Figure 1.

The 3′UTR of DDB2 confers instability to a heterologous reporter gene. (A) Schematic representation of the pTRE-d2EGFP vector used to generate 3′UTR reporter constructs. The location of the tetracycline response element (TRE), minimal cytomegalovirus promoter (CMV), EGFP with a C-terminal PEST sequence (d2EGFP), 3′UTR cloning site and the polyadenylation signal are indicated. (B) HeLa Tet-Off® cells were transfected with the pTRE-d2EGFP vector or vectors containing a 3′UTR corresponding to the indicated nucleotides from the 3′UTR of DDB2. In (B) and (F), doxycycline (1 µg/ml) was added 24 h after transfection and total RNA was collected at the indicated times to monitor loss of d2EGFP expression by qRT-PCR. (C) Stable pools of d2EGFP positive cells were generated through multiple rounds of fluorescence activated cell sorting. (D and E) In all, 1 µg/ml of doxycycline was added to stable cell lines expression d2EGFP with the indicated 3′UTR sequence. At the indicated time, total RNA was collected for qRT-PCR analysis of d2EGFP expression. Each value in (B), (D), (E) and (F) represents the mean (±SEM) determined in several independent experiments. The asterisk and double asterisk indicate the curve is significantly different from the vector control as determined by two-way ANOVA at P < 0.05 and P < 0.0001, respectively.