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

Analysis of cosmid clones of nuclear DNA from Trypanosome brucei shows that the genes for variant surface glycoproteins are clustered in the genome.

Trypanosoma brucei contains more than a hundred genes coding for the different variant surface glycoproteins (VSGs). Activation of some of these genes involves the duplication of the gene (the basic copy or BC) and transposition of the duplicate to an expression site (yielding the expression-linked copy or ELC). We have cloned large fragments of genomic DNA in cosmid vectors in Escherichia coli. Cosmids containing the BCs of genes 117, 118 and 121 were readily obtained, but DNA containing the ELCs was strongly selected against in the cosmid and plasmid cloning systems used. We have analysed the distribution of VSG genes in the genome using probes for the sequences at the edges of the transposed segment which are partially homologous among these genes. In genomic cosmid clone banks, about 9% of all colonies hybridize with probes from the 5'- and 3'-edges of the transposed segment, showing that these sequences are linked in the genome. Moreover, the 117 and 118 BC cosmids contain several additional putative VSG genes in tandem, as deduced from hybridization and sequence analyses. We conclude that the VSG genes are highly clustered and share common sequences at the borders of the transposed segment

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

RNA from the insect trypanosome Crithidia luciliae contains transcripts of the maxi-circle and not of the mini-circle component of kinetoplast DNA.

We have hybridized total cellular RNA of Crithidia luciliae with the kinetoplast DNA of this organism. To allow the discrimination of DNA from mini-circles (2300 base pairs) and maxi-circles (33 000 base pairs), kinetoplast DNA was digested with restriction endonucleases and the fragments were separated by electrophoresis through an agarose gel and transferred to nitrocellulose filters by blotting. No mini-cricle transcripts were found under conditions where maxi-circle fragments showed extensive and specific hybridization. Since maxi-circle sequences are present at less than 1% of the concentration of mini-circle sequences, we conclude that mini-circles may not be transcribed at all. Predominant hybridization with the maxi-circle fragments is obtained with a segment of only 2300--2500 base pairs. The possibility that this segment codes for unusually small mitochondrial ribosomal RNAs is discussed

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

Variations in maxi-circle and mini-circle sequences in kinetoplast DNAs from different Trypanosoma brucei strains.

We have compared a total of 30 recognition sites for eight restriction endonucleases on the 20-kilobase-pair maxi-circle of kinetoplast DNAs from five different Trypanosoma brucei strains. In addition to three polymorphic sites were have found a 5 kilobase-pair region that is not cleaved by any of the eight enzymes and that varies in size over 1 kilobase pair in the strains analysed. Mini-circles from these five strains, digested with endonuclease TaqI or MboII, yield very complex fragment patterns, showing that extensive mini-circle sequence heterogeneity is a common characteristic of these T. brucei strains. The size distribution of mini-circle fragments in these digests was identical for different clones of the 427 strain, but very different for mini-circles from different strains. These results show that maxi-circle sequence is conserved, whereas mini-circle sequence is not. Restriction digests of maxi-circles could be useful in determining how closely two Trypanosoma strains are related, whereas mini-circle digests can serve as sensitive tags for individual strains