bluuebunny/crossref_metadata_2025 huggingface dataset:
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

Addition 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

We 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

We 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

Mercaptoethanol 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

A 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

Two 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

A 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

The 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

A 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

By 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

To 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

We 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

Catabolite 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.
Phylogenetic study of ribosomal DNA of cactophilic Pichia species by restriction mapping
Authors: Shen Randy , Lachance Marc‐André
Published: May 2005

The rDNAs of strains of the cactophilic Pichia species P. amethionina, P. antillensis, P. barkeri, P. cactophila, P. caribaea, P. deserticola, P. heedii, P. kluyveri, P. norvegensis, P. opuntiae, P. pseudocactophila, P. thermotolerans and their varieties and anamorphs were mapped with 15 restriction endonucleases, and compared to P. membranaefaciens and P. salictaria as possible non‐cactophilic relatives. The existence of species complexes among those taxa was confirmed. P. membranaefaciens was a plausible ancestral species, and its closest relative in the cactophilic group was P. deserticola. These two species appeared to be moderately related to P. heedii and to P. barkeri, but the latter was shown clearly to belong to the P. kluyveri complex, in spite of a 6 mol% G+C difference in their nuclear DNAs. P. cactophila and P. pseudocactophila ostensibly emerged from P. norvegensis, a facultatively cactophilic yeast. The P. amethionina, P. cactophila and P. opuntiae species complexes appeared independent from one another and from all other species studied. P. salictaria did not appear to be related to P. amethionina.
Sequence of the open reading frame of the FL01 gene from Saccharomyces cerevisiae
Authors: Teunissen A. W. R. H. , Holub E. , Van Der Hucht J. , Van Den Berg J. A. , Steensma H. Y.
Published: May 2005

The cloned part of the flocculation gene FLO1 of Saccharomyces cerevisiae (Teunissen, A.W.R.H., van den Berg, J.A. and Steensma, H.Y. (1993). Physical localization of the flocculation gene FLO1 on chromosome I of Saccharomyces cerevisiae, Yeast, in press) has been sequenced. The sequence contains a large open reading frame of 2685 bp. The amino acid sequence of the putative protein reveals a serine‐ and threonine‐rich C‐terminus (46%), the presence of repeated sequences and a possible secretion signal at the N‐terminus. Although the sequence is not complete (we assume the missing fragment consists of repeat units), these data strongly suggest that the protein is located in the cell wall, and thus may be directly involved in the flocculation process.
A yeast gene encoding a putative RNA helicase of the “DEAD”‐box family
Authors: Strahl‐Bolsinger Sabine , Tanner Widmar
Published: May 2005

An unknown open reading frame from Saccharomyces cerevisiae was identified and sequenced. The predicted amino acid sequence shows high homology to the DEAD‐box family of proteins. Gene disruption revealed that the gene is not essential for yeast but necessary for normal cell growth.
Endomitotic diploidization of Saccharomyces cerevisiae by heat treatment during spore germination
Authors: Tani Yoshinobu , Tomohiro Yukio , Miyata Akira , Furukawa Kensuke , Hayashida Shinsaku
Published: May 2005

Diploid cells with ability to mate, hereafter referred to as diploid mater cells, were obtained at significant frequencies by the heat treatment of haploid spores at the early germination stage in Saccharomyces cerevisiae heterothallic strain CG5M (a/α diploid cells heterozygous for five auxotrophic markers). The highest frequency (ca. 11%) of diploidization was obtained from viable cells after heat treatment at 55°C for 10 min when spores were precultivated for 30 min in liquid medium to initiate the germination. The diploid mater cells obtained were homozygous for mating type and for the auxotrophic markers. The diploidization of a spore is thus concluded to be due to endomitotic events in germinating heat‐treated spores.
Expression of HIV‐1 nef in yeast: The 27 kDa nef protein is myristylated and fractionates with the nucleus
Authors: Macreadie Ian G. , Ward Alister C. , Failla Paul , Azad Ahmed A. , Grgacic Elizabeth , McPhee Dale
Published: May 2005

