An estradiol‐inducible promoter enables fast,graduated control of gene expression in fission yeast

The fission yeast Schizosaccharomyces pombe lacks a diverse toolkit of inducible promoters for experimental manipulation. Available inducible promoters suffer from slow induction kinetics, limited control of expression levels and/or a requirement for defined growth medium. In particular, no S. pombe inducible promoter systems exhibit a linear dose–response, which would allow expression to be tuned to specific levels. We have adapted a fast, orthogonal promoter system with a large dynamic range and a linear dose response, based on β‐estradiol‐regulated function of the human oestrogen receptor, for use in S. pombe. We show that this promoter system, termed Z₃EV, turns on quickly, can reach a maximal induction of 20‐fold, and exhibits a linear dose response over its entire induction range, with few off‐target effects. We demonstrate the utility of this system by regulating the mitotic inhibitor Wee1 to create a strain in which cell size is regulated by β‐estradiol concentration. This promoter system will be of great utility for experimentally regulating gene expression in fission yeast. Copyright © 2017 John Wiley & Sons, Ltd.     

A homologous cell-free system for studying protein translocation across the endoplasmic reticulum membrane in fission yeast

We report the development of a homologous in vitro assay system for analysing translocation of proteins across the endoplasmic reticulum (ER) membrane of the fission yeast Schizosaccharomyces pombe. Our protocol for preparing an S. pombe extract capable of translating natural messenger RNAs was modified from a procedure previously used for Saccharomyces cerevisiae, in which cells are lysed in a bead-beater. However, we were unable to prepare fission yeast microsomes active in protein translocation using existing budding yeast protocols. Instead, our most efficient preparations were isolated by fractionating spheroplasts, followed by extensive washing and size exclusion chromatography of the crude membranes. Translocation of two ER-targeted proteins, pre-acid phosphatase from S. pombe and prepro-α-factor from S. cerevisiae, was monitored using two distinct assays. First, evidence that a fraction of both proteins was sequestered within membrane-enclosed vesicles was provided by resistance to exogenously added protease. Second, the protected fraction of each protein was converted to a higher molecular weight, glycosylated form; attachment of carbohydrate to the translocated proteins was confirmed by their ability to bind Concanavalin A–Sepharose. Finally, we examined whether proteins could be translocated across fission yeast microsomal membranes after their synthesis was complete. Our results indicate that S. cerevisiae prepro-α-factor can be post-translationally imported into the fission yeast ER, while S. pombe pre-acid phosphatase crosses the membrane only by a co-translational mechanism.     

New vectors for simple and streamlined CRISPR–Cas9 genome editing in Saccharomyces cerevisiae

Clustered regularly interspaced short palindromic repeats (CRISPR)–Cas9 technology is an important tool for genome editing because the Cas9 endonuclease can induce targeted DNA double‐strand breaks. Targeting of the DNA break is typically controlled by a single‐guide RNA (sgRNA), a chimeric RNA containing a structural segment important for Cas9 binding and a 20mer guide sequence that hybridizes to the genomic DNA target. Previous studies have demonstrated that CRISPR–Cas9 technology can be used for efficient, marker‐free genome editing in Saccharomyces cerevisiae. However, introducing the 20mer guide sequence into yeast sgRNA expression vectors often requires cloning procedures that are complex, time‐consuming and/or expensive. To simplify this process, we have developed a new sgRNA expression cassette with internal restriction enzyme sites that permit rapid, directional cloning of 20mer guide sequences. Here we describe a flexible set of vectors based on this design for cloning and expressing sgRNAs (and Cas9) in yeast using different selectable markers. We anticipate that the Cas9–sgRNA expression vector with the URA3 selectable marker (pML104) will be particularly useful for genome editing in yeast, since the Cas9 machinery can be easily removed by counter‐selection using 5‐fluoro‐orotic acid (5‐FOA) following successful genome editing. The availability of new vectors that simplify and streamline the technical steps required for guide sequence cloning should help accelerate the use of CRISPR–Cas9 technology in yeast genome editing. Vectors pT040, pJH001, pML104 and pML107 have been deposited at Addgene ( www.addgene.org ). Copyright © 2015 John Wiley & Sons, Ltd.     

