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.

Identification and characterization of a novel freezing‐inducible gene, li16, in the wood frog Rana sylvatica

“…Total RNA was isolated from liver samples of spring wood frogs that were frozen for 8, 12, or 24 h at −2.5°C by using TRIzol reagent (GIBCO BRL, Bethesda, MD), and then mRNA was prepared using an oligo(dT) cellulose column (New England Biolabs, Beverly, MA) as described previously (7). Aliquots of 2 μg of mRNA from each freezing treatment were combined, and a cDNA library was constructed with the Uni‐ZAP XR vector cDNA synthesis kit (Stratagene, La Jolla, CA).…”

“…Aliquots of 2 μg of mRNA from each freezing treatment were combined, and a cDNA library was constructed with the Uni‐ZAP XR vector cDNA synthesis kit (Stratagene, La Jolla, CA). Differential screening was performed by using 32 P‐labeled single‐stranded total cDNA probes prepared from poly(A) + RNA isolated from liver of control frogs (5°C acclimated) and frogs frozen for 24 h (at −2.5°C), as described previously (7). Plaques that showed an intense signal with the freeze probe but little or no signal with the control probe were collected, and clones were rescued by in vivo excision and were converted to pBluescript circular double‐stranded plasmid according to the manufacturer's instructions (Stratagene).…”

“…Recent research has shown that changes in gene and protein expression are also integral to freezing survival, including both seasonal changes (e.g., glycogen phosphorylase activity, glucose transporters, and ice nucleating proteins all rise in the autumn) and changes that are stimulated directly by freezing (6). For example, initial screening of a cDNA library prepared from liver of frozen wood frogs revealed four genes that were up‐regulated by freezing: α and γ subunits of fibrinogen (7), the mitochondrial ADP/ATP translocase (8), and a 10‐kDa protein, Fr10 (9).…”

Regulation of theRana sylvaticabrevinin-1SY antimicrobial peptide during development and in dorsal and ventral skin in response to freezing, anoxia, and dehydration

“…Previous studies have shown that skin wound healing, such as regeneration of the integument following unnatural ecdysis due to dehydration, is associated with an induction of AMPs (Nakazawa et al, 2003;Bardan et al, 2004;Ohnuma et al, 2006). Additionally, the increased production of fibrinogen, an acute phase protein involved in wound healing, in the liver of R. sylvatica in response to freezing and dehydration (Cai and Storey, 1997) suggests that there is systemic activation of innate immunity possibly as a result of the production or release of DAMPs in response to environmental stress. Therefore, the production of brevinin-1SY in the skin may result from the systemic activation of the innate immune system.…”

Cold acclimation-induced up-regulation of the ribosomal protein L7 gene in the freeze tolerant wood frog, Rana sylvatica

“…The insert was isolated by restriction enzyme digestion of the plasmid DNA and agarose gel-purified with Geneclean III kit (BIO 101, Inc., La Jolla, CA) and was used for the synthesis of 32 P-labeled DNA probe with the random priming method by a protocol adapted from Sambrook et al (1989). Hybridization of Northern blots, autoradiography and X-ray film development were as described previously (Cai and Storey, 1997a;Wu and Storey, 2005). Band intensities were quantified by densitometry using a Scan Jet3C scanner (HewlettPackard, Palo Alto, CA) and Deskscan II v2.2 software (HewlettPackard) to render an autoradiogram into a form recognizable by Imagequant software v3.22 (Innovative Optical Systems Research) which was then used to quantify relative band intensity.…”