The nef gene of human immunodeficiency virus type 1 (HIV‐1) has been expressed in the yeast Saccharomyces cerevisiae to produce native Nef proteins. The proteins of Mr 27 kDa and 25 kDa, produced by translation from the first and second start codons of the nef gene react with human HIV‐1 antisera. Under low‐level steady‐state expression conditions, Nef27 undergoes myristylation and is targeted to the nuclear fraction while Nef25 is not myristylated and not nuclear localized. When produced rapidly and to high levels, Nef27 is initially present in the cytoplasm as a soluble myristylated protein that later fractionates with the nucleus.
Assay of trehalose with acid trehalase purified from Saccharomyces cerevisiae
Authors: Kienle Iris , Burgert Mark Us , Holzer Helmut
Published: May 2005

An enzymatic end‐point assay of trehalose using acid trehalase from yeast is described. After quantitative hydrolysis of trehalose by acid trehalase, the resulting glucose is assayed with the commercially available glucose oxidase/peroxidase dye system. Pre‐existing glucose is determined in a control reaction from which acid trehalase is omitted. When intact cells are analysed for trehalose, pre‐existing glucose can be washed out with ice‐cold water without reducing the trehalose content of the cells. A convenient method for extraction of trehalose from intact yeast cells is heating for 20 min at 95°C followed by centrifugation. The specificity of the assay is determined by the specificity of the acid trehalase preparation used. As described previously (Mittenbühler, K. and Holzer, H., 1988, J. Biol. Chem. 263, 8537–8543; Mittenbühler, K., 1988, Thesis, University of Freiburg), the following sugars and sugar derivatives do not form glucose when incubated with purified acid trehalase: sucrose, cellobiose, mellobiose, raffinose, maltose, lactose, glucose‐6‐phosphate, glucose‐1‐phosphate, galactose. The application of the new trehalose assay to yeast cells grown to different growth stages and at various temperatures is presented.
RIM2, MSI1 and PGI1 and located within an 8 kb segment of Saccharomyces cerevisiae chromosome II, which also contains the putative ribosomal gene L21 and a new putative essential gene with a leucine zipper motif
Authors: Démolis Nadine , Mallet Laurent , Bussereau Françoise , Jacquet Michel
Published: May 2005

We report the DNA sequence of an 8 kb segment localized on the right arm of chromosome II from Saccharomyces cerevisiae. The sequence reveals the presence of eight open reading frames (ORFs). Three of them, YBR1402, YBR1405 and YBR1406 are previously sequenced genes, respectively the RIM2 (replication in mitochondria), MSI1 (multicopy suppressor of IRA1 gene) and PGI1 (phosphoglucoisomerase) genes. The predicted product of the ORF YBR1401 could be the putative yeast ribosomal protein L21. A new essential gene, YBR1403, has been identified by disruption; it possesses a leucine zipper motif.
TA‐repeat microsatellites are closely associated with ARS consensus sequences in yeast chromosome III
Authors: Valle Giorgio
Published: May 2005

Microsatellites are repeats of very short sequences of DNA, interspersed in the genome. In this paper, the occurrence of the two‐base repeat microsatellites has been investigated in the DNA sequence of yeast chromosome III. Only AT‐repeats were found at a significantly high frequency. Some of the regions with the highest concentration of AT‐repeats were located and further analysed, showing a close association with the core consensus of autonomously replicating sequences.
Linguistic analysis of chromosome III DNA sequence of Saccharomyces cerevisiae
Authors: Kalogeropoulos Angelos
Published: May 2005

The analysis of the Saccharomyces cerevisiae chromosome III DNA sequence by computer (‘in silico’) permits the definition of its linguistic characteristics. These characteristics include the designation of non‐randomly occurring oligonucleotides, their distribution along the chromosome, and the distribution of some particular homopolymers. All these elements may contribute to the understanding of the organization of information on the chromosome.
Cloning and sequence of ADP‐ribosylation factor 1 (ARF1) from Schizosaccharomyces pombe
Authors: Erickson F. Les. , Hannig Ernest M. , Krasinskas Alyssa , Kahn Richard A.
Published: May 2005

