UV and arsenate toxicity: a specific and sensitive yeast bioluminescence assay

Abstract

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.

Sequestration of Highly Expressed mRNAs in Cytoplasmic Granules, P-Bodies, and Stress Granules Enhances Cell Viability

“…This is compatible with the proposed role for Ufo1 in maintenance of genome stability [9] and may be indicative of a remodeling of the genome during recovery. These steady state Ufo1 protein values are comparable to results of a study in which we expressed the bacterial reporter genes, luxA and luxB , in yeast from the UFO1 promoter [46]. Luciferase activity was elevated 10-fold in response to 40 mJ/cm 2 UV whereas arsenate or H 2 O 2 led to a threefold elevation of enzyme activity.…”

“…UFO1 is under repression during normal growth conditions [46] and treatment with arsenate led to a fourfold increase in the level of UFO1 mRNA after 15 minutes that stayed high for at least one hour during which the cells were assayed. Treatment with H 2 O 2 gave a threefold increase in mRNA level after 15 minutes followed by a decrease back to the basal level of the untreated control from the 30 minute time point.…”

A pilot study for construction of a new cadmium-sensing yeast strain carrying a reporter plasmid with the <i>JLP1</i> promoter

“…In order to obtain a markedly stronger response by the reporter to Cd, further experi- mental modifications can be considered, such as a change in the reporter gene with Luc or GFP, which may enhance reporter activity, or a change in the β-galactosidase substrate with fluorescent substances including fluorescein di-β-D-glucopyranoside, 4-methylumbelliferyl β-Dgalactopyranoside, and resorufin β-D-galactopyranoside to enhance reporter activity. The use of yeasts with membrane transport gene mutations such as PDR5 and PDR10 may effectively reduce the lowest detection limit of the reporter assay (Bakhrat et al, 2011;Walsh et al, 2005;Ito-Harashima et al, 2015). The reporter activities of the JLP1 promoter were compared with those of the SEO1 and CUP1 promoters, all of which were connected to lacZ in the high-copy plasmids in yeasts with the same genetic background ( Fig.…”

“…The promoter sequences of the CUP1, SEO1, and UFO1 genes have been used to detect heavy metals. Yeast strains possessing CUP1 reporter plasmids respond to copper (Cu) and cadmium (Cd) (Tohoyama et al, 1992), those possessing the SEO1 reporter respond to Cd, arsenic (As), and mercury (Hg) (Park et al, 2007), and those possessing the UFO1 reporter respond to arsenate and ultraviolet light B (UVB) (Bakhrat et al, 2011).…”

Analysis of Environmental Samples with Yeast-Based Bioluminescent Bioreporters

“…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.…”