Conjugation-induced lysis of Schizosaccharomyces pombe

About 15% of the conjugating cells of Schizosaccharomyces pombe were observed to lyse spontaneously during the conjugation process. Lysis occurred at the site of union.     

Integrated map of the Schizosaccharomyces pombe genome

A restriction map of the entire Schizosaccharomyces pombe genome was constructed using two restriction enzymes (BamHI and PstI) that recognize 6 bp. The restriction map contains 420 minimally overlapping clones (miniset) and has 22 gaps. We located 126 genes, marker fragments of DNA (NotI and SfiI linking clones), and 36 transposable elements by hybridization to unique restriction fragments.     

Schizosaccharomyces pombe produces novel pyruvate-containing N-linked oligosaccharides

The large N-linked oligosaccharides released by endo-beta-N-acetylglucosaminidase H from Schizosaccharomyces pombe glycoproteins were analyzed for the presence of noncarbohydrate functional groups. No phosphate, sulfate, or acetate could be detected; however, approximately six molecules of pyruvic acid/molecule were found on 98% of the oligosaccharides. Pyruvate moieties were acetal (ketal)-linked to galactose residues in the R configuration to carbons 4 and 6. This is the first report of pyruvate functional groups being attached to N-linked oligosaccharides in yeast and appears only to be the second documentation of this sugar modification in eukaryotes.     

Enzyme defects in glutamate-requiring strains of Schizosaccharomyces pombe

Among the glutamate-requiring strains of Schizosaccharomyces pombe previously described [1], glu2 and glu3 strains were both shown to lack NAD-specific isocitrate dehydrogenase. glu4 strains were shown to lack glutamine:2-oxoglutarate aminotransferase (GOGAT), and to be defective in ammonia assimilation. The regulation of GOGAT activity in wild-type cells was investigated and was consistent with GOGAT and glutamine synthetase being involved in ammonium assimilation, particularly under conditions of nitrogen limitation.     

A novel mutant allele of Schizosaccharomyces pombe rad26 defective in monitoring S-phase progression to prevent premature mitosis

A semipermissive growth condition was defined for a Schizosaccharomyces pombe strain carrying a thermosensitive allele of DNA polymerase delta (pol delta ts03). Under this condition, DNA polymerase delta is semidisabled and causes a delay in S-phase progression. Using a genetic strategy, we have isolated a panel of mutants that enter premature mitosis when DNA replication is incomplete but which are not defective for arrest in G2/M following DNA damage. We characterized the aya14 mutant, which enters premature mitosis when S phase is arrested by genetic or chemical means. However, this mutant is sensitive to neither UV nor gamma irradiation. Two genomic clones, rad26+ and cds1+, were found to suppress the hydroxyurea sensitivity of the aya14 mutant. Genetic analysis indicates that aya14 is a novel allele of the cell cycle checkpoint gene rad26+, which we have named rad26.a14. cds1+ is a suppressor which suppresses the S-phase feedback control defect of rad26.a14 when S phase is inhibited by either hydroxyurea or cdc22, but it does not suppress the defect when S phase is arrested by a mutant DNA polymerase. Analyses of rad26.a14 in a variety of cdc mutant backgrounds indicate that strains containing rad26.a14 bypass S-phase arrest but not G1 or late S/G2 arrest. A model of how Rad26 monitors S-phase progression to maintain the dependency of cell cycle events and coordinates with other rad/hus checkpoint gene products in responding to radiation damage is proposed.     

