Tec3, a New Developmentally Eliminated DNA Element in Euplotes … · Tec3, a New Developmentally Eliminated DNA Element in Euplotes crassus Mary Ellen Jacobs,1* Adolfo Sa´nchez-Blanco,1

EUKARYOTIC CELL, Feb. 2003, p. 103–114 Vol. 2, No. 11535-9778/03/$08.00�0 DOI: 10.1128/EC.2.1.103–114.2003Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Tec3, a New Developmentally Eliminated DNA Element inEuplotes crassus

Mary Ellen Jacobs,1* Adolfo Sanchez-Blanco,1 Laura A. Katz,2and Lawrence A. Klobutcher1

Department of Biochemistry, University of Connecticut Health Center, Farmington, Connecticut 06030,1 andDepartment of Biological Sciences, Smith College, Northampton, Massachusetts 010632

Received 20 June 2002/Accepted 23 September 2002

More than 100,000 interstitial segments of DNA (internal eliminated sequences [IESs]) are excised from thegenome during the formation of a new macronucleus in Euplotes crassus. IESs include unique sequence DNAas well as two related families of transposable elements, Tec1 and Tec2. Here we describe a new class of E.crassus transposons, Tec3, which is present in 20 to 30 copies in the micronuclear genome. Tec3 elements havelong inverted terminal repeats and contain a degenerate open reading frame encoding a tyrosine-type recom-binase. One characterized copy of Tec3 (Tec3-1) is 4.48 kbp long, has 1.23-kbp inverted terminal repeats, andresides within the micronuclear copy of the ribosomal protein L29 gene (RPL29). The 23 bp at the extreme endsof this element are very similar to those in other E. crassus IESs and, like these other IESs, Tec3-1 is excisedduring the polytene chromosome stage of macronuclear development to generate a free circular form with anunusual junction structure. In contrast, a second cloned element, Tec3-2, is quite similar to Tec3-1 but lacksthe terminal 258 bp of the inverted repeats, so that its ends do not resemble the other E. crassus IES termini.The Tec3-2 element appears to reside in a large segment of the micronuclear genome that is subject todevelopmental elimination. Models for the origins of these two types of Tec3 elements are presented, along witha discussion of how some members of this new transposon family may have come to be excised by the samemachinery that removes other E. crassus IESs.

Ciliated protozoa undergo a massive reorganization of theirgenome during the process of sexual reproduction (20, 52).These organisms contain two types of nuclei: nonexpressedmicronuclei containing conventional chromosomes and tran-scriptionally active macronuclei that contain multiple copies ofsubchromosome-sized DNA molecules. Both types of nucleiare replicated and segregated during asexual reproduction;however, during mating (conjugation), the old macronucleus isdestroyed, and a new one is formed from a mitotic copy of thenewly formed zygotic micronucleus. Extensive genome remod-eling occurs during macronuclear development and includeschromosome fragmentation, de novo telomere formation, andDNA breakage and rejoining.

While all ciliates appear to undergo these various forms ofDNA rearrangement, members of the spirotrich group exhibitextreme forms of macronuclear development (17, 20). For thesubject of this study, the spirotrich Euplotes crassus (also re-ferred to as Moneuplotes crassus [4]), macronuclear develop-ment begins with the endoreplication of micronuclear chromo-somes (20 to 45 h after mating is initiated), resulting in theformation of polytene chromosomes. During this period,�100,000 interstitial DNA segments (internal eliminated se-quences [IESs]) are excised from the chromosomes and flank-ing sequences are rejoined. The polytene chromosome stage isfollowed by the vesicle stage, during which chromosomes arefragmented, telomeres are added to chromosome ends, and

micronucleus-limited DNA begins to be degraded. The finalstage of development includes telomere trimming on macro-nucleus-destined DNA (51) and additional rounds of DNAreplication. The newly generated macronucleus contains lin-ear, highly amplified DNA molecules averaging about 2 kbp inlength and usually containing single genes (39).

In E. crassus, there are two classes of IESs that are elimi-nated during the polytene chromosome stage of development(reviewed in references 17 and 29). The first class is the shortIESs, which are noncoding DNA segments ranging in lengthfrom approximately 30 to 550 bp. As many as 40,000 of theseelements reside within the micronuclear genome. The shortIESs have short inverted terminal repeats and are flanked by5�-TA-3� direct repeats. The second class of IESs includesmembers of the closely related Tec1 and Tec2 transposonfamilies (2, 18, 22, 34). Approximately 10,000 to 14,000 copiesof each element reside within the micronuclear genome of E.crassus. Both Tec1 and Tec2 are 5.3 kbp in length, have�700-bp inverted terminal repeats and, like the short IESs, areflanked by TA direct repeats. The Tec elements contain threedegenerate open reading frames (ORFs), one of which en-codes a “DDE” transposase most similar to those encoded bythe Tc1/Mariner family of transposons (ORF1 [7]), whereasanother (ORF2) is predicted to encode a tyrosine-type recom-binase (7a). However, there is no evidence that any of thesegenes is currently highly transcribed or that functional proteinsare produced (23).

Two lines of evidence indicate that the same machineryexcises the E. crassus short IESs and the Tec1 and Tec2 trans-poson IESs. First, all of these elements share a short terminalconsensus sequence that is similar to the terminal sequences of

* Corresponding author. Mailing address: Department of Biochem-istry, University of Connecticut Health Center, Farmington, CT 06030.Phone: (860) 679-2224. Fax: (860) 679-3408. E-mail: [email protected].

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members of the Tc1/Mariner family of transposons (17, 23).Paramecium IESs have a similar terminal sequence (28), andmutations in the terminal sequence abolish IES excision in thisorganism; these characteristics argue that the terminal se-quence is a key cis-acting element for specifying excision (re-viewed in reference 14). Second, both types of E. crassus IESsgenerate the same excision products. For each one, excision isprecise in that the macronucleus-destined DNA retains a sin-gle copy of the terminal 5�-TA-3� direct repeat (48) and theIES assumes a free circular form with an unusual heteroduplexjunction structure (21, 24, 32, 34, 46).

In this report, we describe a new type of long-inverted-repeat transposon in E. crassus, Tec3, which also undergoesdevelopmental elimination. The Tec3 elements are distinctfrom the Tec1 and Tec2 elements, but at least one familymember (Tec3-1) has termini that are quite similar to those ofthe Tec1 and Tec2 elements. Following excision, this elementforms a free circle with an unusual junction structure thatappears to be identical to those of other characterized E.crassus IESs. A second Tec3 element (Tec3-2) has truncatedinverted repeats and appears to reside in a large block ofdevelopmentally eliminated DNA. Models for the origins ofthese two types of Tec3 elements are discussed.

MATERIALS AND METHODS

E. crassus cell culture, mating, and DNA isolation. E. crassus cells were grownand maintained in artificial seawater by using the alga Dunaliella salina as thefood source as described previously (41), with the exception that Reef Crystals(Aquarium Systems, Mentor, Ohio) served as the base for the artificial seawaterand vitamin B12 was not included. E. crassus strains X1 (mating type III), X2(mating type unknown, but not mating type I or III) (11), CT5 (mating type III),and CT27 (mating type unknown, but not mating type I or III) were used in theanalyses. Matings of strains X1 and X2 as well as of strains CT5 and CT27 werecarried out and the resulting strains were harvested as described previously (41,47). Total cellular DNA (31), micronuclear DNA (35), and developing macro-nuclear DNA (27) were purified as described previously.

