bluuebunny/crossref_metadata_2025_split huggingface dataset:
Flow cytometric analysis of Saccharomyces cerevisiae autolytic mutants and protoplasts
Authors: de la Fuente J. M. , Nombela C. , Sanchez M. , Alvarez A. 
Published: May 2005

AbstractSimple methods, based on the technique of flow cytometry, have been developed for the phenotypic characterization of yeast autolytic mutants and for the analysis of the formation and regeneration of the yeast protoplasts. The expression of lytic mutations determined uptake of the fluorescent dye propidium iodide, which could be carefully monitored by flow cytometry. Mixed populations of lysed and viable cells were precisely quantified and sorted, and the technique was also applied to demonstrate protection from lysis of mutant cells with cell wall defects, in the presence of osmotic stabilizers. Protoplast formation and regeneration was monitored by analysing relative cell size; this was facilitated by the preparation of homogeneous protoplast preparations. The technique of flow cytometry proved superior to other conventional methods for these types of study.
Effects of phosphoglycerate kinase overproduction in Saccheromyces cerevisiae on the physiology and plasmid stability
Authors: van der Aar Paul C. , Röling Wilfred F. M. , Stouthamer Adriaan H. , van Verseveld Henk W. , Raué Hendrik A. , van den Heuvel Joop J. 
Published: May 2005

AbstractIn this report the effects of phosphoglycerate kinase (PGK) overproduction on the physiology and plasmid stability in baker's yeast Saccharomyciae cerevisiae containing the PGK1 gene on an episomal plasmid are described. This examination reveals that there is a preferred intracellular level for this enzyme, amounting to 10–15% of the total soluble protein. Strains containing the plasmid and the host strain were grown in non‐selective batch cultures and continuous culture, under different growth conditons. Plasmid‐containing yeast strains stabilize the copy number of the episomal plasmid at a level at which the PGK concentration is about 12%. This stabilization is due to an equilibrium between normal plasmid loss and selective pressure because of advantages resulting from the increased amount of PGK under glucose‐limited conditions. During respiro‐fermentative growth, PGK‐overproducing cells showed an increased respiration rate and decreased fermentative activity, compared to the host strain.The PGK1 gene can be applied as a direct positive selection marker to obtain a high episomal plasmid stability during growth on glucose. The results are consistent with previously reported data on the physiology and gene stability of PGK‐overproducing yeast cells that contain multiple copies of the PGK1 gene integrated into the genome.
The complete sequence of a 10·8kb fragment to the right of the chromosome III centromere of Saccharomyces cerevisiae
Authors: Biteau Nicolas , Fremaux Christophe , Hebrard Sylvie , Menara Annie , Aigle Michael , Crouzet Marc 
Published: May 2005

AbstractThe complete nucleotide sequence of the D10H fragment (10850 bp) was determined. The D10H fragment is located on the right arm of chromosome III near the centromere and contains the SUF2 gene. Six open reading frames (ORFs) larger than 300 bp were found. One of them is the CIT2 gene encoding the cytoplasmic citrate synthase. The others are new putative genes and show no significant similarly with any known gene. In addition two tRNA genes (Asn and Pro) and a solo delta element were identified. Two ORFs were disrupted; no peculier phenotype was observed.
Thioredoxin genes in Saccharomyces cerevisiae: Map positions of TRX1 and TRX2
Authors: Muller Eric G. D. 
Published: May 2005

AbstractThe two genes encoding thioredoxims in Saccharomyces cerevisiae, TRX1 and TRX2, map to chromosome XII and VII, respectively. From the DNA sequence of the intragenic region TRX1 is 500 bp downstream of PDC1. Tetrad analysis places TRX21·1 cM from ADE3, while a physical map of this region positions TRX2 4·5 kb downstreams of ADE3. The mapping of TRX1 adjacent to PDC1 clarifies previous results (Muller, E. G. D. J. Biol. Chem. 266, 9194–9202, 1991) that suggested a third thioredoxims gene.
Characterization of mutants of the yeast Yarrowia lipolytica defective in acetyl‐coenzyme a synthetase
Authors: Kujau Marian , Weber Herbert , Barth Gerold 
Published: May 2005

