Luc genes: Introduction of colour into bioluminescence assays

Luminescence assays are generally based on measurements of light intensity alone. Inclusion of colour as an additional parameter of the assay could increase the information content. Colour variation in luminescence is particularly prevalent among beetle luciferases. To study the relationship between enzyme structure and colour, luciferases from a Jamalcan click beetle were examined as a model system. These luciferases emit light ranging from green to orange, though their amino acid sequences differ by less than 5%. Through mutation of their respective cDNA clones, the amino acids responsible for the colour variation were identified. These specific amino acids are few, and they act upon colour independently with respect to the enzyme structure. Analysis of their effects indicates that the potential for colour variation among beetle luciferases is greater than is evident among the click beetle luciferase. Because of the subtle changes of enzyme structure that effect colour, luciferases that emit different colours may be useful as paired genetic reporters. They should interact equivalently with the intracellular environment of a host, but could be distinguished by colour in their assay. Such paired reporters could be used to observed simultaneous events, or to provide internal control for luminescence measurements.     

Bioluminescence and chemiluminescence literature: 1987 literature,part III

In studying beetle bioluminescence in the early 1960s, Dr McElroy and his colleagues found that the Jamaican click beetle, Pyrophorus plagiophthalamus, was capable of emitting different colours of light. They further found that the luciferin substrate used by this beetle was the same as that in the firefly, demonstrating that the different colours of bioluminescence were due to differences in the structure of the luciferases. We have recently cloned cDNAs from this beetle species which code for at least four different luciferases. The luciferases are distinguishable by their different colours of bioluminescence when expressed in Escherichia coli. The sequence differences between these different luciferases are few, so the amino acids responsible for the different colours of emission must also be few. Through the construction of hybrid luciferases, by rearranging fragments of the original cDNA clones, we have identified some of these amino acid determinants of colour.     

Comparison of properties of commercially available crystalline native and recombinant firefly luciferases

Commercially available crystalline native and recombinant firefly luciferases were compared. The two types of luciferase had indistinguishable responses to variation in ATP and luciferin concentrations and to omission of reaction components. The time courses of light production, the responses to nucleotide analogues, and the stability of the enzymes under several storage conditions were identical. The native enzyme had a slightly greater specific activity and was more sensitive to trypsin degradation. These differeces are probably attributable to differences in conformation.     

Red-emitting luciferases for bioluminescence reporter and imaging applications

North American firefly Photinus pyralis luciferase, which emits yellow-green light (557 nm), has been adapted for a variety of applications, including gene reporter assays, whole-cell biosensor measurements, and in vivo imaging. Luciferase variants with red-shifted bioluminescence and high specific activity can be paired with green-emitting counterparts for use in dual-color reporter assays or can be used alone for in vivo imaging. Beginning with a previously reported red-emitting thermostable mutant and using mutagenesis techniques, we engineered two luciferases with redder emission maxima while maintaining satisfactory specific activities and thermostability. The novel enzymes were expressed in HEK293 cells, where they performed similarly to Promega’s codon-optimized click beetle red luciferase in model reporter assays. When the firefly luciferase variants were codon-optimized and retested using optimized substrate concentrations, they provided 50- to 100-fold greater integrated light intensities than the click beetle enzyme. These results suggest that the novel enzymes should provide superior performance in dual-color reporter and in vivo imaging applications, and they illustrate the importance of codon optimization for assays in mammalian cells.     

Few substitutions affect the bioluminescence spectra of Phrixotrix (Coleoptera: Phengodidae) luciferases: a site-directed mutagenesis survey.

Phrixotrix (railroad worm) luciferases produce bioluminescence in the green and red regions of the spectrum, depending on the location of the lanterns, and are the only luciferases naturally producing red bioluminescence. Comparison of the luciferase sequences showed a set of substitutions that could be involved in bioluminescence colour determination: (a) unique substitutions in the red luciferase replacing otherwise invariant residues; (b) conserved basic residues in the green-yellow emitting luciferases; and (c) an additional R353 residue in red-emitting luciferase (Viviani et al., 1999). To investigate whether these sites have a functional role in bioluminescence colour determination, we performed a site-directed mutagenesis. Natural substitutions in the region 220-344 and residues in the putative luciferin-binding site were also investigated. With the exception of the previously identified substitution of R215 and T226 (Viviani et al., 2002), which display dramatic red-shift effects on the spectrum of green-yellow-emitting luciferases, only a few substitutions had a moderate effect on the spectrum of the green-emitting luciferase. In contrast, no single substitution affected the spectrum of the red-emitting luciferase. The results suggest that the identity of the active site residues is not so critical for determining red bioluminescence in PxRE luciferase. Rather, the conformation assumed during the emitting step could be critical to set up proper interactions with excited oxyluciferin.     

