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  • Genetics
  • Wiley-Blackwell  (586)
  • American Association for the Advancement of Science (AAAS)  (6)
  • 1995-1999  (592)
  • 1
    Electronic Resource
    Electronic Resource
    Weinheim : Wiley-Blackwell
    Electrophoresis 16 (1995), S. 186-196 
    ISSN: 0173-0835
    Keywords: Genetics ; Two-dimensional electrophoresis ; Denaturing gradient electrophoresis ; Cystic fibrosis ; Mutation ; Breast cancer ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: A major effort in the analysis of DNA currently focuses on identifying genes and their pathological variants underlying disease. Once such disease genes have been isolated a major task of molecular medicine is to identify the spectrum of DNA sequence variations responsible for the aberrant function of such genes. These efforts, however, are hindered by the vast amount of genetic information to scan for variations and the limited capacity of analytical techniques in terms of accuracy and speed. Recently, a number of techniques were developed, so-called “genome scanning” techniques, which allow complete genomes to be analyzed for sequence variation in parallel, i.e., at multiple sites or loci simultaneously rather than serially at predefined loci. Here we present the background and applications of a particular electrophoretic parallel processing approach, generically termed two-dimensional DNA typing. The approach is based on separating DNA fragments by two-dimensional electrophoresis [1], including denaturing gradient gel electrophoresis, thus allowing hundreds of fragments to be simultaneously assessed by comparative analysis for variations in size and sequence. The method is suitable for hybridization analysis with locus-specific and multilocus probes of genomic DNA restriction fragments derived from human and other DNA, and for analysis of polymerase chain reaction (PCR) fragments derived from large genes. Two-dimensional DNA typing has been applied, e.g., in linkage analysis of pedigrees, analysis of tumor genomes for rearrangements, and to scan the cystic fibrosis transmembrane regulator gene for sequence variations such as point mutations.
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  • 2
    Electronic Resource
    Electronic Resource
    Weinheim : Wiley-Blackwell
    Electrophoresis 16 (1995), S. 279-285 
    ISSN: 0173-0835
    Keywords: Genetics ; Linkage(genetics) ; Polymorphism ; Chromosome mapping ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Genomic mismatch scanning (GMS) is a new method of genetic mapping which attempts to purify and map the regions of identity between two complex genomes in a single test. Identical DNA fragments from two genomic sources are enriched in two steps: (i) after reannealing of the two genomes, heterohybrids are purified by using a combination of a restriction methylase and methylation-sensitive endonucleases, (ii) heterohybrids that contain mismatches are nicked in vitro by the E. coli MutHLS mismatch repair system and are eliminated subsequently from the pool, leaving only mismatch-free heterohybrids. The genomic origin of this selected pool of DNA fragments is then mapped in a single hybridization step onto metaphase chromosomes or ordered DNA arrays. The principal advantages of GMS are (i) it approaches the theoretical limit of mapping power and resolution offered by an arbitrarily dense set of completely informative polymorphic markers and (ii) it results in a great increase in the effective number of informative markers without a corresponding increase in the number of individual tests. Thus, it should provide an efficient method for affected-relative-pair linkage mapping and for linkage disequilibrium mapping. In addition, a variation of GMS may allow rapid genomic scanning for regions of homozygosity-by-descent or somatic loss-of-heterozygosity. The feasibility of GMS has been validated in the 15 mb genome of Saccharomyces cerevisiae. This article discusses the principles of GMS, the application to more complex genomes, and the possible uses of GMS.
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  • 3
    ISSN: 0749-503X
    Keywords: yeast ; carboxypeptidase Y ; Saccharomyces cerevisiae ; Schizosaccharomyces pombe ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: Vacuolar carboxypeptidase Y of Saccharomyces cerevisiae (CPYsc) has been expressed in a Schizosaccharomyces pombe strain devoid of the endogenous equivalent peptidase, employing a 2 μ derived plasmid. Immunoblot analysis revealed that CPYsc produced in the fission yeast has a higher molecular mass than mature CPYsc produced by the budding yeast. CPYsc is glycosylated when expressed in S. pombe and uses four N-linked glycosylation sites as shown by endoglycosidase H digestion. Carbohydrate removal leads to a protein moiety which is indistinguishable in size from deglycosylated CPYsc produced by S. cerevisiae. CPYsc isolated from S. pombe soluble extracts is enzymatically active and thus is presumed to undergo correct proteolytic maturation. Subcellular fractionation experiments showed a cofractionation of CPYsc with the S. pombe endoproteinases PrA and PrB, suggesting that the protein is correctly sorted to the vacuole and that these peptidases might be responsible for zymogen activation.
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  • 4
    Electronic Resource
    Electronic Resource
    New York, NY [u.a.] : Wiley-Blackwell
    Yeast 11 (1995), S. 293-300 
    ISSN: 0749-503X
    Keywords: Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
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  • 5
    ISSN: 0749-503X
    Keywords: phosphomannose isomerase ; yeast ; heterologous expression ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: Using a DNA fragment derived from the Saccharomyces cerevisiae phosphomannose isomerase (PMI) structural gene as a probe against a random ordered array library of genomic DNA from the pathogenic fungus Candida albicans, we have cloned the C. albicans PMI 1 gene. This gene, which is unique in the C. albicans genome, can functionally complement PMI-deficient mutants of both S. cerevisiae and Escherichia coli. The DNA sequence of the PMI 1 gene predicts a protein with 64·1% identity to PMI from S. cerevisiae. Sequential gene disruption of PMI 1 produces a strain with an auxotrophic requirement for D-mannose. The heterologous expression of the PMI 1 gene at levels up to 45% of total cell protein in E. coli leads to partitioning of the enzyme between the soluble and particulate fractions. The protein produced in the soluble fraction is indistinguishable in kinetic properties from the material isolated from C. albicans cells. The nucleotide sequence data reported here will appear in the EMBL database under Accession Number X82024.
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  • 6
    Electronic Resource
    Electronic Resource
    New York, NY [u.a.] : Wiley-Blackwell
    Yeast 11 (1995), S. 493-536 
    ISSN: 0749-503X
    Keywords: Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
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  • 7
    ISSN: 0749-503X
    Keywords: genome sequencing ; Saccharomyces cerevisiae ; yeast ; chromosome XIV ; RPD3 ; PAS8 ; KRE1 ; dnaJ ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: The DNA sequence of a 15·4 kb region covering the left arm of chromosome XIV from Saccharomyces cerevisiae was determined. This region contains eight open reading frames (ORFs) which code for proteins of more than 100 amino acids. Three ORFs correspond to the RPD3, PAS8 and KRE1 loci, described previously. Three ORFs show limited homology with known proteins: NO330 with the recessive suppressor of secretory defect SAC1, NO325 with YCR094W identified during chromosome III sequencing; whereas NO315 presents a motif conserved in the dnaJ family. Two ORFs (NO320 and NO325) show no homology to known proteins within the databases screened, but NO320 corresponds to a serine-threonine-rich protein. The sequence has been entered in the EMBL data library under Accession Number Z46259.
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  • 8
    Electronic Resource
    Electronic Resource
    New York, NY [u.a.] : Wiley-Blackwell
    Yeast 11 (1995), S. 615-628 
    ISSN: 0749-503X
    Keywords: Kluyveromyces lactis ; killer plasmid ; gene disruption ; epitope-tagging ; baculovirus over-expression ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: The ORF5 of Kluyveromyces lactis killer plasmid pGKL2 (k2) is capable of encoding a small neutral protein of 18 kDa of as yet unassigned function. Although this ORF is located between two larger ORFs, 4 and 6, which it overlaps, RNA analysis showed that it is transcribed monocistronically. One-step gene disruption of ORF5, via in vivo homologous recombination between native plasmid k2 and a transfer vector employing the Saccharomyces cerevisiae LEU2 gene fused to the k2 UCS5 element, yielded Leu+ transformants at high frequencies. The transformants were found to carry a new recombinant form of k2 with ORF5 replaced by the LEU2 marker, termed rk2, in addition to the wild-type plasmids k1 and k2. Northern analysis detected a plasmid-dependent LEU2 transcript distinct in size and regulation from its nuclear counterpart. Recombinant plasmid, rk2, was unable to displace native k2 during Leu+ selective growth; however rk2 was displaced by k2 during non-selective growth. Thus, ORF5 appears to be an essential gene for plasmid integrity and/or maintenance. The ORF5 product was detected by over-expression of an epitope-tagged allele in the baculovirus system. Western analysis using a monoclonal antibody specific for the epitope tag identified a protein band with apparent molecular weight of 20 kDa, corresponding in size to the predicted product.
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  • 9
    ISSN: 0749-503X
    Keywords: hybridization mapping ; cosmids ; genome analysis ; chromosome XII ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: For the sequencing of the left arm of chromosome XII of Saccharomyces cerevisiae, we fine-mapped the entire 450 kb fragment between the ribosomal DNA (rDNA) and the left telomere. Total yeast DNA in agarose blocks was digested with I-PpoI, which exclusively cuts once in each repeat unit of the rDNA. The resulting fragment was isolated from pulsed-field gels, together with the equally sized chromosome IX. A cosmid library of some 30-fold chromosome coverage was generated from this material, with the cloning efficiency being around 20 000 clones per microgram genomic DNA. The chromosome XII and IX specific clones were identified by complementary hybridizations with the respective chromosomes. For the left arm of chromosome XII, a contiguous cosmid array (contig) with an average map resolution better than 9 kb was generated by clone hybridization procedures. The ordered library serves as a tool for the physical mapping of genetic markers. Also, a minimal set of 15 clones was selected that covers the entire fragment. This subset forms the basis for the generation of a template map of much higher resolution for a directed sequencing of the left arm of chromosome XII.
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  • 10
    ISSN: 0749-503X
    Keywords: 2 μm plasmid ; yeast ; maternal bias ; DNA amplification ; plasmid stability ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: A distributive model has been constructed to describe the maintenance of the native 2 μm and 2 μm-based plasmids in the yeast Saccharomyces cerevisiae. This model includes elements which represent the influence of selection, segregation, replication and amplification on plasmid stability. A computer program has been written in TURBO PASCAL to implement the model and a number of simulation experiments have been carried out. These simulations permitted the choice of a form of the model which is compatible with the available experimental evidence. The form chosen involves an amplification system in which the RAF gene product binds to the Rep1/Rep2 dimer to prevent the latter acting to repress the activity of the FLP gene. At the same time an upper limit (or ‘ceiling’) was imposed on the number of plasmid molecules able to replicate. Maternal bias was accommodated by ‘tagging’ a small proportion of molecules for inheritance by the mother nucleus and these tags being removed (or ‘cleared’) by the Rep1/Rep2 dimers. This final form of the model makes specific predictions about the stability of 2 μm and YEp plasmids in yeast populations and about the distribution of plasmid copy number between cells in such populations. The predictions on stability have been subjected to experimental test and results provide good support for the model.
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  • 11
    Electronic Resource
    Electronic Resource
    New York, NY [u.a.] : Wiley-Blackwell
    Yeast 11 (1995) 
    ISSN: 0749-503X
    Keywords: Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
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  • 12
    ISSN: 0749-503X
    Keywords: chemostat ; mixed substrates ; gluconeogenesis ; glyoxylate cycle ; Saccharomyces cerevisiae ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: Growth efficiency and regulation of key enzyme activities were studied in carbon- and energy-limited chemostat cultures of Saccharomyces cerevisiae grown on mixtures of glucose and ethanol at a fixed dilution rate. Biomass yields on substrate carbon and oxygen could be adequately described as the net result of growth on the single substrates. Activities of isocitrate lyase and malate synthase were not detected in cell-free extracts of glucose-limited cultures. However, both enzymes were present when the ethanol fraction in the reservoir medium exceeded the theoretical minimum above which the glyoxylate cycle is required for anabolic reactions. Fructose-1,6-bisphosphatase activity was only detectable at high ethanol fractions in the feed, when activity of this enzyme was required for synthesis of hexose phosphates. Phospho-enol-pyruvate-carboxykinase activity was not detectable in extracts from glucose-grown cultures and increased with the ethanol fraction in the feed. It is concluded that, during carbon-limited growth of S. cerevisiae on mixtures of glucose and ethanol, biosynthetic intermediates with three or more carbon atoms are preferentially synthesized from glucose. Synthesis of the key enzymes of gluconeogenesis and the glyoxylate cycle is adapted to the cells′ requirement for these intermediates. The gluconeogenic enzymes and their physiological antagonists (pyruvate kinase, pyruvate carboxylase and phosphofructokinase) were expressed simultaneously at high ethanol fractions in the feed. If futile cycling is prevented under these conditions, this is not primarily achieved by tight control of enzyme synthesis.
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  • 13
    ISSN: 0749-503X
    Keywords: Old Yellow Enzyme ; flavoproteins ; Kluyveromyces lactis ; chromosome mapping ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: 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).
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  • 14
    Electronic Resource
    Electronic Resource
    New York, NY [u.a.] : Wiley-Blackwell
    Yeast 11 (1995), S. 1115-1127 
    ISSN: 0749-503X
    Keywords: Saccharomyces cerevisiae ; ribosomes ; ribosomal frameshifting ; L-A dsRNA virus ; Ty ; retrotransposon ; retrovirus ; hungry codons ; polyamines ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: Proper maintenance of translational reading frame by ribosomes is essential for cell growth and viability. In the last 10 years it has been shown that a number of viruses induce ribosomes to shift reading frame in order to regulate the expression of gene products having enzymatic functions. Studies on ribosomal frameshifting in viruses of yeast have been particularly enlightening. The roles of viral mRNA sequences and secondary structures have been elucidated and a picture of how these interact with host chromosomal gene products is beginning to emerge. The efficiency of ribosomal frameshifting is important for viral particle assembly, and has identified ribosomal frameshifting as a potential target for antiviral agents. The availability of mutants of host chromosomal gene products involved in maintaining the efficiency of ribosomal frameshifting bodes well for the use of yeast in future studies of ribosomal frameshifting.
