J. Dayre McNally, Ph.D. Chemistry 2002.

Response to freeze exposure by the wood frog, Rana sylvatica: investigating the freeze induced changes to transcription and translation patterns in heart and liver

In recent years changes to gene and protein expression have been shown to underlie animal adaptation to environmental stresses. Similarly, recent studies have demonstrated significant changes to transcription and translation in wood frog tissues during freeze and thawing. The primary goal of this thesis was to further investigate these changes through the application of cDNA library screening and 2-D PAGE technologies. In total 7 additional genes and one protein were identified, significantly extending the list of known freeze up-regulated genes and proteins. From this expanded list the contractile protein, MLC2; two identifiable heart transcripts (hsp27 and a nac); and the novel liver transcripts (li16 and ddx8) were selected for further investigation. The results implicate the heat shock and hypertrophic gene responses as part of the cellular response to freezing, and reveal that the wood frog heart responds to freezing with changes expected to alter AP-1 mediated gene transcription. Moreover, all of the identified cardiac responses, including specific increases in gene/protein levels and changes to protein phosphorylation states could be linked to the p38 MAPK pathway, confirming that it is the major signaling pathway activated in the wood frog heart following freezing exposure. The two novel liver clones were similarly investigated and studies were carried out to obtain full open reading frames, confirm the protein coding capacity and determine the factors regulating their expression. Although the cellular functions of the novel proteins remain a mystery the results obtained suggest distinctive cellular roles, and on the whole provide further evidence that novel gene development is an important component of acquired freeze tolerance. Secondary to the determination of the specific changes in gene and protein expression patterns following freeze exposure the impact of freezing on the overall rates of transcription and translation in wood frog heart tissue were evaluated. The data from the nuclear run-on assays and western blots analysis (phosphorylation status of eIF2a ), confirmed the presumed suppression of transcription and translation in the wood frog heart during freezing. This discovery highlights the importance of the aforementioned genes and proteins, as their up-regulation takes place against a background of suppressed macromolecular synthesis.