Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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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)

We characterized catabolite repression of the genes encoding xylose utilization in Bacillus megaterium. A transcriptional fusion of xylA encoding xylose isomerase to the spoVG-lacZ indicator gene on a plasmid with a temperature-sensitive origin of replication was constructed and efficiently used for single-copy replacement cloning in the B. megaterium chromosome starting from a single transformant. In the resulting strain, beta-galactosidase expression is 150-fold inducible by xylose and 14-fold repressed by glucose, showing that both regulatory effects occur at the level of transcription. Insertion of a kanamycin resistance gene into xylR encoding the xylose-dependent repressor leads to the loss of xylose-dependent regulation and to a small drop in the efficiency of glucose repression to eightfold. Deletion of 184 bp from the 5' part of the xylA reading frame reduces glucose repression to only twofold. A potential glucose-responsive element in this region is discussed on the basis of sequence similarities to other glucose-repressed genes in Bacillus subtilis. The sequence including the glucose-responsive element is also necessary for repression exerted by the carbon sources fructose and mannitol. Their efficiencies of repression correlate to the growth rate of B. megaterium, as is typical for catabolite repression. Glycerol, ribose, and arabinose exert only a basal twofold repression of the xyl operon, which is independent of the presence of the cis-active glucose-responsive element within the xylA reading frame.
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PMID:Catabolite repression of the xyl operon in Bacillus megaterium. 156 31

We have examined the expression of the gene encoding the iron-protein subunit (Ip) of succinate dehydrogenase in Saccharomyces cerevisiae. The gene had been cloned by us and shown to be subject to glucose regulation (A. Lombardo, K. Carine, and I. E. Scheffler, J. Biol. Chem. 265:10419-10423, 1990). We discovered that a significant part of the regulation of the Ip mRNA levels by glucose involves the regulation of the turnover rate of this mRNA. In the presence of glucose, the half-life appears to be less than 5 min, while in glycerol medium, the half-life is greater than 60 min. The gene is also regulated transcriptionally by glucose. The upstream promoter sequence appeared to have four regulatory elements with consensus sequences shown to be responsible for the interaction with the HAP2/3/4 regulatory complex. A deletion analysis has shown that the two distal elements are redundant. These measurements were carried out by Northern (RNA) analyses of Ip mRNA transcripts as well as by assays of beta-galactosidase activity in cells carrying constructs of the Ip promoter linked to the lacZ coding sequence. These observations on the regulation of mRNA stability were also extended to the mRNA of the flavoprotein subunit of succinate dehydrogenase and in some experiments of iso-1-cytochrome c.
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PMID:Control of mRNA turnover as a mechanism of glucose repression in Saccharomyces cerevisiae. 162 Jan 7

Recombinant DNA and genetic techniques were used to construct Escherichia coli strains SOH92 [phi(cls-lacZ+)] and SOH93 [phi(cls-'lacZ)hyb]. beta-Galactosidase (116 kDa) synthesized by strain SOH92 was primarily present in the particulate fraction. Strain SOH92 produced about 20-fold more beta-galactosidase activity than strain SOH93. Expression of clsphilacZ in both SOH92 and SOH93 was influenced by the terminal electron acceptor (increasing in the order oxygen, nitrate, fumarate) when the cells were cultured in minimal medium with glycerol as the sole carbon source. As strains SOH92 and SOH93 progressed from early to late log growth phase under aerobic conditions in LB broth, clsphilacZ expression increased about 2.5-fold. Fusion strains containing a pss-1 allele had an increased cardiolipin (CL) level, but no corresponding increase in clsphilacZ expression was observed. A cls::Tn10dTet null mutation was introduced into SOH92 and SOH93. The strains produced less CL, but no corresponding changes in clsphilacZ expression were observed. A high copy number plasmid bearing the cls gene had no effect on clsphilacZ expression. Taken together, these results indicate that cls is not subject to autogenous regulation.
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PMID:Genetic regulation of cardiolipin synthase in Escherichia coli. 166 9

