RNA with high template activity from leukaemic myeloblasts (BAI strain A virus-induced avian myeloblastosis). I. Optimal conditions for its isolation and activity in the subcellular protein-synthesizing system.

Abstract

AbstractThree methods of phenol fractionation of metabolically active RNA of leukaemic myeloblasts were compared on the basis of the template activity of the isolated fractions. It was shown that the method of phenol fractionation based on increasing the extraction temperature is most suitable for obtaining a fraction substantially enriched in template active RNA.Standardization of the extraction conditions employed (the use of acetate buffer pH6) led to the separation of RNA of high template activity (65°‐RNA) not contaminated with DNA and highly purified with respect to the presence of other cellular RNAs.The template activity of isolated RNA fractions was tested in the subcellular protein‐synthesizing system from E. coli under the optimal conditions described here with respect to the function of the RNA fraction with the highest template activity (65°‐ RNA).It was found that the 65°‐RNA fraction has characteristic sedimentation properties, a nucleotide composition resembling the DNA of leukaemic myeloblasts and, by its high template activity, meets the requirements of a highly purified preparation of m RNA. Its template activity, tested by incorporation of two amino acids (2160 pmol leucine, 578 pmol phenylalanine/mg RNA) exceeds several‐fold the highest values of template activity so far published for ribonucleic acids isolated from the cells of higher organisms.It was found that the same results can be achieved by applying the method to other types of tissue, such as normal chick liver tissue.