FREEZING-INDUCED GENES IN WOOD FROG (RANA-SYLVATICA) - FIBRINOGEN UP-REGULATION BY FREEZING AND DEHYDRATION

Differential screening of a cDNA library produced from liver of the fr eeze-tolerant wood frog, Rana sylvatica, was used to search for freezi ng-induced genes. Five freezing-responsive cDNA clones representing di fferent genes were isolated when similar to 80,000 plaques of a cDNA l ibrary, prepared from liver of frozen frogs (24 h at -2.5 degrees C), were screened with P-32-labeled total cDNA probes from control(5 degre es C) versus freezing-exposed frogs. Two clones, pBfFR45 and pBfFR04, are reported here in detail and were found to be homologous with the g enes for the alpha- and gamma-subunits of fibrinogen, respectively. Th e clone pBfFR45 carried a 2,305-bp cDNA sequence that was of bipartite structure, containing two open reading frames (ORFs). The first ORF p otentially encoded a 332-residue polypeptide, covering a partial seque nce of the NH2-terminal region of the alpha-chain. The second ORF enco ded a 247-amino acid sequence, covering the whole COOH-terminal region of the alpha-chain; this was highly homologous to the FASORF (fibrino gen-alpha second ORF) of chicken alpha-fibrinogen and the extended alp ha-chain of the human protein. Under control (5 degrees C) conditions, moderate levels of fibrinogen alpha- and gamma-transcripts were exclu sively found in liver. When frogs were given survivable freezing expos ures, levels of these transcripts in liver were highly induced. Transc ription of these genes was also elevated in gut and lung during freezi ng, but mRNA levels in these tissues were lower than in liver. A time course assay confirmed that the transcript levels of both alpha- and g amma-subunit genes were dramatically elevated within the early hours o f freezing and reached a maximum threefold increase over control level s after 8 h of freezing exposure. Two other physiological stresses, wh ole body dehydration and anoxia exposure, mimic individual elements of freezing stress in wood frogs. Northern blot hybridization analysis s howed that:the expression of both the alpha- and gamma-genes was also upregulated in response to dehydration in vivo (20% of total body wate r lost), but both were completely inhibited by anoxia exposure.