Environmental Biosafety Research Institute, Agricultural Biotechnology Centre, Szent-Györgyi Albert str. 4, H-2101 Gödöll, Hungary
Correspondence
János Kiss
kissj@abc.hu
Ferenc Olasz
olasz{at}abc.hu
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ABSTRACT |
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Ildikó Szeverényi and Zita Nagy contributed equally to this work.
Present address: Temasek Life Sciences Laboratory, 1 Research Link, The NUS, Singapore 117604.
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INTRODUCTION |
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Several recent findings indicate that covalently joined IRs separated by a short spacer region (SR) are key structures in the transposition of some ISs and this junction acts as a transposition intermediate for the element. Such junctions have been identified in IS dimers consisting of two directly repeated elements and in minicircles formed by circularization of a single element. IS dimers have been isolated from IS2 (Szeverényi et al., 1996a), IS21 (Reimmann & Haas, 1987
, 1990
; Reimmann et al., 1989
), IS30 (Dalrymple, 1987
; Olasz et al., 1993
, 1997
), IS256 (Prudhomme et al., 2002
) and IS911 (Turlan et al., 2000
), and covalently closed circular IS elements have been described for IS1 (Sekine et al., 1997a
, Turlan & Chandler, 1995
), IS2 (Lewis & Grindley, 1997
), IS3 (Sekine et al., 1994
), IS30 (Kiss & Olasz, 1999
), IS117 (Smokvina et al., 1994
), IS150 (Welz, 1993
; Haas & Rak, 2002
), IS256 (Prudhomme et al., 2002
) and IS911 (Polard et al., 1992
, 1996
). For some of these elements both circular and dimeric forms have been reported, suggesting a possible connection between the formation and transposition of these structures, as described for IS30 (Kiss & Olasz, 1999
).
The IS dimer model developed for Escherichia coli element IS30 (Olasz et al., 1993) is based on reactive IRIR junctions and is able to explain all the transpositional reactions: deletion, inversion, transpositional fusion (cointegration) and simple insertion (Fig. 1
). The key structure of this model is the IS dimer and is produced by site-specific dimerization (SSD) from any replicons carrying two IS elements. The dimer can promote the transpositional rearrangements mentioned above, but most frequently segregates into a replicon carrying only a single IS copy via a site-specific process, called dimer dissolution (DDS). This model is consistent with those based on covalently closed minicircles (Polard et al., 1992
; Lewis & Grindley, 1997
) and accounts for simple insertion on the basis of clear analogy between minicircles and IS-dimer-containing replicons (Kiss & Olasz, 1999
). Similar to the dimer structures, the covalently joined IRs are also separated by a few base pairs in the circular form of the IS elements. These minicircles can be considered as intermediates of simple insertion, as reported for IS1 (Sekine et al., 1997a
; Turlan & Chandler, 1995
), IS2 (Lewis & Grindley, 1997
), IS3 (Sekine et al., 1994
), IS150 (Welz, 1993
; Haas & Rak, 2002
), IS256 (Prudhomme et al., 2002
) and IS911 (Ton-Hoang et al., 1997
, 1998
). The synthetic IS dimer model seems to be applicable for the elements that form both minicircles and dimers (IS2, IS21, IS30, IS256 and IS911); however, its validity might also be extended to additional elements (e.g. IS3, IS150) for which only minicircles have been detected so far.
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METHODS |
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All strains were grown at 37 °C in liquid and solid LuriaBertani (LB) media supplemented as indicated with antibiotics at the following final concentrations: ampicillin (Ap), 150 µg ml-1; chloramphenicol (Cm), 20 µg ml-1; kanamycin (Km), 20 µg ml-1; rifampicin (Rif), 60 µg ml-1.
Plasmids.
