The binding motifs for Ac transposase are absolutely required for excision of Ds1 in maize

A reverse genetic system for studying excision of the transposable elementDs1 in maize plants has been established previously. In this system, theDs1 element, as part of the genome of maize streak virus (MSV), is introduced into maize plants via agroinfection. In the presence of theAc element, excision ofDs1 from the MSV genome results in the appearance of viral symptoms on the maize plants. Here, we used this system to study DNA sequences requiredin cis for excision ofDs1. TheDs1 element contains theAc transposase binding motif AAACGG in only one of its subterminal regions (defined here as the 5′ subterminal region). We showed that mutation of these motifs abolished completely the excision capacity ofDs1. This is the first direct demonstration that the transposase binding motifs are essential for excision. Mutagenesis with oligonucleotide insertions in the other (3′) subterminal region resulted in elements with either a reduced or an increased excision efficiency, indicating that this subterminal region also has an important function.     

Excision of a transposable element from a viral vector introduced into maize plants by agroinfection

The geminivirus maize streak virus (MSV) was used as a vector to introduce the maize transposable element Dissociation (Ds) and to study its excision in maize plants. MSV carrying Ds1 in its genome was introduced into maize plants by agroinfection. Excision of the Ds1 element from the MSV genome was detected only when functions from the transposable element Activator (Ac) were supplied in trans, either endogenously by the recipient maize plant or by co-transformation with Agrobacterium carrying a genomic Ac clone. The excision of Ds1 could easily be visualized by the appearance of viral symptoms induced by the revertant virus. The junction sequences left on the MSV genome after excision revealed 'footprints' typical of transposition as described for maize. From these results, we conclude that transposition functions in our system and that the use of the MSV replicon provides a rapid and simple tool for the investigation of the excision of transposable elements in maize plants.     

Excision of Ds1 from the genome of maize streak virus in response to different transposase-encoding genes

We have previously established a reverse genetic system for studying excision of the transposable element Ds1 in maize plants. Ds1 carried by the genome of maize streak virus (MSV) is introduced into maize plants by agroinfection. Excision of Ds1 from the MSV genome depends on the presence of an active Ac element in the recipient maize plants. With the purpose of exploiting MSV-Ds1 as vector for maize transformation, we studied different genes encoding the transposase (TPase) for their efficiency of activating Ds1 excision. These genes were inserted in the same T-DNA carrying MSV-Ds1 and introduced into maize plants by Agrobacterium-mediated transformation. We showed that the wild-type TPase transcribed by the 2 promoter produced much higher efficiency of Ds1 excision than that transcribed by the Ac promoter. In contrast to what had been observed in tobacco and petunia, the truncated TPase (103–807) lacking the amino-terminal 102 amino acids gave a much more reduced Ds1 excision efficiency than the wild-type TPase when both genes were transcribed by the 2 promoter.     

The binding motifs forAc transposase are absolutely required for excision ofDs1 in maize

A reverse genetic system for studying excision of the transposable elementDs1 in maize plants has been established previously. In this system, theDs1 element, as part of the genome of maize streak virus (MSV), is introduced into maize plants via agroinfection. In the presence of theAc element, excision ofDs1 from the MSV genome results in the appearance of viral symptoms on the maize plants. Here, we used this system to study DNA sequences requiredin cis for excision ofDs1. TheDs1 element contains theAc transposase binding motif AAACGG in only one of its subterminal regions (defined here as the 5′ subterminal region). We showed that mutation of these motifs abolished completely the excision capacity ofDs1. This is the first direct demonstration that the transposase binding motifs are essential for excision. Mutagenesis with oligonucleotide insertions in the other (3′) subterminal region resulted in elements with either a reduced or an increased excision efficiency, indicating that this subterminal region also has an important function.     

A nuclear protein that binds specifically to several maize transposons is not essential for Ds1 excision

Specific binding of plant nuclear proteins to GGTAAA-like motifs in the terminal regions of the transposable elements Ac and Mu1 has been detected in several laboratories. However, the role of these proteins in transposition remains unknown. To test the hypothesis that this binding activity is necessary for transposition, we identified and mutagenized all the binding motifs within the Ds1 element. This analysis enabled us to define more precisely the requirements for binding of the host protein. We then tested the ability of the mutated elements to excise from the maize streak virus (MSV) genome. We found that mutated Ds1 elements that do not bind the host proteins, as determined by gel-shift competition assay, are still capable of undergoing excision in maize, although for one of the maize lines the rate of excision was reduced. Excision of mutated Ds1 elements generated typical excision footprints. These data indicate that binding of host protein(s) to the GGTAAA-like motifs is not essential for Ds1 excision; however, it may contribute to the efficiency of the process. Received: 30 September 1999 / Accepted: 17 January 2000     

