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  • Genetics  (2.228)
  • Wiley-Blackwell  (2.228)
  • Public Library of Science
  • American Institute of Physics (AIP)
  • 1
    Digitale Medien
    Digitale Medien
    New York, NY [u.a.] : Wiley-Blackwell
    Yeast 14 (1998), S. 287-295 
    ISSN: 0749-503X
    Schlagwort(e): Candida maltosa ; Candida albicans ; tRNA splicing gene ; silent genes ; Life and Medical Sciences ; Genetics
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Biologie
    Notizen: 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.
    Zusätzliches Material: 4 Ill.
    Materialart: Digitale Medien
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  • 2
    ISSN: 0749-503X
    Schlagwort(e): Oligotrophic yeasts ; low-nutrient conditions ; starvation ; Cryptococcaceae ; Life and Medical Sciences ; Genetics
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Biologie
    Notizen: 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.
    Zusätzliches Material: 3 Ill.
    Materialart: Digitale Medien
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  • 3
    ISSN: 0749-503X
    Schlagwort(e): Saccharomyces cerevisiae ; mitochondria ; mitochondrial matrix ; homo-oligomeric protein ; Mam33p ; gene disruption ; gC1q-R ; Life and Medical Sciences ; Genetics
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Biologie
    Notizen: 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.
    Zusätzliches Material: 5 Ill.
    Materialart: Digitale Medien
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  • 4
    ISSN: 0749-503X
    Schlagwort(e): phosphofructokinase-1 ; Saccharomyces cerevisiae ; deletion mutants ; reactivation ; assembly ; Life and Medical Sciences ; Genetics
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Biologie
    Notizen: 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.
    Zusätzliches Material: 5 Ill.
    Materialart: Digitale Medien
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  • 5
    ISSN: 0749-503X
    Schlagwort(e): Saccharomyces ; redox ; glycerol ; NADH ; shuttle ; Life and Medical Sciences ; Genetics
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Biologie
    Notizen: 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.
    Zusätzliches Material: 5 Ill.
    Materialart: Digitale Medien
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  • 6
    ISSN: 0749-503X
    Schlagwort(e): modified LFH cassette ; EUROFAN 6-pack analysis ; Life and Medical Sciences ; Genetics
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Biologie
    Notizen: 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.
    Zusätzliches Material: 4 Ill.
    Materialart: Digitale Medien
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  • 7
    Digitale Medien
    Digitale Medien
    New York, NY [u.a.] : Wiley-Blackwell
    Yeast 14 (1998), S. 409-417 
    ISSN: 0749-503X
    Schlagwort(e): transcription factor ; SPT3 sequences ; Schizosaccharomyces pombe ; Kluyveromyces lactis ; Clavispora opuntiae ; Life and Medical Sciences ; Genetics
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Biologie
    Notizen: Spt3 of Saccharomyces cerevisiae is a factor required for normal transcription from particular RNA polymerase II-dependent promoters. As a step towards analysing Spt3 structure-function relationships, we have identified and studied Spt3 homologues from three other yeasts: Kluyveromyces lactis, Clavispora opuntiae and Schizosaccharomyces pombe. Alignment of their predicted amino acid sequences shows an overall identity of 30% between all four homologues and suggests that three conserved domains are present in Spt3. When tested for function in S. cerevisiae, K. lactis SPT3 was shown to fully complement and S. pombe SPT3 to partially complement an spt3 Δ mutation. These data demonstrate that Spt3 is functionally conserved among distantly related yeasts. The new sequences have been entered in GenBank: AF005930 (K. lactis SPT3), AF005932 (C. opuntiae SPT3) and AF005931 (S. pombe SPT3). © 1998 John Wiley & Sons, Ltd.
    Zusätzliches Material: 4 Ill.
    Materialart: Digitale Medien
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  • 8
    Digitale Medien
    Digitale Medien
    New York, NY [u.a.] : Wiley-Blackwell
    Yeast 14 (1998), S. 471-477 
    ISSN: 0749-503X
    Schlagwort(e): ribosomal protein genes ; yeast genome ; Life and Medical Sciences ; Genetics
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Biologie
    Notizen: Screening of the complete genome sequence from the yeast Saccharomyces cerevisiae has enabled us to compile a complete list of the genes encoding cytoplasmic ribosomal proteins in this organism.Putative ribosomal protein genes were selected primarily on the basis of the sequence similarity of their products with ribosomal proteins from other eukaryotic organisms, in particular the rat. These genes were subsequently screened for typical yeast rp-gene characteristics, viz. (1) a high codon adaptation index; (2) their promoter structure and (3) their responses to changes in growth conditions.The yeast genome appears to carry 78 different genes, of which 59 are duplicated, encoding 32 different small-subunit and 46 large-subunit proteins. A new nomenclature for these ribosomal proteins is proposed. © 1998 John Wiley & Sons, Ltd.
    Zusätzliches Material: 1 Ill.
    Materialart: Digitale Medien
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  • 9
    Digitale Medien
    Digitale Medien
    New York, NY [u.a.] : Wiley-Blackwell
    Yeast 14 (1998), S. 335-345 
    ISSN: 0749-503X
    Schlagwort(e): Candida albicans ; arginine permease ; amino acid transport ; affinity chromatography ; functional reconstitution ; Life and Medical Sciences ; Genetics
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Biologie
    Notizen: We have for the first time purified arginine permease from a pathogenic yeast, Candida albicans, to homogeneity by affinity chromatography using L-arginine-linked agarose matrix as affinity column. The purified protein (PP) was of 66 kDa with no subunit structure. Two kinetically distinct binding affinities of PP were evident where high affinity binding (S1) revealed a dependence on acidic pH while pH did not have dramatic effect on low affinity (S2) binding. The specificity of L-arginine binding to PP with regard to other amino acids, structural analogues and inhibitors, was essentially similar to arginine transport observed in the intact cells of C. albicans (Rao et al., 1986). The purified arginine permease was reconstituted into proteoliposomes and its functionality was tested by imposing a valinomycin-induced membrane potential. All the characteristic features of L-arginine transport displayed by the reconstituted system were similar to those observed in intact cells. Thus homogeneous purified arginine permease was also functionally active. © 1998 John Wiley & Sons, Ltd.
    Zusätzliches Material: 8 Ill.
    Materialart: Digitale Medien
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  • 10
    Digitale Medien
    Digitale Medien
    New York, NY [u.a.] : Wiley-Blackwell
    Yeast 14 (1998), S. 383-390 
    ISSN: 0749-503X
    Schlagwort(e): polyphosphatase ; cytosol ; yeast ; purification ; kinetic model ; Mg2+ ; Life and Medical Sciences ; Genetics
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Biologie
    Notizen: A homogenous polyphosphatase preparation was obtained from Saccharomyces cerevisiae cytosol with a 3·8% yield and 3540-fold purification. The enzyme hydrolysed polyphosphate (polyP) with various chain lengths, including polyP3, and split Pi off the end of the chain. It was inactive with respect to ATP, PPi, and p-nitrophenylphosphate. Its specific activity with polyP15 was 283 U/mg protein. The polyphosphatase was a monomeric protein with a molecular mass of 40 kDa. This enzyme was inactive without divalent cations and with Cu2+ and Ca2+. The ability of other divalent cations to activate the enzyme decreased in the following order: Co2+〉Mn2+〉Mg2+〉Zn2+. A kinetic model of the hydrolysis of polyP3 and action of Mg2+ is proposed. © 1998 John Wiley & Sons, Ltd.
    Zusätzliches Material: 5 Ill.
    Materialart: Digitale Medien
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  • 11
    Digitale Medien
    Digitale Medien
    New York, NY [u.a.] : Wiley-Blackwell
    Yeast 14 (1998), S. 401-407 
    ISSN: 0749-503X
    Schlagwort(e): yeast ; Kluyveromyces marxianus ; malic acid transport ; mutants ; Life and Medical Sciences ; Genetics
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Biologie
    Notizen: In malic acid-grown cells of the strains ATCC 10022 and KMS3 of Kluyveromyces marxianus the transport of malic acid occurred by a malate-proton symport, which accepted l-malic, d-malic, succinic and fumaric acids, but not tartaric, malonic or maleic acids. The system was inducible and subjected to glucose repression. Mutants of the strain KMS3, unable to grow in a medium with malic acid, were isolated and checked for their capacity to utilize several carbon sources and to transport dicarboxylic acids by the malate-proton symport. Two distinct clones affected on malate transport were obtained. Both were able to grow on a medium with glycerol or ethanol but not with dl-malic, succinic, oxoglutaric and oxaloacetic acids as the sole carbon and energy sources. However, while one of the mutants (Mal7) displayed activity levels for the enzymes malate dehydrogenase, isocitrate lyase, and phosphoenolpyruvate carboxykinase similar to those of the wild strain, in the other mutant type (Mal6) the activities for the same enzymes were significantly reduced. Plasma membranes from derepressed cells of the wild strain and of the mutants Mal6 and Mal7 were isolated and the protein analysed by SDS-PAGE. The electrophoretic patterns of these preparations differed in a polypeptide with an apparent molecular mass of about 28 kDa, which was absent only in the mutant Mal7. The results indicated that Mal7 can be affected in a gene that encodes a malate carrier in K. marxianus. © 1998 John Wiley & Sons, Ltd.
    Zusätzliches Material: 6 Ill.
    Materialart: Digitale Medien
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  • 12
    Digitale Medien
    Digitale Medien
    New York, NY [u.a.] : Wiley-Blackwell
    Yeast 14 (1998), S. 485-492 
    ISSN: 0749-503X
    Schlagwort(e): Schizosaccharomyces pombe ; protein kinase ; cell flocculation ; PRK1 ; Life and Medical Sciences ; Genetics
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Biologie
    Notizen: We report the isolation and characterization of a protein kinase from the fission yeast Schizosaccharomyces pombe. The proposed Prk1 protein contains 352 amino acids and has significant homology to the Ume5p kinase (also known as Srb10p, Ssn3p and Are1p) of the budding yeast Saccharomyces cerevisiae, a cyclin-dependent kinase involved in regulating the transcription of a diverse set of genes. Disruption of the prk1 gene increases flocculation but does not appear to have any other significant effect on cell behaviour. This defect can be overcome by expressing the UME5 gene, indicating that Prk1 is the fission yeast homologue of Ume5p. The sequence is in the EMBL data library under Accession Number Z98977. © 1998 John Wiley & Sons, Ltd.
    Zusätzliches Material: 5 Ill.
    Materialart: Digitale Medien
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  • 13
    Digitale Medien
    Digitale Medien
    New York, NY [u.a.] : Wiley-Blackwell
    Yeast 14 (1998), S. 431-442 
    ISSN: 0749-503X
    Schlagwort(e): differential display ; S.cerevisiae ; thermotolerance ; repression ; derepression ; gene regulation ; Life and Medical Sciences ; Genetics
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Biologie
    Notizen: We have described the use of differential display of PCR-amplified reverse transcribed mRNA (DDRT-PCR) to survey changes in gene expression profiles induced by heat shock and carbon catabolite derepression in Saccharomyces cerevisiae. It is well established that either of these states elicits thermotolerant phenotypes. An initial analysis conducted on cells of an inherently thermosensitive strain (Ysen) indicated that approximately 10% of the total number of cDNAs detected were either up or down regulated following heat shock at 37°C (30 min) in comparison to control cells (25°C). In addition, whereas 7% of all PCR products were preferentially expressed during derepressive growth, approximately 2% were found to be common to both heat-shocked and derepressed cells. A repeat analysis, performed on all three cell types of Ysen as well as cells of a relatively thermoresistant strain (Yres) yielded 30 differentially displayed cDNA fragments common to heat-shocked and derepressed cells of both strains. Eighteen of these generated signals on Northern blots, of which three were confirmed as regulated. Five amplicons, including one not detected by Northern analysis and another from the derepressed state, were cloned and sequenced. Three of these exhibited homology to S. cerevisiae genes with well-characterized protein products: HSP 90, HXK1and STA1. The remaining two applicons showed nucleotide identity to YTIS11, a homolog of the mammalian TIS11 and putative transcriptional activator, and an orphan gene encoding a hypothetical transmembrane protein belonging to the multi-drug resistance translocase family. Our novel application of DDRT-PCR has identified new and known genes that may be further evaluated as factors involved in stress regulation and has demonstrated the potential of the technique to systematically analyse gene expression in yeast. © 1998 John Wiley & Sons, Ltd.
    Zusätzliches Material: 4 Ill.
    Materialart: Digitale Medien
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  • 14
    Digitale Medien
    Digitale Medien
    New York, NY [u.a.] : Wiley-Blackwell
    Yeast 14 (1998), S. 583-586 
    ISSN: 0749-503X
    Schlagwort(e): Saccharomyces exiguus ; STE3 ; homolog ; sequence analysis ; differentiation of species ; Life and Medical Sciences ; Genetics
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Biologie
    Notizen: 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.
    Zusätzliches Material: 1 Ill.
    Materialart: Digitale Medien
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  • 15
    Digitale Medien
    Digitale Medien
    New York, NY [u.a.] : Wiley-Blackwell
    Yeast 14 (1998), S. 565-571 
    ISSN: 0749-503X
    Schlagwort(e): Saccharomyces cerevisiae ; electroporation ; transformation ; Life and Medical Sciences ; Genetics
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Biologie
    Notizen: 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.
    Zusätzliches Material: 1 Ill.
    Materialart: Digitale Medien
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  • 16
    ISSN: 0749-503X
    Schlagwort(e): multidrug resistance ; CDR1 ; ABC transporter ; baculovirus expression ; C. albicans ; Life and Medical Sciences ; Genetics
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Biologie
    Notizen: 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.
    Zusätzliches Material: 6 Ill.
