bluuebunny/crossref_metadata_2025_split huggingface dataset:
Killer system of Kluyveromyces lactis: The open reading frame 10 of the pGK12 plasmid encodes a putative DNA binding protein
Authors: Tommasino Massimo 
Published: May 2005

AbstractORF 10 of the K2 plasmid from Kluyveromyces lactis encodes a small basic protein (22·3% lysine). The function of its product has been investigated. Western blot analysis, using an antibody against MS2 RNA polymeras/ORF 10 fusion protein, reveals a protein band with an apparent molecular weight of 14 kDa. The protein can bind a DNA‐Sepharose column, and is eluted by 350 mM‐salt. Immunoprecipitation experiments show that the ORF 10 protein coprecipitates with the linear genomic DNAs of the two killer plasmids (K1 and K2). From Western/Southern blot data, it is possible to conclude that the interaction between protein and DNA occurs directly, rather than via other proteins(s). ORF 10 is easily detected by Western blot and its transcript is one of the most abundant of the K2 plasmid, suggesting that this protein may have a structural rather than a regulatory function. This possibility is also suggested by the observed sequeence homology between the ORF 10 protein and the family of histone‐like proteins.
CDC15, an essential cell cycle gene in Saccharomyces cerevisiae, encodes a protein kinased domain
Authors: Schweitzer Bert , Philippsen Peter 
Published: May 2005

AbstractThe cell division cycle gene CDC 15 is essential for the late nucler division in the yeast Saccharomyces cerevisiae. The amino acid sequence of the 974 amino acids/110 kDa CDC 15 gene product, as deduced from the nucletide sequence, includes an aminoterminal protein kinase domain which contains a primary sequence mosaic showing patterns specific for protein serine/theonine kinases besides those for protein tyrosine kinases. Many protein kinases non‐essential for growth are known. CDC 15 represents an essential protein kinase like CDC 7 and CDC 28. A carboxyterminal deletion of 32 amino acids renders the protein inactive.
Optimization of Bacillus α‐amylase production by Saccharomyces cerevisiae
Authors: Ruohonen L. , Penttilä M. , Keränen S. 
Published: May 2005

AbstractProduction of Bacillus amyloliquefaciens α‐amylase by Saccharomyces cerevisiae using the multicopy plasmid pAAH5 and ways of improving the yields of secreted enzyme were studied. In standard non‐buffered medium, α‐amylase was rapidly inactivated but stabilization of the pH at 6 led to stable accumulation of α‐amylase in the culture medium. Removal of 1100 bp of the upstream sequence of the ADH1 promoter present on pAAH5 resulted in delayed but increased α‐amylase production: 29‐fold in selective medium, two‐fold in non‐selective medium. With the original ADH1 promoter, accumulation of α‐amylase in the medium started to level off before the cultures reached stationary phase and was very low when exponentially growing cells were transferred from glucose to ethanol. This coincided with the appearance of a mRNA larger than the α‐amylase messenger. With the shortened promoter, the normal‐size α‐amylase mRNA was detected under all growth conditions and α‐amylase was efficiently secreted into the medium also late in stationary phase and after transfer to ethanol. Highest total yields of α‐amylase were obtained with the short promoter in non‐selective glucose‐containing medium; this may be explained by the greater final cell density obtained. However, the production of α‐amylase per cell mass was higher in ethanol‐containing selective medium. Seventy to eighty per cent of the α‐amylase activity was secreted into the medium independent of the total amount produced.
Induction of a heat‐shock‐type response in Saccharomyces cerevisiae following glucose limitation
Authors: Bataillé Nelly , Régnacq Matthieu , Boucherie Hélian 
Published: May 2005

