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Institute of Cell Biology, Baltzerstrasse 4, CH-3012 Bern, Switzerland
Correspondence
Beatrice Lanzrein
beatrice.lanzrein{at}izb.unibe.ch
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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Primer sequences are available as supplementary material in JGV Online.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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). These viruses are formed in the calyx cells of the wasp's ovary and are injected along with the eggs into the host insects, mostly lepidopteran larvae or eggs (Webb, 1998). Polydnaviruses do not replicate in the lepidopteran host but are essential for survival of the wasp larva within the host; they play an important role in abrogation of the host's immune reaction (Schmidt et al., 2001) and in various aspects of host regulation (Lawrence & Lanzrein, 1993). We are working with the egg-larval parasitoid Chelonus inanitus (Braconidae) and its natural host Spodoptera littoralis (Noctuidae). In this system the bracovirus prevents encapsulation of the parasitoid larva (Stettler et al., 1998), affects feeding and nutritional physiology of the last instar host (Kaeslin et al., 2005) and induces a developmental arrest of the host in the prepupal stage (Soller & Lanzrein, 1996) by interference with the ecdysteroid system (Grossniklaus-Bürgin et al., 1998). Ichnoviruses and bracoviruses have different evolutionary origins (Whitfield & Asgari, 2003). The bracovirus–braconid association arose approximately 74 million years ago in a monophyletic lineage (microgastroid braconids) that consists of over 17 000 species (Whitfield, 2002); much less is known on the origin of the ichnovirus–ichneumonid association. The morphology and formation of viral particles is different between ichnoviruses and bracoviruses. Ichnoviruses are released continuously from calyx cells by budding and their nucleocapsids are surrounded by two membranes (Volkoff et al., 1995). Bracoviruses on the other hand are fully formed in the highly swollen nuclei of calyx cells and are then released by cell lysis; their nucleocapsids are surrounded by one membrane (Wyler & Lanzrein, 2003). Cotesia congregata bracovirus (CcBV) particles contain several nucleocapsids while the particles of the Chelonus inanitus bracovirus (CiV) contain only one (Albrecht et al., 1994). Polydnaviruses are the only viruses that have a segmented genome of double-stranded circular DNA (Webb, 1998) and, for CiV, it was shown that individual segments are singly encapsidated (Albrecht et al., 1994). Their genomes are large and up to now two bracoviruses and one ichnovirus have been fully sequenced (Espagne et al., 2004; Webb et al., 2006). In their proviral form the polydnaviruses are integrated in the wasp's genome. For the two bracoviruses CiV and CcBV it was found that various segments are clustered (Wyder et al., 2002; Belle et al., 2002). It is not known whether proviral segments are separated by spacers, although data obtained by Pasquier-Barre et al. (2002) suggest their existence. In CiV it was shown that viral DNA is replicated only in the proviral form and not after excision (Marti et al., 2003) and excision was shown to begin in the second part of pupal–adult development (Gruber et al., 1996). Similar observations have been reported for the EP1 segment of CcBV (Pasquier-Barre et al., 2002). The process of excision/circularization is proposed to occur through juxtaposition of direct terminal repeats followed by recombination across the repeats; as a consequence the circular segment and the rejoined DNA both contain one repeat (Gruber et al., 1996; Savary et al., 1997). Up to now the excision site loci have been characterized only for two CiV segments (Gruber et al., 1996; Wyder et al., 2002), one CcBV segment (Savary et al., 1997) and one Campoletis sonorensis ichnovirus (CsIV) segment (Rattanadechakul & Webb, 2003). Polydnavirus segments are present in non-equimolar ratios in calyx fluid (Webb, 1998) but it is not known whether this is also reflected in the abundance of the proviral segments. Here, the excision/circularization site of four additional CiV segments was analysed and this led to the identification of an extended excision site motif. Furthermore, analyses of flanking sequences of several proviral CiV segments revealed the existence of spacers between segments. From the orientation of the terminal repeats a model of the spacial organization of the loops formed before segment excision is proposed. In addition, the relative abundance of some segments in both the proviral cluster and the calyx fluid was measured.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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DNA isolation and sequencing.
