A mating deficient and temperature-sensitive lethal mutant of Schizosaccharomyces pombe defines a new fertility locus

Summary A recessive mutant allele, mef1-84, of a novel locus mapping on the left arm of chromosome I, between ade3 and ura1, 5 cM apart from lys5, confers temperature-sensitive growth and mating deficiency at the nonrestrictive temperatures for growth. Two other mutations suppress the phenotype conferred by mef1-84: sts1-1 suppresses the temperature-sensitive growth only, and smd1-35 suppresses both temperature-sensitive growth and mating deficiency.     

Construction and characterization of centric circular and acentric linear chromosomes in fission yeast

Summary Gene disruption and gap repair of chromosomal DNA have been frequently employed techniques in yeast genetics. To extend the possibility of using these gene manipulations for larger genomic regions, we have examined the maximal sizes of chromosomal DNA disrupted or repaired in vivo. Here we report a simple, potentially general, method for selectively deleting a 150 kb region, or gap-filling a 100 kb region, in the fission yeast genome. This enables the generation of acentric linear chromosomes by deletion, or the cloning of large functional centromeric DNAs into circular minichromosomes by gap-filling. The fidelity of the resulting gap-filling is high, judging from partial-digestion mapping of gap-repaired DNAs. By analysing a series of such circular minichromosomes, we conclude that only a part of the repetitive centromeric region, including the central domain, is essential for mitotic and meiotic chromosome segregation. Acentric linear chromosomes, although unstable, could be maintained, indicating that it may be possible to construct an acentric vector for large DNA fragments in this organism.     

Unblocking of meiotic crossing-over between the silent mating-type cassettes of fission yeast,conditioned by the recessive,pleiotropic mutant rik1

Summary The mating-type region of Schizosaccharomyces pombe consists of three subloci: the expressed cassette at mat1, and the silent cassettes at mat2-P and mat3-M. Previous work has shown that the genetically inert spacer region of 15 kb between mat2 and mat3 is completely devoid of meiotic recombination. This crossover blockage is lifted in the recessive mutant rik1. Other properties such as mating-type switching, sporulation efficiency and spore viability are also affected in this pleiotropic mutant. Presumably the wild-type rik1 product is responsible for heterochromatinization throughout the silent domain of the mating-type region.     

Schizosaccharomyces pombe: A novel transport vehicle of functional DNA and mRNA into mammalian antigen-presenting cells

Vaccine vehicles based on recombinant yeasts have become promising candidates for the induction of cellular immune responses. In this study, we investigated the capacity of the fission yeast Sz. pombe for the delivery of functional nucleic acids into murine and human antigen-presenting cells. We demonstrate that Sz. pombe cells effectively induce maturation of human dendritic cells (DC), an important prerequisite for T-cell activation. Further, recombinant fission yeast efficiently delivers functional DNA and mRNA into murine macrophages and human DC resulting in the expression of the model antigen eGFP in these cells. Thus, Sz. pombe suggests itself as a promising candidate for a novel live vaccine.     

Mitotic spindle formation in the absence of Polo kinase

Mitosis is a fundamental process in every eukaryote, in which chromosomes are segregated into two daughter cells by the action of the microtubule (MT)-based spindle. Despite this common principle, genes essential for mitosis are variable among organisms. This indicates that the loss of essential genes or bypass of essentiality (BOE) occurred multiple times during evolution. While many BOE relationships have been recently revealed experimentally, the bypass of essentiality of mitosis regulators (BOE-M) has been scarcely reported, and how this occurs remains largely unknown. Here, by mutagenesis and subsequent evolutionary repair experiments, we isolated viable fission yeast strains that lacked the entire coding region of Polo-like kinase (Plk), a versatile essential mitotic kinase. The BOE of Plk was enabled by specific mutations in the downstream machinery, including the MT-nucleating γ-tubulin complex, and more surprisingly, through down-regulation of glucose uptake, which is not readily connected to mitosis. The latter bypass was dependent on casein kinase I (CK1), which has not been considered as a major mitotic regulator. Our genetic and phenotypic data suggest that CK1 constitutes an alternative mechanism of MT nucleation, which is normally dominated by Plk. A similar relationship was observed in a human colon cancer cell line. Thus, our study shows that BOE-M can be achieved by simple genetic or environmental changes, consistent with the occurrence of BOE-M during evolution. Furthermore, the identification of BOE-M constitutes a powerful means to uncover a hitherto understudied mechanism driving mitosis and also hints at the limitations and solutions for selecting chemotherapeutic compounds targeting mitosis.

