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At least 307 records · Page 17Linked to original sources
PubMed1996

The sea pansy Renilla reniformis luciferase serves as a sensitive bioluminescent reporter for differential gene expression in Candida albicans.

The infectious yeast Candida albicans progresses through two developmental programs which involve differential gene expression, the bud-hypha transition and high-frequency phenotypic switching. To understand how differentially expressed genes are regulated in this organism, the promoters of phase-specific genes must be functionally characterized, and a bioluminescent reporter system would facilitate such characterization. However, C. albicans has adopted a nontraditional codon strategy that involves a tRNA with a CAG anticodon to decode the codon CUG as serine rather than leucine. Since the luciferase gene of the sea pansy Renilla reinformis contains no CUGs, we have used it to develop a highly sensitive bioluminescent reporter system for C. albicans. When fused to the galactose-inducible promoter of GAL1, luciferase activity is inducible; when fused to the constitutive EF1 alpha 2 promoter, luciferase activity is constitutive; and when fused to the promoter of the white-phase-specific gene WH11 or the opaque-phase-specific gene OP4, luciferase activity is phase specific. The Renilla luciferase system can, therefore, be used as a bioluminescent reporter to analyze the strength and developmental regulation of C. albicans promoters.

Animals↗
PubMed1983

Firefly luciferase ATP assay as a screening method for bacteriuria.

A rapid (15 min) test for bacteriuria based on firefly luciferase analysis of bacterial ATP has been evaluated in 2,018 clinical urine specimens. The test procedure involves removal of nonbacterial ATP by treatment of urine with Triton X-100 and apyrase, extraction of bacterial ATP by boiling, and bioluminescent analysis of bacterial ATP by firefly luciferase, using a luminometer. For comparison, the widely used nitrite test was included in the study as an example of an alternative rapid chemical test. The test was set up to distinguish between specimens yielding greater than 10(5) CFU/ml and specimens yielding less than 10(5) CFU/ml. A level of 13.5 nM ATP was chosen to define the limit between negative and positive results. At this discriminatory level, 92% of specimens yielding greater than 10(5) CFU/ml and 88% of specimens yielding less than 10(5) CFU/ml were correctly classified with the luciferase method, whereas corresponding figures for the nitrite test were 55 and 99%, respectively. Of the 12% false luciferase positives, 20% were shown to contain greater than 10(5) CFU/ml on prolonged incubation, thus reducing the false-positive rate to 10%. Of the 8% false luciferase negatives, 65% had low levels of CFU in the range of 10(5) to 10(6).

Adenosine Triphosphate↗
PubMed2002

Replication of subgenomic hepatitis A virus RNAs expressing firefly luciferase is enhanced by mutations associated with adaptation of virus to growth in cultured cells.

Replication of hepatitis A virus (HAV) in cultured cells is inefficient and difficult to study due to its protracted and generally noncytopathic cycle. To gain a better understanding of the mechanisms involved, we constructed a subgenomic HAV replicon by replacing most of the P1 capsid-coding sequence from an infectious cDNA copy of the cell culture-adapted HM175/18f virus genome with sequence encoding firefly luciferase. Replication of this RNA in transfected Huh-7 cells (derived from a human hepatocellular carcinoma) led to increased expression of luciferase relative to that in cells transfected with similar RNA transcripts containing a lethal premature termination mutation in 3D(pol) (RNA polymerase). However, replication could not be confirmed in either FrhK4 cells or BSC-1 cells, cells that are typically used for propagation of HAV. Replication was substantially slower than that observed with replicons derived from other picornaviruses, as the basal luciferase activity produced by translation of input RNA did not begin to increase until 24 to 48 h after transfection. Replication of the RNA was reversibly inhibited by guanidine. The inclusion of VP4 sequence downstream of the viral internal ribosomal entry site had no effect on the basal level of luciferase or subsequent increases in luciferase related to its amplification. Thus, in this system this sequence does not contribute to viral translation or replication, as suggested previously. Amplification of the replicon RNA was profoundly enhanced by the inclusion of P2 (but not 5' noncoding sequence or P3) segment mutations associated with adaptation of wild-type virus to growth in cell culture. These results provide a simple reporter system for monitoring the translation and replication of HAV RNA and show that critical mutations that enhance the growth of virus in cultured cells do so by promoting replication of viral RNA in the absence of encapsidation, packaging, and cellular export of the viral genome.

