bluuebunny/crossref_metadata_2025 huggingface dataset:
Increase in gene expression by respiratory‐deficient mutation
Authors: Kaisho Yoshihiko , Yoshimura Koji , Nakahama Kazuo
Published: May 2005

Respiratory‐deficient mutants (rho− cells) of Saccharomyces cerevisiae produced about 10 times as much human(h‐) lysozyme as did wild‐type strains (rho+ cells) when the GAL10 promoter was used in an expression plasmid with the h‐lysozyme gene. Introduction of intact mitochondria into the rho− cells resulted in a significant decrease in the production of h‐lysozyme, indicating that the rho− mutation increased the expression of the h‐lysozyme gene. The copy number of the expression plasmid was not responsible for the increased expression. The level of h‐lysozyme mRNA in the rho− cells was also much higher than that in the rho+ cells especially at the stationary phase. The increased expression of the h‐lysozyme gene was also observed when a glyceraldehyde‐3‐phosphate dehydrogenase gene promoter and the PHO5 promoter were used in the expression plasmid. The rho− mutation also increased the expression of the PHO5 gene under the control of the HIS5 promoter in a plasmid and the ACT1 gene in the yeast chromosome, but did not increase the expression of the ribosomal RNA gene. In contrast to the rho− mutants, pet mutants did not show higher gene expression compared with wild‐type strains.
Induction of cytochrome P‐450 and catalase activity in Saccharomyces cerevisiae by UV and X‐ray irradiation. Possible role for cytochrome P‐450 in cell protection against oxidative damage
Authors: Morichetti E. , Cundari E. , Del Carratore R. , Bronzetti G.
Published: May 2005

Cytochrome P‐450 was induced both in the diploid wild‐type D7 strain and in two isogenic DNA‐repair‐deficient strains (rad3 and rad56) of Saccharomyces cerevisiae following UV‐ and X‐irradiation. The induction occurred only in logarithmic growth phase cells and it was transient showing a peak 3 h after irradiation. The maximal amount of cytochrome P‐450 was directly proportional to the radiation does applied. Under the same experimental conditions an increase of the catalase activity was also observed, suggesting that activated oxygen species produced by irradiation might be implicated in the induction of both enzymes. The sensitivity to H2O2 of cells containing high cytochrome P‐450 levels was enhanced when this enzyme was specifically inhibited by tetrahydrofuran and metyrapone. This supports the hypothesis that cytochrome P‐450, as well as catalase, might be involved in cell protection against oxidative damage.
Saccharomyces cerevisiae mutants defective in chromosome segregation
Authors: McGrew Jeffrey T. , Xiao Zhixiong , Fitzgerald‐Hayes Molly
Published: May 2005

We have devised a genetic screen to identify trans‐acting factors involved in chromosome transmission in yeast. This approach was designed to potentially identify a subset of genes encoding proteins that interact with centromere DNA. It has been shown that mutations in yeast centromere DNA cause aberrant chromosome segregation during mitosis and meiosis. We reasoned that the function of an altered centromere should be particularly sensitive to changes in factors with which it interacts. We constructed a disomic strain containing one copy of chromosome III with a wild‐type centromere and one copy of chromosome III bearing the SUP11 gene and a mutant CEN3. This strain forms white colonies with red sectors due to nondisjunction of the chromosome bearing the mutant centromere. After mutagenesis we picked colonies that exhibited increased nondisjunction of the mutant chromosome as evidenced by increased red‐white sectoring. Using this approach, we have isolated three trans‐acting chromosome nondisjunction (cnd) mutants that are defective in maintaining chromosomes during mitosis in yeast.
Induction of a heat shock‐type response in fission yeast following nitrogen starvation
Authors: Walker Graeme M. , McWilliams Philip G.
Published: May 2005

When 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

Yeast 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

As 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

We 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

The 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

The 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

We 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

In 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

Three 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

Shuttle 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

Using 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

The 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

Saccharomyces 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

Isolated 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

A 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

We 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

To 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.
Characterization of plasma membrane proton‐ATPase from salt‐tolerant yeast Zygosaccharomyces rouxii cells
Authors: Watanabe Yasuo , Yamaguchi Masahiro , Sanemitsu Yumi , Tamai Youichi
Published: May 2005

