Heterologous modules for efficient and versatile PCR-based gene targeting in Schizosaccharomyces pombe

We describe a straightforward PCR-based approach to the deletion, tagging, and overexpression of genes in their normal chromosomal locations in the fission yeast Schizosaccharomyces pombe. Using this approach and the S. pombe ura4⁺ gene as a marker, nine genes were deleted with efficiencies of homologous integration ranging from 6 to 63%. We also constructed a series of plasmids containing the kanMX6 module, which allows selection of G418-resistant cells and thus provides a new heterologous marker for use in S. pombe. The modular nature of these constructs allows a small number of PCR primers to be used for a wide variety of gene manipulations, including deletion, overexpression (using the regulatable nmt1 promoter), C- or N-terminal protein tagging (with HA, Myc, GST, or GFP), and partial C- or N-terminal deletions with or without tagging. Nine genes were manipulated using these kanMX6 constructs as templates for PCR. The PCR primers included 60 to 80 bp of flanking sequences homologous to target sequences in the genome. Transformants were screened for homologous integration by PCR. In most cases, the efficiency of homologous integration was ≥50%, and the lowest efficiency encountered was 17%. The methodology and constructs described here should greatly facilitate analysis of gene function in S. pombe. © 1998 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.     

Construction of an insertion marker collection of Sz. japonicus (IMACS) for genetic mapping and a fosmid library covering its genome

Measuring relative genetic distances is one of the best ways to locate genetic loci. Here we report the construction of a strains set for genetic mapping in Schizosaccharomyces japonicus, which belongs to the genus Schizosaccharomyces together with the well‐studied fission yeast Sz. pombe. We constructed 29 strains that bear a positive‐negative selection marker at different loci. The marker was inserted every 500 kb in the genome of Sz. japonicus. Each marker thus becomes a ‘scale mark’ of a chromosome that behaves like a yardstick. By determining the genetic distances from the inserted markers, the relative location of a genomic mutation can be determined. We also constructed a fosmid library that covers an entire genome of Sz. japonicus. These tools together would facilitate identification and cloning of the gene. Copyright © 2012 John Wiley & Sons, Ltd.     

AILV1 gene from the yeast Arxula adeninivorans LS3 ­ a new selective transformation marker

The ILV1 gene of the yeast Arxula adeninivorans LS3 (AILV1) has been cloned from a genomic library, characterized and used as an auxotrophic selection marker for transformation of plasmids into this yeast. One copy of the gene is present in the Arxula genome, comprising 1653 bp and encoding 550 amino acids of the threonine deaminase. The protein sequence is similar (60·55%) to that of the threonine deaminase from Saccharomyces cerevisiae encoded by the gene ILV1. The protein is enzymatically active during the whole period of cultivation, up to 70 h. Maximal activities, as well as protein concentrations of this enzyme, were achieved after cultivation times of 20–36 h. The AILV1 gene is a suitable auxotrophic selection marker in transformation experiments using an Arxula adeninivorans ilv1 mutant and a plasmid containing this gene, which is fused into the 25S rDNA of Arxula adeninivorans. One to three copies of the linearized plasmid were integrated into the 25S rDNA by homologous recombination. Transformants resulting from complementation of the ilv1 mutation can be easily and reproducibly selected and in addition are mitotically stable. Therefore, the described system is preferred to the conventional selection for hygromycin B resistance. © 1998 John Wiley & Sons, Ltd.     

Development of episomal vectors carrying a nourseothricin‐resistance marker for use in minimal media for Schizosaccharomyces pombe

In the post‐genomic era, an immediate challenge is to assign biological functions to novel proteins encoded by the genome. This challenge requires the use of a simple organism as a genetic tool and a range of new high‐throughput techniques. Schizosacchromyces pombe is a powerful model organism used to investigate disease‐related genes and provides useful tools for the functional analysis of heterologous genes. To expand the current array of experimental tools, we constructed two series of Sz. pombe expression vectors, i.e. general and Gateway vectors, containing nourseothricin‐resistance markers. Vectors carrying nourseothricin‐resistance markers possess advantages in that they do not limit the parental strains with auxotrophic mutations with respect to availability for use in clone selection and can be used together with vectors carrying nutrient markers in minimal media. We modified the pSLF173, pSLF273 and pSLF373 vectors carrying a triple haemagglutinin epitope (3×HA) and an Ura4 marker. The vectors described here contain the nmt1 promoter with three different episomal expression strengths for proteins fused with 3×HA, EGFP or DsRed at the N‐terminus. These vectors represent an important contribution to the genome‐wide investigation of multiple heterologous genes and for functional and genetic analysis of novel human genes. Copyright © 2013 John Wiley & Sons, Ltd.     

