猴肾vero细胞中MNNG诱发非定标性点突变的序列特异性分析

Sequence specificity analysis of nontargeted point mutation in monkey kidney cells induced by MNNG

我们以穿梭质粒ｐｚ１８９作为探针已在猴肾ｖｅｒｏ细胞中证实了非定标性突变的存在。本实验进一步对突变子质粒中的靶基因ｓｕｐＦｔＲＮＡ基因作序列分析，以揭示非定标性突变的序列特异性。结果表明：８９％（２４／２７）的碱基置换发生在Ｇ：Ｃ碱基对，５９％（１６／２７）为颠换，４１％（１１／２７）为转换，主要为Ｇ：Ｃ→Ｔ：Ａ颠换和Ｇ：Ｃ→Ａ：Ｔ转换；４８％的碱基置换发生在ｓｕｐＦｔＲＮＡ基因的６个位点中，其中４个包含有特征性的邻近ＤＮＡ序列；另有４个位点的碱基置换是迄今为止在自发突变和定标性突变中从未报道的；５个移码突变中有２个发生在位点９９—１０５（ＧＧＴＧＧＧＧ）中，此位点可能为非定标性移码突变的热点。结果提示：非定标性突变有其序列特异性而不同于自发突变和定标性突变，它的形成可能是一种易误性ＤＮＡ聚合酶的作用所致。

The presence of nontargeted mutagenesis in vero cells has been shown byusing a shuttle plasmid pZ189 in this laboratory as reported elsewhere.The target gene supF suppressor tRNA genes in mutant plasmids were sequenced in this work to find thesequence specificity of nontargeted mutagenesis.The results showed that 89%(24/27)ofbase substitutions occurred in G:C base pairs and 59%(16/27) were transversions, 41%(11/27)were transitions. Most of the base substitutions were G:C→T:A and G: C→A:T.Forty-eight percent of base substitutions occurred at 6 sites of supF tRNA gene frequentlyand 4 of these sites have context DNA sequences of 5-TTNN(N is G or C).The basesubstitutions at supF gene sitesT→C at 61,G→T at 70, G→T at 99, and G→C at 103) havenever been reported in Spontaneous and targeted mutagenesis up to now. In addition,in supFgene, 2 of 5 frameshifts occurred at site 99-105,which means that this site might be a hotspot for frameshifts in nontargeted mutagenesis. These results showed that nontargeted mut-ations have sequence specificity which is different from spontaneous and targeted mutations.An error-prone DNA polymerase might be respohsible for nontargeted mutagenesis.