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  • 101
    ISSN: 0749-503X
    Keywords: Saccharomyces cerevisiae ; promoter-substitution cassette ; tetracycline-regulatable promoter ; essential genes ; conditional gene expression ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: 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.
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  • 102
    ISSN: 0749-503X
    Keywords: chemostat cultivation ; Saccharomyces cerevisiae ; carbon source ; transcriptional regulation ; UAS ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: 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.
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  • 103
    ISSN: 0749-503X
    Keywords: LFH deletion cassette ; functional analysis ; chromosome IV ; Saccharomyces cerevisiae ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: 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.
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  • 104
    ISSN: 0749-503X
    Keywords: Candida boidinii ; peroxisome ; peroxisomal proliferation ; peroxisomal membrane proteins ; d-amino acid oxidase ; green fluorescent protein ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: 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.
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  • 105
    ISSN: 0749-503X
    Keywords: anaplerotic reactions ; glyoxylate cycle ; catabolite repression ; Pichia ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: 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.
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  • 106
    ISSN: 0749-503X
    Keywords: Chromosome length ; chromosome VI ; electrophoretic karyotype ; RFLP ; Saccharomyces ; saké yeast ; taxonomy ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: 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.
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  • 107
    Electronic Resource
    Electronic Resource
    New York, NY [u.a.] : Wiley-Blackwell
    Yeast 14 (1998), S. 861-867 
    ISSN: 0749-503X
    Keywords: HIS3 ; TRP1 ; IPP1 ; Pichia pastoris ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: 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.
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  • 108
    ISSN: 0749-503X
    Keywords: Saccharomyces cerevisiae ; chromosome VII ; ribonuclease PH ; HGH1 ; YGR187c ; YGR189c ; YGR194c ; YGR195w ; YGR196c ; YGR198w ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: 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.
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  • 109
    Electronic Resource
    Electronic Resource
    New York, NY [u.a.] : Wiley-Blackwell
    Yeast 14 (1998), S. 813-825 
    ISSN: 0749-503X
    Keywords: Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: 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.
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  • 110
    Electronic Resource
    Electronic Resource
    New York, NY [u.a.] : Wiley-Blackwell
    Yeast 14 (1998), S. 963-967 
    ISSN: 0749-503X
    Keywords: gluconeogenesis ; PEPCK ; Kluyveromyces lactis ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: 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.
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  • 111
    Electronic Resource
    Electronic Resource
    New York, NY [u.a.] : Wiley-Blackwell
    Yeast 14 (1998), S. 905-913 
    ISSN: 0749-503X
    Keywords: carbon dioxide ; cytostasis ; G1 arrest ; meiosis ; sporulation ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: 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.
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  • 112
    ISSN: 0749-503X
    Keywords: S. pastorianus ; S. cerevisiae ; S. bayanus ; chromosome co-existence ; chromosomal rearrangement ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: 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.
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  • 113
    ISSN: 0749-503X
    Keywords: Schizosaccharomyces pombe ; Saccharomyces cerevisiae ; uracil permease ; transmembrane helices ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: 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.
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  • 114
    Electronic Resource
    Electronic Resource
    New York, NY [u.a.] : Wiley-Blackwell
    Yeast 14 (1998), S. 1041-1050 
    ISSN: 0749-503X
    Keywords: Saccharomyces cerevisiae ; STRE ; stress response ; genomics ; bioinformatics ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: 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.
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  • 115
    Electronic Resource
    Electronic Resource
    New York, NY [u.a.] : Wiley-Blackwell
    Yeast 14 (1998), S. 1017-1025 
    ISSN: 0749-503X
    Keywords: Arxula adeninivorans ; AILV1 ; threonine deaminase ; transformation ; homologous integration ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: 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.
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  • 116
    ISSN: 0749-503X
    Keywords: gene disruption ; homologous recombination ; protein A-tagging ; Saccharomyces cerevisiae ; tags ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: 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.
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  • 117
    Electronic Resource
    Electronic Resource
    New York, NY [u.a.] : Wiley-Blackwell
    Yeast 14 (1998), S. 1223-1232 
    ISSN: 0749-503X
    Keywords: Candida utilis ; β-fructofuranosidase ; glycosyl hydrolase ; signal peptide ; sucrose ; polymerase chain reaction ; invertase ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: 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.
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  • 118
    ISSN: 0749-503X
    Keywords: salt-tolerant yeast ; Zygosaccharomyces rouxii ; Na+/H+ antiporter ; functional expression ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: 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.
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  • 119
    Electronic Resource
    Electronic Resource
    New York, NY [u.a.] : Wiley-Blackwell
    Yeast 14 (1998), S. 1307-1310 
    ISSN: 0749-503X
    Keywords: Schizosaccharomyces pombe ; 5-phosphoribosyl-4-carboxamide 5-aminoimidazole (AICAR) transformylase ; inosine monophosphate (IMP) cyclohydrolase ; intrachromosomal recombination ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: 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.
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  • 120
    ISSN: 0749-503X
    Keywords: Candida albicans ; SRST ; systeny ; RAD16 ; LYS2 ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: 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.
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  • 121
    ISSN: 0749-503X
    Keywords: yeast ; Candida utilis ; URA3 ; orotidine 5′-monophosphate decarboxylase ; transformation system ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: 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.
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  • 122
    ISSN: 0749-503X
    Keywords: yeast ; Debaryomyces ; transport ; physiology ; salt tolerance ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: 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.
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  • 123
    ISSN: 0749-503X
    Keywords: World Wide Web ; Saccharomyces Genome Database ; Munich Information Center for Protein Sequences ; Yeast Protein Database ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: 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.
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  • 124
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    Yeast 14 (1998), S. 1529-1566 
    ISSN: 0749-503X
    Keywords: G protein ; mating ; pheromone ; Schizosaccharomyces pombe ; receptor ; yeast ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Additional Material: 7 Ill.
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  • 125
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    Yeast 14 (1998), S. 89-91 
    ISSN: 0749-503X
    Keywords: PCR mutagenesis ; functional domains ; subgenic DNA fragments ; mutagenesis protocol ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: 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.
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  • 126
    ISSN: 0749-503X
    Keywords: Kluyveromyces lactis ; CBF5 ; centromere ; nucleolus ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: 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.
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  • 127
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    Yeast 14 (1998), S. 49-65 
    ISSN: 0749-503X
    Keywords: multidrug resistance ; drug efflux ; MPP+ ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: 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.
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  • 128
    ISSN: 0749-503X
    Keywords: transcription factors ; tRNA biosynthesis ; mitochondrial RNA processing ; ribonuclease P ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: 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.
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  • 129
    ISSN: 0749-503X
    Keywords: Saccharomyces cerevisiae ; yeast ; gene disruption ; S288C ; bacteria-yeast shuttle vectors ; auxotrophic markers ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: 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.
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  • 130
    ISSN: 0749-503X
    Keywords: Saccharomyces cerevisiae ; YAP3 ; KEX2 ; recombinant human albumin ; protease degradation ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: 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.
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  • 131
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    Yeast 14 (1998), S. 443-457 
    ISSN: 0749-503X
    Keywords: evolution ; polyploidy ; gene duplication ; gene order ; LEU2 ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: The extent to which the order of genes along chromosomes is conserved between Saccharomyces cerevisiae and related species was studied by analysing data from DNA sequence databases. As expected, the extent of gene order conservation decreases with increasing evolutionary distance. About 59% of adjacent gene pairs in Kluyveromyces lactis or K. marxianus are also adjacent in S. cerevisiae, and a further 16% of Kluyveromyces neighbours can be explained in terms of the inferred ancestral gene order in Saccharomyces prior to the occurrence of an ancient whole-genome duplication. Only 13% of Candida albicans linkages, and no Schizosaccharomyces pombe linkages, are conserved. Analysis of gene order arrangements, chromosome numbers, and ribosomal RNA sequences suggests that genome duplication occurred before the divergence of the four species in Saccharomyces sensu stricto (all of which have 16 chromosomes), but after this lineage had diverged from Saccharomyces kluyveri and the Kluyveromyces lactis/marxianus species assemblage. © 1998 John Wiley & Sons, Ltd.
