CHARACTERIZATION OF THE INTRON-CONTAINING CITRATE SYNTHASE GENE FROM THE ALKANOTROPHIC YEAST CANDIDA-TROPICALIS - CLONING AND EXPRESSION INSACCHAROMYCES-CEREVISIAE

Citrate synthase, an essential enzyme of the tricarboxylic acid cycle in mitochondria, was purified from acetate-grown Candida tropicalis. R esults from SDS-PAGE and gel filtration showed that this enzyme was a dimer composed of 45-kDa subunits. A citrate synthase cDNA fragment wa s amplified by the 5'-RACE method. Nucleotide sequence analysis of thi s cDNA fragment revealed that the deduced amino acid sequence containe d an extended leader sequence which is suggested to be a mitochondrial targeting signal, as judged from helical wheel analysis. Using this c DNA probe, one genomic citrate synthase clone was isolated from a yeas t lambda EMBL3 library. The nucleotide sequence of the gene encoding C . tropicalis citrate synthase, CtCIT, revealed the presence of a 79-bp intron in the N-terminal region. Sequences essential as yeast splicin g motifs were present in this intron. When the CtCIT gene including it s intron was introduced into Saccharomyces cerevisiae using the promot er UPR-ICL, citrate synthase activity was highly induced, which strong ly indicated that this intron was correctly spliced in S. cerevisiae.