Replication of centromere II of Schizosaccharomyces pombe.

The centromeric DNAs of Schizosaccharomyces pombe chromosomes resemble those of higher eukaryotes in being large and composed predominantly of repeated sequences. To begin a detailed analysis of the mode of replication of a complex centromere, we examined whether any sequences within S. pombe centromere II (cen2) have the ability to mediate autonomous replication. We found a high density of segments with such activity, including at least eight different regions comprising most of the repeated and unique centromeric DNA elements. A physical mapping analysis using two-dimensional gels showed that autonomous replication initiated within the S. pombe sequences in each plasmid. A two-dimensional gel analysis of replication on the chromosomes revealed that the K and L repeat elements, which occur in multiple copies at all three centromeres and comprise approximately 70% of total centromeric DNA mass in S. pombe, are both sites of replication initiation. In contrast, the unique cen2 central core, which contains multiple segments that can support autonomous replication, appears to be repressed for initiation on the chromosome. We discuss the implications of these findings for our understanding of DNA replication and centromere function.     

Effect of 2-deoxyglucose on Schizosaccharomyces pombe

Megnet, Roland (Institut für Allgemeine Mikrobiologie der Universit?t, Bern, Switzerland). Effect of 2-deoxyglucose on Schizosaccharomyces pombe. J. Bacteriol. 90:1032-1035. 1965.-Cultivation of Schizosaccaromyces pombe in a medium containing 2-deoxyglucose (100 mug/ml) results in the death of the cells after an initial period of apparently normal growth. At higher deoxyglucose concentration (400 mug/ml), the cells die immediately after inoculation. Only growing cells are killed, and microscopic inspection of the cultures reveals cell-wall fragments of lysed cells. A mutant resistant to 2-deoxyglucose, which cannot use glucose as a carbon source, was found to be partially deficient in hexokinase. The data constitute evidence for the inhibition of some reaction(s) in the synthesis of cell-wall polysaccharides by metabolites of 2-deoxyglucose in this organism.     

A DNA exonuclease induced during meiosis of Schizosaccharomyces pombe.

In meiotic cells of the fission yeast Schizosaccharomyces pombe, a DNA exonuclease activity increased approximately 5-fold after premeiotic S-phase and decreased to the initial level before the meiotic divisions. We have purified this activity, designated exonuclease I, to near homogeneity. The activity co-purified with a polypeptide with an apparent molecular weight of 36,000. With a linear double-stranded DNA substrate, exonuclease I degraded only the 5'-ended strand from each end to produce 3'-single-stranded tails. The enzyme also acted on nicked circular DNA with comparable affinity. The meiotic induction of exonuclease I and its mode of action, similar to that of recombination-promoting exonucleases from bacteria, suggest that exonuclease I is involved in meiotic homologous recombination in S. pombe.     

Effect of ethanol on the phospholipid and fatty acid content of Schizosaccharomyces pombe membranes

Ethanol at concentrations up to 5% (v/v) had no effect on the growth of Schizosaccharomyces pombe, whereas concentrations over 7.5% were inhibitory. The major membrane phospholipids in S. pombe cells growing aerobically in the absence of added ethanol were phosphatidylinositol, phosphatidylcholine and phosphatidylethanolamine. Oleic acid (18:1) was the main fatty acid. When ethanol (7.5%) was added to aerobically growing cultures, the phosphatidylinositol content increased, whereas the 18:1 content decreased. Similar changes were observed in the membrane phospholipids of cells grown anaerobically without ethanol. However, the presence of ethanol in anaerobically growing cultures had an opposite effect on fatty acids, as the 18:1 content increased. The results support the idea that ethanol tolerance in S. pombe may be connected with a high content of 18:1 fatty acids, and with the ability to maintain a high rate of phospholipid biosynthesis.     

Transport of L-glutamic acid in the fission yeast Schizosaccharomyces pombe.

Transport of L-glutamic acid into the fission yeast Schizosaccharomyces pombe grown to the early stationary phase and preincubated for 60 min with 1% D-glucose is practically unidirectional and is mediated by a single uphill transport system with a KT of 170 microM and Jmax of 4.8 nmol min-1 (mg dry wt.)-1. The system proved to be rather non-specific since all the amino acids transported into the cells acted as potent competitive inhibitors. It has a pH optimum at 3.0-4.0, the accumulation ratio of L-glutamic acid is highest at a suspension density of 0.6-1.0 mg dry wt. per ml and decreases with increasing L-glutamic acid concentrations in the external medium. The system present in the cells after preincubation with D-glucose is unstable and its activity decays after washing the cells with water or after stopping the cytosolic proteinsynthesis with cycloheximide, with a half-time of 24 min in a reaction significantly retarded by phenylmethylsulfonyl fluoride, a serine proteinase inhibitor. The synthesis of the transport protein appears to be repressible by ammonium ions.     

