A protective role of methionine-R-sulfoxide reductase against cadmium in Schizosaccharomyces pombe

The Schizosaccharomyces pombe cells harboring the methionine- R-sulfoxide reductase (MsrB)-overexpressing recombinant plasmid pFMetSO exhibited better growth than vector control cells, when shifted into fresh medium containing cadmium chloride (abbreviated as Cd). Although both groups of cells contained enhanced reactive oxygen species (ROS) and nitric oxide (NO) levels in the presence of Cd, ROS and NO levels were significantly lower in the S. pombe cells harboring pFMetSO than in vector control cells. Conversely, the S. pombe cells harboring pFMetSO possessed higher total glutathione (GSH) levels and a greater reduced/oxidized GSH ratio than vector control cells under the same conditions.     

Hydrolytic activity in the genusSchizosaccharomyces lindner

A total of 22 strains of known species of the genusSchizosaccharomyces Lindner were evaluated by numerical taxonomy based on conventional identification tests. The results of numerical taxonomy were supplemented by a determination of activity of extracellular hydrolytic enzymes, especially 1,3-β-D-glucanase, RNAase and DNAase. All the strains tested were capable of utilizing the 1,4-capable of utilizing the 1,4-α-D-glucan tamarind. Study of life cycles of these organisms showed that extracellular hydrolytic enzymes were present mainly at the time of maturation of asci and release of their walls. Strains forming four-spore asci could be comprised in the single speciesSchizosaccharomyces pombe Lindner as two varieties:S. pombe var.pombe andS. pombe var.malidevorans. The two varieties differ in maltose fermentation. Urease is produced by all strains irrespective of the life cycle phase. A number of hydrolytic enzymes are not produced by the genusSchizosaccharomyces (e.g. amylolytic enzymes) despite the fact that oligomers of the maltose type are utilized. Species of the genusSchizosaccharomyces lack also xylanase, cellulases, mannase, and are incapable of degrading carrageenan and acid polysaccharides.     

Dictyosomes in the yeastSchizosaccharomyces pombe

Schizosaccharomyces pombe log-phase cells were studied by the methods of freeze-etching and ultrathin sectioning. Recognizable dictyosomes consisted of 3–7 cisternae surrounded by vesicles. No distinct tubuli were found on the periphery of the cisternae. Each cell contained several solitary dictyosomes which were most frequently observed near regions of cell-wall growth.     

Crystal structure of the eukaryotic translation initiation factor 2A from Schizosaccharomyces pombe

The eukaryotic translation initiation factor 2A (eIF2A) was identified as a factor that stimulates the binding of methionylated initiator tRNA (Met-tRNA _i ^Met ) to the 40S ribosomal subunit, but its physiological role remains poorly defined. Recently, eIF2A was shown to be involved in unconventional translation initiation from CUG codons and in viral protein synthesis under stress conditions where eIF2 is inactivated. We determined the crystal structure of the WD-repeat domain of Schizosaccharomyces pombe eIF2A at 2.5 Å resolution. The structure adopts a novel nine-bladed β-propeller fold. In contrast to the usual β-propeller proteins, the central channel of the molecule has the narrower opening on the bottom of the protein and the wider opening on the top. Highly conserved residues are concentrated in the positively-charged top face, suggesting the importance of this face for interactions with nucleic acids or other initiation factors.     

The isolation of protoplasts of the fission yeastSchizosaccharomyces byTrichoderma viride and snail enzymes

The formation of protoplasts of the fission yeastsSchizosaccharomyces pombe andSchizosaccharomyces versatilis after the combined application of snail enzymes andTrichoderma viride enzymes in an osmotic stabilizer (0.4m KC1, pH 5.5) was studied by light and electron microscopy. The effect of the enzymes used leads during 30 min to the formation of 100% protoplast population. Using electron microscopy no original walls or wall remnants were detected in the suspension of protoplasts. Protoplasts are viable and in liquid nutrient medium they regenerate cell walls and revert into normal cells. Such a protoplast population may be useful for biochemical study of protoplast metabolism by quantitative methods as well as for the chemical study of regenerating cell walls.     