Plasma membrane was isolated from the salt‐tolerant yeast Zygosaccharomyces rouxii and from Saccharomyces cerevisiae. The ATPase in the plasma membrane of Z. rouxii cells was a typical proton‐ATPase as judged by testing with various ATPase inhibitors. There were slight differences in the pH optima of activities and in the sensitivity to sodium chloride (NaCl) and potassium chloride (KCl) of the ATPase from Z. rouxii and S. cerevisiae. The specific ATPase activity from Z. rouxii was higher in cells grown in a medium containing 2 M‐NaCl than in those not containing NaCl. No in vivo activation by incubation with glucose was observed in Z. rouxii cells and the specific ATPase activity was independent of the growth phase, unlike S. cerevisiae cells.
Intracellular expression of Kluyveromyces lactis toxin γ subunit mimics treatment with exogenous toxin and distinguishes two classes of toxin‐resistant mutant
Authors: Butler Andrew R. , Porter Megan , Stark Michael J. R.
Published: May 2005

The Kluyveromyces lactis toxin is a heterotrimeric protein which irreversible arrests proliferation of sensitive Saccharomyces cerevisiae cells in the G1 phase of the cell cycle. By expressing the γ subunit of the toxin in sensitive yeast cells from a conditional promoter, it was previously demonstrated that it alone is required for inhibition (Tokunaga et al. (1989). Nucleic Acids Res. 17, 3435–3446). Here we show that, like native exogenous toxin, intracellular γ subunit expression promoters a striking arrest of sensitive cells in G1. However, unlike the G1 arrest caused by native toxin, that induced by the γ subunit alone does not result in reduced cellular viability and is fully and rapidly reversible, suggesting that the G1 arrest and the irreversibility of action may reflect different aspects of the toxin's interaction with sensitive cells. We have selected a large number of S. cerevisiae mutants which are highly resistant to the toxin in order to study its mode of action in more detail. Complementation analysis demonstrated that all but one of the mutants were recessive and these defined four separate genes. Members of two complementation groups concurrently acquired resistance to intracellular γ subunit expression, suggesting that they contain a modified toxin target site. The other two genes appear to be required for entry of the γ subunit into the sensitive cell since these mutants, while refractory to exogenous toxin, were fully sensitive to intracellular γ subunit expression.
Cell wall glucomannoproteins of Saccharomyces cerevisiae mnn9
Authors: Van Rinsum Johanna , Klis Frans M. , Ende Herman Van Den
Published: May 2005

Mannoproteins were isolated from Saccharomyces cerevisiae mnn9 mutant cell walls by laminarinase digestion and purified by affinity and anion‐exchange chromatography. The purified mannoprotein fraction contained three predominant proteins with molecular masses of 300 kDa, 220 kDa and 160 kDa. These compounds were absent in an SDS extrct of cell walls or in a hot‐citrate extract of mnn9 cells.

The carbohydrate part of the purified mannoproteins consisted of (N‐acetyl)glucosamine, mannose and glucose in a molar ratio of 1:53:4. O‐Glycosidically linked chains, containing 70% of the mannose, were released by mild β‐elimination. N‐Glycosidically linked chains, representing 80% of the (N‐acetyl)glucosamine and 20% of the mannose, were released by peptide N‐glycosidase F (PNGase F) digestion. Complete degradation of protein by alkaline hydrolysis released besides the N‐ and O‐glycosidically linked chains, another type of carbohydrate chain containing the residual (N‐acetyl)glucosamine, mannose and most of the glucose in a molar ratio of 1:17:18. Glucose was β‐glycosidically linked.

