Detecting interactions in pools of AD strains. (A) Strains expressing LexA (BD) fused to one of three Drosophila proteins (Cdk2, Cdp1, Cdk1) were mated with individual strains expressing the AD fused to Drosophila proteins Cdi2, Cdi3, Cdi5, Cdi12, or CycEI, or no fusion (vector). Diploids were replicated to three kinds of indicator plates: X-Gal, —leu, and —leu X-Gal. Growth on the —leu plate or blue color on the X-Gal plate indicates activation of the LEU2 or lacZ reporters, respectively. The interactions of Cdk2 with Cdi2, Cdi3, Cdi5, and Cdi12 result in strong to weak reporter activation. Cdp1 activates the reporter on its own. The interaction of Cdp1 with Cdi3 is evident from the increase in reporter expression in the presence of AD-Cdi3(cf. Cdp1-vector with Cdp1-Cdi3). CycEI is toxic to yeast and leads to poor growth on all media. The strong interaction between Cdk1 and CycEI can be seen from the high level of lacZ expression, even though the yeast do not grow well. (B) The interactions shown in A in which the strain expressing the interacting AD has been diluted 1/96 by strains expressing noninteractors. Diploids were replicated onto the same three kinds of indicator plates as in A. The matings were performed in duplicate rows. The diluted interactions could not be detected on the X-Gal plates, but were detected on the —leu and —leu X-Gal plates. All plates in A and B were also —his —ura —trp Gal/Raf to select for diploids and to induce expression of the AD protein. Use of —leu X-Gal plates for detecting interactions in pools distinguishes true positives, which are blue, from the rare false positive leu+ colonies, which are not blue.
