a new non-ltr retrotransposon provides evidence for multiple

1995 Oxford University Press Nucleic Acids Research, 1995, Vol. 23, No. 15 2929-2936

A new non-LTR retrotransposon provides evidencefor multiple distinct site-specific elements inCrithidia fasciculata miniexon arraysShu-Chun Teng, Sharon X. Wang and Abram Gabriel*

Department of Molecular Biology and Biochemistry, Rutgers University, Piscataway, NJ 08855, USA

Received April 17, 1995; Revised and Accepted June 23, 1995 GenBank accession no. U19151

ABSTRACT

We have Identified a new member of the family oftrypanosome site-specific retrotransposons, using adegenerate ollgonucleotide PCR strategy. The 9595 bpelement, termed Crithidia retrotransposable element 2(CRE2), was cloned and found to be inserted in thetandemly arrayed miniexon genes of Crithidia fascicu-lata. The element is flanked by 29 bp target siteduplications but lacks the 3' poly dA tract characteris-tic of most other non-long terminal repeat retrotran-sposons. The amino terminal region of the single2518-codon open reading frame contains a putativemetal-binding motif and a prollne-rich region similar togap-like domains of other retrotransposons. Thecarboxy terminal region of this open reading frameshares sequence homology with the reverse transcrip-tase and putative endonuclease regions of threepreviously described trypanosomatid site-specific re-trotransposons. All four of these retrotransposons arespecifically inserted between nucleotides 11 and 12 ofthe highly conserved 39mer sequence of the miniexongene. Most copies of CRE2 and the previously char-acterized CRE1 are located on different sized chromo-somes. Additional CRE-related sequences wereidentified by screening Crithidia libraries. These re-sults suggest that a particular sequence in the C.fasci-culata miniexon repeat is the target for multiple distinctsite-specific retrotransposon insertions.

INTRODUCTION

Retrotransposons are a ubiquitous class of mobile geneticelements. Like retroviruses, retrotransposons encode reversetranscriptases (RT) and replicate via RNA intermediates (1). Onthe basis of their structural organization and the amino acidsimilarities of their encoded RTs, two broad classes of retro-transposons can be distinguished (2,3). One class, typified by theTy elements of the yeast Saccharomyces cerevisiae (4), character-istically contains long terminal repeat (LTR) sequences similar toretroviruses and is thought to replicate via a retrovirus-likestrand-transfer mechanism. The second, more heterogeneous

class has been termed non-LTR retrotransposons and are typifiedby the widely dispersed LINE elements of mammals (2,5). Theseelements lack LTRs, but usually contain variable length terminalstretches of A residues (poly dA tracts) at their 3' ends as well asvariable length target site duplications. Two of these elementshave been shown to transpose via an RNA intermediate (6-8) andfour different elements have been demonstrated to encode RTactivities (9-12). Although most of the elements in this class arewidely dispersed in their respective host genomes (5), twofamilies of site-specific non-LTR retrotransposons have beenidentified, both targeting conserved sequences in tandemlyarrayed genes. In most insects, a fraction of the 28S rRNA genesare interrupted, at specific sites, by Rl and R2 elements (13). ForR2Bm, from Bombyx mod, an element encoded endonuclease(EN) activity and an associated RT activity have been identifiedin vitro and a novel mechanism for initiating reverse transcriptionat the target site has been demonstrated (12).

The genomes of all trypanosomes contain 200-600 copies ofthe tandemly repeated miniexon (or spliced leader) genes whichcode for highly conserved small RNAs that are essentialsubstrates for mRNA rrans-splicing (14). In three species oftrypanosomes, non-LTR retrotransposons have been found tointerrupt a fraction of the miniexon genes [reviewed by Aksoy(15)]. These elements [CRE1 in Crithidia fasciculata (16),SLACS in Trypanosoma brucei{\l) and CZAR in Trypanosomacruzi (18)] are all inserted into the miniexon genes betweennucleotide 11 and 12 of the highly conserved 39mer miniexoncoding sequence. Further, an RT activity encoded by the CRE1open reading frame (ORF) has been demonstrated using a yeastexpression system (9). While the ORFs of the three miniexonassociated retrotransposons show limited sequence identity,phylogenetic analysis has shown that they share a commonancestor, and that this family is at the periphery of the non-LTRretrotransposon tree (3). Given the ubiquity of miniexon genes intrypanosomes, the ancient origins of these organisms (19) and thestriking site-specificity of these three elements, it is of interest toexamine the distribution and genomic organization of this familyof trypanosome site-specific retrotransposons.

