Structure and Origin of a Novel Dimeric Retroposon B1-dID

Dmitri A. Kramerov, Nikita S. Vassetzky

Laboratory of Eukaryotic Genome Evolution, Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, 32 Vavilov St.,Moscow, 117984, Russia

Received: 30 August 1999 / Accepted: 20 March 2000

Abstract. Here we describe a new short retroposonfamily of rodents. Like the primate Alu element consist-ing of two similar monomers, it is dimeric, but the leftand right monomers are different and descend from B1and ID short retroposons, respectively. Such elements(B1-dID) were found in the genomes of Gliridae, Sciu-ridae, Castoridae, Caviidae, and Hystricidae. Nucleotidesequences of this retroposon can be assigned to severalstructural variants. Phylogenetic analysis of B1-dID andrelated sequences suggests a possible scenario of B1-dIDevolution in the context of rodent evolution.

Key words: Repetitive DNA — SINEs — Retro-posons — B1 — ID — Alu — Rodents — Gliridae —Sciuridae — Castoridae

Introduction

Short retroposons, or short interspersed elements(SINEs), are 80–400 bp repeats in eukaryotic DNA thatcan amplify in the genome (Rogers 1985). Short retro-posons carry internal bipartite RNA polymerase III pro-moter (A and B boxes) and are transcribed by this poly-merase at early stages of development and in certaintissues of adult organism (Bachvarova 1988; Grigoryanet al. 1985; Kim et al. 1995). New copies of the elementsappear in the genome in the course of reverse transcrip-tion (retroposition). Most of the retroposons have an A-rich tail at the 38-terminus, and most of the copies are

flanked by 5–20 bp short repeats appearing during theelement’s integration into the genome (Rogers 1985).

According to the present views, amplification of shortretroposons is related to active templates, “master se-quences,” while the rest of their copies is inactive (Dein-inger and Batzer 1993). The master sequences exist for acertain time period until inactivated and replaced byother variants. Hence, there are two processes in theevolution of short retroposons: evolution of the func-tional templates and degradation of their inactive copies(Zuckerkandl et al. 1989).

Short retroposons can influence the functioning ofgenomes, e.g., disturb gene expression during integrationor through unequal crossing-over with the repeats in-volved (Zheng et al. 1992; Makalowski 1995). Singlecopies of short retroposons can control transcription assilencers, enhancers, or insulators (Thorey et al. 1993;Hanke et al. 1995). It remains unclear if short retro-posons are solely parasitic (“selfish”; Doolittle and Sa-pienza 1980; Orgel and Crick 1980) sequences or play acellular (physiological) role (Vidal et al. 1993; Chu et al.1998).

At present we know around 30 short retroposon fami-lies from genomes of various vertebrates and inverte-brates as well as plants (Deininger and Batzer 1993;Okada and Ohshima 1995). SINEs can be divided in twomajor classes by their origin. One descends from 7SLRNA (∼300 nts) and is represented by two families: B1elements in rodents (Krayev et al. 1980) and the Alufamily in primates (Deininger et al. 1981). The Alu ele-ment consists of two monomers with similar nucleotidesequence. Both B1 and Alu lack the internal 155-bp re-gion of 7SL RNA (Ullu and Tschudi 1984). The secondretroposon class is more ample; it originates from certain

Correspondence to:D.A. Kramerov;e-mail;kramerov@genome.eimb.relarn.ru

J Mol Evol (2001) 52:137–143DOI: 10.1007/s002390010142

© Springer-Verlag New York Inc. 2001

tRNAs or their genes (Okada and Ohshima 1995). Fourfamilies of this class were described in rodents: B2 (Kra-merov et al. 1979; Krayev et al. 1982), ID (Kim et al.1994; Sutcliffe et al. 1982), DIP, and MEN (Serdobovaand Kramerov 1998). Their sequences are quite similarwithin the 58-part but are different elsewhere. The IDelement is widespread in rodents, although the number ofits copies in the genome is usually not high: 100–4000(Kim et al. 1994; Kass et al. 1996). It is quite possiblethat the ID element is an evolutionary precursor of othertRNA-derived retroposons in rodents (Serdobova andKramerov 1998); in turn, ID originates from small BC1RNA and more distantly from alanine tRNA (Kim et al.1994).

