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Purification and Characterization of the Circular and Linear Forms of Chrysanthemum Stunt Viroid

Department of Biochemistry, University of Adelaide, Adelaide, South Australia, 5001

A new and rapid extraction procedure is described for the isolation and partial purification of low mol. wt. RNA as well as DNA from 500 g batches of chrysanthemum stunt viroid-infected chrysanthemum. The chrysanthemum stunt viroid was then purified to homogeneity from this extract by one cycle of non-denaturing polyacrylamide slab gel electrophoresis followed by one cycle of denaturing slab gel electrophoresis. The covalently closed circular form and the linear form of the viroid co-migrated on the first gel but were well separated on the second. The yield of circular chrysanthemum stunt viroid was 200 µg/kg of infected chrysanthemum shoots and of the linear form was 35 µg/kg.

³²P-labelled complementary DNA prepared against the circular form was used to show, by hybridization analysis, that the circular and linear forms contained identical nucleotide sequences. The circular and linear forms were equally infectious when inoculated onto Gynura aurantiaca. The linear viroid contained all four nucleotides at the 5'-terminus, about 72% of which had a 5'-phosphate.

Received 11 June 1979; accepted 18 September 1979.

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