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  • 1
    Electronic Resource
    Electronic Resource
    New York, NY [u.a.] : Wiley-Blackwell
    Yeast 14 (1998), S. 287-295 
    ISSN: 0749-503X
    Keywords: Candida maltosa ; Candida albicans ; tRNA splicing gene ; silent genes ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: The tRNA splicing gene SPL1-1 has been cloned and sequenced in Saccharomyces cerevisiae (Kolman and Soll, 1993). Sequence adjacent to the LEU2 gene in Candida maltosa showed some homology to the SPL1-1 gene of S. cerevisiae. This work describes the sequencing of the SPL1 tRNA splicing genes from C. maltosa and C. albicans and the analysis of these genes. Comparison of these sequences and the relationship observed between the LEU2 and SPL1 genes in these yeasts suggests that there may be some synteny amongst various species of yeasts. The coding region of the C. maltosa SPL1 region described in this work differs from previously described partial sequences in that it is a complete uninterrupted open reading frame. Two strains of C. maltosa were each shown to contain different alleles, one uninterrupted open reading frame and one disrupted open reading frame. The sequences have been deposited in the GenBank/EMBL data libraries under Accession Numbers X72940, AF000115, AF000116, AF000117, AF000118, AF000119 and AF000120. © 1998 John Wiley & Sons, Ltd.
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  • 2
    ISSN: 0749-503X
    Keywords: Oligotrophic yeasts ; low-nutrient conditions ; starvation ; Cryptococcaceae ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: Seven oligotrophic yeasts, which can grow in a 104-fold dilution of malt-yeast-glucose-peptone medium (10-4 YM), were mainly isolated from soil. These yeasts belong to the Cryptococcaceae. When inoculated at about 102 cells/ml in 10-4 YM, the isolates grew to 1·4×103-2·4×105 cells/ml after 3 days. Some culture collection yeasts fell into three groups according to their growth characteristics in 10-4 YM, one group showing characteristics of the oligotrophic yeasts. The half-saturation values of uptake by the five isolated oligotrophic yeasts for D-glucose, L-leucine and L-amino acids were 6·0-25·0, 1·7-43·3 and 3·5-21·6 μM, respectively. The oligotrophic yeasts suspended in 10 mM-phosphate buffer (pH 6·0) had high tolerances for starvation, and remained more than 15% viable after 90 days of starvation. © 1998 John Wiley & Sons, Ltd.
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  • 3
    ISSN: 0749-503X
    Keywords: Saccharomyces cerevisiae ; mitochondria ; mitochondrial matrix ; homo-oligomeric protein ; Mam33p ; gene disruption ; gC1q-R ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: Mam33p (mitochondrial acidic matrix protein) is a soluble protein, located in mitochondria of Saccharomyces cerevisiae. It is synthesized as a precursor with an N-terminal mitochondrial targeting sequence that is processed on import. Mam33p assembles to a homo-oligomeric complex in the mitochondrial matrix. It can bind to the sorting signal of cytochrome b2 that directs this protein into the intermembrane space. Mam33p is encoded by an 801 bp open reading frame. Gene disruption did not result in a significant growth defect. Mam33p exhibits sequence similarity to gC1q-R, a human protein that has been implicated in the binding of complement factor C1q and kininogen. © 1998 John Wiley & Sons, Ltd.
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  • 4
    ISSN: 0749-503X
    Keywords: phosphofructokinase-1 ; Saccharomyces cerevisiae ; deletion mutants ; reactivation ; assembly ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: Phosphofructokinase-1 from Saccharomyces cerevisiae is an octameric enzyme comprising two non-identical subunits, α and β, which are encoded by the unlinked genes PFK1 and PFK2. In this paper, assembly and reactivation of the enzyme have been studied in cell-free extracts of single-deletion mutants. In contrast to the previously described lack of phosphofructokinase-1 activity in cell-free extracts of these mutants, we could measure a temporary enzyme activity immediately after lysis of protoplasts. This result supports the assumption that each of the subunits forms an enzyme structure which is active in vivo but not stable after cell disruption.Upon mixing of separately prepared cell-free extracts of both deletion mutants very low activity could be measured. About 40% of the wild-type activity was regained when both mutants were mixed prior to disruption. The reactivation rate could be slightly increased by addition of ATP and fructose 6-phosphate and was found to be a function of the growth state, particularly of the β-subunit-carrying cells. The individual subunits did not interact with Cibacron Blue F3G-A, a biomimetic ligand of phosphofructokinase-1. After reassembly of both subunits in vitro a strong affinity of the reconstituted phosphofructokinase-1 to the dye-ligand was observed.The inability of the subunits to reconstitute under certain conditions seems to result from alterations of the intracellular environment following disruption. These changes give rise to induce an unproductive side reaction like self-aggregation of the subunits.Because reconstitution of phosphofructokinase-1 from S. cerevisiae behaves in a similar way to that of hemoglobin and luciferase, we would speculate a general mechanism for assembly of oligomeric proteins in vivo. © 1998 John Wiley & Sons, Ltd.
