Auffray C, Rougeon F
Eur J Biochem. 1980 Jun;107(2):303-14. doi: 10.1111/j.1432-1033.1980.tb06030.x.
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. gamma 2a and alpha 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 56,000 (gamma 2a) and 60,000 (alpha) in an mRNA-dependent rabbit reticulocyte lysate, whereas L-chain mRNAs yielded polypeptides of molecular weights of 25,000 (gamma) and 27,000 (chi). 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% (gamma 2a), 62% (alpha), for H-chain mRNAs and 60% (chi), for L-chain mRNAs.
本文描述了一种从在小鼠体内产生的浆细胞瘤中大规模纯化轻链（L）和重链（H）mRNA的方法。完整的RNA可从在3M LiCl和6M尿素中匀浆的冷冻肿瘤中以高产率选择性沉淀出来。L链和H链mRNA通过寡聚（dT）-纤维素柱层析进行纯化，然后在防止聚集的条件下进行蔗糖梯度离心，或者在非变性条件下通过高分辨率制备凝胶电泳进行纯化。当L链mRNA沉降为12-S种类时，γ2a和αH链mRNA分别作为主要成分沉降在15.5S和16.5S。在制备凝胶电泳过程中通过连续洗脱分离得到的H链mRNA与L链mRNA和残留的18-S rRNA完全分离，并且在分析性变性凝胶上作为1900±50个核苷酸的单一成分迁移。部分纯化的H链mRNA在依赖mRNA的兔网织红细胞裂解物中被翻译成分子量为56,000（γ2a）和60,000（α）的主要成分，而L链mRNA产生分子量为25,000（γ）和27,000（χ）的多肽。使用特异性抗血清可免疫沉淀高达95%的由纯化mRNA指导的翻译产物。通过相应的cDNA与过量重组质粒DNA的杂交来评估L链和H链mRNA的纯度。结果表明H链mRNA的最低纯度为47%（γ2a）、62%（α），L链mRNA的最低纯度为60%（χ）。
