title: Fast-acting and nearly gratuitous induction of gene expression and protein depletion in Saccharomyces cerevisiae.
abstract: We describe the development and characterization of a system that allows the rapid and specific induction of individual genes in the yeast Saccharomyces cerevisiae without changes in nutrients or temperature. The system is based on the chimeric transcriptional activator Gal4dbd.ER.VP16 (GEV). Upon addition of the hormone β-estradiol, cytoplasmic GEV localizes to the nucleus and binds to promoters containing Gal4p consensus binding sequences to activate transcription. With galactokinase Gal1p and transcriptional activator Gal4p absent, the system is fast-acting, resulting in readily detectable transcription within 5 min after addition of the inducer. β-Estradiol is nearly a gratuitous inducer, as indicated by genome-wide profiling that shows unintended induction (by GEV) of only a few dozen genes. Response to inducer is graded: intermediate concentrations of inducer result in production of intermediate levels of product protein in all cells. We present data illustrating several applications of this system, including a modification of the regulated degron method, which allows rapid and specific degradation of a specific protein upon addition of β-estradiol. These gene induction and protein degradation systems provide important tools for studying the dynamics and functional relationships of genes and their respective regulatory networks.
authors.affiliation: The Lewis-Sigler Institute for Integrative Genomics, Princeton University, Princeton, NJ 08544, USA. rmcisaac@princeton.edu
authors.forename: R Scott
authors.initials: RS
authors.lastname: McIsaac
authors.forename: Sanford J
authors.initials: SJ
authors.lastname: Silverman
authors.forename: Megan N
authors.initials: MN
authors.lastname: McClean
authors.forename: Patrick A
authors.initials: PA
authors.lastname: Gibney
authors.forename: Joanna
authors.initials: J
authors.lastname: Macinskas
authors.forename: Mark J
authors.initials: MJ
authors.lastname: Hickman
authors.forename: Allegra A
authors.initials: AA
authors.lastname: Petti
authors.forename: David
authors.initials: D
authors.lastname: Botstein
mesh_terms: D004958:Estradiol / Q000494:pharmacology; D005686:Galactokinase / Q000235:genetics / Q000378:metabolism; D020869:Gene Expression Profiling; D015966:Gene Expression Regulation, Fungal; D053263:Gene Regulatory Networks; D011401:Promoter Regions, Genetic; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000254:growth & development / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000378:metabolism*; D014157:Transcription Factors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D015533:Transcriptional Activation
chemical_list: D029701:Saccharomyces cerevisiae Proteins; D014157:Transcription Factors; D004958:Estradiol; D005686:Galactokinase