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  • Electronic Resource  (158)
  • 2020-2024
  • 1995-1999  (158)
  • 1860-1869
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  • Genetics  (158)
  • 101
    Electronic Resource
    Electronic Resource
    Sequence Analysis of the Candida albicans ADE2 Gene and Physical Separation of the Two Functionally Distinct Domains of the Phosphoribosylaminoimidazole Carboxylase (1997)
    New York, NY [u.a.] : Wiley-Blackwell
    Yeast 13 (1997), S. 769-776 
    Details
    ISSN: 0749-503X
    Keywords: Candida albicans ; ADE2 gene ; sequencing ; purine biosynthesis ; anti-fungal ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: An ADE2 genomic clone from the pathogenic fungus, Candida albicans, was isolated by complementation of an Escherichia coli purK mutant and the gene was analysed by DNA sequencing. A 1707 bp open reading frame was identified encoding a polypeptide of 569 amino acids with significant homology to all the known yeast ADE2 genes. Sequence homology to both the E. coli purE and purK genes suggests that the C. albicans ADE2 gene is the result of an evolutionary fusion. The amino-acid sequence comparison showed that the N-terminal domain of the Ade2 protein has a 52·5% identity to PurK, whereas the C-terminal domain has a distinct 64·3% identity to PurE. In order to establish the functional relationship of these two regions, deletion mutants of the Ade2 protein were prepared by recombinant expression of the functional domains, which were tested by complementation of their respective E. coli auxotrophs. The sequence described in this paper has been deposited in the EMBL data library under the Accession Number U69606. © 1997 John Wiley & Sons, Ltd.
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  • 102
    Electronic Resource
    Electronic Resource
    Identification and Preliminary Characterization of p31, a New PSTAIRE-related Protein in Fission Yeast (1997)
    New York, NY [u.a.] : Wiley-Blackwell
    Yeast 13 (1997), S. 727-734 
    Details
    ISSN: 0749-503X
    Keywords: p31 ; cdc2 ; PSTAIRE ; fission yeast ; Schizosaccharomyces pombe ; cell division cycle ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: One of the defining characteristics of the catalytic subunit of the cyclin-dependent protein kinases (cdks) is the so-called PSTAIRE motif. Western blots of fission yeast cytosolic extracts using a monoclonal antibody against the PSTAIRE peptide revealed two bands at 34 kDa (p34cdc2) and 31 kDa (p31). Polyclonal antibodies to the C-terminus of p34cdc2 or to the full-length protein recognized the 34 kDa band but not p31. Overexpression of the cdc2+ gene resulted in the increase of the 34 kDa band but not p31. Like p34 the level of p31 revealed no obvious cell cycle regulation but the protein was present in spores where p34cdc2 was barely detectable. p31 expression was unaffected by removal of either phosphate or ammonium from the growth medium, although the level of p34cdc2 was reduced in the absence of phosphate. p31 was not associated with cyclin B, nor was it adsorbed to p13suc1 Sepharose beads, two characteristics of p34cdc2. p31 did, however, interact with p15, the starfish homologue of p13suc1. p31 was present in cells in which cdc2+ was replaced by its budding yeast homologue CDC28. When fission yeast cytosolic extracts were subjected to gel filtration chromatography, p31 eluted in two peaks, one at approximately 100 kDa, the other at approximately 30 kDa. We conclude that p31 is a novel fission yeast PSTAIRE protein and therefore, potentially, a new cdk. © 1997 John Wiley & Sons, Ltd.
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  • 103
    Electronic Resource
    Electronic Resource
    Applications of the Long and Accurate Polymerase Chain Reaction Method in Yeast Molecular Biology: Direct Sequencing of the Amplified DNA and Its Introduction into Yeast (1997)
    New York, NY [u.a.] : Wiley-Blackwell
    Yeast 13 (1997), S. 763-768 
    Details
    ISSN: 0749-503X
    Keywords: Saccharomyces cerevisiae ; long and accurate (LA)-PCR ; homologous recombination ; co-transformation ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: A DNA fragment longer than 10 kb can be amplified by the long and accurate polymerase chain reaction (LA-PCR) method. We demonstrate here applications of this technique in molecular biological studies of Saccharomyces cerevisiae. We have shown that DNA fragments amplified by LA-PCR can be directly used as a template in the chain-termination sequencing protocol, making it possible to quickly identify the DNA insert of yeast genomic library clones. We have also shown that the amplified yeast DNA can easily be introduced into yeast by co-transformation with linearized vector DNA. Overlapping DNA between the amplified yeast fragment and the vector must be more than 20 bp long in order to obtain 90% or more correct recombinant plasmids. These results suggest that simple amplification of yeast clones by LA-PCR can replace the previous procedures of yeast clone recovery, consisting of transformation of Escherichia coli, propagation of plasmids in E. coli and preparation of plasmid DNA. © 1997 John Wiley & Sons, Ltd.
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  • 104
    Electronic Resource
    Electronic Resource
    Characterization of the Checkpoint Gene RAD53/MEC2 in Saccharomyces cerevisiae (1997)
    New York, NY [u.a.] : Wiley-Blackwell
    Yeast 13 (1997), S. 735-745 
    Details
    ISSN: 0749-503X
    Keywords: cell cycle ; checkpoint ; DNA damage ; RAD53 ; protein kinase ; Saccharomyces cerevisiae ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: Saccharomyces cerevisiae cells carrying mutations in RAD53/MEC2 fail to arrest in the S phase when DNA replication is blocked (the S/M checkpoint) or in the G2 phase when DNA is damaged (the G2/M checkpoint). We isolated and determined the DNA sequence of RAD53 and found that it is identical to the SPK1 gene previously identified by Stern et al. (1991). In addition to its checkpoint functions, we show here that RAD53 is essential for cell viability because null mutants are inviable. Weak genomic suppressors of the essential function do arise frequently, though they do not suppress the checkpoint defects of the null mutant. This genetically separates the essential and checkpoint functions. We show genetically that the protein kinase domain is essential for all RAD53-dependent functions tested because a site-specific mutation that inactivates the protein kinase activity results in a mutant phenotype indistinguishable from that of a null mutant. Overexpression of RAD53, or its kinase domain alone, resulted in a delay in cell-cycle progression that required the intact kinase function. The cell-cycle delay did not require any of the checkpoint genes tested (e.g. rad9 or mec1), indicating that the cell-cycle delay is either unrelated to the checkpoint responses, or that it occurs constitutively because RAD53 acts further downstream of the checkpoint genes tested. Finally, elimination of sequences in the promoter region of RAD53 revealed complex regulatory elements. © 1997 John Wiley & Sons, Ltd.
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  • 105
    Electronic Resource
    Electronic Resource
    Cloning and Characterization of the KlDIM1 Gene from Kluyveromyces lactis Encoding the m26A Dimethylase of the 18S rRNA (1997)
    New York, NY [u.a.] : Wiley-Blackwell
    Yeast 13 (1997), S. 777-781 
    Details
    ISSN: 0749-503X
    Keywords: dimethylase ; ribosomal RNA ; Kluyveromcyes lactis ; kasugamycin ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: The KlDIM1 gene encoding the m26A rRNA dimethylase was cloned from a Kluyveromyces lactis genomic library using a PCR amplicon from the Saccharomyces cerevisiae ScDIM1 gene as probe. The KlDIM1 gene encodes a 320-amino acid protein which shows 81% identity to ScDim1p from S. cerevisiae and 25% identity to ksgAp from Escherichia coli. Complementation of the kasugamycin-resistant ksgA-mutant of E. coli lacking dimethylase activity demonstrates that KlDim1p is the functional homologue of the bacterial enzyme. Multiple alignment of dimethylases from prokaryotes and yeasts shows that the two yeast enzymes display distinctive structural motives including a putative nuclear localization signal. © 1997 John Wiley & Sons, Ltd.
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  • 106
    Electronic Resource
    Electronic Resource
    Cloning of the Multicopy Suppressor Gene SUR7: Evidence for a Functional Relationship between the Yeast Actin-binding Protein Rvs167 and a Putative Membranous Protein (1997)
    New York, NY [u.a.] : Wiley-Blackwell
    Yeast 13 (1997), S. 747-761 
    Details
    ISSN: 0749-503X
    Keywords: multicopy suppression ; RVS167 ; RVS161 ; actin cytoskeleton ; budding pattern ; membrane protein ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: The rvs161 and rvs167 mutant cells exhibit several identical phenotypes including sensitivity to several different growth conditions and morphological defects such as alteration of the actin cytoskeleton and budding patterns. The selection of genes that, when overexpressed, are able to suppress the reduced viability upon carbon starvation of the rvs167 mutant strain, has allowed the cloning of the SUR7 gene (Accession Number Z46729x11).We showed that the suppressive ability of the overexpressed SUR7 gene concerns all the rvs167 phenotypes. However, this suppression is only partial since the rvs167-suppressed strain is not of wild-type phenotype. Moreover, SUR7 is also able to suppress partially the phenotypes exhibited by the rvs161 and rvs167 rvs161 mutant strains.The SUR7 gene encodes a putative integral membrane protein with four transmembrane domains. Furthermore, sequence comparisons revealed that Sur7p and two other proteins, Ynl194p and Ydl222p, present significant sequence and structural similarities.Taken together, these results strongly suggest that the Rvs161 and Rvs167 proteins act together in relation with Sur7p. Moreover, the putative transmembranous character of Sur7p suggests a membrane localization of the Rvs function, a localization which is consistent with the different rvs phenotypes and the actin-Rvs167p interaction. © 1997 John Wiley & Sons, Ltd.
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  • 107
    Electronic Resource
    Electronic Resource
    Sequence Analysis of the 33 kb Long Region Between ORC5 and SUI1 from the Left Arm of Chromosome XIV from Saccharomyces cerevisiae (1997)
    New York, NY [u.a.] : Wiley-Blackwell
    Yeast 13 (1997), S. 849-860 
    Details
    ISSN: 0749-503X
    Keywords: Saccharomyces cerevisiae ; chromosome XIV ; ORF analysis ; gene deletion ; dihydropteroate biosynthesis ; cysteine-tRNA synthetase ; CCHC zinc finger protein ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: We have determined the nucleotide sequence of a chromosomal region of 33,016 bp located on the left arm of chromosome XIV from budding yeast between the ORC5 and the SUI1 gene. Subsequent sequence analysis revealed the presence of 18 non-overlapping open reading frames (ORFs) including eight previously identified and sequenced genes (ORC5, ATX1, SIP3, NRD1, RAD50, MPA43, RPA49 and SUI1). Three other ORFs (YNL256w, YNL255c and YNL247w) code for putative proteins with significant homology to proteins from other organisms, while 4 ORFs exhibit only weak homology to known proteins. Three ORFs have no homology with sequences in the databases. The sequences have been deposited in the EMBL database under Accession Number X96722. © 1997 John Wiley & Sons, Ltd.
