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)

U5 small nuclear RNA itself can act as a clastogenic and transforming agent when transfected into cells. In the previous work, the 3' half of the U5 small nuclear RNA first stem structure (designated RNA3S) was capable of driving normal cells into tumorigenic cells when expressed with a poly(A) tail (RNA3S+). This transformation critically depended upon the polypurine sequence GGAGAGGAA in RNA3S+. In this work, we first examined the pre-beta-lactamase and luciferase (model secretory and nonsecretory proteins) translation with the in vitro synthesized RNA3S in rabbit reticulocyte lysate. The capped RNA3S with a poly(A) tail suppressed the translation. In addition, the polypurine sequence played a crucial role in affecting the secretory protein synthesis, indicating a primary action of RNA3S+. Further studies revealed that the oligodeoxynucleotides, corresponding to the polypurine and its antisense sequences, directly contacted 28 S rRNA in ribosome and 7SL RNA in signal recognition particle, respectively, and differentially affected the nascent chain elongation of secretory protein synthesis. These results suggest that RNA3S+ blocks a physiological regulatory function played by signal recognition particle and the ribosome in the secretory protein synthesis and support the idea that the transformation might result from a repressed cellular activity.
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PMID:Effect of transforming RNA on the synthesis of a protein with a secretory signal sequence in vitro. 1033 81

Cell-free translation/translocation systems are broadly applied to examine gene expression and characterize the structure-function relationship of gene products. We present the characterization of Xenopus egg extract (XEE) translocation and processing of proteins synthesized in rabbit reticulocyte lysate. The XEE was prepared from eggs laid by adult female frogs that received serial injections of gonadotropins. The eggs were then dejellied in 2% L-cysteine-HCl and the cytoplasm extracted by centrifugation at 10,000 rpm for 15 min. The in vitro translocation and processing of XEE was examined with a cell-free translation system containing reticulocyte lysate, and appropriate messenger ribonucleic acid (RNA) or complementary deoxyribonucleic acid plasmids with RNA polymerase. Cell-free production of the following proteins were used to assess posttranslational modifications: Escherichia coli beta-lactamase for signal sequence cleavage, Saccharomyces cerevisiae alpha-mating factor for translocation and N-linked glycosylation, the soluble protein luciferase for functional activity, and the membrane-bound human insulin receptor for translation efficiency. All translation products were identified by [35S]-methionine labeling, sodium dodecyl sulfate-polyacrylamide gel electrophoresis and fluorography. The results demonstrate that (1) XEE produces near-complete signal sequence and N-glycosylation processing of proteins synthesized in reticulocyte lysate, (2) XEE contains endoplasmic reticulum-equivalent microsomes, which allows for protein translocation and protease protection, (3) the addition of XEE in the translation reaction does not affect synthesis and chemiluminescence activity of luciferase, (4) XEE is efficient in processing the nascent 160-kDa human insulin receptor precursor, a transmembrane protein, and (5) as compared to canine pancreatic microsomes, XEE translocation efficiency is minimally decreased with the addition of dimethylsulfoxide. These results are the first description of the combined use of XEE with reticulocyte lysate and clearly demonstrate a higher efficiency of translocation and processing compared to canine pancreatic microsomes. This method of cell-free translation and processing allows for more extensive in vitro examination of posttranslational modifications of secretory and membrane-bound proteins.
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PMID:Efficient translocation and processing with Xenopus egg extracts of proteins synthesized in rabbit reticulocyte lysate. 1093 32

This review summarizes recent work on the use of reporter genes to label selected neuronal populations in transgenic mice, with particular emphasis on gonadotropin-releasing hormone (GnRH) neurons. Reporter genes discussed are the lacZ, green fluorescent protein (GFP), luc, and bla genes, which encode the reporter proteins beta-galactosidase, GFP, luciferase, and beta-lactamase, respectively. Targeted transgenic expression of these reporter proteins is obtained by fusing the corresponding reporter gene, with or without a subcellular localization signal, to a cell type- or brain region-specific gene promoter. Mice carrying GnRH promoter-driven reporter genes have proven useful for revealing the promoter elements required for cell type-specific expression of GnRH, the full anatomical profile of the GnRH neuronal network, and its electrophysiological activity, suggesting that similar approaches will assist in elucidating the properties of other neuronal populations as well.
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PMID:Using reporter genes to label selected neuronal populations in transgenic mice for gene promoter, anatomical, and physiological studies. 1116