AbstractThe expression of the glyoxylate cycle enzymes is required for growth of the yeast Yarrowia lipolytica on acetate or fatty acids as sole carbon source. Acetyl‐coenzyme A, which is produced by acetyl‐coenzyme A synthetase (ACS) from acetate, is needed for induction of this expression. Acetate‐non‐utilizing mutants of this yeast were investigated in order to identify mutants which express no or strongly reduced activity of this enzyme. Mutations in gene ICL2 exhibited the strongest effects on the activity. In icl2 mutants, lack of ACS activity resulted in a non‐induced glyoxylate cycle on acetate; however, induction on fatty acids was not affected. Gene ICL2 was identified as the sstructural gene encoding the monomer of ACS. It is shown that a high level of ACS activity is necessary for full expression of the glyoxylate cycle enzymes. Mutations in gene ICL1, which encodes isocitrate lyase, resulted in overproduction of ACS without any growth on acetate. A new gene (GPR1 = glyoxylate pathway rergulation) was detected in which trans‐dominant mutations inhibit expression of ACS and the glyoxylate cycle on acetate as carbon source.
Genetic and molecular analysis of DNA43 and DNA52: Two new cell‐cycle genes in Saccharomyces cerevisiae
Authors: Solomon Natalie A. , Wright Matthew B. , Chang Soo , Buckley Ann M. , Dumas Lawrence B. , Gaber Richard F. 
Published: May 2005

AbstractTwo Saccharomyces cerevisiae genes previously unknown to be required for DNA synthesis have ben identified by screening a collection of temperature‐sensitive mutants. The effects of mutations in DNA43 and DNA52 on the rate of S phase DNA synthesis were detected by monitoring DNA synthesis in synchronous populations that were obtained by isopycnic density centrifugation. dna43‐1 and dna52‐1 cells undergo cell‐cycle arrest at the restrictive temperature (37°C), exhibiting a large‐budded terminal phenotype; the nuclei of arrested cells are located at the neck of the bud have failed to undergo DNA replication. These phenotypes suggest that DNA43 and DNA52 are required for entry into or completion of S phase. DNA43 and DNA52 were cloned by their abilities to suppress the temperature‐sensitive lethal phenotypes of dna43‐1and dna52‐1 cells, respectively. DNA sequence analysis suggested that DNA43 and DNA52 encode proteins of 59.6 and 80.6 kDa, respectively. Both DNA43 and DNA52 are essential for viability and genetic mapping experiments indicate that they represent previously unidentified genes: DNA43 is located on chromosome IX, 32 cM distal from his5 and DNA52 is located on chromosome IV, 0.9 cM from cdc34.
Sequence of the novel essential gene YJU2 and two flanking reading frames located within a 3.2 kb EcoRI fragment from chromosome X of Saccharomyces cerevisiae
Authors: Forrová Helena , Kolarov Jordan , Ghislain Michel , Goffeau André 
Published: May 2005

AbstractWe have isolated and sequenced a novel gene from Saccharomyces cerevisiae coding for an essential protein of unknown function. The gene called YJU2 was borne on a chromosome X fragments shown by hybridization to intact S. cerevisiae chromosomal DNA fractionated by orthogonal pulsed field electrophoresis. Northern hybridization analysis indicated that the 0.8‐kb transcript of YJU2 is expressed in exponential‐phase cells grown in rich medium (data not shown). Figure 1 shows the nucleotide and deduced amino‐acid sequences of the 834‐bp coding region as well as the nucleotide sequences of the 5′ upstream region and of the 3′ downstream region, together with the flanking neighbouring open reading frames (ORFs), YJU1 and YJU3.
Effect of benzoic acid on metabolic fluxes in yeasts: A continuous‐culture study on the regulation of respiration and alcoholic fermentation
Authors: Verduyn Cornelis , Postma Erik , Scheffers W. Alexander , Van Dijken Johannes P. 
Published: May 2005

