Jenkins G J, Chaleshtori M H, Song H, Parry J M
School of Biological Sciences, University of Wales Swansea, Singleton Park, Swansea, SA2 8PP, UK.
Mutat Res. 1998 Sep 20;405(2):209-20. doi: 10.1016/s0027-5107(98)00138-9.
The restriction site mutation (RSM) assay (see Steingrimsdottir et al. [H. Steingrimsdottir, D. Beare, J. Cole, J.F.M. Leal, T. Kostic, J. Lopez-Barea, G. Dorado, A.R. Lehmann, Development of new molecular procedures for the detection of genetic alteration in man, Mutat. Res. 353 (1996) pp. 109-121] for a review) has been developed as a genotypic mutation detection system capable of identifying mutations occurring in restriction enzyme sites of genomic DNA. Here we will report the steps taken to overcome some of the initial problems of the assay, namely the lack of quantitative data and limited sensitivity, the aim being to achieve a methodology suitable for the study of low dose chemical exposures. Quantitative data was achieved in the RSM assay by the inclusion of an internal standard molecule in the PCR amplification stage, thus allowing the calculation of both spontaneous and induced mutation frequencies. The sensitivity of the assay was increased through the discovery that intron sequences of genomic DNA accumulated more mutations in vivo compared to the exons, presumably due to differential selective pressure within genes [G.J.S. Jenkins, I.deG. Mitchell, J.M. Parry, Enhanced restriction site mutation (RSM) analysis of 1, 2-dimethylhydrazine-induced mutations, using endogenous p53 intron sequences, Mutagenesis 12 (1997) pp. 117-123]. This increased sensitivity was examined by applying the RSM assay to analyse the persistence of N-ethyl-N-nitrosourea (ENU)-induced mutations in mice testes. Germ line mutations were sought in testes DNA 3, 10 and 100 days after ENU treatment. Mutations were detected in exons and especially intron regions, the intron mutations were more persistent, still being detected 100 days post-chemical treatment. Assignment of these mutations as ENU induced was complicated in some cases where the spontaneous mutation level was high. This theme of mutation persistence was further investigated by studying the presence of 4-nitroquinoline-1-oxide (4-NQO)-induced DNA mutations in vitro. This study also analysed the relationship between DNA adduct formation and DNA mutation induction by the concurrent RSM analysis and 32P post-labelling analysis of 4-NQO treated human fibroblasts. The results demonstrated that early DNA mutations detected 4 days post-treatment by the RSM assay were probably ex vivo mutations induced by Taq polymerase misincorporation of 4-NQO adducted DNA, due to the maximum levels of 4-NQO adducts being present at this time point. A later mutational peak, after the adduct level had declined, was assumed to be due to DNA sequence changes produced in the fibroblasts by the in vivo processing of DNA adducts.
限制性位点突变（RSM）分析方法（综述见Steingrimsdottir等人[H. Steingrimsdottir, D. Beare, J. Cole, J.F.M. Leal, T. Kostic, J. Lopez-Barea, G. Dorado, A.R. Lehmann，《人类基因改变检测新分子程序的开发》，《突变研究》353 (1996) 第109 - 121页]）已被开发为一种基因型突变检测系统，能够识别基因组DNA限制性酶切位点发生的突变。在此，我们将报告为克服该分析方法最初存在的一些问题所采取的步骤，即缺乏定量数据和灵敏度有限，目的是获得一种适用于低剂量化学暴露研究的方法。通过在PCR扩增阶段加入内标分子，在RSM分析中实现了定量数据，从而能够计算自发突变频率和诱导突变频率。通过发现基因组DNA的内含子序列在体内比外显子积累更多突变，推测是由于基因内不同的选择压力[G.J.S. Jenkins, I.deG. Mitchell, J.M. Parry，《使用内源性p53内含子序列增强对1,2 - 二甲基肼诱导突变的限制性位点突变（RSM）分析》，《诱变》12 (1997) 第117 - 123页]，该分析方法的灵敏度得以提高。通过应用RSM分析方法来分析N - 乙基 - N - 亚硝基脲（ENU）诱导的小鼠睾丸突变的持续性，对这种提高的灵敏度进行了检测。在ENU处理后3天、10天和100天，在睾丸DNA中寻找生殖系突变。在外显子尤其是内含子区域检测到了突变，内含子突变更持久，在化学处理后100天仍能检测到。在某些自发突变水平较高的情况下，将这些突变认定为ENU诱导的情况较为复杂。通过研究4 - 硝基喹啉 - 1 - 氧化物（4 - NQO）在体外诱导的DNA突变的存在情况，对这种突变持续性的主题进行了进一步研究。该研究还通过对4 - NQO处理的人成纤维细胞进行同步RSM分析和32P后标记分析，分析了DNA加合物形成与DNA突变诱导之间的关系。结果表明，RSM分析在处理后4天检测到的早期DNA突变可能是由于Taq聚合酶对4 - NQO加合DNA的错误掺入而在体外诱导的突变，因为此时4 - NQO加合物处于最高水平。在加合物水平下降后的后期突变峰，被认为是由于DNA加合物在体内加工过程中在成纤维细胞中产生的DNA序列变化所致。