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  • 132
    ISSN: 0749-503X
    Keywords: Crabtree effect ; yeast ; biomass ; Kluyveromyces lactis ; oxygen ; pyruvate decarboxylase ; regulation ; fermentation ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: Kluyveromyces lactis is an important industrial yeast, as well as a popular laboratory model. There is currently no consensus in the literature on the physiology of this yeast, in particular with respect to aerobic alcoholic fermentation (‘Crabtree effect’). This study deals with regulation of alcoholic fermentation in K. lactis CBS 2359, a proposed reference strain for molecular studies. In aerobic, glucose-limited chemostat cultures (D=0·05-0·40 h-1) growth was entirely respiratory, without significant accumulation of ethanol or other metabolites. Alcoholic fermentation occurred in glucose-grown shake-flask cultures, but was absent during batch cultivation on glucose in fermenters under strictly aerobic conditions. This indicated that ethanol formation in the shake-flask cultures resulted from oxygen limitation. Indeed, when the oxygen feed to steady-state chemostat cultures (D=0·10 h-1) was lowered, a mixed respirofermentative metabolism only occurred at very low dissolved oxygen concentrations (less than 1% of air saturation). The onset of respirofermentative metabolism as a result of oxygen limitation was accompanied by an increase of the levels of pyruvate decarboxylase and alcohol dehydrogenase. When aerobic, glucose-limited chemostat cultures (D=0·10 h-1) were pulsed with excess glucose, ethanol production did not occur during the first 40 min after the pulse. However, a slow aerobic ethanol formation was invariably observed after this period. Since alcoholic fermentation did not occur in aerobic batch cultures this is probably a transient response, caused by an imbalanced adjustment of enzyme levels during the transition from steady-state growth at μ=0·10 h-1 to growth at μmax. It is concluded that in K. lactis, as in other Crabtree-negative yeasts, the primary environmental trigger for occurrence of alcoholic fermentation is oxygen limitation. © 1998 John Wiley & Sons, Ltd.
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  • 133
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    Yeast 14 (1998), S. 501-515 
    ISSN: 0749-503X
    Keywords: Saccharomyces cerevisiae ; bakers' yeast ; pH homeostasis ; cytoplasmic pH ; vacuolar pH ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: The effects of monovalent cations on the internal pH of yeast were studied. Our former procedure was modified, inducing maximal alkalinization of the cells with 100 mM-NH4OH instead of Tris base. The pH values were lower than reported before (Peña et al., J. Bacteriol. 1995 177, 1017-1022). With glucose as substrate, the internal cytoplasmic pH reached higher values when incubating at an external pH of 6·0, as compared to pH 4·0. Monovalent cations added approximately 5 min after glucose produced a further increase in the internal pH, which was higher at a previous incubation pH of 4·0 than that observed at pH 6·0. The selectivity of the changes followed a similar order to that of the transport system for monovalent cations.When incubating cells with glucose for more than 30 min, the initial changes of the internal pH appeared to be regulated by the cell. However, under the fluorescence microscope, it was observed that pyranine, which was confined to the cytoplasm during the first 15 min, was progressively concentrated in the vacuole. By studying the fluorescence changes of cells electroporated and then incubated with glucose or glucose plus potassium, we could follow the internal pH of this organelle, obtaining values within the range reported by other authors. Also, in cells preincubated with glucose for 60 min, and electroporated afterwards, the fluorescence of pyranine, which only entered the cytoplasm, allowed us to measure the pH of this compartment, showing that it was more alkaline than the vacuole. Moreover, the cytoplasmic pH increased upon addition of glucose or potassium. The vacuolar pH, on the other hand, increased upon addition of potassium after glucose, but decreased upon addition of glucose. In addition, incubation of the cells with glucose with or without pyranine produced vesiculation of the vacuole. © 1998 John Wiley & Sons, Ltd.
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  • 134
    ISSN: 0749-503X
    Keywords: alcohol acetyltransferase ; ATF1 gene ; OLE1 gene ; unsaturated fatty acid ; oxygen ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: 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.
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  • 135
    ISSN: 0749-503X
    Keywords: Pichia pastoris ; expression vectors ; gene regulation ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: 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.
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  • 136
    ISSN: 0749-503X
    Keywords: Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
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  • 137
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    Yeast 14 (1998), S. 1439-1451 
    ISSN: 0749-503X
    Keywords: beginnings of yeast research ; yeasts ; history ; Lavoisier ; Cagniard-Latour ; Kutzing ; Schwann ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Additional Material: 6 Ill.
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  • 138
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    Yeast 14 (1998), S. 1471-1510 
    ISSN: 0749-503X
    Keywords: yeast ; S. cerevisiae ; lipids ; phospholipids ; sterols ; sphingolipids ; fatty acids ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: 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.
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  • 139
    ISSN: 0749-503X
    Keywords: transcription ; microarrays ; expression profiling ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: Open reading frames (6116) of the budding yeast Saccharomyces cerevisiae were PCR-amplified from genomic DNA using 12,232 primers specific to the ends of the coding sequences; the success rate of amplification was 97%. PCR-products were made accessible to hybridization by being arrayed at very high density on solid support media using various robotic devices. Probes made from total RNA preparations were hybridized for the analysis of the transcriptional activity of yeast under various growth conditions and of different strains. Experimental factors that proved critical to the performance, such as different RNA isolation procedures and the assessment of hybridization results, for example, were investigated in detail. Various software tools were developed that permit convenient handling and sound analysis of the large data quantities obtained from transcriptional profiling studies. Comprehensive arrays are being distributed within the European Yeast Functional Analysis Network (EUROFAN) and beyond. © 1998 John Wiley & Sons, Ltd.
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  • 140
    ISSN: 0749-503X
    Keywords: yeast ; PMR1 ; Hansenula polymorpha ; Ca2+-ATPase ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: A gene homologous to Saccharomyces cerevisiae PMR1 has been cloned in the methylotrophic yeast Hansenula polymorpha. The partial DNA fragment of the H. polymorpha homologue was initially obtained by a polymerase chain reaction and used to isolate the entire gene which encodes a protein of 918 amino acids. The putative gene product contains all ten of the conserved regions observed in P-type ATPases. The cloned gene product exhibits 60·3% amino acid identity to the S. cerevisiae PMR1 gene product and complemented the growth defect of a S. cerevisiae pmr1 null mutant in the EGTA-containing medium. The results demonstrate that the H. polymorpha gene encodes the functional homologue of the S. cerevisiae PMR1 gene product, a P-type Ca2+-ATPase. The DNA sequence of the H. polymorpha homologue has been submitted to GenBank with the Accession Number U92083. © 1998 John Wiley & Sons, Ltd.
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  • 141
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    Yeast 14 (1998), S. 1257-1265 
    ISSN: 0749-503X
    Keywords: Candida albicans ; nucleoside transport ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: The nucleoside permease gene (i.e. NUP) from Candida albicans was cloned by complementation of Saccharomyces cerevisiae deficient in nucleoside transport capability. The permease transported adenosine and guanosine and was sensitive to the mammalian nucleoside transport inhibitors: dipyridamole and NBMPR. It did not transport uridine, cytidine, adenine, guanine or uracil. The inability to transport uridine indicated that the NUP gene product was different from the Candida uridine permease, which also transported cytosine and adenosine. The NUP gene coded for a protein of 407 amino acids in size which was approximately the size of the human, Giardia and E. coli nucleoside permeases. It did not, however, exhibit any significant degree of homology with these transporters. The GenBank accession number for the Candida NUP gene is AF016246. © 1998 John Wiley & Sons, Ltd.
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  • 142
    ISSN: 0749-503X
    Keywords: transcription factor ; yeast genetic selection ; bacteriophage λ vector ; Cre-loxP automatic plasmid subcloning ; integration vector ; one hybrid ; target genes ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: We describe the construction of a number of vectors that can be used in yeast genetic selection systems for cloning of transcription factors or other DNA-binding proteins and for identification of the target sites recognized by transcription factors. For transcription factor cloning we have designed an integration vector with two HIS3 reporter gene cassettes to stably integrate reporter gene constructs at the non-essential yeast PDC6 locus. This set of plasmids was tested in a one-hybrid assay with the rice transcription factor Oshox1, a member of the class of homeodomain leucine zipper proteins. A hybrid protein of Oshox1 and the Gal4 transcriptional activation domain was shown to specifically activate a reporter gene construct with upstream Oshox1 binding sites, which had been integrated at the PDC6 locus using the described vector system. Target site identification by genetic selection in yeast employs a transcriptional activator construct and a library of genomic or random DNA fragments upstream of a reporter gene. We have constructed two variants of a bacteriophage λ vector which facilitates the construction of the required reporter gene library because of high cloning efficiency and easy conversion into a yeast/Escherichia coli shuttle vector library by Cre-loxP-mediated automatic subcloning. Tests with Oxhox1 as transcriptional activator demonstrated the usefulness of the deprived reporter gene vector. © 1998 John Wiley & Sons, Ltd.
