Ex Vivo and In Vitro Identification of a Consensus Promoter for VSG Genes Expressed by Metacyclic-Stage Trypanosomes in the Tsetse Fly

Abstract

The trypanosome variant surface glycoprotein (VSG) is first expressed during differentiation to the infective, metacyclic population in tsetse fly salivary glands. Unlike the VSG genes expressed by bloodstream form trypanosomes, metacyclic VSGs (MVSGs) have their own promoters. The scarcity of metacyclic cells has meant that only indirect approaches have been used to study these promoters, and not even their identities have been agreed on. Here, we isolated trypanosomes by dissection from salivary glands and used an approach involving 5 rapid amplification of cDNA ends to identify the transcription start site of three MVSGs. This shows that the authentic start site is that proposed for the MVAT series of MVSGs (K. S. Kim and J. E. Donelson, J. Biol. Chem. 272:24637-24645, 1997). In the more readily accessible procyclic trypanosome stage, where MVSGs are normally silent, we used reporter gene assays and linker scanning analysis to confirm that the 67 bp upstream of the start site is a promoter. This is confirmed further by accurate initiation in a homologous in vitro transcription system. We show also that MVSG promoters become derepressed when tested outwith their endogenous, subtelomeric loci. The MVSG promoters are only loosely conserved with bloodstream VSG promoters, and our detailed analysis of the 1.63 MVSG promoter reveals that its activity depends on the start site itself and sequences 26 to 49 bp and 56 to 60 bp upstream. These are longer than those necessary for the bloodstream promoter.

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

“…The conserved nature of BESs, including the promoter elements, has made this BES TAR cloning project possible. Isolating telomeres containing T. brucei metacyclic ESs (MESs) will prove to be a much greater challenge, as these monocistronic transcription units contain only VSGs and very divergent promoter elements (Ginger et al 2002).…”

Mono-allelic VSG expression by RNA polymerase I in Trypanosoma brucei: Expression site control from both ends?

“…However, the active VSG gene is invariably located next to the telomere within an expression site, with the coding region ending ~200–1800 bp upstream of the telomeric repeats. Metacyclic trypanosomes express a single VSG monocistronically from one of five metacyclic expression sites in which the RNAPI promoter is located ~1–4 kb upstream of the coding region (Ginger et al, 2002; Kolev et al, 2012; Cross et al, 2014). Conversely, BFs express the active VSG from one of fifteen polycistronic “bloodstream expression sites” (BESs) which comprise a tandem array of typically 8–9 expression-site associated genes (ESAGs) and a terminal VSG gene (Figure 1) (Hertz-Fowler et al, 2008).…”

Cell Biology of the Trypanosome Genome

“…They are classified either as those of the initial life cycle stage transmitted by fly bite (metacyclic expression sites [MESs]) or those used later in parasitemia during proliferation in the host bloodstream (bloodstream expression sites [BESs]). Significantly, the genes coding for metacyclic VSG (MVSG), the surface coat protein of the metacyclic life cycle stage, are the only clear examples among protein-coding genes in T. brucei of monocistronic transcription (64,65) and exclusive control at transcription initiation (62).…”