role of micronucleus-limited dna in programmed deletion of ... · mse2.9 is conserved with that of...

EUKARYOTIC CELL, Apr. 2004, p. 288–301 Vol. 3, No. 21535-9778/04/$08.00�0 DOI: 10.1128/EC.3.2.288–301.2004Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Role of Micronucleus-Limited DNA in Programmed Deletion ofmse2.9 during Macronuclear Development of

Tetrahymena thermophilaJeffrey S. Fillingham† and Ronald E. Pearlman*

Department of Biology, York University, Toronto, Ontario, Canada M3J 1P3

Received 8 May 2003/Accepted 12 January 2004

Extensive programmed DNA rearrangements occur during the development of the somatic macronucleusfrom the germ line micronucleus in the sexual cycle of the ciliated protozoan Tetrahymena thermophila. Usingan in vivo processing assay, we analyzed the role of micronucleus-limited DNA during the programmed deletionof mse2.9, an internal eliminated sequence (IES). We identified a 200-bp region within mse2.9 that contains animportant cis-acting element which is required for the targeting of efficient programmed deletion. Our results,obtained with a series of mse2.9-based chimeric IESs, led us to suggest that the cis-acting elements in bothmicronucleus-limited and macronucleus-retained flanking DNAs stimulate programmed deletion to differentdegrees depending on the particular eliminated sequence. The mse2.9 IES is situated within the second intronof the micronuclear locus of the ARP1 gene. We show that the expression of ARP1 is not essential for the growthof Tetrahymena. Our results also suggest that mse2.9 is not subject to epigenetic regulation of DNA deletion,placing possible constraints on the scan RNA model of IES excision.

The ciliated protozoan Tetrahymena thermophila exhibits nu-clear dimorphism, with a mostly transcriptionally silent diploidgerm line nucleus (micronucleus) and a polyploid, transcrip-tionally active somatic nucleus (macronucleus) containedwithin the same cell. When two cells of different mating typesundergo sexual development (conjugation), the micronucleusin each divides meiotically and mitotically to generate a hap-loid gametic nucleus that is reciprocally exchanged and fuseswith that of its partner to form a zygotic nucleus. This zygoticnucleus divides, and from one of the products develops a newmacronucleus. Macronuclear development involves extensiveprogrammed DNA rearrangements, including chromosomefragmentation, DNA amplification, and the site-specific inter-stitial DNA deletion of internal eliminated sequences (IESs)(11, 42). Possible functions of IESs and the reasons for theirelimination from the micronucleus remain unclear. One modeldescribing the possible function of micronucleus-limited se-quences suggests that they may participate in events unique tothe micronucleus, such as mitosis and meiosis (11, 21). Themacronucleus divides during vegetative growth by an amitoticprocess that is devoid of obvious chromosome condensation,and it differs from the micronucleus in the timing of DNAreplication (27). Another model (the two models are not nec-essarily mutually exclusive) proposes that micronucleus-lim-ited DNA is derived from mobile genetic elements (23).

In Tetrahymena, interstitial DNA deletion is responsible forthe elimination of approximately 10 to 15% of the germ linegenome, involving �5,000 single and multicopy elements (41,42). The sizes of IESs in Tetrahymena range from 0.6 kb (3) toover 22 kb (39). Different IESs are generally not conserved in

sequence and are AT-rich, and most are flanked by shortnonconserved direct repeats (42). Alternate forms of rear-rangement have been suggested to exist for approximately 25%of IESs (9), and varying degrees of microheterogeneity areobserved at macronuclear junctions (1, 24, 33). IESs have notyet been found in the coding sequence of Tetrahymena, al-though two are located within introns (10, 20).

Several Tetrahymena IESs have been characterized at themolecular level. The deletion of the tightly linked M and Relements, which map to micronuclear chromosome 4 (4), hasbeen shown to be controlled by flanking cis-acting sequences(5, 19). The M element uses an alternative left boundary,resulting in either a 0.6- or 0.9-kb deletion (3). A 10-bp A5G5

tract is present �45 bp outside of M on both sides of themacronucleus-retained sequence (19). This sequence is alsopresent at the same distance from the alternative left junctionthat results in the smaller deletion. The sequence has beenshown to be necessary and sufficient for M element deletionand to control deletion boundaries at a distance (18). To date,this A5G5 polypurine tract has not been found flanking anyother IES. However, cis-acting sequences in flanking DNAhave been shown to be necessary for the efficient and accuratedeletion of mse2.9 (14, 24) and for the accurate deletion ofTrl1 (34). The controlling sequences for R deletion also flankthe IES on both sides (5). Although the exact identity of thesesequences is unclear, they function in a manner similar to thepolypurine tract of M to specify deletion boundaries at a dis-tance, suggesting that a similar mechanism is utilized for thedeletion of both M and R (5). Flanking sequences to the rightof R can substitute for those flanking the left, although there isno extensive similarity between them (5).

There is evidence that the micronucleus-limited sequence ofthe M element contains cis-acting elements for programmedelimination (41). Yao (41) has discussed the importance of therole of internal promoting sequences (IPS) in M and R ele-

* Corresponding author. Mailing address: Department of Biology,York University, 4700 Keele St., Toronto, Ontario, Canada M3J 1P3.Phone: (416) 736-5241. Fax: (416) 736-5698. E-mail: [email protected].

† Present address: Banting and Best Department of Medical Re-search, University of Toronto, Toronto, Ontario, Canada.

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ment excision. During programmed DNA deletion, the pro-posed function of an IPS is to target an element for deletionwhile cis-acting sequences in macronucleus-retained flankingsequences control the placement of deletion boundaries (11,41). Wuitschick and Karrer (40) have extended this model bydemonstrating that multiple micronucleus-limited fragmentsof the multiple-copy Tlr elements target their own pro-grammed excision, suggesting that multiple redundant elimi-nation targeting signals are distributed through the entire�22-kb element.

To examine possible functions of micronucleus-limitedDNA, Chalker and Yao (8) placed the micronucleus-limitedsequence of the M or R element into high-copy ribosomalDNA (rDNA) in the macronucleus of vegetatively growingcells. When they mated these strains to initiate a new round ofmacronuclear development, they observed an inhibition of theability of exconjugants to delete M or R at its normal chromo-somal locus. This effect was demonstrated to be sequencespecific in that little interference was seen with the deletion ofother IESs. Epigenetic effects on programmed DNA deletionhave also been observed for Paramecium (28).

A 2.9-kb IES, mse2.9 (20), is present within the secondintron of the ARP1 locus, a gene encoding a highly acidicprotein of unknown function containing numerous internalrepeats. mse2.9 has 81% AT content and does not appear tocontain an open reading frame (ORF), and its termini arelocated within TTAT direct repeats. The extensive microhet-erogeneity found at mse2.9 macronuclear junctions initiallysuggested the possibility that mse2.9 could be excised by adifferent mechanism than that for M and R (24). However, ourprevious analysis suggested that the mechanism of action of thecis-acting sequences in macronucleus-retained DNA flankingmse2.9 is conserved with that of M and R elimination, and wehave suggested that the same molecular mechanism is used todelete mse2.9, M, R, and likely the majority of IESs (14).

