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  • Genetics  (1,213)
  • 101
    ISSN: 0749-503X
    Keywords: Saccharomyces cerevisiae ; YAP3 ; KEX2 ; recombinant human albumin ; protease degradation ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: Expression of recombinant human albumin (rHA) in Saccharomyces cerevisiae resulted in secretion of both mature albumin and a 45 kDa degradation product, comprising an N-terminal fragment of rHA with heterogeneous C-termini between residues 403 and 409 (Geisow et al., 1991). Site-directed mutagenesis of the human albumin gene (HA) to change Arg410 to Ala (R410A) caused a significant reduction in the amount of fragment produced. Mutation of the adjacent dibasic site Lys413 Lys414 had little effect in isolation, but in combination with the R410A mutation resulted in a further reduction in the amount of rHA fragment produced. This reduction could be duplicated with nature-identical rHA by disruption of the gene for an aspartyl protease (YAP3), alone or in conjunction with disruption of the KEX2 gene. Disruption of KEX2 alone did not result in any improvement in the degree of degradation of the rHA. Reduced degradation was also observed when an rHA-human growth hormone fusion protein was secreted from a yap3 strain, suggesting that such strains may have a general utility for heterologous protein secretion. © 1998 John Wiley & Sons, Ltd.
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  • 102
    Electronic Resource
    Electronic Resource
    New York, NY [u.a.] : Wiley-Blackwell
    Yeast 14 (1998), S. 443-457 
    ISSN: 0749-503X
    Keywords: evolution ; polyploidy ; gene duplication ; gene order ; LEU2 ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: The extent to which the order of genes along chromosomes is conserved between Saccharomyces cerevisiae and related species was studied by analysing data from DNA sequence databases. As expected, the extent of gene order conservation decreases with increasing evolutionary distance. About 59% of adjacent gene pairs in Kluyveromyces lactis or K. marxianus are also adjacent in S. cerevisiae, and a further 16% of Kluyveromyces neighbours can be explained in terms of the inferred ancestral gene order in Saccharomyces prior to the occurrence of an ancient whole-genome duplication. Only 13% of Candida albicans linkages, and no Schizosaccharomyces pombe linkages, are conserved. Analysis of gene order arrangements, chromosome numbers, and ribosomal RNA sequences suggests that genome duplication occurred before the divergence of the four species in Saccharomyces sensu stricto (all of which have 16 chromosomes), but after this lineage had diverged from Saccharomyces kluyveri and the Kluyveromyces lactis/marxianus species assemblage. © 1998 John Wiley & Sons, Ltd.
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  • 103
    ISSN: 0749-503X
    Keywords: Crabtree effect ; yeast ; biomass ; Kluyveromyces lactis ; oxygen ; pyruvate decarboxylase ; regulation ; fermentation ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: Kluyveromyces lactis is an important industrial yeast, as well as a popular laboratory model. There is currently no consensus in the literature on the physiology of this yeast, in particular with respect to aerobic alcoholic fermentation (‘Crabtree effect’). This study deals with regulation of alcoholic fermentation in K. lactis CBS 2359, a proposed reference strain for molecular studies. In aerobic, glucose-limited chemostat cultures (D=0·05-0·40 h-1) growth was entirely respiratory, without significant accumulation of ethanol or other metabolites. Alcoholic fermentation occurred in glucose-grown shake-flask cultures, but was absent during batch cultivation on glucose in fermenters under strictly aerobic conditions. This indicated that ethanol formation in the shake-flask cultures resulted from oxygen limitation. Indeed, when the oxygen feed to steady-state chemostat cultures (D=0·10 h-1) was lowered, a mixed respirofermentative metabolism only occurred at very low dissolved oxygen concentrations (less than 1% of air saturation). The onset of respirofermentative metabolism as a result of oxygen limitation was accompanied by an increase of the levels of pyruvate decarboxylase and alcohol dehydrogenase. When aerobic, glucose-limited chemostat cultures (D=0·10 h-1) were pulsed with excess glucose, ethanol production did not occur during the first 40 min after the pulse. However, a slow aerobic ethanol formation was invariably observed after this period. Since alcoholic fermentation did not occur in aerobic batch cultures this is probably a transient response, caused by an imbalanced adjustment of enzyme levels during the transition from steady-state growth at μ=0·10 h-1 to growth at μmax. It is concluded that in K. lactis, as in other Crabtree-negative yeasts, the primary environmental trigger for occurrence of alcoholic fermentation is oxygen limitation. © 1998 John Wiley & Sons, Ltd.
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  • 104
    Electronic Resource
    Electronic Resource
    New York, NY [u.a.] : Wiley-Blackwell
    Yeast 14 (1998), S. 501-515 
    ISSN: 0749-503X
    Keywords: Saccharomyces cerevisiae ; bakers' yeast ; pH homeostasis ; cytoplasmic pH ; vacuolar pH ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: The effects of monovalent cations on the internal pH of yeast were studied. Our former procedure was modified, inducing maximal alkalinization of the cells with 100 mM-NH4OH instead of Tris base. The pH values were lower than reported before (Peña et al., J. Bacteriol. 1995 177, 1017-1022). With glucose as substrate, the internal cytoplasmic pH reached higher values when incubating at an external pH of 6·0, as compared to pH 4·0. Monovalent cations added approximately 5 min after glucose produced a further increase in the internal pH, which was higher at a previous incubation pH of 4·0 than that observed at pH 6·0. The selectivity of the changes followed a similar order to that of the transport system for monovalent cations.When incubating cells with glucose for more than 30 min, the initial changes of the internal pH appeared to be regulated by the cell. However, under the fluorescence microscope, it was observed that pyranine, which was confined to the cytoplasm during the first 15 min, was progressively concentrated in the vacuole. By studying the fluorescence changes of cells electroporated and then incubated with glucose or glucose plus potassium, we could follow the internal pH of this organelle, obtaining values within the range reported by other authors. Also, in cells preincubated with glucose for 60 min, and electroporated afterwards, the fluorescence of pyranine, which only entered the cytoplasm, allowed us to measure the pH of this compartment, showing that it was more alkaline than the vacuole. Moreover, the cytoplasmic pH increased upon addition of glucose or potassium. The vacuolar pH, on the other hand, increased upon addition of potassium after glucose, but decreased upon addition of glucose. In addition, incubation of the cells with glucose with or without pyranine produced vesiculation of the vacuole. © 1998 John Wiley & Sons, Ltd.
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  • 105
    ISSN: 0749-503X
    Keywords: alcohol acetyltransferase ; ATF1 gene ; OLE1 gene ; unsaturated fatty acid ; oxygen ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: The ATF1 gene encodes an alcohol acetyl transferase which catalyzes the synthesis of acetate esters from acetyl CoA and several kinds of alcohols. ATF1 expression is repressed by unsaturated fatty acids or oxygen. Analysis using ATF1-lacZ fusion plasmid revealed that ATF1 gene expression is widely repressed by a variety of unsaturated fatty acids, and the degree of ATF1 transcriptional repression varies according to the structure of the unsaturated fatty acids. Interestingly, it was noted that the degree of ATF1 transcriptional repression was related to the melting point of unsaturated fatty acids added to the medium. The OLE1 gene, which encodes Δ-9 fatty acid desaturase, has been reported to be repressed by unsaturated fatty acids. Transcription of OLE1 was also repressed by a wide variety of unsaturated fatty acids under anaerobic conditions. The degree of transcriptional repression of OLE1 was also related to the melting point of the added unsaturated fatty acids. Therefore, it is considered that ATF1 and OLE1 transcription are regulated in response to cell membrane fluidity. As has been reported for OLE1, the repression of ATF1 by unsaturated fatty acids was relieved in a disruptant carrying a faa1 and faa4 double mutation, two fatty acid activation genes. However, the ATF1 transcript in this double gene disruptant was repressed by oxygen. These results suggested that ATF1 transcription was co-regulated by the same mechanism as the OLE1 gene and that unsaturated fatty acids and oxygen repressed the ATF1 transcript by a different regulation pathway. © 1998 John Wiley & Sons, Ltd.
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  • 106
    ISSN: 0749-503X
    Keywords: Pichia pastoris ; expression vectors ; gene regulation ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: The budding yeast Pichia pastoris is an attractive system for exploring certain questions in cell biology, but experimental use of this organism has been limited by a lack of convenient expression vectors. Here we describe a set of compact vectors that should allow for the expression of a wide range of endogenous or foreign genes in P. pastoris. A gene of interest is inserted into a modified pUC19 polylinker; targeted integration into the genome then results in stable and uniform expression of this gene. The utility of these vectors was illustrated by expressing the bacterial β-glucuronidase (GUS) gene. Constitutive GUS expression was obtained with the strong GAP promoter or the moderate YPT1 promoter. The regulatable AOX1 promoter yielded very strong GUS expression in methanol-grown cells, negligible expression in glucose-grown cells, and intermediate expression in mannitol-grown cells. GenBank Accession Numbers are: pIB1, AF027958; pIB2, AF027959; pIB3, AF027960; pIB4, AF027961. © 1998 John Wiley & Sons, Ltd.
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  • 107
    ISSN: 0749-503X
    Keywords: Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
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  • 108
    Electronic Resource
    Electronic Resource
    New York, NY [u.a.] : Wiley-Blackwell
    Yeast 14 (1998), S. 1439-1451 
    ISSN: 0749-503X
    Keywords: beginnings of yeast research ; yeasts ; history ; Lavoisier ; Cagniard-Latour ; Kutzing ; Schwann ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
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  • 109
    Electronic Resource
    Electronic Resource
    New York, NY [u.a.] : Wiley-Blackwell
    Yeast 14 (1998), S. 1471-1510 
    ISSN: 0749-503X
    Keywords: yeast ; S. cerevisiae ; lipids ; phospholipids ; sterols ; sphingolipids ; fatty acids ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: The yeast Saccharomyces cerevisiae is a powerful experimental system to study biochemical, cell biological and molecular biological aspects of lipid synthesis. Most but not all genes encoding enzymes involved in fatty acid, phospholipid, sterol or sphingolipid biosynthesis of this unicellular eukaryote have been cloned, and many gene products have been functionally characterized. Less information is available about genes and gene products governing the transport of lipids between organelles and within membranes, turnover and degradation of complex lipids, regulation of lipid biosynthesis, and linkage of lipid metabolism to other cellular processes. Here we summarize current knowledge about lipid biosynthetic pathways in S. cerevisiae and describe the characteristic features of the gene products involved. We focus on recent discoveries in these fields and address questions on the regulation of lipid synthesis, subcellular localization of lipid biosynthetic steps, cross-talk between organelles during lipid synthesis and subcellular distribution of lipids. Finally, we discuss distinct functions of certain key lipids and their possible roles in cellular processes. © 1998 John Wiley & Sons, Ltd.
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  • 110
    ISSN: 0749-503X
    Keywords: transcription ; microarrays ; expression profiling ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: Open reading frames (6116) of the budding yeast Saccharomyces cerevisiae were PCR-amplified from genomic DNA using 12,232 primers specific to the ends of the coding sequences; the success rate of amplification was 97%. PCR-products were made accessible to hybridization by being arrayed at very high density on solid support media using various robotic devices. Probes made from total RNA preparations were hybridized for the analysis of the transcriptional activity of yeast under various growth conditions and of different strains. Experimental factors that proved critical to the performance, such as different RNA isolation procedures and the assessment of hybridization results, for example, were investigated in detail. Various software tools were developed that permit convenient handling and sound analysis of the large data quantities obtained from transcriptional profiling studies. Comprehensive arrays are being distributed within the European Yeast Functional Analysis Network (EUROFAN) and beyond. © 1998 John Wiley & Sons, Ltd.
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  • 111
    ISSN: 0749-503X
    Keywords: yeast ; PMR1 ; Hansenula polymorpha ; Ca2+-ATPase ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: A gene homologous to Saccharomyces cerevisiae PMR1 has been cloned in the methylotrophic yeast Hansenula polymorpha. The partial DNA fragment of the H. polymorpha homologue was initially obtained by a polymerase chain reaction and used to isolate the entire gene which encodes a protein of 918 amino acids. The putative gene product contains all ten of the conserved regions observed in P-type ATPases. The cloned gene product exhibits 60·3% amino acid identity to the S. cerevisiae PMR1 gene product and complemented the growth defect of a S. cerevisiae pmr1 null mutant in the EGTA-containing medium. The results demonstrate that the H. polymorpha gene encodes the functional homologue of the S. cerevisiae PMR1 gene product, a P-type Ca2+-ATPase. The DNA sequence of the H. polymorpha homologue has been submitted to GenBank with the Accession Number U92083. © 1998 John Wiley & Sons, Ltd.
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  • 112
    Electronic Resource
    Electronic Resource
    New York, NY [u.a.] : Wiley-Blackwell
    Yeast 14 (1998), S. 1257-1265 
    ISSN: 0749-503X
    Keywords: Candida albicans ; nucleoside transport ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: The nucleoside permease gene (i.e. NUP) from Candida albicans was cloned by complementation of Saccharomyces cerevisiae deficient in nucleoside transport capability. The permease transported adenosine and guanosine and was sensitive to the mammalian nucleoside transport inhibitors: dipyridamole and NBMPR. It did not transport uridine, cytidine, adenine, guanine or uracil. The inability to transport uridine indicated that the NUP gene product was different from the Candida uridine permease, which also transported cytosine and adenosine. The NUP gene coded for a protein of 407 amino acids in size which was approximately the size of the human, Giardia and E. coli nucleoside permeases. It did not, however, exhibit any significant degree of homology with these transporters. The GenBank accession number for the Candida NUP gene is AF016246. © 1998 John Wiley & Sons, Ltd.
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  • 113
    ISSN: 0749-503X
    Keywords: transcription factor ; yeast genetic selection ; bacteriophage λ vector ; Cre-loxP automatic plasmid subcloning ; integration vector ; one hybrid ; target genes ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: We describe the construction of a number of vectors that can be used in yeast genetic selection systems for cloning of transcription factors or other DNA-binding proteins and for identification of the target sites recognized by transcription factors. For transcription factor cloning we have designed an integration vector with two HIS3 reporter gene cassettes to stably integrate reporter gene constructs at the non-essential yeast PDC6 locus. This set of plasmids was tested in a one-hybrid assay with the rice transcription factor Oshox1, a member of the class of homeodomain leucine zipper proteins. A hybrid protein of Oshox1 and the Gal4 transcriptional activation domain was shown to specifically activate a reporter gene construct with upstream Oshox1 binding sites, which had been integrated at the PDC6 locus using the described vector system. Target site identification by genetic selection in yeast employs a transcriptional activator construct and a library of genomic or random DNA fragments upstream of a reporter gene. We have constructed two variants of a bacteriophage λ vector which facilitates the construction of the required reporter gene library because of high cloning efficiency and easy conversion into a yeast/Escherichia coli shuttle vector library by Cre-loxP-mediated automatic subcloning. Tests with Oxhox1 as transcriptional activator demonstrated the usefulness of the deprived reporter gene vector. © 1998 John Wiley & Sons, Ltd.
