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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.

The folate pathway represents a powerful target for combating rapidly dividing systems such as cancer cells, bacteria and malaria parasites. Whereas folate metabolism in mammalian cells and bacteria has been studied extensively, it is understood less well in malaria parasites. In two articles, we attempt to reconstitute the malaria folate pathway based on available information from mammalian and microbial systems, in addition to Plasmodium-genome-sequencing projects. In part I, we focused on folate enzymes that are already used clinically as anticancer drug targets or that are under development in drug-discovery programs. In this article, we discuss mammalian folate enzymes that have not yet been exploited as potential drug targets, and enzymes that function in the de novo folate-synthesis pathway of the parasite – a particularly attractive area of attack because of its absence from the mammalian host.

Food fortified with folic acid has been available for consumption in North America for over a decade. This strategy has led to an increase in folate levels in the general population and, more importantly, a significant decrease in the incidence of neural tube defects. However, this increase in folate intake has been associated with a greater risk of cancer disease. Many African countries are now embracing this concept; however, because folate promotes malaria parasite division, as it does in cancer cells, there is a possibility of malaria exacerbation if folate intake is increased. A precedent for such a concern is the now compelling evidence showing that an increase in iron intake can lead to a higher malaria risk; as a result, mass administration of iron in malaria-endemic areas is not recommended. In this article, we review work on the effect of folate on malaria parasites. Although this topic has received little research attention, the available data suggest that the increase in folate concentration could be associated with an increase in malaria infection. Thus, the introduction of food fortification with folic acid in malaria-endemic areas should be attended by precautionary programs to monitor the risk of malaria.

Malaria represents one of the most serious threats to human health worldwide, and preventing and curing this parasitic disease still depends predominantly on the administration of a small number of drugs whose efficacy is continually threatened and eroded by the emergence of drug-resistant parasite populations. This has an enormous impact on the mortality and morbidity resulting from malaria infection, especially in sub-Saharan Africa, where the lethal human parasite species Plasmodium falciparum accounts for approximately 90% of deaths recorded globally. Successful treatment of uncomplicated malaria is now highly dependent on artemisinin-based combination therapies. However, the first cases of artemisinin-resistant field isolates have been reported recently and potential replacement antimalarials are only in the developmental stages. Here, we summarize recent progress in tackling the problem of parasite resistance and discuss the underlying molecular mechanisms that confer resistance to current antimalarial agents as far as they are known, understanding of which should assist in the rational development of new drugs and the more effective deployment of older ones.

Evaluation of: Pearce RJ, Pota H, Evehe MSB et al.: Multiple origins and regional dispersal of resistant dhps in African Plasmodium falciparum malaria. PLoS Med. 6(4), e1000055 (2009). Widespread resistance to current antimalarial drugs is a major factor in the extremely high levels of mortality and disabling illness that still prevail in many developing countries. It is important to understand how frequently resistant malaria parasite strains arise and their patterns of propagation and dispersal across borders and continents. By studying the DNA sequences of both the gene encoding the drug target and its flanking regions, it is possible to collect and map such data, providing a considerable asset in devising and evaluating future strategies of drug use and deployment. In this article, Pearce et al. analyze a large number of parasite samples collected over a decade from countries across Africa, allowing them to present for the first time a detailed picture of the origins and relatively recent spread of resistance to sulfa-drugs, key components of antifolate drug combinations that have been used extensively as part of the antimalarial armory.

The apicomplexan pathogen Cryptosporidium parvum poses major logistical problems in the search for effective drug treatments. These treatments are required urgently because this parasite can cause severe disease and death in immunocompromised individuals and young children. In a recent study, the dependence of Cryptosporidium parasites on a single salvage pathway that leads to essential purine derivatives has been exploited and inhibitors have been identified that selectively target a key enzyme in this salvage process, inosine 5′-monophosphate dehydrogenase.

Despite intensive research extending back to the 1930s, when the first synthetic antimalarial drugs made their appearance, the repertoire of clinically licensed formulations remains very limited. Moreover, widespread and increasing resistance to these drugs contributes enormously to the difficulties in controlling malaria, posing considerable intellectual, technical and humanitarian challenges. A detailed understanding of the molecular mechanisms underlying resistance to these agents is emerging that should permit new drugs to be rationally developed and older ones to be engineered to regain their efficacy. This review summarizes recent progress in analysing the causes of resistance to the major antimalarial drugs and its spread.

