It Takes Two Transposons to Tango Transposable-Element-Mediated Chromosomal Rearrangements

Personal view of the genome project Perspective

45 Goffeau, A. et al. (1997) The Yeast Genome Directory. Nature 48 Leversha, M.A. et al. (1999) A molecular cytogenetic clone to microarrays. Nat. Genet. 20, 207–211 387, 1–105 resource for chromosome 22. Chromosome Res. 7, 571–573 51 Lockhart, D.J. and Winzeler, E.A. (2000) Genomics, gene 46 The C. elegans Sequencing Consortium (1998) Genome 49 Kirsch, I.R. et al. (2000) A systematic, high-resolution expression and DNA arrays. Nature 405, 827–836 sequence of the nematode C. elegans: a platform for linkage of the cytogenetic and physical maps of the human 52 Pandey, A. and Mann, M. (2000) Proteomics to study genes investigating biology. Science 282, 2012–2018 genome. Nat. Genet. 24, 339–340 and genomes. Nature 405, 837–846 47 Adams, M.D. et al. (2000) The genome sequence of 50 Pinkel, D. et al. (1998) High resolution analysis of DNA copy 53 Risch, N.J. (2000) Searching for genetic determinants in the Drosophila melanogaster. Science 287, 2185–2195 number variation using comparative genomic hybridization new millennium. Nature 405, 847–856 It takes two transposons to tango transposable-element-mediated chromosomal rearrangements

Transposable elements (TEs) promote various chromosomal rearrangements more efficiently, and often more specifically, than other cellular processes1–3. One explanation of such events is homologous recombination between multiple copies of a TE present in a genome. Although this does occur, strong evidence from a number of TE systems in bacteria, plants and animals suggests that another mechanism – alternative transposition – induces a large proportion of TE-associated chromosomal rearrangements. This paper reviews evidence for alternative transposition from a number of unrelated but structurally similar TEs. The similarities between alternative transposition and V(D)J recombination are also discussed, as is the use of alternative transposition as a genetic tool.

ince the first description of mobile genetic elements4,5, are TEs that contain sequences encoding other genes in Stransposable elements (TEs) have been found to be addition to transposase, such as genes encoding enzymes associated with chromosomal rearrangements such as responsible for antibiotic resistance. In eukaryotes, all TEs deletions, duplications, inversions, the formation of acen- that transpose by a DNA intermediate are classified as tric fragments and dicentric chromosomes, translocations transposons. Some Class II TEs, such as IS10, IS50, Ac/Ds and recombination of host genomes. This aspect of trans- (Box 1), Tam3, P, hobo and mariner, encode a single posable element function has implications for evolution2,6 transposase gene. Other Class II TEs, such as Tn7, Phage and for understanding several human genomic disorders7,8 Mu, Mutator and En/Spm, encode multiple proteins that and, because of this, the mechanisms involved in trans- catalyse and regulate transposition. poson-mediated chromosomal rearrangements warrant Two possible mechanisms by which TE-associated thorough investigation. chromosomal rearrangements can occur are: (i) indirectly TEs are classified by their sequence structure and trans- by homologous recombination or (ii) directly by an alter- position mechanisms1,3,6. Class I TEs – retroposons and native transposition process. retrotransposons – transpose by an RNA intermediate. The indirect action of TEs promotes chromosomal Retroposons have a structure similar to mRNA; retro- rearrangements by presenting the genome with multiple transposons are structurally similar to retroviruses and are similar, if not identical, sequences between which strand Yasmine H.M. Gray bounded by long terminal repeats (LTR). Class II TEs – transfer can occur. This may occur by recombination of [email protected] insertion sequences (IS elements, Box 1) and transposons – the homologous sequences or by faulty repair of double- Molecular Genetics and transpose by a DNA intermediate catalysed by a trans- strand breaks formed during transposable element Evolution Group, posase enzyme. IS elements and transposons are bounded excision using ectopic homologous sequences as a repair Research School of 3 by terminal inverted repeats (TIR). In addition to the TIR, template . Biological Sciences, additional sequences differentiate the two ends and are Not all the rearrangements observed can be explained Australian National necessary for transposition. In prokaryotes, IS elements by homologous recombination between elements at differ- University, Canberra, contain sequences encoding transposase, and transposons ent locations. For instance, rearrangements have been ACT 2601, Australia.

0168-9525/00/$ – see front matter © 2000 Elsevier Science Ltd. All rights reserved. PII: S0168-9525(00)02104-1 TIG October 2000, volume 16, No. 10 461 Perspective Transposons and chromosomal rearrangements

