Flow process for electroextraction of intracellular enzymes from the fission yeast, Schizosaccharomyces pombe

Flow treatment of the yeast, Schizosaccharomyces pombe, with high intensity electric field pulses released intracellular enzymes such as glyceraldehyde-3-phosphate dehydrogenase and phosphoglycerate kinase. Over 70% of the total activity was liberated within 4 h after pulse application. The optimal field intensities were considerably higher than that needed for irreversible plasma membrane permeabilization.     

Ammonia assimilation in the fission yeast Schizosaccharomyces pombe 972

Glutamine synthetase (GS) activity of Schizosaccharomyces pombe 972 was high in ammonia-limited cultures, low in phosphate-and sulphate-limited cultures and not detected in glucose-limited cultures. When ammonia was pulsed into an ammonia-limited culture then GS activity decreased at a rate faster than that calculated if enzyme synthesis ceased and enzyme was diluted out by growth. Enzyme activity increased in ammonia-starved, phosphate-limited cultures and in the ammonia pulse system when the added ammonia had been utilised. These increases in enzyme activity were prevented by the presence of 100 g/ml cycloheximide. GS activity was inversely related to the intracellular concentration of glutamate.Abbreviations Gs Glutamine synthetase, EC 6.3.1.2 - GOGAT Glutamine: 2-oxo-glutarate amino transferase, EC 2.6.1.53 - GDH Glutamate dehydrogenase, EC 1.4.1.3     

Electron microscopic examination of sporulation-deficient mutants of the fission yeast Schizosaccharomyces pombe

A homothallic haploid strain of the fission yeast Schizosaccharomyces pombe initiates sexual reproduction (mating, meiosis and sporulation) in nitrogen-free sporulation medium. Cellular fine structures of eleven sporulation-deficient mutants (spo2, spo3, spo4, spo5, spo6, spo13, spo14, spo15, spo18, spo19 and spo20) of S. pombe in sporulation medium were examined by serial section-electron microscopy. The striking features of these spo mutants were: 1) the disappearance of the spindle pole bodies (SPBs) after the second meiotic division, and 2) the accumulation of unorganized structures. Based on histochemical staining, these structures were presumably unorganized spore wall precursors. In some mutants (spo3, spo5, spo6, spo19 and spo20), diploid zygotes contained four spore-like bodies which had walls similar to complete spore walls but failed to enclose any nuclei. After completion of the second meiotic division the nuclei were abnormally distributed in zygotic diploid cells. In the spo5, spo13, spo14, spo15 and spo19 mutants, the nuclei remained attached to each other. In spo5 and spo19, the inner membrane of the nuclear envelope was separated, but its outer membrane was shared by two sister nuclei. These observations suggest that the spo⁺ gene products play important roles in spatial and temporal organization of cellular structures during ascospore development.Abbreviations SPB spindle pole body - PTA-Cr phosphotungstic acid and chromic acid - PATAg periodic acid, thiocarbohydrazide and silver proteinate     

Fructose-bisphosphatase-deficient mutants of the yeast Schizosaccharomyces pombe

We showed that in the yeast Schizosaccharomyces pombe, fructose-bisphosphatase is not subject to catabolite inactivation as it was observed in Saccharomyces cerevisiae. However, this enzyme activity is sensitive to catabolite repression in both yeasts. Two mutants lacking completely fructose-bisphosphatase activity were found. They were unable to grow on glycerol medium. They were still respiratory competent and exhibited the ability to derepress partially malate dehydrogenase activity. In glucose exponential phase culture, the parental strain lacks completely the fructosebisphosphatase activity due to catabolite repression. In these conditions, the growth is slowed down only in the mutants eventhough both mutants and their parental strain lack this enzyme activity. Normal sporulation and poor spore germination were observed for one mutant whereas, only in the presence of glucose, normal sporulation and normal spore germination were observed for the second mutant. Mendelian segregation of glycerol growth was found for the well germinating mutant. It is of nuclear heredity. The two mutations appeared to be closely linked.Abbreviations FBPase Fructose-1,6-bisphosphatase - fbp ^- genetic symbol for FBPase deficiency - glr ^- symbol for inability to grow on glycerol A. M. Colson is Research Associate au Fonds National de la Recherche Scientifique     

Mapping autonomously replicating sequence elements in a 73-kb region of chromosome II of the fission yeast, Schizosaccharomyces pombe

Autonomously replicating sequence (ARS) elements are the genetic determinants of replication origin function in yeasts. They can be easily identified as the plasmids containing them transform yeast cells at a high frequency. As the first step towards identifying all potential replication origins in a 73-kb region of the long arm of fission yeast chromosome II, we have mapped five new ARS elements using systematic subcloning and transformation assay. 2D analysis of one of the ARS plasmids that showed highest transformation frequency localized the replication origin activity within the cloned genomic DNA. All the new ARS elements are localized in two clusters in centromere proximal 40 kb of the region. The presence of at least six ARS elements, including the previously reported ars727, is suggestive of a higher origin density in this region than that predicted earlier using a computer based search.     