We have further analyzed the role of micronucleus-limitedDNA during mse2.9 deletion and have identified a 200-bpregion of the mse2.9 micronucleus-limited DNA that containsa cis-acting sequence element which is necessary for the effi-cient targeting of programmed excision. In an attempt to ex-tend mechanistic links with the programmed excision of otherIESs, we assessed the ability of a series of chimeric IESs, withthe mse2.9 flanking sequence bounding a variety of single- andmultiple-copy micronucleus-limited sequences, to be pro-cessed. The differing efficiencies of programmed deletion ofthese chimeric IESs, in combination with an experiment inwhich we removed an important cis-acting sequence from ma-cronucleus-retained DNAs of several of the chimeras, demon-strate that the cis-acting elements in both the eliminated mi-cronucleus-limited sequence and the macronucleus-retainedflanking sequences stimulate programmed excision to differentdegrees, depending upon the IES in question. In addition, wehave investigated the potential epigenetic regulation of mse2.9deletion. Our data suggest that mse2.9 may not be subject tothis type of regulation.

MATERIALS AND METHODS

Cell strains. T. thermophila strains CU428 [Mpr/Mpr (VII, Mps)] and B2086[Mpr�/Mpr� (II, Mps)] from inbreeding line B were provided by J. Gaertig,University of Georgia. Cells were cultured axenically in 1� SPP (1% proteose

peptone, 0.2% glucose, 0.1% yeast extract, 0.003% EDTA:ferric sodium salt) at30°C as described previously (32).

Tetrahymena transformation. Transformation of the Tetrahymena macronu-cleus by microinjection was done essentially as previously described (6, 8, 35, 38).Whole-cell DNA containing the micronucleus-specific sequence cloned into ma-ture rDNA was injected directly into the macronucleus of the cells at a concen-tration of 1 �g/ml under a Zeiss Axiomat 35 inverted microscope and by use ofan Eppendorf micromanipulator and microinjection system. After injection, in-dividual cells were transferred into 200 �l of 1� SPP in a 96-well microtiter plateand were grown at 30°C to saturation. For the screening of transformants, 25 �lfrom each well was replicated into 1� SPP or 1� SPP plus 100 �g of paromo-mycin/ml. This pattern was continued for several days, and transformants wereidentified by growth to saturation in the presence of paromomycin. The proce-dure of Gaertig and Gorovsky (15) was used to electroporate conjugating Tet-rahymena as previously described (14).

Tetrahymena conjugation. Wild-type and transformed strains were mated asdescribed previously (25). After 8 to 10 h of mating, individual mating pairs weretransferred into 30 �l of SPP. Cells were grown to saturation and then screenedfor both sensitivity to paromomycin (100 �g/ml) (Pms) and resistance to 6-meth-ylpurine (15 �g/ml) (Mpr). Whole-cell DNAs were harvested from 10-ml culturesthat were both Pms and Mpr. We also performed this experiment by adding SPPand 6-methylpurine to individual mating pairs at 24 h postmixing. Directly se-lected Mpr clones were screened for Pms and then were grown as describedabove for extraction of whole-cell DNA.

DNA purification and analysis. Whole-cell DNA for microinjection was pu-rified essentially as described by Gaertig and Gorovsky (15) as modified by Li andPearlman (24). For all other experiments, the extraction of whole-cell DNA wasdone essentially according to the method of Gaertig et al. (17), scaled down for10-ml cultures (14). Standard molecular biology techniques were performed asdescribed by Sambrook et al. (36) or by following the supplier’s instructions.Probes for Southern blot analysis were labeled by random priming (36) with[�-32P]dATP (Amersham). DNA-modifying enzymes were obtained from NewEngland Biolabs.

Plasmid construction. The plasmids heh2.2 and heh2.2-9R have been de-scribed previously (14, 24). To make DNA constructs containing mse2.9 flankingmacronucleus-retained sequences surrounding different micronucleus-limitedDNA fragments, we used inverse PCR of heh2.2 in pHSS6 (24), using theprimers msef/r and heh4 (14) to generate a product that was then gel purified,digested with EcoRV, and treated with alkaline phosphatase. This was ligated tothe following EcoRV-digested PCR products amplified with the following primersets (Table 1) and template DNAs: MMICF(0.6 kb) or MMICF(0.9 kb) andMMICR with pCA455-3; RMICF and RMICR with pMY404-21 (plasmidspCA455-3 and pMY404-21 were provided by Douglas Chalker, WashingtonUniversity, St. Louis, Mo.); and H1F and H1R, as well as RTF and RTR, bothwith Tetrahymena whole-cell DNA. All pHSS6-based subclones used for thisstudy were subsequently cloned into the NotI site of pD5H8 or pD5H8N1, aspreviously described (14). To generate chimeric IES clones lacking the A-richflanking sequence, we amplified the respective pHSS6 chimeric IES subclonewith the primers HEH2(ApaI) and HEH6(ApaI) (14) (Table 1). The resultingPCR products were gel purified, digested with ApaI, and ligated at a diluteconcentration. To generate internal deletions in the heh2.2 micronucleus-limitedsequence, we performed inverse PCR, using heh2.2:pHSS6 as the template, withthe indicated primers (Table 1) to replace 200 bp of sequence with EcoRV sites.The ARPKO construct was generated by amplifying the plasmid E-15, whichcarries a 5.5-kb EcoRI fragment containing the entire ARP1 locus (20), withprimers ARP1KOF and ARP1KOR (Table 1), digesting the amplified productwith EcoRV, and ligating it to the 1.4-kb SmaI/EcoRV-digested product ofp4T2-1.

PCR analysis. PCRs were used to amplify junction sequences of processingconstructs obtained by transformation as previously described (14, 24). Long-range PCRs were performed with the Expand Long Template PCR system(Boehringer Mannheim) under the conditions specified by the supplier.

DNA sequencing. Automated cycle sequencing was done with dye-labeleddideoxy terminators and a PE/ABI 373a or 377 sequencer at the Core MolecularBiology Facility, York University, Toronto, Ontario, Canada. PCR products ofmse2.9 junctions were sequenced with the RU4 primer (Table 1).

