INTRODUCTION

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DNA rearrangements have been found in a wide range of living organisms (3) but have reached a remarkable extent in ciliates: deletion of internal sequences, DNA scrambling, and chromosomal fragmentation are developmentally programmed during the sexual phase of their life cycle (33). These unicellular eukaryotes are characterized by the presence of two functionally different nuclei within the same cytoplasm. The polyploid macronucleus, also called the somatic nucleus, is transcribed during vegetative growth and governs the cell phenotype but progressively degenerates during sexual reproduction. The diploid micronucleus is transcriptionally silent during vegetative growth and can be viewed as the germ line nucleus, since it is able to undergo meiosis. In Paramecium aurelia, two successive divisions of the zygotic nucleus take place after fertilization and produce four identical diploid nuclei, two of which become the new micronuclei while the other two differentiate into macronuclei. Macronuclear development extends over two cell cycles (4) and is accompanied by intensive replication to reach the final ploidy of the mature macronucleus, estimated to be between 800 and 1,700N according to previous studies (33, 38). It involves extensive rearrangements of the germ line genome: fragmentation of micronuclear chromosomes coupled to telomere addition to form the macronuclear chromosome ends (13) and precise deletion of internal eliminated sequences (IESs) that interrupt coding and non-coding DNA (reviewed in reference 22).

Paramecium IESs are single-copy sequences of AT-rich noncoding DNA and are flanked by two 5'-TA-3' repeats, one of which is retained in the macronuclear genome after excision (1, 7, 9, 12, 21, 24, 28, 32, 37, 40, 44). Since they were initially identified by sequence comparison of macronuclear and micronuclear versions of a given locus, their sizeranging between 26 and 882 bprepresents, by convention, the whole length of the deleted sequence and formally includes one copy of the flanking 5'-TA-3'. Based on an extrapolation of their frequency within sequenced regions of the genome, their number was estimated to be around 50,000 per haploid germ line genome (12). Paramecium IESs belong to the family of ciliate TA IESs also found in the hypotrichs Euplotes and Oxytricha (18). A statistical analysis led to the identification of a loosely conserved 8-bp consensus (5'-TAYAGYNR-3'), present as inverted repeats at the ends of Paramecium IESs and including the flanking 5'-TA-3' (21). This consensus bears similarities to the ends of Tc1/mariner transposons and of transposon-like Tec elements and some TA IESs of Euplotes (18). It was therefore proposed that IESs have evolved from ancestral transposons but have lost their coding capacity and rely on cellular functions for their excision (22). Genetic studies demonstrated that the terminal 8 bp of IES ends, including the flanking 5'-TA-3', plays a functional role in excision (9, 25, 26). Furthermore, Paramecium IES excision is subjected to a homology-dependent epigenetic control since the presence of an IES in the parental macronucleus was shown in some cases to inhibit excision of the corresponding sequence during development of the macronucleus of the next sexual generation (11, 12).

Several lines of evidence have indicated that excision of TA IESs and transposon-like elements in Oxytricha and Euplotes is accompanied by the formation of extrachromosomal circular molecules, detectable in significant amounts in cells undergoing macronuclear development (19, 23, 46). Under the assumption that such molecules are direct excision products, double-stranded DNA cutting was proposed to initiate the reaction at one (46) or both (19, 23) ends of the excised sequence. In Tetrahymena, a ciliate more closely related to Paramecium than hypotrichs, IESs have also been found, but they differ significantly from TA IESs (22). Extrachromosomal forms of these elements were not detected on Southern blots of DNA from developing macronuclei (2), although putative circularized forms could be amplified by extensive PCR (36, 47). It was therefore proposed that IES excision in Tetrahymena essentially produces unstable excised molecules and that circles are secondary, dead-end products of the reaction. Transient DNA cuts were detected at only one end of these sequences during macronuclear development, indicating that excision might proceed via a transposition-like pathway, including successive DNA transesterification steps, and result primarily in the formation of linear excised molecules (35).

