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

Given the knowledge that replication-deficient adenoviruses can mediate the delivery of unlinked plasmid DNA into eukaryotic cells (K. Yoshimura, M. A. Rosenfeld, P. Seth, and R. G. Crystal, J. Biol. Chem. 268:2300-2303, 1993), this study focuses on the role of receptor-mediated endocytosis in this process. AdCFTR (an E1- E3- adenovirus type 5-based replication-deficient adenovirus containing the 4.5-kb human cystic fibrosis transmembrane conductance regulator cDNA) was added to Cos-7 cells together with plasmid pRSVL (containing the Rous sarcoma virus long terminal repeat promoter followed by the luciferase cDNA), and luciferase activity was quantified as a measure of the expression of the plasmid DNA. When AdCFTR was bound to Cos-7 cells at 4 degrees C and the cells were subsequently incubated at 37 degrees C in the presence of pRSVL, the expression of luciferase activity was increased in proportion to the amount of AdCFTR added, reaching > 10(4)-fold at 3,000 PFU per cell. AdCFTR-mediated increase in pRSVL was inhibited by addition of purified adenovirus fiber but not hexon, suggesting cell surface adenovirus receptors were involved in the cointernalization process. Cell lines with a high number of adenovirus receptors (Cos-7 and HeLa) showed significant AdCFTR-dependent pRSVL expression, while cell lines with low numbers of adenovirus receptors (NIH 3T3 and U-937) showed little. AdCFTR-mediated increase in the expression of pRSVL was prevented when AdCFTR was heat treated and exposed to antibody against adenovirus or when the cointernalization process was evaluated in the presence of chloroquine, conditions all known to prevent adenovirus-mediated disruption of endocytic vesicles. In contrast, the uptake of AdCFTR into Cos-7 cells was not affected by any of these conditions. When AdCFTR was exposed to UV light, its ability to grow in 293 cells was obviated, but AdCFTR-dependent increase in pRSVL expression was minimally reduced. Finally, empty capsids of AdCFTR were able to enhance the delivery and expression of plasmid pRSVL into Cos-7 cells, suggesting that the adenovirus genome is not required for AdCFTR-mediated plasmid cointernalization. Together, these observations suggest that the ability of a replication-deficient recombinantly adenovirus to mediate the cointernalization and expression of plasmids is mediated by the receptor-mediated endocytosis pathway.
 ...
PMID:Mechanism of enhancement of DNA expression consequent to cointernalization of a replication-deficient adenovirus and unmodified plasmid DNA. 750 87

Mitogen-activated protein kinase (MAPK) or extracellular signal-regulated kinase are ubiquitous kinases conserved from fungi to mammals. Their activity is regulated by phosphorylation on both threonine and tyrosine, and they play a crucial role in the regulation of proliferation and differentiation. We report here the cloning of the murine p44 MAP kinase (extracellular signal-regulated kinase 1) gene, the determination of its intron/exon boundaries, and the characterization of its promoter. The gene spans approximately eight kilobases (kb) and can be divided into nine exons and eight introns, each coding region exon containing from one to three of the highly conserved protein kinase domains. Primer extension analysis reveals the existence of two major start sites of transcription located at -183 and -186 base pairs (bp) as well as four discrete start sites for transcription located at -178, -192, -273, and -292 bp of the initiation of translation. However, the start site region lacks TATA-like sequences but does contain initiator-like sequences proximal to the major start sites obtained by primer extension. 1 kb of the promoter region has been sequenced. It contains three putative TATA boxes far upstream of the main start sites region, one AP-1 box, one AP-2 box, one Malt box, one GAGA box, one half serum-responsive element, and putative binding sites for Sp1 (five), GC-rich binding factor (five), CTF-NF1 (one), Myb (one), p53 (two), Ets-1 (one), NF-IL6 (two), MyoD (two), Zeste (one), and hepatocyte nuclear factor-5 (one). To determine the sites critical for the function of the p44 MAPK promoter, we constructed a series of chimeric genes containing variable regions of the 5'-flanking sequence of p44 MAPK gene and the coding region for luciferase. Activity of the promoter, measured by its capacity to direct expression of a luciferase reporter gene, is strong, being comparable with the activity of the Rous sarcoma virus promoter. Progressive deletions of the approximately 1 kb (-1200/-78) promoter region allowed us to define a minimal region of 186 bp (-284/-78) that has maximal promoter activity. Within this context, deletion of the AP-2 binding site reduces by 30-40% the activity of the promoter. Further deletion of this minimal promoter that removes the major start sites (-167/-78) surprisingly preserves promoter activity. This result implicates a major role of this region that contains the Sp1 sites.(ABSTRACT TRUNCATED AT 400 WORDS)
 ...
PMID:The mouse p44 mitogen-activated protein kinase (extracellular signal-regulated kinase 1) gene. Genomic organization and structure of the 5'-flanking regulatory region. 759 46