    Materialart: Digitale Medien
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  • 17
    Digitale Medien
    Digitale Medien
    New York, NY [u.a.] : Wiley-Blackwell
    Yeast 14 (1998), S. 869-875 
    ISSN: 0749-503X
    Schlagwort(e): calmodulin ; CMD1 ; ALG1 ; K. lactis ; EF hand ; Life and Medical Sciences ; Genetics
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Biologie
    Notizen: The KlCMD1 gene was isolated from a Kluyveromyces lactis genomic library as a suppressor of the Saccharomyces cerevisiae temperature-sensitive mutant spc110-124, an allele previously shown to be suppressed by elevated copy number of the S. cerevisiae calmodulin gene CMD1. The KlCMD1 gene encodes a polypeptide which is 95% identical to S. cerevisiae calmodulin and 55% identical to calmodulin from Schizosaccharomyces pombe.Complementation of a S. cerevisiae cmd1 deletion mutant by KlCMD1 demonstrates that this gene encodes a functional calmodulin homologue. Multiple sequence alignment of calmodulins from yeast and multicellular eukaryotes shows that the K. lactis and S. cerevisiae calmodulins are considerably more closely related to each other than to other calmodulins, most of which have four functional Ca2+-binding EF hand domains. Thus like its S. cerevisiae counterpart Cmd1p, the KlCMD1 product is predicted to form only three Ca2+-binding motifs. The KlCMD1 sequence has been assigned Accession Number AJ002021 in the EMBL/GenBank database. © 1998 John Wiley & Sons, Ltd.
    Zusätzliches Material: 3 Ill.
    Materialart: Digitale Medien
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  • 18
    ISSN: 0749-503X
    Schlagwort(e): Saccharomyces cerevisiae ; adrenodoxin reductase ; mitochondria ; essential gene ; Life and Medical Sciences ; Genetics
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Biologie
    Notizen: A yeast gene was found in which the derived protein sequence has similarity to human and bovine adrenodoxin reductase (Nobrega, F. G., Nobrega, M. P. and Tzagoloff, A. (1992). EMBO J. 11, 3821-3829; Lacour, T. and Dumas, B. (1996). Gene 174, 289-292), an enzyme in the mitochondrial electron transfer chain that catalyses in mammals the conversion of cholesterol into pregnenolone, the first step in the synthesis of all steroid hormones. It was named ARH1 (Adrenodoxin Reductase Homologue 1) and here we show that it is essential. Rescue was possible by the yeast gene, but failed with the human gene. Supplementation was tried without success with various sterols, ruling out its involvement in the biosynthesis of ergosterol. Immunodetection with a specific polyclonal antibody located the gene product in the mitochondrial fraction. Consequently ARH1p joins the small group of gene products that affect essential functions carried out by the organelle and not linked to oxidative phosphorylation. © 1998 John Wiley & Sons, Ltd.
    Zusätzliches Material: 3 Ill.
    Materialart: Digitale Medien
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  • 19
    Digitale Medien
    Digitale Medien
    New York, NY [u.a.] : Wiley-Blackwell
    Yeast 14 (1998), S. 633-646 
    ISSN: 0749-503X
    Schlagwort(e): genetic interaction ; membrane traffic ; retrograde transport ; docking complex ; coatomer ; Life and Medical Sciences ; Genetics
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Biologie
    Notizen: Tip20p is an 80 kDa cytoplasmic protein bound to the cytoplasmic surface of the endoplasmic reticulum (ER) by interaction with the type II integral membrane protein Sec20p. Both proteins are required for vesicular transport between the ER and Golgi complex. Recently, sec20-1 was found to be defective in retrograde transport. A collection of temperature-sensitive tip20 mutants are shown to be lethal in combination with ufe1-1, a target SNARE of the ER and ret2-1, yeast δ-COP. A subset of tip20 mutants was found to be lethal in combination with sec20-1, sec21-1, sec22-3 and sec27-1. Since all pairwise combinations of a tip20 mutant, sec20-1, and ufe1-1 are lethal, Tip20p and Sec20p might be part of the docking complex for Golgi-derived retrograde transport vesicles. Since carboxy-terminal tip20 truncations are lethal in combination with mutants in three coatomer subunits, Tip20p might be involved in binding or uncoating of COPI coated retrograde transport vesicles. © 1998 John Wiley & Sons, Ltd.
    Zusätzliches Material: 4 Ill.
    Materialart: Digitale Medien
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  • 20
    ISSN: 0749-503X
    Schlagwort(e): heat shock ; protein-DNA interactions ; transcriptional regulation ; Life and Medical Sciences ; Genetics
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Biologie
    Notizen: The C-terminal hydrophobic repeat (CTR) of heat shock transcription factor (HSF) has been proposed to regulate DNA binding by intramolecular interactions with the leucine zipper motifs present in the HSF trimerization domain. Schizosaccharomyces pombe provides a useful model organism for the study of the regulation of HSF DNA binding because, unlike Saccharomyces cerevisiae, S. pombe hsf is highly heat shock inducible for DNA binding and contains a clear homology to the CTR. We examined the role that the CTR plays in the regulation of S. pombe hsf by constructing isogenic strains bearing deletion and point mutations in the chromosomal copy of hsf. Surprisingly, we found that point mutation of key hydrophobic amino acids within the CTR, as well as full deletion of it, yielded factors that show normal binding at normal growth temperatures and full levels of heat-induced binding. Deletion of the CTR did, however, slightly lower the temperature required for maximal activation. In contrast, a large deletion of the C-terminus, which removes close to a third of the coding sequence, was deregulated and bound DNA at control temperature. Several of the deletion mutants were significantly reduced in their level of expression, yet they showed wild-type levels of DNA binding activity following heat shock. These experiments demonstrate that appropriate regulation of the DNA binding activity of S. pombe hsf is not solely dependent upon the CTR, and imply that a feedback mechanism exists that establishes proper levels of DNA binding following heat shock despite mutations that significantly alter levels of total hsf. © 1998 John Wiley & Sons, Ltd.
    Zusätzliches Material: 7 Ill.
    Materialart: Digitale Medien
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  • 21
    Digitale Medien
    Digitale Medien
    New York, NY [u.a.] : Wiley-Blackwell
    Yeast 14 (1998), S. 747-757 
    ISSN: 0749-503X
    Schlagwort(e): Saccharomyces cerevisiae ; ubiquitin carrier proteins ; cyclin degradation ; functional analysis ; Life and Medical Sciences ; Genetics
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Biologie
    Notizen: UBC11 is the Saccharomyces cerevisiae gene that is most similar in sequence to E2-C, a ubiquitin carrier protein required for the destruction of mitotic cyclins and proteins that maintain sister chromatid cohesion in animal cells and in Schizosaccharomyces pombe. We have disrupted the UBC11 gene and found it is not essential for yeast cell viability even when combined with deletion of UBC4, a gene that has also been implicated in mitotic cyclin destruction. Ubc11p does not ubiquitinate cyclin B in clam cell-free extracts in vitro and the destruction of Clb2p is not impaired in extracts prepared from Δubc11 or Δubc4Δubc11 cells. These results suggest Ubc4p and Ubc11p together are not essential for mitotic cyclin destruction in S. cerevisiae and we can find no evidence to suggest that Ubc11p is the true functional homologue of E2-C. © 1998 John Wiley & Sons, Ltd.
    Zusätzliches Material: 6 Ill.
    Materialart: Digitale Medien
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  • 22
    ISSN: 0749-503X
    Schlagwort(e): Candida albicans ; HIS4 ; complementation ; molecular biology tools ; topological marker ; amino acid biosynthesis general control ; PEX5 ; Life and Medical Sciences ; Genetics
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Biologie
    Notizen: We have isolated the Candida albicans HIS4 (CaHIS4) gene by complementation of a his4-34 Saccharomyces cerevisiae mutant. The sequenced DNA fragment contains a putative ORF of 2514 bp, whose translation product shares a global identity of 44% and 55% to the His4 protein homologs of S. cerevisiae and Kluyveromyces lactis, respectively. Analysis of CaHIS4 sequence suggests that, similarly to S. cerevisiae HIS4, it codes for a polypeptide having three separate enzymatic activities (phosphoribosyl-AMP cyclohydrolase, phosphoribosyl-ATP pyrophosphohydrolase and histidinol dehydrogenase) which reside in different domains of the protein. A C. albicans his4 strain is complemented with this gene when using a C. albicans-S. cerevisiae-Escherichia coli shuttle vector, thus enabling the construction of a host system for C. albicans genetic manipulation. In addition, upstream of the sequenced CaHIS4 sequence, we have found the 3′-terminal half of a gene encoding a PEX5-like protein. The EMBL/DDJB/GenBank Accession Number of this sequence is AJ003115. © 1998 John Wiley & Sons, Ltd.
    Zusätzliches Material: 3 Ill.
    Materialart: Digitale Medien
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  • 23
    ISSN: 0749-503X
    Schlagwort(e): yeast ; elongation factor-3 ; EF-3 ; homolog ; Life and Medical Sciences ; Genetics
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Biologie
    Notizen: A paralog (intraspecies homolog) of the Saccharomyces cerevisiae YEF3 gene, encoding elongation factor-3, has been sequenced in the course of the yeast genome project, and identified by database searching; this gene has been designated HEF3. Bioinformatic and Northern blot analysis indicate that the HEF3 gene is not expressed during vegetative growth. Deletion of the HEF3 gene reveals no growth defects, nor any defects in mating or sporulation. A high copy 2μ clone of HEF3 was constructed, and was shown to be unable to complement a null allele of yef3. Finally, an in vitro assay for ribosome-stimulated ATPase activity was performed with isogenic HEF3 and Δhef3 strains; no difference in biochemical activity could be detected in these strains. From these results, we conclude that the HEF3 gene does not encode a functional homolog of YEF3. © 1998 John Wiley & Sons, Ltd.
    Zusätzliches Material: 4 Ill.
    Materialart: Digitale Medien
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  • 24
    Digitale Medien
    Digitale Medien
    New York, NY [u.a.] : Wiley-Blackwell
    Yeast 14 (1998), S. 1115-1125 
    ISSN: 0749-503X
    Schlagwort(e): Yarrowia lipolytica ; secretion ; pH ; extracellular protease ; Life and Medical Sciences ; Genetics
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Biologie
    Notizen: 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.
    Zusätzliches Material: 5 Ill.
    Materialart: Digitale Medien
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  • 25
    Digitale Medien
    Digitale Medien
    New York, NY [u.a.] : Wiley-Blackwell
    Yeast 14 (1998), S. 1159-1166 
    ISSN: 0749-503X
    Schlagwort(e): cell walls ; protease ; β-glucanase ; lysis ; yeast ; antifungal drugs ; glucan ; mannoprotein ; S. cerevisiae ; C. albicans ; Life and Medical Sciences ; Genetics
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Biologie
    Notizen: 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.
    Zusätzliches Material: 4 Ill.
    Materialart: Digitale Medien
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  • 26
    ISSN: 0749-503X
    Schlagwort(e): carbocyanine fluorescent probes ; membrane potential ; yeast ; cell wall ; Life and Medical Sciences ; Genetics
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Biologie
    Notizen: 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.
    Zusätzliches Material: 7 Ill.
    Materialart: Digitale Medien
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  • 27
    ISSN: 0749-503X
    Schlagwort(e): critical target model ; differential scanning calorimetry ; Saccharomyces cerevisiae ; heat-shock response ; acquired thermotolerance ; thermal stability ; Life and Medical Sciences ; Genetics
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Biologie
    Notizen: 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.
    Zusätzliches Material: 4 Ill.
    Materialart: Digitale Medien
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  • 28
    Digitale Medien
    Digitale Medien
    New York, NY [u.a.] : Wiley-Blackwell
    Yeast 14 (1998), S. 805-812 
    ISSN: 0749-503X
    Schlagwort(e): Dipodascus (Endomyces) magnusii ; genetic transformation ; ribosomal DNA ; autonomous replicating sequence ; electroporation ; Life and Medical Sciences ; Genetics
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Biologie
    Notizen: We have developed the first system for genetic transformation of the multinuclear yeast Dipodascus magnusii. The system is based on a dominant selectable marker and an autonomously replicating sequence. We have constructed a plasmid vector which contains a marker conferring resistance to zeocin and the segment of non-transcribed spacer of D. magnusii ribosomal DNA which supports the autonomous replication of plasmid DNA in yeast cells. Plasmid DNA has been transferred into D. magnusii cells by electroporation. The DNA sequence which is described in this article has been deposited in the EMBL data library under Accession Number Y14587. © 1998 John Wiley & Sons, Ltd.
    Zusätzliches Material: 5 Ill.
    Materialart: Digitale Medien
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  • 29
    Digitale Medien
    Digitale Medien
    New York, NY [u.a.] : Wiley-Blackwell
    Yeast 14 (1998), S. 847-852 
    ISSN: 0749-503X
    Schlagwort(e): Saccharomyces cerevisiae ; 2 micron plasmid ; Flp ; DNA damage ; curing ; Life and Medical Sciences ; Genetics
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Biologie
    Notizen: Powerful mutagenic screens of yeast Saccharomyces cerevisiae have recently been developed which require strains that lack the endogenous 2 micron plasmid (Burns et al., 1994). Here, we describe a simple and reliable method for curing yeast of the highly stable genetic element. The approach employs heterologous expression of a ‘step-arrest’ mutant of the Flp recombinase. The mutant, Flp H305L (Parsons et al., 1988), forms long-lived covalent protein-DNA complexes exclusively at 2 micron-borne recombinase target sites. In vivo, the complexes serve as sites of targeted DNA damage. Using Southern hybridization and a colony color assay for plasmid loss, we show that expression of the mutant enzyme results in the effective elimination of the 2 micron from cells. © 1998 John Wiley & Sons, Ltd.
    Zusätzliches Material: 3 Ill.
