Selection for enhanced germinal excision of Ac in transgenic Arabidopsis thaliana

Gene tagging in Arabidopsis thaliana using the autonomous Ac (Activator) transposable element has so far been hampered by low frequencies of germinal transposition events. Here we describe a procedure by which the frequency of independent germinal reinsertions has been much improved by a process of long-term selection on kanamycin for the continued growth of tissues in which somatic excisions have occurred. Growth on artificial media increased the somatic excision frequency, and the long-term selection procedure channelled somatic transposition events into the germline. This resulted in an overall germinal excision frequency in the progeny of longterm selected plants of 15%, as confirmed by Southern blotting, with 63% of the plants bearing excision events having detectable reinsertions of the Ac element. This compares with a germinal excision frequency of approximately 1% when no long-term selection is employed. However, offspring from individual plants tended to have identical germinal Ac reinsertion patterns, thus the critical parameter for evaluating the system for tagging purposes is the frequency of individual plants yielding offspring with reinsertions, which was 64%. This high frequency, when coupled to the enhanced germinal transposition rate overall, easily allows the generation of a large population of plants with independent reinsertions.     

The<Emphasis Type="Italic"> HY2</Emphasis> gene as an efficient marker for transposon excision in<Emphasis Type="Italic"> Arabidopsis</Emphasis>

Transposable elements can generate germinal and somatic mutations, and hence represent a powerful tool for the analysis of gene function. Transposons from maize have been adapted to mutagenise the genomes of diverse species. The efficiency of these systems partly relies on the ease with which germinal (i.e. germinally transmitted) or somatic excisions can be detected. Here we describe the use of HY2, a gene that codes for an enzyme involved in the biosynthesis of the phytochrome chromophore, to monitor the excision of a Ds gene-trap element in Arabidopsis thaliana. Taking advantage of the altered germination and de-etiolation behaviour of a Ds -tagged hy2 mutant, we have designed an efficient protocol for the recovery of germinal revertants, making HY2 the most precocious excision marker available, to the best of our knowledge. In addition, HY2 is also useful for generating visible sectors in photosynthetic tissues, thanks to the somatic instability of this mutable hy2 allele.Communicated by M.-A. Grandbastien     

Developmental and genetic aspects of Mutator excision in maize

The regulation of excision of Mu elements of the Mutator transposable element family of maize is not well understood. We have used somatic instability of Mu receptor elements from the Bronze 1 and Bronze 2 loci to monitor the frequency and the timing of excision of Mu elements in several tissues. We show that spot size in the aleurone of a bz2::mu1 stock varies between one to approximately 256 cells. This indicates that excision events begin eight divisions prior to full aleurone differentiation and end after the last division of the aleurone. We show that excision is equally biased for late events in all other tissues studied. A locus on chromosome 5 has been identified that affects spot size, possibly by altering the timing of Mu excision. Using somatic excision as an assay of Mutator activity, we found that activity can change in small sectors of the tassel; however, there are no overall activity changes in the tassel during the period of pollen shedding. We also report the recovery of germinal revertants for the bz1::mu1 and bz2::mu1 alleles. One of these revertant alleles was characterized by Southern blot analysis and found to be similar to the progenitor of the mutable allele.     

Somatic excision of the Mu1 transposable element of maize.

The Mu transposons of the Robertsons's Mutator transposable element system in maize are unusual in many respects, when compared to the other known plant transposon systems. The excision of these elements occurs late in somatic tissues and very rarely in the germ line. Unlike the other plant transposons, there is no experimental evidence directly linking Mu element excision and integration. We have analyzed the excision products generated by a Mu1 transposon inserted into the bronze 1 locus of maize. We find that the excision products or 'footprints' left by the Mu1 element resemble those of the other plant transposable elements, rather than those of the animal transposable element systems. We also find some novel types of footprints resembling recombinational events. We suggest that the Mu1 element can promote intrachromosomal crossovers and conversions near its site of insertion, and that this may be another mechanism by which transposons can accelerate the evolution of genomes.     

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.     