We have developed an HSV-1 vector with mutations in the viral IE 3 and VP16 genes that expresses mouse beta nerve growth factor (NGF) from a latency associated transcript (LAT) promoter modified by insertion of a Rous sarcoma virus (RSV) enhancer. The backbone double mutant vector has reduced cytotoxicity compared with a single mutant deleted for IE 3 and is able to express the reporter luciferase gene in rat pheochromocytoma (PC12) cells at low but relatively stable levels in vitro. Intracellular NGF levels of approximately 3 pg per mg of cellular protein were detected in infected PC12 cells for at least 7 days after infection. Furthermore, infected PC12 cells exhibited differentiated morphology marked by neurite outgrowth similar to that found after exposure of PC12 cells to purified 2.5S beta NGF. A persistent level of 0.2 to 0.3 pM of the expressed NGF was detected in the culture media. The NGF-expressing vector also showed reduced cytotoxicity compared with that of the parental virus in infected PC12 cells. In PC12 cells that overexpress the human proto-oncogene bcl-2, the cytotoxicity of both viruses was significantly reduced. These studies demonstrate that the reduced cytotoxicity of the IE 3-VP16 double mutant virus and the increased duration of transgene expression from the RSV-modified LAT promoter permit terminal differentiation of PC12 cells after infection with an NGF-expressing HSV-1 vector.
 ...
PMID:Differentiation of PC12 cells by infection with an HSV-1 vector expressing nerve growth factor. 767 Nov 8

It is reported that cationic liposomes are capable of transfecting embryos in unincubated fertile chicken eggs and that the cationic liposome, TransfectAce, has superior properties to Lipofectin. In order to determine the duration of expression of genes introduced in this way, embryos were transfected with an expression vector encoding the firefly luciferase cDNA under the control of the Rous sarcoma virus long terminal repeat (LTR). Luciferase activity could be observed consistently in day 3 embryos and activity was detectable up to day 8 of incubation. The relative expression of luciferase under the control of different viral promoters was compared in transfected chicken embryo fibroblasts and day 3 embryos. The cytomegalovirus immediate early promoter and the SV40 early promoter directed the highest amount of expression in fibroblasts while the Rous sarcoma virus LTR caused the highest amount of expression in embryos. Chicken embryo fibroblasts were transfected with the luciferase vector in order to examine duration of reporter gene expression in vitro. Luciferase expression was decreased exponentially over a 24-day period after which point luciferase activity could no longer be detected. These data suggest that stable integration of transfected DNA using liposomes is a rare event. Nevertheless, liposome-mediated transfection of embryos is suitable for the examination of promoter activity in vivo and may be a useful method to transfect genes to study embryonic development.
 ...
PMID:In ovo transfection of chicken embryos using cationic liposomes. 779 62

