FIG. 4.

In vitro synthesis and self-cleavage of both polarity RNAs of variant ELVd-2. (A) Diagram of plus and minus DNA templates and of the products generated by transcription with T3 and T7 RNA polymerases, respectively, after restriction with the indicated enzymes. Hatched boxes, vector sequences; filled boxes, RNA polymerase promoters; empty boxes, ELVd sequence. The complete primary transcripts are C⁺ and C⁻ and the cleavage fragments are 5′F⁺, 3′F⁺, 5′F⁻, and 3′F⁻. Positions in the ELVd sequence are shown above the products and their expected sizes in nucleotides are shown below. Self-cleavage sites are indicated by arrowheads. (B) Analysis by denaturing PAGE (in 1× Tris-borate-EDTA-5% polyacrylamide gels containing 8 M urea) and autoradiography of the in vitro transcription (T) and self-cleavage (SC) of the purified primary transcripts incubated at 40°C for 1.5 h in 50 mM Tris-HCl (pH 8)-0.5 mM EDTA, with or without 5 mM MgCl₂. The samples were previously heated in 1 mM EDTA (pH 6) at 95°C for 1 min and snap cooled in ice.