A new pair of CR1-like LINE and tRNA-derived SINE elements in Podarcis sicula genome

Gene 339 (2004) 189 – 198 www.elsevier.com/locate/gene

A new pair of CR1-like LINE and tRNA-derived SINE elements in Podarcis sicula genome Stefania Fantaccione, Consiglia Russo, Patrizia Palomba, Monica Rienzo, Giovanni Pontecorvo* Department of Life Science, II University of Naples, Via Vivaldi 43, 81100, Caserta, Italy Received 12 March 2004; received in revised form 19 March 2004; accepted 29 June 2004 Received by M. D’Urso

Abstract We have identified and characterized a new pair of LINE and SINE elements, called Lucy-1 CR1-like LINE and P.s.1/SINE, respectively, in Podarcis sicula genome. The 3V-tail region in the 3V untranslated region (UTR) of Lucy-1 element is almost identical to the of P.s.1/SINE element. This identity suggests that the P.s.1/SINE element, during evolution, has gained the 3V-end sequence of the Lucy-1 element and has exclusively recruited the enzymatic machinery of its partner CR1 LINE for retroposition. Moreover, the complex molecular organization around Lucy-1 insertion site is discussed and we found that Lucy-1 insertion is associated with the calcium binding transporter gene. Our results confirm that the retrotransposons can be an additional source of genomic diversification and the evolution of the retrotransposable elements can be a vector force shaping genomes by reassorting DNA domains thus forming a new DNA arrangement. D 2004 Elsevier B.V. All rights reserved. Keywords: Non-long terminal repeat retrotransposon; Reverse transcriptase; Long interspersed repeat; Short interspersed repeat; Chicken repeat 1; Repetitive DNA

1. Introduction Retroposons can be divided in two groups: those that do not encode a reverse transcriptase (RTase) and those that do. The former group includes short interspersed repetitive elements (SINEs), processed pseudogenes, and other pseudogenes for small nuclear RNAs (Weiner et al., 1986). The latter group can be further divided into two subgroups on the basis of structure: long terminal repeat (LTR) and non-LTR

Abbreviations: bp, base pairs; kb, kilobase(s) or 1000 bp; dig, digoxigenin; nt, nucleotide; RTase, reverse transcriptase; SINE, short interspersed repetitive elements; LINE, long interspersed repetitive elements; UTR, untranslated region(s); ORF, open reading frame; tDNA, DNA coding for tRNA; LTR, long terminal repeat(s); dNTP, deoxyribonucleoside triphosphate. * Corresponding author. Tel.: +39 0823 274542; fax: +39 0823 274571. E-mail address: [email protected] (G. Pontecorvo). 0378-1119/$ - see front matter D 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.gene.2004.06.051

retrotransposons. Non-LTR retrotransposons are also known as long interspersed repetitive elements (LINEs) (Ogiwara et al., 1999). Almost all LINEs, such as those of the chicken CR1 family and the mammalian L1 family, are moderately to severely 5V-truncated, such that most LINE families consist mainly of 3V-terminal fragments of the full-length element (Terai et al., 1998). The presence of truncated forms suggests that an RTase encoded by a LINE must recognize the 3V-end of the RNA template for initiation of first-strand synthesis (Eickbush, 1992). This model named TPRT (target DNA-primed mechanism of reverse transcription) was verified by Luan et al. (1993) in an elegant experiment with the R2Bm LINE of Bombyx mori. On the other hand, SINEs are non-autonomous retroposons without open reading frames (ORFs) and so lack the machinery necessary to replicate themselves (Van Dellen et al., 2002). In this regard, it has been proposed that SINEs are reverse transcribed by an RTase that is encoded by a