The hepatopancreatic extract of M. mercenaria (hard shelled clam) was found to be a rich source for at least 16 different glycosidases. These glycosidases were successfully employed for the degradation of oligosaccharides, glycolipids, and glycoproteins at analytical as well as preparative levels. The identified glycosidases differ considerably in their stability profiles with respect to time and temperature of storage and presence of glycerol. However, most of the enzymes show higher activity at pH 4.5 than at pH 7.0, and could be bound on a DEAE CL-6B Sepharose anion-exchange column suggesting similar charge characteristics on the protein surface. A Gal beta 1, 3R linkage-specific beta-galactosidase activity has also been detected in the glycosidase-enriched fraction and has been utilized to obtain quantitative conversion of the ganglioside GM1 to GM2 on a preparative scale. The glycosidase-rich extract does not have detectable protease activity at the pH of optimal glycosidase activity (pH 4.5) and, hence, can be safely used for specific hydrolysis of carbohydrate moieties of glycoproteins and glycopeptides. This is the first report to characterize a repertoire of glycosidases from an inexpensive, dependable and convenient source that can be easily employed for compositional studies involving glycoconjugates.
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PMID:Use of exoglycosidases from Mercenaria mercenaria (hard shelled clam) as a tool for structural studies of glycosphingolipids and glycoproteins. 177 74

The mechanisms involved in the translocation of exogenously added genetic information through the cellular cytoplasm and into the nucleus are essentially unknown. Several trans-cytoplasmic translocation systems operate within cells to transport information received by the plasma membrane into the nucleus. Protein kinase C may be functionally involved in many of these translocation mechanisms. In order to explore the involvement of protein kinase C activation in the cytoplasmic translocation of DNA, NIH3T3 fibroblasts were transfected using the calcium-phosphate co-precipitation method with a plasmid containing the lacZ gene and treated with tetradecanoylphorbol 12,13-acetate (TPA) or 1,2-dioctanoylglycerol (DiC8). Addition of TPA or DiC8 immediately after glycerol shock resulted in a 5-7-fold increase in the number of cells expressing beta-galactosidase as well as a concomitant increase in the total amount of beta-galactosidase activity in the population during periods of transient and stable expression. TPA added at later times resulted in lesser increases in the efficiency of transfection. In contrast, TPA added at the time of addition of the calcium-phosphate precipitate inhibited transfection. In support of a role for protein kinase C activation in enhancing DNA transfection, the TPA analog 4 alpha-phorbol 12,13-didecanoate, which does not activate protein kinase C, was ineffective at enhancing transfection. Furthermore, treatment of cells with the protein kinase C inhibitor sphingosine blocked the TPA-mediated increase in transient and stable expression. The results suggest that protein kinase C activation enhances transfection of exogenous DNA through an as yet unknown mechanism.
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PMID:Potentiation of DNA mediated gene transfer in NIH3T3 cells by activators of protein kinase C. 190 Apr 39

Using beta-galactosidase and luciferase as reporter enzymes, we have previously shown that enzymatic inactivation occurring during a heat shock is concomitant with protein insolubilization (Nguyen, V. T., Morange, M., and Bensaude, O. (1989) J. Biol. Chem. 264, 10487-10492). In this paper, we observe that pretreatment of cells with D2O and glycerol, compounds known to stabilize protein structure, leads to a parallel decrease of protein inactivation and insolubilization, suggesting that these two phenomena result most probably from heat-induced protein denaturation. We found that heat shock-promoted inactivation and insolubilization are not irreversible processes, since even in the absence of protein synthesis, beta-galactosidase solubility and luciferase solubility and activity are recovered in vivo after a heat treatment. Cognate heat shock proteins might be involved in this renaturation process.
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PMID:Denaturation of proteins during heat shock. In vivo recovery of solubility and activity of reporter enzymes. 190 89

6-Hydroxy-D-nicotine oxidase (6-HDNO) was expressed in Escherichia coli JM109 cells from the recombinant plasmid pAX-6-HDNO as a beta-galactosidase-6-HDNO fusion protein. Affinity chromatography of the fusion protein on p-aminobenzyl-1-thio-beta-galactopyranoside-agarose and subsequent digestion with protease Xa yielded highly purified apo6-HDNO. Incubation of the purified protein with [14C]FAD demonstrated that flavinylation of apo6-HDNO proceeds autocatalytically. Phosphorylated three-carbon compounds such as glycerol-3-P, which are known to stimulate the formation of the histidyl (N3)-(8 alpha) FAD between apo6-HDNO and FAD (Brandsch, R., and Bichler, V. (1989) Eur. J. Biochem. 182, 125-128), could be replaced in their action by high concentrations of glycerol (45%) or sucrose (20%). These substances apparently induced and stabilized a conformational state of the apoenzyme compatible with covalent attachment of FAD. In the absence of glycerol the apoenzyme readily lost the ability to form holoenzyme at temperatures above 30 degrees C. Holoenzyme formation protected the 6-HDNO polypeptide from this thermal denaturation. Autoflavinylation of 6-HDNO was inhibited by the sulfhydryl reagents dithionitrobenzoate or N-ethylmaleimide. Inhibition was prevented by mercaptoethanol or FAD, but not 6-hydroxy-D-nicotine, the substrate of the holoenzyme. A cysteine-thiol group may therefore be involved in reactions leading to the covalent attachment of FAD to apo6-HDNO. When flavinylation of apo6-HDNO proceeded under anaerobic conditions, the amount of incorporation of [14C]FAD into the polypeptide was indistinguishable from reactions performed in the presence of O2. None of the FAD-derivatives (8-demethyl-FAD, 8-chloro-FAD, and 5-deaza-FAD) could replace FAD in holoenzyme formation. The failure of covalent attachment of 5-deaza-FAD to apo6-HDNO is in agreement with the assumption that the quinone methide form of the isolloxazine ring is an intermediate in the flavinylation reaction.
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PMID:Autoflavinylation of apo6-hydroxy-D-nicotine oxidase. 191 24

