Dihydrofolate Reductase-thymidylate Synthase Research Articles

The origin of the bifunctional dihydrofolate reductasethymidylate synthase isogenes of Arabidopsis thaliana

SummaryIn most prokaryotic and eukaryotic organisms dihydrofolate reductase (DHFR) and thymidylate synthase (TS) are encoded by independent genes. Evidence is presented here that the higher plant Arabidopsis thaliana has two bifunctional DHFR‐TS genes. The structure of the genes, DHFR at the amino terminus and TS at the carboxy terminus, is identical to their organization in protozoa, the only other known organisms with bifunctional genes. Sequence alignments suggest that the bifunctional genes from protozoa and higher plants may have different evolutionary origins. The positions of the introns support the complementary hypothesis that the DHFR domain of the bifunctional plant genes and the monofunctional DHFR gene of vertebrates derive from a common, intron‐containing progenitor, although the structure (bifunctional or monofunctional) of the ancestral gene remains indeterminate. Comparison of the two bifunctional genes of Arabidopsis indicates that the DHFR and TS domains evolved at different rates; each following the evolutionary history of their monofunctional counterparts. In contrast to the DHFR domain, the evolution of the TS domain shows a higher level of nucleotide and amino acid sequence conservation, but a remarkable variability in the intron positions.

The origin of the bifunctional dihydrofolate reductase-thymidylate synthase isogenes of Arabidopsis thaliana.

In most prokaryotic and eukaryotic organisms dihydrofolate reductase (DHFR) and thymidylate synthase (TS) are encoded by independent genes. Evidence is presented here that the higher plant Arabidopsis thaliana has two bifunctional DHFR-TS genes. The structure of the genes, DHFR at the amino terminus and TS at the carboxy terminus, is identical to their organization in protozoa, the only other known organisms with bifunctional genes. Sequence alignments suggest that the bifunctional genes from protozoa and higher plants may have different evolutionary origins. The positions of the introns support the complementary hypothesis that the DHFR domain of the bifunctional plant genes and the monofunctional DHFR gene of vertebrates derive from a common, intron-containing progenitor, although the structure (bifunctional or monofunctional) of the ancestral gene remains indeterminate. Comparison of the two bifunctional genes of Arabidopsis indicates that the DHFR and TS domains evolved at different rates; each following the evolutionary history of their monofunctional counterparts. In contrast to the DHFR domain, the evolution of the TS domain shows a higher level of nucleotide and amino acid sequence conservation, but a remarkable variability in the intron positions.

Primary structure of the dihydrofolate reductase-thymidylate synthase gene from Toxoplasma gondii.

We have determined the primary genomic and cDNA sequences encoding the bifunctional dihydrofolate reductase-thymidylate synthase (DHFR-TS) enzyme of the protozoan parasite Toxoplasma gondii (dihydrofolate reductase, EC 1.5.1.3; thymidylate synthase EC 2.1.1.45). The DHFR-TS gene of T. gondii (strain RH) spans more than 6 kilobases of genomic DNA. Unlike the DHFR-TS genes of other protists, sequences encoding the Toxoplasma protein are interrupted by numerous intervening sequences. Analysis of processed T. gondii DHFR-TS cDNAs reveals a single open reading frame of 1830 nucleotides, predicting a 610-amino acid protein of molecular mass of 69 kilodaltons. Because its nucleotide composition and codon usage are roughly comparable to those observed in "higher" eukaryotes, the Toxoplasma DHFR-TS sequence is particularly useful for assessing evolutionary relationships between eukaryotic species. The predicted amino acid sequence for the DHFR-TS protein shows conservation of the major structural features identified in other DHFR and TS enzymes, while revealing certain differences which may be exploited for the design of novel antifolates for treatment of toxoplasmosis associated with AIDS.

Plasticity in chromosome number and testing of essential genes in Leishmania by targeting.

