bluuebunny/crossref_metadata_2025 huggingface dataset:
Genetic homology between Saccharomyces cerevisiae and its sibling species S. paradoxus and S. bayanus: Electrophoretic karyotypes
Authors: Naumov Gennadi I. , Naumova Elena S. , Lantto Raija A. , Louis Edward J. , Korhola Matti
Published: May 2005

Chromosomal DNAs of many monosporic strains of the biological species Saccharomyces cerevisiae, S. paradoxus and S. bayanus were analysed using contour‐clamped homogeneous electric field electrophgoresis. SSouthern blot hybridization with eight cloned S. cerevisiae genes (ADC1, CUP1, GAL4, LEU2, rDNA, SUC2, TRP1 and URA3) assigned to different chromosomes was used to study homology and chromosomal location of the genes three sibiling species. A comparative study of Ty1, Ty2 and telomere‐associated Y' sequences having multiple chromosomal location was also done.

Chromosome length polymorphism was found in cultured strains of S. cerevisiae. Wild S. cerevisiae and S. paradoxus strains yielded chromosome banding patterns very similar to each other, The karyotype pattern of S. bayanus was readily distinguishable from that of S. cerevisiae and S. paradoxus. Southern blot analysis revealed a low degree of homology between the S. cerevisiae genes studied and the corresponding S. paradoxus and S. bayanus genes. The number of chromosomes appears to be 16 in all three species.
Yeast flocculation: Receptor definition by mnn mutants and concanavalin A
Authors: Stratford Malcolm
Published: May 2005

Yeast flocculation involves the binding of surface lectins on flocculent yeasts, to carbohydrate receptors present as constituents of yeast cell walls. Receptors were investigated by coflocculation of flocculent strains of Saccharomyces cerevisiae, both Flo 1 and NewFlo phenotypes, to known mnn mutants which vary in the wall mannan structure. Strong coflocculation was found with mnn1, mnn4, mnn9 and control strains, while very little coflocculation was found with mnn2 and mnn5 strains. In constrast, aggregation of these muatants by concanavalin A, a lectin with similar sugar inhibition to NewFlo phenotype flocculation, showed strong aggregation of mnn1, mnn4, and mnn5 strains and poor aggregation of mnn2 and mnn9 strains.

The mmn mutant data suggested that flocculation receptorss were the outer‐chain mannan side‐branches, two or three mannose residues in length, confirming an earlier theory based on sugar inhibition data. The similarities and differences between flocculation and concanavalin A aggregation are discussed.
Rag−mutations involved in glucose metabolism in yeast: Isolation and genetic characterization
Authors: Wésolowski‐Louvel M. , Prior C. , Bornecque D. , Fukuhara H.
Published: May 2005

Some natural isolates and many laboratory strains of the yeast Kluyveromyces lactis cannot grow on glucose when respiration is inhibited by antimycin A. The ability or inability to grow on glucose in the absence of mitochondrial respiration has been called Rag+ or Rag− phenotype (resistance to antimycin on glucose, respectively). Rag− strains, unable to grow on glucose in the presence of the respiratory drug, behave as if they were defective in fermentation. The Rag phenotype was first found to be determined by variant alleles of either of the two nuclear genes, RAG1 and RAG2, which code for a low‐affinity glucose transport protein and for phosphoglucose isomerase, respectively. These findings suggested that the Rag− phenotype can be used to obtain mutations of genes involved in glucose metabolism in K. lactis. We thus looked for other Rag− mutants. Seventy‐four mutants were isolated and genetically characterized. All of the mutations were nuclear recessive alleles, defining 11 new complementation groups, which we designate rag3 through rag13.
VYeast sequencing reports. AFG1, a new member of the SEC18–NSF, PAS1, CDC48–VCP, TBP family of ATPases
Authors: Lee Yang‐Ja , Wickner Reed B.
Published: May 2005

We have sequenced a gene that encodes a 377 amino acid putative protein with an ATPase motif typical of the protein family including SEC18p (NSF = N‐ethyl maleimide‐sensitive fusion protein; vesicle‐mediated endoplasmic reticulum to Golgi protein transfer), PAS1p (peroxisome assembly), CDC48p (VCP = valosin‐containing protein; cell cycle) and TBP1 (Tat‐binding protein). This gene, AFG1 for ATPase family, gene, also has substantial homology to these proteins outside the ATPase domain. AFG1 is located on chromosome V imediately centromere‐proximal to M AK10.
Polyamines and cell wall organization in Saccharomyces cerevisiae
Authors: Miret J. J. , Solari A. J. , Barderi Patricia A. , Goldemberg Sara H.
Published: May 2005

Cells of Saccharomyces cerevisiae 179‐5, an ornithine decarboxylase mutant (spe‐1), showed several ultrastructural abnormalities when cultivated in the absence of polyamines. Besides the appearance of microvacuole‐like spaces in the cytoplasm and of deformed nuclei, the most important alterations seemed to be located in the cell wall, which was thicker and of heterogeneous texture, and in the cell membrane, of irregular contour. These modifications could not be evoked by general stress conditions elicited by lack of nutrients. The relative levels of cell wall polysaccharides were altered in polyamine‐deprived organisms, giving an envelope with increased mannan and decreased glucan content; this cell wall was incompletely attacked by the lytic enzyme zymolyase. Polyamine depletion led also to some abnormalities in the budding pattern. The above observations suggest the involvement of polyamines in the correct structure and organization of the yeast cell.
Identification and genetic mapping of CHL genes controlling mitotic chromosome transmission in yeast
Authors: Kouprina N. , Tsouladze A. , Koryabin M. , Hieter P. , Spencer F. , Larionov V.
Published: May 2005

