A temperature-sensitive mutation affecting 28S ribosomal RNA production in mammalian cells

Abstract

We have characterized a temperature-sensitive (ts) mutant of the hamster cell line BHK 21 that appears to have a defect in the processing of ribosomal RNA precursors at 39 degrees . Mutant ts 422E grows at a normal rate at 33 degrees , but upon shift to 39 degrees growth stops after about one cell doubling. The appearance of 28S rRNA and large ribosomal subunits in the cytoplasm of ts 422E at 39 degrees is inhibited by about 95%, when compared to wild-type BHK cells. Production of 18S rRNA and small ribosomal subunits is unaffected. Shift-up experiments show that the defect in 28S rRNA production can be detected as early as 2-3 hr after the shift to 39 degrees . Synthesis of the larger rRNA precursor is normal at high temperature, but the processing appears to be arrested after the formation of 32S rRNA. 32S rRNA accumulates to some extent in the nucleoli of ts 422E. ts 422E cells appear to have a single mutation, directly affecting the conversion of 32S to 28S rRNA. The reduced amount of 28S rRNA in the cytoplasm of ts 422E cells at 39 degrees seems therefore responsible for their inability to grow at this temperature.