REGULATION OF YEAST METHIONINE SULFOXIDE REDUCTASE

Heart, Lung, And Blood Institute

Investigators

Abstract

In mammals, methionine sulfoxide reductase (msrA) activity and msrA gene expression are localized in tissues where high levels of oxidative stress may be expected, such as kidney, retina, neutrophils, macrophages, and brain neurons. The mechanism regulating msrA gene expression during increased oxidative stress is presently unknown. To understand whether the transcriptional regulation of msrA requires specific nuclear proteins, we studied yeast wild-type and msrA null mutants before and after exposure to oxidative stress conditions. Nuclear proteins were isolated from both yeast strains and subjected to partial purification by ammonium sulfate precipitation, DEAE-cellulose, and heparin-affinity chromatography. The various fractions were monitored for DNA binding activity by electrophoretic mobility shift assay using 32P-labeled promoter DNA. The electrophoretic mobility shift assays revealed an increased DNA binding activity in the nuclear extract of the msrA null mutant. Future studies will follow various strategies for identification and cloning of DNA binding proteins and for confirming their functional relevance.