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Multiple Manner Transposons in Flatworms and Hydras Are Related to Those of Insects H. M. Robertson Three flatworm species, the freshwater Dugesia tlgrina and the marine Stylochus zebra and Bdelloura Candida, and two freshwater hydra species, Hydra llttoralls and H. vulgarls, were found to have many distinct representatives of the mariner family of transposable elements In their genomes. In several cases the closest relatives of these mariners are ones known previously from insect genomes, supporting the view that transposons of this family are capable of horizontal transfer across phyla, and hence must be capable of functioning in such diverse host environments. Twenty other Invertebrates representing the major phyla did not appear to have mariners of these kinds in their genomes. From the Department of Entomology, University of Illinois at Urbana-Champaign, 505 S. Goodwin, Urbana, IL 61801.1 thank Ann Grens and Hans Bode for the Hydra vulgaris genomlc DNA, other colleagues listed In Table 1 for various specimens, and Matt Sharkey, Michelle Lepkowltz, Karen Zumpano, and Paul White lor technical assistance. This work was supported by National Science Foundation grant MCB 93-17586. Sequences representing the 20 new distinct types of mariners reported herein (those Indicated with an asterisk In Figure 1) have been submitted to GenBank (accession numbers U51168-U51187). Complete sets of aligned DNA and amlno add sequences are available on request from the author at [email protected]. Journal of Heredity 1997^8:195-201; 0022-1503/97/«5.00 Transposable elements of various kinds have been discovered, mostly by their genetic effects or by accident in sequencing studies, in the genomes of all intensively studied organisms (Berg and Howe 1989). More recently it has become possible using the polymerase chain reaction (PCR) and primers designed to conserved regions of particular families of transposable elements to discover related elements even in organisms not previously subjected to intense genetic or molecular analysis. For example, Flavell et al. (1992) and Voytas et al. (1992) used this "homology PCR" approach to detect members of the copia family of LTR retrotransposons in the genomes of most plants examined. I previously used this approach to detect mariner family elements in diverse insects (Robertson 1993; Robertson and MacLeod 1993). This study revealed several novel aspects of the biology of this family, including the presence in some species of multiple members of the family and the relatively recent horizontal transfer of particular mariners across large taxonomic distances, for example, orders of insects. Garcia-Fernandez et al. (1993) subsequently reported the discovery of a mariner element in the genome of a flatworm, the planarian Dugesia tigrina. This mariner shares 75% amino acid identity with a mariner from an acrobat ant, indicating that it was at some point Involved in a transphyla horizontal transfer. Subsequent work has confirmed that there are approximately 8000 copies of this mariner in the D. tigrina genome, that they differ in sequence by less than 1%, and that no other examined species of Dugesia has similar sequences in its genome (Garcia-Fernandez et al. 1995). All of these observations suggest that this is an active mariner that recently entered the genome of this flatworm after its divergence from Its closest congeners, perhaps by horizontal transfer from an insect host. The mariner family transposons are relatively small (approximately 1300 bp long) with short inverted terminal repeats of about 30 bp bounding a single open reading frame encoding a transposase of about 350 amino acids, features characteristic of the DNA-mediated class of transposable elements (see Haiti 1989; Robertson 1995). This transposase mediates transposition by a cut-and-paste mechanism (Lampe et al. 1996) typical of this class of transposons. The mariner family is part of a larger superfamily that Includes diverse members of the Tel family found in fungi, nematodes, flies, fish, and frogs (Doak et al. 1994; Radice et al. 1994; Robertson 1995; Robertson and Asplund 1996), which In turn Is part of an extended superfamily including elements from ciliates and bacteria, and is distantly related to the bacterial 1S3 transposase and eukaryotlc retroviral and retrotransposon integrase superfamily (Capy et al. 1996; Doak et al. 1994). All of these enzymes likely share a common tertiary structure and catalytic mechanism based on a catalytic D.D35E domain (Craig 1995; Grindley and Leschziner 1995). The mariner family is characterized by transposases with a D.D34D do- 195 Table 1. Animals successfully examined for presence of mariners using homology PCR Phylum Class Porifera Demos ponglae Cinachyra allocladia Cnidaria Hydrozoa Anthozoa Ctenophora Platyhelminthes Turbellarla Annelida Polychaeta Mollusca Gastropoda Arthropoda Crustacea Echlnodermata Echlnoida Species Spongilla sp. Hydra littoralis Hydra vulgaris Pennaria tiarella Campanulana sp. Tubulana sp. Claua sp. Renilla mulleri Leptogorgia virgulata Corynactis califomica Anthopteura eleganlissima Mnemiopsis macrydi Dugesia ligrina Stylochus zebra Bdetloura Candida Sabella melanostigma Diopatra cuprea Corbicula fluminea llyanassa obsolela Anemia salina Procamberus clarkti Daphnia pulex Strongytocentratus purpuratus Heliocidaris erythrogramma Common name PCR Source Yellow ball sponge Freshwater sponge Hydra Hydra Fern hydrold Hydrold Hydrold Club hydrold Sea pansy Sea whip Strawberry anemone Anemone Sea walnut Freshwater flatworm Zebra datworm Horseshoe crab flatworm Feather duster worm Plumed worm Clam Snail Brine shrimp Crayfish Water flea Sea urchin Sea urchin + + + + + - GSML' CBSC* CBSC H. Bode GSML WHMBL' WHMBL WHMBL GSML GSML R. Gillette R. Gillette GSML CBSC GSML GSML GSML GSML J. Gibbons J. Render Pet store R. Gillette M. Lynch R. Raff J. Henry • GSML- Gulf Specimen Marine Laboratories. 