Studies on the introduction and mobility of the maize Activator element in Arabidopsis thaliana and Daucus carota.

We have co-transformed carrot (Daucus carota) and Arabidopsis thaliana with an Agrobacterium tumefaciens non-tumorigenic T-DNA carrying the maize transposable element Activator (Ac) and an Agrobacterium rhizogenes Ri T-DNA. We present evidence that the Ac element transposes in transformed root or root-derived callus cultures of both species. We show that fertile plants can be regenerated from transformed, root-derived callus cultures of Arabidopsis, demonstrating the utility of the Ri plasmid for introducing the maize Ac element into plants. We also present evidence that Ac elements that excise from the transforming T-DNA early after transformation continue to be mobile in carrot root cultures.     

Rapid proliferation of the maize transposable element Activator in transgenic tomato.

We have found that the maize transposable element Activator (Ac) can rapidly proliferate when transformed into tomato plants. The fate of transposed Ac elements in self-pollinated progeny of independent transgenic tomato plants was examined by DNA gel blot hybridizations. When a single copy of Ac was introduced into a transformant, the number of copies usually remained low in subsequent generations. In one lineage, however, the number of Ac elements increased from one to more than 15 copies in only two generations. DNA gel blot analyses indicated that the amplified elements were not grossly rearranged. Amplified copies of Ac resided at unique sites in the genome, and segregation analysis indicated that these sites were not tightly linked at one genetic locus. Taken together, these observations indicate that the mechanism of Ac amplification is associated with transposition.     

Behavior of the maize transposable element Activator in Daucus carota

Summary We previously described the introduction of the maize Ac element in carrot hairy roots where it was shown to transpose. Further studies on this system allowed us to observe complete excision of the element from its original insertion site on the AcT-DNA. Analysis of hairy root subcultures derived from single root tips resolved the chimeric status created by transposition of Ac establishing the unicellular origin of secondary root meristems. Studies with restriction enzymes revealed a specific methylation pattern in the vicinity of the empty donor site left after Ac excision. This process, although not of general occurrence, was probably associated with the excision/transposition process. The activity of Ac in different hairy root transformants as judged by the rate of excision from the original insertion site appeared to be maintained during a period of ca. 2 years, and there was no evidence for bursts of transposition occurring during somatic embryogenesis. The data obtained support the hypothesis that excision is a concerted process involving all the copies of Ac present in a given cell.     

A maize cryptic Ac-homologous sequence derived from an Activator transposable element does not transpose.

Summary Sequences sharing homology to the transposable element Activator (Ac) are prevalent in the maize genome. A cryptic Ac-like DNA, cAc-11, was isolated from the maize inbred line 4Co63 and sequenced. Cryptic Ac-11 has over 90% homology to known Ac sequences and contains an 11 by inverted terminal repeat flanked by an 8 by target site duplication, which are characteristics of Ac and Dissociation (Ds) transposable elements. Unlike the active Ac element, which encodes a transposase, the corresponding sequence in cAc-11 has no significant open reading frame. A 44 by tandem repeat was found at one end of cAc-11, which might be a result of aberrant transposition. The sequence data suggest that cAc-11 may represent a remnant of an Ac or a Ds element. Sequences homologous to cAc-11 can be detected in many maize inbred lines. In contrast to canonical Ac elements, cAc-11 DNA in the maize genome is hypermethylated and does not transpose even in the presence of an active Ac element.     

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.     

Nucleotide sequence of the maize transposable element Mul

A cloned DNA fragment from the maize allele Adhl-S3034 contains all of Mul, an insertion element involved in Robertson's Mutator activity. The element is 1367 base pairs (bp) long and is flanked by nine bp direct repeats of insertion site DNA. It has inverted terminal repeats of 215 and 213 bp showing 95% homology. Within the element are two direct repeats of 104 bp showing 96% homology. Four open reading frames (ORFs) were found, two in each DNA strand. Mul can be divided into two halves, each containing one terminal inverted repeat, an internal direct repeat, and two overlapping ORFs. The GC content of each half is high (70%), while that of a central 60 base portion of the element is low (26%). The central region contains the only sequence resembling the TAATA Goldberg and Hogness eukaryotic promoter signal. Multiple copies of DNA sequences related to Mul found in Mutator maize plants are generally similar in organization to the cloned element. A larger version containing a discrete 300 to 400 base pair insertion was found in some Mutator lines.     

