Construction of a recombinant bacterial plasmid containing a chick pro-alpha2 collagen gene sequence.

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Abstract
A recombinant plasmid containing chick pro-.alpha.2 collagen gene sequences was constructed and cloned in Escherichia coli. Using partially purified collagen mRNA as template, double-stranded DNA was synthesized 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 DNA, 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 .chi. 1776. The cloned recombinant plasmid contained pro-.alpha.2 collagen DNA by its specific hybridization to chick pro-.alpha.2 collagen mRNA, as assayed in an in vitro translation system. A clone containing pro-.alpha.2 collagen DNA was constructed without first obtaining highly purified collagen mRNA.