An Ancient Yeast for Young Geneticists: A Primer on theSchizosaccharomyces pombe Model System
Charles S Hoffman
Valerie Wood
Peter A Fantes
Present address: c/o Professor David Finnegan, Darwin Building, Max Born Crescent, University of Edinburgh, EH9 3BF Edinburgh, United Kingdom
Corresponding author: Biology Department, Boston College, 140 Commonwealth Ave., Chestnut Hill, MA 02467. E-mail:hoffmacs@bc.edu
Issue date 2015 Oct.
This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Abstract
The fission yeastSchizosaccharomyces pombe is an important model organism for the study of eukaryotic molecular and cellular biology. Studies ofS. pombe, together with studies of its distant cousin,Saccharomyces cerevisiae, have led to the discovery of genes involved in fundamental mechanisms of transcription, translation, DNA replication, cell cycle control, and signal transduction, to name but a few processes. However, since the divergence of the two species approximately 350 million years ago,S. pombe appears to have evolved less rapidly thanS. cerevisiae so that it retains more characteristics of the common ancient yeast ancestor, causing it to share more features with metazoan cells. This Primer introducesS. pombe by describing the yeast itself, providing a brief description of the origins of fission yeast research, and illustrating some genetic and bioinformatics tools used to study protein function in fission yeast. In addition, a section on some key differences betweenS. pombe andS. cerevisiae is included for readers with some familiarity with budding yeast research but who may have an interest in developing research projects usingS. pombe.
Keywords: education, fission yeast, forward genetics, genetic screen, Model Organism Database, model system, Primer,Schizosaccharomyces pombe,Saccharomyces cerevisiae
Why IsSchizosaccharomyces pombe a Key Model Organism?
FOR students new to research, it may not be obvious whySchizosaccharomyces pombe andSaccharomyces cerevisiae are such important model organisms. One way to make this point is to describe them as unicellular eukaryotes. As unicellular organisms, they possess many of the same features that in the 1950s and 1960s made the enteric bacteriumEscherichia coli (along with the bacteriophages that infect it) the premier model organism for molecular biology. Since the entire yeast “organism” is composed of a single cell, one can work with extremely large numbers of individuals to discover rare mutants that eventually identify the genes involved in a biological process of interest. One also can alter the composition of the growth medium and vary the growth conditions (e.g., temperature) to allow for the discovery of genes involved in a wide variety of processes. As eukaryotes, these yeasts can be used to study processes that are conserved from yeast to humans but absent from bacteria, such as organelle biogenesis and cytoskeletal organization, or to study mechanisms such as transcription, translation, and DNA replication, in which the eukaryotic components and processes are significantly different from those of their bacterial counterparts.
Molecular genetics studies in these yeasts benefit from three additional features. First, laboratory strains can be either haploid (with one set of chromosomes) or diploid (with two sets of chromosomes). Haploids are especially useful when screening for mutant alleles that produce a desired phenotype. Since most mutant alleles of a gene will be recessive to the wild-type allele owing to a reduction or loss of function, they would not be detected in a diploid strain but are readily observed in a haploid. Subsequent construction of diploid yeast strains allows one to assess whether a mutant allele is dominant or recessive to a wild-type allele. Yeast diploids are also used to place recessive mutant alleles into complementation groups to determine the number of genes identified in a genetic screen. Second, yeasts can maintain autonomous plasmids that reversibly introduce genetic material to modify the behavior of a strain. This facilitates gene cloning by allowing one to screen plasmids carrying different DNA fragments for those that confer a desired effect on the host strain. Finally, yeasts possess highly active homologous recombination mechanisms. To illustrate this point, 1 cM in genetic distance (a measure of meiotic recombination) is approximately 1 million base pairs (bp) in humans (Konget al. 2004) but only ∼2500 bp inS. cerevisiae (Olsonet al. 1986) and ∼6250 bp inS. pombe (Fowleret al. 2014). DNA repair systems in budding yeast can promote recombination during vegetative growth (Orr-Weaveret al. 1981). Linearized DNA introduced into budding yeast cells is treated by the cells as damaged DNA, leading to recombination with the homologous region of the host chromosome. A similar system exists in fission yeast. The ability to recombine homologous sequences allows researchers to construct strains that carry novel combinations of alleles by (1) the direct introduction of foreign or otherwise modified pieces of DNA to a targeted site in the yeast genome and (2) the introduction of sequences into plasmids by cotransforming a linearized plasmid with a piece of DNA that is flanked by sequences homologous to the site of linearization. In these ways, yeasts are genetically pliable organisms whose biology is well conserved in many respects with that of other eukaryotes.
What IsS. pombe?
S. pombe, often known simply as “fission yeast,” is an ascomycete yeast. Ascomycetes are one group of the kingdom Fungi, the other main group being basidiomycetes. Yeasts can belong to either ascomycetes or basidiomycetes but are distinguished from other fungi by their unicellular (as opposed to mycelial) lifestyle and their ability to ferment sugars. The defining characteristic of an ascomycete is that sexual spores are formed within a specialized structure called anascus (Latin for “bag”). In addition toS. pombe andS. cerevisiae, many filamentous fungi familiar as laboratory organisms such asAspergillus andNeurospora are ascomycetes. In contrast, most fungi encountered in the macroscopic world (mushrooms) are basidiomycetes.
On the basis of protein and DNA sequence data, theSchizosaccharomyces genus appears to be an ancient “basal” ascomycete (Taphrinomycetes) whose roots go back to the early radiative evolution of ascomycetes and perhaps close to the split between animals and fungi. This makes the evolutionary distance betweenS. pombe andS. cerevisiae of the same order as the distance between either of these yeasts and mammals (Sipiczki 2000;Heckmanet al. 2001;Sipiczki 2004). However,S. pombe can be thought of as a more “ancient” yeast thanS. cerevisiae based on its biological characteristics because it appears to have undergone fewer evolutionary changes since divergence from the common ancestor. For example,S. cerevisiae has lost many genes (338) that are conserved betweenS. pombe and mammals (Aravindet al. 2000;Wood 2006). Thus, the proteomic content ofS. pombe is closer to that of the common ancestor. Biological similarities betweenS. pombe and mammals are mentioned elsewhere in this Primer. This is a strong argument for using both yeasts as models. If a process is conserved between the two yeasts, it is likely to be more widely conserved. At the same time, mechanistic differences between the two yeasts underscore the potential for functional diversity among higher eukaryotes.
S. pombe is widely distributed around the world and has been isolated from a variety of natural sources.Pombe is the Swahili word for “beer” (or at least a beer-like fermented beverage), andS. pombe is used for its fermentation. A word of warning: in the authors’ experience, beer produced byS. cerevisiae is far more palatable!S. pombe also has been isolated from fruits; from kombucha, a tea product produced by mixed fermentation with yeasts (includingS. pombe) and bacteria; and from molasses, used to produce distilled spirits such as rum and tequila (Gomeset al. 2002). Another use of aSchizosaccharomyces yeast derives from its ability to utilize malic acid and thereby reduce undesirable acidity in wine (Volschenket al. 2003).
Origins ofS. pombe Research
S. cerevisiae has been a companion to humans since the invention of bread making and brewing. In contrast, apart from the relatively minor applications mentioned earlier,S. pombe has not historically had many practical applications. This difference has influenced the ways in which these two model organisms were used in scientific research. Because of interest in improving brewing and baking methods, there is a long history of studyingS. cerevisiae physiology that has focused on the regulation of metabolism. Once genes could be cloned, this led to studies on how gene expression is regulated in response to environmental (growth) conditions and specific genetic changes. Later, researchers investigated cell biological aspects such as cell cycle control, the cytoskeleton, mating processes, and so on. The mass of information about the roles and regulation of genes derived from studies on metabolic genes was exploited in early genetic engineering experiments,e.g., in making expression vectors. In contrast, the main focus inS. pombe has been on basic interest-driven research. Research started in the 1940s and early 1950s in two main areas: the mating-type system, which led to investigation of the sexual cycle, and the growth and division processes that comprise the cell division cycle.
The founder ofS. pombe genetics was Urs Leupold, a Swiss student who visited the Carlsberg Laboratory in Copenhagen during the 1940s. He was introduced to the yeast by Øjvind Winge, head of the Carlsberg Laboratory and “father” ofS. cerevisiae genetics, who suggested the genetic basis ofS. pombe homothallism as the subject for Leupold’s Ph.D. thesis (Leupold 1993). Unfortunately, the strain Leupold was given proved to be infertile, but he later obtained a high-fertility isolate from Delft, Netherlands. This latter isolate proved to contain a homothallic strain and two heterothallic strains apparently derived from it (see later). These strains, 968h90, 972h−, and 975h+, are the ancestor strains for almost all genetic studies onS. pombe. Leupold showed that the differences between the strains were due to alleles of a single genetic locus, now known to be a complex region with silent and expressed genes. Because all three strains derive from a single isolate, the strains used in laboratories around the world are nearly isogenic. This avoids some of the problems experienced inS. cerevisiae studies, in which genetic differences between “wild-type strains” such as S288c and W303 (Ralseret al. 2012) can cause these strains to respond differently to additional mutations (Elionet al. 1991). Leupold and collaborators went on to developS. pombe as a genetically tractable organism by isolating a range of mutants and constructing chromosome maps. His laboratory at the University of Bern, where he became head of the Institute of General Microbiology, remained the mainS. pombe genetics center for several decades.
At around the same time, Murdoch Mitchison, working in Edinburgh, was interested in how single cells grew in mass between cell divisions. He tried various cell types, from bacteria to sea urchin eggs, and settled onS. pombe when he realized that its mode of growth by linear extension and medial division allowed the age of a single cell to be fairly accurately determined by a single cell length measurement (Mitchison 1990). His main interests were in the patterns of increase during the cell cycle of such cellular properties as total cell mass and in the rates of overall protein synthesis of individual proteins and other macromolecules. It was thought that division might be triggered by the accumulation of a molecule to a critical level. Thus, the identification of molecules whose abundance increased during the cell cycle might lead to insight into the control of division (Mitchison 1971). Indeed, such molecules—the mitotic cyclins—were shown some 20 years later to be key regulators of mitosis.
The genetics and cell cycle strands ofS. pombe research came together in the mid-1970s when Paul Nurse, following the pioneering work of Lee Hartwell onS. cerevisiae, undertook the isolation ofS. pombe cell division cycle (cdc) mutants in Mitchison’s laboratory, having first spent time in Leupold’s group, where he learned genetics methods. In the mid-1970s, a series of papers published by Nurse and colleagues described cell cycle mutants and their use in investigating how the cell cycle is controlled (Fantes 1989).
For two decades, theS. pombe field was dominated by the laboratories headed by Leupold and Mitchison and later by their scientific progeny, students and postdoctoral fellows who had worked in their laboratories. Meanwhile, there was a steady accumulation of knowledge aboutS. pombe by European researchers such as Herbert Gutz, Henri Heslot, and Nicola Loprieno (Gutzet al. 1974). A major step forward was taken by Richard Egel, who isolated and characterized mutants unable to undergo meiosis (Breschet al. 1968;Egel 1973). This represented the first use of a genetics approach to study theS. pombe life cycle, and Egel’s mutants formed the basis for many subsequent studies.
During the following decades,S. pombe research expanded worldwide, and this led to the First International Fission Yeast Meeting held in Edinburgh in 1999. Since then,S. pombe has gone from strength to strength, as shown by the increase in fission yeast publications over the years (Figure 1).
Figure 1.
S.pombe publication numbers from 1959 to 2014.
S. pombe Genome Content
In 2002, fission yeast became the sixth eukaryotic model organism to have its genome sequence and annotation published (Woodet al. 2002). The genome sequence provided a robust platform for the ongoing evaluation of genome content, both by the annotation of features on the sequence and by attaching functional annotations to those features. Gradually, a more complete picture ofS. pombe is emerging as new data sets from both small- and large-scale experiments are aggregated to refine the annotated gene structures and assign functional data to them.
Gene complement
A central goal of biological research is to describe fully the orchestrated collection of functions and processes that combine to produce living cells. One way to assess progress is by maintaining and interrogating an accurate “parts list” of the contributing gene products and their attributes. TheS. pombe parts list is maintained by the model organism database (MOD) PomBase (http://www.pombase.org), with 5054 protein-coding genes currently reported (compared to 5821 forS. cerevisiae). This difference in protein number is partly due to the retention of many paralogous pairs (homologous genes within an organism) inS. cerevisiae following what appeared to have been two rounds of whole-genome duplication (WGD) (Wolfe and Shields 1997;Kelliset al. 2004). Despite a lower number of protein-coding genes, fission yeast has a large number of proteins (currently 338) that are conserved in vertebrates (source: PomBase Query Builder using curated fission yeast/budding yeast and fission yeast/human orthologs) but absent from budding yeast, while only 179 genes are conserved between budding yeast and vertebrates that are absent from fission yeast (source: SGD Yeastmine using Compara human orthologs and PomBase curated fission yeast/cerevisiae orthologs supplemented by manual curation). This is a consequence of the increased lineage-specific gene losses in the budding yeast lineage after fission and budding yeasts diverged from a common ancestor (Aravindet al. 2000;Wood 2006).
Of the 5054 known or predicted fission yeast proteins, at this time, 2154 have a published biological role, 2050 have a biological role inferred from an experimentally characterized ortholog (usually from budding yeast), and 850 have no known biological role [inferred from the absence of Gene Ontology (GO) biological process annotation]. A large number of these uncharacterized proteins are conserved outside theSchizosaccharomyces genus; 182 are conserved in mammals, almost 40% (68) of which have no budding yeast ortholog. Tantalizingly, a number of these genes are orthologs of human disease genes or members of the Catalogue of Somatic Mutations in Cancer (COSMIC) of genes with mutations in cancer genomes (Forbeset al. 2014). For example, SPAC652.01 ortholog BLCAP is associated with bladder cancer, and SPAC6G10.10c ortholog MMTAG2 is associated with multiple myelomas. Characterization of these gene products in fission yeast almost certainly will shed light on the functions of human proteins.
