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)

The cationic liposome DOTAP was complexed with plasmid DNA encoding beta-galactosidase in various ratios. As the concentration of DOTAP increased, the DNA became increasingly refractory to staining with ethidium bromide, presumably because the DNA was becoming condensed and being encapsulated by the liposomes. Transfection by DNA-DOTAP complexes at all ratios tested was unaffected by treatment of the complexes with DNase I. This finding has relevance to clinical trials for gene therapy of cystic fibrosis, in which patients are normally removed from treatment with DNase before receiving administration of DNA. We additionally tested the effect of aerosolisation of the liposome-DNA complex and of the DNA alone on the efficiency of in vitro transfection. Aerosolised DNA complexed with fresh DOTAP led to much lower reporter gene expression in Cos 7 cells than non-aerosolised complex, since aerosolisation appeared to destroy almost all of the plasmid. However, complexing the plasmid before passage through the nebuliser did protect most of the DNA from degradation, as reflected in the levels of transfection obtained. These findings contribute towards an overall understanding of both how DNA-cationic liposome complexes are formed and their fate following administration in vivo.
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PMID:Plasmid DNA molecules complexed with cationic liposomes are protected from degradation by nucleases and shearing by aerosolisation. 887 34

Catabolite repression of Bacillus subtilis catabolic operons is supposed to occur via a negative regulatory mechanism involving the recognition of a cis-acting catabolite-responsive element (cre) by a complex of CcpA, which is a member of the GalR-Lacl family of bacterial regulatory proteins, and the seryl-phosphorylated form of HPr (P-ser-HPr), as verified by recent studies on catabolite repression of the gnt operon. Analysis of the gnt promoter region by deletions and point mutations revealed that in addition to the cre in the first gene (gntR) of the gnt operon (credown), this operon contains another cre located in the promoter region (creup). A translational gntR'-'lacZ fusion expressed under the control of various combinations of wild-type and mutant credown and creup was integrated into the chromosomal amyE locus, and then catabolite repression of beta-galactosidase synthesis in the resultant integrants was examined. The in vivo results implied that catabolite repression exerted by creup was probably independent of catabolite repression exerted by credown; both creup and credown catabolite repression involved CcpA. Catabolite repression exerted by creup was independent of P-ser-HPr, and catabolite repression exerted by credown was partially independent of P-ser-HPr. DNase I footprinting experiments indicated that a complex of CcpA and P-ser-HPr did not recognize creup, in contrast to its specific recognition of credown. However, CcpA complexed with glucose-6-phosphate specifically recognized creup as well as credown, but the physiological significance of this complexing is unknown.
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PMID:Catabolite repression of the Bacillus subtilis gnt operon exerted by two catabolite-responsive elements. 910 11

The transcription factor GATA-1, which is expressed in several hematopoietic lineages and multipotential progenitors, is required for the development of red blood cells and platelets. To identify control elements of the mouse GATA-1 gene, we analyzed DNase I hypersensitivity of the locus in erythroid chromatin and the expression of GATA-1/Escherichia coli beta-galactosidase (lacZ) transgenes in mice. Transgenes with 2.7 kb of promoter sequences are expressed infrequently and only within adult (definitive) erythroid cells. We show that inclusion of an upstream hypersensitive site (HS I) markedly enhances the frequency of expressing transgenic lines and activates expression in primitive erythroid cells. This pattern recapitulates the proper pattern of GATA-1 expression during development. By breeding a GATA-1/lacZ transgene into a GATA-1(-) background, we also have shown that the activation or maintenance of GATA-1 expression does not require the presence of GATA-1 itself, thereby excluding simple models of positive autoregulation. The transgene cassette reported here should be useful in directing expression of foreign sequences at the onset of hematopoiesis in the embryo and may assist in the identification of upstream regulators of the GATA-1 gene.
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PMID:An upstream, DNase I hypersensitive region of the hematopoietic-expressed transcription factor GATA-1 gene confers developmental specificity in transgenic mice. 922 98

