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  • Genetics  (2,232)
  • Rehabilitation
  • Strömungsmechanik
  • Wiley-Blackwell  (2,235)
  • Sage Publications  (6)
  • BMJ Publishing Group  (4)
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  • 1
    Publication Date: 2018-02-20
    Description: Aims To identify calcifying epithelial odontogenic tumour (CEOT) mutations in oncogenes and tumour suppressor genes. Methods A panel of 50 genes commonly mutated in cancer was sequenced in CEOT by next-generation sequencing. Sanger sequencing was used to cover the region of the frameshift deletion identified in one sample. Results Missense single nucleotide variants (SNVs) with minor allele frequency (MAF) 〈1% were detected in PTEN , MET and JAK3 . A frameshift deletion in CDKN2A occurred in association with a missense mutation in the same gene region, suggesting a second hit in the inactivation of this gene. APC, KDR, KIT, PIK3CA and TP53 missense SNVs were identified; however, these are common SNVs, showing MAF 〉1%. Conclusion CEOT harbours mutations in the tumour suppressor PTEN and CDKN2A and in the oncogenes JAK3 and MET . As these mutations occurred in only one case each, they are probably not driver mutations for these tumours.
    Keywords: Genetics
    Print ISSN: 0021-9746
    Electronic ISSN: 1472-4146
    Topics: Medicine
    Published by BMJ Publishing Group
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  • 2
    Publication Date: 2018-01-25
    Description: Background Myelodysplasia, infection, restriction of growth, adrenal hypoplasia, genital phenotypes and enteropathy (MIRAGE) syndrome is a recently described congenital disorder caused by heterozygous SAMD9 mutations. The phenotypic spectrum of the syndrome remains to be elucidated. Methods and results We describe two unrelated patients who showed manifestations compatible with MIRAGE syndrome, with the exception of haematological features. Leucocyte genomic DNA samples were analysed with next-generation sequencing and Sanger sequencing, revealing the patients to have two de novoSAMD9 mutations on the same allele (patient 1 p.[Gln695*; Ala722Glu] and patient 2 p.[Gln39*; Asp769Gly]). In patient 1, p.Gln695* was absent in genomic DNA extracted from hair follicles, implying that the non-sense mutation was acquired somatically. In patient 2, with the 46,XX karyotype, skewed X chromosome inactivation pattern was found in leucocyte DNA, suggesting monoclonality of cells in the haematopoietic system. In vitro expression experiments confirmed the growth-restricting capacity of the two missense mutant SAMD9 proteins that is a characteristic of MIRAGE-associated SAMD9 mutations. Conclusions Acquisition of a somatic nonsense SAMD9 mutation in the cells of the haematopoietic system might revert the cellular growth repression caused by the germline SAMD9 mutations (ie, second-site reversion mutations). Unexpected lack of haematological features in the two patients would be explained by the reversion mutations.
    Keywords: Genetics
    Print ISSN: 0022-2593
    Electronic ISSN: 1468-6244
    Topics: Medicine
    Published by BMJ Publishing Group
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  • 3
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    BMJ Publishing Group
    Publication Date: 2018-01-23
    Description: Succinate dehydrogenase (SDH) is a heterotetrameric nuclear encoded mitochondrial protein complex which plays a role in the citric acid cycle and the electron transfer chain. Germline mutations in SDHA are associated with Leigh syndrome. Mutations in SDHB , SDHC and SDHD are found in an increasing number of neoplasms, most notably paragangliomas and wild-type gastrointestinal stromal tumours. SDH deficiency in these tumours has important prognostic implications, and also provides a novel target for molecular therapy. In this article, we outline the structure and function of SDH and provide a summary of its role in various diseases.
