We describe a Saccharomyces cerevisiae bioluminescence assay for UV and arsenate in which bacterial luciferase genes are regulated by the promoter of the yeast gene, UFO1. UFO1 encodes the F-box subunit of the Skp1–Cdc53–F-box protein ubiquitin ligase complex and is induced by DNA damage and by arsenate. We engineered the UFO1 promoter into an existing yeast bioreporter that employs human genes for detection of steroid hormone-disrupting compounds in water bodies. Our analysis indicates that use of an endogenous yeast promoter in different mutant backgrounds allows discrimination between different environmental signals. The UFO1-engineered yeast give a robust bioluminescence response to UVB and can be used for evaluating UV protective sunscreens. They are also effective in detecting extremely low concentrations of arsenate, particularly in pdr5Δ mutants that lack a mechanism to extrude toxic chemicals; however, they do not respond to cadmium or mercury. Combined use of endogenous yeast promoter elements and mutants of stress response pathways may facilitate development of high-specificity yeast bioreporters able to discriminate between closely related chemicals present together in the environment.
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"Since this bioassay is luc-based, D-luciferin must be added. Another S. cerevisiae-based bioreporter has been created to measure arsenate and also UV damage (Bakhrat et al., 2011). This strain is based on the BLYES strain of Sanseverino et al. (2005), containing a constitutive luxCDEfrp plasmid and a luxAB plasmid that has been reengineered to be under control of the UFO1 promoter, which specifically responds to DNA damage by UV light and also arsenate. "
[Show abstract][Hide abstract] ABSTRACT: Living whole-cell bioreporters serve as environmental biosentinels that survey their ecosystems for harmful pollutants and chemical toxicants, and in the process act as human and other higher animal proxies to pre-alert for unfavorable, damaging, or toxic conditions. Endowed with bioluminescent, fluorescent, or colorimetric signaling elements, bioreporters can provide a fast, easily measured link to chemical contaminant presence, bioavailability, and toxicity relative to a living system. Though well tested in the confines of the laboratory, real-world applications of bioreporters are limited. In this review, we will consider bioreporter technologies that have evolved from the laboratory towards true environmental applications, and discuss their merits as well as crucial advancements that still require adoption for more widespread utilization. Although the vast majority of environmental monitoring strategies rely upon bioreporters constructed from bacteria, we will also examine environmental biosensing through the use of less conventional eukaryotic-based bioreporters, whose chemical signaling capacity facilitates a more human-relevant link to toxicity and health-related consequences.
Full-text · Article · May 2013 · Ecological Indicators
[Show abstract][Hide abstract] ABSTRACT: Transcriptome analyses indicate that a core 10%-15% of the yeast genome is modulated by a variety of different stresses. However, not all the induced genes undergo translation, and null mutants of many induced genes do not show elevated sensitivity to the particular stress. Elucidation of the RNA lifecycle reveals accumulation of non-translating mRNAs in cytoplasmic granules, P-bodies, and stress granules for future regulation. P-bodies contain enzymes for mRNA degradation; under stress conditions mRNAs may be transferred to stress granules for storage and return to translation. Protein degradation by the ubiquitin-proteasome system is elevated by stress; and here we analyzed the steady state levels, decay, and subcellular localization of the mRNA of the gene encoding the F-box protein, UFO1, that is induced by stress. Using the MS2L mRNA reporter system UFO1 mRNA was observed in granules that colocalized with P-bodies and stress granules. These P-bodies stored diverse mRNAs. Granules of two mRNAs transported prior to translation, ASH1-MS2L and OXA1-MS2L, docked with P-bodies. HSP12 mRNA that gave rise to highly elevated protein levels was not observed in granules under these stress conditions. ecd3, pat1 double mutants that are defective in P-body formation were sensitive to mRNAs expressed ectopically from strong promoters. These highly expressed mRNAs showed elevated translation compared with wild-type cells, and the viability of the mutants was strongly reduced. ecd3, pat1 mutants also exhibited increased sensitivity to different stresses. Our interpretation is that sequestration of highly expressed mRNAs in P-bodies is essential for viability. Storage of mRNAs for future regulation may contribute to the discrepancy between the steady state levels of many stress-induced mRNAs and their proteins. Sorting of mRNAs for future translation or decay by individual cells could generate potentially different phenotypes in a genetically identical population and enhance its ability to withstand stress.