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Dual Fluorescent Protein (yEGFP/DsRed-Express-2) Bioassay System for Rapid Screening for Chemical Mutagens Based on RNR3 Regulation in Saccharomyces Cerevisiae.

Over the past several years, the most widely used in vitro tests for mutagenicity have been those performed on bacteria. These include the Ames test, SOS, and SOS/umu chromotest. The Ames test is the most commonly used system, but it is not automatable and requires several strains to detect different types of mutagens. Faster alternative tests include the SOS chromotest and the umu test, which are based on SOS promoter-linked Lac Z, which is activated by the SOS response to DNA damage in Escherichia coli and Salmonella typhimurium, respectively. The most important drawback of bacterial tests is that they are not capable of emulating the cell cycle controls or the repair mechanisms found in eukaryotic cells because the gene structure, gene regulation, and metabolism for prokaryotic cells are quite different from those of eukaryotic cells. In this way, the use of prokaryotic cells in mutagenicity screening for chemical substances has certain limitations. Mammalian cell tests such as the single-cell gel electrophoresis assay, V79/HGPRT gene mutation test, and the test of in vitro mammalian cell transformation have no such drawbacks with respect to gene structure, gene regulation, or metabolism. However, the cells require relatively intense experimental culture conditions and their resistance to chemical toxicity is poor. In these ways, these tests are generally unsuitable for large-scale screening. The yeast cell (Saccharomyces cerevisiae) is a specific kind of eukaryotic cell. It has the same advantages as bacteria including its simple genetic background, short cell cycle, fast cultivation, modest nutritional requirements, and strong resistance to harmful chemicals. This excellent yeast model has gene structure, gene regulation, metabolism, and protein post-translational modifications and processes that are similar to those of mammalian cells. As a result, given proper biotechnological techniques, yeast can be an ideal host for construction of recombinant cells for rapid and highthroughput screening of chemical mutagens. DNA damage in eukaryotic cells leads to the gene expression involved in DNA repair. The DNA damage checkpoint pathway responsible for activation of DNA-damage-inducible genes in Saccharomyces cerevisiae yeast has been the focus of intensive research during the past decade. One of the frequently used tools in the DNA damage checkpoint pathway in Saccharomyces has been the gene RNR3. RNR3 is part of the ribonucleotide reductase (RNR) system. Under normal growth conditions, the expression of RNR3 was nearly undetectable in cells. However, this gene is expressed 5to 10-fold, and in some reports more than 100-fold, during DNA breakage and synthesis blockade. These properties of the RNR3 have been used in the identification of a number of important genes involved in the DNA damage caused by chemical products. Some researchers have fused this gene promoter with some reporter genes to construct recombinant yeast cells for screening of chemical mutagens. However, the normalization of reporter genes has routinely been achieved by quantifying total protein of the yeast lysate or on the basis of the optical density (OD600) of the yeast culture, while the optical density measurements can yield highly variable results. The dual luciferase assay is performed by sequentially measuring the firefly and Renilla luciferase activities of the same sample, with the results expressed as the ratio of firefly to Renilla luciferase activity (Fluc/Rluc). Although luciferase