Noncoding RNAs (ncRNAs) include all RNAs other than messenger RNA (mRNA) and are central to a wide range of biological processes, including transcription, translation, gene regulation, and splicing. The ncRNA annotation of the fission yeast genome has increased dramatically in recent years. In addition to the 307 ribosomal RNAs (rRNAs), transfer RNAs (tRNAs), small nuclear RNAs (snRNAs), and small nucleolar RNAs (snoRNAs), there are now 1538 additional ncRNAs annotated in the genome, mainly from RNA sequencing (RNA-Seq) data (Watanabeet al. 2002;Wilhelmet al. 2008;Rhindet al. 2011). Similar extensive intergenic and antisense transcription is detected in budding yeast (Yassouret al. 2010;Goodmanet al. 2013;Pelechanoet al. 2013). A large number of the ncRNAs identified in fission yeast (694) appear to be antisense to protein-coding genes, and some (e.g.,tos1,tos2, andtos3, which are antisense torec7) carry out a regulatory role during sexual differentiation (Molnaret al. 2001;Watanabeet al. 2002), whereas many others display meiosis-specific expression (Bittonet al. 2011). Of the remainder, only a handful, including BORDERLINE/IRC1 (Camet al. 2005;Kelleret al. 2013),mrp1 (Paluh and Clayton 1995),rrk1 (Kruppet al. 1986),sme2 (Watanabe and Yamamoto 1994), andsrp7 (Ribeset al. 1988) are characterized, hinting at a massive potential for future expansion of ncRNA research in fission yeast. Most of the annotated ncRNAs are of low abundance in vegetative cells, similar to tightly repressed meiotic genes, but 187 of them are expressed at ∼1–200 copies/cell (Margueratet al. 2012).
Chromosome organization
TheS. pombe genome size is ∼13.8 Mb compared to the 12.5-Mb genome ofS. cerevisiae. Despite similar genome sizes,S. pombe has only 3 relatively large chromosomes of 5.7, 4.6, and 3.5 Mb compared to 16 smaller chromosomes inS. cerevisiae. Indeed, at 3.5Mb, the smallestS. pombe chromosome (chromosome III) is over twice the length of the largestS. cerevisiae chromosome (chromosome I), which is 1.5 Mb.
Fission yeast has large modular centromeres, more reminiscent of those of higher organisms than of the 125-bp element sufficient for centromere function inS. cerevisiae. Fission yeast centromeres (cen1,cen2, andcen3) are approximately 40, 69, and 110 kb. The centromere structure comprises a nonconserved central core sequence flanked by variable numbers of outer repeats. Many of the proteins that bind toS. pombe centromeres are conserved in mammals but absent from budding yeast, including Swi6 and Chp1 and the RNA interference (RNAi) pathway components (Lorentzet al. 1994;Ekwallet al. 1995;Doeet al. 1998;Volpeet al. 2002). The conservation of centromere features, including size, structure, and multilayered organization, makes fission yeast a valuable model for the study of eukaryotic chromatin remodeling and centromere function.
Subtelomeric regions in both fission yeast and budding yeast are reversibly transcriptionally silent (Aparicioet al. 1991;Nimmoet al. 1998;Halmeet al. 2004) and are enriched for genes encoding membrane transporters, stress-response proteins, and expanded families of species-specific cell surface glycoproteins (Goffeauet al. 1996;Robyret al. 2002;Woodet al. 2002;Hansenet al. 2005). In fission yeast, these regions are very similar to each other (approximately 50–60 kb of the sequence immediately proximal to the telomeric repeats has ∼99% identity among telomeres for most of the region). A similar localization of contingency genes involved in antigenic variation for immune evasion has been observed in parasitic microbes (Barryet al. 2003). Subtelomeric regions therefore may provide the ideal genomic location for eukaryotic cells to test and select for novel species-specific genes required for stress and surface variation in response to changing environmental conditions, and the over-representation of environmentally regulated genes in subtelomeres may be applicable to eukaryotes in general.
Finally, theS. pombe genome contains 262 sequences comprising partial or complete copies of mobile genetic elements (Bowenet al. 2003) derived from LTR retrotransposons. Thirteen full-length active copies of Tf2 exist in the classic laboratory strains, while active copies of Tf1 only occur in wild isolates ofS. pombe (Levinet al. 1990;Levin and Boeke 1992;Hoffet al. 1998). Although these are expressed at low levels under standard laboratory conditions, forced induction of retrotransposon transcription leads to integration of new copies into promoters of RNA pol II–transcribed genes (Guo and Levin 2010). Interestingly, Tf1 integration favors stress-response gene promoters, and once integrated, the Tf1 generally increases expression of the adjacent gene (Guo and Levin 2010;Fenget al. 2013). Thus, these mobile elements may serve as a source of genetic variability to promote adaptation to new conditions of environmental stress.
Genetic Nomenclature
AllS. pombe genes have a systematic identifier, while many, but not all, have community-assigned gene names that bear some similarities to those ofS. cerevisiae in that they follow the convention of three letters and a number written in italics, such aswee1. However, unlikeS. cerevisiae genes,S. pombe genes are always written in lowercase. This contrasts withS. cerevisiae nomenclature, which uses capital letters to designate either wild-type or dominant mutant alleles. ForS. pombe genes, one uses a superscript plus sign to indicate a wild-type allele (wee1+), a hyphen and allele number to indicate a mutant allele (wee1-50), and a Greek delta or a capitalD to indicate a deletion allele (wee1Δ orura4-D18). Alternatively, deletions may be indicated by a pair of colons followed by the selectable marker that was inserted into that locus (geneofinterest 1 deleted with theura4+ gene would begoi1::ura4+), while a tagged allele may be indicated by a hyphen (goi1-GFP).S. pombe proteins are not italicized and are written by a capital letter followed by lowercase letters (Wee1).
Life Cycle
The life cycle ofS. pombe consists of asexual (vegetative) and sexual phases, and these have been described many times (Egel 1989;Fantes 1989;Forsburg and Nurse 1991). Without a doubt,S. pombe’s greatest claim to fame as a model organism comes in the area of cell cycle control—how progress through the vegetative cycle is regulated by internal and external cues (see the sectionNotable Advances from Research on Fission Yeast for the discoveries that led to Lasker and Nobel Prizes for Paul Nurse). Its sexual cycle also has been the subject of many studies, starting in the 1950s with Leupold’s work on mating types and followed by Egel’s pioneering work (Egel 1973,1989;Egel and Egel-Mitani 1974;Leupold 1993).
Mitotic cell cycle
Mitchison’s adoption ofS. pombe as an ideal organism in which to study the cell cycle (Mitchison 1990) was based on the modes of growth and division of the cells.S. pombe cells are cylindrical in shape with roughly hemispherical ends and are about 3.5 µm in diameter during exponential growth on nutrient-rich medium. Newborn cells are about 8 µm long and grow continuously through most of the cell cycle without width change. This means that a good estimate of the age of a cell (time elapsed since birth) can be obtained simply by measuring its length. When cells reach about 15 µm in length, they undergo a closed mitosis (as with most fungi, the nuclear envelope does not break down) with the nucleus close to the center of the cell. Shortly after nuclear division, a transverse septum is laid down medially, which is then cleaved to produce two daughters (Figure 2A and supporting information,File S1). The symmetric mode of division means that the two daughter cells are nearly equal in size, similar to cultured mammalian cells, but quite different from the asymmetric division of budding yeast. The nuclear DNA replicates very soon after mitosis (Nasmythet al. 1979). Thus, the short G1 phase takes place between mitosis and cell separation, with two unreplicated nuclei present in the cell until the septum is cleaved. S phase is coincident with the presence of the septum, so newborn daughter cells are already in G2, containing fully replicated chromosomes. After cytokinesis, cells remain in a G2 phase, which occupies about three-quarters of the cell cycle, leading up to the next mitosis. As such, virtually every haploid cell in a growing population contains two copies of the chromosomal complement. DiploidS. pombe cells are noticeably wider (∼4.4 µm) than haploid cells and undergo cytokinesis at approximately 24 µm in length (Figure 2B andFile S2), making them almost double the volume of haploid cells.
Figure 2.
S. pombe cells and asci. (A) A haploid cell containing a fission plate. (B) Diploid cells. (C) Zygotic asci formed by the mating of two haploid cells. (D) Azygotic asci formed when diploid cells go through meiosis.
When subjected to nutrient starvation in the absence of a mating partner, haploid cells exit the cell cycle and enter stationary phase. One unusual feature ofS. pombe is that while they enter stationary phase from G1 on nitrogen starvation, they do so from G2 upon glucose starvation (Costelloet al. 1986). If a mating partner is present, haploid cells shift from vegetative growth to a sexual cycle (described in the next section). Depending on their mating-type constitution (see next section), diploid cells enter either stationary phase or the sexual cycle in response to nutrient starvation.
Sexual cycle and mating-type system
In addition to vegetative growth,S. pombe has a sexual cycle. Two haploid cells of appropriate mating types (see later) respond to nutrient starvation by mating (conjugation) (Egel and Egel-Mitani 1974;Egel 1989). This involves adhesion of the cells followed by digestion of the walls separating them to form a single fusion cell containing the two parent nuclei (Figure 3 andFile S3). The nuclei then fuse to form a zygote, a single cell containing a single diploid nucleus. Normally, the nucleus enters the meiotic pathway soon after zygote formation, and four haploid nuclei are generated. Subsequently, a spore wall forms around each nucleus to produce an ascus consisting of four spores within the shell of the zygote (Figure 2C andFigure 3H). These zygotic asci typically have a bent shape that reflects the angle between the two cells that fused to generate the zygote (Figure 2C). In contrast, azygotic asci derived from vegetative diploid cells are short and linear (Figure 2D). Whether zygotic or azygotic, the ascus wall lyses on exposure to growth medium, and the spores germinate and develop into vegetative cells that return to mitotic growth.
Figure 3.
Sexual development inS. pombe. Cells of the homothallich90 mating type were plated onto a thin EMM agar pad at 30° and photographed at (A) 0 min, (B) 184 min, (C) 204 min, (D) 254 min, (E) 286 min, (F) 334 min, (G) 446 min, and (H) 560 min. Becauseh90 cells can undergo mating-type switching, which occurs in only one of the two daughter cells, two pairs of daughter are seen to have mated and formed asci.
Truly wild-typeS. pombe strains are of the homothallic mating type referred to ash90, in that they possess three copies of information at the mating-type (mat) locus, one of which is expressed and two of which are transcriptionally silenced (Figure 4).S. pombe h90 strains can transfer genetic information from either silent locus to the expressed locus. This produces a mixture of cells expressing one or the other mating type within a clone to allow mating to take place. This may be beneficial to surviving starvation conditions in the wild by allowing for the creation of spores.S. pombe cells also can be heterothallic such that they require a mating partner of the opposite mating type (Leupold 1950).
Figure 4.
Schematic of theS. pombe mating-type locus.
Diploids
After cells mate, the diploid zygote generally undergoes meiosis and sporulation immediately to produce a zygotic ascus. This depends on the mating-type composition of the diploid:h− andh+ haploid parents will produce anh−/h+ diploid that is meiosis (and sporulation) competent, whereas a diploid that is homozygous for either theh+ orh− mating type is not able to enter meiosis. Mating within anh90 culture or ofh90 cells withh− orh+ cells will also produce a meiosis-competent diploid. The normally transient diploid state of a cell can be “rescued” by transfer to nutrient-rich growth medium. A fraction of such diploid cells will propagate vegetatively as diploids, though rearrangements at themat locus and/or spontaneous meiosis and sporulation are frequent events. The practice of making and using diploidS. pombe strains is discussed further later.
Vive la Difference
It has not escaped our notice that substantially many more people have worked withS. cerevisiae than withS. pombe. Thus, we take this opportunity to point out some biological differences between the two yeasts that one must take into account when transitioning from the lower yeast side to this higher yeast. This Primer is not an attempt to describe all known methods forS. pombe molecular genetics research. We therefore point the reader to several invaluable sources of information. These include two Cold Spring Harbor Laboratory manuals,Experiments with Fission Yeast (Alfaet al. 1993) andFission Yeast: A Laboratory Manual (Haganet al. 2016); the booksMolecular Biology of the Fission Yeast (Nasimet al. 1989) andThe Molecular Biology of Schizosaccharomyces pombe (Egel 2004); volume 33, issue 3 of the journalMethods (edited by Kathleen Gould); and individual methods chapters written by Moreno, Klar, and Nurse (Morenoet al. 1991) and by Forsburg and Rhind (Forsburg and Rhind 2006). There is also a wealth of information regardingS. pombe on Susan Forsburg’s PombeNet website (http://www-bcf.usc.edu/∼forsburg/). If all else fails, or if you just want to get to know us better, you can always e-mail questions to theS. pombe mail list hosted by the European Bioinformatics Institute at pombelist@ebi.ac.uk.
Growth media
It is common for people who have experience withS. cerevisiae to simply use the same growth medium when starting to work withS. pombe. We strongly recommend against the use of YPD (the rich medium used forS. cerevisiae) forS. pombe. Rich medium forS. pombe (YES) is made with only yeast extract, glucose, and trace amounts of histidine, adenine, uracil, lysine, and leucine (Gutzet al. 1974). The peptone in YPD induces lysis ofS. pombe ura4− mutants (Matsuoet al. 2013) and creates a stress in all strains such that they are induced to mate while growing in what should be a rich medium. Thus, YPD does not provide a permissive growth condition forS. pombe. In addition, the standard defined medium used forS. cerevisiae, SC or SD medium, is inferior forS. pombe growth to Edinburgh Minimal Medium (EMM) (Gutzet al. 1974), although the basis of this growth difference is not known.
Ploidy and meiosis
The difficulties of creating and maintaining stable diploid strains ofS. pombe were mentioned earlier.S. cerevisiae cells mate in nutrient-rich conditions but require a strong starvation signal to induce meiosis. In contrast,S. pombe cells require starvation conditions to mate, but nearly all diploid zygotes sporulate immediately (Figure 3 andFile S3). While this is advantageous for tetrad dissection and strain construction, it poses a challenge for experiments that require diploid strains, such as dominance and complementation tests. Diploid strains are also used to introduce a change into the genome that may be lethal or deleterious in a haploid strain, such as a gene deletion. By altering one allele in a diploid, one can first confirm that the change has been introduced before sporulating the diploid to determine the consequence of the change in haploid strains.