RESULTS

A region of mse2.9 micronucleus-limited DNA is requiredfor excision. We previously characterized the role of flankingDNA in an mse2.9 deletion (14) by utilizing the mse2.9-based

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construct heh2.2 (Fig. 1A). The heh2.2 construct lacks theinternal 1.9-kb EcoRI fragment of mse2.9, but when trans-formed into conjugating cells, is processed in a manner iden-tical to that for wild-type mse2.9 (14, 24). Mechanistic analysesof the programmed deletion of mse2.9 have previously beenlimited to the cis-acting elements in macronucleus-retainedflanking sequences. We were therefore interested to determineif any micronucleus-limited DNA of mse2.9 is required for

efficient and accurate programmed excision. The fact thatheh2.2 is efficiently and accurately processed while recapitu-lating the microheterogeneity found at the mse2.9 macro-nuclear locus demonstrates that the internal 1.9-kb EcoRIfragment of mse2.9 is not required for programmed deletion(24). We generated five heh2.2-based constructs, with eachcontaining a 200-bp internal deletion within the 1-kb heh2.2micronucleus-limited sequence, and tested them for their abil-

FIG. 1. Constructs and transformation assay. (A) Organization of T. thermophila DNA at the mse2.9 locus and construction of heh2.2 andheh2.2�19. MAC, macronuclear DNA; MIC, micronuclear DNA; ARP1, acidic repetitive protein gene. The area inside the dotted lines indicatesmicronucleus-limited DNA. Outside of these lines, the macro- and micronuclear sequences are identical. Filled and open boxes denote ARP1 exonsand introns, respectively. The construction of heh2.2 and heh2.2�19 has been described previously (14, 24). The solid line under the unprocessedheh2.2 indicates the probe used for Southern blot analysis of transformants. Intron boundaries that flank mse2.9 are indicated by asterisks.(B) Transformation assay for IES excision. The heh2.2-based sequences described in this study were cloned into pD5H8 and transformed intoconjugating Tetrahymena as previously described (14, 15). H, HindIII; X, XbaI; E, EcoRI; R, EcoRV.

TABLE 1. Oligonucleotides used for amplification in this study

Primer name Sequence (5�33�)

RTRII(RV) ...........................................................................................................................................CATGATATCCTTAGTCTGAGTGAGTCC5R ...........................................................................................................................................................AATAAGATGCAAAGCAGC3R ...........................................................................................................................................................GCTTAAACACAACTATTCRU4........................................................................................................................................................CCATTTTCTAATTTTATAGTTAAGAAA5J.............................................................................................................................................................ATTATAGGTACCATAAAC3J.............................................................................................................................................................GAATTGGTTTATATATTGMMICF(0.9 kb) ....................................................................................................................................CATGATATCTAATTAGTATGGAATAAATTAMMICF(0.6 kb) ....................................................................................................................................CATGATATCTAATTGAAAGGAGGTTGCTATMMICR .................................................................................................................................................CATGATATCAATTATTCATTCATTTTATAATRMICF...................................................................................................................................................CATGATATCGTGATTCAAAAAAATGGTRMICR ..................................................................................................................................................CATGATATCAAGGAAGAAATTTGAGAAH1IESF ..................................................................................................................................................CATGATATCGTACAAAAACGGATTATTAATH1IESR .................................................................................................................................................CATGATATCTTTTGGTCATAATATATTTAAHEHI1 ...................................................................................................................................................CATGATATCGAATTCTTTAAGTTTGTACTTHEHI1REVERSE................................................................................................................................CATGATATCAAGTACAAACTTAAAGAATTCHEHI2 ...................................................................................................................................................CATGATATCTCTTAATTTTAGAAAAGTAAGHEHI2REVERSE................................................................................................................................CATGATATCCTTACTTTTCTAAAATTAAGAHEHI3 ...................................................................................................................................................CATGATATCAGCTTGCTATTTTAAAGATTGHEHI3REVERSE................................................................................................................................CATGATATCCAATCTTTAAAATAGCAAGCTHEHI4 ...................................................................................................................................................CATGATATCTGGTTCTTAGTGCTCATGAATHEHI4REVERSE................................................................................................................................CATGATATCATTCATGAGCACTAAGAACCAHEH2(ApaI)..........................................................................................................................................CATGGGCCCCAATATATAAACCAATTCAATHEH6(ApaI)..........................................................................................................................................CATGGGCCCTTAACACGTTTAAAATAAAAC

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ity to be processed. Four of the constructs were processed withhigh efficiencies, as assayed by Southern blotting (Fig. 2A toD), indicating that the 800 bp from the right boundary of themicronucleus-limited sequence of heh2.2 is dispensable forefficient programmed mse2.9 elimination. The processing wasaccurate in these transformants, and we observed junctionalmicroheterogeneity both within and between transformants(Table 2; data not shown). In contrast, the deletion of 200 bpof micronucleus-limited sequence from the left heh2.2 bound-ary significantly decreased the efficiency of programmed exci-sion (Fig. 2E). Two of the transformants completely failed toprocess the DNA (Fig. 2E, lanes 4 and 6), and the other fourwere significantly altered in the ability to efficiently processmicronucleus-limited DNA (Fig. 2E, lanes 1 to 3 and lane 5).We determined by sequencing of the deletion junctions that

the low level of processing observed for these transformantsdoes represent accurate processing (Table 2; data not shown).

Chimeric IESs with mse2.9 flanking sequences excise withdifferent efficiencies. We previously analyzed the processing ofa series of chimeric IESs that substituted flanking DNA froma variety of IESs for 103 bp of macronucleus-retained DNAflanking the right side of heh2.2 (the removal of this sequenceeliminated accurate programmed excision of heh2.2 [14]). Thedifferential ability of the added sequence to restore processingsuggests that different classes of IES exist in Tetrahymena (14).To extend this observation, we asked whether mse2.9 macro-nucleus-retained flanking sequences are able to effect the ef-ficient processing of other micronucleus-limited sequences.We replaced the 1-kb micronucleus-specific sequence of theheh2.2-based heh2.2�19 clone (14) with a variety of micronu-

FIG. 2. Southern blot analysis of transformants of five heh2.2-based clones with 200-bp internal deletions in micronucleus-limitedDNA (the deleted sequence is represented in the diagram of therespective construct). Whole-cell DNAs from transformants weredigested with HindIII. Southern blots were probed with the 597-bpHindIII-XbaI fragment (Fig. 1A). (A) heh2.2�200internal-1.(B) heh2.2�200internal-2. (C) heh2.2�200internal-3. (D) heh2.2�200internal-4. (E) heh2.2�200internal-5. H, HindIII; X, XbaI; R,EcoRV; E, EcoRI.



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cleus-limited sequences. We used the 0.6- and 0.9-kb forms ofthe M element (3), the 1.1-kb R element (2), the 1.0-kb histoneH1 IES (22), and 1.0 kb of micronucleus-limited DNA thatencodes a portion of the reverse transcriptase (RT) domain ofthe REP element, a multiple-copy Tetrahymena non-long-ter-minal-repeat retrotransposon (14a). The heh2.2�19 internaldeletion clone is missing 25 bp of macronucleus-retained DNAthat is normally found at the right boundary of the heh2.2micronucleus-limited sequence (the net loss is 19 bp since anEcoRV site replaces the 25 bp of deleted sequence [Fig. 1A]).This clone has previously been shown to undergo efficientprogrammed excision, demonstrating that this sequence doesnot function in programmed mse2.9 elimination. Due to thefact that cis-acting elements in macronucleus-retained DNAcontrol the placement of deletion boundaries at a specificdistance, heh2.2�19 deletion junctions are shifted to a micro-nucleus-limited sequence by a distance that is approximatelyequal to the amount deleted (14). Microheterogeneity wasobserved at macronuclear deletion junctions in cells trans-formed with heh2.2�19 (14).

