Title: Increased transport of pteridines compensates for mutations in the high affinity folate transporter and contributes to methotrexate resistance in the protozoan parasite Leishmania tarentolae
Abstract: Article4 May 1999free access Increased transport of pteridines compensates for mutations in the high affinity folate transporter and contributes to methotrexate resistance in the protozoan parasite Leishmania tarentolae Christoph Kündig Christoph Kündig Centre de Recherche en Infectiologie, CHUQ, Pavilon CHUL, 2705 Boulevard Laurier, RC-709, Ste-Foy, Quebec, Canada, G1V 4G2 Search for more papers by this author Anass Haimeur Anass Haimeur Centre de Recherche en Infectiologie, CHUQ, Pavilon CHUL, 2705 Boulevard Laurier, RC-709, Ste-Foy, Quebec, Canada, G1V 4G2 Search for more papers by this author Danielle Légaré Danielle Légaré Centre de Recherche en Infectiologie, CHUQ, Pavilon CHUL, 2705 Boulevard Laurier, RC-709, Ste-Foy, Quebec, Canada, G1V 4G2 Search for more papers by this author Barbara Papadopoulou Barbara Papadopoulou Centre de Recherche en Infectiologie, CHUQ, Pavilon CHUL, 2705 Boulevard Laurier, RC-709, Ste-Foy, Quebec, Canada, G1V 4G2 Search for more papers by this author Marc Ouellette Corresponding Author Marc Ouellette Centre de Recherche en Infectiologie, CHUQ, Pavilon CHUL, 2705 Boulevard Laurier, RC-709, Ste-Foy, Quebec, Canada, G1V 4G2 Search for more papers by this author Christoph Kündig Christoph Kündig Centre de Recherche en Infectiologie, CHUQ, Pavilon CHUL, 2705 Boulevard Laurier, RC-709, Ste-Foy, Quebec, Canada, G1V 4G2 Search for more papers by this author Anass Haimeur Anass Haimeur Centre de Recherche en Infectiologie, CHUQ, Pavilon CHUL, 2705 Boulevard Laurier, RC-709, Ste-Foy, Quebec, Canada, G1V 4G2 Search for more papers by this author Danielle Légaré Danielle Légaré Centre de Recherche en Infectiologie, CHUQ, Pavilon CHUL, 2705 Boulevard Laurier, RC-709, Ste-Foy, Quebec, Canada, G1V 4G2 Search for more papers by this author Barbara Papadopoulou Barbara Papadopoulou Centre de Recherche en Infectiologie, CHUQ, Pavilon CHUL, 2705 Boulevard Laurier, RC-709, Ste-Foy, Quebec, Canada, G1V 4G2 Search for more papers by this author Marc Ouellette Corresponding Author Marc Ouellette Centre de Recherche en Infectiologie, CHUQ, Pavilon CHUL, 2705 Boulevard Laurier, RC-709, Ste-Foy, Quebec, Canada, G1V 4G2 Search for more papers by this author Author Information Christoph Kündig1, Anass Haimeur1, Danielle Légaré1, Barbara Papadopoulou1 and Marc Ouellette 1 1Centre de Recherche en Infectiologie, CHUQ, Pavilon CHUL, 2705 Boulevard Laurier, RC-709, Ste-Foy, Quebec, Canada, G1V 4G2 *Corresponding author. E-mail: [email protected] The EMBO Journal (1999)18:2342-2351https://doi.org/10.1093/emboj/18.9.2342 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Functional cloning led to the isolation of a novel methotrexate (MTX) resistance gene in the protozoan parasite Leishmania. The gene corresponds to orfG, an open reading frame (ORF) of the LD1/CD1 genomic locus that is frequently amplified in several Leishmania stocks. A functional ORF G–green fluorescence protein fusion was localized to the plasma membrane. Transport studies indicated that ORF G is a high affinity biopterin transporter. ORF G also transports folic acid, with a lower affinity, but does not transport the drug analog MTX. Disruption of both alleles of orfG led to a mutant strain that became hypersensitive to MTX and had no measurable biopterin transport. Leishmania tarentolae MTX-resistant cells without their high affinity folate transporters have a rearranged orfG gene and increased orfG RNA levels. Overexpression of orfG leads to increased biopterin uptake and, in folate-rich medium, to increased folate uptake. MTX-resistant cells compensate for mutations in their high affinity folate/MTX transporter by overexpressing ORF G, which increases the uptake of pterins and selectively increases the uptake of folic acid, but not MTX. Introduction Antifolates are inhibitors of the enzyme dihydrofolate reductase (DHFR), which supplies the cell with reduced folates which are essential cofactors used in many one-carbon donor reactions (Schweitzer et al., 1990; Kamen, 1997). Folates are made of three