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Query: EC:3.2.1.23 (beta-galactosidase)
 14,648 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Expression of beta-galactosidase in transcriptional fusions with the pps gene (encoding phosphoenolpyruvate [PEP] synthase), the aceBAK operon (encoding malate synthase, isocitrate lyase, and isocitrate dehydrogenase kinase, respectively), and the phs operon (encoding either thiosulfate reductase or a regulatory protein controlling its expression) was studied in Salmonella typhimurium. beta-Galactosidase synthesis in these strains was repressible either by growth in the presence of glucose or by the presence of a fruR mutation, which resulted in the constitutive expression of the fructose (fru) regulon. Five enzymes of gluconeogenesis (PEP synthase, PEP carboxykinase, isocitrate lyase, malate synthase, and fructose-1,6-diphosphatase) were shown to be repressed either by growth in the presence of glucose or the fruR mutation, while the glycolytic enzymes, enzyme I and enzymes II of the phosphotransferase system as well as phosphofructokinase, were induced either by growth in the presence of glucose or the fruR mutation. Overexpression of the cloned fru regulon genes (not including fruR) resulted in parallel repression of representative gluconeogenic, Krebs cycle, and glyoxylate shunt enzymes. Studies with temperature-sensitive mutants of S. typhimurium which synthesized heat-labile IIIFru proteins provided evidence that this protein plays a role in the regulation of gluconeogenic substrate utilization. Other mutant analyses revealed a complex relationship between fru gene expression and the expression of genes encoding gluconeogenic enzymes. Taken together, the results suggest that a number of genes encoding catabolic, biosynthetic, and amphibolic enzymes in enteric bacteria are transcriptionally regulated by a complex catabolite repression/activation mechanism which may involve enzyme IIIFru of the phosphotransferase system as one component of the regulatory system.
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PMID:Altered transcriptional patterns affecting several metabolic pathways in strains of Salmonella typhimurium which overexpress the fructose regulon. 249 6

The flow of isocitrate through the glyoxylate bypass in Escherichia coli is regulated via the phosphorylation-dephosphorylation of isocitrate dehydrogenase mediated by a bifunctional enzyme: isocitrate dehydrogenase kinase/phosphatase. The aceK gene coding for this enzyme is part of the polycistronic ace operon, which also includes the aceB and aceA genes coding, respectively, for malate synthase and isocitrate lyase, the two glyoxylate bypass enzymes. The complete nucleotide sequence of a 2,214-base-pair DNA fragment containing the aceK gene and its 5' flanking region has been determined. In vivo experiments based on gene expression in a minicell system and protein fusion with beta-galactosidase, as well as in vitro assays with a plasmid-directed transcription-translation coupled system, have shown that the aceK gene extends over 1,731 nucleotides encoding a 66,528-dalton protein. The 5' flanking region presents an unusual intercistronic structural pattern consisting of two consecutive long dyad symmetries, almost identical in sequence, which can yield very stable stem-loop units. These structures are probably responsible for the drastic downshifting in expression observed in acetate-grown bacteria between the aceK gene and the aceA gene located immediately upstream in the ace operon.
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PMID:Nucleotide sequence and expression of the aceK gene coding for isocitrate dehydrogenase kinase/phosphatase in Escherichia coli. 282 8

In this work we have investigated the role of specific peptide sequences for glucose-inactivation of the yeast isocitrate lyase. Thus, different fragments of the ICL1 coding region were fused to the lacZ gene of E. coli to provide a reporter construction. Determinations of beta-galactosidase activities indicated that the decapeptide sequence KTKRNYSARD, located between amino acid residues 37 and 46 of isocitrate lyase, is important for glucose induced proteolytic inactivation. Further experimental evidence was provided by insertion of this sequence into a glucokinase-beta-galactosidase fusion protein, which is not sensitive to glucose regulation. The decapeptide inserted conferred glucose inactivation to this construct, confirming that it is both necessary and sufficient as a signal.
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PMID:Glucose-induced inactivation of isocitrate lyase in Saccharomyces cerevisiae is mediated by an internal decapeptide sequence. 760 10

The ICL1 gene encoding the isocitrate lyase from Saccharomyces cerevisiae was cloned and sequenced. A reading frame of 557 amino acids showing significant similarity to isocitrate lyases from seven other species could be identified. Construction of icl1 null mutants led to growth defects on C2 carbon sources while utilization of sugars or C3 substrates remained unaffected. Using an ICL1-lacZ fusion integrated at the ICL1 locus, a more than 200-fold induction of beta-galactosidase activity was observed after growth on ethanol when compared with glucose-repressed conditions. A preliminary analysis of the ICL1 upstream region identified a 364-bp fragment necessary and sufficient for this regulatory phenotype. Sequence motifs also present in the upstream regions of co-regulated genes were found within this region.
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PMID:Structure and regulation of the isocitrate lyase gene ICL1 from the yeast Saccharomyces cerevisiae. 831 92

We have found that the upstream region of the isocitrate lyase gene (UPR-ICL) from the n-alkane-utilizing yeast Candida tropicalis was functional in Saccharomyces cerevisiae as a novel promoter with nonfermentable carbon sources, such as oleic acid, acetate, ethanol, and glycerol/lactate. The expression of two foreign genes coding for beta-galactosidase from Escherichia coli (LacZ) and glutamate decarboxylase from rat brain was carried out under the control of UPR-ICL. Expression of LacZ was repressed by glucose and enhanced over 300-fold by acetate. When an expression vector pWI3 containing multicloning sites between UPR-ICL and the transcriptional terminator of the isocitrate lyase gene (TERM-ICL) was used, the smaller isoform of glutamate decarboxylase (GAD65) was highly produced in a soluble and active form. These results demonstrate that the novel expression system using UPR-ICL and TERM-ICL from C. tropicalis is useful for the production of heterologous proteins in S. cerevisiae.
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PMID:A novel heterologous gene expression system in Saccharomyces cerevisiae using the isocitrate lyase gene promoter from Candida tropicalis. 886 34