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  • 143
    ISSN: 0749-503X
    Keywords: Yarrowia lipolytica ; cytochrome P450 ; n-alkane metabolism ; n-alkane-inducible gene ; RT-PCR ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: A gene encoding cytochrome P450 involved in n-alkane utilization was cloned from an n-alkane assimilating yeast, Yarrowia lipolytica CX161-1B. The RT-PCR was performed on the mRNA prepared from the cells grown on n-alkane as a template using degenerated PCR primers designed for the conserved amino acid sequences of the CYP52 family. The RT-PCR amplified fragment was then used as a probe to isolate genes coding for P450 of the CYP52 family from the genomic DNA library of the strain CX161-1B. The nucleotide sequence of one of the positive clones was determined. An open reading frame which had the same nucleotide sequence as the RT-PCR-amplified fragment was identified. It was of 523 amino acid residues, 60·2 kDa in molecular mass, and had 30-45% sequence identity with the other members of the CYP52 family of Candida species so far analysed. The expression of the P450 gene that was named as YlALK1 was induced by n-tetradecane and repressed by glycerol. A YlALK1 gene disruptant did not grow well on n-decane, but grew on longer-chain n-alkanes such as hexadecane as a sole carbon source. Introduction of YlALK1 on a plasmid to the disruptant restored the decane assimilation. These results suggest that the YlALK1 gene product is the major P450Alk to metabolize short-chain n-alkanes such as decane and dodecane in Y. lipolytica. © 1998 John Wiley & Sons, Ltd.
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  • 144
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    Yeast 14 (1998), S. 1511-1527 
    ISSN: 0749-503X
    Keywords: Saccharomyces cerevisiae ; oxidative stress ; stress response ; signal transduction ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: All aerobically growing organisms suffer exposure to oxidative stress, caused by partially reduced forms of molecular oxygen, known as reactive oxygen species (ROS). These are highly reactive and capable of damaging cellular constituents such as DNA, lipids and proteins. Consequently, cells from many different organisms have evolved mechanisms to protect their components against ROS. This review concentrates on the oxidant defence systems of the budding yeast Saccharomyces cerevisiae , which appears to have a number of inducible adaptive stress responses to oxidants, such as H2 O2 , superoxide anion and lipid peroxidation products. The oxidative stress responses appear to be regulated, at least in part, at the level of transcription and there is considerable overlap between them and many diverse stress responses, allowing the yeast cell to integrate its response towards environmental stress. © 1998 John Wiley & Sons, Ltd.
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  • 145
    ISSN: 0749-503X
    Keywords: flocculation ; immunolocalization ; mannoprotein ; cell wall ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: 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.
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  • 146
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    Yeast 14 (1998), S. 101-101 
    ISSN: 0749-503X
    Keywords: Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
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  • 147
    ISSN: 0749-503X
    Keywords: Saccharomyces cerevisiae ; amino acid uptake ; ssy mutants ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: 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.
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  • 148
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    Yeast 14 (1998), S. 147-160 
    ISSN: 0749-503X
    Keywords: yeast physiology ; yeast viability ; flow cytometry ; bakers yeast ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: Yeast suspensions were analysed by flow cytometry after dye staining for determination of total and viable cell densities. Results were comparable to traditional colony counting and, in addition, provided further information on the percentage of total cells that were viable. The flow cytometric methods provided results within 20 min whereas colony counts were not available until 36 h. We evaluated a number of fluorescent dyes: ChemChrome Y (CY), oxonol (Ox), propidium iodide (PI), Fungolight and rhodamine 123, for accurate determination of viability of industrial yeast cultures and freshly re-hydrated high activity dried yeast (HADY). PI, Ox and CY gave the most conclusive live/dead discrimination and were the simplest to use. Culturing after dye staining and cell sorting demonstrated that the yeast remained viable after cell sorting and incubation with PI, CY or Ox. The methods, therefore, permit physical selection of individual yeast cells from populations of mixed viability. Sorting demonstrated that PI stained non-culturable cells whilst CY stained culturable cells. Analysis of yeast stained simultaneously with CY and PI or with Ox and PI demonstrated that PI and CY assays were in mutual agreement with respect to viability assessments. The Ox assay was in agreement with CY and PI for live/heat-killed mixtures. However, for re-hydrated HADY, Ox stained a significantly (P≤0·05) higher proportion of cells than did PI. © 1998 John Wiley & Sons, Ltd.
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  • 149
    ISSN: 0749-503X
    Keywords: GPM2 ; GPM3 ; phosphoglycerate mutase ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: Our previous data indicated that GPM1 encodes the only functional phosphoglycerate mutase in yeast. However, in the course of the yeast genome sequencing project, two homologous sequences, designated GPM2 and GPM3, were detected. They have been further investigated in this work. Key residues in the deduced amino acid sequence, shown to be involved in catalysis for Gpm1 (i.e. His8, Arg59, His181) are conserved in both enzymes. Overexpression of the genes under control of their own promoters in a gpm1 deletion mutant did not complement for any of the phenotypes. This could in part be attributed to a lack of expression due to their weak promoters. Higher level expression under the control of the yeast PFK2 promoter partially complemented the gpm1 defects, without restoring detectable enzymatic activity. Nevertheless, deletion of either GPM2 or GPM3, or the two deletions in concert, did not produce any obvious lesions for growth on a variety of different carbon sources, nor did they change the levels of key intermediary metabolites. We conclude that both genes evolved from duplication events and that they probably constitute non-functional homologues in yeast.
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  • 150
    ISSN: 0749-503X
    Keywords: Hansenula polymorpha ; 2 μm DNA ; plasmid partitioning ; nuclear segregation ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: 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.
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  • 151
    ISSN: 0749-503X
    Keywords: methylotrophic yeast ; Pichia methanolica ; DNA transformation ; alcohol oxidase ; vacuolar protease ; protein expression ; fermentation ; human GAD65 ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: 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.
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  • 152
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    Yeast 14 (1998), S. 67-76 
    ISSN: 0749-503X
    Keywords: cellulose degradation ; endo-β-1,4-glucanase ; cellobiohydrolase ; cellodextrinase ; cellobiase ; Saccharomyces cerevisiae ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: 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.
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  • 153
    ISSN: 0749-503X
    Keywords: Paracoccidioides brasiliensis ; chitin synthase ; dimorphic fungi ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: 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.
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  • 154
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    Yeast 14 (1998), S. 171-180 
    ISSN: 0749-503X
    Keywords: gene duplication ; mammalian homologues ; transcript analysis ; mitochondria ; Saccharomyces cerevisiae ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: 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.
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  • 155
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    Yeast 14 (1998), S. 215-224 
    ISSN: 0749-503X
    Keywords: Saccharomyces cerevisiae ; arginine permease ; turnover ; phosphorylation ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: 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.
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  • 156
    ISSN: 0749-503X
    Keywords: Candida albicans ; multidrug resistance ; Fluconazole ; antifungal drugs ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: 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.
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  • 157
    ISSN: 0749-503X
    Keywords: Saccharomyces cerevisiae ; YHR019 ; chromosome VIII ; asparaginyl-tRNA synthetase ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: 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.
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  • 158
    ISSN: 0749-503X
    Keywords: Saccharomyces cerevisiae ; chromosome VII ; FOL2 ; YTA7 ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: 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.
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  • 159
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    Yeast 14 (1998), S. 675-680 
    ISSN: 0749-503X
    Keywords: Candida albicans ; protein secretion ; SEC4 ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: 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.
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  • 160
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    Yeast 14 (1998), S. 687-691 
    ISSN: 0749-503X
    Keywords: Kluyveromyces lactis ; phosphoribosyl-AMP cyclohydrolase ; phosphoribosyl-ATP pyrophosphohydrolase ; histidinol dehydrogenase ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: 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.
