Upstream elements required for expression of nucleoside triphosphate hydrolase genes of Toxoplasma gondii1

Molecular and Biochemical Parasitology 92 (1998) 229 – 239

Upstream elements required for expression of nucleoside triphosphate hydrolase genes of Toxoplasma gondii 1 Valerian Nakaar, David Bermudes 2, Kyong Ran Peck 3, Keith A. Joiner * Department of Internal Medicine 808 LCI, PO Box 208022, Section of Infectious Diseases, Yale Uni6ersity School of Medicine, New Ha6en, CT 06520 -8022, USA Received 23 July 1997; received in revised form 5 November 1997; accepted 5 November 1997

Abstract Nucleoside triphosphate hydrolase is an abundant protein secreted by the obligate protozoan parasite Toxoplasma gondii. The protein has apyrase activity, degrading ATP to the di- and mono-phosphate forms. Because T. gondii is incapable of de no6o synthesis of purines, it is postulated that NTPase may be used by the parasite to salvage purines from the host cell for survival and replication. To elucidate the molecular mechanisms of NTP gene expression, we isolated from the virulent RH strain of T. gondii the putative promoter region of three tandemly repeated NTP genes (NTP1, 2, 3). Using deletion constructs linked to the chloramphenicol acetyl transferase (CAT) reporter gene, we defined an active promoter within the first 220 bp. Sequence analysis of this region reveals the lack of a TATA box, but the promoter region is associated with a sequence which resembles an initiator element (Inr) in the NTP1 and NTP3 genes. This sequence which is similar to other Inrs known to regulate the expression of a wide variety of RNA polymerase II genes, is required for NTP expression. The NTP3 promoter contains sufficient information for developmentally regulated expression of CAT activity when the actively replicating stage tachyzoite differentiates into the dormant bradyzoite form. © 1998 Elsevier Science B.V. All rights reserved. Keywords: Toxoplasma gondii; Nucleoside triphoshate hydrolase cis-elements; Regulation

Abbre6iations: b-gal, b-galactosidase; bp, base pair; CAT, chloramphenicol acetyl transferase; CPRG, chlorophenol red-b-Dgalactopyranoside; DHFR-TS, dihydrofolate reductase-thymidylate synthase; GRA, dense granule antigen; HEPES, N-2-Hydroxyethypiperazine-N’-2-ethanesulfonic acid; Inr, initiator element; kb, kilo base; nt, nucleotide; NTP(ase), nucleoside triphosphate hydrolase gene (enzyme); PARP, procyclic acidic repetitive protein; pol II or III, RNA polymerase II or III; pos, position; SAG, surface antigen; TFIIIC, transcriptional factor IIIC; TLC, thin layer chromatography; TUB, tubulin; UTR, untranslated region; VSG, variable surface antigen; wt, wildtype. * Corresponding author: Tel.: +1 203 7854140; fax: + 1 203 7853864; e-mail: [email protected] 1 Note: Nucleotide sequences data reported in this paper are available in the GenBank™ under the accession number U96965. 2 Present address: Vion Pharmaceuticals, 4 Science Park, New Haven, CT06511, USA. 3 Present address: Samsung Medical Center, Seoul, South Korea. 0166-6851/98/$19.00 © 1998 Elsevier Science B.V. All rights reserved. PII S0166-6851(97)00220-X