Identification of Schizosaccharomyces pombe prenol as dolichol-16,17

Available evidence indicates that transforming growth factor beta (TGFbeta) is produced by bone cells, that production is enhanced by testosterone and dihydrotestosterone, and that TGFbeta is an important modulator of bone formation, induction, and repair. To determine the relative concentrations of isoforms of skeletal TGFbeta, whether orchiectomy alters the concentration of TGFbeta in long bones, and whether alteration is prevented by testosterone replacement, male Sprague-Dawley rats were either sham-operated and given placebo (n = 20) or orchiectomized and given either placebo (n = 20) or 100 mg testosterone (n = 20) by slow-release pellets implanted sc at the back of the neck and killed at 6 weeks. Orchiectomy did not change serum calcium and lowered serum testosterone and serum phosphorus; these reductions were prevented by testosterone replacement. TGFbeta1 in skeletal extracts was much more abundant than TGFbeta2 or TGFbeta3. Orchiectomy reduced skeletal TGFbeta by over 80 percent, and reduction was prevented by testosterone replacement. The relative abundance of the three isoforms of TGFbeta in bone was not influenced by orchiectomy or testosterone replacement, and skeletal messenger RNA of TGFbeta1 and TGFbeta2 was not altered 4 weeks after orchiectomy. Messenger RNA for TGFbeta3 was below the limits of detection. Thus, testosterone deficiency markedly diminishes skeletal TGFbeta, and reduction is prevented by testosterone replacement. The findings support the hypothesis that testosterone and TGFbeta are required for maintenance of the skeleton in male rats.     

Regulation of Schizosaccharomyces pombe Wee1 tyrosine kinase

Wee1 tyrosine kinase regulates mitosis by carrying out the inhibitory tyrosine 15 phosphorylation of Cdc2 M-phase inducing kinase. Schizosaccharomyces pombe Wee1 is a large protein, consisting of a C-terminal catalytic domain of approximately 350 amino acids preceded by a N-terminal domain of approximately 550 residues. The functional properties of the Wee1 N-terminal domain were investigated by expressing truncated forms of Wee1 in S. pombe. Both positive and negative regulatory domains were identified. Sequences important for Wee1 function were mapped to a central region (residues 363-408). This region is not required for kinase activity or nuclear localization, suggesting it may be involved in substrate recognition. The negative regulatory domain resides in the N-terminal third of Wee1, Wee1 constructs lacking this domain are more effective at delaying mitosis than wild-type Wee1. The negative regulatory domain contains clusters of potential Cdc2 phosphorylation sites. Investigations to monitor the abundance of Wee1 mRNA and protein during the cell cycle were also carried out.     

A phosphorylation site mutant of Schizosaccharomyces pombe cdc2p fails to promote the metaphase to anaphase transition

The mammalian cornea receives a dense sensory innervation and a modest sympathetic innervation. The purpose of the current study was to determine if the rat cornea is also innervated by parasympathetic nerves. In the first set of experiments, unilateral combined sympathetic and sensory ocular denervations were performed in adult rats by surgical removal of the superior cervical ganglion and intracranial transection of the trigeminal ophthalmomaxillary nerve. Completeness of the denervation procedure was verified postmortem by a variety of macroscopic and immunohistochemical methods. Five to twelve days later, the corneas were serially sectioned tangential to the ocular surface and processed immunohistochemically with antibodies against the pan-neuronal markers, protein gene product 9.5 (PGP-9.5) and peripherin. In every animal a small, but constant, population of corneal and limbal immunoreactive fibers were unaffected by the surgical denervations and were concluded to derive from parasympathetic ganglia. In the second set of experiments, the origins of the rat corneal innervation were determined by applying the neuroanatomical tracer, wheat germ agglutinin-horseradish peroxidase (WGA-HRP) to the central cornea. Two to four days later, the trigeminal, superior cervical, ciliary, accessory ciliary and pterygopalatine ganglia were sectioned and analysed for the presence of HRP-labeled neurons. Examination of the corneal application site and associated ocular tissues revealed no evidence of tracer spread into neighbouring structures. Small numbers (0-6 per animal) of HRP-labeled neurons were observed in the ipsilateral ciliary and accessory ciliary ganglia of most animals. The results of these carefully controlled studies provide strong anatomical evidence of a modest parasympathetic innervation of the rat cornea.     