5' Untranslated Regions↗
PubMed1993

Heart-specific targeting of firefly luciferase by the myosin light chain-2 promoter and developmental regulation in transgenic mice.

Based on hybridization studies indicating constitutive expression levels of the endogenous myosin light chain-2 (MLC-2) gene in embryonic, fetal, and adult myocardium, a model system for selective targeting of genes to the heart of transgenic mice has been developed. A 2.1-kb DNA fragment of the 5' flanking region of the rat cardiac MLC-2 gene was fused to the firefly luciferase reporter gene and introduced into fertilized mouse oocytes. In four independent transgenic mouse lines, the expression of the MLC-2-luciferase fusion gene was found exclusively in heart muscle. In contrast to the endogenous MLC-2 gene, no luciferase activity was detectable in slow-twitch skeletal muscle or any other tissue of transgenic mice. This result suggests that the 2.1-kb DNA fragment of the 5' flanking region of the cardiac MLC-2 gene contains the regulatory elements required for selective gene expression in cardiac myocytes in vivo. In contrast to the endogenous steady-state MLC-2 expression during development, transgenic luciferase activity was 10-fold higher during embryogenesis, when formation of the ventricular loop and septum takes place. The enhanced luciferase activity in early heart development may suggest a growth-dependent control mechanism, involving either transcriptional or posttranscriptional regulation. In conclusion, this model system with the 2.1-kb ventricle-specific MLC-2 promoter sequence should facilitate the overexpression of gene products in the developing and mature heart muscle and further elucidate molecular mechanisms of myocardial diseases such as cardiomyopathies.

Animals↗
PubMed1997

Expression of a murine gonadotropin-releasing hormone receptor-luciferase fusion gene in transgenic mice is diminished by immunoneutralization of gonadotropin-releasing hormone.

A line of transgenic mice harboring a fusion gene consisting of 1900 bp of proximal 5'-flanking region from the murine GnRH receptor gene linked to the complementary DNA encoding luciferase was established to determine whether this promoter can direct tissue-specific expression in vivo and serve as a model for identifying the molecular mechanisms underlying hormonal regulation of this gene. Of 10 tissues screened, luciferase was detected predominantly in pituitary gland, but also in brain and testis. To assess hormonal regulation, luciferase activity was measured in intact males and ovariectomized females treated with an anti-GnRH serum alone, and in combination with testosterone or 17beta-estradiol. No effect of steroid treatment on transgene expression was detected. However, immunoneutralization of GnRH resulted in decreased serum LH concentrations and suppressed pituitary expression of luciferase. Furthermore, the effects of GnRH antiserum could be prevented by the administration of a noncross-reactive GnRH agonist. Thus, 1900 bp of 5'-flanking DNA from the murine GnRH receptor gene are sufficient to target luciferase expression in transgenic mice to established sites of GnRH receptor gene expression. Furthermore, we suggest that GnRH regulation of GnRH receptor gene expression is mediated by regulatory elements residing within 1900 bp of the 5'-flanking region.

Animals↗
PubMed2005

Enzymatic and genetic characterization of firefly luciferase and Drosophila CG6178 as a fatty acyl-CoA synthetase.