Eight independent chl (chromosome loss) mutants were isolated using yeast haploid strain disomic for chromosome III. In these mutants, chromosome III is lost during mitosis 50‐fold more frequently than in the wild‐type strains. chl mutants are also incapable of stable maintenance of circular and linear artificial chromosomes. Seven of the eight mutations are recessive, and one is semidominant. Complementation tests placed these mutants into six complementation groups (chl11 through chl16). Based on tetrad analysis, chl12, chl14 and chl15 correspond to mutations in single nuclear genes. Tetrad analysis of the other mutants was not possible due to poor spore viability. Complementation analysis was also carried out between collection of chl mutants and ctf mutants (chromosome transmission fidelity) (Spencer et al., 1990). The chl3, chl4, chl8, chl12 and chl15 mutants were unable to complement ctf3, ctf17, ctf12, ctf18 and ctf4, respectively. Three CHL genes were mapped by tetrad analysis. The CHL3 gene is placed on the right arm of chromosome XII, between the ILV5 (33·3 cM) and URA4 (21·8 cM) loci. The CHL10 gene is located on the left arm of chromosome VI, 12·5 cM from the centromere. The CHL15 gene is tightly linked to the KAR3 marker of the right arm of chromosome XVI (8·8 cM). The mapping data indicate that these three genes differ from other genes known to affect chromosome stability in mitosis. Therefore, the total number of the CHL genes identified (including those described by us earlier) is 13 (CHL1–CHL10, CHL12, CHL14 and CHL15).
Stress resistance of yeast cells is largely independent of cell cycle phase
Authors: Elliott Beth , Futcher Bruce
Published: May 2005

Rapidly growing cells of Saccharomyces cerevisiae are sensitive to heat shock, while non‐growing stationary phase cells are highly resistant. We find that slowly growing cells have an intermediate degree of heat shock resistance that can be nearly as great as that of stationary phase cells. This resistance is correlated both with slow growth and with carbon catabolite derepression. Slowly growing cells also showed resistance to Zymolyase digestion of their cell walls. The stress resistance is a property of all the cells in the culture, and cell cycle position makes little difference to the degree of stress resistance. At least some of the properties normally associated with stationary phase cells do not require residence in stationary phase or any other particular compartment of the cell cycle. Stress resistance may be due to a diverse set of physiological adaptations available to cells regardless of their position in the cell cycle. That is, although stress resistance and stationary phase are often correlated, neither is the cause of the other.
Targeting of a heterologous protein to the cell wall of Saccharomyces cerevisiae
Authors: Schreuder Maarten P. , Brekelmans Stephan , Van Den Ende Herman , Klis Frans M.
Published: May 2005

The sexual adhesion protein of Saccharomyces cerevisiae MATα cells, α‐agglutinin, could not be extracted from the cell wall with hot sodium dodecyl sulfate (SDS), but became soluble after digestion of the cell with laminarinase. This indicates that it is intimately associated with cell wall glucan. A fusion protein was constructed consisting of the signal sequence of yeast invertase, guar α‐galactosidase, and the C‐terminal half of the α‐agglutinin. Most of the fusion protein was incorporated in the cell wall. A small amount could be extracted with SDS, but most of it could only be extracted with laminarinase. On the other hand, cells containing a construct consisting of the signal sequence of invertase and α‐galactosidase released most of the α‐galactosidase into the medium and all cell wall‐associated α‐galactosidase was released by SDS. Labelling with antibodies showed that the α‐galactosidase part of the fusion protein was exposed on the surface of the cell wall. The results demonstrate that the C‐terminal half of the α‐agglutinin contains the information needed to incorporate a protein into the cell wall.
A comparative study on the transport of L(‐)malic acid and other short‐chain carboxylic acids in the yeast Candida utilis: Evidence for a general organic acid permease
Authors: Cássio Fernanda , LeñO Cecília
Published: May 2005

Cells of the yeast Candida utilis grown in medium with short‐chain mono‐, di‐ or tricarboxylic acids transported L(‐)malic acid by two transport systems at pH 3·0. Results indicate that probably a proton symport for the ionized form of the acid and a facilitated diffusion for the undissociated form were present. Dicarboxylic acids such as succinic, fumaric, oxaloacetic and α‐ketoglutaric acids were competitive inhibitors of the malic acid for the high‐affinity system, suggesting that these acids used the same transport system. In turn, competitive inhibition uptake studies of labelled carboxylic acid in the low‐affinity range indicated that this system was non‐specific and able to accept not only carboxylic (mono‐, di‐ or tri‐) acids but also some amino acids. Additionally, under the same growth conditions, C. utilis produced two mediated transport systems for lactic acid: a proton symport for the anionic form which appeared to be a common monocarboxylate carrier and a facilitated diffusion system for the undissociated acid displaying a substrate specificity similar to that observed for the low‐affinity dicarboxylic acid transport. The mediated carboxylic acid transport systems were inducible and subjected to repression by glucose. In glucose‐grown cells the undissociated dicarboxylic acids entered the cells slowly by simple diffusion. Repressed glucose‐grown cells were only able to produce both transport systems if an inducer, at low concentration (0·5%, w/v), was present during starvation in buffer. This process was inhibited by the presence of cycloheximide indicating that induction requires de novo protein synthesis. If a higher acid concentration was used, only the low‐affinity transport system was detectable, showing that the high‐affinity system was also repressed by high concentrations of the inducer.
Effects of growth with ethanol on fermentation and membrane fluidity of Saccharomyces cerevisiae
Authors: Lloyd David , Morrell Suzie , Carlsen Helle N. , Degn Hans , James Phillip E. , Rowlands Christopher C.
Published: May 2005

Saccharomyces cerevisiae HSc was grown with ethanol at concentrations up to 10% (v/v). The immediate effects of additions of externally added ethanol on CO2 production and O2 consumption of washed organisms were studied by stopped‐flow membrane inlet quadrupole mass spectrometry. Fermentative activities of organisms grown with ethanol (0–5% v/v) showed similar sensitivities to inhibition by ethanol, whereas those grown with 10% (v/v) ethanol had become protected and were markedly less sensitive.

