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

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 SacI cleavage sites flank the putative promoter in the 4.2-kb viral insert. Deletion in the 1.2-kb SacI fragment obliterated the ability of pATV-6 to synthesize viral RNA. Digestion of the 1.2-kb SacI fragment with PvuI generates two fragments of 0.63 and 0.57 kb. Deletion of the 0.57-kb but not the 0.63-kb PvuI-SacI 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 PvuI-SacI fragment.