INSTABILITY OF CAG AND CTG TRINUCLEOTIDE REPEATS IN SACCHAROMYCES-CEREVISIAE

A quantitative genetic assay was developed to monitor alterations in t ract lengths of trinucleotide repeat sequences in Saccharomyces cerevi siae. Insertion of (GAG)(50) or (CTG)(50) repeats into a promoter that drives expression of the reporter gene ADE8 results in loss of expres sion and white colony color. Contractions within the trinucleotide seq uences to repeat lengths of 8 to 38 restore functional expression of t he reporter, leading to red colony color. Reporter constructs includin g (GAG)(50) or (CTG)(50) repeat sequences were integrated into the yea st genome, and the rate of red colony formation was measured. Both ori entations yielded high rates of instability (4 x 10(-4) to 18 x 10(-4) per cell generation). Instability depended on repeat sequences, as a control harboring a randomized (C,A,G)(50) sequence was at least 100-f old more stable. PCR analysis of the trinucleotide repeat region indic ated an excellent correlation between change in color phenotype and re duction in length of the repeat tracts. No preferential product sizes were observed. Strains containing disruptions of the mismatch repair g ene MSH2, MSH3, or PMS1 or the recombination gene RAD52 showed little or no difference in rates of instability or distributions of products, suggesting that neither mismatch repair nor recombination plays an im portant role in Large contractions of trinucleotide repeats in yeast.