Isoenzyme pattern of malate dehydrogenase during respiratory derepression in Schizosaccharomyces pombe

One isoenzyme of malate dehydrogenase with an isoelectric point of 6.4 was found in glucose-repressed cells of Schizosaccharomyces pombe. During respiratory derepression the activity of this isoenzymes decreased rapidly in vivo. In the course of this inactivation two new forms of malate dehydrogenase with isoelectric points of 6.0 and 5.7 appeared. It has been found that these two enzymic forms disappeared 4 h after the exhaustion of glucose; probably they are degradation products of the isoenzyme present in glucose-repressed cells. Fully derepressed cell of this fission yeast contain one isoenzyme of malate dehydrogenase with an isoelectric point of 5.3. The synthesis of this isoenzyme is initiated at glucose concentrations below 1.5 g/l.     

Cell-wall structure,mitosis and urease activity in Torulopsis species of high GC content

Five Torulopsis species, in which, characteristically, the mean molar percentage of guanine plus cytosine of the DNA bases (GC content) is above 50%, were examined to determine their cell-wall structure; the mode of bud formation; course of mitosis and their urease activity. The two urease-negative species, T. gropengiesseri (% GC 57.1) and T. silvatica (% GC 56.3), were found to possess affinitive properties which characterize perfect ascomycetous yeast species. In the urease-positive species, T. fujisanensis (% GC 65.0–66.1), T. ingeniosa (% GC 55.6) and T. philyla (% GC 63.9), there appeared to be a correlation between pronounced urease activity and the affinitive properties which characterize perfect basidiomycetous yeast species. Pronounced urease activity has, however, also been found in the ascomycetous species Schizosaccharomyces pombe. Consequently neither high GC ratios nor the presence of urease activity when considered individually, can be taken as reliable criteria when attempting to establish the affinity of yeasts the perfect states of which are not known, with the Basidiomycetes. A strain of the ascomycetous species Sclerotinia trifoliorum was examined by electron microscopy for comparative purposes.     

JaponicusDB: rapid deployment of a model organism database for an emerging model species

The fission yeast Schizosaccharomyces japonicus has recently emerged as a powerful system for studying the evolution of essential cellular processes, drawing on similarities as well as key differences between S. japonicus and the related, well-established model Schizosaccharomyces pombe. We have deployed the open-source, modular code and tools originally developed for PomBase, the S. pombe model organism database (MOD), to create JaponicusDB (www.japonicusdb.org), a new MOD dedicated to S. japonicus. By providing a central resource with ready access to a growing body of experimental data, ontology-based curation, seamless browsing and querying, and the ability to integrate new data with existing knowledge, JaponicusDB supports fission yeast biologists to a far greater extent than any other source of S. japonicus data. JaponicusDB thus enables S. japonicus researchers to realize the full potential of studying a newly emerging model species and illustrates the widely applicable power and utility of harnessing reusable PomBase code to build a comprehensive, community-maintainable repository of species-relevant knowledge.     

Fission stories: using PomBase to understand Schizosaccharomyces pombe biology

PomBase (www.pombase.org), the model organism database (MOD) for the fission yeast Schizosaccharomyces pombe, supports research within and beyond the S. pombe community by integrating and presenting genetic, molecular, and cell biological knowledge into intuitive displays and comprehensive data collections. With new content, novel query capabilities, and biologist-friendly data summaries and visualization, PomBase also drives innovation in the MOD community.     