The beta-galactosidase reporter gene is commonly used as a control for transfection efficiency in the promoter reporter assay system. While investigating vasoactive intestinal peptide response elements in the promoter of the prolactin gene, we found that primary pituitary cells from turkey hens highly expressed endogenous beta-galactosidase. Therefore, we developed a new protocol for determining transfection efficiency using the beta-lactamase gene, which is present on many expression vectors. Transcript levels of beta-lactamase were measured by RT-PCR after transfection of different amounts of the pGL3-basic and pGL3-control vectors. A high correlation was observed between the amount of plasmid transfected and beta-lactamase mRNA levels. Although no eukaryotic promoter was present, there was apparently leaky expression of the beta-lactamase gene. Expression of beta-lactamase was independent of expression from the simian virus 40 or turkey prolactin promoters cloned upstream of the luciferase gene.
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PMID:Normalization of transfection efficiency using the beta-lactamase gene of the pGL3 luciferase vector in primary anterior pituitary cells. 1218 85

Molecular markers such as: lacZ (b-galactosidase), xylE (catechol 2,3-dioxygenase), lux (bacterial luciferase), luc (insect luciferase), phoA (alkaline phosphatase), gusA and gurA (beta-glucuronidase), gfp (green fluorescent protein), bla (beta-lactamase) and other antibiotic resistance markers, heavy metals resistance genes are commonly used in environmental microorganisms research (Errampaii et al., 1998; Kohler et al., 1999). Most of these markers require one or more substrates, complex media and/or expensive equipment for detection. The gfp gene is widely used as a marker because of its very useful properties such as high stability, minimal toxicity, non-invasive detection and the ability to generate the green light without addition of external cofactors and without application of expensive equipment. Various applications of that reporter gene were showed starting from monitoring of microorganism's survival in complex biological systems such as activated sludge to biodegradation of chemical compounds in soil. GFP allowed the detection, determination of spatial location and enumeration of bacterial cells from diverse environmental samples such as biofilm and water. The gfp as a biomarker was very useful in monitoring of gene expression and protein localisation in bacterial cells, too. The techniques with using gfp marker promise to supply a better understanding of environmental processes. It can make possible to use that knowledge in designing more effective and more efficient methods of biodegradation of toxic compounds from different environments.
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PMID:Green fluorescent protein as a molecular marker in microbiology. 1258 95

Serratia marcescens encodes an inducible, chromosomal beta-lactamase, ampC. Studies addressing the regulation of inducible ampC genes have focused primarily on Enterobacter cloacae and Citrobacter freundii. The purpose of this study was to clone and sequence the ampC, ampR and intergenic region of S. marcescens and examine both inducible and basal level ampC expression. Sequence analysis of the S. marcescens ampC gene identified an extended 5' untranslated region (UTR) of 126 nucleotides, which formed a prominent stem-loop structure. Induction of ampC expression required AmpR, and the start of transcription was determined using primer extension analysis. In vivo half-life analysis revealed that the half-life of the S. marcescens ampC transcript was 7 min. Confirmation of the in vivo half-life and the role of the stem-loop structure in the 5' UTR was demonstrated by comparing transcript half-life and luciferase expression between a wild-type (WT) and a 5' UTR stem-loop deletion mutant. These data demonstrated that the stem-loop structure was involved in transcript stability. Taken together, these findings indicate that constitutive expression of S. marcescens ampC is regulated by both transcriptional initiation and post-transcriptional events.
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PMID:Analyses of ampC gene expression in Serratia marcescens reveal new regulatory properties. 1265 51

