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

2005 ◽  
Vol 51 (3) ◽  
pp. 269-275 ◽  
Author(s):  
Hey-Jung Park ◽  
Jeong-Su Moon ◽  
Hong-Gyum Kim ◽  
Il-Han Kim ◽  
Kanghwa Kim ◽  
...  
Keyword(s):  
Schizosaccharomyces Pombe ◽  
Fission Yeast ◽  
Genomic Dna ◽  
Homo Sapiens ◽  
Shuttle Vector ◽  
Mrna Level ◽  
Gene Encoding ◽  
Oxidative Stresses ◽  
Solid Media ◽  
Glutamyl Transpeptidase

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.

Characterization and regulation of the γ-glutamyl transpeptidase gene from the fission yeast Schizosaccharomyces pombe

2004 ◽  
Vol 50 (1) ◽  
pp. 61-67 ◽  
Author(s):  
Hey-Jung Park ◽  
Hye-Won Lim ◽  
Kanghwa Kim ◽  
Il-Han Kim ◽  
Eun-Hee Park ◽  
...  
Keyword(s):  
Schizosaccharomyces Pombe ◽  
Fission Yeast ◽  
Rattus Norvegicus ◽  
Genomic Dna ◽  
Homo Sapiens ◽  
Shuttle Vector ◽  
Mrna Level ◽  
Exponential Phase ◽  
Chromosomal Dna ◽  
Glutamyl Transpeptidase

The structural gene for the putative γ-glutamyl transpeptidase (GGT) was isolated from the chromosomal DNA of the fission yeast Schizosaccharomyces pombe. The determined sequence contained 3324 bp and encoded the predicted 630 amino acid sequence of GGT, which resembles counterparts in Homo sapiens, Rattus norvegicus, Saccharomyces cerevisiae, and Escherichia coli. The S. pombe cells harboring the cloned GGT gene showed about twofold higher GGT activity in the exponential phase than the cells harboring the vector only, indicating that the cloned GGT gene was functional. To monitor the expression of the S. pombe GGT gene, we fused the fragment 1085 bp upstream of the cloned GGT gene into the promoterless β-galactosidase gene of the shuttle vector YEp367R to generate the fusion plasmid pGT98. The synthesis of β-galactosidase from the fusion plasmid in S. pombe cells was enhanced by treatments with NO-generating sodium nitroprusside (SN), L-buthionine-(S,R)-sulfoximine (BSO), and glycerol. The GGT mRNA level in the S. pombe cells was increased by SN and BSO. Involvement of Pap1 in the induction of the GGT gene by SN and BSO was observed.Key words: fission yeast, genomic DNA, γ-glutamyl transpeptidase, Pap1, regulation, Schizosaccharomyces pombe.

Stress-dependent regulation of a monothiol glutaredoxin gene from Schizosaccharomyces pombe

2005 ◽  
Vol 51 (7) ◽  
pp. 613-620 ◽  
Author(s):  
Hong-Gyum Kim ◽  
Byung-Chul Kim ◽  
Eun-Hee Park ◽  
Chang-Jin Lim
Keyword(s):  
Schizosaccharomyces Pombe ◽  
Potassium Chloride ◽  
Sodium Nitroprusside ◽  
Fusion Gene ◽  
Mrna Level ◽  
Carbon Sources ◽  
Lacz Gene ◽  
Gene Encoding ◽  
Stress Dependent

Glutaredoxin (Grx) is a small, heat-stable protein acting as a multi-functional glutathione-dependent disulfide oxidoreductase. In this work, a gene encoding the monothiol glutaredoxin Grx4 was cloned from the genomic DNA of the fission yeast Schizosaccharomyces pombe. The determined DNA sequence carries 1706 bp, which is able to encode the putative 244 amino acid sequence of Grx with 27 099 Da. It does not contain an intron, and the sequence CGFS is found in the active site. Grx activity was increased 1.46-fold in S. pombe cells harboring the cloned Grx4 gene, indicating that the Grx4 gene is in vivo functioning. Although aluminum, cadmium, and hydrogen peroxide marginally enhanced the synthesis of β-galactosidase from the Grx4-lacZ fusion gene, NO-generating sodium nitroprusside (0.5 mmol/L and 1.0 mmol/L) and potassium chloride (0.2 mol/L and 0.5 mol/L) significantly enhanced it. The Grx4 mRNA level was also enhanced after the treatment with sodium nitroprusside and potassium chloride. The synthesis of β-galactosidase from the Grx4-lacZ gene was increased by fermentable carbon sources, such as glucose (lower than 2%) and sucrose, but not by nonfermentable carbon sources such as acetate and ethanol. The basal expression of the S. pombe Grx4 gene did not depend on the presence of Pap1. These results imply that the S. pombe monothiol Grx4 gene is genuinely functional and regulated by a variety of stresses.Key words: monothiol glutaredoxin, Pap1, regulation, Schizosaccharomyces pombe, stress response.

