Sub‐Ribosomal Particles of Cultured Chinese‐Hamster Cells

Abstract

Low‐molecular‐weight methylated RNAs exist in the cytoplasm of mechanically or chemically disrupted cultured Chinese hamster ovary cells as nucleoproteins sedimenting in the region of 10—30 S. A differential population of tRNA, representing about 4% tRNA of the cells, is also recovered from this region as is a differential population of aminoacyl synthetases The relationship of these RNA populations to each other and protein moieties of the 10—30‐S sub‐ribosomal region is the subject of this investigation. As regards preparation of the sub‐ribosomal particles, it is found that maximal yield of 10—30‐S material with retention of enzyme activity is achieved when cytoplasm is prepared using the non‐ionic detergent nonidet P‐40. The ratio of protein‐to‐RNA in this region, which contains 2% protein of the cells, is about 17:1. Concentration and resedimentation of fractions within the 10—30‐S region resolve two peaks of approximetaly 18 S and 21 S comprised mainly of protein and a primarily nucleic acid peak of about 20 S which contains most of the tRNA. Low‐molecular‐weight methylated RNAs are recovered from the entire 18—20‐S region with the exception of the largest species, which is preferentially recovered from the 21‐S region. Isopycnic cesium chloride analysis of the 18—20‐S region after formaldehyde fixation resolves most of the RNA from the majority of the protein. The particulate RNA bands at 1.32—1.38g/ml before and at >1.7g/ml after pronase treatment. Sedimentation values of the protein particles are unaffected by ribonuclease or by prolonged storage at‐15 °C or 4 °C. However, tRNA is released as free tRNA as a function of time at 4 °C without concurrent degradation of the 18‐S and 21‐S protein particles and without release of low‐molecular‐weight methylated RNA. Thus, it appears that most of the particle tRNA is unassociated with the major sub‐ribosomal protein particles or with the particles containing low‐molecular‐weight methylated RNA.