Behaviour of the maize transposable element Ac in Arabidopsis thaliana

The somatic and germinal activity of the maize transposable element, Ac, has been analysed in progeny of 43 transformants of A. thaliana using a streptomycin resistance assay to monitor Ac excision. The ability to assay somatic activity enabled, for the first time, a detailed analysis of Ac activity in individual A. thaliana seedlings to be made. The effects of T-DNA copy number, generation, dosage at each locus, flanking sequences and orientation of the element were compared. The most striking observation was the variability in Ac activity in genotypically identical individuals and the poor penetrance of the variegated phenotype. In general, increasing Ac dosage increased both somatic and germinal excision frequencies. The majority of families from individuals selected as inheriting an excision event carried transposed Ac elements re-integrated in different positions in the genome.     

Excision of the maize transposable element Ac in flax callus

The frequency and fidelity of Ac transposition, and that of its non-autonomous derivative Ds, were investigated in flax callus. Flax (Linum usitatissimum var. Antares) hypocotyls were transformed with Agrobacterium Ti plasmid vectors containing the Ac or Ds element inserted within the untranslated leader sequence of a chimaeric neomycin phosphotransferase II gene. Kanamycin resistant tissues were produced as a result of excision of Ac in around 35% of the total number of Ac-containing transformants. In contrast, no excision was observed from transformants containing the Ds element. Whilst Ac appears to have excised completely from T-DNAs, little evidence was found to infer reintegration of the Ac element into the genome.Abbreviations NPT-II/npt-II Neomycin phosphotransferase II - kb Kilobasepairs - bp basepairs - MSO Murashige and Skoog medium - NAA naphthalene acetic acid - BAP 6-benzylaminopurine     

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.     

The maize transposable element Ac is mobile in the legume Lotus japonicus

To evaluate the prospects for transposon mutagenesis in the autogamous diploid legume Lotus japonicus, the behaviour of the maize transposable element Ac was analysed in the progeny of 38 independent transgenic plants. The conditions for monitoring donor site excision using histochemical localization of -glucuronidase activity or the alternative spectinomycin resistance assay were established, and used to follow Ac mobility through two generations. Somatic excision was monitored as variegated cotyledons in the T₂ generation and germinal excision events were scored in segregating T₃ families as complete -glucuronidase-mediated staining of cotyledons or as a fully green spectinomycin-resistant phenotype. Using these assays an average germinal excision frequency of 12% was estimated in the T₃ offspring from variegated plants. The fidelity of the excision assays was ascertained by comparing the frequency of germinal excision to the frequency of Ac reinsertion at new positions of the genome. Transposition of Ac in 42% of the plants and detection of the characteristic Ac insertion/excision footprints suggests that insertion mutagenesis with the autonomous maize Activator element is feasible in Lotus japonicus. Parameters influencing Ac behaviour, such as dosage, position effects and modification of the element itself, were also investigated comparing homozygous and hemizygous plants from the same family and by analysing different transformants.Abbreviations W white - V variegated - FG fully green - FB fully blue - aadA spectinomycin adenyltransferase     

Overproduction of the protein encoded by the maize transposable element Ac in insect cells by a baculovirus vector

Summary The polypeptide encoded in the Activator (Ac) element of Zea mays L. has been expressed in Spodoptera frugiperda insect cells using plasmids which carry the strong polyhedrin promoter of the baculovirus Autographa californica nuclear polyhedrosis virus (AcNPV). Recombinant AcNPVs with the Ac-cDNA integrated and under the control of the viral polyhedrin promoter have been isolated and their genomes have been partly characterized as to the location of the foreign DNA insert. Upon infection of S. frugiperda cells with the recombinant AcNPV, maize Ac element specific messenger RNAs, as well as a newly synthesized polypeptide with an apparent molecular weight of about 116 kDa, have been detected in extracts of recombinant infected cells. This polypeptide is absent from extracts of wild-type infected cells expressing the polyhedrin polypeptide which can be recognized by the presence of nuclear inclusion bodies. Recombinant infected cells lack this protein. The Ac specific polypeptide is detected by antisera, which have been raised against fusion proteins containing Ac sequences synthesized in Escherichia coli, both in immunoprecipitation and in Western blotting experiments. The Ac specific protein is a nuclear phosphoprotein and represents about 1%–2% of the newly synthesized protein.     