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  • 161
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    Yeast 14 (1998), S. 701-710 
    ISSN: 0749-503X
    Keywords: gene recognition ; linear discriminant analysis ; dynamic programming ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: 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.
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  • 162
    ISSN: 0749-503X
    Keywords: Pichia pastoris ; methylotrophic yeasts ; dihydroxyacetone kinase ; DNA sequencing ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: 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.
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  • 163
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    Yeast 14 (1998), S. 681-686 
    ISSN: 0749-503X
    Keywords: Candida albicans ; cell wall protein ; DNA sequence ; hypha-specific ; proline-rich ; glutamine-rich ; serine and threonine-rich ; HWP1 ; RACE ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: 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.
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  • 164
    ISSN: 0749-503X
    Keywords: I-Sce I fragmentation ; yeast ; genome ; cosmids ; colinearity ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: 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.
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  • 165
    ISSN: 0749-503X
    Keywords: pyrolysis mass spectrometry ; Fourier tranform infrared spectroscopy ; chemometrics ; quality assurance ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: 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.
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  • 166
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    Yeast 14 (1998), S. 915-922 
    ISSN: 0749-503X
    Keywords: small GTP-binding proteins ; YPT1 ; YPT6 ; SSD1 ; SLY1 ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: 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.
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  • 167
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    Yeast 14 (1998), S. 977-984 
    ISSN: 0749-503X
    Keywords: aldose reductase ; catalytic mechanism ; coenzyme binding ; sequence comparison ; SDR enzymes ; structure-function relationship ; xylose reductase ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: 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.
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  • 168
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    Yeast 14 (1998), S. 773-781 
    ISSN: 0749-503X
    Keywords: dolichol-PP-GlcNAc2 ; translocation ; endoplasmic reticulum ; alg1 ; exoglucanase ; S. cerevisiae ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: 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.
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  • 169
    ISSN: 0749-503X
    Keywords: esterase ; beer ; brewer's yeast ; enzymatic hydrolysis ; specificity ; Saccharomyces cerevisiae TIP1 gene ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: 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.
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  • 170
    ISSN: 0749-503X
    Keywords: fermentation ; β-galactosidase ; heterologous gene expression ; Kluyveromyces lactis ; lactose-permease ; ribosomal DNA ; whey ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: A diploid strain of Saccharomyces cerevisiae able to metabolize lactose with high efficiency has been obtained. Haploid strains of Saccharomyces able to grow on lactose were constructed by cotransformation with two genes of Kluyveromyces lactis required for the utilization of the sugar, LAC4 and LAC12, encoding β-galactosidase and lactose permease respectively. Both genes were placed under the control of a galactose-inducible promoter and targeted to the rDNA encoding region (RDN1 locus) of the Saccharomyces genome. Lac+ transformants were selected on medium with lactose as the only carbon source. These transformants were mitotically stable, they maintained the Lac+ phenotype after growing in non-selective medium for more than 60 generations, but their growth was slow. We found that this lack of vigour was caused by their genetic background and not by a deficient expression of the heterologous genes. Therefore, their performance could be improved by crossing with a wild-type strain. Among the offspring of the crosses, two strains of opposite mating type were selected and mated to obtain a fast-growing Lac+ diploid. This diploid strain showed the typical fermentative behaviour of S. cerevisiae when it was grown in aerated liquid medium with glucose. In lactose medium, it exhibited a respiro-fermentative metabolism similar to that of K. lactis, with low ethanol production and high biomass yield. © 1998 John Wiley & Sons, Ltd.
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  • 171
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    Yeast 14 (1998), S. 895-903 
    ISSN: 0749-503X
    Keywords: Pichia pastoris ; pulsed-field gel electrophoresis ; chromosome-length polymorphisms ; gene stability ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: We have clearly resolved four chromosomal bands from four Pichia pastoris (Komagataella pastoris) strains by using contour-clamped homogeneous electric field gel electrophoresis. The size of the P. pastoris chromosomal bands ranged from 1·7 Mb to 3·5 Mb and total genome size was estimated to be 9·5 Mb to 9·8 Mb; however, chromosome-length polymorphisms existed among four strains. Thirteen cloned genes isolated from strain GTS115 were assigned to the separated chromosomes, revealing that different hybridization patterns were observed in the AOX2 and URA3 genes among strains. P. pastoris is frequently used as an efficient host for heterologous gene expressions. We analysed chromosomal stability of strain GTS115-derived recombinant cell expressing human serum albumin during serial cultivation under the condition of vegetative and non-selective growth. No chromosomal rearrangements were observed and the expression constructs integrated into the his4 locus on chromosome I were very stable even at 83 generations, suggesting that stable expression would be carried out even in large-scale fermentation. © 1998 John Wiley & Sons, Ltd.
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  • 172
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    Yeast 14 (1998), S. 935-942 
    ISSN: 0749-503X
    Keywords: antifungal drugs ; cytochrome-c oxidase ; gene dosage screening ; lanosterol C-14 demethylase ; overexpression assay ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: The post-genome sequencing era of Saccharomyces cerevisiae is defined by the analysis of newly discovered open reading frames of unknown function. In this report, we describe a genetic method for the rapid identification and characterisation of genes involved in a given phenotype. This approach is based on the ability of overexpressed genomic DNA fragments to cure an induced phenotype in yeast. To validate this concept, yeast cells carrying a yeast DNA library present on multicopy plasmid vectors were screened for resistance to the antifungal drug ketoconazole. Among 1·2 million colonies 13 clones tested positive, including those expressing the lanosterol C-14 demethylase, known to be a cellular target for azole drugs, and the cytochrome-c oxidase of mitochondria, regulating the respiratory chain electron transport. Several other resistant clones were identified, which code for yeast proteins of so far unknown function. These genes may represent potential candidates for antifungal drug effects. Together with the availability of the entire yeast genome sequence, the described genetic screening method is a powerful tool for the effective functional analysis of yeast genes. © 1998 John Wiley & Sons, Ltd.
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  • 173
    ISSN: 0749-503X
    Keywords: ADH ; hypoxic activation ; xylose fermenting yeasts ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: Two Pichia stipitis ADH genes (PsADH1 and PsADH2) were isolated by complementation of a Saccharomyces cerevisiae Adh--mutant. The genes enabled the transformants to grow in the presence of antimycin A on glucose, to use ethanol as sole carbon source and made them sensitive to allylalcohol.The sequences of the genes showed similarities of 70-77% to sequences of ADH genes of Candida albicans, Kluyveromyces lactis, K. marxianus, and S. cerevisiae and about 60% homology to those of Schizosaccharomyces pombe and Aspergillus flavus.Southern hybridization experiments suggested that P. stipitis has only these two ADH genes. Both genes are located on the largest chromosome of P. stipitis.PsADH2 encodes for the ADH activity that is responsible for ethanol formation at oxygen limitation. The gene is regulated at the transcriptional level. Moreover, also in cells grown on ethanol, only PsADH2 transcript was found. PsADH1 transcript was detected under aerobic conditions on fermentable carbon sources.The sequences have been deposited in the EMBL database under the accession numbers Y13238 (PsADH1) and Y13397 (PsADH2). © 1998 John Wiley & Sons, Ltd.
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  • 174
    ISSN: 0749-503X
    Keywords: cell wall ; chitin ; β-glucan ; Saccharomyces cerevisiae ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: A reliable acid hydrolysis method for quantitative determination of the proportion of β-glucan, mannan and chitin in Saccharomyces cerevisiae cell wall is reported together with a simple extraction procedure to quantify within a standard error of less than 2% the proportion of the wall per gram of cell dry mass. This method is an optimized version of Saeman's procedure based on sulfuric acid hydrolysis of complex polysaccharides. It resulted in an almost complete release of glucose, mannose and glucosamine residues from cell wall polysaccharides. After complete removal of sulfate ions by precipitation with barium hydroxide, the liberated monosaccharides were separated and quantified by high performance anion-exchange chromatography with pulsed amperometric detection. The superiority of this method over the hydrolysis in either trifluoroacetic or hydrochloric acid resides in its higher efficiency regarding the release of glucose from β1,6-glucan and of glucosamine from chitin. The sulfuric acid method was successfully applied to determine the β-glucan, mannan and chitin contents in cell walls of genetically well-characterized yeast mutants defective in cell wall biosynthesis, and in Schizosaccharomyces pombe cell walls. The simplicity and reliability of this procedure make it the method of choice for the characterization of cell walls from S. cerevisiae mutants generated in the EUROFAN programme, as well as for other pharmacological and biotechnological applications. © 1998 John Wiley & Sons, Ltd.