Plasmid constructs were cloned into pBluescript KS+ (pKS) (Stratagene), pEMBL19 (Dente et al., 1983) or pJKI88 (Kiss & Olasz, 1999
), respectively. Full IS copies were derived from pAW1326 : : IS plasmids (Szeverényi et al., 1996b
). The cat cassette encoding the CmR gene (chloramphenicol acetyltransferase gene of Tn9) was obtained from pAW302 (Stalder & Arber, 1989
). The joined IRs for C1 and C4 constructs were PCR-amplified from pAW1326 : : IS plasmid templates using the primer pairs oISZ17/oISZ18 for IS3, oISZ23/oISZ24 for IS150 and oISZ19/oISZ20 for IS186 (see Table 1
for primer sequences). The PCR product for IS3 was cloned as a 342 bp HindIII fragment containing the right (10541258 bp) and the left (1138 bp) ends; for IS150, a 560 bp EcoRVTaqI fragment with joined right (11571443 bp) and left (1274 bp) ends was cloned; and for IS186, a 416 bp BamHISmaI fragment with joined right (11481437 bp) and left (1211 bp) ends was cloned. For cloning of C3 constructs, the truncated left ends of the elements were also amplified from the respective pAW1326 : : IS templates (Szeverényi et al., 1996b
) with the primer pairs IS3in/IS3Lout, IS150in/IS150Lout and IS186in/IS186Lout or IS186in2/IS186Lout (for C3 and C3', respectively). The cloned fragments in C3 constructs contained 1138 bp of IS3, 1577 bp of IS150 and 1210 bp of IS186. For cloning the compound transposons in pKS, the cat gene from pAW302 and the fragment of pAW1326 : : IS carrying the full IS copy were used. The whole structures along with flanking regions derived from pAW1326 : : IS were transferred into pJKI88 to establish the low-copy-number version of plasmids carrying the same transposons.
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PCR amplification of right IRleft IR (IRRIRL) junctions.
For detection of the joined IRs from the three IS elements, artificial compound transposons were constructed from each element and the plasmids carrying the transposons were introduced into JM109 cells. Three transformants were grown in 3 ml LB+Km without selection for the resistance marker of the transposon (CmR). Stationary-phase cultures (0·1 ml) were diluted 30-fold with fresh media every 12 h and grown at 37 °C. This step was repeated 10 times (each passage represents about five generations). Plasmid DNA was isolated from the 1st, 3rd, 5th, 7th and 10th passages. The three parallel samples were pooled, RNase-treated and used as templates in PCR reactions performed with the following primers: oISZ18/IS3Lout (IS3), IS150Rout/IS150Lout2 (IS150) and IS186Rout2/IS186Lout (IS186). The cycling included 1 min 94 °C pre-denaturation; 30 cycles of 20 s at 94 °C, 1 min at 55 °C and 3 min at 72 °C; and finally 5 min at 72 °C for terminal elongation. Plasmid samples (pAW1326 : : IS or pAW1326) and genomic DNA purified from JM101 or JM109 strains were also used as templates for the amplification of the abutted IRs using the following primers: oISZ17/oISZ18 (IS3), oISZ23/oISZ24 (IS150) and oISZ19/oISZ20 (IS186). The 30 cycles included 20 s denaturation at 94 °C, 1 min annealing (67 °C for IS3, 60 °C for IS150 and 55 °C for IS186) and 1 (IS3, IS186) or 1·5 min elongation (IS150) at 72 °C. A final elongation was performed for 5 min at 72 °C.
Detection of IS dimerization in C3 structures.
C3 constructs for IS3 and IS186 were introduced into JM109 cells and three transformant colonies were grown in 3 ml LB+Ap. Every 24 h, 30 µl culture was diluted 100-fold with fresh medium and grown further. This step was repeated 10 times (each passage represents about seven generations). Plasmid DNA was purified from the 1st, 3rd, 5th, 7th and 10th passages. The three parallel samples were pooled, RNase-treated and used as templates in PCR amplifications with the following primers: oISZ18/pUCrev (template: C3-IS3) and IS186Rout2/pUCrev (template: C3-IS186). Conditions were 30 cycles of 20 s at 94 °C, 30 s at 55 °C and 1·5 min at 72 °C.
