A PCR-based assay to detect hAT-like transposon sequences in plants

A PCR-based assay to detect hAT-like transposon sequences in plants
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  A PCR-based assay to detect  hAT  -like transposon sequences in plants P. De Keukeleire 1 , S. De Schepper 2 , J. Gielis 3 & T. Gerats 1 * 1 Department of Plant Systems Biology, Faculty of Sciences, Ghent University, K. Ledeganckstraat 35,9000 Ghent, Belgium;  2 Department of Plant Production, Faculty of Applied Biological and Agricultural Sciences, Ghent University, Coupure links 653, 9000 Ghent, Belgium;  3 Geniaal bvba,Nottebohmstraat 8, B-2018 Antwerpen, Belgium; *Present address: Department of Experimental Botany,University of Nijmegen, Toernooiveld 1, 6525 ED Nijmegen, The Netherlands;E-mail: *Correspondence Received 1 December 2002. Resubmitted in revised form and accepted for publication by Pat Heslop-Harrison 10 October 2003 Key words: hAT  -like transposon, phylogeny, plant genomics, polymerase chain reaction (PCR) Abstract Despite their potential as endogenous tools for forward and reverse genetics, members of the  hobo ,  Ac ,  Tam3 (or  hAT  ) superfamily of transposable elements have been characterized in but a limited number of plantspecies.Toexpeditetheirisolation,wedevelopedaPCR-basedassayforthedetectionof  hAT  -liketransposonsequences in plants which was applied to isolate and initially characterize such sequences from  Petuniahybrida ,  Phaseolus vulgaris ,  Bambusa vulgaris ,  Brassica napus  and  Rhododendron simsii  . Introduction Transposable elements have been describedfor virtually all prokaryotes and eukaryotesexamined to date. Based on similarities betweenthe transposase or integrase encoding gene(s),several superfamilies of evolutionary relatedelements can be defined (Capy  et al.  1996). Onesuch superfamily is the  hAT   group, named afterits first isolated members:  hobo  of   Drosophilamelanogaster  (fruit fly),  Ac  of   Zea mays  (maize),and  Tam3  of   Antirrhinum majus  (snapdragon)(Atkinson  et al.  1993). With representativespresently identified in over 30 species, includingfungi, algae, flies, moths, nematodes, fish,mammals, monocots and dicots, the  hAT   super-family appears to be widely distributed in alleukaryotic kingdoms (for reviews, see Kempkenand Windhofer 2001, Rubin  et al.  2001). Several methods have been used to isolate hAT   group-related sequences (and transposableelements in general): elements have been isolatedas spontaneous insertions into previously clonedgenes (e.g. Fedoro¡   et al.  1983), by trapping in aselectable marker gene (e.g. Grappin  et al.  1996), asrepetitive sequences (e.g. Kempken & Kuck 1996),as expressed sequence tags (e.g. Esposito  et al. 1999), by serendipitous sequencing (e.g. Henk et al.  1999), through  in silico  analyses (e.g. Rubin et al.  2001), and by using heterologous probes inlibrary screenings (e.g. MacRae  et al.  1994). Thelatter technique is restricted by the observationthat  hAT   elements do not readily cross-hybridizeacross taxa (e.g. Hartings  et al.  1998). A moresensitive homology-based strategy makes use of the polymerase chain reaction (PCR): degenerateprimers designed on the basis of conservedregions, were used to isolate  hAT  -like elementsfrom several £y and moth species (Atkinson  et al. 1993, DeVault & Narang 1994, Warren  et al. 1995, Handler & Gomez 1996). Although non-degenerate primers were used to isolate  Bg -related Chromosome Research  12 : 117–123, 2004.  117 #  2004  Kluwer Academic Publishers. Printed in the Netherlands  sequences from a number of Gramineae species(Hartings  et al.  1998), and  Ac / Ds -like sequencesfrom  Pennisetum glaucum  (pearl millet) and  Bam-busa multiplex  (hedge bamboo) (Huttley  et al. 1995), a more general assay to detect  hAT  -likesequencesinplantswasnotavailable.The development of such a general PCR assayis not straightforward considering the level of diversity that exists between  hAT  -elements. Withinthe transposases of   hAT  -group members, Rubin et al.  (2001) identi¢ed six conserved blocks (here-after termed Rubin’s blocks A to F) varying in len-gth from 10 to 26 amino acids. However, not allblocks are present in all members; for instanceblocks C, D and F are missing in  Bg , and blocks Cand F are missing in  Tag1 . Phylogenetic analysesof block E, which is most highly conserved,revealed that several subgroups might bedistinguished within the assembly of plant  hAT  -transposases. Among these subgroups are (i) agroup of sequences most similar to the maize  Ac element to which both monocot (e.g.  