The results indicate that β‐glucose is linked to PNGase F‐resistant N‐linked chains present on cell wall mannoproteins. We propose that these chains are responsible for the linkage between mannoproteins and glucan in the cell wall.
Purification and some properties of membrane‐bound and soluble pyrophosphatases of yeast vacuoles
Authors: Lichko Lidiya , Okorokov Lev
Published: May 2005

The membrane‐bound and soluble pyrophosphatase (PPase) activities of Saccharomyces carlsbergenis vacuoles are determined by the functioning of special enzymes and are not due to non‐specific PPi hydrolysis by other vacuolar phosphohydrolases. The molecular mass of the membrane‐bound PPase is apparently 120 000 and its molecule consists of three subunits with Mr = 41 000. Soluble PPase has a molecular mass of about 82 000 and includes three subunits with Mr = 28 000. Both enzymes are glycoproteins. The vacuolar membrane‐bound PPase is a proton pump.
Structure of the yeast endoplasmic reticulum: Localization of ER proteins using immunofluorescence and immunoelectron microscopy
Authors: Preuss Daphne , Mulholland Jon , Kaiser Chris A. , Orlean Peter , Albright Charles , Rose Mark D. , Robbins Phillips W. , Botstein David
Published: May 2005

The endoplasmic reticulum (ER) and other secretory compartments of Saccharomyces cerevisiae have biochemical functions that closely parallel those described in higher eukaryotic cells, yet the morphology of the yeast organelles is quite distinct. In order to associate ER functions with the corresponding cellular structures, we localized several proteins, each of which is expected to be associated with the ER on the basis of enzymatic activity, biological function, or oligosaccharide content. These marker proteins were visualized by immunofluorescence or immunoelectron microscopy, allowing definition of the S. cerevisiae ER structure, both in intact cells and at the ultrastructural level. Each marker protein was most abundant within the membranes that envelop the nucleus and several were also found in extensions of the ER that frequently juxtapose the plasma membrane. Double‐labeling experiments were entirely consistent with the idea that the marker proteins reside within the same compartment. This analysis has permitted, for the first time, a detailed characterization of the ER morphology as yeast cells proceed through their growth and division cycles.
A high‐affinity uptake system for branched‐chain amino acids in Saccharomyces cerevisiae
Authors: Tullin Søren , Gjermansen Claes , Kielland‐Brandt Morten C.
Published: May 2005

In order to isolate mutants with impaired uptake of branched‐chain amino acids, mutants were induced which on complex medium were sensitive to an inhibitor of branched‐chain amino acid biosynthesis. Eighteen mutants of independent origin were found. Ten of them were assayed for branched‐chain amino acid uptake. Three of these were impaired in the uptake of L‐valine, L‐isoleucine and L‐leucine, while the rest were unaffected in uptake of any of the three amino acids. Kinetics of the uptake by one selected mutant and the parental strain S288C were compared to models for one or two systems obeying Michaelis‐Menten kinetics. This analysis suggested that a high‐affinity system for all three amino acids is absent in the mutant, whereas low‐affinity uptake of L‐isoleucine and L‐leucine by one or more systems remains unaffected. Moreover, medium‐affinity uptake components for L‐valine and L‐leucine, not originally seen in the wild type, were identified in the mutant. In the wild type, 10 mM of any of the amino acids L‐alanine, L‐cysteine, L‐isoleucine, L‐leucine, L‐tryptophan and L‐valine reduce uptake of any of the three branched‐chain amino acids. We propose that a permease responsible for high‐affinity uptake of the branched‐chain amino acids in strain S288C is partially or completely inactive in the mutant. Tetrad analysis shows that the phenotype can be ascribed to a single Mendelian gene. The wild‐type allele is denoted BAP1 for branched‐chain amino acid permease. The BAP1‐dependent system is different from the general amino acid permease.
A rapid method for localized mutagenesis of yeast genes
Authors: Muhlrad Denise , Hunter Rosalyn , Parker Roy
Published: May 2005