We attempted to generate homozygous dhfr-ts (dihydrofolate reductase-thymidylate synthase) knockouts in virulent Leishmania major, an asexual diploid protozoan parasite. Transfection of a neo (neomycin phosphotransferase) targeting fragment yielded heterozygous replacement lines with high efficiency. However, second transfections with a hyg (hygromycin B phosphotransferase) targeting fragment in the presence of metabolites shown to rescue homozygous knockouts in attenuated Leishmania did not yield the expected dhfr-ts- thymidine auxotrophs obtained previously with attenuated lines. Molecular karyotype, Southern blot, and flow cytometric DNA content analysis of clonal transfectants revealed three classes: (i) genomic tetraploids, containing two wild-type dhfr-ts chromosomes and one neo and one hyg replacement chromosome; (ii) aneuploid trisomic lines with one wild-type dhfr-ts and one neo and one hyg replacement chromosome; (iii) diploids bearing homologous integration of the targeting fragment without replacement. Aneuploid and tetraploid lines predominated. This confirms the common impression that natural populations of Leishmania are often aneuploid. The remarkable ability of these parasites to undergo and tolerate changes in chromosome number suggests a general method for testing whether genes are essential for growth in vitro, as the ability of Leishmania to simultaneously undergo homologous gene replacement while retaining wild-type genes by increasing chromosome number provides a diagnostic and positive experimental result. Our results show that virulent Leishmania require at least one copy of dhfr-ts and argue that DHFR-TS plays an unanticipated role in addition to its role in the de novo synthesis of thymidine. These results also have implications for genetic tests of the organization of Leishmania populations.

A member of the aldoketo reductase family confers methotrexate resistance in Leishmania.

Methotrexate (MTX)-resistant mutants of the parasitic protozoan Leishmania have been used as models for the mechanism and genetic basis of drug resistance in trypanosomatids and other cells. Three resistance mechanisms to MTX, a dihydrofolate reductase inhibitor, have been described in Leishmania: decreased uptake and accumulation of MTX via the folate/MTX transporter, amplification and overexpression of the dihydrofolate reductase-thymidylate synthase gene, and extrachromosomal amplification of H region DNA. We have now identified hmtxr as the H region gene conferring MTX resistance using a transfection-based approach. Data base searches show that the predicted HMTXr protein is related to members of the polyol dehydrogenase/carbonyl reductase family of aldoketo reductases, whose substrates include polyols, quinones, steroids, prostaglandins, fatty acids, and pterins. We therefore propose that HMTXr is also an oxidoreductase and suggest several biochemical mechanisms of resistance in Leishmania that could be exploited in the design of parasite-specific inhibitors.

Antimalarial activities of oligodeoxynucleotide phosphorothioates in chloroquine-resistant Plasmodium falciparum.

Synthetic oligonucleotides and their chemical modifications have been shown to inhibit viral and cellular gene expression by sequence-specific antisense hybridization to target mRNAs. We now report that oligodeoxynucleotide phosphorothioates and their nuclease-resistant modifications are effective in micromolar and submicromolar concentrations against the growth of both chloroquine-resistant and chloroquine-sensitive strains of Plasmodium falciparum in vitro. Parasitized human erythrocytes were found to be accessible to radioactively labeled oligodeoxynucleotides, whereas the uninfected erythrocytes did not permit any cellular entry of the same compounds. The dihydrofolate reductase-thymidylate synthase gene of P. falciparum was demonstrated to be a good target for sequence-dependent inhibition of plasmodial growth by exogenously administered modified oligonucleotides. The antimalarial activities observed in vitro were identical for chloroquine-sensitive and chloroquine-resistant strains of P. falciparum. The antimalarial activity of oligodeoxynucleotide phosphorothioates is related to sequence complementarity to certain regions of the plasmodial genome as well as to non-sequence-defined activities.