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  • 108
    Electronic Resource
    Electronic Resource
    The Characterization of Two New Clusters of Duplicated Genes Suggests a ‘Lego’ Organization of the Yeast Saccharomyces cerevisiae Chromosomes (1997)
    New York, NY [u.a.] : Wiley-Blackwell
    Yeast 13 (1997), S. 861-869 
    Details
    ISSN: 0749-503X
    Keywords: Saccharomyces cerevisiae ; cluster ; duplication ; genome sequencing ; evolution ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: The systematic sequencing of 42 485 bp of yeast chromosome VII (nucleotides 377948 to 420432) has revealed the presence of 27 putative open reading frames (ORFs) coding for proteins of at least 100 amino acids. The degree of redundancy observed is elevated since five of the 27 ORFs are duplications of a previously identified gene. These duplicated copies may be classified in two types of cluster organization. The first type includes genes sharing a significant level of identity in the amino acid sequences of their predicted protein product. They are recovered on two different chromosomes, transcribed in the same orientation and the distance between them is conserved. The second type of cluster is based on one gene unit tandemly repeated. This duplication is itself repeated elsewhere in the genome. The level of nucleic acid identity is high within the coding sequence and the non-coding region between the two repeats. In addition, the basic gene unit is recovered many times in the genome and is a component of a multigene family of unknown function. These organizations in clusters of genes suggest a ‘Lego organization’ of the yeast chromosomes, as recently proposed for the genome of plants (Moore, 1995). The sequence is deposited in the Yeast Genome Databank under Accession Number from Z72562 to Z72586. © 1997 John Wiley & Sons, Ltd.
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  • 109
    Electronic Resource
    Electronic Resource
    Isolation and Characterization of the Candida albicans PFY1 Gene for Profilin (1997)
    New York, NY [u.a.] : Wiley-Blackwell
    Yeast 13 (1997), S. 871-880 
    Details
    ISSN: 0749-503X
    Keywords: Candida albicans ; profilin ; PFY1 ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: We have isolated the Candida albicans gene for profilin, PFY1. Degenerate oligonucleotide primers based on regions of high homology were utilized to obtain a polymerase chain reaction-amplified copy of the gene. This was then used as a probe to isolate the gene from a C. albicans genomic library. Our studies indicate that the full-length gene is unstable in Escherichia coli. Several clones were sequenced, and the predicted amino acid sequence demonstrated homology with profilin proteins from other organisms, most notably Saccharomyces cerevisiae. Northern analysis revealed that the gene is expressed in C. albicans. Attempts to express the gene in S. cerevisiae cells were unsuccessful until the C. albicans promoter was replaced with an S. cerevisiae promoter. Functional complementation of the gene was demonstrated in S. cerevisiae profilin-requiring cells. Antibodies raised to isolated C. albicans profilin protein recognized a protein of the predicted molecular weight when the gene was expressed in S. cerevisiae cells. The sequence of the C. albicans PFY1 gene has been deposited in the Genome Sequence database under Accession Number L3783. © 1997 John Wiley & Sons, Ltd.
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  • 110
    Electronic Resource
    Electronic Resource
    Characterization of CHS4 (CAL2), a Gene of Saccharomyces cerevisiae Involved in Chitin Biosynthesis and Allelic to SKT5 and CSD4 (1997)
    New York, NY [u.a.] : Wiley-Blackwell
    Yeast 13 (1997), S. 795-807 
    Details
    ISSN: 0749-503X
    Keywords: chitin synthase III ; morphogenesis ; Calcofluor resistance ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: We have cloned CHS4, a gene that complements the resistance to Calcofluor of the Saccharomyces cerevisiae cal2 mutant. We show that CHS4 is allelic to the previously described SKT5 and CSD4 genes. CHS4 encodes a 696 amino acids protein with no potential transmembrane domain. chs4-null mutants are resistant to Calcofluor white and exhibit a considerable reduction in cell wall chitin and in chitin synthase III (CSIII) activity. Biochemical characterization of chitin synthase III from these null mutants indicates that the defect is due to a reduced Vmax of the enzyme. This defect can be overcome in vitro by trypsin treatment of the membrane preparations. Chs4p does not act as a transcriptional or translational regulator of CHS3, the gene coding for the catalytic subunit of CSIII activity, and we therefore propose that Chs4p would be an essential component of the CSIII complex, acting as a post-translational regulator of this activity. In addition to the chitin defect, the chs4 mutant shows a severe defect in mating. © 1997 John Wiley & Sons, Ltd.
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  • 111
    Electronic Resource
    Electronic Resource
    Modulation of Glycerol and Ethanol Yields During Alcoholic Fermentation in Saccharomyces cerevisiae Strains Overexpressed or Disrupted for GPD1 Encoding Glycerol 3-Phosphate Dehydrogenase (1997)
    New York, NY [u.a.] : Wiley-Blackwell
    Yeast 13 (1997), S. 783-793 
    Details
    ISSN: 0749-503X
    Keywords: Saccharomyces cerevisiae ; alcoholic fermentation ; glycerol ; glycerol 3-phosphate dehydrogenase ; redox balance ; metabolic engineering ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: The possibility of the diversion of carbon flux from ethanol towards glycerol in Saccharomyces cerevisiae during alcoholic fermentation was investigated. Variations in the glycerol 3-phosphate dehydrogenase (GPDH) level and similar trends for alcohol dehydrogenase (ADH), pyruvate decarboxylase and glycerol-3-phosphatase were found when low and high glycerol-forming wine yeast strains were compared. GPDH is thus a limiting enzyme for glycerol production. Wine yeast strains with modulated GPD1 (encoding one of the two GPDH isoenzymes) expression were constructed and characterized during fermentation on glucose-rich medium. Engineered strains fermented glucose with a strongly modified [glycerol] : [ethanol] ratio. gpd1Δ mutants exhibited a 50% decrease in glycerol production and increased ethanol yield. Overexpression of GPD1 on synthetic must (200 g/l glucose) resulted in a substantial increase in glycerol production (×4) at the expense of ethanol. Acetaldehyde accumulated through the competitive regeneration of NADH via GPDH. Accumulation of by-products such as pyruvate, acetate, acetoin, 2,3 butane-diol and succinate was observed, with a marked increase in acetoin production. © 1997 John Wiley & Sons, Ltd.
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  • 112
    Electronic Resource
    Electronic Resource
    Characterization of Two New Genes Down-regulated by α-factor (1997)
    New York, NY [u.a.] : Wiley-Blackwell
    Yeast 13 (1997), S. 809-817 
    Details
    ISSN: 0749-503X
    Keywords: Saccharomyces cerevisiae ; pherome ; cell-type regulation ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: To detect genes directly down-regulated by α-factor, 55 000 plaque-forming units of a Saccharomyces cerevisiae λgt10 gene bank were differentially screened with cDNA of cells treated with α-factor for 20 min. Two new genes were detected in this way, called α0·5 and α0·6. The former is transiently down-regulated by α-factor; it is very highly transcribed in late exponential-phase cells. The gene, located on the right arm of chromosome XIII, codes for a 59 amino-acid protein with a signal peptide. The protein has been shown with an antibody to be present in the membrane fraction. The gene has also been cloned as HOR7 (hyperosmolarity-responsive protein; Hirayama et al., 1995). No other homologous sequences have been detected in the yeast genome. α0·6, located on the right arm of chromosome XII, corresponds to the open reading frame YLR110c; it codes for a 133 amino-acid protein containing a signal peptide. Its derived amino-acid sequence is homologous to the N-terminal half of the SED1 gene product. SED1, when overexpressed, is able to suppress a defect in the HDEL receptor coded for by the ERD2 gene (Hardwick and Pelham, 1994); however, α0·6 is not able to do so. The disruption of α0·5 or α0·6 does not lead to a special phenotype. © 1997 John Wiley & Sons, Ltd.
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  • 113
    Electronic Resource
    Electronic Resource
    Cloning, Sequencing and Expression of a Full-Length cDNA Copy of the M1 Double-Stranded RNA Virus from the Yeast, Saccharomyces cerevisiae (1997)
    New York, NY [u.a.] : Wiley-Blackwell
    Yeast 13 (1997), S. 829-836 
    Details
    ISSN: 0749-503X
    Keywords: Saccharomyces cerevisiae ; killer yeast ; killer virus ; M1 dsRNA virus ; M1 cDNA clone ; DNA sequence ; killer gene expression ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: Strains of the budding yeast, Saccharomyces cerevisiae, may contain one or more cytoplasmic viruses with double-stranded RNA (dsRNA) genomes. The killer phenomenon in yeast, in which one cell secretes a killer toxin that is lethal to another cell, is dependent upon the presence of the L-A and M1 dsRNA viruses. The L-A viral genome encodes proteins for the viral capsid, and for synthesis and encapsidation of single-stranded RNA replication cycle intermediates. The M1 virus depends upon the L-A-encoded proteins for its capsid and for the replication of its killer-toxin-encoding genome. A full-length cDNA clone of an M1 genome has been made from a single dsRNA molecule and shown to encode functional killer and killer-immunity functions. The sequence of the clone indicates minor differences from previously published sequences of parts of the M1 genome and of the complete genome of S14 (an internal deletion derivative of M1) but no unreported amino acid variants and no changes in putative secondary structures of the single-stranded RNA. A 118-nucleotide contiguous segment of the M1 genome has not previously been reported; 92 of those nucleotides comprise a segment of A nucleotides in the AU-rich bubble that follows the toxin-encoding reading frame. The GenBank Accession Number for the sequence is U78817; the locus is SCU78817. © 1997 John Wiley & Sons, Ltd.
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  • 114
    Electronic Resource
    Electronic Resource
    Isolation of Three Contiguous Genes, ACR1, ACR2 and ACR3, Involved in Resistance to Arsenic Compounds in the Yeast Saccharomyces cerevisiae (1997)
    New York, NY [u.a.] : Wiley-Blackwell
    Yeast 13 (1997), S. 819-828 
    Details
    ISSN: 0749-503X
    Keywords: Saccharomyces cerevisiae ; gene cloning ; chromosome XVI ; arsenic compounds ; drug resistance ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: A 4·2 kb region from Saccharomyces cerevisiae chromosome XVI was isolated as a yeast fragment conferring resistance to 7 mM-sodium arsenite (NaAsO2), when put on a multicopy plasmid. Homology searches revealed a cluster of three new open reading frames named ACR1, ACR2 and ACR3. The hypothetical product of the ACR1 gene is similar to the transcriptional regulatory proteins, encoded by YAP1, and YAP2 genes from S. cerevisiae. Disruption of the ACR1 gene conduces to an arsenite and arsenate hypersensitivity phenotype. The ACR2 gene is indispensable for arsenate but not for arsenite resistance. The hypothetical product of the ACR3 gene shows high similarity to the hypothetical membrane protein encoded by Bacillus subtilis ORF1 of the skin element and weak similarity to the ArsB membrane protein of the Staphylococcus aureus arsenical-resistance operon. Overexpression of the ACR3 gene confers an arsenite- but not an arsenate-resistance phenotype. The presence of ACR3 together with ACR2 on a multicopy plasmid expands the resistance phenotype into arsenate. These findings suggest that all three novel genes: ACR1, ACR2 and ACR3 are involved in the arsenical-resistance phenomenon in S. cerevisiae. © 1997 John Wiley & Sons, Ltd.
