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W3 AM Toxicogenomics in Environmental Studies
(GRI-1116-947239) Identification of differentially expressed genes in Palaemonetes pugio following xenobiotic exposure using Serial Analysis of Gene Expression (SAGE).
Griffitt, R1, Greig, T3, Chandler, G1, Quattro, J2, 3, 1 University of South Carolina, Environmental Health Sciences Department, Columbia, SC, USA3 Center for Coastal Environmental Health and Biomolecular Research, Charleston, SC, USA2 University of South Carolina, Biological Sciences Department, Columbia, SC, USA
ABSTRACT- We used the novel transcriptomics technique, Serial Analysis of Gene Expression (SAGE) to investigate effects of three xenobiotics on the transcriptional state of the estuarine sentinel species, Palaemonetes pugio. Adult male P. pugio were field collected, acclimated to laboratory conditions, and exposed to experimentally derived, sex-specific 96-hour LC50 concentrations of either the phenylpyrazole insecticide fipronil, the cyclodiene insecticide endosulfan, or the metal cadmium as well as an appropriate control exposure. Each exposure consisted of three replicates of 10 adult males each (30 total per treatment). At the termination of the exposure, surviving P. pugio were removed from the test solution and immediately sacrificed for RNA extraction. Equal amounts of RNA from three randomly chosen survivors were pooled for SAGE library construction. A total of four SAGE libraries have been constructed (CC, FP, ES, Cd exposures). The SAGE technique excises 17-bp ′tags′ from a defined location within mRNA molecules and concatenates them. The concatamers are cloned, sequenced, and the individual tags enumerated. Comparison of tag frequencies across treatments identify which genes are differentially expressed, as well as the magnitude of the effect. As an ′Open′ system, SAGE has certain advantages over more traditional ′Closed′approaches such as microarrays, in that SAGE requires no prior sequence knowledge, and no a priori selection of genes. The ′Control′ and ′Fipronil′ libraries were prioritized for sequencing, and initial results confirm the utility of SAGE as an ecotoxicogenomics tool. To date, 8764 tags representing 863 unique genes from the ′CC′ library, and 4844 tags representing 466 unique genes from the ′FP-exposure′ library have been sequenced, of a targeted 10,000 tags per library. Statistical comparison via the Audic-Claverie test between the two libraries identified 21 genes as being differentially expressed following application of the Bonferroni correction, and also estimated the strength of the response. For example, SAGE identified chaperonin subunit 3 as being upregulated (p-value = 0.02 ), and gamma-glutamyltransferase 3 as strongly downregulated (p-value < 0.001) following Fp exposure.
Key words: SAGE, toxicogenomics, biomarker, Palaemonetes pugio
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