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  • 114
    ISSN: 0749-503X
    Keywords: Yarrowia lipolytica ; cytochrome P450 ; n-alkane metabolism ; n-alkane-inducible gene ; RT-PCR ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: A gene encoding cytochrome P450 involved in n-alkane utilization was cloned from an n-alkane assimilating yeast, Yarrowia lipolytica CX161-1B. The RT-PCR was performed on the mRNA prepared from the cells grown on n-alkane as a template using degenerated PCR primers designed for the conserved amino acid sequences of the CYP52 family. The RT-PCR amplified fragment was then used as a probe to isolate genes coding for P450 of the CYP52 family from the genomic DNA library of the strain CX161-1B. The nucleotide sequence of one of the positive clones was determined. An open reading frame which had the same nucleotide sequence as the RT-PCR-amplified fragment was identified. It was of 523 amino acid residues, 60·2 kDa in molecular mass, and had 30-45% sequence identity with the other members of the CYP52 family of Candida species so far analysed. The expression of the P450 gene that was named as YlALK1 was induced by n-tetradecane and repressed by glycerol. A YlALK1 gene disruptant did not grow well on n-decane, but grew on longer-chain n-alkanes such as hexadecane as a sole carbon source. Introduction of YlALK1 on a plasmid to the disruptant restored the decane assimilation. These results suggest that the YlALK1 gene product is the major P450Alk to metabolize short-chain n-alkanes such as decane and dodecane in Y. lipolytica. © 1998 John Wiley & Sons, Ltd.
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  • 115
    Electronic Resource
    Electronic Resource
    New York, NY [u.a.] : Wiley-Blackwell
    Yeast 14 (1998), S. 1511-1527 
    ISSN: 0749-503X
    Keywords: Saccharomyces cerevisiae ; oxidative stress ; stress response ; signal transduction ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: All aerobically growing organisms suffer exposure to oxidative stress, caused by partially reduced forms of molecular oxygen, known as reactive oxygen species (ROS). These are highly reactive and capable of damaging cellular constituents such as DNA, lipids and proteins. Consequently, cells from many different organisms have evolved mechanisms to protect their components against ROS. This review concentrates on the oxidant defence systems of the budding yeast Saccharomyces cerevisiae , which appears to have a number of inducible adaptive stress responses to oxidants, such as H2 O2 , superoxide anion and lipid peroxidation products. The oxidative stress responses appear to be regulated, at least in part, at the level of transcription and there is considerable overlap between them and many diverse stress responses, allowing the yeast cell to integrate its response towards environmental stress. © 1998 John Wiley & Sons, Ltd.
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  • 116
    ISSN: 0749-503X
    Keywords: flocculation ; immunolocalization ; mannoprotein ; cell wall ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: The yeast FLO genes encode cell surface proteins which are expected to play a major role in the control of flocculation. We have assessed the availability of the Flo proteins at the cell surface during the growth of two flocculent strains, ABXL-1D (FLO1) and STX347-1D (FLO5) using immunological approaches, enzyme-linked immunosorbent assays and immunofluorescence. Our data show that they are not permanently present at the cell surface but that their amount increases during growth. With both strains the flocculation level is tightly correlated to the amount of Flop antigen detected, suggesting that it is the availability of the Flo proteins at the cell surface which determines the flocculation level. Our data are consistent with the idea that the Flo proteins correspond to the flocculation lectins. The differences of flocculation pattern among strains could originate from variations in the regulation of the expression of the FLO genes. Monitoring of the distribution of the Flo proteins during cellular development revealed that they are incorporated essentially in the cell wall of growing buds. Incorporation of the Flo proteins in the cell wall displays a highly polarized aspect, at the bud tip and at the mother-daughter neck junction, which can persist in mature cells. Such a localization could be relevant to constraints of the cell wall incorporation of the mannoproteins. Depending on the regulation of Flop expression and on the incorporation of the proteins in the cell wall, a yeast population can be highly heterogeneous in Flo protein equipment. © 1998 John Wiley & Sons, Ltd.
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  • 117
    Electronic Resource
    Electronic Resource
    New York, NY [u.a.] : Wiley-Blackwell
    Yeast 14 (1998), S. 101-101 
    ISSN: 0749-503X
    Keywords: Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
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  • 118
    ISSN: 0749-503X
    Keywords: Saccharomyces cerevisiae ; amino acid uptake ; ssy mutants ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: In order to identify genes involved in uptake of isoleucine, leucine and valine in Saccharomyces cerevisiae we isolated mutants that, on a complex medium, were sensitive to an inhibitor of the biosynthesis of the branched-chain amino acids. Mutants that in a secondary screen showed reduced uptake of isoleucine, leucine and valine when growing in synthetic complete medium were further characterized. Genetic analysis identified five loci, named ssy1 through ssy5. ssy2 corresponds to the previously characterized bap1 mutation, which we recently have found to be allelic to stp1. ssy1, ssy3 and ssy5 exhibit a reduced uptake of phenylalanine, methionine and threonine, as well. Furthermore, they are resistant to several neutral amino acid analogs. ssy4 only affects uptake of few neutral amino acids and is as sensitive as the wild type to the amino acid analogs tested. It was previously found that a C-terminal truncation of 29 codons of BAP2, which encodes a branched-chain amino acid permease, results in increased uptake of the branched-chain amino acids. We find epistasis of the C-terminally truncated BAP2 gene over the ssy4 mutation, while the other ssy mutations are epistatic over the truncated BAP2 gene. SSY1, SSY3 and SSY5 were cloned from a low-copy genomic library by complementation of the mutants. The SSY3 gene and the SSY5 gene show no significant homology to any sequence in the databases. SSY1 is a member of the major family of genes encoding amino acid permeases in yeast. We discuss possible roles of Ssy1p in amino acid uptake. © 1998 John Wiley & Sons, Ltd.
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  • 119
    Electronic Resource
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    New York, NY [u.a.] : Wiley-Blackwell
    Yeast 14 (1998), S. 147-160 
    ISSN: 0749-503X
    Keywords: yeast physiology ; yeast viability ; flow cytometry ; bakers yeast ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: Yeast suspensions were analysed by flow cytometry after dye staining for determination of total and viable cell densities. Results were comparable to traditional colony counting and, in addition, provided further information on the percentage of total cells that were viable. The flow cytometric methods provided results within 20 min whereas colony counts were not available until 36 h. We evaluated a number of fluorescent dyes: ChemChrome Y (CY), oxonol (Ox), propidium iodide (PI), Fungolight and rhodamine 123, for accurate determination of viability of industrial yeast cultures and freshly re-hydrated high activity dried yeast (HADY). PI, Ox and CY gave the most conclusive live/dead discrimination and were the simplest to use. Culturing after dye staining and cell sorting demonstrated that the yeast remained viable after cell sorting and incubation with PI, CY or Ox. The methods, therefore, permit physical selection of individual yeast cells from populations of mixed viability. Sorting demonstrated that PI stained non-culturable cells whilst CY stained culturable cells. Analysis of yeast stained simultaneously with CY and PI or with Ox and PI demonstrated that PI and CY assays were in mutual agreement with respect to viability assessments. The Ox assay was in agreement with CY and PI for live/heat-killed mixtures. However, for re-hydrated HADY, Ox stained a significantly (P≤0·05) higher proportion of cells than did PI. © 1998 John Wiley & Sons, Ltd.
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  • 120
    ISSN: 0749-503X
    Keywords: GPM2 ; GPM3 ; phosphoglycerate mutase ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: Our previous data indicated that GPM1 encodes the only functional phosphoglycerate mutase in yeast. However, in the course of the yeast genome sequencing project, two homologous sequences, designated GPM2 and GPM3, were detected. They have been further investigated in this work. Key residues in the deduced amino acid sequence, shown to be involved in catalysis for Gpm1 (i.e. His8, Arg59, His181) are conserved in both enzymes. Overexpression of the genes under control of their own promoters in a gpm1 deletion mutant did not complement for any of the phenotypes. This could in part be attributed to a lack of expression due to their weak promoters. Higher level expression under the control of the yeast PFK2 promoter partially complemented the gpm1 defects, without restoring detectable enzymatic activity. Nevertheless, deletion of either GPM2 or GPM3, or the two deletions in concert, did not produce any obvious lesions for growth on a variety of different carbon sources, nor did they change the levels of key intermediary metabolites. We conclude that both genes evolved from duplication events and that they probably constitute non-functional homologues in yeast.
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  • 121
    ISSN: 0749-503X
    Keywords: Hansenula polymorpha ; 2 μm DNA ; plasmid partitioning ; nuclear segregation ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: Insertion of the HindIII-PstI fragment of Saccharomyces cerevisiae 2 μm DNA into the Hansenula polymorpha replicative plasmids decreases plasmid copy number and ensures their distribution to daughter cells at both mitotic and meiotic cell divisions. This suggests that the stabilization effect is caused by the improvement of plasmid partitioning. Deletion analysis revealed that the region of 2 μm DNA sequence responsible for the increase of mitotic stability of H. polymorpha plasmids involves the 2 μm STB locus and adjoining region. Further analysis demonstrated that the stabilization effect may depend on the number of 24-28 bp imperfect repeats which were found in several copies in the STB locus and adjoining region. © 1998 John Wiley & Sons, Ltd.
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  • 122
    ISSN: 0749-503X
    Keywords: methylotrophic yeast ; Pichia methanolica ; DNA transformation ; alcohol oxidase ; vacuolar protease ; protein expression ; fermentation ; human GAD65 ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: We describe a protein expression system in the methylotrophic yeast, Pichia methanolica. Methods for transformation and genetic manipulation of the organism were developed using an ade2 strain and the wild-type ADE2 gene. A vacuolar protease-deficient strain was constructed. Two genes encoding alcohol oxidases were found, yet a single isoform of alcohol oxidase was produced during methanol-fed fermentations. The promoter from this gene was used to drive expression. An integrating plasmid for the cytoplasmic expression of the 65 kDa isoform of human glutamate decarboxylase (human GAD65) was assembled. A strain harboring eight copies of this plasmid expressed enzymatically active human GAD65 at levels approaching 0·5 g/l. Identical amounts were made in Pichia pastoris. The recombinant GAD65 was purified to greater than 90% purity. © 1998 John Wiley & Sons, Ltd.
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  • 123
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    New York, NY [u.a.] : Wiley-Blackwell
    Yeast 14 (1998), S. 67-76 
    ISSN: 0749-503X
    Keywords: cellulose degradation ; endo-β-1,4-glucanase ; cellobiohydrolase ; cellodextrinase ; cellobiase ; Saccharomyces cerevisiae ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: Saccharomyces cerevisiae produces several β-1,3-glucanases, but lacks the multicomponent cellulase complexes that hydrolyse the β-1,4-linked glucose polymers present in cellulose-rich biomass as well as in haze-forming glucans in certain wines and beers. We have introduced into S. cerevisiae a functional cellulase complex for efficient cellulose degradation by cloning the Endomyces fibuliger cellobiase (BGL1) gene and co-expressing it with the Butyrivibrio fibrisolvens endo-β-1,4-glucanase (END1), the Phanerochaete chrysosporium cellobiohydrolase (CBH1) and the Ruminococcus flavefaciens cellodextrinase (CEL1) gene constructs in this yeast. The END1, CBH1 and CEL1 genes were inserted into yeast expression/secretion cassettes. Expression of END1, CBH1 and CEL1 was directed by the promoter sequences derived from the alcohol dehydrogenase II (ADH2), the phosphoglycerate kinase I (PKG1) and the alcohol dehydrogenase I (ADH1) genes, respectively. In contrast, BGL1 was expressed under the control of its native promoter. Secretion of End1p and Cel1p was directed by the signal sequence of the yeast mating pheromone α-factor (MFα1), whereas Cbh1p and Bgl1p were secreted using their authentic leader peptides. The construction of a fur1 ura3 S. cerevisiae strain allowed for the autoselection of this multicopy URA3-based plasmid in rich medium. S. cerevisiae transformants secreting biologically active endo-β-1,4-glucanase, cellobiohydrolase, cellodextrinase and cellobiase were able to degrade various substrates including carboxymethylcellulose, hydroxyethylcellulose, laminarin, barley glucan, cellobiose, polypectate, birchwood xylan and methyl-β-d-glucopyranoside. This study could lead to the development of industrial strains of S. cerevisiae capable of converting cellulose in a one-step process into commercially important commodities. © 1998 John Wiley & Sons, Ltd.
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  • 124
    ISSN: 0749-503X
    Keywords: Paracoccidioides brasiliensis ; chitin synthase ; dimorphic fungi ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: The nucleotide sequence of a chitin synthase gene (CHS2) of the dimorphic fungal human pathogen Paracoccidioides brasiliensis has been determined. The deduced amino acid sequence of Chs2p consists of 1043 residues and is highly homologous to other class II fungal chitin synthases. Computational structural analyses suggest very high similarity to other fungal chitin synthases with a highly variable region at the cytosolic amino-terminal region which may be related to its possible zymogenic nature, and the putative catalytic region close to seven membrane-spanning regions at the carboxyl terminus. The nucleotide sequence of CHS2 and its flanking regions has been submitted to GenBank under Accession Number Y09231. © 1998 John Wiley & Sons, Ltd.
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  • 125
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    Yeast 14 (1998), S. 171-180 
    ISSN: 0749-503X
    Keywords: gene duplication ; mammalian homologues ; transcript analysis ; mitochondria ; Saccharomyces cerevisiae ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: The yeast scERV1 gene is the first representative of a new emerging gene family. Its gene product is essential for the yeast cell and is involved in the biogenesis of mitochondria and the regulation of the cell cycle. Recently the general importance of the gene for the eukaryotic cell was shown by the identification of a structural and functional human homologue. The homologous mammalian ALR (augmenter of liver regeneration) genes from man, mouse and rat are important for different developmental stages of the organism as for example in spermatogenesis and the regeneration of damaged liver organs. Latest research identified an intron with an unusual 3′ branch site in the 5′ region of the yeast scERV1 gene. Analysis of the now available complete genome sequence from Saccharomyces cerevisiae identified a second yeast gene with homologies to scERV1 on chromosome 16. The corresponding gene product has a length of 196 amino acids similar to the 189 residues of the scERV1 protein and exhibits 30% identical amino acid residues in the highly conserved carboxy-terminal part of the polypeptides. Because of the structural similarities the new gene will be termed scERV2 from now on. For the scERV1 gene product it has just been shown that it is associated with yeast mitochondria. Analysis of the amino-terminal part of the putative scERV2 protein also identifies a typical leader sequence for import into mitochondria. The comparison of cDNA and genomic DNA from the scERV2 gene shows that no intron is present in this gene. To investigate the functional relation between the two yeast genes disruption experiments and complementation studies of mutants from scERV1 were performed. In addition the expression of messenger RNA under 15 different growth conditions was investigated by detailed Northern hybridization studies. Both genes show a complex and distinct expression pattern for their transcripts and are highly regulated under different physiological conditions. Moreover correct and efficient splicing of the transcript from the scERV1 gene was found to vary with the physiological state of the yeast cell, as further verified by reverse transcription-polymerase chain reaction analysis of transcripts from galactose-grown yeast cells. © 1998 John Wiley & Sons, Ltd.
