Mapped Ds/T-DNA launch pads for functional genomics in barley

A system for targeted gene tagging and local saturation mutagenesis based on maize transposable elements (Ac/Ds) was developed in barley (Hordeum vulgare L.). We generated large numbers of transgenic barley lines carrying a single copy of the non-autonomous maize Ds element at defined positions in the genome. Independent Ds lines were either generated by activating Ds elements in existing single-copy lines after crossing with AcTPase-expressing plants or by Agrobacterium-mediated transformation. Genomic DNA flanking Ds and T-DNA insertion sites from over 200 independent lines was isolated and sequenced, and was used for a sequence based mapping strategy in a barley reference population. More than 100 independent Ds insertion sites were mapped and can be used as launch pads for future targeted tagging of genes in the vicinity of the insertion sites. Sequence analysis of Ds and T-DNA flanking regions revealed a sevenfold preference of both mutagens for insertion into non-redundant, gene-containing regions of the barley genome. However, whilst transposed Ds elements preferentially inserted adjacent to regions with a high number of predicted and experimentally validated matrix attachment regions (nuclear MARs), this was not the case for T-DNA integration sites. These findings and an observed high transposition frequency from mapped launch pads demonstrate the future potential of gene tagging for functional genomics and gene discovery in barley.     

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.     

Rapid, large-scale generation of Ds transposant lines and analysis of the Ds insertion sites in rice

Rapid, large-scale generation of a Ds transposant population was achieved using a regeneration procedure involving tissue culture of seed-derived calli carrying Ac and inactive Ds elements. In the F(2) progeny from genetic crosses between the same Ds and Ac starter lines, most of the crosses produced an independent germinal transposition frequency of 10-20%. Also, many Ds elements underwent immobilization even though Ac was expressed. By comparison, in a callus-derived regenerated population, over 70% of plants carried independent Ds insertions, indicating transposition early in callus formation. In the remaining population, the majority of plants carried only Ac. Most of the new Ds insertions were stably transmitted to a subsequent generation. An exceptionally high proportion of independent transposants in the regenerated population means that selection markers for transposed Ds and continual monitoring of Ac/Ds activities may not necessarily be required. By analyzing 1297 Ds-flanking DNA sequences, a genetic map of 1072 Ds insertion sites was developed. The map showed that Ds elements were transposed onto all of the rice chromosomes, with preference not only near donor sites (36%) but also on certain physically unlinked arms. Populations from both genetic crossing and tissue culture showed the same distribution patterns of Ds insertion sites. The information of these mapped Ds insertion sites was deposited in GenBank. Among them, 55% of Ds elements were on predicted open-reading frame (ORF) regions. Thus, we propose an optimal strategy for the rapid generation of a large population of Ds transposants in rice.     

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。     

Linked and Unlinked Transposition of a Genetically Marked Dissociation Element in Transgenic Tomato

We have introduced a genetically marked Dissociation transposable element (Ds(neo)) into tomato. In the presence of Ac transposase, Ds(neo) excised from an integrated T-DNA and reinserted at numerous new sites in the tomato genome. The marker genes of Ds(neo) (NPTII) and the T-DNA (HPT) facilitated identification of plants bearing transposon excisions and insertions. To explore the feasibility of gene tagging strategies in tomato using Ds(neo), we examined the genomic distribution of Ds(neo) receptor sites, relative to the location of the donor T-DNA locus. Restriction fragment length polymorphism mapping of transposed Ds(neo) elements was conducted in two tomato families, derived from independent primary transformants each bearing Ds(neo) within a T-DNA at a unique position in the genome. Transposition of Ds(neo) generated clusters of insertions that were positioned on several different tomato chromosomes. Ds(neo) insertions were often located on the same chromosome as the T-DNA donor site. However, no insertion showed tight linkage to the T-DNA. We consider the frequency and distance of Ds(neo) transposition observed in tomato to be well suited for transposon mutagenesis. Our study made use of a novel, stable allele of Ac (Ac3) that we discovered in transgenic tomato. We determined that the Ac3 element bears a deletion of the outermost 5 base pairs of the 5'-terminal inverted repeat. Though incapable of transposition itself, Ac3 retained the ability to mobilize Ds(neo). We conclude that a dual element system, composed of the stable Ac3 trans-activator in combination with Ds(neo), is an effective tool for transposon tagging experiments in tomato.     

