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 Moloney murine leukemia virus (M-MuLV) repressor binding site (RBS) mediates cell-type-specific repression in embryonal carcinoma (EC) cells of expression from several different promoters, including the M-MuLV long terminal repeat promoter. Silencing has been shown to depend on an element normally located in the proviral 5' noncoding region and occurs at the DNA level in the absence of retroviral proteins. Using fragments of the RBS region, we now show that the minimal size of the silencer corresponds to M-MuLV nt 147-163 and overlaps with the retroviral primer binding site region by 17 of its 18 bp. A panel of point mutations within the RBS has been examined to yield a consensus RBS sequence which is consistent with the notion that a previously identified nuclear factor (binding factor A) mediates RBS repression. Viral vectors using neomycin, beta-galactosidase, and luciferase reporters have been employed to show that RBS-mediated repression occurs in EC and embryonal stem, but not in other tested cell types. Repression was observed to occur within 48 hr of infection, prior to when global methylation of proviruses has been reported to occur. Repression also occurred after azacytidine treatment of EC cells, supporting the notion that the RBS functions independently of provirus methylation. However, levels of provirus methylation in selected cells were increased in the presence of a wild-type RBS, and methylation correlated with a secondary stage of virus repression. Thus, the M-MuLV RBS acts directly to control expression in EC cells and also appears to trigger a secondary level of repression which is coincident with provirus methylation.
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PMID:Characterization of the Moloney murine leukemia virus stem cell-specific repressor binding site. 846 Apr 81

The discoidin proteins of Dictyostelium discoideum are highly expressed during development. The Disc I gamma promoter allows the regulation of heterologous protein expression by experimental conditions. We report conditions under which the promoter activity is efficiently repressed during growth in the wildtype strain AX2. In addition we show that a mutant which overexpresses the discoidins also overexpresses the reporter genes beta-galactosidase, luciferase and CAT 10- to 100-fold when these are placed under the control of a Disc I gamma promoter. This system may be generally useful for the overexpression of genes in Dictyostelium, both for functional studies in vivo and for the production of heterologous proteins for purification.
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PMID:Use of a transactive regulatory mutant of Dictyostelium discoideum in a eucaryotic expression system. 846 30

Polycyclic aromatic hydrocarbons such as benzo(a)pyrene diol-epoxide (BPDE-I) cause hepatocellular carcinoma. To identify short-term carcinogen effects, we studied hepatocytes transfected with nonreplicating plasmids, adducted covalently with BPDE-I, varying in promoter structure and encoded reporter gene (beta-galactosidase or luciferase). BPDE inactivated gene expression as a first-order function of BPDE concentration in adduction reactions. No evidence of cytotoxicity, diminished coprecipitation and availability, enhanced nicking of supercoiled forms and reduced cellular uptake, or instability of adducted plasmids was observed. At low BPDE:plasmid ratios, inactivation occurred with 1 adduct/plasmid within a target 23-27% of plasmid bases. Using nuclear extracts and BPDE-adducted G-free cassette-encoding plasmids, the fraction of full-length RNA polymerase II-initiated transcripts also declined as a first-order function of BPDE concentration when approximately 3 adducts were distributed among 48% of plasmid bases. These observations suggest that carcinogens such as BPDE block mRNA transcription along DNA templates by forming limited numbers of persistent adducts at coding or noncoding sites.
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PMID:Inactivation of plasmid reporter gene expression by one benzo(a)pyrene diol-epoxide DNA adduct in adult rat hepatocytes. 848 14

Two reporter genes: the firefly Photinus pyralis luciferase gene and the Escherichia coli beta-galactosidase gene were used for construction and characterization of the five unique recombinant vaccinia strain LIVP viruses expressing these genes in three nonessential regions of the virus genome. We give comparative characteristics of beta-galactosidase and luciferase activities in experiments of transient expression and expression dynamics of reporter genes by different stable recombinant viruses. Both genes are expressed with high efficiency independent on the sites of virus genome localization. It is shown that the TK-, HA- and N-regions of vaccinia virus DNA may be used for inserting foreign genes.
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PMID:[Analysis of reporter gene expression at different segments of the vaccinia virus genome]. 848 70

