Entering the post-genomic era of malaria research

Entering the post-genomic era of malariaresearchPaul Horrocks,1 Sharen Bowman,2 Susan Kyes,3 Andrew P. Waters,4 & Alister Craig5

The sequencing of the genome of Plasmodium falciparum promises to revolutionize the way in which malariaresearch will be carried out. Beyond simple gene discovery, the genome sequence will facilitate the comprehensivedetermination of the parasite’s gene expression during its developmental phases, pathology, and in response toenvironmental variables, such as drug treatment and host genetic background. This article reviews the currentstatus of the P. falciparum genome sequencing project and the unique insights it has generated. We alsosummarize the application of bioinformatics and analytical tools that have been developed for functional genomics.The aim of these activities is the rational, information-based identification of new therapeutic strategies andtargets, based on a thorough insight into the biology of Plasmodium spp.

Keywords: Plasmodium falciparum, genetics; genome, protozoan; base sequence; sequence analysis, DNA.

Bulletin of the World Health Organization, 2000, 78: 1424–1437.

Voir page 1434 le resume en francais. En la pagina 1434 figura un resumen en espanol.

Introduction

The development of genome sequencing technolo-gies over the last five years has resulted in a wealth ofsequence information, culminating in the recentannouncement of a working draft of the humangenome. Pathogen genomes, through their smallersize, have been even more tractable to thesemethodologies and are now well represented ingenome science. Although not always suited as‘‘model’’ organisms, the importance of pathogens inmedicine and agriculture has made the exploitation ofthe sequence databases a high priority. But what doesthe production of long strings of As, Cs, Gs and Tsactually mean in terms of the alleviation of the burdenof disease, particularly in developing countries? Sofar, genome sequencing has largely been the provinceof the developed world, where the resources andscience infrastructure have allowed the formation ofhigh-throughput sequencing centres. However, theuse of sequencing information need not be restrictedin this way, provided that resources for training canbe met.

Probably the most important aspect of thepost-genomic era (i.e. after the sequencing has beencarried out) is analysis of the primary sequence data.This is called bioinformatics and embraces a range oftheoretical analyses aimed at converting the DNAsequence into biological information. A major part ofthis discipline involves the identification of genesthrough a number of processes, from identifyingsimilarities with previously identified genes fromother organisms, to the use of computer-derivedmodels based on existing data. Subsequently comepredictions of biological function and molecularshape (structural genomics), both of which havescope for development.

Knowing the whole sequence of a pathogengenome also allows researchers to investigate thebehaviour of organisms on a much broader basis thanwas previously possible. Now, instead of studying theeffect of drug treatment or differentiation on one ortwo genes, it is possible to study variation in all thegenes at the same time using global transcriptionalanalysis. Protein patterns may also be examined, orthe organism may be genetically modified (transfec-tion), providing a direct link between these biologicaleffectors and gross phenotype. The technology forthese experiments has been developed as a directconsequence of the desire to exploit the genomesequence data.

It is not hard to envisage that the ability toidentify and characterize the genetic blueprint ofpathogens will help us recognize critical elements inthe development and pathogenesis of disease-causing organisms and target our research efforts inthe production of new therapies. For Plasmodium

falciparum, genes and proteins acting at specific stagesin the life cycle can be identified, their roles tested by

1 Post-Doctoral Research Assistant, Molecular Parasitology Group,Institute of Molecular Medicine, John Radcliffe Hospital, Oxford,England.2 Manager, Malaria Sequencing Project, Pathogen Sequencing Unit,Sanger Centre, Wellcome Trust Genome Campus, Hinxton, England.3 Post-Doctoral Research Assistant, Molecular Parasitology Group,Institute of Molecular Medicine, John Radcliffe Hospital, Oxford,England.4 Reader, Department of Parasitology, Leiden University MedicalCentre, Leiden, Netherlands.5 Senior Lecturer, Liverpool School of Tropical Medicine, PembrokePlace, Liverpool L3 5QA, England (email: [email protected]).Correspondence should be addressed to this author.

Ref. No. 00-0825

Special Theme – Malaria

1424 # World Health Organization 2000 Bulletin of the World Health Organization, 2000, 78 (12)

genetic modification and promising candidates usedin vaccine production. Parasite metabolic pathwaysnot present in the host could also be targeted withpotent inhibitors that are non-toxic to humans; andparasite drug-resistance mechanisms could also betargeted, giving existing drugs a longer effectivelifetime. The sequence of the P. falciparum genomewill provide many opportunities for research intomalaria, but this is only a beginning, with thechallenge being to turn those opportunities intoeffective treatments in the field.

The Plasmodium falciparum genome

The genome of P. falciparum consists of three discretecomponents: a linear repeat of a 6 kb element locatedwithin mitochondria; a 35 kb circle within a plastid-like structure (the apicoplast); and 25–30 Mb ofnuclear DNA (genomic DNA). The nuclear DNA isorganized into 14 chromosomes, between 0.75–3.5 Mb in size, as determined by pulse-field gelelectrophoresis (PFGE) and electron microscopiccounts of kinetochore structures. Indirect evidencefor the number of chromosomes also comes fromgenetic studies showing there are 14 linkage groups.

The nuclear genome is organized in a mannertypical of eukaryotes, with linear chromosomes beingbounded at either end with telomeric sequences.Genome plasticity, seen in many parasite isolates andidentified by size polymorphisms on PFGE, isthought to result frequently from deletions andinsertions of DNA within subtelomeric sequences, aregion shown to contain ordered repetitive sequenceelements.

The Plasmodium falciparum GenomeProject Consortium

Prior to the advent of yeast artificial chromosome(YAC) technology, there had been relatively littleaccess to the parasite’s genome, as its extreme [A+T]content rendered inserts unstable in conventionalbacterial plasmid clones. The estimated 80% [A+T]-rich genome could, however, be stably maintainedwithin the pYAC4 construct, as demonstrated by theconstruction of a number of YAC libraries fordifferent P. falciparum clones (1). In 1993 a con-sortium of laboratories distributed throughout theworld established the Wellcome Trust MalariaGenome Mapping Project, with the aim of assem-bling YAC contiguous sequences (contigs) acrosseach chromosome, as well as developing YAC,expressed sequence tag (EST), bioinformatic andgenetic mapping technology (2). This consortiumrealized that sequencing of the entire nuclear DNAwas a real possibility, yet considered the endeavourfraught with difficulties due to the extreme bias inbase content.

Genome sequencing has proved to be a power-ful and efficient approach in accessing the complete

gene complement for organisms as diverse asMycobacterium tuberculosis (3), Saccharomyces cerevisiae (4)and Caenorhabditis elegans (5). The advantages offeredby such a tool in the investigation of human malariaeventually resulted in pilot projects being establishedin 1996 at three high-throughput genome centres, toestablish whether sequencing the entire genome waspossible: the Sanger Centre (England); The Institutefor Genomic Research (TIGR) Malaria Program,Naval Medical Research Centre (NMRC) (USA); andStanford University (USA). The Wellcome Trust, theBurroughs Wellcome Fund, the National Institute ofAllergy and Infectious Diseases and the US Depart-ment of Defence provided funding for the pilotprojects, and following their success these agenciesagreed to fund the entire sequencing effort (6; seeTable 1 for progress). Associated with this work are anumber of other groups supporting the efforts of thehigh-throughput centres in a range of activities,including generation of chromosomal material; addi-tional mapping information; testing bacterial strainsmore tolerant of [A+T]-rich DNA; and the provisionof a repository for P. falciparum reagents (MR4, http://www.malaria.mr4.org/).

A similar strategy is being used by all of the high-throughput sequencing centres, with individual chro-mosomes being excised from pulse-field gels, clonedas small inserts into a double-stranded vector, andsequenced in the forward and reverse directions togenerate read-pair information which is used in gapfilling. Sequence-tagged site and simple sequence-length polymorphism microsatellite markers from theHB3xDd2 genetic cross (7), together with the opticalmap (8) of ordered restriction fragments, are used toposition contigs on each chromosome, or to confirmthat sequence data has been assembled correctly. Inaddition, to help assign and order sequences originat-ing from a particular chromosome region, the groupsat the Sanger Centre and Stanford University use ashotgun skim (1–2 fold coverage) of YAC clonesselected from the chromosomal YAC contigs, gener-ated by the original P. falciparum mapping project. Allthree sequencing centres aim for an error rate of lessthan 1 base in every 10 000 bases.

