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Results for “Luciferases”

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PubMed1993

Monitoring foreign gene expression by a human adenovirus-based vector using the firefly luciferase gene as a reporter.

We have constructed a helper-independent adenovirus type 5-luciferase recombinant (Ad5-Luc 3) containing the firefly luciferase gene flanked by simian virus 40 (SV40) regulatory sequences inserted in the early region 3 (E3) of the Ad5 genome. Expression of luciferase in cells infected with Ad5-Luc3 was relatively efficient. In HeLa cells approximately 20 micrograms luciferase per 10(6) cells was made by 36 h post-infection and a 62 kilo-Dalton (kDa) luciferase band was clearly visible in a [35S]methionine-labeled Ad5-Luc 3-infected cell extract analyzed directly by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). The results of experiments in which cultured cells were infected with Ad5-Luc 3 in the presence or absence of 1-beta-D-arabinofuranosyl cytosine (AraC) showed that the majority of luciferase expression was dependent on viral DNA replication. This suggested that the enzyme was probably translated primarily from mRNA derived from transcripts expressed from the major late promoter of Ad5. An anti-luciferase antibody was raised in a rabbit and used to further characterize the luciferase expressed in HeLa cells infected with Ad5-Luc 3 by immunoprecipitations and Western blot analyses. The half-life of luciferase expressed in HeLa cells infected with Ad5-Luc 3 was calculated to be approximately 6-8 h by pulse chase analysis. Luciferase is likely to be a useful marker for monitoring virus dissemination and gene expression in experimental animals because assays for enzymatic activity are extremely sensitive and backgrounds are low in all tissues. In mice inoculated intraperitoneally (i.p.) with Ad5-Luc 3, luciferase activity was detected in the liver, spleen, kidney, and lung. A single i.p. inoculation of mice with Ad5-Luc 3 was sufficient to raise anti-luciferase antibody and Ad5 neutralizing antibody which persisted for at least 8 weeks. Even in the presence of circulating anti-luciferase and Ad5 neutralizing antibodies, luciferase activity could be detected in the livers, spleens, and kidneys of mice inoculated i.p. a second time with Ad5-Luc 3.

Adenoviridae Infections↗

PubMed1999

Folding and refolding of thermolabile and thermostable bacterial luciferases: the role of DnaKJ heat-shock proteins.

Bacterial luciferases are highly suitable test substrates for the analysis of refolding of misfolded proteins, as they are structurally labile and loose activity at 42 degrees C. Heat-denatured thermolabile Vibrio fischeri luciferase and thermostable Photorhabdus luminescens luciferase were used as substrates. We found that their reactivation requires the activity of the DnaK chaperone system. The DnaKJ chaperones were dispensable in vivo for de novo folding at 30 degrees C of the luciferase, but essential for refolding after a heat-shock. The rate and yield of DnaKJ refolding of the P. luminescens thermostable luciferase were to a marked degree lower as compared with the V. fischeri thermolabile luciferase. The refolding activity of the DnaKJ chaperones was examined at various temperatures. Between 30 and 37 degrees C, the refolding rates of the V. fischeri luciferase decreased and the reaction reached a complete arrest at temperatures above 40 degrees C. The rate of DnaKJ-mediated refolding of the thermostable luciferase at first increased between 30 and 37 degrees C and then decreased at the range of 37-44 degrees C. We observed that the rate of DnaKJ-mediated refolding of the heat-denatured P. luminescens thermostable luciferase, but not of the thermolabile V. fischeri luciferase, decreased during the prolonged incubation at a high (47 degrees C) temperature. The efficiency and reversibility of protein refolding arrest during and after heat-shock strongly depended on the stability of the DnaKJ-denatured luciferase complex. It is supposed that the thermostable luciferase is released during the heat-shock, whereas the thermolabile luciferase remained bound to the chaperone.

Bacterial Proteins↗

PubMed1975

Photoexcited bacterial bioluminescence. Identity and properties of the photoexcitable luciferase.

