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

TLDR

The study presents a large‑scale method for purifying light and heavy chain mRNAs from mouse plasmacytoma tumors. Intact RNA is precipitated from frozen tumors using 3 M LiCl and 6 M urea, then L and H‑chain mRNAs are purified by oligo(dT)-cellulose chromatography followed by either sucrose gradient centrifugation or high‑resolution preparative gel electrophoresis under non‑denaturing conditions, with purity assessed by cDNA hybridization. The purified H‑chain mRNAs sedimented at 15.5 S (γ2a) and 16.5 S (α), migrated as ~1900 nt species, and translated into 56 kDa and 60 kDa proteins with up to 95 % immunoprecipitation, while L‑chain mRNAs sedimented at 12 S and produced 25–27 kDa polypeptides; purity ranged from 47 % to 62 % for H‑chains and 60 % for L‑chains.

Abstract

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.