FIGURE 1. Chromosomal rearrangements caused by homologous recombination

(a) (b) (c) (d)

inversion

trends in Genetics

Homologous recombination between repetitive sequences, such as TEs, can result in chromosomal rearrangement such as deletions, duplications and inversions. Each line represents a DNA double helix. The two sister chromatids of each of the homologous chromosomes are shown. Black ovals denote the centromere. TEs are represented by the thick black line bounded by open and closed arrows, indicating relative orientation of the element. The TE insertion sites are illustrated by open circles or boxes, with each shape representing distinct insertion sites on the chromosome and the equivalent sites on the chromosome(s) without a TE at that site. Homologous recombination requires a minimum of two copies of the repetitive sequence, one at each breakpoint, and is denoted by an ‘X’ in this figure. (a) TEs in same relative orientation on homologous chromosomes result in the formation of chromosomes containing either a deletion or a duplication of the intervening sequence. Both rearrangements are associated with recombination between two homologues. (b) TEs in opposite relative orientation on homologous chromosomes result in the formation of a dicentric chromosome and an acentric fragment. (c) TEs in same relative orientation on one chromosome result in the formation of chromosomes containing either a deletion or a duplication of the intervening sequence, differing from events in A by the lack of recombination between homologues and the net increase or decrease of the TE number. (d) TEs in opposite relative orientation on one chromosome can result in the formation of an inversion between the two TEs. If caused by homologous recombination, deletions and duplications can only be formed by TEs in the same relative orientation and inversions can only be formed by TEs in opposite relative orientation. Another mechanism must be invoked to explain inversions between TEs in the same relative orientation, deletions and duplications between TEs in opposite relative orientations, and all chromosomal rearrangements when a TE is present at only one of the rearrangement breakpoints.

described where an element was found at only one of repair of the double-strand breaks produced during the rearrangement breakpoints in the parental chromo- alternative transposition is analogous to V(D)J recombination some9,10. Some rearrangements described are inconsistent and provides additional evidence supporting the theory with the orientation of the elements present in the chro- that V(D)J recombination is derived from a so-called RAG mosome prior to rearrangement11, such as duplications transposon. between inverted copies of a TE or inversions between TEs in the same relative orientation (Fig. 1). Also, because TEs – a common resource for genome plasticity recombination does not normally occur in Drosophila In order to comprehend complex chromosomal rearrange- melanogaster males12, rearrangements mediated by TEs ments induced by alternative transposition of TEs, one such as P and hobo must occur by another mechanism in must first understand the basics of traditional transpos- the male germ line of Drosophila. ition. The TEs inducing rearrangements described in this The direct action of TEs in promoting chromosomal review are all Class II TEs encoding a single transposase rearrangements is one mechanism that can account for and include prokaryotic IS elements and both prokaryotic rearrangements not caused by homologous recombi- and eukaryotic transposons. Functionally, these TEs share nation. TEs induce chromosomal rearrangements directly a common conservative transposition mechanism, known by an alternative version of the traditional transposition as cut-and-paste, where the first step of transposition is reaction where the TE ends involved come from separate the synapsis of complementary left- and right-TE ends, elements rather than a single element (Fig. 2b). Evidence followed by excision of the ends, target site capture and for similar events has been described for several families of strand transfer1,3. Insertion of the TE into the target mol- TEs, including the IS10/Tn10 elements in bacteria13,14, ecule can occur in either orientation relative to the original Ac/Ds elements in maize and tobacco11,15,16, Tam3 in element, resulting in a simple insertion (Fig. 2a). Repair of Antirrhinum majus (snapdragon)9,10,17–20 and P elements in the double-strand break occurs and can result in for- Drosophila21–24. mation of an excision footprint, regeneration of the TE Rearrangements associated with different TE systems using the sister chromatid as a template, gene conversion, have previously been examined separately. Here, pub- or recombination. lished data from several different systems are reviewed in Chromosomal rearrangements more complex than sim- order to emphasize the fact that the transposon-induced ple insertions result from alternative transposition events rearrangements first described a decade ago in prokary- where complementary ends from separate TEs synapse otes occur by the same mechanism in many eukaryotic TE rather than the traditional synapsis of complementary ends systems. The mechanism is an alternative to the normal from a single TE9–11,13–24. The synapsis of TE ends from sepa- transposition reaction in each system. Furthermore, the rate molecules has been demonstrated in vitro and is referred

