Functional analysis of drug resistance in Plasmodiumfalciparum in the post-genomic era

International Journal for Parasitology 31 (2001) 871±878

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Invited review

Functional analysis of drug resistance in Plasmodium falciparum in the post-genomic era Alan F. Cowman* The Walter and Eliza Hall Institute of Medical Research, Royal Melbourne Hospital, Royal Parade, Melbourne, Victoria 3050, Australia Received 16 October 2000; received in revised form 23 January 2001; accepted 23 January 2001

Abstract Malaria has plagued humans throughout recorded history and results in the death of over 2 million people per year. The protozoan parasite Plasmodium falciparum causes the most severe form of malaria in humans. Chemotherapy has become one of the major control strategies for this parasite; however, the development of drug resistance to virtually all of the currently available drugs is causing a crisis in the use and deployment of these compounds for prophylaxis and treatment of this disease. The genome sequence of P. falciparum is providing the informational base for the use of whole-genome strategies such as bioinformatics, microarrays and genetic mapping. These approaches, together with the availability of a high-resolution genome linkage map consisting of hundreds of microsatellite markers and the advanced technologies of transfection and proteomics, will facilitate an integrated approach to address important biological questions. In this review we will discuss strategies to identify novel genes involved in the molecular mechanisms used by the parasite to circumvent the lethal effect of current chemotherapeutic agents. q 2001 Australian Society for Parasitology Inc. Published by Elsevier Science Ltd. All rights reserved. Keywords: Malaria; Plasmodium falciparum; Genetics; Microarray; Transfection; Bioinformatics; Drug resistance; Chloroquine; Me¯oquine; Quinine

1. Introduction The availability of an increasing amount of the genomic sequence of important infectious agents such as Plasmodium falciparum (Gardner et al., 1998; Bowman et al., 1999) has focused attention on how the data contained in the sequence databases might be interpreted in terms of the structure, function and control of this protozoan parasite. Estimates suggest that there are approximately 6000 genes encoded within the P. falciparum genome (Gardner et al., 1998; Bowman et al., 1999). The proteins encoded by these genes are required for this parasite to survive and ¯ourish in the human host and the mosquito vector. Determining the role for each of these genes will be a formidable task and understanding how the genome functions as a whole in the complex interplay between host and parasite will be an even greater challenge. Importantly, we have to ensure that this large amount of sequence information is utilised ef®ciently to provide the basic knowledge that will underpin an increased understanding of the host-parasite interaction and the development of new antimalarial drugs and vaccines. The development of in vitro continuous culture for P. * Tel.: 161-3-9345-2555; fax: 161-3-9347-0852. E-mail address: [email protected] (A.F. Cowman).

falciparum was a critical technological advance that allowed the analysis of this parasite in the laboratory (Trager and Jensen, 1976). This together with the advent of recombinant DNA techniques in the 1970s ensured a surge in understanding of this parasite and renewed efforts to develop molecularly de®ned vaccines as well as to identify new drug targets. Additionally, we have increased our knowledge of the mechanisms that this parasite uses to evade the lethal effect of many of the current antimalarial drugs used to treat and control malaria. The availability of the genome sequence of P. falciparum has provided the exciting potential to greatly increase our understanding of this parasite and this review will discuss current and new strategies that are now available. Speci®c examples of these molecular strategies will use the analysis of mechanisms of drug resistance in P. falciparum to highlight the exciting new potential of the post-genomic era. 2. The P. falciparum genome Pulsed-®eld gel electrophoresis (Kemp et al., 1985; Van der Ploeg et al., 1985) and EM studies of parasite kinetochores (Prensier and Slomianny, 1986) have identi®ed 14 chromosomes in P. falciparum. These vary in size from 0.6 to 3 Mb and long range chromosome restriction mapping