An examination of the mitomycin C induction of chromosome polymorphisms in cultures of Plasmodium falciparum

Cloned parasites of the FCR3 strain of Plasmodium falciparum that survived treatment with either mitomycin C or ethidium bromide were grown and subloned. The chromosomes of 10 cloned isolates from each population were analysed by contour-clamped homogenous electric field gel electrophoresis. Eight of 10 isolates from the mitomycin C-treated population contained a detectable polymorphic chromosome change while none of the isolates from the ethidium bromide-treated population did. One of the polymorphic changes involved chromosome #4. A pyrimethamine resistant derivative of FCR3, FCR3-D5, that had previously been shown to contain a single nucleotide change in the dihydrofolate reductase-thymidylate synthase (DHFR-TS) gene but no detectable chromosome polymorphisms, was sequentially treated with mitomycin C and a higher concentration of pyrimethamine than the strain had previously been shown to be resistant to in order to determine if there was a correlation between the selection of chromosome #4 polymorphisms and the applied selective pressure. Most of the cloned survivors of this treatment were found to contain chromosome polymorphisms that involved chromosome #4, the chromosome on which the DHFR-TS gene is located. The polymorphic changes were usually different in the different isolates. The selection of mitomycin C-treated, pyrimethamine resistant strains with chromosome #4 polymorphisms will be discussed.

Double targeted gene replacement for creating null mutants.

We have used double gene targeting to create homozygous gene replacements in the protozoan parasite Leishmania major, an asexual diploid. This method uses two independent selectable markers in successive rounds of gene targeting to replace both alleles of an endogenous gene. We developed an improved hygromycin B-resistance cassette encoding hygromycin phosphotransferase (HYG) for use as a selectable marker for Leishmania. HYG-containing vectors functioned equivalently to those containing the neomycin phosphotransferase (NEO) cassette previously used for extrachromosomal transformation or gene targeting. Drug resistances conferred by the NEO and HYG markers were independent, allowing simultaneous selection for both markers. A HYG targeting vector was utilized to replace the single dihydrofolate reductase-thymidylate synthase (DHFR-TS) gene remaining in a line heterozygous for a NEO replacement at the dhfr-ts locus (+/neo), with a targeting efficiency comparable to that seen with wild-type recipients. The resultant dhfr-ts- line (hyg/neo) was auxotrophic for thymidine. The double targeted replacement method will enable functional genetic testing in a variety of asexual diploids, including cultured mammalian cells and fungi such as Candida albicans. Additionally, it may be possible to use Leishmania bearing conditionally auxotrophic gene replacements as safe, improved live vaccines for leishmaniasis.

Simultaneous transient expression assays of the trypanosomatid parasite Leishmania using β-galactosidase and β-glucuronidase as reporter enzymes

We describe a transient transfection protocol for cultured Leishmania major promastigotes, utilizing Escherichia coli genes encoding beta-galactosidase and beta-glucuronidase inserted into an expression vector derived from the dihydrofolate reductase-thymidylate synthase locus. Less than 0.1 pg of either reporter enzyme can be detected with a simple fluorimetric assay, and transfection of 10 micrograms of either reporter construct yields activities at least 100-fold over background. Simultaneous introduction of both constructs showed that the activity of each reporter gene was unaffected by the presence of the other, allowing one reporter construct to serve as a control for experimental variability in test gene constructs containing the second reporter gene. These results show that it is feasible to apply transient expression assays to the identification of cis-acting elements of genes encoding nonabundant mRNAs in the genus Leishmania.

Proteolytic and partial sequencing studies of the bifunctional dihydrofolate reductase-thymidylate synthase from Daucus carota

The bifunctional dihydrofolate reductase-thymidylate synthase (DHFR-TS) of Daucus carota has been further characterized as regards molecular weight, amino acid composition, protease digestion and microsequencing of proteolytic peptides. Data reported in this paper demonstrate that the carrot protein has a calculated Mr of 124,000 thus indicating that, contrarily to what has previously been suggested, it occurs as a dimer of identical subunits. Results of partial amino acid microsequencing show the presence of sequences highly homologous with those of the active sites of both DHFR and TS from other organisms confirming, at the structural level, the bifunctional nature of the carrot protein. As in the case of Leishmania tropica DHFR-TS, incubation of the carrot protein with V8 protease led to a rapid loss of TS activity while retaining that of DHFR. However the pattern of proteolysis did not allow to establish whether the sequence of domains is DHFR-TS as in Leishmania, or vice versa. Low homology of other amino acid sequences, as judged by computer analysis, and absence of common epitopes indicate an apparent divergence between carrot and leishmanian proteins.