小鼠HMGB1启动子荧光素酶报告基因的构建及功能鉴定

利用PCR技术扩增小鼠高迁移率族蛋白1(HMGB1)基因启动子序列,构建小鼠HMGB1启动子荧光素酶报告基因pGL3-basic-HMGB1.经PCR、酶切及测序鉴定后,用脂质体法将pGL3-basic-HMGB1转入巨噬细胞264.7中,并应用萤光素酶测定系统检测其活性.检测结果显示pGL3-basic-HMGB1具有启动子活性.小鼠HMGB1启动子荧光素酶报告基因pGL3-basic-HMGB1的成功构建,为进一步研究HMGB1提供基本材料.     

Novel monomeric luciferase enzymes as tools to study plant gene regulation in vivo

Taking advantage of a specially constructed vector, luciferase LuxA and LuxB subunits were connected in frame to different amino acid linkers to reproduce a series of monomeric luciferase enzymes. A comparison of their activities in E. coli cells demonstrated that the length of the linkers positively affected activity. One luciferase fusion gene was expressed in plant cells, and we showed that this gene activity could be monitored directly without destructive sampling.     

Differential expression of genes of Pseudomonas syringae on leaves and in culture evaluated with random genomic lux fusions

Differential expression of genes of Pseudomonas syringae strain B728a on plants and in culture was assessed by measuring light production by a large collection of mutant strains containing random genomic insertions of a promoterless lux operon. Reporter gene fusions were made using Tn4431 containing lux CDABE from Vibrio fisheri. Light production reproducibly increased seven-fold when n-decanal was added to cells harvested from plant surfaces, to over 800-fold when added to cells cultured on a solidified culture medium, thus increasing the sensitivity of this reporter gene system. One of the 173 mutants tested exhibited significantly higher light production on plants than on solidified culture media compared to other mutants, while one lux fusion-containing strain produced significantly more light on culture media than on plants relative to the other mutants. The plant-inducible genes identified were not required for pathogenicity of this strain. Approximately 2% of the genes of P. syringae are apparently transcribed more actively in cells growing epiphytically on plants than in common culture media indicating that bacterial cells on plants may have substantially different behaviours than that of cultured cells.     

小鼠T-bet启动子荧光素酶报告基因载体的构建及活性鉴定

为了研究T-bet在T细胞中的转录调控机制,并研究其在多发性硬化症中的信号通路,本研究构建小鼠TBX21(编码T-bet)基因启动子区和增强子区萤火虫荧光素酶报告基因载体。在对小鼠TBX21基因5?侧翼区进行详尽生物信息学特征分析后,设计相应引物,用PCR的方法从小鼠基因组中扩增出TBX21基因5?侧翼区–1 000 bp-28 bp片段长为1 028 bp的启动子区(以翻译起始点ATG为+1)和–3 308 bp-–2 000 bp片段长为1308 bp的非编码区保守序列(No-coding conserved sequence,CNS),再用定向克隆的方法将这两个片段定向重组入专门用于启动子活性研究的萤火虫荧光素酶报告基因载体(p GL4.10)中,构建出包含小鼠TBX21基因启动子区和CNS区的萤火虫荧光素酶报告基因载体(p GL4.10-TBX21pr-CNS),电泳与测序鉴定,最后再将p GL4.10-TBX21pr-CNS与内参p RL-TK用lipofectamine 2000共转染293T细胞和Jurkat细胞中,通过双荧光素酶报告基因检测系统鉴定p GL4.10-TBX21pr-CNS的启动子和增强子活性,并用独立样本t检验方法进行统计分析。对照组共转染p GL4.10与内参p RL-TK。结果表明,成功构建出荧光素酶报告基因重组质粒p GL4.10-TBX21pr-CNS。与转染空质粒p RL-TK组相比,293T细胞(P=0.012 2)和Jurkat细胞(P=0.002 2)中转染p GL4.10-TBX21pr-CNS组荧光素酶活性升高。研究结果表明在293T细胞和Jurkat细胞中p GL4.10-TBX21pr-CNS可以表现出启动子活性,为后续小鼠T-bet转录调控研究提供了基本材料。     

