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.
The Schizosaccharomyces pombe msrB(+) gene encoding methionine-R-sulfoxide reductase (MsrB) was cloned into the shuttle vector pRS316 to generate the recombinant plasmid pFMetSO. The msrB(+) mRNA level was significantly increased in the S. pombe cells harboring pFMetSO, indicating that the cloned msrB(+) gene is functioning. In the presence of 0.1 mM L-methionine-(R,S)-sulfoxide, the S. pombe cells harboring pFMetSO could grow normally but the growth of the vector control cells was almost arrested. The S. pombe cells harboring pFMetSO exhibited the enhanced growth on the minimal medium plates with stress-inducing agents, such as hydrogen peroxide, superoxide radical-generating menadione (MD), nitric oxide (NO)-generating sodium nitroprusside (SNP), and cadmium (Cd), when compared with the vector control cells. They also gave rise to the enhanced growth at the high incubation temperature of 37 °C than the vector control cells. The S. pombe cells harboring pFMetSO contained lower reactive oxygen species (ROS) and higher total glutathione (GSH) levels than the vector control cells. In brief, the S. pombe MsrB plays a protective role against oxidative, nitrosative, and thermal stresses, and is involved in diminishing intracellular ROS level.
Hos3 is involved in cellular growth under osmotic stress in Schizosaccharomyces pombe. The recombinant plasmid pYFHos3 harboring the structural gene encoding Hos3 was constructed. The S. pombe cells harboring pYFHos3 contained the increased hos3 (+) mRNA content and exhibited an enhanced growth in high osmotic conditions, such as 1.5 M KCl and 2.5 M D-glucose, compared with the vector control cells. In the presence of hydrogen peroxide (H2O2), superoxide anion-generating menadione (MD) and nitric oxide (NO)-generating sodium nitroprusside (SNP), they could grow better than the vector control cells. In the presence of H2O2, MD and SNP and in the absence of a nitrogen source, the S. pombe cells harboring pYFHos3 contained less elevated NO and reactive oxygen species (ROS) levels than the vector control cells. Collectively, the S. pombe Hos3 also participate in the cellular defense against oxidative, nitrosative and nutritional stresses through down-regulating ROS and NO levels.
The Schizosaccharomyces pombe cells harboring the methionine- R-sulfoxide reductase (MsrB)-overexpressing recombinant plasmid pFMetSO exhibited better growth than vector control cells, when shifted into fresh medium containing cadmium chloride (abbreviated as Cd). Although both groups of cells contained enhanced reactive oxygen species (ROS) and nitric oxide (NO) levels in the presence of Cd, ROS and NO levels were significantly lower in the S. pombe cells harboring pFMetSO than in vector control cells. Conversely, the S. pombe cells harboring pFMetSO possessed higher total glutathione (GSH) levels and a greater reduced/oxidized GSH ratio than vector control cells under the same conditions.
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