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1. Introduction Toxoplasma gondii is an obligate intracellular parasite which can infect most nucleated mammalian cells [1]. It lacks the enzymes necessary for de no6o synthesis of purines and therefore must salvage purines from the host for survival and replication [2,3]. In the host cell, the parasite resides in a membrane bound vacuole, which is freely permeable to small molecules including ATP [4]. The parasite produces an enzyme with dithiol-activated nucleoside triphosphate hydrolase (NTPase) activity [5], which comprises 2% of total parasite protein, and belongs to a new gene family of ATP-diphosphohydrolases [6]. This NTPase is located within the parasite in dense core granules, the contents of which are secreted in high levels into the vacuolar space surrounding the parasite inside infected cells [7,8]. It has been suggested that in this location the NTPase participates in the salvage of purine nucleotides from the host cell [4,7]. While determining the genomic organization of the NTP genes in the virulent RH strain of T. gondii, we found three variably repeated elements both upstream and downstream of three tandemly repeated NTP genes. Interestingly, these are the first tandemly repeated genes identified in T. gondii, which are differentially expressed. It was therefore important to characterize the cis-regulatory sequences controlling transcription. Here we describe the isolation of the NTP promoter regions and the characterization of the sequences involved in the basal and high levels of NTP expression. The NTP promoter is associated with a sequence which resembles an initiator element (Inr). This inr-like sequence is important for expression of NTP genes. A chloramphenicol acetyl transferase (CAT) reporter gene driven by the NTP3 promoter region is developmentally regulated when parasites differentiate from tachyzoites to bradyzoites.

2. Materials and methods

2.1. Sequence analysis Genomic clones for the 5% flanking region of NTP

genes from a genomic phage library of T. gondii were isolated as described previously [7]. The clones were characterized by restriction mapping and Southern blot analysis. Convenient restriction fragments were subcloned into pBlueScript (Stratagene) and sequenced using primers derived from the coding and non-coding regions of NTP genes. Regions upstream of the ATG codon were compared between NTP1, NTP2 and NTP3 using the GCG PILEUP program.

2.2. Cell cultures, growth of parasites Vero cells and human primary human foreskin fibroblasts (HFF) were grown as monolayers in modified Eagle’s minimal medium and a-MEM, respectively, containing heat inactivated 10% fetal calf serum and gentamycin (50 mg ml – 1). Clonal derivatives of the RH strain and the PLK strain of T. gondii were maintained by serial passage in either peritoneum of Swiss Webster mice or by passage in Vero cells or primary HFF in culture. When infected monolayers neared complete lysis by parasites, cells were scraped and parasites isolated by two passages through a 27G needle.

2.3. Transfections, b-galactosidase and CAT assays All plasmids used in the transfection studies were purified on Qiagen ion-exchange columns (Qiagen, Germany). Transient transfection of RH tachyzoites was done essentially as described by Roos et al. [9]. For dual transfections with CAT plasmids and pTUB– bgal [10] (kindly provided by Dr J. Boothroyd), 50 mg of each plasmid DNA was used. Parasites were harvested in 200 ml of 250 mM Tris–HCl, pH 7.8, disrupted in three freeze-thaw cycles, and centrifuged at 12000× g for 5 min, 24 h after transfection. The supernatant was used for subsequent enzyme assays. b-galactosidase (b-gal) activity was determined by using chlorophenol red-b-D-galactopyranosidase (CPRG, Boehringer Mannheim) as a substrate [11]. Cell lysate, 5–20%, from a 10 cm plate or a 25 cm2 flask was incubated at 37°C in 8 mM CPRG, 80 mM sodium phosphate buffer (Na2HP04.2H20 and NaH2P04, pH 7.3), 102 mM b-mercaptoethanol, 9 mM MgCl2, for 10–60

