Transposon in Maize

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a # fi Acta Genetica Sinica , June 2006, 33 (6): 477487 ISSN 0379-4 172

Mutator Transposon in Maize and MULES in the Plant GenomeDIAO Xian-Min''a', Dam on Lisch21. National Millet Improvement Center of China, Institute of Millet Crops. Hehei Academ y of Agricultural and Forestry Sciences,Shijiazhuang 05003 , China;2. University of California at Berkeley, Department of Plant and Microbial Biology, 11 1 Koshland Hall , CA 94720, USA

Abstract: Mutator ( M u ) is by far the most mutagenic plant transposon. The high frequency of transposition and the tendency toinsert into low copy sequences for such transposon have m ade it the primary means by which genes are m utagenized in maize (&amays L.). Mus like elements (MULEs) are widespread among angiosperms and multiple-diverged functional variants can be presentin a single genome. MULEs often capture genetic sequences. These Pack-MuLEs can m obilize thousands of gene fragments, whichmay have had a significant impact on host genome evolution. There is also evidence that MULEs can move between reproductivelyisolated species. Here we present an overview of the discovery, features and utility of Mu transposon. Classification of Mu elementsand future directions of related research are also discussed. Understanding Mu will help us elucidate the dynamic genome.Key words: Mutator; transposon; genome evolution; MULE; Pack-MULE

Transposons or "controlling elements" were dis-covered by B arbara McClintock in maize (Zeamays L.)in the early 1950s, but the theory of transposition wasnot widely accepted until 1970s. Transposable ele-ments of one type or another have been found in allorganisms, including all plants that have been investi-gated. Transposons make up over 50% DNA of thegenome in many species with large genomes. Trans-poson can rearrange genomes and alter individual genestructure and expression as a consequence of transpo-sition, insertion, excision and chromosome breakage.Many transposon system have been studied in China,such as Ac/Ds, Sp dd Sp m, Tourist , Stowaway in maize,Mariner in soybean (Glycine max L.) and Tam inSnapdragon"72'.However, little is known about Muta-ror (simplified as Mu ), which is the hot point of trans-poson research in America. Murator transposon wasdiscovered by Robertson in 1978 from a maize linethat yielded diverse mutations at a frequency muchhigher than the spontaneous mutation frequency ' 3341 .Itwas found that these mutations were caused by theinsertion of Mu transposons. M u elements have be-

come the major tool of gene discovery in maize func-tional genome research and other related fields. Herewe review the discovery, properties, genetic applica-tion of Mu transposon and its role in plant gene andgenome evolution.1 The Discovery ofMu "kansposon

A maize line, known as Mutator, was sent to D.S . Robertson of Iowa State University from the Uni-versity of Wisconsin for detailed analysis of its fre-quent mutations. Genetic experiment by Robertsondemonstrated that not only the heritable mutationfrequency of this line was very high, but a great dealof somatic variation was also observed. Further re-search showed that these mutations were not causedby the previously discovered transposons such asAc/Ds and S p d d S p m . In ACIDSand S p d d S p m lines,only a few autonomous elements are present and theysegregate as a Mendelian element. Transpositions ofthese elements, when they occurred, could be de-tected as changes in the segregation ratios of activity.

Received: 2005-09-2 1 ;Accepted: 2005- 10-08This work was supported by the National Natural Science Foundation of China (No.30370766).@ Corresponding author. E-mail: xm diao@ya hoo.com.cn; Tel: +86-3 1-8767 0697


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478 BffYfw Acta Genetica Sinica Vo1.33 No.6 2006In contrast, 90% of the progeny of an outcross of aMutator line carried active transposons and were con -sistent with a very high duplication rate. Thoseprogenies that did lose activity appeared to do so dueto epigenetic silencing, rather than the segregation ofan autonomous, or controlling element. This wasconfirmed in many later experiments [jq6'.he com-plexity and epigenetic regulation features of this pu-tative transposon system d elayed the discovery of themeans by which the system was regulated.

