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Inhibition of Plasmodium falciparum clag9 gene function by antisense RNA
Molecular and Biochemical Parasitology 110 (2000) 33 – 41 www.elsevier.com/locate/parasitology
Inhibition of Plasmodium falciparum clag9 gene function by antisense RNA Donald L. Gardiner *, Deborah C. Holt, Elizabeth A. Thomas, David J. Kemp, Katharine R. Trenholme Menzies School of Health Research, Casuarina 0812, Darwin, Northern Territory 0812, Australia Received 19 January 2000; received in revised form 7 April 2000; accepted 26 April 2000
Abstract We have previously shown by targeted gene disruption that the clag9 gene of Plasmodium falciparum is essential for cytoadherence to CD36. Here we report inhibition of the function of clag9 by the use of an antisense RNA vector as an alternative to targeted gene disruption. We transfected an antisense construct of clag9 into the P. falciparum clone 3D7 and when the resulting line was cultured in the presence of pyrimethamine it showed 15-fold lower cytoadherence to C32 melanoma cells than the control. Reversion to wildtype upon removal of the introduced plasmid provides direct evidence that the event responsible for the phenotypic change is not at an unrelated site and this approach provides a valuable new tool in malaria transfection technology. © 2000 Elsevier Science B.V. All rights reserved. Keywords: Plasmodim falciparum; Antisense RNA; clag9 ; Cytoadherence
1. Introduction A major advance in studying malaria biology has been the use of transfection technology, utilising vectors containing the Toxoplasma gondii di-
Abbre6iations: clag, cytoadherence linked asexual gene; DHFR, dihydrofolate reductase; KAHRP, knob-associated histidine-rich protein; TGD, targeted gene disruption. Note: Nucleotide sequence data reported in this paper have been submitted to the GenBank™, EMBL and DDJB data bases with the accession number AF055476 * Corresponding author. Tel.: + 61-8-89228074; fax: + 618-89275187. E-mail address: [email protected] (D.L. Gardiner)
hydrofolate reductase thymidylate synthetase gene (Tg DHFR-TS). These vectors confer pyrimethamine resistance as a selectable marker and allow for transient expression of exogenous genes in Plasmodium falciparum [1], targeted disruptions of P. falciparum genes [2,3], and constitutive transgene expression [4]. While other selectable markers such as blasticidin [5] are now becoming available and have the potential to allow the complementation of genes that have been disrupted, as yet there is no published experimental evidence showing that these new vectors can be used in this fashion. Without a positive and negative selection vector, such as those available for T.
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gondii [6], disruption and complementation of genes within the same parasite, as well as sequential disruption of genes within one parasite clone, are not yet possible. As current targeted gene disruptions (TGD) in P. falciparum require 4 – 8 months of continuous culture, alternative methods that may more easily and rapidly answer complex biological questions are required. Here we describe an alternative method that allows inhibition of gene function by the use of an antisense RNA vector and the subsequent restoration of gene function when the antisense vector is removed. This requires the use of one group of the current generation of P. falciparum transfection vectors which allow stable transgene expression in P. falciparum. These vectors consist of two expression cassettes, the first, under the Plasmodium chabaudi DHFR promoter, express the drug resistance gene, while the second expression cassette, under the control of the P. falciparum calmodulin promoter region, allows stable transgene expression [4]. Previous work has shown that loss of cytoadherence to melanoma cells (which express CD36) by P. falciparum infected erythrocytes in vitro, was associated with deletions of the right arm of chromosome 9 in these cell lines [7]. Using positional cloning we located a gene on the right arm of chromosome 9 of the stably cytoadherent P. falciparum line 3D7 which consists of at least nine exons spanning a region of 7 kb [8]. This gene we named clag9 (cytoadherence-linked asexual gene). We performed a TGD of clag9 in P. falciparum line 3D7 [9], and erythrocytes infected with clag9 knockout parasites showed drastically reduced levels of binding to both C32 melanoma cells and to CD36. While this evidence strongly supported the hypothesis that clag9 is essential for cytoadherence to CD36, essential proof of gene function is nevertheless the neutralisation and complementation of the same gene within the same parasite line. As an alternative to targeted gene disruption of clag9, an antisense RNA approach was taken here to confirm the conclusions from the TGD. If feasible, this approach should be of utility in other studies on malaria biology as it allows the phenotype to be re-evaluated after the introduced
genetic change is subsequently removed, providing direct evidence that the phenotypic change is not the result of change at an unrelated site. Antisense RNA inhibition of gene function is thought to operate by a number of possible mechanisms including hybridization between sense and antisense strands [10,11], inactivation due to ribonucleases activated by double stranded RNA [12], or post-transcriptional inhibition [13,14]. Here we report the antisense inhibition of clag9, a gene associated with cytoadherence of P. falciparum infected erythrocytes to C32 melanoma cells and CD36. This phenotype could be measured at 6 weeks after transfection, less than half the minimal time which would have been required for a targeted gene disruption.
2. Materials and methods
2.1. HC1 -AS 6ector construct Oligonucleotides 5X% (AAC TCG AGC TAT AAG TAT GAT GAT AGA AGC TAG) and 3X% (AAC TCG AGT TAA AAA GGA TCA TAA CGA TAA CGT TGC) containing Xho I sites as well as a clag9 sequence were used to amplify a 1.8-kb clag segment containing exons 8 and 9 from 3D7 cDNA by PCR (corresponding to nucleotides 2132–4038, GenBank™ accession number AF055475). The resulting PCR fragment was blunt-end cloned into the Sma I site of pBluescript. It was then excised with Xho I and ligated into the Xho I site of plasmid HC1 (Fig. 1) [4] which is under the control of the calmodulin promoter and allows constitutive transgene expression. Transformants were screened using primers Xho F (GTA TAT TGG GGT GAT GAT AAA ATG) and Xho R (ATA TTT TAT TTA TTA TCA TTC AAG) which span the Xho I site of HC1. In this construct the clag9 exons are inserted in the 3%-5% orientation, 3% to the powerful calmodulin promoter. Clones in which the clag9 fragment was inserted in the antisense orientation to the calmodulin promoter were sequenced to confirm the orientation. The HC7 plasmid contains a small unrelated sequence of P. falciparum DNA inserted into the
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Xho I site of HC1 vector. This vector was obtained from Dr Alan Cowman, and was used as a transfection control. It was electroporated into 3D7 at the same time as the HC1-AS plasmid.
