Molecular Cloning and Nucleotide Sequence of cDNA Encoding the Entire Precursor of Rat Mitochondrial Acetoacetyl-CoA Thiolase1

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Abstract
CDNA clones for rat mitochondria] acetoacetyl-CoA thiolase were isolated and sequenced. The most 5'-extended clone (RT2-6) consisted of 1, 460 bases and contained a 1, 272-base open reading frame encoding a polypeptide of 424 amino acid residues. A coupled in vitro transcription/translation analysis of RT2-6 revealed that RT2-6 encodes the entire precursor of this enzyme. The amino-tenninal sequence and amino acid composition of the purified enzyme agreed with the primary structure deduced from the cDNA. The calculated molecular masses of the precursor and the subunit of the mature enzyme are 44, 694 and 41, 364 Da, respectively. The primary structure of this enzyme was compared with those of four other thiolases (rat mitochondrial and peroxisomal 3-ketoacyl-CoA thiolases, aceto-acetyl-CoA thiolase of Zoogloea ramigera, and cytosolic acetoacetyl-CoA thiolase of Saccharomyces uvarum). Marked homology between any two of them (34–51% identity) indicates that the genes of thiolases have evolved from a common ancestral gene. It has been reported that this enzyme has two isoenzymes A and B. However, the purified isoenzymes were indistinguishable from each other in some analyses. Though 17 independent cDNA clones were isolated, no definite evidence indicating the presence of different cDNAs was found.