The selectable marker human dihydrofolate reductase enables sequential genetic manipulation of the Plasmodium berghei genome

Genetic transformation of malaria parasites has been limited by the number of selectable markers available. For the rodent malaria parasite, Plasmodium berghei, only a single selection marker has been at hand, utilising the dihydrofolate reductase-thymidylate synthase gene from either P. berghei or Toxoplasma gondii to confer resistance to the anti-malarial drug pyrimethamine. Here we report the use of the human dihydrofolate reductase (hDHFR) gene as a new selectable marker, which confers resistance to the antifolate inhibitor WR99210 upon both pyrimethamine sensitive and resistant isolates of P. berghei. Transfection with circular constructs containing the hDHFR gene resulted in the generation of highly resistant parasites containing multiple copies of episomally-maintained plasmids. These parasites showed around a 1000-fold increase in resistance to WR99210 compared to the parental parasites. We were also able to generate and select transgenic parasites harbouring only a single copy of hDHFR targeted into their genome, despite the fact that these parasites showed only a fivefold increase in resistance to WR99210 compared to the parental parasites. Importantly, and for the first time with malaria parasites, the hDHFR gene could be used in conjunction with the existing pyrimethamine selectable markers. This was demonstrated by reintroducing the circumsporozoite (CS) gene into transgenic CS-knockout mutant parasites that contained the P. berghei DHFR-TS selectable marker. The development of hDHFR as a second selectable marker will greatly expand the use of transformation technology in Plasmodium, enabling more extensive genetic manipulation and thus facilitating more comprehensive studies on the biology of the malaria parasite.     

Puromycin-N-acetyltransferase as a selectable marker for use in Plasmodium falciparum

The limited number of selectable markers available for malaria transfection has hindered extensive manipulation of the Plasmodium falciparum genome and subsequently thorough genetic analysis of this organism. In this paper, we demonstrate that P. falciparum is highly sensitive to the drug puromycin, but that transgenic expression of the puromycin-N-acetyltransferase (PAC) gene from Streptomyces alboninger confers resistance to this drug with the IC(50) and IC(90) values increasing approximately 3- and 7-fold, respectively in PAC-expressing parasites. Despite this relatively low level of resistance, parasite populations transfected with the PAC selectable marker and selected directly on puromycin emerged at the same rate post-transfection as human dihydrofolate reductase (hDHFR)-expressing parasites, selected independently with the anti-folate drug WR99210. Transfected parasites generally maintained the PAC expression plasmid episomally at between two and six copies per parasite. We also demonstrate by cycling transfected parasites in the presence and absence of puromycin for several weeks, that the PAC selectable marker can be used for gene-targeting. Since the mode of action of puromycin is distinct from other drugs currently used for the stable transfection of P. falciparum, the PAC selectable marker should also have applicability for use in conjunction with other positive selectable markers, thereby increasing the possibilities for more complex functional studies of this organism.     

Green fluorescent protein as a marker in Plasmodium berghei transformation

We present a new marker that confers both resistance to pyrimethamine and green fluorescent protein-based fluorescence on the malarial parasite Plasmodium berghei. A single copy of the cassette integrated into the genome is sufficient to direct fluorescence in parasites throughout the life cycle, in both its mosquito and vertebrate hosts. Erythrocyte stages of the parasite that express the marker can be sorted from control parasites by flow cytometry. Pyrimethamine pressure is not necessary for maintaining the cassette in transformed parasites during their sporogonic cycle in mosquitoes, including when it is borne by a plasmid. This tool should thus prove useful in molecular studies of P. berghei, both for generating parasite variants and monitoring their behavior.     

Yeast dihydroorotate dehydrogenase as a new selectable marker for Plasmodium falciparum transfection

