Type I collagen is one of a few proteins that is produced constitutively in certain cell types and in a highly regulated manner in others. This grant is designed to understand the molecular basis by which a different cell type can express the identical Alpha1(I) and Alpha2(I) collagen genes in a tissue specific manner. Using osteoblasts in organ culture, primary cultured osteoblasts, osteosarcoma cells and fibroblastic cells, the regulation of type I collagen mRNA will be ascessed using cDNA probes to Alpha1(I) and Alpha2(I) collagen mRNA and dot hybridization. Changes in the cytoplasmic levels of collagen mRNA brought on by PTH, 1,25 dihydroxyvitamin D (1,25(OH)2D3) and insulin will be explained by alteration in the nuclear transcription of the type I collagen genes and the half-life of its cytoplasmic mRNA. In situations where the mRNA change results from a difference in gene transcription, the structure of the promoter region of the gene will be examined. We plan to identify and characterize the 5' flanking region of the Alpha1(I) and Alpha2(I) collagen gene. With these probes subcloned into M13, differences in the domains of DNAse I sensitivity will be contrasted in each cell type. Detailed analysis of the 1,25(OH)2D3 binding site by a filter binding assay and footprinting technique will use purified 1,25(OH)2D3 receptor complex and defined DNA segments from the promoter region. This promoter region will be placed in a gene transfer vector derived from the bovine papilloma virus (BPV). The activity of the collagen promotor will be determined by the production of a marker protein, human metallothionein, that is a part of the BPV vector. The fusion gene will be transferred to the various cell types to determine if the promoter attains the same regulation and conformation as the actual promoter. If the two promoters are similar in the same cell, then the PBV model will be used to map the domain of the promoter or enhancer necessary for cell specific activity and hormone responsiveness. The model has the potential for isolating factors that are associated with the cell specific regulation of the promoter and for reconstituting a regulated in vitro transcription system.
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