The Arabidopsis transposable element Tag1 is widely distributed among Arabidopsis ecotypes

Tag1 is an autonomous transposable element (3.3 kb in length) first identified as an insertion in the CHL1 (NRT1) gene of Arabidopsis thaliana. Tag1 has been found in the Landsberg erecta ecotype of A. thaliana but not in Columbia or WS. In this paper, 41 additional ecotypes were examined for the presence of Tag1. Using an internal Tag1 fragment as probe, we found that DNA from 19 of the 41 ecotypes strongly hybridized to Tag1. Almost all of the Tag1-containing ecotypes had only one or two copies of Tag1 per haploid genome, as determined by Southern blot analysis. The only exception, Bf-1 from Bretagny-sur-Orge, France, had four copies. Two ecotypes, Di-G and S96, gave identical Southern blot patterns to that of Landsberg erecta and were subsequently shown to contain Tag1 at the same two positions found in Landsberg erecta (loci designated as Tag1-2 and Tag1-3). Two other ecotypes, Ag-0 and Lo-1, had a Tag1 element located at Tag1-2 but not at Tag1-3. The distance between these two loci was determined to be 0.37 cM. Analysis of DNA from two related species, A. griffithiana and A. pumila, showed that both species contain sequences that hybridize to Tag1 and that could be amplified with an oligonucleotide specific to the terminal inverted repeats of Tag1. These results show that Tag1 and related elements are present, and may be useful for insertional mutagenesis, in many A. thaliana ecotypes and several Arabidopsis species.     

Characterization of the germinal and somatic activity of the Arabidopsis transposable element Tag1.

Tag1 is an autonomous transposon of Arabidopsis thaliana. The excision behavior of Tag1 during reproductive and vegetative development was examined using CaMV 35STag1-GUS constructs. Germinal reversion frequencies varied from 0 to 27% and correlated with Tag1 copy number. Southern blot and somatic sector analyses indicated that each revertant was derived from an independent excision event, and approximately 75% of the revertants had new Tag1 insertions. Revertants were obtained with similar frequencies from the male and female parents. In flowers, small somatic sectors were observed in siliques, carpels, petals and sepals while stemlike organs (filaments and pedicels) had larger sectors. No sectors encompassing entire flowers or inflorescences were observed, however, indicating that excision occurs late in flower development and rarely in inflorescence meristems. Late excision was also observed during vegetative development with 99.8% of leaves showing small sectors encompassing no more than 20 cells. Roots and cotyledons, however, showed larger sectors that included entire lateral roots and cotyledons. These results indicate that Tag1 can excise in the embryo and all the organs of the plant with the timing of excision being restricted to late stages of vegetative and reproductive development in the shoot.     

Engineering variegated floral patterns in tobacco plants using the Arabidopsis transposable element Tag1

Variegated flower phenotypes were generated using the Arabidopsis transposon Tag1 and the maize R regulatory gene. Tag1 was inserted between the CaMV 35S promoter and the maize R gene and transformed into tobacco plants. In half of the transgenic plants, variegated flower patterns were observed. Each line had a different pattern, with varying intensities with three lines showing only tiny sectors indicative of late excision and one showing large sectors indicative of earlier excision.     

High-frequency excision of transposable element Tc 1 in the nematode Caenorhabditis elegans is limited to somatic cells

Tc 1 transposable elements in the nematode Caenorhabditis elegans undergo excision at high frequency. We show here that this excision occurs primarily or entirely in the somatic tissues of the organism. Absence of germ-line excision is demonstrated by showing that Tc 1 elements are genetically stable; elements at particular genomic sites, as well as the overall number of elements in the genome, were stably maintained during a year of continuous, nonselective propagation. Somatic excision is demonstrated by showing that empty Tc 1 sites arise during a single generation of growth of a synchronous population and are not inherited by the next generation. These results suggest that excision of Tc 1 elements is under the control of tissue-specific factors.     