Calyx DNA was collected and isolated as described in Albrecht et al. (1994). Genomic male or female DNA was obtained with the Wizard Genomic DNA purification kit (Promega). Three adult wasps were homogenized in 600 µl lysis solution with a Polytron (Kinematica) and extracted according to the kit's protocol for animal tissue. For sequencing reactions the BigDye sequencing kit (Applied Biosystems) was used according to the manufacturer's instructions. The PCR products were cleaned with DyeEx columns (Qiagen) and analysed with an ABI PRISM 3100 Genetic Analyzer (Applied Biosystems).
Analysis of excision sites and flanking regions.
To locate the regions in CiV segments that contain the excision site, the method described by Wyder et al. (2002) was applied. Several overlapping primer pairs were designed distributed over the entire segment and PCR was carried out using the Expand Long Template PCR System (Roche). As template either male or female adult C. inanitus DNA was used. The absence of a PCR product with a specific primer pair on male DNA indicates that the excision site is located between the primers. The common motif of the terminal repeats of CiV12 and CiV14 (Wyder et al., 2002) was used to scan the excision site-containing regions by local alignment with the GeneBee multiple alignment software (http://www.genebee.msu.su/services/malign_reduced.html). Inverse PCR (iPCR) was done according to Ochman et al. (1988). This method allows the amplification of unknown sequences flanking already known sequences. Male C. inanitus genomic DNA (1.5–2 µg) harbouring only the proviral form of CiV (Gruber et al., 1996) was digested with an enzyme which cuts near the predicted excision site of a segment and for which the frequency of recognition sites is between 10 and 20 in already known segments. This was CfoI for the left flanking region of CiV14.5 (iPCR1) and the left and right flanking regions of CiV21 (iPCR8 and iPCR10). For the right flanking region of CiV14.5 it was BfaI (iPCR3), for CiV16.8 left and right flanking regions AciI (iPCR7 and iPCR9) and for CiV22.5 right flanking region MspI (iPCR11). iPCR numbers correspond to primers in Supplementary Table S1 (available in JGV Online). For circularization, the fragments were ligated with T4 DNA ligase. The resulting PCR products were gel purified (Wizard SV Gel and PCR Clean-up System; Promega), cloned with the TOPO TA cloning kit (Invitrogen) and sequenced using M13 forward and reverse primers. The sequences obtained were aligned with the corresponding excised/circularized segments using CLUSTAL W (http://www.ebi.ac.uk/clustalw/). The site where an iPCR sequence stops to align to its respective segment represents the excision site. To sequence the rejoined DNA after segment excision primers were designed in the left and right flanking regions (Supplementary Table S1). Adult female C. inanitus DNA was used as template and the resulting PCR products were cloned into the TOPO vector. To search for a common excision site motif shared by all known proviral segments, the MEME Motif Elicitation software was used (Bailey & Elkan, 1994; software at http://meme.sdsc.edu/meme/meme.html). Analysed regions included at least 70 bp of segmental sequence up- or downstream of the excision site and at least 250 bp of proviral flanking sequence. Sequence logos based on the alignment were created with the WebLogo Sequence Logo Generator (Crooks et al., 2004; http://weblogo.berkeley.edu/logo.cgi).
To obtain the sequence of the spacer between CiV14 and CiV22.5, the clone 
1B231, containing CiV14, the complete spacer and a part of CiV22.5 (14FR), was digested with HindIII and the resulting 2 kbp fragment was excised from an agarose gel, cloned into TOPO and sequenced. The resulting sequence was found to overlap with the right end of the proviral CiV14 but did not reach CiV22.5 (Fig. 3a). The left border region of CiV22.5 was thus PCR amplified with primers as indicated in Fig. 3(a), cloned into TOPO and sequenced.
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Real-time PCR.
