Date of Award

8-2011

Document Type

Thesis

Degree Name

Master of Science (MS)

Legacy Department

Environmental Toxicology

Advisor

Baldwin, William S

Committee Member

Bain , Lisa J

Committee Member

Klaine , Stephen J

Committee Member

Turnbull , Matthew W

Abstract

Annotation of the Daphnia pulex nuclear receptors revealed a novel group of three receptors designated NR1L, and named HR97a/b/g because of their similarity to the HR96 receptors involved in xenobiotic detection. We cloned and sequenced the three receptors from a related species often used in aquatic toxicology studies, Daphnia magna, and then analyzed their genomic structure and conducted phylogenetic studies. Phylogenetic studies confirmed that the HR97s do form a distinct group with HR97g being the precursor of HR97a and b. They also confirmed that the HR97 receptors are related to the HR96 (NR1J) receptors, and the VDR/CAR/PXR (NR1I) group. Mining other arthropod genomes revealed that Ixodes also has a NR1L member (IsHR97). Gal4-chimeric plasmids that contain the ligand binding domain of HR97a/b/g were constructed for transactivation assays. Transactivation assays demonstrated that HR97a and HR97g repress basal transcription in the absence of a activator; however, HR97b has constitutive activity. We hypothesized that the HR97's are involved in xenobiotic detoxification. Contrary to our hypothesis, the HR97s are not activated by multiple toxicants. We found that pyriproxyfen and methyl farnesoate activate HR97g. Dose-response studies found that pyriproxyfen and methyl farnesoate have EC50's of 3.4 and 2.2 uM, respectively. The two chemicals are juvenile hormone analogs that have been found to induce the production of males in the otherwise female parthenogenic Daphnia. HR97a and b are also activated by pyriproxyfen. Further, although NR1L is phylogenetically related to NR1I and NR1J, the HR97s do not share significant similarity with the NR1I and NR1J members. We conclude that HR97s are not xenobiotic sensors, but may be involved in juvenile hormone signaling and potentially male production.

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