Stability test for C0C4 structures.
The test system used to determine the activity of different IS-derived structures was composed of five ApR plasmids, each carrying a CmR gene. Plasmids C0C4 for IS3, IS150 and IS186, were transformed into the recA- JM109 strain and three colonies from each transformation were grown overnight in 3 ml LB+Ap. The cultures were subjected to seven passages (except for C3 constructs, which were cultured through 10 passages) under Ap selection as described in the previous section. Plasmid DNA was isolated after each passage, then the 3rd, 7th and 10th (for C3) were used to transform TG2 cells. The transformants were tested for CmR/CmS phenotype by replica plating. The frequency of transposition was calculated as a ratio of CmS ApR/ApR colonies.
Analysis of DDS products.
DNA samples of C5 dimers derived from C4 constructs were digested with an appropriate restriction endonuclease (IS3, StuI; IS150, BglII; IS186, NdeI), for which the intact IS element has a unique cleavage site located outside of the truncated part. JM109 cells were transformed with the digested plasmid population and DNA was purified from at least 30 of the resulting ApR colonies that were very likely to contain products of DDS (C6). The structure of the plasmids was determined by restriction analysis and some of them were also sequenced.
Mating out assay.
Plasmids C0C5 were used to transform the C600 donor strain containing the pOX38Km (Chandler & Galas, 1983) conjugative plasmid. The transformants were selected on LB+Km, Ap, Cm (C1C4) or LB+Km, Ap (C5) plates. Conjugation was carried out on solid LB medium by mixing 0·15 ml O/N donor and 0·05 ml O/N RifR HB101 recipient cultures both washed with 0·9 % NaCl solution to remove antibiotics. Plates were incubated for 6 h at 37 °C, then bacteria were resuspended in 5 ml LB and washed twice with 0·9 % NaCl before plating on LB+Km, Ap, Rif. The frequency of transposition was calculated as a ratio of the KmR ApR RifR and the KmR RifR transconjugants.
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RESULTS |
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Subsequent Southern hybridization and sequence analysis of the PCR fragments confirmed the existence of the IRRIRL junctions for all three elements. Between the abutted IRs, the so-called SR was generally identical with the target duplications that bordered the three elements before joining of the IRs, but some divergence in spacer length was detected (see below).
All three IS elements produce head-to-tail dimers
PCR amplification of the junction fragment of IS3 and IS186 produced extremely weak products, indicating a very small amount of dimer in the template DNA, whereas IS150 resulted in a stronger signal, allowing the isolation of plasmids carrying head-to-tail dimers (Fig. 2). Plasmid DNA samples from the 10th passage of the IS150-based transposon (Fig. 3
a) were transformed into strain TG2 and the transformants were tested by replica plating for the presence or absence of the CmR marker of the transposon. The frequency of the CmS segregants was 9·7±3·3 %. Among 81 plasmid samples prepared from CmS colonies, 64 (79 %) carried a single IS150 copy (monomer), 14 (17 %) represented three types of deletions and 3 (4 %) were a mixed population of plasmids containing the head-to-tail IS150 dimer, monomer and various types of deletions (Fig. 3b, c
, lanes 2). The three isolates containing dimers were re-transformed into TG2 to separate the different plasmid species. More than half of the samples (35/61) extracted from these transformants contained a monomer element (Fig. 3b, c
, lanes 4), five cases were found to be mixed populations similar to the original isolate (Fig. 3b, c
, lanes 3) and various kinds of deletions seem to have occurred in the remaining cases. The failure to isolate the dimer as a unique species suggested that it was highly active. The fragments corresponding to the head-to-tail dimer were isolated from the gel and sequenced. The results confirmed that they carried IRRIRL junctions with a 3 bp SR, proving the capacity of IS150 for dimerization.
