Characterisation of a polymorphic Tc1-like transposable element of the parasitic nematode Haemonchus contortus

Characterisation of a polymorphic Tc1-like transposable element of the parasitic nematode Haemonchus contortus

Molecular and Biochemical Parasitology 102 (1999) 157 – 166 Characterisation of a polymorphic Tc1-like transposa...

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Molecular and Biochemical Parasitology 102 (1999) 157 – 166

Characterisation of a polymorphic Tc1-like transposable element of the parasitic nematode Haemonchus contortus  R. Hoekstra a,*, M. Otsen b, J.A. Lenstra b, M.H. Roos a a

Department of Molecular Recognition, Institute for Animal Science and Health (ID-DLO), P.O. Box 65, 8200 AB Lelystad, The Netherlands b DNA Laboratory, Faculty of Veterinary Medicine, Uni6ersity Utrecht, Utrecht, The Netherlands Received 30 November 1998; received in revised form 4 May 1999; accepted 9 May 1999

Abstract Hctc1, a member of the Tc1-family of transposable elements was isolated from the parasitic nematode Haemonchus contortus. Hctc1 is 1590 bp long, is flanked by 55 bp inverted repeats and carries a single open reading frame of a 340 amino acid transposase-like protein. Hctc1 is similar to Tc1 of Caenorhabditis elegans and elements Tcb1 and Tcb2 of Caenorhabditis briggsae in the inverted terminal repeats, the open reading frame, as well as the target insertion sequence. Furthermore, the copy number of Hctc1 is comparable with the Tc1 copy number in low copy strains of C. elegans. The sequence of Hctc1 is highly variable in H. contortus due to deletions, insertions and point mutations, with at least five distinct length variants of Hctc1. Most of the Hctc1 variation was within rather than between H. contortus populations. The high level of sequence variation is probably due to variation generally found for members of the Tc1-family, as well as a high background level of genetic variation of H. contortus. © 1999 Elsevier Science B.V. All rights reserved. Keywords: Caenorhabditis elegans; Tc1; Inverted repeat; Transposon; Target site; Nematode

1. Introduction The transposable element Tc1 is a useful tool to study the genetics of Caenorhabditis elegans [1]. Tc1 belongs to a family of DNA transpoAbbre6iations: PCR, polymerase chain reaction.  Note: Nucleotide sequence data reported in this paper are available in the EMBL, GenBank™, and DDJB data bases under accession number AF099908. * Corresponding author. Tel.: +31-320-238271; fax: + 31320-238050. E-mail address: [email protected] (R. Hoekstra)

sons in organisms ranging from fungi to vertebrates [2–5]. These elements are spread by vertical and presumably also by horizontal transmission [3,4]. The elements of the Tc1 family have a length of about 1.6 kb and inverted terminal repeats of 20 to more than 400 bp that end with the conserved sequence 5%-CAGT and are flanked by a TA target site duplication. The elements carry an open reading frame encoding a transposase with a conserved DDE motif, which contributes to the catalytic domain of transposases [6,7].

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R. Hoekstra et al. / Molecular and Biochemical Parasitology 102 (1999) 157–166

The transposase encoded by the C. elegans Tc1 transposable element, TC1A, mediates Tc1 transposition in vitro [8]. This is thought to be initiated by double strand cleavage at the ends of the element followed by excision of the transposon and reintegration. The target sequence always consists of a TA dinucleotide, which becomes duplicated by the insertion [8]. Multiple copies of Tc1 are present in the genomes of all tested isolates of C. elegans, but frequent germline transposition has only been observed in certain strains [9 – 11]. The Tc1 copy numbers range from 30 in low copy strains to 200 to more than 500 per haploid genome in high copy strains [12]. The Tc1 transposon has been used as a tool to map, identify and isolate genes and to inactivate target genes in C. elegans. Members of the Tc1 family have also been identified for the closely related nematode Caenorhabditis briggsae [13,14]. Abad et al. [15] concluded on the basis of Southern blotting that the distribution of Tc1-like elements in nematodes was restricted to the family of Rhabditidae, to which the Caenorhabditis species belong. However, we describe here a Tc1-like element, Hctc1, from the parasitic nematode Haemonchus contortus, belonging to the order Strongylida. In contrast to the other organisms from which Tc1-like elements have been characterised, this parasitic nematode species exhibits a high level of genetic variation [16,17]. Characteristics of the Hctc1 elements were therefore determined for different H. contortus populations.

