Expression of the cloned subunits of bacterial luciferase from separate replicons

The lux A and lux B genes of Vibrio harveyi, encoding the alpha and beta subunits of bacterial luciferase, were cloned individually into Escherichia coli in two different compatible plasmids. Active luciferase was formed in an amount equal to that produced in cells carrying a plasmid with the cloned genes on a single fragment.     

Use of bacterial and firefly luciferases as reporter genes in DEAE-dextran-mediated transfection of mammalian cells.

The aim of this study was to compare three different luciferase genes by placing them in a single reporter vector and expressing them in the same mammalian cell type. The luciferase genes investigated were the luc genes from the fireflies Photinus pyralis (PP) and Luciola mingrelica (LM) and the lux AB5 gene, a translational fusion of the two subunits of the bacterial luciferase from Vibrio harveyi (VH). The chloramphenicol acetyltransferase (CAT) gene was also included in this study for comparison. The performances of the assay methods of the corresponding enzymes were evaluated using reference materials and the results of the expressed enzymes following transfection were calculated using calibration curves. All of the bioluminescent assays possess high reproducibility both within and between the batches (less than 15%). The comparison of the assay methods shows that firefly luciferases have the highest detection sensitivity (0.05 and 0.08 amol for PP and LM, respectively) whereas the VH bacterial luciferase has 5 amol and CAT 100 amol. On the other hand, the transfection of the various plasmids shows that the content of the expressed enzyme within the cells is much higher for CAT than for the other luciferase genes. VH luciferase is expressed at very low levels in mammalian cells due to the relatively high temperature of growing of the mammalian cells that seems to impair the correct folding of the active enzyme. PP and LM luciferases are both expressed at picomolar level but usually 10 to 70 times less in content with respect to CAT within the transfected cells. On the basis of these results the overall improvement in sensitivity related to the use of firefly luciferases as reporter genes in mammalian cells is about 30 to 50 times with respect to that of CAT.     

Construction of cloning vectors using the Vibrio harveyi luminescence genes luxA and luxB as markers

A synthetic oligodeoxyribonucleotide harboring four new restriction sites was inserted into the luxB gene of Vibrio harveyi. This insertion did not disrupt the reading frame. An active beta-subunit was synthesized since a plasmid with both the luxA and mutated luxB genes conferred upon Escherichia coli the bacterial luciferase (Lux) phenotype in the presence of an aldehyde. Ligation of a piece of foreign DNA at these new cloning sites in the vector extinguish the Lux phenotype of the transformed bacteria. Therefore, the plasmid was used as a cloning vector, and recombinant DNA-containing bacteria were detected by the loss of bioluminescence. To create more versatile plasmids, the intergenic region of phage f1 was inserted outside of the lux genes. The selection by loss of bioluminescence presents several advantages over the white/blue selection of the lacZ gene on indicator plates.     

Variables Controlling the Expression Level of Exogenous Genes in Dictyostelium

Ectopic expression of genes from recombinant plasmids is commonly used to study gene function. In Dictyostelium, three drug resistance cassettes are commonly used as selectable markers in vectors. We report here a comparative study of the expression of green fluorescent protein (GFP) gene from vectors containing each of the drug-resistant cassettes. The expression was highest in cells transformed with the vectors containing the neomycin-resistant cassette (pDNeoGFP), followed by the hygromycin-resistant cassette (pDHygGFP) and the blasticidin-resistant cassette (pDBsrGFP). The level of GFP expression was directly related to the copy number of the vector in transformants. In turn, the copy number of the vector depended on the drug resistance cassette as well as the concentration of the drug used in selection. In general, cells with higher copy numbers could be selected by a higher drug concentration. The expression of GFP was also affected by the method of transformation. For pDHygGFP, expression of GFP was much higher in cells transformed by electroporation than those transformed by calcium phosphate coprecipitation. However, only a slight difference was observed for pDNeoGFP or pDBsrGFP.     

Variables controlling the expression level of exogenous genes in Dictyostelium.

