Cabral-Neto J B, Gentil A, Cabral R E, Sarasin A
Laboratory of Molecular Genetics, Institut de Recherches Scientifiques sur le Cancer, Villejuif, France.
Mutat Res. 1993 Aug;288(2):249-55. doi: 10.1016/0027-5107(93)90091-s.
Almost all spontaneous point mutations found on a single-stranded shuttle vector after its transfection and replication in monkey cells were located at cytosine residues. In order to understand this very specific type of targeting we have studied the possible implication of uracil residues in the induction of these spontaneous mutations. The single-stranded shuttle vector pCF3A carrying the supF tRNA gene as a mutagenesis target has been allowed to replicate in mammalian COS7 cells, mutations being screened in bacteria using the beta-galactosidase assay. Progenies from untreated DNA and DNA treated with the uracil-DNA glycosylase prior to transfection were analyzed to determine the amount and classes of mutations. While spontaneous mutation frequency was 9.7 x 10(-4) for control DNA, single-stranded vector treated with the E. coli uracil-DNA glycosylase exhibited a reduced mutation frequency of about 30%. The abolished mutations were mainly confined to the cytosine to thymine transitions for which a decrease by a factor of 5 was indeed observed. This finding fits well with the fact that it is usually admitted that uracil pairs with adenine, indicating therefore that approximately 30% of spontaneous mutations observed in our experimental conditions and 80% of C to T transitions may be due to the presence of uracil instead of cytosine.
几乎所有在单链穿梭载体转染并在猴细胞中复制后发现的自发点突变都位于胞嘧啶残基处。为了理解这种非常特殊的靶向类型，我们研究了尿嘧啶残基在这些自发突变诱导中的可能作用。携带supF tRNA基因作为诱变靶点的单链穿梭载体pCF3A已在哺乳动物COS7细胞中复制，使用β-半乳糖苷酶测定法在细菌中筛选突变。分析未处理DNA以及转染前用尿嘧啶-DNA糖基化酶处理的DNA的子代，以确定突变的数量和类别。对照DNA的自发突变频率为9.7×10^(-4)，而用大肠杆菌尿嘧啶-DNA糖基化酶处理的单链载体的突变频率降低了约30%。被消除的突变主要局限于胞嘧啶到胸腺嘧啶的转换，实际上观察到这种转换减少了5倍。这一发现与通常认为尿嘧啶与腺嘌呤配对的事实非常吻合，因此表明在我们的实验条件下观察到的约30%的自发突变和80%的C到T转换可能是由于尿嘧啶而非胞嘧啶的存在。
