Keeping the Soma Free of Transposons: Programmed DNA Elimination in Ciliates*

Many transposon-related sequences are removed from the somatic macronucleus of ciliates during sexual reproduction. In the ciliate Tetrahymena, an RNAi-related mechanism produces small noncoding RNAs that induce heterochromatin formation, which is followed by DNA elimination. Because RNAi-related mechanisms repress transposon activities in a variety of eukaryotes, the DNA elimination mechanism of ciliates might have evolved from these types of transposon-silencing mechanisms. Nuclear dimorphism allows ciliates to identify any DNA that has invaded the germ-line micronucleus using small RNAs and a whole genome comparison of the micronucleus and the somatic macronucleus.

Many transposon-related sequences are removed from the somatic macronucleus of ciliates during sexual reproduction. In the ciliate Tetrahymena, an RNAi-related mechanism produces small noncoding RNAs that induce heterochromatin formation, which is followed by DNA elimination. Because RNAi-related mechanisms repress transposon activities in a variety of eukaryotes, the DNA elimination mechanism of ciliates might have evolved from these types of transposon-silencing mechanisms. Nuclear dimorphism allows ciliates to identify any DNA that has invaded the germ-line micronucleus using small RNAs and a whole genome comparison of the micronucleus and the somatic macronucleus.
Organisms cope with a variety of transposons that invade their genomes. These invaders become residents of the genome as "junk" and "copy and paste" their sequences into the host sequence to expand their copy numbers. Thus, active transposons pose a major problem to genome integrity. Because different transposons use different strategies to mediate their activities, their protein products are not common targets for the repression of transposons. One strategy to silence a variety of transposons using a single mechanism is to target their DNA or RNA products. In many eukaryotes, small noncoding RNAs that are produced by RNAi-related mechanisms induce transposon silencing by repressing the expression of transposon gene products at the transcriptional and/or post-transcriptional levels (reviewed in Refs. 1 and 2). This small noncoding RNA-mediated transposon silencing culminates in programmed DNA elimination in ciliates.
Ciliates are a large heterogeneous group of unicellular protozoans that branched off early in the evolution of the eukaryotic kingdom (Fig. 1A). Most laboratory ciliates belong to the class Oligohymenophorea, including the species Paramecium tetraurelia and Tetrahymena thermophila, or to the class Spirotrichea, including Euplotes crassus, Oxytricha trifallax, and Stylonychia lemnae. (We use only the genus names below.) Most ciliates, including the ones listed above, show nuclear dimorphism by harboring both the germ-line micronucleus (Mic) 2 and the somatic macronucleus (Mac) in a single cell (Fig.  1B) (reviewed in Ref. 3). The Mic is diploid and silent during vegetative growth. In contrast, the Mac is polyploid and transcriptionally active, providing the transcripts that are necessary for growth.
Despite their structural and functional differences, the Mic and Mac are mitotic products of the same nucleus. During vegetative growth, ciliates divide by binary fission, and the two types of nuclei are independently segregated to daughter cells. If nutrients are scarce, ciliates turn to sexual reproduction, which is the conjugation of two cells or autogamy of a single cell, depending on the species. Only conjugation occurs in Tetrahymena, whereas both conjugation and autogamy occur in Paramecium. In both cases, the Mic provides all of the genetic material for the future generation, and the new Mic and Mac of the sexual progeny are derived from the same zygotic nucleus formed by the fertilization of two parental Mic-derived haploid meiotic nuclei (Fig. 1C).