AbstractAddition of benzoate to the medium reservoir of glucose‐limited chemostat cultures of Saccharomyces cerevisiae CBS 8066 growing at a dilution rate (D) of 0.10 h−1 resulted in a decrease in the biomass yield, and an increase in the specific oxygen uptake rate (qO2) from 2.5 to as high as 19.5 mmol g−1h−1. Above a critical concentration, the presence of benzoate led to alcoholic fermentation and a reduction in (qO2) to 13 mmol g−1h−1. The stimulatory effect of benzoate on respiration was dependent on the dilution rate: at high dilution rates respiration was not enhanced by benzoate. Cells could only gradually adapt to growth in the presence of benzoate: a pulse of benzoate given directly to the culture resulted in wash‐out.As the presence of benzoate in cultures growing at low dilution rates resulted in large changes in the catabolic glucose flux, it was of interest of study the effect of benzoate on the residual glucose concentration in the fermenter as well as on the level of some selected enzymes. At D=0.10 h−1, the residual glucose concentration increased proportionally with increasing benzoate concentration. This suggests that modulation of the glucose flux mainly occurs via a change in the entracellular glucose concentration rather than by synthesis of an additional amount of carriers. Also various intracellular enzyme levels were not positively correlated with the rate of respiration. A notable exception was citrate synthase: its level increased with increasing respiration rate.Growth ofS. cerevisiae in ethanol‐limited cultures in the presence of benzoate also led to very high qO2 levels of 19–21 mmol g−1h−1. During growth on glucose as well as on ethanol, the presence of benzoate coincided with an increase in the mitochondrial volume up to one quarter of the total cellular volume.Also with the Crabtree‐negative yeasts Candida utilis, Kluyveromyces marxianus andHansenula polymorpha, growth in the presence of benzoate resulted in an increase in qO2 and, at high concentrations of benzoate, in aerobic fermentation. In contrast to S.Cerevisiae, the highest qO2 of these yeasts when growing at D = 0.10 h−1 in the presence of benzoate was equal to, or lower than the qO2 attainable at μmax without benzoate. Enzyme activities that were repressed by glucose in S. cerevisiae also declined in K.Marxianus when the glucose flux was increased by the presence of benzoate.The maximal aerobic fermentation rate at D = 0.10 h−1 of the Crabtree‐negative yeasts at high benzoate concentrations was considerably lower than for S. cerevisiae. This is probably due to the fact that under aerobic conditions these yeasts are unable to raise the low basal pyruvate decarboxylase level: cultivation without benzoate under oxygen‐limited conditions resulted in rates of alcoholic fermentation and levels of pyruvate decarboxylase comparable to those of S. cerevisiae.
Isolation of new temperature‐sensitive mutants of Saccharomyces cerevisiae deficient in mannose outer chain elongation
Authors: Nagasu Takeshi , Shimma Yoh‐Ichi , Nakanishi Yoko , Kuromitsu Junro , Iwama Kiyoshi , Nakayama Ken‐Ichi , Suzuki Katsunori , Jigami Yoshifumi 
Published: May 2005

AbstractWe have isolated two temperature‐sensitive Saccharomyces cerevisiae mutants which exhibit a deficiency in mannose outer chain elongation of asparagine‐linked oligosaccharide. The size of yeast glycoprotein, secretery form of invertase, of one mutant (ochl) was slightly larger than that of the sec18 mutant at the non‐permissive temperature, while that of the other mutant (och2) was almost the same as that of the sec18 mutant. Unlike sec mutants, the och mutants were not deficient in secretion of invertase. The och1 mutant showed a 2+:2− cosegregation with regard to the temperature sensitivity and mannose outer chain deficiency, suggesting that a single gene designated as OCHI is responsible for these two phenotypes. The och1 mutant stopped its growth at the early stage of bud formation and rapidly lost its viability at the non‐permissive temperature. The och1 mutation was mapped near the ole1 on the left arm of chromosome VII. The och1 mutant cells accumulated the external invertase containing a large amount of core‐like oligosaccharides (Man9–10GlcNAc2) and a small amount of high mannose oligosaccharides (>Man50GlcNAc2) at the non‐permissive temperature. Production of the active form of human tissue‐type plasminogen activator was increased in the och1 mutant compared with the parental strain, suggesting the potential advantage of this mutant for the production of mammalian‐type glycoproteins which lack mannose outer chains in yeast.
The complete sequence of a 9,543 bp segment on the left arm of chromosome III reveals five open reading frames including glucokinase and the protein disulfide isomerase
Authors: Scherens B. , Messenguy F. , Gigot D. , Dubois E. 
Published: May 2005

AbstractWe report here the DNA sequence of a 9·5 kb segment of chromosome III. The sequence was determined by subcloning the segment into subfragments generated by appropriate restriction enzymes followed by oligonucleotide‐directed sequencing. The segment contains at least five open reading frames, YCL311, YCL312, YCL313, YCL314, YCL315. YCL311 and YCL315 extend in the adjacent fragments, A4H and A6C respectively. YCL312 encodes glucokinase, and YCL313 the protein disulfide isomerase. Disruption of YCL311, 314 and 315 by insertion of a URA3 cassette does not lead to a detectable phenotype, whereas disruption of YCL313 provokes cell lethality.
Mercaptoethanol and dithiothreitol decrease the difference of electrochemical proton potentials across the yeast plasma and vacuoar membranes and activate their H+‐ATPases
Authors: Petrov Valery V. , Smirnova Valeria V. , Okorokov Lev A. 
Published: May 2005

