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Query: UNIPROT:P06889 (Mol)
 630,302 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

A plasmid recombination assay, which utilized mutated Vibrio fischeri luciferase genes, cloned in Escherichia coli plasmids was developed. Expression of the recombination product, a functional luxA gene, was assayed by measuring light intensity. This system was used to investigate the effect of E. coli gene functions on lambda Red- and Gam-dependent plasmid recombination. The genetic and physiological requirements for Red- and Gam-dependent plasmid recombination are similar to the conditions which allow synthesis of plasmid linear multimers. Both recombination and linear multimer synthesis are mediated by Red activity in recBrecC and in sbcB mutants and by Gam activity in sbcB and sbcA mutants, but neither recombination nor linear multimer synthesis is mediated by Red or Gam functions in RecBCD+ExoI+ cells. When mediated by Red in sbcB mutants, both recombination and linear multimer synthesis are RecA-independent, and when mediated by Gam, in the same genetic background, both are RecA-dependent. A role for replication in Red- and Gam-mediated plasmid recombination is suggested by the dependence of the recombination activity on DnaB. A model which hypothesizes mutual dependence of linear plasmid multimer synthesis and plasmid recombination by the RecE, RecF and Red pathways is presented. We propose that ends that are produced during this type of replication are recombinogenic in all three pathways and that new rounds of replication are primed by a recombination-dependent invasion of duplex DNA by 3' single strand ends.
J Mol Biol 1988 Sep 20
PMID:Use of a bioluminescence gene reporter for the investigation of red-dependent and gam-dependent plasmid recombination in Escherichia coli K12. 305 84

Bacterial luciferase can be assayed rapidly and with high sensitivity both in vivo and in vitro. Here we demonstrate that the N-terminal hydrophobic domain of the alpha catalytic subunit of the luciferase enzyme is indispensable for enzyme activity, although N-terminal translational fusions with full luciferase activity can be obtained. Bacterial luciferase is therefore ideally suited as a reporter enzyme for gene fusion experiments. A list of vectors for the convenient use of the luciferase marker genes to monitor gene expression in vivo are presented.
Mol Gen Genet 1988 Dec
PMID:The use of the luxA gene of the bacterial luciferase operon as a reporter gene. 307 33

Binding of ligand to the estrogen receptor, a member of the steroid receptor gene family, rapidly increases PRL gene transcription. A 15 base pair core sequence 5'TTGTCACTATGTCCT-3' greater than 1.5 kilobase upstream from the rat PRL gene transcription start site is necessary for receptor binding, demonstrates interaction with the receptor DNA binding domain, and confers estrogen regulation. Transient cotransfection of expression plasmids encoding mutant estrogen receptors with a luciferase reporter plasmid under regulation of the rat PRL estrogen regulatory element were used to investigate the minimal information necessary and sufficient for activation of gene transcription. These analyses confirmed the absolute requirement for the receptor DNA binding domain in positive regulation of transcription, and revealed that removal of amino-terminal domains reduced, but did not abolish transcriptional effects. In contrast, truncation of the receptor immediately carboxy-terminal to the DNA binding domain resulted in constitutive activation of the receptor. The observations that removal of the steroid binding domain results in a constitutively active transcriptional factor, and that the amino-terminal domains are not required for transcriptional effect provides evidence that for two members of the steroid receptor gene family (the glucocorticoid and estrogen receptors), a relatively short DNA binding domain is sufficient for transcriptional activation. These results are likely to be prototypic for other members of this family of transcriptional factors.
Mol Endocrinol 1988 Jan
PMID:A single domain of the estrogen receptor confers deoxyribonucleic acid binding and transcriptional activation of the rat prolactin gene. 339 40

The genes of Photobacterium leiognathi luminescence system were cloned in plasmid pUC18. Escherichia coli cells harboring a recombinant plasmid pPHL1 are luminescent. pPHL1 contains luciferase genes and genes responsible for aldehyde biosynthesis. The luminescence of Escherichia coli is subject to autoinductor regulation similar to the one existing in luminescent bacteria. The 2.7 kb fragment of Photobacterium leiognathi DNA containing the genes for alpha- and beta-luciferase subunits were cloned in pUC19.
Mol Gen Mikrobiol Virusol 1987 Aug
PMID:[Cloning and expression of genes of the luminescence system in Photobacterium leiognathi]. 368 27