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Both dimers and monomers (C5 and C6, Fig. 5) were found among CmS segregants of C3-IS3. The DNA isolates of C5-IS3 dimers seemed to be uniform by restriction analysis, but when they were reintroduced to TG2 cells, the resulting transformants contained C5 dimers or C6 monomers, which supported the idea of C5 being an active intermediate. The isolation of the C5-IS186 dimer was somewhat more problematic, because hundreds of CmS segregants turned out to be C6-type monomers. Pre-screening of 192 CmS transformants by colony PCR that amplifies dimers identified a colony still containing the C5-IS186 structure (demonstrated by sequencing). The plasmid DNA isolated from this colony was a mixed population of the dimer and numerous different segregation products, indicating that C5-IS186 was indeed very unstable.
The IRRIRL junction actively participates in transposition
We developed a test system for comparing the stability of IS-derived constructs containing different parts of IS elements (Fig. 5, C0C4). The system is suitable for the detection of deletions leading to the loss of the CmR marker gene. The constructs carried the following structures: C0, CmR gene without any IS-derived sequence (control to detect spontaneous mutations); C1, CmR gene and a PCR-amplified IRRIRL junction (truncated dimer) without functional transposase production; C2, CmR gene and a single intact IS element (monomer); C3, an intact and a truncated IS copy flanking the CmR gene on both sides; and C4, the full IS element and the truncated dimer flanking the CmR gene on both sides. These plasmids were constructed for all three IS elements, introduced into JM109 cells and the ratio of the plasmid population that had lost the CmR phenotype was determined after the 3rd and 7th passages (Table 2
).
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Detection of transposition activities characteristic for IS dimers
While analysing the structure of plasmids obtained from the CmS segregants of C4 constructs, a C5-type dimer, containing an intact and a truncated IS element, was isolated (IS3, in 11 samples out of 45 analysed; IS150, 22/75; IS186, 49/100). This product presumably arose by SSD or by transposition of the IRRIRL junction to the IR end of the full IS copy, as shown for IS911 (Turlan et al., 2000) and IS30 (Kiss & Olasz, 1999
; Olasz et al., 1997
). The C5 dimers were similar to those that arose from C3 and the SR was identical with the sequence originally separating the truncated IRs in C4 (Fig. 7
). The truncated dimers (C5) derived from C4 constructs (and also from C3) were not the end products of the rearrangements. They could be stabilized by the loss of the full IS element, giving rise to plasmids containing the single truncated element (C6, Fig. 5
). To prove that C6 originated from the dimer, the DNA of C5 isolates was reintroduced into strain TG2 and the plasmid DNA of transformants was analysed. C6-type structures were found among plasmid samples of all three elements, supporting the idea that C6 arose from C5. These results suggested a two-step transition C4
C5
C6, albeit a C4
C6 reaction can also occur, since C6 structures were already present in a high proportion among the CmS segregants of passaged C4 constructs (IS3, 4353 %; IS150, 4671 %; IS186, 1083 %).
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In the case of IS3, the SR in C4-derived C5 structures was GAG (Fig. 7c), corresponding to the triplet in the spacer of the truncated dimer in C4. On the other hand, C3-derived C5-IS3 structures had a CCA spacer (with one exception, where it was a single C), which originally flanked the intact IS element in C3. Further IRRIRL junction sequences were investigated which we found in JM101 or JM109 cells containing the pAW1326 plasmid. We found two samples with an AGT spacer, probably derived from the genomic element IS3A (Umeda & Ohtsubo, 1989
; EMBL/GenBank PRO02 : ECAE137). Four other isolates had a GAG triplet in between the IRs and one sample contained ATG. The origin of these latter spacers remains unknown.