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min, depending on the transfection efficiency. Substrate conversion was determined spectrophotometrically by the increase in absorbance at a wavelength of 570 nm (Titertek Multiskan). Within each series of transfection assays, and based upon the b-gal activity of each extract, the amount of cell lysate used for assaying CAT activity was adjusted accordingly. This normalization was necessary to compensate for transfection efficiency and other experimental variables prior to performing CAT assays. CAT assays were performed with cell extracts adjusted to identical b-gal activity [12]. Extracts were incubated with 3 – 5 ml of D-threo(dichloroacetyl-1-14C) chloramphenicol (NEN) and 40 mM acetyl CoA (Sigma) for 60 – 90 min and separated on thin layer chromatography plates (TLC). CAT assays were quantitated by scanning of the autoradiograms. For stable transfection, PLK strain was transfected with 10:1 of either 1600NTP3 (or 220NTP3) and pc3TUB-bgal [S. Chaturvedi and K.A. Joiner, unpublished]. The construct pc3TUB-bgal was derived from pTUB-bgal in which a 3.6 kb XbaI – HindIII fragment of the dihydrofolate reductase-thymidylate synthase (DHFR-TS) gene from pc3M2M3 [13] was inserted at the NotI site. The DHFR-TS gene confers resistance to pyrimethamine. At 24 h after electroporation, infected monolayers were treated with and maintained in 1 mM pyrimethamine. After 7–10 days of selection, resistant clones were pooled and expanded in culture. Induction of bradyzoite formation was carried out as described [14] with some modifications by Hehl et al. [15]. Confluent monolayers of HFF cells were infected for 4 – 6 h with transgenic PLK lines containing CAT constructs at 5× 104 – 105 parasites per T175 cm flask. Too high an infection can lead to lysis of parasites from the cells within one to a few days after infection. Extracellular parasites were removed by gentle washing of the monolayer with prewarmed medium. The culture medium was replaced with RPMI-1640 containing L-glutamate (Gibco-BRL) switching medium supplemented with 5% fetal bovine serum, 25 mM N-2-Hydroxyethypiperazine-N%-2-ethanesulfonic

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acid (HEPES), 20 mg ml – 1 gentamycin and the pH was adjusted to 8.1 with NaOH. The cells were incubated at 37°C in a CO2 free incubator with replacement of medium every 2 days. The pH of the medium was monitored daily and if necessary adjusted with NaOH. After 3–4 days, parasites were harvested by syringe passage of cells and equal numbers of tachyzoites and bradyzoites were processed. Equal amounts of cell lysate were monitored for b-gal and CAT activities.

2.4. Plasmids, promoter constructs and oligonucleotides The constructs were made by using DNA fragments derived from PCR using the pairs of forward and reverse primers as described in Table 1. The PCR was done in the presence of 1 ng of template, 20 ng of the primers, in reaction buffer (10 mM Tris–HCl pH 8.8, 10 mM KCl, 0.002% (v/v) Tween 20), 200 mM dNTPs, 2.5 mM MgCl2 and 0.5 U of AmpliTaq DNA polymerase (Perkin Elmer). The reaction was cycled 20 times: Denaturation at 90–95°C for 1 min was followed by annealing at 45–50°C for 1 min and elongation at 72°C for 1 min. The CAT constructs were generated by subcloning the PCR fragments from the 5’ flanking regions of NTP1, NTP2, and NTP3 into the basic pCAT construct, which was obtained by replacing the 3’ untranslated region (UTR) PacI–BamHI fragment of pTUB5/CAT [16] with the 1.0 kb PacI–BamHI PCR fragment of NTP3 using the forward and a reverse primers (Table 1). A 500 bp tubulin promoter fragment was then deleted from pTUB5/CAT using NsiI and HindIII restriction sites. The resultant construct is the basic pCAT. To generate the a-tubulin minimal promoter construct 70TUBCAT, a pair of primers (Table 1) one of which is complementary to nucleotide (nt) 26–46 of the CAT coding region was used to generate a 210 bp fragment which was digested with NsiI and HindIII restriction enzymes to generate a 70 bp fragment for insertion into pTUB5/CAT deleted construct. To generate the constructs described in Figs. 2 and 3, the pairs of primers described in

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Table 1 Oligonucleotides used in PCR. Construct

