A transgenic system for generation of transposon Ac/Ds-induced chromosome rearrangements in rice

The maize Activator (Ac)/Dissociation (Ds) transposable element system has been used in a variety of plants for insertional mutagenesis. Ac/Ds elements can also generate genome rearrangements via alternative transposition reactions which involve the termini of closely linked transposons. Here, we introduced a transgene containing reverse-oriented Ac/Ds termini together with an Ac transposase gene into rice (Oryza sativa ssp. japonica cv. Nipponbare). Among the transgenic progeny, we identified and characterized 25 independent genome rearrangements at three different chromosomal loci. The rearrangements include chromosomal deletions and inversions and one translocation. Most of the deletions occurred within the T-DNA region, but two cases showed the loss of 72 kilobase pairs (kb) and 79 kb of rice genomic DNA flanking the transgene. In addition to deletions, we obtained chromosomal inversions ranging in size from less than 10 kb (within the transgene DNA) to over 1 million base pairs (Mb). For 11 inversions, we cloned and sequenced both inversion breakpoints; in all 11 cases, the inversion junctions contained the typical 8 base pairs (bp) Ac/Ds target site duplications, confirming their origin as transposition products. Together, our results indicate that alternative Ac/Ds transposition can be an efficient tool for functional genomics and chromosomal manipulation in rice.     

Genome rearrangements in maize induced by alternative transposition of reversed ac/ds termini

Alternative transposition can induce genome rearrangements, including deletions, inverted duplications, inversions, and translocations. To investigate the types and frequency of the rearrangements elicited by a pair of reversed Ac/Ds termini, we isolated and analyzed 100 new mutant alleles derived from two parental alleles that both contain an intact Ac and a fractured Ac (fAc) structure at the maize p1 locus. Mutants were characterized by PCR and sequencing; the results show that nearly 90% (89/100) of the mutant alleles represent structural rearrangements including deletions, inversions, translocations, or rearrangement of the intertransposon sequence (ITS). Among 37 deletions obtained, 20 extend into the external flanking sequences, while 17 delete portions of the intertransposon sequence. Interestingly, one deletion allele that contains only a single nucleotide between the retained Ac and fAc termini is not competent for further alternative transposition events. We propose a new model for the formation of intertransposon deletions through insertion of reversed transposon termini into sister-chromatid sequences. These results document the types and frequencies of genome rearrangements induced by alternative transposition of reversed Ac/Ds termini in maize.     

Reversed end Ds element: a novel tool for chromosome engineering in Arabidopsis

The maize Ac/Ds transposable element (TE) transposes by a "cut and paste" mechanism. Previous studies in maize showed that when the TE ends are in reversed orientation with respect to each other, alternative transposition reactions can occur resulting in large scale genome rearrangements including deletions and inversions. To test whether similar genome rearrangements can also occur in other plants, we studied the efficacy of such alternative transposition-mediated genome rearrangements in Arabidopsis. Here we present our analysis of 33 independent chromosome rearrangements. Transposition at the reversed ends Ds element can cause deletions over 1 Mbp, and inversions up to 2.4 Mbp in size. We identified additional rearrangements including a reciprocal translocation and a putative ring chromosome. Some of the deletions and inversions are germinally transmitted.     

Genome rearrangements by nonlinear transposons in maize.

Transposable elements have long been considered as potential agents of large-scale genome reorganization by virtue of their ability to induce chromosomal rearrangements such as deletions, duplications, inversions, and reciprocal translocations. Previous researchers have shown that particular configurations of transposon termini can induce chromosome rearrangements at high frequencies. Here, we have analyzed chromosomal rearrangements derived from an unstable allele of the maize P1 (pericarp color) gene. The progenitor allele contains both a full-length Ac (Activator) transposable element and an Ac terminal fragment termed fAc (fractured Ac) inserted in the second intron of the P1-rr gene. Two rearranged alleles were derived from a classical maize ear twinned sector and were found to contain a large inverted duplication and a corresponding deficiency. The sequences at the junctions of the rearrangement breakpoints indicate that the duplication and deletion structures were produced by a single transposition event involving Ac and fAc termini located on sister chromatids. Because the transposition process we describe involves transposon ends located on different DNA molecules, it is termed nonlinear transposition (NLT). NLT can rapidly break and rejoin chromosomes and thus could have played an important role in generating structural heterogeneity during genome evolution.     

