Cloning of a cDNA Encoding Cytosolic Acetoacetyl-Coenzyme A Thiolase from Radish by Functional Expression in Saccharomyces cerevisiae

Abstract

A cDNA coding for radish (Raphanus sativus L.) acetoacetyl-coenzyme A thiolase (AACT) was cloned by complementation of the erg10 mutation affecting AACT in yeast (Saccharomyces cerevisiae). The longest reading frame encodes a protein of 406 amino acids with a predicted relative molecular weight of 42,032, with significant similarities to eukaryotic and prokaryotic thiolases. There is no evidence for the presence of a leader peptide characteristic, e.g. of glyoxysomal thiolase. Yeast transformants expressing the radish AACT gene placed under the control of the GAL1 promoter exhibited a 10-fold higher enzyme activity than a wild-type yeast strain after induction by galactose. This enzyme activity is exclusively localized in the soluble fraction but not in membranes. These data indicate that we have cloned a gene encoding cytoplasmic (biosynthetic) AACT. Genomic DNA gel blot analysis suggests the presence of a single AACT gene, which is expressed in all parts of the seedling. Expression in cotyledons appears to be light-stimulated. We present preliminary evidence that a smaller transcript represents an antisense species being read from the same gene.