Both Sense and Antisense Strands of the LTR of the Schistosoma Mansoni Pao-Like Retrotransposon Sinbad Drive Luciferase Expression

Mol Genet Genomics DOI 10.1007/s00438-006-0181-1

ORIGINAL PAPER

Both sense and antisense strands of the LTR of the Schistosoma mansoni Pao-like retrotransposon Sinbad drive luciferase expression

Claudia S. Copeland · Victoria H. Mann · Paul J. Brindley

Received: 20 August 2006 / Accepted: 4 October 2006 © Springer-Verlag 2006

Abstract Long terminal repeat (LTR) retrotranspo- activity in HeLa cells. The ability of the Sinbad LTR to sons, mobile genetic elements comprising substantial transcribe in both its forward and inverted orientation proportions of many eukaryotic genomes, are so represents one of few documented examples of bidirec- named for the presence of LTRs, direct repeats about tional promotor function. 250–600 bp in length Xanking the open reading frames that encode the retrotransposon enzymes and struc- Keywords Schistosome · Boudicca · Promotor · tural proteins. LTRs include promotor functions as Promoter · Bidirectional · Inverse well as other roles in retrotransposition. LTR retrotransposons, including the Gypsy-like Boudicca and the Pao/BEL-like Sinbad elements, comprise a sub- Introduction stantial proportion of the genome of the human blood Xuke, Schistosoma mansoni. In order to deduce the Eukaryotic genomes generally contain substantial capability of speciWc copies of Boudicca and Sinbad amounts of repetitive sequences, many of which repre- LTRs to function as promotors, these LTRs were sent mobile genetic elements (e.g., Lander et al. 2001; investigated analytically and experimentally. Sequence Holt et al. 2002). These mobile elements have played analysis revealed the presence of TATA boxes, canoni- fundamental roles in host genome evolution (Charles- cal polyadenylation signals, and direct inverted repeats worth et al. 1994; Deininger and Batzer 2002). within the LTRs of both the Boudicca and Sinbad ret- Although less is known about the genome of the platy- rotransposons. Inserted in the reporter plasmid pGL3, helminth parasite Schistosoma than more extensively the LTR of Sinbad drove WreXy luciferase activity in studied genomes such as human and mouse, recent HeLa cells in its forward and inverted orientation. In Wndings suggest that up to half of this genome may be contrast, the LTR of Boudicca did not drive luciferase comprised of repetitive sequences, and much of this repetitive complement will consist of mobile genetic elements (see Brindley 2005). Within this population Communicated by M.-A. Grandbastien. of mobile elements, 15% or more of the schistosome genome may be composed of long terminal repeat C. S. Copeland · V. H. Mann · P. J. Brindley (LTR) retrotransposons (Copeland et al. 2005b). Department of Tropical Medicine, and Interdisciplinary Long terminal repeat retrotransposons (and retrovi- Program in Molecular and Cellular Biology, ruses) are mobile genetic elements that reproduce Tulane University Health Sciences Center, 1430 Tulane Avenue, New Orleans, LA 70112, USA through an RNA intermediate. Typically 5–10 kb in length, their general structure consists of two open C. S. Copeland (&) reading frames (ORFs) Xanked by long direct terminal Center for Medical, Agricultural, and Veterinary repeats of »200–600 bp in length. These LTRs play a Entomology, USDA Agricultural Research Service, 1700 SW 23rd Drive, Gainesville, FL 32608, USA pivotal role in retrotransposition and transcription ini- e-mail: [email protected] tiation. The 5Ј LTR encodes the promotor, which 13 Mol Genet Genomics directs transcription by host cell RNA polymerase II. Retrotransposon LTRs and associated regulatory ele- The LTRs’ roles in retrotransposition include the nine- ments can be considered just as critical as the coding step process of reverse transcription (Gilboa et al. sequences in that they can facilitate transposition of 1979) and the integration of the proviral form of the the retroelement, even when the coding sequence is retrotransposon or retrovirus into the host cell chro- too degraded to encode functional proteins. Transposi- mosomes (Sharon et al. 1994; Hindmarsh and Leis tion of degenerate retrotransposons incapacitated by 1999). LTRs are constituted of three main parts, the internal mutation(s) can be accomplished through the U3 unique region, the repeated (R) region, and the U5 activity of functional proteins encoded by non-mutated unique region (CoYn et al. 1997) (Fig. 1a). The U3 copies, a process known as retrotransposition in trans region includes both promotor and enhancer elements, (Curcio and GarWnkel 1994). and is only found in the 3Ј LTR in the RNA genome of The inXuence of LTRs extends beyond transcription the retrotransposon, since transcription begins down- and transposition of the retrotransposons and retrovi- stream of the U3 in the 5Ј LTR. Transcription begins at ruses themselves. They have also been co-opted by (and deWnes) the 5Ј end of the R region, which is their host genomes as promotors of native genes (Med- repeated at both ends of the transcript. The U5 from strand et al. 2001; Dunn et al. 2003). In some of these the 3Ј LTR is replaced with a poly-A tail, so that in the cases, the LTRs serve as alternative promotors that are transcript, the U5 is only found in the 5Ј LTR (CoYn utilized in addition to the native promotors, and some et al. 1997). The U5 region includes a GT-rich region of these initiate transcription in a tissue speciWc man- that encodes control elements for 3Ј end processing. ner. For example, the human endothelin B receptor The R region is integral to the transfer of DNA synthe- utilizes an LTR derived promotor in addition to its sis between the 5Ј and 3Ј ends of the mRNA template native promotor, but the LTR driven transcripts during reverse transcription. The poly-A tail begins at appear only in placental tissue (Medstrand et al. 2001). the end of the R region of the 3Ј LTR, and by doing so Since expression of LTR retrotransposons has been deWnes the 3Ј boundary of the R region. The polyaden- shown to increase in tissues related to reproduction ylation signal is located either in the 3Ј end of the U3 (Dupressoir and Heidmann 1996; Lecher et al. 1997), region or in the R region. (Polyadenylation signals this pattern of transcription by LTRs acting as promo- found outside the LTRs of retroviruses such as HTLV- tors of native genes is a logical adaptation of function 1 are associated with reverse ORFs (Cavanagh et al. by the host genome. 2006). LTR retrotransposons and simpler retroviruses Schistosomiasis is considered the most important of contain only forward ORFs.) Located at the distal mar- the human helminthiases, and ranks second only to gin of many LTR retrotransposons are small repeats of malaria among all tropical diseases, in terms of mor- sequence TCN-NCA, termed the Dinucleotide bidity and mortality (Chitsulo et al. 2004). Up to 15% Inverted Repeats (DIR) (Bowen and McDonald 1999). of the schistosome genome may be composed of LTR These repeats are the byproducts of the loss of two retrotransposons (Copeland et al. 2005b). Understand- nucleotides (nt) in the initial nucleophilic attack which ing structural and functional aspects of these elements begins the process of integration of the retrotranspo- will enhance the general understanding of the schisto- son into the genome of the host (Hindmarsh and Leis some genome. It is hoped that this will guide improve- 1999). Longer (at least »8–12 bp for retroviruses), ments in the control of schistosomiasis, including the often perfectly inverted terminal sequences (terminal development of vaccines and new antiparasite medica- inverted repeats, or TIRs) are required for recognition tions. In addition to their natural roles as the promo- by integrase during the process of integration into host tors of LTR retrotransposons, LTRs are of interest for genomic DNA (Katzman et al. 1989). their potential in applications related to transgenesis Three other key elements associated with the LTR (i.e., Tanaka et al. 1998). Because of their natural tar- are the primer binding site (PBS), polypurine tract get speciWcity, LTRs of native schistosome retrotrans- (PPT), and encapsidation sequence, psi (). The PBS, posons are of potential use as components of located immediately downstream of the 5Ј LTR, is the transgenesis vectors to facilitate integration into the binding site for the tRNA primer for negative strand schistosome genome. Transduction of Schistosoma DNA synthesis. The PPT is located at the other end of mansoni using retroviral constructs has recently been the retrotransposon, just upstream of the 3Ј LTR, and demonstrated (Kines et al. 2006). Stable integration of is involved in the second (plus strand) DNA strand transgenes into the genome would in turn enable the synthesis during reverse transcription. Downstream of investigation of schistosome gene function and the devel- the PBS and overlapping gag is the encapsidation opment of transgenic schistosomes. The promotor func- sequence (), also known as the packaging signal. tions of LTRs could also be exploited in transgenesis 13 Mol Genet Genomics