building blocks: a pterin moiety which is conjugated to para amino benzoic acid by dihydropteroate synthase (DHPS) and a glutamic acid which is conjugated to dihydropteroate by dihydrofolate synthase to produce dihydrofolate. Dihydrofolate is reduced to tetrahydrofolate by DHFR. Because the DHFR proteins of different organisms share little homology, this enzyme proved to be a valuable target for chemotherapeutic drugs. Various antifolates have been successfully used as anticancer drugs (methotrexate) or in the treatment of bacterial (trimethoprim) or of parasitic infections such as malaria or toxoplasmosis (pyrimethamine) (Schweitzer et al., 1990). No successful antifolate chemotherapy has yet been established against infections with the protozoan parasite Leishmania. Nevertheless, many distinct features in the folate metabolism of this organism have been identified so far, which could prove useful therapeutic targets. By using methotrexate (MTX) as a model antifolate drug, several different resistance mechanisms were identified. Whereas some of them were similar to mechanisms found in cancer cells or bacteria, others turned out to be novel (Borst and Ouellette, 1995; Nare et al., 1997). Gene amplification as part of extrachromosomal elements is commonly seen in response to drug selection in Leishmania (Beverley, 1991; Papadopoulou et al., 1998). Amplification of the dhfr-ts gene encoding the bifunctional dihydrofolate reductase-thymidylate synthase (DHFR-TS) leads to overexpression of the main MTX target enzyme and has been observed in L.major in response to drug selection (Coderre et al., 1983; Ellenberger and Beverley, 1987b). In one case, a combination of overexpression with a point mutation within the L.major DHFR-TS was reported which resulted in a largely increased resistance level (Arrebola et al., 1994). Another locus that is often amplified in several Leishmania species selected for MTX resistance encodes for PTR1 (pterin reductase), an enzyme belonging to the family of short chain dehydrogenase/reductases (Callahan and Beverley, 1992; Papadopoulou et al., 1992). PTR1 is capable of reducing fully or partially oxidized pterins or folates (Bello et al., 1994; Wang et al., 1997). It is believed that overexpression of this enzyme confers MTX resistance by supplying the cell with a sufficient amount of reduced folates, thus by-passing the need for DHFR. Besides the amplification of ptr1 and dhfr-ts genes, reduction of the drug uptake is the second main pathway by which Leishmania resist antifolates. Leishmania have long been believed to be auxotrophic for folates, and during in vitro growth the cells rely mainly on uptake of folates for growth. One common high affinity transporter for folate and MTX has been identified in Leishmania and related parasites and mutations within this gene lead to antifolate resistance (Dewes et al., 1986; Ellenberger and Beverley, 1987b; Kaur et al., 1988; Papadopoulou et al., 1993). These mutations are associated with a large variety of transport phenotypes ranging from a 2-fold decrease in folate/MTX transport to uptake levels below the detection limit. Mutant strains with no apparent measurable folate uptake are able to thrive under laboratory growth conditions. This suggests that Leishmania must be capable of de novo folate synthesis or that folates have alternative routes of entry. The conversion of radiolabeled biopterin (one of the building blocks of folic acid) into reduced folates has been demonstrated in L.donovani (Beck and Ullman, 1991) and is consistent with de novo synthesis. The exact mechanism of this conversion is unknown, but seems to differ from the conventional route via DHPS since incorporation of radiolabeled p-amino benzoic acid could not be detected in L.major (Kovacs et al., 1989), and several DHPS inhibitors are not active against Leishmania (Peixoto and Beverley, 1987; Kaur et al., 1988). By transfecting a L.tarentolae gene bank into wild-type parasites and selecting for MTX resistance, we isolated a novel resistance gene coding for a high affinity membrane biopterin transporter, which also has low affinity for folic acid transport but does not transport MTX. Leishmania tarentolae cells resisting MTX by mutations in their