Characterization of glucose transport inSchizosaccharomyces pombe

Conclusions GHT1 was isolated as suppressor ofd-glucose uptake deficiency ofS. pombe mutant YGS-5. The correspondingS. pombe DNA encodes a putative protein with significant amino acid sequence identity to theS. cerevisiae HXT transporters. Heterologous expression ofGHT1 inS. cerevisiae hxt mutant RE700A (strain HLY709) enabled the mutant to grow ond-glucose as the sole carbon source. HLY709 cells take up hexoses with similar specificity toS. pombe wild strain and accumulate the non-metabolizable analogues of glucose (2DG and 6DG) intracellularly, thus matchingS. pombe wild strain. Southern blot analysis revealed the existence of other putative glucose transporters inS. pombe and the search for related transporter genes inS. pombe genome is in progress.     

Demonstration of an altered phenylalanyl-tRNA synthetase in an analogue-resistant mutant of Aspergillus nidulans

Summary We have isolated and characterized a new class of p-fluorophenylalanine (FPA)-resistant mutant in Aspergillus nidulans using a phenA strain as the wild type, by optimizing the conditions of growth. All four spontaneous mutants selected on a medium containing FPA were found to be recessive to their wild-type alleles in heterozygous diploids. Complementation analyses and linkage data showed that they were allelic and mapped at a single locus (fpaU) in the facA-riboD interval on the right arm of linkage group V. Partial purification and characterization of Phe-tRNA synthetase from wild-type and mutant strains revealed that the mutant enzyme had a greatly reduced ability to activate the analogue. It is suggested that mutation in the fpaU gene brings about a structural alteration in Phe-tRNA synthetase.Abbreviations FPA DL-p-fluorophenylalanine - phenA auxotroph of phenylalanine - Phe-tRNA synthetase phenylalanyl-transfer ribonucleic acid synthetase Current address: Department of Biological Sciences (M/C 066) The University of Illinois at Chicago, Box 4348, Chicago, IL 60680, USA     

prp4 from Schizosaccharomyces pombe, a mutant deficient in pre-mRNA splicing isolated using genes containing artificial introns

Summary We have generated a bank of temperature-sensitive (ts) Schizosaccharomyces pombe mutant strains. About 150 of these mutants were transformed with a ura4 gene containing an artificial intron. We screened these is mutants for mutants deficient in splicing of the ura4 intron. With this approach three mutants were isolated which have a general defect in the splicing process. Two of these mutants fall into the prp1 complementation group and one defines a new complementation group, prp4.     

Genes involved in meiosis and sporulation of a yeast

Summary 300 mutants blocked in meiosis or sporulation were isolated in Schizosaccharomyces pombe and grouped by complementation tests, linkage studies and cytological observation. In total, 5 genes pertaining to meiosis and 18 genes necessary for sporulation could be identified. In addition, a gene participating in dissolution of the separating walls during cell fusion was detected.     

Unique regulation of glyoxalase I activity during osmotic stress response in the fission yeast <Emphasis Type="Italic">Schizosaccharomyces pombe</Emphasis>: neither the mRNA nor the protein level of glyoxalase I increase under conditions that enhance its activity

Glyoxalase I is a ubiquitous enzyme that catalyzes the conversion of methylglyoxal, a toxic 2-oxoaldehyde derived from glycolysis, to S-D-lactoylglutathione. The activity of glyoxalase I in the fission yeast Schizosaccharomyces pombe was increased by osmotic stress induced by sorbitol. However, neither the mRNA levels of its structural gene nor its protein levels increased under the same conditions. Cycloheximide blocked the induction of glyoxalase I activity in cells exposed to osmotic stress. In addition, glyoxalase I activity was increased in stress-activated protein kinase-deficient mutants (wis1 and spc1). We present evidence for the post-translational regulation of glyoxalase I by osmotic stress in the fission yeast.     

