A highly conserved segmental duplication in the subtelomeres of Plasmodium falciparum chromosomes varies in copy number

<p>Abstract</p> <p>Background</p> <p>Segmental duplications (SD) have been found in genomes of various organisms, often accumulated at the ends of chromosomes. It has been assumed that the sequence homology in-between the SDs allow for ectopic interactions that may contribute to the emergence of new genes or gene variants through recombinatorial events.</p> <p>Methods</p> <p><it>In silico </it>analysis of the 3D7 <it>Plasmodium falciparum </it>genome, conducted to investigate the subtelomeric compartments, led to the identification of subtelomeric SDs. Sequence variation and copy number polymorphisms of the SDs were studied by DNA sequencing, real-time quantitative PCR (qPCR) and fluorescent <it>in situ </it>hybridization (FISH). The levels of transcription and the developmental expression of copy number variant genes were investigated by qPCR.</p> <p>Results</p> <p>A block of six genes of >10 kilobases in size, including <it>var</it>, <it>rif</it>, <it>pfmc-2tm </it>and three hypothetical genes (<it>n-, o- </it>and <it>q-gene</it>), was found duplicated in the subtelomeric regions of chromosomes 1, 2, 3, 6, 7, 10 and 11 (SD1). The number of SD1 per genome was found to vary from 4 to 8 copies in between different parasites. The intragenic regions of SD1 were found to be highly conserved across ten distinct fresh and long-term cultivated <it>P. falciparum</it>. Sequence variation was detected in a ≈ 23 amino-acid long hypervariable region of a surface-exposed loop of PFMC-2TM. A hypothetical gene within SD1, the <it>n-gene</it>, encoding a PEXEL/VTS-containing two-transmembrane protein was found expressed in ring stage parasites. The <it>n-gene </it>transcription levels were found to correlate to the number of <it>n-gene </it>copies. Fragments of SD1 harbouring two or three of the SD1-genes (<it>o-gene, pfmc-2tm, q-gene</it>) were also found in the 3D7 genome. In addition a related second SD, SD2, of ≈ 55% sequence identity to SD1 was found duplicated in a fresh clinical isolate but was only present in a single copy in 3D7 and in other <it>P. falciparum </it>lines or clones.</p> <p>Conclusion</p> <p><it>Plasmodium falciparum </it>carries multiple sequence conserved SDs in the otherwise highly variable subtelomeres of its chromosomes. The uniqueness of the SDs amongst plasmodium species, and the conserved nature of the genes within, is intriguing and suggests an important role of the SD to <it>P. falciparum</it>.</p

Default Pathway of var2csa Switching and Translational Repression in Plasmodium falciparum

Antigenic variation is a subtle process of fundamental importance to the survival of a microbial pathogen. In Plasmodium falciparum malaria, PfEMP1 is the major variable antigen and adhesin expressed at the surface of the infected erythrocyte, which is encoded for by members of a family of 60 var-genes. Peri-nuclear repositioning and epigenetic mechanisms control their mono-allelic expression. The switching of PfEMP1 depends in part on variable transition rates and short-lived immune responses to shared minor epitopes. Here we show var-genes to switch to a common gene that is highly transcribed, but sparsely translated into PfEMP1 and not expressed at the erythrocyte surface. Highly clonal and adhesive P. falciparum, which expressed distinct var-genes and the corresponding PfEMP1s at onset, were propagated without enrichment or panning. The parasites successively and spontaneously switched to transcribe a shared var-gene (var2csa) matched by the loss of PfEMP1 surface expression and host cell-binding. The var2csa gene repositioned in the peri-nuclear area upon activation, away from the telomeric clusters and heterochromatin to transcribe spliced, full-length RNA. Despite abundant transcripts, the level of intracellular PfEMP1 was low suggesting post-transcriptional mechanisms to partake in protein expression. In vivo, off-switching and translational repression may constitute one pathway, among others, coordinating PfEMP1 expression

A highly conserved segmental duplication in the subtelomeres of chromosomes varies in copy number-3

–28 hours post invasion. Data was logtransformed and plotted at four-hour intervals for each particular parasite.<p><b>Copyright information:</b></p><p>Taken from "A highly conserved segmental duplication in the subtelomeres of chromosomes varies in copy number"</p><p>http://www.malariajournal.com/content/7/1/46</p><p>Malaria Journal 2008;7():46-46.</p><p>Published online 7 Mar 2008</p><p>PMCID:PMC2279139.</p><p></p

A highly conserved segmental duplication in the subtelomeres of chromosomes varies in copy number-2

Ation of copy numbers and localization of (green) in 3D7, FCR3 and 7G8. Distribution of fluorescent signals at the rim of the parasite nuclei (blue) confirms the position of the SD at the chromosomal ends.<p><b>Copyright information:</b></p><p>Taken from "A highly conserved segmental duplication in the subtelomeres of chromosomes varies in copy number"</p><p>http://www.malariajournal.com/content/7/1/46</p><p>Malaria Journal 2008;7():46-46.</p><p>Published online 7 Mar 2008</p><p>PMCID:PMC2279139.</p><p></p

A highly conserved segmental duplication in the subtelomeres of chromosomes varies in copy number-5

the gene locations on chromosome 1 in 3D7. A black arrow indicates the SD2 found duplicated in UAM25. (B) Genetic organization of the SD2 on the right arm of chromosome 1 in 3D7. Genes found duplicated in UAM25 relative to 3D7, according to CGH data and qPCR, are shown in red. (C) Copy numbers of the SD2 genes in different strains relative to 3D7 parasite confirmed by qPCR.<p><b>Copyright information:</b></p><p>Taken from "A highly conserved segmental duplication in the subtelomeres of chromosomes varies in copy number"</p><p>http://www.malariajournal.com/content/7/1/46</p><p>Malaria Journal 2008;7():46-46.</p><p>Published online 7 Mar 2008</p><p>PMCID:PMC2279139.</p><p></p

Whole-Body Imaging of Sequestration of Plasmodium falciparum in the Rat

The occlusion of vessels by packed Plasmodium falciparum-infected (iRBC) and uninfected erythrocytes is a characteristic postmortem finding in the microvasculature of patients with severe malaria. Here we have employed immunocompetent Sprague-Dawley rats to establish sequestration in vivo. Human iRBC cultivated in vitro and purified in a single step over a magnet were labeled with (99m)technetium, injected into the tail vein of the rat, and monitored dynamically for adhesion in the microvasculature using whole-body imaging or imaging of the lungs subsequent to surgical removal. iRBC of different lines and clones sequester avidly in vivo while uninfected erythrocytes did not. Histological examination revealed that a multiadhesive parasite adhered in the larger microvasculature, inducing extensive intravascular changes while CD36- and chondroitin sulfate A-specific parasites predominantly sequester in capillaries, inducing no or minor pathology. Removal of the adhesive ligand Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1), preincubation of the iRBC with sera to PfEMP1 or preincubation with soluble PfEMP1-receptors prior to injection significantly reduced the sequestration. The specificity of iRBC binding to the heterologous murine receptors was confirmed in vitro, using primary rat lung endothelial cells and rat lung cryosections. In offering flow dynamics, nonmanipulated endothelial cells, and an intact immune system, we believe this syngeneic animal model to be an important complement to existing in vitro systems for the screening of vaccines and adjunct therapies aiming at the prevention and treatment of severe malaria