Hoeijmakers J H, Borst P, van den Burg J, Weissmann C, Cross G A
Gene. 1980 Mar;8(4):391-417. doi: 10.1016/0378-1119(80)90043-8.
We have isolated poly(A)+ RNA from four antigenic variants (117, 118, 121, 221) of one clone of Trypanosoma brucei. Translation of these poly(A)+ RNAs in a rabbit reticulocyte lysate gave rise to proteins that could be precipitated with antisera against homologous variant surface glycoprotein, the protein responsible for antigenic variation in trypanosomes. From the electrophoretic mobility of these in vitro products in sodium dodecyl sulphate (SDS) gels we infer that variant surface glycoproteins (VSGs) are made as pre-proteins, which require trimming to yield mature VSGs. The total translation products from the four poly(A)+ RNAs produced a complex set of bands on SDS gels, which only differed in the region where the variant pre-glycoproteins migrated. The only detectable variation in the messenger RNA populations of these variants is, therefore, in the messenger RNA for variant pre-glycoproteins. We have made duplex DNA copies of these poly(A)+ RNAs, linked the complementary DNA to plasmid pBR322 by GC tailing and cloned this recombinant DNA in Escherichia coli. Colony hybridization with complementary DNA made on poly(A)+ RNA showed that 7--10% of the colonies contained DNA that hybridized only with the homologous probe. Plasmid DNA was isolated from ten such colonies (two or three of each variant complementary DNA), bound to diazobenzyloxymethyl-cellulose (DBM) paper and used to select complementary messenger RNA from total poly(A)+ RNA by hybridization. In eight cases the RNA recovered from the filter gave variant pre-glycoprotein as the predominant product of in vitro translation. Poly(A)+ RNA from each of the variants only hydridized to the homologous complementary DNA in filter hybridizations. Each trypanosome variant, therefore, contains no detectable messenger RNAs for the three heterologous variant-specific glycoproteins tested. We conclude from this lack of cross-hybridization that antigenic diversity in trypanosomes, unlike antibody diversity in mammals, does not involve the linkage of a repertoire of genes for the variable N-terminal half to a single gene for the C-terminal half of the VSGs.
我们从布氏锥虫一个克隆的四个抗原变异体（117、118、121、221）中分离出了多聚腺苷酸[poly(A)]⁺RNA。这些多聚(A)⁺RNA在兔网织红细胞裂解物中进行翻译，产生的蛋白质能够被针对同源变异体表膜糖蛋白的抗血清沉淀，该糖蛋白是锥虫中负责抗原变异的蛋白质。从这些体外产物在十二烷基硫酸钠（SDS）凝胶中的电泳迁移率，我们推断变异体表膜糖蛋白（VSG）是以前体蛋白的形式合成的，需要进行修剪才能产生成熟的VSG。来自这四种多聚(A)⁺RNA的总翻译产物在SDS凝胶上产生了一组复杂的条带，它们仅在变异体前体糖蛋白迁移的区域有所不同。因此，这些变异体的信使RNA群体中唯一可检测到的差异在于变异体前体糖蛋白的信使RNA。我们制备了这些多聚(A)⁺RNA的双链DNA拷贝，通过GC加尾将互补DNA连接到质粒pBR322上，并将这种重组DNA克隆到大肠杆菌中。用基于多聚(A)⁺RNA制备的互补DNA进行菌落杂交，结果显示7% - 10%的菌落含有仅与同源探针杂交的DNA。从十个这样的菌落（每个变异体互补DNA两到三个）中分离出质粒DNA，将其结合到重氮苄氧基甲基纤维素（DBM）纸上，并用于通过杂交从总多聚(A)⁺RNA中选择互补信使RNA。在八个案例中，从滤膜上回收的RNA在体外翻译时产生的主要产物是变异体前体糖蛋白。在滤膜杂交中，每个变异体的多聚(A)⁺RNA仅与同源互补DNA杂交。因此，每个锥虫变异体都不含有针对所测试的三种异源变异体特异性糖蛋白的可检测到的信使RNA。我们从这种缺乏交叉杂交的情况得出结论，锥虫中的抗原多样性与哺乳动物中的抗体多样性不同，它不涉及可变N端一半的基因库与VSG C端一半的单个基因的连接。
