Molecular cloning of the gene for the E1α subunit of the pyruvate dehydrogenase complex from Saccharomyces cerevisiae

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Abstract
The E1.alpha. and E1.beta. 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 .lambda.gt11. The gene encoding the E1.alpha. subunit was unique and localized on a 1.7-kb HindIII fragment from chromosome V. The identity of the gene was confirmed in two years. (a) Expression of the gene in Escherichia coli produced a protein that reacted with the anti-E1.alpha. serum. (b) Gene replacement at the 1.7-kb HindIII fragment abolished both pyruvate dehydrogenase activity and the production of proteins reacting with anti-E1.alpha. 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 E1.alpha. peptide. We propose to call the gene encoding the E1.alpha. subunit of the yeast pyruvate dehydrogenase complex PDA1. Screening of the .lambda.gt11 library using the anti-E1.beta. serum resulted in the reisolation of the RAP1 gene, which was located on chromosome XIV.