Here we describe a new retroposon combining thesetwo classes; it is composed of two monomers, the left(58) one is related to B1, and the right (38) one is relatedto ID.

Materials and Methods

We studied DNA of the following rodent species (their code is given inparentheses). Family Gliridae: forest dormouseDryomys nitedula(Dni); Sciuridae: long-tailed marmotMarmota caudata(Mca) and In-dochinese ground or palm squirrelMenetes berdmorei(Mbe); Casto-ridae: beaverCastor fiber(Cfi); Hystricidae: Indian porcupineHystrixleucura (Hle); and Caviidae: Guinea pigCavia porcellus(Cpo).

DNA was isolated from frozen liver by incubation with protease Kfollowed by phenol/chloroform extraction. Genomic libraries were con-structed in pGEM7f+ (Promega) digested byHind III andEco RI.Thelibraries were screened by a labeled DNA fragment (nucleotides 16–135) of mouse B1. B1-dID DNA of certain species was amplified usingprimers 4 (58GCAYRCCTTTAATCCCAG) and 14 (58CTGGGGAT-KGAACCCAGRG) (Fig. 1). PCR and hybridization conditions weredescribed elsewhere (Serdobova and Kramerov 1998; Kramerov et al.1999). PCR products were cloned into aSma Isite of pGEM7f+. Thecloned DNA fragments were sequenced with the standard M13 primersor primers complementary to the B1 and dID sequences using Se-quenase 2.0 and the USB/Amersham protocols. The B1-dID copy num-ber was estimated from frequency of positive clones in the genomiclibraries of dormouse, marmot, palm squirrel, and beaver. The positiveclones were identified by colony hybridization to the mouse B1 probeand following sequencing of repetitive elements in the isolated clones.The B1-dID copy number for guinea pig and porcupine genomes weredetermined by PCR titration described in detail elsewhere (Kramerov etal. 1999).

The nucleotide sequences were aligned usingGenBeeservice(Brodsky et al. 1991) and manually corrected using theGeneDocpro-gram (Nicholas and Nicholas 1997). The time of active amplification ofB1-dID subfamilies in the genome was calculated as described in Kapi-tonov and Jurka (1996) (Kimura’s two-parameter model [Kimura1981]; CpG sites were excluded and indels were considered as a singlemismatch; the rate of changes was 0.005 / (site × Myr) as synonymoussubstitutions in Sciuridae [Robinson et al. 1997]). Phylogenetic treeswere constructed by the maximum parsimony method with a random-ized order of sequences (100 times) and the bootstrap procedure (100replications), with the help of theDNAPars and SeqBootprograms(Phylogeny Interference Package, version 3.57c) (Felsenstein 1993).

Results

A number of clones were selected from genomic librariesof dormouse, marmot, palm squirrel, beaver, and Guinea

pig by hybridization with mouse B1 DNA. Sequenceanalysis of the clones has revealed copies of short B1-like retroposons in all the above genomes, while in thegenomes of dormouse (Gliridae), marmot, squirrel (Sciu-ridae), and beaver (Castoridae) we found an unusual di-meric retroposon (EMBL accession numbers Y16204–Y16220). Figure 1 presents aligned nucleotide sequencesof this retroposon and their consensus. The left (58) partof this element (135 bp) is similar to mouse and rat B1,whereas the right (38) part is common with the rodent ID(Fig. 1, upper and lower parts, respectively). This se-quence featured a 19-bp deletion as compared to the IDelement. Based on the above structural features of thiselement we called it B1-dID, whered refers to the dele-tion.

The left monomer of B1-dID is present in two vari-ants: one carries a tandem duplication of a 19-bp regionand the other lacks it. Only the first variant was found inthe dormouse genome, while both variants were revealedin the marmot genome (Fig. 1). Note mismatching be-tween the B1-dID and mouse B1 sequences in the du-plication region; the mouse element also has a duplica-tion in this region, but it is longer—29 bp (Ullu andTschudi 1984; Labuda et al. 1991).

In addition to the above deletions, the right monomerhas three nucleotide positions distinct from the Guineapig ID element (marked with ! in Fig. 1). Note the ab-sence of tetranucleotide CCTG specific for the ID ele-ment at the A-rich tail of B1-dID, although four copies(Mbe-86, Mca-02, Mca-34, and Mca-51) carry long C-rich segments at the same position.

