FREQUENT LOSS OF THE ACTIVE-SITE DURING VARIANT SURFACE GLYCOPROTEIN EXPRESSION SITE SWITCHING IN-VITRO IN TRYPANOSOMA-BRUCEI

African trypanosomes undergo antigenic variation of their variant surf ace glycoprotein (VSG) coat to avoid being killed by their mammalian h osts. The active VSG gene is located in one of many telomeric expressi on sites. Replacement of the VSG gene in the active site or switching between expression sites can give rise to a new VSG coat. To study Try panosoma brucei VSG expression site inactivation rather than VSG gene switching, it is useful to have an in vitro negative-selection system independent of the VSG. We have achieved this aim by using a viral thy midine kinase (TK) gene. Following integration of the TK gene downstre am of the 221a VSG expression site promoter, transformant cell lines b ecame sensitive to the nucleoside analog deoxy-2-fluoro-8-D-arabinofur anosyl)-5-iodouracil. These TK trypanosomes were able to revert to res istance at a rate approaching 10(-5) per cell per generation. The majo rity of revertants expressed a new VSG gene even though there had been no selection against the VSG itself. Analysis of these switched varia nts showed that some had shut down TK expression via an in situ expres sion site switch. However, most variants had the complete 221 expressi on site deleted and another VSG expression site activated. We speculat e that a new VSG expression site cannot switch on without inactivation of the old site.