for isolating other DNA glycosylase mutations in E. coli and other organisms.
D000483:Alleles; D010582:Bacteriophage lambda; D002872:Chromosome Deletion; D002874:Chromosome Mapping; D045647:DNA Glycosylases; D004251:DNA Transposable Elements; D004279:DNA, Viral / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics* / Q000302:isolation & purification; D005798:Genes, Bacterial; D008040:Genetic Linkage; D008242:Lysogeny; D009154:Mutation; D009699:N-Glycosyl Hydrolases / Q000235:genetics* / Q000378:metabolism; D051981:Uracil-DNA Glycosidase
D004251:DNA Transposable Elements; D004279:DNA, Viral; D045647:DNA Glycosylases; D009699:N-Glycosyl Hydrolases; D051981:Uracil-DNA Glycosidase