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  • 175
    ISSN: 0749-503X
    Keywords: non-Saccharomyces yeasts ; heterologous gene expression ; autonomously replicating expression vectors ; selective promoter identification ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: We have compared expression systems based on autonomously replicating vectors in the yeasts Saccharomyces cerevisiae, Schizosaccharomyces pombe, Kluyveromyces lactis, Hansenula polymorpha and Yarrowia lipolytica in order to identify a more suitable host organism for use in the expression cloning method (Dalbøge and Heldt-Hansen, 1994) in which S. cerevisiae has traditionally been used. The capacity of the expression systems to secrete active forms of six fungal genes encoding the enzymes galactanase, lipase, polygalacturonase, xylanase and two cellulases was examined, as well as glycosylation pattern, plasmid stability and transformation frequency. All of the examined alternative hosts were able to secrete more active enzyme than S. cerevisiae but the relative expression capacity of the individual hosts varied significantly in a gene-dependent manner. One of the most attractive of the alternative host organisms, Y. lipolytica, yielded an increase which ranged from 4·5 times to more than two orders of magnitude. As the initially employed Y. lipolytica XPR2 promoter is unfit in the context of expression cloning, two novel promoter sequences for highly expressed genes present in only one copy on the genome were isolated. Based on sequence homology, the genes were identified as TEF, encoding translation elongation factor-1α and RPS7, encoding ribosomal protein S7. Using the heterologous cellulase II (celII) and xylanase I (xylI) as reporter genes, the effect of the new promoters was measured in qualitative and quantitative assays. Based on the present tests of the new promoters, Y. lipolytica appears as a highly attractive alternative to S. cerevisiae as a host organism for expression cloning. GenBank Accession Numbers: TEF gene promoter sequence: AF054508; RPS7 gene promoter sequence: AF054509. © 1998 John Wiley & Sons, Ltd.
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  • 176
    ISSN: 0749-503X
    Keywords: BiP ; KAR2 ; Hsp70 ; peptide-binding domain ; secretion ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: Hsp70 is structurally composed of three domains, an amino-terminal ATPase domain, a proximal 18 kDa peptide-binding domain and a distal 10 kDa carboxy-terminal (C-terminal) domain. To dissect the functional significance of the distal 10 kDa domain, and the boundary region between the proximal and distal C-terminal domains of Kar2p in vivo in Saccharomyces cerevisiae, we constructed a series of plasmids which were truncated or had internal deletion mutations in this region. We found that all these mutations are recessive, and that the distal 10 kDa C-terminal domain, including the HDEL ER-retention sequence, is not essential for cell growth, although the major role of this 10 kDa C-terminal domain is due to the function of the HDEL ER-retention signal. We also found that the Kar2p region (Thr492-Thr512), corresponding to the β8-sheet in the peptide-binding domain, which constitutes the bottom plate of the binding pocket in E. coli DnaK, is essential for cell viability, and that the following Kar2p region (Glu513-Lys542), corresponding to α-helices A and B of E. coli DnaK, which was proposed to compose the lid of the binding pocket, is critical but not essential for yeast cell growth. This was further supported by the fact that the latter deletion showed a fully reversible ts phenotype in its growth and only a slight inhibitory effect on the secretion of α-amylase at non-permissive temperature. © 1998 John Wiley & Sons, Ltd.
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  • 177
    ISSN: 0749-503X
    Keywords: Yarrowia lipolytica ; acyl-CoA oxidase ; gene expression ; gene disruption ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: The ACO3 gene, which encodes one of the acyl-CoA oxidase isoenzymes, was isolated from the alkane-utilizing yeast Yarrowia lipolytica as a 10 kb genomic fragment. It was sequenced and found to encode a 701-amino acid protein very similar to other ACOs, 67·5% identical to Y. lipolytica Aco1p and about 40% identical to S. cerevisiae Pox1p. Haploid strains with a disrupted allele were able to grow on fatty acids. The levels of acyl-CoA oxidase activity in the ACO3 deleted strain, in an ACO1 deleted strain and in the wild-type strain, suggested that ACO3 encodes a short chain acyl-CoA oxidase isoenzyme. This narrow substrate spectrum was confirmed by expression of Aco3p in E. coli. © 1998 John Wiley & Sons, Ltd.
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  • 178
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    Yeast 14 (1998), S. 1341-1354 
    ISSN: 0749-503X
    Keywords: Kluyveromyces ; electrophoretic karyotyping ; contour-clamped homogeneous electric field electrophoresis ; chromosome variation ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: The analysis of the electrophoretic chromosome patterns of the species of the genus Kluyveromyces, reveals a high polymorphism in size, number and intensity of bands. Different sets of electrophoresis running conditions were used to establish species-specific patterns and also to detect intraspecific variation. According to their karyotypes, the species of this genus can be divided into two major groups. The first group includes the species K. africanus, K. bacillisporus, K. delphensis, K. lodderae, K. phaffi, K. polysporus and K. yarrowii, composing the so-called ‘Saccharomyces cerevisiae-like’ group, because their karyotypes resemble that of the species S. cerevisiae. The second group comprises the species K. aestuarii, K. blattae, K. dobzhanskii, K. lactis, K. marxianus, K. thermotolerans, K. waltii and K. wickerhamii, whose chromosomal patterns exhibit common characteristics very different to those of the species included in the ‘S. cerevisiae-like’ group. This division is concordant with the position of these species in previous phylogenetic reconstructions. Additionally, the intraspecific analysis of the chromosome patterns show a rich polymorphism in the heterogeneous species K. dobzhanskii, K. lactis, and K. marxianus, which is in concordance with the variability observed with other phenotypic or genetic markers. On the contrary, K. thermotolerans exhibits a homogeneous karyotype indicative of a very low level of chromosomal polymorphism, which is congruent with the reduced variability found in this species with other molecular markers. © 1998 John Wiley & Sons, Ltd.
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  • 179
    ISSN: 0749-503X
    Keywords: Saccharomyces cerevisiae ; functional genomics ; quantitative phenotype ; chemostat ; competition ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: One possible route to the evaluation of gene function is a quantitative approach based on the concepts of metabolic control analysis (MCA). An important first step in such an analysis is to determine the effect of deleting individual genes on the growth rate (or fitness) of S. cerevisiae. Since the specific growth-rate effects of most genes are likely to be small, we employed competition experiments in chemostat culture to measure the proportion of deletion mutants relative to that of a standard strain by using a quantitative PCR method. In this paper, we show that both densitometry and GeneScan™ analysis can be used with similar accuracy and reproducibility to determine the proportions of (at least) two strains simultaneously, in the range 10-90% of the total cell population. Furthermore, we report on a model competition experiment between two diploid nuclear petite mutants, homozygous for deletions in the cox5a or pet191 genes, and the standard strain (ho::kanMX4/ho::kanMX4) in chemostat cultures under six different physiological conditions. The results indicate that competition experiments in continuous culture are a suitable method to distinguish quantitatively between deletion mutants that qualitatively exhibit the same phenotype. © 1998 John Wiley & Sons, Ltd.
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  • 180
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    Yeast 14 (1998), S. 1437-1438 
    ISSN: 0749-503X
    Keywords: Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
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  • 181
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    Yeast 14 (1998), S. 133-146 
    ISSN: 0749-503X
    Keywords: Saccharomyces cerevisiae ; CDC28 gene ; RAD9 gene ; radiation sensitivity ; cell cycle checkpoint ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: CDC28-srm, a non-temperature-sensitive (ts) mutation in the CDC28 gene of Saccharomyces cerevisiae that affects fidelity of mitotic transmission of both mitochondrial and nuclear genetic structures (Devin et al., 1990), also affected cell growth and sensitivity to lethal effects of ionizing radiation. At 30°C CDC28-13, a ts mutation, was without appreciable effects on spontaneous mitochondrial rho--mutagenesis, cell growth and radiation sensitivity, whereas all three cell characteristics mentioned were affected (although to a lesser degree than by CDC28-srm) by CDC28-1, another ts mutation. CDC28-srm was without any significant effect on the rates of spontaneous nuclear gene mutations and γ-ray-induced mitotic recombination. An analysis of double mutants as regards their radiation sensitivity has revealed additive or even synergistic interactions between the CDC28-srm mutation and every one of the rad6-1 and rad52-1 mutations. The rad9Δ allele was found to be epistatic to CDC28-srm. These data suggest that the p34CDC28 protein is involved in the RAD9-dependent feedback control of DNA integrity operating at the cell cycle checkpoints. © 1998 John Wiley & Sons, Ltd.
