Molecular Cloning of Potato Leaf Roll Virus Complementary DNA

Abstract

Potato leaf roll virus (PLRV) was purified from infected leaves of Physalis floridana. The viral RNA was poly(A)-tailed and used to synthesize double-stranded. cDNA and then cloned into the Pstl site of the plasmid pUC9 using oligo(dG)-oligo(dC) tailing methodology. Three initial overlapping clones were selected and used as the source of leftward and rightward probes in colony hybridization experiments to identify additional cloned PLRV cDNAs by "plasmid walking." Three PLRV clones containing cDNA inserts of 3.3, 2.3, and 1.2 kilobase pairs (kbp) were identified. Restriction endonuclease and Southern-blot hybridization analyses indicated that these cDNAs formed an overlapping physical map representing a majority of the viral genome (6 kbp). Each clone was vertified to contain viral cDNA by dot-blot hybridization to PLRV RNA and total RNA isolated from PLRV-infected P. floridana leaves. The composite 5'' to 3'' polarity of PLRV cDNA was established by the use of an M13 strand-specific hybridization probe and overlapping restriction endonuclease sites.