Two methods exist for constructing and maintainingS. pombe diploids. First, theade6-M210 andade6-M216 alleles both confer adenine auxotrophy, but they complement intragenically such that the heterozygous diploid (ade6-M210/ade6-M216) grows in medium lacking adenine. Since these mutations are in the same gene, even when some diploid cells enter meiosis and produce spores, these haploid spores are virtually all adenine auxotrophs because of the tight linkage between theade6-M210 andade6-M216 alleles. This allows one to use Ade+ selection to maintain the diploid strain in the absence of contaminating Ade+ haploid progeny. Second, strains expressing themat2-102 allele can mate with either anh+ orh− heterothallic strain to generate a diploid. The diploid withh+ immediately enters meiosis in the normal way, and sporulation ensues. However, in the diploid withh−, neither haploid parent expresses a functionalmat-Pm gene, which is required for meiosis, so the diploid nucleus persists, generating a stable diploid line. This allows one to select for diploid cells by using two haploid parents carrying complementary selectable markers (e.g., two different auxotrophies). Because meiosis is completely suppressed, there is no possibility of haploid progeny arising by meiotic recombination that would share the same phenotype as the diploid. Spontaneous mitotic haploidization following chromosome loss can occur. This haploidization can be stimulated by exposure to a low concentration of the microtubule-destabilizing drug benomyl (Alfaet al. 1993). Since it occurs in the absence of meiotic recombination, this method can be used to map mutations to one of the threeS. pombe chromosomes, with the caveat that a mitotic recombination event could produce recombinant chromosomes in rare haploid segregants. One can distinguish between the original diploid strain and the viable haploid derivatives by plating for colonies on medium containing the vital stain Phloxine B, which stains dead cells (Gutzet al. 1974;Tange and Niwa 1995). Diploid colonies contain many more dead cells than haploids, so they are dark red on Phloxine B–containing plates, while haploid colonies are light pink. Phloxine B is also be used to detect rare diploids that arise in a culture, presumably as a result of chromosomal nondisjunction. These diploids grow faster than the haploid parent and will overtake a culture if one does not initiate an experiment from a single colony. Such diploids are mating competent but produce mostly inviable aneuploid progeny when crossed with a haploid strain owing to the creation of a triploid zygote.
Homologous recombination
One of the key strengths of bothS. pombe andS. cerevisiae for molecular genetics studies is the ability to introduce DNA into the chromosome via homologous recombination during vegetative growth. This allows one to delete genes, construct translational fusions, or even construct specific mutant alleles. However, the relative ratio of homologousvs. nonhomologous insertion events is lower forS. pombe than forS. cerevisiae. The relatively high proportion of nonhomologous events inS. pombe, in which a small linear DNA molecule integrates at a site in the genome other than at the location targeted by the sequences at the ends of the molecule, can create difficulties. While different laboratories take different approaches to increase the efficiency of targeted DNA insertions, it is generally sufficient to create a PCR product carrying the transforming DNA that has 60 bp of targeting sequences flanking the product (Bähleret al. 1998).
RNAi
UnlikeS. cerevisiae,S. pombe has retained through evolution the genes that encode the RNAi machinery, including the Dcr1 DICER ribonuclease, the Rdp1 RNA-dependent RNA polymerase, and the Ago1 Argonaute family member. Research on RNAi inS. pombe has not only contributed to our understanding of its mechanism and consequences but also helps to explain other biological differences between fission and budding yeasts. Loss of any RNAi genes leads to an increase in the presence of transcripts from the heterochromatic region ofS. pombe centromeres as well as defects in chromosome segregation during mitosis (Volpeet al. 2002,2003). Thus, the absence of RNAi in budding yeast may be linked to loss of large, repetitive centromeres as found inS. pombe, which, in turn, may explain the relatively small size ofS. cerevisiae chromosomes that appear to segregate in mitosis by attachment to a single microtubule (Peterson and Ris 1976). RNAi also appears to play a modest role in regulating expression of Tf2 retrotransposons inS. pombe (Hansenet al. 2005) and the more abundant retrotransposons found in the related fission yeastSchizosaccharomyces japonicus (Rhindet al. 2011). Because RNAi regulates retrotransposon expression in higher organisms (Tabaraet al. 1999), it has been suggested that the loss of RNAi inS. cerevisiae could have led to the increased expression of reverse transcriptase activity that ultimately led to the loss of introns in the genomic DNA (Aravindet al. 2000). This might explain whyS. pombe is more like metazoan cells thanS. cerevisiae with regard to both the presence of introns and the retention of RNAi.
Cell cycle
S. pombe andS. cerevisiae have both contributed greatly to our understanding of events and processes that determine and regulate passage through the eukaryotic cell cycle. It is instructive to compare the cell cycles of the two yeasts to appreciate both their similarities and their differences.S. cerevisiae cells start their cycles in G1 as unbudded cells with unreplicated (1c) DNA content. Prior to “start,” a point in G1, cells are uncommitted to the mitotic cycle or other developmental pathways (Hartwellet al. 1974), while completion of “start” commits the cell to the ongoing mitotic cycle. The G1 phase lasts about one-quarter of the cell cycle and is followed by the S phase, a further third of the cycle. There is arguably no true G2 phase because formation of the mitotic spindle starts during late G1 or early S phase and does not depend on completion of S phase. Thus, the spindle persists through most of the cell cycle, though its final elongation, which separates the chromosomes, takes place well after the end of S phase. Following mitosis, the (larger) mother and (smaller) daughter cells separate, the mother embarking on a new cycle and the daughter becoming a mother cell, giving rise to a new bud. In contrast,S. pombe cells complete their short G1 and S phases very soon after mitosis so that by the time of cell division, virtually every cell is already in G2, and this phase comprises about three-quarters of the cycle (Nasmythet al. 1979). The mitotic spindle is present for only a short period, as in most eukaryotes, and a symmetric cell division ensues. Other genetic (Nurse 1975) and physiologic studies (Fantes and Nurse 1977,1978) indicated the presence of a major regulatory point between G2 and M controlling entry into mitosis.
Initial studies of the cell cycles of the two yeasts did not reveal any similarities in the underlying control mechanisms. Indeed, for a considerable time it was believed that the budding yeast cell cycle was controlled only in G1, while the fission yeast cell cycle was regulated at the G2/M boundary. Therefore, it was surprising whenS. pombe was shown to have a G1 control point despite the shortness of this phase of the cell cycle (Nurse and Bissett 1981). Furthermore, theS. pombe G1 and G2 control points turned out to share a key component, the Cdc2 gene product, later shown to be a protein kinase (Simanis and Nurse 1986). TheS. cerevisiae homolog of Cdc2, Cdc28, was known to be required for G1 progression and was subsequently determined also to be needed at later stages of the cycle. Cdc2 and Cdc28 were shown to be functional homologs in that theCDC28 gene could rescue the defect of acdc2 mutant and vice versa (Beachet al. 1982). Given the major differences between the cell cycles of the two yeasts and the large evolutionary distance between them, Nurse argued that if two disparate yeasts shared the Cdc2/28 function, other eukaryotes such as humans also might do so. This idea was proven by the isolation of a human Cdc2 homolog based on its ability to rescue the cell cycle defect of aS. pombe cdc2 mutant (Lee and Nurse 1987). This discovery was the basis of the awarding of the Nobel Prize to Nurse a decade later, shared with Lee Hartwell for his pioneering studies of cell cycle genes inS. cerevisiae and with Tim Hunt for his identification of the cyclin partner to Cdc2.
Coping with DNA damage during the cell cycle
Cells face intermittent threats to their survival and fitness. One of these is damage to nuclear DNA, which can arise spontaneously or following exposure to one of several environmental agents. Particularly damaging is ionizing radiation, which can cause double-strand DNA breaks. If left unrepaired, these breaks can lead to loss of large chromosomal regions and cell death. A very effective way to repair a broken chromosome is to copy the information from the corresponding sequence on another DNA molecule. This works well for diploid organisms because there are always at least two copies of a given chromosomal region present, but a haploid cell in G1 phase has only a single copy of most sequences. Two quite different strategies have evolved for dealing with double-strand breaks in the two yeasts, and they relate to the different lifestyles of the yeasts.
A pair of haploidS. cerevisiae cells will mate with each other independently of the nutritional environment, provided that they are of opposite mating type, to form a mitotically stable diploid cell that proliferates indefinitely, as long as nutrients are available. Following starvation, the diploid undergoes a meiotic division to generate four ascospores, two of each mating type. On return to growth medium, the ascospores germinate and, unless artificially separated, mate with one another, once again generating diploids. Thus, although laboratoryS. cerevisiae strains can be maintained as haploids, this is not the natural state for the organism. The diploid state ensures that the cell always has two copies of each genomic sequence and can deal effectively with broken chromosomes by copying the intact version of the damaged sequence from the chromosomal homolog. Thus,S. cerevisiae cells can have a long G1 phase and still survive an otherwise lethal DNA-damaging event.
However,S. pombe cells will not mate with one another unless starved. Following starvation for (usually) nitrogen, cells of opposite mating types form a zygote that generally enters meiosis directly, with no extended diploid phase. The four resulting haploid ascospores each will proliferate as a haploid clone. Thus, the default lifestyle ofS. pombe is as a haploid; diploid strains are unstable and difficult to propagate in the laboratory. So how do haploidS. pombe cells survive a double-strand DNA break, given that they have no sister chromosome to copy? The answer is that while the cells only have a single copy of each chromosome, the cells replicate their DNA and enter G2 very early in the cell cycle so that the chromosomes are composed of two sister chromatids. Breakage of one chromatid therefore can be repaired using the intact chromatid as a template. This shows how these yeasts achieve the same goal via two distinct mechanisms.
Genetic Toolkit: As Illustrated by a Plasmid-Based Mutant Screen
Yeast molecular genetics projects commonly follow one of two strategies. The first involves the isolation of strains possessing chromosomal mutations that confer a desired or distinct phenotype (we sometimes find things we were not looking for but cannot ignore), followed by identification of the gene or genes of interest. An excellent description of this approach has been provided in the Primer on the use ofS. cerevisiae (Duinaet al. 2014) and can serve as a template for similar studies inS. pombe. The second strategy involves a plasmid-based screen. This could be a screen of genomic or complementary DNA (cDNA) libraries (Table 1) for plasmids that confer a desired phenotype or a screen to identify mutant alleles of a specific cloned gene of interest. This latter scenario serves as the focus for this Primer onS. pombe molecular genetics methods and tools. While our scenario will involve screening for mutations of a heterologously expressed gene inS. pombe, the same approach can be used to identify mutations of interest in anS. pombe gene carried on a plasmid.
Table 1.S. pombe genomic and cDNA libraries.
| Library | Insert | Marker | Reference |
|---|---|---|---|
| pWH5 (Sau3AI) library | PartialSau3AI digest-genomic—10 kb | LEU2+ | Feilotteret al. 1991 |
| pWH5 (HindIII) library | PartialHindIII digest-genomic—10 kb | LEU2+ | Feilotteret al. 1991 |
| pBG2 library | PartialSau3AI digest-genomic—2.9 kb | his3+ | Ohiet al. 1996 |
| pEA500 library | PartialSau3AI digest-genomic—3.9 kb | his7+ | Wanget al. 2005 |
| pLEV3/SPLE1 | adh1-driven cDNA (>1-kb transcripts) | LEU2+ | Janooet al. 2001 |
| pLEV3/SPLE2 | adh1-driven cDNA (<1.6-kb transcripts) | LEU2+ | Janooet al. 2001 |
| pREP3X library | nmt1-driven cDNA | LEU2+ | Edgar and Norbury, unpublished data |
Four genomic and three cDNA libraries that have been used to cloneS. pombe genes are listed. Information regarding the cloning vector used, the nature of the insert DNA, the selectable marker, and the promoter driving expression of the inserts for the cDNA libraries are given. For genomic libraries, the average size of the inserted DNA is provided. The SPLE1 and SPLE2 cDNA Libraries contain size-selected inserts.
When eminent British naturalist W. T. Pooh was about to search for something, “He thought he would begin the Hunt by looking for Piglet, and asking him what they were looking for before he looked for it” (Milne 1928). Similarly, in forward genetics screens, where the scientific question is “What genetic events confer the desired phenotype?” one must first determine what phenotype is being sought. This phenotype must vary as a function of the activity of the gene of interest. For genes expressed from a plasmid, the level of activity is controlled by plasmid copy number, the strength of the promoter used to drive expression, and the specific activity of the encoded protein. The phenotype also will depend on the host strain used for the screen. When searching for loss-of-function alleles, the level of expression of the target gene is generally not an issue, but when looking for gain-of-function alleles, the level of expression is important because overexpression of the wild-type allele may produce the desired mutant phenotype. In this situation, one must test different expression vectors to identify one that does not alter the relevant phenotype of the cell when expressing the wild-type protein.
In our scenario, you may have discovered through a classical genetics approach (Figure 5) that the target of the drug Now-U-Dead is conserved betweenS. pombe andPlasmodium falciparum, the causative agent for malaria. These studies began by isolatingS. pombe nud1R mutants that are resistant to this drug, which is normally toxic to bothS. pombe andP. falciparum. Further analyses showed that these mutant alleles are genetically linked to each other (all progeny from a cross of any two resistant strains are resistant to Now-U-Dead) (Figure 5) and are dominant to the wild-type allele that is present in strains that are sensitive to Now-U-Dead (i.e.,nud1R/nud1+ heterozygous diploids are resistant to Now-U-Dead). You also tried to isolate strains that are resistant to Now-U-Dead using one of two transposon-insertion strategies (Parket al. 2009;Li and Du 2014). These approaches generate large numbers of mutants for which the presence of the foreign transposon allows rapid identification of the insertion site. However, these approaches failed to produce resistant strains. These results suggest that either the gene or genes in question are essential or that a loss-of-function mutation does not confer resistance to Now-U-Dead.
Figure 5.
Outline for genetic analysis ofnudA gene.
You then identified thenud1+ gene by one of two methods (Figure 5). The choice of method will depend on resources available and likely will change with time as next-generation high-throughput sequencing becomes commonplace (Koboldtet al. 2013). First, high-throughput sequencing of two or more independently isolatednud1R strains identified a single gene that is altered in all these strains relative to the 972 reference genome sequence or your parental strain. A plasmid carrying a candidatenud1R allele confers Now-U-Dead resistance to a strain that is sensitive to Now-U-Dead. Second, candidate genes were identified from a cDNA library (Table 1 provides a list of genomic and cDNA libraries used to cloneS. pombe genes) by their ability to confer resistance to Now-U-Dead when expressed at high levels. The corresponding alleles innud1+ andnud1R strains were sequenced to determine which gene identified in the screen is altered innud1R strains. Finally, after either method of gene identification, you confirm that expression of a candidatenud1R allele, but not thenud1+ allele, in anud1+ strain confers Now-U-Dead resistance.
