Zoe Christodoulou

Trinucleotide repeat amplification and hypermethylation of a CpG island in FRAXE mental retardation

We have cloned the fragile site FRAXE and demonstrate that individuals with this fragile site possess amplifications of a GCC repeat adjacent to a CpG island in Xq28 of the human X chromosome. Normal individuals have 6-25 copies of the GCC repeat, whereas mentally retarded, FRAXE-positive individuals have > 200 copies and also have methylation at the CpG island. This situation is similar to that seen at the FRAXA locus and is another example in which a trinucleotide repeat expansion is associated with a human genetic disorder. In contrast with the fragile X syndrome, the GCC repeat can expand or contract and is equally unstable when passed through the male or female line. These results also have implications for the understanding of chromosome fragility.

A well‐conserved Plasmodium falciparum var gene shows an unusual stage‐specific transcript pattern

The var multicopy gene family encodes Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) variant antigens, which, through their ability to adhere to a variety of host receptors, are thought to be important virulence factors. The predominant expression of a single cytoadherent PfEMP1 type on an infected red blood cell, and the switching between different PfEMP1 types to evade host protective antibody responses, are processes thought to be controlled at the transcriptional level. Contradictory data have been published on the timing of var gene transcription. Reverse transcription‐polymerase chain reaction (RT‐PCR) data suggested that transcription of the predominant var gene occurs in the later (pigmented trophozoite) stages, whereas Northern blot data indicated such transcripts only in early (ring) stages. We investigated this discrepancy by Northern blot, with probes covering a diverse var gene repertoire. We confirm that almost all var transcript types were detected only in ring stages. However, one type, the well‐conserved varCSA transcript, was present constitutively in different laboratory parasites and does not appear to undergo antigenic variation. Although varCSA has been shown to encode a chondroitin sulphate A (CSA)‐binding PfEMP1, we find that the presence of full‐length varCSA transcripts does not correlate with the CSA‐binding phenotype.

Antigenic Variation in Plasmodium falciparum Malaria Involves a Highly Structured Switching Pattern

Many pathogenic bacteria, fungi, and protozoa achieve chronic infection through an immune evasion strategy known as antigenic variation. In the human malaria parasite Plasmodium falciparum, this involves transcriptional switching among members of the var gene family, causing parasites with different antigenic and phenotypic characteristics to appear at different times within a population. Here we use a genome-wide approach to explore this process in vitro within a set of cloned parasite populations. Our analyses reveal a non-random, highly structured switch pathway where an initially dominant transcript switches via a set of switch-intermediates either to a new dominant transcript, or back to the original. We show that this specific pathway can arise through an evolutionary conflict in which the pathogen has to optimise between safeguarding its limited antigenic repertoire and remaining capable of establishing infections in non-naïve individuals. Our results thus demonstrate a crucial role for structured switching during the early phases of infections and provide a unifying theory of antigenic variation in P. falciparum malaria as a balanced process of parasite-intrinsic switching and immune-mediated selection.

PfEMP1 expression is reduced on the surface of knobless Plasmodium falciparum infected erythrocytes

The Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) is a key virulence factor for this species of human malarial parasite. PfEMP1 is expressed on the surface of infected erythrocytes (IEs) and directly mediates adhesion to a variety of host cells. A number of other parasite-encoded proteins are similarly exported to the IE plasma membrane and play an indirect role in this adhesion process through the modification of the erythrocyte cytoskeleton and the formation of electron dense knobs into which PfEMP1 is anchored. Analysis of the specific contribution of knob-associated proteins to adhesion is difficult due to rapid PfEMP1 switching during in vitro culture. Furthermore, these studies typically assume that the level and distribution of PfEMP1 exposed in knobby (K+) and knobless (K–) IEs is unaltered, an assumption not yet supported with data. We describe here the preparation and characterisation of a panel of isogenic K+ and K– parasite clones that express one of two defined PfEMP1 variants. Analysis of the cytoadhesive properties of these clones shows that both static and flow adhesion is reduced in all the K– clones and, further, that this correlates with an approximately 50% reduction in PfEMP1 displayed on the IE surface. However, despite this reduction, the gross distribution of PfEMP1 in K– IEs appears unaltered. These data impact on our current interpretation of the role of knobs in adhesion and the mechanism of trafficking PfEMP1 to the IE surface.

Current status of the Plasmodium falciparum genome project

The Plasmodium falciparum Genome Project is a collaborative effort by many laboratories that will provide detailed molecular information about the parasite, which may be used for developing practical control measures. Initial goals are to prepare an electronically indexed clone bank containing partially sequenced clones representing up to 80% of the parasites genes and to prepare an ordered set of overlapping clones spanning each of the parasites 14 chromosomes. Currently, clones of genomic DNA, prepared as yeast artificial chromosomes, are arranged into contigs covering approximately 70% of the genome of parasite clone 3D7, gene sequence tags are available from more than contigs covering approximately 70% of the genome of parasite clone 3D7, gene sequence tags are available from more than 20% of the parasites genes, and approximately 5% of the parasites genes are tentatively identified from similarity searches of entries in the international sequence databases. A total of > 0.5 Mb of P. falciparum sequence tag data is available. The gene sequence tags are presently being used to complete YAC contig assembly and localize the cloned genes to positions on the physical map in preparation for sequencing the genome. Routes of access to project information and services are described.

