Characterisation of an extrachromosomal DNA element from Theileria annulata

Molecular and Biochemical Parasitology, 38 (1990) 253-260

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Elsevier MOLBIO 01267

C h a r a c t e r i s a t i o n of an e x t r a c h r o m o s o m a l D N A

element from

Theileria annulata Roger Hall 1., Lesley Coggins 2, Susan McKellar 1, Brian Shiels 1 and Andrew Tait I 1Wellcome Unit of Molecular Parasitology, and 2Beatson Institute for Cancer Research, Glasgow, U.K.

(Received 23 June 1989; accepted 15 September 1989)

Extrachromosomal nucleic acid elements are found in all organisms, commonly as organelle, viral or plasmid genomes. In this paper we describe the initial characterisation of a novel 6.5-kb linear, double-stranded extrachromosomal element from Theileria annulata, and a 2.6-kb RNA species. The DNA element is present in different stages of the life cycle and in different stocks of the parasite. Northern blots of total RNA isolated from different stages of the parasite, probed with the purified element, detect three major transcripts, of 1.45, 1.05 and 0.24 kb, present in all life-cycle stages examined. The possible origin and function of this element is discussed, together with its possible use as a transfection vector for the introduction of genes into protozoan cells. Key words: Theileria annulata; Extrachromosomal element

Introduction E x t r a c h r o m o s o m a l nucleic acid elements are i m p o r t a n t carriers of genetic information. T h e y can originate from several different sources, some c o m m o n examples being organelle g e n o m e s , amplified g e n o m i c D N A sequences, viral g e n o m e s and plasmids. Such elements have been f o u n d in all types of organisms studied, including that extraordinarily diverse g r o u p the p r o t o z o a . Within the p r o t o z o a , examples of e x t r a c h r o m o s o m a l elements include the k D N A of the kinetoplastida [1], several examples of viruses [2], the amplified linear episomes bearing ribosomal D N A ( r D N A ) genes in Tetrahymena and o t h e r p r o t o z o a [3-5], the a u t o n o m o u s l y replicating r D N A circular plasmids in the schizopyrenid a m o e b a e [6,7], and Correspondence address: Andrew Tait, Wellcome Unit of Molecular Parasitology, Bearsden Road, Glasgow, G61 1QH, U.K. *Present address; Dept. of Biology, University of York, Heslington, York, YO1 5DD, U.K. Abbreviations: Con A, concanavalin A; SSC, saline sodium

citrate.

the amplification of dihydrofolate reductase genes on circular elements in m e t h o t r e x a t e resistant Leishmania [8]. In m a n y cases the e x t r a c h r o m o somal element has been d e m o n s t r a t e d to carry important genetic information to the advantage or detriment of the host cell. T h e characterisation of these elements in parasitic organisms is of particular importance for two principle reasons: on the one hand, a knowledge of their function m a y shed light on particular nuances of the biology of the parasite which m a y be exploited in devising new control measures: on the o t h e r hand such elements m a y f o r m the basis for designing stable vectors for introducing r e c o m b i n a n t D N A molecules into these i m p o r t a n t organisms. To these ends, we are characterising extrachrom o s o m a l elements of the bovine p r o t o z o a n parasite Theileria annulata. H e r e we describe a 6.5kb linear, double-stranded, e x t r a c h r o m o s o m a l D N A element which occurs in high-molecularweight D N A preparations. W e report our findings on the presence of this element in different life-cycle stages of the parasite and in parasites from different geographic locations, as well as d o c u m e n t i n g the expression of genes e n c o d e d by this element. W e also show preliminary data

0166-6851/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)

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demonstrating the occurrence of an as yet uncharacterised 2.6-kb RNA species in the same DNA preparations. Materials and Methods