The SRs in three C5-IS150 isolates were AGG, which may have derived from the junction in C4, while the spacer of dimers originating from the low-copy-number compound transposon showed variability: one of them had an AAG spacer, which flanked the IS elements in the transposon, while the AGG spacer may represent an unusual dimerization process. Additional samples were sequenced from the pAW1326 : : IS150 isolates. The majority of them contained AGG, but we found AAG, GTA and ACC triplets, as well. The AAG spacer corresponds to the target duplication bordering the element in pAW1326 : : IS150 and GTA flanks the IRL of the genomic IS150 copy (EMBL/GenBank PRO02 : ECAE433), while AGG and ACC do not match the bases bordering IS150 in pAW1326 : : IS150 or those that delimit the chromosomal copy.
Three C5-IS186 isolates derived from the C4 structure all contained the same 10 bp spacer, GGCTCGATCC, that corresponded to the SR in the IRRIRL junction and the flanking sequences of the full IS186 copy on C4. To identify the origin of SR in C5 structures we constructed two almost identical C3-IS186 plasmids, which differed only in a single GA change that was introduced to the sequence adjacent to the truncated element (C3'-IS186, Fig. 7a
). This enabled us to trace the origin of SR in the junction. The amplified IRRIRL junctions in the C3 and C3' plasmid populations showed considerable diversity (Fig. 7c
). However, it was clear that the SRs were derived from the sequences flanking either the left or the right IR in C3' structures. Further junctions amplified from one pAW1326 : : IS186 DNA template comprised the 10 bp spacer GGCTCGATCC, which was identical with the target duplication flanking the element in pAW1326 : : IS186.
SR is lost during DDS
The bordering sequence in C6 structures may provide information on the mechanism of dissolution of C5 dimers, therefore C6 was isolated and sequenced. The flanking sequences in C6-IS3 (Fig. 9a) differed from the SR of C5-IS3, indicating the loss of the spacer during DDS. This corresponds to the results obtained for dissolution of the (IS30)2 dimer (Olasz et al., 1993
). However, the bordering sequences in different C6-IS3 samples were not identical. Only in one case (out of 14) was the single IR end flanked by the original CCA target duplication. In the remaining cases the IR was bordered by sequences generated from the original flanking region of C5 by a short (15 bp) deletion. For IS150, the bordering sequence in C6 was identical to the flanking region of the intact IS element in C5 (AAG) in two out of six isolates. In the remaining cases an additional base (G), presumably originating from the SR, was found right beside the IR end (Fig. 9b
). The sequence bordering the left end of the IS186 copy in six isolates of the C5 structure was identical with that of the SR; therefore, the origin of the sequence flanking the truncated IS copy in C6 derivatives could not be determined.
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DISCUSSION |
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Although the C5 structure for both IS3 and IS150 was the most active species in intermolecular transposition, the C4-borne C5 structure of IS186 showed only as much activity as the truncated dimer in C1 (Fig. 8), whereas both C3-IS186 and C4-IS186 were more active than C5. The fact that C1, C4 and C5 structures, all of which contain an IRRIRL junction, and C3, which has a potential to form such junction, were more active than C2 containing an IS186 monomer, clearly indicates that the IRRIRL junction represents an active transpositional intermediate. The observations that (i) both C3 and C4 segregated into C6 with high frequency, (ii) the C5-type molecules could hardly be isolated among segregants of C3, (iii) the only C3-borne C5 isolate was not uniform, and (iv) C5 structure could not be recovered by re-transformation of this isolate all suggest that IS186 may behave similarly to the other two elements. The formation of C5-IS186 dimers results in the appearance of a variety of structures differing in their SR (Fig. 7c
) and possibly in their transposition activity. However, the only structure we could recover by re-transformation was a C5 derivative carrying a 10 bp SR, presumably because it was the least active.
