We have developed a yeast-based genetic system that is capable of detect- ing the interaction of two proteins. This system uses reconstitution of the activity of a transcriptional activator, the yeast GAL4 protein, as its assay. Two GAL4 hybrid proteins are constructed: one contains the GAL4 DNA-binding domain fused to a protein X and the other contains the GAL4 activation domain fused to a protein Y. Interaction between proteins X and Y leads to the transcriptional activation of a reporter gene under the regulation of GAL4. Our goal is to develop this system such that a viral oncoprotein forms part of one hybrid and total human cDNA is used to construct the other hybrid. This new methodology should result in the identification of additional cellular proteins that bind to these oncoproteins. The advantages of this system include the ability to screen gene libraries to identify proteins present in the cell at very low levels and the immediate availability of the gene encoding an interacting protein. In addition, the powerful genetics available in yeast may make it feasible to detect rare mutations in the interacting proteins that affect the interaction. Our specific goals are: 1) To reconstruct test oncoprotein/cellular protein interactions in this system and to optimize the assay in yeast; 2) To construct appropriate plasmid vectors, yeast strains and cDNA libraries; 3) To use these libraries to identify novel interacting cellular proteins; 4) To obtain the complete cDNAs and DNA sequences encoding these novel proteins, and to prepare specific antibodies to detect the proteins; 5) To assay these proteins for possible functions; 6) To identify possible homologs of these human proteins in yeast; 7) To identify the specific interacting domains, and to obtain and assay conditional mutations that eliminate the interaction.
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