The results of the transformations with heh2.2�19�Mmic(0.6 kb) and heh2.2�19�Mmic(0.9 kb) are shown in Fig. 3A.heh2.2�19�Mmic(0.6 kb) did not process efficiently. Only oneof the six transformants analyzed showed efficient and accurateprogrammed deletion (Fig. 3A, lane 4), while four of the sixhad mostly unprocessed DNA (Fig. 3A, lanes 1, 3, 5, and 6).One of the transformants showed a combination of accuratelyprocessed DNA and an aberrantly processed smaller fragment(Fig. 3A, lane 2). Thus, the mse2.9 flanking sequences are ableto direct accurate, but inefficient, processing of the 0.6-kb Melement. A similar result was obtained for the heh2.2�19�Mmic(0.9 kb) chimera (Fig. 3A, lanes 7 to 12). Five of the sixtransformants contained a low level of accurately processedconstruct along with significant amounts of unrearranged DNA(Fig. 3A, lane 7 and lanes 9 to 12). One transformant showedalmost exclusively unrearranged DNA (Fig. 3A, lane 8). There-fore, mse2.9 flanking sequences are able to direct a low level ofprocessing of both forms of the M element. We verified thatthe excision products observed at the expected size repre-sented accurate deletions by amplifying and directly sequenc-

ing macronuclear junctions from transformants (Table 3). Thesequence data indicated that while the proper left macro-nuclear boundary was utilized, the right macronuclear bound-ary in these transformants was shifted into a micronucleus-limited sequence of the chimeric IES by a distance of 16 to 21bp (Table 3) (this is a consequence of the internal deletion inthe construct [14]). These transformants exhibited a low levelof accurate processing, and microheterogeneity of the macro-nuclear junctions was observed between different transfor-mants (Table 3). The results of transformations with theheh2.2�19�H1IES (Fig. 3B) construct echoed those of the Melement constructs. The H1 IES was processed with a lowefficiency in several transformants (Fig. 3B, lanes 1 to 3 andlane 6) and was completely unprocessed in two others (Fig. 3B,lanes 4 and 5). We verified that the excision products observedat the expected size represented accurate deletions (Table 3).Microheterogeneity was observed between different transfor-mants and within the same transformants (Table 3; data notshown). The heh2.2�19�Rmic IES was processed more effi-ciently than the two previous chimeric IESs, with all transfor-mants displaying efficient processing (Fig. 3C, lanes 1 to 6).Programmed excision was accurate as well as efficient, andjunctional microheterogeneity was observed between as well aswithin transformants (Table 3; data not shown).

To test the effect of placing a multiple-copy micronucleus-limited sequence between mse2.9 flanking sequences, we gen-erated a chimeric IES with the heh2.2�19 flanking sequenceand 1.0 kb of an RT domain of ORF2 of the REP element, amicronucleus-limited multiple-copy non-long-terminal-repeatretrotransposon (14a). The heh2.2�19�REP transformantsexcised the micronucleus-limited sequence as efficiently(Fig. 3D, lanes 1 to 6) as wild-type heh2.2�19 (14) andheh2.2�19�Rmic (Fig. 3C). The excision was also accurate,and we observed microheterogeneity both between differenttransformants (Table 3) and within the same transformant(data not shown). In addition to the correctly excised DNA,each transformant contained a significant amount of a smallerproduct that could represent the aberrant deletion of a largerfragment than was expected (Fig. 3D).

TABLE 2. Sequences of macronuclear deletion junctions from transformants of heh2.2 constructs containing 200-bp micronucleus-limitedinternal deletions

Construct Sequencea

heh2.2�200 internal-1 1-3, 6 ..............................................TTTCTAGATttatttattcaa.......ataaaaGTACAAACTTAAAGAATTCGATATCGTTTTheh2.2�200 internal-1 5......................................................TTTCTAGATTTATttattcaa.......ataaaagtACAAACTTAAAGAATTCGATATCGTTTTheh2.2�200 internal-2 3-5...................................................TTTCTAGATTTATTtattcaa.......ttattattatattattAAAAAAATATTheh2.2�200 internal-3 4......................................................TTTCTAGATTTATTtattcaa.......ttattattatattattAAAAAAATATTheh2.2�200 internal-3 6......................................................TTTCTAGATTTATTtattcaa.......ttattattatattATTAAAAAAATATTheh2.2�200 internal-4 2......................................................TTTCTAGATTTATttattcaa.......ttattattatatTATTAAAAAAATATTheh2.2�200 internal-4 3......................................................TTTCTAGATTTATttattcaa.......ttattattatattATTAAAAAAATATTheh2.2�200 internal-4 4......................................................TTTCTAGATTTATTtattcaa.......ttattattatattATTAAAAAAATATTheh2.2�200 internal-4 6......................................................TTTCTAGATttatttattcaa.......ttattattATATTATTAAAAAAATATTheh2.2�200 internal-5 5......................................................TTTCTAGATGATatctcttaa.......ttattattatattATTAAAAAAATATTheh2.2-9R background

heh2.2�200 internal-1 3, 6..............................................TTTCTAGATTTATttattcaa.......ataaaagtacAAACTTAAAGAATTCGATATCGTTTTheh2.2�200 internal-1 2, 4, 5 .........................................TTTCTAGATttatttattcaa.......ataaaaGTACAAACTTAAAGAATTCGATATCGTTTT

a The right junctions are shifted into mse2.9 micronucleus-limited sequences by �19 bp in the heh2.2�200internal-1 transformants, as the deletion constructs weresynthesized by using the same right primer as the heh2.2�19 construct (14). XbaI restriction sites are in italics, and underlined nucleotides indicate repetitions of thebases at either end of the micronucleus-limited DNA which make the precise deletion boundaries unclear. Uppercase sequences represent the macronucleus-retainedsequence at deletion junctions, while lowercase letters represent the eliminated micronucleus-limited sequence.



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FIG. 3. Southern blot analysis of transformants of heh2.2�19-based IES chimeras. Whole-cell DNAs from transformants were digested withHindIII, except those of the heh2.2�19�Rmic series, which were digested with NotI. Southern blots were probed as described for Fig. 2.(A) heh2.2�19�M(0.6 kb) and heh2.2�19�M(0.9 kb). (B) heh2.2�19�H1IES. (C) heh2.2�19�Rmic. (D) heh2.2�19�REP. H, HindIII; X, XbaI;R, EcoRV.