Although the Mic and Mac are produced from one zygotic nucleus, their genetic contents and their ploidies are different. These differences are caused by extensive programmed DNA rearrangements and endoreplication of chromosomes in the newly formed Mac during each round of sexual reproduction (reviewed in Ref. 3). The programmed DNA rearrangements include DNA elimination, chromosome breakage, and DNA unscrambling of the new Mac genome to produce a fully functional, highly gene-dense, polyploid somatic nucleus (Fig. 2). DNA elimination and chromosome breakage are common among many ciliates, whereas DNA unscrambling is restricted to some classes of ciliates, such as Oxytricha and Stylonychia. In Tetrahymena, chromosome breakage happens at conserved sequences of ϳ15 bp in length, and new telomeres are added to these breakage sites, which produces ϳ200 Mac chromosomes from the five chromosomes that are present in the haploid Mic genome (4). These new chromosomes are eventually endoreplicated to a copy number of ϳ45. Strikingly, some spirotrichous ciliates, such as Oxytricha, process the germ-line genome to generate Mac nanochromosomes that range in size from a few hundred base pairs to ϳ15 kb, with an average of ϳ2 kb, and typically contain a single gene. These short Mac chromosomes are endoreplicated to up to 1000 copies.
Although the Mac has more DNA than the Mic due to endoreplication, the Mac contains less genomic information than the Mic. The downsizing of the genome in the Mac is caused by DNA elimination. An estimated portion of 34% of the germ-line Mic genome is eliminated from the newly developed Mac by DNA elimination in Tetrahymena, 3  Tetrahymena, the vast majority of the DNA elimination events are deletions of internal DNA segments, named internal eliminated sequences (IESs), followed by ligation of their flanking Mac-destined sequences (MDSs). Elimination followed by telomere addition to the flanking sequences is also observed in other ciliates, such as Paramecium.
The stretches of DNA eliminated contain many transposonderived sequences (4 -11), indicating that DNA elimination in ciliates may remove transposons from the transcriptionally active somatic Mac. In recent years, advances have been made in identifying the molecular mechanisms that regulate DNA elimination, and similarities to transposon-silencing mechanisms of other eukaryotes have been revealed. In this minireview, we discuss our current understanding of how DNA elimination in ciliates, mainly in Tetrahymena, is regulated by small noncoding RNAs, and we identify the possible relationship between transposon silencing and DNA elimination.

Epigenetic Regulation of DNA Elimination
Approximately 6000 IESs exist in the Mic genome of Tetrahymena, and they vary in size between 0.6 and 22 kb (12,13). IESs and the DNA flanking them have highly diverse sequences (14 -16). Despite this divergence, IESs are precisely excised in two aspects: 1) identical IESs are removed in different individuals and in different generations, and 2) the boundaries occur within a few to several base pairs, although some IESs have alternative boundaries. Because the processes are specific and reproducible, some mechanism must clearly define IESs. In nutrient-rich conditions, Tetrahymena proliferates asexually by binary fission, in which the Mic and Mac divide independently (step 1). If nutrients are scarce, Tetrahymena turns to sexual reproduction, called conjugation (step 2). Soon after pair formation, the morphology of the Mic changes to a crescent form during its meiotic prophase (step 3). Subsequently, the Mic undergoes meiosis, one of the four meiotic products divides once mitotically to produce two haploid pronuclei in each cell, and three other meiotic products are degraded (step 4). One of the two pronuclei is exchanged by the conjugating cells (step 5), and the pronuclei fuse to give rise to the diploid zygotic nucleus (step 6). The zygotic nucleus divides twice mitotically (step 7), and two of the products differentiate to Macs, whereas the others differentiate to Mics (step 8). The parental Mac becomes pyknotic and is resorbed. After pair separation, one Mic is degraded (step 9). If nutrients are available, the Mic divides, and cytokinesis segregates Macs and Mics, giving rise to Tetrahymena cells with one Mic and one Mac that grow vegetatively (step 10). The process of DNA elimination involves epigenetic information that is transmitted from the parental Mac to the new Mac (reviewed in Ref. 17). In both Paramecium and Tetrahymena, the introduction or retention of a Mic-specific sequence in the parental Mac results in the retention of the same sequence in the newly formed somatic Mac (18,19). Conversely, removing an MDS from the Mac causes ectopic elimination of the sequence from the new Mac in Paramecium (20). A foreign (neomycin resistance) gene introduced into the Mic is eliminated from the new Mac in Tetrahymena (21,22). Therefore, DNA elimination is probably not determined by primary sequences of eliminated DNAs or their flanking regions, but it seems that any DNA sequence that exists only in the Mic is eliminated from the Mac. These results led to the hypothesis that certain sequence-specific information is transferred from the parental Mac to the new Mac to define eliminated DNAs. Advances in the field indicate that DNA elimination depends on small noncoding RNA as the transmitter of this epigenetic information.