Purification and characterization of phosphatidylglycerolphosphate synthase from Schizosaccharomyces pombe

The enzyme CDP-diacylglycerol:sn-glycerol-3-phosphate 3-phosphatidyltransferase (phosphatidylglycerolphosphate synthase; PGPS4; EC 2.7.8.5) is located in the mitochondrial inner membrane and catalyzes the committed step in the cardiolipin branch of phospholipid synthesis. Previous studies revealed that PGPS is the most highly regulated enzyme in cardiolipin biosynthesis in both Saccharomyces cerevisiae and Schizosaccharomyces pombe. In this work, we report the purification to homogeneity of PGPS from S. pombe. The enzyme was solubilized from the mitochondrial membrane of S. pombe with Triton X-100. The solubilized enzyme, together with the associated detergent and intrinsic lipids, had a molecular mass of 120 kDa, as determined by gel filtration. The enzyme was further purified using salt-induced phase separation, gel filtration, and ionic exchange, hydroxylapatite, and affinity chromatographies. The procedure yielded a homogeneous protein preparation, evidenced by both SDS-polyacrylamide gel electrophoresis (PAGE) and agarose isoelectric focusing under nondenaturing conditions. The purified enzyme had an apparent molecular mass of 60 kDa as determined by SDS-PAGE. The enzyme showed a strong dependence on lipid cofactors for activity in vitro. While both phosphatidic acid and CDP-diacylglycerol appeared to be activators, the most significant activation was observed with cardiolipin. The possible physiological significance of the lipid cofactor effect is discussed. This is the first purification of a eucaryotic PGPS enzyme to date, and the first purification of a phospholipid biosynthetic enzyme from S. pombe.     

Characterization of a Holliday junction-resolving enzyme from Schizosaccharomyces pombe

The rearrangement and repair of DNA by homologous recombination involves the creation of Holliday junctions, which are cleaved by a class of junction-specific endonucleases to generate recombinant duplex DNA products. Only two cellular junction-resolving enzymes have been identified to date: RuvC in eubacteria and CCE1 from Saccharomyces cerevisiae mitochondria. We have identified a protein from Schizosaccharomyces pombe which has 28% sequence identity to CCE1. The YDC2 protein has been cloned and overexpressed in Escherichia coli, and the purified recombinant protein has been shown to be a Holliday junction-resolving enzyme. YDC2 has a high degree of specificity for the structure of the four-way junction, to which it binds as a dimer. The enzyme exhibits a sequence specificity for junction cleavage that differs from both CCE1 and RuvC, and it cleaves fixed junctions at the point of strand exchange. The conservation of the mechanism of Holliday junction cleavage between two organisms as diverse as S. cerevisiae and S. pombe suggests that there may be a common pathway for mitochondrial homologous recombination in fungi, plants, protists, and possibly higher eukaryotes.     

Stage-specific activity of the Leishmania major CRK3 kinase and functional rescue of a Schizosaccharomyces pombe cdc2 mutant

The effects of polyamines and related compounds on the development of drug/antibiotic resistance in a variety of bacterial strains were studied. Methods employed included standard toxicity assays, modified Ames tests for mutation frequencies and antimutagenic effects, prophage induction assays, and recA-lacZ and ada-lacZ induction assays. Using these methods, we have shown that the polyamines produce strong antimutagenic effects against EMS and MMS-induced antibiotic resistance. Spermidine also seems to have antimutagenic potential against 4NQO-induced mutations. DNA fidelity assays suggest that polyamines play a vital role in DNA synthesis, and several polyamines prevent the development of resistance to dihydrostreptomycin. The polyamine putrescine appears to be required for streptomycin action and also enhances the activity of some antibiotics (e.g., neomycin, kanamycin) but shows no enhancing effect on tetracycline or erythromycin. The potential significance of these studies for infectious diseases and tumor therapy is discussed.     