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  • 115
    Electronic Resource
    Electronic Resource
    Obituary: In Memory of Michael Ciriacy (1997)
    New York, NY [u.a.] : Wiley-Blackwell
    Yeast 13 (1997), S. 881-882 
    Details
    ISSN: 0749-503X
    Keywords: Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: No Abstract
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  • 116
    Electronic Resource
    Electronic Resource
    A Set of Vectors with a Tetracycline-Regulatable Promoter System for Modulated Gene Expression in Saccharomyces cerevisiae (1997)
    New York, NY [u.a.] : Wiley-Blackwell
    Yeast 13 (1997), S. 837-848 
    Details
    ISSN: 0749-503X
    Keywords: Saccharomyces cerevisiae ; expression vectors ; transcriptional activator ; tetracycline-regulatable promoter ; function analysis ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: A set of Saccharomyces cerevisiae expression vectors has been developed in which transcription is driven by a hybrid tetO-CYC1 promoter through the action of a tetR-VP16 (tTA) activator. Expression from the promoter is regulated by tetracycline or derivatives. Various modalities of promoter and activator are used in order to achieve different levels of maximal expression. In the presence of antibiotic in the growth medium at concentrations that do not affect cell growth, expression from the tetO promoter is negligible, and upon antibiotic removal induction ratios of up to 1000-fold are observed with a lacZ reporter system. With the strongest system, overexpression levels comparable with those observed with GAL1-driven promoters are reached. For each particular promoter/tTA combination, expression can be modulated by changing the tetracycline concentration in the growth medium. These vectors may be useful for the study of the function of essential genes in yeast, as well as for phenotypic analysis of genes in overexpression conditions, without restrictions imposed by growth medium composition. © 1997 by John Wiley & Sons, Ltd.
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  • 117
    Electronic Resource
    Electronic Resource
    Stationary-Phase Gene Expression in Saccharomyces cerevisiae During Wine Fermentation (1997)
    New York, NY [u.a.] : Wiley-Blackwell
    Yeast 13 (1997), S. 903-915 
    Details
    ISSN: 0749-503X
    Keywords: wine yeasts ; alcoholic fermentation ; stationary phase ; nitrogen limitation ; Northern analysis ; heat-shock promoter ; HSP30::lacZ fusion ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: Genetic engineering of wine yeast strains requires the identification of gene promoters specifically activated under wine processing conditions. In this study, transcriptional activation of specific genes was followed during the time course of wine fermentation by quantifying mRNA levels in a haploid wine strain of Saccharomyces cerevisiae grown on synthetic or natural winery musts. Northern analyses were performed using radioactive probes from 19 genes previously described as being expressed under laboratory growth conditions or on molasses in S. cerevisiae during the stationary phase and/or under nitrogen starvation. Nine genes, including members of the HSP family, showed a transition-phase induction profile. For three of them, mRNA transcripts could be detected until the end of the fermentation. Expression of one of these genes, HSP30, was further studied using a HSP30::lacZ fusion on both multicopy and monocopy expression vectors. The production of β-galactosidase by recombinant cells was measured during cell growth and fermentation on synthetic and natural winery musts. We showed that the HSP30 promoter can induce high gene expression during late stationary phase and remains active until the end of the wine fermentation process. Similar expression profiles were obtained on five natural winery musts. © 1997 John Wiley & Sons, Ltd.
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  • 118
    Electronic Resource
    Electronic Resource
    Identification of Genes with Nutrient-controlled Expression by PCR-mapping in the Yeast Saccharomyces cerevisiae (1997)
    New York, NY [u.a.] : Wiley-Blackwell
    Yeast 13 (1997), S. 973-984 
    Details
    ISSN: 0749-503X
    Keywords: differential display ; gene expression ; nutritional control ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: We have used RNA fingerprinting by the mRNA Differential Display technique to identify new genes in the yeast Saccharomyces cerevisiae, expression of which is controlled by specific nutrient conditions. mRNA was isolated from cells grown on glucose medium into exponential and stationary phase, and from cells starved for nitrogen on glucose-containing medium. To avoid interference with the large number of glucose-repressible genes, a glucose-repression-deficient strain was used. Twenty different sets of arbitrary primers chosen at random were used for PCR-amplification of reverse transcriptase generated cDNAs, which resulted in six highly reproducible gene expression patterns. The validity of the approach was confirmed by sequencing PCR products of genes with known expression patterns, SUP44/RPS4, CTT1, SSA3, HSP30 and HSP104, and genes with related functions, TEF1 and TEF3, encoding translation elongation factors. In all cases the specificity of the responses was confirmed by Northern blot analysis. The results show that the PCR-mapping method is highly useful for the identification of new genes expressed under specific conditions in the yeast S. cerevisiae © 1997 John Wiley & Sons, Ltd.
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  • 119
    Electronic Resource
    Electronic Resource
    GCR1-Dependent Transcriptional Activation of Yeast Retrotransposon Ty2-917 (1997)
    New York, NY [u.a.] : Wiley-Blackwell
    Yeast 13 (1997), S. 917-930 
    Details
    ISSN: 0749-503X
    Keywords: yeast retrotransposon ; GCR1 ; transcription ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: Transcription of Saccharomyces cerevisiae Ty2-917 retrotransposon depends on regulatory elements both upstream and downstream of the transcription initiation site. An upstream activation sequence (UAS) and a downstream enhancer stimulate transcription synergistically. Here we show that activation by both of these sites depends on the GCR1 product, a transcription factor which also regulates the genes encoding yeast glycolytic enzymes. Eliminating GCR1 causes a 100-fold decrease in transcription of Ty2-917. Activation by the isolated Ty2-917 UAS also strongly depends on GCR1. Unexpectedly, GCR1-dependent activation by the Ty2-917 enhancer is strongly position-dependent. Activation by the enhancer in its normal position within the transcription unit depended strongly on GCR1, but eliminating GCR1 reduced activation only three-fold when the enhancer was moved upstream of the transcribed region. Gel mobility shift and DNaseI protection assays indicated that GCR1 binds specifically to multiple sites within the Ty2-917 UAS and enhancer regions. © 1997 John Wiley & Sons, Ltd.
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    The Saccharomyces cerevisiae MFS Superfamily SGE1 Gene Confers Resistance to Cationic Dyes (1997)
    New York, NY [u.a.] : Wiley-Blackwell
    Yeast 13 (1997), S. 891-902 
    Details
    ISSN: 0749-503X
    Keywords: yeast ; Sacchromyces cerevisiae ; PDR ; MFS ; 10-N-nonyl acridine orange ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: A gene from Saccharomyces cerevisiae whose overexpression confers resistance to 10-N-nonyl acridine orange (NAO) has been isolated. This cationic dye binds acidic phospholipids and more specifically cardiolipin (Petit, J. M., Maftah, A., Ratinaud, M. H. and Julien, R. Eur. J. Biochem. 209, 267-273, 1992). The isolated gene was found to be identical to SGE1, a partial multicopy suppressor of the gal11 mutation (Amakasu, H., Suzuki, Y., Nishizawa, M. and Fukasawa, T. Genetics 134, 675-683, 1993), that also confers crystal violet resistance to a supersensitive strain (Ehrenhofer-Murray, A. E., Wurgler, F. E. and Sengstag, C. Mol. Gen. Genet. 244, 287-294, 1994). The data presented in this paper show that the SGE1 gene product, a member of the major facilitator superfamily, confers a pleiotropic drug-resistance phenotype when present in high copy number. The results also demonstrate that Sge1p acts as an extrusion permease whose specificity seems restricted to dye molecules possessing a large unsaturated domain that stabilizes a permanent positive charge such as NAO, crystal violet, ethidium bromide or malachite green. © 1997 John Wiley & Sons, Ltd.
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    Phylogenetic Relationships of Fungal Cytochromes c (1997)
    New York, NY [u.a.] : Wiley-Blackwell
    Yeast 13 (1997), S. 985-990 
    Details
    ISSN: 0749-503X
    Keywords: cytochrome c ; evolution ; phylogenetics ; Saccharomyces cerevisiae ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: The CYC1 gene encoding cytochrome c in the yeast Candida albicans was cloned by complementation of a cytochrome c-deficient mutant of Saccharomyces cerevisiae, and its DNA sequence was determined. The analysis of the amino acid sequences of cytochrome c from 14 fungal species and two isoforms from S. cerevisiae revealed sequences unique to fungi, and revealed a phylogenetic relationship with a pronounced divergence between Schizosaccharomyces pombe and other ascomycetous budding yeast. The C. albicans CYC1 cytochrome c sequence has been assigned Accession Number U57896 in the GenBank/EMBL database. © 1997 John Wiley & Sons, Ltd.
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    Isolation and Characterization of the KlHEM1 Gene in Kluyveromyces lactis (1997)
    New York, NY [u.a.] : Wiley-Blackwell
    Yeast 13 (1997), S. 961-971 
    Details
    ISSN: 0749-503X
    Keywords: Kluyveromyces lactis ; HEM1 ; 5-aminolevulinate synthase ; transcription regulation ; heme-responsive element ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: The KlHEM1 gene from Kluyveromyces lactis encodes a functional 5-aminolevulinate synthase (δALA synthase), as confirmed by complementation of a hem1 mutant Saccharomyces cerevisiae strain, homology search, and detection of a 2·3 kb transcript. The gene is highly homologous to the ScHEM1 gene, and the sequence of the promoter region contains a complex combination of putative regulatory signals. Some of them are related to phospholipid biosynthesis, glycolytic metabolism, and regulation by carbon source. Transcription of KlHEM1 increased significantly in response to limited oxygen, and only slightly with the change from repressed (glucose) to derepressed conditions (glycerol). The δALA synthase from K. lactis contains, in the amino-terminal region, two heme-responsive elements that are not present in the protein from Saccharomyces cerevisiae. The complete nucleotide sequence has been entered in the EMBL data library under Accession Number X92944. © 1997 John Wiley & Sons, Ltd.