    Materialart: Digitale Medien
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  • 30
    Digitale Medien
    Digitale Medien
    New York, NY [u.a.] : Wiley-Blackwell
    Yeast 14 (1998), S. 985-1000 
    ISSN: 0749-503X
    Schlagwort(e): Mig1 repressor ; glucose repression ; Saccharomyces cerevisiae ; Life and Medical Sciences ; Genetics
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Biologie
    Notizen: A major mediator of glucose repression in yeast is Mig1, a zinc finger protein that binds to a GC-rich recognition sequence found upstream of many glucose-repressible genes. Because these Mig1 sites are found upstream of genes under different modes of regulation, we studied regulation of transcription mediated by an isolated Mig1 site placed upstream of a reporter gene under control of UASCYC1. The Mig1 site responded appropriately to glucose control and regulatory mutations, including snf1, reg1, cyc8, and tup1, mimicking the behavior of the SUC2 gene. Deletion of the MIG1-coding gene reduced but did not eliminate glucose repression mediated by the Mig1 site. Complete loss of repression was seen in a mig1 mig2 double mutant. When the UASCYC1 was replaced by UASADH1 in the reporter plasmid, the Mig1 site activated transcription under most conditions. Mutations of the two Mig1 binding sites in the SUC2 promoter resulted in loss of activation of SUC2 expression. These results suggest the presence of an unknown activator or activators that binds to the Mig1 site. The activator is not any of the proteins previously proposed to bind to this site, including Mig1, Mig2, Msn2, or Msn4. Band shift assays showed that Mig1 is the major protein in yeast cell extracts that binds to the Mig1 site in vitro. This binding is not regulated by glucose or mutations in CYC8 or TUP1. © 1998 John Wiley & Sons, Ltd.
    Zusätzliches Material: 5 Ill.
    Materialart: Digitale Medien
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  • 31
    Digitale Medien
    Digitale Medien
    New York, NY [u.a.] : Wiley-Blackwell
    Yeast 14 (1998), S. 1001-1006 
    ISSN: 0749-503X
    Schlagwort(e): succinate dehydrogenase ; SDH1 ; SDH1b ; Life and Medical Sciences ; Genetics
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Biologie
    Notizen: Transformation of the respiratory-defective mutant (E264/U2) of Saccharomyces cerevisiae with a yeast genomic library yielded two different plasmids capable of restoring the ability of the mutant to grow on non-fermentable substrates. One of the plasmids (pG52/T3) contained SDH1 coding for the flavoprotein subunit of mitochondrial succinate dehydrogenase. The absence of detectable succinate dehydrogenase activity in mitochondria of E264/U2 and the lack of complementation of the mutant by an sdh11null strain indicated a mutation in SDH1. The second plasmid (pG52/T8) had an insert with reading frame (YJL045w) of yeast chromosome X coding for a homologue of SDH1. Subclones containing the SDH1 homologue (SDH1b), restored respiration in E264/U2 indicating that the protein encoded by this gene is functional. The expression of the two genes was compared by assaying the β-galactosidase activities of yeast transformed with plasmids containing fusions of lacZ to the upstream regions of SDH1 and SDH1b. The 100-500 times lower activity measured in transformants harbouring the SDH1b-lacZ fusion indicates that the isoenzyme encoded by SDH1b is unlikely to play an important role in mitochondrial respiration. This is also supported by the absence of any obvious phenotype in cells with a disrupted copy of SDH1b. © 1998 John Wiley & Sons, Ltd.
    Zusätzliches Material: 2 Ill.
    Materialart: Digitale Medien
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  • 32
    ISSN: 0749-503X
    Schlagwort(e): Saccharomyces cerevisiae ; promoter-substitution cassette ; tetracycline-regulatable promoter ; essential genes ; conditional gene expression ; Life and Medical Sciences ; Genetics
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Biologie
    Notizen: A promoter-substitution cassette has been constructed that allows one-step substitution of chromosomal gene promoters for the tetracycline-regulatable tetO promoter in yeast cells, which uses kanMX4 as selective marker for geneticin resistance. Oligonucleotides for PCR amplification of the cassette are designed to allow homologous recombination through short flanking regions of homology with the upstream sequences of the chromosomal gene, upon transformation of target cells. By testing three essential genes of chromosome XV (YOL135c, YOL142w and YOL144w), the system causes tetracycline-dependent conditional growth of the cells, being modulatable by intermediate concentrations of the effector. Analysis of terminal phenotypes of the promoter-substituted cells in the presence of the antibiotic may facilitate functional analysis of essential orphan genes. © 1998 John Wiley & Sons, Ltd.
    Zusätzliches Material: 4 Ill.
    Materialart: Digitale Medien
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  • 33
    ISSN: 0749-503X
    Schlagwort(e): chemostat cultivation ; Saccharomyces cerevisiae ; carbon source ; transcriptional regulation ; UAS ; Life and Medical Sciences ; Genetics
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Biologie
    Notizen: To identify common regulatory sequences in the promoters of genes, transcription of 31 genes of Saccharomyces cerevisiae was analysed during the transient response to a glucose pulse in a chemostat culture. mRNA levels were monitored during the subsequent excess glucose, ethanol and acetate phases, while other conditions were kept constant. This setup allowed a direct comparison between regulation by glucose, ethanol and acetate.Genes with identical regulation patterns were grouped to identify regulatory elements in the promoters. In respect to regulation on glucose four classes were identified: no transcription under any of the conditions tested, no difference in regulation on glucose, induced on glucose and repressed on glucose. In addition, genes were found that were repressed or induced on ethanol or acetate. Sequence alignment of genes with similar regulation patterns revealed five new, putative regulatory promoter elements. (i) The glucose-inducible fermentation genes PDC1 and ADH1 share the sequence ATACCTTCSTT. (ii) Acetate-repression might be mediated by the decamer CCCGAG RGGA, present in the promoters of ACS2 and ACR1. (iii) A specific element (CCWTTSRNCCG) for the glyoxylate cycle was present in seven genes studied: CIT2, ICL1, MLS1, MDH2, CAT2, ACR1 and ACH1. These genes were derepressed on ethanol or acetate. (iv) The sequence ACGTSCRGAATGA was found in the promoters of the partially ethanol-repressed genes ACS1 and YAT1. (v) Ethanol induction, as seen for ACS2, ADH3 and MDH1, might be mediated via the sequence CGGSGCCGRAG. © 1998 John Wiley & Sons, Ltd.
    Zusätzliches Material: 5 Ill.
    Materialart: Digitale Medien
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  • 34
    ISSN: 0749-503X
    Schlagwort(e): LFH deletion cassette ; functional analysis ; chromosome IV ; Saccharomyces cerevisiae ; Life and Medical Sciences ; Genetics
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Biologie
    Notizen: We report here the construction of six deletion mutants and the analysis of their basic phenotype. Deletion cassettes containing the KanMX4 marker module and long flanking regions homologous to the target locus were constructed for each of the six open reading-frames (ORFs YDL088c, YDL087c, YDL086w, YDL085w, YDL084w and YDL082w) located on chromosome IV. Sporulation and tetrad analysis of heterozygous deletant strains revealed that, in the FY1679 genetic background, ORFs YDL088c, YDL087c and YDL084w are essential genes for vegetative growth whereas YDL086w, YDL085w and YDL082w are non-essential. ydl088cΔ and ydl084wΔ haploid strains are viable in the CEN. PK2 genetic background although ydl084wΔ grows at a slower rate than the wild type. Complementation tests by corresponding cognate genes confirmed that gene inactivation was responsible for these growth defects. © 1998 John Wiley & Sons, Ltd.
    Zusätzliches Material: 1 Ill.
    Materialart: Digitale Medien
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  • 35
    ISSN: 0749-503X
    Schlagwort(e): Candida boidinii ; peroxisome ; peroxisomal proliferation ; peroxisomal membrane proteins ; d-amino acid oxidase ; green fluorescent protein ; Life and Medical Sciences ; Genetics
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Biologie
    Notizen: A methylotrophic yeast, Candida boidinii, was grown on various combinations of peroxisome-inducing carbon source(s) (PIC(s)), i.e. methanol, oleate and d-alanine, and the regulation of peroxisomal proteins (both matrix and membrane ones) and organelle proliferation were studied. This regulation was followed (1) at the protein or enzyme level by means of the peroxisomal enzyme activity and Western analysis; (2) at the mRNA level by Northern analysis; and (3) at the organelle level by direct observation of peroxisomes under a fluorescent microscope. Peroxisomal proliferation was followed in vivo by using a C. boidinii strain producing a green fluorescent protein having peroxisomal targeting signal 1. When multiple PICs were used for cell growth, C. boidinii induced specific peroxisomal proteins characteristic of all PIC(s) present in the medium, responding to all PIC(s) simultaneously. Thus, these PICs were considered to induce peroxisomal proliferation independently and not to repress peroxisomes induced by other PICs. Next, the sensitivity of the peroxisomal induction to glucose repression was studied. While the peroxisomal induction by methanol or oleate was completely repressed by glucose, the d-alanine-induced activities of d-amino acid oxidase and catalase, Pmp47, and the organelle proliferation were not. These results indicate that peroxisomal proliferation in yeasts is not necessarily sensitive to glucose repression. Lastly, this regulation was shown to occur at the mRNA level. © 1998 John Wiley & Sons, Ltd.
    Zusätzliches Material: 8 Ill.
    Materialart: Digitale Medien
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  • 36
    Digitale Medien
    Digitale Medien
    New York, NY [u.a.] : Wiley-Blackwell
    Yeast 14 (1998), S. 647-654 
    ISSN: 0749-503X
    Schlagwort(e): anaplerotic reactions ; glyoxylate cycle ; catabolite repression ; Pichia ; Life and Medical Sciences ; Genetics
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Biologie
    Notizen: We have cloned and characterized a gene encoding pyruvate carboxylase from the methylotrophic yeast Pichia pastoris. Disruption of this gene produced inability to grow in minimal medium with glucose as carbon source and ammonium as nitrogen source. Growth was possible with aspartate or glutamate as nitrogen source. The gene PpPYC1 expressd from its own promoter was able to rescue the phenotype of Saccharomyces cerevisiae mutants devoid of pyruvate carboxylase.In a P. pastoris strain carrying a disrupted PpPYC1 gene we have isolated spontaneous mutants able to grow in non-permissive conditions. In a mutant strain grown in glucose several enzymes sensitive to catabolite repression were derepressed. The strain also had elevated levels of glutamate dehydrogenase (NAD) both in repressed and derepressed conditions. The sequence of the PpPYC1 gene has been entered in the EMBL nucleotide sequence databank: Accession Number Y11106. © 1998 John Wiley & Sons, Ltd.
    Zusätzliches Material: 3 Ill.
    Materialart: Digitale Medien
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  • 37
    ISSN: 0749-503X
    Schlagwort(e): Chromosome length ; chromosome VI ; electrophoretic karyotype ; RFLP ; Saccharomyces ; saké yeast ; taxonomy ; Life and Medical Sciences ; Genetics
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Biologie
    Notizen: Previous studies have revealed that chromosome VI of saké yeasts is much larger than that of the other strains of Saccharomyces cerevisiae. Southern analysis using segments of chromosome VI of a laboratory strain as probes suggested that the nucleotide sequence of a major portion of this chromosome is conserved, but considerable diversity was found in the distal parts in the other strains. Physical maps also indicated that differences in length of chromosome VI were mainly due to differences in its ends. NotI was found to generate 9 kb and/or 16 kb fragments from the left telomere of chromosome VI in most saké yeasts, but no fragment in the case of AB972. SfiI produced one or two 30-50 kb fragments from the right end of this chromosome in all saké yeasts tested, but produced a 20 kb fragment in the case of AB972. All S. cerevisiae strains not employed in saké brewing were the same as AB972 in these respects. S. paradoxus had one NotI site in chromosome VI, while S. bayanus had two, one of which is possibly common to both species. The SfiI site mentioned above was present in chromosome VI of all species, while that of S. bayanus and S. paradoxus each had a second site distinct from the other. Chromosome VI of S. pastorianus was not distinguishable from that of S. bayanus. © 1998 John Wiley & Sons, Ltd.
    Zusätzliches Material: 5 Ill.
    Materialart: Digitale Medien
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  • 38
    Digitale Medien
    Digitale Medien
    New York, NY [u.a.] : Wiley-Blackwell
    Yeast 14 (1998), S. 861-867 
    ISSN: 0749-503X
    Schlagwort(e): HIS3 ; TRP1 ; IPP1 ; Pichia pastoris ; Life and Medical Sciences ; Genetics
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Biologie
    Notizen: The Pichia pastoris TRP1 and HIS3 genes were cloned by complementation of the Saccharomyces cerevisiae trp1 and his3 mutants, respectively, and their nucleotide sequence was determined. The P. pastoris TRP1 gene includes an open reading frame (ORF) of 714 nucleotides corresponding to a polypeptide of 237 amino acids whose sequence shares about 40% identity with that of TRP1 encoding proteins in other yeast species. DNA sequencing showed that an ORF of 858 nucleotides, encoding a protein of 285 amino acids with high homology to inorganic pyrophosphatases (IPP1), is located downstream of the P. pastoris TRP1 gene. Both genes converge in this chromosomal region, showing a genetic organization analogous to that found in the Kluyveromyces lactis genome. The P. pastoris HIS3 gene possesses an ORF of 675 nucleotides, encoding a polypeptide of 224 amino acids which shows 74·1% identity to the homologous S. cerevisiae protein. The hexameric consensus GCN4 binding sequence (TGACTC), characteristic of many amino acid biosynthetic genes, is present in the promoter region. The TRP1 and IPP1 sequences were deposited in the EMBL databank under Accession Number AJ001000. The Accession Number of the HIS3 gene is U69170. © 1998 John Wiley & Sons, Ltd.
    Zusätzliches Material: 5 Ill.
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  • 39
    ISSN: 0749-503X
    Schlagwort(e): Saccharomyces cerevisiae ; chromosome VII ; ribonuclease PH ; HGH1 ; YGR187c ; YGR189c ; YGR194c ; YGR195w ; YGR196c ; YGR198w ; Life and Medical Sciences ; Genetics
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Biologie
    Notizen: We have deleted six different ORFs of unknown function located on the right arm of Saccharomyces cerevisiae chromosome VII; namely, YGR187c/HGH1, YGR189c, YGR194c, YGR195w, YGR196c and YGR198w. No basic phenotypes could be attributed to the strains deleted in any of genes YGR187c/HGH1, YGR189c, YGR194c and YGR196c. These deletants did not show mating, sporulation or growth defects under any of the conditions tested. However, spores bearing deletions in either the YGR195w or YGR198w genes were unable to develop into macroscopical colonies. The YGR195w gene product shows significant homology with bacterial ribonuclease PH, an enzyme hitherto undescribed in yeasts, and its deletion causes a loss of viability after one to three rounds of cell division. Overexpression of this gene, using a tetracycline-regulatable promoter system, did not cause any effect on the cells. Contrary to what has been reported for prokaryotic homologs, this enzyme could play an essential role in yeast cell biology. The product encoded by the other essential ORF, YGR198w, shows no significant homology with any protein of known function in the databases. Spores bearing the deletion usually germinate and give rise to microcolonies of 50-100 non-viable cells. © 1998 John Wiley & Sons, Ltd.