Once a section of chromosome sequence isfinished it is analysed to identify a number of features,including putative protein-coding regions, tRNAsand repetitive sequences. Database searches areperformed to identify similarities to protein andEST sequences. Further analyses are performed todetermine whether the predictions have proteindomains, signal sequences, putative membrane-spanning regions or any other distinctive features.A number of computing tools such as Hexamer/Genefinder (R. Durbin, P. Green, L. Hillier, unpub-lished software, 1998)), and GlimmerM (9) areavailable to assist in the identification of protein-coding regions, but many other gene predictionprograms exist or are currently being developed.

These tools are efficient at identifying single,large, open reading frames (ORFs) or genes with twoor three relatively large exons. However, the current

1425Bulletin of the World Health Organization, 2000, 78 (12)

Entering the post-genomic era of malaria research

generation of gene prediction software producesconflicting data when predicting multi-exon genes,particularly those with small exons (10, 11). Theseconflicting gene predictions are currently being testedexperimentally by reverse-transcription polymerasechain reaction (RT–PCR) assays (Fig. 1). Annotationof current and future P. falciparum sequences istherefore an ongoing process, with predictions andannotations being refined as more information, suchas the RT–PCR and EST sequencing data, becomesavailable.

Analysis of the Plasmodiumfalciparum genome

A total of 424 predicted protein-coding genes and3 tRNA genes have been identified on chromosomes2 and 3, the two chromosomes for which sequencinghas been completed (12, 13). Approximately 37%(158 genes) of these genes have a readily identifiablehomologue in another species. Such similarity allowsa function to be implied, with many of thoseidentified being involved in parasite metabolism.Interestingly, a comparison of the predicted proteinsequences with their homologues in other speciesshowed that the majority of P. falciparum proteinshave insertions of low complexity sequences, oftenruns of a single amino acid residue (typicallyasparagine, lysine or glutamic acid), or tandem arraysof a short peptide repeat sequence. Examples of suchregions have been identified previously, and arepolymorphic between different parasite isolates.

P. falciparum contains two organelles thought tohave arisen through endosymbiotic events: mito-chondria (14) and apicoplasts (15, 16). The uniquenature of the apicoplast and the essential functionsthat it carries out make it a prime candidate forantimalarial drug development. Like many extra-nuclear elements, many of the genes for the organellefunction have become nuclear encoded. Examina-tion of the predicted proteins encoded on chromo-somes 2 and 3 identified several that have a putativeapicoplast signal sequence. This indicates that theapicoplast contains type II fatty acid synthasesystems, typically associated with bacterial and plantplastids (17, 18). This supports the hypothesis thatthe apicoplast is algal in origin and provides a veryspecific target for rational drug design.

Other proteins likely to play a critical role inparasite metabolism are also easily identified fromsearches of the P. falciparum databases. Using thisapproach, Jomaa et al. (19) identified two P. falciparum

proteins with similarity to enzymes involved in the1-deoxy-D-xylulose-5-phosphate pathway of isopre-noid synthesis, which is used in green algae and somebacteria, but not in animals. Antibiotics developed toinhibit this pathway have already been shown to haveantimalarial activity in a rodent model system and inP. falciparum culture in vitro.

Database searches permit the ‘‘virtual’’ identi-fication of P. falciparum homologues to proteins fromother species, but comparative analysis of sequenceorganization (12, 13) has played a significant role inrevealing subtelomeric coding sequences that wouldotherwise have gone unnoticed. A closer examinationof the four available P. falciparum subtelomericsequences shows that the order of both repetitivesequences and the variant multigene family membersare conserved (Fig. 2). Members of the var, rif, stevorand Pf60 multigene families are present at all fourtelomeres, and new telomere-associated multigenefamilies have also been identified (conserved telomere-encoded proteins (CTPs)). While var, Pf60 and stevor

had already been relatively well-characterized, rif

required a more detailed analysis.Var comprises a highly polymorphic gene

family of roughly 50 copies per haploid genome(20–22). It codes for PfEMP-1 (P. falciparum

erythrocyte membrane protein-1) variants expressedon the surface of red blood cells, from the late ringstages of infection through schizogony. Each var

gene is composed of two exons (Fig. 3). Exon 1 codesfor the highly variable extracellular portion ofPfEMP-1, including between one and seven Duffy-binding-like (DBL) domains, with at least onecysteine-rich interdomain region (CIDR) (23). The3’ end of exon 1 codes for the semiconservedtransmembrane region, and exon 2 codes for thesemiconserved cytoplasmic region of the protein.PfEMP-1 is involved in cytoadherence of infectedred cells to a range of different host receptors onendothelial cells and in binding to uninfected redcells, forming rosettes. The cytoadherent or rosettingphenotype of an individual parasite-infected red cell

Table 1. Progress summary for the Malaria Genome Project

Chromosome Size (Mb) Statusa

14b 3.4 Late Closure13c 3.2 Closure12d 2.4 Late Closure11b 2.4 Closure10b 2.1 Closure

9c 1.8 Shotgun in progress8c 1.7 Shotgun in progress7c 1.7 Shotgun in progress6c 1.6 Shotgun complete5c 1.4 Closure4c 1.2 Late Closure3c 1.06 Finished2b 0.95 Finished1c 0.7 Late Closure

a Shotgun = random phase of sequencing where no selection of clones is used.

Closure = the shotgun phase leaves gaps in the chromosomal sequence and these haveto be filled using combinatorial approaches based on the sequence information flankingthe gaps. Sometimes the sequence data do exist but have to be extracted manually fromthe computer database.

Late Closure = sometimes a small number of gaps remain which are refractory to high-throughput sequencing methodologies and these need highly focussed approaches to fill them.b The Institute for Genomic Research, Malaria Program, Naval Medical Research Centre.c The Sanger Centre.d Stanford University.

1426 Bulletin of the World Health Organization, 2000, 78 (12)


has been correlated with severity of disease anddisease pathology (reviewed in 24).

The Pf60 family members contain a 3’ exonhighly similar to the var exon 2 sequence (25, 26), andhave multiple possible 5’ exon organizations. OnePf60 protein, 6.1, is expressed in the nucleus of lateasexual blood stage parasites, is composed of 7 exons,and employs a mechanism for reading through aninternal stop codon (27). Hybridization-based analy-sis of Pf60 suggested there were 140 copies perhaploid genome, but as the sequences are highlysimilar to var, an adjusted estimate would be roughly90 Pf60 genes per haploid genome.

Rif was originally described as an ˜1-kb DNAfragment repeated in the P. falciparum genome, ananomalous open reading frame (ORF) with no logicalmethionine start site (28). There was little point inhunting for upstream exons, since the dogma at thattime was that most P. falciparum genes did not haveintrons. The sequence was eventually spottedbetween two var genes (22), and from early releasesof genome sequences rif was recognized as a relativeof stevor (29). This analysis identified the smallupstream exon for both stevor and rif sequences

(Fig. 3), and showed that both sequence types containpredicted transmembrane regions, which wouldorient the predicted protein as a loop on the outersurface of a cell membrane. The rif sequences aredistinct from, and much more polymorphic than,stevor with an estimated 200 copies of rif per haploidgenome (13, 30), and roughly thirty-four members ofthe stevor family (29).

