Use of the rpoB gene to determine the specificity of base substitution mutations on the Escherichia coli chromosome
Use of the rpoB gene to determine the specificity of base substitution mutations on the Escherichia coli chromosome
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DOI：
10.1016/s1568-7864(03)00024-7
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发表时间：
2003-05-13
期刊：
影响因子：
3.8
通讯作者：
Miller, JH
中科院分区：
文献类型：
--
作者：
Garibyan, L;Huang, T;Miller, JH
大肠杆菌rpoB基因的突变通过改变RNA聚合酶的亚基而导致对抗生素利福平（Rif（r））的抗性。先前的研究已经确定了rpoB基因中的39个单碱基替换，导致37 ℃时的Rif（r），以及另外两个导致温度敏感细胞的突变。我们已经扩展了这项工作，并确定了额外的30个单碱基取代，导致Rif（r）表型。有了这些突变，rpoB/Rif（r）系统现在允许监测分布在24个编码位置中的37个位点（碱基对）的37度处的69个碱基替换。六种可能的碱基取代中的每一种由8-17个突变表示。超过90%的突变在rpoB基因的足够小的区域内，以允许用单对寡核苷酸引物进行PCR扩增，然后用单个引物进行测序，从而快速分析许多突变。可以使用另外的引物对监测剩余的突变。为了校准该系统，我们对rpoB中自发发生的或由具有已知特异性的不同诱变剂和诱变剂产生的500多个突变进行了测序。这些结果表明，rpoB/Rif（r）是一个准确和易于使用的检测系统，并提供了允许分析发生在染色体上而不是染色体外元件上的突变的优点。mutS、mutT、mutY、M突变体以及诱变剂甲磺酸乙酯（EMS）、紫外线（UV）照射、2-氨基嘌呤（2AP）、5-氮杂胞苷（5AZ）和顺铂（CPT）给出了根据其特征特异性预测的结果。不同序列背景的数量足以揭示自发mutS、2-氨基嘌呤、紫外光、5-氮杂胞苷和顺铂突变谱中的显著热点。顺铂的分布特别引人注目，68%的突变是由单个位点的A：T --> T：A颠换引起的。由于许多微生物中RNA聚合酶的关键区域的保守性，使用Rif（r）/rpoB系统可能是研究没有发育良好的遗传系统的微生物中的突变过程的通用方法。(C)2003 Elsevier Science B. V.保留所有权利。
Mutations in the rpoB gene of Escherichia coli result in resistance to the antibiotic rifampicin (Rif(r)) by altering the subunit of RNA polymerase. Previous studies have identified 39 single base substitutions in the rpoB gene that lead to Rif(r) at 37 degreesC and an additional two mutations that result in temperature sensitive cells. We have extended this work and identified an additional 30 single base substitutions that result in the Rif(r) phenotype. With these mutations the rpoB/Rif(r) system now allows the monitoring of 69 base substitutions at 37degrees at 37 sites (base pairs) distributed among 24 coding positions. Each of the six possible base substitutions is represented by 8-17 mutations. More than 90% of the mutations are within a small enough region of the rpoB gene to allow PCR amplification with a single pair of oligonucleotide primers, followed by sequencing with a single primer, leading to rapid analysis of numerous mutations. The remaining mutations can be monitored using an additional primer pair. To calibrate this system we sequenced over 500 mutations in rpoB occurring spontaneously or generated by different mutagens and mutators with known specificity. These results show that rpoB/Rif(r) an accurate and easy to employ detection system, and offers the advantage of allowing analysis of mutations occurring on the chromosome rather than on an extrachromosomal element. The mutS, mutT, mutY, M mutators, as well as the mutagenic agents ethyl methanesulfonate (EMS), ultraviolet (UV) irradiation, 2-aminopurine (2AP), 5-azacytidine (5AZ), and cisplatin (CPT) gave results predicted by their characterized specificities. The number of different sequence contexts is sufficient to reveal significant hotspots among the spontaneous mutS, 2-aminopurine, ultraviolet light, 5-azacytidine, and cisplatin mutational spectra. The cisplatin distribution is particularly striking, with 68% of the mutations resulting from an A:T --> T:A transversion at a single site. Because of the conservation of key regions of RNA polymerase among many microorganisms, using the Rif(r)/rpoB system may be a general method for studying mutational processes in microorganisms without well developed genetic systems. (C) 2003 Elsevier Science B.V. All rights reserved.