Figure 3.

Dicistronic mRNAs containing synthetic IRESes based on multiple linked copies of nucleotides 133–141 of the Gtx 5′ UTR. (A) Schematic representation of the dicistronic constructs used in this analysis is indicated. Constructs are based on the RPh vector and contain 25 nt of the mouse β-globin 5′ UTR sequence (β) immediately upstream of the initiation codon, represented as a thick black line. (Inset) One, five, or ten copies of the Gtx IRES module (Gtx133–141) are indicated as gray bars; constructs with multiple copies are spaced 9 nt apart by one of three spacer sequences, SI, SII, or SIII, shown as a thick black bar, an open bar, and a thin line, respectively. Constructs were transfected into N2a cells, and luciferase activities were normalized for transfection efficiency (see Materials and Methods). Numbers in parentheses represent SEM. (B) Analysis of dicistronic constructs containing the CAT gene as the second cistron. The constructs used in this assay are based on the RCh vector. Inserts include the EMCV IRES and (Gtx133–141)₁₀(SI)₉β. Constructs were transfected into N2a cells and assayed for Renilla (R) and CAT (C) activities. CAT activities in the table were determined by liquid scintillation assay and, because CAT activity of the RCh vector was indistinguishable from a negative control, numbers were normalized to the activity of EMCV/RCh. An autoradiogram of a TLC assay of CAT activity is also shown. Lane 1, negative control; lanes 2–4, transfections with RCh; lanes 5–7, transfections with EMCV/RCh (EMCV); lanes 8–10, transfections with [Gtx133–141)₁₀(SI)₉β/RCh (Gtx(10X)]. Each lane represents an independent transfection. Ch represents ¹⁴C chloramphenicol; bCh represents butyrylated ¹⁴C chloramphenicol and is the product of CAT enzyme activity.