Nucleotide and deduced amino acid sequence of the alpha subunit of yeast pyruvate dehydrogenase

Abstract

A 413-base cDNA insert encoding a portion of the alpha subunit of pyruvate dehydrogenase (E 1α; EC 1.2.4.1) from Saccharomyces cerevisiae was isolated from a λgtll cDNA library by immunoscreening and by hybridization with an oligonucleotide probe which corresponded to the amino acid sequence around the phosphorylation site of E 1α. 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 E 1α. 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 E 1α 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 ( M r = 42,703). Hybridization analysis showed that the size of the mRNA is about I.4 kilobases.