PLANT PHYSIOLOGY , Vol 111, Issue 4 1097-1107, Copyright © 1996 by American Society of Plant Biologists
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BIOCHEMISTRY AND ENZYMOLOGY |
Cloning of a cDNA Encoding Cytosolic Acetoacetyl-Coenzyme A Thiolase from Radish by Functional Expression in Saccharomyces cerevisiae
K. U. Vollack and T. J. Bach
Botanisches Institut II, Universitat Karlsruhe, D-76128 Karlsruhe, Germany (K.-U.V.)
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.