AbstractThe protein pattern of yeast cells which have arrested proliferation in response to glucose exhaustion is drastically different from that of exponentially growing cells (Boucherie, 1985). In this study, we used two‐dimensional gel electrophoresis to characterize the protein events responsible for these alterations. We found that the induction of heat‐shock proteins is one of the major events responsible for these changes. This induction accounts for the synthesis of 18 of the 35 novel polypeptides observed in glucose‐limited cells. It was shown to occur in combination with two other protein events: the derepression of carbon catabolite repressed proteins, which accounts for the synthesis of the other novel polypeptides, and an arrest of the synthesis of almost all the proteins present in exponentially growing cells.The time course of each of these events was determined by carrying out a detailed analysis of the pattern of proteins synthesized at vaious stages of a culture exhausting its glucose supply, and by the measurement of the rate of synthesis of individual polypeptides. The results showed in particular that the synthesis of most of the heat‐shock proteins synthesized in glucose‐limited cells was induced closely before glucose exhaustion, and that this synthesis was transient, climaxing by the time glucose was exhausted. Under the culture condition investigated, the entry into stationary phase associated with glucose limitation began several hours before glucose exhaustion. It was thus concluded that the observed induction of heat‐shock proteins is directly related to the nutritional limitation and is independent from the arrest of cell proliferation.
The YDp plasmids: A uniform set of vectors bearing versatile gene disruption cassettes for Saccharomyces cerevisiae
Authors: Berben Gilbert , Dumont Jacques , Gilliquet Véronique , Bolle Paul‐André , Hilger François 
Published: May 2005

AbstractThe YDp plasmids (Yeast Disruption plasmids) are pUC9 vectors bearing a set of yeast gene disruption cassettes, all uniform in structure and differing only in the selectable marker used (HIS3, LEU2, LYS2, TRP1 or URA3). The markers, surrounded by translational termination codons, are embedded in the slightly modified sequence of the pUC9 multiple cloning sites.
The determinants of heat‐shock element‐directed lacZ expression in Saccharomyces cerevisiae
Authors: Kirk Niall , Piper Peter W. 
Published: May 2005

AbstractHeat‐shock induction of heat‐shock protein genes is due to a specific promoter element (the heat‐shock element, HSE). This study used lacZ under HSE control (HSE‐lacZ) to characterize HSE activity in Saccharomyces cerevisiae cells of different physiological states and differing genetic backgrounds.In batch fermentations HSE‐lacZ induction by heat shock was maximal in exponential growth, and showed marked decline with the approach to stationary phase. Expression in the absence of heat shock was unaffected by growth phase, indicating that the growth‐dependent expression of many yeast heat‐shock genes uses promoter elements in addition to the HSE. Heat‐induced expression was strongly influenced by the temperature at which cultures were grown. While basal, uninduced expression was constant during growth at different temperatures to 30°C, induction by transfer to 39°C was reduced by increases in growth temperature as low as 18–24°C. Maximal HSE‐lacZ induction (30‐ to 50‐fold) was in cultures grown at low temperatures (18–24°C), then heat shocked at 39°C. Ethanol was a poor inducer. Mutations having little effect on HSE‐lacZ expression included a respiratory petite; ubi4 (which inactivates the polyubiquitin gene); also ubc4 and ubc5 (which each inactivate one of the ubiquitin ligases involved in degradation of aberrant protein). pep4‐3 increased both basal and induced β‐galactosidase about two‐fold, probably because of slower turnover of this enzyme in pep4‐3 strains.
The control of trehalose biosynthesis in Saccharomyces cerevisiae: Evidence for a catabolite inactivation and repression of trehalose‐6‐phosphate synthase and trehalose‐6‐phosphate phosphatase
Authors: François Jean , Neves Maria‐José , Hers Henri‐Géry 
Published: May 2005

AbstractDuring diauxic growth of yeast in glucose‐rich medium, the accumulation of trehalose started well after complete exhaustion of glucose from the medium. The accumulation of the disaccharide was concomitant with a resumption of cell growth on the ethanol accumulated in the medium, but not with a degrdation of glycogen which occurred as soon as glucose had been consumed. In contrast, in a mutant deficient in phosphoenlpyruvate carboxykinase, the synthesis of trehalose coincided exactly with the degradation of glycogen. Upon inoculation of stationary phase wild‐type cells into a glucose medium, the activites of trehalose‐6‐phosphate (Tre6P) synthase and Tre6P phosphatase dropped in parallel to reach only 15% of their initial values after 3 h, and only recovered their original values as cell re‐entered staionary phase. In the presence of cycloheximide, the decrease in Tre6P synthase and Tre6P phosphatase activities was restricted to 50–60%, the remaining decrease being inhibited by the drug. Furthermore, the reappearance of the enzyme activities following transfer of cells to an acetate medium was blocked by cycloheximide. It was also shown that loss of activity of these two enzymes required a combination of metabolizable sugars together with a nitrogen source. Low activities of Tre6P synthase and Tre6P phosphatase were measured in mutants with increased adenylate cyclase activity (RAS2ala18val19 mutants). Moreover, derepression of these enzymes at the approach of stationary phase was prevented in a pde2 mutant when it was cultivated in the presence of exogenous cyclic nucleotide. The mechanism of this effect is not clear, but may involve a transcriptional regulation by cAMP of the genes encoding these proteins.
The use of proline as a nitrogen source causes hypersensitivity to, and allows more economical use of 5FOA in Saccharomyces cerevisiae
Authors: McCusker John H. , Davis Ronald W. 
Published: May 2005