Forward primer

pCAT

5%-GTAGCCGCGATGTCTCTGGG-3%

Reverse primer

5%-GCTAGGATCCGCTACTGCTGGGTGCCTAACC-3 1600NTP3 5%-GCGCCACACTAGTGTAGGCCTCAGC-3% 5%-GCAACCACATGATGCCTACCTATAAACG-3% 1045NTP3 5%-CGTCCACAAACCGGTTCCGC-3% 5%-GCAACCACATGATGCCTACCTATAAACG-3% 620NTP3 5%-CTCGCCCGTAATCTGCTCGG-3% 5%-GCAACCACATGATGCCTACCTATAAACG-3% 22ONTP3 5%-CTCGGCCTCGAGCACGCGCACGTCC-3% 5%-GCAACCACATGATGCCTACCTATAAACG-3% 60NTP3 5%-GACGATCGACGCGTCTTGCC-3% 5%-GCAACCACATGATGCCTACCTATAAACG-3% 1045NTP1 5%-CGTCCACAAACCGGTTCCGC-3% 5%-GCAACCACATGATGCCTACCTATAAACG-3% 620NTP2 5%-CTCGCCCGTAATCTGCTCGG-3% 5%-GCAACCACATGATGCCTACCTATAAACG-3% 720NTP2 5%-GCGAATGAATTCACGCATGTCCCGG-3% 5%-CAGACATGCATGACGCAGTTACTTGGTG-3% 70TUBCAT 5%-CACAATAACTTGTGTGAAG-3% 5%-TTGGGATATATCAACGGTGG-3% E1-TUBCAT 5%-CATGTGTCTGTTCCCACACCAAGTAAC-3% 5%-GCAACCACATGATGCCTACCTATAAACG-3% E3-TUBCAT 5%-GGACCGGAAGTGCGTGCCGACCACC-3% 5%-GTGAATTGAAAATAGGACAGAATCGTC-3% 620/220mut 5%-AGCTGCTGTTTCTTTTTTTTGACGATCGAC-3% 5%-GTCGATCGTCAAAAAAAAGAAACAGCAGCT-3%

Table 1 were used. To mutate the putative Inr sequence in constructs 620mut and 220mut, a pair of mutated primers (Table 1) were used in PCR with the same conditions as described above. PCR fragments were gel purified using either Gene Clean kit (Sun Bio II Science) or QiAquick gel extraction kit (Qiagen) before cloning. The constructs were verified by restriction mapping or sequencing. In the case of 620mut and 220mut the entire 5’ flanking or most of it was sequenced to ensure that there were no additional mutations outside the desired region.

3. Results

3.1. Organization of genomic and promoter regions of the NTP locus We have previously reported the isolation of three genes for NTP (NTP1, 2, 3), two of which encode functional gene products [7]. We have now obtained the complete sequence of the 5’ and 3’ flanking regions for NTP1, NTP2 and NTP3. The organization of the genomic and intergenic regions of NTP genes is depicted schematically in Fig. 1A. Sequence comparison showed a very high degree of homology suggesting that they may use

common elements to regulate transcription. Five separate elements (E1 to E5) are variably present in the 5’ flanking regions. Interestingly, NTP2, for which no cDNA was recovered [7], lacks a proximal element (E1) which is found immediately upstream of NTP1 and NTP3. This suggests that the lack of NTP2 expression may be related to its promoter structure. The second (E2) of the three repeated elements is common to all the genes while the third element (E3) includes a tRNAAla gene which is absent in NTP1. Further upstream, a fourth region (E4) is partially conserved in all three genes. The fifth region (E5) of : 800 bp is conserved and corresponds to the 3’ region of NTP1, NTP2 and NTP3. A comparison of the first 1560 nt of the 5’ flanking region of the three genes is displayed in Fig. 1B.

