Construction of an EBV-derived shuttle vector for studying the influence of transcription on mutagenesis.
We have constructed an EBV-derived shuttle vector, pF1-EBV, which replicates in human cells as an extrachromosomal element. The structural sequences of the gene encoding the bacterial xanthine-guanine-phosphoribosyltransferase (gpt) were fused to the promoter and presumptive control region of the mouse metallothionein I (MT-I) gene. Human 293 cells transformed with the recombinant plasmid synthesized gpt mRNA and the expression of the gene was inducible by zinc. The gpt gene offers a convenient system of selection for mutant plasmids by transformation into the appropriate gpt- E. coli strain. A clonal cell line created by establishment of the pF1-EBV shuttle vector showed a spontaneous gpt- frequency of 2.10(-5). An increase in mutation frequency above background was induced by mutagenizing this cell line with the alkylating agent N-methyl-N-nitrosourea (MNU). The recombinant molecule that we have constructed should provide a tool for studying the role of gene expression in DNA repair and mutagenesis.
Palombo
F
F
Laboratory of Comparative Toxicology and Ecotoxicology, Istituto Superiore di Sanità, Rome, Italy.
Dogliotti
E
E
D000818:Animals; D015152:Blotting, Northern; D002478:Cells, Cultured; D004247:DNA / Q000032:analysis / Q000302:isolation & purification; D004926:Escherichia coli / Q000235:genetics; D005822:Genetic Vectors; D004854:Herpesvirus 4, Human / Q000235:genetics*; D006801:Humans; D007041:Hypoxanthine Phosphoribosyltransferase / Q000235:genetics; D007668:Kidney / Q000166:cytology / Q000187:drug effects; D008668:Metallothionein / Q000235:genetics; D008770:Methylnitrosourea / Q000633:toxicity; D051379:Mice; D009154:Mutation; D009693:Nucleic Acid Hybridization; D010957:Plasmids; D011401:Promoter Regions, Genetic; D012313:RNA / Q000235:genetics; D011995:Recombination, Genetic; D014158:Transcription, Genetic; D014162:Transfection
D012313:RNA; D008770:Methylnitrosourea; D004247:DNA; D008668:Metallothionein; D007041:Hypoxanthine Phosphoribosyltransferase