To date, it is uncertain where and when theSTEVOR proteins are expressed (31), although therelative lack of polymorphism suggests that they arenot likely to be expressed on the surface of red bloodcell. However, for rif the large number of highlypolymorphic copies suggested a location exposed tohost immune/selective pressures. Indeed, it wasshown that rif sequences code for clonally variant35–44 kD RIFIN proteins expressed on the surface ofinfected red blood cells (30), and that antibodies to theproteins are detectable in sera from immune indivi-duals (32). Although RIFINs and PfEMP-1 share thesame cellular localization, RIFIN function remainsunclear. While var genes are transcribed during all ringstages of development, and rif genes are only expressedfor a short time at the late-ring/early pigmented



trophozoite stage, both proteins are detected on thered cell surface at roughly the same time (earlytrophozoites) (25, 33). Further studies are necessary todetermine whether these proteins are functionallylinked as well as physically linked within the genome.

The final gap to be sequenced on chromosome3 covered a region of extreme [A+T] composition:97.3% for 2.6kb. Subsequent comparison with thesequence of chromosome 2 identified a region similarin both composition and length. Both [A+T]-richregions occur in gene-sparse areas of the chromo-somes, forming part of the longest intergenic regionon chromosome 3. Closer analysis revealed thepresence of chromosome-specific repetitive se-quences. Thus, their structure and extreme [A+T]composition suggest these regions are candidatecentromeres. [A+T]-rich central cores are present inSaccharomyces cerevisiae and Schizosaccharomyces pombe

centromeres and the latter contain complex, chro-mosome-specific sequences. Although, to date, theseregions have not been demonstrated to function asP. falciparum centromeres, a third example of thisstructure has recently been identified on chromo-some 1.

Functional genomics

The availability of an increasing number of pathogengenome sequences has strengthened the impetus for‘‘global’’ investigation. The paradigm for thesestudies has been set by yeast researchers, who havehad access to the full genome sequence of S. cerevisiaesince 1996 (4). Although implicit in genomicsequencing, the development of techniques to studyfunctional parameters across whole genomes hasevolved into a new field of research termed‘‘functional genomics’’. For P. falciparum, a numberof technical difficulties remain, but researchers are

already applying functional genomics to the biologyof this parasite.

Bioinformatics

Bioinformatics covers both sequencing and func-tional genomics in converting raw genome sequencedata into useful information for the biologist.Probably the most important aspects are the searchengines that can screen vast amounts of informationfor significant similarities between sequences, and thealgorithms used to predict protein-coding regions(see above). As far as the search engines areconcerned, several sites exist on the web that willallow the user to search for homology to a testsequence (see Table 2), returning the results asmultiple sets of alignments. Although there are someerrors in defining genes ‘‘in silico’’ (i.e, a gene modelpredicted by computer), it is clear that thesetechniques have generated an enormous number ofpotential gene functions that can be tested in thelaboratory. Perhaps the easiest forms of this are theannotations of complete chromosome sequencesthat are based on high-quality sequence information.The resulting tables of genes can be divided intothose that have confirmed function in P. falciparum;sequences with homology to known genes in otherorganisms; sequences with homology to genes ofunknown function in other organisms; and se-quences specific to P. falciparum of unknownfunction. One of the underlying assumptions is thatthe annotated sequence contains all the genes andthat the predictions for the coding sequence arecorrect. While this is largely true, there has been somediscussion about ‘‘missing’’ genes and the incorrectidentification of splice sites. Therefore, an essentialpart of any bioinformatic system is the provision ofsoftware for browsing genomic sequence data so that



independent laboratories can make their own judge-ments on coding sequences.

Access to information is possible through therelease of sequence data prior to publication, whichhas already made a significant contribution to malariaresearch. However, different web sites containdifferent parts of the sequence information fromdifferent chromosomes. The site at the NationalCenter for Biotechnology Information (NCBI, seeTable 2) has attempted to collate all the availableinformation for P. falciparum (as well as that for otherPlasmodium species and the related apicomplexanparasite Toxoplasma gondii). A recent development,funded by the Burroughs Wellcome Fund, has esta-blished a full database (http://www.PlasmoDB.org).In its current form, PlasmoDB provides: views offinished and annotated sequence in both web-basedand CD format; a relational database that shouldfacilitate entry and analysis of expression data; a‘‘BLASTable’’ database containing the most recentgenomic sequence information (finished and un-finished); text-queriable results from a completeBLAST search of all P. falciparum data against theGenBank and EMBL databases; and various otherdata-mining tools. In addition to the obvious interestof PlasmoDB to the malaria research community, theUNDP/World Bank/WHO Special Programme forResearch and Training in Tropical Diseases has madea commitment to provide on-line ‘‘help-desk’’support for PlasmoDB (and other parasite data-

bases), through the training of scientists/bioinfor-maticians from developing countries.

Microarrays

Microarrays are high-density arrays of DNA targets onglass or filter supports. These have been used inseveral studies, most notably for S. cerevisiae. There areessentially two formats for the DNA targets: DNAfragments (usually generated by PCR) or oligonucleo-tides. PCR-derived targets for P. falciparum have beenproduced from genome sequence data (D. Carrucci,personal communication, 1998), or by the amplifica-tion of sequences from a mung bean nuclease library.In the mung bean nuclease library study (34), arrayswere made from a random library and screened with

Table 2. Plasmodium falciparum bioinformatic web-site URLs

http://www.sanger.ac.uk/Projects/P_falciparum (Sequence data)http://www.tigr.org/tdb/edb/pfdb/pfdb.html (Sequence data)http://sequence-www.stanford.edu/group/malaria/index.html (Sequence data)

http://www.PlasmoDB.org (‘Official’ database of the Plasmodium GenomeConsortium)http://www.ncbi.nlm.nih.gov/Malaria (Genetics/bibliography/sequence data)http://www.ebi.ac.uk/parasites/parasite-genome.html (General site/proteomics)

http://sites.huji.ac.il/malaria/(Metabolic pathways)



mRNA from asexual (trophozoite) and sexual (game-tocyte) stages. The RNAs were labelled with differentfluorochromes and allowed to hybridize to the arrays.Genes transcribed during asexual stages were labelledgreen, those transcribed during sexual stages red, andthose transcribed during both stages yellow (green +red). From this single experiment the authorsidentified several developmentally regulated genes,including some which had been previously identified,confirming the efficacy of this approach.

The potential for these types of experiments isconsiderable, but will require careful data handlingand analysis. Hayward et al. (34) identified theircandidate genes by sequencing the inserts afterhybridization, but once the genome has beencompletely sequenced the address of each ORF willbe stored as part of the array information. An arraycovering all of the genes in the P. falciparum genomein duplicate would have approximately 12 000–14 000 spots; therefore every experiment wouldgenerate at least 14 000 quantitative data points perprobe, in addition to the array baseline information. Itis not hard to see that without a highly efficientdatabase, the efficiency and accuracy of any analysiswould be greatly reduced, particularly since transcrip-tional changes are likely to be defined by ‘‘clusters’’ ofgenes (35), which makes the contribution ofindividual genes difficult to interpret. The databasemust be accessible by the research community to takefull advantage of the results and, as seen for C. elegans(http://www.wormbase.org), a centralized databaseis essential.

Transcriptional analysis for P. falciparum is alsobeing attempted by Serial Analysis of Gene Expres-sion (SAGE, 36; D. Wirth, personal communication,1998)). This technique uses PCR amplification ofmRNA to derive short sequence tags that are uniqueto each gene. The tags are concatenated into longstrings and cloned into suitable vectors. By sequen-cing many tags, it is possible not only to identify whatgene is being expressed, but also to quantify the levelof expression by recording the number of timesspecific sequence tags are present. The handling ismore complicated than hybridization to arrays, butthe major advantage of this technique is that relativelysmall quantities of material can be used, which isparticularly important where samples may be limited(e.g. field samples). For both techniques it isimportant that technology transfer is carried out,including not only the mechanics such as roboticsand image analysis, but also an appreciation of theassumptions and limitations. For example, neithertechnique can detect the products of alternativemRNA splicing events.