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  • 126
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    Yeast 14 (1998), S. 215-224 
    ISSN: 0749-503X
    Keywords: Saccharomyces cerevisiae ; arginine permease ; turnover ; phosphorylation ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: To study the post-translational fate of arginine permease (Can1p), the gene coding for this transport protein was placed behind a constitutive promoter of plasma membrane ATPase (PMA1) and furnished with a Myc tag. In exponential-phase cells the amount of Can1p is constant, although turnover can be demonstrated. A rapid decrease in transport activity during the early stationary phase is paralleled by a corresponding net degradation of the protein. The amount of Can1p present in exponential cells grown on various nitrogen sources is the same, except in arginine-grown cells, in which the amount of the protein is markedly lower. This occurs solely when arginine serves as nitrogen source but not as an immediate consequence of, for example, arginine addition to cells growing on other nitrogen sources. It was demonstrated that Can1p is phosphorylated. Since Can1p expression under the PMA1 promoter is glucose-dependent, the amount of the permease expressed in high-glucose-grown cells is higher than in low-glucose-grown ones. Only a part of the Can1p overexpressed in high-glucose-grown cells is phosphorylated, while in low-glucose-grown cells the phosphorylated form probably represents the majority of Can1p. The permease phosphorylation or dephosphorylation is not related to transinhibition. © 1998 John Wiley & Sons, Ltd.
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  • 127
    ISSN: 0749-503X
    Keywords: Candida albicans ; multidrug resistance ; Fluconazole ; antifungal drugs ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: The Candida albicans CDR1 gene encodes a member of the ABC-type family of multidrug transporters which has been shown to be involved in azole resistance. Using an in-frame gene fusion between the CDR1 open reading frame and the green fluorescent protein allele yEGFP3, an optimized derivative for its use in C. albicans, we show here how the CDR1-yEGFP3 gene expression is induced in response to azoles as well as to other structurally unrelated drugs like cycloheximide. Moderate increases were observed for calcofluor, canavanine, 5′-fluorcytosine, cilofungin and caffeine, while no induction was found for the antifungals benomyl and amphotericin B or hydrogen peroxide at subinhibitory concentrations. The use of confocal microscopy enabled us to localize the Cdr1p fusion protein at the cell periphery, thus suggesting a cytoplasmic membrane localization. These results suggest deregulation of CDR1 gene as a putative mechanism for the generation of azole resistance in this clinically important pathogenic fungus. © 1998 John Wiley & Sons, Ltd.
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  • 128
    ISSN: 0749-503X
    Keywords: Saccharomyces cerevisiae ; YHR019 ; chromosome VIII ; asparaginyl-tRNA synthetase ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: Exploiting the asparagine auxotrophy of the Saccharomyces cerevisiae mutant strain 8556a, we have isolated the gene for the cytosolic asparaginyl-tRNA synthetase (AsnRS) of S. cerevisiae, by functional complementation of the mutation affecting this strain. The isolated gene could be identified to the open reading frame YHR019, called DED81, located on chromosome VIII. The mutant gene from the 8556a strain, asnrs--1, was amplified from genomic DNA by PCR. This gene contains a point mutation, leading to the replacement of a glycine residue by a serine in a region of the protein probably important for the asparaginyl-adenylate recognition. The protein encoded by YHR019 is very similar to cytosolic AsnRS from other eukaryotic sources. In a phylogenetic analysis based on AsnRS sequences from various organisms, the eukaryotic sequences were clustered. Expression of YHR019 in Escherichia coli demonstrated that a yeast AsnRS activity was produced. The recombinant enzyme was purified to homogeneity in three chromatography steps. We showed that the recombinant S. cerevisiae AsnRS was able to charge unfractionated yeast tRNA, but not E. coli tRNA, with asparagine. © 1998 John Wiley & Sons, Ltd.
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  • 129
    ISSN: 0749-503X
    Keywords: Saccharomyces cerevisiae ; chromosome VII ; FOL2 ; YTA7 ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: In the framework of the EU programme for systematic sequencing of the Saccharomyces cerevisiae genome we determined the sequence of a 9359 bp fragment of the right arm of chromosome VII. Five open reading frames (ORFs) of at least 300 nucleotides were found in this region. YGR267c encodes a protein with significant similarity to the enzyme GTP-cyclohydrolase I, that controls the first step in the biosynthetic pathway leading to various pterins and shows a high degree of sequence conservation from bacteria to mammals. We have recently demonstrated (Nardese et al., 1996) that YGR267c corresponds to the FOL2 gene, previously localized in the same chromosomal region by genetic mapping. The protein deduced from YGR270w belongs to the superfamily of putative ATPases associated with diverse cellular activities. It corresponds to the YTA7 gene, a member of a set of yeast genes coding for putative ATPases with high similarity to constituents of the 26S protease. The three ORFs YGR266w, YGR268c and YGR269w encode putative products of unknown function, with neither significant similarity to proteins in databases nor recognizable domains. YGR268c and YGR269w are partially overlapping ORFs: YGR268c seems to correspond to a real gene, whereas YGR269w is probably a fortuitous ORF. The sequence has been entered in the EMBL data library under Accession Number Y07893. © 1998 John Wiley & Sons, Ltd.
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  • 130
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    Yeast 14 (1998), S. 675-680 
    ISSN: 0749-503X
    Keywords: Candida albicans ; protein secretion ; SEC4 ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: The SEC4 gene product is a major component of the protein secretion machinery. More specifically, it is believed to play a pivotal role in targeting and fusion of secretory vesicles to the plasma membrane. Its recently described implication with the Saccharomyces cerevisiae Rho3p, which is required for directing growing points during bud formation, has prompted us to investigate the role and function of Sec4p in the morphological changes of the yeast pathogen Candida albicans. We have therefore cloned the C. albicans SEC4 gene. It encodes a 210 amino acids long protein sharing up to 75% homology to the S. cerevisiae homolog, when conserved changes are allowed. Its RNA is constitutively expressed in C. albicans grown under various physiological conditions. We also show that it can functionally complement a S. cerevisiae sec4 thermosensitive mutant. The sequence of the C. albicans SEC4 gene has been deposited in GenBank under Accession Number AF017183. © 1998 John Wiley & Sons, Ltd.
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  • 131
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    Yeast 14 (1998), S. 687-691 
    ISSN: 0749-503X
    Keywords: Kluyveromyces lactis ; phosphoribosyl-AMP cyclohydrolase ; phosphoribosyl-ATP pyrophosphohydrolase ; histidinol dehydrogenase ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: The Kluyveromyces lactis HIS4 gene was cloned by complementation of a Saccharomyces cerevisiae his4 mutant. Sequence analysis revealed a 2388 bp open reading frame encoding a single polypeptide predicted to encompass three distinct enzymatic activities (phosphoribosyl-AMP cyclohydrolase, phosphoribosyl-ATP pyrophosphohydrolase and histidinol dehydrogenase). This structural organization is strikingly similar to that of the His4 proteins from S. cerevisiae and Pichia pastoris. Transcript analysis detected a single mRNA species of 2.5 kb. The EMBL accession number of this gene is Y09503. © 1998 John Wiley & Sons, Ltd.
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  • 132
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    Yeast 14 (1998), S. 701-710 
    ISSN: 0749-503X
    Keywords: gene recognition ; linear discriminant analysis ; dynamic programming ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: A special program developed by the authors, called Pombe, identifies protein coding regions in the Schizosaccharomyces pombe genome. Linear discriminant analysis was applied to predict 5′-terminal, internal, 3′-terminal exons (coding-exon) and introns. The accuracy of the prediction was tested by cross verifications. The sensitivity, specificity and correlation coefficient for the internal exon prediction were 98·5%, 99·9% and 98·3% respectively at the nucleotide level. Open reading frames were studied and used to predict intron-less genes: 99·0% of such genes were identified with correct stopping sites. The gene structure was determined by dynamic programming and the prediction achieved 97·0% correlation coefficient at the nucleotide level. The program is available at http://clio.cshl.org/genefinder. © 1998 John Wiley & Sons, Ltd.
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  • 133
    ISSN: 0749-503X
    Keywords: Pichia pastoris ; methylotrophic yeasts ; dihydroxyacetone kinase ; DNA sequencing ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: Dihydroxyacetone kinase (DAK) is essential for methanol assimilation in methylotrophic yeasts. We have cloned the DAK gene from Pichia pastoris by functional complementation of a mutant that was unable to grow on methanol. An open reading frame of 1824 bp was identified that encodes a 65·3 kDa protein with high homology to DAK from Saccharomyces cerevisiae. Although DAK from P. pastoris contained a C-terminal tripeptide, TKL, which we showed can act as a peroxisomal targeting signal when fused to the green fluorescent protein, the enzyme was primarily cytosolic. The TKL tripeptide was not required for the biochemical function of DAK because a deletion construct lacking the DNA encoding this tripeptide was able to complement the P. pastoris dakΔ mutant. Peroxisomes, which are essential for growth of P. pastoris on methanol, were present in the dakΔ mutant and the import of peroxisomal proteins was not disturbed. The dakΔ mutant grew at normal rates on glycerol and oleate media. However, unlike the wild-type cells, the dakΔ mutant was unable to grow on methanol as the sole carbon source but was able to grow on dihydroxyacetone at a much slower rate. The metabolic pathway explaining the reduced growth rate of the dakΔ mutant on dihydroxyacetone is discussed. The nucleotide sequence reported in this paper has been submitted to GenBank with Accession Number AF019198. © 1998 John Wiley & Sons, Ltd.
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  • 134
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    Yeast 14 (1998), S. 681-686 
    ISSN: 0749-503X
    Keywords: Candida albicans ; cell wall protein ; DNA sequence ; hypha-specific ; proline-rich ; glutamine-rich ; serine and threonine-rich ; HWP1 ; RACE ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: A previously isolated partial cDNA encoding a cell wall protein antigen found on hyphal surfaces of the opportunistic fungal pathogen, Candida albicans (Staab et al., 1996) was used to clone the complete hyphal wall protein 1 gene (HWP1). Hyphal forms of C. albicans invade mucosal surfaces of immunocompromised patients such as those with AIDS. HWP1 consisted of an open reading frame predicting an acidic protein (pI of 3·37) with a calculated molecular size of 61,122. The antigenic domain was located in the N-terminal third of the protein. The remainder of the protein contained abundant hydroxy amino acids, and terminated with a string of 15 amino acids typical of sequences specifying post-translational modification with glycosylphosphatidylinositol (6PI). The analyses suggested that Hwp1 is a glucan-linked protein with serine/threonine-rich regions that are predicted to function in extending a ligand-binding domain into the extracellular space. The nucleotide sequence reported in this paper has been submitted to GenBank/EMBL databank with Accession Number U64206. © 1998 John Wiley & Sons, Ltd.
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  • 135
    ISSN: 0749-503X
    Keywords: I-Sce I fragmentation ; yeast ; genome ; cosmids ; colinearity ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: The high resolution complete physical maps of chromosomes VII and XV were constructed to form the basis for sequencing these chromosomes as part of the European systematic sequencing programme of the yeast genome, using a unique cosmid library from strain FY1679, and an original top-down mapping strategy involving I-Sce I chromosome fragmentation. A total of 138 and 196 cosmid clones were used to construct the maps for VII and XV, respectively, forming two unique contigs that cover the entirety of chromosomes (1091 kb each), except the telomeric repeats. Colinearity of the cosmid inserts with yeast DNA was verified, and the physical maps were eventually compared with the independently generated genetic maps. © 1998 John Wiley & Sons, Ltd.
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  • 136
    ISSN: 0749-503X
    Keywords: pyrolysis mass spectrometry ; Fourier tranform infrared spectroscopy ; chemometrics ; quality assurance ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: Two rapid spectroscopic approaches for whole-organism fingerprinting - pyrolysis mass spectrometry (PyMS) and Fourier transform infrared spectroscopy (FT-IR) - were used to analyse 22 production brewery Saccharomyces cerevisiae strains. Multivariate discriminant analysis of the spectral data was then performed to observe relationships between the 22 isolates. Upon visual inspection of the cluster analyses, similar differentiation of the strains was observed for both approaches. Moreover, these phenetic classifications were found to be very similar to those previously obtained using genotypic studies of the same brewing yeasts. Both spectroscopic techniques are rapid (typically 2 min for PyMS and 10 s for FT-IR) and were shown to be capable of the successful discrimination of both ale and lager yeasts. We believe that these whole-organism fingerprinting methods could find application in brewery quality control laboratories. © 1998 John Wiley & Sons, Ltd.
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  • 137
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    Yeast 14 (1998), S. 915-922 
    ISSN: 0749-503X
    Keywords: small GTP-binding proteins ; YPT1 ; YPT6 ; SSD1 ; SLY1 ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: Ypt6p, the yeast homologue of human RAB6, is required for protein trafficking at elevated temperatures. Biochemical data provide evidence that Ypt6p plays a role in an early step(s) of the secretory pathway: from ER to Golgi, or from cis to medial Golgi, or both. Here we show that overexpression of YPT1 suppresses the growth and secretion defects of a ypt6 temperature-sensitive (ts) strain. SLY1-20, encoding a dominant mutant allele that suppresses the lethal effect of YPT1, also suppresses the growth defect of a ypt6 ts strain. Conversely, SSD1, isolated as a suppressor of ypt6 ts, can suppress the growth defect of a ypt1 ts allele. These data suggest that Ypt6p has some redundant function with Ypt1p. However, overexpression of Ypt6p is toxic to a ypt1 ts strain, although it does not affect the growth of wild-type cells, suggesting that Ypt6p may sequester proteins shared with Ypt1p. This genetic evidence confirms the conclusion that Ypt6p is involved in an early step of the secretory pathway. © 1998 John Wiley & Sons, Ltd.
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  • 138
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    Yeast 14 (1998), S. 977-984 
    ISSN: 0749-503X
    Keywords: aldose reductase ; catalytic mechanism ; coenzyme binding ; sequence comparison ; SDR enzymes ; structure-function relationship ; xylose reductase ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: Yeast xylose (aldose) reductases are members of the aldo-keto reductase family of enzymes which are widely distributed in a variety of other organisms. In yeasts, these enzymes catalyse the first step of xylose metabolism where xylose is converted to xylitol. In the past 16 years, xylose reductases from yeasts able to ferment or utilize xylose have been isolated and studied mainly because of their importance in xylose bioconversions. In recent years, genes encoding xylose reductases from several yeasts have been cloned and sequenced. A comparison of the primary sequences of yeast xylose reductases with the much better characterized human aldose reductase and human aldehyde reductase reveals that the yeast enzymes are hybrids between aldo-keto reductases and the short chain dehydrogenases/reductases families of enzymes. Why this is so and its evolutionary significance is presently not known. This short review will critically examine the structure and function information that can be gleaned from the sequence comparison. Several interesting questions arise from the sequence comparison and these can provide fruitful areas for further investigations. © 1998 John Wiley & Sons, Ltd.