6 CBSC: Carolina Biological Supply Company. 'WHMBL Woods Hole Marine Biological Laboratory. main (that is, a conserved aspartic acid separated by a somewhat variable distance from two additional conserved aspartic acids usually 34 amino acids apart) (Doak et al. 1994; Robertson 1995). In this study an additional pair of fully degenerate oligonucleotide primers designed to regions of the transposase gene encoding the amino acids surrounding the first two aspartic acids of this domain were employed in combination with the original mariner primers (Robertson 1993) to provide additional confidence that mariners encoding these conserved regions would be detected. Using this homology PCR approach at least five distinct kinds of mariner family elements were discovered in the genome of D. tigrina, two other flatworms were found to have additional kinds of mariners in their genomes, and two hydras also have multiple mariners in their genomes. The closest relatives of these mariners are often in an insect, indicating again that transphyla horizontal transfers must have taken place. Materials and Methods The species examined and the sources from which they were obtained are listed in Table 1. Genomic DNA was extracted from parts of specimens of large organisms such as the anemones, individual animals of small species such as the sea walnut, or from multiple Individuals such as the hydra species using denaturation of proteins with SDS, precipitation of proteins with high salt, phenol extraction of proteins, and ethanol precipitation of the DNA (Sambrook et al. 1989). Samples of the extracted DNA were run on agarose gels to estimate their concentration by visualization with ethidium bromide stainIng and illumination with UV light. In addition to the original fully degenerate PCR primers, called MAR-124F and MAR-276R (Robertson 1993), two new primers were designed to regions encoding the conserved amino acid motifs (V1L)T(CGM)DE (KT)W and DNARPH(TV1), where the amino acids in parentheses are common al- ternatives that were taken into account in the design of the primers. The primers are MAR-159F-5'TNACNDKNGAYGARAMNTGG and MAR-249R-5' RYR TGN GGN SKN GCR TTRTC, respectively, where the number refers to the amino acid at the 3' end of the primer in the Drosophila mauritiana mariner 1 transposase, and the letter refers to the orientation (N = A,C,G,T; D = A,G,T; K = G,T; M = A,C; S = G,C; R = A,G; Y = C,T). Amplifications were performed with all four possible combinations of these primers from 1-10 ng of genomic DNA in 12.5M.1 reactions for the screens. For cloning, only the MAR-124F and MAR276R combination was used in 50 \i\ reactions from about 5 ng DNA to amplify the central half of the transposase gene (about 450 bp of these 1300 bp elements). PCR, visualization of products, band purification, cloning, and sequencing were performed as described in Robertson and MacLeod (1993). Sequences were aligned with those previously obtained and those published by others, and a representative set of the closest relatives and representatives of the known major and minor subfamilies was chosen for phylogenetic analysis. Maximum parsimony, as implemented by the computer program PAUP v3.1.1 for the Macintosh (Swofford 1993), was employed for the phylogenetic analysis, using the Heuristic algorithm with random addition of sequences, tree bisection and reconnection branch swapping, and 10 iterations. All amino acids were weighted equally, and gaps and frameshifts were excluded from the analysis. A bootstrap analysis with 200 replications and heuristic searches was performed on a reduced dataset (see results). Results Twenty-six species representing many of the major phyla and classes of invertebrates beyond the Insecta were successfully examined (Table 1). Only the three planarians examined and the two hydra species were positive, and each was positive with all four combinations of PCR primers, that is, they yielded at least one Figure 1. Alignment of the conceptual translations of mariner transposase PCR fragments from Qatworms and hydras with those of Insects, other Invertebrates, and humans. Sequences generated for this study are Indicated with an asterisk (only one sequence Is shown when two or more were very similar to each other). The sequences are ordered following the phylogenetic tree In Figure 2, with subfamilies separated by spaces for clarity. The positions of the first two conserved aspartic acids of the D.D34D motif, which are In the conserved blocks of amino adds to which the MAR-159F and MAR-249R primers were designed, are highlighted In bold, and are underlined in the C elegans Tel sequence. The consensus sequence for the mariner family was derived by majority rule of the 15 subfamilies shown here (majority rule within subfamilies too). The Tel consensus sequence is for the entire family from Robertson and Asplund (1996). Dashes Indicate gaps Introduced to maintain alignment, an asterisk In the sequences Indicates an encoded stop codon, a pound sign indicates a frameshift Introduced to maintain an aligned reading frame, and a "m" after the clone name Indicates that at least one of the frameshifts Involved removal of a small Insertion In that clone. 196 The Journal oi Heredity 1997.88(3) Alignment numb«ra Duyuia. tigrina. marl Acrobat.ant.29.3 Houaa.ant.16.2 Cecropia.moth.l.2 Staphylinid.bwtle.2 •Hydra.littoralis.l * Stylochus.cobra.4m European.oarvig.5.6 1 11 21 KDEBXQQIUJDACLSLLSRNKADP NDAQKEQRLEACLSLLSRNKTEP NQKQHDNRFEACISLLSRHXSEP SKSNLQTRVDCSVTLLNRHHNEa NENQXNRRrKVSSAIJJJlHNNDP NDJQjmRRTEVSSALLIJUnCNDP NENQKKHRYEVSSSPFLjmKDDP •Hydra.littoralis.3 Silverflsh.8.4 Totranychua.urtica*.1 SIDHjmQRLTICTSLLSRHNlEP IPDIECNRADTYVTLLSCLHKEP TECQKOHCVSICWSLLLRNETAN 31 41 51 FljnUVTCDEKWIKYDNRKRSSQ FLI'IVTCDKKWIKYIWIWBSSQ FLHRIVTCDEKWILFIWRKRSAS imRIITCDEKKILYDNRKRSSQFLDRIVTCDK1OCILVEHRRRSAQ FICRIVTCDnonLyiWRKRSAQ FLDRvVTCDEKXVLyNNRRRSAQ LLDRIVTCDKKI,ILYI»nUlRSGQ FLDQIVTCDE1O*1LYIWQRRPAQFLDRLLTCDEKWTVYNNTKRCYH FLDRKLTCDQKWVLYEVPRRRyH FLRNLVTSDKKWIEYNNTSRKLH D.mauritiana.marl M. occidentalis. taarl 'Hydra.vulgaris. 