Transposition of reversed Ac element ends generates novel chimeric genes in maize

The maize Activator/Dissociation (Ac/Ds) elements are members of the hAT (hobo, Ac, and Tam3) superfamily of type II (DNA) transposons that transpose through a “cut-and-paste” mechanism. Previously, we reported that a pair of Ac ends in reversed orientation is capable of undergoing alternative transposition reactions that can generate large-scale chromosomal rearrangements, including deletions and inversions. We show here that rearrangements induced by reversed Ac ends transposition can join the coding and regulatory sequences of two linked paralogous genes to generate a series of chimeric genes, some of which are functional. To our knowledge, this is the first report demonstrating that alternative transposition reactions can recombine gene segments, leading to the creation of new genes.     

Intra-chromosomal rearrangements generated by Cre-lox site-specific recombination.

Chromosomal rearrangements are useful genetic and breeding tools but are often difficult to detect and characterize. To more easily identify and define chromosome deletions and inversions, we have used the bacteriophage P1 Cre-lox site-specific recombination system to generate these events in plants. This involves three steps: (i) the introduction of two lox sites into one locus in a plant genome, including one site within a modified Ds transposon; (ii) Ac transposase-mediated transposition of the Ds-lox element to a new locus on the same chromosome; (iii) Cre-mediated site-specific recombination between the two lox sites that bracket a chromosome segment. We report the production of a deletion and three inversion events in tobacco. The utility of chromosomal segments bracketed by lox sites for targeted manipulation and cloning is discussed.     

Transposition of reversed Ac element ends generates chromosome rearrangements in maize

In classical "cut-and-paste" transposition, transposons are excised from donor sites and inserted at new locations. We have identified an alternative pathway in which transposition involves the 5' end of an intact Ac element and the 3' end of a nearby terminally deleted fAc (fractured Ac). The Ac and fAc elements are inserted at the maize p1 locus on chromosome 1s in the same orientation; the adjacent ends of the separate elements are thus in reversed orientation with respect to each other and are separated by a distance of approximately 13 kb. Transposition involving the two ends in reversed orientation generates inversions, deletions, and a novel type of local rearrangement. The rearrangement breakpoints are bounded by the characteristic footprint or target site duplications typical of Ac transposition reactions. These results demonstrate a new intramolecular transposition mechanism by which transposons can greatly impact genome evolution.     

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.     

Macrotransposition and other complex chromosomal restructuring in maize by closely linked transposons in direct orientation

Several observations indicate that compatible ends of separate, yet closely linked, transposable elements (TEs) can interact in alternative transposition reactions. First, pairs of TEs cause chromosome breaks with frequencies inversely related to the intertransposon distance. Second, some combinations of two TEs produce complex rearrangements that often include DNA adjacent to one or both elements. In pairs of TEs in direct orientation, alternative reactions involving the external ends of the two TEs should lead to the transposition of a macrotransposon consisting of both elements plus the intervening chromosomal segment. Such macrotransposons have been hypothesized previously based on deletions, but no macrotransposon insertions have been recovered. To detect macrotransposition, we have analyzed heritable chromosomal rearrangements produced by a chromosome-breaking pair of Ac and Ds elements situated 6.5 kb apart in direct orientation in a part of the maize (Zea mays) genome dispensable for viability. Here, we show that the postulated macrotransposon can excise and reinsert elsewhere in the genome. In addition, this transposon pair produces other complex rearrangements, including deletions, inversions, and reshuffling of the intertransposon segment. Thus, closely linked TE pairs, a common transposition outcome in some superfamilies, are adept at restructuring chromosomes and may have been instrumental in reshaping plant genomes.     

Fusion of reverse-oriented Ds termini following abortive transposition in Arabidopsis: implications for the mechanism of Ac/Ds transposition

We studied the products of alternative transposition reactions that utilize reverse-oriented Ds termini as substrates. In this configuration, Ds transposition can generate genome rearrangements including deletions, inversions, and reciprocal translocations. In approximately half of the transposition products recovered in Arabidopsis, the termini of the reversed ends Ds element were ligated together. The sequences at these fused-end junctions suggest that the excised transposon termini form covalently closed hairpin structures. These results shed new light on the mechanism of Ac/Ds transposition.     