High-density arrays are not exclusively for usein transcriptional analysis, but also have applicationsin genome analysis, particularly as oligonucleotidearrays. The high degree of specificity of oligonucleo-tide hybridization allows a high level of discrimina-tion, including single nucleotide substitutions.However, their use is precluded prior to thecompletion of the P. falciparum sequencing project

due to the cost of the chemical synthesis. Oneexample of this type of approach comes again fromS. cerevisiae (37), in which allelic variation throughoutthe genomes of two strains of yeast was identifiedsolely by hybridization of their genomic DNA tooligonucleotide arrays. The application of thistechnology to natural populations of parasites willbe a powerful tool for molecular epidemiology.However, information from many P. falciparum

genomes will need to be collated to fully cover thetrue extent of highly variant and recombinogenicgenes such as var.

Proteomics and structural genomics

One of the greatest technical challenges in functionalgenomics concerns the analysis of protein expression(proteomics). Although genome-wide transcriptionalanalysis has produced very useful information, it isclear that the correlation between mRNA and proteinlevels is not perfect (38). Techniques for identifyingproteins in general have developed rapidly over thelast few years. However, the major advance withregard to genome sequencing has come through theuse of mass spectrometry for protein analysis andcharacterization, by combining the mass (and there-fore amino acid composition) of peptide fragmentswith a database of all the available peptide combina-tions derived from the genome sequence data (see 39for review). Using these techniques it is possible toidentify individual proteins from complex mixturesresolved by two-dimensional (2-D) electrophoresisor chromatography. This, allied with improvementsin the reproducibility of 2-D electrophoresis, hasfacilitated the differential analysis of protein compo-sition from two populations of cells.

Applications of this technology, for example inthe analysis of the effect of drug treatments, have animportant place in malaria research. Some practicaldifficulties also remain, particularly for membraneproteins, which are poorly represented using classicalproteomic techniques (40). However, technologycontinues to improve (41) and the reward will be aclear definition of the spectrum of proteins involvedin critical biological processes in the parasite.

Protein–protein interactions play an importantrole in cell biology. The tools to investigate thisphenomenon, again, have been developed with yeastthrough the use of the ‘‘two-hybrid’’ system. In this,ORFs are fused to the Gal4 transcription-activationdomain in one yeast strain and screened by matingwith a second strain in which specific codingsequences have been fused with the Gal4 DNA-binding domain. Positive protein interactions arescored by the ability of the yeast progeny to grow onselective media. This procedure has recently beenexpanded to enable researchers to carry out acomprehensive analysis of the protein–protein inter-actions of approximately 6000 yeast ORFs (42),generating novel information as well as confirmingprevious specific screens. The extension of this



technique to pathogen genome research will producea further layer of functional information.

One of the goals of computational biology is topredict the biological function of a protein fromprimary sequence information. Currently, the clearestmanifestation of this is in structural genomics inwhich the three-dimensional structure of proteins isstudied in the context of the amino acid sequence.While much still needs to be done, the increasingnumber of resolved crystal structures for biologicalmolecules has already resulted in the generation ofrules/motifs that can be applied when searching forstructural information (43). As more informationbecomes available, including the integration offunctional data in terms of active residues, thesecomputer algorithms will improve.

Metabolomics and vaccinomics

One of the many outcomes from functionalgenomics has been the production of a newvocabulary to cover the many applications ofmetabolomics and vaccinomics. Thus, the entiremRNA and protein complement of an organism havebeen termed the transcriptome and the proteome,respectively. In a similar vein, the use of genomicinformation to facilitate studies of metabolic pro-cesses has been termed metabolomics (44, 45).Clearly, the use of sequence similarities to identifycomponents of known pathways will have a hugeimpact on research in this area, but functional screenshave also been proposed. For example, by expressingall the predicted ORFs from a genome as hetero-logous fusion proteins it might be possible to screenfor specific enzyme functions. This resource couldalso be used to determine host protective immuneresponses through immunization with protein pools.Related studies have been called vaccinomics, inwhich DNA vaccine plasmids containing ORFs(derived from the sequencing project) are used toimmunize experimental animals and, in the case ofpathogens, to screen for protection (46). Not onlycould this lead to the identification of vaccinecandidates, but the resulting sera could also be usedto determine the cellular location of each protein.

As the number of sequenced genomes in-creases and researchers become more accustomed tothinking in ‘‘global’’ terms, the amount of informa-tion generated by functional genomics will expandrapidly. The challenges will then be to developsystems to analyse this information and to maintain afocus on the biological issues. Clearly, for malaria thiswould be well served through studies on the parasitesthemselves, facilitated by genetic crosses (47) and thetechnology of genetic manipulation (see below).

Transfection of Plasmodium falciparumTransfection, or the introduction of exogenous DNAinto the organism in vivo, potentially provides one of

the most powerful tools for the analysis of theparasite’s genome, allowing us to specifically addressquestions relating to gene function. However, prior tothe initial demonstration of luciferase expression in thechicken malaria model, P. gallinaceum, the ability tomodify or disrupt components of the P. falciparum

genome had eluded researchers (48). Following thisreport, transfection of P. falciparum was successfullycarried out when a plasmid bearing the reporter geneencoding chloramphenicol acetyltransferase (CAT)was introduced into the readily cultured, intra-erythrocytic stages (49). Stable transfection of aplasmid bearing a pyrimethamine selectable marker(mdhfr-ts, a mutant dihydrofolate reductase–thymidinesynthetase gene), and its subsequent integration viahomologous recombination into the genome, soonfollowed (50). The plasmids described in these initialreports, as well as a transgene expression system(expression of a reporter gene from a plasmid stablymaintained by virtue ofmdhfr-ts (51)) have provided thebasis for all the subsequent studies in P. falciparum.

Using both CAT and luciferase reporter genes,a series of studies have addressed the structure andfunction of P. falciparum transcriptional units. P. falci-parum promoters were shown to conform to aclassical bipartite structure: a basal promoter regu-lated by upstream regulatory factors, with transcriptstability directed by 3’ regulatory sequences (52–57).Also, the nuclear context in which promoters areplaced play a key role in their function, suggestingthat phenomena such as stage-specific gene expres-sion, and switching of expression between membersof the var multigene family, may rely on epigeneticfactors, such as chromatin assembly (58, 59).

More advanced work, utilizing gene disruptionand allelic replacement, has formed the basis of aseries of recent reports that examined aspects ofP. falciparum biology, such as cytoadhesion, gameto-genesis and drug resistance (Table 3). Disruption ofthe gene encoding the knob-associated histidine-richprotein (KAHRP), located within electron-denseknob structures on the surface of infected erythro-cytes, showed that this protein is important in theirformation (51). Follow-up studies demonstrated thatthe knob structure is critical in supporting PfEMP-1protein during its interaction with host receptorsunder fluid flow conditions. Pfg27, a proteinexpressed early following a parasite’s commitmentto sexual differentiation, is also essential sincedisruption of this gene resulted in the abortion oftransfectants early in gametogenesis, and resulted inhighly vacuolated and morphologically disruptedparasites (60).

The roles of mutations within genes conferringresistance to a wide range of antimalarial drugs havealso been investigated by altering ORFs rather thandisrupting them (61). And the roles of dihydropteroatesynthase in sulfadoxine resistance, and P-glycoproteinhomologue 1 (Pgh-1) in multidrug resistance, wereelegantly demonstrated by introducing mutationsassociated with drug resistance in epidemiologicalstudies into parasites bearing a drug-sensitive back-



ground (62). Moreover, these studies have also beenused to demonstrate the reverse situation, wheremutations of cg2, a candidate chloroquine resistancegene, did not confer resistance in a sensitive parasitebackground, prompting investigations that identified amore promising candidate (D. Fiddock, T. Wellems,personal communication, 1999).

The primary limitation with this type ofexperimental strategy is that the intraerythrocyticstage parasites investigated are haploid. Targeteddisruption of essential genes inevitably result in thedeath of the parasite; moreover, any effect on parasiteviability will place that proportion of the population ata growth disadvantage. This may be partially overcomeby selection, or by the presence of alternative pathwaysthat can overcome the growth disadvantage.

