Abbott P J
Mutat Res. 1985 Jan-Mar;145(1-2):25-34. doi: 10.1016/0167-8817(85)90036-7.
A plasmid containing the STR operon has been modified in vitro (i) by irradiation with UV light, (ii) by reaction with ethyl methanesulphonate (EMS), (iii) by reaction with N-acetoxy-2-acetylaminofluorene (AcO-AAF), (iv) by reaction with (+/-)trans-benzo[a]pyrene-7, 8-dihydrodiol-9,10-epoxide (BPDE), and (v) by heating at 70 degrees C to produce apurinic sites. Suitably modified plasmid DNA was then used to transform both repair-proficient and repair-deficient strains of Escherichia coli, and the mutation frequency in the plasmid-encoded rspL+ gene measured. The influence of host mutations in the uvrB+, recA+, umuC+ and lexA+, genes on the mutation frequency have been investigated. Transformation into a uvrB strain significantly decreased survival and increased the level of mutations observed for UV- and AcO-AAF-modified plasmid DNA, while only a small increase in mutation frequency was seen with EMS-modified DNA and no increase in mutation frequency with plasmid DNA containing apurinic sites. Mutagenesis in UV- and BPDE-modified DNA (and probably also DNA containing apurinic sites) was totally dependent on he recA+ gene product, while EMS and AcO-AAF induced mutagenesis was only partially independent on the recA+ gene. Transformation of UV- or BPDE-modified DNA into a umuC or lexA strain, on the other hand, showed no change in mutation frequency from that observed with wild-type strain. Pre-irradiation of the wild-type host with UV light before transformation led to a significant increase in mutation frequency for UV- and BPDE-modified plasmid DNA. These results are discussed in terms of mutational or recombinational pathways which may be available to act on modified plasmid DNA, and suggest that the majority of the mutational events measured in this system are due to recombination between homologous regions on the plasmid and chromosomal DNA.
一个含有STR操纵子的质粒已在体外进行了如下修饰：(i) 用紫外线照射；(ii) 与甲磺酸乙酯（EMS）反应；(iii) 与N-乙酰氧基-2-乙酰氨基芴（AcO-AAF）反应；(iv) 与（±）反式苯并[a]芘-7,8-二氢二醇-9,10-环氧化物（BPDE）反应；以及(v) 在70℃加热以产生脱嘌呤位点。然后，将经过适当修饰的质粒DNA用于转化大肠杆菌的修复 proficient 和修复缺陷菌株，并测量质粒编码的rspL+基因中的突变频率。研究了uvrB+、recA+、umuC+和lexA+基因中的宿主突变对突变频率的影响。转化到uvrB菌株中显著降低了存活率，并增加了紫外线和AcO-AAF修饰的质粒DNA观察到的突变水平，而EMS修饰的DNA仅导致突变频率略有增加，含脱嘌呤位点的质粒DNA则未观察到突变频率增加。紫外线和BPDE修饰的DNA（可能还有含脱嘌呤位点的DNA）中的诱变完全依赖于recA+基因产物，而EMS和AcO-AAF诱导的诱变仅部分独立于recA+基因。另一方面，将紫外线或BPDE修饰的DNA转化到umuC或lexA菌株中，突变频率与野生型菌株相比没有变化。在转化前用紫外线对野生型宿主进行预照射，导致紫外线和BPDE修饰的质粒DNA的突变频率显著增加。这些结果根据可能作用于修饰质粒DNA的突变或重组途径进行了讨论，并表明在该系统中测量到的大多数突变事件是由于质粒和染色体DNA同源区域之间的重组所致。
