Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:1.14.14.3 (luciferase)
 38,195 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Bacterial luciferase, derived from a fusion of the luxA and luxB genes of Vibrio harveyi, has been expressed at very high levels in caterpillars and insect cells. The coding sequence for luciferase was inserted into vectors developed in our laboratory which were designed to expedite screening of recombinant virus. These vectors contained the beta-galactosidase indicator gene under control of immediate early (IE1), early (ETL), or very late (P10) promoters and a cloning site for inserting the fused luciferase gene next to the polyhedrin promoter. Recombinant baculoviruses containing the luciferase gene as well as the beta-galactosidase gene could be easily selected when Bluo-gal (beta-galactosidase indicator) was included in the plaque assays. Using cells derived from the fall armyworm (Spodoptera frugiperda), luciferase was strongly expressed very late in infection (48-72 h). The bacterial luciferase assay was sufficiently sensitive that light production could be detected from an extract of a single cell. In addition, live insects, including the cabbage looper (Trichoplusia ni) and saltmarsh caterpillar (Estigmene acrea) were infected by mixing recombinant baculovirus into their diet. Cabbage loopers (with an average wet weight of 223 mg) produced at least 195 micrograms of active luciferase and levels of synthesis peaked between 96-120 h. The results indicate that bacterial luciferase may be used as a reporter of gene expression in insects.
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PMID:Bacterial luciferase produced with rapid-screening baculovirus vectors is a sensitive reporter for infection of insect cells and larvae. 130 4

The purpose of this study was to determine whether plasmid DNA is able to persist in nondividing or slowly dividing brain cells in vivo. A new cationic lipid formulation which contains 70 mol% of DOTMA (N[1-(2,3-dioleyloxy)propyl]-N, N, N-trimethylammonium) and 30 mol% of cholesterol was used to transfect reporter genes into fetal brain cells in culture that were then transplanted into adult host brains. Gene expression was localized both to glial and neuronal cells after transfection of fetal brain cells with pRSVLac-Z, the gene coding for Escherichia coli beta-galactosidase protein. After the transfection of pRSVL plasmid which contains the firefly luciferase gene into fetal brain cells that were transplanted, substantial amounts of luciferase and pRSVL DNA were present in the host brains for 1 to 2 months. These results have implications for intracerebral viral infections and gene therapy of brain disorders.
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PMID:Persistence of plasmid DNA and expression in rat brain cells in vivo. 131 Dec 68

Expression of the Vibrio fischeri luminescence genes (luxR and luxICDABEG) in Escherichia coli requires autoinducer (N-3-oxohexanoyl homoserine lactone) and LuxR protein, which activate transcription of luxICDABEG (genes for autoinducer synthase and the luminescence enzymes), and cyclic AMP (cAMP) and cAMP receptor protein (CRP), which activate transcription of the divergently expressed luxR gene. In E. coli and in V. fischeri, the autoinducer-LuxR protein-dependent induction of luxICDABEG transcription (called autoinduction) is delayed by glucose, whereas it is promoted by iron restriction, but the mechanisms for these effects are not clear. To examine in V. fischeri control of lux gene expression by autoinducer, cAMP, glucose, and iron, lux::Mu dI(lacZ) and lux deletion mutants of V. fischeri were constructed by conjugation and gene replacement procedures. beta-Galactosidase synthesis in a luxC::lacZ mutant exhibited autoinduction. In a luxR::lacZ mutant, complementation by the luxR gene was necessary for luminescence, and addition of cAMP increased beta-galactosidase activity four- to sixfold. Furthermore, a luxI::lacZ mutant produced no detectable autoinducer but responded to its addition with induced synthesis of beta-galactosidase. These results confirm in V. fischeri key features of lux gene regulation derived from studies with E. coli. However, beta-galactosidase specific activity in the luxI::lacZ mutant, without added autoinducer, exhibited an eight- to tenfold decrease and rise back during growth, as did beta-galactosidase and luciferase specific activities in the luxR::lacZ mutant and luciferase specific activity in a delta(luxR luxICD) mutant. The presence of glucose delayed the rise back in beta-galactosidase and luciferase specific activities in these strains, whereas iron restriction promoted it. Thus, in addition to transcriptional control by autoinducer and LuxR protein, the V. fischeri lux system exhibits a cell density-dependent modulation of expression that does not require autoinducer, LuxR protein, or known lux regulatory sites. The response of autoinducer-LuxR protein-independent modulation to glucose and iron may account for how these environmental factors control lux gene expressions.
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PMID:Cell density-dependent modulation of the Vibrio fischeri luminescence system in the absence of autoinducer and LuxR protein. 131 12