In the 1980s, using this Mututor line as mutagenfor gene tagging became popular. A new kind oftransposon inserted into an allele of alcohol dehydro-genase 1 and was cloned by Bennetzen et ~ 1 ' ~ ' .ow weknow that this transposon is a nonautonomo us memberof the Mu transposon family, which was named M u l .Characterization of Mu1 and using it as a probe notonly identified a new kind of transposon but alsoopened a new era of efficient maize functional genomeresearch. This new transposon was named Mututorbecause it came from Mutator maize line. Schnable etul. [*I identified a Cy/rcy transposon system from anindependent maize line with similar transposion andgenetic properties of Mu system, which was laterdemonstrated to be Mu7 of the Mu family. In the early1990s, a minimal version of the Mutator line was usedto isolate the regulatory transposon for the Mututorsystem. This regulator, which was isolated independ-ently in three different laboratories, was designated asMuDR, in honor of Don Robertson. The isolation ofthe autonomous two-element, MUDR 9, lo], the creationof a minimal Mututor line [ ' 'I and an engineered res-cue-Mu element have made it possible for a sys-tematically elucidation of this super transposo n family.

2 The Components and Gene Structure ofMuDR and Mu Transposon FamilyAll m aize Mu-transposable elements are regulated

by MuDR, which is the autonomous element; MuDRcarries two genes, mudrA and mudrB (Fig. 1). ThemudrA transcript encodes a 120 kDa protein, MU RA,which is the transposase of the family. MU RA containsa domain with high similarity to several bacterial

transposase, which was used as evidence for its veryearly origin I 3 ' . The mudrB major transcript encodes a23 kDa protein (MURB) that is not similar to any se-quences in public database outside m aize and its closerelatives. Although the precise function of MURB re-mains enigmatic, both deletion derivatives and trans-genic experiments suggest that MURB is required forM u insertions, especially germinally transmitted inser-tions L'4 ' . Only two maize lines with active Mu DR ele-ments have been identified so far, but all maize linescarry MuDR elements derivatives, or homologousMuDR sequences (hMuDRs), whose coding sequencesare 80%-99% identical to those of MuDR. Surprisingly,these hMuDRs can be expressed at the transcriptionlevel though they are not associated with Mututor ac-tivity and they might play an as-yet-unknown functionin Mututor regulation j1 .

Unlike Ac/Ds an d S p d d S p m transpson systems,which mostly comprise of the autonomous elementand its deletion derivatives, the Mu transposon systemcontains complex family elements, includingautonomous M u D R , homologous M u D R elements andmany variants with similar TIRs. All Mu transposonelements share similar -170-220 bp TIRs; the lengthof direct repeat sequence flanlung the inserted ele-ments is 9 bp. Many Mu elements contain additionaldirect or inverted repeat sequen ces. Although deletionderivatives are the most common source of Ds ele-ment from Ac and of dSpm elements from Spm, mostMu elements internal sequences are unrelated to thetranposase-coding sequence of Mu DR, or to eachother (Fig. I ) . In most cases, the internal sequen ces ofM u elements are part of a host gene and it seems thatMu TIRs can capture host functional sequence by sofar unknown mechanism. These kinds of elements arecalled Puck-MuLEs I s 1 . In some species, such as rice(Oryza sativu L.), there can be thousands of inde-pendently derived Puck-MuLEs.

Jittery is a newly isolated and characterizedmember of M u element, whose transposase-codingsequence is only a 3.9 kb gene with high identity tomudrA but not to mudrB. The length of direct hostsequence duplication flanking the Jittery insertions isalso 9 bp, but its 177 bp TIRs are unrelated to that of


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DIAO Xian-Min ef al.: Murator Transposon in Maize and MULES n the Plant Genome 479

Fig. 1 The Mutator transposon family in maize and the engineered RescueMuAll classes of Mu elements share similar terminal inverted repeats (TIRs; Black ends), but each class has a unique internal sequencethat shares similarity to various host functional protein coding sequences, as indicated in the figure. Mu1 is similar to MRS-A(Mu-related sequence A). Mu3 is similar to an Arubidopsis protein and Mugo nashi protein. Mu4 includes a sequence 95% identicalto a maize expressed sequence, accession No . BG466445. Mu7 is similar to Arubidopsis protein accession No. NP-192120. Mu8contains a sequence with similarity to pgpl protein from Aruhidopsis. RescueMu is an engineered element based on Mu1 used as agene-tagging tool, into which unique tag sequence, pBluescript and selectable maker w ere inserted. For MuDR, the position of mudrAand mudrB are indicated. The figure is a modified version from Lisch (2002) [I7.