As a first step in investigating the DNA transactions and the putative enzymatic machinery participating in Paramecium IES excision, we used a quantitative PCR approach to study the timing of IES elimination. Our data indicate that it starts after three rounds of DNA replication have taken place in the developing macronuclei (anlagen) and is completed by the time of the first cell division (the karyonidal division), when the two anlagen segregate into daughter cells. These experiments allowed the concomitant detection of abundant, circularized forms of two IESs. The structure of the circle junctions suggests that Paramecium IES excision may involve DNA cutting at the flanking 5'-TA-3' dinucleotide.

	MATERIALS AND METHODS

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Paramecium strains and growth conditions. P. tetraurelia stocks 51 and d4.2 are entirely homozygous strains carrying the A⁵¹ and A²⁹ alleles, respectively, of the gene encoding surface antigen A (39). Both carry the G⁵¹ allele of the paralogous gene encoding surface antigen G. Culture conditions were as described previously (11). Unless otherwise stated, cells were grown at 27°C. Under starvation conditions, macronuclear development was induced by conjugation of reactive cells of complementary mating types or through a self-fertilization process called autogamy. Autogamy was monitored by staining cells with a 20:1 (vol/vol) mix of carmine red (0.5% in 45% acetic acid) and fast green (1% in ethanol) or by 4',6-diamidino-2-phenylindole (DAPI) staining.

Synchronization of macronuclear development. Reactive cells were mixed and incubated at room temperature for 1 h 45 min to allow the first conjugating pairs to form. Rich medium (2 volumes) was added, and the cells were incubated at 27°C for 45 min to stop further conjugation: only 30% of the cells were involved in the formation of stable synchronous pairs. These were hand sorted and transferred into rich medium. A fixed number of 60 cells were picked at the indicated time points from the time of exconjugant separation until the completion of karyonidal division and treated with 1% Nonidet P-40 (NP-40) for 10 min at 65°C before being subjected to PCR analysis (26).

Quantitative PCR analysis of total genomic DNA. Aliquots (10 µl) of NP-40-lysed cells (representing 10 cells) were subjected to PCR amplification in a Perkin-Elmer Cetus DNA thermal cycler, using 1 U of Tfl DNA polymerase (Promega) and 10 to 100 pmol of each primer in a final volume of 25 µl of commercial buffer (pH 9) (Promega) supplemented with 1.25 mM MgSO₄. For IES 51A2591, 100 pmol of primer 51A3 and 10 pmol of primer 51A-3' (Table 1) were used in the following amplification program: 6 cycles of 60 s at 92°C, 60 s at 54°C, and 60 s at 74°C, and 20 cycles of 20 s at 92°C, 20 s at 54°C, and 60 s at 74°C. For IES 51G4404, 10 pmol of each primer (51G3 and 51G6) (Table 1) was used and the program was 6 cycles of 60 s at 92°C, 60 s at 63°C, and 60 s at 74°C, followed by 30 cycles of 20 s at 92°C, and 75 s at 63°C; a final termination cycle (3 min at 72°C) was included for both IESs. To ensure that amplification yields were proportional to the input DNA concentration, serial dilutions of total genomic DNA extracted from a 400-ml vegetative culture of P. tetraurelia stock speedy (mating-type E) were amplified with the desired set of PCR primers. The resulting products were analyzed by Southern blot hybridization with a ³²P-labeled oligonucleotide and quantified using a Fuji phosphorimager. The number of amplification cycles for each primer set was chosen to give a good signal/input proportionality within a 1- to 15-fold range of input DNA concentrations, starting from the equivalent of the mean micronuclear signal obtained from two independent NP-40-treated samples of 10 vegetative cells (because of the lack of synchrony of vegetative cultures, this signal may vary within a 2-fold range between two different control samples). These conditions gave a perfectly proportional signal for IES 51A2591, while saturation was observed for IES 51G4404 at input DNA concentrations above 15 equivalents of the vegetative micronuclear signal. This could lead to an underestimation of the highest amplification factors obtained for IES 51G4404.

