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Malaria still poses one of the most serious threats to human health worldwide and the prevailing lack of effective, clinically licensed, vaccines means that prophylaxis and treatment depend heavily on a small number of compounds whose efficacies are progressively compromised at varying rates by the inevitable emergence of drug-resistant parasite populations. Of these antimalarials, those inhibiting steps in folate metabolism, along with chloroquine, are the oldest synthetic compounds, with origins dating back three-quarters of a century. Despite widespread parasite resistance, the antifolates still play an important role in malaria control, and our understanding of the underlying mechanisms of folate metabolism and genesis of drug resistance has increased considerably over the last twenty years. Folate de novo synthesis in the parasite, interconversion of active folate derivatives and their utilisation as multifunctional cofactors involve numerous enzymes, although only two of these have ever served as targets of clinical antimalarial inhibitors. The current application of antifolates, resistance to this class of drugs, new insights into folate metabolism in the parasite, its potential for providing novel targets of inhibition and some of the questions that are still outstanding are reviewed here.

Folate salvage by Plasmodium falciparum is an important source of key cofactors, but little is known about the underlying mechanism. Using synchronised parasite cultures, we observed that uptake of this dianionic species against the negative-inward electrochemical gradient is highly dependent upon cell-cycle stage, temperature and pH, but not on mono- or divalent metal ions. Energy dependence was tested with different sugars; glucose was necessary for folate import, although fructose was also able to function in this role, unlike sugars that cannot be processed through the glycolytic pathway. Import into both infected erythrocytes and free parasites was strongly inhibited by the anion-channel blockers probenecid and furosemide, which are likely to be acting predominantly on specific folate transporters in both cases. Import was not affected by high concentrations of the antifolate drugs pyrimethamine and sulfadoxine, but was inhibited by the close folate analogue methotrexate. The pH optimum for folate uptake into infected erythrocytes was 6.5-7.0. Dinitrophenol and nigericin, which strongly facilitate the equilibration of H(+) ions across biological membranes and thus abolish or substantially reduce the proton gradient, inhibited folate uptake profoundly. The ATPase inhibitor concanamycin A also greatly reduced folate uptake, further demonstrating a link to ATP-powered proton transport. These data strongly suggest that the principal folate uptake pathway in P. falciparum is specific, highly regulated, dependent upon the proton gradient across the parasite plasma membrane, and is likely to be mediated by one or more proton symporters.

The thymidylate cycle in Plasmodium falciparum is essential for cell growth and replication, and dihydrofolate reductase (DHFR), a key enzyme in this cycle, is the target of important antimalarial drugs such as pyrimethamine and cycloguanil. Following previous work, where we found no evidence of upregulation of the dhfr-ts gene upon challenge with pyrimethamine, we investigated the expression at the protein level of the bifunctional gene product, which also carries thymidylate synthase (TS) activity. Challenge of parasite cultures with fluoro-substituted bases that are specific TS inhibitors at levels close to the IC(50) resulted in five to seven-fold increases in enzyme level, as monitored by both DHFR and TS activities, while pyrimethamine and another DHFR-binding inhibitor, WR99210, induced smaller but still significant increases of approximately three-fold. However, when parasites were challenged with tetracycline, an antimalarial not directed at the folate pathway, although an increase was consistently seen above untreated controls, this was at a level of approximately 1.8-fold. These increases reflect enhanced synthesis of the DHFR-TS enzyme, rather than liberation of a latent activity, as they were completely abolished if cultures were pre-incubated with cycloheximide to block de novo protein synthesis. Moreover, none of the above antimalarial drugs was found to significantly alter absolute levels of the dhfr-ts mRNA under the conditions of challenge used. We conclude that, in common with mammalian systems, where a similar phenomenon has been reported, malaria parasites are able to significantly relieve translational constraint when faced with antifolate drug challenge. The data indicate that there is a specific component in addition to a low-level non-specific increment, and that binding to the TS domain of the DHFR-TS protein appears to be better able to relieve this constraint than binding to the DHFR domain.

