INVITRO SYNTHESIS AND CHARACTERIZATION OF DNA COMPLEMENTARY TO CADANG-CADANG-ASSOCIATED RNA

The anomalous viroid-like RNA associated with cadang-cadang disease of coconut palms (ccRNA-I) has been cleaved by treatment with the single-strand specific nuclease S1, polyadenylated and used as a template for the oligo(dT) primed synthesis of complementary DNA (cDNA) by the avian myeloblastosis virus reverse transcriptase. The efficiency of synthesis was low, with only 3 to 4.5 ng of cDNA synthesized from 2 μg of RNA. Most of the cDNA was in the 4S size class. A R0t(1/2) value of 1 x 10-3 mol. s/1 was obtained when this cDNA was hybridized with ccRNA-I, consistent with ccRNA-I representing a unique species of mol. wt. approx. 100,000. The maximum hybridization value obtained with ccRNA-I was approx. 50%; the S1 nuclease resistance of the cDNA after self-annealing was approx. 7%. The melting behaviour of the homologous hybrids provided evidence for the specificity of base-pairing with no evidence of mismatching. The cDNA has been shown to be a specific probe for cadang-cadang associated RNA. It has been used to demonstrate that ccRNA-I and ccRNA-2 have common nucleotide sequences, the ccRNA-I is uniquely associated with diseased and not healthy palms and that it has no significant homology with high mol. wt. RNA or DNA from diseased palms. The value of the cDNA as a diagnostic probe for ccRNA-I in crude nucleic acid extracts has been demonstrated.