Analysis by molecular hybridization of the RNAs transcribed by a cell-free 
fraction from avocado infected with avocado sunblotch viroid (ASBV) demonstrated 
the presence of newly synthesized viroid-specific sequences, most of which were 
of the same polarity as the mature infectious viroid RNA. Treatment of the 
cell-free fraction with DNase reduced the total synthesis of RNA considerably, 
but it did not influence that of the ASBV-specific RNAs, indicating that the 
latter were transcribed on an RNA template. Inhibition studies with 
alpha-amanitin showed that the synthesis of ASBV-specific RNAs was not affected 
by concentrations of 1 and 200 micrograms/ml of the drug, which typically 
inhibit RNA polymerase II and III, respectively, from most animal and plant 
systems. These results suggest that either RNA polymerase I or an unidentified 
RNA polymerase activity resistant to alpha-amanitin, acting on an RNA template, 
plays a role in the replication of ASBV, whereas for the rest of the viroids 
studied so far it appears that RNA polymerase II is involved. Analysis by 
polycrylamide gel electrophoresis under partially and fully denaturing 
conditions of the ASBV-specific RNAs synthesized in vitro showed that they 
contain unit and longer than unit length viroid strands, probably associated in 
complexes with single- and double-stranded regions. The structural properties of 
these complexes are similar to those of the RNAs accumulating in vivo in 
viroid-infected tissues, which are the postulated replicative intermediates of 
the rolling-circle mechanism proposed for viroid synthesis.