发光甲虫与生物荧光

生物荧光是活体生物自身可以发光的有趣生命现象。具有这一现象的生物存在于生物四界中,但目前关于这一现象的研究报道主要来自于昆虫,尤其是以萤火虫为代表的发光甲虫的研究。文章对发光甲虫的分类地位、生物荧光发生的原理、发光器官的类型、闪光的“开关”机制、生物荧光的生物学意义及其相关行为学研究进展等进行了详细介绍。此外,还简要提及了荧光生物及其荧光酶的应用。这对了解及探讨生物荧光现象、加强对中国的发光甲虫及其它发光生物的研究及保护利用具有一定的借鉴作用。     

Evaluation and Comparison of the GUS, LUC and GFP Reporter System for Gene Expression Studies in Plants

Abstract: The detailed analysis of the expression pattern of a plant gene can give important clues about its function in plant development, cell differentiation and defence reactions. Gene expression studies have been greatly facilitated by the employment of proteins like β-glucuronidase (GUS), green fluorescent protein (GFP), and firefly luciferase (LUC) as reporters of gene activity. The application of reporter genes in plants, specifically in the field of gene expression studies, has expanded over the years from a mere tool to quantify (trans) gene expression in tissue samples, to real-time imaging of in planta promoter dynamics. To correctly interpret the activity that is given by each reporter, it is important to have a good understanding of the intrinsic properties of the different reporter proteins. Here we discuss those properties of GUS, LUC and GFP that are of interest in gene expression studies.     

Improved Expression of Novel Red- and Green-emitting Luciferases of Phrixothrix Railroad Worms in Mammalian Cells

Luciferases are widely used for the quantitative monitoring of gene expression in a variety of organisms. We successfully expressed novel red- and green-emitting luciferases of Phrixothrix railroad worms in mammalian cells in combination with the Kozak sequence and the CAG promoter. The characteristic properties of these luciferases indicate that they are appropriate reporter genes for the simultaneous monitoring of two gene expressions.     

Low-light imaging technology in the life sciences

Photon imaging is an increasingly important technique for the measurement and analysis of chemiluminescence and bioluminescence. New high-performance low-light level imaging systems have recently become available for the life science. These systems use advances in camera design and digital image processing and are now being used for a wide range of luminescence applications. They offer good sensitivity for photon detection and large dynamic range, and are suitable for quantitative analysis. This is achieved using a range of software techniques including image arithmetic, histogramming or summing regions of interest, feature extraction and multiple image processing for kinetics or assay screening. Improvements in imageprocessing hardware and software have increased the usefulness of these systems in the biosciences. Low-light imaging is a rapid and non-invasive method for the sensitive detection and analysis of luminescent assays. As such it offers a powerful and sensitive tool for investigating processes, both at the cellular level (luc and lux reporter genes, intracellular signalling) and for measurement of macro samples (immunoassays, gels and blots, tissue sections).     

Prior flea beetle herbivory affects oviposition preference and larval performance of a potato beetle on their shared host plant

Abstract 1. The herbaceous plant Solanum carolinense (L.) (Solanaceae) is host to a number of specialist insects, including the leaf-feeding beetles Epitrix fuscula (Crotch) and Leptinotarsa juncta (Germar) (Coleoptera: Chrysomelidae). Potted individuals of S. carolinense were subjected to one of two treatments: exposure to herbivory by E. fuscula or exclusion of all herbivores. The effects of E. fuscula herbivory on larval performance and oviposition preference of L. juncta were investigated.
2. Although the masses of the L. juncta pupae did not differ between the two treatments, larvae feeding on damaged plants developed more slowly than those feeding on undamaged plants.
3. In both paired leaf choice trials and whole plant choice trials, larvae of L. juncta showed no preference for undamaged versus damaged hosts.
4. In a field transplant experiment, adult L. juncta females showed slight feeding preferences and strong oviposition preferences for undamaged plants versus plants that had been fed on by E. fuscula .
5. The results are discussed with reference to their implications for plant-mediated competition among herbivores and constraints on the evolution of plant resistance.     

