DETECTING PROTEIN INTERACTIONS BY A YEAST GENETIC SYSTEM

SPECIFIC PROTEIN- PROTEIN INTERACTIONS ARE CENTRAL TO MANY CELLULAR FUNCTIONS. DETECTION OF THESE INTERACTIONS HAS GENERALLY BEEN BY CO-IMMUNOPRECIPITATION ANALYSES. THESE METHODS HAVE DRAWBACKS DERIVING FROM THE NATURE OF THIS TECHNIQUE, INCLUDING THE DIFFICULTY OF CLONING GENE SEQUENCES FOR NEWLY DETECTED PROTEINS. A YEAST-BASED GENETIC SYSTEM TO STUDY PROTEIN INTERACTIONS HAS BEEN DESCRIBED, WHICH IS APPLICABLE TO PROTEINS FROM ANY ORGANISM. THIS SYSTEM DETECTS THE IN VIVO INTERACTION OF TWO HYBRID PROTEINS USING YEAST PLASMIDS THAT ENCODE CHIMERIC GENES. THIS PROTEIN INTERACTION RECONSTITUTES A YEAST TRANSCRIPTIONAL ACTIVATOR (GAL4) FUNCTION RESULTING IN ACTIVATION OF A REPORTER GENE VISUALIZED AS COLOR OUTPUT. THE PHASE I AIMS ARE TO EVALUATE THE SYSTEM'S SENSITIVITY TO DETECT PROTEIN INTERACTION OF DIFFERENT AFFINITIES USING THE P53-SV40 TANTIGEN INTERACTION. PREVIOUSLY CHARACTERIZED MUTATIONS WILL BE INTRODUCED INTO THE SV40 T ANTIGEN, ANALYZED WITH THIS SYSTEM AND COMPARED TO PUBLISHED CO- IMMUNOPRECIPITATION RESULTS. THE PHASE II OBJECTIVE IS TO EXTEND THIS METHOD TO WIDESPREAD USE BY COMMERCIALIZ- ING KITS FOR IDENTIFICATION OF NOVEL INTERACTIONS BETWEEN ANY PROTEIN UNDER STUDY AND THOSE ENCODED BY A CDNA/RANDOM GENOMIC LIBRARY. THE DISCOVERY OF SUCH INTERACTIONS IS CRITICAL TO THE UNDERSTANDING OF DISEASES RESULTING FROM ABNORMALITIES IN EITHER SIGNAL TRANSDUCTION OR CELLULAR ORGANIZATION.