The high hemeozoin (beta-hemeatin) content of Plasmodium falciparum lysates imposes severe limitations on the analysis of the malarial proteome, in particular compromising the loading capacities of two-dimensional gels. Here we report on the adaptation of a recently developed solution-phase isoelectric focusing-based fractionation technique as a prefractionation strategy for efficient containment of hemeoglobin-derived products and complexity reduction, to facilitate the high-resolution gel-based quantitative analysis of plasmodial lysates.

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.

Synthesis de novo, acquisition by salvage and interconversion of purines and pyrimidines represent the fundamental requirements for their eventual assembly into nucleic acids as nucleotides and the deployment of their derivatives in other biochemical pathways. A small number of drugs targeted to nucleotide metabolism, by virtue of their effect on folate biosynthesis and recycling, have been successfully used against apicomplexan parasites such as Plasmodium and Toxoplasma for many years, although resistance is now a major problem in the prevention and treatment of malaria. Many targets not involving folate metabolism have also been explored at the experimental level. However, the unravelling of the genome sequences of these eukaryotic unicellular organisms, together with increasingly sophisticated molecular analyses, opens up possibilities of introducing new drugs that could interfere with these processes. This review examines the status of established drugs of this type and the potential for further exploiting the vulnerability of apicomplexan human pathogens to inhibition of this key area of metabolism.

The lethal species of malaria parasite, Plasmodium falciparum, continues to exact a huge toll of mortality and morbidity, particularly in sub-Saharan Africa. Completion of the genome sequence of this organism and advances in proteomics and mass spectrometry have opened up unprecedented opportunities for understanding the complex biology of this parasite and how it responds to drug challenge and other interventions. This review describes recent progress that has been made in applying proteomics technology to this important pathogen and provides a look forward to likely future developments.

The glycine-cleavage complex (GCV) and serine hydroxymethyltransferase represent the two systems of one-carbon transfer that are employed in the biosynthesis of active folate cofactors in eukaryotes. Although the understanding of this area of metabolism in Plasmodium falciparum is still at an early stage, we discuss evidence that genes and transcription products of the GCV are present and expressed in this parasite. The potential role of the GCV and its relevance to the life cycle and pathogenesis of the malaria erythrocytic stages are also considered. According to its expression profile, the GCV seems to be particularly active in gametocytes. The GCV enzyme dihydrolipoamide dehydrogenase has two isoforms encoded by two different genes. It has been demonstrated recently that both genes are functional, with one of them identified as being part of a pyruvate dehydrogenase complex that is present exclusively in the apicoplast of Plasmodium species. The other isoform probably forms part of the Plasmodium GCV. The GCV is the first enzyme complex involved in folate metabolism in this parasite that can be assumed, with a good degree of certainty, to be located in the mitochondria.

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.

The ability to measure accurately comparative levels of protein expression after drug challenge, metabolic stress, developmental programming or other perturbation represents one of the most important goals in post-genomics malaria research. We describe here a simple and robust quantitative methodology that is ideally suited to in vitro experiments designed to study changes in the proteome of the most important of the human parasites, the lethal species Plasmodium falciparum. The metabolic labelling technique we have developed uses parasite uptake of heavy isotope-containing isoleucine during normal growth followed by two-dimensional separation of individual proteins and mass spectrometry. The method is applicable to essentially each of the approximately 5300 proteins of P. falciparum predicted from the completed genome sequence, permitting facile identification and accurate comparative quantification of labelled peptides from any of these proteins synthesized by in vitro cultures subjected to different stimuli. We demonstrate its application to the study of cell cycle changes, where we observe divergent patterns of protein and reported transcript levels indicative of modulation at the translational level. Our data also provide evidence for significant levels of post-translational modification in the parasite, and we measure differences among variants of phosphoethanolamine N-methyltransferase and actin-I across the cell cycle. We have also monitored parasite responses to equipotent doses of the clinical antimalarial inhibitors pyrimethamine and tetracycline and observed differential effects for a number of proteins unrelated to likely targets of these drugs.