PCR detection of IES circular junctions. Primer sets 51G7 plus 51G8 and 51A1 plus 51A9 (Table 1) were used for the detection of the junctions between the ends of IES 51G4404 and 51A2591, respectively (Table 1). For 10 µl of NP-40 cell lysates, the PCR conditions were 6 cycles of 60 s at 92°C, 60 s at 54°C, and 60 s at 72°C followed by 36 cycles of 20 s at 92°C, 20 s at 54°C, and 60 s at 72°C, in a final volume of 25 µl. When 1 µl of total genomic DNA was used as a template, the initial 6 cycles were followed by 32 cycles of 20 s at 92°C, 20 s at 54°C, and 60 s at 72°C. For all PCR amplifications, 10 pmol of each primer was used and a final termination step of 3 min at 72°C was included.

DNA manipulations. 51Amic and 51Gmic phages harbor the micronuclear A⁵¹ and G⁵¹ genes, respectively (12). Plasmid pRA593 is a pBR322 derivative carrying a minitransposon derived from bacterial insertion sequence IS911. In the presence of IS911 transposase, the minitransposon is excised in vivo as a 411-bp covalently closed supercoiled minicircle (31). A 1-kb DNA ladder (Gibco BRL) was used as a linear size standard. All oligonucleotides (Eurobio or Eurogentec) are listed in Table 1.

Southern blotting. For the identification of extrachromosomal IESs, equivalents of 1 µl of a total genomic DNA preparation at an optical density at 260 nm of 1,200 were treated with 20 µg of Rnase A per ml and loaded on a 1.5% agarose (Appligene)-1.5% NuSieve (FMC) gel in 1× Tris-borate-EDTA (TBE) buffer. PCR amplification products were analyzed on 3% NuSieve (FMC) or 1.5% agarose gels (Appligene) in 1× TBE buffer. Alkaline transfer onto Hybond N+ membranes (Amersham), hybridization with ³²P-labeled DNA fragments, and washing conditions were as described previously (12), while hybridization with ³²P-labeled oligonucleotides was carried out for 1 h at 60°C followed by slow cooling to 30°C and washing at 35°C in 2× SSC (0.3 M NaCl, 30 mM sodium citrate)-0.1% sodium dodecyl sulfate.

	RESULTS

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Timing of IES excision. In Paramecium, the formation of a new macronucleus in rich medium takes approximately 22 h from the time the zygotic nucleus has divided twice, and extensive DNA amplification was reported to occur in the developing macronucleus during this period (4). To investigate precisely the timing of IES excision relative to DNA amplification, macronuclear development in strain speedy was synchronized through conjugation of reactive cells of complementary mating types. After cell mixing, early-conjugating pairs that formed within 1 h 45 min were picked and transferred into rich medium. Separation of exconjugants occurred within a 1-h interval about 6 h 30 min after mixing, and karyonidal division took place within a 4-h period around 18 h after mixing. Since fragments of the degenerating parental macronucleus coexist with the anlagen over the entire period of macronuclear development (4), it was impossible, in total-DNA preparations, to distinguish the new macronuclear junctions generated by IES excision from the sequences of the parental macronucleus. Instead, we analyzed the micronuclear junctions between IESs and their flanking macronuclear-destined sequences.

