Figure 3

Y2H screen for homodimers. (A) Yeast cells harboring bait and prey plasmids were resuspended in sterile water and dropped onto protein interaction selective yeast medium. The positive control shows the interaction of the p53 and SV40-large T-antigen (Y2H internal controls) and 1 of the 22 found homodimers in this study. A negative interaction obtained in the screening is also shown. To test for the presence of both plasmids, the yeast cells were also plated on plasmid-selective medium lacking tryptophan and leucine. Y2H positive colonies can grow on synthetic complete yeast media lacking histidine, adenine, tryptophan and leucine. (B) Flowchart showing the results of retesting candidate homodimers under stringent selection conditions. Fifty-nine candidate homodimers were retransformed into AH109 as follows: i) AD-ORFs + DB empty vector; ii) DB-ORFs + empty AD vector; and iii) DB-ORFs + AD-ORFs. The transformations were plated on nutritionally selective medium deficient for tryptophan, leucine, histidine, and adenine, plus 10 mM 3-AT. All 22 homodimers were confirmed as "clean homodimers." The numbers of each type of autoactivator are indicated.