9-氨基吖啶诱导LacZ靶基因突变分子机制的研究

Molecular mechanism of LacZ gene mutagenesis induced by 9-aminoacridine

目的 使用我室新构建的含LacZ靶基因的λgt11DNA体外重包装致突变检测系统 ,研究了移码突变剂 9 氨基吖啶 (9 AA)诱发突变及分子突变谱 ,并进一步对该系统的可行性进行了评价。方法 用 9 AA直接处理λgt11DNA ,经体外重包装为完整噬菌体、铺皿 ,通过X gal和IPTG检测突变率 ,使用DNA测序了解阳性克隆DNA分子改变情况。结果 9 AA能明显影响噬菌体的存活率和诱发DNA突变 ,突变率呈明显剂量 反应关系 ;9 AA主要诱发移码突变 ,移码突变主要集中在Gs、Cs、As、Ts重复区内且突变频率随碱基重复长度增加而增加 ;9 AA同时还能诱发碱基置换 ,碱基置换主要表现为颠换 ,而碱基转换主要发生于Gs碱基。结论 该致突变检测系统不仅可以快速 ,灵敏地筛选出外来化合物的遗传毒性 ,而且可以深入分析外来化合物致突变分子机制。

Objective To analyze the mechanism of molecular mutagenesis induced by the 9 aminoacridine (9 AA) with the molecular mutation detective system of λ gt11DNA with LacZ gene and to evaluate this detective system. Methods The 9 AA damaged λ gt11DNA was added to Lambda packaging extracts and then the repacked Lambda phage were cultured in E.coli Y1090 on a selective plate pre supplemented with X Gal and IPTG. The positive cloned DNA molecule was determined with DNA sequencer. Results 9 AA had an obvious effect on the survival rate of the Lambda phage and induce DNA mutation in a dose effect manner. The 9 AA induced not only frameshift mutations, but also base substitutions. The frameshift mutation mainly occurred in the repeats of As, Cs, Gs. and Ts, and the frequency elevated linearly with the increasing amount of As, Cs, Gs, and Ts in the repeat. The 9 AA induced base substitutions were mainly base transversion, and the base transition was only in Gs repeat. Conclusion With this mutagenesis detective system, the genetic toxicity of exogenous compound can be easily investigated and the mechanism of molecular mutagenesis can also be obtained.