Isolation of mRNA from KB‐Cells by Affinity Chromatography on Polyuridylic Acid Covalently Linked to Sepharose

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Abstract
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.