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PubMedDate not supplied

Optical imaging of Renilla luciferase, synthetic Renilla luciferase, and firefly luciferase reporter gene expression in living mice.

We have recently demonstrated that Renilla luciferase (Rluc) is a promising bioluminescence reporter gene that can be used for noninvasive optical imaging of reporter gene expression in living mice, with the aid of a cooled charged couple device (CCD) camera. In the current study, we explore the expression of a novel synthetic Renilla luciferase reporter gene (hRluc) in living mice, which has previously been reported to be a more sensitive reporter than native Rluc in mammalian cells. We explore the strategies of simultaneous imaging of both Renilla luciferase enzyme (RL) and synthetic Renilla luciferase enzyme (hRL):coelenterazine (substrate for RL/hRL) in the same living mouse. We also demonstrate that hRL:coelenterazine can yield a higher signal when compared to Firefly luciferase enzyme (FL): D-Luciferin, both in cell culture studies and when imaged from cells at the surface and from lungs of living mice. These studies demonstrate that hRluc should be a useful primary reporter gene with high sensitivity when used alone or in conjunction with other bioluminescence reporter genes for imaging in living rodents.

Animals↗

PubMed1994

Preparation of P-flavin-bound and P-flavin-free luciferase and P-flavin-bound beta-subunit of luciferase from Photobacterium phosphoreum.

P-flavin-bound luciferase, P-flavin-free luciferase, and P-flavin-bound beta-subunit of luciferase were prepared from Photobacterium phosphoreum using hydrophobic interaction chromatography after conventional purification using DEAE-cellulose chromatography and gel-filtration. The P-flavin-bound luciferase preparation contained about 20% P-flavin-free luciferase not removable by the present procedure. Since the specific activity of the P-flavin-bound luciferase preparation was about 20% of that of the P-flavin-free luciferase, it was concluded that the P-flavin-bound luciferase is an enzyme-product complex and has no more luciferase activity. Unlike the absorption spectrum of FP390 or other flavoproteins, that of P-flavin-bound luciferase preparation has a high absorption peak around 370 nm and resembles the spectrum synthesized by superposing the P-flavin-free luciferase spectrum on the P-flavin-bound beta-subunit spectrum: the P-flavin-bound beta-subunit spectrum is similar to that of FP390, while that of P-flavin-free luciferase has an absorption peak around 370 nm but practically no peak around 450 nm. In addition, P-flavin-free luciferase exhibits a weak but distinct NADH-FMN oxidoreductase activity. These results suggest that a prosthetic group, which absorbs around 370 nm, binds to the luciferase and that this compound is required to yield P-flavin; and they support the hypothesis that the physiological function of bacterial luciferase is to produce P-flavin. Furthermore, the presence of P-flavin-bound beta-subunit of the luciferase in the cell extract supports the hypothesis that physiological function of the lux operon is the biosynthesis of FP390 including its prosthetic group.

Amino Acid Sequence↗

PubMedDate not supplied

A new firefly luciferase with bimodal spectrum: identification of structural determinants of spectral pH-sensitivity in firefly luciferases.

Fireflies emit flashes in the green-yellow region of the spectrum for the purpose of sexual attraction. The bioluminescence color is determined by the luciferases. It is well known that the in vitro bioluminescence color of firefly luciferases can be shifted toward the red by lower pH and higher temperature; for this reason they are classified as pH-sensitive luciferases. However, the mechanism and structural origin of pH sensitivity in fireflies remains unknown. Here we report the cloning of a new luciferase from the Brazilian twilight active firefly Macrolampis sp2, which displays an unusual bimodal spectrum. The recombinant luciferase displays a sensitive spectrum with the peak at 569 nm and a shoulder in the red region. Comparison of the bioluminescence spectra of Macrolampis, Photinus and Cratomorphus firefly luciferases shows that the distinct colors are determined by the ratio between green and red emitters under luciferase influence. Comparison of Macrolampis luciferase with the highly similar North American Photinus pyralis luciferase (91%) showed few substitutions potentially involved with the higher spectral sensitivity in Macrolampis luciferase. Site-directed mutagenesis showed that the natural substitution E354N determines the appearance of the shoulder in the red region of Macrolampis luciferase bioluminescence spectrum, helping to identify important interactions and residues involved in the pH-sensing mechanism in firefly luciferases.