Genetic manipulation of Plasmodium falciparum in culture through transfection has provided numerous insights into the molecular and cell biology of this parasite. The procedure is rather cumbersome, and is limited by the number of drug-resistant markers that can be used for selecting transfected parasites. Here we report a new selectable marker that could allow multiple transfections. We have taken advantage of our finding that a critical function of the mitochondrial electron transport chain (mtETC) in the erythrocytic stages of P. falciparum is the regeneration of ubiquinone as co-substrate of dihydroorotate dehydrogenase (DHODH), and that transgenic P. falciparum expressing ubiquinone-independent DHODH from yeast (yDHODH) are resistant to all mtETC inhibitors. We assessed the possibility of using yDHODH as a positive selectable marker for transfections of P. falciparum, including its use in gene disruption strategies. We constructed a transfection vector designed for gene disruption, termed pUF-1, containing the yDHODH gene as the positive selection marker in combination with a previously described fused yeast cytosine deaminase-uracil phosphoribosyl transferase gene as a negative selection marker. Transfection of the D10 strain followed by selection with atovaquone yielded positively selected parasites containing the plasmid, demonstrating that yDHODH can be used as a selective marker. Atovaquone, however, could not be used for such selection with the Dd2 strain of P. falciparum. On the other hand, we demonstrated that yDHODH transgenic parasites could be selected in both strains by Plasmodium DHODH-specific triazolopyrimidine-based inhibitors. Thus, selection with DHODH inhibitors was superior in that it successfully selected transgenic Dd2 parasites, as well as yielded transgenic parasites after a shorter period of selection. As a proof of concept, we have successfully disrupted the type II vacuolar proton-pumping pyrophosphatase gene (PfVP2) in P. falciparum by double crossover recombination, showing that this gene is not essential for the survival of blood stage parasites.     

Transformation of Rhizopus niveus using a bacterial blasticidin S resistance gene as a dominant selectable marker

Summary Rhizopus niveus has been transformed to blasticidin S resistance by vectors containing the bacterial blasticidin S resistance gene under the control of a Rhizopus promoter. Southern analysis of the total DNA from transformants indicated that the introduced DNA was rearranged, and that one of the transformants harbored extrachromosomal plasmids with rearranged DNA. Using this transformation system, the introduction of pUBSR101, a plasmid carrying the Escherichia coli lacZ gene fused to the promoter and the N-terminal region of the R. niveus aspartic proteinase-II (RNAP-II) gene, resulted in an increase of -galactosidase activity in the cell extract, indicating expression of the lacZ fusion gene in R. niveus. This is the first report of a transformation system for filamentous fungi using the blasticidin S resistance gene as a dominant selectable marker.     

A marker epitope of attenuated Plasmodium berghei

Plasmodium berghei XAT is an irradiation-induced, permanent attenuated derivative from high-virulence P. berghei NK65. Monoclonal antibodies against XAT were developed. By immunofluorescence screening, one monoclonal antibody was identified that was reactive with XAT at the schizont stage but not with NK65 nor with any other stage of intra-erythrocytic development of either parasite. The monoclonal antibody precipitated a 240-kD molecule from metabolically labeled XAT antigens. This molecule was thought to be a marker epitope of the attenuated parasite.     

Ape Plasmodium parasites as a source of human outbreaks

Recent studies have revealed a remarkable molecular diversity of Plasmodium parasites in great apes in Africa, as well as parasite exchange events between these primates and humans. We review the different points of view proposed on the origin of human malaria, and discuss ape Plasmodium parasites as a source of human outbreaks.     

Extensive heterozygosity at four microsatellite loci flanking Plasmodium vivax dihydrofolate reductase gene

Only limited but contrasting reports are available on microsatellites based population structure of Plasmodium vivax. Further, there is complete lack of information on microsatellites in the flanking regions of the P. vivax drug resistance genes to trace the origin and spread of the drug resistant vivax malaria. Therefore, we scanned +/-300 kb flanking sequences of the P. vivax dihydrofolate reductase (pvdhfr) gene for di-nucleotide microsatellite repeats with minimum of 8 unit array length. Only 13 such repeats were detected in this region as compared to 738 di-nucleotide repeats present in +/-300 kb flanking region of P. falciparumdhfr gene. We have analyzed here the nucleotide sequence of 110 Indian P. vivax isolates for four of these di-nucleotide microsatellites (two in the nearest regions at -38.83 kb and +6.15 kb, and two in the farthest regions at -230.54 kb and +283.28 kb). All the four microsatellites were found to be highly polymorphic in the population where number of alleles varied from 4 to 10 with the median values of 9-11 at these loci. The expected heterozygosity (He) at these loci ranged from 0.50 to 0.82. We did not find any association between pvdhfr mutations and the flanking microsatellite alleles. There was a regional variation in the microsatellites polymorphism which was not associated with the reported prevalent rates of drug resistance or malaria transmission. In conclusion, the level of microsatellite polymorphism in P. vivax is as high as in P. falciparum. These results will be valuable in understanding the evolutionary history of the pvdhfr alleles as well as for designing the malaria control strategies.     