A gene encoding a homologue of the ADP‐ribosylation factor (ARF) family of small GTP binding proteins was cloned from a Schizosaccharomyces pombe cDNA library by a functional screen of suppressors of sensitivity to 3‐aminotriazole in a gcn3 null strain of Saccharomyces cerevisiae. Two independent isolates each contained the full coding region of the ARF1 gene. The encoded SpARF1 protein has a predicted molecular weight of 20 618 and is 88% and 79% identical to human and S. cerevisiae ARF1 proteins, respectively. As independent isolates were obtained, this effect of the SpARF1 appears to be a real phenomenon, but cannot currently be easily understood within the context of the evidence for a role(s) for ARF proteins in the protein secretory pathway.
Comparison of the biochemical and biological functions of tyrosine phosphatases from fission yeast, budding yeast and animal cells
Authors: Hannig Gerhard , Ottilie Sabine , Schievella Andrea R. , Erikson Raymond L.
Published: May 2005

In a previous communication, we have shown that two protein tyrosine phosphatases (PTPases) from fission yeast, pyp1+ and pyp2+, act as novel inhibitors of mitosis upstream of the wee1+ lmik1+ pathway (Ottilie et al., 1992). Here we describe that both genes possess intrinsic PTPase activity as judged by in vitro PTPase assays using 32P‐labeled Raytide as a substrate, and that 32P‐labeled p107wee1 is an in vitro substrate for pyp1. To compare the biological activity of pyp1 and pyp2 to that of other known PTPases, we expressed the budding yeast PTP1 and human placental phosphatase 1B (PTP1B) genes in either a cdc25–22 or wee1–50 genetic background and established that, in contrast to pyp1+ and pyp2+, Saccharomyces cerevisiae PTP1 and human PTP1B complement the cdc25 mutant, opposing the wee1+ lmik1+ pathway.
The nucleotide sequence of a 2·1 kb fragment from chromosome VI of Saccharomyces cerevisiae identifies a tRNAGly gene, part of a delta element and a palindromic sequence
Authors: Van Heusden G. Paul H. , De Koning Wim J. , Van Der Aart Quirina J. M. , Van Den Berg Johan A. , Steensma H. Yde
Published: May 2005

The nucleotide sequence was determined of a 2·1 kb DNA fragment located at approximately 35 kb to the right of the centromere of chromosome VI from Saccharomyces cerevisiae. Analysis revealed the presence of a tRNAGLy gene, part of a delta element and a remarkable palindromic sequence. The longest open reading frame found encodes a putative protein of 195 amino acids. Although the fragment was isolated by hybridization to a human diacylglycerol kinase cDNA, no evidence was obtained for the presence of a gene encoding diacylglycerol kinase.
The complete sequence of a 6794 bp segment located on the right arm of chromosome II of Saccharomyces cerevisiae. Finding of a putative dUTPase in a yeast
Authors: Doignon Francois , Biteau Nicolas , Aigle Michel , Crouzet Marc
Published: May 2005

The DNA sequence of a 6794 bp fragment located at about 100 kb from the right telomere of chromosome II from Saccharomyces cerevisiae has been determined. Sequence analysis reveals five open reading frames. One is the ARO4 gene encoding the 3‐deoxy‐D‐arabinoheptulosonate 7‐phosphate synthase. Another presents strong homology with the S5 ribosomal protein from bacteria. The open reading frame YBR1705 shows significant homology with dUTPase, suggesting for the first time the existence of such an enzyme in S. cerevisiae.
The ERG3 gene in Saccharomyces cerevisiae is required for the utilization of respiratory substrates and in heme‐deficient cells
Authors: Smith Steven J. , Parks Leo W.
Published: May 2005

