These authors contributed equally to this work.
Identification of a cis-acting DNA–protein interaction implicated in singular var gene choice in Plasmodium falciparum
Version of Record online: 4 SEP 2012
© 2012 Blackwell Publishing Ltd
Volume 14, Issue 12, pages 1836–1848, December 2012
How to Cite
Brancucci, N. M. B., Witmer, K., Schmid, C. D., Flueck, C. and Voss, T. S. (2012), Identification of a cis-acting DNA–protein interaction implicated in singular var gene choice in Plasmodium falciparum. Cellular Microbiology, 14: 1836–1848. doi: 10.1111/cmi.12004
Re-use of this article is permitted in accordance with the Terms and Conditions set out at http://wileyonlinelibrary.com/onlineopen#OnlineOpen_Terms
- Issue online: 19 NOV 2012
- Version of Record online: 4 SEP 2012
- Accepted manuscript online: 14 AUG 2012 10:23AM EST
- Manuscript Accepted: 8 AUG 2012
- Manuscript Revised: 6 AUG 2012
- Manuscript Received: 27 JUL 2012
- Swiss National Science Foundation. Grant Numbers: PP00A-110835, PP00P3_130203
Supporting experimental procedures
Fig. S1. Southern analysis of gDNA isolated from parasites presented in Fig. 2.
A. Autoradiographs of Southern blots showing episomal maintenance or plasmid integration into the endogenous kahrp locus in 3D7/pBKmin and 3D7/pBKmin-RI. gDNA was digested with BglII and HindIII. Blots were probed with a radiolabelled kahrp fragment. E, episomal; I, integrated.
B. Autoradiographs of Southern blots showing episomal maintenance or plasmid integration into the endogenous kahrp locus in 3D7/pBC1Kmin, 3D7/pBC2Kmin and 3D7/pBC3Kmin. gDNA was digested with BglII and HindIII. Blots were probed with a radiolabelled kahrp fragment.
C. Schematic map of the endogenous kahrp locus.
D–F. Schematic maps of the integration events in 3D7/pBKmin (D), 3D7/pBC1Kmin and 3D7/pBC2Kmin (E) and 3D7/pBC3Kmin (F). BglII and HindIII restriction sites and length of the corresponding fragments are indicated.
Fig. S2. Transcriptional initiation form an alternative upsC upstream TSS. The promoters in pBC and pBC4 are schematically depicted on top. Semi-quantitative analysis of protein and transcript abundance by Western and Northern blot in a time-course experiment. Total protein and RNA were harvested simultaneously from synchronized 3D7/pBC and 3D7/pBC4 parasites at three consecutive time points during intra-erythrocytic development (ring stages, 8–18 hpi; late ring stages/early trophozoites, 16–26 hpi; late trophozoites/early schizonts, 24–34 hpi). Expression of hDHFR-GFP and GAPDH (loading control) was detected with anti-GFP and anti-GAPDH antibodies respectively (upper panels). Steady-state hdhfr-gfp and hsp86 (loading control) transcripts were detected using radiolabelled hdhfr and hsp86 probes respectively.
Fig. S3. Competition EMSAs. All EMSAs were carried out using radiolabelled MEE2 and parasite nuclear extract.
A. Mutational analysis of MEE2. Competition was carried out in presence of a 25- and 100-fold molar excess of unlabelled DNA. The nucleotide sequences of wild-type and mutated MEE2 elements are indicated on the right. The ATAGATTA core motif is underlined. Mutated 8mers are highlighted in red.
B. Competition of the MEE2 complex by a MEE2-related upsB sequence element. Competition was carried out in presence of a 25-, 100-, 250- and 500-fold molar excess of unlabelled DNA. The nucleotide sequences of wild-type and scrambled MEE2 and the MEE2-related upsB element are indicated on the right. The ATAGATTA core motif is underlined. The differences in the upsB-derived motif compared with MEE2 are highlighted in red.
C. The ATAGATTA core motif is not sufficient for complex formation. Competition was carried out in presence of a 25-, 100- and 500-fold molar excess of unlabelled DNA. The ATAGATTA core motif is underlined. The nucleotide sequences of wild-type and scrambled MEE2 and two unrelated sequence elements that contain the ATAGATTA core motif are indicated on the right.
Fig. S4. The MEE2 core motif occurs in a conserved position upstream of 44 var genes.
A. The schematic shows the presence and relative position of the (A/T)(A/T)(A/T)GA(A/T)TA consensus sequence found upstream of 44 var genes. This motif forms the core of the 47 bp MEE2 element that is bound by a nuclear factor in a sequence-specific manner (see Figs 4 and S3). Red boxes indicate the position of the motif in each upstream region. Numbers on the right represent the position of the first nucleotide of the motif relative to the translation initiation ATG. Gene accession numbers were retrieved from PlasmoDB version 7.2 (http://www.plasmoDB.org) and are indicated on the left. The colour code clusters var genes into the different var gene subgroups upsA, upsB, upsC, upsE, upsB/C and upsB/A (Lavstsen et al., 2003).
B. Alignment of MEE2-related sequences that are centred around the (A/T)(A/T)(A/T)GA(A/T)TA core consensus element in 44 var upstream regions. The original MEE2 motif identified upstream of the upsC var gene PFL1960w is shown as the first sequence in the alignment. The local context of the MEE2-related core motifs shows a high level of sequence similarity that includes a prominent upstream poly-dT stretch. Gene accession numbers are indicated on the left and are colour-coded as in Fig. S4A. Orientation of the motif is indicated on the right (+, upper strand; −, lower strand). The red bar on top highlights the position of the core motif.
Table S1. All primers used in this study are listed. Restriction sites are indicated in bold.
Please note: Wiley Blackwell is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.