We have developed a simple procedure for the localized mutagenesis of yeast genes. In this technique the region of interest is first amplified under mutagenic chain reaction (PCR) conditions. Cotransformation of the PCR product with a gapped plasmid containing homology to both ends the PCR procuct allows in vivo recombination to repair the gap with the mutagenized DNA. This procedure is efficient, allows trageting of specific regions for mutagenesis, and requires no subcloning steps in Escherichia coli.
IMP2, a nuclear gene controlling the mitochondrial dependence of galactose, maltose and raffinose utilization in Saccharomyces cerevisiae
Authors: Donnini Claudia , Lodi Tiziana , Ferrero Iliana , Puglisi Pier Paolo
Published: May 2005

The IMP2 gene of Saccharomyces cerevisiae is involved in the nucleo‐mitochondrial control of maltose, galactose and raffinose utilization as shown by the inability of imp2 mutants of grow on these carbon sources in respiratory‐deficient conditions or in the presence of ethidium bromide and erythromycin. The negative phenotype cannot be scored in the presence of inhibitors of respiration and oxidative phosphorylation, indicating that the role of the mitochondria in the utilization of the above‐mentioned carbon sources in imp2 mutants is not at the energetical level.

Mutantions in the IMP2 gene also confer many phenotypic alterations in respiratory‐sufficient conditions, e.g. leaky phenotype on oxidizable carbon sources, sensitivity to heat shock and sporulation deficiency.

The IMP2 gene has been cloned, sequenced and disrupted. The phenotype of null imp2 mutants is indistinguishable from that of the originally isolated mutant.
Identification of a Saccharomyces cerevisiae homolog of the SNF2 transcrioptional regulator in the DNA sequence of an 8·6 kb region in the LTE1‐CYS1 interval on the left arm of chormosome I
Authors: Clark Michael W. , Zhong Wu Wei , Keng Teresa , Storms Reginald K. , Barton Arnold , Kaback David B. , Bussey Howard
Published: May 2005

The DNA sequence of an 8·6 kb region of the left arm of chromosome I has been determined. This region, between the LTEL and CYS1 loci, is approximately 40 kb from centromere. There are six potential open‐reading frames (ORFs), Provisionally nemed YAL001‐006 within this fragment of chromosome I. Four of these ORFs can be aligned with Previously indentified FUN transcripts: FUN28 with YAL006, FUN29 with YAL004, FUN30 with YAL001 and FUN31 with YAL002. The YAL001 ORF shows significant homology to the SNF2 transcriptional regulator. A region of the DNA contains an extensive repeat of the bases C‐A‐T positioned in the 5′ terminus of the YAL004 promoter region.
Sequence of the sup61‐RAD18 region on chromosome III of Saccharomyces cerevisiae
Authors: Benit Paule , Chanet Roland , Fabre Francis , Faye Gerard , Fukuhara Hiroshi , Sor Frederic
Published: May 2005

A 7965 bp DNA segment from the right arm of chromosome III of Saccharomyces cerevisiae, encompassing the sup61 and RAD18 genes, was sequenced. Four new open reading frames were found in this DNA fragment. One of them YCR103, is 51% homologous with the G10 gene product of Xenopus laevis.
Architectural features of pre‐mRNA introns in the fission yeast Schizosaccharmyces pombe
Authors: Prabhala Ganesh , Rosenberg George H. , Käufer Norbert F.
Published: May 2005

The architectural features of 73 introns found in 36 genes of the fission yeast Schizosaccharomyces pombe have been compiled and tabulated. The intron features of Saccharomyces cerevisia and other eukaryotes. The results that S. pombe displays quite different architectural features than the budding yeast S. cerevisiae. However, particularly in the 3′ region, S. pombe introns also appear to differ from mammalian introns.
The complete sequence of K3B, a 7·9 kb fragment between PGK1 and CRY1 on chromosome III, reveals the presence of seven open reading frames
Authors: Bolle Paul‐André , Gilliquet Véronique , Berben Gilbert , Dumont Jacques , Hilger François
Published: May 2005

We have determined the nucleotide sequence of a segment from chromosome III fo Saccharomyces cerevisiae extending over 7·9 kb between the PGK1 and CRY1 loci.