190

S. Fantaccione et al. / Gene 339 (2004) 189–198

partner LINE in the same genome (Ogiwara et al., 1999). This hypothesis is based on the observation that the 3V-tail sequences of certain SINEs are almost identical to those of certain LINEs (Van Dellen et al., 2002) and that this sequence organization is related to the acquisition of retropositional activity (Okada et al., 1997). SINEs transcription, however, occurs independently of the LINE partner. The SINEs typically have an internal promoter for RNA Polymerase III (Humphrey, 1995). Almost all SINEs reported to date appear to have a tRNA-derived region (Van Dellen et al., 2002); these tRNA-derived SINEs are not simple pseudogenes for tRNAs, but have a composite structure with a region homologous to a tRNA, a tRNAunrelated region, and an AT-rich region (Okada and Hamada, 1997). There are many examples of the phenomenon whereby the 3V-tail region of certain tRNA-derived SINEs is identical to the 3V portion of the 3V untranslated regions (UTRs) of certain LINEs. The tortoise Pol III/SINE and the turtle PsCR1 LINE were first described as a pair of SINEs and LINEs with the same 3V-tail region (Kajikawa et al., 1997). Other examples have also been identified such as the ruminant BovtA SINE and BovB LINE (Szemraj et al., 1995), the mammalian MIR SINE and LINE2 (Smit, 1996), and the cichlid AFC SINE and CiLINE2 (Terai et al., 1998). Recently, Gilbert and Labuda (1999) reported that two MIRlike SINEs share their 3V-tail regions with those of the CR1 LINE and BovB LINE, respectively. These findings suggest that several families of SINEs were generated by recombination between a tRNA-derived precursor and a pre-existing LINE at a certain stage of evolution (Ogiwara et al., 1999). The interspersed repetitive DNA family CR1, originally identified in the domestic chicken (Gallus gallus) by Stumph et al. (1983), is present in the genome of all vertebrates. CR1 elements are the predominant non-LTR retrotransposons in the genome of birds (Chen et al., 1991) and CR1-related (CR1-like elements) are present in the genome of reptiles (Vandergon and Reitman, 1994), amphibia (Kajikawa et al., 1997), fish (Poulter et al., 1999), invertebrates (Drew and Brindley, 1997) and mammals (Jurka, 2000). Thus, it is likely that CR1 elements arose early in evolution and became the predominant retrotransposons in certain organisms (Haas et al., 2001). The 3V-end of CR1 elements consists of repeats (usually two or three) of an 8-base pair (bp) sequence and, unlike most non-LTR retrotransposons, does not contains an A- or ATrich region. Terminating intermediate transcription prematurely, the 5V-ends are heterogeneous, extending a variable distance upstream, with most elements b400-bp in length, as compared with the predicted full-length of at least 5 kilobase (kb). To date, the longest CR1 known is 2.3 kb and includes part of an ORF that is homologous to the RTase of other non-LTR retrotransposons (Vandergon and Reitman, 1994). Here we report the structural characterization of novel CR1-like LINE and tRNA-derived SINE elements from

Podarcis sicula (P.s.) genome, providing further evidence that LINEs might be a source of the enzymatic machinery required for retroposition of SINEs. These LINE and SINE retrotransposons, which we have named Lucy-1 CR1-like LINE and P.s.1/SINE (P. sicula 1), respectively, are the first retrotransposable elements to be reported from P.s. genome. Moreover, the complex molecular organization around the Lucy-1 insertion is discussed. Furthermore, Lucy-1 element sequence exhibited strong identity to several CR1 elements found in Aves and Reptiles, suggesting that the CR1 family originated before the divergence of Aves and Reptiles (Vandergon and Reitman, 1994).

2. Materials and methods 2.1. Sample collection and isolation of genomic DNA Reptilian P.s. (Reptilia, Lacertidae) were collected in the South-Italy (Caserta). Frozen or fresh livers from P.s. were used for genomic DNA extraction by the standard proteinase K/SDS method (Maniatis et al., 1982). 2.2. PCR amplification and sequencing Amplification of the retrotransposable elements was carried out in 50 Al volumes in Gene Amp PCR 9600 System using the following conditions: one cycle (94 8C for 5 min) followed by 30 cycles (94 8C for 30 s, 50 8C for 1 min, 75 8C for 40 s) and one cycle (75 8C for 7 min). PCR reactions were performed with approximately 150 ng template DNA, 100 nM each deoxyribonucleoside triphosphate (dNTP) (Roche), 200 nM each primer and 2.5 units Taq DNA Polymerase with its appropriate buffer. The oligonucleotide primers were synthesized from Roche Molecular Biology. One primer is homologous to Homo sapiens Werner syndrome gene stretch found in a repeat sequence DNA from a Tc1-like transposon (Pontecorvo et al., 2000). The other is homologous to Tc1 transposase gene stretch. Fragments of approximately 400 and 700 bp obtained as PCR products were isolated from an 1.2% agarose gel using QIAGEN GEL Purification Kit (QIAGEN, Hilden), as well as all fragments that were purified in the procedures described in this report. A portion of the 406bp eluted fragment was labelled with digoxigenin (dig) by a random priming method (DIG High Prime-Roche) and used as a hybridization probe (probe 1) in Southern blot analysis. 2.3. DNA sequence analysis Sequencing reactions were performed by Big Dye Terminator method using ABI 310 Genetic Analyzer (Perkin Elmer) and polymer POP-6 according to the manufacturerVs

S. Fantaccione et al. / Gene 339 (2004) 189–198

191

3. Results 3.1. Identification of CR1-like LINE and tRNA-derived SINE elements from the genome of P.s.

Fig. 1. Schematic representation of the different primers used in the Inverse PCR strategy and their respective positions on R.E.1 sequence.

