Genetically modified yeast cells with human regulatory elements h

Genetically modified yeast cells with human regulatory elements have been successfully used also in assessing estrogenicity [7, 21-25] and androgenicity of compounds [26] and detecting cell Site URL List 1|]# wall-disturbing agents [27].In this study we introduce an alternative approach for the assessment of non-specific toxicity of several chemicals, even in real-time. Our toxicity assay is based on S. cerevisiae transformed with a modified firefly (Photinus pyralis) luciferase gene (luc) as a reporter for genetic response. The luc gene is inserted into the plasmid pRS316/GPD-PGK between the constitutive promoter GPD and PGK terminator. The plasmid produces light constitutively [21]. Firefly luciferase catalyses the following reaction: Luc + D-luciferin + ATP �� oxyluciferin + AMP + CO2 + PPi + light.

The resulting luminescence (yellowish light) can be measured very sensitively in real-time. In our assays, we use D-luciferin substrate at a pH of 5.0, because in the modified firefly luciferase the last three amino acids of the enzyme have been truncated. The natural peroxisomal targeting signal (Ser-Lys-Leu) [28] lacking from the C-terminus of the enzyme results in a cytoplasmic expression, which leads to high level of light emission. Under such conditions, where full length firefly luciferase is used [20] D-luciferin must traverse cytoplasmic and peroxisomal membranes to give light emission. This together with a D-luciferin substrate at a pH of 3.0 results in a low level of light emission [20].

We have previously shown that keeping the yeast cells at pH of 5.

0 increases the light emission and the growth rate, providing more viable cells for toxicity measurement [29]. Furthermore, an assay can be done in a multi-well plate and the light Batimastat emission produced by luciferase can be measured simply by adding D-luciferin substrate after an exposure of few hours or even in real-time. We show in this study that the acute toxicity of several model compounds representing completely different kinds of molecular families or structures against eukaryotic organisms can now be performed with light-emitting intact yeast cells on the contrary to Hollis et al. [20].2.?Results and Discussion2.1.

Bioluminescence assayIn this GSK-3 work, we estimated the toxicity of selected chemicals by exposing genetically modified yeast cells and measuring the luminescence produced in the presence of D-luciferin. In our study, the response to different chemicals varied a lot from activation to complete inhibition of light emission depending on the concentrations used. The toxicity of two compounds, 5,6-benzoflavone and rapamycin were monitored continuously in real-time.

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