The fluidity of subcellular membrane fractions was measured by determination of the temperature dependence of the rotational order parameter of the spin label 5‐doxyl stearic acid (free radical) by electron spin resonance. Mitochondria prepared from yeasts grown with 0, 7 and 9% (v/v) ethanol showed similar overall fluidity, although differences in temperature‐dependent behaviour indicate altered lipid composition or lateral phase separations. On the other hand the microsomal fraction from organisms grown with 9% ethanol showed a remarkable increase in fluidity. These data suggest that the protective effects of growth with ethanol near the limit of tolerance on fermentative activities may arise from altered plasma membrane fluidity properties.
Theoretical analysis of the effects of mitotic crossover in large yeast populations
Authors: Krafzig Dirk , Klawonn Frank , Gutz Herbert
Published: May 2005

In a diploid yeast population which is heterozygous for a given marker, A1A2, mitotic crossover (mit. c.o.) between the centromere and the marker will give rise to homozygous daughter cells, A1A1 and A2A2. Since this causes a decrease in the frequency of A1A2 cells, mit. c.o. is an important population genetic process in vegetatively propagated yeast cultures. The effect of mit. c.o. is counteracted by mutations and, in the case of heterosis, by selection. We present a mathematical analysis of these interactions.
GUT2, a gene for mitochondrial glycerol 3‐phosphate dehydrogenase of Saccharomyces cerevisiae
Authors: Rønnow Birgitte , Kielland‐Brandt Morten C.
Published: May 2005

A gut2 mutant of Saccharomyces cerevisiae is deficient in the mitochondrial glycerol 3‐phosphate dehydrogenase and hence cannot utilize glycerol. Upon transformation of a gut2 mutant strain with a low‐copy yeast genomic library, hybrid plasmids were isolated which complemented the gut2 mutation. The nucleotide sequence of a 3·2 kb PstI‐XhoI fragment complementing a gut2 mutant strain is presented. The fragment reveals an open reading frame (ORF) encoding a polypeptide with a predicted molecular weight of 68·8 kDa. Disruption of the ORF leads to a glycerol non‐utilizing phenotype. A putative flavin‐binding domain, located at the amino terminus, was identified by comparison with the amino acid sequences of other flavoproteins. The cloned gene has been mapped both physically and genetically to the left arm of chromosome IX, where the original gut2 mutation also maps. We conclude that the presented ORF is the GUT2 gene and propose that it is the structural gene for the mitochondrial glycerol 3‐phosphate dehydrogenase.
Ultrastructural changes in yeast following heat shock and recovery
Authors: Webster Deborah L. , Watson Kenneth
Published: May 2005

The effects of heat shock and heat stress on the ultrastructure of Saccharomyces cerevisiae is reported. Following a mild heat shock, referred to as an increase in temperature from 25°C to 37°C for 30 min, we observed contraction of the nucleolus, formation of electron‐dense particles (90 nm) in mitochondria and heat‐shock granules (30–40 nm) in the cytoplasm. The electron‐dense particles in the mitochondria were similar in appearance to those previously reported in plant cells exposed to elevated temperatures. In a heat‐sensitive yeast strain, the nucleolus was severely aggregated after a mild heat shock, a treatment which hardly affected relatively more heat‐resistant strains. The nucleolus was aggregated in all strains after a more severe heat stress (50°C for 2 or 4 min). When cells were observed during a recovery period after heat stress it was found that nucleolar ultrastructure was regained more rapidly in cells that were previously heat shocked compared to cells that were stressed directly with no prior heat shock.
Sequencing and functional analysis of a 32 560 bp segment on the left arm of yeast chromosome II. Identification of 26 open reading frames, including the KIP1 and SEC17 genes
Authors: Scherens Bart , El Bakkoury Mohamed , Vierendeels Fabienne , Dubois Evelyne , Messenguy Francine
Published: May 2005

We report here the DNA sequence of a segment (α1006.13: YBLO5) of chromosome II of Saccharomyces cerevisiae, extending over 32·5 kb. The segment contains 26 open reading frames (ORFs) from YBLO501 to YBLO526. YBL0505 corresponds to the SEC17 gene and YBL0521 to the KIP1 gene. YBL0516 contains an intron, YBL0513 shows homology with the RAT protein phosphatase and YBL0526 contains a zinc‐finger motif. Disruption of 14 genes by insertion of a URA3 cassette has been performed and these mutants were analysed for their mating and sporulation ability, and for their growth on different carbon sources. YBL0515 and YBL0526 ORFs seem to be involved in the sporulation process.
XI. Yeast sequencing reports. Analysis of an 11·7 kb DNA fragment of chromosome XI reveals a new tRNA gene and four new open reading frames including a leucine zipper protein and a homologue to the yeast mitochondrial regulator ABF2
Authors: Purnelle Bénédicte , Skala Jacek , van Dyck Luc , Goffeau André
Published: May 2005

We report the nucleotide sequence of an 11·7 kb fragment from the left arm of Saccharomyces cerevisiae chromosome XI. Analysis reveals a new tRNA for valine and four unknown open reading frames among which YKL245 shows homology with a yeast mitochondrial regulatory protein and YKL244, YKL246 and YKL247 are unknown.
Selectable marker replacement in Saccharomyces cerevisiae
Authors: Vidal Marc , Gaber Richard F.
Published: May 2005