As with other retroposons (Rogers 1985), most ofB1-dID elements are framed with 5–19 bp direct repeats(underlined in Fig. 1). This confirms that B1-dID is asingle element capable of retroposition rather than tworetroposons randomly integrated nearby. B1-dID copiesare terminated with quite variable A-rich sequences (Fig.1), which characterizes almost all retroposon families(Rogers 1985). The two parts of this element (B1 anddID) are also linked with an A-rich sequence of variablelength containing the AAA(A)T motif.

The sequences presented significantly differ fromeach other and the consensus sequence (Fig. 1). How-ever, one also notes more homogeneous and similar el-ements. These include all five sequenced dormouse B1-dIDs and a significant portion (seven) of the squirrelelements (grouped in the upper part of Fig. 1). We des-ignated this group of variants as gsB1-dID (Gliridae/Sciuridae B1-dID).

We found two more B1-dID sequences in the ge-nomes of marmot and Guinea pig by similarity search inthe data blank (EMBL AC: Z13234 and X60129). Inboth cases the structure of these genomic repeats was notconsidered in the original publications. These two ele-ments were combined with five partially (not shownin Fig. 1) and all completely sequenced B1-dID copies

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to construct the consensus sequence of this element(Fig. 1).

In addition to the gsB1-dID we found a variant des-ignated as proB1-dID. It lacks the above deletion in theID monomer and, apparently, is closer to the B1-dIDprecursor. This is confirmed by the estimated time oftheir active amplification: 35 and 41 Myr for gs- andproB1-dID, respectively. Only 1 out of 14 sequencedmarmot copies can be assigned to this type (Mca-53); allthree sequenced beaver elements proved to be proB1-dIDs, despite a small internal deletion in one of thesecopies (Cfi-35) (Fig. 1).

The number of copies of this retroposon as well as theproB1-dID: gsB1-dID ratio in the genome varies amongthe rodent families. According to our estimates, B1-dIDelements are abundant in the genomes of dormice andsquirrels (0.4–1 × 105 copies) by contrast to the genomesof beaver (5 × 103), porcupine (5 × 102) and Guinea pig(50). Here we present PCR sequences from the genomesof porcupine (Hystricidae) and Guinea pig (Caviidae)obtained with primers shown in Fig. 1. Figure 2 presentsaligned nucleotide sequences of the cloned PCR productstogether with gsB1-dID and proB1-dID consensus se-quences. Hence, the genomes of porcupine and Guineapig carry copies of B1-dID although these copies arehighly divergent and differ from the gsB1-dID variant.

Most of the B1-dID copies in the genomes of porcu-pine and Guinea pig lack the 19-bp internal duplicationin the left (B1) monomer. However, some of them (Hle-04, Hle-04d, and Cpo-76) carry additional sequences thatpresumably result from an 8- or 10-bp duplication,whereas one copy from the Guinea pig genome (Cpo-57)has a duplication similar to that of the classical B1 ele-ment (Fig. 2) Judging from the right monomer structure,the majority of B1-dIDs in porcupine and Guinea pig canbe assigned to the typical B1-dIDs, although proB1-dIDvariants also occur (Cpo-102, Hle-15, and Hle-37). Notethat B1 and especially ID monomer sequences are moredivergent in these species as compared to dormice andsquirrels.

We tried to infer the phylogenetic relations betweenB1-dID by constructing a phylogenetic tree of com-pletely sequenced copies using the maximal parsimonymethod with the bootstrap procedure for the B1 and IDmonomers (Fig. 3A and B, respectively). Precursor B1(PB1, Quentin 1994) and the Guinea pig BC1 RNA (Kimet al. 1994) were introduced as the outgroups for B1 andID trees, respectively.

The B1 monomer tree is clearly divided into twobranches corresponding to the gsB1-dID (all Dni andMbe as well as Mca-01, Mca-02, Mca-34, Mca-51, andMca-64) and the other sequences. On the ID monomertree one can see that the proB1-dID elements (Cfi-06,Cfi-14, Cfi-35, and Mca-53) are neighboring the ances-tral sequences. The rest of the sequences branched intotwo groups containing different variants of B1-dID. All

branching points discussed have significant bootstrapvalues (at least 75).