Effects of seven different mutations in the pho1 gene on enzymatic activity, glycosylation and secretion of acid phosphatase in Schizosaccharomyces pombe

Summary Structural gene mutants of the cell-surface glycoprotein acid phosphatase of Schizosaccharomyces pombe were analysed to define structural determinants that are responsible for enzymatic activity, N-glycosylation and secretion. All seven defined mutations cause a single amino acid substitution in the mature acid phosphatase protein and destroy the enzymatic activity. The mutational lesions are distributed throughout the pho1 gene. A ser to phe substitution at position 349 abolishes enzymatic activity only and does not affect glycosylation and secretion. Two mutations create a new N-glycosylation site by substitution of pro at position 56 by phe and ser, respectively. This new site is apparently used in the mutants. Their core-glycosylated acid phosphatase is slightly larger than that of the wild type. Overglycosylation seems not to affect secretion. Four different mutations (a gly to asp substitution at position 281 and ser to phe substitutions at positions 150, 271 and 277) cause intracellular accumulation of enzymatically inactive core-glycosylated acid phosphatase precursor. These mutational lesions apparently block transport of acid phosphatase from the endoplasmic reticulum to the Golgi apparatus.     

Involvement of small heat shock proteins,trehalose, and lipids in the thermal stress management in <Emphasis Type="Italic">Schizosaccharomyces pombe</Emphasis>

Changes in the levels of three structurally and functionally different important thermoprotectant molecules, namely small heat shock proteins (sHsps), trehalose, and lipids, have been investigated upon heat shock in Schizosaccharomyces pombe. Both α-crystallin-type sHsps (Hsp15.8 and Hsp16) were induced after prolonged high-temperature treatment but with different kinetic profiles. The shsp null mutants display a weak, but significant, heat sensitivity indicating their importance in the thermal stress management. The heat induction of sHsps is different in wild type and in highly heat-sensitive trehalose-deficient (tps1Δ) cells; however, trehalose level did not show significant alteration in shsp mutants. The altered timing of trehalose accumulation and induction of sHsps suggest that the disaccharide might provide protection at the early stage of the heat stress while elevated amount of sHsps are required at the later phase. The cellular lipid compositions of two different temperature-adapted wild-type S. pombe cells are also altered according to the rule of homeoviscous adaptation, indicating their crucial role in adapting to the environmental temperature changes. Both Hsp15.8 and Hsp16 are able to bind to different lipids isolated from S. pombe, whose interaction might provide a powerful protection against heat-induced damages of the membranes. Our data suggest that all the three investigated thermoprotectant macromolecules play a pivotal role during the thermal stress management in the fission yeast.     

Effects of Nickel Chloride on the Erythrocytes and Erythrocyte Immune Adherence Function in Broilers

This study was conducted to investigate the immune adherence function of erythrocytes and erythrocyte induced by dietary nickel chloride (NiCl₂) in broilers fed on a control diet and three experimental diets supplemented with 300, 600, and 900 mg/kg NiCl₂ for 42 days. Blood samples were collected from five broilers in each group at 14, 28, and 42 days of age. Changes of erythrocyte parameters showed that total erythrocyte count (TEC), hemoglobin (Hb) contents, and packed cell volume (PCV) were significantly lower (p?p?p?p?+/K⁺-ATPase) and calcium adenosine triphosphatase (Ca²⁺-ATPase) activities were significantly decreased (p?p?2-treated groups. The results of erythrocyte immune adherence function indicated that erythrocyte C_3b receptor rosette rate (E-C_3bRR) was significantly decreased (p?p?p?p?2 in excess of 300 mg/kg caused anemia and impaired the erythrocytic integrity, erythrocytic ability to transport oxygen, and erythrocyte immune adherence function in broilers. Impairment of the erythrocytes and erythrocyte immune adherence function was one of main effect mechanisms of NiCl₂ on the blood function.     

