Figure 6.

Translation of nonstop mRNA is generally repressed. (A) Yeast cells were grown on SC-Leu medium, and HIS3 mRNAs were detected by Northern blotting with DIG-labeled GFP probe. Lanes 1 and 2: W303; lanes 3 and 4: YIT888 (ski2Δ); lanes 5 and 6: YIT890 (ski3Δ); lanes 7 and 8: YIT929 (ski7Δ). Cells were transformed with pIT810 (pGFP, odd lanes) or pIT811 (pgfp-ns, even lanes). (B) Yeast cells as described in (A) were grown on SC-Leu medium, and proteins from cell extracts equivalent to 0.1 OD₆₀₀ were subjected to 12% SDS–PAGE and analyzed by immunoblot analysis with anti-GFP antibodies. (C) W303 cells harboring pIT810 (pGFP) or pIT811 (pgfp-ns) were grown on SC-Leu medium and cell extracts were prepared after the addition of 0.1 mg/ml CHX. Cell extracts equivalent to 40 A₂₆₀ were resolved by velocity sedimentation on 10–50% sucrose gradients. RNA samples prepared from the indicated fractions were analyzed by Northern blotting with DIG-labeled GFP probe.