bluuebunny/crossref_metadata_2025_split huggingface dataset:
Induction of a heat shock‐type response in fission yeast following nitrogen starvation
Authors: Walker Graeme M. , McWilliams Philip G. 
Published: May 2005

AbstractWhen cells of the fission yeast, Schizosaccharomyces pombe, are incubated in medium devoid of a nitrogen source, they accelerate into cell division and differentially synthesize two polypeptides at 46 and 27 kD (named p46 and p27) after a delay of about an hour. The synthesis of p46 and p27 is transient. These proteins have no obvious cell cycle connection since they are also evident in nitrogen‐starved (but not accelerated) cells of the temperature‐sensitive mutant of S. pombe, wee 1·50h−. We infer from this that p46 and p27 are synthesized as a direct result of nutritional stress. The possibility that p46 and p27 represent examples of general environmental stress proteins was investigated by comparing nitrogen starvation with the heat‐shock response in S. pombe. Heat‐shock analysis of cells revealed the existence of two proteins of similar Mr to p46 and p27. In addition, nitrogen‐starved cells acquired thermotolerance in a manner similar to heat‐shocking cells. We suggest that nitrogen starvation in fission yeast induces a subset of the total array of heat‐shock proteins.
Yeast flocculation: Cationic inhibition
Authors: Stratford Malcolm , Brundish Helen M. 
Published: May 2005

AbstractYeast flocculation was inhibited by high concentrations of a number of salts. Calcium and magnesiun salts were potent inhibitors and cesium salts were least effective. Partial inhibitors by different salts additive and were completely reversible by salt removal. Inhibition by salts was time dependent; prolonged incubation increased the degree of inhibition. Salt inhibition was partly caused by the action of salts lowering the buffer pH value, and partly caused by chaotropic inhibition of proteins on the surfaces of flocculent cells. Flocculation receptors on non‐flocculent cells were ubaffected by high salt concentration. At sub‐inhibitory salt concentrations, there was an enhancement of flocculation in both rate and extent.
Peroxisome‐deficient mutants of Hansenula polymorpha
Authors: Cregg James M. , Van Klei Ida J. , Sulter Grietje J. , Veenhuis Marten , Harder Wim 
Published: May 2005

AbstractAs a first step in a genetic approach towards understanding peroxisome biogenesis and function, we have sought to isolate mutants of the methylotrophic yeast Hansenula polymorpha which are deficient in peroxisomes. A collection of 260 methanol‐utilization‐defective strains was isolated and screened for the ability to utilize a second compound, ethanol, the metabolism of which involves peroxisomes. Electron microscopical investigations of ultrathin sections of selected pleiotropic mutants revealed two strains which were completely devoid of peroxisomes. In both, different peroxisomal matrix enzymes were active but located in the cytosol; these included catalase, alcohol oxidase, malate synthase and isocitrate lyase.Subsequent backcrossing experiments revealed that for all crosses involving both strains, the methanol‐ and ethanol utilizing‐deficient phenotypes segregated independently of each other, indicating that different gene mutations were responsible for these phenotypes. The phenotype of the backcrossed peroxisome‐deficient derivates was identical: defective in the ability to utilize methanol but capable of growth on other carbon sources, including ethanol.The mutations complemented and therefore were recessive mutations in different genes.
Cloning and sequencing of the malate synthase gene from Hansenula polymorpha
Authors: Bruinenberg Paul G. , Blaauw Mieke , Kazemier Bert , Ab Geert 
Published: May 2005

AbstractWe have cloned the MAS gene, encoding the microbody matrix enzyme malate synthase (EC 4.1.3.2.) from the methylotrophic yeast Hensenula polymorphia. The gene was isolated by screening of a genomic library with a mixed‐sequence probe, based on the partial amino acid sequence of the purified enzyme. The nucleotide sequence of a 2·4‐kilobase stretch of DNA covering the MAS gene was determined. The gene contains an open reading frame 555 amino acids, amounting to a calculated molecular mass of 63 254 for the encoded protein. Comparison of the amino acid sequence with the malate synthase sequences of Escherichia coli, Brassica napus L. and Cucumis sativus L. Clearly establishes the homology of all four proteins. Compared to the soluble enzyme from E. coli, the malate synthases from H. polymorpha and both plant species, which are located in the microbodies, have a short carboxy‐terminal extension. In the plant malate synthases, the extension is probably involved in routing to the microbodies, since it contains the potential peroxisomal targeting signal, Ser‐Arg/Lys‐Leu, at the carboxy terminus. The H. polymorpha enzyme terminates with similar amino acids, but their sequence, Ser‐Leu‐Lys, does not conform to any of the known peroxisomal targeting signals.
Oscillatory CO2 evolution in glycolysing yeast extracts
Authors: Das Joachim , Timm Hartmut , Busse Heinrich‐Gustav , Degn Hans 
Published: May 2005