6 Silv«rfish.8.5 Houaa.ant.16.4 * Hydra. vulgari s. 5m •Dugaaia.tigrina.6 •Hydra.vulgari a.2 Sand.tly.24.2 •Hydra.llttoralia.2 SilvarCiah.S.e NKRQKERRKOTCEILLSRY13UCS SERQKEVRLTVCRELLSRYKNKS NERQHENRKNTCEILIJUIHIRXS TDROHEKRKNVCEILLQRFEJUCS NKRQQENRKIKCEMLLQRHCRKS NKRQQEH'KTiaOajQXQERKG KPRDVERRFCHSEMLLNIUaaaCP KPRD-vmFAFSKYCLKCHQRKS KAIDVERRLSTCTLLLQRHQKKS KERDIEJUU.\rrCEMlXHRFERKS KVRUVERRJCAICELLLQRQSRXZ rLHRrVTODEKWIFFVNPKRKKS n,YRIITSDEKKIYYian<niKRS VLHRIVTODEKK1YFKKPKRKKS FLHMVTODElOfrYFQJPRRiat FLHRIVTCDEJCKIFFDNPiauatS FHHIIITCDKMIFFENPIUIKKS FLHCIVTCDEKWIHYIWPKRQRS FLYRrVTOBEKWVNYItfiUtiUtXI, FLHRIVTCDEKWIRYEHPKJUOCS FXHRrVTOTEianYFIWPVRTTH FLHRIITGDEKWIHYENPKIWKA Honoy. b«a.4.2 Ichnaumonid.wasp.28.2 Droaophila.eracta.marl •Dugesia.tigrina.1 KErHLTQRINSCDIJJUaiSENDP TTRNLISRIEICDTLIjaUOamP TOKHLLERINACDMLLJIRNELDP TNVNKSRRSSICOILLNRNLTDP FLKRLITGDEKHWYNNIKRKRS WSRPRESAaTTSFJ^IHRJCKVIXLVWWDm-^rVYITXIJ>PiroTmSV-^ FLURUTGDErWIKYTNVlCRiaiS WLKPCIVPQTTTKPELTASKVMLSVWWDMK—GIVYYEILEPOQTVDSO-LYCQ-QLTRLQEAIQK-ItRPKLVHRKS--IEFHHDNARPHTSLinTlQKI.TEF OHEILLHPP CLKBMVTOOEKWITYDNIKRKR KRESSQTVAKPOLTARKVLLCVHKDWK—0IIHYIXLPYOQTUJST-IYC*-RLDRLK0*lDQ-KRI'ELAHRKO--VVTHQI)NARPHTSLHrRQKLRKL OHEVLSHPP FT£RrVTGDBKWVLYENPOlFKQ----KI£Via^PTBTPKPNIJIClUCVUX:iW»mR--aiI IPOETrTAE-VYCE-QLHRLRI»MTT-KCPCTJJJRNR--VILQHD«ARPHAAKKAQRXLKEL---OKEVLPHPA 'Hydra.11ttoralis.4m European.«arwig.5.1 •Hydra.vulgaria.Bm •Hydra.vulgaris.1 •Dugasia.tigrina.5 NKVSKfflOlLQIAAQHLARH'AIRSHKQRFLY'IVLGDEIOCLYIHMKQRXE NEVHKENRLQIAAQHLARHRATRCNKHRFT.YRIITCDiraCLYVNl(KHBKE HEVKKENRT'IAAQQFARHRAKRCHKQRFLYRTVTaTKRWiLYVNIKQKKE NEKNKEKRLQTAAQHLACHRATHCDKORFLYRIITGDEKWCLYVNVltQRKE TWQKDIOU^IAAQNIJRHQCTHGHRQRrLYRIVTGOEKWCLYVNMK'RKE *VAP<nwPTPRVlWDLYPlUn»IICVV«D«E--SKIHWE--IRNATVNKE-LY#A-QLRRINEAKRL-KRPDQ*O* HVAP<ro»PTPRVTQDI£MCKTMLa3«KD«re--SKniKH<LFjaiASVH]^-LYIA-QI£RVNEAMCa.-KRPriRQGQ WVAPOTTraPRV*QDiaPKKIMSVY(WVra--SlCnn«EMLERSATVNEE-LYI^ )T/AP<aTPKPRViaroLHPKKA)aCV>*roWE--SMVHKQ(LERNAIVNKE-IYIA-QLNCVNEAIRI,-iaCTDRQOQ "VSMEKPIUTaiaU)iaPIOtTMICIW'Dire--GIJBMEMFGi™ifrVSlCN-LYXA-*LYRVNEAIQ(3-KRPDRQO<) VTLLHDHARPHVAQWKTAFQEL EWEVLQHLP VTLLHDNARPHVAQWKTALQEL EHEVLQHPP VTLLHDHVRTHIAQWKTALQEP---EHEVLKHPP VTLLHMNARPHIAO\A/KTALQEI. EWEVLQHPP VTLLYDNARPHTAKTVKIALQEL OWEILQHPP TLPSLTAKLTFUVISSLSPOVDH CLDQUTanEKWVLYANHHRQAQ WIOEOQTPCDVPKLGLHPKKSMLSVWWOVD- -OPSVWELLPEGEIITGE-THSR-QLRHLKKTVDR- SALQDKK VYFQH1OJAQPHVAKQVKEKLA1CY tafTILLQPP TOANLDrLVDnSLSLLILHOADR WLDPLITODEltWVLYiaOIHRRAQ WiaEOETPaDAAKPDUlPKKVMLSVWWSVY—CPIYRBLLPDSKIITGG-LYII-'PRNLKKvTNR-SPLMDKQ LYFQQBNARPYCLXOVLQELVRR OWKVLLYPP C.elagans.mar2 SASQKLTRVNVCTQLLTFRJIKFT) WLNNLVTCDIKWVLYVNHSRKRQ- HLPIOEKOIPTPKPDLHPKKIMICVHWCVQ—SPVHKELLPTNKTITAD-YYCA-QLDRVAEKTNO-KYElt LYFLHIHARPHVAKXTFQKLQDL OHTVLPHPP C.elogans.marl SDSQXILLCDLSLELLTRKRTTD KVltDIITaBDKWVLYVSHTRKKE HVPVlSTATPOIJt*EUjaU0niSiaPJ>SK--<J7ISREIAPDFATINAC-LYCI-*LEKVVHAHRL-HRPROSK LLLLHDHARPHTTF1CTRQKLQTV OIQILSYPS SMJNQERR1J1ACISLLSKERTTD HLDTIVTODEKWCLYVNWIUaiS KVDKCTPSOAQPKPEIHOKKLMLCVLMIJVS—aVTYWEHU!PNQTINAE-LYCT-QLQia.VCTISQ-RRPNLEK IRnJnWTRPHTAlQfrREXIJlQL RHEVLIHPP HOBO.sapians.marl C.«l*gans.mar4 C.alogans.mar3 •Stylochua.aabra.2 •Stylochus.sebra.5 Silvarfiah.8.2 •Dugasia.tigrina.4 •Dugesia. t i g r i n a . 11 61 71 81 91 101 111 121 131 141 151 161 WLDPDEPPKHCPKRKVH0KX1MVTVWWSSY—GVIHYDFHVPOTSITSD-VYCS-QU3DM(Iiaj^-TOP10OTmLT--PIIiHlWAPJ>HSA]uVTVAKL<XX. GLETLRHPP WLDRAEPPIOiCEKRCIHQlaUJfVTVWWSAS--OVTHHSFIffiPOTSIl»V-WCK-QIJ>I^^ ETFPHPS KLDKHESPKHCPKQKIHQXKLMVSVKWTDS—OirYRTFIJUOTSITAE-IYCS-QIJ3EMaRIJU-iaCPRIJ2JRD3--PIIAQIWARPHvAXNTLLKL<)SL HLETLLHPA KmSOYPVKSCPiaUCLTQlaaj.VSVHWT3A—CVViryS7UCSOQTITXD-IYCQ-QLQTllKKKIJUk-I^PM,VNRSR--PIJiHl»UaUVTW3QTTTiaj)EL QLECLSQPP HLDRDOAPQHTPlCPAIJIQKKVKvTVWCVA—(^TIHH£rLNPGETITAE-ryCQ-QIDKlfflQiaAW-HCPRLVNia(O--PIIXHIWARPHVAQPTLQKLMQt. OYETLPHPA KI^RDEAPQH7PKPNLHQ1OtVHVTV»*SAA--GI^n£HSPTin^ETITAZ-KYCQ-QIDETHQiaJK:-lC OYETUPHPP tOJ3M»APRHPT!a>PI^QXKVHLTV>mSS#-^UHYSFTiIAGOTrcM-ireCQ-Q UJ3TDEPPRHFPKXKTH0XTTKVTVWHSAA--<WIHYNFI*P«miAI^ WJ>REEAFlCHrPlU>HIJlQXKVKVTVWWSAA--QLIHYSFTjn«OTITSE-]WA(>-QIDEim^ OYEVLPHLP KLSPDDPIPKTPKPNLHiaWVLLCIWWrTA—OVVHyEUJ^OQTIT<3L-vySA-QLQRvm3LIXV-I«PALVHIUffl--VIXIJffiNARPHTvTlVTQDKLQSL GWESLPHPP m.VSHEPVPK--IQQNNIKEDLLCVW-TAR—OrVHWELI^AGQIIMAT-VYCO-QI^SVHElUjnf-IJtPAIJJ)CEO--VILLQI»>AKPHVXRKimffyLLL CHETLLHPP HLDKVQANUIAKPDIHGKXVUCnfWSaP—OWHYQfLKPOQTIDAB-LyVN-QL IDKIXE-1OIPAKVNREH--VVYLHDNARPHVAKIVKEKLLAL KTOVLPHJ'P YVDKWPATCTARPNRFCTUnWU^WWIX»--OVTYYIIiXPOETVOT^ WVSPOTPAEKSVTUWRraUCTHI/rVY*n)QR--<mYHKUJU>OETVOTA-RYQ^ WVNP<WPSTCTJ«PDRrauc™iXVRWDQK--aVVraEIJJUaBTV>m>-R HLAPOEAOPSTTRPNRrVLKTMLCVWKDQC—0VVYTELIJU«ETlOITI>-RYR0-QIINUrajU,IE-iaU^m'RRHCK-VII«3HlWAPSHTAJ^VQDTLKTP HvSPGEAVT>STSRHDRFVRinMLCvWKDYR--<r/VVYEIJJU<3ETVIK3E-RYRQ-QIJNUJHMJE-IWPDWAR^ WVSPO0PSTLTAKTNR7GKKRKVPVWWDQK—GIVYYEIXEP(^9fIHSE-RY*0-QIIHIJfflSLIE-N'LEW10^RHKK-VILQHDNA*PHKSKDVIO)TLNAL YVKPGQPAKSTPKPNIHOLKVNLCIWWDQK—CVLYYniKSO<^ITOE-LYIlQ-QIJRLK0AIAa-iaiPEWrTimEK-LIFHin»IARPHVAVVVKNYLENA NVTPI>QPSTSTA*PNIHIAKVTL*IF>nHm--EILYYEIJT^SQTITGD-FYOA-QIJnu^lUCTIVB-KIlPEYVIRHEV-IIUnuWARP^^ WOU<aaCGKQKAKPNIHGSrNMLCIWM)QE--<»VVHEVlJCSNESrroE-LYRK-QLIOUJJRAIja-iai^ YVDPGAPVKPTPKRNIHGNKALLSIWWDQI—OVVYYmjJU'l(ETIDCP-LYRL-QLFlUJUC«.EE-la«>EYAERHDK-IILQHl>NWlPHVCKVVQTALKTI WVXPOEPGPSQPKRDIHCAKVHLCIWWDMX—GVVYYELWKreTKWE-LYRR-QIJOUJtOMJ^-TRPEWENRHDK-LrVQHDNARPHVAKPVKTYLENV O»ILLPHPP EHEILQHPP KKDVLSHPP EHEELPHLP OWEILPHPP OWEVLCHPL OHEVLPBPL Silverfiah.8.3 TELQAKCRVEICBQLIJWPHSDQ nfKKXVTSDZKKIHLIKHSRQKR )T/PYDQAPPPVPCQERF<na^VllLCvMHNFQ--AIIHYEFVPHCRAIDAE-LYCE-QvERVYEKLKE-KYPALIRRKC—ALLOQBHAKPHTAKKTKKKFEEL—DCVBILPHPP TEEOASKRVKICROLLSNPLDDR LHKHJVTSDYKKVYLVQHHRDKR WVKHOQETPPSVPKQDRFDiatVMCLHMJFI- -OrVHTELVPNORAVNAI-LYCQ-QLERVYDKLKX-HYPTLINRKR- -AIJIMIIHAKPHTARKTKDCTAEV- -DCVSVLPHPA IQIQTEKRFETCQKLLENPRDDR FUUIIVTCDEIWVYFSNPDQQN* *LDAGQHAKPVAKRDRFSPJtALLCVS>MPE—OVIHFELVLMSRTIDAD-LYCA-KLDRMYAALOE-KYPALINRKR--VliQQBKAKPHTAKQTXEKIKNL ELLPHQA TFAQAQHRXDTCKKLLQNSHHER WmatWTCDElWTYFSNPNKENQ HLDPOQEAYPVAKRDRFSKKVMLCVWHNFE—CVIHTELVAmnUkINAE-LYTD-QIiRMYTALCK-KYPALINIlKO--VILQQONASPHTAALTRRlUEEI.