Measuring antibiotic-induced killing relies on time-consuming biological tests. The firefly luciferase gene (luc) was successfully used as a reporter gene to assess antibiotic efficacy rapidly in slow-growing Mycobacterium tuberculosis. We tested whether luc expression could also provide a rapid evaluation of bactericidal drugs in Streptococcus gordonii. The suicide vectors pFW5luc and a modified version of pJDC9 carrying a promoterless luc gene were used to construct transcriptional-fusion mutants. One mutant susceptible to penicillin-induced killing (LMI2) and three penicillin-tolerant derivatives (LMI103, LMI104, and LMI105) producing luciferase under independent streptococcal promoters were tested. The correlation between antibiotic-induced killing and luminescence was determined with mechanistically unrelated drugs. Chloramphenicol (20 times the MIC) inhibited bacterial growth. In parallel, luciferase stopped increasing and remained stable, as determined by luminescence and Western blots. Ciprofloxacin (200 times the MIC) rapidly killed 1.5 log10 CFU/ml in 2-4 hr. Luminescence decreased simultaneously by 10-fold. In contrast, penicillin (200 times the MIC) gave discordant results. Although killing was slow (< or = 0.5 log10 CFU/ml in 2 hr), luminescence dropped abruptly by 50-100-times in the same time. Inactivating penicillin with penicillinase restored luminescence, irrespective of viable counts. This was not due to altered luciferase expression or stability, suggesting some kind of post-translational modification. Luciferase shares homology with aminoacyl-tRNA synthetase and acyl-CoA ligase, which might be regulated by macromolecule synthesis and hence affected in penicillin-inhibited cells. Because of resemblance, luciferase might be down-regulated simultaneously. Luminescence cannot be universally used to predict antibiotic-induced killing. Thus, introducing reporter enzymes sharing mechanistic similarities with normal metabolic reactions might reveal other effects than those expected.
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PMID:Antibiotic-dependent correlation between drug-induced killing and loss of luminescence in Streptococcus gordonii expressing luciferase. 1282 Jul 96

Neurotrophins are a family of secreted proteins that play an important role in the development, differentiation, and survival of neurons. Studies also suggest that aberrant neurotrophin signaling may play a role in processes underlying disease states such as schizophrenia, Alzheimer's disease, and depression. Whereas the development of agents that selectively stimulate neurotrophin signaling has proven to be difficult, compounds have been identified that potentiate neurotrophin 3 (NT-3)-mediated activation of trk A. In the present studies, we extend those initial observations to identify compounds that also potentiate NT-3-mediated activation of trk B. Compound potentiation of NT-3 was observed using several readouts of transfected and endogenous trk receptor activity, including trk receptor phosphorylation, mitogen-activated protein kinase phosphorylation, reporter assay activity (beta-lactamase and luciferase), cell survival and neurite extension assays. Studies using chimeric trk receptors demonstrated that the extracellular domain is essential for compound potentiation and rule out interaction with intracellular signaling molecules as a mechanism of compound activity. Thus, the present studies demonstrate that trk B receptor activity can be potentiated by small-molecule compounds via the extracellular domain of the receptor and provide reagents for further evaluating the role of NT-3-mediated trk A and trk B activity in vivo.
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PMID:Identification and characterization of compounds that potentiate NT-3-mediated Trk receptor activity. 1639 50

A Klebsiella pneumoniae strain, KU6500, which showed resistance to extended-spectrum beta-lactams and produced the plasmid-encoded AmpC beta-lactamase CMY-4, was identified from clinical isolates in Japan. The aim of this study was to identify the mechanism of the high-level expression of blaCMY-4. Sequence analysis indicated that the promoter element of Citrobacter freundii was conserved, but the insertion sequence ISEcp1 coding with the putative promoter element, was inserted into the AmpR binding site. We determined the influence of the promoter on blaCMY-4 expression and beta-lactam resistance. Two recombinant plasmids containing the entire blaCMY-4 gene, with or without the ISEcp1-mediated promoter sequences, were constructed and named pMWampC and pMWISEcp1, respectively. Escherichia coli DH5alpha (pMWISEcp1) was resistant to almost all beta-lactams tested and E. coli DH5alpha (pMWampC) was susceptible to all, except for cephalothin. In addition, the activity of each promoter was measured by subcloning the element into a promoterless luciferase plasmid pGL3-Basic vector. The expression of the putative promoter of ISEcp1 was 18.9-fold higher than that of C. freundii. These results suggest that the putative promoter element of ISEcp1 is necessary for the high-level expression of blaCMY-4 to confer resistance to extended-spectrum cephalosporins.
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PMID:Resistance to gram-negative organisms due to high-level expression of plasmid-encoded ampC beta-lactamase blaCMY-4 promoted by insertion sequence ISEcp1. 1733 24

Ribozymes are RNA molecules capable of associating with other RNA molecules through base-pairing and catalyzing various reactions involving phosphate group transfer. Of particular interest to us is the well known ribozyme from Tetrahymena thermophila capable of catalyzing RNA splicing in eukaryotic systems, chiefly because of its potential use as a gene therapy agent. In this article we review the progress made towards visualizing the RNA splicing mediated by the Tetrahymena ribozyme in single living mammalian cells with the beta-lactamase reporter system and highlight the development made in imaging RNA splicing with the luciferase reporter system in living animals.
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PMID:Visualizing RNA splicing in vivo. 1746 Jul 89


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