The Schizosaccharomyces pombe cfr1+ gene participates in mating through a new pathway that is independent of fus1+

Conjugation is a complex event directed to ensure the transfer of genetic material, which is achieved by the union of two cells. In fungi, success of this relevant process requires digestion of the cell wall at the point where both cells have agglutinated and, later, the union of the plasma membranes and nuclei from the mating partners. In order to gain information about cell fusion, we have cloned and disrupted the cfr1+ gene from the fission yeast Schizosaccharomyces pombe. cfr1+ gene is slightly induced at the beginning of mating but Cfr1p protein is degraded soon after the cells are transferred to nitrogen-lacking medium. cfr1Delta mutants present a defect in cell fusion owing to a failure in the digestion of the cell walls between the two parental cells. Finally, cytological and genetic analyses show that cfr1+ acts in a new pathway involved in conjugation that is independent of fus1+, the only gene that has been found to be specifically required for cell fusion during mating in the fission yeast.     

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

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

Helicase activity is only partially required for Schizosaccharomyces pombe Rqh1p function

The RecQ-related family of DNA helicases is required for the maintenance of genomic stability in organisms ranging from bacteria to humans. In humans, mutation of three RecQ-related helicases, BLM, WRN and RecQL4, cause the cancer-prone and premature ageing diseases of Bloom syndrome, Werner's syndrome and Rothmund-Thompson syndrome, respectively. In the fission yeast Schizosaccharomyces pombe, disruption of the rqh1(+) gene, which encodes the single Sz. pombe RecQ-related helicase, causes cells to display reduced viability and elevated levels of chromosome loss. After S-phase arrest or DNA damage, cells lacking rqh1(+) function display elevated levels of homologous recombination and defective chromosome segregation. Here we show that, like other RecQ family members, the Rqh1p protein displays 3' to 5' DNA helicase activity. Interestingly, however, unlike other RecQ family members, the helicase activity of Rqh1p is only partially required for its function in recovery from S-phase arrest or DNA damage. We also report that high cellular levels of Rqh1p result in lethal chromosome segregation defects, while more moderate levels of Rqh1p cause significantly elevated rates of chromosome loss. This suggests that careful regulation of RecQ-like protein levels in eukaryotic cells is vital for maintaining genome stability.     

A truncated derivative of nmt 1 promoter exhibits temperature-dependent induction of gene expression in Schizosaccharomyces pombe

Despite increasing exploitation of Schizosaccharomyces pombe as a model system there is a lack of convenient vectors for research and application. Expression with the commonly used promoter, nmt 1, requires a laborious regime involving the removal of repressor, thiamine, from a growing culture and further growth for 18 h to achieve maximum expression, thus underlining the need for more user-friendly promoters. We report here the isolation and characterization of a truncated derivative of the nmt 1 promoter having novel induction characteristics: it is induced by shift of growth temperature from 36 degrees C to 25 degrees C, achieving maximum expression within 3 h. Similar features of expression were observed with the reporter genes GFP and beta-galactosidase, a native gene, cdc 18, and a commercially important foreign therapeutic protein, streptokinase. The new promoter element offers additional advantages, such as lack of deleterious effect on cell viability and potential ability to express toxic proteins. These features make the new promoter a potentially better alternative to nmt 1, both as a research tool and for expression of commercially important proteins in Sz. pombe, and suggest the possibility of using similar approaches to design promoters with novel and useful properties.     

A 'marker switch' approach for targeted mutagenesis of genes in Schizosaccharomyces pombe

The completion of the Schizosaccharomyces pombe genome sequencing project has led to a dramatic acceleration of gene characterization in this system. Once a gene has been identified, the challenge then comes in using reverse genetics to generate a range of mutants in this gene of interest so that the powerful genetics and wealth of genetic backgrounds available in Sz. pombe can be exploited to study the function of the newly identified molecule. Beyond simple PCR-tagging approaches, the high frequency with which illegitimate recombination occurs in Sz. pombe has made the manipulation of some loci complex, time consuming and a process of trial and error. Here we describe a simple 'marker switch' approach that enables the rapid selection of integration events at the locus of interest from an excessive background of integration at heterologous sites. We use the generation of temperature-sensitive mutations in the plo1(+) gene to validate this approach.     

Construction and characterization of a series of vectors for Schizosaccharomyces pombe

A set of vectors was created to allow cloning and expression studies in Schizosaccharomyces pombe. These vectors had a uniform backbone with an efficient Sz. pombe ARS, ARS3002, but different selectable markers--his3+, leu1+, ade6+ and ura4+. The vectors functioned efficiently as autonomously replicating plasmids that could also be converted into integrating vectors. The ura4+-containing vector was used to construct a Sz. pombe genomic library.     

