Isolation and characterization of a 15-kilobase genomic sequence coding for part of the Pro alpha 2 chain of sheep type I collagen

Abstract

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.