Recently we found that firefly luciferase is a bifunctional enzyme, catalyzing not only the luminescence reaction but also long-chain fatty acyl-CoA synthesis. Further, the gene product of CG6178 (CG6178), an ortholog of firefly luciferase in Drosophila melanogaster, was found to be a long-chain fatty acyl-CoA synthetase and dose not function as a luciferase. We investigated the substrate specificities of firefly luciferase and CG6178 as an acyl-CoA synthetase utilizing a series of carboxylic acids. The results indicate that these enzymes synthesize acyl-CoA efficiently from various saturated medium-chain fatty acids. Lauric acid is the most suitable substrate for these enzymes, and the product of lauroyl CoA was identified with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). Phylogenetic analysis indicated that firefly luciferase and CG6178 genes belong to the group of plant 4-coumarate:CoA ligases, and not to the group of medium- and long-chain fatty acyl-CoA synthetases in mammals. These results suggest that insects have a novel type of fatty acyl-CoA synthetase.

Acyl Coenzyme A↗
PubMed2006

Luciferases as reporter genes.

Luciferase is the ideal reporter gene to provide temporal and spatial information on promoter activity in Arabidopsis and other eukaryotes; the noninvasive detection of luminescence and short half-life of luciferase activity allow repeated measurements of individual seedlings over several days to assay dynamic changes in gene expression. Transgenic or transiently transformed plants with a luciferase gene under the control of a promoter of interest are required. Detection of the low level of luminescence produced by the luciferase gene in Arabidopsis requires the use of low light detecting charge-coupled device (CCD) cameras or scintillation counters. This chapter contains protocols on assaying and imaging luciferase in vivo and the automation for high-resolution timecourses.

Arabidopsis↗
PubMed2000

Activities of the bimodal fluorescent protein produced by Photobacterium phosphoreum strain bmFP in the luciferase reaction in vitro.

The activity of the bimodal fluorescent protein (bmFP) (lambda max, 488 and 517 nm) in the in vitro luciferase reaction has been studied. The bmFP that is produced by Photobacterium phosphoreum strain bmFP is a dimer of two homologous subunits binding four riboflavin 5'-phosphate (FMN)-myristate chromophores. The addition of bmFP to the luciferase reaction in the presence of the lumazine protein prevented the lumazine protein-induced blue shift in the emission band. The bmFP reduced electrochemically serves as a substrate in the luciferase reaction in the absence of added FMN, resulting in light emission with a single maximum at about 487 nm. The bmFP was also active in lieu of FMN in the NADH/FMN oxidoreductase (flavin reductase)-luciferase coupled bioluminescence reaction in the absence of added FMN. In the coupled reaction, bioluminescence with the isolated bmFP chromophore was weaker than that with the holo-bmFP. After bmFP was used in luciferase reactions initiated either chemically or electrochemically, it was still capable of emitting bimodal fluorescence.

Bacterial Proteins↗
PubMed2000

Luciferase and urate may act as antioxidant defenses in larval Pyrearinus termitilluminans (Elateridae: Coleoptera) during natural development and upon 20-hydroxyecdysone treatment.

Insects show unique adaptations to cope with oxidative challenges during larval development, metamorphosis and adulthood. Our previous findings suggested that bioluminescence may act as an auxiliary oxygen-detoxifying mechanism in larvae of Pyrearinus termitilluminans (Elateridae: Coleoptera). We now study the antioxidant status in larval P. termitilluminans, evaluated in terms of levels of chemical and enzymatic antioxidant defenses, as compared to luciferase activity in the prothorax (intensely bright) and abdomen (dim) of the larvae, during natural- and 20-hydroxyecdysone (20-HE)-induced development. In the prothorax, relative total SOD activities in small (< 1 cm), medium (1-2 cm) and large (> 2 cm) larvae were 1.00:0.53:0.32. Catalase activity also decreased with development (1.00:0.69:0.55). In contrast, prothorax luciferase activities and urate content increased with ratios of 1.0:2.2:2.5 and 1:15:97, respectively. No increases were found in the level of prothorax lipid and protein oxidation. In the abdomen, luciferase activity decreased markedly with development (1.00:0.33:0.17), as did other antioxidant enzymes, including dehydroascorbate reductase (1.00:0.59:0.17) and levels of lipid peroxidation products and protein carbonyls. Similar variations were observed in antioxidant enzyme activities when the larvae were treated with 20-HE, except for prothorax catalase. As observed in natural larval growth, luciferase activity was augmented (two-fold in prothorax) upon steroid treatment, and the levels of thiobarbituric acid-reactive substances were magnified in both segments. The increase of luciferase activity and a higher urate content in the prothorax during larval development may reflect metabolic adaptations to keep levels of oxyradicals low in order to compensate for decreased antioxidant enzyme activities.