AbstractThe rate of formation of carbon dioxide in cytoplasmic yeast extracts in an open system with continuous infusion of glucose was measured by membrane inlet mass spectrometry during glycolytic oscillations. The rate of CO2 production rose in the first third of each cycle to a maximum of about 100 μmol per ml yeast extract per hour and subsequently diminished to a final level of about 50 μmol per h. Measurements of the NADH light absorption under the same conditions revealed oscillations of relaxations type. The phase of high CO2 production could be related to the phase of the high NADH level, giving evidence that the flux in glycolysis is increased during the phase of high NADH concentration. Only half of the amount of injected glucose was metabolized to CO2 during the sustained oscillation, although free glucose did not accumulate.
The hydrophilic and acidic N‐terminus of the integral membrane enzyme phosphatidylserine synthase is required for efficient membrane insertion
Authors: Sperka‐Gottlieb Constanze , Fasch Evelyn‐V. , Kuchler Karl , Bailis Adam M. , Henry Susan A. , Paltauf Fritiz , Kohlwein Sepp D. 
Published: May 2005

AbstractThe product of the yeast CHO1 gene, phosphatidylserine synthase (PSS), is an integral membrane protein that catalyses a central step in cellular phospholipid biosynthesis. A 1·2 kb fragment containing the regulatory and structural components of the CHO1 gene was sequenced. Transcription initiation in wild‐type cells was found to occur between −1 and −15 relative to the first ATG of a large open reading frame capable of encoding a 30 804 molecular weight protein. This translation initiation site was active in vivo and in vivo in a hetrologous system. In both cases it supported production of a protein of approximately 30 000 molecular weight. A second potential translation initiation site was detected 225 or 228 bases dowstream from the first ATG. This second site was active in vitro where it supported production of a protein of 22 400 molecular weight. A subclone, lacking the 5′ regulatory region and the subsequence encoding the first 12 amino acids of the large open reading frame, allowed translation in vivo starting at the second ATG. The resulting protein was 22 000 moleculare weight, lacked the 74 N‐terminal amino acids and was capable of complementing the choline auxotroy of a cho1 null‐mutant. In transformants carrying this construct, PSS activity and 22 kDa protein was found to be associated with membrane fractions corresponding to mitochondria and endoplasmic reticulum. However, most of the truncated PSS protein accumulated in the cytosol in an inactive form. A hybrd‐protein containing the 63 N‐terminal amino acids of PSS protein accumulated in the cytosol in a active form. A hybrid‐protein containing the 63 N‐terminal amino acids of PSS fused to mouse dihydrofolate reductase was found exclusively in the cytosol when expressed in wild‐type yeast. Thus, the hydrophilic, highly acidic N‐terminus of PSS is required for efficient membrane insertion but does not appear to contain sequences required for a targeting to the membrane compartment.
Excessive membrane development following exposure of the methylotrophic yeast Hansenula polymorpha to oleic acid‐containing media
Authors: Veenhuis Marten , Kram Anita M. , Kunau Wolf H. , Harder Wim 
Published: May 2005

AbstractWe studied the physiological responses of Hansenula polymorpha during adaptation of cells to oleic acid‐containing media. Growth experiments indicated that the organism was unable to use oleic acid as the sole source of carbon and energy. However, upon incubation of glucose‐grown cells in mineral media containing oleic acid, activities of various enzymes of the β‐oxidation pathway were induced. These enzymes were localized in microbodies together with alcohol oxidase. Furthermore, a drastic increase in phospholipid content of the cells was observed; this was due to a rapid proliferation of membranes. These consisted of a variable number of membranous layers which were continuous with the peroxisomal membrane. Upon continued incubation, the membrane proliferations extended and large compartments were formed. This process was dependent on the presence of peroxisomes in the cells since it was not observed in peroxisome‐deficient mutant strains of H. polymorpha. The newly formed membranous compartments differed from peroxisomes since they did not contain peroxisomal matrix proteins; these were confined to the single enlarged organelle which was incorporated in the membranous structure and characterized by a large alcohol oxidase crystalloid. The membranous compartments are considered to be whole entities since they could not be separated from the peroxisomes by common cell fraction methods; also they were degraded entirely after a shift of cells to glucose‐excess condition.Freeze fracturing reveled that the substructure of the membranes greatly resembled that of normal peroxisomal membranes. Since a distinct enhancement of different peroxisomal membrane proteins was observed during the initial hours after the shift, we assume that exposure of H. polymorpha to acid lead to a drastic overproduction of peroxisomal membranes.
Sequence and genetic analysis of NHP2: A moderately abundant high mobility group‐like nuclear protein with an essential function in Saccharomyces cerevisiae
Authors: Kolodrubetz David , Burgum Alex 
Published: May 2005

