Figure 5.

Use of the transgenic trypanosome line 3174 to characterize VSG switching events. The telomeric region of the actively transcribed expression site in 3174 bloodstream cells is indicated. VSG221 and the most proximal ESAG (ESAG1) are separated by resistance markers for hygromycin (Hyg) and G418 (Neo), which are in turn separated by a stretch of 70-bp repeats; transcription of the site is depicted by a dashed arrow, and the position of primers used to assay for the presence of the genes within the expression site are indicated by small arrows. Three different switching events are observed in this trypanosome strain. In an in situ switch, another expression site (containing an unknown VSG; X) is transcriptionally activated and the VSG221 site is inactivated. These cells are sensitive to Hygromycin (Hyg^s) and G418 (G418^s), and each marker gene can be amplified by PCR (Hyg+, Neo+, 221+). A long range gene conversion event from another expression site deletes all the marker genes from the VSG221 site, and replaces them with VSGX, these are Hyg^s and G418^s, and the genes cannot be amplified by PCR (Hyg⁻, Neo⁻, 221⁻). In a gene conversion event extending from the 70-bp repeats to the 3′ end of VSG221, VSGX replaces only the 221 and Neo markers; these switchers are hygromycin resistant (Hyg^R) and G418^s, and are Hyg⁺, Neo⁻ , 221⁻ in PCR.