TABLE 3. Sequences of macronuclear junctions from transformants of chimeric IESs

Construct Sequencea

heh2.2�19�M (0.6kb) 4............................................................TTTCTAGatgatatc.......attaaTTATAAAATGAATGAATAATTGATATCGTTTTAheh2.2�19�M (0.9kb) 1............................................................TTTCTAGATgatatc.......attaattATAAAATGAATGAATAATTGATATCGTTTTAheh2.2�19�M (0.9kb) 2............................................................TTTCTAGATGAtatc.......attaattatAAAATGAATGAATAATTGATATCGTTTTAheh2.2�19�M (0.9kb) 3............................................................TTTCTAGATGatatc.......attaattaTAAAATGAATGAATAATTGATATCGTTTTAheh2.2�19�M (0.9kb) 4............................................................TTTCTAGatgatatc.......attaaTTATAAAATGAATGAATAATTGATATCGTTTTAheh2.2�19�M (0.9kb) 5............................................................TTTCTAGATGatatc.......attaaTTATAAAATGAATGAATAATTGATATCGTTTTAheh2.2�19�Hl IES 2.................................................................TTTCTAGATGAtatc.......gtcgtttaaATATATTATGACCAAAAGATATCGTTTTAheh2.2�19�Hl IES 4.................................................................TTTCTAGatgatatc.......gtcgtTTAAATATATTATGACCAAAAGATATCGTTTTAheh2.2�19�Hl IES 5.................................................................TTTCTAGATGatatc.......gtcgtttaAATATATTATGACCAAAAGATATCGTTTTAheh2.2�19�R element 2...........................................................TTTCTAGATGatatc.......atctaTTTTTCTCAAATTTCTTCCTTGATATCGTTTTAheh2.2�19�R element 4...........................................................TTTCTAGATGAtatc.......atctattttTCTCAAATTTCTTCCTTGATATCGTTTTAheh2.2�19�R element 5...........................................................TTTCTAGATGATatc.......atctatttttCTCAAATTTCTTCCTTGATATCGTTTTAheh2.2�19�R element 6...........................................................TTTCTAGatgatatc.......atctatTTTTCTCAAATTTCTTCCTTGATATCGTTTTAheh2.2�19�REP 2.....................................................................TTTCTAGATGatatc.......tacatAAAGGACTCACTCAGACTAAGGATATCGTTTTAheh2.2�19�REP 3.....................................................................TTTCTAGATGATatc.......tacatAAAGGACTCACTCAGACTAAGGATATCGTTTTA

a The right deletion junctions are shifted into micronucleus-limited DNA by approximately 19 bp, as the chimeric IESs contain flanking sequence derived from theheh2.2�19 construct (14). XbaI and EcoRV restriction sites are in italics and macronuclear-retained IES sequences not originating from mse2.9 are bold. Underlinednucleotides indicate repetitions of the bases at either end of the micronucleus-limited DNA which make the precise deletion boundaries unclear. Uppercase sequencesrepresent the macronucleus- retained sequence at deletion junctions, while lowercase letters represent the eliminated micronucleus-limited sequence.



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Removal of an A-rich sequence in mse2.9 flanking sequenceaffects excision of heterologous IESs. We previously identifiedan �10-bp A-rich sequence in the DNA flanking the right sideof chromosomal mse2.9 that is essential for accurate mse2.9processing (14). We replaced this A-rich region, 47 to 61 bp tothe right boundary of chromosomal mse2.9, with an ApaI sitein several of the chimeric constructs to generate theheh2.2�19�cis series. As a control, we removed the sequencefrom heh2.2�19, and as expected (14), abolished all accurateprocessing (Fig. 4A). A low level of aberrantly processed DNAwas observed in these transformants (Fig. 4A, lanes 2 to 4 andlane 7). In addition, when the A-rich sequence was removedfrom heh2.2�19�Mmic(0.9 kb) (Fig. 4B), very little processingwas observed. We did not observe accurate R element excisionfrom heh2.2�19�cis�Rmic (Fig. 4C). However, three of thesix transformants displayed significant levels of aberrantly pro-cessed DNA (Fig. 4C, lanes 3 to 5) which we confirmed did notrepresent accurate processing by PCR amplification and se-quencing of transformant deletion junctions. Similarly, we ob-served no accurate processing of heh2.2�19�cis�REP for the

four transformants analyzed (Fig. 4D, lanes 1 to 4). Only twoof the four contained a significant amount of unrearrangedDNA (Fig. 4D, lanes 3 and 4). A variety of fragments wereobserved in all of the transformants that likely represent ab-errantly processed DNAs (Fig. 4D). In addition, two of thefour transformants contained significantly less hybridizingDNA than was expected for rDNA-based heh2.2 constructs(Fig. 4D, compare lanes 1 and 2 with lane C, which contains anequivalent amount of NotI-digested DNA from theheh2.2�19�cis�Rmic transformant shown in Fig. 4C, lane 5).

Expression of ARP1 is not essential for growth of Tetrahy-mena. The mse2.9 micronucleus locus is within the secondintron of ARP1, a gene of unknown function (20). We previ-ously demonstrated that all necessary macronucleus-retainedcis-acting sequences necessary for mse2.9 excision are con-tained within intronic sequences (14). Since mse2.9 is locatedin the second intron of ARP1, there is a possibility that theinhibition or interference of programmed mse2.9 deletioncould affect Arp1p functioning. We addressed this by generat-ing an ARP1 knockout strain to determine if its expression is

FIG. 4. Southern blot analysis of transformants of heh2.2�19�cis-based IES chimeras. Whole-cell DNAs from transformants were digestedwith HindIII, except those of the heh2.2�19�Rmic�47-61 and heh2.2�19�RTM�47-61 series, which were both digested with NotI. All Southernblots were probed as described for Fig. 2. (A) heh2.2�19�47-61. (B) heh2.2�19�M(0.9kb)�47-61. (C) heh2.2�19�Rmic�47-61.(D) heh2.2�19�REP�47-61. Lane C contains an equivalent amount of NotI-digested DNA from the heh2.2�19�cis�Rmic transformant of panelC, lane 5. H, HindIII; X, XbaI; R, EcoRV; E, EcoRI; A, ApaI.



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essential for the growth of Tetrahymena. Using gene replace-ment by homologous recombination, we generated somaticARP1 knockout strains with a construct containing a neomycinresistance cassette that replaces almost the entire ARP1 codingsequence (Fig. 5A). The wild-type allele was replaced entirelywith the knockout allele by phenotypic assortment (Fig. 5B),indicating that ARP1 expression is not essential for the vege-tative growth of Tetrahymena. Simple modular architectureresearch tool (SMART) analysis of the ARP1p predicted pro-tein sequence indicated the presence of a hydrophobic signalpeptide in the N-terminal 20 amino acids, suggesting thatArp1p may be a secreted protein.

