Determination of DNA sequence changes induced by ethyl methanesulfonate in human cells, using a shuttle vector system.

Abstract

The DNA sequence changes for 54 mutations induced in human cells by the alkylating agent ethyl methanesulfonate are reported. The mutations were obtained by using a shuttle vector system with the bacterial lacl gene as the target. Of the 54 mutations obtained, 53 were G:C to A:T transitions.Recent advances in molecular biology have led to the development of recombinant DNA vectors designed to provide information about the nature of the mutations induced by various mutagens and carcinogens in mammalian cells (1,24,15,20,21,25). These shuttle vector systems comprise plasmids with the ability to replicate in both mammalian and bacterial cells and with genes in which mutations can be scored in Escherichia coli. After such vectors have been transfected into mammalian cells and mutagenesis has occurred, plasmid DNA is purified and returned to E. coli for ease of detection and analysis of mutations.We have devised such a system (15) by using the lacI gene of E. coli as the mutational target and portions of simian virus 40 for replication in human cells (13). The spontaneous frequency for mutations in lacI after passage of the shuttle vector through the human cells is 3.4 x 10-7/base pair. This value is approximately 2 orders of magnitude higher than the spontaneous mutation frequency expected for a chromosomal gene in human cells (26). The elevated mutation frequency is apparently a result of mutations incurred during transfection (1,15,16,20,21). However, the spontaneous mutation frequency per base pair in this lacI shuttle system is at least 1 order of magnitude lower than that of any other simian virus 40-based shuttle system described to date. It is feasible to obtain a mutation frequency above the background level in the shuttle vector by treating the human cells with a mutagen shortly after transfection with the vector DNA. The entire mutagenesis process thus takes place in the human cell environment. Shortly after mutagenesis the vector DNA is purified from the human cells and returned to E. coli, where the mutations can be rapidly detected.The well-characterized genetics of the lacI gene make it possible to determine the DNA sequence changes involved in base substitution mutations by using genetic techniques alone (4, 5). With this system, nonsense mutations are assigned to 1 of over 80 mutational sites by a combination of deletion mapping, pattern of suppression, and comparison with a library of all the possible nonsense sites. Because the entire lacI sequence is known, determining the identity of each nonsense site genetically is equivalent to determining the sequence change by biochemical methods. Also, the DNA sequence surrounding each mutational site is automatically revealed.Recently we used this lacI shuttle system to determine the nature of the mutations induced by UV light in human cells * Corresponding author.(14). Here we used the system to examine the mutagenic specificity of a member of another major group of mutagens, the alkylating agents. We chose ethyl methanesulfonate (EMS; Sigma Chemical Co.)...

Direct selection for mutations affecting specific splice sites in a hamster dihydrofolate reductase minigene.

“…Mutations at 5 of the 7 less-well-conserved positions were found; more often than not, the phenotype was severe, with little or no correctly spliced transcripts detected (NS79, 42,8,91, and 90 at -3, -2, -1, +3, and +4, respectively). No mutants were isolated that affected positions +5 or +6.…”

“…EMS is VOL. 13,1993 on April 27, 2019 by guest http://mcb.asm.org/ Downloaded from 296 CHEN AND CHASIN expected to alkylate G residues (67) and is known to induce predominantly G. C to A. T transitions in mammalian cells (6,42). Indeed, the two guanine bases at donor positions -1 and +1 on the nontranscribed strand (top strand in Fig.…”

Genetic definition of ras effector elements.

“…EMS has been shown in cultured mammalian cells to induce G-to-A transitions almost exclusively (26).…”

Chemically induced mutagenesis in a shuttle vector with a low-background mutant frequency.

“…0.1%) of mutations. By using a lacI-SV40 shuttle vector in the human 293 cell line, Calos and coworkers (5,6) observed a significant increase in the mutant frequency of the lacI gene after treatment of cells containing the vector with ultraviolet light or ethyl methanesulfonate.…”