AbstractThe use of proline as a nitrogen soucre causes hypersensitivity to 5‐fluoro‐orotic acid (5FOA) and allows up to 40‐fold less of this drug to be used to select for the loss of URA3 function in Saccharomyces cerevisiae. 5FOA hypersensitivity is presumably due to the absence of nitrogen catabolite repression when proline is substituted for (NH4)2SO4 as a nitrogen source. There are two constraints to the use of the proline–5FOA combination: (1) S288c genetic background strains are hypersensitive to 5FOA when grown in proline as a nitrogen source but at least one other genetic background is resistant to low levels of 5FOA under these conditions. (2) The addition of some nutritional supplements confers phenotypic resistance to the 5FOA–proline combination.
A family of low and high copy replicative, integrative and single‐stranded S. cerevisiae/E. coli shuttle vectors
Authors: Bonneaud Nathalie , Ozier‐Kalogeropoulos Odile , Li Guoya , Labouesse Michel , Minvielle‐Sebastia Lionel , Lacroute Francois 
Published: May 2005

AbstractWe describe a set of replicative, integrative and single‐stranded shuttle vectors constructed from the pUC19 plasmid that we use routinely in our experiments. They bear a yeast selectable marker: URA3, TRP1 or LEU2. Replicative vectors carrying different yeast replication origins have been constructed in order to have plasmids based on the same construction with a high or low copy number per cell and with different mitotic stabilities. All the vectors are small in size, provide a high yield in Escherichia coli and efficiently transform Saccharomyces cerevisiae. These plasmids have many of the unique sites of the pUC19 multicloning region and many of them allow for the screening of plasmids with an insert by alpha‐complementation. The nucleotide sequence of each of them is completely known.
The sequence of 8·8 kb of yeast chromosome III cloned in lambda PM3270 contains an unusual long ORF (YCR601)
Authors: Rodriguez Francesco , Martegani Enzo , Mauri Isabella , Alberghina Lilia 
Published: May 2005

AbstractWe have determined the nucleotide sequence of a segment of chromosome III contained in the right part of the lambda PM3270 clone, for a total of 8824 bp. This sequence contains an unusual long open reading frame, YCR601, of 6501 bp that encodes for a protein of 2167 amino acids that show no homology with other known proteins. YCR601 was disrupted by internal deletion and insertion of LEU2 gene and is a non‐essential gene, however it is transcribed during vegetative growth yielding a polyadenylated mRNA of approximately 7 kb.
Yeast mutant affected for viability upon nutrient starvation: Characterization and cloning of the RVS161 gene
Authors: Crouzet Marc , Urdaci Maria , Dulau Laurent , Aigle Michel 
Published: May 2005

AbstractIn yeast, nutrient starvation leads to entry into stationary phase. Mutants that do not respond properly to starvation conditons have been isolated in Saccharomyces cerevisiae. Among them the rvs161 mutant (RVS for Reduced Viability upon Starvation) is sensitive to carbon, nitrogen and sulphur starvation. When these nutrients are depleted in the medium, mutant cells show cellular viability loss with morphological changes. The mutation rvs161‐1 is very pleiotropic, and besides the defects in stationary phase entry, the mutant strain presents other alterations: sensitivity to high salt concentrations, hypersensitivity to amino acid analogs, no growth on lactate or acetate medium. The addition of salts or amino acid analogs leads to the same morphological defects observed in starved cells, suggesting that the gene could be implicated mainly in the control of cellular viability. The gene RVS161 was cloned; it codes for a 30,252 daltons protein. No homology was detected with the proteins contained in the databases. Moreover, Southern analysis revealed the presence of other sequences homologous to the RVS161 gene in the yeast genome.
Four major transcriptional responses in the methionine/threonine biosynthetic pathway of Saccharomyces cerevisiae
Authors: Mountain Harry A. , Byström Anders S. , Larsen Jörgen Tang , Korch Christopher 
Published: May 2005