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  • 139
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    Yeast 14 (1998), S. 773-781 
    ISSN: 0749-503X
    Keywords: dolichol-PP-GlcNAc2 ; translocation ; endoplasmic reticulum ; alg1 ; exoglucanase ; S. cerevisiae ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: Transfer of truncated oligosaccharides to yeast exoglucanase (Exg) in Saccharomyces cerevisiae alg1 has been investigated. When incubated at the non-permissive temperature, alg1 cells secreted into the culture medium, in addition to the exoglucanase glycoforms secreted by wild type, underglycosylated forms as well as material with ionic properties of the non-glycosylated enzyme. As expected, none of the latter had affinity towards concanavalin A, but part of it bound to wheat germ agglutinin (WGA), suggesting that it contained, in addition to non-glycosylated Exg, glycoforms carrying non-reducing terminal GlcNAc. Only the WGA-bound material could be labelled with galactosyltransferase; furthermore, the label could be released by treatment with peptide-N4-N-acetyl-β-glucosamine asparagine amidase. These results unambiguously demonstrate that GlcNAc2 can be transferred from dolichol-PP-GlcNAc2 to one or both sequons of yeast Exg. Accordingly, they support previous observations suggesting that this early intermediate is able to translocate in vivo in order to make its sugar portion accessible to the oligosaccharyltransferase in the lumen of the endoplasmic reticulum. © 1998 John Wiley & Sons, Ltd.
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  • 140
    ISSN: 0749-503X
    Keywords: esterase ; beer ; brewer's yeast ; enzymatic hydrolysis ; specificity ; Saccharomyces cerevisiae TIP1 gene ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: An extracellular esterase was isolated from the brewer's yeast, Saccharomyces carlsbergensis. Inhibition by diisopropyl fluorophosphate shows that the enzyme has a serine active site. By mass spectrometry, the molecular weight of the enzyme was 16·9 kDa. The optimal pH for activity was in the range of four to five. Esterase activity was found in beer before pasteurization, and a low level of activity was still present after pasteurization. Caprylic acid, which is present in beer, competitively inhibited the esterase. The substrate preference towards esters of p-nitrophenol indicated that the enzyme prefers esters of fatty acids from four to 16 carbon atoms. The esterase has lipolytical activity; olive oil (C-18:1), which is a classical substrate for lipase, was hydrolysed. N-terminal sequence analysis of the esterase yielded a sequence which was identical to the deduced amino acid sequence of the S. cerevisiae TIP1 gene. The esterase preparation did not appear to contain significant amounts of other proteins than Tip1p, indicating that the TIP1 gene is the structural gene for the esterase. © 1998 John Wiley & Sons, Ltd.
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  • 141
    ISSN: 0749-503X
    Keywords: fermentation ; β-galactosidase ; heterologous gene expression ; Kluyveromyces lactis ; lactose-permease ; ribosomal DNA ; whey ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: A diploid strain of Saccharomyces cerevisiae able to metabolize lactose with high efficiency has been obtained. Haploid strains of Saccharomyces able to grow on lactose were constructed by cotransformation with two genes of Kluyveromyces lactis required for the utilization of the sugar, LAC4 and LAC12, encoding β-galactosidase and lactose permease respectively. Both genes were placed under the control of a galactose-inducible promoter and targeted to the rDNA encoding region (RDN1 locus) of the Saccharomyces genome. Lac+ transformants were selected on medium with lactose as the only carbon source. These transformants were mitotically stable, they maintained the Lac+ phenotype after growing in non-selective medium for more than 60 generations, but their growth was slow. We found that this lack of vigour was caused by their genetic background and not by a deficient expression of the heterologous genes. Therefore, their performance could be improved by crossing with a wild-type strain. Among the offspring of the crosses, two strains of opposite mating type were selected and mated to obtain a fast-growing Lac+ diploid. This diploid strain showed the typical fermentative behaviour of S. cerevisiae when it was grown in aerated liquid medium with glucose. In lactose medium, it exhibited a respiro-fermentative metabolism similar to that of K. lactis, with low ethanol production and high biomass yield. © 1998 John Wiley & Sons, Ltd.
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  • 142
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    Yeast 14 (1998), S. 895-903 
    ISSN: 0749-503X
    Keywords: Pichia pastoris ; pulsed-field gel electrophoresis ; chromosome-length polymorphisms ; gene stability ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: We have clearly resolved four chromosomal bands from four Pichia pastoris (Komagataella pastoris) strains by using contour-clamped homogeneous electric field gel electrophoresis. The size of the P. pastoris chromosomal bands ranged from 1·7 Mb to 3·5 Mb and total genome size was estimated to be 9·5 Mb to 9·8 Mb; however, chromosome-length polymorphisms existed among four strains. Thirteen cloned genes isolated from strain GTS115 were assigned to the separated chromosomes, revealing that different hybridization patterns were observed in the AOX2 and URA3 genes among strains. P. pastoris is frequently used as an efficient host for heterologous gene expressions. We analysed chromosomal stability of strain GTS115-derived recombinant cell expressing human serum albumin during serial cultivation under the condition of vegetative and non-selective growth. No chromosomal rearrangements were observed and the expression constructs integrated into the his4 locus on chromosome I were very stable even at 83 generations, suggesting that stable expression would be carried out even in large-scale fermentation. © 1998 John Wiley & Sons, Ltd.
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  • 143
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    Yeast 14 (1998), S. 935-942 
    ISSN: 0749-503X
    Keywords: antifungal drugs ; cytochrome-c oxidase ; gene dosage screening ; lanosterol C-14 demethylase ; overexpression assay ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: The post-genome sequencing era of Saccharomyces cerevisiae is defined by the analysis of newly discovered open reading frames of unknown function. In this report, we describe a genetic method for the rapid identification and characterisation of genes involved in a given phenotype. This approach is based on the ability of overexpressed genomic DNA fragments to cure an induced phenotype in yeast. To validate this concept, yeast cells carrying a yeast DNA library present on multicopy plasmid vectors were screened for resistance to the antifungal drug ketoconazole. Among 1·2 million colonies 13 clones tested positive, including those expressing the lanosterol C-14 demethylase, known to be a cellular target for azole drugs, and the cytochrome-c oxidase of mitochondria, regulating the respiratory chain electron transport. Several other resistant clones were identified, which code for yeast proteins of so far unknown function. These genes may represent potential candidates for antifungal drug effects. Together with the availability of the entire yeast genome sequence, the described genetic screening method is a powerful tool for the effective functional analysis of yeast genes. © 1998 John Wiley & Sons, Ltd.
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  • 144
    ISSN: 0749-503X
    Keywords: ADH ; hypoxic activation ; xylose fermenting yeasts ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: Two Pichia stipitis ADH genes (PsADH1 and PsADH2) were isolated by complementation of a Saccharomyces cerevisiae Adh--mutant. The genes enabled the transformants to grow in the presence of antimycin A on glucose, to use ethanol as sole carbon source and made them sensitive to allylalcohol.The sequences of the genes showed similarities of 70-77% to sequences of ADH genes of Candida albicans, Kluyveromyces lactis, K. marxianus, and S. cerevisiae and about 60% homology to those of Schizosaccharomyces pombe and Aspergillus flavus.Southern hybridization experiments suggested that P. stipitis has only these two ADH genes. Both genes are located on the largest chromosome of P. stipitis.PsADH2 encodes for the ADH activity that is responsible for ethanol formation at oxygen limitation. The gene is regulated at the transcriptional level. Moreover, also in cells grown on ethanol, only PsADH2 transcript was found. PsADH1 transcript was detected under aerobic conditions on fermentable carbon sources.The sequences have been deposited in the EMBL database under the accession numbers Y13238 (PsADH1) and Y13397 (PsADH2). © 1998 John Wiley & Sons, Ltd.
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  • 145
    ISSN: 0749-503X
    Keywords: cell wall ; chitin ; β-glucan ; Saccharomyces cerevisiae ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: A reliable acid hydrolysis method for quantitative determination of the proportion of β-glucan, mannan and chitin in Saccharomyces cerevisiae cell wall is reported together with a simple extraction procedure to quantify within a standard error of less than 2% the proportion of the wall per gram of cell dry mass. This method is an optimized version of Saeman's procedure based on sulfuric acid hydrolysis of complex polysaccharides. It resulted in an almost complete release of glucose, mannose and glucosamine residues from cell wall polysaccharides. After complete removal of sulfate ions by precipitation with barium hydroxide, the liberated monosaccharides were separated and quantified by high performance anion-exchange chromatography with pulsed amperometric detection. The superiority of this method over the hydrolysis in either trifluoroacetic or hydrochloric acid resides in its higher efficiency regarding the release of glucose from β1,6-glucan and of glucosamine from chitin. The sulfuric acid method was successfully applied to determine the β-glucan, mannan and chitin contents in cell walls of genetically well-characterized yeast mutants defective in cell wall biosynthesis, and in Schizosaccharomyces pombe cell walls. The simplicity and reliability of this procedure make it the method of choice for the characterization of cell walls from S. cerevisiae mutants generated in the EUROFAN programme, as well as for other pharmacological and biotechnological applications. © 1998 John Wiley & Sons, Ltd.
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  • 146
    ISSN: 0749-503X
    Keywords: non-Saccharomyces yeasts ; heterologous gene expression ; autonomously replicating expression vectors ; selective promoter identification ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: We have compared expression systems based on autonomously replicating vectors in the yeasts Saccharomyces cerevisiae, Schizosaccharomyces pombe, Kluyveromyces lactis, Hansenula polymorpha and Yarrowia lipolytica in order to identify a more suitable host organism for use in the expression cloning method (Dalbøge and Heldt-Hansen, 1994) in which S. cerevisiae has traditionally been used. The capacity of the expression systems to secrete active forms of six fungal genes encoding the enzymes galactanase, lipase, polygalacturonase, xylanase and two cellulases was examined, as well as glycosylation pattern, plasmid stability and transformation frequency. All of the examined alternative hosts were able to secrete more active enzyme than S. cerevisiae but the relative expression capacity of the individual hosts varied significantly in a gene-dependent manner. One of the most attractive of the alternative host organisms, Y. lipolytica, yielded an increase which ranged from 4·5 times to more than two orders of magnitude. As the initially employed Y. lipolytica XPR2 promoter is unfit in the context of expression cloning, two novel promoter sequences for highly expressed genes present in only one copy on the genome were isolated. Based on sequence homology, the genes were identified as TEF, encoding translation elongation factor-1α and RPS7, encoding ribosomal protein S7. Using the heterologous cellulase II (celII) and xylanase I (xylI) as reporter genes, the effect of the new promoters was measured in qualitative and quantitative assays. Based on the present tests of the new promoters, Y. lipolytica appears as a highly attractive alternative to S. cerevisiae as a host organism for expression cloning. GenBank Accession Numbers: TEF gene promoter sequence: AF054508; RPS7 gene promoter sequence: AF054509. © 1998 John Wiley & Sons, Ltd.
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  • 147
    ISSN: 0749-503X
    Keywords: BiP ; KAR2 ; Hsp70 ; peptide-binding domain ; secretion ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: Hsp70 is structurally composed of three domains, an amino-terminal ATPase domain, a proximal 18 kDa peptide-binding domain and a distal 10 kDa carboxy-terminal (C-terminal) domain. To dissect the functional significance of the distal 10 kDa domain, and the boundary region between the proximal and distal C-terminal domains of Kar2p in vivo in Saccharomyces cerevisiae, we constructed a series of plasmids which were truncated or had internal deletion mutations in this region. We found that all these mutations are recessive, and that the distal 10 kDa C-terminal domain, including the HDEL ER-retention sequence, is not essential for cell growth, although the major role of this 10 kDa C-terminal domain is due to the function of the HDEL ER-retention signal. We also found that the Kar2p region (Thr492-Thr512), corresponding to the β8-sheet in the peptide-binding domain, which constitutes the bottom plate of the binding pocket in E. coli DnaK, is essential for cell viability, and that the following Kar2p region (Glu513-Lys542), corresponding to α-helices A and B of E. coli DnaK, which was proposed to compose the lid of the binding pocket, is critical but not essential for yeast cell growth. This was further supported by the fact that the latter deletion showed a fully reversible ts phenotype in its growth and only a slight inhibitory effect on the secretion of α-amylase at non-permissive temperature. © 1998 John Wiley & Sons, Ltd.
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  • 148
    ISSN: 0749-503X
    Keywords: Yarrowia lipolytica ; acyl-CoA oxidase ; gene expression ; gene disruption ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: The ACO3 gene, which encodes one of the acyl-CoA oxidase isoenzymes, was isolated from the alkane-utilizing yeast Yarrowia lipolytica as a 10 kb genomic fragment. It was sequenced and found to encode a 701-amino acid protein very similar to other ACOs, 67·5% identical to Y. lipolytica Aco1p and about 40% identical to S. cerevisiae Pox1p. Haploid strains with a disrupted allele were able to grow on fatty acids. The levels of acyl-CoA oxidase activity in the ACO3 deleted strain, in an ACO1 deleted strain and in the wild-type strain, suggested that ACO3 encodes a short chain acyl-CoA oxidase isoenzyme. This narrow substrate spectrum was confirmed by expression of Aco3p in E. coli. © 1998 John Wiley & Sons, Ltd.
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  • 149
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    Yeast 14 (1998), S. 1341-1354 
    ISSN: 0749-503X
    Keywords: Kluyveromyces ; electrophoretic karyotyping ; contour-clamped homogeneous electric field electrophoresis ; chromosome variation ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: The analysis of the electrophoretic chromosome patterns of the species of the genus Kluyveromyces, reveals a high polymorphism in size, number and intensity of bands. Different sets of electrophoresis running conditions were used to establish species-specific patterns and also to detect intraspecific variation. According to their karyotypes, the species of this genus can be divided into two major groups. The first group includes the species K. africanus, K. bacillisporus, K. delphensis, K. lodderae, K. phaffi, K. polysporus and K. yarrowii, composing the so-called ‘Saccharomyces cerevisiae-like’ group, because their karyotypes resemble that of the species S. cerevisiae. The second group comprises the species K. aestuarii, K. blattae, K. dobzhanskii, K. lactis, K. marxianus, K. thermotolerans, K. waltii and K. wickerhamii, whose chromosomal patterns exhibit common characteristics very different to those of the species included in the ‘S. cerevisiae-like’ group. This division is concordant with the position of these species in previous phylogenetic reconstructions. Additionally, the intraspecific analysis of the chromosome patterns show a rich polymorphism in the heterogeneous species K. dobzhanskii, K. lactis, and K. marxianus, which is in concordance with the variability observed with other phenotypic or genetic markers. On the contrary, K. thermotolerans exhibits a homogeneous karyotype indicative of a very low level of chromosomal polymorphism, which is congruent with the reduced variability found in this species with other molecular markers. © 1998 John Wiley & Sons, Ltd.
    Additional Material: 6 Ill.
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  • 150
    ISSN: 0749-503X
    Keywords: Saccharomyces cerevisiae ; functional genomics ; quantitative phenotype ; chemostat ; competition ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: One possible route to the evaluation of gene function is a quantitative approach based on the concepts of metabolic control analysis (MCA). An important first step in such an analysis is to determine the effect of deleting individual genes on the growth rate (or fitness) of S. cerevisiae. Since the specific growth-rate effects of most genes are likely to be small, we employed competition experiments in chemostat culture to measure the proportion of deletion mutants relative to that of a standard strain by using a quantitative PCR method. In this paper, we show that both densitometry and GeneScan™ analysis can be used with similar accuracy and reproducibility to determine the proportions of (at least) two strains simultaneously, in the range 10-90% of the total cell population. Furthermore, we report on a model competition experiment between two diploid nuclear petite mutants, homozygous for deletions in the cox5a or pet191 genes, and the standard strain (ho::kanMX4/ho::kanMX4) in chemostat cultures under six different physiological conditions. The results indicate that competition experiments in continuous culture are a suitable method to distinguish quantitatively between deletion mutants that qualitatively exhibit the same phenotype. © 1998 John Wiley & Sons, Ltd.
