Isolation and characterization of the antifreeze protein messenger RNA from the winter flounder

The messenger RNA coding for the serum antifreeze protein of the winter flounder (Pseudopleuronectes americanus) was extracted from the liver polysomes of fish caught in November and was purified by oligo(dT)-cellulose chromatography and sucrose density gradient centrifugation. The length of the mRNA was 520 nucleotides measured by agarose gel electrophoresis in the pesence of methyl mercury. It sedimented between trout protamine mRNA (6 S) and rabbit globin globin mRNA (9-10 S) on sucrose density gradients. The purified antifreeze protein mRNA assayed in the mRNA-dependent reticulocyte lysate translation system incorporated 10 times as much alanine as arginine into a single translation product. the molecular weight of the primary translation product was 11,700 estimated by sodium dodecyl sulfate-gel electrophoresis. Antifreeze protein mRNA was the only major mRNA species present in liver polysomes of fish caught in November but was absent in the liver polysomes of fish caught in April. This finding emphasizes the seasonal nature of antifreeze protein production in the winter flounder. A 32P-labeled cDNA prepared from the antifreeze protein mRNA was hybridized against total cellular RNAs from flounder liver. These RNA excess hybridizations showed that antifreeze protein mRNA is several orders of magnitude more abundant in liver in mid-November than in late August. This suggests that transcriptional control is involved in the seasonal expression of the antifreeze protein gene.