FIG. 1.

vhs induces preferential endonucleolytic cleavage in the vicinity of the 3′ boundary of the EMCV IRES. A 2.3-kb runoff transcript of pCITE-1 was added to RRLs containing pretranslated vhs (lanes marked vhs) and to control RRL (lanes marked RC), and samples were removed at the indicated times (in minutes). RNA was extracted from each sample, resolved on a 1% agarose–6% formaldehyde gel, and transferred to a Nytran Plus membrane. (A) Capped, internally labeled RNA. The closed and open arrowheads indicate the ca. 1,800- and 600-nt fragments, respectively. (B) 5′-Cap-labeled RNA. (C) Capped, unlabeled RNA. The radioactive signals in panels A and B were detected by autoradiography. The RNA products in panel C were detected by hybridization to a 5′-labeled oligonucleotide probe complementary to residues 729 to 746 of the pCITE transcript, followed by autoradiography. The numbers to the right of panels A and B indicate the sizes of RNA markers (lanes marked M) in nucleotides. (D) Diagram of the pCITE transcript indicating the approximate position(s) of the cleavage site(s) (arrows).