2.2. Parasite culture and electroporation P. falciparum clone 3D7, obtained from D. Walliker, was cultured essentially as described [15]. Ring-stage parasites were subjected to electroporation in the presence of 150 mg plasmid HC1-AS DNA as described [1,4] and cultured in the presence of 0.1 mM pyrimethamine for 10
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weeks. Briefly, 200 mg of plasmid DNA was ethanol precipitated and resuspended in 30 ml TE (10 mM Tris–HCl, pH 8.0, 1 mM EDTA pH 8.0), then mixed gently with 770 ml of cytomix [1]. A 30-ml culture of 3D7 containing 3% synchronous ring stage parasites was pelleted and 400 ml of packed red cells added to the DNA-cytomix solution. The total volume of 1.4 ml was transferred to a sterile 0.4-cm cuvette and electroporated at 2.5 kV, 200 V and 25 mF in a BioRad gene Pulser II. Fresh medium and red cells were then added and the parasites grown under standard conditions for 2 days prior to the addition of pyrimethamine.
2.2.1. Cytoadherence assays Melanoma cell binding assays were carried out using a published method [16]. 2.3. Southern blotting and PCR analysis
Fig. 1. Antisense construct HC1-AS does not integrate the P. falciparum genome. (A) Antisense construct HC1-AS based on vector HC1. Small arrows show position of PCR primers, large arrows indicate vector sequence. Note Cso 46 and Xho R are divergent. HSP86 3%, P. falciparum Heat shock protein 86 3% untranslated region; 5X%-3X%, clag9 exon 8 and 9; Cam 5%, P. falciparum calmodulin 5% untranslated region; PcDT 5%, P. chabaudi dihydofolate reductase 5% untranslated region; Tg DHFR-TS, T. gondii dihydrofolate reductase thymidilate synthase gene; HRP2 3%, P. falciparum histidine rich protein 2 3% untranslated region. (B) Integration test. M, l/Hind III. Lanes 1–4: 3D7 DNA; lanes 5, 6: 3D7.HC1-AS DNA 9 weeks; lanes 9–12: 3D7.HC1-AS DNA 15 weeks. Lanes 1, 5, 9: primers Cso 46 + Xho R, circular vector test; lanes 2, 6, 10: primers Xho F+Xho R vector insert test. Lanes 3, 7, 11: primers 5X%+3X% clag gene test. Lanes 4, 8, 12: primers S2E5+ Xho R integration test. PCR product will only be produced in lanes 8 and 12 if the vector has integrated into chromosome 9 as primer S2E5 lies 5% to the region of clag inserted into the HC1 vector.
P. falciparium chromosome blocks were prepared [17] and the DNA digested with restriction endonucleases as described [18]. Briefly blocks were dialysed overnight at room temperature in 20 ml TE, then preincubated for 30 min at room temperature in 100 ml of the appropriate restriction enzyme buffer containing 10 mg ml − 1 acetylated BSA and 0.01% Triton X-100. The blocks were then transferred to fresh buffer containing 20–30 U of the appropriate restriction enzyme and incubated at the optimum temperature for each enzyme for 4–6 h. Blocks were then stored at 4°C until analysed by either pulsed field gel electrophoresis (PFGE), or standard agarose gel electrophoresis. Digested chromosomes analysed by PFGE were run for 16 h at 5.4 V cm − 1 with a pulse time of 7 s. After electrophoresis the gels were subjected to short wave UV irradiation for 2 min and the DNA was denatured in alkaline solution for 45 min, followed by neutralization for 45 min. DNA was transferred to Hybond N+ membrane (Amersham) by dry blotting overnight. The filters were fixed by baking at 80°C for 2 h. DNA probes labelled with a-32P-dATP were produced by random priming of purified PCR amplified DNA, according to the manufacturer’s specifications (Giga Prime-Bresatec).
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Genomic DNA for PCR analysis was extracted from 200-ml aliquots of culture at \ 1% parasitemia and 5% hematocrit using a QIAamp Kit (Qiagen). In all subsequent PCR reactions, 1 ml of extract was used.
2.4. Northern blotting Total RNA was prepared according to the method of Kyes et al. [19]. Then 10 – 20 mg of total RNA was heated to 65°C for 10 min, and separated by gel electrophoresis on a 1% TBE gel, containing 10 mM guanidine thiocyanate. After electrophoresis the gel was soaked in 50 mM NaOH for 30 min, then transferred to Hybond N + by dry blotting overnight. The blot was air-dried. Prehybrization, hybridization and washing were performed using a MaxiNorthern Kit (Ambion). The Northern blot was probed with a purified PCR product amplified from a pBluescipt clone containing 3D7 clag9 cDNA corresponding to nucleotides 2132– 4038 using M13 forward and reverse primers. This product was labelled with a-32P-dATP produced by random priming, according to the manufacturer’s specifications (Giga Prime-Bresatec). The probe was hybridized overnight at 41°C in hybridization buffer containing formamide (Ambion). The filter was washed and then exposed to film for 2 days. The filter was stripped then re-probed using a probe which would bind specifically to the antisense RNA. This probe was produced by PCR. Purified PCR product as described above, primed with only the 5X% primer, was cycled five times, in the presence of dNTPs containing a-32P-dATP. The probe was hybridized to the filter as outlined above.
2.5. Western blotting A total of 50 ml of gelatin selected trophozoite stage parasites was extracted on ice for 30 min with 2 ml of ice-cold TTP (PBS containing 1% Triton X-100 and protease inhibitors (Complete Mini, Boehringer Mannheim). The suspension was vortexed vigorously every 10 min, then centrifuged at 7500× g for 10 min and the supernatant discarded. The pellet was washed once in
TTP. The pellet was then resuspended in 150 ml of 2% SDS/PBS with protease inhibitors and incubated for 45 min at room temperature with frequent vortexing and pipetting to solubilize the pellet. The solution was centrifuged for 15 min at 7500× g and the supernatant kept at − 70°C until used. After 20 ml of the Triton X-100 insoluble/SDS soluble fraction was added to 2 ml of 10× loading buffer, it was heated to 100°C for 10 min. This was then fractionated under denaturing conditions on a 6% Tris-glycine polyacrylamide gel. The fractionated protein was then transferred to nitrocellulose by wet blotting and blocked overnight in 5% skim milk in TNT (Tris-buffered saline/Tween 20). Antisera to clag9, produced in mice, as described previously [9], was used at 1/2000 dilution as the primary antibody. The secondary antibody was an HRP-labelled anti-mouse antibody (Transduction Laboratories) used at a 1/5000 dilution.