Insertion and excision of Caenorhabditis elegans transposable element Tc1.

The transposable element Tc1 is responsible for most spontaneous mutations that occur in Caenorhabditis elegans variety Bergerac. We investigated the genetic and molecular properties of Tc1 transposition and excision. We show that Tc1 insertion into the unc-54 myosin heavy-chain gene was strongly site specific. The DNA sequences of independent Tc1 insertion sites were similar to each other, and we present a consensus sequence for Tc1 insertion that describes these similarities. We show that Tc1 excision was usually imprecise. Tc1 excision was imprecise in both germ line and somatic cells. Imprecise excision generated novel unc-54 alleles that had amino acid substitutions, amino acid insertions, and, in certain cases, probably altered mRNA splicing. The DNA sequences remaining after Tc1 somatic excision were the same as those remaining after germ line excision, but the frequency of somatic excision was at least 1,000-fold higher than that of germ line excision. The genetic properties of Tc1 excision, combined with the DNA sequences of the resulting unc-54 alleles, demonstrated that excision was dependent on Tc1 transposition functions in both germ line and somatic cells. Somatic excision was not regulated in the same strain-specific manner as germ-line excision was. In a genetic background where Tc1 transposition and excision in the germ line was not detectable, Tc1 excision in the soma still occurred at high frequency.     

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.     

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.     

Germline excision of the transposable element Tc1 in C. elegans.

We have examined eight germline revertants generated by the excision of Tc1 from a site within the unc-22 gene of Caenorhabditis elegans. A rich variety of rearrangements accompanied Tc1 excision at this site, including transposon 'footprints', deletions of sequences flanking the insertion site and direct nontandem duplications of flanking DNA. With only modest modification the double-strand gap repair model for transposition, recently proposed by Engles and coworkers (Cell 62: 515-525 1990), can explain even the most complex of these rearrangements. In light of this model rearrangements of the target site accompanying transposition/excision may not be the end result of imprecise excision of the element. Instead, these rearrangements may be the result of imprecise repair of the double-strand gap by the host replication and repair machinery. Sequences surrounding an insertion site influence the fidelity of gap repair by this machinery. This may lead to a number of possible resolutions of a double-strand gap as documented here for a Tc1 site in unc-22.     

A non-autonomous insect piggyBac transposable element is mobile in tobacco

The piggyBac transposable element, originally isolated from a virus in an insect cell line, is a valuable molecular tool for transgenesis and mutagenesis of invertebrates. For heterologous transgenesis in a variety of mammals, transfer of the piggyBac transposable element from an ectopic plasmid only requires expression of piggyBac transposase. To determine if piggyBac could function in dicotyledonous plants, a two-element system was developed in tobacco (Nicotiana tabacum) to test for transposable element excision and insertion. The first transgenic line constitutively expressed piggyBac transposase, while the second transgenic line contained at least two non-autonomous piggyBac transposable elements. Progeny from crosses of the two transgenic lines was analyzed for piggyBac excision and transposition. Several progeny displayed excision events, and all the sequenced excision sites exhibited evidence of the precise excision mechanism characteristic of piggyBac transposase. Two unique transposition insertion events were identified that each included diagnostic duplication of the target site. These data indicate that piggyBac transposase is active in a dicotyledonous plant, although at a low frequency.     

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.     

The transposon Tip100 from the common morning glory is an autonomous element that can transpose in tobacco plants

The mutable flaked or a (flaked) (a(f)) line of the common morning glory (Ipomoea purpurea) displays white flowers with colored flakes, and the a(f) mutation is caused by the insertion of a transposable element named Tip100 into the CHS-D gene for anthocyanin biosynthesis. The 3.9-kb Tip100 element belongs to the Ac/Ds family and contains an ORF encoding a polypeptide of 808 amino acids. The frequency and timing of flower variegation vary in different a(f) lines, and a genetic element termed Modulator has been postulated to affect the variegation pattern. Since the pattern of flower variegation is determined by the frequency and timing of excision of Tip100 from the CHS-D gene, we wished to determine whether Tip100 is an autonomous element that is itself capable of transposition in a heterologous host. To do this, we introduced the element into the genome of tobacco plants by Agrobacterium-mediated transformation. The intact Tip100 element was able to excise from its original position in the chromosome and reinsert into new sites in the tobacco genome, whereas an internal deletion derivative was not. Based on these results, we conclude that Tip100 is an autonomous element. We also discuss the nature of the putative Modulator element affecting flower and leaf variegation in various mutable lines of the morning glory.     