Interdependence of cell cycle events inSchizosaccharomyces pombe. Terminal phenotypes of cell division cycle mutants arrested during dna synthesis and nuclear division

Summary Temperature-sensitive cell division cycle (cdc) mutants of the fission yeastSchizosaccharomyces pombe, previously characterized as defective in nuclear division were examined by thin section electron microscopy. All of the mutants failed to enter mitosis, rather they accumulated at one of four distinct terminal phenotypes. Class one were arrested with a nucleus rectangular in cross-section and a laterally situated spindle pole body (SPB). The second group had spherical or rectangular nuclei with a single SPB. The sole member of the third group wascdc 27. K 3, which had a spherical crenated nucleus with a single SPB from which microtubules emerged and extended into the cytoplasm. Allelic variants ofcdc 25 comprised the fourth group all of which displayed aberrant nuclear morphologies. Utilizing this ultrastructural data together with a knowledge of the transition points of these mutants a model for the interdependence of certain cell cycle event is proposed in which the initiation of DNA synthesis is uncoupled from the replication and separation of the SPB. This paper also provides new information on SPB structure inS. pombe. This is discussed in connection with the transient assembly of both spindle and cytoplasmic microtubules.     

Urea and thiourea transport in Aspergillus nidulans

Wild-type Aspergillus nidulans has an active transport system specific for urea which concentrates urea at least 50-fold relative to the extracellular concentration. It is substrate concentration dependent, with an apparent K _m of 3×10^–5 m for urea. Competition studies and the properties of mutants indicate that thiourea is taken up by the same system as urea. Thiourea is toxic at 5mm to wild-type cells of Aspergillus nidulans. Mutants, designated ureA1 to ureA16, resistant to thiourea have been isolated, and transport assays and growth tests show that they are specifically impaired in urea transport. The mutant ureA1 has a higher K _m value than the wild type for thiourea uptake. The ureA locus has been assigned to linkage group VIII. ureA1 is recessive for thiourea resistance while semidominant for the low uptake characteristic. The urea uptake system is under nitrogen regulation, with l-glutamine as the probable effector. The mutants, meaA8 and gdhA1, which are insensitive to ammonium control of many nitrogen-regulated metabolic systems, are also insensitive to ammonium control of urea uptake, but both are sensitive to l-glutamine regulation.Formerly at the Department of Genetics, University of Glasgow, Glasgow, Scotland.     

Genetic control of lysine permeases in Saccharomycopsis lipolytica

In order to obtain strains of Saccharomycopsis lipolytica impaired in the active transport of l-lysine, mutants resistant to a mixture of l-canavanine, l-4-5-transdehydrolysine and l-S-amino ethylcysteine, taken either all three or two by two, were isolated. These compounds were shown previously to be competitive inhibitors of l-lysine uptake.The resistance patterns and excretion capacity of the mutants were established. All mutants behaved as monogenic. Recombination tests indicated that four genes at least were involved. All mutants were impaired in both high and low affinity l-lysine transport systems.Several hypotheses on the functions of these genes are put forward and discussed.     

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.     

D-Gluconate is an alternative growth substrate for cultivation of Schizosaccharomyces pombe mutants

Schizosaccharomyces pombe cells grow on d-gluconate as the sole carbon and energy source. d-Gluconate is taken up in symport with protons by a specific symporter, pH being the sole driving force. d-Gluconate uptake is independent of the sugar transporting system (e.g. for d-glucose) and of . The carrier is expressed constitutively, and its activity is not subject to glucose repression. Hence, d-gluconate is a suitable carbon and energy source for growth, when d-glucose or other hexoses have to be eliminated e.g. for selection of mutants deficient in hexose transport.Abbreviations 2-DG 2-deoxy-d-glucose - CCCP carbonylcyanide m-chlorophenylhydrazone - pH pH-gradient - electrical potential difference across the plasma membrane - SD standard deviation - SEM standard error of the mean - TPP⁺ tetraphenylphosphonium     

Effect of hydrogen peroxide on sugar transport inSchizosaccharomyces pombe. Absence of membrane lipid peroxidation

Stationary unaerated cells ofS. pombe containing endogenous substrates but not energized by any exogenous ones take up 2-deoxy-d-glucose, 6-deoxy-d-glucose,d-xylose andd-arabinose actively over diffusion equilibrium. The active uptake is inhibited by 20–100 mmol/L H₂O₂ which causes an increase inK _T but has no effect onJ _max. This “competitive inhibition” indicates that H₂O₂ affects directly the sugar binding sites of the transporters. The ATP-binding site of the plasma membrane H⁺-ATPase is also affected by 100 mmol/L H₂O₂; theK _T decreases 7-fold,J _max about 2.5-fold. These effects are not likely to be mediated by membrane lipid peroxidation which appears to be lacking inS. pombe, and this lack may be one of the reasons for the high resistance of this yeast to H₂O₂. Because of thisS. pombe represents a suitable system for studying direct effects of oxidants on membrane proteins.     

