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。     

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.     

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.     

The transposition pattern of the Ac element in tobacco cultured cells.

We investigated physical distances and directions of transposition of the maize transposable element Ac in tobacco cultured cells. We introduced a T-DNA construct that carried a non-autonomous derivative of Ac (designated dAc-I-RS) that included sites for cleavage by restriction endonuclease MluI. Another cleavage site was also introduced into the T-DNA region outside of the dAc-I-RS transposable element. The tobacco cultured cell line BY-2 was transformed with the T-DNA and several transformed lines that had a single copy of the T-DNA at a different chromosomal location were isolated. These lines were co-cultured with Agrobacterium tumefaciens cells that carried a cDNA for the Ac transposase gene under the control of various promoters. Sublines of cultured cells in which dAc-I-RS had been transposed, were isolated. The genomic DNAs of these sublines were isolated and digested with MluI. Sizes of DNA segments generated by digestion were determined by pulse-field gel electrophoresis. Our results showed that 20 to 70% of transposition events had occurred within several hundreds kilo-base pairs (kb) on the same chromosome. These results demonstrate that the Ac-Ds element preferentially transposed to regions near the original site in a tobacco chromosome. In addition, the present results are an example of asymmetric transposition as demonstrated by the distance of transposition on the chromosome.     

Mutational analysis of the inverted repeats of Tn3

The transposase protein and the terminal inverted repeat sequences of the prokaryotic transposon Tn3 are essential for transposition. In order to determine the sequences within the inverted repeat necessary for transposition and interaction with transposase, we have constructed a series of mini-Tn3s in which specific mutations have been introduced into the inverted repeats. The effects of these mutations on transposition have been assayed in vivo using a mating-out transposition assay. Several single base-pair mutations within the transposase binding site reduce transposition frequency. Mutations that affect transposition show a greater effect when present in both inverted repeats than when present in only one inverted repeat.     

Tipping the balance between replicative and simple transposition

The bacterial insertion sequence IS903 has the unusual ability to transpose both replicatively and non-replicatively. The majority of products are simple insertions, while co-integrates, the product of replicative transposition, occur at a low frequency (<0.1% of simple insertions). In order to define the critical steps that determine the outcome of IS903 transposition, we have isolated mutants that specifically increase the rate of replicative transposition. Here we show that the nucleotide immediately flanking the transposon influences both overall transposition frequency and co-integrate formation. In particular, when the 3'-flanking nucleotide is A, co-integrates are increased 500-fold compared with a 3' C. In addition, we have isolated five transposase mutants that increase replicative transposition. These residues are close to the catalytic residues and are thus likely to be part of the active site. These are the first transposase mutations described that affect the product of transposition. Our results are consistent with the hypothesis that a delay in cleavage of the 5'-flanking DNA will increase the effective half-life of the 3'-nicked transposon intermediate and consequently enhance co-integrate formation.     

Independence of excision frequency and transposition frequency of P element in Drosophila melanogaster.

Although a family of transposon, P elements, are used as tools for molecular genetics in Drosophila melanogaster, the molecular details and mechanism of their mobilization process have not been studied extensively. In particular, the relationship between excision and transposition is little understood. We have previously produced a transgenic fly with a P element insertion that is nonautonomous (stable without transposase) and is highly-transposable in the presence of transposase. Using this insertion, we traced its mobilizations following introduction of a stable transposase source. We found a strain that has a 26-bp tandem repeat at the end of the original P element insertion. The 26-bp repeat reduced the frequency of excision although the frequency of transposition was not altered. Our results indicate independence of transposition from excision and importance of terminal repeat in excision.     

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.     

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.     

