Genetic control of the cell division cycle in the fission yeast Schizosaccharomyces pombe

Differentiated cells have been established in monolayer culture from adult rat liver and their ultrastructural and biochemical features characterized after 20-30 generations. Hepatocytes were isolated by enzyme perfusion of the liver followed by treatment with papain, which allowed cultures to be established more readily and to be cloned at an early stage. Ultrastructural studies indicated that the cells were derived largely from hepatic parenchymal cells. The cells showed structural modifications during primary culture but were stable thereafter. The cultured cells retained some differentiated functions unique to liver cells, including the synthesis of ornithine form arginine and the secretion of serum proteins, albumin, chi- and beta-globulins.     

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.     

Bax- and Bak-induced cell death in the fission yeast Schizosaccharomyces pombe

The dental profession faces educational, scientific, and ethical challenges in orofacial pain and headache. Past educational deficiencies are being addressed with guidance and recommendations from the AADS, the ADA, and the AAOP. With education and further research, many dental ethical questions in TMD will be resolved. The educational process must continue with a solid foundation in scientific basis provided in university settings. The appropriate use of TMD diagnostic machines, treatment modalities, and management of perpetuating factors such as sleep will evolve with the new knowledge of scientific discovery. These are some of the many challenges of orofacial pain and headache disorders that warrant special consideration.     

Effect of ethanol on the sterols of the fission yeast Schizosaccharomyces pombe

We report a child who was thought to suffer a non-accidental injury. The parents were unable to convince the child abuse team of their innocence. The eruption of lucent teeth established the diagnosis of osteogenesis imperfecta type IVB.     

Analysis of the ntp1+ gene, encoding neutral trehalase in the fission yeast Schizosaccharomyces pombe

We report a clinicopathologic feature of primary cutaneous T-cell lymphoma (CTCL) in a five-year-old boy with increasing swelling of his cheek since two years of age. Histologically, an infiltrate of atypical lymphoid cells with mature T-cell phenotype and clonality was prominent from the dermis to the subcutaneous tissue of the cheek. Although little effect was seen with aggressive multidrug-combined chemotherapy, therapy with interferon-alpha and steroids achieved a prolonged remission. This patient may provide important clues to understanding the clinicopathologic feature of rare primary CTCL in young children.     

Identification of Myo3, a second type-II myosin heavy chain in the fission yeast Schizosaccharomyces pombe

We cloned the myo3+ gene of Schizosaccharomyces pombe which encodes a type-II myosin heavy chain. myo3 null cells showed a defect in cytokinesis under certain conditions. Overproduction of Myo3 also showed a defect in cytokinesis. Double mutant analysis indicated that Myo3 genetically interacts with Cdc8 tropomyosin and actin. Myo3 may be implicated in cytokinesis and stabilization of F-actin cables. Moreover, the function of Myo2 can be replaced by overexpressed Myo3. We observed a modest synthetic interaction between Myo2 and Myo3. Thus, Myo2 and Myo3 seem to cooperate in the formation of the F-actin ring in S. pombe.     

Molecular, functional and evolutionary characterization of the gene encoding HMG-CoA reductase in the fission yeast, Schizosaccharomyces pombe

The synthesis of mevalonate, a molecule required for both sterol and isoprene biosynthesis in eukaryotes, is catalysed by 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase. Using a gene dosage approach, we have isolated the gene encoding HMG-CoA reductase hmgl+, from the fission yeast Schizosaccharomyces pombe (Accession Number L76979). Specifically, hmgl+ was isolated on the basis of its ability to confer resistance to lovastatin, a competitive inhibitor of HMG-CoA reductase. Gene disruption analysis showed that hmgl+ was an essential gene. This result provided evidence that, unlike Saccharomyces cerevisiae, S. pombe contained only a single functional HMG-CoA reductase gene. The presence of a single HMG-CoA reductase gene was confirmed by genomic hybridization analysis. As observed for the S. cerevisiae HMGlp, the hmgl+ protein induced membrane proliferations known as karmellae. A previously undescribed 'feed-forward' regulation was observed in which elevated levels of HMG-CoA synthase, the enzyme catalysing the synthesis of the HMG-CoA reductase substrate, induced elevated levels of hmgl+ protein in the cell and conferred partial resistance to lovastatin. The amino acid sequences of yeast and human HMG-CoA reductase were highly divergent in the membrane domains, but were extensively conserved in the catalytic domains. We tested whether the gene duplication that produced the two functional genes in S. cerevisiae occurred before or after S. pombe and S. cerevisiae diverged by comparing the log likelihoods of trees specified by these hypotheses. We found that the tree specifying post-divergence duplication had significantly higher likelihood. Moreover, phylogenetic analyses of available HMG-CoA reductase sequences also suggested that the lineages of S. pombe and S. cerevisiae diverged approximately 420 million years ago but that the duplication event that produced two HMG-CoA reductase genes in the budding yeast occurred only approximately 56 million years ago. To date, S. pombe is the only unicellular eukaryote that has been found to contain a single HMG-CoA reductase gene. Consequently, S. pombe may provide important opportunities to study aspects of the regulation of sterol biosynthesis that have been difficult to address in other organisms and serve as a test organism to identify novel therapies for modulating cholesterol synthesis.     