Selectable markers integrated by the ‘gamma’ deletion method (Sikorski and Hieter, 1989) can be efficiently replaced in vivo with other markers by transformation with homologous plasmids. Transformation frequencies in experiments designed to replace original selectable markers with an alternate marker were high and molecular analysis confirmed that all transformants that exhibited the expected phenotypes (loss of the original prototrophy and gain of the alternate prototrophy) resulted from homologous recombination between plasmid sequences at the target locus. This technique involves no plasmid construction and greatly facilitates the generation of yeast cells containing multiple gene disruptions.
Positioning of cell growth and division after osmotic stress requires a map kinase pathway
Authors: Brewster Jay L. , Gustin Michael C.
Published: May 2005

The yeast Saccharomyces cerevisiae has a genetic program for selecting and assembling a bud site on the cell cortex. Yeast cells confine their growth to the emerging bud, a process directed by cortical patches of actin filaments within the bud. We have investigated how cells regulate budding in response to osmotic stress, focusing on the role of the high osmolarity glycerol response (HOG) pathway in mediating this regulation. An increase in external osmolarity induces a growth arrest in which actin filaments are lost from the bud. This is followed by a recovery phase in which actin filaments return to their original locations and growth of the original bud resumes. After recovery from osmotic stress, haploid cells retain an axial pattern of bud site selection while diploids change their bipolar budding pattern to an increased bias for forming a bud on the opposite side of the cell from the previous bud site. Mutants lacking the mitogen‐activated protein (MAP) kinase encoded by HOG1 or the MAP kinase kinase encoded by PBS2 (previously HOG4) show a similar growth arrest after osmotic stress. However, in the recovery phase, the mutant cells (a) do not restart growth of the original bud but rather start a new bud, (b) fail to restore actin filaments to the original bud but move them to the new one, and (c) show a more random budding pattern. These defects are elicited by an increase in osmolarity and not by other environmental stresses (e.g., heat shock or change in carbon source) that also cause a temporary growth arrest and shift in actin distribution. Thus, the HOG pathway is required for repositioning of the actin cytoskeleton and the normal spatial patterns of cell growth after recovery from osmotic stress.
Isolation of a CDC25 family gene, MSI2/LTE1, as a multicopy suppressor of ira1
Authors: Shirayama Masaki , Matsui Yasushi , Tanaka Kazuma , Toh‐E Akio
Published: May 2005

We have identified MS12 as a gene of Saccharomyces cerevisiae which, when on a multicopy vector, suppresses the heat shock sensitivity caused by the loss of the IRA1 product, a negative regulator of the RAS protein. The multicopy MSI2 also suppresses the heat shock sensitivity of cells with the RAS2val19 mutation but not those with the bcy1 mutation, suggesting that the MSI2 protein may interfere with the activity of the RAS protein. The sequence analysis of MSI2 reveals that it is identical to LTE1 belonging to the CDC25 family: CDC25, SCD25 and BUD5, each of which encodes a guanine nucleotide exchange factor for the ras superfamily gene products. Deletion of the entire MSI2 coding region reveals that MSI2 is not essential but the disruptant shows a cold‐sensitive phenotype. Under the non‐permissive conditions, more than 70% of the msi2 disruptants arrested at telophase as large budded cells with two nuclei divided completely and elongated spindles, indicating that the msi2 deletion is a cell division cycle mutation. These results suggest that MSI2 is involved in the termination of M phase and that this process is regulated by a ras superfamily gene product.
A search for an essential function of the replication origin ARS1 in the life cycle of Saccharomyces cerevisiae
Authors: Kirpekar Finn , Friis Jørgen , Gulløv Kay
Published: May 2005

We have investigated the significance of the chromosomal replication origin, ARS1, during the entire life cycle of yeast. This was done by substituting the chromosomal copy with a series of ars1 deletion mutants. It was shown that the ARS1 replication origin is not essential for mitotic or premeiotic DNA replication since no effect on growth, chromosomal loss rate and spore viability was observed in the ars1 mutant strains. We conclude that replication origins are abundantly, present in the yeast genome and that the removal of a single replication origin is compensated for by replication forks emanating from neighbouring origins.
Mating pheromone‐induced expression of the MAT1‐Pm gene of Schizosaccharomyces pombe: Identification of signalling components and characterization of upstream controlling elements
Authors: Aono Toshiya , Yanai Hiroyuki , Miki Futaba , Davey John , Shimoda Chikashi
Published: May 2005

Transcription of the mat1‐Pm gene of Schizosaccharomyces pombe controlling entry into meiosis is stimulated by the mating pheromone, M‐factor. We have studied its expression by monitoring β‐galactosidase activity in cells carrying a plasmid‐borne mat1‐Pm/lacZ fusion construct. Stimulation required the M‐factor receptor (Map3) and other proteins (Gpa1, Byr1, Byr2 and Spk1) thought to be involved in propagating the pheromone signal within the cell. Mutational activation of gpa1 encoding an α subunit of the receptor‐coupled heterotrimeric G protein causes full expression of mat1‐Pm even in the absence of pheromone, suggesting that Gpa1 is a key signal transmitter. Furthermore, an activated ras1val17 mutant exhibited a much stronger level of induction than wild‐type cells, though full expression needs M‐factor treatment. Deletion analysis of the mat1‐Pm promoter region identified a stretch of 21 bp that is shown to play a critical role in controlling expression. This region lies just upstream of a TATA‐like box and contains a TR‐box (TTCTTTGTTY) motif which is the recognition site of a putative transcription factor Ste11. Point mutations in the TR‐box motif abolished the expression of mat1‐Pm/lacZ. Almost no expression of mat1‐Pm was detected in a ste11 deletion mutant, whereas overproduction of Ste11 greatly increased the expression.
A fission yeast gene encoding a protein that preferentially associates with curved DNA
Authors: Yamada Hisami , Mori Hideki , Momoi Hiroyuki , Nakagawa Yoshiyuki , Ueguchi Chiharu , Mizuno Takeshi
Published: May 2005