Amino Acid Sequence↗

PubMed2000

Bioluminescence color determinants of Phrixothrix railroad-worm luciferases: chimeric luciferases, site-directed mutagenesis of Arg 215 and guanidine effect.

Chimeric proteins were produced using the green light-emitting luciferase of Phrixothrix vivianii (PxGr: lambda max = 548 nm) and the red light-emitting luciferase of Phrixothrix hirtus (PxRe: lambda max = 623 nm). Constructs containing residues 1-344 of the red light-emitting luciferase with residues 345-545 of the green light emitting one emitted red light (PxReGr; lambda max = 613 nm), while the reverse emitted green light (PxGrRe; lambda max = 552 nm). From these results we conclude that the region 1-344 determines the color of bioluminescence (BL) in railroad-worm luciferases, and that residues above 344 are not involved. The substitution R215S in the green light-emitting luciferase (PxGr) resulted in a approximately 40 nm redshift on the BL spectrum (lambda max = 585 nm) and an associated decrease of activity, whereas the same mutation in PxRe luciferase had little effect. Guanidine was shown to cause blueshifts in the BL spectra and stimulate the activity of the red-emitting luciferases (from lambda max = 623 to lambda max = 600 nm) and in PxGr R215S (from lambda max = 585 to lambda max = 560 nm) mutant luciferase, but not in the green-emitting luciferases, suggesting that guanidine can simulate positively charged residues involved in BL color determination.

Animals↗

PubMed2002

[Inhibition of Luciferase expression in mammalian cells by AAV vector plasmid mediated Luciferase shRNA].

OBJECTIVE: Constructing a plasmid containing the shRNA of luciferase to suppress the expression of luciferase in BHK-21 cell. METHODS: A 334 bp human U6 snRNA promoter was amplified from human genomic DNA by PCR and ligated to a 21 bp reverse repeated motif of luciferase target sequence with 9 bp spacer and AAV plasmid pSNAV. The recombinant pSNAV/U6/Luc plasmid cotransfected with pMAMneoLuc or transfected luciferase cell line to detect the effect of luciferase expression separately. RESULTS: pSNAV/U6/luc suppresses the luciferase expression from pMAMneoLuc by 50% and luciferase cell line by 70%. CONCLUSIONS: The results showed that the short hairpin RNA of luciferase can efficiently suppress its expression in BHK-21.

Cell Line↗

PubMed1994

Common structural features of the luxF protein and the subunits of bacterial luciferase: evidence for a (beta alpha)8 fold in luciferase.

The amino acid sequence identity and potential structural similarity between the subunits of bacterial luciferase and the recently determined structure of the luxF molecule are examined. The unique beta/alpha barrel fold found in luxF appears to be conserved in part in the luciferase subunits. From secondary structural predictions of both luciferase subunits, and from structural comparisons between the protein product of the luxF gene, NFP, and glycolate oxidase, we propose that it is feasible for both luciferase subunits to adopt a (beta alpha)8 barrel fold with at least 2 excursions from the (beta alpha)8 topology. Amino acids conserved between NFP and the luciferase subunits cluster together in 3 distinct "pockets" of NFP, which are located at hydrophobic interfaces between the beta-strands and alpha-helices. Several tight turns joining the C-termini of beta-strands and the N-termini of alpha-helices are found as key components of these conserved regions. Helix start and end points are easily demarcated in the luciferase subunit protein sequences; the N-cap residues are the most strongly conserved structural features. A partial model of the luciferase beta subunit from Photobacterium leiognathi has been built based on our crystallographically determined structure of luxF at 1.6 A resolution.

Alcohol Oxidoreductases↗

PubMed1997

The use of Renilla luciferase, Oplophorus luciferase, and apoaequorin as bioluminescent reporter protein in the presence of coelenterazine analogues as substrate.

To investigate the use of various luciferases as reporter protein, the substrate specificity of recombinant Renilla luciferase, Oplophorus luciferase and recombinant apoaequorin was examined using 23 kinds of coelenterazine analogues as the substrate. The intensity of luminescence was generally highest with Oplophorus luciferase and lowest with apoaequorin, but varied widely by the substrate used. A very high level of light intensity was obtained when the luminescence reactions of e-coelenterazine and v-coelenterazine were catalyzed by Renilla luciferase, strongly suggesting the usefulness of recombinant Renilla luciferase as a highly sensitive reporter protein. Oplophorus luciferase can be an equally sensitive reporter protein when its gene is obtained.