Fig. 1 Generalized structure of an LTR (a) and sequence alignments of 5Ј and 3Ј LTRs of (b) Sinbad and (c) Boudic- ca. Dinucleotide inverted repeats (DIRs) are marked with solid underline (red in online version). Seven nucleotide terminal inverted repeats in the Sinbad LTR are identi- Wed with double underline (orange in online version). Possible transcription initiation sites (TATA and CAAT sequences) are indicated with round dotted underline (green in online version). Polyadeny- lation signals (AATAAA or AATATA) are marked with square dotted underline (blue in online version). A GT rich region in Sinbad’s LTR is marked with dashed underline (purple in online version)

systems if the LTRs have been demonstrated to be of Boudicca and Sinbad and their ability to drive WreXy capable of initiating transcription. luciferase expression in mammalian cells. We cloned Boudicca and Sinbad are discrete LTR retrotranspo- both forward and reverse versions of each LTR into sons recently characterized from the genome of S. the luciferase expression vector pGL3, transformed mansoni (Copeland et al. 2003, 2005a). The 386 bp HeLa cells with the constructs, and investigated the long LTRs of the copy of Sinbad characterized by capacity and performance of the LTRs of Boudicca Copeland et al. (2005a) were identical in sequence, and Sinbad in driving luciferase expression in these implying recent insertion and competence to carry out cells. their roles in transcription initiation and integration (Bowen and McDonald 1999). The LTRs of the copy of Boudicca characterized in Copeland et al. (2003) Materials and methods were 88.4% identical, with the 327 bp long 5Ј LTR representing an apparently more intact version, as com- Construction of LTR-luciferase expression vectors pared with the consensus of several fragments of the LTRs of other copies of Boudicca (Copeland et al. The 5Ј LTRs of Boudicca and Sinbad were ampliWed 2003). Here we investigated the structure of the LTRs from clones of a BAC library representing an eightfold

