FIG. 2.

Ribozyme expression cassettes. (A) Schematic representation of the δRz expression strategy. The secondary structures of the cis (hammerhead) and trans (delta) (shaded areas) ribozymes are shown. Transcription of the cis-trans-cis cassette produces a chimeric RNA that self-cleaves at two specific sites (HH₁ and HH₂), releasing the trans-acting δRz (δRz). The arrows indicate the hammerhead (HH₁ and HH₂) cleavage sites. (B) In vitro transcription of the cis-trans-cis δRz constructs. Expected RNA products generated by self-cleavage of the hammerhead motifs during transcription are shown. The positions of the full-length precursor (Pre-δRz, 205 nt), intermediates (127 and 158 nt), and fully processed transcript (HH₁, 78 nt; HH₂, 47 nt; δRz, 80 nt) are shown. The positions of restriction enzyme sites used for cloning are indicated. (C) Autoradiogram of a denaturing 8% polyacrylamide gel containing in vitro transcripts of the three cis-trans-cis δRz expression cassettes. The area enclosed in a box was taken from the lower part of the same autoradiogram to show the relative intensity of the small band (47 nt) corresponding to the released HH₂.