Tightly regulated, beta-estradiol dose-dependent expression system for yeast.
We have refined the regulated expression of UASGAL1, 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 was monitored by the expression of an integrated UASGAL10-lacZ reporter and by immunoblot analysis of a UASGAL1-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 induction can be achieved; and (iii) induction or depletion studies can be conducted independent of carbon source in gal4 delta strains. In addition, any UASGAL1,10 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 useful to researchers investigating the functions of essential genes, dominant negative mutants, mitochondrial genes, and viral, plant, and mammalian genes in yeast assay systems.
Gao
C Y
CY
University of Massachusetts, Amherst, MA, USA.
Pinkham
J L
JL
D002244:Carbon / Q000502:physiology; D004268:DNA-Binding Proteins; D004305:Dose-Response Relationship, Drug; D004958:Estradiol / Q000494:pharmacology*; D005656:Fungal Proteins / Q000037:antagonists & inhibitors / Q000096:biosynthesis / Q000235:genetics*; D015966:Gene Expression Regulation, Fungal / Q000187:drug effects*; D018390:Gene Targeting; D005822:Genetic Vectors / Q000138:chemical synthesis / Q000378:metabolism; D006801:Humans; D011887:Raffinose / Q000502:physiology; D011959:Receptors, Estradiol / Q000037:antagonists & inhibitors / Q000096:biosynthesis / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000138:chemical synthesis / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000187:drug effects / Q000235:genetics* / Q000254:growth & development / Q000821:virology; D029701:Saccharomyces cerevisiae Proteins; D015534:Trans-Activators / Q000037:antagonists & inhibitors / Q000096:biosynthesis / Q000235:genetics; D014157:Transcription Factors / Q000037:antagonists & inhibitors / Q000096:biosynthesis / Q000235:genetics*
D004268:DNA-Binding Proteins; D005656:Fungal Proteins; C052130:GAL4 protein, S cerevisiae; C070633:Gal-VP16; D011959:Receptors, Estradiol; D011993:Recombinant Fusion Proteins; D029701:Saccharomyces cerevisiae Proteins; D015534:Trans-Activators; D014157:Transcription Factors; D004958:Estradiol; D002244:Carbon; D011887:Raffinose