    Keywords: Genetics
    Print ISSN: 0021-9746
    Electronic ISSN: 1472-4146
    Topics: Medicine
    Published by BMJ Publishing Group
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  • 4
    Publication Date: 2018-04-28
    Description: Background Chitayat-Hall syndrome, initially described in 1990, is a rare condition characterised by distal arthrogryposis, intellectual disability, dysmorphic features and hypopituitarism, in particular growth hormone deficiency. The genetic aetiology has not been identified. Methods and results We identified three unrelated families with a total of six affected patients with the clinical manifestations of Chitayat-Hall syndrome. Through whole exome or whole genome sequencing, pathogenic variants in the MAGEL2 gene were identified in all affected patients. All disease-causing sequence variants detected are predicted to result in a truncated protein, including one complex variant that comprised a deletion and inversion. Conclusions Chitayat-Hall syndrome is caused by pathogenic variants in MAGEL2 and shares a common aetiology with the recently described Schaaf-Yang syndrome. The phenotype of MAGEL2 -related disorders is expanded to include growth hormone deficiency as an important and treatable complication.
    Keywords: Genetics
    Print ISSN: 0022-2593
    Electronic ISSN: 1468-6244
    Topics: Medicine
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  • 5
    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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  • 6
    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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  • 7
    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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  • 8
    Electronic Resource
    Electronic Resource
    New York, NY [u.a.] : Wiley-Blackwell
    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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  • 9
    Electronic Resource
    Electronic Resource
    New York, NY [u.a.] : Wiley-Blackwell
    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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  • 10
    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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  • 11
    Electronic Resource
    Electronic Resource
    New York, NY [u.a.] : Wiley-Blackwell
    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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  • 12
    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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  • 13
    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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  • 14
    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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  • 15
    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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  • 16
    Electronic Resource
    Electronic Resource
    New York, NY [u.a.] : Wiley-Blackwell
    Yeast 14 (1998), S. 985-1000 
    ISSN: 0749-503X
    Keywords: Mig1 repressor ; glucose repression ; Saccharomyces cerevisiae ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: A major mediator of glucose repression in yeast is Mig1, a zinc finger protein that binds to a GC-rich recognition sequence found upstream of many glucose-repressible genes. Because these Mig1 sites are found upstream of genes under different modes of regulation, we studied regulation of transcription mediated by an isolated Mig1 site placed upstream of a reporter gene under control of UASCYC1. The Mig1 site responded appropriately to glucose control and regulatory mutations, including snf1, reg1, cyc8, and tup1, mimicking the behavior of the SUC2 gene. Deletion of the MIG1-coding gene reduced but did not eliminate glucose repression mediated by the Mig1 site. Complete loss of repression was seen in a mig1 mig2 double mutant. When the UASCYC1 was replaced by UASADH1 in the reporter plasmid, the Mig1 site activated transcription under most conditions. Mutations of the two Mig1 binding sites in the SUC2 promoter resulted in loss of activation of SUC2 expression. These results suggest the presence of an unknown activator or activators that binds to the Mig1 site. The activator is not any of the proteins previously proposed to bind to this site, including Mig1, Mig2, Msn2, or Msn4. Band shift assays showed that Mig1 is the major protein in yeast cell extracts that binds to the Mig1 site in vitro. This binding is not regulated by glucose or mutations in CYC8 or TUP1. © 1998 John Wiley & Sons, Ltd.
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  • 17
    ISSN: 0749-503X
    Keywords: Saccharomyces cerevisiae ; gene cloning ; gene disruption ; functional analysis ; chromosome XVI ; translation ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: A 7·24 kb genomic DNA fragment from the yeast Saccharomyces cerevisiae chromosome XVI was isolated by complementation of a new temperature-sensitive mutation tsa1. We determined the nucleotide sequence of this fragment located on the right arm of chromosome XVI. Among the three, complete open reading frames: YPR041w, YPR042c and YPR043w contained within this fragment, the gene YPR041w was shown to complement the tsa1 mutation and to correspond to the TIF5 gene encoding an essential protein synthesis initiation translation factor. The YPR042c gene encodes a hypothetical protein of 1075 amino acids containing four putative transmembrane segments and is non-essential for growth. The gene YPR043c encoding the 10 kDa product, highly similar to the human protein L37a from the 60S ribosomal subunit, was found to be essential and a dominant lethal. We conclude that three tightly linked yeast genes are involved in the translation process. © 1998 John Wiley & Sons, Ltd.
