Luciferase reporter

Molecular weight. The molecular weight of C. hilgendorfii luciferase reported in the past varies considerably across a range of 50,000-80,000 (Chase and Langridge, 1960 Shimomura etal., 1961, 1969 Tsuji and Sowinski, 1961 Tsuji et al., 1974) it appears most likely to be 60,000-70,000. The luciferase is an acidic protein with an isoelectric point of 4.35 (Shimomura et al., 1961). The absorption spectrum of luciferase is that of a simple protein without any prosthetic group, showing a peak at 280 nm. Absorbance value at 280 nm of a 0.1% luciferase solution is approximately 0.96 (Shimomura etal., 1969). 

Kendall, J. M., et al. (1996). Recombinant apoaequorin acting as a pseudo-luciferase reports micromolar changes in the endoplasmic reticulum free Ca2+ of intact cells. Biochem. J. 318 383-387. 

The test system was considerably less sensitive to endosulfan when mouse ER, rather than human ER, was used to mediate (3-gal activity (Ramamoorthy et al. 1997). In similar assays, endosulfan at 10 jM had no effect on (3-gal activity in yeast Saccharomyces) transfected with either the human or rainbow trout ER (Andersen et al. 1999). In addition, no effect was observed on transcriptional activation of HeLa cells transfected with plasmids containing an estrogen receptor as a responsive element (Shelby et al. 1996). Endosulfan also did not induce transient reporter gene expression in MCF-7 human breast cancer cells at an incubation concentration of 2.5 pM (Andersen et al. 1999). Maximum endosulfan-induced ER-mediated luciferase reporter gene expression occurred in vitro in a T47D human breast adenocarcinoma cell line at approximately 10 pM, while 50% expression of luciferase occurred at about 5.9 pM the maximum expression was approximately 59% of the effect from exposure to 0.03 nM estradiol (0.00003 pM) (Legler et al. 1999). Luciferase expression from combined treatment with endosulfan and dieldrin was additive over concentrations ranging from 3 to 8 pM. 

Legler J, van den Brink C, Brouwer A, et al. 1998. Assessment of (anti-)estrogenic compounds using a stably transfected luciferase reporter gene assay in the human T47-D breast cancer cell line. Organohalogen Compounds 37 265. 

FIGURE 20.2 The involvement of RARE on apo-lO -lycopenoic acid-transactivated RARp expression. Upper panel Diagram of the RARp reporter vector with wild type and mutated R AREs. Lower panel HeLa cells transfected with the RARp reporter vector and an internal control vector were treated with 5 pmol/I. of apo-lO -lycopenoic acid or 1 pmol/L of all-trans retinoic acid for 24h. Luciferase activities were measured by dual-luciferase reporter system. Values are means of SEM of three replicate assays., statistically significantly different, as compared with control in the same group, P < 0.05. (Adapted from Lian, F. et al., Carcinogenesis, 28, 1567, 2007. With permission.)... 

Recent studies have demonstrated that lithium (and to a lesser extent VPA) produces, at therapeutically relevant concentrations, complex alterations in basal and/or stimulated DNA-binding of 12-o-tetradecanoyl-phorbol 13-acetate (TPA) response element (TRE) to AP-1 transcription factors. These alterations are produced not only in human SH-SY5Y cells in vitro, but also in rodent brain following chronic, in vivo administration [5, 7, 15-21]. Corresponding to an increase in basal AP-1 DNA-binding activity, lithium and VPA have been shown to increase the expression of a luciferase reporter gene driven by an SV40 promoter that contains TREs in a time- and concentration-dependent fashion. Mutations in the TRE... 

Cells are lysed for Firefly and Renilla luciferase assays using the Dual-Luciferase Reporter Assay system (Promega), following the manufacturer s instructions. We use a multimode microplate reader with automatic injectors (FLUOROstar Optima from BMG Labtech, OfFenburg, Germany) for luminescence measurements. 

Hardin We can rescue locomotor activity rhythms with a static per RNA level. What is important here are the protein levels. The other answer we need to find for this story is looking in antennae with anti-PER antibodies to see whether the protein is cychng. The other experiment is to use a luciferase reporter to see whether there are rhythms in PER expression. 

Fig. 19. The Cx43 promoter region. Partial analysis of the human Cx43 gene extending from -360 to the site of fusion where De Leon et al. [1994] inserted the luciferase reporter gene at position +143. The transcription start site is numbered -1 [redrawn from De Leon et al., 1994]. The TATA box and the putative AP-1-binding sequence are underlined.

The estrogenic and antiestrogenic activities of several PBDE congeners and three hydroxylated PBDEs were tested in vitro using human breast cell line assays based on estrogen receptor (ER)-dependent luciferase reporter gene expression (Mccrts et al. 2001). The hydroxylated PBDEs,... 

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