You subsequently showed thatnud1+ is an essential gene (Figure 5). This information is present in the PomBase database; although had the database suggested thatnud1Δ cells are viable, you should still confirm this independently (Guoet al. 2013). This is done by deleting one copy of the gene in a diploid strain, inducing sporulation, dissecting tetrads, and determining that haploid progeny carrying thenud1Δ deletion are nonviable. The fact thatnud1+ is essential suggests that Now-U-Dead killsS. pombe by inactivating or partially inactivating Nud1. You also showed that theP. falciparum nudA gene, whose sequence is homologous tonud1+, can replacenud1+ to keep anud1Δ strain alive (Figure 5). (Anud1Δ/nud1+ heterozygous diploid strain with a plasmid that expressesP. falciparum nudA produced viablenud1Δ progeny that carry the plasmid.) To facilitate malaria research, you would like to identify mutations in theP. falciparum nudA gene that confer resistance to Now-U-Dead with the aim of developing other drugs targeting NudA that remain effective against the mutant NudA protein. In this way, drug resistance would be much less likely to arise in patients who receive a combination of drugs that target different sites in NudA.
SinceS. pombe Nud1 is believed to be inactivated by Now-U-Dead, the host strain can benud1+, and you can screen for Now-U-Dead resistance among transformants that carry an autonomously replicating plasmid expressing theP. falciparum nudA gene. ManyS. pombe plasmids use theS. cerevisiae LEU2+ gene, which complements a mutation in theS. pombe leu1 gene (Beach and Nurse 1981), orS. pombe biosynthetic pathway genes such asura4+,his7+,his3+, andlys2+ (Bach 1987;Apolinarioet al. 1993;Burke and Gould 1994;Hoffman and Hoffman 2006). In contrast, drug selection markers, which do not require host strains to possess auxotrophic mutations, are used more often as templates to generate PCR products for integration into theS. pombe genome to delete or tag target genes (Bähleret al. 1998;Tastoet al. 2001;Satoet al. 2005). Unlike inS. cerevisiae, centromeric plasmids are not used inS. pombe becauseS. pombe centromeres are too large to incorporate into traditional cloning vectors (Clarkeet al. 1986). Therefore,S. pombe plasmids are present in multiple copies per cell, which contributes to the level of expression of the cloned gene, as does the strength of the promoter used to drive expression (Table 2).
Table 2. Promoters used inS. pombe research.
| Promoter | Strength | Regulation | Other comments | Reference |
|---|---|---|---|---|
| nmt1 | Strong | Regulateda | Moderate expression even when repressed. | Forsburg 1993;Maundrell 1993 |
| nmt81 | Moderate | Regulateda | Weak expression even when repressed. | Forsburg 1993;Maundrell 1993 |
| nmt41 | Weak | Regulateda | Forsburg 1993;Maundrell 1993 | |
| urg1 | Strong | Regulatedb | Not regulated when on a plasmid. | Watsonet al. 2011,2013 |
| adh1 | Strong | Constitutive | Russell and Hall 1983;McLeodet al. 1987;Forsburg 1993 | |
| tif471 | Moderate | Constitutive | De Medeiroset al. 2015 | |
| lys7 | Weak | Constitutive | De Medeiroset al. 2015 |
nmt-based promoters are repressed by thiamine (vitamin B1), but induction is relatively slow.
urg1 promoter is rapidly induced by exogenous uracil.
To clone theP. falciparum nudA gene into anS. pombe expression vector, use PCR to amplifynudA from a cDNA clone or library with primers whose 5′ ends contain 45–60 bp of sequence found on either side of a unique restriction site that is downstream of a promoter in a replicativeS. pombe expression vector such as pART1 (McLeodet al. 1987) or pREP1 (Maundrell 1993) (Figure 6). To promote homologous recombination, 45 bp of targeting sequence is sufficient for integrating DNA into plasmids, while 60 bp is safer for chromosomal insertions. The 3′ ends of these primers anneal to the ends of thenudA open reading frame, so the final product is thenudA gene flanked by sequences that target the insertion of this DNA into the cloning vector. Cotransforming anS. pombe strain with the PCR product and the linearized expression vector produces plasmids with the PCR product inserted into the vector by gap repair (Kostrubet al. 1998). These transformants form colonies on the defined medium EMM lacking leucine as a result of expression of theLEU2+ selectable marker. However,adh1 andnmt1 (in the presence or absence of thiamine) express NudA at such high levels that even transformants expressing the wild-type allele display some resistance to Now-U-Dead. In contrast, transformants expressing NudA from the moderate-strengthnmt41 promoter in pREP41, when grown in the presence of thiamine, remain sensitive to Now-U-Dead. This identifies a level of NudA expression that will facilitate the isolation and characterization of alleles ofnudA that are resistant to Now-U-Dead (Figure 5).
Figure 6.
Gap-repair cloning of thenudA gene. ThenudA gene is amplified by PCR using oligonucleotides that target its insertion into the linearized expression vector pART1 by gap repair transformation. This places thenudA open reading frame under the control of theS. pombe adh1 promoter to drive its expression in fission yeast.
Next, generate PCR products containing a low number of random mutations, including ones that produce drug-resistant proteins. PCR using the FailSafe PCR System (Epicentre Biotechnologies) produces both transition (purine-to-purine) and transversion (purine-to-pyrimidine) mutations to generate a greater diversity of amino acid substitution mutations than transition mutations alone (Iveyet al. 2010). This PCR reaction is carried out usingnudA cDNA template and oligonucleotides that will target insertion into a linearized pREP41 vector, and the product is cotransformed with the linearized pREP41 vector. Once Leu+ transformants are visible on the plate (generally after 4–6 days of growth at 30°), replica plate them to medium containing Now-U-Dead to identify drug-resistant transformants. It may be useful to include the vital stain Phloxine B in the growth medium in case the cells in compound-sensitive colonies undergo a few rounds of division before dying, which may mask the fact that the cells have died.
Since Now-U-Dead resistance could arise from a chromosomal mutation in theS. pombe nud1+ gene, you must confirm that compound resistance is plasmid conferred. This can be done by rescuing the plasmids from yeast transformants intoE. coli, isolating the plasmids, and retransforming the original host strain (all transformants should now be compound resistant). You can also test whether loss of the plasmid in the yeast transformant restores sensitivity to Now-U-Dead by growing transformants on rich medium [yeast extract medium with supplements (YES)] to allow for mitotic loss of plasmids and streaking for single colonies on YES medium to produce colonies that either lack or retain plasmids. These colonies are then replica plated to YES medium, EMM lacking leucine (to identify Leu− colonies that have lost plasmid), and medium containing Now-U-Dead (to determine whether the Leu− colonies have regained compound sensitivity). Once plasmids have been shown to confer compound resistance, you can sequence the mutant alleles to determine what residue or residues have been altered in the mutant sequence. If alleles carry multiple mutations, you may need to perform site-directed mutagenesis to create single amino acid substitution alleles and test their ability to confer drug resistance. This can be done using the CRISPR/Cas9 system, which has been used recently to directly editS. pombe genomic sequences (Jacobset al. 2014).
While there is not a crystal structure for the NudA protein, you may be able to use the program SWISS-MODEL to predict its tertiary structure and to identify the location of the altered residues (Schwedeet al. 2003). In this way, you can identify a likely binding site for the compound that may provide guidance for the design of other compounds that target NudA and thus remain effective against forms of NudA that are resistant to Now-U-Dead. Additionally, if there is more than one compound that targets NudA, you can screen for resistant alleles to the individual compounds. These alleles then can be used to profile resistance to the various compounds to develop a drug combination to treat malaria patients so as to prevent the selection for drug-resistant strains ofP. falciparum.
In addition to usingS. pombe to detect alleles ofP. falciparum nudA that are resistant to Now-U-Dead, you may study the function of theS. pombe Nud1 protein directly. Sincenud1+ is essential, you could conduct a plasmid-based screen to identify temperature-sensitivenud1 alleles to investigate what happens as cells lose Nud1 function. However, Now-U-Dead can be used for this purpose without the need for temperature-sensitive alleles. Instead, you will look for proteins that physically interact with Nud1. While there are many ways to do this, we will consider just two of them. First, you could take a two-hybrid screening approach (Chienet al. 1991) to look forS. pombe proteins that directly bind Nud1. To do this, you construct a translational fusion between thenud1+ open reading frame and theS. cerevisiae Gal4 DNA-binding domain in a plasmid such as pAS1 that can replicate in eitherE. coli orS. cerevisiae (Durfeeet al. 1993). You then coexpress this “bait” plasmid, which carries aTRP1+ selectable marker, together with a library ofLEU2+-marked “prey” plasmids (the insert DNA is randomly shearedS. pombe cDNA to create a collection of translational fusions to the Gal4 transcriptional activation domain) in anS. cerevisiae strain such as YRG-2 (Durfeeet al. 1993). If the bait and prey proteins interact in a transformant, they form a protein complex that stimulates transcription of reporter genes.
Alternatively, you could look for proteins that form a complex with Nud1, via direct or indirect interactions, by constructing a tandem affinity protein (TAP)–tagged version of Nud1 using cassettes that tag either the N- or C-terminus of the protein (Tastoet al. 2001) at thenud1+ locus inS. pombe. The tagged Nud1 protein then can be isolated together with its binding partners by applying sequentially two different affinity purification methods, leading to a highly purified protein complex. You can determine the individual binding partners or detect all copurified proteins via mass spectrometry (Tastoet al. 2001). Either approach helps to identify the role of Nud1 by revealing binding partners that may have been studied by other laboratories.
Using PomBase as a Research Tool
The rapidly increasing information available regarding individual gene products and the growing trend toward functional dissection of large numbers of genes in a single study have made the fission yeast MOD PomBase (http://www.pombase.org/) an increasingly important tool for data mining to generate hypotheses, design experiments, and analyze data (Woodet al. 2012;Mcdowallet al. 2014). The main tools provided by PomBase are summarized inTable 3.
Table 3. PomBase tools and resources.
| Tool/Resource | Purpose |
|---|---|
| Advanced search Query history | Find all gene products with a specific feature (e.g., otology term, intron number, domain). Combine the output of searches (add, intersect, subtract). |
| Gene list search | Upload a user-defined list to compare with other lists. |
| GO slim | Access lists of genes annotated to “broad” biological processes. |
| Motif search | Identify lists of proteins matching user-defined amino acid sequence motifs. |
| Ensembl browser | Access sequence features in the context of the genome (e.g., polyadenylation sites, transcriptome data, nucleosome position). |
| Comparative genomics | Synteny views (S. octosporus,S. japonicas genomes). |
| Compara | Search for orthologs/paralogs in Fungi, or in a pan-taxonomic comparison (eukaryotes), using Compara in the Ensembl browser. |
PomBase is structured around gene pages that provide comprehensively curated data for each gene product. Here we describe the use of PomBase to find tentative clues for the cellular role of the elusive Nud1, identified in your studies. Although a minimal amount of curated data is available for previously unstudied genes such asnud1+, it is possible to gain some insights into the function of Nud1 from the GO, phenotype, protein domain, expression, and interaction data presented on PomBase.
The GO controlled vocabulary is used to consistently describe the gene product attributes of molecular functions, biological processes, and cellular component (localization or complex) (Ashburneret al. 2000). Because neither Nud1 nor its orthologs have been studied previously, no molecular function or biological process data are available. However, a genome-wide localization study using a GFP library provides cellular component (localization) data for ∼85% of fission yeast protein-coding gene products (Matsuyamaet al. 2006). You see that Nud1 is found in the mitochondria (along with 757 other gene products).
The gene page also provides phenotype data using the Fission Yeast Phenotype Ontology (FYPO) together with supporting information describing alleles and experimental conditions (Harriset al. 2013). Analysis of a genome-wide deletion data set provides null mutant viability for >90% of protein-coding genes (Kimet al. 2010). In addition, a visual screen of this deletion resource for mutants affecting cell cycle (detected by effects on cell length) and cell shape has provided broad morphology phenotype annotations for many gene products (representing both viable cells and the terminal phenotypes of nonviable deletions) (Hayleset al. 2013). You see that deletion ofnud1+ produces the phenotype “nonviable tapered cell,” similar to that of 134 other gene deletions.
Because mutations in functionally related genes often confer the same phenotype, studying a list of the genes coannotated to your ontology term of interest may shed some light on the biological role of Nud1. It could be even more informative to survey the list of genes that share these two annotations (cellular component “mitochondrion” and phenotype “nonviable tapered cell”). PomBase provides a tool (http://www.pombase.org/spombe/query/builder) that allows curated lists to be rapidly intersected, added to, or subtracted from. The intersect between cellular component “mitochondrion” and phenotype “nonviable tapered cell” shows that 95% of the genes with phenotype “nonviable tapered cell” encode mitochondrial proteins. In addition to GO terms and phenotypes, a wide range of other features can be queried, including intron number, protein length, modifications, protein families, and domains.
The protein motifs and domains predicted by the InterPro Consortium databases can be powerful functional indicators (Mitchellet al. 2015). Even if your protein of interest only contains a domain of unknown function (DUF), it may be a member of a superfamily with a known fold, which can indicate some broad classification such as a catalytic activity or transport function.
The “quantitative gene expression” data on the gene page provides transcript expression level data for all gene products. The protein level during vegetative growth or quiescence is also available for most proteins (Margueratet al. 2012;Carpyet al. 2014). This expression level and the presence or absence at specific cell cycle or life cycle stages may give insight into possible roles of your gene of interest.
Physical and genetic interactions are curated in collaboration with BioGRID (Chatr-Aryamontriet al. 2013) and displayed on the PomBase gene pages, and they are also available via network diagrams using esyN (Beanet al. 2014). Physical interactions can link a gene product to processes based on the roles of interacting partners. There are no high-throughput physical interaction data sets for fission yeast at the time of this writing, so it is highly unlikely that there would be any physical interaction data for an unstudied gene. However, genome-wide genetic interaction data sets are becoming available (Ryanet al. 2012).
Other information presented on the gene pages includes protein features, protein modifications, human andS. cerevisiae orthologs, disease associations, and quantitative protein and RNA expression levels. A link from each gene page provides access to an Ensembl browser hosting sequence-based features including nucleosome positioning, polyadenylation sites, and transcriptomics data displayed in the context of the genome sequence.
Using PomBase for Data Mining
Although the primary use of PomBase is to look up information on specific genes and their products, MODs are becoming increasingly pivotal during data analyses or for data mining to identify candidate genes of interest before embarking on any experiments. Ontologies provide a critical component of this data-mining capability because the ontology structure [reviewed inWashington and Lewis (2008)] enables powerful database querying. Increasingly, gene lists are generated as the output of a functional genomics experiment (e.g., differentially expressed genes or the results of a phenotypic screen). You can import any user-defined gene list into the PomBase Query Builder and combine it with any of the available biological queries to evaluate the intersection of any feature with your list and to identify attributes that may be over-represented in your gene list relative to the whole genome.