462 TIG October 2000, volume 16, No. 10 Transposons and chromosomal rearrangements Perspective

14,24 to as bimolecular synapsis (Box 1) . Figure 2b depicts an FIGURE 2. Traditional versus alternative transposition alternative transposition event, in steps equivalent to those depicted in Figure 2a for traditional transposition. Once the hybrid element is formed (Box 1), the chemi- (a) (b) cal steps of the alternative transposition reaction are identical to those of a normal transposition reaction. Excision of the hybrid element forms two double-strand breaks. (i) The ‘excised’ hybrid element may reinsert into the genome. The remaining double-strand breaks at the site of hybrid element excision may be repaired. Transposase is required for alternative transposition to occur. However, in contrast to the excision of an intact TE, one end of each (ii) of the TEs in a hybrid element remains covalently bound to a large chromosomal fragment. The type of rearrangements produced by alternative transposition depends on the type of DNA molecules involved – either linear or circular – for both donor and * target, and on the location of the target site relative to * the ends involved in alternative synapsis. The types of ** (iii) rearrangements observed depend on the viability of the resulting chromosome structure in the species being examined. Detailed examples of various types of rearrangements can be found in the original publications9–11,13–24. ** * Figure 2b examines an alternative transposition event (iv) * where the complementary ends involved are from homologous elements on sister chromatids, with an insertion target site located on the same chromosome arm on the homologue as the element ends forming the hybrid element. This scenario results in the formation either of an acentric fragment and a dicentric chromosome or of recombinant chromosomes with recombinants containing (v) a reciprocal deletion/duplication. Most of the reported chromosomal rearrangements (Box 1) consistent with alternative transposition are of deletions, duplications and inversions. Such a bias in the types of observed events could be due to higher frequency of occurrence or viability. (vi) While Figure 2b details one type of rearrangement that can be formed by alternative transposition, Figure 3 contains a schematic summary of sixteen possible classes trends in Genetics of rearrangement caused by alternative transposition. Notably, an inversion is produced if the hybrid element inserts into one of the chromosome arms involved in for- The basic steps of transposition are shown as discrete steps for illustration purposes. Each line mation of the hybrid element (Classes 2Ј and 3Ј in Fig. 3). represents a DNA double helix. The two sister chromatids of each of the homologous chromosomes are Inversions formed by alternative transposition will contain shown. Black ovals denote the centromere. Complementary left- and right-ends of the TE are shown as both copies of the target site duplication on a single chro- open or closed triangles, respectively. The original target site duplications are shown as open circles. mosome. One of the target site duplications is located The new target site duplications are shown as open boxes. Asterisks denote the double-strand breaks within the inverted segment and, therefore, the duplicated that are repaired and can result in formation of an excision footprint, regeneration of the TE using the target sites are in inverse complementary orientation, sister chromatid as a template, gene conversion or recombination. (a) Traditional cut-and-paste transposition – complementary TE ends from an intact element synapse, excise and reinsert into a new rather than the direct orientation found flanking normal target site. The TE can insert in either of two orientations relative to the directionality of the original transposon insertions. The experimental determination of insertion. In the case of traditional transposition, either insertion orientation results in a simple a number of independent inversion events containing the insertion. (b) Alternative transposition – the first step in alternative transposition is the synapsis of predicted structure, including the target site duplication complementary TE ends from seperate TEs to form a hybrid element. In the case illustrated here, the typical of TE insertion events, was instrumental in demon- complementary TE ends are derived from homologous elements on sister chromatids. Once bimolecular strating that alternative transposition does occur in synapsis occurs, excision, insertion of the hybrid element into the new target site and repair of the eukaryotes22. An animated diagram of the formation of an double-strand breaks occurs by the same mechanisms as in traditional transposition. Because the inversion caused by alternative transposition can be found hybrid element remains covalently bound to the chromosome, different insertion orientations result in at http://www.wisc.edu/genestest/CATG/engels/Pelements/ different types of chromosomal rearrangements. In the example shown here, one insertion orientation HEIinv.html. results in formation of an acentric fragment and a dicentric chromosome, while the other insertion orientation results in the formation of recombinant chromosomes. Note that the recombinant A translocation event could result if the insertion target chromosomes in this example also contain a reciprocal deletion or duplication of the genomic segment site is on a different chromosome from that which the between the original and new target sites. All chromosomal rearrangements resulting from alternative TE ends forming the hybrid element originate (Fig. 4). transposition have two distinctive structures consistent with a TE insertion event. First, one of the Specifically, precise reciprocal translocations result when breakpoints in the rearrangement should be at the terminus of a functional TE end. Second, a target site caused by alternative transposition. Translocations were duplication should be produced. The two copies of the target site duplication will be situated on two amongst the first observed TE-mediated chromosomal different chromosomes in many of the resulting rearrangements.

TIG October 2000, volume 16, No. 10 463 Perspective Transposons and chromosomal rearrangements

FIGURE 3. Many types of chromosomal rearrangement can result from a single TE insertion

i ii iii 1 2 Alternative transposition results in sixteen classes of rearrangement when the insertion target site is on the same chromosome arm as the TEs involved in * * forming the hybrid element. The diversity of possible rearrangements formed by 3 4 alternative transposition is in contrast to the specificity of rearrangements 5 6 formed by homologous recombination (Fig. 1). The basic steps, (i)–(vi), of traditional and alternative transposition as shown in Figure 2 are repeated here 7 8 illustrating the process and outcomes of alternative transposition for target sites in the eight zones possible relative to the TEs involved in forming the hybrid element. Each line represents a DNA double helix. The two sister chromatids of iv 1 2 each of the homologous chromosomes are shown. Black ovals denote the * * * * centromere. Complementary left- and right-ends of the TE are shown as open or * * * * closed triangles, respectively. The original target site duplications are shown as 3 4 open circles. The new target site is shown as open boxes. Asterisks denote the double-strand breaks that are repaired and can result in formation of an excision footprint, regeneration of the TE using the sister chromatid as a template, gene conversion, or recombination. Symbols: ⌬ = deletion, ⌺ = duplication. * * * *