Summary Folate metabolism in Plasmodium falciparum is the target of important antimalarial agents. The biosyn- thetic pathway converts GTP to polyglutamated deriv- atives of tetrahydrofolate (THF), essential cofactors for DNA synthesis. Tetrahydrofolate can also be acquired by salvage mechanisms. Using a transfec- tion system adapted to studying this pathway, we investigated modulation of dihydropteroate syn- thase (DHPS) activity on parasite phenotypes. Dihy- dropteroate synthase incorporates p- aminobenzoate (pABA) into dihydropteroate, the precursor of dihy- drofolate. We were unable to obtain viable parasites where the dhps gene had been truncated. However, parasites where the protein was full-length but mutated at two key residues and having < 10% of normal activity were viable in folate-supplemented medium. Metabolic labelling showed that these para- sites could still convert pABA to polyglutamated folates, albeit at a very low level, but they could not survive on pABA supplementation alone. This degree of disablement in DHPS also abolished the synergy of the antifolate combination pyrimethamine/sulfa- doxine. These data indicate that DHPS activity above a low but critical level is essential regardless of the availability of salvageable folate and formally prove the role of this enzyme in antifolate drug synergy and folate biosynthesis in vivo . However, we found no evidence of a significant role for DHPS in folate sal- vage. Moreover, when biosynthesis was compro- mised by the absence of a fully functional DHPS, the parasite was able to compensate by increasing flux through the salvage pathway.

Pyrimethamine acts against malarial parasites by selectively inhibiting their dihydrofolate reductase-thymidylate synthase. Resistance to pyrimethamine in Plasmodium falciparum is due to point mutations in the DHFR domain, initially at residue 108 (S108N), with additional mutations imparting much greater resistance. Our previous work, the development of a simple rational drug design strategy to overcome such resistance, used suitable meta-substituents in the pyrimethamine framework to avoid the unfavorable steric clash with mutant side chains at position 108. Interestingly, the meta-chloro analog of pyrimethamine not only overcame the resistance due to S108N, but also that contributed by the more remote mutation, C59R. The present work improves on this by means of other meta-substituents. Against wild type DHFR, double mutant types A16V + S108T and C59R + S108T, and the highly pyrimethamine/cycloguanil-resistant quadruple-mutant form N51I + C59R + S108N + I164L, pyrimethamine itself gave Ki values of 1.5, 2.4, 72.3 and 859 nM, respectively. The meta-substituted analogs, especially the meta-bromo analog, were much more powerful inhibitors of these DHFRs, including the quadruple-mutant form (meta-bromo analog, Ki 5.1 nM). For comparison, the dihydropyrazine antifolate, WR99210, gave Ki values of 0.9, 3.2, 0.8 and 0.9 nM, respectively. Ki values were also measured against recombinant human DHFR, as were their activities against the growth of Plasmodium falciparum cultures bearing the double mutations (FCB and K1 strains) and quadruple mutation (V1/S) and the wild type (3D7). The meta-analogs were highly active against all of these, with the meta-bromo again being the strongest, having an IC50 of 37 nM against V1/S, compared to >5000 nM for pyrimethamine itself and 1.1 nM for WR99210.

Folate metabolism in Plasmodium falciparum is essential for cell growth and replication, and the target of important antimalarial agents. The pathway comprises a series of enzymes that convert GTP to derivatives of tetrahydrofolate, which are cofactors in one-carbon transfer reactions. We investigated the expression of five of the genes encoding these enzymes by quantitative reverse transcription-polymerase chain reaction (qRT-PCR) using a threshold detection technique. We followed changes in mRNA levels as parasites progress through the erythrocytic cell cycle and examined this process in two cloned lines of diverse origins, as well as under stress conditions, induced by either removal of important metabolites or challenge by folate enzyme inhibitors. Although conventionally regarded as performing housekeeping functions, these genes show disparate levels of and changes in expression through the cell cycle, but respond quite uniformly to folate pathway-specific stress factors, with no evidence of feedback at the transcriptional level. Overall, the two genes involved in the thymidylate cycle (encoding dihy-drofolate reductase-thymidylate synthase, dhfr-ts, and serine hydroxymethyltransferase, shmt) gave the most abundant transcripts. However, only the latter showed major variation across the cell cycle, with a peak around the time of onset of DNA replication, possibly indicative of a regulatory function.

