Host cell reactivation of CAT-expression vectors as a method to assay for cloned DNA-repair genes.
We demonstrate the feasibility of using passive host-cell reactivation of a shuttle-vector pRSVcat to detect cloned DNA-repair genes. As models, a transient expression vector, pRSVdenV, and a positive-selection vector, pRSVdenV/SVgpt, were constructed containing the T4 coliphage denV gene, coding for an ultraviolet-specific endonuclease, under promotion of the Rous sarcoma virus (RSV) long-terminal repeat. Cotransfection of one or three copies of pRSVdenV per UV-irradiated pRSVcat molecule into xeroderma pigmentosum (XP) cells (XP12Ro[M1]) resulted in a dramatic increase in transient expression of chloramphenicol acetyl transferase (CAT) activity. XP clones stable transformed by pRSVdenV/SVgpt but not the parent cell line rescued CAT activity from this UV-irradiated reporter gene. The ability to express CAT activity from a UV-irradiated pRSVcat correlated with the presence of the structural denV gene as detected by Southern blot analysis. Post-UV irradiation colony-forming ability and DNA nucleotide excision-repair synthesis were partially restored in XP clones which rescued CAT activity. These results demonstrate the feasibility of using the cloned denV gene with its well characterized pyrimidine cyclobutane dimer-specific endonuclease activity to reconstitute UV-induced DNA repair in human cells deficient in DNA repair. Measuring CAT expression from pRSVcat affords a rapid, sensitive procedure to screen for functional cloned DNA-repair genes and to test mutant cells for defects in DNA repair.
Henderson
E E
EE
Department of Microbiology and Immunology, Temple University School of Medicine, Philadelphia, PA 19140.
Valerie
K
K
Green
A P
AP
de Riel
J K
JK
D000595:Amino Acid Sequence; D001483:Base Sequence; D002478:Cells, Cultured; D015500:Chloramphenicol O-Acetyltransferase / Q000235:genetics; D003001:Cloning, Molecular / Q000379:methods; D004260:DNA Repair; D005814:Genes, Viral; D005822:Genetic Vectors; D006801:Humans; D008969:Molecular Sequence Data; D010957:Plasmids; D014983:Xeroderma Pigmentosum / Q000235:genetics
D015500:Chloramphenicol O-Acetyltransferase