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  • 15
    ISSN: 0749-503X
    Keywords: S. cerevisiae ; flow cytometry ; dynamics of the cell cycle ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: Studies on the dynamics of growth of single eukaryotic cells and their relationships with cell cycle regulations are generally carried out following cell synchronization procedures or, on a relatively low number of cells, by time-lapse studies. Establishment of both time-lapse studies and synchronous cell populations usually requires elaborate experimental efforts and is prone to perturb the physiological state of the cell.In this paper we use a new flow cytometric approach which allows, in asynchronous growing Saccharomyces cerevisiae populations, tagging of both the cell age and the cell protein content of a cohort of daughter cells at the different cell cycle set points. Since the cell protein content is a good estimation of the cell size, it is possible to follow the kinetics of the cell size increase during cell cycle progression. The experimental findings obtained indicate an exponential increase of the cell size during growth, that the daughter and the parent subpopulations grow with the same specific growth rate, that the average cell size increase rate of each individual cell is almost identical to the specific growth rate of the overall population and provide the opportunity to estimate the cell cycle length for the daughter cell population as well as the identification of the complex structure of asynchronously growing yeast populations.
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  • 16
    Electronic Resource
    Electronic Resource
    New York, NY [u.a.] : Wiley-Blackwell
    Yeast 11 (1995), S. 1215-1222 
    ISSN: 0749-503X
    Keywords: Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
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  • 17
    ISSN: 0749-503X
    Keywords: S. cerevisiae ; nuclear membrane ; endoplasmic reticulum ; ploidy ; cell division ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: We have cloned a new gene, SCP160, from Saccharomyces cerevisiae, the deduced amino acid sequence of which does not exhibit overall similarity to any known yeast protein. A weak resemblance between the C-terminal part of the Scp160 protein and regulatory subunits of cAMP-dependent protein kinases from eukaryotes as well as the pstB protein of Escherichia coli was observed. The SCP160 gene resides on the left arm of chromosome X and codes for a polypeptide of molecular weight around 160 kDa. By immunofluorescence microscopy the Scp160 protein appears to be localized to the nuclear envelope and to the endoplasmic reticulum (ER). However, no signal sequence or membrane-spanning region exists, suggesting that the Scp160 protein is attached to the cytoplasmic surface of the ER-nuclear envelope membranes. Disruption of the SCP160 gene is not lethal but results in cells of decreased viability, abnormal morphology and increased DNA content. This phenotype is not reversible by transformation with a plasmid carrying the wild-type gene. Crosses of SCP160 deletion mutant strains among each other or with unrelated strains lead to irregular segregation of genetic markers. Taken together the data suggest that the Scp160 protein is required during cell division for faithful partitioning of the ER-nuclear envelope membranes which in S. cerevisiae enclose the duplicated chromosomes.
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  • 18
    ISSN: 0749-503X
    Keywords: arginine regulation ; ARG1 ; ARG8 ; CPA1 ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: Repression or induction of the genes involved in arginine biosynthesis or catabolism, respectively, both require participation of the ArgRp/Mcm1p regulatory complex. Our previous work showed that those opposite effects were mediated by a similar arginine-responsive element of 23 nucleotides (that we now call ARC, for ARginine Control) situated close to the start of transcription in the repressed promoters and far upstream of the TATA-element in the induced promoters.To define more precisely the sequence and position requirements of the ARC element, we have now characterized by mutagenesis the promoter elements of the arginine-repressible ARG1 and ARG8 genes. We also identify a functional ARC in the CPA1 promoter, thereby confirming, in agreement with our previous mRNA pulse-labelling data, the participation of a transcriptional component in the arginine regulation of that gene otherwise submitted to a translational regulation.From the 12 ARC elements now characterized, we have derived a consensus sequence and show that such a synthetic element is able to mediate ArgRp/Mcm1p-dependent arginine regulation.An important new finding illustrated by ARG1 and CPA1, is that contrary to what all the previous data suggested, repression can be mediated by ARC elements located far upstream of the TATA-box. The new data suggest that the arginine repressor might inhibit transcription in an active process.
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  • 19
    ISSN: 0749-503X
    Keywords: Ashbya ; Eremothecium ; Holleya ; Kluyveromyces ; Metschnikowia ; Nematospora ; Saccharomyces ; Crustaceae ; Diplopoda ; Heteroptera ; Saccharomycetaceae ; phylogeny ; taxonomy ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: Phylogenetic relationships between species from the genera Kluyveromyces and Saccharomyces and representatives of the Metschnikowiaceae (Holleya, Metschnikowia, Nematospora) including the two filamentous phytopathogenic fungi Ashbya gossypii and Eremothecium ashbyii were studied by comparing the monosaccharide pattern of purified cell walls, the ubiquinone system, the presence of dityrosine in ascospore walls, and nucleotide sequences of ribosomal DNA (complete 18S rDNA, ITS1 and ITS2 region). Based on sequence information from both ITS regions, the genera Ashbya, Eremothecium, Holleya and Nematospora are closely related and may be placed in a single genus as suggested by Kurtzman (1995; J. Industr. Microbiol. 14, 523-530). In a phylogenetic tree derived from the ITS1 and ITS2 region as well as in a tree derived from the complete 18S rDNA gene, the genus Metschnikowia remains distinct. The molecular evidence from ribosomal sequences suggests that morphology and ornamentation of ascospores as well as mycelium formation and fermentation should not be used as differentiating characters in family delimitation. Our data on cell wall sugars, ubiquinone side chains, dityrosine, and ribosomal DNA sequences support the inclusion of plant pathogenic, predominantly filamentous genera like Ashbya and Eremothecium or dimorphic genera like Holleya and Nematospora with needle-shaped ascospores within the family Saccharomycetaceae. After comparison of sequences from the complete genes of the 18S rDNA the genus Kluyveromyces appears heterogeneous. The type species of the genus, K. polysporus is congeneric with the genus Saccharomyces. The data of Cai et al. (1996; Int. J. Syst. Bacteriol. 46, 542-549) and our own data suggest to conserve the genus Kluyveromyces for a clade containing K. marxianus, K. dobzhanskii, K. wickerhamii and K. aestuarii, which again can be included in the family Saccharomycetaceae. The phylogenetic age of the Metschnikowiaceae and Saccharomycetaceae will be discussed in the light of coevolution. © 1997 John Wiley & Sons, Ltd.
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  • 20
    ISSN: 0749-503X
    Keywords: Saccharomyces cerevisiae ; carbamylphosphate synthetase ; mutational analysis ; CPA1 ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: The role of selected amino acid residues located in the putative catalytic domain and of two conserved histidine residues within the small subunit of the carbamylphosphate synthetase (CPS) specific to the arginine biosynthesis pathway of the yeast Saccharomyces cerevisiae was studied using site-directed mutagenesis to change all residues to aspartic acid. Carbamylphosphate synthesis catalysed by modified CPS was tested in vivo. The C264D, H307D and H349D mutants were unable to grow on minimal medium, indicating the importance of these three residues for efficient CPS activity, whereas, four other mutated residues located in the catalytic site (including a proline residue) do not affect the growth rate. These results in comparison to those obtained with the CPS of Escherichia coli, implicate residues Cys 264 and His 349 in the glutaminase catalytic activity, and His 307 in the binding of glutamine to the active site. Using these three defective mutants, we investigated the in vivo utilization of ammonia by CPS. C264D and H307D mutants are able to use ammonia as a substrate when provided in sufficiently high concentrations (up to 200 mm). The H349D mutant, however, did not grow even at ammonium sulfate concentrations above 400 mm, suggesting that this substitution is critical to NH3-dependent CPS activity although the ammonia binding site is presumably located within the large subunit of the enzyme. © 1997 by John Wiley & Sons, Ltd.
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  • 21
    ISSN: 0749-503X
    Keywords: gene disruption ; functional analysis ; chromosome X ; Saccharomyces cerevisiae ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: We describe here the construction of six deletion mutants and their basic phenotypic analysis. Six open reading frames (ORFs) from chromosome X, YJR039w, YJR041c, YJR043c, YJR046w, YJR053w and YJR065c, were disrupted by deletion cassettes with long (LFH) or short (SFH) flanking regions homologous to the target locus. The LFH deletion cassette was made by introducing into the kanMX4 marker module two polymerase chain reaction (PCR) fragments several hundred base pairs (bp) in size homologous to the promoter and terminator regions of a given ORF. The SFH gene disruption construct was obtained by PCR amplification of the kanMX4 marker with primers providing homology to the target gene. The region of homology to mediate homologous recombination was about 70 bp. Sporulation and tetrad analysis revealed that ORFs YJR041c, YJR046w and YJR065c are essential genes. Complementation tests by corresponding cognate gene clones confirmed this observation. The non-growing haploid segregants were observed under the microscope. The yjr041cΔ haploid cells gave rise to microcolonies comprising about 20 to 50 cells. Most yjr046wΔ cells were blocked after one or two cell cycles with heterogeneous bud sizes. The yjr065cΔ cells displayed an unbudded spore or were arrested before completion of the first cell division cycle with a bud of variable size. The deduced protein of ORF YJR065c, that we named Act4, belongs to the Arp3 family of actin-related proteins. Three other ORFs, YJR039w, YJR043c and YJR053w are non-essential genes. The yjr043cΔ cells hardly grew at 15°C, indicating that this gene is required for growth at low temperature. Complementation tests confirmed that the disruption of YJR043c is responsible for this growth defect. In addition, the mating efficiency of yjr043cΔ and yjr053wΔ cells appear to be moderately a ffected. © 1997 John Wiley & Sons, Ltd.
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  • 22
    Electronic Resource
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    New York, NY [u.a.] : Wiley-Blackwell
    Yeast 13 (1997), S. 1375-1381 
    ISSN: 0749-503X
    Keywords: Candida albicans ; prolyl tRNA synthetase ; CaPRS ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: We have isolated a 4·0-kb fragment from a genomic library of Candida albicans which contained two open reading frames (ORFs). One of them is homologous to a prolyl-tRNA synthetase that catalyses the charging of a specific tRNA by proline (CaPRS). A deduced sequence of 575 amino acids representing a polypeptide of 66·2 kDa was determined. A FASTA search indicated that the CaPRSp had an overall similarity of 54·4% with the product of a Saccharomyces cerevisiae ORF (YER087) and 43·8% with the prolyl-tRNA synthetase of Escherichia coli (COLIPRO). Consensus Class II aminoacyl-tRNA synthetase sequences were identified by the PROSITE program. CaPRS was localized to chromosome R of the C. albicans genome and CaPRS DNA hybridized to a major RNA transcript of 1·7 kb under all conditions tested. The CaPRS sequence submitted to the EMBL data library is available under Accession Number U86341.© 1997 John Wiley & Sons, Ltd.
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  • 23
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    New York, NY [u.a.] : Wiley-Blackwell
    Yeast 13 (1997), S. 1243-1250 
    ISSN: 0749-503X
    Keywords: Saccharomyces ; phylogeny ; ribosomal RNA ; systematics ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: Sequences of two internally transcribed spacer regions between 18S and 28S rRNA genes were determined to assess the phylogenetic relationship in the strains belonging to the genus Saccharomyces. The sequences of S. bayanus and S. pastorianus were quite similar, but not identical. Two phylogenetic trees constructed by the neighbor-joining method showed that all the species examined were distinguished from one another. The Saccharomyces sensu stricto species: S. cerevisiae, S. bayanus, S. paradoxus and S. pastorianus, were closely related and far from the Saccharomyces sensu lato species including S. barnetti, S. castellii, S. dairensis, S. exiguus, S. servazzii, S. spencerorum and S. unisporus, and an outlying species, S. kluyveri. © 1997 John Wiley & Sons, Ltd.
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  • 24
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    Yeast 13 (1997), S. 1347-1355 
    ISSN: 0749-503X
    Keywords: yeast metabolism ; metabolite extraction ; metabolic engineering ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: A simple and reliable method for the efficient inactivation of metabolism and for quantitative metabolite extraction from yeast cells is presented. It is based on the use of a boiling solution made of 75% ethanol (volume/final volume) buffered with 70 mm-Hepes (final concentration), pH 7·5, to guarantee the stability throughout the whole procedure of a large variety of metabolites, including all glycolytic intermediates, nucleotides, pyridine nucleotides and organic acids compounds. The extraction is fast, requiring only 3 min incubation of yeast cells in the ethanol-buffered mixture maintained at 80°C. It can be carried out either directly by spraying the cells into the boiling mixture, or after quenching the whole culture in 60% methanol kept at -40°C. Extracts are subsequently concentrated by evaporation under partial vacuum and the residue is resuspended in a small volume of water. This concentration step and the use of a highly sensitive analytical method allow us to quantify metabolites in less than 10 mg dry weight cells. This method, which can be applied to other fungi, could be very helpful for the determination of true metabolites in mutants generated through the EUROFAN programme and for metabolic flux analysis. © 1997 John Wiley & Sons, Ltd.
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  • 25
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    Yeast 13 (1997), S. 1357-1362 
    ISSN: 0749-503X
    Keywords: Suppressor tRNA ; UGA codon ; 5′-flanking sequence ; gene copy number ; fidelity of translation ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: The Saccharomyces cerevisiae haploid genome includes six copies of the gene encoding tRNATrp which are scattered on five chromosomes. Other, non-functional tDNATrp fragments also occur in the genome. The segments of all six genes which encode the 72-nucleotide mature tRNAas well as a 34-nucleotide intervening sequence, are identical. However, the 5′ and 3′ flanking sequences diverge virtually at the boundaries of the coding region. We have used an assay based on suppression of UGA mutations by multi-copy clones of tDNATrp to search for functional differences among these genes. Previous studies with one tDNATrp had demonstrated that moderate suppression of a UGA mutation, leu2-2, resulted from introduction of a multi-copy clone of the gene. Attempts to use this assay to select tDNATrp clones from a yeast genomic library yielded only four of the six different clones. The other two genes were amplified by PCR and cloned in pRS202, a 2 μ vector also used for the genomic library. Plasmids bearing the six tRNA genes were transformed into S. cerevisiae strain JG369.3B and scored for their ability to suppress the leu2-2 mutation as well as his4-260, another UGA marker. Two of the six tRNATrp clones were unable to suppress either marker, two evidenced weak suppression of the Leu auxotrophy, and two were able to suppress both markers. Growth rates in liquid media requiring suppression were measured for cell lines carrying each of the clones. Differences greater than 50-fold were observed in media lacking histidine. An evolutionary tree based on 5′-flanking sequence corresponds reasonably well with suppressor activity, while a similar analysis of 3′-flanking sequence does not. This suggests that the functional differences are based on divergence in the 5′-flanking sequences of the tRNATrp genes. © 1997 John Wiley & Sons, Ltd.
