Telomeric Expression Sites Are Highly Conserved in Trypanosoma brucei

Abstract

Subtelomeric regions are often under-represented in genome sequences of eukaryotes. One of the best known examples of the use of telomere proximity for adaptive purposes are the bloodstream expression sites (BESs) of the African trypanosome Trypanosoma brucei. To enhance our understanding of BES structure and function in host adaptation and immune evasion, the BES repertoire from the Lister 427 strain of T. brucei were independently tagged and sequenced. BESs are polymorphic in size and structure but reveal a surprisingly conserved architecture in the context of extensive recombination. Very small BESs do exist and many functioning BESs do not contain the full complement of expression site associated genes (ESAGs). The consequences of duplicated or missing ESAGs, including ESAG9, a newly named ESAG12, and additional variant surface glycoprotein genes (VSGs) were evaluated by functional assays after BESs were tagged with a drug-resistance gene. Phylogenetic analysis of constituent ESAG families suggests that BESs are sequence mosaics and that extensive recombination has shaped the evolution of the BES repertoire. This work opens important perspectives in understanding the molecular mechanisms of antigenic variation, a widely used strategy for immune evasion in pathogens, and telomere biology.

Transcription is initiated on silent variant surface glycoprotein expression sites despite monoallelic expression in Trypanosoma brucei

“…Most sequences were present in 1-11 copies, except for G1, which was present in 30-40 copies. Because G1 is present in both FM162566 and FM162572 (33), and FM162566 is the active ES in this isolate, the relatively high proportion of this PCR product was in keeping with the higher transcription efficiency in the dominant ES (36) and with the more efficient processing of transcripts from the active ES (29). Here again, 11-17 outliers (12-19% of the total sequences) were generated.…”

“…29 were not represented, possibly because different trypanosome strains were analyzed. As previously noticed (29,33,34) these sequences can be classified as evolving one from another. Not classified in these groups are 11 different sequences (11.7% of the total) generated by loner mutations that possibly could originate from sequencing/PCR mistakes (Table 1, column 2).…”

“…The sequences were numbered according to the nomenclature adopted in ref. 29 (Table 1 and Table S1, column 1), and correspondence was made to the nomenclature of Hertz-Fowler et al (33) (Table S2). All sequences reported by Hertz-Fowler et al (33) were recovered.…”

“…According to Vanhamme et al (29), the large transcript heterogeneity found in some antigenic variants at the beginning of the expression site was lost during downstream progression from the promoter. Notably, between 3 and 5 kbp downstream from the promoter the heterogeneity was only ∼2, corresponding to the transcripts from the two homologous genes, ESAG6 (expression site associated gene 6) and ESAG7 (expression site associated gene 7), located in tandem in all ESs except BES8/ TAR64 (FM162575) (33,34). Single-cell RT-PCR analysis of the ESAG6/7 region was performed in the MiTat isolate.…”

“…Single-cell RT-PCR analysis of the ESAG6/7 region was performed in the MiTat isolate. First, a genomic control generated four sequences found by both Vanhamme et al (29) and Hertz-Fowler et al (33) and three sequences that were found only by Vanhamme et al (29) (Table S1, column 2). After serial dilution, cloning and sequencing, single-cell RT-PCR generated a major product (77/90 sequences), a minor type (2/90 sequences), and 11 different outliers (Table S1, column 3).…”