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Protein Engineering vol. 3 no. 8 pp. 733-737, 1990
© 1990 Oxford University Press

RESEARCH-ARTICLE

Construction of a novel artificial-ribozyme-releasing plasmid

Kazunari Taira1, Masanao Oda, Hideaki Shinshi, Hidekatsu Maeda and Kensuke Furukawa2

Fermentation Research Institute, Agency of Industrial Science and Technology MITI, Tsukuba Science City 305, Japan 2Department of Agricultural Chemistry, Faculty of Agriculture, Kyushu University Fukuoka 812, Japan

1To whom correspondence should be addressed

A novel ‘active-ribozyme-releasing system’ was constructed, taking advantage of the consensus sequence of a new class of ribozyme. An active ribozyme sequence, targeted for the SFWl gene (a yeast suppressor gene for flocculation) was fused just downstream of the T7 promoter. The 3' terminus of the Arst ribozyme was designed to be trimmed by the second ribozyme connected to the downstream of the first active ribozyme. In vitro experiments revealed that the active ribozyme targeted to SFLl was successfully released by the action of the second rhzyme, subsequently cleaving the SFL1 mRNA at the predetermined site. Since the first active ribozyme with a defined 3'-terminus can be produced even when a circular DNA is used as a template, this kind of construct has a potential to release an ‘active ribozyme’ tailored to destroy a target gene (RNA) in vivo. Moreover, the second ribozyme in this construct can be utilized as a universal pseudo-terminator for generation of any RNA transcripts inserted in place of the cassette portion of the first ribozyme.

Keywords: ribozymes/RNA cleavage/SFLl gene/3'-processor/universal terminator

Received December 15, 1989; accepted April 26, 1990.


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