5 * *6 * * rearrangements25 but have not featured prominently in the recent literature. The paucity of reports describing TE- 7 8 induced translocations could be due to restriction of target site choice by the physical constraints of the hybrid element being tethered to chromosome arms. Alternatively, v 1 243 even if the translocation event occurs readily, gametes pro- * * * * duced are not likely to be viable unless the complementing * * * * translocated chromosomes segregate together. The transposition of non-TE genomic sequences could result if complementary ends from two different elements 1' 2' 3' 4' * * * * on the same chromosome arm separated by the exogenous * * * * sequence combine to form a hybrid element (Fig. 5). Such events are known to occur readily in prokaryotic systems. For instance, compound transposons such as Tn10 are 8 5 6 7 formed when two copies of an IS element, IS10 in the * * * * * * * * case of Tn10, transpose as a unit with the intervening sequence. Evidence for similar events involving closely linked Ac/Ds elements in plants has been published11,26,27. 5' 6' 7' 8' Recently, successful excision and transposition of a * * * * macrotransposon has been observed in Drosophila con- * * * * sisting of two mariner elements (Box 1) surrounding exogenous sequences. The inner ends of each mariner element had been mutated and were no longer able to serve as substrates for transposase (Lozovskaya and Hartl, personal communication). Such eukaryotic macrotrans- vi posons might be regarded as analogous to bacterial trans- 1 2 3 4 ∆ posons. Several more complex rearrangements could be ∆ ∆ ∆ ∆ envisioned and probably do occur, although more complex rearrangements are also more likely to be non-viable. Given that alternative transposition events are expected 1' 2' 3' 4' to undergo the same molecular reactions as occur during classical transposition, one would expect that the double- strand breaks created during hybrid element excision inversion inversion would also undergo repair, as does the double-strand break formed by the excision of an intact TE. Rearranged 5 6 7 8 chromosomes result from this process, referred to as

Σ ∆ Σ ∆ Hybrid-element Excision Repair (HER) in the P element 22,28,29 ∆ Σ ∆ Σ system . Strong evidence is available from Ac/Ds in maize and tobacco where excision footprints have been found at the rearrangement breakpoint of complex 5' 6' 7' 8' rearrangements shown to have been mediated by alternative transposition11,15,16. The many examples of transposase-dependent excision and insertion events involving hybrid elements, as well as repair of double-strand breaks formed by the excision trends in Genetics of hybrid elements, strongly support the occurrence of

464 TIG October 2000, volume 16, No. 10 Transposons and chromosomal rearrangements Perspective

alternative transposition. The only difference between tra- FIGURE 4. Formation of precise reciprocal translocations ditional and alternative transposition is the choice of TE ends to synapse. Bimolecular synapsis cannot explain all TE-mediated rearrangements not explained by homologous recombination. True one-ended transpositions and adjacent inverted duplications reported in snapdragon18,30,31 and Drosophila32 are just some of the rearrangements fitting this description. The purpose of this review is to demon- strate the ubiquity of alternative transposition in a number of TE systems, without diminishing the role other mechanisms in TE-mediated genome rearrangement.

Regulation of alternative transposition * In addition to similarities in structure and transposition * mechanism, described above, the IS10, hAT elements – such as Ac/Ds and Tam3 – and P elements show other similarities that must be considered in the context of their effects on alternative transposition, including regulation mechanisms, choice of insertion site and complexity of TE structure. Regulation of transposition is similar in P elements, the hAT superfamily (Box 1) and IS10, although not all regu- latory mechanisms have been demonstrated for all the elements examined1,3. Relevant to alternative transposition is the regulation of transposition by the methylation state of the element. Both traditional and alternative transpositions of IS10 and Ac/Ds elements have been shown to be regulated by methylation16,27,33. Only hemi-methylated sequences at the transposase binding sites are recognized by transposase, thus restricting transposition to immedi- ately after passage of a DNA replication fork27,33 and providing an additional regulation mechanism in the recogni- tion of TE end complementarity16. D. melanogaster does not display obvious differential methylation12 and this trends in Genetics regulation mechanism is therefore unlikely to affect P or other elements in Drosophila. Insertion of a hybrid element into a separate chromosome results in the formation of exact reciprocal Another important factor affecting the types of chro- translocations, regardless of the target site. A viable zygote could result if the reciprocal products of mosome rearrangements caused by alternative transpos- hybrid-element-mediated translocation segregated into the same germ cell and no deleterious gene ition is the location of hybrid element insertion. Here, the interruption occurred at the breakpoints. issue of physical constraint of the hybrid element should be considered. Deletions extending over at least 100 kb have been described23. It is therefore reasonable to expect In terms of TE structures that promote alternative that duplications or inversions of over 100 kb are also transposition, an inverse correlation between the complex- formed. No absolute limit has yet been established for the ity of element structure and the formation of chromoso- distance between the locations of hybrid element excision mal rearrangements has been established for Ac/Ds and P and insertion, so the possibility remains that alternative elements21,27. Ac elements and State-II Ds elements, which transposition is not physically constrained by the covalent are simple deletion derivatives of Ac, produce high rates tether between the hybrid element and the remaining of transposition. Conversely, State-I Ds elements have chromosome arms. This supports the possibility of hybrid complicated structures and produce high levels of element insertion into a separate chromosome, resulting in chromosome breakage with very little transposition27. the formation of exact reciprocal translocations (Fig. 4). Chromosomal rearrangements associated with alternative In terms of preference of the insertion distance, both P transposition of P elements have also been shown to be and Ac/Ds elements have been shown to transpose to more likely to occur with more complex presentation of closely linked sites and to sites in close proximity of other functional element ends21. One explanation is that, unlike P and Ac/Ds elements more often than if insertion sites intact elements that can undergo either traditional or were chosen at random27,34. The preference for insertion alternative transposition, disjointed element ends may into nearby target sites would decrease the possibility of be recognized by transposase only as part of a hybrid large deletions and duplications, as well as translocations element. – an aspect that may be crucial to the viability of alterna- Clearly, intact TEs undergo traditional transposition tive transpositions. Also, the choice of a target site close more frequently than alternative transposition. However, to an existing element may explain the number of intact TEs do participate in the formation of hybrid el- rearranged chromosomes derived from progenitor chro- ements that then undergo alternative transposition. The mosomes containing elements at both rearrangement relative frequency of traditional versus alternative trans- breakpoints. position can be determined by examining systems in which

