Mutagenesis by 06 meG residues within codon 12 of the human Ha-ras proto-oncogene in monkey cells

摘要

The first or/and the second guanines of the human Haras codon 12 (normally GGC) were substituted by O6 meG residues and the modified sequence was subsequently introduced into an SV4O-based shuttle vector able to replicate in both simian cells and bacteria. After replication in simian COS7 cells (proficient in O6-alkyl-guanine transferase), plasmid DNA was extracted and mutations were screened in E.coli DH5α cells. The vast majority of the mutations induced by O6 meG were G-A transitions The mutation frequency observed at the second guanine of codon 12 (12G2 position: 3.75% ± 0.4) was higher than the one observed at the first guanine (12G1 position: 1.09% ± 0.6).This difference was confirmed by the results obtained when two adjacent 0 meG residues were positioned within codon 12.The higher mutation frequency observed for the 12G2 position could be attributed to differential repair or/and variation in polymerase fidelity. These results are in agreement with animal experiments where alkylating agents gave rise to mutation on G2 position of codon 12.