Analysis of functienal domain and expression in bone, cartilage and muscle of minor collagen

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1) Initially, to determine the transcription initiation site of mouse α3 (V) gene, Oligo-Cap methods was used. It was located about 100 bp upstream of ATG codon. We used structure-function analysis of the core promoter region to elucidate the transcriptional features of the gene. The core promoter was defined within about 300 bp immediately upstream from the transcription start site by transient transfection experiments. In this region, we identified two nuclear-factor binding sites by electrophoretic mobility shift assay (EMSAs): BS1 (-130 to -110), BS2 (-190 to -170). Oligonucleotide competition and supershift assays revealed that CBF,/NF-Y specifically bind to BS1.The N-terminal globular domain of the pro-α3 (V) chain has a unique structure that contains a highly basic segment of 23 amino acids. The peptide showed a specific adhesive feature to osteosarcoma cells but not to chondrosarcoma cells. This feature was derived from four heparin binding sites in the segment. When the peptid … More e adhere to the cell, actin fiber was formed, which was inhibited Rho-kinase inhibitor, Y27632.2) We have characterized the proximal promoter of the human α1 (III) collagen gene (COL3A1). Transient transfection assays indicated that the segment from -96 to -34 was necessary to activate transcription. EMSAs showed that the multiple proteins form the DNA-protein complex in different combinations depending on the cell types. The region of -79 to -63 bp was critical for DNA protein complex formation. This sequence was contained in the B element of mouse al (III) collagen gene (Col3a1) reported by other researcher.3) Collagen XXIV is a newly discovered and poorly characterized member of the fibril-forming family of collagen molecules, which is expressed predominantly in bone tissue. Cell transfection experiments in combination with DNA binding assay demonstrated that Co124a1 promoter activity was under control of an upstream cis-acting element, which was recognized by specific combination of c jun, CREB1, ATF1, and ATF2 dimers. Less