Expression of a chimeric ribozyme gene results in endonucleolytic cleavage of target mRNA and a concomitant reduction of gene expression in vivo.
The subclass of catalytic RNAs termed ribozymes cleave specific target RNA sequences in vitro. Only circumstantial evidence supports the idea that ribozymes may also act in vivo. In this study, ribozymes with a hammerhead motif directed against a target sequence within the mRNA of the neomycin phosphotransferase gene (npt) were embedded into a functional chimeric gene. Two genes, one containing the ribozyme and the other producing the target, were cotransfected into plant protoplasts. Following in vivo expression, a predefined cleavage product of the target mRNA was detected by ribonuclease protection. Expression of both the ribozyme gene and the target gene was driven by the CaMV 35S promoter. Concomitant with the endonucleolytic cleavage of the target mRNA, a complete reduction of NPT activity was observed. An A to G substitution within the ribozyme domain completely inactivates ribozyme-mediated hydrolysis but still shows a reduction in NPT activity, albeit less pronounced. Therefore, the reduction of NPT activity produced by the active ribozyme is best explained by both hydrolytic cleavage and an antisense effect. However, the mutant ribozyme--target complex was more stable than the wildtype ribozyme--target complex. This may result in an overestimation of the antisense effect contributing to the overall reduction of gene expression.
Steinecke
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P
Max-Planck-Institut für Züchtungsforschung, Köln, FRG.
Herget
T
T
Schreier
P H
PH
D000595:Amino Acid Sequence; D001483:Base Sequence; D002678:Chimera; D015870:Gene Expression; D019868:Kanamycin Kinase; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D009838:Oligodeoxyribonucleotides; D010770:Phosphotransferases / Q000235:genetics / Q000378:metabolism; D010942:Plant Viruses / Q000201:enzymology / Q000235:genetics*; D010947:Plants, Toxic; D011523:Protoplasts / Q000502:physiology; D012313:RNA / Q000235:genetics; D015347:RNA Probes; D016337:RNA, Catalytic / Q000235:genetics* / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism*; D019244:RNA, Satellite; D014026:Nicotiana / Q000201:enzymology / Q000235:genetics*
D009838:Oligodeoxyribonucleotides; D015347:RNA Probes; D016337:RNA, Catalytic; D012333:RNA, Messenger; D019244:RNA, Satellite; D012313:RNA; D010770:Phosphotransferases; D019868:Kanamycin Kinase