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  • 26
    ISSN: 0749-503X
    Keywords: Saccharomyces cerevisiae ; short ORFs ; computational ORF verification ; ORF properties ; sequence similarity ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: We have analysed short open reading frames (between 150 and 300 base pairs long) of the yeast genome (Saccharomyces cerevisiae) with a two-step strategy. The first step selects a candidate set of open reading frames from the DNA sequence based on statistical evaluation of DNA and protein sequence properties. The second step filters the candidate set by selecting open reading frames with high similarity to other known sequences (from any organism). As a result, we report ten new predicted proteins not present in the current sequence databases. These include a new alcohol dehydrogenase, a protein probably related to the cell cycle, as well as a homolog of the prokaryotic ribosomal protein L36 likely to be a mitochondrial ribosomal protein coded in the nuclear genome. We conclude that the analysis of short open reading frames leads to biologically interesting discoveries, even though the quantitative yield of new proteins is relatively low. © 1997 John Wiley & Sons, Ltd.
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  • 27
    ISSN: 0749-503X
    Keywords: ornithine decarboxylase ; SPE1 gene ; Candida albicans ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: The gene encoding ornithine decarboxylase, SPE1, from the pathogenic yeast Candida albicans has been isolated by complementation of an ornithine decarboxylase-negative (spe1Δ) strain of Saccharomyces cerevisiae. Four transformants, three of which contain plasmids with the SPE1 gene, were isolated by selection on polyamine-free medium. The C. albicans ornithine decarboxylase (ODC) showed high homology with other eukaryotic ODCs at both the amino acid and nucleic acid levels. The GenBank accession number for this gene is U85005. © 1997 John Wiley & Sons, Ltd.
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  • 28
    ISSN: 0749-503X
    Keywords: GPI-anchor ; GPI-attachment site ; yeast ; Ascomycetes ; fungi ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: Use of the Von Heijne algorithm allowed the identification of 686 open reading frames (ORFs) in the genome of Saccharomyces cerevisiae that encode proteins with a potential N-terminal signal sequence for entering the secretory pathway. On further analysis, 51 of these proteins contain a potential glycosyl-phosphatidylinositol (GPI)-attachment signal. Seven additional ORFs were found to belong to this group. Upon examination of the possible GPI-attachment sites, it was found that in yeast the most probable amino acids for GPI-attachment are asparagine and glycine. In yeast, GPI-proteins are found at the cell surface, either attached to the plasma-membrane or as an intrinsic part of the cell wall. It was noted that plasma-membrane GPI-proteins possess a dibasic residue motif just before their predicted GPI-attachment site. Based on this, and on homologies between proteins, families of plasma-membrane and cell wall proteins were assigned, revealing 20 potential plasma-membrane and 38 potential cell wall proteins. For members of three plasma-membrane protein families, a function has been described. On the other hand, most of the cell wall proteins seem to be structural components of the wall, responsive to different growth conditions. The GPI-attachment site of yeast slightly differs from mammalian cells. This might be of use in the development of anti-fungal drugs. © 1997 John Wiley & Sons, Ltd.
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  • 29
    ISSN: 0749-503X
    Keywords: Saccharomyces cerevisiae ; LYS80gene ; α-ketoglutarate ; apparent repression ; pleiotropic factor ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: In Saccharomyces cerevisiae, an intermediate of the lysine pathway, α-aminoadipate semialdehyde (αAASA), acts as a coinducer for the transcriptional activation of LYS genes by Lys14p. The limitation of the production of this intermediate through feedback inhibition of the first step of the pathway results in apparent repression by lysine. Previously, the lys80 mutations, reducing the lysine repression and increasing the production of lysine, were interpreted as impairing a repressor of LYS genes expression. In order to understand the role of Lys80p in the control of the lysine pathway, we have analysed the effects of mutations epistatic to lys80 mutations. The effects of lys80 mutations on LYS genes expression were dependent on the integrity of the activation system (Lys14p and αAASA). The increased production of lysine in lys80 mutants appeared to result from an improvement of the metabolic flux through the pathway and was correlated to an increase of the α-ketoglutarate pool and of the level of several enzymes of the tricarboxylic acid cycle. The LYS80 genes has been cloned and sequenced; it turned out to be identical to gene MKS1 cloned as a gene encoding a negative regulator of the RAS-cAMP pathway. We conclude that Lys80p is a pleiotropic regulatory factor rather than a specific repressor of LYS genes. © 1997 John Wiley & Sons, Ltd.
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  • 30
    ISSN: 0749-503X
    Keywords: acyl-CoA binding protein ; ACB1 ; Saccharomyces cerevisiae ; Saccharomyces carlsbergensis ; Saccharomyces monacensis ; brewing yeasts ; hybrid yeast ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: Saccharomyces carlsbergensis is an amphiploid, and it has previously been suggested that the genomes of S. carlsbergensis originate from S. cerevisiae and S. monacensis. We have cloned the ACB1 genes encoding the acyl-CoA binding protein (ACBP) from S. carlsbergensis, S. cerevisiae and S. monacensis. Two genes were found in S. carlsbergensis and named ACB1 type 1 and type 2, respectively. The type 1 gene is identical to the S. cerevisiae ACB1 gene except for three substitutions, one single base pair deletion and one double base pair insertion, all located in the promoter region. The type 2 gene is completely identical to the S. monacensis ACB1 gene. These findings substantiate the notion that S. carlsbergensis is a hybrid between S. cerevisiae and S. monacensis.Both ACB1 type 1 and type 2 are actively transcribed in S. carlsbergensis and transcription is initiated at sites identical to those used for transcriptional initiation of the ACB1 genes in S. cerevisiae and S. monacensis, respectively. Two polyadenylation sites, spaced 225 bp apart, are present in the S. cerevisiae ACB1 gene. The upstream polyadenylation site is used exclusively during exponential growth, whereas both sites are utilized during later stages of growth. All sequence information is listed under EMBL Accession Numbers Y08687, Y08688, Y08689 and Y08690. © 1997 John Wiley & Sons, Ltd.
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  • 31
    ISSN: 0749-503X
    Keywords: peroxisome biogenesis ; peroxisomal protein import ; peroxisomal membrane protein ; peroxisome proliferation ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: Pex3p has been implicated in the biosynthesis of the peroxisomal membrane of the yeast Hansenula polymorpha. Here we show that in the initial stages of a sharp increase in Pex3p levels, induced in batch cultures of cells of a constructed H. polymorpha strain, which contained seven copies of PEX3 under control of the alcohol oxidase promoter (WT::PAOX.PEX37x), strongly interfered with normal peroxisome proliferation. Ultrastructural studies demonstrated that in such cells numerous small peroxisomes had developed, which were absent in wild-type controls. These organelles, which contained typical peroxisomal matrix and membrane proteins (alcohol oxidase, catalase, Pex3p, Pex10p and Pex14p), showed a relatively low density (1·18 g cm-3) after sucrose gradient centrifugation of WT::PAOX.PEX37x homogenates, compared to normal peroxisomes (1·23 g cm-3). We furthermore demonstrated that these early induced, small peroxisomes were protected against glucose-induced proteolytic degradation and did not fuse to form larger organelles. Remarkably, the induction of these small peroxisomes was paralleled by a partial defect in matrix protein import, reflected by the mislocalization of minor amounts of alcohol oxidase protein in the cytosol. However, when the cells were subsequently placed under conditions in which the synthesis of a new matrix enzyme (amine oxidase) was induced while simultaneously the excessive proliferation was repressed (by repression of the PAOX), amine oxidase protein was selectively incorporated into these organelles. This indicated that the small peroxisomes had regained a normal protein import capacity. Based on these results we argue that peroxisome proliferation and matrix protein import are coupled processes in H. polymorpha. © 1997 John Wiley & Sons, Ltd.
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  • 32
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    Yeast 13 (1997), S. 1535-1545 
    ISSN: 0749-503X
    Keywords: DNA repair ; GFP ; RAD54 ; recombination ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: The green fluorescent protein (GFP) of Aequorea victoria is now an established marker for gene expression and subcellular localization in budding yeast. Relatively high expression (greater than 2500 copies per cell) of GFP is required for direct microscopic visualization. This report provides a method for studying the expression of less highly expressed genes by the analysis of crude cell extracts - a simple and cheap alternative to the fluorescent activated cell sorter (FACS). The utility of this marker is demonstrated in a study of the expression of the RAD54 gene. It is shown that the induction of the RAD54 promoter leads to the accumulation of Rad54p and of GFP and that the fluorescence induction is correctly regulated. This method should allow the screening of large numbers of novel gene disruptants for their effects on RAD54 expression and so identify trans-acting factors involved in the cellular response to DNA damage. © 1997 John Wiley & Sons, Ltd.
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  • 33
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    Yeast 14 (1998), S. 147-160 
    ISSN: 0749-503X
    Keywords: yeast physiology ; yeast viability ; flow cytometry ; bakers yeast ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: Yeast suspensions were analysed by flow cytometry after dye staining for determination of total and viable cell densities. Results were comparable to traditional colony counting and, in addition, provided further information on the percentage of total cells that were viable. The flow cytometric methods provided results within 20 min whereas colony counts were not available until 36 h. We evaluated a number of fluorescent dyes: ChemChrome Y (CY), oxonol (Ox), propidium iodide (PI), Fungolight and rhodamine 123, for accurate determination of viability of industrial yeast cultures and freshly re-hydrated high activity dried yeast (HADY). PI, Ox and CY gave the most conclusive live/dead discrimination and were the simplest to use. Culturing after dye staining and cell sorting demonstrated that the yeast remained viable after cell sorting and incubation with PI, CY or Ox. The methods, therefore, permit physical selection of individual yeast cells from populations of mixed viability. Sorting demonstrated that PI stained non-culturable cells whilst CY stained culturable cells. Analysis of yeast stained simultaneously with CY and PI or with Ox and PI demonstrated that PI and CY assays were in mutual agreement with respect to viability assessments. The Ox assay was in agreement with CY and PI for live/heat-killed mixtures. However, for re-hydrated HADY, Ox stained a significantly (P≤0·05) higher proportion of cells than did PI. © 1998 John Wiley & Sons, Ltd.
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  • 34
    ISSN: 0749-503X
    Keywords: GPM2 ; GPM3 ; phosphoglycerate mutase ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: Our previous data indicated that GPM1 encodes the only functional phosphoglycerate mutase in yeast. However, in the course of the yeast genome sequencing project, two homologous sequences, designated GPM2 and GPM3, were detected. They have been further investigated in this work. Key residues in the deduced amino acid sequence, shown to be involved in catalysis for Gpm1 (i.e. His8, Arg59, His181) are conserved in both enzymes. Overexpression of the genes under control of their own promoters in a gpm1 deletion mutant did not complement for any of the phenotypes. This could in part be attributed to a lack of expression due to their weak promoters. Higher level expression under the control of the yeast PFK2 promoter partially complemented the gpm1 defects, without restoring detectable enzymatic activity. Nevertheless, deletion of either GPM2 or GPM3, or the two deletions in concert, did not produce any obvious lesions for growth on a variety of different carbon sources, nor did they change the levels of key intermediary metabolites. We conclude that both genes evolved from duplication events and that they probably constitute non-functional homologues in yeast.
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  • 35
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    Yeast 14 (1998), S. 287-295 
    ISSN: 0749-503X
    Keywords: Candida maltosa ; Candida albicans ; tRNA splicing gene ; silent genes ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: The tRNA splicing gene SPL1-1 has been cloned and sequenced in Saccharomyces cerevisiae (Kolman and Soll, 1993). Sequence adjacent to the LEU2 gene in Candida maltosa showed some homology to the SPL1-1 gene of S. cerevisiae. This work describes the sequencing of the SPL1 tRNA splicing genes from C. maltosa and C. albicans and the analysis of these genes. Comparison of these sequences and the relationship observed between the LEU2 and SPL1 genes in these yeasts suggests that there may be some synteny amongst various species of yeasts. The coding region of the C. maltosa SPL1 region described in this work differs from previously described partial sequences in that it is a complete uninterrupted open reading frame. Two strains of C. maltosa were each shown to contain different alleles, one uninterrupted open reading frame and one disrupted open reading frame. The sequences have been deposited in the GenBank/EMBL data libraries under Accession Numbers X72940, AF000115, AF000116, AF000117, AF000118, AF000119 and AF000120. © 1998 John Wiley & Sons, Ltd.
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  • 36
    ISSN: 0749-503X
    Keywords: Oligotrophic yeasts ; low-nutrient conditions ; starvation ; Cryptococcaceae ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: Seven oligotrophic yeasts, which can grow in a 104-fold dilution of malt-yeast-glucose-peptone medium (10-4 YM), were mainly isolated from soil. These yeasts belong to the Cryptococcaceae. When inoculated at about 102 cells/ml in 10-4 YM, the isolates grew to 1·4×103-2·4×105 cells/ml after 3 days. Some culture collection yeasts fell into three groups according to their growth characteristics in 10-4 YM, one group showing characteristics of the oligotrophic yeasts. The half-saturation values of uptake by the five isolated oligotrophic yeasts for D-glucose, L-leucine and L-amino acids were 6·0-25·0, 1·7-43·3 and 3·5-21·6 μM, respectively. The oligotrophic yeasts suspended in 10 mM-phosphate buffer (pH 6·0) had high tolerances for starvation, and remained more than 15% viable after 90 days of starvation. © 1998 John Wiley & Sons, Ltd.
