A shuttle vector plasmid for studying carcinogen-induced point mutations in mammalian cells

• Published in: Gene Vol. 38; no. 1; pp. 233 - 237
• Main Authors: Seidman, Michael M., Dixon, Kathleen, Razzaque, Abdur, Zagursky, Robert J., Berman, Michael L.
• Format: Journal Article
• Language: English
• Published: Lausanne Elsevier B.V 1985; Amsterdam Elsevier; New York, NY
• Subjects: Animals; Biological and medical sciences; Carcinogens - pharmacology; Cell Line; Cercopithecus aethiops; Chemical mutagenesis; DNA Replication; E. colihost; Genetic Vectors; Medical sciences; mutagenesis; Mutation - drug effects; Plasmids; Recombinant DNA; RNA, Transfer - genetics; Simian virus 40 - genetics; Suppression, Genetic; SV40 virus; Toxicology; ‘poison’ region of pBR322; UV; ‘poison’ region of pBR322; ori; Recombinant DNA; mutagenesis; bp; Ap; Tc; IG region; DMEM; R; XGal; RF; bla; E. colihost; IPTG; DEAE; SV40 virus; Cell culture; Mutagenicity testing; Escherichia coli; Polyomavirus; Monkey; Papovaviridae; Kidney; Suppressor tRNA; Virus; Vertebrata; Mammalia; Mutagenesis; Point mutation; Primates; Shuttle vector; Bacteria; Escherichieae; Enterobacteriaceae
• ISSN: 0378-1119; 1879-0038
• DOI: 10.1016/0378-1119(85)90222-7

We have constructed a shuttle vector plasmid for studying mutagenesis in mammalian cells. The plasmid replicates in cell lines permissive for SV40 virus as well as in the bacterium Escherichia coliand carries a bacterial suppressor tRNA gene ( supF) that can serve as a mutagenesis marker. The plasmid replicates as efficiently as SV40 virus in African Green Monkey kidney CV1 cells, indicating that all traces of the inhibitory sequences normally found in pBR322 and its derivatives have been removed. The design of the plasmid and the small size of the mutagenesis target gene decrease the probability of recovering spontaneous deletion mutations that have been shown to occur at high frequency during passage in mammalian cells. The frequency of spontaneous-mutant plasmids recovered after passage in CV1 cells is substantially lower than with other vectors described previously. When the plasmid DNA is treated with UV radiation before passage in CV1 cells, mutants are observed at a frequency about 20-fold above the spontaneous background.