Department of Biochemistry and Molecular Biology
DNA Repair; DNA, Fungal; DNA-Binding Proteins; Fungal Proteins; Genes, Reporter; Genetic Techniques; Mutagenesis; Nucleic Acid Heteroduplexes; Phenotype; Polymerase Chain Reaction; Promoter Regions (Genetics); Rad52 DNA Repair and Recombination Protein; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins; Sequence Analysis, DNA; Sequence Deletion; Trinucleotide Repeats
Life Sciences | Medicine and Health Sciences
A quantitative genetic assay was developed to monitor alterations in tract lengths of trinucleotide repeat sequences in Saccharomyces cerevisiae. Insertion of (CAG)50 or (CTG)50 repeats into a promoter that drives expression of the reporter gene ADE8 results in loss of expression and white colony color. Contractions within the trinucleotide sequences to repeat lengths of 8 to 38 restore functional expression of the reporter, leading to red colony color. Reporter constructs including (CAG)50 or (CTG)50 repeat sequences were integrated into the yeast genome, and the rate of red colony formation was measured. Both orientations 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-fold more stable. PCR analysis of the trinucleotide repeat region indicated an excellent correlation between change in color phenotype and reduction in length of the repeat tracts. No preferential product sizes were observed. Strains containing disruptions of the mismatch repair gene 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 important role in large contractions of trinucleotide repeats in yeast.
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Citation: Mol Cell Biol. 1997 Jun;17(6):3382-7.
Molecular and cellular biology
Miret, Juan J.; Pessoa-Brandao, Luis; and Lahue, Robert S., "Instability of CAG and CTG trinucleotide repeats in Saccharomyces cerevisiae" (1997). Open Access Articles. 1450.