Phosphatidylethanolamine Is Required for Normal Cell Morphology and Cytokinesis in the Fission Yeast Schizosaccharomyces pombe

To investigate the contributions of phosphatidylethanolamine to the growth and morphogenesis of the fission yeast Schizosaccharomyces pombe, we have characterized three predicted genes in this organism, designated psd1, psd2, and psd3, encoding phosphatidylserine decarboxylases, which catalyze the conversion of phosphatidylserine to phosphatidylethanolamine in both eukaryotic and prokaryotic organisms. S. pombe mutants carrying deletions in any one or two psd genes are viable in complex rich medium and synthetic defined minimal medium. However, mutants carrying deletions in all three psd genes (psd1-3Δ mutants) grow slowly in rich medium and are inviable in minimal medium, indicating that the psd1 to psd3 gene products share overlapping essential cellular functions. Supplementation of growth media with ethanolamine, which can be converted to phosphatidylethanolamine by the Kennedy pathway, restores growth to psd1-3Δ cells in minimal medium, indicating that phosphatidylethanolamine is essential for S. pombe cell growth. psd1-3Δ cells produce lower levels of phosphatidylethanolamine than wild-type cells, even in medium supplemented with ethanolamine, indicating that the Kennedy pathway can only partially compensate for the loss of phosphatidylserine decarboxylase activity in S. pombe. psd1-3Δ cells appear morphologically indistinguishable from wild-type S. pombe cells in medium supplemented with ethanolamine, but when cultured in nonsupplemented medium, they produce high frequencies of abnormally shaped cells as well as cells exhibiting severe septation defects, including multiple, mispositioned, deformed, and misoriented septa. Our results demonstrate that phosphatidylethanolamine is essential for cell growth and for normal cytokinesis and cellular morphogenesis in S. pombe, and they illustrate the usefulness of this model eukaryote for investigating potentially conserved biological and molecular functions of phosphatidylethanolamine.Phosphatidylethanolamine (PE) is a major phospholipid component of cell membranes in both prokaryotic and eukaryotic organisms (34, 35). There are three distinct pathways for PE synthesis in eukaryotic cells: (i) decarboxylation of phosphatidylserine (PS) via reactions catalyzed by PS decarboxylase (PSD) enzymes; (ii) the CDP-ethanolamine branch of the Kennedy pathway, which converts ethanolamine to PE (34); and (iii) acylation of lysophosphatidylethanolamine (21, 29), a reaction that in the budding yeast Saccharomyces cerevisiae is catalyzed by the enzyme Ale1 (22). Genetic studies have demonstrated that PE is essential for cell viability in S. cerevisiae, although the minimal threshold of PE required for cell growth in this organism can apparently be provided by any of the routes of PE synthesis listed above (22). In contrast, the results of mouse knockout experiments indicate that both PSD- and Kennedy pathway-catalyzed pathways for PE synthesis are essential for embryonic development (9, 28, 35).While PE is present in most, if not all, eukaryotic cell membranes, it is particularly enriched in the membranes of mitochondria (32, 35, 37). Indeed, S. cerevisiae mutants carrying a null mutation in the PSD1 gene, which encodes a mitochondrially localized PSD, exhibit phenotypes indicative of mitochondrial dysfunction, as do cells derived from mouse embryos carrying a disruption of the Psid gene, which encodes a protein highly homologous in structure to S. cerevisiae Psd1 (28, 32). A second PSD enzyme in S. cerevisiae, encoded by the PSD2 gene, is localized to Golgi and vacuolar membranes (33, 37). Consistent with a role in vacuolar function, PE has been implicated in the process of autophagy by genetic studies utilizing S. cerevisiae vacuolar targeting mutants and by studies showing that Atg8, a ubiquitin-like protein required for yeast autophagy, is conjugated to PE, as are several related mammalian proteins (19, 20, 27).Interestingly, studies utilizing a streptavidin-conjugated form of the PE-binding peptide cinnamycin demonstrated that PE is enriched at cell division sites in S. cerevisiae, the fission yeast Schizosaccharomyces pombe, and mammalian cells (6, 11). Moreover, streptavidin-conjugated cinnamycin was shown to inhibit the disassembly of the contractile ring and the completion of cytokinesis in cultures of Chinese hamster ovary cells, and a PE-deficient cell line from the same species was found to arrest growth in cytokinesis with an intact contractile ring (7). PE has also been shown to be enriched at the growing ends of interphase S. pombe cells and at the emerging bud cortex in dividing cells of S. cerevisiae, findings that implicate PE in processes controlling polarized cell growth (11).Although S. pombe mutants defective in enzymes that directly catalyze PE synthesis have not been described previously, we recently showed that mutants carrying a null mutation in the PS synthase gene pps1 are ethanolamine auxotrophs that exhibit severe morphology- and cytokinesis-defective phenotypes under ethanolamine-limited growth conditions (17). These findings implicated PE in the regulation of cellular morphogenesis and cytokinesis in S. pombe. To investigate the biological functions of PE in S. pombe, in particular its contributions to the control of cell morphology and cytokinesis, we have in the present study generated and characterized mutants carrying null mutations in three open reading frames predicted to encode PSD enzymes in this organism. In this paper, we describe the phenotypes of S. pombe PSD-null mutants, which demonstrate central roles for PE in the regulation of cell morphology and cytokinesis in this model eukaryote.     