Molecular biological techniques. The recombinant LEMIC micronuclear li-brary, constructed from E. crassus strain G1 (3), was screened by hybridizationwith radiolabeled probes (10, 42). DNA was isolated from recombinant bacte-riophage as described previously (42). Plasmid DNAs were isolated from bacte-rial cells by using a Magic Miniprep DNA purification system (Promega Corp.,Madison, Wis.) according to the manufacturer’s instructions. PCR products wereisolated from low-melting-point agarose (GIBCO BRL Life Technologies, Inc.,Rockville, Md.) gels as described previously (40) and cloned by using a TOPOTA cloning kit for sequencing (Invitrogen, Carlsbad, Calif.). DNA restrictiondigestion, dephosphorylation, and ligation were carried out under conditionsrecommended by the enzyme suppliers (New England Biolabs, Beverly, Mass.,and GIBCO BRL Life Technologies) or with commonly used protocols (42).DNAs were analyzed by electrophoresis on agarose or low-melting-point agarosegels prepared and run in a buffer containing 89 mM Trizma, 89 mM H3BO3, and2 mM disodium EDTA-H2O.

PCR. All oligonucleotides were purchased from GIBCO BRL Life Technol-ogies, and PCR was carried out with a model PTC-150 MiniCycler (MJ Re-search, Cambridge, Mass.). For amplification of the micronuclear RPL29 locus,Hercules polymerase (Stratagene, La Jolla, Calif.) was used under conditionsrecommended by the manufacturer. Twenty-five cycles of amplification werecarried out with oligonucleotides LRPL-4 (5�-CGATCTACCTTATTATCACG-3�) and RFr29-1 (5�-TCTTTCCTAGTATTTGGATG-3�) and 0.5 �g of E. cras-sus strain X1 total cellular DNA as the substrate. A cycle consisted of 92°C for30 s, 50°C for 30 s, and 68°C for 8 min.

PCR also was carried out with KlenTaq DNA polymerase (Sigma-Aldrich, St.Louis, Mo.) essentially under the conditions described by the manufacturer and100 ng of substrate DNA. Excision of the Tec3-1 element was assessed witholigonucleotide primers LRPL-4 and RFr29-1. Twenty-five cycles of amplifica-tion were carried out with total DNA from vegetative and developing cells, witha cycle consisting of 95°C for 1 min, 49°C for 1 min, and 72°C for 1 min. Fordetection of the excised Tec3-1 circle junction, PCR amplification was carried

out with primers Tec3.1cirR (5�-TCTGAAGGACGGCATAATTA-3�) andTec3.1cirL (5�-GCTGAAGGATGTCCTAATCG-3�). Thirty cycles of amplifica-tion were performed, with a cycle consisting of 95°C for 1 min and 51°C for 1 min.

To obtain additional copies of the core regions of Tec3 elements, three sep-arate PCRs were performed with total cellular DNA and sets of primers designedto give overlapping products. The sequences of the primer pairs were based oneither the Tec3-1 sequence or a combination of the Tec3-1 and Tec3-2 sequencesand were as follows: Int6 (5�-GCTACTGTGTACCATGCAAC-3�) and Int7(5�-GCCTTGAAGACAAGAATGTC-3�), F4 (5�-TGCCTCTWGTRAACTTTTCR-3�, where W is A or T and R is A or G) and B4 (5�-GTCWAGAGYMGAAGAGGATA-3�, where Y is C or T and M is A or C), and F5 (5�-ATTKSGARRTCTCCTTTCCC-3�, where S is G or C and K is T or G) and B5 (5�-YCCAARTCTYTYCTCTSTRC-3�). Twenty-five cycles of amplification wereperformed, with a cycle consisting of 95°C for 30 s, 1 min at the annealingtemperature, and 72°C for 1 min. The annealing temperatures were 54, 49, and51°C for the Int6-Int7, F4-B4, and F5-B5 primer combinations, respectively.

DNA sequencing and analysis. Sequencing was performed at the University ofConnecticut Health Center Molecular Core Facility by using a Taq DyeDeoxytermination cycle sequencing kit (Perkin-Elmer Corp., Norwich, Conn.). Forsequencing of the Tec3-containing clones (Mic.RPL-29-2 and PhCl.4), restrictionfragments were subcloned into the pBluescript SK(�) phagemid (Stratagene).The subclones were then sequenced from their termini with primers that werecomplementary to vector sequences, followed by sequencing with primers thatwere complementary to internal regions of the subclones. Both strands of theentire Mic.RPL-29-2 insert and the Tec3-2 transposon in PhCl.4 were sequenced,while only one strand of the PhCl.4 region containing the Tec1 elements wassequenced. Sequences were compiled and analyzed by using SeqEd version 1.0.3software (Applied Biosystems, Inc., Foster City, Calif.) and MacVector version4.1.4 software (Accelrys, Princeton, N.J.).

Database searches were performed by using BLAST version 2.2.1 (1). DNAand protein sequences were aligned by using the ClustalW program (16, 50),accessed on the Baylor College of Medicine (Houston, Tex.) Human GenomeSequencing Center Search Launcher site (43), and edited by using SeqVu version1.1 (Garvan Institute of Medical Research, Sydney, New South Wales, Austra-lia). Default parameters were used in the alignments of tyrosine recombinaseproteins, but some manual adjustments were made to conform to the alignmentsof Nunes-Duby et al. (38). The additional tyrosine recombinase proteins ana-lyzed were obtained from GenBank and were as follows (with designations andaccession numbers given in parentheses): E. crassus Tec1 (Ecra Tec1, 397763), E.crassus Tec2 (Ecra Tec2, 397768), bacteriophage lambda integrase (Lambda 95Int, 138569), P1-like virus recombinase CRE (P1 CRE, 132262), Autographacalifornica nucleopolyhedrovirus very late expression factor vlf1 (AcN vlf1,1175103), Acholeplasma phage L2 integrase/recombinase-like protein (PL2 Int,9626516), bacteriophage phi CTX integrase (PhiCTX Int, 4063818), Escherichiacoli integrase XerD (Ecoli XerD, 139819), Clostridium butyricum hypotheticalprotein (Cbut hypo, 481912), Methanocaldococcus jannaschii integrase/recombi-nase (Mjan Int, 15668543), Bergeyella zoohelcum integrase (Bzoo Int, 557887),Haemophilus influenzae Rd putative integrase/recombinase (HinfInt, 16273329),Streptococcus pneumoniae Tn1545 integrase (Spne TnInt, 47463), E. coli fimbriaregulatory protein (Ecoli FimB, 729489), Methanothermobacter thermautotrophi-cus integrase/recombinase protein (Mthe Int, 7428936), Lactobacillus leichman-nii XerC recombinase (Llei XerC, 1359910), Staphylococcus aureus recombinaseXerD (Saur XerD, 3747042), Rickettsia prowazekii integrase/recombinase XerD(Rpro XerD, 7443326), Vibrio cholerae integrase/recombinase XerD (VchoXerD, 11355590), Nostoc sp. recombinase XisA (Nostoc XisA, 20141864),Anabaena sp. recombinase XisC (Anab XisC, 1094355), E. coli integrase/recom-binase XerC (Ecoli XerC, 14917067), Bacillus thuringiensis Tn4430 resolvaseTnPI (Bthur TnPI, 135957), S. aureus transposon Tn554 TnPA (Saur TnPA,135955), S. aureus phage �11 integrase (�11 Int, 166159), Enterobacter phageP22 integrase (P22 Int, 138565), bacteriophage P21 integrase (P21 Int, 138558),and Streptomyces ambofaciens integrase (Samb Int, 124698). Patterns of variationwere analyzed by using MacClade version 4.0 software (Sinauer Associates,Sunderland, Mass.).