AbstractGenes encoding enzymes in the threonin/methionine biosynthetic pathwa were cloned and used to investigate their transcriptional response to signals known to affect gene expression on the basis of enzyme specific‐activities. Four major responses were evident: strong repression by methionine of MET3, MET5 and MET14, as previously described for MET3, MET2 and MET25; weak repression by methionine of MET6; weak stimulation by methionine but no response to threonine was seen for THR1, HOM2 and HOM3; no response to any of the signals tested, for HOM6 and MES1. In a BOR3 mutant, THR1, HOM2 and HOM3 mRNA levels were increased slightly. The stimulation of transcription by methionine for HOM2, HOM3 and THR1 is mediated by the GCN4 gene product and hence these genes are under the general amino acid control. In addition to the strong repression by methionine, MET5 is also regulated by the general control.
Electroporation‐stimulated recombination in yeast
Authors: Higgins David R. , Strathern Jeffrey N. 
Published: May 2005

AbstractSaccharomyces cerevisiae cells treated by high voltage and made transformation‐competent (electroporation) are also made hyper‐recombinational as determined by an assay that measures interchromosomal mitotic recombination between chromosome III homologs, each containing mutant heteroallelic copies of the trp1 and his3 genes. There is a 10‐fold stimulation of Trp+ and 21‐fold stimulation of His+ prototrophs. Although this stimulation coincides with conditions for maximal transformation competence it is independent of the presence of transforming plasmid DNA. Electroporation does not increase the reversion frequency of these mutations, nor is there a stimulation in Ty transposition. Among the electroporation‐stimulated Trp+ and His+ recombinants there is no dramatic difference in the pattern of events: that is to say that, while there is an increase in the number of recombinants, the distribution of gene conversion and cross–over events among the stimulated recombinants is not significantly altered compared to spontaneously arising Trp+ and His+ recombinants. This electroporation‐stimulated recombination is abolished in an isogenic rad52 mutant strain consistent with the increase in Trp+ and His+ prototrophs being the result of a stimulation of a RAD52‐dependent recombination pathway.
The cysteine transport system of Saccharomyces cerevisiae
Authors: Ono Bun‐Ichiro , Naito Kazuhide 
Published: May 2005

AbstractAlthough Saccharomyces cerevisiae strains had different cysteine uptake activities, they revealed monophasic uptake kinetics and had the same KT(83·3 μM). The optimal pH of cysteine uptake was between 4·5 and 5·0, but the activity was quickly lost if cells were kept in buffer. When the activity was measured in the growth medium, it increased in the presence of EDTA and greatly decreased in the presence of mercuric chloride. Thioglycol as well as metabolic inhibitors such as dinitropherol and azide were inhibitory. Homocysteine and methionine were competitive and non‐competitive inhibitors, respectively. Cysteamine and cysteic acid were not inhibitory. From these observations, we conclude that the system mediating uptake of cysteine is specific (we thus name it the cysteine transport system) and that the cysteine transport system recognizes not only the SH‐group but also amino‐ and carboxyl‐groups.In wild‐type strains the cysteine transport system was derepressed only when the cells were incubated without any sulfur source. On the other hand, in cysteine‐dependent mutants, cysteine uptake activity increased with increase of exogenous supply of cysteine, glutathione or methionine. From this result, we suspect that the cellular cysteine level is the limiting factor for biosynthesis of the cysteine transport system in cysteine‐dependent strains.
Participation of proteinase yscA in the in vitro formation of the smaller subunit of glycogen phosphorylase in extracts of Saccharomyces cerevisiae
Authors: Ziegler Karl‐Josef , Schwartz Arnold C. 
Published: May 2005

AbstractAnalyses by sodium dodecyl sulphate–polyacrylamide gradient‐gel electrophoresis and Western blotting of the proteins from boiled cell samples from all growth phases of yeast cultures, and from all stages of extract preparation indicate that the smaller subunit (s‐monomer), which is found in purified glycogen phosphorylase (EC 2.4.1.1) from baker's yeast, is not present in the living cell. It is observed in extracts of Saccharomyces carlsbergensis and S. cerevisiae after incubation at ambient temperatures or even after storage in the frozen state at −25°C. Its formation is sensitive towards pepstatin A, and it is absent from extracts of several mutants of S. cerevisiae that do not contain active proteinase yscA (EC 3.4.33.6). When purified proteinase yscA‐deficient strains are grown with a reduced amount of complex nitrogen compounds, the slightly smaller sc‐monomer is formed in their extracts. This event must be attributed to a different proteinase, since it is sensitive towards p‐hydroxymercuriphenylsulphonate, but not towards pepstatin A. The N‐terminal amino acid of the sc‐monomer was found to be blocked, as in the case of the native 1‐monomer, but not the s‐monomer.
On the level of plasmid‐bearing cells in transformed cultures of Saccharomyces cerevisiae
Authors: Guerrini Anna Maria , Boglione Clara , Ascenzioni Fiorentina , Donini Pierluigi 
Published: May 2005

