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  • 1
    Electronic Resource
    Electronic Resource
    Molecular cloning of a gene involved in glucose sensing in the yeast Saccharomyces cerevisiae (1993)
    Oxford, UK : Blackwell Publishing Ltd
    Molecular microbiology 8 (1993), S. 0 
    Details
    ISSN: 1365-2958
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: Cells of the yeast Saccharomyces cerevisiae display a wide range of glucose-induced regulatory phenomena, including glucose-induced activation of the RAS-adenylate cyclase pathway and phosphatidylinositol turnover, rapid post-translational effects on the activity of different enzymes as well as long-term effects at the transcriptional level. A gene called GGS1 (for General Glucose Sensor) that is apparently required for the glucose-induced regulatory effects and several ggs1 alleles (fdp1, byp1 and cif1) has been cloned and characterized. A GGS1 homologue is present in Methanobacterium thermoautotrophicum. Yeast ggs1 mutants are unable to grow on glucose or related readily fermentable sugars, apparently owing to unrestricted influx of sugar into glycolysis, resulting in its rapid deregulation. Levels of intracellular free glucose and metabolites measured over a period of a few minutes after addition of glucose to cells of a ggsi1Δ strain are consistent with our previous suggestion of a functional interaction between a sugar transporter, a sugar kinase and the GGS1 gene product. Such a glucose-sensing system might both restrict the influx of glucose and activate several signal transduction pathways, leading to the wide range of glucose-induced regulatory phenomena. Deregulation of these pathways in ggs1 mutants might explain phenotypic defects observed in the absence of glucose, e.g. the inability of ggs1 diploids to sporulate.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1111/j.1365-2958.1993.tb01638.x
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  • 2
    Electronic Resource
    Electronic Resource
    Optimisation of a host/vector system for heterologous gene expression by Hansenula polymorpha (1991)
    Springer
    Current genetics 19 (1991), S. 81-87 
    Details
    ISSN: 1432-0983
    Keywords: Methylotrophic yeast ; Integration ; Origin of replication ; Plant α-galactosidase
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary For the methylotrophic yeast, Hansenula polymorpha, expression vectors with different origins of replication have been constructed in order to analyse their influence on transformation and integration efficiency. The constructed plasmids are identical except for their origin of replication, which involve, respectively, that of the Saccharomyces cerevisiae 2-μm plasmid and a H. polymorpha ARS sequence (HARS2). A plasmid with no origin of replication served as a control. The plasmids also contained the α-galactosidase expression cassette, consisting of the Cyamopsis tetragonoloba α-galactosidase gene, the H. polymorpha methanol oxidase promoter and terminator, and the S. cerevisiae invertase signal sequence. The transformation frequencies of the expression vectors containing the 2-μm and the HARS2 origins of replication, and no origin of replication, were 2,50 and 15 per μg of DNA respectively, which demonstrates the negative effect of the 2-μm sequence on the transformation frequency. Autonomously replicating plasmids could be isolated from the transformants obtained with the plasmid containing either the 2-μm or the HARS2 sequence. Integration of the 2-μm based plasmid into the H. polymorpha genome could not be established using a standard procedure. This is in contrast with transformants containing a plasmid bearing the HARS2 sequence or else with no origin of replication, which shows that the 2-μm sequence negatively influences the integration of the expression vector into the H. polymorpha genome. Integration of expression plasmids occurred in 50% of the analysed integrants on the homologous methanol oxidase locus, and tandem integration was favoured. The level of specific mRNA, and the expression of the α-galactosidase protein by these integrants, was proportional to the number of integrated copies of the expression plasmid in the H. polymorpha genome.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00326287
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  • 3
    Electronic Resource
    Electronic Resource
    Analysis of glucose repression in Saccharomyces cerevisiae by pulsing glucose to a galactose-limited continuous culture (1992)
    New York, NY [u.a.] : Wiley-Blackwell
    Yeast 8 (1992), S. 1077-1087 
    Details
    ISSN: 0749-503X
    Keywords: Yeast ; glucose repression ; continuous culture ; transcriptional regulation ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: In this study, glucose repression in Saccharomyces cerevisiae was analysed under defined physiological conditions, at both the molecular and physiological levels, by pulsing glucose to a galactose-limited continuous culture. During this pulse of glucose, the galactose feed was kept constant. Directly after the glucose pulse, carbon dioxide production increased while oxygen consumption remained constant, demonstrating that the surplus of glucose had been consumed by means of fermentation. The direct accumulation of galactose in the medium after the glucose pulse indicated that the consumption of galactose had been stopped instantaneously. Galactose uptake experiments revealed that the galactose transporter was still present but apparently was incapable of galactose uptake, which could be due to inhibition of the galactose transporter by glucose. The total concentration of cAMP increased from 5 nmol g-1 at t = 0 to 25 nmolg-1 at t = 1·5 min. After 2 min the concentration of cAMP gradually decreased again to the normal level. Within 2 min after the addition of glucose, the transcription of the GAL genes and SUC2 was inhibited. In addition, the transcription of the HXK1 gene, encoding hexokinase isoenzyme 1, was also inhibited, which demonstrates that the HXK1 gene is regulated at the transcriptional level comparable with invertase.
    Additional Material: 5 Ill.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1002/yea.320081210
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  • 4
    Electronic Resource
    Electronic Resource
    Regulation of glycolytic enzymes and the crabtree effect in galactose-limited continuous cultures of Saccharomyces cerevisiae (1993)
    New York, NY [u.a.] : Wiley-Blackwell
    Yeast 9 (1993), S. 787-795 
    Details
    ISSN: 0749-503X
    Keywords: S. cerevisiae ; glucose repression ; carbon metabolism ; continuous culture ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: In order to determine whether the changes in the activities and mRNA levels of enzymes involved in intermediary carbon metabolism previously observed in glucose-limited continuous cultures (Sierkstra et al., 1992a) were glucose specific, we have analysed their regulation in a galactose-limited continuous culture of Saccharomyces cerevisiae. The Vmax of the galactose uptake system was shown to be dilution rate (D) dependent, comparable with the high-affinity glucose uptake. The maximum uptake was observed at D 0·2 h-1 (0·25 mmol min-1 per g) and the minimum uptake (0·1 mmol min-1 per g) at D 0·05 h-1 and 0·3 h-1. The aerobic fermentation of galactose occurred at D 0·275-0·3 h-1 which is identical to the results obtained in glucose-limited continuous cultures of this strain. Because galactose is not a repressing carbon source, this demonstrates that the Crabtree effect is not mediated by, or in any way related to glucose repression. Moreover, invertase and hexokinase I mRNA levels (both subject to glucose repression at the transcriptional level) were present when the yeast produced ethanol in galactose- and glucose-limited continuous cultures. In glucose-limited continuous cultures a decrease in alcohol dehydrogenase (I and II) mRNA levels and activity and phosphoglucomutase activity was observed with increasing dilution rates. In addition, at D 0·3 h-1, when the yeast produced ethanol, glucose-6-phosphate dehydrogenase and pyruvate decarboxylase were induced and a decrease in respiration was observed. The fact that the same changes were seen in a galactose-limited culture and that invertase and hexokinase I mRNA levels were present demonstrates that glucose is not specifically involved in the regulation of these enzymes. The changes observed are therefore caused by the growth rate of the organism, the glycolytic flux (e.g. alcohol dehydrogenase) or the overflow metabolism when the yeast produces ethanol (e.g. pyruvate decarboxylase).
    Additional Material: 5 Ill.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1002/yea.320090713
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