M u D R . Despite limited investigation has been doneon the transposition activity of Jittery, the results sofar obtained demonstrated that i t is an autonomoustransposable element. Jittery, like M u D R causes ahigh frequency of late somatic reversion, but like de-letion derivatives that lack mudrB, it is not associatedwith new insertions I 6 .

In addition to the members of Mu elements foundin maize, many M u-like elements (MULES)have beenidentified in both dicots and monocots. Mu transposonfamily is characterized by the presence of many diver-sified and potentially functional variants, and its veryhigh levels of activity in maize, which,complicatedanalysis of its origin and evolution.

3 Transposition Activity ofMuData related to Mu transposition activity are

largely a product of investigations on maize, althoughMULES in Arnbidopsis had been confirmed to be ac-tive in D D M I mutant background I . Two main typesof transpositions occur with maize Mu elements. Oneis somatic excision, or excision and insertion late dur-ing development in somatic cells and tissues, the other

one is insertions occurring in germinal cells, which isthe source of heritable new mutations in Murutor lines.M u excisions have been investigated extensively withanthocyanin reporter alleles, such as a 1-mum2, e-cause pigmented spots can easily be visualized andquantified. Most somatic excisions occur in the latedevelopment stage, within the last two or three celldivisions of a given lineage, though early excisionscan occasionally occur. The most visible result of so-matic excision is revertant sectors, resulting from theexcision of the inserted transposon and restoration ofthe a1 pigmentation function. Two kinds of somaticexcision have been documented for Mu elements, thecut-only and the cut-and-paste. The excised trans-posons of the cut-only form were failed in insertinginto other sites of the host genome. Cut-and-pastetransposition is the typical mode of transposition ofDN A transposons and is often associated with reinser-tion elsewhere in the genome. But excision of A d D sand S p d d S pm elements typically result in minorchanges to the host sequence duplication created ontransposon insertion. In contrast, Mu element excisioncreate deletions and additions of the host gene, *

In contrast to high frequency of somatic revertant


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480 Acta Genetica Sinica Vo1.33 No.6 2006

mutations, germinally transmitted revertants are ex-tremely rare in Mututor lines, which suggest that exci-sions are prevented by some kind of mechanism incells that give rise to In minim al Mututorlines with just o ne copy of Mu1 initially inserted intothe ul-mum2 allele and one copy of MuDR, from10%-20%of progeny contain more than one copyof Mu1 or MuDR"'] and many experiments con-formed that the existing M u insertion segregate in aMendelian fasion. These experiments reveal that newgerminal insertions must happen without the loss ofthe existing Mu insertion; in another word, som e kindof duplicative or replicative MuDR transposition hap-pens during the process of de~elopment[~'g 1 . Thoughgerminal duplicative insertion can occur throughoutthe development, which mostly begins late in sporo-phyte cell divisions, where transposition events createsmall clusters of gam etes carrying the same mutations.Germinal transposition continue through meiosis tothe last mitotic divisions of the gametophytes, and theafter meiosis insertion generates sperm with d ifferentmutations [33221.The frequency of germinally transmit-ted insertions depends on the Mututor line used, butthere are clear position effects on both cis and transactivity of individua l MuDR elem ents "I

Many Mu-induced mutations are suppressible.The mutant phenotype in these cases arises only in thepresence of active MuDR elements, probably due tosteric effects introduced by the presence of the trans-posase bound to the transposon TIRs. Consistent withthis model, many suppressible Mu insertions are inpromoter regions. However, M u insertion into intronscan also be suppressible 1231, and it is likely that Musuppression will turn out to be more complex than asimple model of steric hindrance would suggest.

Many investigations have demonstrated that Mutransposons generally insert into single copy orlow-copy-number regions of the genom e whichmakes it a powerful tool for maize functional gene tag-ging. By analyzing the sequences that flanking the 88RescueMu insertions, Dietrich and co-authors demon-strated that 69%of these sequences were genes and only4% were repetitive retrotranspo~ons[~~~.much recentexperiment also with RescueMu shows that the rate of