Parasite material. Three stocks of T. annulata were used in this study: T. annulata Ankara, from Turkey [9], T. annulata Hissar, from India [10]; and T. annulata Gharb, from Morocco (Ouhelli, H. (1985), Th6se de Doctorat des Sciences INP, Toulouse, France). Piroplasms were prepared as described by Shiels et al. [11]. Briefly, blood (400 ml) was taken from calves exhibiting parasitaemias of 50-95%, into 70 ml of Glascodine's anticoagulant and preservative (GAP); 14.3 mM citric acid/89.4 mM tri-sodium citrate/14.1 mM sodium dihydrogen orthophosphate/193.3 mM glucose/3.26 mM adenine-HCl (Glascodine, J. (1989) Ph.D. Thesis, Edinburgh University). The blood was either processed immediately or was stored at 4°C for up to one week without obvious signs of parasite degeneration. The blood was depleted of white blood cells by removing the buffy coat and passage over a CF11 column (Whatman) followed by ammonium chloride lysis of erythrocytes [12] in order to release the piroplasms, which were harvested by centrifugation. Contamination of purified piroplasms with leucocytes was routinely less than 0.001% (determined by light microscopy). Macroschizont-infected bovine leucocytes were maintained in RPMI-1640 medium plus 15% heat-inactivated foetal calf serum at 37°C in 5% CO 2 in air, as described previously [13]. Clones from such cell lines were prepared by limiting dilution [14]. The uninfected cell lines, BL20 (a non-viral lymphosarcoma) [15] and BAE (a bovine aortic endothelial line) [16] were maintained under identical conditions to the infected cell lines. Concanavalin A (ConA) blasts were prepared as described by Shiels et al. [17]. Sporozoite-infected salivary glands were dissected from ticks (Hyalomma anatolicum anatolicum) fed for two days, as described [18]. Nucleic acid extraction, gel electrophoresis, hybridisation procedures and probes. High-molecularweight DNA was prepared by standard proce-

dures [19]. Briefly, piroplasm or macroschizontinfected leucocyte pellets were resuspended in 10 vols, lysis buffer (1 x SSC, 1% Sarkosyl, 100 txg ml-I proteinase K) and incubated for a minimum of 4 h at 60°C. The nucleic acid was very gently extracted three times with phenol/chloroform (1:1), the phenol phase being removed by puncturing the bottom of the tube to avoid shearing the DNA in the aqueous phase. After an extraction with ether, the DNA was poured into a dialysis bag and dialysed against 3 x 2 1 TE (10 mM Tris, pH 7.4, 1 mM EDTA) for 24 h. The 6.5-kb element was purified by cutting the band out of a preparative agarose gel and adsorbing it to glassmilk, using a commercially available kit, exactly as described by the manufacturer (Gene-Clean, BI0 101). The large mitochondrial rDNA probe SPP144 [20] is from the sea urchin, Strongylocentrus purpuratus, and was kindly supplied by Dr. H. Jacobs. The probes for genomic rDNA genes is a HindIII fragment of clone )t 104 from Trypanosoma brucei (Hide, G., (1988) Ph.D. Thesis, Edinburgh University) was a kind gift from Geoff Hide. RNA was prepared from all life-cycle stages by the method described by Williamson et al. [21]. Electrophoresis of both RNA and DNA, and hybridisation using randomly primed DNA was also as described by Williamson et al. [21]. Reduced stringency hybridisations with SPP144 and X 104 insert were performed at 50°C and 55°C, respectively, in the buffers described [21], followed by 3 washes in 2 x SSC, 0.1% SDS at the temperature of hybridisation.