RNAi-related Mechanism Is Required for DNA Elimination
As we describe below, DNA elimination in Tetrahymena is regulated by an RNAi-related mechanism. RNAi-related pathways are unified by their dependence on base pairing interactions between small RNAs (20 -30 nucleotides (nt)) and target sequences and by their common use of Argonaute family proteins (23). In metazoans, there are two subfamilies of Argonaute proteins, Ago and Piwi proteins (24). The Ago proteins associate with small RNAs (siRNAs and microRNAs) that are produced by Dicer family proteins from various endogenous and exogenous dsRNAs. In contrast, Piwi proteins interact with small RNAs (piRNAs) that are produced by a Dicer-independent mechanism from endogenous single-stranded RNAs. piR-NAs from all organisms studied so far are 2Ј-O-methylated at their 3Ј termini by conserved homologs of the RNA methyltransferase Hen1. However, 2Ј-O-methylation is not limited to piRNAs, and some Dicer-produced small RNAs are also 2Ј-Omethylated by Hen1 homologs (25)(26)(27).
Twelve Argonaute proteins, three Dicer proteins, and a single Hen1 homolog have been identified in Tetrahymena (28 -31). Small RNAs called scan RNAs (scnRNAs) are expressed exclusively during the sexual reproduction of Tetrahymena (32). The length of scnRNAs was originally suggested to be ϳ28 nt (32); however, recent sequencing analyses by our group indicate that most of them range from 28 to 30 nt, with a peak at 29 nt. 4 Although the production of scnRNAs depends on the Dicer protein Dcl1p (28,29), they interact with the Piwi protein Twi1p (32) and are stabilized by 2Ј-O-methylation at their 3Ј termini, which is mediated by the Hen1 homolog Hen1p (31). Thus, Tetrahymena has an RNAi-related mechanism that involves both Piwi and Dicer proteins. Twi1p and Dcl1p are indispensable for DNA elimination, and Hen1p is required for efficient DNA elimination (28,31,32). These findings indicate that scnRNAs play a pivotal role in DNA elimination. Recently, two Dicer proteins, Dcl2 and Dcl3, and two Piwi proteins, Ptiwi01 and Ptiwi09, were identified as the functional counterparts of Dcl1p and Twi1p, respectively, in Paramecium (33,34). Double knockdown of these Dicer or Piwi proteins causes the loss of scnRNAs (ϳ25 nt in Paramecium) and defects in DNA elimination. Therefore, the mechanism of scnRNA-directed DNA elimination is probably conserved among oligohymenophorean ciliates.

DNA Elimination in Ciliates Requires Concerted Action of Different Nuclei
Three types of nuclei are involved in the pathway of DNA elimination: the Mic, the parental Mac, and the developing new Mac. Although this knowledge is based on studies of Paramecium and Tetrahymena, we will focus mainly on the molecular pathway of DNA elimination in Tetrahymena.

