Molecular mechanisms controlling type I collagen gene expression are highly complex in that, besides the cell-specific expression of basal levels, this gene is controlled by calcitropic hormones (1,25- dihydroxyvitamin D3 and parathyroid hormone) in some cell types but not others. The proposed experiments are based on the preliminary data from the laboratories of applicant's sponsor and advisor, indicating that 1,25-D inhibits type I collagen synthesis in bone cells at the level of transcription. The model system proposed in this study includes rat osteosarcoma (ROS 17/2.8) cells transfected either transiently with chimeric plasmids containing marker gene, or permanently with bovine papilloma virus (BPV), both carrying cloned alpha-1(I) regulatory genomic sequences. Since preliminary data showed that the -3.6 kb alpha-1(I) genomic fragment induced high basal expression of marker gene and 1,25- D-dependent inhibition of the collagen promoter, the first specific aim of these experiments is to determine whether the recently cloned alpha-1(I) first intron sequences have an additive efect on basal expression and/or 1,25-D-dependent regulation of the 3.6 kb fragment. Furthermore, deletion analysis will be used to identify the smallest fragments (cis-active elements) within the -3.6 kb and the first intron sequences responsible for strong basal expression and inhibition by 1,25-D. This cis-active fragments will also be joined to a constitutive promoter, such as thymidine kinase (TK), in order to demonstrate their role in conferring high basal activity and hormone responsivity. Finally, trans-acting nuclear factors from untreated and 1,25-D-treated ROS 17/2.8 cells that bind to a specific DNA sequences and presumably regulate alpha-1(I) promoter, will be detected both in vitro, using gel retardation and exonuclease protection assays in crude nuclear extracts, and in vivo, using BPV vector and exonuclease protection assay in stable transfection experiments.
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