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has identi®ed a large amount of size polymorphism that occurs predominantly in the subtelomeric regions (Corcoran and Kemp, 1986; Corcoran et al., 1986). The subtelomeric regions have been shown to contain a series of complex repeats and variation in the number of these repeats can cause chromosome size polymorphisms among different strains (Oquendo et al., 1986; Corcoran et al., 1988). Additionally, chromosome breakage and healing events create a large amount of size polymorphism leading to deletion of telomeric regions and juxtaposition of genes or parts of genes directly to the newly derived telomere (Pologe and Ravetch, 1988; Cappai et al., 1989). Although there is a high degree of chromosomal size polymorphism in the P. falciparum genome this is usually con®ned to the subtelomeric region (Corcoran et al., 1988). At least one exception involves ampli®cation events of DNA surrounding the pfmdr1 gene on chromosome 5 and this is associated with alterations in drug resistance phenotypes (Foote et al., 1989). The full genomic sequence of chromosomes 2 and 3 from P. falciparum have been completed and contain 210 and 215 predicted protein-coding genes, respectively (Gardner et al., 1998; Bowman et al., 1999). Additionally a putative centromere has been identi®ed in both chromosomes that has a core region of about 2 kilobases with an extremely high (adenosine 1 thymidine) composition and arrays of tandem repeats (Bowman et al., 1999). A large proportion of the P. falciparum genome is available as shotgun sequencing reads and assembled contigs from the Sanger Centre (http:// www.sanger.ac.uk/Projects/P_falciparum), TIGR (http:// www.tigr.org/tab/mdb/pfdb/pfdb.html) and Stanford University (http://sequence-www.stanford.edu/group/ malaria/index.html) databases. The challenge will be to utilise the genomic sequence databases to identify the function of each gene and their role in speci®c phenotypes such as drug resistance.

3. Current knowledge in drug resistance 3.1. Antifolate drugs The antifolate drugs, along with chloroquine, have been the mainstays of antimalarial treatment over the 50 years. There are two groups of antifolates, ®rstly; the dihydrofolate reductase (DHFR) inhibitors of which pyrimethamine and proguanil are the most important and secondly; the sulphones and sulphonamides. The DHFR inhibitor pyrimethamine is usually used in combination with the sulphonamide, sulphadoxine due to a marked synergistic effect (Chulay et al., 1984). Unfortunately, resistance to the combination sulphadoxine and pyrimethamine is widespread in South East Asia and India (Ghosh et al., 1992) and is growing in Africa (Bloland et al., 1996). Folate is an essential co-factor for the malaria parasite and blockage of its synthesis results in decreased synthesis