Electrophoretic analysis of plasma membrane proteins of wild-type and differentiation-deficient mutant strains ofPodospora anserina

In the fungusPodospora anserina, themodC mutations inhibit the sexual female organ differentiation. Previous results have suggested that the plasma membrane of this mutant may be altered. Proteins solubilized from highly purified plasmalemma from a wild-type and threemodC mutant strains were studied by one- and two-dimensional electrophoresis. Isoelectric focusing revealed in the wild-type strain about 80 polypeptide spots whose molecular weight ranged from 15,000 to 80,000 daltons; 65% of the spots were between 20,000 and 60,000 daltons, and 60% were in the 6.5–7.5 pH zone. The only difference inprotein noted between wild-type and the threemodC mutants was that the threemodC mutants lacked a plasmalemma protein of M_r=42,000 and of pI=6.7. These results suggest a close relationship between plasma membrane proteins and differentiation in this eukaryotic organism.     

Mutations in the ArsA ATPase that restore interaction with the ArsD metallochaperone

The ArsA ATPase is the catalytic subunit of the ArsAB As(III) efflux pump. It receives trivalent As(III) from the intracellular metallochaperone ArsD. The interaction of ArsA and ArsD allows for resistance to As(III) at environmental concentrations. A quadruple mutant in the arsD gene encoding a K2A/K37A/K62A/K104A ArsD is unable to interact with ArsA. An error-prone mutagenesis approach was used to generate random mutations in the arsA gene that restored interaction with the quadruple arsD mutant in yeast two-hybrid assays. A number of arsA genes with multiple mutations were isolated. These were analyzed in more detail by separation into single arsA mutants. Three such mutants encoding Q56R, F120I and D137V ArsA were able to restore interaction with the quadruple ArsD mutant in yeast two-hybrid assays. Each of the three single ArsA mutants also interacted with wild type ArsD. Only the Q56R ArsA derivative exhibited significant metalloid-stimulated ATPase activity in vitro. Purified Q56R ArsA was stimulated by wild type ArsD and to a lesser degree by the quadruple ArsD derivative. The F120I and D137V ArsAs did not show metalloid-stimulated ATPase activity. Structural models generated by in silico docking suggest that an electrostatic interface favors reversible interaction between ArsA and ArsD. We predict that mutations in ArsA propagate changes in hydrogen bonding and salt bridges to the ArsA–ArsD interface that affect their interactions.     

