bluuebunny/crossref_metadata_2025_split huggingface dataset:
Mutational analysis of Schizosaccharomyces pombe U4 snRNA by plasmid exchange
Authors: Dandekar Thomas , Tollervey David 
Published: May 2005

AbstractWe have developed a system for testing mutations by plasmid exchange in the fission Schizosaccharomyces pombe. This system has been used to test the requirement for different regions of the small nuclear RNA U4 in S. pombe. Surprisingly, five of seven deletion and substitution mutations tested in different regions of U4 prevent the accumulation of the mutant RNA. Substitution of the U4 sequence in stem 1 of the U4/U6 interaction doamain allows accumuation of the mutant U4, but does not supports viability. Two sequences with homology to the Sm binding site are found in the 3′ region of S. pombe U4; substitution of the 3′ sequence of the two does not interfere with accumulation or function of U4, indicating that the 5′ sequence is the functional Sm‐binding site.
The telomere‐associated MAL3 locus of Saccharomyces is a tandem array of repeateed genes
Authors: Michels Corinne A. , Read Evan , Nat Karyl , Charron Maureen J. 
Published: May 2005

AbstractSaccharomyces strains capable of fermenting maltose contain any one of five telomere‐associated MAL loci. Each MAL locus is a complex of three genes encoding the three functions required to ferment maltos: maltose permease (GENE 1), maltase (GENE 2) and the MAL trans‐activator (GENE 3). All five loci have been cloned and all are highly sequence homologous over least a 9‐0 kbp region containing these GENEs (Charron et al., Genetics 122, 307–331, 1989). Our initial studies of strians carrying the MAL3 locus indicated the presence of linked, repeated MLA‐homologous sequences (Michels and Needleman, Mol. Gen. Genet. 191, 225–230, 1983). Here we report our analysis of the centromere‐proximal MAL3‐linked sequence and show that the complete MAL3 locus spans approximately 40 kbp and consists of tandemly arrayed, partial repeats of the three GENE sequences described above. In addition, the structure of the MAL3 locus is compared to that of three partially functional alleles of MAL3. These alleles were shown to contain only MAL31 and MAL32 and their structure suggests that they resulted from MAL3 deletions removing the sequences centromere‐proximal of MAL31. The amplification and rerrangement of the telomer‐linked MAL3 sequences are discussed in the context of studies on other telemere‐associated sequences from yeast and other species.
MOL1, a Sacchromyces cerevisiae gene that is highly expressed in early stationary phase during growth on molasses
Authors: Praekelt U. M. , Meacock P. A. 
Published: May 2005

AbstractWe have isolated a new Saccharamyces cerevisiae gene, MOL1, that is transiently expressed at high levels in the early stationary phase of batch cultures growing on industrial molasses medium. The DNA sequence of the M O L I gene (for MOLasses‐inducible) with its flanking regions was determined (EMBL accession number X61669). It encodes a polypeptide of M, 35 kDa that is closely related to stress‐inducible proteins of similar size frow two Fusarium species. Unlike ST135 of Fusarium, MOL1 is not induced by ethanol or heat shock. MOL1 expression is absent in rich (YP) medium, and only very low levels of expression are detectable in minimal (YNB) medium. The gene is not essential, and a MOL1 distruption strain showed no apparent phenotype undera variety of growth conditions.The 5′ region of MOL1 contains the complete sequence previously determined for the SUF4 locus, encoding a tRNA‐gly (UCC) gene, which has been mapped to chromosome VII.
II. Yeast sequencing reports. Sequence of a 12·7 kb segment of yeast chromosome II identifies a PDR‐like gene and several new open reading frames
Authors: Delaveau TH. , Jacq C. , Perea J. 
Published: May 2005

AbstractA 12,684 bp DNA fragment, between FUS3 and the centromere, from the left arm of chromosome II of Saccharomyces cerevisiae was sequenced as part of the European project to sequence the whole chromosome. This segment contains at least five complete new open reading frames (ORFs) and the beginning (191 first 5′ codons) of an ORF whose putative translational product is highly similar to the multidrug resistance PDR1 gene previously characterized by Balzi et al. (1987) on chromosome VII.
Schizosaccharomyces pombe mitochondria: Morphological, respiratory and protein import characteristics
Authors: Moore Anthony L. , Walters Andrew J. , Thorpe Julian , Fricaud Anne‐Catherine , Watts Felicity Z. 
Published: May 2005