Electron microscopy. A sample of the 6.5-kb element, purified from an agarose gel, was prepared for electron microscopy by the diffusion method of Lang and Mitani [22]. 20-1xl drops of 0.5 M ammonium acetate, pH 7.5, 50 ~g m1-1 cytochrome c were placed on a parafilm sheet in a petri dish, and 1-2 txl DNA (5-10 ng), with or without the addition of the circular plasmid pIC20R [23], was added to each drop. After 10-15 min, a collodion-coated grid was touched to the surface of each drop and rinsed in 0.25 M ammonium acetate pH 7.5. Grids were stained with uranyl acetate, rotary-shadowed with platinumpalladium, and examined with a Philips EM300

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electron microscope. Length measurements were carried out on molecules in electron micrographs, with a Summagraphics digitising tablet and a Jandel Sigmascan programme, using open circular plC-20R molecules (2.716 kb) as an internal size standard.

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Identification o f extrachromosomal D N A and R N A species in genomic D N A preparations. When high-molecular-weight D N A from T. annulata Hissar was subjected to electrophoresis through a 0.6% agarose gel, two bands were revealed in the separation zone, in addition to the non-resolved bulk genomic D N A (Fig. 1, lane 4). These bands have sizes of 6.5 kb and 2.6 kb. To ascertain the chemical nature of these extrachromosomal nucleic acid species, an aliquot of the high-molecular-weight D N A preparation was digested with boiled RNase A and then electrophoresed through an agarose gel (Fig. 1, lane 3). The upper 6.5-kb band was clearly resistant to the RNase A, strongly suggesting that it was D N A . By contrast, the 2.6-kb band was clearly sensitive, and is therefore defined as R N A . To check that the RNase had the correct specificity, a sample of R N A markers was digested simultaneously; it was totally degraded (Fig. 1, lane 1), whilst a sample of D N A markers also incubated with the enzyme remained completely intact (Fig. 1, lane 2). A sample of R N A markers incubated without enzyme remained intact (Fig. 1, lane 6) as compared to a similar sample loaded without incubation (Fig. 1, lane 7), demonstrating that there was no non-specific degradation of R N A during the incubation period. Further evidence that the 6.5-kb band was D N A was provided by the fact that, when it was isolated from a gel, it could be labelled by the standard 'random priming' labelling technique (Figs. 3 and 5). In contrast, the isolated 2.6-kb band could not be labelled under these conditions (data not shown), supporting the conclusion that it was R N A .

The 6.5.-kb molecule is finear and doublestranded. We examined the isolated 6.5-kb molecule by electron microscopy (Fig. 2A). Our results revealed that the molecule was linear; a very

2.6-~

Fig. 1. Ethidium bromide-stained 0.6% gel showing the presence of extrachromosomal elements in high-molecularweight piroplasm DNA from T. annulata Hissar. Lanes 1, 6 and 7 contain 4 I~g of RNA markers (BRL). Lanes 2, 5 and 8 contain 2 I~gDNA markers (1-kb ladder, BRL). Lanes 3 and 4 contain 15 I~gpiroplasm DNA from T. annulata Hissar. The samples in lanes 1-3 were incubated for 8 h at 37°C with 25 ixg ml-~ boiled RNase A, whilst those in lanes 4-6 were incubated under identical conditions but without RNase. The samples in lanes 7 and 8 were not incubated prior to loading the gel. The numbers on the left of the figure refer to sizes in kb. The series of bars on the right of the figure denote markers of the following sizes, in ascending order, 0.506/0.516, 1.018, 1.635, 2.036, 3.045, 4.072, 5.090, 6.108, 7.126 kb. occasional circular molecule was observed (not shown), which could be a covalently-closed circular form, or just fortuitous juxtaposition of free ends. The element adopted an extended form in the absence of formamide and it was of similar width to pIC-20R, suggesting that it is a duplex. This was confirmed by the restriction map analysis (Fig. 2B), which also revealed that it was not palindromic in nature. Its size was calculated to be 6.30 - 0.18 kb (n=30), by length comparison with adjacent plC-20R molecules; this is in good agreement with the estimate obtained by gel analysis.

