The two-hybrid system (THS) (1 ) is a molecular genetic screen that detects protein-protein interactions. The protein specified by the yeastGAL4gene 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 ofGAL1, GAL2, andGAL7to stimulate transcription. The screen involves two plasmids; one carries theGAL4BDsequence fused, in-frame, to a sequence coding for a “bait” protein, and the other carriesGAL4ADsequences, 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 aGAL1, GAL2, orGAL7promoter. 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 theGAL4AD:cDNAplasmid can be recovered, and the positive cDNA isolated and characterized.