Antifolate drugs that target the biosynthesis and processing of essential folate cofactors are widely used for treatment of chloroquine-resistant falciparum malaria. Salvage of pre-formed folate can strongly compromise the efficacy of these drugs in vitro and the availability of folate from the human host in natural infections also influences therapeutic outcomes. To investigate how different parasite lines respond to the presence of exogenous folate, we measured the effect of the latter on the susceptibility of parasites to sulfa-drug blockage of folate biosynthesis, utilising the parents and 22 progeny of the HB3-Dd2 genetic cross of Plasmodium falciparum, together with selected unrelated lines. Complete linkage of the folate utilisation phenotype was observed to a DNA sequence of 48.6 kb lying between nucleotide positions 738,489 and 787,058 of chromosome 4 and encompassing the dihydrofolate reductase-thymidylate synthase (dhfr-ts) gene locus. Examination of the putative ORFs on this fragment upstream (3) and downstream (4) of dhfr-ts revealed no plausible candidate genes for folate processing. Similarly, a marked heterogeneity in the 5'-UTR regions of Dd2 and HB3, manifest as a directly repeated 256 bp sequence in the former, also did not correlate with the folate utilisation phenotype nor apparently influence levels of dhfr-ts transcripts or protein products. By contrast, the nature of the coding sequence of the dhfr domain appeared to play a direct role, with the single mutant (S108N) HB3-type utilising folic acid much less efficiently than other allelic variants. We also compared the processing of exogenous folic acid, folinic acid and p-aminobenzoic acid (pABA) in metabolic labelling studies of HB3 and Dd2. These support the view that DHFR is likely to have a low-level folate reductase activity as well as its normal function of reducing dihydrofolate to tetrahydrofolate, and that a significant hurdle in the utilisation of exogenous folic acid is the initial reduction of fully oxidised folic acid to dihydrofolate, an activity that the single mutant enzyme found in HB3 is postulated to perform particularly poorly. This would mirror earlier studies indicating that the DHFR activity of HB3 is also compromised relative to other variants.

With the near-completion of the genome sequence of Plasmodium falciparum, further understanding of this major human pathogen urgently requires more effective genetic tools. These must include faster and more reliable gene replacement or gene knockout techniques, essential for the analysis of gene function. We describe a serial system which uses the blasticidin S deaminase (bsd) gene of Aspergillus and the neomycin phosphotransferase II (neo) gene from transposon Tn5 as selectable markers for, respectively, transient transfection of malaria parasites and the selection of stable integrants. Challenge with blasticidin S (BS) enriches the parasite population transiently expressing the bsd gene, laying the foundation for the subsequent, much less frequent, integration event. Positive selection for this rare event is enormously facilitated by fusing the neo gene in frame to the replacement or knockout targeting gene. The sequence employed for the targeting (the polymorphic pppk-dhps gene of P. falciparum, as a model system) is truncated at the 5' end with no promoter located upstream, therefore neo cannot be expressed without specifically integrating within the genomic copy of the target gene. After BS selection, the culture is immediately exposed to geneticin (G418), leading to an apparently homogenous population of mutant parasites. As well as excluding spurious integrants at non-targeted sequences, this system greatly reduces the lengthy selection period for obtaining the desired mutants by eliminating the drug-on and drug-off cycles for the production of stable integrants, which are normally required by the single marker systems currently in use for transfection of malaria parasites.