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  • 37
    ISSN: 0749-503X
    Keywords: Saccharomyces cerevisiae ; mitochondria ; mitochondrial matrix ; homo-oligomeric protein ; Mam33p ; gene disruption ; gC1q-R ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: Mam33p (mitochondrial acidic matrix protein) is a soluble protein, located in mitochondria of Saccharomyces cerevisiae. It is synthesized as a precursor with an N-terminal mitochondrial targeting sequence that is processed on import. Mam33p assembles to a homo-oligomeric complex in the mitochondrial matrix. It can bind to the sorting signal of cytochrome b2 that directs this protein into the intermembrane space. Mam33p is encoded by an 801 bp open reading frame. Gene disruption did not result in a significant growth defect. Mam33p exhibits sequence similarity to gC1q-R, a human protein that has been implicated in the binding of complement factor C1q and kininogen. © 1998 John Wiley & Sons, Ltd.
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  • 38
    ISSN: 0749-503X
    Keywords: phosphofructokinase-1 ; Saccharomyces cerevisiae ; deletion mutants ; reactivation ; assembly ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: Phosphofructokinase-1 from Saccharomyces cerevisiae is an octameric enzyme comprising two non-identical subunits, α and β, which are encoded by the unlinked genes PFK1 and PFK2. In this paper, assembly and reactivation of the enzyme have been studied in cell-free extracts of single-deletion mutants. In contrast to the previously described lack of phosphofructokinase-1 activity in cell-free extracts of these mutants, we could measure a temporary enzyme activity immediately after lysis of protoplasts. This result supports the assumption that each of the subunits forms an enzyme structure which is active in vivo but not stable after cell disruption.Upon mixing of separately prepared cell-free extracts of both deletion mutants very low activity could be measured. About 40% of the wild-type activity was regained when both mutants were mixed prior to disruption. The reactivation rate could be slightly increased by addition of ATP and fructose 6-phosphate and was found to be a function of the growth state, particularly of the β-subunit-carrying cells. The individual subunits did not interact with Cibacron Blue F3G-A, a biomimetic ligand of phosphofructokinase-1. After reassembly of both subunits in vitro a strong affinity of the reconstituted phosphofructokinase-1 to the dye-ligand was observed.The inability of the subunits to reconstitute under certain conditions seems to result from alterations of the intracellular environment following disruption. These changes give rise to induce an unproductive side reaction like self-aggregation of the subunits.Because reconstitution of phosphofructokinase-1 from S. cerevisiae behaves in a similar way to that of hemoglobin and luciferase, we would speculate a general mechanism for assembly of oligomeric proteins in vivo. © 1998 John Wiley & Sons, Ltd.
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  • 39
    ISSN: 0749-503X
    Keywords: Saccharomyces ; redox ; glycerol ; NADH ; shuttle ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: Maintenance of a cytoplasmic redox balance is a necessity for sustained cellular metabolism. Glycerol formation is the only way by which Saccharomyces cerevisiae can maintain this balance under anaerobic conditions. Aerobically, on the other hand, several different redox adjustment mechanisms exist, one of these being the glycerol 3-phosphate (G3P) shuttle. We have studied the importance of this shuttle under aerobic conditions by comparing growth properties and glycerol formation of a wild-type strain with that of gut2Δ mutants, lacking the FAD-dependent glycerol 3-phosphate dehydrogenase, assuming that the consequent blocking of G3P oxidation is forcing the cells to produce glycerol from G3P. To impose different demands on the redox adjustment capability we used various carbon sources having different degrees of reduction.The results showed that the shuttle was used extensively with reduced substrate such as ethanol, whereas the more oxidized substrates lactate and pyruvate, did not provoke any activity of the shuttle. However, the absence of a functional G3P shuttle did not affect the growth rate or growth yield of the cells, not even during growth on ethanol. Presumably, there must be alternative systems for maintaining a cytoplasmic redox balance, e.g. the so-called external NADH dehydrogenase, located on the outer side of the inner mitochondrial membrane. By comparing the performance of the external NADH dehydrogenase and the G3P shuttle in isolated mitochondria, it was found that the former resulted in high respiratory rates but a comparably low P/O ratio of 1·2, whereas the shuttle gave low rates but a high P/O ratio of 1·7.Our results also demonstrated that of the two isoforms of NAD-dependent glycerol 3-phosphate dehydrogenase, only the enzyme encoded by GPD1 appeared important for the shuttle, since the enhanced glycerol production that occurs in a gut2Δ strain proved dependent on GPD1 but not on GPD2. © 1998 John Wiley & Sons, Ltd.
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  • 40
    ISSN: 0749-503X
    Keywords: modified LFH cassette ; EUROFAN 6-pack analysis ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: Six open reading frames (ORFs) of unknown function from Saccharomyces cerevisiae chromosome XV, three from the left and three from the right arm, were deleted in two diploid strains by the short flanking homology method (Wach et al., 1994). Transformants were selected as Geneticin (G418)-resistant colonies and correct integration of the kanMX4 cassette was checked by colony PCR. Following sporulation of the diploids, tetrads were dissected and scored for the segregation of the G418-resistant marker. We have developed a widely applicable method for the construction of gap repair plasmids to obtain the cognate clones for each of the disrupted ORFs. The 5′- and 3′-flanks of the ORF in question are linked by a unique restriction endonuclease. When the plasmid is cut at this site it can be used to obtain, by selection for the appropriate antibiotic resistance, long flanking homology (LFH) cassettes containing the cognate clone or the disrupted allele. The LFH cassette containing the cognate clone or the disrupted allele can be released from the gap-repaired plasmid by cutting at the inserted flanking restriction sites. One of the six ORFs (YOR319w) corresponds to an essential gene whose product is part of the spliceosome complex. Haploid as well as homozygous and heterozygous diploid disruptant strains for each of the five non-essential ORFs were subjected to growth test on different media at 15°C, 30°C and 37°C. Disruption of YOR322c causes osmotically sensitive growth on YEPD at 37°C and the product of YOL091w appears to play a role in sporulation since the homozygous diploid disruptant has lost the ability to sporulate. © 1998 John Wiley & Sons, Ltd.
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  • 41
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    Yeast 14 (1998), S. 583-586 
    ISSN: 0749-503X
    Keywords: Saccharomyces exiguus ; STE3 ; homolog ; sequence analysis ; differentiation of species ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: Homologs of Saccharomyces cerevisiae STE3, a-factor receptor gene were detected from S. exiguus NFRI 3539 by low stringency Southern hybridization. This strain might have at least two types of homolog. One of these homologs, designated as e-STE3 was cloned. Its nucleotide sequence revealed 60% identity to STE3. The putative protein coding region consisted of 453 amino acid residues. The amino acid sequence identity between STE3 and e-STE3 was 62%, and that of the N-terminal 303 amino acid residues considered to be the pheromone binding domain was 79%. The e-STE3 sequence submitted to the DDBJ/EMBL/GenBank data libraries is available under Accession Number AB003086. © 1998 John Wiley & Sons, Ltd.
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  • 42
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    Yeast 14 (1998), S. 565-571 
    ISSN: 0749-503X
    Keywords: Saccharomyces cerevisiae ; electroporation ; transformation ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: Pretreatment of yeast cells with lithium acetate (LiAc) and dithiothreitol (DTT) enhances the frequency of transformation by electroporation. The method shows improvements of 6-67-fold in wild-type strains derived from commonly used Saccharomyces cerevisiae genetic backgrounds. In addition, 15-300-fold improvement in transformation frequency was achieved with several mutant strains of S. cerevisiae that transformed poorly by conventional procedures. Both DTT and lithium acetate were necessary for maximal transformation frequencies. Pretreatment with lithium and DTT also resulted in an ∼3·5-fold increase in the electroporation transformation frequency of the pathogenic fungus Candida albicans. © 1998 John Wiley & Sons, Ltd.
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  • 43
    ISSN: 0749-503X
    Keywords: multidrug resistance ; CDR1 ; ABC transporter ; baculovirus expression ; C. albicans ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: Cdr1p, an ATP-binding cassette transporter from the pathogenic yeast Candida albicans, confers resistance to several unrelated drugs including anti-Candida drugs (Prasad et al., 1995b). We demonstrate that the deletion of 237 bp (79 aa) from the 3′ end of CDR1 (which encompasses the transmembrane domain (TM) 12 of the putative transporter) did not result in the total loss of its ability to efflux cytotoxic agents. While the expression of ΔCDR1 in yeast resulted in impaired sensitivity to drugs like cycloheximide, anisomycin, sulfomethuron methyl and antifungal nystatin, its ability to confer resistance remained unaltered to drugs such as o-phenanthroline, 4-nitroquinoline-N-oxide, cerulenin, azoles, oligomycin, erythromycin, and benomyl. Similar to human MDR1p, Cdr1p might also have localized drug binding sites in TM 12, but that might not be the case for all the drugs. The TM 12 deletion also did not lead to any significant impairment in NTPase activities. Both ATPase and UTPase activities of complete Cdr1p and ΔCdr1p were not significantly altered, as was the case with respect to their ability to efflux Rh123 and steroid hormone like [3H]-β-estradiol. To further dissect the functionality of Cdr1p, its truncated version was overexpressed in a baculovirus-insect cell expression system. The synthesis of ΔCdr1p in Sf9 cells was temporally regulated as a function of the baculovirus polyhedrin gene promoter. The Sf9 derived ΔCdr1p was ∼130 kDa, which was lower than the expected size, probably due to the differences in glycosylation. This, however, did not affect the functionality of ΔCdr1p. The deletion of TM 12 did not affect the targeting of the protein and ΔCdr1p was exclusively localized in plasma membrane of Sf9 cells as detected by immunofluorescence. The expression of ΔCdr1p in the baculovirus-insect expression system generated a high drug-stimulated plasma membrane-bound ATPase activity which was not demonstrable when ΔCdr1p was expressed in yeast. © 1998 John Wiley & Sons, Ltd.
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  • 44
    ISSN: 0749-503X
    Keywords: lys1+ gene ; Schizosaccharomyces pombe ; α-aminoadipate reductase ; peptide synthetase ; lysine biosynthesis ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: The 4·2 kbp lys1+ gene of Schizosaccharomyces pombe encoding the large subunit of α-aminoadipate reductase (EC1.2.1.31), an enzyme specific to lysine synthesis in higher fungi, was completely sequenced at the nucleotide level from pLYS1H. The S. pombe lys1+ gene product consists of 1415 amino acid residues and has a putative molecular weight of 155·8 kDa. The encoded protein converts α-aminoadipic acid to α-aminoadipate-δ-semialdehyde by an ATP-mediated adenylation. Analysis of the sequence showed that the putative protein encoded by lys1+ shares strong homology with the peptide antibiotic synthetases which also use an adenylation step. The sequence data reported in this paper have been submitted to GenBank database (Washington DC, USA) under the Accession Number U15923. © 1998 John Wiley & Sons, Ltd.
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  • 45
    ISSN: 0749-503X
    Keywords: Apiotrichum curvatum ; cDNA sequence ; DnaJ protein ; cytosolic Hsp70s ; ribosome association ; Sis1 protein ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: DnaJ proteins have been localized in different intracellular compartments of eukaryotes. In Apiotrichum curvatum, a fat-storing yeast, we found a DnaJ homolog associated with ribosomes and large cytosolic complexes as well. Using a plant DnaJ probe and a cDNA library constructed from poly(A)+ -RNA of A. curvatum grown on oleate we isolated a SIS1 cDNA coding for a 39·5 kDa protein. The putative protein contains neither a zinc finger motif nor a CAAX motif but is characterized by a J-domain at the N-terminal region and a large G-rich region in the middle part of the molecule. Heat shock applied for 1 h resulted in a pronounced but transient increase of the SIS1 mRNA. An antiserum was raised against the bacterially expressed protein. Cell fractions from A. curvatum were further separated by sedimentation centrifugation on sucrose gradients. Analysing the sub-fractions, we detected Sis1p mainly associated with ribosomes, and with particles sedimenting at approximately 200S. Hsp70 was found to be associated with the 200S fraction. The respective cytosolic A. curvatum Hsp70 cDNA was cloned and sequenced. High salt conditions caused the removal of Hsp70 and Sis1p from the 200S complexes. Mild RNase treatment of the 200S fraction afforded monosomes and 200S complexes unaffected by RNase. Heat shock led to a pronounced increase in the rate of de novo synthesis. However, due to the large pools of Sis1p on ribosomes and large cytosolic complexes, the increase in gene activation did not lead to a significant change of the total amount of Sis1p. Accession numbers are: Y12079 for ACHSP70 and Y12080 for ACSIS1. © 1998 John Wiley & Sons, Ltd.
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  • 46
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    Yeast 14 (1998), S. 443-457 
    ISSN: 0749-503X
    Keywords: evolution ; polyploidy ; gene duplication ; gene order ; LEU2 ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: The extent to which the order of genes along chromosomes is conserved between Saccharomyces cerevisiae and related species was studied by analysing data from DNA sequence databases. As expected, the extent of gene order conservation decreases with increasing evolutionary distance. About 59% of adjacent gene pairs in Kluyveromyces lactis or K. marxianus are also adjacent in S. cerevisiae, and a further 16% of Kluyveromyces neighbours can be explained in terms of the inferred ancestral gene order in Saccharomyces prior to the occurrence of an ancient whole-genome duplication. Only 13% of Candida albicans linkages, and no Schizosaccharomyces pombe linkages, are conserved. Analysis of gene order arrangements, chromosome numbers, and ribosomal RNA sequences suggests that genome duplication occurred before the divergence of the four species in Saccharomyces sensu stricto (all of which have 16 chromosomes), but after this lineage had diverged from Saccharomyces kluyveri and the Kluyveromyces lactis/marxianus species assemblage. © 1998 John Wiley & Sons, Ltd.