In this paper, we describe the identification and analysis ofCRE2, a second site-specific non-LTR retrotransposon foundwithin the miniexon arrays of the insect parasite C.fasciculata.While CRE2 shares 43% identity at the amino acid level with

To whom correspondence should be addressed

Downloaded from https://academic.oup.com/nar/article-abstract/23/15/2929/1111929by gueston 10 February 2018

2930 Nucleic Acids Research, 1995, Vol. 23, No. 15

Consensus

cu3..CREl..SLACS. .CZAR..CRE3R2Bm. .LI.2..

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Figure 1. Augnment of the predicted amino acid sequence of the CRE2 RT region with those of the three completely sequenced trypanosome non-LTRretrotransposons (CREl, SLACS and CZAR) and two typical non-LTR retrotransposons [R2Bm, a 28S rDNA specific element from B.mori (40), LI 2, a dispersedelement from Homo sapiens (41)]. Partial sequence information from CRE3 is also included. Conserved residues amongst the trypanosome non-LTRretrotransposons are shown above the alignment Identical residues in all of these non-LTR retrotransposons are in bold print. The residues used to design thedegenerate PCR primers, RAG 19 (plus strand) and RAG 20 (minus strand), are designated by arrows. The numbers to the left of each sequence refer to the numberof residues from the amino terminus of the ORF containing the RT region.

CREl in its RT domain and inserts at precisely the same locationas CRE1, its size and structure are quite distinct CRE1 and CRE2appear to be part of a larger family of site-specific non-LTRretrotransposons in C.fasciculata that have invaded their host'sminiexon array.

MATERIALS AND METHODS

Crithidia culturing and preparation of nucleic acids

In this paper, C.fasciculata refers to American Type CultureCollection (ATCC) strain 50083, unless otherwise specified.Crithidia fasciculata (Paul Englund) was the gift of Dr PaulEnglund (Johns Hopkins School of Medicine) and was used in ourprevious study (16) to isolate CREl. The following Crithidiastrains were purchased from ATCC: 11745,12857,12858,30251,30254, 30255, 30256, 30258, 30817, 30818, 30862, 30969,50083, 50118, 50211. Crithidia culturing and preparation ofnucleic acids were carried out as previously described. Crithidiaclones were prepared by plating diluted samples from liquidcultures onto brain heart infusion (Difco) nutrient agar plates, aspreviously described (16). Since C.fasciculata (ATCC 50083) isa cloned strain, this cloning procedure is equivalent to thesubcloning procedure used in our previous study (16).

Primers and degenerate PCR strategy

A pair of degenerate oligonucleotides were designed on the basisof conserved amino acid sequences in the RT regions of CREl,SLACS and CZAR (Fig. 1). The underlined portions of the two

degenerate primers, RAG 19 (5'-GGAATT CCN GGN (T/OTNGA(T/O GGN TGG AC-3") and RAG 20 (5'-GCTCTAGAGCC NA(A/G1 NAC CAT NCC (T/OTG-3^ corresponded tonucleotides 1743-1762 and 2286-2303 of CREl (16),4383-4402 and 4920-4937 of SLACS (17) and 4736-4755 and5273-5290 of CZAR (18). EcoRl and Xbal sites were placed atthe respective primer 5' ends to facilitate cloning of the PCRproduct. A standard PCR reaction contained 50 pmol of eachprimer and 1 u.g of Crithidia genomic DNA in a final volume of25 \L\ (20). The initial cycle was denaturation at 95°C for 1 min,annealing at 37°C for 2 min and subsequent synthesis at 72°C for3 min. The following 29 cycles consisted of denaturation at 95°Cfor 1 min, annealing at 50°C for 2 min and subsequent synthesisat 72°C for 3 min.

Cloning methods

The PCR products were cloned into EcoRl and Xbal digestedpBluescript IIKS+ (pBSKS) (Stratagene). All Crithidia librarieswere constructed by ligating gel-purified, size-selected restrictionenzyme digested Crithidia genomic DNA into correspondinglyrestriction enzyme digested and alkaline phosphatase-treatedpBSKS. After transformation into Escherichia coli strain TGI orJS5, colonies were lifted onto nitrocellulose filters (Schleicherand Schuell) and screened with appropriate probes. pBSCRE2-3'was isolated from a 6.7 kb size-selected ///VidlU-digestedCrithidia DNA library hybridized with the 32P-labeled CRE2PCR product probe (Fig. 3, probe A). pBSCRE2-y4/xzI wasisolated from a 3.5 kb size-selected Apal-digested Crithidia DNA


Nucleic Acids Research, 1995, Vol. 23, No. 15 2931

library hybridized with probe B (Fig. 3) obtained frompBSCRE2-3'. pBSCRE2-5' was isolated from a 3.4 kb size-selected //indlll-digested Crithidia DNA library hybridized withprobe C (Fig. 3) obtained from pBSCRE2-/\paI. pBSCRE2FLwas isolated from a 10 kb size-selected fispEI-digested CrithidiaDNA library hybridized with probe D (Fig. 3) obtained frompBSCRE2-3'.