Phase 1: Production of scnRNA in Mic
The Mic is transcriptionally inert throughout most of the life cycle, and it is activated for transcription exclusively during its meiotic prophase (Fig. 3, Phase 1) (35,36). Mic transcription most likely produces noncoding RNAs because the transcripts are derived from regions that have no predicted open reading frames and have heterogeneous ends. Here, we call these transcripts Mic noncoding RNAs (mic-ncRNAs). The Dicer protein Dcl1p localizes to the Mic during meiotic prophase and meiosis. The loss of DCL1 results in the accumulation of mic-ncRNAs and the disappearance of scnRNAs (28,29). This finding indicates that mic-ncRNAs are the precursor of scnRNAs and that Dcl1p processes mic-ncRNA to form scnRNA in the Mic. Consistent with this, mic-ncRNAs are transcribed bidirectionally (37) and potentially form double-stranded RNAs. Because all of the Mic sequences studied to date produce mic-ncRNAs (37,38), scnRNAs may cover the entire Mic genome. The results of our recent large-scale scnRNA sequencing study support this view. 5 Chromatin restructuring and specific protein localization may be involved in mic-ncRNA transcription. The Mic-specific linker histone MLH-␦ is phosphorylated only during the early conjugation stages, and it has been suggested that this phosphorylation induces the decondensation of the Mic chromosomes, which may make these chromosomes transcriptionally competent (39). In addition, the dramatic morphological change of the Mic from a compact round shape to an elongated crescent shape during meiotic prophase may contribute to the decompaction of the chromosomes. The transcriptional machinery, such as the subunits of DNA-dependent RNA polymerase II and the TATA-binding protein, is excluded from the Mic in most life stages but localizes to the Mic during meiotic prophase (40,41). The Mac and Mic have distinct nuclear pore proteins and utilize different sets of karyopherins (42,43). These differences may act as the basis for nuclear specific protein localization. The Mic-specific transportation system may be temporary modified during meiotic prophase and allows the basic transcriptional machinery to localize to the Mic.

Phase 2: Maturation of Argonaute-scnRNA Complex
The scnRNAs that are produced in the Mic are subsequently exported to the cytoplasm and loaded into the Argonaute protein Twi1p (Fig. 3, Phase 2) (32,44). The mechanisms of the cytoplasmic export and loading of scnRNAs are unknown. Because Dicer produces scnRNAs from long dsRNAs, scnRNAs are double-stranded when they are loaded into Twi1p. Like many conserved Argonaute proteins, Twi1p harbors intrinsic endoribonucleolytic ("slicer") activity to cleave one strand of the scnRNA duplex, leading to removal of the "passenger" strand from the complex (45).
In mid-conjugation stages, the Twi1p-scnRNA complexes translocate from the cytoplasm to the parental Mac. In the absence of Dcl1p, which is required for the production of scn-RNAs, or in the absence of Twi1p slicer activity, which occurs in a TWI1 mutant strain, the Twi1p protein remains in the cytoplasm (45). Therefore, loading and passenger strand removal of scnRNAs are essential for Mac localization of the Twi1p-scn-RNA complexes, suggesting a mechanism that selectively transports Twi1p complexed with single-stranded scnRNA. This selective transport is supported by the Twi1p-binding protein Giw1p. Giw1p is indispensable for the nuclear localization of Twi1p and binds to Twi1p only when it is complexed with single-stranded scnRNA (45). The Twi1p-scnRNA complex may change its conformation via passenger strand removal, and after this conformational change, the Twi1p-scnRNA complex may be specifically recognized and transported to the Mac by Giw1p. Similar conformational changes have been reported in a bacterial Argonaute protein (46).
The RNA methyltransferase Hen1p mediates the 2Ј-Omethylation of scnRNAs at their 3Ј termini (31). Loss of Hen1p completely abolishes scnRNA methylation, resulting in a gradual reduction of the accumulation and length of scnRNAs and a defect in DNA elimination. Therefore, Hen1p-mediated 2Ј-Omethylation stabilizes scnRNAs and ensures DNA elimination in Tetrahymena. Because Hen1p methylates only singlestranded RNAs in vitro, scnRNAs can be 2Ј-O-methylated by Hen1p only after the Twi1p-scnRNA complex releases the passenger strand. Hen1p is localized to the parental Mac. Therefore, the 2Ј-O-methylation of scnRNA may occur after the Twi1p-siRNA complex is imported into the parental Mac (Fig.  3, Phase 2). In summary, the "maturation" of the Twi1p-scn-RNA complex is achieved via four consecutive steps: 1) loading of the scnRNA duplex into Twi1p, 2) endoribonucleolytic cleavage and removal of the passenger strand of scnRNA, 3) translocation of the Twi1p-scnRNA complex into the Mac, and 4) 2Ј-O-methylation of scnRNA by Hen1p.