The fragment contains seven open reading frames, YCR241, YCR242, YCR243, YCR244, YCR245, YCR246 and YCR247, of more than 70 codons. The study of the effects of a global disruption of YCR242, YCR243, YCR244, YCR245 and YCR247 shows that they are not essential for growth division.
The peroxisomal import signal of amine oxidase from the yeast Hansenula polymorpha is not universal
Authors: De Hoop M. J. , Valkema R. , Kienhuis C. B. M. , Hoyer M. A. , Ab G.
Published: May 2005

Amine oxidase from the yeast Hansenula polymorpha is a peroxisomal protein. The signal for routing of the protein into peroxisomes has not been identified yet. Expression of a mutant amine oxidase in H. Polymorpha has revealed that the C‐terminal sequence, which possesses an internal SRL tripeptide, is not involved in targeting (Faber et al., unpublished). We have explored heterologous expression of the amine oxidase gene (AMO) in Saccharomyces cerevisiae to investigate the conservation of peroxisomal targeting pathways between yeasts. Surprisingly, wide‐type amine oxidase is not recognized as a peroxisomal protein by S. cerevisiae. The enzyme, which was fully active and acumulated to levels similar to those found in H. polymorpha, stayed entirely in the cytosol. However, fusing a SKL or a SRL sequence to the C‐terminus forced the protein at least partially into peroxisomes of the heterologous host. These data suggest that the functional targeting sequence of amine oxidase may differ from the C‐terminal peroxisomal targeting signal S/C/A‐K/R/H‐L (Gould et al., 1989). Contrary to the established tripeptide motif, the amine oxidase targeting signal appears not to be conserved between the different yeast species.
Mutations in phosphofructokinases alter the control characteristics of glycolysis in vivo in Saccharomyces cerevisiae
Authors: Lloyd David , James Christopher J. , Maitra Pabitra K.
Published: May 2005

Ethanol and CO2 production from gluecose by non‐proliferating suspensions of aerobicaly‐grown, glucose‐derepressed wild‐type Sacharomyces cerevisiae is inhibited by O2; monitoring by mass spectrometry provides a direct method for measurement of the Pasteur effect.

Under aerobic conditons, that part of the CO2 evolved equivalent to the O2 consumed, is produced by respiration: subtraction of this respiratory CO2 from the total gives, CO2 produced by aerobic glycolysis. Pasteur quotients (anaerobic CO2/aerobic glycolytic CO2) were within the range 1.2 to 3.0. The Pasteur effect was not observed in the presence of carbonyl cyanid m‐chlorophenylhydrazone, an uncoupler of mitochondrial energy metabolism, or in a ρ cytoplasmic petite mutant. A ‘non‐allosteric’ mutant with an altered regulatory subunit of phosphofructokinase showed no Pasteur effect. Strains bearing a nonsense mutation pfk1 in the catalytic subnit of soluble phosphofructokinase (PFKI) also showed no Pasteur effect; the residual fermentative activity of this strain was dependent on PFKII, the particulate phosphofructokinase. A double mutant lacking both PFKI and glucose‐6‐phosphat dehydrogenase showed similar characteristics to those of the single pfk1 mutant; this indicates that the hexose monophosphate shunt is not acting to bypass the phosphofructokinase block. A ‘hyper‐allosteric’ mutant altered in the regulatory subunit encoded by the gene PFK2 showed characteristics of glucose fermentation and ethanol oxidation very similar to those of wild‐type organisms. These results indicate that either of the two phosphofructokinases can cary out glycolysis.
A Ser/Thr‐rich multicopy suppressor of a cdc24 bud emergence defect
Authors: Bender Alan , Pringle John R.
Published: May 2005

MSB2 was identified previously as a multicopy suppressor of a temerature‐sensitive mutation in CDC24, a gene required for polarity establishment and bud formation in Saccharomyces cerevisiae. The inferred MSB2 product contains 1306 amino acids, 42% of which are Ser or Thr. Its Ser+Thr‐richnes and hydrophobicity profile suggest that Msb2p may be an integral membrane protein containing a long, periplasmic, N‐terminal domain and a short, cytoplasmic, C‐terminal domain. Cells that lack MSB2 display no obvious mutant phenotypes. MSB2 is located between the centromere and KSS1 on the right arm of chromosome VII. Although physical mapping suggests that MSB2 and LEU1 (on the left arm of chromosome VII) are approximately 40 kb apart, the genetic map distance observed between leul and msb2 :: URA3 marker was only 2.3 cM.
Isolation and sequence analysis of the gene encoding translation elongation factor 3 from Candida albicans
Authors: Di Domenico B. J. , Lupisella J. , Sandbaken M. , Chakraburtty K.
Published: May 2005