Folate metabolism in the human malaria parasite Plasmodium falciparum is an essential activity for cell growth and replication, and the target of an important class of therapeutic agents in widespread use. However, resistance to antifolate drugs is a major health problem in the developing world. To date, only two activities in this complex pathway have been targeted by antimalarials. To more fully understand the mechanisms of antifolate resistance and to identify promising targets for new chemotherapies, we have cloned genes encoding as yet uncharacterised enzymes in this pathway. By means of complementation experiments using 1-carbon metabolism mutants of both Escherichia coli and Saccharomyces cerevisiae, we demonstrate here that one of these parasite genes encodes both dihydrofolate synthetase (DHFS) and folylpolyglutamate synthetase (FPGS) activities, which catalyse the synthesis and polyglutamation of folate derivatives, respectively. The malaria parasite is the first known example of a eukaryote encoding both DHFS and FPGS activities in a single gene. DNA sequencing of this gene in antifolate-resistant strains of P. falciparum, as well as drug-inhibition assays performed on yeast and bacteria expressing PfDHFS--FPGS, indicate that current antifolate regimes do not target this enzyme. As PfDHFS--FPGS harbours two activities critical to folate metabolism, one of which has no human counterpart, this gene product offers a novel chemotherapeutic target with the potential to deliver a powerful blockage to parasite growth.

Toxoplasma gondii is an important cause of AIDS-related opportunistic infection, manifest as toxoplasmic encephalitis. The clinical treatment of choice is the synergistic combination of antifolate agents, pyrimethamine and sulphadiazine, of which the latter targets the parasite's dihydropteroate synthase (DHPS) activity. Here, we describe the isolation of the gene encoding this activity in T. gondii. The nucleotide sequence contains an open reading frame interrupted by five introns, which encodes a protein of 664 amino acids with an Mr of 72 991. Sequence analysis revealed that, in addition to DHPS, the predicted protein contains a second enzyme function, hydroxymethyldihydropterin pyrophosphokinase (PPPK). This enzyme immediately precedes DHPS in the folate biosynthetic pathway. The bifunctional arrangement of the T. gondii pppk-dhps gene is the same as that observed in the related protozoan parasite, Plasmodium falciparum, and confirms previous biochemical data that these activities were inseparable. Recently, specific mutations within conserved motifs of the DHPS gene of P. falciparum have been identified which give rise to sulphonamide drug resistance. Analysis of seven clinical isolates of T. gondii did not reveal any similar mutations in this limited sample of organisms that had been subjected to drug pressure.

Malaria parasites switch to sexual development after a period of vegetative growth in the host's erythrocytes. This switch, vital for parasite transmission to mosquitoes, is little understood at the genetic level. Likely candidates for developmental control are the alpha- and beta-tubulin subunits required for microtubule assembly. We report here that the transcription of the alpha- and beta-tubulin genes in Plasmodium falciparum show a radically different pattern of transcription in the sexual and sexual phases of parasite growth. Our studies lead to the conclusion that three transcripts of the beta-tubulin gene differ by sequences in their 5'- or 3'-untranslated regions.

