Assaying synthetic ribozymes in plants: high-level expression of a functional hammerhead structure fails to inhibit target gene activity in transiently transformed protoplasts
| Autor: | Laurent Mazzolini, Nicole Lescure, M. Axelos, Pierre Yot |
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| Rok vydání: | 1992 |
| Předmět: |
Hammerhead ribozyme
Transcription Genetic Molecular Sequence Data Arabidopsis DNA Recombinant GUS reporter system Plant Science Transformation Genetic Gene expression Genetics Escherichia coli Gene silencing RNA Catalytic RNA Messenger Cloning Molecular Cells Cultured Glucuronidase Messenger RNA Reporter gene biology Base Sequence Protoplasts fungi Ribozyme food and beverages RNA General Medicine biology.organism_classification Blotting Northern Molecular biology biology.protein Nucleic Acid Conformation Agronomy and Crop Science Plasmids |
| Zdroj: | Plant molecular biology. 20(4) |
| ISSN: | 0167-4412 |
| Popis: | A hammerhead ribozyme designed against the mRNA coding for the Escherichia coli beta-glucuronidase (GUS) reporter enzyme was constructed. The synthetic ribozyme appeared able to correctly cleave in vitro the target RNA. This catalytic molecule was then assayed for in vivo activity in plant protoplasts. Plasmids coding either for the ribozyme or for the GUS target gene were cotransfected into the cells by the PEG-calcium procedure and GUS gene expression monitored following transient expression by measuring the intracellular GUS enzymatic activity. Expression of the ribozyme to high molar excess over the GUS transcript did not lead to any significant decrease of GUS activity in the transfected protoplasts. Insertion of the ribozyme sequence in the 3'-untranslated region of the GUS mRNA also had no detectable effect on GUS reporter gene expression whereas the corresponding RNA appeared able to self-cleave in vitro. These results indicate that the ability of ribozymes to perform catalytic cleavage of their substrate mRNA in vitro is essential but clearly not sufficient to ensure that efficient inhibition of the corresponding target gene will occur upon endogenous expression of this catalytic RNA in the plant cell. |
| Databáze: | OpenAIRE |
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