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  • 182
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    Yeast 14 (1998), S. 189-194 
    ISSN: 0749-503X
    Keywords: branched-chain amino acids ; aminotransferase ; Schizosaccharomyces pombe ; Saccharomyces cerevisiae ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: The Saccharomyces cerevisiae genes for the cytosolic and mitochondrial branched-chain amino-acid aminotransferases (BCAT) were isolated recently. These genes show significant homology to mammalian ECA39, originally isolated as a gene regulated by the c-myc oncogene. We now report the isolation of the Schizosaccharomyces pombe eca39/BCAT gene. The S. pombe protein shows 47-52% identity to other eukaryotic BCAT proteins isolated from S. cerevisiae, nematode, mouse and man. A genetic growth assay for BCAT activity was established using an S. cerevisiae strain disrupted in both BCAT isoenzymes. Consequently, the activity of the S. pombe BCAT was demonstrated by genetic and biochemical means. Possible applications of BCAT-encoding genes as selection markers in yeast transformation are proposed. The sequence has been deposited in the GenBank data library under Accession Number U88029. © 1998 John Wiley & Sons, Ltd.
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  • 183
    ISSN: 0749-503X
    Keywords: recombinant DNA ; K. lactis genomic library ; pCXJ22 ; arginine biosynthesis ; KlARG8 ; mitochondrial transformation ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: A recombinant plasmid was isolated from a Kluyveromyces lactis genomic DNA library which complements a Saccharomyces cerevisiae arg8 mutant defective in the gene encoding acetylornithine aminotransferase. The complementation activity was found to reside within a 2.0 kb DNA fragment. Nucleotide sequence analysis revealed an open reading frame able to encode a 423-residue protein sharing 68·1% and 35·0% sequence identities with the products of the ARG8 and argD genes of S. cerevisiae and Escherichia coli. That the cloned gene, KlARG8, is the functional equivalent of S. cerevisiae ARG8 was supported by a gene disruption experiment which showed that K. lactis strains carrying a deleted chromosomal copy of KlARG8 are auxotrophic for arginine. The nucleotide sequence of KlARG8 has been submitted to GenBank under Accession Number U93209.
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  • 184
    ISSN: 0749-503X
    Keywords: Escherichia coli ; diacylglycerol kinase ; lipase ; Saccharomyces cerevisiae ; TGL2 ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: Escherichia coli cells with a disrupted diacylglycerol kinase gene are unable to grow on media containing arbutin due to a lethal accumulation of diacylglycerol. In order to isolate genes from the yeast Saccharomyces cerevisiae involved in diacylglycerol metabolism we complemented an E. coli diacylglycerol kinase disruptant with a yeast genomic library and transformants were selected capable of growing in the presence of arbutin. Using this method, a gene (TGL2) was isolated coding for a protein resembling lipases from Pseudomonas. After expression of the TGL2 gene in E. coli, lipolytic activity towards triacylglycerols and diacylglycerols with short-chain fatty acids could be measured. Therefore, it is very likely that the TGL2 gene can complement the E. coli diacylglycerol kinase disruptant, because it encodes a protein that degrades the diacylglycerol accumulated after growth in the presence of arbutin. Disruption of the TGL2 gene in S. cerevisiae did not result in a detectable phenotype. The role of the Tgl2 protein in lipid degradation in yeast is still unclear. The nucleotide sequence published here has been submitted to the EMBL sequence data bank and is available under accession number X98000. © 1998 John Wiley & Sons, Ltd.
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  • 185
    ISSN: 0749-503X
    Keywords: elongation factor 3 ; YEF3 homolog ; ATPase ; ABC cassette ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: Yeast and other fungi contain a soluble elongation factor 3 (EF-3) which is required for growth and protein synthesis. EF-3 contains two ABC cassettes, and binds and hydrolyses ATP. We identified a homolog of the YEF3 gene in the Saccharomyces cerevisiae genome database. This gene, designated YEF3B, is 84% identical in protein sequence to YEF3, which we will now refer to as YEF3A. YEF3B is not expressed during growth under laboratory conditions, and thus cannot rescue growth of YEF3A deletion strains. However, YEF3B can take the place of YEF3A in vivo when expressed from the YEF3A or ADH1 promoters. The products of the YEF3A and YEF3B genes, EF-3A and EF-3B, respectively, were expressed from the ADH1 promoter and purified. Both factors possessed basal and ribosomal-stimulated ATPase activity, and had similar affinity for yeast ribosomes (103 to 113 nm). Km values for ATP were similar, but the Kcat values differed significantly. Ribosome-dependent ATPase activity of EF-3A was more efficient than EF-3B, since the Kcat and Kcat/Km values for EF-3A were about two-fold higher; however, the difference in Kcat/Km values between the two factors was small for basal ATPase activity. © 1998 John Wiley & Sons, Ltd.
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  • 186
    ISSN: 0749-503X
    Keywords: marine yeast ; superoxide dismutase ; Debaryomyces hansenii ; cloning ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: Cu-Zn superoxide dismutase (SOD-1) is a ubiquitously occurring eukaryotic enzyme with a variety of important effects on respiring organisms. A gene (dhsod-1) encoding a Cu-Zn superoxide dismutase of the marine yeast Debaryomyces hansenii was cloned using mRNA by the RT-PCR technique. The deduced amino-acid sequence shows ∼70% homology with that of cytosolic superoxide dismutase from Saccharomyces cerevisiae and Neurospora crassa, as well as lower homologies (between 55 and 65%) with the corresponding enzyme of other eukaryotic organisms, including human. The gene sequence encodes a protein of 153 amino acids with a calculated molecular mass of 15·92 kDa, in agreement with the observed characteristics of the purified protein from D. hansenii. The dhsod-1 sequence has been deposited in the public data library of the NCBI under Accession Number AFO 16383. © 1998 John Wiley & Sons, Ltd.
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  • 187
    ISSN: 0749-503X
    Keywords: gene disruption ; functional analysis ; Saccharomyces cerevisiae ; G418-resistance ; sticky-end PCR ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: The disruption of eight novel genes was realized in two genetic backgrounds. Among these open reading frames, NO333, NO348 and NO364 presented homologies with other proteins of yeast or other organisms, whereas NO320, NO325, NO339, NO384 and NO388 showed no similarity with any protein. Tetrad analysis of heterozygous deletant strains revealed that NO348, NO364 and NO388 are essential genes for vegetative growth, whereas NO320, NO325, NO333, NO339 and NO384 are non-essential. Basic phenotypic analyses of the non-lethal deletant strains as suggested in the six-pack B0 programme did not reveal any significant differences between parental and mutant strains. © 1998 John Wiley & Sons, Ltd.
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  • 188
    ISSN: 0749-503X
    Keywords: hexokinase PII ; glycolysis ; Tps1 ; fermentation ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: In the yeast Saccharomyces cerevisiae a novel control exerted by TPS1 (=GGS1=FDP1=BYP1=CIF1=GLC6=TSS1)-encoded trehalose-6-phosphate synthase, is essential for restriction of glucose influx into glycolysis apparently by inhibiting hexokinase activity in vivo. We show that up to 50-fold overexpression of hexokinase does not noticeably affect growth on glucose or fructose in wild-type cells. However, it causes higher levels of glucose-6-phosphate, fructose-6-phosphate and also faster accumulation of fructose-1,6-bisphosphate during the initiation of fermentation. The levels of ATP and Pi correlated inversely with the higher sugar phosphate levels. In the first minutes after glucose addition, the metabolite pattern observed was intermediate between those of the tps1Δ mutant and the wild-type strain. Apparently, during the start-up of fermentation hexokinase is more rate-limiting in the first section of glycolysis than phosphofructokinase. We have developed a method to measure the free intracellular glucose level which is based on the simultaneous addition of d-glucose and an equal concentration of radiolabelled l-glucose. Since the latter is not transported, the free intracellular glucose level can be calculated as the difference between the total d-glucose measured (intracellular+periplasmic/extracellular) and the total l-glucose measured (periplasmic/extracellular). The intracellular glucose level rose in 5 min after addition of 100 mm-glucose to 0·5-2 mm in the wild-type strain, ±10 mm in a hxk1Δ hxk2Δ glk1Δ and 2-3 mm in a tps1Δ strain. In the strains overexpressing hexokinase PII the level of free intracellular glucose was not reduced. Overexpression of hexokinase PII never produced a strong effect on the rate of ethanol production and glucose consumption. Our results show that overexpression of hexokinase does not cause the same phenotype as deletion of Tps1. However, it mimics it transiently during the initiation of fermentation. Afterwards, the Tps1-dependent control system is apparently able to restrict properly up to 50-fold higher hexokinase activity. © 1998 John Wiley & Sons, Ltd.