Aequorin↗

PubMed2003

The modified firefly luciferase reporter gene (luc+) but not Renilla luciferase is induced by all-trans retinoic acid in MCF-7 breast cancer cells.

Luciferase genes are widely used as reporters to analyze promoter and regulatory elements. We found that a luciferase reporter gene vector with a modified firefly luciferase gene (luc+), but not Renilla luciferase (Rluc), was induced by all-trans retinoic acid (tRA) in the MCF-7 breast cancer cell line. tRA (5 x 10(-6) M) increased luciferase activity of the pGL3 promoter vector (containing luc+) up to approximately 3.8-fold in MCF-7 cells, but not in LNCaP prostate cancer cells or JEG-3 choriocarcinoma cells. Chimeric plasmids were constructed and showed that tRA-induction required the luc+ gene, but not any specific promoter or vector sequence. Time course and dose-response studies of tRA-induction indicated that longer treatment (> 24h) and higher tRA dose (> 10(-6) M) were required for luc+ induction compared with those for a positive retinoic acid response element (maximum induction at 6 h and 10(-8) M tRA). Studies with the translation inhibitor, cycloheximide, indicated the half-life of the luc+ protein was increased from 9.7 +/- 1.5 to 22.1 +/- 3.1 h with tRA treatment. Other retinoids, TTNPB, a retinoic acid receptor beta/gamma-specific ligand, and a retinoid X receptor ligand, did not significantly increase luc+ expression. Caution is needed in analysis of retinoid responsive gene regulation with the luciferase reporter system in MCF-7 cells, especially at high retinoid concentrations.

Animals↗

PubMed2004

Cloning and characterization of the cDNA for the Brazilian Cratomorphus distinctus larval firefly luciferase: similarities with European Lampyris noctiluca and Asiatic Pyrocoelia luciferases.

Studies on firefly (Lampyridae) luciferases have focused on nearctic species of Photinus and Photuris and Euroasiatic species of Lampyris, Luciola, Hotaria, and Pyrocoelia. Despite accounting for the greatest diversity of fireflies in the world, no molecular studies have been carried out on the highly diverse genera from the neotropical region. Here we report the luciferase cDNA cloning for the larva of the Brazilian firefly Cratomorphus distinctus. The cDNA has 1978 bp and codes for a 547-residue-long polypeptide. Noteworthy, sequence comparison as well as functional properties show the highest degree of similarity with Lampyris noctiluca (93%) and Pyrocoelia spp. (91%) luciferases, suggesting a close phylogenetic relationship despite the geographical distance separating these species. The bioluminescence emission spectrum peaks at 550 nm and, as expected, is sensitive to pH, shifting to 605 nm at pH 6. The kinetic properties of the recombinant luciferase were similar to those of other firefly luciferases.

Animals↗

PubMed2000

Haemoglobin interferes with the ex vivo luciferase luminescence assay: consequence for detection of luciferase reporter gene expression in vivo.

The luciferase reporter gene is a useful tool for determining the efficacy of transfection of plasmid DNA and adenovirus-mediated gene transfer in vivo. However, we report here that the haemoglobin present in tissue samples can mask the detection of the luciferase activity and lead to underestimation of the luciferase levels. We evaluated the degree of interference in different organ samples of mice and investigated the possibilities for removal of haemoglobin from tissue samples by: (1) perfusion of the whole animal; (2) different hypotonic treatments lysing preferentially red blood cells; and (3) chromatographic separation. Removal of haemoglobin resulted in significantly improved detection of luciferase activity from tissue samples. The results indicate that the luciferase activity determined in tissue samples may not reflect the actual level of reporter gene expression, if contaminating blood is not taken into consideration.

Adenoviridae↗

PubMed1994

The turnover of bacterial luciferase is limited by a slow decomposition of the ternary enzyme-product complex of luciferase, FMN, and fatty acid.