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  • 151
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    New York, NY [u.a.] : Wiley-Blackwell
    Yeast 14 (1998), S. 1437-1438 
    ISSN: 0749-503X
    Keywords: Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
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  • 152
    Electronic Resource
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    New York, NY [u.a.] : Wiley-Blackwell
    Yeast 14 (1998), S. 133-146 
    ISSN: 0749-503X
    Keywords: Saccharomyces cerevisiae ; CDC28 gene ; RAD9 gene ; radiation sensitivity ; cell cycle checkpoint ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: CDC28-srm, a non-temperature-sensitive (ts) mutation in the CDC28 gene of Saccharomyces cerevisiae that affects fidelity of mitotic transmission of both mitochondrial and nuclear genetic structures (Devin et al., 1990), also affected cell growth and sensitivity to lethal effects of ionizing radiation. At 30°C CDC28-13, a ts mutation, was without appreciable effects on spontaneous mitochondrial rho--mutagenesis, cell growth and radiation sensitivity, whereas all three cell characteristics mentioned were affected (although to a lesser degree than by CDC28-srm) by CDC28-1, another ts mutation. CDC28-srm was without any significant effect on the rates of spontaneous nuclear gene mutations and γ-ray-induced mitotic recombination. An analysis of double mutants as regards their radiation sensitivity has revealed additive or even synergistic interactions between the CDC28-srm mutation and every one of the rad6-1 and rad52-1 mutations. The rad9Δ allele was found to be epistatic to CDC28-srm. These data suggest that the p34CDC28 protein is involved in the RAD9-dependent feedback control of DNA integrity operating at the cell cycle checkpoints. © 1998 John Wiley & Sons, Ltd.
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  • 153
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    Yeast 14 (1998), S. 189-194 
    ISSN: 0749-503X
    Keywords: branched-chain amino acids ; aminotransferase ; Schizosaccharomyces pombe ; Saccharomyces cerevisiae ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: The Saccharomyces cerevisiae genes for the cytosolic and mitochondrial branched-chain amino-acid aminotransferases (BCAT) were isolated recently. These genes show significant homology to mammalian ECA39, originally isolated as a gene regulated by the c-myc oncogene. We now report the isolation of the Schizosaccharomyces pombe eca39/BCAT gene. The S. pombe protein shows 47-52% identity to other eukaryotic BCAT proteins isolated from S. cerevisiae, nematode, mouse and man. A genetic growth assay for BCAT activity was established using an S. cerevisiae strain disrupted in both BCAT isoenzymes. Consequently, the activity of the S. pombe BCAT was demonstrated by genetic and biochemical means. Possible applications of BCAT-encoding genes as selection markers in yeast transformation are proposed. The sequence has been deposited in the GenBank data library under Accession Number U88029. © 1998 John Wiley & Sons, Ltd.
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  • 154
    ISSN: 0749-503X
    Keywords: recombinant DNA ; K. lactis genomic library ; pCXJ22 ; arginine biosynthesis ; KlARG8 ; mitochondrial transformation ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: A recombinant plasmid was isolated from a Kluyveromyces lactis genomic DNA library which complements a Saccharomyces cerevisiae arg8 mutant defective in the gene encoding acetylornithine aminotransferase. The complementation activity was found to reside within a 2.0 kb DNA fragment. Nucleotide sequence analysis revealed an open reading frame able to encode a 423-residue protein sharing 68·1% and 35·0% sequence identities with the products of the ARG8 and argD genes of S. cerevisiae and Escherichia coli. That the cloned gene, KlARG8, is the functional equivalent of S. cerevisiae ARG8 was supported by a gene disruption experiment which showed that K. lactis strains carrying a deleted chromosomal copy of KlARG8 are auxotrophic for arginine. The nucleotide sequence of KlARG8 has been submitted to GenBank under Accession Number U93209.
    Additional Material: 3 Ill.
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  • 155
    ISSN: 0749-503X
    Keywords: Escherichia coli ; diacylglycerol kinase ; lipase ; Saccharomyces cerevisiae ; TGL2 ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: Escherichia coli cells with a disrupted diacylglycerol kinase gene are unable to grow on media containing arbutin due to a lethal accumulation of diacylglycerol. In order to isolate genes from the yeast Saccharomyces cerevisiae involved in diacylglycerol metabolism we complemented an E. coli diacylglycerol kinase disruptant with a yeast genomic library and transformants were selected capable of growing in the presence of arbutin. Using this method, a gene (TGL2) was isolated coding for a protein resembling lipases from Pseudomonas. After expression of the TGL2 gene in E. coli, lipolytic activity towards triacylglycerols and diacylglycerols with short-chain fatty acids could be measured. Therefore, it is very likely that the TGL2 gene can complement the E. coli diacylglycerol kinase disruptant, because it encodes a protein that degrades the diacylglycerol accumulated after growth in the presence of arbutin. Disruption of the TGL2 gene in S. cerevisiae did not result in a detectable phenotype. The role of the Tgl2 protein in lipid degradation in yeast is still unclear. The nucleotide sequence published here has been submitted to the EMBL sequence data bank and is available under accession number X98000. © 1998 John Wiley & Sons, Ltd.
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  • 156
    ISSN: 0749-503X
    Keywords: elongation factor 3 ; YEF3 homolog ; ATPase ; ABC cassette ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: Yeast and other fungi contain a soluble elongation factor 3 (EF-3) which is required for growth and protein synthesis. EF-3 contains two ABC cassettes, and binds and hydrolyses ATP. We identified a homolog of the YEF3 gene in the Saccharomyces cerevisiae genome database. This gene, designated YEF3B, is 84% identical in protein sequence to YEF3, which we will now refer to as YEF3A. YEF3B is not expressed during growth under laboratory conditions, and thus cannot rescue growth of YEF3A deletion strains. However, YEF3B can take the place of YEF3A in vivo when expressed from the YEF3A or ADH1 promoters. The products of the YEF3A and YEF3B genes, EF-3A and EF-3B, respectively, were expressed from the ADH1 promoter and purified. Both factors possessed basal and ribosomal-stimulated ATPase activity, and had similar affinity for yeast ribosomes (103 to 113 nm). Km values for ATP were similar, but the Kcat values differed significantly. Ribosome-dependent ATPase activity of EF-3A was more efficient than EF-3B, since the Kcat and Kcat/Km values for EF-3A were about two-fold higher; however, the difference in Kcat/Km values between the two factors was small for basal ATPase activity. © 1998 John Wiley & Sons, Ltd.
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  • 157
    ISSN: 0749-503X
    Keywords: marine yeast ; superoxide dismutase ; Debaryomyces hansenii ; cloning ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: Cu-Zn superoxide dismutase (SOD-1) is a ubiquitously occurring eukaryotic enzyme with a variety of important effects on respiring organisms. A gene (dhsod-1) encoding a Cu-Zn superoxide dismutase of the marine yeast Debaryomyces hansenii was cloned using mRNA by the RT-PCR technique. The deduced amino-acid sequence shows ∼70% homology with that of cytosolic superoxide dismutase from Saccharomyces cerevisiae and Neurospora crassa, as well as lower homologies (between 55 and 65%) with the corresponding enzyme of other eukaryotic organisms, including human. The gene sequence encodes a protein of 153 amino acids with a calculated molecular mass of 15·92 kDa, in agreement with the observed characteristics of the purified protein from D. hansenii. The dhsod-1 sequence has been deposited in the public data library of the NCBI under Accession Number AFO 16383. © 1998 John Wiley & Sons, Ltd.
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  • 158
    ISSN: 0749-503X
    Keywords: gene disruption ; functional analysis ; Saccharomyces cerevisiae ; G418-resistance ; sticky-end PCR ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: The disruption of eight novel genes was realized in two genetic backgrounds. Among these open reading frames, NO333, NO348 and NO364 presented homologies with other proteins of yeast or other organisms, whereas NO320, NO325, NO339, NO384 and NO388 showed no similarity with any protein. Tetrad analysis of heterozygous deletant strains revealed that NO348, NO364 and NO388 are essential genes for vegetative growth, whereas NO320, NO325, NO333, NO339 and NO384 are non-essential. Basic phenotypic analyses of the non-lethal deletant strains as suggested in the six-pack B0 programme did not reveal any significant differences between parental and mutant strains. © 1998 John Wiley & Sons, Ltd.
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  • 159
    ISSN: 0749-503X
    Keywords: hexokinase PII ; glycolysis ; Tps1 ; fermentation ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: In the yeast Saccharomyces cerevisiae a novel control exerted by TPS1 (=GGS1=FDP1=BYP1=CIF1=GLC6=TSS1)-encoded trehalose-6-phosphate synthase, is essential for restriction of glucose influx into glycolysis apparently by inhibiting hexokinase activity in vivo. We show that up to 50-fold overexpression of hexokinase does not noticeably affect growth on glucose or fructose in wild-type cells. However, it causes higher levels of glucose-6-phosphate, fructose-6-phosphate and also faster accumulation of fructose-1,6-bisphosphate during the initiation of fermentation. The levels of ATP and Pi correlated inversely with the higher sugar phosphate levels. In the first minutes after glucose addition, the metabolite pattern observed was intermediate between those of the tps1Δ mutant and the wild-type strain. Apparently, during the start-up of fermentation hexokinase is more rate-limiting in the first section of glycolysis than phosphofructokinase. We have developed a method to measure the free intracellular glucose level which is based on the simultaneous addition of d-glucose and an equal concentration of radiolabelled l-glucose. Since the latter is not transported, the free intracellular glucose level can be calculated as the difference between the total d-glucose measured (intracellular+periplasmic/extracellular) and the total l-glucose measured (periplasmic/extracellular). The intracellular glucose level rose in 5 min after addition of 100 mm-glucose to 0·5-2 mm in the wild-type strain, ±10 mm in a hxk1Δ hxk2Δ glk1Δ and 2-3 mm in a tps1Δ strain. In the strains overexpressing hexokinase PII the level of free intracellular glucose was not reduced. Overexpression of hexokinase PII never produced a strong effect on the rate of ethanol production and glucose consumption. Our results show that overexpression of hexokinase does not cause the same phenotype as deletion of Tps1. However, it mimics it transiently during the initiation of fermentation. Afterwards, the Tps1-dependent control system is apparently able to restrict properly up to 50-fold higher hexokinase activity. © 1998 John Wiley & Sons, Ltd.
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  • 160
    ISSN: 0749-503X
    Keywords: sphingolipids ; hydroxylase ; cytochrome b5 ; CSG1 ; CSG2 ; calcium ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: Saccharomyces cerevisiae mutants lacking Scs7p fail to accumulate the inositolphosphorylceramide (IPC) species, IPC-C, which is the predominant form found in wild-type cells. Instead scs7 mutants accumulate an IPC-B species believed to be unhydroxylated on the amide-linked C26-fatty acid. Elimination of the SCS7 gene suppresses the Ca2+-sensitive phenotype of csg1 and csg2 mutants. The CSG1 and CSG2 genes are required for mannosylation of IPC-C and accumulation of IPC-C by the csg mutants renders them Ca2+-sensitive. The SCS7 gene encodes a protein that contains both a cytochrome b5-like domain and a domain that resembles the family of cytochrome b5-dependent enzymes that use iron and oxygen to catalyse desaturation or hydroxylation of fatty acids and sterols. Scs7p is therefore likely to be the enzyme that hydroxylates the C26-fatty acid of IPC-C. © 1998 John Wiley & Sons, Ltd.
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  • 161
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    Yeast 14 (1998), S. 359-369 
    ISSN: 0749-503X
    Keywords: SKSI ; snf3 ; low glucose ; over-expression ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: The SKS1 gene was originally identified as a multicopy suppressor of the growth defect of snf3 null mutations on low glucose concentrations. Snf3p is required for the rapid induction of HXT2 during growth on low substrate concentrations. Loss of Snf3p leads to a dramatic delay in expression of HXT2. Adaptation to low substrate concentrations does not occur in snf3 sks1 double null mutant strains, suggesting that SKS1 is required for the glucose-dependent expression of HXT2 in the absence of Snf3p activity. Over-expression of SKS1 leads to over-expression of Hxt2p, thus explaining the mechanism of suppression of the snf3 defect. SKS1 defines a novel, Snf3p-independent pathway for the expression of Hxt2p. Under certain growth conditions, over-expression of SKS1 itself leads to a growth defect which is diminished in snf3 hxt2 double mutants. This suggests that over-expression of Hxt2p at physiologically inappropriate times is detrimental to the cells. © 1998 John Wiley & Sons, Ltd.
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  • 162
    ISSN: 0749-503X
    Keywords: α1-antitrypsin ; Saccharomyces cerevisiae ; Hansenula polymorpha ; Pichia pastoris ; glycosylation ; secretion ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: Human α1-antitrypsin (α1-AT) is a major serine protease inhibitor in plasma, secreted as a glycoprotein with a complex type of carbohydrate at three asparagine residues. To study glycosylation of heterologous proteins in yeast, we investigated the glycosylation pattern of the human α1-AT secreted in the baker's yeast Saccharomyces cerevisiae and in the methylotrophic yeasts, Hansenula polymorpha and Pichia pastoris. The partial digestion of the recombinant α1-AT with endoglycosidase H and the expression in the mnn9 deletion mutant of S. cerevisiae showed that the recombinant α1-AT secreted in S. cerevisiae was heterogeneous, consisting of molecules containing core carbohydrates on either two or all three asparagine residues. Besides the core carbohydrates, variable numbers of mannose outer chains were also added to some of the secreted α1-AT. The human α1-AT secreted in both methylotrophic yeasts was also heterogeneous and hypermannosylated as observed in S. cerevisiae, although the overall length of mannose outer chains of α1-AT in the methylotrophic yeasts appeared to be relatively shorter than those of α1-AT in S. cerevisiae. The α1-AT secreted from both methylotrophic yeasts retained its biological activity as an elastase inhibitor comparable to that of α1-AT from S. cerevisiae, suggesting that the different glycosylation profile does not affect the in vitro activity of the protein. © 1998 John Wiley & Sons, Ltd.
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  • 163
    ISSN: 0749-503X
    Keywords: lys1+ gene ; Schizosaccharomyces pombe ; α-aminoadipate reductase ; peptide synthetase ; lysine biosynthesis ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: The 4·2 kbp lys1+ gene of Schizosaccharomyces pombe encoding the large subunit of α-aminoadipate reductase (EC1.2.1.31), an enzyme specific to lysine synthesis in higher fungi, was completely sequenced at the nucleotide level from pLYS1H. The S. pombe lys1+ gene product consists of 1415 amino acid residues and has a putative molecular weight of 155·8 kDa. The encoded protein converts α-aminoadipic acid to α-aminoadipate-δ-semialdehyde by an ATP-mediated adenylation. Analysis of the sequence showed that the putative protein encoded by lys1+ shares strong homology with the peptide antibiotic synthetases which also use an adenylation step. The sequence data reported in this paper have been submitted to GenBank database (Washington DC, USA) under the Accession Number U15923. © 1998 John Wiley & Sons, Ltd.