Ectopic expression of genes from recombinant plasmids is commonly used to study gene function. In Dictyostelium, three drug resistance cassettes are commonly used as selectable markers in vectors. We report here a comparative study of the expression of green fluorescent protein (GFP) gene from vectors containing each of the drug-resistant cassettes. The expression was highest in cells transformed with the vectors containing the neomycin-resistant cassette (pDNeoGFP), followed by the hygromycin-resistant cassette (pDHygGFP) and the blasticidin-resistant cassette (pDBsrGFP). The level of GFP expression was directly related to the copy number of the vector in transformants. In turn, the copy number of the vector depended on the drug resistance cassette as well as the concentration of the drug used in selection. In general, cells with higher copy numbers could be selected by a higher drug concentration. The expression of GFP was also affected by the method of transformation. For pDHygGFP, expression of GFP was much higher in cells transformed by electroporation than those transformed by calcium phosphate coprecipitation. However, only a slight difference was observed for pDNeoGFP or pDBsrGFP.     

Molecular biology of bacterial bioluminescence.

The cloning and expression of the lux genes from different luminescent bacteria including marine and terrestrial species have led to significant advances in our knowledge of the molecular biology of bacterial bioluminescence. All lux operons have a common gene organization of luxCDAB(F)E, with luxAB coding for luciferase and luxCDE coding for the fatty acid reductase complex responsible for synthesizing fatty aldehydes for the luminescence reaction, whereas significant differences exist in their sequences and properties as well as in the presence of other lux genes (I, R, F, G, and H). Recognition of the regulatory genes as well as diffusible metabolites that control the growth-dependent induction of luminescence (autoinducers) in some species has advanced our understanding of this unique regulatory mechanism in which the autoinducers appear to serve as sensors of the chemical or nutritional environment. The lux genes have now been transferred into a variety of different organisms to generate new luminescent species. Naturally dark bacteria containing the luxCDABE and luxAB genes, respectively, are luminescent or emit light on addition of aldehyde. Fusion of the luxAB genes has also allowed the expression of luciferase under a single promoter in eukaryotic systems. The ability to express the lux genes in a variety of prokaryotic and eukaryotic organisms and the ease and sensitivity of the luminescence assay demonstrate the considerable potential of the widespread application of the lux genes as reporters of gene expression and metabolic function.     

Construction and testing of a bacterial luciferase reporter gene system forin Vivo measurement of nonsense suppression inStreptomyces

A reporter gene system, based on luciferase genes from Vibrio harvei, was constructed for measurement of translation nonsense suppression in Streptomyces. Using the site-directed mutagenesis the TCA codon in position 13 of the luxB gene was replaced by all of the three stop codons individually. By cloning of luxA and luxB genes under the control of strong constitutive Streptomyces promoter ermE* in plasmid pUWL201 we created Wluxl with the wild-type sequence and pWlux2, pWlux3 and pWlux4 plasmids containing TGA-, TAG- and TAA-stop codons, respectively. Streptomyces lividans TK 24 was transformed with the plasmids and the reporter system was tested by growth of the strain in the presence of streptomycin as a translation accuracy modulator. Streptomycin increased nonsense suppression on UAA nearly 10-fold and more than 20-fold on UAG. On the other hand, UGA, the most frequent stop signal in Streptomyces, the effect was negligible.     

Construction and testing of an intron-containing luciferase reporter gene from<Emphasis Type="Italic">Renilla reniformis</Emphasis>