One of the main uses of GO annotation is to analyze lists of genes or summarize experimental results by GO Enrichment analysis or GO slimming. GO Enrichment describes the identification of any statistically over-represented GO terms in a list of genes using one of the many GO Enrichment tools (e.g.,http://geneontology.org/ on the GO website). A GO slim is a set of broad GO terms used to provide a summary of the GO annotations for genes in a data set [e.g., by the GO Term Mapper tool (http://go.princeton.edu/cgi-bin/GOTermMapper)]. GO slimming also can be used to summarize an organism’s total functional capabilities; a GO biological process slim classification for the entire fission yeast gene product set is on the PomBase website (www.pombase.org/browse-curation/fission-yeast-go-slim-terms).
Notable Advances from Research on Fission Yeast
The yeastsS. pombe andS. cerevisiae were propelled to the forefront of research to characterize basic cellular mechanisms because they are (1) single-cell organisms, (2) eukaryotes, (3) able to be maintained as a haploid or diploid, (4) able to maintain autonomously replicating plasmids, and (5) organisms with highly active homologous recombination machinery that facilitates targeted changes to the genome. Research usingS. pombe has made significant contributions to our understanding of biology in several key areas.
Cell cycle
S. pombe research has played a unique and highly prominent role in understanding the cell cycle. Nurse combined Mitchison’s interest in the cell cycle with Leupold’s genetic approaches to identify genes that play essential roles in cell cycle progression (Nurse 1975;Nurseet al. 1976). The best known and most important outcome of this work was the cloning of thecdc2+ cyclin-dependent kinase gene (Beachet al. 1982) and the critical demonstration that the human homolog could supply this function to acdc2ts temperature-sensitive mutant (Lee and Nurse 1987). The mutant screens that identifiedcdc2+ also led to the discovery of several genes whose products interact with or act on the Cdc2 kinase, such as Cdc25 [the phosphatase that activates Cdc2 by dephosphorylation of a crucial phosphotyrosine residue (Russell and Nurse 1986)] and Cdc13 (Nurseet al. 1976;Fisher and Nurse 1996). A separate study led to the isolation of mutants with reduced cell size, nearly all of which had a defectivewee1 gene, later shown to encode the tyrosine kinase that inhibits Cdc2 activity until cells are ready to pass from G2 to M phase (Nurse 1975;Nurse and Thuriaux 1980;Russell and Nurse 1986,1987;Gould and Nurse 1989). Furthermore, the importance of cell size in cell cycle control was supported by the direct demonstration that cell size at division is homeostatically controlled. A cell that is larger or smaller than normal at birth will undergo division at close to the normal size, so nearly all the deviation from normal is corrected within the same cycle (Fantes 1977).
Strains carrying mutations in othercdc genes facilitated studies of important processes in addition to entry to mitosis: because they displayed defects in DNA replication, septum formation/septation, and cytokinesis. For example, Cdc1, Cdc6/Pol3, and Cdc27 are subunits of DNA polymerase delta (MacNeillet al. 1996), while Cdc18, Mcm2 (Cdc19), Mcm4 (Cdc21), Cdc23, and Cdc24 all play roles in either regulation of S phase or DNA replication itself (Coxonet al. 1992;Kellyet al. 1993;Forsburg and Nurse 1994;Aveset al. 1998;Gouldet al. 1998). Cdc7, Cdc11, Cdc14, and Cdc16 are required for function of the septation initiation network (SIN) that monitors the completion of mitosis to regulate the start of cytokinesis (McCollum and Gould 2001). Finally, Cdc3 (profilin), Cdc8 (tropomyosin), Cdc12 (formin), and Cdc15 are all required for cytokinesis (Fankhauseret al. 1993;Balasubramanianet al. 1994;Changet al. 1997). Thus, the study of mutations that cause cell cycle defects inS. pombe formed the basis of many distinct areas of research.
In addition to requiring dedicated gene products, progress through the cell cycle is influenced by both intrinsic and extrinsic conditions. There exist several cell cycle checkpoints at which the cell determines whether or not conditions have been met to allow progression to the next phase of the cell cycle. Several related DNA checkpoints act at various cell cycle stages (Carr 2004), while the spindle checkpoint acts to regulate exit from mitosis (Hauf 2013). Similar checkpoints exist in all eukaryotes, and many of the components are conserved.
DNA checkpoints detect damaged or unreplicated DNA and block cell cycle progress until the damage is repaired or replication is completed, when progress resumes. InS. pombe, the best understood is the G2/M checkpoint that prevents entry into mitosis while damaged DNA is present. Mutants defective in this checkpoint function have been isolated in both budding and fission yeast by their sensitivity to DNA-damaging agents such as radiation or agents that block replication such as hydroxyurea (Nasim and Smith 1975;Enochet al. 1992). In outline, the checkpoint system works as follows: sites of damaged DNA are recognized by binding of checkpoint protein complexes, which activates a cascade of signaling components, ultimately preventing entry into mitosis. Gene products involved in binding to damaged or unreplicated DNA include the Rad3 protein kinase (Bentleyet al. 1996) and its accessory factor Rad26 (Edwardset al. 1999). Further complexes involved in binding to sites of damage contain Rad17 (Griffithset al. 1995), Hus1 (Kostrubet al. 1997), Rad1 (Sunnerhagenet al. 1990), Rad9 (Murrayet al. 1991), and other proteins. Rad3 then interacts with other components, including the effector kinase Chk1 (Walworthet al. 1993;Al-Khodairyet al. 1994), whose activation results in increased tyrosine phosphorylation of Cdc2, which prevents entry into mitosis. The increase in Cdc2 phosphorylation appears to involve both downregulation of the Cdc25 phosphatase and upregulation of Wee1 (Furnariet al. 1997;Boddyet al. 1998;Raleigh and O’Connell 2000).
DNA checkpoints that act at other cycle stages—e.g., to check that the DNA is properly replicated in S phase—share many of the components and complexes involved in the G2/M checkpoint, but there are also elements unique to one or other system. For instance, the effector kinase in the S-phase checkpoint is Cds1 (Murakami and Okayama 1995;Raleigh and O’Connell 2000) rather than Chk1 (Furnariet al. 1997;Boddyet al. 1998). Many of these components and mechanisms are conserved in mammalian cells, along with the functions of many more proteins involved in the detection and responses to DNA damage or stalled DNA replication (Sancaret al. 2004).
A significant difference between fission and budding yeast is that the final target of the G2/M checkpoint in budding yeast is not Cdc28 (the functional homolog of Cdc2) but Pds1, a protein whose destruction is required for exit from metaphase and entry into anaphase (Sanchezet al. 1999). The controls in mammalian cells are in this respect similar to those inS. pombe. The G2/M checkpoint is more complex than inS. pombe, but a major part of the G2 delay following DNA damage is due to a reduction in Cdc25 activity. This leads to an increased level of Cdc2/Cdk1 tyrosine phosphoryation on the equivalent residue to that ofS. pombe Cdc2 and hence its inactivation. In response to DNA damage in G1, Cdc25A is phosphorylated and degraded so that it is unable to activate Cdk2–cyclin E, the cyclin complex required to initiate S phase (Sancaret al. 2004).
The observation that the nutritional composition of the growth medium influences cell cycle progress is an old one (Fantes and Nurse 1977), but until recently, the mechanism has not been clear. The fortuitous isolation of a mutant in which cell size was greatly affected by nutritional status (Ogden and Fantes 1986) led to the discovery and elucidation of the Sty1/Spc1 stress-activated MAP kinase signaling pathway that affects the rate of passage through the G2/M control. This nutritional control involves signaling through the TOR system and the Polo-like kinase Plo1, which acts indirectly to regulate the tyrosine phosphorylation status and hence activity of Cdc2 (Petersen and Nurse 2007;Hartmuth and Petersen 2009). In addition to influencing progress at the G2/M boundary, the Sty1/Spc1 pathway transduces nitrogen and glucose starvation stress as well as osmotic, oxidative, and heat stress to generate a spectrum of cellular responses (Chenet al. 2003).
Chromosome dynamics
S. pombe also has been a key model for the study of biological mechanisms in the area of chromosome dynamics. Possibly owing to the large sizes of theS. pombe chromosomes, mechanisms associated with chromosome replication, recombination, and segregation during meiosis and mitosis are areas in whichS. pombe research has played a central role. Mitsuhiro Yanagida and coworkers identified proteins required for proper chromosomal pairing and segregation in mitosis by isolatingcut mutants that formed a septum prior to chromosome segregation (Hiranoet al. 1986;Samejimaet al. 1993). Yanagida’s contributions to this field have been recognized with several awards, including the Imperial Prize and Japan Academy Prize in 2003 and the Order of Culture Prize (Japan) in 2011. He was elected as a foreign member of the Royal Society (United Kingdom) in 2000 and a foreign associate of the National Academy of Sciences (United States) in 2011.
BecauseS. pombe centromeres are large, repetitive structures, similar to those of mammalian cells and unlikeS. cerevisiae centromeres, research on chromatid cohesion at the centromeres (Watanabe and Nurse 1999;Kitajimaet al. 2004), kinetochores and their interactions with the mitotic spindle (Goshimaet al. 1999), and centromere clustering and movement during mitosis (Funabikiet al. 1993) have been central to developing our understanding of these processes in metazoan chromosomes. For example, twoS. pombe Shugoshin homologs, Sgo1 and Sgo2, connect the spindle checkpoint proteins to sister chromatid cohesion (Kitajimaet al. 2004). This work has prompted studies of vertebrate Shugoshin-like proteins to show that they are involved in the same interactions as inS. pombe (Salicet al. 2004;Kitajimaet al. 2005) and in chromosomal instability associated with various forms of cancer (Kahyoet al. 2011;Yamadaet al. 2012).
Similarly, studies of the structure and behavior of telomeres at the ends of these large chromosomes have led to important discoveries about teleomeric proteins (Cooperet al. 1997;Baumann and Cech 2001;Chikashige and Hiraoka 2001;Martin-Castellanoset al. 2005) involved in controlling the clustering and positioning of telomeres during meiosis that contribute to proper chromosome segregation as well as the pairing of chromosomes for homologous recombination.
In addition,S. pombe continues to be a key model organism for the study of DNA replication, with many of the originalcdc genes being involved in various aspects of DNA replication. Current studies are examining the locations of origins of replication, the timing of origin firing, the regulation of replication fork movement, and the response to events such as replication fork stalling.S. pombe origins of replication are somewhat like those of higher eukaryotes in their lack of a conserved sequence element (Mojardinet al. 2013), thus makingS. cerevisiae an outlier in this regard. Along with studies of DNA replication mechanisms, it has been shown that gene expression is affected by whether a DNA replication fork passes through the gene in the sense or antisense direction and that this is controlled by the location of surrounding origins, the timing of origin firing, and the presence of binding sites for the Sap1 or Reb1 proteins that block replication fork progression (Sanchez-Gorostiagaet al. 2004;Krings and Bastia 2005;Mejia-Ramirezet al. 2005). Replication fork arrest is also used to control the direction of replication through a site in the mating locus (Figure 4) that carries an imprint that controls which daughter cell will undergo a mating switch event (Dalgaard and Klar 2001). Further studies of replication origins, DNA replication, and the interplay between DNA replication and mating-type switching or gene expression inS. pombe undoubtedly will lead to new insights that will inform our understanding of mammalian molecular mechanisms.
Chromatin, epigenetics, and gene expression
BothS. pombe andS. cerevisiae have been important organisms for the study of epigenetic control of gene expression. Expression of genes present at themat2-P andmat3-M mating-type loci (Figure 4) and in telomeric and subtelomeric regions is silenced by a variety of mechanisms that ultimately affect the chromatin in these regions (Egelet al. 1984;Thon and Klar 1992;Thonet al. 1994;Allshireet al. 1995;Ekwallet al. 1996). In addition, the large heterochromatic centromeres ofS. pombe have provided researchers with a third region in which to study silencing mechanisms (Allshireet al. 1994,1995). Studies of heterochromatin formation atS. pombe centromeres have helped to establish a “histone code” that associates specific posttranslational modifications of histones with the transcriptional state of the associated chromatin (Jenuwein and Allis 2001). InS. pombe, Clr4 methylation of lysine 9 of histone H3 leads to the recruitment of the chromodomain protein Swi6 to nucleosomes containing the H3K9 mark (Nakayamaet al. 2001). The establishment of transcriptionally silent heterochromatin also requires the function of the RNAi RITS complex (Volpeet al. 2002;Verdelet al. 2004), and Clr4, Swi6, and components of the RITS complex have been found to colocalize throughout the genome at heterochromatic regions (Camet al. 2005). For an in-depth review of the many studies into the mechanisms and consequences of chromatin remodeling atS. pombe centromeres, seeAllshire and Ekwall (2015). Finally, ncRNAs can affect chromatin remodeling and gene expression in an RNAi-independent manner. For example, transcriptional activation of the glucose-repressedfbp1+ gene involves the transient and stepwise expression of three ncRNAs that facilitate chromatin remodeling in the promoter region and the eventual expression of the mRNA (Hirotaet al. 2008). Given the complexity of chromatin remodeling mechanisms at various regions of theS. pombe genome and the importance of this to both regulated gene expression and the maintenance of heterochromatic regions of the genome, this will remain a vibrant area of research for years to come.
Conclusions
Over the past 60 years,S. pombe has risen from relative obscurity as a research organism to one of the two major yeast model systems, together withS. cerevisiae. Early research focused on studies of sexual development and the cell cycle, but with the increasing number of scientists drawn to this organism, there has been a significant expansion of the research areas in whichS. pombe work has made substantial contributions. This trend undoubtedly will continue in the years to come. Along with the classical yeast genetics approaches described in theS. cerevisiae Primer (Duinaet al. 2014) and here, the use of global expression studies (Mataet al. 2002;Chenet al. 2003;Margueratet al. 2012) and screens of theS. pombe deletion collection (Kimet al. 2010) creates large data sets that are accessible through MODs such as PomBase. This provides newcomers to fission yeast research with the ability to generate hypotheses regarding previously unstudied genes so that they can create research projects that will add to our understanding of the biology ofS. pombe. Such new knowledge will, in turn, provide new insights into the biology of nonmodel organisms such as pathogens and ourselves.
Acknowledgments
We thank Mitsuhiro Yanagida, Masayuki Yamamoto, Chikashi Shimoda, Amar Klar, Gerry Smith, and Paul Russell for sharing their recollections on the history and growth ofS. pombe research. Special thanks are extended to Richard Egel for his help and encyclopedic knowledge of research activities. We thank Henry Levin, Midori Harris, and Antonia Lock for comments on various sections of this Primer. Valerie Wood is supported by the Wellcome Trust grant 104967/Z/14/Z.