3.2. Identification of NTP1 and NTP3 promoter acti6ity To determine the minimal sequence required for promoter activity, transient expression assays were performed in tachyzoites using deletion constructs as described in Fig. 2. Fragments from the 5’ flanking sequence of NTP3 were used to direct transcription of a promoterless plasmid containing the bacterial CAT reporter gene (pCAT). To

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correct for variation in transfection efficiency, we cotransfected parasites with pTUB-bgal [10]. The plasmid TUB-bgal contains the lacZ gene under the control of the a-tubulin promoter. After transfection, parasites were harvested and monitored for CAT activity. The amount of protein lysate used to measure CAT activity was first normalized against the expression of the cotransfected b-gal gene. Construct 1600NTP3, which contains full length NTP3 5’ flanking region, was as active as the control, pTUB/5CAT (Fig. 2). Similarly, 1045NTP3 and 620NTP3 containing truncations of this region promoted comparable levels of CAT activity relative to the 1600NTP3, suggesting that sequences upstream of position 620 are not required for expression. The deletion construct 220NTP3 resulted in about a 10-fold reduction in CAT activity when compared to wildtype (wt) expression, while construct 60NTP3 resulted in almost complete loss of CAT activity. We next determined whether NTP1 and NTP2 upstream regions also contained promoter activity (Fig. 2). To this end, we made similar constructs using the strategy as described above. Constructs 1045NTP1 and 620NTP1 promoted comparable CAT activity. Similar to NTP3 deletion constructs, sequences upstream of nt 620 are not essential for NTP1 reporter activity. On the other

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hand, 720NTP2, which lacks the E1 element found immediately upstream of NTP1 and NTP3 genes (Fig. 1), promoted little or no CAT activity. This suggests that the E1 element is important for promoting reporter activity. Thus, deletion analysis of the 5’ flanking regions of NTP1 and NTP3 shows that sequences beyond position 620 are not required for high CAT activity and that 220 bp is the minimum sequence required to drive the expression of the CAT gene. It was interesting to find tRNA genes in the 5’ flanking regions of NTP2 and NTP3 genes, suggesting that they might have a regulatory role in the expression of NTP genes (Fig. 1). This is supported by the observation that in other protozoan parasites such as the trypanosomes, tRNA genes are necessary for the expression of their downstream companion genes [17]. To directly test it’s role, we linked the tRNAAla gene to the CAT reporter under the control of a minimal tubulin promoter, 70TUBCAT [15]. This construct E3-TUBCAT, activated CAT activity at least 10-fold over the minimal promoter, while activity driven by the E1 element (E1-TUBCAT) was 20-fold higher than the 70TUBCAT (Fig. 3). Our results suggest that tRNA gene contains elements which can enhance tubulin promoter activity. However, in their native location they have no appreciable effect on reporter NTP promoter activity.

Fig. 1. Sequence comparison of NTP promoter region. The upstream intergenic region of the NTP gene cluster was compared using the GCG PILEUP (Wisconsin Computer Group). The tandemly repeated organization of the NTP1, 2 and 3 dictates that the upstream region of NTP2 and NTP3 is downstream of NTP1 and NTP2. The sequence compared extends back from the initiating ATG. (A) Scheme of the intergenic and genomic organization of the tandemly repeated NTP1, NTP2 and NTP3 genes. Five distinct elements are recognized and are depicted as E1, E2, E3, E4 and E5. The arrows show the tRNAAla gene and the putative poly (A) signals. (B) Sequence comparison of the first 1560 bp of the intergenic region of NTP1, 2 and 3. Hyphens (-) indicate gaps while colons (:) denote identity with NTP3 sequence. Non-homologous sequences are indicated by lower case. Conserved motifs are underlined. The A-box sequence of tRNAAla is derived from the consensus T6 RGCNNA6 GYGG6 and the B-box from GG6 TTCGANTC6 C {where the underlined is the invariant nucleotide(s), R =purine, Y= pyrimidine and N= any nucleotide [28]}. The Inr is derived from the consensus PyPyA(+ 1)N(T/A)PyPy (where Py = pyrimidine, N = any nucleotide, and A +1 = transcription start site; also see text for details). Arrows show the 5’ end of E1, E2, and E3, as described in (A) relative to the ATG. The GenBank™ accession number for NTP1, NTP2 and NTP3 is U96965.

