Characterization of the intron-containing citrate synthase gene from the alkanotrophic yeast Candida tropicalis: cloning and expression in Saccharomyces cerevisiae

• Published in: Archives of microbiology Vol. 168; no. 1; pp. 8 - 15
• Main Authors: Ueda, M, Sanuki, S, Kawachi, H, Shimizu, K, Atomi, H, Tanaka, A
• Format: Journal Article
• Language: English
• Published: Heidelberg Springer 01.07.1997; Berlin
• Subjects: Amino Acid Sequence; Base Sequence; Biological and medical sciences; Candida - genetics; Candida tropicalis; Citrate (si)-Synthase - genetics; Cloning, Molecular; DNA, Fungal - analysis; DNA, Fungal - isolation & purification; Fundamental and applied biological sciences. Psychology; Gene Expression; Genes, Fungal; Genes. Genome; Introns; Mitochondria - genetics; Mitochondria - metabolism; Molecular and cellular biology; Molecular genetics; Molecular Sequence Data; Promoter Regions, Genetic; Recombinant Proteins - biosynthesis; Restriction Mapping; Saccharomyces cerevisiae; Saccharomyces cerevisiae - genetics; Sequence Alignment; Sequence Analysis, DNA; Sequence Homology, Amino Acid; Yeast; Splicing; Enzyme; Gene organization; Lyases; Gene expression; Fungi; Oxo-acid-lyases; Characterization; Carbon-carbon lyases; Citrate (si)-synthase; Gene; Intron; Candida tropicalis; Ascomycetes; Fungi Imperfecti; Molecular cloning; Saccharomyces cerevisiae; Thallophyta; Structural analysis
• ISSN: 0302-8933; 1432-072X
• DOI: 10.1007/s002030050463

Citrate synthase, an essential enzyme of the tricarboxylic acid cycle in mitochondria, was purified from acetate-grown Candida tropicalis. Results from SDS-PAGE and gel filtration showed that this enzyme was a dimer composed of 45-kDa subunits. A citrate synthase cDNA fragment was amplified by the 5'-RACE method. Nucleotide sequence analysis of this cDNA fragment revealed that the deduced amino acid sequence contained an extended leader sequence which is suggested to be a mitochondrial targeting signal, as judged from helical wheel analysis. Using this cDNA probe, one genomic citrate synthase clone was isolated from a yeast 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 splicing motifs were present in this intron. When the CtCIT gene including its intron was introduced into Saccharomyces cerevisiae using the promoter UPRpICL, citrate synthase activity was highly induced, which strongly indicated that this intron was correctly spliced in S. cerevisiae.