During the late fall and winter, the winter flounder produces a family of unique antifreeze proteins (AFP) to prevent the lethal formation of ice crystals in its blood. Winter flounder possess the ability to induce AFP mRNA synthesis in vitro by lowering the ambient temperature of the fish from 18 degrees centigrade in the summer months (when AFP synthesis is at a minimum) to 4 degrees centigrade. More importantly, this cold induction of AFP mRNA synthesis has been investigated by the PI in vitro in isolated liver tissue and in nuclear preparations isolated from liver tissue. In addition, the PI has sequenced a cDNA clone of an AFP mRNA and with it, demonstrated a family of AFP genes in the winter flounder genome. The cloned AFP genes have been transferred to a variety of fish cell lines. Expression of AFP genes in transgenic cells has been established. Future studies will be focused on generation of cyprinid loach transgenic fish with cloned AF gene by several transfection methods including electroporation. Tissue-specific expression of the AF genes in offspring of transgenic fish following various acclimations will be extensively studied. Basic studies such as these are of the greatest importance to our facing global problems of biodiversity. They investigate the survival of species under less then maximal conditions.//