AbstractA technique is described for the isolation and purification of intact, respiratory‐competent mitochondria from Sachizo‐saccharomyces pombe. The purified mitochondria are capabel of oxidizing NADH and succinate as resporatory substrates. indicating the presence of succinate dehydorgenase and an NADH dehydrogenase located on the outer suface of the inner membrane. Mitochondria display good respiratory control with an ADP/O ratio of <2. Respiratory activity is linearly dependent upon the redox poise of the quinone pool, suggesting the presence of an unbranched repiratory pathway to molecular oxygen. Immunogold labelling using antisera raised against mitochodria proteins (SSPI, SSCI, and PHSPI) from three different species, namely S. pombe, Saccharomyces cerevisiane and the plant Pisum sativum respectively, has been used to investigate the presence and ultrastructure of the mitochondria isolated by this procedure. The immunocytochemistry was carried out using cells containing wild‐type levels of SSPI protein and cells over‐expressing the protein. These results also demonstrate the capacity of mitochondria to import increased levels of protein in vivo. In vitro import experiments using COXIV‐DHFR indicate that purified S. pombe mitochondria can efficiently import this precursor, and that protein translocation is dependent upon an oxidizable substrate and a membrane potential.
XI. Yeast sequencing reports. The sequence of a 9·3 kb segment located on the left arm of the yeast chromosome XI reveals five open reading frames including the CCE1 gene and putative products related to MYO2 and to the ribosomal protein L10
Authors: Pascolo Steve , Ghazvini Marjan , Boyer Jeanne , Colleaux Laurence , Thierry Agnès , Dujon Bernard 
Published: May 2005

AbstractWe report the wequence of a 9·3 kb DNA segment of chromosome XI of Saccharomyces cerevisiae, located between the MAK11 locus and the centromere. This sequence contains four long open reading frames (ORFs). YKL160, YKL162, YKL164, YKL165 and part of another ORF, YKL166, covering altogether 90% of the entire sequence. One of these ORFs YKL164, corresponds to CCE1. Translation products of two other ORFs, YKL160 and YKL165, exhibit homology with previously known S. cerevisiae proetins: the robosomal protein L10, and the MYO2 gene product, respectively.
RNA delivery in Saccharomyces cerevisiae using electroporation
Authors: Everett Jeffrey G. , Gallie Daniel R. 
Published: May 2005

AbstractAn efficient delivery method for introducing in vitro synthesized RNA into yeast into has been developed using electroporation. Spheroplast preparation, electroporation, and subsequent expression analysis can be accomplished within a single day. The use of introduced mRNA constructs avoids any complications due to nuclear regulation and is particularly suited for cytoplasmic regulatory studies. Moreover, this technique is useful for introduicing those RNas that connot be made in vivo, Such as poly (A)−mRNAs or RNAs with base modifications. We demonstrate that the Escherichia coli GUS gene and the firefly Luc gene are both excellent reporter genes for RNA electroporation.
Cloning and disruption of a gene required for growth on acetate but not on ethanol: The acetyl‐coenzyme a synthetase gene of Saccharmoyces cerevisiae
Authors: De Virgilio Claudio , Bürckert Niels , Barth Gerold , Neuhaus Jean‐Marc , Boller Thomas , Wiemken Andres 
Published: May 2005

AbstractA DNA fragment of Saccharomyces cerevisiae with high homology to the acetyl‐coenzyme A (acetyl‐CoA) synthetase genes of Aspergillus nidulans and Neurospora crassa has been cloned, sequenced and mapped to chromsome I. It contains an open reading frame of 2139 nucleotides, encoding a predicted gene product of 79·2 kDa. In contrast to its ascomycete homologs, there are no introns in the coding sequence. The first ATG codon of the open reading frame is in an unusual context for a translational start site, while the next ATG, 24 codons dowunstream, is in a more conventional context. Possible implications of two alternative traslational start sites for the celular lacalization of the enzyme are disussed. A stable mutant of this gene, obtained by the gene disruptiona technique, had the same low basal acitivity of acetyl‐CoA synthetase as wild‐type cells when grown on glucose but completely lacked the strong increase in activity upon entering the stationary phase, providing direct proof that the gene encodes an inducible acetlyl‐CoA synthetase (ACSI) of yeast. As expected, the mutant was unable to grow on acetate as sole carbon source. Nevertheless, it showed normal induction os isocitrate lyase on acetate media, indicating that activity of acetyl‐CoA sytheatase is dispensable for incuctionof the glyoxylate cycle in S. cerevisiae. Surprisingly, disruption of the ACSI gene did not affect growth on media containing ethanol as the sole crbon source. Demonstrating that threre are alternative pathways pathways leading to acetyl‐CoA under these condintions.
Fluorocytosine causes uncoupled dissipation of the proton gradient and behaves as an imperfect substrate of the yeast cytosine permease
Authors: Hopkins P. , Shaw R. , Acik L. , Oliver Simon , Eddy A. A. 
Published: May 2005