Sce3, a suppressor of the Schizosaccharomyces pombe septation mutant cdc11, encodes a putative RNA-binding protein

In the fission yeast Schizosaccharomyces pombe, the cdc11 gene is required for the initiation of septum formation at the end of mitosis. The sce3 gene was cloned as a multi-copy suppressor of the heat-sensitive mutant cdc11-136. When over-expressed, it rescues all mutants of cdc11 and also a heat-sensitive allele of cdc14, but not the cdc14 null mutant. Deletion shows that sce3 is not essential for cell proliferation. It encodes a putative RNA-binding protein which shows homology to human eIF4B. Immunolocalisation indicates that Sce3p is located predominantly in the cytoplasm. Elevated expression of sce3 increases the steady-state level of cdc14 mRNA. Possible mechanisms of its action are discussed.     

Energy-dependent uptake of calcium by the yeast Schizosaccharomyces pombe

1. In resting cells of the fission yeast Schizosaccharomyces pombe, the uptake of calcium is stimulated by the addition of 90 mM glucose in the presence as in the absence of respiration and inhibited by Antimycin A in the absence of exogenous carbon source. This uptake therefore requires fermentative or respiratory metabolic energy. 2. The calcium uptake by S. pombe exhibits saturation kinetics and high affinity for calcium. At external pH 4.5, the apparent Km is 45 muM ca2+ 400 muM of other divalent cations exert competitive inhibitions of calcium uptake in the following order of affinities: Sr2+ greater than Mn2+ greater than Co2+ greater than Mg2+. Inhibition by KCl is also observed but is of non-competitive type and requires high concentrations of the order of 40 mM. 3. At 30 degrees C, the uptake rate of calcium is about 10-times higher at pH 8925 than at pH 4.0. An extrusion of 45Ca2+, the rate of which is estimated to be lower than one-fifth of the uptake, is observed in the presence of glucose when the external pH is acid. 4. At external pH 4.5, low concentrations of lanthanum chloride, ruthenium red and hexamine cobaltichloride are inhibitory for the uptake of calcium by the yeast cells. 5. In presence of Antimycin A, the uncouplers: NaN3, dinitrophenol, and concentrations of crobonylcyanide m-chlorophenylhydrazone higher than 80 muM inhibit the calcium uptake by glycolysing cells. In the presence of glucose, the K+ ionophore Dio-9 dnhances severalfold the uptake of calcium even at 2 degrees C. 6. It is concluded that S. pombe possess an active transport system for low concentrations of calcium. This transport seems to be dependent on an electric potential (negative inside) across the cellular membrane.     

The centromere enhancer mediates centromere activation in Schizosaccharomyces pombe

The centromere enhancer is a functionally important DNA region within the Schizosaccharomyces pombe centromeric K-type repeat. We have previously shown that addition of the enhancer and cen2 centromeric central core to a circular minichromosome is sufficient to impart appreciable centromere function. A more detailed analysis of the enhancer shows that it is dispensable for centromere function in a cen1-derived minichromosome containing the central core and the remainder of the K-type repeat, indicating that the critical centromeric K-type repeat, like the central core, is characterized by functional redundancy. The centromeric enhancer is required, however, for a central core-carrying minichromosome to exhibit immediate centromere activity when the circular DNA is introduced via transformation into S. pombe. This immediate activation is probably a consequence of a centromere-targeted epigenetic system that governs the chromatin architecture of the region. Moreover, our studies show that two entirely different DNA sequences, consisting of elements derived from two native centromeres, can display centromere function. An S. pombe CENP-B-like protein, Abp1p/Cbp1p, which is required for proper chromosome segregation in vivo, binds in vitro to sites within and adjacent to the modular centromere enhancer, as well as within the centromeric central cores. These results provide direct evidence in fission yeast of a model, similar to one proposed for mammalian systems, whereby no specific sequence is necessary for centromere function but certain classes of sequences are competent to build the appropriate chromatin foundation upon which the centromere/kinetochore can be formed and activated.     