Genealogical analysis of Tec3 clone and PCR product sequences was donewith the neighbor-joining (NJ) algorithm in Paup*4.0, with distances estimatedby the Tamura-Nei model (45) and variability in rates among sites estimated bya gamma parameter with an � value of 0.5. Genealogies of tyrosine recombinaseamino acid sequences were constructed by using several algorithms in order toassess the impact of different evolutionary models on relationships among se-quences. These analyses were done with 95 amino acids corresponding to theboxes and patches characterized by Nunes-Duby et al. (38; for the alignment andregions analyzed, see the supplemental figure at www.science.smith.edu/departments/Biology/lkatz/align.html) and included the cloned Tec3-1 element, a

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consensus sequence (conTec3-2) derived from Tec3-2, and the PCR clonesgenerated from the tyrosine recombinase regions of other Tec3 elements. Ge-nealogies were constructed by using the NJ algorithm with mean characterdistances and maximum parsimony (MP) analyses with 25 additional randomsequences in Paup* 4.0 (Sinauer Associates). Bootstrap support was calculatedby using 100 replicates for both models. Maximum-likelihood (ML) analyseswere performed through quartet puzzling by using the JTT model (25), allowingfor variation in rates among sites, as implemented by Tree-puzzle 5.0 (44).Resulting puzzle quartet (PZ) support values are reported.

Southern and dot blot hybridizations. DNA restriction fragments and PCRproducts used as hybridization probes were labeled with [�-32P]dATP by therandom hexamer priming method (10). Southern blotting was carried out asdescribed previously (5, 33). Hybridization was performed with a high-efficiencyhybridization system (HS 114; Molecular Research Center, Cincinnati, Ohio)essentially as described by the manufacturer. Blots were routinely washed twiceat room temperature for 5 min in 1� SSC (1� SSC is 0.15 M NaCl plus 0.015 Msodium citrate), twice at 65°C for 30 min in 1� SSC, and twice at 65°C for 30 minin 0.5� SSC or 0.1� SSC. All SSC buffers contained 1% sodium dodecyl sulfate.

For the dot blot analysis to determine the Tec3 copy number, DNA sampleswere boiled for 5 min before being spotted in triplicate on Nytran membranes(Schleicher & Schuell, Keene, N.H.) by using a Hybri-Dot (GIBCO BRL LifeTechnologies) filtration manifold as described by the manufacturer. Blots con-tained 10 �g of E. crassus strain X2 total vegetative-cell DNA, along with 11, 33,and 109 pg of a 2-kbp EcoRI restriction fragment derived from the Tec3-2element as copy number standards. The amounts of the 2-kbp EcoRI fragmentwere chosen to be equivalent to 10, 30, and 100 copies of the Tec3 element/cellin a 10-�g sample of total cellular DNA, based on a DNA content of 50 pg foran E. crassus cell (30). Sufficient salmon sperm DNA was added to the standardsamples to ensure that DNA gram amounts equal to those of the E. crassussamples were applied to the slots. Following hybridization with the 2-kbp EcoRIfragment as the probe, the filters were washed as described for Southern blots;the final washes were done with 0.5� SSC at 65°C. The amount of probe boundwas then determined by using an Instant Imager (Packard BioScience Co.,Meriden, Conn.).

Nucleotide sequence accession numbers. Sequences determined here weredeposited in GenBank under accession numbers AY115662 (Mic.RPL-29-2),AY115671 (PhCl.4), and AY115663 to AY115670 (Tec3 PCR product clones).

RESULTS

A Tec3 element interrupts the micronuclear copy of theribosomal protein L29 gene. In a previous study (27), inversePCR was used to isolate and clone the ends and flankingregions of the micronuclear copy of the ribosomal protein L29gene (RPL29). To obtain the complete micronuclear copy ofthe RPL29 gene, we attempted to carry out PCR with a pair ofoligonucleotide primers complementary to the regions flankingthe locus (LRPL-4 and RFr29-1) (Fig. 1). Repeated standardPCRs with this pair of primers, as well as other combinationsof primers, failed to give a specific PCR product. Since themacronuclear chromosome containing the RPL29 gene is only553 bp long (plus telomeres) (19), the failure to obtain a PCRproduct suggested that the micronuclear copy of the locuscontained one or more IESs. To investigate this possibility, wecarried out PCR with the above two primers under conditionsthat would allow for the amplification of large products (seeMaterials and Methods). A single product of 5.3 kbp wasobtained (Fig. 1A) and inserted into the TOPO TA vector togenerate clone Mic.RPL-29-2.

Complete sequencing of Mic.RPL-29-2 revealed that it con-tained a perfect match to all of the sequences present in ma-cronuclear RPL29, with the exception of telomeric repeats.However, the micronuclear copy of RPL29 contains a single4,483-bp IES inserted within the fourth codon prior to thetermination codon of the RPL29 ORF. Like all other E. crassusIESs, it is bounded by a 5�-TA-3� dinucleotide repeat, one copyof which is retained in the RPL29 macronuclear DNA mole-cule (Fig. 1B). The RPL29 element has large, imperfect (90%identical) inverted repeats of 1.23 kbp at its ends, surroundinga central core region of 2.02 kbp (Fig. 1B). These structural

FIG. 1. Isolation and characterization of Tec3-1. (A) Agarose gel containing PCR products obtained from E. crassus strain X1 total DNA withprimers LRPL-4 and RFr29-1 (lane PCR), along with size markers (lane M). (B) Maps of micronuclear clone Mic.RPL-29-2 and the macronuclearchromosome containing the RPL29 gene. Sequences forming macronuclear RPL29 are shown as cross-hatched rectangles, flanking micronuclearDNA is shown as narrow lines, and the Tec3-1 element is shown as a grey rectangle. The arrows inside the grey rectangle indicate the invertedrepeats of Tec3-1, and “-T” represents the telomeres of the macronuclear DNA molecule. The positions of oligonucleotides LRPL-4 and RFr29-1,which were used as the PCR primers in panel A, and of primers Tec3-1cirL and Tec3-1cirR (see Fig. 4) are indicated below the micronuclear map,with arrowheads denoting the direction in which they prime DNA synthesis

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features, as well as other data presented below, indicate thatthe IES in the micronuclear RPL29 locus is a transposableelement. Based on the previous naming of elements in E.crassus, we refer to this family of elements as Tec3 (transposonof E. crassus 3) and to the particular element in the RPL29locus as Tec3-1. In other analyses, it has been found that thereis a perfect duplication of the first 258 bp of the RPL29 locus,along with at least 316 bp of sequence that is a nearly perfectmatch to left flanking DNA, located �6.5 kbp upstream of themicronuclear region represented in clone Mic.RPL-29-2 (A.Sanchez-Blanco and L. A. Klobutcher, unpublished results).However, the remainder of the RPL29 locus, including theregion interrupted by the Tec3-1 element, is not present in theintervening DNA.

