In vitro Synthesis and Characterization of DNA Complementary to Cadang-cadang-associated RNA

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Abstract
The anomalous viroid-like RNA associated with cadang-cadang disease of coconut palms (ccRNA-1) was cleaved by treatment with the single-strand specific nuclease S1, polyadenylated and used as a template for the oligo(dT) primed synthesis of complementary (c)DNA by the avian myeloblastosis virus reverse transcriptase. The efficiency of synthesis was low, with only 3-4.5 ng of cDNA synthesized from 2 .mu.g of RNA. Most of the cDNA was in the 4S size class. A R0t1/2 [R0 = initial concentration, t1/2 = half hybridization time] value of 1 .times. 10-3 mol s/l was obtained when this cDNA was hybridized with ccRNA-1, consistent with ccRNA-1 representing a unique species of MW about 100,000. The maximum hybridization value obtained with ccRNA-1 was about 50%; the S1 nuclease resistance of the cDNA after self-annealing was about 7%. The melting behavior of the homologous hybrids provided evidence for the specificity of base-pairing with no evidence of mismatching. The cDNA was a specific probe for cadang-cadang associated RNA. It was used to demonstrate that ccRNA-1 and ccRNA-2 have common nucleotide sequences, that ccRNA-1 is uniquely associated with diseased and not healthy palms and that it has no significant homology with high MW RNA or DNA from diseased palms. The value of the cDNA as a diagnostic probe for ccRNA-1 in crude nucleic acid extracts was demonstrated.