single copy insertion is about 66%,which is consistentwith the previous result Both Ac/Ds and S p d d S p mtend to transpose to genetically linked sites. In contrast,Mututor transposes to unlinked sites, an advantage forwhole genome mutagenesis applications. However,Hardeman and ChandleJ271ound that certain classes ofMu elements predominantly targeted certain genes,which implies that some M u elements may have inser-tion affinities. The 5WTR region or the promoter regionof 818 gene show ed a strong preference for Mu elementinsertion, 62 of 75 insertions of g18 gene targeted in thisregion, suggesting that 5'UTR targeting might be a fea-ture of Mu insertion. But introns and other regions ofgenes are also often targeted by Mu elements'251. naly-sis of 339 target site duplications (TSDs) created by Muinsertions also showed some degree of sequence prefer-ence, the weak consensus for Mu insertion wasCTCB(G/C)(A/C)(G/A)(A/G)C. Furthermore, se-quences immediately linked to TSDs also showed con-servation; the consensus sequence of 5' of the TSD isCCT and that of the 3' of the TSD is AGG Mu-targetedsequences were found to be GC rich relative to the restof the maize genome 2" However, many M u insertionsites do not have the consensus sequence.

Four types of assays are usually used to demonstrateMututor system activity, including examination of somaticinstability of reporter alleles (such as sectored leaves,kernels or anthers), special enzym es to detect m ethylationof diagnostic restriction sites in the TIRs Hinf I for Mu1and Sac I for MuDR), the detection of new insertions inprogeny plants, and an elevated forward mutation fre-quency resulting from new germinal insertion^'^'. Whichmethod should be used depends on needs of the user.Early observations of Mututor suggested that the loss ofactivity was due to epigenetic silencing, rather than sim-ple segregation of a regulatory transposon. Two modelsfor silencing had been proposed: ectopic paring betweenhomologous TIRs and posttranscriptional RNA-basedsilencing '52g'. Until recently, the detailed initiation andprocess of Mu family silencing still remains enigmatic. Adominant locus that can initiate Mututor silencing, Mukiller (Muk), was cloned. This locus was dem onstrated tobe an inverted duplication of a partially deleted autono-mous MuDR element. Muk produces a d r A hairpin


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transcript that is processed into smal l RNAs that targetsmudrA for silencing. This, in turn, esults in transcrip-tional silencing of mudrA, and subsequently mudrB. Mukprovides the first example of a natural occurrence of de-rivative that is able to initiate heritable silencing of anactive transposon family '299". This discovery not onlyclarified our understanding of M u epigenetic silencing,but also gave us new tools for investigation of bothtranscriptional and posttranscriptional regulation ofgene expression.

It is suggested that the initial silencing of Mu ac -tivity is followed by detectable TIRs methylati~n'~'.Indeed, 5'-methylation of cytosines within TIRs is adiagnostic feature of Mu transposons silencing state.In typical Mutator lines or complex lines, both Mu DRand nonautonomous element, such as M u l , can bemethylated gradually in the progeny of a linage or atdifferent development stage of an individual plant.The methylation of MuDR is accompanied by tran-scriptional silencing and loss of activity [ sx31 i . Inminimal lines with single copy of Mu DR, both MuDRand the nonautonomous elements are unmethylated.Methylation of nonautonomous elements occurs ifMuDR element is lost due to genetic segregation. Insuch cases that Mu DR is restored genetically, the me-thylation of nonautonomous elements is lost, sug-gesting that it represents a default state that occurs inthe absence of the transposase"'. 321 . This default me-thylation is dependent on at least two mutations thatwere discovered due to their effects on paramutation.In mop1 (mediator of paramutation) mutants, para-mutatable alleles of several color genes are epigen-etically activated and nonautonomous Mu elementsTable 1 List of someMu agging cloned m aize functional genes

are hypomethylated. Mu DR elements that had beensilenced by Mu kil ler are also hypomethylated in amop1 mutant background. If this element is main-tained in a mutant background for multiple genera-tions, one of the two genes encoded by Mu DR, thetransposase mudrA becomes reactivated. Further, thesecond gene, mudrB, remains methylated and si-lenced, suggesting that, although the two genes hadboth been silenced by Muk, maintenance of that si-lenced state is mediated by different factors'331.Si-lenced M u elements in typical lines can also be reac-tivated by gamma and U V radiation of seeds and pol-len, and radiation treatments are more efficient onlines within one or two generations of silencing thanthose lines that had kept many generations of silencestate'". Singer et al. ["I found that Mutatur-like ele-ments ( M U L E S ) n Arabidopsis genome become de-methylated and active in the chromatin-remodelingmutant ddml (Decrease in DNA Methylation), whichleads to loss of heterochromatic DNA methylation.

