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.

scholarly journals ACEI of Trichoderma reesei Is a Repressor of Cellulase and Xylanase Expression

2003 ◽  
Vol 69 (1) ◽  
pp. 56-65 ◽  
Author(s):  
Nina Aro ◽  
Marja Ilmén ◽  
Anu Saloheimo ◽  
Merja Penttilä
Keyword(s):  
Trichoderma Reesei ◽  
Culture Media ◽  
Carbon Sources ◽  
Cellulase Production ◽  
The Novel ◽  
Hypocrea Jecorina ◽  
Gene Encoding ◽  
Gene Coding ◽  
Better Than

ABSTRACT We characterized the effect of deletion of the Trichoderma reesei (Hypocrea jecorina) ace1 gene encoding the novel cellulase regulator ACEI that was isolated based on its ability to bind to and activate in vivo in Saccharomyces cerevisiae the promoter of the main cellulase gene, cbh1. Deletion of ace1 resulted in an increase in the expression of all the main cellulase genes and two xylanase genes in sophorose- and cellulose-induced cultures, indicating that ACEI acts as a repressor of cellulase and xylanase expression. Growth of the strain with a deletion of the ace1 gene on different carbon sources was analyzed. On cellulose-based medium, on which cellulases are needed for growth, the Δace1 strain grew better than the host strain due to the increased cellulase production. On culture media containing sorbitol as the sole carbon source, the growth of the strain with a deletion of the ace1 gene was severely impaired, suggesting that ACEI regulates expression of other genes in addition to cellulase and xylanase genes. A strain with a deletion of the ace1 gene and with a deletion of the ace2 gene coding for the cellulase and xylanase activator ACEII expressed cellulases and xylanases similar to the Δace1 strain, indicating that yet another activator regulating cellulase and xylanase promoters was present.

scholarly journals Site-directed RNA repair of endogenous Mecp2 RNA in neurons

2017 ◽  
Vol 114 (44) ◽  
pp. E9395-E9402 ◽  
Author(s):  
John R. Sinnamon ◽  
Susan Y. Kim ◽  
Glen M. Corson ◽  
Zhen Song ◽  
Hiroyuki Nakai ◽  
...  
Keyword(s):  
Rna Editing ◽  
Protein Function ◽  
Rna Binding ◽  
Catalytic Domain ◽  
Mrna Level ◽  
Gene Encoding ◽  
Rna Repair ◽  
Mecp2 Protein ◽  
Localization Signal

Rett syndrome (RTT) is a debilitating neurological disorder caused by mutations in the gene encoding the transcription factor Methyl CpG Binding Protein 2 (MECP2). A distinct disorder results from MECP2 gene duplication, suggesting that therapeutic approaches must restore close to normal levels of MECP2. Here, we apply the approach of site-directed RNA editing to repair, at the mRNA level, a disease-causing guanosine to adenosine (G > A) mutation in the mouse MeCP2 DNA binding domain. To mediate repair, we exploit the catalytic domain of Adenosine Deaminase Acting on RNA (ADAR2) that deaminates A to inosine (I) residues that are subsequently translated as G. We fuse the ADAR2 domain, tagged with a nuclear localization signal, to an RNA binding peptide from bacteriophage lambda. In cultured neurons from mice that harbor an RTT patient G > A mutation and express engineered ADAR2, along with an appropriate RNA guide to target the enzyme, 72% of Mecp2 mRNA is repaired. Levels of MeCP2 protein are also increased significantly. Importantly, as in wild-type neurons, the repaired MeCP2 protein is enriched in heterochromatic foci, reflecting restoration of normal MeCP2 binding to methylated DNA. This successful use of site-directed RNA editing to repair an endogenous mRNA and restore protein function opens the door to future in vivo applications to treat RTT and other diseases.

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 Genetic and Physiological Characterization of Fructose-1,6-Bisphosphate Aldolase and Glyceraldehyde-3-Phosphate Dehydrogenase in the Crabtree-Negative Yeast Kluyveromyces lactis

2022 ◽  
Vol 23 (2) ◽  
pp. 772
Author(s):  
Rosaura Rodicio ◽  
Hans-Peter Schmitz ◽  
Jürgen J. Heinisch
Keyword(s):  
Kluyveromyces Lactis ◽  
Regulation Of Gene Expression ◽  
Carbon Sources ◽  
Pentose Phosphate ◽  
Gene Encoding ◽  
Physiological Characterization ◽  
Genes Encoding ◽  
Fructose 1,6 Bisphosphate