3. Results
3.1. Transfection Approximately 4 weeks after transfection, drug resistant parasites were obtained in the cultures electroporated with either the HC7 or HC1-AS plasmids. The line electroporated with the HC1AS plasmid was designated 3D7.HC1-AS and maintained under drug selection. The line electroporated with the HC7 plasmid was designated 3D7.HC7 and also maintained under drug selection. After 10 weeks of continuous culture in the presence of pyrimethamine, 3D7.HC1-AS was split into two cultures. One culture was maintained on drug selection (3D7.HC1-AS) while the other, designated 3D7.HC1-ASp-, had drug pressure removed to facilitate loss of the episomally carried vector.
3.2. PCR and Southern blot analysis shows that integration has not occurred Line 3D7.HC1-AS was tested by PCR and by Southern blotting after pulsed field electrophore
D.L. Gardiner et al. / Molecular and Biochemical Parasitology 110 (2000) 33–41
Fig. 2. Southern blotting experiments to test for the presence of episomal and integrated forms of HC1-AS. (A) 3D7 (lane 1) and 3D7.HC1-AS (lane 2) genomic DNA digested with Bgl II (which linearises the HC1-AS plasmid) was fractionated by agarose gel electophoresis, blotted and probed for the clag9 insert present in the plasmid. (B) Genomic DNA digested with Bgl I was fractionated by pulsed field electrophoresis and probed for the 5% region of clag9 that is not present in the plasmid. 3D7 (lane 1) and 3D7.HC1-AS (lane 2) have an 180-kb Bgl I fragment whereas a 3D7 clone with a vector integrated into the clag9 gene (lane 3) has a 70-kb fragment as the integrated vector introduces an additional Bgl I site.
sis to examine whether the vector had integrated into the clag9 gene. This construct was not designed for a targeted gene disruption and contained an intact 3% end of the clag9 gene; hence if it did integrate into clag9, one of the two resulting recombinant copies would have been a complete gene (although with promoters at each end). The PCRs demonstrated the presence of the construct HC1-AS and of an intact chromosomal clag9 gene (Fig. 1). However a test for homologous recombination between a region of clag9 5% to that included in HC1-AS and a HC1 vector specific sequence was negative (Fig. 1). The presence of plasmid was also demonstrated by hybridization of a clag9 segment present in the construct to DNA from 3D7 and from HC1-AS after digestion with Bgl II (Fig. 2A). Hybridization to HC1-AS was far stronger than to 3D7, demonstrating multiple copies of the plasmid. Furthermore, the 180-kb fragment detected by Southern blotting after pulsed field electrophoresis of a Bgl I digest of HC1-AS by a probe for
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clag9 that was not present in the construct was identical to that in 3D7 (Fig. 2B). An increase in size of the 3D7.HC1-AS line fragment would have been expected if the 8-kb plasmid had integrated in the chromosomal copy of clag9. In contrast, as expected, a 70-kb fragment was detected in another line with a plasmid integrated into the clag9 gene [9] because that construct, unlike the HC1AS construct, contains a Bgl I site (Fig. 2B). As the PCRs were carried out on DNA prepared from 3D7.HC1-AS at 9 and 15 weeks of culture and the blot was carried out on DNA prepared at 23 weeks, we conclude that the plasmid had not integrated during the course of these experiments, but continued replicating as an episome.
3.3. A no6el clag9 RNA species in the transfection line To confirm that antisense clag9 RNA was being produced, 3D7, 3D7.HC7, 3D7.HC1-AS, and 3D7.HC1-ASp- derived RNA was probed with the sequence corresponding to the clag9 insert in the vector. As a region of clag9 corresponding to the cDNA sequence of exon 8 and 9 was used for the probe, this would detect both sense and antisense mRNA strands (Fig. 3A). All four lines produced clag9 mRNA as shown by the band of 5 kb on the Northern blot, although this band appears to be less intense in the 3D7.HC1-AS line. In addition there was a second more intense band of 2 kb only present in the 3D7.HC1-AS line. This band corresponds to the anticipated size of the mRNA signal that would be expected given the 1.8-kb size of the clag9 insert in the vector. After 10 weeks without drug selection, 3D7.HCASp- does not contain this second band, consistent with the loss of the vector and hence antisense mRNA production over time. This was confirmed using a probe that was complementary only to the antisense mRNA, and, as expected only the lower band hybridized to the probe after the original blot was stripped (Fig. 3B)
3.4. Reduction in CLAG9 protein expression Previous work has shown that CLAG9 migrates as an 220-kDa protein in the Triton
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X-100 insoluble/SDS soluble fraction of extracted trophozoite stage parasites [9]. Using antibodies generated by DNA vaccination of mice [9] the CLAG9 signal was greatly diminished in the 3D7.HC1-AS line when compared to the parental 3D7 line, or the 3D7.HC1-ASp- (10 weeks without drug) line, again confirming that the antisense mRNA had greatly inhibited production of the CLAG9 protein, and that removal of the vector allows restoration of the gene product CLAG9 (Fig. 4).
Table 1 Inhibition of cytoadherence to melanoma cells after transfection of 3D7 with antisense construct HC1-ASa Parasite line
3D7.HC1-AS 3D7.HC7 3D7
Weeks post transfection 6
10
15
20
36 91 ND 108 9 4
18 9 2 ND 187 96
109 2 ND ND
16 9 5 141 94 188 9 6
Weeks without pyrimethamine 5
3.5. Decrease in cytoadherence in the antisense line 3D7.HC1-AS was tested for cytoadherence to C32 melanoma cells after 6 weeks of culture with pyrimethamine. In duplicate experiments it initially showed threefold lower binding to
Fig. 3. Northern blot of total RNA extracted from 3D7, 3D7.HC7, 3D7.HC1-AS, and 3D7.HC1-ASp-. (A) The blot was probed with the randomly primed region of clag9 present in the antisense vector HC1-AS: 3D7.HC7 (lane 1), 3D7.HC1ASp- (lane 2), 3D7.HC1-AS (lane 3) and 3D7 (lane 4). (B) The blot was probed with clag9 cDNA complementary to only the antisense mRNA produced by the HC1-AS vector: 3D7.HC7 (lane 1), 3D7HC1-ASp- (lane 2), 3D7.HC1-AS (lane 3) and 3D7 (lane 4).
