No abstract

In our previous study, the first structural gene (GGTI) encoding g-glutamyl transpeptidase was cloned and characterized from the fission yeast Schizosaccharomyces pombe, and its transcription, using the GGTI-lacZ fusion gene, containing the 1,085 bp upstream region from the translational initiation point, was found to be enhanced by sodium nitroprusside and L-buthionine-(S,R)-sulfoximine (BSO). In the present work, regulation of the GGTI gene was further elucidated. Non-fermentable carbon sources, such as acetate and ethanol, markedly enhanced the synthesis of beta-galactosidase from the GGTI-lacZ fusion gene. However, its induction by non-fermentable carbon sources appeared to be independent of the presence of the Pap1 protein. Nitrogen starvation also gave rise to induction of GGTI gene expression in a Pap1-independent manner. The three additional fusion plasmids, carrying 754, 421 and 156 bp regions, were constructed. The sequence responsible for the induction by non-fermentable carbon sources and nitrogen starvation was identified to exist within a -421 bp region of the GGTI gene. Taken together, the S. pombe GGTI gene is regulated by non-fermentable carbon sources and nitrogen starvation.

Glutaredoxin (Grx), also known as thioltransferase (TTase), is an enzyme that catalyzes the reduction of a variety of disulfide compounds, including protein disulfides, in the presence of reduced glutathione. A second gene encoding Grx (Grx2) was cloned from the chromosomal DNA of the fission yeast Schizosaccharomyces pombe. The determined DNA sequence contains 1645 bp which is able to encode a polypeptide of 110 amino acids with a molecular mass of 12.2 kDa. The genomic DNA consists of 4 exons and 3 introns. The isolated gene was found to produce functional glutaredoxin that could accelerate the growth of the fission yeast, and is highly expressed at the mid- and late exponential phases. Aluminum, cadmium and hydrogen peroxide marginally enhanced the synthesis of beta-galactosidase from the Grx2-lacZ fusion gene. Shifts to lower concentrations (0.2, 0.4 or 0.8%) of D-glucose significantly enhanced the synthesis of beta-galactosidase from the Grx2-lacZ fusion gene. And shifts to sucrose (0.2, 0.4, 0.8 or 1.6%) as a sole carbon source markedly enhanced the synthesis of beta-galactosidase from the Grx2-lacZ fusion gene, the degree of which was inversely dependent on concentration. However, nonfermentable carbon sources reduced the expression of the Grx2 gene due to their growth arrest. The transcription factor Pap1 is not involved in the basal expression and induction of the Grx2 gene. The Grx2 protein was subcellularly localized in the nucleus of the yeast cells. Our results indicate that the Grx2 protein, located in the nucleus, is linked with the yeast growth, and that the gene is regulated by carbon sources.

To elucidate the physiological roles and regulation of a protein disulfide isomerase (PDI) from the fission yeast Schizosaccharomyces pombe, the full-length PDI gene was ligated into the shuttle vector pRS316, resulting in pPDI10. The determined DNA sequence carries 1,636 bp and encodes the putative 359 amino acid sequence of PDI with a molecular mass of 39,490 Da. In the amino acid sequence, the S. pombe PDI appears to be very homologous to A. thaliana PDI. The S. pombe cells harboring pPDI10 showed increased PDI activity and accelerated growth, suggesting that the cloned PDI gene is functioning and involved in the yeast growth. The 460 bp upstream region of the PDI gene was fused into promoterless beta-galactosidase gene of the shuttle vector YEp367R to generate pYUPDI10. The synthesis of beta-galactosidase from the PDI-lacZ fusion gene was enhanced by oxidative stress, such as superoxide anion and hydrogen peroxide. It was also induced by some non-fermentable and fermentable carbon sources. Nitrogen starvation was able to enhance the synthesis of beta-galactosidase from the PDI-lacZ fusion gene. The enhancement by oxidative stress and fermentable carbon sources did not depend on the presence of Pap1. The PDI mRNA levels were increased in both Pap1-positive and Pap1-negative cells treated with glycerol. Taken together, the S. pombe PDI gene is involved in cellular growth and response to nutritional and oxidative stress.