Presence of deletion clone heh2.2-9R in the macronucleus ofconjugating cells does not affect programmed mse2.9 deletion.We previously established that mechanistic links exist in theprogrammed excision of several different IESs (14). The resultsof the present study suggest that mechanistic links may extendto include a requirement for sequence elements within micro-nucleus-limited DNA. Chalker and Yao (8) showed that byectopically loading the macronucleus of growing cells withmicronucleus-limited DNA and then forcing them to mate, itwas possible to interfere with the subsequent deletion of thehomologous IES in the next round of macronuclear develop-ment. Similarly, we attempted to interfere with programmedmse2.9 deletion. Since this effect required only micronucleus-limited DNA, not macronucleus-retained flanking sequences,we performed an experiment with heh2.2�19�Mmic(0.6 kb),which we have shown is not processed efficiently when intro-duced into conjugating cells (Fig. 3A). We purified whole-cellDNA from a transformant that completely failed to delete the

IES and microinjected it into the macronucleus of growingCU428 and B2086 strains of Tetrahymena (Fig. 6). The trans-forming DNA was mature, linear rDNA that carried unproc-essed heh2.2�19�Mmic(0.6 kb) DNA. CU428 is a hetero-karyon that carries a homozygous dominant allele forresistance to 6-methylpurine (Mpr) in its micronucleus. SinceCU428 does not carry the Mpr allele in its macronucleus, it issensitive to 6-methylpurine (Mps), allowing the direct selectionof all exconjugants by their Mpr phenotype. Southern blottingof the whole-cell DNA isolated from the microinjected trans-formants showed that the transformants carried the injectedDNA at high copy numbers in the macronucleus (Fig. 7A).Individual mating pairs were cloned from conjugating B2086:heh2.2�19�Mmic(0.6 kb) and CU428:heh2.2�19�Mmic(0.6kb) transformants. Whole-cell DNAs were isolated from ex-conjugant cultures that successfully completed conjugation andwere subjected to Southern blotting to analyze their ability toexcise the M element (Fig. 7B). The cells containing theheh2.2�19�Mmic(0.6 kb) construct in their macronuclei pro-duced exconjugants that were significantly inhibited in the abil-ity to excise the M element (Fig. 6B). The M element is alwaysexcised efficiently from exconjugants of wild-type matings (3).This result confirmed that the epigenetic inhibition of DNAdeletion is not a strain-specific phenomenon in Tetrahymenaand that the presence of a nonhomologous mse2.9 flankingsequence does not interfere with the M-induced inhibition ofM deletion. Chalker and Yao (8) previously showed that al-though this epigenetic effect was largely sequence specific, theprogrammed deletion of unrelated IESs could be affected to adegree. To see if this was the case for the heh2.2�19�Mmic

FIG. 5. ARP1 expression is not essential for growth. (A) Design ofan ARP1 knockout construct. The probe for Southern blot analysis isindicated by a solid bar. The solid arrow represents the 1.4-kb EcoRV/SmaI neo cassette from p4T21 (16). (B) Southern blot analysis ofHaeIII-digested whole-cell DNAs purified from wild-type CU428(C) and two ARP1 knockout strains. Transformed strains were grownfor �100 generations in 100 �g of paromomycin [lanes 1(�) and2(�)]/ml, at which point single cells from each transformant wereisolated and grown for �100 generations in 1� SPP [lanes 1() and2()]. H, HindIII; X, XbaI; E, EcoRI.



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(0.6 kb) construct, we examined the processing of the closelylinked R element (Fig. 7C) and of unlinked mse2.9 (Fig. 7D).Although programmed excision of the linked R element wasaffected to a small degree (Fig. 7C, lanes 3, 4, 8, 9, 13, and 14),that of unlinked mse2.9 was not affected (Fig. 7D). Althoughthe clone used for this assay contained mse2.9 flanking se-quence, there was no effect on programmed mse2.9 deletion.

To examine whether mse2.9 is subject to the epigeneticeffect on DNA deletion, we performed the experiment usingthe heh2.2-9R deletion clone that was previously shown not toprocess (24). We could not use wild-type heh2.2 for this ex-periment, as unprocessed heh2.2 is never observed in transfor-mants (24; J. Fillingham and R. Pearlman, unpublished data).In contrast, Tetrahymena cells transformed with the heh2.2-9Rconstruct contain in their macronucleus the full micronuclearsequence of heh2.2 (minus 9 bp at the right boundary) clonedinto their rDNA. We generated Tetrahymena strains B2086 andCU428, which carry the heh2.2-9R clone at high copy numbersin the macronucleus (Fig. 8A). Individual mating pairs fromconjugating B2086:heh2.2-9R and CU428:heh2.2-9R transfor-mants were cloned as described above. Whole-cell DNAs wereisolated from exconjugant cultures and subjected to Southernblotting for an analysis of their ability to excise mse2.9 (Fig.8B), the M element (Fig. 8C), and the R element (Fig. 8D). Incontrast to the results observed for the heh2.2�19�Mmic(0.6kb) transformants, we saw no significant effect on the deletion

FIG. 6. Schematic outline of epigenetic experiment. See the textfor details.

FIG. 7. The M element within mse2.9 flanking sequence inhibits its own programmed excision. (A) Southern blot analysis of HindIII-digestedwhole-cell DNAs from strains of B2086 (B) and CU428 (C) microinjected with whole-cell DNA containing heh2.2�19�M(0.6 kb) in rDNA andprobed as described for Fig. 2. (B) Southern blot analysis of whole-cell DNAs purified from exconjugant clones digested with HindIII and probedfor M element excision. (C) Southern blot analysis of whole-cell DNAs purified from exconjugant clones digested with EcoRI and BglII and probedfor R element excision. *, aberrant processing. (D) Southern blot analysis of HaeIII-digested whole-cell DNAs probed for mse2.9 excision asdescribed for Fig. 2.



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of the mse2.9 locus (Fig. 8B) or the unlinked M and R ele-ments (Fig. 8C and D).

Presence of heh2.2-9R in the macronucleus of conjugating cellsdoes not affect programmed deletion of heh2.2�200internal-1in a processing assay. To address the possibility that its chro-mosomal position could influence the potential epigenetic reg-ulation of mse2.9 deletion, we used conjugating B2086:heh2.2-9Rand CU428:heh2.2-9R (Fig. 8A) in a transformation assay (Fig.9A). If the underlying mechanism of the epigenetic effect wascommon to all IESs, we might have observed an epigeneticeffect on heh2.2 deletion in a different chromosomal environ-ment, in this case the �21-kb rDNA minichromosome. Weelectroporated the heh2.2�200internal-1 clone, which pro-cesses accurately and efficiently in wild-type B2086 and CU428conjugations, into conjugating B2086:heh2.2-9R and CU428:heh2.2-9R (Fig. 2A). In this case, transformants were selectedwith paromomycin and 6-methylpurine to ensure the exclusivesurvival of exconjugants (Fig. 9A). An analysis of these trans-formants showed that they accurately processed theheh2.2�200internal-1 constructs with the same efficiency andaccuracy as wild-type cells (Fig. 9B; Table 2). This result indi-cates that the programmed deletion of mse2.9 may not besubject to epigenetic effects on its programmed deletion.