directions. Sequences were analysed and compared with the GenBank-NCBI DNA database using the Blast Network service. Sequence multiple alignments were performed with the Clustal W software at Infobiogen (http:// www.infobiogen.fr/services/deambulum). 2.4. Southern blot analysis of digested genomic DNA Genomic DNAs from P.s. were digested with 20 U/Ag of several restriction endonucleases (EcoRI, HindIII, AluI, NsiI, HaeIII). The resulting fragments were separated on 1% agarose gel and Southern transferred to nylon membrane (Hybond-N+, Amersham) under standard conditions (Maniatis et al., 1982). Hybridization was performed at 65 8C and the filter detected with a DIG Detection Kit from Roche. 2.5. Further characterization of the Lucy-1 element by Inverse PCR Inverse PCR was performed by initial digestion of 1 Ag of lizard genomic DNA with NsiI which cut once within the 406-bp sequence. After 2 and 4 h digestion, restriction fragments were purified by phenol–chloroform extraction (1:1) and precipitated with ethanol in the presence of 0.3 M NaCl. A total of 40 ng NsiI-digested genomic DNA were circularized by ligation at 16 8C for 16 h (T4 DNA LigaseRoche). Inverse PCR reactions were cycled as follows: 4 min at 94 8C followed by 30 cycles of 94 8C 30 s, 55.4 8C 1 min or 59.5 8C 1 min, 75 8C 40 s and then 7 min at 75 8C. The inverse primers were designed manually from the lizard 406-bp sequence. To selectively amplify 5V-region flanking to the 406-bp sequence the following primers were used: 5V-CCACTGTCAAAAAGCTCTTCC-3V (INV1) and 5V-GGACTCTCCTTCCTTGGAGGA-3V (INV2), designed upstream of the NsiI restriction site. On the other hand, to screen for region located downstream of the 406-bp sequence the following primers were used: 5V-TGTGGTCAAGCTCCTCCTTGG-3V (INV3) and 5V-GCCCAGCTCATAGGTGTGCAT-3V (INV4), designed downstream of the NsiI restriction site. The positions of primers are shown in Figs. 1 and 3. PCR products were sized on 1.0% agarose gel and purified from gel prior to sequencing or cloning. Sequencing was performed either directly on PCR products derived from 3V-Inverse PCR and via cloning of 5V-Inverse PCR products into pGEM-T Easy vector (Promega).

To isolate retrotransposable elements, we first performed PCR strategy using lizard genomic DNA with the previously described primers (see Section 2.2). To our surprise, we have verified that these primers are able to amplify transposable and retrotransposable elements of different eukariotic organisms (data unpublished). These results could be explained by some highly conserved DNA stretches in the different evolutionary lines of these mobile genetic elements. The resulting products included at least 5 discrete bands superimposed on a smear of PCR products ranging in length from about 0.4 to 1.6 kb (Fig. 2). Of five sequenced PCR products, only two, 406- and 737-bp bands, were selected for further studies (GenBank accession no. AY620369 and AY620370). These bands are named by us Retrotransposable Element 1 (R.E.1) and Retrotransposable Element 2 (R.E.2), respectively, and their sequences were used to search the GenBank database using the BLAST algorithm. 3.1.1. R.E.1 characterization The R.E.1 contains a CR1-like LINE element (from position 51 to 233 nt, Fig. 3), which we have called Lucy-1. Lucy-1 element exhibits strong identity to Anolis carolinensis rhodopsin gene (Kawamura and Yokoyama, 1994; Kajikawa et al., 1997; GenBank accession no. L31503.1). In fact, position at nt 51–140 of the R.E.1 sequence shares 84% identity with the region comprised between position at nt 8832 and 8743 of the clone L31503.1, whereas the stretch nt 173–233 shows 87% identity as with the region comprised between position at nt 8707–8647 as with region comprised between position at nt 9706–9644. The latter is located in the 3V-flanking of A. carolinensis rhodopsin gene. Interestingly, the regions of rhodopsin gene overlapping to Lucy-1 element are included in the two

Fig. 2. P.s. genomic DNA PCR amplification. Lanes M and 1 indicate respectively DNA size-standard and PCR products. Arrows indicate the PCR products described in this paper.

192

S. Fantaccione et al. / Gene 339 (2004) 189–198

Fig. 3. Nucleotide sequence of R.E.1. Matches with A. carolinensis rhodopsin gene are in bold. The Inverse PCR sequences primers are indicated by arrows.

sequences (bases 9015–8640 and 9715–9645) containing characteristic CR1 3V-ends (Vandergon and Reitman, 1994). Further, the 3V-tail region of Lucy-1 element is similar to 3VUTR of PsCR1 element from Platemys spixii (Kajikawa et al., 1997) (Fig. 4) and to 3V-tail region of consensus sequence of the CR1-like elements from Crotalinae snake genomes (Nobuhisa et al., 1998). As do the majority of other CR1-like LINE elements, Lucy-1 ends in the characteristic octameric repeat sequences (AACTCTATGATTCTAT), whereas the CR1-like elements found in Crotalinae snake contain at their 3V-ends 6-bp repeated sequences (TTCTGA)2 (Nobuhisa et al., 1998). Since the 3Vend region of a LINE transcript is thought to serve as a recognition site for the RTase encoded by the LINE (Luan and Eickbush, 1995), the strongly conserved tail region of the Lucy-1 might be critical for its retroposition. Analysis of the deduced amino acid sequence of the Lucy-1 shows an partial reverse transcriptase ORF that shares strong homol-