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  • 47
    ISSN: 0749-503X
    Keywords: Crabtree effect ; yeast ; biomass ; Kluyveromyces lactis ; oxygen ; pyruvate decarboxylase ; regulation ; fermentation ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: Kluyveromyces lactis is an important industrial yeast, as well as a popular laboratory model. There is currently no consensus in the literature on the physiology of this yeast, in particular with respect to aerobic alcoholic fermentation (‘Crabtree effect’). This study deals with regulation of alcoholic fermentation in K. lactis CBS 2359, a proposed reference strain for molecular studies. In aerobic, glucose-limited chemostat cultures (D=0·05-0·40 h-1) growth was entirely respiratory, without significant accumulation of ethanol or other metabolites. Alcoholic fermentation occurred in glucose-grown shake-flask cultures, but was absent during batch cultivation on glucose in fermenters under strictly aerobic conditions. This indicated that ethanol formation in the shake-flask cultures resulted from oxygen limitation. Indeed, when the oxygen feed to steady-state chemostat cultures (D=0·10 h-1) was lowered, a mixed respirofermentative metabolism only occurred at very low dissolved oxygen concentrations (less than 1% of air saturation). The onset of respirofermentative metabolism as a result of oxygen limitation was accompanied by an increase of the levels of pyruvate decarboxylase and alcohol dehydrogenase. When aerobic, glucose-limited chemostat cultures (D=0·10 h-1) were pulsed with excess glucose, ethanol production did not occur during the first 40 min after the pulse. However, a slow aerobic ethanol formation was invariably observed after this period. Since alcoholic fermentation did not occur in aerobic batch cultures this is probably a transient response, caused by an imbalanced adjustment of enzyme levels during the transition from steady-state growth at μ=0·10 h-1 to growth at μmax. It is concluded that in K. lactis, as in other Crabtree-negative yeasts, the primary environmental trigger for occurrence of alcoholic fermentation is oxygen limitation. © 1998 John Wiley & Sons, Ltd.
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  • 48
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    Yeast 14 (1998), S. 501-515 
    ISSN: 0749-503X
    Keywords: Saccharomyces cerevisiae ; bakers' yeast ; pH homeostasis ; cytoplasmic pH ; vacuolar pH ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: The effects of monovalent cations on the internal pH of yeast were studied. Our former procedure was modified, inducing maximal alkalinization of the cells with 100 mM-NH4OH instead of Tris base. The pH values were lower than reported before (Peña et al., J. Bacteriol. 1995 177, 1017-1022). With glucose as substrate, the internal cytoplasmic pH reached higher values when incubating at an external pH of 6·0, as compared to pH 4·0. Monovalent cations added approximately 5 min after glucose produced a further increase in the internal pH, which was higher at a previous incubation pH of 4·0 than that observed at pH 6·0. The selectivity of the changes followed a similar order to that of the transport system for monovalent cations.When incubating cells with glucose for more than 30 min, the initial changes of the internal pH appeared to be regulated by the cell. However, under the fluorescence microscope, it was observed that pyranine, which was confined to the cytoplasm during the first 15 min, was progressively concentrated in the vacuole. By studying the fluorescence changes of cells electroporated and then incubated with glucose or glucose plus potassium, we could follow the internal pH of this organelle, obtaining values within the range reported by other authors. Also, in cells preincubated with glucose for 60 min, and electroporated afterwards, the fluorescence of pyranine, which only entered the cytoplasm, allowed us to measure the pH of this compartment, showing that it was more alkaline than the vacuole. Moreover, the cytoplasmic pH increased upon addition of glucose or potassium. The vacuolar pH, on the other hand, increased upon addition of potassium after glucose, but decreased upon addition of glucose. In addition, incubation of the cells with glucose with or without pyranine produced vesiculation of the vacuole. © 1998 John Wiley & Sons, Ltd.
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  • 49
    ISSN: 0749-503X
    Keywords: yeast ; S. cerevisiae ; genetics ; karyotypes ; chromosomal rearrangements ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: In order to study meiotic segregation of chromosome length polymorphism in yeast, we analysed the progeny of a cross involving two laboratory strains FL100trp and YNN295. Analysis of the parental strains led us to detect an important length polymorphism of chromosomes I and III in FL100trp. A reciprocal translocation involving 80 kb of the left arm of chromosome III and 45 kb of the right arm of chromosome I was shown to be the cause for the observed polymorphism in this strain. The characterization of the translocation breakpoints revealed the existence of a transposition hot-spot on chromosome I: the sequence of the translocation joints on chromosomes I and III suggests that the mechanism very likely involved homologous recombination between Ty2 transposable elements on each chromosome. Analysis of FL100, FL200 and FL100trp ura, which are related to FL100trp, shows that this reciprocal translocation is present in some of the strains of the FL series, whereas the parental strain FL100 does not carry the same rearrangement. We evidenced instead the duplication of 80 kb of chromosome III on chromosome I and a deletion of 45 kb of the right arm of chromosome I in this strain, indicating that secondary events might have taken place and that the strain currently named FL100 is not the common ancestor of the FL series. © 1998 John Wiley & Sons, Ltd.
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  • 50
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    Yeast 14 (1998), S. 665-674 
    ISSN: 0749-503X
    Keywords: histidine kinase ; phosphorylation ; signal transduction ; gene ; two-component ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: We have cloned and analysed the sequence of a putative histidine kinase, two-component gene (CaHK1) from Candida albicans. This gene encodes a 2471 amino acid protein (Cahk1p) with an estimated molecular mass of 281·8 kDa. A homology search of Cahk1p with other proteins in the databases showed that Cahk1p exhibits the greatest homology at its C-terminus with both the sensor and regulator components of prokaryotic and eukaryotic two-component histidine kinases. A further analysis of this homology showed that the Cahk1p possessed both sensor and regulator domains in the same polypeptide. Also, Cahk1p is likely to be a soluble protein. The sensor kinase domain of Cahk1p contains conserved motifs that are characteristic of all histidine kinase proteins, including the putative histidine which is believed to be autophosphorylated during activation, ATP binding motifs and others (F- and N-motifs), with unknown function. The Cahk1p regulator domain also contains conserved aspartate and lysine residues and the putative aspartate, which is secondarily phosphorylated by the autophosphorylated histidine. Finally, according to the codon usage frequency of the CaHK1 gene in comparison with other genes from C. albicans, there would appear to be a low level of expression of the gene. The accession number for the described sequence is AF013273, as filed in the EMBL/GenBank/DDBJ database. © 1998 John Wiley & Sons, Ltd.
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  • 51
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    Yeast 14 (1998), S. 655-664 
    ISSN: 0749-503X
    Keywords: Saccharomyces cerevisiae ; PCR-based disruption ; YOL113w ; YOL100w ; YOL107w ; YOR267c ; YGL196w ; YGL194c ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: Using PCR to construct disruption cassettes, null alleles of six genes have been created in Saccharomyces cerevisiae. In a FY1679 background, no defects were detected in any of the haploid deletion mutants with respect to growth, gross morphology, or mating. A diploid FY1679-derived Δygl194c/Δygl194c homozygous disruptant displayed reduced sporulation. In contrast to the lack of phenotypic consequences of Δyol100w disruptions in the FY1679 background, in the CEN.PK2 strain even a heterozygous disruption of the same gene caused striking effects, very slow vegetative growth and highly impaired sporulation. Tetrad analysis showed YOL100w to be an essential gene in this strain. A copy of the YGL194c or the YOL100w wild-type gene borne on a centromeric episomal plasmid was introduced into a corresponding disruption mutant strain, and in both cases was found to partially complement the defects. © 1998 John Wiley & Sons, Ltd.
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  • 52
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    Yeast 14 (1998), S. 1115-1125 
    ISSN: 0749-503X
    Keywords: Yarrowia lipolytica ; secretion ; pH ; extracellular protease ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: The secretion and maturation of the acid extracellular protease (AXP) of the yeast Yarrowia lipolytica have been characterized using antiserum raised against this enzyme. A 42 kDa pro-enzyme form of AXP was identified from lysates of radiolabelled Y. lipolytica cells and found to contain no N-linked carbohydrate moieties. Using pulse-chase immune precipitation it was demonstrated that the AXP precursor was secreted into the extracellular medium where, under conditions of low pH, it underwent autocatalytic activation forming the mature enzyme. Conversion of the AXP pro-form in the presence of the protease inhibitor pepstatin indicated that an intramolecularly-catalysed reaction mechanism was involved in AXP maturation. Further evidence supporting the role of autocatalytic processing came from the side-chain specificity of mature AXP towards the oxidized B-chain of insulin. © 1998 John Wiley & Sons, Ltd.
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  • 53
    ISSN: 0749-503X
    Keywords: cell walls ; protease ; β-glucanase ; lysis ; yeast ; antifungal drugs ; glucan ; mannoprotein ; S. cerevisiae ; C. albicans ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: The rate of formation of spheroplasts of yeast can be used as an assay to study the structural integrity of cell walls. Lysis can be measured spectrophotometrically in hypotonic solution in the presence of Zymolyase, a mixture of cell wall-digesting enzymes. The optical density of the cell suspension decreases as the cells lyse. We optimized this assay with respect to enzyme concentration, temperature, pH, and growth conditions for several strains of Saccharomyces cerevisiae. The level of variability (standard deviation) was 1-5% between trials where the replications were performed on the same culture using enzyme prepared from the same lot, and 5-15% for different cultures of the same strain. This assay can quantitate differences in cell wall structure (1) between exponentially growing and stationary phase cells, (2) among different S. cerevisiae strains, (3) between S. cerevisiae and Candida albicans, (4) between parental and mutated lines, and (5) between drug- or chemically-treated cells and controls. © 1998 John Wiley & Sons, Ltd.
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  • 54
    ISSN: 0749-503X
    Keywords: carbocyanine fluorescent probes ; membrane potential ; yeast ; cell wall ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: Membrane-potential-dependent accumulation of diS-C3(3) in intact yeast cells in suspension is accompanied by a red shift of the maximum of its fluorescence emission spectrum, λmax, caused by a readily reversible probe binding to cell constituents. Membrane depolarization by external KCl (with or without valinomycin) or by ionophores causes a fast and reproducible blue shift. As the potential-reporting parameter, the λmax shift is less affected by probe binding to cuvette walls and possible photobleaching than, for example, fluorescence intensity. The magnitude of the potential-dependent red λmax shift depends on relative cell-to-probe concentration ratio, a maximum shift (572→582 nm) being found in very thick suspensions and in cell lysates. The potential therefore has to be assessed at reasonably low cell (≤5×106 cells/ml) and probe (10-7 M) concentrations at which a clearly defined relationship exists between the λmax shift and the potential-dependent accumulation of the dye in the cells. The redistribution of the probe between the medium and yeast protoplasts takes about 5 min, but in intact cells it takes 10-30 min because the cell wall acts as a barrier, hampering probe penetration into the cells. The barrier properties of the cell wall correlate with its thickness: cells grown in 0·2% glucose (cell wall thickness 0·175±0·015 μm, n=30) are stained much faster and the λmax is more red-shifted than in cells grown in 2% glucose (cell wall thickness 0·260±0·043 μm, n=44). At a suitable cell and probe concentration and under standard conditions, the λmax shift of diS-C3(3) fluorescence provides reliable information on even fast changes in membrane potential in Saccharomyces cerevisiae. © 1998 John Wiley & Sons, Ltd.
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  • 55
    ISSN: 0749-503X
    Keywords: critical target model ; differential scanning calorimetry ; Saccharomyces cerevisiae ; heat-shock response ; acquired thermotolerance ; thermal stability ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: Hyperthermic cell killing profiles of Saccharomyces cerevisiae cells were biphasic and a shoulder (phase 1) was followed by an exponential killing (phase 2). Assuming that (i) the rate of thermal damage in particular macromolecules or their assemblies limits the rate of hyperthermic cell killing (the critical target model), and (ii) the damages of two families of targets are lethal independently, we built a ‘dual critical target model’ in order to interpret the biphasic cell killing.Time-courses of temperature-programmed fractional survival were traced for S. cerevisiae cells in exponentially growing phase, heat shocked, and in stationary phase. Non-linear curve-fitting of the time-courses by using the dual critial target model provided the Arrhenius parameters of denaturation of the two families of targets. The cells were killed more slowly in phase 1 than in phase 2. Arrest in stationary phase, not heat shock, stabilizes the family of targets that is critical to phase 1 death. On the other hand, both heat-shock response and arrest in stationary phase stabilizes the other family of targets that, in addition to the previous one, is responsible for phase 2 death. © 1998 John Wiley & Sons, Ltd.
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  • 56
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    Yeast 14 (1998), S. 1439-1451 
    ISSN: 0749-503X
    Keywords: beginnings of yeast research ; yeasts ; history ; Lavoisier ; Cagniard-Latour ; Kutzing ; Schwann ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
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  • 57
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    Yeast 14 (1998), S. 1069-1087 
    ISSN: 0749-503X
    Keywords: amines and amides ; biodetergents ; biocides ; bioremediation ; biosensors ; Candida rugosa ; carbohydrate esters ; cosmetics and perfumery ; food and flavour ; immobilisation ; isoenzymes ; lipases ; molecular biology ; pharmaceuticals ; single cell protein ; specificity ; tanning ; ultra-structure ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: This review describes how the versatile Candida rugosa lipases (CRL) have extended the frontiers of biotechnology. As evidenced by the current literature, CRL claims more applications than any other biocatalyst. This review comprises a detailed discussion on the molecular biology of CRL, its versatile catalytic reactions, broad specificities and diverse immobilization strategies. It also discusses its role in the food and flavour industry, the production of ice cream and single cell protein, biocatalytic resolution of life-saving pharmaceuticals, carbohydrate esters and amino acid derivatives unobtainable by conventional chemical synthesis, potent biocide making, biosensor modulations, eco-friendly approach and bioremediation, biosurfactants in detergent making, and recently, cosmetics and perfumery. © 1998 John Wiley & Sons, Ltd.