AbstractIn order to determine the biological functions of moderately abundant, high mobility group (HMG)‐like nuclear proteins, a genetic approach has been taken. The gene for one such protein, NHP2, has been cloned and characterized from Saccharomyces cerevisiae. NHP2 has been called ‘HMG‐like’ because of the physical/chemical properties it shares with the HMG proteins from higher eukaryotic cells. However, nucleotide sequence analysis revealed that NHP2 could encode a 17·1 kilodalton basic protein which was not significantly homologous to any previously sequenced HMG proteins. Thus NHP2 defines a new member of the HMG class of proteins. A search of protein databases showed that the amino acid sequence of NHP2 shared significant identities with two ribosomal proteins; the acidic ribosomal protein S6 from Halobacterium marismorium and protein L7a from mammals. The biological relevance of these homologies is nuclear since previous biochemical results indicated that NHP2 was not a ribosomal protein. S1 nuclease analysis indicated that the gene contained no introns but had multiple transcription initiation sites 20 to 40 bases before the ATG codon. Finally, NHP2 has been shown to have a critical role in the cell; when a diploid yeast strain deleted of one copy of the NHP2 gene was sporulated and dissected, only half of the spores grew into normal colonies. The rest of the spors germinated, but only formed microcolonies containing 12 to 40 cells. None of the spores which grew into normal‐sized colonies contained the mutant NHP2 gene, thus demonstrating that the NHP2 protein has an essential physiological function.
Production of the STA2‐encoded glucoamylase in Saccharomyces cerevisiae is subject to feed‐back control
Authors: Suntsov N. I. , Kuchin S. V. , Neystat M. A. , Mashko S. V. , Benevolensky S. V. 
Published: May 2005

AbstractThree modes of production of the extracellular glucoamylase (GA) in Saccharomyces cerevisiae have been identified: repressed, basal and induced. The repressed mode is found with cells grown in rich media containing non‐limiting concentrations of monosaccharides or disaccharides, including GA‐hydrolysable maltose, as a sole carbon source. Both the basal and the induced modes (spanned by some seven‐fold difference in the rate of GA production) can be displayed by either glucose‐limited or glycerol‐ plus ethanol‐consuming cultures: the induced mode is switched over to the basal one due to a feed‐back inhibition by extracellularly accumulated GA. It is proposed that the feed‐back control involved in GA production can be attenuated by starch which can thus ‘induce’ higher rates of GA production compared to the basal mode.
Constitutive expression of the Saccharomyces cerevisiae CUP1 gene in Kluyveromyces lactis
Authors: Macreadie Ian G. , Horaitis Ourania , Vaughan Paul R. , Des Clark‐Walker G. 
Published: May 2005

AbstractShuttle plasmids, pE1.CUP1B and pE1.CUP1E of 10·6 kb, have been constructed between the metallothioneinencoding CUP1 gene of Saccharomyces cerevisiae and a vector capable of replication in Kluyveromyces lactis. Introduction of these plasmids into K. lactis confers resistance to copper as well as to cadmium and silver. Resistance to these latter metal ions, in the absence of induction by copper, suggested that the CUP1 gene is constitutively expressed in the foreign background. Introduction of the lacZ reporter gene from Escherichia coli into a cloning site downstream from the CUP1 promoter showed that expression of this gene is constitutive in K. lactis but in S. cerevisiae induction by copper is necessary. Sequences upstream from the CUP1 promoter are involved in the constitutive expression since deletion of 91 nucleotides from this region abolishes metal resistance. It is suggested that a K. lactis protein, normally involved in activating transcription of the resident CUP1 gene in the presence of copper, can promote transcription in the absence of metal ion by binding to the upstream activation sequence of the introduced CUP1 gene.
Chemostat studies of microsomal enzyme induction in Saccharomyces cerevisiae
Authors: Stansfield I. , Cliffe K. R. , Kelly S. L. 
Published: May 2005