The Tec3-1 element is organizationally similar to the previ-ously characterized Tec1 and Tec2 E. crassus transposon IESs(21, 24, 34), but they appear to be distinct elements on thebasis of a number of criteria. First, Tec3-1 is shorter than theTec1 and Tec2 elements, which are both 5.3 kbp long. Second,the Tec1 and Tec2 elements have shorter inverted repeats of�700 bp. Third, there is little primary sequence similaritybetween Tec3-1 and Tec 1 or Tec2. The latter was assessed bycarrying out dot matrix comparisons of the various elements.When Tec3-1 was compared to either Tec1 (Fig. 2A) or Tec2(data not shown), no sequence similarity was evident. In con-trast, as initially shown by Jahn et al. (18), Tec1-1 and Tec2display significant similarity when compared by using the sameparameters (Fig. 2B).

Despite this lack of overall sequence similarity, the terminiof Tec3-1 are quite similar to those of the Tec1 and Tec2elements (Fig. 2C). Of the first 23 positions at the ends of theTec1 and Tec2 elements, 16 are universally or nearly univer-sally conserved among members of the two transposon families(Fig. 2C) (24). The ends of Tec3-1 are identical at all of thesepositions and are similar to the consensus sequences of Tec1and Tec2 at other, less conserved terminal positions (Fig. 2C).This region includes the first 9 bp (including the flanking 5�-TA-3� direct repeats), which are identical in all three elements.

Excision of Tec3-1 during macronuclear development. Pre-vious studies with both E. crassus and Paramecium suggestedthat the terminal sequences of IESs in these organisms play animportant role in specifying their developmental excision (re-viewed in references 14 and 20). Thus, the similarity of theTec3-1 termini to the ends of the Tec1 and Tec2 elementssuggested that Tec3-1 might be removed by the same excisionmachinery during macronuclear development. To test this pro-posal, two types of analyses were carried out.

First, we used a PCR procedure to determine whetherTec3-1 is excised from the RPL29 locus during the same de-velopmental period as that during which the Tec1 and Tec2IESs are removed. PCR was performed with total DNA iso-lated from cells at different stages of development and witholigonucleotide primers LRPL-4 and RFr29-1, which bind tosequences flanking the micronuclear copy of RPL29 (Fig. 1B).These primers will amplify the micronuclear copy of the RPL29locus with or without the Tec3-1 element up until the chromo-some fragmentation stage of macronuclear development butwill not amplify the macronuclear chromosome containing theRPL29 gene. PCR amplification was carried out with vegeta-tive whole-cell DNA and DNA isolated at various times after

mixing of cells of complementary mating types to initiate con-jugation and macronuclear development (Fig. 3A). Develop-ing-cell DNAs at 19 and 22 h yielded only a 5.3-kbp PCRproduct representing the micronuclear RPL29 locus containingthe Tec3-1 element. While the vegetative-cell and 16-h DNApreparations, which contained fewer copies of the micro-nuclear RPL29 locus, did not produce detectable amounts ofthe 5.3-kbp product in this analysis, we have observed theexpected products under different experimental conditions(Fig. 1A and data not shown). Beginning at 25 h, an 0.8-kbpband was also detected, representing the micronuclear RPL29locus lacking the Tec3-1 element (Fig. 3A). The 0.8-kbp signalincreased at 28 h, concomitant with the reduction of the largerPCR product representing the Tec3-1-containing forms. Theseresults indicate that Tec3-1 is excised from the RPL29 locus

FIG. 2. Dot matrix comparisons of Tec elements. (A and B) Dotplot matrix comparisons of Tec1-1 (GenBank accession no. L03359)and the Tec3-1 sequence (A) and of Tec1-1 and Tec2-1 (GenBankaccession no. L03360) (B). The comparisons were carried out by usingMacVector DNA sequence analysis software, with a window size of 30bases and a minimum score of 65%. (C) Comparison of the terminalsequence of Tec3-1 to the consensus terminal sequences of the Tec1and Tec2 transposons (24). The terminal 5�-TA-3� direct repeat isindicated in italic type, and positions that are identical in all elementsare underlined.


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during the early polytene chromosome stage of macronucleardevelopment, the same period during which excision of theTec1 and Tec2 transposon IESs begins (21, 34). To confirmthat Tec3-1 is indeed excised at the same time, Southern hy-bridization analysis of the same developing-cell DNA prepa-rations was carried out with a Tec1-specific probe. Free, extra-chromosomal circular and linear forms of Tec1 also were firstdetected at 25 h of development (Fig. 3B).

As a second means of assessing whether Tec3-1 excisionmight be mediated by the same machinery as that which re-moves the Tec1 and Tec2 transposon IESs, we characterized itsexcision product. Previous studies indicated that both E. cras-sus small IESs and Tec1 and Tec2 transposon IESs exist as freecircles following their developmental excision (21, 24, 32, 34,46). The junctions, or joining points, of these circles are un-usual: they contain two copies of the terminal 5�-TA-3� directrepeat separated by 10 bases that are derived from the macro-nucleus-destined DNA immediately flanking the IES. The cen-tral six bases of this junction region appear to be a heterodu-plex, with one DNA strand being derived from right flanking

sequences and the other being derived from left flanking se-quences (for models of the excision process, see references 24and 32). To determine whether Tec3-1 excision produces cir-cles with this unusual junction structure, PCR was performedwith primers that were complementary to regions 72 bp fromthe ends of the Tec3-1 element and that directed DNA syn-thesis toward the ends of the element (Tec3-1cirL and Tec3-1cirR; Fig. 1B). These primers should produce a PCR productof �150 bp only if the ends of the Tec3-1 element are joinedto form a circle following excision. Because Tec3-1 has in-verted repeats at its ends, the PCR product produced from thecircle junction will be palindromic. As such structures are dif-ficult to amplify by PCR, the two primers were chosen to becomplementary to a region of the Tec3-1 inverted repeats thatwas not perfectly complementary. PCR amplification of DNAisolated from developing macronuclei at 30 and 40 h produceda product of the expected size (Fig. 4A), while a control PCRwith vegetative-cell DNA produced no detectable product(Fig. 4A).

