ANALYSIS OF TRYPANOSOMA-BRUCEI VSG EXPRESSION SITE SWITCHING IN-VITRO

Trypanosoma brucei can undergo antigenic variation by switching betwee n distinct telomeric variant surface glycoprotein gene (vsg) expressio n sites (ESs) or by replacing the active vsg. DNA rearrangements have often been associated with ES switching, but it is unclear if such rea rrangements are necessary or whether ES inactivation always accompanie s ES activation. To explore these issues, we derived ten independent c lones, from the same parent, that had undergone a similar vsg activati on event. This was achieved in the absence of an immune response, in v itro, using cells with selectable markers integrated into an ES. Nine of the ten clones had undergone ES switching. Such heritable changes i n transcription state occurred at a frequency of approximately 6 x 10( -7). Comparison of switched and un-switched clones highlighted the dyn amic nature of T. brucei telomeres, but changes in telomere length wer e not specifically associated with ES switching. Mapping within and be yond the ESs revealed no detectable DNA rearrangements, indicating tha t rearrangements are not necessary for ES activation/inactivation. Exa mination of individual cells indicated that ES activation consistently accompanied inactivation of the previously active ES. In some cases, however, we found cells that appeared to have efficiently established the switched state but which subsequently, at a frequency of approxima tely 2 x 10(-3), generated cells expressing both pre- and post-switch vsgs. These results show that ES activation/inactivation is usually a coupled process but that cells can inherit a propensity to uncouple th ese events. (C) 1997 Elsevier Science B.V.