FIG. 2.

Cap-independent translation mediated by the p27 5′-UTR. Bicistronic mRNAs encoding CAT upstream of luciferase were expressed in proliferating D6P2T cells. Cells were transfected with either pGL2CAT/Luc (vector), which has a short spacer region between the CAT and luciferase coding regions, pGL2CAT/Luc-p27UTR, which carries the region encoding the mouse p27 5′-UTR inserted within the spacer region, pGL2CAT/Luc-p27UTR(rev), which carries the region encoding the mouse p27 5′-UTR inserted in reverse orientation within the spacer region, or pGL2CAT/Luc-TR-UTR, which carries 200 bp of the TR 5′-UTR inserted within the spacer region. After cell lysis, CAT and luciferase (LUC) activities were determined for each extract (A), and the relative level of cap-independent translation was estimated from the luciferase/CAT ratio (B). The luciferase/CAT ratio was determined by dividing the relative light units (luciferase) by the percentage of substrate converted to the acetylated form (CAT). (C) D6P2T cells were transfected with either pGL2CAT/Luc (vector), pGL2CAT/Luc-p27UTR, or pcBiP, which carries the BIP 5′-UTR inserted between CAT and luciferase in the plasmid pcDNA1. The cells were harvested 1 day after transfection and assayed for CAT and luciferase activities. All data represent the mean of a minimum of three replicates plus or minus the standard error.