DETECTION OF CLEAVAGE PRODUCTS FROM AN IN-VIVO TRANSCRIBED CIS HAIRPIN RIBOZYME IN TURNIPS USING THE CAMV PLANT-VIRUS

In order to examine ribozyme (Rz) activity in vivo, we have adapted a virus to deliver Rz to plants, DNA fragments that code for both active and mutant cis-hairpin Rz were cloned into the double-stranded DNA plant virus, cauliflower mosaic virus (CaMV). These Rz constructs successfully infected Brassica campestris I apa (turnip), The plants that were infected with the active-Rz construct showed, on average, a one-week delay in the appearance of viral symptoms, when compared to the mutant-Rz control, Since CaMV replicates through reverse transcription of a full-length RNA intermediate, Rz cloned into the CaMV DNA should be transcribed within this viral RNA, If these Rz constructs cleave, the amount of intact virus RNA should be reduced, resulting in attenuated viral symptoms, In addition, lysate RNase protection assays showed fragments corresponding to the sizes of both the 5' and 3' cis cleavage products in the active Rz tissue, No cleavage products were observed from plant tissue infected with the mutant Rz, Both the attenuated systemic viral symptoms and the cleavage products from the protection assay strongly support in vivo transcription and cleavage of this hairpin Rz, This is the first report of an in vivo transcribed Rz showing cleaved products by direct RNA analysis (non-PCR) in plants or animals.