Genetic assays for measuring rates of (CAG).(CTG) repeat instability in Escherichia coli.
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 >or=25 repeats in the chloramphenicol acetyltransferase (CAT) gene of pBR325 resulted in a chloramphenicol-sensitive (Cm(s)) phenotype. When (CAG)25 comprised the leading template strand, deletion of one or more repeats resulted in a chloramphenicol resistant (Cm(r)) phenotype at a rate of 4 x 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 Cm(r) 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 x 10(-9) to 1 x 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.
Hashem
Vera I
VI
Laboratory of DNA Structure and Mutagenesis, Center for Genome Research, Institute of Biosciences and Technology, Texas A&M University System Health Science Center, Houston, TX 77030-3303, USA.
Rosche
William A
WA
Sinden
Richard R
RR
D020137:Base Pair Mismatch; D001483:Base Sequence; D004260:DNA Repair; D004261:DNA Replication; D004926:Escherichia coli / Q000235:genetics*; D008969:Molecular Sequence Data; D010957:Plasmids; D011995:Recombination, Genetic; D017384:Sequence Deletion; D018911:Trinucleotide Repeats