Gentil A, Cabral-Neto J B, Mariage-Samson R, Margot A, Imbach J L, Rayner B, Sarasin A
Laboratory of Molecular Genetics, Institut de Recherches Scientifiques sur le Cancer, Villejuif, France.
J Mol Biol. 1992 Oct 20;227(4):981-4. doi: 10.1016/0022-2836(92)90513-j.
Abasic sites are common DNA lesions produced either spontaneously or as a consequence of the action of some genotoxic agent. The mutagenic properties of a unique abasic site replicated in mammalian cells have been studied using a shuttle vector. A plasmid, able to replicate both in mammalian cells and in bacteria, carrying a unique abasic site chemically synthesized has been constructed. After replication in mammalian cells, plasmid DNA was recovered and used to transform bacteria. Mutants were screened without selection pressure by differential hybridization with a labelled oligonucleotide and their DNA was sequenced. A mutation frequency ranging from 1% to 3% was found, depending on the base originally inserted during the vector construction, opposite the abasic site. All the sequenced mutants correspond to single base-pair substitutions targeted at the abasic site. We observed a deficit in guanine incorporation opposite the abasic site, while the three other bases were incorporated with a similar efficiency. The mutational potency of abasic sites was observed without any voluntary preconditioning treatment of mammalian cells in order to induce "SOS" like conditions.
无碱基位点是常见的DNA损伤，可自发产生或由某些基因毒性剂的作用导致。已使用穿梭载体研究了在哺乳动物细胞中复制的独特无碱基位点的诱变特性。构建了一种质粒，它能够在哺乳动物细胞和细菌中复制，并携带化学合成的独特无碱基位点。在哺乳动物细胞中复制后，回收质粒DNA并用于转化细菌。通过与标记的寡核苷酸进行差异杂交，在没有选择压力的情况下筛选突变体，并对其DNA进行测序。根据载体构建过程中最初插入到无碱基位点对面的碱基不同，发现突变频率在1%到3%之间。所有测序的突变体都对应于针对无碱基位点的单碱基对替换。我们观察到在无碱基位点对面鸟嘌呤掺入不足，而其他三种碱基的掺入效率相似。在没有对哺乳动物细胞进行任何诱导“ SOS”样条件的自愿预处理的情况下，观察到了无碱基位点的诱变能力。
