Construction and characterization of a 2.5-kilobase procollagen clone.

Authors:
  • HLH Lehrach
    H Lehrach
  • AFA M Frischauf
    A M Frischauf
  • DHD Hanahan
    D Hanahan
  • JWJ Wozney
    J Wozney
  • FFF Fuller
    F Fuller
  • RCR Crkvenjakov
    R Crkvenjakov
  • HBH Boedtker
    H Boedtker
  • PDP Doty
    P Doty
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Abstract
Recombinant bacterial plasmids were constructed by inserting double-stranded chicken procollagen c[complementary]DNA sequences linked to chemically synthesized decanucleotides containing HindIII sites into the HindIII site of pBR322. After transformation of Escherichia coli .chi. 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 3 recombinant plasmids, pCg10, pCg13 and pCg45, were 1200, 2200 and 2550 base pairs long, respectively. Their sequence homology was established by restriction mapping and cross/hybridization of nick-translated plasmids to Southern blots of Hpa II fragments of the inserts. pCg45 has been positively identified as containing the pro .alpha.2 collagen sequence by partial determination of the DNA sequence of its ends: it has a short thymine-rich sequence at 1 end and a sequence coding for residues 478-499 in the chicken .alpha.2 chain at the other.

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