The mae1 gene of Schizosaccharomyces pombe encodes a permease for malate and other C4 dicarboxylic acids

The mae1 gene of the yeast Schizosaccharomyces pombe was identified on the basis of its ability to complement a mutant defective in the transport of malic acid. Analysis of the DNA sequence revealed an open reading frame of 1314 base pairs, encoding a polypeptide of 438 amino acids with a predicted molecular weight of 49 kDa. A hydropathy profile of the predicted amino acid sequence revealed a protein with ten membrane-spanning or associated domains and hydrophilic N- and C- termini. The predicted secondary structure of the protein is similar to models proposed for other integral mambrane proteins from both prokaryotes and eukaryotes. The S. pombe mae1 gene encodes a single mRNA of 1·5 kb. The mae1 gene is expressed constitutively and is not subject to catabolite repression as was previously reported for the malate permease systems of Candida utilis and Hansenula anomala. The mae1 gene was mapped 2842 bp 5′ to the MFm1 gene on chromosome I. Transport assays revealed that the mae1 gene encodes a permease involved in the uptake of L-malate, succinate and malonic acid. The sequence of the S. pombe mae1 gene is available in GenBank under Accession Number U21002.     

Transport of L-leucine in the fission yeast Schizosaccharomyces pombe

Transport of L -leucine into Schizosaccharomyces pombe cells from the stationary phase of growth (after preincubation for 60 min with 1% glucose) proceeds uphill, practically unidirectionally, and is mediated by at least two systems: a high-affinity system with a K_T of 0·045 mmol 1^?1 and J_max of 3·3 nmol min^?1 (mg dry weight)^?1 and a low-affinity system with a K_T of 1·25 mmol 1^?1 and J_max of 16·0 nmol min^?1 (mg dry weight)^?1. The high-affinity system has a pH optimum at 3.2, the accumulation ratio is highest at a cell density of 2–4 mg dry weight per ml and decreases with increasing leucine concentration. Transport of leucine by the high-affinity system is strongly inhibited by proton conductors, ammonium ions and by most amino acids, but only L -phenylalanine, L -isoleucine, L -valine and L -cysteine behave as fully competitive inhibitors. Systems of L -leucine transport in S. pombe are not constitutive. Transport activity appears only after preincubation of cells with a suitable source of energy. If cycloheximide is added during preincubation with glucose, no transport systems for leucine are synthesized. After removal of glucose, the activity of transport systems decays with a half-time of about 20 min. The presence of cyclic AMP increases the initial rate of leucine uptake only in cells preincubated with glucose and in the absence of cycloheximide.     

Mutational analysis of the gene for Schizosaccharomyces pombe RNase MRP RNA,mrp1, using plasmid shuffle by counterselection on canavanine

Reverse genetics in fission yeast is hindered by the lack of a versatile established plasmid shuffle system. In order to screen efficiently and accurately through plasmid-borne mutations in the essential gene for the RNA component of RNase MRP, mrp1, we have developed a system for plasmid shuffling in fission yeast using counterselection on canavanine. The system takes advantage of the ability of the Saccharomyces cerevisiae CAN1 gene to complement a Schizosaccharomyces pombe can1-1 mutation. Two general use plasmids were constructed that allow directional cloning and initial selection for histidine before counterselection by canavanine. The strain constructed for plasmid shuffling carries auxotrophic markers for ade6, leu1, ura4 and his3 along with the can1-1 mutation. Using this system we examined several partial deletions and point mutations in conserved nucleotides of Schizosaccharomyces pombe RNase MRP RNA for their ability to complement a chromosomal deletion of the mrp1 gene. The degree of background canavanine resistance as well as plasmid–plasmid recombination encountered in these experiments was sufficiently low to suggest that the system we have set up for counterselection by canavanine in fission yeast using multicopy plasmids will be widely useful.     

Characterization of end4+, a gene required for endocytosis in Schizosaccharomyces pombe

To understand endocytic trafficking in Schizosaccharomyces pombe, we constructed an end4 disruption mutant. The end4+ gene encodes a protein homologous to Sla2p/End4p, which is essential for the assembly and function of the cytoskeleton and endocytosis in Saccharomyces cerevisiae. We characterized the fission yeast mutant end4 Delta as well as ypt7 Delta, which is deficient in vacuolar fusion and, hence, endocytosis. The delivery of FM4-64 to the vacuolar membrane, accumulation of Lucifer yellow CH and internalization of plasma membrane protein Map3-GFP were inhibited in the end4 mutant. Deletion of end4 resulted in pleiotropic phenotypes consistent with F-actin depolarization, including high temperature sensitivity, abnormal morphology and mating defects. Extensive missorting of carboxypeptidase Y was detected in the ypt7 mutant; however, little missorting was detected in the end4 mutant. These results indicate that End4p is essential for the internalization process and Ypt7p affects endocytosis at a post-internalization step after the intersection of the endocytic and the vacuolar protein-sorting pathways in fission yeast.     

