A genetic strategy to eliminate self-activator baits prior to high-throughput yeast two-hybrid screens

Large-scale sequencing projects have predicted high numbers of gene product s for which no functional information is yet available. Hence, large-scale projects, such as gene knockouts, gene expression profiles, and protein-int eraction mapping, are currently under way to initiate the understanding of the Function of these gene products. The high-throughput strategies that ar e currently being developed to generate protein-interaction maps include au tomated versions of the yeast two-hybrid system. These strategies rely oil the large-scale construction of DNA-binding domain/protein-of-interest hybr id constructs [DB-X baits]. An inherent problem of large-scale two-hybrid s ystems is that a high percentage of cloned sequences encode polypeptides th at, when fused to DB, can activate transcription in the absence of any two- hybrid-interacting partner protein. Here, we describe and validate a geneti c strategy that efficiently eliminates such self-activator baits prior to s creening procedures. The strategy is based on a negative-growth selection a nd is compatible with high-throughput settings.