An approach to classifying sequence tags sampled from Plasmodium falciparum var genes.
Ontology highlight
ABSTRACT:
SUBMITTER: Bull PC
PROVIDER: S-EPMC1906845 | biostudies-literature | 2007 Jul
REPOSITORIES: biostudies-literature
Publications
An approach to classifying sequence tags sampled from Plasmodium falciparum var genes.
Bull Peter C PC Kyes Sue S Buckee Caroline O CO Montgomery Jacqui J Kortok Moses M MM Newbold Chris I CI Marsh Kevin K
Molecular and biochemical parasitology 20070325 1
Similar Datasets
Project description:The antigenic variation and cytoadherence of Plasmodium falciparum-infected erythrocytes are modulated by a family of variant surface proteins encoded by the var multigene family. The var genes occur on multiple chromosomes, often in clusters, and 50 to 150 genes are estimated to be present in the haploid parasite genome. Transcripts from var genes have been previously mapped to internal chromosome positions, but the generality of such assignments and the expression sites and mechanisms that control switches of var gene expression are still in early stages of investigation. Here we describe investigations of closely related var genes that occur in association with repetitive elements near the telomeres of P. falciparum chromosomes. DNA sequence analysis of one of these genes (FCR3-varT11-1) shows the characteristic two-exon structure encoding expected var features, including three variable Duffy binding-like (DBL) domains, a transmembrane sequence, and a carboxy-terminal segment thought to anchor the protein product in knobs at the surface of the parasitized erythrocyte. FCR3-varT11-1 cross-hybridizes with var genes located close to the telomeres of many other P. falciparum chromosomes, including a transcribed gene (FCR3-varT3-1) in chromosome 3 of the P. falciparum FCR3 line. The relatively high level transcription from this gene shows that the polymorphic chromosome ends of P. falciparum, which have been proposed to be transcriptionally silent, can be active expression sites for var genes. The pattern of the FCR3-varT11-1 and FCR3-varT3-1 genes are variable between different P. falciparum lines, presumably due to DNA rearrangements. Thus, recombination events in subtelomeric DNA may have a role in the expression of novel var forms.
| S-EPMC231785 | biostudies-other
Project description:Var genes encode the major surface antigen (PfEMP1) of the blood stages of the human malaria parasite Plasmodium falciparum. Differential expression of up to 60 diverse var genes in each parasite genome underlies immune evasion. We compared the diversity of the DBLalpha domain of var genes sampled from 30 parasite isolates from a malaria endemic area of Papua New Guinea (PNG) and 59 from widespread geographic origins (global). Overall, we obtained over 8,000 quality-controlled DBLalpha sequences. Within our sampling frame, the global population had a total of 895 distinct DBLalpha "types" and negligible overlap among repertoires. This indicated that var gene diversity on a global scale is so immense that many genomes would need to be sequenced to capture its true extent. In contrast, we found a much lower diversity in PNG of 185 DBLalpha types, with an average of approximately 7% overlap among repertoires. While we identify marked geographic structuring, nearly 40% of types identified in PNG were also found in samples from different countries showing a cosmopolitan distribution for much of the diversity. We also present evidence to suggest that recombination plays a key role in maintaining the unprecedented levels of polymorphism found in these immune evasion genes. This population genomic framework provides a cost effective molecular epidemiological tool to rapidly explore the geographic diversity of var genes.
| S-EPMC1828697 | biostudies-literature
Project description:BackgroundGuanine-rich nucleic acid sequences are capable of folding into an intramolecular four-stranded structure called a G-quadruplex. When found in gene promoter regions, G-quadruplexes can downregulate gene expression, possibly by blocking the transcriptional machinery. Here we have used a genome-wide bioinformatic approach to identify Putative G-Quadruplex Sequences (PQS) in the Plasmodium falciparum genome, along with biophysical techniques to examine the physiological stability of P. falciparum PQS in vitro.ResultsWe identified 63 PQS in the non-telomeric regions of the P. falciparum clone 3D7. Interestingly, 16 of these PQS occurred in the upstream region of a subset of the P. falciparum var genes (group B var genes). The var gene family encodes PfEMP1, the parasite's major variant antigen and adhesin expressed at the surface of infected erythrocytes, that plays a key role in malaria pathogenesis and immune evasion. The ability of the PQS found in the upstream regions of group B var genes (UpsB-Q) to form stable G-quadruplex structures in vitro was confirmed using 1H NMR, circular dichroism, UV spectroscopy, and thermal denaturation experiments. Moreover, the synthetic compound BOQ1 that shows a higher affinity for DNA forming quadruplex rather than duplex structures was found to bind with high affinity to the UpsB-Q.ConclusionThis is the first demonstration of non-telomeric PQS in the genome of P. falciparum that form stable G-quadruplexes under physiological conditions in vitro. These results allow the generation of a novel hypothesis that the G-quadruplex sequences in the upstream regions of var genes have the potential to play a role in the transcriptional control of this major virulence-associated multi-gene family.