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  • 5
    ISSN: 0749-503X
    Keywords: Saccharomyces ; redox ; glycerol ; NADH ; shuttle ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: Maintenance of a cytoplasmic redox balance is a necessity for sustained cellular metabolism. Glycerol formation is the only way by which Saccharomyces cerevisiae can maintain this balance under anaerobic conditions. Aerobically, on the other hand, several different redox adjustment mechanisms exist, one of these being the glycerol 3-phosphate (G3P) shuttle. We have studied the importance of this shuttle under aerobic conditions by comparing growth properties and glycerol formation of a wild-type strain with that of gut2Δ mutants, lacking the FAD-dependent glycerol 3-phosphate dehydrogenase, assuming that the consequent blocking of G3P oxidation is forcing the cells to produce glycerol from G3P. To impose different demands on the redox adjustment capability we used various carbon sources having different degrees of reduction.The results showed that the shuttle was used extensively with reduced substrate such as ethanol, whereas the more oxidized substrates lactate and pyruvate, did not provoke any activity of the shuttle. However, the absence of a functional G3P shuttle did not affect the growth rate or growth yield of the cells, not even during growth on ethanol. Presumably, there must be alternative systems for maintaining a cytoplasmic redox balance, e.g. the so-called external NADH dehydrogenase, located on the outer side of the inner mitochondrial membrane. By comparing the performance of the external NADH dehydrogenase and the G3P shuttle in isolated mitochondria, it was found that the former resulted in high respiratory rates but a comparably low P/O ratio of 1·2, whereas the shuttle gave low rates but a high P/O ratio of 1·7.Our results also demonstrated that of the two isoforms of NAD-dependent glycerol 3-phosphate dehydrogenase, only the enzyme encoded by GPD1 appeared important for the shuttle, since the enhanced glycerol production that occurs in a gut2Δ strain proved dependent on GPD1 but not on GPD2. © 1998 John Wiley & Sons, Ltd.
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  • 6
    ISSN: 0749-503X
    Keywords: modified LFH cassette ; EUROFAN 6-pack analysis ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: Six open reading frames (ORFs) of unknown function from Saccharomyces cerevisiae chromosome XV, three from the left and three from the right arm, were deleted in two diploid strains by the short flanking homology method (Wach et al., 1994). Transformants were selected as Geneticin (G418)-resistant colonies and correct integration of the kanMX4 cassette was checked by colony PCR. Following sporulation of the diploids, tetrads were dissected and scored for the segregation of the G418-resistant marker. We have developed a widely applicable method for the construction of gap repair plasmids to obtain the cognate clones for each of the disrupted ORFs. The 5′- and 3′-flanks of the ORF in question are linked by a unique restriction endonuclease. When the plasmid is cut at this site it can be used to obtain, by selection for the appropriate antibiotic resistance, long flanking homology (LFH) cassettes containing the cognate clone or the disrupted allele. The LFH cassette containing the cognate clone or the disrupted allele can be released from the gap-repaired plasmid by cutting at the inserted flanking restriction sites. One of the six ORFs (YOR319w) corresponds to an essential gene whose product is part of the spliceosome complex. Haploid as well as homozygous and heterozygous diploid disruptant strains for each of the five non-essential ORFs were subjected to growth test on different media at 15°C, 30°C and 37°C. Disruption of YOR322c causes osmotically sensitive growth on YEPD at 37°C and the product of YOL091w appears to play a role in sporulation since the homozygous diploid disruptant has lost the ability to sporulate. © 1998 John Wiley & Sons, Ltd.
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  • 7
    Electronic Resource
    Electronic Resource
    New York, NY [u.a.] : Wiley-Blackwell
    Yeast 14 (1998), S. 409-417 
    ISSN: 0749-503X
    Keywords: transcription factor ; SPT3 sequences ; Schizosaccharomyces pombe ; Kluyveromyces lactis ; Clavispora opuntiae ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: 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.
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  • 8
    Electronic Resource
    Electronic Resource
    New York, NY [u.a.] : Wiley-Blackwell
    Yeast 14 (1998), S. 471-477 
    ISSN: 0749-503X
    Keywords: ribosomal protein genes ; yeast genome ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: 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.
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  • 9
    Electronic Resource
    Electronic Resource
    New York, NY [u.a.] : Wiley-Blackwell
    Yeast 14 (1998), S. 335-345 
    ISSN: 0749-503X
    Keywords: Candida albicans ; arginine permease ; amino acid transport ; affinity chromatography ; functional reconstitution ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: 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.
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  • 10
    ISSN: 0749-503X
    Keywords: polyphosphatase ; cytosol ; yeast ; purification ; kinetic model ; Mg2+ ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: 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.
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