Properties of photoexcitable luciferase are compared with those of luciferase, both isolated from the bacterium Beneckea harveyi. The proteins have the same molecular weight, are similarly charged at pH 8, and can be inactivated, with comparable efficiencies, by antibodies against either pure luciferase (a heterodimeric protein) or individual subunits thereof. Compared with luciferase, photoexcitable luciferase has a broader pH range for optimal activity, is more stable under acidic conditions, is less stable under alkaline conditions, and is more resistant at neutral pH to inactivation by heat, urea, and trypsin; A flavine-like chromophore, designated B, can be isolated from photoexcitable luciferase. The binding of B to luciferase restores all the properties characteristic of photoexcitable luciferase. Moreover, photoexcitable luciferases from mutants selected to have heat labile luciferases are also thermally unstable. It is concluded that photoexcitable luciferase actually consists of a luciferase-B complex which is conformationally distinct from luciferase under certain conditions.

Animals↗

PubMed2006

Active-site properties of Phrixotrix railroad worm green and red bioluminescence-eliciting luciferases.

The luciferases of the railroad worm Phrixotrix (Coleoptera: Phengodidae) are the only beetle luciferases that naturally produce true red bioluminescence. Previously, we cloned the green- (PxGR) and red-emitting (PxRE) luciferases of railroad worms Phrixotrix viviani and P. hirtus[OLE1]. These luciferases were expressed and purified, and their active-site properties were determined. The red-emitting PxRE luciferase displays flash-like kinetics, whereas PxGR luciferase displays slow-type kinetics. The substrate affinities and catalytic efficiency of PxRE luciferase are also higher than those of PxGR luciferase. Fluorescence studies with 8-anilino-1-naphthalene sulfonic acid and 6-p-toluidino-2-naphthalene sulfonic acid showed that the PxRE luciferase luciferin-binding site is more polar than that of PxGR luciferase, and it is sensitive to guanidine. Mutagenesis and modelling studies suggest that several invariant residues in the putative luciferin-binding site of PxRE luciferase cannot interact with excited oxyluciferin. These results suggest that one portion of the luciferin-binding site of the red-emitting luciferase is tighter than that of PxGR luciferase, whereas the other portion could be more open and polar.

Animals↗

PubMed1995

Thermostability of a nuclear-targeted luciferase expressed in mammalian cells. Destabilizing influence of the intranuclear microenvironment.

Protein denaturation and aggregation are most likely the cause for the noxious effects of heat shock. There are some indications that the nucleus is one of the most sensitive cellular compartments. To test the possibility that the intranuclear microenvironment might be detrimental to the heat stability of proteins, we compared the in situ thermal stability of a reporter protein localized in the nucleus or in the cytoplasm. A recombinant firefly (Photynus pyralis) luciferase carrying a point mutation in the C-terminal domain remains in the cytoplasm (cyt-luciferase). A nuclear localization sequence was fused to the N-terminal domain of cyt-luciferase; the resulting nuc-luciferase was efficiently targeted to the cell nucleus. In both cases, decreased luciferase activity and solubility were found in lysates from heat-shocked cells. These characteristics were taken as an indication of thermal denaturation in situ. The heat-inactivated luciferases were partially reactivated during recovery after stress, indicating the capacity of both the cytoplasmic and nuclear compartments to reassemble proteins from an aggregated state. Although both the nuc- and the cyt-luciferases were heat inactivated at similar rates in vitro, nuc-luciferase was more susceptible to thermal denaturation in situ compared to cyt-luciferase. This observation suggests that the microenvironment of an intracellular compartment may modulate the thermal stability of proteins. The local concentration might be one element of this microenvironment affecting the heat-stability of proteins. In cells made thermotolerant by a priming shock, the thermal inactivation of the recombinant luciferases occurred at a slower rate during a second challenging stress. However, this decreased thermal sensitivity was less pronounced for the nuc-luciferase (threefold) than for the cyt-luciferase (sevenfold). The nuclear luciferase might become a useful tool to investigate the action of molecular chaperones in the nucleus.