ERG3 is the structural gene in Saccharomyces cerevisiae for the sterol Δ5 desaturase that introduces the C5=6 unsaturation in ergosterol biosynthesis. The ERG3 gene has been mapped on chromosome XII, 13·7 centimorgans from GAL2 toward SPT8. The essentially of the gene is dependent on the conditions used for the cultivation of the mutants. Insertionally inactivated mutants of ERG3 fail to grow without ‘sparking’ levels of Δ5 sterols in heme‐deficient cells, and are unable to grow on the respiratory substrates glycerol and ethanol.
Transformation in the methylotrophic yeast Pichia methanolica utilizing homologous ADE1 and heterologous Saccharomyces cerevisiae ADE2 and LEU2 genes as genetic markers
Authors: Hiep Tran Tuan , Noskov Vladimir N. , Pavlov Yuri I.
Published: May 2005

Development of transformation systems for methylotrophic yeasts is the starting point for research aimed at developing molecular genetics of these genera and will be the key to their further successful use in biotechnology. We transformed Pichia methanolica using selector genes ADE2 and LEU2 from Saccharomyces cerevisiae and ADE1 (homologue of S. cerevisiae ADE2 gene) from P. methanolica which was cloned and sequenced in our laboratory (Hiep et al., 1991). Lithium transformation of P. methanolica strains was inefficient with intact plasmids. Linearization of plasmids at a unique restriction site within the ADE1 gene prior to transformation substantially increased its frequency. Transformation with linear ADE1, ADE2 or LEU2 gene fragments was even more effective. Introduced DNA fragments either circularized in vivo, irrespective of the structures of their ends, giving unstable transformants; or integrated at different sites of the host genome. Using this transformation system, we obtained a disruption of the ADE1 gene on the chromosome by inserting the S. cerevisiae LEU2 gene. The disruption mutation ade1::LEU2 was used to study the mechanism of intragenic recombination in P. methanolica.
Fluorescent staining with bromocresol purple: A rapid method for determining yeast cell dead count developed as an assay of killer toxin activity
Authors: Kurzweilová Helena , Sigler Karel
Published: May 2005

A method is described for detecting yeast cells with plasma membrane damage, based on cell staining with bromocresol purple (BCP) which has a convenient fluorescence after entering the cells at pH below 5·2. The method was used to determine the activity of Saccharomyces cerevisiae pore‐forming killer toxin K1 in commonly used lethal units. The BCP test is rapid and as precise as the plating test.
Sequence and function analysis of a 2·73 kb fragment of Saccharomyces cerevisiae chromosome II
Authors: Miosga Thomas , Zimmermann Friedrich K.
Published: May 2005

The nucleotide sequence of a fragment of 2728 base pairs of Saccharomyces cerevisiae chromosome II has been determined. The sequence contains two open reading frames, one of them being incomplete. Deletion mutants of YBR11.21 are viable. YBR11.20 is identical to the recessive omnipotent suppressor SUP45 (SUP1).
A series of yeast shuttle vectors for expression of cDNAs and other DNA sequences
Authors: Brunelli Joseph P. , Pall Martin L.
Published: May 2005

Expression/shuttle vectors for the yeast Saccharomyces cerevisiae have usually been large plasmids with only one or a small number of sites that are suitable for cloning and expression. We report here the construction and properties of a series of 12 expression vectors with multiple (four to eight) unique sites in their polylinkers which allow directional cloning and expression of DNA sequences under four different promoters. Eleven of these plasmids replicate at high copy number in Escherichia coli, and all have the yeast TRP1 gene, and the 2 μm origin including REP3 sequence, allowing selection and high copy number replication in yeast. Six of the plasmids are designed for the construction and selection of cDNA libraries from various eukaryotic organisms, allowing directional cloning and expression of cDNAs. All of these six have similar polylinkers containing a unique promoter proximal EcoRI site and a unique promoter distal XhoI site, allowing for directional cloning and expression of ‘ZAP’‐type cDNAs. cDNAs that complement a wide variety of yeast mutants can be selected from libraries constructed in this way. The four alternative promoters, ADH2, PGK, GAL10 and SV40 were compared for their relative activity, both in E. coli and in yeast. All yeast promoters showed substantial activity in E. coli with ADH2 showing the highest activity. ADH2 also was well‐regulated in yeast, showing very high relative activity under derepressing conditions. cDNAs selected by genetic complementation from libraries constructed in these vectors should be easily subclonable into other vectors, allowing expression in different eukaryotic organisms, DNA sequencing or site‐directed mutagenesis.
A series of yeast/Escherichia coli λ expression vectors designed for directional cloning of cDNAs and cre/lox‐mediated plasmid excision
Authors: Brunelli Joseph P. , Pall Martin L.
Published: May 2005

