Inactivation of the maize transposable element Activator (Ac) is associated with its DNA modification.

The Activator (Ac) element at the waxy locus (wx-m7 allele) has the ability to undergo changes in its genetic activity and cycles between an active and inactive phase. Comparison of active Ac elements at several loci and the inactive Ac at wx-m7 by Southern blot analysis revealed that the inactive Ac sequence was not susceptible to digestion by the methylation sensitive enzyme PvuII while active elements were susceptible to PvuII digestion. Restriction digest comparisons between the clones of the active and inactive Ac elements were indistinguishable. Further analyses with the enzymes SstII and the methylation sensitive and insensitive isoschizomers EcoRII and BstNI showed the inactive Ac sequence was methylated at these sites, whereas the active Ac was hypomethylated. Although the active Ac at the wx-m7 allele in different genetic backgrounds showed differences in the Ac DNA modification pattern, at least a fraction of genomic DNA contained Ac sequences that were unmethylated at all of the internal sites we assayed. These data may suggest a role for DNA modification in the ability of Ac to transpose from the waxy locus and to destabilize unlinked Ds elements.     

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。     

Ac-immobilized, a stable source of Activator transposase that mediates sporophytic and gametophytic excision of Dissociation elements in maize

We have identified and characterized a novel Activator (Ac) element that is incapable of excision yet contributes to the canonical negative dosage effect of Ac. Cloning and sequence analysis of this immobilized Ac (Ac-im) revealed that it is identical to Ac with the exception of a 10-bp deletion of sequences at the left end of the element. In screens of approximately 6800 seeds, no germinal transpositions of Ac-im were detected. Importantly, Ac-im catalyzes germinal excisions of a Ds element resident at the r1 locus resulting in the recovery of independent transposed Ds insertions in approximately 4.5% of progeny kernels. Many of these transposition events occur during gametophytic development. Furthermore, we demonstrate that Ac-im transactivates multiple Ds insertions in somatic tissues including those in reporter alleles at bronze1, anthocyaninless1, and anthocyaninless2. We propose a model for the generation of Ac-im as an aberrant transposition event that failed to generate an 8-bp target site duplication and resulted in the deletion of Ac end sequences. We also discuss the utility of Ac-im in two-component Ac/Ds gene-tagging programs in maize.     

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.     

Variegation patterns caused by excision of the maize transposable element Dissociation (Ds) are autonomously regulated by allele-specific Activator (Ac) elements and are not due to trans-acting modifier genes

The Ac elements present in the unstable wxm7 and wx-m9 alleles of maize trigger different patterns of Ds excision in trans. To determine whether this differential regulation is a feature of the Ac alleles themselves or is mediated by genetically distinct factors, maize plants heterozygous for the wx-m7 and wx-m9 alleles were crossed to tester strains homozygous for Ds reporter alleles. Kernels showing the variegation pattern characteristic for the Ac elements carried in the wx-m7 and wx-m9 alleles were found to be present in the ratios expected from the genetic constitution of the strains. The aleurone variegation caused by excision of the Ds reporter element and the endosperm variegation caused by excision of Ac from the wx-m7 and wx-m9 alleles themselves segregated with the original wx-m alleles. In addition, stable Wx and wx derivatives of wx-m9 that have lost Ac no longer exert any trans effect on the wx-m7 allele (and vice versa). Therefore it is concluded that the observed variegation patterns are autonomously determined by specific trans effects of the particular Ac element.     

Novel, developmentally specific control of Ds transposition in maize

Plants form their gametes late in somatic development and, as a result, often pass somatic mutations on to their progeny. Classic examples of this process are the germinal revertants of unstable, Ac/Ds transposon-induced kernel mutations in maize: frequent and early reversion events during somatic development are generally correlated with a high frequency of revertant gametes. We have characterized a Ds allele of the maize waxy(wx) gene, wx-m5:CS7, for which the correlation between somatic and germinal reversion frequencies no longer holds. The ability of wx-m5:CS7 (CS7) to produce revertant gametes is suppressed ∼100-fold in comparison with a second Ds allele, wx-m5:CS8 (CS8), which has an identical insertion at Wx and the same frequent and early somatic reversion pattern in endosperm. The excision of Ds from wx is not reduced 100-fold in the somatic tissues of CS7 plants as compared with CS8 plants. Suppressed formation of CS7 revertant gametes is independent of the Ac transposase source and is heritably passed to the embryos of progeny kernels; however, frequent and early somatic reversion is observed again in endosperms of these progeny kernels. This suppression appears to be caused by a dominant mutation in a trans-acting product that can suppress the germinal reversion of other Ds-induced alleles as well; the mutation is tightly linked to Wx but is not in the CS7 Ds itself. Taken together, the data suggest a novel mode of developmental control of Ac/Ds elements by the host plant, suppressing element excision in the shoot meristem. Received: 16 December 1996 / Accepted: 4 March 1997     

Somatic and germinal activities of maize Activator (Ac) transposase mutants in transgenic tobacco

The properties have been investigated of two deletion derivatives of the transposase protein (TPase) of maize transposable element Ac in transgenic tobacco. The wild-type and mutant TPases were expressed as fusions to the cauliflower mosaic virus 35S promoter. A deletion of 102 amino acids from the N-terminus, TPase(103–807), induces Ds excisions from a SPT::Ds reporter locus with a higher frequency than the wild-type TPase. The increased transpositional activity of TPase(103–807) is a dominant trait, as seedlings coexpressing truncated and wild-type TPase show the characteristic TPase(103–807) variegation pheno-type. A transpositionally inactive TPase deletion derivative lacking 188 amino acids from the N-terminus inhibits the transpositional activity of the wild-type TPase.     

