Sobel M E, Yamamoto T, Adams S L, DiLauro R, Avvedimento V E, de Crombrugghe B, Pastan I
Proc Natl Acad Sci U S A. 1978 Dec;75(12):5846-50. doi: 10.1073/pnas.75.12.5846.
A recombinant plasmid containing chick pro-alpha2 collagen gene sequences has been constructed and cloned in Escherichia coli. Using partially purified collagen mRNA as template, we synthesized double-stranded DNA by the successive action of reverse transcriptase (RNA-directed DNA nucleotidyltransferase) from avian myeloblastosis virus and the Klenow A fragment of E. coli DNA polymerase I. From this complex mixture of double-stranded DNAs, a specific 200-base-pair restriction fragment was generated by cleavage with the restriction endonucleases BamHI and EcoRI. These enzymes also make unique cuts in the plasmid vector pBR322. The restriction fragment was inserted into pBR322 via these BamHI and EcoRI sites and cloned in E. coli chi1776. The cloned recombinant plasmid was shown to contain pro-alpha2 collagen DNA by its specific hybridization to chick pro-alpha2 collagen mRNA, as assayed in an in vitro translation system. Thus, a clone containing pro-alpha2 collagen DNA was constructed without first obtaining highly purified collagen mRNA.
一种含有鸡原α2胶原蛋白基因序列的重组质粒已构建并克隆于大肠杆菌中。以部分纯化的胶原蛋白mRNA为模板，我们通过禽成髓细胞瘤病毒的逆转录酶（RNA指导的DNA核苷酸转移酶）和大肠杆菌DNA聚合酶I的Klenow A片段的相继作用合成了双链DNA。从这种双链DNA的复杂混合物中，用限制性内切酶BamHI和EcoRI切割产生了一个特定的200碱基对的限制性片段。这些酶在质粒载体pBR322中也进行独特的切割。该限制性片段通过这些BamHI和EcoRI位点插入到pBR322中，并克隆于大肠杆菌chi1776中。如在体外翻译系统中所检测的，通过其与鸡原α2胶原蛋白mRNA的特异性杂交，表明克隆的重组质粒含有原α2胶原蛋白DNA。因此，在未首先获得高度纯化的胶原蛋白mRNA的情况下构建了一个含有原α2胶原蛋白DNA的克隆。