ogy to the carboxy-terminal region of the ORF2 protein in the CR1 LINE elements. The best database matches to Lucy-1 sequence are with G. gallus reverse transcriptase pol-like—72% positivity, from position 66–140 of Lucy1—(Silva and Burch, 1989) and with the SR1 non-LTR retrotransposon from the human blood fluke Schistosoma mansoni—67% positivity, from position 66–131—(Drew and Brindley, 1997). A modest identity is found with the P. spixii CR1–ORF2—60% positivity, from position 51– 149—(Kajikawa et al., 1997) and Branchiostoma floridae endonuclease/RT ORF (65% positivity, from position 63– 140) which is the first evidence of retrotransposable elements in lower chordates (Albalat et al., 2003). Moreover, Lucy-1, of 183-bp in length, is 5V-truncated. It is flanked by perfect direct 5-bp repeats representing the duplication target site that occurred when this element transposed to its genomic location (Fig. 4). This usually occurs for most CR1 elements (Silva and Burch, 1989).

Fig. 4. Structure of Lucy-1 element. The 3VUTR sequence of the Lucy-1 element with turtle PsCR1 (Kajikawa et al., 1997) is aligned using CLUSTAL W program; stars indicate identical nucleotides and gaps ( ) have been introduced to maximize homology. The highly conserved region is in bold. The 8-bp direct repeats are indicated by arrows and the Lucy-1 insertion site (CAGGA) is shown. The sequence 51–150 (from the 5V-end of R.E.1 sequence) indicates the carboxy-terminal RT region.

S. Fantaccione et al. / Gene 339 (2004) 189–198

3.1.2. R.E.2 characterization Here, we have also identified a new SINE element designated P.s.1/SINE that is included in R.E.2 sequence (Fig. 5A). The P.s.1/SINE element is 116-bp in length and includes a 67-bp domain, from 510 to 576 nt. This is homologous to the tRNALys gene from several organisms (Fig. 5C), as reported for most other SINE elements (Okada, 1991; Ogiwara et al., 1999). This tRNALys-related region contains two sequences promoter of RNA Polymerase III

193

(Fig. 5B). Moreover, the sequence from 586 to 626 nt, located downstream of tRNA-related domain, is homologous to A. carolinensis rhodopsin gene intron IV (87% identity with L31503.1 clone from 9680 to 9640 nt). This stretch of 41-bp represents the tRNA-unrelated region characteristic of the SINE elements (Fig. 5C). Both, Lucy1 CR1-like LINE and P.s.1/SINE elements are very similar to clone L31503.1 (9680–9640 nt) of intron IV rhodopsin gene.

Fig. 5. (A) R.E.2 nucleotide sequence containing the P.s.1/SINE element. tRNALys-related region is underlined and tRNA-unrelated region is in bold. (B) Box A and box B indicate the consensus sequences of promoters for RNA Polymerase III (Galli et al., 1981). (C) Schematic representation of the composite structure of the P.s.1/SINE element.

194

S. Fantaccione et al. / Gene 339 (2004) 189–198

Fig. 6. The 3V-end of 3VUTR Lucy-1 and the tRNA-unrelated region of P.s.1/SINE are aligned. Identical nucleotides are indicated by stars. The nucleotides in Lucy-1 and P.s.1/SINE elements are numbered from the 5V-terminus of R.E.1 and R.E.2 sequences, respectively.

3.1.3. The 3V-ends of the Lucy-1 and P.s.1/SINE elements are similar The sequence of 36-bp at the 3V-end tail region of the Lucy-1 is similar to the tRNA-unrelated region of the P.s.1/ SINE element and both sequences are strongly conserved, as shown in Fig. 6. Moreover, the 3V-terminal repeated sequences of P.s.1/SINE are not simple a AT-rich repeats, but they include sequences similar to the Lucy-1 element imperfect octameric repeats. To examine the genomic organization of the Lucy-1 CR1-like LINE and P.s.1/SINE elements, a Southern blot of genomic DNAs was hybridized with probe 1, under highstringency conditions. As shown in Fig. 7, the probe detected the smear bands in each of the digested P.s. genomic DNAs, suggesting that both elements are widespread in the genome of this species. In contrast, when probe 1 (control) was hybridized to a Southern blot containing human, mouse, bat, frog and fish DNA, no hybridization is evident.

showed that the 700-bp product, named by us INV A, contains a sequence (from position 20 to 70 nt) similar to peroximal Ca2+-dependent carrier-like protein mRNA found in Rattus norvegicus (84% identity; GenBank accession no. XM227597.1). Moreover, INV A includes a 90-nt sequence (from position 21 to 111 nt with 80% identity) similar to calcium binding transporter mRNA found in H. sapiens (GenBank accession no. AF123303). We also found these homologies by a deduced amino acid sequence analysis of INV A. In fact, high scores were obtained with highly conserved domain (accession no. PF00153) of the Ca2+dependent mitochondrial carrier superfamily (Mashima et al., 2003).

