Attempts will be made to express our 1,25-(OH)2D3 receptor cDNA using expression vectors. Quantities of 1,25-(OH)2D3 receptor will be generated, purified, and used to study 3-dimensional structure. Continued work on the calcium binding protein and osteocalcin genes using a reporter gene system will be emphasized to deduce vitamin D-responsive elements. By co-transfecting cDNA of receptor and reporter gene system with a D-responsive element, necessary components of the responsive element and the 1,25-(OH)2D3 receptor can be deduced. Using differential hybridization, the vitamin D-responsive genes in intestine, bone, and HL-60 cells will be identified. Cloning of the corresponding cDNA and their genes will facilitate searching for D-responsive elements. The possible presence of D-responsive elements in the oncogenes and their relationship to the mechanism of action of 1,25-(OH)2D3 in HL-60 will be examined. Homologous regulation of 1,25-(OH)2D3 receptor will be pursued by completing the cloning experiments of the 1,25-(OH)2D3 receptor gene and searching for a responsive element. Work will continue on the role of vitamin D and reproduction. Synthesis of analogs of 1,25-(OH)2D3 that possess either special, singular or antagonistic activity in one or more systems will continue. Attempts will be made to verify the expression of retinoic acid receptors in tissues and cells responsive to vitamin A. Differential hybridization will be used to locate retinoic acid responsive genes, and attempts to identify retinoic acid responsive elements made. Retinoylation of proteins through retinoyl Co-A will be examined as a possible mechanism of gene regulation. A possible role of retinoic acid in stimulating NGF or NGF receptor or other regulatory genes in neuroblastoma and chick dorsal root ganglia will be examined to begin investigation of the role of the retinoids in development of neural tissue. An attempt will be made to determine the role of vitamin A in control of B-lymphocyte activity by determining if vitamin A deficiency prevents lipid 4A or surface IGM-mediated stimulation of B-lymphocyte growth and differentiation. The role of vitamin A compounds in the responsible signal transduction systems involved will be studied. Vitamin K-dependent carboxylase will be purified to homogeneity and studied for mechanism and specificity. A vitamin K-epoxide reductase system will be investigated and the cell biology of prothrombin biosynthesis will be studied.
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