AbstractMercaptoethanol and dithiothreitol (DTT) inhibited the acidification of external medium by by Saccharomyces Carlsbergensis cells and protoplasts during glucose oxidation. The inhibition was also observed when cells were incubated with mercaptoethanol or when mercaptoethanol and DTT were used to prepare protolasts. Experiments with S. carlsbergensis plasma membrene vesicles and vacuoles showed these thiol reagents to inhibitATP‐dipendent generation of ΔpH and Em across plasma membrane vesicles and vacuoles but to activate their H+‐ATPases. Mercaptoethanol and DTT are suggested to de‐energize plasmalemma as well as tonoplast by increasing their H+‐permeability and to disturb the cell ion homeostasis.
A screening procedure for the intracellular expression of native proteins by Saccharomyces cerevisiae: Discrimination of diphtheria toxin‐resistant mutants
Authors: Donovan Maura G. , Veldman Sarah A. , Bodley James W. 
Published: May 2005

AbstractA genetal method is described for screening Saccharomyces cerevisiae colonies for the intracellular expression of native proteins. Colonies are replicated onto nitrocellulose membranes and yeast walls are removed enzymatically. The resulting spheroplasts are rapidly lysed by placing chromatography paper soaked in hypotonic buffer on the membranes. Intracellular proteins released by spheroplast lysis are bound in situ to the nitrocllulose under non‐denaturing conditions and potentially can be examined using enzymatic or immunologeic methods. For example, in the present study colonies were screened for the presence of elongation factor 2 (EF‐2) that can be [32P]ADP‐ribosylated by diphtheria toxin and [32P]NAD+. Recognition by the toxin requires the presence in EF‐2 of the unique posttranslationally modifed histidine derivative, dipthamide. The procedure described here reliably discriminates between wild‐type yeast colonies and mutant colonies that do not synthesize diphthamide. In addition to faciliating the study of dipthamide biosynthesis in yeast, the more general application of this procedure of this procedure will enable the screening of colonies with assays that require native proteins.
RHO gene products, putative small GTP‐binding proteins, are importnat for activation of the CAL1/CDC43 gene product, a protein geranylgeranyltransferase in Saccharomyces cerevisiae
Authors: Qadota Hiroshi , Ishii Isao , Fujiyama Asao , Ohya Yoshikazu , Anraku Yasuhiro 
Published: May 2005

AbstractTwo multicopy suppressors of the call‐1 mutation in the yeast Saccharomyces cerevisiae have been isolated and characterized. They are identical to the yeast RHO1 and RHO2 genes, which encode putative small GTP‐binding proteins. Multiple copies of either RHO gene suppressed temperature‐sensitive growth of the call‐1 mutant but did not suppress the call null mutant. Genetic analysis suggests that overproduction of either RHO gene product acts for activation of the CAL1 gene product.
XI. Yeast sequencing reports. DNA sequencing and analysis of a 24·7 kb segment encompassing centromere CEN11 of Saccharomyces cerevisiae reveals nine previously unknown open reading frames
Authors: Düsterhöft Andreas , Philippsen Peter 
Published: May 2005

AbstractA 24·7 kb segment of the cosmid clone pUKG047 containing a Sau3AI‐partial fragment from the centromere region of Saccharomyces cerevisiae chromosome XI was sequenced and analysed. A mexed strategy of directed methods including exonuclease III nested deletion, restriction fragment subcloning and oligonucleotide‐directed sequences was carried out. Exclusive use waas made of the Applied Biosystems Taq DyeDeoxy™ Terminator Cycle technology and a laser‐based ABI373A sequencing system for reactions, gel electrophoresis and automated reading. A total of 12 open reading frames (ORFs) was found. Nine new ORFs (YK102 to YK110) were identified, three of which (YK102, YK107, YK108)showed homologies to proteins of known function from other organisms. In addition, sequence analysis reveled three recently functionally characterized genes (MET14, VPS/SPO15, PAP1), which could be joined to the earlier published CEN11 region.
II. Yeast sequencing reports. The sequence of an 8 kb segment on the left arm of chromosome II from Saccharomyces cerevisiae identifies five new open reading frames of unknown functions, two tRNA genes and two transposable elements
Authors: Skala Jacek , Van Dyck Luc , Purnelle Bénédicte , Goffeau André 
Published: May 2005

AbstractThe DNA sequence of an 8079 bp ClaI fragment located at 40 kb from the centromere on the left arm of chromosome II from Saccharomyces cerevisiae has been determined. Sequence analysis reveals five new oen reading frames, tRNAGly and tRNALeu genes as well as sigma and truncated delta elements. The disruption of the three larger open reading frames shows that they are not essential for mitotic growth.
Yeast sequencing reports. LEU2 gene homolog in Kluyveromyces lactis
Authors: Zhang Ying‐Pei , Chen Xin‐Jie , Li Yu‐Yang , Fukuhara Hiroshi 
Published: May 2005

