Directed mutagenesis method for analysis of mutagen specificity: application to ultraviolet-induced mutagenesis.
Directed mutagenesis method for analysis of mutagen specificity: application to ultraviolet-induced mutagenesis.
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用于分析诱变剂特异性的定向诱变方法：应用于紫外线诱导诱变。
DOI：
10.1073/pnas.80.1.237
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发表时间：
1983
影响因子：
11.1
通讯作者：
Livneh,Z
中科院分区：
文献类型：
--
作者：
Livneh,Z
建立了一种分析诱变剂特异性的定向诱变方法。该方法基于在给定标记基因内的特定片段处构建被诱变剂损伤的质粒，然后筛选突变质粒并对损伤片段进行核苷酸序列分析。通过使用该方法，质粒pXf 3在四环素抗性（泰特）基因中的BamHI-Sph I区段处被UV辐射特异性破坏，并用于转化SOS诱导的大肠杆菌。14氨苄青霉素抗性，四环素敏感的突变体的pXf 3分离和进行序列分析。数据揭示了转换，颠换，移码突变和缺失的诱导。单碱基变化均位于嘧啶序列内，缺失位于多聚嘧啶序列的直接重复之间。此外，还发现了突变质粒，在受损片段内没有突变。在这些情况下，泰特基因的其他部分中的“非靶向诱变”可能是突变表型的原因。该方法适用于任何与DNA反应的诱变剂，也可用于遗传学分析，作为一种突变DNA特定片段的手段。
A directed mutagenesis method has been developed for the analysis of mutagen specificity. The method is based on the construction of a plasmid damaged by the mutagen at a specific segment within a given marker gene, followed by screening for mutant plasmids and nucleotide sequence analysis of the damaged segment. By using this method plasmid pXf3 has been specifically damaged by UV radiation at the BamHI-Sph I segment in the tetracycline resistance (tet) gene(s) and used to transform SOS-induced Escherichia coli. Fourteen ampicillin-resistant, tetracycline-sensitive mutants of pXf3 were isolated and subjected to sequence analysis. The data revealed the induction of transitions, a transversion, a frameshift mutation, and deletions. The single base changes all were located within runs of pyrimidines, and the deletions mapped between direct repeats of polypyrimidine tracts. In addition, mutant plasmids were found with no mutation within the damaged segment. In these cases it is likely that "untargeted mutagenesis" in other portions of the tet gene(s) is responsible for the mutant phenotype. The method can be applied to any mutagen that reacts with DNA, and it also can be used for genetic analysis as a means to mutate specific segments of DNA.