Methionine induces sexual development in the fission yeast Schizosaccharomyces pombe via an ste11-dependent signalling pathway

Methionine added to minimal medium overcomes the repressing effects of ammonium and cyclic AMP (cAMP) on sexual development and efficiently induces mating and sporulation in homothallic strains of Schizosaccharomyces pombe. In heterothallic strains it induces G1 arrest when cells enter stationary phase. We show that methionine reduces the intracellular cAMP pool and induces the expression of at least two cAMP-repressible genes, including fbp1 and ste11. The easiest interpretation of the results is that methionine induces sexual development via a cAMP-dependent ste11 signalling pathway.     

Cytoplasmic ribosomal protein genes of the fission yeast Schizosaccharomyces pombe display a unique promoter type: a suggestion for nomenclature of cytoplasmic ribosomal proteins in databases

We identified 34 new ribosomal protein genes in the Schizosaccharomyces pombe database at the Sanger Centre coding for 30 different ribosomal proteins. All contain the Homol D-box in their promoter. We have shown that Homol D is, in this promoter type, the TATA-analogue. Many promoters contain the Homol E-box, which serves as a proximal activation sequence. Furthermore, comparative sequence analysis revealed a ribosomal protein gene encoding a protein which is the equivalent of the mammalian ribosomal protein L28. The budding yeast Saccharomyces cerevisiae has no L28 equivalent. Over the past 10 years we have isolated and characterized nine ribosomal protein (rp) genes from the fission yeast S.pombe . This endeavor yielded promoters which we have used to investigate the regulation of rp genes. Since eukaryotic ribosomal proteins are remarkably conserved and several rp genes of the budding yeast S.cerevisiae were sequenced in 1985, we probed DNA fragments encoding S.cerevisiae ribosomal proteins with genomic libraries of S.pombe . The deduced amino acid sequence of the different isolated rp genes of fission yeast share between 65 and 85% identical amino acids with their counterparts of budding yeast.     

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.     

Multiple orientation-dependent, synergistically interacting, similar domains in the ribosomal DNA replication origin of the fission yeast, Schizosaccharomyces pombe

Previous investigations have shown that the fission yeast, Schizosaccharomyces pombe, has DNA replication origins (500 to 1500 bp) that are larger than those in the budding yeast, Saccharomyces cerevisiae (100 to 150 bp). Deletion and linker substitution analyses of two fission yeast origins revealed that they contain multiple important regions with AT-rich asymmetric (abundant A residues in one strand and T residues in the complementary strand) sequence motifs. In this work we present the characterization of a third fission yeast replication origin, ars3001, which is relatively small ( approximately 570 bp) and responsible for replication of ribosomal DNA. Like previously studied fission yeast origins, ars3001 contains multiple important regions. The three most important of these regions resemble each other in several ways: each region is essential for origin function and is at least partially orientation dependent, each region contains similar clusters of A+T-rich asymmetric sequences, and the regions can partially substitute for each other. These observations suggest that ars3001 function requires synergistic interactions between domains binding similar proteins. It is likely that this requirement extends to other fission yeast origins, explaining why such origins are larger than those of budding yeast.     

Cdm1, the smallest subunit of DNA polymerase d in the fission yeast Schizosaccharomyces pombe, is non-essential for growth and division