PCR runs were performed in 96-well optical reaction plates (Applied Biosystems) with the SYBR Green I Reaction System (Eurogentec). PCRs were done on a ABI PRISM 7000 Sequence Detection System (Applied Biosystems), using the following thermal profile: an initial denaturing step of 95 °C for 10 min was followed by 40 cycles of 95 °C for 10 s and 60 °C for 60 s. As templates, 1 ng calyx DNA or 300 ng male genomic DNA were used. Measurements were done in duplicate in at least three independent runs. For the determination of relative abundances, the standard curve method was used. Standard curves for viral segments were obtained by measuring five dilutions of segments cloned into pSP65 (CiV12, CiV14, CiV14.5, CiV16.8) or pBluescript (CiV21); for CiV22.5, a 11 641 bp part of the segment cloned into pCRII-TOPO (Invitrogen) was used. Standard curves for flanking spacers were obtained with pCRII-TOPO clones containing part of CiV12 left and right flanking spacers and the complete spacer between CiV14 and CiV22.5 (Sp14/22.5). For the detection of circular CiV segments in the calyx DNA, primers spanning excision/circularization sites were designed. Because of site-specific primer design constraints, the length of these PCR products varied between 134 and 178 bp; for the detection of the proviral segments and flanking spacers, primers were designed to give PCR products from 50 to 60 bp in length. For the detection of each proviral segment (CiV12, CiV14 and CiV22.5), the left and right spacers of CiV12 and the spacer SpCiV14/CiV22.5, an additional set of primer pairs was designed to give PCR products between 50 and 60 bp in length. As an exception, the alternative primer pair detecting CiV12 right flanking spacer gave a product of 178 bp (Supplementary Table S1). As the clones used for standard curves had different lengths, the molecule numbers were not equal in a given concentration (ng µl–1) of DNA. To correct this, quotients resulting from divisions of the length of each cloned target sequence plus vector by the length of the complete cloned CiV16.8 plus vector were used to adjust the calculated relative target abundances. The fractional cycle number of threshold value (Ct value) of a sample at a given constant threshold (for all samples in the same PCR run) was inserted into the equation of the corresponding standard curve fit. Resulting values are expressed as mean percentages+SD, relative to proviral or excised CiV16.8, respectively. CiV16.8 was chosen as a reference because of its intermediate abundance.
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RESULTS AND DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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; Wyder et al., 2002). To identify general characteristics of CiV excision/circularization sites, we analysed additional segments. For CiV16.8 the region containing the excision site had been narrowed down to 1855 bp (Wyder et al., 2002). For the segments CiV14.5 (Genbank accession no. AJ319654
[GenBank]
) and CiV21 (accession no. AJ627175
[GenBank]
), the region containing the excision site was narrowed down to 1.3 kb and 600 bp, respectively. The unknown excision sites and flanking sequences of CiV14.5, CiV16.8 and CiV21 could then be identified by sequencing the iPCR products of the proviral border regions (including at least 70 bp of segmental sequence, the excision site and at least 250 bp of proviral flanking sequence). A BLAST search with the newly sequenced segment CiV22.5 (Genbank accession no. AM261426
[GenBank]
) revealed that a part of the segment was identical to a stretch of sequence of the male genomic clone 1B231 (Wyder et al., 2002). As this clone contains the complete proviral segment CiV14 including right flanking sequence, CiV22.5 must be on the right-hand side of CiV14 in the proviral cluster. Aligning this lambda clone to the excised CiV22.5 led to the identification of the excision site. The right flanking region of CiV22.5 was analysed by iPCR. The rejoined DNA after excision of each segment was PCR-amplified and the product was cloned and sequenced. The results obtained in this (CiV14.5, CiV16.8, CiV21 and CiV22.5) and a previous analysis (CiV12 and CiV14) are shown in Fig. 1. Fig. 1(a) shows schematically the proviral integrated form of a segment (1), the formation of a loop and juxtaposition of the direct terminal repeats before excision (2) and the excised segment (3a) along with the rejoined DNA (3b). Fig. 1(b) shows an alignment of 34 nucleotides around the excision/circularization site of the six segments, namely the circular segments, the left and right flanking sequences of the proviral segments and the rejoined DNA. For all segments except CiV22.5, left and right is defined by aligning the repeats according to their terminal GCT. As the terminal repeats of CiV22.5 are inverted in relation to CiV14 in the provirus, the reverse complementary sequence is depicted to fit the alignment (see Fig. 3b
). All newly sequenced segments were found to have direct terminal repeats (Fig. 1, red) with sequence similarities to each other and to CiV12 and CiV14. All have a similar length (12 to 17 bp) and a GCT at the end. A comparison of the sequences of the repeats in the proviral and excised/circular form and the rejoined DNA supports the hypothesis that excision of segments occurs through a recombinational event which involves juxtaposition of the direct repeats [Fig. 1a (2)]. As a result, one terminal repeat of the proviral segment ends up in the circle and the other in the rejoined DNA [Fig. 1a (3)] (Gruber et al., 1996). Recombination through direct terminal repeats associated with rejoining of DNA has also been shown for the EP1 segment of the CcBV (Savary et al., 1997; Pasquier-Barre et al., 2002) and segment B of CsIV (Rattanadechakul & Webb, 2003).