View larger version (27K): [in this window] [in a new window]   FIG. 1.   Excision of IES 51A2591 and of the internal 28-bp IES in synchronized exconjugant cells. (A) Schematic diagram of IES 51A2591, with the internal 28-bp IES indicated by a solid box. The oligonucleotides used in the study are represented by arrowheads. (B) Southern blot analysis of the PCR products amplified from synchronized exconjugant cells with primers 51A3 and 51A-3'. Samples were run on a 1.5% agarose gel and revealed with 32P-labeled oligonucleotide 51A1. Indicated time points refer to the time following the mixing of reactive cells. Two independent samples of 10 vegetative cells were used as controls for the micronuclear signal. (C) Quantitative analysis of the excision timing of IES 51A2591. The signals shown in panel B were quantified and normalized relative to the value obtained for the 6 h 30 min time point. (D) Prolonged electrophoresis of the samples shown in panel B on a 1.5% agarose gel. IES 51A2591-derived fragments were revealed by Southern blot hybridization with oligonucleotide 51A1. The 400-bp band lacking the 28-bp internal IES is marked by an asterisk. (E) Same blot as in panel D, but hybridized with oligonucleotide 51A28.

View larger version (24K): [in this window] [in a new window]   FIG. 2.   Timing of IES 51G4404 excision and detection of joined IES ends in synchronized exconjugant cells. (A) Diagram of the PCR strategies used for the amplification of chromosomal IES 51G4404 prior to its excision (primers 51G3 and 51G6 [black arrowheads]) and for the detection of putative IES circular forms (primers 51G7 and 51G8 [white arrowheads]). (B) Southern blot analysis of the PCR products amplified from synchronized exconjugant cells with primers 51G3 and 51G6. Samples were revealed with 32P-labeled oligonucleotide 51G8 after electrophoresis on a 3% NuSieve gel. Indicated time points refer to the time following the mixing of reactive cells. Two independent samples of 10 vegetative cells were used as controls for the micronuclear signal. (C) Quantitative analysis of the timing of IES 51G4404 excision. The signals shown in panel B were quantified and normalized relative to the value obtained for the 6 h 30 min time point. (D) PCR amplification from the same NP-40-treated synchronized exconjugant cells as in panel B, using divergent primers 51G7 and 51G8 (see Materials and Methods). PCR products were run on a 3% NuSieve gel and revealed by ethidium bromide staining. The lane order is as in panel B, except that only one vegetative control was used.

Excision of a 28-bp IES internal to IES 51A2591. Prolonged electrophoresis of the PCR products amplified with primers 51A3 and 51A-3' in the experiment described above (Fig. 1B) revealed an approximately 400-bp fragment migrating ahead of the major 423-bp species in the 12- and 14-h samples and hybridizing with probe 51A1 (Fig. 1D). Excision of a 28-bp sequence located within IES 51A2591 (Fig. 1A) and exhibiting the characteristics of an IES has previously been observed when excision of IES 51A2591 was abolished by a point mutation at one end (26) or when it was epigenetically inhibited by the parental macronucleus (12). The absence of the 28-bp sequence from the 400-bp PCR product in Fig. 1D was assayed by hybridization with oligonucleotide 51A28, which includes the 28-bp sequence and three upstream adjacent nucleotides (Table 1). This failed to reveal the 400-bp fragment (Fig. 1E), suggesting that this additional band arises through excision of the internal 28-bp IES in the normal course of macronuclear development.

Joining of the ends of excised IESs. While the previous experiments allowed the identification of the time interval during which IESs are excised, it was of interest to investigate whether putative excision products could be detected. Following a strategy already described for other ciliates (22), divergent primers 51G7 and 51G8 hybridizing within IES 51G4404 were used to selectively amplify molecules in which both IES ends would have been covalently linked, as expected for a circular form of the excised sequence (Fig. 2A). PCR amplification products obtained from the same synchronized cell lysates as in the previous experiment were revealed by ethidium bromide staining (Fig. 2D). While no PCR product was detected from the vegetative control sample or at early time points (6 h 30 min to 12 h), a major product migrating just above the 220-bp size marker, hence exhibiting the expected size for IES 51G4404, was amplified from the 14-h sample and at all later time points (16 to 22 h). A species with a slightly higher molecular weight was also apparent: although its structure remains unclear, it most probably arose from the saturating PCR conditions used and accumulated with increasing numbers of amplification cycles (data not shown). Strikingly, IES excision (Fig. 2B) and the joining of ends (Fig. 2D) appeared to be closely linked in time, since both started to be detected between 12 and 14 h after cell mixing. DAPI staining of 14-h exconjugant cells revealed that anlagen were already clearly visible at this stage (Fig. 3).