Hybridization, wash conditions and probe preparations

Hybridizations were carried out at as previously described (16)and consisted of three 20 min incubations of filters in 0.1 x SSC,0.1% SDS at 65°C. Hybridization probes were prepared by gelisolating restriction enzyme digested or PCR amplified DNAfragments and labeling them by the random primer method (21).The CRE2 fragments used for probe preparation are shown inFigure 3. An 828 bp EcoRV-EcoRV fragment from CRE1spanning nucleotides 1002-1830 (16) was used to prepare theCRE1 probe. The miniexon probe was prepared from a 198 bpPCR product spanning nucleotides 181-378 (22).

1 2 3 4 5 6 7 8 9 10 11 12

kb

23 •9 . 4 -

6.6 •

4.4-

2 . 3 -2 . 0 -

1.35-

0.6-

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Sequencing strategy

The complete DNA sequence of CRE2 was obtained by firstsubcloning the 2.3 kb HindUl-Clal, the 3.1 kb KpnlSacl, the3.0 kb Clal-EcoKl and the 1.4 kb £coRI-//<>idIII DNA restric-tion fragments from pBSCRE2-3' (Fig. 3) into the multiplecloning site of pBSKS. A series of bidirectional nested deletionsof these subclones as well as of pBSCRE2-5', was constructedusing exonuclease III (Erase-a-Base; Promega) according to themanufacturer's directions. Dideoxy sequencing reactions wereperformed using cycle sequencing protocols recommended byBethesda Research Laboratory (BRL). All of the subclonejunctions and sequence gaps were sequenced using a variety ofspecifically synthesized internal oligonucleotides (Applied Bio-systems 392 DNA/RNA synthesizer) as primers. All parts of the10 kb element were sequenced completely on both strands.

Copy number estimation

Southern blot analysis of Aval digested Crithidia genomic DNAwas performed using the miniexon probe. The signal intensitiesof the unique sized Aval fragments corresponding to the miniexonrepeat, the 5' end of CRE1 and the 5' end of CRE2 werequantitated by phosphorimage analysis. Relative band intensitiesprovide an estimate of the percentage of miniexon repeatsinterrupted by each element This fraction was multiplied by thepreviously derived copy number of miniexon repeats (22,23) togive an estimate of the CRE1 and CRE2 copy numbers.

CHEF gel electrophoresis

Crithidia cultures were grown until late log phase (2-5 x 107/ml).Crithidia chromosomal blocks were prepared by mixing equalvolumes of Crithidia cells with 1% low melting agarose (FMCBioProducts) at a final concentration of 109 cells/ml as previouslydescribed (16). Pulsed-field gel electrophoresis was performedusing the contour-clamped homogeneous electric field-dynami-cally regulated CHEF-DR III system (Bio-Rad). Chromosomeswere separated on a 1% agarose gel in 0.5x TBE buffer (45 mMTris, 45 mM boric acid, 1 mM EDTA, pH 8.3) at 14°C for 43.7 h

Figure 2. Southern blot analysis of the genomic organization of CRE2 withinC.fasciculata. For each lane, 3 |ig of genomic DNA was digested withrestriction enzymes that either cut once (HindHI, lane \;Ava\, lane 2; Smal,lane 3; Styl, lane 4) or not at all (PvuW, lane 5; Sspl, lane 6; Accl, lane 7; AflU,lane 8; Apa\, lane 9; ApaU, lane 10; BamYU, lane 11; Ban\, lane 12) within theminiexon gene repeat, separated on a 0.7% agarose gel and transferred to anylon filter (GeneScreen Plus, Du Pont). The filter was hybridized with theCRE2 PCR product probe. Size markers (kb) are shown at the left.

at 6.0 V/cm (200 V) using a 120°linear switch time ramp.

Computer analysis

included angle with a 80-104 s

Sequences were compiled using Geneworks (IntelliGenetics Inc.)and analyzed using programs in either the Genetics ComputerGroup (GCG) (24) or Geneworks package. Database searcheswere performed via the National Center for BiotechnologyInformation (NCBI; Bethesda, MD) blast network service.