Plasmodium falciparum: Evidence for a DNA methylation pattern

The methylation status of the adenine and cytosine residues in the genome of Plasmodium falciparum was studied using restriction enzymes exhibiting differential activity dependent on the methylation state of these residues in their recognition site. The gene coding for the enzyme dihydrofolate reductase-thymidylate synthase was studied for that purpose. No methylated adenine residues were observed in this gene in four strains tested. However, partial methylation of cytosine residues was observed in all strains. This methylation occurred at a specific site of the gene and was of the eukaryotic type, namely at a CpG sequence.

Hybridization arrest of the cell-free translation of the malarial dihydrofolate reductase/thymidylate synthase mRNA by anti-sense oligodeoxyribonucleotides

In order to inhibit the in vitro translation of Plasmodium falciparum mRNA coding for the bifunctional enzyme dihydrofolate reductase-thymidylate synthase (DHFR-TS), oligodeoxynucleotides (ODNs) were directed against the translation initiation site or a site in the TS-coding region. In both cases considerable hybridization arrest, i.e. greater than 50% inhibition, was only achieved if the lengths of the ODNs to the two regions were 30 and 39 nucleotides, respectively, or longer. The ODN with the highest efficiency was a 49-mer directed against the TS-coding region (OTS49); 45 microM was sufficient to inhibit the expression of DHFR-TS by almost 90%. In this case the synthesis of DHFR-TS was interrupted at the binding site of OTS49 by a RNase H-independent mechanism. The resulting polypeptide was smaller (55 kDa) than one subunit of the native protein (71 kDa) and lacked TS activity.

Heterologous expression of active thymidylate synthase-dihydrofolate reductase from Plasmodium falciparum

The coding sequence of the bifunctional thymidylate synthase-dihydrofolate reductase (TS-DHFR) from a moderately pyrimethamine-resistant strain (HB3) of Plasmodium falciparum was assembled in a pUC expression vector. The coding sequence possesses unique Nco1 and Xba1 sites which flank 243 bp of the DHFR gene that include all point mutations thus far linked to pyrimethamine resistance. Wild-type (3D7) and highly pyrimethamine-resistant (7G8) TS-DHFRs were made from this vector by cassette mutagenesis using Nco1-Xba1 fragments from the corresponding cloned TS-DHFR genes. Catalytically active recombinant TS-DHFRs were expressed in Escherichia coli, albeit at low levels. Both TS and DHFR coeluted upon gel filtration and copurified upon affinity and anion exchange chromatography. Gel filtration and SDS-PAGE indicated that the enzyme was a dimer with identical 67-kDa subunits, characteristic of protozoan TS-DHFRs. Amino-terminal sequencing gave 10 amino acids which perfectly matched the sequence predicted from the nucleotide sequence. The recombinant TS-DHFR was purified to homogeneity by 10-formylfolate affinity chromatography followed by Mono Q FPLC. The inhibition properties of pyrimethamine toward the purified recombinant enzymes show that the point mutations are the molecular basis of pyrimethamine resistance in P. falciparum.

Gene replacement in parasitic protozoa

Trypanosomatid protozoa frequently cause severe diseases in humans. Many molecules likely to have a role during the infectious cycle have been identified, yet proof of their function is often lacking. We describe studies in Leishmania major of homologous gene targeting, a powerful method for testing gene function in other organisms. Following introduction of a construct containing dihydrofolate reductase-thymidylate synthase (dhfr-ts) flanking sequences fused to neomycin phosphotransferase, 45% of the colonies contained the planned homologous replacement; this frequency rose to nearly 100% in transfections using low amounts of DNA. Integrative transfection in Leishmania thus resembles that of Saccharomyces cerevisiae in giving predominantly homologous events. To facilitate studies of folate metabolism and chemotherapy the sole dhfr-ts copy in a heterozygous deletion line was replaced, yielding lines that were functionally DHFR-TS-. Although most genes are diploid in trypanosomatids, methods exploiting the high frequency of homologous recombination should permit complete replacement of any parasite gene.

