THE ISOLATION OF PLASMIDS CONTAINING DNA COMPLEMENTARY TO MESSENGER RNA FOR VARIANT SURFACE GLYCOPROTEINS OF Trypanosoma brucei

SUMMARY 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 con*Present address: Laboratory of Medical Microbiology, Mauritskade 57, Amsterdam (The Netherlands) Abbreviations: bp, base pairs; cDNA, complementary DNA; DBM paper, diazobenzyloxymethyl-cellulose paper; kd, kilodaltons; rRNA, ribosomal RNA; SDS, sodium dodecyl sulphate; VSG, variant surface glycoprotein.