Fig 7. var2csa expression in response to chaetocin treatment is a true switching event and maintains mutually exclusive expression.

The transgenic parasite line DC-J contains a modified var gene that encodes the drug resistance marker blasticidin-S-deaminase (bsd). Thus upon selection with blasticidin, these parasites are forced to express bsd and consequently silence the rest of the var gene family. DC-J parasites were grown under different conditions and the var gene expression profile determined by Q-RT-PCR and displayed as pie charts. (A) Growth of DC-J in the presence of blasticidin results in stable expression of bsd which remains the dominant var gene transcript detectable. (B) Growth of DC-J in the absence of blasticidin but in the presence of chaetocin results in induction of var2csa expression. var2csa has become the dominant var gene expressed by this population after 6 weeks of exposure to chaetocin. (C) Growth of DC-J parasites in the absence of blasticidin selection results in switching to several other var genes, including var2csa. After 6 weeks of growth in the absence of selection, both var2csa and a third var gene (PF3D7_071200, shown in purple) have become prominently expressed. (D) DC-J grown in the presence of both blasticidin and chaetocin results in stable expression of bsd for 4 weeks, after which the population fails to grow and eventually dies out. (E) Parasite growth charts for populations treated with blasticidin, chaetocin, blasticidin + chaetocin or untreated. Parasitemias were calculated daily by blood smear and three weeks are shown. Populations were allowed to replicate until they reached ~3% parasitemia, at which time the population density was reduced to 0.5% (red arrow) and the culture allowed to continue. By the end of week 5, the population exposed to both blasticidin and chaetocin failed to replicate efficiently (blue arrow) and the population died out by week 6. Individual copy number values for each transcript are shown in S7–S11 Figs.