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    Vacuole Segregation in the Saccharomyces cerevisiae vac2-1 Mutant: Structural and Biochemical Quantification of the Segregation Defect and Formation of New Vacuoles (1997)
    New York, NY [u.a.] : Wiley-Blackwell
    Yeast 13 (1997), S. 999-1008 
    Details
    ISSN: 0749-503X
    Keywords: Saccharomyces cerevisiae ; vacuole biogenesis ; phenotypic lag ; optical trapping ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: The conditional vacuolar segregation mutant vac2-1 [Shaw and Wickner (1991) EMBO J. 10,1741-1748] shifted to non-permissive temperature (37°C), forms large-budded cells without a vacuole in the bud, and daughter cells without an apparent vacuole. Some cells still contain normal segregation structures. Structural and biochemical quantification of the segregation defect showed that (i) about 10% of the full-grown buds did not contain a vacuole, (ii) about 15% of the small cells washed out of a population growing in an elutriation chamber at 37°C, did not contain a visible vacuole, and (iii) 15% of the cells per generation lost carboxypeptidase Y activity after proteinase A depletion. Thus, 10-15% of the daughter cells did not inherit vacuolar structures or vacuolar proteolytic activity from the mother cell. To investigate the fate of vacuole-less daughters, these cells were isolated by optical trapping. The isolated cells formed colonies on agar plates that consisted of cells with normal vacuoles, both at 23 and 37°C. Thus, the vacuole-less cells that failed to inherit proteolytic activities from the mother cell apparently give rise to progeny containing structurally normal vacuoles. Time-lapse experiments showed that vacuole-less daughter cells formed vacuolar vesicles that fused into a new vacuole within 30 min. Although new buds only emerged after a vacuole had formed in the mother cell, the temporary lack of a vacuole had little effect on growth rate. The results suggest that an alternative pathway for vacuole formation exists, and that yeast cells may require a vacuole of some minimal size to initiate a new round of budding. © 1997 by John Wiley & Sons, Ltd.
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    Two New S-Phase-Specific Genes from Saccharomyces cerevisiae (1997)
    New York, NY [u.a.] : Wiley-Blackwell
    Yeast 13 (1997), S. 1029-1042 
    Details
    ISSN: 0749-503X
    Keywords: S phase ; silencing ; telomere ; ASF ; SIR3 ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: Two new yeast genes, ASF1 (Anti-Silencing Function) and ASF2, as well as a C-terminal fragment of SIR3, were identified as genes that derepressed the silent mating type loci when overexpressed. ASF2 overexpression caused a greater derepression than did ASF1. ASF1 overexpression also weakened repression of genes near telomeres, but, interestingly, ASF2 had no effect on telomeric silencing. Sequences of these two genes revealed open reading frames of 279 and 525 amino acids for ASF1 and ASF2, respectively. The ASF1 protein was evolutionarily conserved. MCB motifs, sequences commonly present upstream of genes transcribed specifically in S phase, were found in front of both genes, and, indeed, both genes were transcribed specifically in the S phase of the cell cycle. While an asf2 mutant was viable and had no obvious phenotypes, an asf1 mutant grew poorly. Neither mutant exhibited derepression of the silent mating type loci. The asf1 mutant was sensitive to methyl methane sulfonate, slightly UV-sensitive and somewhat deficient in minichromosome maintenance. It also lowered the restrictive temperature of a cdc13ts mutant. These phenotypes suggested a role for ASF1 in DNA repair and chromosome maintenance. The GenBank accession numbers for the ASF1 and ASF2 sequences are L07593 and L07649, respectively. © 1997 by John Wiley & Sons, Ltd.
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    Tandemly Repeated 147 bp Elements Cause Structural and Functional Variation in Divergent MAL Promoters of Saccharomyces cerevisiae (1997)
    New York, NY [u.a.] : Wiley-Blackwell
    Yeast 13 (1997), S. 1135-1144 
    Details
    ISSN: 0749-503X
    Keywords: yeast ; MAL6 ; divergent promoter ; repeats ; nucleosomes ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: We have studied four novel MAL promoters isolated from a single strain of bakers' yeast. Within these promoters we have identified up to five tandem 147 bp repeats located between the MAL UAS region and the MALT TATA box. These repeats strongly reduce MALT (maltose permease) gene expression but only weakly reduce MALS (maltase) gene expression. Insertion of the 147 bp elements into the heterologous CYC1 promoter reduced expression when located between the CYC1 UAS and the TATA box, but not when located upstream of the UAS. We propose that these naturally occurring repeats have evolved as a mechanism to lower the level of MALT expression relative to MALS expression, thus avoiding possible toxic effects associated with over-expression from multiple copies of the permease gene. Accession numbers are: WIG1, U86359; WIG3, U86360; WIG4, U86361; WIG5, U86362. © 1997 by John Wiley & Sons, Ltd.
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    Heterologous HIS3 Marker and GFP Reporter Modules for PCR-Targeting in Saccharomyces cerevisiae (1997)
    New York, NY [u.a.] : Wiley-Blackwell
    Yeast 13 (1997), S. 1065-1075 
    Details
    ISSN: 0749-503X
    Keywords: PCR-targeting ; pFA plasmids ; HIS3 heterologous auxotrophic marker ; geneticin ; green fluorescence protein ; kanMX ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: We have fused the open reading frames of his3-complementing genes from Saccharomyces kluyveri and Schizosaccharomyces pombe to the strong TEF gene promotor of the filamentous fungus Ashbya gossypii. Both chimeric modules and the cognate S. kluyveri HIS3 gene were tested in transformations of his3 S. cerevisiae strains using PCR fragments flanked by 40 bp target guide sequences. The 1·4 kb chimeric Sz. pombe module (HIS3MX6) performed best. With less than 5% incorrectly targeted transformants, it functions as reliably as the widely used geniticin resistance marker kanMX. The rare false-positive His+ transformants seem to be due to non-homologous recombination rather than to gene conversion of the mutated endogenous his3 allele. We also cloned the green fluorescent protein gene from Aequorea victoria into our pFA-plasmids with HIS3MX6 and kanMX markers. The 0·9 kb GFP reporters consist of wild-type GFP or GFP-S65T coding sequences, lacking the ATG, fused to the S. cerevisiae ADH1 terminator. PCR-synthesized 2·4 kb-long double modules flanked by 40-45 bp-long guide sequences were successfully targeted to the carboxy-terminus of a number of S. cerevisiae genes. We could estimate that only about 10% of the transformants carried inactivating mutations in the GFP reporter. © 1997 by John Wiley & Sons, Ltd.
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    Impairment of Peroxisome Degradation in Pichia methanolica Mutants Defective in Acetyl-CoA Synthetase or Isocitrate Lyase (1997)
    New York, NY [u.a.] : Wiley-Blackwell
    Yeast 13 (1997), S. 1043-1052 
    Details
    ISSN: 0749-503X
    Keywords: peroxisome ; autophagic degradation ; ethanol metabolism ; methyltrophic yeast ; Pichia methanolica ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: Single recessive mutations of the methylotrophic yeast Pichia methanolica acs1, acs2, acs3 and icl1 affecting acetyl-CoA synthetase and isocitrate lyase, and growth on ethanol as sole carbon and energy source, caused a defect in autophagic peroxisome degradation during exposure of methanol-grown cells to ethanol. As a control, a mutation in mdd1, which resulted in a defect of the ‘malic’ enzyme and also prevented ethanol utilization, did not prevent peroxisome degradation. Peroxisome degradation in glucose medium was unimpaired in all strains tested. Addition of ethanol to methanol-grown cells of acs1, acs2, acs3 and icl1 mutants led to an increase in average vacuole size. Thickening of peroxisomal membranes and tight contacts between groups of peroxisomes and vacuoles were rarely observed. These processes proceeded much more slowly than in wild-type or mdd1 mutant cells incubated under similar conditions. No peroxisomal remnants were observed inside vacuoles in the cells of acs1, acs2, acs3 and icl1 mutants after prolonged cultivation in ethanol medium. We hypothesize that the acs and icl mutants are defective in synthesis of the true effector - presumably glyoxylate - of peroxisome degradation in ethanol medium. Lack of the effector suspends peroxisome degradation at an early stage, namely signal transduction or peroxisome/vacuole recognition. Finally, these defects in peroxisome degradation resulted in mutant cells retaining high levels of alcohol oxidase which further led to increased levels of acetaldehyde accumulation upon incubation of mutant cells with ethanol. © 1997 by John Wiley & Sons, Ltd.
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    Advancement through Mitosis requires rae1 Gene Function in Fission Yeast (1997)
    New York, NY [u.a.] : Wiley-Blackwell
    Yeast 13 (1997), S. 1167-1179 
    Details
    ISSN: 0749-503X
    Keywords: Schizosaccharomyces pombe ; rae1-1 mutant ; mitosis ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: Growth of the rae1-1 mutant of Schizosaccharomyces pombe at restrictive temperature results in accumulation of poly(A)+ RNA in the nucleus and a cell cycle arrest at the G2/M boundary. We demonstrate here that rae1 function is required for a process other than mRNA export which is essential for advancement through mitosis. Cells lacking rae1 function arrest with elevated Cdc2p kinase levels at a step before the formation of a mitotic spindle and without separation of the spindle pole bodies. Rae1p was localized to the nuclear periphery, consistent with a role in nucleocytoplasmic trafficking, which could include protein import. We propose a model where rae1 functions in cell cycle progression through trafficking of proteins required for mitosis. © 1997 John Wiley & Sons, Ltd.
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    Ribosomal Protein L9 is the Product of GRC5, a Homolog of the Putative Tumor Suppressor QM in S. cerevisiae (1997)
    New York, NY [u.a.] : Wiley-Blackwell
    Yeast 13 (1997), S. 1155-1166 
    Details
    ISSN: 0749-503X
    Keywords: rpL9 ; QM homolog ; yeast ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: Genes encoding members of the highly conserved QM family have been identified in eukaryotic organisms from yeast to man. Results of previous studies have suggested roles for QM in control of cell growth and proliferation, perhaps as a tumor suppressor, and in energy metabolism. We identified recessive lethal alleles of the Saccharomyces cerevisiae QM homolog GRC5 that increased GCN4 expression when present in multiple copies. These alleles encode truncated forms of the yeast QM protein Grc5p. Using a functional epitope-tagged GRC5 allele, we localized Grc5p to a 60S fraction that contained the large ribosomal subunit. Two-dimensional gel analysis of highly purified yeast ribosomes indicated that Grc5p corresponds to 60S ribosomal protein L9. This identification is consistent with the predicted physical characteristics of eukaryotic QM proteins, the highly biased codon usage of GRC5, and the presence of putative Rap1p-binding sites in the 5′ sequences of the yeast GRC5 gene. © 1997 John Wiley & Sons, Ltd.
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    A Review of Phenotypes in Saccharomyces cerevisiae (1997)
    New York, NY [u.a.] : Wiley-Blackwell
    Yeast 13 (1997), S. 1099-1133 
    Details
    ISSN: 0749-503X
    Keywords: Saccharomyces cerevisiae ; functional analysis ; phenotypic screening ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: A summary of previously defined phenotypes in the yeast Saccharomyces cerevisae is presented. The purpose of this review is to provide a compendium of phenotypes that can be readily screened to identify pleiotropic phenotypes associated with primary or suppressor mutations. Many of these phenotypes provide a convenient alternative to the primary phenotype for following a gene, or as a marker for cloning a gene by genetic complementation. In many cases a particular phenotype or set of phenotypes can suggest a function for the product of the mutated gene. © 1997 John Wiley & Sons, Ltd.
