Molecular cloning of potato spindle tuber viroid (PSTV) cDNA synthesized by enzymatic elongation of PSTV-specific DNA primers: A general strategy for viroid cloning

Different cDNAs were synthesized by primer extension from the RNA of the severe strain KF 440 of potato spindle tuber viroid (PSTV) with the aid of reverse transcriptase using three PSTV-specific DNA molecules as primers. The cDNAs were made double-stranded and cloned into plasmid pBR 322. Various overlapping subgenomic DNA fragments were prepared from these clones and recombined in two different ways. In both cases a PSTV DNA copy was obtained which represented the entire PSTV RNA genome. The sequence of the DNA of one of the resulting full-length clones was identical with the original PSTV isolate, whereas the other clone showed one nucleotide change. On the basis of these results the advantages and problems of different strategies for the molecular cloning of the circular viroid RNA genome are discussed.