Ca2+/calmodulin-dependent protein kinase II (CaM kinase II) is one of the most abundant protein kinases in the brain and has a broad substrate specificity [M.K. Bennett, N.E. Erondu, M.B. Kennedy, Purification and characterization of a calmodulin-dependent protein kinase that is highly concentrated in brain, J. Biol. Chem. 258 (1983) 12735-12744 [1]; J.R. Goldenring, B. Gonzalez, J.S. McGuire, Jr., R.J. DeLorenzo, Purification and characterization of a calmodulin-dependent kinase from rat brain cytosol able to phosphorylate tubulin and microtubule-associated proteins, J. Biol. Chem. 258 (1983) 12632-12640 [4]; M.B. Kennedy, P. Greengard, Two calcium/calmodulin-dependent protein kinases, which are highly concentrated in brain, phosphorylate protein I at distinct sites, Proc. Natl. Acad. Sci. U.S.A. 78 (1981) 1293-1297 [10]; T. Yamauchi, H. Fujisawa, Evidence for three distinct forms of calmodulin-dependent protein kinases from rat brain, FEBS Lett. 116 (1980) 141-144 [20]; T. Yamauchi, H. Fujisawa, Purification and characterization of the brain calmodulin-dependent protein kinase (kinase II), which is involved in the activation of tryptophan 5-monooxygenase, Eur. J. Biochem. 132 (1983) 15-21 [21]]. The alpha and beta isoforms of CaM kinase II are known to be expressed almost exclusively in the brain [P.I. Hanson, H. Schulman, Ca2+/calmodulin-dependent protein kinases, Annu. Rev. Biochem. 61 (1992) 559-601 [7]]. To elucidate the cellular function of CaM kinase II, we introduced cDNA of wild-type CaM kinase II alpha- or beta-isoform, and of mutant alpha-isoform (Ala-286 kinase) into two different types of neuroblastoma, Neuro2a (Nb2a) and NG108-15, thus generating cell lines stably producing elevated levels of these kinases. The mutant alpha-isoform is markedly suppressed in its autophosphorylation by replacement of Thr-286 with Ala [Y.-L. Fong, W.L. Taylor, A.R. Means, T.R. Soderling, Studies of the regulatory mechanism of Ca2+/calmodulin-dependent protein kinase II. Mutation of threonine 286 to alanine and aspartate, J. Biol. Chem. 264 (1989) 16759-16763 [3]; P.I. Hanson, M.S. Kapiloff, L.L. Lou, M.G. Rosenfeld, H. Schulman, Expression of a multifunctional Ca2+/calmodulin-dependent protein kinase and mutational analysis of its autoregulation, Neuron 3 (1989) 59-70 [6]; S. Ohsako, H. Nakazawa, S. Sekihara, A. Ikai, T. Yamauchi, Role of Threonine-286 as autophosphorylation site for appearance of Ca2+-independent activity of calmodulin-dependent protein kinase II alpha subunit, J. Biochem. 109 (1991) 137-143 [15]]. We provided evidence that CaM kinase II played a role in regulating neurite outgrowth and growth cone motility in these cells, and that the autophosphorylation is essential for the kinase to sufficiently exert its cellular function in vivo [Y. Goshima, S. Ohsako, T. Yamauchi, Overexpression of Ca2+/calmodulin-dependent protein kinase II in Neuro2a and NG108-15 neuroblastoma cell lines promotes neurite outgrowth and growth cone motility, J. Neurosci. 13 (1993) 559-567 [5]]. Neurite outgrowth was further stimulated by treatment with 1-(5-isoquinolinesulfonyl)-2-methylpiperazine (H-7) or chelerythrine, inhibitors of protein kinase C [T. Nomura, K. Kumatoriya, Y. Yoshimura, T. Yamauchi, Overexpression of alpha and beta isoforms of Ca2+/calmodulin-dependent protein kinase II in neuroblastoma cells-H-7 promotes neurite outgrowth, Brain Res. 766 (1997) 129-141 [14]]. The morphological change stimulated with protein kinase inhibitors was rapid and was greater in the beta than alpha cells. Some substrates of CaM kinase II related to neurite outgrowth were detected in cells overexpressing the kinase stimulated with H-7. These results suggest that CaM kinase II and protein kinase C play an important role in the control of cell change. (c) 1998 Elsevier Science B.V. All rights reserved.     

Increased thermal stability of the enzyme content in permeabilized whole cells from the fission yeast Schizosaccharomyces pombe by exogenous trehalose and other compounds

Cells of the fission yeast Schizosaccharomyces pombe were permeabilized by treatment with toluene-ethanol. The permeabilized cells lost the bulk of the internal trehalose pool while most of the alkaline phosphatase, invertase, alpha-glucosidase, or neutral trehalase activities located inside the cells remained unaffected. This system was used as an in situ assay to determine the involvement of trehalose in enzyme protection during thermal treatments. The addition of trehalose to suspensions of permeabilized cells resulted in a sugar-dependent thermoprotection of the internal marker enzymes. This approach demonstrates that in whole cells of the fission yeast trehalose plays a physiological role as a protective molecule against thermal denaturation of cellular enzymes.     

