Fig. 4. (A) Primer extension analysis with oligonucleotides PIII (left) and PIV (right) using as template the monomeric linear ASBVd RNAs obtained from band x (lane X), or the ASBVd (–) linear RNA generated by self-cleavage during in vitro transcription of the linearized pAS19d plasmid containing a dimeric ASBVd insert (lane S). Sequencing ladders were obtained with the same oligonucleotides and recombinant plasmids containing a monomeric ASBVd insert. Arrowheads indicate the extension product and the nucleotide position at their 5′ end in the written sequence. (B) Location on the secondary structure predicted for the ASBVd (–) circular monomeric RNA of the 5′ termini (marked by arrowheads) mapped by primer extension. Continuous lines denote positions to which PIII and PIV are complementary. Numbering is the same as in the (+) polarity.