Boyd C D, Tolstoshev P, Schafer M P, Trapnell B C, Coon H C, Kretschmer P J, Nienhuis A W, Crystal R G
J Biol Chem. 1980 Apr 10;255(7):3212-20.
DNA fragments, prepared by partial Eco RI digestion of fetal sheep liver genomic DNA, were used to prepare a "library" of amplified genomic sequences with the lambda vector Charon 4A. Several recombinant plaques were identified by their ability to hybridize to 32P-labeled cDNA prepared from fetal sheep tendon type I procollagen mRNA. Two of these recombinant DNA bacteriophages (SpC3 and SpC7) were identified as containing procollagen pro alpha 2 gene sequences by their ability to specifically anneal to procollagen pro alpha 2 mRNA. Restriction endonuclease and hybridization to a cloned pro alpha 2 cDNA demonstrated that approximately half (2.5 kilobases) of the pro alpha 2 mRNA sequence is distributed over 15 kilobases of genomic DNA. Restriction maps of SpC3 and SpC7 demonstrated that these two DNA fragments contain overlapping sequences of the pro alpha 2 gene. Electron microscopy and R-loop analysis of SpC3 revealed that at least 12 to 16 intervening sequences are distributed throughout the length of this gene fragment.
通过对胎羊肝脏基因组DNA进行部分Eco RI酶切制备的DNA片段，用于构建一个用λ载体Charon 4A扩增的基因组序列“文库”。通过与从胎羊I型原胶原mRNA制备的32P标记的cDNA杂交的能力，鉴定出了几个重组噬菌斑。通过它们与原胶原proα2 mRNA特异性退火的能力，确定其中两个重组DNA噬菌体（SpC3和SpC7）含有原胶原proα2基因序列。限制性内切酶分析以及与克隆的proα2 cDNA杂交表明，proα2 mRNA序列的大约一半（2.5千碱基）分布在15千碱基的基因组DNA上。SpC3和SpC7的限制性图谱表明，这两个DNA片段包含proα2基因的重叠序列。对SpC3的电子显微镜和R环分析表明，至少12至16个间隔序列分布在该基因片段的全长上。