The maize transposable element Ac is mobile in the legume Lotus japonicus

To evaluate the prospects for transposon mutagenesis in the autogamous diploid legume Lotus japonicus, the behaviour of the maize transposable element Ac was analysed in the progeny of 38 independent transgenic plants. The conditions for monitoring donor site excision using histochemical localization of -glucuronidase activity or the alternative spectinomycin resistance assay were established, and used to follow Ac mobility through two generations. Somatic excision was monitored as variegated cotyledons in the T₂ generation and germinal excision events were scored in segregating T₃ families as complete -glucuronidase-mediated staining of cotyledons or as a fully green spectinomycin-resistant phenotype. Using these assays an average germinal excision frequency of 12% was estimated in the T₃ offspring from variegated plants. The fidelity of the excision assays was ascertained by comparing the frequency of germinal excision to the frequency of Ac reinsertion at new positions of the genome. Transposition of Ac in 42% of the plants and detection of the characteristic Ac insertion/excision footprints suggests that insertion mutagenesis with the autonomous maize Activator element is feasible in Lotus japonicus. Parameters influencing Ac behaviour, such as dosage, position effects and modification of the element itself, were also investigated comparing homozygous and hemizygous plants from the same family and by analysing different transformants.Abbreviations W white - V variegated - FG fully green - FB fully blue - aadA spectinomycin adenyltransferase     

Reactivation of the Mutator transposable element system following gamma irradiation of seed

Summary The bz2-mu1 allele contains a 1.4 kb Mu element insertion in the open reading frame of the bronze-2 locus. This insertion suppresses gene activity. In an active Mutator line, however, the bz2-mu1 allele shows high somatic instability resulting in numerous purple spots of full gene activity against a beige background in the aleurone tissue of the kernel; restoration of gene activity results from excision of the Mu element. In contrast, in plants with an inactive Mutator system, uniformly bronze kernels are found, and the Mu element at bz2-mu1 is stabilized. Accompanying a loss of somatic instability, this Mu element, as well as the Mu elements elsewhere in the genome, have an increased level of DNA modification. Spontaneous reactivation of somatic instability in inactive Mutator lines rarely occurs; however, reactivation can be induced with gamma irradiation. Reactivated plants regain both the spotted kernel phenotype indicative of element excision from the bz2-mu1 reporter allele and diagnostic restriction sites within the Mu elements indicative of a hypomethylated state. The reactivated plants transmit these characters to their progeny. These data support the hypothesis that genomic shock can elicit cryptic transposable element activities in maize. Possible mechanisms for inactivation and reactivation of the Mutator transposable element system are also discussed.     

Mu transposable elements are structurally diverse and distributed throughout the genusZea

Summary The Robertson's Mutator stock of maize exhibits a high mutation rate due to the transposition of theMu family of transposable elements. All characterizedMu elements contain similar 200-bp terminal inverted repeats, yet the internal sequences of the elements may be completely unrelated. Non-Mutator stocks of maize have a 20–100-fold lower mutation rate relative to Mutator stocks, yet they contain multiple sequences that hybridize to theMu terminal inverted repeats. Most of these sequences do not cohybridize to internal regions of previously clonedMu elements. We have cloned two such sequences from the maize line B37, a non-Mutator inbred line. These sequences, termedMu4 andMu5, have an organization characteristic of transposable elements and possess 200-bpMu terminal inverted repeats that flank internal DNA, which is unrelated to other clonedMu elements.Mu4 andMu5 are both flanked by 9-bp direct repeats as has been observed for otherMu elements. However, we have no direct evidence that they have recently transposed because they have not been found in known genes. Although the internal regions ofMu4 andMu5 are not related by sequence similarity, both elements share an unusual structural feature: the terminal inverted repeats extend more than 100 bp internally fromMu-similar termini. The distribution of these elements in maize lines and related species suggests thatMu elements are an ancient component of the maize genome. Moreover, the structure of theMu termini and the fact thatMu termini are found flanking different internal sequences leads us to speculate thatMu termini once may have been capable of transposing as independent entities.     