3D7.HC1-ASp-
–
–
26 9 3
10 143 9 3
a P. falciparum clone 3D7 was electroporated with HC1-AS or HC7 and cultured in 0.1 mM pyrimethamine. After synchronization by gelatin flotation parasites were grown to 5–8% parasitemia and cytoadherence to melanoma cells was then measured as described [16]. Results are shown as parasitized cells bound per 100 melanoma cells. Experiments were carried out in duplicate and mean values 9S.D. are shown.
melanoma cells than did the parental clone 3D7 (Table 1). Over the next 4 weeks binding decreased to 15-fold lower, using, as an additional control, 3D7 transfected with an unrelated plasmid HC7 (which contains an unrelated sequence in the same vector) at the same time as HC1-AS, and cultured in parallel to the control (Table 1). This is consistent with the evidence that clag9 is essential for cytoadherence to C32 melanoma cells. To confirm that the lowered binding was determined by the presence of the plasmid we cultured 3D7.HC1-AS in the absence of pyrimethamine for several weeks. Under these conditions it has been demonstrated that plasmid, and hence pyrimethamine resistance, is eventually lost, unless the plasmid has integrated into the chromosome. After 4 weeks the line 3D7.HC1-ASpexhibited 2.6-fold greater binding to melanoma cells than 3D7.HC1-AS cultured throughout this time with pyrimethamine (Table 1) and by 10 weeks it had regained a level of cytoadherence comparable to that of 3D7 (Table 1). At this time no plasmid could be detected by PCR in 3D7.HC1-ASp-. When transferred back into medium containing pyrimethamine after 10 weeks
D.L. Gardiner et al. / Molecular and Biochemical Parasitology 110 (2000) 33–41
it also failed to grow. This result, in conjunction with both the Western and Northern blot results, strongly supports the conclusion that the presence of plasmid HC1-AS is responsible for the change in phenotype. This change can be attributed to clag9 sequence rather than other plasmid sequences as 3D7.HC7 retained cytoadherence (Table 1). The slow loss of function below 2.6fold and the slow regain of function can be readily explained by amplification of the plasmid which occurs under prolonged selection (Fig. 2A) and would presumably result in higher intracellular levels of antisense RNA.
3.6. The antisense line was knob positi6e Another possibility to exclude was that 3D7.HC1-AS had undergone a subtelomeric deletion of the region of chromosome 2 containing the KAHRP gene and that the loss of ability to bind was due to its conversion to a knobless phenotype [20]. This was highly unlikely as a gelatin separation was always employed to enrich the parasitized cells before binding was measured, and it has been established that this selects for cells which are knob positive [21]. Further, the KAHRP gene was present as determined by PCR (data not shown). In order to establish that
Fig. 4. Western blot of SDS soluble/Triton X-100 insoluble fraction of gelatin selected trophozoites from 3D7, 3D7.HC1AS, and 3D7.HC1-ASp- parasite lines: 3D7 (lane 1), 3D7.HC1-AS (lane 2), and 3D7.HC1-ASp- (lane 3) probed with antisera generated by DNA vaccination of mice [9].
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3D7.HC1-AS continued to produce knobs it was examined by transmission electron microscopy. All 3D7.HC1-AS cells examined had characteristic electron-dense knobs whereas these were not present in the knobless control B8 (data not shown).
4. Discussion We have used the transfection system recently developed for P. falciparum [1–4] in order to introduce an antisense construct of clag9 into 3D7. This is the first time that this approach has been reported in P. falciparum. It presents a major advantage over targeted gene disruptions in that the phenotype could first be determined at 6 weeks. Such an experiment is presumed to rely on the formation of intracellular RNA duplexes with authentic clag9 mRNA resulting in the inhibition of translation of the clag9 mRNA. We conclude this to be the most likely hypothesis as substantial quantities of clag9 mRNA were present (Fig. 3A). The RNA duplexes in turn may affect other processes such as splicing and transport. Nevertheless the observed inhibition strongly supports the conclusion that clag9 function is essential for cytoadherence to melanoma cells. An alternative explanation that must be considered in studies on cytoadherence is that switching of 6ar genes in the line under study has led to loss of cytoadherence. There are several reasons why it is highly unlikely that switching could have played any significant role in the observed phenotypes. As it is clear that multiple transfectants are generated during electroporation ([22], and our unpublished observations), this line must result from multiple independent transfection events and the majority of these cells would then have had to switch off the relevant 6ar gene. No such switching off of binding was observed in transfected 3D7 cells over considerably longer periods of culture than those employed here ([2], and our unpublished observations). Further, 3D7.HC7 was generated simultaneously and cultured for the same time but nevertheless retained cytoadherence (Table 1).
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D.L. Gardiner et al. / Molecular and Biochemical Parasitology 110 (2000) 33–41
Nevertheless this possibility must be excluded unequivocally and is currently being addressed. The most significant reason for being confident that the ablation of cytoadherence was due to clag9 antisense inhibition is that the antisense approach has allowed us to re-evaluate the phenotype after subsequently removing the plasmid. The fact that reversion to wildtype levels of cytoadherence accompanied reversion to pyrimethamine sensitivity provides a very direct line of evidence that it was not due to events at some second site. This confirms results obtained by conventional targeted disruption of the clag9 gene [9]. Our antisense RNA approach could become an important new tool in malaria research, in particular if suitable controllable promoters such as a tetracycline inducible promoter, can be utilized [23]. That the phenotype could be determined 6 weeks after electroporation is an important consideration. Integration of the plasmid in a targeted gene disruption may not occur until between 8 and 20 weeks after electroporation and it is usually necessary to clone the line after integration has been confirmed, making the whole process lengthy and time consuming. However, why integration of the HC1-AS construct did not eventually occur in this case is not clear. Inhibition of gene function in P. falciparum by antisense vectors will allow us to increase our knowledge of the biology of this most important human pathogen, and further our understanding of the genes involved in the pathogenesis of this organism.
Acknowledgements The authors would like to thank Sue Keyes and Chris Newbold for their advice on Northern blotting analysis, and Alan Cowman for the HC1 vector. This work was supported by a generous donation from Mark Nicholson and Alice Hill, the Australian National Health and Medical Research Council, the Northern Territory Government and the Tudor Foundation.