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  • 189
    ISSN: 0749-503X
    Keywords: sphingolipids ; hydroxylase ; cytochrome b5 ; CSG1 ; CSG2 ; calcium ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: Saccharomyces cerevisiae mutants lacking Scs7p fail to accumulate the inositolphosphorylceramide (IPC) species, IPC-C, which is the predominant form found in wild-type cells. Instead scs7 mutants accumulate an IPC-B species believed to be unhydroxylated on the amide-linked C26-fatty acid. Elimination of the SCS7 gene suppresses the Ca2+-sensitive phenotype of csg1 and csg2 mutants. The CSG1 and CSG2 genes are required for mannosylation of IPC-C and accumulation of IPC-C by the csg mutants renders them Ca2+-sensitive. The SCS7 gene encodes a protein that contains both a cytochrome b5-like domain and a domain that resembles the family of cytochrome b5-dependent enzymes that use iron and oxygen to catalyse desaturation or hydroxylation of fatty acids and sterols. Scs7p is therefore likely to be the enzyme that hydroxylates the C26-fatty acid of IPC-C. © 1998 John Wiley & Sons, Ltd.
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  • 190
    Electronic Resource
    Electronic Resource
    New York, NY [u.a.] : Wiley-Blackwell
    Yeast 14 (1998), S. 359-369 
    ISSN: 0749-503X
    Keywords: SKSI ; snf3 ; low glucose ; over-expression ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: The SKS1 gene was originally identified as a multicopy suppressor of the growth defect of snf3 null mutations on low glucose concentrations. Snf3p is required for the rapid induction of HXT2 during growth on low substrate concentrations. Loss of Snf3p leads to a dramatic delay in expression of HXT2. Adaptation to low substrate concentrations does not occur in snf3 sks1 double null mutant strains, suggesting that SKS1 is required for the glucose-dependent expression of HXT2 in the absence of Snf3p activity. Over-expression of SKS1 leads to over-expression of Hxt2p, thus explaining the mechanism of suppression of the snf3 defect. SKS1 defines a novel, Snf3p-independent pathway for the expression of Hxt2p. Under certain growth conditions, over-expression of SKS1 itself leads to a growth defect which is diminished in snf3 hxt2 double mutants. This suggests that over-expression of Hxt2p at physiologically inappropriate times is detrimental to the cells. © 1998 John Wiley & Sons, Ltd.
    Additional Material: 7 Ill.
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  • 191
    ISSN: 0749-503X
    Keywords: α1-antitrypsin ; Saccharomyces cerevisiae ; Hansenula polymorpha ; Pichia pastoris ; glycosylation ; secretion ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: Human α1-antitrypsin (α1-AT) is a major serine protease inhibitor in plasma, secreted as a glycoprotein with a complex type of carbohydrate at three asparagine residues. To study glycosylation of heterologous proteins in yeast, we investigated the glycosylation pattern of the human α1-AT secreted in the baker's yeast Saccharomyces cerevisiae and in the methylotrophic yeasts, Hansenula polymorpha and Pichia pastoris. The partial digestion of the recombinant α1-AT with endoglycosidase H and the expression in the mnn9 deletion mutant of S. cerevisiae showed that the recombinant α1-AT secreted in S. cerevisiae was heterogeneous, consisting of molecules containing core carbohydrates on either two or all three asparagine residues. Besides the core carbohydrates, variable numbers of mannose outer chains were also added to some of the secreted α1-AT. The human α1-AT secreted in both methylotrophic yeasts was also heterogeneous and hypermannosylated as observed in S. cerevisiae, although the overall length of mannose outer chains of α1-AT in the methylotrophic yeasts appeared to be relatively shorter than those of α1-AT in S. cerevisiae. The α1-AT secreted from both methylotrophic yeasts retained its biological activity as an elastase inhibitor comparable to that of α1-AT from S. cerevisiae, suggesting that the different glycosylation profile does not affect the in vitro activity of the protein. © 1998 John Wiley & Sons, Ltd.
    Additional Material: 7 Ill.
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  • 192
    ISSN: 0749-503X
    Keywords: lys1+ gene ; Schizosaccharomyces pombe ; α-aminoadipate reductase ; peptide synthetase ; lysine biosynthesis ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: The 4·2 kbp lys1+ gene of Schizosaccharomyces pombe encoding the large subunit of α-aminoadipate reductase (EC1.2.1.31), an enzyme specific to lysine synthesis in higher fungi, was completely sequenced at the nucleotide level from pLYS1H. The S. pombe lys1+ gene product consists of 1415 amino acid residues and has a putative molecular weight of 155·8 kDa. The encoded protein converts α-aminoadipic acid to α-aminoadipate-δ-semialdehyde by an ATP-mediated adenylation. Analysis of the sequence showed that the putative protein encoded by lys1+ shares strong homology with the peptide antibiotic synthetases which also use an adenylation step. The sequence data reported in this paper have been submitted to GenBank database (Washington DC, USA) under the Accession Number U15923. © 1998 John Wiley & Sons, Ltd.
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  • 193
    ISSN: 0749-503X
    Keywords: Apiotrichum curvatum ; cDNA sequence ; DnaJ protein ; cytosolic Hsp70s ; ribosome association ; Sis1 protein ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: DnaJ proteins have been localized in different intracellular compartments of eukaryotes. In Apiotrichum curvatum, a fat-storing yeast, we found a DnaJ homolog associated with ribosomes and large cytosolic complexes as well. Using a plant DnaJ probe and a cDNA library constructed from poly(A)+ -RNA of A. curvatum grown on oleate we isolated a SIS1 cDNA coding for a 39·5 kDa protein. The putative protein contains neither a zinc finger motif nor a CAAX motif but is characterized by a J-domain at the N-terminal region and a large G-rich region in the middle part of the molecule. Heat shock applied for 1 h resulted in a pronounced but transient increase of the SIS1 mRNA. An antiserum was raised against the bacterially expressed protein. Cell fractions from A. curvatum were further separated by sedimentation centrifugation on sucrose gradients. Analysing the sub-fractions, we detected Sis1p mainly associated with ribosomes, and with particles sedimenting at approximately 200S. Hsp70 was found to be associated with the 200S fraction. The respective cytosolic A. curvatum Hsp70 cDNA was cloned and sequenced. High salt conditions caused the removal of Hsp70 and Sis1p from the 200S complexes. Mild RNase treatment of the 200S fraction afforded monosomes and 200S complexes unaffected by RNase. Heat shock led to a pronounced increase in the rate of de novo synthesis. However, due to the large pools of Sis1p on ribosomes and large cytosolic complexes, the increase in gene activation did not lead to a significant change of the total amount of Sis1p. Accession numbers are: Y12079 for ACHSP70 and Y12080 for ACSIS1. © 1998 John Wiley & Sons, Ltd.
    Additional Material: 7 Ill.
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  • 194
    ISSN: 0749-503X
    Keywords: yeast ; S. cerevisiae ; genetics ; karyotypes ; chromosomal rearrangements ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: In order to study meiotic segregation of chromosome length polymorphism in yeast, we analysed the progeny of a cross involving two laboratory strains FL100trp and YNN295. Analysis of the parental strains led us to detect an important length polymorphism of chromosomes I and III in FL100trp. A reciprocal translocation involving 80 kb of the left arm of chromosome III and 45 kb of the right arm of chromosome I was shown to be the cause for the observed polymorphism in this strain. The characterization of the translocation breakpoints revealed the existence of a transposition hot-spot on chromosome I: the sequence of the translocation joints on chromosomes I and III suggests that the mechanism very likely involved homologous recombination between Ty2 transposable elements on each chromosome. Analysis of FL100, FL200 and FL100trp ura, which are related to FL100trp, shows that this reciprocal translocation is present in some of the strains of the FL series, whereas the parental strain FL100 does not carry the same rearrangement. We evidenced instead the duplication of 80 kb of chromosome III on chromosome I and a deletion of 45 kb of the right arm of chromosome I in this strain, indicating that secondary events might have taken place and that the strain currently named FL100 is not the common ancestor of the FL series. © 1998 John Wiley & Sons, Ltd.