Different organisms have different sets of essential genes for their viability and propagation (1, 2). This indicates that most “essential” genes are context dependent and can become dispensable during evolution. Plausibly, a loss of essentiality is compensated for by manifestation of an alternative, currently “masked,” or far less active mechanism to ensure a similar cellular activity. Many experimental efforts have been made to recapitulate the molecular diversity found in nature (3). Large-scale systematic surveys have been recently conducted in budding and fission yeasts, in which a number of bypass-of-essentiality (BOE) events have been identified (4–7). In these studies, suppressors were screened for haploid strains, in which an essential gene was experimentally disrupted. For 9 to 27% of the essential gene disruptants, a mutation or overexpression of other gene(s) or chromosomal gain makes the strain viable, indicating that essentiality depends on genetic background and that BOE could indeed occur at a certain frequency. However, in most cases, the underlying mechanism remains unexplored. It is also unclear why BOE is rarely observed in certain processes, such as mitotic cell division.Mitotic cell division is controlled by many essential genes in a given cell type (8–10). Evolutionary evidence of BOE is clearly visible for this fundamentally critical biological process. One striking example is the centrosome, which is assembled by the action of many essential proteins in animal, fungal, and algal species and plays a vital role in cell division and cellular motility (11). However, the centrosome and most of its components have been lost in land plants, and yet, plant cells undergo spindle assembly and chromosome segregation at high fidelity (12). Kinetochore components, such as the Constitutive Centromere Associated Network (CCAN); spindle microtubule (MT)–associated proteins, such as TPX2, augmin, and mitotic motors; and cell cycle regulators, such as anaphase-promoting complex/cyclosome, are among other examples. They are not universally conserved or essential factors (12–15). Despite the evidence of BOE for almost all the genes involved in mitosis, only a limited number of cases can be found in experimental BOE screening. For example, two random BOE screenings in fission yeast encompassing 23 mitotic genes have identified only a single protein, Cnp20/CENP-T, despite the fact that >20% BOE has been observed for mitosis-unrelated genes (6, 7). The BOE of cnp20 is conceivable, as CENP-T functions in parallel with CENP-C for kinetochore assembly (16, 17). Another known bypass of essentiality of mitosis regulators (BOE-M) in fission yeast is MT plus end–directed kinesin-5/Cut7, which is required for bipolar spindle formation through force generation on spindle MTs. The viability of cut7Δ was restored when the opposing minus end–directed kinesin-14/Pkl1 was simultaneously deleted. Thus, the balance of forces applied to spindle MTs is critical (18–20). However, many other essential mitotic genes have no apparent functionally redundant or counteracting factors, and whether these relationships are general mechanisms of BOE-M is unclear. Essential mitotic genes are potential targets of cancer chemotherapy (21); it is also important to understand the BOE that underlies drug resistance.In this study, we found that the essentiality of the sole Polo-like kinase (Plk) in fission yeast (Plo1) can be bypassed. Plo1, similar to human Plk1, is assumed to be essential for spindle MT formation and spindle bipolarization. However, these essential processes were restored in the absence of Plo1 by multiple independent mechanisms that increase MT nucleation and stabilization, one of which involved a remarkably simple change in glucose concentration in the culture medium and depended on casein kinase I (CK1). Thus, our study uncovered an unexpected alternative mechanism of spindle MT formation and further implies that more BOE-M can be recapitulated in the experimental system.     

A fission yeast strain expressing human CDC25A phosphatase: a tool for selectivity studies of pharmacological inhibitors of CDC25

Fission yeast is a simple eukaryotic model organism in which many aspects of cell cycle control can be explored. We examined by homologous recombination whether the human CDC25A phosphatase could substitute for the function of the fission yeast Cdc25. We first show: (a) that CDC25A efficiently replaces the endogenous Cdc25 mitotic inducer for vegetative growth and (b) that CDC25A is able to partially restore a functional checkpoint in response to both ionising and UV irradiation, but not a DNA replication checkpoint. We then describe a simple assay in which we demonstrate that growth of the humanised CDC25A strain is strongly repressed in a CDC25-dependent manner by BN2003, a potent chemical inhibitor of CDC25 belonging to the benzothiazoledione family. The ease of manipulation of fission yeast humanised CDC25 cells and the simplicity of the above assay offer a powerful tool with which to investigate the specificity of pharmacological inhibitors of CDC25.Communicated by M. Yamamoto     

Regulation of pho1-encoded acid phosphatase of Schizosaccharomyces pombe by adenine and phosphate

Summary Expression of pho1-encodd acid phosphatase of Schizosaccharomyces pombe has been reported to be regulated by phosphate. In this communication we show that it is also regulated by adenine. Starving adenine auxotrophic strains for adenine leads to a drastic increase of the enzymatic activity while adenine represses this activity. Full repression by adenine only occurs when phosphate is not growth limiting and vice versa. Regulation occurs at the level of mRNA. We isolated adenine non-repressible mutants. They define four genes (anr1, anr2, anr3, and anr5) which are involved in adenine-dependent pho1 expression. All anr mutants are also phosphate nonrepressible. These results indicate that the generation and/or transduction of the intracellular signal responsible for pho1 repression is simultaneously dependent on both adenine and phosphate.     

Triploid meiosis and aneuploidy in Schizosaccharomyces pombe: an unstable aneuploid disomic for chromosome III

Summary Triploid meiosis in crosses between haploid and diploid strains of the fission yeast Schizosaccharomyces-pombe was investigated by tetrad analyses. Viability of the segregants was low. Only about 10% of the total tetrads contained four colony-forniing spores and most of these segregated into two haploids and two diploids. The remaining tetrads were rather normal in germination but were defective in colony formation. More than 50% of the total tetrads had no colony-forming spores while about 30% contained 1–3 colony-forming spores. Among the colony-forming segregants from the defective tetrads, a class of aneuploids disomic for the shortest chromosome III was obtained. In such aneuploid cells combined with a cold-sensitive ß-tubulin mutation, an additional short chromosome corresponding to chromosome III was observed by DAPI staining when the cells were incubated at a restrictive temperature. The other classes of aneuploids appeared able to be to germinate but not to enter into vegetative growth. Detailed genetical analyses indicated that recombination frequencies near the centromere were strikingly different between triploid and normal diploid meiosis.