Proposals for a consortium of laboratories,similar to the EUROFAN network for S. cerevisiae, tosystematically ‘‘knock-out’’ every gene identified bythe genome project have considered a wide variety ofissues, such as poor parasite viability, low transfectionefficiency, the numbers of laboratories able to createmutants, and the unit cost per gene knock-out (63).Although there are still many issues to be resolved, ithas been agreed that some form of systematicanalysis of the parasite’s gene complement shouldtake place. However, whether each laboratory wouldbe allocated a share of the genome, or whether athematic approach is used (based on a laboratory’sinterest in a particular subject, such as erythrocyte

invasion, cytoadhesion or gametocytogenesis) re-mains to be determined.

Malaria model systems

The lack of in vitro culture systems for mostP. falciparum developmental stages and the ethicalconsiderations necessary for the use of New Worldmonkeys and chimpanzees highlight the advantagesoffered by model malaria parasite systems. Transfec-tion of animal malaria models has opened up theentire parasite’s life cycle, including those in theinvertebrate mosquito host, for critical analysis in amanner not possible with human parasites (64).Moreover, the efficiency of the transfection process,facilitating double-cross over gene replacements, andthe rapid selection of transfectants in vivo, haveresulted in the rapid accumulation of new insightsinto Plasmodium spp. biology.

Following the first reports of transfection inthe rodent malarial model P. berghei in 1995 (65),disruptions of the genes encoding circumsporozoiteprotein (CS) (66) and thrombospondin-relatedanonymous protein (TRAP) (67) demonstrated thevalue of this approach in the investigation offunction: apart from their roles in host cell recogni-tion and invasion, new roles in sporozoite formationand gliding motility were attributed to CS and TRAP,respectively. Recurrent biological themes have

Table 3. Genes modified or ‘‘knocked out’’ in Plasmodium

Plasmodium gene targeteda Phenotype Reference

Pb CS Inhibition of sporozoite formation, loss of infectivity 66Pb TRAP Sporozoites fail to glide and show reduced infectivity 67Pb, Pf CTRP Ookinetes non-motile and fail to invade midgut epithelia and develop into oocysts 83–85Pf KAHRP Infected-red blood cells ‘‘knobless’’ and have reduced binding to CD36 under 51

flow conditionsPF G27 Total (3’ disruption) or significant (5’ disruption) inhibition of gametocytogenesis 60

Plasmodium gene modified Manipulation and phenotype Reference

Pf DHPS mutation Introduction of field-observed mutations give rise to sulfadoxine 61resistance when introduced

Pf pgh1 replacement Introduction of field observed mutations give rise to mefloquine, halofantrine 62and quinine resistance. Also resistance to chloroquine in a strain-specific manner.

Pb TRAP Replacement with Pf TRAP. Sporozoites can glide, invade salivary glands 86and are infectious

Pb TRAP Replacement with Pf TRAP mutants. TRM- sporozoites do not glide or invade 86salivary glands, but remain infectious. Amut sporozoites do not invade salivaryglands, but remain infectious

Pb TRAP Replacement with Pb TRAP mutant. Cyt ~S and ~L sporozoites do not glide 87normally, cannot invade salivary glands and are not infectious. Cyt/MIC-2sporozoites can glide, invade salivary glands and are infectious

Pf Acyl Carrier Protein Green fluorescent protein tag demonstrates need for bipartite leader 80sequence to correctly target protein to apicoplast and conservation of leaderwithin apicoplasts.

Pf MSP1 Replaced C terminal with that of P. chabaudi MSP-1 demonstrates conservation 88of function and potential for immune variation.

a Except where indicated, the targeted loci have been disrupted after integration. Genes targeted in P. berghei are indicated by Pb or PBand genes targeted in P. falciparum are indicated by Pf or PF.



already been demonstrated. For example, CTRP, aTRAP homologue, has been shown to have a role inookinete motility and thus in the ability to infectmosquitoes.

The readily available and immunologically well-characterized animal hosts allow a full range of hostparasite interactions to be investigated. Following thesuccess of this work, transfection of two primatemalaria models has been established at the Biochem-ical Primate Research Centre in the Netherlands.Both P. knowlesi (68) and P. cynomolgi (69) have beentransfected with entirely heterologous plasmid con-structs bearing a Toxoplasma gondii DHFR-TS drugselectable marker controlled by P. berghei regulatorysequences. One particular advantage of primatemalaria models is that they allow investigation oftransfected parasites within both their natural andartificial hosts. Within strict ethical limitations, theuse of these systems facilitates meaningful analyses ofhost–parasite interactions and will allow the mecha-nisms governing the host immune response to beexamined, as well as providing an experimental modelfor evaluating vaccines.

Animal models share many features with theirhuman malarial counterparts, such as life cycles, host-cell restrictions and immune responses. For example,P. cynomolgi infection of macaques is a particularlygood model for P. vivax infection, sharing preferencefor reticulocytes and hypnozoite formation (69).Although we have a substantial pool of knowledgefor animal malaria models, particularly with respect tohomologous vaccine antigens, we understand com-paratively little about these systems at the molecularlevel. Software for sequencing EST libraries, as wellas low-coverage genomic shotgun sequencing, havealready provided a fundamental boost to theapplication of animal models to human malariabiology and should be supported with larger-scalesequencing activities. Use of animal models wouldallow intensive programmatic approaches to genefunction to be considered through the use of knock-out/tagging methods.

Where species ofPlasmodium, such as P. knowlesiand P. cynomolgi, provide models for malaria infectionand immunity, the closely related apicomplexanT. gondii provides a model for many aspects ofPlasmodium biology (70). Transfection systems arewell developed in this parasite, with high-efficiencytransformation and a number of markers for bothpositive and negative selection (71). This experimen-tally tractable system has been instrumental intackling issues such as plastid structure and function(72), host cell invasion, drug resistance, virulence andgene expression. Unlike Plasmodium spp., whichexclusively integrate DNA molecules through homo-logous recombination, T. gondii also permits non-homologous integration, allowing the entire genometo be tagged for a phenotype-based approach to genefunction. For this,Plasmodiumwill require a transposon-based system of simple recognition site specificity,such as the Drosophila mariner element, which

efficiently transfers into a number of eukaryoticgenomes including Leishmania major (73).

Both low- and high-technology developmentsare needed to continue a systematic assault on thePlasmodium genome, such as improved culture meth-ods for certain human and model bloodstage parasites,gamete development and efficient sporozoite culture.These, together with advances in transfection tech-nology, such as new markers, methods to increasetransfection efficiencies and adoption of the Tet-repressor system, will combine to make transfection amore reliable and sensitive tool. However, only thecoupling of these advances with the knowledge of theparasite‘s entire gene complement will allow theadvances to be fully exploited.

Comparative genomics

Mention has already been made of the difficulties thatcan be encountered when annotating a completegenome, particularly assigning function to unknowngenes and correctly identifying genes that containnumerous exons. Ultimately, the availability of a fullyor extensively sequenced genome from a relatedspecies will be an invaluable tool that will facilitate anaccurate assessment of the coding potential of agenome (74). Studies on the chromosomes of rodentmalaria species demonstrated that there was little, ifany, difference in gene linkage (75), and that largeconserved linkage groups could be detected betweenthese rodent malarias and P. falciparum (76). Limited,but more detailed, analysis revealed that genomeorganization is highly conserved in the internal non-subtelomeric regions of the chromosome (77, 78).

Thus, the expectation is that alignment oforthologous chromosome regions will reveal detailedintron–exon boundaries, and help identify ortholo-gous, but polymorphic, antigen-coding genes, cen-tromeres and species-specific genes. In this lastregard, comparison with a more distant apicomplexangenome may also prove valuable. Although geneorder may not be well conserved, many salientfeatures of apicomplexans will be revealed throughgenome comparisons, for example the apicoplast(79–81). The ease of transformation of Toxoplasma

gondii and its suitability for cell biological studies,combined with comparative genomics, provides anexcellent opportunity to illuminate malaria biology,develop drug targets (19, 82) and possibly vaccines.

Conclusion

The ability to completely sequence genomes has had ahuge impact on the way in which scientific research isconducted. As the number of pathogen genomes thathave been completed increases, the impetus todevelop techniques that utilize this information hasincreased. In parallel with the P. falciparum genomeproject, techniques such as transfection, microarraysand proteomics, as well as bioinformatic analysis, are



already being developed and applied to fundamentalbiological questions. The combination of these tools isexpected to provide a predictive platform from whichto launch hypothesis-driven biological research.

