Comprehensive genetic analysis of transcription factor pathways using a dual reporter gene system in budding yeast.
The development and application of genomic reagents and techniques has fuelled progress in our understanding of regulatory networks that control gene expression in eukaryotic cells. However, a full description of the network of regulator-gene interactions that determine global gene expression programs remains elusive and will require systematic genetic as well as biochemical assays. Here, we describe a functional genomics approach that combines reporter technology, genome-wide array-based reagents and high-throughput imaging to discover new regulators controlling gene expression patterns in Saccharomyces cerevisiae. Our strategy utilizes the synthetic genetic array (SGA) method to systematically introduce promoter-GFP (green fluorescent protein) reporter constructs along with a control promoter-RFP (red fluorescent protein) gene into the array of approximately 4500 viable yeast deletion mutants. Fluorescence intensities from each reporter are assayed from individual colonies arrayed on solid agar plates using a scanning fluorimager and the ratio of GFP to RFP intensity reveals deletion mutants that cause differential GFP expression. We are exploiting this screening approach to construct a detailed map describing the interplay of regulators controlling the eukaryotic cell cycle. The method is extensible to any transcription factor or signalling pathway for which an appropriate reporter gene can be devised.
Kainth
Pinay
P
Banting & Best Department of Medical Research, University of Toronto, 160 College Street, Toronto, Ont. M5S3E1E1, Canada.
Sassi
Holly Elizabeth
HE
Peña-Castillo
Lourdes
L
Chua
Gordon
G
Hughes
Timothy R
TR
Andrews
Brenda
B
D053263:Gene Regulatory Networks / Q000235:genetics*; D017930:Genes, Reporter / Q000235:genetics*; D016681:Genome, Fungal; D049452:Green Fluorescent Proteins / Q000378:metabolism; D008164:Luminescent Proteins; D011401:Promoter Regions, Genetic / Q000235:genetics*; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000378:metabolism; D014157:Transcription Factors / Q000235:genetics* / Q000378:metabolism; D000097573:Red Fluorescent Protein
D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins; D014157:Transcription Factors; D000097573:Red Fluorescent Protein