256 hybridisation to some higher molecular weight bands, possibly suggesting the existence of multimers of this element. The element is evidently not present in uninfected cow cells of various types (lanes 16-19). In addition, the Northern analysis shown below (Fig. 5) demonstrated that the element was present in sporozoites. Thus, it appears to have a ubiquitous distribution, both geographically and in the different life-cycle stages of the parasite.

P I I

S I

R H I I

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lkb

Fig. 2. Demonstration that the 6.5-kb element is a linear double-stranded DNA molecule. (A) Electron micrograph of the purified 6.5-kb element from T. annulata Hissar. The 6.5kb element was purified from a gel and viewed under the electron microscope. The element is labelled with an (e). The circular molecules labelled with a (p) are pIC-20R plasmid molecules, added as a size marker. Bar represents 0.5 ~zm.(B) Restriction map of the 6.5-kb element from T. annulata Ankara. The enzyme abbreviations are P, PvuII; S, SphI; R, EcoRI; H, HindIII; K, KpnI. Geographical and life-cycle distribution o f the 6.5kb element. When the isolated 6.5-kb molecule (T. annulata Hissar) was used to probe a Southern blot (Fig. 3) containing D N A from macroschizont-infected leucocytes, the element was clearly seen in 4 cell lines from T. annulata Hissar (consisting of a parental uncloned line, lane 1, and three derived clones, lanes 2-4), and 7 cell lines from T. annulata Ankara (parental line, lane 5, and derived clones, lanes 6-11). In addition, the element hybridised to a T. annulata Hissar macroschizont-infected cell line propagated in a bovine lymphosarcoma (BL20, lane 13), whilst the uninfected cells clearly did not possess the element (lane 16). The presence of the element in the piroplasm stage of T. annulata Hissar has already been established (Fig. 1), and it was shown also to be present in the piroplasm D N A extracted from T. annulata Ankara and T. annulata Gharb (lanes 14 and 15). In these tracks there was

The 6.5-kb element does not carry mitochondrial r D N A sequences. To ascertain if the 6.5-kb element carried mitochondrial r D N A sequences, high-molecular-weight T. annulata Hissar D N A was separated in two lanes on the same agarose gel and Southern-blotted (Fig. 4). One filter was hybridised with a highly conserved large mitochondrial rDNA [20] probe under reduced stringency (lane 2), whilst the replica filter was hybridised to the T. annulata Hissar element (lane 1). The mitochondrial r D N A probe did not hybridise to the element, but did recognise sequences in the high-molecular-weight DNA. Thus, the 6.5kb element does not encode mitochondrial rDNA. The 6.5-kb element is transcribed into several different R N A species, which are not r R N A , in three life-cycle stages. Northern blot analysis (Fig. 5) revealed that the 6.5-kb element is expressed as 3 major transcripts of 1.45, 1.05 and 0.24 kb in the sporozoite (lanes 6, 9 and 10), macroschizont (lane 7), and piroplasm (lane 8) life-cycle stages. Other minor transcripts were also observed. The band at 6.5 kb observed on these blots was shown to be the element itself, since it was susceptible to digestion with RNAase-free DNAase, whilst the other bands (transcripts) were unaffected (compare lanes 9 and 10). Hybridisation of the same filter as shown in lanes 5-8, at reduced stringency, with a trypanosome r D N A probe demonstrated that this element does not encode the genes for cytoplasmically located rRNA (lanes 1-4). This is clearly evident, since the patterns of hybridisation with the r D N A probe are totally different to that observed with the element, in all samples. More specifically the large and small parasite rRNA subunits have sizes of 3.4 and 1.75 kb, respectively (lanes 2 and 4), which do not