We searched for fission yeast (Schizosaccharomyces pombe) proteins that preferentially bind to a synthetic curved DNA sequence, by means of a DNA‐binding gel shift assay in the presence of an excess amount of a non‐curved DNA sequence as a competitor. We identified such a protein in S. pombe. The protein, thus purified, has an apparent molecular weight of 42 000, as estimated by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis. It was suggested that this protein (42 K‐protein) recognizes and binds to a curved DNA structure in a given nucleotide sequence, although it also binds to a non‐curved DNA sequence with lower affinity. As its putative coding sequence, a 1·9‐kilobase genomic DNA from S. pombe was cloned and sequenced. Sequencing of a cDNA clone also revealed the existence of an open reading frame, with no intron, encoding a 381‐amino‐acid protein with a calculated molecular mass, 41 597. This protein appears to be located in the nucleus. The predicted protein sequence revealed that the 42 K‐protein exhibits no significant similarity to any other known proteins, except to a hypothetical protein of Caenorhabditis elegans.
II. Yeast sequencing reports. Sequence analysis of a 31 kb DNA fragment from the right arm of Saccharomyces cerevisiae chromosome II
Authors: Van Der Aart Quirina J. M. , Ydesteensma H. , Barthe Christophe , Doignon François , Aigle Michel , Crouzet Marc
Published: May 2005

The nucleotide sequence of a 31 352 bp fragment from chromosome II of Saccharomyces cerevisiae has been determined and analysed. The fragment originates from the right arm of chromosome II, located between the GAL7,10,1 and the PHO3,5 loci, at a distance of about 130 kb from the centromere. The sequence contains a tRNA tandem repeat and 17 open reading frames (ORFs) larger than 100 amino acids. One of them extends into adjacent DNA and is incomplete. The two tRNA genes, coding for a tRNAasp and a tRNAarg, and three of the ORFs, had been sequenced previously, i.e. HSP26, SEC18, and UBC4. Four other ORFs showed similarity with yeast genes; amino acid transporter genes, the RAD54, SNF2 and STH1 family, the SPS2 gene and the bromodomain of SPT7, respectively. Two showed homology with sequences from other organisms, i.e. with a Plasmodium falciparum gene encoding a surface antigen and with a gene from Saimirine herpes virus respectively. Three ORFs, YBR0726, YBR0735 and YBR0740 are completely contained in YBR0727, YBR0734 and YBR0739 respectively, and thus probably do not represent real genes. Two ORFs, YBR0727 and YBR0745 most likely contain an intron. The sequences have been deposited in the EMBL data library under Accession Number X76294.
Glucose metabolism and ethanol production in adh multiple and null mutants of Kluyveromyces lactis
Authors: Saliola Michele , Bellardi Silvia , Marta Isabella , Falcone Claudio
Published: May 2005

Four genes coding for alcohol dehydrogenase (ADH) activities were identified in Kluyveromyces lactis. Due to the presence in this yeast of multiple ADH isozymes, mutants in the individual genes constructed by gene replacement yielded no clear phenotype. We crossed these mutants and developed a screening procedure which allowed us to identify strains lacking several ADH activities. The analysis of the adh triple mutants revealed that each activity confers to the cell the ability to grow on ethanol as the sole carbon source. On the contrary, adh null strains failed to grow on this substrate, indicating that no other important ADH activities are present in K. lactis cells. In the adh null mutants we also found a residual production of ethanol, as has been reported to be the case in Saccharomyces cerevisiae. This production showed a ten‐fold increase when the K1ADHI activity was reintroduced in the null mutant and cells were cultivated under oxygen‐limiting conditions. Differently from S. cerevisiae, glycerol is poorly accumulated in K. lactis adh null mutants.
Molecular characterization of a gene that confers 2‐deoxyglucose resistance in yeast
Authors: Sanz Pascual , Randez‐Gil Francisca , Prieto José Antonio
Published: May 2005

We have isolated a gene whose expression enables yeast cells to overcome the inhibition of growth produced by the presence of 2‐deoxyglucose. The gene contains an open reading frame of 738 bp that may code for a protein of 27 100 Da. Cells carrying this gene contain high levels of a specific 2‐deoxyglucose‐6‐phosphate phosphatase. The expression of this phosphatase is increased by the presence of 2‐deoxyglucose and is constant along the growth curve. The sequence reported here has the GenBank accession number U03107.
Expression and in vivo determination of firefly luciferase as gene reporter in Saccharomyces cerevisiae
Authors: Vieites J. M. , Navarro‐García F. , Pérez‐Díaz R. , Pla J. , Nombela C.
Published: May 2005

The LUC gene coding for Photinus pyralis firefly luciferase was cloned in different yeast episomal plasmids in order to assess its possibilities as an in vivo reporter gene. Activity of the enzyme in transformed cells in vivo was measured by following light emission and assay conditions optimized in intact cells, with regard to oxygen concentration, temperature, cell concentration in assay mixtures and external ATP concentration. Among the factors tested, light emission was drastically influenced by the external pH in the assay (which resulted in a ten‐fold amplification signal) and by substrate permeability. The growth phase of the cells was also important for the level of activity detected. Cloning of firefly luciferase gene under the control of different yeast‐regulated promoters (ADH1, GAL1–10) enabled us to measure their strength which correlated well with previously described data. We conclude that firefly luciferase is an adequate gene reporter for the in vivo sensitive determination of gene expression and promoter strength in yeast.
The TYE7 gene of Saccharomyces cerevisiae encodes a putative bHLH‐LZ transcription factor required for Tyl‐mediated gene expression
Authors: Löhning Corinna , Ciriacy Michael
Published: May 2005

