Shaobo Wu, Ph.D. Biology 1999

Differential gene expression under environmental stress in the freeze tolerant wood frog, Rana sylvatica



Freeze survival of wood frog, Rana sylvatica, involves adaptations including control over extracellular ice formation, production of glucose cryoprotectant, and resistance to freezing-caused intracellular dehydration and ischemia. Gene expression associated with stress survival was investigated in this freeze tolerant species. Freeze-inducible genes were found by differential screening of frog brain and liver cDNA libraries; these included mitochondrial genes [encoding ATPase subunit 6 & 8, 16S rRNA and NADH-ubiquinone oxidoreductase subunit 4 (ND4)], the phosphoglycerate kinase 1 (PGK1) gene, and genes whose products are involved in translational processes [acidic ribosomal phosphoprotein (P0), elongation factor 1 gamma subunit (EF-1 gamma)]. Another ribosome-associated gene, encoding ribosomal protein L7 (RPL7), was identified in skin via differential display of polymerase chain reaction (DD-PCR). This gene was up-regulated in skin of cold-acclimated frogs and brain of freeze-exposed frogs. Freezing stimulated the upregulation of the above genes in selected frog organs. Tissue-specific gene expression also occurred in frog brain and liver in response to anoxia or dehydration. Anoxia stimulated P0, PGK1, RPL7, 16S rRNA and ATPase 6 & 8 gene expression and modulated ND4and EF-l gamma expression, whereas dehydration enhanced the expression of genes such as PGK1.Upregulation of genes whose products are directly involved in energy generation (PGK1, ATPase subunit 6 & 8, ND4) and whose products are related to protein biosynthesis suggested that maintenance of minimal ATP levels and functional translation machinery may be critical for freezing survival. Freezing-induced ischemia may be a primary signal that triggers the upregulation of most of the isolated genes. However, low temperature seemed to play a role in the expression of ribosome associated protein genes, whereas freezing-related cell water stress may also regulate other selected genes (e.g. PGK1).Immunoblotting confirmed that elevated PGK1 transcripts resulted in increased enzyme protein and showed the potential physiological significance of up-regulated genes in response to stress. Immunoblotting also showed the elevation of Ca2+/ca1modulin-dependent protein kinase and phosphatase under freezing, anoxia and/or dehydration stresses which suggests that a Ca2~signaling pathway plays a role in stress-mediated gene expression. These cellular responses may play an important role in survival of environmental stress.