4 Genetic Application of M u TransposonTransposon tagging is one of the main methods

used in functional genome research. A d D s taggingsystem has not only been successfully used in maizebut also in rice and Arabidupsis [34i. The advantages ofM u tagging are that it moves to any chromosome, itcauses a very high mutation rate, insertions tend to beinto or near genes, and most loci appear to be poten-tial targets. Using this transposon system, many maizegenes have been cloned and characterized, some ofwhich are listed in Table 1.

No. Gene or allele Possible function Database code Reference1 su I Kernel developm ent related AY290 402 WI2 Rough sheath 1 Leaf sheath development related L44133 P6 13 APETALA2 Spikelet development related AF048900 WI4 Ligueless3 Architecture of ligule AF4 57 125 [3815 Knotted1 Leaf development AY3 12169 P I6 Viviparous1 Induction of embryo development NO01635 ~4017 A d h l Oxygen starvation response X04049 [71


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482 B.ff%#@Acta Genetica Sinica Vo1.33 No.6 2006Targeted mutagenesis was the initial application

in typical Mutator lines, because it is relatively easyfor scientists to recover mutations in well-studiedgenes that con fer visible phenotypes [51. For loci witha recessive loss-of-function allele, wild-type plant withactive Mu were crossed to the homozygous recessivetester stock, mutant individuals in the F1 progenywould be expected to carry a Mu-tagged allele fromthe Mu-active parent. For loci with dominantgain-of-function alleles, Mu-active lines homozygousfor the dominant allele need to be established first, andthen crossed to a normal test line. Normal individualsof the F, most likely contained a Mu-inserted disrup-tion of the dominant allele. Because Mu causes a highmutation rate, large populations of Mu active lineshave been screened to find new phenotypes of interest.

Three big project of Mu-tagging maize func-tional genome research had been carried out in theUnited States. The Trait Utility System for Corn(TUSC) was implemented by Pioneer Hi-Bred, Inc.,which was based on DNA samples of -45 000Mu-active individual plants and the available ofmaize EST sequences. Using a primer running outfrom the Mu TIR sequence and a primer designed onthe basis of an EST of interest, population screeningcan be used to find individuals with a Mu insertioninto or near the gene of interest. Progeny analysis canthen be used to determine which plants transmitted aheritable mutant allele. Maize-Targeted Mutagenesis(MTM) is a US National Science Foundation fundedproject for public maize research service. With thesame strategy to TUSC, MTM uses PCR to screenfrom a population of -46 000 M u active plants andprovide users with seed for phenotypic characteriza-tion. RescueMu was another project carried out byjoint co-operation of Stanford University, UC Berke-ley and other institutes, which involves screeningprogeny of RescueMu transformed maize plants. Thisapproach has the virtue of combining genomic se-quencing and functional genomics into a single step,in which RescueMu insertions provide not only amutation but also a cloned allele for sequencin g.

Since M u transposon had been effectively used

in maize functional genome research, it would beadvantageous if we use this system in other plantspecies for gene tagging as well. Th e most direct wayto do this is to create a heterologous M u transposontagging in other species. Unfortunately, althoughmudrA and mudrB have been successfully trans-formed into rice, and the transformed genes can beinherited for a few generations, transcription ofmudrA an d mudrB was not detected (unpublisheddata). So we need more knowledge about Mu epige-netic regulation and transferring gene silencing.Transposition active MULEs have been observed inArabidopsis D D M l mutant I and a fungal speciesFusurium oxysporum [411, suggesting that transposi-tionally active M u elements do exist in organisms inaddition to maize. Thus, screening for active andmutagenic MULEs may be another option for M utagging in species su ch as rice.