View larger version (124K): [in this window] [in a new window]   FIG. 3.   DAPI staining of a 14-h exconjugant cell. The anlagen (arrows) are surrounded by the parental macronuclear fragments.

View larger version (29K): [in this window] [in a new window]   FIG. 4.   Direct detection of circular forms of the excised IESs in autogamous cells. (A) Diagram of IESs 51A2591 and 51G4404, with the corresponding oligonucleotide primers represented by arrowheads. (B) PCR amplification of circular junctions from autogamous cells. Primer sets 51A1 plus 51A9 and 51G7 plus 51G8 were used for the detection of a junction between the ends of IES 51A2591 (left panel) and IES 51G4404 (right panel), respectively (see Materials and Methods). The t = 0 time point was arbitrarily defined as stated in the text, and other time points refer to later stages of autogamy (see the text for details), up to karyonidal division (kar. div.). Electrophoresis was carried out on a 3% NuSieve gel. The major amplification products are indicated by their sizes, and the 342-bp minor band corresponding to the form lacking the 28-bp IES internal to IES 51A2591 is indicated by an asterisk. (C) Southern blot analysis of uncut genomic DNA from autogamous cells. Electrophoresis was carried out on a 1.5% agarose-1.5% NuSieve gel. Chromosomal (I) and extrachromosomal (II) forms of IES 51A2591 (left panel) were revealed after hybridization with a 370-bp PCR fragment specific for the IES sequence and amplified with primers 51A3 and 51A4. A 236-bp fragment, specific for IES 51G4404 and amplified with primers 51G5 and 51G6, was used as a probe in the right panel. The central panel shows ethidium bromide staining of supercoiled (C) or linear (L) forms of a 411-bp control minicircle (see Materials and Methods), electrophoresed on the same gel. (D) Restriction maps of the micronuclear regions around IES 51A2591 (left) and IES 51G4404 (right). Chromosomal IESs are drawn as black boxes, and the 28-bp IES inside IES 51A2591 is shown as a white square. The corresponding circular IES molecules are not drawn to scale. Restriction enzymes: B, BsaI; D, DdeI; H, HinfI; Xm, XmnI. (E) DdeI and exonuclease III (exo III) treatment of total genomic DNA from 100% autogamous cells. DNA extracted from autogamous cells at t = 12 h 15 min and t = 21 h was treated with DdeI and, where indicated, incubated for 2 h at 37°C with 200 U of exonuclease III. Electrophoresis and hybridization probes were as in panel C. IESs are shown as black boxes on the diagrams of their corresponding micronuclear DdeI fragments.

Circular forms of the excised IESs. The molecules responsible for the amplification signal obtained with divergent PCR primers (Fig. 2D and 4B) could be circular forms or direct tandem repeats of the IESs. To distinguish between these two alternatives, total unrestricted DNA extracted from cells at different stages of autogamy was directly analyzed by Southern blot hybridization with DNA probes corresponding to IESs 51A2591 or 51G4404 (Fig. 4C).