In higher eukaryotes, pteridine cofactors such as tetrahydro-biopterin (BH4) are essential for a range of enzyme-catalysed reactions, including hydroxylation of aromatic amino acids and cleavage of ether lipids by specific monooxygenases, as well as production of nitric oxide by NO synthase [1]. Among the protozoa, it has long been known that trypanosomatids are pteridine auxotrophs that salvage the necessary molecules from the host organism [2]. Although pterins (i.e. naturally occurring pteridines with 2-amino and 4-oxy substitutions on the pteridine ring) are recognised as a growth factor for trypanosomatids, their precise functions in these organisms are not clear, but may include roles in dealing with oxidative stress and in parasite differentiation [3,4]. Interestingly, there is also evidence that Leishmania can synthesise folates from biopterin by a route that differs from the standard folate biosynthetic pathway found in other microorganisms [5]. These observations led us to consider a possible role for pterin metabolism in the apicomplexan parasites. The conventional route to pterin synthesis, lacking in trypanosomatids, initially involves the conversion of GTP to 7,8-dihydroneopterin triphosphate by GTP cyclohydrolase I (GTPC; EC 3.5.4.16), an enzyme that is present in both P. falciparum [6] and T. gondii (Smith, Hyde and Sims, unpublished data). This product can then be utilised by many microorganisms (but not higher eukaryotes, other than plants) in a biosynthetic pathway leading to tetrahydrofolate and its derivatives, which are also key enzyme cofactors, or it can be acted upon by 6-pyruvoyl-tetrahydropterin synthase (PTPS; EC 4.2.3.12) and sepiapterin reductase (SR; EC 1.1.1.153) to yield BH4. Although much has been learnt about folate biosynthesis and salvage in P. falciparum [7] and to a lesser extent, in T. gondii [8], almost nothing is known about the pterin content of these organisms and any metabolic role such molecular species might play. The only annotated gene in the complete P. falciparum genome sequence hinting at the existence of pterin metabolism is PFF1360w, putatively encoding a PTPS orthologue, although there is no obvious orthologue of SR. However, we discovered an unannotated gene located between PF11_0095 and PF11_0096 whose predicted product bore histidine motifs separated by 16 residues characteristic of pterin-4a-carbinolamine dehydratase (PCD; EC 4.2.1.96) (Fig. 1). PCD is an enzyme that in many organisms is essential for the recycling of the BH4 moiety, which is oxidised to the dihydro- level when acting as a cofactor for amino acid hydroxylations (e.g. Phe to Tyr, Tyr to L-dopa, Trp to 5-hydroxytryptamine) and other reactions. PCD executes a dehydration step, removing as water an –OH group introduced onto the 4a position of the pterin ring in the first step of BH4 utilisation (Fig. 2). This gene may have been missed in the original annotation, as it is very short, with a coding length of 324 bp split by a 142 bp intron between codons 39 and 40. We also found an equivalent gene in the T. gondii database with a 315 bp ORF and a single intron (271 bp) in the same relative position (between codons 44 and 45) as in the P. falciparum gene (Fig. 1). Fig. 1 Alignment of pterin-4a-carbinolamine dehydratase (PCD) sequences. Pf, P. falciparum (this work, accession no. {"type":"entrez-nucleotide","attrs":{"text":"DQ223776","term_id":"80973347","term_text":"DQ223776"}}DQ223776); Tg, T. gondii (this work, {"type":"entrez-nucleotide","attrs":{"text":"DQ223777","term_id":"80973349","term_text":"DQ223777"}} ... Fig. 2 Recycling of tetrahydrobiopterin (BH4) via the pterin-4a-carbinolamine dehydratase (PCD) activity after acting as a cofactor in the hydroxylation of phenylalanine by phenylalanine hydroxylase (PAH). The redox cycle is completed by dihydropterin reductase ... To test the function of a putative PCD gene biochemically is not straightforward as the carbinolamine substrate is not readily available. We therefore took advantage of the fact that phenylalanine hydroxylation systems are relatively rare in prokaryotes to set up a microbiological complementation test in an Escherichia coli tyrosine auxotroph [9]. Thus, E. coli lacks both phenylalanine hydroxylase and PCD genes, but does have a dihydropteridine reductase activity (DHPR; EC 1.6.99.7), which is necessary for completion of a pterin recycling pathway (Fig. 2). However, phenylalanine hydroxylase and PCD genes both occur in certain bacteria including the gamma-proteobacterium Pseudomonas aeruginosa [10]. Thus, when transformed into E. coli, the products of these two genes can combine with the endogenous DHPR to establish a pterin recycling assay with the necessary positive controls. Our strategy was thus to assay for recycling by the successful synthesis of Tyr from Phe, dependent upon the introduced P. aeruginosa phenylalanine hydroxylase and PCD activities, and then test the putative P. falciparum and T. gondii PCD genes for function by substituting them for the P. aeruginosa PCD gene in the positive control. Without pterin recycling, the synthesis of Tyr is inadequate for viability. After PCR amplification of the P. falciparum and T. gondii genes from cDNA libraries, the products were cloned into pGEM-T or pGEM-T Easy (Promega) in such a way that they were positioned downstream of the lacZ promoter with a stop codon in-frame with the lac gene situated shortly before the PCD ATG start codon. This was to truncate the synthesis of the beta-galactosidase alpha-peptide product. We were thus depending upon translational reinitiation at the start codon of a correctly orientated PCD gene in order to observe activity. To complete the recycling system, the P. aeruginosa phenylalanine hydroxylase gene (phhA) was introduced into the E. coli host (JP2255) on a compatible pACYC177-based plasmid, pJSll [9]. For both P. falciparum and T. gondii, clones were tested for their ability to rescue the E. coli mutant to a degree comparable to that observed with the P. aeruginosa positive control, as judged by growth rate on the minimal medium agar plates containing phenylalanine (Fig. 3A and B). All positive clones were found to contain the putative PCD gene orientated in the sense direction with respect to the lacZ promoter. To further confirm that the activity was not spurious but was a direct result of the genes we had inserted, the T. gondii ORF was reversed in the plasmid while the P. falciparum gene was cut with BstBI and the two-base overhang filled in with Klenow polymerase, causing the PCD reading frame to be disrupted about half-way into the coding sequence. Neither of these constructs was able to rescue the E. coli auxotroph (Fig. 3C and D). We thus conclude that the genes we have identified in P. falciparum and T. gondii indeed encode PCD activity. Fig. 3 Complementation tests in the E. coli tyrosine auxotroph JP2255 [9] of the putative P. falciparum and T. gondii pcd genes. Panels A and B: sector 1, positive control carrying Pseudomonas phhB plus Pseudomonas phhA genes; 2, negative control carrying Pseudomonas ... Our observations point to a new area of metabolism that merits investigation in these apicomplexan organisms. It is not yet clear what role pterins might play in these parasites, although observations made in trypanosomatids are suggestive [4]. As found in E. coli and P. aeruginosa, although both P. falciparum and T. gondii appear to have a gene encoding PTPS, albeit functionally untested, there seems to be no equivalent of an SR-encoding gene whose product could convert 6-pyruvoyltetrahydropterin to BH4, at least in the case of P. falciparum. It has been suggested in the case of the bacterial systems that one or more pterin cofactors other than BH4, such as tetrahydroneopterin, might be involved in the recycling reaction described above [9]. The feasibility of this is reinforced by the fact that chemical studies of the rat PCD enzyme have demonstrated a lack of sensitivity to the nature of the 6-substituent, and to the stereochemistry of this or the 4a-hydroxyl group [11], indicative of a flexible binding pocket. Interestingly, it has also been shown in humans that the PCD protein plays a second role as a transcriptional regulator (known as DCoH) in the nucleus [12], although this may be a function only found in higher organisms. PCD genes appear to be quite widely distributed in bacteria, but the known enzymes that use a pterin cofactor, such as the hydroxylases described above, are much less common. Thus there probably remain to be discovered in bacteria, and possibly in apicomplexans, other enzymes that produce a carbinolamine intermediate requiring recycling via PCD. The identification and functional demonstration of a pterin recycling activity reported here is a first step in exploring the likely importance of pterin metabolism in P. falciparum and T. gondii.