An IPTG‐inducible derivative of the fission yeast nmt promoter

We here describe an IPTG‐inducible system that reveals that the lac repressor alone can function as a potent transmodulator to regulate gene expression in the fission yeast, Schizosaccharomyces pombe. This expression system is a derivative of the Sz. pombe nmt promoter, which normally is strongly repressed by thiamine. With appropriate positioning of a lac operator site (lacO) downstream of the TATA‐box, we show that gene expression from a chimeric nmt::lacO promoter can be regulated by the lac repressor up to two orders of magnitude in response to IPTG. The chimeric nmt::lacO promoter is rapidly induced and when GFP is used as a reporter; almost full induction is achieved 40 min after the addition of IPTG. Like the wild‐type nmt promoter, the chimeric nmt::lacO is repressed by thiamine. This allows expression in a short and defined window, e.g. the S‐phase of a synchronized cell population, by first adding IPTG to turn on expression, followed by addition of thiamine to switch off expression. Copyright © 2015 John Wiley & Sons, Ltd.     

Measuring oxidative DNA damage and DNA repair using the yeast comet assay

Chromosomal DNA damage can be a result of several processes and agents of endogenous or exogenous origin. These cause strand breaks or oxidized bases that lead to strand breaks, which relax the normally supercoiled genomic DNA and increase its electrophoretic mobility. The extent of DNA damage can be assessed by single cell gel electrophoresis, where the chromosomal DNA migration distance correlates with the extent of DNA damage. This technique has been used for a variety of applications with several organisms, but only a few studies have been reported for Saccharomyces cerevisiae. A possible reason for this absence is that low cellular DNA content could hamper visualization. Here we report an optimization of the comet assay protocol for yeast cells that is robust and sensitive enough to reproducibly detect background DNA damage and oxidative damage caused by hydrogen peroxide. DNA repair was observed and quantified as diminishing comet tail length with time after oxidative stress removal in a process well described by first‐order kinetics with a tail length half‐life of 11 min at 37 °C. This is, to our knowledge, the first quantitative measurement of DNA repair kinetics in S. cerevisiae by this method. We also show that diet antioxidants protect from DNA damage, as shown by a three‐fold decrease in comet tail length. The possibility of assessment of DNA damage and repair in individual cells applied to the model organism S. cerevisiae creates new perspectives for studying genotoxicity and DNA repair. Copyright © 2010 John Wiley & Sons, Ltd.     

The essential function of Rrs1 in ribosome biogenesis is conserved in budding and fission yeasts

The Rrs1 protein plays an essential role in the biogenesis of 60S ribosomal subunits in budding yeast (Saccharomyces cerevisiae). Here, we examined whether the fission yeast (Schizosaccharomyces pombe) homologue of Rrs1 also plays a role in ribosome biogenesis. To this end, we constructed two temperature‐sensitive fission yeast strains, rrs1‐D14/22G and rrs1‐L51P, which had amino acid substitutions corresponding to those of the previously characterized budding yeast rrs1‐84 (D22/30G) and rrs1‐124 (L61P) strains, respectively. The fission yeast mutants exhibited severe defects in growth and 60S ribosomal subunit biogenesis at high temperatures. In addition, expression of the Rrs1 protein of fission yeast suppressed the growth defects of the budding yeast rrs1 mutants at high temperatures. Yeast two‐hybrid analyses revealed that the interactions of Rrs1 with the Rfp2 and Ebp2 proteins were conserved in budding and fission yeasts. These results suggest that the essential function of Rrs1 in ribosome biogenesis may be conserved in budding and fission yeasts. Copyright © 2015 John Wiley & Sons, Ltd.     

