Polysome profile analysis of PLE-containing mRNA. Cos-1 cells were transfected with firefly luciferase expression plasmids without (control) or with a PLE (+PLE). Post-nuclear extracts were applied to a linear 10%–45% sucrose gradient and centrifuged at 225,000g for 2 h at 4°C in the direction indicated by the arrow. (A) Fractions of 0.25 mL were collected from the bottom and the absorbance profile at 254 nm is shown. (B) The sedimentation of control and PLE-containing (+PLE) luciferase mRNA was determined by RNase protection assay of RNA recovered from even-numbered fractions, and radioactivity was determined by PhosphorImager analysis. (C) The radioactivity in each RPA fraction in B was normalized to the total counts to yield a percent distribution of luciferase mRNA across the gradient.
