THE REVERSE 2-HYBRID SYSTEM - A GENETIC SCHEME FOR SELECTION AGAINST SPECIFIC PROTEIN PROTEIN INTERACTIONS/

The yeast two-hybrid system is a powerful experimental approach for th e characterization of protein/ protein interactions. A unique strength of the yeast two-hybrid system is the provision for genetic selection techniques that enable the identification of specific protein/protein interactions. We now report the development of a modified yeast two-h ybrid system which enables genetic selection against a specific protei n/protein interaction. This reverse two-hybrid system utilizes a yeast strain which is resistant to cycloheximide due to the presence of a m utant cyh2 gene. This strain also contains the wild-type CYH2 allele u nder the transcriptional control of the Gall promoter. Expression of t he wild-type Gal4 protein is sufficient to restore growth sensitivity to cycloheximide, Growth sensitivity towards cycloheximide is also res tored by the coexpression of the avian c-Rel protein and its I kappa B alpha counterpart, p40, as Gal4 fusion proteins. Restoration of growt h sensitivity towards cycloheximide requires the association of c-Rel and p40 at the Gall promoter and correlates with the ability of the c- Rel/p40 interaction to activate expression from the Gall promoter. A g enetic selection scheme against specific protein/protein interactions may be a valuable tool for the analysis of protein/protein interaction s.