TIG October 2000, volume 16, No. 10 465 Perspective Transposons and chromosomal rearrangements

FIGURE 5. Compound transposons (prokaryotes) and BOX 1. Glossary of terms macrotransposons (eukaryotes) Ac/Ds – family of transposons first described in the 1940s by Barbara McClintock in maize. Originally described as two separate elements, Activator (Ac) and Dissociation (Ds), molecular analysis has subsequently revealed that Ds elements are, in fact, derivatives of Ac elements. Bimolecular synapsis – synapsis of complementary TE ends from separate molecules. Chromosomal rearrangement – rearrangement of the linear sequence of chromosomes including transposition, duplication, deletion, inversion or translocation of nucleic acid segments. hAT superfamily – group of eukaryotic transposons that have related transposase genes. The superfamily name is derived from the hobo, Ac/Ds and Tam3 elements, which were the first ‘members’ of this superfamily recognized to have similar transposases. The level of protein similarity ranges between 20 and 60%. The superfamily now includes ** a number of other transposons, including Ascot-1 (from the fungus Ascobolus immersus) hermes (first described in the housefly Musca domestica), hermit (first described in Queensland fruit fly, Bactrocera tryoni ), hopper (from the oriental fruit fly, Bactrocera dorsalis), restless (first described in fungus Tolypocladium inflatum) and Tfo1 (from the fungus Fusarium oxysporum). * * Hybrid element – the unit of DNA consisting of complementary TE ends from separate elements that have synapsed and can undergo excision, target site capture and insertion by the same mechanism as normal transposition. Insertion sequence (IS) – prokaryotic TEs that transpose by a DNA intermediate and contain only sequences necessary for transposition (termini and transposase gene). Some IS elements can form the termini of prokaryotic transposons, such as IS10 forming the ends of Tn10 or IS50 forming the ends of Tn5. mariner – transposon first isolated from Drosophila and since shown to exist in a number of species, including humans. All mariner elements duplicate the 2bp sequence, TA, upon insertion and contain a D,D, (35) D catalytic triad, rather than the D, D, (35) E motif shared by most other transposases. Several subfamilies of mariner have been described, with each subfamily containing elements with highly conserved transposase proteins. trends in Genetics RAG transposon – proposed structure from which originated the signal ends and RAG1/RAG2 proteins involved in V(D)J Ends from two copies of a TE in the same chromosome arm can associate to form a compound recombination. For further discussion on the model for evo- transposon (e.g. with IS10/Tn10). Similar structures have been called macrotransposons when Ac/Ds lution of V(D)J recombination from a transposon insertion elements are involved. The resulting structure allows intervening ectopic sequences to be placed in a event, see Reference 40. new genomic context. This figure illustrates that transposition of the macrotransposon to the V(D)J recombination – the process by which V, D and J cod- homologous chromosome can result in duplication of the sequences between the two TEs. In the case of ing segments are spliced together in somatic cells of the Tn10, a tetracycline resistance gene is contained in the ectopic sequence. Similar structures have been immune system to produce a diverse range of antibodies. constructed using mariner elements with the inner inverted repeats mutated. These ‘mariner sandwiches’ have been shown to excise and transpose (E. Lozovskaya and D. Hartl, pers. commun.).

IS10 elements, Tn10 also confers an evolutionary advan- an intact TE is also known to participate in the formation tage to the bacterial cell in which it is located. of a hybrid element. The IS10/Tn10 elements constitute An extended superfamily of TEs consisting of elements one convenient system for this analysis. An IS10 element that transpose by a cut-and-paste mechanism, and which transposes about once per 103 cell generations. may also undergo alternative transposition-induced Rearrangements associated with alternative association of rearrangements, could be an appropriate category for TEs the ends from the two IS10 elements that form the Tn10 such as IS1 and IS50. These TEs have demonstrated termini13 occur about once per 105 cell generations35. cut-and-paste transposition as well as cointegrate for- When compared with IS10 transposition and complex mation36–38 that can now be understood as aberrant trans- rearrangements consistent with alternative transposition, position events rather than a true cointegrate. In fact, an Tn10 transposition, which occurs about once every 107 inversion resulting in replication of the 9bp IS1 target site cell generations35, may be viewed as a specific result of and IS1 elements at the rearrangement breakpoints39 aberrant synapsis of IS10 ends. Because Tn10 contains corresponds exactly with the sequence structure predicted sequences conferring antibiotic resistance between the two by the alternative transposition model.