To further characterize the circle junction, the �150-bpPCR product was gel purified, reamplified by PCR, and di-rectly sequenced with oligonucleotide Tec3-1cirR as the se-quencing primer. The resulting sequencing chromatogram con-firmed that the PCR product was indeed derived from DNAmolecules in which the left and right ends of Tec3-1 werejoined and that the predicted type of circle junction waspresent (Fig. 4B) (note that circle junctions from other Tec3elements were not observed, presumably because the primerswere chosen to match divergent positions of the Tec3-1 in-verted repeats). The junction region contained a pair of 5�-TA-3� direct repeats separated by 10 bases. The central sixbases of the junction region were ambiguous in the chromato-gram; at each position, two peaks were present, a result thatone would expect if the PCR product were derived from asubstrate DNA molecule containing a heteroduplex region.The sequences of the six ambiguous positions can be inter-preted to indicate that two types of PCR products were gen-erated: one with the six bases derived from left flanking se-quences and the other with the six bases derived from rightflanking sequences (Fig. 4B and C). To demonstrate that thiswas the case, the PCR products were inserted into a plasmidvector, and four individual clones were isolated and sequenced.Two classes of clones were obtained. For two clones (Fig. 4Cand D, class I), the junction region could be viewed as beingformed by the joining of the eighth base distal to the right5�-TA-3� direct repeat of Tec3-1 to the second base upstreamof the left 5�-TA-3� repeat. For the other two clones, thejunction was such that the second base to the right of theelement was joined to the eighth base to the left of the elementto form the circle junction (Fig. 4C and D, class II). Theseresults parallel those obtained in similar analyses of the circlejunctions of E. crassus small IESs (32). While the results do notprove that a heteroduplex is present at the circle junction, thetwo observed classes of clones are consistent with a circlejunction in which the central six bases are a heteroduplex. Weconclude from these analyses that Tec3-1 is excised at the sametime of macronuclear development as Tec1 and Tec2 and thatit forms a free circular form with a structure that appears to beidentical to those of other excised IESs in E. crassus.

Identification and characterization of additional Tec3 ele-

FIG. 3. Developmental excision of Tec3-1. (A) Timing of Tec3-1excision from the RPL29 locus. Shown is a Southern blot containingthe PCR amplification products obtained from strain CT5 and CT27vegetative whole-cell DNAs and whole-cell DNAs isolated at varioustimes after mating of the CT5 and CT27 cell lines (lanes representing16, 19, 22, 25, and 28 h). The blot was hybridized with the 804-bp PCRproduct generated from the micronuclear RPL29 locus after Tec3-1excision with primers RFr29-1 and LRPL-4 (Fig. 1B). Hybridizationsignals at 5.3 kbp represent the RPL29 locus with the Tec3-1 element(�Tec3-1), while the 0.8-kbp signal represents the locus followingexcision (�Tec3-1). (B) Southern blot of the same undigested, whole-cell DNAs as those used as the substrates for PCR following hybrid-ization with a 1.6-kb HindIII/SalI restriction fragment derived from theTec1 region of clone PhCl.4 (see Fig. 7B). The Tec1 elements inmicronuclear DNA are indicated (Mic), along with the positions offree supercoiled circles (SC), relaxed circles (RC), and linear forms(L).


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ments in the micronuclear genome. Other studies were carriedout to determine whether additional copies of the Tec3 ele-ment were present in the micronucleus and to further charac-terize the element family. To assess the abundance of Tec3elements, we carried out a dot blot hybridization analysis (Fig.

5A). Hybridization filters were prepared with a known amountof E. crassus total cellular DNA as well as with various amountsof a cloned 2-kbp EcoRI fragment derived from a Tec3 ele-ment (Fig. 5B), which served as copy number standards. Thefilters were then hybridized with the same 2-kbp EcoRI frag-ment and washed at moderate stringency, and the amount ofbound radioactivity was determined. Repeated analyses of thistype indicated that there are only 20 to 30 copies of Tec3residing within the micronuclear genome. This number is sig-nificantly smaller that those of the E. crassus Tec1 and Tec2elements, which are each present at 5,000 to 7,000 copies perhaploid micronuclear genome (2, 21, 34). Southern hybridiza-tion of total cellular DNA with a variety of Tec3-derivedprobes indicated that all copies of the element are eliminatedduring the formation of the macronucleus (data not shown).

To obtain additional copies of Tec3, the E. crassus LEMICmicronuclear library (2) was screened with a hybridizationprobe derived from the core region of Tec3 elements. Thisscreening resulted in the identification of clone PhCl.4, whichcontains an �15.8-kbp insert harboring a Tec3 element. A9.2-kbp region of the clone containing the Tec3 element(Tec3-2) was subcloned and completely sequenced (Fig. 5B).Tec3-2 is quite similar to Tec3-1 but also displays two signifi-cant differences. The core region of Tec3-2 lacks a block of 171bp that is present in Tec3-1 (Fig. 5C) but is otherwise 68%identical in sequence. The second significant difference in theelements concerns their termini. Tec3-2 is bounded by 5�-TA-3� direct repeats, but its inverted repeats are only 980 bplong. The Tec3-2 inverted repeats are quite similar to theinnermost regions of the Tec3-1 inverted repeats, but the distal258 bp of the 1.23-kbp Tec3-1 inverted repeats are missingfrom Tec3-2 (Fig. 5C). As a result of these terminal trunca-tions, the ends of the Tec3-2 element show no significant sim-ilarity to the Tec1 or Tec2 terminal consensus sequence.

The Tec3-2 element also appears to differ from Tec3-1 inthat it does not reside within macronucleus-destined DNA.Southern hybridization analysis with the flanking regions ofTec3-2 as probes against total cellular DNA showed no stronghybridization to macronucleus-sized DNA (data not shown).Thus, Tec3-2 appears to reside within a segment of the micro-nuclear genome that is eliminated during development. Thisconclusion is further supported by the sequence analysis, whichidentified two incomplete copies of the Tec1 element adjacentto Tec3-2 (Fig. 5B). One copy of the Tec1 element appears tobe inserted within another (in inverse orientation), a phenom-enon previously observed for the E. crassus Tec2 element (34).Thus, the entire insert of clone PhCl.4 may represent part of alarge region of the micronuclear genome that is eliminatedduring development. Frels et al. (12) previously found thatmost Tec1 and Tec2 transposons in non-macronucleus-des-tined regions of the genome are both replicated and eliminatedlater in macronuclear development than are elements in ma-cronucleus-destined DNA. They suggested that such elementsmay not be excised as IESs but instead are removed in thecontext of other eliminated DNA. We suspect that this is thecase for Tec3-2, as its truncated inverted terminal repeats lackthe conserved termini that appear to be necessary for excisionas an IES.

