A ribozyme that enhances gene suppression in tobacco protoplasts.
Site-directed mutagenesis has been used to produce two hammerhead ribozyme molecules targeting the chloramphenicol acetyltransferase gene (CAT). One ribozyme has a single catalytic domain between two 12-nucleotide arms that can hybridize 5' and 3' of the GUC target site of the CAT RNA transcript. The second ribozyme is a full-length antisense RNA with four catalytic domains inserted along the length, each targeting a specific GUC site within the CAT mRNA. Our results show that both ribozymes can produce almost equivalent rates of cleavage of the CAT mRNA in vitro (T1/2 of 18 or 15 min, respectively). In tobacco protoplasts we show consistently greater gene suppression in the presence of the long ribozyme molecule, compared with the equivalent antisense (22% gene reduction for antisense compared with 44% with the long ribozyme). These results suggest that hammerhead ribozymes may be developed for the inactivation of gene activity in plant cells.
Perriman
R
R
CSIRO Division of Plant Industry, Canberra, Australia.
Graf
L
L
Gerlach
W L
WL
D001665:Binding Sites; D002478:Cells, Cultured; D015500:Chloramphenicol O-Acetyltransferase / Q000235:genetics; D018274:Electroporation; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010947:Plants, Toxic; D011523:Protoplasts / Q000166:cytology / Q000378:metabolism; D016372:RNA, Antisense / Q000235:genetics / Q000378:metabolism*; D016337:RNA, Catalytic / Q000235:genetics / Q000378:metabolism*; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism*; D013489:Suppression, Genetic; D014026:Nicotiana / Q000235:genetics; D014158:Transcription, Genetic
D016372:RNA, Antisense; D016337:RNA, Catalytic; D012333:RNA, Messenger; D015500:Chloramphenicol O-Acetyltransferase