3.2. Further characterization of the Lucy-1 CR1-like LINE element To investigate the genomic organization around the Lucy-1 insertion site we performed Inverse PCR strategy. 3.2.1. Determination of 3V-flanking region Products ranging in length from about 0.5 to 0.7 kb were obtained from 3V-Inverse PCR (Fig. 8A). Sequence analysis

Fig. 7. Southern hybridization of the Lucy-1 CR1-like LINE and P.s.1/ SINE elements. P.s. genomic DNAs are digested with different enzymes (lane 1, EcoRI; lane 2, HindIII; lane 3, AluI; lane 4, NsiI; lane 5, HaeIII) and hybridized with probe 1. The subsequent lanes (from 6 to 10) contain EcoRI digested of 8 Ag genomic DNA samples isolated from, in order, Homo sapiens, Mus musculus, Pipistrellus kuhli, Rana esculenta and Salmo trout. DNA size marker is into lane M.

Fig. 8. (A) Inverse PCR of NsiI-digested P.s. genomic DNA ligated: lane M, DNA size standard; lane 1, 5V-Inverse PCR from 2 h digestion; lane 2, 5V-Inverse PCR from 4h digestion; lanes 3–4, 3V-Inverse PCR from 2 h digestion; lanes 5–6, 3V-Inverse PCR from 4 h digestion. Arrows indicate the positions of INV A (700 bp) and INV B (430 bp). (B) A PCR reamplification of INV B (Lanes: R, PCR reamplification pattern; m, DNA size-standard).

S. Fantaccione et al. / Gene 339 (2004) 189–198

195

To investigate genomic organization around the Lucy-1 insertion site, we evaluated the homology between INV A sequence and calcium binding transporter (cbt) gene found in R. norvegicus (gene=LOC310791; GenBank accession no. NW047627). Results obtained using the Clustal W program suggest that an 101-nt sequence downstream of Lucy-1 element is homologous to a part of the 68 exon of gene (23691–23781 nt) and the homologous region should be further extended up to the GT/AG splicing rule (Fig. 9A).

highly conserved in four sequenced clones, as shown in Fig. 10. This 72 bp-region is homologous to the 5V-portion of the 68 exon of cbt gene up to nucleotide 23631 (Fig. 9A). The results indicate that the homologous region should be further extended in both the 5V- and 3V-flanking regions of the Lucy-1. Thus, we concluded that Lucy-1 CR1-like LINE element was inserted into the exon of an ancestor cbt gene (Fig. 9B).

3.2.2. Determination of 5V-flanking region Products ranging in length from about 0.45 kb were obtained from 5V-Inverse PCR (Fig. 8A). A 430-bp band, named by us INV B, was reamplified and cloned (Fig. 8B). The complete nucleotide sequence of four clones and a monomer consensus sequence were determined (Fig. 10). Remarkably, structure analysis revealed that INV B sequence consensus contains 13 copies of 27-bp direct repeats, flanked at 5V-end by sequence of 72-bp that is

4. Discussion We provide a new example, in P.s. genome, among the pairs of SINEs and LINEs proposed. Our results add further support to the model of the way in which non-autonomous SINEs might have recruited the enzymatic machinery for their retroposition during evolution (Luan and Eickbush, 1995; Moran et al., 1996). In fact, as proposed by Ogiwara et al. (1999), an RTase encoded by a LINE recognizes the

Fig. 9. (A) Alignment of the 5V- and 3V-flanking sequences of the Lucy-1 and the 68 exon of the calcium binding transporter gene (accession number NW047627). Stars indicate identical nucleotides and gaps ( ) have been introduced to maximize homology. The GT/AG splicing rule is indicated by arrow. (B) Molecular organization around Lucy-1 insertion site. The stars indicate the 13 copies of 27-bp direct repeats. V and VI indicated above the open boxes show the introns, while 6 below the black box shows the 68 exon.

196

S. Fantaccione et al. / Gene 339 (2004) 189–198

Fig. 10. INV B consensus sequence. The 72-bp region is boxed. The monomeric unit (27 bp) is indicated by arrow. Dots indicate nucleotides from all sequenced clones identical to those of the consensus sequence.