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Inhibition of Plasmodium falciparum clag9 gene function by antisense RNA Donald L. Gardiner *, Deborah C. Holt, Elizabeth A. Thomas, David J. Kemp, Katharine R. Trenholme Menzies School of Health Research, Casuarina 0812, Darwin, Northern Territory 0812, Australia Received 19 January 2000; received in revised form 7 April 2000; accepted 26 April 2000
Abstract We have previously shown by targeted gene disruption that the clag9 gene of Plasmodium falciparum is essential for cytoadherence to CD36. Here we report inhibition of the function of clag9 by the use of an antisense RNA vector as an alternative to targeted gene disruption. We transfected an antisense construct of clag9 into the P. falciparum clone 3D7 and when the resulting line was cultured in the presence of pyrimethamine it showed 15-fold lower cytoadherence to C32 melanoma cells than the control. Reversion to wildtype upon removal of the introduced plasmid provides direct evidence that the event responsible for the phenotypic change is not at an unrelated site and this approach provides a valuable new tool in malaria transfection technology. © 2000 Elsevier Science B.V. All rights reserved. Keywords: Plasmodim falciparum; Antisense RNA; clag9 ; Cytoadherence
1. Introduction A major advance in studying malaria biology has been the use of transfection technology, utilising vectors containing the Toxoplasma gondii di-
Abbre6iations: clag, cytoadherence linked asexual gene; DHFR, dihydrofolate reductase; KAHRP, knob-associated histidine-rich protein; TGD, targeted gene disruption. Note: Nucleotide sequence data reported in this paper have been submitted to the GenBank™, EMBL and DDJB data bases with the accession number AF055476 * Corresponding author. Tel.: + 61-8-89228074; fax: + 618-89275187. E-mail address: [email protected] (D.L. Gardiner)
hydrofolate reductase thymidylate synthetase gene (Tg DHFR-TS). These vectors confer pyrimethamine resistance as a selectable marker and allow for transient expression of exogenous genes in Plasmodium falciparum [1], targeted disruptions of P. falciparum genes [2,3], and constitutive transgene expression [4]. While other selectable markers such as blasticidin [5] are now becoming available and have the potential to allow the complementation of genes that have been disrupted, as yet there is no published experimental evidence showing that these new vectors can be used in this fashion. Without a positive and negative selection vector, such as those available for T.
0166-6851/00/$ - see front matter © 2000 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 6 - 6 8 5 1 ( 0 0 ) 0 0 2 5 4 - 1
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gondii [6], disruption and complementation of genes within the same parasite, as well as sequential disruption of genes within one parasite clone, are not yet possible. As current targeted gene disruptions (TGD) in P. falciparum require 4 – 8 months of continuous culture, alternative methods that may more easily and rapidly answer complex biological questions are required. Here we describe an alternative method that allows inhibition of gene function by the use of an antisense RNA vector and the subsequent restoration of gene function when the antisense vector is removed. This requires the use of one group of the current generation of P. falciparum transfection vectors which allow stable transgene expression in P. falciparum. These vectors consist of two expression cassettes, the first, under the Plasmodium chabaudi DHFR promoter, express the drug resistance gene, while the second expression cassette, under the control of the P. falciparum calmodulin promoter region, allows stable transgene expression [4]. Previous work has shown that loss of cytoadherence to melanoma cells (which express CD36) by P. falciparum infected erythrocytes in vitro, was associated with deletions of the right arm of chromosome 9 in these cell lines [7]. Using positional cloning we located a gene on the right arm of chromosome 9 of the stably cytoadherent P. falciparum line 3D7 which consists of at least nine exons spanning a region of 7 kb [8]. This gene we named clag9 (cytoadherence-linked asexual gene). We performed a TGD of clag9 in P. falciparum line 3D7 [9], and erythrocytes infected with clag9 knockout parasites showed drastically reduced levels of binding to both C32 melanoma cells and to CD36. While this evidence strongly supported the hypothesis that clag9 is essential for cytoadherence to CD36, essential proof of gene function is nevertheless the neutralisation and complementation of the same gene within the same parasite line. As an alternative to targeted gene disruption of clag9, an antisense RNA approach was taken here to confirm the conclusions from the TGD. If feasible, this approach should be of utility in other studies on malaria biology as it allows the phenotype to be re-evaluated after the introduced
genetic change is subsequently removed, providing direct evidence that the phenotypic change is not the result of change at an unrelated site. Antisense RNA inhibition of gene function is thought to operate by a number of possible mechanisms including hybridization between sense and antisense strands [10,11], inactivation due to ribonucleases activated by double stranded RNA [12], or post-transcriptional inhibition [13,14]. Here we report the antisense inhibition of clag9, a gene associated with cytoadherence of P. falciparum infected erythrocytes to C32 melanoma cells and CD36. This phenotype could be measured at 6 weeks after transfection, less than half the minimal time which would have been required for a targeted gene disruption.
2. Materials and methods
2.1. HC1 -AS 6ector construct Oligonucleotides 5X% (AAC TCG AGC TAT AAG TAT GAT GAT AGA AGC TAG) and 3X% (AAC TCG AGT TAA AAA GGA TCA TAA CGA TAA CGT TGC) containing Xho I sites as well as a clag9 sequence were used to amplify a 1.8-kb clag segment containing exons 8 and 9 from 3D7 cDNA by PCR (corresponding to nucleotides 2132–4038, GenBank™ accession number AF055475). The resulting PCR fragment was blunt-end cloned into the Sma I site of pBluescript. It was then excised with Xho I and ligated into the Xho I site of plasmid HC1 (Fig. 1) [4] which is under the control of the calmodulin promoter and allows constitutive transgene expression. Transformants were screened using primers Xho F (GTA TAT TGG GGT GAT GAT AAA ATG) and Xho R (ATA TTT TAT TTA TTA TCA TTC AAG) which span the Xho I site of HC1. In this construct the clag9 exons are inserted in the 3%-5% orientation, 3% to the powerful calmodulin promoter. Clones in which the clag9 fragment was inserted in the antisense orientation to the calmodulin promoter were sequenced to confirm the orientation. The HC7 plasmid contains a small unrelated sequence of P. falciparum DNA inserted into the
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Xho I site of HC1 vector. This vector was obtained from Dr Alan Cowman, and was used as a transfection control. It was electroporated into 3D7 at the same time as the HC1-AS plasmid.