AbstractAt‐pH 5–6 ATP‐depleted washed cell preparations of strain NC233‐10b[pII4‐9], in which the cytosine permease was overexpressed, absorbed cytosine, hypoxanthine or fluorocytosine stoichiometrically with, respectively, about 1, 1·4 and 5 proton equivalents. The cellular pH fell proportionately. The membrane depolarization caused by each compound was assayed in the presence of glucose with a voltage‐sensitive dye and increased in the same order. Fluorocytosine significantly lowered the growth yield that a ‘petite’ strain of the yeast formed at limiting glucose concentrations. At pH 5·6 with extracellular [K+] below 1 mM, each of the three substrates was accumulated about 200‐fold from a dilute solution at the expense of the proton gradient. This concentration ratio corresponds to a solute gradient (Δμs) of 13 kJ mol−1. Raising [K+]0 systematically lowered the substrate accumulation ratio and ΔμH. The mean ratio Δμs/ΔμH was 0·82 all three substrates. It was concluded that whereas the behaviour of cytosine approximated to that expected for a symport of unit proton stoichiometry, the absorption of protons with fluorocytosine and, to a lesser extent, hypoxanthine, was only partly conserved as useful work. A possible mechanism of this novel phenomenon is outlined.
Analysis of glucose repression in Saccharomyces cerevisiae by pulsing glucose to a galactose‐limited continuous culture
Authors: Sierkstra Laurens N. , Nouwen Nico P. , Verbakel John M. A. , Verrips C. Theo 
Published: May 2005

AbstractIn this study, glucose repression in Saccharomyces cerevisiae was analysed under defined physiological conditions, at both the molecular and physiological levels, by pulsing glucose to a galactose‐limited continuous culture. During this pulse of glucose, the galactose feed was kept constant. Directly after the glucose pulse, carbon dioxide production increased while oxygen consumption remained constant, demonstrating that the surplus of glucose had been consumed by means of fermentation. The direct accumulation of galactose in the medium after the glucose pulse indicated that the consumption of galactose had been stopped instantaneously. Galactose uptake experiments revealed that the galactose transporter was still present but apparently was incapable of galactose uptake, which could be due to inhibition of the galactose transporter by glucose. The total concentration of cAMP increased from 5 nmol g−1 at t = 0 to 25 nmolg−1 at t = 1·5 min. After 2 min the concentration of cAMP gradually decreased again to the normal level. Within 2 min after the addition of glucose, the transcription of the GAL genes and SUC2 was inhibited. In addition, the transcription of the HXK1 gene, encoding hexokinase isoenzyme 1, was also inhibited, which demonstrates that the HXK1 gene is regulated at the transcriptional level comparable with invertase.
Functional expression of bacterial β‐glucuronidase and its use as a reporter system in the yeast Yarrowia lipolytica
Authors: Bauer Ronald , Paltauf Fritz , Kohlwein Sepp D. 
Published: May 2005

AbstractThe use of β‐glucuronidase (β‐GUS) as a reporter and sensitive detection system for Yarrowia lipolytica was studied. The Escherichia coli gusA gene was expressed under control of the homologous LEU2 promoter in a transcriptional fusion. An NcoI restriction site was introduced at the translational start‐ATG, conserving the most favorable context for initiation of translation. The chimeric LEU2′‐gusA gene was integrated into the LEU2 locus by homologous recombination. The β‐GUS assay was very sensitive and highly reproducible, using the cytosolic fraction or a total cell extract as the source of enzyme. In a leucine‐free medium, β‐GUS activity was at a high, constant level, independent of growth phase. In transformants grown on complete medium, β‐GUS activity was reduced about three‐fold, but doubled during logarithmic growth. No intrinsic β‐GUS activity was detectable in untransformed Y. lipolytica and no effect of β‐GUS expression on growth was obseved. β‐GUS‐producing Y. lipolytica cells could be directly detected on media plates containing X‐gluc (5‐bromo‐4‐chloro‐3‐indolyl‐β‐D‐glucuronide).
QCR9, the nuclear gene encoding a small subunit of the mitochondrial cytochrome bc1 complex, maps to the right arm of chromosome VII in Saccharomyces cerevisiae
Authors: Phillips John D. , Trumpower Bernard L. 
Published: May 2005