common high affinity folate/MTX transporter showed an increase in the activity of their biopterin transporter. Results Functional cloning of the novel MTX resistance gene orfG Drug resistance genes in Leishmania are usually isolated by analyzing mutants selected for resistance by increasing drug concentrations (Borst and Ouellette, 1995). In order to isolate new resistance genes, we used functional cloning which was initially set up to study genes involved in lipophosphoglycan biosynthesis (Descoteaux et al., 1994). Wild-type L.tarentolae cells were transfected with a genomic cosmid bank and plated on MTX-containing plates (see Materials and methods). An identical cosmid called cMM4 was found in five of the transfectants obtained by functional cloning. Retransfection of the cosmid cMM4 into L.tarentolae TarII wild type (WT) restored the MTX resistance and this transfectant showed an increase of its EC50 by ∼10-fold when compared with wild-type cells (Figure 1A). The level of resistance conferred by cMM4 differs from L.tarentolae cells transfected with cosmids containing either the dhfr-ts or ptr1 gene (Figure 1A), suggesting the presence of a novel MTX resistance gene on cosmid cMM4. The novelty of the resistance gene was confirmed by hybridization experiments since neither a ptr1 nor a dhfr-ts probe hybridized to cMM4 (not shown). The cosmid cMM4 was digested with either BglII, NheI or SpeI, and subcloned into the Leishmania expression vector pSPY-hyg (Papadopoulou et al., 1994b). After transfection in TarII WT, three different restriction fragments, a 6 kb BglII, a 6.8 kb SpeI and an 8.5 kb NheI fragment were associated with MTX resistance. A 2.3 kb BglII–NheI fragment was the smallest segment common to all three fragments (Figure 1B). Transfection of this fragment conferred a similar level of MTX resistance as the original cosmid cMM4 (Figure 1A). Figure 1.Functional cloning of a novel Leishmania MTX resistance gene. (A) Profile of MTX resistance of TarII WT (○) and transfected with dhfr-ts (▵), cMM4 (▪), the 2.3 kb BglII–NheI fragment of cMM4 (●) and ptr1 (□). The cells were grown in SDM 79 medium supplemented with 5% heat-inactivated FBS. (B) Partial physical map of the orfG region of L.mexicana. Below the map, the restriction fragments associated with MTX resistance when transfected on a multicopy expression vector are depicted. B, BglII; N, NheI; S, SpeI. (C) Hydrophobicity plot of ORF G of L.mexicana (Kyte and Doolittle, 1982). Putative transmembrane segments are underlined and numbered. The sequence of the L.mexicana orfG gene can be found under the DDBJ/EMBL/GenBank accession No. AF078929. (D) Confocal laser scanning microscopy of L.tarentolae overexpressing the ORF G–GFP fusion protein, showing the membrane location of ORF G. Download figure Download PowerPoint DNA sequence analysis of the 2.3 kb BglII–NheI fragment (DDBJ/EMBL/GenBank accession No. AF078929) revealed an open reading frame (ORF) of 1893 bp with the TAG stop codon being part of the NheI site used for the subcloning of the gene. The ORF shared 88% identity with ORF G of L.donovani. ORF G was first described as part of LD1/CD1 amplicons spontaneously occurring in various Leishmania species (Myler et al., 1994). ORF G also shares considerable homology with ESAG10 of Trypanosoma brucei, a protein with unknown function encoded by an expression site-associated gene (Gottesdiener, 1994). The orfG gene was identified as a MTX resistance gene by functional cloning. It is possible that during selection we may have selected for a point mutation within the gene which is responsible for the observed resistance phenotype. To address this possibility, the orfG genes of L.mexicana, L.donovani and L.tarentolae were cloned in a Leishmania expression vector. Upon transfection, all these genes produced a similar level of MTX resistance as observed with the original cosmid cMM4 (not shown). These experiments indicated that the wild-type orfG genes from at least three different Leishmania species are able to confer antifolate resistance. Hydrophobicity analysis of ORF G suggested the presence of 12 putative transmembrane segments (Figure 1C). Most of these transmembrane domains contain one or more hydrophilic amino acid