CHARACTERIZATION OF MUTATIONS IN THE LEXA GENE OF ESCHERICHIA COLI K-12.
AuthorPETERSON, KENNETH RICHARD.
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PublisherThe University of Arizona.
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AbstractThe lexA41 (formerly tsl-l) mutant was previously isolated as a UV-resistant, temperature-sensitive derivative of its UV-sensitive lexA3(Ind⁻) parent. Cells exhibit a so-called "split-phenotype", a phenomenon in which only a subset of the SOS responses can be detected physiologically following inducing treatments. In this work, lexA41 has been cloned and sequenced; the mutant gene retains the lexA3 mutation (Gly to Asp at position 85) and has a second mutation, lexA41 (Ala to Thr at position 132). LexA41 protein is not cleaved by the RecA protein-catalyzed pathway in vivo, but the mutant protein is degraded by the Lon protease at both 32° and 42°C. β-galactosidase activities of lac fusions to thirteen different SOS promoters were measured at 30° and 42° to determine levels of expression and were found to vary considerably. LexA41 protein is deficient in repressor function. The temperature sensitive phenotype is due to increased expression of sulA, which encodes a division inhibitor, at 42°. Excision repair genes, including uvrA, uvrB and uvrD, are constitutively expressed at 30° accounting for the UV resistance of the lexA41 mutant, but the SOS mutagenesis operon, umuDC, is not adequately derepressed explaining the failure to induce mutagenesis in this background. This differential expression of SOS genes gives a plausible explanation of the "split-phenotype" associated with lexA41. In another set of experiments, I have examined the level of expression of the SOS regulon in cells lacking DNA adenine methylase activity (dam⁻). Mud (Ap, lac) fusions to several SOS operons (recA, lexA, uvrA, uvrB, uvrD, sulA, dinD, and dinF) were found to express higher levels of (beta)-galactosidase in dam⁻ strains than in isogenic dam⁺ strains. The attempted construction of dam⁻ strains that were also mutant in one of several SOS genes indicated that viability of methylase-deficient strains correlates with the inactivation of the SOS repressor (LexA protein). Consistent with this, the wild-type functions of two LexA-repressed genes (recA and ruv) appear to be required for viability of dam⁻ strains.
Degree ProgramMolecular and Cellular Biology