The Bacillus subtilis xyl operon encoding enzymes for xylose utilization is repressed in the absence of xylose and in the presence of glucose. Transcriptional fusions of spoVG-lacZ to this operon show regulation of beta-galactosidase expression by glucose, indicating that glucose repression operates at the level of transcription. A similar result is obtained when glucose is replaced by glycerol, thus defining a general catabolite repression mechanism. A deletion of xylR, which encodes the xylose-sensitive repressor of the operon, does not affect glucose repression. The cis element mediating glucose repression was identified by Bal31 deletion analysis. It is confined to a 34 bp segment located at position +125 downstream of the xyl promoter in the coding sequence for xylose isomerase. Cloning of this segment in the opposite orientation leads to reduced catabolite repression. The homology of this element to various proposed consensus sequences for catabolite repression in B. subtilis is discussed.
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PMID:Catabolite repression of the operon for xylose utilization from Bacillus subtilis W23 is mediated at the level of transcription and depends on a cis site in the xylA reading frame. 192 70

Saccharomyces cerevisiae chromosomes end with the sequence C2-3A(CA)1-4, commonly abbreviated as C1-3A. These sequences can function as upstream activators of transcription (UAS's) when placed in front of a CYC1-lacZ fusion gene. When C1-3A sequences are placed between the GAL1,10 UAS and the CYC1-lacZ fusion, the C1-3A UAS still functions and the amount of beta-galactosidase produced in cells grown on glucose is as much or more than that for cells grown on either glycerol medium, or cells grown on glucose medium containing a plasmid with just the C1-3A UAS. These data indicate that the UAS is immune from glucose repression from the upstream GAL1,10 UAS. Because C1-3A sequences are bound in vitro by the transcription factor RAP1, the UAS activity of yeast telomere sequences was compared with that of a similar UAS from the tightly regulated ribosomal protein gene RP39A, which also contains a RAP1 binding site. While transcription from the ribosomal protein gene UAS was responsive to cell density, the amount of transcription from the C1-3A UAS was nearly the same at all cell densities tested. These data show that the transcriptional activation by C1-3A sequences is not regulated by cell density.
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PMID:Properties of the transcriptional enhancer in Saccharomyces cerevisiae telomeres. 211 Jun 55

The ugp promoter (pugp) responsible for expression of the binding-protein-dependent sn-glycerol-3-phosphate transport system in Escherichia coli was cloned into a small multicopy plasmid pTER5, a derivative of pBR322, between the transcription terminators rpoCt and tL1. The resulting expression vector, pPH3, permits convenient insertion of structural genes containing their own translational-initiation regions, into the multiple-cloning site derived from the pUC19 plasmid. The efficiency and regulatory properties of pugp were measured using xylE and lacZ as reporter genes, which code for the corresponding enzymes catechol-2,3-dioxygenase (C23O) and beta-galactosidase (beta Gal), respectively. Enzyme activities were virtually completely repressed in the presence of excess inorganic phosphates (Pi) and high concentrations of glucose. Maximal induction was observed at limiting Pi (less than 0.1 mM) and normal levels of glucose (0.2-0.4%). The maximum expression of the pugp-directed beta Gal synthesis was approx. 80% of that directed by strong ptac. When the xylE gene was maximally expressed, the induced enzyme constituted approx. 50% of total cellular protein as judged by laser densitometry following sodium dodecyl sulfate-polyacrylamide-gel electrophoresis. These results suggest the usefulness of the pugp in expression vectors for strong, but controlled, expression of cloned genes in E. coli. This Pi controlled vector can be adapted to large-scale fermentation by using Pi-limiting growth conditions.
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PMID:A novel phosphate-regulated expression vector in Escherichia coli. 237 33


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