Nucleotide and deduced amino acid sequence of the alpha subunit of yeast pyruvate dehydrogenase.
A 413-base cDNA insert encoding a portion of the alpha subunit of pyruvate dehydrogenase (E1 alpha; EC 1.2.4.1) from Saccharomyces cerevisiae was isolated from a lambda gt11 cDNA library by immunoscreening and by hybridization with an oligonucleotide probe which corresponded to the amino acid sequence around the phosphorylation site of E1 alpha. This cDNA was subcloned, sequenced and used as a probe to isolate two additional cDNA inserts which were subcloned and sequenced. These overlapping clones comprised the carboxyl-terminal part of E1 alpha. To identify the missing nucleotide sequence, the polymerase chain reaction was used to amplify yeast genomic DNA with synthetic oligonucleotide primers based on the amino-terminal sequence of E1 alpha and the 5' end of one of the cDNA clones. Three DNA fragments were isolated and sequenced. The composite nucleotide sequence has an open reading frame of 1260 nucleotides encoding a putative presequence of 33 amino acids and a mature protein of 387 amino acids (Mr = 42,703). Hybridization analysis showed that the size of the mRNA is about 1.4 kilobases.
Behal
R H
RH
Clayton Foundation Biochemical Institute, University of Texas, Austin 78712.
Browning
K S
KS
Reed
L J
LJ
D000595:Amino Acid Sequence; D001483:Base Sequence; D004271:DNA, Fungal / Q000235:genetics*; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D015345:Oligonucleotide Probes; D016133:Polymerase Chain Reaction; D011768:Pyruvate Dehydrogenase Complex / Q000235:genetics*; D015183:Restriction Mapping; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics
D004271:DNA, Fungal; D046911:Macromolecular Substances; D015345:Oligonucleotide Probes; D011768:Pyruvate Dehydrogenase Complex