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    Inducible Membranes in Yeast: Relation to the Unfolded-Protein-Response Pathway (1997)
    New York, NY [u.a.] : Wiley-Blackwell
    Yeast 13 (1997), S. 1211-1229 
    Details
    ISSN: 0749-503X
    Keywords: endoplasmic reticulum ; IRE1 ; KAR2 ; phospholipids ; yeast ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: Overproduction of an endoplasmic reticulum (ER)-resident membrane protein (cytochrome P450 52A3) and of a secretory protein (invertase) was used to study the regulation of the luminal ER protein Kar2p under conditions that lead to ER proliferation and secretory overload, respectively. In both cases we found (i) a significant increase of Kar2 protein and mRNA levels, (ii) a transcriptional regulation based on the function of the 22 bp unfolded-protein-response element of the KAR2 promoter and (iii) an essential role of the transmembrane kinase Ire1p for upregulation of KAR2 gene expression. These results show that the same mechanism operates when KAR2 induction is triggered by overproduction of cytochrome P450 or invertase and that this mechanism shares the known features of the unfolded-protein-response pathway. Disruption of the IRE1 gene resulted in a marked decrease of the invertase protein levels produced. In contrast, a functional IRE1 gene was not required to reach high-level production of the integral membrane protein cytochrome P450 52A3. Moreover, IRE1 gene disruption did not prevent P450-induced ER proliferation. We suggest that Ire1p-mediated KAR2 induction is, in the case of cytochrome P450 52A3 overproduction, a process which follows on ER proliferation, thereby monitoring the increase of ER size and adjusting the level of Kar2p accordingly. © 1997 John Wiley & Sons, Ltd.
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    A rapid and reliable method for metabolite extraction in yeast using boiling buffered ethanol (1997)
    New York, NY [u.a.] : Wiley-Blackwell
    Yeast 13 (1997), S. 1347-1355 
    Details
    ISSN: 0749-503X
    Keywords: yeast metabolism ; metabolite extraction ; metabolic engineering ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: A simple and reliable method for the efficient inactivation of metabolism and for quantitative metabolite extraction from yeast cells is presented. It is based on the use of a boiling solution made of 75% ethanol (volume/final volume) buffered with 70 mm-Hepes (final concentration), pH 7·5, to guarantee the stability throughout the whole procedure of a large variety of metabolites, including all glycolytic intermediates, nucleotides, pyridine nucleotides and organic acids compounds. The extraction is fast, requiring only 3 min incubation of yeast cells in the ethanol-buffered mixture maintained at 80°C. It can be carried out either directly by spraying the cells into the boiling mixture, or after quenching the whole culture in 60% methanol kept at -40°C. Extracts are subsequently concentrated by evaporation under partial vacuum and the residue is resuspended in a small volume of water. This concentration step and the use of a highly sensitive analytical method allow us to quantify metabolites in less than 10 mg dry weight cells. This method, which can be applied to other fungi, could be very helpful for the determination of true metabolites in mutants generated through the EUROFAN programme and for metabolic flux analysis. © 1997 John Wiley & Sons, Ltd.
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    The homeodomain protein Pho2p binds at an A/T-rich segment flanking the binding site of the basic-helix-loop-helix protein Pho4p in the yeast PHO promoters (1997)
    New York, NY [u.a.] : Wiley-Blackwell
    Yeast 13 (1997), S. 1299-1308 
    Details
    ISSN: 0749-503X
    Keywords: Saccharomyces cerevisiae ; adenine/thymine-rich segment ; PHO regulon ; Pho2p ; PHO promoter ; upstream activation site (UAS) ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: Transcription of the genomic PHO5, PHO81 and PHO84 genes of the PHO regulon requires Pho4p and Pho2p activity, whereas transcription of PHO8 is directed by Pho4p alone. Pho4p binds to two 9-bp motifs, 5′-GCACGTGGG-3′ (type 1, e.g. UASp2 of PHO5 and site D of PHO84) and 5′-GCACGTTTT-3′ (type 2, e.g. UASp1 of PHO5 and site E of PHO84) in the PHO promoter. Experiments were performed to evaluate the ability of these 9-bp motifs to function as upstream activation sites (UASs) by insertion of various 36-bp fragments bearing the 9-bp motif in a CYC1-lacZ fusion gene. No expression of the lacZ gene was detected with the 36-bp fragment bearing UASp2 of PHO5, whereas similar 36-bp fragments bearing UASp1 of PHO5 and sites D and E of PHO84 showed UAS activity in response to Pi concentration in the medium and to the pho2 mutation. The Pho2p-responsive UASs are flanked by one or two copies of an A/T-rich segment, whereas UASp2 is not. Gel retardation and competition experiments performed using a T7-Pho2p-His chimeric protein showed that Pho2p binds to the 36-bp fragments bearing A/T-rich segment(s) but not appreciably to the 36-bp fragments not bearing such segment(s). Thus, the A/T segments flanking the PHO UASs are Pho2p binding sites and play an important role in PHO regulation. © 1997 John Wiley & Sons, Ltd.
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    Purification and characterization of phosphofructokinase from the yeast Kluyveromyces lactis (1997)
    New York, NY [u.a.] : Wiley-Blackwell
    Yeast 13 (1997), S. 1309-1317 
    Details
    ISSN: 0749-503X
    Keywords: phosphofructokinase ; protein purification ; molecular and kinetic properties ; Kluyveromyces lactis ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: Phosphofructokinase from Kluyveromyces lactis was purified by 180-fold enrichment, elaborating the following steps: cell disruption, polyethylene glycol precipitation, affinity chromatography, size exclusion chromatography on Sepharose 6B and on Bio-Sil SEC 400 and ion exchange chromatography. The homogeneous enzyme exhibits a molecular mass of 845±20 kDa as determined by sedimentation equilibrium measurements and a specific activity of 100 units/mg protein. The apparent sedimentation coefficient was found to be s20,C=20·7±0·6 S and no significant dependence on the protein concentration was observed in a range from 0·2 to 8 mg protein/ml. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed two bands corresponding to molecular masses of 119±5 kDa and 102±5 kDa, respectively. Thus, the enzyme assembles as octamer composed of two types of subunits. From Western blot analysis applying subunit-specific monoclonal antibodies raised against Saccharomyces cerevisiae phosphofructokinase and from the determination of the N-terminal amino acid sequence, the conclusion was drawn that the 102 kDa-subunit corresponds to the β-subunit of the S. cerevisiae enzyme. In contrast to bakers' yeast phosphofructokinase, the K. lactis enzyme exhibits no cooperativity with respect to the substrate fructose 6-phosphate. Both activators AMP and fructose 2,6-bisphosphate decrease the Michaelis constant with respect to this substrate. The enzyme from K. lactis is also inhibited by ATP. Fructose 2,6-bisphosphate or AMP diminish the ATP-inhibition. In contrast to the phosphofructokinase from S. cerevisiae, where fructose 2,6-bisphosphate turned out to be more efficient than AMP, both activators exert similar effects on the K. lactis enzyme. © 1997 John Wiley & Sons, Ltd.
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    Measurement of nuclear DNA content in fission yeast by flow cytometry (1997)
    New York, NY [u.a.] : Wiley-Blackwell
    Yeast 13 (1997), S. 1329-1335 
    Details
    ISSN: 0749-503X
    Keywords: cdc mutant ; DNA content ; fission yeast ; flow cytometry ; non-nuclear staining ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: Cell division cycle (cdc) mutants of Schizosaccharomyces pombe are arrested at specific points in the cell cycle when grown at restrictive temperature. Flow cytometry of such cells reveals an anomalous increase in the DNA fluorescence signal, which represents a problem in experiments designed to determine the cell cycle arrest point. The increased fluorescence signal is due to cytoplasmic constituents and has been attributed to mitochondrial DNA synthesis (S. Sazer and S. W. Sherwood, J. Cell Sci.97: 509-516, 1990). Here we have studied the cdc10 mutant by flow cytometry using different DNA-binding fluorochromes and found no evidence that the increased fluorescence signal was caused by mitochondrial DNA synthesis. To determine more accurately the nuclear DNA content we have developed a novel method to remove most of the cytoplasmic material by exposing the cells to Triton X-100 and hypotonic conditions after cell wall digestion. The DNA fluorescence from cells treated in this way was more constant with time of incubation at restrictive temperature in spite of a considerable increase in cell size. With this method we could determine that the recently isolated temperature sensitive orp1 mutant is arrested with a 1C DNA content. Premature and abnormal mitosis (‘cut’) could be observed for the orp1 mutant after only 4 h at restrictive temperature. © 1997 John Wiley & Sons, Ltd.
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    Lys80p of Saccharomyces cerevisiae, previously proposed as a specific repressor of LYS genes, is a pleiotropic regulatory factor identical to Mks1p (1997)
    New York, NY [u.a.] : Wiley-Blackwell
    Yeast 13 (1997), S. 1337-1346 
    Details
    ISSN: 0749-503X
    Keywords: Saccharomyces cerevisiae ; LYS80gene ; α-ketoglutarate ; apparent repression ; pleiotropic factor ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: In Saccharomyces cerevisiae, an intermediate of the lysine pathway, α-aminoadipate semialdehyde (αAASA), acts as a coinducer for the transcriptional activation of LYS genes by Lys14p. The limitation of the production of this intermediate through feedback inhibition of the first step of the pathway results in apparent repression by lysine. Previously, the lys80 mutations, reducing the lysine repression and increasing the production of lysine, were interpreted as impairing a repressor of LYS genes expression. In order to understand the role of Lys80p in the control of the lysine pathway, we have analysed the effects of mutations epistatic to lys80 mutations. The effects of lys80 mutations on LYS genes expression were dependent on the integrity of the activation system (Lys14p and αAASA). The increased production of lysine in lys80 mutants appeared to result from an improvement of the metabolic flux through the pathway and was correlated to an increase of the α-ketoglutarate pool and of the level of several enzymes of the tricarboxylic acid cycle. The LYS80 genes has been cloned and sequenced; it turned out to be identical to gene MKS1 cloned as a gene encoding a negative regulator of the RAS-cAMP pathway. We conclude that Lys80p is a pleiotropic regulatory factor rather than a specific repressor of LYS genes. © 1997 John Wiley & Sons, Ltd.
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    Saccharomyces carlsbergensis contains two functional genes encoding the Acyl-CoA binding protein, one similar to the ACB1 gene from S. cerevisiae and one identical to the ACB1 gene from S. monacensis (1997)
    New York, NY [u.a.] : Wiley-Blackwell
    Yeast 13 (1997), S. 1409-1421 
    Details
    ISSN: 0749-503X
    Keywords: acyl-CoA binding protein ; ACB1 ; Saccharomyces cerevisiae ; Saccharomyces carlsbergensis ; Saccharomyces monacensis ; brewing yeasts ; hybrid yeast ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: Saccharomyces carlsbergensis is an amphiploid, and it has previously been suggested that the genomes of S. carlsbergensis originate from S. cerevisiae and S. monacensis. We have cloned the ACB1 genes encoding the acyl-CoA binding protein (ACBP) from S. carlsbergensis, S. cerevisiae and S. monacensis. Two genes were found in S. carlsbergensis and named ACB1 type 1 and type 2, respectively. The type 1 gene is identical to the S. cerevisiae ACB1 gene except for three substitutions, one single base pair deletion and one double base pair insertion, all located in the promoter region. The type 2 gene is completely identical to the S. monacensis ACB1 gene. These findings substantiate the notion that S. carlsbergensis is a hybrid between S. cerevisiae and S. monacensis.Both ACB1 type 1 and type 2 are actively transcribed in S. carlsbergensis and transcription is initiated at sites identical to those used for transcriptional initiation of the ACB1 genes in S. cerevisiae and S. monacensis, respectively. Two polyadenylation sites, spaced 225 bp apart, are present in the S. cerevisiae ACB1 gene. The upstream polyadenylation site is used exclusively during exponential growth, whereas both sites are utilized during later stages of growth. All sequence information is listed under EMBL Accession Numbers Y08687, Y08688, Y08689 and Y08690. © 1997 John Wiley & Sons, Ltd.