2. Materials and methods

2.1. Nematode populations Three outbred H. contortus populations with different origins were used for this research; SE from Great Britain, RHS6 from Zimbabwe, and CAVR from Australia [18 – 20]. RHS6 and CAVR have been selected for resistance to the chemotherapeutics levamisole and ivermectin, respectively. In addition two inbred populations were used; ISE, inbred for 90% by consecutive infections with offspring from single female worms starting with population SE, and IRE,

inbred for 90% from the benzimidazole resistant population RE4, which was selected for benzimidazole resistance from population SE [21]. Nematodes were bred and maintained as described [18].

2.2. Isolation and sequence analysis of Hctc1 Isolation of genomic DNA from pooled L3 larvae of H. contortus was performed as described [18]. The Hctc1 element was isolated from the populations SE and RHS6. The degenerate primers, H2 (5%-GNTCNGTNATGGTNTGGGG-3%) and H3 (5%-TCNGGNGAYTGNGANGGC-3%), were used to amplify an internal fragment of the Tc1 homologue in a touch-down PCR reaction with 2 ng of genomic H. contortus DNA as template (Fig. 1). The PCR was carried out using AmpliTaq Gold DNA Polymerase (Perkin Elmer) according to the manufacturer’s protocol in the GeneAmp PCR System 9600 (Perkin Elmer). The PCR profile was: 94°C, 10 min, then ten cycles of 94°C for 30 s, 65°C for 30 s with a decrease of 1°C/cycle and 72°C for 50 s, followed by 25 similar cycles with a constant annealing temperature kept at 53°C. The PCR products were separated by agarose gel electrophoresis and the fragment of the expected length (240 bp) was purified using the Qiaex II Gel Extraction Kit (Qiagen). About 1 ng of the purified fragment was used as template DNA in a touch-down PCR with primers H2 and H3 as described above. The resulting PCR fragment was cloned using the TA Cloning Kit (Invitrogen) and subsequently sequenced from both directions with vector primers. Genomic DNA partially digested with Sau3A I and ligated to vectorette linkers was used as template DNA in PCR reactions with vectorette primers [22,23] combined with successively H7, H11 and H13 for the 5% part and H6, H8 and H10 for the 3% part (Fig. 1). The sequence and location of the primers are given in Fig. 1. Resulting PCR fragments of approximately 200– 600 bp were cloned as described above. Generally two to four clones originating from either population were sequenced from both directions. The sequences were combined to construct the Hctc1 consensus sequence. The sequence of full length

R. Hoekstra et al. / Molecular and Biochemical Parasitology 102 (1999) 157–166 Fig. 1. Consensus DNA sequence of Hctc1 and deduced amino acid sequence of the encoded hypothetical protein Hctc1A. The inverted terminal repeats are underlined. Arrows indicate the primers used to isolate and analyse Hctc1. The position of degenerate primers H2 and H3 is indicated by a dotted arrow.



R. Hoekstra et al. / Molecular and Biochemical Parasitology 102 (1999) 157–166

Hctc1 was obtained from two cloned 1.6 kb PCR fragments using H12 as single primer and 2 ng of genomic ISE DNA as template. The putative target sequence of Hctc1 was determined using primers H13 and H10 in combination with the vectorette system. From populations ISE, IRE, CAVR and RHS6 an equal number of target sequences was isolated and sequenced. Nucleotide sequences were determined on an automatic Applied Biosystems 373A DNA sequencer by using the PRISM Dye Terminator Cycle Sequencing Ready Reaction Kit (Applied Biosystems). Further analysis of DNA sequences was performed with the Lasergene Biocomputing Software for Windows (DNASTAR, Inc).

