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(P581) A Yeast Assay for Monitoring Estrogen Mimics.
Passos, Ana*,1, Moura, Natalia1, Cavaco, Jose1, Power, Deborah1, Canario, Adelino1, 1 Universidade do Algarve, Faro, Portugal
ABSTRACT- A yeast-based assay is being developed and will be used to monitor the presence of estrogen mimics in Portuguese coastal waters and rivers, including pristine reference waters and those subject to industrial or urban pollution. This assay consists in transforming Saccharomyces cerevisiae with an expression plasmid, containing the coding region of the sea bream (Sparus aurata) estrogen receptor- (sbER-) and a reporter plasmid, containing two hormone response elements (ERE) linked to the yeast proximal iso-1-cytochrome c gene (CYC1) promoter located upstream to the Escherichia coli gene for -galactosidase, lacZ, reporter gene. The coding region of the sbER- was successfully cloned in the expression plasmid and the receptor protein was, highly and stably, expressed in yeast with a maximum of expression occurring at 10 hours after induction of expression. To confirm that the sbER produced in yeast was able to bind estrogen with high affinity, ligand binding assays were carried out using estradiol as ligand. Yeast sbER- bound estradiol with high affinity with a dissociation constant (Kd) of 144.6 pM and a maximum binding capacity of 65.04 pM. After validation, this assay will be a useful and powerful tool for screening of natural waters.
Key words: estrogen mimics, yeast assay, estrogen receptor, sea bream
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