5 Occurrence and Classification of M uElements in Other Plant Genom esIt has been demonstrated that homologs ofmudrA exist in many plant genomes, but homologs

of mudrB have only been observed i n Z . luxurians,Z. diploperennis and a few other species closely re-lated to maize[421.Mu-like elements are genericallyreferred to as M U L E S [ ] .Autonomous MULEs showvarying degrees of similarity with mudrA in maize.So far intact element of MULEs , including TIRs andTSDs, had been cloned and characterized fromArubidopsis, Brassic napus, rice, sorghum (Sorghumbicolor L.), sugar cane (Saccharum oficinarum L.),wheat (Triticum aestivum L.), barley (Hordeum vul-gare L.), foxtail millet (Seturia italica L.) and a fewbamboo species (Bambuseae) species[532-44. Lisch etal . [421 loned and characterized mudrA sequencesfrom 28 plant species. Cluster analysis based on theunclear tide identity of the cloned fragments showeda discrepancy in the relationship between phylogenyof the sample species and the evolution phylogeny ofthose MULEs . Eisen et al. I 3 found that the sequenceof transposase mudrA was similar to transposase ofsome insertion sequence (IS) in a group of bacteria


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species such as Mycobacterium bovis. These suggestthat the M u transposon family might have had a veryearly origin. The genomes of fungus do have MULEsand even a transpositional active one w as characterizedI 4 I 1 . To date no MULEs have been reported in animals.

Not only do we know that MULEs are ubiqui-tous in plant genomes so far tested, but analysis ofpublic EST data demonstrate that MULEs in manyplants, including rice, wheat, sugar cane and barleyare transcripted. Thus, transcriptional active MULEsmaybe a common property of plant gen~mes'~*].os tMULEs contain mudrA genes that are well divergedfrom that in maize, but many of those MULEs havegood open-reading frames with evidence for selectionat the amino acid level, consistent with continuedfunctionality. Conservation of TIR sequences witheach other and targeted side duplication (TSD ) is alsoconstant with continued transposition activity. Manyduplicated copies of one kind MULEs were found inthe sequenced rice genome (unpublished observation),and we found several MULEs of Setaria viridis werehighly polymorphic among different genotypes of thesame species by Southern blot (unpublished data),suggesting activity in this species. Database searchusing the protein sequence of MURA reveals thatproteins with high similarity to MURA exist inArabodopsis, potato (Salanum tuberosum L.), tomato(Lycopersicon esculentum M.) and many other plantspecies [ 3 2 1 .Yoshida 1451 found a rice MULE expressedin callus subcultured with praline and even found atranscriptionally active M U L E in rice somaclonallines. Three instances of transpositionally activeMULEs have been reported, including Jittery inmaize, AtMul in Arabidopsis DDM mutant and Hop1in the fungus Fusarium oxysporum. Jittery resemblesMurator in the length of the element's TIRs, the sizeof the target site duplication, and the makeup of itstransposase, but differs from MuDR in that it encodesa single MURA-like protein. Jittery also differs fromMutator elements in the high frequency with which itexcises to produce germinal revertants and in its lowcopy number in most maize lines and maize relativesexamined. However, Jittery cannot be considered as abona fide transposon in its present host line, because

i t does not reinsert in the genome [ I 6 ' . Together, thesedata suggest that MULEs may be transpositionallyactive now or in the near past in many plant genomes.Thus, it is quite likely that active Mu systems in otherplant species may be as useful as Mutator does inmaize.

MULEs can be widely diverged from each othereven they come from the same genome. Jittery, whichis an active M U L E in maize, is m ore similar to MULESin rice and Arabidopsis than it is to MuDR [16]. Re-markably, two of MULEs in Arabidopsis with highsimilarity to Jittery appear to be host functional genesrather than transposons; mutations in those genes causedefects in the far-red-light response pathway , Inthis case, it appears that the transposase has been re-cruited by the host to perform a novel function.

The distribution of any given group of MULEs inthe grasses is patchy, suggesting the possibility ofhorizontal gene transfer. Diao and colleagues'491 ounda MULE from Setaria with high similarity to a MULEfrom rice; the nucleotide identity between the twoelements is as high as 90% including correspondingintron sequences. Given the 50-60 million years ofevolution divergence that separate Setaria and rice'501,the high degree of similarity of non-coding sequencescan only be explained by horizontal transfer[491. his isthe first well-documented examp le of horizontal trans-fer of any nuclear-encoded genes between higherplants. It is clear that the evolution of M u super familyis markedly different from their hosts.

