An Escherichia coli plasmid-based, mutational system in which supF mutants are selectable: insertion elements dominate the spontaneous spectra.
A new system is described to determine the mutational spectra of mutagens and carcinogens in Escherichia coli; data on a limited number (142) of spontaneous mutants is presented. The mutational assay employs a method to select (rather than screen) for mutations in a supF target gene carried on a plasmid. The E. coli host cells (ES87) are lacI- (am26), and carry the lacZ delta M15 marker for alpha-complementation in beta-galactosidase. When these cells also carry a plasmid, such as pUB3, which contains a wild-type copy of supF and lacZ-alpha, the lactose operon is repressed (off). Furthermore, supF suppression of lacIam26 results in a lactose repressor that has an uninducible, lacIS genotype, which makes the cells unable to grow on lactose minimal plates. In contrast, spontaneous or mutagen-induced supF- mutations in pUB3 prevent suppression of lacIam26 and result in constitutive expression of the lactose operon, which permits growth on lactose minimal plates. The spontaneous mutation frequency in the supF gene is approximately 0.7 and approximately 1.0 x 10(-6) without and with SOS induction, respectively. Spontaneous mutations are dominated by large insertions (67% in SOS-uninduced and 56% in SOS-induced cells), and their frequency of appearance is largely unaffected by SOS induction. These are identified by DNA sequencing to be Insertion Elements; IS1 dominates, but IS4, IS5, gamma-delta and IS10 are also obtained. Large deletions also contribute significantly (19% and 15% for -SOS and +SOS, respectively), where a specific deletion between a 10 base pair direct repeat dominates; the frequency of appearance of these mutations also appears to be unaffected by SOS induction. In contrast, SOS induction increases base pairing mutations (13% and 27% for -SOS and +SOS, respectively). The ES87/pUB3 system has many advantages for determining mutational spectra, including the fact that mutant isolation is fast and simple, and the determination of mutational changes is rapid because of the small size of supF.
Rodriguez
H
H
Department of Biology, Boston University, MA 02215.
Snow
E T
ET
Bhat
U
U
Loechler
E L
EL
D001483:Base Sequence; D004252:DNA Mutational Analysis / Q000379:methods*; D004251:DNA Transposable Elements; D004926:Escherichia coli / Q000235:genetics*; D005798:Genes, Bacterial; D016153:Genes, Suppressor; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional; D010957:Plasmids; D012329:RNA, Bacterial / Q000235:genetics; D012343:RNA, Transfer / Q000235:genetics; D017384:Sequence Deletion; D014170:Transformation, Genetic
D004251:DNA Transposable Elements; D012329:RNA, Bacterial; D012343:RNA, Transfer