Animals↗
PubMedDate not supplied

Effects of iodide on the fluorescence and activity of the hydroperoxyflavin intermediate of Vibrio harveyi luciferase.

The 4a-hydroperoxy-4a,5-dihydroFMN intermediate (II or HFOOH) of Vibrio harveyi luciferase is known to transform from a low quantum yield IIx to a high quantum yield (lambdamax 485 nm, uncorrected) IIy fluorescent species on exposure to excitation light. Similar results were observed with II prepared from the alphaH44A luciferase mutant, which is very weak in bioluminescence activity. Because of the rapid decay of the alphaH44A II, its true fluorescence was obscured by the more intense 520 nm fluorescence (uncorrected) from its decay product oxidized flavin mononucleotide (FMN). Potassium iodide (KI) at 0.2 M was effective in quenching the FMN fluorescence, leaving the 485 nm fluorescence of II from both the wild-type (WT) and alphaH44A luciferase readily detectable. For both II species, the luciferase-bound peroxyflavin was well shielded from KI quenching. KI also enhanced the decay rates of both the WT and alphaH44A II. For alphaH44A, the transformation of IIx to IIy can be induced by KI in the dark, and it is proposed to be a consequence of a luciferase conformational change. The WT II formed a bioluminescence-inactive complex with KI, resulting in two distinct decay time courses based on absorption changes and decreases of bioluminescence activity of II.

Absorption↗
PubMed2005

A luciferase transgenic mouse model: visualization of prostate development and its androgen responsiveness in live animals.

Numerous mouse models of prostate carcinogenesis have been developed, but hitherto there has been no model in which the prostate gland could be imaged in live animals. The transgenic model generated here targeted mouse prostate gland using a firefly luciferase enzyme under the control of a small but highly active and specific supra prostate-specific antigen (sPSA) promoter. We evaluated postnatal prostate development, involution and androgen-induced restoration of prostate growth in adult transgenic mice using bioluminescence imaging. Results of our study showed that: (i) the prostate gland of male offspring did not yield a significant bioluminescence signal until after sexual maturity. Luciferase was detected in the luminal epithelial cells of the ventral and dorsolateral lobes of the prostate gland and caput epididymis, with little or no activity in 18 other organs evaluated. (ii) While a constant high level of bioluminescence was detected in the mouse prostate from 5 to 35 weeks of age, a slight drop in bioluminescence was detected at 36 to 54 weeks. (iii) Upon castration, the luciferase activity signal associated with mouse prostate detected by a cooled charge-coupled device camera was dramatically reduced. This signal could be rapidly restored to pre-castration levels after androgen administration. Androgen-induced luciferase activity subsided to nearly basal levels 5 days following the last injection. These data demonstrate that a bioluminescent mouse model with luciferase activity restricted to the prostate gland under the control of a (sPSA) promoter can be used on a real-time basis in live animals to investigate the development and responsiveness of the prostate gland to exogenously administered androgen. This model can be extended to detect the responsiveness of the prostate gland to therapy and used as a founder strain to visualize tumors in hosts with different genetic backgrounds.

Androgens↗
PubMed2006

Estrogenic endpoints in fish early life-stage tests: luciferase and vitellogenin induction in estrogen-responsive transgenic zebrafish.

