Screening for Protein-Protein Interactions in the Yeast Two-Hybrid System in Embryonic Stem Cells

The two-hybrid system (THS) (1 ) is a molecular genetic screen that detects protein-protein interactions. The protein specified by the yeast GAL4 gene activates the transcription of genes involved in galactose metabolism. It has two functional domains, a DNA binding domain, Gal4BD , and a transcriptional activating domain, Gal4AD , which interact with DNA sequences in the promoter regions of GAL1, GAL2 , and GAL7 to stimulate transcription. The screen involves two plasmids; one carries the GAL4 BD sequence fused, in-frame, to a sequence coding for a “bait” protein, and the other carries GAL4 AD sequences, fused to “prey” sequences from a cDNA library. The two plasmids are introduced, typically by transformation, into a yeast strain carrying a reporter gene coupled to a GAL1, GAL2 , or GAL7 promoter. If the proteins encoded by the bait and prey sequences interact to allow correct positioning of the Gal4AD and GaL4BD moieties, the reporter gene is activated. Transformants are plated on medium that allows the detection of reporter gene activation. The plasmid carrying the GAL4 AD :cDNA plasmid can be recovered, and the positive cDNA isolated and characterized.