RESEARCH PROGRAMS

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1. Antifreeze Proteins. The antifreeze proteins (AFPs) are a group of proteins that have the unique property of inhibiting ice crystal formation and ice recrystallization (Fig. 1). These proteins are essential for the survival of many organisms living in icy environment. These proteins are therefore important and interesting models to study protein-ice interaction, biochemical adaptation and gene expression by environmental factors.

Protein structure and function. We are the first group to report the isolation and the primary structure of the different fish AFPs including the winter flounder, shorthorn sculpin, ocean pout and sea raven and their subsequent cDNA cloning (for detail references, please see Yang and Hew, European J. Biochemistry, vol 203, 33-42,1992). These studies established their structural diversity and allowed us to classify them into different types of AFPs. We are the first to report the three dimensional structure of a type I AFP (Fig. 2) from winter flounder using X ray crystallography (Yang et al., Nature 333, 232-237, 1988) and the first to carry out extensive structure-function study using peptide synthesis and site directed mutagenesis. The x ray structure of the type III ocean pout AFP (Fig. 4) has also been resolved and a model of a type II herring AFP (Fig. 3) was constructed based on its homology with the C type lectins. The availability of these models will allow us to carry extensive mutagenesis study to examine the structure and function of these proteins. More recently, our laboratories also discovered a novel, intracellular AFP from the winter flounder and shorthorn sculpin. These new AFPs are different from other secretory AFPs reported earlier in that they lacked signal peptides and are intracellularly localized. The occurrence of both the intracellular and extracellular AFPs has raised many interesting questions on the relative role of those proteins in freeze protection, their differential regulation and evolution. These questions are now being actively pursued in the laboratory. Because of our work, many laboratories are now actively pursuing the application of these proteins in the food and dairy industry, cryopreservation of cells and tissues, cold storage of human platelet cells and the development of cold/freeze resistant transgenic plans and animals in addition to basic research.

Control of gene expression. Using the winter flounder as a model, we are the first to examine the role of the environmental and endocrine factors in controlling AFP gene expression on a seasonal level. We have demonstrated that growth hormone (GH) inhibits AFP gene expression. On a molecular level, we further show that the intron of the AFP gene contains a liver-specific enhancer. The role of an enhancer element and some of the cis acting elements mediating AFP gene transcription were mapped (Chan et al., European J. Biochem, vol 247, 44-51,1997). Two transcription factors, C/EBP and a presumptive Antifreeze Enhancer Binding Protein (AEP) bind to the enhancer sequence and activate AFP gene expression. A working model (Fig. 5) illustrating the control mechanisms for regulating flounder AFP gene expression is being tested.

2. Molecular endocrinology of fish pituitary hormones. The pituitary gland contains several polypeptide hormones important in somatic growth, reproduction, homeostasis, lactation, adaptation and other functions. We are one of the first groups to take a molecular approach to the studies of comparative endocrinology. We were the first to clone the fish gonadotropin (GTH), prolactin and several fish transcription factors important to pituitary functions which include pit-1, Isl-1and steroidogenic factor-1 (SF-1). More recently we are first to demonstrate that the synergistic interaction of steroidogenic factor-1 and estrogen receptor are sufficient to confer the tissue and cell type specific expression of the GTHII gene (Le Drean et al., Molecular Endocrinology, vol 10, 217-229,1996). A model (Fig. 6) showing the interaction of these factors in activating GTHII gene promoter is included. We are now focusing on the structural features important in protein-protein interaction and the functional domains important for transcriptional regulation.

3. Transgenic fish. We are the first group to explore the use of transgenic fish in basic research and biotech applications. We invented the microinjection technique via the micropyle and the development of "All Fish" gene constructs for the use in aquaculture. We have successfully produced transgenic salmon harboring the antifreeze protein gene (for freeze resistance), growth hormone (for growth enhancement) and lysozyme (for disease resistance). Our growth hormone-transgenic salmon was the first to exhibit 5 to 10 fold enhanced growth rate (Du et al., BioTechnology, vol 10(2), 176-181, 1992, Super salmon (Fig. 7) and was also the first transgenic fish issued by the U.S patent office (1996). Because of our work, a biotech. company (A/F Protein Inc.) is actively marketing the technology and the transgenic salmon worldwide. Presently, we are focusing methodology improvement for the gene transfer methods and the development of other commercially important fish species.

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Sept. 2000