Molecular genetic analysis of U2AF59 in Schizosaccharomyces pombe: differential sensitivity of introns to mutational inactivation

The large subunit of the mammalian U2AF heterodimer (U2AF65) is essential for splicing in vitro. To expand our understanding of how this protein functions in vivo, we have created a null allele of the gene encoding the Schizosaccharomyces pombe ortholog, U2AF59, and employed it in a variety of genetic complementation assays. First, analysis of an extensive series of double amino acid substitutions indicates that this splicing factor is surprisingly refractory to mutations. Second, despite extensive structural conservation, we find that metazoan large subunit orthologs cannot substitute in vivo for fission yeast U2AF59. Third, because the activity of U2AF65 in vitro involves binding to the 3' polypyrimidine tract, we examined the splicing of introns containing or lacking this feature in a U2AF59 mutant described here as well as a previously isolated temperature-sensitive mutant (Potashkin et al., 1993, Science 262:573-575). Our data indicate that all four introns tested, including two that lack extensive runs of pyrimidines between the branchpoint and 3' splice site, show splicing defects upon shifting to the nonpermissive condition. In all cases, splicing is blocked prior to the first transesterification reaction in the mutants, consistent with the role inferred for human U2AF65 based on in vitro experiments.     

Characterization of the geranylgeranyl transferase type I from Schizosaccharomyces pombe

Transport from the TGN to the basolateral surface involves a rab/N-ethylmaleimide-sensitive fusion protein (NSF)/soluble NSF attachment protein (SNAP)/SNAP receptor (SNARE) mechanism. Apical transport instead is thought to be mediated by detergent-insoluble sphingolipid-cholesterol rafts. By reducing the cholesterol level of living cells by 60-70% with lovastatin and methyl-beta-cyclodextrin, we show that the TGN-to-surface transport of the apical marker protein influenza virus hemagglutinin was slowed down, whereas the transport of the basolateral marker vesicular stomatitis virus glycoprotein as well as the ER-to-Golgi transport of both membrane proteins was not affected. Reduction of transport of hemagglutinin was accompanied by increased solubility in the detergent Triton X-100 and by significant missorting of hemagglutinin to the basolateral membrane. In addition, depletion of cellular cholesterol by lovastatin and methyl-beta-cyclodextrin led to missorting of the apical secretory glycoprotein gp-80, suggesting that gp-80 uses a raft-dependent mechanism for apical sorting. Our data provide for the first time direct evidence for the functional significance of cholesterol in the sorting of apical membrane proteins as well as of apically secreted glycoproteins.     

A Schizosaccharomyces pombe artificial chromosome large DNA cloning system

The feasibility of using the fission yeast, Schizosaccharomyces pombe , as a host for the propagation of cloned large fragments of human DNA has been investigated. Two acentric vector arms were utilized; these carry autonomously replicating sequences ( ars elements), selectable markers ( ura4(+) or LEU2 ) and 250 bp of S. pombe terminal telomeric repeats. All cloning was performed between the unique sites in both vector arms for the restriction endonuclease Not I. Initially the system was tested by converting six previously characterized cosmids from human chromosome 11p13 into a form that could be propagated in S.pombe as linear episomal elements of 50-60 kb in length. In all transformants analysed these cosmids were maintained intact. To test if larger fragments of human DNA could also be propagated total human DNA was digested with Not I and size fractionated by pulsed field gel electrophoresis (PFGE). Fractions of 100-1000 kb were ligated to Not I-digested vector arms and transformed into S.pombe protoplasts in the presence of lipofectin. Prototrophic ura+leu+transformants were obtained which upon examination by PFGE were found to contain additional linear chromosomes migrating at between 100 and 500 kb with a copy number of 5-10 copies/cell. Hybridization analyses revealed that these additional bands contained human DNA. Fluorescent in situ hybridization (FISH) analyses of several independent clones indicated that the inserts were derived from single loci within the human genome. These analyses clearly demonstrate that it is possible to clone large fragments of heterologous DNA in fission yeast using this S.p ombe artificial chromosome system which we have called SPARC. This vector-host system will complement the various other systems for cloning large DNA fragments.     