Nucleotide sequence accession number

The sequence of CRE2 reported here has been given GenBankaccession no. U19151.

RESULTS

Identification of a PCR product homologous to the RTdomains of trypanosome site-specific retrotransposons

We designed a pair of degenerate oligonucleotides based on twohighly conserved blocks of amino acids in the RT regions ofCRE1, SLACS and CZAR, and separated by 188 amino acids(Fig. 1). In control experiments, the primer pair successfullyamplified an -580 bp fragment using either genomic DNA fromC.fasciculata (Paul Englund) or cloned copies of CRE1, SLACSand CZAR (data not shown). We then used the primer pair toamplify DNA from a variety of Crithidia strains. Although mostreactions resulted in a smear of bands, a particularly strong bandat -580 bp was observed using DNA from Crithidia species



ME Tn An

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P1

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1kb

Probes

DNA

Protein

Figure 3. Structure and restriction map of CRE2, derived from sequence data. The solid lines (A-F) correspond to the restriction fragments used as probes as follows:A, the PCR fragment; B, 339-bp CRE2-/y;ndIII-Kp/iI fragment from pBSCR£2-3'; C, 2.2 kb CRE2^aI-tfmdIII fragment from pBSCRE2-/tpaI; D, 1.6 kbCRE2-Nsil-Nsi\ fragment; E, 1.2 kb CRE2-Pst\-Psl\ fragment; F, 375 bp CRE2-EcoRl-Dral fragment. CRE2 is flanked by miniexon (ME) genes (hatched boxes)and 29 bp target site duplications ( • ) at both ends. Poly dA (An) and poly dT (Tn) runs are found in the 5' and 3' untranslated regions. The location of the PCR productused to clone the 3' portion of CRE2 is marked (Q). The location of the single long CRE2 ORF is indicated below the restriction map and the position of the potentialgag-like, EN-like and RT domains are shown. Restriction sites used for cloning and probes are as follows: A, Apa\; B, BspEl; C, Cla\; D, DraV, E, EcoRl; H, Hindlll;K, Kpnl; N, Nsil; P, Pstl; S, Sad; X, Xbal.

ATCC 50083. (This strain has since been redesignated C.fascicu-lata based on comparative restriction fragment length poly-morphism analysis of its 18S rDNA gene region; L. S. Diamondand C. G. Clark, pers. comm.). We cloned this PCR product anddetermined the sequence of three independent clones. Thesequence of one clone was identical to CRE1. The two otherclones were identical to each other and clearly related to CRE1,SLACS and CZAR (Fig. 1). We therefore postulated that the PCRproduct came from a related retrotransposon, which we havedubbed Crithidia retrotransposable element 2 (CRE2).

Cloning of the 10 kb element from Crithidia genomicDNA libraries

Crithidia fasciculata (ATCC 50083) was used for all furtheranalyses. DNA was digested with a variety of restriction enzymesand hybridized with the CRE2 PCR product probe. Thehybridization pattern was complex, usually with one predominantband and several minor bands present in each lane (Fig. 2). Thisfinding suggests that the CRE2 PCR probe cross-hybridizes withadditional genomic sequences, even at the high stringency that weemployed. Of note, none of the observed bands matched thepredicted restriction map for CRE1.

In order to clone a full-length CRE2 element, we focused firston the 6.7 kb Hindlll fragment (Fig. 2, lane 1). We reasoned thatif CRE2 was site-specifically inserted into the miniexon array, the6.7 kb fragment might include flanking miniexon sequences,since a single HindlU site is present in each C.fasciculataminiexon gene repeat. We obtained multiple positive clones byscreening a size-selected library from Crithidia genomic DNAwith the CRE2 PCR product as a probe. Upon sequencing one ofthese clones, we found that the 6.7 kb fragment contained a longORF extending from the 5' end of the fragment through -80% ofits sequence and including the previously sequenced PCRamplified region. The 3' end of the fragment consisted of 42 basesof the C.fasciculata miniexon gene. We designated these clonesas pBSCRE2-3' (see Materials and Methods).

Based on this sequence data we reasoned that CRE2 is indeedassociated with the miniexon array but contains an internalHindlU site. To obtain the 5' end of the element, we first isolated

overlapping clones from a 3.5 kb Apa\ fragment Crithidiagenomic library (pBSCRE2-A/?aI clones). The 5' end of theseclones, as well as a miniexon-specific probe were used to screena 3.4 kb Hindlll fragment Crithidia genomic library. Wesequenced the entire 3.4 kb Hindlll fragment from a single cloneand found that it contained an ORF extending through -65% ofits sequence to the 3' end of the fragment. Its 5' end sequence wasnearly identical to the previously sequenced miniexon gene ofC.fasciculata (22). The ORF within the 3.4 kb fragment wascontiguous with the ORF within the 6.7 kb fragment. Thestructure and relevant restriction map of the entire cloned CRE2element is shown in Figure 3.