We describe a newRenilla reniformis luciferase reporter gene,RiLUC, which was designed to allow detection of luciferase activity in studies involvingAgrobacterium-based transient expression studies. TheRLUC gene was altered to contain a modified intron from the castor bean catalase gene while maintaining consensus eukaryotic splicing sites recognized by the plant spliceosome.RLUC andRiLUC reporter genes were fused to the synthetic plant SUPER promoter. Luciferase activity within agrobacteria containing the SUPER-RLUC construct increased during growth in culture. In contrast, agrobacteria harboring the SUPER-RiLUC gene fusion showed no detectable luciferase activity. Agrobacteria containing these gene fusions were cotransformed with a compatible normalization plasmid containing a cauliflower mosaic virus 35S promoter (CaMV) joined to the firefly luciferase coding region (FiLUC) and infused into tobacco leaf tissues through stomatal openings. The kinetics of luciferase production from theRLUC orRiLUC reporters were consistent, with expression of theRiLUC gene being limited to transiently transformed plant cells.RiLUC activity from the reporter gene fusions was measured transiently and within stably transformed tobacco leaf tissues. Analysis of stably transformed tobacco plants harboring either reporter gene fusion showed that the intron altered neither the levels of luciferase activity nor tissue-specific expression patterns driven by the SUPER promoter. These results demonstrate that theRiLUC reporter gene can be used to monitor luciferase expression in transient and stable transformation experiments without interference from contaminating agrobacteria.     

<Emphasis Type="Italic">Renilla</Emphasis> luciferase-<Emphasis Type="Italic">Aequorea</Emphasis> GFP (Ruc-GFP) fusion protein,a novel dual reporter for real-time imaging of gene expression in cell cultures and in live animals

Light-emitting reporter proteins play an increasing role in the study of gene expression in vitro and in vivo. Here we present a ruc-gfp fusion gene construct generated by fusing a cDNA for Renilla luciferase (ruc) in-frame with a cDNA encoding the "humanized" GFP (gfp) from Aequorea. A plasmid containing the fusion gene construct was successfully transformed into, and expressed in, mammalian cells. The transformed cells exhibited both Renilla luciferase activity in the presence of coelenterazine and GFP fluorescence upon excitation with UV light. Spectrofluorometry of cells containing the Ruc-GFP fusion protein, in the absence of wavelengths capable of exciting GFP fluorescence but in the presence of the luciferase substrate, coelenterazine, showed an emission spectrum with two peaks at 475 nm and 508 nm. These two peaks correspond to the emission maximum of Renilla luciferase at 475 nm and that of GFP at 508 nm. The peak at 508 nm generated in the presence of coelenterazine alone (without UV excitation) is the result of intramolecular energy transfer from Renilla luciferase to Aequorea GFP. Southern analysis of genomic DNA purified from transformed Chinese hamster ovary (CHO) cells and fluorescence in situ hybridization (FISH) to metaphase chromosomes confirmed the integration of the ruc-gfp fusion gene on a single chromosome. The bifunctional Ruc-GFP fusion protein allows the detection of gene expression at the single-cell level based on green fluorescence, and in a group of cells based on luminescence emission. Furthermore, animal experiments revealed that light emission from the Ruc-GFP fusion protein can be detected externally in the organs or tissues of live animals bearing the gene construct.     

地衣芽胞杆菌FLP/FRT基因编辑系统的构建及验证

地衣芽胞杆菌有效的基因编辑工具有限,为了拓展和丰富其基因编辑手段,在地衣芽胞杆菌中构建一个抗性标记可重复使用的FLP/FRT基因编辑系统,并通过敲除α-淀粉酶基因amyL、蛋白酶基因aprE及敲入外源透明颤菌血红蛋白基因vgb对该系统进行初步验证。首先以温敏质粒pNZT1为载体分别构建amyL和aprE基因敲除质粒pNZTT-AFKF和pNZTT-EFKF,两个敲除质粒各自包含针对目标基因的同源臂、抗性基因及同向的FRT位点;将敲除质粒转化地衣芽胞杆菌并经过两次同源交换过程实现目标基因的敲除;最后导入一个FLP重组酶表达质粒通过FLP/FRT系统的重组作用介导抗性基因的回收。为进一步验证本系统的实用性及编辑效率,构建了包含透明颤菌血红蛋白编码基因vgb表达盒及基因组丙酮酸甲酸裂解酶编码基因pflB敲除盒的重组质粒pNZTK-PFTF-vgb,并以此进行外源基因vgb在基因组上的定向敲入。结果显示,成功敲除amyL及aprE并回收了抗性标记卡那霉素基因,敲除后淀粉酶活和蛋白酶活分别减少95.3%和80.4%;vgb基因成功整合入基因组pflB位点并回收了抗性标记四环素基因,并利用荧光定量PCR技术检测到vgb的整合表达。文中首次建立了一个适用于地衣芽胞杆菌的、抗性标记可重复使用的FLP/FRT基因编辑系统,并进行了基因敲除及基因敲入验证,为地衣芽胞杆菌遗传改造提供了良好的方法参考。     

