Molecular cloning of DNA complementary to tobacco vein mottling virus RNA

RNA isolated from tobacco vein mottling virus (TVMV) was used as a template for avian myeloblastosis virus RNA-dependent DNA polymerase, primed with oligo(dT). The largest single-stranded cDNA synthesized was 10 kb, the same as the viral RNA. This material was converted to double-stranded cDNA with Escherichia coli DNA polymerase I and digested with restriction endonuclease HindIII. The cDNA fragments were ligated to HindIII-digested plasmid pBR322 and the product used to transform E. coli strain DG-75. Clones containing recombinant plasmids were selected by ampicillin resistance, and those containing TVMV RNA sequences were selected by colony hybridization with a single-stranded cDNA probe. Four different sizes of recombinant plasmid were reproducibly observed. The inserted DNA portion could be excised in each case with HindIII. The lengths of inserted DNA were 3.0, 1.85, 1.1, and 0.72 kb. A similar procedure was used with PstI-digested cDNA and pBR322. A single type of recombinant plasmid, containing a DNA insertion of 1.85 kb, was reproducibly observed. Hybridization with TVMV RNA confirmed that the five inserted DNA segments were derived from the viral RNA. Hybridization of each recombinant plasmid with the others established that each of the cloned HindIII fragments was unique and that one of them overlapped the cloned PstI fragment. The cloned cDNA fragments were ordered by establishing a restriction map of the cDNA. Together the cloned cDNA fragments account for over 80% of the viral genome.