Iron controls luminescence in Vibrio fischeri by an indirect but undefined mechanism. To gain insight into that mechanism, the involvement of cyclic AMP (cAMP) and cAMP receptor protein (CRP) and of modulation of DNA levels in iron control of luminescence were examined in V. fischeri and in Escherichia coli containing the cloned V. fischeri lux genes on plasmids. For V. fischeri and E. coli adenylate cyclase (cya) and CRP (crp) mutants containing intact lux genes (luxR luxICDABEG), presence of the iron chelator ethylenediamine-di(o-hydroxyphenyl acetic acid) (EDDHA) increased expression of the luminescence system like in the parent strains only in the cya mutants in the presence of added cAMP. In the E. coli strains containing a plasmid with a Mu dl(lacZ) fusion in luxR, levels of beta-galactosidase activity (expression from the luxR promoter) and luciferase activity (expression from the lux operon promoter) were both 2-3-fold higher in the presence of EDDHA in the parent strain, and for the mutants this response to EDDHA was observed only in the cya mutant in the presence of added cAMP. Therefore, cAMP and CRP are required for the iron restriction effect on luminescence, and their involvement in iron control apparently is distinct from the known differential control of transcription from the luxR and luxICDABEG promoters by cAMP-CRP. Furthermore, plasmid and chromosomal DNA levels were higher in E. coli and V. fischeri in the presence of EDDHA. The higher DNA levels correlated with an increase in expression of chromosomally encoded beta-galactosidase in E. coli and with a higher level of autoinducer in cultures of V. fischeri. These results implicate cAMP-CRP and modulation of DNA levels in the mechanism of iron control of the V. fischeri luminescence system.
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PMID:Mechanism for iron control of the Vibrio fischeri luminescence system: involvement of cyclic AMP and cyclic AMP receptor protein and modulation of DNA level. 132 97

As an approach to gene therapy for the respiratory manifestations of cystic fibrosis (CF), in vivo plasmid-mediated direct transfer of the normal CF transmembrane conductance regulator (CFTR) gene to the airway epithelium was investigated in mice. To evaluate the feasibility of this strategy, pRSVL, a plasmid composed of a firefly luciferase gene driven by the Rous sarcoma virus long terminal repeat (RSV-LTR), along with cationic liposomes was instilled into the trachea of C57BI/6NCR mice. With administration of 200-400 micrograms plasmid DNA, luciferase expression could be detected in the mouse lung homogenates for at least 4 wk. With this background, a CFTR expression plasmid vector (pRSVCFTR) constructed by replacing the luciferase cDNA from pRSVL with the normal human CFTR cDNA was evaluated in vivo in mice. Intratracheal instillation of pRSVCFTR with cationic liposomes followed by analysis of mouse lung RNA by polymerase chain reaction amplification (after conversion of mRNA to cDNA) using a RSV-LTR specific sense primer and a human CFTR-specific antisense primer demonstrated human CFTR mRNA transcripts from one day to 4 wk after instillation. Further, in vivo evaluation of beta-galactosidase activity after intratracheal administration of an E. coli lacZ gene expression plasmid vector directed by the cytomegalovirus promoter (pCMV beta) demonstrated that the airway epithelium was the major target of transfer and expression of the exogenous gene. These observations demonstrate successful plasmid-mediated gene transfer to the airway epithelium in vivo. This strategy may be feasible as a form of gene therapy to prevent the pulmonary manifestations of CF.
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PMID:Expression of the human cystic fibrosis transmembrane conductance regulator gene in the mouse lung after in vivo intratracheal plasmid-mediated gene transfer. 137 20

