Open AccessIsolation of mRNA from KB‐Cells by Affinity Chromatography on Polyuridylic Acid Covalently Linked to Sepharose
Author(s) -
Lindberg Uno,
Persson Torgny
Publication year - 1972
Publication title -
european journal of biochemistry
Language(s) - English
Resource type - Journals
eISSN - 1432-1033
pISSN - 0014-2956
DOI - 10.1111/j.1432-1033.1972.tb02527.x
Subject(s) - polysome , messenger rna , sepharose , rna , cytoplasm , ribosomal rna , microbiology and biotechnology , affinity chromatography , ribosome , covalent bond , biochemistry , biology , chemistry , enzyme , gene , organic chemistry
Messenger RNA from polysomes of KB‐cells was isolated by affinity chromatography on columns of polyuridylic acid covalently linked to Sepharose. The mRNA molecules were retained by the resin apparently via their poly(A) segments by base pairing to the poly(U). Ribosomal RNA and transfer RNA were not retained by the columns and were thus removed from the mRNA. The mRNA was recovered to an extent of 90% and apparently in intact form. This method allows studies of mRNA resulting from unabated synthesis and was used here in studies of the size distribution of different classes of cytoplasmic poly(A)‐containing RNA. The presence of poly(A)‐containing RNA in the non‐polysomal fractions of the cytoplasm was demonstrated. This putative mRNA was shown to constitute about 30% of the total cytoplasmic poly(A)‐containing RNA. Also by using the poly (U)‐Sepharose technique it was demonstrated that actinomycin D at low concentration, 0.04 μg/ml, supresses the appearance of mRNA on polysomes to an extent of 50%. This low concentration of the drug was previously though to effect only ribosomal RNA synthesis.