Striated muscle is the only tissue found to be capable of taking up and expressing reporter genes that are transferred in the form of plasmid DNA. Thus, direct gene transfer is a potential method of gene therapy for the primary inherited myopathies. However, results to date have had insufficient and too variable expression to consider using direct gene transfer in human trials. We have determined that much of the variability of expression is due to nonuniform distribution of substances injected into skeletal muscle in vivo, and have developed a model to ameliorate this. Preinjection of muscles with a relatively large volume of hypertonic sucrose improves the distribution of injected substances and results in significantly less variable expression of reporter genes for luciferase or beta-galactosidase; the coefficient of variation for mean luciferase activity was reduced from about 120% to 25%. Expression is not directly proportional to dose, but is more so if the muscles are preinjected with sucrose than not. Expression is higher and less variable if DNA is injected in a larger than a smaller volume. The choice of promoter appears to be particularly important. Luciferase reporter gene expression from the SV40 promoter was transient and low, whereas expression driven by the Rous sarcoma virus (RSV) promoter was high and sustained, such that a 1,000-fold difference in expression could be observed. The mechanism of gene uptake is still unknown, but our findings indicate that fibers damaged by the injection procedure do not take up and express plasmid DNA.
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PMID:Direct gene transfer into skeletal muscle in vivo: factors affecting efficiency of transfer and stability of expression. 849 24

Electroporation mediated DNA transfer into fish eggs has been improved by using a train of square pulses. Fertilized eggs of African catfish (Clarias gariepinus), zebrafish (Brachydanio rerio) and rosy barb (Barbus conchonius) were dechorionated enzymatically followed by application of pulses. Efficiency of plasmid DNA delivery was significantly increased by applying multiple pulses on dechorionated eggs. Optimization of physical parameters such as field strength, pulse width and pulse numbers resulted in reproducible transient expression in 25-50% of embryos and larvae by using the firefly luciferase and the E. coli beta-galactosidase (lacZ) genes both driven by CMV IE1 promoter. Temporal luciferase expression was assayed using both qualitative (sheet film) and quantitative (scintillation counting) methods in developing embryos and fry in vivo. Spatial expression of lacZ was assayed by histochemical staining. A number of embryos revealed foreign gene product also localised in the vegetal pole of the embryo.
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PMID:Efficient transient expression system based on square pulse electroporation and in vivo luciferase assay of fertilized fish eggs. 850 55

The striated muscle of the tongue provides a readily accessible site for the introduction of DNA expression vectors. Parameters were established to use the striated muscle of the tongue as a model system for the examination of gene expression following the direct injection of DNA constructs bearing gene promoter sequences controlling the expression of reporter genes. Plasmid expression vectors were used that contained either constitutive or muscle-specific promoters directing the transcription of reporter genes. Chloramphenicol acetyltransferase (CAT), luciferase, and beta-galactosidase (lacZ) were used as the reporter genes to detect the promoter-specific expression of the injected DNA. The expression of the injected plasmids was directly correlated with the mass of injected DNA and the time of incubation following the injection. Maximal levels of reporter gene expression were observed seven days after the injection, and the expression was maintained for more than two months following injection. Simultaneous injection of two individual expression vectors bearing either CAT or luciferase reporter genes resulted in a dose-dependent level of expression for each of the plasmids. The linearity of the coexpression provided a means to normalize DNA uptake and analyze promoter efficiency. The troponin C-fast enhancer linked to its own promoter directed significantly more CAT expression than an enhancerless SV40 promoter-CAT plasmid, demonstrating that different promoter strengths could be determined in the mouse tongue muscle in vivo. This model system represents a convenient means to approach the functional analysis of muscle gene promoters in vivo.
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PMID:Direct DNA injection into mouse tongue muscle for analysis of promoter function in vivo. 851 70

We recently have developed a unique cytoplasmic transient gene expression system based on cotransfection of target cells with bacteriophage T7 RNA polymerase (RNAP) and plasmid DNA vectors containing a T7 autogene. Because this T7 system is self-initiating, self-maintaining, and requires no cellular factors for transcription, it is therefore likely to function in any mammalian cell with any gene both in vitro and, more importantly, in vivo. In this study we demonstrate that the T7 DNA vector and T7 RNAP could be efficiently codelivered to cultured cells by lipofection. Different target genes were expressed by the T7 system in a wide variety of mammalian cells including several tumor cell lines. Gene expression could be detected in more than 30% of the cells of some tumor cell lines transiently transfected by the T7 vector. Average activity of the reporter enzyme (luciferase) expressed by a transfected cell was relatively constant regardless of the cell line used. When a T7-luciferase vector was directly injected into various tissues of mice without the use of liposomes, luciferase activity could be found in the injected liver, muscle, brain and tail connective tissues. The luciferase levels expressed by the T7 system were found to be up to 200-fold higher, depending upon the injected tissues, than levels achieved with a traditional nuclear gene expression vector. Direct tumor injection with a T7-beta-galactosidase (beta-gal) construct resulted in beta-gal gene expression in tumor cells near the injection sites. In addition, direct injection of the T7 system in mice did not generate detectable quantities of antibodies against the T7 RNAP. These results suggest that this gene expression system may be useful in many different medical applications such as cancer gene therapies and DNA vaccination, where transient but rapid and efficient gene expression is required.
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PMID:A novel nonviral cytoplasmic gene expression system and its implications in cancer gene therapy. 854 82