AbstractUsing cells grown in a chemostat at steady state, the levels of various components of the microsomal electron transport chain of Saccharomyces cerevisiae were examined. Cytochrome P450 haemoprotein levels measured in cells grown in medium with a dissolved oxygen concentration of 15% were induced between 10‐ and 20‐fold over levels in cells grown in medium containing 70% dissolved oxygen concentation. An increase in the dilution rate of a culture growing in medium containing 15% dissolved oxygen resulted in an increase in the residual glucose concentration of the medium. This was paralleled by an increase in the microsomal levels of cytochrome P450. The rise could not be attributed either to increases in the concentration of ethanol in the chemostat or to an increase in the proportion of energy generated using fermentative pathways. However, this effect was not observed in cells grown in an oxygen concentration of 70%. Cytochrome b5 haemoprotein levels were also induced approximately three‐fold by reducing the dissolved oxygen concentration from 70% to 15%. Changes in the medium glucose concentration from 0·03% to 1·6% (w/v) had no effect on the levels of this enzyme. Conversely, levels of cytochrome P450 NADPH reductase appeared lower in cells grown in 15% as opposed to 70% dissolved oxygen concentration. Northern slot blot analysis of total RNA extracted from chemostat‐grown cells, probed with a C‐14 sterol demethylase cytochrome P450 gene (cytochrome P450 LIA1), revealed a pattern of message induction which matched that of the cytochrome P450 haemoprotein, indicating that control of the levels of this enzyme was at least partially transcriptional. Qualitative examination of combined cytochrome P450 apoprotein and haemoprotein levels using Western blot analysis revealed a similar pattern of induction to that observed with Northern blotting.
The product of the KIN1 locus in Saccharomyces cerevisiae is a serine/threonine‐specific protein kinase
Authors: Lamb Allen , Tibbetts Michael , Hammond Charlotte I. 
Published: May 2005

AbstractThe catalytic domain (30 kDa) of all protein kinases can be aligned for maximum homology, thereby revealing both invariant and highly conserved residues. The KIN1 locus from Saccharomyces cerevisiae was isolated by hybridization to a degenerate oligonucleotide encoding the conserved protein kinase domain, DVWSFG. The predicted amino acid sequence revealed significant homology to the catalytic domain of protein kinases. Using antibodies raised against a bacterial LacZ/KIN1 fusion protein, we have identified by immunoprecipitation the yeast KIN1 gene product as a 145 000 dalton protein (p145KIN1). In exponentially growing yeast cells, the KIN1 protein is phosphorylated primarily on serine residues. The gene product of KIN1 was shown to be a serine/threonine‐specific protein kinase in immune complexes, as detrmined by the transfer of label from [γ‐32P]ATP to either pp145KIN1 or to an exogenously added substrate, α‐casein. The optimal metal ion concentration in this assay was 20 mM‐MnCl2. Subsequent phosphoamino acid analysis of the radiolabelled product, pp145KIN1, demonstrated that this autophosphorylation was specific for serine/threonine residues. There is no apparent difference between wild‐type cells and cells containing a disrupted KIN1 gene. The biochemical characterization of protein kinases in simple eukaryotes such as yeast will aid us in detrmining the role of phosphorylation in cellular growth and physiology.
Incorporation of unsaturated fatty acids by Saccharomyces cerevisiae: Conservation of fatty‐acyl saturation in phosphatidylinositol
Authors: Pilkington Bridget J. , Rose Anthony H. 
Published: May 2005

AbstractSaccharomyces cerevisiae was grown anaerobically in media supplemented with myritoleic 14:1(9c), palmitoleic 16:1(9c), oleic 18:1(9c), linoleic 18:2(9,12c), γ‐linolenic 18:3(9,12,15c) or eicosenoic 20:1(11c) acid. Cells from exponential‐phase cultures contained approximately the same proportions of the major phospholipid classes, namely phosphatidycholine, phosphatidylethanolamine, phosphatidylinositol and phosphatidylserine, the greatest differences being detected in cells grown in the presence of 14:1(9c) or 20:1(11c) acids. The extent to which phospholipids from cells were enriched with residues of the exogenously supplied acid varied from 52% in cells grown in the presence of 14:1(9c) acid to 13% in cells grown in media supplemented with 20:1(11c) acid. Analysis of the fatty‐acyl composition of the four major phospholipid classes revealed that the degree of unsaturation varied considerably in three of the classes, while phosphatidylinositol conserved a high degree of saturation. The possible significance of the latter finding in relation to the physiological role of phosphatidylinositol in the plasma membrane is discussed.
Transport of organic acid anions and guanosine into vacuoles of Saccharomyces pastorianus
Authors: Kulakovskaya T. V. , Matys S. V. , Okorokov L. A. 
Published: May 2005

