A Lallzyme MMX‐based rapid method for fission yeast protoplast preparation

Fungal cells including yeasts are surrounded by cell wall that counteracts turgor pressure and prevents cell lysis. Many yeast experiments, including genetic manipulation of sterile strains, morphogenesis studies, nucleic acid isolation and many others, require mechanical breakage or enzymatic removal of the cell wall. Some of these experiments require the generation of live cells lacking cell walls, called protoplasts, that can be maintained in osmostabilized medium. Enzymatic digestion of cell wall proteoglycans is a commonly used method of protoplast preparation. Currently existing protocols for fission yeast cell wall digestion are time consuming and not very efficient. We developed a new rapid method for fission yeast protoplast preparation that relies on digesting cell walls with Lallzyme MMX commercial enzyme mix, which produces protoplasts from all cells in less than 10 min. We demonstrate that these protoplasts can be utilized in three commonly used fission yeast protocols. Thus, we provide the fission yeast community with a robust and efficient plasmid extraction method, a new protocol for diploid generation and an assay for protoplast recovery that should be useful for studies of morphogenesis. Our method is potentially applicable to other yeasts and fungi. Copyright © 2013 John Wiley & Sons, Ltd.     

Evaluation of phylogenetic relationships among fission yeast by nDNA/nDNA reassociation and conventional taxonomic criteria

The genus Schizosaccharomyces comprises a small, somewhat heterogeneous group of yeast species which have in common a unique mode of vegetative reproduction by cross-wall formation without constriction as well as a certain degree of osmophilia, most strains having been isolated from habitats of high sugar concentration. This study evaluated inter- and intraspecific relationships utilizing nDNA/nDNA optical reassociation and by the analysis of physiological profiles of several strains of each species. Results demonstrate that the genus should be divided into three species: Schiz. japonicus, Schiz. octosporus and Schiz. pombe.     

An estradiol‐inducible promoter enables fast,graduated control of gene expression in fission yeast

The fission yeast Schizosaccharomyces pombe lacks a diverse toolkit of inducible promoters for experimental manipulation. Available inducible promoters suffer from slow induction kinetics, limited control of expression levels and/or a requirement for defined growth medium. In particular, no S. pombe inducible promoter systems exhibit a linear dose–response, which would allow expression to be tuned to specific levels. We have adapted a fast, orthogonal promoter system with a large dynamic range and a linear dose response, based on β‐estradiol‐regulated function of the human oestrogen receptor, for use in S. pombe. We show that this promoter system, termed Z₃EV, turns on quickly, can reach a maximal induction of 20‐fold, and exhibits a linear dose response over its entire induction range, with few off‐target effects. We demonstrate the utility of this system by regulating the mitotic inhibitor Wee1 to create a strain in which cell size is regulated by β‐estradiol concentration. This promoter system will be of great utility for experimentally regulating gene expression in fission yeast. Copyright © 2017 John Wiley & Sons, Ltd.     

Schizosaccharomyces japonicus: the fission yeast is a fusion of yeast and hyphae

The clade of Schizosaccharomyces includes 4 species: S. pombe, S. octosporus, S. cryophilus, and S. japonicus. Although all 4 species exhibit unicellular growth with a binary fission mode of cell division, S. japonicus alone is dimorphic yeast, which can transit from unicellular yeast to long filamentous hyphae. Recently it was found that the hyphal cells response to light and then synchronously activate cytokinesis of hyphae. In addition to hyphal growth, S. japonicas has many properties that aren't shared with other fission yeast. Mitosis of S. japonicas is referred to as semi‐open mitosis because dynamics of nuclear membrane is an intermediate mode between open mitosis and closed mitosis. Novel genetic tools and the whole genomic sequencing of S. japonicas now provide us with an opportunity for revealing unique characters of the dimorphic yeast. © 2013 The Author. Yeast Published by John Wiley & Sons Ltd.     