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.

Polymerase chain reaction amplification and DNA sequencing of the Toxoplasma gondii dihydropteroate synthase gene (dhps) identified 4 alleles among parasite populations from 32 cases of human toxoplasmosis. Heterologous expression and enzyme assay reveal that 3 of these alleles encode sulfadiazine (Sdz)-sensitive enzymes. The fourth, generating a highly Sdz-resistant enzyme, differs from 1 of the other 3 at only a single residue (407) of DHPS. Of interest, a fifth allele, found in a laboratory-induced Sdz-resistant line, also differs from another of these 3 drug-sensitive forms by the same single mutation that affects residue 407 of DHPS. Significantly, residues corresponding to DHPS-407 are implicated in sulfonamide resistance in other microorganisms. The human-derived allelic form encoding the Sdz-resistant enzyme was found in T. gondii associated with a fatal infection, and its presence within clinical material may have implications for sulfonamide use, particularly in cases of toxoplasmosis in which the initial response to drug treatment is poor.

Chemotherapy and chemoprophylaxis are the principal means of combating malaria parasite infections in the human host. In the last 75 years, since the introduction of synthetic antimalarials, only a small number of compounds have been found suitable for clinical usage, and this limited armoury is now greatly compromised by the spread of drug-resistant parasite strains. Our current knowledge of the molecular mechanisms underlying resistance in the lethal species Plasmodium falciparum is reviewed here.

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.

Although the folate metabolic pathway in malaria parasites is a major chemotherapeutic target, resistance to currently available antifolate drugs is an increasing problem. This pathway, however, includes a number of enzymes that, to date, have not been characterized despite their potential for clinical exploitation. As a step towards evaluation of additional targets in this pathway, we report the isolation and characterization of 3 new genes that encode homologues of GTP cyclohydrolase I (GTP-CH), dihydrofolate synthase/folylpolyglutamate synthase (DHFS/FPGS) and serine hydroxymethyltransferase (SHMT). The genes encoding GTP-CH and SHMT are unambiguously assigned to chromosome 12, while that for DHFS/FPGS is tentatively assigned to chromosome 13. All 3 genes are expressed in blood-stage parasites, yielding transcripts of which only ca 60–70% is accounted for by coding sequence. All 3 of the proteins predicted to be encoded by these genes display sequence differences compared to the human host homologues that may be of functional significance. These data bring the complement of cloned genes that encode activities in the pathway to seven, leaving only the gene encoding dihydroneopterin aldolase (DHNA) to be identified in the route from GTP to folate synthesis and folate turnover in the thymidylate cycle.

The antifolate combination pyrimethamine/sulphadoxine (PYR/SDX; Fansidar) is frequently used to combat chloroquine-resistant malaria. Its success depends upon pronounced synergy between the two components, which target dihydrofolate reductase (DHFR) and dihydropteroate synthetase (DHPS) in the folate pathway. This synergy permits clearance of parasites resistant to either drug alone, but its molecular basis is still unexplained. Plasmodium falciparum can use exogenous folate, which is normally present in vivo, bypassing SDX inhibition of DHPS and, apparently, precluding synergy under these conditions. However, we have measured parasite inhibition by SDX/PYR combinations in assays in which folate levels are strictly controlled. In parasites that use exogenous folate efficiently, SDX inhibition can be restored by levels of PYR significantly lower than those required to inhibit DHFR. Isobolograms show that the degree of synergy between PYR and SDX is highly dependent upon prevailing folate concentrations and are indicative of PYR acting to block folate uptake and/or utilization. No significant synergy was observed at physiological drug levels when PYR/SDX acted on purified DHFR, whether wild type or mutant. We conclude that the primary basis for antifolate synergy in these organisms arises from PYR targeting a site (or sites) in addition to DHFR, which restores DHPS as a relevant target for SDX.