Saccharomyces cerevisiae Mus81–Mms4 and Rad52 can cooperate in the resolution of recombination intermediates

Mus81 is a well‐conserved DNA structure‐specific endonuclease which belongs to the XPF/Rad1 family of proteins that are involved in DNA nucleotide excision repair. Mus81 forms a heterodimer with a non‐catalytic subunit, Mms4, in Saccharomyces cerevisiae (Eme1/EME1 in Schizosaccharomyces pombe and mammals). Recent evidence shows that Mus81 functions redundantly with Sgs1, a member of the ubiquitous RecQ family of DNA helicases, to process toxic recombinant intermediates. In budding yeast, homologous recombination is regulated by the Rad52 epistasis group of proteins, including Rad52, which stimulates the main steps of DNA sequence‐homology searching. Mus81 was proven to act in the Rad52‐dependent pathway. Here, we demonstrate that Rad52 and Mus81–Mms4 possesses a functional interaction; the presence of Rad52 significantly enhances the endonuclease activity of Mus81–Mms4 on a broad range of its preferred synthetic substrates. Furthermore, this functional interaction is demonstrated to be species specific. We fragmented Rad52 and found that the N‐terminal fragment from the 86th to 169th amino acid residue, which belongs to DNA‐binding and self‐association domains, can stimulate Mus81–Mms4 endonuclease. These results strongly support the notion that Rad52 and Mus81–Mms4 collaborate and work jointly in processing of homologous recombination intermediates.     

MPR1 as a novel selection marker in Saccharomyces cerevisiae

L ‐Azetidine‐2‐carboxylic acid (AZC) is a toxic four‐membered ring analogue of L ‐proline that is transported into cells by proline transporters. AZC and L ‐proline in the cells are competitively incorporated into nascent proteins. When AZC is present in a minimum medium, misfolded proteins are synthesized in the cells, thereby inhibiting cell growth. The MPR1 gene has been isolated from the budding yeast Saccharomyces cerevisiae Σ1278b as a multicopy suppressor of AZC‐induced growth inhibition. MPR1 encodes a novel acetyltransferase that detoxifies AZC via N‐acetylation. Since MPR1 is absent in the laboratory strain of S. cerevisiae S288C, it could be a positive selection marker that confers AZC resistance in the S288C background strains. To examine the usefulness of MPR1, we constructed some plasmid vectors that harboured MPR1 under the control of various promoters and introduced them into the S288C‐derived strains. The expression of MPR1 conferred AZC resistance that was largely dependent on the expression level of MPR1. In an additional experiment, the galactose‐inducible MPR1 and ppr1⁺, the fission yeast Schizosaccharomyces pombe homologue of MPR1, were used for gene disruption by homologous recombination, and here AZC‐resistant colonies were also successfully selected. We concluded that our MPR1–AZC system provides a powerful tool for yeast transformation. Copyright © 2009 John Wiley & Sons, Ltd.     

Effects of phleomycin-induced DNA damage on the fission yeast Schizosaccharomyces pombe cell cycle

The effect of phleomycin, a bleomycin-like antibiotic, has been investigated in the fission yeast, Schizosaccharomyces pombe. We report that in response to phleomycin-induced DNA damage, growth was inhibited and S. pombe cells arrested in the G2-phase of the cell cycle. DNA repair mutants rad9 and rad17 did not arrest and were hypersensitive to phleomycin. Cell cycle mutants that entered mitosis without monitoring the completion of DNA replication also displayed an increased sensitivity to this DNA-damaging agent. Thus, phleomycin could be used as a tool in the fission yeast S. pombe model system for the study of DNA damage and cell cycle checkpoints, or as a new selective agent.     

