Cloning and Molecular Characterization of the cDNA for the Brazilian Larval Click-beetle Pyrearinus termitilluminans Luciferase

Viviani, Vadim R.; Silva, A. C. R.; Perez, Guillin; Santelli, Roberto Vicente; Bechara, Etelvino José Henriques; Reinach, Fernando C.

doi:10.1562/0031-8655(1999)070<0254:camcot>2.3.co;2

Cited by 43 publications

(48 citation statements)

References 12 publications

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“…To assess the timing of mitosis, our experiments required that we image luminescence so that we could visualize a cell splitting into daughter cells. We used green luciferase (PTGRm), identified in the luminescent Brazilian click beetle Pyrearinus termitilluminans, as our reporter because it is 21 times brighter than the widely used firefly luciferase (pGL3-basic) (37).…”

Section: Resultsmentioning

confidence: 99%

Circadian-independent cell mitosis in immortalized fibroblasts

Yeom

Pendergast

Ohmiya

et al. 2010

Proc. Natl. Acad. Sci. U.S.A.

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Two prominent timekeeping systems, the cell cycle, which controls cell division, and the circadian system, which controls 24-h rhythms of physiology and behavior, are found in nearly all living organisms. A distinct feature of circadian rhythms is that they are temperaturecompensated such that the period of the rhythm remains constant (∼24 h) at different ambient temperatures. Even though the speed of cell division, or growth rate, is highly temperature-dependent, the cell-mitosis rhythm is temperature-compensated. Twenty-fourhour fluctuations in cell division have also been observed in numerous species, suggesting that the circadian system is regulating the timing of cell division. We tested whether the cell-cycle rhythm was coupled to the circadian system in immortalized rat-1 fibroblasts by monitoring cell-cycle gene promoter-driven luciferase activity. We found that there was no consistent phase relationship between the circadian and cell cycles, and that the cell-cycle rhythm was not temperature-compensated in rat-1 fibroblasts. These data suggest that the circadian system does not regulate the cell-mitosis rhythm in rat-1 fibroblasts. These findings are inconsistent with numerous studies that suggest that cell mitosis is regulated by the circadian system in mammalian tissues in vivo. To account for this discrepancy, we propose two possibilities: (i) There is no direct coupling between the circadian rhythm and cell cycle but the timing of cell mitosis is synchronized with the rhythmic host environment, or (ii) coupling between the circadian rhythm and cell cycle exists in normal cells but it is disconnected in immortalized cells.O rganization of physiology and behavior into specific time domains is a fundamental property of nearly all living organisms. Anticipation of periodic changes in the environment presumably increased survival and reinforced the development of endogenous circadian oscillators (1). Because cell division is critical to the survival of unicellular organisms and the integrity of DNA is susceptible to UV irradiation, the progression of the cell cycle was probably also strongly affected by daily changes in the environment. Indeed, multiple studies have measured diurnal fluctuations in cell division such that mitosis occurs at a specific time of day in numerous species ranging from unicellular organisms (2) to humans (3-5). These data suggest that the circadian and cell cycles may be coupled in vivo.Circadian rhythms are self-sustained oscillations in physiology and behavior with endogenous periods of ≈24 h that can be synchronized, or entrained, to environmental cues such as the light/dark cycle or temperature (6). In mammals, the master circadian clock is located in the suprachiasmatic nuclei (SCN) of the anterior hypothalamus. Genes that are important for circadian timekeeping are expressed not only in the SCN but also in many peripheral tissues, including fibroblasts (7-11). Immortalized embryonic fibroblasts exhibit circadian rhythms of gene expression (12) and, using singlecell imaging...

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Section: Resultsmentioning

confidence: 99%

Circadian-independent cell mitosis in immortalized fibroblasts

Yeom

Pendergast

Ohmiya

et al. 2010

Proc. Natl. Acad. Sci. U.S.A.

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“…Although the active site of firefly luciferase was originally thought to be hydrophobic (15), more recent results suggest that it is amphiphilic (16). Click beetle luciferases, which produce different colours, have also been cloned (17,18). Mutagenesis studies identified the residues important for the bioluminescence activity and colours of these luciferases (19)(20)(21)(22)(23)(24).…”

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confidence: 99%

Active-Site Properties of Phrixotrix Railroad Worm Green and Red Bioluminescence-Eliciting Luciferases

Viviani¹,

Arnoldi²,

Balan³

et al. 2006

The Journal of Biochemistry

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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 luciferinbinding 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 redemitting luciferase is tighter than that of PxGR luciferase, whereas the other portion could be more open and polar.

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“…These fragments were inserted into the modified pcDNA4/V5-His (B) vector. (Nakajima et al, 2010;Viviani et al, 1999).…”

Section: Time-lapse Bioluminescence Imaging Of P53 Translocation Inmentioning

confidence: 99%

Dynamic monitoring of p53 translocation to mitochondria for the analysis of specific inhibitors using luciferase‐fragment complementation

Noda

Awais

et al. 2017

Biotech & Bioengineering

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Intracellular protein translocation plays a pivotal role in regulating complex biological processes, including cell death. The tumor suppressor p53 is a transcription factor activated by DNA damage and oxidative stress that also translocates from the cytosol into the mitochondrial matrix to facilitate necrotic cell death. However, specific inhibitors of p53 mitochondrial translocation are largely unknown. To explore the inhibitors of p53, we developed a bioluminescent probe to monitor p53 translocation from cytosol to mitochondria using luciferase fragment complementation assays. The probe is composed of a novel pair of luciferase fragments, the N-terminus of green click beetle luciferase CBG68 (CBGN) and multiple-complement luciferase fragment (McLuc1). The combination of luciferase fragments showed significant luminescence intensity and high signal-to-background ratio. When the p53 connected with McLuc1 translocates from cytosol into mitochondrial matrix, CBGN in mitochondrial matrix enables to complement with McLuc1, resulting in the restoration of the luminescence. The luminescence intensity was significantly increased under hydrogen peroxide-induced oxidative stress following the complementation of CBGN and McLuc1. Pifithrin-μ, a selective inhibitor of p53 mitochondrial translocation, prevented the mitochondrial translocation of the p53 probe in a concentration-dependent manner. Furthermore, the high luminescence intensity made it easier to visualize the p53 translocation at a single cell level under a bioluminescence microscope. This p53 mitochondrial translocation assay is a new tool for high-throughput screening to identify novel p53 inhibitors, which could be developed as drugs to treat diseases in which necrotic cell death is a major contributor.

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Cloning and Molecular Characterization of the cDNA for the Brazilian Larval Click-beetle Pyrearinus termitilluminans Luciferase

Cited by 43 publications

References 12 publications

Circadian-independent cell mitosis in immortalized fibroblasts

Circadian-independent cell mitosis in immortalized fibroblasts

Active-Site Properties of Phrixotrix Railroad Worm Green and Red Bioluminescence-Eliciting Luciferases

Dynamic monitoring of p53 translocation to mitochondria for the analysis of specific inhibitors using luciferase‐fragment complementation

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