    Zusätzliches Material: 2 Ill.
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  • 40
    Digitale Medien
    Digitale Medien
    New York, NY [u.a.] : Wiley-Blackwell
    Yeast 14 (1998), S. 813-825 
    ISSN: 0749-503X
    Schlagwort(e): Life and Medical Sciences ; Genetics
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Biologie
    Notizen: We describe the quantitative monitoring of TATA-binding protein (TBP) localization and expression in living Saccharomyces cerevisiae cells. We replaced the endogenous TBP with a green fluorescent protein (GFP) · TBP fusion, which was imaged quantitatively by laser scanning confocal microscopy (LSCM). When GFP · TBP expression was altered by using various promoters, the levels measured by LSCM correlated well with the levels determined by immunoblot of whole cell extract protein. These results show that GFP · TBP imaging not only offers a method of measurement equivalent to a more conventional technique but also provides real-time quantitation in living cells and subcellular localization information. Time-lapse confocal imaging of GFP · TBP in mitotic yeast cells revealed that it remains localized to the nucleus and displays an asymmetric distribution (1:0·7) between mother and daughter cells. Based on this and data from a mutant which underexpresses GFP · TBP, we suggest that intracellular levels of TBP are near rate-limiting for growth and viability. © 1998 John Wiley & Sons, Ltd.
    Zusätzliches Material: 5 Ill.
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  • 41
    Digitale Medien
    Digitale Medien
    New York, NY [u.a.] : Wiley-Blackwell
    Yeast 14 (1998), S. 963-967 
    ISSN: 0749-503X
    Schlagwort(e): gluconeogenesis ; PEPCK ; Kluyveromyces lactis ; Life and Medical Sciences ; Genetics
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Biologie
    Notizen: The KlPCK1 gene encoding phosphoenolpyruvate carboxykinase (PEPCK; ATP-dependent) was cloned from the Kluyveromyces lactis genome using a PCR amplicon from Saccharomyces cerevisiae PCK1 gene as a probe. A DNA fragment of about 4·8 kb containing KlPCK1 complemented PEPCK activity of the mutant of S. cerevisiae defective in PEPCK. The KlPCK1 gene has an open reading frame of 1629 bp (543 amino acids). The KlPCK1 nucleotide sequence and deduced amino acid sequence showed 76% and 84% homologies to those of S. cerevisiae PCK1, respectively. Multiple alignment of ATP-dependent PEPCK genes shows highly conserved regions. The nucleotide sequence of KlPCK1 has been submitted to the DDBJ/GenBank/EMBL data bank with Accession Number U88575. © 1998 John Wiley & Sons, Ltd.
    Zusätzliches Material: 2 Ill.
    Materialart: Digitale Medien
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  • 42
    Digitale Medien
    Digitale Medien
    New York, NY [u.a.] : Wiley-Blackwell
    Yeast 14 (1998), S. 905-913 
    ISSN: 0749-503X
    Schlagwort(e): carbon dioxide ; cytostasis ; G1 arrest ; meiosis ; sporulation ; Life and Medical Sciences ; Genetics
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Biologie
    Notizen: Limitation of nutrients allows yeast cells to arrest proliferation at G1 phase of the cell cycle and to enter the so-called stationary phase. We show here another pathway for cytostasis, which is associated with extracellular accumulation of bicarbonate and the resulting alkalisation of medium during the proliferation of cells respiring acetate. Alkalisation of medium by addition of bicarbonate or alkaline buffers ceased proliferation at G1 phase of logarithmically growing cells and caused a severe drop in G1-cyclin (CLN1 and CLN2) mRNAs. The arrested cells were heat-shock resistant, suggesting that the cells entered the stationary phase. Cells confluently grown on acetate re-entered into the cell cycle after acidification of the culture medium. These results indicate that external alkalisation is a primary cause of the cytostasis. The alkali-induced G1 arrest was shown to be cyclic AMP (cAMP)-independent using mutant cells which lack a functional Ras/cAMP signaling pathway. Alkalisation of medium also stimulated meiosis and sporulation in rich acetate medium, confirming our previous proposal that environmental alkalisation but not nitrogen limitation is a key condition for entry into meiosis and sporulation. © 1998 John Wiley & Sons, Ltd.
    Zusätzliches Material: 7 Ill.
    Materialart: Digitale Medien
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  • 43
    Digitale Medien
    Digitale Medien
    New York, NY [u.a.] : Wiley-Blackwell
    Yeast 14 (1998), S. 923-933 
    ISSN: 0749-503X
    Schlagwort(e): S. pastorianus ; S. cerevisiae ; S. bayanus ; chromosome co-existence ; chromosomal rearrangement ; Life and Medical Sciences ; Genetics
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Biologie
    Notizen: The bottom fermenting yeasts in our collection were classified as Saccharomyces pastorianus on the basis of their DNA relatedness. The genomic organization of bottom fermenting yeast was analysed by Southern hybridization using eleven genes on chromosome IV, six genes on chromosome II and five genes on chromosome XV of S. cerevisiae as probes. Gene probes constructed from S. cerevisiae chromosomes II and IV hybridized strongly to the 820-kb chromosome and the 1500-kb chromosome of the bottom fermenting yeast, respectively. Five gene probes constructed from segments of chromosome XV hybridized strongly to the 1050-kb and the 1000-kb chromosomes. These chromosomes are thought to be S. cerevisiae-type chromosomes. In addition, these probes also hybridized weakly to the 1100-kb, 1350-kb, 850-kb and 700-kb chromosome. Gene probes constructed from segments including the left arm to TRP1 of chromosome IV and the right arm of chromosome II hybridized to the 1100-kb chromosome of S. pastorianus. Gene probes constructed using the right arm of chromosome IV and the left arm of chromosome II hybridized to the 1350-kb chromosome of S. pastorianus. These results suggested that the 1100-kb and 1350-kb chromosomes were generated by reciprocal translocation between chromosome II and IV in S. pastorianus. Three gene probes constructed using the right arm of chromosome XV hybridized weakly to the 850-kb chromosome, and two gene probes from the left arm hybridized weakly to the 700-kb chromosome. These results suggested that chromosome XV of S. cerevisiae was rearranged into the 850-kb and 700-kb chromosomes in S. pastorianus. These weak hybridization patterns were identical to those obtained with S. bayanus. Therefore, two types of chromosome co-exist independently in bottom fermenting yeast: one set which originated from S. bayanus and another set from S. cerevisiae. This result supports the hypothesis that S. pastorianus is a hybrid of S. cerevisiae and S. bayanus. © 1998 John Wiley & Sons, Ltd.
    Zusätzliches Material: 5 Ill.
    Materialart: Digitale Medien
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  • 44
    ISSN: 0749-503X
    Schlagwort(e): Schizosaccharomyces pombe ; Saccharomyces cerevisiae ; uracil permease ; transmembrane helices ; Life and Medical Sciences ; Genetics
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Biologie
    Notizen: The uracil permease gene of Schizosaccharomyces pombe was cloned and sequenced. The deduced protein sequence shares strong similarities with five open reading frames from Saccharomyces cerevisiae, namely the uracil permease encoded by the FUR4 gene, the allantoin permease encoded by DAL4, a putative uridine permease (YBL042C) and two unknown ORFs YOR071c and YLR237w.A topological model retaining ten transmembrane helices, based on predictions and on experimental data established for the uracil permease of S. cerevisiae by Galan and coworkers (1996), is discussed for the four closest proteins of this family of transporters. The sequence of the uracil permease gene of S. pombe has been deposited in the EMBL data bank under Accession Number X98696. © 1998 John Wiley & Sons, Ltd.
    Zusätzliches Material: 2 Ill.
    Materialart: Digitale Medien
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  • 45
    Digitale Medien
    Digitale Medien
    New York, NY [u.a.] : Wiley-Blackwell
    Yeast 14 (1998), S. 1041-1050 
    ISSN: 0749-503X
    Schlagwort(e): Saccharomyces cerevisiae ; STRE ; stress response ; genomics ; bioinformatics ; Life and Medical Sciences ; Genetics
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Biologie
    Notizen: Stress response elements (STREs, core consensus AG4 or C4T) have been demonstrated previously to occur in the upstream region of a number of genes responsive to induction by a variety of stress signals. This stress response is mediated by the homologous transcription factors Msn2p and Msn4p, which bind specifically to STREs. Double mutants (msn2 msn4) deficient in these transcription factors have been shown to be hypersensitive to severe stress conditions. To obtain a more representative overview of the set of yeast genes controlled via this regulon, a computer search of the Saccharomyces cerevisiae genome was carried out for genes, which, similar to most known STRE-controlled genes, exhibit at least two STREs in their upstream region. In addition to the great majority of genes previously known to be controlled via STREs, 69 open reading-frames were detected. Expression patterns of a set of these were examined by grid filter hybridization, and 14 genes were examined by Northern analysis. Comparison of the expression patterns of these genes demonstrates that they are all STRE-controlled although their detailed expression patterns differ considerably. © 1998 John Wiley & Sons, Ltd.
    Zusätzliches Material: 2 Ill.
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  • 46
    Digitale Medien
    Digitale Medien
    New York, NY [u.a.] : Wiley-Blackwell
    Yeast 14 (1998), S. 1017-1025 
    ISSN: 0749-503X
    Schlagwort(e): Arxula adeninivorans ; AILV1 ; threonine deaminase ; transformation ; homologous integration ; Life and Medical Sciences ; Genetics
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Biologie
    Notizen: The ILV1 gene of the yeast Arxula adeninivorans LS3 (AILV1) has been cloned from a genomic library, characterized and used as an auxotrophic selection marker for transformation of plasmids into this yeast. One copy of the gene is present in the Arxula genome, comprising 1653 bp and encoding 550 amino acids of the threonine deaminase. The protein sequence is similar (60·55%) to that of the threonine deaminase from Saccharomyces cerevisiae encoded by the gene ILV1. The protein is enzymatically active during the whole period of cultivation, up to 70 h. Maximal activities, as well as protein concentrations of this enzyme, were achieved after cultivation times of 20-36 h.The AILV1 gene is a suitable auxotrophic selection marker in transformation experiments using an Arxula adeninivorans ilv1 mutant and a plasmid containing this gene, which is fused into the 25S rDNA of Arxula adeninivorans. One to three copies of the linearized plasmid were integrated into the 25S rDNA by homologous recombination. Transformants resulting from complementation of the ilv1 mutation can be easily and reproducibly selected and in addition are mitotically stable. Therefore, the described system is preferred to the conventional selection for hygromycin B resistance. © 1998 John Wiley & Sons, Ltd.
    Zusätzliches Material: 5 Ill.
    Materialart: Digitale Medien
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  • 47
    ISSN: 0749-503X
    Schlagwort(e): gene disruption ; homologous recombination ; protein A-tagging ; Saccharomyces cerevisiae ; tags ; Life and Medical Sciences ; Genetics
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Biologie
    Notizen: Gene disruption and tagging can be achieved by homologous recombination in the yeast genome. Several PCR-based methods have been described towards this end. However these strategies are often limited in their applications and/or their efficiencies and may be technically demanding. Here we describe two plasmids for C-terminal tagging of proteins with the IgG binding domain of the Staphyloccocus aureus protein A. We also present simple and reliable strategies based on PCR to promote efficient integration of exogenous DNA into the yeast genome. These simple methods are not limited to specific strains or markers and can be used for any application requiring homologous recombination such as gene disruption and epitope tagging. These strategies can be used for consecutive introduction of various constructs into a single yeast strain. © 1998 John Wiley & Sons, Ltd.
    Zusätzliches Material: 2 Ill.
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  • 48
    Digitale Medien
    Digitale Medien
    New York, NY [u.a.] : Wiley-Blackwell
    Yeast 14 (1998), S. 1223-1232 
    ISSN: 0749-503X
    Schlagwort(e): Candida utilis ; β-fructofuranosidase ; glycosyl hydrolase ; signal peptide ; sucrose ; polymerase chain reaction ; invertase ; Life and Medical Sciences ; Genetics
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Biologie
    Notizen: The gene INV1 encoding invertase from the yeast Candida utilis has been cloned using a homologous PCR hybridization probe, amplified with two sets of degenerate primers designed considering sequence comparisons between yeast invertases. The cloned gene was sequenced and found to encode a polypeptide of 533 amino acids that contain a 26 amino-acid signal peptide and 12 potential N-glycosylation sites. The nucleotide sequences of the 5′ and 3′ non-coding regions were found to contain motifs probably involved in initiation, regulation and termination of gene transcription. The amino-acid sequence shows significant identity with other yeast, bacterial and plant β-fructofuranosidases. The INV1 gene from C. utilis was able to complement functionally the suc2 mutation of S. cerevisiae. The sequence presented here has been deposited in the EMBL data library under Accession Number Y12659. © 1998 John Wiley & Sons, Ltd.
    Zusätzliches Material: 6 Ill.
    Materialart: Digitale Medien
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  • 49
    ISSN: 0749-503X
    Schlagwort(e): salt-tolerant yeast ; Zygosaccharomyces rouxii ; Na+/H+ antiporter ; functional expression ; Life and Medical Sciences ; Genetics
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Biologie
    Notizen: We reported in our previous paper on the characterization of the Na+/H+-antiporter gene (ZSOD2) closely related to the salt-tolerance of yeast Zygosaccharomyces rouxii. In the present paper, we have cloned a second gene (ZSOD22) of Na+/H+ antiporter from Z. rouxii. The deduced amino acid sequence of Zsod22p was highly homologous to that of Zsod2p, Sod2p from Schizosaccharomyces pombe, and Nha1p from Saccharomyces cerevisiae. The open reading frames (ORFs) from ZSOD2 or ZSOD22 were inserted into a yeast expression vector pYES2, and their constructs (pZSOD2 and pZSOD22) were used to transform the salt-sensitive S. cerevisiae. pZSOD2- or pZSOD22-harboring-recombinant S. cerevisiae cells showed increases in salt tolerance. However, the Z. rouxii disruptant of ZSOD22 did not show any phenotypes related to salt tolerance or osmotolerance, unlike that of ZSOD2. The transcriptional expression of ZSOD22 was not observed by Northern blot analysis even in Z. rouxii cells subjected to NaCl-shock. From these results we conclude that although Z. rouxii includes at least two copies of the Na+/H+-antiporter gene (ZSOD2 and ZSOD22), ZSOD2 encodes a functional product as an antiporter and ZSOD22 is poorly transcribed, if at all. The nucleotide sequence data of ZSOD22 will appear in the DDBJ, EMBL and GenBank nucleotide sequence databases with the following accession number: AB010106. © 1998 John Wiley & Sons, Ltd.