Ac ,  Pac1 )and dicot elements ( Tam3 ,  Slide ) belong; (ii) the Tag1  and (iii)  Bg  subgroups, and (iv) an appar-ently distinct group of   Tag2 -like sequences exclu-sively found in  Arabidopsis . Elements belonging tothese di¡erent subgroups might very well coexistin one plant species. Examples are the  Ac  and Bg  elements of   Z. mays  and  Tag1  and  Tag2  of  A. thaliana . Also in rice have two separateparalogs of   hAT  -like sequences been identi¢ed(Mao et al. 2000).Here we report on the development of aPCR-based assay to detect  hAT  -like transposonsequences in plants, and the isolation and initialcharacterization of such sequences from ¢veadditionalplantspecies. Materials and methods DNA amplification Polymerase chain reactions were performed in0.2ml Ultra-Thin polypropylene PCR reactiontubes (Biozym) on a GeneAmp PCR System9600 (Perkin-Elmer). Reaction mixturescontained 50ng to 250ng of genomic DNA,0.4 m mol/L forward primer, 0.2 m mol/L reverseprimer, 2 units AmpliTaq (Perkin-Elmer),1  PCR buffer II [Perkin-Elmer, i.e. 10mmol/LTris-HCl, pH 8.3 (at 25  C); 50mmol/L KCl],1.5mmol/L MgCl 2  (Perkin-Elmer), and0.2mmol/L of dATP, dCTP, dGTP and dTTPeach (Pharmacia Biotech) in a final reactionvolume of 30 m l. The cycling parameters were:initial denaturation for 2:30 min at 94  C,followed by 45 cycles of 30s at 94  C, 60s at46  C, 90s at 72  C, with a final extension for15min at 72  C. To achieve a manual hot start,the addition of MgCl 2  was postponed until afterthe initial denaturation; during addition thereaction mixture was held at 80  C. Both forward and reverse primers were de-generate. The sequence of the forward primer was5 0 -CA(C/T)GTI(A/C)GITG(C/T)IIITG(C/T)CA(C/T)AT(A/C/T)(C/T)T-3 0 , the sequence of thereverse primer was 5 0 -AAIGCI(C/G)I(C/T)TCI(C/G)(A/T)IGC(A/C/G/T)AC(A/C/G/T)GT-3 0 ,whereIdenotesdeoxyinosine.For all plant species tested, negative controlreactions were performed with only one of bothprimers, and in the absence of template genomicDNA. In all cases major PCR products were notobserved in single-primer reactions. 10 m l of thereaction products was separated by electrophoresisthrough a 1.5% (w/v) agarose gel, containing10 m g/ml ethidium bromide and visualized on aUVtransilluminator. Sequence analysis Amplified fragments were ligated into a pGEM-Tvector (Promega) and transformed into  E. coli  XL1blue or DH5 a  cells (Hanahan 1983).Recombinant clones were identified (Sandu  et al. 1989) and sequenced (Sanger  et al.  1977) usingfluorescent dye terminators and AmpliTaq FS(cat. no. 402079) in a cycle sequencing protocolaccording to the recommendations of themanufacturer (PE Applied Biosystems, ABI373Aand ABI377 automatic DNA sequencer). Splice sites were predicted with the NetPlant-Gene2v2.4 algorithm (Hebsgaard  et al.  1996).Implied protein sequences encoded by theampli¢ed fragments and possible frame shifts wereidenti¢ed by FastY comparison (Pearson  et al. 1997) of their nucleotide sequence to the equi-valent regions of the  ZmAc ,  Tag2  and  Tam3 transposase amino acid sequences. Blocks of ungapped aligned regions were identi¢ed using 118  P. De Keukeleire et al.  Dialign2 (Morgenstern 1999). Phylogenetic treeswere constructed using the neighbor-joiningroutine in Clustal W (Thompson  et al.  1994),Kimura corrections for multiple substitutions wereapplied and 1000 bootstrap resamplings wereperformed. Trees were drawn using TreeView(Page1996). Results Given the level of divergence among plant  hAT  -group members we decided to conceptuallyrestrict the PCR assay to the  Ac  subgroup. Aforward primer which targets the HVRC(I/A)-CHIL motive in Rubin’s block B, and a reverseprimer directed towards the TVASE(S/R)AFmotive in block E were developed to fit the maize Ac  and snapdragon  Tam3  elements. Althoughadditional  Ac -subgroup members are known(Rubin  et al.  2001), only these two elements werechosen for primer development because theirautonomous function has been demonstrated(Baker  et al.  1986, Martin  et al.  1989). PCRreaction mixture compositions and temperatureprofile conditions were established in which theseprimers amplify the predicted 953-bp fragmentfrom a  Tam3  plasmid clone (  pAmp8 ). Underthese conditions, two fragments are amplifiedfrom  A. majus  (line 164) genomic DNA (Figure1). Larger insertion-relatives of the reference Tam3  (S-CHS) element present in the plasmidclone, have been characterized (Yamashita  et al. 