Plasmids with E2 epitope tags: tagging modules for N‐ and C‐terminal PCR‐based gene targeting in both budding and fission yeast,and inducible expression vectors for fission yeast

A single‐step PCR‐based epitope tagging enables fast and efficient gene targeting with various epitope tags. This report presents a series of plasmids for the E2 epitope tagging of proteins in Saccharomyces cerevisiae and Schizosaccharomyces pombe. E2Tags are 10‐amino acids (epitope E2a: SSTSSDFRDR)‐ and 12 amino acids (epitope E2b: GVSSTSSDFRDR)‐long peptides derived from the E2 protein of bovine papillomavirus type 1. The modules for C‐terminal tagging with E2a and E2b epitopes were constructed by the modification of the pYM‐series plasmid. The N‐terminal E2a and E2b tagging modules were based on pOM‐series plasmid. The pOM‐series plasmids were selected for this study because of their use of the Cre–loxP recombination system. The latter enables a marker cassette to be removed after integration into the loci of interest and, thereafter, the tagged protein is expressed under its endogenous promoter. Specifically for fission yeast, high copy pREP plasmids containing the E2a epitope tag as an N‐terminal or C‐terminal tag were constructed. The properties of E2a and E2b epitopes and the sensitivity of two anti‐E2 monoclonal antibodies (5E11 and 3F12) were tested using several S. cerevisiae and Sz. pombe E2‐tagged strains. Copyright © 2009 John Wiley & Sons, Ltd.     

A multiplex culture system for the long‐term growth of fission yeast cells

Maintenance of long‐term cultures of yeast cells is central to a broad range of investigations, from metabolic studies to laboratory evolution assays. However, repeated dilutions of batch cultures lead to variations in medium composition, with implications for cell physiology. In Saccharomyces cerevisiae, powerful miniaturized chemostat setups, or ministat arrays, have been shown to allow for constant dilution of multiple independent cultures. Here we set out to adapt these arrays for continuous culture of a morphologically and physiologically distinct yeast, the fission yeast Schizosaccharomyces pombe, with the goal of maintaining constant population density over time. First, we demonstrated that the original ministats are incompatible with growing fission yeast for more than a few generations, prompting us to modify different aspects of the system design. Next, we identified critical parameters for sustaining unbiased vegetative growth in these conditions. This requires deletion of the gsf2 flocculin‐encoding gene, along with addition of galactose to the medium and lowering of the culture temperature. Importantly, we improved the flexibility of the ministats by developing a piezo‐pump module for the independent regulation of the dilution rate of each culture. This made it possible to easily grow strains that have different generation times in the same assay. Our system therefore allows for maintaining multiple fission yeast cultures in exponential growth, adapting the dilution of each culture over time to keep constant population density for hundreds of generations. These multiplex culture systems open the door to a new range of long‐term experiments using this model organism. © 2017 The Authors. Yeast published by John Wiley & Sons, Ltd.     

Spatial organization of the Schizosaccharomyces pombe genome within the nucleus

The fission yeast Schizosaccharomyces pombe is a useful experimental system for studying the organization of chromosomes within the cell nucleus. S. pombe has a small genome that is organized into three chromosomes. The small size of the genome and the small number of chromosomes are advantageous for cytological and genome‐wide studies of chromosomes; however, the small size of the nucleus impedes microscopic observations owing to limits in spatial resolution during imaging. Recent advances in microscopy, such as super‐resolution microscopy, have greatly expanded the use of S. pombe as a model organism in a wide range of studies. In addition, biochemical studies, such as chromatin immunoprecipitation and chromosome conformation capture, have provided complementary approaches. Here, we review the spatial organization of the S. pombe genome as determined by a combination of cytological and biochemical studies. Copyright © 2016 John Wiley & Sons, Ltd.     

