The annotated genomes of organisms define a 'blueprint' of their possible gene products. Post-genome analyses attempt to confirm and modify the annotation and impose a sense of the spatial, temporal and developmental usage of genetic information by the organism. Here we describe a large-scale, high-accuracy (average deviation less than 0.02 Da at 1,000 Da) mass spectrometric proteome analysis of selected stages of the human malaria parasite Plasmodium falciparum. The analysis revealed 1,289 proteins of which 714 proteins were identified in asexual blood stages, 931 in gametocytes and 645 in gametes. The last two groups provide insights into the biology of the sexual stages of the parasite, and include conserved, stage-specific, secreted and membrane-associated proteins. A subset of these proteins contain domains that indicate a role in cell-cell interactions, and therefore can be evaluated as potential components of a malaria vaccine formulation. We also report a set of peptides with significant matches in the parasite genome but not in the protein set predicted by computational methods.

Plasmodium falciparum sporozoites that develop and mature inside an Anopheles mosquito initiate a malaria infection in humans. Here we report the first proteomic comparison of different parasite stages from the mosquito—early and late oocysts containing midgut sporozoites, and the mature, infectious salivary gland sporozoites. Despite the morphological similarity between midgut and salivary gland sporozoites, their proteomes are markedly different, in agreement with their increase in hepatocyte infectivity. The different sporozoite proteomes contain a large number of stage specific proteins whose annotation suggest an involvement in sporozoite maturation, motility, infection of the human host and associated metabolic adjustments. Analyses of proteins identified in the P. falciparum sporozoite proteomes by orthologous gene disruption in the rodent malaria parasite, P. berghei, revealed three previously uncharacterized Plasmodium proteins that appear to be essential for sporozoite development at distinct points of maturation in the mosquito. This study sheds light on the development and maturation of the malaria parasite in an Anopheles mosquito and also identifies proteins that may be essential for sporozoite infectivity to humans.

Pfs48/45, a member of a Plasmodium-specific protein family, displays conformation-dependent epitopes and is an important target for malaria transmission-blocking (TB) immunity. To design a recombinant Pfs48/45-based TB vaccine, we analyzed the conformational TB epitopes of Pfs48/45. The Pfs48/45 protein was found to consist of a C-terminal six-cysteine module recognized by anti-epitope I antibodies, a middle four-cysteine module recognized by anti-epitopes IIb and III, and an N-terminal module recognized by anti-epitope V antibodies. Refolding assays identified that a fragment of 10 cysteines (10C), comprising the middle four-cysteine and the C-terminal six-cysteine modules, possesses superior refolding capacity. The refolded and partially purified 10C conformer elicited antibodies in mice that targeted at least two of the TB epitopes (I and III). The induced antibodies could block the fertilization of Plasmodium falciparum gametes in vivo in a concentration-dependent manner. Our results provide important insight into the structural organization of the Pfs48/45 protein and experimental support for a Pfs48/45-based subunit vaccine.Malaria parasites are spread in the human population by Plasmodium-infected Anopheles mosquitoes. After a blood meal on infected humans, mosquitoes become infected by ingesting a sexual form of the malaria parasite called gametocytes. Subsequent sporogonic development in the mosquito can be prevented by the presence of anti-malaria, transmissionblocking (TB) 3 antibodies in the ingested blood meal (1-3). Pfs48/45 is a TB vaccine candidate that belongs to a family of malaria-specific proteins that contain conserved motifs with four-or six-cysteine residues (4). Pfs48/45 plays a key role in parasite fertilization (5), and monoclonal antibodies (mAbs) against Pfs48/45 prevent fertilization (6). Anti-Pfs48/45 antibodies are present in human sera from endemic areas, and there is evidence that the presence of anti-Pfs48/45 antibodies in natural sera correlates with TB activity (7). The induction of antibodies after natural infection, as observed in the field, creates the highly beneficial potential of vaccine boosting in the endemic setting. At least four different epitopes (I, IIb, III, and V) on Pfs48/45 are targeted by monoclonal antibodies that block or reduce malaria transmission (8, 9). Apart from epitope V, the TB epitopes are sensitive to reducing agents, which indicates the importance of the disulfide bridges and hence the conformational dependence of these epitopes (8, 10).To design a Pfs48/45-based TB vaccine, we aimed to delineate and characterize the TB epitopes of Pfs48/45. Protease digestion of the native protein, expression in Escherichia coli of truncations, cysteine mutations, and refolding assays were evaluated by immunological analysis of the TB epitopes. An N-terminally truncated protein with higher refolding efficiency was identified, partially purified, and tested in mice immunization experiments. The elicited antibodies recognized the native Pfs48/45 protein, competed ...

A new DNA polymorphism assay was developed in 1990 that is based on the amplification by the polymerase chain reaction (PCR) of random DNA segments, using single primers of arbitrary nucleotide sequence. The amplified DNA fragments, referred to as RAPD markers, were shown to be highly useful in the construction of genetic maps ("RAPD mapping"). We have now adapted the RAPD assay to tomato. Using a set of 11 oligonucleotide decamer primers, each primer directed the amplification of a genome-specific "fingerprint" of DNA fragments. The potential of the original RAPD assay to generate polymorphic DNA markers with a given set of primers was further increased by combining two primers in a single PCR. By comparing "fingerprints" of L. esculentum, L. pennellii, and the L. esculentum chromosome 6 substitution line LA1641, which carries chromosome 6 from L. pennellii, three chromosome 6-specific RAPD markers could be directly identified among the set of amplified DNA fragments. Their chromosomal position on the classical genetic map of tomato was subsequently established by restriction fragment length polymorphism (RFLP) linkage analysis. One of the RAPD markers was found to be tightly linked to the nematode resistance gene Mi.

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