Tightly regulated, β-estradiol dose-dependent expression system for yeast

Abstract

We have refined the regulated expression of UAS(GAL1), 10(-driven) genes in yeast by modifying a vector encoding the β-estradiol inducible activator, GAL4.ER.VP16 (GEV). The expression of GEV was placed under the regulation of the low-level, constitutive MRP7 promoter, and β-estradiol-regulated expression was monitored by the expression of an integrated UAS(GAL10)-lacZ reporter and by immunoblot analysis of a UAS(GAL1)-regulated gene product. Target gene expression regulated by low levels of GEV has several advantages over the standard galacrose-inducible expression systems. (i) Most importantly, the target gene expression is undetectable in the absence of hormone; (ii) target gene expression is β-estradiol dose-dependent, and variable levels of target gene expression from low to several hundred-fold induction can be achieved; and (iii) induction or depletion studies can be conducted independent of carbon source in gal4Δ strains. In addition, any UAS(GAL1,10) expression construct can be used without modification of the target gene or many gal4Δ host strains, and GEV vectors are compatible with other inducible yeast expression systems. This method may be useful to researchers investigating the functions of essential genes, dominant negative mutants, mitochondrial genes, and viral, plant, and mammalian genes in yeast assay systems.