of pyrimidines and consequently arrest of DNA replication, decreased conversion of glycine to serine and decreased methionine synthesis. Enzymes within this synthetic pathway in the malaria parasite have been useful targets for chemotherapeutic attack (Peters, 1987). The main enzyme targets have been the linkage of p-aminobenzoic acid (pABA) to synthesise 7,8-dihydropteroate by dihydropteroate synthetase (DHPS) and the reduction of dihydrofolate to tetrahydrofolate by DHFR. The mechanism of resistance to pyrimethamine in ®eld isolates of P. falciparum has been shown to be primarily due to mutations in the DHFR enzyme that lower the af®nity of binding of these drugs (Cowman et al., 1988; Peterson et al., 1988; Sirawaraporn et al., 1990). Previous analysis of the enzyme kinetics of DHFR from P. falciparum lines resistant to pyrimethamine suggested that altered properties of the enzyme could account for pyrimethamine resistance (McCutchan et al., 1984; Dieckmann and Jung, 1986; Walter, 1986; Chen et al., 1987). Sequencing of the dhfr gene from pyrimethamine-resistant and -sensitive P. falciparum isolates identi®ed important amino acid changes that confer resistance (Cowman et al., 1988; Peterson et al., 1988; Snewin et al., 1989; Zolg et al., 1989). A Ser to Asn change at position 108 in DHFR is the key event in the development of resistance and subsequent mutations at position 52 or 58 can result in greatly increased levels. This has been supported by the expression of recombinant DHFR enzymes and analysis of their inhibition constant (Ki) for pyrimethamine (Sirawaraporn et al., 1990). The presence of a Asn at position 108 in DHFR appears to confer on P. falciparum approximately 10-fold resistance to pyrimethamine, whereas a Asn at position 108 plus a Cys at position 52 increases the level of resistance to 100-fold when compared with that of sensitive isolates. Con®rmation of the central role of mutations in the DHFR enzyme was obtained by transfection of the mutant dhfr gene into P. falciparum to obtain parasite lines that were resistant to the expected levels of pyrimethamine (Wu et al., 1996). Sulfa drugs are structural analogues of p-aminobenzoic acid (pABA) which is required for the de novo biosynthesis of folate co-factors from guanosine-5 0 -triphosphate (GTP) (Woods, 1940; Miller, 1944). The target of the sulfa drugs is the enzyme DHPS which catalyses the conversion of 6hydroxymethyl-7,8-dihydropterin pyrophosphate and pABA to 7,8-dihydropteroate (Triglia et al., 1997). In P. falciparum the DHPS enzyme is encoded by a bifunctional enzyme with 6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase (PPPK) at the N-terminus and DHPS at the Cterminus (Triglia and Cowman, 1994; Brooks et al., 1994). Sequence analysis of the dhps gene from drug-sensitive and -resistant P. falciparum strains identi®ed mutations linked to the resistance phenotype. The importance of the mutations in the dhps gene has been con®rmed by the analysis of a genetic cross showing that they segregate with the sulfadoxine-resistance phenotype (Brooks et al., 1994). Further analysis of the progeny from the genetic cross has shown

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that folate in the culture medium could in¯uence the level of sulfadoxine-resistance (folate effect) (Wang et al., 1999). These data pointed to a second gene other than dhps that could regulate the levels of folate enabling the parasite to utilise exogenous folate to a higher extent than in other parasite strains, and therefore show increased resistance to the effects of sulfadoxine. This suggests that the protein encoded by the second gene may be involved in folate uptake and/or utilisation. Expression and puri®cation of functional PPPK-DHPS enzyme from P. falciparum in Escherichia coli has shown that the mutations identi®ed in DHPS enzymes from sulfadoxine-resistant parasites mediate resistance by decreasing sulfadoxine af®nity for the enzyme (Triglia et al., 1997). Measurement of the Ki of sulfadoxine for the PPPK-DHPS enzymes containing the different mutations found in different ®eld isolates of P. falciparum showed that this parameter varied almost three orders of magnitude between the sulfadoxine-sensitive DHPS enzyme and the enzyme from the most highly resistant parasites. Transfection of the different mutant alleles of pppk-dhps has directly determined the role of dhps mutations on sulphadoxine resistance (Triglia et al., 1998). Plasmid constructs containing truncated mutant dhps alleles have been integrated into the genome of a sulphadoxine-sensitive parasite resulting in allelic exchange and expression of mutant DHPS enzyme. Determination of the sulphadoxine susceptibility of the transfected parasites showed that mutant dhps alleles conferred sulfadoxine resistance on P. falciparum and not only proves the role of the mutations but also determined the relative contribution of the different mutations on the ®nal drug resistance phenotype. These results further suggest that resistance to high levels of sulfadoxine in P. falciparum has arisen by an accumulation of mutations consistent with its occurrence in most dhps alleles from resistant isolates. The level of resistance obtained by transfection of the mutant dhps alleles and measured in in vitro cultures parallels closely the result seen both for the kinetic parameters of the corresponding puri®ed enzymes and the natural isolates in culture. The mechanisms of resistance used by P. falciparum to evade the lethal effects of pyrimethamine and sulfadoxine is now well known. This understanding has come about through the use of a multi-targeted approach using a combination of genetics, enzymology and transfection. However, it appears that there may be a second gene that mediates the folate effect that has been show to reduce the level of sulfadoxine resistance in the presence of folate (Wang et al., 1999). To gain a complete understanding of the folate effect and its role in sulfadoxine resistance it will be essential to identify the relevant gene so that biochemical analyses can answer this outstanding question. 3.2. Chloroquine and other quinine-like drugs Current evidence suggests that the antimalarial activity of