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Fig. 1. (Continued)

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Fig. 2. Functional analysis of NTP1, NTP2 and NTP3 5’ flanking regions by transient transfection of T. gondii tachyzoites. Various fragments from the NTP upstream regions were cloned upstream of the promoterless CAT gene in pCAT. Tachyzoites were cotransfected with the CAT construct and pTUB-bgal. Extracts were prepared 24 h post transfection and assayed for reporter activity. CAT activity from each construct was normalized to the b-gal activity from a cotransfected internal control plasmid pTUB-bgal as described in Section 2. Acetylated products of chloramphenicol were separated on a TLC and the spots were quantitated by densitometric scanning of autoradiogram. This is a representative assay of CAT activity in which the constructs were assayed at least twice.

Deletion analysis suggested that 220NTP3 contains the minimal sequence necessary for CAT activity. Inspection of this region did not show any of the promoter elements found associated with most RNA polymerase II (pol II) transcribed genes. However, the sequence TCAGTTT (at position 75–81 upstream of the ATG) in Fig. 1 is very similar to an Inr which has been identified in a number of eukaryotic genes (Fig. 4A) including the T. gondii surface antigen 1 (SAG1) gene. To test whether this Inr-like sequence is an important regulatory element, we introduced a single cluster of point mutations in the sequence TCAGTTT converting it to TTTCTTT (mutations are underlined) in construct 620NTP3 to generate 620mut (Fig. 4B). The effect of this mutation was monitored by CAT activity in transient transfection. This mutation reduced CAT activity to 37% of 620NTP3. However, when the mutations were introduced in the context of 220NTP3 to generate 220mut, this reduced CAT activity to less than 2%

of 620NTP3 activity. This indicates that the Inrlike sequence is necessary for the basal activity of NTP3 gene and that the trinucleotide CAG is important for its function.

3.3. NTP3 CAT expression is down regulated in bradyzoites NTP is actively expressed in tachyzoites, but is not detected in the sporozoite stage (M. White, personal communication). Expression in bradyzoites has not been examined before. Northern blot analysis has shown that NTP transcripts are abundant in tachyzoites [18]. We therefore sought to test whether an exogenous CAT gene driven by the NTP3 promoter is regulated when tachyzoites of the PLK strain differentiate to bradyzoites. For this analysis, we used 1600NTP3 and 220NTP3 constructs to transfect rapidly dividing tachyzoites. In the same transfection we included pc3TUBb-gal. The latter construct served two

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Fig. 3. NTP3 upstream sequences can enhance a heterologous a-tubulin promoter. Fragments from the NTP3 upstream regions were cloned upstream of the minimal tubulin promoter, 70TUBCAT. The CAT activity from each construct was normalized to the b-gal activity from a cotransfected internal control plasmid TUB-bgal. Spots corresponding to acetylated products of chloramphenicol were separated by TLC and quantitated by densitometric scanning of the autoradiogram. The CAT activities are the mean ( 9 SD) from two experiments. The transcription start site of the tubulin gene is marked by an arrow.

purposes: First, the b-gal gene which is driven by the tubulin promoter has been shown to be constitutively expressed in both the tachyzoite and bradyzoite stages [19] and thus it conveniently served as a control of differentiation. Second, the DHFR-TS gene was used as a positive delectable marker. Resistant clones were pooled and induced to differentiate to bradyzoites at pH 8.1, as described above. CAT and b-gal activities were simultaneously monitored from each cell extract. CAT activity was expressed as a ratio between inducible NTP-CAT activity and the constitutive b-gal activity. Construct 1600NTP3 was about nine times less active in bradyzoites than in tachyzoites (Fig. 5). Similarly, 220NTP3 CAT activity was down regulated 5-fold in bradyzoites. Taken together, these results indicate that 220 bp of NTP3 promoter region contains necessary and sufficient information for the developmentally down regulated expression of CAT when tachyzoites differentiate into bradyzoites.

