Differential regulation of the mitochondrial ADP/ATP translocase gene in wood frogs under freezing stress

The gene Aat coding for ADP/ATP translocase (AAT) was cloned from liver of the freeze-tolerant wood frog, Rana sylvatica, via differential screening of a cDNA library from liver of frozen frogs and using probes from control versus frozen frogs. Sequence analysis showed that clone pBfFR07 bearing the AAT cDNA contained a 1318-bp insert with one full length open reading frame. The deduced amino acid sequence included 317 residues, with 81-86% identities to mammalian AAT. A 1750-nt transcript from the Aat gene was detected using pBfFR07 probe and a putative frog AAT of over 30 kDa was visualized by immunoblotting using a polyclonal antibody raised against chicken AAT. Analysis of liver samples from a time course of freezing showed a maximal 4.5-fold increase in mRNA after 8 h with AAT protein peaking in 24-h frozen frogs. Freezing also induced Aat expression in bladder and lung. In liver, mRNA expression also responded positively to anoxia stress but not to experimental dehydration of the animals. These results suggest that AAT induction during freezing may be stimulated by the ischemia that develops when plasma freezes; changes in AAT may contribute to stabilizing energetics in mitochondrial versus cytosolic pools over freeze/thaw cycles