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  • 164
    ISSN: 0749-503X
    Keywords: Apiotrichum curvatum ; cDNA sequence ; DnaJ protein ; cytosolic Hsp70s ; ribosome association ; Sis1 protein ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: DnaJ proteins have been localized in different intracellular compartments of eukaryotes. In Apiotrichum curvatum, a fat-storing yeast, we found a DnaJ homolog associated with ribosomes and large cytosolic complexes as well. Using a plant DnaJ probe and a cDNA library constructed from poly(A)+ -RNA of A. curvatum grown on oleate we isolated a SIS1 cDNA coding for a 39·5 kDa protein. The putative protein contains neither a zinc finger motif nor a CAAX motif but is characterized by a J-domain at the N-terminal region and a large G-rich region in the middle part of the molecule. Heat shock applied for 1 h resulted in a pronounced but transient increase of the SIS1 mRNA. An antiserum was raised against the bacterially expressed protein. Cell fractions from A. curvatum were further separated by sedimentation centrifugation on sucrose gradients. Analysing the sub-fractions, we detected Sis1p mainly associated with ribosomes, and with particles sedimenting at approximately 200S. Hsp70 was found to be associated with the 200S fraction. The respective cytosolic A. curvatum Hsp70 cDNA was cloned and sequenced. High salt conditions caused the removal of Hsp70 and Sis1p from the 200S complexes. Mild RNase treatment of the 200S fraction afforded monosomes and 200S complexes unaffected by RNase. Heat shock led to a pronounced increase in the rate of de novo synthesis. However, due to the large pools of Sis1p on ribosomes and large cytosolic complexes, the increase in gene activation did not lead to a significant change of the total amount of Sis1p. Accession numbers are: Y12079 for ACHSP70 and Y12080 for ACSIS1. © 1998 John Wiley & Sons, Ltd.
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  • 165
    ISSN: 0749-503X
    Keywords: yeast ; S. cerevisiae ; genetics ; karyotypes ; chromosomal rearrangements ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: In order to study meiotic segregation of chromosome length polymorphism in yeast, we analysed the progeny of a cross involving two laboratory strains FL100trp and YNN295. Analysis of the parental strains led us to detect an important length polymorphism of chromosomes I and III in FL100trp. A reciprocal translocation involving 80 kb of the left arm of chromosome III and 45 kb of the right arm of chromosome I was shown to be the cause for the observed polymorphism in this strain. The characterization of the translocation breakpoints revealed the existence of a transposition hot-spot on chromosome I: the sequence of the translocation joints on chromosomes I and III suggests that the mechanism very likely involved homologous recombination between Ty2 transposable elements on each chromosome. Analysis of FL100, FL200 and FL100trp ura, which are related to FL100trp, shows that this reciprocal translocation is present in some of the strains of the FL series, whereas the parental strain FL100 does not carry the same rearrangement. We evidenced instead the duplication of 80 kb of chromosome III on chromosome I and a deletion of 45 kb of the right arm of chromosome I in this strain, indicating that secondary events might have taken place and that the strain currently named FL100 is not the common ancestor of the FL series. © 1998 John Wiley & Sons, Ltd.
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  • 166
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    Yeast 14 (1998), S. 665-674 
    ISSN: 0749-503X
    Keywords: histidine kinase ; phosphorylation ; signal transduction ; gene ; two-component ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: We have cloned and analysed the sequence of a putative histidine kinase, two-component gene (CaHK1) from Candida albicans. This gene encodes a 2471 amino acid protein (Cahk1p) with an estimated molecular mass of 281·8 kDa. A homology search of Cahk1p with other proteins in the databases showed that Cahk1p exhibits the greatest homology at its C-terminus with both the sensor and regulator components of prokaryotic and eukaryotic two-component histidine kinases. A further analysis of this homology showed that the Cahk1p possessed both sensor and regulator domains in the same polypeptide. Also, Cahk1p is likely to be a soluble protein. The sensor kinase domain of Cahk1p contains conserved motifs that are characteristic of all histidine kinase proteins, including the putative histidine which is believed to be autophosphorylated during activation, ATP binding motifs and others (F- and N-motifs), with unknown function. The Cahk1p regulator domain also contains conserved aspartate and lysine residues and the putative aspartate, which is secondarily phosphorylated by the autophosphorylated histidine. Finally, according to the codon usage frequency of the CaHK1 gene in comparison with other genes from C. albicans, there would appear to be a low level of expression of the gene. The accession number for the described sequence is AF013273, as filed in the EMBL/GenBank/DDBJ database. © 1998 John Wiley & Sons, Ltd.
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  • 167
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    Yeast 14 (1998), S. 655-664 
    ISSN: 0749-503X
    Keywords: Saccharomyces cerevisiae ; PCR-based disruption ; YOL113w ; YOL100w ; YOL107w ; YOR267c ; YGL196w ; YGL194c ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: Using PCR to construct disruption cassettes, null alleles of six genes have been created in Saccharomyces cerevisiae. In a FY1679 background, no defects were detected in any of the haploid deletion mutants with respect to growth, gross morphology, or mating. A diploid FY1679-derived Δygl194c/Δygl194c homozygous disruptant displayed reduced sporulation. In contrast to the lack of phenotypic consequences of Δyol100w disruptions in the FY1679 background, in the CEN.PK2 strain even a heterozygous disruption of the same gene caused striking effects, very slow vegetative growth and highly impaired sporulation. Tetrad analysis showed YOL100w to be an essential gene in this strain. A copy of the YGL194c or the YOL100w wild-type gene borne on a centromeric episomal plasmid was introduced into a corresponding disruption mutant strain, and in both cases was found to partially complement the defects. © 1998 John Wiley & Sons, Ltd.
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  • 168
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    Yeast 14 (1998), S. 623-631 
    ISSN: 0749-503X
    Keywords: Saccharomyces cerevisiae ; bicarbonate ; meiosis ; sporulation ; respiration ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: Meiosis and sporulation in the yeast Saccharomyces cerevisiae requires social communication, mediated by an extracellular factor which is secreted from cells during sporulation and accumulates in a cell density-dependent manner. We show here genetic and biochemical analyses supporting our conclusion that the extracellular factor is bicarbonate acting as an alkali to elevate extracellular pH. Sporulation defects of mdh1 (mitochondrial malate dehydrogenase) mutants and of wild-type cells at low density were rescued extracellularly by addition of bicarbonate or other alkaline solutions to raise medium pH. Addition of bicarbonate (or alkalization of medium) raised steady-state levels of mRNA in respiration-deficient mdh1 mutants and inhibited proliferation of wild-type cells at low density. These results indicate that the two conditions (respiration competency and high cell density), required for meiosis and sporulation, are essential for extracellular accumulation of bicarbonate and resulting alkalization of medium. © 1998 John Wiley & Sons, Ltd.
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  • 169
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    Yeast 14 (1998), S. 617-622 
    ISSN: 0749-503X
    Keywords: Saccharomyces cerevisiae ; extracellular factor ; meiosis ; sporulation ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: Meiosis and sporulation in the yeast Saccharomyces cerevisiae has been classically viewed as an example of unicellular, eukaryotic differentiation that occurs in response to nutritional starvation. We present evidence that S. cerevisiae produces an extracellular factor(s), called meiosis-promoting factor (MEP), that is required, in addition to starvation conditions, for efficient meiosis and sporulation. This factor is secreted and accumulates in a cell density-dependent fashion such that cells at a low density sporulate poorly under conditions in which cells at a high density sporulate efficiently. Conditioned medium from sporulating cells at a high density contains a small anionic molecule that has cytostatic activity and stimulates sporulation of cells at low density under a normal starvation condition. These results indicate that MEP-mediated social communication between cells is required for meiosis and sporulation. © 1998 John Wiley & Sons, Ltd.
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  • 170
    ISSN: 0749-503X
    Keywords: epitope tagging ; green fluorescent protein ; functional analysis ; overexpression studies ; gene deletion ; gene truncation ; polymerase chain reaction ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: An important recent advance in the functional analysis of Saccharomyces cerevisiae genes is the development of the one-step PCR-mediated technique for deletion and modification of chromosomal genes. This method allows very rapid gene manipulations without requiring plasmid clones of the gene of interest. We describe here a new set of plasmids that serve as templates for the PCR synthesis of fragments that allow a variety of gene modifications. Using as selectable marker the S. cerevisiae TRP1 gene or modules containing the heterologous Schizosaccharomyces pombe his5+ or Escherichia coli kanr gene, these plasmids allow gene deletion, gene overexpression (using the regulatable GAL1 promoter), C- or N-terminal protein tagging [with GFP(S65T), GST, or the 3HA or 13Myc epitope], and partial N- or C-terminal deletions (with or without concomitant protein tagging). Because of the modular nature of the plasmids, they allow efficient and economical use of a small number of PCR primers for a wide variety of gene manipulations. Thus, these plasmids should further facilitate the rapid analysis of gene function in S. cerevisiae. © 1998 John Wiley & Sons, Ltd.
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  • 171
    ISSN: 0749-503X
    Keywords: fission yeast ; gene deletions ; gene truncations ; overexpression studies ; epitope tagging ; polymerase chain reaction ; gene expression ; green fluorescent protein ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: We describe a straightforward PCR-based approach to the deletion, tagging, and overexpression of genes in their normal chromosomal locations in the fission yeast Schizosaccharomyces pombe. Using this approach and the S. pombe ura4+ gene as a marker, nine genes were deleted with efficiencies of homologous integration ranging from 6 to 63%. We also constructed a series of plasmids containing the kanMX6 module, which allows selection of G418-resistant cells and thus provides a new heterologous marker for use in S. pombe. The modular nature of these constructs allows a small number of PCR primers to be used for a wide variety of gene manipulations, including deletion, overexpression (using the regulatable nmt1 promoter), C- or N-terminal protein tagging (with HA, Myc, GST, or GFP), and partial C- or N-terminal deletions with or without tagging. Nine genes were manipulated using these kanMX6 constructs as templates for PCR. The PCR primers included 60 to 80 bp of flanking sequences homologous to target sequences in the genome. Transformants were screened for homologous integration by PCR. In most cases, the efficiency of homologous integration was ≥50%, and the lowest efficiency encountered was 17%. The methodology and constructs described here should greatly facilitate analysis of gene function in S. pombe. © 1998 John Wiley & Sons, Ltd.
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  • 172
    ISSN: 0749-503X
    Keywords: Saccharomyces cerevisiae ; gene cloning ; gene disruption ; functional analysis ; chromosome XVI ; translation ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: A 7·24 kb genomic DNA fragment from the yeast Saccharomyces cerevisiae chromosome XVI was isolated by complementation of a new temperature-sensitive mutation tsa1. We determined the nucleotide sequence of this fragment located on the right arm of chromosome XVI. Among the three, complete open reading frames: YPR041w, YPR042c and YPR043w contained within this fragment, the gene YPR041w was shown to complement the tsa1 mutation and to correspond to the TIF5 gene encoding an essential protein synthesis initiation translation factor. The YPR042c gene encodes a hypothetical protein of 1075 amino acids containing four putative transmembrane segments and is non-essential for growth. The gene YPR043c encoding the 10 kDa product, highly similar to the human protein L37a from the 60S ribosomal subunit, was found to be essential and a dominant lethal. We conclude that three tightly linked yeast genes are involved in the translation process. © 1998 John Wiley & Sons, Ltd.
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  • 173
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    Yeast 14 (1998), S. 1007-1016 
    ISSN: 0749-503X
    Keywords: nicotine ; diphenylamine ; astaxanthin biosynthesis ; Xanthophyllomyces dendrorhous ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: The effect of nicotine and diphenylamine on astaxanthin biosynthesis in Xanthophyllomyces dendrorhous was studied. The effects were analysed under standard and low temperature conditions. It was found that 10 mm-nicotine inhibits the cyclization of lycopene and de novo protein synthesis was not needed to reverse the inhibition. The oxidation of β-carotene was irreversibly inhibited by 10 μM-diphenylamine while the dehydrogenation of phytoene was reversibly inhibited by 60 μM-diphenylamine. The simultaneous exposure to low temperature (4°C) overcomes the inhibition of β-carotene oxidation at low diphenylamine concentration. © 1998 John Wiley & Sons, Ltd.
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  • 174
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    Yeast 14 (1998), S. 1069-1087 
    ISSN: 0749-503X
    Keywords: amines and amides ; biodetergents ; biocides ; bioremediation ; biosensors ; Candida rugosa ; carbohydrate esters ; cosmetics and perfumery ; food and flavour ; immobilisation ; isoenzymes ; lipases ; molecular biology ; pharmaceuticals ; single cell protein ; specificity ; tanning ; ultra-structure ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: This review describes how the versatile Candida rugosa lipases (CRL) have extended the frontiers of biotechnology. As evidenced by the current literature, CRL claims more applications than any other biocatalyst. This review comprises a detailed discussion on the molecular biology of CRL, its versatile catalytic reactions, broad specificities and diverse immobilization strategies. It also discusses its role in the food and flavour industry, the production of ice cream and single cell protein, biocatalytic resolution of life-saving pharmaceuticals, carbohydrate esters and amino acid derivatives unobtainable by conventional chemical synthesis, potent biocide making, biosensor modulations, eco-friendly approach and bioremediation, biosurfactants in detergent making, and recently, cosmetics and perfumery. © 1998 John Wiley & Sons, Ltd.
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  • 175
    ISSN: 1530-0358
    Keywords: Inflammatory bowel disease ; Crohn's disease ; Ulcerative colitis ; Genetics ; Family history
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract BACKGROUND: Although the etiology of inflammatory bowel disease (IBD) is unknown, there is increasing evidence that genetic predisposition plays a major etiologic role. To provide the framework for gene identification using a positional cloning approach, ascertainment of families with multiple affected members and careful documentation of pedigrees are essential. Objective: To report the initial findings of the IBD Genetics Project of the Mount Sinai Hospital IBD Research Unit. METHODS: All records of patients with ulcerative colitis and Crohn's disease followed at the Mount Sinai Hospital IBD Unit were reviewed. A questionnaire was sent to all patients to ascertain those with a family history of IBD. Patients with a presumed family history were contacted by a research assistant, and after confirmation of diagnosis, relevant clinical information, pedigrees, and consent to contact family members were obtained. Blood for DNA and cell line preparation were collected from affected and nonaffected family members. RESULTS: Of 2,504 patients registered in the IBD database, 231 (9.2 percent) were found to have an affected family member: 96 of 964 (10 percent) with Crohn's disease (CD) and 135 of 1,540 (8.8 percent) with ulcerative colitis (UC). A mean of 2.4 family members were affected. In families in which the proband had CD, 82.3 percent had only two affected family members, 78.1 percent had only family members affected with CD, and 82.3 percent had only first-degree family members affected. In families in which the proband had UC, 70.4 percent had only two affected family members, 71.1 percent had only family members affected with UC, and 65.2 percent had only first-degree family members affected. In the 231 families, there were 103 sibling pairs: 46 percent with CD, 28 percent with UC, and 26 percent with CD/UC. CONCLUSION: These data suggest that approximately 10 percent of IBD patients have affected family members, with the rate being similar in UC and CD. Future research is directed to genome scanning and linkage analysis in this cohort of patients.
