Trypanosoma brucei survives in the mammalian blood-stream by regularly changing its variant surface glycoprotein (VSG) coat. The active VSG gene is located in a telomeric expression site, and coat switching occurs either by replacing the transcribed VSG gene or by changing the expression site that is active. To determine whether VSG expression site control requires promoter-specific sequences, we replaced the active VSG expression site promoter in bloodstream-form T. brucei with a ribosomal DNA (rDNA) promoter. These transformants were fully infective in laboratory animals, and the rDNA promoter, which is normally constitutively active, was efficiently inactivated and reactivated in the context of the VSG gene expression site. As there is no sequence similarity between the VSG expression site promoter and the rDNA promoter, VSG expression site control does not involve sequences specific to the VSG expression site promoter. We conclude that an epigenetic mechanism, such as telomeric silencing, is involved in VSG expression site control in bloodstream-form T. brucei.

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.

In Trypanosoma brucei, transcription by RNA polymerase II and 5 capping of messenger RNA are uncoupled: a capped spliced leader is trans spliced to every RNA. This decoupling makes it possible to have protein-coding gene transcription driven by RNA polymerase I. Indeed, indirect evidence suggests that the genes for the major surface glycoproteins, variant surface glycoproteins (VSGs) in bloodstream-form trypanosomes, are transcribed by RNA polymerase I. In a single trypanosome, only one VSG expression site is maximally transcribed at any one time, and it has been speculated that transcription takes place at a unique site within the nucleus, perhaps in the nucleolus. We tested this by using f luorescence in situ hybridization. With probes that cover about 50 kb of the active 221 expression site, we detected nuclear transcripts of this site in a single f luorescent spot, which did not colocalize with the nucleolus. Analysis of marker gene-tagged active expression site DNA by f luorescent DNA in situ hybridization confirmed the absence of association with the nucleolus. Even an active expression site in which the promoter had been replaced by an rDNA promoter did not colocalize with the nulceolus. As expected, marker genes inserted in the rDNA array predominantly colocalize with the nucleolus, whereas the tubulin gene arrays do not. We conclude that transcription of the active VSG expression site does not take place in the nucleolus.

We have analyzed DNA of EUGLENA: gracilis for the presence of the unusual minor base beta-D-glucosyl-hydroxymethyluracil or J, thus far only found in kinetoplastid flagellates and in DIPLONEMA: Using antibodies specific for J and post-labeling of DNA digests followed by two-dimensional thin-layer chromatography of labeled nucleotides, we show that approximately 0.2 mole percent of EUGLENA: DNA consists of J, an amount similar to that found in DNA of Trypanosoma brucei. By staining permeabilized EUGLENA: cells with anti-J antibodies, we show that J is rather uniformly distributed in the EUGLENA: nucleus, and does not co-localize to a substantial extent with (GGGTTA)(n) repeats, the putative telomeric repeats of EUGLENA: Hence, most of J in EUGLENA: appears to be non-telomeric. Our results add to the existing evidence for a close phylogenetic relation between kinetoplastids and euglenids.