In order to obtain a full-length element, we took advantage ofthe fact that the miniexon genes of C.fasciculata contain a uniqueBspEl site (22) which is not found in CRE2 (Fig. 3). We thereforeselected eight copies of full-length 10 kb CRE2 from asize-selected BspEl Crithidia genomic library. To determine ifour composite sequence differed from the full-length clones at thelevel of restriction fragment length polymorphisms (RFLP), wedigested all of the clones with eleven restriction enzymes andcompared the results to our sequence and to each other. All cuttingsites were consistent with the sequence and no RFLPs were foundamongst individual clones (data not shown).

Genomic library screening reveals additional CRE-likeclones

In the course of screening the several size-selected Crithidiagenomic libraries required to obtain full-length CRE2, multipleindependent clones were selected and characterized by theirrestriction enzyme digestion pattern. Two of the sevenpBSCRE2-/lpaI clones had restriction patterns which differedfrom the others. Sequence data from a 197 base region justupstream of the Apal cloning site in these two clones indicatedthat they were identical to one another but only 93% identical tothe CRE2 sequence at the nucleotide level. All of the changeswere simple base substitutions and none interrupted the openreading frame.

While analyzing the initial pBSCRE2-3' clones, we identifiedan additional distinct element which had a completely different



23-1

9.4-

6.6-

4.4-

Probe:

3 4 5 6 7 8 P 1 2 3 4 5 6 7 8 P I 2 3 4 5 6 7

CREl CRE2 5' CRE2 3'

Figure 4. Clonal instability of the CREl and CRE2 restriction patterns. Three micrograms of DNA from each clone was digested with EcoRi, fractionated through0.4% agarose, transferred to a nylon filter and equivalent filters were hybridized with (A) a CREl probe that spans the single CREl EcoRi site; (B) a CRE2 5' probe(probe E in Fig. 3); or (C) a CRE2 3' probe (probe F in Fig. 3). Size markers (kb) are shown at the left. Lanes: P, the parental stock; 1-8, individual clones. The arrowand arrowhead point to specific bands demonstrating linkage of CREl and CRE2 copies (see text for details).

set of restriction enzyme digestion patterns compared with theothers. In order to determine if this represented a relatedretrotransposon or an unrelated but cross-hybridizing sequence,we attempted to amplify the cloned copy using our degeneratePCR primers. The resulting 580 base PCR product was clonedand sequenced. The predicted amino acid sequence of thisfragment was 40% identical to CREl and 47% identical to CRE2over a 191 amino acid stretch. We have tentatively named thiselement CRE3 (Fig. 1). These observations indicate that theC.fasciculata genome is home to several related retrotransposonswhich may account for the lighter bands noted in the Southernblot in Figure 2. Of note, the size of the strongest band in each laneof Figure 2 is consistent with the derived sequence of CRE2.

The 10 kb element shows features of a site-specificnon-LTR retrotransposon

Features of CRE2 are presented in Figure 3. The total length of thesequenced region is 10 053 base pairs. Miniexon gene repeatsequences are present at both ends. The nucleotides 1-416represent a nearly exact copy of the previously determined miniexongene repeat from nucleotide 1 to 416. Nucleotides 10 012-10 053consist of a duplication of the 3' 29 nucleotides of miniexonsequence, followed by the next 13 nucleotides in the miniexongene repeat. Thus, as with the previously characterized CRE1 (16),the inserted element is accompanied by 29 base target siteduplications, beginning at base 11 of the miniexon sequence (22).

From nucleotides 416-10 010 is a 9595 bp region with nohomology to the miniexon. A single long ORF, predicted toencode a 2518 amino acid protein, occupies 79% of the element,extending from nucleotide 1260 to 8812. CRE2 contains long 5'and 3' non-coding regions. Although the CRE2 3' untranslatedregion does not end in a 3' poly dA tract, as has been observedwith the three other trypanosome site-specific elements, it doeshave two internal poly dA tracts, spanning nucleotides9115-9132 and 9333-9344. In the 5' untranslated region an

extended T tract runs from nucleotide 734 to 777 and another Atract is present between nucleotides 1236 and 1260. The singlelong ORF is immediately preceded by a run of 24-25 dA residuesin different CRE2 clones. In the two CRE2 variants identified inthe pBSCRE2-/4/?aI clones, this poly dA stretch consisted of 32and 35 residues. The CRE2 ORF is in the same orientation as thedirection of transcription of the miniexon units. The predictedmolecular weight of this polypeptide, initiated from the first ATGcodon, is 277 kDa.

