THE AIM OF THIS PROJECT IS TO DEVELOP CLONING VECTORS THAT DIRECT THE HIGH-LEVEL SYNTHESIS AND SECRETION OF HETEROLOGOUS PROTEIN PRODUCTS IN THE YEAST SACCHAROMYCES CEREVISIAE.

THE AIM OF THIS PROJECT IS TO DEVELOP CLONING VECTORS THAT DIRECT THE HIGH-LEVEL SYNTHESIS AND SECRETION OF HETEROLOGOUS PROTEIN PRODUCTS IN THE YEAST SACCHAROMYCES CEREVISIAE. A CRITICAL COMPONENT NEEDED FOR SUCH VECTORS IS A DNA SEQUENCE CODING FOR A SECRETION SIGNAL RECOGNIZED BY YEAST. THE GENE FOR THE SECRETED K2 KILLER TOXIN OF S. CEREVISIAE WILL BE ANALYZED TO DETERMINE IF PORTIONS OF THIS GENE CAN BE USED AS THE SECRETION SIGNAL IN THE VECTORS. CDNA CLONES DERIVED FROM THE DOUBLE-STRANDED RNA RESPONSIBLE FOR K2 TOXIN PRODUCTION WILL BE ANALYZED BY SEQUENCING AND BY EXPRESSION IN YEAST CELLS TO IDENTIFY CLONES THAT CODE FOR THE TOXIN PROTEIN. TO DETERMINE WHETHER THE TOXIN SEQUENCES WILL DIRECT THE SECRETION OF HETEROLOGOUS PRODUCTS, VARIOUS FRAGMENTS OF THE TOXIN CDNAS WILL THEN BE FUSED WITH MAMMALIAN GENES, PLACED INTO YEAST EXPRESSION VECTORS, AND TRANSFORMED INTO YEAST CELLS TO ASSAY FOR EXPRESSION AND SECRETION OF THE FOREIGN PROTEINS. IF SECRETION IS DETECTED, THEN A SECOND PHASE OF THE PROJECT WILL BEGIN. THIS PHASE WILL PRIMARILY INVOLVE A MORE DETAILED ANALYSIS OF THE TOXIN GENE, IN ORDER TO OPTIMIZE THE TOXIN ELEMENTS INCORPORATED INTO THE VECTORS.