Differential gene expression in response to sub-zero temperatures in the freeze tolerant wood frog, Rana sylvatica
Exposure to subzero freezing temperatures causes a variety of biochemical changes in the freeze-tolerant wood frog, Rana sylvatica. Cellular responses to freezing commence a mere five minutes after the initial formation of epidermal ice. To obtain a general overview of differential protein expression in response to freezing, in vitro translation of mRNA isolated from tissues (liver, brain, heart, muscle, kidney and gut) was conducted and compared for control (5°C), frozen (24 hours at -2.5°C) and recovered (24 hour thaw at 5°C) wood frogs; 35S-labeled proteins were separated by sodium dodecyl sulphate polyacrylamide gel electrophoresis and located by fluorography. Results showed changes in the translatable mRNA population of freezing exposed wood frogs. Differential protein expression was greatest in the comparison of liver from control-versus frozen frogs but other tissues each showed one or two differentially expressed proteins in either the frozen or thawed states. Subsequently, isolation of a specific cDNA clone, the R. sylvatica liver glucose transporter (glut2), was attempted via heterologous probing of the liver cDNA library from frozen frogs with a human glut2 cDNA fragment. Potential clones were chosen from a primary screening and further purification was achieved with secondary and tertiary screens. Analysis of the partial nucleic acid sequence of fifteen potential positive clones using the GenBank database did not reveal the presence of the wood frog glut2 cDNA. In another effort to find a wood frog glut2, TA cloning, a PCR based method, was attempted. A PCR product was amplified using a variety of degenerate primers and one product was cloned into a T-vector. The insert of the resulting recombinant plasmid was sequenced and comparison of this with the GenBank database did not reveal its identity as the liver glucose transporter.