Adams S L, Alwine J C, de Crombrugghe B, Pastan I
J Biol Chem. 1979 Jun 25;254(12):4935-8.
Two recombinant plasmids containing chick collagen DNA sequences have been used to characterize messenger RNAs for pro-alpha1 (type I) and pro-alpha2 collagen. Poly(A)-containing RNA from chick embryo calvaria and long bones, tissues which are very active in collagen synthesis, were electrophoresed on agarose gels containing methylmercuric hydroxide and transferred to diazobenzyloxymethyl paper; these covalently bound RNAs were hybridized to 32P-labeled pro-alpha1 or pro-alpha2 collagen DNA sequences derived from the recombinant plasmids. The pro-alpha1 collagen probe identified two RNAs, a major species of 5000 bases and a minor species of 7100 bases; the pro-alpha2 collagen probe hybridized to a major species very similar in size to the pro-alpha1 mRNA, about 5200 bases, and a minor species of 5700 bases. It is possible that the 7100 and 5700 base RNAs represent precursors of pro-alpha1 and pro-alpha2 collagen mRNA, respectively. When similar hybridization experiments were performed with RNA from chick embryo fibroblasts, both the pro-alpha1 and pro-alpha2 collagen mRNAs were observed, as well as their corresponding larger species. With RNAs from fibroblasts transformed by Rous sarcoma virus, however, the levels of all RNA species which hybridized with the pro-alpha1 and pro-alpha2 collagen DNA probes were significantly reduced.
两种含有鸡胶原蛋白DNA序列的重组质粒已被用于鉴定I型前α1胶原蛋白和前α2胶原蛋白的信使RNA。从鸡胚颅骨和长骨中提取的含poly(A)的RNA，这些组织在胶原蛋白合成中非常活跃，在含有氢氧化甲基汞的琼脂糖凝胶上进行电泳，然后转移到重氮苄氧基甲基纸上；这些共价结合的RNA与来自重组质粒的32P标记的前α1或前α2胶原蛋白DNA序列杂交。前α1胶原蛋白探针鉴定出两种RNA，一种主要的5000个碱基的种类和一种次要的7100个碱基的种类；前α2胶原蛋白探针与一种大小与前α1 mRNA非常相似的主要种类杂交，约5200个碱基，以及一种5700个碱基的次要种类。7100和5700个碱基的RNA可能分别代表前α1和前α2胶原蛋白mRNA的前体。当用鸡胚成纤维细胞的RNA进行类似的杂交实验时，观察到了前α1和前α2胶原蛋白mRNA及其相应的较大种类。然而，在用劳氏肉瘤病毒转化的成纤维细胞的RNA中，与前α1和前α2胶原蛋白DNA探针杂交的所有RNA种类的水平都显著降低。
