Synthesis of RNA complementary to potato spindle tuber viroid using Qβ replicase

Abstract

Qβ replicase accepts potato spindle tuber viroid (PSTV) as template for RNA synthesis under two different sets of conditions. Comparatively high concentrations of unmodified PSTV template in the presence of Mn 2+ direct the synthesis of a heterogeneous mixture of RNAs. The largest products appear by polyacrylamide gel analysis to be nearly complete copies of PSTV. Primer-dependent polynucleotide phosphorylase can be used to add a poly(cytidylic acid) sequence to the 3′-terminus of PSTV. When this modified RNA is used as template for Qβ replicase in the presence of Mg 2+, a high-molecular-weight RNA product is synthesized. The poly(guanylic acid) sequence at the 5′-terminus of the product provides a potential means for separating template and product. Both methods yield an RNA product that is complementary to PSTV, as measured by RNA · RNA hybridization. The specificity of the product containing the 5′terminal poly(guanylic acid) sequence is also indicated by its poor hybridization to a variety of unrelated viral RNAs. The apparent sequence complexities of the various complementary RNA products are identical and approximately eightfold greater than the estimated molecular weight of PSTV.