Base Sequence↗

PubMed2001

Characterization of the binding of Photobacterium phosphoreum P-flavin by Vibrio harveyi Luciferase.

The isolated Photobacterium phosphoreum luciferase is associated with a bound flavin designated P-flavin and tentatively identified as 6-(3"-myristic acid)-FMN. Since FMN and myristic acid are products of the normal luciferase reaction, we explored the possibility that P-flavin can also be bound by luciferase from other luminous bacteria and serve as an active site probe. P-flavin has never been detected in Vibrio harveyi cells. We found that the V. harveyi luciferase binds P. phosphoreum P-flavin, at a ratio of 1 P-flavin per luciferase alphabeta dimer, and with concomitant absorption spectral perturbation of P-flavin, fluorescence quenching of P-flavin and luciferase, and activity inhibition of luciferase. Isolated P-flavin can be fully reduced photochemically. V. harveyi luciferase bound the oxidized P-flavin with a K(d) (or K(i) competitively against decanal) of 0.1-0.16 microM, which is three orders of magnitude lower than the K(d) for FMN binding but similar to that of reduced FMN binding. The reduced P-flavin exhibited a K(i) (competitively against the reduced FMN substrate) of 0.16 microM, also similar to the K(d) for reduced FMN. Hence, the covalent attachment of myristic acid to FMN greatly and preferentially enhanced the binding of oxidized P-flavin. The dissociation of P-flavin was slow in comparison with the binding of reduced FMN and decanal substrates. Modification of the alphaCys106 near the active site by N-ethylmaleimide can be retarded by P-flavin. These findings indicate that P-flavin is potentially a superb active site probe for luciferase. We hypothesize that P-flavin is a by-product of luciferase generated by a side reaction which is trivial with the V. harveyi luciferase but significant in the P. phosphoreum luciferase-catalyzed reaction.

Binding Sites↗

PubMed1999

Modification of luciferase to be a substrate for plant aspartic proteinase.

The possibility of using firefly luciferase as a substrate for an aspartic proteinase was explored. Several amino acid modifications to the C-terminus of the luciferase were created on the basis of the known substrate of the Arabidopsis thaliana aspartic proteinase, pro-(barley lectin). One luciferase with the sequence Arg-Asp-Gly-Val-Phe-Ala-Ala instead of the native Arg-Glu-Ile-Leu-Ile-Lys-Ala at position -15 to -9 relative to the C-terminus of native luciferase was found to possess 17% of the original luciferase activity. When this modified luciferase was incubated with the aspartic proteinase, a specific loss in activity occurred that was not observed with the original luciferase. However, both enzymes seemed very sensitive to the acidic conditions required for aspartic proteinase activity. The other versions of luciferase with different numbers of pro-(barley lectin) amino acids were not active luciferases. This provided information on the structural requirements of the C-terminal portion of the protein for luciferase activity. The luciferase proteins were also monitored during the digestion by using Western blots and some were shown to be substrates for the aspartic proteinase. Contrary to what had been expected, the modified luciferase that incorporated the pro-(barley lectin) sequences was not simply cleaved at the engineered site but at additional positions in the protein. The Arabidopsis aspartic proteinase cleaved two other standard protein substrates at many sites, suggesting that this proteinase could have a role in the degradation of proteins in addition to processing propeptides in plants.

Amino Acid Sequence↗

PubMed2005

Inhibitory effects associated with use of modified Photinus pyralis and Renilla reniformis luciferase vectors in dual reporter assays and implications for analysis of ISGs.