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  • 58
    ISSN: 0749-503X
    Keywords: transcription ; microarrays ; expression profiling ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: Open reading frames (6116) of the budding yeast Saccharomyces cerevisiae were PCR-amplified from genomic DNA using 12,232 primers specific to the ends of the coding sequences; the success rate of amplification was 97%. PCR-products were made accessible to hybridization by being arrayed at very high density on solid support media using various robotic devices. Probes made from total RNA preparations were hybridized for the analysis of the transcriptional activity of yeast under various growth conditions and of different strains. Experimental factors that proved critical to the performance, such as different RNA isolation procedures and the assessment of hybridization results, for example, were investigated in detail. Various software tools were developed that permit convenient handling and sound analysis of the large data quantities obtained from transcriptional profiling studies. Comprehensive arrays are being distributed within the European Yeast Functional Analysis Network (EUROFAN) and beyond. © 1998 John Wiley & Sons, Ltd.
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  • 59
    ISSN: 0749-503X
    Keywords: yeast ; PMR1 ; Hansenula polymorpha ; Ca2+-ATPase ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: A gene homologous to Saccharomyces cerevisiae PMR1 has been cloned in the methylotrophic yeast Hansenula polymorpha. The partial DNA fragment of the H. polymorpha homologue was initially obtained by a polymerase chain reaction and used to isolate the entire gene which encodes a protein of 918 amino acids. The putative gene product contains all ten of the conserved regions observed in P-type ATPases. The cloned gene product exhibits 60·3% amino acid identity to the S. cerevisiae PMR1 gene product and complemented the growth defect of a S. cerevisiae pmr1 null mutant in the EGTA-containing medium. The results demonstrate that the H. polymorpha gene encodes the functional homologue of the S. cerevisiae PMR1 gene product, a P-type Ca2+-ATPase. The DNA sequence of the H. polymorpha homologue has been submitted to GenBank with the Accession Number U92083. © 1998 John Wiley & Sons, Ltd.
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  • 60
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    Yeast 14 (1998), S. 1257-1265 
    ISSN: 0749-503X
    Keywords: Candida albicans ; nucleoside transport ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: The nucleoside permease gene (i.e. NUP) from Candida albicans was cloned by complementation of Saccharomyces cerevisiae deficient in nucleoside transport capability. The permease transported adenosine and guanosine and was sensitive to the mammalian nucleoside transport inhibitors: dipyridamole and NBMPR. It did not transport uridine, cytidine, adenine, guanine or uracil. The inability to transport uridine indicated that the NUP gene product was different from the Candida uridine permease, which also transported cytosine and adenosine. The NUP gene coded for a protein of 407 amino acids in size which was approximately the size of the human, Giardia and E. coli nucleoside permeases. It did not, however, exhibit any significant degree of homology with these transporters. The GenBank accession number for the Candida NUP gene is AF016246. © 1998 John Wiley & Sons, Ltd.
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  • 61
    ISSN: 0749-503X
    Keywords: transcription factor ; yeast genetic selection ; bacteriophage λ vector ; Cre-loxP automatic plasmid subcloning ; integration vector ; one hybrid ; target genes ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: We describe the construction of a number of vectors that can be used in yeast genetic selection systems for cloning of transcription factors or other DNA-binding proteins and for identification of the target sites recognized by transcription factors. For transcription factor cloning we have designed an integration vector with two HIS3 reporter gene cassettes to stably integrate reporter gene constructs at the non-essential yeast PDC6 locus. This set of plasmids was tested in a one-hybrid assay with the rice transcription factor Oshox1, a member of the class of homeodomain leucine zipper proteins. A hybrid protein of Oshox1 and the Gal4 transcriptional activation domain was shown to specifically activate a reporter gene construct with upstream Oshox1 binding sites, which had been integrated at the PDC6 locus using the described vector system. Target site identification by genetic selection in yeast employs a transcriptional activator construct and a library of genomic or random DNA fragments upstream of a reporter gene. We have constructed two variants of a bacteriophage λ vector which facilitates the construction of the required reporter gene library because of high cloning efficiency and easy conversion into a yeast/Escherichia coli shuttle vector library by Cre-loxP-mediated automatic subcloning. Tests with Oxhox1 as transcriptional activator demonstrated the usefulness of the deprived reporter gene vector. © 1998 John Wiley & Sons, Ltd.
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  • 62
    ISSN: 0749-503X
    Keywords: Yarrowia lipolytica ; cytochrome P450 ; n-alkane metabolism ; n-alkane-inducible gene ; RT-PCR ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: A gene encoding cytochrome P450 involved in n-alkane utilization was cloned from an n-alkane assimilating yeast, Yarrowia lipolytica CX161-1B. The RT-PCR was performed on the mRNA prepared from the cells grown on n-alkane as a template using degenerated PCR primers designed for the conserved amino acid sequences of the CYP52 family. The RT-PCR amplified fragment was then used as a probe to isolate genes coding for P450 of the CYP52 family from the genomic DNA library of the strain CX161-1B. The nucleotide sequence of one of the positive clones was determined. An open reading frame which had the same nucleotide sequence as the RT-PCR-amplified fragment was identified. It was of 523 amino acid residues, 60·2 kDa in molecular mass, and had 30-45% sequence identity with the other members of the CYP52 family of Candida species so far analysed. The expression of the P450 gene that was named as YlALK1 was induced by n-tetradecane and repressed by glycerol. A YlALK1 gene disruptant did not grow well on n-decane, but grew on longer-chain n-alkanes such as hexadecane as a sole carbon source. Introduction of YlALK1 on a plasmid to the disruptant restored the decane assimilation. These results suggest that the YlALK1 gene product is the major P450Alk to metabolize short-chain n-alkanes such as decane and dodecane in Y. lipolytica. © 1998 John Wiley & Sons, Ltd.
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  • 63
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    Yeast 14 (1998), S. 1511-1527 
    ISSN: 0749-503X
    Keywords: Saccharomyces cerevisiae ; oxidative stress ; stress response ; signal transduction ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: All aerobically growing organisms suffer exposure to oxidative stress, caused by partially reduced forms of molecular oxygen, known as reactive oxygen species (ROS). These are highly reactive and capable of damaging cellular constituents such as DNA, lipids and proteins. Consequently, cells from many different organisms have evolved mechanisms to protect their components against ROS. This review concentrates on the oxidant defence systems of the budding yeast Saccharomyces cerevisiae , which appears to have a number of inducible adaptive stress responses to oxidants, such as H2 O2 , superoxide anion and lipid peroxidation products. The oxidative stress responses appear to be regulated, at least in part, at the level of transcription and there is considerable overlap between them and many diverse stress responses, allowing the yeast cell to integrate its response towards environmental stress. © 1998 John Wiley & Sons, Ltd.
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  • 64
    ISSN: 0749-503X
    Keywords: ADH ; hypoxic activation ; xylose fermenting yeasts ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: Two Pichia stipitis ADH genes (PsADH1 and PsADH2) were isolated by complementation of a Saccharomyces cerevisiae Adh--mutant. The genes enabled the transformants to grow in the presence of antimycin A on glucose, to use ethanol as sole carbon source and made them sensitive to allylalcohol.The sequences of the genes showed similarities of 70-77% to sequences of ADH genes of Candida albicans, Kluyveromyces lactis, K. marxianus, and S. cerevisiae and about 60% homology to those of Schizosaccharomyces pombe and Aspergillus flavus.Southern hybridization experiments suggested that P. stipitis has only these two ADH genes. Both genes are located on the largest chromosome of P. stipitis.PsADH2 encodes for the ADH activity that is responsible for ethanol formation at oxygen limitation. The gene is regulated at the transcriptional level. Moreover, also in cells grown on ethanol, only PsADH2 transcript was found. PsADH1 transcript was detected under aerobic conditions on fermentable carbon sources.The sequences have been deposited in the EMBL database under the accession numbers Y13238 (PsADH1) and Y13397 (PsADH2). © 1998 John Wiley & Sons, Ltd.
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  • 65
    ISSN: 0749-503X
    Keywords: Candida maltosa ; codon usage ; cytochrome P450 ; protein degradation ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: We demonstrate that serine instead of leucine is specified by the CUG codon in the yeast Candida maltosa. Evidence for this deviation from the universal genetic code was obtained by means of in vitro translation experiments. Depending on the cell-free system used, either serine, in the C. maltosa system, or leucine, in the control with the conventional wheat germ system, was found to be incorporated into the translation products of artificial CUG-containing mRNAs. Moreover, we were able to transfer the non-universal decoding of CUG to the wheat germ system by adding a tRNA fraction isolated from C. maltosa. This finding indicates the presence in C. maltosa of an unusual serine tRNA that recognizes CUG. As a consequence of the altered genetic code, expression in Saccharomyces cerevisiae of C. maltosa cytochrome P450 genes required an exchange of their CTG triplets by TCT encoding serine in order to produce the authentic proteins. In contrast, heterologous expression of the original C. maltosa genes resulted in the formation of still active but unstable enzymes probably subject to selective proteolysis in the host cells.
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  • 66
    ISSN: 0749-503X
    Keywords: Candida maltosa ; codon usage ; heterologous gene expression ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: An alkane-assimilating yeast Candida maltosa had been studied in order to establish systems suitable for biotransformation of hydrophobic compounds. However, functional expression of heterologous genes tested for this purpose had not been successful in several cases. On the other hand, it had been reported that the codon CUG, a universal leucine codon, is read as serine in C. cylindracea. The same altered codon usage had also been suggested by in vitro experiments in some Candida yeasts which are phylogenetically closely related to C. maltosa.In this study we have shown that the failure in functional expression of a heterologous gene is due to the fact that the codon CUG is read as serine in C. maltosa. This conclusion was drawn from the following experimental results: (1) when a cytochrome P450 gene of C. maltosa containing a CTG codon was expressed in C. maltosa, the corresponding amino acid was found to be serine, and not leucine; (2) a tRNA gene with an almost identical structure to that of the tRNA SerCAG gene of C. albicans could be isolated from the genome of C. maltosa; (3) the Saccharomyces cerevisiae URA3 gene, which has one CTG codon, could not complement the ura3 mutation of C. maltosa as itself, but when the CTG codon was changed to another leucine codon, CTC, the mutated gene could complement the ura3 mutation.The last result is the first example of succeeding in functional expression of a heterologous gene in Candida species having an altered codon usage by changing the CTG codon in the gene to another codon.The nucleotide sequence datum reported in this paper will appear in the GSDB, DDBJ, EMBL and NCBI nucleotide sequence databases with the Accession Number D26074.
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  • 67
    ISSN: 0749-503X
    Keywords: Saccharomyces cerevisiae ; chromosome II ; GAL7 ; GAL10 ; leucine zipper ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: The nucleotide sequence of a fragment of 7200 base pairs of Saccharomyces cerevisiae chromosome II has been determined. The sequence contains three open reading frames (ORFs). Two genes for galactose metabolism, GAL7 and part of the GAL10 coding region, are localized on the fragment. Comparison to the previously published sequence data showed several differences, leading to changes in the amino acid sequences of GAL7 and GAL10.One new ORF, YBR0224, was detected, coding for a protein with 918 amino acids. Comparison to the DNA and protein data bases showed no significant homologies. The protein has some interesting features pointing to a function involved in transcription regulation: a leucine zipper motif, a highly acidic region, possibly involved in transcription activation and a putative nuclear localization signal. Deletion analysis showed that the gene is essential when deleted in strain W303. Spores could germinate and form microcolonies, but efforts to propagate the colonies failed. Deletion of this gene in a different genetic background (strain M5) led to very poor-growing mutant strains with cells showing aberrant cellular morphologies.The nucleotide sequence of the fragment has been deposited in the EMBL-database under the Accession Number X81324.
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  • 68
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    Yeast 11 (1995), S. 111-119 
    ISSN: 0749-503X
    Keywords: Pichia sorbitophila ; halotolerance ; osmoregulation ; glycerol transport ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: Pichia sorbitophila is a halotolerant yeast capable of surviving to extracellular NaCl concentrations up to 4 M in mineral medium when glucose or glycerol are the only carbon and energy sources. Evidence is presented here that glycerol, the main compatible solute this yeast accumulates so as to maintain osmotic balance, is actively co-transported with protons. This transport system was shown to be constitutive, not needing induction by either glycerol or salt, and was not repressible by glucose. In glucose- or glycerol-grown cells, a simple diffusion was detectable, and iterative calculations were performed to calculate kinetic parameters, in the presence and in the absence of NaCl. At 25°C, pH 5·0, in glucose-grown cells these were: Km = 0·81 ± 0·11 mM and Vmax = 634·2 ± 164·8 μmol h-1 per g (glycerol); Km = 1·28 ± 0·60 mM and Vmax = 558·6 · 100·6 μmol h-1 per g (protons). Correspondent stoichiometry was approximately 1, either for these conditions or in the presence of 1 M-NaCl. An increase in acumulation capacity was evident when different concentrations of NaCl were present. This capacity was shown to be dependent on ΔpH and membrane potential, consistently with an electrogenic character. We suggest that the main role of this system is in osmoregulation, by keeping glycerol accumulated inside the cells, compensating for leakage, due to its liposoluble character.
