Freezing-induced genes in wood frog (Rana sylvatica): fibrinogen upregulation by freezing and dehydration

Abstract

Differential screening of a cDNA library produced from liver of the freeze-tolerant wood frog, Rana sylvatica, was used to search for freezing-induced genes. Five freezing-responsive cDNA clones representing different genes were isolated when approximately 80,000 plaques of a cDNA library, prepared from liver of frozen frogs (24 h at -2.5 degrees C), were screened with 32P-labeled total cDNA probes from control (5 degrees C) versus freezing-exposed frogs. Two clones, pBfFR45 and pBfFR04, are reported here in detail and were found to be homologous with the genes for the alpha- and gamma-subunits of fibrinogen, respectively. The clone pBfFR45 carried a 2,305-hp cDNA sequence that was of bipartite structure, containing two open reading frames (ORFs). The first ORF potentially encoded a 332-residue polypeptide, covering a partial sequence of the NH2-terminal region of the alpha-chain. The second ORF encoded a 247-amino acid sequence, covering the whole COOH-terminal region of the alpha-chain; this was highly homologous to the FASORF (fibrinogen-alpha second ORF) of chicken alpha-fibrinogen and the extended alpha-chain of the human protein. Under control (5 degrees C) conditions, moderate levels of fibrinogen alpha- and gamma-transcripts were exclusively found in liver. When frogs were given survivable freezing exposures, levels of these transcripts in liver were highly induced. Transcription of these genes was also elevated in gut and lung during freezing, but mRNA levels in these tissues were lower than in liver. A time course assay confirmed that the transcript levels of both alpha- and gamma-subunit genes were dramatically elevated within the early hours of freezing and reached a maximum threefold increase over control levels after 8 h of freezing exposure. Two other physiological stresses, whole body dehydration and anoxia exposure, mimic individual elements of freezing stress in wood frogs. Northern blot hybridization analysis showed that the expression of both the alpha- and gamma-genes was also upregulated in response to dehydration in vivo (20% of total body water lost), but both were completely inhibited by anoxia exposure.