Although formation and dissolution of the joined IRs share similar basic characteristics, the different ISs show important differences. One of these features is the difference in the formation of the spacer between the joined IRs during SSD. In the dimers of IS3 or IS150 the length of the spacer was generally the same as that of the target duplication generated by the respective element, while IS186 produced IRRIRL junctions with divergent spacer length (Fig. 7c). The sequence of the SRs, where it was traceable, always derived from the flanking regions of one or the other IR taking part in dimerization, as has been reported for other elements [IS1 (Sekine et al., 1997a
, Turlan & Chandler, 1995
); IS3 (Sekine et al., 1994
); IS30 (Olasz et al., 1993
); IS911 (Polard et al., 1992
)]. The C4-derived C5 dimers of all three ISs contained the same SRs that separated the truncated IRs rather than the bases flanking the intact element in C4, whereas C3-borne IRRIRL junctions behaved differently. In IS3 junctions the spacer always derived from the target duplication bordering the full IS copy in C3 and never from the bordering region of the truncated one, while in IS186 junctions the SRs derived either from the flanking sequence of the full or the truncated copies. The different origin of SRs in C3- or C4-borne dimers may reflect the differences in the molecular processes that can occur when two IRs (C3) or an IRRIRL junction and a third IR (C4) take part in the dimerization.
It can be assumed that the length of the SR depends on the site of the staggered cuts adjacent to the reacting IRs. This cleavage site in the cases of IS30 (Olasz et al., 1997, 1998
) and IS911 (Ton-Hoang et al., 1998
; Turlan et al., 2000
) proved to be at the same distance from the end of IRs both in dimerization or integration into the target site, resulting in the same length of the spacer and of the target duplication. On the other hand, the spacer in (IS2)2 (Szeverényi et al., 1996a
) and in (IS21)2 (Reimmann & Haas, 1987
) does not correspond to the target duplication characteristic for these elements. In these cases the cleavage site is different in SSD and integration into the target DNA. These deviations can play a role in the regulation of transposase gene expression, especially in the formation of new hybrid promoters, as summarized for IS911 (Ton-Hoang et al., 1997
) and proposed for IS30 (Dalrymple, 1987
). Although there are no obvious hybrid promoters (-35 promoter box in the IRR and correctly positioned -10 box in the IRL) in the joined IRs of IS3 (Duval-Valentin et al., 2001
), IS150 and IS186, all of these elements can generate promoters by insertion, as shown by their entrapment in pAW1326 (Szeverényi et al., 1996b
). Furthermore, the target specificity may also play a role in the formation of spacers in IS186 junctions. The flanking DNA of the truncated element in C3-IS186 contains hotspot-like sequences for IS186 that differ only in one position from the consensus G3N6C3 target site of the element (Keller, 1996
; SaiSree et al., 2001
), which may influence the dimerization reaction and consequently the length of SR. The fact that different flanking sequences of IS186 influenced the length of the resulting SR (Fig. 7c
) supports the role of flanking sequences in spacer formation via dimerization. It is worth noting that IS186 generates a variable length of target duplication via insertion as well (Sengstag et al., 1986
).
The ability of joining the left and right IRs of an element accompanies the potential to form both dimers and minicircles. The primary isolates of IRRIRL junctions (i.e. those generated by PCR from the compound transposons and pAW1326 or pAW1326 : : IS templates) could have originated either from tandemly repeated or circular IS elements. The fact that joined ends for IS3, IS150 and IS186 were also detectable in bacteria containing pAW1326 without IS elements suggests that several amplified IRRIRL junctions might have derived from minicircles of genomic IS copies.
