Properties of RNA polymerases of healthy and citrus exocortis viroid-infected Gynura aurantiaca DC

Abstract

The properties of the RNA polymerases from Gynura aurantiaca DC infected with the citrus exocortis viroid have been studied and compared to the RNA polymerases from healthy tissue. The optimal Mg 2+ and Mn 2+ concentrations for the polymerase activity have been determined. The incorporation of [ 3H]UMP into acid-insoluble material in isolated nuclei was dependent upon the presence of all four ribonucleotides, and was inhibited by actinomycin D and deoxyribonuclease. The product was ribonuclease-sensitive. Addition of exogenous substrate in the form of purified CEV, ssRNA of bean-pod mottle virus (BPMV), and dsRNA of bacteriophage ∅ 6 to intact nuclei pretreated with deoxyribonuclease did not effect the [ 3H]UMP incorporation level. The solubilized RNA polymerases were separated on DEAE-cellulose columns into three fractions: I, IIa, and IIb. I and IIa prefer native-DNA as template, IIb prefers denatured-DNA. The three enzyme fractions lack specificity: CEV-RNA, BPMV-RNA, and ∅ 6 dsRNA are accepted as substrates. The products of the reaction using CEV as a substrate and fractions I and IIa as enzyme source have been analyzed by polyacrylamide-gel electrophoresis and are of low molecular weight (about 5S and smaller).