Cellular levels of diadenosine tetraphosphate (Ap4A) and adenosine tetraphospho-guanosine (Ap4G) were specifically measured during the cell cycle of Physarum polycephalum by a high-pressure liquid chromatographic method. Ap4A was also measured indirectly by a coupled phosphodiesterase-luciferase assay. No cell cycle-specific changes in either Ap4A or Ap4G were detected in experiments involving different methods of assay, different strains of P. polycephalum, or different methods of fixation of macroplasmodia. Our results on Ap4A are in contrast with those reported previously (C. Weinmann-Dorsch, G. Pierron, R. Wick, H. Sauer, and F. Grummt, Exp. Cell Res. 155:171-177, 1984). Weinmann-Dorsch et al. reported an 8- to 30-fold increase in Ap4A in early S phase in P. polycephalum, as measured by the phosphodiesterase-luciferase assay. We also measured levels of Ap4A, Ap4G, and ATP in macroplasmodia treated with 0.1 mM dinitrophenol. Ap4A and Ap4G transiently increased three- to sevenfold after 1 h and then decreased concomitantly with an 80% decrease in the level of ATP after 2 h in the presence of dinitrophenol. These results do not support the hypothesis that Ap4A is a positive pleiotypic activator that modulates DNA replication, but they are consistent with the hypothesis proposed for procaryotes that Ap4A and Ap4G are signal nucleotides or alarmones of oxidative stress (B.R. Bochner, P.C. Lee, S.W. Wilson, C.W. Cutler, and B.N. Ames, Cell 37:225-232, 1984).
Mol Cell Biol 1986 Apr
PMID:In vivo levels of diadenosine tetraphosphate and adenosine tetraphospho-guanosine in Physarum polycephalum during the cell cycle and oxidative stress. 378 60

The nucleotide sequence of the luciferase gene from the firefly Photinus pyralis was determined from the analysis of cDNA and genomic clones. The gene contains six introns, all less than 60 bases in length. The 5' end of the luciferase mRNA was determined by both S1 nuclease analysis and primer extension. Although the luciferase cDNA clone lacked the six N-terminal codons of the open reading frame, we were able to reconstruct the equivalent of a full-length cDNA using the genomic clone as a source of the missing 5' sequence. The full-length, intronless luciferase gene was inserted into mammalian expression vectors and introduced into monkey (CV-1) cells in which enzymatically active firefly luciferase was transiently expressed. In addition, cell lines stably expressing firefly luciferase were isolated. Deleting a portion of the 5'-untranslated region of the luciferase gene removed an upstream initiation (AUG) codon and resulted in a twofold increase in the level of luciferase expression. The ability of the full-length luciferase gene to activate cryptic or enhancerless promoters was also greatly reduced or eliminated by this 5' deletion. Assaying the expression of luciferase provides a rapid and inexpensive method for monitoring promoter activity. Depending on the instrumentation employed to detect luciferase activity, we estimate this assay to be from 30- to 1,000-fold more sensitive than assaying chloramphenicol acetyltransferase expression.
Mol Cell Biol 1987 Feb
PMID:Firefly luciferase gene: structure and expression in mammalian cells. 382 27

To provide for bioluminescence measurements of the enzymatic activities of dehydrogenases, disturbing contaminants were removed from a bacterial luciferase extract by chromatography, using Blue Sepharose CL-6B, a cross-linked agarose to which Cibacrone Blue F3G-A is covalently attached. This compound has a strong affinity to the dinucleotide fold, which is a region in enzymes binding NAD(H) or NADP(H). In contrast to the absorbed dehydrogenases, both luciferase and oxidoreductase were easily eluted and appeared close to the main bulk of UV-absorbing but analytically less important material. A rapid recording of the elution of luciferase was accomplished with a new electrochemical bioluminescence assay. Due to this and the early elution of the desired material, it could be chromatographed, recognized and collected in less than two hours. Thereby the light-yielding capacity of the sensitive material was well preserved. For bioluminescence assay solutions composed of pooled oxidoreductase-luciferase fractions, FMN and a long chain aldehyde were prepared and supplemented with NAD+ and either lactate, malate or 3-hydroxybutyrate. The analyses were carried out in a single step performance by adding the enzyme sample to the luciferase solution. Minute amounts of lactate dehydrogenase, malate dehydrogenase and 3-hydroxybutyrate dehydrogenase yielded a linear light response permitting assay in the lower part of the femtomole region. In case a dehydrogenase does not occur as a contaminant of a commercial luciferase preparation, purification with Cibacrone Blue can be omitted as demonstrated for glucose-6-phosphate dehydrogenase.(ABSTRACT TRUNCATED AT 250 WORDS)
Mol Cell Biochem 1983
PMID:Single-step bioluminescence analyses of enzymes, using Cibacrone Blue chromatography for removal of interfering dehydrogenases. 663 14