Transposition of the maize transposable element Ac in barley (Hordeum vulgare L.)

Transposition of the maize autonomous element Ac (Activator) was investigated in barley (Hordeum vulgare L.) with the aim of developing a transposon tagging system for the latter. The Ac element was introduced into meristematic tissue of barley by microprojectile bombardment. Transposon activity was then examined in the resulting transgenic plants. Multiple excision events were detected in leaf tissue of all plant lines. The mobile elements generated empty donor sites with small DNA sequence alterations, similar to those found in maize. Reintegration of Ac at independent genomic loci in somatic tissue was demonstrated by isolation of new element-flanking regions by AIMS-PCR (amplification of insertion-mutagenized sites). In addition, transmission of transposed Ac elements to progeny plants was confirmed. The results indicate that the introduced Ac element is able to transpose in barley. This is a first step towards the establishment of a transposon tagging system in this economically important crop.     

Excision of the maize transposable element Ac in periclinal chimeric leaves of 35S-Ac-rolC transgenic aspen-Populus

The transposable element Ac from maize, in combination with the phenotypic selectable marker rolC, was employed in transformation experiments of a hybrid aspen clone. A number of transgenic clones exhibited light-green sectors on green leaves. In vitro regeneration from leaves showing a high number of light-green spots resulted in R2 plants, which also showed light-green sectored leaves. However, only one out of 385 regenerated plants obtained showed green leaves. Both PCR and northern analysis indicated Ac excision and restoration of rolC expression. In Southern blot analysis of this green plant additional bands were observed as compared to the original R1 plant. The occurrence of these bands and a suggested Ac excision in the non-green L1-epidermal layer leading to periclinal chimerism of this plant is discussed.     

Introduction and transposition of the maize transposable element Ac in rice (Oryza sativa L.).

Summary To develop a transposon tagging system in an important cereal plant, rice (Oryza sativa L.), the maize transposable element Ac (Activator) was introduced into rice protoplasts by electroporation. We employed a phenotypic assay for excision of Ac from the selectable hph gene encoding resistance to hygromycin B. Southern blot analysis of hygromycin B-resistant calli showed that the Ac element can transpose from the introduced hph gene into the rice chromosomes. Sequence analysis of several Ac excision sites in the hph gene revealed sequence alterations characteristic of the excision sites of this plant transposable element. The Ac element appears to be active during development of transgenic rice plants from calli. Moreover, hybridization patterns of different leaves from the same plant indicated that some Ac elements are stable whereas others are able to transpose further during development of leaves. The results indicate that the introduced Ac element can transpose efficiently in transgenic rice plants.     

Establishment of a gene tagging system in Arabidopsis thaliana based on the maize transposable element Ac

Summary An Ac-derived, two-component transposable element system has been developed and analyzed with respect to its use in Arabidopsis thaliana. This system consists of an immobilized Ac element (Ac clipped wing, Ac_cl) as the source of transactivating transposase and a nonautonomous Ds element, Ds_A, which is inserted into a chimaeric neomycinphosphotransferase gene used as excision marker. After separate introduction of Ac_c1 and Ds_A into Arabidopsis thaliana, progeny analysis of crosses between five different Ac_cl lines and seven different Ds_A lines shows that: (1) different Ac_cl lines differ greatly in their capacity to transactivate Ds_A; (2) different Ds_A lines do not differ significantly with respect to Ds_A transactivation by one Ac_cl line; (3) reintegration of excised Ds_A elements, both at (genetically) linked and unlinked sites, occurs in about 50% of the excision events; and (4) plants with a high rate of somatic excisions can be used as source of new Ds_A transpositions, allowing the creation of a large number of independent Ds_A insertions.     

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.