The global regulator Lrp (leucine-responsive regulatory protein), in some cases modulated by its co-regulator leucine, has been shown to regulate more than 40 genes and operons in Escherichia coli. Leucine modulates Lrp regulation of leucine-responsive operons. The level of sensitivity of these operons to leucine varies greatly, but the basis for this variation is only partially understood. One operon controlled by Lrp that is relatively insensitive to leucine is gltBDF, which includes genes specifying the large (GltB) and small (GltD) subunits of glutamate synthase. Earlier gel mobility shift assays have demonstrated that Lrp binds to a fragment of DNA containing the gltBDF promoter region. To further define the nature of this Lrp-gltBDF interaction, DNase I footprinting experiments were performed. The results indicate that Lrp binds cooperatively to three sites quite far upstream, spanning the region from -140 to -260 base-pairs relative to the start of transcription. Phased hypersensitivity is observed throughout the entire binding region, suggesting that Lrp bends the DNA. To determine the relative importance of these three sites for the transcriptional activation of gltBDF, a series of site-directed mutations was generated. The effects of these mutations on Lrp binding were determined both by DNase I footprinting and by quantitative mobility shift assays, while their effects on transcription in vivo were examined by measuring beta-galactosidase activity levels of chromosomal gltB::lacZ operon fusions. Our results indicate that all three sites are required for maximal gene expression, as is the proper phasing of the sites with one another and with the start of transcription. Our results suggest that Lrp binds a central palindromic site, interacting predominantly with the major groove of its DNA target, and that additional dimers bind to flanking sites to form a nucleoprotein activation complex.
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PMID:A nucleoprotein activation complex between the leucine-responsive regulatory protein and DNA upstream of the gltBDF operon in Escherichia coli. 923 18

To dissect the cis-regulatory elements of the murine Msx-1 promoter, which lacks a conventional TATA element, a putative Msx-1 promoter DNA fragment (from -1282 to +106 base pairs (bp)) or its congeners containing site-specific alterations were fused to luciferase reporter and introduced into NIH3T3 and C2C12 cells, and the expression of luciferase was assessed in transient expression assays. The functional consequences of the sequential 5' deletions of the promotor revealed that multiple positive and negative regulatory elements participate in regulating transcription of the Msx-1 gene. Surprisingly, however, the optimal expression of Msx-1 promoter in either NIH3T3 or C2C12 cells required only 165 bp of the upstream sequence to warrant detailed examination of its structure. Therefore, the functional consequences of site-specific deletions and point mutations of the cis-acting elements of the minimal Msx-1 promoter were systematically examined. Concomitantly, potential transcriptional factor(s) interacting with the cis-acting elements of the minimal promoter were also studied by gel electrophoretic mobility shift assays and DNase I footprinting. Combined analyses of the minimal promoter by DNase I footprinting, electrophoretic mobility shift assays, and super shift assays with specific antibodies revealed that 5'-flanking regions from -161 to -154 and from -26 to -13 of the Msx-1 promoter contains an authentic E box (proximal E box), capable of binding a protein immunologically related to the upstream stimulating factor 1 (USF-1) and a GC-rich sequence motif which can bind to Sp1 (proximal Sp1), respectively. Additionally, we observed that the promoter activation was seriously hampered if the proximal E box was removed or mutated, and the promoter activity was eliminated completely if the proximal Sp1 site was similarly altered. Absolute dependence of the Msx-1 minimal promoter on Sp1 could be demonstrated by transient expression assays in the Sp1-deficient Drosophila cell line cotransfected with Msx-1-luciferase and an Sp1 expression vector pPacSp1. The transgenic mice embryos containing -165/106-bp Msx-1 promoter-LacZ DNA in their genomes abundantly expressed beta-galactosidase in maxillae and mandibles and in the cellular primordia involved in the formation of the meninges and the bones of the skull. Thus, the truncated murine Msx-1 promoter can target expression of a heterologous gene in the craniofacial tissues of transgenic embryos known for high level of expression of the endogenous Msx-1 gene and found to be severely defective in the Msx-1 knock-out mice.
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PMID:A minimal murine Msx-1 gene promoter. Organization of its cis-regulatory motifs and their role in transcriptional activation in cells in culture and in transgenic mice. 927 25

