Development of yeast-based fluorogenic biosensors for detection of DNA-damaging agents in environmental samples.
Susanna Boronat, Benjamín Piña
Molecular and Cellular Biology, IBMB-CSIC, Jordi Girona, 18, Barcelona, 08034, Spain
Yeast-based bioassays are becoming widespread tools for detection and quantification of environmental stressors. We are interested in establishing a collection of reporter yeast strains for detection of different kinds of environmental stress (genotoxic, metals, metalloids, oxidants, etc.) based on the yeast stress response pathways. We have already established three yeast strains based on the transcriptional activation by DNA damage of the RNR3, RAD54 and PHR1 genes, involved in different pathways in the response to DNA damage. Diploid strains have been constructed by replacing ORFs of these genes. Since yeast strains are diploid, replacement of ORFs by the reporter gene does not result in the loss of the ability to repair DNA. Transcriptional activation is measured by the enzymatic activity of the E. coli gene Gus and expressed as the slope of fluorescence vs. time. This approach makes the method automatizable and control by user is not necessary. We have analyzed the RNR3 mRNA copy number by real-time RT-PCR in MMS treated cells and correlated it with the slope units. The detection limit for MMS using the RNR3-GUS strain is 30 µg/ml in 4 hours treatment and corresponds to 10 -4 slope units/cell, which equals 3-4 x 10 -3 RNR3 mRNA copies/cell. An advantage of this integrated system is its easy transfer to yeast strains with interesting features for commercialization. We will present and discuss data of the application of these strains to the analysis of environmental samples.