Engineering of monomeric bacterial luciferases by fusion of luxA and luxB genes in Vibrio harveyi

Olsson, Olof; Escher, Alan; Sandberg, Göran; Schell, Jeff; Koncz, Csaba; Szalay, Aladar A.

doi:10.1016/0378-1119(89)90194-7

Cited by 51 publications

(29 citation statements)

References 33 publications

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“…The 934-bp band corresponded to a transcript that had been spliced twice, having had the chimeric intron in addition to the majority of the RL ORF and XIAP-derived sequence removed. Translation of this transcript should result in the formation of a RL-FL fusion protein that lacks RL activity but likely exhibits FL activity (Olsson et al 1989;Eu and Andrade 2001). Both smaller mRNAs should facilitate efficient cap-dependent translation of FL.…”

Section: Demonstrating Ires Elements In Eukaryotic Mrnasmentioning

confidence: 99%

Demonstrating internal ribosome entry sites in eukaryotic mRNAs using stringent RNA test procedures

Eden¹,

Byrd²,

Sherrill³

et al. 2004

RNA

135

159

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Section: Demonstrating Ires Elements In Eukaryotic Mrnasmentioning

confidence: 99%

Demonstrating internal ribosome entry sites in eukaryotic mRNAs using stringent RNA test procedures

Eden¹,

Byrd²,

Sherrill³

et al. 2004

RNA

135

159

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“…In fact there were about 30 photoprotein fusions and conjugates reported between 1988 and 2000 (63)(64)(65). The first such fusion was that of the Vibrio harveyi luxA and luxB into the luxAB (luxF) fusion, which was expressed as a monomer in E. coli, Bacillus, yeast and plant systems (30,66). A luxAB fusion has also been made from the luciferase of Photorhabdus luminescens (67).…”

Section: Fusionsmentioning

confidence: 99%

“…Olsson et al (1988) have shown that fusion gene products can be added to the luxA of Vibrio harveyi as long as the Nterminal hydrophobic sequences of the a-subunit are preserved intact, in order to retain enzymatic activity (29). Olsson et al (1989) constructed luxAB and luxBA fusions of the V. harveyi luciferase genes and expressed them in E. coli (Fig. 1) and in calli of Nicotiana tabacum, indicating their possible application as reporter genes in eukaryotic cells (30).…”

Section: Imaging Of Luciferase Expression In Transformed Single Cellsmentioning

confidence: 99%

“…Olsson et al (1989) constructed luxAB and luxBA fusions of the V. harveyi luciferase genes and expressed them in E. coli (Fig. 1) and in calli of Nicotiana tabacum, indicating their possible application as reporter genes in eukaryotic cells (30). The authors showed that luxAB has higher expression levels than luxBA.…”

Section: Imaging Of Luciferase Expression In Transformed Single Cellsmentioning

confidence: 99%

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Imaging of light emission from the expression of luciferases in living cells and organisms: a review

2002

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Luciferases are enzymes that emit light in the presence of oxygen and a substrate (luciferin) and which have been used for real-time, low-light imaging of gene expression in cell cultures, individual cells, whole organisms, and transgenic organisms. Such luciferin-luciferase systems include, among others, the bacterial lux genes of terrestrial Photorhabdus luminescens and marine Vibrio harveyi bacteria, as well as eukaryotic luciferase luc and ruc genes from firefly species (Photinus) and the sea panzy (Renilla reniformis), respectively. In various vectors and in fusion constructs with other gene products such as green fluorescence protein (GFP; from the jellyfish Aequorea), luciferases have served as reporters in a number of promoter search and targeted gene expression experiments over the last two decades. Luciferase imaging has also been used to trace bacterial and viral infection in vivo and to visualize the proliferation of tumour cells in animal models.

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“…It should be noted that the physiological substrate of luciferase is thought to be n-tetradecanal (37), which is not generally used for experimental work because of its poor stability and low solubility (30). The applicability of bacterial luciferase as a reporter enzyme in eukaryotic systems has been enhanced by fusing the luxA and luxB genes, which encode the two subunits of bacterial luciferase (6,20,42,43). Successful application of this fused bacterial luciferase in yeast (6) and plant (34) and quinones or by nitric oxide (2,16,27,36,40,41).…”

mentioning

confidence: 99%

Intracellular generation of superoxide as a by-product of Vibrio harveyi luciferase expressed in Escherichia coli

González-Flecha

Demple

1994

J Bacteriol

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Luciferase genes are widely used as reporters of gene expression because of the high sensitivity of chemiluminescence detection and the possibility of monitoring light production in intact cells. We engineered fusions of the Escherichia coli soxS promoter to the luciferase structural genes (luxAB) from Vibrio harveyi. Since soxS transcription is positively triggered by the activated SoxR protein in response to agents such as paraquat that generate intracellular superoxide, we hoped to use this construct as a sensitive reporter of redox stress agents. Although a soxR+ soxS'::luxAB fusion exhibited a paraquat-inducible synthesis of luciferase, a smaller increase was consistently observed even in the absence of known soxRS inducers. This endogenous induction was soxR dependent and was further characterized by introducing a plasmid carrying the luciferase structural genes without the soxS promoter into a strain carrying a soxS'::lacZ fusion in the bacterial chromosome. These cells exhibited increased 1-galactosidase expression as they grew into mid-log phase. This increase was ascribed to luciferase activity because 0-galactosidase induction was suppressed (but not eliminated) when the substrate n-decanal was present in the medium. The soxS'::luxAB plasmid transformed superoxide dismutasedeficient strains very poorly under aerobic conditions but just as efficiently as a control plasmid under anaerobic conditions. The production of hydrogen peroxide, the dismutation product of superoxide anion, was significantly increased in strains carrying bacterial luciferase and maximal in the absence of n-decanal. Taken collectively, these data point to the generation of significant amounts of intracellular superoxide by bacterial luciferase, the possible mechanism of which is discussed. In addition to providing insights into the role of superoxide in the activation of the SoxR protein, these results suggest caution in the interpretation of experiments using luciferase as a reporter of gene expression.Luciferases have been widely used as reporter genes to determine temporal and tissue-specific regulation of promoters in both prokaryotes (bacterial luciferase) (10, 13, 22) and eukaryotes (firefly luciferase) (1, 18). Bacterial luciferase is also the target gene in a mutagenicity assay (50) and has been used to quantitate long-chain aldehydes (49), protease activity (3), and intracellular concentrations of NADH, NADPH, and flavin mononucleotide (FMN) (46).Bacterial luciferase catalyzes the FMNH2-dependent oxidation of n-decanal (29)(30)(31)35). Both the substrate used experimentally (n-decanal) and the measurable product (photons of blue-green light) can cross cell membranes rapidly, which allows luciferase activity to be monitored continuously without damage to the cell. It should be noted that the physiological substrate of luciferase is thought to be n-tetradecanal (37), which is not generally used for experimental work because of its poor stability and low solubility (30). The applicability of bacterial luciferase as a reporte...

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Engineering of monomeric bacterial luciferases by fusion of luxA and luxB genes in Vibrio harveyi

Cited by 51 publications

References 33 publications

Demonstrating internal ribosome entry sites in eukaryotic mRNAs using stringent RNA test procedures

Demonstrating internal ribosome entry sites in eukaryotic mRNAs using stringent RNA test procedures

Imaging of light emission from the expression of luciferases in living cells and organisms: a review

Intracellular generation of superoxide as a by-product of Vibrio harveyi luciferase expressed in Escherichia coli

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