13 Mol Genet Genomics coverage of the S. mansoni genome in DH10B Escheri- gel-puriWed PCR products and the promotorless lucif- chia coli cells (Le Paslier et al. 2000). BAC clone 53-J-5 erase expression vector pGL3-Basic (Promega) were and 33-N-3 have been shown previously to contain cop- digested with Kpn I and Hind III. The ends of the line- ies of Boudicca (Copeland et al. 2003) and Sinbad arized pGL3 vector were dephosphorylated by incu- (Copeland et al. 2005a), respectively. Primers were bating with Antarctic Phosphatase (New England designed from the S. mansoni genomic DNA just BioLabs, Beverly, MA, USA) for 30 min at 37°C fol- upstream of the 5Ј LTR of each retrotransposon (fwd) lowed by heat inactivation for 5 min at 65°C. The and the untranslated region just downstream of the 5Ј digested LTRs were ligated into the dephosphorylated, LTR (rev). The 5Ј LTR of Boudicca was ampliWed linearized pGL3 plasmid after which the ligation prod- from BAC 53-J-5 and the 5Ј LTR of Sinbad was ampli- ucts were employed to transform electrocompetent E. Wed from BAC 33-N-3, using as the templates BAC coli cells (EP-Max 10B Competent Cells, Bio-Rad, plasmid DNA isolated from transformed E. coli using Hercules, CA, USA) by electroporation (Bio-Rad the Phaseprep kit from Sigma (St. Louis, MO USA). GenePulser Xcell). Plasmids were puriWed from trans- DNA was allowed to denature at 95°C, followed by 30 formed cells and the sequence of the inserts deter- denaturation-annealing-extension cycles of 95°C mined (Davis Sequencing, Davis, CA, USA) to (1 min), 56°C (1 min), and 72°C (1 min). Primers conWrm identity and orientation of the retrotransposon included restriction sites for Kpn 1 and Hind III to LTR inserts. allow cloning of forward and reverse LTRs. Four sets of primers were designed; two with the Kpn 1 and Hind Sequence analysis and alignment of LTR sequences III sites positioned such that the LTR would be cloned into an expression vector in the forward orientation, Nucleotide sequence data were analyzed using SeqMan and two with restriction sites positioned such that the (DNAstar Inc., Madison, WI, USA) and VecScreen LTR would be cloned in the inverted orientation (NCBI) software. The LTR sequences have been (Fig. 2a) Primer sequences are shown in Table 1. assigned GenBank accession numbers; AY965072, The PCR products were isolated using the Wizard AY965073, AY965074, and AY965075. Sequence align- system (Promega, Madison, WI, USA), after which ments were accomplished with ClustalW (Thompson

A Amp B

F1 ori

pGL3-Basic

Luc+ (no promotor)

Amp

F1 ori pGL3-Control (SV40) pGL3-Sinbad LTR

Luc+ pGL3-Boudicca LTR pGL3-GST-28A

Promotor

pG Boudic Boudic Si Sinbad, invGST28-A Amp L3-basic nbad

ca ca, inv. F1 ori pGL3-Sinbad LTR, . Inverse Luc+ pGL3-Boudicca LTR, Inverse

Promotor

Fig. 2 a Luciferase expression vector constructs. Promotors the promotorless luciferase expression vector (negative control), (LTRs) were cloned into a multiple cloning site 5Ј of the lucifer- pGL3 with the Boudicca LTR as promotor, pGL3 with an invert- ase coding sequence. The pGL3 Basic construct does not have a ed Boudicca LTR as promotor, pGL3 with the Sinbad LTR as promotor for the luciferase gene. b Mean luciferase activity, mea- promotor, pGL3 with an inverted Sinbad LTR as promotor, sured in relative light units per mg total protein (RLU/mg pro- pGL3 with the S. mansoni glutathione-S-transferase promotor, tein). From left to right, constructs measured are: pGL3-Basic, GST28-A (positive control)

13 Mol Genet Genomics

Table 1 Primers used for PCR ampliWcation of forward and inverse LTRs of Boudicca and Sinbad Retrotransposon LTR Primer Sequence orientation

Boudicca Forward fwd 5Ј-CGGGGTACCTGTAGCTGTAAATAGTTCCCC-3Ј Boudicca Forward rev 5Ј-CCCAAGCTTTGAGGATACGACTAGTATTCTA-3Ј Boudicca Inverse fwd 5Ј-CGGGGTACCTGAGGATACGACTAGTATTCTA-3Ј Boudicca Inverse rev 5Ј-CCCAAGCTTTGTAGCTGTAAATAGTTCCCC-3Ј Sinbad Forward fwd 5Ј-CGGGGTACCAAAGACGCAAAGAGATGGTT-3Ј Sinbad Forward rev 5Ј-CCCAAGCTTATCCAGTGTGAACACAGAACC-3Ј Sinbad Inverse fwd 5Ј-CGGGGTACCATCCAGTGTGAACACAGAACC-3Ј Sinbad Inverse rev 5Ј-CCCAAGCTTAAAGACGCAAAGAGATGGTT-3Ј

et al. 1994) and MacVector software (Accelrys, San which they were incubated for 24 h at 37°C under 5%

Diego, CA, USA). CO2 in air. The assay was replicated four times.

