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Differential Display Analyses of the Amphipod Hyalella Azteca Exposed to Ethynylestradiol at Three Different Trophic Levels
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| Abstract: | A study was conducted to determine if differential display could be used to detect differences in gene expression in the amphipod, Hyalella azteca. In a study of synthetic estrogen attenuation in different aquatic media, amphipods were exposed to 20 ng/L 17 a-ethynylestradiol in mesocosms at low (total N and P, 0.77 and 0.015 mg/L, respectively), medium (total N and P, 1.09 and 0.022 mg/L), and high (total N and P, 1.99 and 0.32 mg/L) nutrient levels. Amphipods were deployed on the bottom and below the surface of mesocosms with and without 17 a-ethynylestradiol for 2 days. PVC couplers (4 x 6 cm) enclosed at each end with 250 micron (#60) Nitex mesh were used as exposure chambers. Amphipods were then recovered and stored in RNAlater (Ambion) until needed for RNA isolation. RNA samples were pooled across replicate nutrient levels into groups exposed at the top and bottoms of the mesocosms. Total RNA was isolated and reverse transcribed using an anchored oligo-dT primer. cDNAs were subsequently amplified in a PCR reaction using fluorescent TMR-labeled anchored oligo-dTprimers and one of several arbitrary decamers. The PCR products were separated on a polyacrylamide gel and visualized with a fluorescent scanner. Several bands were identified that exhibited differential expression between amphipods exposed to 17 a-ethynylestradiol and controls. These bands were excised from the gel, and the cDNAs were re-amplified and sequenced. We plan to verify differential expression through semi-quantitative PCR using primers generated from the cDNA sequences. These techniques promise to expand the use of amphipods as biomonitors of contaminants in sediments and water. |
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| Citation: | Flick, R., D. L. Lattier, J. M. Lazorchak, M. E. Smith, D. E. Williams, S. E. Campbell, F. J. Denoyelles, T. V. Reddy, D. W. Graham, and G. P. Toth. Differential Display Analyses of the Amphipod Hyalella Azteca Exposed to Ethynylestradiol at Three Different Trophic Levels. Presented at Society of Environmental Toxicology and Chemistry, Baltimore, MD, November 11-15, 2001. |
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| Contact: |
Linda Ransick - (513) 569-7395 or ransick.linda@epa.gov
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| Division: |
Ecological Exposure Research Division |
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| Branch: |
Molecular Ecology Research Branch |
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| Product Type: |
Abstrct/Oral |
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| Presented: |
11/11/2001 |
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