Regulation of antifreeze polypeptide biosynthesis in the winter flounder (Pseudopleuronectes americanus)

Fourney, Ronald Mitchell (1984) Regulation of antifreeze polypeptide biosynthesis in the winter flounder (Pseudopleuronectes americanus). Doctoral (PhD) thesis, Memorial University of Newfoundland.

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Abstract

The heterogeneity of antifreeze polypeptides (AFP) in the Newfoundland winter flounder Pseudopleuronectes americanus was analyzed by reverse phase high performance liquid chromatography (HPLC) and by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Seven AFP components could be readily, resolved. Five of the components were similar in molecular weight (3,300) and amino acid composition. Two of the AFP components were larger (4,500) and contained valine. The two major components (components 6 and 8) were identical to those reported earlier from our laboratories (Davies et al., Proc. Natl. Acad. Sci., USA 79, 335, 1982). -- Serum AFP were isolated from winter flounder collected from Nova Scotia, New Brunswick and Long Island (New York) and compared to the AFP found in Newfoundland winter flounder. These peptides were analysed by reverse phase HPLC, and showed nearly identical elution profiles to that of Newfoundland AFP. Individual components from New Brunswick and Long Island had antifreeze activity and agreed with the activity measurements previously established for the Newfoundland AFP. Amino acid analyses of the major AFP components from the different flounder populations indicated that they had similar compositions. A minor variation which included valine in one AFP component was noted in the New Brunswick sample suggesting that a limited amount of genetic polymorphism may be present in the winter flounder population from New Brunswick. In general, there appears to be little or no variation in the structure of the AFP from winter flounder inhabiting different geographical habitats. In view of the similarity of the serum AFP components and in light of recent information on the structure and genomic organization of AFP genes, it is likely that the winter flounder AFP components are products of an AFP gene family. -- The seasonal synthesis of AFP in winter flounder is influenced by photoperiod and comes under the control of the pituitary gland. The effects of hypophysectomy (hypex) and long day length on the accumulation of antifreeze messenger RNA (mRNA) in the liver were examined. Hypophysectony resulted in a significant decrease in serum freezing temperature, and increases in liver weights, total liver Poly (A)⁺ RNA and AFP mRNA accumulation. The identity of the AFP mRNA in hypex animals was confirmed by gel electrophoresis, cell-free translation and Northern blot hybridization techniques. Cytoplasmic-dot hybridization analysis indicated that the AFP mRNA level in hypex fish approximated that observed in winter animals actively synthesizing AFP. These experiments utilized a nick-translated CT5, an antifreeze protein cDNA clone. An increase in AFP mRNA was detectable as early as the first day after hypophysectomy and by day 7 reached 25% of the level found in fish actively synthesizing AFP mRNA during the winter months. Since AFP mRNA is found at very low levels in the control flounder, this suggests that its accumulation after hypophysectomy depends on accelerated transcription. The pituitary gland appears to regulate the liver AFP mRNA level by a negative transcriptional control mechanism. -- The effect of photoperiod on the seasonal accumulation of winter flounder AFP mRNA in the liver was examined. Flounder maintained under conditions of 15 h long day length have both a delayed appearance and decreased accumulation of AFP mRNA. AFP mRNAs were identified and their concentrations measured by a cytoplasmic dot-hybridization method and Northern blot hybridization utilizing a nick-translated E₃, an antifreeze genomic clone. December flounder maintained under conditions of long day length demonstrated the most significant decrease in AFP mRNA levels. It was estimated that these fish contained less than 0.6% the AFP mRNA normally found in control December fish. The seasonal fluctuation of AFP mRNA in both experimental and control fish match closely, but preceed, the rise and fall of plasma AFP levels. These results suggest that long day length suppresses the rate of transcription of antifreeze genes, and support the hypothesis that photoperiod may act as the initial cue for entraining the precise activation of AFP synthesis possibly through a pituitary mediator. A model for the regulation of winter flounder AFP gene expression is presented.

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