Transfection of HeLa cells Luciferase assay

The HeLa cell line was purchased from ATCC Luciferase activity was determined using the Lucifer- (Manassas, VA, USA) and maintained as recom- ase Assay System from Promega. Media were removed mended by the vendor. HeLa cells were transfected from transfected cells, after which 400 l of Luciferase with pGL3 plasmids using the TransFectin lipid Cell Culture Lysis Reagent (CCLR, Promega: 25 mM reagent system (Bio-Rad). About 5 £ 105 cells were Tris–phosphate (pH 7.8), 2 mM DTT, 2 mM 1,2-diami- seeded onto 60 mm diameter plates (triplicates of nocyclohexane-N,N,N’,N’-tetraacetic acid, 10% glyc- experimental groups, along with negative and positive erol, 1% Triton X-100) was added to each plate to lyse controls) and cultured overnight in Dulbecco’s Modi- the HeLa cells. The lysate was removed and half of it Wed Eagle’s Medium (DMEM) supplemented with 1% was used for the luciferase assay (the other half was penicillin/Streptomycin and 10% fetal calf serum. One stored at ¡80°C for subsequent protein measurement). microgram plasmid DNA of each pGL3 construct was One hundred microliters of Luciferase Assay Reagent diluted in 50 l serum free DMEM medium and 15 l (Promega) (luciferin substrate in ATP/Mg2+ buVer) TransFectin lipid reagent (Bio-Rad) was added to 35 l was added to 100 l cell lysate immediately before serum free DMEM. The diluted DNA and diluted lipid measuring the luciferase activity. Luciferase activity reagent were then combined and incubated at room was measured using a luminometer (Sirius, Berthold temperature (RT) for 20 min. The overnight culture Detection Systems, Bad Wildbad, Germany), with medium was removed from the cells and replaced with units of output (raw readings) in Relative Light Units 400 l serum free medium and the diluted DNA-Trans- (RLU). Two 100 l samples from each plate (above) Fectin complexes (100 l). Seven treatment groups were analyzed, and the RLU results from these two were included: Boudicca LTR; Boudicca LTR in readings were averaged to yield the raw RLU readings inverse orientation; Sinbad LTR; Sinbad LTR in for each plate. inverse orientation; S. mansoni GST28-A promoter; SV40-pGL3 control; and pGL3 Basic (Fig. 2a) [the S. Measurement of total protein mansoni GST28-A-pGL3 construct included a 1.3 kb fragment of the promoter region, exons 1 and 2, and Total protein per sample was measured using the BCA intron 1 of the gene encoding glutathione-S-transferase Protein Assay Reagent Kit (Pierce, Rockford, IL, 28 kDa (GST28) of S. mansoni. This construct, kindly USA). A standard curve was prepared by diluting provided by Dr. Edward Pearce, University of Pennsyl- known concentrations of bovine serum albumin (BSA) vania, was included here as a positive control promoter in CCLR. Two replicates of each standard and experi- of an endogenous schistosome gene known to be capa- mental sample were incubated in a cupric sulfate based ble of driving luciferase expression in HeLa cells (P. J. reagent provided in the BCA kit (0.08% cupric sulfate, Brindley et al., unpublished data)]. The cells were incu- 0.1 M sodium hydroxide, and undisclosed proportions bated at 37°C for 4 h in the presence of the transfection of sodium carbonate, sodium bicarbonate, bicinchoni- reagents, with gentle rocking. Subsequently, 2.5 ml of nic acid and sodium tartrate) at 37°C for 30 min, and DMEM with 10% serum was added to each plate, after then were cooled at RT. Absorbances at wavelength