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), the same two sequence variants (ATA and TAC) were found for the circular form and the rejoined DNA (Wyder et al., 2002). This suggests that recombination may happen at various positions either before the ATA or TAC variants (CTA in CiV12, TA in CiV14) or afterwards (GCTT), as illustrated in Fig. 1(b) (asterisks and crosses). For the terminal repeats of the segments CiV14.5, CiV16.8 and CiV22.5, no such variants were found and the recombination must take place within the terminal AGCT (CiV14.5 and 22.5) or terminal TTATAGCTT (CiV16.8). In the latter, the right terminal repeat has a C in the middle which is present in the circle but missing in the left repeat and the rejoined DNA. In contrast, segment CiV21 was found to behave similarly to CiV12 and CiV14 and to occur in two variants. Variant ‘a’ results from recombination within the terminal AGCT while variant ‘b’ results from recombination at the T (seventh last position of the repeat) whereby an additional T is added in the circular form which is lost in the rejoined DNA. A comparison between all CiV segments indicates that the terminal GCT is a preferred site of recombination. Interestingly, the direct terminal repeats of the EP1 segment of the CcBV also contain a GCT (Savary et al., 1997) as well as the segment B junction locus of the CsIV (Rattanadechakul & Webb, 2003). In five of six analysed CiV segments recombination appears to take place without loss or addition of nucleotides (Fig. 1b). In EP1 of CcBV, the only other bracovirus segment analysed in this respect, loss of one nucleotide during the recombination process was reported (Savary et al., 1997), but recent data do not support this finding (G. Periquet and J. M. Drezen, personal communication).
As shown in Fig. 1(b) all known proviral segments have a similar terminal repeat, whereby the similarity between the related segments CiV12 and CiV14 (Wyder et al., 2002) is the highest. When the border regions of all six segments were scanned with MEME (as mentioned above, border regions included at least 70 bp of segmental sequence, the excision site and at least 250 bp of proviral flanking sequence), the program found a common motif of 31 bp in seven of the 12 border regions, namely CiV12 left and right, CiV14 left and right, CiV14.5 left and right and CiV21 left. Interestingly, the motif was rooted at the nucleotide quadruplet GCTT/A which is always found at the end of the terminal repeats (positions 28–31 in Fig. 2). The five border regions not automatically included by MEME were then manually added to the MEME sequence block, using GCTT/A as a terminal anchor sequence and including upstream nucleotides to a total of 31 to match the length of the block. The sequence logos produced with all 12 sequences are shown in Fig. 2 and indicate the existence of an extended excision site motif which is larger than the direct terminal repeat. The highest level of conservation is seen within the terminal repeat: at positions 28, 29 and 30 there is always GCT, at positions 18–20 near the beginning of the repeat mostly AAT and at position 23 mostly T. In the remainder of the extended excision site motif the level of conservation is lower. Nevertheless, there is a high conservation of AT at positions 1 and 2 as well as 12 and 13. When the possibility of random occurrence of the extended excision site motif was calculated (according to D'Haeseleer, 2006) it was found that it may appear every 2.9x106 nucleotides by coincidence. As CiV segments have sizes around 9 to 30 kbp, the occurrence of this motif at both ends of every proviral segment is highly significant and suggests that it is a specific recognition site for proteins involved in circularization and excision. For the EP1 segment of the CcBV, part of the terminal repeat was shown to have similarity to a Hin recombinase binding site (Savary et al., 1997). Such a similarity was not found in the six CiV segments analysed here. When a stretch of 60 nucleotides within the EP1 excision region was used to make comparisons with all other CcBV segments, 11 identical nucleotides including a GCT at the end were found in the presumptive terminal repeats of eight out of the 29 segments; furthermore, a search with the same 60 nucleotide stretch revealed the same 11 nucleotides in some segments of the Cotesia plutellae BV and the Microplitis demolitor BV (G. Periquet and J. M. Drezen, personal communication). However, in none of these segments has the actual excision site been identified. The similarity of CiV excision sites with these other bracoviruses appears to be restricted to the GCT.