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chloroquine acts by inhibition of the detoxi®cation of haematin inside the parasite food vacuole. Haem is released as a by-product of haemoglobin digestion and is sequestered as haemozoin, a crystalline-like structure (Slater, 1992). Chloroquine appears to inhibit the haem polymerisation process causing a build-up of toxic haematin/chloroquine complex (Slater and Cerami, 1992; Dorn et al., 1995; Sullivan and Meshnick, 1996) that is lethal to the parasite by a mechanism that is not fully understood. Chloroquine-resistant malaria parasites accumulate less chloroquine than do parasites that are sensitive to the drug (Fitch, 1970; Verdier et al., 1985; Yayon et al., 1985; Krogstad et al., 1989, 1992). Decreased accumulation of chloroquine in resistant parasites may be explained by altered chloroquine±haematin binding parameters suggesting that the parasite has devised a mechanism that reduces the access of chloroquine to haematin (Bray et al., 1998). Recent measurements of food vacuole pH in parasitised red blood cells has suggested that it is more acidic in chloroquine-resistant parasites although this work needs to be con®rmed (Dzekunov et al., 2000; Ursos et al., 2000). These data lead to an interesting hypothesis suggests that a more acidic pH in the food vacuole will favour formation of insoluble haematin decreasing the soluble haematin with which chloroquine can interact. This mechanism would essentially decrease access of chloroquine to soluble haematin as suggested previously (Bray et al., 1998, 1999a,b). It has been known for some time that lysosomotrophic agents such as verapamil can modulate chloroquine-resistance in P. falciparum to levels of that observed in chloroquine-sensitive parasites. A number of mechanisms have been suggested to explain this, including inhibition of a chloroquine ef¯ux pump (Krogstad et al., 1988); however, the most likely hypothesis involves altering the ability of chloroquine to interact with free haematin (Bray et al., 1998, 1999a,b; Dzekunov et al., 2000; Ursos et al., 2000). This could occur by resetting the food vacuolar pH back to the levels found in chloroquine-sensitive parasites either by a direct effect on pH or interaction with factors involved in pH regulation. These studies suggest that the protein(s) involved in mediating chloroquine resistance may play a role in regulating pH of the food vacuole. Analysis of a genetic cross between chloroquine-resistant and chloroquine-sensitive parental P. falciparum cloned lines has mapped a region of chromosome 7 that segregates with the chloroquine-resistance phenotype (Wellems et al., 1990; Su et al., 1997). Analysis of this region identi®ed a number of potential genes, such as cg2, involved in this phenotype; however, a role for this gene has been ruled out by transfection to replace the gene with the identi®ed mutations (Fidock et al., 2000a). No alterations in the level of chloroquine resistance was obtained suggesting that the polymorphisms identi®ed in this gene were closely linked to another gene that was responsible. Recently, a closely linked gene called pfcrt was identi®ed that contained a polymorphism that was present in all chloroquine-resistant