2.2. Parasite culture and electroporation P. falciparum clone 3D7, obtained from D. Walliker, was cultured essentially as described [15]. Ring-stage parasites were subjected to electroporation in the presence of 150 mg plasmid HC1-AS DNA as described [1,4] and cultured in the presence of 0.1 mM pyrimethamine for 10
35
weeks. Briefly, 200 mg of plasmid DNA was ethanol precipitated and resuspended in 30 ml TE (10 mM Tris–HCl, pH 8.0, 1 mM EDTA pH 8.0), then mixed gently with 770 ml of cytomix [1]. A 30-ml culture of 3D7 containing 3% synchronous ring stage parasites was pelleted and 400 ml of packed red cells added to the DNA-cytomix solution. The total volume of 1.4 ml was transferred to a sterile 0.4-cm cuvette and electroporated at 2.5 kV, 200 V and 25 mF in a BioRad gene Pulser II. Fresh medium and red cells were then added and the parasites grown under standard conditions for 2 days prior to the addition of pyrimethamine.
2.2.1. Cytoadherence assays Melanoma cell binding assays were carried out using a published method [16]. 2.3. Southern blotting and PCR analysis
Fig. 1. Antisense construct HC1-AS does not integrate the P. falciparum genome. (A) Antisense construct HC1-AS based on vector HC1. Small arrows show position of PCR primers, large arrows indicate vector sequence. Note Cso 46 and Xho R are divergent. HSP86 3%, P. falciparum Heat shock protein 86 3% untranslated region; 5X%-3X%, clag9 exon 8 and 9; Cam 5%, P. falciparum calmodulin 5% untranslated region; PcDT 5%, P. chabaudi dihydofolate reductase 5% untranslated region; Tg DHFR-TS, T. gondii dihydrofolate reductase thymidilate synthase gene; HRP2 3%, P. falciparum histidine rich protein 2 3% untranslated region. (B) Integration test. M, l/Hind III. Lanes 1–4: 3D7 DNA; lanes 5, 6: 3D7.HC1-AS DNA 9 weeks; lanes 9–12: 3D7.HC1-AS DNA 15 weeks. Lanes 1, 5, 9: primers Cso 46 + Xho R, circular vector test; lanes 2, 6, 10: primers Xho F+Xho R vector insert test. Lanes 3, 7, 11: primers 5X%+3X% clag gene test. Lanes 4, 8, 12: primers S2E5+ Xho R integration test. PCR product will only be produced in lanes 8 and 12 if the vector has integrated into chromosome 9 as primer S2E5 lies 5% to the region of clag inserted into the HC1 vector.
P. falciparium chromosome blocks were prepared [17] and the DNA digested with restriction endonucleases as described [18]. Briefly blocks were dialysed overnight at room temperature in 20 ml TE, then preincubated for 30 min at room temperature in 100 ml of the appropriate restriction enzyme buffer containing 10 mg ml − 1 acetylated BSA and 0.01% Triton X-100. The blocks were then transferred to fresh buffer containing 20–30 U of the appropriate restriction enzyme and incubated at the optimum temperature for each enzyme for 4–6 h. Blocks were then stored at 4°C until analysed by either pulsed field gel electrophoresis (PFGE), or standard agarose gel electrophoresis. Digested chromosomes analysed by PFGE were run for 16 h at 5.4 V cm − 1 with a pulse time of 7 s. After electrophoresis the gels were subjected to short wave UV irradiation for 2 min and the DNA was denatured in alkaline solution for 45 min, followed by neutralization for 45 min. DNA was transferred to Hybond N+ membrane (Amersham) by dry blotting overnight. The filters were fixed by baking at 80°C for 2 h. DNA probes labelled with a-32P-dATP were produced by random priming of purified PCR amplified DNA, according to the manufacturer’s specifications (Giga Prime-Bresatec).
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D.L. Gardiner et al. / Molecular and Biochemical Parasitology 110 (2000) 33–41
Genomic DNA for PCR analysis was extracted from 200-ml aliquots of culture at \ 1% parasitemia and 5% hematocrit using a QIAamp Kit (Qiagen). In all subsequent PCR reactions, 1 ml of extract was used.
2.4. Northern blotting Total RNA was prepared according to the method of Kyes et al. [19]. Then 10 – 20 mg of total RNA was heated to 65°C for 10 min, and separated by gel electrophoresis on a 1% TBE gel, containing 10 mM guanidine thiocyanate. After electrophoresis the gel was soaked in 50 mM NaOH for 30 min, then transferred to Hybond N + by dry blotting overnight. The blot was air-dried. Prehybrization, hybridization and washing were performed using a MaxiNorthern Kit (Ambion). The Northern blot was probed with a purified PCR product amplified from a pBluescipt clone containing 3D7 clag9 cDNA corresponding to nucleotides 2132– 4038 using M13 forward and reverse primers. This product was labelled with a-32P-dATP produced by random priming, according to the manufacturer’s specifications (Giga Prime-Bresatec). The probe was hybridized overnight at 41°C in hybridization buffer containing formamide (Ambion). The filter was washed and then exposed to film for 2 days. The filter was stripped then re-probed using a probe which would bind specifically to the antisense RNA. This probe was produced by PCR. Purified PCR product as described above, primed with only the 5X% primer, was cycled five times, in the presence of dNTPs containing a-32P-dATP. The probe was hybridized to the filter as outlined above.
2.5. Western blotting A total of 50 ml of gelatin selected trophozoite stage parasites was extracted on ice for 30 min with 2 ml of ice-cold TTP (PBS containing 1% Triton X-100 and protease inhibitors (Complete Mini, Boehringer Mannheim). The suspension was vortexed vigorously every 10 min, then centrifuged at 7500× g for 10 min and the supernatant discarded. The pellet was washed once in
TTP. The pellet was then resuspended in 150 ml of 2% SDS/PBS with protease inhibitors and incubated for 45 min at room temperature with frequent vortexing and pipetting to solubilize the pellet. The solution was centrifuged for 15 min at 7500× g and the supernatant kept at − 70°C until used. After 20 ml of the Triton X-100 insoluble/SDS soluble fraction was added to 2 ml of 10× loading buffer, it was heated to 100°C for 10 min. This was then fractionated under denaturing conditions on a 6% Tris-glycine polyacrylamide gel. The fractionated protein was then transferred to nitrocellulose by wet blotting and blocked overnight in 5% skim milk in TNT (Tris-buffered saline/Tween 20). Antisera to clag9, produced in mice, as described previously [9], was used at 1/2000 dilution as the primary antibody. The secondary antibody was an HRP-labelled anti-mouse antibody (Transduction Laboratories) used at a 1/5000 dilution.