Oscillations of redox states in synchronously dividing cultures of Acanthamoeba castellanii and Schizosaccharomyces pombe.

The redox state of the mitochondria of Acanthamoeba castellanii and Schizosaccharomyces pombe was assessed with a flying-spot fluorometer (Chance et al. 1978. Am. J. Physiol. 235:H 809) that provides excitation appropriate for oxidized flavoprotein or reduced pyridine nucleotide. Fluorescence signals could be resolved from the thin films of cultures that were only one cell deep. In both organisms anoxia was associated with an increased pyridine nucleotide and decreased flavoprotein fluorescence. The addition of mitochondrial uncoupling agents increased the flavoprotein fluorescence and the fluorometer was able to resolve uncoupler-sensitive and uncoupler-insensitive fractions of S. pombe cultures. In both synchronous and asynchronous cultures of A. castellanii and S. pombe the mitochondrial redox state oscillates with a period of 4.5 +/- 1.0 min. Oscillations with much longer period, of the order of an hour, are observed in synchronous cultures and these oscillations correlate with similar oscillations in respiratory rate, uncoupler sensitivity, and adenine nucleotide pool sizes. The results are consistent with the hypothesis that synchronous cultures of A. castellanii and S. pombe oscillate between the ADP-limited (state 4) and ADP-sufficient (state 3) respiratory states, i.e., exhibit in vivo respiratory control.     

The Birt-Hogg-Dube and tuberous sclerosis complex homologs have opposing roles in amino acid homeostasis in Schizosaccharomyces pombe

Birt-Hogg-Dube (BHD) is a tumor suppressor gene disorder characterized by skin hamartomas, cystic lung disease, and renal cell carcinoma. The fact that hamartomas, lung cysts, and renal cell carcinoma can also occur in tuberous sclerosis complex (TSC) suggests that the BHD and TSC proteins may function within a common pathway. To evaluate this hypothesis, we deleted the BHD homolog in Schizosaccharomyces pombe. Expression profiling revealed that six permease and transporter genes, known to be down-regulated in Deltatsc1 and Deltatsc2, were up-regulated in Deltabhd, and levels of specific intracellular amino acids known to be low in Deltatsc1 and Deltatsc2 were elevated in Deltabhd. This "opposite" profile was unexpected, given the overlapping clinical phenotypes. The TSC1/2 proteins inhibit Rheb in mammals, and Tsc1/Tsc2 inhibit Rhb1 in S. pombe. Expression of a hypomorphic allele of rhb1(+) dramatically increased permease expression levels in Deltabhd but not in wild-type yeast. Loss of Bhd sensitized yeast to rapamycin-induced increases in permease expression levels, and rapamycin induced lethality in Deltabhd yeast expressing the hypomorphic Rhb1 allele. In S. pombe, it is known that Rhb1 binds Tor2, and Tor2 inhibition leads to up-regulation of permeases including those that are regulated by Bhd. Our data, therefore, suggest that Bhd activates Tor2. If the mammalian BHD protein, folliculin, similarly activates mammalian target of rapamycin, it will be of great interest to determine how mammalian target of rapamycin inhibition in BHD patients and mammalian target of rapamycin activation in TSC patients lead to overlapping clinical phenotypes.     

Effect of amino acid starvation on glucose transport in two archaeal organisms

When protein synthesis is arrested by amino acid starvation, Escherichia coli wild-type strains show stringent control (SC) over stable RNA (sRNA) accumulation as well as a large number of other growth-related processes. One of the events under SC is transport of metabolites. Thus, under amino acid starvation, E. coli fails to accumulate the non-metabolizable glucose analog alpha-methyl-D-glucoside, whereas isogenic relaxed strains continue to take up this glucose analog. Unlike the Bacteria, most wild-type archaeal strains show relaxed control of sRNA accumulation, although a number of stringent strains have been identified. In order to determine whether stringency in the Archaea affects physiological events different from sRNA accumulation, transport of glucose analogs was examined under amino acid starvation in two stringent archaeal strains, Haloferax volcanii and Sulfolobus acidocaldarius. The experiments were performed with 2-deoxy-D-glucose, which was shown to be transported, but metabolized very limitedly. Unlike E. coli, H. volcanii and S. acidocaldarius continued to transport 2-deoxy-D-glucose under amino acid starvation. Thus, in both Archaea glucose analog transport is not under SC, as it is in E. coli.     