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strains analysed, suggesting that the protein encoded by this gene plays an important role in chloroquine resistance (Fidock et al., 2000b). Analysis of the pfcrt gene using transfection in P. falciparum has shown that this gene plays an important role in the mechanism of chloroquine resistance (Fidock et al., 2000b). Additionally, there was circumstantial evidence that suggests the pfmdr1 gene, which encodes the P-glycoprotein homologue 1 (Pgh1), is linked to the mechanism of chloroquine resistance (Foote et al., 1989, 1990). The pfmdr1 gene encodes a protein of 162 kDa (Cowman et al., 1991) and has a typical structure shared by members of the ATP binding cassette (ABC) family of transporters that includes the P-glycoproteins (Endicott and Ling, 1989). Polymorphisms have been identi®ed in the pfmdr1 gene that are present in many, but not all, chloroquine resistant isolates analysed (Foote et al., 1990). Recently, it has been shown by transfection using gene replacement that the mutations modulate the level of chloroquine resistance in concert with a second gene(s) (Reed et al., 2000). The second gene is likely to be pfcrt although this has not yet been functionally tested. Interestingly, insertion of the polymorphisms into the wild-type pfmdr1 gene by transfection had an effect on resistance and sensitivity to me¯oquine, halofantrine and quinine providing direct proof that mutations in pfmdr1 can encode resistance to these antimalarials (Reed et al., 2000). The same mutations in¯uence the level of sensitivity to the structurally unrelated compound, artemisinin. Functional analysis of the Pgh1 protein in Chinese hamster ovary (CHO) cells has shown that this protein can affect the pH of the lysosomal compartment causing a chloroquine-sensitive phenotype due to increased accumulation of the drug into the lysosomes (Van Es et al., 1994a,b). The increased drug accumulation was a result of increased acidity of the lysosome causing increased accumulation of the weak base drug (Van Es et al., 1994b). This has suggested that Pgh1 in P. falciparum may be involved in regulation of pH in the food vacuole and therefore any alterations in Pgh1 would have an effect on accumulation of weak base drugs (Cowman, 1991). This was con®rmed by transfection of a pfmdr1 allele, that had been previously linked to the chloroquine resistance phenotype (Foote et al., 1990), into CHO cells (Van Es et al., 1994a). The transfectant CHO cells expressing the mutant Pgh1 were not sensitive to chloroquine as the pH of the lysosomes was the same as the untransfected parental cells (Van Es et al., 1994b). This suggests that mutations in the Pgh1 protein alter its function and affect the accumulation of antimalarials such as chloroquine and the other quinine-like antimalarials. There is still much to understand with respect to the mechanism of resistance of P. falciparum to chloroquine and other quinine-like drugs. Importantly, two genes pfcrt (Fidock et al., 2000b) and pfmdr1 (Reed et al., 2000) have been identi®ed that can play a role in resistance and sensitivity to these drugs. It will be necessary to understand the function of the proteins encoded by these genes so that we

can elucidate their role in the mechanism of resistance to these drugs. It is also very likely that there are other genes that can modulate the level of resistance and sensitivity to these drugs. The P. falciparum genome sequence together with the new advanced technologies for analysing whole genomes will play an important role in the mapping and identi®cation of these genes. This will facilitate a complete understanding of the mechanisms of resistance to this group of antimalarials that will be important for the development of strategies to circumvent them. 4. Approaches to identify novel genes involved in drug resistance It is clear that the availability of a large portion of the genome sequence from P. falciparum will cause a shift in emphasis from genomic structure and gene cloning to the correlation between genomics and proteomics and the biological signi®cance of this information. Outlined below are some of the important approaches that are being used to identify the genes and proteins involved in important parasite phenotypes, and I have used the example of drug resistance to illustrate particular applications. Our understanding of the mechanisms that P. falciparum uses to evade the lethal effect of antimalarial drugs has increased greatly over the last 20 years. Despite this increased knowledge there are still many unanswered questions. A number of genes have been identi®ed that play a role in resistance to some of the important antimalarial drugs but it is likely that there are currently unknown genes that can modify the level of drug resistance. For example, there are P. falciparum isolates from Thailand that are highly resistant to some or all of the currently used antimalarial drugs (Price et al., 1999). It will be important to identify the basis of these resistance phenotypes so that we have the molecular methodology to more easily monitor drug resistance in malaria endemic areas. 4.1. Complementation using transfection The ability to transfect (Wu et al., 1995) and express transgenes in P. falciparum (Crabb et al., 1997) has been an important technological breakthrough that has increased our ability to determine the role of different proteins in phenotypes such as drug resistance. Allelic replacement of the dhps and pfmdr1 gene has shown that they play an important role in resistance to sulfa and quinoline-containing antimalarials respectively (Triglia et al., 1998; Reed et al., 2000). Functional testing by transfection of genes that are suspected of an involvement in drug resistance mechanisms is relatively straightforward; however, it may be possible to consider direct selection of unknown genes that confer drug resistance phenotypes from genomic libraries by functional complementation. Functional complementation of drug resistance would involve constructing genomic or cDNA libraries in appro-

