Yeast-based assay for the measurement of positive and negative influences on microsatellite stability.
The evolutionary conservation of mismatch repair and Saccharomyces cerevisiae as a model system have been exploited for monitoring the influence of everyday beverages and the antineoplastic agent, hydroxyurea, on the stability of regions of highly repetitive DNA known as microsatellites. Two different reporter systems are compared for sensitivity and reproducibility by measuring the extent of frame slippage events occurring in microsatellite regions in wild-type and mismatch repair-compromised yeast strains. Increased frame slippage results in increased reporter gene expression and hence represents instability within the repetitive region, whereas a decrease or no significant change indicates the faithful replication of the original assay plasmid, suggesting a beneficial or neutral effect of the test component. A significant outcome of this study was the identification of the protective influence exerted by the green tea catechin (-)-epigallocatechin-3-gallate (EGCG) against microsatellite instability, which is in agreement with the hypothesis that EGCG is the major chemopreventive ingredient of green tea. Immunological detection can also be used in conjunction with the green fluorescent protein (GFP) version of the assay system to identify compounds, such as hydroxyurea, which increased microsatellite instability. The system has the potential for development as a high-throughput assay for wider application.
Marden
Aileen
A
School of Life Sciences, Heriot-Watt University, Edinburgh, UK.
Walmsley
Richard M
RM
Schweizer
Lilian M
LM
Schweizer
Michael
M
D015153:Blotting, Western; D002392:Catechin / Q000031:analogs & derivatives / Q000494:pharmacology; D004261:DNA Replication / Q000187:drug effects; D042822:Genomic Instability / Q000187:drug effects*; D006918:Hydroxyurea / Q000494:pharmacology; D018895:Microsatellite Repeats / Q000187:drug effects*; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000254:growth & development; D013662:Tea
D013662:Tea; D002392:Catechin; C045651:epigallocatechin gallate; D006918:Hydroxyurea