Oxylipin Associated Co‐Flocculation in Yeasts

According to the lectin‐theory, the yeast Schizosaccharomyces pombe lacks the specific receptors (α‐mannans) necessary to facilitate co‐flocculation with Saccharomyces cerevisiae species. In this study we demonstrate oxylipin associated co‐flocculation between Sacch. cerevisiae and S. pombe strains using differential cell staining, immunofluoresence and ultrastructural studies. Using a 3‐hydroxy (OH) oxylipin specific antibody coupled to a fluorescing compound, 3‐OH oxylipins were found to be present on the cell surfaces of Sacch. cerevisiae and S. pombe. The presence of 3‐OH oxylipins was confirmed using gas chromatography‐mass spectrometry. Strikingly, when acetylsalicylic acid (aspirin), a 3‐OH oxylipins inhibitor, was added to Sacch. cerevisiae which was then mixed with S. pombe strains grown in complex media, co‐flocculation was significantly inhibited. We conclude that aspirin‐sensitive 3‐OH 8:0 is probably involved in co‐flocculation.     

Vacuolar carboxypeptidase Y of Saccharomyces cerevisiae is glycosylated,sorted and matured in the fission yeast Schizosaccharomyces pombe

Vacuolar carboxypeptidase Y of Saccharomyces cerevisiae (CPY^sc) has been expressed in a Schizosaccharomyces pombe strain devoid of the endogenous equivalent peptidase, employing a 2 μ derived plasmid. Immunoblot analysis revealed that CPY^sc produced in the fission yeast has a higher molecular mass than mature CPY^sc produced by the budding yeast. CPY^sc is glycosylated when expressed in S. pombe and uses four N-linked glycosylation sites as shown by endoglycosidase H digestion. Carbohydrate removal leads to a protein moiety which is indistinguishable in size from deglycosylated CPY^sc produced by S. cerevisiae. CPY^sc isolated from S. pombe soluble extracts is enzymatically active and thus is presumed to undergo correct proteolytic maturation. Subcellular fractionation experiments showed a cofractionation of CPY^sc with the S. pombe endoproteinases PrA and PrB, suggesting that the protein is correctly sorted to the vacuole and that these peptidases might be responsible for zymogen activation.     

A new inducible CRISPR-Cas9 system useful for genome editing and study of double-strand break repair in Candida glabrata

In recent years, the CRISPR-Cas9 system has proven extremely useful for genome editing in many species, including the model yeast Saccharomyces cerevisiae and other yeast species such as Candida glabrata. Inducible CRISPR-Cas9 systems have the additional advantage of allowing to separate the transformation step of the organism by the CRISPR-Cas9 system, from the cutting and repair steps. This has indeed been developed in S. cerevisiae, where most inducible expression systems rely on the GAL promoters. Unfortunately, C. glabrata is gal⁻ and lacks the GAL genes, like many other yeast species. We report here the use of a vector expressing cas9 under the control of the MET3 promoter, with the guide RNA cloned into the same plasmid. We show that it can be used efficiently in C. glabrata, for both described outcomes of CRISPR-Cas9-induced chromosome breaks; nonhomologous end joining in the absence of a homologous repair template; and homologous recombination in the presence of such a template. This system therefore allows easy editing of the genome of C. glabrata, and its inducibility may allow identification of essential genes in this asexual yeast, where spore lethality cannot be observed, as well as the study of double-strand break repair.     

Discovery of genes involved in mitosis,cell division,cell wall integrity and chromosome segregation through construction of Schizosaccharomyces pombe deletion strains

The fission yeast model system Schizosaccharomyces pombe is used to study fundamental biological processes. To continue to fill gaps in the Sz. pombe gene deletion collection, we constructed a set of 90 haploid gene deletion strains covering many previously uncharacterized genes. To begin to understand the function of these genes, we exposed this collection of strains to a battery of stress conditions. Using this information in combination with microscopy, proteomics and mini‐chromosome loss assays, we identified genes involved in cell wall integrity, cytokinesis, chromosome segregation and DNA metabolism. This subset of non‐essential gene deletions will add to the toolkits available for the study of biological processes in Sz. pombe. Copyright © 2016 John Wiley & Sons, Ltd.