IS903 transposase mutants that suppress defective inverted repeats

The inverted repeats (IRs) of the insertion element IS903 are composed of two functional regions. An inner region, consisting of basepairs 6-18, is the transposase binding site. The outer region (positions 1-3) is not contacted during initial transposase binding, but is essential for efficient transposition. We have examined the interaction of the IR with the transposase by isolating transposase suppressors of IR mutations. These suppressors define two patches within the N-terminus of the protein. One class of suppressors, which rescued the majority of outer IR mutants tested, contained mutations in close proximity to an aspartate residue (D121) believed to form part of the catalytic DDE motif, suggesting that their suppressive effect is in the positioning of the catalytic site at the terminus of the transposon. The hypertransposition phenotype of mutant VA119 is also consistent with this hypothesis. The second class was more allele specific and preferentially suppressed a mutation at position 3 of the IR. Finally, we showed that mutations at the termini of the IR elevate the frequency of cointegrate formation by IS903. Other outer IR mutations did not have this effect. These data are consistent with the terminal bases of the transposon playing multiple and distinct roles in transposition.     

DNA sequence requirements for hobo transposable element transposition in Drosophila melanogaster

We have conducted a structure and functional analysis of the hobo transposable element of Drosophila melanogaster. A minimum of 141 bp of the left (L) end and 65 bp of the right (R) end of the hobo were shown to contain sequences sufficient for transposition. Both ends of hobo contain multiple copies of the motifs GGGTG and GTGGC and we show that the frequency of hobo transposition increases as a function of the copy number of these motifs. The R end of hobo contains a unique 12 bp internal inverted repeat that is identical to the hobo terminal inverted repeats. We show that this internal inverted repeat suppresses transposition activity in a hobo element containing an intact L end and only 475 bp of the R end. In addition to establishing cis-sequences requirements for transposition, we analyzed trans-sequence effects of the hobo transposase. We show a hobo transposase lacking the first 49 amino acids catalyzed hobo transposition at a higher frequency than the full-length transposase suggesting that, similar to the related Ac transposase, residues at the amino end of the transposase reduce transposition. Finally, we compared target site sequences of hobo with those of the related Hermes element and found both transposons have strong preferences for the same insertion sites.     

Construction and uses of a new transposable element whose insertion is able to produce gene fusions with the neomycin-phosphotransferase-coding region of Tn903

We describe the construction of a transposable element derived from the Mu phage that upon insertion is able to create a gene fusion between the region of Tn903 coding for neomycin phosphotransferase (NPT I), which confers resistance to aminoglycosides including kanamycin (KmR), neomycin and G418, and the control elements of the gene where the insertion occurs. A chloramphenicol (Cm) transacetylase gene (cat) that confers resistance to Cm is present in the transposon so that transposition events can be monitored even when no active fusions with the nptI coding region occur. The transposase gene is deleted and, therefore, this transposon is perfectly stable upon insertion. The properties of this new transposable element were studied by obtaining gene fusions between the Escherichia coli L-arabinose operon and 'nptI gene. In some of them the KmR phenotype is induced by arabinose. Insertions of this element in cloned fragments of the T-DNA region of Agrobacterium rhizogenes were also isolated. Some of them confer a KmR phenotype upon its E. coli carriers, which indicates that portions of the T-DNA are expressed in these cells.     

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.     

piggyBac can bypass DNA synthesis during cut and paste transposition

DNA synthesis is considered a defining feature in the movement of transposable elements. In determining the mechanism of piggyBac transposition, an insect transposon that is being increasingly used for genome manipulation in a variety of systems including mammalian cells, we have found that DNA synthesis can be avoided during piggyBac transposition, both at the donor site following transposon excision and at the insertion site following transposon integration. We demonstrate that piggyBac transposon excision occurs through the formation of transient hairpins on the transposon ends and that piggyBac target joining occurs by the direct attack of the 3'OH transposon ends on to the target DNA. This is the same strategy for target joining used by the members of DDE superfamily of transposases and retroviral integrases. Analysis of mutant piggyBac transposases in vitro and in vivo using a piggyBac transposition system we have established in Saccharomyces cerevisiae suggests that piggyBac transposase is a member of the DDE superfamily of recombinases, an unanticipated result because of the lack of sequence similarity between piggyBac and DDE family of recombinases.     

