Molecular analysis of paramutant plants of Antirrhinum majus and the involvement of transposable elements

Paramutation is observed when the Antirrhinum majus lines 44 and 53 are crossed. These two lines both have insertions at the nivea locus, which encodes chalcone synthase (chs). The allele niv-53 carries the transposable element Tam1 in the promoter region of the chs gene; niv-44 carries the element Tam2 within the gene. The Tam1 element has previously been extensively characterised. Here the Tam2 element is further characterised, and the arrangement of the nivea locus in paramutant plants is analysed. The complete sequence of Tam2, and that of a partial cDNA complementary to it, have been determined. The cDNA is probably transcribed from a different copy of Tam2 from that present at the nivea locus, and does not encode a functional protein. Genomic Southerns of F1 plants from the 53/44 cross show that no major rearrangements are consistently associated with paramutation at the nivea locus of A. majus. The isolation from a paramutant plant arising from a 53/44 cross of an allele (niv-4432) resulting from the excision of Tam2 is reported. The excision of Tam2 resulted in a 32 bp deletion of chs gene sequences. Plants homozygous for the new niv-4432 allele have white flowers and are still paramutagenic, demonstrating that Tam2 need not be present at the nivea locus for paramutation to occur. Different interactions between Tam1 and Tam2 are discussed, and a possible model for paramutation is presented.     

Allelic interactions at the nivea locus of Antirrhinum.

Most null alleles at the nivea (niv) locus are recessive to Niv+ and, when homozygous, give white flowers rather than the red of the wild type. In contrast, the niv-571 allele is semidominant; although it gives white flowers when homozygous, very pale flowers result when this allele is heterozygous with NIV+. We showed that in heterozygotes, niv-571 acts in trans to inhibit expression of its Niv+ homology 25-fold to 50-fold. The inhibition is reversible after meiosis and partially reversible somatically. The niv-571 allele carries a transposable element Tam3 insertion and three truncated copies of the niv gene, one copy being in inverse orientation. Analysis of two further niv alleles, niv-572 and niv-527, showed that excision of Tam3 from niv-571 does not affect the ability of the allele to repress Niv+ and that one truncated niv copy alone is insufficient to confer semidominance. The detailed structures of various semidominant niv alleles suggest that their effects in trans are not readily explained by production of antisense RNA but are more easily reconciled with a direct recognition/interaction between homologous genes, reminiscent of cosuppression and transvection phenomena described in other systems.     

Pigmentation mutants produced by transposon mutagenesis in Antirrhinum majus

New pigmentation mutants were generated by transposon mutagenesis in Antirrhinum majus, in three previously described loci, nivea, delila and incolorata, and two new loci, daphne and olive. The wild-type olive gene is required for the production of dark-green leaves, and the daphne gene for the synthesis of flavones. Five out of the six mutants were both germinally and somatically unstable, indicating that they resulted from transposon insertions. Molecular analysis of the mutant at nivea (niv-600) showed that it was caused by insertion of a new transposon, Tam4. The sequence of Tam4 suggests that it is unable to transpose autonomously and that it is related to Tam1 and Tam2. All three of these transposons have identical inverted repeats, produce 3 bp target duplications, leave similar excision footprints and share at one end a 600-700 bp region containing many palindromic copies of a motif sequence, possibly required in cis for transposition. The somatic excision of Tam4 in niv-600 is at a very low rate compared to germinal excision but it can be activated by crossing to lines carrying derivative alleles of a Tam1 insertion at niv. Molecular analysis of four different pigmentation mutants has shown that insertions of Tam1, Tam2, Tam3 and Tam4 have been obtained, illustrating the potential of general transposon mutagenesis for trapping and isolating new transposons as well as for tagging genes.     

Large-scale chromosomal restructuring is induced by the transposable element tam3 at the nivea locus of antirrhinum majus

The transposable element, Tam3, gives rise to large-scale (greater than 1 kb) chromosomal rearrangements at a low frequency, when it is inserted at the nivea locus of Antirrhinum majus. Although some deletions may result from imprecise excision of Tam3, rearrangements involving deletion, dispersion and inverted duplication of flanking sequences, where Tam3 remains in situ, have also been identified. These rearrangements have been mapped at the molecular level, and the behavior of Tam3 following rearrangement has been observed. It is clear that Tam3 has enormous potential to restructure chromosomes through successive rounds of large-scale rearrangements. The mechanisms by which such rearrangements might arise are discussed.     