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  • 176
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    Journal of molecular medicine 75 (1997), S. 502-511 
    ISSN: 1432-1440
    Keywords: Key words T helper 1/T helper 2 ; Genetics ; Interleukin 12 ; Allergy ; Autoimmunity ; Leishmania
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract  We examined the effect of genetic background on Th1/Th2 development. We discuss data demonstrating that genetic background is an important determinant of interleukin-12 (IL-12) responsiveness and the potential implications for disease progression in murine experimental leishmaniasis. Genetic analysis of the differential control of IL-12 responsiveness led to the identification of a controlling locus on the middle portion of murine chromosome 11. This genetic region (or its human counterpart, 5q31) has been associated with increased disease susceptibilities for several atopic, infectious, and autoimmune disorders. We discuss potential roles for genetic control of IL-12 responsiveness in the development of these diseases.
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  • 177
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    Annals of hematology 74 (1997), S. 131-134 
    ISSN: 1432-0584
    Keywords: Key words Hodgkin's disease ; Sex ; Age ; Genetics
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract  Familial Hodgkin's disease (FHD) is estimated to represent approximately 4.5% of all cases of Hodgkin's disease (HD). Shared environmental factors, such as Epstein-Barr virus and other viral agents, and genetic determinants have all been proposed to explain familial aggregation of HD. In order to compare the characteristic features of FHD with those of the much more common sporadic form, we reviewed 28 articles on FHD, published between 1972 and 1995, and analyzed in further detail data from 18 papers, reporting on a total of 328 patients. The male-to-female ratio of the FHD population examined was 1.5, similar to that reported for sporadic HD, and lower than the one suggested for FHD by some authors. On the other hand, a significant difference was found between sporadic and familial HD according to age at diagnosis; that is, only one major peak between 15 and 34 years was present in the group of patients with FHD. Further investigation of FHD in young adulthood may provide insight into the hypothesis of a genetic or infectious etiology of the disease.
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  • 178
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    Sexual plant reproduction 10 (1997), S. 13-21 
    ISSN: 1432-2145
    Keywords: Key words Soybean ; Male sterility ; Genetics ; Callase ; Callose
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract  Genetic and cytological studies were conducted with a new male-sterile, female-fertile soybean [Glycine max (L.) Merr.] mutant. This mutant was completely male sterile and was inherited as a single-recessive gene. No differences in female or male gamete transmission of the recessive allele were observed between reciprocal cross-pollinations in the F1 or F2 generations. This mutant was not allelic to any previously identified soybean genic male-sterile mutants: ms1, ms2, ms3, ms4, ms5, or ms6. No linkage was detected between sterility and flower color (W1 locus), or between sterility and pubescence color (T1 locus). Light microscopic and cytological observations of microsporogenesis in fertile and sterile anthers were conducted. The structure of microspore mother cells (MMC) in male-sterile plants was identical to the MMCs in male-fertile plants. Enzyme extraction analyses showed that there was no callase activity in male-sterile anthers, and this suggests that sterility was caused by retention of the callose walls, which normally are degraded around tetrads at the late tetrad stage. The tapetum from male-sterile anthers also showed abnormalities at the tetrad stage and later stages, which were expressed by an unusual formation of vacuoles, and by accumulation of densely staining material. At maturity, anthers from sterile plants were devoid of pollen grains.
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  • 179
    ISSN: 1432-2072
    Keywords: Key words Naltrexone ; Alcohol ; Genetics ; Social drinkers ; Family history of alcoholism ; Biphasic Alcohol Effects Scale
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract We investigated specific subjective effects of naltrexone pretreatment or placebo during various intervals on the breath alcohol level (BAL) curve in nonalcoholic volunteers. Fifteen high-risk (social drinkers with an alcoholic father) and 14 low-risk (no alcoholic relatives in at least two generations) subjects were tested in a double-blind, placebo-controlled study of the effects of 50 mg oral naltrexone on response to a moderate dose of alcohol. Dependent measures included subjective stimulation and sedation subscales from the Biphasic Alcohol Effects Scale (BAES) and mood subscales from the Profile of Mood States (POMS). At rising BALs, high-risk subjects showed a naltrexone-related attenuation of BAES stimulation. This effect was not evident in low-risk subjects, who directionally showed the opposite effect, although nonsignificant. For both groups, there were no significant naltrexone-related effects for BAES sedation; however, naltrexone did affect several POMS scales on alcohol response, such as decreased vigor, and increased fatigue, tension, and confusion. Confusion was significantly elevated for the high-risk group during rising BALs of the naltrexone session. The results suggest a differential response to naltrexone, based on paternal history of alcoholism and level of stimulation experienced during alcohol drinking.
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  • 180
    ISSN: 1432-1459
    Keywords: Key words Parkinson’s disease ; Genetics ; Mitochondrial DNA
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract To date, five single base pair changes of the mitochondrial DNA have been reported to occur either exclusively or with increased frequency in Caucasian patients with Parkinson’s disease (PD) and it has been postulated that these mutations might be causally related to the observed inhibition of mitochondrial respiratory chain function in PD. To evaluate these findings, we analysed the frequency of all five polymorphisms in 100 cases of pathologically proven cases of PD. We were either unable to detect the previously described polymorphisms in our series or found them to be present with the same frequency among controls. Our data do not support the hypothesis of an involvement of the mitochondrial DNA in the pathogenesis of PD.
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  • 181
    ISSN: 1432-2072
    Keywords: Key words Startle ; Prepulse inhibition ; Inbred strains ; Haloperidol ; Catalepsy ; Schizophrenia ; Genetics
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract The acoustic startle response (ASR), prepulse inhibition (PPI) of the ASR and the effects of haloperidol on the ASR and PPI were examined in C57BL/6J (B6) and DBA/2 (D2) inbred mouse strains and their F1 and F2 progeny. The startle stimulus was a 60-ms, 110-dB, 10-kHz tone; the prepulse stimuli were 20-ms white noise bursts at 56, 68 and 80 dB against a 50-dB background presented 100-ms before the startle pulse. The B6 strain showed modest PPI (25–40%); in contrast, the D2 strain showed on average no PPI and numerous individuals showed prepulse augmentation (PPA). The F2 progeny showed an intermediate PPI; however, the extreme values ranged from 200% PPA to essentially 100% PPI. Haloperidol in dose-dependent fashion, increased PPI in both the B6 and D2 strains; the threshold dose was in the range of 0.1–0.2 mg/kg. Raclopride (0.3 mg/kg), clozapine (2 mg/kg) and risperidone (0.4 mg/kg) also increased PPI in both strains. The effects of haloperidol (0.4 mg/kg) on PPI in 140 F2 progeny were examined. For all prepulse intensities, there were highly significant (r 〉 0.80) and negative correlations between baseline PPI and the haloperidol-induced change in PPI. Thus, those animals that showed the greatest PPA showed the greatest haloperidol-induced increase in PPI. There was, however, significant variance in the haloperidol response; plots of the regression residuals showed the most and least responsive animals differed by almost 100% in effect on PPI. The F2 progeny were subsequently phenotyped for haloperidol-induced catalepsy. There was no association between the variation in effects on catalepsy and PPI. However, it was observed that those individuals with the poorest baseline PPI were catalepsy non-responsive.
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  • 182
    ISSN: 1432-2072
    Keywords: Key words Mouse ; Inbred strains ; Behavior ; Genetics ; Locomotion ; Open field activity ; Learning ; Memory ; Aggression ; Parental behaviors ; Acoustic startle ; Prepulse inhibition ; Alcohol ; Nicotine ; Cocaine ; Opiates ; Haloperidol ; Diazepam ; Breeding ; Embryonic stem cell lines ; Transgenic ; Knockouts ; Null mutation
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract  Choosing the best genetic strains of mice for developing a new knockout or transgenic mouse requires extensive knowledge of the endogenous traits of inbred strains. Background genes from the parental strains may interact with the mutated gene, in a manner which could severely compromise the interpretation of the mutant phenotype. The present overview summarizes the literature on a wide variety of behavioral traits for the 129, C57BL/6, DBA/2, and many other inbred strains of mice. Strain distributions are described for open field activity, learning and memory tasks, aggression, sexual and parental behaviors, acoustic startle and prepulse inhibition, and the behavioral actions of ethanol, nicotine, cocaine, opiates, antipsychotics, and anxiolytics. Using the referenced information, molecular geneticists can choose optimal parental strains of mice, and perhaps develop new embryonic stem cell progenitors, for new knockouts and transgenics to investigate gene function, and to serve as animal models in the development of novel therapeutics for human genetic diseases.
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  • 183
    ISSN: 1432-2072
    Keywords: Key words Cocaine ; Operant behavior ; Genetics ; Mice
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract  The availability of numerous genetically homogenous mouse strains permits the analysis of genetic influences on behavior and also behavioral sensitivity (responsivity) to drugs of abuse. The current study was conducted to characterize discriminated operant responding for food in four inbred strains (Balb/cByJ, DBA/2J, C57BL/6J, SJL/J), an F1 Hybrid (C57BL/6×SJL), and one outbred strain (CD1) of mouse. The effect of cocaine on this operant behavior was also examined. Initially, all animals were trained to nosepoke for food on a continuous reinforcement schedule. The minimum response requirement for reinforcement was increased every 5 days until the animals were responding on an FR-15 schedule of reinforcement. All strains increased operant responding as the schedule of reinforcement was raised. However, significant differences in response rate and discrimination learning were observed among the various strains of mice. Cocaine administration reduced operant responding for food in Balb/cByJ, C57BL/6J, C57BL/6×SJL/J and CD1 mice at a dose of 15.0 mg/kg, whereas higher doses were required in DBA/2J mice (30.0 mg/kg) and SJL/J mice (56.0 mg/kg). These results suggest that operant performance and the effect of cocaine on this behavior is differentially influenced by genetic make-up.
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  • 184
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    Journal of mathematical biology 35 (1997), S. 294-320 
    ISSN: 1432-1416
    Keywords: Key words: Natural selection ; Homoclinic chaos ; Genetics
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Mathematics
    Notes: Abstract.  We modify a simple mathematical model for natural selection originally formulated by Robert M. May in 1983 by permitting one homozygote to have a larger selective advantage when rare than the other, and show that the new model exhibits dynamical chaos. We determine an open region of parameter space associated with homoclinic points, and prove that there are infinite sequences of period-doubling bifurcations along selected paths through parameter space. We also discuss the possibility of chaos arising from imbalance in the homozygote fitnesses in more realistic biological situations, beyond the constraints of the model.
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  • 185
    ISSN: 1432-2242
    Keywords: Key words Stress proteins ; Heat tolerance ; Chloroplast ; Genetics
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract  We have used a combination of molecular and classical genetic approaches to delineate the relationship between a specific HSP member and cell viability under heat stress. Using recombinant inbred lines (RILs) of wheat, derived from a cross of the thermotolerant cultivar ‘Mustang’ and the thermosusceptible cultivar ‘Sturdy,’ we have identified a unique HSP and a differentially expressed cDNA sequence, both related to the plastid-localized HSP26 gene family, that are closely associated with acquired thermotolerance in wheat. An isoform of HSP26 was synthesized under heat stress in all examined thermotolerant RILs and ‘Mustang’, and was absent in all examined thermosusceptible RILs and ‘Sturdy.’ Using a modified differential-display method, we have also identified a gene-specific cDNA sequence that is similar to other known members of the wheat HSP26 gene family and is selectively expressed in ‘Mustang’ and most of the examined thermotolerant RILs, but not expressed in ‘Sturdy’ and all the thermosusceptible RILs. These results suggest a genetic linkage between the acquired thermotolerance trait and the differential expression of a unique member of the HSP26 gene family.
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  • 186
    ISSN: 1432-2242
    Keywords: Key words Extreme virus resistance ; Potyviruses ; Genetics ; Genes Rysto and Ra ; Broad-spectrum resistance
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract  Extreme resistance to the potato V potyvirus (PVV) was found in four potato cultivars that contain Ry genes from Solanum stoloniferum. When plants of these cultivars, were inoculated by grafting in shoot tips from PVV-infected tomato plants, necrotic symptoms developed in some cultivars, although a full hypersensitive reaction was not elicited, while other cultivars were symptomless. PVV replication was not detected in any of the inoculated plants by ELISA, an infectivity assay of leaf extracts by manual inoculation to Nicotiana benthamiana indicator plants, or by ‘return grafting’ of shoot tips taken from newly developed shoots of the potato plants to virus-free indicator plants of tomato. These methods readily detected PVV infection in inoculated plants of cv ‘Flourball’, which does not contain an Ry gene and is susceptible, and in cvs ‘Maris Piper’ and ‘Dr Macintosh’, which contain gene Nv conditioning a hypersensitive reaction to inoculation. One of the Ry-containing cultivars, ‘Barbara’, has been previously shown to contain two genes that control extreme resistance, defined as no viral replication in intact plants, to the potyviruses potato viruses Y and A (PVY and PVA). These genes are: Ry sto , which conditions resistance to PVY and PVA, and gene Ra, which conditions resistance to PVA only. It was found that in genotypes from a progeny of the cross ‘Barbara’ (Ry sto /Ra)בFlourball’ (ry/ra), extreme resistance to PVV segregated with gene Ry sto . It is proposed that either gene Ry sto conditions broad-spectrum extreme resistance to the distinct potyviruses PVY, PVA, and PVV or that Ry sto represents a family of genetically closely linked genes each controlling resistance to a specific virus.
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  • 187
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    Theoretical and applied genetics 94 (1997), S. 1092-1096 
    ISSN: 1432-2242
    Keywords: Key words Fragaria×ananassa ; Genetics ; Inheritance ; Red core ; Red stele
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract   Phytophthora fragariae var. fragariae is the causal agent of red stele (red core) root rot in strawberry (Fragaria spp.). The inheritance of resistance to one isolate of this fungus was studied in 12 segregating populations of F.×ananassa derived from crosses between four resistant cultivars (‘Climax’, ‘Redgauntlet’, ‘Siletz’, and ‘Sparkle’) and three susceptible cultivars (‘Blakemore’, ‘Glasa’, and ‘Senga’ Sengana’). The analysis clearly supports the hypothesis of a single segregating dominant resistance gene. It is proposed that this gene be designated Rpf2.
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  • 188
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    Child psychiatry & human development 27 (1997), S. 139-150 
    ISSN: 1573-3327
    Keywords: ADHD ; Tourette Syndrome ; Genetics ; Gene Selection ; Conduct Disorder
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract The evidence is reviewed to support the concept that many disruptive, childhood and adolescent behavioral disorders including ADHD, Tourette syndrome, learning disabilities, substance abuse, oppositional defiant disorder and conduct disorder, are part of a spectrum of inter-related behaviors that have a strong genetic component, are polygenically inherited, share a number of genes in common that affect dopamine, serotonin and other neurotransmitters, and are transmitted from both parents. Some of the implications of this hypothesis in relation to diagnosis and treatment are reviewed, including the possibility that the genes involved may be increasing in frequency.