Peroxisome proliferators cause a rapid and coordinated transcriptional activation of genes encoding the enzymes of the peroxisomal beta-oxidation pathway in rats and mice. Cis-acting peroxisome proliferator responsive elements (PPREs) have been identified in the 5'-flanking region of H202-producing rat acyl-CoA oxidase (ACOX) gene and in other genes inducible by peroxisome proliferators. To gain more insight into the purported nonresponsiveness of human liver cells to peroxisome volume density and in the activity of the beta-oxidation enzyme system, we have previously cloned the human ACOX gene, the first and rate-limiting enzyme of the peroxisomal beta-oxidation system. We now present information on a regulatory element for the peroxidase proliferator-activated receptor (PPAR)/retinoid X receptor (RXR) heterodimers. The PPRE, consists of AGGTCA C TGGTCA, which is a direct repeat of hexamer half-sites interspaced by a single nucleotide (DR1 motif). It is located at -1918 to -1906 base pairs upstream of the transcription initiation site of this human ACOX gene. This PPRE specifically binds to baculovirus-expressed recombinant rat PPAR alpha/RXR alpha heterodimers. In transient transfection experiments, the maximum induction of luciferase expression by ciprofibrate and/or 9-cis-retinoic acid is dependent upon cotransfection of expression plasmids for PPAR alpha and RXR alpha. The functionally of this human ACOX promoter was further demonstrated by linking it to a beta-galactosidase reporter gene or to a rat urate oxidase cDNA and establishing stably transfected African green monkey kidney (CV1) cell lines expressing reporter protein. The human ACOX promoter has been found to be responsive to peroxisome proliferators in CV1 cells stably expressing PPAR alpha, whereas only a basal level of promoter activity is detected in stably transfected cells lacking PPAR alpha. The presence of a PPRE in the promoter of this human peroxisomal ACOX gene and its responsiveness to peroxisome proliferators suggests that factors other than the PPRE in the 5'-flanking sequence of the human ACOX gene may account for differences, if any, in the pleiotropic responses of humans to peroxisome proliferators.
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PMID:Identification of a peroxisome proliferator-responsive element upstream of the human peroxisomal fatty acyl coenzyme A oxidase gene. 856 72

This study was directed toward initial comparison and characterization of the activities of the human steroid 21-hydroxylase gene (CYP21) and pseudogene (CYP21P) promoters. DNA fragments containing the promoter regions of CYP21 and CYP21P were amplified and cloned into promoterless luciferase reporter plasmids either containing or lacking an enhancer element. Cells of the nonsteroidogenic COS-1 cell line, and the steroidogenic Y-1 cell line were transiently transfected with these recombinant plasmids and a beta-galactosidase cotransfection control plasmid. Cellular lysates were analyzed for luciferase and beta-galactosidase activities. In the nonsteroidogenic system, transfectants with either the CYP21 or CYP21P upstream sequence in enhancer containing plasmids showed a 2.3 fold increase (p < .001) in light production over controls. In the steroidogenic Y-1 cell system, these same CYp21 and CYP21P transfectants showed a 14.3 (+/- 0.8) and 5.2 (+/- 0.6) fold increase in luciferase activity respectively (p < .001) Transfections with recombinant reporter plasmids lacking an enhancer produced light emission which was not significantly different than controls. These observations indicate that 1.) one or more of the 35 nucleotide differences between the CYP21 and CYP21P upstream regions alters a DNA recognition site important for transcriptional activation of this gene in steroidogenic cells, 2.) the steroidogenic milieu has a stimulatory effect on both CYP21 and CYP21P promoter activities, and 3.) based on the minimal promoter activity observed in either cell type transfected with constructs lacking an enhancer element, both of these promoter sequences are enhancer dependent under constitutive conditions in both steroidogenic and nonsteroidogenic cells.
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PMID:Constitutive human steroid 21-hydroxylase promoter gene and pseudogene activity in steroidogenic and nonsteroidogenic cells with the luciferase gene as a reporter. 858 28


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