The ade6 gene of the fission yeast Schizosaccharomyces pombe has the same chromatin structure in the chromosome and in plasmids.

We have analysed the chromatin structure of the ade6 gene of Schizosaccharomyces pombe and its flanking regions both in the chromosome and in plasmids. The chromatin structure is independent of the chromosomal or extrachromosomal location. The ade6 gene contains eight precisely positioned nucleosomes on the 5' half, 'not positioned' nucleosomes around the 3' end and a nuclease-sensitive promoter region. Precisely positioned nucleosomes, but no nuclease-sensitive region were also detected on the ura4 gene in the chromosome and on a plasmid. The results show that S. pombe chromosomal and extrachromosomal genes have chromatin structures similar to those of S. cerevisiae and higher eukaryotes.     

Vacuolar carboxypeptidase Y of Saccharomyces cerevisiae is glycosylated,sorted and matured in the fission yeast Schizosaccharomyces pombe

Vacuolar carboxypeptidase Y of Saccharomyces cerevisiae (CPY^sc) has been expressed in a Schizosaccharomyces pombe strain devoid of the endogenous equivalent peptidase, employing a 2 μ derived plasmid. Immunoblot analysis revealed that CPY^sc produced in the fission yeast has a higher molecular mass than mature CPY^sc produced by the budding yeast. CPY^sc is glycosylated when expressed in S. pombe and uses four N-linked glycosylation sites as shown by endoglycosidase H digestion. Carbohydrate removal leads to a protein moiety which is indistinguishable in size from deglycosylated CPY^sc produced by S. cerevisiae. CPY^sc isolated from S. pombe soluble extracts is enzymatically active and thus is presumed to undergo correct proteolytic maturation. Subcellular fractionation experiments showed a cofractionation of CPY^sc with the S. pombe endoproteinases PrA and PrB, suggesting that the protein is correctly sorted to the vacuole and that these peptidases might be responsible for zymogen activation.     

Effects of phleomycin-induced DNA damage on the fission yeast Schizosaccharomyces pombe cell cycle

The effect of phleomycin, a bleomycin-like antibiotic, has been investigated in the fission yeast, Schizosaccharomyces pombe. We report that in response to phleomycin-induced DNA damage, growth was inhibited and S. pombe cells arrested in the G2-phase of the cell cycle. DNA repair mutants rad9 and rad17 did not arrest and were hypersensitive to phleomycin. Cell cycle mutants that entered mitosis without monitoring the completion of DNA replication also displayed an increased sensitivity to this DNA-damaging agent. Thus, phleomycin could be used as a tool in the fission yeast S. pombe model system for the study of DNA damage and cell cycle checkpoints, or as a new selective agent.     

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

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

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.     

Measurement of nuclear DNA content in fission yeast by flow cytometry

Cell division cycle (cdc) mutants of Schizosaccharomyces pombe are arrested at specific points in the cell cycle when grown at restrictive temperature. Flow cytometry of such cells reveals an anomalous increase in the DNA fluorescence signal, which represents a problem in experiments designed to determine the cell cycle arrest point. The increased fluorescence signal is due to cytoplasmic constituents and has been attributed to mitochondrial DNA synthesis (S. Sazer and S. W. Sherwood, J. Cell Sci.97: 509–516, 1990). Here we have studied the cdc10 mutant by flow cytometry using different DNA-binding fluorochromes and found no evidence that the increased fluorescence signal was caused by mitochondrial DNA synthesis. To determine more accurately the nuclear DNA content we have developed a novel method to remove most of the cytoplasmic material by exposing the cells to Triton X-100 and hypotonic conditions after cell wall digestion. The DNA fluorescence from cells treated in this way was more constant with time of incubation at restrictive temperature in spite of a considerable increase in cell size. With this method we could determine that the recently isolated temperature sensitive orp1 mutant is arrested with a 1C DNA content. Premature and abnormal mitosis (‘cut’) could be observed for the orp1 mutant after only 4 h at restrictive temperature. © 1997 John Wiley & Sons, Ltd.     