| S-EPMC2736202 | biostudies-literature
Project description:Most severe Plasmodium falciparum infections are experienced by young children. Severe symptoms are precipitated by vascular sequestration of parasites expressing a particular subset of the polymorphic P. falciparum erythrocyte membrane protein 1 (PfEMP1) adhesion molecules. Parasites binding human endothelial protein C receptor (EPCR) through the CIDRα1 domain of certain PfEMP1 were recently associated with severe malaria in children. However, it has remained unclear to which extend the EPCR-binding CIDRα1 domains epitomize PfEMP1 expressed in severe malaria. Here, we characterized the near full-length transcripts dominating the var transcriptome in children with severe malaria and found that the only common feature of the encoded PfEMP1 was CIDRα1 domains. Such genes were highly and dominantly expressed in both children with severe malarial anaemia and cerebral malaria. These observations support the hypothesis that the CIDRα1-EPCR interaction is key to the pathogenesis of severe malaria and strengthen the rationale for pursuing a vaccine or adjunctive treatment aiming at inhibiting or reducing the damaging effects of this interaction.
| S-EPMC4967939 | biostudies-literature
Project description:The enormous diversity and complexity of var genes that diversify rapidly by recombination has led to the exclusion of assembly of these genes from major genome initiatives (e.g., Pf6). A scalable solution in epidemiological surveillance of var genes is to use a small 'tag' region encoding the immunogenic DBLα domain as a marker to estimate var diversity. As var genes diversify by recombination, it is not clear the extent to which the same tag can appear in multiple var genes. This relationship between marker and gene has not been investigated in natural populations. Analyses of in vitro recombination within and between var genes have suggested that this relationship would not be exclusive. Using a dataset of publicly-available assembled var sequences, we test this hypothesis by studying DBLα-var relationships for four study sites in four countries: Pursat (Cambodia) and Mae Sot (Thailand), representing low malaria transmission, and Navrongo (Ghana) and Chikwawa (Malawi), representing high malaria transmission. In all study sites, DBLα-var relationships were shown to be predominantly 1-to-1, followed by a second largest proportion of 1-to-2 DBLα-var relationships. This finding indicates that DBLα tags can be used to estimate not just DBLα diversity but var gene diversity when applied in a local endemic area. Epidemiological applications of this result are discussed.
| S-EPMC10060044 | biostudies-literature
Project description:One of the major virulence factors of the malaria causing parasite is the Plasmodium falciparum encoded erythrocyte membrane protein 1 (PfEMP1). It is translocated to It the membrane of infected erythrocytes and expressed from approximately 60 var genes in a mutually exclusive manner. Switching of var genes allows the parasite to alter functional and antigenic properties of infected erythrocytes, to escape the immune defense and to establish chronic infections. We have developed an efficient method for isolating VAR genes from telomeric and other genome locations by adapting transformation-associated recombination (TAR) cloning, which can then be analyzed and sequenced. For this purpose, three plasmids each containing a homologous sequence representing the upstream regions of the group A, B, and C var genes and a sequence homologous to the conserved acidic terminal segment (ATS) of var genes were generated. Co-transfection with P. falciparum strain ITG2F6 genomic DNA in yeast cells yielded 200 TAR clones. The relative frequencies of clones from each group were not biased. Clones were screened by PCR, as well as Southern blotting, which revealed clones missed by PCR due to sequence mismatches with the primers. Selected clones were transformed into E. coli and further analyzed by RFLP and end sequencing. Physical analysis of 36 clones revealed 27 distinct types potentially representing 50% of the var gene repertoire. Three clones were selected for sequencing and assembled into single var gene containing contigs. This study demonstrates that it is possible to rapidly obtain the repertoire of var genes from P. falciparum within a single set of cloning experiments. This technique can be applied to individual isolates which will provide a detailed picture of the diversity of var genes in the field. This is a powerful tool to overcome the obstacles with cloning and assembly of multi-gene families by simultaneously cloning each member.