AbstractA DNA fragment that can complement the leu2 mutation of Saccharomyces cerevisiae was cloned from the genomic library of Kluyveromyces lactis.The nucleotide sequence revealed an open reading frame of 362 codons, 75% homologous to S. cerevisiae LEU2 gene. The upstream region contained a CCGGAACCGG sequence identical to the site of leucine‐specific control of LEU2. Further upstream, there is a partial open reading frame homologous to rat ribosmal protien L7.
Yeasts have a four‐fold variation in ribosomal DNA copy number
Authors: Maleszka R. , Clark‐Walker G. D. 
Published: May 2005

AbstractBy employing pulsed‐field gel electrophoresis we have determined the size of the rDNA cluster in wild‐type yeast strains representing genera of Candida, Kluyveromyces, Pachysolen, Schizosaccharomyces and Torulaspora. Although the genome size of the examined species is similar (12·3–13·9 Mb), at least a four‐fold variation has been observed between the lowest amount of rDNA repeats in P. tannophilus (28) and the highest in C. glabrata and S. poombe (> 115).In two species the rDNA cluster is represented by two loci, residing either in one (S. pombe) or two chromosomes (C. glabrata).
Human catalase is imported and assembled in peroxisomes of Saccharomyces cerevisiae
Authors: De Hoop M. J. , Holtman W. L. , Ab G. 
Published: May 2005

AbstractTo study the conservation of peroxisomal targeting signals, we have determined the intracellular localization of human peroxisomal catalase when expressed in yeast. Using immunofluorescence, differential centrifugation and immunoelectron microscopy, we show that the protein is targeted to the peroxisomes of the heterologous cell and assembled in its active tetrameric form. These data show the conservation of the catalase targeting signal and import specificity between human and yeast peroxisomes.
Cloning and genetic characterization of a calcium‐ and phospholipid‐binding protein from Saccharomyces cerevisiae that is homologous to translation elongation factor‐1γ
Authors: Kambouris Nicholas G. , Burke Daniel J. , Creutz Carl E. 
Published: May 2005

AbstractWe have isolated a gene (CAM1) from the yeast Saccharomyces cerevisiae that encodes a protein homologous to the translational cofactor elongation factor‐1γ (EF‐1γ) first identified in the brine shrimp Artemia salina. The predicted Cam1 amino acid sequence consists of 415 residues that share 32% identity with the Artemia protein, increasing to 72% when conservative substitutions are included. The calculated Mr of Cam1p (47 092 Da) is in close agreement with that of EF‐1γ (Mr = 49 200 Da), and hydropathy plots of each protein exhibit strikingly similar profiles. Disruption of the CAM1 locus yields four viable meiotic progeny, indicating that under normal growth conditions the Cam1 protein is non‐essential. Attempts to elicit a translational phenotype have been unsuccessful. Since EF‐1γ participates in the regulation of a GTP‐binding protein (EF‐1α), double mutants with cam1 disruptions and various mutant alleles of known GTP‐binding proteins were constructed and examined. No evidence was found for an interaction of CAM1 with TEF1, TEF2, SEC4, YPT1, RAS1, RAS2, CDC6, ARF1, ARF2 or CIN4. The possibility that Cam1p may play a redundant role in the regulation of protein synthesis or another GTP‐dependent process is discussed.
The role of ALA‐S and ALA‐D in regulating porphyrin biosynthesis in a normal and a HEM R+ mutant strain of Saccharomyces cerevisiae
Authors: García S. Correa , Moretti M. Bermúdez , Cardalda C. , Rossetti M. V. , del C. Batlle A. M. 
Published: May 2005

AbstractCatabolite repression and derepression on δ‐aminolevulinate synthase (ALA‐S) and δ‐aminolevulinate dehydratase (ALA‐D) in a normal yeast strain, D27, and its derived D27/C6 (HEM R+) were investigated. ALA‐S and ALA‐D activities and intracellular ALA (I‐ALA) at different physiological states of the cells were measured. In YPD medium, under conditions of repression and when glucose was exhausted, both strains behaved identically as if the mutation was not expressed. In YPEt medium, however, both ALA‐S and ALA‐D activities were higher than in YPD, but the I‐ALA content and the enzymic activity profiles shown by the two strains were quite different. It appears, therefore, that the mutation causes a deregulation of ALA‐S, so that its activity is kept at a high level throughout the cell cycle. This would explain the increased levels of cytochromes present in the mutant. This mutation may affect some regulatory aspect of ALA formation and renders an ALA‐S of high activity; moreover, this enzyme species seems to be more stable than in the normal strain.