13 Mol Genet Genomics

562 nm (OD592) were determined with a spectropho- apparently include a similar GT-rich region (Fig. 1b, c). tometer (Bio-Rad, SmartSpec 3000) within 10 min of The LTRs of both retrotransposons include DIRs, but each other. The average OD592 reading for the blank only Sinbad’s LTR includes more extended inverted standard (CCLR only) was subtracted from the aver- repeats. The locations of these regulatory signals and age reading for each sample to yield the individual the segment lengths they imply are consistent with the absorbance readings. The standard curve was used to structures of the LTRs of many retroviruses (CoYn calculate total protein for each cell lysate sample. Indi- et al. 1997), but the Sinbad LTR includes more recog- vidual luciferase RLU values for each sample were nizable signals similar to those of retroviral LTRs. then divided by total calculated protein for each sample to yield RLU/mg protein for each sample. RLU/mg Luciferase assay results for the three samples of each experimental group were determined along with standard error of Luciferase activity and total protein was measured for mean values. seven triplicate sets of transfected cells, in each of four replicates of the assay. These included triplicates for each experimental condition, Boudicca LTR forward, Results Boudicca LTR inverted, Sinbad LTR forward, and Sin- bad LTR inverted, and for three controls (Fig. 2a). All Structural analysis of retrotransposon LTRs constructs were sequenced prior to their use in the assay and were identical to the expected sequences, in A BLAST search of the non-redundant nt database GenBank accession numbers AY965072, AY965073, revealed a highly conserved region in the Sinbad LTR AY965074, and AY965075. The pGL3 Control SV40 between nt positions »200 and 230. As this region con- driven construct produced strong levels of luciferase tains a potential TATA box (Fig. 1b), this probably activity (about 100 £ that of pGL3-Basic, as expected represents the transcription start site, in the vicinity of based on data from Promega) (not shown) while the the U3/R boundary (CoYn et al. 1997). This indicated GST28-A driven construct produced moderately that the lengths of the U3 and the R + U5 region of the strong activity (Fig. 2b). The pGL3-Basic construct Sinbad LTR were each »200 bp. A BLAST search produced weak, though detectable, activity. In general, with the Boudicca LTR also revealed a conserved both the forward and inverted Boudicca LTR con- region, between nt positions »170 and 200. This was structs drove only weak or negligible luciferase activ- one of the predicted positions for a potential TATA ity, with levels similar to those from the promotorless, box for the LTR of Boudicca (Fig. 1c), and so this rep- pGL3 basic, control. In contrast, the Sinbad LTR for- resents the probable location for the transcription start ward construct drove luciferase activity at levels com- site for Boudicca. Further, this indicated that the parable to those seen with the S. mansoni GST28-A length of Boudicca’s U3 and R + U5 regions are construct (Fig. 2b). In addition, surprisingly, the Sin- »150 bp each in length. In addition to the polymerase bad LTR inverted construct drove luciferase activity at binding sites, canonical polyadenylation signals of a similar level to the Sinbad LTR forward construct sequence AATAAA (CoYn et al. 1997) and the vari- (Fig. 2b). These Wndings showing luciferase activity ant AATATA were apparent in the LTRs of both ret- with both forward and inverse orientation versions of rotransposons. the Sinbad LTR construct were observed consistently, The LTRs of both Boudicca and Sinbad exhibited in each of the four replicates of the experiment (not many of the hallmark features of LTRs of retroviruses. shown). The polyadenylation signal for the Boudicca LTR is located about 10–40 bp downstream of the putative PBS (at nts 208–213) placing it either in the R region, Discussion as is seen in HIV-1 and other retroviruses, or at the 3Ј end of the U3, as is seen in HTLV-1 and other retrovi- This study illustrated not only the ability of Sinbad’s ruses (CoYn et al. 1997) (Fig. 1a, c). Sinbad’s poly- LTR to create active transcripts, but also showed that adenylation signal is located at 265–270 bp, about 40– it is capable of functioning bidirectionally. The Boud- 70 bp downstream of the putative PBS, probably in the icca LTR, in contrast, did not appear to be functional R region. In addition, the Sinbad LTR also includes a in HeLa cells, though it may be capable of transcrip- GT-rich region 12 bp downstream of the forward poly- tion in diVerent systems (although luciferase activity adenylation signal (TTTGGAGTTTTGGTT, nts 283– driven by the Boudicca LTR was signiWcantly greater 297 of the LTR). The LTR of Boudicca does not in the inverse versus forward orientation, the level of 13 Mol Genet Genomics luciferase activity was low compared to that of the pos- signal may be due to mutations in the Boudicca 5Ј LTR itive control). accumulated over time. The LTRs of other, more Transcription of LTR retrotransposons can be tissue recently integrated copies of Boudicca may be found to speciWc (Awasaki et al. 1996; Dupressoir and Heid- be permissive enough to drive robust levels of expres- mann 1996; Lecher et al. 1997), and genomic Xanking sion in mammalian and other non-schistosome cells. sequence can have a major impact on retrotransposon In contrast to Boudicca, the Sinbad promotor exhib- promotor activity (Lavie et al. 2006). Boudicca tran- ited levels of expression approaching those of the schis- scription could therefore be aVected by the absence of tosome GST28-A promotor. The LTRs of this copy of the natural cellular and genomic context of copies inte- Sinbad were 100% identical, suggesting recent inser- grated in the schistosome genome. Since numerous tion and lack of mutation. The present Wndings indicate transcripts of both Boudicca and Sinbad have been that the LTR of the Sinbad retrotransposon is func- detected in both larval and adult stages of S. mansoni tional as a promotor. Interestingly, retrotransposons (Copeland et al. 2003, 2004, 2005a), it is likely that closely related to Sinbad have been found in verte- LTRs of both retrotransposons are active within schis- brates (Wsh) as well as platyhelminths (Copeland et al. tosome tissues. The Boudicca transcripts seen in these 2005a), whereas retrotransposons closely related to previous studies, however, may have been driven by Boudicca have only been found in platyhelminths (Bae LTRs of diVerent copies of the Boudicca retrotranspo- et al. 2001; Bae and Kong 2003) and insects (Abe et al. son. The structural analysis shown in Fig. 1 suggested 2000; Tubio et al. 2005). This may reXect less stringent that the copy of Boudicca examined here may not host speciWcity in terms of transcription factors and include fully functional LTRs. Alternatively, the fail- other host speciWc elements on the part of Sinbad. ure of the Boudicca LTR to drive luciferase activity in Whereas LTR driven expression of luciferase was HeLa could indicate that either the LTR may not be not surprising, expression of luciferase driven by the active in heterologous (mammalian) cells and/or that it inverted Sinbad LTR (antisense) was unanticipated. cannot drive transcription from the WreXy luciferase Analysis of the LTR showed that it was not palin- gene. Interestingly, the LTR of the same copy of Boud- dromic and sequencing of the construct conWrmed that icca examined here can drive green Xuorescent protein the insert represented a single LTR in the inverted ori- (GFP) expression in adult schistosomes transfected by entation. While apparently uncommon, other promo- particle bombardment (B. H. Kalinna, personal com- tors have been shown to function both in the forward munication), a Wnding that suggests that the absence of and inverse orientation, including the LTR of the IAP luciferase activity in HeLa cells could reXect tissue murine endogenous retroviruses (Maksakova et al. speciWcity, perhaps schistosome or molluscan tissue 2006). Some promotors, such as the Drosophila H2A- speciWcity, rather than simple lack of promotor func- H2B spacer (Crayton et al. 