RNA with high template activity from leukaemic myeloblasts (Bai strain a virus-induced avian myeloblastosis) II. Sedimentation profiles of template activity and of rapid 32P labelling

RNA preparations made from leukaemic myeloblasts by fractionating extraction in a two-phase system with phenol-0.1 M sodium acetate (pH 6.0), using successively increasing temperatures, were analyzed by centrifugation in a sucrose gradient. The RNA of the individual fractions was precipitated with soluble RNA as carrier, and the template activity of the fractions was determined in a subcellular protein-synthesizing system from E. coli. Distribution of rapidly labelled material in similar preparations 2 h after an intravenous administration of 32P-orthophosphatewas also investigated. A comparison of the sedimentation profiles obtained showed that cytoplasmic RNAs extracted at 25°C showed only low template activity in the region of 15–30S. Preparations isolated at 45°C contained the two precursors of ribosomal RNA, 45S and 35S, exhibiting a pronounced template activity. The final extraction at 65°C yielded preparations of messenger RNA of outstanding purity, their main component sedimenting in the region of 18S and another significant portion sedimenting heterogeneously over a wide range from 18 to 50S. In the presence of all the cofactors required for protein synthesis, RNA extracted at 65°C was able to bind one or more E. coli ribosomes. At the same time, this RNA was rapidly degraded.