Control of variant surface glycoprotein gene-expression sites in Trypanosoma brucei

Abstract

Trypanosoma brucei has 20 similar telomeric-expression sites for variant surface glycoprotein genes. Expression sites appear to be controlled at the level of transcription initiation, resulting in only one site being active at any time. Switching between expression sites occurs at a low rate. To analyse the switching mechanism, we used trypanosomes with two expression sites tagged with two different drug-resistance genes and selected these on agarose plates containing both drugs. Double-resistant clones arose at a low frequency of 10 -7 per cell, but these behaved as if they rapidly switched between the two tagged expression sites and lost double resistance in the absence of selection. Using in situ hybridization we found that only 10% of the double-resistant cells had two fluorescent spots corresponding to transcribed expression sites. Our results suggest that: (i) a double expressor is not a stable intermediate in expression site switching; (ii) expression sites are not independently switched on and off; and (iii) expression sites can be in a 'pre-active' silent state from which they can be readily activated.

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

“…Under these conditions it was impossible to express more than two ESs at a time, and this expression was found to be unstable (31). This observation suggested an alternative model in which ES expression is controlled at the level of transcription initiation from one fully active and one preactive ES per cell (30).…”

“…On the other hand, experiments tagging ESs with antibiotic-resistance genes showed that RNA expression can be detected from two ES, although the expression was unstable (30). This result gave rise to a model in which transcription is initiated on only two ESs, one being fully active and a second being preactive (moderately transcribed) (31). This model is equally able to account for the finding of transcripts from multiple ESs within a trypanosome population (29), provided the preactive ES differs among individual cells.…”

Bloodstream Form-specific Up-regulation of Silent VSG Expression Sites and Procyclin in Trypanosoma brucei after Inhibition of DNA Synthesis or DNA Damage

“…18). Stable maximal activation of two ESs does not appear possible (19). Selection for double ES expressors using selectable markers inserted immediately downstream of different ES promoters results in trypanosomes that have one ES maximally active and another ES partially up-regulated, or that rapidly switch between two different ESs (19,20).…”

“…Stable maximal activation of two ESs does not appear possible (19). Selection for double ES expressors using selectable markers inserted immediately downstream of different ES promoters results in trypanosomes that have one ES maximally active and another ES partially up-regulated, or that rapidly switch between two different ESs (19,20). Possibly these rapidly switching trypanosomes alternate between a privileged subnuclear location that has been called an expression site body (ESB), a pol I transcriptional body specific to bloodstream form T. brucei (21).…”

Telomere as an Important Player in Regulation of Microbial Pathogen Virulence

“…It is therefore hypothesized that ESB, enriched with RNAP I, can only accommodate one B-ES, which would effectively limit the number of active B-ES to one. In support of this view, when two different B-ESs were tagged with selective markers immediately downstream of their respective promoters and forced to be active simultaneously, the two B-ESs appear to switch back and force rapidly and locate next to each other in the nucleus, presumably competing for available RNAP I at ESB (Chaves et al 1999). …”