AbstractIsolated vacuoles of the yeast Saccharomyces pastorianus accumulate citrate, α‐ketoglutarate, malate and guanosine. This accumulation is Mg ATP‐dependent and inhibited by protonophores. The ionophores monensin and A23187 (electroneutral Men+/nH+‐exchange) inhibit guanosine accumulation but fail to block citrate uptake. Mg2+ ions (2 mM) increase the values of both Δ\documentclass{article}\pagestyle{empty}\begin{document}$ \tilde \mu $\end{document}H+ components and stimulate the uptake of all the above compounds. Ca2+ ions (1 mM), hyperpolarizing the yeast vacuolar membrane and dissipating the pH gradient, inhibit guanosine uptake and stimulate that of citrate. It is concluded that guanosine is transported into yeast vacuoles by an H+/guanosine antiporter while citrate, malate and α‐ketoglutarate are translocated by a uniporter(s) at the expense of the membrane potential (positive inside).
Isolation and characterization of Schizosaccharomyces pombe mutants lacking aminopeptidase activity
Authors: Arbesu MA José , Gascon Santiago , Suarez‐Rendueles Paz 
Published: May 2005

AbstractA mutant strain of the fission yeast Schizosaccharomyces pombe defective in aminopeptidase I was isolated by screening for lack of activity against the chromogenic substrate lysine‐β‐naphthlamide in isolated colonies. Tetrad dissection of sporulated diploids heterozygous for the wild‐type and mutant allele resulted in a 2:2 segregation of mutant and wild‐type phenotype indicating a single chromosomal gene mutation. Gene dosage experiments indicated that the mutation might reside in the structural gene of aminopeptidase I. No vital consequences of aminopeptidase I deficiency on cell life and sporulation could be detected. However, the enzyme seems to be involved in protein degradation under conditions of nutrient deprivation.
The ade6 gene of the fission yeast Schizosaccharomyces pombe has the same chromatin structure in the chromosome and in plasmids
Authors: Bernardi Fabio , Koller Theo , Thoma Fritz 
Published: May 2005

AbstractWe have analysed the chromatin structure of the ade6 gene of Schizosaccharomyces pombe and its flanking regions both in the chromosome and in plasmids. The chromatin structure is independent of the chromosomal or extrachromosomal location. The ade6 gene contains eight precisely positioned nucleosomes on the 5′ half, ‘not positioned’ nucleosomes around the 3′ end and a nuclease‐sensitive promoter region. Precisely positioned nucleosomes, but no nuclease‐sensitive region were also detected on the ura4 gene in the chromosome and on a plasmid. The results show that S. pombe chromosomal and extrachromosomal genes have chromatin structures similar to those of S. cerevisiae and higher eukaryotes.
Cyclic variations in the permeability of the cell wall of Saccharomyces cerevisiae
Authors: De Nobel Johannes G. , Klis Frans M. , Ram Arthur , Van Unen Hans , Priem Jan , Munnik Teun , Ende Herman Van Den 
Published: May 2005

AbstractTo study cell‐cycle‐related variations in wall permeability of Saccharomyces cerevisiae, two approaches were used. First, an asynchronous culture was fractionated by centrifugal elutriation into subpopulations containing cell of increasing size. The subpopulations represented different stages of the cell cycle as judged by light microscopy. Cell wall porosity increased when these subpopulations became enriched with budded cells. Secondly, synchronous cultures were obtained by releasing MATa cells from alpha‐factor induced G1‐arrest. These cultures grew synchronously for at least two generations. The cell wall porosity incresed sharply in these cultures, shortly before buds became visible and was maximal during the initial stages of bud growth. It decreased in cells which had completed nuclear migration and before abscission of the bud had occurred. The porosity reached its lowest value during abscission and in unbudded cells.We examined the incorporation of mannoproteins into the wall during the cell cycle. SDS‐extractable mannoproteins were incorporated continuously. However, the incorporation of glucanase‐extractable mannoproteins, which are known to affect cell wall porosity, showed cyclic oscillations and reached its maximum after nuclear migration. This coincided with a rapid decrease in cell wall porosity, indicating that glucanase‐extractable mannoproteins might contribute to this decrease.