Inhibition of transient gene expression in Chinese hamster ovary cells by cyclobutane dimers and (6-4) photoproducts in transfected ultraviolet-irradiated plasmid DNA.
Using a transient gene expression assay to measure host cell reactivation, the effects of cyclobutane dimer and noncyclobutane dimer uv photoproducts on expression of a reporter gene were examined in normal and repair-deficient Chinese hamster ovary (CHO) cell lines. Ultraviolet damage in plasmid pRSV beta gal DNA, containing the Escherichia coli beta-galactosidase gene, resulted in reduced reporter gene expression in both uv-hypersensitive mutant CHO cell lines UV5 and UV61 relative to wild-type, parental AA8 cells. However, the effects of uv irradiation of transfected plasmid DNA on gene activity were reduced in UV61, a mutant with normal (6-4) photoproduct repair, compared to UV5, which is deficient in (6-4) photoproduct repair; this reduction correlated with the intermediate uv-hypersensitivity of UV61. Selective removal of cyclobutane dimers by in vitro photoreactivation of uv-irradiated plasmid DNA prior to transfection substantially increased reporter gene activity in both uv-hypersensitive mutant cell lines. This increase was significantly greater in UV61 than in UV5, consistent with UV5 being deficient in repair of both (6-4) photoproducts and cyclobutane dimers. These results suggest that unrepaired (6-4) photoproducts in transfected pRSV beta gal plasmid DNA are responsible for a significant fraction of the reduction in transient gene expression observed in recipient uv-hypersensitive CHO cell mutants.
Mitchell
D L
DL
University of Texas M.D. Anderson Cancer Center, Science Park, Research Division, Smithville 78957.
Vaughan
J E
JE
Nairn
R S
RS
D000818:Animals; D002460:Cell Line; D006224:Cricetinae; D003412:Cricetulus; D003503:Cyclobutanes / Q000494:pharmacology*; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D005260:Female; D005696:Galactosidases / Q000235:genetics*; D005786:Gene Expression Regulation / Q000528:radiation effects*; D005796:Genes / Q000528:radiation effects; D005798:Genes, Bacterial / Q000528:radiation effects; D010053:Ovary; D010782:Photolysis; D010957:Plasmids / Q000528:radiation effects*; D014162:Transfection; D014466:Ultraviolet Rays; D001616:beta-Galactosidase / Q000096:biosynthesis / Q000235:genetics*
D003503:Cyclobutanes; D005696:Galactosidases; D001616:beta-Galactosidase