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  • 69
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    Yeast 11 (1995), S. 169-177 
    ISSN: 0749-503X
    Keywords: Saccharomyces cerevisiae ; chromosome ; ATCase ; URA2 ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: A prototroph revertant (Rev9) selected from an ATCase- mutant of the URA2 gene containing three nonsense mutations was shown to contain two ATCase coding sequences. We cloned both ATCase coding areas to show that the duplicated locus (dl9) was the only functional one. Its size corresponded roughly to the second half of the URA2 wild-type gene. Sequence analysis of the 5′ end of dl9 indicated that this duplicated sequence was inserted within the intergenic region close to the MRS3 gene and was transcribed from an unknown promoter divergently from the MRS3 gene. The event leading to the revertant strain Rev9 included a rearrangement that increased the size of chromosome X by about 60 kb. In agreement with such a rearrangement, recombination was undetectable in the vicinity of the locus dl9. Genetic mapping confirms that the MRS3 gene is 2 cM distal to the URA2 gene on the right arm of chromosome X.
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  • 70
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    Yeast 11 (1995) 
    ISSN: 0749-503X
    Keywords: Life and Medical Sciences ; Genetics
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  • 71
    ISSN: 0749-503X
    Keywords: fission yeast ; cell cycle ; phleomycin ; DNA damage ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: The effect of phleomycin, a bleomycin-like antibiotic, has been investigated in the fission yeast, Schizosaccharomyces pombe. We report that in response to phleomycin-induced DNA damage, growth was inhibited and S. pombe cells arrested in the G2-phase of the cell cycle. DNA repair mutants rad9 and rad17 did not arrest and were hypersensitive to phleomycin. Cell cycle mutants that entered mitosis without monitoring the completion of DNA replication also displayed an increased sensitivity to this DNA-damaging agent. Thus, phleomycin could be used as a tool in the fission yeast S. pombe model system for the study of DNA damage and cell cycle checkpoints, or as a new selective agent.
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  • 72
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    Yeast 11 (1995), S. 343-353 
    ISSN: 0749-503X
    Keywords: Yeast ; Hansenula polymorpha ; plasmid ; transformation ; ARS sequence ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: During studies of integrative transformation in Hansenula polymorpha, it was found that transformants with plasmids possessing the LEU2 gene of H. polymorpha were frequently unstable and lost plasmids while growing on non-selective medium. These transformants possessed reorganized plasmids capable of replication in H. polymorpha. Two such plasmids were isolated and characterized. It was shown that they contain additional DNA segments which were not present in the original plasmid used for transformation. Southern hybridization analysis carried out with labeled DNA probes derived from these segments showed that they consisted of H. polymorpha DNA. The hybridization patterns indicated that corresponding sequences were homologous to several chromosomal regions. These chromosomal DNA segments apparently carried H. polymorpha autonomous replicating sequences (HARS), since plasmids bearing them could transform H. polymorpha with high efficiency and were maintained in transformants in an autonomous state. Sequence analysis of one such captured chromosomal fragment revealed several eight- to ten-base AT-rich blocks similar to the presumed HARS sequence defined by Roggenkamp et al. (1986). Analogous reorganization was also observed with respect to integrative plasmids carrying the TRP3 and HIS3 genes of H. polymorpha and the ADE2 gene of Saccharomyces cerevisiae as selectable markers.
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  • 73
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    Yeast 11 (1995), S. 391-392 
    ISSN: 0749-503X
    Keywords: Life and Medical Sciences ; Genetics
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  • 74
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    Yeast 11 (1995), S. 425-433 
    ISSN: 0749-503X
    Keywords: Yarrowia lipolytica ; orotate phosphoribosyl transferase ; nucleotide sequence ; transcription ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: 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.
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  • 75
    ISSN: 0749-503X
    Keywords: Phosphoinositide-specific phospholipase C ; PLC-δ ; Schizosaccharomyces pombe ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: 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.
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  • 76
    ISSN: 0749-503X
    Keywords: Phospholipid biosynthesis ; transcriptional regulatory genes ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: 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.
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  • 77
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    Yeast 11 (1995), S. 219-224 
    ISSN: 0749-503X
    Keywords: glucose repression ; SUC2 expression ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: Mutations in the SNF8 gene impair derepresson of the SUC2 gene, encoding invertase, in response to glucose limitation of Saccharomyces cerevisiae. We report here the cloning of the SNF8 gene by complementation. Sequence analysis predicts a 26 936-dalton product. Disruption of the chromosomal locus caused a five-fold decrease in invertase derepression, defective growth on raffinose, and a sporulation defect in homozygous diploids. Genetic analysis of the interactions of the snf8 null mutation with spt6/ssn20 and ssn6 suppressors distinguished SNF8 from the groups, SNF1, SNF4 and SNF2, SNF5, SNF6. Notably, the snf8 ssn6 double mutants were extremely sick. Mutations of SNF8 and SNF7 showed similar phenotypes and genetic interactions, and the double mutant combination caused no additional phenotypic impairment. These findings suggest that SNF7 and SNF8 are functionally related. The complete nucleotide sequence of SNF8 has been deposited in GenBank under accession number U10361.
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  • 78
    ISSN: 0749-503X
    Keywords: Ca2+ sensitive mutants ; Saccharomyces cerevisiae ; P-type ATPases ; Cu2+ ; CCC2 ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: We have isolated, sequenced, mapped and disrupted a gene, CCC2, from Saccharomyces cerevisiae. This gene displays non-allelic complementation of the Ca2+-sensitive phenotype conferred by the csg1 mutation. Analysis of the CCC2p amino acid sequence reveals that it encodes a member of the P-type ATPase family and is most similar to a subfamily thought to consist of Cu2+ transporters, including the human genes that mutate to cause Wilson disease and Menkes disease. The ability of this gene, in two or more copies, to reverse the csg1 defect suggests that Ca2+-induced death of csg1 mutant cells is related to Cu2+ metabolism. Cells without CCC2 require increased Cu2+ concentrations for growth. Therefore CCC2p may function to provide Cu2+ to a cellular compartment rather than in removal of excess of Cu2+. The sequence of CCC2 is available through GenBank under accession number L36317.
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  • 79
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    Yeast 11 (1995), S. 327-336 
    ISSN: 0749-503X
    Keywords: yeast ; Hanseniaspora uvarum ; respiration ; fermentation ; chemostat culture ; glucose metabolism ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: The physiology of Hanseniaspora uvarum K5 was studied in glucose-limited chemostat cultures and upon glucose pulse. Up to a dilution rate of 0·28 h-1, glucose was completely metabolized in biomass and CO2. Above this value, increase in the dilution rate was accompanied by sequential production of metabolites (glycerol, acetate and ethanol) and decrease in cell yield. Similar results were observed upon glucose pulse. From the enzyme activities (pyruvate dehydrogenase, pyruvate decarboxylase, NAD and NADP-dependent acetaldehyde dehydrogenases, acetyl coenzyme A synthetase and alcohol dehydrogenase) and substrate affinities, the following conclusions were drawn with respect to product formation of cells: (1) pyruvate was preferentially metabolized via pyruvate dehydrogenase, when biomass and CO2 were the only products formed; (2) acetaldehyde formed by pyruvate decarboxylase was preferentially oxidized in acetate by NADP-dependent aldehyde dehydrogenase; acetate accumulation results from insufficient activity of acetyl-CoA synthetase required for the complete oxidation of acetate; (3) acetaldehyde was oxidized in ethanol by alcohol dehydrogenase, in addition to acetate production.
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  • 80
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    Yeast 11 (1995), S. 701-711 
    ISSN: 0749-503X
    Keywords: spontaneous reversion rate ; limiting metabolite content ; adaptive mutagenesis ; heterogeneity of revertants ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: The appearance dynamics of spontaneous revertants in adenine- or leucine-auxotrophic haploid Saccharomyces cerevisiae strains has been studied using mathematical simulation methods. In the case of adenine auxotrophs an increase in the number of revertants with decreasing metabolite content is found to result mainly from increasing the rate of intragenic suppressor mutations. In the case of leucine auxotrophs revertants result from increasing the appearance of mutants formed at similar rates under different cultivation conditions. In the latter case the appearance of mutant colonies increases with decreasing size of colonies of the initial auxotrophic cells. The last can simulate so-called adaptive mutagenesis. The heterogeneity of revertants appearing in different time periods is described.
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  • 81
    ISSN: 0749-503X
    Keywords: Saccharomyces cerevisiae ; GLC7 ; protein phosphatase ; mitosis ; MET3 promoter ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: Saccharomyces cerevisiae possesses a single essential gene (GLC7) encoding protein phosphatase 1 (PP1). Elevated expression of this gene from the GAL1 promoter is highly detrimental to the cell, causing a growth defect and aberrant bud morphology, which leads to cells exhibiting long, extended buds. By comparison, expression of GLC7 from the weaker MET3 promoter was without significant effect on either growth or morphology. However, repression of GLC7 expression from the MET3 promoter in cells where the MET3-GLC7 fusion was the sole source of PP1 resulted in a mitotic delay. Such cultures showed a massive decrease in the rate of proliferation in conjunction with a significant increase in the proportion of large, budded cells. 4′,6-diamidino-2-phenylindole dihydrochloride (DAPI) staining and anti-tubulin immunofluorescence analysis of these cells revealed that many were blocked in mitosis, with a short spindle and DAPI-stained material stretched between the mother and daughter cell within the bud neck. These results support a role for PP1 in the completion of mitosis in S. cerevisiae.
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  • 82
    ISSN: 0749-503X
    Keywords: red ade mutations ; Kluyveromyces lactis ; ADE1 ; ADE2 ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: Seventy-six red adenine mutants of Kluyveromyces lactis were isolated. By complementation they could be assigned to two groups with 31 and 45 mutants. Transformation of several strains from each group with plasmids containing the Saccharomyces cerevisiae ADE1 or ADE2 gene showed that the largest group was ade2 and the other group was ade1. Several previously isolated ‘ade1’ mutants were classified to either group and given new gene and allele numbers. ADE1 was localized at chromosome III, closely linked to the mating type gene, making it a convenient marker for mating type. ADE2 was localized at chromosome V.
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  • 83
    ISSN: 0749-503X
    Keywords: heat shock ; promoter ; nucleosome positioning ; Saccharomyces cerevisiae ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: We present the upstream sequences of HSP82 and HSC82, two closely related, but differentially regulated, heat-shock genes of Saccharomyces cerevisiae. Several dozen potential regulatory elements are identified within each upstream region; interestingly, only a few are conserved between the two genes. These include a consensus heat-shock element, an upstream repressor element, and a consensus TATA element. A search for motifs known actively to position nucleosomes in vitro revealed that such sequences are three- to seven-fold enriched within each promoter; a comparable enrichment is seen near the 3′ end of each transcription unit. Located ∼ 1100 bp upstream of HSC82 is an open reading frame (ORF) of 255 amino acids; ∼ 800 bp upstream of HSP82 is an ORF of 132 amino acids. The latter ORF contains several conserved ankyrin motifs and appears to be expressed under normal growth conditions. Finally, we show by clamped homogeneous electric field gel electrophoresis that the two genetic loci map to different chromosomes: HSP82 to chromosome XVI and HSC82 to chromosome XIII. The sequences have been deposited in the GenBank database under Accession Numbers U20323 and U20349.
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  • 84
    ISSN: 0749-503X
    Keywords: yeast genome ; chromosome VII ; histidine permease ; glyceraldehyde-3-phosphate dehydrogenase ; pyruvate dehydrogenase ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: We report the sequence of a 9037 bp fragment from the right arm of Saccharomyces cerevisiae chromosome VII. Analysis of the sequence revealed four complete open reading frames (ORFs), namely G7572, G7576, G7579 and G7584. The first three corresponded, respectively, to the previously cloned genes: HIP1, coding for a high-affinity histidine-specific permease, TDH1, one of the known genes coding for glyceraldehyde-3-phosphate dehydrogenase and ODPX, which encodes a precursor of protein X, a component of the pyruvate dehydrogenase complex. The ORF G7584 showed 35·8% identity with a hypothetical protein of Caenorhabditis elegans chromosome 3. The reported sequence has been deposited in the EMBL data library under Accession Number X82408.
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  • 85
    ISSN: 0749-503X
    Keywords: Saccharomyces ; yeast protein map ; protein identification ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: This publication marks the beginning of the construction of a gene-protein index that relates proteins which are resolved on the two-dimensional protein map of Saccharomyces cerevisiae with their corresponding genes. We report the identification of 36 novel polypeptide spots on the yeast protein map. They correspond to the products of 26 genes. Together with the polypeptide spots previously identified, this raises to 41 the number of genes whose products have been identified on the protein map. The proteins identified here are concerned with four major areas of yeast cellular physiology: carbon metabolism, heat shock, amino acid biosynthesis and purine biosynthesis. Given the molecular weight and isoelectric point of the identified proteins, and the codon-usage bias of the corresponding genes, it can be estimated that 25 to 35% of all the soluble yeast proteins are detectable under the labelling and running gel conditions used in this study.
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  • 86
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    Yeast 11 (1995), S. 1027-1029 
    ISSN: 0749-503X
    Keywords: pulsed-field gels ; chromosome preparation ; yeasts ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: A modified procedure is proposed for chromosomal DNA extraction based on a cell-wall lytic enzyme never applied before in pulsed field gel electrophoresis. Protoplasting efficiency is retained under very challenging conditions for enzyme activity, such as those required for non-Saccharomyces yeasts often characterized by cell walls highly resistant to lysis.
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  • 87
    ISSN: 0749-503X
    Keywords: Saccharomyces cerevisiae ; chromosome XV ; ARG8 gene ; CDC33 gene ; riboflavin synthase ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: The DNA sequence of a 9873 bp fragment located near the left telomere of chromosome XV has been determined. Sequence analysis reveals seven open reading frames. One is the ARG8 gene coding for N-acetylornithine aminotransferase. Another corresponds to CDC33, which codes for the initiation factor 4E or cap binding protein. The open reading frame AOE169 can be considered as the putative gene for the Saccharomyces cerevisiae riboflavin synthase beta chain, since its translation product shows strong homology with four prokaryotic riboflavin synthase beta chains. The nucleotide sequence reported here has been submitted to the EMBL data library under the Accession Number X84036.
