Identification of mutator genes and mutational pathways in Escherichia coli using a multicopy cloning approach.

We searched for genes that create mutator phenotypes when put on to a multicopy plasmid in Escherichia coli. In many cases, this will result in overexpression of the gene in question. We constructed a random shotgun library with E.

Polymerases leave fingerprints: analysis of the mutational spectrum in Escherichia coli rpoB to assess the role of polymerase IV in spontaneous mutation.

We compared the distribution of mutations in rpoB that lead to rifampin resistance in strains with differing levels of polymerase IV (Pol IV), including strains with deletions of the Pol IV-encoding dinB gene, strains with a chromosomal copy of dinB, strains with the F'128 plasmid, and strains with plasmid amplification of either the dinB operon (dinB-yafNOP) or the dinB gene alone. This analysis identifies several hot spots specific to Pol IV which are virtually absent from the normal spontaneous spectrum, indicating that Pol IV does not contribute significantly to mutations occurring during exponential growth in liquid culture.

Developing a genetic system in Deinococcus radiodurans for analyzing mutations.

We have applied a genetic system for analyzing mutations in Escherichia coli to Deinococcus radiodurans, an extremeophile with an astonishingly high resistance to UV- and ionizing-radiation-induced mutagenesis. Taking advantage of the conservation of the beta-subunit of RNA polymerase among most prokaryotes, we derived again in D. radiodurans the rpoB/Rif(r) system that we developed in E.

Competition between MutY and mismatch repair at A x C mispairs In vivo.

We show that the MutY protein competes with the MutS-dependent mismatch repair system to process at least some A. C mispairs in vivo, converting them to G. C pairs.

Use of the rpoB gene to determine the specificity of base substitution mutations on the Escherichia coli chromosome.

Mutations in the rpoB gene of Escherichia coli result in resistance to the antibiotic rifampicin (Rif(r)) by altering the beta subunit of RNA polymerase. Previous studies have identified 39 single base substitutions in the rpoB gene that lead to Rif(r) at 37 degrees C and an additional two mutations that result in temperature sensitive cells. We have extended this work and identified an additional 30 single base substitutions that result in the Rif(r) phenotype.