Induction of Winter Flounder Antifreeze Protein Messenger RNA at 4 C in vivo and in vitro

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Abstract
Northern blot hybridization analysis of RNA from fish acclimated to different temperatures revealed that the inducibility of antifreeze protein messenger RNA (AF mRNA) accumulation in winter flounder varies with fish from different seasons. A high accumulation of AF mRNA was observed in fish maintained at 4 C, but not 18 C, during the fall (Oct.-Dec.). This low temperature requirement was specific for AF mRNA, since low temperature was not required for the accumulation of a femalespecific mRNA (mRNA♀). No accumulation of AF mRNA was detected in either cold- or warm-acclimated fish during the spring and summer (Feb.-Aug.), although an effect of temperature on the decline of AF mRNA in females was noted. The synthesis of AF mRNA in vitro was investigated by Northern blot analysis and by hybridization of 32P-labeled RNA from liver slices to AF cDNA. This synthesis in vitro also varied with the season. Liver slices from October and January fish synthesized AF mRNA in vitro at 4 C in preference to 18 C, but no synthesis of AF mRNA was detected at either low or high temperature in liver slices from February and March fish. The results are interpreted in terms of a proposed model for AF gene regulation. AF mRNA synthesis occurs preferentially at low temperature in fish that have been primed by an endogenous clock mechanism. The in vitro evidence suggests that temperature shifts lead progressively to changes in AF mRNA synthesis.