Isolation of the repertoire of VSG expression site containing telomeres of Trypanosoma brucei 427 using transformation-associated recombination in yeast

Abstract

Trypanosoma brucei switches between variant surface glycoproteins (VSGs) allowing immune escape. The active VSG is in one of many telomeric bloodstream form VSG expression sites (BESs), also containing expression site-associated genes (ESAGs) involved in host adaptation. The role of BES sequence diversity in parasite virulence can best be understood through analysis of the full repertoire of BESs from a given T. brucei strain. However, few BESs have been cloned, as telomeres are highly underrepresented in standard libraries. We devised a strategy for isolating the repertoire of T. brucei 427 BES-containing telomeres in Saccaromyces cerevisiae by using transformation-associated recombination (TAR). We isolated 182 T. brucei 427 BES TAR clones, 167 of which could be subdivided into minimally 17 BES groups. This set gives us the first view of the breadth and diversity of BESs from one T. brucei strain. Most BESs ranged between 40 and 70 kb (average, 57 ± 17 kb) and contained most identified ESAGs. Phylogenetic comparison of the cohort of BES promoter and ESAG6 sequences did not show similar trees, indicating rapid evolution most likely mediated by sequence exchange between BESs. This cloning strategy could be used for any T. brucei strain, facilitating research on the biodiversity of telomeric gene families and host-pathogen interactions.

The central roles of telomeres and subtelomeres in antigenic variation in African trypanosomes

“…A few bacterial artificial chromosomes containing complete or partial BESs have been sequenced (Berriman et al 2002). More recently, transformation-associated recombination in yeast, which isolates complete chromosome ends, has been used to generate stable clones representing at least 17 BESs from the Lister 427 strain (Becker et al 2004). This approach is being used also to isolate specific chromosome ends by cloning via the final known sequences in current chromosome assemblies (E. Louis, personal communication).…”

“…In this case, they may have had a role, through retrotransposition, in early evolution of the silent archive, and more recently through homologous recombination, resulting in the moving and inversion of subarrays. Rapid diversification of other subtelomeric families, most likely by recombination, is apparent also in trypanosome BESs (Becker et al 2004).…”

Telomeres in fungi

“…In these cells such 'contingency genes' are often found in subtelomeric domains, with subtelomeric chromatin silencing regulating which are expressed. This is the case not only in protozoan pathogens such as Plasmodium falciparum (Hernandez-Rivas et al 1997) and Trypanosoma brucei (Chaves et al 1999;Becker et al 2004) but in fungal pathogens too.…”

“…Clones that lost CYH2 can be selected and characterised. This method has been used successfully to clone the six chromosome ends of Schizosaccharomyces pombe (Sharp and Louis, personal communication), as well as the telomeres of Pneumocystis carinii (Underwood et al 1994(Underwood et al , 1996, Trypanosoma brucei (Becker et al 2004) and others.…”

“…Telomere-associated recombination (TAR) cloning (Larionov et al 1996;Noskov et al 2003;Becker et al 2004) is a strategy for targeting a specific chromosome end using an anchor sequence (500bp) unique to that end. The anchor sequence is cloned into a half-YAC vector between a negative selectable marker (CYH2) and the rest (URA3, CEN4, ARS1, and telomeric repeat array).…”