Database searching revealed that the greatest similarities to thepredicted amino acid sequence of CRE2 were the three previouslydescribed trypanosomatid retrotransposons, CREl, SLACS andCZAR. The alignment of homology extends from codon 1517 to2499 (nucleotides 5808-8756) which approximately covers thecarboxy terminal 1000 residues of the 2518 amino-acid ORF. Theoverall level of amino acid identity in this extensive region is onthe order of 30%. The homology region includes two potentialmetal-binding motifs which correspond to the presumptive ENregion (codon 1517-1583: Cx2Cx2oHx4Hx17Cx2Cxi3HxiC,where x represents any amino acid) as well as a large domain(codon 1865-2116) corresponding to the eight segments of RThomology found in other retrotransposons (3) (Fig. 1). The fourelements continue to share -30% identity almost to their verycarboxy termini. To underscore the relationship of the fourtrypanosome elements, it is noteworthy that the residue betweenthe tyrosine and the first aspartic acid in the highly conservedYXDD box region of the RT domain (beginning at codon 2047)is either a leucine or isoleucine for all four of the trypanosome-sitespecific elements, whereas this residue is an alanine in nearly allother non-LTR retrotransposons examined to date.

Two regions of similarity exist between CRE2 and the firstORFs of SLACS and CZAR. First, the amino terminus of CRE2is very prolinerich(12% in the first 450 amino acids). While theprolines lack regular spacing in the overall sequence, one set ofproline residues in CRE2 conforms to a consensus SH3 binding



motif (25). A high density of prolines has been observed atanalogous locations in many other retrotransposons (26,27). Whileno specific function has been assigned to the proline rich region forany of these elements, one potential role would be in bindingtogether protein subunits to form an aggregate or even a regularstructure like a core particle (28). The second notable sequencefeature in the amino terminal region of CRE2 is a putativemetal-binding domain beginning at codon 405 and consisting ofthe sequence CX3HX8HX4H. This domain was identified in ourdatabase search as matching a region of SLACS that had beenpreviously noted to be a potential metal-binding domain (18) andmight correspond functionally to the DNA binding domainobserved in the gag region of retroviruses and many retrotranspo-sons (29-31).

Genomic instability of CRE1 and CRE2

In our previous study of CRE1 genomic organization, using theC.fasciculata (Paul Englund) isolate, we concluded that CRE1rapidly rearranges within the miniexon array, based on the variablerestriction enzyme digestion pattern of these elements derived fromcloned and subcloned lines (16). In light of our current observationthat the miniexon array is more complex than originally surmised,we sought to determine whether our initial observation could beextended to another strain of C.fasciculata and whether theserearrangements involved both CRE1 and CRE2.

We therefore isolated 20 clones from our C.fasciculata culture,prepared genomic DNA from each clone and digested the DNAfrom each clone with £coRI, a restriction enzyme that cuts bothCRE1 and CRE2 once per element, but which does not cut withinthe miniexon gene repeat. After electrophoretically separating theDNA and transferring it to a nylon filter, we hybridized the DNAwith either CRE1 or CRE2 probes. Using these probes, eight ofthe 20 clones showed variation from the parental pattern. Theseclonal alterations involved the appearance and disappearance ofindividual bands, as well as changes in the relative intensity ofexisting bands. The eight individual clones which containedobvious rearrangements relative to the parental stock (P) areshown in Figure 4. For example, using a CRE1-specific probe, anew band of -8 kb is observed in lane 3 and a band of -7.5 kb ismissing from lanes 4 and 5. This result extends our previousfindings and suggests that CRE1 rearrangements are a generalphenomenon in C.fasciculata.

Hybridizing an equivalent filter with a probe that is specific tothe 5' end of CRE2 (probe E in Fig. 3), it appeared that the CRE2pattern in all clones was identical to the parental stock andconsisted of five invariant bands (Fig. 4B). However, when aprobe specific to the 3' end of CRE2 (probe F in Fig. 3) washybridized to an equivalent filter, a different and less stable bandpattern was observed (Fig. 4C). Specifically, a band is absent inthree clones (lane 2, 5 and 8) and a new band is apparent in oneclone (lane 4). Of note, the four clones from the original 20examined which contain CRE2 rearrangements represent a subsetof the eight having CRE1 rearrangements. Thus it appears thatboth CRE1 and CRE2 are markers for a process that rearrangesthe miniexon arrays and which could be based on transpositionaland/or recombinational mechanisms.

