Mutagenesis in the lacI gene target of E. coli: improved analysis for lacI(d) and lacO mutants

Mutation Research
Sarah J Swerdlow, R M Schaaper

Abstract

The lacI gene of Escherichia coli has been a highly useful target for studies of mutagenesis, particularly for analysis of the specificity (spectrum) of mutations generated under a variety of conditions and in various genetic backgrounds. The gene encodes the repressor of the lac operon, and lacI-defective mutants displaying constitutive expression of the operon are readily selected. DNA sequencing of the lacI mutants has often been confined to the N-terminal region of the protein, as it presents a conveniently short target with a high density of detectably mutable sites. Mutants in this region are easily selected due to their dominance in a genetic complementation test (lacI(d) mutants). A potential complication in these studies is that constitutive expression of lac may also arise due to mutations in the lac operator (lacO mutants). Under some conditions, for example when analyzing spontaneous mutations, lacO mutants can comprise a very high fraction of the constitutive mutants due to a strong base-substitution hotspot in the lac operator. Such mutational hot spots diminish the return of the sequencing effort and do not yield significant new information. For this reason, a procedure to eliminate the lacO mutants prior to DNA ...Continue Reading

References

Nov 15, 1992·Proceedings of the National Academy of Sciences of the United States of America·A R OllerR M Schaaper
Nov 1, 1988·Proceedings of the National Academy of Sciences of the United States of America·R M Schaaper
Sep 1, 1987·Proceedings of the National Academy of Sciences of the United States of America·R M Schaaper, R L Dunn
Jan 1, 1985·Gene·R M SchaaperB W Glickman
Jun 1, 2000·Proceedings of the National Academy of Sciences of the United States of America·K A Datsenko, B L Wanner
Apr 27, 2001·Toxicology Letters·R C SillsM L Cunningham

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Citations

Aug 28, 2019·Proceedings of the National Academy of Sciences of the United States of America·Griffin ChureRob Phillips

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