J.N. Amritha De Croos, Ph.D. Biology, 2003

Gene and protein regulation in liver of the freeze tolerant wood frog, Rana sylvatica.



The wood frog Rana sylvaticahas the ability to tolerate whole body freezing in nature. Biochemical mechanisms supporting freezing survival have been widely studied in this species, but much remains to be explored. The present research demonstrates the involvement of specific gene and protein level changes in the response to freezing by liver. Screening of a cDNA library prepared from liver of R. sylvatica identified a freeze-responsive clone containing a 1370 nt sequence with an open reading frame of 360 amino acids. Sequence analysis revealed 84-86% identity with the mammalian inorganic phosphate carrier (PiC) that spans the inner mitochondrial membrane. Northern blot analysis showed that pic transcript levels increased over time during freezing, reaching >70-fold up-regulation after 24 hours frozen. Transcript levels were also assessed under freezing-related stresses with results showing a strong increase inpic transcript levels during dehydration (9.0-fold in 40% dehydrated frogs) but not under anoxia. Western blotting revealed elevated PiC protein over a time course of freeze-thaw whereas other mitochondrial carriers of the same family were not affected. Southern blot analysis showed that the increase in PiC was not due to an increase in mitochondrial numbers. Analysis of polysome profiles revealed disaggregation of polysomes into translationally less active monosomes upon freezing. PiC transcripts segregated into monosomes in control liver, but would aggregate into actively transcribing polysomes during freezing. Western blot analysis showed that the activity of specific transcription factors in frogs is temporally controlled. The transcription factors CREB and NFk B were significantly activated after two hours of freezing exposure whereas protein kinase B (Akt) was activated after 24 hours of freezing. Screening of a cDNA microarray identified an anti-apoptotic factor that is up-regulated during freezing in liver. Western blot analysis showed time dependent changes in anti-apoptotic activity mitigated by significant changes in AP-2a , BAX-inhibitor and p53 protein levels. These results suggest that freeze tolerance in R. sylvatica involves control of gene and protein levels through the up-regulation of selected transcripts and the activation of kinases and transcription factors. This gene and protein control, or regulation, combined with specific anti-apoptotic actions, allows hepatocyte survival of the multiple stresses imposed by freezing.