MOLECULAR-CLONING OF THE GENE FOR THE E1-ALPHA SUBUNIT OF THE PYRUVATE-DEHYDROGENASE COMPLEX FROM SACCHAROMYCES-CEREVISIAE

The E1α and E1β subunits of the pyruvate dehydrogenase complex from the yeast Saccharomyces cerevisiae were purified. Antibodies raised against these subunits were used to clone the corresponding genes from a genomic yeast DNA library in the expression vector λgt11. The gene encoding the E1α subunit was unique and localized on a 1.7‐kb HindIII fragment from chromosome V. The identity of the gene was confirmed in two ways. (a) Expression of the gene in Escherichia coli produced a protein that reacted with the anti‐E1α serum. (b) Gene replacement at the 1.7‐kb HindIII fragment abolished both pyruvate dehydrogenase activity and the production of proteins reacting with anti‐E1α serum in haploid cells. In addition, the 1.7‐kb HindIII fragment hybridized to a set of oligonucleotides derived from amino acid sequences from the N‐terminal and central regions of the human E1α peptide. We propose to call the gene encoding the E1α subunit of the yeast pyruvate dehydrogenase complex PDA1. Screening of the λgt11 library using the anti‐E1β serum resulted in the reisolation of the RAP1 gene, which was located on chromosome XIV. Copyright © 1990, Wiley Blackwell. All rights reserved