4. Discussion The development of transfection assays for T. gondii in the past couple of years has facilitated the study of gene expression [13,20,21]. However, compared to other eukaryotes very little is known about the regulation of gene expression in protozoan parasites in general and of Apicomplexans in

particular. Although there has been no systematic study, the evidence from several laboratories indicates that transcription of mRNA genes in T. gondii is likely to follow the vertebrate paradigm in which pol II transcribes all mRNA encoding genes [16,22,23] [L. Moulton and D. Roos, unpublished]. NTP transcripts are very abundant in T. gondii [18], which is consistent with the prevalence of the NTPase enzyme in the parasite. It is likely that NTP expression is controlled at the level of transcription, probably because the genes for NTP must be expressed at very high levels. However, control through mRNA stability cannot be ruled out. As a first step in defining transcriptional control, we have identified the cis-regulatory elements which are important in the expression of NTP genes. From the deletion analysis of NTP1 and NTP3, sequences between nt 220 and 620 appear to be essential for high activity of the gene whereas, sequences between nt 60 and 220 support low level of expression of NTP3. Examination of the latter region revealed no canonical eukaryotic promoter elements, such as TATA, CAAT or SP1 motifs. These elements are typical of most eukaryotic genes, and are required for their basal transcription. The sequence TCAGTTT at pos. 75–81 is a perfect match to the Inr consensus, which is found in an increasingly large and important group of genes that lack TATA elements. Initially characterized as a

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Fig. 4. Mutation of putative Inr diminishes CAT activity. (A) Prevalence of Inr consensus in natural pol II promoters. Except for NTP1 and NTP3, the transcription start site for all the indicated genes is A + 1. In the case of T. gondii SAG1, there are two start sites both mapped to within 4 nt which include an A residue [36].The sequence shown here corresponds to the minor site of transcription initiation. The major site is part of a 27 bp repeat termed the ‘selector of initiation’ [16]. TdT, terminal deoxynucleotidyltransferase [24,37]; AdML, Adenovirus type 2 major late [37]; Vb, T-cell receptor, variable chain b [38]; LFA 1a, human lymphocyte function associated antigen-1 [39]; TRHR, human thyrotropin-releasing hormone receptor [40]; ACHE, mouse acetylcholinesterase [41]; BCHE, human and rabbit butyrylcholinesterase [42]; APP, human amyloid b-protein precursor [43]; SAG1, T. gondii, surface antigen 1 [16]; NTP1 and 3, T. gondii nucleoside triphosphate hydrolase 1 and 3. (B) A cluster mutation was introduced by changing the sequence TCAGTTT to TTTCTTT in constructs 620NTP3 and 220NTP3 to give 620mut and 220mut respectively. The open box represents the wt Inr and the black box, the mutated Inr. The CAT activity from each construct was normalized to the b-gal activity from a cotransfected internal control plasmid pTUB-bgal. Acetylated products of chloramphenicol were separated on a TLC and the spots were quantitated by densitometric scanning of the autoradiogram. The CAT activities are the mean ( 9 SD) from two experiments.

pyrimidine-rich sequence, it has been reported that a stretch of pyrimidines in the Inr is not always necessary for efficient transcription. Thus, an approximate Inr consensus sequence, PyPyA(+1)N(T/A)PyPy, was proposed [24,25]. By mutating this sequence we show that this element is important for NTP3 expression. In other studies it has been demonstrated that the Inr is important for efficient transcription from a

variety of promoters [26,27]. However, there is no evidence that this putative Inr participates in NTP gene transcription. In addition, two other potential regulatory elements AGAGACGC and GGAGAGG located between nt 1453 and 1491 of NTP3 5’ flanking region (and also present at similar locations in NTP1) resemble the conserved sequence A/TGAGAGC. This sequence has recently been identified as a common sequence up-

