title: Description of a new amplifiable shuttle vector for mutagenesis studies in human cells: application to N-methyl-N'-nitro-N-nitrosoguanidine-induced mutation spectrum.
abstract: In order to analyze the mechanisms of mutagenesis in human cells, we have established a human 293 cell-derived line containing a permanent mutagenesis target, the bacterial lacZ' gene, on an episomal EBV/SV40-based shuttle vector. This plasmid was maintained at a low copy number per cell which rendered it closer to an endogenous gene as compared to the usual transient shuttle vectors. Transient amplification of vectors, inside the host cell due to expression of the SV40 T-antigen, allowed the recovery of a large number of bacterial colonies transformed by plasmids extracted from human cells. Mutations produced in human host cells on the lacZ' locus were easily and rapidly scored and identified in bacteria using the blue/white color assay. Over a 6-month period in culture, we have shown that the lacZ' gene exhibited a low background frequency of point mutations (< 4.8 x 10(-6)). The efficiency of our system for detecting genotoxic-induced mutations was investigated by treating cells with a potent mutagen, the direct alkylating agent N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). A significant increase (< 230-fold) in the frequency of single-base substitutions was observed after MNNG treatment. In total, 63 MNNG-induced independent mutations were characterized. All substitutions but one involved G:C base pairs with 89% being G:C to A:T transitions which is consistent with the MNNG mutagenic specificity already reported in bacteria and mammalian cells. Mutations were distributed along the two strands of the lacZ' gene and there was no obvious influence of either the 5' or the 3' flanking base near the G:C to A:T transition sites. The low spontaneous point mutation frequency on the mutagenesis locus and the ability to detect induced point mutations indicate that this system could be readily used in human mutagenesis studies at the molecular level.
authors.affiliation: Laboratory of Molecular Genetics, Institut de Recherches Scientifiques sur le Cancer, Villejuif, France.
authors.forename: A
authors.initials: A
authors.lastname: Stary
authors.forename: C F
authors.initials: CF
authors.lastname: Menck
authors.forename: A
authors.initials: A
authors.lastname: Sarasin
mesh_terms: D001483:Base Sequence; D002460:Cell Line; D004247:DNA; D005822:Genetic Vectors; D004854:Herpesvirus 4, Human / Q000235:genetics; D006801:Humans; D008769:Methylnitronitrosoguanidine / Q000633:toxicity*; D008969:Molecular Sequence Data; D009152:Mutagenicity Tests / Q000379:methods*; D010957:Plasmids; D013539:Simian virus 40 / Q000235:genetics; D001616:beta-Galactosidase / Q000235:genetics
chemical_list: D008769:Methylnitronitrosoguanidine; D004247:DNA; D001616:beta-Galactosidase