3V-tail region of LINEs the same way it may recognize the 3V-tail region of SINEs during retroposition. Fig. 6 shows comparisons nucleotide sequences between the tail regions of the Lucy-1 CR1-like LINE and that of the P.s.1/SINE. Several observations indicate that the two elements constitute another pair of SINE and LINE sharing the same tail region. First, we demonstrated that the 3V-end of 3VUTR Lucy-1 element and the tRNA-unrelated region of P.s.1/SINE are almost identical in the P.s. genome. This identity suggests that the enzymatic machinery responsible for the retroposition of the P.s.1/SINE might be the same as the one responsible for the retrotransposition of Lucy-1 CR1-like LINE. Moreover, the Lucy-1 ends in 8-bp 3V-tandem repeats that are nearly identical to the characteristic 3V-terminal repeats of the CR1 and CR1-like elements. In particular, the direct repeats of Lucy-1 are also found in the 3V-tail region of our P.s.1/SINE element, unlike most of tRNA-derived SINEs having an AT-rich region at their 3V-termini. This AT-rich tail probably reflects the participation of other cellular components in the integration of SINEs (Kajikawa et al., 1997). We hypothesize that during evolution the P.s.1/SINE element has gained the 3V-end sequence of the Lucy-1 element and has exclusively recruited the enzymatic machinery of its partner CR1 LINE for retroposition. These findings provide further evidence that when there is a simple tRNA-derived SINE family, a LINE family, which has the same 3V-end as the tRNA-derived SINE family, must always be present in the genome of the same organism (Ohshima et al., 1996). In this regard, we propose a possible mechanism for the generation of P.s.1/SINE element based on its chimerical

structure. In fact, P.s.1/SINE has a composite structure, with a tRNALys-related region possessing two promoter sequences of RNA Polymerase III and a tRNA-unrelated region homologous to the 3V-tail region of Lucy-1. Several families of SINEs are generated by recombination between a mature tRNALys and a pre-existing LINE at a certain stage of evolution (Okada and Hamada, 1997; Ogiwara et al., 1999). This model is based on the observation that the tRNArelated regions of these families of SINEs end with the sequence CCA, which is added post-transcriptionally to tRNAs and is not encoded in their respective genes (Okada and Hamada, 1997). On the other hand, the tRNA-related region of our P.s.1/SINE element does not end with the CCA sequence. This suggests that P.s.1/SINE may have come by recombination from an already-existing tRNA pseudogene, unlike most tRNA-derived SINEs. Our hypothesis is in agreement with Daniels and Deininger (1983) who proposed that SINEs may have been generated from tDNAs that accumulated mutations, which did not hinder the intrinsic functions of tRNAs. An example of this SINEs organization is provided by BovtA. This appears to have a structure that reflects recombination of two independent units, namely, a tRNA pseudogene, with promoter activity for RNA polymerase, and BovA (Okada and Hamada, 1997). Another example of this architecture is the galago type II family, in which a tRNA pseudogene and the primate Alu monomer have been recombined (Daniels and Deininger, 1983; Okada and Hamada, 1997). Moreover, we investigated the genomic organization around the Lucy-1 insertion site in P.s. genome. As shown previously, we found that the Lucy-1 flanking regions, in both upstream and downstream sites, are contiguously homologous to 68 exon of cbt gene found in R. norvegicus.

S. Fantaccione et al. / Gene 339 (2004) 189–198

The 101-nt sequence downstream of Lucy-1 element is homologous to a part of the 68 exon of gene up to the GT/ AG splicing rule. In connection with this, a segment of 72bp in the 5V-flanking region of Lucy-1 is also found to be homologous to the 5V-portion of 68 exon of cbt gene up to nucleotide 23631. These facts suggest that Lucy-1 CR1-like LINE element at one time transposed into a cbt gene, belonging to Ca2+-dependent mitochondrial carrier superfamily. Moreover, compared to cbt gene we found that Lucy-1 CR1-like LINE element is inserted in the opposite direction. An example of this architecture is provided by two CR1-like LINE sequences (CR1-S and CR1-L) that are tandemly inserted in the opposite direction as the PLA2 gene (Fujimi et al., 2002). There is no specific biological significance for the opposite direction of the gene target. The literature reports that the retrotransposons sculpt vertebrate genomes and behave as insertional mutagens either by distrupting exons or by inserting into introns, leading to mis-splicing (Deininger et al., 2003). Narita et al. (1993) reported that insertion of a 5V-truncated L1 element into the 3V-end of exon 44 of the dystrophin gene resulted in skipping of the exon during splicing in a case of Duchenne muscular dystrophy (DMD). In fact, these elements create a diverse set of genomic changes during and after their integration that are subject to population influences and major changes in amplification potential of different elements with evolutionary time (Deininger et al., 2003). An interesting view is to consider that the composition of any genome is not static and must inevitably change with time (Mighell et al., 2000). The retrotransposition is essential in the evolution of complex genomes and contributes to the generation of new pseudogenes. The majority of retrotransposed genes are inactivated to processed pseudogenes, but in a few instances the retrotransposon is maintained as a functional, intronless gene. Two intronless retrotransposons, wPGK-1 and PGK-2, were derived from the functional, intron-containing human PGK-1 gene. wPGK-1 is a typical processed pseudogene, but PGK-2 generates transcripts that are translated into a protein with a high homology to that encoded by PGK-1 (Mighell et al., 2000). It is noteworthy that the retrotransposons can be an additional source of genomic diversification and we showed that the evolution of the transposable elements can be a vector driving evolution by shaping genomes reassorting DNA domains and amplifying DNA (Pontecorvo et al., 2000). Unlike to CR1 elements reported to date, we found that the 5V-flanking region of Lucy-1 element consists of tandemly arranged arrays with basic repeats around 27-bp long. The short direct repeats likely originated during or after a retrotransposition event and a duplication mechanism could give rise to the tandem array. In fact, during evolution the inactivated transposable elements continue to accumulate mutations following DNA amplification by tandem duplication resulting in satellite DNA (Pontecorvo et al., 2000).