Distribution of Unlinked Transpositions of a Ds Element from a T-DNA Locus on Tomato Chromosome 4

In maize, receptor sites for unlinked transpositions of Activator (Ac) elements are not distributed randomly. To test whether the same is true in tomato, the receptor sites for a Dissociation (Ds) element derived from Ac, were mapped for 26 transpositions unlinked to a donor T-DNA locus on chromosome 4. Four independent transposed Dss mapped to sites on chromosome 4 genetically unlinked to the donor T-DNA, consistent with a preference for transposition to unlinked sites on the same chromosome as opposed to sites on other chromosomes. There was little preference among the nondonor chromosomes, except perhaps for chromosome 2, which carried seven transposed Dss, but these could not be proven to be independent. However, these data, when combined with those from other studies in tomato examining the distribution of transposed Acs or Dss among nondonor chromosomes, suggest there may be absolute preferences for transposition irrespective of the chromosomal location of the donor site. If true, transposition to nondonor chromosomes in tomato would differ from that in maize, where the preference seems to be determined by the spatial arrangement of chromosomes in the interphase nucleus. The tomato lines carrying Ds elements at known locations are available for targeted transposon tagging experiments.     

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。     

Transposon tagging in diploid strawberry

Fragaria vesca was transformed with a transposon tagging construct harbouring amino terminally deleted maize transposase and EGFP (Ac element), NPTII, CaMV 35S promoter (P35S) driving transposase and mannopine synthase promoter (Pmas) driving EGFP (Ds element). Of 180 primary transgenics, 48 were potential launch pads, 72 were multiple insertions or chimaeras, and 60 exhibited somatic transposition. T(1) progeny of 32 putative launch pads were screened by multiplex PCR for transposition. Evidence of germ-line transposition occurred in 13 putative launch pads; however, the transposition frequency was too low in three for efficient recovery of transposants. The transposition frequency in the remaining launch pads ranged from 16% to 40%. After self-pollination of the T(0) launch pads, putative transposants in the T(1) generation were identified by multiplex PCR. Sequencing of hiTAIL-PCR products derived from nested primers within the Ds end sequences (either P35S at the left border or the inverted repeat at the right border) of T(1) plants revealed transposition of the Ds element to distant sites in the strawberry genome. From more than 2400 T(1) plants screened, 103 unique transposants have been identified, among which 17 were somatic transpositions observed in the T(0) generation. Ds insertion sites were dispersed among various gene elements [exons (15%), introns (23%), promoters (30%), 3' UTRs (17%) as well as intergenically (15%)]. Three-primer (one on either side of the Ds insertion and one within the Ds T-DNA) PCR could be used to identify homozygous T(2) transposon-tagged plants. The mutant collection has been catalogued in an on-line database.     

Germinal Transpositions of the Maize Element Dissociation from T-DNA Loci in Tomato

We have analyzed the pattern of germinal transpositions of artificial Dissociation (Ds) transposons in tomato. T-DNA constructs carrying Ds were transformed into tomato, and the elements were trans-activated by crossing to lines transformed with a stabilized Activator (sAc) that expressed the transposase gene. The sAc T-DNA carried a GUS gene to monitor its segregation. The Ds elements were inserted in a marker gene so that excision from the T-DNA could be monitored. The Ds elements also carried a genetic marker that was intended to be used for reinsertion selection of the elements after excision. Unfortunately, this gene was irreversibly inactivated on crossing to sAc. Germinal excision frequencies of Ds averaged 15-40%, but there was large variation between and within plants. Southern hybridization analysis of stable transposed Ds elements indicated that although unique transpositions predominate, early transposition events can lead to large clonal sectors in the germline of developing plants and to sibling offspring carrying the same transposition event. Multiple germinal transpositions from three different loci were examined for uniqueness, and 15 different transpositions were identified from each of three T-DNA loci that carried a single independent Ds. These were mapped relative to the donor T-DNA loci, and for each locus 70-80% of the transposed elements were closely linked to the donor site.     

Frequency and pattern of transposition of the maize transposable element Ds in transgenic rice plants

Two kinds of T-DNA constructs, I-RS/dAc-I-RS and Hm(R)Ds, carrying a non-autonomous transposable element of Ac of maize were introduced into rice plants by Agrobacterium-mediated gene transfer. Six transgenic rice plants identified as containing a single copy of the element were crossed with two transgenic rice plants carrying a gene for Ac transposase under the control of the cauliflower mosaic virus 35S promoter. In F2 progenies, excision of the element was detected by PCR analysis and re-integration of the element was investigated by Southern blot analysis. The frequency of the excision of the element was found to vary from 0 to 70% depending on the crossing combination. The frequency of the number of individual transposition events out of the total number of F2 plants with germinal excision was 44% in one crossing combination and 38% in the other. In the most efficient case, 10 plants with independent transposition were obtained out of the 49 F2 plants tested. Linkage analysis of the empty donor site and the transposed Ds-insertion site in F3 plants demonstrated that one of five Ds-insertion sites was not linked to the empty donor site. The transgenic rice obtained in this study can be used for functional genomics of rice.     

