CONSTRUCTION OF DNA-SEQUENCES COMPLEMENTARY TO RAT ALPHA-1 AND ALPHA-2 COLLAGEN MESSENGER-RNA AND THEIR USE IN STUDYING THE REGULATION OF TYPE-I COLLAGEN-SYNTHESIS BY 1,25-DIHYDROXYVITAMIN-D
CONSTRUCTION OF DNA-SEQUENCES COMPLEMENTARY TO RAT ALPHA-1 AND ALPHA-2 COLLAGEN MESSENGER-RNA AND THEIR USE IN STUDYING THE REGULATION OF TYPE-I COLLAGEN-SYNTHESIS BY 1,25-DIHYDROXYVITAMIN-D
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DOI：
10.1021/bi00320a049
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发表时间：
1984-01-01
期刊：
影响因子：
2.9
通讯作者：
KREAM, B
中科院分区：
文献类型：
--
作者：
GENOVESE, C;ROWE, D;KREAM, B
将来自胎鼠颅骨的I型胶原mRNA用作合成cDNA [互补DNA]的模板，随后将其插入质粒载体pBR 322的Pst I位点并克隆。对含有I型胶原特异性序列的三种重组质粒进行了表征：p α 1 R1为1600 bp [碱基对]，跨度约为1000 bp。α 1（I）和p α 1 R2的三螺旋区内的500个氨基酸残基大小为900 bp，覆盖了α 1（I）胶原mRNA的整个3 ″非编码区和约一半的C-末端前肽区。第三个重组体p α 2 R2为1500 bp，含有对整个3 ″非编码和C-末端前肽区特异的α 2（I）序列。部分核酸序列分析表明，与大鼠cDNA相似区域的氨基酸和核苷酸同源性从高到低依次为小鼠>人>鸡。在0.8%琼脂糖中电泳后的mRNA的北方杂交揭示了当与相应的cDNA探针杂交时，α 1（I）（4.7和5.7 kb）和α 2（I）（4.2和4.5 kb）胶原mRNA的两种明显不同的MW模式特征。尽管高度的序列同源性，从大鼠或人类的DNA探针产生了显着降低杂交信号时，用作种间杂交探针比其相应的mRNA。用大鼠cDNA探针进行斑点杂交，测定胎鼠颅骨I型胶原mRNA的含量。1，25-二羟维生素D3 [1，25-（OH）2D 3]降低了位于颅骨中央骨段的成骨细胞的胶原合成和I型胶原mRNA水平，但对骨膜中的细胞没有影响。此外，1，25-（OH）2D 3似乎以协调的方式调节α 1（I）和α 2（I）胶原mRNA的水平。
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.