AbstractWe present here mapping data for QCR9, a nuclear gene encoding a subunit of the ubiquinol‐cytochrome c oxidoreductase complex. Deletion of QCR9 results in the inability of cells to grow on non‐fermentable carbon sources at 37°C. Thus, qcr9 mutants can be scored by growing cells on YPE/G at 37°C, or followed by the URA3 marker, which was inserted when making the qcr9 deletion strain, JDP1. The location of QCR9 on the right arm of chromosome VII with respect to the previously mapped genes ADE3, SER2 and PET54 is given.
The complete sequence of a 19,482 bp segment located on the right arm of Chromosome II from Saccharomyces cerevisiae
Authors: Doignon Francois , Biteau Nicolas , Crouzet Marc , Aigle Michel 
Published: May 2005

AbstractWe report here the sequence of a 19,482 bp DNA segment of chromosome II of Saccharomyces cerevisiae. The fragment contains 16 open reading frames (ORFs) covering 74% of the sequence. Four predicted products present homology with known proteins. The ORF YBR1732 exhibits a strong homology to serine hydroxymethyl transferase; the best score is 53·1% identity in 458 amino acids overlap with the serine hydroxymethyl transferase from rabbit liver. YBR1724, which shows homology with riboflavin synthase of Bacillus subtilis, is probably the RIB5 gene implied in riboflavine synthesis and mapped in this region. YBR1733 is homologous to rab protein and YBR1728 is presumably a GTPase activating protein.
Sequence of a cytochrome c gene from Kluyveromyces lactis and its upstream region
Authors: Picos M. Angeles Freire , Torres Ana M. Rodriguez , Ramil Elvira , Cerdan M. Esperanza , Breunig Karin D. , Hollenberg Cornelis P. , Zitomer Richard S. 
Published: May 2005

AbstractThe complete sequence of a cytochrome c gene from Kluyveromyces lactis including its upstream region is reported. Sequence of the translated open reading frame is discussed in terms of cytochrome c structural requirements. Putative regulatory signals in the upstream region are described and compared with reported sequences which modulate the expression of respiratory‐related yeast genes.
Molecular genetics in Saccharomyces kluyveri: The HIS3 homolog and its use as a selectable marker gene in S. kluyveri and Saccharomyces cerevisiae
Authors: Weinstock Keith G. , Strathern Jeffrey N. 
Published: May 2005

AbstractWe cloned the Saccharomyces kluyveri HIS3 homolog, k‐HIS3, and made a partial deletion of the gene. The k‐HIS3 gene complemented a HIS3 deletion in S. cerevisiae. The DNA sequences of the open reading frames (ORFs) of the HIS3 homologs are 70% identical at the DNA level and 83% identical at the deduced amino acid level. The ORF upstream of the k‐HIS3 gene is related to the PET56 gene of S. cerevisiae found upstream of the HIS3 gene of S. cerevisiae. The ORF downstream from the k‐HIS3 gene is not related to the DED1 gene found downstream of the HIS3 gene in S. cerevisiae.
Physical localization of yeast CYS3, a gene whose product resembles the rat γ‐cystathionase and Escherichia coli cystathionine γ‐synthase enzymes
Authors: Barton Arnold B. , Kaback David B. , Clark Michael W. , Keng Teresa , Ouellette B. F. Francis , Storms Reg K. , Zeng Bin , Zhong Wuwei , Fortin Nathalie , Delaney Shawn , Bussey Howard 
Published: May 2005

AbstractWe have cloned, sequenced and physically mapped the CYS3 gene of Saccharomyces cerevisiae. This gene can complement the cys3–1 allele, and disruptions at this locus lead to cysteine auxotrophy. The predicted CYS3 product is closely related (46% identical) to the rat cystathionine γ‐lyase (Erickson et al., 1990), but differs in lacking cysteine residues. These results provide further evidence that the S288C strain of yeast resembles mammals in synthesizing cysteine solely via a trans‐sulfuration pathway. The CYS3 product was found to have strong homology to three other enzymes involved in cysteine metabolism: the Escherichia coli metB and metC products and the S. cerevisiae MET25 gene product. The trans‐sulfuration enzymes appears to form a diverged family and carry out related functions from bacteria to mammals.
Secretion of mouse α‐amylase from fission yeast Schizosaccharomyces pombe: Presence of chymostatin‐sensitive protease activity in the culture medium
Authors: Tokunaga Masao , Kawamura Akiko , Yonekyu Sayuri , Kishida Masao , Hishinuma Fumio 
Published: May 2005