2004), and the mammalian tion [Boudicca-like retrotransposons may be present in ACACA and TADA2L shared gene promotor (Tra- the genomes of some snail hosts of schistosomes, but vers et al. 2005), function bidirectionally in nature, ini- do not appear to be present in the human genome tiating transcription for coding sequences on either (Copeland et al. 2006)]. Further experiments using side. In addition, 3Ј LTRs have been shown to produce whole schistosomes or cell lines such as Bge, a line antisense transcripts encoding reverse ORFs in retrovi- derived from embryonic Biomphalaria glabrata snail ruses (Cavanagh et al. 2006). Bidirectional promotors cells (Yoshino et al. 1998), may help to clarify whether have also been produced synthetically in order to the Boudicca LTR is capable of functioning in non- express two transgenes in a coordinated manner human tissues. Alternatively, expression may be con- (Amendola et al. 2005). Promotors that function natu- struct-speciWc, with this LTR capable of driving GFP rally only in the forward direction have also been expression but not luciferase expression. Despite the shown to exhibit some transcription in the reverse success with GFP expression, the LTRs of the 53-J-5 direction in vitro (e.g., Johnson and Friedmann 1990). copy of Boudicca are only 88.4% identical. Although Although expression driven by the antisense multi-copy analysis of other Boudicca LTRs indicates sequence of the LTR of Sinbad may seem to suggest a that the 3Ј LTR is degraded and the 5Ј LTR (investi- natural function for this phenomenon, how this might gated here) is similar to the consensus (Copeland et al. confer an increase in Wtness on an LTR retrotranspo- 2003), the more intact 5Ј LTR nonetheless does not son remains unclear (no obvious reverse orientation represent the promotor of a recently integrated retro- ORFs extend from the 3Ј LTR of Sinbad; all reverse transposon. In addition, the lack of terminal inverted orientation ORFs are short, and are not located adja- repeats beyond the DIRs and the lack of a recogniz- cent to the 3Ј LTR). Although the co-evolution of a able GC-rich area downstream of the polyadenylation multi-copy mobile element with a Xanking host gene 13 Mol Genet Genomics seems unlikely, L1 elements encode antisense promo- Amendola M, Venneri MA, BiY A, Vigna E, Naldini L (2005) tors in their 5Ј UTRs that can drive the transcription of Coordinate dual-gene transgenesis by lentiviral vectors carrying synthetic bidirectional promoters. Nat Biotechnol adjacent genes (Speek 2001), and as mentioned above 23:108–116 the LTR of IAP can drive ectopic gene expression in Awasaki T, Juni N, Yoshida KM (1996) An eye imaginal disc-spe- an antisense direction. Perhaps the LTR of Sinbad is ciWc transcriptional enhancer in the long terminal repeat of derived from the bidirectional promotor of a host gene the tom retrotransposon is responsible for eye morphology mutations of Drosophila ananassae. Mol Gen Genet pair, or from the LTR of a retrovirus encoding reverse 251:161–166 reading frames; LTR retrotransposons are thought to Bae YA, Kong Y (2003) Divergent long-terminal-repeat retro- have evolved through the acquisition of both genes and transposon families in the genome of Paragonimus wester- LTRs from exogenous sources (Xiong and Eickbush mani. Korean J Parasitol 41:221–231 Bae YA, Moon SY, Kong Y, Cho SY, Rhyu MG (2001) CsRn1, a 1990; McClure 1991; Malik et al. 2000). novel active retrotransposon in a parasitic trematode, Clonor- Functionality of the antisense sequence of the Sinbad chis sinensis, discloses a new phylogenetic clade of Ty3/gypsy- LTR could be due to a relatively recent acquisition of a like LTR retrotransposons. Mol Biol Evol 18:1474–1483 bidirectional promotor, or could be rooted in some as Bowen NJ, McDonald JF (1999) Genomic analysis of Caenor- habditis elegans reveals ancient families of retrovirus-like yet unknown function for the retrotransposon. Func- elements. Genome Res 9:924–935 tional studies clarifying the deWnitive polyadenylation Brindley PJ (2005) The molecular biology of schistosomes. signals, as well as the deWnitive transcription initiation Trends Parasitol 21:533–536 sites, would be useful in investigating these possibilities. Cavanagh MH, Landry S, Audet B, Arpin-Andre C, Hivin P, Pare ME, Thete J, Wattel E, Marriott SJ, Mesnard JM, Bar- In addition, investigation of the activity of the LTRs of beau B (2006) HTLV-I antisense transcripts initiating in the other Pao/BEL elements might clarify whether bidirec- 3ЈLTR are alternatively spliced and polyadenylated. Retro- tional LTRs are unique to Sinbad or are characteristic virology 3:15 of Pao/BEL clade retrotransposons at large. Charlesworth B, Sniegowski P, Stephan W (1994) The evolutionary dynamics of repetitive DNA in eukaryotes. Nature The ability of retrotransposon and retroviral promo- 371(6494):215–220 tors to function bidirectionally has important implica- Chitsulo L, Loverde P, Engels D (2004) Schistosomiasis. Nat Rev tions for genome function and evolution. This ability Microbiol 2:12–13 Y expands the known roles of retrotransposons as inser- Co n JM, Hughes SH, Varmus HE (1997) Retroviruses. Cold Ј Spring Harbor Laboratory Press, Plainview, NY tional mutagens and generators of transcripts 3 of the Copeland CS, Brindley PJ, Heyers O, Michael SF, Johnston DA, 3Ј LTR to now include the possibility of generation of Williams DJ, Ivens A, Kalinna BH (2003) Boudicca, a retro- transcripts 5Ј of the 5Ј LTR. Promotors capable of virus-like, LTR retrotransposon from the genome of the hu- X functioning bidirectionally should thus be capable of man blood uke, Schistosoma mansoni. J Virol 77:6153–6166 V Copeland CS, Heyers O, Kalinna BH, Bachmair A, Stadler PF, a ecting upstream genes or regulatory regions in addi- Hofacker IL, Brindley PJ (2004) Structural and evolutionary tion to those downstream, enabling LTR retrotranspo- analysis of the transcribed sequence of Boudicca, a Schisto- sons with these promotors to transcribe genetic soma mansoni retrotransposon. Gene 329:103–114 material on either side of their insertion sites. The Copeland CS, Laha T, Brindley PJ (2005b) Schistosome Long terminal repeat retrotransposons. In: Brindley PJ (ed) Mobile investigation of the promotor function of other LTRs, genetic elements in metazoan parasites. Eurekah Press/Lan- as well as analyses of insertion sites of genes or regula- des Bioscience, Georgetown, TX, pp315–323 tory elements upstream of bidirectionally functional Copeland CS, Lewis FA, Brindley PJ (2006) IdentiWcation of the Boudicca and Sinbad retrotransposons in the genome of the LTRs, can be expected to shed light on how bidirec- X X human blood uke Schistosoma haematobium. Mem Inst tional LTRs may in uence genome function and Osv Cruz 101(5):565–571 genome evolution. Copeland CS, Mann VH, Morales ME, Kalinna BH, Brindley PJ (2005a) The Sinbad retrotransposon from the genome of the Acknowledgments We thank Dr. Edward Pearce for the provi- human blood Xuke, Schistosoma mansoni, and the distribu- sion of the GST28-A-pGL3 construct. We thank Tulane Univer- tion of related Pao-like elements. BMC Evol Biol 5:20 sity for Dissertation Fellowship support for CSC. PJB is a Crayton ME, Ladd CE, Sommer M, Hampikian G, Strausbaugh recipient of a Burroughs Welcome Fund scholar award in Molec- LD (2004) An organizational model of transcription factor ular Parasitology. binding sites for a histone promoter in D. melanogaster. In Silico Biol 4:537–548 Curcio MJ, GarWnkel DJ (1994) Heterogeneous functional Ty1 elements are abundant in the Saccharomyces cerevisiae References genome. Genetics 136:1245–1259 Deininger PL, Batzer MA (2002) Mammalian retroelements. Ge- Abe H, Ohbayashi F, Shimada T, Sugasaki T, Kawai S, Mita K, nome Res 12(10):1455–1465 Oshiki T (2000) Molecular structure of a novel gypsy-Ty3- Dunn CA, Medstrand P, Mager DL (2003) An endogenous retro- like retrotransposon (Kabuki) and nested retrotransposable viral long terminal repeat is the dominant promoter for hu- elements on the W chromosome of the silkworm Bombyx man beta1,3-galactosyltransferase 5 in the colon. Proc Natl mori. Mol Gen Genet 263:916–924 Acad Sci USA 100:12841–12846 13 Mol Genet Genomics

