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RNA with high template activity from leukaemic myeloblasts (Bai strain a virus‐induced avian myeloblastosis) II. Sedimentation profiles of template activity and of rapid 32 P labelling
Author(s) -
Veprek L.,
Trávníček M.,
Ríman J.
Publication year - 1967
Publication title -
international journal of cancer
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 2.475
H-Index - 234
eISSN - 1097-0215
pISSN - 0020-7136
DOI - 10.1002/ijc.2910020507
Subject(s) - rna , ribosome , phenol extraction , centrifugation , ribosomal rna , microbiology and biotechnology , biochemistry , messenger rna , sedimentation coefficient , biology , cytoplasm , cell fractionation , chemistry , enzyme , gene
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 32 P‐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.