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Show THE UNIVERSITY OF UTAH RESEARCH POSTERS ON THE HILL 2007 Functional Characterization of a Highly Conserved RNA Structure Bryndon Hatch and Michael Howard Department of Human Genetics I am currently performing research under the direction of Dr. Howard to show that a predicted RNA structure within Sps2 mRNA has an important function in the redefinition of the UGA stop codon to encode the Selenocysteine amino acid during the translation of selenoproteins. These proteins perform important enzymatic oxidation-reduction reactions in the body and mutations in the genes that encode for these proteins have been linked with myopathies such as Rigid Spine Muscular Dystrophy and Multiminicore Disease. I am still working on the project which I began at the beginning of the schoolyear. Work has been slow and tedious, as results have not been as expected. Due to a poor yield of results from the work done until the beginning of the summer semester, it is necessary to redo the experiment in order to achieve better results. I designed mutations in the Sps2 gene which would test the functionality of various parts of the predicted stem loop structure. The mutations will be produced by performing multi-stage PCR with primers containing the mutation sequences and the Sel D 0 template. The PCR products will be purified, cut with restriction enzymes, inserted into p2luc vectors. The p2luc vectors contain two different luciferases, which produce enzymes that emit light when translation occurs. The vectors will then cloned by transfecting them into E. coli. A PCR screen will be done to select for the colonies containing vectors with the desired inserts. Sequencing will be performed on the vectors from the selected clones to test if the vectors contained inserts with the correct DNA sequence. After the correct clones are made, the functionality of the structure in Sps2 will be tested by performing translation of the clones in cultured mammalian cells and then measuring the activity ratios of the two luciferase enzymes by use of a luminometer. The results are expected to show that the mutations in the Sps2 stem loop structure decrease the levels of selenocysteine insertion. The reduced insertion levels will be indicative of the involvement of the mRNA structure in the redefinition of the UGA stop codon to cause selenocysteine insertion. Further tests will be able to identify specific sequences within the structure that are especially important that may indicate sites where accessory proteins bind in order to instruct the ribo-some to redefine the UGA codon to insert Selenocysteine instead of terminating translation. This research is supported by funding from The Muscular Dystrophy Association and The National Institute of Health grant NS051792 (MTH). Bryndon Hatch is supported by funding from The University of Utah, Undergraduate Research Opportunities Program. 92 |