Plasmodium falciparum var gene expression is developmentally controlled at the level of RNA polymerase II-mediated transcription initiation

The Plasmodium falciparum var gene family codes for a major virulence factor in this most lethal of human malaria parasites. A single var protein variant type is expressed on each infected red blood cell, with antigenic variation allowing progeny parasites to escape host immune detection. The control of mutually exclusive var gene expression in the parasite relies on in situ epigenetic changes. Whether control of expression occurs at transcription initiation or post transcription, however, remains to be established. Recent evidence supports existence of a unique var transcription site at the nuclear periphery containing the dominantly expressed var gene, although silent var genes can colocalize to the same region. We demonstrate here that exclusive var gene expression is controlled at the level of transcription initiation during ring stages and that var genes are transcribed by RNA polymerase II. This represents another example where P. falciparum differs from the paradigm for antigenic variation, Trypanosoma brucei.

Origins of the fragile X syndrome mutation.

The fragile X syndrome is a common cause of mental impairment. In view of the low reproductive fitness of affected males, the high incidence of the syndrome has been suggested to be the result of a high rate of new mutations occurring exclusively in the male germline. Extensive family studies, however, have failed to identify any cases of a new mutation. Alternatively, it has been suggested that a selective advantage of unaffected heterozygotes may, in part, explain the high incidence of the syndrome. Molecular investigations have shown that the syndrome is caused by the amplification of a CGG trinucleotide repeat in the FMR-1 gene which leads to the loss of gene expression. Further to this, genetic studies have suggested that there is evidence of linkage disequilibrium between the fragile X disease locus and flanking polymorphic markers. More recently, this analysis has been extended and has led to the observation that a large number of fragile X chromosomes appear to be lineage descendants of founder mutation events. Here, we present a study of the FRAXAC1 polymorphic marker in our patient cohort. We find that its allele distribution is strikingly different on fragile X chromosomes, confirming the earlier observations and giving further support to the suggestions of a fragile X founder effect.

Functional Identification of the Plasmodium Centromere and Generation of a Plasmodium Artificial Chromosome

Summary The artificial chromosome represents a useful tool for gene transfer, both as cloning vectors and in chromosome biology research. To generate a Plasmodium artificial chromosome (PAC), we had to first functionally identify and characterize the parasites centromere. A putative centromere (pbcen5) was cloned from chromosome 5 of the rodent parasite P. berghei based on a Plasmodium gene-synteny map. Plasmids containing pbcen5 were stably maintained in parasites during a blood-stage infection with high segregation efficiency, without drug pressure. pbcen5-containing plasmids were also stably maintained during parasite meiosis and mitosis in the mosquito. A linear PAC (L-PAC) was generated by integrating pbcen5 and telomere into a plasmid. The L-PAC segregated with a high efficiency and was stably maintained throughout the parasites life cycle, as either one or two copies. These results suggest that L-PAC behaves like a Plasmodium chromosome, which can be exploited as an experimental research tool.

Acquisition of naturally occurring antibody responses to recombinant protein domains of Plasmodium falciparum erythrocyte membrane protein 1

BackgroundAntibodies targeting variant antigens expressed on the surface of Plasmodium falciparum infected erythrocytes have been associated with protection from clinical malaria. The precise target for these antibodies is unknown. The best characterized and most likely target is the erythrocyte surface-expressed variant protein family Plasmodium falciparum erythrocyte membrane protein 1 (Pf EMP1).MethodsUsing recombinant proteins corresponding to five domains of the expressed A4 var gene, A4 Pf EMP1, the naturally occurring antibody response was assessed, by ELISA, to each domain in serum samples obtained from individuals resident in two communities of differing malaria transmission intensity on the Kenyan coast. Using flow cytometry, the correlation in individual responses to each domain with responses to intact A4- infected erythrocytes expressing A4 Pf EMP1 on their surface as well as responses to two alternative parasite clones and one clinical isolate was assessed.ResultsMarked variability in the prevalence of responses between each domain and between each transmission area was observed, as wasa strong correlation between age and reactivity with some but not all domains. Individual responses to each domain varied strikingly, with some individuals showing reactivity to all domains and others with no reactivity to any, this was apparent at all age groups. Evidence for possible cross-reactivity in responses to the domain DBL4γ was found.ConclusionIndividuals acquire antibodies to surface expressed domains of a highly variant protein. The finding of potential cross-reactivity in responses to one of these domains is an important initial finding in the consideration of potential vaccine targets.

Effect of var gene disruption on switching in Plasmodium falciparum

The molecular mechanisms underpinning switching of variant antigens on the surface of Plasmodium falciparum‐infected erythrocytes are poorly understood. We tested the hypothesis that insertional disruption of the A4var gene, one of two var genes located within the subtelomeric region of one end of chromosome 13, would result in a preferential switch in transcription to the adjacent R29var gene upon rosette selection. In this way, we aimed to mimic the preferential transcription of R29var in rosetting R29 parasites, a parasite line in which the A4var gene is deleted through a chromosome end truncation. Initial analysis of the knock‐out parasite lines shows that the insertional disruption of the A4var gene prevents A4 PfEMP1 expression, but that switching transcription to other var gene variants is unaffected. Furthermore, analysis of var transcription in the knock‐out parasite line during rosette selection shows that, rather than facilitating a switch to R29var gene transcription, this event was suppressed in the transfectants . These data, and the implications for epigenetic transcriptional control of var genes, are discussed.