Removal of UV photoproducts from an adenovirus-encoded reporter gene following infection of unirradiated and UV-irradiated human fibroblasts.
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Ad5HCMVsp1lacZ is a recombinant nonreplicating adenovirus containing the lacZ gene under the control of the human cytomegalovirus immediate early promoter. Previous reports show that lacZ expression for UV-irradiated Ad5HCMVsp1lacZ is greater in nucleotide excision repair (NER) proficient compared to NER deficient human fibroblasts from patients with xerodermapigmentosum (XP) and Cockaye's syndrome (CS) and that pre-UV-treatment of normal fibroblasts results in an enhanced expression of the lacZ gene from UV-irradiated Ad5HCMVsp1lacZ. We have used a quantitative PCR technique to examine whether UV photoproducts are actually removed from the lacZ gene following infection of human fibroblasts with UV-irradiated Ad5HCMVsp1lacZ. Primers flanking a 2.6-kb region of the lacZ reporter gene were added to equal amounts of DNA extracted from Ad5HCMVsp1lacZ infected cells and amplified by PCR using radiolabelled nucleotides as substrates. Results show a significant removal of UV photoproducts in normal human fibroblasts, but a reduced removal in NER deficient XP and CS cells and an enhanced removal in pre-UV-treated normal fibroblasts.
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