Chromosome Breakage by Pairs of Closely Linked Transposable Elements of the Ac-Ds Family in Maize

Chromosome breaks and hence chromosomal rearrangements often occur in maize stocks harboring transposable elements (TEs), yet it is not clear what types of TE structures promote breakage. We have shown previously that chromosomes containing a complex transposon structure consisting of an Ac (Activator) element closely linked in direct orientation to a terminally deleted or fractured Ac (fAc) element have a strong tendency to break during endosperm development. Here we show that pairs of closely linked transposons with intact ends, either two Ac elements--a common product of Ac transposition--or an Ac and a Ds (Dissociation) element, can constitute chromosome-breaking structures, and that the frequency of breakage is inversely related to intertransposon distance. Similar structures may also be implicated in chromosome breaks in other eukaryotic TE systems known to produce chromosomal rearrangements. The present findings are discussed in light of a model of chromosome breakage that is based on the transposition of a partially replicated macrotransposon delimited by the outside ends of the two linked TEs.     

Alternative Transposition Generates New Chimeric Genes and Segmental Duplications at the Maize p1 Locus

The maize Ac/Ds transposon family was the first transposable element system identified and characterized by Barbara McClintock. Ac/Ds transposons belong to the hAT family of class II DNA transposons. We and others have shown that Ac/Ds elements can undergo a process of alternative transposition in which the Ac/Ds transposase acts on the termini of two separate, nearby transposons. Because these termini are present in different elements, alternative transposition can generate a variety of genome alterations such as inversions, duplications, deletions, and translocations. Moreover, Ac/Ds elements transpose preferentially into genic regions, suggesting that structural changes arising from alternative transposition may potentially generate chimeric genes at the rearrangement breakpoints. Here we identified and characterized 11 independent cases of gene fusion induced by Ac alternative transposition. In each case, a functional chimeric gene was created by fusion of two linked, paralogous genes; moreover, each event was associated with duplication of the ∼70-kb segment located between the two paralogs. An extant gene in the maize B73 genome that contains an internal duplication apparently generated by an alternative transposition event was also identified. Our study demonstrates that alternative transposition-induced duplications may be a source for spontaneous creation of diverse genome structures and novel genes in maize.     

Molecular analysis of rice plants harboring an Ac/Ds transposable element-mediated gene trapping system

In rice, limited efforts have been made to identify genes by the use of insertional mutagens, especially heterologous transposons such as the maize Ac/Ds. We constructed Ac and gene trap Ds vectors and introduced them into the rice genome by Agrobacterium-mediated transformation. In this report, rice plants that contained single and simple insertions of T-DNA were analysed in order to evaluate the gene-tagging efficiency. The 3' end of Ds was examined for putative splicing donor sites. As observed in maize, three splice donor sites were identified at the 3' end of the Ds in rice. Nearly 80% of Ds elements were excised from the original T-DNA sites, when Ac cDNA was expressed under a CaMV 35S promoter. Repetitive ratoon culturing was performed to induce new transpositions of Ds in new plants derived from cuttings. About 30% of the plants carried at least one Ds which underwent secondary transposition in the later cultures. Eight per cent of transposed Ds elements expressed GUS in various tissues of rice panicles. With cloned DNA adjacent to Ds, the genomic complexities of the insertion sites were examined by Southern hybridization. Half of the Ds insertion sites showed simple hybridization patterns which could be easily utilized to locate the Ds. Our data demonstrate that the Ac/Ds-mediated gene trap system could prove an excellent tool for the analysis of functions of genes in rice. We discuss genetic strategies that could be employed in a large scale mutagenesis using a heterologous Ac/Ds family in rice.     

Ac transposition is impaired by a small terminal deletion

In maize, the P1-vv allele specifies variegated pericarp and cob pigmentation, and contains an Ac transposable element inserted in the second intron of the P1-rr gene. Starting from P1-vv, we recovered a new allele, called P1-vv5145, which gives an extremely light variegated pericarp and cob phenotype. The P1-vv5145 allele contains an Ac element ( Ac5145) at the same position and in the same orientation as in the progenitor P1-vv allele; however, the P1-vv5145 allele has a 2-bp deletion which removes the last nucleotide (A) from the 3' end of the Ac element, and an adjacent flanking nucleotide (C) from the p1 intron. In crosses with a Ds tester stock, P1-vv5145 shows a normal ability to induce Ds transposition; however, Ac excision from P1-vv5145 is 3800-fold less frequent than from the progenitor P1-vv allele. Our results demonstrate that the alteration of the 3' terminal base strongly impairs Ac transposition. The P1-vv5145 allele thus provides a relatively stable source of Ac transposase for controlling Ds transposition in genetic experiments. In addition, we describe two further alleles ( P1-ww7B8, P1-ww9A146-3) that contain deletions of Ac and flanking p1 gene sequences. These latter deletions are larger and involve the 5' end of the the Ac element. A model is proposed to explain the formation of one-sided deletions as a consequence of Ac transposition during replication of the element.     