The small GTP-binding protein Rho1 is a multifunctional protein that regulates actin localization, cell polarity, and septum formation in the fission yeast Schizosaccharomyces pombe

BACKGROUND: The small GTP-binding protein Rho has been shown to regulate the formation of the actin cytoskeleton in animal cells. We have previously isolated two rho genes, rho1+ and rho2+, from the fission yeast Schizosaccharomyces pombe in order to investigate the function of Rho using genetic techniques. In this paper, we report the cellular function of Rho1. RESULTS: We found that Rho1 is essential for cell viability and cell polarity using gene disruption and by exogenous expression of botulinum C3 ADP-ribosyltransferase. In cells expressing either a constitutively active Rho1 or a dominant-negative Rho1, actin patches were delocalized. Both the cell wall and secondary septum were thick and stratified in cells expressing the constitutively active Rho1, while the cell wall of cells expressing the dominant-negative Rho1 seemed to be loosely organized. Furthermore, inactivation of Rho1 is apparently required for the separation of daughter cells. Cell fractionation studies suggested that Rho1 is predominantly membrane-bound. Moreover, we observed that Rho1 is localized to the cell periphery and to the septum. CONCLUSIONS: Rho1 is involved in actin patch localization, the control of cell polarity, the regulation of septation, and cell wall synthesis.     

Global control of meiotic recombination genes by Schizosaccharomyces pombe rec16 (rep1)

1. The influence of circulating 5-hydroxytryptamine (serotonin) on small intestinal motility was investigated in healthy volunteers. 2. Small intestinal motility was studied by means of a constantly perfused multi-channel manometry tube, connected to a computer system. 3. Intravenous infusions of either 5-hydroxytryptamine at increasing doses or saline were given over a period of 4 h. 4. 5-Hydroxytryptamine infusion dose-dependently increased plasma 5-hydroxytryptamine from approximately 2 to 10 and 25 nmol/l respectively, as well as urinary excretions of 5-hydroxytryptamine and 5-hydroxyindole acetic acid, a major 5-hydroxytryptamine metabolite. 5. The number of phase III of the migrating motor complex originating in the small intestine was dose-dependently increased by 5-hydroxytryptamine, and found to correlate to the plasma concentration of 5-hydroxytryptamine. The fraction of phase III also increased at the expense of phase II activity. In addition, 5-hydroxytryptamine increased the motility index, propagation velocity of phase III activity and the amplitude of contractions during phase III. 6. Whereas the low dose of 5-hydroxytryptamine (15 nmol.min-1.kg-1) had no haemodynamic effects, an increase in heart rate by approximately 20 beats/min, without change in blood pressure, was observed at the higher dose (60 nmol.min-1.kg-1). Respiratory parameters did not change during infusion of 5-hydroxytrytamine at either dose. 7. In conclusion, elevation of circulating 5-hydroxytryptamine by intravenous infusion results in more frequent and faster propagating migrating motor complexes in the human small intestine during the inter-digestive period.     

Human and mouse homologs of the Schizosaccharomyces pombe rad17+ cell cycle checkpoint control gene

In the cat, the nucleus retroambiguus (NRA) projects to expiratory motoneurons in the brainstem and spinal cord. Recently, it has been demonstrated that the NRA sends fibers to a specific set of motoneurons in the lumbosacral cord, which pathway is thought to play a crucial role in mating behavior. The question is whether such projections exist in the hamster, because the female of this species displays a very distinctive receptive behavior. In the hamster, lumbosacral cord injections of wheat germ agglutinin-horseradish peroxidase (WGA-HRP) combined with hemisection 1 or 2 segments rostral to injection sites in three of the five cases demonstrated retrogradely labeled neurons in the NRA at levels 1.0-2.25 mm caudal to the obex, contralateral to the injection sites. Injections of WGA-HRP into the NRA and adjoining reticular formation revealed that NRA fibers crossed the midline in the caudal medulla and descended in the contralateral lateral and ventrolateral funiculi to terminate bilaterally, but mainly contralaterally, in the motoneuronal cell groups of the abdominal wall and iliopsoas muscles. NRA projections to levels caudal to lumbar segment 5 were virtually absent. Electron microscopic examination revealed that, of the 162 labeled NRA terminal profiles found in the ultrathin sections, 144 (89%) made monosynaptic contacts with retrogradely labeled dendrites of iliopsoas motoneurons. These NRA terminals formed asymmetrical synapses and contained spherical vesicles indicative of an excitatory function. The results indicate that, in the hamster, direct contralateral NRA projections exist to iliopsoas motoneurons. A concept is discussed in which this pathway plays a crucial role in mating behavior.