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Chris J Janse

Publications and source records attributed to Chris J Janse.

25 records · Page 2Linked to original sources
PubMed2004

A Plasmodium berghei reference line that constitutively expresses GFP at a high level throughout the complete life cycle.

Green fluorescent protein (GFP) is a well-established reporter protein for the examination of biological processes. This report describes a recombinant Plasmodium berghei, PbGFPCON, that constitutively expresses GFP in a growth responsive manner in its cytoplasm from a transgene that is integrated into the genome and controlled by the strong promoter from a P. berghei elongation factor-1alpha gene. All life cycle forms of PbGFPCON except for male gametes can be easily visualized by fluorescent microscopy. PbGFPCON showed similar growth characteristics to wild type P. berghei parasites throughout the whole life cycle and can therefore be used as a reference line for future investigations of parasite-host cell interactions. The principle of automated fluorescence-based counting and sorting of live parasites from host cell backgrounds and different parasite forms from complex mixtures such as asynchronous blood stages is established. PbGFPCON allows the visualization and investigation of live parasite stages that are difficult and labor-intensive to observe, such as the liver and mosquito stages. PbGFPCON can be employed to establish the phenotype of independent mutant parasites. With the recent development of a second, independent selectable marker in P. berghei, PbGFPCON is a useful tool to investigate the effect of further genetic modifications on host-parasite interactions.

Animals↗
PubMed2004

Gene targeting demonstrates that the Plasmodium berghei subtilisin PbSUB2 is essential for red cell invasion and reveals spontaneous genetic recombination events.

The Plasmodium merozoite proteases involved in the crucial process of erythrocyte invasion are promising targets for novel malaria control strategies. We report here the characterization of the subtilisin-like protease SUB2 from the rodent parasites Plasmodium berghei and Plasmodium yoelii, leading the way to in vivo functional studies of this enzyme. The kinetics of expression and subcellular localization imply a central role for SUB2 in erythrocyte invasion. Through the use of gene targeting strategies, we assessed the relevance of P. berghei SUB2 for the intraerythrocytic cycle. The selection of recombinant Pbsub2-TrimycDuoXpress-tagged parasites and the proper expression of the modified coding region demonstrate that the Pbsub2 locus is accessible to genetic modifications. However, Pbsub2 knock-out parasites were not recovered, confirming the importance of PbSUB2 for P. berghei merozoite stages, and supporting the fact that its Plasmodium falciparum SUB2 orthologue is an attractive drug target candidate. Finally, we identify revertant parasites that have lost the integrated selection cassette while conserving a Pbsub2-tagged gene. These spontaneous reversion events should overcome the scarcity of selectable markers available for this parasite, giving access to multiple gene tagging strategies, which, together with the validation of a TrimycDuoXpress tag, would represent valuable new tools for studying the biology of P. berghei.

Amino Acid Sequence↗
PubMed2004

Real-time, in vivo analysis of malaria ookinete locomotion and mosquito midgut invasion.

Invasion of the Anopheles mosquito midgut by the Plasmodium ookinete is a critical step in the malaria transmission cycle. We have generated a fluorescent P. berghei transgenic line that expresses GFP in the ookinete and oocyst stages, and used it to perform the first real-time analysis of midgut invasion in the living mosquito as well as in explanted intact midguts whose basolateral plasma membranes were vitally stained. These studies permitted detailed analysis of parasite motile behaviour in the midgut and cell biological analysis of the invasion process. Throughout its journey, the ookinete displays distinct modes of motility: stationary rotation, translocational spiralling and straight-segment motility. Spiralling is based on rotational motility combined with translocation steps and changes in direction, which are achieved by transient attachments of the ookinete's trailing end. As it moves from the apical to the basal side of the midgut epithelium, the ookinete uses a predominant intracellular route and appears to glide on the membrane in foldings of the basolateral domain. However, it traverses serially the cytoplasm of several midgut cells before entering and migrating through the basolateral intercellular space to access the basal lamina. The invaded cells commit apoptosis, and their expulsion from the epithelium invokes wound repair mechanisms including extensive lamellipodia crawling. A 'hood' of lamellipodial origin, provided by the invaded cell, covers the ookinete during its egress from the epithelium. The flexible ookinete undergoes shape changes and temporary constrictions associated with passage through the plasma membranes. Similar observations were made in both A. gambiae and A. stephensi, demonstrating the conservation of P. berghei interactions with these vectors.

Amino Acid Sequence↗
PubMed2003

Malaria parasites lacking eef1a have a normal S/M phase yet grow more slowly due to a longer G1 phase.

Eukaryotic elongation factor 1A (eEF1A) plays a central role in protein synthesis, cell growth and morphology. Malaria parasites possess two identical genes encoding eEF1A (eef1aa and eef1ab). Using pbeef1a-Plasmodium berghei mutants that lack an eEF1a gene, we demonstrate that the level of eEF1A production affects the proliferation of blood stages and parasite fitness. Pbeef1a- parasites can complete the vertebrate and mosquito phases of the life cycle, but the growth phase of the asexual blood stages is extended by up to 20%. Analysis of the cell cycle by flow cytometry as well as transcriptional analyses revealed that the duration of the S and M phases and the number of daughter cells produced were not detectably affected, but that the G1 phase is elongated. Thus, as in budding yeast, a growth threshold must be achieved by blood-stage Plasmodium parasites to permit transition from G1 into S/M phase. Initial analyses indicate that transcriptional events associated with gametocyte development were not remarkably retarded. Insight into protein synthesis and its influence on cell proliferation might be used to generate slow-growing (attenuated) parasites.

Animals↗
PubMed2002

Genome sequence and comparative analysis of the model rodent malaria parasite Plasmodium yoelii yoelii.

Species of malaria parasite that infect rodents have long been used as models for malaria disease research. Here we report the whole-genome shotgun sequence of one species, Plasmodium yoelii yoelii, and comparative studies with the genome of the human malaria parasite Plasmodium falciparum clone 3D7. A synteny map of 2,212 P. y. yoelii contiguous DNA sequences (contigs) aligned to 14 P. falciparum chromosomes reveals marked conservation of gene synteny within the body of each chromosome. Of about 5,300 P. falciparum genes, more than 3,300 P. y. yoelii orthologues of predominantly metabolic function were identified. Over 800 copies of a variant antigen gene located in subtelomeric regions were found. This is the first genome sequence of a model eukaryotic parasite, and it provides insight into the use of such systems in the modelling of Plasmodium biology and disease.

Animals↗
PubMed2002

Topology and replication of a nuclear episomal plasmid in the rodent malaria Plasmodium berghei.

The rodent malaria Plasmodium berghei is one of a small number of species of Plasmodium that can currently be genetically transformed through experimentally controlled uptake of exogenous DNA by bloodstage parasites. Circular DNA containing a selectable marker replicates and is maintained under selection pressure in a randomly segregating episomal form during the first weeks after transformation. In this study, using pulsed field gel electrophoresis and ionising radiation, we show that in dividing asexual blood stage parasites the episomes are completely converted, within 2 weeks post-infection, into non-rearranged circular concatamers ranging in size between about 9 and 15 copies of the monomer. These occur as slow-moving aggregates held together by radiation-sensitive linkers consisting partly of single-stranded DNA. The process generating these complexes is not clear but 2D gel analysis showed that Cairns-type replication origins were absent and it seems most likely that the initial concatamerisation takes place using a rolling circle mechanism followed by circularisation through internal recombination. We propose a model in which continued rolling circle replication of the large circular concatamers and the recombinational activity of the tails of the rolling circles could lead to the formation of the large aggregates.

Animals↗