Development of near-infrared firefly luciferin analogue reacted with wild-type and mutant luciferases

Interestingly, only the D-form of firefly luciferin produces light by luciferin–luciferase (L–L) reaction. Certain firefly luciferin analogues with modified structures maintain bioluminescence (BL) activity; however, all L-form luciferin analogues show no BL activity. To this date, our group has developed luciferin analogues with moderate BL activity that produce light of various wavelengths. For in vivo bioluminescence imaging, one of the important factors for detection sensitivity is tissue permeability of the number of photons emitted by L–L reaction, and the wavelengths of light in the near-infrared (NIR) range (700–900 nm) are most appropriate for the purpose. Some NIR luciferin analogues by us had performance for in vivo experiments to make it possible to detect photons from deep target tissues in mice with high sensitivity, whereas only a few of them can produce NIR light by the L–L reactions with wild-type luciferase and/or mutant luciferase. Based on the structure–activity relationships, we designed and synthesized here a luciferin analogue with the 5-allyl-6-dimethylamino-2-naphthylethenyl moiety. This analogue exhibited NIR BL emissions with wild-type luciferase (λ_max = 705 nm) and mutant luciferase AlaLuc (λ_max = 655 nm).     

Integrating biogeographic and genetic approaches to investigate the history of bioluminescent colour alleles in the Jamaican click beetle, Pyrophorus plagiophthalamus

Bioluminescent colour in the Jamaican click beetle, Pyrophorus plagiophthalamus, is an ideal system for studies moving from gene to landscape to gain a holistic understanding of the molecular, ecological, and historical bases for adaptation. Previous studies have established the genetics of bioluminescent colour variation in the beetle to the level of the nucleotide base pair in the target gene luciferase. Three different luciferase colour alleles affecting ventral light organ colour [yellow-green (vYG), yellow (vYE), and orange (vOR)] were found segregating in P. plagiophthalamus populations. These alleles differ from each other in a number of replacement mutations (14 total), the majority of which (11) have a measurable effect on colour. Phylogenetic analysis revealed a long-term adaptive trend on Jamaica towards longer wavelength bioluminescence, culminating in the most recently derived vOR allele. Here, we further investigate the historical and geographic context of adaptive colour evolution by testing a vicariance model for the origins of the extant ventral light organ polymorphism: that the vOR allele arose and differentiated in an isolated deme on the east side of Jamaica before spreading westward. Comparisons of colour phenotypes, luciferase coding sequences, the third intron of the gene, mtDNA, and microsatellite data provided evidence for past population subdivision on Jamaica and ongoing gene flow, as has been found for other island endemics. However, the pattern of differentiation supported the allopatric divergence of vYG and vYE alleles. The vOR gene appears to have arisen relatively recently from a vYE precursor and postdates the period of major biogeographic isolation. We discuss the implications of the results for discerning ecological causation in the adaptive sequence from nucleotide to landscape to population change for bioluminescent colour.     

A new indicator cell line established to monitor bovine foamy virus infection

In order to improve the accuracy for quantitating the bovine foamy virus (BFV) in vitro, we developed a baby hamster kidney cell (BHK)-21-derived indicator cell line containing a plasmid that encodes the firefly luciferase driven by the BFV long terminal repeat promoter (LTR, from −7 to 1012). The BFV titer could be determined by detecting the luciferase expression since the viral trans-activator BTas protein activates the promoter activity of the LTR. One clone, designated BFVL, was selected from ten neomycin-resistant clones. BFVL showed a specific and inducible dose- and time-dependent luciferase activity in response to BFV infection. Although the changes in luciferase activity of BFVL peaked at 84 h post infection, it was possible to differentiate infected and uninfected cells at 48 h post infection. A linear relationship was established between the multiplicity of infection (MOI) of BFV and the activated ratio of luciferase expression in BFVL. Moreover, the sensitivity of the BFVL-based assay for detecting infectious BFV was 10,000 times higher than the conventional CPE-based assay at 48 h post infection. These findings suggest that the BFVL-based assay is rapid, easy, sensitive, quantitative and specific for detection of BFV infection.