Figure 5.

The XbaI–HaeIII site acts as a stability element within a heterologous transcript. (a) Stable subclones of Jurkat/D1.1 cells expressing Renilla luciferase with (solid line) or without (broken line) the CD40 ligand (CD40L) XbaI–HaeIII stability region were analysed for messenger RNA stability by the addition of 50 μg/ml 5,6-dichlorobenzimidizole 1-m-d-ribofuranoside (DRB) over an 8-hr time course. Cells (5 × 10⁶) were collected for each time-point, RNA was extracted and triplicate samples were quantified using quantitative polymerase chain reaction (qPCR). The fractions of total RNA remaining at each time-point are plotted. Results are the average of three independent experiments and curve-fitting was performed by non-linear regression. (b) Cytoplasmic extracts of stable subclones of Jurkat/D1.1 cells expressing Renilla luciferase with (dark rectangles, pRLABC) and without (light rectangles, pRLSV40) the CD40L XbaI–HaeIII region were fractionated into polysome-associated and non-associated fractions by centrifugation at 130 000 g. The pellet was further washed with high salt to release loosely associated proteins. Analysis of Renilla luciferase RNA (left graph) and 28S RNA (right graph) was carried out using qPCR.