In Saccharomyces cerevisiae, expression of a gene adjacent to the retrotransposon Ty1 is often mediated by Ty‐internal sequences. We have identified novel mutants, tye7, which are affected in Ty1‐mediated expression of ADH2 through a Ty1 sequence distal to the 5′ long terminal repeat sequence. The TYE7 gene has been isolated and characterized. It encodes a 33 kDa protein whose N‐terminal third is extremely rich in serine residues (28%). Within its C‐terminal sequence, a remarkable similarity to Myc and Max proteins can be found. Thus, TYE7 is a potential member of the basic region/helix‐loop‐helix/leucine‐zipper protein family. TYE7 function is not essential for growth. It may primarily function as a transcriptional activator in Ty1‐mediated gene expression, as has been confirmed by the activation of reporter gene expression by a LexA‐TYE7 hybrid protein. ADH2 activation by defined Ty1 derivatives revealed that TYE7 acts positively through the more distal Ty1 enhancer element (region D), and negatively in a region between A (the 5′ proximal enhancer element) and D.
A novel structural form of the 2 micron plasmid of the yeast Saccharomyces cerevisiae
Authors: Petes Thomas D. , Williamson D. H.
Published: May 2005

DNA was isolated from cells of Saccharomyces cerevisiae incubated under conditions that enriched for DNA replication intermediates. A novel form of the 2 μm plasmid was detected, in which two monomeric or dimeric circles were joined by a linear double‐stranded segment of variable length. We suggest that this molecule is a consequence of site‐specific recombination within a dimeric DNA molecule during DNA replication. The existence of this molecule provides supporting physical evidence for a variant of the model of 2 μ plasmid amplification first proposed by Futcher (1986).
Assessment of pheromone production and response in fission yeast by a halo test of induced sporulation
Authors: Egel Richard , Willer Martin , Kjærulff Søren , Davey John , Nielsen Olaf
Published: May 2005

We describe a rapid, sensitive and semi‐quantitative plate assay for monitoring pheromone activity in the fission yeast Schizosaccharomyces pombe. It is based on the observation that meiosis requires stimulation by pheromone and exploits diploid strains that will only sporulate after addition of exogenous pheromone. The tester strains are heterozygous for mating type, are non‐switching, and are mutated in one of the early subfunctions (either mat1‐Mc or mat1‐Pc), so that meiosis is only induced after exposure to exogenous pheromone (M‐factor or P‐factor, respectively). Pheromone activity is assessed as an iodine‐positive halo of sporulation surrounding the pheromone source, and the width of the halo is related to the amount of pheromone being produced. The assay is sufficiently sensitive to monitor the low amount of M‐factor produced by an M mam1 strain, and its sensitivity towards P‐factor is greatly increased by using a hyper‐sensitive tester strain lacking the Sxa2 protease that is believed to degrade this pheromone. We also demonstrate that the production of P‐factor is very much stimulated by exposure of P cells to M‐factor.
Transcript map of two regions from chromosome XI of Saccharomyces cerevisiae for interpretation of systematic sequencing results
Authors: Fairhead Cécile , Dujon Bernard
Published: May 2005

A detailed and systematic transcript map is a first and necessary step to characterize new genes revealed by systematic sequencing. Chromosome XI of Saccharomyces cerevisiae contains 331 open reading frames (ORFs) of which 44% are of unknown function (Dujon et al., 1994). As a first study towards complete transcript analysis of chromosome XI, we have extracted RNA from three isogenic strains (a, α and 2 n) grown in three standard laboratory media, and have analysed them using contiguous probes covering two regions of 17 and 19 kilobases, respectively. All 20 predicted ORFs in the sequences correspond to expressed genes, six of which have no predicted function. Four short ORFs which were suspected as not being real genes on the basis of their sequence are not expressed in our growth conditions. An additional transcript which does not correspond to a large ORF was found. Steady‐state RNA level of most ORFs is 10 to 100 times than that of the actin gene, only three are transcribed in comparable amounts. Three ORFs show variable levels of transcripts in the different growth conditions, all patterns being different from one another. Extrapolation of these results to systematic transcript analysis of chromosome XI and other yeast chromosomes is presented.
Generation of glycerophospholipid molecular species in the yeast Saccharomyces cerevisiae. Fatty acid pattern of phospholipid classes and selective acyl turnover at sn‐1 and sn‐2 positions
Authors: Wagner S. , Paltauf F.
Published: May 2005