--EAIEIAPHPA TIDQKQQRIDOSEQCLELSBQNRTD FFCRYITMDETHLHHHTPESNRQ-SAEWTARDEPTPKRGKTQQSACKFHASV7WDTH—OIIFIDYLEltCKTINSD-YYIA-LLERLKDEIAK-ltRPHIjaWK VLFHQONAPCHKSHNTHIjaNEL GFEU.PHPP TFiaUtLQRVTIDSERCFQLLTRMTPQ FFRRYVTIDETHLHHYTPESNRO- SAEWTATGEPSPKRGKTQKSADKVMASVFWnAH—CIIFIDYLEKQKTINSD-YYHA-LLERLKVEIAA-KKPHMKKXK VSFHHDIAPCHKSLRTHAKIHEL GTELLPHPP HPDQLQTOASLSMEILNKWDQDPEA- - -FLRRIVTCDETWLYQYNPEDKAQ- SKQWLPROaSOPVKAKVDHSRAItVlfATVFWDAQ- -GILLVDFLEOQRMITSA- YYES- VLRKLAKALAE-KRPOKLHQR— - VLLHHIMAPAHSSHQTRAILREF- - -RWEIIRHPP rVAQKQKRVEICEQLMQOYSENPTE FFERLVTVDEIWFLYETPEKKRQ-SIEHRHTCSPRPlOtARJIOIJVHKQHAAvrRDQE—OILLVEWLPPKTSIDSE-SYCS-SLH'LRRHIQQ-RRQaiOIORO VLLQHDNAQPHVSHQTIATVHEL CrSVLTHPP Tetranychua-urticae.6 PPCQHEHJWKACRFNLQMHRKTRE LIQRTISIDETWVSLYKEPKKEO-AKCWYYPDEQPEEVPRWIHOOaQ€LI]«nO)YN-^IA7TEIiPEinTTraK!-TYK3-FI^RHVIUn(I/;-T^ SDANKELRIRICNELLVXFOINN ITJRLITVDET>TCYKRNEBTFS<>-TKCY«Aa«nSRTTNVSRTLTPEKSIJWFFTOCK--<WV^^ •Hydra.vulgaris.3 Horn.fly.3.4 Homo.sapions.nar2 •Bdalloura.Candida.1 TDQNR1CNRVETCKENLAFFRNSPWR •Hydra.littoralis.5m Tortricid. moth. 19 .1 Bombyx. nori. mar 1 C.elegans.marS mariner family consensi Tel family conaansus D.hydoi.Hinos P.oxysporum.impala D.aelanogaster.Bari S.salar.Tasl D.melanogastor.S C.elegana.Tc3 C.elegana.Tel • LWLLHDNARPHKHQWREHLERH EITLMHHPP NLPSLPHPP tCDIITQffiUfFYIJlQVaHXSA-NASmOEaESPRTVVRCDRF^PraMFYIFT^ NELQKERRVDHCKFKIDKFDAGEKK NVYDILTaDETXLYNYDPETKRO-STWKFEDEPTPTKCRRTRSTQKQKVCSFFCKT—OHIATWLENQHTVNSE-HYVS- ICAPKVLSAMCDKRPKSCTRH LLWHHDNAAPBTSARTHDYFSSK SEAAKIKRKDRAHNLLRRFRHC K R C LL It RL A TLRQIOaaiLQKARERKSWrQR TPLQARKRUMAIRYQCI TPTNKTTOLR7ALEYVKKPLD QNRHXNARUlFATAHaDraRT SKINIEXRHSFAVSMIDHAEE-SAOHKlJUU^ErAlQOnOT SKICNlUtARVAKAKAHLRWJRQ AHRKVLFTTEKIFCIEQSFNTQNDRVYAK TQPNSKVQRT<^PK0Iir^A0ITAr«in'PIJFVPQ0IIWl«m-NYIJ)-lOJtTELMPWVK-KHFKKTlt R TCCEKW N HO K H VHWD G L T Y L KRP W H DESKF FO V R P T K GCGSVXVWGCF G L I O Y I L L S L QWIWIIFSDEAKFDVSVGiyrRKR--VIRiaiSETYHKICLKRTTK-FPASTHVl«X3tS--AKGU;iaiHFIEGTVNAE-KYIN^ DWRRVKHSDECHVRRCQGMRPTW TFLSPREPYAYRMF]aU/3alRQMFWAAFa--HRSRTPLVPLVDiaNAIGIYKL-YSFILPWFLQS-G FWFNILWrOESAFQVQGSYSKHF !EUCNNQKHlJUlQPTimi^axrnmFWXI^--YYCFGDLvTIE»nj)(^-OYLi-IUlNHAFTSGN-RIJPTTE FWRNVU«SDETiaEIJXaOrtlHRY--VWRKKaEAYKPlQmiPTHKHGGGSIKIJ*GCFA--AGGT<yUJ^ YVffiDVIFCDETnDOJTOTIGPSR--VWRJWI^ALETWIIPTIKrorLSVia»CCIS--SHGVCTaj^IESTlflU NWSKVWSDEKKPXLDGPDOCRY YWRDLRKEPMVr3RRNFOOaTVHVWQAJT--EKKKLEIQrVS3KllNST-DYQN-VLELELSKYLJ*-HYSRKD EWAKHIMSaESKFNLFGSDGNSH—VRRIVGSRYSPIW(X:PTViaiGGOSVMVl»KFT--STSJCPIJUa(KIW3RF-QYEN-IFETTMRP»*AL-QNVaRO HTFQQDOAPAHKHKNVOAWCESNPPljnAFNQWPP LHDNARPH L L HPP F FQQBHD H L WP FTFQQDCASSHTAKRTXNWLQYN OHEVUMPS DIFMHDNASVHTARIVKALLEEL OVDLMTHPP KILQQm)APCHKORIPTKFIjn)L---MLAVIJ>WPP WVFQMDNDPKHTSKWAIMIJUW---KVKVLEWPS FRFQQDNATIHVSNSTRDYFTJJt FVTMBNDPKHTSLHVRSW7QRR HISILPHPP KINLLEWPA HVHLLDWPS band of the appropriate size. The other 21 species usually yielded at least some PCR products from at least one combination of primers; however, these were not of the appropriate size. Occasionally no products were obtained, yet the PCR was not inhibited because "primer-dimer" product was observed at about 50 bp length. A few DNA samples, for example, from sponges, appeared always to inhibit the PCR because no "primer-dimer" product was observed, presumably because of inhibiting contaminants that copurified with the DNA, and these were excluded from the study completely. Alignments of the conceptual amino acid translations of the plasmid subclones of PCR products with representative clones from the earlier PCR screen, as well as the equivalent region of several published full-length sequences, are presented in Figure 1. The previously published fulllength sequences are the original active Dr. mauritiana mariner 1 (Medhora et al. 1991), the D. tigrina mariner 1 (Garcia-Fernandez et al. 1993, 1995), the Metaseiulus occidentalis mariner 1 from a mite (Jeyaprakash et al. 1995), the Dr. erecta mariner 1 (Lohe et al. 1995), the Caenorhabditis elegans mariner 1 and 2 elements (Robertson 1995; Robertson and Asplund 1996; Sedensky et al. 1994), and the Bombyx mori mariner 1 (Robertson and Asplund 1996). The C. elegans mariner 3, 4, and 5 sequences are from consensuses of additional mariners in C. elegans cosmid sequences in the public databases. Homo sapiens mariner 1 is a confident consensus derived from multiple PCR, cDNA, and genomlc sequences (Auge-Gouillou et al. 1995; Morgan 1995; Robertson HM and Zumpano KL, manuscript in preparation; Smit and Riggs 1996), while H. sapiens mariner 2 is a preliminary consensus sequence (Robertson et al. 1996) derived from published genomic sequences (Oosumi et al. 1995; Reiter et al. 1996), and cDNA and genomic sequences from the public databases. These mariners are all named, and abbreviated where necessary, following the convention of Robertson and Asplund (1996). Finally, two PCR clone sequences from the mite Tetranychus urticae are included because they represent divergent lineages (Robertson, in press). The alignment is essentially similar to that employed in Robertson and MacLeod (1993) except that additional gaps were needed to align the basal B. mori marl and C. elegans mar5 sequences at alignment positions 86-87, 110, and 158-160. In addition, alignment with seven sequences chosen from the Tel sister