A series of Saccharomyces cerevisiae/Escherichia coli λ/plasmid expression vectors have been constructed which allow easy excision of the plasmid sequences from λ. Features of six are described, and two designated λPG15 and λAD5, are characterized in detail. Transcription of cloned sequences is controlled by the alternative promoters, ADH2, PGK, GAL10 and SV40 early, and by the CYC1 transcriptional terminator. Unique EcoRI and XhoI restriction sites in the intervening polylinker make these λ vectors compatible for directional cloning of ‘ZAP’‐synthesized cDNAs. Inserted DNAs have been previously shown to have high levels of the genetic activity in both S. cerevisiae and E. coli, allowing these vectors to be used for genetic complementation in both species. Plasmid recovery from the λ vector is mediated by the activity of the cre‐encoded enzyme upon lox sequences flanking the plasmid and adjoining the λ arms. The plasmids contain the yeast 2 μm origin and E. coli pBR322 origin, the URA3 or TRP1 yeast selectable markers, and ampicillin‐resistance marker in E. coli. The usefulness of the λPG15 and the λAD5 cloning vectors was demonstrated by constructing large Neurospora crassa cDNA libraries. The λPG15–N. crassa library was used to infect purE, purC and trpC mutants of E. coli, and complemented and/or suppressed prototrophic colonies were selected. The flexibility and power of this system for cloning of cDNAs is discussed.
Sequencing and analysis of 51·6 kilobases on the left arm of chromosome XI from Saccharomyces cerevisiae reveals 23 open reading frames including the FAS1 gene
Authors: Wiemann Stefan , Voss Hartmut , Schwager Christian , Rupp Thomas , Stegemann Joseph , Zimmermann Jürgen , Grothues Dietmar , Sensen Christoph , Erfle Holger , Hewitt Neil , Banrevi Andrea , Ansorge Wilhelm
Published: May 2005

We have sequenced two segments containing a total of 51·6 kb of the left arm from chromosome XI of Saccharomyces cerevisiae. The first segment of 38·5 kb contains 18 open reading frames (ORFs) of more than 100 amino acid residues. Five ORFs encode known yeast genes, including the fatty acid synthase gene (FAS1). Three new yeast genes were discovered with homologies to non‐yeast genes and ten new genes without homologies to any known sequences. The second segment of 13 kb contains five ORFs with two known yeast genes and three unknown genes. The sequences from cosmid pUKG041 were obtained entirely with the walking primer strategy resulting in a very low overall sequence redundancy of 2·8 and an average reading length of 443 bases.
Low‐Affinity glucose carrier gene LGT1 of Saccharomyces cerevisiae, a homologue of the Kluyveromyces lactis RAG1 gene
Authors: Prior C. , Fukuhara H. , Blaisonneau J. , Wesolowski‐Louvel M.
Published: May 2005

A low‐affinity glucose transporter gene of Saccharomyces cerevisiae was cloned by complementation of the rag1 mutation in a strain of Kluyveromyces lactis defective in low‐affinity glucose transport. Gene sequence and effects of null mutation in S. cerevisiae were described. Data indicated that there are multiple genes for low‐affinity glucose transport.
The sequence of a 17.5 kb DNA fragment on the left arm of yeast chromosome XI identifies the protein kinase gene ELM1, the DNA primase gene PRI2, a new gene encoding a putative histone and seven new open reading frames
Authors: Purnelle Bénédicte , Tettelin Hervé , Van Dyck Luc , Skala Jacek , Goffeau André
Published: May 2005