    Zusätzliches Material: 3 Ill.
    Materialart: Digitale Medien
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  • 50
    Digitale Medien
    Digitale Medien
    New York, NY [u.a.] : Wiley-Blackwell
    Yeast 14 (1998), S. 1307-1310 
    ISSN: 0749-503X
    Schlagwort(e): Schizosaccharomyces pombe ; 5-phosphoribosyl-4-carboxamide 5-aminoimidazole (AICAR) transformylase ; inosine monophosphate (IMP) cyclohydrolase ; intrachromosomal recombination ; Life and Medical Sciences ; Genetics
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Biologie
    Notizen: We have cloned and sequenced the Schizosaccharomyces pombe ade10 gene encoding 5-phosphoribosyl-4-carboxamide 5-aminoimidazole transformylase inosine monophosphate cyclohydrolase. The sequence has an uninterrupted open reading frame of 1755 nucleotides corresponding to 585 amino acid residues. The deduced amino acid sequence shows a high degree of similarity to the purH gene product of many species, including Saccharomyces cerevisiae, human, chicken and Escherichia coli. Moreover our data indicate that intrachromosomal recombination in Schiz. pombe is enhanced if the ade10 gene product is defective. The sequence has been submitted to the EMBL data library under Accession Number Y16419. © 1998 John Wiley & Sons, Ltd.
    Zusätzliches Material: 2 Ill.
    Materialart: Digitale Medien
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  • 51
    ISSN: 0749-503X
    Schlagwort(e): Candida albicans ; SRST ; systeny ; RAD16 ; LYS2 ; Life and Medical Sciences ; Genetics
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Biologie
    Notizen: Single-read sequences from both ends of 415 3-kb average size genomic DNA fragments of Candida albicans were compared with the complete sequence data of Saccharomyces cerevisiae. Comparison at the protein level, translated DNA against protein sequences, revealed 138 sequence tags with clear similarity to S. cerevisiae proteins or open reading frames. One case of synteny was found for the open reading frames of RAD16 and LYS2, which are adjacent to each other in S. cerevisiae and C. albicans. © 1998 John Wiley & Sons, Ltd.
    Zusätzliches Material: 3 Ill.
    Materialart: Digitale Medien
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  • 52
    ISSN: 0749-503X
    Schlagwort(e): yeast ; Candida utilis ; URA3 ; orotidine 5′-monophosphate decarboxylase ; transformation system ; Life and Medical Sciences ; Genetics
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Biologie
    Notizen: The URA3 gene of Candida utilis encoding orotidine-5′-phosphate decarboxylase enzyme was isolated by complementation in Escherichia coli pyrF mutation. The deduced amino-acid sequence is highly similar to that of the Ura3 proteins from other yeast and fungal species. An extensive analysis of the family of orotidine-5′-phosphate decarboxylase is shown. The URA3 gene of C. utilis was able to complement functionally the ura3 mutation of Saccharomyces cerevisiae. The sequence presented here has been deposited in the EMBL data library under Accession Number Y12660. © 1998 John Wiley & Sons, Ltd.
    Zusätzliches Material: 2 Ill.
    Materialart: Digitale Medien
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  • 53
    Digitale Medien
    Digitale Medien
    New York, NY [u.a.] : Wiley-Blackwell
    Yeast 14 (1998), S. 1355-1371 
    ISSN: 0749-503X
    Schlagwort(e): yeast ; Debaryomyces ; transport ; physiology ; salt tolerance ; Life and Medical Sciences ; Genetics
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Biologie
    Notizen: Debaryomyces hansenii showed an increased growth in the presence of either 1m KCl or 1m NaCl and a low acidification of the medium, higher for the cells grown in the presence of NaCl. These cells accumulated high concentrations of the cations, and showed a very fast capacity to exchange either Na+ or K+ for the opposite cation. They showed a rapid uptake of 86 Rb+ and 22 Na+ . 86 Rb+ transport was saturable, with Km and Vmax values higher for cells grown in 1m NaCl. 22 Na+ uptake showed a diffusion component, also higher for the cells grown with NaCl. Changes depended on growth conditions, and not on further incubation, which changed the internal ion concentration. K+ stimulated proton pumping produced a rapid extrusion of protons, and also a decrease of the membrane potential. Cells grown in 1m KCl showed a higher fermentation rate, but significantly lower respiratory capacity. ATP levels were higher in cells grown in the presence of NaCl; upon incubation with glucose, those grown in the presence of KCl reached values similar to the ones grown in the presence of NaCl. In both, the addition of KCl produced a transient decrease of the ATP levels. As to ion transport mechanisms, D. hansenii appears to have (a) an ATPase functioning as a proton pump, generating a membrane potential difference which drives K+ through a uniporter; (b) a K+/H+ exchange system; and (c) a rapid cation/cation exchange system. Most interesting is that cells grown in different ionic environments change their studied capacities, which are not dependent on the cation content, but on differences in their genetic expression during growth. © 1998 John Wiley & Sons, Ltd.
    Zusätzliches Material: 10 Ill.
    Materialart: Digitale Medien
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  • 54
    ISSN: 0749-503X
    Schlagwort(e): World Wide Web ; Saccharomyces Genome Database ; Munich Information Center for Protein Sequences ; Yeast Protein Database ; Life and Medical Sciences ; Genetics
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Biologie
    Notizen: The completion of the Saccharomyces cerevisiae genome sequencing project11 and the continued development of improved technology for large-scale genome analysis have led to tremendous growth in the amount of new yeast genetics and molecular biology data. Efficient organization, presentation, and dissemination of this information are essential if researchers are to exploit this knowledge. In addition, the development of tools that provide efficient analysis of this information and link it with pertinent information from other systems is becoming increasingly important at a time when the complete genome sequences of other organisms are becoming available. The aim of this review is to familiarize biologists with the type of data resources currently available on the World Wide Web (WWW). © 1998 John Wiley & Sons, Ltd.
    Zusätzliches Material: 7 Ill.
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  • 55
    Digitale Medien
    Digitale Medien
    New York, NY [u.a.] : Wiley-Blackwell
    Yeast 14 (1998), S. 1529-1566 
    ISSN: 0749-503X
    Schlagwort(e): G protein ; mating ; pheromone ; Schizosaccharomyces pombe ; receptor ; yeast ; Life and Medical Sciences ; Genetics
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Biologie
    Zusätzliches Material: 7 Ill.
    Materialart: Digitale Medien
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  • 56
    Digitale Medien
    Digitale Medien
    New York, NY [u.a.] : Wiley-Blackwell
    Yeast 14 (1998), S. 89-91 
    ISSN: 0749-503X
    Schlagwort(e): PCR mutagenesis ; functional domains ; subgenic DNA fragments ; mutagenesis protocol ; Life and Medical Sciences ; Genetics
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Biologie
    Notizen: Here we describe a method for performing a PCR-driven random mutagenesis of 100 bp DNA fragments that yields mutations at a useful frequency. The method is a modification of the manganese ion substitution PCR technique, and neither creates ‘hot-spots’ nor favors transition mutations over transversions. © 1998 John Wiley & Sons, Ltd.
    Zusätzliches Material: 2 Tab.
    Materialart: Digitale Medien
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  • 57
    ISSN: 0749-503X
    Schlagwort(e): Kluyveromyces lactis ; CBF5 ; centromere ; nucleolus ; Life and Medical Sciences ; Genetics
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Biologie
    Notizen: The gene coding for the centromere-binding factor 5 (CBF5) of Kluyveromyces lactis has been isolated by hybridization of a Saccharomyces cerevisiae CBF5 DNA probe to a K. lactis library. The amino acid sequence of KlCbf5 is highly homologous, 88% identity, to ScCbf5, but also to the rat protein Nap57 (64% identity). The main difference between both yeast proteins and the rat protein is the presence of a lysine-rich domain with KKE/D repeats in the C-terminal part of the protein. These repeats are thought to be involved in binding of the protein to microtubules. Deletion of the KKE/D domain in KlCbf5 however, has no discernible effect on growth on rich medium, sensitivity to the microtubule-destabilizing drug benomyl or segregation of a reporter plasmid. On the other hand, insertion of two leucine residues adjacent to the KKE domain increases the loss rate of a reporter plasmid. In both yeasts complementation of a lethal CBF5 disruption with the heterologous gene results in a slight increase in benomyl sensitivity. A possible role of CBF5 in chromosome segregation will be discussed. © 1998 John Wiley & Sons, Ltd.
    Zusätzliches Material: 5 Ill.
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  • 58
    Digitale Medien
    Digitale Medien
    New York, NY [u.a.] : Wiley-Blackwell
    Yeast 14 (1998), S. 49-65 
    ISSN: 0749-503X
    Schlagwort(e): multidrug resistance ; drug efflux ; MPP+ ; Life and Medical Sciences ; Genetics
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Biologie
    Notizen: In this study, we report the further characterization of the Saccharomyces cerevisiae crystal violet-resistance protein Sge1. Sge1 is a highly hydrophobic 59 kDa protein with 14 predicted membrane-spanning domains. It shares homologies with several drug-resistance proteins and sugar transporters of the major facilitator superfamily. Here, we have demonstrated that Sge1 is not only a crystal violet-resistance protein, but that it also confers resistance to ethidium bromide and methylmethane sulfonate. Disruption of SGE1 leads to increased sensitivity towards all three compounds, thus designating Sge1 as a multiple drug-resistance protein. Subcellular fractionation as well as immunolocalization on whole yeast cells demonstrated that Sge1 was tightly associated with the yeast plasma membrane. Furthermore, Sge1 was highly enriched in preparations of yeast plasma membranes. In analogy to other multidrug-resistance proteins, we suggest that Sge1 functions as a drug export permease. © 1998 John Wiley & Sons, Ltd.
    Zusätzliches Material: 6 Ill.
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  • 59
    ISSN: 0749-503X
    Schlagwort(e): transcription factors ; tRNA biosynthesis ; mitochondrial RNA processing ; ribonuclease P ; Life and Medical Sciences ; Genetics
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Biologie
    Notizen: RPM2 is a Saccharomyces cerevisiae nuclear gene required for normal cell growth yet the only known function of Rpm2p is as a protein subunit of yeast mitochondrial RNase P, an enzyme responsible for the 5′ maturation of mitochondrial tRNAs. Since mitochondrial protein synthesis in S. cerevisiae is not essential for viability, RPM2 must provide another function in addition to its known role as a mitochondrial tRNA processing enzyme. During a search for RPM2 homologues from Kluyveromyces lactis, we recovered a K. lactis gene that compensates for the essential function but not the RNase P function of RPM2. We have named this gene SEF1 (Suppressor of the Essential Function). DNA sequence analysis of SEF1 reveals it contains a Zn(2)-Cys(6) binuclear cluster motif found in a growing number of yeast transcription factors. The SEF1 homologue of S. cerevisiae also compensates for the essential function of RPM2. The two proteins share 49% identity and 72% amino acid sequence similarity. The SEF1 sequence has been deposited in the GenBank data library under accession number U92898. © 1998 John Wiley & Sons, Ltd.
    Zusätzliches Material: 4 Ill.
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  • 60
    ISSN: 0749-503X
    Schlagwort(e): Saccharomyces cerevisiae ; yeast ; gene disruption ; S288C ; bacteria-yeast shuttle vectors ; auxotrophic markers ; Life and Medical Sciences ; Genetics
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Biologie
    Notizen: A set of yeast strains based on Saccharomyces cerevisiae S288C in which commonly used selectable marker genes are deleted by design based on the yeast genome sequence has been constructed and analysed. These strains minimize or eliminate the homology to the corresponding marker genes in commonly used vectors without significantly affecting adjacent gene expression. Because the homology between commonly used auxotrophic marker gene segments and genomic sequences has been largely or completely abolished, these strains will also reduce plasmid integration events which can interfere with a wide variety of molecular genetic applications. We also report the construction of new members of the pRS400 series of vectors, containing the kanMX, ADE2 and MET15 genes. © 1998 John Wiley & Sons, Ltd.
    Zusätzliches Material: 8 Ill.
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  • 61
    ISSN: 0749-503X
    Schlagwort(e): Saccharomyces cerevisiae ; YAP3 ; KEX2 ; recombinant human albumin ; protease degradation ; Life and Medical Sciences ; Genetics
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Biologie
    Notizen: Expression of recombinant human albumin (rHA) in Saccharomyces cerevisiae resulted in secretion of both mature albumin and a 45 kDa degradation product, comprising an N-terminal fragment of rHA with heterogeneous C-termini between residues 403 and 409 (Geisow et al., 1991). Site-directed mutagenesis of the human albumin gene (HA) to change Arg410 to Ala (R410A) caused a significant reduction in the amount of fragment produced. Mutation of the adjacent dibasic site Lys413 Lys414 had little effect in isolation, but in combination with the R410A mutation resulted in a further reduction in the amount of rHA fragment produced. This reduction could be duplicated with nature-identical rHA by disruption of the gene for an aspartyl protease (YAP3), alone or in conjunction with disruption of the KEX2 gene. Disruption of KEX2 alone did not result in any improvement in the degree of degradation of the rHA. Reduced degradation was also observed when an rHA-human growth hormone fusion protein was secreted from a yap3 strain, suggesting that such strains may have a general utility for heterologous protein secretion. © 1998 John Wiley & Sons, Ltd.
    Zusätzliches Material: 3 Ill.
