The ovalbumin gene: Cloning of a complete ds-cDNA in a bacterial plasmid

Abstract The ovalbumin structural gene was purified and amplified by cloning in the bacterial strain χ1776 using the tetracycline-resistant plasmid pMB9. To prepare this recombinant plasmid, a full-length copy of the mRNA was first synthesized with avian myeloblastosis virus (AMV) reverse transcriptase. A double-stranded copy of the DNA was then synthesized and joined to the plasmid by the polyA-polydT “tailing” procedure. Transformed colonies containing ovalbumin DNA were selected by in situ hybridization with 32 P-cRNA ov . Digestion of the chimeric clones with HhaI , which does not cleave the ovalbumin DNA, can be used to calculate the size of the inserted DNA. One chimeric plasmid, pOv230, contained a 1950 base pair insert, which is longer than the 1850 bases of the mRNA ov . The vast majority of this DNA is complementary to ovalbumin gene sequences, since a complete DNA copy of the mRNA ov (cDNA ov = 1850 bp) is protected from S 1 nuclease digestion (>95%) by pOv230. The ramaining bases are probably comprised of the poly(dA-dT) linkers. Multiple restriction enzyme digests of the inserted ovalbumin DNA demonstrated that sites present in the double-stranded ovalbumin DNA are preserved in the chimeric plasmid and allowed us to determine the orientation of the inserted ovalbumin DNA. Finally, the chimeric plasmid pOv230 is able to transform χ1776 with an efficiency similar to the parent plasmid pMB9.