Acyl chains linked to phospholipids of the yeast, Saccharomyces cerevisiae, are mainly C16:1 and C18:1 accompanied by minor amounts of C14:0, C16:0 and C18:0. In view of this rather simple fatty acid composition, the question arose whether in yeast, as in higher eukaryotes, fatty acyl groups were characteristically distributed among the sn‐1 and sn‐2 positions of distinct phospholipid classes. Analysis of fatty acids linked to the sn‐1 and sn‐2 positions of the major phospholipids showed that indeed saturated fatty acyl groups predominated in the sn‐1 positions. While the percentage of saturated fatty acids was low (10%) in phosphatidylcholine (PtdCho) and phosphatidylethanolamine (PtdEtn) from cells grown on rich medium, it was higher in phosphatidylserine (PtdSer) (25%) and highest in phosphatidylinositol (PtdIns) (41%). Oleate was mainly linked to position sn‐2, while palmitoleate predominated in position sn‐1. Striking differences in the fatty acid distribution of phospholipids that are metabolically closely related (e.g. PtdSer and PtdEtn, PtdEtn and PtdCho, and PtdIns and PtdSer) suggest that pathways must exist for the generation of distinct phospholipid molecular species within the different phospholipid classes. The highly selective incorporation of exogenous [14C]palmitic acid (90%) and [3H]oleic acid (99%) into the sn‐2 position of PtdCho, and the preferential incorporation of these fatty acids into the sn‐2 position of PtdEtn (70 and 90%, respectively, for palmitic and oleic acid) are compatible with the postulate that phospholipase A2‐mediated deacylation followed by reacylation of the lysophospholipids is involved in the generation of phospholipid species in yeast.
Genome renewal: A new phenomenon revealed from a genetic study of 43 strains of Saccharomyces cerevisiae derived from natural fermentation of grape musts
Authors: Mortimer Robert K. , Romano Patrizia , Suzzi Giovanna , Polsinelli Mario
Published: May 2005

We have analyzed by genetic means 43 strains of Saccharomyces that had been isolated from fermenting grape musts in Italy. Twenty eight of these strains were isolated from 28 cellars in the Region of Emilia Romagna. The other 15 strains came from 5 fermentations at four cellars near the city of Arpino, which is located south and east of Rome.

We found that 20 of the 28 strains from Emilia Romagna were heterozygous at from one to seven loci. The balance were, within the limits of our detection, completely homozygous. All these strains appeared to be diploid and most were homozygous for the homothallism gene (HO/HO). Spore viability varied greatly between the different strains and showed an inverse relation with the degree of heterozygosity.

Several of the strains, and in particular those from Arpino, yielded asci that came from genetically different cells. These different cells could be interpreted to have arisen from a heterozygote that had sporulated and, because of the HO gene, yielded homozygous diploid spore clones. We propose that natural wine yeast strains can undergo such changes and thereby change a multiple heterozygote into completely homozygous diploids, some of which may replace the original heterozygous diploid. We call this process ‘genome renewal’.
The nucleotide sequence and initial characterization of pyruvate decarboxylase from the yeast Hanseniaspora uvarum
Authors: Holloway Paul , Subden Ronald E.
Published: May 2005

We have isolated a pyruvate decarboxylase (PDC) gene from the yeast Hanseniaspora uvarum using the Saccharomyces cerevisiae PDC1 gene as a probe. The nucleotide sequence of this gene was determined and compared to PDC genes from yeast and other organisms. The H. uvarum PDC gene is more than 70% identical to the S. cerevisiae PDC isozymes and possesses a putative thiamine diphosphate binding site. The PDC enzyme was purified and partially characterized. The H. uvarum PDC was very similar to other known PDCs; the Km for pyruvate was 0·75 mM, and the enzyme is a homotetramer with subunits of Mr = 57 000. The sequence has been submitted to GenBank under Accession No. U13635.
Transcriptional regulation of the Saccharomyces cerevisiae HXK1, HXK2 and GLK1 genes
Authors: Herrero P. , Galíndez J. , Ruiz N. , Martínez‐Campa C. , Moreno F.
Published: May 2005

In Saccharomyces cerevisiae, the transcriptional regulation of most glycolytic genes has been extensively studied. By contrast, little is known about the transcriptional control of the three glucose‐phosphorylating enzymes, although this catalytic reaction has an important role in the regulation of cell metabolism. In this paper, we describe the transcriptional regulation of the HXK1, HXK2 and GLK1 genes in the hope of revealing differences in the steady‐state levels of mRNA associated with a particular carbon source used in the culture medium. Our results provide evidence supporting a differential expression of the three genes depending on the carbon source used for growth. We have also studied the induction and repression kinetics of mRNA expression for the HXK1, HXK2 and GLK1 genes.
Molecular cloning of the plc1+ gene of Schizosaccharomyces pombe, which encodes a putative phosphoinositide‐specific phospholipase C
Authors: Andoh Tomoko , Yoko‐O Takehiko , Matsui Yasushi , Toh‐E Akio
Published: May 2005

Exploiting the polymerase chain reaction, we have isolated a gene that encodes a putative phosphoinositide‐specific phospholipase C (PLC) of the fission yeast Schizosaccharomyces pombe. Inspection of the nucleotide sequence of the gene revealed an open reading frame that can encode a polypeptide of 899 amino acid residues with a calculated molecular mass of 102 kDa. This putative polypeptide contains both the X and Y regions that are conserved among three classes of mammalian PLC, and also contains a presumptive Ca2+‐binding site (an E‐F hand motif). The structure of the putative protein is most similar to that of the δ class of PLC isozymes. To investigate the role of this gene, designated plc1+, gene disruption was carried out by interrupting the coding region with the ura4+ marker. Growth of plc1 cells was temperature‐sensitive in rich medium, and cells could not grow in synthetic medium. Expression of the PLC1 gene of Saccharomyces cerevisiae suppressed the growth defect phenotype of plc1− cells, a strong suggestion that the plc1+ gene encodes PLC. The PLC1 sequence appears in the public data libraries, DDBJ GenBank, EMBL under the following Accession Number: D38309.
Physical map locations of the phospholipid biosynthetic structural and regulatory genes of Saccharomyces cerevisiae
Authors: Anderson Melanie S. , Kanipes Margaret I. , Jackson John C. , Yates Jonathan , Henry Susan A. , Lopes John M.
Published: May 2005