Dupressoir A, Heidmann T (1996) Germ line-speciWc expression M, Gibbs RA, Muzny DM, Scherer SE, Bouck JB, Sodergren of intracisternal A-particle retrotransposons in transgenic EJ, Worley KC, Rives CM, Gorrell JH, Metzker ML, Naylor mice. Mol Cell Biol 16:4495–4503 SL, Kucherlapati RS, Nelson DL, Weinstock GM, Sakaki Y, Gilboa E, Mitra SW, GoV S, Baltimore D (1979) A detailed mod- Fujiyama A, Hattori M, Yada T, Toyoda A, Itoh T, Kawagoe el of reverse transcription and tests of crucial aspects. Cell C, Watanabe H, Totoki Y, Taylor T, Weissenbach J, Heilig 18:93–100 R, Saurin W, Artiguenave F, Brottier P, Bruls T, Pelletier E, Hindmarsh P, Leis J (1999) Retroviral DNA integration. Micro- Robert C, Wincker P, Smith DR, Doucette-Stamm L, Ru- biol Mol Biol Rev 63:836–843 benWeld M, Weinstock K, Lee HM, Dubois J, Rosenthal A, Holt RA, Subramanian GM, Halpern A, Sutton GG, Charlab R, Platzer M, Nyakatura G, Taudien S, Rump A, Yang H, Yu J, Nusskern DR, Wincker P, Clark AG, Ribeiro JM, Wides R, Wang J, Huang G, Gu J, Hood L, Rowen L, Madan A, Qin Salzberg SL, Loftus B, Yandell M, Majoros WH, Rusch DB, S, Davis RW, Federspiel NA, Abola AP, Proctor MJ, Myers Lai Z, Kraft CL, Abril JF, Anthouard V, Arensburger P, RM, Schmutz J, Dickson M, Grimwood J, Cox R, Olson MV, Atkinson PW, Baden H, de Berardinis V, Baldwin D, Benes Kaul R, Raymond C, Shimizu N, Kawasaki K, Minoshima S, V, Biedler J, Blass C, Bolanos R, Boscus D, Barnstead M, Evans GA, Athanasiou M, Schultz R, Roe BA, Chen F, Pan Cai S, Center A, Chaturverdi K, Christophides GK, Chrystal H, Ramser J, Lehrach H, Reinhardt R, McCombie WR, de MA, Clamp M, Cravchik A, Curwen V, Dana A, Delcher A, la Bastide M, Dedhia N, Blocker H, Hornischer K, Nordsiek Dew I, Evans CA, Flanigan M, Grundschober-Freimoser A, G, Agarwala R, Aravind L, Bailey JA, Bateman A, Batzog- Friedli L, Gu Z, Guan P, Guigo R, Hillenmeyer ME, Hladun lou S, Birney E, Bork P, Brown DG, Burge CB, Cerutti L, SL, Hogan JR, Hong YS, Hoover J, Jaillon O, Ke Z, Kodira Chen HC, Church D, Clamp M, Copley RR, Doerks T, Eddy C, Kokoza E, Koutsos A, Letunic I, Levitsky A, Liang Y, Lin SR, Eichler EE, Furey TS, Galagan J, Gilbert JG, Harmon JJ, Lobo NF, Lopez JR, Malek JA, McIntosh TC, Meister S, C, Hayashizaki Y, Haussler D, Hermjakob H, Hokamp K, Miller J, Mobarry C, Mongin E, Murphy SD, O’Brochta DA, Jang W, Johnson LS, Jones TA, Kasif S, Kaspryzk A, Ken- Pfannkoch C, Qi R, Regier MA, Remington K, Shao H, Sha- nedy S, Kent WJ, Kitts P, Koonin EV, Korf I, Kulp D, Lancet rakhova MV, Sitter CD, Shetty J, Smith TJ, Strong R, Sun J, D, Lowe TM, McLysaght A, Mikkelsen T, Moran JV, Mul- Thomasova D, Ton LQ, Topalis P, Tu Z, Unger MF, Walenz der N, Pollara VJ, Ponting CP, Schuler G, Schultz J, Slater G, B, Wang A, Wang J, Wang M, Wang X, Woodford KJ, Smit AF, Stupka E, Szustakowski J, Thierry-Mieg D, Thi- Wortman JR, Wu M, Yao A, Zdobnov EM, Zhang H, erry-Mieg J, Wagner L, Wallis J, Wheeler R, Williams A, Zhao Q, Zhao S, Zhu SC, Zhimulev I, Coluzzi M, della Wolf YI, Wolfe KH, Yang SP, Yeh RF, Collins F, Guyer MS, Torre A, Roth CW, Louis C, Kalush F, Mural RJ, Myers Peterson J, Felsenfeld A, Wetterstrand KA, Patrinos A, EW, Adams MD, Smith HO, Broder S, Gardner MJ, Morgan MJ, de Jong P, Catanese JJ, Osoegawa K, Shizuya Fraser CM, Birney E, Bork P, Brey PT, Venter JC, Weiss- H, Choi S, Chen YJ, International Human Genome enbach J, Kafatos FC, Collins FH, HoVman SL (2002) The Sequencing Consortium (2001) Initial sequencing and analy- genome sequence of the malaria mosquito Anopheles sis of the human genome. Nature 409(6822):860–921 gambiae. Science 298(5591):129–149 Lavie L, Maldener E, Brouha B, Meese EU, Mayer J (2006) The Johnson P, Friedmann T (1990) Limited bidirectional activity of human L1 promoter: variable transcription initiation sites two housekeeping gene promoters: human HPRT and PGK. and a major impact of upstream Xanking sequence on pro- Gene 88:207–213 moter activity. Genome Res 14(11):2253–2260 Katzman M, Katz RA, Skalka AM, Leis J (1989) The avian retro- Le Paslier MC, Pierce RJ, Merlin