The milk yeast Kluyveromyces lactis degrades glucose through glycolysis and the pentose phosphate pathway and follows a mainly respiratory metabolism. Here, we investigated the role of two reactions which are required for the final steps of glucose degradation from both pathways, as well as for gluconeogenesis, namely fructose-1,6-bisphosphate aldolase (FBA) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). In silico analyses identified one gene encoding the former (KlFBA1), and three genes encoding isoforms of the latter (KlTDH1, KlTDH2, KlGDP1). Phenotypic analyses were performed by deleting the genes from the haploid K. lactis genome. While Klfba1 deletions lacked detectable FBA activity, they still grew poorly on glucose. To investigate the in vivo importance of the GAPDH isoforms, different mutant combinations were analyzed for their growth behavior and enzymatic activity. KlTdh2 represented the major glycolytic GAPDH isoform, as its lack caused a slower growth on glucose. Cells lacking both KlTdh1 and KlTdh2 failed to grow on glucose but were still able to use ethanol as sole carbon sources, indicating that KlGdp1 is sufficient to promote gluconeogenesis. Life-cell fluorescence microscopy revealed that KlTdh2 accumulated in the nucleus upon exposure to oxidative stress, suggesting a moonlighting function of this isoform in the regulation of gene expression. Heterologous complementation of the Klfba1 deletion by the human ALDOA gene renders K. lactis a promising host for heterologous expression of human disease alleles and/or a screening system for specific drugs.

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.

Expression of the atf1+ gene is upregulated in fission yeast under nitrosative and nutritional stresses

2009 ◽  
Vol 55 (11) ◽  
pp. 1323-1327 ◽  
Author(s):  
S.-H. Song ◽  
B.-M. Kim ◽  
C.-J. Lim ◽  
Y.S. Song ◽  
E.-H. Park
Keyword(s):  
Nitric Oxide ◽  
Schizosaccharomyces Pombe ◽  
Sodium Nitroprusside ◽  
Fission Yeast ◽  
Fusion Gene ◽  
Lacz Fusion ◽  
Transcriptional Level ◽  
Mrna Levels ◽  
Nitrogen Depletion ◽  
Rt Pcr

This work was designed to assess regulation of the atf1+ gene in the fission yeast Schizosaccharomyces pombe under nitrosative and nutritional stresses, using the atf1+–lacZ fusion gene and RT–PCR. Nitric oxide (NO)-generating sodium nitroprusside (SNP; 10 µmol/L) and nitrogen depletion significantly enhanced synthesis of β-galactosidase from the atf1+–lacZ fusion gene in S. pombe Pap1-positive KP1 cells, but not in S. pombe Pap1-negative TP108-3C cells. SNP (10 µmol/L) and nitrogen depletion also caused a significant increase in atf1+ mRNA levels in Pap1-positive cells, but not in Pap1-negative cells. Depletion of glucose marginally increased synthesis of β-galactosidase from the fusion gene in S. pombe Pap1-positive cells. Taken together, the S. pombe atf1+ gene is upregulated by nitrosative and nutritional stresses on a transcriptional level, possibly via the mediation of Pap1.

scholarly journals Partitioning of the Nuclear and Mitochondrial tRNA 3′-End Processing Activities between Two different Proteins in Schizosaccharomyces pombe

2013 ◽  
Vol 288 (38) ◽  
pp. 27415-27422 ◽  
Author(s):  
Xiaojie Zhang ◽  
Qiaoqiao Zhao ◽  
Ying Huang
Keyword(s):  
Schizosaccharomyces Pombe ◽  
Short Form ◽  
Temperature Sensitive ◽  
Mitochondrial Trna ◽  
Gene Encoding ◽  
Protein Levels ◽  
Long Form ◽  
Trnase Z ◽  
Target Effects

tRNase Z is an essential endonuclease responsible for tRNA 3′-end maturation. tRNase Z exists in a short form (tRNase ZS) and a long form (tRNase ZL). Prokaryotes have only tRNase ZS, whereas eukaryotes can have both forms of tRNase Z. Most eukaryotes characterized thus far, including Saccharomyces cerevisiae, Caenorhabditis elegans, Drosophila melanogaster, and humans, contain only one tRNase ZL gene encoding both nuclear and mitochondrial forms of tRNase ZL. In contrast, Schizosaccharomyces pombe contains two essential tRNase ZL genes (trz1 and trz2) encoding two tRNase ZL proteins, which are targeted to the nucleus and mitochondria, respectively. Trz1 protein levels are notably higher than Trz2 protein levels. Here, using temperature-sensitive mutants of trz1 and trz2, we provide in vivo evidence that trz1 and trz2 are involved in nuclear and mitochondrial tRNA 3′-end processing, respectively. In addition, trz2 is also involved in generation of the 5′-ends of other mitochondrial RNAs, whose 5′-ends coincide with the 3′-end of tRNA. Thus, our results provide a rare example showing partitioning of the nuclear and mitochondrial tRNase ZL activities between two different proteins in S. pombe. The evolution of two tRNase ZL genes and their differential expression in fission yeast may avoid toxic off-target effects.

scholarly journals Formation of a Carboxy-Terminal Domain Phosphatase (Fcp1)/TFIIF/RNA Polymerase II (pol II) Complex in Schizosaccharomyces pombe Involves Direct Interaction between Fcp1 and the Rpb4 Subunit of pol II

2002 ◽  
Vol 22 (5) ◽  
pp. 1577-1588 ◽  
Author(s):  
Makoto Kimura ◽  
Hisako Suzuki ◽  
Akira Ishihama
Keyword(s):  
Rna Polymerase ◽  
Schizosaccharomyces Pombe ◽  
Rna Polymerase Ii ◽  
Direct Interaction ◽  
Gene Encoding ◽  
Pol Ii ◽  
Terminal Domain ◽  
Ctd Phosphatase