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  • 18
    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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  • 19
    Electronic Resource
    Electronic Resource
    New York, NY [u.a.] : Wiley-Blackwell
    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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  • 20
    Electronic Resource
    Electronic Resource
    New York, NY [u.a.] : Wiley-Blackwell
    Yeast 14 (1998), S. 1001-1006 
    ISSN: 0749-503X
    Keywords: succinate dehydrogenase ; SDH1 ; SDH1b ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: Transformation of the respiratory-defective mutant (E264/U2) of Saccharomyces cerevisiae with a yeast genomic library yielded two different plasmids capable of restoring the ability of the mutant to grow on non-fermentable substrates. One of the plasmids (pG52/T3) contained SDH1 coding for the flavoprotein subunit of mitochondrial succinate dehydrogenase. The absence of detectable succinate dehydrogenase activity in mitochondria of E264/U2 and the lack of complementation of the mutant by an sdh11null strain indicated a mutation in SDH1. The second plasmid (pG52/T8) had an insert with reading frame (YJL045w) of yeast chromosome X coding for a homologue of SDH1. Subclones containing the SDH1 homologue (SDH1b), restored respiration in E264/U2 indicating that the protein encoded by this gene is functional. The expression of the two genes was compared by assaying the β-galactosidase activities of yeast transformed with plasmids containing fusions of lacZ to the upstream regions of SDH1 and SDH1b. The 100-500 times lower activity measured in transformants harbouring the SDH1b-lacZ fusion indicates that the isoenzyme encoded by SDH1b is unlikely to play an important role in mitochondrial respiration. This is also supported by the absence of any obvious phenotype in cells with a disrupted copy of SDH1b. © 1998 John Wiley & Sons, Ltd.
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  • 21
    ISSN: 0749-503X
    Keywords: nicotine ; diphenylamine ; astaxanthin biosynthesis ; Xanthophyllomyces dendrorhous ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: The effect of nicotine and diphenylamine on astaxanthin biosynthesis in Xanthophyllomyces dendrorhous was studied. The effects were analysed under standard and low temperature conditions. It was found that 10 mm-nicotine inhibits the cyclization of lycopene and de novo protein synthesis was not needed to reverse the inhibition. The oxidation of β-carotene was irreversibly inhibited by 10 μM-diphenylamine while the dehydrogenation of phytoene was reversibly inhibited by 60 μM-diphenylamine. The simultaneous exposure to low temperature (4°C) overcomes the inhibition of β-carotene oxidation at low diphenylamine concentration. © 1998 John Wiley & Sons, Ltd.
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  • 22
    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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  • 23
    ISSN: 0749-503X
    Keywords: Candida albicans ; HIS4 ; complementation ; molecular biology tools ; topological marker ; amino acid biosynthesis general control ; PEX5 ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: We have isolated the Candida albicans HIS4 (CaHIS4) gene by complementation of a his4-34 Saccharomyces cerevisiae mutant. The sequenced DNA fragment contains a putative ORF of 2514 bp, whose translation product shares a global identity of 44% and 55% to the His4 protein homologs of S. cerevisiae and Kluyveromyces lactis, respectively. Analysis of CaHIS4 sequence suggests that, similarly to S. cerevisiae HIS4, it codes for a polypeptide having three separate enzymatic activities (phosphoribosyl-AMP cyclohydrolase, phosphoribosyl-ATP pyrophosphohydrolase and histidinol dehydrogenase) which reside in different domains of the protein. A C. albicans his4 strain is complemented with this gene when using a C. albicans-S. cerevisiae-Escherichia coli shuttle vector, thus enabling the construction of a host system for C. albicans genetic manipulation. In addition, upstream of the sequenced CaHIS4 sequence, we have found the 3′-terminal half of a gene encoding a PEX5-like protein. The EMBL/DDJB/GenBank Accession Number of this sequence is AJ003115. © 1998 John Wiley & Sons, Ltd.