Here we report the physical map locations of five genes required for phospholipid biosynthesis in Saccharomyces cerevisiae. These include four structural genes (INO1, CHO2, OP13 and PIS1) and one global negative regulatory gene (UME6). Collectively, this information completes the mapping of all phospholipid biosynthetic structural and regulatory genes identified to date.
Cloning and sequencing of the URA5 gene from the yeast Yarrowia lipolytica
Authors: Sánchez Manuel , Prado Marciano , Iglesias Francisco J. , Domínguez Angel
Published: May 2005

The URA5 gene of Yarrowia lipolytica encoding the orotate phosphoribosyl transferase (OPRTase, EC2.4.2.10) was isolated by target integration in a mutant strain originally named ura2.21. The nucleotide sequence of the gene predicts a protein with high similarities with the OPRTases from Saccharomyces cerevisiae, Podospora anserina and Escherichia coli and to a lesser extent with that of Dictyostelium discoideum. The transcription start point has been mapped by primer extension analysis and indicates the existence of a long leader sequence in the corresponding mRNA. Northern‐blot hybridization revealed the URA5 transcript to be approximately 0·94 kb. Deletion of the URA5 gene in Y. lipolytica produced a leaky phenotype similar to the one described for the ura5 mutation in S. cerevisiae. The URA5 gene of Y. lipolytica was able to complement functionally the ura5 mutation of S. cerevisiae. The sequence presented here has been submitted to the EMBL data library under Accession Number Z22571.
Transcriptional regulation of flocculation genes inSaccharomyces cerevisiae
Authors: Teunissen Aloys W. R. H. , Van Den Berg Johan A. , Yde Steensma H.
Published: May 2005

Northern analysis showed that DNA from the flocculation geneFLO1hybridized to mRNA molecules of 4·8 kb. This transcript was specific for theFLO1gene at the right end of chromosome I since disruption of this gene resulted in the disappearance of the transcript. We further found an absolute correlation between flocculation and the presence of transcripts hybridizing toFLO1DNA, both in various flocculent and non‐flocculent strains and in cells from the non‐flocculating and flocculating stages of growth. In all cases transcripts were present in flocculating and absent from non‐flocculating cultures. From these results we conclude that theFLO1gene is transcriptionally regulated.

Mutations inTUP1orSSN6cause flocculation. Several transcripts hybridizing toFLO1DNA were present in the mutants but not in the corresponding wild‐type strains. Disruption of theFLO1gene in thetup1andssn6strains showed that one of the transcripts corresponded to theFLO1gene. Disruption ofFLO1did not abolish flocculation completely but only reduced it, indicating that at least two flocculation genes, includingFLO1, are activated or derepressed by mutations in theTUP1/SSN6regulatory cascade.
Nucleotide sequence and chromosomal localization of the gene encoding the old yellow enzyme from Kluyveromyces lactis
Authors: Miranda Manuel , Ramírez Jorge , Guevara Soledad , Ongay‐Larios Laura , Peña Antonio , Coria Roberto
Published: May 2005

A 6·6 kb genomic DNA fragment from the yeast Kluyveromyces lactis was isolated. Sequence analysis of this fragment revealed the presence of two incomplete open reading frames (ORFs) in one strand, one coding for the carboxyl terminus of the plasma membrane H+‐ATPase and the other for the amino terminus of an unidentified product. In the complementary strand, a full‐length ORF which encodes for a protein homologous to the yeast NADPH‐dependent Old Yellow Enzyme was found. The deduced amino acid sequence of this ORF predicts a protein of 398 residues with 84% similarity in its full length to OYE1 from Saccharomyces carlsbergensis and OYE2 from Saccharomyces cerevisiae. In addition, an internal region showed considerable similarity to the bile acid‐inducible polypeptide from Eubacterium sp., to the NADH oxidase from Thermoanaerobium brockii, to the trimethylamino dehydrogenase from bacterium W3A1 and to the estrogen‐binding protein from Candida albicans, suggesting a functional or structural relationship between them. Inactivation of the KYE1 (Kluyveromyces Yellow Enzyme) gene by deletion of 0·6 kb fragment between positions +358 and +936 produced viable cells with a slight increase in their generation time. Haploid cells carrying the disrupted allele showed one‐third of the NADPH oxidase activity, compared to wild‐type cells. Southern blotting analysis of digested DNA and chromosomes separated by contour‐clamped homogeneous electric field electrophoresis from K. lactis indicated that this is a single‐copy gene and it is localized on chromosome II, whose molecular size has been estimated to be approximately 1·3 Mb. The sequence reported in this paper has been deposited in the GenBank data base (Accession No. L37452).
The DNA sequence of a 7941 bp fragment of the left arm of chromosome VII of Saccharomyces cerevisiae contains four open reading frames including the multicopy suppressor gene of the pop2 mutation and a putative serine/threonine protein kinase gene
Authors: Coglievina Maristella , Bertani Iris , Klima Raffaella , Zaccaria Paolo , Bruschi Carlo Vito
Published: May 2005

We report the sequence of a 7941 bp DNA fragment from the left arm of chromosome VII of Saccharomyces cerevisiae which contains four open reading frames (ORFs) of greater than 100 amino acid residues. ORF biC834 shows 100% bp identity with the recently identified multicopy suppressor gene of the pop2 mutation (MPT5); its deduced protein product carries an eight‐repeat domain region, homologous to that found in the hypothetical regulatory YGL023 protein of S. cerevisiae and the Pumilio protein of Drosophila. ORF biE560 protein exhibits patterns typical of serine/threonine protein kinases, with which it shares high degrees of homology. The complete nucleotide sequence was submitted to the EMBL Data Library under Accession Number X83690.