ABSTRACT In transcriptional regulation, RNA polymerase II (pol II) interacts and forms complexes with a number of protein factors. To isolate and identify the pol II-associated proteins, we constructed a Schizosaccharomyces pombe strain carrying a FLAG tag sequence fused to the rpb3 gene encoding the pol II subunit Rpb3. By immunoaffinity purification with anti-FLAG antibody-resin, a pol II complex containing the Rpb1 subunit with a nonphosphorylated carboxyl-terminal domain (CTD) was isolated. In addition to the pol II subunits, the complex was found to contain three subunits of a transcription factor TFIIF (TFIIFα, TFIIFβ, and Tfg3) and TFIIF-interacting CTD-phosphatase Fcp1. The same type of pol II complex could also be purified from an Fcp1-tagged strain. The isolated Fcp1 showed CTD-phosphatase activity in vitro. The fcp1 gene is essential for cell viability. Fcp1 and pol II interacted directly in vitro. Furthermore, by chemical cross-linking, glutathione S-transferase pulldown, and affinity chromatography, the Fcp1-interacting subunit of pol II was identified as Rpb4, which plays regulatory roles in transcription. We also constructed an S. pombe thiamine-dependent rpb4 shut-off system. On repression of rpb4 expression, the cell produced more of the nonphosphorylated form of Rpb1, but the pol II complex isolated with the anti-FLAG antibody contained less Fcp1 and more of the phosphorylated form of Rpb1 with a concomitant reduction in Rpb4. This result indicates the importance of Fcp1-Rpb4 interaction for formation of the Fcp1/TFIIF/pol II complex in vivo.

Overexpression of a metacaspase gene stimulates cell growth and stress response inSchizosaccharomyces pombe

2007 ◽  
Vol 53 (8) ◽  
pp. 1016-1023 ◽  
Author(s):  
Hye-Won Lim ◽  
Su-Jung Kim ◽  
Eun-Hee Park ◽  
Chang-Jin Lim
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Stress Response ◽  
Cell Growth ◽  
Schizosaccharomyces Pombe ◽  
Sodium Nitroprusside ◽  
Fission Yeast ◽  
Recombinant Plasmid ◽  
Mrna Level ◽  
Yeast Cells ◽  
Yeast Saccharomyces Cerevisiae

A unique gene named pca1+, encoding a metacaspase, was cloned from the fission yeast Schizosaccharomyces pombe and was used to create a recombinant plasmid, pPMC. The metacaspase mRNA level was markedly elevated in the fission yeast cells harboring the plasmid pPMC. Overexpressed Pca1+appeared to stimulate the growth of the fission yeast cells instead of arresting their growth. Its expression was enhanced by stress-inducing agents such as H2O2, sodium nitroprusside, and CdCl2, and it conferred cytoprotection, especially against CdCl2. However, such protection was not reproducible in the budding yeast Saccharomyces cerevisiae harboring pPMC. Taken together, these results propose that Pca1+may be involved in the growth and stress response of the fission yeast.

Stress-dependent regulation of Pbh1, a BIR domain-containing protein, in the fission yeast

2006 ◽  
Vol 52 (12) ◽  
pp. 1261-1265 ◽  
Author(s):  
Nam-Chul Cho ◽  
Hyun-Jung Kang ◽  
Hye-Won Lim ◽  
Byung-Chul Kim ◽  
Eun-Hee Park ◽  
...  
Keyword(s):  
Schizosaccharomyces Pombe ◽  
Fission Yeast ◽  
Nitrosative Stress ◽  
Nitrogen Starvation ◽  
Fusion Gene ◽  
Shuttle Vector ◽  
Upstream Region ◽  
Basal Expression ◽  
Stress Dependent ◽  
Bir Domain

To elicit the physiological roles of Pbh1, a baculoviral IAP repeat (BIR) domain-containing protein, in Schizosaccharomyces pombe, we investigated if Pbh1 expression is regulated by stress. The upstream region (1221 bp) of the pbh1 gene was fused into the promoterless β-galactosidase gene of the shuttle vector YEp367R, and the resultant fusion plasmid was named pPbh04. The synthesis of β-galactosidase from the pbh1-lacZ fusion gene was markedly enhanced by sodium nitroprusside (SNP) generating nitric oxide. The basal expression of the pbh1 gene required the presence of Pap1. Pap1 also mediated the induction of the pbh1 gene by SNP and nitrogen starvation. Pap1-dependent induction of the pbh1 gene by SNP was confirmed by the enhanced level of the pbh1 mRNA in Pap1-positive cells but not in Pap1-negative cells. Taken together, it was demonstrated that the pbh1 genes are positively regulated by nitrosative and nitrogen starvation stresses in Pap1-dependent manner.Key words: fission yeast, nitrosative stress, nutritional stress, nitrogen starvation, Pap1, Pbh1, regulation, Schizosaccharomyces pombe.

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