Deubiquitinating activity of Sdu1, a putative member of the PPPDE peptidase family, in Schizosaccharomyces pombe

2013 ◽  
Vol 59 (12) ◽  
pp. 789-796 ◽  
Author(s):  
Yunsik Kim ◽  
Hannah Jo ◽  
Chang-Jin Lim
Keyword(s):  
Schizosaccharomyces Pombe ◽  
Vector Control ◽  
Sodium Nitroprusside ◽  
Shuttle Vector ◽  
Activity Levels ◽  
Amino Acid Residues ◽  
Gene Encoding ◽  
Nitric Oxide Level ◽  
Dsrna Viruses ◽  
Putative Member

The Schizosaccharomyces pombe sdu+ gene encoding a putative member of the PPPDE (Permuted Papain fold Peptidases of DsRNA viruses and Eukaryotes) superfamily was cloned into an Escherichia coli – yeast shuttle vector pRS316, resulting in the recombinant plasmid pYSTP. The determined nucleotide sequence carries 1207 bp, which would encode a protein of 201 amino acid residues. The S. pombe cells harboring pYSTP contained higher sdu1+ mRNA and deubiquitinating activity levels than the vector control cells, indicating that the sdu1+ gene is functioning. They exhibited a better growth in normal rich medium than the vector control cells. When shifted into the fresh medium containing hydrogen peroxide, menadione, or sodium nitroprusside, the S. pombe cells harboring pYSTP were able to grow reasonably well, while the growth of the vector control cells was arrested. The reactive oxygen species and total glutathione levels of the S. pombe cells harboring pYSTP were lower and higher than those of the vector control cells under the same stressful conditions, respectively. They exhibited a lower nitric oxide level than the vector control cells when subjected to sodium nitroprusside. Taken together, the sdu1+ gene encodes an actual protein having deubiquitinating activity and is involved in the response against oxidative and nitrosative stresses in S. pombe.

scholarly journals Efficient targeted integration at leu1-32 and ura4-294 in Schizosaccharomyces pombe.

Genetics ◽  
1994 ◽  
Vol 136 (3) ◽  
pp. 849-856 ◽  
Author(s):  
J B Keeney ◽  
J D Boeke
Keyword(s):  
Homologous Recombination ◽  
Schizosaccharomyces Pombe ◽  
Fission Yeast ◽  
Gene Conversion ◽  
Blot Analysis ◽  
Genomic Dna ◽  
Plasmid Vector ◽  
Homologous Integration ◽  
Targeted Integration ◽  
Chromosomal Loci

Abstract Homologous integration into the fission yeast Schizosaccharomyces pombe has not been well characterized. In this study, we have examined integration of plasmids carrying the leu1+ and ura4+ genes into their chromosomal loci. Genomic DNA blot analysis demonstrated that the majority of transformants have one or more copies of the plasmid vector integrated via homologous recombination with a much smaller fraction of gene conversion to leu1+ or ura4+. Non-homologous recombination events were not observed for either gene. We describe the construction of generally useful leu1+ and ura4+ plasmids for targeted integration at the leu1-32 and ura4-294 loci of S. pombe.

scholarly journals The Gene Encoding γ-Glutamyl Transpeptidase II in the Fission Yeast Is Regulated by Oxidative and Metabolic Stress

BMB Reports ◽  
2005 ◽  
Vol 38 (5) ◽  
pp. 609-618 ◽  
Author(s):  
Hyun-Jung Kang ◽  
Byung-Chul Kim ◽  
Eun-Hee Park ◽  
Ki-Sup Ahn ◽  
Chang-Jin Lim
Keyword(s):  
Fission Yeast ◽  
Metabolic Stress ◽  
Gene Encoding ◽  
Glutamyl Transpeptidase

Analysis of the Schizosaccharomyces pombe cyclin puc1: evidence for a role in cell cycle exit

1994 ◽  
Vol 107 (3) ◽  
pp. 601-613 ◽  
Author(s):  
S.L. Forsburg ◽  
P. Nurse
Keyword(s):  
Schizosaccharomyces Pombe ◽  
Fission Yeast ◽  
Sexual Development ◽  
Mitotic Cycle ◽  
Nitrogen Starvation ◽  
Cell Cycle Exit ◽  
Gene Encoding ◽  
Yeast Saccharomyces Cerevisiae ◽  
Protein Blocks

The puc1+ gene, encoding a G1-type cyclin from the fission yeast Schizosaccharomyces pombe, was originally isolated by complementation in the budding yeast Saccharomyces cerevisiae. Here, we report the molecular characterization of this gene and analyse its role in S. pombe. We fail to identify any function of this cyclin at the mitotic G1/S transition in S. pombe, but demonstrate that it does function in exit from the mitotic cycle. Expression of the puc1+ gene is increased during nitrogen starvation, and puc1 affects the timing of sexual development in response to starvation. Overexpression of the puc1 protein blocks sexual development, and rescues pat1ts cells, which would otherwise undergo a lethal meiosis. We conclude that puc1 contributes to negative regulation of the timing of sexual development in fission yeast, and functions at the transition between cycling and non-cycling cells.

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