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    Deviant Pex3p Levels affect Normal Peroxisome Formation in Hansenula polymorpha: A Sharp Increase of the Protein Level Induces the Proliferation of Numerous, Small Protein-import Competent Peroxisomes (1997)
    New York, NY [u.a.] : Wiley-Blackwell
    Yeast 13 (1997), S. 1449-1463 
    Details
    ISSN: 0749-503X
    Keywords: peroxisome biogenesis ; peroxisomal protein import ; peroxisomal membrane protein ; peroxisome proliferation ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: Pex3p has been implicated in the biosynthesis of the peroxisomal membrane of the yeast Hansenula polymorpha. Here we show that in the initial stages of a sharp increase in Pex3p levels, induced in batch cultures of cells of a constructed H. polymorpha strain, which contained seven copies of PEX3 under control of the alcohol oxidase promoter (WT::PAOX.PEX37x), strongly interfered with normal peroxisome proliferation. Ultrastructural studies demonstrated that in such cells numerous small peroxisomes had developed, which were absent in wild-type controls. These organelles, which contained typical peroxisomal matrix and membrane proteins (alcohol oxidase, catalase, Pex3p, Pex10p and Pex14p), showed a relatively low density (1·18 g cm-3) after sucrose gradient centrifugation of WT::PAOX.PEX37x homogenates, compared to normal peroxisomes (1·23 g cm-3). We furthermore demonstrated that these early induced, small peroxisomes were protected against glucose-induced proteolytic degradation and did not fuse to form larger organelles. Remarkably, the induction of these small peroxisomes was paralleled by a partial defect in matrix protein import, reflected by the mislocalization of minor amounts of alcohol oxidase protein in the cytosol. However, when the cells were subsequently placed under conditions in which the synthesis of a new matrix enzyme (amine oxidase) was induced while simultaneously the excessive proliferation was repressed (by repression of the PAOX), amine oxidase protein was selectively incorporated into these organelles. This indicated that the small peroxisomes had regained a normal protein import capacity. Based on these results we argue that peroxisome proliferation and matrix protein import are coupled processes in H. polymorpha. © 1997 John Wiley & Sons, Ltd.
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  • 139
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    Green fluorescent protein as a reporter for the DNA damage-induced gene RAD54 in Saccharomyces cerevisiae (1997)
    New York, NY [u.a.] : Wiley-Blackwell
    Yeast 13 (1997), S. 1535-1545 
    Details
    ISSN: 0749-503X
    Keywords: DNA repair ; GFP ; RAD54 ; recombination ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: The green fluorescent protein (GFP) of Aequorea victoria is now an established marker for gene expression and subcellular localization in budding yeast. Relatively high expression (greater than 2500 copies per cell) of GFP is required for direct microscopic visualization. This report provides a method for studying the expression of less highly expressed genes by the analysis of crude cell extracts - a simple and cheap alternative to the fluorescent activated cell sorter (FACS). The utility of this marker is demonstrated in a study of the expression of the RAD54 gene. It is shown that the induction of the RAD54 promoter leads to the accumulation of Rad54p and of GFP and that the fluorescence induction is correctly regulated. This method should allow the screening of large numbers of novel gene disruptants for their effects on RAD54 expression and so identify trans-acting factors involved in the cellular response to DNA damage. © 1997 John Wiley & Sons, Ltd.
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  • 140
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    Suitability of replacement markers for functional analysis studies inSaccharomyces cerevisiae (1997)
    New York, NY [u.a.] : Wiley-Blackwell
    Yeast 13 (1997), S. 1563-1573 
    Details
    ISSN: 0749-503X
    Keywords: Saccharomyces cerevisiae ; functional analysis ; gene replacement ; competition experiments ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: The complete yeast sequence contains a large proportion of genes whose biological function is completely unknown. One approach to elucidating the function of these novel genes is by quantitative methods that exploit the concepts of metabolic control analysis. An important first step in such an analysis is to determine the effects of deleting individual genes on the growth rate (or fitness) of Saccharomyces cerevisiae. Since the specific growth-rate effects of most genes are likely to be small, they are most readily determined by competition against a standard strain in chemostat cultures where the true steady state demanded by metabolic control analysis may be achieved. We have constructed two different standard strains in which the HO gene is replaced by either HIS3 or kanMX. We demonstrate that HO is a selectively neutral site for gene replacement. However, there is a significant marker effect associated with HIS3 which, moreover, is dependent on the physiological conditions used for the competition experiments. In contrast, the kanMX marker exhibited only a small effect on specific growth rate (≤±4%). These data suggest that nutritional markers should not be used to generate deletion mutants for the quantitative analysis of gene function in yeast but that kanMX replacements may be used, with confidence, for such studies. © 1997 John Wiley & Sons, Ltd.
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    Phytopathogenic Filamentous (Ashbya, Eremothecium) and Dimorphic Fungi (Holleya, Nematospora) with Needle-shaped Ascospores as New Members Within the Saccharomycetaceae (1997)
    New York, NY [u.a.] : Wiley-Blackwell
    Yeast 13 (1997), S. 945-960 
    Details
    ISSN: 0749-503X
    Keywords: Ashbya ; Eremothecium ; Holleya ; Kluyveromyces ; Metschnikowia ; Nematospora ; Saccharomyces ; Crustaceae ; Diplopoda ; Heteroptera ; Saccharomycetaceae ; phylogeny ; taxonomy ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: Phylogenetic relationships between species from the genera Kluyveromyces and Saccharomyces and representatives of the Metschnikowiaceae (Holleya, Metschnikowia, Nematospora) including the two filamentous phytopathogenic fungi Ashbya gossypii and Eremothecium ashbyii were studied by comparing the monosaccharide pattern of purified cell walls, the ubiquinone system, the presence of dityrosine in ascospore walls, and nucleotide sequences of ribosomal DNA (complete 18S rDNA, ITS1 and ITS2 region). Based on sequence information from both ITS regions, the genera Ashbya, Eremothecium, Holleya and Nematospora are closely related and may be placed in a single genus as suggested by Kurtzman (1995; J. Industr. Microbiol. 14, 523-530). In a phylogenetic tree derived from the ITS1 and ITS2 region as well as in a tree derived from the complete 18S rDNA gene, the genus Metschnikowia remains distinct. The molecular evidence from ribosomal sequences suggests that morphology and ornamentation of ascospores as well as mycelium formation and fermentation should not be used as differentiating characters in family delimitation. Our data on cell wall sugars, ubiquinone side chains, dityrosine, and ribosomal DNA sequences support the inclusion of plant pathogenic, predominantly filamentous genera like Ashbya and Eremothecium or dimorphic genera like Holleya and Nematospora with needle-shaped ascospores within the family Saccharomycetaceae. After comparison of sequences from the complete genes of the 18S rDNA the genus Kluyveromyces appears heterogeneous. The type species of the genus, K. polysporus is congeneric with the genus Saccharomyces. The data of Cai et al. (1996; Int. J. Syst. Bacteriol. 46, 542-549) and our own data suggest to conserve the genus Kluyveromyces for a clade containing K. marxianus, K. dobzhanskii, K. wickerhamii and K. aestuarii, which again can be included in the family Saccharomycetaceae. The phylogenetic age of the Metschnikowiaceae and Saccharomycetaceae will be discussed in the light of coevolution. © 1997 John Wiley & Sons, Ltd.
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    In vivo Mutational Analysis of Highly Conserved Amino Acid Residues of the Small Subunit Cpa1p of the Carbamylphosphate Synthetase of Saccharomyces cerevisiae (1997)
    New York, NY [u.a.] : Wiley-Blackwell
    Yeast 13 (1997), S. 1021-1028 
    Details
    ISSN: 0749-503X
    Keywords: Saccharomyces cerevisiae ; carbamylphosphate synthetase ; mutational analysis ; CPA1 ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: The role of selected amino acid residues located in the putative catalytic domain and of two conserved histidine residues within the small subunit of the carbamylphosphate synthetase (CPS) specific to the arginine biosynthesis pathway of the yeast Saccharomyces cerevisiae was studied using site-directed mutagenesis to change all residues to aspartic acid. Carbamylphosphate synthesis catalysed by modified CPS was tested in vivo. The C264D, H307D and H349D mutants were unable to grow on minimal medium, indicating the importance of these three residues for efficient CPS activity, whereas, four other mutated residues located in the catalytic site (including a proline residue) do not affect the growth rate. These results in comparison to those obtained with the CPS of Escherichia coli, implicate residues Cys 264 and His 349 in the glutaminase catalytic activity, and His 307 in the binding of glutamine to the active site. Using these three defective mutants, we investigated the in vivo utilization of ammonia by CPS. C264D and H307D mutants are able to use ammonia as a substrate when provided in sufficiently high concentrations (up to 200 mm). The H349D mutant, however, did not grow even at ammonium sulfate concentrations above 400 mm, suggesting that this substitution is critical to NH3-dependent CPS activity although the ammonia binding site is presumably located within the large subunit of the enzyme. © 1997 by John Wiley & Sons, Ltd.
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    Who's Who among the Saccharomyces cerevisiae Actin-Related Proteins? A Classification and Nomenclature Proposal for a Large Family (1997)
    New York, NY [u.a.] : Wiley-Blackwell
    Yeast 13 (1997), S. 1053-1058 
    Details
    ISSN: 0749-503X
    Keywords: Actin-related proteins ; S. cerevisiae ; genome ; alignment ; nomenclature ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: Inspection of the complete Saccharomyces cerevisiae genome sequence and analysis of the actin-related proteins (ARPs) found therein revealed seven proteins, in addition to the previously designated actin-related proteins Arp1, Arp2 and Arp3, which contained substantial blocks of conservation relative to a chosen sub-set of actins. We have ordered the new ARPs relative to this group of actins and propose to name the more distantly related ARP members, according to their amino acid identity and similarity, Arp4-Arp10. Most of these proteins appear to represent the first example of new classes of ARPs, each of which may have specific localization(s) and cellular function(s). Recently reported ARPs from other species have also been included in the phylogenetic tree derived from the overall alignment of 29 actins and 28 ARPs. © 1997 by John Wiley & Sons, Ltd.