1998), and possibly the larger fragment producedon genomic DNA of   A. majus  is the amplifica-tion product of such an element. When tested on genomic DNA of   P. hybrida (line W137),  Phyllostachys edulis ,  B. vulgaris  and Sasa veitchii  , major PCR products were obtainedof a size corresponding to the smaller of the two A. majus  fragments (Figure 1). Major productswith a size similar to the larger  A. majus  fragment,were obtained with  P. vulgaris ,  Phaseolus acuti- folius  and  B. napus . With  R. simsii  , cultivar‘Hellmut Vogel’, major fragments of both sizeclasses were ampli¢ed. Plasmid clones of the P. hybrida ,  P. vulgaris ,  B. vulgaris ,  B. napus (which were termed  hATpehy1 ,  hATphvu1 ,  hAT-bavu1 ,  hATbrna1  respectively) and  R. simsii   majorPCR products were sequenced. In the case of   R.simsii  , three di¡erent fragments were cloned fromthe PCR mixture: two sequences, termed  hATrosi1 and  hATrosi2 , have a size which corresponds tothe smaller of the two major products; onesequence, termed  hATrosi3 , corresponds to thelarger product. For all clones, FastY comparisons(Pearson  et al.  1997) revealed sequence similaritiesto the equivalent regions of the  ZmAc1 ,  Tag2  and Tam3  transposases. Coding frames appear intactin the case of   hATbavu1 ,  hATpehy1  and  hATrosi3 ,but are interrupted by a single stop codon in thecase of   hATrosi1  and  hATrosi2 , and by severalstop codons and frame shifts in the case of  hATbrna1 and hATphvu1 .The primer annealing sites span an intron in themaize  Ac  element (Kunze  et al.  1987), an intronat a di¡erent position in the  Arabidopsis Tag2 element (Henk  et al.  1999), but no introns arepresent within this region in the snapdragon  Tam3 element (Hehl  et al.  1991). The extra length of the hATphvu1 ,  hATbrna1  and  hATrosi3  fragmentssuggests these also contain an intron. The  Net-PlantGene2  algorithm (Hebsgaard  et al.  1996)predicts a 92-bp intron in the case of   hATbrna1 , Figure 1.  Agarose gel electrophoresis analysis of the PCRproducts obtained on genomic DNA of various plant species,including  Petunia hybrida  (Pehy),  Phaseolus vulgaris  (Phvu), Phaseolus acutifolius  (Phac),  Phyllostachys edulis  (Phed), Bambusa vulgaris  (Bavu),  Sasa veitchii   (Save),  Rhododendronsimsii   (Rosi) and  Brassica napus  (Brna). The reaction on Antirrhinum majus  (Anma) genomic DNA is included as apositive control.  M : molecular size marker. A PCR-based assay to detect hAT-like transposon sequences in plants  119  and a 98-bp intron in the case of   hATphvu1 (con¢dence levels for splice donor and acceptorsites are better than 92%; branch point scoresare   4.07, respectively   2.58). These putativeintrons start at exactly the same nucleotideposition from the 5 0 forward primer end in bothsequences, and are situated in the region of thesingle intron in the  Arabidopsis Tag2  element(Figure 2a). Upon removal of these putativeintrons from the sequence, FastY comparisonsidentify an apparently intact coding frame in thecase of   hATphvu1 , while this frame is still inter-rupted by a single frame shift in the case of  hATbrna1 . The putative translation product of theORF present in the  hATrosi3  sequence appears tocontain an extra 30 amino acids which fail to alignand which are located approximately at the posi-tion of intron-2 in the  Zea mays Ac  element(Figure 2a). The  NetPlantGene2  algorithm predictsthe  Ac  intron (con¢dence levels for splice donorand acceptor sites are better than 87%; the branchpoint score is   2.79), but does not identify anintron in the region corresponding to the extrasequencein hATrosi3 .To further investigate the sequence similaritiesbetween the isolated fragments mutually and withestablished  hAT  -group representatives, ungappedaligned regions were identi¢ed within their putativetransposase amino acid sequence (Figure 2b) andused to infer a tree-based phylogeny (Figure 2c).Although  Tag2 -like elements were previouslyrestricted to  Arabidopsis  (Rubin  et al.  2001), thenewly isolated  hATrosi1 ,  hATbrna1  and  hATphvu1 appear to form a distinct branch with  Tag2 , whilenone of the ampli¢ed fragments displays phylo-genetic a⁄nity to  Tam3 . The recovery of   Tag2 -likesequences is unexpected, as the PCR primers usedin the assay were not designed to ¢t the HVRCSAHIL motive present in Rubin’s block B of   Tag2 .The tree also illustrates the divergence of the threesequences isolated from  R. simsii  : even within thecontiguous blocks, which are thought to encode thecore