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Screening in Yeast With a Bacterial/Yeast Two-Hybrid System 277Table 2Interpretation of Primary Isolates’ BehaviorObserved phenotypeAuxotrophic Colorimetricreporter reporter InterpretaionGlu Gal Glu Gal Conservative Optimistic Recommendation− + − + Very good sign Work with thoseclones first(+) + (+) + Bait is mutated or • GAL1 promoter Take a smallits expression is is slightly leaky number ofupregulated, • both bait and clones (six orcausing a high prey are very less) for confirbackgroundof stable mation oftranscriptional • interaction occurs interaction;activation with high affinity store the rest− + − − Yeast mutation Some bait-interactor If all otheroccurred that combinations are candidates fail,favors growth or known to check thesetranscriptional preferentially clones (or redoactivation on activate one the screen withgalactose reporter versus different bait,medium another library, andso on)All other phenotypes Contamination/ Something really Trashplasmidnewrearrangements/mutationsFrom http://www.fccc.edu/research/labs/golemis/interactiontrapinwork.html.and specific interaction with the bait used to select them, using a strategy asshown in Fig. 6. If a large number of positives are obtained, the subsequent characterizationsteps require prioritization. In this case, select up to about 24–48 independentcolonies (preferably, those growing the soonest after plating on selectivemedia) for the first round of assessment, while maintaining master plates ofadditional positives at 4°C. This first analysis set will be tested for specificity ofinteraction (i.e., for their ability to bind unrelated baits in addition to the originalone) and screened by polymerase chain reaction (PCR)/restriction digest analysisand/or sequencing to establish whether clusters of frequently isolated cDNAs areobtained: such clusters are generally a good indication for a specific interaction.