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    The Effect of a Suppressed rad52 Mutation on the Suppression of rad6 by srs2 (1997)
    New York, NY [u.a.] : Wiley-Blackwell
    Yeast 13 (1997), S. 1059-1064 
    Details
    ISSN: 0749-503X
    Keywords: Saccharomyces cerevisiae ; DNA repair ; UV damage ; RAD52 ; proteosome ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: We have cloned a suppressor of a temperature-sensitive rad52 allele and found it to be a mutation in PUP1, a gene encoding a protease subunit of the 20s proteasome. This identity prompted us to examine the interrelationship among PUP1, RAD52, SRS2 and RAD6 because srs2 mutations not only suppress some rad52 mutations but also suppress deletions of rad6, a gene encoding a protein in the ubiquination-dependent proteolysis pathway. We have found that while srs2 suppresses the UV sensitivity of rad6 in the presence of RAD52, srs2 cannot suppress rad6 when the temperature-sensitive allele of rad52 is present. This inability of srs2 to suppress rad6 is irrespective of the incubation temperature or whether pup1 is suppressing the temperature-sensitive rad52 mutation. © 1997 by John Wiley & Sons, Ltd.
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    Specific Labelling of Cell Wall Proteins by Biotinylation. Identification of Four Covalently Linked O-mannosylated Proteins of Saccharomyces cerevisiae (1997)
    New York, NY [u.a.] : Wiley-Blackwell
    Yeast 13 (1997), S. 1145-1154 
    Details
    ISSN: 0749-503X
    Keywords: cell wall ; mannosylation ; KEX2 ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: Intact Saccharomyces cerevisiae cells were biotinylated with the non-permeable sulfosuccinimidyl-6-(biotinamido)hexanoate reagent. Twenty specifically labelled cell wall proteins could be extracted and visualized on SDS gels via streptavidin/horseradish peroxidase. Nine cell wall proteins were released by SDS extraction under reducing conditions and were designated Scw1-9p for (soluble cell wall proteins); five proteins were released from SDS-extracted cell walls by laminarinase (Ccw1-5p for covalently linked cell wall proteins) and six with mild (30 mm-NaOH, 4°C, 14 h) alkali treatment (Ccw6-11p). N-terminal sequences of the Ccw proteins 6, 7, 8 and 11 showed that these cell wall proteins are members of the PIR gene family (predicted proteins with internal repeats), CCW6 being identical to PIR1 and CCW8 to PIR3. Single gene disruptions of all four genes did not yield a phenotype. In the CCW11 disruption the Ccw11p as well as the laminarinase-extracted Ccw5 protein was missing. The new cell wall proteins are O-mannosylated, contain a Kex2 processing site, but no C-terminal GPI anchor sequence. © 1997 John Wiley & Sons, Ltd.
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  • 146
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    Disruption of Six Novel Yeast Genes Reveals Three Genes Essential for Vegetative Growth and One Required for Growth at Low Temperature (1997)
    New York, NY [u.a.] : Wiley-Blackwell
    Yeast 13 (1997), S. 1181-1194 
    Details
    ISSN: 0749-503X
    Keywords: gene disruption ; functional analysis ; chromosome X ; Saccharomyces cerevisiae ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: We describe here the construction of six deletion mutants and their basic phenotypic analysis. Six open reading frames (ORFs) from chromosome X, YJR039w, YJR041c, YJR043c, YJR046w, YJR053w and YJR065c, were disrupted by deletion cassettes with long (LFH) or short (SFH) flanking regions homologous to the target locus. The LFH deletion cassette was made by introducing into the kanMX4 marker module two polymerase chain reaction (PCR) fragments several hundred base pairs (bp) in size homologous to the promoter and terminator regions of a given ORF. The SFH gene disruption construct was obtained by PCR amplification of the kanMX4 marker with primers providing homology to the target gene. The region of homology to mediate homologous recombination was about 70 bp. Sporulation and tetrad analysis revealed that ORFs YJR041c, YJR046w and YJR065c are essential genes. Complementation tests by corresponding cognate gene clones confirmed this observation. The non-growing haploid segregants were observed under the microscope. The yjr041cΔ haploid cells gave rise to microcolonies comprising about 20 to 50 cells. Most yjr046wΔ cells were blocked after one or two cell cycles with heterogeneous bud sizes. The yjr065cΔ cells displayed an unbudded spore or were arrested before completion of the first cell division cycle with a bud of variable size. The deduced protein of ORF YJR065c, that we named Act4, belongs to the Arp3 family of actin-related proteins. Three other ORFs, YJR039w, YJR043c and YJR053w are non-essential genes. The yjr043cΔ cells hardly grew at 15°C, indicating that this gene is required for growth at low temperature. Complementation tests confirmed that the disruption of YJR043c is responsible for this growth defect. In addition, the mating efficiency of yjr043cΔ and yjr053wΔ cells appear to be moderately a ffected. © 1997 John Wiley & Sons, Ltd.
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    Deviant Pex3p Levels affect Normal Peroxisome Formation in Hansenula polymorpha: High Steady-state Levels of the Protein fully Abolish Matrix Protein Import (1997)
    New York, NY [u.a.] : Wiley-Blackwell
    Yeast 13 (1997), S. 1437-1448 
    Details
    ISSN: 0749-503X
    Keywords: peroxisome biogenesis ; peroxisomal protein import ; peroxisomal membrane protein ; PEX gene regulation ; Hansenula polymorpha ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: PEX3 encodes a 52 kDa peroxisomal membrane protein (PMP), essential for peroxisome biogenesis in the yeast Hansenula polymorpha. The relation between Pex3p levels and peroxisome formation was studied in wild type (WT) and Δpex3 strains expressing additional copies of PEX3 under control of a substrate-inducible promoter, namely the strong alcohol oxidase (PAOX) or the weaker amine oxidase (PAMO) promoter. In glucose-grown Δpex3 cells, containing PAOX. PEX3, Pex3p was undetectable and peroxisomes were absent. After induction of these cells on methanol, peroxisomes were rapidly formed. At Pex3p levels up to 7-10 times the values observed in WT controls normal peroxisomes were present. However, at further enhanced Pex3p levels a general matrix protein import defect was observed. This phenomenon was paralleled by aberrant peroxisome assembly and the formation of numerous small vesicles. These vesicles contained Pex3p, together with other H. polymorpha PMPs, but lacked the major matrix proteins which has accumulated in the cytosol. The implications of our results on PEX3 gene regulation and functioning of the peroxisomal matrix protein import machinery in H. polymorpha are discussed. © 1997 John Wiley & Sons, Ltd.
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    Two-dimensional electrophoretic separation of yeast proteins using a non-linear wide range (pH 3-10) immobilized pH gradient in the first dimension; reproducibility and evidence for isoelectric focusing of alkaline (pI 〉7) proteins (1997)
    New York, NY [u.a.] : Wiley-Blackwell
    Yeast 13 (1997), S. 1519-1534 
    Details
    ISSN: 0749-503X
    Keywords: proteome analysis ; 2D-PAGE ; protein identification ; protein expression ; S. cerevisiae ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: The proteome of the yeast Saccharomyces cerevisiae was analysed by two-dimensional (2D) polyacrylamide gel electrophoresis utilizing a non-linear immobilized pH gradient (3-10) in the first-dimensional separation. Cells were labelled by [35S]methionine incorporation in the respiro-fermentative phase during exponential growth on glucose. Gels were run, visualized with phosphoimager technology and all resolved proteins automatically quantified. Proteins were well resolved over the whole pH interval, and evidence for isoelectric focusing on the basic side of the pattern was generated by sequencing of some spots, revealing the 2D positions of Tef1p, Pgk1p, Gpm1p, Tdh1p and Shm2p. Roughly 25% of the spots were resolved at the alkaline side of the pattern (pI〉7). The position reproducibility was high and in the range 1-2 mm in the x-and y-dimension, respectively. No quantitative variation was linked to a certain size or charge class of resolved proteins, and the average quantitative standard deviation was 17±11%. The obtained immobilized pH gradient based pattern could easily be compared to the old ampholine-based 2D pattern, and the previously reported identifications could thus be transferred. Our yeast pattern currently contains 43 known proteins, all identified by protein sequencing. Utilizing these identified proteins, relevant pI and Mr scales in the pattern were constructed. Normalization of the expression of identified spots by compensating for the number of methionine residues a protein contains allowed stoichiometric comparisons. The most dominant proteins under these growth conditions were Tdh3p, Fba1p, Eno2p and Tef1p/Tef2p, all being expressed at more than 500 000 copies per cell. The differential carbon source response during exponential growth on either glucose, galactose or ethanol was examined for the alkaline proteins identified by micro-sequencing in this study. © 1997 John Wiley & Sons, Ltd.
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    Guest Editorial: 1997 ushers in an era of yeast functional genomics (1997)
    New York, NY [u.a.] : Wiley-Blackwell
    Yeast 13 (1997), S. 1501-1503 
    Details
    ISSN: 0749-503X
    Keywords: Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: No Abstract
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    Surface Properties of Top- and Bottom-Fermenting Yeast (1997)
    New York, NY [u.a.] : Wiley-Blackwell
    Yeast 13 (1997), S. 931-943 
    Details
    ISSN: 0749-503X
    Keywords: brewing yeast ; fermentation ; cell surface ; flocculation ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: The surface physico-chemical properties (hydrophobicity, electrophoretic mobility, chemical composition) of a large set of top- and bottom-fermenting brewing yeasts, harvested in the exponential and stationary growth phases, have been investigated. Bottom- and top-fermenting strains showed different surface properties. Top strains were generally more hydrophobic than bottom strains, due to higher surface protein concentrations. Bottom strains possessed higher surface phosphate concentrations. The different profiles of electrophoretic mobility versus pH for top and bottom strains could be explained by modelling the surface charge according to the surface chemical composition as given by X-ray photoelectron spectroscopy. For bottom strains, the electrical properties were mainly controlled by phosphate, resulting in a low isoelectric point (pH 2 or below) and an electrophoretic mobility that did not become much more negative above pH 4. For the top strains, they were mainly determined by the balance of protonated amino- and carboxylate groups in proteins, which gave a high isoelectric point (pH 4) and an electrophoretic mobility changing greatly with pH in the range of 2 to 7. No difference in surface properties was found between flocculating and non-flocculating strains, or between cells from the exponential and stationary growth phases, even for strains where flocculation occurred during the transition from one growth phase to the other. © 1997 John Wiley & Sons, Ltd.
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    Mapping of ure1, ure2 and ure3 Markers in Fission Yeast (1997)
    New York, NY [u.a.] : Wiley-Blackwell
    Yeast 13 (1997), S. 1195-1197 
    Details
    ISSN: 0749-503X
    Keywords: Schizosaccharomyces pombe ; mapping ; urease ; yeast ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: The following urease genes of the fission yeast Schizosaccharomyces pombe have been mapped by induced haploidization and tetrad analysis - ure1: chromosome arm III-L; ure2 and ure3: chromosome arm I-R. The previously determined tps19-rad1 interval (11-12 cM) has been increased to 18 cM. A convenient medium for rapidly scoring the ure gene markers of fission yeast was developed. © 1997 John Wiley & Sons, Ltd.
