Protection against cadmium toxicity in yeast by alcohol dehydrogenase.

A cDNA expression library from Schizosaccharomyces pombe was transformed into Saccharomyces cerevisiae to screen for genes capable of conferring cadmium resistance to S. cerevisiae cells. The cDNA library was cloned into the S. cerevisiae expression vector pDB20 which is designed to express cDNAs via the constitutively-expressed promoter of the gene for alcohol dehydrogenase I (ADH1). Terminator and polyadenylation signals are also provided by the ADH1 gene. Cadmium resistant colonies were shown to arise by a recombination event leading to the exchange of the S. pombe DNA with the chromosomal ADH1 gene and a consequent dramatic increase in the ADH1 gene expression due to the high copy number of the plasmid. The overexpression of ADH1 effectively buffered the cells for cadmium ions by formation of Cd-ADH.     

Versatile use of Schizosaccharomyces pombe plasmids in Saccharomyces cerevisiae

The two model yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe appear to have diverged 1000 million years ago. Here, we describe that S.?pombe vectors can be propagated efficiently in S.?cerevisiae as pUR19 derivatives, and the pREP and pJR vector series carrying the S.?cerevisiae LEU2 or the S.?pombe ura4(+) selection marker are maintained in S.?cerevisiae cells. In addition, genes transcribed from the S.?pombe nmt1(+) promoter and derivatives are expressed in budding yeast. Thus, S.?pombe vectors can be used as shuttle vectors in S.?cerevisiae and S.?pombe. Our finding greatly facilitates the testing for functional orthologs of protein families and simplifies the cloning of new S.?pombe plasmids by using the highly efficient in vivo homologous recombination activity of S.?cerevisiae.     

A set of loxP marker cassettes for Cre-mediated multiple gene disruption in Schizosaccharomyces pombe

For functional analysis, the presence of gene families and isoenzymes often makes it necessary to delete more than one gene, while the number of marker genes is limited in Schizosaccharomyces pombe. Here we describe a loxP-flanked ura4(+) cassette and Cre recombinase vector for a Cre-loxP-mediated marker removal procedure in S. pombe. This loxP-ura4-loxP cassette can be used for disruption of hmt1(+) as a model target gene. We have constructed two vectors which express Cre recombinase under the control of the nmt1 or nmt41 promoter. Excisive recombination at loxP sites in the chromosome was promoted efficiently and accurately when the Cre recombinase was expressed under the control of the nmt41 promoter. In addition, ura4(+) could be excised from the genome by Cre recombinase, when a single loxP site was adjacent to ura4. The use of the Cre-loxP system proved to be a practical strategy to excise a marker gene for repeated use in S. pombe.     

An inducible expression vector for both fission and budding yeast

We have developed a vector system for inducible gene expression in both fission yeast (Schizosaccharomyces pombe) and budding yeast (Saccharomyces cerevisiae). The autonomously replicating expression vector contains multiple glucocorticoid response elements, rendering a linked promoter inducible 20-70-fold by glucocorticoid hormones in the presence of the mammalian glucocorticoid receptor. A polylinker with several unique cloning sites allows insertion of cDNAs of interest. Glucocorticoids are gratuitous signalling molecules in yeast, exerting little or no effect on the expression of genes other than those fused to the regulated promoter.     

A vector system for genomic FLAG epitope-tagging in Schizosaccharomyces pombe

The fission yeast Schizosaccharomyces pombe is a popular model organism to study various cellular processes, although research tools available for S. pombe are relatively inadequate. To facilitate genetic and biochemical investigation in S. pombe, we report here a system of vectors for genomic FLAG epitope-tagging. These vectors enable us to amplify gene-targeting fragments for integration into specific loci of the S. pombe genome. All vectors in this report were designed to express FLAG epitope-tagged proteins from their endogenous genomic loci. Vectors for N-terminal FLAG epitope-tagging allow us to control protein expression levels using the wild-type nmt1 promoter, its weaker derivatives, and the urg1 promoter. These vectors are available with various antibiotic markers including kanMX6, hphMX6, natMX6 and bleMX6, and the his3(+) marker. Vectors for C-terminal FLAG epitope-tagging were designed to express FLAG-fusion proteins under the control of their native promoters at their own genomic loci, allowing us to characterize protein functions under physiological conditions. These vectors are available with kanMX6, hphMX6, nat-MX6 and bleMX6 markers. The series of vectors described in this report should prove useful for protein studies in fission yeast.     

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

The structural gene for the putative gamma-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 beta-galactosidase gene of the shuttle vector YEp367R to generate the fusion plasmid pGT98. The synthesis of beta-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.     

Effect of human immunodeficiency virus type 1 protein R (vpr) gene expression on basic cellular function of fission yeast Schizosaccharomyces pombe.

The human immunodeficiency virus type 1 (HIV-1) Vpr protein affects cell morphology and prevents proliferation of human cells by induction of cell cycle G2 arrest. In this study, we used the fission yeast Schizosaccharomyces pombe as a model system to investigate the cellular effects of HIV-1 vpr gene expression. The vpr gene was cloned into an inducible fission yeast gene expression vector and expressed in wild-type S. pombe cells, and using these cells, we were able to demonstrate the specific Vpr-induced effects by induction and suppression of vpr gene expression. Induction of HIV-1 vpr gene expression affected S. pombe at the colonial, cellular, and molecular levels. Specifically, Vpr induced small-colony formation, polymorphic cells, growth delay, and cell cycle G2 arrest. Additionally, Vpr-induced G2 arrest appeared to be independent of cell size and morphological changes. The cell cycle G2 arrest correlated with increased phosphorylation of p34cdc2, suggesting negative regulation of mitosis by HIV-1 Vpr. Treatment of Vpr-induced cell with a protein phosphatase inhibitor, okadaic acid, transiently suppressed cell cycle arrest and morphological changes. This observation implicates possible involvement of protein phosphatase(s) in the effects of Vpr. Together, these data showed that the HIV-1 Vpr-induced cellular changes in S. pombe are similar to those observed in human cells. Therefore, the S. pombe system is suited for further investigation of the HIV-1 vpr gene functions.     

Heat shock-inducible expression vectors for use in Schizosaccharomyces pombe

A new, heat shock-inducible expression system based on an endogenous hsp16+ promoter was developed for use in the fission yeast Schizosaccharomyces pombe. Analysis of GFP expression profiles indicated that a 1.2-kb segment of the hsp16+ promoter region was sufficient to drive expression of heterologous protein. The hsp16+ promoter was found to be activated not only by heat shock but also by other stresses including cadmium, ethanol, and oxidative stress. Two expression vectors, pHIL and pHIU, were constructed using the 1.2-kb hsp16+ promoter for inducible gene expression in Sch. pombe. This new expression system utilizes a simple induction protocol and promises to be a useful tool for analyzing gene expression in Sch. pombe.     

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

The first gene encoding gamma-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.