Figure 1.

The ntl ribozyme expression vector and schematic secondary structure of ntl ribozyme RNA. (A) Ntl ribozyme expression vector pT₇vaRz. The vector pT₇vaRz(ntl) was constructed first by an insertion of a ribozyme sequence (Rz) against the ntl mRNA into a va I RNA expression cassette; the cassette containing the ribozyme sequence (va-Rz-loop-va’) subsequently was cloned into a pTM-1 expression vector (27) lacking the encephalomyocarditis virus internal ribosome entry site sequence in such a way that the expression of Rz is under the control of both a T₇ promoter (P_T7) and an adenovirus va I internal promoter, a pol III promoter (upstream va). The P_T7-driven transcription stops at the T₇ terminator (T_T7) whereas the va I-driven transcription terminates at the 3′ end of the loop. (B) The sequences flanking Rz(ntl) were designed in such a way that the 5′ and 3′ ends of the ntl ribozyme form a stable stem structure (18). Both the ribozyme and its targeted, complementary ntl mRNA sequences, as well as the cleavage site (435), also are shown.