A point mutation in codon 540 of the dihydropteroate synthetase (dhps) gene affecting sulfadoxine resistance has previously been found in parasites from patients with Plasmodium falciparum infection. Here, we investigated 4 methods of identifying this mutation in clinical specimens and established a reliable quantitative assay to estimate the percentage of resistant type in mixed infections. A diagnostic PCR assay based on allele-specific amplification was developed, which clearly typed the clinical specimens examined. The mutation in codon 540 introduces an additional FokI cleavage site which provided a second method to differentiate mutant from wild type, where the former gives rise to 2 characteristic fragments of 538 and 326 bp that are absent from the latter. To calibrate quantitatively the ratio of alleles in mixed samples, we constructed artificial mixes containing 2 plasmid DNAs, one carrying the mutation and the other a wild-type insert. When 32P-labelling was employed, the allele-specific PCR assay could detect the level of resistant type in a mixture down to 0.1-1%, while for the restriction enzyme/PCR analysis, the figure was approximately 10%. Furthermore, neither fluorescent dye-labelled terminator nor dye-labelled primer cycle sequencing was able to detect the mutant allele if it was present at less than 20-30%. We conclude that the allele-specific PCR assay is the most sensitive method of detecting the codon 540 mutation in P. falciparum dhps, and the method of choice for estimating the composition of mixed samples.

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.

Selected reaction monitoring mass spectrometry has been combined with the use of an isotopically labelled synthetic protein, made up of proteotypic tryptic peptides selected from parasite proteins of interest. This allows, for the first time, absolute quantification of proteins from Plasmodium falciparum. This methodology is demonstrated to be of sufficient sensitivity to quantify, even within whole cell extracts, proteins of low abundance from the folate pathway as well as more abundant "housekeeping" proteins.

Background The folate pathway enzyme serine hydroxymethyltransferase (SHMT) converts serine to glycine and 5,10-methylenetetrahydrofolate and is essential for the acquisition of one-carbon units for subsequent transfer reactions. 5,10-methylenetetrahydrofolate is used by thymidylate synthase to convert dUMP to dTMP for DNA synthesis. In Plasmodium falciparum an enzymatically functional SHMT (PfSHMTc) and a related, apparently inactive isoform (PfSHMTm) are found, encoded by different genes. Here, patterns of localization of the two isoforms during the parasite erythrocytic cycle are investigated. Methods Polyclonal antibodies were raised to PfSHMTc and PfSHMTm, and, together with specific markers for the mitochondrion and apicoplast, were employed in quantitative confocal fluorescence microscopy of blood-stage parasites. Results As well as the expected cytoplasmic occupancy of PfSHMTc during all stages, localization into the mitochondrion and apicoplast occurred in a stage-specific manner. Although early trophozoites lacked visible organellar PfSHMTc, a significant percentage of parasites showed such fluorescence during the mid-to-late trophozoite and schizont stages. In the case of the mitochondrion, the majority of parasites in these stages at any given time showed no marked PfSHMTc fluorescence, suggesting that its occupancy of this organelle is of limited duration. PfSHMTm showed a distinctly more pronounced mitochondrial location through most of the erythrocytic cycle and GFP-tagging of its N-terminal region confirmed the predicted presence of a mitochondrial signal sequence. Within the apicoplast, a majority of mitotic schizonts showed a marked concentration of PfSHMTc, whose localization in this organelle was less restricted than for the mitochondrion and persisted from the late trophozoite to the post-mitotic stages. PfSHMTm showed a broadly similar distribution across the cycle, but with a distinctive punctate accumulation towards the ends of elongating apicoplasts. In very late post-mitotic schizonts, both PfSHMTc and PfSHMTm were concentrated in the central region of the parasite that becomes the residual body on erythrocyte lysis and merozoite release. Conclusions Both PfSHMTc and PfSHMTm show dynamic, stage-dependent localization among the different compartments of the parasite and sequence analysis suggests they may also reversibly associate with each other, a factor that may be critical to folate cofactor function, given the apparent lack of enzymic activity of PfSHMTm.