A 17.5 kb DNA fragment of chromosome XI, located between the genetic loci mif2 and mak11 was sequenced and analysed. Ten open reading frames were identified. Two of them are the previously sequenced genes ELM1 and PRI2, two (YKL253 and YKL256) show homologies to proteins from other organisms and one (YKL262) to yeast and mouse histone.
Analysis of the rad3‐101 and rad3‐102 mutations of saccharomyces cerevisiae: Implications for structure/function of rad3 protein
Authors: Montelone Beth A. , Malone Robert E.
Published: May 2005

The mutations rad3‐101 and rad3‐102 (formerly rem1‐1 and rem1‐2) of the essential RAD3 gene of Saccharomyces cerevisiae confer a phenotype of semidominant enhancement of spontaneous mitotic recombination and mutation frequencies, but not extreme sensitivity to ultraviolet (UV) light. These properties differ from the previously published observations of other rad3 mutations, which are very UV‐sensitive but do not alter recombination frequencies significantly. We have located the position of DNA sequence changes from wild‐type RAD3 to the rad3‐101 and rad3‐102 mutations and have demonstrated that these sequence changes are necessary and sufficient to confer the (Rem−) mutant phenotype when transferred into otherwise wild‐type RAD3 plasmids. The Rem− mutations are not located in the same region. It is possible that the two regions of the gene in which these mutations map define portions of the molecule which are in contact when folded in the native configuration. To begin to test this hypothesis, we have constructed two double mutant alleles, one with rad3‐101 and rad3‐102, and one with the UV‐sensitive rad3‐1 mutation and rad3‐102. We find that plasmids carrying these double mutant alleles of RAD3 are no longer able to confer a hyper‐recombinational phenotype and do not complement the UV‐sensitivity of the excision‐defective rad3‐2 allele. We conclude that the double mutant alleles are non‐functional for excision repair, and may be null. We have also constructed new rad3 alleles by oligonucleotide‐directed mutagenesis and have tested their effects on spontaneous mutation and mitotic recombination and on UV repair.
Cloning, nucleotide sequence and functions of XPR6, which codes for a dibasic processing endoprotease from the yeast Yarrowia lipolytica
Authors: Enderlin Carol S. , Ogrydziak David M.
Published: May 2005

Yarrowia lipolytica DO613, carrying the xpr6‐13 mutation, secretes an inactive precursor of alkaline extracellular protease that has not been cleaved after the Lys‐Arg at the end of the pro‐region. Compared to wild type, DO613 membrane preparations had significantly reduced ability to cleave after Lys‐Arg of an artificial substrate. The XPR6 gene was cloned by complementation by screening for restoration of production of alkaline protease activity. Sequencing of a 3735 base pair SalI‐SphI XPR6 fragment revealed a large open reading frame with a coding capacity of 976 amino acids (molecular weight, 110 016). The deduced amino acid sequence had significant homology to Saccharomyces cerevisiae Kex2p, a processing endoprotease that cleaves after pairs of basic amino acids. Disruption of the XPR6 gene was not lethal, but it resulted in several phenotypic changes. First, essentially no mature alkaline extracellular protease was produced indicating that the low levels produced by strains carrying previously isolated xpr6 alleles were due to leaky mutations. Second, mating type B strains carrying the disrupted XPR6 gene did not mate, but mating type A strains did. Third, the XPR6 disruption strains grew poorly on rich media at pH 5·5 and above. Cells remained physically attached after budding and continued to bud forming large dog balloon‐like structures. In addition, these structures aggregated forming visible clumps in liquid culture. These growth aberrations were largely eliminated by growing cells in medium at pH 4. Fourth, no mycelial forms were observed regardless of the pH.
Structural modification of spindle pole bodies during meiosis II is essential for the normal formation of ascospores in Schizosaccharomyces pombe: Ultrastructural analysis of spo mutants
Authors: Hirata Aiko , Shimoda Chikashi
Published: May 2005

