Graduate Thesis Or Dissertation
 

Stress induced differential gene expression in the brain of juvenile steelhead trout, (Oncorhynchus Mykiss)

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  • Gene expression profiles of tissues and cell-lines can be powerful tools for documenting the genetic response to a particular treatment, such as stressors. However, there is a paucity of information on the genetic stress response in the brain. Therefore, we attempted to profile gene expression in the brain of juvenile steelhead trout (Oncorhynchus mykiss) in response to stressors commonly encountered in aquaculture settings and similar to those encountered in hydropower dam mitigation efforts. We subjected fish to a combined out-of-water and low-water stressor totaling three hours. Plasma stress response factors indicate that fish were undergoing a physiological stress response after 3 hours of continuous stressor. We utilized suppression subtractive hybridization to identify cDNA fragments up- or down-regulated in the brain upon completion of the stressor. Forward and reverse subtractions, and sub-cloning of the purified PCR products yielded 59 clones all of which were sequenced. Sequenced cDNA fragments were subjected to BLASTn and BLASTx searches over the course of one year. Fragments fell into the following functional categories: those associated with ATP generation, signal transduction, ion transport, translational machinery, DNA packaging and mobilization, cell structure, and cDNA fragments with cryptic function. Of the 59, 12 were selected for further analysis, and 5 were confirmed to be differentially expressed by northern hybridization. The differentially expressed genes included cytochrome b, NADH dehydrogenase subunit 2, ATPsynthase subunit 6, a cDNA fragment with unknown function, and neuron specific gene 1. Our results present a first attempt to profile gene expression in the brain of fish and demonstrate the power of molecular tools at capturing large amounts of biological information without having to target any one particular gene. A gene expression profile of the brain consequent to stress provides a catalog of responses at a given time point. This catalog can then be used to isolate full-length cDNAs, localize mRNAs in the brain or other tissue, as probes to determine expression patterns and time courses of gene expression in other tissues, and for the quantification of cDNA molecules with real time PCR.
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