fam- 198 The Journal oi Heredity 199788(3) ily for the outgroup (see Robertson 1995; Robertson and Asplund 1996) required another alignment gap at position 105 to accommodate the impala sequence from the fungus Fusarium oxysporum (Langin et al. 1995). A phylogenetic tree of the relationships of these mariners based on this dataset is shown In Figure 2. The robustness of these relationships was evaluated In several ways. First, a bootstrap analysis was conducted on a reduced dataset using only the representative flatworm and hydra sequences in Figure 1. This analysis confirms earlier findings that the relationships of the subfamilies with each other cannot be established confidently from these approximately 150 amino acid fragments (Robertson and MacLeod 1993)(Figure 2). However, when the available full-length transposase sequences are analyzed the basal positions of the mori and irritans subfamilies can be established with confidence (Robertson and Asplund 1996). It seems likely that the new mariner subfamilies represented by the Bdelloura Candida and H. littoralis 5 clones are also correctly placed basally given the sequence peculiarities and length variants they share with the mori and Irritans subfamilies (Figure 1). Second, the placement of most of the flatworm and hydra sequences within particular subfamilies Is very confident, with bootstrap support for these placements generally above the 90% level, even based only on these 150 amino acid fragments, and these placements are also strongly supported by the length variants which were not Included in the phylogenetic analysis (Figure 1). Third, relationships within subfamilies are not completely shown here because many hundreds of additional sequences are now available. However, the closest insect mariners and other relatives are shown for each flatworm and hydra mariner, and these are confident relationships when analyzed within each subfamily (Robertson et al., In press) and often have bootstrap support in this analysis (Figure 2). Amplifications from D. tigrina DNA with the original PCR primer pair yielded a discrete PCR fragment of about 500 bp, and 13 clones derived from it were sequenced. Six are essentially the same as the mariner Garcia-Fernandez et al. (1993, 1995) reported (at least 97% amino acid identity; clones Dugesla.tigrina.2, 7, 10, 12, 13, and 14). As noted earlier, these clones cluster phylogenetically in the cecropia subfamily of mariner elements, having 75% identity with those from an acrobat ant (e.g., clone acrobat.ant.29.3). The remaining seven clones derive from five other quite distinct kinds of mariner elements. Two clones (Dugesia.tlgrina.6 and 8) represent a mariner belonging to the mauritiana subfamily. One clone (Dugesia.tigrina.l) may form a basal branch of the mellifera subfamily, while another (Dugesla.tigrina.5) is a basal lineage of the capitata subfamily. Two more clones (Dugesia.tigrina.il and 15) join a single further clone (Dugesia.tigrina.4) in forming a new subfamily, the lineata subfamily, of elements with similarity to the silverfish.8.2 clone from Ctenolepisma lineata (sharing 66% identity with each other and 48% with the sllverfish.8.2 clone). The second flatworm examined, Stylochus zebra, which Is a free-living marine species, also yielded a single PCR band from which four clones were sequenced that represent at least three distinct mariners. One clone (Stylochus.zebra.4) belongs in the cecropia subfamily where it is most closely related to clones from a staphylinid beetle (74% identity), the European earwig (65% identity), and a set of H. littoralis clones (see below, 76% identity). Another clone (Stylochus.zebra.2) does not cluster in any of the major subfamilies previously Identified in arthropods, and indeed has amino acid length differences from all previously determined clones (Figure 1). It is therefore thought to represent a novel subfamily of mariners, and tentatively clusters phylogenetically with a series of mariners from the nematode C. elegans. The last two clones (Stylochus.zebra.5 and 6) belong in the newly recognized lineata subfamily, sharing with the other members of this subfamily an unusual feature in having 35 amino acids between the second and third aspartic acids of the conserved catalytic domain of these transposases (the first D is that of the conserved DE(KT)W motif, the second is in the DNA motif, while the third is nine amino acids beyond the sequences in Figure 1). That this difference is not too unusual Is shown by the spacing of 37 amino acids in B. mori marl and C. elegans mar5. The third flatworm examined, Bd. Candida, which is a commensal on horseshoe crabs, again yielded a single PCR band, but this time all five clones sequenced yielded essentially the same sequence (98% identity). This sequence is quite distinct from all other known mariners, both in that it is at least one amino acid longer than all others and in having only 25-37% amino acid identity with other mariners Dugesla.tlgrlna.10 Dugesla.tlgrlna.13 Dugesia.tianna.mar1 Dugesla.ffgrina.2 Dugesia.tfgrina.7 Dugesia.tfgrina.12 Dugesla.tigrina.14 Acrobat.ant.29.3 House.ant.16.2 moth. 1.2 )eetle.2 Hydra.flttoralis.1 HyUra.iittoralis.13 Hydra.littoralis.12m Stylochus.zebra.4m — European.earwig.5.6 — Homo.sapiens.mari OA Hydra.littoralls.3 2 4 r *- Hydra.lfttoralfs.7 Hydra.llttoralls.9 —— Silverfish.8.4 etranychus.urticae.1 _i D.mauritiana.marl M.occidentalis.marl ilgarls _ih/erfish!B.5 House.ant.16.4 Hydra.vulgarls.5m [5_r Dugesla.tlgrlna.6 ia,tigrina.8 cecropia subfamily J mauritiana subfamily Hydra Silverfish.8.6 Honey.bee4.2 mellifera lchneumonid.wasp.28.2 lhd subfamily Drosophila.erecta.man Dugesia.tigrlna.1 ••r dra.littoralls.4m opean.earwig.5.1 ar]s.8m capitata g subfamily dra.vulgaris.1 24 ' — H/dra.vulg"arls.4 ' iarls.4 Dugesia.tlgrina.5 40 i— C.elegans.mar4 