MULEs form a highly com plex and broadly diver-sified family of transposon. All Mu-related elements sofar found in plant genomes can be classified into fourgroups: the first includes MuDR and hMuDRs as statedin the second part of the review. Many of these elementscontain point m utations but are transc riptionally ex-pressed and may p lay a role in the epigene tic regulationof the family '51. The second group includes elementsthat have mudrA homologs and long distinct TIRs fromMuDR, such as Jittery in maize and Sf4 in Setaria. Thethird group of Mu super family includes those elementsthat share similar TIRs with a MULE encoding mudrAbut that carry internal sequences that are unrelated to the

[46-481


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484 B%%%! Acta Genetica Sinica Vo1.33 No.6 2006transposase. Examples of these include some of thenonautonmous elements in maize. Their internal se-quences are captured fragments of host genes. Theseelements are known as Pack-MULES, and they can bepresent in large numbers in any given genome"5,5',521.The fourth group includes transposons that canymudrA-like genes but lack long TIRs. Individual in-stances of this class of e lemen ts may represent cooptedmudrA genes, genes such as Farl in Arabidopsis [46,471.6 Mu Elements in Plant Genome and G ene

EvolutionThe genom e of all plant species is in a state of dy-namic equilibrium between evolution and stability, but

of course stability is always temporary. The diverse ac-tivity of transposons has been demonstrated to be animportant factor affecting on the evolution of genes andgenomes 11953v541.ransposon can induce gene silencingor reactivation, gene or genome restructuring includingdeletion, duplication, reversion and translocation. Theaccumulation of such changes undoubtedly has had aprofound impact on genome evolution .Mu transposons are the most active, most com-plex and m ost ubiquitous DNA transposable element inplant genomes, suggesting a special role in gene andgenome evolution. Unlike Ac/Ds and SpddSpm,whose activity result in relatively minor changes to thehost sequence, Mu activity can result in a wide rangeof changes [I2]. It has demonstrated that MuDR ele-ments in different loci can show distinct frequencies oftransposition, suggesting that this class of elements isparticularly sensitive to their local environment. It isclear that Mu elements can evolve a novel function,such as in the case of the Arabidopsis Farl gene .The most recent discovery of the impact of M u ele-ments on plant genome evolution is Pack-MULES,which contain fragments of genes. With systematic andcareful analysis, Jiang er aL[553561und more than 3 OOOPack-MULES in the rice genome, and Pack-MULESalso had been seen in maize and Arabidopsis. Thelarge number of Pack-MULES and their presence inmultiple genomes suggest that they may be a majorcomponent of most or all flowering plant genomes.

W 4 7 1

Gene fragments from different rice genes were foundtogether in -23% of Pack-MULES in rice, suggesting ameans by which hybrid genes could be created due tothe activity of a transposon. At least 5% ofPack-MULES were found to be expressed, as evi-denced by full-length cDNAs, with an identical DNAsequence match. H ence, by the criterion of expressionat the RNA level, many of these Pack-MULES are al-ready new genes. The ability of Pack-MULES to cap-ture gene fragments combined with the possibility ofhorizontal transfer as documented by Diao er ~ 1 . ' ~ ~ 'also suggest a means by which gene fragments couldbe moved between species.

Although we have made some progress in under-standing of the MuDR/Mu superfamily, many interestingpuzzles remains to be explored for future research. Themost intriguing questions include the detailmechanism ofMu transposition and the epigenetic regulation of Musilencing and reactivation. The solution for such questionswill help us establish Mu tagging system in other plantsthan maize and w idening its application in plant genetics.The frequent transposition and interaction with host ge-nomes make the evolution study of transposons difficult,but analysis of this fascinating super family of elem entsand a clear understanding of their evolution will certainlyhelp us understand the dynamics of gene and genomeevolution. Pack-MULE structure is ubiquitous in plantgenomes and predicted to be a mechansism for the crea-tion of novel but we know nothing about themechanism of fragments acquisition and so far no in-stances of newly created genes in this way have beenreported. As stated by Shapiro of Chicago Un i~e rsit y"~ '"transposable elements are the key to a 21st centuryview of evolution", advances in our understanding ofthe MULE super family will for certainly help us to il-luminate both transposon and genome evo lution.Acknowledgement: We thank Hui Zhi of NationalMillet Improvement Center of China for figurepreparation.References:[ l ] Bennetzn J L. Transposable element contributions to

plant gene and genome evolution. Plunf Mol Biol, 2000,


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