The ability of microorganisms to degrade L-tyrosine to phenol, pyruvate, and ammonia is catalyzed by the inducible enzyme L-tyrosine phenol lyase (EC 4.1.99.2). To investigate possible mechanisms for how the synthesis of this enzyme is regulated, a variety of biochemical and genetic procedures was used to analyze transcription from the tpl promoter of Citrobacter freundii ATCC 29063 (C. braakii). By computer analysis of the region upstream of the tpl structural gene, two segments of DNA bearing strong homology to the known operator targets of the TyrR protein of Escherichia coli were detected. A DNA fragment of 509 bp carrying these operator targets plus the presumptive tpl promoter was synthesized by PCR and used to construct a single-copy tpl-lacZ reporter system. The formation of beta-galactosidase in strains carrying this reporter system, which was measured in E. coli strains of various genotypes, was strongly dependent on the presence of a functional TyrR protein. In strains bearing deletions of the tyrR gene, the formation of beta-galactosidase was reduced by a factor of 10. Several mutationally altered forms of TyrR were deficient in their abilities to activate the tpl promoter. The pattern of loss of activation function was exactly parallel to the effects of the same tyrR mutations on the mtr promoter, which is known to be activated by the TyrR protein. When cells carrying the tpl-lacZ reporter system were grown on glycerol, the levels of beta-galactosidase were 10- to 20-fold higher than those observed in glucose-grown cells. The effect was the same whether or not TyrR-mediated stimulation of the tpl promoter was in effect. By deleting the cya gene, it was shown that the glycerol effect was attributable to stimulation of the tpl promoter by the cyclic AMP (cAMP)-cAMP reporter protein system. A presumptive binding site for this transcription factor was detected just upstream of the -35 recognition hexamer of the tpl promoter. The transcriptional start point of the tpl promoter was determined by chemical procedures. The precise locations of the TyrR binding sites, which were established by DNase I footprinting, agreed with the computer-predicted positions of these regulatory sites. The two TyrR operators, which were centered at coordinates -272.5 and -158.5 with respect to the transcriptional start point, were independently disabled by site-directed mutagenesis. When the upstream operator was altered, activation was completely abolished. When the downstream operator was altered, there was a fourfold reduction in reporter enzyme levels. The tpl system presents a number of intriguing features not previously encountered in TyrR-activated promoters. First among these is the question of how the TyrR protein, bound to widely separated operators, activates the tpl promoter which is also widely separated from the operators.
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PMID:The tpl promoter of Citrobacter freundii is activated by the TyrR protein. 929 52

DtxR is a dimeric, sequence-specific, DNA-binding protein that functions as an iron-dependent, negative global regulator in Corynebacterium diphtheriae. Under high-iron conditions, DtxR represses the synthesis of diphtheria toxin, corynebacterial siderophore, and other components of the high-affinity iron uptake system. Three DtxR-regulated promoter/operators designated tox, IRP1, and IRP2 were reported previously. In this study, we identified and characterized three additional DtxR-regulated promoter/operators from C. diphtheriae designated IRP3, IRP4, and IRP5. When beta-galactosidase was expressed from these three new promoter/ operators in Escherichia coli containing dtxR+ on pDSK29, enzyme levels were 5- to 30-fold lower during high-iron growth than during low-iron growth. In gel shift assays, the mobility of DNA fragments containing each promoter/operator decreased in the presence of purified DtxR and Co2+. In footprinting assays, DtxR protected 36-, 35-, and 30-bp regions of IRP3, IRP4, and IRP5, respectively, from cleavage by DNase I. In the 19-bp core of each promoter/operator, 12 or 13 bp matched the consensus for the DtxR-binding site. The putative polypeptides encoded by the open reading frames (ORFs) downstream from IRP3 and IRP4 were homologous, respectively, to several bacterial transcriptional regulators and to the deduced polypeptide encoded by an ORF located between the E. coli genes for primosomal replication protein N and adenine phosphoribosyltransferase. The putative polypeptide encoded by the ORF downstream from IRP5 was not homologous to any sequence in the protein database at the National Center for Biotechnology Information. When the ORFs downstream from IRP3 and IRP4 were expressed under the control of the phage T7 promoter in E. coli, polypeptide products of the predicted sizes were detected in small amounts by sodium dodecyl sulfate-polyacrylamide gel electrophoresis.
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PMID:Identification and characterization of three new promoter/operators from Corynebacterium diphtheriae that are regulated by the diphtheria toxin repressor (DtxR) and iron. 931 37