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priate vectors, transfecting these into drug sensitive strains of P. falciparum and directly selecting for drug resistance. There are a number of drawbacks to this potential strategy that need to be considered. Firstly, drug resistant phenotypes may involve a number of genes that may either confer partial resistance or modulate the level of resistance already established. It would be possible to circumvent these problems by transfecting the libraries into P. falciparum strains that are resistant to medium levels of drug so that genes involved in modulating resistance could be isolated. Secondly, the transfection ef®ciency in P. falciparum is still relatively low and requires major improvement to make strategies such as this viable. Current estimates of stable transfection ef®ciencies suggest that 100±200 parasites in each experiment take up plasmids that are initially maintained as episomes (Reed et al., 2000). The estimated stable transfection ef®ciency is 10 26 which is still far too low to contemplate direct selection of genes encoding drug resistance phenotypes (Reed et al., 2000). It will be essential to pursue strategies for increasing the ef®ciency of transfection in P. falciparum so that functional complementation with genomic and cDNA libraries becomes a reality. 4.2. Microarrays and proteomics The phenotype of an organism is largely determined by the genes that it expresses and can be modulated by external pressures such as drug selection. The altered phenotypes can be mediated either by mutations in speci®c genes or alterations in their level of expression. The expressed genes in a cell have been de®ned as the `transcriptome' which describes both the identity of the genes and the level of expression at the time of sampling (Velculescu et al., 1997). Knowing the pattern of genes expressed in a cell can provide strong clues about their functional roles. DNA microarrays consisting of all of the known genes in the P. falciparum genome will provide a practical and economical tool for analysing gene expression of the genome. This involves printing portions of the 6000 genes from the genome sequence onto a single glass microscope slide in a high-density array (DeRisi et al., 1997). Hybridisation of cDNA probes made from mRNA of P. falciparum will provide a large amount of information with respect to timing of their expression. Genes that play a role in drug resistance phenotypes will be expressed in the trophozoite stage as many of the major antimalarials, such as chloroquine, act against this stage of the asexual life cycle (Peters, 1987). Mutations in genes such as dhfr and dhps are responsible for resistance to antifolate drugs and these alterations would not be detected using the normal hybridisation techniques for microarrays (DeRisi et al., 1997). However, microarray analysis will detect differences in expression of genes and therefore will enable identi®cation of those genes that are upregulated in response to selective pressure such as drug use. Therefore, comparison between a drug-resistant and -sensitive parasite line would identify