Chromosomal rearrangements and point mutations in the DHFR-TS gene of Plasmodium chabaudi under antifolate selection

Selection of the rodent malaria Plasmodium chabaudi with low levels of the antifolate drug pyrimethamine has previously been shown by us to result in duplication of the dihydrofolate reductase-thymidylate synthase (DHFR-TS) gene by a duplication of chromosome 7 and subsequent rearrangements. We have selected this resultant parasite line with large doses of pyrimethamine and analysed the DHFR-TS gene and chromosomes for any changes. Increased drug pressure has resulted in reappearance of a chromosome with the same structure as chromosome 7 from DS the parent line. Sequencing of the DHFR gene from each of the chromosomes has identified a single point mutation that results in a serine to asparagine change at position 106. This is the equivalent mutation that has been identified as the key residue in the mechanism of resistance to pyrimethamine in Plasmodium falciparum. There is no apparent increase in transcription of the DHFR-TS gene and the large increase in resistance is most likely a result of the mutation in the DHFR gene.

Thymidylate synthase-dihydrofolate reductase in protozoa

In protozoa, thymidylate synthase (TS) and dihydrofolate reductase (DHFR) exist on the same polypeptide. The DHFR domain is on the amino terminus, TS is on the carboxy terminus, and the domains are separated by a junction peptide of varying size depending on the source. The native protein is a dimer of two such subunits and is 110–140 kDa. Most studies of bifunctional TS-DHFR have been performed with the protein from anti-folate resistant strains of Leishmania major, which show amplification of the TS-DHFR gene and overproduction of the bifunctional protein. The Leishmania TS-DHFR has also been highly expressed in heterologous systems. There is extensive communication between domains, and channeling of the H 2folate product of TS to DHFR. Antifolates commonly used to treat microbial infections are poor inhibitors of L. major DHFR. However, selective inhibitors of L. major vs human DHFR have been found. The TS-DHFR from Plasmodium falciparum has also been cloned and sequenced. Interestingly, pyrimethamine-resistant strains of P. falciparum have a common point mutation in the DHFR coding sequence which causes decreased binding of the folate analog. A detailed knowledge of the structure and function of protozoan TS-DHFRs will soon be available.

Amino acids in the dihydrofolate reductase-thymidylate synthase gene of Plasmodium falciparum involved in cycloguanil resistance differ from those involved in pyrimethamine resistance.

Cycloguanil, the active metabolite of the antimalarial drug proguanil, is an inhibitor of dihydrofolate reductase as is another antimalarial, pyrimethamine. Its use has been limited by the rapid development of resistance by parasites around the world. We have determined the cycloguanil- and pyrimethamine-sensitivity status of 10 isolates of Plasmodium falciparum and have sequenced in all these isolates the dihydrofolate reductase (DHFR; 5,6,7,8-tetrahydrofolate: NADP+ oxidoreductase, EC 1.5.1.3) portion of the DHFR-thymidylate synthase (TS; 5,10-methylenetetrahydrofolate: dUMP C-methyltransferase, EC 2.1.1.45) gene. Instead of the known serine-to-asparagine change at position 108 that is important in pyrimethamine resistance, a serine-to-threonine change at the same position is found in cycloguanil-resistant isolates along with an alanine-to-valine change at position 16. We conclude that pyrimethamine and cycloguanil resistance most commonly involve alternative mutations at the same site. However, we also have identified a parasite with a unique set of changes that results in resistance to both drugs.

Stable transfection of the human parasite Leishmania major delineates a 30-kilobase region sufficient for extrachromosomal replication and expression.