Phase 3: Selection of scnRNAs in Parental Mac
Although scnRNAs complementary to both MDSs and Micspecific sequences (mostly IESs in Tetrahymena) are produced in the early conjugation stage (Phase 1), the population of scn-RNAs is gradually enriched in the second class during the midconjugation stages (38,44). This process is called scnRNA selection. As described above, Twi1p-scnRNA complexes are localized to the parental Mac during the mid-conjugation stages (Phase 2). Because only scnRNAs that are complemen-tary to MDSs are selectively down-regulated in this process, some mechanism must specifically degrade scnRNAs complementary to the genomic DNA (or transcripts therefrom) in the parental Mac in a genome-wide fashion.
The interaction between scnRNAs and long noncoding transcripts in the parental Mac (parental Mac noncoding RNA (pmac-ncRNA)) probably plays an important role in scnRNA selection in Paramecium and Tetrahymena. In Tetrahymena, the Twi1p-binding putative RNA helicase Ema1p is essential for scnRNA selection. Ema1p is required for the interaction of nascent transcripts and scnRNA-Twi1p complexes in the parental Mac (38). In Paramecium, down-regulation of pmac-ncRNA by RNAi induces DNA elimination at the corresponding site (34), and in Tetrahymena, injected dsRNAs complementary to MDS regions result in aberrant DNA elimination of these MDS from the new Mac (21). These results indicate that changing the population of either scnRNAs or long noncoding RNAs is deleterious for correct DNA elimination, and scnRNA selection depends on base pairing between scnRNAs and nascent pmac-ncRNA in the parental Mac (Fig. 3, Phase 3).
The mechanism that mediates scnRNA/pmac-ncRNA interactions to regulate scnRNA selection is not well understood. The interaction may induce the degradation of scnRNAs. Alternatively, it has been proposed that pmac-ncRNAs serve as molecular sponges that sequester scnRNAs by base pairing. Because scnRNAs complementary to some MDSs are lost during scnRNA selection in Tetrahymena (38), degradation of scn-RNAs is most likely the responsible mechanism in Tetrahymena. It has been suggested that two Gly-Trp repeatcontaining proteins, Wag1p and CnjBp from Tetrahymena (47), as well as Nowa1p and Nowa2p from Paramecium (48), are involved in transnuclear cross-talk between the parental Mac and the new Mac. More unknown proteins may be important during scnRNA selection, and the identification and characterization of such proteins would enable us to understand the process mechanistically.

Phase 4: scnRNA-induced Heterochromatin Formation in New Mac
Immediately following the formation of the new Mac, Twi1p is transferred from the parental Mac into the new Mac, where the Twi1p-scnRNA complex is required for the accumulation of methylated histone H3 at Lys-9 (H3K9me) and Lys-27 (H3K27me) (Fig. 3, Phase 4). Similar to pmac-ncRNAs, the new Mac expresses long noncoding RNAs (38). Here, we call these transcripts new Mac noncoding RNAs (nmac-ncRNAs). The Twi1p-associated RNA helicase Ema1p is required for the interaction between Twi1p and nascent nmac-ncRNA and for the accumulation of the histone modifications (38), indicating that scnRNA/nmac-ncRNA interactions are involved in the induction of H3K9me/H3K27me.
The Enhancer of zeste-like histone methyltransferase Ezl1p is responsible for the accumulation of H3K9me and H3K27me (49). Ezl1p is essential for DNA elimination. In addition, Tetrahymena strains expressing a histone H3 mutant in which Lys-9 is replaced with glutamine (H3K9Q) have a defect in DNA elimination (50). In the H3K9Q mutant, H3K27me is still accumulated, arguing that H3K27me is upstream of H3K9me (49).
