Bait binds prey

Transcription activated

Transcription activated

Two-hybrid analysis was developed in yeast and is being used to generate a complete list of interactions between all 6000 or so yeast proteins. It is thus necessary to examine 6,000 x 6,000 combinations. To examine these potential interactions, each open reading frame in the yeast genome was amplified by PCR and cloned into two separate vectors, one carrying the DBD domain and one with the AD domain. Thus each yeast protein is tested as both bait and prey. The vectors are designed to give inframe gene fusions of each ORF with the DBD domain and AD domains of a suitable transcriptional activator, such as GAL4 (Fig. 26.18). One vector has a multiple cloning site downstream of the GAL4-DBD and thus gives a 3'-fusion of GAL4-DBD to protein X (GAL4-DBD-X). The other vector has its MCS upstream of the GAL4-AD and gives a 5'-fusion of GAL4-AD and protein Y (Y-GAL4-AD).

The bait and prey fusion plasmids are transformed into yeast cells of different mating types. This results in two sets of approximately 6000 transformants. All possible matings are carried out between the two sets using a laboratory robot to manipulate the colonies. When the two yeast mate, the diploid cell will have a bait plasmid

Bacterial origin

Yeast Bait R

°rigin^ vector S

Promoter oí

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