Fig. 4.

Northern blot hybridization analysis of transgenic Arabidopsis expressing dimeric (+) and (-) HSVd RNAs. Viroid-enriched RNA preparations, obtained by chromatography on nonionic cellulose, were fractionated by single denaturing PAGE in duplicated gels and blotted to nylon membranes that were hybridized with ³²P-labeled riboprobes for detecting the (+) and (-) HSVd strands (A and B, respectively). Lanes 1, control of HSVd-infected cucumber; lanes 2, control of nontransformed Arabidopsis; lanes 3 and 4, two independent transgenic Arabidopsis expressing dimeric (-) HSVd RNA; and lanes 5, transgenic Arabidopsis expressing dimeric (+) HSVd RNA. Positions of linear RNA markers, with their size in nucleotides, are indicated on the left. Positions of circular and linear HSVd RNAs are indicated on the right in A. Both riboprobes were equalized in acid-precipitable counts, and the films were exposed for the same time. For facilitating detection of HSVd strands accumulating at lower levels, the volume of applied extract was 10-fold higher in lanes 3 and 4 in A, and in lanes 1 and 5 in B.