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1B231 situated at a distance of about 2 kb from CiV14 in the proviral cluster (Wyder et al., 2002 and Fig. 3). This spacer between CiV14 and CiV22.5 was sequenced and found to have a length of 2065 bp, including both terminal repeats (Genbank accession no. AM261418
[GenBank]
). This observation about the proviral location of CiV22.5 is in agreement with earlier data showing that probe 14FR hybridized strongly with a circular segment of approximately 22 kbp (Wyder et al., 2002). This is the first identification of a spacer separating two proviral segments of a polydnavirus. Gene prediction with Softberry software suggested the presence of a gene with a deduced product of 57 amino acids and two exons on the spacer. However, protein and DNA BLAST searches gave no significant similarities and PCR on C. inanitus cDNA of female late pupae, a stage when replication of viral DNA and formation of virions is very intense (Marti et al., 2003; Wyler & Lanzrein, 2003), did not give a product (data not shown). The male genomic clone 2B211 contains a large portion of CiV14 and left flanking sequences; Southern blots with probe FL from this clone had indicated that CiV14 is flanked on the left side by an approximately 30 kbp segment (Wyder et al., 2002). This clone was digested with HindIII, and the 3 kbp fragment containing the left border of CiV14 and flanking sequence was cloned and sequenced. This allowed the identification of the spacer on the left side of CiV14 (Sp14L, Genbank accession no. AM261417
[GenBank]
). Sequence comparisons with the excision site logo of Fig. 2 suggest that the excision site of the approx. 30 kbp neighbouring segment is located at a distance of 1299 bp or 1640 bp, whereby the site at 1299 bp had higher similarity. PCR experiments with host haemolymph as template (data not shown; for experimental approach see below) support that the neighbouring approx. 30 kbp segment begins at one of these sites. The right flanking spacer of CiV22.5 was analysed by iPCR and 1147 bp could be sequenced. No indication of an excision site of a neighbouring segment was detected within this region, suggesting that this spacer is longer than 1159 bp. Interestingly the terminal repeats of CiV14 and CiV22.5 have an opposite orientation (Fig. 3b, top). A hypothetical model of the putative loop formation preceding excision of CiV14 and CiV22.5 is illustrated in Fig. 3b bottom; it proposes a model of how spacers could play a role in the spacial organization of the proviral cluster. In view of the demonstration of spacers between proviral segments, the earlier data on amplification of proviral, excised and rejoined DNA (Marti et al., 2003) can be reinterpreted; they show that spacers are coamplified with proviral sequences and that rejoined spacers appear simultaneously with excised viral segments. Whether amplification is from the chromosomally integrated form or from an excised macrolocus containing the proviral cluster is not known, as the borders of the cluster have not yet been identified.
Parts of additional spacers were sequenced by the iPCR approach: for CiV14.5 left and right 263 and 475 bp (Genbank accession nos AM261419
[GenBank]
and AM261420
[GenBank]
), for CiV16.8 426 and 330 bp (accession nos AM261421
[GenBank]
and AM261422
[GenBank]
) and for CiV21 511 and 312 bp (accession nos AM261423
[GenBank]
and AM261424
[GenBank]
). The partial spacer sequence of CiV12 left was already known, namely 624 bp (Genbank accession no. Z58830
[GenBank]
, pos. 1–624) as well as that of CiV12 right, namely 402 bp (accession no. Z58831
[GenBank]
, pos. 56–461) and were taken from Gruber et al. (1996). The flanking sequences of these segments were also scanned with the common motif of the terminal repeats, but, with the exception of CiV12 left flanking sequence, no indication of an excision site could be found. The potential terminal repeat/excision site on the CiV12 left flanking sequence was found at a distance of 159 bp from CiV12. To analyse the fate of spacer sequences and to exclude the possibility that they represent very small segments, additional experiments were carried out. Fig. 4(a) shows two possible states of the spacer Sp14/22.5. In state X, a situation after excision of CiV14 and CiV22.5 is illustrated, whereby the spacer Sp14/22.5 comes to lie between the left flanking spacer of CiV14 (Sp14L) and the right flanking spacer of CiV22.5 (Sp22.5R) in the rejoined DNA. In state Y, the spacer Sp14/22.5 would be excised and represent a mini segment and CiV14 left and CiV22.5 right flanking spacers would lie next to each other. With primers as indicated in Fig. 4(a) and adult female C. inanitus DNA as template, the existence of the two possible states was investigated. Fig. 4(b) shows a PCR product of the expected size (about 2.6 kbp) for state X but none for state Y (expected product size 520 bp). Sequencing of the 2.5 kbp PCR product confirmed that it consists of parts of Sp14L, the entire spacer Sp14/22.5 and parts of Sp22.5R. These data indicate that Sp14/22.5 is not excised. An additional PCR product of approx. 