We investigated the regulation of expression of a gene encoding malate synthase (MS) of an n-alkane-utilizable yeast Candida tropicalis in the yeast Saccharomyces cerevisiae, where its expression is highly induced by acetate. By comparing levels of gene expression in cells grown on glucose, acetate, lactate, and oleic acid, we found that the increase in gene expression was due to a glucose repression-derepression mechanism. In order to obtain information concerning the regulation of the gene expression, a fusion gene which consists of the 5'-upstream region of MS-2 (UPR-MS-2) and the lacZ gene (encoding Escherichia coli beta-galactosidase), was introduced into S. cerevisiae, and beta-galactosidase activities were measured with cells grown on glucose or acetate. Deletion analysis of UPR-MS-2 revealed that the region between -777 and -448 (against the translation initiation codon) enhanced the level of gene expression in both glucose- and acetate-grown cells. In this region, sequences which resemble binding sites of Rap1p/Grf1p/Tufp, a global transcription activator, were found at seven locations and one was found for another pleiotropic activator Abf1p. The result also suggested the presence of multiple upstream repression sequences (URSs), which function specifically in glucose-grown cells, in the region between -368 and -126. In the repressing region, there were three tandem C(A/T)CTCCC sequences and also a putative binding site of Mig1p, a transcriptional repressor which mediates glucose repression of several other genes. When MIG1 gene of S. cerevisiae was disrupted, the expression of the UPR-MS-2-lacZ gene in glucose-grown cells increased approx. 10-fold. Furthermore, the effect of deletion of a putative Mig1p binding site was abolished in the MIG1-disrupted strain, suggesting Mig1p binds to this site and brings about glucose repression. When the SNF1 gene was disrupted, the high level gene expression observed in acetate-grown cells bearing UPR-MS-2 was abolished. This indicated that derepression of UPR-MS-2 -mediated gene expression was dependent on Snf1p, as is the case of genes encoding isocitrate lyase and gluconeogenic enzymes in S. cerevisiae.
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PMID:Analysis of carbon source-regulated gene expression by the upstream region of the Candida tropicalis malate synthase gene in Saccharomyces cerevisiae. 900 61

Carbon catabolite repression by the CreA-transcriptional repressor is widespread in filamentous fungi, but the mechanism by which glucose triggers carbon catabolite repression is still poorly understood. We investigated the hypothesis that the growth rate on glucose may control CreA-dependent carbon catabolite repression by using glucose-limited chemostat cultures and the intracellular beta-galactosidase activity of Aspergillus nidulans, which is repressed by glucose, as a model system. Chemostat cultures at four different dilution rates (D = 0.095, 0.068, 0.045 and 0.015 h-1) showed that formation of beta-galactosidase activity is repressed at the two highest Ds, but increasingly derepressed at the lower Ds, the activity at 0.015 h-1 equalling that in derepressed batch cultures. Chemostat cultures with the carbon catabolite derepressed A. nidulans mutant strain creADelta4 revealed a dilution-rate independent constant beta-galactosidase activity of the same range as that found in the wild-type strain at D = 0.015 h-1. Two other enzymes--isocitrate lyase, which is almost absent on glucose due to a CreA-independent mechanism; and galactokinase, which is formed constitutively and independent of CreA--were measured as controls. They were formed at constant activity at each dilution rate, both in the wild-type strain as well as in the carbon catabolite derepressed mutant strain. We conclude that the growth rate on glucose is a determinant of carbon catabolite repression in A. nidulans, and that below a certain growth rate carbon catabolite derepression occurs.
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PMID:CreA-mediated carbon catabolite repression of beta-galactosidase formation in Aspergillus nidulans is growth rate dependent. 1515 74

Recombinant yeast cells intracellularly overexpressing three different enzymes were permeabilized with alcohol under various conditions. The effects of enzyme stability in alcohol and enzyme molecular weight on the activities of permeabilized cells and enzyme leakage during incubation were examined. Saccharomyces cerevisiae YPH250 overexpressing glyoxalase I (GloI), S. cerevisiae MT8-1 overexpressing isocitrate lyase (ICL), and Pichia pastoris GS115 overexpressing beta-galactosidase (beta-gal) were used as model recombinant yeast systems. In all cases, the percentage of alcohol used for the treatment significantly affected the activity of permeabilized whole cell biocatalysts; cells showed high activity when treated with 40% isopropyl alcohol. The activity of whole cell biocatalysts was also significantly affected by the stability of the enzyme in alcohol solution; permeabilized yeast cells overexpressing ICL, which had low stability, showed rather low activity. Although the enzyme leakage from permeabilized cells was rather low in all cases, the molecular weight of the enzyme appeared to affect the extent of enzyme leakage during incubation. Permeabilized cells of P. pastoris overexpressing beta-gal (540 kDa) retained particularly high activity during incubation and could be used as an immobilized whole cell biocatalysts.
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PMID:Preparation of high-activity whole cell biocatalysts by permeabilization of recombinant yeasts with alcohol. 1623 97