POLΗ基因反义阻断细胞系的建立及POLΗ在MNNG引起的非定标性突变中的作用  

Establishment of a cell line with antisense-blocked POLH and the role of POLH in alkylating agent MNNG induced nontargeted mutagenesis

作　　者：罗月球[1] 杨军[1] 余应年[1] 

机构地区：[1]浙江大学医学院病理生理教研室,浙江杭州310031

出　　处：《浙江大学学报（医学版）》2003年第5期398-402,共5页Journal of Zhejiang University(Medical Sciences)

基　　金：<国家重点基础研究发展规划> (973)(2 0 0 2 CB5 12 90 1);国家自然科学基金资助 (30 170 810 )

摘　　要：目的 :通过建立 POLΗ反义阻断细胞系 FL - POLΗ-,研究 POLΗ的功能。方法 :将 POLΗ基因的 14 73～ 2 131片段反向克隆到哺乳动物细胞表达载体 p MAMneo- amp-中 ,将重组表达质粒转染 FL细胞 ,G4 18筛选 ,获得 POLΗ基因被表达阻断的哺乳动物细胞系 FL - POLΗ-。采用穿梭质粒 p Z189介导的突变实验 ,观察在 POLΗ基因表达被阻断细胞系中进行复制时的自发突变频率和 MNNG引起的诱发突变频率。结果 :成功建立细胞系 FL -POLΗ-。穿梭质粒 p Z189在 FL- POLΗ-细胞中复制后 ,其 Sup F t RNA基因的自发突变频率为 13.5× 10 -4,而对照细胞 FL和 FL- M分别为 4 .9× 10 -4和 3.7× 10 -4。同时 ,质粒在接触过 MNNG的 FL- POLΗ-细胞中复制 ,其 Sup Ft RNA基因的非定标性突变频率不发生。结论 :POLΗ在维持哺乳类细胞的遗传稳定性中起重要作用 ,且参与哺乳动物细胞中 MNNG引起的非定标性突变。Objective: To investigate the function of POLΗ(polymerase eta) through establishment of the POLΗ gene-blocked cell line FL-POLΗ-. Methods: A mammalian expression vector expressing antisense POLΗ gene fragment pMAMneo-amp-POLΗ- was constructed by cloning the 1473～2131 fragment of POLΗ gene into the mammalian expression vector pMAMneo-amp- in antisense orientation. The FL cells were transfected with this antisense RNA expressing vector and selected by G418. The mutation assay was conducted using the shuttle plasmid pZ189. Results: The spontaneous mutation frequency of SupF tRNA gene in the plasmid replicated in the FL-POLΗ- was 13.5×10 -4, while it was 4.9×10 -4 and 3.7×10 -4 in the control cells FL and FL-M, respectively. The nontargeted mutation frequency of SupF tRNA gene decreased in the plasmid replicated in these cell lines pretreated with MNNG. Conclusion: POLH plays an important role in maintenance of genetic stability and genesis of nontargeted mutation.

关 键 词：POLH基因 N-甲基-N'-硝基-N-亚硝基胍 非定标性突变 DNA突变分析 

分 类 号：R346[医药卫生—基础医学]