466 TIG October 2000, volume 16, No. 10 Transposons and chromosomal rearrangements Perspective

Aberrant transposition and evolution preciation of the parallels between V(D)J recombination Many TEs were discovered because of the mutations they and alternative transposition will hopefully result in cause. In some cases this was due to simple insertion or experiments that will elucidate the factors controlling excision of the element. In other cases complex traits were the balance between transposition and chromosomal observed, such as chromosome breakage-fusion-bridge rearrangements. cycles due to Ac/Ds elements in maize and hybrid dysgenesis due to P elements in D. melanogaster, which can now Alternative transposition as a genetic tool be explained by the alternative transposition mechanism Several genetic tools have been developed using TEs, reviewed here. including transposon tagging and transposon-mediated The deleterious effects of alternative transposition are transformation. Here, alternative transposition is balanced by the evolutionary advantages conferred by the proposed as an additional method in the repertoire of ability to rearrange genomic information. Mobility of TEs TE-based genetic manipulation. is thought to increase in times of environmental and Alternative transposition can be used to delete regions of genomic stress. Increases in traditional transposition chromatin adjacent to the TE both in vitro and in vivo (Fig. are likely to be accompanied by increases in alternative 2b). In prokaryotes, TE-induced deletions are a well-estab- transposition, as occurs during P-M hybrid dysgenesis. lished technique. Recently, the EZ::TN™, KAN-2 insertion Although the majority of chromosomal rearrangements kit has been commercialized by Epicentre Technologies. would be deleterious, occasional genome shuffling may This kit is based upon in vitro transposition of Tn5 elements result in increased fitness. Could the increased capacity for (with IS50 ends) and can be used to created deletions and genome evolution constitute a selective advantage allow- inversions adjacent to the Tn5 element43,44. ing TEs to persist? Such a process may be particularly rel- In eukaryotes, deletions have been induced adjacent to evant when the survival of a species is challenged, and intact Tam3 elements in snapdragon and P elements in could be the evolutionary basis for increased TE mobility Drosophila in vivo23,34,45–47. The advantage of this method under stressful conditions. over traditional mutagenesis techniques, such as use of the mutagen ethyl methanesulfonate (EMS), is that only the Similarities with V(D)J recombination targeted gene is affected. The advantage over current TE Several parallels have been observed between transpos- imprecise excision techniques is that one endpoint of the ition and V(D)J recombination40–42. The coding joints deletion is defined. Isolation of deletion events and the formed between V, D and J segments have structures simi- direction of the deletion are easily accomplished by screen- lar to the footprints found at TE excision repair sites40. ing for recombinant phenotypes. Nested deletions from a Also, the finding that the signal end fragment can trans- few basepairs to several hundred kilobases in length, with pose and create a 5bp target site duplication upon inser- the breakpoint at the TE end being constant, can be iso- tion further supports the concept of a RAG transposon lated and the resulting differences in gene expression and (Box 1)41,42. Furthermore, and most important in respect protein function examined. to parallels with alternative transposition, the 12/23 signal The usefulness of this method in D. melanogaster is a end pairing rule is reminiscent of the TE end specificity, direct result of the availability of the P element insertion with complementary left- and right-TE ends required for libraries and techniques using a stable transposase source synapsis, end cleavage and transposition40. to control target P element activity34,48–52. Available Some of the chromosomal rearrangements induced by through the Berkeley Drosophila Genome Project (BDGP), alternative transposition – specifically, repair after hybrid the libraries are a collection of stocks, each containing a element excision – are analogous to those occurring single P element insertion. A large proportion of D. during V(D)J recombination (Box 1). Just as the comple- melanogaster genes have been disrupted, not all producing mentary TE ends involved in alternative synapsis form a visible phenotypes. Insertion libraries of Ac/Ds and hybrid element, the complementary 12-signal and 23- En/Spm elements exist in the plant model organism, signal ends of the RAG transposon can synapse in any Arabidopsis thaliana53,54. TE insertion libraries are also number of combinations, regardless of linearity on being developed in other genomes and could be used for the chromosome. In V(D)J recombination, excision of the rapid isolation of deletions by similar methods. the hybrid element – the signal ends – is repaired to form the coding joint, providing for a diverse and flexible Acknowledgements immune system. The following question is then raised. I am grateful to J.A. Sved, M.M. Tanaka, W.R. Engels, Why does V(D)J recombination (repair of alternative J.D.G. Jones and G.J. Cost for the insights that con- transposition) occur much more frequently than transpos- tributed to this work. J. Gibson, D. Jones, N. McCarthy, ition of the intact RAG transposon or rearrangements in B. Dixon, E. Tchoubrieva and three anonymous reviewers which the signal ends reinsert into the genome? provided valuable and much appreciated review of the The same question could be asked from another per- manuscript. I thank D. Hartl and E. Lozovskaya for perspective. Why do some transposons undergo alternative mission to mention unpublished results. Y.H.M.G. is sup- transposition far less frequently than others? The ap- ported by the Australian Research Council.

