Stress-dependent regulation of the gene encoding gamma-glutamylcysteine synthetase from the fission yeast.
Glutathione (GSH), an important antioxidant involved in stress response, is synthesized in two sequential reactions. Gamma-glutamylcysteine synthetase (GCS) catalyzes the first step in GSH biosynthesis, which is usually known to be rate-limiting. In this work, regulatory patterns of the GCS gene from the fission yeast Schizosaccharomyces pombe have been investigated. The 607 bp upstream region from the translational initiation point was amplified by the two synthetic primers. The amplified DNA was ligated into the BamHI/HindIII site of the shuttle vector YEp367R to generate the fusion plasmid pUGCS101. The GCS-lacZ fusion gene construct was confirmed by restriction mapping and nucleotide sequencing. The GCS-lacZ fusion gene was used to study effects of various agents on the transcription of the GCS gene. The synthesis of beta-galactosidase from the fusion plasmid pUGCS101 was enhanced by metals, oxidative and nitrosative stresses, and glutathione-depleting agents. The GCS mRNA level in the wildtype S. pombe cells was significantly elevated by the treatment with sodium nitroprusside or menadione, which was detected by RT-PCR. It was also induced by low concentrations of glucose and sucrose. These results suggest that the expression of S. pombe GCS gene is regulated by various stresses and carbon sources.
Kim
Su-Jung
SJ
Division of Life Sciences, Kangwon National University, Chuncheon 200-701, Korea.
Park
Eun-Hee
EH
Lim
Chang-Jin
CJ
D002244:Carbon / Q000378:metabolism; D002455:Cell Division / Q000187:drug effects; D015245:Deoxyribonuclease BamHI / Q000235:genetics / Q000378:metabolism; D015966:Gene Expression Regulation, Fungal; D005947:Glucose / Q000378:metabolism / Q000494:pharmacology; D005721:Glutamate-Cysteine Ligase / Q000187:drug effects / Q000235:genetics* / Q000378:metabolism; D005978:Glutathione / Q000378:metabolism; D008670:Metals / Q000378:metabolism / Q000494:pharmacology; D009569:Nitric Oxide / Q000378:metabolism; D020030:Nitric Oxide Donors / Q000494:pharmacology; D009599:Nitroprusside / Q000494:pharmacology; D018384:Oxidative Stress; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D012568:Schizosaccharomyces / Q000201:enzymology / Q000235:genetics*; D029702:Schizosaccharomyces pombe Proteins / Q000187:drug effects / Q000235:genetics* / Q000378:metabolism; D015265:Site-Specific DNA-Methyltransferase (Adenine-Specific) / Q000235:genetics / Q000378:metabolism; D013395:Sucrose / Q000378:metabolism / Q000494:pharmacology; D013481:Superoxides / Q000378:metabolism; D024483:Vitamin K 3 / Q000494:pharmacology; D001616:beta-Galactosidase / Q000235:genetics / Q000378:metabolism
D008670:Metals; D020030:Nitric Oxide Donors; D011993:Recombinant Fusion Proteins; D029702:Schizosaccharomyces pombe Proteins; D013481:Superoxides; D009599:Nitroprusside; D009569:Nitric Oxide; D013395:Sucrose; D024483:Vitamin K 3; D002244:Carbon; C090528:DNA modification methylase HindIII; D015265:Site-Specific DNA-Methyltransferase (Adenine-Specific); D015245:Deoxyribonuclease BamHI; D001616:beta-Galactosidase; D005721:Glutamate-Cysteine Ligase; D005978:Glutathione; D005947:Glucose