Yeast reporter gene assays

The yeast reporter gene assays not only assess for the interaction of the chemical with the hormone receptor, but also the ability of that receptor-chemical ligand interaction to activate the hormone DNA response element. It should be realized, however, that most of these systems have been developed with human and mammalian hormone receptors and differences in ligand potencies can occur between different animal species. A comprehensive review of in vitro assays for measuring estrogenic activity, and some of the issues of comparability, is provided by Zacharewski (1997). 

Yeast reporter gene assays and MCF-7 cell-based proliferation assays (E-screen) are particularly popular. The E-screen, in which proliferation of human breast cancer cells (MCF-7) is measured as a response to estrogen, has also been used to determine the estrogenicity of sewage effluent and surface water (Kdrner et al., 2001). 

Cell based assays for NRs range from reporter gene assays to in vivo recruitment assays. The most reported of these is the GAL4 reporter assay. This assay takes advantage of the fact that the GAL4 response element of yeast does not exist in mammalian systems. 

Hepatotoxicity—enzyme release and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazodium bromide (MTT) assay on fresh or cryopreserved (human) hepatocytes Endocrine disruption—yeast and mammalian reporter gene assay (oestrogenicity and adrogenicity)... 

Reporter gene assays that measure ER binding-dependant transcriptional and translational activity. The yeast-based assays fall within this group. The vast majority of yeast-based assays are based on S. cerevisiae containing a stably transfected human estrogen, receptor (hER), although there are several variations in strain and detection method (NICEATM, 2002)... 

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. 

Figure 5.2. High-throughput mating assay for two-hybrid protein interaction screening. Yeast strains containing individual bait and prey clones are combined in a well and allowed to mate. Diploids are then selected and scored for a protein-protein interaction using the selection provided by the transcriptional reporter gene.

Fig. 7.1. Different yeast n-hybrid systems that have been developed to study protein-protein, protein-DNA, protein-RNA, and protein - small molecule interactions. A. In the original version of the Y2H system, transcriptional activation of the reporter gene is reconstituted by recruitment of the activation domain (AD) to the promoter region through direct interaction of protein X and Y, since protein X is fused to a DNA-binding domain (DBD) and protein Y is fused to the AD [1], B. In the Y1 H assay, the AD is fused directly to the DBD [109]. This assay can be used to screen either DBDs that can bind to a specific DNA sequence or the in vivo binding site for a given DBD. C.

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