Schizosaccharomyces malidevorans sp.n., a yeast decomposing L-malic acid

A new yeastSchizosaccharomyces malidevorans sp.n. is described. It resemblesSchizosaccharomyces pombe but differs from it in appearance of the spores and inability to ferment maltose. It decomposesl-malic acid completely in all grape juice and synthetic media tested, but not thed-isomer. During fermentation a copious evolution of hydrogen sulphide occurs.     

The isolation and analysis of one functional type ofpyr-3 mutant inNeurospora

Twenty-seven pyrimidine-requiring mutants were isolated as suppressors of anarg-3 mutant. All 27 are deficient for ATCase activity and show linkage to thecol-4 marker located on linkage group IV. Analyses of prototroph frequencies resulting from crosses of the new mutants to previously mappedpyr-3 mutants indicate that this functional type ofpyr-3 mutant is restricted to one region of the genetic map. Complementation studies with 11 of the new mutants further extend and subdivide the complementation map of thepyr-3 locus.This work was supported in part by National Institutes of Health, Public Health Service Grant GM 15137-01 and by National Science Foundation Grant GB5998.     

Repair of cyclobutane pyrimidine dimers and 6-4 photoproducts in the fission yeast Schizosaccharomyces pombe

We have measured repair of both of the major lesions induced by ultraviolet irradiation (cyclobutane pyrimidine dimers and 6-4 photoproducts) in wild-type Schizosaccharomyces pombe and in selected rad mutants, including mutants with deletions in genes from the main phenotypic groups. We find that rad13Δ, rad15 and rad16Δ, which are the S. pombe homologues of the excision-defective Saccharomyces cerevisiae rad2, rad3 and rad1, respectively, repair lesions somewhat more slowly than the wild type, but still have considerable repair capacity. rad2Δ, also a presumed excision-defective mutant, behaves similarly. radS and rad9δ, which belong to different phenotypic groups, repair lesions at the same rate as wild-type cells. These findings provide new evidence that S. pombe has a second repair system for removing ultraviolet damage, which is absent in S. cerevisiae. Surprisingly, this second mechanism repairs lesions very efficiently; its possible nature is discussed.     

Metabolism of d-xylose in Schizosaccharomyces pombe cloned with a xylose isomerase gene

Summary The Escherichia coli xylose isomerase gene was transformed into Schizosaccharomyces pombe for direct d-xylose utilization. In order to understand d-xylose metabolism and determine the limiting factors on d-xylose utilization by the transformed yeast, d-xylose transport, xylose isomerization, and xylulose phosphorylation were investigated. The results indicated that low activity of xylose isomerization in the cloned yeast was the limiting step for d-xylose fermentation. An in vitro study showed that yeast proteases decreased xylose isomerase activity. Xylitol, a by-product of d-xylose fermentation, had no effect on the activity of xylose isomerase.     

Production and characteristics of the exocellular polysaccharide in mutant strains ofXanthomonas fuscans

Production of the exocellular polysaccharide of the phytopathogenic bacteriumXanthomonas fuscans was investigated with respect to its possible use in utilization of industrial wastes containing lactose. Six stablelac ⁺ mutants were obtained after the treatment withN-methyl-N′-nitroso-N′-nitroguanidine. The mutants were compared with the parent strain. Morphological and cultivation characteristics, as well as production of the exooellular polysaccharide were compared. The production was found to be maximal during the stationary phase of growth in strains cultivated under submerged conditions. Gas chromatography revealed that the polysaccharide of the parent strain is formed by α- and β-D-glucose and α- and β-d-mannose with a small amount ofd-ribose and 6-deoxy-l-mannose. Composition of the polysaccharides produced by the mutant strains (lac ⁺) does not qualitatively differ from that of the parent strain. However, they were found to contain a higher quantity ofd-mannose, which is favourable for their industrial utilization.     