Analysis of splice donor and acceptor site function in a transposable gene trap derived from the maize element Activator

Gene trap vectors have been used in insertional mutagenesis in animal systems to clone genes with interesting patterns of expression. These vectors are designed to allow the expression of a reporter gene when the vector inserts into a transcribed region. In this paper we examine alternative splicing events that result in the expression of a GUS reporter gene carried on a Ds element which has been designed as a gene trap vector for plants. We have developed a rapid and reliable method based on PCR to study such events. Many splice donor sites were observed in the 3′ Ac border. The relative frequency of utilisation of certain splice donor and acceptor sites differed between tobacco and Arabidopsis. A higher stringency of splicing was observed in Arabidopsis.     

R-stippled maize as a transposable element system

The I-R element at the R locus destabilizes kernel pigmentation giving the variegated pattern known as stippled ( R-st). In trans linkage phase with R-st the element was shown to act as a modifier of stippled, intensifying seed spotting in parallel with effects of the dominant linked modifier M-st. Presence of I-R in the genome was, therefore, shown to be detectable as a modifier of R-st. When this test was used, new modifiers resembling M-st were often detected following mutations of R-st to the stable allele R-sc. Such mutations evidently occurred by transposition of I-R away from the R locus to a site where it was identifiable as a modifier. M-st may be such a transposed I-R. Analysis of mutations to R-sc during the second (sperm-forming) mitosis in pollen grains showed that some of the transposed I-R elements were linked with R, whereas others assorted independently. Their strengths varied from barely discernible to a level equal to M-st. Overreplication frequently accompanied transposition at the sperm-forming mitosis, leading to transposed I-R elements in both the mutant and nonmutant sperm.     

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     

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.     

The generation of Mutator transposable element subfamilies in maize

The mobile DNAs of the Mutator system of maize (Zea mays) are exceptional both in structure and diversity. So far, six subfamilies of Mu elements have been discovered; all Mu elements share highly conserved terminal inverted repeats (TIRs), but each sub-family is defined by internal sequences that are apparently unrelated to the internal sequences of any other Mu subfamily. The Mu1/Mu2 subfamily of elements was created by the acquisition of a portion of a standard maize gene (termed MRS-A) within two Mu TIRs. Beside the unusually long (185–359 bp) and diverse TIRs found on all of these elements, other direct and inverted repeats are often found either within the central portion of a Mu element or within a TIR.Our computer analyses have shown that sequence duplications (mostly short direct repeats interrupted by a few base pairs) are common in non-autonomous members of the Mutator, Ac/Ds, and Spm(En) systems. These duplications are often tightly associated with the element-internal end of the TIRs. Comparisons of Mu element sequences have indicated that they share more terminal components than previously reported; all subfamilies have at least the most terminal 215 bp, at one end or the other, of the 359-bp Mu5 TIR. These data suggest that many Mu element subfamilies were generated from a parental element that had termini like those of Mu5. With the Mu5 TIRs as a standard, it was possible to determine that elements like Mu4 could have had their unusual TIRs created through a three-step process involving (1) addition of sequences to interrupt one TIR, (2) formation of a stem-loop structure by one strand of the element, and (3) a subsequent DNA repair/gene conversion event that duplicated the insertion(s) within the other TIR. A similar repair/conversion extending from a TIR stem into loop DNA could explain the additional inverted repeat sequences added to the internal ends of the Mu4 and Mu7 TIRs. This same basic mechanism was found to be capable of generating new Mu element subfamilies. After endonucleolytic attack of the loop within the stem-loop structure, repair/conversion of the gap could occur as an intermolecular event to generate novel internal sequences and, therefore, a new Mu element subfamily. Evidence supporting and expanding this model of new Mu element subfamily creation was identified in the sequence of MRS-A.     

Transposition of the maize transposable element dSpm in transgenic sugar beets

Transgenic plants and cell lines of sugar beet carrying Spm/dSpm system of maize transposable elements have been obtained by Agrobacterium-mediated transformation. A heterologous system of mobile elements Spm/dSpm remains active in the genome of sugar beet that permit of transposon-based gene tagging and obtaining of marker-free transgenic sugar beet.