Figure 9.

H5R mRNAs associate with polyribosomes. Lysates of cells infected for 2 h in the presence of hydroxyurea were subjected to polyribosome gradients. Each fraction was subjected to Northern blotting with the use of a specific probe to H5R mRNA. The positions of the 40S, 60S, and 80S ribosomal subunits and the polyribosome containing fractions as well as the top (10% sucrose) and the bottom (50% sucrose) of the gradient are indicated. (A) The analysis of untreated cell lysates shows that a substantial amount of H5R mRNA associates with the heavy polyribosome-containing fractions. (B) The lysate was treated with 25 mM EDTA before centrifugation to disintegrate polyribosomes. (C) Infected cells were treated with 100 μg/ml puromycin before lysis to release ribosomes from mRNAs. In both B and C H5R mRNAs can no longer be detected in the fractions expected to contain polyribosomes. The panels on the right indicate the corresponding OD254 read-out of the different fractions.