Bacterial luciferase catalyzes the conversion of reduced flavin mononucleotide, O2, and fatty aldehyde to FMN, H2O, and fatty acid with light being emitted at a very low rate characterized by the decay of luminescence in single turnover flash assays. The present studies have now revealed that the decomposition of the ternary complex of luciferase with FMN and myristic acid occurs at a rate 10-15 times slower than the decay of luminescence and that functional luciferase is only regenerated after the release of flavin. In contrast, the rate of FMN dissociation and recovery of activity observed with a binary luciferase-FMN complex was more rapid indicating that release of fatty acid played the critical role in determining the rate of FMN dissociation from the ternary complex. Decomposition of the ternary complex was shown to occur by an ordered process involving the slow release of fatty acid followed by the more rapid release of flavin. The present results suggest that the rate-limiting step for the turnover of luciferase occurs subsequent to emission of light and would be the controlling step under conditions (e.g. in cells) with continuous light emission.

Enzyme Stability↗

PubMed2000

Secretional luciferase of the luminous shrimp Oplophorus gracilirostris: cDNA cloning of a novel imidazopyrazinone luciferase(1).

The deep-sea shrimp Oplophorus gracilirostris secretes a luciferase that catalyzes the oxidation of coelenterazine to emit blue light. The luciferase (M(r) approx. 106000) was found to be a complex composed of 35 kDa and 19 kDa proteins, and the cDNAs encoding these two proteins were cloned. The expression of the cDNAs in bacterial and mammalian cells indicated that the 19 kDa protein, not the 35 kDa protein, is capable of catalyzing the luminescent oxidation of coelenterazine. The primary sequence of the 35 kDa protein revealed a typical leucine-rich repeat sequence, whereas the catalytic 19 kDa protein shared no homology with any known luciferases including various imidazopyrazinone luciferases.

Amino Acid Motifs↗

PubMed2000

Parathyroid hormone and prostaglandin E2 preferentially increase luciferase levels in bone of mice harboring a luciferase transgene controlled by elements of the pro-alpha1(I) collagen promoter.

Type I collagen is the major extracellular protein in bone, tendons, ligaments, and skin. DNA elements of the mouse pro-alpha1 (I) collagen promoter were shown to drive the bone-selective expression of a luciferase transgene. We examined whether this expression can be used to evaluate the effect of anabolic agents on bone formation in vivo. Treatment of either intact males, intact females, or ovariectomized (ovx) mice with 80 microg/kg/day of human parathyroid hormone (hPTH), for 5 to 11 days increased luciferase levels in tibiae by two- to threefold compared with vehicle-treated mice. The increases were tissue specific, as no changes in skin luciferase expression were observed. Treatment with prostaglandin E2, a potent bone anabolic agent, for 11 days also increased expression of the transgene in bone, but not in skin. Treatment with dihydrotestosterone (DHT) for 11 days increased luciferase activity in skin, but not in bone. Histomorphometric analysis revealed that 28-day treatment with PTH increased bone formation; 60-day treatment of OVX mice with DHT did not. These findings show a correlation between bone formation and the expression of a transgene driven by DNA elements of the mouse pro-alpha1 (I) collagen promoter, suggesting that this expression can be used as an indicator and provide a faster readout for the ability of agents to stimulate bone formation in this mouse strain.

Animals↗

PubMed1985

Cloning of firefly luciferase cDNA and the expression of active luciferase in Escherichia coli.

A cDNA library was constructed from firefly (Photinus pyralis) lantern poly(A)+ RNA, using the Escherichia coli expression vector lambda gt11. The library was screened with anti-P. pyralis luciferase (Photinus luciferin:oxygen 4-oxidoreductase, EC 1.13.12.7) antibody, and several cDNA clones expressing luciferase antigens were isolated. One clone, lambda Luc1, contained 1.5 kilobase pairs of cDNA that hybridized to a 1.9- to 2.0-kilobase band on a nitrocellulose blot of electrophoretically fractionated lantern RNA. Hybridization of the cloned cDNA to lantern poly(A)+ RNA selected an RNA that directed the in vitro synthesis of a single polypeptide. This polypeptide comigrated with luciferase on NaDodSO4/PAGE and produced bioluminescence upon the addition of luciferin and ATP. A 1.8-kilobase-pair cDNA was isolated by probing the firefly cDNA library with the cDNA from lambda Luc1. This cDNA contained sufficient coding information to direct the synthesis of active firefly luciferase in E. coli.

Animals↗

PubMed1976

Studies on luciferase from Photobacterium phosphoreum. VIII. FMN-H2O2 initiated bioluminescence and the thermodynamics of the elementary steps of the luciferase reaction.