Resistance of Plasmodium falciparum to antifolate chemotherapy is a significant problem where combinations such as Fansidar (pyrimethamine–sulfadoxine; PYR–SDX) are used in the treatment of chloroquine-resistant malaria. Antifolate resistance has been associated with variant sequences of dihydrofolate reductase (DHFR) and dihydropteroate synthetase (DHPS), the targets of PYR and SDX respectively. However, while the nature and distribution of mutations in the dhfr gene are well established, this is not yet the case for dhps. We have thus examined by DNA sequence analysis 141 field samples from several geographical regions with differing Fansidar usage (West and East Africa, the Middle East and Viet Nam) to establish a database of the frequency and repertoire of dhps mutations, which were found in 60% of the samples. We have also simultaneously determined from all samples their dhfr sequences, to better understand the relationship of both types of mutation to Fansidar resistance. Whilst the distribution of mutations was quite different across the regions surveyed, it broadly mirrored our understanding of relative Fansidar usage. In samples taken from individual patients before and after drug treatment, we found an association between the more highly mutated forms of dhps and/or dhfr and parasites that were not cleared by antifolate therapy. We also report a novel mutation in a Pakistani sample at position 16 of DHFR (A16S) that is combined with the familiar C59R mutation, but is wild-type at position 108. This is the first observation in a field sample of a mutant dhfr allele where the 108 codon is unchanged.