This study incorporated specific endpoints for estrogenic activity in the early life-stage (ELS) test, as described in Guideline 210 of the Organization for Economic Cooperation and Development and traditionally used for toxicity screening of chemicals. A transgenic zebrafish model expressing an estrogen receptor-mediated luciferase reporter gene was exposed to ethinylestradiol (EE2), and luciferase activity as well as vitellogenin (VTG) was measured. Concentrations of EE2 were tested at 1, 3, or 10 ng/L for 30 d from fertilization or during only the last 4 d with dimethylsulfoxide (DMSO) as presolvent (0.01%). Exposure to EE2 induced no toxic effects. Mean body weights were significantly higher in groups exposed for 30 d in the presence of DMSO, but condition factors were not affected. Significant luciferase and VTG induction occurred following 30-d exposure (3 and 10 ng EE2/L), while only VTG levels were affected in the 4-d exposure (10 ng EE2/L). This study demonstrated the usefulness of incorporating estrogenic endpoints in the OECD ELS test, fitting the requirements for screening estrogenic activity of chemicals. Quantitative measurement of both VTG and luciferase activity proved to be rapid and sensitive. Additional value of using transgenic zebrafish lies in combining VTG measurement with the more mechanistic approach of luciferase induction in one experiment.

Animals↗
PubMed2002

Detecting protein-protein interactions using Renilla luciferase fusion proteins.

We have developed a novel system designated the luciferase assay for protein detection (LAPD) to study protein-protein interactions. This method involves two protein fusions, a soluble reporter fusion and a fusion for immobilizing the target protein. The soluble reporter is an N-terminal Renilla luciferase fusion protein that exhibits high Renilla luciferase activity. Crude cleared lysates from transfected Cos1 cells that express the Renilla luciferase fusion protein can be used in binding assays with immobilized target proteins. Following incubation and washing, target-bound Renilla luciferase fusion proteins produce light from the coelenterazine substrate, indicating an interaction between the two proteins of interest. As proof of the principle, we reproduced known, transient protein-protein interactions between the Cdc42 GTPase and its effector proteins. GTPase Renilla fusion proteins produced in Cos1 cells were tested with immobilized recombinant GST-N-WASP and CEP5 effector proteins. Using this assay, we could detect specific interactions of Cdc42 with these effector proteins in approximately 50 min. The specificity of these interactions was demonstrated by showing that they were GTPase-specific and GTP-dependent and not seen with other unrelated target proteins. These results suggest that the LAPD method, which is both rapid and sensitive, may have research and practical applications.

Amino Acid Sequence↗
PubMed1999

Model of the active site of firefly luciferase.

A model for the spatial structure of firefly luciferase--ATP--luciferin complex is suggested using the coordinates of unliganded luciferase and the enzyme--substrate complex of the adenylating subunit of gramicidin S synthetase known from the literature. Conformational changes in luciferase can occur during substrate binding resulting in a relative orientation of two luciferase domains similar to that in case of the AMP--phenylalanine--synthetase complex. The model is consistent with data on the physicochemical properties of firefly luciferase and its complexes with the substrates.

Adenosine Triphosphate↗
PubMed2002

Improvement of chemically-activated luciferase gene expression bioassay for detection of dioxin-like chemicals.