AbstractWe have constructed two secretion vectors for Schizosaccharomyces pombe using an SV40 promoter and the secretion signals of the pGKL killer toxin complex derived from Kluyveromyces lactis. Although indigenous secretory glycoproteins tend to accumulate in the periplasmic space of S. pombe, we have succeeded in the secretion of mouse α‐amylase into the culture medium. The efficiency of secretion, processing pattern, stability and culture conditions for mouse α‐amylase were studied in S. pombe. The 128 kDa killer secretion signal was more effective in directing secretion of mouse α‐amylase than the 28 kDa killer secretion signal. We detected a chymostatin‐sensitive protease activity in the culture medium of S. pombe, which digests mouse α‐amylase secreted into the culture medium. The addition of 5 μg/ml chymostatin was shown to protect mouse α‐amylases from this degradation.
Rapid identification and characterization of peroxisomal assembly mutants in Yarrowia lipolytica
Authors: Nuttley William M. , Brade Anthony M. , Eitzen Gary A. , Glover John R. , Aitchison John D. , Rachubinski Richard A. , Gaillardin Claude 
Published: May 2005

AbstractWe describe the isolation and characterization of peroxisomal assembly mutants in the genetically manipulable yeast Yarrowia lipolytica (pay mutants). These mutants were initially identified as oleic acid‐non‐utilizers by their inability to grow on oleic acid, the utilization of which requires peroxisomal β‐oxidation enzymes. Identification of a subset of oleic acid‐non‐utilizers as pay mutants was obtained by a rapid immunofluorescence procedure using antibodies to the peroxisomal targeting signal Ser‐Lys‐Leu‐CO2H. Punctate structures characteristic of peroxisomes were not detected in pay mutants using this technique. This rapid identification by immunofluorescence should be generally applicable to the selection of peroxisomal assembly mutants in other yeasts. To take advantage of the pay mutant system, we constructed a genomic library in the autonomously replicating vector pINA445 and developed an efficient and rapid electroporation procedure for the functional complementation of these mutants. We have been successful in functionally complementing two independent pay mutants. Molecular analysis of these and other complementing genes will allow for characterization of some of the cellular elements involved in peroxisomal assembly.
Increased transformation levels in intact cells of Saccharomyces cerevisiae aculeacin A‐resistant mutants
Authors: Gallego Carme , Casas Celia , Herrero Enrique 
Published: May 2005

AbstractSeveral Saccharomyces cerevisiae mutants resistant to the cell wall synthesis inhibitor aculeacin A exhibit higher transformation levels than the parental strain. Mutant acr2 has been studied in more detail. It is transformed up to ten‐fold more efficiently than the wild‐type strain with episomal, centromeric and integrative plasmids, and dimethyl sulfoxide has an additive effect improving transformation efficiency. Transformation with linear DNA molecules is not as much affected in acr2 cells. The observed effects may be caused by the altered plasma membrane composition of the mutants.
Absence of cell wall chitin in Saccharomyces cerevisiae leads to resistance to Kluyveromyces lactis killer toxin
Authors: Takita Marco A. , Castilho‐Valavicius Beatriz 
Published: May 2005

AbstractKluyveromyces lactis killer toxin causes sensitive strains of a variety of yeasts to arrest at the G1 stage of the cell cycle, and to lose viability. We describe here the isolation and characterization of a class of recessive mutations in Saccharomyces cerevisiae that leads to toxin resistance and a temperature‐sensitive phenotype. These mutant cells arrest growth at 37°C with a characteristic phenotype of elongated buds. Cloning of the gene complementing these defects revealed it to be CAL1, coding for chitin synthase 3 activity. Calcofluor staining of the mutant cells indicated that chitin is absent both at 23°C and 37°C. Given that the CAL1 activity is responsible for the synthesis of most of chitin in yeast cells, and that in its absence the cells are viable but resistant to the killer toxin, our results strongly suggest that chitin might represent the receptor for this killer toxin.
Purification and characterization of aminopeptidase yspI from Schizosaccharomyces pombe
Authors: Arbesú Ma José , Valle Eulalia , Suárez‐Rendueles Paz 
Published: May 2005

AbstractAminopeptidase yspI was purified to apparent homogeneity from the fission yeast Schizosaccharomyces pombe. The molecular mass of the native enzyme was estimated to be 184 kDa by gel filtration chromatography. A value of 92 kDa was calculated after sodium dodecyl sulfate–polyacrylamide gel electrophoresis. The enzyme is thus a dimer with two identical subunits. Optimum pH for cleavage of synthetic aminoacyl‐4‐nitroanilides is 7·0. Mercury ions, EDTA and chloroquine were found to be potent inhibitors of aminopeptidase yspI activity. Substrate specificity studies indicate that the purified enzyme cleaves L‐lysine‐4‐nitroanilide with high efficiency.