    Materialart: Digitale Medien
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  • 62
    Digitale Medien
    Digitale Medien
    New York, NY [u.a.] : Wiley-Blackwell
    Yeast 14 (1998), S. 443-457 
    ISSN: 0749-503X
    Schlagwort(e): evolution ; polyploidy ; gene duplication ; gene order ; LEU2 ; Life and Medical Sciences ; Genetics
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Biologie
    Notizen: 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.
    Zusätzliches Material: 4 Ill.
    Materialart: Digitale Medien
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  • 63
    ISSN: 0749-503X
    Schlagwort(e): Crabtree effect ; yeast ; biomass ; Kluyveromyces lactis ; oxygen ; pyruvate decarboxylase ; regulation ; fermentation ; Life and Medical Sciences ; Genetics
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Biologie
    Notizen: 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.
    Zusätzliches Material: 5 Ill.
    Materialart: Digitale Medien
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  • 64
    Digitale Medien
    Digitale Medien
    New York, NY [u.a.] : Wiley-Blackwell
    Yeast 14 (1998), S. 501-515 
    ISSN: 0749-503X
    Schlagwort(e): Saccharomyces cerevisiae ; bakers' yeast ; pH homeostasis ; cytoplasmic pH ; vacuolar pH ; Life and Medical Sciences ; Genetics
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Biologie
    Notizen: 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.
    Zusätzliches Material: 10 Ill.
    Materialart: Digitale Medien
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  • 65
    ISSN: 0749-503X
    Schlagwort(e): alcohol acetyltransferase ; ATF1 gene ; OLE1 gene ; unsaturated fatty acid ; oxygen ; Life and Medical Sciences ; Genetics
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Biologie
    Notizen: The ATF1 gene encodes an alcohol acetyl transferase which catalyzes the synthesis of acetate esters from acetyl CoA and several kinds of alcohols. ATF1 expression is repressed by unsaturated fatty acids or oxygen. Analysis using ATF1-lacZ fusion plasmid revealed that ATF1 gene expression is widely repressed by a variety of unsaturated fatty acids, and the degree of ATF1 transcriptional repression varies according to the structure of the unsaturated fatty acids. Interestingly, it was noted that the degree of ATF1 transcriptional repression was related to the melting point of unsaturated fatty acids added to the medium. The OLE1 gene, which encodes Δ-9 fatty acid desaturase, has been reported to be repressed by unsaturated fatty acids. Transcription of OLE1 was also repressed by a wide variety of unsaturated fatty acids under anaerobic conditions. The degree of transcriptional repression of OLE1 was also related to the melting point of the added unsaturated fatty acids. Therefore, it is considered that ATF1 and OLE1 transcription are regulated in response to cell membrane fluidity. As has been reported for OLE1, the repression of ATF1 by unsaturated fatty acids was relieved in a disruptant carrying a faa1 and faa4 double mutation, two fatty acid activation genes. However, the ATF1 transcript in this double gene disruptant was repressed by oxygen. These results suggested that ATF1 transcription was co-regulated by the same mechanism as the OLE1 gene and that unsaturated fatty acids and oxygen repressed the ATF1 transcript by a different regulation pathway. © 1998 John Wiley & Sons, Ltd.
    Zusätzliches Material: 3 Ill.
    Materialart: Digitale Medien
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  • 66
    Digitale Medien
    Digitale Medien
    New York, NY [u.a.] : Wiley-Blackwell
    Yeast 14 (1998), S. 783-790 
    ISSN: 0749-503X
    Schlagwort(e): Pichia pastoris ; expression vectors ; gene regulation ; Life and Medical Sciences ; Genetics
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Biologie
    Notizen: The budding yeast Pichia pastoris is an attractive system for exploring certain questions in cell biology, but experimental use of this organism has been limited by a lack of convenient expression vectors. Here we describe a set of compact vectors that should allow for the expression of a wide range of endogenous or foreign genes in P. pastoris. A gene of interest is inserted into a modified pUC19 polylinker; targeted integration into the genome then results in stable and uniform expression of this gene. The utility of these vectors was illustrated by expressing the bacterial β-glucuronidase (GUS) gene. Constitutive GUS expression was obtained with the strong GAP promoter or the moderate YPT1 promoter. The regulatable AOX1 promoter yielded very strong GUS expression in methanol-grown cells, negligible expression in glucose-grown cells, and intermediate expression in mannitol-grown cells. GenBank Accession Numbers are: pIB1, AF027958; pIB2, AF027959; pIB3, AF027960; pIB4, AF027961. © 1998 John Wiley & Sons, Ltd.
    Zusätzliches Material: 1 Ill.
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  • 67
    ISSN: 0749-503X
    Schlagwort(e): Life and Medical Sciences ; Genetics
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Biologie
    Materialart: Digitale Medien
    Standort Signatur Erwartet Verfügbarkeit
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  • 68
    Digitale Medien
    Digitale Medien
    New York, NY [u.a.] : Wiley-Blackwell
    Yeast 14 (1998), S. 1439-1451 
    ISSN: 0749-503X
    Schlagwort(e): beginnings of yeast research ; yeasts ; history ; Lavoisier ; Cagniard-Latour ; Kutzing ; Schwann ; Life and Medical Sciences ; Genetics
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Biologie
    Zusätzliches Material: 6 Ill.
    Materialart: Digitale Medien
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  • 69
    Digitale Medien
    Digitale Medien
    New York, NY [u.a.] : Wiley-Blackwell
    Yeast 14 (1998), S. 1471-1510 
    ISSN: 0749-503X
    Schlagwort(e): yeast ; S. cerevisiae ; lipids ; phospholipids ; sterols ; sphingolipids ; fatty acids ; Life and Medical Sciences ; Genetics
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Biologie
    Notizen: The yeast Saccharomyces cerevisiae is a powerful experimental system to study biochemical, cell biological and molecular biological aspects of lipid synthesis. Most but not all genes encoding enzymes involved in fatty acid, phospholipid, sterol or sphingolipid biosynthesis of this unicellular eukaryote have been cloned, and many gene products have been functionally characterized. Less information is available about genes and gene products governing the transport of lipids between organelles and within membranes, turnover and degradation of complex lipids, regulation of lipid biosynthesis, and linkage of lipid metabolism to other cellular processes. Here we summarize current knowledge about lipid biosynthetic pathways in S. cerevisiae and describe the characteristic features of the gene products involved. We focus on recent discoveries in these fields and address questions on the regulation of lipid synthesis, subcellular localization of lipid biosynthetic steps, cross-talk between organelles during lipid synthesis and subcellular distribution of lipids. Finally, we discuss distinct functions of certain key lipids and their possible roles in cellular processes. © 1998 John Wiley & Sons, Ltd.
    Zusätzliches Material: 5 Ill.
    Materialart: Digitale Medien
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  • 70
    ISSN: 0749-503X
    Schlagwort(e): transcription ; microarrays ; expression profiling ; Life and Medical Sciences ; Genetics
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Biologie
    Notizen: 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.
    Zusätzliches Material: 7 Ill.
    Materialart: Digitale Medien
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  • 71
    ISSN: 0749-503X
    Schlagwort(e): yeast ; PMR1 ; Hansenula polymorpha ; Ca2+-ATPase ; Life and Medical Sciences ; Genetics
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Biologie
    Notizen: 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.
    Zusätzliches Material: 5 Ill.
    Materialart: Digitale Medien
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  • 72
    Digitale Medien
    Digitale Medien
    New York, NY [u.a.] : Wiley-Blackwell
    Yeast 14 (1998), S. 1257-1265 
    ISSN: 0749-503X
    Schlagwort(e): Candida albicans ; nucleoside transport ; Life and Medical Sciences ; Genetics
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Biologie
    Notizen: 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.
    Zusätzliches Material: 5 Ill.
    Materialart: Digitale Medien
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  • 73
    Digitale Medien
    Digitale Medien
    New York, NY [u.a.] : Wiley-Blackwell
    Yeast 14 (1998), S. 1407-1416 
    ISSN: 0749-503X
    Schlagwort(e): transcription factor ; yeast genetic selection ; bacteriophage λ vector ; Cre-loxP automatic plasmid subcloning ; integration vector ; one hybrid ; target genes ; Life and Medical Sciences ; Genetics
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Biologie
    Notizen: 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.
    Zusätzliches Material: 3 Ill.
    Materialart: Digitale Medien
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  • 74
    Digitale Medien
    Digitale Medien
    New York, NY [u.a.] : Wiley-Blackwell
    Yeast 14 (1998), S. 1387-1397 
    ISSN: 0749-503X
    Schlagwort(e): Yarrowia lipolytica ; cytochrome P450 ; n-alkane metabolism ; n-alkane-inducible gene ; RT-PCR ; Life and Medical Sciences ; Genetics
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Biologie
    Notizen: 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.
    Zusätzliches Material: 6 Ill.
    Materialart: Digitale Medien
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  • 75
    Digitale Medien
    Digitale Medien
    New York, NY [u.a.] : Wiley-Blackwell
    Yeast 14 (1998), S. 1511-1527 
    ISSN: 0749-503X
    Schlagwort(e): Saccharomyces cerevisiae ; oxidative stress ; stress response ; signal transduction ; Life and Medical Sciences ; Genetics
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Biologie
    Notizen: 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.
    Materialart: Digitale Medien
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  • 76
    ISSN: 0749-503X
    Schlagwort(e): flocculation ; immunolocalization ; mannoprotein ; cell wall ; Life and Medical Sciences ; Genetics
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Biologie
    Notizen: The yeast FLO genes encode cell surface proteins which are expected to play a major role in the control of flocculation. We have assessed the availability of the Flo proteins at the cell surface during the growth of two flocculent strains, ABXL-1D (FLO1) and STX347-1D (FLO5) using immunological approaches, enzyme-linked immunosorbent assays and immunofluorescence. Our data show that they are not permanently present at the cell surface but that their amount increases during growth. With both strains the flocculation level is tightly correlated to the amount of Flop antigen detected, suggesting that it is the availability of the Flo proteins at the cell surface which determines the flocculation level. Our data are consistent with the idea that the Flo proteins correspond to the flocculation lectins. The differences of flocculation pattern among strains could originate from variations in the regulation of the expression of the FLO genes. Monitoring of the distribution of the Flo proteins during cellular development revealed that they are incorporated essentially in the cell wall of growing buds. Incorporation of the Flo proteins in the cell wall displays a highly polarized aspect, at the bud tip and at the mother-daughter neck junction, which can persist in mature cells. Such a localization could be relevant to constraints of the cell wall incorporation of the mannoproteins. Depending on the regulation of Flop expression and on the incorporation of the proteins in the cell wall, a yeast population can be highly heterogeneous in Flo protein equipment. © 1998 John Wiley & Sons, Ltd.
    Zusätzliches Material: 4 Ill.
    Materialart: Digitale Medien
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  • 77
    Digitale Medien
    Digitale Medien
    New York, NY [u.a.] : Wiley-Blackwell
    Yeast 14 (1998), S. 101-101 
    ISSN: 0749-503X
    Schlagwort(e): Life and Medical Sciences ; Genetics
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Biologie
    Materialart: Digitale Medien
    Standort Signatur Erwartet Verfügbarkeit
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  • 78
    Digitale Medien
    Digitale Medien
    New York, NY [u.a.] : Wiley-Blackwell
    Yeast 14 (1998), S. 103-114 
    ISSN: 0749-503X
    Schlagwort(e): Saccharomyces cerevisiae ; amino acid uptake ; ssy mutants ; Life and Medical Sciences ; Genetics
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Biologie
    Notizen: In order to identify genes involved in uptake of isoleucine, leucine and valine in Saccharomyces cerevisiae we isolated mutants that, on a complex medium, were sensitive to an inhibitor of the biosynthesis of the branched-chain amino acids. Mutants that in a secondary screen showed reduced uptake of isoleucine, leucine and valine when growing in synthetic complete medium were further characterized. Genetic analysis identified five loci, named ssy1 through ssy5. ssy2 corresponds to the previously characterized bap1 mutation, which we recently have found to be allelic to stp1. ssy1, ssy3 and ssy5 exhibit a reduced uptake of phenylalanine, methionine and threonine, as well. Furthermore, they are resistant to several neutral amino acid analogs. ssy4 only affects uptake of few neutral amino acids and is as sensitive as the wild type to the amino acid analogs tested. It was previously found that a C-terminal truncation of 29 codons of BAP2, which encodes a branched-chain amino acid permease, results in increased uptake of the branched-chain amino acids. We find epistasis of the C-terminally truncated BAP2 gene over the ssy4 mutation, while the other ssy mutations are epistatic over the truncated BAP2 gene. SSY1, SSY3 and SSY5 were cloned from a low-copy genomic library by complementation of the mutants. The SSY3 gene and the SSY5 gene show no significant homology to any sequence in the databases. SSY1 is a member of the major family of genes encoding amino acid permeases in yeast. We discuss possible roles of Ssy1p in amino acid uptake. © 1998 John Wiley & Sons, Ltd.
    Zusätzliches Material: 3 Ill.
    Materialart: Digitale Medien
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  • 79
    Digitale Medien
    Digitale Medien
    New York, NY [u.a.] : Wiley-Blackwell
    Yeast 14 (1998), S. 147-160 
    ISSN: 0749-503X
    Schlagwort(e): yeast physiology ; yeast viability ; flow cytometry ; bakers yeast ; Life and Medical Sciences ; Genetics
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Biologie
    Notizen: 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.
    Zusätzliches Material: 4 Ill.
    Materialart: Digitale Medien
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  • 80
    Digitale Medien
    Digitale Medien
    New York, NY [u.a.] : Wiley-Blackwell
    Yeast 14 (1998), S. 203-213 
    ISSN: 0749-503X
    Schlagwort(e): GPM2 ; GPM3 ; phosphoglycerate mutase ; Life and Medical Sciences ; Genetics
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Biologie
    Notizen: 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.
    Zusätzliches Material: 3 Ill.
