Ohta T, Watanabe-Akanuma M, Tokishita S, Yamagata H
School of Life Science, Tokyo University of Pharmacy and Life Science, 1432-1 Horinouchi, Hachioji, Tokyo 192-0392, Japan.
Mutat Res. 1999 Mar 15;440(1):59-74. doi: 10.1016/s1383-5718(99)00005-4.
We previously reported the development of mutation-specific Escherichia coli B tester strains WP3101 to WP3106 from strain WP2uvrA. In this study we constructed their pKM101-containing derivatives WP3101P to WP3106P, and further isolated their rfa derivatives WP4101-WP4106 and WP4101P-WP4106P. The six kinds of F' plasmids (lacI-, lacZ-, proAB+), each of which carries a different lacZ allele, contained in the above strains were originally derived from E. coli K-12 strains CC101-CC106. All the tester strains show Lac- and Trp- phenotype. Assays for transitions and transversions are based upon Lac+ reversion of a specific mutation located within the lacZ gene on an F' plasmid. The trpE65(ochre) allele in the same strains enables them to be used for Trp+ reversion assays as well. In the present paper, we evaluated the sensitivity, specificity, and usefulness of the newly developed tester strains. Strains WP3101P-WP3106P were highly sensitive to determine mutational profile of heterocyclic amines with S9 mix-mediated metabolic activation and most of the oxidative mutagens and free radical generators tested. Every type of base-pair substitutions induced by 2-amino-3,4-dimethylimidazo[4,5-f]quinoline (MeIQ) or 5-diazouracil were detected in strains WP3101P-WP3106P, while A:T-->C:G and G:C-->A:T mutations induced by MeIQ, and A:T-->C:G, G:C-->A:T, and G:C-->C:G by 5-diazouracil were not detected in pKM101-free tester strains. In pKM101-carrying strains, cumene hydroperoxide induced all types of base substitutions, while formaldehyde preferentially induced G:C-->T:A transversions. Phenazine methosulfate induced predominantly G:C-->A:T transitions and G:C-->T:A transversions, while H2O2 induced predominantly G:C-->T:A and A:T-->T:A transversions. Introduction of the rfa mutation considerably enhanced sensitivity to bulky mutagens such as polycyclic aromatic compounds. All six possible base substitutions induced by 9, 10-dimethyl-1,2-benzanthracene (DMBA) were detected in tester strains WP4101P-WP4106P. In conclusion, our tester strains WP3101P-WP3106P and WP4101P-WP4106P permitted rapid and simple detection of specific mutations induced by variety of mutagens.
我们之前报道了从WP2uvrA菌株开发出突变特异性大肠杆菌B测试菌株WP3101至WP3106。在本研究中，我们构建了它们含pKM101的衍生物WP3101P至WP3106P，并进一步分离出它们的rfa衍生物WP4101 - WP4106和WP4101P - WP4106P。上述菌株中所含的六种F'质粒（lacI - ，lacZ - ，proAB + ），每种都携带不同的lacZ等位基因，最初源自大肠杆菌K - 12菌株CC101 - CC106。所有测试菌株均表现出Lac - 和Trp - 表型。转换和颠换分析基于F'质粒上lacZ基因内特定突变的Lac + 回复突变。同一菌株中的trpE65（赭石）等位基因使其也可用于Trp + 回复突变分析。在本文中，我们评估了新开发的测试菌株的敏感性、特异性和实用性。菌株WP3101P - WP3106P对于在S9混合物介导的代谢活化下测定杂环胺的突变谱以及大多数测试的氧化诱变剂和自由基产生剂具有高度敏感性。在菌株WP3101P - WP3106P中检测到了由2 - 氨基 - 3,4 - 二甲基咪唑[4,5 - f]喹啉（MeIQ）或5 - 重氮尿嘧啶诱导的每种碱基对替换类型，而在不含pKM101的测试菌株中未检测到由MeIQ诱导的A:T→C:G和G:C→A:T突变，以及由5 - 重氮尿嘧啶诱导的A:T→C:G、G:C→A:T和G:C→C:G突变。在携带pKM101的菌株中，氢过氧化异丙苯诱导了所有类型的碱基替换，而甲醛优先诱导G:C→T:A颠换。硫酸吩嗪主要诱导G:C→A:T转换和G:C→T:A颠换，而过氧化氢主要诱导G:C→T:A和A:T→T:A颠换。rfa突变的引入显著增强了对多环芳烃等大分子诱变剂的敏感性。在测试菌株WP4101P - WP4106P中检测到了由9,10 - 二甲基 - 1,2 - 苯并蒽（DMBA）诱导的所有六种可能的碱基替换。总之，我们的测试菌株WP3101P - WP3106P和WP4101P - WP4106P能够快速简单地检测由多种诱变剂诱导的特定突变。