Sulphur-based antimalarial drugs targeted at dihydropteroate synthetase (DHPS) are frequently used in synergistic combination with inhibitors of dihydrofolate reductase (DHFR) to combat chloroquine-resistant malaria. We have previously shown that lines of Plasmodium falciparum resistant to the most commonly used sulpha drug, sulphadoxine, carry point mutations in the DHPS coding region, relative to the sequence of sensitive strains (Brooks et al., Eur. J. Biochem. 224 (1994) 397–405). We have now developed PCR diagnostic assays based on allele-specific amplification that are able to detect such mutations. The four tests described can reliably discriminate all of the mutations observed to alter codons 436, 581 and 613, yielding allele-specific amplification products of different sizes in each case. Moreover, by careful adjustment of primer length and the degree of mismatch to target and non-target alleles, we were able to standardise all four tests to a single set of PCR conditions, allowing all possible mutations to be monitored simultaneously on one thermocycler. These assays should prove invaluable in further assessing the contribution of specific base changes in the DHPS gene of the parasite to the sulphadoxine resistance phenotype and to the clinical failure of the sulphadoxine/pyrimethamine combination Fansidar.

We have developed a recombinant system that directs the functional expression from Escherichia coli of both dihydrofolate reductase-thymidylate synthetase (DHFR-TS) and the isolated DHFR domain from Plasmodium falciparum. Both products are inhibitory to a number of E. coli cell lines to the extent that cell growth ceases immediately upon induction. This dramatic inhibition is not seen in strain AB1899, in which amounts of plasmodial protein of up to 100 times the basal E. coli TS level can be accumulated. However, as well as the full-length DHFR-TS molecule, smaller proteins carrying an intact TS substrate-binding site are produced. These represent ca. 60-75% of the total plasmodial protein expressed and are observed in every E. coli strain examined. We show that they are not derived by degradation of the parent DHFR-TS molecule, but can be correlated with the sizes of proteins expected to be produced if erroneous initiation of translation were occurring at 3 internal methionine residues.

We report the isolation and sequencing of genomic clones encompassing the entire alpha-tubulin II gene from the human malaria parasite Plasmodium falciparum. This gene is closely related to, but significant different from the alpha-tubulin I gene that we have described previously. These two genes represent the entire complement of alpha-tubulin sequences in this organism and are expressed in a stage-specific manner. The alpha-II gene is present as a single copy and encodes a tubulin molecule with a predicted length of 450 amino acid residues (49.7 kDa). Like the alpha-I gene, it contains two introns, which are in identical positions to those of alpha-I, but are about one-third smaller. The deduced alpha-II protein is very similar to alpha-tubulin I (94.2% amino acid identity), except for notable differences across residues 40-45. In addition, unlike the great majority of alpha-tubulin genes (including alpha-I), alpha-II does not encode a terminal tyrosine residue. Using pulsed field gel electrophoresis we demonstrate that the two alpha-tubulin genes, together with the single beta-tubulin gene, are unlinked, all residing on different chromosomes. We assign alpha-I to chromosome 9, alpha-II to chromosome 4 and beta-tubulin to chromosome 10.