residues that are predicted to form amphiphilic α-helices or β-strands, a structure that is typical for type IV integral membrane proteins (Singer, 1990). It has been suggested that members of this class of membrane proteins act as aqueous channels through the membrane and mediate specific transport of small hydrophilic molecules. To confirm the membrane location of ORF G, we constructed an ORF G–green fluorescent protein (ORF G–GFP) fusion. This fusion has the same activity as the intact ORF G since it confers the same level of MTX resistance (not shown) and it has the same pteridine transport properties (Figure 2). This suggests that the cellular location of the overexpressed ORF G–GFP is similar to that of the overexpressed ORF G. The localization of the ORF G–GFP fusion was studied by confocal microscopy (Figure 1D). Uniform staining of the plasma membrane was observed. In addition, the fusion protein was also detected within an intracellular compartment at the base of the flagellum that probably corresponds to the flagellar pocket. Figure 2.Accumulation of radiolabeled pteridines in Leishmania wild-type and MTX-resistant cells. The transport studies with TarII WT (□), TarII transfected with orfG (▪), MTX 100.5 (▵) and MTX 1000.6 (○) were carried out as described in Materials and methods. (A) [3H]MTX accumulation using 150 nM of the drug. (B) [3H]MTX accumulation using varying concentrations of the drug. (C) [3H]folate accumulation using 150 nM of substrate. (D) [3H]folate accumulation using varying concentrations of the substrate. (E) [3H]biopterin accumulation; TarII transfected with an orfG–GFP fusion (●). Transport experiments with each individual cell line have been done at least three times and similar results were obtained consistently. Download figure Download PowerPoint Pteridine transport properties of MTX-resistant L.tarentolae and of an orfG transfectant Computer analysis of ORF G suggested that it can specifically transport hydrophilic molecules. One possible way to increase the level of resistance would be an accelerated extrusion of the drug outside the cell (Borst and Ouellette, 1995). To test this possibility we measured the steady-state accumulation of [3H]MTX in wild-type and MTX mutant cells as well as in an orfG transfectant (Figure 2A). We have previously described two classes of L.tarentolae MTX/folate transporter mutants with a decreased uptake of 50 and >95%, respectively (Papadopoulou et al., 1993). As reported in the past, the mutant MTX 100.5 showed a 2-fold decrease in uptake while no MTX uptake could be measured in mutant MTX 1000.6 (Figure 2A). The accumulation of folic acid in wild-type and MTX mutant cells was very similar to the kinetics of MTX uptake although some folate uptake could be detected in MTX1000.6 (Figure 2C), further suggesting that Leishmania has a common folate/MTX transporter (Ellenberger and Beverley, 1987b). Overexpression of orfG led to no significant change in MTX accumulation compared with wild-type levels, indicating no involvement of ORF G in MTX export (Figure 2B). The sensitivity of Leishmania to antifolate drugs is heavily influenced by the concentration of exogenous folates (Beverley, 1991; Papadopoulou and Ouellette, 1993). Therefore, a selective increase of folate import compared with MTX uptake is another possible mechanism by which the putative transmembrane transporter ORF G could confer MTX resistance. Under standard conditions, the orfG transfectant showed no significant increase in folate transport compared with wild-type cells (Figure 2C). At higher concentrations of folate, however, ORF G effectively transported folic acid (Figure 2D). ORF G seems therefore to correspond to a low affinity folate transporter that can discriminate between MTX and folic acid (Figure 2B and D). We also tested the possible involvement of ORF G in the transport of pterins using radioactive biopterin. Biopterin enables growth of Leishmania cells in a defined folate-deficient medium (Kaur et al., 1988; Beck and Ullman, 1990; Bello et al., 1994; Papadopoulou et al., 1994a). The uptake of biopterin was shown to be transport mediated (Beck and Ullman, 1990), and radiolabeled biopterin can be incorporated into reduced folate (Beck and