100 ' C.elegans.mar3 C.elegans.mar2 C.elegans.maM Stylocnus.zebra.2 tylochus.zebra.5 >tylochus.zebra.6 lineata Silverfish.8.2 Dugesia.tigrlna.11 subfamily Dugesia.tlqrina.15 Dugesla.tlgrlna.4 Hydra.vulgarls.3 irritans Horn.fly.3.4 Homo.sapiens.mar2 subfamily Silverfish.8.3 Tetranychus.urticae.6 Bdelloura.candida.2 elloura.candida.3 elloura.candida.4 elloura.candida.6 elloura.candlda.1 Hydra.littoralls.5m Bombyx.mori.mar1 C.elegans.mar5 I mori subfamily C.elegans.Td C.elegans.Tc3 D.melanogaster.S S.salar.Tssi D.melanogaster.Bari F.oxysporum.impara D.hydei.Minos Tc1 family Figure 2. Phylogenetlc relationships of the mariner family transposons based on the conceptual translations of their transposases In Figure 1. The Tel family sequences were defined as the outgroup. The major subfamilies are Indicated with their names. Branch lengths In number ol amlno add changes are shown only for those branches supporting nodes present in the "semistrlct" consensus of the 144 equally parsimonious trees of 3123 steps. This tree Is an arbitrary representative of those 144 trees, shown in complete detail to Indicate the levels of divergence between sequences (branch lengths determined with the accelerated transformation option). Values In smaller font below branches are the bootstrap values above 75* (percent of trees out of 200 containing that branch). The sequences obtained for this study are highlighted In bold. Robertson • Multiple Manners in Ratworms and Hydras 1 9 9 (excluding B. mori marl and C. elegans mar5, since these mariners share just 1425% identity with other mariners in this region of the transposase). It therefore represents a novel subfamily of mariner elements. These results, together with the detection of at least five mariners in the genome of the nematode C. elegans (Garcla-Fernandez et al. 1993; Robertson 1995; Sedensky et al. 1994; see above), suggested that mariner elements might be widespread in nonarthropod invertebrates, so a screen of a variety of other invertebrates was conducted (Table 1). Surprisingly, only the two freshwater hydra species examined were found to be positive (Table 1), each again yielding a single PCR band. When these were subcloned and a set of clones were sequenced they again yielded a variety of distinct mariner sequences. Eleven clones from H. littoralis yielded at least five distinct sequences. Three clones (Hydra.littoralis.l, 12, and 13) cluster phylogenetlcally in the cecropia subfamily where they share 92% amino acid identity with clones from a staphylinid beetle. Another three clones (Hydra.littoralis.3, 7, and 9) belong to a distinct lineage of this subfamily and do not share particularly close sequence identity with any other clones in the dataset. A single clone (Hydra.littoralis.4) clusters in the capltata subfamily with 83% amino acid identity to two clones from the European earwig. Another clone (Hydra.llttoralis.2) clusters within the mauritiana subfamily, while the final clone (Hydra.llttoralis.5) Is colinear with the unclassified clones from a tortricid moth, but its amino acid sequence is quite different (only 30% identity). A similar set of clones was obtained from a locally collected hydra, presumably of the same or a closely related species (data not shown). In contrast, the eight clones sequenced from H. vulgaris yielded a quite different set of at least five distinct mariners, most not closely related to those obtained from H. littoralis. Three clones (Hydra.vulgaris.2, 5, and 6) cluster in the mauritiana subfamily, each apparently representing a distinct type of mariner, with the Hydra.vulgaris.6 clone sharing 75% amino acid identity with the canonical mariner element from Dr. mauritiana. A single clone (Hydra.vulgaris.3) clusters within the irritans subfamily where it shares 70% amino acid identity with clones from the horn fly, Haematobia irritans. Finally, four clones (Hydra.vulgaris.l, 4, 8, and 9) cluster within the capitata subfamily. 2 0 0 The Journal of Heredity 1997:88(3) Discussion All of the results herein are based on amplifications using the PCR, so it is first useful to evaluate how certain we can be that these PCR fragments represent actual mariner elements in the genomes of these animals. Several observations give substantial support to this conclusion. First, in previous work of this kind the fidelity of the PCR was confirmed in that few errors and no chimeric clones were obtained in a test amplification from a defined mixture of clones (Robertson 1993). Second, this PCR approach recovered fragments that correspond to the mariner previously found in the D. tigrina genome (Garcia-Fernandez et al. 1993, 1995), with little more sequence divergence than is present among the genomic clones they characterized, and no evidence of chimeric clones with any of the other five mariners in the genome of this flatworm. Third, 12 PCR fragments from the previous PCR screens in insects have subsequently been used as probes to clone the corresponding fulllength mariners from genomic libraries (Lampe DJ, Soto-Adames FN, and Robertson HM, manuscript in preparation; Robertson and Lampe 1995), confirming that these PCR fragments usually represent mariners that can be recovered from these genomes. Fourth, most of the published full-length mariners have been shown, by in situ hybridization to polytene chromosomes or their insertion into genes that are clearly from the host genome, to be part of the host genome (rather than from a parasite or some other contaminant). We can therefore be confident that all of these PCR fragments accurately represent mariner elements in the genomes of these animals. These results extend the host range of mariner family transposons considerably, however, they also suggest that most of the species examined here do not have mariner elements In their genomes, at least not that can be