Abstract
Ribozyme expression cassettes were constructed which generate trimmed, trans-acting ribozymes from longer transcripts through the action of a downstream cis-acting ribozyme. This self-processing system produces small, well-defined trans-acting ribozymes with minimal, nonproductive, intramolecular structure. These cassettes also permit direct comparison of different ribozyme expression vectors without the need to compensate for different transcription initiation and termination sequences. Expression cassettes were created that contain a T7 promoter and that encode a single trans-acting ribozyme followed by either a hammerhead, hairpin, or hepatitis delta virus cis-acting ribozyme. All three ribozyme motifs function efficiently when transcribed in vitro, although slight differences are observed in the efficiency of self-processing for the different motifs. When transiently expressed in cultured mouse cells, the same specific cleavage products are observed. In addition, the relative efficiencies of in vitro self-processing between the three ribozyme constructs was maintained in vivo. Thus, the cellular milieu does not differentially alter the activity of the three ribozyme motifs. Detection of ribozyme-catalyzed RNA cleavage products from cultured cells is direct proof of ribozyme action in vivo.
Highlights
These cassettes permit direct comparison of differ- quences (Fedor and Uhlenbeck, 1990)
One objective of this study was to createxpression vectors that produce ribozymes with littleor no extraneous sequence, thereby minimizing nonwhen transcribed in vitro, slight differences productive folding of the ribozymes. This objective has been are observedin the efficiency of self-processingfor the accomplished through the action of downstream, cis-cleaving different motifsW. hen transiently expressedin cultured ribozymes which free the trans-actingribozymes from extranemouse cells, the same specific cleavage products are ob- ous sequences
OST7-1 cells appears to be strikingly similar to the extent of self-processing in vitro (Fig. 4,In Vitro +MgCl,versus Cellular)
Summary
Annealing of the primer to its complementary RNA sequence. The primer was extended using Superscript I1 reverse transcriptase Construction of Self-processingRibozyme Expression Cassettes-To The reaction was terminated by adding an equal volume of 2 x formprepare DNA inserts that encodeself-processingribozyme cassettes, amide gel loading buffer and freezing on crushed dry ice. The samples partially overlapping top- and bottom-strand oligonucleotides(60-90 wereresolved on a 10%polyacrylamide sequencing gel. HindIII-digested pucl and transformed into E. coli strain DH5a using tured separately inastandard cleavagebuffer (containing 50 mM standard protocols(Maniatis et al, 1982).The identity of positive clones Tris.HC1, pH 7.5, and 10 mM MgCl,) by heating to 90 "C for 2 min and was confirmedby sequencing small-scaleplasmid preparations. Larger scale preparations of plasmid DNA for use as in vitro tran- of 5 pl were taken at regular time intervals, quenched by adding an scription templates and intranfections were prepared using the proto- equal volume of 2 x formamidegel loading buffer, and frozen on dry ice. In order to prepare 5' end-labeled transcripts, standard transcription reactions
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