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  • 24
    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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  • 25
    Electronic Resource
    Electronic Resource
    New York, NY [u.a.] : Wiley-Blackwell
    Yeast 14 (1998), S. 1069-1087 
    ISSN: 0749-503X
    Keywords: amines and amides ; biodetergents ; biocides ; bioremediation ; biosensors ; Candida rugosa ; carbohydrate esters ; cosmetics and perfumery ; food and flavour ; immobilisation ; isoenzymes ; lipases ; molecular biology ; pharmaceuticals ; single cell protein ; specificity ; tanning ; ultra-structure ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: This review describes how the versatile Candida rugosa lipases (CRL) have extended the frontiers of biotechnology. As evidenced by the current literature, CRL claims more applications than any other biocatalyst. This review comprises a detailed discussion on the molecular biology of CRL, its versatile catalytic reactions, broad specificities and diverse immobilization strategies. It also discusses its role in the food and flavour industry, the production of ice cream and single cell protein, biocatalytic resolution of life-saving pharmaceuticals, carbohydrate esters and amino acid derivatives unobtainable by conventional chemical synthesis, potent biocide making, biosensor modulations, eco-friendly approach and bioremediation, biosurfactants in detergent making, and recently, cosmetics and perfumery. © 1998 John Wiley & Sons, Ltd.
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  • 26
    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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  • 27
    ISSN: 0749-503X
    Keywords: yeast ; elongation factor-3 ; EF-3 ; homolog ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: A paralog (intraspecies homolog) of the Saccharomyces cerevisiae YEF3 gene, encoding elongation factor-3, has been sequenced in the course of the yeast genome project, and identified by database searching; this gene has been designated HEF3. Bioinformatic and Northern blot analysis indicate that the HEF3 gene is not expressed during vegetative growth. Deletion of the HEF3 gene reveals no growth defects, nor any defects in mating or sporulation. A high copy 2μ clone of HEF3 was constructed, and was shown to be unable to complement a null allele of yef3. Finally, an in vitro assay for ribosome-stimulated ATPase activity was performed with isogenic HEF3 and Δhef3 strains; no difference in biochemical activity could be detected in these strains. From these results, we conclude that the HEF3 gene does not encode a functional homolog of YEF3. © 1998 John Wiley & Sons, Ltd.
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  • 28
    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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  • 29
    Electronic Resource
    Electronic Resource
    New York, NY [u.a.] : Wiley-Blackwell
    Yeast 14 (1998), S. 1115-1125 
    ISSN: 0749-503X
    Keywords: Yarrowia lipolytica ; secretion ; pH ; extracellular protease ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: The secretion and maturation of the acid extracellular protease (AXP) of the yeast Yarrowia lipolytica have been characterized using antiserum raised against this enzyme. A 42 kDa pro-enzyme form of AXP was identified from lysates of radiolabelled Y. lipolytica cells and found to contain no N-linked carbohydrate moieties. Using pulse-chase immune precipitation it was demonstrated that the AXP precursor was secreted into the extracellular medium where, under conditions of low pH, it underwent autocatalytic activation forming the mature enzyme. Conversion of the AXP pro-form in the presence of the protease inhibitor pepstatin indicated that an intramolecularly-catalysed reaction mechanism was involved in AXP maturation. Further evidence supporting the role of autocatalytic processing came from the side-chain specificity of mature AXP towards the oxidized B-chain of insulin. © 1998 John Wiley & Sons, Ltd.
