Molecular cloning and characterization of potato spindle tuber viroid cDNA sequences

Abstract

Double-stranded c[complementary]DNA was synthesized from a polyadenylated potato spindle tuber viroid (PSTV) template and inserted in the PstI endonuclease site of plasmid pBR322 by using the oligo(dC).cntdot.oligo(dG)-tailing procedure. Tetracycline-resistant ampicillin-sensitive transformants contained sequences complementary to PSTV [32P]cDNA, and one recombinant clone (pDC-29) contains a 460-base-pair insert. This cloned double-stranded PSTV cDNA contains the cleavage sites for six restriction endonucleases predicted by the published primary sequence of PSTV, and one additional site each for AvaI, HaeIII, HpaII and SmaI. The additional AvaI, HpaII and SmaI sites are explained by the presence of a 2 cdC-C-C-G-G-G sequence in the cloned double-stranded cDNA. The largest fragment released by HaeIII digestion contains approximately 360 base pairs. These results suggest that almost the entire sequence of PSTV was cloned, but the sequence cloned differs slightly from that published. Hybridization probes derived from pDC-29 insert allowed detection and preliminary characterization of RNA molecules having the same size as PSTV, but the opposite polarity. This RNA is present during PSTV replication in infected tomato cells.