Comparative analysis of non-random DNA repair following Ac transposon excision in maize and Arabidopsis

Ac/Ds transposable elements often leave short DNA rearrangements, or 'footprints,' at the sites where they excise. Previous studies at the maize waxy ( wx ) gene suggest that the DNA repair that forms transposon footprints is not random. Each excision site consistently displays a different, predominant repair product suggesting flanking DNA may influence footprint formation. We have expanded these studies to show that predominant end-joining products also form in association with Ac/Ds excision in Arabidopsis and that chromosomal location of the Ac -containing construct does not appear to influence this repair. The predominant repair product is identical in both maize and Arabidopsis for Ac elements with the same adjacent DNA sequences. However, a broader range of minor footprint types is observed in Arabidopsis , including footprints that are rare in maize, suggesting potential differences in the host proteins involved in either transposition, repair or both. The data also suggest that the sequences influencing footprint formation are within 39 bp 5' and 18 bp 3' of the transposon. These studies demonstrate that transgenic Ac/Ds -containing plants will be useful tools in dissecting plant DNA repair processes.     

Adjacent Sequences Influence DNA Repair Accompanying Transposon Excision in Maize

Mobile elements transposing via DNA intermediates often leave small rearrangements, or ``transposon footprints,' at sites where they excise. Each excision event leaves its own footprint and, at any given site, these vary in size and sequence. Footprint formation involves DNA repair of sequences flanking the element. We have analyzed the footprints formed by a 2-kb Ds element excising from six different sites in exons of the maize waxy (Wx) gene. We find that groups of footprints left at individual sites are surprisingly nonrandom; different excision products predominate consistently at each site. Less frequent footprints left by each insertion appear related to the predominant type. The data suggest that flanking sequences affect the DNA repair processes associated with element excision. Two models have been proposed to explain footprint formation, one featuring a 5' exonuclease and the other featuring hairpin loop formation and an endonuclease. Our data have interesting implications for both these models. Evidence is also presented to support the presence of a separate excision mechanism that can remove Ac/Ds elements without leaving any footprint and that operates in parallel with the footprint-forming mechanism.     

The role of subterminal sites of transposable element Ds of Zea mays in excision

Transposition depends on DNA sequences located at or near the termini of the transposon. In the maize transposable element Ds, these sequences were studied by site-directed mutagenesis followed by a transient excision assay in Petunia protoplasts. The transposase-binding AAACGG motifs found in large numbers in the element are important, but none of them is in itself indispensable, for excision. However, mutation of an isolated motif at the 3 end considerably reduced excisability. The inverted termini were confirmed to be indispensable. Point mutations in regions outside the inverted termini of Ds and not located in the transposase-binding motifs had, in some cases, a pronounced effect on excision frequency. The implications of these findings are discussed.     

水稻基因组上一个Ds切离及其双位点插入行为的分子鉴定

玉米转座元件Ac/Ds是hAT转座子家族的成员, 导入水稻基因组后具有转座活性, 尽管转座机制还不完全清楚, 但它们通常经保守的非复制型“剪切-粘贴”过程转座。研究表明, 在Ac编码的转座酶作用下, Ds从原位点切离后常优先重新插入到连锁位点。文章利用TAIL-PCR技术从水稻一个Ds插入突变体及其回复突变体中分离Ds侧翼序列, 结合生物信息学分析方法, 对Ds在突变体上插入位点、回复突变体内切离足迹和重新插入位点进行了分子鉴定。结果显示, 突变体中Ds从3号染色体切离后, 在原插入位点残留了8 bp足迹序列(CATCATGA), 引起Ds标记基因外显子和内含子数目增加, 从而影响基因结构。切离后的Ds重新插入回复突变体第2和第6号染色体上, 分别编码烟草胺氨基转移酶和衰老相关蛋白的2个基因的编码区。因此, 典型的“剪切-粘贴”机制不能完全解释Ds的转座行为, Ds转座存在“剪切-复制-粘贴”的特点。     

The Ds1 controlling element family in maize andTripsacum

Summary Hybridization experiments indicated that the maize genome contains a family of sequences closely related to the Ds1 element originally characterized from theAdh1-Fm335 allele of maize. Examples of these Ds1-related segments were cloned and sequenced. They also had the structural properties of mobile genetic elements, i.e., similar length and internal sequence homology with Ds1, 10- or 11-bp terminal inverted repeats, and characteristic duplications of flanking genomic DNA. All sequences with 11-bp terminal inverted repeats were flanked by 8-bp duplications, but the duplication flanking one sequence with 10-bp inverted repeats was only 6 bp. Similar Ds1-related sequences were cloned fromTripsacum dactyloides. They showed no more divergence from the maize sequences than the individual maize sequences showed when compared with each other. No consensus sequence was evident for the sites at which these sequences had inserted in genomic DNA.     

