Construction and Characterization of a 2.5-Kilobase Procollagen Clone

• Published in: Proceedings of the National Academy of Sciences - PNAS Vol. 75; no. 11; pp. 5417 - 5421
• Main Authors: Lehrach, Hans, Frischauf, Anna Maria, Hanahan, Douglas, Wozney, John, Fuller, Forrest, Crkvenjakov, Radomir, Boedtker, Helga, Doty, Paul
• Format: Journal Article
• Language: English
• Published: United States National Academy of Sciences of the United States of America 01.11.1978; National Acad Sciences
• Subjects: Animals; Base Sequence; Biochemistry; Bone and Bones - metabolism; Cellulose nitrate; Chick Embryo; Collagens; Complementary DNA; Digestion; DNA Restriction Enzymes; DNA, Recombinant - metabolism; Escherichia coli - metabolism; Gels; Messenger RNA; Molecular Weight; Nucleotides; Plasmids; Procollagen - biosynthesis; Restriction mapping; RNA, Messenger - metabolism; Transformation, Genetic
• ISSN: 0027-8424; 1091-6490
• DOI: 10.1073/pnas.75.11.5417

Recombinant bacterial plasmids have been constructed by inserting double-stranded chicken procollagen cDNA sequences linked to chemically synthesized decanucleotides containing HindIII sites into the HindIII site of pBR322. After transformation of Escherichia coli χ 1776, colonies were selected by ampicillin resistance and recombinants containing procollagen sequences were identified by colony hybridization to 32P-labeled procollagen cDNA. The inserts from three recombinant plasmids, pCg10, pCg13, and pCg45, were 1200, 2200, and 2550 base pairs long, respectively. Their sequence homology has been established by restriction mapping and crosshybridization of nick-translated plasmids to Southern blots of Hpa II fragments of the inserts. pCg45 has been positively identified as containing the pro α 2 collagen sequence by partial determination of the DNA sequence of its ends: it has a short thymine-rich sequence at one end and a sequence coding for residues 478-499 in the chicken α 2 chain at the other end.