Initial searches of the GenBank database with the core re-gions of the Tec3-1 and Tec3-2 elements indicated that they

FIG. 4. Characterization of excised Tec3-1 circles. (A) Southernblot containing the PCR amplification products obtained from strainX1 and X2 vegetative-cell DNAs and developing macronuclear DNAsisolated at 30 and 40 h after mating of the two strains with primersTec3-1cirL and Tec3-1cirR (Fig. 1). The blot was hybridized with aplasmid subclone containing a 0.98-kbp EcoRI restriction fragmentfrom the left end of clone Mic.RPL-29-2, which includes the left end ofthe Tec3-1 element. (B) DNA sequencing chromatogram from thebulk analysis of the 153-bp PCR product derived from the Tec3-1circles. The junction region, containing six ambiguous positions, isshown. See the description of panel C for an explanation of colors.(C) Sequences at the ends of the Tec3-1 element. The Tec3-1 sequenceis shown in red, and the left flanking and right flanking sequences thatwill form macronuclear RPL29 are shown in green and blue, respec-tively. The 5�-TA-3� repeats flanking the element are underlined, andthe brackets illustrate which bases are joined to form the two types ofPCR products obtained. (D) Sequences of the two classes of clonesobtained from PCR amplification of the Tec3-1 circle junction. Colorcoding is as described above, with double underlining indicating theproposed heteroduplex region of the circle junction.


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contain degenerate ORFs encoding proteins similar to thecatalytic domain of a number of prokaryotic tyrosine recom-binases (38). However, the analysis was complicated becausethe putative ORF regions encompassed the 171-bp region inTec3-1 that is absent in Tec3-2 (Fig. 5C) and by the presenceof multiple stop codons and possible frameshifts. Thus, tomore thoroughly address the potential coding functions ofTec3 elements, we sought to determine a consensus sequencefor the core region. Based on the sequences of the core regionsin Tec3-1 and Tec3-2, three pairs of oligonucleotide primerswere designed to amplify by PCR three overlapping segmentsof the core region (Fig. 5C). For each pair of primers, two tofour cloned PCR products were generated and sequenced.Each of the PCR products showed some differences in se-quence from the Tec3-1 and Tec3-2 elements and from eachother, indicating that they were derived from independent el-ements.

The PCR-generated cloned sequences are more similar toTec3-2 than to Tec3-1. For example, Fig. 6A shows a genea-logical analysis of the PCR clones generated with primers Int6and Int7 and the corresponding regions of Tec3-1 and Tec3-2.The average pairwise difference between Tec3-1 and the re-maining sequences, calculated from 465 overlapping base pairs(excluding gaps) by using the Tamura-Nei model (45) to cor-rect for multiple hits, is 0.193 0.004. This level of divergenceis reflected by the 65 fixed differences between Tec3-1- andTec3-2-like sequences (Fig. 6A). In contrast, the average pair-wise difference between the Int6-Int7 PCR-generated clonesand Tec3-2 is 0.054 0.018. All of the available sequence data

were used to derive a consensus sequence for the core regionof the Tec3 elements, which was used in subsequent databasesearches described below. This analysis also indicates that theextra 171 bp in Tec3-1 relative to Tec3-2 likely represent aninsertion, as all of the PCR-generated clones lacked this re-gion.

Tec3 elements encode a tyrosine recombinase. BLASTsearches of databases were performed with conceptual trans-lations of the core region consensus sequence to identify po-tential proteins encoded by the Tec3 elements. Only the leftend of the core region produced significant matches in theBLAST search, detecting numerous tyrosine recombinases(38). The best matches (e values of 10�4) were to homologsof the XerC and XerD proteins, which are involved in resolv-ing the products of DNA replication in a number of bacterialspecies. An alignment of the putative consensus Tec3 proteinwith the best matches in the databases is shown in Fig. 7. Theputative Tec3 protein displays significant sequence similarity tothe 180-amino-acid region which has been defined as the ty-rosine recombinase domain (amino acids 175 through 355 ofthe bacteriophage � Int protein [38]). The sequence similarityis primarily limited to the highly conserved box I and II regionsand the three more poorly conserved patch regions that aresignatures of the tyrosine recombinase family (Fig. 7). In ad-dition, the putative Tec3 protein contains the four box I and IIresidues (R-H-R-Y) that are almost universally conservedamong tyrosine recombinases and that participate in catalysis(38). While these results provide a strong indication that Tec3elements contain a tyrosine recombinase gene, it appears that

FIG. 5. Additional Tec3 elements in the micronuclear genome. (A) Determination of Tec3 copy number. An autoradiograph of a dot blothybridized with a 2-kbp EcoRI restriction fragment from Tec3-2 (see panel B) is shown. The blot contains 10 �g of E. crassus strain X2 total cellularDNA and amounts of the 2-kbp EcoRI fragment from Tec3-2 that were calculated to be equivalent to 10, 30, and 100 copies of the element percell (STDS.) (see Materials and Methods). (B) Map of a portion of the insert in recombinant clone PhCl.4. The Tec3-2 and Tec1 elements areshown as open rectangles, with arrows indicating inverted repeats. Grey regions represent other micronucleus-limited sequences flanking theelements. E, EcoRI; H, HindIII; S, SalI. (C) Comparison of the Tec3-1 and Tec3-2 elements, along with the positions of the three pairs of PCRprimers (Int6-Int7, F5-B5, and F4-B4) that were used to amplify additional segments of Tec3 from genomic DNA. The positions of the defectivetyrosine recombinase ORF (Y-recomb.) and two other long ORFs are indicated as arrows below the map. The 171-bp insertion within the tyrosinerecombinase ORF of the Tec3-1 element is indicated in grey.


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the gene is no longer functional in a significant fraction of theelements, as even the Tec3 consensus sequence has three stopcodons (Fig. 7).

Tyrosine recombinase genes are typically not found in eu-karyotic transposons. Nonetheless, a recent analysis indicatedthat the ORF2 regions of the Tec1 and Tec2 transposons alsoencode tyrosine recombinases (7a). As we have noted, there islittle shared sequence similarity between Tec3 and the Tec1 orTec2 element at the DNA level. The Tec3 tyrosine recombi-nase also appears to be distantly related to that of Tec1 orTec2, as the Tec1 and Tec2 proteins were not among the 50best matches (e values, 0.14) in the BLAST searches. Tofurther explore this issue, we carried out a genealogical anal-ysis of 26 proteins that represent the major subclasses of thetyrosine recombinase family and the tyrosine recombinases ofTec1, Tec2, and Tec3-1. Also included in the analysis were aconsensus sequence (conTec3-2) derived from the Tec3-2 ty-rosine recombinase region and the four related Int6-Int7 PCR-generated clones (Fig. 6A). The resulting genealogy revealsonly limited phylogenetic information from tyrosine recombi-nases from eukaryotic, bacterial, viral, and archaeal genomes,as evidenced by the short length of branches at deep nodes andthe relatively low bootstrap support at most nodes (Fig. 6B).There is strong support, as indicated by bootstrap values of�99%, for the sister relationship of the E. crassus Tec1 andTec2 ORF2 proteins and for the E. crassus Tec3-1 and con-Tec3-2 tyrosine recombinases in the NJ and MP analyses (Fig.6B). In contrast, the ML analyses provide limited support forthe sister status of Tec1 and Tec2 (PZ value, 68%) and ofTec3-1 and conTec3-2 (PZ value, 50%).