OBJECTIVE: To improve the chemically-activated luciferase expression (CALUX) bioassay for detection of dioxin-like chemicals (DLCs) based on the toxicity mechanisms of DLCs. METHODS: A recombinant vector was constructed and used to transfect human hepatoma (HepG2). The expression of this vector was 10-100 folds higher than that of pGL2 used in previous experiments. The transfected cells showed aromatic hydrocarbon receptor (AhR)-meditated luciferase gene expression. The reliability of luciferase induction in this cell line as a reporter of AhR-mediated toxicity was evaluated, the optimal detection time was examined and a comparison was made by using the commonly used ethoxyresoufin-O-deethylase (EROD) activity induction assay. RESULTS: The results suggested that the luciferase activity in recombinant cells was peaked at about 4 h and then decreased to a stable activity by 14 h after TCDD treatment. The detection limit of this cell line was 0.11 pmol/L, or 10-fold lower than in previous studies, with a linear range from 1 to 100 pmol/L, related coefficient of 0.997, and the coefficient of variability (CV) of 15-30%. CONCLUSION: The luciferase induction is 30-fold more sensitive than EROD induction, the detection time is 68 h shorter and the detection procedure is also simpler.

Biological Assay↗
PubMedDate not supplied

Firefly luciferase gene transmission and expression in transgenic medaka (Oryzias latipes).

Plasmids containing the luciferase gene from the firefly (Photinus pyralis) fused to the Chinese hamster metallothioneine I promoter (ChMTI) were microinjected into the pronuclei of medaka (Oryzias latipes) eggs, which were then artificially inseminated. Evidence of integration into the genome was gained from observation of germ-line transmission in a mendelian fashion from the F1 to the F2 generation. However, gene expression (light emission) could not be demonstrated in the established transgenic line. In a separate program, transient expression of gene constructs containing the luciferase gene fused to various promoters was compared in medaka embryos. Plasmids were microinjected into pronuclei, and homogenates from 3-day-old embryos were measured for light emission using a luminometer. Among the various promoters tested (SV40, RSV-LTR, ChMTI, HSP70, and mouse albumin), the highest levels of luciferase gene expression were observed in gene constructs containing ChMTI and HSP70 gene promoters. Expression in these two constructs was significantly increased following administration of ZnSO4 or heat treatment, respectively. Plasmids were also introduced into goldfish fibroblast-like cells in vitro, in which enzymatically active luciferase was transiently expressed. Assaying for expression of luciferase provided a rapid and sensitive method for monitoring promoter activity. The potential usefulness of this fish species for cancer research is discussed based on accumulated information from carcinogenesis studies.

Animals↗
PubMed1992

The carboxyl-terminal tripeptide serine-lysine-leucine of firefly luciferase is necessary but not sufficient for peroxisomal import in yeast.

Firefly luciferase is imported into peroxisomes in insects, mammals, plants, and yeast, which implies that the mechanism of protein translocation into peroxisomes has been conserved during eukaryotic evolution. The carboxyl-terminal tripeptide serine-lysine-leucine in luciferase acts as a peroxisomal import signal in mammalian cells. We have investigated whether this tripeptide is also involved in translocation of firefly luciferase into peroxisomes in yeast (Saccharomyces cerevisiae). We show by gene fusion experiments that the carboxyl-terminal 104 amino acids of luciferase can direct a heterologous protein to yeast peroxisomes. Luciferase mutant proteins were tested for their ability to be imported into yeast peroxisomes in vivo. We demonstrate that mutations in the carboxyl-terminal serine-lysine-leucine tripeptide abolish translocation of the protein into yeast peroxisomes. However, when a passenger protein was tagged at its carboxyl terminus with this tripeptide the fusion protein did not go to peroxisomes. These results indicate that, in yeast, the tripeptide is necessary but not sufficient for peroxisomal import.

Animals↗
PubMedDate not supplied

Optimized use of the firefly luciferase assay as a reporter gene in mammalian cell lines.

The enzymatic activity of firefly luciferase provides a sensitive, rapid means to assay transcriptional activity of regulated activation sequences of DNA when fused to the protein coding sequence of the luciferase gene. We have developed several modifications of the luciferase assay and have characterized certain parameters of the assay, resulting in optimization of conditions for the preparation and storage of cell lysates and establishment of substrate concentrations. These findings should be useful to investigators interested in applying the luciferase assay to studies of the transcriptional activity of test DNAs incorporating the luciferase reporter.

Animals↗