The RCK1 and RCK2 protein kinase genes from Saccharomyces cerevisiae suppress cell cycle checkpoint mutations in Schizosaccharomyces pombe

The protein kinase-encoding genes RCK1 and RCK2 from Saccharomyces cerevisiae have been identified as suppressors of Schizosaccharomyces pombe cell cycle checkpoint mutations. Upon expression of these genes, radiation resistance is partially restored in S. pombe mutants with checkpoint deficiencies, but not in mutants with DNA repair defects. Some checkpoint mutants are sensitive to the DNA synthesis inhibitor hydroxyurea, and this sensitivity is also suppressed by RCK1 and RCK2. The degree of suppression can be modulated by varying expression levels. Expression of RCK1 or RCK2 in S. pombe causes cell elongation and decelerated growth. Cells expressing these genes have a single nucleus and a 2n DNA content. We conclude that these genes act in S. pombe to prolong the G2 phase of the cell cycle.     

Isolation and characterization of temperature-sensitive mutations in the gene (rpb3 ) for subunit 3 of RNA polymerase II in the fission yeast Schizosaccharomyces pombe

Subunit 3 (Rpb3) of eukaryotic RNA polymerase II is a homologue of the α subunit of prokaryotic RNA polymerase, which plays a key role in subunit assembly of this complex enzyme by providing the contact surfaces for both β and β′ subunits. Previously we demonstrated that the Schizosaccharomyces pombe Rpb3 protein forms a core subassembly together with Rpb2 (the β homologue) and Rpb11 (the second α homologue) subunits, as in the case of the prokaryotic α₂β complex. In order to obtain further insight into the physiological role(s) of Rpb3, we subjected the S. pombe rpb3 gene to mutagenesis. A total of nine temperature-sensitive (Ts) and three cold-sensitive (Cs) S. pombe mutants have been isolated, each (with the exception of one double mutant) carrying a single mutation in the rpb3 gene in one of the four regions (A–D) that are conserved between the homologues of eukaryotic subunit 3. The three Cs mutations were all located in region A, in agreement with the central role of the corresponding region in the assembly of prokaryotic RNA polymerase; the Ts mutations, in contrast, were found in all four regions. Growth of the Ts mutants was reduced to various extents at non-permissive temperatures. Since the metabolic stability of most Ts mutant Rpb3 proteins was markedly reduced at non-permissive temperature, we predict that these mutant Rpb3 proteins are defective in polymerase assembly or the mutant RNA polymerases containing mutant Rpb3 subunits are unstable. In accordance with this prediction, the Ts phenotype of all the mutants was suppressed to varying extents by over-expression of Rpb11, the pairing partner of Rpb3 in the core subassembly. We conclude that the majority of rpb3 mutations affect the assembly of Rpb3, even though their effects on subunit assembly vary depending on the location of the mutation considered.     

Identification and properties of UDP-glucose: Cyanidin-3-O-glucosyltransferase isolated from petals of the red campion (Silene dioica)

An enzyme catalyzing the transfer of the glucosyl moiety of UDP-glucose to the 3-hydroxyl group of cyanidin has been demonstrated in petal extracts of Silene dioica mutants with cyanidin-3-O-glucoside in the petals. This transferase activity was also present in young rosette leaves and calyces of these plants. The highest glucosyltransferase activity was found in petals of opening flowers of young plants. The enzyme was purified ninetyfold by PVP and Sephadex chromatography. The glucosyltransferase had a pH optimum of 7.5, had a “true K_m value” of 4.1×10^?4 m for UDP-glucose and 0.4×10^?4 m for cyanidin chloride, and was not stimulated by divalent metal ions. Both p-chloromercuribenzoate and HgCl₂ inhibited the enzyme activity. Pelargonidin chloride and delphinidin chloride at reduced rates also served as substrates. The enzyme did not catalyze the glucosylation of the 3-hydroxyl group of flavonols or the 5-hydroxyl group of anthocyanins. ADP-glucose could not serve as a glucosyl donor. The results of Sephadex G150 chromatography suggest that the glucosyltransferase can exist as dimer of about 125,000 daltons and as active monomers of 60,000 daltons. The genetic control of the glucosyltransferase activity is discussed.