Cloning of avian tumor virus DNA fragments in plasmid pBR322: evidence for efficient transcription in E. coli from a virus-coded promoter.

Abstract Two avian tumor virus DNA fragments of 4.2 and 3.2 kb were inserted into pBR322 in the two possible orientations for each fragment. In the Escherichia coli host cells, RNA polymerase initiates transcription of large quantities (up to 0.5 to 1% of total E. coli RNA) of virus-specific RNA in the recombinant plasmids carrying the 4.2-kb fragment (pATV-6) but not in pATV-2 which contains the 3.2-kb fragment. Two Sac I cleavage sites flank the putative promoter in the 4.2-kb viral insert. Deletion in the 1.2-kb Sac I fragment obliterated the ability of pATV-6 to synthesize viral RNA. Digestion of the 1.2-kb Sac I fragment with Pvu I generates two fragments of 0.63 and 0.57 kb. Deletion of the 0.57-kb but not the 0.63-kb Pvu I- Sac I fragment completely eliminated the ability of the recombinant to synthesize viral RNA. These results strongly suggest that viral RNA in E. coli transcription is indeed initiated at a size present in the viral genome and that this site is localized in the 0.57-kb Pvu I- Sac I fragment.