Comparison of the bands observed using different CRE probesprovides potential information about the organization and linkageof CRE 1 and CRE2 copies within the miniexon arrays. Only threebands in Figure 4 are at corresponding positions for both CRE1

and CRE2 (e.g. arrows and arrowheads in Fig. 4), indicating thatmost copies of these two elements are not closely linked. Further,the finding of bands smaller than 10 kb (i.e. full length CRE2) inFigure 4C that do not correspond to bands in either Figure 4A orB suggests that non-miniexon, non-CREl sequences are presentdownstream of some copies of CRE2. These results underscorethe complexity of insertions within the miniexon arrays.

Organization of CRE1 and CRE2 within the genome

We have estimated the copy number of CRE2 elements associatedwith the miniexon repeat to be -6 per genome (data not shown).This number is derived from our observation that — 1% of theminiexon genes are interrupted by CRE2 and from previousstudies that estimated the total number of miniexon repeats to be-500-600 (22,23). We also estimated the copy number of CRE1in this strain to be -15-20 per genome. These estimates correlatewell widi the number of £coRI fragments that hybridize witheither CRE 1 or CRE2 probes (Fig. 4).

The primary sequence of our 10 kb element indicates that CRE2is inserted within a miniexon gene repeat Previous work frommultiple laboratories has shown that the miniexon genes intrypanosomes are organized as arrays of tandemly duplicatedrepeats (16,18,32). We sought to determine whether all copies ofCRE2 are associated with miniexon arrays. Therefore, weseparated Crithidia chromosomes by contour-clamped homogene-ous field gel electrophoresis (CHEF) and performed Southern blotanalysis on the separated chromosomes, using several uniqueprobes. As shown in Figure 5, the CRE2 probe hybridizesprimarily to a single size class of chromosome (-940 kb), with asecond size class of chromosome (-825 kb) showing a weakerCRE2 hybridization signal. A CRE1 probe reveals a reciprocalhybridization pattern compared with CRE2, while a miniexonprobe shows roughly equivalent levels of hybridization to both sizeclasses of chromosomes. Thus, CRE1 and CRE2 elements are notwidely dispersed throughout the Crithidia genome, but rather arerestricted to the chromosomes containing the miniexon genearrays. Further, the miniexon arrays on the two chromosomes arenot equivalent with respect to either the type or number ofassociated retrotransposons. These results correlate with thelimited linkage of CRE1 and CRE2 copies observed by conven-tional restriction digest analysis (Fig. 4).

DISCUSSION

In this paper we have demonstrated that the C.fasciculataminiexon gene array is interrupted by at least two different butclearly related genetic elements, which both have propertiessuggesting that they are retrotransposons. While we had previouslyidentified the 3.6 kb CRE1, inserted site-specifically into afraction of the C.fasciculata miniexon genes (16), it is nowapparent that the 9.6 kb CRE2 is also inserted at precisely thesame location in a subset of the miniexon gene repeats.

Crithidia retrotransposable elements 1 and 2 share -30%sequence identity over an -1000 amino acid region that includesthe putative EN domain and the RT domain. However, beyondthis sequence similarity the two elements are structurally distinctWhereas CRE2 has an 844 base 5' UTR, CRE1 has no apparent5' UTR. Likewise, while CRE2 has a 1200 base 3' UTR, CRE1has only a 60 base 3' UTR. As opposed to CRE 1, CRE2 lacks thevariable length 3' terminal poly dA tracts which are characteristic



WClt ~—

1120-

945-915-

815-785-

Probe CRE1 CRE2

Figure 5. Gcnomic linkage of miniexon, CRE1 and CRE2. Crithidiafasciculatachromosomes were separated by clamped homogeneous electric field (CHEF)gel electrophoresis. After transfer to a nylon filter, the blots were hybridized withminiexon (ME), CRE1 orCRE2 (probe E in Fig. 3) probes. Filters were strippedand exposed for sufficient time periods to demonstrate removal of probe, beforerehybridizaOon. Size markers (kb) are shown at the left

of most non-LTR retrotransposons (1,2). These significantstructural differences underscore our unexpected finding that twoevolutionarily diverged transposable elements share the sameinsertion site within the same organism.