Since the SR derives from the flanking sequences of the original IS copy, this allows us to determine the ancestry of some IRRIRL junctions isolated. When the junctions were amplified from pAW1326 : : IS186 DNA templates, the abutted IRs were separated by the GGCTCGATCC sequence, which was identical with the direct repeat bordering the element in pAW1326 : : IS186. This suggested that the junction was formed by the inserted element via dimerization or circularization. The same origin could be assumed for the IRRIRL junction of IS150 with the AAG spacer, while GTA could derive from the genomic IS150 copy. Similarly, the abutted IRs of IS3 separated by GAG must have derived from the IS3A element of E. coli. Numerous SRs of unidentified origin were found in the IRRIRL junctions of IS3 and IS150, as was also reported for IS1 and IS3 circles (Sekine et al., 1994, 1997b
). This indicates that at any time the bacterial population carries transpositional intermediates deriving from new insertions, which may be present only in subclones of the cells. The other explanation for this phenomenon may be the ability of these IS elements to undergo multiple cycles of rearrangements, e.g. successive cycles of circularization and dimerization, which has been reported for IS30 (Kiss & Olasz, 1999
).
Our experiments provide evidence that DDS takes place in IS3, IS150 and IS186 dimers similarly to those described by Olasz et al. (1993) and Turlan et al. (2000)
. However, this process also has some unique features in IS3 and IS150 (Fig. 9
). In the dissolution of the C5-IS3 dimer, the bases that directly flanked the IRL of the full element (the original target duplication) were lost in many cases and the bases positioned originally 25 bp upstream from the IRL bordered the remaining IR end in the C6 product (Fig. 9a
). This phenomenon may provide an explanation for the interesting fact that only 4 out of the 13 complete IS3 copies available in the GenBank database are delimited by the common 3 bp direct repeat. The others flanked by different sequences presumably underwent a dimerizationdissolution cycle, which could alter either of the bordering sequences, similar to those presented in Fig. 9a
. In C6-IS150 isolates the flanking bases were preserved, but an additional base probably originating from the SR in C5 was detected. One may conclude that for some ISs the bordering sequence of the remaining element is not affected during DDS [IS2 (Szeverényi et al., 1996a
); IS30 (Olasz et al., 1993
); IS911 (Turlan et al., 2000
); IS186, this work], while other elements, like IS3 and IS150, generate a few base pair deletions or insertions, respectively, in the flanking DNA (Fig. 9
).
Our findings reported here extend the number of IS elements, in which an active dimer-type intermediate was detected, to eight (IS2, IS3, IS21, IS30, IS150, IS186, IS256 and IS911). These elements are dispersed in five IS families [IS3, IS4, IS21, IS30 and IS256 (Mahillon & Chandler, 1998)]. However, additional experimental evidence is required to determine the overall occurrence of this intermediate in other IS elements, and further potential members may be found among the elements for which minicircle formation has already been reported [e.g. IS1 (Sekine et al., 1997a
; Turlan & Chandler, 1995
)]. It has been suggested that covalently closed IS circles and dimers could be alternative representations of the same transpositional pathway (Lewis & Grindley, 1997
; Kiss & Olasz, 1999
). Recent studies (Berger & Haas, 2001
; Prudhomme et al., 2002
; Turlan et al., 2000
) seem to further support this possibility.
ISs that have been reported to produce dimers and/or minicircles are scattered over at least seven IS families: IS1, IS3, IS4, IS21, IS30, IS110 and IS256 (Mahillon & Chandler, 1998). However, the presence of covalently attached transposon ends and their participation in the integration of mobile DNA is not restricted to bacteria, they can also be found in eukaryotic organisms, for example: Tc1 (Rose & Snutch, 1984
; Ruan & Emmons, 1984
), copia (Flavell & Ish-Horowicz, 1983
) and the P element (Roiha et al., 1988
) of Drosophila; Mu1 transposon (Sundaresan & Freeling, 1987
) and Ac/Ds of maize (Gorbunova & Levy, 1997
). The formation and transposition of abutted ends of mobile elements may reflect another common hallmark of these entities. It should be mentioned, however, that several verified models exist which explain the transposition/integration processes differently and further investigations in this field will contribute to a deeper insight into transposition processes.
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ACKNOWLEDGEMENTS |
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Received 12 November 2002;
revised 30 January 2003;
accepted 4 February 2003.