Honors Theses


Carissa M. Krane, Ph.D.



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Honors Thesis


Cope’s Gray Treefrog, Dryophytes chysoscelis, seasonally tolerates freezing. During this process, it is subject to cellular stress from factors such as metabolic starvation, DNA damage, toxin accumulation (from cellular metabolism), and the risk of protein misfolding. Preliminary transcriptome data indicates that many mRNA transcripts vary in relative abundance within hepatocytes of D. chrysoscelis during discrete periods of warm acclimation, cold acclimation, freezing, and thawing. Physiological changes that occur in this frog are likely the result of epigenetic regulation—an alteration in gene expression that does not influence DNA sequence. This modification in gene expression can be observed by the subsequent change in relative abundance of mRNA transcripts. This thesis investigates the relative abundance of the hepatic mRNA transcripts for (1) NDUF7, a methyltransferase gene that mediates some epigenetic regulation by transferring methyl groups (global regulation) and (2) ACADV and BFAR–genes that mediate stress responses contributing to freeze tolerance (local regulation). All of the genes selected are orthologous counterparts to those found in humans. Based on preliminary transcriptome data, we hypothesized that methyltransferases important to the freezing process will be differentially regulated (either up-regulated or down-regulated) as compared to the warm condition, whereas stress genes that enhance the survivability of the frogs during freezing will be up-regulated and those that are metabolically costly will be downregulated. These results demonstrate trends in mRNA expression within four biological groups (warmacclimated, cold-acclimated, frozen, and freshly thawed) that may be relevant to the freezing process. mRNA was isolated from livers from frogs of the four biological group. cDNA was synthesized and the relative abundance of each transcript was identified using RTqPCR techniques and compared to the abundance of a housekeeper gene Dc LS-14, which served as a control. The results from the experimental groups were analyzed using ΔΔCt logarithm to calculate the fold change. The results showed that all in genes were down-regulated in expression relative to the warm-acclimated control. NDUF7 showed respective fold changes of -5.82 (cold), -4.40 (frozen), and -6.54(thawed). ACADV demonstrated negative fold changes of -3.74, -3.06, and -3.68, while BFAR had fold changes of -1.14, -1.13, and -1.78 respectively. These data indicate that global down-regulation of gene expression at the transcript level may be an important energy conservation mechanism necessary for surviving freezing and thawing in Cope’s gray treefrog.

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