The structural gene encoding translation elongation factor 3 (EF‐3) has been cloned from a Candida albicans genomic library by hybrization to a Saccharomyces cerevisiae probe containing the Saccharomyces gene, YEF3 (Sandbaken et al., 1990b). The sequences were shown to be functionally homologous to the Saccharamyces gene by three criteria: (1) a Saccharomyces strain transformed with a high copy plasmid containing CaEF3 sequences overprodues the EF‐3 peptide two‐fold; (2) extracts from this strain exhibit a two‐fold increase in the Ef‐3 catalysed, ribosome‐dependent ATPase activity (Kamath and Chakraburtty, 1988); and (3) the Candida gene complements a Saccharomyces null mutant. The coding region, identified by DNA sequencing, indicates that CaEF3 encodes a 1050 amino acid polypeptide having a potential molecular weight of 116 865 Da. This protein shows 77% overall identity to the Saccharomyces YEF3 gene, with a significantly greater identity (94%) concentrated in the region of the protein thought to contain the catalytic domain of EF‐3 (Sandaken et al., 1990a). The upstream non‐coding region contains T‐rich regions typical of many yeast genes and several potential RAPI/GRFI elements shown to regulate expression of a number of translational genes (Mager, 1988). The data confirm a high degree of conservation for EF‐3 among the two organisms.
Molecular analysis of yeast chromosome II between CMD1 and LYS2: The excision repair gene RAD16 located in this region belongs to a novel group of double‐finger proteins
Authors: Mannhaupt Gertrud , Stucka Rolf , Ehnle Susanne , Vetter Irene , Feldmann Horst
Published: May 2005

We have analysed a region some 30 kb centromere distal form PHO5 on the right arm of yeast chromosome II and determined the nucleotide sequence of a 8.95 kb DNA segment from this region. By this analysis we were able to derive the precise location and the transcriptional orientation of CMD1, ALG1, SSN6 and LYS2. An open reading frame of 2370 bp was locatlized between SSN6 and LYS2, which has recently been identified (Schild et al., 1991) to be the RAD16 gene. The putative gene product, 790 amino acids in length, reveals several interesting freatures. It contains a nuclear target singnature and shares several blocks of similarity with the yeast recombinational repair protein RAD54 and the nuclear factor SNF2 (SW12), which is required for teh transcriptioal activation of a number of yeast genes. The similarity blocks in these three proteins are reminiscent of those found in the helicase superfamily. Furthrmore, RAD16 contains a novel ‘double‐finger’ motif, which has been encountered in a variety of proteins from different organisms that are suggested to interact with DNA and are involved in diverse functions including site‐specific recombination, DNA repair, and transcriptional regulation. The putative gene product of RAD16 then is the first example of a proteins in which the novel double‐finger motif is found to be combined with a poteintial DNA helicase framework.
Efficient selection of phleomycin‐resistant Saccharomyces cerevisiae transformants
Authors: Wenzel Thibaut. J. , Migliazza Anna , Yde Steensma H. , Van Den Berg Johan A.
Published: May 2005

The recently dsecribed dominant yeast marker Tn5ble confers phleomycin resistance on the yeast Saccharomyces cerevisiae (Gatignol, Baron and Tiraby, 1987. Mol. Gen. Genet. 207, 342–348). Incubation in non‐selective medium prior to selection is critical, however, for getting phleomycin‐resistant transformants. A 6‐h incubation period was found to give optimal transformation frequencies, up to 105 transformants/μg plasmid, comparable to selection for uracil prototrophy (Ura+).