28] Cloning of yeast genes coding for glycolytic enzymes

Abstract

Publisher Summary This chapter discusses the cloning of yeast genes coding for glycolytic enzymes. Large changes in the specific activity of yeast glycolytic enzymes occur with changes in the growth environment of the cells. The kinetics of yeast glycolytic enzyme synthesis after a shift from the growth on acetate to glucose as a carbon source suggests that the induction of these enzymes is coordinately regulated. One approach to understanding the coordinate regulation of glycolytic genes is to isolate and characterize the structural genes that encode glycolytic enzymes. Most of the glycolytic enzymes of yeast are present at high intracellular concentration as are the messenger ribonucleic acids (mRNAs) that encode them. mRNAs coding for enolase, phosphoglycerate kinase, and glyceraldehyde-3-phosphate dehydrogenase have been isolated by preparative polyacrylamide gel electrophoresis of total cellular poly(A)-containing mRNA. This chapter describes a rapid method for isolating Escherichia coli (E. coli) transformants containing hybrid plasmid deoxyribonucleic acid (DNA) composed of a bacterial plasmid vector and a segment of yeast genomic DNA containing a glycolytic enzyme structural gene. Radioactively labeled complementary DNA (cDNA) synthesized from partially purified mRNA is used as a hybridization probe for the selection of a transformant. The isolation of a yeast glyceraldehyde-3-phosphate dehydrogenase structural gene illustrates the cloning procedure.