The malaria parasite Plasmodium falciparum depends primarily on nutrient sources from its human host. Most compounds, such as glucose, purines, amino acids, as well as cofactors and vitamins, are abundantly available in the host cell, and can be readily salvaged by the parasite. However, in some cases the parasite can also synthesize cofactors de novo in reactions that appear to be essential. Importantly, the three biosynthetic pathways that produce vitamins B 1 , B 6 and B 9 are absent from the host, but are well established in P. falciparum . This review summarizes and updates the current knowledge of vitamin B de novo synthesis and salvage in P. falciparum and focuses on their potential as targets for drug intervention.

Dihydroneopterin aldolase (FolB) catalyzes conversion of dihydroneopterin to 6-hydroxymethyldihydropterin (HMDHP) in the classical folate biosynthesis pathway. However, folB genes are missing from the genomes of certain bacteria from the phyla Chloroflexi , Acidobacteria , Firmicutes , Planctomycetes , and Spirochaetes . Almost all of these folB -deficient genomes contain an unusual paralog of the tetrahydrobiopterin synthesis enzyme 6-pyruvoyltetrahydropterin synthase (PTPS) in which a glutamate residue replaces or accompanies the catalytic cysteine. A similar PTPS paralog from the malaria parasite Plasmodium falciparum is known to form HMDHP from dihydroneopterin triphosphate in vitro and has been proposed to provide a bypass to the FolB step in vivo. Bacterial genes encoding PTPS-like proteins with active-site glutamate, cysteine, or both residues were accordingly tested together with the P. falciparum gene for complementation of the Escherichia coli folB mutation. The P. falciparum sequence and bacterial sequences with glutamate or glutamate plus cysteine were active; those with cysteine alone were not. These results demonstrate that PTPS paralogs with an active-site glutamate (designated PTPS-III proteins) can functionally replace FolB in vivo. Recombinant bacterial PTPS-III proteins, like the P. falciparum enzyme, mediated conversion of dihydroneopterin triphosphate to HMDHP, but other PTPS proteins did not. Neither PTPS-III nor other PTPS proteins exhibited significant dihydroneopterin aldolase activity. Phylogenetic analysis indicated that PTPS-III proteins may have arisen independently in various PTPS lineages. Consistent with this possibility, merely introducing a glutamate residue into the active site of a PTPS protein conferred incipient activity in the growth complementation assay, and replacing glutamate with alanine in a PTPS-III protein abolished complementation.

The geographic distribution of the following enzyme systems is described in the rat heart (left and right ventricles) and in different skeletal muscles (soleus, plantaris, and red and white gastrocnemius): xanthine oxidase and dehydrogenase, creatine kinase isoenzymes, lactate dehydrogenase isoenzymes, and the free radical scavenger enzymes superoxide dismutase, glutathione reductase, and glutathione peroxidase. No substantial difference in enzyme activities was observed between the left and right ventricles. Skeletal muscles showed a clear distinction between enzyme activities depending on their composition of oxidative fibers and glycolytic fibers.

Folate metabolism in malaria parasites is a long-standing, clinical target for chemotherapy and prophylaxis. However, despite determination of the complete genome sequence of the lethal species Plasmodium falciparum, the pathway of de novo folate biosynthesis remains incomplete, as no candidate gene for dihydroneopterin aldolase (DHNA) could be identified. This enzyme catalyses the third step in the well-characterized pathway of plants, bacteria, and those eukaryotic microorganisms capable of synthesizing their own folate. Utilizing bioinformatics searches based on both primary and higher protein structures, together with biochemical assays, we demonstrate that P. falciparum cell extracts lack detectable DHNA activity, but that the parasite possesses an unusual orthologue of 6-pyruvoyltetrahydropterin synthase (PTPS), which simultaneously gives rise to two products in comparable amounts, the predominant of which is 6-hydroxymethyl-7,8-dihydropterin, the substrate for the fourth step in folate biosynthesis (catalysed by 6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase; PPPK). This can provide a bypass for the missing DHNA activity and thus a means of completing the biosynthetic pathway from GTP to dihydrofolate. Supported by site-directed mutagenesis experiments, we ascribe the novel catalytic activity of the malarial PTPS to a Cys to Glu change at its active site relative to all previously characterized PTPS molecules, including that of the human host.