Luciferase reporter constructs are widely used for analysis of gene regulation when characterizing promoter and enhancer elements. We report that the recently developed codon-modified Renilla luciferase construct included as an internal standard for cotransfection must be used with great caution with respect to the amount of DNA transfected. Also, the dual-luciferase reporter vectors encoding Photinus pyralis firefly or Renilla reniformis luciferase showed a linear increase in dose-response with increasing amounts of transfected DNA, but at higher levels of transfected DNA, a reduction in expressed levels of luciferase activity resulted. In addition, treatment with type I interferon (IFN) was found to significantly reduce levels of P. pyralis firefly and Renilla luciferase activity. In contrast, cells transfected with a green fluorescent protein (GFP) reporter construct showed no significant IFN-associated change. The reduction in luciferase activity resulting from IFN treatment was not due to IFN-mediated cytotoxicity, as no change in cellular propidium iodide (PI) staining was observed by flow cytometry. IFN treatment did not alter the levels of firefly luciferase activity in cell culture supernatants or the luciferase mRNA levels determined by quantitative real-time RT-PCR analysis. Based on these results, it is probable that the IFN-induced reduction in levels of luciferase activity detected in reporter assays occurs via a posttranscriptional mechanism. Thus, it is important to be aware of these complications when using luciferase reporter systems in general or for analyzing cytokine-mediated responsive regulation of target genes, particularly by the type I IFNs.

Animals↗

PubMed2001

Thr226 is a key residue for bioluminescence spectra determination in beetle luciferases.

The comparison of click beetle and railroadworm luciferases (pH-insensitive) with firefly luciferases (pH-sensitive) showed a set of conserved residues differing between the two groups which could be involved with the bioluminescence spectra pH sensitivity. The substitution C258V in Pyrocoelia miyako (Pml) firefly luciferase and V255C in Ragophthalmus ohbai railroad worm luciferase (Rol) had no effect on the bioluminescence spectra. Substitution of Thr226 in the green-light-emitting luciferases of Rol and Pyrearinus termitilluminans (Pyt) click beetle luciferases resulted in red-shifts (12 to 35 nm), whereas the substitution T226N in the red-light-emitting luciferase of Phrixothrix hirtus (PhRE) railroadworm resulted in a 10 nm blue-shift. In PmL the substitution N230S resulted in a typical red mutant (lambda(max) = 611 nm). The bioluminescence spectrum of all these luciferase mutants did not show altered pH-sensitivity nor considerably changed half-bandwidth in relation to the wild-type luciferases. Altogether present data suggest that Thr226 is an important residue for keeping active-site core in both groups of beetle luciferases. The mechanism for bioluminescence color determination between pH-sensitive and pH-insensitive luciferases could be different.

Amino Acid Sequence↗

PubMed2006

The influence of insertion of a critical residue (Arg356) in structure and bioluminescence spectra of firefly luciferase.

The firefly bioluminescence reaction, which uses luciferin, Mg-ATP, and molecular oxygen to yield an electronically excited oxyluciferin, is carried out by luciferase and visible light is emitted. The bioluminescence color of firefly luciferases is determined by the luciferase structure and assay conditions. Among different beetle luciferases, those from Phrixothrix railroad worm emit either yellow or red bioluminescence colors. Sequence alignment analysis shows that the red-emitter luciferase from Phrixothrix hirtus has an additional Arg residue at 353, which is absent in firefly luciferases. We report here the construction and purification of a mutant at residue Arg(356), which is not conserved in beetle luciferases. By insertion of an additional residue (Arg(356)) using site-specific insertion mutagenesis in a green-emitter luciferase (Lampyris turkestanicus) the color of emitted light was changed to red and the optimum temperature of activity was also increased. Insertion of this Arg in an important flexible loop showed changes of the bioluminescence color and the luciferase reaction took place with relatively retention of its basic kinetic properties such as Km and relative activity. Comparison of native and mutant luciferases using homology modeling reveals a significant conformational change of the flexible loop in the red mutant. Movement of flexible loop brought about a new ionic interaction concomitant with a change in polarity of the emitter site, thereby leading to red emission. It is worthwhile to note that the increased optimum temperature and emission of red light might make mutant luciferase a suitable reporter for the study of gene expression and bioluminescence imaging.

