hygromycin-a and Trypanosomiasis--African

Trypanosoma brucei undergoes antigenic variation by changing its Variant Surface Glycoprotein (VSG) coat. Although there are up to a thousand VSG genes, only one is transcribed at a time from a telomeric VSG expression site. Switching can involve DNA rearrangements exchanging the active VSG gene, or transcriptional activation of a new expression site and transcriptional silencing of the old one. Determining the mechanism mediating a switch has not always been easy, as the many virtually identical copies of VSG gene expression sites complicate transcriptional analysis. To overcome this problem, we have used bloodstream form T. brucei with a single copy VSG gene in an active expression site marked with a hygromycin resistance gene. We allowed these transformants to undergo switching of the active VSG gene, via three different experimental methods. We were able to select large numbers of switched trypanosomes from a single infected mouse using a new microtitre-dish based procedure developed for this purpose. The drug sensitivity of the switched trypanosomes allowed us to determine the transcriptional state of the marked expression site, and polymerase chain reaction (PCR) amplification was used to determine whether the single copy drug resistance gene and VSG gene present in the marked expression site had been retained. These studies showed that telomere exchange, which has been considered rare, can in some cases be an important mechanism of VSG gene switching. We describe 4 telomere exchange events between the active VSG 221 expression site and 4 different chromosomes.

Topics: Animals; Antigenic Variation; Cinnamates; Cloning, Molecular; Drug Resistance; Gene Conversion; Genes, Protozoan; Hygromycin B; Immunization; Mice; Mice, Inbred BALB C; Rats; Rats, Sprague-Dawley; Telomere; Transformation, Genetic; Trypanosoma brucei brucei; Trypanosomiasis, African; Variant Surface Glycoproteins, Trypanosoma