2.3. Southern and dot blot analysis To detect length variants of Hctc1 in different H. contortus populations first a PCR product was obtained using H12 as a single primer and 2 ng of genomic population DNA as template. The PCR was carried out in a 20 ml volume using the Expand High Fidelity PCR System (Boehringer Mannheim) according to the manufacturer’s protocol. The PCR profile was 94°C for 2 min, then ten cycles of 94°C for 15 s, 55°C for 30 s, 68°C for 3.5 min, then 94°C for 15 s, 55°C for 30 s, 68°C for 3.5 min with elongation time increasing 20 s/cycle for 20 cycles followed by 7 min at 72°C. PCR products were separated by electrophoresis on a 0.9% agarose gel and transferred to a nylon membrane. The blot was hybridised to a labelled H2 – H3 fragment of a clone derived from population SE. Hybridisation conditions were as [18]. A phosphorimager screen was exposed to the membrane and analysed with the Molecular Dynamics Storm system. The Hctc1 copy number was determined by spotting RNase treated genomic DNA of different populations and, as a reference, linear plasmid DNA carrying the H2 – H3 fragment derived from the SE population, in twofold dilutions and hybridising with the Hctc1-specific probe as described above.

3. Results

3.1. Isolation and characterisation of Hctc1 Conservation of amino acid sequences in transposases of Tc1, the related Tcb1 and Tcb2 elements of C. briggsae and members of the Tc1 family of fish and insects allowed the design of two degenerate oligonucleotides to isolate Hctc1 from the H. contortus populations, SE and RHS6. These primers H2 and H3, directed the amplification of a fragment with the expected length. Sequence analysis of the PCR fragments showed a significant homology with Tc1. Next, the 5% and 3% flanks of the Tc1-like element and the target sequences were isolated from both populations using the vectorette system, an anchor-based PCR [22,23] (Fig. 1). A Hctc1 consensus sequence of 1590 nucleotides was deduced from the combined sequences (Fig. 1). This consensus sequence was confirmed by high similarity with two full length Hctc1 elements amplified by using H12 as single primer (results not shown). The element has a similarity of 37–39% to Tc1, Tcb1 and Tcb2. Hctc1 carries one open reading frame encoding a putative 343 amino acid protein, Hctc1A. In contrast to the transposase genes carried by Tc1, Tcb1 and Tcb2, no intron was found in the Hctc1A gene. The Hctc1A gene displayed a 43– 45% similarity at the nucleotide level and 47– 51% at the amino acid level with the genes carried by Tc1, Tcb1 and Tcb2 among which a similarity of 62–64% is found (Fig. 2). The similarity with the conceptual proteins of other isolated members of the Tc1 family is lower than 32% at amino acid level. The DDE motif which contributes to the catalytic domain of Tc1/ mariner has been conserved in Hctc1A. The clear homology between Hctc1A and the transposases of Tc1, Tcb1 and Tcb2 does not include the N-terminal part of 47 amino acids, which is highly variable between the four deduced nematode proteins. Hctc1 is bounded by perfect inverted repeats of 55 bp, that end with the highly conserved sequence 5%-CAGT (Fig. 1). The inverted termi-

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nal repeat of Hctc1 resembles the Tc1 inverted terminal repeat both in length, 55 versus 54 bp, and in sequence (Fig. 3). The sequence similarity of 45% is mainly due to conservation of nucleotides 1–12 and two conserved blocks of five nucleotides between nucleotides 31 and 49. The inverted terminal repeats of Tcb1 and Tcb2 also show sequence similarity with the Hctc1 repeat, however the length of the repeats, 80 bp, is deviating. The non-coding regions of Hctc1 between the inverted terminal repeats are T-rich, like in Tc1, Tcb1 and Tcb2, but have no clear similarity to the comparable Caenorhabditis sequences.