The 17-kb Tam1 element of Antirrhinum majus induces a 3-bp duplication upon integration into the chalcone synthase gene

The DNA sequence of the termini and the flanking regions of the 17-kb transposable element Tam1 was determined. Tam1 is integrated in the chalcone synthase gene of the niv-53 mutant of Antirrhinum majus. The element has a 13-bp perfect inverted repeat at its termini and appears to induce a 3-bp duplication of the target site upon integration. The DNA sequence of a niv⁺ revertant was analyzed and found to differ from the wild-type sequence by an additional 2 bp that seem to derive from the target site duplication. Stretches of homologous sequences have been found between the ends of Tam1, within each terminus of the element, and between the termini and target site sequences. Structural similarities between the ends of Tam1 and the Spm-18 element of Zea mays reflect a possible horizontal spread of a common progenitor.     

Genome juggling by transposons: Tam3-induced rearrangements in Antirrhinum majus

Transposable elements are well known for their ability to generate large- and small-scale rearrangements of the sequences flanking their insertion sites. These include deletions, inversions, and duplications. Tam3, a transposon from the Snapdragon (Antirrhinum majus), is highly active in the generation of such rearrangements. We have analysed a number of Tam3-induced rearrangements at the nivea (niv) locus by Southern blotting, cloning, and sequence determination. The data obtained from these analyses have led to an understanding of the mechanisms by which these complex alleles were formed. We have shown that the primary rearrangements usually occur without excision of the element and therefore result from aberrant transposition attempts. Subsequent rearrangements may occur on excision of the element. Finally, we suggest how the analysis of such rearrangements may not only provide information about Tam3 transposition but also show how transposon-induced rearrangements may influence the structure and function of the genome as a whole.     

Resistance to gap repair of the transposon Tam3 in Antirrhinum majus: a role of the end regions

The extremely homogeneous organization of the transposon family Tam3 in Antirrhinum majus is in sharp contrast to the heterogeneity of the copies constituting many other transposon families. To address the issue of the Tam3 structural uniformity, we examined two possibilities: (1) recent invasion of Tam3 and (2) failure of gap repair, which is involved in conversion from autonomous forms to defective forms. The phylogenetic analysis of 17 Tam3 copies suggested that the invasion of Tam3 into the Antirrhinum genome occurred at least 5 mya, which is sufficiently long ago to have produced many aberrant copies by gap repair. Thus, we investigated gap repair events at the nivea(recurrens:Tam3) (niv(rec)::Tam3) allele, where Tam3 is actively excised. We show here that the gap repair of de novo somatic Tam3 excision was arrested immediately after initiation of the process. All of the identified gap repair products were short stretches, no longer than 150 bp from the ends. The Tam3 ends have hairpin structures with low free energies. We observed that the gap repair halted within the hairpin structure regions. Such small gap repair products appear to be distributed in the Antirrhinum genome, but are unlikely to be active. Our data strongly suggest that the structural homogeneity of Tam3 was caused by immunity to gap repair at the hairpins in both of the end regions. The frequency of extensive gap repair of de novo excision products in eukaryotic transposons was found to be correlated with the free energies of the secondary structures in the end regions. This fact suggests that the fates of transposon families might depend on the structures of their ends.     

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.     

Interaction between the Tam1 and Tam2 transposable elements of Antirrhinum majus

Summary Two stable derivatives of the highly unstable niv-53::Tam1 allele of Antirrhinum majus were analysed. In both derivatives the Tam1 element is integrated at the same site and in the same orientation as in the parental niv-53::Tam1 allele. In both cases the Tam1 element was found to carry a 5 bp deletion (CACTA) in one of its termini. This explains the excision deficiency of these two alleles of Tam1, niv-53::Tam1-46 and niv-53::Tam1-49. Niv-44::Tam2, another stable nivea mutation, carries the 5 kb element Tam2, which is not a derivative of Tam1 but possesses identical terminal inverted repeats. When the stable lines 46 and 49 were corssed with line 44, suprisingly, a high number of the flowers in the F₁ displayed a variegated phenotype. Sequence analysis of two germinal revertants isolated from the heterozygote niv-53::Tam1-46/niv-44::Tam2 shows excision of the Tam2 element. This indicates that Tam2 is a defective element, which can be complemented by an active Tam1 element. However, the variegated F₁ phenotype observed is not inherited monofactorially. Variegation is seen only at particular times of development of the F₁ plants. These phenomena seem to involve both the Tam1 and Tam2 transposable elements.     