Cloning, characterization and regulation of a protein disulfide isomerase from the fission yeast Schizosaccharomyces pombe

To elucidate the physiological roles and regulation of a protein disulfide isomerase (PDI) from the fission yeast Schizosaccharomyces pombe, the full-length PDI gene was ligated into the shuttle vector pRS316, resulting in pPDI10. The determined DNA sequence carries 1,636 bp and encodes the putative 359 amino acid sequence of PDI with a molecular mass of 39,490 Da. In the amino acid sequence, the S. pombe PDI appears to be very homologous to A. thaliana PDI. The S. pombe cells harboring pPDI10 showed increased PDI activity and accelerated growth, suggesting that the cloned PDI gene is functioning and involved in the yeast growth. The 460 bp upstream region of the PDI gene was fused into promoterless β-galactosidase gene of the shuttle vector YEp367R to generate pYUPDI10. The synthesis of β-galactosidase from the PDI–lacZ fusion gene was enhanced by oxidative stress, such as superoxide anion and hydrogen peroxide. It was also induced by some non-fermentable and fermentable carbon sources. Nitrogen starvation was able to enhance the synthesis of β-galactosidase from the PDI–lacZ fusion gene. The enhancement by oxidative stress and fermentable carbon sources did not depend on the presence of Pap1. The PDI mRNA levels were increased in both Pap1-positive and Pap1-negative cells treated with glycerol. Taken together, the S. pombe PDI gene is involved in cellular growth and response to nutritional and oxidative stress.     

Expression of human cytochrome p450 3A4 gene in Schizosaccharomyces pombe

Heterologous expression systems can be utilized to great advantage in the study of cytochrome P450 enzymes. P450 3A4 is one of the major forms of cytochrome P450 found in liver. It is also involved in the metabolism of numerous widely used drugs and xenobiotics. In the present study human liver cytochrome P450 3A4 gene was transferred into the fission yeast Schizosaccharomyces pombe via two different S. pombe expression vectors carrying thiamine repressible promoter — nmt1 (pREP42) and constitutive promoter — adh1 (pART1). Heterologously expressed cytochrome P450 3A4 was detected in the cells grown in minimal (EMM) or rich medium (YEL) containing 0.5% (w/v) glucose. A typical cytochrome P450 peak for 3A4 was observed at 448 nm in microsomal fraction. The presence of heterologous expression of 3A4 form was also determined by SDS-PAGE and it molecular mass was identified as 52 kDa. The enzyme activity was confirmed by HPLC analysis, using testosterone as substrate.     

Assignment of ten DNA repair genes from Schizosaccharomyces pombe to chromosomal NotI restriction fragments

Summary Ten DNA repair (rad) genes from the fission yeast, Schizosaccharomyces pombe were mapped to the 17 NotI fragments of the three chromosomes. Nine of the genes map to chromosome I, but there is no evidence for significant clustering.     

Studies on inositol-mediated expression of MAL gene encoding maltase and phospholipid biosynthesis in Schizosaccharomyces pombe

In this study, the effects of inositol addition on expression of the MAL gene encoding maltase and phosphatidylinositol (PI) biosynthesis in Schizosaccharomyces pombe (a naturally inositol-requiring strain) were examined. We found that specific maltase activity was at its maximum when the concentration of added inositol reached 6 μg ml⁻¹ in a synthetic medium containing 2.0% (w/v) glucose. When the concentration of added inositol was 1 μg ml⁻¹ in the medium, repression of MAL gene expression occurred at glucose concentration higher than 0.2% (w/v). However, when S. pombe was cultured in the synthetic medium containing 6 μg ml⁻¹, repression of maltase gene expression occurred only at initial glucose concentration above 1.0% (w/v). More mRNA encoding maltase was detected in the cells grown in the medium with 6 μg ml⁻¹ inositol than in those grown in the same medium with 1 μg ml⁻¹ inositol. These results demonstrate that higher inositol concentrations in the synthetic medium could derepress MAL gene expression in S. pombe. PI content of the yeast cells grown in the synthetic medium with 6 μg ml⁻¹ of inositol was higher than that of the yeast cells grown in the same medium with 1 μg ml⁻¹ of inositol. This means that PI may be involved in the derepression of MAL gene expression in S. pombe.     