Discussion

The family of genomic repeats presented is the fifthexample of dimeric retroposons containing 7SL RNA-derived nucleotide sequences. In addition to the well-known Alu element in primates composed of two 7SL-derived monomers (Deininger et al. 1981), this groupincludes the type II SINE in galagoOtolemur crassicau-datus (Daniels and Deininger 1985) and the MEN ele-ment in palm squirrelMenetes berdmorei(Serdobovaand Kramerov 1998). Interestingly, mouse has a lowcopy number retroposon RSINE2 also composed of ID-and B1-like monomers (Repbase Update, http://www.girinst.org/Repbase_Update.html). In these threeelements the left monomers are tRNA-derived se-quences. The structure of B1-dID element demonstratesthe inverse order with 7SL-derived sequences on the leftand tRNA-derived sequences on the right. So, it is thefirst SINE where a 7SL RNA–related monomer precedesa tRNA-related one. Because we know no examples ofdimeric retroposons formed from two tRNA-derived se-quences, it is possible that the 7SL-derived elements tendto associate either with their homologs or tRNA-derivedsequences. Such association may provide for a moreefficient amplification of the resulting retroposons rela-tive to the initial monomeric elements. According to ourobservations, B1-dID elements in dormice and squir-rels far exceed the monomeric B1 and ID in number ofcopies in the genome (by one or two orders of magni-tude; data not shown), indicating that the majority ofB1-dID copies appeared by amplification of master B1-dID sequence(s) rather than by independent acts of in-sertions or recombinations between separate B1 and IDelements.

A particularly high number of B1-dID copies (0.4–1 ×105) is present in the genomes of dormice and squirrels(which explains the surprisingly high number of ID ele-ments in squirrel estimated by dot-hybridization; Kass etal. 1996). Note that the variant with both the deletion inthe ID monomer and the duplication in the B1 monomer(gsB1-dID) is specific for these rodents. It is quitepossible that these structural rearrangements pro-vided for an amplification explosion of this retroposonin the genomes of dormice and squirrels. At least, wefound no 19-bp duplication in the B1 monomer in thegenomes of porcupine and Guinea pig and no specificdeletion in the ID monomer in the genomes of beaver; atthe same time the number of B1-dID copies in theirgenomes was significantly lower than in dormice andsquirrels.

We propose the following scenario of B1-dID originand evolution illustrated by phylogenetic trees presented

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in Fig. 3. At the first stage a B1 element without theinternal duplication was fused to the common ID ele-ment. The resulting proB1-dID had moderate capacityfor amplification (taking place∼ 41 Myr ago) and smallstructural rearrangements had no dramatic effect on it. Ata certain time period the 19-bp fragment was deletedfrom the ID monomer (Fig. 3B) and, finally, the 19-bpduplication occurred in the B1 monomer (Fig. 3A),which gave rise to the gsB1-dID element and providedfor its active amplification (∼ 35 Myr ago) in the ge-nomes of dormice and squirrels.

It was interesting to relate B1-dID evolution to theevolution of rodents. While studying the distribution ofthis element in the genomes using the PCR technique, wefailed to find it in the genomes of Muridae, Cricetidae,Spalacidae, Zapodidae, and Dipodidae (Kramerov et al.1999). On the basis of this fact and the data obtained in

this work we conclude that (1) the above families, con-stituting the group of myomorphous rodents, differenti-ated from other rodents quite early, before the appear-ance of the proB1-dID element; and (2) by contrast tothe traditional viewpoint (Romer 1966; Wahlert et al.1993), dormice with a typical B1-dID element cannotbe assigned to this group but should be grouped withthe squirrels, thus supporting alternative views of mor-phologists (Carrol 1988) and molecular taxonomists(Nedbal et al. 1996) on evolutionary relations of rodentfamilies.

Acknowledgments. We are grateful to Drs. E.A. Liapunova, E.Panova, and O. Likhnova for providing animal tissues, and Drs. I.K.Gogolevskaya, O.R. Borodulina, and E.S. Grigorian for help in someexperiments. The work was supported by the International ScienceFoundation (N4E000 and N4E300) and the Russian Foundation forBasic Research (99-04-49177 and 99-04-49178).

Fig. 3. Phylogenetic trees of B1(A)and ID (B) monomers of B1-dIDelements (maximum parsimonymethod). Bootstrap probability is givenon the right of the corresponding node.For other explanations, see text.

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