Figure 7.

Gag sedimentation and nuclease protection of Ty1 mRNA in wild type and loc1Δ strains. (A) Total cell extracts from BY4742 WT or a loc1Δ mutant were centrifuged through a 7–47% continuous sucrose gradient. Ten micrograms of protein extract (input) and equal volumes of each fraction (15 μl/lane) were subjected to western blot analysis using Gag antiserum (TY-tag), which recognizes Gag isoforms and Gag-p45. Fractions containing the highest concentrations of Gag, as determined by densitometry tracing, are underlined. Sucrose gradient analysis was repeated three times and a representative experiment is shown here. (B) Ty1 mRNA packaging as monitored by sensitivity to the nuclease benzonase. Equal aliquots of whole cell extract from BY4742 WT and a loc1Δ mutant were incubated with (+) or without (−) benzonase. To detect Ty1 mRNA, total RNA extracted from these samples was analyzed by northern blot analysis. Nuclease protection assays were repeated three times and a representative experiment is shown. WT protection ranged from 26.5 to 31.4%, and loc1Δ protection ranged from 16.1 to 18.8%. ACT1 was used as a control to confirm RNA degradation in the benzonase treated samples.