Molecular evidence that chromosome breakage by Ds elements is caused by aberrant transposition.

The transposable Dissociation (Ds) element of maize was first discovered as a site of high-frequency chromosome breakage. Because both Ds-mediated breakage and transposition require the presence of the Activator (Ac) element, it has been suggested that chromosome breakage may be the outcome of an aberrant transposition event. This idea is consistent with the finding that only complex structures containing multiple Ds or Ac and Ds elements have been correlated with chromosome breakage. In this report, we describe two chromosome-breaking maize alleles that contain pairs of closely linked but separate Ds elements inserted at the Waxy locus. A polymerase chain reaction assay was utilized to isolate intermediates in the breakage process. The DNA sequence of these intermediates reveals deletions and base pair changes consistent with transposon footprints that may represent the junctions between fused sister chromatids. These results provide direct molecular evidence that chromosome breakage is the result of aberrant transposition events.     

Pattern of somatic transposition in a high copy Ac tomato line

A transgenic tomato line containing between eight and ten copies per genome of an exceptionally active maize transposable element Ac has previously been described. Southern analyses indicated that these elements are somatically active in these plants. In order to characterize further the pattern of somatic transposition in this line, 24 independent Ac insertion events from a single plant were cloned. In 21 cases, Ac inserted into single copy genomic DNA while in three cases Ac inserted into sequences present at two to four copies per genome; none of the insertions occurred into more highly repetitive DNA. The chromosomal locations of 20 insertion sites were determined by RFLP mapping and a pattern of small dispersed clusters emerged. Thirteen of the 20 insertion sites were linked to at least one other insertion site but these were distributed over nine of the 12 tomato chromosomes. Only one Ac insertion was linked to the T-DNA locus. The structural integrity of these Ac elements was examined and no evidence of deletions or other rearrangements suggestive of Ds elements was found. The implications of these findings with respect to the use of Ac as a transposon tag in heterologous species are discussed.     

Origination of Ds elements from Ac elements in maize: evidence for rare repair synthesis at the site of Ac excision.

Although it has been known for some time that the maize transposon Ac can mutate to Ds by undergoing internal deletions, the mechanism by which these mutations arise has remained conjectural. To gain further insight into this mechanism in maize we have studied a series of Ds elements that originated de novo from Ac elements at known locations in the genome. We present evidence that new, internally deleted Ds elements can arise at the Ac donor site when Ac transposes to another site in the genome. However, internal deletions are rare relative to Ac excision footprints, the predominant products of Ac transposition. We have characterized the deletion junctions in five new Ds elements. Short direct repeats of variable length occur adjacent to the deletion junction in three of the five Ds derivatives. In the remaining two, extra sequences or filler DNA is inserted at the junction. The filler DNAs are identical to sequences found close to the junction in the Ac DNA, where they are flanked by the same sequences that flank the filler DNA in the deletion. These findings are explained most simply by a mechanism involving error-prone DNA replication as an occasional alternative to end-joining in the repair of Ac-generated double-strand breaks.     

Large-scale systematic study on stability of the Ds element and timing of transposition in rice

Activator/Dissociation (Ac/Ds) transposon mutagenesis is a widely used tool for gene identification; however, several reports on silencing of the Ac/Ds element in starter lines and in stable transposants question the applicability of such an approach in later generations. We have performed a systematic analysis on various aspects of the silencing phenomenon in rice (Oryza sativa ssp. japonica cv. Nipponbare). High somatic and germinal transposition frequencies observed in earlier generations were maintained as late as T4 and T5 generations; thus the propagation of parental lines did not induce transposon silencing. Moreover, the stably transposed Ds element was active even at the F5 generation, since Ac could remobilize the Ds element as indicated by the footprint analysis of several revertants. Expression of the bar gene was monitored from F3 to F6 generations in >1,000 lines. Strikingly, substantial transgene silencing was not observed in any of the generations tested. We analyzed the timing of transposition during rice development and provide evidence that Ds is transposed late after tiller formation. The possibility, that the independent events could be the result of secondary transposition, was ruled out by analyzing potential footprints by reciprocal PCR. Our study validates the Ac/Ds system as a tool for large-scale mutagenesis in rice, since the Ds elements were active in the starter and insertion lines even in the later generations. We propose that harvesting rice seeds using their panicles is an alternative way to increase the number of independent transposants due to post-tillering transposition.     

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。