| S-EPMC3049791 | biostudies-literature
Project description:The Plasmodium falciparum variant surface antigen PfEMP1 expressed on the surface of infected erythrocytes is thought to play a major role in the pathology of severe malaria. As the sequence pool of the var genes encoding PfEMP1 expands there are opportunities, despite the high degree of sequence diversity demonstrated by this gene family, to reconstruct full-length var genes from small sequence tags generated from patient isolates. To test whether this is possible we have used a set of recently laboratory adapted ICAM-1-binding parasite isolates to generate sequence tags and, from these, to identify the full-length PfEMP1 being expressed by them. In a subset of the strains available we were able to produce validated, full-length var gene sequences and use these to conduct biophysical analyses of the ICAM-1 binding regions.
| S-EPMC5818487 | biostudies-literature
Project description:The most polymorphic gene family in P. falciparum is the ∼60 var genes distributed across parasite chromosomes, both in the subtelomeres and in internal regions. They encode hypervariable surface proteins known as P. falciparum erythrocyte membrane protein 1 (PfEMP1) that are critical for pathogenesis and immune evasion in Plasmodium falciparum. How var gene sequence diversity is generated is not currently completely understood. To address this, we constructed large clone trees and performed whole genome sequence analysis to study the generation of novel var gene sequences in asexually replicating parasites. While single nucleotide polymorphisms (SNPs) were scattered across the genome, structural variants (deletions, duplications, translocations) were focused in and around var genes, with considerable variation in frequency between strains. Analysis of more than 100 recombination events involving var exon 1 revealed that the average nucleotide sequence identity of two recombining exons was only 63% (range: 52.7-72.4%) yet the crossovers were error-free and occurred in such a way that the resulting sequence was in frame and domain architecture was preserved. Var exon 1, which encodes the immunologically exposed part of the protein, recombined in up to 0.2% of infected erythrocytes in vitro per life cycle. The high rate of var exon 1 recombination indicates that millions of new antigenic structures could potentially be generated each day in a single infected individual. We propose a model whereby var gene sequence polymorphism is mainly generated during the asexual part of the life cycle.
| S-EPMC4270465 | biostudies-literature
Project description:A genes-first approach to genome sequencing is described which efficiently generates gene sequence tags from genomic DNA. Mung bean nuclease (EC 3.1.30.1) cleaves the genomic DNA of many organisms before and after genes and within some introns. Analysis of gene sequence tags prepared from mung bean nuclease-digested Plasmodium falciparum DNA demonstrates that this method has several advantages over the popular cDNA expressed sequence tag approach. To date, 673 sequence tags containing over 215 kb of sequence have been generated from 400 clones. Sixty clones (15%) have significant similarity to sequences in the protein and translated nucleic acid data bases. These represent 51 unique genes, of which only 5 encode previously known P. falciparum proteins. The identified proteins include those expressed in erythrocytic, exoerythrocytic, and gametocytic stages of the parasite. Thirty percent of clones identified appear to carry complete coding regions. The spacer DNA separating genes is rarely cloned. These gene sequence tags will form a useful data base from which to initiate projects to develop new therapeutics, vaccines, and strategies to control human malaria.
| S-EPMC47673 | biostudies-other
Project description:Parasite-encoded variant surface antigens (VSAs) like the var gene-encoded Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) family are responsible for antigenic variation and infected red blood cell (RBC) cytoadhesion in P. falciparum malaria. Parasites causing severe malaria in nonimmune patients tend to express a restricted subset of VSA (VSA(SM)) that differs from VSA associated with uncomplicated malaria and asymptomatic infection (VSA(UM)). We compared var gene transcription in unselected P. falciparum clone 3D7 expressing VSA(UM) to in vitro-selected sublines expressing VSA(SM) to identify PfEMP1 responsible for the VSA(SM) phenotype. Expression of VSA(SM) was accompanied by up-regulation of Group A var genes. The most prominently up-regulated Group A gene (PFD1235w/MAL7P1.1) was translated into a protein expressed on the infected RBC surface. The proteins encoded by Group A var genes, such as PFD1235w/MAL7P1.1, appear to be involved in the pathogenesis of severe disease and are thus attractive candidates for a vaccine against life-threatening P. falciparum malaria.
| S-EPMC2211911 | biostudies-literature