Ullman, 1991). Wild-type L.tarentolae cells were shown to accumulate [3H]biopterin in a time-dependent manner (Figure 2E). Using standard conditions, under which no increase in folate transport could be detected (Figure 2C), the accumulation of [3H]biopterin was increased 10-fold in the orfG transfectant compared with wild-type cells and a similar increase was observed with the ORF G–GFP fusion (Figure 2E). MTX 100.5, a cell line with a 2-fold reduced MTX/folate uptake, accumulated biopterin at the same rate as wild-type cells. Interestingly, the cell line MTX 1000.6, without any detectable activity of its high affinity MTX/folate transporter, showed a biopterin accumulation that was several times greater than the wild-type level (Figure 2E). The uptake of biopterin in L.tarentolae is probably mediated by an active transport mechanism as no accumulation could be measured when the cells were incubated on ice (Figure 3A). This was substantiated by the lack of biopterin accumulation in cells treated with the metabolic inhibitors sodium azide (20 mM) and 2,4-dinitrophenol (5 mM) (Figure 3A). Similar concentrations of inhibitors were shown to inhibit active folate uptake (Ellenberger and Beverley, 1987a) and active efflux of arsenite (Dey et al., 1994) in Leishmania. To characterize further the biopterin transport properties of ORF G, we measured the rate of uptake of biopterin in a wild-type cell and in an orfG transfectant while varying biopterin concentration. Wild-type cells exhibit uptake of biopterin with high affinity and an apparent Km of 4.9 μM (Figure 3B; Table I). A similarly high affinity biopterin with an apparent Km of 4.7 μM was observed for the orfG transfectant while its Vmax was increased by >10-fold (Figure 3B; Table I). The increase in the rate of uptake (Table I) correlates well with the levels of the steady-state accumulation of biopterin observed in wild-type cells and orfG transfected cells (Figure 2E). Figure 3.Characterization of the biopterin transporter ORF G. (A) Biopterin transport (500 nM) in L.tarentolae wild-type cells (○) or incubated on ice (□), or in the presence of 20 mM sodium azide (●) or of 5 mM 2,4-dinitrophenol (▪). (B) Lineweaver–Burk analysis of biopterin transport in TarII WT cells (○) or Tar II cells transfected with orfG (▪). Apparent Km and Vmax values found in Table I were determined from the intercepts of the x- and y-axes. Download figure Download PowerPoint Table 1. Biochemical characteristics of pteridine transport in L.tarentolae Cells Biopterin Folate Km (μM) Vmax (pmol/min/ 109 cells) Km (μM) Vmax (pmol/min/ 109 cells) Tar II wild type 4.9 1.28 0.26 7.6 orfG transfectant 4.7 17.1 0.45 8.1 Wild-type Leishmania cells also have a high affinity folate transporter with a Km value of 0.7 μM for L.major (Ellenberger and Beverley, 1987a) and 0.23 μM in L.donovani (Kaur et al., 1988). We have performed Lineweaver–Burk analysis of folate transport in L.tarentolae and found an apparent Km of 0.26 μM in wild-type cells (Table I). Similar kinetic parameters were observed for folate transport in L.tarentolae orfG transfectant (Table I), indicating that ORF G is not the high affinity folate transporter. Nonetheless, the L.tarentolae ORF G can transport folic acid (Figure 2D), at least when folate concentration is at 6 μM. Attempts to characterize the ORF G-mediated folate transport activity in more detail were unsuccessful mainly for technical reasons (see Materials and methods). Although a precise Km value could not be determined for folate transport mediated by ORF G, results indicated clearly that ORF G contributes only marginally, at least at low folate concentration, to folate transport (Figure 2D; Table I). Overall, our transport studies indicated that ORF G is a high affinity active biopterin transporter and a low affinity folate transporter, but does not transport the drug analog MTX (Figure 2B and D). Phenotype of the L.tarentolae orfG knock-out mutant To characterize further the role of ORF G in the pteridine metabolism of Leishmania, an orfG-null mutant was generated. The first allele was disrupted using an hygromycin phosphotransferase (hyg) expression cassette (Papadopoulou et al., 1994b). Southern blot analysis indicated that orfG is