ABSTRACTCHARACTERIZATION OF TWO LARGE GENE FAMILIES IN THE SEA LAMPREYBySteven ChangThis dissertation employed molecular biology and bioinformatics to examine two large gene families in the sea lamprey, Petromyzon marinus. An integrative approach was used to define these gene families in order to ensure the validity of the size and members of each gene family. There are two chapters: Chapter 1 examines chemosensory gene expression in a specialized part of the olfactory system and Chapter 2 studies the expression of detoxification genes in the liver and gills in response to the lampricide, 3-trifluoromethyl-4-nitrophenol (TFM).CHEMORECEPTOR GENESFor this dissertation, I will restrict chemoreception to the detection of chemical signals in the nose (note: chemoreception includes taste), and is accomplished by detection of odorants in the environment by specialized sensory cells in the main olfactory epithelium (MOE). In certain tetrapods, a second sensory epithelium is also found in the nose, called the vomeronasal organ (VNO). Canonically, each epithelium represents the start of different olfactory pathways, which govern different behavioral responses. Each epithelium expresses different classes of chemoreceptor (CR) genes; the MOE expresses odorant receptors (ORs) and trace amine-associated receptors (TAARs), while the VNO expresses ORs, vomeronasal type-1 and type-2 receptors (V1Rs and V2Rs). The sea lamprey olfactory organ has one nostril and so has one nasal capsule, which is divided into two spatially distinct regions: the main olfactory epithelium (MOE) and the accessory olfactory organ (AOO). The MOE has been well studied in lampreys but the function of the AOO has eluded description for over 100 years. Based on other research and due to its proximity to the MOE, we hypothesized that the AOO represents an ancestral VNO. If this AOO is indeed an ancestral VNO, we expect a different connectivity to the central nervous system than from the MOE, and would expect expression of pheromone receptors (V1Rs and V2Rs). CR expression in the MOE and AOO of sea lamprey were examined. The differential expression of CR genes between the two epithelia was determined and the connectivity of the main and accessory epithelia was determined using neural tract tracing. Quantitative PCR confirmed and quantified the differential expression of specific genes in the main and accessory olfactory epithelia.CYTOCHROME P450 GENESThe second gene family to be explored is the cytochrome P450 family. P450 genes encode for steroidogenic or detoxification enzymes that are inducible by a substrate. As part of the strategy for controlling sea lamprey populations TFM is applied to streams. Very little is known at the molecular level of how TFM works to kill sea lamprey larvae, but based on responses by other organisms to xenobiotic substances, our hypothesis is that P450 genes are induced by exposure to TFM. P450 genes were predicted from the sea lamprey genome and larvae were exposed to TFM and gill and liver tissues were harvested over an 8-hour time course. Expression was confirmed using high-throughput sequencing and quantitative PCR. The immediate goal was to determine which P450 genes are induced by exposure to TFM. Alternatively, we generated a list of predicted Phase II detoxification enzymes in the event that P450 genes showed no difference in expression. The long-term goal is to use that knowledge to design more efficient and specific lampricides.
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