These data indicate that either H3K9me alone or both H3K9me and H3K27me are required for DNA elimination. Ezl1p may display a dual specificity for both the H3K9 and H3K27 positions. Alternatively, Ezl1p may catalyze only H3K27me, and this modification may be required for catalysis of H3K9me by another methyltransferase.
Twi1p is required for the accumulation of H3K9me and H3K27me, whereas Ezl1p is not required for the accumulation of scnRNAs (49,50). This finding indicates that Twi1p is upstream of the Ezl1p-mediated H3K9me/H3K27me. Ezl1p may be recruited to the chromatin sites, where Twi1p-scnRNA complexes interact with nascent nmac-ncRNAs. Because scn-RNAs complementary to Mic-limited sequences are enriched in the parental Mac (Phase 3), the scnRNA/nmac-ncRNA interaction can specifically recruit Ezl1p to induce the histone modifications only on IESs. The molecular mechanism underlying this recruitment is unknown because no direct interaction between Twi1p and Ezl1p has been detected.
The chromodomain proteins Pdd1p and Pdd3p recognize H3K9me and H3K27me (49,51), and Pdd1p is necessary for the formation of a tight heterochromatic structure and for DNA elimination (52). Tethering Pdd1p to an ectopic chromatin site is sufficient to trigger DNA elimination at the target site, indicating that the chromatin localization of Pdd1p is sufficient to recruit all downstream factors necessary for DNA elimination (51). Therefore, the structure of heterochromatin, but not scn-RNAs or H3K9me/H3K27me, may directly trigger DNA elimination. Pdd1p and Pdd3p, together with H3K9me/H3K27me and IESs, localize to perinuclear heterochromatic foci, where the final DNA excision process is believed to take place (53,54). Other proteins that are necessary for DNA elimination, such as Pdd2p and Lia1p, also localize to these heterochromatic structures (55)(56)(57), but their contribution in the process of DNA elimination needs to be determined.

Phase 5: DNA Excision in New Mac
The piggyBac transposase-like proteins Pgm and Tpb2p in Paramecium and Tetrahymena, respectively (58,59), are indispensable for DNA elimination (Fig. 3, Phase 5). Although they are structurally similar to piggyBac transposases, the genes encoding these proteins are in the Mac. No additional transposon-related sequences exist in proximity to these genes. Therefore, these transposases were most likely domesticated during the evolution of these ciliates. Recombinant Tpb2p shows endonucleolytic cleavage activity, producing DNA double-strand breaks possessing 4-base 5Ј-protruding ends (59). This structure has been observed at IES boundaries during DNA elimination in vivo (60). How these piggyBac-like proteins are directed to cleavage sites has not been identified. Interestingly, no consensus sequence at IES boundaries has been found in Tetrahymena, but Paramecium IESs have loosely conserved 8-bp consensus sequences (5Ј-TAYAGYNR-3Ј) at their boundaries (reviewed in Ref. 3). Studies on cis-acting elements either in flanking Mac-retained DNA or inside IESs have revealed that they help determine the excision boundaries and the efficiency of DNA elimination (61)(62)(63)(64)(65). These cis-elements may regulate the recruitment of the piggyBac-like proteins. Alternatively, these proteins may directly recognize some heterochromatin component. Further biochemical studies of the piggyBac-like proteins, including their DNA-and chromatin-binding properties and the sequence specificity of the endonucleolytic DNA cleavage reaction, will help us understand how the boundaries of IESs are determined.
In the spirotrich Oxytricha, TBE (telomere-bearing element) family transposases are necessary for DNA elimination (66). Interestingly, these transposases are evolutionarily unrelated to the piggyBac transposases and are encoded in TBE transposons in Mic-limited sequences. Therefore, different classes of ciliates may have independently acquired abilities to utilize transposon-derived enzymes for DNA elimination. This hypothesis suggests that the mechanisms of DNA elimination of oligohymenophorean and spirotrichous ciliates are evolutionarily unrelated.