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genes that differ in the level of expression. These could be analysed in more detail to determine if they are involved in the drug resistance phenotypes. Comparative microarray analysis of the transcriptome of two different lines of P. falciparum that differ in drug resistance phenotypes will be complex because of the polymorphic antigens encoded within the genome of different isolates (Kemp et al., 1987). However, the genes encoding these polymorphic antigens are usually located in subtelomeric regions of chromosomes and it is likely that proteins involved in drug resistance will function in house keeping roles such as folate metabolism and pH regulation. Genes encoding these proteins will be highly conserved and any increased expression detected by microarray analysis may be relevant to the phenotype. The proof that any candidate genes identi®ed by these approaches contribute to the drug resistant phenotypes will still require rigorous functional testing using transfection and biochemical analysis. Another approach to identify proteins involved in phenotypic differences between P. falciparum involves analysis of protein expression. The `transcriptome' de®nes the expression of RNA molecules in a given cell and similarly the `proteome' de®nes the expression of all of the proteins (Shevchenko et al., 1996). The ability to identify proteins by in-gel tryptic digestion and mass spectrometry after separation with two-dimensional gel electrophoresis will be a powerful approach that can link information from the proteome to the genome sequence. Differences in expression of proteins between different parasite lines of P. falciparum can be identi®ed and tryptic peptides sequenced and the corresponding gene identi®ed from the genome database. Such approaches will be important for identifying alterations in protein expression between cloned lines where one has been altered in some way such as disruption of a gene that has some phenotypic effect on the parasite. However, it will be dif®cult to compare different strains of P. falciparum due to the high degree of polymorphism in many of the antigens (Kemp et al., 1987). It may be possible to limit this dif®culty by analysing puri®ed organelles such as food vacuoles (Saliba et al., 1998). Most of the proteins associated with this organelle would be highly conserved and any alterations in expression or migration properties in two-dimensional gel electrophoresis are likely to have been selected. This may be a useful way to identify any novel proteins involved in drug resistance phenotypes as the target of antimalarials such as chloroquine appear to be in the food vacuole. 4.3. Genetics and microsatellite markers Genetic mapping is a powerful tool that can be used to identify genes in organisms such as P. falciparum that contribute to heritable phenotypes such as drug resistance. Analysis of two genetic crosses for P. falciparum has enabled mapping of the genes responsible for pyrimethamine and sulfadoxine resistance and additionally, a locus

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has been identi®ed that is important in the chloroquineresistance phenotype (Peterson et al., 1988; Wang et al., 1997; Wellems et al., 1990; Su et al., 1997). This approach is assisted by high resolution linkage maps using polymorphic markers that can be scored at hundreds of loci spread throughout the genome to de®ne the pattern of inherited alleles known as a haplotype for each parasite clone analysed. This enables the identi®cation of chromosomal regions that harbour genes contributing to particular phenotypes in genetic crosses or outbreed populations in the absence of speci®c biochemical knowledge of the trait. A major contribution to the power of genetic mapping in P. falciparum has been the recent development of a highresolution genetic linkage map consisting of over 800 polymorphic microsatellite markers (Su et al., 1999; Su and Wellems, 1999). These microsatellite markers are simple repeats, found in all eukaryotes, varying in length and de®ning multiple alleles in parasite populations. A genetic linkage map de®ned by a comprehensive set of microsatellite markers together with the P. falciparum genome sequence will be an important tool that will enable much easier identi®cation of candidate genes involved in particular phenotypes such as drug resistance. The ®nal proof of the role of any such gene in these phenotypes will still require rigorous functional tests such as complementation using transfection (Triglia et al., 1998; Reed et al., 2000). It has been suggested that mutations contributing to drug resistance may be mapped by detection of allelic associations in populations of P. falciparum (Ferdig and Su, 2000). The development of phenotypes such as drug resistance are very recent events increasing the likelihood that linkage disequilibrium between a gene associated with the drug resistance trait and the closely associated microsatellite markers would still be maintained. To increase the potential of this strategy being successful parasite populations from areas of low-transmission or regions to which resistance has recently spread could be used (Ferdig and Su, 2000). 5. Concluding remarks The full genome sequence of P. falciparum is fast nearing completion and this information will provide an immensely valuable resource to drive a greater understanding of this parasite and its interactions with the human host. This together with the availability of technologies that enable analyses of gene function and genetic perturbations on a whole genome scale will rapidly yield a wealth of information in important phenotypes such as the example of drug resistance used here. Perhaps the greatest challenge will be to develop ef®cient ways of organizing, distributing, interpreting, and obtaining insights from the large increase in the ¯ow of data that will emanate from these new experiments. Importantly, the post-genomic era of P. falciparum will not only provide us with the information and tools to increasingly understand this parasite but also to develop vaccines

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