The vav gene is expressed in all hematopoietic but few other cell types. To explore its unusual compartment-wide regulation, we cloned the murine gene, sequenced its promoter region, identified DNase I hypersensitive (HS) sites in the chromatin, and tested their promoter activity with a beta-galactosidase (beta-gal) reporter gene in cell lines and transgenic mice. Whereas fibroblasts had no HS sites, a myeloid and an erythroid cell line contained five, located 0.2 kb (HS1), 1.9 kb (HS2), and 3.6 kb (HS3) upstream from the transcription start and 0.6 kb (HS4) and 10 kb (HS5) downstream. A vav DNA fragment including HS1 promoted beta-gal expression in a myeloid but not a fibroblast line. Expression in leukocytes of transgenic mice also required HS2 and HS5. Only hematopoietic organs contained beta-gal, but virtually all beta-gal+ cells were B or T lymphocytes. Expression was always variegated (mosaic), and the proportion of beta-gal+ cells declined with lymphoid maturation and animal age. Thus, these vav regulatory elements promoted hematopoietic-specific expression in vivo, at least in lymphocytes, but the transgene was sporadically silenced. Maintaining pan-hematopoietic expression may require additional vav elements or an alternative reporter.
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PMID:Transcriptional regulation of vav, a gene expressed throughout the hematopoietic compartment. 942 94

The catBCA operon of Pseudomonas putida encodes enzymes involved in the catabolism of benzoate. Transcription of this operon requires the LysR-type transcriptional regulator CatR and an inducer molecule, cis,cis-muconate. Previous gel shift assays and DNase I footprinting have demonstrated that CatR occupies two adjacent sites proximal to the catBCA promoter in the presence of the inducer. We report the presence of an additional binding site for CatR downstream of the catBCA promoter within the catB structural gene. This site, called the internal binding site (IBS), extends from +162 to +193 with respect to the catB transcriptional start site and lies within the catB open reading frame. Gel shift analysis and DNase I footprinting determined that CatR binds to this site with low affinity. CatR binds cooperatively with higher affinity to the IBS in the presence of the two upstream binding sites. Parallel in vivo and in vitro studies were conducted to determine the role of the internal binding site. We measured beta-galactosidase activity of catB-lacZ transcriptional fusions in vivo. Our results suggest a probable cis-acting repressor function for the internal binding site. Site-directed mutagenesis of the IBS verified this finding. The location of the IBS within the catB structural gene, the cooperativity observed in footprinting studies, and phasing studies suggest that the IBS likely participates in the interaction of CatR with the upstream binding sites by looping out the intervening DNA.
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PMID:Transcriptional repression mediated by LysR-type regulator CatR bound at multiple binding sites. 957 87

We have investigated CFTR specific intestinal expression by transfection assays in mouse cultured fibroblasts and transimmortalized intestinal crypt m-ICc12 cells using the beta-galactosidase gene linked to rat CFTR non-coding regions. Two constructs were studied, one encompassing a 5.3 kb region 5' to the gene where numerous duodenum-specific DNase I hypersensitive sites (DHSs) were previously mapped and the other including a 1.3 kb 3' region in which novel DHSs had been identified. In transient transfection assays, transgenes were expressed in m-ICc12 cells but not in fibroblasts. In m-ICc12 cells, the pattern of expression of the chromosomally integrated transgenes paralleled the endogenous expression of CFTR and beta-galactosidase activity was detected in cells containing villin and forming domes. Thus, a 6.6 kb region encompassing 5' and 3' non-coding parts of rat CFTR is able to drive specific expression of a reporter gene in cultured mouse intestinal cells having kept a crypt phenotype.
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PMID:CFTR regions containing duodenum specific DNase I hypersensitive sites drive expression in intestinal crypt cells but not in fibroblasts. 975 29


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