In order to characterize the morphological steps defined by sporulation (spo) genes during the formation of ascospores in the fission yeast Schizosaccharomyces pombe, we performed an electron microscopic study of the ultrastructure of the spindle pole body (SPB) and of the development of the forespore membrane during the second meiotic division (meiosis II) in sporulation‐deficient (spo) mutants (spo4, spo5, spo14 and spo18). No difference was found in terms of the function and the structure of the SPB during the first meiotic division (meiosis I) between the four mutants and wild‐type cells. However, during meiosis II, the spo4 and spo18 mutants underwent nuclear division but in neither case were the SPBs modified nor were forespore membranes formed. The SPBs of the spo18 mutant diminished in size after meiosis II and eventually disappeared after 18 h in sporulation medium. By contrast, the SPBs of the spo4 mutant remained unchanged even after an 18‐h incubation. The outer plaques of SPBs of spo5 and spo14 mutants were sufficiently modified to allow them to initiate development of the forespore membrane, but the membrane had an abnormally expanded lumen and did not enclose the nuclei during meiosis II. The spo5 mutant produced anucleate spore‐like bodies while the spo14 mutant formed unorganized structures with irregular peripheries which, presumably, contained spore‐wall precursors, instead of anucleate spore‐like bodies. We conclude that the modification of the SPB is essential for the formation of ascospores and at least two genes (spo5 and spo14) participate in the development of the forespore membrane. The defective phenotypes define discrete steps in the development of ascospores, which proceeds via steps defined by the mutant spo4, spo18, spo14 and spo5 genes respectively. Our observations provide further substantial evidence that the SPB plays a pivotal role in the normal development of ascospores in yeasts.
A general model of yeast energy metabolism in aerobic chemostat culture
Authors: Castrillo J. I. , Ugalde U. O.
Published: May 2005

The pattern of energy metabolism of different types of yeasts (obligate aerobes and facultative anaerobes) in aerobic chemostat cultures has been evaluated and interpreted on the basis of a coupling of metabolic fluxes between glycolytic and oxidative components.

A model has been formulated which defines glycolytic and oxidative subunits through which the substrate C‐flux (gram‐atom g−1 h−1) is calculated, stating that a relative imbalance between glycolytic flux and subsequent oxidative steps alone is sufficient to account for the onset of oxidoreductive metabolism in any type of yeast, irrespective of the maximum respiratory capacity. The model is able to reproduce the patterns of behaviour reported for the different types of yeasts, and the individual features of each strain are explained on the basis of metabolic differences which are defined by a set of normalized parameters. The model can be applied to different substrates and conditions, providing a methodological basis for more detailed studies of the steps controlling yeast energy metabolism.
XI. Yeast sequencing reports. Sequence analysis of a 3·5 kb EcoRI fragment from the left arm of Saccharomyces cerevisiae chromosome XI reveals the location of the MBR1 gene and a sequence related to a GTPase‐activating protein
Authors: James Carolyn M. , Gent Manda E. , Oliver Stephen G.
Published: May 2005

We present the DNA sequence analysis of a region covering a 3·5 kb EcoRI fragment from the left arm of chromosome XI from Saccharomyces cerevisiae. This region contains five open reading frames (ORFs) which code for proteins of greater than 100 amino acids. ORF YKL425 codes for the previously sequenced Mbr1 (Valens et al., 1991; Daignan‐Fornier et al., 1993) which participates in mitochondrial biogenesis. YKL424 has identity with a GTPase‐activating protein of higher eukaryotes. The three remaining ORFs have no identity to known proteins within the databases screened and are not assigned ORF numbers as they are completely contained with ORFs YKL424 and YKL425. This sequence has been entered in the EMBL Data Library under Accession Number X75561.
XV. Yeast sequencing reports. A gene from Saccharomyces cerevisiae which codes for a protein with significant homology to the bacterial 3‐phosphoserine aminotransferase
Authors: Belhumeur Pierre , Fortin Nathalie , Clark Michael W.
Published: May 2005