A maize cryptic Ac-homologous sequence derived from an Activator transposable element does not transpose.

Summary Sequences sharing homology to the transposable element Activator (Ac) are prevalent in the maize genome. A cryptic Ac-like DNA, cAc-11, was isolated from the maize inbred line 4Co63 and sequenced. Cryptic Ac-11 has over 90% homology to known Ac sequences and contains an 11 by inverted terminal repeat flanked by an 8 by target site duplication, which are characteristics of Ac and Dissociation (Ds) transposable elements. Unlike the active Ac element, which encodes a transposase, the corresponding sequence in cAc-11 has no significant open reading frame. A 44 by tandem repeat was found at one end of cAc-11, which might be a result of aberrant transposition. The sequence data suggest that cAc-11 may represent a remnant of an Ac or a Ds element. Sequences homologous to cAc-11 can be detected in many maize inbred lines. In contrast to canonical Ac elements, cAc-11 DNA in the maize genome is hypermethylated and does not transpose even in the presence of an active Ac element.     

Germinal and somatic products of Mu1 excision from the Bronze-1 gene of Zea mays

Summary Germinal and somatic excision products of Mu1 from the insertion allele bz::mu1 were selectively amplified from maize cob tissue. The sequence of these footprints often included deletions at the target site, suggesting that substantial exonucleolytic degradation occurs upon excision of the element. In addition to deletions of target site sequences, single base insertions were also found. The isolation of an excision product including a 4 by inverted duplication of the target site provides evidence that the double-stranded chromosomal break generated by Mu excision may be terminated by a covalently closed hairpin structure. The majority of excision products, however, do not include inverted duplications of target site sequences, suggesting that such structures are the result of occasional repair activities, rather than an essential step in the mechanism of Mu excision. The sequence of the Mu insertion sites of the bz::mu1 and bz::mu2 alleles is also presented.     

A novel class of <Emphasis Type="Italic">Helitron</Emphasis>- related transposable elements in maize contain portions of multiple pseudogenes

We recently described a maize mutant caused by an insertion of a Helitron type transposable element (Lal, S.K., Giroux, M.J., Brendel, V., Vallejos, E. and Hannah, L.C., 2003, Plant Cell, 15: 381–391). Here we describe another Helitron insertion in the barren stalk1 gene of maize. The termini of a 6525 bp insertion in the proximal promoter region of the mutant reference allele of maize barren stalk1 gene (ba1-ref) shares striking similarity to the Helitron insertion we reported in the Shrunken-2 gene. This insertion is embedded with pseudogenes that differ from the pseudogenes discovered in the mutant Shrunken-2 insertion. Using the common terminal ends of the mutant insertions as a query, we discovered other Helitron insertions in maize BAC clones. Based on the comparison of the insertion site and PCR amplified genomic sequences, these elements inserted between AT dinucleotides. These putative non-autonomous Helitroninsertions completely lacked sequences similar to RPA (replication protein A) and DNA Helicases reported in other species. A blastn analysis indicated that both the 5 and 3 termini of Helitrons are repeated in the maize genome. These data provide strong evidence that Helitron type transposable elements are active and may have played an essential role in the evolution and expansion of the maize genome.     

Transposon excision from an atypical site: a mechanism of evolution of novel transposable elements

The role of transposable elements in sculpting the genome is well appreciated but remains poorly understood. Some organisms, such as humans, do not have active transposons; however, transposable elements were presumably active in their ancestral genomes. Of specific interest is whether the DNA surrounding the sites of transposon excision become recombinogenic, thus bringing about homologous recombination. Previous studies in maize and Drosophila have provided conflicting evidence on whether transposon excision is correlated with homologous recombination. Here we take advantage of an atypical Dissociation (Ds) element, a maize transposon that can be mobilized by the Ac transposase gene in Arabidopsis thaliana, to address questions on the mechanism of Ds excision. This atypical Ds element contains an adjacent 598 base pairs (bp) inverted repeat; the element was allowed to excise by the introduction of an unlinked Ac transposase source through mating. Footprints at the excision site suggest a micro-homology mediated non-homologous end joining reminiscent of V(D)J recombination involving the formation of intra-helix 3' to 5' trans-esterification as an intermediate, a mechanism consistent with previous observations in maize, Antirrhinum and in certain insects. The proposed mechanism suggests that the broken chromosome at the excision site should not allow recombinational interaction with the homologous chromosome, and that the linked inverted repeat should also be mobilizable. To test the first prediction, we measured recombination of flanking chromosomal arms selected for the excision of Ds. In congruence with the model, Ds excision did not influence crossover recombination. Furthermore, evidence for correlated movement of the adjacent inverted repeat sequence is presented; its origin and movement suggest a novel mechanism for the evolution of repeated elements. Taken together these results suggest that the movement of transposable elements themselves may not directly influence linkage. Possibility remains, however, for novel repeated DNA sequences produced as a consequence of transposon movement to influence crossover in subsequent generations.     

