Open AccessMolecular cloning of the gene for the E1α subunit of the pyruvate dehydrogenase complex from Saccharomyces cerevisiae
Author(s) -
STEENSMA H. Yde,
HOLTERMAN Lennart,
DEKKER Iris,
SLUIS Cees A.,
WENZEL Thibaut J.
Publication year - 1990
Publication title -
european journal of biochemistry
Language(s) - English
Resource type - Journals
eISSN - 1432-1033
pISSN - 0014-2956
DOI - 10.1111/j.1432-1033.1990.tb19186.x
Subject(s) - microbiology and biotechnology , biology , gene , pyruvate dehydrogenase complex , genomic library , saccharomyces cerevisiae , biochemistry , peptide sequence , enzyme
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 Hin dIII 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 Hin dIII 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 Hin dIII 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.