Although there is little support at deep nodes, there is noevidence that the diverse E. crassus Tec sequences form amonophyletic group. The NJ analysis of aligned box and patchamino acids places the E. crassus Tec1 and Tec2 sequences ina clade with integrase genes from the bacterium Haemophilusinfluenzae (Fig. 6B, Hinf Int) and the lambda and P21 phages(Fig. 6B, Lambda Int and P21 Int), although NJ bootstrapvalues are less than 50% for these relationships and thesenodes are unresolved in both the MP and the ML (puzzle)analyses. In contrast, the Tec3-1 and conTec3-2 sequences donot appear to be closely related to any known tyrosine recom-binase (Fig. 6B). The results of a Kishino-Hasegawa test (26)that compared the most parsimonious tree with one constrain-ing all of the Tec elements to be monophyletic were not sig-nificant, indicating that we cannot reject a single origin for E.crassus Tec tyrosine recombinases based on these analyses.The only other well-supported nodes in the genealogy, basedon bootstrap and PZ support values of �70%, are for tworecombinase genes isolated from cyanobacteria (Fig. 6B, Nos-toc XisA and Anab XisC), two phage integrase genes (Fig. 6B,Lambda Int and P21 Int), E. coli and V. cholerae XerD se-quences, and a large clade containing diverse XerC, XerD, andXerC- or XerD-like proteins from bacteria and archaea. Anal-yses of the highly conserved box regions alone or of alternativealignments had no significant impact on the resulting geneal-ogy (data not shown). There is a relatively high divergence(�19%, excluding gaps) between the Tec3-1 and conTec3-2tyrosine recombinase sequences, indicating that these twoclasses of sequences have coexisted within the genome of E.crassus for quite some time (Fig. 6B).

FIG. 6. Genealogical analyses. (A) Genealogy of Tec3-1, Tec3-2,and four cloned PCR products generated with primers Int6 and Int7(Int6/Int7.2-Int6/Int7.5), determined by NJ analysis of nucleotide se-quences, excluding gaps. Numbers above branches represent NJ boot-strap support values; branches with less than 50% bootstrap supportare not labeled. Branch lengths are proportional to the numbers ofchanges, and the numbers of unambiguous changes are indicated be-low branches in italic type. (B) Genealogy of tyrosine recombinases,determined by NJ analysis of amino acid sequences. Numbers abovebranches represent bootstrap support values estimated by NJ and MPanalyses and PZ support values, in that order. Branches with less than50% bootstrap support are not labeled. Branch lengths are propor-tional to the numbers of changes in each branch. GenBank numbersand further details on algorithms and models are described in Mate-rials and Methods. For the alignment of the proteins and regionsincluded in the analyses, see the supplemental figure at www.science.smith.edu/departments/Biology/lkatz/align.html.


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While the remainder of the Tec3 core region generated nosignificant BLAST matches, other coding regions are likely tobe present. Two relatively long regions that are uninterruptedby stop codons are present in the core region consensus se-quence (ORF2 and ORF3 in Fig. 5C; bases 3310 to 2641 and3034 to 3657 in Mic.RPL-29-2, respectively). ORF3 would betranscribed in the orientation opposite that of the tyrosinerecombinase-coding region and so might encode a second pro-tein. ORF2 would be transcribed in the same direction as thetyrosine recombinase-coding region, but it is in a differentreading frame. As a result, ORF2 might also encode a separateprotein. However, many tyrosine recombinases have N-termi-nal regions that are not conserved among family members, andit is possible that ORF2 represents such an N-terminal regionthat has been separated from the Tec3 tyrosine recombinase bya frameshift mutation or by the presence of an intron.

DISCUSSION

In this study, we have identified a small family of trans-posons in E. crassus that differ significantly from the previouslyidentified Tec1 and Tec2 elements. One copy of the Tec3family (Tec3-1) interrupts the RPL29 locus. Tec3-1 is 4.48 kbp

long and has 1.23-kbp terminal inverted repeats. A secondTec3 family member (Tec3-2) resides in a region of the ge-nome that contains Tec1 elements. Tec3-2 shares a �1.9-kbpcore region with Tec3-1 but lacks 258 bp of the inverted ter-minal repeats. Which of these two end structures is generallycharacteristic of the Tec3 element family is unclear at present,as we have been unable to isolate additional copies of elementtermini by using a number of PCR-based procedures. All cop-ies of the element family are eliminated during the process ofmacronuclear development.

The Tec3-1 element residing within the micronuclear RPL29locus behaves as an IES. It is excised during the early polytenechromosome stage of macronuclear development, the sameperiod during which removal of the Tec1 and Tec2 IESs begins(21, 34). In addition, the results indicate that Tec3-1 excisionproducts are likely identical to those of other Euplotes IESs.Tec3-1 is precisely excised, resulting in an RPL29 macro-nuclear DNA molecule that retains one copy of the terminal5�-TA-3� direct repeat of the element and forms a circle fol-lowing excision. Moreover, the data indicate that the Tec3-1element forms a circle following excision which appears tohave the same type of heteroduplex junction structure as otherE. crassus IESs. These results are significant in that Tec3-1

FIG. 7. Comparison of the Tec3 tyrosine recombinase consensus sequence with the XerC- or XerD-like proteins of Methanothermobacteriumthermoautotrophicum (Methano.; GenBank accession no. 7428936), L. leichmannii (Lactobac.), S. aureus (Staphyloc.), R. prowazekii (Rickettsia),and V. cholerae (Vibrio). The alignment was obtained by using ClustalW, but with some manual adjustments. Positions which are identical in themajority of the proteins are shaded. The conserved box and patch regions characteristic of the tyrosine recombinase protein family (38) are boxed,and the nearly universally conserved residues involved in catalysis are indicated by arrows.


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shares little sequence similarity with the Tec1 and Tec2 ele-ments, except for its termini, where 16 of 23 bp are identical toTec1 and Tec2. These terminal sequences, which are also sim-ilar to the consensus sequences for the E. crassus short IESs,Paramecium short IESs, and the Tc1/Mariner transposons (17,28), have been suggested to play a major role in specifying IESexcision. As the termini are the only discernible feature sharedamong the E. crassus IESs, the similarity in the timing andproducts of Tec3-1 excision provides additional evidence thatthese sequences play a major role in specifying IES removal.

The results also indicate that the IES terminal sequence maybe a bipartite structure. In a comparison of the ends of thethree E. crassus transposons, a terminal conserved block of 9bp is separated from a second conserved block of 6 bp by anAT-rich 8-bp region (Fig. 2C). The two shared blocks repre-sent nearly universally conserved regions of members of theTec1 and Tec2 element families (24), arguing that they play arole in some function important for the elements. This bipar-tite structure is reminiscent of the V(D)J recombination signalsequences (RSS) that specify gene rearrangement in the ver-tebrate immune system. The RSS are comprised of a conservedheptamer and nonamer that are separated by either 12 or 23 bp(reviewed in reference 13). Intriguingly, a number of lines ofevidence suggest that the V(D)J recombination system evolvedfrom a transposable element (reviewed in reference 13), andsome similarity between the RSS heptamer and the termini ofthe Tc1/Mariner family of transposons has been noted (8).