Effect of amino acid starvation on UV sensitivity ofLactobacillus acidophilus cells

InLactobacillus acidophilus cultures UV irradiated in the exponential phase of growth, the dosesurvival curve was of the simple exponential type, without any shoulder. If the bacteria were subjected to amino acid starvation prior to irradiation, an shoulder corresponding to a quasi-treshold dose (D_q) of about 780 ergs/mm² appeared in the curve. The administration of protein or RNA-synthesia inhibitors prior to irradiation had the same effect. The effect of pre-irradiation amino acid starvation was abolished by simultaneous thymidine starvation. It was likewise abolished if amino acid starvation was followed by incubation in the presence of amino acids (without thymidine) and then by irradiation of the cells. Post-irradiation amino acid starvation did not lead to the formation of an shoulder but if combined with thymidine starvation it did. It can be concluded from the results that post-irradiation repair processes are facilitated or promoted if, during the post-irradiation interval DNA synthesis is delayed. This delay represents a compensation of the pre-irradiation increase of cellular DNA-content, taking place during inhibition of proteosynthesis. The postirradiation administration of caffeine did not abolish the formation of the shoulder induced by pre-irradiation amino acid starvation; on the contrary, it induced its formation even in exponentially growing, irradiated control bacteria.     

The initiation of cell wall synthesis in parasynchronous cultures of Schizosaccharomyces pombe

Summary Schizosaccharomyces pombe has been grown in parasynchronous culture to study the synthesis of cell wall material. After a lag period of 2.5h following inoculation the cells began to grow, as measured by optical density, dry weight and cell size. The cell number remained constant until 4.5h after inoculation when approximately 70% of the population divided synchronously. Immunofluorescence studies of the growing cells have shown that new wall material is inserted at the cell apices from 2.5 h after inoculation; this result is supported by radio-isotope labelling data which indicated that synthesis of new cell wall material also commenced 2.5 h after inoculation. The incorporation experiments also demonstrated an interruption in cell wall synthesis during the cell separation stage. The composition of the cell wall material varied during the growth cycle, with maximum nitrogen levels at inoculation and following cell division. No serological differences could be detected in the cell walls during the growth cycle.     

Nonspecific acid phosphatase from Schizosaccharomyces pombe. Purification and physical chemical properties.

Repressible nonspecific acid phosphatase from Schizosaccharomyces pombe was purified to apparent homogeneity, as ascertained from ultracentrifugal, electrophoretic, and chromatographic data. The native protein has a molecular weight of 383,000 as determined by sucrose density gradient centrifugation and 381,000 as determined by gel filtration. The native protein can be dissociated in the presence of 8 M urea-1% sodium dodecyl sulfate into sub-units possessing an approximate molecular weight of 104,000. Neutral sugars account for about 66% of the total molecular weight and contribute to the high solubility and some of the other physical properties of this enzyme. Purified enzyme preparations have a Km for 4-nitrophenyl phosphate of 0.17 mM and a broad substrate specificity, but do not show diesterase activity. Phosphate and sulfate are competitive inhibitors. The enzyme is inactivated at neutral and alkaline pH and at relatively low temperatures. Mannose and galactose was found as the main components of the carbohydrate moiety; glucosamine was present in lower amounts. The amino acid analysis revealed a high content of aspartate, threonine, and serine; no sulfhydryl group could be detected. Pi is released in stoichiometric amount (1 mol per enzyme monomer) on protein digestion.     

Derepression of amino acid transport by amino acid starvation in rat hepatoma cells.

Amino acid starvation causes an adaptive increase in the initial rate of transport of selected neutral amino acids in an established line of rat hepatoma cells in tissue culture. After a lag of 30 min, the initial rate of transport of alpha-aminoisobutyric acid (AIB) increases to a maximum after 4 to 6 h starvation of 2 to 3 times that seen in control cells. The increased rate of transport is accompanied by an increase in the Vmax and a modest decrease in the Km for this transport system, and is reversed by readdition of amino acids. The enhancement is specific for amino acids transported by the A or alanine-preferring system (AIB, glycine, proline); uptake of amino acids transported by the L or leucine-preferring system (threonine, phenylalanine, tyrosine, leucine) or the Ly+ system for dibasci amino acids (lysine) is decreased under these conditions. Amino acids which compete with AIB for transport also prevent the starvation-induced increase in AIB transport; amino acids which do not compete fail to prevent the enhancement. Paradoxically threonine, phenylalanine, tryptophan, and tyrosine, which do not compete with AIB for transport, block the enhancement of transport upon amino acid starvation. The starvation-induced enhancement of amino acid transport does not appear to be the result of a release from transinhibition. After 30 min of amino acid starvation, AIB transport is either unchanged or slightly decreased even though amino acid pools are already depleted. Furthermore, loading cells with high concentrations of a single amino acid following a period of amino acid starvation fails to prevent the enhancement of AIB transport, whereas incubation of the cells with the single amino acid for the entire duration of amino acid starvation prevents the enhancement; intracellular amino acid pools are similar under both conditions. The enhancement of amino acid transport requires concomitant RNA and protein synthesis, consistent with the view that the adaptive increase reflects an increased amount of a rate-limiting protein involved in the transport process. Dexamethasone, which dramatically inhibits AIB transport in cells incubated in amino acid-containing medium, both blocks the starvation-induced increase in AIB transport, and causes a time-dependent decrease in transport velocity in cells whose transport has previously been enhanced by starvation.