of conserved amino acids, identity (% ID) andsimilarity (% SIM) scores are only between 41%and 50%, and 63% (for all pairwise comparisons)respectively. These scores are below those observedwith  hAT  -like transposon sequences from otherspecies: the blocks of the dicot  hATrosi2  are 65%identical, 80% similar to the monocot  hATbavu1 ;top scores for  hATrosi1  are found with  Tag2  (59%ID, 75% SIM), and a BlastP analysis (Altschul et al.  1997) reveals the  hATrosi3  blocks are mostsimilar to a transposase ORF from  Arabidopsis ( gi  15228784;58%ID,70%SIM). Discussion Using a newly-developed PCR-based assay, wehave detected  hAT   group-related sequences in P. acutifolius ,  P. edulis  and  S. veitchii  , and inaddition isolated from  P. hybrida  ( hATpehy1 ), P. vulgaris  ( hATphvu1 ),  B. vulgaris  ( hATbrna1 ), R. simsii   ( hATrosi1-3 ) and  B. napu s ( hATbrna1 ).Only in  hATbrna1 ,  hATpehy1  and  hATrosi3  doesthe amplified portion of the transposase-encodingORF appear to be intact. In the case of  hATbrna1 ,  hATphvu1 ,  hATrosi1  and  hATrosi2 this coding frame is interrupted by a stop codonor frame shift, indicating these fragmentsprobably do not represent functional transposasegenes. The exact nature of the putative intronsequence will codetermine the functionality of  hATphvu1 . hAT  -group elements usually exist within thegenome as heterogeneous families of one or a fewautonomous, transposase-providing elements andmany non-autonomous members. The latter areoften internal deletion derivatives of the former.For their mobility they rely on the provision of atransposase in trans. In addition, a genome maycontain immobile members, due to mutations infor example the transposase recognition sites,or even entire fossil families, which have beeninactivated during the course of evolution (Kunze et al.  1997). Although the presented PCR assayprovides a rapid method for the isolation of   hAT  group-related sequences from a range of species,even in the absence of obvious sequence aberra-tions, it thus cannot provide conclusive informa-tion on their functionality. Moreover, theidenti¢cation of a transposase-providing elementwithin the multitude of non-autonomous familymembersmaynotbestraightforward.Although less pronounced than in the case of   Ac and  Bg  of   Z. mays  (Rubin  et al.  2001),  Tag1  and Tag2  of   A. thaliana  (Henk  et al. , 1999) and twoparalogs identi¢ed in  O. sativa  (Mao  et al.  2000),the sequence divergence between the three di¡erent R. simsii   fragments could point toward the 120  P. De Keukeleire et al.  Figure 2.  ( A ) Schematic representation of the structural similarities between the fragments isolated by means of the PCR assay andthe transposase genes of the  A. thaliana Tag2 ,  Z. mays ZmAc  and  A. majus Tam3 hAT  -group members. Bars A  F mark the positionsof Rubin’s blocks (Rubin  et al.  2001) in  Tag2 . Open boxes indicate exons (thick border) or exon fragments (thin border), connectinglines represent introns.  hATphvu1  and  hATbrna1  contain a putative intron (open box with question mark) situated in the region of thesingle intron of   Tag2 . An extra sequence, which fails to align is present in  hATrosi3  approximately at the equivalent position of thesecond intron of   ZmAc . /: frameshift introduced in  hATbrna1  to maintain sequence similarity. In the case of   hATrosi1  and  hATrosi2 the transposase-encoding reading frames are interrupted by a stop codon (*, resp. **). Grey boxes delineate blocks of ungappedaligned regions (Block 1 to 5; cf. Figure 2b). ( B ) Ungapped aligned blocks identified within the (deduced) amino acid sequences of fragments isolated by means of the PCR assay and representative  hAT  -group transposases. 15228784 (genbank identifier) and  Pac1 are  hAT  -like transposases from  A. thaliana  and  Pennisetum glaucum , respectively. Identical amino acid residues are in inverted type,chemically similar residues are indicated with a grey background. ( C ) Phylogenetic tree derived by the neighbor joining method onthe ungapped aligned blocks, illustrating the heterogeneity of sequences derived from  R. simsii   ( hATrosi1-3 ), the phylogenetic affinityof   hATbrna1 ,  hATrosi2  and  hATphvu1  for the  A. thaliana Tag2 , and the absence of sequences similar to  Tam3 . The horizontal barrepresents the number of substitutions per site; bootstrap percentages are indicated. A PCR-based assay to detect hAT-like transposon sequences in plants  121
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