A strategy for the determination of RNA autolysis in carrot protoplasts
Over the past eight years, several examples of catalytic RNAs have been identified and shown to cleave. In this study, a self-cleaving RNA cassette, derived from the "hairpin" catalytic RNA, will be used to test for in vivo autolysis of this RNA in carrot protoplasts. Plasmids for use as gene expression vectors in plants were constructed. These plasmids contained either the chloramphenicol acetyl transferase (CAT) gene or the hairpin cassette at the 3’ end of the CAT gene. Upon electroporation of these vectors into carrot protoplasts, cleavage of the hairpin RNA cassette could not be identified by northern analysis. An alternate system for this detection is considered, which involves SI nuclease analysis of plants stably transformed with the hairpin cassette.