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stream of TUB1 and SAG1 genes and is critical for the expression of dense granule genes of T. gondii [21]. A/TGAGAGC is part of the core of the 27 bp repeat element which functions as a selector of transcription initiation in the SAG1 gene [16]. However, functional analysis indicates that the sequences upstream of position 620 in NTP1 and NTP3, which include these motifs, are not required for transcription of the NTP1 and NTP3 genes. This together with the finding that Inr-like sequence is essential for NTP gene expression, suggest that the cis acting DNA elements necessary for NTP gene expression may be different from those of SAG1 and GRA genes. The tRNAAla gene does not affect NTP promoter activity but can activate a heterologous tubulin promoter when closely juxtaposed to it. Thus, it is likely that the lack of effect of tRNA on NTP promoter activity is distance related. In most eukaryotes, mRNA and tRNA are synthe-

Fig. 5. Developmentally regulated expression of NTP CAT constructs in a stable transfection of T. gondii. PLK strain was cotransfected with either 220NTP3 or 1600NTP3 (from Fig. 2) and pc3TUB-bgal. Pyrimethamine resistant clones were pooled and induced to differentiate to bradyzoites at pH 8.1 as described in Section 2. Representative assay of two experiments showing activity expressed as a ratio of inducible CAT activity and constitutive b-gal activity at 72 h post infection from 20% of total cell lysate. Enzyme activity was measured in both tachyzoites and bradyzoites.

sized by pol II and III respectively, and thus the activation of the tubulin promoter by the tRNA gene underscores the combinatorial nature of the DNA elements which comprise the transcriptional machinery. This enhancing activity on the tubulin promoter is likely to be mediated by the A and B box elements of the tRNA gene (Fig. 1), which are the binding sites for transcription factor TFIIIC [28], but this factor has not yet been identified in T. gondii. Differentiation of T. gondii is accompanied by metabolic changes as well as stage specific expression of genes [29,30]. The high level of gene expression mediated by the NTP3 promoter is reduced when tachyzoites differentiate into the slow growing stages of the parasite. It is reasonable to assume that tachyzoites that actively synthesize DNA are more likely to require the synthesis of NTPase and other essential enzymes for purine salvage than the slowly dividing bradyzoites. Additional genes, including SAG1 and SAG2, are also abundantly expressed in the tachyzoite stage but are down regulated during conversion to bradyzoites, while a number of specific antigens are only expressed in bradyzoites [31,32]. The development of an in vitro differentiation system [14] has permitted the recent general identification of promoter regions for bradyzoite genes [19,33], although no details of bradyzoite promoter structure are yet defined. The constructs 1600NTP3 and 220NTP3 behave differently in transient and stable transfections. It is likely that the chromosomal context in which these plasmids have been intergrated influences their expression. This is not unprecedented, for in trypanosomes, the variable surface antigen (VSG) and the procyclin acidic repetitive protein (PARP) activities appear to depend strongly on the chromosomal context of their promoters [34] similiar to the ‘position’ effects operating in yeast telomeres [35]. Thus far we have been unable to disrupt the function of NTPase by gene knock out experiments, most likely due to the essential nature of the NTP gene (V. Nakaar and K.A. Joiner, unpublished). We plan to explore other avenues for regulating the expression of NTP genes and as a prerequisite for these alternative strategies, it is

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important to understand the regulatory mechanisms of NTP gene expression, which the data reported herein provide.

Acknowledgements We are grateful to John Boothroyd, Stanford University, Palo Alto for kindly providing pTUB5/CAT and pTUB-bgal. We thank Christian Tschudi and Tim Stedman for critical comments on the manuscript. This investigation was supported by a Public Health Service Grant AI 31808 (to K.A.J.) and by Samsung Medical Center, Seoul, South Korea (K.R.P.). K.A.J. is a Burroughs Wellcome Fund Scholar in Molecular Parasitology.

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