The isolation of plasmids containing DNA complementary to messenger RNA for variant surface glycoproteins of Trypanosoma brucei.

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-termina

The protein that binds to DNA base J in trypanosomatids has features of a thymidine hydroxylase

© 2007 The Author et al. This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/ by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. The definitive version was published in Nucleic Acids Research 35 (2007): 2107-2115, doi:10.1093/nar/gkm049.Trypanosomatids contain an unusual DNA base J (ß-D-glucosylhydroxymethyluracil), which replaces a fraction of thymine in telomeric and other DNA repeats. To determine the function of base J, we have searched for enzymes that catalyze J biosynthesis. We present evidence that a protein that binds to J in DNA, the J-binding protein 1 (JBP1), may also catalyze the first step in J biosynthesis, the conversion of thymine in DNA into hydroxymethyluracil. We show that JBP1 belongs to the family of Fe2+ and 2-oxoglutarate-dependent dioxygenases and that replacement of conserved residues putatively involved in Fe2+ and 2-oxoglutarate-binding inactivates the ability of JBP1 to contribute to J synthesis without affecting its ability to bind to J-DNA. We propose that JBP1 is a thymidine hydroxylase responsible for the local amplification of J inserted by JBP2, another putative thymidine hydroxylase.This work was funded by a grant from the Netherlands Organization for Scientific Research and Chemical Sciences (NWO-CW) to P.B., NIH grant A1063523 to R.S. and NIH grant GM063584 to R.P.H

Biochemistry of variant antigens

Meeting: Conference on Recent Advances in the Knowledge of Pathogenicity of Trypanosomes, 20-23 Nov. 1978, Nairobi, KEIn IDL-329

Systematic study of sequence motifs for RNA trans splicing in Trypanosoma brucei

10.1128/MCB.25.21.9586-9594.2005Molecular and Cellular Biology25219586-9594MCEB

A variant surface glycoprotein of Trypanosoma brucei is synthesized with a hydrophobic carboxy-terminal extension from purified glycoprotein.

Sequential expression of variant surface glycoproteins (VSGs) enables the parasitic protozoan Trypanosoma brucei to evade the immune response of its mammalian hosts. Studies of several VSGs, which have been isolated as soluble molecules following disruption of cells in the absence of detergent, have indicated extensive amino acid diversity and the absence of a hydrophobic segment which might serve to anchor the carboxy terminus to the membrane. The carboxy-terminal tryptic peptides of six VSGs have recently been characterized and shown to be glycosylated. Three of these VSGs terminated with a glycosylated aspartate or asparagine residue (Asx), suggesting that the VSG was cleaved following synthesis and glycosylation and before characterization. We present here nucleotide sequence data which suggest that the primary translation product of one VSG gene contains a hydrophobic tail at the carboxy terminus which is not found on the isolated, mature glycoprotein. The data also predict that the glycosylated residue is aspartic acid rather than the anticipated asparagine

The genes for variant antigens in trypanosomes.

We have studied the mechanism of antigenic variation by using DNA complementary to the messenger RNAs for four variant surface glycoproteins of Trypanosoma brucei. Pure complementary DNAs were obtained by cloning as recombinant DNA in Escherichia coli. Using these complementary DNAs as hybridization probes, we have analyzed the genes for these variant surface glycoproteins. The results provide new information on the origin and evolution of antigenic variation, and on the mechanism involved in switching from one antigenic type to another