Recently, Plasmodium falciparum bearing dihydrofolate reductase (DHFR) I164L was isolated from Africa. Quadruple mutations containing I164L confer high-level resistance to antifolate antimalarials. We prospectively measured the effect of co-trimoxazole (CTX) prophylaxis on P. falciparum antifolate resistance development among HIV-infected persons. HIV-positive patients with CD4 cell count350 cells/microl (n=692) received CTX; HIV-positive patients with CD4 cell countor = 350 cells/microl (n=336) and HIV-negative patients (n=132) received multivitamins. Malaria microscopy-positive samples (n=413) and selected microscopy-negative/PCR-positive samples (n=76) were analysed for DHFR mutations at baseline and during six months follow up. We identified I164L in 14 patients. Seven were malaria microscopy-positive: two failed sulfadoxine-pyrimethamine (SP). Among seven microscopy-negative/PCR-positive patients, none developed patent infections with I164L. I164L was not associated with high-level SP resistance or poor outcome among adults living where malaria is highly endemic. Surveillance to monitor spread of I164L is critical, especially among children and pregnant women, who are potentially a source for I164L amplification.

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.

Resistance to antifolate drugs such as pyrimethamine is widespread among malaria parasites of the most pathogenic species Plasmodium falciparum. These drugs inhibit the dihydrofolate reductase activity of the dihydrofolate reductase-thymidylate synthetase (DHFR-TS) bifunctional enzyme. This review examines work done to characterize the enzyme, the cloning of plasmodial DHFR-TS genes, chromosomal mapping studies of these genes by pulsed-field gel electrophoresis, and the structural insights into the mechanism of drug resistance that have been gained by comparing genes from drug-sensitive parasites with those from drug-resistant strains that have arisen in the field or after the experimental induction.

Despite intensive research extending back to the 1930s, when the first synthetic antimalarial drugs made their appearance, the repertoire of clinically licensed formulations remains very limited. Moreover, widespread and increasing resistance to these drugs contributes enormously to the difficulties in controlling malaria, posing considerable intellectual, technical and humanitarian challenges. A detailed understanding of the molecular mechanisms underlying resistance to these agents is emerging that should permit new drugs to be rationally developed and older ones to be engineered to regain their efficacy. This review summarizes recent progress in analysing the causes of resistance to the major antimalarial drugs and its spread.

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.

In the past 21 years, a modest increase in the range of antimalarial drugs approved for clinical use has been complemented by a more impressive expansion in the analysis and understanding of the molecular mechanisms underlying resistance to these agents. Such resistance is a major factor in the increasing difficulty in controlling malaria, and important developments during this period are recounted here.

New inhibitors are urgently needed to overcome the burgeoning problem of drug resistance in the treatment of Plasmodium falciparum infection. Targeting the folate pathway has proved to be a powerful strategy for drug development against rapidly multiplying systems such as cancer cells and microorganisms. Antifolates have long been used for malaria treatment but, despite their success, much less is known about parasite folate metabolism than about that of the human host. In this article, we focus on folate enzymes used clinically as anticancer drug targets, in addition to those that have potential to be used as drug targets, for which there are inhibitors at various stages of development. We discuss how this information could lead to the identification of new targets in malaria parasites.

The ability to measure accurately comparative levels of protein expression after drug challenge, metabolic stress, developmental programming or other perturbation represents one of the most important goals in post-genomics malaria research. We describe here a simple and robust quantitative methodology that is ideally suited to in vitro experiments designed to study changes in the proteome of the most important of the human parasites, the lethal species Plasmodium falciparum. The metabolic labelling technique we have developed uses parasite uptake of heavy isotope-containing isoleucine during normal growth followed by two-dimensional separation of individual proteins and mass spectrometry. The method is applicable to essentially each of the approximately 5300 proteins of P. falciparum predicted from the completed genome sequence, permitting facile identification and accurate comparative quantification of labelled peptides from any of these proteins synthesized by in vitro cultures subjected to different stimuli. We demonstrate its application to the study of cell cycle changes, where we observe divergent patterns of protein and reported transcript levels indicative of modulation at the translational level. Our data also provide evidence for significant levels of post-translational modification in the parasite, and we measure differences among variants of phosphoethanolamine N-methyltransferase and actin-I across the cell cycle. We have also monitored parasite responses to equipotent doses of the clinical antimalarial inhibitors pyrimethamine and tetracycline and observed differential effects for a number of proteins unrelated to likely targets of these drugs.

