[Establishment of a cell line with antisense-blocked POLH and the role of POLH in alkylating agent MNNG induced nontargeted mutagenesis].
To investigate the function of POLH(polymerase eta) through establishment of the POLH gene-blocked cell line FL-POLH(-).



A mammalian expression vector expressing antisense POLH gene fragment pMAMneo-amp-POLHA (-) was constructed by cloning the 1473 - 2131 fragment of POLH 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.



The spontaneous mutation frequency of SupF tRNA gene in the plasmid replicated in the FL-POLH(-) was 13.5 x 10(-4), while it was 4.9x10(-4) and 3.7x10(-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.



POLH plays an important role in maintenance of genetic stability and genesis of nontargeted mutation.
Luo
Yue-qiu
YQ
Department of Pathophysiology, College of Medicine, Zhejiang University, Hangzhou 310031, China.
Yang
Jun
J
Yu
Ying-nian
YN
D016375:Antisense Elements (Genetics) / Q000494:pharmacology*; D002460:Cell Line; D004259:DNA-Directed DNA Polymerase / Q000235:genetics / Q000502:physiology*; D008769:Methylnitronitrosoguanidine / Q000633:toxicity*; D016296:Mutagenesis; D000099270:Y-Family DNA Polymerases
D016375:Antisense Elements (Genetics); D004259:DNA-Directed DNA Polymerase; D008769:Methylnitronitrosoguanidine; C112338:Rad30 protein; D000099270:Y-Family DNA Polymerases