Isolation of a full-length complementary DNA coding for human E1 alpha subunit of the pyruvate dehydrogenase complex.
A 1.5-kilobase cDNA clone for human pyruvate dehydrogenase E1 was isolated from a lambda gt11 expression library by screening with polyclonal antiserum to the E1 alpha subunit of the porcine pyruvate dehydrogenase complex, a polyclonal antibody against bovine pyruvate dehydrogenase complex and a synthetic oligonucleotide based on the known amino acid sequence of the amino-terminal of the bovine pyruvate dehydrogenase-E1 alpha subunit. Nucleotide sequence analysis of the cDNA revealed a 5'-untranslated sequence of 72 nucleotides, a translated sequence of 1170 nucleotides, and a 3'-untranslated sequence of 223 nucleotides with a poly(A) tail. The cDNA structure predicts a leader sequence of 29 amino acids and a mature protein of 362 amino acids comprising an amino-terminal peptide identical to that of the bovine E1 alpha subunit and three serine phosphorylation sites whose sequence was also identical to those in the bovine E1 alpha subunit. The translated sequence for the mature protein differs substantially from that described by Dahl et al. (Dahl, H. H., Hunt, S. M., Hutchison, W. M., and Brown, G. K. (1987) J. Biol. Chem. 262, 7398-7403) by virtue of a frameslip between bases 390 and 594. This amended sequence is confirmed by the presence of additional restriction sites for the enzymes NaeI and HaeII at the beginning and end, respectively, of this section. The leader sequence is typical for mitochondrial enzymes being composed of a combination of neutral and basic residues. The amino acid composition is strikingly similar to that of the bovine protein. This cDNA clone hybridizes with a 1.8-kilobase mRNA on a Northern blot analysis of human fibroblasts, and a second minor band of 4.4 kilobases is also detected.
De Meirleir
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L
Department of Pediatrics and Biochemistry, University of Toronto, Ontario, Canada.
MacKay
N
N
Lam Hon Wah
A M
AM
Robinson
B H
BH
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D001483:Base Sequence; D006528:Carcinoma, Hepatocellular / Q000201:enzymology / Q000235:genetics; D004247:DNA / Q000302:isolation & purification*; D006801:Humans; D008113:Liver Neoplasms / Q000201:enzymology / Q000235:genetics; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D008970:Molecular Weight; D011768:Pyruvate Dehydrogenase Complex / Q000235:genetics*; D014407:Tumor Cells, Cultured / Q000201:enzymology
D000596:Amino Acids; D046911:Macromolecular Substances; D011768:Pyruvate Dehydrogenase Complex; D004247:DNA