Sequence of the junctions on IES circles. To determine the nucleotide sequence of the junctions between circularized IES ends, the PCR fragments in Fig. 2D and 4B were sequenced on both strands with the primers used for their amplification. For each IES, total PCR products from synchronized exconjugants (Fig. 2D, t = 14 to 22 h) or autogamous cells (Fig. 4B, t = 5 h 45 min to karyonidal division) were pooled and purified from residual unlabeled primers. En masse PCR-mediated sequencing allowed the identification of a unique and unambiguous sequence for each IES (Fig. 5A for IES 51A2591 and Fig. 5B for IES 51G4404). In both cases, IES ends were precisely joined, with a single copy of the flanking 5'-TA-3' repeat at the junction. Sequencing of the gel-purified major amplification product from IES 51A2591 (the 370-bp species in Fig. 4B, left panel) revealed that it carried the 28-bp internal IES (data not shown), in agreement with its observed size on agarose gels (Fig. 4B). The very faint PCR band migrating ahead of this major 370-bp species (Fig. 4B, left panel) was also gel purified and sequenced with the same primers; it was shown to correspond to a 342-bp molecule with the same junction between the ends of IES 51A2591 but from which the internal 28-bp IES had been excised, leaving one 5'-TA-3' dinucleotide at the new junction (data not shown).

View larger version (46K): [in this window] [in a new window]   FIG. 5.   Sequence of IES circles. Sequences of the ends (capital letters) and their flanking macronucleus-destined regions (lowercase letters) of IES 51A2591 (A) and IES 51G4404 (B) are shown at the top of each panel. The 5'-TA-3' dinucleotide between joined IES ends is boxed in the diagram representing the circularized form of each IES. For purposes of clarity, only the structure of the major circular species observed for IES 51A2591 is shown in panel A (see the text for details). Arrowheads represent the divergent primers used for PCR amplification and sequencing of the circle junctions. Sequencing reactions were performed on both strands, and portions of the sequencing gels encompassing the joined ends of each IES are shown on the right. The nucleotide sequence of each junction is displayed at the bottom.

	DISCUSSION

Top Abstract Introduction Materials and Methods Results Discussion References

While the timing of IES excision in other ciliates has been known for a long time (2, 42, 45), this type of study has been delayed for Paramecium because of the difficulty in obtaining large amounts of cells synchronized for macronuclear development. We have overcome this problem by using manual sorting of early-conjugating pairs coupled to quantitative PCR. For the IESs studied here, excision was shown to start 12 to 14 h after the mixing of reactive cells and was essentially completed at 16 h. Beginning 14 h after initiation of conjugation, excised IES circles were formed, with the joined IES ends separated by a single copy of the flanking 5'-TA-3' dinucleotide. A chromosomal junction resulting from the excision of a short IES inserted within a larger one was also transiently detected during the same time interval.

IES excision and DNA replication. IES excision in Paramecium seems to take place during a specific period of the first cell cycle following micronuclear meiosis. This is based on the observations that the same elimination timing (12 to 14 h after initiation of conjugation) was observed for two IESs within different surface antigen genes and for a third, short IES (28 bp). Previous cytological analysis of macronuclear development following conjugation indicated that three doublings of the anlage DNA content take place before the 12-h time point (4). This would correspond to an 8-fold amplification of the IESs, in agreement with our observation that IESs 51A2591 and 51G4404 are amplified 7- and 12-fold, respectively, prior to their excision. Care must be taken, however, in the quantitative comparison of our data with those of Berger (4) because the specific contribution of the developing macronuclei cannot be distinguished from that of the new micronuclei in our experiments and because no information is available on the timing of micronuclear replication during the first cell cycle following conjugation. In addition, Berger's work provided an estimation of the overall DNA doubling rate within the anlage, but particular genomic loci could be amplified at different rates. On the other hand, we cannot exclude that early but rare IES excision events take place before the 12-h time point, which would decrease the apparent rate of IES replication. The results presented here nevertheless imply that up to 16 copies of each IES may be present in one anlage before their excision. This could allow significant variability in the excision products obtained from a single germ line sequence within the same macronucleus. Alternative rearrangement patterns have indeed been reported for several IESs in vegetative clones arising from unique macronuclear differentiation events (1, 9, 12).

