Molecular cloning of the gene for the E1 alpha subunit of the pyruvate dehydrogenase complex from Saccharomyces cerevisiae.
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 identify of the gene was confirmed in two ways. (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.
Department of Microbiology and Enzymology, Delft University of Technology, The Netherlands.
H Y
HY
Steensma
L
L
Holterman
I
I
Dekker
C A
CA
van Sluis
T J
TJ
Wenzel
D000595:Amino Acid Sequence; D003001:Cloning, Molecular; D004271:DNA, Fungal / Q000235:genetics / Q000302:isolation & purification; D015247:Deoxyribonuclease HindIII; D005800:Genes, Fungal; D006801:Humans; D015151:Immunoblotting; D008969:Molecular Sequence Data; D009693:Nucleic Acid Hybridization; D011768:Pyruvate Dehydrogenase Complex / Q000235:genetics* / Q000302:isolation & purification; D015183:Restriction Mapping; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics; D012689:Sequence Homology, Nucleic Acid; D013172:Spores, Fungal / Q000032:analysis
D004271:DNA, Fungal; D011768:Pyruvate Dehydrogenase Complex; D015247:Deoxyribonuclease HindIII