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  • 88
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    Yeast 11 (1995), S. 1093-1101 
    ISSN: 0749-503X
    Keywords: gene expression ; glycolysis ; GCR ; S. cerevisiae ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: A new essential gene of Saccharomyces cerevisiae was found upstream of GCR1. Its cloning and sequencing predict a 280 amino acid protein (32 577 Da). The predicted protein is fairly hydrophobic, and a search of the database did not identify any homologous proteins. A LEU2 disruption at codon 104 was lethal, but disruption at codon 221 showed a temperature-sensitive conditional growth phenotype. Abnormalities were observed in some glycolytic enzyme levels. The sequence has been submitted to GenBank-EMBL-DDBJ under Accession Number D29645.
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    Yeast 11 (1995), S. 1113-1113 
    ISSN: 0749-503X
    Keywords: Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
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  • 90
    Electronic Resource
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    New York, NY [u.a.] : Wiley-Blackwell
    Yeast 11 (1995), S. 1129-1138 
    ISSN: 0749-503X
    Keywords: Saccharomyces cerevisiae ; excision repair ; recombination ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: In this study we show that the previously described uvs112 (uvs12) mutation blocks one of the steps of the excision repair pathway. The properties of this mutation permit the assignment of the UVS112 gene to the RAD3 epistasis group. It was established that the uvs112 mutation caused a 2·5-fold reduction in the number of recombinants produced by conversion and also significantly increased the frequency of mitotic crossing-over in interplasmid recombination. Tetrad analysis placed the UVS112 gene on the left arm of chromosome IX, approximately 20 cM from HIS5. The analysis of mitotic recombination revealed that UVS112 lies between HIS6 and HIS5, and is an allele of the RAD25 gene.
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  • 91
    ISSN: 0749-503X
    Keywords: Saccharomyces cerevisiae ; chromosome II ; yeast ; functional analysis ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: In a coordinated approach, several laboratories sequenced Saccharomyces cerevisiae chromosome II during the European BRIDGE project. Here we report on the sequence and functional analysis of a 7217 bp fragment located on the right arm of chromosome II between RPB5 and CDC28. The fragment contains four open reading frames probably encoding proteins of 79·2 kDa (corresponding gene YBR156c), 12·1 kDa (YBR157c), 62·7 kDa (YBR158w) and 38·7 kDa (YBR159w). All four open reading frames encode new proteins, as concluded from data base searches. The respective genes were destroyed by gene replacement in one allele of diploid cells. After sporulation and tetrad analysis, the resulting mutant haploid strains were investigated. No phenotype with respect to spore germination, viability, carbohydrate utilization, and growth was found for YBR157c, encoding the smallest open reading frame investigated. Gene replacement within the YBR156c gene encoding a highly basic and possibly nuclear located protein was lethal. Ybr158 revealed similarities to the Grr1 (Cat80) protein with respect to the leucine-rich region. Cells harboring a mutation in the YBR158w gene showed strongly reduced growth as compared to the wild-type cells. The protein predicted from YBR159w shared 33% identical amino acid residues with the human estradiol 17-beta-hydroxysterol dehydrogenase 3. Haploid ybr159c mutants were only able to grow at reduced temperatures, but even under these conditions the mutants grew slower than wild-type strains. The DNA sequence was deposited at the EMBL data base with accession numbers Z36025 (YBR156c), Z36026 (YBR157c), Z36027 (YBR158w) and Z36028 (YBR159w).
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  • 92
    ISSN: 0749-503X
    Keywords: Saccharomyces cerevisiae ; 2-deoxyglucose ; 2-deoxyglucose-6 phosphate phosphatase ; DOGR1 ; DOGR2 ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: Saccharomyces cerevisiae contains two genes (DOGR1 and DOGR2) that are able to confer 2-deoxyglucose resistance when they are overexpressed. These genes are very similar, sharing 92% identity at the protein level. They code for two isoenzymes with 2-deoxyglucose-6 phosphate (2-DOG-6P) phosphatase activity. These enzymes have been purified and characterized. DogR1p shows an optimum pH of 6, an optimum temperature of 30°C and a KM on 2-DOG-6P of 17 mM. DogR2p shows a similar optimum pH, but the optimum temperature is 40°C and it exhibits a KM on 2-DOG-6P of 41 mM. Both enzymes require 10 mM-MgCl2 for maximal activity and they are inhibited by inorganic phosphate.
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  • 93
    ISSN: 0749-503X
    Keywords: gene disruption ; fusion PCR ; Saccharomyces cerevisiae ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: We adapted a fusion polymerase chain reaction (PCR) strategy to synthesize gene disruption alleles of any sequenced yeast gene of interest. The first step of the construction is to amplify sequences flanking the reading frame we want to disrupt and to amplify the selectable marker sequence. Then we fuse the upstream fragment to the marker sequence by fusion PCR, isolate this product and fuse it to the downstream sequence in a second fusion PCR reaction. The final PCR product can then be transformed directly into yeast. This method is rapid, relatively inexpensive, offers the freedom to choose from among a variety of selectable markers and allows one to construct precise disruptions of any sequenced open reading frame in Saccharomyces cerevisiae.
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  • 94
    ISSN: 0749-503X
    Keywords: Saccharomyces cerevisiae ; chromosome XIV ; serine/threonine protein kinase ; rp gene ; plasma membrane protein ; replication factor ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: We have determined the nucleotide sequence of a 30 kb fragment of chromosome XIV of Saccharomyces cerevisiae. The sequence revealed the presence of 19 open reading frames (ORFs) longer than 300 bp. NO422 and NO425 correspond to the split ribosomal protein genes encoding S16A and rp28, respectively, NO450 displays a striking similarity with serine/threonine protein kinase genes, in particular with STE20, and therefore may encode a novel member of this protein family. NO453 is the longest ORF in this DNA segment, having a size of 4908 bp, but its function is not yet known. NO530 encodes the plasma membrane protein Mid1p and NO533 corresponds to the gene coding for a 40 kDa subunit of replication factor C. The remaining ORFs show weak or no homology with proteins in the data bases. The sequence has been submitted to the EMBL data library under Accession Number U23084.
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  • 95
    Electronic Resource
    Electronic Resource
    New York, NY [u.a.] : Wiley-Blackwell
    Yeast 11 (1995), S. 1345-1351 
    ISSN: 0749-503X
    Keywords: Saccharomyces cerevisiae ; O-glycosylation ; dolichol phosphate ; PMT gene family ; glycosyltransferase ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: Two genes PMT3 and PMT4 were identified by polymerase chain reaction of genomic DNA using primers derived from regions of high homology between the products of three genes PMT1, PMT2 of Saccharomyces cerevisiae and part of a PMT1 related sequence of Kluyveromyces lactis. Pmt1p and Pmt2p are mannosyltransferases involved in the transfer of a mannosyl residue from dolichyl phosphate-D-mannose (Dol-P-Man) to seryl and threonyl residues in proteins. The products encoded by the PMT3 and PMT4 genes have almost identical hydropathy profiles in comparison to PMT1 and PMT2: a hydrophobic N- and C-terminal third each with multiple potential transmembrane helices and a central hydrophillic part.The predicted Pmt3p contains 753 amino acids, four potential N-glycosylation sites and it is significantly homologous to Pmt1p, Pmt2p and Pmt4p. Pmt4p contains 762 amino acids and two potential N-glycosylation sites. Northern blot analysis showed a single mRNA transcript of PMT3 and PMT4 of 2·8 kb. Thus PMT3 and PMT4 are two new members of the PMT gene family. The pmt4 null mutant the pmt3 pmt4 double null mutant, but not pmt3 null mutant, showed a small but significant shift of chitinase due to under glycosylation of the enzyme. The triple disruption pmt2 pmt3 pmt4 and the quadruple disruption result in a lethal phenotype. The EMBL data library Accession Number for PMT3 is X83797 and for PMT4 X83798.
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  • 96
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    Electronic Resource
    New York, NY [u.a.] : Wiley-Blackwell
    Yeast 13 (1997), S. 931-943 
    ISSN: 0749-503X
    Keywords: brewing yeast ; fermentation ; cell surface ; flocculation ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: The surface physico-chemical properties (hydrophobicity, electrophoretic mobility, chemical composition) of a large set of top- and bottom-fermenting brewing yeasts, harvested in the exponential and stationary growth phases, have been investigated. Bottom- and top-fermenting strains showed different surface properties. Top strains were generally more hydrophobic than bottom strains, due to higher surface protein concentrations. Bottom strains possessed higher surface phosphate concentrations. The different profiles of electrophoretic mobility versus pH for top and bottom strains could be explained by modelling the surface charge according to the surface chemical composition as given by X-ray photoelectron spectroscopy. For bottom strains, the electrical properties were mainly controlled by phosphate, resulting in a low isoelectric point (pH 2 or below) and an electrophoretic mobility that did not become much more negative above pH 4. For the top strains, they were mainly determined by the balance of protonated amino- and carboxylate groups in proteins, which gave a high isoelectric point (pH 4) and an electrophoretic mobility changing greatly with pH in the range of 2 to 7. No difference in surface properties was found between flocculating and non-flocculating strains, or between cells from the exponential and stationary growth phases, even for strains where flocculation occurred during the transition from one growth phase to the other. © 1997 John Wiley & Sons, Ltd.
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  • 97
    Electronic Resource
    Electronic Resource
    New York, NY [u.a.] : Wiley-Blackwell
    Yeast 13 (1997), S. 1077-1090 
    ISSN: 0749-503X
    Keywords: Saccharomyces cerevisiae ; chromosome VII ; sequence ; snRNA ; SNR10 ; SNR39 ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: The nucleotide sequences of five major regions from chromosome VII of Saccharomyces cerevisiae have been determined and analysed. These regions represent 203 kilobases corresponding to approximately one-fifth of the complete yeast chromosome VII. Two fragments originate from the left arm of this chromosome. The first one of about 15·8 kb starts approximately 75 kb from the left telomere and is bordered by the SKI8 chromosomal marker. The second fragment covers the 72·6 kb region between the chromosomal markers CYH2 and ALG2. On the right chromosomal arm three regions, a 70·6 kb region between the MSB2 and the KSS1 chromosomal markers and two smaller regions dominated by the KRE11 marker and another one in the vicinity of the SER2 marker were sequenced.We found a total of 114 open reading frames (ORFs), 13 of which were completely overlapping with larger ORFs running in the opposite direction.A total of 44 yeast genes, the physiological functions of which are known, could be precisely mapped on this chromosome.Of the remaining 57 ORFs, 26 shared sequence homologies with known genes, among which were 13 other S. cerevisiae genes and five genes from other organisms. No homology with any sequence in the databases could be found for 31 ORFs.Furthermore, five Ty elements were found, one of which may not be functional due to a frame shift in its Ty1B amino acid sequence.The five chromosomal regions harboured five potential ARS elements and one sigma element together with eight tRNA genes and two snRNAs, one of which is encoded by an intron of a protein-coding gene. © 1997 by John Wiley & Sons, Ltd.
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  • 98
    ISSN: 0749-503X
    Keywords: yeast ; MAL6 ; divergent promoter ; repeats ; nucleosomes ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: We have studied four novel MAL promoters isolated from a single strain of bakers' yeast. Within these promoters we have identified up to five tandem 147 bp repeats located between the MAL UAS region and the MALT TATA box. These repeats strongly reduce MALT (maltose permease) gene expression but only weakly reduce MALS (maltase) gene expression. Insertion of the 147 bp elements into the heterologous CYC1 promoter reduced expression when located between the CYC1 UAS and the TATA box, but not when located upstream of the UAS. We propose that these naturally occurring repeats have evolved as a mechanism to lower the level of MALT expression relative to MALS expression, thus avoiding possible toxic effects associated with over-expression from multiple copies of the permease gene. Accession numbers are: WIG1, U86359; WIG3, U86360; WIG4, U86361; WIG5, U86362. © 1997 by John Wiley & Sons, Ltd.
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  • 99
    Electronic Resource
    Electronic Resource
    New York, NY [u.a.] : Wiley-Blackwell
    Yeast 13 (1997), S. 1195-1197 
    ISSN: 0749-503X
    Keywords: Schizosaccharomyces pombe ; mapping ; urease ; yeast ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: The following urease genes of the fission yeast Schizosaccharomyces pombe have been mapped by induced haploidization and tetrad analysis - ure1: chromosome arm III-L; ure2 and ure3: chromosome arm I-R. The previously determined tps19-rad1 interval (11-12 cM) has been increased to 18 cM. A convenient medium for rapidly scoring the ure gene markers of fission yeast was developed. © 1997 John Wiley & Sons, Ltd.
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  • 100
    ISSN: 0749-503X
    Keywords: Candida albicans ; Saccharomyces cerevisiae ; ribosomal proteins ; RPL39 ; RPS7 ; RPL29 ; hyphal morphogenesis ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: Genes encoding the Candida albicans ribosomal proteins L39 and S7 (RPL39, RPS7) were isolated and sequenced. From RPL39 cDNA a single intron interrupting the fifth codon in the genomic sequence could be deduced. Two homologous RPL39 genes in Saccharomyces cerevisiae contain a single intron in a conserved position. In contrast, C. albicans RPS7 was found to lack an intron, while both S. cerevisiae homologs are interrupted by single introns. The deduced L39 and S7 proteins contained 67% and 83% identical residues compared to the S. cerevisiae homologs. During hyphal induction the RPL39, RPS7 and RPL29 transcript levels increased three- to six-fold relative to ribosomal RNA, while ACT1 and RPS33 control transcripts were not regulated extensively. As suggested by unaltered transcript stabilities during hyphal induction, this regulation occurs on the transcriptional level; a conserved 18 bp palindromic sequence (5′-TTAGGGCTATAGCCCTAA-3′), which is present in the promoter regions of the RPL39 and RPS7 genes, may be involved in regulation. © 1997 John Wiley & Sons, Ltd.
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