title: Transient expression of a plasmid gene, a tool to study DNA repair in human cells: defect of DNA repair in Cockayne syndrome; one thymine cyclobutane dimer is sufficient to block transcription.
abstract: Transfected recombinant DNA with regulatory elements such as eukaryotic promoter and termination sites is transiently expressed in human fibroblast cells. Utilizing an expression vector containing the simian virus 40 (SV 40) early control region followed by the E. coli chloramphenicol acetyltransferase (CAT) gene, we investigated the ability of normal, Xeroderma pigmentosum and Cockayne Syndrome cells to repair UV lesions in transfected DNA. Fibroblasts from Xeroderma pigmentosum patients which cannot excise pyrimidine cyclobutane dimers were unable to restore expression of UV irradiated CAT gene. An UV dose inducing one thymine cyclobutane dimer in the transcribed strand of the CAT gene blocked its transcription in these repair deficient cells. Normal cell were able to repair the lesions in transfected DNA during an incubation period of about 40 h and in this way could overcome the UV block. In several fibroblast cell lines from patients suffering from Cockayne Syndrome expression of UV damaged CAT gene was restored significantly less than in normal fibroblasts, indicating that Cockayne Syndrome is associated with a UV repair defect.
mesh_terms: D000477:Alkylating Agents; D002795:Choline O-Acetyltransferase / Q000378:metabolism; D002877:Chromosomes, Human / Q000378:metabolism; D003057:Cockayne Syndrome / Q000235:genetics* / Q000378:metabolism; D003432:Cross-Linking Reagents; D004247:DNA / Q000378:metabolism; D004260:DNA Repair / Q000528:radiation effects; D004392:Dwarfism / Q000235:genetics*; D005347:Fibroblasts / Q000378:metabolism; D005786:Gene Expression Regulation; D006801:Humans; D010957:Plasmids; D011740:Pyrimidine Dimers / Q000494:pharmacology*; D014158:Transcription, Genetic; D014162:Transfection; D014466:Ultraviolet Rays; D014983:Xeroderma Pigmentosum / Q000235:genetics / Q000378:metabolism
chemical_list: D000477:Alkylating Agents; D003432:Cross-Linking Reagents; D011740:Pyrimidine Dimers; D004247:DNA; D002795:Choline O-Acetyltransferase