Characterization of a second gene encoding γ-glutamyl transpeptidase from Schizosaccharomyces pombe

• Published in: Canadian journal of microbiology Vol. 51; no. 3; pp. 269 - 275
• Main Authors: Park, Hey-Jung, Moon, Jeong-Su, Kim, Hong-Gyum, Kim, Il-Han, Kim, Kanghwa, Park, Eun-Hee, Lim, Chang-Jin
• Format: Journal Article
• Language: English
• Published: Ottawa, Canada NRC Research Press 01.03.2005; National Research Council of Canada
• Subjects: Amino Acid Sequence; Animals; Base Sequence; Biological and medical sciences; Cloning, Molecular; Escherichia coli; Fundamental and applied biological sciences. Psychology; gamma-Glutamyltransferase - chemistry; gamma-Glutamyltransferase - genetics; gamma-Glutamyltransferase - metabolism; Gene Expression Regulation, Fungal; Growth, nutrition, metabolism, transports, enzymes. Molecular biology; Heat-Shock Response; Humans; Microbiology; Molecular Sequence Data; Mycology; Oxidative Stress; Rats; Rattus norvegicus; Saccharomyces cerevisiae; Schizosaccharomyces - enzymology; Schizosaccharomyces - genetics; Schizosaccharomyces - growth & development; Schizosaccharomyces pombe; Schizosaccharomyces pombe Proteins - chemistry; Schizosaccharomyces pombe Proteins - genetics; Schizosaccharomyces pombe Proteins - metabolism; Sequence Analysis, DNA; Enzyme; Transferases; Sequence alignment; Gene expression; Aminoacyltransferases; Survival; Stress; γ-Glutamyltransferase; Fungi; Polymerase chain reaction; Regulation(control); Schizosaccharomyces pombe; Ascomycetes; Thallophyta
• ISSN: 0008-4166; 1480-3275
• DOI: 10.1139/w04-137

The first gene encoding γ-glutamyl transpeptidase (GGTI) of the fission yeast has previously been characterized, and its expression was found to be regulated by various oxidative stress-inducing agents. In this work, a second gene, encoding GGTII, was cloned and characterized from the fission yeast Schizosaccharomyces pombe. The structural gene encoding GGTII was amplified from the genomic DNA of the fission yeast and ligated into the shuttle vector pRS316 to generate the recombinant plasmid pPHJ02. The determined sequence contains 3040 bp and is able to encode the putative 611 amino acid sequence of GGTII, which resembles the counterparts of Saccharomyces cerevisiae, Homo sapiens, Rattus norvegicus, and Escherichia coli. The DNA sequence also contains 940-bp upstream and 289-bp downstream regions of the GGTII gene. The Schizosaccharomyces pombe cells harboring plasmid pPHJ02 showed about 4-fold higher GGT activity in the exponential phase than the cells harboring the vector only, indicating that the cloned GGTII gene is functional. The S. pombe cells containing the cloned GGTII gene were found to contain higher levels of both intracellular glutathione (GSH) content and GSH uptake. The S. pombe cells harboring plasmid pPHJ02 showed increased survival on solid media containing hydrogen peroxide, diethylmaleate, aluminum chloride, cadmium chloride, or mercuric chloride. The GGTII mRNA level was significantly elevated by treatment with GSH-depleting diethylmaleate. These results imply that the S. pombe GGTII gene produces functional GGTII protein and is involved in the response to oxidative stresses in S. pombe cells.Key words: fission yeast, genomic DNA, γ-glutamyl transpeptidase, regulation, Schizosaccharomyces pombe, stress response.