Yang Hanjing, Wolff Erika, Kim Mandy, Diep Amy, Miller Jeffrey H
Department of Microbiology, Immunology and Molecular Genetics, and the Molecular Biology Institute, 1602 Molecular Sciences Building, 405 Hilgard Avenue, University of California, Los Angeles, CA 90095, USA.
Mol Microbiol. 2004 Jul;53(1):283-95. doi: 10.1111/j.1365-2958.2004.04125.x.
We searched for genes that create mutator phenotypes when put on to a multicopy plasmid in Escherichia coli. In many cases, this will result in overexpression of the gene in question. We constructed a random shotgun library with E. coli genomic fragments between 3 and 5 kbp in length on a multicopy plasmid vector that was transformed into E. coli to screen for frameshift mutators. We identified a total of 115 independent genomic fragments that covered 17 regions on the E. coli chromosome. Further studies identified 12 genes not previously known as causing mutator phenotypes when overproduced. A striking finding is that overproduction of the multidrug resistance transcription regulator, EmrR, results in a large increase in frameshift and base substitution mutagenesis. This suggests a link between multidrug resistance and mutagenesis. Other identified genes include those encoding DNA helicases (UvrD, RecG, RecQ), truncated forms of the DNA mismatch repair protein (MutS) and a primosomal component (DnaT), a negative modulator of initiation of replication/GATC-binding protein (SeqA), a stationary phase regulator AppY, a transcriptional regulator PaaX and three putative open reading frames, ycgW, yfjY and yjiD, encoding hypothetical proteins. In addition, we found three genes encoding proteins that were previously known to cause mutator effects under overexpression conditions: error-prone polymerase IV (DinB), DNA methylase (Dam) and sigma S factor (RpoS). This genomic strategy offers an approach to identify novel mutator effects resulting from the multicopy cloning (MCC) of specific genes and therefore complementing the conventional gene inactivation approach to finding mutators.
我们寻找那些在大肠杆菌中导入多拷贝质粒时会产生突变体表型的基因。在许多情况下，这会导致相关基因的过表达。我们构建了一个随机鸟枪法文库，其中包含长度在3至5千碱基对之间的大肠杆菌基因组片段，这些片段位于一个多拷贝质粒载体上，该载体被转化到大肠杆菌中以筛选移码突变体。我们总共鉴定出115个独立的基因组片段，它们覆盖了大肠杆菌染色体上的17个区域。进一步的研究鉴定出12个基因，这些基因在过量表达时以前并不被认为会导致突变体表型。一个惊人的发现是，多药耐药转录调节因子EmrR的过量表达会导致移码和碱基替换诱变的大幅增加。这表明多药耐药与诱变之间存在联系。其他鉴定出的基因包括那些编码DNA解旋酶（UvrD、RecG、RecQ）、DNA错配修复蛋白（MutS）的截短形式和一个引发体成分（DnaT）、复制起始的负调节因子/GATC结合蛋白（SeqA）、一个稳定期调节因子AppY、一个转录调节因子PaaX以及三个假定的开放阅读框，即ycgW、yfjY和yjiD，它们编码假定的蛋白质。此外，我们还发现了三个基因，它们编码的蛋白质在以前已知在过表达条件下会导致诱变效应：易出错的聚合酶IV（DinB）、DNA甲基化酶（Dam）和σS因子（RpoS）。这种基因组策略提供了一种方法来鉴定由特定基因的多拷贝克隆（MCC）产生的新型诱变效应，因此补充了寻找突变体的传统基因失活方法。