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  • 189
    ISSN: 1573-6792
    Keywords: EEG normality ; Brain topography ; Twins ; Genetics
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Summary The influence of genetic relatedness on the similarity degree of topographical EEG parameters was studied in a sample of 26 sets of monozygotic (MZ) and 46 sets of dizygotic (DZ) twins. All 144 subjects were healthy, primary school children, aged 7–15 years, 69 boys and 75 girls. Correlation coefficients were calculated for 50 quantitative EEG parameters of paired values obtained at each of 16 active electrode sites, in four groups of paired tracings: 1. MZ twins, 2. DZ twins, 3. The autocorrelated (A) group formed by correlating the spectral parameters from the same subjects in two different analyzed sequences, 4. The random (R) control group of 1200 unrelated pairs formed from DZ twin pairs. Sets of MZ twins and A group showed the highest degrees of similarity of spectral parameters over all brain areas except for significant differences only for some background features over posterior regions. In contrast, highly significant differences in topographic parameters were evident in comparison of MZ sets with DZ sets, particularly when MZ sets were compared with DZ subsets of opposite sex. Both number and degree of significant differences increased progressively in comparisons with groups 3 vs 2,1 vs 4, and 3 vs 4. The data gave strong evidence for a complex polygenic determination of normal human EEG topography.
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  • 190
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    Yeast 13 (1997), S. 1077-1090 
    ISSN: 0749-503X
    Keywords: Saccharomyces cerevisiae ; chromosome VII ; sequence ; snRNA ; SNR10 ; SNR39 ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: The nucleotide sequences of five major regions from chromosome VII of Saccharomyces cerevisiae have been determined and analysed. These regions represent 203 kilobases corresponding to approximately one-fifth of the complete yeast chromosome VII. Two fragments originate from the left arm of this chromosome. The first one of about 15·8 kb starts approximately 75 kb from the left telomere and is bordered by the SKI8 chromosomal marker. The second fragment covers the 72·6 kb region between the chromosomal markers CYH2 and ALG2. On the right chromosomal arm three regions, a 70·6 kb region between the MSB2 and the KSS1 chromosomal markers and two smaller regions dominated by the KRE11 marker and another one in the vicinity of the SER2 marker were sequenced.We found a total of 114 open reading frames (ORFs), 13 of which were completely overlapping with larger ORFs running in the opposite direction.A total of 44 yeast genes, the physiological functions of which are known, could be precisely mapped on this chromosome.Of the remaining 57 ORFs, 26 shared sequence homologies with known genes, among which were 13 other S. cerevisiae genes and five genes from other organisms. No homology with any sequence in the databases could be found for 31 ORFs.Furthermore, five Ty elements were found, one of which may not be functional due to a frame shift in its Ty1B amino acid sequence.The five chromosomal regions harboured five potential ARS elements and one sigma element together with eight tRNA genes and two snRNAs, one of which is encoded by an intron of a protein-coding gene. © 1997 by John Wiley & Sons, Ltd.
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  • 191
    ISSN: 0749-503X
    Keywords: Saccharomyces cerevisiae ; endoplasmic reticulum ; chaperone ; unfolded protein response ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: RNAP-I, an aspartic proteinase from a filamentous fungus Rhizopus niveus, is secreted very efficiently in Saccharomyces cerevisiae. It is synthesized first as a precursor form with signal sequence and prosequence in its amino-terminus. Our previous study indicated that the prosequence of RNAP-I had important roles in its correct folding and secretion in yeast, and that a prosequence-deleted derivative of RNAP-I, Δpro, was not secreted but was retained and degraded in the yeast endoplasmic reticulum (ER). In the present study, we show that the accumulation of Δpro in the yeast ER caused elevated synthesis of ER resident chaperones, indicating that Δpro is recognized as an unfolded protein species in the ER. Our biochemical data demonstrated that Δpro formed aggregates which contained BiP, but not protein disulfide isomerase (PDI), in the ER. Immunoelectron microscopical analysis revealed that the Δpro aggregates were indeed visible as electron-dense regions in the ER and nuclear envelope. Such ‘chaperone-associated misfolded protein bodies’ were observed for the first time in yeast. Morphologies of the ER and nucleus were drastically altered by the accumulation of the Δpro aggregates. The ER lost its flat cisternal shape; the ER lumen extended aberrantly and the ER membrane irregularly proliferated. The misfolded Δpro proteins are probably sorted from the ordinary ER lumen to form the aggregates so that the ER function would not be grossly impaired, and the dilated ER may represent an ER subcompartment where the Δpro aggregates are degraded. © 1997 by John Wiley & Sons, Ltd.
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  • 192
    ISSN: 0749-503X
    Keywords: Saccharomyces cerevisiae ; chromosome VI ; expression profiles ; systematic analyses ; Northern hybridization ; YFL012w ; YFR032c ; YFL059w ; YFR011c ; novel genes ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: Chromosome VI of Saccharomyces cerevisiae contains 126 open reading frames (ORFs), and the functions of proteins encoded by 80 ORFs are still unknown. In this report, we have systematically examined the expression profiles of all 126 ORFs on chromosome VI under five kinds of growth conditions by quantitative Northern hybridization. A series of Northern analyses and reverse transcription polymerase chain reactions have revealed that more than 64 novel ORFs are transcribed. Two ORFs (YFL059w and YFR011c) are specifically expressed in the presence of galactose. Two ORFs (YFL012w and YFR032c) are specifically transcribed in sporulation. Six ORFs (YFL049w, YFL035c, YFL010c, YFR006w, YFR010w and YFR017c) are abundantly expressed in many growth conditions. © 1997 John Wiley & Sons, Ltd.
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  • 193
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    Yeast 13 (1997), S. 1231-1242 
    ISSN: 0749-503X
    Keywords: yeast ; gpd ; codon usage ; carotenoid ; astaxanthin ; splicing ; phylogeny ; evolution ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: The glyceraldehyde-3-phosphate dehydrogenase (GPD; EC1.2.1.12)-encoding gene (gpd) was isolated from a genomic library of Phaffia rhodozyma CBS 6938. Unlike some other eukaryotic organisms the gpd gene is represented by a single copy in P. rhodozyma. The complete nucleotide sequence of the coding, as well as the flanking non-coding regions was determined. The nucleotide sequence of gpd predicted six introns and a polypeptide chain of 339 amino acids. The codon usage in the gpd gene of P. rhodozyma was highly biased and was significantly different from the codon usage in other yeasts. Phylogenetic analysis of different yeasts and filamentous asco- and basidiomycetes gpd sequences indicated that the gpd gene of P. rhodozyma forms a cluster with the corresponding genes of filamentous basidiomycetes. © 1997 John Wiley & Sons, Ltd.
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  • 194
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    Yeast 13 (1997), S. 1243-1250 
    ISSN: 0749-503X
    Keywords: Saccharomyces ; phylogeny ; ribosomal RNA ; systematics ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: Sequences of two internally transcribed spacer regions between 18S and 28S rRNA genes were determined to assess the phylogenetic relationship in the strains belonging to the genus Saccharomyces. The sequences of S. bayanus and S. pastorianus were quite similar, but not identical. Two phylogenetic trees constructed by the neighbor-joining method showed that all the species examined were distinguished from one another. The Saccharomyces sensu stricto species: S. cerevisiae, S. bayanus, S. paradoxus and S. pastorianus, were closely related and far from the Saccharomyces sensu lato species including S. barnetti, S. castellii, S. dairensis, S. exiguus, S. servazzii, S. spencerorum and S. unisporus, and an outlying species, S. kluyveri. © 1997 John Wiley & Sons, Ltd.
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  • 195
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    Yeast 13 (1997), S. 1265-1274 
    ISSN: 0749-503X
    Keywords: Saccharomyces cerevisiae ; transcription ; regulation ; translation factor ; psi factor ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: SUP35 and SUP45 encode translational release factors in the yeast Saccharomyces cerevisiae. In addition, Sup35p is related to the cytoplasmically inherited prion-like phenotype [PSI+]. The vital cellular role of Sup35p and Sup45p prompted us to study the regulation of transcription of the corresponding genes. Since the [PSI] state of the yeast strain affects the abundance of Sup35p and Sup45p, both [PSI+] and [psi-] variants were included in these analyses. It turned out that SUP35 and SUP45 transcript levels are regulated by nutritional changes and stress in a way strikingly similar to those of ribosomal protein genes. The [PSI] state did not influence the respective transcript levels nor their regulation, although HSP12 (as a monitor of general stress-responsive) gene expression appeared to differ in the two variant strains. The transcription activation sites of SUP35 and SUP45 were mapped using deletion analysis of the respective promoter-reporter fusion genes. The UAS in both cases was found to consist of an Abf1p-site and a T-rich element. Also in this respect SUP35 and SUP45 show a notable resemblance with ribosomal protein genes. Evidence was found that SUP35 in addition harbors a potential internal promoter element which became active after progressive 5′-deletion removing the first of the three in-frame ATGs. © 1997 John Wiley & Sons, Ltd.
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  • 196
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    Yeast 13 (1997), S. 1357-1362 
    ISSN: 0749-503X
    Keywords: Suppressor tRNA ; UGA codon ; 5′-flanking sequence ; gene copy number ; fidelity of translation ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: The Saccharomyces cerevisiae haploid genome includes six copies of the gene encoding tRNATrp which are scattered on five chromosomes. Other, non-functional tDNATrp fragments also occur in the genome. The segments of all six genes which encode the 72-nucleotide mature tRNAas well as a 34-nucleotide intervening sequence, are identical. However, the 5′ and 3′ flanking sequences diverge virtually at the boundaries of the coding region. We have used an assay based on suppression of UGA mutations by multi-copy clones of tDNATrp to search for functional differences among these genes. Previous studies with one tDNATrp had demonstrated that moderate suppression of a UGA mutation, leu2-2, resulted from introduction of a multi-copy clone of the gene. Attempts to use this assay to select tDNATrp clones from a yeast genomic library yielded only four of the six different clones. The other two genes were amplified by PCR and cloned in pRS202, a 2 μ vector also used for the genomic library. Plasmids bearing the six tRNA genes were transformed into S. cerevisiae strain JG369.3B and scored for their ability to suppress the leu2-2 mutation as well as his4-260, another UGA marker. Two of the six tRNATrp clones were unable to suppress either marker, two evidenced weak suppression of the Leu auxotrophy, and two were able to suppress both markers. Growth rates in liquid media requiring suppression were measured for cell lines carrying each of the clones. Differences greater than 50-fold were observed in media lacking histidine. An evolutionary tree based on 5′-flanking sequence corresponds reasonably well with suppressor activity, while a similar analysis of 3′-flanking sequence does not. This suggests that the functional differences are based on divergence in the 5′-flanking sequences of the tRNATrp genes. © 1997 John Wiley & Sons, Ltd.
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  • 197
    ISSN: 0749-503X
    Keywords: Saccharomyces cerevisiae ; short ORFs ; computational ORF verification ; ORF properties ; sequence similarity ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: We have analysed short open reading frames (between 150 and 300 base pairs long) of the yeast genome (Saccharomyces cerevisiae) with a two-step strategy. The first step selects a candidate set of open reading frames from the DNA sequence based on statistical evaluation of DNA and protein sequence properties. The second step filters the candidate set by selecting open reading frames with high similarity to other known sequences (from any organism). As a result, we report ten new predicted proteins not present in the current sequence databases. These include a new alcohol dehydrogenase, a protein probably related to the cell cycle, as well as a homolog of the prokaryotic ribosomal protein L36 likely to be a mitochondrial ribosomal protein coded in the nuclear genome. We conclude that the analysis of short open reading frames leads to biologically interesting discoveries, even though the quantitative yield of new proteins is relatively low. © 1997 John Wiley & Sons, Ltd.
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  • 198
    ISSN: 0749-503X
    Keywords: ornithine decarboxylase ; SPE1 gene ; Candida albicans ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: The gene encoding ornithine decarboxylase, SPE1, from the pathogenic yeast Candida albicans has been isolated by complementation of an ornithine decarboxylase-negative (spe1Δ) strain of Saccharomyces cerevisiae. Four transformants, three of which contain plasmids with the SPE1 gene, were isolated by selection on polyamine-free medium. The C. albicans ornithine decarboxylase (ODC) showed high homology with other eukaryotic ODCs at both the amino acid and nucleic acid levels. The GenBank accession number for this gene is U85005. © 1997 John Wiley & Sons, Ltd.
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  • 199
    ISSN: 0749-503X
    Keywords: GPI-anchor ; GPI-attachment site ; yeast ; Ascomycetes ; fungi ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: Use of the Von Heijne algorithm allowed the identification of 686 open reading frames (ORFs) in the genome of Saccharomyces cerevisiae that encode proteins with a potential N-terminal signal sequence for entering the secretory pathway. On further analysis, 51 of these proteins contain a potential glycosyl-phosphatidylinositol (GPI)-attachment signal. Seven additional ORFs were found to belong to this group. Upon examination of the possible GPI-attachment sites, it was found that in yeast the most probable amino acids for GPI-attachment are asparagine and glycine. In yeast, GPI-proteins are found at the cell surface, either attached to the plasma-membrane or as an intrinsic part of the cell wall. It was noted that plasma-membrane GPI-proteins possess a dibasic residue motif just before their predicted GPI-attachment site. Based on this, and on homologies between proteins, families of plasma-membrane and cell wall proteins were assigned, revealing 20 potential plasma-membrane and 38 potential cell wall proteins. For members of three plasma-membrane protein families, a function has been described. On the other hand, most of the cell wall proteins seem to be structural components of the wall, responsive to different growth conditions. The GPI-attachment site of yeast slightly differs from mammalian cells. This might be of use in the development of anti-fungal drugs. © 1997 John Wiley & Sons, Ltd.
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  • 200
    ISSN: 0749-503X
    Keywords: Saccharomyces cerevisiae ; metal ion toxicity ; vacuole ; protein sorting ; gene regulation ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: The metal ions, Cu2+/+ and Fe3+/2+, are essential co-factors for a wide variety of enzymatic reactions. However, both metal ions are toxic when hyper-accumulated or maldistributed within cells due to their ability to generate damaging free radicals or through the displacement of other physiological metal ions from metalloproteins. Although copper transport into yeast cells is apparently independent of iron, the known dependence on Cu2+ for high affinity transport of Fe2+ into yeast cells has established a physiological link between these two trace metal ions. In this study we demonstrate that proteins encoded by genes previously demonstrated to play critical roles in vacuole assembly or acidification, PEP3, PEP5 and VMA3, are also required for normal copper and iron metal ion homeostasis. Yeast cells lacking a functional PEP3 or PEP5 gene are hypersensitive to copper and render the normally iron-repressible FET3 gene, encoding a multi-copper Fe(II) oxidase involved in Fe2+ transport, also repressible by exogenous copper ions. The inability of these same vacuolar mutant strains to repress FET3 mRNA levels in the presence of an iron-unresponsive allele of the AFT1 regulatory gene are consistent with alterations in the intracellular distribution or redox states of Fe3+/2+ in the presence of elevated extracellular concentrations of copper ions. Therefore, the yeast vacuole is an important organelle for maintaining the homeostatic convergence of the essential yet toxic copper and iron ions. © 1997 John Wiley & Sons, Ltd.
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