The best way to overcome immunorejection in heterologous myoblast transfer (HMT) is by the use of immunodeficient and/or highly immunosuppressed mice as hosts. The same may be attained by autologous myoblast transfer (AMT). In this paper, we describe myoblast transfer in mdx and normal mice where the donor myogenic cells originated from highly inbred litter mates that are considered to be isogenic and thus the procedure is analogous to AMT. The myoblasts were marked in vitro with Rous Sarcoma Virus (RSV)-luciferase (Lux) or RSV-beta-galactosidase (LacZ) reporter genes through transduction mediated by an autonomously replication-defective recombinant human adenovirus. This permitted us to follow their fate after transplantation. mdx and normal mice were irradiated with 20 Gray gamma rays; necrosis and regeneration were induced by intramuscular notexin prior to myoblast injection. In both mdx and normal mice, the expression of luciferase rapidly declined after the injection implying that a large portion of the injected myoblasts were lost by 48 hr, due to undetermined cause(s). The surviving, injected myoblasts well-mosaicized large groups of host fibers but only in the immediate vicinity of the injection. Substantial expression of the reporter gene continued up to 1 month post-transplantation in normal mice, but there was a gradual decline and eventual disappearance of the reporter gene expression in mdx mice. This latter phenomenon was due to the ongoing intense necrosis of muscle fibers in mdx. There was no evidence of immunorejection. These experiments indicate that even in the absence of immunorejection, myoblast transfer suffers from important negative features: major loss of myoblasts within 48 hr after the injection and lack of significant spread of the injected cells from the injection site in the host muscle. These factors, plus the limited proliferative and fusion capacity of Duchenne muscular dystrophy (DMD) myoblasts, make them less than an ideal vector for the dystrophin cDNA for dystrophin gene replacement therapy in DMD.
 ...
PMID:Gene transfer into skeletal muscles by isogenic myoblasts. 794 44

The critical physiological functions of the liver make hepatocytes important targets for therapeutic gene delivery. This study reports significant gene expression following direct injection of plasmid DNA into the livers of rats and cats. Transfection was characterized using luciferase and Lac Z expression from plasmids with the cytomegalovirus immediate early promoter/enhancer (CMV IE) or the Rous sarcoma virus long terminal repeat (RSV LTR). Dexamethasone treatment enhanced and prolonged transfected gene expression, possibly by activating gene expression. Southern analysis of total DNA extracted from liver at various times following injection detected persistent unintegrated plasmid DNA which maintained a prokaryotic methylation pattern. This study demonstrates the feasibility of direct DNA injection in the experimental analysis of hepatic gene expression in vivo.
 ...
PMID:Dexamethasone enhancement of gene expression after direct hepatic DNA injection. 796 86

We have optimized a lipospermine-based transfection method for introducing genes into intact vertebrate embryos in vivo. The method employs small amounts of the cationic lipid Transfectam (DOGS), in a concentrated (40 mM) ethanolic solution, to compact and to transfer exogenous genes into chick embryos during the early stages of development (< 36 h of incubation). Plasmid vectors containing the reporter gene luciferase were used to follow the time course of expression. Luciferase activity was detected as early as 12 h post-transfection and was highest at this time. Enzyme activity then decreased over the next two days and was usually undetectable by 72-h post-transfection. To follow the spatial expression of the exogenous genes, a Rous sarcoma virus (RSV)-beta-galactosidase vector was used. When the transfection complex was applied externally around the developing embryo, the main site of expression was the cardiac tissue. Expression could be targeted to the nervous system by micro-injecting the DNA/DOGS (DNA/dioctadecylamidoglycylspermine) complex into the developing brain. The results show that reporter genes can be efficiently expressed in both the developing central nervous system and heart. This raises the possibility that lipospermines can be used to transfer functional genes into embryos during defined periods of development and also to deliver genes in other species and in other in vivo contexts.
 ...
PMID:Temporal and spatial expression of lipospermine-compacted genes transferred into chick embryos in vivo. 818 24