During the sequencing of the gene GSP2 from Saccharomyces cerevisiae, we have encountered an adjacent open reading frame having strong homology to the 3‐phosphoserine aminotransferase (E.C.2.6.1.52) from other organisms. In this report, we present the sequence for this yeast SERC, and evidence that its deletion from the yeast genome leads to serine dependency. The sequence has been deposited in the GenBank data library under Accession Number L20917.
Use of β‐lactamase as a secreted reporter of promoter function in yeast
Authors: Cartwright Charles P. , Li Yu , Zhu Yun‐Song , Kang Yoon‐Se , Tipper Donald J.
Published: May 2005

K1 preprotoxin is the 316 residue precursor of the K1 killer toxin secreted by the yeast Saccharomyces cerevisiae. The SPβla reporter consists of the mature, secreted form of β‐lactamase (βla) fused to S and P, two fragments of preprotoxin. S is the N‐terminal 34 residues, including the secretion signal. P, a 67 residue ‘processing’ segment with three sites for N‐glycosylation, terminates in a Lys Arg site for cleavage by the Kex2 protease. Expression of SPβla in yeast results in efficient secretion, processing by signal peptidase and glycosylation in the endoplasmic reticulum, producing proßla. Kex2 cleavage of proßla in the lumen of a late Golgi compartment releases βla, which accumulates stably in culture media buffered at pH 5·8–7. The half‐life of secretion is 11 min at 30°C; 10–12% of the total activity in exponential‐phase cells is intracellular, mostly in the form of proßla, indicating that transit from the endoplasmic reticulum to the Golgi is rate limiting. We have used SPβla expression in single‐ and multi‐copy vectors to compare the PGK, GAL1, GAL10, PHO5 and CUP1 promoters under varying nutritional conditions. In exponential‐phase cells, secretion of βla over a 40‐fold range and up to several μg/ml was proportional to transcript level, demonstrating that SPβla can be employed as a convenient secreted reporter of promoter function in yeast.
I. Yeast sequencing reports. Isolation and characterization of the LEU2 gene of Hansenula polymorpha
Authors: Agaphonov Michael. O. , Poznyakovski Andrei I. , Bogdanova Aliona I. , Ter‐Avanesyan Michael D.
Published: May 2005

A DNA fragment carrying the LEU2 gene of methylotrophic yeast Hansenula polymorpha was isolated by complementation of the leuB mutation of Escherichia coli. The nucleotide sequence of the isolated DNA fragment contains an open reading frame of 363 codons, coding for a protein 80% identical to the LEU2 gene product of Saccharomyces cerevisiae. Further downstream, there is a partial reading frame with no obvious similarity to known proteins. The LEU2 gene of H. polymorpha cannot complement the leu2 mutation of S. cerevisiae. The sequence has been entered in the EMBL data library under the Accession Number U00889.
I. Yeast sequencing reports. Sequencing of chromosome I of Saccharomyces cerevisiae: Analysis of the 42 kbp SP07‐CENI‐CDC15 region
Authors: Clark Michael W. , Keng Teresa , Storms Reg K. , Zhong Wuwei , Fortin Nathalie , Zeng Bin , Delaney Shawn , Ouellette B. F. Francis , Barton Arnold B. , Kaback David B. , Bussey Howard
Published: May 2005

Determination of the DNA sequence and preliminary functional analysis of a 42 kbp centromeric section of chromosome I have been completed. The section spans the SPO7‐CEN1‐CDC15 loci and contains 19 open reading frames (ORFs). They include an apparently inactive Ty1 retrotransposon and eight new ORFs with no known homologs or function. The remaining ten genes have been previously characterized since this part of the yeast genome has been studied in an unusually intensive manner. Our directed sequencing allows a complete ordering of the region. The sequence has been deposited in the GenBank data library under Accession Number L22015.