A DETAILED MUTATIONAL ANALYSIS OF THE VSG GENE-EXPRESSION SITE PROMOTER

The African trypanosome Trypanosoma brucei is a protozoan parasite tha t causes the disease African sleeping sickness. This parasite avoids t he host's immune response by the process of antigenic variation, or by sequentially expressing antigenically different cell-surface coat pro teins. These proteins, called variant surface glycoproteins (VSGs), ar e expressed from a specific locus, the VSG gene expression site (ES). In an attempt to understand expression of VSG genes, we expanded on ea rlier investigations of the promoter that controls the large VSG gene expression site transcription unit. We studied VSG ES promoter functio n both in transient transfection assays, and after stable integration at a chromosomal locus. Analysis of closely spaced deletion mutants sh owed that the minimum VSG ES promoter fragment that gives full activit y is extremely small, and mapped precisely to a fragment that contains no more than - 67 bp 5' to the putative transcription initiation site . The promoter lacked an upstream control element, or UCE, an element found al the PARP promoter, and al most eukaryotic Pol I promoters. Fu rthermore, linker scanning mutagenesis demonstrated that the VSG ES pr omoter contains at least two essential regulatory elements, including sequences within the region - 67/ - 60 and the region - 35/ -20, both numbered relative to the initiation site. An altered promoter with mut ated nucleotides surrounding the transcription initiation site still d irected wild-type levels of expression. In this study, the results wer e similar for both insect and bloodstream form trypanosomes, suggestin g that the same basic machinery for expression from the VSG ES promote r is found in both stages of the parasite.