We describe a stably transformed cell line (BHKSINLuc2) that contains a defective Sindbis virus genome under the control of a Rous sarcoma virus promoter and the luciferase gene downstream of the viral subgenomic RNA promoter. This cell line expresses high levels of luciferase activity following infection with Sindbis virus and provides a sensitive assay for titering variants of Sindbis virus that lack the structural protein genes, in particular, Sindbis virus replicons that express heterologous proteins. Cell lines such as this may be of value for detection of positive-strand RNA viruses.
 ...
PMID:A cell line that expresses a reporter gene in response to infection by Sindbis virus: a prototype for detection of positive strand RNA viruses. 825 75

A rare germ-line polymorphism in codon 47 of the p53 gene replaces the wild-type proline (CCG) with a serine (TCG). Restriction analysis of 101 human samples revealed the frequency of the rare allele to be 0% (n = 69) in Caucasians and 4.7% (3/64, n = 32) among African-Americans. To investigate the consequence of this amino acid substitution, a cDNA construct (p53 mut47ser) containing the mutation was introduced into a lung adenocarcinoma cell line (Calu-6) that does not express p53. A growth suppression similar to that obtained after introduction of a wild-type p53 cDNA construct was observed, in contrast to the result obtained by introduction of p53 mut143ala. Furthermore, expression of neither p53 mut47ser nor wild-type p53 was tolerated by growing cells. In transient expression assays, both mut47ser and wild-type p53 activated the expression of a reporter gene linked to a p53 binding sequence (PG13-CAT) and inhibited the expression of the luciferase gene under the control of the Rous sarcoma virus promoter (RSVluc). In the same assay, mut143ala did not activate the expression of PG13-CAT and produced only a slight inhibitory effect on RSVluc. These findings indicate that the p53 variant with a serine at codon 47 should be considered as a rare germ-line polymorphism that does not alter the growth-suppression activity of p53.
 ...
PMID:Functional studies of a germ-line polymorphism at codon 47 within the p53 gene. 835 80

FET cells are well differentiated human adenocarcinoma cells whose growth is partially inhibited (50-60%) by transforming growth factor-beta 1 (TGF-beta 1). In exponentially growing cultures, TGF-beta 1 induces the expression of transforming growth factor-alpha (TGF-alpha) by 3-fold. To determine whether this induction is the result of increased TGF-alpha promoter activity, FET cells were transiently transfected with a plasmid containing 2816 base pairs of the 5'-flanking region of the TGF-alpha gene linked to luciferase. Transfected FET cells treated with growth-inhibitory concentrations of TGF-beta 1 (10 ng/ml) showed up to a 10-fold increase in luciferase activity. The increase in luciferase activity was dose dependent through the normal physiological range of TGF-beta 1 (0.5-20 ng/ml), saturating at 10 ng/ml. This effect was also TGF-alpha promoter specific, inasmuch as the Rous sarcoma virus long terminal repeat used as a control remained relatively insensitive to the effects of TGF-beta 1. By using progressively smaller portions of the TGF-alpha promoter region, the TGF-beta 1-responsive element was mapped between base pairs -77 and -201 of the 5'-flanking region. TGF-beta 1 treatment also affected epidermal growth factor receptor levels. FET cells treated with TGF-beta 1 (10 ng/ml) for 48 h showed a 20% decrease in the number of epidermal growth factor receptors and a 2-fold increase in the number of high affinity epidermal growth factor receptors on their surface. These results indicate that TGF-beta 1 acts as a positive regulator of TGF-alpha transcription, and they suggest a possible mechanism by which these cells circumvent the growth-inhibitory effects of TGF-beta 1.
 ...
PMID:Transforming growth factor-beta 1 induces transforming growth factor-alpha promoter activity and transforming growth factor-alpha secretion in the human colon adenocarcinoma cell line FET. 835 33


<< Previous 1 2 3 4 5 6 7 Next >>