Purification of Mouse Immunoglobulin Heavy‐Chain Messenger RNAs from Total Myeloma Tumor RNA

FEBS Journal - Tập 107 Số 2 - Trang 303-314 - 1980
Charles Auffray1, François Rougeon1
1Groupe de Biologie Moléculaire du Gène, Equipe de Recherche Associée N° 201 du Centre National de la Recherche Scientifique, Unité de Génie Génétique, Institut Pasteur, 28 Rue du Docteur-Roux, F-75724 Paris-Cedex 15, France

Tóm tắt

A procedure is described for the large‐scale purification of light (L) and heavy (H) chain mRNAs from plasmacytomas produced in mice. Intact RNA is selectively precipitated in high yield from frozen tumors homogenized in 3 M LiCl and 6 M urea. L and H‐chain mRNAs were purified by oligo(dT)‐cellulose chromatography and either sucrose gradient centrifugation in conditions preventing aggregation or by means of high‐resolution preparative gel electrophoresis under non‐denaturing conditions. γ2a and α H‐chain mRNAs sedimented as major components at 15.5 S and 16.5 S respectively, when L‐chain mRNAs sedimented as 12‐S species. H‐chain mRNAs isolated by continuous elution during preparative gel electrophoresis were completely separated from both L‐chain mRNA and residual 18‐S rRNA, and migrated as single components of 1900 ± 50 nucleotides on analytical denaturing gels. The partially purified H‐chain mRNAs were translated into major components of molecular weights of 56000 (γ2a) and 60000 (α) in an mRNA‐dependent rabbit reticulocyte lysate, whereas L‐chain mRNAs yielded polypeptides of molecular weights of 25000 (λ) and 27000 (к). Up to 95% of the translation products directed by the purified mRNAs were immunoprecipitated using specific antisera. The purity of L and H‐chain mRNAs was assessed by hybridization of corresponding cDNAs with excess recombinant plasmid DNA. The results indicated a minimum purity of 47% (γ2a), 62% (α), for H‐chain mRNAs and 60% (к), for L‐chain mRNAs.

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FEBS Journal

Tập 107 Số 2

303-314

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