Post-integration behavior of a Minos transposon in the malaria mosquito Anopheles stephensi

Transposable elements represent important tools to perform functional studies in insects. In Drosophila melanogaster, the remobilization properties of transposable elements have been utilized for enhancer-trapping and insertional mutagenesis experiments, which have considerably helped in the functional characterization of the fruitfly genome. In Anopheles mosquitoes, the sole vectors of human malaria, as well as in other mosquito vectors of disease, the use of transposons has also been advocated to achieve the spread of anti-parasitic genes throughout field populations. Here we report on the post-integration behavior of the Minos transposon in both the germ-line and somatic tissues of Anopheles mosquitoes. Transgenic An. stephensi lines developed using the piggyBac transposon and expressing the Minos transposase were tested for their ability to remobilize an X-linked Minos element. Germ-line remobilization events were not detected, while somatic excisions and transpositions were consistently recovered. The analysis of these events showed that Minos activity in Anopheles cells is characterized by unconventional functionality of the transposon. In the two cases analyzed, re-integration of the transposon occurred onto the same X chromosome, suggesting a tendency for local hopping of Minos in the mosquito genome. This is the first report of the post-integration behavior of a transposable element in a human malaria vector. Christina Scali and Tony Nolan contributed equally to the work.     

Plant transposable elements generate the DNA sequence diversity needed in evolution

Two germinal and 16 somatic reversion events induced by the Enhancer (En) transposable element system at the wx-8::Spm-I8 allele of Zea mays were cloned and studied by sequence analysis. Excision of the Spm-I8 receptor element from the wx gene results in various mutant DNA sequences. This leads to altered gene products, some of which are still capable of restoring the wild-type phenotype. Possible 'foot-print' sequences that may have arisen by the excision of transposable elements were observed when intron sequences of the wild-type (wx+) and mutant (wx-m8) alleles of the wx gene were compared. The sequence divergence generated by visitation of a locus by plant transposable elements is discussed with respect to the molecular evolution of the new gene functions.     

The maize autonomous element Activator (Ac) shows a minimal germinal excision frequency of 0.2%–0.5% in transgenic Arabidopsis thaliana plants

Summary The autonomous mobile element Activator from Zea mays was introduced into Arabidopsis thaliana via Agrobacterium-mediated gene transfer. The use of a chimaeric construct, where the Ac element is located in the leader of the neomycin phosphotransferase (NPT II) gene, enabled the excision of Ac to be monitored by assaying for the reconstitution of NPT II gene activity. Using this approach, the transpositional activity of AC was initially studied in primary transformants. About 50% of the regenerating Ac transformants showed evidence for excision of the element. Reintegration of Ac was confirmed by Southern blot analysis. Transposition events are transmitted to the F1 generation with a minimal frequency of 0.3%. In a few exceptional cases they are detected in a high proportion of the F1 generation. Seedlings from the F2 and F3 generations were assayed for the rate of germinal excisions by scoring for kanamycin resistance. The minimal frequency of germinal excision events amounts to 0.2%–0.5% and hence allows the use of the Ac element for gene tagging purposes in A. thaliana.     

Factors affecting the excision frequency of the maize transposable element Ds in Arabidopsis thaliana

A two-element transposon system based on the maize elements Ac and Ds is currently being used for insertional mutagenesis in Arabidopsis. With the aim of making this system as efficient as possible we have continued to analyse several parameters which affect Ds activity in Arabidopsis. The influence of genomic position on Ds excision has been analysed in five lines carrying Ds integrated in different genomic locations. Differences in both somatic and germinal excision were observed between the different lines. The relationship between somatic and germinal excision, the timing of excision events and environmental influences on transposition frequency have been investigated. The effect of varying dosage of the different elements was also analysed. A strong positive dosage effect was observed for the transposase source, but not for the Ds element. Analysis of germinal excision events showed that the majority of them occurred very late in the development of the plant, resulting in the majority of Ds transpositions being independent events.     