RNA was isolated from trophozoites, schizonts and mixed populations of Plasmodium falciparum. 5% of the total was poly(A+) message, of average length 1.2 kb (10-12 kb maximum) and a poly(A) content of 10%. The mRNA fractions could be translated in vitro by reticulocyte lysates supplemented either with exogenous or P. falciparum tRNA. The patterns from two independent isolates, one cloned (T9-94) and one uncloned (K1) were virtually identical. Major translation products from 16-230 kDa have been measured. The most abundant is lactate dehydrogenase (34.8 kDa). Trophozoite mRNA codes principally for proteins of less than or equal to 93 kDa, while schizont mRNA codes for additional proteins of higher molecular mass. There are marked similarities between the in vitro translation products and proteins synthesised in vivo in synchronous cultures. A number of schizont mRNA translation products (principally those of 230, 203, 185, 170, 115, 101 and 71 kDa) are specifically precipitated without post-translational modification by sera from humans exposed to malaria. A cDNA library has been constructed in phage lambda from total poly(A+) RNA and partially characterised. About 10% of the clones derive from abundant mRNA sequences. Putative actin clones have been isolated from this library and the parasite actin mRNA sized at approx. 2.8 kb.

We show that the l -(+)-lactate dehydrogenase (EC 1.1.1.27; l -lactate; NAD+-oxidoreductase) of Plasmodium falciparum (LDH-P) is encoded in the parasite genome. A monoclonal antibody (McAb 7.2) has been shown to bind the LDH-P subunit which has an apparent molecular mass of 35 kDa. A polyclonal antiserum raised against affinity purified LDH-P has been used to isolate cDNA clones containing LDH-P epitopes from a λgt11Tn5 expression library. DNA sequence analysis of one clone, λLDH-P.1, reveals a single open reading frame which shows a degree of homology to the N-terminal domain of known LDH amino acid sequences.

We have constructed a number of oligonucleotide probes and tested their utility in identifying various genes in Plasmodium falciparum. The probe sequences were based on known conserved regions of proteins from other organisms, coupled with an analysis of the codon usage of the parasite. By using long single oligonucleotides, we have successfully isolated the DHFR-TS gene, two actin genes and two tubulin genes from the K1 (Thailand) isolate of P. falciparum. We compare these single probes to multiply-redundant short oligonucleotide probes and to heterologous probes. We also present a detailed quantitative analysis of optimal probe design, and of how this approach can best be implemented as a general method of isolating plasmodial genes.

A detailed analysis of P190, a major surface associated protein of Plasmodium falciparum erythrocytic stages has been undertaken. We have demonstrated that this protein is recognised by two monoclonal antibodies, one of which recognises a constant feature (2.2) and one a variable feature (7.3). Cell free protein synthesis demonstrates that the variable 7.3 epitope is encoded in the structural gene for P190. The 7.3 epitope is only present on late trophozoites and schizonts whilst the 2.2 epitope is present on all erythrocytic stages. Labelling of synchronised cultures demonstrates that P190 is made only from 30 h onwards, (i.e. by trophozoites and schizonts). By pulse chase analysis we show that P190 undergoes processing and is lost at release/re-invasion, correlating with a lack of 7.3 immunofluorescence reactivity on rings. Sera from Nigeria recognise P190 from a Thai isolate of malaria. They also react with purified P190 in a micro-ELISA assay. A model for the role of P190 in re-invasion is presented, and the possible clinical significance of this protein is discussed.

Ribosomal RNA isolated from the malaria parasite Plasmodium falciparum consists of two species with molecular weight of 1.49 +/- 0.09 X 10(6) and 0.78 +/- 0.02 X 10(6) present in equimolar quantities. Their molecular weights are comparable with those of other protozoa but quite distinct from those of the human host. The overall base composition of the rRNA (40% G+C) is close to that of the rodent parasite Plasmodium berghei, unlike the latter, however, P. falciparum has no nick in the RNA from its large ribosomal subunit.

The DNA of Plasmodium falciparum has been purified and fragmented with the restriction endonucleases Eco RI and Hin dIII. The fragments have been incorporated in vitro into derivatives of bacteriophage λ to make libraries in which most of the parasite DNA is represented. By Southern hybridisation we have been able to recover from these libraries specific clones containing (a) repetitive DNA sequences, (b) rRNA gene(s) and (c) sequences homologous to an actin gene probe. Parasite DNA from two independent sources differs markedly in the pattern of its repetitive DNA visualised by hybridisation to our repetitive clone. By contrast, the rRNA genes of the two isolates prove to be carried on identically sized fragments.