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MRI: Acquisition of Automated DNA Sequencer for Undergraduate Research and Training

A grant has been awarded to Dr. Evans at Lake Superior State University to acquire an automated Genetic Analyzer. The equipment will advance faculty research and undergraduate research training involving DNA sequencing and DNA fingerprinting. The goals of the project are as follows: 1) to discover how photoreceptor opsin gene expression changes during retinal development in a variety of teleost fish species. 2) to characterize the cross-breeding (hybridization) occurring between pink salmon and chinook salmon in the St. Marys River. 3) to determine if specific DNA markers can establish whether Great Lakes sea lamprey return to their natal streams to spawn. 4) to provide research training in DNA sequencing for Biology and Clinical Laboratory Science majors, and to train students majoring in Criminalistics, and Fisheries & Wildlife Management in DNA fingerprinting techniques.<br/>Three faculty projects will incorporate undergraduate research training: 1) Spectral sensitivity in the eye is mainly determined by the opsin proteins in the photoreceptor cells of the retina. In the winter flounder, these opsin proteins change as the animals go through metamorphosis. Before a cell can produce a protein, messenger RNA (mRNA) must be copied from the cell's DNA. At selected developmental stages, fish retinas will be examined for opsin mRNA expression, photoreceptor morphology and cell specific opsin expression. Using "polymerase chain reaction" (PCR), the opsin genes will be amplified and then sequenced using the Genetic Analyzer. Once sequences are identified, mRNA tags will be created to follow the change in photoreceptor opsin as the fish retina transforms from larval to juvenile state. 2) Hybrids between Chinook and pink salmon are frequently encountered in the St Marys River, but apparently these hybrids only result from crossbreeding between Chinook females and pink salmon males. Why the hybrids are occurring this way is unclear. Mitochondria are only inherited from the mother; thus DNA fingerprints created using mitochondrial DNA (mtDNA) will allow the maternal species of the hybrids to be identified. Using the Genetic Analyzer, a database of mtDNA fingerprints will be established for the chinook, pink and "pinook" salmon hybrids to determine if the hybrid crosses are truly restricted. If so, further studies will examine the mechanisms involved. 3) Sea lamprey are a serious parasite on native Great Lakes fishes. Protein (isozyme) analysis has indicated that lamprey return to their natal spawning grounds. The current study will test the utility of using DNA fingerprints of lamprey populations to assess the gene flow between populations. Population genetic surveys commonly create DNA fingerprints using microsatellite DNA, which are unique regions of the DNA. Such methods have been used for many fish populations, but not for lamprey. The Genetic Analyzer will be used create DNA fingerprints for the lamprey using microsatellite DNA markers. The results from these genetic analyses will be compared with the previous isozyme studies to assess their usefulness for monitoring lamprey life cycles. 4) Undergraduate students will be hired as research assistants on these projects. In addition, several courses will incorporate DNA sequencing for Biology and Clinical Laboratory Science majors and DNA fingerprinting technology for Criminalistics, and Fisheries & Wildlife Management majors. Both techniques will be available for students to use in their senior thesis research. <br/>The project contributes to several fields of basic science research. 1) The neural retina of the eye is an accessible part of the central nervous system where structure and function are clearly related. Understanding how the retina is "built" gives insight into how other neural networks develop. The teleost fish retina is a typical vertebrate retina and presents a useful model for understanding normal development (i.e., how cells divide and differentiate into specific tissues). In addition, the change in opsin gene expression in the winter flounder eye may answer questions of how cell-cell interactions determine a cell's fate. 2) In their natural habitat, there are few opportunities for Chinook and pink salmon to crossbreed, but in the St. Marys River, the number of 'pinook' hybrids appear to be increasing. In the laboratory, pink and chinook crosses have been made in both directions suggesting no physiological barriers to hybridization. The presence of restricted crossbreeding in the wild may be a result of behavioral barriers such as territoriality or courtship rituals. 3) Sea lampreys are a major problem in the Great Lakes, and a solution may reside in understanding their life history and spawning behavior. Protein analysis indicates that lamprey do return to the region of their birth to spawn. Use of microsatellite DNA markers to create DNA fingerprints is a method that can be automated, allowing fast, routine analysis of gene flow. A better understanding of lamprey population genetics may allow these populations to be brought under control in the future. 4) Student learning, by active participation, is central at Lake Superior State University, and faculty consider their personal research interests to be a mechanism to stimulate student curiosity. DNA technology is becoming increasingly prominent in our everyday lives. It is important that we not only train students how to perform these techniques for research careers, but also train them to recognize when these methods are being applied and interpreted correctly.

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