Amino Acid Sequence↗

PubMed1987

Firefly luciferase gene: structure and expression in mammalian cells.

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.

Animals↗

PubMed1993

Response of human CYP1-luciferase plasmids to 2,3,7,8-tetrachlorodibenzo-p-dioxin and polycyclic aromatic hydrocarbons.

The cytochrome P4501 gene family consists of two members, CYP1A1 and CYP1A2, that are induced by halogenated hydrocarbons and polycyclic aromatic hydrocarbons. The human CYP1 promoters and 5'-flanking sequences were cloned into luciferase expression vectors to develop cell lines that stably express luciferase activity in response to CYP1 gene induction. Plasmids were initially tested in transient transfection assays. Transient transfections resulted in high-level expression of luciferase by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) from both the CYP1 expression vectors, pLUC1A1 and pLUC1A2. In dose-response experiments, 10 nM TCDD caused a maximal induction of pLUC1A2-directed luciferase activity that was 10-fold over control. Maximal pLUC1A1-directed luciferase activity was 65-fold over control in cells treated with 10 nM TCDD. Stable integration of CYP1-luciferase-neo plasmids, pL1A1N and pL1A2N, into the human hepatoma cell line, HepG2, was achieved by selection with G418. G418-resistant colonies were isolated for both pL1A1N and pL1A2N plasmids. The pL1A2N transfectants showed basal-level luciferase activity, but were nonresponsive to treatment with 10 nM TCDD. These results are in contrast to the observed induction of pLUC1A2-mediated luciferase expression in transient transfection experiments. Stable integration of the human CYP1A2 gene sequences appears to silence the transcriptional activation by TCDD. The pL1A1N transfectants showed inducible luciferase activity and one cell line, referred to as 101L, was used to establish dose-response relationships for TCDD and various polycyclic aromatic hydrocarbons. Maximal induction occurred after treatment with 100 nM TCDD, 10 microM 3-methylcholanthrene, 50 microM benz[a]anthracene, and 50 microM benzo[a]pyrene. These studies illustrate the use of the CYP1A1-luciferase cell line for the study of structure-activity relationships.

Cell Line↗

PubMed1991

Avian retroviral expression of luciferase.

Biologically active replication-competent (subgroups A, B, and C) and replication-defective Rous sarcoma virus-derived vectors containing the cDNA encoding firefly luciferase as a reporter gene were constructed. In these retroviral vectors, luciferase is expressed from a spliced subgenomic mRNA. A biologically active replication-defective UR2 virus-derived vector expressing the reporter gene as a gag-luciferase fusion protein from an unspliced genomic mRNA was also constructed. The luciferase reporter gene was used because it lacks homology with chicken genomic sequences and because a rapid and sensitive direct enzymatic assay is available to monitor luciferase expression in retrovirus-infected cells. The levels of luciferase expression in luciferase recombinant retrovirus-infected chicken embryo fibroblasts are greater than 10(3) higher than that detected in uninfected cells or in cells infected with retroviral vectors carrying other genes. Endpoint dilution titration experiments demonstrated that one infected cell can be detected in a background of 10(3) uninfected cells. The vectors are stable in tissue culture and high level expression of the unselected luciferase reporter gene is maintained. The vectors were used to express luciferase in chicken embryos, demonstrating the potential utility of luciferase as a reporter in vivo.

Animals↗

PubMed2006

Firefly luciferase terminally degraded by mild heat exposure: implications for reporter assays.