The structure of CRE2 is more similar to SLACS and CZARthan it is to CRE 1. Although SLACS and CZAR both encode twoORFs while CRE2 encodes a single long ORF, their respectivecoding capacities are comparable and the amino terminus ofCRE2 shares domains with SLACS and CZAR not found inCRE1. Given the sizable differences in coding capacity of CRE 1and CRE2 and the finding that CRE1 lacks the equivalent of theamino terminus of CRE2, it is an intriguing possibility that CRE2provides structural, gag-like, proteins in trans to CRE1 whichmight be important for its replication. Of note, since cloningCRE2, we have identified CRE2 by Southern blot analysis inthree additional C.fasciculata isolates including C.fasciculata(Paul Englund) (data not shown).

Crithidia retrotransposable element 2 is the fourth site-specifictrypanosome retrotransposon whose sequence has been deter-mined. Despite limited sequence identity, these four elementsshare specific sequence and structural features, as well as acommon insertion site, that distinguish them as members of adiscrete family of non-LTR retrotransposons. Interestingly, twocompletely distinct families of site-specific retrotransposons, Rland R2, are inserted at different conserved sites in a fraction of therRNA genes of most insects. As with the trypanosome elements,the R1 and R2 elements from a wide variety of insect species haveindependently diverged in their individual sequences whilemaintaining their target site specificity. Given this analogoussituation, it is of interest that only partial counterparts to the unusualrelationship of CRE1 and CRE2 have been observed in the moreextensively studied site-specific insect elements. For example, in

their survey of Rl and R2 elements in a wide variety of insects,Eickbush et al. sequenced the 3' insertion sites from several speciesand found instances of species containing multiple, distinctinsertions into the same sequence within the 28S rDNA array,suggesting that divergent families of related elements compete forthe same insertion site in certain insect hosts (33). In a moredetailed study of Anopheles gambiae, Besansky et al. identifiedtwo variant forms of Rl that insert at precisely the same site(different from the common Rl site) in a subset of the 28 rRNAgenes of this organism (34). However these two elements, dubbedRT1 and RT2, are much more closely related to one another insequence and structure than are CRE1 and CRE2.

We observed that CRE1 and CRE2 copies are unequallyrepresented in the two observed miniexon arrays (Fig. 4) and thateach element predominates on a different size class of chromosome(Fig. 5). Without independent markers for these two chromosomeswe cannot determine whether these classes represent two non-homologous chromosomes that happen to contain miniexon arraysor homologous but variably sized chromosomes. However, thelack of significant linkage between individual copies of CRE 1 andCRE2 suggests that interchromosomal recombination betweenminiexon arrays in this strain is infrequent, at least in areas of thearray where CREs are present. The chromosome-specific distribu-tion of CREs may be analogous to the situation observed in manystrains of D.melanogaster, where R1 insertions are common on theX chromosome rDNA array but rare on the Y chromosome rDNAcluster (35,36). Our observation is also consistent with thepossibility that the two elements evolved in separate host strainsand were brought into the same genome, more recently, via amating event.

In the course of cloning CRE2 we identified portions of anadditional CRE-like element as well as a variant form of CRE2.Within the limited regions of these elements that were analyzed atthe sequence level, the former showed - 50% identity to CRE2 atthe amino acid level (CRE3, Fig. 1) while the latter showed -93%identity to CRE2 at the nucleotide level. Further, both appear tomaintain an open reading frame, despite multiple base substitu-tions. In both S.cerevisiae and Schizosacchammyces pombe, twodistinct families of retrotransposons derived from commonancestors have been characterized at the sequence level (4,37,38).In both cases, the related elements have the unusual property ofsharing near identity in certain portions of their genomes, but littleor no homology in other regions. Further analysis of the multipleelements identified in the C.fasciculata genome will determine theprecise nature of the sequence relationships that exist among theseretrotransposons.

Insertion site specificity is increasingly recognized as a propertyof many transposable elements, requiring interactions with host-encoded proteins. Recent in vitro studies with yeast Ty3 integraseindicates that position-specific insertion involves interactionsbetween this protein and yeast RNA polymerase III transcriptionfactors (39). Analysis of the association between the gene productsof CRE-related elements and proteins present in the Crithidiaminiexon gene locus may elucidate the basis for this site-specificsystem.

ACKNOWLEDGEMENTS

We thank S. Aksoy for SLACS and CZAR clones, G. Clark andL. Diamond for discussions on the origin and taxonomy ofCrithidia species, J. Berg for discussion of the putative metal-x



binding domains, C. C. Lin for technical assistance and E. Mulesfor critical reading of the manuscript. The work was supported,in part, by the Charles and Johanna Busch Endowment and theLucille P. Markey Charitable Trust. A.G. is a Lucille P. MarkeyScholar.

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a new non-ltr retrotransposon provides evidence for multiple

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