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  • 30
    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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  • 31
    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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  • 32
    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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  • 33
    ISSN: 0749-503X
    Keywords: cell walls ; protease ; β-glucanase ; lysis ; yeast ; antifungal drugs ; glucan ; mannoprotein ; S. cerevisiae ; C. albicans ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: The rate of formation of spheroplasts of yeast can be used as an assay to study the structural integrity of cell walls. Lysis can be measured spectrophotometrically in hypotonic solution in the presence of Zymolyase, a mixture of cell wall-digesting enzymes. The optical density of the cell suspension decreases as the cells lyse. We optimized this assay with respect to enzyme concentration, temperature, pH, and growth conditions for several strains of Saccharomyces cerevisiae. The level of variability (standard deviation) was 1-5% between trials where the replications were performed on the same culture using enzyme prepared from the same lot, and 5-15% for different cultures of the same strain. This assay can quantitate differences in cell wall structure (1) between exponentially growing and stationary phase cells, (2) among different S. cerevisiae strains, (3) between S. cerevisiae and Candida albicans, (4) between parental and mutated lines, and (5) between drug- or chemically-treated cells and controls. © 1998 John Wiley & Sons, Ltd.
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  • 34
    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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  • 35
    ISSN: 0749-503X
    Keywords: carbocyanine fluorescent probes ; membrane potential ; yeast ; cell wall ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: Membrane-potential-dependent accumulation of diS-C3(3) in intact yeast cells in suspension is accompanied by a red shift of the maximum of its fluorescence emission spectrum, λmax, caused by a readily reversible probe binding to cell constituents. Membrane depolarization by external KCl (with or without valinomycin) or by ionophores causes a fast and reproducible blue shift. As the potential-reporting parameter, the λmax shift is less affected by probe binding to cuvette walls and possible photobleaching than, for example, fluorescence intensity. The magnitude of the potential-dependent red λmax shift depends on relative cell-to-probe concentration ratio, a maximum shift (572→582 nm) being found in very thick suspensions and in cell lysates. The potential therefore has to be assessed at reasonably low cell (≤5×106 cells/ml) and probe (10-7 M) concentrations at which a clearly defined relationship exists between the λmax shift and the potential-dependent accumulation of the dye in the cells. The redistribution of the probe between the medium and yeast protoplasts takes about 5 min, but in intact cells it takes 10-30 min because the cell wall acts as a barrier, hampering probe penetration into the cells. The barrier properties of the cell wall correlate with its thickness: cells grown in 0·2% glucose (cell wall thickness 0·175±0·015 μm, n=30) are stained much faster and the λmax is more red-shifted than in cells grown in 2% glucose (cell wall thickness 0·260±0·043 μm, n=44). At a suitable cell and probe concentration and under standard conditions, the λmax shift of diS-C3(3) fluorescence provides reliable information on even fast changes in membrane potential in Saccharomyces cerevisiae. © 1998 John Wiley & Sons, Ltd.
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  • 36
    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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  • 37
    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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  • 38
    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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  • 39
    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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  • 40
    Electronic Resource
    Electronic Resource
    New York, NY [u.a.] : Wiley-Blackwell
    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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  • 41
    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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  • 42
    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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  • 43
    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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  • 44
    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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  • 45
    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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  • 46
    Electronic Resource
    Electronic Resource
    New York, NY [u.a.] : Wiley-Blackwell
    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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  • 47
    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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  • 48
    Electronic Resource
    Electronic Resource
    New York, NY [u.a.] : Wiley-Blackwell
    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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  • 49
    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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  • 50
    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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  • 51
    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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  • 52
    Electronic Resource
    Electronic Resource
    New York, NY [u.a.] : Wiley-Blackwell
    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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  • 53
    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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  • 54
    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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  • 55
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    New York, NY [u.a.] : Wiley-Blackwell
    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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  • 56
    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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  • 57
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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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  • 58
    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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  • 59
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    New York, NY [u.a.] : Wiley-Blackwell
    Yeast 14 (1998), S. 287-295 
    ISSN: 0749-503X
    Keywords: Candida maltosa ; Candida albicans ; tRNA splicing gene ; silent genes ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Note