Circadian control of heat tolerance in stationary phase cultures of Schizosaccharomyces pombe

The capacity of stationary phase cultures of Schizosaccharomyces pombe to survive a heat treatment at 55°C is controlled by a circadian rhythm. In a synchronizing light-dark-cycle this rhythm shows a stable phase relationship to the onset of light. In continuous darkness it persists for several cycles without marked damping. The free-running period of about 27 h at 30°C is only slightly longer at 20°C, hence temperature-compensated. These results indicate that S. pombe is a suitable experimental organism for further research into both heat tolerance and circadian rhythms.     

Dissociation of meiotic and mitotic roles of the fission yeast cdc2 gene

Summary The fission yeastcdc2 gene is pleiotropic, functioning both in the cell division cycle and in meiosis. Here we show thatcdc2 is allelic totws1, a previously isolated meiotic gene. Dissociation of meiotic and mitotic roles of the gene is also demonstrated by finding mutant alleles specifically altered in only one of the two processes.     

Molecular identification and characterization of peptide: N-glycanase from Schizosaccharomyces pombe

Peptide:N-glycanase (PNGase) is an enzyme responsible for deglycosylation of misfolded glycoproteins in so-called endoplasmic reticulum-associated degradation (ERAD) system. In this study, we reported the molecular identification and characterization of SpPNGase (Schizosaccharomyces pombe PNGase). Enzymatic analysis revealed that SpPNGase deglycosylated the misfolded glycoproteins and distinguished native and denatured high-mannose glycoproteins in vitro. The deglycosylation activity was lost with the addition of chelating agent EDTA and was not restored by re-addition of metal ions. By construction of deletion mutant, we confirmed that N-terminal α-helix of SpPNGase was responsible for the protein-protein interaction. Combining the results from ternary structure prediction and dendrogram analysis, we suggested that the N-terminal α-helices of PNGase are derived from evolutionary motif/peptide fusion.     

Radiation induction of delayed recombination in Schizosaccharomyces pombe

Ionizing radiation is known to induce delayed chromosome and gene mutations in the descendants of the irradiated tissue culture cells. Molecular mechanisms of such delayed mutations are yet to be elucidated, since high genomic complexity of mammalian cells makes it difficult to analyze. We now tested radiation induction of delayed recombination in the fission yeast Schizosaccharomyces pombe by monitoring the frequency of homologous recombination after X-irradiation. A reporter with 200 bp tandem repeats went through spontaneous recombination at a frequency of 1.0 x 10(-4), and the frequency increased dose-dependently to around 10 x 10(-4) at 500 Gy of X-irradiation. Although the repair of initial DNA damage was thought to be completed before the restart of cell division cycle, the elevation of the recombination frequency persisted for 8-10 cell generations after irradiation (delayed recombination). The delayed recombination suggests that descendants of the irradiated cells keep a memory of the initial DNA damage which upregulates recombination machinery for 8-10 generations even in the absence of DNA double-strand breaks (DSBs). Since radical scavengers were ineffective in inhibiting the delayed recombination, a memory by continuous production of DNA damaging agents such as reactive oxygen species (ROS) was excluded. Recombination was induced in trans in a reporter on chromosome III by a DNA DSB at a site on chromosome I, suggesting the untargeted nature of delayed recombination. Interestingly, Rad22 foci persisted in the X-irradiated population in parallel with the elevation of the recombination frequency. These results suggest that the epigenetic damage memory induced by DNA DSB upregulates untargeted and delayed recombination in S. pombe.     

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.     

Effect of cytochalasin A on actin distribution in the fission yeastSchizosaccharomyces pombe studied by fluorescent and electron microscopy

Summary Actin distribution and ultrastructure of the fission yeastSchizosaccharomyces pombe treated with cytochalasin A (CA) were investigated by fluorescence microscopy using rhodamine-conjugated phalloidin (rh-ph) and freeze substitution electron microscopy. Among the cytochalasins tested, CA was most effective and at 5 g/ ml inhibited the appearance of the actin ring at the cell equator at the stage prior to septum formation and the accumulation of actin dots at the septum-forming site both in wild-type cells and the mutantcdc 11, which is defective in septum formation at restrictive temperature. Freeze substitution electron microscopy of CA-treated cells revealed the displacement and morphological alteration of cytoplasmic vesicles and dictyosomes within 30 min and the appearance of dense bodies in the cytoplasm. A sub-population of cytoplasmic vesicles and dictyosomes were insensitive to CA and maintained their original structure. An electron less dense layer containing filamentous material was noted beneath the plasma membrane and thought to be the area of heavy actin patches stained with rh-ph at the cells ends. These results suggest that CA disrupted an actin network that normally maintains the organization of the secretory pathway involving dictyosomes and vesicles.