Transposition Pattern of the Maize Element Ds in Arabidopsis Thaliana

As part of establishing an efficient transposon tagging system in Arabidopsis using the maize elements Ac and Ds, we have analyzed the inheritance and pattern of Ds transposition in four independent Arabidopsis transformants. A low proportion (33%) of plants inheriting the marker used to monitor excision contained a transposed Ds. Selection for the transposed Ds increased this to at least 49%. Overall, 68% of Ds transpositions inherited with the excision marker were to genetically linked sites; however, the distribution of transposed elements varied around the different donor sites. Mapping of transposed Ds elements that were genetically unlinked to the donor site showed that a proportion (3 of 11 tested) integrated into sites which were still physically linked.     

Development of a simple transient assay for Ac/Ds activity in cells of intact barley tissue

The development of a barley ( Hordeurn vulgare L.) transformation system made it possible to consider the use of maize Activator/Dissociation ( Ac/Ds ) transposable elements for gene tagging in transgenic barley plants. However, barley transformation is time-consuming, and therefore a simple transient assay for Ac/Ds activity in intact barley tissues was developed to test the components of a proposed gene tagging system, prior to their stable introduction into plants. In this assay, barley scutellar tissue is co-transformed with constructs containing the maize Ac transposase gene and an Escherichia coli uid A reporter gene ( Gus ), the expression of which is interrupted by a maize Ds element. In transformed barley scutellar cells, Ac transposase-mediated excision of the Ds element generates a functional Gus gene, leading to histochemically detectable GUS activity. Characterization of the excision products showed that they had a pattern of nucleotide deletions and/or transversions similar to that found in maize and other heterologous plant systems. In addition, although contrary to the situation observed in heterologous dicot systems, efficient Ds excision in barley, a heterologous monocot system, appears to be inversely associated with Ac copy number, a finding similar to the Ac dosage effects observed in maize. The transient assay was used to demonstrate functional transposase activity in barley callus lines stably transformed with an Ac transposase gene.     

A hyperactive transposase of the maize transposable element activator (Ac)

Activator/Dissociation (Ac/Ds) transposable elements from maize are widely used as insertional mutagenesis and gene isolation tools in plants and more recently also in medaka and zebrafish. They are particularly valuable for plant species that are transformation-recalcitrant and have long generation cycles or large genomes with low gene densities. Ac/Ds transposition frequencies vary widely, however, and in some species they are too low for large-scale mutagenesis. We discovered a hyperactive Ac transposase derivative, AcTPase(4x), that catalyzes in the yeast Saccharomyces cerevisiae 100-fold more frequent Ds excisions than the wild-type transposase, whereas the reintegration frequency of excised Ds elements is unchanged (57%). Comparable to the wild-type transposase in plants, AcTPase(4x) catalyzes Ds insertion preferentially into coding regions and to genetically linked sites, but the mutant protein apparently has lost the weak bias of the wild-type protein for insertion sites with elevated guanine-cytosine content and nonrandom protein-DNA twist. AcTPase(4x) exhibits hyperactivity also in Arabidopsis thaliana where it effects a more than sixfold increase in Ds excision relative to wild-type AcTPase and thus may be useful to facilitate Ac/Ds-based insertion mutagenesis approaches.     

Resources for targeted insertional and deletional mutagenesis in Arabidopsis

The maize transposons Activator (Ac) and Dissociation (Ds) are active in many monocots and dicots, including Arabidopsis. We describe a new Ac-derived transposon construct, designated the Ds-loxP T-DNA, which can be used for both insertional and deletional mutagenesis. There are loxP sites in both orientations on both the transposon and the donor site T-DNA and an arrangement of marker genes that permits selection of transposition events, as well as deletions and inversions extending from the donor site to a transposon reinserted on either side of it. We show that Cre-mediated deletions and inversions occur at a high frequency. The tendency of Ac-Ds transposons to reinsert near the donor site can be used to target both insertional and deletional mutagenesis, but efficient exploitation of this property requires a library of mapped marked donor sites distributed in the genome. We have created a population of independent Ds T-DNA transformants and we have mapped an initial set of 75 Ds T-DNA integration sites. We assessed the potential efficiency of targeted mutagenesis by detecting Ds reinsertion events at several loci over a 400 kb interval from each of two donor sites with different Ds T-DNA constructs. The distribution of reinsertion sites is similar around the two tested loci, with roughly 10, 4, and ca. 1% of reinsertions detected within 1-2 kb of sites 10, 100, and 200-400 kb from the donor site, respectively. To facilitate the use of this targeted mutagenesis system. we have constructed a searchable database of the mapped Ds T-DNA integration sites.