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.     

The behaviour of the autonomous maize transposable element En/Spm in Arabidopsis thaliana allows efficient mutagenesis

The behavior of the autonomous maize transposable element En/Spm of maize was studied in Arabidopsis. Transgenic Arabidopsis plants carrying En-1 elements were propagated for 12 generations using a single seed descent procedure. The distribution and activity of the En-1 element was monitored using Southern DNA hybridisations in generations 1, 6 and 12. In the first generation the highest number of En-1 insertions per line was 7, which increased to 20 in generation 12. The average number of En-1 insertions increased only slightly in the population, due to a gradual accumulation of segregants that lost the transposable element. During the development of the En-1 mutagenised population the element remained active even in the high-copy lines. In situ hybridisation demonstrated that multiple En-1 insertions were distributed over all Arabidopsis chromosomes. From the initial En-1 mutagenised populations many unstable gene mutations were recovered, indicating that En-1 can be used as a efficient tool for gene tagging in Arabidopsis.     

Trans-activation of an artificial dTam3 transposable element in transgenic tobacco plants

In Antirrhinum majus only autonomous Tam3 transposons have been characterized. We investigated whether an artificial dTam3 element, with a deletion in the presumptive transposase coding region, can be trans-activated in tobacco by an activator Tam3 element, which was immobilized by the deletion of one inverted repeat. A phenotypic assay based on restored hygromycin resistance demonstrates that a dTam3 element harbouring a bacterial plasmid can be trans-activated with a low frequency. Molecular analysis confirms that the dTam3 element has been excised from the HPTII marker gene. Reintegration of the dTam3 element into the tobacco genome is detected only in one out of six hygromycin-resistant plants analysed. PCR analysis of empty donor sites shows that excision of the dTam3 element in tobacco results in rearrangements (deletions and additions), that have been shown to be characteristic of Tam3 excision in the original host Antirrhinum majus. This trans-activation assay allowed us to establish that, in contrast to what has been detected in Antirrhinum majus, a periodical temperature shift down to 15°C does not enhance dTam3 transposition in regenerating tobacco calli.     

Somatic excision of transposable element Tc1 from the Bristol genome of Caenorhabditis elegans.

We investigated the ability of the transposable element Tc1 to excise from the genome of the nematode Caenorhabditis elegans var. Bristol N2. Our results show that in the standard lab strain (Bristol), Tc1 excision occurred at a high frequency, comparable to that seen in the closely related Bergerac strain BO. We examined excision in the following way. We used a unique sequence flanking probe (pCeh29) to investigate the excision of Tc1s situated in the same location in both strains. Evidence of high-frequency excision from the genomes of both strains was observed. The Tc1s used in the first approach, although present in the same location in both genomes, were not known to be identical. Thus, a second approach was taken, which involved the genetic manipulation of a BO variant, Tc1(Hin). The ability of this BO Tc1(Hin) to excise was retained after its introduction into the N2 genome. Thus, we conclude that excision of Tc1 from the Bristol genome occurs at a high frequency and is comparable to that of Tc1 excision from the Bergerac genome. We showed that many Tc1 elements in N2 were apparently functionally intact and were capable of somatic excision. Even so, N2 Tc1s were prevented from exhibiting the high level of heritable transposition displayed by BO elements. We suggest that Bristol Tc1 elements have the ability to transpose but that transposition is heavily repressed in the gonadal tissue.