1.3 kbp was seen (Fig. 4b, asterisk); it was also sequenced and found to result from unspecific annealing of the left primer within the spacer Sp14/22.5. To verify further that the spacer Sp14/22.5 is not excised and circularized an additional experiment was carried out. Two primers at the left and right end of Sp14/22.5 were designed, pointing towards the proviral segments CiV14 and CiV22.5 (SpccLL and SpccRR, see Supplementary Table S1). These would produce a PCR product of 164 bp if Sp14/22.5 exists in excised circularized form. As a positive control the same primer pair was used on BstXI-digested and religated DNA from a TOPO clone containing the complete Sp14/22.5 as insert. BstXI restriction sites are located left and right of the insert on the TOPO vector. As a consequence, religation of the mixture produces circularized inserts with about 140 additional nucleotides from the vector. Fig. 4(c) (1) shows that no 164 bp PCR product was found with calyx fluid DNA as template; this again indicates that Sp14/22.5 is not excised and circularized. On the other hand, products of the expected sizes were seen with the primers SpccLL and SpccRR and the circularized insert as template [Fig. 4c (2)] as well as with primers on CiV14 and calyx fluid DNA as template [Fig. 4c (3)]. Furthermore, the presence of viral segments and spacer sequences was compared between female C. inanitus DNA and haemolymph of parasitized hosts. Fig. 5 shows the results obtained for Sp14/22.5 as well as left and right flanking spacers of CiV12 and CiV16.8. Primers to detect Sp14/22.5 were 14R22.5Lf/r, primers to detect CiV12 left and right spacers were 12Lf/r and 12Rf/r, and to detect CiV16.8 left and right spacers the primers were 16.8Lf/r and 16.8Rf/r. For details see Supplementary Table S1. The PCR products obtained indicate that only viral segments are detectable in host haemolymph (Fig. 5a, b and c lane 1) but no spacer sequences (Fig. 5a, b and c lanes 3 and 5). Spacer sequences were only seen in female wasp DNA (Fig. 5a, b and c lanes 2 and 4). The same observations were made with CiV14 left spacer and CiV14.5 left and right spacers (data not shown). Taken together, the results of Figs 4 and 5 show that segments in the proviral cluster are flanked by non-segmental spacer DNA which is not excised and not injected into the host.
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); this was also observed in CiV (Albrecht et al., 1994; Marti et al., 2003). In previous publications it was shown that CiV segments are clustered in the wasp genome (Wyder et al., 2002) and that they are amplified only in the proviral form but not after excision and circularization (Marti et al., 2003). This suggests that abundant segments are present in multiple copies in the proviral cluster. Here we measured and compared the relative abundance of six proviral and excised segments as well as that of three spacers. In Fig. 6, the results obtained by real-time PCR are summarized. CiV12 shows the highest relative abundance in both calyx fluid and male genomic DNA. CiV16.8 has the second highest abundance, while CiV14, CiV14.5, CiV21 and CiV22.5 show values between 7 and 25 % compared with CiV16.8. For each analysed segment the abundances of its proviral and excised form varied to a similar extent relative to CiV16.8; this indicates that in the proviral form abundant segments are present in higher copy numbers than less abundant segments. This is the first analysis to address this point in any polydnavirus. It is not clear whether variable proviral segment abundance is a general feature of polydnaviruses or restricted to bracoviruses or CiV. The findings are in agreement with the earlier observation that amplification of CiV DNA is restricted to the proviral form (Marti et al., 2003). The spacer Sp14/22.5 was found to have almost the same relative abundance as the flanking segments. CiV12 left and right flanking spacers were present with about a third of the abundance of CiV12. This could mean that the abundant segment CiV12 occurs in the proviral cluster with more than one different left and right flanking spacer and that we identified only one on each side. This is in agreement with the finding that the quantity of rejoined DNA (representing the rejoined spacers described above) was lower than that of the excised segment in the case of CiV12 but not CiV14 (Marti et al., 2003). Nothing is yet known about the position of the various segments within the proviral cluster and whether segments present in multiple copies are grouped together within the cluster or are distributed. An answer to this question could only be obtained by sequencing the entire proviral cluster.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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Ochman, H., Gerber, A. S. & Hartl, D. L. (1988). Genetic applications of an inverse polymerase chain reaction. Genetics 120, 621–623.
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Received 20 July 2006;
accepted 9 October 2006.
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