References 4 McClintock, B. (1947) Cytogenetic studies of maize and New Engl. J. Med. 332, 941–944 1 Berg, D.E. and Howe, M.M. (1989) Mobile DNA, American neurospora. Carnegie Institute of Washington Year Book 46, 8 Lupski, J.R. (1998) Genomic disorders: structural features of Society For Microbiology 146–152 the genome can lead to DNA rearrangements and human 2 Lim, J.K. and Simmons, M.J. (1994) Gross chromosome 5 McClintock, B. (1948) Mutable loci in maize. Carnegie disease traits. Trends Genet. 14, 417–422 rearrangements mediated by transposable elements in Institute of Washington Year Book 47, 155–169 9 Lister, C. and Martin, C. (1989) Molecular analysis of a Drosophila melanogaster. BioEssays 16, 269–275 6 Finnegan, D.J. (1989) Eukaryotic transposable elements and transposon-induced deletion of the nivea locus in Antirrhinum 3 Saedler, H. and Gierl, A. (1996) Transposable Elements, genome evolution. Trends Genet. 5, 103–107 majus. Genetics 123, 417–425 Springer-Verlag 7 Schwartz, R.S. (1995) Molecular medicine: jumping genes. 10 Lister, C. et al. (1993) Transposon-induced inversion in

TIG October 2000, volume 16, No. 10 467 Perspective Transposons and chromosomal rearrangements

Antirrhinum modifies nivea gene expression to give a novel 25 McClintock, B. (1950) Mutable loci in maize. Carnegie Institute Escherichia coli. J. Biol. Chem. 273, 8376–8381 flower color pattern under the control of cycloidearadialis. Plant of Washington Year Book 49, 157–167 40 Fugmann, S.D. et al. (2000) The RAG proteins and V(D)J Cell 5, 1541–1553 26 Ralston, E. et al. (1989) Chromosome-breaking structure in recombination: Complexes, ends, and transposition. Annu. Rev. 11 Weil, C.F. and Wessler, S.R. (1993) Molecular evidence that maize involving a fractured Ac element. Proc. Natl. Acad. Sci. U. Immunol. 18, 495–527 chromosome breakage by Ds elements is caused by aberrant S. A. 86, 9451–9455 41 Agrawal, A. et al. (1998) Transposition mediated by RAG1 and transposition. Plant Cell 5, 515–522 27 Kunze, R. (1996) The maize transposable element Activator (Ac). RAG2 and its implications for the evolution of the immune 12 Ashburner, M. (1989) Drosophila: A Laboratory Handbook, Cold In Transposable Elements (Saedler, H. and Gierl, A., eds.), pp. system. Nature 394, 744–751 Spring Harbor Laboratory Press 161–194, Springer-Verlag 42 Hiom, K. et al. (1998) DNA transposition by the RAG1 and RAG2 13 Roberts, D.E. et al. (1991) IS10 promotes adjacent deletions at 28 Gloor, G.B. and Lankenau, D.H. (1998) Gene conversion in proteins: a possible source of oncogenic translocations. Cell 94, low frequency. Genetics 128, 37–44 mitotically dividing cells: a view from Drosophila. Trends Genet. 463–470 14 Chalmers, R.M. and Kleckner, N. (1996) IS10/Tn10 transposition 14, 43–46 43 Goryshin, I.Y. and Reznikoff, W.S. (1998) Tn5 in vitro efficiently accommodates diverse transposon end 29 Lankenau, D.H. and Gloor, G.B. (1998) In vivo gap repair in transposition. J. Biol. Chem. 273, 7367–7374 configurations. EMBO J. 15, 5112–5122 Drosophila: a one-way street with many destinations. BioEssays 44 York, D. et al. (1998) Simple and efficient generation in vitro of 15 English, J. et al. (1993) A genetic analysis of DNA sequence 20, 317–327 nested deletions and inversions: Tn5 intramolecular requirements for Dissociation state I activity in tobacco. Plant 30 Martin, C. et al. (1988) Large-scale chromosomal restructuring transposition. Nucleic Acids Res. 26, 1927–1933 Cell 5, 501–514 is induced by the transposable element Tam3 at the nivea locus 45 Ingram, G.C. et al. (1998) The Antirrhinum ERG gene encodes a 16 English, J.J. et al. (1995) Aberrant transpositions of maize of Antirrhinum majus. Genetics 119, 171–184 protein related to bacterial small GTPases and is required for double Ds-like elements usually involve Ds ends on sister 31 Coen, E.S. and Carpenter, R. (1988) A semi-dominant allele, niv- embryonic viability. Curr. Biol. 8, 1079–1082 chromatids. Plant Cell 7, 1235–1247 525, acts in trans to inhibit expression of its wild-type 46 Ingram, G.C. et al. (1997) Dual role for fimbriata in regulating 17 Martin, C. et al. (1988) Large-scale chromosomal restructuring homologue in Antirrhinum majus. EMBO J. 7, 877–883 floral homeotic genes and cell division in Antirrhinum. EMBO J. is induced by the transposable element Tam3 at the nivea locus 32 Delattre, M. et al. (1995) Prevalence of localized rearrangements 16, 6521–6534 of Antirrhinum majus. Genetics 119, 171–184 vs. transpositions among events induced by Drosophila P 47 Gray, Y.H.M. et al. (1998) Structure and associated mutational 18 Martin, C. and Lister, C. (1989) Genome juggling by element transposase on a P transgene. Genetics 141, effects of the cysteine proteinase (CP1) gene of Drosophila transposons: Tam3-induced rearrangements in Antirrhinum 1407–1424 melanogaster. Insect Mol. Biol. 7, 291–293 majus. Dev. Genet. 10, 438–451 33 Kleckner, N. et al. (1996) Tn10 and IS10 transposition and 48 Rørth, P. (1996) A modular misexpression screen in Drosophila 19 Coen, E.S. et al. (1986) Transposable elements generate novel chromosome rearrangements: mechanism and regulation in vivo detecting tissue-specific phenotypes. Proc. Natl. Acad. Sci. U. S. spatial patterns of gene expression in Antirrhinum majus. Cell and in vitro. In Transposable elements (Saedler, H. and Gierl, A., A. 93, 12418–12422 47, 285–296 eds.), pp. 49–82, Springer-Verlag 49 Rørth, P. et al. (1998) Systematic gain-of-function genetics in 20 Robbins, T.P. et al. (1989) A chromosome rearrangement 34 Engels, W.R. (1996) P elements in Drosophila. In Transposable Drosophila. Development 125, 1049–1057 suggests that donor and recipient sites are associated during Elements (Saedler, H. and Gierl, A., eds), pp. 103–124, Springer- 50 Rubin, G.M. (1998) The Drosophila genome project: a progress Tam3 transposition in Antirrhinum majus. EMBO J. 8, 5–13 Verlag report. Trends Genet. 14, 340–343 21 Svoboda, Y.H.M. et al. (1995) P-element-induced male 35 Kleckner, N. (1990) Regulating Tn10 and IS10 transposition. 51 Spradling, A.C. et al. (1999) The BDGP gene disruption project: recombination can be produced in Drosophila melanogaster by Genetics 124, 449–454 single P-element insertions mutating 25% of vital Drosophila combining end-deficient elements in trans. Genetics 139, 36 Tomcsanyi, T. et al. (1990) Intramolecular transposition by a genes. Genetics 153, 135–177 1601–1610 synthetic IS50 (Tn5) derivative. J. Bacteriol. 172, 6348–6354 52 Spradling, A.C. et al. (1995) Gene disruptions using P 22 Gray, Y.H.M. et al. (1996) P-element-induced recombination in 37 Turlan, C. and Chandler, M. (1995) IS1-mediated intramolecular transposable elements: an integral component of the Drosophila melanogaster: Hybrid element insertion. Genetics rearrangements: formation of excised transposon circles and Drosophila genome project. Proc. Natl. Acad. Sci. U. S. A. 92, 144, 1601–1610 replicative deletions. EMBO J. 14, 5410–5421 10824–10830 23 Preston, C.R. et al. (1996) Flanking duplications and deletions 38 Lichens-Park, A. and Syvanen, M. (1988) Cointegrate formation 53 Parinov, S. et al. (1999) Analysis of flanking sequences from associated with P-induced male recombination in Drosophila. by IS50 requires multiple donor molecules. Mol. Gen. Genet. 211, Dissociation insertion lines: A database for reverse genetics in Genetics 144, 1623–1638 244–251 Arabidopsis. Plant Cell 11, 2263–2270 24 Beall, E.L. and Rio, D.C. (1997) Drosophila P-element 39 Badía, J. et al. (1998) A rare 920-kilobase chromosomal 54 Tissier, A.F. et al. (1999) Multiple independent defective transposase is a novel site-specific endonuclease. Genes Dev. inversion mediated by IS1 transposition causes constitutive suppressor-mutator transposon insertions in Arabidopsis: a 11, 2137–2151 expression of the yiaK-S operon for carbohydrate utilization in tool for functional genomics. Plant Cell 11, 1841–1852