Folate metabolism in Plasmodium falciparum is the target of important antimalarial agents. The biosynthetic pathway converts GTP to polyglutamated derivatives of tetrahydrofolate (THF), essential cofactors for DNA synthesis. Tetrahydrofolate can also be acquired by salvage mechanisms. Using a transfection system adapted to studying this pathway, we investigated modulation of dihydropteroate synthase (DHPS) activity on parasite phenotypes. Dihydropteroate synthase incorporates p-aminobenzoate (pABA) into dihydropteroate, the precursor of dihydrofolate. We were unable to obtain viable parasites where the dhps gene had been truncated. However, parasites where the protein was full-length but mutated at two key residues and having10% of normal activity were viable in folate-supplemented medium. Metabolic labelling showed that these parasites could still convert pABA to polyglutamated folates, albeit at a very low level, but they could not survive on pABA supplementation alone. This degree of disablement in DHPS also abolished the synergy of the antifolate combination pyrimethamine/sulfadoxine. These data indicate that DHPS activity above a low but critical level is essential regardless of the availability of salvageable folate and formally prove the role of this enzyme in antifolate drug synergy and folate biosynthesis in vivo. However, we found no evidence of a significant role for DHPS in folate salvage. Moreover, when biosynthesis was compromised by the absence of a fully functional DHPS, the parasite was able to compensate by increasing flux through the salvage pathway.

We sought to determine the SAG2 genotypes of Toxoplasma gondii associated with cases of acute human toxoplasmosis in England and Wales. The samples examined were collected from a wide range of cases including congenital infections, AIDS and immunosuppressed patients and were derived from a number of different tissues. Parasite DNA was detected by PCR amplification without the need for prior template purification, and SAG2 genotype was determined by both restriction enzyme analysis and direct DNA sequencing of the PCR amplification products. Parasites of both SAG2 type I and type II genotypes were seen with approximately equal frequency amongst the samples examined. Neither of these genotypes was found to be more frequently associated with a particular clinical presentation or sample tissue. Unexpectedly, we found clear evidence of mixed (SAG2 type I+type II) infections in approximately the same number of samples as were seen to be associated with either type I or II alone. Our use of direct DNA sequencing rather than simple restriction analysis was essential for the detection of mixed infections since incomplete restriction digestion of samples containing a single parasite type was occasionally observed. It is possible that the presence of more than one type of parasite in single samples might be related to our recent demonstration that mixtures of SAG2 type I and type II parasites are present in a significant proportion of commercial meat preparations. Moreover, the presence of mixed infections in single patients might offer a direct molecular method of assessing risk factors for infection.

Folate metabolism in Plasmodium falciparum is essential for cell growth and replication, and the target of important antimalarial agents. The pathway comprises a series of enzymes that convert GTP to derivatives of tetrahydrofolate, which are cofactors in one-carbon transfer reactions. We investigated the expression of five of the genes encoding these enzymes by quantitative reverse transcription-polymerase chain reaction (qRT-PCR) using a threshold detection technique. We followed changes in mRNA levels as parasites progress through the erythrocytic cell cycle and examined this process in two cloned lines of diverse origins, as well as under stress conditions, induced by either removal of important metabolites or challenge by folate enzyme inhibitors. Although conventionally regarded as performing housekeeping functions, these genes show disparate levels of and changes in expression through the cell cycle, but respond quite uniformly to folate pathway-specific stress factors, with no evidence of feedback at the transcriptional level. Overall, the two genes involved in the thymidylate cycle (encoding dihy-drofolate reductase-thymidylate synthase, dhfr-ts, and serine hydroxymethyltransferase, shmt) gave the most abundant transcripts. However, only the latter showed major variation across the cell cycle, with a peak around the time of onset of DNA replication, possibly indicative of a regulatory function.