Luciferase reporter constructs are an accurate method of assessing gene promoter activity and vectors constitutively expressing luciferase are useful in quantifying transfection efficiency. Common methodologies for examining the induction of the heat shock (stress) response require exposure of cells transfected with luciferase-expressing vectors to a mild heat stress. Here we re-examine the under-recognised phenomenon that luciferase is exquisitely sensitive to small temperature changes. In cells subjected to mild heat exposure following transfection with both luciferase and beta-galactosidase reporter vectors, a marked reduction in luciferase activity was observed compared with beta-galactosidase activity. On exposing recombinant firefly luciferase to small increases in temperature in vitro, a time and temperature dependent decrease in luciferase activity was demonstrated. Loss of luciferase activity following mild heat exposure will result in misinterpretation of reporter activity. This vastly underappreciated effect is worthy of further emphasis and luciferase reporter vectors should be used with caution in protocols that involve exposure to temperatures outside the physiological range.

Cell Line, Tumor↗

PubMed2004

Identity of the emitter in the bacterial luciferase luminescence reaction: binding and fluorescence quantum yield studies of 5-decyl-4a-hydroxy-4a,5-dihydroriboflavin-5'-phosphate as a model.

The excited state of 4a-hydroxy-4a,5-dihydroFMN has been postulated to be the emitter in the bacterial bioluminescence reaction. However, while the bioluminescence quantum yield of the luciferase emitter is about 0.16, chemiluminescence and fluorescence quantum yields of earlier flavin models mimicking the luciferase emitter were no more than 10(-5). To further examine the proposed chemical identity of the luciferase emitter, 5-decyl-4a-hydroxy-4a,5-dihydroFMN was prepared as a new flavin model. Both the wild-type Vibrio harveyi luciferase and a catalytically active alphaC106A mutant formed complexes with the flavin model at a 1:1 molar ratio with K(d) values at 2.4 and 1.2 microM, respectively. This flavin model inhibited the activity of both luciferases, suggesting that it was bound to the enzyme active center. While the free flavin model was itself only very weakly fluorescent, its binding to either luciferase species resulted in markedly enhanced fluorescence, peaking at 440 nm. The fluorescence quantum yields of 5-decyl-4a-hydroxy-4a,5-dihydroFMN bound to wild-type and alphaC106A luciferases were 0.08 and 0.05, respectively, which are about 50% of the respective emitter bioluminescence quantum yields of these two luciferases. The present findings clearly demonstrated that the luciferase active site was suitable for marked enhancement of fluorescence of 4a-hydroxyflavin and, hence, provides a strong support to the proposed identity of 4a-hydroxy-4a,5-dihydroFMN, in its exited state, as the luciferase emitter.

Binding Sites↗

PubMed1999

Cloning, sequence analysis, and expression of active Phrixothrix railroad-worms luciferases: relationship between bioluminescence spectra and primary structures.

Phrixothrix railroad-worms emit yellow-green light through 11 pairs of lateral lanterns along the body and red light through two cephalic lanterns. The cDNAs for the lateral lanterns luciferase of Phrixothrix vivianii, which emit green light (lambda max= 542 nm), and for the head lanterns of P. hirtus, which emit the most red-shifted bioluminescence (lambda max= 628 nm) among luminescent beetles, were cloned. Positive clones which emitted green (PvGR: lambda max= 549 nm) and red (PhRE: lambda max= 622 nm) bioluminescence were isolated. The lucifereases coded by PvGR (545 amino acid residues) and PhRE (546 amino acid residues) cDNAs share 71% identity. PvGR and PhRE luciferases showed 50-55% and 46-49% identity with firefly luciferases, respectively, and 47-49% with click-beetle luciferases. PhRE luciferase has some unique residues which replace invariant residues in other beetle luciferases. The additional residue Arg 352 in PhRE, which is deleted in PvGR polypeptide, seems to be another important structural feature associated with red light production. As in the case of other railroad-worms and click-beetle luciferases studied, Phrixothrix luciferases do not undergo the typical red shift suffered by firefly luciferases upon decreasing pH, a property which might be related to the many amino acid residues shared in common between railroad-worm and click-beetle luciferase.

Amino Acid Sequence↗

PubMed1999

Control of luminescence decay and flavin binding by the LuxA carboxyl-terminal regions in chimeric bacterial luciferases.

