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

We have refined the regulated expression of UAS(GAL1), (10)-driven genes in yeast by modifying a vector encoding the beta -estradiol inducible activator GAL4.ER.VP16 (GEV). The expression of GEV was placed under the regulation of the low-level, constitutive MRP7 promoter and beta -estradiol-regulated expression Iras 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 galactose-inducible expression systems. (i) Most importantly the target gene expression is undetectable in the absence of hormone; (ii) target gene expression is beta -estradiol dose-dependent, and variable levels of target gene expression from low to several hundred-fold indiction can be achieved; and (iii) induction or depletion studies can be conducted independent of carbon source in gal4 Delta strains. In addition, any UAS(GAL,10) expression construct can be used without modification of the target gene or many gal4 Delta host strains, and GEV vectors are compatible with other inducible yeast expression systems. This method may be Icseful to researchers investigating the funcitons of essential genes, dominant negative mutants, mitochondrial genes, and viral, plant, and mammalian genes in yeast assay systems.