ENU 诱导带 LacZ 靶基因的 λgt11 DNA 突变分子机理的初步研究

Mutation Study of λ gt11 DNA with LacZ Induced by ENU

采用ＥＮＵ（乙基亚硝基脲）作用于裸露的λｇｔ１１ＤＮＡ，经体外重包装，转染宿主菌ＥｃｏｌｉＹ１０９０，在含底物Ｘ－ｇａｌ，诱导剂ＩＰＴＧ的选择性培养基上铺皿，发现被处理的λｇｔ１１ＤＮＡ除了使噬菌体存活率下降外，还出现了靶基因“ＬａｃＺ”较高频率的突变。其中以二甲基亚砜（ＤＭＳＯ）为溶剂，当存活率分别为３５×１０－３、１６×１０－３和５５×１０－４时，相应的突变率依次为１１×１０－３、３２×１０－３和５２×１０－３，ＤＭＳＯ溶剂对照突变率则＜５０×１０－５。对ＥＮＵ诱导的５个阳性突变体进行了扩增，以ＰＣＲ产物为模板，采用正向引导，对阳性突变体靶基因ＬａｃＺ进行了部分测序，在被测序的２６０ｂｐ范围内，发现了９个位点的碱基突变。碱基突变的类型有颠换（６７％）、转换（１１％）和移码突变（２２％）。颠换主要以Ａ→Ｔ、Ｇ→Ｃ为主。似乎胞嘧啶（Ｃ）更易发生突变（占４３％）。

To construct molecular mutation detective system of λ DNA with LacZ, naked λ gt11 DNA was treated with mutagen ENU (Ethylnitro sourea) The ENU-damaged DNA was added to Lambda packaging extracts and the resulting phage were grown in host E coli Y1090 on a selective plate containing substract X-gal and inducer IPTG Under these conditions, the results showed that the higher the viability ratio was, the lower the frequency of clear-plaque mutants occured In our study, when survival ratios of the host cell survival ratio were 3 5×10 -3 、1 6×10 -3 and 5 5×10 -4 respectively, the mutation ratio were 1 1×10 -3 、3 2×10 -3 and 5 2×10 -3 accordingly, and the mutation ratio by DMSO (negative control ) was below 5 0×10 -5 260 bases from ENU induced LacZ gene were subjected to DNA sequence analysis There were several mutation sites: transversion (6, 67%), transition(1, 11%), frame shift(2, 22%)(both were insert mutation) Transversions mainly consisted of A→T, G→C Among the four bases, cytosine seemed to be more sensitive to ENU (43%)