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Supplementary Discussion
A recent study demonstrated that deletion of the intron in cis was required for episomal upsC
activation1. The plasmid used (pVDH+int) carried a single expression cassette consisting of
an upsC promoter driving expression of hdhfr, followed by the hrp2 terminator sequence and
a var gene intron. In the 3D7/upsCRI transfectants described here the presence of the intron
was not inhibitory to episomal activation of upsC since Southern blot analysis revealed no
difference in the structure of silenced and activated pHBupsCRI episomes (Supplementary
Fig. 1). This discrepancy between the two studies might be explained by the differences in
both transfection strategy and vector design. The transfection strategy used by Gannoun-Zaki
et al.1 was entirely dependant on an active upsC promoter expressing the hdhfr gene since
parasites were challenged with WR directly after transfection. In contrast, we used a second
drug-selectable marker gene (bsd), driven by the constitutive hsp86 promoter rather than the
upsC promoter, to select for transfected parasites. This approach allowed us to investigate
upsC activity in parasites maintaining the pHBupsCRI episome as stably replicating
extrachromosomal chromatin. We therefore believe that our approach reflects the regulation
of endogenous var genes more accurately than the study by Gannoun-Zaki et al.1. An
alternative explanation may be gained from the fact that we failed repeatedly to select for
blasticidin-S-resistant parasites transfected with pHBupsCI which, as for the pVDH+int
vector used in Gannoun-Zaki et al.1, contains the intron but lacks rep20 repeats to target
episomes to telomeric clusters. Hence, it is possible that the nuclear periphery plays a key
role in unlocking inhibitory interactions between upsC and the intron. This hypothesis is
supported by the finding that upsC var genes naturally reside in this nuclear subcompartment
and there are no indications to date that activation of endogenous var genes requires deletion
of the intron. In summary, we conclude that upsC var genes can be activated in the presence
of the intron in cis.
1
We investigated var transcription by Northern blot analysis and showed that activation of a
plasmid-borne upsC promoter resulted in the repression of endogenous var gene transcription
in three independent transgenic parasites; this effect was observed with both episomal and
chromosomal upsC promoters. Furthermore we confirmed the absence of var transcripts at
the phenotype level by static cytoadherence assays and flow cytometry. Intriguingly, the
study by Gannoun-Zaki et al. reported that activation of the episomal upsC promoter had no
effect on endogenous var transcription1. The authors investigated var transcription by realtime RT-PCR using an unbiased primer pair to amplify the semi-conserved PfEMP1
DBL1
-encoding sequence at the 5’ end of var genes2. It is well documented
that RT-PCR, in contrast to Northern blot analysis, detects not only transcription of fulllength var genes but also the relaxed transcription of many var genes in ring and trophozoite
stage parasites resulting in the presence of prematurely terminated and probably nonfunctional var transcripts2-4. It is therefore possible that in the study by Gannoun-Zaki et al.1
relaxed transcription of multiple var genes was detected rather than transcription of fulllength var genes.
1. Gannoun-Zaki, L., Jost, A., Mu, J., Deitsch, K. W. & Wellems, T. E. A silenced
Plasmodium falciparum var promoter can be activated in vivo through spontaneous
deletion of a silencing element in the intron. Eukaryot Cell 4, 490-2 (2005).
2. Taylor, H. M., Kyes, S. A., Harris, D., Kriek, N. & Newbold, C. I. A study of var gene
transcription in vitro using universal var gene primers. Mole. Biochem. Parasit. 105, 1323 (2000).
3. Duffy, M. F. et al. Broad analysis reveals a consistent pattern of var gene transcription in
Plasmodium falciparum repeatedly selected for a defined adhesion phenotype. Mol
Microbiol 56, 774-88 (2005).
4. Duffy, M. F. et al. Transcription of multiple var genes by individual, trophozoite-stage
Plasmodium falciparum cells expressing a chondroitin sulphate A binding phenotype. Mol
Microbiol 43, 1285-93. (2002).
2