197

Finally, we argue that the similarity to several CR1 and CR1-like elements, as shown previously, suggests a close phylogenetic relationship between the Lucy-1 element and the bB subfamilyQ of CR1 elements. In fact, Vandergon and Reitman (1994) proposed that the chicken CR1 elements group into at least six subfamilies (A, B, C, D, E and F). Our results are in agreement with the evolutionary model of CR1 family proposed by Vandergon and Reitman (1994) who postulate that CR1s in both Avian and Reptilian species suggest an origin for this element before the divergence of these Vertebrate classes. Further studies are needed to better understand the way CR1 LINEs and SINEs elements could influence the structure and evolution of higher eukaryote genomes.

References Albalat, R., Permanyer, J., Canestro, C., Martinez-Mir, A., GonzalezAngulo, O., Gonzalez-Duarte, R., 2003. The first non-LTR retrotransposon characterised in the cephalochordate amphioxus, Bf CR1, shows similarities to CR1-like elements. Cell. Mol. Life Sci. 60, 803 – 809. Chen, Z.Q., Ritzel, R.G., Lin, C.C., Hodgetts, R.B., 1991. Sequence conservation in avian CR1: an interspersed repetitive DNA family evolving under functional constraints. Proc. Natl. Acad. Sci. U. S. A. 88, 5814 – 5818. Daniels, G.R., Deininger, P.L., 1983. A second major class of Alu family repeated DNA sequences in a primate genome. Nucleic Acids Res. 11, 7595 – 7610. Deininger, P.L., Moran, J.V., Batzer, M.A., Kazazian Jr., H.H., 2003. Mobile elements and mammalian genome evolution. Curr. Opin. Genet. Dev. 13, 651 – 658. Drew, A.C., Brindley, P.J., 1997. A retrotransposon of the non-long terminal repeat class from the human blood fluke Schistosoma mansoni. Similarities to the chicken-repeat-1-like elements of vertebrates. Mol. Biol. Evol. 14, 610 – 692. Eickbush, T.H., 1992. Transposing without ends: the non-LTR retrotransposable elements. New Biol. 4, 430 – 440. Fujimi, T.J., Tsuchiya, T., Tamiya, T., 2002. A comparative analysis of invaded sequences from group IA phospholipase A2 provides evidence about the divergence period of genes groups and snake families. Toxicon 40, 873 – 884. Galli, G., Hofstetter, H., Birnstiel, M.L., 1981. Two conserved sequence blocks within eukaryotic tRNA genes are major promoter elements. Nature 294, 626 – 631. Gilbert, N., Labuda, D., 1999. CORE-SINEs: eukaryotic short interspersed retroposing elements with common sequence motifs. Proc. Natl. Acad. Sci. U. S. A. 96, 2869 – 2874. Haas, N.B., Grabowski, J.M., North, J., Moran, J.V., Kazazian, H.H., Burch, J.B., 2001. Subfamilies of CR1 non-LTR retrotransposon have different 5V UTR sequences but are otherwise conserved. Gene 265, 175 – 183. Humphrey, G.W., 1995. SINEs and trans-acting factors. In: Maraia, R.J. (Ed.), The Impact of Short Interspersed Elements (SINEs) on the Host Genome. R.G. Landes, Austin, TX, pp. 197 – 222. Jurka, J., 2000. Repbase update: a database and an electronic journal of repetitive elements. Trends Genet. 16, 418 – 420. Kajikawa, M., Ohshima, K., Okada, N., 1997. Determination of the entire sequence of turtle CR1: the first open reading frame of the turtle CR1 element encodes a protein with a novel zinc finger motif. Mol. Biol. Evol. 14, 1206 – 1217. Kawamura, S., Yokoyama, S., 1994. Cloning of the rhodopsin-encoding gene from the rod-less lizard Anolis carolinensis. Gene 149, 267 – 270.