Construction and Application of Efficient Ac-Ds Transposon Tagging Vectors in Rice

Transposons are effective mutagens alternative to T-DNA for the generation of insertional mutants in many plant species including those whose transformation is inefficient. The current strategies of transposon tagging are usually slow and labor-intensive and yield low frequency of tagged lines. We have constructed a series of transposon tagging vectors based on three approaches: (i) Ac TPase controlled by glucocorticoid binding domain/VP16 acidic activation domain/Gal4 DNA-binding domain (GVG) chemical-inducible expression system; (ii) deletion of Ac TPase via Cre- lox site-specific recombination that was initially triggered by Ds excision; and (iii) suppression of early transposition events in transformed rice callus through a dual-functional hygromycin resistance gene in a novel Ds element ( HPT-Ds ). We tested these vectors in transgenic rice and characterized the transposition events. Our results showed that these vectors are useful resources for functional genomics of rice and other crop plants. The vectors are freely available for the community.     

Formation of a nucleoprotein complex containing Tn7 and its target DNA regulates transposition initiation

Tn7 insertion into its specific target site, attTn7, is mediated by the proteins TnsA, TnsB, TnsC and TnsD. The double-strand breaks that separate Tn7 from the donor DNA require the Tns proteins, the transposon and an attTn7 target DNA, suggesting that a prerequisite for transposition is the formation of a nucleoprotein complex containing TnsABC+D, and these DNAs. Here, we identify a TnsABC+D transposon-attTn7 complex, and demonstrate that it is a transposition intermediate. We demonstrate that an interaction between TnsB, the transposase subunit that binds to the transposon ends, and TnsC, the target DNA-binding protein that controls the activity of the transposase, is essential for assembly of the TnsABC+D transposon-attTn7 complex. We also show that certain TnsB residues are required for recombination because they mediate a TnsB-TnsC interaction critical to formation of the TnsABC+D transposon-attTn7 complex. We demonstrate that TnsA, the other transposase subunit, which also interacts with TnsC, greatly stabilizes the TnsABC+D transposon-attTn7 complex. Thus multiple interactions between the transposase subunits, TnsA and TnsB, and the target-binding transposase activator, TnsC, control Tn7 transposition.     

The Mod(mdg4) component of the Su(Hw) insulator inserted in the P transposon can repress its mobility in Drosophila melanogaster

Transposable element P of Drosophila melanogaster is one of the best-characterized eukaryotic transposons. Successful transposition requires the interaction between transposase complexes at both termini of the P element. Here we found that insertion of one or two copies of the Su(Hw) insulator in the P transposon reduces the frequency of its transposition. Inactivation of a Mod(mdg4) component of the Su(Hw) insulator suppresses the insulator effect. Thus, the Su(Hw) insulator can modulate interactions between transposase complexes bound to the ends of the P transposon in germ cells.     

T-DNA and transposon tagging in aspen

Abstract: We have investigated the somatic activity of the maize Activator (Ac) element in haploid and diploid aspen with the objective of developing an efficient transposon-based system for gene isolation in the model tree species Populus. It was shown that Ac is reinserted, frequently into or near coding regions in aspen, and therefore can be used for gene tagging studies. A number of phenotypic variants were also found following transformation of constructs harbouring the rolC gene. Comparative analyses of T-DNA flanking regions of variants and wild type lines indicate that T-DNA insertion has occurred in or near coding regions. However, the frequency of T-DNA insertion into genes is about one half of the frequency of Ac insertion hitting coding sequences. The results obtained give a proof-of-concept for transposon tagging in a tree system. Given the long generation cycles in tree species, gene tagging strategies are practical only to obtain dominant gain-of-function mutants that do not require selfing or test crossing. In order to obtain recessive loss-of-function mutants, we have regenerated haploid lines from immature pollen. These lines were successfully transformed with a construct containing the rolC transgene from Agrobacterium rhizogenes and Ac element from maize. The results indicate that Ac is also active in haploid aspen and hence can be used in general for gene tagging in trees.