To delineate segments of the genome of the human protozoan parasite Leishmania major necessary for replication and expression, we developed a vector (pR-NEO) which can be reproducibly introduced into L. major. This DNA was derived from a 30-kilobase extrachromosomal amplified DNA bearing the dihydrofolate reductase-thymidylate synthase gene, with the coding region for neomycin phosphotransferase substituted for that of dihydrofolate reductase-thymidylate synthase and a bacterial origin of replication and selectable marker added. G418-resistant lines were obtained at high efficiency by electroporation of pR-NEO (approaching 10(-4) per cell), while constructs bearing an inverted neo gene or lacking Leishmania sequences did not confer resistance. pR-NEO replicated in L. major and gave rise to correctly processed transcripts bearing the trans-spliced miniexon. Molecular karyotype analysis showed that in some lines pR-NEO DNA exists exclusively as an extrachromosomal circle, a finding supported by the rescue of intact pR-NEO after transformation of Escherichia coli. These data genetically localize all elements required in cis for DNA replication, transcription, and trans splicing to the Leishmania DNA contained within pR-NEO DNA and signal the advent of stable transfection methodology for addressing molecular phenomena in trypanosomatid parasites.

Stable Transfection of the Human Parasite Leishmania major Delineates a 30-Kilobase Region Sufficient for Extrachromosomal Replication and Expression

To delineate segments of the genome of the human protozoan parasite Leishmania major necessary for replication and expression, we developed a vector (pR-NEO) which can be reproducibly introduced into L. major. This DNA was derived from a 30-kilobase extrachromosomal amplified DNA bearing the dihydrofolate reductase-thymidylate synthase gene, with the coding region for neomycin phosphotransferase substituted for that of dihydrofolate reductase-thymidylate synthase and a bacterial origin of replication and selectable marker added. G418-resistant lines were obtained at high efficiency by electroporation of pR-NEO (approaching 10(-4) per cell), while constructs bearing an inverted neo gene or lacking Leishmania sequences did not confer resistance. pR-NEO replicated in L. major and gave rise to correctly processed transcripts bearing the trans-spliced miniexon. Molecular karyotype analysis showed that in some lines pR-NEO DNA exists exclusively as an extrachromosomal circle, a finding supported by the rescue of intact pR-NEO after transformation of Escherichia coli. These data genetically localize all elements required in cis for DNA replication, transcription, and trans splicing to the Leishmania DNA contained within pR-NEO DNA and signal the advent of stable transfection methodology for addressing molecular phenomena in trypanosomatid parasites.

Detection of pyrimethamine resistance in Plasmodium falciparum by mutation-specific polymerase chain reaction

The point mutation at nucleotide 323 within the dihydrofolate reductase-thymidylate synthase gene of Plasmodium falciparum, which distinguishes pyrimethamine-sensitive from drug-resistant isolates, can be discriminated by the polymerase chain reaction using mutation-specific primers. The technique makes use of the principle that short oligonucleotides with a perfect match at their 3′ ends, complementary to the mutation to be detected, will initiate the polymerization by Taq polymerase far more efficiently than primers with a single mismatch in this position. The Asn-108 codon was detected using a primer of 17 nucleotides with an adenosine at its 3′ end, the Thr-108 codon with a 14-mer primer ending with a cytosine and the Ser-108 codon with a 16-mer containing guanidine at the critical 3′ end. By selecting appropriate counterprimers, the size of the amplification products is either indicative of pyrimethamine-resistant parasites of the 7G8 type, or of pyrimethamine-sensitive parasites of the FCR-3 type or 3D7 type. The fragments obtained can be easily separated in a single lane after agarose gel electrophoresis. Coded P. falciparum DNA samples were typed unambiguously using these primers as were reconstituted parasitized blood samples stored as high salt lysates. Sensitivity, speed and specificity make this assay a realistic alternative to in vitro drug testing to monitor the resistance of P. falciparum to inhibitors of the dihydrofolate reductase.