Bacterial luciferase from Photobacterium phosohoreum was found to produce bioluminescence on reaction with FMN and H2O2 in the presence of aldehyde. This luminescence is presumably produced by the same X1 intermediate as that found in FMNH2-O2 initiated luminescence. From the ratio of the light intensities of the FMN-H2O2 initiated reactions, we calculated the association constant of the reaction, luciferase+FMN+H2O2in equilibriumX1, and estimated its temperature dependence. From these results we calculated the thermodynamic parameters of the reaction, luciferase+FMNH2+O2in equilibriumX1. We found that the free energy level of X1 is only 3.2 kcal below that of fr-e FMN and H2O2. We also estimated the thermodynamic parameters of other steps of the luminescent reaction. The values obtained showed that the formation of X1 from luciferase, FMNH2 and O2 involves a positive entropy change, but the intermediate is in a state stabilized against decomposition. Results also suggest a considerable degree of electronic rearrangement on formation of the excited-state molecule from the X1-aldehyde complex.

Aldehydes↗

PubMed1978

Studies on luciferase from Photobacterium phosphoreum. XI. Interaction of 8-substituted FMNH2 with luciferase.

The interaction of bacterial luciferase from Photobacterium phosphoreum with reduced flavin was investigated using various 8-substituted FMNH2 analogs. Flavins tested were FMNH2 and FMNH2 substituted at the 8 position with HO-, CH3O-, C2H5O-, Cl-, Br-, I-, H2N-, (CH3)HN-, and (ch3)2n. 8-ch30-, c2h5o-, cl-, and Br-FMNH2 showed luminescent activity in the luciferase reaction with emission peaks at various wavelengths. 8-HO- and I-FMNH2 were competitive inhibitors toward FMNH2 in the luminescent reaction. 8-Amino analogs of FMNH2 showed no luminescent or inhibitor activity. The dissociation constant of the luciferase-FMNH2 analog complex was determined kinetically as a substrate or inhibitor constant. A contribution of the imino group at position 5 in the isoalloxazine ring to the FMNH2 binding to luciferase was suggested by a Hammett plot of the dissociation constants.

Flavin Mononucleotide↗

PubMed1994

[Kinetics of luciferase gene expression from fireflies Photinus pyralis and Luciola mingrelica in Escherichia coli cells. I. Kinetics of the transformation of recombinant luciferase into enzymatically-active conformers].

The Photinus pyralis and Luciola mingrelica luciferases genes expression has been studied on the pME61 or pJG lambda plasmids with a thermoinducible lambda Pr promoter in the E. coli strain CA using three independent methods: SDS gel electrophoresis to quantify the synthesized luciferase protein, EIA to quantify the enzyme native conformer and activity measurements. The cultures were incubated by the temperature schemes 28 degrees-42 degrees-21 degrees C and 28 degrees-21 degrees C. The maximal amount of the Ph. pyralis protein reached 4.5%, while that of L. mingrelica luciferase was 4.1% of the total amount of the cellular proteins after 10-hr incubation. The amount of the native conformer and the luciferase activity began to grow after a long induction period, reaching the maximal level by hour 50-60 after the thermoinduction. The increase in the enzyme activity correlated well with the increase in the ATP content in the cell. The observed "low-temperature induction" of the enzyme activity is interpreted as a protein transition to an active conformation. This transformation is considerably behind the protein biosynthesis process. Intracellular metabolic reactions have been shown to play an active part in these conformational changes.

Animals↗

PubMed2002

Preparation of luciferase-bacterial magnetic particle complex by artificial integration of MagA-luciferase fusion protein into the bacterial magnetic particle membrane.

MagA is an iron-translocating protein found in the membranes of magnetic bacterium. Luciferase-bacterial magnetic particle (BMP) complexes were prepared by artificially inserting MagA-luciferase fusion proteins into the membranes of BMPs from Magnetospirillum magneticum strain AMB-1. Fusion proteins were from recombinant Escherichia coli membranes. MagA-Luc fusion proteins were integrated by sonication in vitro. Successful integration of fusion proteins was confirmed by luciferase luminescence on BMPs. Maximum luminescence was obtained after sonication for 3 min with a solution containing 300 mM NaCl, and is 18 times higher compared with recombinant Luc-BMPs generated using previously reported gene fusion techniques.

Artificial Gene Fusion↗