Footnotes
Communicating editor: E. A. De Stasio
Supporting information is available online atwww.genetics.org/lookup/suppl/doi:10.1534/genetics.115.181503/-/DC1
Literature Cited
- Alfa C., Fantes P., Hyams J., McLeod M., Warbrick E., 1993. Experiments with Fission Yeast.Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. [Google Scholar]
- al-Khodairy F., Fotou E., Sheldrick K. S., Griffiths D. J., Lehmann A. R., et al. , 1994. Identification and characterization of new elements involved in checkpoint and feedback controls in fission yeast.Mol. Biol. Cell5: 147–160. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Allshire R. C., Ekwall K., 2015. Epigenetic regulation of chromatin states in Schizosaccharomyces pombe.Cold Spring Harb. Perspect. Biol.7: a018770. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Allshire R. C., Javerzat J. P., Redhead N. J., Cranston G., 1994. Position effect variegation at fission yeast centromeres.Cell76: 157–169. [DOI] [PubMed] [Google Scholar]
- Allshire R. C., Nimmo E. R., Ekwall K., Javerzat J. P., Cranston G., 1995. Mutations derepressing silent centromeric domains in fission yeast disrupt chromosome segregation.Genes Dev.9: 218–233. [DOI] [PubMed] [Google Scholar]
- Aparicio O. M., Billington B. L., Gottschling D. E., 1991. Modifiers of position effect are shared between telomeric and silent mating-type loci in S. cerevisiae.Cell66: 1279–1287. [DOI] [PubMed] [Google Scholar]
- Apolinario E., Nocero M., Jin M., Hoffman C. S., 1993. Cloning and manipulation of the Schizosaccharomyces pombe his7+ gene as a new selectable marker for molecular genetic studies.Curr. Genet.24: 491–495. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Aravind L., Watanabe H., Lipman D. J., Koonin E. V., 2000. Lineage-specific loss and divergence of functionally linked genes in eukaryotes.Proc. Natl. Acad. Sci. USA97: 11319–11324. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Ashburner M., Ball C. A., Blake J. A., Botstein D., Butler H., et al. , 2000. Gene Ontology: tool for the unification of biology. The Gene Ontology Consortium.Nat. Genet.25: 25–29. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Aves S. J., Tongue N., Foster A. J., Hart E. A., 1998. The essential Schizosaccharomyces pombe cdc23 DNA replication gene shares structural and functional homology with the Saccharomyces cerevisiae DNA43 (MCM10) gene.Curr. Genet.34: 164–171. [DOI] [PubMed] [Google Scholar]
- Bach M. L., 1987. Cloning and expression of the OMP decarboxylase gene URA4 from Schizosaccharomyces pombe.Curr. Genet.12: 527–534. [DOI] [PubMed] [Google Scholar]
- Bähler J., Wu J. Q., Longtine M. S., Shah N. G., McKenzie A., 3rd, et al. , 1998. Heterologous modules for efficient and versatile PCR-based gene targeting inSchizosaccharomyces pombe.Yeast14: 943–951. [DOI] [PubMed] [Google Scholar]
- Balasubramanian M. K., Hirani B. R., Burke J. D., Gould K. L., 1994. The Schizosaccharomyces pombe cdc3+ gene encodes a profilin essential for cytokinesis.J. Cell Biol.125: 1289–1301. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Barry J. D., Ginger M. L., Burton P., McCulloch R., 2003. Why are parasite contingency genes often associated with telomeres?Int. J. Parasitol.33: 29–45. [DOI] [PubMed] [Google Scholar]
- Baumann P., Cech T. R., 2001. Pot1, the putative telomere end-binding protein in fission yeast and humans.Science292: 1171–1175. [DOI] [PubMed] [Google Scholar]
- Beach D., Durkacz B., Nurse P., 1982. Functionally homologous cell cycle control genes in budding and fission yeast.Nature300: 706–709. [DOI] [PubMed] [Google Scholar]
- Beach D., Nurse P., 1981. High-frequency transformation of the fission yeast Schizosaccharomyces pombe.Nature290: 140–142. [DOI] [PubMed] [Google Scholar]
- Bean D. M., Heimbach J., Ficorella L., Micklem G., Oliver S. G., et al. , 2014. esyN: network building, sharing and publishing.PLoS One9: e106035. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Bentley N. J., Holtzman D. A., Flaggs G., Keegan K. S., DeMaggio A., et al. , 1996. The Schizosaccharomyces pombe rad3 checkpoint gene.EMBO J.15: 6641–6651. [PMC free article] [PubMed] [Google Scholar]
- Bitton D. A., Grallert A., Scutt P. J., Yates T., Li Y., et al. , 2011. Programmed fluctuations in sense/antisense transcript ratios drive sexual differentiation in S. pombe.Mol. Syst. Biol.7: 559. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Boddy M. N., Furnari B., Mondesert O., Russell P., 1998. Replication checkpoint enforced by kinases Cds1 and Chk1.Science280: 909–912. [DOI] [PubMed] [Google Scholar]
- Bowen N. J., Jordan I. K., Epstein J. A., Wood V., Levin H. L., 2003. Retrotransposons and their recognition of pol II promoters: a comprehensive survey of the transposable elements from the complete genome sequence of Schizosaccharomyces pombe.Genome Res.13: 1984–1997. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Bresch C., Muller G., Egel R., 1968. Genes involved in meiosis and sporulation of a yeast.Mol. Gen. Genet.102: 301–306. [DOI] [PubMed] [Google Scholar]
- Burke J. D., Gould K. L., 1994. Molecular cloning and characterization of the Schizosaccharomyces pombe his3 gene for use as a selectable marker.Mol. Gen. Genet.242: 169–176. [DOI] [PubMed] [Google Scholar]
- Cam H. P., Sugiyama T., Chen E. S., Chen X., FitzGerald P. C., et al. , 2005. Comprehensive analysis of heterochromatin- and RNAi-mediated epigenetic control of the fission yeast genome.Nat. Genet.37: 809–819. [DOI] [PubMed] [Google Scholar]
- Carpy A., Krug K., Graf S., Koch A., Popic S., et al. , 2014. Absolute proteome and phosphoproteome dynamics during the cell cycle of Schizosaccharomyces pombe (fission yeast).Mol. Cell. Proteomics13: 1925–1936. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Carr A. M., 2004. Checkpoint controls halting the cell cycle, pp. 41–56 inThe Molecular Biology of Schizosaccharomyces pombe: Genetics,Genomics,and Beyond, edited by Egel R.Springer, Berlin. [Google Scholar]
- Chang F., Drubin D., Nurse P., 1997. cdc12p, a protein required for cytokinesis in fission yeast, is a component of the cell division ring and interacts with profilin.J. Cell Biol.137: 169–182. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Chatr-Aryamontri A., Breitkreutz B. J., Heinicke S., Boucher L., Winter A., et al. , 2013. The BioGRID interaction database: 2013 update.Nucleic Acids Res.41: D816–823. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Chen D., Toone W. M., Mata J., Lyne R., Burns G., et al. , 2003. Global transcriptional responses of fission yeast to environmental stress.Mol. Biol. Cell14: 214–229. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Chien C. T., Bartel P. L., Sternglanz R., Fields S., 1991. The two-hybrid system: a method to identify and clone genes for proteins that interact with a protein of interest.Proc. Natl. Acad. Sci. USA88: 9578–9582. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Chikashige Y., Hiraoka Y., 2001. Telomere binding of the Rap1 protein is required for meiosis in fission yeast.Curr. Biol.11: 1618–1623. [DOI] [PubMed] [Google Scholar]
- Clarke L., Amstutz H., Fishel B., Carbon J., 1986. Analysis of centromeric DNA in the fission yeast Schizosaccharomyces pombe.Proc. Natl. Acad. Sci. USA83: 8253–8257. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Cooper J. P., Nimmo E. R., Allshire R. C., Cech T. R., 1997. Regulation of telomere length and function by a Myb-domain protein in fission yeast.Nature385: 744–747. [DOI] [PubMed] [Google Scholar]
- Costello G., Rodgers L., Beach D., 1986. Fission yeast enters the stationary phase G0 state from either mitotic G1 or G2.Curr. Genet.11: 119–125. [Google Scholar]
- Coxon A., Maundrell K., Kearsey S. E., 1992. Fission yeast cdc21+ belongs to a family of proteins involved in an early step of chromosome replication.Nucleic Acids Res.20: 5571–5577. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Dalgaard J. Z., Klar A. J., 2001. A DNA replication-arrest site RTS1 regulates imprinting by determining the direction of replication at mat1 in S. pombe.Genes Dev.15: 2060–2068. [DOI] [PMC free article] [PubMed] [Google Scholar]
- de Medeiros A. S., Kwak G., Vanderhooft J., Rivera S., Gottlieb R., et al. , 2015. Fission yeast-based high-throughput screens for PKA pathway inhibitors and activators,pp. 77–91 in Chemical Biology: Methods and Protocols, edited by Hempel J. E., Williams C. H., Hong C. C.Springer, New York. [DOI] [PubMed] [Google Scholar]
- Doe C. L., Wang G., Chow C., Fricker M. D., Singh P. B., et al. , 1998. The fission yeast chromo domain encoding gene chp1(+) is required for chromosome segregation and shows a genetic interaction with alpha-tubulin.Nucleic Acids Res.26: 4222–4229. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Duina A. A., Miller M. E., Keeney J. B., 2014. Budding yeast for budding geneticists: a primer on the Saccharomyces cerevisiae model system.Genetics197: 33–48. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Durfee T., Becherer K., Chen P. L., Yeh S. H., Yang Y., et al. , 1993. The retinoblastoma protein associates with the protein phosphatase type 1 catalytic subunit.Genes Dev.7: 555–569. [DOI] [PubMed] [Google Scholar]
- Edwards R. J., Bentley N. J., Carr A. M., 1999. A Rad3-Rad26 complex responds to DNA damage independently of other checkpoint proteins.Nat. Cell Biol.1: 393–398. [DOI] [PubMed] [Google Scholar]
- Egel R., 1973. Commitment to meiosis in fission yeast.Mol. Gen. Genet.121: 277–284. [Google Scholar]
- Egel R., 1989. Mating-type genes, meiosis and sporulation, pp. 31–73 in Molecular Biology of the Fission Yeast, edited by Nasim A., Young P., Johnson B. F.Academic Press, San Diego. [Google Scholar]
- Egel R., 2004. The Molecular Biology of Schizosaccaromyces pombe: Genetics,Genomics,and Beyond. Springer, Berlin. [Google Scholar]
- Egel R., Beach D. H., Klar A. J., 1984. Genes required for initiation and resolution steps of mating-type switching in fission yeast.Proc. Natl. Acad. Sci. USA81: 3481–3485. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Egel R., Egel-Mitani M., 1974. Premeiotic DNA synthesis in fission yeast.Exp. Cell Res.88: 127–134. [DOI] [PubMed] [Google Scholar]
- Ekwall K., Javerzat J. P., Lorentz A., Schmidt H., Cranston G., et al. , 1995. The chromodomain protein Swi6: a key component at fission yeast centromeres.Science269: 1429–1431. [DOI] [PubMed] [Google Scholar]
- Ekwall K., Nimmo E. R., Javerzat J. P., Borgstrom B., Egel R., et al. , 1996. Mutations in the fission yeast silencing factors clr4+ and rik1+ disrupt the localisation of the chromo domain protein Swi6p and impair centromere function.J. Cell Sci.109: 2637–2648. [DOI] [PubMed] [Google Scholar]
- Elion E. A., Brill J. A., Fink G. R., 1991. FUS3 represses CLN1 and CLN2 and in concert with KSS1 promotes signal transduction.Proc. Natl. Acad. Sci. USA88: 9392–9396. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Enoch T., Carr A. M., Nurse P., 1992. Fission yeast genes involved in coupling mitosis to completion of DNA replication.Genes Dev.6: 2035–2046. [DOI] [PubMed] [Google Scholar]
- Fankhauser C., Marks J., Reymond A., Simanis V., 1993. The S. pombe cdc16 gene is required both for maintenance of p34cdc2 kinase activity and regulation of septum formation: a link between mitosis and cytokinesis?EMBO J.12: 2697–2704. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Fantes P. A., 1977. Control of cell size and cycle time in Schizosaccharomyces pombe.J. Cell Sci.24: 51–67. [DOI] [PubMed] [Google Scholar]
- Fantes P. A., 1989. Cell cycle controls, pp. 127–204 in Molecular Biology of the Fission Yeast, edited by Nasim A., Young P., Johnson B. F.Academic Press, San Diego. [Google Scholar]
- Fantes P. A., Nurse P., 1977. Control of cell size at division in fission yeast by a growth-modulated size control over nuclear division.Exp. Cell Res.107: 377–386. [DOI] [PubMed] [Google Scholar]
- Fantes P. A., Nurse P., 1978. Control of the timing of cell division in fission yeast. Cell size mutants reveal a second control pathway.Exp. Cell Res.115: 317–329. [DOI] [PubMed] [Google Scholar]
- Feilotter H., Nurse P., Young P. G., 1991. Genetic and molecular analysis of cdr1/nim1 in Schizosaccharomyces pombe.Genetics127: 309–318. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Feng G., Leem Y. E., Levin H. L., 2013. Transposon integration enhances expression of stress response genes.Nucleic Acids Res.41: 775–789. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Fisher D. L., Nurse P., 1996. A single fission yeast mitotic cyclin B p34cdc2 kinase promotes both S-phase and mitosis in the absence of G1 cyclins.EMBO J.15: 850–860. [PMC free article] [PubMed] [Google Scholar]
- Forbes S. A., Beare D., Gunasekaran P., Leung K., Bindal N., et al. , 2014. COSMIC: exploring the world’s knowledge of somatic mutations in human cancer.Nucleic Acids Res. 10.1093/nar/gku1075. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Forsburg S. L., 1993. Comparison of Schizosaccharomyces pombe expression systems.Nucleic Acids Res.21: 2955–2956. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Forsburg S. L., Nurse P., 1991. Cell cycle regulation in the yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe.Annu. Rev. Cell Biol.7: 227–256. [DOI] [PubMed] [Google Scholar]
- Forsburg S. L., Nurse P., 1994. The fission yeast cdc19+ gene encodes a member of the MCM family of replication proteins.J. Cell Sci.107: 2779–2788. [DOI] [PubMed] [Google Scholar]