The transcription of the luminescence (lux) system of Vibrio fischeri is regulated by the LuxR protein and an autoinducer. We previously showed that apart from these regulatory elements, the transcription of the lux system is negatively controlled by the LexA protein and positively controlled by the HtpR protein (sigma 32). This study was conducted in order to elucidate the mode of action of the HtpR protein. Using luxR-lacZ fused genes, we showed that the HtpR protein is essential for the maximum expression of beta-galactosidase activity in Escherichia coli lac mutant cells. Using this construct, we also demonstrated that luxR is preferentially expressed toward the end of the logarithmic phase of growth. Starvation and addition of ethanol significantly advanced the appearance of beta-galactosidase activity in htpR+ cells. The luminescence system of E. coli htpR+ cells harboring the pChv1 plasmid with a deletion in the luxI gene is induced in the presence of low and constant concentrations (150 pg/ml) of the inducer only at a late stage of the logarithmic phase of growth. When the cellular LuxR content is reduced, following 23 generations of exponential growth in Luria broth, a mid-log-phase culture does not respond to the inducer (150 pg/ml). On the basis of the above observations we suggest that the HtpR protein controls the formation of V. fischeri LuxR protein. Preliminary findings indicate that the HtpR protein acts through the chaperonins GroESL. E. coli htpR/pChv1 cells retained their full level of in vivo and in vitro luciferase activities in the presence of multiple copies of groESL genes. The possibility that GroESL proteins stabilize the native form of LuxR protein is discussed.
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PMID:Formation of the LuxR protein in the Vibrio fischeri lux system is controlled by HtpR through the GroESL proteins. 142 36

We compare the transfection efficiency of plasmid DNA encoding either luciferase or beta-galactosidase encapsulated in pH-sensitive liposomes or non-pH-sensitive liposomes or DNA complexed with cationic liposomes composed of dioleoyloxypropyl-trimethylammonium:dioleoylphosphatidyl-eth anolamine (1:1, w/w) (Lipofectin) and delivered into various mammalian cell lines. Cationic liposomes mediate the highest transient transfection level in all cell-lines examined. pH-sensitive liposomes, composed of cholestryl hemisuccinate and dioleoylphosphatidylethanolamine at a 2:1 molar ratio, mediate gene transfer with efficiencies that are 1 to 30% of that obtained with cationic liposomes, while non-pH-sensitive liposome compositions do not induce any detectable transfection. Cationic liposomes mediate a more rapid uptake of plasmid DNA, to about an eightfold greater level than that obtained with pH-sensitive liposomes. The higher uptake of DNA mediated by Lipofectin accounts for part of its high transfection efficiency. Treatment of cells with chloroquine, ammonium chloride, or monensin decreases (threefold) transfection using pH-sensitive liposomes and either has no effect on or enhances cationic liposome-mediated transfection. Therefore plasma membrane fusion is not the only mechanism available to cationic liposomes; in certain cell lines DNA delivery via endocytosis is a possible parallel pathway and could augment the superior transfection efficiency observed with cationic liposomes.
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PMID:Delivery of plasmid DNA into mammalian cell lines using pH-sensitive liposomes: comparison with cationic liposomes. 144 19

