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.     

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.     

General purpose tagging vectors for fission yeast

We have designed a series of vectors for use in the fission yeast Schizosaccharomyces pombe that allow fusion of any protein of interest to a triple HA epitope or a GST domain. The HA epitope may be placed at the N terminus or the C terminus under three different versions of the nmt1 promoter, to allow varying levels of gene expression. The GST tag may be placed at the N terminus or C terminus under control of a fully active nmt1 promoter. This family of vectors has compatible restriction sites and modular design, so that the protein under study may be exchanged easily between different plasmids. Using the Cdc19p protein as a test case, we have demonstrated that these plasmids can express functional tagged proteins in the fission yeast cell.     

酵母作为外源基因表达系统的研究进展

甲醇营养型酵母和裂殖酵母作为外源基因表达的有效系统,正引起人们广泛研究。甲醇营养型酵母具有易诱导调控、适用于高密生长、能高效表达外源蛋白的特点。裂殖酵母有许多与高等真核细胞相似的特点,是研究真核分子生物学和真核基因表达有用的工具。本文综述了这两个酵母表达系统的特点。     

A novel copper-regulated promoter system for expression of heterologous proteins in Schizosaccharomyces pombe

The increasing use of the fission yeast Schizosaccharomyces pombe as a model organism for elucidating the mechanisms of critical biological processes such as cell-cycle control, DNA replication, and stress-mediated signal transduction has fostered the development and utilization of expression systems for gene function analysis. Using the promoter of the ctr4(+) copper transporter gene from S. pombe, we created a series of vectors, named pctr4(+)-X, which regulate the expression of heterologous genes as a function of copper availability. In this system, the addition of copper ions at levels that are non-toxic to yeast cells represses gene expression, while copper deprivation strongly induces gene expression. Conveniently, changes of growth medium or carbon sources are not required to shut down or induce gene expression. The Cu-starvation-mediated inducible expression system is rapid, producing heterologous proteins within 3 h, with sustained expression of proteins that persists for several hours. The pctr4(+)-X expression vectors harbor unique restriction sites constructed in-frame to DNA sequences encoding for epitope tags, which facilitate the detection or purification of the heterologous proteins using commercially available antibodies and affinity columns. Furthermore, the pctr4(+)-X copper-regulatable protein expression vectors have been constructed with three different selectable markers, offering more versatility for studying gene function in fission yeast.     

A yeast model for the study of human DFNA5, a gene mutated in nonsyndromic hearing impairment

A mutation in human DFNA5 is associated with autosomal dominant nonsyndromic hearing impairment. The function of DFNA5 protein remains unknown and no experimental model has been described so far. Here we describe fission yeast Schizosaccharomyces pombe as a model organism for studying the function of heterologously expressed DFNA5. We have expressed wild-type as well as mutant DFNA5 alleles under control of regulatable nmt1 promoter. Yeast cells tolerated expression of wild-type DFNA5, while expression of the mutant DFNA5 allele, which is responsible for nonsyndromic autosomal dominant hearing impairment, led to cell cycle arrest. We identified new rat and horse DFNA5 homologues and we describe a domain of homology shared between DFNA5 and the Mcm10 family of DNA replication proteins. Genetic interactions between heterologously expressed DFNA5 and a fission yeast cdc23 (mcm10) mutant support a possible link between DFNA5 and Mcm10 proteins.     

Transcriptional regulation of glutathione synthetase in the fission yeast Schizosaccharomyces pombe

Glutathione (GSH), an important antioxidant involved in the stress response, is synthesized in two sequential reactions involving glutamylcysteine synthetase (GCS), followed by glutathione synthetase (GS). Expression of the unique GS gene in the fission yeast Schizosaccharomyces pombe was previously found to be regulated by nitric oxide and by L-buthionine-(S,R)-sulfoximine (BSO), a specific inhibitor of GCS. In this work, expression of S. pombe GS gene is shown to be induced by menadione (MD), which generates superoxide. The responsible DNA sequence between -365 and -234 bp from the translation start site, was convinced using five GS-lacZ fusion plasmids. Expression of GS gene is also induced by low glucose, fructose and disaccharides, apparently dependent on Pap1 protein; GS mRNA increases in low concentrations of glucose in wild type S. pombe but not in Pap1-negative cells. Although nonfermentable carbon sources such as acetate and ethanol stimulate expression of GS gene, they also arrest the growth of the yeast cells. These results indicate that the biosynthesis of glutathione is regulated by superoxide radicals and carbon source limitation.     

Expression of human cytochrome p450 3A4 gene in Schizosaccharomyces pombe

Heterologous expression systems can be utilized to great advantage in the study of cytochrome P450 enzymes. P450 3A4 is one of the major forms of cytochrome P450 found in liver. It is also involved in the metabolism of numerous widely used drugs and xenobiotics. In the present study human liver cytochrome P450 3A4 gene was transferred into the fission yeast Schizosaccharomyces pombe via two different S. pombe expression vectors carrying thiamine repressible promoter — nmt1 (pREP42) and constitutive promoter — adh1 (pART1). Heterologously expressed cytochrome P450 3A4 was detected in the cells grown in minimal (EMM) or rich medium (YEL) containing 0.5% (w/v) glucose. A typical cytochrome P450 peak for 3A4 was observed at 448 nm in microsomal fraction. The presence of heterologous expression of 3A4 form was also determined by SDS-PAGE and it molecular mass was identified as 52 kDa. The enzyme activity was confirmed by HPLC analysis, using testosterone as substrate.     

Choosing and using Schizosaccharomyces pombe plasmids

A wide range of plasmids has been developed for molecular studies in the fission yeast Schizosaccharomyces pombe. This includes general purpose episomes, expression vectors, epitope tagging plasmids, and integration vectors. This review describes the typical features of S. pombe vectors, including replication origins, positive and negative selection markers, and constitutive and inducible promoter systems. We will also discuss vectors with epitope tags and how these can be used to modify episomal or endogenous gene sequences. Considerations for choosing and using a plasmid are presented and specialized methods are described.     

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.     

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.