Central to this expectation is continuingfinancial support for the further development oftechniques and resources to exploit the genomesequence. Developments that will provide insightinto parasite biology are publicly accessible relational

databases, and detailed comparative and surveyanalyses. It must be emphasized that the overall goalof such costly enterprises is to work towards a curefor malaria, and the contribution of this technologicalapproach could be immense. The current challengeto the research community is to continue thedissemination of this technology and to focus effortson the development of new drugs and vaccines. n

Resume

La recherche sur le paludisme s’engage dans l’ere postgenomiqueLe developpement des techniques de sequencage dugenome a permis de recueillir au cours de ces cinqdernieres annees une masse d’informations sur cessequences, qui ont abouti a l’annonce faite recemmentqu’on etait parvenu a obtenir une premiere version detravail du genome humain. Les genomes des germespathogenes, du fait de leur taille reduite, ont ete encoreplus faciles a analyser avec ces techniques et sontdesormais bien representes dans la genomique. Bienqu’ils ne puissent pas toujours servir de micro-organismes « modeles », l’importance des germespathogenes en medecine et en agriculture ont fait del’exploitation des bases de donnees relatives auxsequences dont ils sont constitues une priorite de toutpremier ordre. Mais que signifie reellement la productionde ces longues series d’A-C-G-T sur le plan de lareduction de la charge de morbidite, en particulier dansles pays en developpement ? Jusqu’ici, le sequencage dugenome a surtout ete la specialite du monde developpe,ou les ressources et les infrastructures scientifiques enplace ont permis la creation de centres de sequencage ahaut rendement. Toutefois, l’utilisation des resultats dece sequencage n’a aucune raison d’etre ainsi limitee,pour autant qu’on puisse disposer de ressources pour laformation.

L’analyse des donnees des sequences primairesest probablement l’aspect le plus important de l’erepostgenomique (c’est-a-dire de celle qui va succeder ausequencage proprement dit). C’est ce qu’on appelle labio-informatique, qui englobe toute une serie d’analysestheoriques visant a convertir les sequences d’ADN eninformations biologiques. Une part importante de cettediscipline a trait a l’identification des genes par le biaisd’un certain nombre de processus, depuis l’analyse dessimilitudes qu’ils presentent avec des genes deja connusd’autres organismes, jusqu’a l’emploi de modelesinformatises bases sur les donnees existantes. Viennentensuite les previsions que l’on peut faire en matiere defonction biologique et de forme moleculaire (genomique

structurelle), qui toutes deux ont un potentiel dedeveloppement important.

Le fait de connaıtre l’ensemble des sequences dugenome d’un germe pathogene permet egalement auxchercheurs d’etendre beaucoup plus qu’auparavant leurchamp d’investigation du comportement de ces germes.Desormais, au lieu d’etudier l’effet d’un traitementmedicamenteux ou la differenciation sur un ou deuxgenes, il est possible d’etudier simultanement lesvariations presentees par l’ensemble des genes aumoyen d’une analyse transcriptionnelle globale. On peutegalement examiner les protidogrammes, ou modifiergenetiquement le micro-organisme (transfection), of-frant ainsi un lien direct entre ces effecteurs biologiqueset le phenotype macroscopique. La mise au point destechniques necessaires a ces experiences est uneconsequence directe du desir d’exploiter les donneesdu sequencage genomique.

Il n’est pas difficile de prevoir que la possibilited’identifier et de caracteriser le schema d’organisationgenetique des germes pathogenes nous permettrad’identifier les elements essentiels a leur developpementet a leur pathogenese et de cibler nos efforts de recherchesur la production de nouveaux traitements. En ce quiconcerne Plasmodium falciparum, on peut identifier lesgenes et les proteines qui agissent lors de stadesparticuliers du cycle evolutif de l’hematozoaire, determinerleur role au moyen de modifications genetiques et utiliserceux qui sont prometteurs pour la production de vaccins.Les voies metaboliques parasitaires, absentes chez l’hote,pourraient egalement servir de cible a des inhibiteurspuissants qui ne soient pas toxiques pour l’homme ; enfin,on pourrait cibler les mecanismes de la pharmacoresis-tance des plasmodies et allonger ainsi la duree d’efficacitedes medicaments existants. Le sequencage du genome deP. falciparum offrira de nombreuses possibilites derecherche sur le paludisme, mais il ne s’agit la que d’undebut, l’objectif etant de transformer ces possibilites entraitements efficaces sur le terrain.

Resumen

La investigacion del paludismo en la era posgenomicaEl desarrollo de las tecnologıas de secuenciacion delgenoma a lo largo de los ultimos cinco anos ha generadouna gran cantidad de informacion sobre secuencias, queha culminado en el reciente anuncio de un borrador del

genoma humano. Debido a su menor tamano, losgenomas de microorganismos patogenos se hanprestado aun mas a esas metodologıas, y actualmenteestan bien representados en el mundo de la genomica.



Aunque no siempre sirven como microorganismos«modelo», la trascendencia de esos patogenos para lamedicina y la agricultura ha hecho que la explotacion delas bases de datos de secuencias se convierta en una granprioridad. ¿Pero como puede repercutir realmente laidentificacion de largas cadenas de A, C, G y T en elobjetivo de reducir la carga de morbilidad, en particularen los paıses en desarrollo? Hasta el momento, lasecuenciacion de genomas se ha circunscrito en su granmayorıa al mundo desarrollado, donde los recursos y lainfraestructura cientıfica han permitido crear centros desecuenciacion muy productivos. No obstante, el uso de lainformacion sobre las secuencias no tiene por que quedarlimitado de ese modo, siempre y cuando haya recursospara formacion.

El aspecto mas importante de la era posgenomica,es decir, la que comienza al completarse la secuenciacion,es probablemente el analisis de las secuencias. Estecampo, lo que se conoce como bioinformatica, comprendeuna serie de analisis teoricos cuyo fin es convertir lasecuencia de ADN en informacion biologica. Una parteimportante de esta disciplina implica la identificacion degenes mediante una serie de procedimientos, que vandesde la determinacion de las similitudes con otros genesdescritos anteriormente hasta el uso de modelosinformaticos basados en los datos existentes. Posterior-mente se hacen predicciones sobre la funcion biologica yla forma molecular (genomica estructural), cosas ambasque abren amplias perspectivas de desarrollo.

El conocimiento de la secuencia completa delgenoma de un patogeno tambien permite a loscientıficos investigar el comportamiento de los micro-

organismos con una base mas amplia. Ahora, en lugar deestudiar el efecto de un tratamiento farmacologico o dela diferenciacion en uno o dos genes, es posible estudiarla variacion simultanea de todos los genes utilizando elanalisis transcripcional global. Se puede asimismoexaminar la distribucion de las proteınas o modificargeneticamente el microorganismo (transfeccion), yestablecer ası una relacion directa entre esos efectoresbiologicos y el fenotipo general. La tecnologıa para llevara cabo esos experimentos es consecuencia directa delinteres por explotar las secuencias genomicas.

Cabe prever que la capacidad para identificar ycaracterizar la huella genetica de los agentes patogenosayudara a reconocer elementos clave del desarrollo y losmecanismos patogenicos de los microorganismoscausantes de enfermedades, y a centrar nuestrasinvestigaciones en el desarrollo de nuevos tratamientos.En el caso de Plasmodium falciparum, es posibleidentificar los genes y las proteınas que actuan endeterminadas etapas del ciclo de vida, verificar sufuncion mediante tecnicas de modificacion genetica, yutilizar candidatos prometedores en la produccion devacunas. Las vıas metabolicas del parasito que no estanpresentes en el huesped son otra posible diana deinhibidores potentes no toxicos para el ser humano;ademas, se podrıa interferir en los mecanismos defarmacorresistencia del parasito, y alargar ası la vidaeficaz de los medicamentos actuales. La secuencia delgenoma de P. falciparum brindara muchas oportunida-des para investigar el paludismo, pero esto es solo elprincipio: el reto es convertir esas oportunidades entratamientos eficaces sobre el terreno.

