The reverse two-hybrid system: a genetic scheme for selection against specific protein/protein interactions.

AUTOR(ES)

Leanna, C A

RESUMO

The yeast two-hybrid system is a powerful experimental approach for the 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-hybrid system which enables genetic selection against a specific protein/protein interaction. This reverse two-hybrid system utilizes a yeast strain which is resistant to cycloheximide due to the presence of a mutant cyh2 gene. This strain also contains the wild-type CYH2 allele under the transcriptional control of the Gal1 promoter. Expression of the wild-type Gal4 protein is sufficient to restore growth sensitivity to cycloheximide. Growth sensitivity towards cycloheximide is also restored by the coexpression of the avian c-Rel protein and its I kappa B alpha counterpart, p40, as Gal4 fusion proteins. Restoration of growth sensitivity towards cycloheximide requires the association of c-Rel and p40 at the Gal1 promoter and correlates with the ability of the c-Rel/p40 interaction to activate expression from the Gal1 promoter. A genetic selection scheme against specific protein/protein interactions may be a valuable tool for the analysis of protein/protein interactions.

Documentos Relacionados

• A bacterial two-hybrid selection system for studying protein–DNA and protein–protein interactions
• A new version of the two-hybrid assay for detection of protein-protein interactions.
• Reverse two-hybrid and one-hybrid systems to detect dissociation of protein-protein and DNA-protein interactions.
• The yeast two-hybrid system: forward and reverse.
• Genetic characterization of a mammalian protein-protein interaction domain by using a yeast reverse two-hybrid system.