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Fig. 3. Life-cycle and geographic distribution of the 6.5-kb element. The figure shows a Southern blot of high-molecular-weight DNA from various sources, probed with the 6.5-kb element from T. annulata Hissar. Lanes containing macroschizont-infected leucocyte DNA and uninfected bovine cell DNA were loaded with 20 izg. Lanes containing piroplasm DNA were loaded with 3 txg. Lane 1 contains DNA from T. annulata Hissar 46-P, a line of macroschizont-infected leucocytes, whilst lanes 2-4 contain DNA from cloned macroschizont lines T. annulata Hissar 46-2, 46-3 and 46-4, respectively, which are derived from 7". annulata Hissar 46-P. Lane 5 contains DNA from T. annulata Ankara 46-P, a macroschizont-infected leucocyte line, and lanes 6-11 contain derived clones T. annulata Ankara 46-A, 46-2, 46-3, 139-D4, 139-D6 and 139-E5, respectively. Lanes 12, 14 and 15 contain DNA from piroplasms purified from T. annulata isolates Hissar, Ankara, and Gharb respectively. Lane 13 contains DNA from a T. annulata Hissar-infected bovine lymphosarcoma (BL20). Lanes 16-19 contain uninfected bovine DNA extracted from BL20, Con A blasts, aortic endothelial cells and calf thymus (Sigma). m a t c h any of the sizes of the transcripts hybridising to the element. Furthermore, the r D N A probe shows no hybridisation to the element itself at 6.5 kb, which we have already d e m o n s t r a t e d to be a c o n t a m i n a n t in our R N A (see above).

Discussion W e have described the discovery of an extrac h r o m o s o m a l D N A e l e m e n t in T. annulata. It is a h o m o g e n e o u s 6.5-kb linear d o u b l e - s t r a n d e d molecule. W e have also o b s e r v e d an R N A molecule of 2.6 kb, which requires further characterisation. In particular, it will be interesting to determine the relationship, if any, b e t w e e n these two molecules. T h e restriction m a p of the 6.5-kb element d e m o n s t r a t e s that it is not palindromic. T h e element does not consist of r D N A , as shown by the N o r t h e r n analysis (Fig. 5), since n o n e of the transcripts c o r r e s p o n d to the parasite r R N A mole-

cules of 3.4 and 1.75 kb, size estimates close to those calculated for Theileria parva r R N A [24]. This is supported by the fact that a T. brucei r D N A p r o b e failed to hybridise to the element (which is k n o w n to be a c o n t a m i n a n t in our R N A preparations) in both the N o r t h e r n blot analysis shown in Fig. 5 and in S o u t h e r n blot experiments (data not shown). These facts distinguish this elem e n t from the linear r D N A elements that occur in a range of lower eukaryotes including Tetrah y m e n a and other p r o t o z o a [3-5], and the slime moulds Physarum [5,25] and Dictyostelium [5,26]. By the same tokens it is clearly distinguished from the r D N A bearing circular plasmids of the schizopyrenid amoebae [6,7]. It is present in all stages of the parasite e x a m i n e d (sporozoite, macroschizont and piroplasm), and occurs in three geographically distinct stocks, originating from Turkey, India and M o r o c c o . T h e origin of the 6.5-kb element remains unclear, and currently we have not d e t e r m i n e d w h e t h e r it is a specifically ampli-

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Fig. 4. Demonstration that the 6.5-kb element does not encode mitochondrial r D N A sequences. Lanes 1 and 2 are autoradiograms of filters obtained by transfer of D N A from adjacent tracks on the same original gel, each containing 5 ~g T. annulata Hissar piroplasm D N A . Lane 1 was probed with the T. annulata Hissar 6.5-kb element at high stringency. Lane 2 was probed with a large mitochondrial r D N A sequence, SpP144, from sea urchin [20] at reduced stringency.

fled genomic sequence, or an autonomously replicating episome. Our data demonstrate that the 6.5-kb element does not encode a sequence homologous to mitochondrial r D N A , demonstrating that it is not a very small mitochondrial genome. We cannot, however, rule out the possibility that it is a mitochondrial plasmid. Furthermore, the 6.5-kb element is not specifically associated with drug resistance, which has been shown in the case of methotrexate resistance in Leishmania, to induce specific amplification of the genes for dihydrofolate reductase [8]. A viral origin of this element is possible, as there have been several reports of viruses in parasitic protozoa (reviewed in ref. 2), the best studied being the doublestranded R N A viruses of Giardia lamblia [27] and

