Duncan B K
J Bacteriol. 1985 Nov;164(2):689-95. doi: 10.1128/jb.164.2.689-695.1985.
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.
基于尿嘧啶糖基化酶降解含有尿嘧啶-DNA的生物体染色体的能力，设计了两种尿嘧啶-DNA糖基化酶（ung）突变选择程序，以获得一系列定义明确的ung等位基因。在富集程序中，从感染了DNA中含有尿嘧啶的λpKanr噬菌体的大肠杆菌培养物中选择溶原菌。（这些尿嘧啶-DNA噬菌体是通过在缺乏dUTPase和尿嘧啶-DNA糖基化酶的宿主细胞上生长制备的。）溶原性Kanr群体中尿嘧啶糖基化酶缺陷型突变体的富集倍数为10⁴。在噬菌体自杀选择程序中，λpung⁺噬菌体无法在染色体中含有尿嘧啶-DNA的dut ung细胞上形成噬菌斑，所有后代均为λpung⁻。通过使用这些程序分离出缺失、插入（ung::Mu和ung::Tn10）、无义及错义突变体。三个插入突变体的提取物未检测到酶活性。所有其他突变体分离株的尿嘧啶糖基化酶比活性均低于正常水平的1%。先前通过N-甲基-N'-硝基-N-亚硝基胍诱变获得的ung-1等位基因产生的尿嘧啶糖基化酶活性约为正常量的0.02%。未观察到ung-1和ung::Tn10等位基因之间存在明显的表型差异。溶原菌选择程序的变体可能有助于在大肠杆菌和其他生物体中分离其他DNA糖基化酶突变。
