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.     

André Goffeau's imprinting on second generation yeast “genomologists”

All authors of the present paper have worked in labs that participated to the sequencing effort of the Saccharomyces cerevisiae reference genome, and we owe to this the fact that we have all chosen to work on genomics of yeasts. S. cerevisiae has been a popular model species for genetics since the 20th century as well as being a model for general eukaryotic cellular processes. Although it has also been used empirically in fermentation for millennia, there was until recently, a lack of knowledge about the natural and evolutionary history of this yeast. The achievement of the international effort to sequence its genome was the foundation for understanding many eukaryotic biological processes but also represented the first step towards the study of the genome and ecological diversity of yeast populations worldwide. We will describe recent advances in yeast comparative and population genomics that find their origins in the S. cerevisiae genome project initiated and pursued by André Goffeau.     

A series of yeast shuttle vectors for expression of cDNAs and other DNA sequences

Expression/shuttle vectors for the yeast Saccharomyces cerevisiae have usually been large plasmids with only one or a small number of sites that are suitable for cloning and expression. We report here the construction and properties of a series of 12 expression vectors with multiple (four to eight) unique sites in their polylinkers which allow directional cloning and expression of DNA sequences under four different promoters. Eleven of these plasmids replicate at high copy number in Escherichia coli, and all have the yeast TRP1 gene, and the 2 μm origin including REP3 sequence, allowing selection and high copy number replication in yeast. Six of the plasmids are designed for the construction and selection of cDNA libraries from various eukaryotic organisms, allowing directional cloning and expression of cDNAs. All of these six have similar polylinkers containing a unique promoter proximal EcoRI site and a unique promoter distal XhoI site, allowing for directional cloning and expression of ‘ZAP’-type cDNAs. cDNAs that complement a wide variety of yeast mutants can be selected from libraries constructed in this way. The four alternative promoters, ADH2, PGK, GAL10 and SV40 were compared for their relative activity, both in E. coli and in yeast. All yeast promoters showed substantial activity in E. coli with ADH2 showing the highest activity. ADH2 also was well-regulated in yeast, showing very high relative activity under derepressing conditions. cDNAs selected by genetic complementation from libraries constructed in these vectors should be easily subclonable into other vectors, allowing expression in different eukaryotic organisms, DNA sequencing or site-directed mutagenesis.     

RRD1, a component of the TORC1 signalling pathway,affects anaesthetic response in Saccharomyces cerevisiae

The molecular mechanisms of action of volatile anaesthetics remain unknown despite clinical use for over 150 years. While many effects of these agents have been characterized, clear insight into how these effects relate to the physiological state of anaesthesia has not been established. Volatile anaesthetics arrest cell division in Saccharomyces cerevisiae in a manner that parallels the anaesthetic actions of these drugs in mammals. To gain additional insight into the cellular activities of these drugs, we isolated genes that, when present on multi‐copy plasmids, render S. cerevisiae resistant to the volatile anaesthetic isoflurane. One of these genes, RRD1, encodes a subunit of the Tap42p–Sit4p–Rrd1p phosphatase complex that functions in the target of rapamycin complex 1 (TORC1) signalling pathway. In addition, we show that mutations in two other genes encoding components of the TORC1 pathway, GLN3 and URE2, also affect yeast anaesthetic response. These findings suggest that TORC1‐mediated signalling is involved in cellular response to volatile anaesthetics in S. cerevisiae. Copyright © 2009 John Wiley & Sons, Ltd.     

Puromycin‐ and methotrexate‐resistance cassettes and optimized Cre‐recombinase expression plasmids for use in yeast

Here we expand the set of tools for genetically manipulating Saccharomyces cerevisiae. We show that puromycin‐resistance can be achieved in yeast through expression of a bacterial puromycin‐resistance gene optimized to the yeast codon bias, which in turn serves as an easy‐to‐use dominant genetic marker suitable for gene disruption. We have constructed a similar DNA cassette expressing yeast codon‐optimized mutant human dihydrofolate reductase (DHFR), which confers resistance to methotrexate and can also be used as a dominant selectable marker. Both of these drug‐resistant marker cassettes are flanked by loxP sites, allowing for their excision from the genome following expression of Cre‐recombinase. Finally, we have created a series of plasmids for low‐level constitutive expression of Cre‐recombinase in yeast that allows for efficient excision of loxP‐flanked markers. Copyright © 2015 John Wiley & Sons, Ltd.     

