Genetic assays for measuring rates of (CAG)·(CTG) repeat instability in Escherichia coli

Vera I. Hashem, William A. Rosche, Richard R. Sinden

Research output: Contribution to journalArticlepeer-review

38 Scopus citations

Abstract

Genetic selection assays were developed to measure rates of deletion of one or more (CAG)·(CTG) repeats, or an entire repeat tract, in Escherichia coli. In-frame insertions of ≥25 repeats in the chloramphenicol acetyltransferase (CAT) gene of pBR325 resulted in a chloramphenicol-sensitive (Cms) phenotype. When (CAG)25 comprised the leading template strand, deletion of one or more repeats resulted in a chloramphenicol resistant (Cmr) phenotype at a rate of 4×10-2 revertants per cell per generation. The mutation rates for plasmids containing (CAG)43 or (CAG)79 decreased significantly. When (CTG)n comprised the leading template strand the Cmr mutation rates were 100-1000 lower than for the opposite orientation. As an initial application of this assay, the effects of mutations influencing mismatch repair and recombination were examined. The methyl directed mismatch repair system increased repeat stability only when (CTG)n comprised the leading template strand. Replication errors made with the opposite repeat orientation were apparently not recognized. For the (CAG)n leading strand orientation, mutation rates were reduced as much as 3000-fold in a recA- strain. In a second assay, out-of-frame mutation inserts underwent complete deletion at rates ranging from about 5×10-9 to 1×10-7 per cell per generation. These assays allow careful quantitation of triplet repeat instability in E. coli and provide a way to examine the effects of mutations in replication, repair, and recombination on repeat instability.

Original languageEnglish (US)
Pages (from-to)25-37
Number of pages13
JournalMutation Research - Fundamental and Molecular Mechanisms of Mutagenesis
Volume502
Issue number1-2
DOIs
StatePublished - May 22 2002

Keywords

  • Mismatch repair
  • Repeat instability
  • Replication slippage
  • Spontaneous mutation
  • Template slippage
  • Triplet repeats

ASJC Scopus subject areas

  • Molecular Biology
  • Genetics
  • Health, Toxicology and Mutagenesis

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