Pbtert deletion and selection of tert- mutants.

<p>(A) Schematic representation of the construct used to delete the <i>tert</i> gene. The construct, containing the <i>Tgdhfr-ts</i> selectable marker (SM) cassette, targets the <i>tert</i> gene at the flanking regions (red) by double cross-over integration. The red arrows indicate primers used for diagnostic PCR to confirm correct disruption of <i>tert</i>. Boxes correspond to lanes on the PCR gels in (B), (D) and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0108930#pone.0108930.s001" target="_blank">Fig. S1A</a>. (B) Diagnostic PCR of uncloned parasites transfected with a DNA construct to delete the <i>tert</i> gene. Parasites were collected and analysed directly after transfection and selection with pyrimethamine (parent populations). Diagnostic PCR shows the presence of parasites with correct disruption of the <i>tert</i> gene. In all experiments (1065, 1078, 1138, 1207, 1217) the 5′ and 3′ integration fragments (lanes 5′, 3′), as well as the <i>Tgdhfr-ts</i> fragment (lane SM) were amplified. However, all populations contained parasites with a wild type <i>tert</i> gene as shown by amplification of the wild type <i>tert</i> fragment (lane wt). The primer pairs used are shown in (A) and expected fragment sizes in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0108930#pone.0108930.s004" target="_blank">Table S2</a>. <i>pbs21</i>-specific primers were used as a positive control for all the PCR reactions (“+”). The water control is marked as “-“. (C) Southern analysis of separated chromosomes using the 3′UTR <i>Pbdhfr-ts</i> probe shows only in experiment 1065 and 1217 hybridisation with chromosome 14 on which the <i>tert</i> gene is located. This probe recognizes the endogenous <i>Pbdhfr-ts</i> gene on chromosome 7 in all populations and additional chromosomes in experiments 1078, 1138, 1207 (possible episomal construct signal). (D) Diagnostic PCR of uncloned and propagated parasites transfected with a DNA construct to delete the <i>tert</i> gene. The parent parasite populations of experiment 1065, 1207 and 1217 [see (B)] were propagated in mice (m0  =  mouse 0, m1  =  mouse 1) for another 1–2 weeks. Parasite populations collected were analysed by diagnostic PCR for the presence of parasites with correct disruption of the <i>tert</i> gene [primers same as in (B)]. In all populations no parasites with a disrupted <i>tert</i> gene could be detected by diagnostic PCR after 1 week (1207 all populations) or after two weeks of propagation (1065 uncl.2, 1217 uncl.2 m0 and 1217 uncl.2 m1).</p

Pbtert gene structure (A) and PbTERT (B) and PbTR (C) expression.

<p>(A) The <i>tert</i> gene of <i>P. berghei</i> and homology (percentage identity) of TERT proteins in different <i>Plasmodium</i> species. Sequencing of the gap between two adjacent <i>tert</i> gene models available in PlasmoDB revealed a sequence duplication of 57 nt (19aa). The complete Pb<i>tert</i> gene encodes a protein of 2312aa, which is comparable to the size of other <i>Plasmodium tert</i> genes. (B) Western analysis of PbTERT protein in mixed blood stages. Two bands with a size between 150 and 250 kDa were detected (expected size of the TERT protein is ∼240 kDa). (C) Northern analysis of Telomerase-associated RNA (TR) in different blood stages of <i>P. berghei</i>. RNA was hybridized with a probe recognizing TR (upper panel) (the expected size of TR is 2 kb) and as a loading control with a probe recognizing <i>large subunit ribosomal RNA</i> (expected size 0.8 kb). The “% loading” refers to the quantity of the loading control signal detected for each stage relative to the “late trophozoite” lane which is set as 100%.</p

<i>P. berghei</i> telomere characterisation.

<p>(A) Determination of telomere length by Telomere Restriction Fragment (TRF) analysis. Left Panel: Southern analysis of separated chromosomes of <i>P. berghei</i> (Pb), <i>P. chabaudi</i> (Pc), <i>P. vinckei</i> (Pv) and <i>P. yoelii</i> (Py) showing hybridization of all chromosomes to a telomere-specific probe. The same probe was used for TRF analysis (middle, right panels). Middle panel: Southern analysis of digested <i>P. yoelii</i> (size control) and <i>P. berghei</i> gDNA probed with the telomeric probe showing the characteristic “smeared” hybridisation pattern in TRF analysis. Right panel: The average telomere length was measured as the highest peak of the signal intensity along the smear. Using the molecular marker (“M”, grey line) as a size reference (relevant marker bands sizes are noted on the graph), the mean telomere length was estimated to be ∼2500 bp and ∼950 bp for <i>P. yoelii</i> (blue line) and <i>P. berghei</i> (red line), respectively. Complete digestion of gDNA was confirmed by hybridisation with a 5′ <i>d-type small unit ribosomal RNA</i> probe. (B) Fluorescence <i>in situ</i> hybridisation with a telomere-specific probe. Fixed late blood stages of <i>P. berghei</i>. The telomeric probe (1.5 kb) was labelled with fluorescein (green). Hoechst (blue) was used for nuclear staining. The size bar is 5 µm.</p

A cascade of DNA-binding proteins for sexual commitment and development in Plasmodium

Commitment to and completion of sexual development are essential for malaria parasites (protists of the genus Plasmodium) to be transmitted through mosquitoes. The molecular mechanism(s) responsible for commitment have been hitherto unknown. Here we show that PbAP2-G, a conserved member of the apicomplexan AP2 (ApiAP2) family of DNA-binding proteins, is essential for the commitment of asexually replicating forms to sexual development in Plasmodium berghei, a malaria parasite of rodents. PbAP2-G was identified from mutations in its encoding gene, PBANKA_143750, which account for the loss of sexual development frequently observed in parasites transmitted artificially by blood passage. Systematic gene deletion of conserved ApiAP2 genes in Plasmodium confirmed the role of PbAP2-G and revealed a second ApiAP2 member (PBANKA_103430, here termed PbAP2-G2) that significantly modulates but does not abolish gametocytogenesis, indicating that a cascade of ApiAP2 proteins are involved in commitment to the production and maturation of gametocytes. The data suggest a mechanism of commitment to gametocytogenesis in Plasmodium consistent with a positive feedback loop involving PbAP2-G that could be exploited to prevent the transmission of this pernicious parasite

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A cascade of DNA-binding proteins for sexual commitment and development in Plasmodium

Commitment to and completion of sexual development are essential for malaria parasites (protists of the genus Plasmodium) to be transmitted through mosquitoes1. The molecular mechanism(s) responsible for commitment have been hitherto unknown. Here we show that PbAP2-G, a conserved member of the apicomplexan AP2 (ApiAP2) family of DNA-binding proteins, is essential for the commitment of asexually replicating forms to sexual development in Plasmodium berghei, a malaria parasite of rodents. PbAP2-G was identified from mutations in its encoding gene, PBANKA_143750, which account for the loss of sexual development frequently observed in parasites transmitted artificially by blood passage. Systematic gene deletion of conserved ApiAP2 genes in Plasmodium confirmed the role of PbAP2-G and revealed a second ApiAP2 member (PBANKA_103430, here termed PbAP2-G2) that significantly modulates but does not abolish gametocytogenesis, indicating that a cascade of ApiAP2 proteins are involved in commitment to the production and maturation of gametocytes. The data suggest a mechanism of commitment to gametocytogenesis in Plasmodium consistent with a positive feedback loop involving PbAP2-G that could be exploited to prevent the transmission of this pernicious parasite