Microbioassays using bacteria or enzymes are increasingly applied to measure chemical toxicity in the environment. Attractive features of these assays may include low cost, rapid response to toxicants, high sample throughput, modest laboratory equipment and space requirements, low sample volume, portability, and reproducible responses. Enzymatic tests rely on measurement of either enzyme activity or enzyme biosynthesis. Dehydrogenases are the enzymes most used in toxicity testing. Assay of dehydrogenase activity is conveniently carried out using oxidoreduction dyes such as tetrazolium salts. Other enzyme activity tests utilize ATPases, esterases, phosphatases, urease, luciferase, beta-galactosidase, protease, amylase, or beta-glucosidase. Recently, the inhibition of enzyme (beta-galactosidase, tryptophanase, alpha-glucosidase) biosynthesis has been explored as a basis for toxicity testing. Enzyme biosynthesis was found to be generally more sensitive to organic chemicals than enzyme activity. Bacterial toxicity tests are based on bioluminescence, motility, growth, viability, ATP, oxygen uptake, nitrification, or heat production. An important aspect of bacterial tests is the permeability of cells to environmental toxicants, particularly organic chemicals of hydrophobic nature. Physical, chemical, and genetic alterations of the outer membrane of E. coli have been found to affect test sensitivity to organic toxicants. Several microbioassays are now commercially available. The names of the assays and their basis are: Microtox (bioluminescence), Polytox (respiration), ECHA Biocide Monitor (dehydrogenase activity), Toxi-Chromotest (enzyme biosynthesis), and MetPAD (enzyme activity). An important feature common to these tests is the provision of standardized cultures of bacteria in freeze-dried form. Two of the more recent applications of microbioassays are in sediment toxicity testing and toxicity reduction evaluation. Sediment pore water may be assayed directly or solvents may be used to extract the toxicants. Some of the solvents used for extraction of organic chemicals are themselves toxic to bacteria (e.g., dichloromethane), requiring exchange with a less toxic solvent (e.g., ethanol, methanol, DMSO). A modification of the Microtox test allows direct assay of solid-phase samples such as sediments. The toxicity reduction evaluation (TRE) must be carried out at wastewater treatment plants whose effluents fail toxicity standards. The TREs require numerous and repeated toxicity assays, thus favoring application of microbioassays. Presently, no single microbioassay can detect all categories of environmental toxicants with equal sensitivity. Therefore, a battery of tests approach is recommended. The differential sensitivity of alternative tests may, in fact, be exploited. Further research is needed to construct strains of genetically engineered microorganisms or isolate microorganisms or enzymes that respond to specific classes of toxicants. These can be combined into batteries appropriate for different environments or test objectives.
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PMID:Bacterial and enzymatic bioassays for toxicity testing in the environment. 150 75

Iron influences luminescence in Vibrio fischeri; cultures iron-restricted for growth rate induce luminescence at a lower optical density (OD) than faster growing, iron-replete cultures. An iron restriction effect analogous to that in V. fischeri (slower growth, induction of luminescence at a lower OD) was established using Escherichia coli tonB and tonB+ strains transformed with recombinant plasmids containing the V. fischeri lux genes (luxR luxICDABEG) and grown in the presence and absence of the iron chelator ethylenediamine-di(o-hydroxylphenyl acetic acid) (EDDHA). This permitted the mechanism of iron control of luminescence to be examined. A fur mutant and its parent strain containing the intact lux genes exhibited no difference in the OD at induction of luminescence. Therefore, an iron-binding repressor protein apparently is not involved in iron control of luminescence. Furthermore, in the tonB and in tonB+ strains containing lux plasmids with Mu dI(lacZ) fusions in luxR, levels of beta-galactosidase activity (expression from the luxR promoter) and luciferase activity (expression from the luxICDABEG promoter) both increased by a similar amount (8-9 fold each for tonB, 2-3 fold each for tonB+) in the presence of EDDHA. Similar results were obtained with the luxR gene present on a complementing plasmid. The previously identified regulatory factors that control the lux system (autoinducer-LuxR protein, cyclic AMP-cAMP receptor protein) differentially control expression from the luxR and luxICDABEG promoters, increasing expression from one while decreasing expression from the other. Consequently, these results suggest that the effect of iron on the V. fischeri luminescence system is indirect.
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PMID:Iron control of the Vibrio fischeri luminescence system in Escherichia coli. 151 May 56

Complexes containing plasmid DNA, transferrin-polylysine conjugates, and polylysine-conjugated peptides derived from the N-terminal sequence of the influenza virus hemagglutinin subunit HA-2 have been used for the transfer of luciferase or beta-galactosidase marker genes to K562 cells, HeLa cells, and BNL CL.2 hepatocytes. These DNA complexes mimic the entry of viruses into cells, as they contain functions for (i) the packaging of the nucleic acid with polylysine, (ii) the attachment to the cell and receptor-mediated endocytosis with transferrin as a ligand, and (iii) the release from endosomes by using membrane-disrupting influenza peptides. The presence of these influenza peptide conjugates in the DNA complexes renders the complexes active in membrane disruption in a liposome leakage assay and results in a substantial augmentation of the transferrin-polylysine-mediated gene transfer.
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PMID:Influenza virus hemagglutinin HA-2 N-terminal fusogenic peptides augment gene transfer by transferrin-polylysine-DNA complexes: toward a synthetic virus-like gene-transfer vehicle. 151 16


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