198

S. Fantaccione et al. / Gene 339 (2004) 189–198

Luan, D.D., Eickbush, T.H., 1995. RNA template requirements for target DNA-primed reverse transcription by the R2 retrotransposable element. Mol. Cell Biol. 15, 3882 – 3891. Luan, D.D., Korman, M.H., Jakubczak, J.L., Eickbush, T.H., 1993. Reverse transcription of R2Bm RNA is primed by a nick at the chromosomal target site: a mechanism for non-LTR retrotransposition. Cell 72, 595 – 605. Maniatis, T., Fritsch, E.F., Sambrook, J., 1982. Molecular Cloning, A Laboratory Manual. Cold Spring Harbor Laboratory, New York. Mashima, H., Ueda, N., Ohno, H., Suzuki, J., Ohnishi, H., Yasuda, H., Tsuchida, T., Kanamaru, C., Makita, N., Iiri, T., Omata, M., Kojima, I., 2003. A novel mitochondrial Ca2+-dependent solute carrier in the liver identified by mRNA differential display. J. Biol. Chem. 278, 9520 – 9527. Mighell, A.J., Smith, N.R., Robinson, P.A., Markham, A.F., 2000. Vertebrate pseudogenes. FEBS Lett. 468, 109 – 114. Moran, J.V., Holmess, S.E., Naas, T.P., DeBerardinis, R.J., Boeke, J.D., Kazazian Jr., H.H., 1996. High frequency retrotransposition in cultured mammalian cells. Cell 87, 917 – 927. Narita, N., Nishio, H., Kitoh, Y., Ishikawa, Y., Ishikawa, Y., Minami, R., Nakamura, H., Matsuo, M., 1993. Insertion of a 5V truncated L1 element into the 3Vend of exon 44 of the dystrophin gene resulted in skipping of the exon during splicing in a case of Duchenne muscolar dystrophy. J. Clin. Invest. 91, 1862 – 1867. Nobuhisa, I., Ogawa, T., Deshimaru, M., Chijiwa, T., Nakashima, K.I., Chuman, Y., Shimohigashi, Y., Fukumaki, Y., Hattori, S., Ohno, M., 1998. Retrotransposable CR1-like elements in Crotalinae snake genomes. Toxicon 36, 915 – 920. Ogiwara, I., Miya, M., Ohshima, K., Okada, N., 1999. Retropositional parasitism of SINEs on LINEs: identification of SINEs and LINEs in elasmobranchs. Mol. Biol. Evol. 16, 1238 – 1250. Ohshima, K., Hamada, M., Terai, Y., Okada, N., 1996. The 3V ends of tRNA-derived short interspersed repetitive elements are derived from the 3Vends of long interspersed repetitive elements. Mol. Cell Biol. 16, 3756 – 3764. Okada, N., 1991. SINEs: short interspersed repeated elements of the eukaryotic genome. Trends Ecol. Evol. 6, 358 – 361.

Okada, N., Hamada, M., 1997. The 3V ends of tRNA-derived SINEs originated from the 3Vends of LINEs: a new example from the bovine genome. J. Mol. Evol. 44, S52 – S56. Okada, N., Hamada, M., Ogiwara, I., Ohshima, K., 1997. SINEs and LINEs share common 3Vsequences: a review. Gene 205, 229 – 243. Pontecorvo, G., De Felice, B., Carfagna, M., 2000. A novel repeated sequence DNA originated from a Tc1-like transposon in water green frog Rana esculenta. Gene 261, 205 – 210. Poulter, R., Butler, M., Ormandy, J., 1999. A LINE element from the pufferfish (fugu) Fugu rubripes which shows similarity to the CR1 family of non-LTR retrotransposon. Gene 227, 169 – 179. Silva, R., Burch, J.B., 1989. Evidence that chicken CR1 elements represent a novel family of retrotransposon. Mol. Cell Biol. 9, 3563 – 3566. Smit, A.F.A., 1996. The origin of interspersed repeats in the human genome. Curr. Opin. Genet. Dev. 6, 743 – 748. Stumph, W.E., Baez, M., Beattie, W.G., Tsai, M.J., O’Malley, B.W., 1983. Characterization of deoxyribonucleic acid sequence at the 5V and 3V borders of the 100 kilobase pair ovalbumin gene domain. Biochemistry 22, 306 – 315. Szemraj, J., Plucienniczak, G., Jaworski, J., Plucienniczak, A., 1995. Bovine Alu-like sequences mediate transposition of a new site-specific retroelement. Gene 152, 261 – 264. Terai, Y., Takahashi, K., Okada, N., 1998. SINE cousins: the 3V-end tails of two oldest and distantly related families of SINEs with the same genealogical origin. Mol. Biol. Evol. 15, 1460 – 1471. Van Dellen, K., Field, J., Wang, Z., Loftus, B., Samuelson, J., 2002. LINEs and SINE-like elements of the protist Entamoeba histolytica. Gene 297, 229 – 239. Vandergon, T.L., Reitman, M., 1994. Evolution of chicken repeat 1 (CR1): evidences for ancient subfamilies and multiple progenitors. Mol. Biol. Evol. 11, 886 – 898. Weiner, A.M., Deininger, P.L., Efstratiadis, A., 1986. Nonviral retroposons: genes, pseudogenes, and transposable elements generated by the reverse flow of genetic information. Annu. Rev. Biochem. 55, 631 – 661.