Isolierung und Charakterisierung einer cDNA fuer die cytosolische Acetoacetyl-CoA-Thiolase aus Raphanus sativus L. durch Komplementierung einer Saccharomyces cerevisiae-Mutante

Abstract

For the first time this study presents the successful cloning and functional characterization of a plant gene coding for cytosolic acetoacyl-CoA thiolase (cAACT). For the cloning of cAACT from radish, yeast mutants (erg10A), deficient in cAACT enzyme activity and therefore auxotrophic for ergosterol, were used to isolate the clones bearing the radish cAACT cDNA by functional complementation. The radish cDNA library was converted into the integrated plasmid vector pYEUra3, which has been transformed into a Saccharomyces cerevisae strain with a mutation in the endogenous cAACT gene (erg10A). Positive radish cAACT clones have been indentified by the restoration of wild-type yeast growth characteristics. Using the cAACT probe under highly stringent conditions, a gel blot analysis of differentially digested genomic DNA gave strong evidence for the radish gene being organized as a single copy gene, which most likely is interrupted by several introns. (WEN)SIGLEAvailable from TIB Hannover: RN 5800(30) / FIZ - Fachinformationszzentrum Karlsruhe / TIB - Technische InformationsbibliothekDEGerman