Tam3 produces a suppressible allele of the DAG locus of Antirrhinum majus similar to Mu-suppressible alleles of maize

Tam3 from Antirrhinum majus belongs to the Ac/Ds family of transposable elements. An allele of the DAG locus of Antirrhinum ( dag ::Tam3), which is required for chloroplast development and leaf palisade differentiation, has been generated by Tam3 insertion into the untranslated leader sequence of the gene. This allele gives rise to a cold-sensitive phenotype, where mutant tissue containing wild-type revertant somatic sectors is observed in the leaves of plants grown at 15°C, while leaves of plants grown at 25°C appear near wild-type. The temperature sensitivity of dag ::Tam3 results from expression of the DAG locus responding to the activity of the transposable element, the transposition of which is very sensitive to growing temperature. Genetic suppression of Tam3 transposition, using the STABILISER locus, also results in suppression of the dag mutant phenotype. dag ::Tam3 represents a Tam3-suppressible allele similar to those described for Mu transposons in maize. Suppression of the dag mutant phenotype in response to element inactivation appears to result from use of an alternative promoter at the 3' end of the Tam3 element. The production of suppressible alleles by an Ac-like element is discussed in relation to the mutagenic potential of plant transposons in producing complex genetic diversity.     

The transposable element Tam3 of Antirrhinum majus generates a novel type of sequence alterations upon excision

Summary The 3.5 kb transposable element, Tam3, has been shown to cause somatic and germinal instability at the nivea locus, which encodes chalcone synthase, of Antirrhinum majus. Molecular cloning and sequence analysis of the niv-98::Tam3 allele revealed that the termini of Tam3 consist of 12 bp perfect inverted repeats. Tam3 is integrated in the promoter region of the chalcone synthase gene and generates an 8 bp duplication of target sequences upon integration. DNA sequencing of a niv ⁺x revertant, niv-164, revealed a new type of sequence alteration upon excision: the duplications are displaced by ten nucleotides generated from adjacent sequences. Structural similarities of Tam3 and the maize elements Ac/Ds suggest that these elements belong to a common family.Dedicated to Professor Georg Melchers to celebrate his 50-year association with the journal     

Phenotypic effects of short-range and aberrant transposition in Antirrhinum majus

We describe two novel ways in which changes in gene expression in Antirrhinum majus may arise as a consequence of the Tam3 transposition mechanism. One involves excision of Tam3 from the nivea gene promoter and insertion of two new Tam3 copies 3.4 kb and 2.1 kb away, on either side of the excision site. One of the new insertions is in the nivea coding region and completely blocks production of an active gene product. This allele probably arose by a symmetrical double transposition, following chromosome replication. The second case involves a small deletion at one end of Tam3 in the pallida gene, flanked by a sequence typical of a Tam3 excision footprint. This suggests that the end of Tam3 was cleaved at an early step in an attempted transposition and re-ligated back to its original flanking sequence. The alteration restores some expression to the pallida gene, suggesting that the ends of the intact Tam3 element contain components which can actively inhibit gene expression. The implications of these findings for the mechanism of Tam3 transposition and for the effects of Tam3 on host gene expression are discussed.     

Homogeneity in the structure of the medaka fish transposable element Tol2

The hAT family is a group of transposable elements of the terminal inverted repeat class, which includes Ac of maize, hobo of Drosophila and Tam3 of Antirrhinum (snapdragon). All the members of this family so far examined are known to comprise complete and defective copies, with a good correspondence to autonomous and non-autonomous elements, respectively. Internal deletion is the most common cause of defective copies. Tol2, a transposable element of the medaka fish Oryzias latipes, is a member of the hAT family. We examined, mainly by the genomic Southern blot analysis, variation in the structure of copies of this element, and revealed that there are few or no internally deleted copies. This situation is unusual in a member of the hAT family. Possible causes of this anomaly are discussed.     

