Figure 3.

Flow chart for the reverse RRS. Cdc25-2 cells are cotransfected with the membrane bait and a cDNA library fused to Ras. The expression of the bait and Ras prey proteins are designed under the control of the Met and Gal1 promoters, respectively. Transformants are selected on glucose minimal plates lacking leucine and uracil at 24°C. After 5–7 days, plates are replica plated onto galactose-containing plates either containing or lacking methionine and incubated at 36°C. Transformants that exhibit efficient growth on the plates lacking methionine and no growth on the plate containing methionine are selected on a glucose plate and grown at 24°C. The selected transformants are retested for their methionine-dependent growth. Those transformants that pass this secondary methionine-dependent test are considered candidates and are further pursued. DNA is extracted from candidates and the library plasmid is identified by a restriction digest. The DNA is used to retransform Cdc25-2 cells with either the specific bait or non-specific bait.