Ultimately, the lesions produced by the DNA excision must be repaired. In Paramecium, two core components of the nonhomologous end-joining pathway, Lig4p (DNA ligase IV) and Xrcc4p, are required for the repair of IES excision sites (67). Whether a similar non-homologous end-joining pathway is involved in healing DNA double-strand breaks after DNA elimination in other ciliates remains undetermined.

Why Do Ciliates Perform DNA Elimination?
As described above, an RNAi-related mechanism is required for the formation of the heterochromatin state that precedes DNA elimination in Tetrahymena. Because the RNAi machinery is also required for heterochromatin formation and subsequent transcriptional gene silencing in other eukaryotes, the molecular mechanism regulating IES elimination in Tetrahymena (and potentially in Paramecium) is probably evolutionarily related to RNAi-directed heterochromatin formation. In a wide variety of eukaryotes, transposons are silenced by RNAirelated mechanisms. For example, piRNAs mediate both transcriptional and post-transcriptional silencing of transposons in animals (reviewed in Ref. 1). siRNAs induce RNA-directed DNA methylation, which represses transcription from transposons in plants (reviewed in Ref. 2). Because many sequences that are removed by DNA elimination in ciliates are related to transposable elements, it has been suggested that one of the roles of DNA elimination is to remove transposons from the transcriptionally active Mac (8,21). Therefore, DNA elimination probably evolved as an RNAi-directed and heterochromatin-mediated transposon-silencing pathway. Nuclear dimorphism and the epigenetic regulation of DNA elimination by transnuclear scnRNAs allow ciliates to recognize transposons in their germ line even without any previous experience of their invasion.
On the other hand, transposons may have benefitted from DNA elimination as well. The main "goal" of transposons is to maximize their copy number. At the same time, transposons should colonize hosts without killing them because they rely on the host environment as their home. Therefore, there must be some limit to transposon activity, and they cannot occupy genome regions that are essential for the viability of the host. Because transposons are kept in the transcriptionally inactive Mic and removed from the active Mac in ciliates, transposons might be able to multiply without disturbing any cellular activ-ities in the nuclear dimorphism system. We speculate that the programmed DNA elimination system in ciliates is a strategy of transposons to increase their copy number in an unlimited manner in the host germ line. Ciliated protozoans may be "hijacked" as vehicles of transposons, and the programmed DNA elimination may be used as a colonization strategy by these molecular parasites.

Conclusions
DNA elimination in the oligohymenophorean ciliates Tetrahymena and Paramecium is regulated by scnRNAs that are produced by an RNAi-related mechanism. We hypothesize that the scnRNA-mediated comparison of the complete Mic and Mac genomes may act to identify the DNA sequences to be eliminated. The detailed molecular mechanisms regulating biogenesis and selection of scnRNAs, scnRNA-induced heterochromatin formation, and heterochromatin-mediated DNA excision are still emerging. We hope that established genetic and genomic tools in these model ciliates, such as gene deletion, RNAi techniques, and fully sequenced genomes, will help solve these questions in the near future.
DNA elimination of oligohymenophorean ciliates is potentially a small RNA-directed transposon-silencing mechanism. Based on previous studies, all of the genes required for DNA elimination are also required to produce viable sexual progeny in Tetrahymena and Paramecium. Thus, removal of transposons from the transcriptionally active Mac by DNA elimination may be necessary for the survival of both host ciliates and transposons. DNA elimination and nuclear dimorphism may have evolved as a result of an evolutionary race between transposons and host cells. Ciliates consist of a large group of eukaryotes and include many morphologically and physiologically divergent species. Investigating the DNA elimination processes of different classes of ciliates may illuminate how host/ transposon conflicts shape complex biological pathways.