Trichomonas vaginalis [28]. If this element is viral, it is clearly different from either of these since it is composed of DNA. However, no viral particles have been observed to date, either in the cytoplasm or culture supernatants of T. annulata. The presence of viruses in Theileria spp. has been tentatively suggested before [29], in relation to the transformation of leucocytes induced by the macroschizont, although no evidence has been presented. It is tempting to speculate that this element may be involved in this transformation process, and direct transfection assays will be performed to test this possibility. It is arguable, however, that this element has nothing to do with transformation, since it is expressed at all stages, suggesting that transcription is essential throughout the life-cycle rather than being specific to the stages of the parasite involved in lymphocyte transformation. If the transcripts are essential throughout the life-cycle, then curing the parasite of this element would provide a novel strategy for parasite control. Until the sequence of the element is determined, and the function of the genes and their relationships to known genes is defined, it is difficult to assess these possibilities. It is also possible that the 6.5-kb element could be used as a stably replicating vector for introducing recombinant D N A into T. annulata. This is a much needed area of research in the parasitic protozoa but only a few isolated reports of successful transfections exist [30-32], the most promising to date being the work on Leptomonas by Bellafatto and Cross [32]. Theileria presents a particular problem in this respect, since for a large part of its life-cycle it is intracellular. An alternative and complementary strategy would be to utilise this element as a vector for extracellular protozoa; such an approach would rely upon the properties of stability and autonomous replication being sustainable in an heterologous environment and on the introduction of a selective marker. In this regard it will be interesting to analyse the termini, to discover what features they possess to promote their stability, and to compare them with known telomeric sequences.

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-'

Fig. 5. Demonstration that the 6.5-kb element is transcribed in three life-cycle stages, and does not encode rRNA, by Northern blotting. All samples consist of total RNA. Lanes 1 and 5 contain 10 txg BL20 RNA. Lanes 2 and 6 contain 5 I~g sporozoite RNA from T. annulata Ankara. Lanes 3 and 7 contain 10 izg of T. annulata Hissar macroschizont-infected BL20 RNA. Lanes 4 and 8 contain 5 ixg T. annulata Hissar piroplasm RNA. Lanes 9 and 10 contain 5 txg T. annulata Hissar sporozoite RNA. The RNA in lane 10 was pre-incubated for 1 h at 25°C, with 1 unit of RNase-free DNAase (BCL), prior to loading. The filter containing lanes 5-8 was probed first at high stringency with the 6.5-kb element, and then after stripping was reprobed at reduced stringency with a T. brucei rDNA probe, h 104 (lanes 1-4). The filter containing lanes 9 and 10 was probed with the 6.5-kb element at high stringency. The numbered arrows denote sizes in kb.

Acknowledgements W e w o u l d like to t h a n k P. B e c k , J. Glascodine, A. W a l k e r , J. F l e t c h e r , S. W i l l i a m s o n , D. B r o w n a n d L. Bell for their assistance a n d enc o u r a g e m e n t t h r o u g h o u t this work. T h a n k s are due to A f s h a n F a i r l e y for t y p i n g the m a n u s c r i p t

a n d to A l a n M a y for the p h o t o g r a p h y . This work was s u p p o r t e d by the W e l l c o m e T r u s t ( R . H . , S.M., B.S. a n d A . T . ) a n d the C a n c e r R e s e a r c h Campaign (L.C.). The support provided by all the staff of the P r o t o z o o l o g y Division, C e n t r e for T r o p i c a l V e t e r i n a r y M e d i c i n e ( E d i n b u r g h ) is gratefully a c k n o w l e d g e d .

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