CLONING OF A CDNA-ENCODING CYTOSOLIC ACETOACETYL-COENZYME-A THIOLASE FROM RADISH BY FUNCTIONAL EXPRESSION IN SACCHAROMYCES-CEREVISIAE

A cDNA coding for radish (Raphanus sativus L.) acetoacetyl-coenzyme A thiolase (AACT) was cloned by complementation of the erg10 mutation af fecting AACT in yeast (Saccharomyces cerevisiae). The longest reading frame encodes a protein of 406 amino acids with a predicted relative m olecular 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 tr ansformants expressing the radish AACT gene placed under the control o f the GAL1 promoter exhibited a 10-fold higher enzyme activity than a wild-type yeast strain after induction by galactose. This enzyme activ ity is exclusively localized in the soluble fraction but not in membra nes. These data indicate that we have cloned a gene encoding cytoplasm ic (biosynthetic) AACT. Genomic DNA gel blot analysis suggests the pre sence of a single AACT gene, which is expressed in all parts of the se edling. Expression in cotyledons appears to be light-stimulated. We pr esent preliminary evidence that a smaller transcript represents an ant isense species being read from the same gene.