Molecular and functional characterization of Slide, anAc-like autonomous transposable element from tobacco

A new transposable element of tobacco, Slide, was isolated from thetl mutant line, which shows somatic instability, after its transposition into a locus encoding nitrate reductase (NR). The Slide-124 element is 3733 bp long and its coding sequences show similarities with conserved domains of the transposases ofAc, Tam3 andhobo. Excision from the NR locus is detectable in somatic leaf tissues and Slide mobility is triggered by in vitro tissue culture. Slide excision events create footprints similar to those left byAc and Tam3. Tobacco lines derived from thetl mutant line seem characterized by unmethylated copies of a few members of the highly repetitive Slide family. Slide mobility was monitored in transient expression assays. In wild-type tobacco protoplasts, the complete Slide element, as well as a defective copy, is able to excise. The complete Slide element, but not the defective version, is able to excise in protoplasts of the heterologous species lettuce (Lactuca sativa). These results show that Slide carries the functions required for its own mobility, and represents the first autonomousAc-like element characterized inSolanaceae species.     

Modified P Elements That Mimic the P Cytotype in Drosophila Melanogaster

Activity of the P family of transposable elements in Drosophila melanogaster is regulated primarily by a cellular condition known as P cytotype. It has been hypothesized that P cytotype depends on a P element-encoded repressor of transposition and excision. We provide evidence in support of this idea by showing that two modified P elements, each with lesions affecting the fourth transposase exon, mimic most of the P cytotype effects. These elements were identified by means of two sensitive assays capable of detecting repression by a single P element. One assay makes use of cytotype-dependent gene expression of certain P element insertion mutations at the singed bristle locus. The other measures suppression of transposase activity from the unusually stable genomic P element, delta 2-3(99B), that normally produces transposase in both germinal and somatic tissues. The P cytotype-like effects include suppression of snw germline hypermutability, snw somatic mosaicism, pupal lethality, and gonadal dysgenic sterility. Unlike P cytotype, however, there was no reciprocal cross effect in the inheritance of repression.     

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.     

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.     

Increased Ac excision (iae): Arabidopsis thaliana mutations affecting Ac transposition

The maize transposable element Ac is highly active in the heterologous hosts tobacco and tomato, but shows very much reduced levels of activity in Arabidopsis . A mutagenesis experiment was undertaken with the aim of identifying Arabidopsis host factors responsible for the observed low levels of Ac activity. Seed from a line carrying a single copy of the Ac element inserted into the streptomycin phosphotransferase (SPT) reporter fusion, and which displayed typically low levels of Ac activity, were mutagenized using gamma rays. Nineteen mutants displaying high levels of somatic Ac activity, as judged by their highly variegated phenotypes, were isolated after screening the M₂ generation on streptomycin-containing medium. The mutations fall into two complementation groups, iae1 and iae2 , are unlinked to the SPT::Ac locus and segregate in a Mendelian fashion. The iae1 mutation is recessive and the iae2 mutation is semi-dominant. The iae1 and iae2 mutants show 550- and 70-fold increases, respectively, in the average number of Ac excision sectors per cotyledon. The IAE1 locus maps to chromosome 2, whereas the SPT:: Ac reporter maps to chromosome 3. A molecular study of Ac activity in the iae1 mutant confirmed the very high levels of Ac excision predicted using the phenotypic assay, but revealed only low levels of Ac re-insertion. Analyses of germinal transposition in the iae1 mutant demonstrated an average germinal excision frequency of 3% and a frequency of independent Ac re-insertions following germinal excision of 22%. The iae mutants represent a possible means of improving the efficiency of Ac/Ds transposon tagging systems in Arabidopsis , and will enable the dissection of host involvement in Ac transposition and the mechanisms employed for controlling transposable element activity.     

Somatic and Germinal Mobility of the RescueMu Transposon in Transgenic Maize

RescueMu, a Mu1 element containing a bacterial plasmid, is mobilized by MuDR in transgenic maize. Somatic excision from a cell-autonomous marker gene yields >90% single cell sectors; empty donor sites often have deletions and insertions, including up to 210 bp of RescueMu/Mu1 terminal DNA. Late somatic insertions are contemporaneous with excisions, suggesting that "cut-and-paste" transposition occurs in the soma. During reproduction, RescueMu transposes infrequently from the initial transgene array, but once transposed, RescueMu is suitable for high throughput gene mutation and cloning. As with MuDR/Mu elements, heritable RescueMu insertions are not associated with excisions. Both somatic and germinal RescueMu insertions occur preferentially into genes and gene-like sequences, but they exhibit weak target site preferences. New insights into Mu behaviors are discussed with reference to two models proposed to explain the alternative outcomes of somatic and germinal events: a switch from somatic cut-and-paste to germinal replicative transposition or to host-mediated gap repair from sister chromatids.     