DISCUSSION

A cis-acting sequence in micronucleus-limited DNA is re-quired for targeting of efficient programmed mse2.9 deletion.We have identified a 200-bp region of micronuclear sequenceat the left side of the micronucleus-limited sequence in heh2.2

that contains an important cis-acting element for the pro-grammed deletion of mse2.9. The fact that a low level ofaccurate programmed elimination occurs in cells transformedwith this construct suggests that the sequence does not func-tion to control the placement of deletion boundaries, as hasbeen shown for cis-acting sequences in the DNA flankingmse2.9 (14, 24). Yao (41) has discussed the importance of therole of IPSs in IES excision. IPSs are described as sequenceelements existing within micronucleus-limited DNA that arerequired in cis for programmed deletion. We suggest that the200-bp stretch of mse2.9 comprises or contains an IPS. The 200bp of micronucleus-limited sequence at the left side of heh2.2has also been shown to contain a cis-acting sequence elementthat, in the absence of a wild-type heh2.2 macronucleus-re-tained flanking sequence, directs alternate processing (24). Itwill be informative to examine a possible relationship betweenthese two functionally different cis-acting elements to see, forexample, if they are genetically separable. In addition, the factthat a low level of processing was observed in some transfor-mants using this construct indicates the possible presence of anadditional IPS within the remaining 800 bp of micronucleus-limited heh2.2 sequence.

cis-Acting elements in flanking sequences and micronucle-us-limited DNA have different IES-specific abilities to promoteprogrammed excision. The current model of IES excision sug-gests that IPSs target elements for deletion while the cis-actingsequences in macronucleus-retained flanking sequences con-trol the proper placement of deletion boundaries (11, 41). Theresults of experiments described here and in our previous study(14) suggest that the programmed excision of mse2.9 follows

FIG. 8. Presence of the heh2.2-9R clone in parental strains does not interfere with programmed DNA deletion. (A) Analysis of microinjectedparental strains as described for Fig. 6A. (B) Analysis of mse2.9 structure as described for Fig. 9. (C) Analysis of M element as described for Fig.6B. (D) Analysis of R element as described for Fig. 6C.



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an identical pattern and that both of these functionally distinctclasses of cis-acting sequences are required for efficient andaccurate programmed excision. Using chimeric IESs withmse2.9 macronucleus-retained flanking sequences surroundingdifferent micronucleus-limited sequences, we observed vari-ability in the efficiency of programmed excision. Chimeric IESswith the mse2.9 flanking sequence surrounding versions of theM element and the histone H1 IES are processed with rela-tively low efficiencies, while the R element, mse2.9 (24), andthe RT domain from the REP element are processed withrelatively high efficiencies. We suggest that the variable effi-ciencies of processing of these constructs in transformants area direct result of cis-acting elements within the micronucleus-limited sequence of the different IESs. Specifically, we suggestthat the micronucleus-limited sequence targets its pro-grammed deletion with an efficiency that depends on the par-ticular micronucleus-limited sequence in question. For exam-ple, relative to mse2.9, both forms of the M element and theH1 IES do not strongly target their own excision. Alternatively,the R element, mse2.9, and the REP element target theirprogrammed excision relatively strongly.

The relative strength of cis-acting signals in micronucleus-limited DNA is not the only determinant of the efficiency ofprocessing. The efficiency of processing also depends on thestrength of cis-acting sequences in macronucleus-retainedflanking DNA that are utilized to determine deletion bound-aries. For example, it has been shown that the R element doesnot process with a high efficiency when using its own flankingsequences in the processing assay (5) and occasionally exhibitssome unrearranged DNA at its wild-type locus (Fillingham andPearlman, unpublished data). Since we suggested that the Rmicronucleus-limited sequence targets its deletion with a rel-atively high efficiency, we hypothesize that the stimulation ofprogrammed deletion by the micronucleus-limited sequence of

a particular IES is modified by the relative strength of thecis-acting sequences in its flanking macronucleus-retainedDNA. Thus, the R element itself strongly targets its excision,but it has relatively weak cis-acting signals in its macronucleus-retained flanking sequences. The relative weakness of the Relement flanking sequence is demonstrated by the fact that theR flanking sequence only weakly rescues a processing-deficientheh2.2 construct (14). This may also explain the functionalredundancy observed in the R flanking sequence (5): since Rmicronucleus-limited DNA strongly enhances its own pro-grammed deletion relative to M and H1 IESs, it is more com-pelled to use the cryptic cis-acting sequences in macronucleus-retained flanking DNA as they become available. Consistentwith this, we observed a larger amount of aberrant processingin heh2.2�19�Rmic�cis transformants (Fig. 4C) than inheh2.2�19�M(0.9 kb)�cis transformants (Fig. 4B). Thus, IESswith relatively strong targeting cis-acting signals in their micro-nucleus-limited DNA are more likely to utilize cryptic cis-acting signals in the macronucleus-retained sequence.

Relative to those of mse2.9 and the R element, the micro-nucleus-limited sequences of the M element and the H1 IESdo not contain strong targeting cis-acting signals. However, ourprevious results suggested that the relative strengths of thecis-acting elements in their flanking sequences may differ (14).The H1 IES is predicted to contain stronger cis-acting se-quences than is the M or R element (14). One inconsistency ofthis model for describing the behavior of cis-acting elements inIES excision is the in vivo behavior of the M element. The Melement has not been observed in its unprocessed form at itschromosomal locus in wild-type strains (3, 8). However, wild-type M element constructs are frequently not processed withhigh efficiencies in processing assays (18, 19), a fact that is inagreement with our interpretation of the relative strengths ofthe cis-acting elements in the M element DNA. Thus, there

FIG. 9. Presence of heh2.2-9R in the macronucleus of parental strains does not interfere with processing of heh2.2�200internal-1 in processingassay. (A) Cartoon showing the modified transformation assay. (B) Southern blot analysis of HindIII-digested whole-cell DNAs from transformantsof heh2.2�200internal-1 probed as described for Fig. 2.



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may be an additional degree of regulation of M element exci-sion at its chromosomal locus.

The multiple-copy RT domain of the REP fragment pro-cesses with a relatively high efficiency within the heh2.2�19flanking sequence, and we suggest that it targets its own pro-grammed excision with a high efficiency. Accordingly, transfor-mants of the heh2.2�19�REP�cis construct process theirDNA aberrantly, and several of the transformants do not con-tain significant amounts of unrearranged heh2.2�19�REP�cisDNA (Fig. 3D). We suggest that the reason for this is thattargeting of cis-acting signals in the REP element DNA stim-ulates its processing to such a high degree that it forces the useof cryptic cis-acting sequences in macronucleus-retained flank-ing DNA to an even greater degree than the R element. Thebehavior of the heh2.2�19�REP�cis construct in the process-ing assay echoes that of several Tlr-based constructs used byWuitschick and Karrer (40). In their analysis of the require-ment for micronucleus-limited DNA during programmed ex-cision of the multiple-copy Tlr element, they demonstratedthat Tlr DNA was efficiently deleted when its flanking se-quences were replaced with DNA from regions of the genomethat are not normally associated with rearrangement (40). Aninterpretation of this result is that Tlr element micronucleus-limited DNA contains targeting cis-acting signals that verystrongly enhance programmed excision and that in the absenceof strong flanking sequences it will, like REP element DNA inthe context of heh2.2�19�cis, force processing through the useof any cryptic flanking sequence it can find. Wuitschick andKarrer (40) interpreted their data to suggest that there is astrong correlation between sequence copy number and DNAelimination. In support of this, Yao et al. (43) demonstratedthat programmed DNA elimination can be induced by increas-ing the copy number of a particular sequence. Using this cri-terion, we expect that the REP element, which is present in theTetrahymena micronucleus at an estimated copy number of 40to 175 (14a), similar to the Tlr element, contains targetingcis-acting signals in its micronucleus-limited DNA that stronglyenhance programmed excision. Wuitschick and Karrer (40)have also demonstrated that the Trl multiple-copy family ofmicronucleus-limited sequences contains elimination targetingsignals that are likely distributed throughout the entire se-quence. We suggest that targeting cis-acting signals are simi-larly distributed throughout the REP element DNA. It is pos-sible that IESs that are present as a single copy in themicronucleus, such as mse2.9, contain different numbers ofIPSs that promote excision, while the entire sequence of mul-tiple-copy micronucleus-limited DNA functions as an IPS.Thus, the multiple-copy REP element and the Tlr familystrongly enhance their own deletion as a consequence of theirrepeated nature in the micronucleus.