Differential uptake of fumarate by Candida utilis and Schizosaccharomyces pombe

The dicarboxylic acid fumarate is an important intermediate in cellular processes and also serves as a precursor for the commercial production of fine chemicals such as l-malate. Yeast species differ remarkably in their ability to degrade extracellular dicarboxylic acids and to utilise them as their only source of carbon. In this study we have shown that the yeast Candida utilis effectively degraded extracellular fumarate and l-malate, but glucose or other assimilable carbon sources repressed the transport and degradation of these dicarboxylic acids. The transport of both dicarboxylic acids was shown to be strongly inducible by either fumarate or l-malate while kinetic studies suggest that the two dicarboxylic acids are transported by the same transporter protein. In contrast, Schizosaccharomyces pombe effectively degraded extracellular l-malate, but not fumarate, in the presence of glucose or other assimilable carbon sources. The Sch. pombe malate transporter was unable to transport fumarate, although fumarate inhibited the uptake of l-malate. Received: 15 March 2000 / Received revision: 4 July 2000 / Accepted: 9 July 2000     

Analogue-resistant and auxotrophic mutants ofMethanobacterium thermoautotrophicum

The death rate ofMethanobacterium thermoautotrophicum strain Marburg upon exposure toN-methyl-N-nitro-N-nitrosoguanidine under anaerobic conditions was of the same order of magnitude as the death rates that have been reported forEscherichia coli. Cultures of the methanogenic bacterium, mutagenized by nitrosoguanidine-treatment and grown under non-selective conditions, yielded mutants resistant toDL-ethionine (30 mM) or to 2-bromoethane sulfonic acid (3.8 mM). No mutants were observed in untreated controls. Among 1500 clones obtained from nitrosoguanidine-treated cell suspensions there were 6 mutants requiring a single growth factor each, namelyl-leucine,l-phenylalanine, thiamine (2 mutants) or adenosine (2 mutants). Three mutant-strains were studied in more detail. They were genetically stable (no revertants among 10⁹ cells), and wild type growth rates were restored by 5 mml-leucine, 0.4 mM adenosine and 0.03 mM thiamine, respectively.Abbreviations 2-BES 2-bromoethanesulfonic acid - MIC minimum inhibitory concentration     

Isolation of the DLD gene of Saccharomyces cerevisiae encoding the mitochondrial enzyme D-lactate ferricytochrome c oxidoreductase

In Saccharomyces cerevisiae the utilization of lactate occurs via specific oxidation of l- and d-lactate to pyruvate catalysed by l-lactate ferricytochrome c oxidoreductase (L-LCR) (EC 1.1.2.3) encoded by the CYB2 gene, and d-lactate ferricytochrome c oxidoreductase (D-LCR) (EC 1.1.2.4), respectively. We selected several lactate^– pyruvate⁺ mutants in a cyb2 genetic background. Two of them were devoid of D -LCR activity (dld mutants, belonging to the same complementation group). The mutation mapped in the structural gene. This was demonstrated by a gene dosage effect and by the thermosensitivity of the enzyme activity of thermosensitive revertants. The DLD gene was cloned by complementation for growth on d-, l-lactate in the strain WWF18-3D, carrying both a CYB2 disruption and the dld mutation. The minimal complete complementing sequence was localized by subcloning experiments. From the sequence analysis an open reading frame (ORF) was identified that could encode a polypeptide of 576 amino-acids, corresponding to a calculated molecular weight of 64000 Da. The deduced protein sequence showed significant homology with the previously described microsomal flavoprotein l-gulono--lactone oxidase isolated from Rattus norvegicus, which catalyses the terminal step of l-ascorbic acid biosynthesis. These results are discussed together with the role of L-LCR and D-LCR in lactate metabolism of S. cerevisiae.     

Production ofl-phenylalanine by double auxotrophic mutants ofArthrobacter globiformis: Effect of temperature,trace salts and inoculum dose

Three tryptophan-plus-tyrosine double auxotrophic mutants were isolated from a biotin-requiring glutamate-producingArthrobacter globiformis. The mutants were found to producel-phenylalanine in a mineral salt medium. Further improvement ofl-phenylalanine production was achieved by isolation of mutants resistant to β-2-thienylalanine from these double auxotrophs. Temperature of 30 °C and a 4% inoculum dose were found to be optimum for phenylalanine production. Addition of some trace salts does not enhance phenylalanine yield. Under optimal cultural conditions one mutant yielded 6.8 g phenylalanine per L medium in flask culture.