Tec3 tyrosine recombinase gene. The Tec3 elements containa defective ORF that appears to have encoded a tyrosinerecombinase protein. Tyrosine recombinases are a broad fam-ily of proteins that carry out a variety of functions, includingviral integration, DNA inversion, transposition, and the reso-lution of catenated DNA circles (see, e.g., reference 38). All ofthe analyzed Tec3 ORFs are defective by virtue of the presenceof indels and/or stop codons, making it unlikely that the pre-dicted tyrosine recombinase protein plays a role in the IESexcision process at present. It seems more likely that it wasinvolved in the transposition of Tec3 elements. Resolvasegenes are found in prokaryotic elements that transpose via areplicative mechanism (reviewed in reference 15). The re-solvases are usually members of the distinct family of serinerecombinases, but some elements encode a tyrosine recombi-nase that functions as a resolvase (see, e.g., reference 36).During replicative transposition, a cointegrate structure formsthat links the transposon donor DNA with the target DNA.The resolvase carries out site-specific recombination betweensites within the two transposon copies to resolve the donor andtarget DNAs. Resolvase genes have not been identified yet ineukaryotic transposons. As discussed by Doak et al. (7a), theresolution of replicative transposition products in eukaryoticgenomes with multiple linear chromosomes and multiple trans-poson copies would likely give rise to a variety of deleteriouschromosome rearrangements, unless a mechanism existed thatrestricts recombination to the donor and target copies of thetransposon. Thus, while it is possible that the Tec3 tyrosinerecombinase served as a resolvase, a replicative transpositionmechanism does pose some problems.

An alternative is that the Tec3 tyrosine recombinase wasinvolved in the initial steps of transposition. A tyrosine recom-binase protein mediates the excision and transposition of the

Tn916 and Tn1545 conjugative transposons (49), so that it isconceivable that the Tec3 tyrosine recombinase played a sim-ilar role in the multiplication of this element. Intriguingly, theexcised circular form of Tn916, which is believed to be a trans-position intermediate, has a heteroduplex region at its junctionthat is derived from the sequences originally flanking the ele-ment (6). Although the structure of the Tn916 circular junctiondiffers somewhat from that of the E. crassus circular small IESsand Tec transposons, these are the only examples that we areaware of in which excised circular forms of elements haveheteroduplex junctions.

Our genealogical analysis indicates that the Tec3 tyrosinerecombinase is not closely related to those of the Tec1 andTec2 transposons. Moreover, the phylogenetic analyses failedto provide strong support for the association of the Tec3 ty-rosine recombinase with any of the functionally distinct sub-families of tyrosine recombinases. The possibility of elevatedmutation rates in E. crassus Tec3 sequences is likely part of theexplanation for the lack of resolution in our genealogies. Al-though these analyses did not provide insight into the functionof the Tec3 protein, it is clear that it is more closely related toprokaryotic members of the group than to eukaryotic members(e.g., the yeast FLP recombinase proteins), which differ signif-icantly in sequence (9, 38). In fact, the yeast sequences are sodivergent that they could not be unambiguously aligned withthe prokaryotic, viral, and ciliate sequences and were not in-cluded in our analyses. The high level of divergence betweenciliate Tec tyrosine recombinases and other eukaryotic tyrosinerecombinases may be an indication of lateral transfer of Tecelements from a prokaryote or virus to Euplotes. In nature,ciliates meet their nutritional requirements by engulfing othermicroorganisms, including prokaryotes, and it has been sug-gested that such food organisms might be the source of at leastsome ciliate transposons (29).

Possible origins of multiple developmentally excised trans-poson families. Our studies establish the Tec3 element as an-other family of sequences that undergo developmental elimi-nation in E. crassus, with at least one family member (Tec3-1)behaving as an IES. The previously described Tec1 and Tec2elements behave in a similar manner, raising the question ofhow these various elements are related. The Tec1 and Tec2elements are very similar elements, so that these families mayhave diverged from one another following the invasion of themicronuclear genome by an ancestral element. However, thisputative ancestral element is unlikely to have also given rise tothe Tec3 element. The Tec3 element shares little sequencesimilarity at the DNA level with the Tec1 and Tec2 elementsand appears to lack a gene encoding a Tc1/Mariner-like trans-posase, which both Tec1 and Tec2 possess (7). Furthermore,even though all of the elements have a tyrosine recombinaseORF, our analyses indicate that the Tec1 or Tec2 protein isdistantly related to the Tec3 protein. Thus, we suggest that theTec3 element independently invaded the E. crassus micro-nuclear genome.

If so, how have members of these different element familiescome to be subject to the same developmental excision system?It was recently suggested that ciliate IES removal represents ameans of ridding the genome of repetitive DNA elements,possibly serving as a defense against the deleterious events oftransposable elements (20, 37, 52). It was also proposed that


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transposable elements were responsible for establishing theexcision system, an event which would serve to enhance theirsurvival in the host (29). Whatever the origin and purpose ofthe excision system, it appears to be a sequence-specific pro-cess in E. crassus, as the majority of IESs share similar termini.

Scenarios for the origin of the Tec3 element in the E. crassusmicronucleus need to explain the two types of terminal struc-tures observed for the Tec3-1 and Tec3-2 family members.While there are a number of possible explanations, one inter-esting possibility is that some Tec3 elements acquired theirterminal sequences from Tec1 or Tec2 by an “end-swapping”process (Fig. 8). In this scheme, the original Tec3 element thatentered the micronuclear genome possessed the shorter in-verted terminal repeats of Tec3-2. This original element wouldhave been under the selective constraints typical for trans-posons in most organisms, as it could not be developmentallyexcised. However, during limited expansion in the micro-nuclear genome, a Tec3 element might have inserted within aTec1 or Tec2 element. Insertions of transposons within trans-posons are found in many organisms. Indeed, in the micro-nuclear clone harboring Tec3-2, an insertion of one Tec1 ele-ment into another was found (Fig. 5B), and Krikau and Jahn(34) previously described a Tec2 transposon residing withinanother. Once such an insertion of a Tec3 element into Tec1 orTec2 occurred, sequence deletions may have resulted in acomposite element primarily composed of Tec3 sequences butwith Tec1 or Tec2 termini. By virtue of acquiring Tec1 or Tec2termini, this composite element would now be subject to theIES excision process, enhancing its chances of survival in thehost genome. In addition, since terminal sequences are typi-cally the cis-acting sequences required for transposition, end-swapping may also have allowed the Tec3 element to transpose

by using Tec1 or Tec2 transposase in trans. This transposonend-swapping hypothesis is attractive in that it provides astraightforward means for disparate transposons to fall underthe same system of developmental removal.

ACKNOWLEDGMENTS

We thank Tom Doak and Glenn Herrick for assistance in the align-ment of tyrosine recombinase proteins and for helpful discussions.

This work was supported by National Science Foundation grantMCB-9816765 to L. A. Klobutcher and a National Science FoundationCAREER award to L. A. Katz.

REFERENCES

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Tec3, a New Developmentally Eliminated DNA Element in Euplotes … · Tec3, a New Developmentally Eliminated DNA Element in Euplotes crassus Mary Ellen Jacobs,1* Adolfo Sa´nchez-Blanco,1