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Nucleic Acids Research, 1992, Vol. 20, No. 4 831-837
© 1992

Articles

Selective cleavage of closely-related mRNAs by synthetic ribozymes

Malcolm J. Bennett* and Julie V. Cullimore+

Department of Biological Sciences, University of Warwick Coventry CV4 7AL, UK

*To whom correspondence should be addressed

Received October 24, 1991. Revised January 28, 1992. Accepted January 28, 1992.

In Phaseolus vulgaris L. (French bean) glutamine synthetase (GS) is encoded by four closely-related genes termed gln-{alpha}, gln-ß, gln-{gamma} and gln-{delta}. We have constructed and characterised In vitro a number of hammerhead ribozymes designed to cleave individual RNAs encoded by these genes. The three ribozymes, termed J1, J2 and J3, were targeted to cleave RNA at the start of the {gamma} and ß, and the middle of the {gamma}, GS open reading frames respectively. All three ribozymes successfully discriminated between the four ({alpha}, ß, {gamma} and {delta}) highly homologous sequences, even though the targeted sites of cleavage shared up to 18 out of 22 identical bases with other gene family members. The rlbozyme-medlated cleavage reactions were Mg2+ dependent and enhanced at higher temperatures, although the J1 ribozyme retained considerable activity at physiological temperatures. Both J1 and J2 demonstrated a time-dependent cleavage of their targeted GS RNAs, although these two ribozymes differed markedly in their ability to cleave multiple substrate molecules. The rate of cleavage by J1 was found to be reduced in the presence of related GS RNAs and by total leaf poly(A) RNAs. The implications of these results for ribozyme activity in vivo are discussed.

+Present address: Laboratoire de Biologic Moleculaire des Relations Plantes-Microorganismes, INRA-CNRS Bp 27, F-31326 Castanet-Tolosan Cedex, France


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