Allelic polymorphism in the Plasmodium vivax dihydrofolate reductase gene among Indian field isolates

In total, 129 Plasmodium vivax isolates from different geographical areas in India were analysed for point mutations in the P. vivax dihydrofolate reductase gene that were associated with pyrimethamine resistance. A gradual increase in the frequency of mutant genotypes was observed from north to south (p <0.0001). In the northern region (Delhi, Panna and Nadiad), the wild-type genotype was most prevalent, while the mutant genotype predominated in the coastal regions of southern India (Navi Mumbai, Goa and Chennai). Isolates from the Car-Nicobar islands showed only mutant genotypes. The differential geographical pattern of mutations may be associated with the transmission pattern.     

Cloning of the nitrate reductase gene of Stagonospora (Septoria) nodorum and its use as a selectable marker for targeted transformation

The nitrate reductase gene (NIA1) of the phytopathogenic fungus Stagonospora (Septoria) nodorum has been cloned from a cosmid library by homologous hybridisation with a PCR-generated probe. A 6.7-kb fragment carrying the NIA1 gene was subcloned and partially characterised by restriction mapping. Sequencing of the gene indicated a high degree of homology, both at the nucleotide and amino-acid levels, with nitrate reductase genes of other filamentous fungi. Furthermore, consensus regulatory signals thought to be involved in the control of nitrogen metabolism are present in the 5′ flanking region. The cloned NIA1 gene has been used to develop a gene-transfer system based on nitrate assimilation. Stable nia1 mutants of S. nodorum defective in nitrate reductase were isolated by virtue of their resistance to chlorate. These were transformed back to nitrate utilisation with the wild-type S. nodorum NIA1 gene. Southern analyses revealed that transformation occurred as a result of the integration of transforming DNA into the fungal genome; in all cases examined, integration was targeted to the homologous sequence. Received: 30 March / 9 June 1998     

Analysis of dihydrofolate reductase gene amplification in a methotrexate-resistant human tumor cell line

Detailed cytogenetic and molecular biologic studies have been performed on the human KB tumor cell line and four methotrexate-resistant subclones. Results are presented, demonstrating that the gene encoding the target enzyme dihydrofolate reductase is increasingly amplified in progressively methotrexate-resistant subclones, and that dihydrofolate reductase sequences are localized to a homogeneously staining region on chromosome 10q.     

Genetic variations of the dihydrofolate reductase gene of Plasmodium vivax in Mandalay Division, Myanmar

Dihydrofolate reductase (DHFR; EC1.5.1.3) is a known target enzyme for antifolate agents, which are used as alternative chemotherapeutics for chloroquine-resistant malaria. Mutations in the dhfr gene of Plasmodium vivax are thought to be associated with resistance to the antifolate drugs. In this study, we have analyzed genetic variations in the dhfr genes of clinical isolates of P. vivax (n=21) in Myanmar, to monitor antifolate resistance in this country. Sequence variations within the entire dhfr gene were highly restricted to codons from 57 to 117, and the GGDN tandem repeat region. Double (S58R and S117N/T) or quadruple mutations (F57L/I, S58R, T61M, and S117N/T), which may be closely related to the drug resistance, were recognized in most of the isolates (20/21 cases). Our results suggest that antifolate-resistant P. vivax is becoming widespread in Myanmar, as it also is in the neighboring countries in Southeast Asia. It appears that the drug resistance situation may be worsening in the country.Byoung-Kuk Na and Hyeong-Woo Lee have contributed equally to the work.     

Antifolate screening using yeast expressing Plasmodium vivax dihydrofolate reductase and in vitro drug susceptibility assay for Plasmodium falciparum

The presence of homologous point mutations in the dhfr gene in Plasmodium vivax and Plasmodium falciparum is associated with resistance to antifolate drugs. The spread of antifolate resistance encouraged research for novel antifolate drugs active against both wild-type and dhfr-mutant strains of malaria parasites. Because P. vivax cannot be easily maintained in culture, we transformed a Saccharomyces cerevisiae DHFR-deleted mutant to express wild-type P. vivax dhfr gene and its mutant forms. Twenty-five dicyclic and tricyclic 2,4-diaminopyrimidine derivatives were screened. Six quinazoline compounds showed selective inhibition of yeast transformants expressing P. vivax dhfr genes. The 50% inhibitory concentration (IC(50)) of these six compounds was determined against field isolates of P. falciparum. Our results suggest that a close relationship between the yeast assay based on expression of P. vivax dhfr genes and the in vitro test using P. falciparum parasites in culture is a promising initial step for drug screening.     