Linear DNA plasmid pPK2 of Pichia kluyveri: distinction between cytoplasmic and mitochondrial linear plasmids in yeasts

The linear plasmids frequently found in plants and filamentous fungi are associated with mitochondria or chloroplasts. In contrast, all the linear plasmids known in yeasts are cytoplasmic elements. From a strain of the yeast Pichia kluyveri, we have isolated a new linear plasmid, pPK2, which was found to be associated with mitochondria. This 7·1 kilobase pairs‐long DNA contained only two genes, which code for DNA and RNA polymerases, as judged from their nucleotide sequences translated by a mitochondrial genetic code. When we examined several recently isolated yeast plasmids for their subcellular localization, we found that two linear plasmids, pPH1 from Pichia heedii, as well as pPK1 from another strain of P. kluyveri, were also localized in mitochondria. These plasmids are the first examples of mitochondria‐associated linear plasmids in yeast. All other linear plasmids we examined were of cytoplasmic origin. Whilst the cytoplasmic type linear plasmids were efficiently eliminated by ultraviolet irradiation of host cells, the mitochondria‐associated plasmids were highly resistant. The mitochondrial pPK2 plasmid was rapidly lost by treatment of the host cells with ethidum bromide. Copyright © 1999 John Wiley & Sons, Ltd.     

Transformation-associated recombination between diverged and homologous DNA repeats is induced by strand breaks

Rearrangements within plasmid DNA are commonly observed during transformation of eukaryotic cells. One possible cause of rearrangements may be recombination between repeated sequences induced by some lesions in the plasmid. We have examined the mechanisms of transformation-associated recombination in the yeast Saccharomyces cerevisiae using a plasmid system which allowed the effects of physical state and/or extent of homology on recombination to be studied. The plasmids contain homologous or diverged (19%) repeats of the URA3 genes (from S. cerevisiae or S. carlsbergensis) separated by the genetically detectable ADE2 colour marker. Recombination during transformation for covalently closed circular plasmids was over 100-fold more frequent than during mitotic growth. The frequency of recombination is partly dependent on the method of transformation in that procedures involving lithium acetate or spheroplasting yield higher frequencies than electroporation. When present in the repeats, unique single-strand breaks that are ligatable, as well as double-strand breaks, lead to high levels of recombination between diverged and identical repeats. The transformation-associated recombination between repeat DNAs is under the influence of the RAD52 and RAD1 genes.     

The budding yeast life cycle: More complex than anticipated?

The budding yeast, Saccharomyces cerevisiae, has served as a model for nearly a century to understand the principles of the eukaryotic life cycle. The canonical life cycle of S. cerevisiae comprises a regular alternation between haploid and diploid phases. Haploid gametes generated by sporulation are expected to quickly restore the diploid phase mainly through inbreeding via intratetrad mating or haploselfing, thereby promoting genome homozygotization. However, recent large population genomics data unveiled that heterozygosity and polyploidy are unexpectedly common. This raises the interesting paradox of a haplo‐diplobiontic species being well‐adapted to inbreeding and able to maintain high levels of heterozygosity and polyploidy, thereby suggesting an unanticipated complexity of the yeast life cycle. Here, we propose that unprogrammed mating type switching, heterothallism, reduced spore formation and viability, cell–cell fusion and dioecy could play key and uncharted contributions to generate and maintain heterozygosity through polyploidization.     

The Saccharomyces cerevisiae enolase‐related regions encode proteins that are active enolases

In addition to two genes (ENO1 and ENO2) known to code for enolase (EC4.2.1.11), the Saccharomyces cerevisiae genome contains three enolase‐related regions (ERR1, ERR2 and ERR3) which could potentially encode proteins with enolase function. Here, we show that products of these genes (Err2p and Err3p) have secondary and quaternary structures similar to those of yeast enolase (Eno1p). In addition, Err2p and Err3p can convert 2‐phosphoglycerate to phosphoenolpyruvate, with kinetic parameters similar to those of Eno1p, suggesting that these proteins could function as enolases in vivo. To address this possibility, we overexpressed the ERR2 and ERR3 genes individually in a double‐null yeast strain lacking ENO1 and ENO2, and showed that either ERR2 or ERR3 could complement the growth defect in this strain when cells are grown in medium with glucose as the carbon source. Taken together, these data suggest that the ERR genes in Saccharomyces cerevisiae encode a protein that could function in glycolysis as enolase. The presence of these enolase‐related regions in Saccharomyces cerevisiae and their absence in other related yeasts suggests that these genes may play some unique role in Saccharomyces cerevisiae. Further experiments will be required to determine whether these functions are related to glycolysis or other cellular processes. Copyright © 2012 John Wiley & Sons, Ltd.     

