Rapid mutational analysis of regulatory loci in Escherichia coli K-12 using bacteriophage M13.
Rapid mutational analysis of regulatory loci in Escherichia coli K-12 using bacteriophage M13.
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DOI：
10.1073/pnas.81.12.3801
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发表时间：
1984-06
影响因子：
11.1
通讯作者：
K. Wertman;J. W. Little;D. Mount
中科院分区：
文献类型：
--
作者：
K. Wertman;J. W. Little;D. Mount
制备了噬菌体 M13mp8 的衍生物，命名为 M13mp8 /P，其中细菌基因的启动子和 NH2 末端密码子可以与 β-半乳糖苷酶的一部分融合，从而产生易于评分的表型。由于插入的启动子的转录仍然对宿主调节系统有反应，因此可以很容易地筛选诱变的噬菌体以寻找具有异常调节表型的分离株，并通过双脱氧序列分析来确定突变变化。通过分离lexA和recA两个基因调控区的大量突变，证明了该方法的可行性。在recA的单个LexA阻遏物结合位点和lexA的两个结合位点以及可能影响翻译效率的其他位点中均发现了改变重组噬菌体表型的碱基取代。我们的结果表明，这种方法通常可用于转录和翻译调控元件的突变分析。
A derivative of bacteriophage M13mp8 , designated M13mp8 /P, was prepared in which the promoter and NH2-terminal codons of bacterial genes may be fused to a portion of beta-galactosidase, resulting in an easily scorable phenotype. Because transcription from the inserted promoter remains responsive to the host regulatory system, it is simple to screen mutagenized phage for isolates with aberrant regulatory phenotypes and to determine the mutational changes by dideoxy sequence analysis. The feasibility of the method was demonstrated by isolation of a large number of mutations in the regulatory regions of two genes, lexA and recA. Base substitutions that altered the phenotype of recombinant phage were identified both in the single LexA repressor binding site of recA and in the two binding sites of lexA, as well as in other sites that likely affect translational efficiency. Our results suggest that this approach will be generally useful for mutational analysis of transcriptional and translational regulatory elements.