葡萄相关酵母多样性及其菌种鉴定的研究进展

本文对葡萄相关酵母菌种的种类及其多样性、酵母菌群在不同环境和酿造过程中的动态变化以及传统和现代菌种鉴定方法的特点等方面进行综合阐述,为筛选具有地域特征、发酵性能良好的野生酵母菌种提供一定的理论依据。      

Basic methods for fission yeast

The fission yeast Schizosaccharomyces pombe is a popular model system, and has been particularly influential in studies of the cell cycle and chromosome dynamics. Despite its differences from Saccharomyces cerevisiae, the tools and methods for fission yeast are conceptually similar to those used in budding yeast. Here, we present basic methods sufficient for a beginner in this system to carry out most required manipulations for genetic analysis or molecular biology.     

中国本土葡萄酒酵母种群多样性分布的研究进展

酵母菌是葡萄酒酿造过程中最重要的微生物菌群,较好的认识葡萄酒相关酵母种群的分布规律对于生产具有特色的高品质葡萄酒具有重要意义.文中论述了中国葡萄酒相关酵母资源的分布情况,以期为本地葡萄酒生产提供理论指导.     

The cyclophilin repertoire of the fission yeast Schizosaccharomyces pombe

The cyclophilin repertoire of the fission yeast Schizosaccharomyces pombe is comprised of nine members that are distributed over all three of its chromosomes and range from small single-domain to large multi-domain proteins. Each cyclophilin possesses only a single prolyl-isomerase domain, and these vary in their degree of consensus, including at positions that are likely to affect their drug-binding ability and catalytic activity. The additional identified motifs are involved in putative protein or RNA interactions, while a novel domain that is specific to SpCyp7 and its orthologues may have functions that include an interaction with hnRNPs. The Sz. pombe cyclophilins are found throughout the cell but appear to be absent from the mitochondria, which is unique among the characterized eukaryotic repertoires. SpCyp5, SpCyp6 and SpCyp8 have exhibited significant upregulation of their expression during the meiotic cycle and SpCyp5 has exhibited significant upregulation of its expression during heat stress. All nine have identified members in the repertoires of H. sapiens, D. melanogaster and A. thaliana. However, only three identified members in the cyclophilin repertoire of S. cerevisiae with SpCyp7 identifying a fourth protein that is not a member of the recognized repertoire due to its possession of a degenerate prolyl-isomerase domain. The cyclophilin repertoire of Sz. pombe therefore represents a better model group for the study of cyclophilin function in the higher eukaryotes.     

The essential function of Rrs1 in ribosome biogenesis is conserved in budding and fission yeasts

The Rrs1 protein plays an essential role in the biogenesis of 60S ribosomal subunits in budding yeast (Saccharomyces cerevisiae). Here, we examined whether the fission yeast (Schizosaccharomyces pombe) homologue of Rrs1 also plays a role in ribosome biogenesis. To this end, we constructed two temperature‐sensitive fission yeast strains, rrs1‐D14/22G and rrs1‐L51P, which had amino acid substitutions corresponding to those of the previously characterized budding yeast rrs1‐84 (D22/30G) and rrs1‐124 (L61P) strains, respectively. The fission yeast mutants exhibited severe defects in growth and 60S ribosomal subunit biogenesis at high temperatures. In addition, expression of the Rrs1 protein of fission yeast suppressed the growth defects of the budding yeast rrs1 mutants at high temperatures. Yeast two‐hybrid analyses revealed that the interactions of Rrs1 with the Rfp2 and Ebp2 proteins were conserved in budding and fission yeasts. These results suggest that the essential function of Rrs1 in ribosome biogenesis may be conserved in budding and fission yeasts. Copyright © 2015 John Wiley & Sons, Ltd.