AbstractLC1, a YIP5‐derived plasmid containing a human DNA fragment with ARS activity in yeast, has been used to study the replication of ARS plasmids in Saccharomyces cerevisiae. ARS plasmids carried in yeast hosts are normally mitotically unstable. In transformed cultures the fraction of cells that contain plasmid, measured by plating on selective media, is lower than would be expected from measured rates of plasmid loss. In the case of S. cerevisiae carrying either the plasmid LC1 or YRP17, the assay yields values of the order of 10–20% or 30–50% respectively. We have found that by doing a double nutritional upshift that involves conditioned medium and casamino acids, a population of cells can be defined that carry plasmid but are unable to grow on media that select for the plasmid marker. Thus the total fraction of cells that can be shown to contain plasmid increases to greater than 70%. To distinguish between the inability of plasmid to replicate in these cells and lack of expression of the selectable gene, cultures grown from single cells were analysed for the presence of plasmid DNA. In a substantial fraction of the population, plasmid DNA could be detected only by polymerase chain reaction and not by standard blotting and hydribization. These results suggest that plasmid is unable to replicate in these cells. Growth kinetics experiments with transformed cultures are consistent with the notion that only a small fraction of the cells contains plasmid capable of replication upon dilution into selective medium. Possible explanations for the phenomena observed are discussed.
Cloning and disruption of the LEU2 gene of Kluyveromyces marxianus CBS 6556
Authors: Bergkamp Ronald J. M. , Geerse Ruud H. , Verbakel John M. A. , Musters Wouter , Planta Rudi J. 
Published: May 2005

AbstractThe LEU2 gene, coding for β‐isopropylmalate dehydrogenase, of the yeast Kluyveromyces marxianus was isolated and sequenced. An open reading frame, coding for a protein with a molecular weight of 38kDa was found. Comparison of the deduced amino acid sequence of the LEU2 gene with the corresponding enzymes of three other yeasts and two thermophilic bacteria, revealed extensive sequence similarities. The cloned gene could complement a leuB mutation of Escherichia coli and a leu2 mutation of Saccharomyces cerevisiae. Using orthogonal field alternation gel electrophoresis, the genomic copy of the gene was found to be located at chromosome VI or VII. Analysis of the 5′‐untranslated region indicated the presence of a putative binding site for the LEU3 protein, which is involved in the leucine‐specific regulation of transcription. We show that the cloned gene can be used for the construction of a non‐reverting K. marxianus leu2 mutant.
The nucleotide sequence of a third cyclophilin‐homologous gene from Saccharomyces cerevisiae
Authors: Franco L. , Jiménez A. , Demolder J. , Molemans F. , Fiers W. , Contreras R. 
Published: May 2005

AbstractThe nucleotide sequence of a 1558 bp DNA fragment from the right arm of chromosome III of Saccharomyces cerevisiae contains an open reading frame of 954 nucleotides with coding potential for a protein with high similarity to the ubiquitous cyclophilins which are both peptidyl‐prolyl cis‐trans isomerases and cyclosporin A‐binding proteins. It should, therefore, represent the third gene (SCC3) of this kind from S. cerevisiae. SCC3 is present in a single copy in the genome of S. cerevisiae and results in a constitutively expressed 1·2 kb transcript during cell growth. Its putative protein product (Scc3) contains two hydrophobic cores, one at the amino terminal, 20 amino acids long, which could serve as a signal peptide, and the other one at the carboxyl end with a structure similar to a transmembrane helix. These findings suggest that Scc3 could be a secretory or, more likely, a transmembrane protein. The only cyclophilin with similar structure to that of Scc3 is ninaA from Drosophila melanogaster, a transmembrane protein which seems to be implicated in the correct folding and/or intercalation of rhodopsin in the endoplasmic reticulum of the fly photoreceptors (Stamnes, M. A. et al., Cell 65, 219–227, 1991). In addition, the amino and the carboxy regions of Scc3 and ninaA share a significant level of homology, which suggests that they have a similar function, albeit for different target proteins.