F, Hirai H, Wu W, Williams viral integration protein cleaves the terminal sequences of DL, Johnston D, LoVerde PT, Le Paslier D (2000) Construc- linear viral DNA at the in vivo sites of integration. J Virol tion and characterization of a Schistosoma mansoni bacterial 63:5319–5327 artiWcial chromosome library. Genomics 65:87–94 Kines KJ, Mann VH, Morales ME, Shelby BD, Kalinna BH, Go- Lecher P, Bucheton A, Pelisson A (1997) Expression of the Dro- bert GN, Chirgwin SR, Brindley PJ (2006) Transduction of sophila retrovirus gypsy as ultrastructurally detectable parti- Schistosoma mansoni by vesicular stomatitis virus glycopro- cles in the ovaries of Xies carrying a permissive Xamenco tein-pseudotyped Moloney murine leukemia retrovirus. Exp allele. J Gen Virol 78:2379–2388 Parasitol 112:209–220 Maksakova IA, Romanish MT, Gagnier L, Dunn CA, van de Lander ES, Linton LM, Birren B, Nusbaum C, Zody MC, Bald- Lagemaat LN, Mager DL (2006) Retroviral elements and win J, Devon K, Dewar K, Doyle M, FitzHugh W, Funke R, their hosts: insertional mutagenesis in the mouse germ line. Gage D, Harris K, Heaford A, Howland J, Kann L, Le- PLoS Genet 2:e2 hoczky J, LeVine R, McEwan P, McKernan K, Meldrim J, Malik HS, HenikoV S, Eickbush TH (2000) Poised for contagion: Mesirov JP, Miranda C, Morris W, Naylor J, Raymond C, evolutionary origins of the infectious abilities of invertebrate Rosetti M, Santos R, Sheridan A, Sougnez C, Stange– retroviruses. Genome Res 10:1307–1318 Thomann N, Stojanovic N, Subramanian A, Wyman D, Rog- McClure MA (1991) Evolution of retroposons by acquisition or ers J, Sulston J, Ainscough R, Beck S, Bentley D, Burton J, deletion of retrovirus-like genes. Mol Biol Evol 8:835–856 Clee C, Carter N, Coulson A, Deadman R, Deloukas P, Medstrand P, Landry JR, Mager DL (2001) Long terminal re- Dunham A, Dunham I, Durbin R, French L, Grafham D, peats are used as alternative promoters for the endothelin B Gregory S, Hubbard T, Humphray S, Hunt A, Jones M, receptor and apolipoprotein C-I genes in humans. J Biol Lloyd C, McMurray A, Matthews L, Mercer S, Milne S, Chem 276:1896–1903 Mullikin JC, Mungall A, Plumb R, Ross M, Shownkeen R, Sharon G, Burkett TJ, GarWnkel DJ (1994) EYcient homologous Sims S, Waterston RH, Wilson RK, Hillier LW, McPherson recombination of Ty1 element cDNA when integration is JD, Marra MA, Mardis ER, Fulton LA, Chinwalla AT, Pe- blocked. Mol Cell Biol 14:6540–6551 pin KH, Gish WR, Chissoe SL, Wendl MC, Delehaunty KD, Speek M (2001) Antisense promoter of human L1 retrotranspo- Miner TL, Delehaunty A, Kramer JB, Cook LL, Fulton RS, son drives transcription of adjacent cellular genes. Mol Cell Johnson DL, Minx PJ, Clifton SW, Hawkins T, Branscomb Biol 21:1973–1985 E, Predki P, Richardson P, Wenning S, Slezak T, Doggett N, Tanaka AS, Tanaka M, Komuro K (1998) A highly eYcient meth- Cheng JF, Olsen A, Lucas S, Elkin C, Uberbacher E, Frazier od for the site-speciWc integration of transfected plasmids

13 Mol Genet Genomics

into the genome of mammalian cells using puriWed retroviral Tubio JMC, Naveira H, Costas J (2005) Structural and evolution- integrase. Gene 216:67–76 ary analyses of the Ty3/gypsy group of LTR retrotranspo- Thompson JD, Higgins DG, Gibson TJ (1994) CLUSTAL W: sons in the genome of Anopheles gambiae. Mol Biol Evol improving the sensitivity of progressive multiple sequence 22:29–39 alignment through sequence weighting, position-speciWc gap Xiong Y, Eickbush T (1990) Origin and evolution of retroele- penalties and weight matrix choice. Nucleic Acids Res ments based upon their reverse transcriptase sequences. 22:4673–4680 EMBO J 9:3353–3362 Travers MT, Cambot M, Kennedy HT, Lenoir GM, Barber MC, Yoshino TP, Wu XJ, Liu HD (1998) Transfection and heat-induc- Joulin V (2005) Asymmetric expression of transcripts de- ible expression of molluscan promoter-luciferase reporter rived from the shared promoter between the divergently ori- gene constructs in the Biomphalaria glabrata embryonic ented ACACA and TADA2L genes. Genomics 85:71–84 snail cell line. Am J Trop Med Hyg 59:414–420