Three different NAD(P)H-FMN reductases were extracted from Beneckea harveyi MB-20 cells and separated by DEAE-Sephadex A50 column chromatography. Further purification was achieved by affinity chromatography. In determinations of Km values for NADH, NADPH, and FMN, these three reductases exhibited different specificities and kinetic parameters. One reductase utilizes NADH, whereas a second one utilizes NADPH as the preferred substrate. The third, a newly described reductase species, exhibits about the same reaction rates with NADH and NADPH. The reaction mechanisms of the three enzyme forms have been deduced by steady state kinetic analysis. The highly pure (based on gel electrophoresis) NADPH-FMN reductase still exhibited a low (approximately 2%) activity for NADH, which activity was increased upon storage at 4 degrees but suppressed completely by the replacement of the phosphate buffer with sodium citrate buffer. This high specificity of NADPH-FMN reductase for NADPH under these conditions is useful for the assay of NADPH, notably in systems coupled to bacterial luciferase.
Mol Cell Biochem 1982 May 14
PMID:Specificities and properties of three reduced pyridine nucleotide-flavin mononucleotide reductases coupling to bacterial luciferase. 698 Oct 58

Bioluminescence photokinetic assay of NADP+ is described, using the glucose-6-phosphate dehydrogenase reaction for conversion to its reduced form and subsequent measurement of this with luciferase extracts of Vibria fisherii. the analyses were applied to the determination of the activity of minute amounts of glutathione reductase using NADP+ as measurable product and for nucleotide assay in cell samples of 0.5--10 microgram dry weight. The sensitivity was sufficient for determining 0.5 picomoles NADP+. Previously, FMN, NADH, NAD+ and NADH have been analysed with the bacterial luciferase system. Its applicability has not been extended by the assay of NADP+.
Mol Cell Biochem 1980 Aug 29
PMID:Photokinetic microanalysis of NADP+, using bacterial luciferase. 744 56

We report the construction, characterization, and use of luciferase reporters to test the ability of antisense oligonucleotides to inhibit RNA splicing. beta-Globin and adenovirus introns were inserted into a luciferase cDNA, and luciferase expression was analyzed in transiently transfected cells. The adenovirus reporter expressed large amounts of luciferase, but two beta-globin constructs were inactive. RNA analyses determined that the beta-globin pre-mRNAs were not spliced. Mutagenesis of the beta-globin 5' splice site, branchpoint, and 3' splice site sequences to the adenovirus intron sequences promoted maximal splicing and luciferase activity; reciprocal changes in all three elements of the adenovirus intron eliminated luciferase activity. Wild-type and 3' splice site mutated adenovirus reporters were used to determine the ability of phosphorothioate deoxy and 2' methoxy oligonucleotides to inhibit splicing. RNase H activating oligodeoxynucleotides were better inhibitors of wild-type adenovirus expression than were 2' methoxy analogues. However, 2' methoxy oligonucleotides specific for the branchpoint were more effective inhibitors of splicing of adenovirus transcript containing the beta-globin branchpoint and 3' splice site. We suggest that pre-mRNAs with weak splice sites are potential targets for oligonucleotides that inhibit splicing by occupancy rather than cleavage of the transcripts.
Mol Pharmacol 1995 Nov
PMID:Inhibition of splicing of wild-type and mutated luciferase-adenovirus pre-mRNAs by antisense oligonucleotides. 747 22


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