- Forsburg S. L., Rhind N., 2006. Basic methods for fission yeast.Yeast23: 173–183. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Fowler K. R., Sasaki M., Milman N., Keeney S., Smith G. R., 2014. Evolutionarily diverse determinants of meiotic DNA break and recombination landscapes across the genome.Genome Res.24: 1650–1664. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Funabiki H., Hagan I., Uzawa S., Yanagida M., 1993. Cell cycle-dependent specific positioning and clustering of centromeres and telomeres in fission yeast.J. Cell Biol.121: 961–976. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Furnari B., Rhind N., Russell P., 1997. Cdc25 mitotic inducer targeted by chk1 DNA damage checkpoint kinase.Science277: 1495–1497. [DOI] [PubMed] [Google Scholar]
- Goffeau A., Barrell B. G., Bussey H., Davis R. W., Dujon B., et al. , 1996. Life with 6000 genes.Science274: 546–547. [DOI] [PubMed] [Google Scholar]
- Gomes F. C., Pataro C., Guerra J. B., Neves M. J., Correa S. R., et al. , 2002. Physiological diversity and trehalose accumulation in Schizosaccharomyces pombe strains isolated from spontaneous fermentations during the production of the artisanal Brazilian cachaca.Can. J. Microbiol.48: 399–406. [DOI] [PubMed] [Google Scholar]
- Goodman A. J., Daugharthy E. R., Kim J., 2013. Pervasive antisense transcription is evolutionarily conserved in budding yeast.Mol. Biol. Evol.30: 409–421. [DOI] [PubMed] [Google Scholar]
- Goshima G., Saitoh S., Yanagida M., 1999. Proper metaphase spindle length is determined by centromere proteins Mis12 and Mis6 required for faithful chromosome segregation.Genes Dev.13: 1664–1677. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Gould K. L., Burns C. G., Feoktistova A., Hu C. P., Pasion S. G., et al. , 1998. Fission yeast cdc24(+) encodes a novel replication factor required for chromosome integrity.Genetics149: 1221–1233. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Gould K. L., Nurse P., 1989. Tyrosine phosphorylation of the fission yeast cdc2+ protein kinase regulates entry into mitosis.Nature342: 39–45. [DOI] [PubMed] [Google Scholar]
- Griffiths D. J., Barbet N. C., McCready S., Lehmann A. R., Carr A. M., 1995. Fission yeast rad17: a homologue of budding yeast RAD24 that shares regions of sequence similarity with DNA polymerase accessory proteins.EMBO J.14: 5812–5823. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Guo Y., Levin H. L., 2010. High-throughput sequencing of retrotransposon integration provides a saturated profile of target activity in Schizosaccharomyces pombe.Genome Res.20: 239–248. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Guo Y., Park J. M., Cui B., Humes E., Gangadharan S., et al. , 2013. Integration profiling of gene function with dense maps of transposon integration.Genetics195: 599–609. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Gutz H., Heslot H., Leupold U., Loprieno N., 1974. Schizosaccharomyces pombe, pp. 395–446 in Handbook of Genetics, edited by King R. C.Plenum Press, New York. [Google Scholar]
- Hagan I. M., Carr A. M., Grallert A., Nurse P., 2016. Fission Yeast: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. [Google Scholar]
- Halme A., Bumgarner S., Styles C., Fink G. R., 2004. Genetic and epigenetic regulation of the FLO gene family generates cell-surface variation in yeast.Cell116: 405–415. [DOI] [PubMed] [Google Scholar]
- Hansen K. R., Burns G., Mata J., Volpe T. A., Martienssen R. A., et al. , 2005. Global effects on gene expression in fission yeast by silencing and RNA interference machineries.Mol. Cell. Biol.25: 590–601. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Harris M. A., Lock A., Bahler J., Oliver S. G., Wood V., 2013. FYPO: the fission yeast phenotype ontology.Bioinformatics29: 1671–1678. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Hartmuth S., Petersen J., 2009. Fission yeast Tor1 functions as part of TORC1 to control mitotic entry through the stress MAPK pathway following nutrient stress.J. Cell Sci.122: 1737–1746. [DOI] [PubMed] [Google Scholar]
- Hartwell L. H., Culotti J., Pringle J. R., Reid B. J., 1974. Genetic control of the cell division cycle in yeast.Science183: 46–51. [DOI] [PubMed] [Google Scholar]
- Hauf S., 2013. The spindle assembly checkpoint: progress and persistent puzzles.Biochem. Soc. Trans.41: 1755–1760. [DOI] [PubMed] [Google Scholar]
- Hayles J., Wood V., Jeffery L., Hoe K. L., Kim D. U., et al. , 2013. A genome-wide resource of cell cycle and cell shape genes of fission yeast.Open Biol.3: 130053. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Heckman D. S., Geiser D. M., Eidell B. R., Stauffer R. L., Kardos N. L., et al. , 2001. Molecular evidence for the early colonization of land by fungi and plants.Science293: 1129–1133. [DOI] [PubMed] [Google Scholar]
- Hirano T., Funahashi S., Uemura T., Yanagida M., 1986. Isolation and characterization of Schizosaccharomyces pombe cutmutants that block nuclear division but not cytokinesis.EMBO J.5: 2973–2979. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Hirota K., Miyoshi T., Kugou K., Hoffman C. S., Shibata T., et al. , 2008. Stepwise chromatin remodelling by a cascade of transcription initiation of non-coding RNAs.Nature456: 130–134. [DOI] [PubMed] [Google Scholar]
- Hoff E. F., Levin H. L., Boeke J. D., 1998. Schizosaccharomyces pombe retrotransposon Tf2 mobilizes primarily through homologous cDNA recombination.Mol. Cell. Biol.18: 6839–6852. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Hoffman R. L., Hoffman C. S., 2006. Cloning the Schizosaccharomyces pombe lys2+ gene and construction of new molecular genetic tools.Curr. Genet.49: 414–420. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Ivey F. D., Taglia F. X., Yang F., Lander M. M., Kelly D. A., et al. , 2010. Activated alleles of the Schizosaccharomyces pombe gpa2+ Gα gene identify residues involved in GDP-GTP exchange.Eukaryot. Cell9: 626–633. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Jacobs J. Z., Ciccaglione K. M., Tournier V., Zaratiegui M., 2014. Implementation of the CRISPR-Cas9 system in fission yeast.Nat. Commun.5: 5344. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Janoo R. T., Neely L. A., Braun B. R., Whitehall S. K., Hoffman C. S., 2001. Transcriptional regulators of the Schizosaccharomyces pombe fbp1 gene include two redundant Tup1p-like corepressors and the CCAAT binding factor activation complex.Genetics157: 1205–1215. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Jenuwein T., Allis C. D., 2001. Translating the histone code.Science293: 1074–1080. [DOI] [PubMed] [Google Scholar]
- Kahyo T., Iwaizumi M., Shinmura K., Matsuura S., Nakamura T., et al. , 2011. A novel tumor-derived SGOL1 variant causes abnormal mitosis and unstable chromatid cohesion.Oncogene30: 4453.–. [DOI] [PubMed] [Google Scholar]
- Keller C., Kulasegaran-Shylini R., Shimada Y., Hotz H. R., Buhler M., 2013. Noncoding RNAs prevent spreading of a repressive histone mark.Nat. Struct. Mol. Biol.20: 994.–. [DOI] [PubMed] [Google Scholar]
- Kellis M., Birren B. W., Lander E. S., 2004. Proof and evolutionary analysis of ancient genome duplication in the yeast Saccharomyces cerevisiae.Nature428: 617.–. [DOI] [PubMed] [Google Scholar]
- Kelly T. J., Martin G. S., Forsburg S. L., Stephen R. J., Russo A., et al. , 1993. The fission yeast cdc18+ gene product couples S phase to START and mitosis.Cell74: 371.–. [DOI] [PubMed] [Google Scholar]
- Kim D. U., Hayles J., Kim D., Wood V., Park H. O., et al. , 2010. Analysis of a genome-wide set of gene deletions in the fission yeast Schizosaccharomyces pombe.Nat. Biotechnol.28: 617.–. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Kitajima T. S., Hauf S., Ohsugi M., Yamamoto T., Watanabe Y., 2005. Human Bub1 defines the persistent cohesion site along the mitotic chromosome by affecting Shugoshin localization.Curr. Biol.15: 353.–. [DOI] [PubMed] [Google Scholar]
- Kitajima T. S., Kawashima S. A., Watanabe Y., 2004. The conserved kinetochore protein shugoshin protects centromeric cohesion during meiosis.Nature427: 510.–. [DOI] [PubMed] [Google Scholar]
- Koboldt D. C., Steinberg K. M., Larson D. E., Wilson R. K., Mardis E. R., 2013. The next-generation sequencing revolution and its impact on genomics.Cell155: 27.–. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Kong X., Murphy K., Raj T., He C., White P. S., et al. , 2004. A combined linkage-physical map of the human genome.Am. J. Hum. Genet.75: 1143.–. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Kostrub C. F., al-Khodairy F., Ghazizadeh H., Carr A. M., Enoch T., 1997. Molecular analysis of hus1+, a fission yeast gene required for S-M and DNA damage checkpoints.Mol. Gen. Genet.254: 389.–. [DOI] [PubMed] [Google Scholar]
- Kostrub C. F., Lei E. P., Enoch T., 1998. Use of gap repair in fission yeast to obtain novel alleles of specific genes.Nucleic Acids Res.26: 4783.–. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Krings G., Bastia D., 2005. Sap1p binds to Ter1 at the ribosomal DNA of Schizosaccharomyces pombe and causes polar replication fork arrest.J. Biol. Chem.280: 39135.–. [DOI] [PubMed] [Google Scholar]
- Krupp G., Cherayil B., Frendewey D., Nishikawa S., Soll D., 1986. Two RNA species co-purify with RNase P from the fission yeast Schizosaccharomyces pombe.EMBO J.5: 1697.–. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Lee M. G., Nurse P., 1987. Complementation used to clone a human homologue of the fission yeast cell cycle control gene cdc2.Nature327: 31.–. [DOI] [PubMed] [Google Scholar]
- Leupold U., 1950. Die Vererbung von Homothallie und Heterothallie bei Schizosaccharomyces pombe.C.R. Trav. Lab. Carlsberg. Ser. Physiol.24: 381.–. [Google Scholar]
- Leupold U., 1993. The origin ofSchizosaccharomyces pombe genetics, pp. 125.– in The Early Days of Yeast Genetics, edited by Hall M. N., Linder P.Cols Spring Harbor Laboratory Press, Cold Spring Harbor, NY. [Google Scholar]
- Levin H. L., Boeke J. D., 1992. Demonstration of retrotransposition of the Tf1 element in fission yeast.EMBO J.11: 1145–1153. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Levin H. L., Weaver D. C., Boeke J. D., 1990. Two related families of retrotransposons from Schizosaccharomyces pombe.Mol. Cell. Biol.10: 6791–6798. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Li J., Du L. L., 2014. piggyBac transposon-based insertional mutagenesis for the fission yeast Schizosaccharomyces pombe.Methods Mol. Biol.1163: 213–222. [DOI] [PubMed] [Google Scholar]
- Lorentz A., Ostermann K., Fleck O., Schmidt H., 1994. Switching gene swi6, involved in repression of silent mating-type loci in fission yeast, encodes a homologue of chromatin-associated proteins from Drosophila and mammals.Gene143: 139–143. [DOI] [PubMed] [Google Scholar]
- MacNeill S. A., Moreno S., Reynolds N., Nurse P., Fantes P. A., 1996. The fission yeast Cdc1 protein, a homologue of the small subunit of DNA polymerase delta, binds to Pol3 and Cdc27.EMBO J.15: 4613–4628. [PMC free article] [PubMed] [Google Scholar]
- Marguerat S., Schmidt A., Codlin S., Chen W., Aebersold R., et al. , 2012. Quantitative analysis of fission yeast transcriptomes and proteomes in proliferating and quiescent cells.Cell151: 671–683. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Martin-Castellanos C., Blanco M., Rozalen A. E., Perez-Hidalgo L., Garcia A. I., et al. , 2005. A large-scale screen in S. pombe identifies seven novel genes required for critical meiotic events.Curr. Biol.15: 2056–2062. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Mata J., Lyne R., Burns G., Bahler J., 2002. The transcriptional program of meiosis and sporulation in fission yeast.Nat. Genet.32: 143–147. [DOI] [PubMed] [Google Scholar]
- Matsuo Y., Nishino K., Mizuno K., Akihiro T., Toda T., et al. , 2013. Polypeptone induces dramatic cell lysis in ura4 deletion mutants of fission yeast.PLoS One8: e59887. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Matsuyama A., Arai R., Yashiroda Y., Shirai A., Kamata A., et al. , 2006. ORFeome cloning and global analysis of protein localization in the fission yeast Schizosaccharomyces pombe.Nat. Biotechnol.24: 841–847. [DOI] [PubMed] [Google Scholar]
- Maundrell K., 1993. Thiamine-repressible expression vectors pREP and pRIP for fission yeast.Gene123: 127–130. [DOI] [PubMed] [Google Scholar]
- McCollum D., Gould K. L., 2001. Timing is everything: regulation of mitotic exit and cytokinesis by the MEN and SIN.Trends Cell Biol.11: 89–95. [DOI] [PubMed] [Google Scholar]
- McDowall M. D., Harris M. A., Lock A., Rutherford K., Staines D. M., et al. , 2014. PomBase 2015: updates to the fission yeast database. Nucleic Acids Res. 10.1093/nar/gku1040. [DOI] [PMC free article] [PubMed]
- McLeod M., Stein M., Beach D., 1987. The product of the mei3+ gene, expressed under control of the mating-type locus, induces meiosis and sporulation in fission yeast.EMBO J.6: 729–736. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Mejia-Ramirez E., Sanchez-Gorostiaga A., Krimer D. B., Schvartzman J. B., Hernandez P., 2005. The mating type switch-activating protein Sap1 Is required for replication fork arrest at the rRNA genes of fission yeast.Mol. Cell. Biol.25: 8755–8761. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Milne A. A., 1928. The House at Pooh Corner.E. P. Dutton, New York. [Google Scholar]
- Mitchell A., Chang H. Y., Daugherty L., Fraser M., Hunter S., et al. , 2015. The InterPro protein families database: the classification resource after 15 years.Nucleic Acids Res.43: D213–221. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Mitchison J. M., 1971. The control of division, pp. 201–250 in The Biology of the Cell Cycle. Cambridge University Press, Cambridge, UK. [Google Scholar]
- Mitchison J. M., 1990. My favourite cell: the fission yeast, Schizosaccharomyces pombe.BioEssays12: 189–191. [DOI] [PubMed] [Google Scholar]
