反义阻断REV3基因表达对细胞自发突变和MNNG诱导突变的影响

Influence of Antisense Suppression of REV3 Gene Expression on Spontaneous and MNNG-induced Mutation Frequencies of HPRT Gene

目的 :通过反义核酸技术研究REV3基因对次黄嘌呤鸟嘌呤磷酸核糖基转移酶基因HPRT的自发突变频率及烷化剂甲基硝基亚硝基胍 (methy1-nitro -nitrosoguanidine ,MNNG)的诱发突变频率的影响。方法 :用REV3基因反义核酸表达质粒pMAM -REV3-转染人羊膜上皮细胞 (FL) ,建立了由地塞米松诱导下的REV3基因表达可被反义阻断的细胞系 (FL-REV3-) ,并应用经典的筛选HPRT基因位点的突变子的实验进行REV3基因对HPRT基因位点的自发与诱发突变影响的检测研究。结果 :在自发突变率的检测中 ,FL细胞系的自发突变率比它的诱发突变率低 3 0 1倍 (P <0 0 5 ) ,而在FL -REV3-细胞系中始终未能筛选到自发突变子 ;用 0 .4 μmol/LMNNG分别处理FL -REV3-和FL细胞系 ,前者诱发突变率比后者低 6 0 2倍 (P <0 0 5 )。结论 :REV3基因不仅参与细胞自发突变 ,而且参与细胞的诱发突变 。

Objective:With antisense RNA technique to observe the underlying effect of REV3 gene on spontaneous and MNNG(methyl-nitro-nitrosoguanidine)-induced mutation frequencies of HPRT locus Methods:The eukaryotic expression plasmid pMAM-REV3 - expressing antisense fragment to REV3 gene was transferred into FL cell to establish the FL-REV3 - cell line With classical mutant selection protocol,spontaneous and MNNG-induced mutants of HPRT gene locus were checked Results:The spontaneous mutation frequency of FL cell line was 3 01 times lower than MNNG-induced mutation frequency( P <0 05),no spontaneous mutant was ever found in FL-REV3 - cell line In observing the induced mutation frequency,it has been found that the mutation frequency of FL-REV3 - cell line was 60 2 times lower than that of the FL cell line( P <0 05) Conclusion:It is clear that REV3 gene is involved not only in spontaneous mutation ,but in the induced mutation as well REV3 gene may play a critical role in the DNA translesion repair of the cells after being exposured to DNA damage agents