References

1. Triglia T, Kemp DJ. Large fragments of Plasmodium falciparumDNA can be stable when cloned in yeast artificial chromosomes.Molecular and Biochemical Parasitology, 1991, 44: 207–211.

2. The Wellcome Trust Malaria Genome MappingConsortium. The Plasmodium falciparum Genome Project: aresource for researchers. Parasitology Today, 1995, 11: 1–4.

3. Cole ST et al. Deciphering the biology of Mycobacteriumtuberculosis from the complete genome sequence. Nature, 1998,393: 537–544.

4. Goffeau A et al. Life with 6000 genes. Science, 1996,274: 563–567.

5. The C. elegans Sequencing Consortium. Genome sequenceof the nematode C. elegans: a platform for investigating biology.Science, 1998, 282: 2012–2018.

6. Hoffman SL et al. Funding for malaria genome sequencing.Nature, 1997, 387: 647.

7. Su XZ, Wellems TE. Plasmodium falciparum: assignment ofmicrosatellite markers to chromosomes by PFG-PCR. ExperimentalParasitology, 1999, 91: 367–369.

8. Lai Z et al. A shotgun optical map of the entire Plasmodiumfalciparum genome. Nature Genetics, 1999, 23: 309–313.

9. Salzberg SL et al. Interpolated Markov models for eukaryoticgene finding. Genomics, 1999, 59: 24–31.

10. Pertea M, Salzberg SL, Gardner MJ. Finding genes inPlasmodium falciparum. Nature, 2000, 404: 34.

11. Lawson D, Bowman S, Barrell B. Finding genes in Plasmodiumfalciparum. Nature, 2000, 404: 34–35.

12. Gardner MJ et al. Chromosome 2 sequence of the humanmalaria parasite Plasmodium falciparum. Science, 1998,282: 1126–1132.

13. Bowman S et al. The complete nucleotide sequence ofchromosome 3 of Plasmodium falciparum. Nature, 1999,400: 532–538.

14. Feagin JE. The extrachromosomal DNAs of apicomplexanparasites. Annual Review of Microbiology, 1994, 48: 81–104.

15. Wilson RJ, Williamson DH. Extrachromosomal DNA in theApicomplexa. Microbiology and Molecular Biology Reviews,1997, 61: 1–16.

16. Kohler S et al. A plastid of probable green algal origin inApicomplexan parasites. Science, 1997, 275: 1485–1489.

17. McFadden GI, Roos DS. Apicomplexan plastids as drug targets.Trends in Microbiology, 1999, 7: 328–333.

18. Calas M et al. Antimalarial activity of compounds interfering withPlasmodium falciparum phospholipid metabolism: comparisonbetween mono- and bisquaternary ammonium salts. Journalof Medicinal Chemistry, 2000, 43: 505–516.

19. Jomaa H et al. Inhibitors of the nonmevalonate pathway ofisoprenoid biosynthesis as antimalarial drugs. Science, 1999,285: 1573–1576.

20. Baruch DI et al. Cloning the P. falciparum gene encodingPfEMP1, a malarial variant antigen and adherence receptor on thesurface of parasitized human erythrocytes. Cell, 1995, 82: 77–87.

21. Smith JD et al. Switches in expression of Plasmodium falciparumvar genes correlate with changes in antigenic and cytoadherencephenotypes of infected erythrocytes. Cell, 1995, 82: 101–110.

22. Su X-Z et al. The large diverse gene family var encodes proteinsinvolved in cytoadherence and antigenic variation of Plasmodiumfalciparum infected erythrocytes. Cell, 1995, 82: 89–100.



23. Buffet PA et al. Plasmodium falciparum domain mediatingadhesion to chondroitin sulfate A: a receptor for human placentalinfection. Proceedings of the National Academy of Sciences ofthe United States of America, 1999, 96: 12743–12748.

24. Newbold C et al. Cytoadherence, pathogenesis and the infectedred cell surface in Plasmodium falciparum. International Journalof Parasitology, 1999, 29: 927–937.

25. Carcy B et al. A large multigene family expressed during theerythrocytic schizogony of Plasmodium falciparum. Molecularand Biochemical Parasitology, 1994, 68: 221–233.

26. Bonnefoy S et al. Evidence for distinct prototype sequenceswithin the Plasmodium falciparum Pf60 multigene family.Molecular and Biochemical Parasitology, 1997, 87: 1–11.

27. Bischoff E et al. A member of the Plasmodium falciparumPf60 multigene family codes for a nuclear protein expressed byreadthrough of an internal stop codon. Molecular Microbiology,2000, 35: 1005–1016.

28. Weber JL. Interspersed repetitive DNA from Plasmodiumfalciparum. Molecular and Biochemical Parasitology, 1988,29: 117–124.

29. Cheng Q et al. stevor and rif are Plasmodium falciparummulticopy gene families which potentially encode variant antigens.Molecular and Biochemical Parasitology, 1998, 97: 161–176.

30. Kyes SA et al. Rifins: a second family of clonally variant proteinsexpressed on the surface of red cells infected with Plasmodiumfalciparum. Proceedings of the National Academy of Sciencesof the United States of America, 1999, 96: 9333–9338.

31. Limpaiboon T et al. Characterization of a Plasmodiumfalciparum epitope recognized by a monoclonal antibody withbroad isolate and species specificity. Southeast Asian Journalof Tropical Medicine and Public Health, 1990, 21: 388–396.

32. Fernandez V et al. Small, clonally variant antigens expressed onthe surface of the Plasmodium falciparum-infected erythrocyte areencoded by the rif gene family and are the target of humanimmune responses. Journal of Experimental Medicine, 1999,190: 1393–1404.

33. Kyes S, Pinches R, Newbold C. A simple RNA analysis methodshows var and rif multigene family expression patterns inPlasmodium falciparum. Molecular and Biochemical Parasitology,2000, 105: 311–315.

34. Hayward RE et al. Shotgun DNA microarrays and stage-specificgene expression in Plasmodium falciparum malaria. MolecularMicrobiology, 2000, 35: 6–14.

35. Eisen MB et al. Cluster analysis and display of genome-wideexpression patterns. Proceedings of the National Academyof Sciences of the United States of America, 1998, 95:14863–14868.

36. Velculescu VE et al. Serial analysis of gene expression. Science,1995, 270: 484–487.

37. Winzeler EA et al. Direct allelic variation scanning of theyeast genome. Science, 1998, 281: 1194–1197.

38. Gygi SP et al. Correlation between protein and mRNAabundance in yeast. Molecular and Cellular Biology, 1999,19: 1720–1730.

39. Yates JR, III. Mass spectrometry. From genomics to proteomics.Trends in Genetics, 2000, 16: 5–8.

40. Rabilloud T et al. Analysis of membrane proteins by two-dimensional electrophoresis: comparison of the proteins extractedfrom normal or Plasmodium falciparum-infected erythrocyteghosts. Electrophoresis, 1999, 20: 3603–3610.

41. Link AJ et al. Direct analysis of protein complexes using massspectrometry. Nature Biotechnology, 1999, 17: 676–682.

42. Uetz P et al. A comprehensive analysis of protein-proteininteractions in Saccharomyces cerevisiae. Nature, 2000,403: 623–627.

43. Teichmann SA, Chothia C, Gerstein M. Advances in structuralgenomics. Current Opinions in Structural Biology, 1999, 9:390–399.

44. Tweeddale H, Notley-McRobb L, Ferenci T. Effect of slowgrowth on metabolism of Escherichia coli, as revealed by globalmetabolite pool (‘‘pmetabolome’’) analysis. Journal ofBacteriology, 1998, 180: 5109–5116.

45. Kell DB, King RD. On the optimization of classes for theassignment of unidentified reading frames in functional genomicsprogrammes: the need for machine learning. Trends inBiotechnology, 2000, 18: 93–98.