New vectors for combinatorial deletions in yeast chromosomes and for gap-repair cloning using ‘split-marker’ recombination

New tools are needed for speedy and systematic study of the numerous genes revealed by the sequence of the yeast genome. We have developed a novel transformation strategy, based on ‘split-marker’ recombination, which allows generation of chromosomal deletions and direct gene cloning. For this purpose, pairs of yeast vectors have been constructed which offer a number of advantages for large-scale applications such as one-step cloning of target sequence homologs and combinatorial use. Gene deletions or gap-repair clonings are obtained by cotransformation of yeast by a pair of recombinant plasmids. Gap-repair vectors are based on the URA3 marker. Deletion vectors include the URA3, LYS2 and kanMX selection markers flanked by I-SceI sites, which allow their subsequent elimination from the transformant without the need for counter-selection. The application of the ‘split-marker’ vectors to the analysis of a few open reading frames of chromosome XI is described.     

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.     

Designed construction of recombinant DNA at the ura3Δ0 locus in the yeast Saccharomyces cerevisiae

Recombinant DNAs are traditionally constructed using Escherichia coli plasmids. In the yeast Saccharomyces cerevisiae, chromosomal gene targeting is a common technique, implying that the yeast homologous recombination system could be applied for recombinant DNA construction. In an attempt to use a S. cerevisiae chromosome for recombinant DNA construction, we selected the single ura3Δ0 locus as a gene targeting site. By selecting this single locus, repeated recombination using the surrounding URA3 sequences can be performed. The recombination system described here has several advantages over the conventional plasmid system, as it provides a method to confirm the selection of correct recombinants because transformation of the same locus replaces the pre‐existing selection marker, resulting in the loss of the marker in successful recombinations. In addition, the constructed strains can serve as both PCR templates and hosts for preparing subsequent recombinant strains. Using this method, several yeast strains that contained selection markers, promoters, terminators and target genes at the ura3Δ0 locus were successfully generated. The system described here can potentially be applied for the construction of any recombinant DNA without the requirement for manipulations in E. coli. Interestingly, we unexpectedly found that several G/C‐rich sequences used for fusion PCR lowered gene expression when located adjacent to the start codon. Copyright © 2013 John Wiley & Sons, Ltd.     

Induction of a heat shock-type response in fission yeast following nitrogen starvation

When cells of the fission yeast, Schizosaccharomyces pombe, are incubated in medium devoid of a nitrogen source, they accelerate into cell division and differentially synthesize two polypeptides at 46 and 27 kD (named p46 and p27) after a delay of about an hour. The synthesis of p46 and p27 is transient. These proteins have no obvious cell cycle connection since they are also evident in nitrogen-starved (but not accelerated) cells of the temperature-sensitive mutant of S. pombe, wee 1·50h^?. We infer from this that p46 and p27 are synthesized as a direct result of nutritional stress. The possibility that p46 and p27 represent examples of general environmental stress proteins was investigated by comparing nitrogen starvation with the heat-shock response in S. pombe. Heat-shock analysis of cells revealed the existence of two proteins of similar Mr to p46 and p27. In addition, nitrogen-starved cells acquired thermotolerance in a manner similar to heat-shocking cells. We suggest that nitrogen starvation in fission yeast induces a subset of the total array of heat-shock proteins.     