Isolation of synthetic lethal mutants of ras1 in Schizosaccharomyces pombe

Ras proteins are membrane-associated guanine nucleotide-binding proteins that serve as molecular switches for signal transduction pathways in a diverse array of organisms. Various cellular factors are known to interact with Ras proteins. In order to find the novel cellular factors that are associated with Ras function, we have constructed synthetic lethal mutants of the ras1+ gene in Schizosaccharomyces pombe and used them to identify the genes that are functionally dependent on the Ras1. We first constructed S. pombe strains in which chromosomal ras1+ gene is placed under the nmt1 promoter that is regulated by thiamine. This strain shows ras1+ phenotype in the absence of thiamine, whereas it shows ras1- phenotype in the presence of thiamine. Second, we mutated the constructed strains with ultraviolet light (UV) and selected two synthetic lethal mutants that could not grow when Ras1 function was repressed (ras1-). One of the mutants, KSC3, showed a swollen cell shape, aberrant deposition of septum materials, and aberrant nuclei. The other mutant, KSC4, showed sensitivity to hyper-osmolarity when Ras1 function is absent. These mutants, however, grow normally when Ras1 is expressed (ras1+). These two novel synthetic lethal mutants of ras1 provide the means to isolate the corresponding genes that function in association with Ras1 in S. pombe. Screening of a genomic library of S. pombe complementing the mutant phenotype allowed us to identify several novel genes associated with Ras1 of S. pombe.     

Dynamics of cell wall formation in fission yeast, Schizosaccharomyces pombe

Studies on the dynamics of surface and intracellular structures during cell wall formation from the reverting protoplast of Schizosaccharomyces pombe were reviewed, and the correlation between cell wall formation and actin cytoskeleton, which is the most important conductor of the mechanism, is described in this paper. A close spatial and temporal relationship between actin cytoskeleton and cell wall formation was found by using wild type and actin point-mutant cps8 of S. pombe. Concomitant with the cell wall formation, dynamic behavior of the intracellular secretion machinery, especially the Golgi apparatus and secretory vesicles, was analyzed by three-dimensional reconstruction of 40 to 80 serial sections at five reverting stages. Total reverting protoplast volume increased by 3.8 and 4.3 times at 3 and 5 h, respectively, and the volume of the Golgi apparatus in the corresponding stages increased 2.3- and 2. 5-fold over the same periods. The number of secretory vesicles also markedly increased by 3.4 and 5.8 times over that of the corresponding reverting protoplasts. Actin point-mutant cps8 cells have abnormal structure in the cell wall and septum, and the distribution pattern of the actin cytoskeleton during the reversion process was different from wild-type protoplasts. The profiles of actin showed one or two thick cables and patches in the cytoplasm which remained throughout reversion. The development of crosslinkage of the glucan fibrils which are beta-1,3-glucan in nature on the reverting protoplast surface was defective; the glucan networks consisted of thin, rope-shaped fibrils up to 30 nm in width which formed a ribbon-shape 200 nm wide in wild-type reverting protoplasts. The intrafibrillar space is not filled with amorphous particles of alpha-galactomannan in nature. The secretion machinery was seen to have a similar profile as the wild type. The above results suggest that actin cytoskeleton may control secretion of beta-1,6-glucan and other cell wall substances such as alpha-glucan and alpha-galactomannan rather than beta-1,3-glucan. Study of the role of actin cytoskeleton in the cell wall formation is contributing to the development of antifungal agents together with basic cell biology.