detected with the four combinations of PCR primers used here. This result does not mean that they do not have any mariners in their genomes, since we already know of several mariners that would not be amplified even with these four primer combinations [e.g., the C. elegans mar2 and mar5, and the B. mori marl (Robertson and Asplund 1996)]. Nevertheless it seems reasonable to conclude that they do not have mariners of the major subfamilies known so far. We recently used a similar homology PCR approach to examine many of these spe- cies and others for members of the sister Tel family and found multiple members in H. vulgaris and positive PCR amplifications from many of the others, indicating that this family is even more widespread than are mariners (Avancini et al. 1996). These flatworm and hydra mariner sequences exhibit several features of mariner elements that were recognized in the earlier insect screen. First, most species have multiple different kinds of mariner elements. Several insects had up to six different kinds, so the finding of at least six kinds In D. tigrina and five in H. littoralis is not unusual. Second, these PCR fragments represent mariners in a range of evolutionary states from probably active elements, such as the D. tigrina elements identified by Garcia-Fernandez et al. (1993, 1995), the Hydra.littoralis.1 and 3 type elements, and the B. Candida elements, to mariners that clearly are highly defective, having accumulated many mutations including stop codons and frameshifts, such as all the members of the capltata subfamily. Third, three of these sequences (Stylochus.zebra.2, Hydra.littoralis.5, and the B. Candida mariner) represent mariners that are so distinct from all the known subfamilies, both on the basis of amino acid divergence and length differences, that they probably represent additional subfamilies of mariners, which might be rare or might be common in organisms not yet screened, or might have sequences that make them difficult to amplify with these particular PCR primers. These three new subfamilies, together with the C. elegans mariners, bring the total number of subfamilies of mariners to 15. Finally, as was the case for the insect mariners, the phylogenetic relationships of these mariners clearly indicate that they have been involved in both ancient and more recent horizontal transfers across great taxonomic distances, in this case phyla of animals. The most extreme example is the presence in H. littoralis of mariners with 92% amino acid identity with mariners in a staphylinid beetle, Carpelimus sp., and both share 81% amino acid identity with the consensus sequence of the Hsmarl element in our genome. The existence of these mariners in these species has been confirmed by cloning and sequencing of full-length copies from their genomes (Robertson HM, Walden KKO, and Lampe DJ, unpublished results). Many of the other flatworm and hydra elements share 60-80% amino acid identity with elements from insects. In several other cases the closest relatives of hydra ele- ments are in Datworms, and vice versa. It is difficult to envisage how these sequences could be so similar except as a result of horizontal transfer across these phyla. The alternative explanation of vertical inheritance for the past 600-800 million years with conservation of these sequences in some species and loss in all others examined is unlikely for several reasons. First, mariner elements generally appear to evolve rapidly and effectively neutrally within particular hosts, the only strong conservation of the transposase gene being observed when comparing elements from different species (Robertson and Lampe 1995). Second, the similarity of these mariners across phyla is comparable to or greater than the similarity of extremely conserved genes shared by species from these three phyla. For example, an actin from H. uulgaris has 94% identity with that of Dr. melanogaster (Fisher and Bode 1989), the Na,K-ATPase alpha subunit of H. vulgaris has 70% identity with that of Dr. melanogaster (Canfield et al. 1992), and for a variety of homeobox genes from hydras and flatworms only the homeobox Itself is in fact generally alignable with those of insects, and then only 60-80% identical (e.g., Schummer et al. 1992). How these horizontal transfers might occur remains a mystery; indeed, the occurrence of transfers across phyla of animals makes the mechanism(s) even more enigmatic. The screen of marine cnidaria and other marine invertebrates was conducted in part to examine the possibility that mariners might be restricted to terrestrial and freshwater taxa. However, the presence of multiple mariners in the two marine flatworms, 5. zebra and Bd. Candida, refutes that possible ecological connection between D. tigrina, hydras, and insects. The fact that such transfers are possible nevertheless implies that these various mariner elements belonging to a variety of different subfamilies are all capable of functioning in the cellular environments of their diverse hosts. The transposition mechanism for mariners is therefore likely to be independent of species-specific host factors, and indeed we have recently developed a completely in vitro transposition system using an irritans subfamily mariner from the horn fly, H. irritans, that requires no host factors (Lampe et al. 1996). Similar results have been obtained for the related Tel element (Vos et al. 1996). It is therefore reasonable to expect that versions of some of these mariner transposons, and perhaps any member of the Tcl-mariner superfamily, might be developed as genetic tools for a wide diversity of animals. 