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  • 81
    ISSN: 0749-503X
    Schlagwort(e): Hansenula polymorpha ; 2 μm DNA ; plasmid partitioning ; nuclear segregation ; Life and Medical Sciences ; Genetics
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Biologie
    Notizen: Insertion of the HindIII-PstI fragment of Saccharomyces cerevisiae 2 μm DNA into the Hansenula polymorpha replicative plasmids decreases plasmid copy number and ensures their distribution to daughter cells at both mitotic and meiotic cell divisions. This suggests that the stabilization effect is caused by the improvement of plasmid partitioning. Deletion analysis revealed that the region of 2 μm DNA sequence responsible for the increase of mitotic stability of H. polymorpha plasmids involves the 2 μm STB locus and adjoining region. Further analysis demonstrated that the stabilization effect may depend on the number of 24-28 bp imperfect repeats which were found in several copies in the STB locus and adjoining region. © 1998 John Wiley & Sons, Ltd.
    Zusätzliches Material: 4 Ill.
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  • 82
    ISSN: 0749-503X
    Schlagwort(e): methylotrophic yeast ; Pichia methanolica ; DNA transformation ; alcohol oxidase ; vacuolar protease ; protein expression ; fermentation ; human GAD65 ; Life and Medical Sciences ; Genetics
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Biologie
    Notizen: We describe a protein expression system in the methylotrophic yeast, Pichia methanolica. Methods for transformation and genetic manipulation of the organism were developed using an ade2 strain and the wild-type ADE2 gene. A vacuolar protease-deficient strain was constructed. Two genes encoding alcohol oxidases were found, yet a single isoform of alcohol oxidase was produced during methanol-fed fermentations. The promoter from this gene was used to drive expression. An integrating plasmid for the cytoplasmic expression of the 65 kDa isoform of human glutamate decarboxylase (human GAD65) was assembled. A strain harboring eight copies of this plasmid expressed enzymatically active human GAD65 at levels approaching 0·5 g/l. Identical amounts were made in Pichia pastoris. The recombinant GAD65 was purified to greater than 90% purity. © 1998 John Wiley & Sons, Ltd.
    Zusätzliches Material: 8 Ill.
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  • 83
    Digitale Medien
    Digitale Medien
    New York, NY [u.a.] : Wiley-Blackwell
    Yeast 14 (1998), S. 67-76 
    ISSN: 0749-503X
    Schlagwort(e): cellulose degradation ; endo-β-1,4-glucanase ; cellobiohydrolase ; cellodextrinase ; cellobiase ; Saccharomyces cerevisiae ; Life and Medical Sciences ; Genetics
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Biologie
    Notizen: Saccharomyces cerevisiae produces several β-1,3-glucanases, but lacks the multicomponent cellulase complexes that hydrolyse the β-1,4-linked glucose polymers present in cellulose-rich biomass as well as in haze-forming glucans in certain wines and beers. We have introduced into S. cerevisiae a functional cellulase complex for efficient cellulose degradation by cloning the Endomyces fibuliger cellobiase (BGL1) gene and co-expressing it with the Butyrivibrio fibrisolvens endo-β-1,4-glucanase (END1), the Phanerochaete chrysosporium cellobiohydrolase (CBH1) and the Ruminococcus flavefaciens cellodextrinase (CEL1) gene constructs in this yeast. The END1, CBH1 and CEL1 genes were inserted into yeast expression/secretion cassettes. Expression of END1, CBH1 and CEL1 was directed by the promoter sequences derived from the alcohol dehydrogenase II (ADH2), the phosphoglycerate kinase I (PKG1) and the alcohol dehydrogenase I (ADH1) genes, respectively. In contrast, BGL1 was expressed under the control of its native promoter. Secretion of End1p and Cel1p was directed by the signal sequence of the yeast mating pheromone α-factor (MFα1), whereas Cbh1p and Bgl1p were secreted using their authentic leader peptides. The construction of a fur1 ura3 S. cerevisiae strain allowed for the autoselection of this multicopy URA3-based plasmid in rich medium. S. cerevisiae transformants secreting biologically active endo-β-1,4-glucanase, cellobiohydrolase, cellodextrinase and cellobiase were able to degrade various substrates including carboxymethylcellulose, hydroxyethylcellulose, laminarin, barley glucan, cellobiose, polypectate, birchwood xylan and methyl-β-d-glucopyranoside. This study could lead to the development of industrial strains of S. cerevisiae capable of converting cellulose in a one-step process into commercially important commodities. © 1998 John Wiley & Sons, Ltd.
    Zusätzliches Material: 3 Ill.
    Materialart: Digitale Medien
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  • 84
    ISSN: 0749-503X
    Schlagwort(e): Paracoccidioides brasiliensis ; chitin synthase ; dimorphic fungi ; Life and Medical Sciences ; Genetics
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Biologie
    Notizen: The nucleotide sequence of a chitin synthase gene (CHS2) of the dimorphic fungal human pathogen Paracoccidioides brasiliensis has been determined. The deduced amino acid sequence of Chs2p consists of 1043 residues and is highly homologous to other class II fungal chitin synthases. Computational structural analyses suggest very high similarity to other fungal chitin synthases with a highly variable region at the cytosolic amino-terminal region which may be related to its possible zymogenic nature, and the putative catalytic region close to seven membrane-spanning regions at the carboxyl terminus. The nucleotide sequence of CHS2 and its flanking regions has been submitted to GenBank under Accession Number Y09231. © 1998 John Wiley & Sons, Ltd.
    Zusätzliches Material: 2 Ill.
    Materialart: Digitale Medien
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  • 85
    Digitale Medien
    Digitale Medien
    New York, NY [u.a.] : Wiley-Blackwell
    Yeast 14 (1998), S. 171-180 
    ISSN: 0749-503X
    Schlagwort(e): gene duplication ; mammalian homologues ; transcript analysis ; mitochondria ; Saccharomyces cerevisiae ; Life and Medical Sciences ; Genetics
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Biologie
    Notizen: The yeast scERV1 gene is the first representative of a new emerging gene family. Its gene product is essential for the yeast cell and is involved in the biogenesis of mitochondria and the regulation of the cell cycle. Recently the general importance of the gene for the eukaryotic cell was shown by the identification of a structural and functional human homologue. The homologous mammalian ALR (augmenter of liver regeneration) genes from man, mouse and rat are important for different developmental stages of the organism as for example in spermatogenesis and the regeneration of damaged liver organs. Latest research identified an intron with an unusual 3′ branch site in the 5′ region of the yeast scERV1 gene. Analysis of the now available complete genome sequence from Saccharomyces cerevisiae identified a second yeast gene with homologies to scERV1 on chromosome 16. The corresponding gene product has a length of 196 amino acids similar to the 189 residues of the scERV1 protein and exhibits 30% identical amino acid residues in the highly conserved carboxy-terminal part of the polypeptides. Because of the structural similarities the new gene will be termed scERV2 from now on. For the scERV1 gene product it has just been shown that it is associated with yeast mitochondria. Analysis of the amino-terminal part of the putative scERV2 protein also identifies a typical leader sequence for import into mitochondria. The comparison of cDNA and genomic DNA from the scERV2 gene shows that no intron is present in this gene. To investigate the functional relation between the two yeast genes disruption experiments and complementation studies of mutants from scERV1 were performed. In addition the expression of messenger RNA under 15 different growth conditions was investigated by detailed Northern hybridization studies. Both genes show a complex and distinct expression pattern for their transcripts and are highly regulated under different physiological conditions. Moreover correct and efficient splicing of the transcript from the scERV1 gene was found to vary with the physiological state of the yeast cell, as further verified by reverse transcription-polymerase chain reaction analysis of transcripts from galactose-grown yeast cells. © 1998 John Wiley & Sons, Ltd.
    Zusätzliches Material: 5 Ill.
    Materialart: Digitale Medien
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  • 86
    Digitale Medien
    Digitale Medien
    New York, NY [u.a.] : Wiley-Blackwell
    Yeast 14 (1998), S. 215-224 
    ISSN: 0749-503X
    Schlagwort(e): Saccharomyces cerevisiae ; arginine permease ; turnover ; phosphorylation ; Life and Medical Sciences ; Genetics
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Biologie
    Notizen: To study the post-translational fate of arginine permease (Can1p), the gene coding for this transport protein was placed behind a constitutive promoter of plasma membrane ATPase (PMA1) and furnished with a Myc tag. In exponential-phase cells the amount of Can1p is constant, although turnover can be demonstrated. A rapid decrease in transport activity during the early stationary phase is paralleled by a corresponding net degradation of the protein. The amount of Can1p present in exponential cells grown on various nitrogen sources is the same, except in arginine-grown cells, in which the amount of the protein is markedly lower. This occurs solely when arginine serves as nitrogen source but not as an immediate consequence of, for example, arginine addition to cells growing on other nitrogen sources. It was demonstrated that Can1p is phosphorylated. Since Can1p expression under the PMA1 promoter is glucose-dependent, the amount of the permease expressed in high-glucose-grown cells is higher than in low-glucose-grown ones. Only a part of the Can1p overexpressed in high-glucose-grown cells is phosphorylated, while in low-glucose-grown cells the phosphorylated form probably represents the majority of Can1p. The permease phosphorylation or dephosphorylation is not related to transinhibition. © 1998 John Wiley & Sons, Ltd.
    Zusätzliches Material: 9 Ill.
    Materialart: Digitale Medien
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  • 87
    ISSN: 0749-503X
    Schlagwort(e): Candida albicans ; multidrug resistance ; Fluconazole ; antifungal drugs ; Life and Medical Sciences ; Genetics
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Biologie
    Notizen: The Candida albicans CDR1 gene encodes a member of the ABC-type family of multidrug transporters which has been shown to be involved in azole resistance. Using an in-frame gene fusion between the CDR1 open reading frame and the green fluorescent protein allele yEGFP3, an optimized derivative for its use in C. albicans, we show here how the CDR1-yEGFP3 gene expression is induced in response to azoles as well as to other structurally unrelated drugs like cycloheximide. Moderate increases were observed for calcofluor, canavanine, 5′-fluorcytosine, cilofungin and caffeine, while no induction was found for the antifungals benomyl and amphotericin B or hydrogen peroxide at subinhibitory concentrations. The use of confocal microscopy enabled us to localize the Cdr1p fusion protein at the cell periphery, thus suggesting a cytoplasmic membrane localization. These results suggest deregulation of CDR1 gene as a putative mechanism for the generation of azole resistance in this clinically important pathogenic fungus. © 1998 John Wiley & Sons, Ltd.
    Zusätzliches Material: 4 Ill.
    Materialart: Digitale Medien
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  • 88
    ISSN: 0749-503X
    Schlagwort(e): Saccharomyces cerevisiae ; YHR019 ; chromosome VIII ; asparaginyl-tRNA synthetase ; Life and Medical Sciences ; Genetics
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Biologie
    Notizen: Exploiting the asparagine auxotrophy of the Saccharomyces cerevisiae mutant strain 8556a, we have isolated the gene for the cytosolic asparaginyl-tRNA synthetase (AsnRS) of S. cerevisiae, by functional complementation of the mutation affecting this strain. The isolated gene could be identified to the open reading frame YHR019, called DED81, located on chromosome VIII. The mutant gene from the 8556a strain, asnrs--1, was amplified from genomic DNA by PCR. This gene contains a point mutation, leading to the replacement of a glycine residue by a serine in a region of the protein probably important for the asparaginyl-adenylate recognition. The protein encoded by YHR019 is very similar to cytosolic AsnRS from other eukaryotic sources. In a phylogenetic analysis based on AsnRS sequences from various organisms, the eukaryotic sequences were clustered. Expression of YHR019 in Escherichia coli demonstrated that a yeast AsnRS activity was produced. The recombinant enzyme was purified to homogeneity in three chromatography steps. We showed that the recombinant S. cerevisiae AsnRS was able to charge unfractionated yeast tRNA, but not E. coli tRNA, with asparagine. © 1998 John Wiley & Sons, Ltd.
    Zusätzliches Material: 3 Ill.
    Materialart: Digitale Medien
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  • 89
    ISSN: 0749-503X
    Schlagwort(e): Saccharomyces cerevisiae ; chromosome VII ; FOL2 ; YTA7 ; Life and Medical Sciences ; Genetics
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Biologie
    Notizen: In the framework of the EU programme for systematic sequencing of the Saccharomyces cerevisiae genome we determined the sequence of a 9359 bp fragment of the right arm of chromosome VII. Five open reading frames (ORFs) of at least 300 nucleotides were found in this region. YGR267c encodes a protein with significant similarity to the enzyme GTP-cyclohydrolase I, that controls the first step in the biosynthetic pathway leading to various pterins and shows a high degree of sequence conservation from bacteria to mammals. We have recently demonstrated (Nardese et al., 1996) that YGR267c corresponds to the FOL2 gene, previously localized in the same chromosomal region by genetic mapping. The protein deduced from YGR270w belongs to the superfamily of putative ATPases associated with diverse cellular activities. It corresponds to the YTA7 gene, a member of a set of yeast genes coding for putative ATPases with high similarity to constituents of the 26S protease. The three ORFs YGR266w, YGR268c and YGR269w encode putative products of unknown function, with neither significant similarity to proteins in databases nor recognizable domains. YGR268c and YGR269w are partially overlapping ORFs: YGR268c seems to correspond to a real gene, whereas YGR269w is probably a fortuitous ORF. The sequence has been entered in the EMBL data library under Accession Number Y07893. © 1998 John Wiley & Sons, Ltd.
    Zusätzliches Material: 1 Ill.
    Materialart: Digitale Medien
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  • 90
    Digitale Medien
    Digitale Medien
    New York, NY [u.a.] : Wiley-Blackwell
    Yeast 14 (1998), S. 675-680 
    ISSN: 0749-503X
    Schlagwort(e): Candida albicans ; protein secretion ; SEC4 ; Life and Medical Sciences ; Genetics
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Biologie
    Notizen: The SEC4 gene product is a major component of the protein secretion machinery. More specifically, it is believed to play a pivotal role in targeting and fusion of secretory vesicles to the plasma membrane. Its recently described implication with the Saccharomyces cerevisiae Rho3p, which is required for directing growing points during bud formation, has prompted us to investigate the role and function of Sec4p in the morphological changes of the yeast pathogen Candida albicans. We have therefore cloned the C. albicans SEC4 gene. It encodes a 210 amino acids long protein sharing up to 75% homology to the S. cerevisiae homolog, when conserved changes are allowed. Its RNA is constitutively expressed in C. albicans grown under various physiological conditions. We also show that it can functionally complement a S. cerevisiae sec4 thermosensitive mutant. The sequence of the C. albicans SEC4 gene has been deposited in GenBank under Accession Number AF017183. © 1998 John Wiley & Sons, Ltd.