- Mojardin L., Vazquez E., Antequera F., 2013. Specification of DNA replication origins and genomic base composition in fission yeasts.J. Mol. Biol.425: 4706–4713. [DOI] [PubMed] [Google Scholar]
- Molnar M., Parisi S., Kakihara Y., Nojima H., Yamamoto A., et al. , 2001. Characterization of rec7, an early meiotic recombination gene in Schizosaccharomyces pombe.Genetics157: 519–532. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Moreno S., Klar A., Nurse P., 1991. Molecular genetic analysis of fission yeast Schizosaccharomyces pombe.Methods Enzymol.194: 795–823. [DOI] [PubMed] [Google Scholar]
- Murakami H., Okayama H., 1995. A kinase from fission yeast responsible for blocking mitosis in S phase.Nature374: 817–819. [DOI] [PubMed] [Google Scholar]
- Murray J. M., Carr A. M., Lehmann A. R., Watts F. Z., 1991. Cloning and characterisation of the rad9 DNA repair gene from Schizosaccharomyces pombe.Nucleic Acids Res.19: 3525–3531. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Nakayama J., Rice J. C., Strahl B. D., Allis C. D., Grewal S. I., 2001. Role of histone H3 lysine 9 methylation in epigenetic control of heterochromatin assembly.Science292: 110–113. [DOI] [PubMed] [Google Scholar]
- Nasim A., Smith B. P., 1975. Genetic control of radiation sensitivity in Schizosaccharomyces pombe.Genetics79: 573–582. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Nasim A., Young P., Johnson B. F., 1989. Molecular Biology of the Fission Yeast. Academic Press, San Diego. [Google Scholar]
- Nasmyth K., Nurse P., Fraser R. S., 1979. The effect of cell mass on the cell cycle timing and duration of S-phase in fission yeast.J. Cell Sci.39: 215–233. [DOI] [PubMed] [Google Scholar]
- Nimmo E. R., Pidoux A. L., Perry P. E., Allshire R. C., 1998. Defective meiosis in telomere-silencing mutants of Schizosaccharomyces pombe.Nature392: 825–828. [DOI] [PubMed] [Google Scholar]
- Nurse P., 1975. Genetic control of cell size at cell division in yeast.Nature256: 547–551. [DOI] [PubMed] [Google Scholar]
- Nurse P., Bissett Y., 1981. Gene required in G1 for commitment to cell cycle and in G2 for control of mitosis in fission yeast.Nature292: 558–560. [DOI] [PubMed] [Google Scholar]
- Nurse P., Thuriaux P., 1980. Regulatory genes controlling mitosis in the fission yeast Schizosaccharomyces pombe.Genetics96: 627–637. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Nurse P., Thuriaux P., Nasmyth K., 1976. Genetic control of the cell division cycle in the fission yeast Schizosaccharomyces pombe.Mol. Gen. Genet.146: 167–178. [DOI] [PubMed] [Google Scholar]
- Ogden J. E., Fantes P. A., 1986. Isolation of a novel type of mutation in the mitotic control of Schizosaccharomyces pombe whose phenotypic expression is dependent on the genetic background and nutritional environment.Curr. Genet.10: 509–514. [DOI] [PubMed] [Google Scholar]
- Ohi R., Feoktistova A., Gould K. L., 1996. Construction of vectors and a genomic library for use with his3- deficient strains of Schizosaccharomyces pombe.Gene174: 315–318. [DOI] [PubMed] [Google Scholar]
- Olson M. V., Dutchik J. E., Graham M. Y., Brodeur G. M., Helms C., et al. , 1986. Random-clone strategy for genomic restriction mapping in yeast.Proc. Natl. Acad. Sci. USA83: 7826–7830. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Orr-Weaver T. L., Szostak J. W., Rothstein R. J., 1981. Yeast transformation: a model system for the study of recombination.Proc. Natl. Acad. Sci. USA78: 6354–6358. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Paluh J. L., Clayton D. A., 1995. Schizosaccharomyces pombe RNase MRP RNA is homologous to metazoan RNase MRP RNAs and may provide clues to interrelationships between RNase MRP and RNase P.Yeast11: 1249–1264. [DOI] [PubMed] [Google Scholar]
- Park J. M., Evertts A. G., Levin H. L., 2009. The Hermes transposon of Musca domestica and its use as a mutagen of Schizosaccharomyces pombe.Methods49: 243–247. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Pelechano V., Wei W., Steinmetz L. M., 2013. Extensive transcriptional heterogeneity revealed by isoform profiling.Nature497: 127–131. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Petersen J., Nurse P., 2007. TOR signalling regulates mitotic commitment through the stress MAP kinase pathway and the Polo and Cdc2 kinases.Nat. Cell Biol.9: 1263–1272. [DOI] [PubMed] [Google Scholar]
- Peterson J. B., Ris H., 1976. Electron-microscopic study of the spindle and chromosome movement in the yeast Saccharomyces cerevisiae.J. Cell Sci.22: 219–242. [DOI] [PubMed] [Google Scholar]
- Raleigh J. M., O’Connell M. J., 2000. The G(2) DNA damage checkpoint targets both Wee1 and Cdc25.J. Cell Sci.113: 1727–1736. [DOI] [PubMed] [Google Scholar]
- Ralser M., Kuhl H., Ralser M., Werber M., Lehrach H., et al. , 2012. The Saccharomyces cerevisiae W303–K6001 cross-platform genome sequence: insights into ancestry and physiology of a laboratory mutt.Open Biol.2: 120093. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Rhind N., Chen Z., Yassour M., Thompson D. A., Haas B. J., et al. , 2011. Comparative functional genomics of the fission yeasts.Science332: 930–936. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Ribes V., Dehoux P., Tollervey D., 1988. 7SL RNA from Schizosaccharomyces pombe is encoded by a single copy essential gene.EMBO J.7: 231–237. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Robyr D., Suka Y., Xenarios I., Kurdistani S. K., Wang A., et al. , 2002. Microarray deacetylation maps determine genome-wide functions for yeast histone deacetylases.Cell109: 437–446. [DOI] [PubMed] [Google Scholar]
- Russell P. R., Hall B. D., 1983. The primary structure of the alcohol dehydrogenase gene from the fission yeast Schizosaccharomyces pombe.J. Biol. Chem.258: 143–149. [PubMed] [Google Scholar]
- Russell P., Nurse P., 1986. cdc25+ functions as an inducer in the mitotic control of fission yeast.Cell45: 145–153. [DOI] [PubMed] [Google Scholar]
- Russell P., Nurse P., 1987. Negative regulation of mitosis by wee1+, a gene encoding a protein kinase homolog.Cell49: 559–567. [DOI] [PubMed] [Google Scholar]
- Ryan C. J., Roguev A., Patrick K., Xu J., Jahari H., et al. , 2012. Hierarchical modularity and the evolution of genetic interactomes across species.Mol. Cell46: 691–704. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Salic A., Waters J. C., Mitchison T. J., 2004. Vertebrate shugoshin links sister centromere cohesion and kinetochore microtubule stability in mitosis.Cell118: 567–578. [DOI] [PubMed] [Google Scholar]
- Samejima I., Matsumoto T., Nakaseko Y., Beach D., Yanagida M., 1993. Identification of seven new cut genes involved in Schizosaccharomyces pombe mitosis.J. Cell Sci.105: 135–143. [DOI] [PubMed] [Google Scholar]
- Sancar A., Lindsey-Boltz L. A., Unsal-Kacmaz K., Linn S., 2004. Molecular mechanisms of mammalian DNA repair and the DNA damage checkpoints.Annu. Rev. Biochem.73: 39–85. [DOI] [PubMed] [Google Scholar]
- Sanchez Y., Bachant J., Wang H., Hu F., Liu D., et al. , 1999. Control of the DNA damage checkpoint by chk1 and rad53 protein kinases through distinct mechanisms.Science286: 116––1171. [DOI] [PubMed] [Google Scholar]
- Sanchez-Gorostiaga A., Lopez-Estrano C., Krimer D. B., Schvartzman J. B., Hernandez P., 2004. Transcription termination factor reb1p causes two replication fork barriers at its cognate sites in fission yeast ribosomal DNA in vivo.Mol. Cell. Biol.24: 398–406. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Sato M., Dhut S., Toda T., 2005. New drug-resistant cassettes for gene disruption and epitope tagging in Schizosaccharomyces pombe.Yeast22: 583–591. [DOI] [PubMed] [Google Scholar]
- Schwede T., Kopp J., Guex N., Peitsch M. C., 2003. SWISS-MODEL: an automated protein homology-modeling server.Nucleic Acids Res.31: 3381–3385. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Simanis V., Nurse P., 1986. The cell cycle control gene cdc2+ of fission yeast encodes a protein kinase potentially regulated by phosphorylation.Cell45: 261–268. [DOI] [PubMed] [Google Scholar]
- Sipiczki M., 2000. Where does fission yeast sit on the tree of life?Genome Biol.1: 1011. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Sipiczki M., 2004. Fission yeast phylogenesis and evolution, pp. 431–443 inThe Molecular Biology of Schizosaccharomyces pombe: Genetics,Genomics,and beyond, edited by Egel R.Springer, Berlin. [Google Scholar]
- Sunnerhagen P., Seaton B. L., Nasim A., Subramani S., 1990. Cloning and analysis of a gene involved in DNA repair and recombination, the rad1 gene of Schizosaccharomyces pombe.Mol. Cell. Biol.10: 3750–3760. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Tabara H., Sarkissian M., Kelly W. G., Fleenor J., Grishok A., et al. , 1999. The rde-1 gene, RNA interference, and transposon silencing in C. elegans.Cell99: 123–132. [DOI] [PubMed] [Google Scholar]
- Tange Y., Niwa O., 1995. A selection system for diploid and against haploid cells in Schizosaccharomyces pombe.Mol. Gen. Genet.248: 644–648. [DOI] [PubMed] [Google Scholar]
- Tasto J. J., Carnahan R. H., McDonald W. H., Gould K. L., 2001. Vectors and gene targeting modules for tandem affinity purification in Schizosaccharomyces pombe.Yeast18: 657–662. [DOI] [PubMed] [Google Scholar]
- Thon G., Cohen A., Klar A. J., 1994. Three additional linkage groups that repress transcription and meiotic recombination in the mating-type region of Schizosaccharomyces pombe.Genetics138: 29–38. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Thon G., Klar A. J., 1992. The clr1 locus regulates the expression of the cryptic mating-type loci of fission yeast.Genetics131: 287–296. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Verdel A., Jia S., Gerber S., Sugiyama T., Gygi S., et al. , 2004. RNAi-mediated targeting of heterochromatin by the RITS complex.Science303: 672–676. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Volpe T. A., Kidner C., Hall I. M., Teng G., Grewal S. I., et al. , 2002. Regulation of heterochromatic silencing and histone H3 lysine-9 methylation by RNAi.Science297: 1833–1837. [DOI] [PubMed] [Google Scholar]
- Volpe T., Schramke V., Hamilton G. L., White S. A., Teng G., et al. , 2003. RNA interference is required for normal centromere function in fission yeast.Chromosome Res.11: 137–146. [DOI] [PubMed] [Google Scholar]
- Volschenk H., van Vuuren H. J., Viljoen-Bloom M., 2003. Malo-ethanolic fermentation in Saccharomyces and Schizosaccharomyces.Curr. Genet.43: 379–391. [DOI] [PubMed] [Google Scholar]
- Walworth N., Davey S., Beach D., 1993. Fission yeast chk1 protein kinase links the rad checkpoint pathway to cdc2.Nature363: 368–371. [DOI] [PubMed] [Google Scholar]
- Wang L., Griffiths K., Zhang Y. H., Ivey F. D., Hoffman C. S., 2005. Schizosaccharomyces pombe adenylate cyclase suppressor mutations suggest a role for cAMP phosphodiesterase regulation in feedback control of glucose/cAMP signaling.Genetics171: 1523–1533. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Washington N., Lewis S., 2008. Ontologies: scientific data sharing made easy.Nature Education1: 5. [Google Scholar]
- Watanabe T., Miyashita K., Saito T. T., Nabeshima K., Nojima H., 2002. Abundant poly(A)-bearing RNAs that lack open reading frames in Schizosaccharomyces pombe.DNA Res.9: 209–215. [DOI] [PubMed] [Google Scholar]
- Watanabe Y., Nurse P., 1999. Cohesin Rec8 is required for reductional chromosome segregation at meiosis.Nature400: 461–464. [DOI] [PubMed] [Google Scholar]
- Watanabe Y., Yamamoto M., 1994. S. pombe mei2+ encodes an RNA-binding protein essential for premeiotic DNA synthesis and meiosis I, which cooperates with a novel RNA species meiRNA.Cell78: 487–498. [DOI] [PubMed] [Google Scholar]
- Watson A. T., Daigaku Y., Mohebi S., Etheridge T. J., Chahwan C., et al. , 2013. Optimisation of the Schizosaccharomyces pombe urg1 expression system.PLoS One8: e83800. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Watson A. T., Werler P., Carr A. M., 2011. Regulation of gene expression at the fission yeast Schizosaccharomyces pombe urg1 locus.Gene484: 75–85. [DOI] [PubMed] [Google Scholar]
- Wilhelm B. T., Marguerat S., Watt S., Schubert F., Wood V., et al. , 2008. Dynamic repertoire of a eukaryotic transcriptome surveyed at single-nucleotide resolution.Nature453: 1239–1243. [DOI] [PubMed] [Google Scholar]
- Wolfe K. H., Shields D. C., 1997. Molecular evidence for an ancient duplication of the entire yeast genome.Nature387: 708–713. [DOI] [PubMed] [Google Scholar]
- Wood V., 2006. How to get the most from fission yeast genome data: a report from the 2006 European Fission Yeast Meeting computing workshop.Yeast23: 905–912. [DOI] [PubMed] [Google Scholar]
- Wood V., Gwilliam R., Rajandream M. A., Lyne M., Lyne R., et al. , 2002. The genome sequence of Schizosaccharomyces pombe.Nature415: 871–880. [DOI] [PubMed] [Google Scholar]
- Wood V., Harris M. A., McDowall M. D., Rutherford K., Vaughan B. W., et al. , 2012. PomBase: a comprehensive online resource for fission yeast.Nucleic Acids Res.40: D695–699. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Yamada H. Y., Yao Y., Wang X., Zhang Y., Huang Y., et al. , 2012. Haploinsufficiency of SGO1 results in deregulated centrosome dynamics, enhanced chromosomal instability and colon tumorigenesis.Cell Cycle11: 479–488. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Yassour M., Pfiffner J., Levin J. Z., Adiconis X., Gnirke A., et al. , 2010. Strand-specific RNA sequencing reveals extensive regulated long antisense transcripts that are conserved across yeast species.Genome Biol.11: R87. [DOI] [PMC free article] [PubMed] [Google Scholar]