Critical regions of the Vibrio fischeri luxR protein defined by mutational analysis.

Expression of Vibrio fischeri luminescence genes requires an inducer, termed autoinducer, and a positive regulatory element, the luxR gene product. A plasmid containing a tac promoter-controlled luxR was mutagenized in vitro with hydroxylamine, and luxR mutant plasmids were identified by their inability to complement a luxR deletion mutation in trans. Sixteen luxR mutant plasmids were obtained, ten of which encoded full-length but inactive luxR gene products as demonstrated by a Western immunoblot analysis. The effects of 1 of the 10 mutations could be overcome by the addition of autoinducer at a high concentration. The mutations in each of the 10 mutant plasmids that directed the synthesis of an inactive LuxR protein were identified by DNA sequencing. Of the 10 proteins encoded by the mutant luxR plasmids, 9 differed from the normally active LuxR in only a single amino acid residue. The amino acid residue substitutions in the proteins encoded by the nine mutant luxR genes clustered in two regions. One region around the middle of the polypeptide encoded by luxR was hypothesized to represent an autoinducer-binding domain, and the other region towards the carboxy terminus of the gene product was hypothesized to constitute a lux operator DNA-binding domain or a lux operator DNA recognition domain.     

Nucleotide sequence, expression, and properties of luciferase coded by lux genes from a terrestrial bacterium

The lux genes required for expression of luminescence have been cloned from a terrestrial bacterium, Xenorhabdus luminescens, and the nucleotide sequences of the luxA and luxB genes coding for the alpha and beta subunits of luciferase determined. The lux gene organization was closely related to that of marine bacteria from the Vibrio genus with the luxD gene being located immediately upstream and the luxE downstream of the luciferase genes, luxAB. A high degree of homology (85% identity) was found between the amino acid sequences of the alpha subunits of X. luminescens luciferase and the luciferase from a marine bacterium, Vibrio harveyi, whereas the beta subunits of the two luciferases had only 60% identity in amino acid sequence. The similarity in the sequences of the alpha subunits of the two luciferases was also reflected in the substrate specificities and turnover rates with different fatty aldehydes supporting the proposal that the alpha subunit almost exclusively controls these properties. The luciferase from X. luminescens was shown to have a remarkably high thermal stability being stable at 45 degrees C (t 1/2 greater than 3 h) whereas V. harveyi luciferase was rapidly inactivated at this temperature (t 1/2 = 5 min). These results indicate that the X. luminescens lux system may be the bacterial bioluminescent system of choice for application in coupled luminescent assays and expression of lux genes in eukaryotic systems at higher temperatures.     

Toxicity of the bacterial luciferase substrate, n-decyl aldehyde, to Saccharomyces cerevisiae and Caenorhabditis elegans

This study determined that the bacterial luciferase fusion gene (luxAB) was not a suitable in vivo gene reporter in the model eukaryotic organisms Saccharomyces cerevisiae and Caenorhabditis elegans. LuxAB expressing S. cerevisiae strains displayed distinctive rapid decays in luminescence upon addition of the bacterial luciferase substrate, n-decyl aldehyde, suggesting a toxic response. Growth studies and toxicity bioassays have subsequently confirmed, that the aldehyde substrate was toxic to both organisms at concentrations well tolerated by Escherichia coli. As the addition of aldehyde is an integral part of the bacterial luciferase activity assay, our results do not support the use of lux reporter genes for in vivo analyses in these model eukaryotic organisms.