Formation of excised IES circles. This study demonstrates the developmentally programmed formation of excised double-stranded circular forms for two Paramecium IESs, which appear to result from the excision reaction. Indeed, their timing of appearance coincides with the dissociation of IESs from their chromosomal flanking sequences. Also, they are quite abundant in developing macronuclei, as suggested by their detection on Southern blots of DNA from autogamous cells. This point distinguishes Paramecium IESs from those of Tetrahymena, for which putative circularized forms could hardly be amplified by PCR (36, 47) and were not detected on Southern blots of DNA from developing macronuclei (2). As was proposed for Euplotes IESs (23, 41), Paramecium IES circles could be directly generated by the excision reaction. It should be noted, however, that trace amounts of molecules with the electrophoretic mobility expected for linear forms of the IESs are detected early during autogamy. Although the exact structure of these minor species remains to be established, this raises the possibility that IES excision primarily produces linear molecules that could be precursors to the circles. The relative abundance of the circles and their homogeneous nucleotide sequence would then imply that circularization is an efficient and precise reaction, involving little or no degradation of linear DNA ends. In a first attempt to investigate whether circular molecules are direct products of the excision reaction, the radioactive signals detected on the Southern blots in Fig. 4C were quantified to monitor the conversion of the chromosomal signal into the extrachromosomal signal. This indicated that the amount of extrachromosomal circles remained smaller than the maximum amplification level of the chromosomal IES (data not shown). However, it is worth pointing out that IES circles are not amplified in the final macronucleus, as indicated by their absence from vegetative cells. Therefore, since no information is available concerning the stability of the circular molecules (i.e., whether they are actively degraded or simply diluted out during subsequent cell divisions), one should be cautious in interpreting this result in terms of the circles being (or not being) primary excision products.

Mechanism of IES excision. The relatively easy detection of extrachromosomal Paramecium IESs on Southern blots will be very helpful in further studies of the molecular mechanism of excision and in the identification of the different intermediates of the reaction. The present study has focused on the analysis of the abundant circular forms of the excised IESs. IES excision in Paramecium may formally be viewed as a recombination between short DNA repeats, in which one copy of the repeated 5'-TA-3' dinucleotide is found between joined IES ends on the excised circle while the other is retained at the new junction formed on the macronuclear chromosome (hereafter referred to as the donor molecule, by analogy to other DNA excision systems, such as those involved in cut-and-paste transposition). Similar joining of the donor and excised molecules was previously reported only for TBE1 transposon-like elements in Oxytricha (46). TBE1 circle junctions carry a single copy of the 3-bp target site duplication, joined on each side to the terminal 3' dG of each end. However, recent data have indicated that the nucleotide facing this dG in the duplex circle is not always its complement, which can result in a mispaired position on both sides of the 3-bp target sequence (K. Williams, T. Doak, and G. Herrick, personal communication). The double-stranded DNA junctions created during IES excision in Paramecium therefore appear to be unprecedented in ciliates. More generally, transposase-induced excision of DNA transposons moving via a cut-and-paste pathway (reviewed in reference 34) generates double-stranded breaks on the donor molecule, which is either lost, imprecisely repaired to give characteristic DNA footprints, or repaired by gene conversion with a homologous sequence as a template. In these systems, the transposon is generally excised as a linear molecule. In the particular case of Tc1/mariner transposable elements, the integrated copies of the transposon are flanked by repeated 5'-TA-3' dinucleotides but the excised linear form does not contain any part of these repeats (30). Transposase-induced formation of a DNA minicircle, in which the joined ends of the excised sequence are separated by one copy of the 3-bp repeat initially flanking the element, was demonstrated for bacterial insertion sequence IS911, but the fate of the donor molecule is unclear (31). Finally, in mammalian V(D)J recombination, both the circular excised molecule and the resealed donor molecule have been analyzed (reviewed in reference 5). While the signal joints on the excised molecules result from the precise fusion of conserved heptamer sequences, rejoining of the coding ends on the donor molecule is accompanied by nucleotide addition or removal.