Antibacterial responses in Drosophila are the focus of several recent studies. The caspase encoding gene dredd, functions in an antibacterial pathway probably with imd and relish1,2. This conclusion is supported by results from Stöven et al., who show that Relish processing and activation requires a functional dredd gene3. Two members of a Drosophila I␬B kinase complex, the kinase DmIKK␤ and the structural factor DmIKK␥,`` are required for antibacterial gene induction by LPS, regulate Relish phosphorylation and processing but are not required for Toll-mediated antifungal gene expression4. Mutations in the DmIKK␥ gene block Relish-dependent immune induction of the genes encoding antibacterial peptides after infection5. Dredd, DmIKK␤, DmIKK␥, Imd and Relish may define a pathway that mediates Drosophila antibacterial responses. Finally, recent results show that the Jak–Stat signalling cascade regulates the expression of complement-like proteins in the Drosophila fat body after infection6.

References 1 Elrod-Erickson, M. et al. (2000) Interactions between the cellular and humoral immune responses in Drosophila. Curr. Biol. 10 (13), 781–784 2 Leulier, F. et al. The Drosophila caspase Dredd is required to resist Gram-negative bacterial infection. EMBO R. (in press) 3 Stöven, S. et al. Activation of the Drosophila NF-␬B factor Relish by rapid endoproteolytic cleavage. EMBO R. (in press) 4 Silverman, N. et al. (2000) A Drosophila I␬B kinase complex required for Relish cleavage and antibacterial immunity. Genes Dev. (in press) 5 Rutschmann, S. et al. Role of Drosophila IKK␥ in a Toll-independent antibacterial immune response. Nat. Immun. (in press) 6 Lagueux, M. et al. (2000) Constitutive expression of a novel complement like protein in Toll and Jak gain-of-function mutants of Drosophila. Proc. Natl. Acad. Sci. U. S. A. (in press)

468 TIG October 2000, volume 16, No. 10