IN-SITU ANALYSIS OF A VARIANT SURFACE GLYCOPROTEIN EXPRESSION-SITE PROMOTER REGION IN TRYPANOSOMA-BRUCEI

In Trypanosoma brucei, the active variant surface glycoprotein genes ( vsg) are located at telomeric expression sites (ES), whose expression is highly regulated during the life cycle. In the procyclic form, all ESs are repressed. In the bloodstream form, where antigenic variation occurs, only one of approximately 20 ESs is active at a given lime. We have investigated chromatin structure and DNA sequence around the ES promoter to identify cis-acting regulatory regions. A marker gene, ins erted 1 kb downstream of the ES promoter, was used as a specific probe to map the position of nuclease hypersensitive sites. A prominent hyp ersensitive site was detected within the core promoter. This site was present in both active and inactive ES promoters, suggesting that a pr otein complex is bound to the promoter irrespective of its transcripti onal state. However, none of the regions showed differential nuclease sensitivity between active and inactive transcriptional states. A syst ematic deletion analysis of the sequences surrounding the active ES pr omoter in situ confirmed the absence of cis-regulatory elements. We fi nd that only 70 bp within the ES promoter are necessary to support ES regulation. Analysis of the reporter activities in an inactive bloodst ream-form ES revealed the existence of an intermediate promoter activi ty in some clones, but we never observed full activation of more than one ES. The vsg mRNA from this intermediate ES was expressed less effi ciently. (C) 1998 Elsevier Science B.V. All rights reserved.