SHUTTLE-VECTOR MUTAGENESIS BY AFLATOXIN B-1 IN HUMAN-CELLS - EFFECTS OF SEQUENCE CONTEXT ON THE SUPF MUTATIONAL SPECTRUM
SHUTTLE-VECTOR MUTAGENESIS BY AFLATOXIN B-1 IN HUMAN-CELLS - EFFECTS OF SEQUENCE CONTEXT ON THE SUPF MUTATIONAL SPECTRUM
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DOI：
10.1016/0027-5107(94)90025-6
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发表时间：
1994-04-15
期刊：
影响因子：
--
通讯作者：
ANDERSON, A
中科院分区：
文献类型：
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作者：
COURTEMANCHE, C;ANDERSON, A
利用大鼠肝微粒体激活黄曲霉毒素B-1，对携带大肠杆菌supF基因的pS189穿梭载体和相关特征载体pSP189进行体外修饰。在没有Mg2+离子的情况下进行体外培养，质粒降解最小化。将修饰的质粒转染到人Ad293细胞中，然后回收并电穿孔到大肠杆菌MBM7070中进行突变体鉴定。体外修饰质粒的点突变频率在自发背景水平上显著增加。突变质粒通过dna序列分析进行了表征。绝大多数黄曲霉毒素b1诱导的突变是碱基置换，主要是G:C到T:A的转换。anatoxin B-1诱导的pS189 supF基因突变谱与先前在转染Ad293细胞的cDNA表达载体合成的细胞色素P450 1A2 （CYP1A2）激活的anatoxin B-1诱导的pS189 supF基因突变谱非常相似。而与pS189携带相同supF基因但周围序列不同的pSP189，其光谱与pS189存在显著差异；这表明序列上下文对诱变特异性的影响可以在几十个碱基对的距离上起作用。
Rat-liver microsomes were used to activate aflatoxin B-1 for in vitro modification of the pS189 shuttle vector and the related signature vector pSP189, both of which carry the Escherichia coli supF gene as a mutational target. Plasmid degradation was minimized by carrying out the in vitro incubations in the absence of Mg2+ ions. Modified plasmids were transfected into human Ad293 cells, then recovered and electroporated into E. coli MBM7070 for mutant identification. Point mutation frequencies for in vitro modified plasmids were dramatically increased over the spontaneous background level. Mutant plasmids were characterized by DNA-sequence analysis. The vast majority of aflatoxin B-1-induced mutations were base substitutions, mostly G:C to T:A transversions. The spectrum of anatoxin B-1-induced mutations in the pS189 supF gene was very similar to that observed previously for the pS189 supF gene with anatoxin B-1 activated by cytochrome P450 1A2 (CYP1A2) synthesized from a cDNA expression vector within transfected Ad293 cells. However, the spectrum for pSP189, which carries the same supF gene as pS189, but with different surrounding sequences, exhibited some notable differences from that of pS189; this suggests that sequence context effects on mutagenic specificity can operate over distances of tens of base pairs.