Molecular Analysis of the Maize Wx-B3 Allele Indicates That Precise Excision of the Transposable Ac Element Is Rare

The somatic and germinal behavior of the maize wx-B3 mutation indicates that this Ac allele rarely reverts. Endosperms containing wx-B3 display tiny and infrequent Wx revertant sectors while no significant reversion is detected when wx-B3 pollen is stained with I/KI. Previous studies of other transposable element alleles that revert infrequently have implicated low levels of element excision. Unlike these other alleles, the wx-B3 Ac element is indistinguishable from fully active Ac elements with respect to its structure, and its ability to transpose from the Wx gene or to trans-activate a Ds element. Characterization of somatic and germinal excision events lead us to conclude that excision of the wx-B3 Ac element almost always produces null alleles. Furthermore, the excellent correlation between the position of the wx-B3 mutation on the physical and genetic maps indicates that the Ac insertion is the only lesion of wx-B3. As a result, precise excision of this Ac should restore Wx function. The fact that revertant sectors and pollen grains are rare indicates that precise excision of Ac is also rare. The finding that the wx-B3 reversion frequency is comparable whether wx-B3 is hemizygous or over a wx allele with a wild-type insertion site illustrates a fundamental difference between the excision mechanisms of Ac and Drosophila P elements.     

Construction of an inducible transposon,INAc, to develop a gene tagging system in higher plants

An inducible transposable element, termed INAc (inducible Activator), was constructed for development of a gene tagging system in higher plants. The advantage of such an inducible element is that, unlike the native transposon, its excision can be induced at any time during plant development and the resulting mutants are stable after removal of the inducer. A fusion of the SA inducible promoter (PR-1a) with the Ac transposase gene was inserted together with a hygromycin resistance gene between ca. 400 bp sequences from each end of the maize Ac element, yielding INAc. The INAc element was introduced into tobacco and tomato plants. A high frequency of spontaneous transposition was apparent in primary transformed tomato calli but not in tobacco calli. Treatment of tobacco plants with salicylic acid induced transposition of INAc in both somatic and germinal tissue, with germinal transposition events being revealed by characterization of the progeny of transformed plants whose flowers were exposed to SA. The INAc element thus exhibits potential for development of an inducible transposon system suitable for gene isolation in heterologous plant species.     

Transactivation of Ds elements in plants of lettuce (Lactuca sativa)

The maize transposable element, Activator (Ac), is being used to develop a transposon mutagenesis system in lettuce, Lactuca sativa. In this paper, we describe somatic and germinal transactivation of Ds by chimeric transposase genes in whole plants. Constructs containing either the Ds element or the Ac transposase open reading frame (ORF) were introduced into lettue. The Ds element was located between either the 35S or the Nos promoter and a chimeric spectinomycin resistance gene (which included a transit peptide), preventing expression of spectinomycin resistance. The genomic coding region of the Ac transposase was expressed from the 35S promoter. Crosses were made between 104 independent R₁ plants containing Ds and three independent R₁ plants expressing transposase. The excision of Ds in F₁ progenies was monitored using a phenotypic assay on spectinomycin-containing medium. Green sectors in one-third of the F₁ families indicated transactivation of Ds by the transposase at different developmental stages and at different frequencies in lettuce plants. Excision was confirmed using PCR and by Southern analysis. The lack of green sectors in the majority of F₁ families suggests that the majority of T-DNA insertion sites are not conducive to excision. In subsequent experiments, the F₁ plants containing both Ds and the transposase were grown to maturity and the F₂ seeds screened on medium containing spectinomycin. Somatic excision was again observed in several F₂ progeny; however, evidence for germinal excision was observed in only one F₂ family.