Construction of DNA sequences complementary to rat .alpha.1 and .alpha.2 collagen mRNA and their use in studying the regulation of type I collagen synthesis by 1,25-dihydroxyvitamin D

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Abstract
Type I collagen mRNA from fetal rat calvaria was used as a template for the synthesis of a cDNA [complementary DNA] that was subsequently inserted in the PstI site of the plasmic vector pBR322 and cloned. Three recombinant plasmids containing type I collagen specific sequences were characterized: p.alpha.1R1 is 1600 bp [base pairs] and spans .apprx. 500 amino acid residues within the triple helical region of .alpha.1(I) and p.alpha.1R2 is 900 bp in size and covers the entire 3'' noncoding and about half of the C-terminal propeptide region of .alpha.1(I) collagen mRNA. The third recombinant p.alpha.2R2 is 1500 bp and contains .alpha.2(I) sequences specific for the entire 3'' noncoding and C-terminal propeptide region. Partial nucleic acid sequence data revealed that the decreasing order of amino acid and nucleotide homology to similar regions of the rat cDNA was mouse > human > chick. Northern hybridization of mRNA after electrophoresis in 0.8% agarose revealed 2 distinctly different MW patterns characteristic of .alpha.1(I) (4.7 and 5.7 kb) and .alpha.2(I) (4.2 and 4.5 kb) collagen mRNA when hybridized with the corresponding cDNA probe. Despite the high degree of sequence homology, DNA probes from rat or human produced a significantly reduced hybridization signal when used as an interspecies hybridization probe than to its corresponding mRNA. The rat cDNA probes were used in a dot hybridization assay to measure the type I collagen mRNA content in the fetal rat calvaria. The 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] reduced collagen synthesis and type I collagen mRNA levels in osteoblasts located in the central bone segment of calvaria but had no effect on cells in the periosteum. Furthermore, 1,25-(OH)2D3 appeared to regulate the levels of the .alpha.1(I) and .alpha.2(I) collagen mRNA in a coordinated manner.