    Zusätzliches Material: 4 Ill.
    Materialart: Digitale Medien
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  • 91
    Digitale Medien
    Digitale Medien
    New York, NY [u.a.] : Wiley-Blackwell
    Yeast 14 (1998), S. 687-691 
    ISSN: 0749-503X
    Schlagwort(e): Kluyveromyces lactis ; phosphoribosyl-AMP cyclohydrolase ; phosphoribosyl-ATP pyrophosphohydrolase ; histidinol dehydrogenase ; Life and Medical Sciences ; Genetics
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Biologie
    Notizen: The Kluyveromyces lactis HIS4 gene was cloned by complementation of a Saccharomyces cerevisiae his4 mutant. Sequence analysis revealed a 2388 bp open reading frame encoding a single polypeptide predicted to encompass three distinct enzymatic activities (phosphoribosyl-AMP cyclohydrolase, phosphoribosyl-ATP pyrophosphohydrolase and histidinol dehydrogenase). This structural organization is strikingly similar to that of the His4 proteins from S. cerevisiae and Pichia pastoris. Transcript analysis detected a single mRNA species of 2.5 kb. The EMBL accession number of this gene is Y09503. © 1998 John Wiley & Sons, Ltd.
    Zusätzliches Material: 2 Ill.
    Materialart: Digitale Medien
    Standort Signatur Erwartet Verfügbarkeit
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  • 92
    Digitale Medien
    Digitale Medien
    New York, NY [u.a.] : Wiley-Blackwell
    Yeast 14 (1998), S. 701-710 
    ISSN: 0749-503X
    Schlagwort(e): gene recognition ; linear discriminant analysis ; dynamic programming ; Life and Medical Sciences ; Genetics
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Biologie
    Notizen: A special program developed by the authors, called Pombe, identifies protein coding regions in the Schizosaccharomyces pombe genome. Linear discriminant analysis was applied to predict 5′-terminal, internal, 3′-terminal exons (coding-exon) and introns. The accuracy of the prediction was tested by cross verifications. The sensitivity, specificity and correlation coefficient for the internal exon prediction were 98·5%, 99·9% and 98·3% respectively at the nucleotide level. Open reading frames were studied and used to predict intron-less genes: 99·0% of such genes were identified with correct stopping sites. The gene structure was determined by dynamic programming and the prediction achieved 97·0% correlation coefficient at the nucleotide level. The program is available at http://clio.cshl.org/genefinder. © 1998 John Wiley & Sons, Ltd.
    Zusätzliches Material: 4 Ill.
    Materialart: Digitale Medien
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  • 93
    ISSN: 0749-503X
    Schlagwort(e): Pichia pastoris ; methylotrophic yeasts ; dihydroxyacetone kinase ; DNA sequencing ; Life and Medical Sciences ; Genetics
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Biologie
    Notizen: Dihydroxyacetone kinase (DAK) is essential for methanol assimilation in methylotrophic yeasts. We have cloned the DAK gene from Pichia pastoris by functional complementation of a mutant that was unable to grow on methanol. An open reading frame of 1824 bp was identified that encodes a 65·3 kDa protein with high homology to DAK from Saccharomyces cerevisiae. Although DAK from P. pastoris contained a C-terminal tripeptide, TKL, which we showed can act as a peroxisomal targeting signal when fused to the green fluorescent protein, the enzyme was primarily cytosolic. The TKL tripeptide was not required for the biochemical function of DAK because a deletion construct lacking the DNA encoding this tripeptide was able to complement the P. pastoris dakΔ mutant. Peroxisomes, which are essential for growth of P. pastoris on methanol, were present in the dakΔ mutant and the import of peroxisomal proteins was not disturbed. The dakΔ mutant grew at normal rates on glycerol and oleate media. However, unlike the wild-type cells, the dakΔ mutant was unable to grow on methanol as the sole carbon source but was able to grow on dihydroxyacetone at a much slower rate. The metabolic pathway explaining the reduced growth rate of the dakΔ mutant on dihydroxyacetone is discussed. The nucleotide sequence reported in this paper has been submitted to GenBank with Accession Number AF019198. © 1998 John Wiley & Sons, Ltd.
    Zusätzliches Material: 6 Ill.
    Materialart: Digitale Medien
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  • 94
    Digitale Medien
    Digitale Medien
    New York, NY [u.a.] : Wiley-Blackwell
    Yeast 14 (1998), S. 681-686 
    ISSN: 0749-503X
    Schlagwort(e): Candida albicans ; cell wall protein ; DNA sequence ; hypha-specific ; proline-rich ; glutamine-rich ; serine and threonine-rich ; HWP1 ; RACE ; Life and Medical Sciences ; Genetics
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Biologie
    Notizen: A previously isolated partial cDNA encoding a cell wall protein antigen found on hyphal surfaces of the opportunistic fungal pathogen, Candida albicans (Staab et al., 1996) was used to clone the complete hyphal wall protein 1 gene (HWP1). Hyphal forms of C. albicans invade mucosal surfaces of immunocompromised patients such as those with AIDS. HWP1 consisted of an open reading frame predicting an acidic protein (pI of 3·37) with a calculated molecular size of 61,122. The antigenic domain was located in the N-terminal third of the protein. The remainder of the protein contained abundant hydroxy amino acids, and terminated with a string of 15 amino acids typical of sequences specifying post-translational modification with glycosylphosphatidylinositol (6PI). The analyses suggested that Hwp1 is a glucan-linked protein with serine/threonine-rich regions that are predicted to function in extending a ligand-binding domain into the extracellular space. The nucleotide sequence reported in this paper has been submitted to GenBank/EMBL databank with Accession Number U64206. © 1998 John Wiley & Sons, Ltd.
    Zusätzliches Material: 2 Ill.
    Materialart: Digitale Medien
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  • 95
    ISSN: 0749-503X
    Schlagwort(e): I-Sce I fragmentation ; yeast ; genome ; cosmids ; colinearity ; Life and Medical Sciences ; Genetics
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Biologie
    Notizen: The high resolution complete physical maps of chromosomes VII and XV were constructed to form the basis for sequencing these chromosomes as part of the European systematic sequencing programme of the yeast genome, using a unique cosmid library from strain FY1679, and an original top-down mapping strategy involving I-Sce I chromosome fragmentation. A total of 138 and 196 cosmid clones were used to construct the maps for VII and XV, respectively, forming two unique contigs that cover the entirety of chromosomes (1091 kb each), except the telomeric repeats. Colinearity of the cosmid inserts with yeast DNA was verified, and the physical maps were eventually compared with the independently generated genetic maps. © 1998 John Wiley & Sons, Ltd.
    Zusätzliches Material: 4 Ill.
    Materialart: Digitale Medien
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  • 96
    Digitale Medien
    Digitale Medien
    New York, NY [u.a.] : Wiley-Blackwell
    Yeast 14 (1998), S. 885-893 
    ISSN: 0749-503X
    Schlagwort(e): pyrolysis mass spectrometry ; Fourier tranform infrared spectroscopy ; chemometrics ; quality assurance ; Life and Medical Sciences ; Genetics
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Biologie
    Notizen: Two rapid spectroscopic approaches for whole-organism fingerprinting - pyrolysis mass spectrometry (PyMS) and Fourier transform infrared spectroscopy (FT-IR) - were used to analyse 22 production brewery Saccharomyces cerevisiae strains. Multivariate discriminant analysis of the spectral data was then performed to observe relationships between the 22 isolates. Upon visual inspection of the cluster analyses, similar differentiation of the strains was observed for both approaches. Moreover, these phenetic classifications were found to be very similar to those previously obtained using genotypic studies of the same brewing yeasts. Both spectroscopic techniques are rapid (typically 2 min for PyMS and 10 s for FT-IR) and were shown to be capable of the successful discrimination of both ale and lager yeasts. We believe that these whole-organism fingerprinting methods could find application in brewery quality control laboratories. © 1998 John Wiley & Sons, Ltd.
    Zusätzliches Material: 5 Ill.
    Materialart: Digitale Medien
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  • 97
    Digitale Medien
    Digitale Medien
    New York, NY [u.a.] : Wiley-Blackwell
    Yeast 14 (1998), S. 915-922 
    ISSN: 0749-503X
    Schlagwort(e): small GTP-binding proteins ; YPT1 ; YPT6 ; SSD1 ; SLY1 ; Life and Medical Sciences ; Genetics
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Biologie
    Notizen: Ypt6p, the yeast homologue of human RAB6, is required for protein trafficking at elevated temperatures. Biochemical data provide evidence that Ypt6p plays a role in an early step(s) of the secretory pathway: from ER to Golgi, or from cis to medial Golgi, or both. Here we show that overexpression of YPT1 suppresses the growth and secretion defects of a ypt6 temperature-sensitive (ts) strain. SLY1-20, encoding a dominant mutant allele that suppresses the lethal effect of YPT1, also suppresses the growth defect of a ypt6 ts strain. Conversely, SSD1, isolated as a suppressor of ypt6 ts, can suppress the growth defect of a ypt1 ts allele. These data suggest that Ypt6p has some redundant function with Ypt1p. However, overexpression of Ypt6p is toxic to a ypt1 ts strain, although it does not affect the growth of wild-type cells, suggesting that Ypt6p may sequester proteins shared with Ypt1p. This genetic evidence confirms the conclusion that Ypt6p is involved in an early step of the secretory pathway. © 1998 John Wiley & Sons, Ltd.
    Zusätzliches Material: 4 Ill.
    Materialart: Digitale Medien
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  • 98
    Digitale Medien
    Digitale Medien
    New York, NY [u.a.] : Wiley-Blackwell
    Yeast 14 (1998), S. 977-984 
    ISSN: 0749-503X
    Schlagwort(e): aldose reductase ; catalytic mechanism ; coenzyme binding ; sequence comparison ; SDR enzymes ; structure-function relationship ; xylose reductase ; Life and Medical Sciences ; Genetics
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Biologie
    Notizen: Yeast xylose (aldose) reductases are members of the aldo-keto reductase family of enzymes which are widely distributed in a variety of other organisms. In yeasts, these enzymes catalyse the first step of xylose metabolism where xylose is converted to xylitol. In the past 16 years, xylose reductases from yeasts able to ferment or utilize xylose have been isolated and studied mainly because of their importance in xylose bioconversions. In recent years, genes encoding xylose reductases from several yeasts have been cloned and sequenced. A comparison of the primary sequences of yeast xylose reductases with the much better characterized human aldose reductase and human aldehyde reductase reveals that the yeast enzymes are hybrids between aldo-keto reductases and the short chain dehydrogenases/reductases families of enzymes. Why this is so and its evolutionary significance is presently not known. This short review will critically examine the structure and function information that can be gleaned from the sequence comparison. Several interesting questions arise from the sequence comparison and these can provide fruitful areas for further investigations. © 1998 John Wiley & Sons, Ltd.
    Zusätzliches Material: 2 Ill.
    Materialart: Digitale Medien
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  • 99
    Digitale Medien
    Digitale Medien
    New York, NY [u.a.] : Wiley-Blackwell
    Yeast 14 (1998), S. 773-781 
    ISSN: 0749-503X
    Schlagwort(e): dolichol-PP-GlcNAc2 ; translocation ; endoplasmic reticulum ; alg1 ; exoglucanase ; S. cerevisiae ; Life and Medical Sciences ; Genetics
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Biologie
    Notizen: Transfer of truncated oligosaccharides to yeast exoglucanase (Exg) in Saccharomyces cerevisiae alg1 has been investigated. When incubated at the non-permissive temperature, alg1 cells secreted into the culture medium, in addition to the exoglucanase glycoforms secreted by wild type, underglycosylated forms as well as material with ionic properties of the non-glycosylated enzyme. As expected, none of the latter had affinity towards concanavalin A, but part of it bound to wheat germ agglutinin (WGA), suggesting that it contained, in addition to non-glycosylated Exg, glycoforms carrying non-reducing terminal GlcNAc. Only the WGA-bound material could be labelled with galactosyltransferase; furthermore, the label could be released by treatment with peptide-N4-N-acetyl-β-glucosamine asparagine amidase. These results unambiguously demonstrate that GlcNAc2 can be transferred from dolichol-PP-GlcNAc2 to one or both sequons of yeast Exg. Accordingly, they support previous observations suggesting that this early intermediate is able to translocate in vivo in order to make its sugar portion accessible to the oligosaccharyltransferase in the lumen of the endoplasmic reticulum. © 1998 John Wiley & Sons, Ltd.
    Zusätzliches Material: 7 Ill.
    Materialart: Digitale Medien
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  • 100
    ISSN: 0749-503X
    Schlagwort(e): esterase ; beer ; brewer's yeast ; enzymatic hydrolysis ; specificity ; Saccharomyces cerevisiae TIP1 gene ; Life and Medical Sciences ; Genetics
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Biologie
    Notizen: An extracellular esterase was isolated from the brewer's yeast, Saccharomyces carlsbergensis. Inhibition by diisopropyl fluorophosphate shows that the enzyme has a serine active site. By mass spectrometry, the molecular weight of the enzyme was 16·9 kDa. The optimal pH for activity was in the range of four to five. Esterase activity was found in beer before pasteurization, and a low level of activity was still present after pasteurization. Caprylic acid, which is present in beer, competitively inhibited the esterase. The substrate preference towards esters of p-nitrophenol indicated that the enzyme prefers esters of fatty acids from four to 16 carbon atoms. The esterase has lipolytical activity; olive oil (C-18:1), which is a classical substrate for lipase, was hydrolysed. N-terminal sequence analysis of the esterase yielded a sequence which was identical to the deduced amino acid sequence of the S. cerevisiae TIP1 gene. The esterase preparation did not appear to contain significant amounts of other proteins than Tip1p, indicating that the TIP1 gene is the structural gene for the esterase. © 1998 John Wiley & Sons, Ltd.
    Zusätzliches Material: 4 Ill.
    Materialart: Digitale Medien
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