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.     

Novel, developmentally specific control of Ds transposition in maize

Plants form their gametes late in somatic development and, as a result, often pass somatic mutations on to their progeny. Classic examples of this process are the germinal revertants of unstable, Ac/Ds transposon-induced kernel mutations in maize: frequent and early reversion events during somatic development are generally correlated with a high frequency of revertant gametes. We have characterized a Ds allele of the maize waxy(wx) gene, wx-m5:CS7, for which the correlation between somatic and germinal reversion frequencies no longer holds. The ability of wx-m5:CS7 (CS7) to produce revertant gametes is suppressed ～100-fold in comparison with a second Ds allele, wx-m5:CS8 (CS8), which has an identical insertion at Wx and the same frequent and early somatic reversion pattern in endosperm. The excision of Ds from wx is not reduced 100-fold in the somatic tissues of CS7 plants as compared with CS8 plants. Suppressed formation of CS7 revertant gametes is independent of the Ac transposase source and is heritably passed to the embryos of progeny kernels; however, frequent and early somatic reversion is observed again in endosperms of these progeny kernels. This suppression appears to be caused by a dominant mutation in a trans-acting product that can suppress the germinal reversion of other Ds-induced alleles as well; the mutation is tightly linked to Wx but is not in the CS7 Ds itself. Taken together, the data suggest a novel mode of developmental control of Ac/Ds elements by the host plant, suppressing element excision in the shoot meristem.     

Gametophyte-specific transposition of the maize Ds element in transgenic tobacco

To assess the potential advantages of a transposon-tagging system based on gametophyte-specific transposition a fusion between the anther-specific Arabidopsis thaliana apg promoter and the maize Ac transposase gene was constructed and introduced into tobacco. The ability of this transposase source to activate Ds transposition in a developmentally controlled manner was monitored by crossing to plants harbouring the cell autonomous excision marker gene construct, Ds —SPT. A number of fully green, streptomycin-resistant seedlings resulting from germinal transposition events were observed in the progeny of apg -TPase x Ds —SPT F1 plants. Streptomycin-resistant sectors were not observed in either F₁ seedlings or F₂ progeny, indicating a complete lack of somatic excision. Further crosses of apg —TPase sources to plants containing Ds—bar herbicide selection excision marker constructs gave reproducible gametophytic excision frequencies of up to 0.3%. Sequencing of Ds excision sites from F₂ seedlings derived from single F₁ plants revealed various sequence alterations in the original Ds insertion 'footprint' indicative of independent Ds excision events. Independent re-insertion was confirmed by Southern analysis of F₂ siblings. It is concluded that apg -controlled Ac transposase expression activates male gametophyte-specific Ds transposition.     

Variegation patterns caused by excision of the maize transposable element Dissociation (Ds) are autonomously regulated by allele-specific Activator (Ac) elements and are not due to trans-acting modifier genes

The Ac elements present in the unstable wxm7 and wx-m9 alleles of maize trigger different patterns of Ds excision in trans. To determine whether this differential regulation is a feature of the Ac alleles themselves or is mediated by genetically distinct factors, maize plants heterozygous for the wx-m7 and wx-m9 alleles were crossed to tester strains homozygous for Ds reporter alleles. Kernels showing the variegation pattern characteristic for the Ac elements carried in the wx-m7 and wx-m9 alleles were found to be present in the ratios expected from the genetic constitution of the strains. The aleurone variegation caused by excision of the Ds reporter element and the endosperm variegation caused by excision of Ac from the wx-m7 and wx-m9 alleles themselves segregated with the original wx-m alleles. In addition, stable Wx and wx derivatives of wx-m9 that have lost Ac no longer exert any trans effect on the wx-m7 allele (and vice versa). Therefore it is concluded that the observed variegation patterns are autonomously determined by specific trans effects of the particular Ac element.     

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.     

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.     

Microhomology-dependent end joining and repair of transposon-induced DNA hairpins by host factors in Saccharomyces cerevisiae

The maize, cut-and-paste transposon Ac/Ds is mobile in Saccharomyces cerevisiae, and DNA sequences of repair products provide strong genetic evidence that hairpin intermediates form in host DNA during this transposition, similar to those formed for V(D)J coding joints in vertebrates. Both DNA strands must be broken for Ac/Ds to excise, suggesting that double-strand break (DSB) repair pathways should be involved in repair of excision sites. In the absence of homologous template, as expected, Ac excisions are repaired by nonhomologous end joining (NHEJ) that can involve microhomologies close to the broken ends. However, unlike repair of endonuclease-induced DSBs, repair of Ac excisions in the presence of homologous template occurs by gene conversion only about half the time, the remainder being NHEJ events. Analysis of transposition in mutant yeast suggests roles for the Mre11/Rad50 complex, SAE2, NEJ1, and the Ku complex in repair of excision sites. Separation-of-function alleles of MRE11 suggest that its endonuclease function is more important in this repair than either its exonuclease or Rad50-binding properties. In addition, the interstrand cross-link repair gene PSO2 plays a role in end joining hairpin ends that is not seen in repair of linearized plasmids and may be involved in positioning transposase cleavage at the transposon ends.     

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.     

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.