3.2. Target sequence of Hctc1 In order to reveal the Hctc1 flanking sequences, 21 different sequences flanking the 5%end and 16 different sequences flanking the 3%-end of Hctc1 of four different H. contortus populations were analysed. Each Hctc1 insertion is flanked by TA dinucleotides. A weak symmetrical consensus ANATAKNT of the target site can be deduced from the flanking sequences (Fig. 4). This resembles the better defined consensus sequence CAYATATRTG of the target site of Tc1 [23]. Particularly the positions − 5 and + 5 show a strong bias in base composition for both Hctc1 and Tc1.


3.3. Copy number of Hctc1 The copy number of Hctc1 in different H. contortus populations was roughly estimated by dot blot analysis. Different dilutions of RNase treated genomic DNA and linear plasmid DNA carrying a H2-H3 fragment of Hctc1 were hybridised with a labelled H2–H3 fragment of Hctc1, and subsequently hybridisation intensities were compared (Fig. 5). About 25 ng of genomic DNA hybridised equally as 0.03 ng of plasmid DNA with in total 0.25/2.9kb × 0.03 ng= 0.0025 ng of H2–H3 fragment to the probe. Since this equals to 0.016 ng of full length Hctc1, about 0.065% of the genomic DNA of different populations was estimated to be related with Hctc1, corresponding to 40 copies/100 Mb.

3.4. Hctc1 sequence 6ariation A comparison of nucleotide and deduced amino acid sequences of the different cloned PCR fragments of Hctc1 of populations SE and RHS6 revealed sequence variation (Fig. 6). The nucleotide similarities range from 62 to 99%. The heterogeny is due to nucleotide differences, deletions and insertions. In the region between the 5% inverted repeat and the coding region many deletions ranging in length from 1 to 111

Fig. 2. Comparison of the deduced amino acid sequence of Hctc1A with Tc1A and the putative proteins of C. briggsae elements Tcb1 and Tcb2 [13,14,26,32]. Residues of the DDE motif are indicated with asterisks. Identical amino acids are indicated with dots. Gaps in the alignment are represented by dashes.


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Fig. 3. Comparison of the sequences of the inverted terminal repeats of the elements Hctc1, Tc1, Tcb1 and Tcb2. Identical nucleotides are indicated with dots.

bp, and two insertions were found. In the inverted repeats, the coding region and the 3% noncoding region the variation between the clones is only based on nucleotide differences or deletions of 1 –11 bp, which in a few cases lead to a shift in the Hctc1A reading frame. Only four of the seven sequenced clones contain a start codon at the expected position. None of the variants is strictly typical for the SE or RHS6 population and no clustering of clones per population was apparent. To examine the length variation between Hctc1 elements in more detail, PCR products generated by primer H12, specific for the inverted terminal repeats, were analysed by Southern hybridisation with an internal part of the Hctc1A gene as probe (Fig. 7). Sequence analysis confirmed that the main fragment of approximately 1.6 kb corresponds to the full-length Hctc1 element (results not shown). Five smaller or larger Hctc1-related fragments are detected. The different H. contortus populations generally share the approximately five length variants of Hctc1.

4. Discussion Hctc1 has all typical features of the members of the Tc1 family: 1590 bp between two TA dinucleotides are bordered by 55-bp inverted repeats that end with a 5%-CAGT sequence, and carry one open reading frame highly similar to transposases with a conserved DDE motif. Within the Tc1 family Hctc1 is most similar to Tc1, Tcb1 and Tcb2, which in a phylogenetic analysis have been grouped together [4]. Since the Tc1 phylogeny is consistent with the species