1995. Horizontal transmission, vertical Inactlvatlon, and stochastic loss of mariner-like transposable elements. Mol Biol Evol 12:62-72. Medhora M, Maruyama K, and Hartl DL, 1991. Molecular and functional analysis of the mariner mutator element Most In Drosophila. Genetics 128:311-318. Morgan GT, 1995. Identification In the human genome of mobile elements spread by DNA-medlated transposition. J Mol Blol 254:1-5. References Oosuml T, Belknap WR, and Garllck B, 1995. Mariner transposons in humans. Nature 378:672. Auge-Goulllou C, Bigot Y, Pollet N, Hamelln M-H, Meunier-Rotlval M, and Periquet G, 1995. Human and other mammalian genomes contain transposons of the mariner family. Fed Eur Blochem Soc Lett 368:541-546. Radice AD, Buga) B, Fitch DHA, and Emmons SW, 1994. Widespread occurrence of the Tel transposon family. 7c/-llke transposons from teleost fish. Mol Gen Genet 244:606-621. Avanclnl RMP, Walden KKO, and Robertson HM, 19%. The genomes of most animals have multiple members of the Tel family of transposable elements. Genetlca98: 131-140. Reiter LT, Murakami T, Koeuth T, Pentao L, Muzny DM, Glbbs RA, and Lupsld JR, 1996. A recombination hotspot responsible for two Inherited peripheral neuropathies Is located near a mariner transposon-llke element. Nat Genet 12:288-297. Berg DE and Howe MM, 1989. Mobile DNA. Washington, D.C.: American Society for Microbiology. Canfield VA, Xu K-Y, D'Aqulla T, Shyjan AW, and Levenson R, 1992. Molecular cloning and characterization of Na,K-ATPase from Hydra vulgarw. Implications for enzyme evolution and ouabaln sensitivity. New Blol 4: 339-348. Capy P, Vltallj R, Langln T, Hlguet D, and Bazln C, 1996. Relationships between transposable elements based upon the integrase-transposase domains: Is there a common ancestor? J Mol Evol 42:359-368. Craig NL, 1995. Unity in transposition reactions Science 270:253-254. Doak TG, Doerder FP, Jahn CL, and Herrick G, 1994. A proposed superfamlry of transposase-related genesnew members in transposon-llke elements of ciliated protozoa and a common "D35E" motif. Proc Natl Acad Sci USA 91*12-946. Fisher DA and Bode HR, 1989. Nucleotlde sequence of an actln-encodlng gene from Hydra attcnuatcr. structural characteristics and evolutionary Implications. Gene 84: 55-64. Flavell AJ, Dunbar E, Anderson R, Pearce SR, Hartley R, and Kumar A, 1992. Tyl<opia group retrotransposons are ubiquitous and heterogeneous In higher plants. Nucleic Acids Res 14:3639-3644. Garcla-Fernandez J, Bayascas-Ramfrez JR, Marfany G, Mufloz-Marmol AM, Casall A, Bagufla J, and Sal6 E, 1995. High copy number of highly similar marfner-like transposons in planarian (Platyhelmlnthe): evidence for a trans-phyla horizontal transfer. Mol Blol Evol 12: 421^)31. Garcla-Femandez J, Marfany G, Bagufla J, and Sal6 E, 1993. Infiltration of mariner elements. Nature 364:109110. Grlndley ND and Leschzlner AE, 1995. DNA transposition: from a black box to a color monitor. Cell 83:10631066. Hartl DL, 1989. Transposable element mariner In Drosophila species. In: Mobile DNA (Berg DE and Howe MM, eds). Washington, D.C.: American Society for Microbiology; 5531-5536. Jeyaprakash A and Hoy MA, 1995. Complete sequence of a mariner transposable element from the predatory mite Metaseiulus ocadentatis Isolated by an inverse PCR approach. Insect Mol Blol 4:31-39. Lampe DJ, Churchill MEA, and Robertson HM 1996. A purified mariner transposase Is sufficient to mediate transposition in vitro. EMBO J 155470-5479. Robertson HM, 1993. The mariner transposable element Is widespread in Insects. Nature 362241-245. Robertson HM, 1995. The Tcl-mariner superfamily of transposons in animals. J Insect Physlol 41:99-105. Robertson HM, In press Mariner transposable elements in mites. Proc K Int Cong Acarol. Robertson HM and Asplund ML, 1996. Bmmarl: a basal lineage of the manner family of transposable elements In the silkworm moth, Bombyx mori. Insect Biochem Mol Biol 26545-954. Robertson HM and Lampe DJ, 1995. Recent horizontal transfer of a mariner transposable element between Dlptera and Neuroptera. Mol Blol Evol 12:850-862. Robertson HM and MacLeod EG, 1993. Five major subfamilies of mariner transposable elements In Insects, Including the Mediterranean fruit fly, and related arthropods. Insect Mol Blol 2:125-139. Robertson HM, Soto-Adames FN, Walden KKO, Avanclni RMP, and Lampe DJ, In press. The manner transposons of animals: horizontally jumping genes. In: Horizontal tranlers of genes: Implications and consequences. (Syvanen M, ed). Proc 1996 Falllen Leaf Lake Conf. Robertson HM, Zumpano KL, Lone AR, and Hartl DL, 1996. Reconstructing the ancient mariners of humans Nat Genet 12:360-361. Sambrook J, Frltsch EF, and Manlatls T, 1989. Molecular cloning. A laboratory manual, 2nd ed. Cold Spring Harbor, New York; Cold Spring Harbor Laboratory Press. Schummer M, Scheurlen I, Schaller C, and Galliot B, 1992. HOM/HOX homeobox genes are present In hydra (Chlorohydra viridissima) and are differentially expressed during regeneration. EMBO J 11:1815-1823. Sedensky MM, Hudson SJ, Everson B, and Morgan PG, 1994. Identification of a mariner-like repetitive sequence In C elegans. Nucleic Acids Res 221719-1723. Smit AFA and Rlggs AD, 1996. riggers and other DNA transposon fossils In the human genome. Proc Natl Acad Scl USA 93:1443-1448. Swoflord DL, 1993. PAUP: phylogenetic analysis using parsimony, version 3.1.1. Washington, D.C.: Smithsonian Institution. Vos JC, De Baere 1, and Plasterk RHA, 1996. Transposase Is the only nematode protein required for In vitro transposition of Tel. Genes Dev 10:755-761. Voytas DF, Cummlngs MP, Konieczny A, Ausobel FM, and Rodermel SR, 1992. Copio-like retrotransposons are ubiquitous among plants. Proc Natl Acad Scl USA 89: 7124-7128. Langln T, Capy P, and Daboussl M-J, 1995. The transposable element impala, a fungal member of the Tclmariner superfamily. Mol Gen Genet 246:19-28. Received May 20, 1996 Accepted September 23, 1996 Lohe AR, Mortyama EN, Udholm D-A, and Hartl DL, Corresponding Editor Ross Maclntyre Robertson • Multiple Mariners in Flatworms and Hydras 2 0 1
