FIG. 5.

Identification of a sequence with a CTE-like activity in the MusD 3′ untranslated region. (A) Rationale of the assay and structure of the reporter pDM128/PL vector. pDM128/PL contains a cat gene flanked by donor and acceptor splice sites (SD and SA) and placed under the control of a simian virus 40 promoter and polyadenylation signal (prom, pA). Sequences to be tested for CTE activity are inserted as indicated. The presence of a CTE should promote the export of unspliced RNA from the nucleus of cells transfected with the reporter plasmid, leading to a detectable CAT activity. The absence of CTE-like activity should lead to the export of spliced RNA and a lack of CAT activity. (B) 293T cells were transiently transfected with pDM128/PL vectors containing the indicated MusD fragments, placed in the forward or reverse orientation (termini are indicated by nucleotide positions in the MusD sequence, and orientation is indicated by arrows). At 48 h posttransfection, cells were lysed and CAT activity was determined by using [¹⁴C]chloramphenicol as described in Materials and Methods. The mean ratio of CAT activity between pDM128/PL containing a MusD (or control MPMV CTE) sequence versus the empty vector (no CTE) was calculated from two to four independent experiments.