46. Hoffman SL et al. From genomics to vaccines: malaria asa model system. Nature Medicine, 1998, 4: 1351–1353.

47. Su X et al. A genetic map and recombination parameters ofthe human malaria parasite Plasmodium falciparum. Science,1999, 286: 1351–1353.

48. Goonewardene R et al. Transfection of the malaria parasiteand expression of firefly luciferase. Proceedings of the NationalAcademy of Sciences of the United States of America, 1993,90: 5234–5236.

49. Wu Y et al. Transfection of Plasmodium falciparum within humanred blood cells. Proceedings of the National Academy of Sciencesof the United States of America, 1995, 92: 973–977.

50. Wu Y, Kirkman LA, Wellems TE. Transformation ofPlasmodium falciparum malaria parasites by homologousintegration of plasmids that confer resistance to pyrimethamine.Proceedings of the National Academy of Sciences of the UnitedStates of America, 1996, 93: 1130–1134.

51. Crabb BS et al. Targeted gene disruption shows that knobsenable malaria-infected red cells to cytoadhere under physio-logical shear stress. Cell, 1997, 89: 287–296.

52. Crabb BS, Cowman AF. Characterization of promoters andstable transfection by homologous and nonhomologous recom-bination in Plasmodium falciparum. Proceedings of the NationalAcademy of Sciences of the United States of America, 1996,93: 7289–7294.

53. Horrocks P, Kilbey BJ. Physical and functional mapping of thetranscriptional start sites of Plasmodium falciparum proliferatingcell nuclear antigen. Molecular and Biochemical Parasitology,1996, 82: 207–215.

54. Horrocks P, Dechering K, Lanzer M. Control of geneexpression in Plasmodium falciparum. Molecular & BiochemicalParasitology, 1998, 95: 171–181.

55. Horrocks P, Lanzer M. Mutational analysis identifies a fivebase pair cis-acting sequence essential for GBP130 promoteractivity in Plasmodium falciparum. Molecular and BiochemicalParasitology, 1999, 99: 77–87.

56. Dechering KJ et al. Isolation and functional characterizationof two distinct sexual-stage-specific promoters of the humanmalaria parasite Plasmodium falciparum. Molecular andCellular Biology, 1999, 19: 967–978.

57. Golightly LM et al. 3’ UTR elements enhance expressionof Pgs28, an ookinete protein of Plasmodium gallinaceum.Molecular and Biochemical Parasitology, 2000, 105: 61–70.

58. Deitsch KW, del Pinal A, Wellems TE. Intra-clusterrecombination and var transcription switches in the antigenicvariation of Plasmodium falciparum. Molecular and BiochemicalParasitology, 1999, 101: 107–116.

59. Horrocks P, Lanzer M. Differences in nucleosomal organizationover episomally located plasmids coincides with aberrantpromotor activity in P. falciparum. Parasitology International,1999, 48: 55–61.

60. Lobo CA et al. Disruption of the Pfg27 locus by homologousrecombination leads to loss of the sexual phenotype inP. falciparum. Molecular Cell, 1999, 3: 793–798.

61. Triglia T et al. Allelic exchange at the endogenous genomic locusin Plasmodium falciparum proves the role of dihydropteroatesynthase in sulfadoxine-resistant malaria. EMBO Journal, 1998,17: 3807–3815.

62. Reed MB et al. Pgh1 modulates sensitivity and resistance tomultiple antimalarials in Plasmodium falciparum. Nature, 2000,403: 906–909.



63. Craig AG, Waters AP, Ridley RG. Malaria Genome ProjectTask Force – A post-genomic agenda for functional analysis.Parasitology Today, 1999, 15: 211–214.

64. Waters AP et al. Transfection of malaria parasites. Methods,1997, 13: 134–147.

65. van Dijk MR, Waters AP, Janse CJ. Stable transfection ofmalaria parasite blood stages. Science, 1995, 268: 1358–1362.

66. Menard R et al. Circumsporozoite protein is required fordevelopment of malaria sporozoites in mosquitoes. Nature,1997, 385: 336–340.

67. Sultan AA et al. TRAP is necessary for gliding motility andinfectivity of Plasmodium sporozoites. Cell, 1997, 90: 511–522.

68. van der Wel AM et al. Transfection of the primate malariaparasite Plasmodium knowlesi using entirely heterologousconstructs. Journal of Experimental Medicine, 1997, 185:1499–1503.

69. Kochen C, van der Wel A, Thomas AW. Plasmodiumcynomolgi: Transfection of blood stage parasites usingheterologous DNA constructs. Experimental Parasitology, 1999,93: 58–60.

70. Roos DS et al. Transport and trafficking: Toxoplasma as amodel for Plasmodium. Novartis Foundation Symposium, 1999,226: 176–195.

71. Soete M, Hettman C, Soldati D. The importance of reversegenetics in determining gene function in apicomplexan parasites.Parasitology, 1999, 118: S53–61.

72. Roos DS et al. Origin, targeting, and function of the api-complexan plastid. Current Opinions in Microbiology, 1999,2: 426–432.

73. Beverley SM, Turco SJ. Lipophosphoglycan (LPG) and theidentification of virulence genes in the protozoan parasiteLeishmania. Trends in Microbiology, 1998, 6: 35–40.

74. Rubin GM et al. Comparative genomics of the eukaryotes.Science, 2000, 287: 2204–2215.

75. Janse CJ et al. Conserved location of genes on polymorphicchromosomes of four species of malaria parasites. Molecularand Biochemical Parasitology, 1994, 68: 285–296.

76. Carlton JM et al. Gene synteny in species of Plasmodium.Molecular and Biochemical Parasitology, 1998, 93: 285–294.

77. Vinkenoog R et al. Malaria parasites contain two identicalcopies of an elongation factor 1 alpha gene. Molecular andBiochemical Parasitology, 1998, 94: 1–12.

78. van Lin LHM, Janse CJ, Waters AP. The conserved genomeorganization of non-falciparum malaria species: the need to knowmore. International Journal of Parasitology, 2000, 30: 357–370.

79. Waller RF et al. Nuclear-encoded proteins target to the plastidin Toxoplasma gondii and Plasmodium falciparum. Proceedingsof the National Academy of Sciences of the United States ofAmerica, 1998, 95: 12352–12357.

80. Waller RF et al. Protein trafficking to the plastid of Plasmodiumfalciparum is via the secretory pathway. EMBO Journal, 2000,19: 1794–1802.

81. Striepen B et al. Expression, selection, and organellar targetingof the green fluorescent protein in Toxoplasma gondii. Molecularand Biochemical Parasitology, 1998, 92: 325–338.

82. Roos DS. The apicoplast as a potential therapeutic targetin Toxoplasma and other apicomplexan parasites: some additionalthoughts. Parasitology Today, 1999, 15: 41.

83. Dessens JT et al. CTRP is essential for mosquito infectionby malaria ookinetes. EMBO Journal, 1999, 18: 6221–6227.

84. Yuda M, Sakaida H, Chinzei Y. Targeted disruption of thePlasmodium berghei CTRP gene reveals its essential role inmalaria infection of the vector mosquito. Journal of ExperimentalMedicine, 1999, 190: 1711–1716.

85. Templeton TJ, Kaslow DC, Fidock DA. Developmental arrestof the human malaria parasite Plasmodium falciparum withinthe mosquito midgut via CTRP gene disruption. MolecularMicrobiology, 2000, 36: 1–9.

86. Wengelnik K et al. The A-domain and the thrombospondin-related motif of Plasmodium falciparum TRAP are implicated inthe invasion process of mosquito salivary glands. EMBO Journal,1999, 18: 5195–5204.

87. Kappe S et al. Conservation of a gliding motility and cell invasionmachinery in Apicomplexan parasites. Journal of Cell Biology,1999, 147: 937–944.

88. O’Donnell RA et al. Functional conservation of the malariavaccine antigen MSP-119 across distantly related Plasmodiumspecies. Nature Medicine, 2000, 6: 91–95.



Entering the post-genomic era of malaria research

Documents

Transcript of Entering the post-genomic era of malaria research