Primers‐4‐Yeast: a comprehensive web tool for planning primers for Saccharomyces cerevisiae

The budding yeast Saccharomyces cerevisiae is a key model organism of functional genomics, due to its ease and speed of genetic manipulations. In fact, in this yeast, the requirement for homologous sequences for recombination purposes is so small that 40 base pairs (bp) are sufficient. Hence, an enormous variety of genetic manipulations can be performed by simply planning primers with the correct homology, using a defined set of transformation plasmids. Although designing primers for yeast transformations and for the verification of their correct insertion is a common task in all yeast laboratories, primer planning is usually done manually and a tool that would enable easy, automated primer planning for the yeast research community is still lacking. Here we introduce Primers‐4‐Yeast, a web tool that allows primers to be designed in batches for S. cerevisiae gene‐targeting transformations, and for the validation of correct insertions. This novel tool enables fast, automated, accurate primer planning for large sets of genes, introduces consistency in primer planning and is therefore suggested to serve as a standard in yeast research. Primers‐4‐Yeast is available at: http://www.weizmann.ac.il/Primers‐4‐Yeast Copyright © 2013 John Wiley & Sons, Ltd.     

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.     

Saccharomyces cerevisiae Shuttle vectors

Yeast shuttle vectors are indispensable tools in yeast research. They enable cloning of defined DNA sequences in Escherichia coli and their direct transfer into Saccharomyces cerevisiae cells. There are three types of commonly used yeast shuttle vectors: centromeric plasmids, episomal plasmids and integrating plasmids. In this review, we discuss the different plasmid systems and their characteristic features. We focus on their segregational stability and copy number and indicate how to modify these properties. Copyright © 2017 John Wiley & Sons, Ltd.     

Comparative proteomic analysis of Rhodosporidium toruloides during lipid accumulation

Intracellular lipid accumulation is a common biological process for some eukaryotic microorganisms under specific growth conditions, yet study on this phenomenon at an ‘‐omics’ level remains rare. In this study we induced lipid accumulation by the oleaginous yeast Rhodosporidium toruloides by transferring cells into a nitrogen‐limited medium and performed a comparative and semi‐quantitative proteomic analysis of cell samples obtained thereafter by a 2D‐LC–MS/MS approach. A total of 184 proteins were identified, based on the database of yeast Saccharomyces cerevisiae. Semi‐quantitative analysis suggested that 46 proteins were notably changed during the lipid production process. Among them, seven, three and four proteins were significantly upregulated only at the late stage, the early stage and both stages, respectively. There were 26 proteins drastically downregulated at both stages. The majority of the downregulated proteins are related to protein metabolism and carbohydrate metabolism, whereas the upregulated proteins are mainly involved in alternative nitrogen sources metabolism and lipid biosynthesis. Our data indicated that a nitrogen deficiency environment had a key impact on cellular metabolism that likely stimulated the lipid accumulation process by R. toruloides. This work provids valuable information for further exploration of the molecular mechanism of cellular lipid metabolism and should be of great interest in oleaginous microorganisms engineering. Copyright © 2009 John Wiley & Sons, Ltd.     

Reagents for investigating MAPK signalling in model yeast species

Here we present a set of resources (bacterial expression plasmids and antibodies) for the interrogation of proteins involved in yeast MAPK signalling. We constructed bacterial protein expression plasmids for 25 proteins involved in MAPK signalling in budding yeast. From these constructs we expressed and purified proteins and generated rabbit polyclonal antibodies against 13 proteins in the pheromone MAPK pathway. We verified the specificity of the antibodies and employed them to follow pathway proteins in cells stimulated with pheromone. We show that these reagents can be used to detect pheromone‐induced post‐translational modifications and changes in the oligomeric state of pathway proteins. In addition to recognizing their target proteins in Saccharomyces cerevisiae, these antibodies allow the detection of predicted orthologues in the distant evolutionary relatives Kluyveromyces lactis and Schizosaccharomyces pombe. These antibodies are new tools for investigating MAPK signalling in model yeast species and may be useful for studying MAPK signalling in higher eukaryotes. Copyright © 2010 John Wiley & Sons, Ltd.     

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.