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  • 195
    Electronic Resource
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    New York, NY [u.a.] : Wiley-Blackwell
    Yeast 14 (1998), S. 665-674 
    ISSN: 0749-503X
    Keywords: histidine kinase ; phosphorylation ; signal transduction ; gene ; two-component ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: We have cloned and analysed the sequence of a putative histidine kinase, two-component gene (CaHK1) from Candida albicans. This gene encodes a 2471 amino acid protein (Cahk1p) with an estimated molecular mass of 281·8 kDa. A homology search of Cahk1p with other proteins in the databases showed that Cahk1p exhibits the greatest homology at its C-terminus with both the sensor and regulator components of prokaryotic and eukaryotic two-component histidine kinases. A further analysis of this homology showed that the Cahk1p possessed both sensor and regulator domains in the same polypeptide. Also, Cahk1p is likely to be a soluble protein. The sensor kinase domain of Cahk1p contains conserved motifs that are characteristic of all histidine kinase proteins, including the putative histidine which is believed to be autophosphorylated during activation, ATP binding motifs and others (F- and N-motifs), with unknown function. The Cahk1p regulator domain also contains conserved aspartate and lysine residues and the putative aspartate, which is secondarily phosphorylated by the autophosphorylated histidine. Finally, according to the codon usage frequency of the CaHK1 gene in comparison with other genes from C. albicans, there would appear to be a low level of expression of the gene. The accession number for the described sequence is AF013273, as filed in the EMBL/GenBank/DDBJ database. © 1998 John Wiley & Sons, Ltd.
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  • 196
    Electronic Resource
    Electronic Resource
    New York, NY [u.a.] : Wiley-Blackwell
    Yeast 14 (1998), S. 655-664 
    ISSN: 0749-503X
    Keywords: Saccharomyces cerevisiae ; PCR-based disruption ; YOL113w ; YOL100w ; YOL107w ; YOR267c ; YGL196w ; YGL194c ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: Using PCR to construct disruption cassettes, null alleles of six genes have been created in Saccharomyces cerevisiae. In a FY1679 background, no defects were detected in any of the haploid deletion mutants with respect to growth, gross morphology, or mating. A diploid FY1679-derived Δygl194c/Δygl194c homozygous disruptant displayed reduced sporulation. In contrast to the lack of phenotypic consequences of Δyol100w disruptions in the FY1679 background, in the CEN.PK2 strain even a heterozygous disruption of the same gene caused striking effects, very slow vegetative growth and highly impaired sporulation. Tetrad analysis showed YOL100w to be an essential gene in this strain. A copy of the YGL194c or the YOL100w wild-type gene borne on a centromeric episomal plasmid was introduced into a corresponding disruption mutant strain, and in both cases was found to partially complement the defects. © 1998 John Wiley & Sons, Ltd.
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  • 197
    Electronic Resource
    Electronic Resource
    New York, NY [u.a.] : Wiley-Blackwell
    Yeast 14 (1998), S. 623-631 
    ISSN: 0749-503X
    Keywords: Saccharomyces cerevisiae ; bicarbonate ; meiosis ; sporulation ; respiration ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: Meiosis and sporulation in the yeast Saccharomyces cerevisiae requires social communication, mediated by an extracellular factor which is secreted from cells during sporulation and accumulates in a cell density-dependent manner. We show here genetic and biochemical analyses supporting our conclusion that the extracellular factor is bicarbonate acting as an alkali to elevate extracellular pH. Sporulation defects of mdh1 (mitochondrial malate dehydrogenase) mutants and of wild-type cells at low density were rescued extracellularly by addition of bicarbonate or other alkaline solutions to raise medium pH. Addition of bicarbonate (or alkalization of medium) raised steady-state levels of mRNA in respiration-deficient mdh1 mutants and inhibited proliferation of wild-type cells at low density. These results indicate that the two conditions (respiration competency and high cell density), required for meiosis and sporulation, are essential for extracellular accumulation of bicarbonate and resulting alkalization of medium. © 1998 John Wiley & Sons, Ltd.
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  • 198
    Electronic Resource
    Electronic Resource
    New York, NY [u.a.] : Wiley-Blackwell
    Yeast 14 (1998), S. 617-622 
    ISSN: 0749-503X
    Keywords: Saccharomyces cerevisiae ; extracellular factor ; meiosis ; sporulation ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: Meiosis and sporulation in the yeast Saccharomyces cerevisiae has been classically viewed as an example of unicellular, eukaryotic differentiation that occurs in response to nutritional starvation. We present evidence that S. cerevisiae produces an extracellular factor(s), called meiosis-promoting factor (MEP), that is required, in addition to starvation conditions, for efficient meiosis and sporulation. This factor is secreted and accumulates in a cell density-dependent fashion such that cells at a low density sporulate poorly under conditions in which cells at a high density sporulate efficiently. Conditioned medium from sporulating cells at a high density contains a small anionic molecule that has cytostatic activity and stimulates sporulation of cells at low density under a normal starvation condition. These results indicate that MEP-mediated social communication between cells is required for meiosis and sporulation. © 1998 John Wiley & Sons, Ltd.
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  • 199
    ISSN: 0749-503X
    Keywords: epitope tagging ; green fluorescent protein ; functional analysis ; overexpression studies ; gene deletion ; gene truncation ; polymerase chain reaction ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: An important recent advance in the functional analysis of Saccharomyces cerevisiae genes is the development of the one-step PCR-mediated technique for deletion and modification of chromosomal genes. This method allows very rapid gene manipulations without requiring plasmid clones of the gene of interest. We describe here a new set of plasmids that serve as templates for the PCR synthesis of fragments that allow a variety of gene modifications. Using as selectable marker the S. cerevisiae TRP1 gene or modules containing the heterologous Schizosaccharomyces pombe his5+ or Escherichia coli kanr gene, these plasmids allow gene deletion, gene overexpression (using the regulatable GAL1 promoter), C- or N-terminal protein tagging [with GFP(S65T), GST, or the 3HA or 13Myc epitope], and partial N- or C-terminal deletions (with or without concomitant protein tagging). Because of the modular nature of the plasmids, they allow efficient and economical use of a small number of PCR primers for a wide variety of gene manipulations. Thus, these plasmids should further facilitate the rapid analysis of gene function in S. cerevisiae. © 1998 John Wiley & Sons, Ltd.
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  • 200
    ISSN: 0749-503X
    Keywords: fission yeast ; gene deletions ; gene truncations ; overexpression studies ; epitope tagging ; polymerase chain reaction ; gene expression ; green fluorescent protein ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: We describe a straightforward PCR-based approach to the deletion, tagging, and overexpression of genes in their normal chromosomal locations in the fission yeast Schizosaccharomyces pombe. Using this approach and the S. pombe ura4+ gene as a marker, nine genes were deleted with efficiencies of homologous integration ranging from 6 to 63%. We also constructed a series of plasmids containing the kanMX6 module, which allows selection of G418-resistant cells and thus provides a new heterologous marker for use in S. pombe. The modular nature of these constructs allows a small number of PCR primers to be used for a wide variety of gene manipulations, including deletion, overexpression (using the regulatable nmt1 promoter), C- or N-terminal protein tagging (with HA, Myc, GST, or GFP), and partial C- or N-terminal deletions with or without tagging. Nine genes were manipulated using these kanMX6 constructs as templates for PCR. The PCR primers included 60 to 80 bp of flanking sequences homologous to target sequences in the genome. Transformants were screened for homologous integration by PCR. In most cases, the efficiency of homologous integration was ≥50%, and the lowest efficiency encountered was 17%. The methodology and constructs described here should greatly facilitate analysis of gene function in S. pombe. © 1998 John Wiley & Sons, Ltd.
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