利用粟酒裂殖酵母生长法定量测定肌醇浓度

利用本实验室分离得到的一株天然的肌醇合成缺陷型的粟酒裂殖酵母 (Schizosaccha romycespombe)特异地依赖外部肌醇生长的特性 ,研究了该菌株在含有不同浓度肌醇的合成培养基中的生长情况 ,发现在一定的肌醇浓度范围内 ,菌株的生物量随肌醇浓度增大而增大 ,且呈线性关系 ,由此建立了一套微生物法定量测定肌醇浓度的方法。我们发现在固定的培养温度、振荡速度等外部条件下 ,当接种量OD60 0 为 1 0、葡萄糖浓度为 4 0 %时 ,在肌醇浓度 0～ 5mg/L范围内 ,培养 52h后 ,肌醇测定标准曲线最为理想 ,R值为 0 9973。应用该方法对标准肌醇样品的测定得到了较好的结果     

Molecular cloning of the plc1+ gene of Schizosaccharomyces pombe,which encodes a putative phosphoinositide-specific phospholipase C

Exploiting the polymerase chain reaction, we have isolated a gene that encodes a putative phosphoinositide-specific phospholipase C (PLC) of the fission yeast Schizosaccharomyces pombe. Inspection of the nucleotide sequence of the gene revealed an open reading frame that can encode a polypeptide of 899 amino acid residues with a calculated molecular mass of 102 kDa. This putative polypeptide contains both the X and Y regions that are conserved among three classes of mammalian PLC, and also contains a presumptive Ca²⁺-binding site (an E-F hand motif). The structure of the putative protein is most similar to that of the δ class of PLC isozymes. To investigate the role of this gene, designated plc1⁺, gene disruption was carried out by interrupting the coding region with the ura4⁺ marker. Growth of plc1 cells was temperature-sensitive in rich medium, and cells could not grow in synthetic medium. Expression of the PLC1 gene of Saccharomyces cerevisiae suppressed the growth defect phenotype of plc1^? cells, a strong suggestion that the plc1⁺ gene encodes PLC. The PLC1 sequence appears in the public data libraries, DDBJ GenBank, EMBL under the following Accession Number: D38309.     

Isolation and characterization of Schizosaccharomyces pombe fragile mutants

Three Schizosaccharomyces pombe fragile mutants requiring the presence of an osmotic stabilizer to grow, that lyse when transferred into hypotonic solutions and that secrete to the extracellular medium more protein than the parental strain were isolated. In the three mutants, the fragile phenotype segregated in a Mendelian fashion, indicating a single chromosomal gene mutation, and behaved as a recessive character. By complementation analysis, the three fragile mutants fell in a single complementation group, defining the same gene (SRB1). Mutations of this gene are responsible for alterations in the cells such as fragile character, increase in the cell wall porosity, changes in the cell morphology and floc-forming ability. The study of the three srb1 alleles indicated that the degree of these alterations is proportional to a significant decrease in the galactomannan fraction of the mutants cell wall. The data presented in this report suggest that the product of the SRB1 gene is critical for the maintenance of the integrity and structure of Sz. pombe cell wall.     

The Essential Schizosaccharomyces pombe gpi1+ Gene Complements a Bakers' Yeast GPI Anchoring Mutant and is Required for Efficient Cell Separation

The Schizosaccharomyces pombe gpi1⁺ gene was cloned by complementation of the Saccharomyces cerevisiae gpi1 mutant, which has temperature-sensitive defects in growth and glycosyl phosphatidylinositol (GPI) membrane anchoring of protein, and which is defective in vitro in the first step in GPI anchor assembly, the formation of N -acetylglucosaminyl phosphatidylinositol (GlcNAc-PI). S. pombe gpi1⁺ encodes a protein with 29% identity to amino acids 87–609 of the S. cerevisiae protein, and is the functional homolog of the S. cerevisiae Gpi1 protein, for it restores [³H]inositol-labelling of protein and in vitro GlcNAc-PI synthetic activity to both S. cerevisiae gpi1 and gpi1::URA3 cells. Disruption of gpi1⁺ is lethal. Haploid Δgpi1⁺::his7⁺ spores germinate, but proceed through no more than three rounds of cell division, many cells ceasing growth as binucleate, septate cells with thickened septa. These results indicate that GPI synthesis is an essential function in fission yeast, and suggest that GPI anchoring is also required for completion of cytokinesis. The nucleotide sequence reported will appear in the GenBank Nucleotide Sequence database under the Accession Number U77355.©1997 John Wiley & Sons, Ltd.     

Characterization of vps33+, a gene required for vacuolar biogenesis and protein sorting in Schizosaccharomyces pombe

From the fission yeast Schizosaccharomyces pombe we have identified and deleted vps33, a gene encoding a homologue of VPS33, which is required for vacuolar biogenesis in S. cerevisiae cells. When the vps33(+) gene is disrupted, Sz. pombe strains are temperature-sensitive for growth and contain numerous small vesicular structures stained with FM4-64 in the cells. Deletion of the Sz. pombe vps33(+) gene results in pleiotropic phenotypes consistent with the absence of normal vacuoles, including missorting of vacuolar carboxypeptidase Y, various ion- and drug-sensitivities, and sporulation defects. These results are consistent with Vps33p being necessary for the morphogenesis of vacuoles and subsequent expression of vacuolar functions in Sz. pombe cells.