Upregulation of a novel gene by freezing exposure in the freeze-tolerant wood frog (Rana sylvatica).

A novel gene responsive to freezing exposure was identified among five cDNA clones obtained through differential screening of a cDNA library constructed from liver of frozen wood frogs. The cDNA sequence of this gene, cloned in the recombinant plasmid, pBfFR14, showed no homology to any genes available in the Genbank database. The clone, designated as Fr10, carried a 457 bp cDNA sequence and contained a single open reading frame that could potentially encode a small protein of 90 amino acids with a molecular weight of about 10 kDa, named FR10. The putative protein contained a highly hydrophobic N-terminal region (21 residues) that carries a potential nuclear exporting signal (NES) sequence, LALVVLVIAISGL, similar to the NES found in PKI, an inhibitor of protein kinase A (PKA). A single mRNA transcript with a size of 550 nt was detected when the insert of the pBfFR14 was used as a probe against the Northern blot containing total RNA isolated from wood frog organs. RNA blotting analysis for gene expression in eight organs showed that transcription of the gene was highly induced by 24 h of freezing exposure at -2.5 degrees C in liver and gut, moderately elevated in heart, lung, brain and bladder but showed no change in skeletal muscle and decreased in kidney. A time-course analysis for freezing regulation of gene expression in liver showed that transcript levels were increased by 2-fold in 1 h of freezing exposure and the levels continued to increase up to 3.5-fold over the control after 24 h of freezing exposure, but had returned to control levels after 24 h thawing at 5 degrees C. Gene expression in liver was also up-regulated by whole animal dehydration at 5 degrees C but strongly down-regulated by anoxia exposure, indicating that the gene may respond to cell volume regulatory signals in vivo during natural freezing.