phylogeny, horizontal transmission of the elements is not likely to have occurred [24,25]. The inverted repeats of the nematode elements share homologous regions, while other Tc1-like elements share only the terminal 5%-CAGT sequence [2,4]. The predicted proteins encoded by these four nematode elements are also similar, although the N-terminal 47 amino acids show little homology. The N-terminal 68 amino acids of Tc1A constitute a DNA binding domain that binds to base pairs 12-25 of the inverted terminal repeats, while amino acids 69–142 form another DNA binding domain that binds to base pairs 7–12 [7]. The similarity of the first 12 basepairs of the inverted terminal repeats in the four nematode elements and of the predicted proteins starting from amino acid 47 suggests that the binding of the C-terminal domain is structurally conserved in the related nematode Tc1-like elements. Conversely, the divergence of the N-terminal amino acids of the predicted proteins and of the nucleotides 13–31 of the inverted terminal repeats suggests that the binding of the N-terminal domain is not conserved in the four nematodes. This observation is surprising, since the variation in putative proteins of members of the Tc1-family in insects and fish is clearly lower [3,4]. Moreover, the 39 N-terminal amino acids from C. elegans are for 31% identical to a N-terminal domain of the prokaryotic IS30 transposase, implying an ancient origin of this DNA binding domain [26]. An analysis of Hctc1 flanking sequences reveals a consensus sequence that is similar to the Tc1 target site. In C. elegans Tc1 has clear preference for target sites with a symmetrical sequence, which is suggested to be a reflection of orientation independent insertion [27]. The resemblance of the target site preference of Tc1

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Fig. 4. The frequency of occurrence of nucleotides of 21 fragments flanking the 5%-end and 16 fragments flanking the 3%-end of Hctc1 and the deduced consensus of the putative insertion site of Hctc1.

and Hctc1 is an indication for the functional conservation of the encoded proteins. The copy number of Hctc1 was estimated to be about 40 per 100 Mb haploid genome. This is comparable with the Tc1 copy number of 27 – 31 for the 100 Mb haploid genome of five low-copy strains of C. elegans [12]. A high level of sequence variation is found between the sequences of the clones carrying fragments of the Hctc1 elements: the similarity ranged from 62–99%. Since no clustering of clones per population was found, the heterogeneity of the Hctc1 sequence is not due to differences between populations, but rather to variation within populations. Deletions, insertions or nucleotide differences in Tc1-like elements have been described repeatedly [28 – 31], but with a higher similarity of the Tc1-like elements (92 – 99%). The variability between the Hctc1 clones may reflect the high degree of genetic variation of the strongylid nematodes [16,17]. The different populations share also various length variants of elements that hybridise with a Hctc1 probe and can be amplified with primers specific for the inverted repeat. Levitt et al. (1989) observed that a Southern blot analysis of different C. elegans strains also revealed length variants which are smaller than Tc1, due to internal deletions. Further sequence analysis is required to reveal the structure of the Hctc1 variants. The identification of the H. contortus transposon may provide access to the methodology developed with C. elegans for the generation of

genetic markers and the elucidation of gene function.

Acknowledgements The work was supported by the Netherlands Organization for the Advancement of Research SLW-STW 790.43.805. We thank J. Tibben for expert technical assistance.

Fig. 5. Dot blot hybridisation for determination of the abundance of Hctc1 in H. contortus populations RHS6, CAVR, IRE and ISE. Genomic DNA of the populations, spotted in doubling dilutions from 25 ng (A) to 3.1 ng (D) and linear plasmid DNA of the clone carrying a 0.25 bp H2-H3 fragment of the SE population (total length plasmid: 2.9 kb), spotted in doubling dilutions from 1.28 ng (A) to 0.01 ng (H) was hybridised with a probe of the H2 – H3 fragment of the SE population.

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Fig. 6. Comparison of clones of the 5%-end of Hctc1; 3 clones, S1 – S3, from population SE, and 4 clones, R1–R4, from population RHS6. The Hctc1 consensus sequence is indicated. The inverted terminal repeat and the putative start codon are underlined. Identical nucleotides are indicated with dots. Gaps in the alignment are represented by dashes.

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Fig. 7. Southern analysis of PCR products, amplified using primer H12, of H. contortus populations CAVR, RHS6, IRE and ISE hybridised with the H2–H3 fragment of the SE population as Hctc1-specific probe.

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