3. Results
3.1. Transfection Approximately 4 weeks after transfection, drug resistant parasites were obtained in the cultures electroporated with either the HC7 or HC1-AS plasmids. The line electroporated with the HC1AS plasmid was designated 3D7.HC1-AS and maintained under drug selection. The line electroporated with the HC7 plasmid was designated 3D7.HC7 and also maintained under drug selection. After 10 weeks of continuous culture in the presence of pyrimethamine, 3D7.HC1-AS was split into two cultures. One culture was maintained on drug selection (3D7.HC1-AS) while the other, designated 3D7.HC1-ASp-, had drug pressure removed to facilitate loss of the episomally carried vector.
3.2. PCR and Southern blot analysis shows that integration has not occurred Line 3D7.HC1-AS was tested by PCR and by Southern blotting after pulsed field electrophore
D.L. Gardiner et al. / Molecular and Biochemical Parasitology 110 (2000) 33–41
Fig. 2. Southern blotting experiments to test for the presence of episomal and integrated forms of HC1-AS. (A) 3D7 (lane 1) and 3D7.HC1-AS (lane 2) genomic DNA digested with Bgl II (which linearises the HC1-AS plasmid) was fractionated by agarose gel electophoresis, blotted and probed for the clag9 insert present in the plasmid. (B) Genomic DNA digested with Bgl I was fractionated by pulsed field electrophoresis and probed for the 5% region of clag9 that is not present in the plasmid. 3D7 (lane 1) and 3D7.HC1-AS (lane 2) have an 180-kb Bgl I fragment whereas a 3D7 clone with a vector integrated into the clag9 gene (lane 3) has a 70-kb fragment as the integrated vector introduces an additional Bgl I site.
sis to examine whether the vector had integrated into the clag9 gene. This construct was not designed for a targeted gene disruption and contained an intact 3% end of the clag9 gene; hence if it did integrate into clag9, one of the two resulting recombinant copies would have been a complete gene (although with promoters at each end). The PCRs demonstrated the presence of the construct HC1-AS and of an intact chromosomal clag9 gene (Fig. 1). However a test for homologous recombination between a region of clag9 5% to that included in HC1-AS and a HC1 vector specific sequence was negative (Fig. 1). The presence of plasmid was also demonstrated by hybridization of a clag9 segment present in the construct to DNA from 3D7 and from HC1-AS after digestion with Bgl II (Fig. 2A). Hybridization to HC1-AS was far stronger than to 3D7, demonstrating multiple copies of the plasmid. Furthermore, the 180-kb fragment detected by Southern blotting after pulsed field electrophoresis of a Bgl I digest of HC1-AS by a probe for
37
clag9 that was not present in the construct was identical to that in 3D7 (Fig. 2B). An increase in size of the 3D7.HC1-AS line fragment would have been expected if the 8-kb plasmid had integrated in the chromosomal copy of clag9. In contrast, as expected, a 70-kb fragment was detected in another line with a plasmid integrated into the clag9 gene [9] because that construct, unlike the HC1AS construct, contains a Bgl I site (Fig. 2B). As the PCRs were carried out on DNA prepared from 3D7.HC1-AS at 9 and 15 weeks of culture and the blot was carried out on DNA prepared at 23 weeks, we conclude that the plasmid had not integrated during the course of these experiments, but continued replicating as an episome.
3.3. A no6el clag9 RNA species in the transfection line To confirm that antisense clag9 RNA was being produced, 3D7, 3D7.HC7, 3D7.HC1-AS, and 3D7.HC1-ASp- derived RNA was probed with the sequence corresponding to the clag9 insert in the vector. As a region of clag9 corresponding to the cDNA sequence of exon 8 and 9 was used for the probe, this would detect both sense and antisense mRNA strands (Fig. 3A). All four lines produced clag9 mRNA as shown by the band of 5 kb on the Northern blot, although this band appears to be less intense in the 3D7.HC1-AS line. In addition there was a second more intense band of 2 kb only present in the 3D7.HC1-AS line. This band corresponds to the anticipated size of the mRNA signal that would be expected given the 1.8-kb size of the clag9 insert in the vector. After 10 weeks without drug selection, 3D7.HCASp- does not contain this second band, consistent with the loss of the vector and hence antisense mRNA production over time. This was confirmed using a probe that was complementary only to the antisense mRNA, and, as expected only the lower band hybridized to the probe after the original blot was stripped (Fig. 3B)
3.4. Reduction in CLAG9 protein expression Previous work has shown that CLAG9 migrates as an 220-kDa protein in the Triton
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X-100 insoluble/SDS soluble fraction of extracted trophozoite stage parasites [9]. Using antibodies generated by DNA vaccination of mice [9] the CLAG9 signal was greatly diminished in the 3D7.HC1-AS line when compared to the parental 3D7 line, or the 3D7.HC1-ASp- (10 weeks without drug) line, again confirming that the antisense mRNA had greatly inhibited production of the CLAG9 protein, and that removal of the vector allows restoration of the gene product CLAG9 (Fig. 4).
Table 1 Inhibition of cytoadherence to melanoma cells after transfection of 3D7 with antisense construct HC1-ASa Parasite line
3D7.HC1-AS 3D7.HC7 3D7
Weeks post transfection 6
10
15
20
36 91 ND 108 9 4
18 9 2 ND 187 96
109 2 ND ND
16 9 5 141 94 188 9 6
Weeks without pyrimethamine 5
3.5. Decrease in cytoadherence in the antisense line 3D7.HC1-AS was tested for cytoadherence to C32 melanoma cells after 6 weeks of culture with pyrimethamine. In duplicate experiments it initially showed threefold lower binding to
Fig. 3. Northern blot of total RNA extracted from 3D7, 3D7.HC7, 3D7.HC1-AS, and 3D7.HC1-ASp-. (A) The blot was probed with the randomly primed region of clag9 present in the antisense vector HC1-AS: 3D7.HC7 (lane 1), 3D7.HC1ASp- (lane 2), 3D7.HC1-AS (lane 3) and 3D7 (lane 4). (B) The blot was probed with clag9 cDNA complementary to only the antisense mRNA produced by the HC1-AS vector: 3D7.HC7 (lane 1), 3D7HC1-ASp- (lane 2), 3D7.HC1-AS (lane 3) and 3D7 (lane 4).