Novel episomal vectors and a highly efficient transformation procedure for the fission yeast Schizosaccharomyces japonicus

Schizosaccharomyces japonicus is a fission yeast for which new genetic tools have recently been developed. Here, we report novel plasmid vectors with high transformation efficiency and an electroporation method for Sz. japonicus. We isolated 44 replicating segments from 12 166 transformants of Sz. japonicus genomic fragments and found a chromosomal fragment, RS1, as a new replicating sequence that conferred high transformation activity to Sz. japonicus cells. This sequence was cloned into a pUC19 vector with ura4⁺ of Sz. pombe (pSJU11) or the kan gene on the kanMX6 module (pSJK11) as selection markers. These plasmids transformed Sz. japonicus cells in the early‐log phase by electroporation at a frequency of 123 cfu/µg for pSJK11 and 301 cfu/µg for pSJU11, which were higher than previously reported autonomously replicating sequences. Although a portion of plasmids remained in host cells by integration into the chromosome via RS1 segment, the plasmids could be recovered from transformants. The plasmid copy number was estimated to be 1.88 copies per cell by Southern blot analysis using a Sz. pombe ura4⁺ probe. The plasmid containing ade6⁺ suppressed the auxotrophic growth of the ade6‐domE mutant, indicating that the plasmid would be useful for suppressor screening and complementation assays in Sz. japonicus. Furthermore, pSJU11 transformed Sz. pombe cells with the same frequency as the pREP2 plasmid. This study is a report to demonstrate practical use of episomal plasmid vectors for genetic research in Sz. japonicus. RS1 has been submitted to the DDBJ/EMBL/GenBank database (Accession No. AB547343). Copyright © 2010 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.     

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.     

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.     

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.     

Multipurpose vectors designed for the fast generation of N- or C-terminal epitope-tagged proteins

In this paper are described a set of new high-copy-number yeast vectors, which are specially designed for the conditional expression of epitope-tagged proteins in vivo. One of the major advantages of these plasmids is that they allow polymerase chain reaction-amplified open reading frames to be automatically fused in frame with the epitope-coding sequence, avoiding longer procedures such as site-directed mutagenesis. This heterologous construction can be realized either at the 5′-end of the coding sequence, in the pYeF1 vector, or at its 3′-end, in pYeF2, generating N- or C-terminal tagged proteins, respectively. Moreover, to increase the usefulness of the method, derivatives of the two basic URA3-borne pYeF1 and pYeF2 were constructed, carrying either the HIS3 or TRP1 gene as a marker of selection. These vectors could be of use for the purpose of functional analysis of the newly discovered genes resulting from the systematic sequencing of the yeast genome. Here, we present results showing the functional expression and the efficient immunoprecipitation of the epitope-tagged Rna15 protein, which is involved in Saccharomyces cerevisiae mRNA stability.     

Yeasts have a four-fold variation in ribosomal DNA copy number

By employing pulsed-field gel electrophoresis we have determined the size of the rDNA cluster in wild-type yeast strains representing genera of Candida, Kluyveromyces, Pachysolen, Schizosaccharomyces and Torulaspora. Although the genome size of the examined species is similar (12·3–13·9 Mb), at least a four-fold variation has been observed between the lowest amount of rDNA repeats in P. tannophilus (28) and the highest in C. glabrata and S. poombe (> 115). In two species the rDNA cluster is represented by two loci, residing either in one (S. pombe) or two chromosomes (C. glabrata).     

Molecular,functional and evolutionary characterization of the gene encoding HMG-CoA reductase in the fission yeast,Schizosaccharomyces pombe

The synthesis of mevalonate, a molecule required for both sterol and isoprene biosynthesis in eukaryotes, is catalysed by 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase. Using a gene dosage approach, we have isolated the gene encoding HMG-CoA reductase, hmg1+, from the fission yeast Schizosaccharomyces pombe (Accession Number L76979). Specifically, hmg1+ was isolated on the basis of its ability to confer resistance to lovastatin, a competitive inhibitor of HMG-CoA reductase. Gene disruption analysis showed that hmg1+ was an essential gene. This result provided evidence that, unlike Saccharomyces cerevisiae, S. pombe contained only a single functional HMG-CoA reductase gene. The presence of a single HMG-CoA reductase gene was confirmed by genomic hybridization analysis. As observed for the S. cerevisiae HMG1p, the hmg1+ protein induced membrane proliferations known as karmellae. A previously undescribed ‘feed-forward’ regulation was observed in which elevated levels of HMG-CoA synthase, the enzyme catalysing the synthesis of the HMG-CoA reductase substrate, induced elevated levels of hmg1+ protein in the cell and conferred partial resistance to lovastatin. The amino acid sequences of yeast and human HMG-CoA reductase were highly divergent in the membrane domains, but were extensively conserved in the catalytic domains. We tested whether the gene duplication that produced the two functional genes in S. cerevisiae occurred before or after S. pombe and S. cerevisiae diverged by comparing the log likelihoods of trees specified by these hypotheses. We found that the tree specifying post-divergence duplication had significantly higher likelihood. Moreover, phylogenetic analyses of available HMG-CoA reductase sequences also suggested that the lineages of S. pombe and S. cerevisiae diverged approximately 420 million years ago but that the duplication event that produced two HMG-CoA reductase genes in the budding yeast occurred only approximately 56 million years ago. To date, S. pombe is the only unicellular eukaryote that has been found to contain a single HMG-CoA reductase gene. Consequently, S. pombe may provide important opportunities to study aspects of the regulation of sterol biosynthesis that have been difficult to address in other organisms and serve as a test organism to identify novel therapies for modulating cholesterol synthesis.     