part of a 3.5 kb PstI fragment in wild-type cells (Figure 4A). After longer exposure we could detect several weaker fragments hybridizing to an orfG probe. One of these, which was not affected by the construction of orfG mutants, is visible in Figure 4A (marked by an asterisk). Analysis of the hygromycin-resistant cell pool showed the appearance of two additional fragments of 2.7 and 1.8 kb, which is consistent with the introduction of an additional PstI site within the hyg marker into orfG (Figure 4A). Leishmania is diploid and this makes it necessary to inactivate the second allele which can conveniently be done by loss of heterozygosity (Gueiros-Filho and Beverley, 1996). By increasing the selection pressure with hygromycin B, we observed an increase of the strength of the hybridization signal of the mutant fragments of 2.7 and 1.8 kb over the wild-type 3.5 kb fragment (Figure 4A, lane 3), suggesting that we have enriched for double disruptants within the cell pool. Cloning of this culture led to the isolation of the orfG-null mutant IIorfGYhygro.2, in which both alleles of orfG were disrupted by the hyg resistance marker (Figure 4A, lane 4). Figure 4.Analysis of a L.tarentolae orfG-null mutant. (A) Southern blot analysis of an orfG-null mutant. Total DNA was digested with PstI and hybridized to an intragenic orfG probe. Lane 1, TarII WT; lane 2, total population of orfGYhygro mutants; lane 3, orfGYhygro mutants selected with high levels of hygromycin B; lane 4, IIorfGYhygro.2, an orfG-null mutant. An orfG homologous gene is marked by an asterisk. A partial physical map of the orfG region of L.tarentolae wild-type and the orfG-null mutant is shown. Fragments obtained after PstI digestion are depicted below the map. P, PstI; C, Csp45I. (B) Measurement of biopterin accumulation. TarII WT (▪), IIorfGYhygro.2 (○), IIorfGYhygro.2 transfected with orfG (□). (C) MTX resistance of TarII WT (▪), IIorfGYhygro.2 (○) and IIorfGYhygro.2 transfected with orfG (□). Download figure Download PowerPoint No biopterin accumulation could be measured in IIorfGYhygro.2 (Figure 4B), but by introducing an expression vector carrying the orfG wild-type gene into the orfG null mutant we were able to revert the transport phenotype of the mutant (Figure 4B). Inactivation of the high affinity biopterin transporter ORF G decreases the EC50 to MTX by ∼4-fold compared with wild-type cells (Figure 4C). By overexpressing orfG on a plasmid in the orfG null mutant, we were able to reverse the hypersensitivity to MTX and produce a level of resistance which is close to that we have observed in wild-type cells overexpressing orfG (Figures 1A and 4C). An orfG null mutant of L.tarentolae is viable in culture and shows only a very small delay in its growth rate compared with wild-type cells. ORF G is therefore not essential for growth of L.tarentolae in culture medium, although it seems to be the only high affinity transporter for biopterin. Overexpression of ORF G in folate transport mutants The folate transport-deficient mutant MTX 1000.6 accumulates biopterin at a rate ∼3–5 times higher compared with wild-type cells (Figure 2C). As gene amplification is a common mechanism by which Leishmania survive drug challenge (Beverley, 1991; Papadopoulou et al., 1998), we tested whether amplification of orfG was responsible for increased biopterin transport in L.tarentolae MTX 1000.6 mutants. A 3.5 kb PstI fragment is recognized by an orfG probe in wild-type cells (Figure 5A, lane 1). The same probe also recognizes one copy of the orfG gene family (marked with an asterisk in Figure 5A). Gene amplification or DNA rearrangements could neither be detected in the mutant MTX 100.5 (Figure 5A, lane 2), nor in MTX 1000.4 or MTX 1000.5 (not shown), mutants in which folate/MTX transport was only reduced by 2-fold (Papadopoulou et al., 1993) (Figure 2A and C). However, novel non-amplified PstI fragments at a size of ∼4.8 kb hybridized to an orfG probe in mutants MTX 1000.6, but also in mutants MTX 1000.3 and MTX 1000.7 (Figure 5A, lanes 3–5). No measurable high affinity MTX/folate transport can be detected in the latter three mutants (Papadopoulou et al., 1993). The precise rearrangements may differ between the three mutants as the sizes