Bacterial luciferases (LuxAB) can be readily classed as slow or fast decay luciferases based on their rates of luminescence decay in a single turnover assay. Luciferases from Vibrio harveyi and Xenorhabdus (Photorhabdus) luminescens have slow decay rates, and those from the Photobacterium genus, such as P. (Vibrio) fischeri, P. phosphoreum, and P. leiognathi, have rapid decay rates. By generation of an X. luminescens-based chimeric luciferase with a 67 amino acid substitution from P. phosphoreum LuxA in the central region of the LuxA subunit, the "slow" X. luminescens luciferase was converted into a chimeric luciferase, LuxA(1)B, with a significantly more rapid decay rate. Two other chimeras with P. phosphoreum sequences substituted closer to the carboxyl terminal of LuxA, LuxA(2)B and LuxA(3)B, retained the characteristic slow decay rates of X. luminescens luciferase but had weaker interactions with both reduced and oxidized flavins, implicating the carboxyl-terminal regions in flavin binding. The dependence of the luminescence decay on concentration and type of fatty aldehyde indicated that the decay rate of "fast" luciferases arose due to a high dissociation constant (K(a)) for aldehyde (A) coupled with the rapid decay of the resultant aldehyde-free complex via a dark pathway. The decay rate of luminescence (k(T)) was related to the decanal concentration by the equation: k(T) = (k(L)A + k(D)K(a))/(K(a) + A), showing that the rate constant for luminescence decay is equal to the decay rate via the dark- (k(D)) and light-emitting (k(L)) pathways at low and high aldehyde concentrations, respectively. These results strongly implicate the central region in LuxA(1)B as critical in differentiating between "slow" and "fast" luciferases and show that this distinction is primarily due to differences in aldehyde affinity and in the decomposition of the luciferase-flavin-oxygen intermediate.

Aldehydes↗

PubMed2001

A new beta-lactoglobulin-based vector targets luciferase cDNA expression to the mammary gland of transgenic mice.

A beta-lactoglobulin (BLG)/luciferase gene vector (p907), composed of a luciferase intronless gene inserted between the second and sixth BLG exons was constructed. Stable transfections of CID-9 cells with this vector, as well as with a series of additional vectors, were performed to define regulatory regions within the BLG sequence, and the contribution of the SV40 polyadenylation (PA) site to luciferase expression. A relatively low level of luciferase activity was supported by vector p907. It was partially rescued by vector p906, in which the BLG 3' region, downstream of the luciferase cDNA, was replaced with the SV40 PA site. Flanking the SV40 region of vector p906, at its 3' end, with BLG sequences of exon 6/intron 6/exon 7 and the 3' region of the gene resulted in vector p904. This vector supported the highest luciferase activity, 10 times or 2.5 times higher than that measured in cells transfected with vectors p907 and p906, respectively. The induced activity supported by vector p904 is attributed to interaction between the SV40 PA site and elements of the distal part of the BLG 3' flanking sequences. The BLG 5' regulatory region of vector p904 encompasses a 3-kb promoter sequences. Deletion of 935 bp of its proximal end resulted in a 60% decrease in luciferase activity. Reduced activity was also seen with vector p915 lacking sequences of exon 1/intron 1/exon 2. This decrease could not be rescued with heterologous sequences of insulin intron 1, inserted upstream of the luciferase cDNA. Two sets of transgenic mice carrying vectors p907 and p904 were generated. Vector p907 supported only marginal luciferase activity in the mammary gland of all transgenic mice tested and luciferase RNA could not be detected by northern analysis. In contrast, 50% of the transgenic mice carrying vector p904 expressed luciferase RNA in the mammary gland and tissue-specific, hormonal-dependent activity was determined. However, the new p904 vector was not able to insulate the transgene from surrounding host DNA sequences, as reflected by its copy number-independent manner of expression. Nevertheless, vector p904 may represent a valuable tool for the expression of cDNAs in the mammary gland of transgenic animals.

3' Flanking Region↗