Construction of a recombinant bacterial plasmid containing a chick pro-α2 collagen gene sequence

Abstract

A recombinant plasmid containing chick pro-α2 collagen gene sequences has been constructed and cloned in Escherichia coli. Using partially purified collagen mRNA as template, the authors synthesized double-stranded DNA 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 DNAs, a specific 200-base-pair restriction fragment was generated by cleavage with the restriction endonuclease BamHI and EcoRI. These enzymes also make unique cuts in the plasmid vector pBR322. The restriction fragment was inserted into pBR322 via these Bam HI and EcoRI sites and cloned in E. coli χ1776. The cloned recombinant plasmid was shown to contain pro-α2 collagen DNA by its specific hybridization to chick pro-α2 collagen mRNA, as assayed in an in vitro translation system. Thus, a clone containing pro-α2 collagen DNA was constructed without first obtaining purified collagen mRNA.