of the rearranged fragments differ slightly. All three mutants showing rearrangement within the orfG region also demonstrated an increase in biopterin transport (Figure 5C). All mutants that resist MTX by a mutation in their high affinity MTX/folate transport are compensating by increasing the activity of the high affinity biopterin transporter. Figure 5.Overexpression of ORF G in MTX-transport mutants. (A) Southern blot analysis of total DNA of L.tarentolae TarII WT and MTX-transport mutants: total DNA was digested with PstI and hybridized with an intragenic orfG probe. Lane 1, TarII WT; lane 2, MTX 100.5; lane 3, MTX 1000.3; lane 4, MTX 1000.6; lane 5, MTX 1000.7. (B) Analysis of orfG RNA by Northern blot. Total RNA was hybridized to an intragenic orfG fragment of L.mexicana and re-hybridized to an α-tubulin gene probe to monitor the amount of RNA layered in each lane. Lane 1, TarII WT; lane 2, MTX 1000.6; lane 3, TarII orfG transfectant. (C) Measurement of biopterin accumulation. TarII WT (▪), MTX 1000.3 (○), MTX 1000.6 (□), MTX 1000.7 (▵). Download figure Download PowerPoint The increase in biopterin transport in the mutants was not due to orfG gene amplification but to an increased steady-state accumulation of orfG RNA by ∼5-fold in MTX 1000.6 (Figure 5B, lane 2). In addition to the increased amount of RNA, we also noted that the orfG RNA in MTX 1000.6 (at least part of it) is larger than the wild-type RNA, which could possibly be a result of the described genomic rearrangement. The RNA of the transfectant also differs in size (Figure 5B, lane 3), but this is not surprising as only the protein-coding region of ORF G (without its own RNA maturation sequences) was cloned in a Leishmania expression vector. Overexpression of ORF G in MTX 1000.6 not only increases biopterin uptake but also augments uptake of folate when a high folate concentration was present in the transport assay (Figure 2D). The increase of the steady-state orfG RNA level in MTX 1000.6 is commensurate with the high biopterin transport activity of this mutant. In one previous report, the RNA of orfG was shown to be increased following translocation of an orfG segment into the ribosomal locus (Lodes et al., 1995). To test whether this also occurred in the mutant MTX 1000.6, the chromosomes of wild-type and MTX 1000.6 cells were separated by TAFE and hybridized to an orfG and a ribosomal DNA probe. The orfG gene is part of a 2.1 Mb chromosome while ribosomal RNA genes are part of 1.5 and 1.8 Mb chromosomes. We could detect neither any gross gene rearrangements nor translocation of orfG into the ribosomal locus (not shown). Discussion Isolation of a new MTX resistance gene by functional cloning Leishmania often resists in vitro drug selection by amplifying specific portions of its genome as part of extrachromosomal elements (Beverley, 1991; Papadopoulou et al., 1998). Characterization of amplicons derived from MTX-resistant Leishmania led to the identification of dhfr-ts and ptr1 genes. The presence of repeated sequences flanking the resistance genes enhanced greatly the frequency with which gene amplification events are selected in Leishmania (Grondin et al., 1996). It is therefore likely that genes lacking such repeated flanking regions will not be amplified while selecting for stepwise increased resistance. To identify such genes, we introduced a Leishmania cosmid genomic library into wild-type cells and selected for genes which confer MTX resistance when overexpressed from a multicopy vector. The orfG gene was isolated in this way and constitutes a novel resistance gene. Functional cloning can therefore serve as a useful complementary approach to mutant analysis for the isolation of resistance genes. Using functional cloning, but screening for the ability of a L.donovani MTX-resistant mutant to thrive in folate-deficient medium supplemented with biopterin, the group of S.Beverley (Washington University, St Louis, MO) has independently isolated orfG and shown that it can transport biopterin (Moore and Beverley, Woods Hole Molecular Parasitology Meeting 1996, abstract 107, cited in Segovia and Ortiz, 1997). ORF G is present in a chromosomal region called the LD1/CD1 locus (Stuart, 1991; Segovia and Ortiz, 1997