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    Characterization and Regulation of the Genes Encoding Ribosomal Proteins L39 and S7 of the Human Pathogen Candida albicans (1997)
    New York, NY [u.a.] : Wiley-Blackwell
    Yeast 13 (1997), S. 1199-1210 
    Details
    ISSN: 0749-503X
    Keywords: Candida albicans ; Saccharomyces cerevisiae ; ribosomal proteins ; RPL39 ; RPS7 ; RPL29 ; hyphal morphogenesis ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: Genes encoding the Candida albicans ribosomal proteins L39 and S7 (RPL39, RPS7) were isolated and sequenced. From RPL39 cDNA a single intron interrupting the fifth codon in the genomic sequence could be deduced. Two homologous RPL39 genes in Saccharomyces cerevisiae contain a single intron in a conserved position. In contrast, C. albicans RPS7 was found to lack an intron, while both S. cerevisiae homologs are interrupted by single introns. The deduced L39 and S7 proteins contained 67% and 83% identical residues compared to the S. cerevisiae homologs. During hyphal induction the RPL39, RPS7 and RPL29 transcript levels increased three- to six-fold relative to ribosomal RNA, while ACT1 and RPS33 control transcripts were not regulated extensively. As suggested by unaltered transcript stabilities during hyphal induction, this regulation occurs on the transcriptional level; a conserved 18 bp palindromic sequence (5′-TTAGGGCTATAGCCCTAA-3′), which is present in the promoter regions of the RPL39 and RPS7 genes, may be involved in regulation. © 1997 John Wiley & Sons, Ltd.
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    Molecular Cloning of Chromosome I DNA from Saccharomyces cerevisiae: Characterization of the 54 kb Right Terminal CDC15-FLO1-PHO11 Region (1997)
    New York, NY [u.a.] : Wiley-Blackwell
    Yeast 13 (1997), S. 1251-1263 
    Details
    ISSN: 0749-503X
    Keywords: Saccharomyces cerevisiae ; chromosome I ; yeast genome ; subtelomeric ; transcription ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: Gene density near the ends of Saccharomyces cerevisiae chromosomes is much lower than on the rest of the chromosome. Non-functional gene-fragments are common and a high proportion of the sequences are repeated elsewhere in the genome. This sequence arrangement suggests that the ends of chromosomes play a structural rather than a coding role and may be analogous to the highly repeated heterochromatic DNA of higher organisms. In order to evaluate the function of the ends of S. cerevisiae chromosomes, the rightmost 54-kb of DNA from chromosome I was investigated. The region contains 16 open reading frames (ORFs) and two tRNA genes. Gene-disruption studies indicated that none of these genes are essential for growth on rich or minimal medium, mating or sporulation. In contrast to the central region where 80% of the genes are transcribed when cells are grown on rich medium, only seven ORFs and the two tRNA genes appeared to produce transcripts. Six of the transcribed ORFs were from the centromere-proximal part of the region, leaving the rightmost 35-kb with only a single sequence that is transcribed during vegetative growth. Two genes located 3 and 10-kb from the chromosome I telomere are almost identical to two genes located somewhat further from the chromosome VIII telomere. Surprisingly, the chromosome VIII copies were transcribed while the chromosome I genes were not. These results suggest that the chromosome I genes may be repressed by a natural telomere position effect. The low level of transcription, absence of essential genes as well as the repetitive nature of these sequences are consistent with their having a structural role in chromosome function. © 1997 John Wiley & Sons, Ltd.
    Additional Material: 2 Ill.
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    Isolation of a putative prolyl-tRNA synthetase (CaPRS) gene from Candida albicans (1997)
    New York, NY [u.a.] : Wiley-Blackwell
    Yeast 13 (1997), S. 1375-1381 
    Details
    ISSN: 0749-503X
    Keywords: Candida albicans ; prolyl tRNA synthetase ; CaPRS ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: We have isolated a 4·0-kb fragment from a genomic library of Candida albicans which contained two open reading frames (ORFs). One of them is homologous to a prolyl-tRNA synthetase that catalyses the charging of a specific tRNA by proline (CaPRS). A deduced sequence of 575 amino acids representing a polypeptide of 66·2 kDa was determined. A FASTA search indicated that the CaPRSp had an overall similarity of 54·4% with the product of a Saccharomyces cerevisiae ORF (YER087) and 43·8% with the prolyl-tRNA synthetase of Escherichia coli (COLIPRO). Consensus Class II aminoacyl-tRNA synthetase sequences were identified by the PROSITE program. CaPRS was localized to chromosome R of the C. albicans genome and CaPRS DNA hybridized to a major RNA transcript of 1·7 kb under all conditions tested. The CaPRS sequence submitted to the EMBL data library is available under Accession Number U86341.© 1997 John Wiley & Sons, Ltd.
    Additional Material: 6 Ill.
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  • 155
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    The ALD6 gene of Saccharomyces cerevisiae encodes a cytosolic, Mg2+-activated acetaldehyde dehydrogenase (1997)
    New York, NY [u.a.] : Wiley-Blackwell
    Yeast 13 (1997), S. 1319-1327 
    Details
    ISSN: 0749-503X
    Keywords: aldehyde dehydrogenase ; gene cloning ; gene deletion ; protein family ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: The deduced translation product of an open reading frame on the left arm of chromosome XVI of Saccharomyces cerevisiae, with the systematic name of YPL061w, is 500 amino acids in length and shares significant homology with aldehyde dehydrogenases. Amino acids 2 to 16 of the protein encoded by YPL061w were found to be identical to the N-terminal 15 amino acids of the purified cytosolic, Mg2+-activated acetaldehyde dehydrogenase (ACDH) of S. cerevisiae. This enzyme is thought to be involved in the production of acetate from which cytosolic acetyl-CoA is then synthesized. Deletion of YPL061w was detrimental to the growth of haploid strains of yeast; an analysis of one deletion mutant revealed a maximum specific growth rate (in complex medium containing glucose) of one-third of that displayed by the wild-type strain. Mutants deleted in YPL061w were also unable to use ethanol as a carbon source. As expected, the cytosolic, Mg2+-activated ACDH activity had been lost from the mutants, although the mitochondrial, K+-activated ACDH was readily detected. YPL061w has been registered with the name of ALD6 in the Saccharomyces Genome Database and the nucleotide sequence submitted to GenBank as part of accession number U39205. © 1997 John Wiley & Sons, Ltd.
    Additional Material: 4 Ill.
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  • 156
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    Molecular cloning of TvDAO1, a gene encoding a D-amino acid oxidase from Trigonopsis variabilis and its expression in Saccharomyces cerevisiae and Kluyveromyces lactis (1997)
    New York, NY [u.a.] : Wiley-Blackwell
    Yeast 13 (1997), S. 1399-1408 
    Details
    ISSN: 0749-503X
    Keywords: Trigonopsis variabilis ; D-amino acid oxidase ; heterologous gene expression ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: The DAO1 gene of Trigonopsis variabilis encoding a D-amino acid oxidase (EC 1.4.3.3) was isolated from genomic clones selected for their specific hybridization to synthetic oligodeoxyribonucleotide probes based on regions of the enzyme that have been conserved through evolution. The nucleotide sequence of the gene predicts a protein with similarities to human, pig, rabbit, mouse and Fusarium solani D-amino acid oxidases. The open reading frame of the T. variabilis DAO1 gene was interrupted by an intron. The Dao1p sequence displays two regions, one in the N-terminal section - the FAD binding site - and the other near the C-terminal region that contains conserved signatures found in all the D-amino acid oxidases. The three C-terminal amino acids suggest that the enzyme may be located in peroxisomes. Northern blot experiments showed that no transcriptional activation occurred in the presence of D-methionine. The cDNA encoding Dao1p was expressed in Saccharomyces cerevisiae and Kluyveromyces lactis. Both yeast species are able to synthesize a functional enzyme under the control of the GAL1 promoter. In K. lactis, up to six times more enzyme units per gram of dry weight are produced with a multicopy plasmid in comparison with the wild-type strain of T. variabilis. The yeast expression system we describe may constitute an alternative source for the production of D-amino acid oxidases at industrial level. The sequence presented here has been submitted to the EMBL data library under Accession Number Z50019. © 1997 John Wiley & Sons, Ltd.
    Additional Material: 5 Ill.
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  • 157
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    The 2DWG meta-database of two-dimensional electrophoretic gel images on the Internet (1997)
    Weinheim : Wiley-Blackwell
    Electrophoresis 18 (1997), S. 2759-2773 
    Details
    ISSN: 0173-0835
    Keywords: World Wide Web ; Internet ; Two-dimensional polyacrylamide gel electrophoresis ; Databases ; Meta-database ; Proteins ; Genetics ; Image analysis ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: The 2DWG meta-database is a searchable database of two-dimensional (2-D) electrophoretic gel images found on the Internet. A meta-database contains information about locating data in other databases - but not that data itself. This database was constructed because of a need for an enriched set of World Wide Web (WWW) locations (URLs) of 2-D gel images on the Internet. These gel images are used in conjunction with the National Cancer Institute (NCI) Flicker Server to manipulate and visually compare 2-D gel images across the Internet. User's gels may also be compared with those in the database. The 2DWG is organized as a spreadsheet table with each gel image being represented by a row sorted by tissue type. Data for each gel includes tissue type, species, cell-line, image URL, database URL, gel protocol, organization URL, image properties, map URL if it exists, etc. The 2DWG may be searched to find relevant subsets of gels. Searching is done using the dbEngine - a WWW database search engine which accesses selected rows of gels from the full 2DWG table. The 2DWG meta-database is accessible on the WWW at http://www-lecb.ncifcrf.gov/2dwgDB/ and the NCI Flicker server at http://www-lecb.ncifcrf.gov/flicker/.
    Additional Material: 7 Ill.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1002/elps.1150181510
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    Angiotensinogen gene variation in a population case-control study of preeclampsia/eclampsia in Australians and Chinese (1997)
    Weinheim : Wiley-Blackwell
    Electrophoresis 18 (1997), S. 1646-1649 
    Details
    ISSN: 0173-0835
    Keywords: Angiotensinogen ; Preeclampsia ; Genetics ; Australian ; Chinese ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Preeclampsia/eclampsia (PE/E) is a common disease of human pregnancy with a strong genetic component. The etiology of PE/E is unknown. Two recent reports indicated that the angiotensinogen gene (AGT) could be involved in susceptibility to PE/E. We performed a population-based case-control study in Australian and Chinese populations to investigate whether AGT is a good candidate gene for PE/E. A microsatellite polymorphism within AGT was typed as well as a molecular variant T235 (Met→Thr) of AGT using allele-specific PCR and allele-induced restriction site PCR. The allele distributions of the microsatellite and the variant T235 of AGT were significantly different between the two ethnic groups. However, no significant allele associations were found with disease when comparing PE/E patients and controls in Australian or Chinese populations, which is in contrast to the two earlier reports. The results suggest that the contribution of AGT to the occurrence of PE/E is small, if anything, and is not constant across populations.
    Additional Material: 3 Ill.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1002/elps.1150180929
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