A comparative study of Tam3 and Ac transposition in transgenic tobacco and petunia plants

Transposition of the Anthirrinum majus Tam3 element and the Zea mays Ac element has been monitored in petunia and tobacco plants. Plant vectors were constructed with the transposable elements cloned into the leader sequence of a marker gene. Agrobacterium tumefaciens-mediated leaf disc transformation was used to introduce the transposable element constructs into plant cells. In transgenic plants, excision of the transposable element restores gene expression and results in a clearly distinguishable phenotype. Based on restored expression of the hygromycin phosphotransferase II (HPTII) gene, we established that Tam3 excises in 30% of the transformed petunia plants and in 60% of the transformed tobacco plants. Ac excises from the HPTII gene with comparable frequencies (30%) in both plant species. When the -glucuronidase (GUS) gene was used to detect transposition of Tam3, a significantly lower excision frequency (13%) was found in both plant species. It could be shown that deletion of parts of the transposable elements Tam3 and Ac, removing either one of the terminal inverted repeats (TIR) or part of the presumptive transposase coding region, abolished the excision from the marker genes. This demonstrates that excision of the transposable element Tam3 in heterologous plant species, as documented for the autonomous element Ac, also depends on both properties. Southern blot hybridization shows the expected excision pattern and the reintegration of Tam3 and Ac elements into the genome of tobacco plants.     

Cloning and Characterization of a Foldback DNA Element from Antirrhinum majus

A fold back sequence linked to a copy of the transposable element, Tam2, has been Isolated, from a genomic library constructed using partially digested and size-fractionated Antirrhinum DNA Into λ EMBL4 vector, by using Tam2 specific probe. The fold back sequence visualized by heteroduplex analysis reveals a typical stem-loop structure. The foldback element is around 1.2 kb with an average of 227 bp of Inverted termini forming the stem.     

Transposable elements generate novel spatial patterns of gene expression in Antirrhinum majus

The pallida gene of A. majus encodes a product required for the synthesis of red flower pigment. We have shown that the unstable pallida(recurrens) mutation is due to the insertion of the Tam3 transposable element near the promoter of the gene. Imprecise excision of Tam3 alters pallida gene expression and generates new spatial patterns or different intensities of flower pigmentation. Distinct spatial patterns may also result from rearrangements induced by Tam3 that alter the relative position of the pallida gene. Changes in Tam3 structure or position result in new unstable phenotypes. These findings suggest that genes may be rendered genetically hypervariable as a consequence of transposable element insertion and excision.     

PCR detection of excision suggests mobility of the medaka fish Tol1 transposable element in the frog Xenopus laevis

Tol1 is a DNA-based transposable element identified in the medaka fish Oryzias latipes and a member of the hAT (hobo/Activator/Tam3) transposable element family. Its mobility has already been demonstrated in the human and mouse, in addition to its original host species. This element is thus expected to be useful in a wide range of vertebrates as a genomic manipulation tool. Herein, we show that the Tol1 element can undergo excision in the African clawed frog Xenopus laevis, a major model organism for vertebrate genetics and developmental biology. An indicator plasmid carrying a Tol1 element was injected into 2- or 4-cell-stage embryos together with either a helper plasmid coding for the full-length Tol1 transposase or a modified helper plasmid yielding a truncated protein, and recovered from tailbud-stage embryos. Deletion of the Tol1 region of the indicator plasmid was observed in the experiment with the full-length transposase, and not in the other case. The deletion was associated with various footprint sequences at breakpoints, as frequently observed with many DNA-based transposable elements. These results indicate that the Tol1 element was excised from the indicator plasmid by catalysis of the transposase, and suggest that the Tol1 element is mobile in this frog species.     

Immobilized copies with a nearly intact structure of the transposon Tam3 in Antirrhinum majus: implications for the cis-element related to the transposition

 In this study we have focused on two copies of the transposon Tam3 isolated from an Antirrhinum majus plant which has flower variegation due to the excision of Tam3 from the nivea locus. These two copies possess a high homology, over 95%, to an active Tam3 element found in the nivea ^{recurrence:Tam3} allele. Although somatic excision of the Tam3 copy from the nivea locus can be detected at 15°C by Southern blotting, neither of the two copies showed any sign of the excision. Both of the immobilized copies were also found in five varieties from different A. majus sources, all of which contain common fragments. The results suggest that the two copies have been fixed in the genomes of many A. majus varieties. Structural differences between these immobilized copies and the known active copy were mainly observed in the subterminal regions, including the terminal inverted repeats. The immobility of the two Tam3 copies might be due to mutations within the end regions of essential cis-elements in Tam3 transposition, as reported for Ac and En/Spm. Received: 30 June 1997 / Accepted: 5 August 1997