TELOMERE EXCHANGE CAN BE AN IMPORTANT MECHANISM OF VARIANT SURFACE GLYCOPROTEIN GENE SWITCHING IN TRYPANOSOMA-BRUCEI

Trypanosoma brucei undergoes antigenic variation by changing its Varia nt Surface Glycoprotein (VSG) coat. Although there are up to a thousan d VSG genes, only one is transcribed at a lime from a telomeric VSG ex pression site. Switching can involve DNA rearrangements exchanging the active VSG gene, or transcriptional activation of a new expression si te and transcriptional silencing of the old one. Determining the mecha nism mediating a switch has not always been easy. as the many virtuall y identical copies of VSG gene expression sites complicate transcripti onal analysis. To overcome this problem, we have used bloodstream form T. brucei with a single copy VSG gene in an active expression site ma rked with a hygromycin resistance gene. We allowed these transformants to undergo switching of the active VSG gene, via three different expe rimental methods. We were able to select large numbers of switched try panosomes from a single infected mouse using a new microtitre-dish bas ed procedure developed for this purpose. The drug sensitivity of the s witched trypanosomes allowed us to determine the transcriptional state of the marked expression site, and polymerase chain reaction (PCR) am plification was used to determine whether the single copy drug resista nce gene and VSG gene present in the marked expression site had been r etained. These studies showed that telomere exchange, which has been c onsidered rare, can in some cases be an important mechanism of VSG gen e switching. We describe 4 telomere exchange events between the active VSG 221 expression site and 4 different chromosomes.