Journal of Molecular Biology
Volume 69, Issue 3, 28 August 1972, Pages 373-376, IN5, 377-386
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Isolation of messenger-like RNA from immunochemically separated polyribosomes

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Abstract

A method for the isolation of a given messenger RNA from mammalian cells has been developed. This method consists of (i) specific immunochemical precipitation of polyribosomes synthesizing the protein, and (ii) extraction of the mRNA coding for this protein from the polyribosome/antigen/antibody complex.

From a study of the kinetics of 32P incorporation into the cytoplasmic RNA of MOPC 149 mouse plasmacytoma cells, which secrete an immunoglobulin κ-type light chain, it was concluded that within 30 minutes two species of RNA were predominantly labelled, i.e. transfer RNA and a class of polydisperse RNA considered to be mRNA. Polyribosomes labelled with 32P were isolated from MOPC 149 plasmacytomas and treated with an excess of F(ab′)2 fragments prepared from either a specific (rabbit anti-MOPC 149 L-chain) or a control (rabbit anti-goat IgG) antiserum. Optimum amounts of the respective antigens were added to the soluble complexes as carriers to yield maximum precipitation. A comparison of the 32P activity in the precipitates revealed that L-chain-bearing polyribosomes were specifically isolated. A rapidly labelled species of RNA, having a sedimentation coefficient of 11 to 12 s and a base composition of 48 moles of G + C per cent, was isolated from the precipitated polyribosomes. These properties can be directly correlated with the size and composition of the MOPC 149 L-chain and suggest that this RNA molecule is the mRNA coding for this L-chain.

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    This work was initiated in the Department of Chemistry, McGill University and completed in the Department of Immunology, University of Manitoba. One of us (T.L.D.) was the recipient of a postgraduate scholarship from the National Research Council of Canada. This study was supported by a grant from the Medical Research Council of Canada and, in part, by the Banting Research Foundation.

    Present address: Department of Biology, Massachusetts Institute of Technology, Cambridge, Mass. U.S.A.

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