Somatic Variegation and Germinal Mutability Reflect the Position of Transposable Element Dissociation within the Maize R Gene

The R gene regulates the timing and tissue-specificity of anthocyanin deposition during maize development. The Ac/Ds system of transposable elements was used to induce insertional mutants of the R-sc:124 allele during two cycles of mutagenesis. Of 43 unstable, spotted-aleurone mutants generated, 42 contain inserts of the Ds6 transposable element differing only in the position and orientation of the element. The remaining mutant, r-sc:m1, contained an insert of a Ds element of the approximate size of the Ds1 transposable element. The patterns of somatic variegation of these mutants, resulting from excision of Ds, define a spectrum of phenotypes ranging from sparse to dense variegation. The sparsely variegated mutants produce few germinal revertants but relatively many stable null derivative alleles; densely variegated mutants produce many germinal revertants and few stable null derivatives. Molecular analysis shows that the sparsely variegated alleles are caused by Ds6 insertions in protein coding regions of R-sc:124 whereas the densely variegated mutants result from insertions in introns or in flanking regions of the gene. The excision rate of Ds6 from R, estimated as the proportion of R genomic DNA restriction fragments lacking the element, was uniform regardless of position, orientation or whether the element was inserted in R-sc:124 or another R allele. The excision rate was greater, however, for the mutable alleles involving the Ds element from r-sc:m1. These data indicate that, although the excision rates are uniform for a given Ds element, the somatic and germinal mutability patterns of alleles associated with that element vary widely and depend primarily on the position of the transposable element within coding or noncoding regions of the gene.     

Ac/Ds转座系统在水稻转化群体中的转座活性及转座子插入位点旁侧序列的分析

利用本实验室构建的转Ac(AcTPase)及Ds(Dissociation)的水稻(Oryza sativa L.)转化群体,配置了Ac×Ds的杂交组合354个.检测了转基因植株的T-DNA插入位点右侧旁邻序列,研究了Ac/Ds转座系统在水稻转化群体中的转座活性.结果表明,有些转化植株T-DNA插入位点相同或相距很近,插入位点互不相同的占65.4%.检测到T-DNA可插入到编码蛋白的基因中.在Ac×Ds的F2代中,Ds因子的转座频率为22.7%.对Ac×Ds杂交子代中Ds因子旁侧序列的分析,进一步表明了Ds因子在水稻基因组中的转座活性,除了从原插入位点解离并转座到新的位点之外,还有复制--转座和不完全切离等现象.获得的旁侧序列中,有些序列与GenBank中的数据没有同源性,目前有2个DNA片段在GenBank登录.探讨了构建转座子水稻突变体库进行水稻功能基因组学研究的策略.     

Ac／Ds转座系统在水稻转化群体中的转座活性及转座子插入位点旁侧序列的分析

利用本实验室构建的转Ac(Ac TPase)及Ds(Dissociation)的水稻(Oryza sativa L.)转化群体,配置了Ae×Ds的杂交组合354个。检测了转基因植株的T-DNA插入位点右侧旁邻序列,研究了Ac/Ds转座系统在水稻转化群体中的转座活性。结果表明,有些转化植株T-DNA插入位点相同或相距很近,插入位点互不相同的占65.4％。检测到T-DNA可插入到编码蛋白的基因中。在Ac×Ds的F2代中,Ds因子的转座频率为22.7％。对Ac×Ds杂交子代中Ds因子旁侧序列的分析,进一步表明了Ds因子在水稻基因组中的转座活性,除了从原插入位点解离并转座到新的位点之外,还有复制——转座和小完全切离等现象。获得的旁侧序列中,有些序列与GenBank中的数据没有同源性,目前有2个DNA片段在GenBank登录。探讨了构建转座子水稻突变体库进行水稻功能基因组学研究的策略。