Deletion of mse2.9 is not epigenetically inhibited. The epi-genetic inhibition of IES excision has been described for bothParamecium (30) and Tetrahymena (8). The effect is sequencespecific in that interference is observed at the IES locus that isectopically represented in the macronucleus. The inhibitoryeffect is observed with micronucleus-limited DNA with noflanking sequence, not when the macronucleus-destined flank-ing sequence is used by itself. Two models have been presentedto describe the epigenetic effect on DNA deletion. Meyer andDuharcourt (29) have argued that the sequence specificity ob-

served was likely the result of a trans-nuclear communicationbetween the parental macronucleus and the developing anla-gen. They pointed out that the epigenetic inhibition of DNAdeletion resembled homology-dependent gene silencing inplants, at the time an enigmatic phenomenon (26), and sug-gested that the molecule mediating this communication was anucleic acid that could act as a template to guide IES excision.Chalker and Yao (8) suggested that IESs play a passive role inthis epigenetic effect, titrating trans-acting factors that are spe-cific for individual IESs. The recently developed scan RNAmodel (31) provides a mechanistic framework to understandthis epigenetic effect and brings together the two models. Tran-scription of an IES during meiosis results in the generation ofsmall scan RNAs (scnRNAs) that travel to and scan the pa-rental macronucleus for the presence of homologous se-quences. In this model, the parental macronuclear sequencetitrates (8) the specific trans-acting scnRNAs, leaving thosethat are unpaired to travel to the anlagen, where a nucleic acid,the scnRNA, guides IES excision (29).

Our results indicate that, similar to the case in Paramecium(12), the epigenetic inhibition of IES excision may not be auniversal phenomenon in Tetrahymena. We were able to in-hibit the programmed excision of the M element by using achimeric M element but were unable to inhibit that of mse2.9by using the heh2.2-9R deletion clone. The chromosomal po-sition of ARP1 presents one potential problem in interpretingthe results of this experiment. If ARP1 were an essential geneand the retention of mse2.9 in the macronucleus interferedwith processing of the ARP1 mRNA, then there would beselection for any cells containing deleted mse2.9. In this case,the apparent lack of an epigenetic effect on mse2.9 processingwould be artifactual. However, this is not the case, as we havedemonstrated that the expression of ARP1 is not essential forgrowth. In addition, by using Southern blot analysis, we ana-lyzed the whole-cell DNAs extracted after a B2086:heh2.2-9Rand CU428:heh2.2-9R conjugation had finished but before se-lection for growth. We observed no effect on the programmeddeletion of mse2.9 (data not shown).

One additional caveat to our interpretation of the lack of anepigenetic effect on programmed mse2.9 excision is that it ispossible that the full 2.9-kb IES is required as a template forthe titration of scnRNAs. We have attempted to perform thisexperiment using the full 2.9-kb IES cloned into rDNA buthave had difficulty generating the required strains with appro-priate macronuclear copy numbers (Fillingham and Pearlman,unpublished data). To attempt to address this issue, wechanged the chromosomal context, testing the ability of theheh2.2�200internal-1 construct to process by using conjugatingcells loaded with heh2.2-9R in the macronucleus. These cellsloaded with heh2.2-9R in the macronucleus contained all ofthe micronucleus-limited sequence that is present in theheh2.2�200internal-1 construct. If scnRNAs direct mse2.9 ex-cision, this construct should not have processed in this mutantbackground, as the required scnRNAs would have been ti-trated and therefore not available to guide processing.

There is a precedent for the differences between mse2.9 andthe M and R elements. The meiosis-specific transcription of aparticular IES is proposed to comprise the first step of thescnRNA model. Chalker and Yao (7) described two classes ofmicronucleus-limited sequence defined by their temporal tran-



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scription profiles during early conjugation. The first class, rep-resented by the M and R elements, begins to be transcribed inearly meiosis and is transcribed through to the time periodcorresponding to early macronuclear development (7). Theother class is represented by the multicopy pTt2512 elementand is transcribed strongly, not in meiosis, but in the timeperiod corresponding to early macronuclear development.Chalker and Yao have placed mse2.9 into this latter class (7).According to the scnRNA model, if an IES is not transcribedduring meiosis, it cannot be processed to a scnRNA and con-sequently will not be available to scan the parental macronu-cleus. We suggest that IESs transcribed solely during earlymacronuclear development may require a different targetingmechanism than that of the first class of IESs (M and Relements). Taverna et al. (37) used chromatin immunoprecipi-tation to show that mse2.9 is associated with the methyl-H3K9modification as well as with Pdd1p during the time periodcorresponding to IES excision. This result is consistent withour previous conclusion that a common mechanism is used todelete IESs from Tetrahymena (14). We suggest that Tetrahy-mena uses more than one mechanism to generate the methyl-H3K9 modification, which then attracts a common IES exci-sion machinery. The programmed deletion of mse2.9 isunaffected by the addition of the histone deacetylase inhibitortrichostatin A (TSA) during macronuclear development (13).It is tempting to speculate that the different response of mse2.9to TSA than those of M and R elements is related to ourobservation that mse2.9 is not subject to epigenetic inhibitionof deletion. Could scnRNAs be required at a subset of IESs totarget histone deacetylation? Our results raise the possibilitythat mse2.9 may not require histone deacetylation for the gen-eration of the methyl-H3K9 epigenetic marker.

ACKNOWLEDGMENTS

We thank Anita Samardzic for expert technical assistance. We alsothank Nora Tsao and Emina David for helpful discussions throughoutthe course of this work. We thank Olga Ornatsky and John McDermott(York University, Toronto, Ontario, Canada) for help with microin-jection. DNA sequencing was done by Lee Wong (Core MolecularBiology Facility, York University).

This work was supported by a grant from the Canadian Institutes ofHealth Research (CIHR) to R.E.P. J.S.F. was supported by a CIHRstudentship and the York University President’s Dissertation Scholarship.

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