Murine model for assessment of Plasmodium falciparum transmission-blocking vaccine using transgenic Plasmodium berghei parasites expressing the target antigen Pfs25

Currently, there is no animal model for Plasmodium falciparum challenge to evaluate malaria transmission-blocking vaccines based on the well-established Pfs25 target antigen. The biological activity of transmission-blocking antibodies is typically assessed using an assay known as the membrane feeding assay (MFA). It is an in vitro method that involves mixing antibodies with cultured P. falciparum gametocytes and feeding them to mosquitoes through an artificial membrane followed by assessment of infection in the mosquitoes. We genetically modified Plasmodium berghei to express Pfs25 and demonstrated that the transgenic parasites (TrPfs25Pb) are susceptible to anti-Pfs25 antibodies during mosquito-stage development. The asexual growth kinetics and mosquito infectivity of TrPfs25Pb were comparable to those of wild-type parasites, and TrPfs25Pb displayed Pfs25 on the surface of ookinetes. Immune sera from nonhuman primates immunized with a Pfs25-based vaccine when passively transferred to mice blocked transmission of TrPfs25Pb to Anopheles stephensi. Furthermore, mice immunized with Pfs25 DNA vaccine and challenged with TrPfs25Pb displayed reduced malaria transmission compared to mice immunized with wild-type plasmid. These studies describe development of an animal malaria model alternative to the in vitro MFA and show that the model can facilitate P. falciparum transmission-blocking vaccine evaluation based on the target antigen Pfs25. We believe that an animal model to test transmission-blocking vaccines would be superior to the MFA, since there may be additional immune factors that synergize the transmission-blocking activity of antibodies in vivo.     

Cloning of a yeast dihydrofolate reductase gene in Escherichia coli

Summary The dihydrofolate reductase gene of Saccharomyces cerevisiae has been isolated by selection of trimethoprim resistant Escherichia coli transformed with a gene bank of yeast DNA in plasmid pBR322. From 9.2 kilobase pair BamHI DNA fragment this gene has been localized to a 1.76 kb fragment, the restriction map of which appears different from those reported for the E. coli and the mouse dihydrofolate reductase genes.The enzyme encoded by the chimeric plasmid was established as yeast dihydrofolate reductase by its sensitivity to antifolates in vivo through growth studies and in vitro by enzyme assay. Since, the expression of this gene occurs independent of its orientation within the chimeric plasmid, the 1.76 kb fragment may contain functional regulatory sequences in addition to the structural sequences for yeast dihydrofolate reductase.This work was carried out in part at Merck & Co., Rahway, New Jersey, USA and at Southern Biotech, Inc., Tampa, Florida. USA     

Bialaphos resistance as a dominant selectable marker in Neurospora crassa

Summary Conidia of Neurospora crassa are sensitive to the herbicide bialaphos at concentrations of 160 mg/1 in Westergaard's or Fries' minimal media. Plasmid pJA4 was constructed by inserting a truncated bar gene from Streptomyces hygroscopicus fused to the his-3 promoter from N. crassa into pUC19. The bar gene in plasmid pJA4 confers resistance to bialaphos when transformants are selected on a medium containing bialaphos. The bar gene can be used as an additional dominant selectable marker for transformation of fungi.     

Cloning of the Aspergillus oryzae 5-aminolevulinate synthase gene and its use as a selectable marker

The hemA gene encoding 5-aminolevulinate synthase, the first enzyme in heme biosynthesis, was cloned from Aspergillus oryzae and evaluated as a selectable marker for the transformation of filamentous fungi. Deletion of the hemA gene resulted in a lethal phenotype that could be rescued either by the supplementation of culture media with 5-aminolevulinic acid (ALA) or by transformation with the wild-type hemA gene, but not by growth on rich media, nor by the addition of exogenous heme. Transformation of a hemA deletion strain with the hemA gene linked to a lipase expression cassette yielded ALA prototrophs expressing lipase. The hemA gene can therefore be used as a selectable marker for the transformation of A. oryzae. Received: 16 March 2000 / Accepted: 18 July 2000     

Phleomycin resistance as a dominant selectable marker in CHO cells

The Tn5 and the Streptoalloteichus hindustanus (Sh) ble genes conferring resistance to bleomycin-phleomycin antibiotics have been cloned into a mammalian vector under the RSV-LTR promoter. The resulting plasmids, pUT506 and pUT507 respectively, were used to transfect CHO cells by either the calcium phosphate or the recently described polybrene-DMSO method. Phleomycin- or bleomycin-resistant clones arose with a higher frequency after transfection with pUT507, and pUT507 transfectants were more resistant to both antibiotics than pUT506 transfectants. Phleomycin resistance in pUT507 transfectants was stable and associated with integration of plasmid sequences in genomic DNA. The Sh ble gene, which confers a dominant phleomycin-resistance phenotype, should provide a useful transferable selectable marker in CHO cells as well as in other animal cell lines.