A new method to efficiently induce a site-specific double-strand break in the fission yeast Schizosaccharomyces pombe

Double‐strand DNA breaks are a serious threat to cellular viability and yeast systems have proved invaluable in helping to understand how these potentially toxic lesions are sensed and repaired. An important method to study the processing of DNA breaks in the budding yeast Saccharomyces cerevisiae is to introduce a unique double‐strand break into the genome by regulating the expression of the site‐specific HO endonuclease with a galactose inducible promoter. Variations of the HO site‐specific DSB assay have been adapted to many organisms, but the methodology has seen only limited use in the fission yeast Schizosaccharomyces pombe because of the lack of a promoter capable of inducing endonuclease expression on a relatively short time scale (~1 h). We have overcome this limitation by developing a new assay in which expression of the homing endonuclease I‐PpoI is tightly regulated with a tetracycline‐inducible promoter. We show that induction of the I‐PpoI endonuclease produces rapid cutting of a defined cleavage site (> 80% after 1 h), efficient cell cycle arrest and significant accumulation of the checkpoint protein Crb2 at break‐adjacent regions in a manner that is analogous to published findings with DSBs produced by an acute exposure to ionizing irradiation. This assay provides an important new tool for the fission yeast community and, because many aspects of mammalian chromatin organization have been well‐conserved in Sz. pombe but not in S. cerevisiae, also offers an attractive system to decipher the role of chromatin structure in modulating the repair of double‐stranded DNA breaks. Copyright © 2012 John Wiley & Sons, Ltd.     

Sticky-end polymerase chain reaction method for systematic gene disruption in Saccharomyces cerevisiae

We describe a new procedure for the generation of plasmids containing a large promoter and terminator region of a gene of interest, useful for gene disruption. In a two-step polymerase chain reaction (PCR), a fragment, corresponding to the terminator and promoter regions separated by a 16 bp sequence containing a rare restriction site (e.g. AscI), is synthesized (T-P fragment). This PCR fragment is cloned in vectors presenting a rare blunt-end cloning site and a yeast marker for selection in Saccharomyces cerevisiae (TRP1, HIS3 and KanMX). The final plasmids are used directly for gene disruption after linearization by the enzyme (e.g. AscI) specific for the rare restriction site. This approach was used to disrupt three open reading frames identified during the sequencing of COS14–1 from chromosome XIV of S. cerevisiae.     

The 50:50 method for PCR‐based seamless genome editing in yeast

The ability to edit the yeast genome with relative ease has contributed to the organism being a model eukaryote for decades. Most methods for deleting, inserting or altering genomic sequences require transformation with DNA that carries the desired change and a selectable marker. One‐step genome editing methods retain the selectable marker. Seamless genome editing methods require more steps and a marker that can be used for both positive and negative selection, such as URA3. Here we describe the PCR‐based 50:50 method for seamless genome editing, which requires only two primers, one PCR with a URA3 cassette, and a single yeast transformation. Our method is based on pop‐in/pop‐out gene replacement and is amenable to the facile creation of genomic deletions and short insertions or substitutions. We used the 50:50 method to make two conservative loss‐of‐function mutations in MATα1, with results suggesting that the wild‐type gene has a new function outside of that presently known. Copyright © 2013 John Wiley & Sons, Ltd.