3D7.HC1-ASp-
–
–
26 9 3
10 143 9 3
a P. falciparum clone 3D7 was electroporated with HC1-AS or HC7 and cultured in 0.1 mM pyrimethamine. After synchronization by gelatin flotation parasites were grown to 5–8% parasitemia and cytoadherence to melanoma cells was then measured as described [16]. Results are shown as parasitized cells bound per 100 melanoma cells. Experiments were carried out in duplicate and mean values 9S.D. are shown.
melanoma cells than did the parental clone 3D7 (Table 1). Over the next 4 weeks binding decreased to 15-fold lower, using, as an additional control, 3D7 transfected with an unrelated plasmid HC7 (which contains an unrelated sequence in the same vector) at the same time as HC1-AS, and cultured in parallel to the control (Table 1). This is consistent with the evidence that clag9 is essential for cytoadherence to C32 melanoma cells. To confirm that the lowered binding was determined by the presence of the plasmid we cultured 3D7.HC1-AS in the absence of pyrimethamine for several weeks. Under these conditions it has been demonstrated that plasmid, and hence pyrimethamine resistance, is eventually lost, unless the plasmid has integrated into the chromosome. After 4 weeks the line 3D7.HC1-ASpexhibited 2.6-fold greater binding to melanoma cells than 3D7.HC1-AS cultured throughout this time with pyrimethamine (Table 1) and by 10 weeks it had regained a level of cytoadherence comparable to that of 3D7 (Table 1). At this time no plasmid could be detected by PCR in 3D7.HC1-ASp-. When transferred back into medium containing pyrimethamine after 10 weeks
D.L. Gardiner et al. / Molecular and Biochemical Parasitology 110 (2000) 33–41
it also failed to grow. This result, in conjunction with both the Western and Northern blot results, strongly supports the conclusion that the presence of plasmid HC1-AS is responsible for the change in phenotype. This change can be attributed to clag9 sequence rather than other plasmid sequences as 3D7.HC7 retained cytoadherence (Table 1). The slow loss of function below 2.6fold and the slow regain of function can be readily explained by amplification of the plasmid which occurs under prolonged selection (Fig. 2A) and would presumably result in higher intracellular levels of antisense RNA.
3.6. The antisense line was knob positi6e Another possibility to exclude was that 3D7.HC1-AS had undergone a subtelomeric deletion of the region of chromosome 2 containing the KAHRP gene and that the loss of ability to bind was due to its conversion to a knobless phenotype [20]. This was highly unlikely as a gelatin separation was always employed to enrich the parasitized cells before binding was measured, and it has been established that this selects for cells which are knob positive [21]. Further, the KAHRP gene was present as determined by PCR (data not shown). In order to establish that
Fig. 4. Western blot of SDS soluble/Triton X-100 insoluble fraction of gelatin selected trophozoites from 3D7, 3D7.HC1AS, and 3D7.HC1-ASp- parasite lines: 3D7 (lane 1), 3D7.HC1-AS (lane 2), and 3D7.HC1-ASp- (lane 3) probed with antisera generated by DNA vaccination of mice [9].
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3D7.HC1-AS continued to produce knobs it was examined by transmission electron microscopy. All 3D7.HC1-AS cells examined had characteristic electron-dense knobs whereas these were not present in the knobless control B8 (data not shown).
4. Discussion We have used the transfection system recently developed for P. falciparum [1–4] in order to introduce an antisense construct of clag9 into 3D7. This is the first time that this approach has been reported in P. falciparum. It presents a major advantage over targeted gene disruptions in that the phenotype could first be determined at 6 weeks. Such an experiment is presumed to rely on the formation of intracellular RNA duplexes with authentic clag9 mRNA resulting in the inhibition of translation of the clag9 mRNA. We conclude this to be the most likely hypothesis as substantial quantities of clag9 mRNA were present (Fig. 3A). The RNA duplexes in turn may affect other processes such as splicing and transport. Nevertheless the observed inhibition strongly supports the conclusion that clag9 function is essential for cytoadherence to melanoma cells. An alternative explanation that must be considered in studies on cytoadherence is that switching of 6ar genes in the line under study has led to loss of cytoadherence. There are several reasons why it is highly unlikely that switching could have played any significant role in the observed phenotypes. As it is clear that multiple transfectants are generated during electroporation ([22], and our unpublished observations), this line must result from multiple independent transfection events and the majority of these cells would then have had to switch off the relevant 6ar gene. No such switching off of binding was observed in transfected 3D7 cells over considerably longer periods of culture than those employed here ([2], and our unpublished observations). Further, 3D7.HC7 was generated simultaneously and cultured for the same time but nevertheless retained cytoadherence (Table 1).
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D.L. Gardiner et al. / Molecular and Biochemical Parasitology 110 (2000) 33–41
Nevertheless this possibility must be excluded unequivocally and is currently being addressed. The most significant reason for being confident that the ablation of cytoadherence was due to clag9 antisense inhibition is that the antisense approach has allowed us to re-evaluate the phenotype after subsequently removing the plasmid. The fact that reversion to wildtype levels of cytoadherence accompanied reversion to pyrimethamine sensitivity provides a very direct line of evidence that it was not due to events at some second site. This confirms results obtained by conventional targeted disruption of the clag9 gene [9]. Our antisense RNA approach could become an important new tool in malaria research, in particular if suitable controllable promoters such as a tetracycline inducible promoter, can be utilized [23]. That the phenotype could be determined 6 weeks after electroporation is an important consideration. Integration of the plasmid in a targeted gene disruption may not occur until between 8 and 20 weeks after electroporation and it is usually necessary to clone the line after integration has been confirmed, making the whole process lengthy and time consuming. However, why integration of the HC1-AS construct did not eventually occur in this case is not clear. Inhibition of gene function in P. falciparum by antisense vectors will allow us to increase our knowledge of the biology of this most important human pathogen, and further our understanding of the genes involved in the pathogenesis of this organism.
Acknowledgements The authors would like to thank Sue Keyes and Chris Newbold for their advice on Northern blotting analysis, and Alan Cowman for the HC1 vector. This work was supported by a generous donation from Mark Nicholson and Alice Hill, the Australian National Health and Medical Research Council, the Northern Territory Government and the Tudor Foundation.
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